1 I Volume 167 Number 1 THE BIOLOGICAL BULLETIN e AUG 29 1984 PUBLISHED BY ^ i MARINE BIOLOGIC Ay^A^QRATORY THE f3SS. Editorial Board Robert B. Barlow, Jr., Syracuse University Michael G. O'Rand, Laboratories for Cell Biology, . ^ ^ ..^ University of North Carolina at Chapel Hill Wallis H. Clark, Jr., University of California at Davis Ralph S. Quatrano, Oregon State University at ^ ,- ^ ., • • rr-, J Corvallis David H. Evans, University of Florida ^.-K. GoviND, Scarborough Campus, University Lionel L Rebhun, University of Virginia of Toronto Dorothy M. Skinner, Oak Ridge National Judith P. Grassle, Marine Biological Laboratory Laboratory Harlyn O. Halvorson, Brandeis University John D. Strandberg, Johns Hopkins University Maureen R. Hanson, University of Virginia John M. Teal, Woods Hole Oceanographic ,, . Institution Ronald R. Hoy, Cornell University Samuel S. Koide, The Population Council, ^- ^f "^°^ Whittaker Boston University Rockefeller University Marine Program and Manne Biological Laboratory Frank J. Longo, University of Iowa George M. Woodwell, Ecosystems Center. Marine Biological Laboratory Charlotte P. Mangum, The College of William and Mary Seymour Zigman, University of Rochester Editor: CHARLES B. METZ, University of Miami AUGUST, 1984 Printed and Issued by LANCASTER PRESS, Inc. PRINCE &. LEMON STS. LANCASTER. PA. New: MBL Library Serials Publications List Complete serial holdings of the combined libraries of the Marine Biological Laboratory and the Woods Hole Oceanographic Institution. — 1 983 Edition — 292 pages, softcover— $10.°° per copy Order From: Library Marine Biological Laboratory Woods Hole, Massachusetts 02543 THE BIOLOGICAL BULLETIN The Biological Bulletin is published six times a year by the Marine Biological Laboratory, MBL Street, Woods Hole, Massachusetts 02543. Subscriptions and similar matter should be addressed to The Biological Bulletin, Marine Biological Laboratory, Woods Hole, Massachusetts. Single numbers, $13.00. Subscription per volume (three issues), $32.50 ($65.00 per year for six issues). Communications relative to manuscripts should be sent to Dr. Chades B. Metz, Editor, or Pamela Clapp, Assistant Editor, at the Marine Biological Laboratory, Woods Hole, Massachusetts 02543 between May 1 and October 1, and at the Institute For Molecular and Cellular Evolution, University of Miami, 521 Anastasia, Coral Gables, Florida 33134 during the remainder of the year. Postmaster: Send address changes to The Biological Bulletin, Marine Biological Laboratory, Woods Hole, MA 02543. Copyright © 1984, by the Marine Biological Laboratory Second-class postage paid at Woods Hole, MA, and additional mailing offices. ISSN 0006-3185 INSTRUCTIONS TO AUTHORS The Biological Bulletin accepts outstanding original research reports of general interest to biologists throughout the world. Papers are usually of intermediate length (10-40 manuscript pages). Very short papers (less than 10 manuscript pages including tables, figures, and bibliography) will be published in a separate section entitled "Short Reports." A limited number of solicited review papers may be accepted after formal review. A paper will usually appear within four months after its acceptance. The Editorial Board requests that manuscripts conform to the requirements set below; those manuscripts which do not conform will be returned to authors for correction before review. 1 . Manuscripts. Manuscripts, including figures, should be submitted in triplicate. (Xerox copies of photographs are not acceptable for review purposes.) 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Citations should include complete titles and inclusive pagination. Journal abbreviations should normally follow those of the U. S. A. Standards Institute (USASI), as adopted by Biological Abstracts and Chemical Abstracts, with the minor differences set out below. The most generally useful list of biological journal titles is that published each year by Biological Abstracts (biosis List of Serials; the most recent issue). Foreign authors, and others who are accustomed to using The World List of Scientirc Periodicals, may find a booklet published by the Biological Council of the U.K. (obtainable from the Institute of Biology, 41 Queen's Gate, London, S.W.7, England, U.K.) useful, since it sets out the World List abbreviations for most biological journals with notes of the USASI abbreviations where these differ. Chemical Abstracts publishes quarterly supplements of additional abbreviations. The following points of reference style for The Biological Bulletin differ from USASI (or modified World List) usage: A. Journal abbreviations, and book titles, all underlined (for italics) B. All components of abbreviations with initial capitals (not as European usage in World List e.g. J. Cell. Comp. Physiol. NOT / cell. comp. Physiol.) C. All abbreviated components must be followed by a period, whole word components must not {i.e. J. Cancer Res.) D. Space between all components {e.g. J. Cell. Comp. Physiol., not J. Cell. Comp. Physiol.) E. Unusual words in journal titles should be spelled out in full, rather than employing new abbreviations invented by the author. For example, use Rit Visindafjelags Islendinga without abbreviation. F. All single word journal titles in full {e.g. Veliger. Ecology, Brain). G. The order of abbreviated components should be the same as the word order of the complete title {i.e. Proc. and Trans, placed where they appear, not transposed as in some Biological Abstracts listings). H. A few well-known international journals in their preferred forms rather than World List or USASI usage {e.g. Nature, Science, Evolution NOT Nature, Lond., Science, N.Y.; Evolution, Lancaster, Pa.) 6. Reprints, charges. The Biological Bulletin has no page charges. However, authors will be requested to help pay printing charges of manuscripts that are unusually costly due to length or numbers of tables, figures, or formulae. Reprints may be ordered at time of publication and normally will be delivered about two to three months after the issue date. Authors (or delegates or foreign authors) will receive page proofs of articles shortly before publication. They will be charged the current cost of printers' time for corrections to these (other than corrections of printers' or editors' errors). 11 THE BIOLOGICAL BULLETIN PUBLISHED BY THE MARINE BIOLOGICAL LABORATORY Editorial Board Robert B. Barlow, Jr., Syracuse University Wallis H. Clark, Jr., University of California at Davis David H. Evans, University of Florida C. K. GoviND, Scarborough Campus, University of Toronto Judith P. Grassle, Marine Biological Laboratory Harlyn O. Halvorson, Brandeis University Maureen R. Hanson, University of Virginia Ronald R. Hoy, Cornell University Samuel S. Koide, The Population Council, Rockefeller University Frank J. Longo, University of Iowa Charlotte P. Mangum, The College of William and Mary Michael G. O'Rand, Laboratories for Cell Biology, University of North Carolina at Chapel Hill Ralph S. Quatrano, Oregon State University at Corvallis Lionel I. Rebhun, University of Virginia Dorothy M. Skinner, Oak Ridge National Laboratory John D. Strandberg, Johns Hopkins University John M. Teal, Woods Hole Oceanographic Institution J. Richard Whittaker, Boston University Marine Program and Marine Biological Laboratory George M. Woodwell, Ecosystems Center, Marine Biological Laboratory Seymour Zigman, University of Rochester Editor: CHARLES B. METZ, University of Miami AUGUST, 1984 Printed and Issued by LANCASTER PRESS, Inc. PRINCE &. LEMON STS. LANCASTER, PA. The Biological Bulletin is issued six times a year at the Lancaster Press, Inc., Prince and Lemon Streets, Lancaster, Penn- sylvania. Subscriptions and similar matter should be addressed to The Biological Bulletin, Marine Biological Laboratory, Woods Hole, Massachusetts. Single numbers, $13.00. Subscription per volume (three issues), $32.50 ($65.00 per year for six issues). Communications relative to manuscripts should be sent to Dr. Charles B. Metz, Marine Biological Laboratory, Woods Hole, Mas- sachusetts 02543 between May 1 and October 1 , and to Dr. Charles B. Metz, Institute For Molecular and Cellular Evolution, University of Miami, 521 Anastasia, Coral Gables, Florida 33134 during the remainder of the year. The Biological Bulletin (ISSN 0006-3185) Postmaster: Send address changes to The Biological Bulletin, Marine Biological Laboratory, Woods Hole, MA 02543. Second-class postage paid at Woods Hole, MA, and additional mailing offices. LANCASTER press, INC.. LANCASTER, PA. THE MARINE BIOLOGICAL LABORATORY Eighty-sixth Report, for the Year 1983 — Ninety-sixth Year I. Trustees and Standing Committees 1 II. Members of the Corporation 5 1. Life Members 5 2. Regular Members 7 3. Associate Members 25 III. Certificate of Organization 28 IV. Articles of Amendment 29 V. Bylaws 30 VI. Report of the Director 34 VII. Report of the Treasurer and the Controller 45 VIII. Report of the Librarian 55 IX. Educational Programs 55 1 . Summer 55 2. January 65 3. Spring 66 4. Short Courses 67 X. Research and Training Programs 72 1 . Summer 72 2. Year-round 79 XI. Honors 85 XII. Institutions Represented 87 XIII. Laboratory Support Staff 91 I. TRUSTEES Including Action of the 1983 Annual Meeting Officers Prosser Gifford, Chairman of the Board of Trustees, Woodrow Wilson International Center for Scholars, Smithsonian Building, Washington, DC 20560 Denis M. Robinson, Honorary Chairman of the Board of Trustees, High Voltage Engineering Corporation, Burlington, Massachusetts 01830 Robert Mainer, Treasurer, The Boston Company, One Boston Place, Boston, Massachusetts 02106 Paul R. Gross, President of the Corporation and Director of the Laboratory. Marine Biological Laboratory, Woods Hole, Massachusetts 02543 David D. Potter, Clerk, Harvard Medical School, Cambridge, Massachusetts 02138 Copyright © 1984, by the Marine Biological Laboratory Library of Congress Card No. A38-5 1 8 (ISSN 0006-3185) MARINE BIOLOGICAL LABORATORY Emeriti John B. Buck, National Institutes of Health AURIN Chase, Princeton University Anthony C. Clement, Emory University Kenneth S. Cole, San Diego, California Arthur L. Colwin, University of Miami Laura Colwin, University of Miami D. Eugene Copeland, Marine Biological Laboratory Sears Crowell, Indiana University Alexander T. Daignault, W. R. Grace Company Harry Grundfest, Columbia University (deceased 10/83) Thru Hayashi, Miami, Florida Hope Hibbard, Oberlin College Lewis Kleinholz, Reed College Maurice Krahl, Tucson, Arizona Douglas Marsland, Cockysville, Maryland Charles B. Metz, University of Miami Harold H. Plough, Amherst, Massachusetts C. Ladd Prosser, University of Illinois John S. Rankin, Ashford, Connecticut Meryl Rose, Waquoit, Massachusetts George T. Scott, Woods Hole, Massachusetts Mary Sears, Woods Hole, Massachusetts Carl C. Speidel, University of Virginia (no mailings) Albert Szent-Gyorgyi, Marine Biological Laboratory W. Randolph Taylor, University of Michigan George Wald, Harvard University Class OF 1987 Edward A. Adelberg, Yale University James M. Clark, Shearson/American Express Harold Gainer, National Institutes of Health William Golden, New York, New York Hans Kornberg, University of Cambridge Laszlo Lorand, Northwestern University Carol Reinisch, Tufts University Howard A. Schneiderman, Monsanto Company Sheldon J. Segal, The Rockefeller Foundation Class OF 1986 George H. A. Clowes, Jr., Cancer Research Institute Gerald Fischbach, Washington University John E. Hobbie, Ecosystems Center Edward A. Kravitz, Harvard Medical School RODOLFO Llinas, New York University Thomas Reese, National Institutes of Health D. Thomas Trigg, Wellesley, Massachusetts Nancy Sabin Wexler (elected 2/84) J. Richard Whittaker, Marine Biological Laboratory Class OF 1985 Robert W. Ashton, Gaston Snow Beekman and Bogue Geno a. Ballotti (elected 2/84) TRUSTEES AND STANDING COMMITTEES Harlyn O. Halvorson, Brandeis University John G. Hildebrand, Columbia University Thomas J. Hynes, Jr., Meredith & Grew, Inc. Shinya Inoue, Marine Biological Laboratory Richard P. Mellon, Richard King Mellon Foundation (resigned 8/83) John W. Moore, Duke University W. D. Russell-Hunter, Syracuse University Evelyn Spiegel, Dartmouth College Class OF 1984 Clay Armstrong, University of Pennsylvania Robert B. Barlow, Jr., Syracuse University Joel P. Davis, Seapuit, Inc. Judith Grassle, Marine Biological Laboratory Holger Jannasch, Woods Hole Oceanographic Institution Benjamin Kaminer, Boston University Brian Salzberg, University of Pennsylvania W. Nicholas Thorndike, Boston, Massachusetts Richard W. Young, Houghton Mifflin Company STANDING COMMITTEES EXECUTIVE Committee of the Board of Trustees Prosser Gifford* Paul R. Gross* Robert Mainer* John E. Hobbie, 1986 Edward A. Kravitz, 1986 Harlyn O. Halvorson, 1985 J. Richard Whittaker, 1985 John G. Hildebrand, 1984 Benjamin Kaminer, 1984 Buildings and Grounds Committee Francis Hoskin, Chairman Lawrence B. Cohen A. Farmanfarmaian Alan Fein Daniel Gilbert Clifford Harding, Jr. Donald B. Lehy* Philip Person Robert Prusch Thomas Reese Evelyn Spiegel Capital Development Committee Richard W. Young, Chairman Joel P. Davis Prosser Gifford* William T. Golden Paul R. Gross* Harlyn O. Halvorson Robert Mainer* Carol Gannon Salguero* D. Thomas Trigg Employee Relations Committee Catherine Norton, Chairman Ed Enos William Evans John Helfrich John MacLeod Carol Wagner 4 marine biological laboratory Financial Policy and Planning Committee George H. A. Clowes, Jr., Chairman Robert Mainer Robert Ashton W. Nicholas Thorndike Thomas Hynes J. Richard Whittaker Housing, Food Service, and Day Care Committee Jelle Atema, Chairman Mona Gross Daniel Alkon Thomas Reese Nina Allen Brian Salzberg Robert B. Barlow, Jr. Homer P. Smith* Gail Burd Susan Szuts Instruction Committee Judith Grassle, Chairman Bruce Peterson Randall S. Alberte Brian Salzberg John Dowling Herbert Schuel Alan Fein Andrew Szent-Gyorgyi George Pappas Investment Committee W. Nicholas Thorndike, Chairman William T. Golden John Arnold Maurice Lazarus Prosser Gilford* Robert Mainer* Library Joint Management Committee Paul R. Gross, Chairman Joe Kjebala Edward A. Adelberg John Speer George Grice John Steele Library Joint Users Committee Edward A. Adelberg, Chairman Shinya Inoue Wilfred Bryan John Schlee John Dowling Fredric Serchuk Frederick Grassle Oliver Zarriou Marine Resources Committee Sears Crowell, Chairman Jack Levin Carl J. Berg Anne F. O'Melia June Harrigan John S. Rankin Bill Jeffery John Valois* Izja Lederhendler Jonathan Wittenberg Louis Leibovitz Radiation Committee Paul DeWeer, Chairman Louis Kerr* Richard L. Chappell Anthony Liuzzi Sherwin Cooperstein Joseph Neary Daniel Grosch . Harris Ripps trustees and standing committees Research Services Committee Raymond Stephens, Chairman Bryan Noe Jella Atema Barry O'Neil* Robert Barlow, Jr. Bruce Peterson Robert Goldman Birgit Rose John Hildebrand Joel Rosenbaum Samuel S. Koide Sidney Tamm Raymond Lasek Research Space Committee J. Richard Whittaker, Chairman Rodolfo Llinas Clay Armstrong Laszlo Lorand Arthur Dubois Eduardo Macagno Robert Goldman Jerry Melillo David Landowne Alan Pearlman Hans Laufer Joel Rosenbaum Safety Committee John Hobbie, Chairman E. F. MacNichol, Jr. Daniel Alkon Barry O'Neil* Eugene Copeland Raymond Stephens Louis Kerr Paul Steudler Alan Kuzirian Frederick Thrasher Donald Lehy * ex officio II. MEMBERS OF THE CORPORATION Including Action of the 1983 Annual Meeting Life Members Abbott, Marie, 259 High St., R.D. 2, Coventry, CT 06238 Adolph, Edward F., University of Rochester, School of Medicine and Dentistry, Rochester, NY 14642 Beams, Harold W., Department of Zoology, University of Iowa, Iowa City, I A 53342 Behre, Ellinor, Black Mountain, NC 28711 Bertholf, Lloyd M., Westminster Village #2114, 2025 E. Lincoln St., Bloomington, IL 61701 Bishop, David W., Department of Physiology, Medical College of Ohio, C. S. 10008, Toledo, OH 43699 Bold, Harold C, Department of Botany, University of Texas, Austin, TX 78712 Bridgman, a. Josephine, 715 Kirk Rd., Decatur, GA 30030 Burbanck, Madeline P., Box 15134, Atlanta, GA 30333 Burbanck, William D., Box 15134, Atlanta, GA 30333 Burdick, C. Lalor, 900 Barley Drive, Barley Mill Court, Wilmington, DE 19807 Carpenter, Russell L., 60 Lake St., Winchester, MA 01890 Chase, Aurin, Professor of Biology Emeritus, Princeton University, Princeton, NJ 08540 Cheney, Ralph H., 45 Coleridge Drive, Falmouth, MA 02540 (deceased 3/84) Clarke, George L., 44 Juniper Rd., Belmont, MA 02178 Clement, Anthony C, Department of Biology, Emory University, Atlanta, GA 30322 (de- ceased 6/84) 6 MARINE BIOLOGICAL LABORATORY Cole, Kenneth S., 2404 Loring St., San Diego, CA 92109 (deceased 4/84) CoLWiN, Arthur, 320 Woodcrest Rd., Key Biscayne, FL 33149 COLWIN, Laura, 320 Woodcrest, Key Biscayne, FL 33149 COPELAND, D. E., 41 Fern Lane, Woods Hole, MA 02543 COSTELLO, HELEN M., 507 Monroe St., Chapel Hill, NC 27514 Crouse, Helen, Institute of Molecular Biophysics, Florida State University, Tallahassee, FL 32306 DiLLER, Irene C, 2417 Fairhill Ave., Glenside, PA 19038 DiLLER, William F., 2417 Fairhill Ave., Glenside, PA 19038 Elliott, Alfred M., 2345 Tarpon Rd., Naples, FL 33992 Ferguson, James K. W., 56 Clarkehaven St., Thomhill, Ontario L4J 2B4 Canada Fraenkel, Gottfried S., Department of Entomology, University of Ilhnois, 320 Morrill Hall, Urbana, IL 61801 Fries, Erik F. B., 3870 Leafy Way, Miami, FL 33133 GiLMAN, Lauren C, Department of Biology, University of Miami, PO Box 24918, Coral Gables, FL 33124 Graham, Herbert, 36 Wilson Road, Woods Hole, MA 02543 Green, James W., Department of Physiology, Rutgers University, Piscataway, NJ 08854 Grundfest, Harry, Department of Neurology, College of Physicians and Surgeons, Columbia University, New York, NY 10032 (deceased 10/83) GUTTMAN, Rita, 75 Henry St., Brooklyn, NY 1 1210 (deceased 10/83) Hamburger, Viktor, Professor Emeritus, Washington University, St. Louis, MO 63 1 30 Hamilton, Howard L., Department of Biology, University of Virginia, Charlottesville, VA 22901 Hibbard, Hope, 143 East College St., Apt. 309, Oberiin, OH 44074 Hisaw, F. L. 5925 SW Plymouth Drive, Corvallis, OR 97330 Hollaender, Alexander, Associated Universities, Inc. 1717 Massachusetts Ave., NW, Washington, DC 20036 Humes, Arthur, Marine Biological Laboratory, Woods Hole, MA 02543 Johnson, Frank H., Department of Biology, Princeton University, Princeton, NJ 08540 Kaan, Helen, 62 Locust St., Falmouth, MA 02540 Kahler, Robert, P.O. Box 423, Woods Hole, MA 02543 KiLLE, Frank R., 1111 S. Lakemont Ave., #444, Winter Park, FL 32792 Kleinholz, Lewis, Department of Biology, Reed College, Portland, OR 97202 Levine, Rachmiel, 2024 Canyon Rd., Arcadia, CA 91006 Lochhead, John H., 49 Woodlawn Rd., London SW 6 6PS, England, U. K. Lynn, W. Gardner, Department of Biology, Catholic University of America, Washington, DC 20017 Magruder, Samuel R., 270 Cedar Lane, Paducah, KY 42001 Manwell, Reginald, D., Syracuse University, Lyman Hall, Syracuse, NY 13210 Marsland, Douglas, Broadmead N12, 13801 York Rd., Cockeysville, MD 21030 Miller, James A., 307 Shorewood Drive, E. Falmouth, MA 02536 Milne, Lorus J., Department of Zoology, University of New Hampshire, Durham, NH 03824 Moore, John A., Department of Biology, University of Cahfomia, Riverside, CA 92521 MOUL, E. T., 43 F. R. Lillie Rd., Woods Hole, MA 02543 Nace, Paul F., 5 Bowditch Road, Woods Hole, MA 02543 Nachmanshon, David, Department of Neurology, College of Physicians and Surgeons Co- lumbia University, New York, NY 10032 (deceased 11/83) Page, Irving H., Box 516, Hyannisport, MA 02647 Plough, Harold H., 31 Middle St., Amherst, MA 01002 POLLISTER, A. W., Box 23, Dixfield, ME 04224 Pond, Samuel E., P.O. Box 63, E. Winthrop, ME 04343 Prosser, C. Ladd, Department of Physiology and Biophysics, University of Illinois, Urbana, IL 61801 Prytz, Margaret McDonald, 21 McCouns Lane, Oyster Bay, NY 11771 Rankin, John S., Jr., Box 97, Ashford, CT 06278 Renn, Charles E., Route 2, Hempstead, MD 21074 MEMBERS OF THE CORPORATION 7 Reznikoff, Paul, 1 1 Brooks Rd., Woods Hole, MA 02543 (deceased 3/84) Richards, A. Glenn, Department of Entomology, Fisheries and Wildlife, University of Min- nesota, St. Paul, MN 55101 Richards, Oscar W., Pacific University, Forest Grove, OR 97462 SCHARRER, Berta, Department of Anatomy, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461 SCHMITT, F. O., Room 16-512, Massachusetts Institute of Technology, Cambridge, MA 02139 Shemin, David, Department of Biochemistry and Molecular Biology, Northwestern University, Evanston, IL 60201 Sichel, Elsa, 4 Whitman Rd., Woods Hole, MA 02543 (deceased 12/83) SONNENBLICK, B. P., Department of Zoology and Physiology, Rutgers University, 195 University Ave., Newark, NJ 07102 Speidel, CarlC, 1873 Field Rd., Charlottesville, VA 22903 (no mailings) Steinhardt, Jacinto, 1508 Spruce St., Berkeley, CA 94709 Stunkard, Horace W., American Museum of Natural History, Central Park West at 79th St., New York, NY 10024 Taylor, W. Randolph, Department of Biology, University of Michigan, Ann Arbor, MI 48109 TeWinkel, Lois E., 4 Sanderson Ave., Northampton, MA 01060 Tracer, William, The Rockefeller University, 1230 York Ave., New York, NY 10021 Travis, Dorothy F., 35 Coleridge Drive, Falmouth, MA 02540 (deceased 10/83) Wald, George, Higgins Professor of Biology Emeritus, Harvard University, Cambridge, MA 02138 Wichterman, Ralph, 3 1 Buzzards Bay Ave., Woods Hole, MA 02543 Young, D. B., 1137 Main St., N. Hanover, MA 02357 ZiNN, Donald J., P.O. Box 589, Falmouth, MA 02541 Zorzoli, Anita, Department of Botany, Vassar College, Poughkeepsie, NY 12601 Zweifach, Benjamin W., c/o Ames, University of CaUfomia, La Jolla, CA 92037 Regular Members Ache, Barry W., Whitney Marine Laboratory, University of Florida, Rt. 1 Box 121, St. Augustine, FL 32084 Acheson, George H., 25 Quissett Ave., Woods Hole, MA 02543 Adams, James A., Department of Biological Sciences, Tennessee State University 3500 John Merritt Blvd., Nashville, TN 37203 Adelberg, Edward A., Department of Human Genetics, Yale University Medical School, P.O. Box 3333, New Haven, CT 06510 Afzelius, Bjorn, Wenner-Gren Institute, University of Stockholm, Stockholm, Sweden Alberte, Randall S., University of Chicago, Barnes Laboratory, 5630 S. Ingleside Ave., Chicago, IL 60637 Albright, John T., 7 Siders Pond Rd., Falmouth, MA 02540 Alkon, Daniel, Section on Neural Systems, Laboratory of Biophysics, NIH, Marine Biological Laboratory, Woods Hole, MA 02543 Allen, Garland E., Department of Biology, Washington University, St. Louis, MO 63130 Allen, NinaS., Department of Biology, Wake Forest University, Box 7325, Reynolds Station, Winston-Salem, NC 27109 Allen, Robert D., Department of Biology, Dartmouth College, Hanover, NH 03755 Alscher, Ruth, Department of Biology, Manhattanville College, Purchase, NY 10577 Amatniek., Ernest, 4797 Boston Post Rd., Pelham Manor, NY 10803 Anderson, Everett, Department of Anatomy, LHRBB, Harvard Medical School, Boston, MA 02115 Anderson, J. M., Cornell University, Emerson Hall, Ithaca, NY 14850 Armet-Kjbel, Christine, Biology Department, University of Massachusetts — Boston, Boston, MA 02125 Armstrong, Clay M., Department of Physiology, Medical School, University of Pennsylvania, Philadelphia, PA 19174 8 MARINE BIOLOGICAL LABORATORY Armstrong, Peter B., Department of Zoology, University of California, Davis, CA 95616 Arnold, John M., Pacific Biomedical Research Center, University of Hawaii, 41 Ahui St., Honolulu, HI 96813 Arnold, William A., 102 Balsam Rd., Oak Ridge, TN 37830 ASHTON, Robert W., Gaston Snow Beekman and Bogue, 14 Wall St., New York, NY 10005 Atema, Jelle, Marine Biological Laboratory, Woods Hole, MA 02543 Atwood, Kimball C, 100 Haven Ave., Apt. 21-E, New York, NY 10032 Augustine, George, Jr., Department of Biology, University of California, Los Angeles, CA 90024 Austin, Mary L., 506'/2 N. Indiana Ave., Bloomington, IN 47401 Bacon, Robert, P.O. Box 723, Woods Hole, MA 02543 Baker, Robert G., New York University Medical Center, 550 First Ave., New York, NY 10016 Baldwin, Thomas O., Department of Biochemistry and Biophysics, Texas A&M University, College Station, TX 77843 Ballotti, Geno a., Permanent Charity Fund of Boston, Boston Place, Boston. MA 02106 Bang, Betsy, 76 F. R. Lillie Rd., Woods Hole, MA 02543 Barker, Jeffery L., NIH, Bldg. 36 Room 2002, Bethesda, MD 20205 Barlow, Robert B., Jr., Institute for Sensory Research, Syracuse University, Merrill Lane, Syracuse, NY 13210 Bartell, Clelmer K., 2000 Lake Shore Drive, New Orleans, LA 70122 Barth, Lucena J., 26 Quissett Ave., Woods Hole, MA 02543 Bartlett, James H., Department of Physics, Box 1921, University of Alabama, University, AL 35486 Battelle, Barbara-Anne, National Eye Institute, NIH, Bethesda, MD 20205 Bauer, G. Eric, Department of Anatomy, University of Minnesota, Minneapolis, MN 55414 Beauge, Luis Alberto, Instituto de Investigacion Medica, Casilla de Correo 389, 5000 Cordoba, Argentina Beck, L. V., School of Experimental Medicine, Department of Pharmacology, Indiana Uni- versity, Bloomington, IN 47401 Begg, David A., LHRRB, Harvard Medical School, 45 Shattuck St., Boston, MA 021 15 Bell, Eugene, Department of Biology, Massachusetts Institute of Technology, 77 Massachusetts Ave., Cambridge, MA 02139 Bennett, M. V. L., Albert Einstein College of Medicine, Department of Neuroscience, 1300 Morris Park Ave., Bronx, NY 10461 Bennett, Miriam F., Department of Biology, Colby College, Waterville, ME 04901 Berg, Carl J., Jr., Marine Biological Laboratory, Woods Hole, MA 02543 Berne, Robert W., University of Virginia, School of Medicine, Charlottesville, VA 22908 Bernheimer, Alan W., New York University, College of Medicine, New York, NY 10016 (Life Member 10/83) Bezanilla, Francisco, Department of Physiology, University of California, Los Angeles, CA 90052 BiGGERS, John D., Department of Physiology, Harvard Medical School, Boston, MA 02115 Bishop, Stephen H., Department of Zoology, Iowa State University, Ames, lA 50010 Bodian, David, Department of Otolaryngolgy, Johns Hopkins University, 1721 Madison St., Baltimore, MD 21206 Boettiger, Edward G., 29 Juniper Point, Woods Hole, MA 02543 BOGORAD, Lawrence, The Biological Laboratories, Harvard University, Cambridge, MA 02 1 38 Boolootian, Richard A., Science Software Systems, Inc., 11899 W. Pico Blvd., W. Los Angeles, CA 90064 BOREI, Hans G., Department of Zoology, University of Pennsylvania, Philadelphia, PA 19174 Borgese, Thomas A., Department of Biology, Lehman College, CUNY, Bronx, NY 10468 BORISY, Gary G., Laboratory of Molecular Biology, University of Wisconsin, Madison, WI 53715 BoscH, Herman F., Whipple Hill, Richmond, NH 03470 BOTKIN, Daniel, Department of Biology, University of California, Santa Barbara, CA 93106 BOWEN, Vaughn T., 652 Knox Rd., Strafford, Wayne, PA 19087 MEMBERS OF THE CORPORATION 9 Bowles, Francis P., P.O. Box 674, Woods Hole, MA 02543 BOYER, Barbara C, Department of Biology, Union College, Schneclady, NY 12308 Brinley, F. J., Neurological Disorders Program, NINCDS, 716 Federal Building, Belhesda, MD 20205 Brown, Joel E., Department of Ophthalmology, Box 8096 Sciences Center, Washington University, 660 S. Euclid Ave., St. Louis, MO 63110 Brown, Stephen C, Department of Biological Sciences, SUNY, Albany, NY 12222 Buck, John B., NIH, Laboratory of Physical Biology, Room 1 12, Building 6 Bethesda, MD 20205 BURDICK, Carolyn J., Department of Biology, Brooklyn College, Brooklyn, NY 11210 Burger, Max, Department of Biochemistry, Biocenter of the University of Basel, Klingel- bergstrasse 70, CH-4056 Basel, Switzerland BURKY, Albert, Department of Biology, University of Dayton, Dayton, OH 45649 BURSTYN, Harold Lewis, 523 National Center, U. S. Geological Survey, Reston, VA 22092 BuRSZTAJN, Sherry, Neurology Department — Program in Neuroscience, Baylor College of Medicine, Houston, TX 77030 Bush, Louise, 7 Snapper Lane, Falmouth, MA 02540 Candelas, Graciela C, Department of Biology, University of Puerto Rico, Rio Piedras, PR 00931 Cariello, Lucio, Stazione Zoologica, Villa Comunale, Napoli, Italy Carlson, Francis D., Department of Biophysics, Johns Hopkins University, Baltimore, MD 21218 Case, James, Department of Biological Sciences, University of California, Santa Barbara, CA 93106 Cassidy, J. D., University of Illinois at Chicago Circle Department of Biological Sciences, Box 4348, Chicago, IL 60680 Cebra, John J., Department of Biology, Leidy Labs, G-6, University of Pennsylvania, Phil- adelphia, PA 19174 Chaet, Alfred B., University of West Florida, Pensacola, FL 32504 Chambers, Edward L., Department of Physiology and Biophysics, University of Miami, School of Medicine, P.O. Box 520875, Miami, FL 33152 Chang, Donald C, Department of Physiology, Baylor College of Medicine, 1200 Moursund, Houston, TX 77030 Chappell, Richard L., Department of Biological Sciences, Hunter College, Box 210, 695 Park Ave., New York, NY 10021 Chauncey, Howard H., 30 Falmouth St., Wellesley Hills, MA 02181 Child, Frank M., Department of Biology, Trinity College, Hartford, CT 06106 CiTKOWiTZ, Elna, 410 Livingston St., New Haven, CT 0651 1 Clark, A. M., 48 Wilson Rd., Woods Hole, MA 02543 Clark, Eloise E., Vice President for Academic Affairs, Bowling Green State University, Bowling Green, OH 43403 Clark, Hays, 26 Deer Park Drive, Greenwich, CT 06830 Clark, James M., Shearson/American Express, 14 Wall St., New York, NY 10005 Clark, Wallis H., Jr., Aquacuhure Program, Room 243, Department of Animal Science, University of California, Davis, CA 95616 Claude, Philippa, Primate Center, Capitol Court, Madison, WI 53706 Clayton, Roderick K., Cornell University, Section of Genetics, Development and Physiology, Ithaca, NY 14850 Clowes, George H. A., Jr., The Cancer Research Institute, 194 Pilgrim Rd., Boston, MA 02215 Clutter, Mary, Cellular and Physiological Biosciences Section, National Science Foundation, 1800 G St., NW, Washington, DC 20550 Cobb, Jewell P., President, California State University, Fullerton, CA 92634 Cohen, Adolph I., Department of Ophthalmology, School of Medicine, Washington University, 660 S. Euclid Ave., St. Louis, MO 631 10 Cohen, Carolyn, Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, MA 02154 10 MARINE BIOLOGICAL LABORATORY Cohen, Lawrence B., Department of Physiology, Yale University, 333 Cedar St., New Haven, CT 06510 Cohen, Seymour S., Department of Pharmacological Science, SUNY, Stony Brook, NY 11790 Cohen, William D., Department of Biological Sciences, Hunter College, 695 Park Ave., New York, NY 10021 Cole, Jonathan J., Institute for Ecosystems Studies, Cary Arboretum, Millbrook, NY 12545 Coleman, Annette W., Division of Biology and Medicine, Brown University, Providence, RI 02912 Collier, Jack R., Department of Biology, Brooklyn College, Brooklyn, NY 1 1210 Collier, MarjorieMcCann, Biology Department, Saint Peter's College, Kennedy Boulevard, Jersey City, NJ 07306 Cook, Joseph A., The Edna McConnell Clark Foundation, 250 Park Ave., New York, NY 10017 COOPERSTEIN, S. J., University of Connecticut, School of Medicine, Farmington Ave., Far- mington, CT 06032 Corliss, JohnO., Department of Zoology, University of Maryland, College Park, MD 20742 Cornell, Neal W., 6428 Bannockbum Drive, Bethesda, MD 20817 CORNMAN, Ivor, IOA Orchard St., Woods Hole, MA 02543 Corson, David Wesley, Jr., Laboratory of Sensory Physiology, Marine Biological Laboratory, Woods Hole, MA 02543 Costello, Walter J., College of Medicine, Ohio University, Athens, OH 45701 Couch, Ernest F., Department of Biology, Texas Christian University, Fort Worth, TX 76129 Cremer-Bartels, Gertrud, Universitats Augenklinik, 44 Munster, West Germany Crippa, Marco, Faculte des Sciences, Universite de Geneve, 20, quai Emest-Ansermet, Geneve 4, Switzerland Crow, Terry J., Department of Physiology, University of Pittsburgh, School of Medicine, Pittsburgh, PA 15261 Crowell, Sears, Department of Biology, Indiana University, Bloomington, IN 4740 1 Daignault, Alexander T., W. R. Grace Company, 1114 Avenue of the Americas, New York, NY 10036 Dan, Katsuma, Professor Emeritus, Tokyo Metropolitan Union, Meguro-ku, Tokyo, Japan David, John R., Seeley G. Mudd Building, Room 504, 250 Longwood Ave., Boston MA 02115 David, Roberta A., Seeley G. Mudd Building, Room 504, 250 Longwood Ave., Boston, MA 02115 Davis, Bernard D., Bacterial Physiology Unit, Harvard Medical School, 25 Shattuck St., Boston, MA 02115 Davis, Joel P., Seapuit, Inc., P.O. Box G, Osterville, MA 02655 Daw, Nigel W., 78 Aberdeen Place, Clayton, MO 63105 DeGroof, Robert C, RR#1 Box 343, Green Lane, PA 18054 DeHaan, Robert L., Department of Anatomy, Emory University, Atlanta, GA 30322 DeLanney, Louis E., Institute for Medical Research, 2260 Clove Drive, San Jose, CA 95128 DePhillips, Henry A., Jr., Department of Chemistry, Trinity College, Hartford, CT 06106 DeTerra, Noel, Marine Biological Laboratory, Woods Hole, MA 02543 Dettbarn, Wolf-Dietrich, Department of Pharmacology, School of Medicine, Vanderbilt University, Nashville, TN 37127 DeWeer, Paul J., Department of Physiology, School of Medicine, Washington University, SL Louis, MO 63110 DiSCHE, Zacharias, Eye Institute, College of Physicians and Surgeons, Columbia University, 639 W. 165 St., New York, NY 10032 Dixon, Keith E., School of Biological Sciences, Flinders University, Bedford Park, South Australia DowDALL, Michael J., Department of Biochemistry, University Hospital and Medical School, Nottingham N672 UH, England, U. K. Dowling, John E., The Biological Laboratories, Harvard University, 16 Divinity Ave., Cam- bridge, MA 02138 MEMBERS OF THE CORPORATION 1 1 Dubois, Arthur Brooks, John B. Pierce Foundation Laboratory, 290 Congress Ave., New Haven, CT 06519 Dudley, Patricia L., Department of Biological Sciences, Barnard College, Columbia University, New York, NY 10027 Dunham, Philip B., Department of Biology, Syracuse University, Syracuse, NY 13210 Ebert, James D., Office of the President, Carnegie Institution of Washington 1530 P St., NW, Washington, DC 20008 Eckberg, William R., Department of Zoology, Howard University, Washington, DC 20059 Eckert, Roger O., Department of Zoology, University of California, Los Angeles, CA 90024 Edds, Kenneth T., Department of Anatomical Sciences, SUNY, Buffalo, NY 14214 Edds, Louise, College of Osteopathic Medicine, Grosvenor Hall, Ohio University, Athens, OH 45701 Eder, Howard a., Albert Einstein College of Medicine, 1300 Morris Park Ave. Bronx, NY 10461 Edwards, Charles, Department of Biological Sciences, SUNY, Albany, NY 12222 Egyud, Laszlo G., P.O. Box 342, Woods Hole, MA 02543 Ehrenstein, Gerald, NIH, Bethesda, MD 20205 Ehrlich, Barbara E., Department of Physiology, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461 EiSEN, Arthur Z., Chief of Division of Dermatology, Washington University, St. Louis, MO 63110 Elder, Hugh Young, Institute of Physiology, University of Glasgow, Glasgow, Scotland, U. K. Elliott, Gerald P., The Open University Research Unit, Foxcombe Hall, Berkeley Rd., Boars Hill, Oxford, England, U. K. Epel, David, Hopkins Marine Station, Pacific Grove, CA 93950 Epstein, Herman T., Department of Biology, Brandeis University, Waltham, MA 02154 Erulkar, Solomon D., 318 Kent Rd., Bala Cynwyd, PA 19004 EssNER, Edward S., Kresge Eye Institute, Wayne State University, 540 E. Canfield Ave., Detroit, MI 48201 Failla, Patricia M., Office of the Director, Argonne National Laboratory, Argonne, IL 60439 Farmanfarmaian, a.. Department of Physiology and Biochemistry, Rutgers University, New Brunswick, NJ 08903 Faust, Robert G., Department of Physiology, Medical School, University of North Carolina, Chapel Hill, NC 27514 Fein, Alan, Laboratory of Sensory Physiology, Marine Biological Laboratory Woods Hole, MA 02543 Feldman, Susan C, Department of Anatomy, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, 100 Bergen St., Newark, NJ 07103 Ferguson, F. P., National Institute of General Medical Science, NIH, Bethesda, MD 20205 Fessenden, Jane, Marine Biological Laboratory, Woods Hole, MA 02543 FINKELSTEIN, ALAN, Albert Einstein College of Medicine, 1 300 Morris Park Ave., Bronx, NY 10461 FISCHBACH, Gerald, Department of Anatomy and Neurobiology, Washington University School of Medicine, St. Louis, MO 631 10 FiSCHMAN, Donald A., Department of Cell Biology and Anatomy, Cornell University Medical College, 1300 York Ave., New York, NY 10021 Fisher, J. Manery, Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada M5S 1A8 FiSHMAN, Harvey M., Department of Physiology, University of Texas Medical Branch, Gal- veston, TX 77550 Flanagan, Dennis, Scientific American, 415 Madison Ave., New York, NY 10017 Fox, Maurices., Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02138 Franzini, Clara, Department of Biology G-5, School of Medicine, University of Pennsylvania, Philadelphia, PA 19174 1 2 MARINE BIOLOGICAL LABORATORY Frazier, Donald T., Department of Physiology and Biophysics, University of Kentucky Medical Center, Lexington, KY 40536 Freeman, Alan R., Department of Physiology. Temple University, 3420 N. Broad St., Phil- adelphia, PA 19140 Freeman, Gary L., Department of Zoology, University of Texas, Austin, TX 78172 French, Robert J., Department of Biophysics, University of Maryland, School of Medicine, Baltimore, MD 21201 Freygang, Walter J., Jr., 6247 29th St., NW, Washington, DC 20015 Fulton, Chandler, M., Department of Biology, Brandeis University, Waltham, MA 02154 FURSHPAN, Edwin J., Department of Neurophysiology, Harvard Medical School, Boston, MA 02115 FUSELER, John W., Department of Cell Biology, University of Texas Medical Branch, 53233 Harry Hines Blvd., Dallas, TX 75235 FUTRELLE, Robert P., Department of Genetics and Development, 5 1 5 Morrill Hall, University of IlHnois, 505 S. Goodwin Ave., Urbana, IL 68101 Fye, Paul, P.O. Box 309, Woods Hole, MA 02543 Gabriel, Mordecai, Department of Biology, Brooklyn College, Brooklyn, NY 11210 Gainer, Harold, Section of Functional Neurochemistry, NIH, Bldg. 36 Room 2A21, Bethesda, MD 20205 Galatzer-Levy, Robert M., Room 1819, 55 East Washington Street, Chicago, IL 60602 Gall, Joseph G., Carnegie Institution, 115 West University Parkway, Baltimore, MD 21210 Gascoyne, Peter, Marine Biological Laboratory, Woods Hole, MA 02543 Gelfant, Seymour, Department of Dermatology, Medical College of Georgia, Augusta, GA 30904 Gelperin, Alan, Department of Biology, Princeton University, Princeton, NJ 08540 German, James L., Ill, The New York Blood Center, 310 East 67th St., New York, NY 10021 GiBBS, Martin, Institute for Photobiology of Cells and Organelles, Brandeis University, Wal- tham, MA 02154 Gibson, A. Jane, Wing Hall, Cornell University, Ithaca, NY 14850 GiFFORD, Prosser, The Wilson Center, Smithsonian Building, 1000 Jefferson Drive, SW, Washington, DC 20590 Gilbert, Daniel L., NIH, Laboratory of Biophysics, NINCDS, Bldg. 36, Room 2A-29, Bethesda, MD 20205 GiUDiCE, Giovanni, Via Archirafi 22, Palermo, Italy Glusman, Murray, Department of Psychiatry, Columbia University, 722 W. 168th St., New York, NY 10032 Golden, William T., 40 Wall St., New York, NY 10005 Goldman, David E., 63 Loop Rd., Falmouth, MA 02540 Goldman, Robert D., Department of Cell Biology and Anatomy, Northwestern University, 303 E. Chicago Ave., Chicago, IL 6061 1 Goldsmith, Paul K., 551 1 Oakmont Avenue, Bethesda, MD 20034 Goldsmith, Timothy H., Department of Biology, Yale University, New Haven, CT 06520 Goldstein, Moise H., Jr., Johns Hopkins University, School of Medicine, 720 Rutland Ave., Baltimore, MD 21205 Goodman, Lesley Jean, Department of Zoology and Comparative Physiology, Queen Mary College, Mile End Road, London, El 4NS, England, U. K. GOTTSCHALL, GERTRUDE Y., 315 E. 68th St., 9-M, New York, NY 10021 GOUDSMIT, Esther, M., Department of Biology, Oakland University, Rochester, MI 48063 Gould, Robert Michael, Institute for Basic Research in Developmental Disabilities, 1050 Forest Hill Rd., Staten Island, NY 10314 Gould, Stephen J., Museum of Comparative Zoology, Harvard University, Cambridge, MA 02138 Govind, C. K., Zoology Department — Scarborough, University of Toronto, 1265 Military Trail, West Hill, Ontario, Canada, MIC 1A4 Grant, Philip, Department of Biology, University of Oregon, Eugene, OR 97403 Grass, Albert, The Grass Foundation, 77 Reservoir Rd., Quincy, MA 02170 MEMBERS OF THE CORPORATION 1 3 Grass, Ellen R., The Grass Foundation, 77 Reservoir Rd., Quincy, MA 02170 Grassle, Judith, Marine Biological Laboratory, Woods Hole, MA 02543 Green, Jonathan P., Department of Biology, Roosevelt University, 430 S. Michigan Avenue, Chicago, IL 60605 Greenberg, Everett Peter, Department of Microbiology, Stocking Hall, Cornell University, Ithaca, NY 14853 Greenberg, Michael J., C. V. Whitney Laboratory, Rt. 1, Box 121, St. Augustine, PL 32086 Greif, Roger L., Department of Physiology, Cornell University, Medical College New York, NY 10021 Griffin, Donald R., The Rockefeller University, 1230 York Ave., New York, NY 10021 Grosch, Daniels., Department of Genetics, Gardner Hall, North Carolina State University, Raleigh, NC 27607 Gross, Paul R., President and Director, Marine Biological Laboratory, Woods Hole, MA 02543 Grossman, Albert, New York University, Medical School, New York, NY 10016 Gunning, A. Robert, P.O. Box 165, Falmouth, MA 02541 GwiLLiAM, G. P., Department of Biology, Reed College, Portland, OR 97202 Hall, Linda M., Department of Genetics, Albert Einstein College of Medicine, 1 300 Morris Park Ave., Bronx, NY 10461 Hall, Zack W., Department of Physiology, University of California, San Francisco, CA 94143 Halvorson, Harlyn O., Rosenstiel Basic Medical Sciences Research Center, Brandeis Uni- versity, Waltham, MA 02154 Hamlett, Nancy Virginia, Department of Biology, Swarthmore College, Swarthmore, PA 19081 Hanna, Robert B., College of Environmental Science and Forestry, SUNY, Syracuse, NY 13210 Harding, Clifford V., Jr., Kresge Eye Institute, Wayne State University, 540 E. Canfield. Detroit, MI 48201 Harosi, Ferenc I., Laboratory of Sensory Physiology, Marine Biological Laboratory, Woods Hole, MA 02543 Harrigan, June F., Laboratory of Biophysics, Marine Biological Laboratory, Woods Hole. MA 02543 Harrington, Glenn W., Department of Microbiology, School of Dentistry, University of Missouri, 650 E. 25th St., Kansas City, MO 64108 Haschemeyer, Audrey E. V., Department of Biological Sciences, Hunter College, 695 Park Ave., New York, NY 10021 Hastings, J. W., The Biological Laboratories, Harvard University, Cambridge, MA 02138 Hayashi, Teru, 7105 SW 112 PL, Miami, FL 33173 Hayes, Raymond L., Jr., Howard University, College of Medicine, 520 W St., NW, Washington. DC 20059 Henley, Catherine, 5225 Pooks Hill Rd., #1 127 North, Bethesda, MD 20034 Herndon, Walter R., University of Tennessee, 506 Andy Holt Tower, Knoxville, TN 37916 Hessler, Anita Y., 5795 Waveriy Ave., La Jolla, CA 92037 Heuser, John, Department of Biophysics, Washington University, School of Medicine, St. Louis, MO 63110 HiATT, Howard H., Harvard University, School of Public Health, 677 Huntington Ave., Boston, MA 02 1 1 5 HiGHSTEiN, Stephen M., Department of Otolaryngology, Washington University, St. Louis, MO 631 10 Hildebrand, John G., Department of Biological Sciences, Fairchild Center #913, Columbia University, New York, NY 10027 HiLLis-COLiNVAUX, Llewellya, Department of Zoology, The Ohio State University 484 W 12th Ave., Columbus, OH 43210 HiLLMAN, Peter, Department of Biology, Hebrew University, Jerusalem, Israel Hinegardner, Ralph T., Division of Natural Sciences, University of California, Santa Cruz, CA 95064 14 MARINE BIOLOGICAL LABORATORY HiNSCH, Gertrude, W., Department of Biology, University of South Florida, Tampa, FL 33620 HOBBIE, John E., Ecosystems Center, Marine Biological Laboratory, Woods Hole, MA 02543 Hodge, Alan J., Marine Biological Laboratory, Woods Hole, MA 02543 Hodge, Charles, IV, P.O. Box 4095, Philadelphia, PA 19118 Hoffman, Joseph, Department of Physiology, School of Medicine, Yale University, New Haven, CT 06515 Hoffman, Richard J., Department of Zoology, Iowa State University, Ames, lA 50011 HOLLYFIELD, JOE G., Baylor School of Medicine, Texas Medical Center, Houston, TX 77030 Holtzman, Eric, Department of Biological Sciences, Columbia University, New York, NY 10017 HOLZ, George G., Jr., Department of Microbiology, SUNY, Syracuse, NY 13210 HOSKIN, Francis C. G., Department of Biology, Illinois Institute of Technology, Chicago, IL 60616 Houghton, Richard A., Ill, Ecosystems Center, Marine Biological Laboratory, Woods Hole, MA 02543 Houston, Howard E., 2500 Virginia Ave., NW, Washington, DC 20037 HowARTH, Robert, Ecosystems Center, Marine Biological Laboratory, Woods Hole, MA 02543 Hoy, Ronald R., Section of Neurobiology and Behavior, Cornell University, Ithaca, NY 14850 Hubbard, Ruth, The Biological Laboratories, Harvard University, Cambridge, MA 02 1 38 Hufnagel, Linda A., Department of Microbiology, University of Rhode Island, Kingston, RI 02881 Hummon, William D., Department of Zoology, Ohio University, Athens, OH 45701 Humphreys, Susie H., Kraft Research and Development, 801 Waukegan Rd., Glenview, IL 60025 Humphreys, Tom D., University of Hawaii, PBRC, 41 Ahui St., Honolulu, HI 96813 Hunter, Bruce W., Box 321, Lincoln Center, MA 01773 Hunter, Robert D., Department of Biological Sciences, Oakland University, Rochester, NY 48063 HuNZiKER, Herbert E., Esq., P.O. Box 547, Falmouth, MA 02541 Hurwitz, Charles, Basic Science Research Lab, Veterans Administration Hospital, Albany, NY 12208 Hurwitz, Jerard, Albert Einstein College of Medicine, Department of Molecular Biology, 1300 Morris Park Avenue, Bronx, NY 10461 Huxley, Hugh E., Medical Research Council, Laboratory of Molecular Biology, Cambridge, England, U. K. Hynes, Thomas J., Jr., Meredith and Grew, Inc., 125 High Street, Boston, MA 021 10 ILAN, Joseph, Department of Anatomy, Case Western Reserve University, Cleveland, OH 44106 Ingoglia, Nicholas, Department of Physiology, New Jersey Medical School, 100 Bergen St., Newark, NJ 07103 Inoue, Saduykj, McGill University Cancer Centre, Department of Anatomy, 3640 University St., Montreal, PQ, H3A 2B2, Canada Inoue, Shiny a. Marine Biological Laboratory, Woods Hole, MA 02543 ISENBERG, Irving, Department of Biochemistry and Biophysics, Oregon State University, Corvallis, OR 97331 ISSADORIDES, MARIETTA R., Department of Psychiatry, University of Athens, Monis Petraki 8, Athens, 140, Greece ISSELBACHER, KURT J., Massachusetts General Hospital, Boston, MA 021 14 IzzARD, Colin S., Department of Biological Sciences, SUNY, Albany, NY 12222 Jacobson, AntoneG., Department of Zoology, University of Texas, Austin, TX 78712 Jaffe, Lionel, Marine Biological Laboratory, Woods Hole, MA 02543 Jahan-Parwar, Behrus, Worcester Foundation for Experimental Biology, 222 Maple Ave., Shrewsbury, MA 01545 Jannasch, Holger W., Woods Hole Oceanographic Institution, Woods Hole, MA 02543 MEMBERS OF THE CORPORATION 15 Jeffery, William R., Department of Zoology, University of Texas, Austin, TX 78712 Jenner, Charles E., Department of Zoology, University of North Carolina, Chapel Hill, NC 27514 Jones, Meredith L., Division of Worms, Museum of Natural History, Smithsonian Institution, Washington, DC 20560 JOSEPHSON, Robert K., School of Biological Sciences, University of California, Irvine, CA 92664 Kabat, E. a.. Department of Microbiology, College of Physicians and Surgeons Columbia University, 630 West 168th St., New York, NY 10032 Kaley, Gabor, Department of Physiology, Basic Sciences Building, New York Medical College, Valhalla, NY 10595 Kaltenbach, Jane, Department of Biological Sciences, Mount Holyoke College, South Hadley, MA 01075 Kaminer, Benjamin, Department of Physiology, School of Medicine, Boston University, 80 East Concord St., Boston, MA 021 18 Kammer, Ann E., Division of Biology, Kansas State University, Manhatten, KS 66506 Kane, Robert E., University of Hawaii, PBRC, 41 Ahui St., Honolulu, HI 96813 Kaneshiro, EdnaS., Department of Biological Sciences, University of Cincinnati, Cincinnati, OH 45221 Kaplan, Ehud, The Rockefeller University, 1230 York Ave., New York, NY 10021 Karakashian, Stephen J., Apt. 16-F, 165 West 91st St., New York, NY 10024 Karush, Fred, Department of Microbiology, School of Medicine, University of Pennsylvania, Philadelphia, PA 19174 Katz, George M., Fundamental and Experimental Research, Merck, Sharpe and Dohme Rahway, NJ 07065 Kean, Edward L., Department of Ophthalmology and Biochemistry, Case Western Reserve University, Cleveland, OH 44101 Kelley, Darcy Brisbane, Department of Biological Sciences, 1018 Fairchild, Columbia University, New York, NY 10032 Kelly, Robert E., Department of Anatomy, College of Medicine, University of IlHnois, P.O. Box 6998, Chicago, IL 60680 Kemp, Norman E., Department of Zoology, University of Michigan, Ann Arbor, MI 48104 Kendall, John P., Faneuil Hall Associates, One Boston Place, Boston, MA 02108 Keynan, Alexander, Hebrew University, Jerusalem, Israel Kingsbury, John M., Department of Botany, Cornell University, Ithaca, NY 14853 Kirschenbaum, Donald, Department of Biochemistry, SUNY, 450 Clarkson Ave., Brooklyn, NY 11203 Klein, Morton, Department of Microbiology, Temple University, Philadelphia, PA 19122 Klotz, I. M., Department of Chemistry, Northwestern University, Evanston, IL 60201 KOIDE, Samuel S., Population Council, The Rockefeller University, 66th St. and York Ave., New York, NY 10021 Konigsberg, Irwin R., Department of Biology, Gilmer Hall, University of Virginia, Char- lottesville, VA 22903 Kornberg, Hans, Department of Biochemistry, University of Cambridge, Tennis Court Rd., Cambridge, CB2 7QW, England, U. K. Kosower, Edward M., Ramat-Aviv, Tel Aviv, 69978 Israel Krahl, M. E., 2783 W. Casas Circle, Tucson, AZ 85741 Krane, Stephen M., Massachusetts General Hospital, Boston, MA 021 14 Krassner, Stuart M., Department of Developmental and Cell Biology, University of Cal- ifornia, Irvine, CA 92717 Krauss, Robert, FASEB, 9650 Rockville Pike, Bethesda, MD 20205 Kravitz, Edward A., Department of Neurobiology, Harvard Medical School, 25 Shattuck St., Boston, MA 02115 Kriebel, Mahlon E., Department of Physiology, B.S.B., Upstate Medical Center, 766 Irving Ave., Syracuse, NY 13210 Krieg, Wendell J. S., 1236 Hinman, Evanston, IL 60602 16 MARINE BIOLOGICAL LABORATORY Kristan, William B., Jr., Department of Biology B-022, University of California San Diego, San Diego, CA 92093 KUHNS, William J., University of North Carolina, 512 Faculty Lab Office, Bldg. 231-H, Chapel Hill, NC 27514 KUSANO, KiYOSHi, Illinois Institute of Technology, Department of Biology, 3300 South Federal St., Chicago, IL 60616 Laderman, Aimlee, P.O. Box 689, Woods Hole, MA 02543 LaMarche, Paul H., Eastern Maine Medical Center, 489 State St., Bangor, ME 04401 Landis, Dennis M. D., Department of Neurology, Massachusetts General Hospital, Boston, MA 02114 Landis, Story C, Department of Neurobiology, Harvard Medical School, Boston, MA 021 15 Landowne, David, Department of Physiology, University of Miami, R-430, P.O. Box 016430, Miami, FL 33101 Langford, George M., Department of Physiology, Medical Sciences Research Wing 206H, University of North Carolina, Chapel Hill, NC 27514 Laser, Raymond J., Case Western Reserve University, Department of Anatomy, Cleveland, OH 44106 Laster, Leonard, University of Oregon, Health Sciences Center, Portland, OR 97201 Laufer, Hans, Biological Sciences Group U-42, University of Connecticut, Storrs, CT 06268 Lauffer, Max a.. Department of Biophysics, University of Pittsburgh, Pittsburgh, PA 15260 Lazarow, Jane, 221 Woodlawn Ave., St. Paul, MN 55105 Lazarus, Maurice, Federated Department Stores, Inc., 50 Comhill, Boston, MA 02108 Leadbetter, Edward R., Biological Sciences Group U-42, University of Connecticut, Storrs, CT 06268 Lederberg, Joshua, President, The Rockefeller University, New York, NY 10021 Lederhendler, IzjA I., Laboratory of Biophysics, Marine Biological Laboratory, Woods Hole, MA 02543 Lee, John J., Department of Biology, City College of CUNY, Convent Ave. and 138th St., New York, NY 10031 LeFevre, PaulG., Box 339, Shoreham, NY 11786 Leibovitz, Louis, Laboratory for Marine Animal Health, Marine Biological Laboratory, Woods Hole, MA 02543 Leighton, Joseph, 1201 Waverly Rd., Gladwyne, PA 19035 Leighton, Stephen, NIH, Bldg. 13 3W13, Bethesda, MD 20205 Lenher, Samuel, 50-C Cokesbury Village, Hockessin, DE 19707 Lerman, Sidney, Laboratory for Ophthalmic Research, Emory University, Atlanta, GA 30322 Lerner, Aaron B., Yale University, School of Medicine, New Haven, CT 06510 Levin, Jack, Clinical Pathology Service, VA Hospital- 1 1 3A, 4150 Clement St., San Francisco, CA 94120 Levinthal, Cyrus, Department of Biological Sciences, Columbia University, 908 Schermerhom Hall, New York, NY 10027 Levitan, Herbert, Department of Zoology, University of Maryland, College Park, MD 20742 Ling, Gilbert, 307 Berkeley Road, Marion, PA 1 9066 LiPiCKY, Raymond J., Laboratory of Biophysics, NIH, Bldg. 36 Room 2A29, Bethesda, MD 20205 LiSMAN, John E., Department of Biology, Brandeis University, Waltham, MA 02 1 54 Liuzzi, Anthony, Department of Physics, University of Lowell, Lowell, MA 01854 Llinas, RodolfoR., Department of Physiology and Biophysics, New York University Medical Center, 550 First Ave., New York, NY 10016 LOEWENSTEIN, WERNER R., Department of Physiology and Biophysics, University of Miami, P.O. Box 016430, Miami, FL 33101 LOEWUS, Frank, A., Department of Argicultural Chemistry, Washington State University, Pullman, WA 99164 Loftreld, Robert B., Department of Biochemistry, School of Medicine, University of New Mexico, 900 Stanford, NE, Alburquerque, NM 87105 London, Irving M., Massachusetts Institute of Technology, Cambridge, MA 02139 MEMBERS OF THE CORPORATION 17 LONGO, Frank J., Department of Anatomy, University of Iowa, Iowa City, lA 52442 LORAND, Laszlo, Department of Biochemistry and Molecular Biology, Northwestern University, Evanston, IL 60201 LURIA, Salvaix)RE., Massachusetts Institute of Technology, Department of Biology, Cambridge, MA 02139 Lynch, Clara J., 4800 Fillmore Ave., Alexandria, VA 2231 1 Macagno, Eduardo R., 1003B Fairchild, Columbia University, New York, NY 10022 MacNichol, E. F., Jr., Laboratory of Sensory Physiology, Marine Biological Laboratory, Woods Hole MA 02543 Maglott, Donna R. S., Department of Zoology, Howard University, Washington, DC 20059 Mainer, Robert, The Boston Company, One Boston Place, Boston, MA 02108 Malkiel, Saul, Allergic Diseases, Inc., 130 Lincoln St., Worcester, MA 01605 Manalis, Richards., RR #10, 400N, Columbia City, IN 47625 Mangum, Charlotte P., Department of Biology, College of William and Mary, Williamsburg, VA 23185 Margulis, Lynn, Department of Biology, Boston University, 2 Cummington St., Boston, MA 02215 Marinucci, Andrew C, Department of Civil Engineering, Princeton University, Princeton, NJ 08544 Marsh, Julian B., Department of Biochemistry and Physiology, Medical College of Penn- sylvania, 3300 Henry Ave., Philadelphia, PA 19129 Martin, Lowell V., Marine Biological Laboratory, Woods Hole, MA 02543 Maser, Morton, 100 Hackmatak Way, Falmouth, MA 02540 Mastroianni, Luigi, Jr., Department of Obstetrics and Gynecology, University of Pennsyl- vania, Philadelphia, PA 19174 Mathews, Rita, W., c/o A. J. Johnson, New York University Medical Center, 550 First Ave., New York, NY 10016 Matteson, Donald R., Department of Physiology, G4, School of Medicine, University of Pennsylvania, Philadelphia, PA 19104 Mautner, Henry G., Department of Biochemistry and Pharmacology, Tufts University, 136 Harrison Ave., Boston, MA 02 1 1 1 Mauzerall, David, The Rockefeller University, 1230 York Ave., New York, NY 10021 Mazia, Daniel, Hopkins Marine Station, Pacific Grove, CA 93950 McCann, Frances, Department of Physiology, Dartmouth Medical School, Hanover, NH 03755 McClosky, Lawrence R., Department of Biology, Walla Walla College, College Place, WA 99324 McLaughlin, Jane A., P.O. Box 187, Woods Hole, MA 02543 McMahon, Robert F., Department of Biology, Box 19498, University of Texas, ArUngton, TX 76019 Meedel, Thomas, Boston University Marine Program, Marine Biological Laboratory. Woods Hole, MA 02543 Meinertzhagen, Ian A., Department of Psychology, Life Sciences Center, Dalhousie Uni- versity, Halifax, Nova Scotia, B3H 451, Canada Meinkoth, Norman A., Department of Biology, Swarthmore College, Swarthmore, PA 19081 Meiss, Dennis E., Department of Biology, Clark University. Worcester, MA 01610 Melillo Jerry a.. Ecosystems Center, Marine Biological Laboratory. Woods Hole, MA 02543 Mellon, Deforest, Jr., Department of Biology, University of Virginia, Charlottesville, VA 22903 Mellon, Richard P., P.O. Box 187, Laughlintown, PA 15655 Menzel, Randolf, Institut fir Tierphysiologie, Free Universitat of Berlin, 1000 Berlin 41, Federal Republic of Germany Metuzals, Janis, Department of Anatomy, Faculty of Medicine, University of Ottawa, Ottawa, Ontario, KIN 9A9, Canada Metz, Charles B., Institute of Molecular and Cellular Evolution, University of Miami, 521 Anastasia Ave., Carol Gables, FL 33134 18 MARINE BIOLOGICAL LABORATORY Milkman, Roger, Department of Zoology, University of Iowa, Iowa City, lA 52242 Mills, Eric L., Institute of Oceanography, Dalhousie University, Halifax, Nova Scotia Mills, Robert, 56 Worcester Court, Falmouth, MA 02540 Mitchell, Ralph, Pierce Hall, Harvard University, Cambridge, MA 02138 Miyamoto, David M., Department of Biology, Seton Hall University, South Orange, NJ 07079 MiZELL, Merle, Department of Biology, Tulane University, New Orleans, LA 70 11 8 MONROY, Alberto, Stazione Zoologica, Villa Communale, Napoli, Italy Moore, John W., Department of Physiology, Duke University Medical Center, Durham, NC 27710 Moore, Lee E., Department of Physiology and Biophysics, University of Texas, Medical Branch, Galveston, TX 77550 Moran, Joseph F., Jr., 23 Foxwood Drive, RR #1, Eastham, MA 02642 (resigned 10/83) MORIN, James G., Department of Biology, University of Cahfomia, Los Angeles, CA 90024 MORRELL, Frank, Department of Neurological Sciences, Rush Medical Center, 1753 W. Congress Parkway, Chicago, IL 60612 Morrill, John B., Jr., Division of National Sciences, New College, Sarasota, FL 33580 Morse, Richard S., 193 Winding River Rd., Wellesley, MA 02181 Morse, Robert W., Box 574, N. Falmouth, MA 02556 Morse, Stephen Scott, Department of Biological Sciences, Rutgers University, Nelson Bio- logical Laboratories, New Brunswick, NJ 08903 Moscona, a. a., Department of Biology, University of Chicago, 920 East 58th St., Chicago, IL 60637 Mote, Michael I., Department of Biology, Temple University, Philadelphia, PA 19122 Mountain, Isabel, Vinson Hall #112, 6251 Old Dominion Drive, McLean, VA 22101 MUSACCHIA, Xavier J., Graduate School, University of Louisville, Louisville, KY 40292 Nabrit, S. M., 686 Beckwith St., SW, Atlanta, GA 30314 Naka, Ken-ichi, National Institute for Basic Biology, Okazaki 444, Japan Nakajima, Shigehiro, Department of Biological Sciences, Purdue University, West Lafayette, IN 47907 Nakajima, Yasuko, Department of Biological Sciences, Purdue University, West Lafayette, IN 47907 Narahashi, Toshio, Department of Pharmacology, Medical Center, Northwestern University, 303 East Chicago Ave., Chicago, IL 6061 1 Nasatir, Maimon, Department of Biology, University of Toledo, Toledo, OH 43606 Nelson, Leonard, Department of Physiology, Medical College of Ohio, Toledo, OH 43699 Nelson, Margaret C, Section on Neurobiology and Behavior, Cornell University, Ithaca, NY 14850 NiCHOLLS, John G., Department of Neurobiology, Stanford University, Stanford, CA 94305 Nicosia, Santo V., Department of OB-GYN, Division of Reproductive Biology, University of Pennsylvania, Philadelphia, PA 19174 Nielsen, Jennifer B. K., Merck, Sharp & Dohme Laboratories, Bldg. 50-G, Room 226, Rahway, NJ 07065 NOE, Bryan D., Department of Anatomy, Emory University, Atlanta, GA 30345 Obaid, Ana Lia, Department of Physiology and Pharmacy, University of Pennsylvania, 400 1 Spruce St., Philadelphia, PA 19104 Ochoa, Severo, 530 East 72nd St., New York, NY 10021 Odum, Eugene, Department of Zoology, University of Georgia, Athens, GA 30701 Oertel, Don ATA, Department of Neurophysiology, University of Wisconsin, 283 Medical Science Bldg., Madison, WI 53706 O'Herron, Jonathan, Lazard Freres and Company, 1 Rockefeller Plaza, New York, NY 10020 Olins, Ada L., University of Tennessee — Oak Ridge, Graduate School of Biomedical Sciences, Oak Ridge National Laboratory, Biology Division, P.O. Box Y, Oak Ridge, TN 37830 Olins, Donald E., University of Tennessee — Oak Ridge, Graduate School of Biomedical Sciences, Oak Ridge National Laboratory, Biology Division, P.O. Box Y, Oak Ridge, TN 37830 MEMBERS OF THE CORPORATION 19 O'Melia, Anne F., George Mason University, 4400 University Drive, Fairfax, VA 22030 Olson, John M., Institute of Biochemistry, Odense University, Campusvej 55, DK 5230 Odense M, Denmark (resigned 1/84) OSCHMAN, James L., Marine Biological Laboratory, Woods Hole, MA 02543 Palmer, John D., Department of Zoology, University of Massachusetts, Amherst, MA 01002 Paltl Yoram, Department of Physiology and Biophysics, Israel Institute of Technology, 12 Haaliya St., BAT-GALIM, POB 9649, Haifa, Israel Pant, Harish C, Laboratory of Preclinical Studies, National Institute on Alcohol Abuse and Alcoholism, 12501 Washington Ave., Rockville, MD 20852 Pappas, George D., Department of Anatomy, College of Medicine, University of Illinois, 808 South Wood St., Chicago, IL 60612 Pardee, Arthur B., Department of Pharmacology, Harvard Medical School, Boston, MA 02115 Pardy, Rosevelt L., School of Life Sciences, University of Nebraska, Lincoln, NE 68588 Parmentier, James L., Department of Anesthesiology, Anesthesiology Research Laboratory, 284 Cancer Center/Clinical Bldg., University of South Alabama, Mobile, AL 36688 Passano, Leonard M., Department of Zoology, Birge Hall, University of Wisconsin, Madison, WI 53706 Pearlman, Alan L., Department of Physiology, School of Medicine, Washington University, Sl Louis, MO 631 10 Pederson, Thoru, Worcester Foundation for Experimental Biology, Shrewsbury, MA 01545 Perkins, C. 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Pettibone, Marian H., Division of Worms, W-213, Smithsonian Institution, Washington, DC 20560 Pfohl, Ronald J., Department of Zoology, Miami University, Oxford, OH 45056 Pierce, Sidney K., Jr., Department of Zoology, University of Maryland, College Park, MD 20740 Pollard, Harvey B., NIH, F Building 10, Room 10B17, Bethesda, MD 20205 Pollard, Thomas D., Department of Cell Biology and Anatomy, Johns Hopkins University, 725 North Wolfe St., Baltimore, MD 21205 Pollock, Leland W., Department of Zoology, Drew University, Madison, NJ 07940 Porter, Beverly H., 14433 Taos Court, Wheaton, MD 20906 Porter, Keith R., 748 Eleventh St., Boulder, CO 80302 Potter, David, Department of Neurobiology, Harvard Medical School, Boston, MA 02 11 5 Potter, H. David, P.O. Box 2286, Bloomington, IN 47401 Potts, William T., Department of Biology, University of Lancaster, Lancaster, England, U. K. POUSSART, Denis, Department of Electrical Engineering, Universite Laval. 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Box 2000, Rahway, NJ 07065 Reynolds, George T., Department of Physics, Jadwin Hall, Princeton University, Princeton, NJ 08540 Rice, Robert V., Carnegie Mellon Institute, 4400 Fifth Ave., Pittsburgh, PA 15213 Rickles, Frederick R., University of Connecticut, School of Medicine, VA Hospital, New- ington, CT06111 RiPPS, Harris, Department of Ophthalmology, New York University School of Medicine, 550 First Ave., New York, NY 10016 Roberts, JohnL., Department of Zoology, University of Massachusetts, Amherst, MA 01002 Robinson, Denis M., High Voltage Engineering Corporation, Burlington, MA 01803 ROCKSTEIN, Morris, 335 Fluvia Ave., Miami, FL 33134 Ronkin, Raphael R., 3212 McKJnley St., NW, Washington, DC 20015 ROSBASH, Michael, Rosenstiel Center, Department of Biology, Brandeis University, Waltham, MA 02154 Rose, Birgit, Department of Physiology R-430, University of Miami School of Medicine, P.O. Box 016430, Miami, FL 33152 Rose, S. 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Box 152, Woods Hole, MA 02543 Segal, Sheldon J., Population Division, The Rockefeller Foundation, 1 133 Avenue of the Americas, New York, NY 10036 Seliger, Howard H., Johns Hopkins University, McCollum-Pratt Institute. Baltimore, MD 21218 22 MARINE BIOLOGICAL LABORATORY Selman, Kelly, Department of Anatomy, College of Medicine, University of Florida, Gaines- ville, FL 32601 Senft, Joseph, 378 Fairview St., Emmaus, PA 18049 Shanklin, Douglas R., P.O. Box 1267, Gainesville, FL 32602 Shapiro, Herbert, 6025 North 13th St., Philadelphia, PA 19141 Shaver, GaiusR., Ecosystems Center, Marine Biological Laboratory, Woods Hole, MA 02543 Shaver, JohnR., Department of Zoology, Michigan State University, E. Lansing, MI 48823 Shepard, David C, P.O. Box 44, Woods Hole, MA 02543 Shepro, David, Department of Biology, Boston University, 2 Cummington St., Boston, MA 02215 Sherman, I. W., Division of Life Sciences, University of CaHfomia, Riverside, CA 92502 Shilo, Moshe, Department of Microbiological Chemistry, Hebrew University, Jerusalem, Israel Shoukimas, Jonathan J., Marine Biological Laboratory, Woods Hole, MA 02543 Siegel, Irwin, M., Department of Ophthalmology, New York University Medical Center, 550 First Avenue, New York, NY 10016 Siegelman, Harold W., Department of Biology, Brookhaven National Laboratory, Upton, NY 11973 Sjodin, Raymond A., Department of Biophysics, University of Maryland, Baltimore, MD 21201 Skinner, Dorothy M., Oak Ridge National Laboratory, Biology Division, Oak Ridge, TN 37830 Sloboda, Roger D., Department of Biological Sciences, Dartmouth College, Hanover, NH 03755 Smith, Homer P., Marine Biological Laboratory, Woods Hole, MA 02543 Smith, Michael A., Colombo Campus, P.O. Box 1490, Colombo 3, Sri Lanka Smith, Paul F., P.O. Box 264, Woods Hole, MA 02543 Smith, Ralph L, Department of Zoology, University of California, Berkeley, CA 94720 Sorenson, Albert L., Albert Einstein College of Medicine, Department of Physiology, 1300 Morris Park Avenue, Bronx, NY 10461 Sorenson, Martha M., Depto de Bioquimica-RFRJ, Centro de Ciencias da Saude-I.C.B., Cidade Universitaria-Fundad, Rio de Janeiro, Brasil 21.910 Speck, William T., Case Western Reserve University, Department of Pediatrics, Cleveland, OH 44106 Spector, a.. College of Physicians and Surgeons, Columbia University, Black Bldg., Room 1516, New York, NY 10032 Speer, John W., Marine Biological Laboratory, Woods Hole, MA 02543 Spiegel, Evelyn, Department of Biological Sciences, Dartmouth College, Hanover, NH 02755 Spiegel, Melvin, Department of Biological Sciences, Dartmouth College, Hanover, NH 02755 Spray, David C, Albert Einstein College of Medicine, Department of Neurosciences, 1300 Morris Park Avenue, Bronx, NY 10461 Steele, John Hyslop, Woods Hole Oceanographic Institution, Woods Hole, MA 02543 Steinacher, Antoinette, Department of Biophysics, The Rockefeller University, New York, NY 10021 Steinberg, Malcolm, Department of Biology, Princeton University, Princeton, NJ 08540 Stephens, GroverC, Department of Developmental and Cell Biology, University of California, Irvine, CA 92717 Stephens, Raymond E., Marine Biological Laboratory, Woods Hole, MA 02543 Stetten, DeWitt, Jr., Senior Scientific Advisor, NIH, Bldg. 16, Room 118, Bethesda, MD 20205 Stokes, Darrell R., Department of Biology, Emory University, Atlanta, GA 30322 Stracher, Alfred, Downstate Medical Center, SUNY, 450 Clarkson Ave., Brookyln, NY 11203 Strehler, Bernard L., 2235 25th St., #217, San Pedro, CA 90732 Stuart, AnnE., Department of Physiology, Medical Sciences Research Wing 206H, University of North Carolina, Chapel Hill, NC 27514 MEMBERS OF THE CORPORATION 23 Summers, William C, Huxley College, Western Washington University, Bellingham, WA 98225 SussMAN, Maurice, Department of Life Sciences, University of Pittsburgh, Pittsburgh, PA 15260 SWENSON, Randolphe P., JR., Department of Physiology G-4, University of Pennsylvania, Philadelphia, PA 19174 (resigned 1/84) SzABO, George, Harvard School of Dental Medicine, 188 Longwood Avenue, Boston, MA 02115 Szamier. R. 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Richard, Boston University Marine Program, Marine Biological Laboratory, Woods Hole, MA 02543 WiERCiNSKi, Floyd J., 21 Glenview Road, Glenview, IL 60025 (Life Member 10/83) WiGLEY, Roland L., 35 Wilson Road, Woods Hole, MA 02543 WiLBER, Charles G., Department of Zoology, Colorado State University, Fort Collins, CO 80523 Wilson, DarcyB., Department of Pathology, School of Medicine, University of Pennsylvania, Philadelphia, PA 19174 Wilson, Edward O., Department of Zoology, Harvard University, Cambridge, MA 02138 Wilson, T. Hastings, Department of Physiology, Harvard Medical School, Boston, MA 021 15 Wilson, Walter L., Department of Biology, Oakland University, Rochester, MI 48063 WiTKOVSKY, Paul, Department of Ophthalmology, New York University Medical Center, 550 First Ave., New York, NY 10016 Wittenberg, Jonathan B., Department of Physiology and Biochemistry, Albert Einstein College, 1300 Morris Park Ave., New York, NY 10016 Wolf, Don P., Department of OB-GYN, University of Texas Health Sciences Center, 6431 Fannin, Houston, TX 77030 Wolfe, Ralph, Department of Microbiology, 131 Burrill Hall, University of Illinois, Urbana, IL 61801 WooDWELL, George M., Ecosystems Center, Marine Biological Laboratory, Woods Hole, MA 02543 MEMBERS OF THE CORPORATION 25 WORGUL, Basil V., Department of Ophthalmology, Columbia University, 630 West 168th St., New York, NY 10032 Wu, Chau Hsiung, Department of Pharmacology, Northwestern University Medical School, 203 E. Chicago Ave., Chicago, IL 6061 1 Wyttenbach, Charles R., Department of Physiology and Cell Biology, University of Kansas, Lawrence, KS 66045 Yeh, Jay Z., Department of Pharmacology, Northwestern University Medical School, 303 E. Chicago Ave., Chicago, IL 6061 1 Young, Richard, Houghton Mifflin Company, One Beacon St., Boston, MA 02108 ZiGMAN, Seymour, School of Medicine and Dentistry, University of Rochester, 260 Crittenden Blvd., Rochester, NY 14620 Zucker, Robert S., Department of Physiology, University of California, Berkeley, CA 94720 Associate Ackroyd, Dr. and Mrs. Frederick W. Adelberg, Dr. and Mrs. Edward A. Allen, Miss Camilla K. Allen, Drs. Robert D. and Nina S. Amberson, Mrs. William R. Anderson, Drs. James L. and Helene M. Armstrong, Dr. and Mrs. Samuel C. Arnold, Dr. and Mrs. John M. Atwood, Dr. and Mrs. Kimball C. Ball, Mrs. Eric G. Ballantine, Dr. and Mrs. H. T., Jr. Bang, Mrs. Frederik B. Banks, Mr. and Mrs. William L. Barrows, Mrs. Albert W. 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Van Alan Clarke, Dr. Barbara J. Clement, Dr. and Mrs. A. C. Clowes Fund, Inc. Clowes, Dr. and Mrs. Alexander W. Clowes, Mr. Allen W. Clowes, Dr. and Mrs. G. H. A., Jr. COBURN, Mr. Lawrence Coleman, Dr. and Mrs. John CONNELL, Mr. and Mrs. W. J. CoPELAND, Mrs. D. Eugene Copeland, Mr. and Mrs. Preston S. CosTELLO, Mrs. Donald P. Crain, Mr. and Mrs. Melvin, C. Cramer, Mr. and Mrs. Ian D. W. Crane, Mrs. John Crane, Josephine B., Foundation Crane, Mr. Thomas Cross, Mr. and Mrs. Norman C. Crossley, Mr. and Mrs. Archibald M. Crowell, Dr. and Mrs. Sears Daignault, Mr. and Mrs. Alexander T. Daniels, Mr. and Mrs. Bruce G. Davis, Mr. and Mrs. Joel F. Day, Mr. and Mrs. Pomeroy Di Berardino, Dr. Marie A. Dickson, Dr. William A. Drummond, Mr. and Mrs. A. H., Jr. Dubois, Dr. and Mrs. Arthur B. Dunkerley, Mr. and Mrs. H. Gordon DuPoNT, Mr. a. Felix, Jr. Ebert, Dr. and Mrs. James D. Egloff, Dr. and Mrs. F. R. L. Eppel, Mr. and Mrs. Dudley Evans, Mr. and Mrs. Dudley 26 MARINE BIOLOGICAL LABORATORY EwiNG, Dr. and Mrs. Gifford C. Ferguson, Dr. and Mrs. James J., Jr. Fine, Dr. and Mrs. Jacob Fisher, Mrs. B. C. Fisher, Mr. Frederick S., Ill Fisher, Dr. and Mrs. Saul H. Francis, Mr. and Mrs. Lewis W., Jr. Friendship Fund Fries, Dr. and Mrs. E. F. B. Fye, Dr. and Mrs. Paul M. Gabriel, Dr. and Mrs. Mordecai L. Gaiser, Dr. and Mrs. David W. Garheld, Miss Eleanor Garrey, Dr. Walter E. Gellis, Dr. and Mrs. Sydney German, Dr. and Mrs. James L., Ill Gifford, Mr. John A. Gifford, Dr. and Mrs. Prosser Gilbert, Dr. and Mrs. Daniel L. Gilbert, Mrs. Carl J. GiLDEA, Dr. Margaret C. L. Gillette, Mr. and Mrs. Robert S. Glass, Dr. and Mrs. H. Bentley Glazebrook, Mrs. James R. Glusman, Dr. and Mrs. Murray Goldman, Dr. and Mrs. Allen, S. Goldstein, Dr. and Mrs. Moise H., Jr. Goodwin, Mr. and Mrs. Charles Grant, Dr. and Mrs. Philip Grassle, Mrs. J. F. Green, Miss Gladys M. Greene, Mr. and Mrs. William C. Greer, Mr. and Mrs. W. H., Jr. Grosch, Dr. and Mrs. Daniel S. Gross, Mrs. Paul C. Gruson, Mrs. Martha R. Gunning, Mr. and Mrs. Robert Haakonsen, Dr. Harry O. Halvorson, Dr. and Mrs. Harlyn O. Handler, Mrs. Philip Harvey, Dr. and Mrs. Richard B. Hassett, Mr. and Mrs. Charles Hastings, Dr. and Mrs. J. Woodland Henley, Dr. Catherine Hersey, Mrs. George L. HiATT, Dr. and Mrs. Howard Hill, Mrs. Samuel E. Hilsinger, Mr. and Mrs. Arthur Hirschfield, Mrs. Nathan B. HoBBiE, Dr. and Mrs. John Hocker, Mr. and Mrs. Lon Hoffman, Rev. and Mrs. Charles Horwitz, Dr. and Mrs. Norman H. Houston, Mr. and Mrs. Howard E. Howard, Mr. and Mrs. L. L. Huettner, Dr. and Mrs. Robert J. Hynes, Mr. and Mrs. Thomas j; Jr. iNOufe, Dr. and Mrs. Shinya Ireland, Mrs. Herbert A. IssoKSON, Mr. and Mrs. Israel IvENS, Dr. Sue Jackson, Miss Elizabeth B. Jaffe, Dr. and Mrs. Ernest R. Janney, Mrs. F. Wistar Jewett, G. F., Foundation Jewett, Mr. and Mrs. G. F., Jr. Jones, Mr. and Mrs. Frederick, III Jordan, Dr. and Mrs. Edwin, P. Kaan, Dr. Helen W. Kahler, Mr. and Mrs. George A. Kahler, Mr. and Mrs. Robert W. Kaminer, Dr. and Mrs. Benjamin Karush, Dr. and Mrs. Fred Keith, Mrs. Jean R. Kelleher, Mr. and Mrs. Paul R. Kendall, Mr. Richard E. Keosian, Mrs. Jessie KiEN, Mr. and Mrs. Pieter Kinnard, Mrs. L. Richard KiVY, Dr. and Mrs. Peter Kohn, Dr. and Mrs. Henry I. Koller, Dr. and Mrs. Lewis R. KuFFLER, Mrs. Stephen W. Laderman, Mr. and Dr. Aimlee Ezra Lash, Dr. and Mrs. James Laster, Dr. and Mrs. Leonard Laufer, Dr. and Mrs. Hans LaVigne, Mrs. Richard J. Lawrence, Mr. Frederick V. Lawrence, Mr. and Mrs. William Lazarow, Mrs. Arnold Leatherbee, Mrs. John H. Lemann, Mrs. Lucy B. Lenher, Dr. and Mrs. Samuel Levine, Dr. and Mrs. Rachmiel Lewis, Mr. John T. Little, Mrs. Elbert LoEB, Mrs. Robert F. LovELL, Mr. and Mrs. Hollis R. Lowe, Dr. and Mrs. Charles W. lowengard, mrs. joseph Mackey, Mr. and Mrs. William K. MacLeish, Mrs. Margaret MacNary, Mr. and Mrs. B. Glenn MacNichol, Dr. and Mrs. Edward F., Jr. Maher, Miss Annie Camille Marsland, Dr. Douglas Martyna, Mr. and Mrs. Joseph C. Marvin, Dr. Dorothy H. Maser, Dr. and Mrs. Morton Mastroianni, Dr. and Mrs. Luigi, Jr. Mather, Mr. and Mrs. Frank J., Ill Matthiessen, Mr. and Mrs. G. C. MEMBERS OF THE CORPORATION 27 McCusKER, Mr. and Mrs. Paul T. McElroy, Mrs. Nella W. McLane, Mrs. T. Thorne Meigs, Mr. and Mrs. Arthur Meigs, Dr. and Mrs. J. Wister Melillo, Dr. and Mrs. Jerry M. Mellon, Richard King, Trust Mellon, Mr. and Mrs. Richard P. Mendelson, Dr. Martin Menke, Dr. W. J. Metz, Dr. and Mrs. Charles B. Meyers, Mr. and Mrs. Richard Miller, Dr. Daniel A. MixTER, Mr. and Mrs. William J., Jr. Montgomery, Dr. and Mrs. Charles H. Montgomery, Dr. and Mrs. Raymond P. MooG, Dr. Florence Moore, Mr. and Mrs. Berrien, III Moore, Dr. and Mrs. John A. Morse, Mr. and Mrs. Charles, L., Jr. Morse, Mr. and Mrs. Richard S. MouL, Dr. and Mrs. Edwin T. Nace, Dr. and Mrs. Paul Nelson, Dr. Pamela Newton, Mr. and Mrs. William F. NiCKERSON, Mr. and Mrs. Frank L. Norman, Mr. and Mrs. Andrew E. Norman Foundation NoRRis, Mr. John, Esq. O'Herron, Mr. and Mrs. Jonathan Ortins, Mr. and Mrs. Armand O'SuLLiVAN, Dr. Renee Bennett Pappas, Dr. and Mrs. George D. Park, Mrs. Franklin A. Parmenter, Miss Carolyn L. Pendergast, Mrs. Claudia Pendleton, Dr. and Mrs. Murray E. Pennington, Miss Anne H. Perkins, Mr. and Mrs. Courtland D. Person, Dr. and Mrs. Philip Peterson, Mr. and Mrs. E. Gunnar Peterson, Mr. and Mrs. E. Joel Peterson, Mr. Raymond W. Philippe, Mr. and Mrs. Pierre Porter, Dr. and Mrs. Keith R. Press, Drs. Frank and Billie Prosser, Dr. and Mrs. C. Ladd PSYCHOYOS, Mr. Alexandre Putnam, Mr. Allan Ray Putnam, Mr. and Mrs. William A., Ill Raymond, Dr. and Mrs. Samuel Reynolds, Dr. and Mrs. George Reznikoff, Mrs. Paul RICCA, Dr. and Mrs. Renato A. RiGGS, Mr. and Mrs. Lawrason, III Riina, Mr. and Mrs. John R. RoBB, Mrs. Alison A. Robertson, Mrs. C. Stuart Robertson, Dr. and Mrs. C. W. Robinson, Dr. and Mrs. Denis M. Rogers, Mrs. Julian Root, Mrs. Walter S. RosLANSKY, Mr. and Mrs. John Ross, Dr. Virginia Roth, Mr. Stephen RowE, Mr. and Mrs. William S. Rubin, Dr. Joseph Rugh, Mrs. Roberts Russell, Mr. and Mrs. Henry D. Ryder, Mr. and Mrs. Francis C. Saunders, Dr. and Mrs. John W. Saunders, Mrs. Lawrence Saunders, Lawrence Fund Sawyer, Mr. and Mrs. John E. Saz, Mrs. Ruth L. SCHLESINGER, DR. AND MRS. R. WALTER Scott, Dr. and Mrs. George T. Scott, Mr. and Mrs. Norman E. Sears, Mr. and Mrs. Harold B. Segal, Dr. and Mrs. Sheldon J. Senft, Dr. and Mrs. Alfred Shapiro, Mrs. Harriet S. Shemin, Dr. and Mrs. David Shepro, Dr. and Mrs. David Smith, Mrs. Homer P. Smith, Mr. Van Dorn C. Snider, Mr. Eliot Solomon, Dr. and Mrs. A. K. Specht, Mrs. Heinz Spiegel, Dr. and Mrs. Melvin Steele, Mrs. M. Evelyn Steinbach, Mrs. H. Burr Stetson, Mrs. Thomas J. Stetten, Dr. DeWitt, Jr. Stewart, Mr. and Mrs. Peter Stunkard, Dr. Horace Sturtevant, Mrs. A. H. Swanson, Dr. and Mrs. Carl P. SwoPE, Dr. and Mrs. Gerard L. SwoPE, Mrs. Gerard, Jr. Taylor, Dr. and Mrs. W. Randolph TiETJE, Mr. and Mrs. Emil D., Jr. TiMMiNS, Mrs. William Tolkan, Mr. and Mrs. Norman N. Trager, Mrs. William Trigg, Mr. and Mrs. D. Thomas Troll, Dr. and Mrs. Walter TuLLY, Mr. and Mrs. Gordon F. Ulbrich, Mrs. Mary Steinbach Valois, Mr. and Mrs. John Veeder, Mrs. Ronald A. Waksman, Dr. and Mrs. Byron H. 28 MARINE BIOLOGICAL LABORATORY Ward, Dr. Robert T. Ware, Mr. and Mrs. J. Lindsay Watt, Mr. and Mrs. John B. Watterson, Dr. Ray Weisberg, Mr. and Mrs. Alfred M. Wheeler, Dr. and Mrs. Paul S. Whitney, Mr. and Mrs. Geoffrey G. Jr. WiCHTERMAN, DR. AND MRS. RALPH WlCKERSHAM, Mr. AND MRS. A. A. TiLNEY WlCKERSHAM, MR. AND MRS. JaMES H.. Jr. WiLHELM, Dr. Hazel, S. WiTMER, Dr. and Mrs. Enos E. WOLHNSOHN, Mr. and MrS. WOLFE WooDWELL, Dr. and Mrs. George M. Yntema, Mrs. Chester L. Zinn, Dr. and Mrs. Donald J. ZiPF, Dr. Elizabeth ZwiLLiNG, Mrs. Edgar III. CERTinCATE OF ORGANIZATION (On File in the Office of the Secretary of the Commonwealth) No. 3170 We. Alpheus Hyatt, President, William Stanford Stevens, Treasurer, and William T. Sedgwick, Edward G. Gardiner, Susan Mims and Charles Sedgwick Minot being a majority of the Trustees of the Marine Biological Laboratory in compliance with the requirements of the fourth section of chapter one hundred and fifteen of the Public Statutes do hereby certify that the following is a true copy of the agreement of association to constitute said Corporation, with the names of the subscribers thereto: We, whose names are hereto subscribed, do, by this agreement, associate ourselves with the intention to constitute a Corporation according to the provisions of the one hundred and fifteenth chapter of the Public Statutes of the Commonwealth of Massachusetts, and the Acts in amendment thereof and in addition thereto. The name by which the Corporation shall be known is THE MARINE BIOLOGICAL LAB- ORATORY. The purpose for which the Corporation is constituted is to establish and maintain a laboratory or station for scientific study and investigations, and a school for instruction in biology and natural history. The place within which the Corporation is established or located is the city of Boston within said Commonwealth. The amount of its capital stock is none. In Witness Whereof, we have hereunto set our hands, this twenty seventh day of February in the year eighteen hundred and eighty-eight, Alpheus Hyatt, Samuel Mills, William T. Sedgwick, Edward G. Gardiner, Charles Sedgwick Minot, William G. Farlow, William Stanford Stevens, Anna D. Phillips, Susan Mims, B. H. Van Vleck. That the first meeting of the subscribers to said agreement was held on the thirteenth day of March in the year eighteen hundred and eighty-eight. In Witness Whereof, we have hereunto signed our names, this thirteenth day of March in the year eighteen hundred and eighty-eight, Alpheus Hyatt, President, William Stanford Stevens, Treasurer, Edward G. Gardiner, William T. Sedgwick, Susan Mims, Charles Sedgwick Minot. BYLAWS 29 (Approved on March 20, 1888 as follows: / hereby certify that it appears upon an examination of the within written certificate and the records of the corporation duly submitted to my inspection, that the requirements of sections one, two and three of chapter one hundred and fifteen, and sections eighteen, twenty and twenty-one of chapter one hundred and six, of the Public Statutes, have been complied with and I hereby approve said certificate this twentieth day of March A.D. eighteen hundred and eighty-eight. CHARLES ENDICOTT Commissioner of Corporations) IV. ARTICLES OF AMENDMENT (On File in the Office of the Secretary of the Commonwealth) We. James D. Ebert, President, and David Shepro, Clerk of the Marine Biological Laboratory, located at Woods Hole, Massachusetts 02543, do hereby certify that the following amendment to the Articles of Organization of the Corporation was duly adopted at a meeting held on August 15, 1975, as adjourned to August 29, 1975, by vote of 444 members, being at least two-thirds of its members legally qualified to vote in the meeting of the corporation: VOTED: That the Certificate of Organization of this corporation be and it hereby is amended by the addition of the following provisions: "No Officer, Trustee or Corporate Member of the corporation shall be personally liable for the payment or satisfaction of any obligation or liabilities incurred as a result of, or otherwise in connection with, any commitments, agreements, activities or affairs of the corporation. "Except as otherwise specifically provided by the Bylaws of the corporation, meetings of the Corporate Members of the corporation may be held anywhere in the United States. "The Trustees of the corporation may make, amend or repeal the Bylaws of the corporation in whole or in part, except with respect to any provisions thereof which shall by law, this Certificate or the bylaws of the corporation, require action by the Corporate Members." The foregoing amendment will become effective when these articles of amendment are filed in accordance with Chapter 1 80, Section 7 of the General Laws unless these articles specify, in accordance with the vote adopting the amendment, a later effective date not more than thirty days after such filing, in which event the amendment will become effective on such later date. In Witness whereof and Under the Penalties of Perjury, we have hereto signed our names this 2nd day of September, in the year 1975, James D. Ebert, President; David Shepro, Clerk. (Approved on October 24, 1975, as follows: I hereby approve the within articles of amendment and, the filing fee in the amount of $10 having been paid, said articles are deemed to have been filed with me this 24th day of October, 1975. PAUL GUZZI Secretary of the Commonwealth) 30 MARINE BIOLOGICAL LABORATORY V. BYLAWS OF THE CORPORATION OF THE MARINE BIOLOGICAL LABORATORY (Revised August 11, 1978) I. (A) The name of the Corporation shall be The Marine Biological Laboratory. The Cor- poration's purpose shall be to establish and maintain a laboratory or station for scientific study and investigation, and a school for instruction in biology and natural history. (B) Marine Biological Laboratory admits students without regard to race, color, sex, national and ethnic origin to all the rights, privileges, programs and activities generally accorded or made available to students in its courses. It does not discriminate on the basis of race, color, sex, national and ethnic origin in employment, administration of its educational policies, admissions policies, scholarship and other programs. II. (A) The members of the Corporation ("Members") shall consist of persons elected by the Board of Trustees, upon such terms and conditions and in accordance with such procedures, not inconsistent with law or these Bylaws, as may be determined by said Board of Trustees. Except as provided below, any Member may vote at any meeting either in person or by proxy executed no more than six months prior to the date of such meeting. Members shall serve until their death or resignation unless earlier removed with or without cause by the affirmative vote of two-thirds of the Trustees then in office. Any member who has attained the age of seventy years or has retired from his home institution shall automatically be designated a Life Member provided he signifies his wish to retain his membership. Life Members shall not have the right to vote and shall not be assessed for dues. (B) The Associates of the Marine Biological Laboratory shall be an unincorporated group of persons (including associations and corporations) interested in the Laboratory and shall be organized and operated under the general supervision and authority of the Trustees. III. The officers of the Corporation shall consist of a Chairman of the Board of Trustees, President, Director, Treasurer and Clerk, elected or appointed by the Trustees as set forth in Article IX. IV. The Annual Meeting of the Members shall be held on the Friday following the Second Tuesday in August in each year at the Laboratory in Woods Hole, Massachusetts, at 9:30 a.m. Subject to the provisions of Article VIII(2), at such meeting the Members shall choose by ballot six Trustees to serve four years, and shall transact such other business as may properly come before the meeting. Special meetings of the Members may be called by the Chairman or Trustees to be held at such time and place as may be designated. V. Twenty five Members shall constitute a quorum at any meeting. Except as otherwise required by law or these Bylaws, the affirmative vote of a majority of the Members voting in person or by proxy at a meeting attended by a quorum (present in person or by proxy) shall constitute action on behalf of the Members. VI. (A) Inasmuch as the time and place of the Annual Meeting of Members are fixed by these Bylaws, no notice of the Annual Meeting need be given. Notice of any special meeting of Members, however, shall be given by the Clerk by mailing notice of the time and place and purpose of such meeting, at least 1 5 days before such meeting, to each Member at his or her address as shown on the records of the Corporation. (B) Any meeting of the Members may be adjourned to any other time and place by the vote of a majority of those Members present or represented at the meeting, whether or not such Members constitute a quorum. It shall not be necessary to notify any Member of any adjournment. BYLAWS 3 1 VII. The Annual Meeting of the Trustees shall be held promptly after the Annual Meeting of the Corporation at the Laboratory in Woods Hole, Massachusetts. Special meetings of the Trustees shall be called by the Chairman, the President, or by any seven Trustees, to be held at such time and place as may be designated. Notice of Trustees' meetings may be given orally, by telephone, telegraph or in writing; and notice given in time to enable the Trustees to attend, or in any case notice sent by mail or telegraph to a Trustee's usual or last known place of residence, at least one week before the meeting shall be sufficient. Notice of a meeting need not be given to any Trustee if a written waiver of notice, executed by him before or after the meeting is filed with the records of the meeting, or if he shall attend the meeting without protesting prior thereto or at its commencement the lack of notice to him. VIII. (A) There shall be four groups of Trustees: ( 1 ) Trustees (the "Corporate Trustees") elected by the Members according to such procedures, not inconsistent with these Bylaws, as the Trustees shall have determined. Except as provided below, such Trustees shall be divided into four classes of six, one class to be elected each year to serve for a term of four years. Such classes shall be designated by the year of expiration of their respective terms. (2) Trustees ("Board Trustees") elected by the Trustees then in office according to such procedures, not inconsistent with these Bylaws, as the Trustees shall have determined. Except as provided below, such Board Trustees shall be divided into four classes of three, one class to be elected each year to serve for a term of four years. Such classes shall be designated by the year of expiration of their respective terms. It is contemplated that, unless otherwise de- termined by the Trustees for good reason. Board Trustees shall be individuals who have not been considered for election as Corporate Trustees. (3) Trustees ex officio, who shall be the Chairman, the President, the Director, the Treasurer, and the Clerk. (4) Trustees emeriti who shall include any Member who has attained the age of seventy years (or the age of sixty five and has retired from his home institution) and who has served a full elected term as a regular Trustee, provided he signifies his wish to serve the Laboratory in that capacity. Any Trustee who qualifies for emeritus status shall continue to serve as a regular Trustee until the next Annual Meeting whereupon his office as regular Trustee shall become vacant and be filled by election by the Members or by the Board, as the case may be. The Trustees ex officio and emeriti shall have all the rights of the Trustees, except that Trustees emeriti shall not have the right to vote. (B) The aggregate number of Corporate Trustees and Board Trustees elected in any year (excluding Trustees elected to fill vacancies which do not result from expiration of a term) shall not exceed nine. The number of Board Trustees so elected shall not exceed three and unless otherwise determined by vote of the Trustees, the number of Corporate Trustees so elected shall not exceed six. (C) The Trustees and Officers shall hold their respective offices until their successors are chosen in their stead. (D) Any Trustee may be removed from office at any time with or without cause, by vote of a majority of the Members entitled to vote in the election of Trustees; or for cause, by vote of two-thirds of the Trustees then in office. A Trustee may be removed for cause only if notice of such action shall have been given to all of the Trustees or Members entitled to vote, as the case may be, prior to the meeting at which such action is to be taken and if the Trustee so to be removed shall have been given reasonable notice and opportunity to be heard before the body proposing to remove him. (E) Any vacancy in the number of Corporate Trustees, however arising, may be filled by the Trustees then in office unless and until filled by the Members at the next Annual Meeting. Any vacancy in the number of Board Trustees may be filled by the Trustees. (F) A Corporate Trustee or a Board Trustee who has served an initial term of at least 2 years duration shall be eligible for re-election to a second term, but shall be ineligible for re- election to any subsequent term until two years have elapsed after he last served as Trustee. 32 MARINE BIOLOGICAL LABORATORY IX. (A) The Trustees shall have the control and management of the affairs of the Corporation. They shall elect a Chairman of the Board of Trustees who shall be elected annually and shall serve until his successor is selected and qualified and who shall also preside at meetings of the Corporation. They shall elect a President of the Corporation who shall also be the Vice Chairman of the Board of Trustees and Vice Chairman of meetings of the Corporation, and who shall be elected annually and shall serve until his successor is selected and qualified. They shall annually elect a Treasurer who shall serve until his successor is selected and qualified. They shall elect a Clerk (a resident of Massachusetts) who shall serve for a term of 4 years. Eligibility for re-election shall be in accordance with the content of Article VIII (F) as applied to Corporate or Board Trustees. They shall elect Board Trustees as described in Article VIII (B). They shall appoint a Director of the Laboratory for a term not to exceed five years, provided the term shall not exceed one year if the candidate has attained the age of 65 years prior to the date of the appointment. They may choose such other officers and agents as they may think best. They may fix the compensation and define the duties of all the officers and agents of the Corporation and may remove them at any time. They may fill vacancies occurring in any of the offices. The Board of Trustees shall have the power to choose an Executive Committee from their own number as provided in Article X, and to delegate to such Committee such of their own powers as they may deem expedient in addition to those powers conferred by Article X. They shall from time to time elect Members to the Corporation upon such terms and conditions as they shall have determined, not inconsistent with law or these Bylaws. (B) The Board of Trustees shall also have the power, by vote of a majority of the Trustees then in Office, to elect an Investment Committee and any other committee and, by like vote, to delegate thereto some or all of their powers except those which by law, the Articles of Organization or these Bylaws they are prohibited from delegating. The members of any such committee shall have such tenure and duties as the Trustees shall determine; provided that the Investment Committee, which shall oversee the management of the Corporation's endowment funds and marketable securities, shall include the Chairman of the Board of Trustees, the Treasurer of the Corporation, and the Chairman of the Corporation's Budget Committee, as ex officio members, together with such Trustees as may be required for not less than two-thirds of the Investment Committee to consist of Trustees. Except as otherwise provided by these Bylaws or determined by the Trustees, any such committee may make rules for the conduct of its business; but, unless otherwise provided by the Trustees or in such rules, its business shall be conducted as nearly as possible in the same manner as is provided by these Bylaws for the Trustees. X. (A) The Executive Committee is hereby designated to consist of not more than ten members, including the ex officio Members (Chairman of the Board of Trustees, President, Director and Treasurer); and six additional Trustees, two of whom shall be elected by the Board of Trustees each year, to serve for a three-year term. (B) The Chairman of the Board of Trustees shall act as Chairman of the Executive Committee, and the President as Vice Chairman. A majority of the members of the Executive Committee shall constitute a quorum and the affirmative vote of a majority of those voting at any meeting at which a quorum is present shall constitute action on behalf of the Executive Committee. The Executive Committee shall meet at such times and places and upon such notice and appoint such sub-committees as the Committee shall determine. (C) The Executive Committee shall have and may exercise all the powers of the Board during the intervals between meetings of the Board of Trustees except those powers specifically withheld from time to time by vote of the Board or by law. The Executive Committee may also appoint such committees, including persons who are not Trustees, as it may from time to time approve to make recommendations with respect to matters to be acted upon by the Executive Committee or the Board of Trustees. (D) The Executive Committee shall keep appropriate minutes of its meetings and its action shall be reported to the Board of Trustees. BYLAWS 33 (E) The elected Members of the Executive Committee shall constitute as a standing "Com- mittee for the Nomination of Officers," responsible for making nominations, at each Annual Meeting of the Corporation, and of the Board of Trustees, for candidates to fill each office as the respective terms of office expire (Chairman of the Board, President, Director, Treasurer, and Clerk). XI. A majority of the Trustees, the Executive Committee, or any other committee elected by the Trustees shall constitute a quorum; and a lesser number than a quorum may adjourn any meeting from time to time without further notice. At any meeting of the Trustees, the Executive Committee, or any other committee elected by the Trustees, the vote of a majority of those present, or such different vote as may be specified by law, the Articles of Organization or these Bylaws, shall be sufficient to take any action. XII. Any action required or permitted to be taken at any meeting of the Trustees, the Executive Committee or any other committee elected by the Trustees as referred to under Article IX may be taken without a meeting if all of the Trustees or members of such committee, as the case may be, consent to the action in writing and such written consents are filed with the records of meetings. The Trustees or members of the Executive Committee or any other committee appointed by the Trustees may also participate in meeting by means of conference telephone, or otherwise take action in such a manner as may from time to time be permitted by law. XIII. The consent of every Trustee shall be necessary to dissolution of the Marine Biological Laboratory. In case of dissolution, the property shall be disposed of in such a manner and upon such terms as shall be determined by the affirmative vote of two-thirds of the Board of Trustees then in office. XIV. These Bylaws may be amended by the affirmative vote of the Members at any meeting, provided that notice of the substance of the proposed amendment is stated in the notice of such meeting. As authorized by the Articles of Organization, the Trustees, by a majority of their number then in office, may also make, amend, or repeal these Bylaws, in whole or in part, except with respect to (a) the provisions of these Bylaws governing (i) the removal of Trustees and (ii) the amendment of these Bylaws and (b) any provisions of these Bylaws which by law, the Articles of Organization or these Bylaws, requires action by the Members. No later than the time of giving notice of the meeting of Members next following the making, amending or repealing by the Trustees of any Bylaw, notice thereof stating the substance of such change shall be given to all Corporation Members entitled to vote on amending the Bylaws. Any Bylaw adopted by the Trustees may be amended or repealed by the Members entitled to vote on amending the Bylaws. XV. The account of the Treasurer shall be audited annually by a certified public accountant. XVI. The Corporation will indemnify every person who is or was a trustee, officer or employee of the Corporation or a person who provides services without compensation to an Employee Benefit Plan maintained by the Corporation, for any liability (including reasonable costs of defense and settlement) arising by reason of any act or omission affecting an Employee Benefit Plan maintained by the Corporation or affecting the participants or beneficiaries of such Plan, including without limitation any damages, civil penalty or excise tax imposed pursuant to the Employee Retirement Income Security Act of 1974; provided, (1) that the Act or omission shall have occurred in the course of the person's service as trustee or officer of the Corporation or within the scope of the employment of an employee of the Corporation 34 MARINE BIOLOGICAL LABORATORY or in connection with a service provided without compensation to an Employee Benefit Plan maintained by the Corporation, (2) that the Act or omission be in good faith as determined by the Corporation (whose determination made in good faith and not arbitrarily or capriciously shall be conclusive), and (3) that the Corporation's obligation hereunder shall be offset to the extent of any otherwise applicable insurance coverage, under a policy maintained by the Cor- poration or any other person, or other source of indemnification. VI. REPORT OF THE DIRECTOR . . . The future is neither ahead nor behind, on one side or another. Nor is it dark or light. It is contained within ourselves; it is drawn from ourselves; its evil and its good are perpetually within us. The future that we seek from oracles, whether it be war or peace, starvation or plenty, disaster or happiness, is not forward to be come upon. Rather its gestation is now . . . — Loren Eiseley Introduction Three changes have led to another one: a change in the form of this Report. First among them was the interest of Trustees in additional meetings, one to be held early in the summer. That interest caused action in February, 1984: scheduling of a meeting for (the 8th and 9th) June, 1984. A particular argument in favor was the need for more time for review of MBL research and scientific policy. The second change followed establishment of the Laboratory's industrial liaison program, whose activities will begin in the summer of 1984. As a part of this program (dubbed the ISP, Interactive Science Program), we have established a database for all MBL investigators and faculty, randomly accessible via their affiliations and research activities. When it is correct and in full use, the database will allow not only periodic rectification of the files, but also inclusion of abstracts on significant research outcomes. It will be printed out in its entirety for the 1984 Decennial Review visitors, and doubtless for many others. Finally, there is a current effort by our Public Information staff to issue new publications on MBL research, some for fund-raising and other external distribution, and some for internal readers. One result of these actual and proposed changes will be a comprehensive review, in 1984, of all research and training programs at the MBL, and the likely decision to issue a separate report or reports. There is too much of it to fit in the August Biological Bulletin. The purposes of this Director's Report will necessarily change. It is, after all, primarily an internal document, read (presumably) by some Corporation members and Trustees; but because of its form of publication and its location amidst legal, statistical, financial, and other membership data, it does not and cannot effectively serve the important purpose of representing MBL science. Yet the Biological Bulletin is the MBL's own journal; and its publication of the Annual Report is widely perceived to be important. That cannot change. Some form of Director's Report must be incorporated; it will continue to be seen by many subscribers, but it will be read for the most part by active members of the MBL family. I have chosen, therefore, to try a somewhat different use for it than the yearly history, by Department and by program: to address MBL people quite directly; to avoid discussion of the content of research or teaching, except as these may be immediate issues of policy, saving review of annual accomplishments for other pub- lications; and finally, to address issues of MBL policy and organization with a good deal more frankness than is typical of the alumni magazines produced by college REPORT OF THE DIRECTOR 35 publications offices. This first trial is concerned with progress and problems. It may entail some risk; but I have never known the truth to cause real trouble when it is truth about something excellent. Is Progress Necessary? Readers of about my age may remember a volume by Thurber and White, entitled "Is Sex Necessary?" Its distinguishing feature was the absence of sex. Such is not my intention in choosing the heading for this section. I raise the question about the necessity of progress because it is sometimes raised, as such or by implication, within the Corporation. In the time-honored way, the force of the question is blurred by demurrers that argue, not the necessity, but the definition. That fools few listeners. Most of us have a pretty good idea of the difference between rest and motion. Most questions attack the idea that it is practical, or necessary, or even in good taste, to move. "Why not," such questions really ask, "stand still?" "After all, things are not so very bad just now; and they were much better some time ago." It is a view with which I can sympathize in principle. I see no progress in elec- tronically-amplified popular music, for example, because it's bad for the Organ of Corti, and most of it isn't music anyway. Nor do I believe that political TV "debates" are debates; that "found objects" are art; that computer games are more than pinball machines; that Transactional Analysis is more effective than reading fiction; or that "computer-literacy" is any kind of literacy. But the MBL is different. I put the matter bluntly: the MBL of the nineteen- fiflties could not hope to live in the nineteen-eighties. Most excuses for our financial support in those good old years — and I mean excuses, not the truth (that the MBL was and is the world's most productive Annual Congress of Biology) — have evaporated. With important exceptions such as squid, sea animals, and plants for research can now be shipped to inland locations and used there with some inconvenience. Scientific meetings, and opportunities for travel to them, have multiplied, despite declining grant support. There are several times as many biomedical disciplines and working scientists today as there were in 1950; since the MBL hasn't grown in size to match, the percentage of top people in each field who can be MBL regulars has necessarily declined: we still have a disproportionate share, but we don't any longer have, as we could once claim, nearly all of them. No place has. The facilities requirements for biomedical science, e.g., equipment and buildings, have grown beyond all expectation. It becomes more difficult and expensive, year by year, to equip MBL laboratories for the advanced work that must be done and taught in them. We are still far ahead of the game, and our courses remain unique in quality; but other institutions are moving aggressively into the business in most MBL fields. Nor can we go back to teaching descriptive, undergraduate-level courses. We couldn't charge the necessary tuition; there would be no grant support for them; and there is strong objection to the idea in many quarters, for it would mean giving up the intellectual frontier we occupy. It is necessary, in short, for the MBL to understand the times; and to keep, not just abreast, but ahead of them. Change, with minimum dislocation of good, ongoing work; and with minimum hurt to persons, but change measured and according to plan. There is nothing new in this. Old organizations retain support because the public sees their value in simple and immediate terms, not as imponderables or with nostalgia. The situation might be represented by that universal device for measuring car- diovascular competence: the analytical treadmill. Biomedical science is a treadmill. 36 MARINE BIOLOGICAL LABORATORY For reasons qualitative as well as quantitative, its tilt up is increasing year by year. Doing it, one can't just stop and stand still. Stopping means falling off. That was not true as late as 1 960, but it is true today. Therefore progress is necessary. We must ask how much progress we are making in the key domains of MBL operations, and what problems there are. Why, for example, too little progress is one area, if that is the judgment; why signs of decUning progress elsewhere, even if we are still on track; what are the interactions and contradictions between progress in one domain and the problems of others? These are the kinds of issues I hope to identify below: not to discuss them comprehensively, for that is disallowed by available space and in- appropriate for publication with data meant to cover just the year 1983. But I do want to identify some domains and the tensions between progress and problems. I hope that readers among our colleagues may be set to thinking, on their own, about the subject. Research The broad measures of our progress in research are seen in many places: Director's Reports of the past few years; statistical data presented to the Corporation and to our donors; the very many publications and achievements, recognized by elections, public honors, and awards, of current members of this community. There are other measures of MBL excellence, a little less direct but nonetheless compelling for those who understand the machinery of science and its support. One example will suffice: the funding of research grant proposals and its relationship to demand for MBL space and accommodations, summer and year-round. Under prevailing policies of government agencies that support most MBL science, the number of grants of all types awarded each year has been stabilized at what is necessarily a smaller figure that than of earlier years. Although total dollars awarded per grant have risen, purchasing power of the grant dollar has declined. Yet the two direct indicators of success in grant-getting, under the tough peer-reviews that determine it, show the MBL's position, unlike that of many research universities, to be strength- ening. Grants to the MBL have been rising, over the past few years, at the remarkable annual rate of 20%. While the number of grants and — more important — the percentage of applicants receiving grants in all fields of MBL interest have stabilized nationally at lower values than in the 1970's, the MBL remains chock-full in summer. The number of requests for year-round research facilities at the MBL has risen or remained level each year since 1978. Despite the absence of any general system of tenure for scientists, people want to make full-time MBL careers; and many are leaders in their fields. MBL scientists are therefore drawn from a sub-population of the nation's best- qualified biologists: those "approved" applicants who also succeed in getting funded. The dollar squeeze has damaged fine programs elsewhere, and hurt — often unjustly — some distinguished investigators. But the MBL continues to get a full share of the survivors, who must be doing, on the whole, highest-quality science. The squeeze has had, moreover, the interesting (and sometimes painful) consequence that new or younger investigators may get some form of funding preference over the established. Those younger ones, too, want to come to the MBL. A study of recent applicants for MBL laboratory space shows that the funding squeeze, far from reducing the MBL family to a core of the old and established, has in fact led to some turnover and an encouraging number of new and younger applicants. But there are consequent problems and there will be more. The treadmill will not stop or be lowered to horizontal. REPORT OF THE DIRECTOR 37 MBL science is the best science; and thus it gets more remote by the month from the microscope, the finger bowls, and the aquaria that could equip an MBL investigator during the early decades. Younger people and new disciplines make demands — legitimate ones — that were unanticipated even ten years ago. They need such unheard- of facilities (for the old MBL) as a proper vivarium for mammals, because, for example, they depend upon immunology and cell culture for the antibodies that are routine tools of cytology and neurobiology. They need larger, more sophisticated, and better- supported facilities for recombinant DNA technology than we have. Computer-assisted imaging and image processing have revolutionized light and electron microscopy. But visiting investigators cannot all bring such gear or the attached technicians with them: it must be here and functional for summer research. As these facilities are established for the year-round programs, they require more space, energy, and trained support staff. Tinker shops are vanishing; skilled instrument makers, shops, and assistants are in rising demand. We must have production-scale mariculture of species essential for research, against the certainty that some of those will become too hard to fish for, and because specimen health and genetics must be defined, as they are not in the wild. If the MBL is to continue to represent — as has been its mission — the best current thought and techniques in biology, then it will have to spend much more, not less, in support of research over the next few years. If it does not, the best science will be done elsewhere: by former MBL investigators who find that they must stay at home in the summers, despite the losses implied, or by those investigators going to other, less comprehensive institutions, equipped, however, for the technical support of their research specialties. Unless we find better methods of recovering the costs of research support and services — including, of course, library and administration — the notable progress made toward placing and keeping the MBL where it belongs on the intellectual ladder of biology will stop. For the reasons mentioned, that means that the sign of progress will be reversed, not merely zeroed. Education Kingsley Amis has described (using a very impolite name) the mind-set of certain persons with persistent "views" as a pyramid. The base of this structure I see as at the top, which is composed of many light-weight pieces; each one is an attitude, a slogan, or a canned argument. From top to bottom, the pyramid's pieces get weightier, because the arguments they contain become more personal. At the bottom is the point, and the point is heavy indeed: its argument is always personal, and it may be selfish. The supernatural feature of this inverted pyramid is that it is not unstable: the disproportionate mass of the point on the ground keeps it stuck upright. Discourse with persons of such a mind-set can be fun if you know in advance the composition of their point. You can be sure that even if discussion starts at the top of the pyramid, it will end at the bottom, firmly planted in terra firma. I think that Mr. Amis has made an important discovery about cognition. We'll return to it shortly. Judged against the threats to quality science education that have damaged teaching programs in many fine institutions, the MBL has succeeded remarkably well over the past ten years. It has inched forward on the treadmill. Our internationally-known summer courses show nothing like the declines of interest, applicant numbers, and applicant quality that have plagued graduate education elsewhere, to which editorials in Nature, Science, and publications of the NSF attest.* The mandated regular turnover * A case, of more than superficial relevance, in point: Science-Education doctorates. It is well known that the number of doctorates granted in natural science (hfe sciences, physical sciences, and mathematics) 38 MARINE BIOLOGICAL LABORATORY and updating of MBL course leadership and content goes on, despite pressures from within and without. New courses have been established, e.g., Neural Systems, Par- asitism, Microbial Ecology; and they have all succeeded by every strong measure of success. The January Semester, among off-season programs, has vanished, more or less, because (1) the "free January" has vanished at nearly all of our feeder institutions, and (2) undergraduates are over-committed for tuition payments to their own colleges. But the off-season short course program has grown in quality and recognition. Non- MBL media coverage is good, independent witness. It is now receiving the imaginative input, not only of our own scientists, but also of research and marketing staff from industry, and most recently of clinical specialties (such as neurosurgery) to which MBL strengths are relevant. Taken in toto. the educational program is more than ever, more even than at the founding, inseparable from the rest of the MBL. It is in fact an equal contributor, with independent research, to the values and character of this place. None of this would have been predictable with confidence in, say, 1974. We have made much progress. Returning now to the mind-set pyramid, it is the main problem inherent in, and working to diminish or reverse, progress. The pyramid's point is different for each category of argument against MBL education as it has evolved. The resulting problem is the same, however, for the effort to sustain orderly operations. Below are some non-imaginary bases (up in the air) and their points (anchored, like grounding rods, in the soil). I admit to coloring them a little for emphasis. "There are thousands of students out there who could pay realistic tuition fees to study beginning marine invertebrate zoology, marine botany, natural history, if and when you were to return the MBL to its proper and traditional role of teaching those subjects." The point: "Descriptive biology is still important; and that is what I know about. I want a shot at being an MBL faculty member, with all the rights and privileges appertaining thereunto." "Make 'em pay!" (Students a meaningful tuition fee; Faculty for the research laboratories provided them gratis). The point: "I don't see any profit to myself, or my independent research, in the summer courses. Each one is an expensive little world. I don't want to compete with them for facilities and services. I especially don't want my grant money supporting them in luxury." "We (the course faculty) bring enormous amounts of money to the MBL." The point: "I know the tuition income and the direct-cost budgets for the grants we have; I don't believe the MBL; as I disbelieve my own university, about indirect costs. There is bureaucracy, wasting money, everywhere." "The provision of services and materials to the courses (independent investigators) should be more (less) centralized." The point: "Mr. (Ms. ), of the MBL support staff seems unaware that my needs are of the highest priority." peaked in 1972 and has been declining ever since. But Science-Education doctorates are not the same thing: these are usually granted, after an interval of practical experience, to school and college teachers who have taken some time off for advanced study and the writing of a dissertation. Holders of such degrees are generally headed for Department chairmanships in secondary schools and toward liberal arts colleges. In 1982, the number of doctorates granted had fallen by 60 ijercent from the peak year, also 1972. The change reflects very complex market conditions, but one of its implications is clear: the demand for instruction in science at the secondary school and junior college level has fallen drastically, even more so than the number of students now enrolling for graduate study in pure science. The 60 percent fall is an underestimate: more than 22 percent of those earning Science-Education doctorates last year were foreigners, most of them holding temporary visas. Note that if, by a miracle, this situation were turned around next year, the half-time for a significant effect upon graduate studies would be of the order of eight years. These data are taken from the NSFs Mosaic. Vol. 15, No. 1, 1984. REPORT OF THE DIRECTOR 39 "Well," the reader may say, if he has followed this far, "what of that? You are describing no more than ordinary human behavior. Why is that a particular problem?" My response would be that while it is not a heart-stopper among institutional problems, since we have managed it successfully and will continue to do so, it is particular for the MBL's educational program. Perhaps by discussing it, this small one can be disposed of The MBL educational program is unique in the world. Among courses providing advanced training in the biomedical sciences, with full-time exposure of students to the most talented faculty and sophisticated instrumentation, those of the MBL summer and short-course programs, taken as a whole, are irreplaceable; an international re- source. It would be a violation of the MBL's historic mission to water them down in any way, even upon the questionable hope of tuition income better matched to costs. Program quality has continued to rise, or at least been maintained, despite (1) increased total costs; (2) decreased grant support for direct costs; (3) under-recovery or non-recovery of the indirect costs; and (4) the need for greater expenditures on faculty support, in order to maintain faculty quality in the face of resort-area living expenses. The strains are felt and they cause vibration at the tops of some pyramids. The real problem causing strain tends to remain unaddressed, except by the few admin- istrators and course directors who work regularly at it. I repeat: without the kind of educational program we have, the MBL would be a different, and a lesser place. The quality of MBL research is in no small measure a result of the combined contributions of independent investigation and the teaching program. That's progress. Entirely new ways of paying for that program must be found if the MBL is to go forward, or at least maintain position, on the treadmill. That's the problem. Finances I will deal simply with this issue, which is, of those taken up, the most complicated. It is also the least related to those scientific values which we, the Corporation and biologist-Trustees, have been educated to deal with. I can do so because there is no need to provide even a summary of progress; data in this and its predecessor volumes, notably the financial statements and reports of financial officers, summarize it well. The MBL has made good progress in controlling expenses; in keeping its operating budgets decently close to balance over the past few years; and in managing its small but critically important investments. We have the will and the means to keep it thus. But in the very means there is a problem that must grow every year until we find new ones. I state it as transparently as possible, without doing violence to important details published elsewhere for the trained, or inquiring eye. Let me start with data from a real, but here unnamed, research university. This institution receives Federal agency support, as we do, for research and certain kinds of educational programs {e.g.. Training Grants). It is among the distinguished scientific institutions. It has average indirect costs for a mid-sized city without a housing shortage. They are never fully recovered, but the recoverable costs are reimbursed by annually-negotiated agency payments figured as a percentage of total direct costs granted. The allocated share of the university's expenses for energy, maintenance, and technical services, payroll management, financial oversight, libraries, clerical and administrative services, becomes the "pooled sponsored research cost." This university has three different pooled cost rates in 1984. One is for the Medical Center; one for the campus that includes the College and most graduate education programs; and one for a large and specialized engineering development laboratory. The approved rates are, respectively, 50%, 70%, 40 MARINE BIOLOGICAL LABORATORY and 53% of direct costs. This year, actual recoveries are slightly lower, as a result of a small concession on the 70% component alone. Let us now make a realistic and very conservative comparison, using the MBL's situation. In a recent year, for which we have full and audited data, the MBL received direct cost payments, on grants and contracts under its full administration, of about $2.97 million. These grants were almost entirely to year-round programs and for some of the courses. Now, the MBL's operating expenses are not solely for work supported by in-house grants. We provide plant, services, housing, library, and so on for the summer population as well. In fact it is much larger than the year-round population, and its grants are made to other institutions. There is no easy way, then, to estimate the direct costs represented by all the grants held by all summer investigators, let alone to allocate correctly the fraction spent during an investigator's months at the MBL. (For example, accounting for and payment of salaries may remain a re- sponsibility of the home institution, but purchases made through the MBL, services such as supplying animals, insuring building safety and security, acquiring equipment, and housing accommodation are all handled here.) An educated guess as to the direct-cost dollar value of summar research at the MBL can nevertheless be made. I have done so, showing the numbers, at a recent Trustees' meeting. Reducing that last educated guess by about half, I still get an allocated direct cost of about $6 million. The total effective direct cost of grant- supported work at the MBL was therefore, in that recent year, more than $9 million. That year the MBL spent $3.6 million on science support services; which amount was reduced by direct income to departments and from tuition fees to about $2 million. The equivalent of "pooled costs," in other words, amounted to roughly 22% of total direct costs, for research and grant-supported instruction. Total indirect costs actually recovered, as laboratory rental fees, came to $1.6 milhon, i.e., 18%. This is to be compared with the cost recoveries given earher for an average research university. Such data are extractable in proper detail from published financial statements. They have the same outcome every year: the cost of research and advanced education at the MBL is lower than it is at other institutions performing comparable work; the fraction recovered by the MBL is notably lower than that recovered by peer orga- nizations. For the year in question, our minimum under-recovery was several hundred thousand dollars. I stress "minimum," because some of the support expense was managed by use of private funds. The difference was made up, the budget balanced, more or less, as it has been for the past several years, with investment income and gifts. That is a dangerous course to follow in perpetuity; not only because of increasing uncertainties of private philanthropy, but because it is a waste. It is a waste of the preparation of this and any future MBL Director and Executive Committee to give nearly full time and imagination to soliciting gifts, when they should be given to scientific work and leadership. Worse, it is a waste of privately generated funds, because they could be used for new research initiatives and facilities, rather than to pay routine bills. There are very few independent laboratories that are as mature, scientifically and culturally, as the MBL. The MBL ought also to be mature in the financial sense that its peer organizations are; i.e., in possession of cost-recovery systems that make at least partial sense in relation to the volume and quality of work done. Such maturity is yet to be attained. In order for it to be attained, we require more than imaginative and expert technical work on the part of financial staff and paid outside advisors: we need clear understanding of the problem by all Trustees and Corporation members, and an unselfish willingness work toward financial systems that are just for the Lab- oratory as a whole, as well as for its scientists individually. The symbol of today's situation might be, better than a treadmill, the more REPORT OF THE DIRECTOR 4 1 familiar one in Woods Hole of a small cruising sailboat (hull speed: 6.0 knots) heading into maximum current in the Hole (5.8 knots). The two possible outcomes — making it into Vineyard Sound or ending on the rocks east of Devil's Foot — remain a toss- up unless something is done. The wise skipper starts his engine. Operating Range and "Ruggedizing" The foregoing, which contains perhaps too much alternation between the sublime and the ridiculous, does so consciously. At the heart of the message I want to com- municate is the issue of operating range for the machine — or organism — that is the MBL. If I have given the impression that the problems discussed are an immediate threat to this Laboratory, then I renounce it. That is not the intention. It is, rather, to suggest that the environment in which this machine is expected to work is different from what it was, and that it will continue to change, within reasonable predictable limits, in the decade ahead. The environment of concern is mainly external: competing organizations; the pool of funds for science and instruction; the composition and personnel of new disciplines with which we are involved. But the internal environment — the MBL family itself — is also to be considered, although much less urgently than the external one. Self-regulating machines must be designed to work within environmental limits considerably wider than a few percentage points about the mean. Thus a decent on- board computer for an automobile should be able to withstand, not only normal road vibrations at speed, but an occasional pothole. Its thermal environmental limits should be from well below freezing to at least 11 5°F. The operating range for computers aboard the space shuttle will be far broader, obviously: otherwise they would fail during the first few seconds of flight. The design of such machines entails many principles, but three are followed universally and consciously: high component reliability, with some redundancy; "rug- gedizing" (awful word!); and simplification. These principles are often conflicting. The machine's components should, for example, have operating limits at least a little wider than those chosen, with a suitable safety factor, for the complete machine. There should be redundancy, either by du- plication of components or via the possibility of circuit-switching, in case of component or connection fails. But what about simplicity? Is it better (or less expensive in the end) to provide a backup chip for each one in service, with all the complications ensuing, or to avoid the complications by choosing a fail-safe chip in the first place, at much greater cost and bulk, but with simplification? There is no formal answer. That's what engineering is about: you find one by a combination of thought and empirical testing. "Ruggedizing" is even more subtle. Basically, it requires that connections among parts, and the housing for all, have wider operating limits even than the components. So you plate circuit boards with noble metals; use carbon fiber and plastics to insulate and hold things together; mount chassis in rubber or something better; and make the external switches moisture- and idiot-proof But each of those, unless done with thought and care, can end by being more costly than all the components, or by introducing the probability of damage to the components during assembly. I have tried, in the earlier sections and in what follows, to catalyze objective thinking about the MBL, as though it were a machine or a designable organism; to suggest some of the parameters of its present and future environments, external and internal; and to invite more collaboration among Corporation members and Trustees 42 MARINE BIOLOGICAL LABORATORY in the work of design. For design there must be, if only for retrofitting (to add one more industrial cliche), however excellent the machine may be for its purposes at this time. Nobody in his right mind would judge the MBL a weak machine for its present purposes and time. It is one-of-a-kind, and it's working well. But we are responsible for seeing to it that the MBL works in future time, and that components designed into it are compatible with the future environment. We are responsible for "ruggedizing" it, so that the heat and vibrations, inside and outside, which are a part of the human condition, have no chance of driving it into malfunction. Governance: The Future Within Us Academic governance, as the writer of an essay on the subject argued recently at the very start of the piece, is the least (inherently) interesting thing that goes on in a university. It is nevertheless a matter of great practical importance: almost but not quite as important as the teaching and research. And as a matter of fact, governance is of passionate interest among students and faculty. (For the MBL, read students, faculty, and investigators.) Denial of that interest is commonplace; but those elected to serve as, e.g., Trustees are visibly and justifiably proud of it, as are the elected to any exclusive professional body. Those not nominated or elected to such bodies, and yet deserving of it — and there are many such — are hurt by the neglect; they feel pain for a while each year. One doesn't need direct avowals as evidence of pride or hurt: the nature and tone of debate over causes celebres tells the story. Why should the MBL, with its large collection of talented scientists, be different from the rest of Academe? The interest in governance animates academic debates over process, rights, wrongs, responsibilities, even of style, with an emotional content much higher than that of legislatures and Boards of Directors in the outside world. Thus, the best efforts of TV and the press cannot invest with the academic sense of righteousness a debate on, say, town meeting versus mayoral systems for town government. The citizenry have other fish to fry. When the issue is merely selection of one over another local candidate for office, many sensible ones forbear to vote; counting that both a privilege of democracy and a vote. I do not necessarily approve: I merely note it. But, in a faculty meeting, as Corporation members know from home, there is no telling why and on what subject the flash-point may be reached. It may be as weighty a topic as "Are we falling behind the competition in faculty salaries?"; or as seemingly- innocuous as the proposal from Women's Studies for a seminar on "Women in Cosmology." You never can tell. A few pyramids totter on their points, and the fur flies. It's real and it is positive. Whatever the reasons, professors care about governance. The great thing is to keep that care channeled, applied toward progress. Progress has been made here at the MBL. The original Certificate of Organization, dated the 20th March, 1888, was a simple but effective document. Since then there have been numerous revisions and amendments; no single one of them was, in retrospect, other than wise. The last Bylaw revisions of significance were made as recently as 1978. In the formal sense, then, progress in governance — the adjustment of rules and processes to needs of the times — has been steady. The emergent system is unique, just as the institution it serves is unique. But despite care about governance, and progress, problems remain to be solved. Trustees of academic organizations are usually accomplished persons and are not paid members of the community whose well-being is, literally, in their trust. The general idea — one that has worked magnificently for American higher education — is that distinguished outsiders have the wisdom, the technical knowledge, and most important, the objectivity to be responsible, ultimately, for institutional policy; to REPORT OF THE DIRECTOR 43 Stand up for it to the faculty as well as to the public. It is sometimes said, in orienting new university Trustees to their tasks, that they have only two real jobs: ( 1 ) to select, compensate, and monitor the performance of the Chief Executive Officer; and (2) to "balance the present against the future," in respect of institutional assets, their grov^h, and their utilization. These two jobs take time, study, and unselfish devotion. To the extent that my own experience as a university Trustee and as Trustee of research laboratories other than the MBL is normal, the minimum job seems to call for three or four regular meetings per year, each one lasting at least a day and a half. Preparation for a meeting requires a week's evenings of reading and writing, and sometimes many telephone conversations. Every Trustee has visiting committee responsibility, which calls for two to four more working days on location per year, and very much more study; in this case of work of the unit or administrative function visited. Finally, because getting and managing gifts of money is the transcendent concern of scholarly organizations (scholars not normally being heavy earners), and because some Trustees are specialists in money (they have a great deal of it; or can earn it; or manage it professionally; or are close to other people who have a great deal of it), financial policy and action usually originate with the Trustees in consultation with the Chief Executive Officer. Implementation is the job of others: professional assistants (e.g., Vice Presidents) to the C.E.O. In our system at the MBL there is a significant departure from that pattern. It is that twenty-four, at least, of the thirty-six Trustees are — in effect — working members of the faculty; dedicated scholars. They are specifically not outsiders. They have a high personal stake in the day-to-day operations of the place. Their views of the work of the Laboratory are technically expert, and not infrequently narrow. It could not be otherwise. MBL Board Trustees, of course, are more like those of other institutions; but the tone and style of the whole Board's activities tend to be set by the majority. The basic system has worked for nearly a century, which means that no responsible member of the community is going to propose re-writing the Bylaws. It has worked, and it has changed a little, from time to time, to accommodate to external pressures. So far, so good: that is progress. The system was established in 1888 to run "... a laboratory or station for scientific study and investigations, and a school for instruction in biology and natural history." The clear understanding was that it would be a summer school and station. Woods Hole having been judged uninhabitable the rest of the year. The idea that biologists of the station would also be its owners and policy- managers, under no required constraint of advice from outsiders, was critical to survival of the place during the early decades. The idea was fought over; and happily it triumphed. The job of the resident MBL staflP was to take care of the scientists during the summer; and to take care of the plant, such as it was, during the long months when the scientists were back at home. To be sure, there were indispensable resident managers, the most important of whom must surely be our own Homer Smith: but the clear principle was that high- level policy is made by non-residents, while residents take care of the place, making such lower-level policy as may be needed for the provision of services, the payment of staflr(not scientists), and the maintenance of order. A great scientific research hotel, open for the summers. No derogation is implied: it was the world's best of its kind, but still a hotel. The firm decision to stop being a summer-scientific research hotel was made almost a decade ago. No member of the present Administration, except for Mr. Smith, was here or had any direct role in that decision; hence I can be objective about it, with neither pride in the decision nor the urge to disclaim responsibility for it. I believe that it was not only the right decision, but the only possible one. I have 44 MARINE BIOLOGICAL LABORATORY offered grounds for that belief, in writing, and shall not repeat them here. Suffice it to say that the MBL, functioning as a year-round scientific organization, with extended programs (not just laboratories for rent) and a long-term development plan adopted by the Trustees (in 1979), stepped onto the treadmill of competition. Competition with other free-standing laboratories, with universities and colleges, public and private, and with scholarly organizations of every other kind. Competition for people, rec- ognition, and support. Many accommodations to that reality have already been made. Quietly, but fairly effectively, procedures have been established for assembling a resident administration (never a mistake-free undertaking, and always traumatic for people already on the scene), which the MBL did not really have until the late 1970's. The job is far from done, but it is being done. Year-round scientific programs have been established. Some have quit but more have flourished, without any reduction of the historic commitment to summer research and teaching. This, too, has not been mistake-free, and trauma has been known to occur at times. Yet the year-round programs exist and do the place honor, as I have shown in earlier Director's Reports. Procedures for regular and searching review of their performance have been established and are being refined. The problem of managing the differences between year-round labo- ratories and those on location, i.e., summer laboratories, has been met head-on and is being solved, with responsible help of the Research Space Committee and the Executive Committee, neither of which had any such responsibility under the original plan of governance. We have found several millions of dollars, these past five years, for urgent physical rehabilitation of scientific facilities; and we have spent considerable sums on im- provements of housing. Having stepped onto the treadmill, the MBL has managed not to fall off or slip back: we could, today, even tolerate some (unexpected) upward tilt of the bed. Nevertheless the problems must be faced and solved. Changes in governance of the kind just noted are too much of a patchwork and are not widely understood. Some of them need to be codified. Some need to be extended; some should be reversed. The MBL needs more internal communication; informed guidance; technical man- agement skills; and effective decision-making processes that are not available within the existing system, and which cannot simply be bought or imposed from above. There is no "above." We, the Corporation, are not just the faculty and day-to-day users of the place: we are also the above. No simple handing-out of duties to the existing Board — not even to the twelve Board Trustees — can do the job. Our team of elected officers has insufficient depth in the line positions. They control tens of millions of dollars, in market- value, worth of assets; and an annual volume of ordinary business of the order often milhon. The responsibilities of a thin Administration are great: it is perhaps too personal to describe them as "crushing;" but their undeniable weight is felt by too few persons and there is no practical way, in our system, to spread them over all MBL Trustees, let alone among the more than six hundred non-resident Corporation members. It could be done in 1960; it can't be done today. I will not now discuss solutions to such problems; and we have already rejected Draconian measures, such as rewriting the Bylaws. But I am sure that there are solutions. I hope to be allowed in the next year or two to discuss them with those who care about the MBL. The aim here has been not to suggest specific devices by which we can stay on the treadmill and even gain on it, but rather to argue that such devices are needed, despite the fact that we have not fallen off; and to assert that they exist. They can vary greatly in form. Most versions require no fiddling with the Bylaws. No version requires denial of the MBL's historic missions in science and education. REPORT OF THE DIRECTOR 45 or allowing program separatism, or the imposition of rules from outside. That was all rejected ninety years ago. But before specific versions are proposed and argued, there must be real agreement among members of our MBL family that (1) we have made progress, and (2) its continuance requires the possibility of regular changes in governance. It would be the most important of all attractions for the next MBL Director, if he or she is to be the person of quality this great Laboratory deserves, to find that agreements ( 1 ) and (2) have been reached; that the way is clear to manage, as management must be done, a successful scientific enterprise of the late Twentieth and early Twenty-first centuries. Only a Pangloss expects progress without problems. But problems arising out of necessary progress are usually soluble, once their existence is recognized, the alternatives are studied honestly, and their costs and benefits weighed with care. Even nation- states behave that way; once in a great while. VIL REPORT OF THE TREASURER AND THE CONTROLLER "Sweet are the uses of adversity . . ." — Shakespeare Your Treasurer recalls that his father, an incurable optimist, would look upon his son's trying moments as "maturing experiences." This supportive comfort often would be accompanied by a Hoosier proverb, which observes that the same rain that rots the fallen log deepens the roots of the growing tree. Financially speaking, the MBL had some "maturing experiences" in 1982, cul- minating in a deficit. The $161,000 excess of expenses over income that year was a modest setback relative to the magnitude of the total budget. Nevertheless, 1982's problem served to strengthen our resolve in 1983 to add momentum to various efforts to increase revenues while intensifying the disciplines of expense management. We are pleased to report that these management actions not only improved the balance between receipts and disbursements, but in fact produced a surplus of $47,000. To understand 1983's achievement, one must look behind the figures. MBL's commitment to excellence has given the institution a stamina superior to the condition of many struggling not-for-profit organizations. Despite cutbacks in federal funding for research, MBL's laboratories continue to enjoy high levels of occupancy. The impHcation is that the calibre of the investigators applying for MBL laboratory space is such as to make them relatively more successful in their competition for available funds. Course enrollments, too, have remained high and continue to confirm the excellence of the MBL's educational and training programs. Thus, the historic quality of MBL activities have enabled it to sustain or increase revenues in most all categories. Specifically, Grants and Restricted Projects increased 1 2 percent, from last year's $3,476,000 to $3,894,000 in 1983. Laboratory fee income was up 15 percent compared with the previous year. Private gifts increased from $883,000 to $1,199,000, a 33 percent gain. Progress on the revenue side was matched with achievements in expense control. In the unrestricted budget, operating expenditures in support of research increased a modest $220,000, an increment of less than 6 percent over 1982. A switch from a service bureau's accounting system to an internally operated system, installed on our own recently acquired computer, resulted in faster turn-around on accounting and control reports. One consequence of improved management information was a highly successful effort to reduce Accounts Receivable in the over 90 days category by more than 50 percent. Throughout all operating areas, MBL employees once again dem- onstrated uncommon resourcefulness in finding ways to control costs while giving strong support to the MBL's purposes. 46 MARINE BIOLOGICAL LABORATORY Two balance sheet categories deserve comment. Fixed Assets are slightly reduced from the 1982 figure, reflecting the fact that the recent pace of new construction declined in 1983 so that fewer new facilities and improvements were put "on the books" last year. This coupled with the normal provision for depreciation, produced an expected decline in the book value of Fixed Assets. The Fund Balance also shows a modest decrease from $164,000 to $149,000, which largely is the result of capitalizing the acquisition of a new computer ($63,600) and renovating Devil's Lane housing ($13,700). Both projects were directly charged to the Fund Balance. We enter 1984 with more reasons for optimism than we had in 1983's early months. Although the outlook for research funding is no brighter, we feel a bit more comfortable with the MBL's ability to attract funded research to its laboratories. We see encouraging evidence that the arguments employed in our fund raising efforts are persuasive. We have better management tools with which to control expenses and to analyze the effectiveness of our operations. For all of these reasons, we expect to be in a position to give renewed attention to several key objectives for 1984. Increased participation by the Corporation's membership and Trustees in the development of the MBL's endowment will be one such important objective. Income from invested funds and from trusts benefitting the MBL are important sources of support for our purposes, accounting for 5.6 percent of 1983 income. Nevertheless, our present endowment is not an adequate cushion against financial adversity as its income is entirely consumed by day-to-day operations and therefore unavailable for support to new scientific initiatives or needed improvements to our facilities. With respect to endowment development, we are most anxious to leverage the challenge grant received from the Andrew W. Mellon Foundation. Successful matching of this grant will add $2.5 million to endowment funds available for library support. Also in connection with fund raising efforts, two feasibility projects will be com- pleted in 1984. One is studying the physical alternatives and associated costs for the development of a new Marine Resources Center. The second is addressing the feasibility of alternative solutions for additional housing, including the possibility of completing the Swope Center. Nineteen-eighty-four also will see continued review of MBL's real estate holdings and the policies that have governed the role played by these assets in our financial strategy. Although not generating income, the MBL's undeveloped land in Woods Hole continues to appreciate very significantly because of its scarcity, and holding costs are inconsequential. Our policy to date has been to view this land as an asset to be held in reserve against the possibility of a future financial contingency or to be utilized in some manner consistent with or supportive to the MBL's scientific and educational purposes. A third important objective for 1984 financial management will be the conclusion of the development and installation of a revised overhead recovery system. Although the need for a new approach has been recognized for some time, the process of reaching internal consensus while negotiating for approvals by cognizant agencies has required careful evaluation of alternatives and thorough preparation of supporting material. We are confident that a more rational approach will increase the MBL's overhead recovery, and of course, that provides a strong incentive to press forward, but at the same time any new system must be fair and equitable to all parties concerned. Finally, and no less importantly, we will be striving again in 1984 to achieve the income and expense goals of a balanced budget. Put differently, we prefer not to have any "maturing experiences" in 1984. Respectfully submitted, John Speer Robert Mainer Controller Treasurer REPORT OF THE TREASURER AND CONTROLLER 47 00 On VO ON Ol r- o ON^ ■ — OO r^l ir> r^ IT) m r<-i CO — NO On 00 ■/-! rn 2, Tt m 00 NO NO — O NO NO NO o oo NO O — o 00 o rn O On t^ r^ o c-t m' ro ■^ -^ OO ■/"! >/~l O CM m o O O O NO r "~- l\ Ov O. "«< 03 oi, P K. NO '^ t\ (J Ov ►-, ^^ 9 :^ •-^ '-J "O ^ l\ "> On (J :^ 3 ^ ?J I*, ;5: >^ On k! ^^ k 'N-) t\ On (^4 00 O ro "Tl u-i /-) NO r^ OO Tf O <^) r<-i On On rj- NO NO On «^ On Tt OO o ■ ^^ "^ rj (N t^ _ rj ON ON (N IT) m 00 (N a o d c ID ft; + a X *J , a H 03 i d < _3 o 2. « u o c OS 3 Lb o c > •c 6 .^ ■£ c op O ca c ■? 5: :i2 £ o. uu 48 MARINE BIOLOGICAL LABORATORY Coopers &Ly brand cerlified public accountants To the Trustees of Marine Biological Laboratory Woods Hole, Massachusetts We Laboratory as current funds the year then generally ace tests of the we considered and reported year ended De for comparatl have examined the balance sheet of December 31, 1983 and the re revenue and expenses and change ended. Our examination was mad epted auditing standards and, ac accounting records and such othe necessary in the circumstances. upon the financial statements of ceraber 31, 1982 which condensed ve purposes only. of Marine Biological lated statements of s in fund balances for e in accordance with cordingly, included such r auditing procedures as We previously examined the Laboratory for the statements are presented In our opinion, the financial statements referred to above present fairly the financial position of Marine Biological Laboratory at December 31, 1983 and its current funds revenue and expenses and the changes in fund balances for the year then ended, in conformity with generally accepted accounting principles, applied on a basis consistent with that of the preceding year. ^oopcjvb % CAubnomA Boston, Massachusetts June 7, 198'4 REPORT OF THE TREASURER AND CONTROLLER 48a <3^ o oo in oo ■* Tt O «n o m ■* so Ov 1^ t~ <-s< OS so oo_ "'1 r-_ r-i rn >n ■* rl Tt vO o so T 1^ OS, "^i O _J _^ ^ o oo 1^ •r> w^ W~l t~- oo SO 1 O fN rn sD SO r~i ro Tj- (rt •* OS OO _ T t~- f .M so — • Os_ so »n r»i •*' oo Vl ■ so r-_ r- t (N OS __ O •<*• so sO_ r-_ •X rs| p--' so 0^ O H < 00 oi On C -^ 03 c/n T3 < H C UJ X 01 00 <* OD as U z < < e Q ^ «J c V) ^ ^ a C 05 K 1 X u 3 T3 li. lU T3 2 C u a ^ .§ T3 C •^ m * u 4> «5 e o ca u a c c/l T3 C u 3 t O 4> o 0= c_» i? < Q u u s »^ « s •n "> **- c •c S T3 C ,3 C u E o ■a C i« O obob.S T3 T3 T3 ^ a> i> •O -O T3 C C C i 5 K ^ ^ -^j *-* c c u v t fc 3 3 U U a> a> u c c c D D D "N 00 >A> 00 OS so Ov — sO OO O OS ' so t^ ro •* O -is (O •= " X) c ''I •o ^ ^ C I- >- a .2 <2 M u u g E E c o o too ^ B s. o " T3 c UJ u c C4 c ,3 C -o Q o c ^ .y ■i 3 °^ 3 a 4> c v. o ■c ■§ X) u •o 'C c ts ,3 c a> w- D ex: C CD Ou ca e ,3 i 1 a> (A c -^ iS — * ■« ■o — u E u O OS — ■* o o — so Tt •/^ "/^ rn n a> o -s c o- c •S 3 U c eg c o fc 3 c K5 (0 U. •o E a j: c p S « 8 U S < •S 8 t; .•e u! s = " 8 C ~ 3 ■^ '^ s^ U z c c '^ E s u D. .2 O 3 Ji z ? ^ t;;'g-S 8 « I M ot s ^'1 I c ~ E 3 3 ♦i • ™ 4J ^ (/3 > C- (/) 3 o H 48b MARINE BIOLOGICAL LABORATORY •^ m ^ -^ ^r\ — r-'^mo — O^ •oooso ONr-r-— Tj-ONi/^o r-' r^i ON ON oo' r-' *o rn r-' — ' o' _f*-i__ — Ooo— ooONOor- »/~ioofn (Nrsif'^ — ^cNO^oo NO r-j NO so ON ■^, «r> W-1 •W vl O NO r*-i ^ w^ rs ro r-' *»e ON — •ri r~ oo — |N-t „ o pr, «o — oo oo fs o M^ O oo NO so <-o r-^ o — r-; rn I--' — o oo' r-i fs o< ^' ^ ^ rK o rs rsi On o ■^, rr), On ON_ ~~ r-' r—oorn noo*— r^r^^ — v-i nono— r-~^o^<^^^s^-^m■^t■ r-i oo r--_^ — — o- 00 '^ r^ oo rj <^^»/^ ONt/^O^Oi^-^iOir- 00^— rN O H O OQ < H-l ^ z si H Z u z o c/3 z uu Ui < C/5 00 - I T5 5 C £ CO "*; m oo ON 6 O Q C £2 I J3 i-i ,o 00 *r\ 00 oo On^ ■^r — oo r- — ^ fN — t rsi VI r~ fs no^ r^' <*{ ON o o — r-i oo m r4 3 — r-i O NO ON^ fS 1 NO r^' r«^ *0 so' 1 O ON «/^ — — O r*^ 'T m rj ON — f*^ vi r- r- r- oo •^ r- r- oo TT ^, On' m' *r{ r- ON OO •^^ r^ ^ Sf*i ^ r-i rN — o' v^ <^ ^ 00 On "/^ On '-' ^ - 8.E eS. :al Bulletin h services resources It s s I. 3 c *> X > IS =1 6 1 c c i c •— 00 II Library Biologic Researe Marine ii "3 a Q^ H M c X 5^ S c = N— ' ™ ti 4J 3 C c 1» V) •o c W) c B > o -^ ■S ■— 00 r^ " = c i (O c ^ U 3 I « c = 1- o 5 -c - o e c o 5 .-5 S i3 o Q Q _l CQ Oi O c c 3.1.2 S S 2 y i: V g c o S <0. 2 o H Q. E o REPORT OF THE TREASURER AND CONTROLLER 49 i ■§ ^ ■? 2 vO •o (N ON w-i oo ■4 iri m r> r- fN -"S- •^ t- o O NO 00' TJ-' — r- r~-_^ On r<-i ^^ NO 00 (N ^__^ ON 'I- — 2, NO 00 «/^ 00 NO 1^ 00 no' m NO -^ o — r-' ri rn 00 o 00 nd' ^ , 00 m — cnI rsi NO On «-i — ■ •o c ,3 13 g 3 o •a ■c y c c 2 DO u > c ■§5 I/) ^ 6 00 (U 6 ?^ oa v, 1^ .t: .- a 3 H I e X c •o c o w c o .2 " o o C C O a c o c o o o '35 c 8. 2 I 00 (/) N Jj — X c (/> O c c ,3 QO c 4> Z ■3 s: » •o u X 5 2 o c o c CO s: a ?6 i CO 50 MARINE BIOLOGICAL LABORATORY 00 =3 2; ^ 0\ ■« as _ oo t^ On o •^ o fN Os 00 oo «-) ■* — rri .t; •^ c m >5 00 5b ■n oi, rvT V» ■Q ^ .U so v! <^ *-* ^ so c 00 :i «« o o o r-' so n Tj- 00 00 so' Os' OS so as ■n so (^ 00 •*' fN 00 o so SO (N OS ^.-^ — ro Os (N 0^ so' 00' (^4 — — ^^ (N ^-^ Vi ,_^ 00 OS m ^« m ri Os' OS rt 00 c 6 « to c c s B c > ^ C > !U c^« u» T3 >» ,3 00 13 ■•5 ■0 '3 ■O 1 fe c DOO £ CeJ < H u c C op 12 S c ^ 2 u z i3 4 O .id X 3 c 1) 03 -.V- (1 A 3 4J a- j= PQ CO a 1 c a n I/) u ^-* o c 00 c >. c « a S o u u ca /-> ^ O On On t^ — r- ly^ 00 ON m rs| •o oo 1^ ^- iri — oo rn CNl oo On — N£> rt O ro O 'I- On rn NO (N NO NO rn rsi oo O >n' TT O iri «^ ^^ w-m O ^ >n ■o r-' r J «n' o NO rn O «N rs I On Tj- Tj- r'l 00 On >r) ro NO O ■^ >r) On fN NO •/"! ro OO OO rn r-' Tj- On "n NO — ' r- NO OO Tt — ' ■^' 00 o On 00 o 00 o o On_^ •n no' NO ^ NO ■^_^ oo' Vi On r*^ 00 NO On w-i T — (^ NO -^ — '"n ■^^ r-_ fN p--_ in r-~ 00 m' oo' tN no' r--' o' On r- -^ fN o — r- "«■» r^cn m'Tt >n rsf m C 00 IZ ^ 'N-l o "" OO On (U _' _3 rn "S u > u w X) « H Sq o « O t« *" p^ t« o u ^6 «« t H<2 H Ji 3 6 " o C3 O 52 C 00 -s c F .~ U ^ E >5 o i: ■»»»* — c/3 § s ^ s H o c -M — V-, NO On n rn r^ r- >n NO t^ O «n On NO O -^ ■ E o c F. "■ <" C (U o 5 X o C «= t? ^ «i c o « o a s E &E D (J U C/3 1> E 5 §^ o •n o -^ o i^ o O m (N >n •^ on' t*^ NO rr PO 00 On o ■^ t- o ■^ On — ^ — r~ NO m ^^ ON ^« rn rn r^ in NO — NO NO ^^ c T3 "o O a o H c '•5 o *-* 1/3 3 (J •c 3 o « E >^ C o c a> E «^ (A > _c 13 *-• o H s: s: 5 s: = t O C ,o Xl l*-i qj c o S<2 < a o T3 CO ^ 3 u ^ I C c k. 1/1 ca o ^ B O 1 o B u t: 3 T3 U N ? S g :§ ifl T3 C ,3 (U o Ct5 D 54 MARINE BIOLOGICAL LABORATORY At December 31, 1983 the following summarizes the participation of the various funds in the investment pool. Unexpended income of endowment $ 43,178 Unrestricted endowment 1,716,309 Restricted endowment 682,475 Unrestricted quasi-endowment 846,880 Restricted quasi-endowment 2,788.073 Retirement 1.263.477 $7,340,392 REPORT OF THE LIBRARIAN 55 VIII. REPORT OF THE LIBRARIAN This past year all the Library collections were brought back to Lillie and reshelved in new locations so that now, after two years of minor confusion, the collection is once more easily accessible to all users. The Rare Books/Archives area on the first floor is a handsome suite of three rooms, one a comfortable Discussion Room, furnished with rugs and Louis Agassiz's large table in the center. The books and archival material are secured in an environmentally controlled area. Fortunately, we now have an Archivist, Ruth Davis, who is organizing and cataloging all Class photographs, cor- respondence. Meeting minutes, and other archival material. We would be delighted to receive any material of this nature that may be in personal collections of MBL Corporation members. In March of 1983 we initiated a most comprehensive Survey of the USE made of our entire Journal collection. Cathy Norton of the Library Staff is the Project Coordinator and the funding for this project came from the Rockefeller Foundation. It included purchase of a computer terminal and printer for the Library in order to process all the data received from the Study. Every Journal issue that was received from the first of March to the end of December was monitored each time it was used, and all bound journals in the stacks were marked each time they were returned to the shelves. It involved an inordinate amount of record-keeping the results of which will be available in the Fall of 1984, and in the next Annual Report. One most interesting tentative fact; MBL's BIOLOGICAL BULLETIN was referred to over 1,000 times during the ten month survey and was the tenth "most used" journal in the collection of 4,763 separate journal titles. A second part of the Survey is a USER study which will continue through the summer of 1984. On a number of unannounced days all doors to the Library are locked with the exception of two entrances. All users on that day pass by a desk where they are registered as to institution affiliation. This information will be analyzed at the end of September. Discussions were held with the National Marine Fisheries Service during the year concerning the incorporation of their library collection with ours in the Main Library. Space has been provided in the Book and Journal stack area for this eventuality. IX. EDUCATIONAL PROGRAMS SUMMER Biology of Parasitism Course Director David, John, Harvard School of Public Health/Harvard Medical School Other faculty, staff, and lecturers ASKANASE, Philip, Yale University Caulreld, John, Harvard Medical School Chang, Kwang-Poo, Chicago Medical School COLTEN, Harvey, Harvard Medical School Cross, George, The Rockefeller University David, Roberta, Harvard Medical School Dessein, Alain, Harvard Medical School DwYER, Dennis, NIH Elsbach, Peter, New York University Englund, Paul, Johns Hopkins University 56 MARINE BIOLOGICAL LABORATORY Fearon, Douglas. Harvard Medical School GiTLER, Carlos, Weizmann Institute of Science, Israel Harn, Donald, Harvard Medical School Ku, Albert, Harvard Medical School Landfear, Scott, Harvard School of Public Health LoDiSH, Harvey, Massachusetts Institute of Technology Marsden, Philip, University of Brasilia, Brazil McLafferty, Martha, Harvard School of Public Health Metzger, Henry, NIH Miller, Louis, NIH Nelson, George, Liverpool School of Tropical Medicine, England, U. K. NUSSENSWEIG, Ruth, New York University Pereira, Miercio, Tufts New England Medical Center Perkjns, Margaret, The Rockefeller University Pftfferkorn, Elmer, Dartmouth College PlESSENS, Willy, Harvard University Pratt, Dianne, Harvard Medical School RiFKiN, Mary, The Rockefeller University Roberts, Bryan, Harvard Medical School RossiGNOL, Philippe, Harvard School of Public Health Sher, Alan, NIH Sherman, Irwin, University of California at Riverside Spielman, Andrew, Harvard School of Public Health Swiston, Linda, Mount Holyoke College Wilson, Darcy, University of Pennsylvania Wirth, Dyann, Harvard School of Public Health Wyler, David, Tufts University Medical School Students ' Anaya-Velazquez, Luis, Center for Research and Advanced Studies of National Polytechnical Institute, Mexico Bhasin, Virendra, The Rockefeller University Chavez, Larry, The University of New Mexico Cseko, Yara, Fundacao Oswaldo Cruz, Brazil DOBBELAERE, DiRK, International Laboratory for Research on Animal Diseases, Kenya Ekapanyakul, Galayanee, Mahidol University, Thailand Fairfield, Alexandra, Cornell University Medical College Flisser, Ana, Instituto de Investigaciones Biomedicas, Universidad Nacional Autonoma de Mexico Goodman, Howard, Massachusetts General Hospital Krakow, Jessica, Johns Hopkins University School of Medicine Percy, Amy, University of California, Los Angeles RiVAS-LOPEZ, Luis, Instituto de Inmunologia y Biologia Microbiana, Spain Romero, Guillermo, Instituto de Medicina Tropical "Alexander von Humboldt", Peru Schwarz, Ralph, Deutsche Forschungsgemeinschaft, West Germany Sibley, Laurence, Louisiana State University Wyman, Claire, Johns Hopkins School of Hygiene and Public Health Embryology Course Directors Brandhorst, Bruce, McGill University, Canada Jeffery, William, University of Texas ' All summer students listed completed the formal course program. Asterisk indicates those completing post-course research sessions. EDUCATIONAL PROGRAMS 57 Other faculty, staff, and lecturers Angerer, Robert, University of Rochester Bates, William, University of Texas BiGGERS, John, Harvard Medical School Brodeur, Bonnie, University of Texas Brodeur, Richard. University of Texas Brower, Daniel, University of California, Irvine Brown, Donald, Carnegie Institute Broyles, Robert, University of Oklahoma College of Medicine Capco, David, Massachusetts Institute of Technology Chambers, Edward, University of Miami Crain, William, Worcester Foundation Cross, Nicholas, University of California, Davis Davidson, Eric, California Institute of Technology Desantis, Rosaria, Naples Marine Station, Italy Drago, Salvatore, University of Toronto EiSEN, Andrew, University of Pennsylvania Elinson, Richard, University of Toronto Emerson, Julie, University of California, San Francisco Epel, David, Hopkins Marine Station Etkjn, Larry, University of Tennessee Goldsmith, Marian, University of Rhode Island Gould, Meredith, University of California at San Diego GuRDON, John, University of Cambridge Henry, Jonathan, University of Texas HiLLE, Merrill, University of Washington HOSHI, MOTONORI, Nagoya University, Japan Humphreys, Thomas, University of Hawaii Jacobson, Alan, University of Massachusetts Medical School Jaffe, Laurinda, University of Connecticut Health Center Jaffe, Lionel, Purdue University/Marine Biological Laboratory Kalthoff, Klaus, University of Texas Klein, William, Indiana University Masui, Yoshio, University of Toronto Maxson, Ellen, Stanford University Maxson, Robert, Stanford University Melton, Douglas, Harvard University Miller, John, University of Calgary, Canada MoHUN, Timothy, University of Cambridge, U. K. Moon, Randy, Caltech Morrow, Laura, University of Texas Nelson, Ellen, University of Texas NucciTELLi, Richard, University of California, Davis Nusselein-Volhard, Christiana, University of Tubingen, West Germany Pederson, Thoru, Worcester Foundation for Experimental Biology Phillips, Carey, University of California, Berkeley Phillips, Eric, University of Texas Pinto, Angelo, University of Toronto Raff, Rudy, Indiana University Rankin, Mary Ann, University of Texas Robinson, Kenneth, University of Connecticut ROSBASH, Michael, Brandeis University RUDERMAN, Joan, Harvard Medical School Sardet, Christian, ViUefranche, France Schlicter, Lyanne, University of Connecticut 58 MARINE BIOLOGICAL LABORATORY ScHULTZ, Gilbert, University of Calgary, Canada SCHULTZ, Thomas, University of Texas ScoRELD, Virginia, Hopkins Marine Station Shettles, Brewer, University of Texas Spradling, Al, Carnegie Institution Spray, David, Albert Einstein College of Medicine Trinkaus, J. P., Yale University Vacquier, Victor, University of California, San Diego Wassarman, Paul, Harvard Medical School Weischaus, Eric, Princeton University Whitaker, Michael, University College, London, England, U. K. Whittaker, J. Richard, Boston University Marine Program/Marine Biological Laboratory Wilson, Linda, University of Texas Winkler, Matt, University of Texas Woodland, Hugh, University of Warwick Ziomek, Carol, Worcester Foundation Students ' Bao, Cheng- Yuan, Case Western Reserve University *Beach, Rebecca, University of Connecticut, Storrs *Begovac, Paul, University of Rorida College of Medicine *vON Brunn, Albrecht, University of Texas, Austin *Chung, Margaret, Tufts University School of Medicine *CoNLON, Ronald, McGill University, Canada *Dearolf, Charles, The Johns Hopkins University *Halsell, Susan, University of Texas/Patterson Laboratories *HowLETT, Sarah, University of Cambridge, England, U. K. *Klein, Karen, University of lUinois *LuNDMARK, Cathy, University of California, Berkeley *Lynch, Eileen, The Rockefeller University *Lyons, Gary, University of Pennsylvania School of Medicine Margules, Deborah, University of Michigan *MiLLER, Mill, Tulane University *Nagy, Lisa, University of California, Berkeley *OSHiRO, DiANNE, University of Virginia *Perez-Grau, Lluis, European Molecular Biology Laboratory, West Germany *ROMANO, Charles, University of Massachusetts, Amherst *Singer, Susan, Rensselaer Polytechnic Institute *Stevens, Mary, University of California at Irvine *SWALLA, BiLLiE, University of Iowa *Wang, Allan, University of Hawaii, Manoa Marine Ecology Course Directors Teal, John, Woods Hole Oceanographic Institution Valiela, Ivan, Boston University Marine Program/Marine Biological Laboratory Other faculty, staff, and lecturers Alberte, Randall, University of Chicago Anderson, Donald, Woods Hole Oceanographic Institution Banta, Gary, Boston University Marine Program/Marine Biological Laboratory Caron, David, Woods Hole Oceanographic Institution Connell, Joseph, University of California, Santa Barbara EDUCATIONAL PROGRAMS 59 Dacey, John, Woods Hole Oceanographic Institution D'AVANZO, Charlene, Hampshire College Dennison, William, University of Chicago Frank, Peter, University of Oregon Gallagher, Eugene, Marine Biological Laboratory GiBLiN, Ann, Woods Hole Oceanographic Institution Gilbert, Patricia, Woods Hole Oceanographic Institution Grassle, Frederick, Woods Hole Oceanographic Institution HOBBIE, John, Marine Biological Laboratory HowARTH, Robert, Marine Biological Laboratory Jannasch, Holger, Woods Hole Oceanographic Institution Jefferies, Robert, University of Toronto, Canada KOEHL, MiMi, University of California, Berkeley Lambertsen, R., University of Florida Levinton, Jeffrey, SUNY, New Paltz Madin, Laurence, Woods Hole Oceanographic Institution Mann, Roger, Woods Hole Oceanographic Institution Marsh, J., University of Guam Nixon, Scott, University of Rhode Island Odum, William, University of Virginia Peterson, Susan, Woods Hole Oceanographic Institution Revelas, Gene, SUNY, Stony Brook Rietsma, Carol, SUNY, New Paltz Sanders, Howard, Woods Hole Oceanographic Institution Stoecker, Diane, Woods Hole Oceanographic Institution Welschmeyer, N., Harvard University WOODWELL, George, Marine Biological Laboratory Wynes, David, Mount Desert Island Biological Laboratory Students ' *Ackerman, Josef, SUNY, Stony Brook Berggren, Ruth, Oberlin College Cooler, Sue, Northeastern University Donaldson, Jack, New College of the University of South Rorida *EVANS, Ann, Virginia Institute of Marine Science/College of WiUiam and Mary Feddeler, William, Wayne State University Frey, Jonathan, Ohio Wesleyan University Goldberg, Sandra, Tufts University HoGUET, Nancy, Barnard College Kessing, Bailey, New College of the University of South Florida Lasta, Mario, Instituto de Biologia Marina y Pesquera "Alte. Stomi," Argentina *LiEBMAN, Matthew, SUNY, Stony Brook McCormick, Deborah, University of Alaska, Anchorage Neill, Christopher, Louisiana State University Pascual, Marcela, Instituto de Biologia Marina y Pesquera "Alte. Storni," Argentina Perkins, Eleanor, Marine Biological Laboratory Prochazka, Karan, Cambridge, Massachusetts Slough, Debra, Butler University Tamse, Armando, Boston University Marine Program/Marine Biological Laboratory Wagenbach, Gary, Carleton College Microbial Ecology Course Director Halvorson, Harlyn, Brandeis University 60 MARINE BIOLOGICAL LABORATORY Other faculty, staff, and lecturers Alexander, Martin, Cornell University Atwood, Kimball, Columbia University AusiCH, Rodney, Standard Oil of Indiana BOSTIAN, Keith, Brown University Castenholz, Richard, University of Oregon Cavanaugh, Colleen, Harvard University Cronin, John, University of Illinois Davis, Bernard, Harvard University School of Medicine DwoRKiN, Martin, University of Minnesota Gray, T. R. G., University of Essex, England, U. K. Greenberg, E. Peter, Cornell University Hansen, Richard, Gray Freshwater Biological Institute HOBBIE, John, Marine Biological Laboratory Humphrey, Arthur, Air Products and Chemicals, Inc. Jannasch, Holger, Woods Hole Oceanographic Institution Keynan, Alexander, Hebrew University, Jerusalem, Israel Kornberg, Hans, Cambridge University, England, U. K. Leadbetter, Edward, University of Connecticut, Storrs MoRTENSON, Leonard, Exxon Research and Engineering Company Ornston, L. N., Yale University PiERSON, Beverly, University of Puget Sound Poindexter, Jeanne, Public Health Research Institute, New York, NY Rich, Alex, Massachusetts Institute of Technology Romesser, James, Dupont Corporation Ruby, Edward, University of Southern California Schaechter, M., Tufts University Shilo, Moshe, Life Science Institute of Hebrew University, Jerusalem, Israel Vincent, Walter, University of Delaware Waterbury, John, Woods Hole Oceanographic Institution WosE, Carl, University of Illinois Wolfe, Ralph, University of Illinois Students ' Ackerman, Eugene, University of Arkansas BOYER, Joseph, Virginia Institute of Marine Science/College of William and Mary Decho, Alan, Louisiana State University Escher, Andreas, Montana State University College of Engineering Harten, James, Vanderbilt University Hartzell, Patricia, University of Illinois JOUPER, ASA, Gothenberg University, Sweden KiEFT, Thomas, New Mexico Highlands University Malmcrona-Friberg, Karin, Gothenberg University, Sweden MUEHLSTEIN, LISA, Wright State University Noll, Kenneth, University of Illinois Robertson, Charles, Skidaway Institute of Oceanography Santoro, Nicholas, Ohio State University Skladany, George, Clemson University Sutherland, Dale, Creighton University Sutton, William, Auburn University Wrenn, Brian, University of Miami Rosenstiel School of Marine and Atmospheric Sciences educational programs 61 Neural Systems and Behavior Course Directors Hoy, Ronald, Cornell University Macagno, Eduardo, Columbia University Other faculty, staff, and lecturers Alkon, Daniel, NINCDS/Marine Biological Laboratory Bennet-Clark, Henry, Oxford University, England, U. K. Calabrese, Ronald, Harvard University Carew, Thomas, Yale University Chalre, Martin, Columbia University Flanagan, Thomas, Cold Spring Harbor Laboratory Getting, Peter, University of Iowa Gould, James, Princeton University Grinvald, Amiram, Weizmann Institute, Israel Haerter, Ursula, Columbia University Harris-Warrick, Ronald, Cornell University Hopkins, Carl, Cornell University Kandel, Eric, Columbia College of Physicians and Surgeons Kelley, Darcy, Columbia University Kravitz, Edward, Harvard Medical School Kristan, William, University of California, San Diego Lent, Charles, Brown University Marder, Eve, Brandeis University Marler, Peter, The Rockefeller University McVey, Margaret, The Rockefeller University Menzel, Randolf, Free University of Berlin, West Germany Murphey, Rodney, SUNY, Albany MuSACCHiO, MiCHELE, Columbia University Nelson, Margaret, Cornell University NiCHOLLS, John, Stanford University School of Medicine/Biocenter, Basel, Switzerland Rehder, Vincent, Institut fur Neurobiologie, West Germany Sahley, Christine, Yale University Sassoon, David, Columbia University Segil, Neil, Columbia University Silver, Rae, Columbia University Stewart, Randy, Columbia University WiESEL, Torsten, Rockefeller University ZiPSER, Birgit, Cold Spring Harbor Laboratory Students ' *Baptista, Carlos, University of Lisbon, Portugal Beason, Robert, SUNY, Geneseo *BiRD, Edythe, Yale University BORST, Alexander, Institut fur Genetik und Mikrobiologie, West Germany Coon, Steven, University of Maryland *Delaney, Kerry, Princeton University *Gaide, Michael, Free University of Berlin, West Germany Gilliam, David, University of Colorado KUTERBACH, DEBORAH, SUNY, Stony Brook Lander, Eric, Harvard University Lannoo, Michael, Dalhousie University, Nova Scotia, Canada Lesk, Mark, The Weizmann Institute of Science, Israel 62 MARINE BIOLOGICAL LABORATORY Li, Christine, Harvard University Noble, Michael, St. Mary's Hospital Medical School, University of London, U. K. RiSKA, Diane, University of California, Los Angeles Sims, Stephen, Columbia University Smith, Jeffrey, Harvard School of Public Health Stevens, Craig, University of Illinois, Chicago Weiner, George, Harvard University *WoRDEN, Mary, University of Chicago Neurobiology Course Directors HiLDEBRAND, JOHN, Columbia University Reese, Thomas, NINCDS/NIH/Marine Biological Laboratory Other faculty, staff, and lecturers Armstrong, Clay, University of Pennsylvania Battelle, Barbara, National Eye Institute, NIH Bentley, David, University of California at Berkeley Bray, Dennis BuRD, Gail, The Rockefeller University Constantine-Patton, Martha, Princeton University Christakis, Nicholas, Yale University DuNLAP, Kathleen, Tufts University Medical School Evans, Peter, University of Cambridge, England, U. K. Fischbach, Gerald, Washington University School of Medicine FuRSHPAN, Edwin, Harvard Medical School GoY, Michael, Harvard Medical School Hall, Linda, Albert Einstein College of Medicine HUTTNER, SuSANNE, University of California, Los Angeles Jacobson, Marcus, University of Utah College of Medicine Kachar, Bechara, NINCDS, NIH Kent, Karla, Columbia University KiNNANE, Janet, Marine Biological Laboratory LaFratta, James, Harvard Medical School Landis, Dennis, Massachusetts General Hospital Landis, Story, Harvard Medical School Marler, Jenni, McGill University, Canada Matsumoto, Steven, Harvard Medical School MiCHAUD, Jayne, Marine Biological Laboratory NiCAiSE, Ghislain, Universite Claude Bernard, France NiCAiSE, Mari, Universite Claude Bernard, France NiSHi, Rae, Harvard Medical School O'CONNELL, Maureen, NINCDS, NIH O'Lague, Paul, University of California, Los Angeles Patterson, Paul, Harvard Medical School Potter, David, Harvard Medical School PURVES, Dale, Washington University School of Medicine Raviola, Elio, Harvard Medical School Reese, Barbara, NINCDS, NIH ROSENBLUTH, JACK, New York University School of Medicine Salzberg, Brian, University of Pennsylvania SCHNAPP, Bruce, NINCDS, NIH/Marine Biological Laboratory Shotton, David, University of Oxford, England, U. K. Stevens, John, Toronto Western Hospital, Canada EDUCATIONAL PROGRAMS 63 Thoenen, Hans, Max Planck Institute for Psychiatry, West Germany Walrond, John, NINCDS, NIH/Marine Biological Laboratory WiESEL, TORSTEN, Harvard Medical School WiLLARD, Mark, Washington University School of Medicine ZiGMOND, Richard, Harvard Medical School Students ' Baetge, Greg, Columbia University Boardman, Ian, University of Pennsylvania BODMER, Rolf, Friedrich Miescher Institut, Switzerland De Santis, Amedeo, Stazione Zoologica de Napoli, Italy Goodman, Linda, The Albert Einstein College of Medicine Marsh, Terry, National Jewish Hospital and Research Center New, John, Wesleyan University Peinado, Alejandro, Columbia University Ruano-Arroyo, Gualberto, Yale University School of Medicine SCHRANK, Ethan, University of North Carolina at Chapel Hill WiTTEN, Jane, University of Chicago Physiology Course Director ROSENBAUM, Joel, Yale University Other faculty, staff, and lecturers ACKERS, Gary, Johns Hopkins University Allewell, Norma, Wesleyan University Altman, Sidney, Yale University AusuBEL, Fred, Harvard University Baltimore, David, Massachusetts Institute of Technology Begg, David, Harvard Medical School Bloodgood, Robert, University of Virginia Borisy, Gary, University of Wisconsin Brinkley, William, Baylon University Medical School Burgess, David, University of Miami Centonze, Vicky, Dartmouth College Chisholm, Rex, Massachusetts Institute of Technology CONDEELIS, John, Albert Einstein College of Medicine Dreyfus, Gideon, Northwestern University FiNDLY, Craig, Yale University Gall, Joseph, Yale University Gerace, Larry, Johns Hopkins University Medical School Goldman, Robert, Northwestern University Medical School Greene, Kathleen, Northwestern University Medical School Hereford, Lynna, Brandeis University HoTANi, Q-Chan, University Kyoto, Japan Hunt, Timothy, Cambridge University, England, U. K. INOUE, Shinya, Marine Biological Laboratory Johnson, Ross, University of Minnesota Jones, Jonathan, Northwestern University Medical School Kaumeyer, John, University of Pennsylvania Kilmartin, John, Medical Research Council, England, U. K. KuczMARSKi, Edward, Northwestern University Kumar, Ajit, George Washington University Medical Center KURIYAMA, Ryoko, University of Wisconsin 64 MARINE BIOLOGICAL LABORATORY KuwABARA, Patricia, University of Pennsylvania Lazarides, Elias, California Institute of Technology Lefebvre, Peter, University of Minnesota LoDiSH, Harvey, Massachusetts Institute of Technology May, Gregory, Yale University McCarthy, Michael, Wesleyan University Mitchell, David, Yale University Mooseker, Mark, Yale University Murray, Andrew, Harvard University Pant, Harish, National Institute on Alcohol Abuse and Alcoholism Reid, Martha, Earlham College Rich, Alexander, Massachusetts Institute of Technology Rosenthal, Eric, Harvard University Medical School RuSHFORTH, Alice, Earlham College SCHACHMAN, HOWARD, University of California, Berkeley Sheetz, Michael, University of Connecticut Medical School SiLFLOW, Carolyn, University of Minnesota Sjostak, Jack, Dana Farber Cancer Research Center/Harvard University Sloboda, Roger, Dartmouth College Sluder, Kip, Worcester Foundation Snell, William, University of Texas, Southwest Medical School Steffen, Pamela, Wesleyan University Stroud, Robert Tamm, Sidney, Boston University Marine Program Taylor, D. Lansing, Carnegie-Mellon University Thompson, Thomas, University of Virginia Tighman, Shirley Tilney, Lewis, University of Pennsylvania Tobin, Sally, Washington University Travis, Jeffrey, Yale University Tytell, Michael, Wake Forest University Vallee, Richard, Worcester Foundation Weinberg, Eric, University of Pennsylvania Zackroff, Robert, Northwestern University Medical School Students ' *Bornslaeger, Elayne, University of Pennsylvania Bruehl, Charles, Northwestern University *Carson, Monica, University of Pennsylvania *CoNRAD, Patricia, University of Massachusetts, Amherst *CoYNE, Robert, Harvard University *CuPO, James, University of Rochester *Diener, Dennis, University of Kansas *Garrett, Esther, George Washington University *George, Elizabeth, University of Virginia *GiLBERT, Susan, Dartmouth College *Green, Philip, University of North CaroHna *Greer, Karen, Yale University Happel, Anne, Harvard University Healy, Judith, Harvard University *HiLL, David, Loyola University/Foster McGaw Hospital HONMA, Mary, Harvard University *Intres, Richard, Wesleyan University *Jones, Stephanie, Vanderbilt University *JoYCE, Catherine, University of Minnesota *Kelly, Thomas, Jr., University of North Carolina, Chapel Hill *LuM, Richard, University of Hawaii, Manoa EDUCATIONAL PROGRAMS 65 *MiLGRAM, Amanda, Johns Hopkins University *Pagliaro, Leonard, Wesleyan University Rodriguez, Olga, University of Puerto Rico, Rio Piedras *Shupe, Kathleen, University of Rochester *TiTUS, Margaret, Brandeis University *TuCKER, Richard, University of CaHfomia, Davis *Ward, Eric, Washington University Wordeman, Linda, University of CaHfomia, Berkeley *Wright, Connie, The George Washington University Medical Center JANUARY Behavior Course Director Atema, Jelle, Boston University Marine Program/Marine Biological Laboratory Other faculty, staff, and lecturers Alkon, Daniel, NIH/Marine Biological Laboratory Barlow, Robert, Syracuse University Institute for Sensory Research Brisbin, I. Lehr, Savannah River Ecology Program Bryant, Bruce, Boston University Marine Program/Marine Biological Laboratory Callard, Gloria, Boston University Canick, Jacob, Brown University/Woman and Infants Hospital Dethier, Vincent, University of Massachusetts Dolphin, William, Boston University Dorsey, Ellie, Payne Laboratories Erskine, Mary, Massachusetts Institute of Technology Fay, Richard, Loyola University of Chicago Parmly Hearing Institute Ferme, Paula, Boston University Marine Program Francis, Elizabeth, Bates College Eraser, Jean, Boston University Handrich, Linda, Boston University Marine Program Hausfater, Glen, Cornell University Jacklett, Jon, SUNY, Albany Johnson, Bruce, Boston University Marine Program Kamil, Al, University of Massachusetts Kreithen, Mel, University of Pittsburgh Kroodsma, Donald, University of Massachusetts Langbauer, William, Boston University Marine Program/Marine Biological Laboratory LiEM, Karel, Harvard University Museum of Comparative Zoology MOLLER, Peter, American Museum of Natural History Payne, Katy, Lincoln, Massachusetts Payne, Roger, Lincoln, Massachusetts RiSTAU, Carolyn, The Rockefeller University Stuart, Alastair, University of Massachusetts SuLZMAN, Frank, SUNY, Binghamton Tayak, Peter, Woods Hole Oceanographic Institution Traniello, James, Boston University Trott, Thomas, Boston University Marine Program/Marine Biological Laboratory Wilcox, Stimson, SUNY, Binghamton Students Carter, Stephanie, Boston University Chu, Kevin, Boston University Marine Program Einolf, David, University of Delaware College of Marine Studies 66 MARINE BIOLOGICAL LABORATORY Handrich, Linda, Boston University Marine Program Hutchinson, Linda, Boston University Kesaris, Alex, University of Connecticut Leibensperger, Laura, Boston University Murray-Brown, Mark, Boston University Neidhardt, Peter, Bucknell University Plass, Karen, University of Wisconsin, Madison Comparative Pathology of Marine Invertebrates Course Directors Bang, Betsy Garrett, Johns Hopkins University School of Hygiene & PubUc Health/ Marine Biological Laboratory Reinisch, Carol, Tufts University School of Veterinary Medicine Other faculty, staff, and lecturers ASKENASE, Philip, Yale University Campbell, David, Johns Hopkins University School of Hygiene & Public Health DucKLOW, Hugh, Columbia University Elston, Ralph, Battelle Marine Research Laboratory Farley, Austin, Oxford Marine Research Laboratory Frazier, John, Johns Hopkins University School of Hygiene & Public Health Leibovitz, Louis, Marine Biological Laboratory Leonard, Leslie, Johns Hopkins University School of Hygiene & Public Health Levin, Jack, University of California, San Francisco MiCHAELSON, Edward, Harvard University School of Public Health Pearce, John, National Marine Fisheries Service Prendergast, Robert, Johns Hopkins Hospital ROSENWASSER, LENNY, Tufts University School of Medicine Sinderman, Carl, National Marine Fisheries Service Sparks, Alfred, University of Washington School of Fisheries Stephens, Raymond, Marine Biological Laboratory Stewart, James, Fisheries Research Branch, Nova Scotia, Canada Strandberg, John, Johns Hopkins University School of Medicine Whittaker, J. Richard, Boston University Marine Program/Marine Biological Laboratory Students Ayvazian, Suzanne, University of Lowell Callahan, Joyce, Stonehill College Campbell, Walton, Stamford, Connecticut Fisher, William, University of California, Davis Fore, Stephanie, St. Andrews Presbyterian College GiUDiCE, GiNA, Immaculata College Jansen, Maura, Virginia Institute of Marine Science/College of William and Mary Kanungo, Kalpataru, Western Connecticut State University Lima, Gail, Tufts University Margosian, Arlene, Trent University, Canada Ruano, Francisco, Instituto Nacional de Investigacao das Pescas, Portugal TiCE, Kimberly, Southhampton College SPRING Biophysics of Neural Function Course Director Alkon, Daniel, NINCDS, NIH/Marine Biological Laboratory EDUCATIONAL PROGRAMS 67 Other faculty, staff, and lecturers Adelman, William, Jr., NINCDS, NIH/Marine Biological Laboratory Atwood, Harold, University of Toronto Barlow, Robert, Jr., Syracuse University Brightman, Milton, NINCDS, NIH Connor, John, Bell Laboratories DeFIelice, Louis, Emory University School of Medicine DOWLING, John, Harvard University Farley, Joseph, Princeton University Gilbert, Charles, Harvard Medical School GoviND, C. K., University of Toronto, Canada Jacklet, Jon, SUNY, Albany Kaplan, Ehud, The Rockefeller University Kravitz, Edward, Harvard Medical School Llinas, Rodolfo, New York University Medical Center Moore, John, University of Massachusetts Pappas, George, University of Illinois Potter, David, Harvard Medical School Rasmussen, Howard, Yale University School of Medicine Raymond, Stephen, Massachusetts Institute of Technology Shepherd, Gorix)N, Yale University School of Medicine Weiss, Thomas, Massachusetts Institute of Technology Students Apfeldorf, William, Yale University School of Medicine Bassi, Carl, Vanderbilt University Earnest, Thomas, Boston University Feinman, Richard, SUNY, Downstate Medical Center Grayson, Carolyn, University of Toronto, Canada Herron, Paul, University of Massachusetts Howard, Heidi, Marlboro College Jacobson, Samuel, Massachusetts Eye and Ear Infirmary/Harvard Medical School Johnson, Karen, The University of Texas Medical Branch Moss, Anthony, Boston University Marine Program/Marine Biological Laboratory Saltzman, Charles, University of North Carolina School of Medicine Smith, Dolores, Tulane University School of Medicine Sullivan, John, Mount Sinai School of Medicine Unnikrishnan, K. p., Syracuse University Vining, Elizabeth, Iowa State University Weiss, David, Baylor College of Medicine/Texas Medical Center short courses Analytical and Quantitative Light Microscopy in Biology, Medicine, and Materials Sciences Course Director Inoue, Shinya, Marine Biological Laboratory Other faculty, staff, and lecturers Amato, Philip, Carnegie Mellon University Benck, Ray, Cohu, Inc. Chiasson, Richard, Olympus Corporation of America Duffy, Jack, Fran M. Valenti, Inc. Ellis, Gordon, University of Pennsylvania 68 MARINE BIOLOGICAL LABORATORY Grogan, Tom, GYYR Hansen, Eric, Dartmouth College School of Engineering Hayes, Thomas, University of North CaroUna HiNSCH, Jan, E. Leitz, Inc. Keller, Ernst, Carl Zeiss, Inc. Kennedy, Wayne, GYYR Kerr, Louis, Marine Biological Laboratory Kleifgan, Gerald, DAGE— MTI Laws, Brian, Crimson Camera Technical Sales, Inc. Lutz, Douglas, Marine Biological Laboratory MiCHAUD, Jayne, Marine Biological Laboratory Olwell, Patricia, E. Leitz, Inc. Presley, Philip, Carl Zeiss, Inc. Pulliam, Harry, Nikon, Inc. RiKUKAWA, Katsuji, Nikon, Inc. Salmon, Edward, University of North Carolina Scott, Eric, Venus Scientific Taylor, D. Lansing, Carnegie Mellon University Taylor, Richard, Colorado Video Thomas, Paul, DAGE— MTI Wang, Robert, Imaging Technology Wick, Robert, Carl Zeiss, Inc. Woodcock, Peter, Cari Zeiss, Inc. Woodward, Bertha, Marine Biological Laboratory Students BoYARSKY, Gregory, Yale University Clarke, Margaret, Albert Einstein College of Medicine FosKETT, J. Kevin, NIH Fuller, Margaret, Indiana University Holden, Cheryl, Research Triangle Institute Im, Michael, Johns Hopkins Hospital Jensen, Peter, NIH Johnson, Carl, Harvard University McConnell, Dennis, University of Florida Pagliaro, Leonard, Wesleyan University Safranyos, Richard, The University of Western Ontario, Canada Saft, Mallory, University of Health Sciences/The Chicago Medical School SCHOENWOLF, GARY, University of Utah School of Medicine SiZTO, Ning-Leung, Yale University School of Medicine SUNDBERG, Marshall, University of Wisconsin Tietge, Joseph, University of Wyoming Basic Immunohistochemical Techniques in Tissue Sections and Whole Mounts Course Directors Beltz, Barbara, Harvard Medical School BuRD, Gail, The Rockefeller University Other faculty, staff, and lecturers Bibee, Mike, Carl Zeiss, Inc. Enneking, Kitty, Hacker Instruments Heintz, John, The Rockefeller University Merikas, Lewis, Hacker Instruments EDUCATIONAL PROGRAMS 69 Presley, Philip, Carl Zeiss, Inc. Tracy, Cheryl, Harvard Medical School Wanless-Dorn, ViCKi, Immuno Nuclear Corporation Students Battista, Arthur, New York University Medical School Claassen, Dale, Kansas State University Davis, Norman, University of Connecticut/The Biological Sciences Group Heimberg, Carolyn, Boystown National Institute Henderson, Judith, SUNY, Buffalo Lysakowski, Anna, University of Illinois Medical Center Newkjrk, Robert, Tennessee State University Prevette, David, Bowman Gray School of Medicine Richards, Ann, Burroughs Wellcome Company Schmied, Robert, Columbia University Sloley, Brian, University of Waterloo Smith, Louis, Baylor College of Medicine Mariculture: Culture of Marine Invertebrates FOR Research Purposes Course Director Berg, Carl, Jr., Marine Biological Laboratory Other faculty, staff, and lecturers Alatalo, Philip, Marine Biological Laboratory Bower, Carol, Institute for Aquarium Studies Capo, Thomas, Marine Biological Laboratory Capuzzo, Judith, Woods Hole Oceanographic Institution Doyle, Roger, Dalhousie University FujiTA, Rodney, Marine Biological Laboratory Garibaldi, Louis, New York Aquarium GuiLLARD, Robert, Bigelow Laboratories Hanlon, Roger, Marine Biomedical Institute Harrigan, June, Marine Biological Laboratory Hughes, John, Massachusetts State Lobster Hatchery Kerr, Louis, Marine Biological Laboratory Leibovitz, Louis, Marine Biological Laboratory Mann, Roger, Woods Hole Oceanographic Institution Marcus, Nancy, Woods Hole Oceanographic Institution Spotte, Stephen, Mystic Marinelife Aquarium SULKJN, Stephen, Horn Point Laboratory Turner, David, Institute for Aquarium Studies Students Al-Yamani, Faiza, University of Miami BORRERO, Francisco, University of South Carolina Castelli, Maurizio, Virginia Institute of Marine Science Checa, Miguel, Aquamundo, Peru CORBITT, Michael, Sea Farms of Connecticut DeFreese, Duane, Florida Institute of Technology Detwyler, Robert, Norwich University Febry, Ricardo, University of Miami Landeau, Laurie, Philadelphia, Pennsylvania Landy, Ronald, New York State College of Veterinary Medicine/Cornell University 70 MARINE BIOLOGICAL LABORATORY Latson, F. Edgar, Central Park Animal Hospital LuBZENS, Esther, Israel Oceanographic and Limnological Research Ltd., Israel MisiTANO, David, National Marine Fisheries Service Mladenov, Philip, Mount Allison University Nadeau, Lloyd, Marine Biological Laboratory RuANO, Francisco, Northeast Fisheries Center Stewart, V. Ann, Magnolia, Massachusetts SziKLAS, Robert, Wauwinet Shellfish Company Wyatt, Jeffrey, The University of Rochester Medical Center Young- Wallace, Nina, Wallace and Company Optical Microscopy and Imaging in the Biomedical Sciences Course Director Allen, Robert, Dartmouth College Other faculty, staff, and lecturers Amato, Philip, Carnegie Mellon University Ashmead, Robert, Nikon Instrument Division, Nikon, Inc. Balcom, Richard, Olympus Corporation of America Bibee, Michael, Carl Zeiss, Inc. Chiasson, Richard, Olympus Corporation of America Clayton, Cary, Instrumentation Marketing Corporation Cowan, Diane, Boston University Marine Program/Marine Biological Laboratory FUJIWAKE, Dr., Hamamatsu Photonics, K.K., Japan Hansen, Eric, Dartmouth College School of Engineering Hayden, John, Dartmouth College Hinsch, Jan, E. Leitz, Inc. Izzard, Colin, SUNY, Albany Kleifgen, Jerome, DAGE — MTl Knutrud, Paul, Interactive Video Systems MoNGiELLO, John, A. O. Reichert Scientific Orndorff, Kenneth, Dartmouth College Presley, Phil, Carl Zeiss, Inc. Saporetti, Tony, Interactive Video Systems Taylor, D. Lansing, Carnegie Mellon University Students Aufderheide, Karl, Texas A&M University Banker, Gary, Albany Medical College of Union University Bevan, Rosemary, University of Vermont School of Medicine Bridgman, Paul, NIH BuCKLAND-NiCKS, JOHN, University of Alberta, Canada Endo, Burton, Agricultural Research Service/Plant Protection Institute Frankel, Richard, Massachusetts Institute of Technology Holdren, Dale, University of Washington Hulbert, William, University of Alberta, Canada Huxley, Virginia, University of California, Davis KusuMi, Akihiro, Princeton University Pawley, James, HVEM Laboratory Rakowski, Robert, Washington University School of Medicine Shepherd, Gordon, Yale University School of Medicine Welsh, Michael, University of Iowa Hospitals educational programs 7 1 Protein Analysis by Polyacrylamide Gel Electrophoresis Course Directors Stephens, Raymond, Boston University School of Medicine/Marine Biological Laboratory ZwEiDLER, Alfred, The Institute for Cancer Research Other faculty, staff, and lecturers Good, Michael, Marine Biological Laboratory Kerr, Louis, Marine Biological Laboratory Masure, H. Robert, Boston University School of Medicine Students Adams, Susan, VA Medical Center, Kansas City, Missouri Brower, Danny, University of California, Irvine Campenot, Robert, Cornell University Chepko, Gloria, Albert Einstein School of Medicine Chou, Ta-Hsu, Michigan Cancer Foundation Donady, J. James, Wesleyan University FUSELER, John, University of Texas Health Science Center, Dallas Ganz, Peter, Brigham and Women's Hospital Hayhome, Barbara, University of Nebraska, Omaha Kazura, James, University Hospitals Koopmans, Henry, Columbia University KuHNS, William, University of North Carolina LiSMAN, John, Brandeis University Liu, H. Mei, The Miriam Hospital McGrath, Ann, VA Hospital, San Francisco, California Pearson, James, The Upjohn Company RiPPS, Harris, New York University School of Medicine Roesijadi, Guri, Battelle Marine Research Laboratory Troncoso, Juan, Johns Hopkins University Walter, Anne, NIH — National Heart, Lung and Blood Institute Small Computers in Biomedical Research Course Director Palmer, Larry, University of Pennsylvania School of Medicine Other faculty, staff, and lecturers Cowan, Diane, Boston University Marine Program/Marine Biological Laboratory Jones, Judson, University of Pennsylvania School of Medicine Peachey, Lee, University of Pennsylvania Students Bruce, Richard, Highlands Biological Station Chen, Lee, University of California Cheng, Toni, Marine Biological Laboratory Herman, Lawrence, New York Medical College • Hester, Kelly, Texas A&M University College of Medicine Jacobson, Samuel, Bascom Palmer Eye Institute Kuhns, William, University of North Carolina School of Medicine KusuMi, Akjhiro, Princeton University Moran, John, The Upjohn Company Sheet, Michael, The Miriam Hospital SuNDELL, Cynthia, University of Pennsylvania 72 MARINE BIOLOGICAL LABORATORY X. RESEARCH AND TRAINING PROGRAMS SUMMER Principal Investigators Adams, James A., Tennessee State University Alberte, Randall S., University of Chicago, Barnes Laboratory Allen, Nina S., Dartmouth College Allen, Robert D., Dartmouth College Anderson, Peter A. V., Whitney Laboratory Armstrong, Clay M., University of Pennsylvania Armstrong, Peter B., University of California Arnold, John M., Kewalo Marine Laboratory, Pacific Biomedical Research Center Bamburg, James R., Colorado State University Barlow, Robert B., Jr., Syracuse University Batten, Bruce E., Tufts Medical School Beauge, Luis, Instituto de Investigacion Medica, Argentina Begenisich, Ted B., University of Rochester Medical Center Bennett, Michael V. L., Albert Einstein College of Medicine BORGESE, Thomas A., Lehman College, City University of New York Boron, Walter F., Yale University School of Medicine Boss, W. P., North Carolina State University BOYER, Barbara C, Union College Brodwick, Malcolm S., University of Texas Medical Branch Brown, Joel E., SUNY, Stony Brook Browne, Carole L., Wake Forest University Browne, Robert A., Wake Forest University BuRDiCK, Carolyn J., Brooklyn College Burger, Max M., University of Basel, Switzerland BuRSZTAJN, Sherry, Baylor College of Medicine Chang, Donald C, Baylor College of Medicine Chappell, Richard L., Hunter College Charlton, Milton P., University of Toronto, Canada Cohen, Lawrence B., Yale University School of Medicine Cohen, William D., Hunter College Cooperstein, Sherwin J., University of Connecticut Health Center De Weer, Paul, Washington University School of Medicine Dunham, Phillip B., Syracuse University Eaton, Douglas C, University of Texas Medical Branch ECKERT, Barry S., SUNY, Buffalo Edds, Kenneth T., SUNY, Buffalo Ehrenstein, Gerald, NIH Farmanfarmaian, a., Rutgers University Festoff, Barry W., VA Medical Center FiSHMAN, Harvey M., University of Texas Medical Branch French, Robert J., University of Maryland Gilbert, Daniel L., NIH Goldman, Robert D., Northwestern University Medical School GoviND, C. K., Scarborough College Grinvald, Amiram, Weizmann Institute of Science Haimo, Leah T., University of California, Riverside Harrington, John P., University of Alaska Haschemeyer, Audrey, E. V., Hunter College Hepler, Peter K., University of Massachusetts Highstein, Stephen, Albert Einstein College of Medicine RESEARCH AND TRAINING PROGRAMS 73 Humphreys, Tom, University of Hawaii Kaminer, Benjamin, Boston University School of Medicine Kao, C. Y., SUNY, Downstate Medical Center INGOGLIA, Nicholas A., UMDNJ — New Jersey Medical School Kuriyama, Ryoko, University of Wisconsin Landowne, David, University of Miami Langford, George M., The School of Medicine, University of North Carolina Lasek, Raymond J., Case Western Reserve University Laufer, Hans, University of Connecticut Levin, Jack, Veterans Administration Hospital, San Francisco LiPiCKY, John, Food & Drug Administration Llinas, Roexdlfo, New York University Medical Center Longo, Frank J., University of Iowa Loewenstein, Werner R., University of Miami School of Medicine Matsumura, Fumio, Michigan State University Metuzals, J., University of Ottawa, Canada Maglott, Donna R., Howard University Mitchell, Ralph, Harvard University Miyamoto, David M., Seton Hall University Morrell, Frank, Rush Medical College Mullins, L. F., University of Maryland Nagel, Ronald L., Albert Einstein College of Medicine Narahashi, Toshio, Northwestern University Medical School Nelson, Leonard, Medical College of Ohio NoE, Bryan D., Emory University Obaid, Ana Lia, University of Pennsylvania School of Dental Medicine Olins, Donald E., University of Tennessee O'Melia, Anne F., George Mason University Oxford, Gerry S., University of North Carolina, Chapel Hill Pappas, George D., University of Illinois, Chicago Pierce, Sidney K., University of Maryland POZNANSKY, Mark J., University of Alberta, Canada Pratt, Melanie M., University of Miami School of Medicine PUMPLIN, David W., University of Maryland, Baltimore QuiGLEY, James P., SUNY, Downstate Medical Center Rakowski, Robert F., Washington University School of Medicine Reynolds, George T., Princeton University Ripps, Harris, New York University School of Medicine Ross, William Noel, New York Medical College Russell, John M., University of Texas Medical Branch Saffo, Mary Beth, Swarthmore College Sahley, Christie L., Yale University Salzberg, Brian M., University of Pennsylvania Sanger, Joseph W., University of Pennsylvania School of Medicine Schneider, E. Gayle, University of Nebraska Medical Center ScHUEL, Herbert, SUNY, Buffalo ScoHELD, Virginia L., Stanford University School of Medicine Segal, Sheldon J., Rockefeller Foundation Selman, Kelly, University of Florida Silver, Robert Benjamin, University of Health Sciences, Chicago Medical School Sheetz, Michael, University of Connecticut Health Center Shemin, David, Northwestern University Speck, William T., Rainbow Babies & Children's Hospital Spiegel, Evelyn, Dartmouth College Spiegel, Melvin, Dartmouth College Stanley, Elis F., Johns Hopkins Medical School 74 MARINE BIOLOGICAL LABORATORY Stuart, Ann E., University of North Carolina Szent-Gyorgyi, Andrew G., Brandeis University Tasakj, Ichiji, National Institute of Mental Health Tashiro, Jay Shiro, Kenyon College Bezanilla, Francisco, University of California, Los Angeles Taylor, Robert E., NIH Treistman, Steven N., Worcester Foundation for Experimental Biology Trinkaus, John Philip, Yale University Troll, Walter, New York University Medical Center Tucker, Edward B., Vassar College Tytell, Michael, Bowman Gray School of Medicine Wallace, Robin A., Whitney Marine Laboratory Weissman, Gerald, New York University Medical Center WoLNiAK, Stephen M., University of Maryland Yeh, Jay Z., Northwestern University Medical School ZiGMAN, Seymour, University of Rochester School of Medicine & Dentistry Library Readers Adelberg, Edward A., Yale Medical School Albright, John T., Harvard School of Dental Medicine Alkon, Daniel, Marine Biological Laboratory Armett-Kibel, Christine, University of Massachusetts, Boston Anderson, Everett, Harvard Medical School Armstrong, Margaret, University of California Bang, Betsy G., Marine Biological Laboratory Barenholz Yechezkel, Hebrew University, Jerusalem, Israel Bean, Charles P., General Electric Research and Development Center Becker, Frederick F., M. D. Anderson Hospital & Tumor Institute Beidler, Lloyd, Florida State University Berg, Paul, Stanford University School of Medicine Bourne, Donald W., Woods Hole Oceanographic Institution BoYER, John F., Union College Broyles, Robert H., University of Oklahoma Health Sciences Center Brown, Frank, Woods Hole, Massachusetts Buck, John, NIH Candelas, Grasiela C, Universidad de Puerto Rico Carlson, Francis, John Hopkins University Carriere, Rita, SUNY, Downstate Medical Center Chatterjee, Deb K., University of Illinois Child, Frank M., Trinity College Clark, Arnold, University of Delaware Cohen, Seymour S., SUNY, Stony Brook Collier, Jack R., Brooklyn College Collier, Marjorie M., Saint Peter's College Couch, Ernest F., Texas Christian University Cowling, Vincent F., SUNY, Albany Crowley, William F., Massachusetts General Hospital Dessner, Daniel A., Kenyon College Dettbarn, Wolf-D., Vanderbilt University Medical Center Duncan, Thomas, Marine Biological Laboratory Dunn, Stephen, Harvard University Ebert, James, Carnegie Institute of Washington Edds, Louise L., Ohio University Eder, Howard A., Albert Einstein College of Medicine Ellner, Jerrold, University Hospitals, Cleveland, Ohio Epel, David, Stanford University RESEARCH AND TRAINING PROGRAMS 75 Feingold, David S., New England Medical Hospital Fisher, Saul, New York University School of Medicine Flaherty, Claire V., Fairleigh Dickenson University Flisser, Anna, Institute de Investigaciones Biomedicas, Mexico Freinkel, Norbet, Northwestern University Medical School Friedler, Gladys, Boston University School of Medicine Frost, John Kingsbury, John Hopkins School of Medicine Gabriel, Mordecai L., Brooklyn College German, James L., The New York Blood Center Goldstein, Lester, University of Kentucky Goldstein, Moise H., John Hopkins University Goode, Dennis, University of Maryland Grant, Philip, University of Oregon Grosch, Daniel S., North Carolina State University Grossman, Albert, New York University Medical Center Guttenplan, Joseph B., New York University College of Dentistry Han, Jon, Kenyon College Harding, Clifford V., Kresge Eye Institute of Wayne State University Haubrich, Robert R., Denison University Hernandez-Nicaise, Mari-Luz, Universite Claude Bemand, France Hubbard, Ruth, Harvard University HuFNAGEL, Linda, University of Rhode Island Ilan, Joseph D., Case Western Reserve University School of Medicine Inoue, Sadayukj, McGill University INOUE, Shinya, Marine Biological Laboratory /University of Pennsylvania Issenberg, Irvin, Oregon State University Johnson, Michael, Harvard Medical School Jones, Megan, Harvard University Kane, Robert E., University of Hawaii Kaltenbach, Jane C, Mount Holyoke College Kawai, Masataka, Columbia University KiMANi, Robinson Gauchuhi, Clinical Research Centre, Nairobi, Kenya Kirschenbaum, Donald M., College of Medicine, SUNY KosowER, Edward M., Tel- Aviv University, Israel Kravitz, Edward A., Harvard Medical School Laderman, Aimlee D., Smithsonian Institution Lazarow, Paul B., The Rockefeller University Lee, John J., City College of New York Leighton, Joseph, The Medical College of Pennsylvania Levine, Rachmiel, City of Hope Medical Center, California Levine, Walter G., Albert Einstein College of Medicine Levitz, Mortimer, New York University Medical Center Lo, Woo-KUEN, Kresge Eye Institute of Wayne State University LoRAND, Laszlo, Northwestern University Maienschein, Jane, Arizona State University Marine Research, Falmouth, Massachusetts Maser, Morton, Woods Hole, Massachusetts Mautner, Henry G., Tufts University School of Medicine Mauzerall, David, The Rockefeller University Meinertzhagen, I. A., Dalhousie University, Nova Scotia Micikas, Lynda, Temple University Miller, Daniel G., PMI-Strang Clinic Mitchell, James B., Moravian College Mizell, Merle, Tulane University Monsanto Company, St. Louis, Missouri Monroy, Alberto, Stazione Zoologica, Napoli, Italy Moore, John W., Duke University 76 MARINE BIOLOGICAL LABORATORY Morse, Stephen, Rutgers University MusiCK, Jim, Ultra Pure Laboratories, Salt Lake City O'Rand, Angela, Duke University OscHMAN, James, Woods Hole, Massachusetts Orme-Johnson, Nanette Roberts, Tufts University Pederson, Thoru, Worcester Foundation for Experimental Biology Peisach, Jack, Albert Einstein College of Medicine Person, Philip, V. A. Hospital, Brooklyn, New York QuATTROCHi, James J., Ohio State University College of Medicine Reiner, John M., Albany Medical Center Rice, Robert, Carnegie-Mellon University Rosenbluth, Raja, Simon Eraser University, Canada Rowland, Lewis P., Neurological Institute, New York RUESS, Lynne, Kenyon College RusHFORTH, Norman B., Case Western Reserve University Russell-Hunter, W. D., Syracuse University Saunders, John W., SUNY, Albany Schwartz, Martin, University of Maryland, Baltimore County Schrater, Fa ye. Smith College Shemin, David, Northwestern University Shephard, Frank, Deep Sea Research Shepro, David, Boston University Sherman, Irwin W., University of California Singer, Maxine, NIH Sluder, Greenfield, Worcester Foundation for Experimental Biology Snyder, Judith, University of Denver SONNENBLICK, B. P., Rutgers University Speck, William, Case Western Reserve University School of Medicine Spector, a., Columbia University Speigel, Mel, Dartmouth College Stephen, Michael J., Rutgers University Stephens, Raymond, Marine Biological Laboratory Stunckard, Horace, American Museum of Natural History SussMAN, Maurice, University of Pittsburgh Tashiro, Jay S., Kenyon College Trager, William, The Rockefeller University TwEEDELL, Kenyon S., University of Notre Dame Wainio, Walter, Rutgers University Wangh, Lawrence, Brandeis University Warren, Leonard, Instar Institute Webb, H. Marguerite, Woods Hole, Massachusetts Webster, Leslie T., Case Western Reserve Medical School Weidner, Earl, Louisiana State University Wheeler, George E., Brooklyn College WiLBER, Charles G., Colorado State University Wittenberg, Beatrice A., Albert Einstein College of Medicine Wittenberg, Jonathan B., Albert Einstein College of Medicine Wolken, Jerome J., Carnegie-Mellon University WoRTHiNGTON, C. R., Camegie-Mellon University Zacks, Sumner I., Miriam Hospital Zeleski, Ilene, Deep Sea Research Zimmerman, Morris, Merck, Sharp & Dohme Research Laboratories Other Research Personnel Alliegro, Mark, SUNY, Buffalo Anderson, Cathleen, Syracuse University RESEARCH AND TRAINING PROGRAMS 77 Augustine, George, University of California, Los Angeles Baker, Robert G., New York University Medical Center Beach, Robert L., University of Virginia Medical Center Betchaku, Teiichi, Yale University School of Medicine Blumer, Jefprey, Case Western Reserve University Bookman, Richard, University of Pennsylvania Boss, Wendy, University of North Carolina Bower, James, New York University Medical Center Brady, Scott, Case Western Reserve University Breitwieser, Gerda E., University of Texas Medical Branch Brown, Douglas, Dartmouth College Caputo, Carlo, National Institutes of Mental Health Garden, M., University of Ottawa, Canada Cariello, Lucio, Stazione Zoologica, Naples, Italy Chung, Margaret P., Tufts University Medical School Clapin, D. F., University of Ottawa, Canada Clark, John M., University of Massachusetts Cohen, Rochelle S., University of lUinois DeFelice, Louis, Emory University Desimone, Douglas, Dartmouth College Dennison, William C, University of Chicago Dessner, Daniel, Kenyon College DiPOLO, RiNALDO, Medica Instituto d'Investigacion, Argentina Dowling, John E., Harvard University Eagles, P. A., University of London, U. K. Ehring, G. R., Northwestern University School of Medicine EiSEN, Andrew, Children's Hospital, Boston Eisenberg, Robert, Rush Medical College Eisner, D., University College, London, U. K. Ellner, Jerrold, University Hospital, Cleveland Fath, Karl, Case Western Reserve University Feinman, Richard D., Downstate Medical Center, Brooklyn Feldman, Susan C, New Jersey Medical School Fennelly, G. J., New York University FONG, Peying, Yale University School of Medicine Frank, Dodie, Case Western Reserve University Gainer, Harold, N.I.H. George, Ted, Case Western Reserve University Gilbert, Susan, Dartmouth College GiUDiTTA, Antonio, International Institute of Genetics and Biophysics, Italy Goldberg, Jay M., University of Chicago Goldman, Anne E., Northwestern University Gould, Robert, New York Institute of Basic Research for Mental Retardation Gregory, William, Albert Einstein College of Medicine Grizzle, Raymond, Rutgers University Hagelstein, Eric B., Northwestern University School of Medicine Haronian, Grace, University of Connecticut Heiple, Jeanne, The Rockefeller University Hill, W. David, Bowman Gray School of Medicine HiRAi, Setsuro, Rockefeller Foundation HoLLOWAY, Stephen, Northwestern University School of Medicine Horn, Lyle W., Temple University Hu, Shi Ling, Downstate Medical Center, Brooklyn Huang, James, Northwestern University School of Medicine Jacobsen, Ronald, Georgia Institute of Technology Jaslove, S., Albert Einstein College of Medicine Joseph-Silverstein, Jacquelyn, Hunter College 78 MARINE BIOLOGICAL LABORATORY Kao, Peter N., Columbia University College of Physicians and Surgeons Kaplan, Ehud, The Rockefeller University Kaupp, Benjamin, KiSHiMOTO, Takeo, National Institute for Basic Biology, Japan Kjelleberg, Staffen, Gothenburg University, Denmark KoiDE, Samuel S., Rockefeller Foundation Krawthamer, Victor, New York Medical College Leuchtag, H. Richard, University of Texas Medical Branch Levis, Richard, Rush Medical College Lewenstein, Lisa, New York Medical College Mackin, Robert, Emory University Marcum, James A., Massachusetts Institute of Technology Margolis, Jonathan, Swarthmore College Marsh, James A., University of Guam Martz, Dean, Case Western Reserve University Matteson, Donald R., University of Pennsylvania MiSEVic, Gradimir, University of Basel, Switzerland Morrell, Leyla deToledo, Rush Medical College Moss, Roberta, SUNY, Buffalo Nadeau, Joseph, The Jackson Laboratories Nakaye, Toshio, National Institutes of Mental Health Olins, Ada L., University of Tennessee Orbach, Harry, Yale University School of Medicine Pant, Harish, National Institutes on Alcohol Abuse and Alcoholism Paul, D., Harvard Medical School Paxhia, Teresa, University of Rochester School of Medicine Pearce, Joanne, Scarborough College Persell, Roger, Mercy College Pochapin, Mark, University of Pennsylvania PopiELA, Heinz, Virginia Medical Center PoussART, Denis, Universite Laval, Quebec, Canada Prior, David J., University of Kentucky QuiNN, R., University of Maryland Renninger, George, University of Guelph, Canada Requena, Jamie, Venezuelan Institute for Scientific Investigation Rich, Abby, New York University Medical Center Ringer, Steven, Rainbow Babies and Childrens Hospital, Cleveland Rose, Birgit, University of Miami School of Medicine Rosenbaum, Faye, Rush Medical College ROZDZIAL, MOSHE, University of California Rubel, Edwin W., University of Virginia Ruben, Carlos, Pacific Biomedical Research Center, Hawaii Rubin, Leona, SUNY RUESS, Lynne, Kenyon College Salzman, C, Albert Einstein College of Medicine Sanger, Jean M., University of Pennsylvania School of Medicine Sarma, Vidya, University of Maryland Sato, Elmei, Dartmouth College ScHUEL, Regina, SUNY, Buffalo Scruggs, Virginia M., Northwestern University School of Medicine Senseman, David M., University of Pennsylvania Slaughter, Sabrina, Tennessee State University Smith, Catherine, University of Rochester Medical School Smith, Stephan, Yale University Socci, Robin, Rutgers University Soderhall, Kenneth, University of Uppsala, Sweden RESEARCH AND TRAINING PROGRAMS 79 Spray, David C, Albert Einstein College of Medicine Steinacker, Antoinette, Albert Einstein College of Medicine Stimers, Joseph, University of California, Los Angeles Stockbridge, Norman, New York Medical College SuGiMORi, M., New York University School of Medicine Szamier, Bruce, Marine Biological Laboratory Szentkiralyi-Szent-Gyorgyi, Eva, Brandeis University Tanguy, Joelle, Laboratoire de Neurobiologie Taylor, LaVentrice, University of North Carolina Taylor, D. L., Carnegie Mellon University TiFFERT, T., University of Maryland Torres, Rafael, University of California at Los Angeles Vale, Ronald, Stanford University Varner, Judith, Klingelbergstrasse Biocenter, Switzerland VOSSHALL, Leslie, Syracuse University Walch, Marianne, Harvard University Walton, Alan J., Open University, England, U. K. Wang, Linfang, Rockefeller Foundation Weiss, J., Northwestern University School of Medicine White, Richard, Albert Einstein College of Medicine White, Michael M., University of California, Los Angeles Whittembury, Jonathan, University Peruana, Cayetano WiENS, T. J., University of Manitoba, Canada Wirtz, K. W. a.. State University of Utrecht, Netherlands Wu, Chau H., Northwestern University School of Medicine Yamamoto, Daisuke, Northwestern University School of Medicine YoKO, Karen, Northwestern University School of Medicine Zakevicius, Jane, New York University School of Medicine Zavilowitz, J., Albert Einstein College of Medicine Zecevic, Dejan, Institute of Biological Research, Belgrade, Yugoslavia YEAR-ROUND PROGRAMS (All of Marine Biological Laboratory unless otherwise indicated) Boston University Marine Program (BUMP) Staff (of Boston University unless otherwise indicated) Allen, Sarah Atema, Jelle Cogswell, Charlotte, University of Connecticut Cowan, Diane Crowther, Robert D'AvANZO, Charlene, Hampshire College GoviND, C. K., University of Toronto, Canada Hahn, Dorothy Hartman, Jean, University of Connecticut Humes, Arthur LoESCHER, Jane Meedel, Thomas Miyamoto, David Nakamura, Shogo Pearce, Joanne, University of Toronto, Canada RiETSMA, Carol, SUNY, New Paltz ScHWALBE, Karen 80 MARINE BIOLOGICAL LABORATORY Tamm, Sidney Tamm, Signhild Taylor, Margery Valiela, Ivan Van Etten, Richard VoiGHT, Rainer Whittaker, J. Richard, Director Graduate Students Banta, Gary Barshaw, Diana Bryant, Donald Buchsbaum, Robert Caraco, Nina Chu, Kevin Cohen, Rosalind Costa, Joseph Coulter, Douglas Ferme, Paola Foreman, Kenneth FujiTA, Rodney Goehringer, Dale Goddard, Kathryn Hall, Valerie Handrich, Linda Hettenbach, Gail Howes, Brian Johnson, Bruce Lavalli, Kari Merill, Carl Moss, Anthony Neidinger, Richard Poole, Alan Tamse, Armando Trott, Thomas Webb, Jacqueline White, David Wilson, John Wood, Susan Undergraduates Alceste, Cesar Chase, James Gadzik, Mary Beth Glick, Stephen Iannazzi, Ruth KoENiG, Patricia Kyriazi, Constant Maybaum, Hillary McPhie, Donna Miller, Cynthia Sierra, Evelyn Taricano, Diane Warren, Lisa Wisgirda, Mary Developmental and Reproductive Biology Laboratory Gross, Paul R., Director O'LouGHLiN, John T. Laboratory of Biophysics Adelman, William, J., Jr., Chief Section on Neural Membranes Staff (of NINCDS-NIH unless otherwise indicated) Adelman, William J., Jr., Chief Clay, John R. Defelice, Louis J., Emory University FOHLMEISTER, JuRGEN F., University of Minnesota Goldman, David E., SUNY, Binghamton Hodge, Alan J., Marine Biological Laboratory Leonard, Dorothy A. Mueller, Ruthanne, Marine Biological Laboratory Rice, Robert V., Carnegie-Mellon University Stanley, Elis, Johns Hopkins University Tyndale, Clyde L., Marine Biological Laboratory RESEARCH AND TRAINING PROGRAMS 81 VOLKMAN, Mary, Marine Biological Laboratory Waltz, Richard B., Marine Biological Laboratory Section on Neural Systems Staff Alkon, Daniel L., Chief Acosta-Urquidi, Juan Coulter, Douglas, Boston University Farley, Joseph, Princeton University FoRMAN, Robin Gart, Serge, University of Vermont GOH, Yasumasa Harrigan, June, Marine Biological Laboratory Hay, Bruce, University of California Jacklet, John, SUNY, Albany KuziRiAN, Alan M. Kuzirian. Jeanne Lederhendler, Izja, Marine Biological Laboratory Leighton, Stephen, NIH Neary, Joseph T., Marine Biological Laboratory Richards, William, Princeton University Saicaibara, Manabu Shoukimas, Jonathan J. Stulman, James Tengelsen, Leslie WOOLF, Thomas, University of Chicago Laboratory of Carl J. Berg, Jr. Staff Adams, Nancy L. Alatalo, Philip Berg, Carl J., Jr. MiTTON, Jeftrey B., University of Colorado Orr, Katherine S. Laboratory of Carol L. Reinisch Staff Leavitt, Dale, Tufts University School of Veterinary Medicine Reinisch, Carol L., Tufts University School of Veterinary Medicine Sakamoto, Hidemi, Tufts University School of Veterinary Medicine Visiting Investigators Charles, Ann, Tufts University School of Veterinary Medicine Farley, Austin, National Marine Fisheries Service Laboratory of D. Eugene Copeland Staff Block, Barbara, Duke University Copeland, D. Eugene 82 marine biological laboratory Laboratory of Eric Kandel Staff BiDWELL, Joseph, Howard Hughes Medical Institute Capo, Thomas, Howard Hughes Medical Institute Gagosian, Susan, Howard Hughes Medical Institute Good, Michael, Howard Hughes Medical Institute Kandel, Eric, Columbia University Nadeau, Lloyd, Howard Hughes Medical Institute Paige, John A., Howard Hughes Medical Institute Schwartz, James H., Columbia University Laboratory of Felix Strumwasser Staff Lovely, Karen ViELE, Daniel P. McIntyre, Joseph Strumwasser, Felix, California Institute of Technology Laboratory of J. Richard Whittaker Staff Crowther, Robert Loescher, Jane L. Meedel, Thomas H. Whittaker, J. Richard, Boston University/Marine Biological Laboratory Visiting Investigators Miyamoto, David, Seton Hall University Laboratory of Judith P. Grassle Staff Gelfman, Cecilia Grassle, Judith P. Mills, Susan Staff Laboratory of Marine Animal Health Abt, Donald, University of Pennsylvania Leibovitz, Louis, Cornell University, Director Moniz, Polly C. OsoFSKY, Norman RiCKARD, Charles, Cornell University ScHOTT, Edward F. Tamse, Catherine T. Laboratory of Noel De Terra Staff De Terra, Noel Moss, Ann research and training prcx3rams 83 Laboratory of Osamu Shimomura Staff Nemeth, Edward Shimomura, Akemi Shimomura, Osamu, Boston University School of Medicine Visiting Investigators Anctil, Michel, University of Montreal, Canada La, Sung, William Paterson College of New Jersey Laboratory of Raymond E. Stephens Staff Shoukimas, Jonathan J. Stephens, Raymond E., Marine Biological Laboratory/Boston University School of Medicine Stommel, Elijah, Marine Biological Laboratory/Boston University School of Medicine Laboratory of Sensory Physiology Staff Collins, Barbara Ann Cook, Patricia B. Cornwall, Carter, Boston University School of Medicine Corson, D. Wesley Fein, Alan Harosi, Ferenc L Hashimoto, Yoko, Tokyo Women's Medical College, Japan Levine, Joseph S. LiPETZ, Leo, Ohio State University MacNichol, Edward F., Jr., Director Mansreld, Richard, Boston University School of Medicine Payne, Richard SZUTS, Ete Zoltan Laboratory of Shinya Inoue Staff Akins, Robert E., University of Pennsylvania Brown, Carolyn R., University of Pennsylvania Inoue, Shinya, Marine Biological Laboratory/University of Pennsylvania Inoue, Theodore D., Cornell University LuTZ, Douglas A., University of Pennsylvania Woodward, Bertha M. Visiting Investigators Ellis, Gordon W., University of Pennsylvania Horn, Edward, University of Pennsylvania Okazaki, Kayo, Tokyo, Japan Otter, Timothy, Albert Einstein School of Medicine Schweitzer, Peter, University of Pennsylvania TiLNEY, Lewis G., University of Pennsylvania Woodruff, Richard L, West Chester State College 84 MARINE BIOLOGICAL LABORATORY' Sra#(ofNIH) Andre\\s. Brian Cheng. Tony KaCHAR. BECHAR-A Kjnnane. Janet MiCHAUD, Ja^'NE Nemeth. Edward Laboratory of Thomas S. Reese O'CoNNELL. Maureen Philbin. Linda M. Reese. Barbara Reese. Thomas S. ScHNAPP. Bruce Walrond, John National Foundation for Cancer Research Staff Gasco^'ne, Peter R. C. McLaughlin. Jane a. Mean^ . Richard A. Pethig. Ronald. University College of North Wales. U. K. Szent-Gyorg^i. Albert. Director National \'ibr.-\ting Probe Facility' Staff Jaffe. Lionel. Purdue University. Director SCHEFFE^'. Carl E. Shipley. Alan M. I 'isiting Investigators Goodall. Harr^'. University of Cambridge. U. K. Robinson. Kenneth. University of Connecticut Saltzman, Charles. Universitv of Nonh Carolina The Ecosystems Center Staff Bergquist. Berit Boone. Richard BoRETOs. Diane Cole. Jonathan Corliss. Teresa D'Aquilla, Andrea Dauk-as. Paula DuNGAN. Jennifer Garritt. Robert GiBLiN. .An-ne E. Gordon. E>oria Helfrich. John V. K. Hobbie. John E. Houghton, Richard A. Howarth. Robert W. Juers. David KlJOWSKJ, VOYTEK Larssen. Cheryl Macaluso. Marianne Mann, Alicja Marintjcci. Andrew C. Marino. Roxanne Martyna. Jonathan Matherlv. Walter J. Melillo. Jerry M. Merkel. Susan Montgomery . Ell^n T. Montgomery Mar'*' Louise Moss. .Ann Nadelhoffer. Kntjte Palm. Cheryl A. Peterson. Bruce J. R-ask. Susan Sechokla, Elizabeth Shaver. Gaius R. Steudler. Paul a. Stone. Thomas A. Turner. .Andrea Woodwell. George M.. Director RESEARCH AND TRAINING PROGRAMS 85 Trainees Cavanaugh, Colleen, Harvard University Sampou, Peter, University of Rhode Island XI. HONORS Friday Evening Lectures PiTTENDRiGH, COLiN, Hopkins Marine Station, Stanford University, 24 June, "The Structure and Evolution of Circadian Programs" Kravitz, Edward A., Harvard University School of Medicine, 1 July, Lang Lecture, "The Well-Modulated Lobster: Neurohormones and Aspects of Lobster Behavior" Gurdon, John B., M. R. C. Laboratory of Molecular Biology, 8 July, "Clones of Frogs and Some Principles of Development" Land, Edwin H., Rowland Institute for Science, 15 July, "Recent Advances in Retinex Theory and Some Implications for Cortical Computations" PURVES, Dale, Washington University School of Medicine, 21, 22 July, Forbes Lectures, I. "Formation and Maintenance of Synaptic Connections Between Neurons: Quantitative Aspects:" II. "Formation and Maintenance of Synaptic Connections Between Neurons: Qualitative Aspects" Berg, Paul, Stanford University School of Medicine, 29 July, "The Prospects of Gene Re- placement Therapy in Human Disease" Kaminer, Benjamin, Boston University School of Medicine, 5 August, "Albert Szent-Gyorgyi: Search and Discovery" Jeffery, William R., University of Texas, 19 August, E. E. Just Lecture, "Control of Egg Polarity: New Dimensions to an Old Problem" Jannasch, Holger, Woods Hole Oceanographic Institution, 26 August, "Plant Life in the Deep Sea" Associates' Lecture Wilson, E. O., Harvard University, 12 August, "The Social Life of Ants" Special Lectures Smith, Federick E., Harvard University. 13 July, Charles A. Lindbergh Lecture in Ecology, "Niche Theory, Genetic Systems, and the Survival of Species" Bethe, Hans A., The Floyd R. Newman Laboratory of Nuclear Science, Cornell University, 31 August, "The Arms Race" Steps Toward Independence Fellows Bursztajn, Sherry, Baylor College of Medicine Gadsby, David C, The Rockefeller University Kuriyama, Ryoko, University of Wisconsin Obaid, Ana Lia, University of Pennsylvania School of Dental Medicine Pratt, Melanie M., University of Miami School of Medicine Sahley, Christie L., Yale University Schneider, E. Gayle, University of Nebraska Medical Center Silver, Robert, University of Chicago Medical School Wolniak, Stephen, University of Maryland Biology Club of New York Ackerman, Josef D., SUNY, Stony Brook Berggren, Ruth, Oberlin College g6 MARINE BIOLOGICAL LABORATORY COBLER, Sue, Northeastern University Prochazka, Karan, Cambridge, Massachusetts Gary N. Calkins Memorial Scholarship COBLER, Sue, Northeastern University Frances S. Claff Memorial Scholarship Feddeler, William, Wayne State University Edwin Grant Conklin Memorial Scholarship Lyons, Gary LucRETiA Crocker Scholarship Cobler, Sue, Northeastern University Kessing, Bailey, New College Tamse, Armando, Boston University Founders Scholarships These Scholarships were given in memory of: W. E. Garrey S. O. Mast L. V. Heilbrunn E. Witschi Recipients: BODMER, Rolf, Friedrich Miesler Institute, Switzerland Von Brunn, Albrecht, University of Texas at Austin Cobler, Sue, Northeastern University DeSantis, Amedeo, Stazione Zoologica, Naples, Italy Gaide, Michael, Free University of BerUn, West Germany Kessing, Bailey, New College Lasta, Mario, Institute of Marine Biology, Argentina Liebman, Matthew, SUNY, Stony Brook Pascual, Marcela, Institute of Marine Biology, Argentina Perez-Grau, Lluis, European Molecular Biology Laboratory, West Germany Prochazka, Karan, Cambridge, Massachusetts Tamse, Armando, Boston University Irene P. Goldring Scholarships DeSantis, Amedeo, Stazione Zoologica, Naples, Italy Peng, Yan-yi, Northwestern University Aline D. Gross Scholarship Worden, Mary, University of Chicago Merkel H. Jacobs Scholarship Cobler, Sue, Northeastern University HONORS 87 Ernest Everett Just Fellowships in Biology JosiAH Macy, Jr. Foundation Chavez, Larry, University of New Mexico Ruano-Arroyo, Gualberto, Yale University Taylor, LaVentrice D., University of North Carolina Arthur Klorfein Fund Baptista, Carlos, University of Lisbon, Portugal Conlon, Ronald, McGill University Gaide, Michael, Free University of Berlin, West Germany Lesk, Mark, Weizmann Institute, Israel Lyons, Gary, University of Pennsylvania School of Medicine Noble, Michael, University of London, U. K. Stephen W. Kuffler Fellowships Gadsby, David, The Rockefeller University Sahley, Christie L., Yale University F. R. LiLLiE Fellowship Gurdon, J. B., M. R. C. Laboratory of Molecular Biology, Cambridge, England Allen, R. Memhard Scholarships Kessing, Bailey, New College Lyons, Gary, University of Pennsylvania School of Medicine James S. Mountain Memorial Fund, Inc. Scholarship BORNSLAGER, Elayne, University of Pennsylvania Green, Philip, University of North Carolina Ward, Eric, Washington University Wright, Connie, George Washington University Medical Center Herbert W. Rand Fellowship Soderhall, Kenneth, Institute of Physiological Botany, University of Uppsala Society of General Physiologists Bodmer, Rolf, Friedrich Miescher Institute, Switzerland Diener, Dennis, University of Kansas Noble, Michael, University of London, U. K. XIII. INSTITUTIONS REPRESENTED U.S.A. Alaska, University of Bascom Palmer Eye Institute Albert Einstein College of Medicine Battelle Marine Research Laboratory American Museum of Natural History Baylor College of Medicine M. D. Anderson Hospital and Tumor Institute Bell Laboratories Arizona State University Bigelow Laboratories 88 MARINE BIOLOGICAL LABORATORY Boston University Boston University School of Medicine Bowman Gray School of Medicine Boystown National Institute Brandeis University Brigham and Women's Hospital Bucknell University Burroughs Wellcome Company California Institute of Technology California, University of, Davis California, University of, Irvine California, University of, Los Angeles California, University of, San Francisco California, Veterans Administration Hospital, San Francisco Carnegie Institute of Washington Carnegie-Mellon University Case Western Reserve University Case Western Reserve University School of Medicine Central Park Animal Hospital Chicago, University of Children's Hospital City of Hope Medical Center Cohu, Inc. Colorado, University of Colorado State University Colorado Video Columbia University Columbia University College of Physicians and Surgeons Connecticut, Sea Farms of Connecticut, University of Connecticut, University of. Health Center Cornell University Crimson Camera Technical Sales, Inc. DAGE-MTI Dartmouth College Dartmouth College School of Engineering Deep Sea Research Delaware, University of Delaware, University of. College of Marine Studies Denison University Denver, University of Duke University Emory University Emory University School of Medicine Fairleigh Dickenson University Dana Farber Cancer Research Center Florida Institute of Technology Florida State University Florida, University of General Electric Research and Development Center George Mason University Georgia Institute of Technology Hampshire College Hacker Instruments Harvard Medical School Harvard School of Dental Medicine Harvard University Harvard University School of Public Health Hawaii, University of Highlands Biological Station Hopkins Marine Station, Stanford University Horn Point Laboratory Howard University Howard Hughes Medical Institute Hunter College Illinois, University of Illinois, University of. Medical Center Imaging Technology Immaculata College Immuno Nuclear Corporation Indiana University Instar Institute Institute for Aquarium Studies Institute for Cancer Research, The Instrumentation Marketing Corporation Interactive Video Systems Iowa, University of Iowa, University of. Hospitals Jackson Laboratories, The Johns Hopkins Hospital Johns Hopkins Medical School Johns Hopkins University Johns Hopkins University School of Medicine Johns Hopkins University School of Hygiene and Public Health Kansas State University Kentucky, University of Kenyon College Kewalo Marine Laboratory Kresge Eye Institute Leitz, Inc. Lowell, University of Louisiana State University INSTITUTIONS REPRESENTED 89 Marine Biomedical Institute Marine Research, Inc. 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School of Fisheries Washington University School of Medicine Wauwinet Shellfish Company Wayne State University Wesleyan University West Chester State College Western Connecticut State University Whitney Marine Laboratory William and Mary, College of William Paterson College Wisconsin, University of Wisconsin, University of, Madison Woods Hole Oceanographic Institution Worcester Foundation for Experimental Biology Wyoming, University of Ultra Pure Laboratories Union College Union University University Hospitals Yale University Yale University Medical School Carl Zeiss, Inc. FOREIGN INSTITUTIONS Alberta, University of, Canada Basel, University of, Switzerland Biozentrum der Universitat, Switzerland Brasilia, University of, Brazil Calgary, University of, Canada Cambridge, University of, England, U. K. Clinical Research Centre, Kenya Dalhousie University, Canada Deutsche Forschungsgemeinschaft, West Germany Essex, University of, England, U. K. European Molecular Biology Laboratory, West Germany Fisheries Research Branch, Canada Simon Eraser University, Canada Free University of Berlin, West Germany Fundacao Oswaldo Cruz, Brazil Gothenberg University, Sweden Guam, University of, Guam Guelph, University of, Canada INSTITUTIONS REPRESENTED 91 Hamamatsu Photonics, Japan Hebrew University, Israel Institut fur Genetik und Mikrobiologie, West Germany Institut fur Neurbiologie, West Germany Institute of Biological Research. Yugoslavia Institute of Marine Biology, Argentina Institute de Biologia Marina y Pesquera "Alte. Storni," Argentina Institute de Inmunologia y Biologia Micro- biana, Spain Institute de Investigacion Medica, Argentina Institute de Investigaciones Biemedicas, Universidad Nacienal Autenema de Mexico, Mexico Institute de Medicina Tropical "Alexander von Humboldt," Peru Institute Nacienal de Investigacao das Pescas, Portugal Israel Oceanegraphic and Limnological Re- search Ltd., Israel International Institute of Genetics and Bio- physics, Italy International Laboratory for Research on An- imal Diseases, Kenya KJingelbergstrasse Biocenter, Switzerland Life Science Institute of Hebrew University, Israel Lisbon, University of, Portugal Liverpool School of Tropical Medicine, England, U. K. London, University of, England, U. K. Mahidol University, Thailand Manitoba, University of, Canada Max-Planck Institute, West Germany McGill University, Canada Medical Research Council, England, U. K. M. R. C. Laboratory of Molecular Biology, England, U. K. Friedrich Miescher Institut, Switzerland Montreal, University of, Canada Nagoya University, Japan Naples Marine Station, Italy National Institute for Basic Biology, Japan National Polytechnical Institute, Center for Research and Advanced Studies, Mexico Open University, England, U. K. Ottawa, University of, Canada Oxford, University of, England, U. K. Stazione de Zoologica, Naples, Italy Tel-Aviv University, Israel Tokyo Women's Medical College, Japan Toronto, University of, Canada Toronto Western Hospital, Canada Trent University, Canada Tubingen, University of. West Germany Universite Claude Bernard, France Universite Laval, Canada University College, England, U. K. University College of North Wales, U. K. University Kyoto, Japan Uppsala, University of, Sweden Utrecht, State University of, Netherlands Venezuelan Institute for Scientific Investiga- tion, Venezuela Waterloo, University of, Canada Weizmann Institute of Science, Israel Western Ontario, University of, Canada XIII. LABORATORY SUPPORT STAFF Including Persons Who Joined or Left the Staff Duiing 1983 Biological Bulletin Metz, Charles B., Editor Clapp, Pamela L. MouNTFORD, Rebecca J. Buildings and Grounds Gunning, A. Robert, Superintendent Lehy, Donald B., Superintendent Anderson, Lewis B. Averett, Donald L. Baldic, David Baldic, Irene Berrios, Hector Berrios, Jose R. Bourgoin, Lee Broderick, Madeline Carini, Robert J. Collins, Paul J. Costa, Robert A. DuTRA, Steven J. Enos, Glenn R. Evans, Frances G. FuGLiSTER, Charles K. Geggatt, Richard E. Gonsalves, Walter W., Jr. Illgen, Robert F. KuiL, Elisabeth Lewis, Ralph H. 92 MARINE BIOLOGICAL LABORATORY LocHHEAD, William M. LovERiNG, Richard A. LuNN, Alan G. MacLeod, John B. Mills, Stephen A. Pells, Stanley RoMiZA, Carl St. Jean, Simone Smart, Merilyn A. Thrasher, Frederick Varao, John deVeer, Robert L. Ward, Frederick Weeks, Gordon W. Whittaker, William Controller's Office Speer, John W., Controller BiNDA, Ellen F. Campbell, Ruth B. Davis, Doris C. 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Diane Child, Malcolm Early, Julie Enos, Edward G., Jr. Enos, Joyce EusEBio, Shawn Nadeau, Lloyd Fisher, Harry T., Jr. Moniz, Priscilla Murphy, Charles F. Smith, A. Dickson Trapasso, Bruno Varao, John Public Information Office Shreeve, James M., Public Information Officer Ashmore, Jill M. MacInnes, Arch Research Services O'Neil, Barry T., Department Head Barnes, Franklin D. Barnes, John S. Evans, William Colder, Linda M. Colder, Robert J. Martin, Lowell V. Nichols, Francis H., Jr. Sadowski, Edward SiLVA, Mark S. Sylvia, Frank E. Copeland, D. Eugene, Special Consultant to Electron Microscope Laboratory Kerr, Louis M. 1983 Summer Support Staff Anderson, Janice A. Ashmore, Michael W. BiNDA, John H. Black, Robert W. Bowin, Bret R. Brunelli, Carol Anne Brunette, Lisa Burnett, Lynn Callagy, Annemarie Croney, Michaela a. Daniels, Judith A. Donohoe, William P. DowLiNG, Christopher T. Dressel, Douglas M. Engles, Christopher R. FiSCHBACH, ELISSA D. Coodman, Elizabeth Hahn, Erika Hanson, Anthony Irish, Bradford G. Laufer, Leonard Lauther, Gary B. Lee, James M. Lunn, Jeffery R. Mandych, Alexander K. Martyna, Jonathan W. Maxwell, Brett A. Mellon, Armour Oppenheim, Bert RooNEY, Mark C. Rooney, Michele N. Sandler, Bennett ScuTT, Diane H. SwopE, Stephen P. Tarbell, Leslie J. Valois, Francis X. Van Kooy, Dana Wetzel, Ernest Whittaker, William A. Wyttenbach, Ann G. Wyttenbach, Robert A. Zacks, Susan Reference: Biol Bull. 167: 94-1 19. (August, 1984) DEVELOPMENT OF ASYMMETRY IN THE NEUROMUSCULAR SYSTEM OF LOBSTER CLAWS C. K. GOVIND Department of Zoology, Scarborough Campus. University of Toronto, West Hill. Ontario. Canada. MIC 1A4 Abstract The paired claws of the lobster Homarus americanus which are symmetrical in form and function in the larval and early juvenile stages gradually transform into a slender, fast-acting cutter claw and a stout, slow-acting crusher claw during later juvenile and adult stages. Correspondingly changes occur in the neuromuscular system of the claws. The paired claw-closer muscles are initially symmetrical in their fiber composition and consist of a band of fast fibers sandwiched on either side by slow fibers. During development one of the muscles transforms into a cutter with a majority of fast fibers and a small ventral band of slow fibers and the other muscle transforms into a crusher with only slow fibers. The firing pattern of the juvenile fast closer excitor motoneuron consisting of high frequency, long duration bursts, is essentially retained in the adult crusher but changed in the adult cutter to low frequency, short duration bursts. In the paired juvenile closer muscles almost all fibers receive mixed innervation from both fast and slow axons whereas in the adult cutter muscle in- nervation by the fast axon predominates while in the crusher both are equitably distributed. The development of asymmetry in the closer muscle is regulated by impulse-mediated muscle tension though how the neural asymmetry arises is unknown, but amenable to experimentation. Introduction The body plan of many higher animals from annelids to vertebrates is characterized by symmetry of the left and right sides. Within this bilaterally symmetrical organization, however, asymmetries arise manifested most dramatically by cerebral lateralization in humans (Corballis and Morgan, 1978), vocalization in songbirds (Nottebohm, 1977), and cheliped laterality in crustaceans (Przibram, 1901). Despite the tremendous interest throughout the ages in human laterality we still do not understand how it or any of the other biological asymmetries in the animal world is acquired. One of the more compelling hypotheses put forward by Corballis and Morgan (1978) attributes cerebral lateralization to a left-right maturational gradient. According to this scheme both sides are equipotent initially, but mature at different rates subsequently, with the left leading and at the same time suppressing the right; thereby resulting in the left cerebral hemisphere being dominant for speech and verbal processes while the right deals with non-verbal input. This also explains why when the left side is damaged or lesioned the right side is more disposed to take over its function than vice-versa. Among certain songbirds sectioning of the left hypoglossal nerve, but not the right, severely disrupts the singing pattern and demonstrates the lateralization of singing which includes not only the efferent pathway but the associated nuclei in the brain (Nottebohm, 1977). However, the right side can take over control of singing if the Received 31 May 1984; accepted 5 June 1984. 94 DEVELOPMENT OF ASYMMETRY 95 left hypoglossus is sectioned before the onset of spring song suggesting that both sides have the capacity for singing but its expression is normally limited to the left side. These examples of asymmetry point to a bias during development which may be profitably studied among crustaceans such as the lobster Homarus americanus. The paired chelipeds or claws of the adult lobster are asymmetric in form and function consisting of a greatly enlarged, slow acting, and powerful crusher claw either on the left or right side, and a more slender, fast-acting cutter claw on the opposite side (Herrick, 1895). Yet in the larval and early juvenile stages the paired claws are symmetrical and equipotent. The neuromuscular system within these claws has received considerable attention because of their relative simplicity: there are only two muscles each innervated by few motoneurons (Wiersma, 1955). It is the development of asymmetry in the neuromuscular system of the paired claws that is reviewed here as part of an ongoing study to understand the biological basis of asymmetry. Development of Asymmetric Claws The natural history of the east coast lobster Homarus americanus is given in narrative detail in the two voluminous works of Herrick (1895, 1911). The adult female bears eggs every second year. Following a molt usually in July she copulates with a male and subsequently extrudes fertilized eggs. These eggs are carried attached to the swimmerets until the following spring when they hatch as myesid larvae. All three larval stages are planktonic, swimming by means of fan-shaped expodites on their thoracic appendages (Neal et al, 1976). In all three larval stages the paired claws are symmetrical in form (Fig. 1 ) and slightly larger than the walking legs. They have a few conspicuous sensory bristles but do not have any teeth on their biting surfaces which is characteristic of the adults. At the molt to the 4th stage, which is the first juvenile stage, the animal transforms to a diminutive lobster in that it loses the expodites, and now swims by means of its swimmerets located on the abdomen. At LARVA EARLY JUVENILE LATE JUVENILE ADULT 1.5mm 3mm 10mm 120mm Figure 1. Development of the paired claws of the lobster beginning from a symmetrical condition in the larval (1st) and an early juvenile (4th) stage to an increasingly asymmetrical condition in a late juvenile (12th) and an adult stage. Magnification: larva 13X; early juvenile 7X; late juvenile 2X; adult 0.2X. 96 C. K. GOVIND the same time the claws elongate disproportionately compared to the walking legs and are held extended in front of the animal. The biting surfaces in particular are covered with sensory hair and show the first signs of dentition, usually a single central tooth on the poUex. The paired claws in the 4th, 5th, and 6th stage are symmetrical in form but begin to differentiate in the succeeding stages with the putative cutter claw remaining long and slender and the putative crusher becoming short and stout. The other characteristic change concerns the central tooth on the pollex which remains sharp and narrow (incisor-like) in the cutter while becoming rounded and broad (molar-like) in the crusher. The differentiation in external morphology continues until in the adult the paired claws consist of a distinct cutter and crusher claw. Indeed the claws appear to continue elaborating their distinct external form as the asymmetry becomes even more striking in large adults. It is known that the claws grow in a positive allometric fashion compared to the rest of the body (Lang et ai, 1977c) throughout the life of the lobster. Less is known about the development of functional differentiation between the paired claws. Casual observation in the larval stage show the claws to be used in grasping food. This is supplemented in the early juvenile stages by "meral display" in which the paired claws are held extended and open in a threatening or defensive posture. In these early stages the claw closes at a variety of speeds ranging from approximately 50 to 400 ms (Hill and Govind, 1984). Both claws display this range of closing speeds. It is only in late juveniles and early adults that a clear distinction in closing speeds occurs between the asymmetric claws (Govind and Lang, 1974, 1 979). Now the cutter claw displays both fast and slow closing speeds while the crusher closes only slowly. In isolated claws stimulation of the fast closer excitor (FCE) axon with 2 impulses 6.5 ms apart closes the cutter claw in 20 ms while in the crusher claw the homologous axon required 8 impulses, 5 ms apart to cause closing in 90 ms. The closing behavior fatigues more readily and at a lower frequency of stimulation of the FCE in the cutter than in the crusher claw. An essentially similar differentiation in closing behavior was seen between the crusher and cutter claws with stimulation of the slow excitor (SCE) axon. Thus tonic contractions were observed at a lower stimulus frequency and they fatigued more rapidly in the cutter than in the crusher claw. Overall closing of the crusher claw is much slower and more powerful than in its cutter counterpart v^dth stimulation of the homologous motoneurons. The difference in closing behaviors between the paired asymmetric claws is seen in all sizes of adult lobsters including some very large animals, thus suggesting that the functional dif- ferentiation is maintained throughout the life span of the lobster. How this functional dissimilarity develops will be traced by examining the muscular and neural substrates governing claw behavior. Development of Muscle Asymmetry The lobster claw represents a relatively simple motor system having only two antagonistic muscles (Fig. 2). Both muscles are bipinnate in form and run the length of the propus. The opener muscle is relatively small occupying 10% of the claw muscle mass while the massive closer makes up the other 90%. The opener muscle is situated distally and its contraction opens the dactyl: the closer muscle closes the dactyl on the pollex. Most of the work on the development of asymmetry has been done on the closer muscle because it is responsible for the striking differences in closing behavior of the cutter and crusher claws. On the other hand such differences are not obvious in the opening behavior and consequently the opener muscle has received scant attention. DEVELOPMENT OF ASYMMETRY 97 Dactylopodite Opener muscle Propodite Figure 2. Cut-away diagram of an adult cutter claw showing the location and relative size of the antagonistic opener and closer muscles (from Govind and Lang, 1974). Closer muscle The development of the closer muscle in the paired claws has been extensively investigated especially v/ith regard to its fiber composition using contractile, structural, histochemical, and biochemical characteristics. The overall picture obtained from all these studies is the symmetry in fiber composition of the paired muscles in the larval and early juvenile stages with the gradual differentiation into a cutter muscle with predominantly fast fibers and some slow fibers and a crusher muscle with all slow fibers. Structural properties. Unlike vertebrate muscle in which the different fiber types of fast-twitch and slow-twitch have a uniform sarcomere length (SL) of 2-4 ^m, crustacean muscle has a wide range of SL from 2-20 nm (Govind and Atwood, 1982). Early studies by Atwood and his collaborators (reviewed by Atwood, 1967, 1973) established that short SL (2-4 ^m) fibers are fast-contracting while long SL (>6 ^m) fibers are slow-contracting. Using this scheme the fiber composition of the paired closer muscle was determined during development (Jahromi and Atwood, 1971; Gou- dey and Lang, 1974; Lang et ai, 1977a, b, c; Govind and Lang, 1978; Costello and Lang, 1979) and is summarized in Table I and Figure 3. The grouping of sarcomeres into the three categories of short <4 /xm, intermediate 4-6 /nm, and long >6 fim, in Table I was based on the prevailing dogma that these represented respectively fast, 98 C. K. GOVIND Table I Fiber composition based on sarcomere length of the paired claw closer muscles during development of the lobster % of fiber types based on sarcomere length (nm)* Claw 1 (cutter) Claw II (crusher) Length of Inter- Inter- No. of animal Fast mediate Slow Fast mediate Slow Stage animals (mm) <4 4-6 >6 <4 4-6 >6 Larval 1 3 7.5 39 58 3 29 68 3 2 3 8.5 43 35 2 40 56 4 3 5 10 54 45 21 25 54 21 Early Juvenile 4 7 12 36 5 59 26 3 71 5 5 14 50 1 49 27 1 72 Late Juvenile 6 4 16 56 44 21 1 78 11 2 32 73 1 26 23 1 76 13 1 39 64 36 100 15 1 55 82 18 4 96 Adult ? 1 250 63 37 4 96 * The number of fibers sampled from each closer muscle was 30 for the larval stages, 60 for the juvenile 4th stage, and 90 for the remainder (from Lang et ai, 1977a, b; and Govind and Lang, 1978). intermediate, and slow fiber types (Atwood, 1967, 1973). According to the scheme the first two larval stages have predominantly short and intermediate SL fibers. There is a substantial increase in the number of long SL fibers at the 3rd (larval) stage, and again at the 4th and 5th (early juvenile) stages. These increases occur at the expense of the intermediate SL fibers so that by the 5th stage there are few fibers of intermediate SL. These data show a lengthening of the SL from intermediate to long during development of the larval and early juvenile stages. Such lengthening of the sarcomeres appears to be a normal process of crustacean muscle development (Bittner, 1968; Govind et al, 1974; Bittner and Traut, 1978). Over and above this growth-related process, the closer muscle shows two distinct populations of relatively short and long SL fibers in the larval and early juvenile stages (Table I). This dichotomy is graphically represented in the histograms of SL (Fig. 3) where for the larval stage, the fibers separate into the categories of <4 nva and >6 ^m. Development of the closer muscle up to the early (4th and 5th) juvenile stages shows a distinct separation of short and long SL fibers. This distribution is seen in both of the paired muscles. The asymmetry between the paired muscles occurs in the succeeding juvenile stages. Beginning with the 6th stage the population of short SL (<4 ytm) increases in one of the paired claws while the population of long SL (>6 nm) fibers increases in the other claw. As a result of these changes in SL, the cutter muscle ends up with predominantly (60-80%) short SL fibers and the remainder long SL fibers while the crusher muscle ends up with all long SL fibers. The asymmetry of the paired closer muscles characteristic of the adult is usually established by the 1 3th stage which represents the first year of juvenile development. It takes between 5-7 years for lobsters to mature into adults (Hughes et ai, 1972). DEVELOPMENT OF ASYMMETRY 99 30 -| 25- 20- 15- 10- 5- CLOSER I (CUTTER) 1st Stage 1 — \ — I — I — I — 1 CLOSER n (CRUSHER) m 1 — I — I — I — I u z LU a: a: D U U o u a: 35-] 30- 25 20- 15- 10 5 50 -| 45- 40- 35- 30- 25- 20- 15- 10- 5- 50 45- 40- 35- 30- 25- 20- 15 10 5-1 4th Stage JI T — r ■^^1 — \ — nil — \ — 1 — I 6th Stage q- -r — r J 1 — I 13th Stage -P- ffK ^^ R- ^ 1 I I I I r 4 12 3 4 5 6 n — I — I — I — I — F^ 789 10 11 123456 SARCOMERE LENGTH (urn) 3_ 7 8 9 10 11 Figure 3. Histograms of percent occurrence of muscle fiber types based on sarcomere length in the paired claw closer muscles during development as represented by a larval (1st) and several juvenile (4th. 6th, 13th) stages. Number of fibers sampled for each claw is 30 for the 1st stage, 60 for the 4th stage, and 90 for the remaining stages (from Lang et al.. 1977a, b, Govind and Lang, 1978). 100 C. K. GOVIND Stage 4th stage claw I claw 5th stage DEVELOPMENT OF ASYMMETRY 101 The transformation of the paired claw closer muscles from the symmetrical to the asymmetrical condition involves the acquisition of short SL, presumably fast, fibers in the putative cutter claw and of long SL, presumably slow, fibers in the putative crusher claw. Since no evidence for degenerating fibers has been found, the changeover to short and long SL fibers in the appropriate claws must be due to the transformation of existing fibers. The transformation from short to long SL fibers in the development of the crusher claw may be explained by the lengthening of sarcomeres: a process which has been amply demonstrated among crustacean muscle fibers. However, the transformation of long to short SL fibers during development of the cutter claw is not as easily explained. They could arise by longitudinal splitting of existing short SL fibers; a mechanism which has been suggested to account for growth of a lobster leg muscle (El-Haj et ai, 1984), or the short SL fibers could arise by transverse splitting of sarcomeres at their H-bands as has been shown to occur in an adult crab muscle (Jahromi and Charlton, 1978). Histochemical properties. Among vertebrates determination of muscle fiber types using enzyme histochemistry for the detection of myofibrillar adenosinetriphosphatase (ATPase) activity is well established (review by Burke, 1981). Such histochemical techniques have only more recently been applied to crustacean muscle (Ogonowski and Lang, 1979) where fast muscle stains more intensely than slow since the specific activity of myofibrillar ATPase of crustacean fast muscle is two to three times greater than that of slow muscle (Hajek et al, 1973; Lehman and Szent-Gyorgyi, 1975). The differentiation of fiber types in the paired closer muscles was followed in a larval and several juvenile stages (Fig. 4) (Ogonowski et ai, 1980). In the 3rd larval stage the paired muscles were symmetrical in their fiber composition consisting of a central band of dark-staining fast fibers sandwiched by light-staining, slow fibers on the dorsal and ventral surfaces. Histochemistry of the 1st and 2nd stage larval claws revealed little if any staining for ATPase in the muscles suggesting that the fibers had little (if any) of this enzyme in these early developmental stages. The symmetry in fiber composition between the paired muscles is also seen in the juvenile 4th stage, though occasionally slight asymmetries in the width of the central dark-staining band are present (Fig. 4). In the 5th stage one of the claws has the central fast band consistently broader than that of its counterpart claw. This is the putative cutter claw where the fast fibers continue to be elaborated over most of the closer muscle except for a narrow ventral strip in the succeeding juvenile stages until the process is completed by the 9th- 10th stage. The other claw differentiates into the crusher by the expansion of slow fibers dorsally and ventrally and the diminution of the central fast band until about the 1 3th stage when the muscle is composed of all slow fibers. Thus at the end of the first year of development the paired closer muscles are differentiated into their asymmetric condition. The cutter muscle has predominantly fast fibers and a small ventro-lateral band of slow while the crusher muscle has all slow fibers. Among the slow fibers in both claws there is a small sub-population located distally which are slower than the remaining majority (Kent and Govind, 1981.) The development of asymmetry in the paired closer muscles from a symmetric condition occurs by the transformation of slow to fast fibers in the putative cutter claw and of fast to slow in the putative crusher claw. This is suggested by the observation of fibers with an intermediate staining intensity than that characteristic of fast and Figure 4. Representative cross-sections stained for myofibrillar ATPase activity showing distribution of fast (dark-staining) and slow (light-staining) fibers during development of the paired closer muscles in a larval (3rd) and several juvenile (4th, 5th, 7th, 13th) stage lobsters. The small dorsally situated opener muscle retains its slow (light-staining) character throughout development (from Ogonowski el al.. 1980). 102 C. K. GOVIND slow fibers. Thus in the putative cutter muscle these intermediate type fibers were found in the dorsal region which is destined to become fast while in the putative crusher muscle they were found in the central region which is destined to become slow. (Fig. 4). Such changeovers in the enzymatic profile of fibers have been shown to occur between the fast-twitch and slow-twitch fibers in vertebrate muscle and to be under the direction of the innervating motoneurons (reviewed by Guth, 1968; Gutmann, 1976; Harris, 1974; Jolesz and Sreter, 1981). Biochemical properties. The protein composition of the closer muscle in juvenile and adult lobsters has been examined using gel electrophoresis (Costello and Govind, 1984). Adult fast and slow muscle have several proteins in common and these are listed along with their molecular weights as follows: (Fig. 5) myosin heavy chain (HC, 154K), two myosin light chains (LCI, 20K in doublet form and LC2, 16K), actin (A, 4 IK), tropomyosin (TM, 34K), and a protein tentatively identified as a-actinin at 92K. Another major protein tentatively identified as paramyosin (P) differs in molecular weight between fast and slow muscle at 99K and 96K respectively. Apart from these common proteins, adult fast and slow muscle have proteins unique to A. s I ? B. g 1- 5 5 Crt o o < —1 _l u. Ui (/) I-' H cr o O o 5 5 p H 5 -J _j ^'^ < _i H K a: H q: o o o o o ->f niii..t»» Hi ^* wr ^^ HC 200K- 105K-^ 66K-»- F-75K -F-48K «■»> «M*- TNT -S-47K ^ ^^ ^., 24K-^ ^TNT -TM -F-26K -S-25K LCi ^LC, 17K*- *- W ^S-17K -LC2 -S-13K LCz TNI(F-26K) TN|(S-25K) TNG ♦ • Figure 5. Electrophoretic protein patterns of fast and slow muscle from the cutter (CT) and crusher (CR) claw closer muscle of an adult lobster. A, Whole myofibrillar homogenate showing the common proteins such as myosin heavy chain (HC), two myosin light chains (LCI, LC2), actin (A), paramyosin (P), tropomyosin (TM), troponin-C (TNC), and troponin-T (TNT). Proteins unique to fast (F) and slow (S) muscle are so indicated at their respective molecular weights. B, Myosin extract showing heavy and light chains. C, Troponin-tropomyosin extract showing several unique proteins and troponin-I (TNI) (from Costello and Govind, 1984). DEVELOPMENT OF ASYMMETRY 103 themselves. There were three such bands in the electrophoretic pattern for fast muscle seen at F-75K, F-48K and F-26K in Figure 5 and four unique bands for slow muscle at S-47K (doublet form), S-25K, S17K, and S-13K. One of these unique proteins in each fiber type, F-26K in fast muscle and S-25K in slow muscle corresponds to the regulatory protein troponin-I (Fig. 5) Other regulatory proteins include troponin-T (TNT) normally masked by actin and troponin-C (TNC) and tropomyosin (TM) which are common to both fast and slow muscle (Fig. 5). The earliest stage during development of the paired closer muscles examined electrophoretically was the 4th juvenile stage when the muscles are symmetric in fiber composition. At this stage the closer has almost all of the major proteins common to both adult fast and slow muscle viz. myosin heavy and light chains, actin, para- myosin, and tropomyosin (Fig. 6). A high molecular weight protein at 290K which is common to both types of adult muscle is lacking in the 4th stage muscle. More significantly, however, is the lack of all proteins unique to fast muscle (F-75K, F-48K, and F-26K) and of one protein unique to slow muscle (S-13K) in this juvenile muscle. Furthermore the slow muscle protein S-47K is present in a singlet form in the 4th stage muscle and not in the doublet form characteristic of the adult muscle. These missing proteins are present in the 10th stage muscle, except for S-13K which is still absent in the cutter slow muscle. The major proteins common to both fast and slow muscle are present in the first juvenile form (4th stage) as are also three of the four proteins unique to slow muscle. a b c d e f -: tt 290K HC F-75K F-48K S-47K S-17K LC2 S-13K Figure 6. Differentiation of the electrophoretic protein pattern of the claw closer muscles. Lane a: undifferentiated muscle of juvenile 4th stage. Lanes b, c: differentiated cutter (fast and slow fibers) and crusher (slow fibers) muscles respectively of juvenile 10th stage. Lanes d, e, f: fully differentiated cutter fast, cutter slow, and crusher slow muscles respectively of an adult. Abbreviations as in Figure 5 (from Costello and Govind, 1984). 104 C. K. GOVIND During juvenile development the missing unique proteins of fast and slow muscle are expressed. Since some of these unique fast proteins (F-26K, and F-75K) are tentatively identified as troponin I and troponin-tropomyosin complex respectively, their belated appearance suggests a gradual maturation of the regulatory mechanism governing contraction of fast muscle. Moreover, the appearance of these unique proteins during juvenile development not only signals the activation of new genes but underscores the fact that the muscle fibers are differentiating into their adult character. These biochemical studies do not address the question of how and when the paired closer muscles become asymmetric. In order to answer these questions, in- dividual fibers or at most a small group of fibers taken from areas of the closer muscle known to be either fast or slow according to structural and histochemical tests would have to be analyzed for their protein composition in the first year of development i.e., from the 4th to the 13th stage. This will reveal the protein composition of fibers which are transforming from fast to slow and vice versa. Contractile properties. The contractile behavior of individual fibers in the adult cutter and crusher muscles has revealed a wide spectrum which has been conveniently grouped into fast, slow, and intermediate types (Jahromi and Atwood, 1971; Costello and Govind, 1983a). Thus fast-follower fibers have a rapid rise to peak tension which is maintained at a plateau and a rapid decay (Fig. 7). Slow-follower fibers show a gradual and continual increase in tension with a decay phase that is equally slow. The intermediate fibers showed a mixture of the tension properties of fast and slow fibers by having an initial rapid rise time followed by a slower rise time. The wide range of contractile behavior encompassed by these three arbitrary categories is seen in the rise time of fibers which extends between 50 to 800 ms for both adult muscles. In larval and early juvenile lobsters the rise time of fibers was between 50 to 400 ms. The slower rise times characteristic of the adult fibers is not present in the 4th juvenile stage and must be acquired during subsequent juvenile development. Indeed there are few slow-follower type fibers in the 2nd, 3rd, and 4th stage muscle, the majority being intermediate and fast-follower types. In the differentiation to asymmetric muscles the slow fiber population increases at the expense of the intermediate fibers in the cutter claw and of the fast fibers in the crusher claw judging by the distribution of these three fiber types during development (Table I). FAST INTERMEDIATE SLOW ^1 2 3 O cn LU X CO ID cr o L B Figure 7. Contractile responses of single muscle fibers (upper trace) to short, 800 ms, depolarizing pulses (lower trace) in cutter and crusher claw closer muscles showing representative fast, intermediate, and slow types. Calibration: vertical 5 mg; horizontal, 400 ms. (from Costello and Govind, 1983a). DEVELOPMENT OF ASYMMETRY 105 As a result, the adult crusher muscle has more intermediate and slow fibers and less fast fibers than its cutter counterpart. This would account for the fact that in the intact animal both claws display a wide range of movements from brief, rapid twitches to prolonged, slow contractions (Costello et ai, 1984). The asymmetry in contractile types between the paired muscles is therefore in the relative proportion of the three types and not in the fiber types themselves. Given this fact it is interesting to correlate the contractile behavior of these fibers with their SL, ATPase activity and innervation in order to obtain a more comprehensive picture of muscle asymmetry. Such a correlation (Table II) made for groups of fibers, shows broad agreement with the idea that fast-contracting fibers have high ATPase levels, low oxidative capacities, and short SL, while slow-contracting ones have low ATPase levels, high oxidative capacities and long SL. On an individual basis this three-way correlation does not necessarily hold; e.g., in the crusher all fibers have long SL, low ATPase levels yet can contract rapidly. Finally, when the motor innervation of these bundles of fibers is considered with their other prof)erties, the bundles are seen to be functionally specialized, some for fast, brief contractions (such as the cutter dorsal and proximal bundles) and others for slower, more sustained contractions (such as the central distal bundles). Opener muscle As an antagonist to the closer muscle, the opener muscle elevates the dactyl in preparation for the closing action. As such it performs a necessary function, consid- erably limited in scope, which is reflected by the small size of the muscle compared to the closer. Not surprisingly it has received little attention in the adult claws and none whatsoever during development of the claws. Structural properties. The frequency histogram of SL from the adult muscles (Fig. 8) shows a range between 6-9 ^lva for the cutter and 9-1 1 ^m for the crusher, with no overlap between them (Govind et al., 1981). Though the SL of fibers in both adult muscles is >6 ^m, the mean SL of the cutter muscle at 8 nm is significantly shorter than that of its counterpart muscle at 10 fim (Fig. 8). Clearly the paired muscles are Table II Correlation of contractile (rise time) histochemical (ATPase and oxidative capacity) and structural (sarcomere length) properties of closer muscle in different regions of the paired cutter and crusher claws (from Costello and Govind. 1983a) Rise time ATPase Oxidative Sarcomere (ms) activity capacity length Cutter muscle dorsal 95 high low short proximal 80 mixed mixed short ventral 236 low high long proximal ventral 326 low high long central distal 489 very low very high long Crusher muscle dorsal 232 low high long proximal 189 low high long ventral 256 low high long proximal ventral 379 low high long central distal 554 very low very high long 106 C. K. GOVIND o O o o 50 -| 45- 40- 35- 30- 25- 20- 15- 10- 5- CUTTER "T" 6 CRUSHER 1^ 6 8 9 10 11 6 7: SARCOMERE LENGTH (urn) 9 10 11 Figure 8. Histogram of percent occurrence of muscle fiber types based on sarcomere length in the paired claw opener muscles of an adult lobster. Number of fibers sampled is 158 for each claw (from Govind et al., 1981). asymmetric in SL though the asymmetry is much more subtle than that seen for the closer muscle. Histochemical properties. Since cross-sections of the entire claw were taken for histochemistry of the closer muscle (Fig. 3), the opener muscle was always included. The opener muscle showed low specific activity of myofibrillar ATPase typical of slow muscle judging by the light staining compared to the fast fibers of the cutter closer muscle. The staining pattern remains virtually unchanged between the paired claws during development resulting in the symmetry seen in the adult cutter and crusher muscles. Proximal slow fibers of both opener and closer muscles in both claws stain less intensely for ATPase than the remainder (Kent and Govind, 1981), which suggests subdivision within the slow category. Biochemical properties. The electrophoretic protein pattern of the opener muscle is similar in both adult cutter and crusher claws and resembles the pattern of the slow fibers from the closer muscle (cf. Fig. 5). Thus all the unique proteins of the slow fibers of the closer muscle are found in the opener muscle as well as those represented by the bands at S-47K, S-25K, S-17K, and S-13K. The only difference is the presence of an unidentified protein at 122K which is not found in the closer muscle. Development of Neuronal Asymmetry The innervation of the limb muscles in crustaceans is well established from the classical work of Wiersma (1961). The claw closer muscle is supplied by three motor axons (two excitors and an inhibitor) which are well-characterized in the adult and whose development has been followed. The claw opener muscle receives only two DEVELOPMENT OF ASYMMETRY 107 axons (an excitor and an inhibitor) which have not been as well studied as the closer axons. In addition there are the large numbers of different sensory receptors landscaping the claw for which little information is available. Motoneurons to closer muscle Number and type. The two excitor axons to the adult muscle are differentiated into a fast closer excitor (FCE) and a slow closer excitor (SCE) on the basis of the contractions they evoke (Wiersma, 1955). Though these contractions are qualitatively similar between the paired claws, those to the cutter are more rapid and fatigue more readily than those to the crusher (Govind and Lang, 1974, 1979). Thus the homologous motoneurons are asymmetric in the adult. Excitatory innervation of the closer muscle is present at the time of hatching with well-defined neuromuscular terminals containing synaptic vesicles and presynaptic dense bars (Fig. 9) denoting active sites of transmitter release at synapses (King and Govind, 1980). The number and type of excitatory axons is however not known in these 1st larval stages. Two excitor axons are physiologically identifiable in the 2nd larval stage with one of them being reminiscent of the FCE (Hill and Govind, 1984). By the 3rd larval stage, the two axons are sufficiently well-differentiated to b>e rec- ognizable as putative FCE and SCE axons and their physiological identity is firmly established in the succeeding juvenile stages (Costello et ai, 1981), though exactly when the homologous motoneurons diverge into cutter and crusher types is not known. Morphologically the motoneurons mature within the first year of development so that by the 10th juvenile stage they resemble their adult counterparts (Fig. 10) (Hill and Govind, 1983). The general form for both FCE and SCE neurons is similar, consisting of an antero-ventrally located soma from which a single neurite rises ver- tically to the dorsal surface of the ganglion. The neurite courses diagonally across the ganglion to the second root which it enters as an axon. Dendritic branches which Figure 9. Excitatory neuromuscular terminal (E) recognized by spherical synaptic vesicles (v) adjacent to an inhibitory terminal (1) which contains irregularly-shaped vesicles in the juvenile 4th stage claw closer muscle. These two types of terminals also occur in the larval 1st stage and adult muscle. Magnification 20,000X. (from King and Govind, 1980). 108 C. K. GOVIND anterior connective Figure 10. Camera lucida drawings of cobalt-filled motoneurons of paired FCE and SCE motoneurons in juvenile lobsters with cutter (right side) and crusher (left side) claws. Magnification, 40X. (from Hill and Govind, 1983). arise from the neurite and are restricted to their respective hemiganglia, differ between FCE and SCE neurons. The SCE has a much more elaborate dendritic field than the FCE. Thus, whereas the FCE has only a distal dendritic field of two primary branches, the SCE has a distal field of several primary branches and a proximal field as well. In view of the striking asymmetry in behavior, external form and muscle composition of the paired claws, there was surprisingly no asymmetry between the homologous motoneurons. A closer inhibitor (CI) axon is present in the 1st larval stage muscle (Fig. 9) (King and Govind, 1980) judging from the occurrence of neuromuscular terminals populated by ellipsoid-shaped synaptic vesicles which are characteristic of inhibitory terminals (Atwood et al, 1972). Whether there is a single CI cannot be deduced from this type of morphological evidence. In the juvenile 4th stage, however, a single class of inhibitory junctional potentials (ijp) is seen with stimulation of the closer nerve suggesting the presence of a single CI (Costello et al, 1981). The adult closer muscles receive a lone CI (Hill and Govind, 1981) which is seen to be shared with several other cheliped muscles (Hill and Lang, 1979). Distribution. In the juvenile 4th, 5th, and 6th stages, the innervation pattern of the FCE and SCE axons is similar between the paired muscles (Table III) (Lang et al, 1980; Costello et al, 1981). The majority of fibers receive both axons while a small number receive each axon exclusively. In the adult the pattern is dramatically different between cutter and crusher muscles. Most of the fibers in the cutter receive Table III Distribution of innervation by FCE and SCE axons in claw closer muscle of juvenile lobsters where the pattern is similar between the paired claws and in adult lobsters where the pattern differs between the paired (cutter and crusher) claws (from Costello et al., 1981). % innervation FCE SCE FCE + SCE Stage 4 Stage 5 Stage 6 Aduh cutter Adult crusher 9 18 9 64 15 5 5 9 16 18 86 77 82 20 67 DEVELOPMENT OF ASYMMETRY 109 FCE only, a few receive either the SCE only or both SCE and FCE together. In contrast, most of the crusher fibers receive both axons, while a few receive either FCE or SCE exclusively. This signifies a clear change in the innervation patterns between juvenile and adult muscles. Since the paired juvenile muscles are symmetrical in their innervation they may be regarded as being undifferentiated compared to the adult cutter and crusher muscles which have their own peculiar innervation pattern representing the differentiated condition. From the undifferentiated juvenile state where the large majority (80%) of fibers received both FCE and SCE axons, selective elimination of SCE would result in the adult distribution of 64% FCE innervation in the cutter. Synaptic elimination, on a smaller scale, of the FCE elements would give rise to the adult value of 16% SCE innervation. Similar processes would operate in finalizing the innervation to the crusher muscle where synapse elimination would affect fewer fibers since only a small number are supplied exclusively by each axon. According to this scheme the final pattern of innervation is refined by selective elim- ination of cutter FCE or SCE synapses from an initial (juvenile) condition where both axons are present. There may well be alternative methods for achieving the adult innervation patterns, such as the generation of new synapses, though the proposed mechanism is the most parsimonious one. The distribution of the CI axon has not been mapped out for either developing (juvenile) or adult lobsters. In the few instances where CI has been detected, it was found on fibers with SCE innervation (Costello et al, 1981; Hill and Govind, 1982). Synaptic properties. In adult lobsters the neuromuscular synapses provided by the FCE and SCE axons differ in their physiological and fine structural properties. Thus the ampHtude of the excitatory junctional potential (ejp) at 1 Hz stimulation is generally larger for the FCE than for the SCE synapses (Fig. 1 1 ) (Govind and Lang, 1974; Costello et al, 1981). Conversely the degree of facilitation of the ejps calculated as the ratio of the ejp amplitude at 10 and 1 Hz is greater for the SCE than the FCE synapses. The SCE synapses were more fatigue-resistant and showed better recovery following fatigue than their FCE counterparts. FCE fine structure is relatively simple in having small-diameter terminals each with few synaptic vesicles, a single long synapse and little if any postsynaptic apparatus (Hill and Govind, 1981). The SCE innervation is more complex in having a wide size range of terminals each with many synaptic vesicles, several short synapses, and an extensive postsynaptic apparatus. The above data shows a clear distinction between neuromuscular synapses of the FCE and SCE axons. 1 Hz /X 10 Hz L LU O CO Figure 1 1. Synaptic properties represented by the amplitude of the ejp at 1 Hz stimulation and its degree of facilitation at 10 Hz for the FCE and SCE axons in an adult cutter closer muscles. Vertical calibration, FCE, 5 mV; SCE 4 mV. Horizontal calibration, 20 ms. (Costello el al.. 1981). 110 C. K. GOVIND The physiological data reveals no differences in homologous synapses between cutter and crusher claws except perhaps that the FCE synapses show a greater maximal EJP amplitude in the cutter compared to the crusher claw. The other difference between the paired claws is that synapses of both axons tended to be more fatigue- resistant in the crusher compared to the cutter claw (Govind and Lang, 1974). The development of neuromuscular synapses from the excitatory axons has been studied using electrophysiology and electron microscopy. In a few recordings made from the larval 2nd stage muscle, large ejps of approximately 10 and 20 mV were characteristic of putative SCE and FCE synapses respectively (Hill and Govind, 1984). These large synaptic potentials of the FCE axon often produced secondary regenerative responses. In a larger sampling of synapses from the larval 3rd stage the mean ejp size was 6 mV with a range of 4 to 10 mV for the FCE synapses. The SCE synapses were considerably smaller with a mean of 3 mV and a range of 2 to 4 mV. The amount of facilitation was similar for the two types of synapses. However, the FCE synapses often produced regenerative responses and displayed fewer transmission failures than the SCE synapses. In the juvenile 4th, 5th, and 6th stages (Costello et ai, 1981) ejps of the FCE displayed a wide range in amplitude though the mean size was similar to the larval and adult forms signifying that they had reached their final condition. On the other hand, ejps of the SCE axon in these juvenile stages had as narrow a range of amplitudes as those in the larval stage. The wider spread in ejp amplitude typical of the adult SCE synapses must presumably come with maturation. The other difference between FCE and SCE synapses in the juvenile lobsters is the fact that the former synapses are much more fatigue-resistant than the latter; a situation which is exactly the reverse of that found in adult lobsters. In terms of the size of the ejp, synapses differentiate into FCE and SCE types early in the larval stages while the properties of facilitation and fatigue-sensitivity mature later during juvenile development. Structural aspects of the development of excitatory synapses were examined by serial section electron microscopy of the closer muscle in a larval 1st stage, a juvenile 4th stage, and an adult lobster (King and Govind, 1980). No attempt was made to determine whether the terminals belonged to the FCE or SCE axons. There was a tremendous proliferation of excitatory innervation from the 1 st larval stage where it was restricted to four discrete locations over the entire muscle to individual muscle fibers in the adult. Concomitantly there is a ten-fold and significant increase in the mean size of synapses between larval and adult stages (Table IV). The mean size of terminals varied considerably among the three stages examined and showed no con- sistent trend. On the other hand, the presynaptic dense bars, representing active sites of transmitter release were consistently similar in size and were found in the majority (>60%) of synapses. Synaptic development therefore consists of an increase in number and size of excitatory synapses which occur in tandem with the increase in mass of the closer muscle. From within this overall pattern of synaptic development, there is a need to distinguish between FCE and SCE synapses in order to understand how their final distribution within the closer muscle forms. A start has been made in this direction by examining physiologically identified FCE and SCE terminals in juvenile lobsters (Hill and Govind, 198 1). The FCE innervation is relatively simple, consisting of small terminals each with a single synapse, few synaptic vesicles and limited post- synaptic apparatus. In contrast the SCE innervation is more complex, having larger and more variable terminals each with several short synapses, many synaptic vesicles, and an extensive postsynaptic apparatus. Firing patterns. The in vivo activity of the FCE and SCE axons during reflex closing of the claws was analyzed in one to three-year-old juvenile lobsters with DEVELOPMENT OF ASYMMETRY 111 Table IV Quantitative comparison of excitatory nerve terminals, synapses, and presynaptic dense bars in the claw closer muscle of a larval (1st stage), juvenile (4th stage), and adult lobster (from King and (iovind. 1980) 1st stage 4th stage Adult Nerve terminals: Length of muscle fiber serially sectioned (^m) 10.23 11.12 12.05 Total number 6 5 5 Mean surface area (nm^) 34.63 53.73 19.49 (X ± S.E.M.) ±10.93 ±32.93 ±10.55 Synapses: Number completely sectioned 29 51 15 Mean number per terminal 4.83 10.20 3.20 (x ± S.E.M.) ±1.79 ±5.04 ±0.89 Mean surface area (^lTn^) 0.079 0.136 0.805 (X ± S.E.M.) ±0.010 ±0.012 ±0.174 Presynaptic dense bars: Total number 22 40 13 Mean surface area (mhi^) 0.018 0.016 0.017 (X ± S.E.M.) ±0.005 ±0.002 ±0.002 Mean number per synapse 0.759 0.784 0.867 (X ± S.E.M.) ±0.128 ±0.081 ±0.236 dimorphic claws (Costello et ai, 1984). While the dactyl was free to move, the rest of the claw and the animal was immobilized in order to permit recordings of ejps from the closer muscle fibers. Under these conditions, the FCE fired only during rapid closing at a lower frequency and duration than the SCE which fired only during slow closing of the cutter claw (Table V). However, the crusher FCE and SCE axons were active during fast and slow closing respectively and their firing patterns were similar. This similarity was also found between the two axons during maintained closing of the crusher. Thus a clear distinction is found between FCE and SCE axons in the cutter but not in the crusher claw. When the homologous motoneurons are compared, the FCE of the crusher has a significantly higher firing frequency and burst duration than its cutter counterpart during fast closing of the claw (Table V). The homologous SCEs, however, displayed Table V In v'wo firing frequency and burst duration of FCE and SCE axons during fast, slow, and maintained closing of cutter and crusher claws of intact lobsters (from Costello et al., 1984) Claw type Closing behavior Motoneuron type Frequency (Hz) X ± S.D. Burst duration (ms) X ± S.D. Cutter fast FCE 2± 2 55 ± 26 46 Cutter slow SCE 37 ±24 361 ± 143 51 Cutter maintained SCE 15 ± 9 1963 ± 1351 31 Crusher fast FCE 37 ±27 215 ± 94 44 Crusher maintained FCE 10 ± 9 1010 ± 630 13 Crusher slow SCE 31 ± 12 406 ± 175 17 Crusher maintained SCE 18 ± 13 1278 ± 707 23 112 C. K. GOVIND a close similarity in their firing patterns during slow closing. Maintaining the closed claw was achieved by the FCE and SCE axons in the crusher but only by the SCE in the cutter claw though all three axons were similar in their firing patterns. Thus the FCE alone showed an asymmetry in firing patterns between cutter and crusher claws in intact juvenile lobsters. In contrast to the above, the in vitro activity of the adult motoneurons shows a clear asymmetry between FCE and SCE in both claws and between both homologs (Govind and Lang, 1981). Activity of the motoneurons was recorded from their respective somata in response to electrical stimulation of the mixed nerve roots in an isolated claw-ganglion preparation. In both claws, the FCE fired at a lower frequency and for a shorter time than the SCE. When homologous somata were examined, the crusher FCE and SCE produced higher frequencies and longer bursts of spikes than their cutter counterparts (Fig. 12). Since this asymmetry was found in response to both sensory stimulation via the 2nd nerve root and depolarization of the soma it could have both an extrinsic (sensory) and intrinsic (built-in) origin. The motoneurons also produce a distinct pattern of paired impulses (Costello et al, 1981; Govind and Hill, 1982) which are functionally more effective in generating muscle tension than uniformly spaced impulses of the same average frequency (Ripley and Wiersma, 1953; Govind and Lang, 1974). In intact juvenile lobsters, paired impulses with interpulse intervals of between 8 to 1 3 ms, were found for both FCE and SCE axons in both claws. The only indication of asymmetry in this firing pattern FCE B SCE Cutter soma Crusher soma J D *»t4- Closer nerve Cutter soma Closer nerve Crusher soma Figure 12. Firing patterns of homologous FCE and SCE neurons recorded either from their somata (A, B) in response to sensory stimulation via the 2nd nerve root or from the closer nerve (C, D) in response to depolarization of their somata. For each homologous pair, the crusher motoneuron shows a greater response than its cutter counterpart. Vertical calibration, 4 mV in A; 10 mV in B; 1 ^A in C lower trace; 2 ^A in C upper trace and D. Horizontal calibration, 40 ms in A, B, D lower trace; 100 ms in C, D upper trace (from Govind and Lang, 1981). DEVELOPMENT OF ASYMMETRY 1 1 3 was for the homologous FCE axon which produced paired impulses almost all the time in the crusher but only 25% of the time in the cutter. The homologous SCE axons resembled each other in producing paired impulses 60% of the time. In isolated claw-ganglion preparations of adult lobsters, depolarization of the soma gave rise to paired impulses in FCE and SCE motoneurons thereby strongly implicating an en- dogenous mechanism for the generation of this patterned activity. The development of the characteristic firing patterns for the FCE motoneurons alone has been examined in intact juveniles (Costello and Govind, 1983b; unpub.) where closing has been reflexly evoked. In the juvenile 4th and 5th stage the homologous FCE fire at a similar frequency of 100-150 Hz for 150-200 ms. In the subsequent juvenile stages till about the 12th stage, both the frequency and duration of firing decreases dramatically in the putative cutter claw to <10 Hz for <50 ms which approximates the adult condition. In the putative crusher the duration fluctuates between 100-200 ms while the frequency gradually decreases to <50 Hz which is reminiscent of the adult condition. The activity patterns of the homologous FCE have essentially matured by the time the lobster is a year old. The closer muscles have a timetable similar to that of the FCEs in achieving their final composition of fiber types. Whether there is any causal relationship between the development of asymmetry in the firing patterns of the homologous FCE mo- toneurons and in the fiber composition of the closer muscles cannot be deduced from this correlation. However, transformation of fast fibers to slow and the resultant diflferentiation of a crusher muscle with all slow fibers can be prevented by denervation or tenotomy in the early juvenile stages (Govind, 1981; Govind and Kent, 1982). Since these treatments reduce or eliminate active muscle tension mediated by motor impulses, they implicate the motoneurons in directing the differentiation of muscle fiber types. On the other hand, these experiments also suggest that it may be the overall level of active muscle tension which transforms fast fibers to slow. Consequently, the trigger for muscle transformation may well reside in the level of motoneuronal activity of both excitors, FCE and SCE, and the inhibitor, CI, axons. The claw muscle experiencing the higher level of motor activity would become the crusher while its counterpart muscle would become the cutter. Motoneurons to the opener muscle The innervation to this muscle in lobsters has received scant attention compared to the very extensive studies of the homologous muscle in crayfish (reviewed by Atwood, 1976). The discovery of subtle asymmetries in the neuromuscular system in the opener muscle between cutter and crusher claws (Govind et ai, 1981) has initiated a more detailed current investigation (G. Kass-Simon and K. Mearow, unpub.) which provides the basis for most of the comments given here unless otherwise acknowledged. Number and type. The opener muscle in the limbs of decapod crustaceans is supplied by an excitor (OE) and inhibitor (OI) motoneuron (Wiersma, 1961). While the OE also innervates the stretcher muscle in the next proximal segment, the OI is a private motoneuron. However, more recent evidence suggests that the CI also innervates the opener muscle (T. J. Wiens, pers. comm.). The OE is reminiscent of a slow excitor axon as it does not cause rapid opening of the dactyl and is more fatigue-resistant. The development of excitatory and inhibitory innervation has not been examined in the lobster though both types of synapses are present immediately after hatching in the homologous opener muscle in the crayfish (Atwood and Kwan, 1976). 114 C. K. GOVIND Distribution. Being the only excitor axon, OE may be expected to innervate all fibers of the adult opener muscle. The presence of OI and CI however has not been detected in all fibers examined suggesting a regional distribution of innervation for each of these two axons which may account for the fact that they were not recognized as separate axons in the past. Synaptic properties. Generally the amplitude of the ejps are small ranging from < 1 mV to 5 m V; many being visible only after a bout of high frequency stimulation designed to produce facilitation and summation. All of the excitatory synapses showed moderate to strong facilitation with repeated stimulation. The OI synapses also gave small junctional potentials which were either hyperpolarizing or depolarizing in sign. These ejps exerted considerable postsynaptic inhibition judging from the fact that they reduced the size of the ejp considerably. Almost complete elimination of the ejp occurred occasionally suggesting pre-synaptic inhibition similar to that found in the homologous motoneurons in the crayfish opener muscle. In terms of their phys- iology the OE and OI synapses in lobster resemble their counterparts in crayfish (Atwood and Bittner, 1971). Consequently they may also resemble them in ultra- structure which has been extensively described in crayfish (Jahromi and Atwood, 1974). Firing patterns. The crusher OE has a higher frequency of firing and longer burst duration than its cutter counterpart in response to nerve root stimulation in isolated ganglia of adult lobsters (Govind et al, 1981). The crusher OE is also more resistant to fatigue when stimulated repetitively than the cutter OE. This asymmetry in firing patterns between homologous OE motoneurons in vitro forebodes a similar asymmetry in the intact lobster. The ontogeny of these firing patterns is unknown. Sensory neurons In a singular attempt to document asymmetry in the sensory system between paired cutter and crusher claws, the number and size of axons was determined in the nerve roots to a juvenile lobster (Govind and Pearce, 1984). The nerve roots are mixed, containing both sensory and motor axons. However, since the motor axons are bilaterally constant and relatively few in number, the majority of axons in the nerve roots are sensory. The total numbers of axons in the first root were approximately 16,000 for the crusher and 13,000 for the cutter; which gave a crusher-cutter ratio of 1.22. For the second root the counts were 119,000 for the crusher and 124,000 for the cutter which gave a ratio of 0.96. The slight asymmetries in the roots proved not to be significant in random samples from homologous regions. Furthermore, a representative sampling of the axon diameters showed a parallel distribution in all size classes between crusher and cutter claws. Consequently, there does not appear to be a asymmetry in the numbers and sizes of sensory axons between the paired claws in a juvenile lobster. However, in adults the external dimorphism between the paired claws is much more pronounced than in the juvenile and there is the possibility that the sensory system may be asymmetric. Similarly, no differences in the distribution of four different types of cuticular hair organs were detected between cutter and crusher claws of "subadult" lobsters (Solon and Cobb, 1980). These four cuticular hair organs which are regarded as mechanosensory in function differ basically in the length of the sensilla: type I are the longest (70-130 txm), type II slightly smaller at 30-60 ^lm, type III still smaller but located in a raised protuberance, and type IV are simply 1 nm long conical hairs occurring in clusters. Types II and III differed in distribution between dorsal and ventral sides and among different areas of the claw. More interesting was their dis- DEVELOPMENT OF ASYMMETRY 115 tribution in a juvenile lobster with symmetrical claws. Type IV receptors were just as ubiquitous in the juvenile as in the subadult. Type III receptors, however, had a lower density in the juvenile than in the subadult denoting the addition of these hair organs during growth of the claws. On the other hand, types I and II with a higher density in the juvenile than in the subadults are apparently not added during growth. These differences in density of particular types of hair organs between juveniles and subadults may reflect changes in behavior during development which have been documented previously (Lang et ai, 1977c). Comparison with other Asymmetric Systems Claw asymmetry is not uncommon among crustaceans and it may be instructive to review how it arises during development in fiddler crabs and how it is maintained during regeneration in snapping shrimps. Adult male fiddler crabs have a hypertrophied major claw used for courtship and defence and a minor claw used for feeding and grooming (reviewed by Crane, 1977). The asymmetry in external form is matched by an asymmetry in muscle mass (Rhodes, 1977), soma size, and dendritic field of the motoneurons (Young and Govind, 1983), and in the numbers of sensory axons (Govind and Pearce, 1984). Early in development the paired claws are symmetrical. The asymmetry develops following the loss of one of the paired claws during a critical period which extends from the megalopa to a young crab stage (Morgan, 1923, 1924; Yamaguchi, 1977). If both claws are removed at this stage then no major claw develops; if both are kept intact during this critical period, then paired major claws develop. Consequently the loss of a claw during development triggers the remaining one to differentiate into a major claw in male fiddler crabs. Once the claw asymmetry is established it remains fixed and removal of either major or minor claw will cause the same type to regenerate. This is similar to the situation in lobsters but unlike that in snapping shrimps where claw laterality is not fixed in the adult. The major or snapper claw in Alpheid shrimps is used in defence when it ejects a jet of water on closing and at the same time makes a loud popping sound; the minor or pincer claw is used for feeding and grooming. In adult shrimps, autotomy of the snapper results in the regeneration of a pincer in its place while the existing pincer transforms into a snapper (Prizbram, 1901; Wilson, 1903). This pincer to snapper transformation involves several changes: a hypertrophy and differentiation in the external form, a hypertrophy of the motoneuron somata to the closer muscle (Mellon et al, 1981), a hypertrophy of the closer muscle and the transformation of its fast fibers to slow, and an increase in facilitation of the excitatory neuromuscular synapses (Stephens and Mellon, 1979). Transformation is either prevented if the nerve to the pincer is transected at the time of snapper removal (Wilson, 1 903) or promoted if the nerve to the snapper alone is transected (Mellon and Stephens, 1978). The pincer can be regarded as an undifferentiated snapper which is arrested in its devel- opment by the existing contralateral snapper. Once this inhibition is removed by autotomy of the snapper or transection of its nerve, the pincer completes its differ- entiation to a snapper and at the same time arrests the development of the newly regenerating claw to a pincer type. Clearly, the maintenance of claw asymmetry and its reversal in adult snapping shrimps is under neural control (Mellon, 1981). This is similar to how claw type is determined in juvenile lobsters (Govind, 1981) where denervation of one claw causes the contralateral one to become the crusher (Govind and Kent, 1982). However, in lobsters, once claw asymmetry is determined during juvenile development, it remains fixed throughout adult life whereas in snapping shrimps it can be altered in the adult. 116 c. k. govind Future Prospects One aspect of our fascination with asymmetric systems, whether it be cerebral dominance in humans or claw lateralization in lobsters, lies in being able to understand how it arises from a bilaterally symmetrical body plan. Such a goal is feasible in the neuromuscular system of the lobster claw because certain features of this system are of advantage in studying development. First the lobster has a protracted period of development, consisting of a 9-1 1 month embryonic period, a two- week larval period, and a 5-7 year juvenile period (Herrick, 1895, Hughes et ai, 1972) which is divided into discrete stages by the molt cycle. All these stages can be reared in the laboratory (Hughes et al, 1974; Lang, 1975). Second, there are only two muscles, the antagonistic opener and closer, which make up the claw. Third, each muscle is innervated by few motoneurons: two excitors and an inhibitor in the closer and a single excitor and two inhibitors in the opener (Wiersma, 1961; T. J. Wiens, pers. comm.). Fourth, and perhaps most important of all, is the fact that claw laterality is determined during a critical two-week period of juvenile development, between the 4th and 5th stages, when the claws can be experimentally manipulated (Emmel, 1908; Lang et al, 1978). Indeed, manipulations such as tenotomy of the opener or closer muscle, or de- nervation can suppress the differentiation of a crusher claw, resulting in lobsters with paired cutter claws (Govind and Kent, 1982). In terms of the fiber composition of the closer muscle, this means that fast fibers are prevented from transforming to slow because of a lack of nerve-mediated muscle tension. Since there are only three mo- toneurons to the closer muscle, each uniquely identifiable, it is possible to examine the influence of each on muscle development. Experimental manipulations of these motoneurons such as selective deletion or electrical stimulation during the critical juvenile period, should pinpoint the role of motoneurons in the differentiation of muscle fiber types. The experiments proposed above would test the hypothesis that it is the difference in motor output in the paired claws that determines laterality. The claw receiving the greater overall motor output during the critical developmental period transforms its fast fibers to slow and becomes the crusher muscle with all slow fibers. In the absence of a certain level of motor output this transformation is prevented and the closer muscle remains with predominantly fast fibers (Lang et al, 1978) characteristic of the cutter muscle which is presumably the primitive condition. As a corollary, by controlling the motor output to the juvenile undifferentiated muscle we should be able to produce a crusher not only on a prescribed side but on both sides. These experiments, currently in progress, would explain how the asymmetry in muscle fiber composition arises during development. There is still a need to explain the asymmetry in firing patterns of the homologous motoneurons, specifically, to what extent are they due to the intrinsic (cable) properties of the motoneurons or to extrinsic (synaptic) influences. This will necessitate examining the electrical properties of the homologous motoneurons in the juvenile stages and their synaptic input. If the asymmetry in firing patterns is influenced by the synaptic input we would need to explore its nature and number. This involves primarily the sensory system of the claws though ascending and descending inputs within the ganglion can also influence the motoneuron firing patterns. Apart from the above experiments revolving around the sensory system and gan- glion there is the need to explain the differences in the distribution of the homologous motoneurons onto the closer muscles. From the juvenile condition where the majority of fibers in both muscles are innervated by both excitatory axons, the cutter closer muscle has predominantly FCE innervation while the crusher muscle has predomi- DEVELOPMENT OF ASYMMETRY 117 nantly mixed, FCE and SCE, innervation (Costello et ai, 1981). Can this asymmetry in the pattern of synaptic connections be explained by the selective elimination of synapses in the developing cutter muscle, as has been seen in a lobster abdominal muscle (Stephens and Govind, 1981). An equally challenging task would be to un- derstand why such an asymmetry arises: is it due to competition between the mo- toneurons or is it influenced by the muscle fiber properties? 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An analysis of the functional differences between the contractions of the adductor muscles in the thoracic legs of the lobster Homarus americanus. Arch. Neerl. Zool. 11: 1-13. Wiersma, C. A. G. 1961. The neuromuscular system. Pp. 191-240 in The Physiology of Crustacea, T. H. Waterman, ed. Academic Press, New York. Wilson, E. B. 1903. Notes on the reversal of asymmetry in the regeneration of the chelae in Alpheus hererochelis. Biol. Bull. 4: 197-210. Yamaguchi, T. 1977. Studies on the handedness of the fiddler crab, Uca lactea. Biol. Bull. 152: 424-436. Young, R. E., and C. K. Govind. 1983. Neural asymmetry in male fiddler crabs. Brain Res. 280: 251- 262. Reference: Biol. Bull. 167: 120-123. (August, 1984) APPARENT ABSENCE OF GAP JUNCTIONS IN TWO CLASSES OF CNIDARIA G. O. MACKJE*, P. A. V. ANDERSONf, AND C. L. SINGLA* t C. V. Whitney Laboratory and Department of Physiology. University of Florida, St. Augustine. Florida 32086, and* Biology Department, University of Victoria, Victoria. British Columbia, Canada V8W 2Y2 Abstract Study of the literature and new observations by electron microscopy suggest that gap junctions are absent in the anthozoa and scyphozoa, but present in the hydrozoa. While this may help to explain the marked electrophysiological differences known to exist between the hydrozoa and the other two groups, it raises questions about how intercellular metabolic communication is achieved in the groups lacking gap junctions. Discussion In many tissues of metazoans from Hydra to the mammals, cell interiors are directly linked by aqueous channels represented structurally by the channels of gap junctional particles, or connexons (Unwin and Zamphighi, 1980). The diameter of the channel, determined by probing with fluorescent molecules, is estimated to be 16-20 A in mammals and 20-30 A in insects (Schwarzmann et al, 1981). Gap junctions are widely believed to be responsible for electrical and dye coupling and for the transmission of electrical signals within various excitable tissues. While final proof is still lacking, gap junctions probably play an important role in tissue homeostasis by allowing permeant molecules to equilibrate throughout groups of coupled cells, in transport of nutrients from cell to cell, and in the dissemination of regulatory molecules (reviewed by Loewenstein, 1981). These regulatory functions are thought to be especially important in embryonic and differentiating tissues where gap junctions are frequently found, along with electrical coupling. Despite the circumstantial nature of much of the evidence for metabolic cooperation in cells joined by gap junctions, there can be little question that the first appearance of gap junctions in early metazoans represented a major organizational advance. The fact that sponges remain at the cellular rather than the tissue level (Hyman, 1940) may be due in large part to their apparent "genetic incapacity to produce gap junctions" (Mackie, 1984). The lowest metazoans to have gap junctions are the cnidarians, specifically members of the class Hydrozoa. Evidence from conventional transmission electron microscopy, lanthanum staining, and freeze fracture work shows these junctions to be structurally closely similar to those of higher animals (Hand and Gobel, 1972; King and Spencer, 1979). Gap junctions are present in electrically coupled glandular epithelium (Mackie, 1976), simple epithelia (Josephson and Schwab, 1974; Satterlie and Spencer, 1983), myoepitheUa (Chain et al, 1981; Satterlie and Spencer, 1983) and between certain (but not all) neurons (Spencer and Satterlie, 1980; Spencer, 1981). Dye coupling has been demonstrated in several of these cases. The most obvious function for gap junctions in hydrozoans is as a pathway for impulse transmission both between coupled neurons and between the cells in electrically Received 30 May 1984; accepted 5 June 1984. 120 GAP JUNCTIONS IN CNIDARIA 121 excitable epithelia which provide the non-nervous conduction pathways which are such a striking feature of hydrozoans (reviewed by Anderson, 1980). Whether they serve a role in metabolic communication is as much an open question here as in other groups. Morphogenetic regulatory molecules have been identified in Hydra but it is still not known if they spread within the epithelia, within nerves, or extracellularly (reviewed by Bennett et ai, 1981). Ever since the earliest days of electrical recording from cnidarians it has been clear that the hydrozoans stand sharply apart from the other cnidarians in their electrophysiological characteristics. Josephson (1974) characterizes the dichotomy as follows: "The anthozoans and scyphozoans examined have what might be termed conventional electrophysiology. Signals recorded with extracellular electrodes from conducting systems and muscles are small, generally well under 1 mv, and critically dependent on electrode placement. This is . . . what one would expect for activity in diffuse fibers in a nerve net or thin muscle sheets." In the hydrozoans, on the other hand, conducting systems produce "large electrical signals, typically 1-10 mv. The size of these potentials and their insensitivity to small changes in electrode position indicate that they are generated by blocks of electrogenic epithelia." The cubomedusae, sometimes treated as a fourth cnidarian class (Werner, 1975; Passano, 1982) exhibit electrophysiological responses of the scyphozoan-anthozoan type (Sat- terlie, 1979; Satterlie and Spencer, 1979). How are we to account for the existence within one phylum of groups having such profoundly different electrical profiles? In considering this question, it struck us that while the hydrozoan ultrastructure literature is replete with reports of gap junctions, we could recall no such reports from other cnidarian groups. A survey of the literature and discussions with colleagues bears this out. No one to our knowledge has found gap junctions in any cnidarian outside the hydrozoa. Their absence, with few exceptions {e.g., Anderson and Schwab, 1981) has excited no comment. To satisfy ourselves that the lack of such reports does not simply reflect the use of differing techniques, we have examined tissues from various scyphozoans and anthozoans using a standard procedure (Singla, 1978) that has revealed gap junctions in many hydrozoans. The scyphozoan tissues examined were taken from the arms and tentacles of Haliclystus steinegeri, Thaumatoscyphus atlanticus, and the gonads of Cyanea capillata and Rhopilema verrilli. Developing embryos and planulae of Cyanea were also examined. For anthozoans, tentacles from the sea anemones Aiptasia pulchella and Corynactis califomica were investigated. In none of these tissues were gap junctions observed. Taking these findings at face value, we can immediately see how the electro- physiological differences between hydrozoans and other groups might arise. In hy- drozoans, gap junctions would provide close coupling and ready spread of depolar- izations within epithelia, whether as propagative events or as local potentials spreading decrementally from neuroeffector junctions. The simultaneous depolarization of such groups of cells would, as Josephson suggested, generate large extracellular signals. The lack of such spread would account for the "conventional electrophysiology" of other cnidarians. There is no evidence for electrical coupling between cells in anthozoans or scy- phozoans. Intracellular recordings from one scyphozoan nerve net, the motor nerve net of Cyanea capillata, indicate that there is no coupling between the neurons. Instead, the synapses appear to be chemical (Anderson, unpub.). It has been suggested that the slow conduction systems (SS 1, SS 2) of corals and sea anemones such as Calliactis (reviewed by McFariane, 1982) are neuroid systems of the hydrozoan type, and Shelton (1975) developed a computer model for the SS 1 based on the assumption of electrical coupling in the ectoderm, but none of the workers in this field would 122 G. O. MACKIE ET AL. claim that there is any direct evidence for coupUng or even for the involvement of the epithelia as the slow conduction pathways. The failure to observe gap junctions in these groups could merely mean that the junctions are very small, consisting of isolated connexons, or small groups of them. If this were so, the membranes of adjacent cells should show frequent close contacts. Study of the material does not support such a picture. Alternatively, junctions other than gap junctions {e.g., septate junctions) might provide for electrical communication between cells. Certainly such a possibility cannot be excluded a priori, but the available evidence is most easily explained on the assumptions that gap junctions are absent and that coupling, if it exists, must be very loose. We come then to the hypothesis, which is also a conclusion from existing data, that among the Cnidaria only the hydrozoans have gap junctions. Many questions inevitably arise. Did the common ancestor of the Cnidaria have gap junctions, which survive only in the one class? If so, what led to their elimination in the other classes? If on the other hand the common ancestor lacked gap junctions and they were a hydrozoan invention, does this establish a hydrozoan as ancestor for all higher metazoa? The gap junction is firmly established as a pathway for electrical communication and, in many cases, transmission of impulses, but what of its supposed role in metaboHc communication? Despite their apparent lack of gap junctions the scyphozoans and the anthozoans are no less well organized histologically than the hydrozoans. Pre- sumably, in the absence of direct pathways between cells, tissue communication could still be achieved by interaction of signaling molecules embedded in the membranes of adjacent cells, or by humoral signaling between the cells composing the epithelia (Loewenstein, 1984). Or, finally, tissue regulation could be achieved indirectly by trophic influences from nerves, as in the maintenance of vertebrate skeletal muscle (reviewed by Dennis, 198 1 ). Nutrient transport could be largely extracellular, or could involve amoebocytes. These cells are present in the anthozoa and scyphozoa but are absent in hydrozoans, though in the latter interstitial cells are believed to assume some of the same functions (Chapman, 1974). Any useful hypothesis should suggest experiments by which it can be tested. The obvious need highlighted by the arguments presented here is for verification of the two fundamental propositions, namely that gap junctions are truly absent from the tissues of scyphozoans and anthozoans and that their cells consequently have little if any capabiUty for direct electrical or metaboUc communication. If these propositions prove to be true, we will be in a much better position to explain the electrophysiological dichotomy that exists in the phylum, and to plan experiments which might elucidate the mechanisms of metabolic communication within the Cnidaria. ACKNOWLEDGMENTS Supported by N.S.F. Grant BNS 82-09849 to P.A.V.A. and by N.S.E.R.C. Grant A-1427 toG.O.M. LITERATURE CITED Anderson, P. A. V. 1980. Epithelial conduction: its properties and functions. Prog. Neitrobiol. 15: 161- 203. Anderson, P. A. V., and W. E. Schwab. 1981. The organization and structure of nerve and muscle in the jellyfish Cyanea capillata (Coelenterata:Scyphozoa). J. Morphol. 170: 383-399. Bennett, M. V. L., D. C. Spray, and A. L. Harris. 1981. Gap junctions and development. Trends in Neurosci. 4: 159-163. Chain, B. M., Q. Bone, and P. A- V. Anderson. 1981. Electrophysiology of a myoid epithelium in Chelophyes (Coelenterata: Siphonophora) / Comp. Physiol. 143: 329-328. GAP JUNCTIONS IN CNIDARIA 123 Chapman, D. M. 1974. Cnidarian histology. Pp. 1-92 in Coelenierate Biology. L. Muscatine and H. M. Lenhoff, eds. Academic Press, New York. Dennis, M. J. 1981. Development of the neuromuscular junction. Inductive interactions between cells. Ann. Rev. Neurosci. 4: 43-68. Hand, A. R., and S. Gobel. 1972. 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Propagated spikes and secretion in a coelenterate glandular epithelium. / Gen. Physiol. 68: 313-325. Mackie, G. O. 1984. Introduction to the diploblastic level. Pp. 43-46 in Biology of the Integument. Vol. 1, J. Bereiter-Hahn, A. G. Matoltsy, and K. S. Richards, eds. Springer Verlag. Heidelberg. McFarlane, I. D. 1982. Calliactis parasitica. Pp. 243-265 in Electrical Conduction and Behaviour in 'Simple' Invertebrates. G. A. B. Shelton, ed. Clarendon Press, Oxford. Passano, C. M. 1982. Scyphozoa and Cubozoa. Pp. 149-202 in Electrical Conduction and Behaviour in 'Simple' Invertebrates, G. A. B. Shelton, ed. Clarendon Press, Oxford. Satterlie, R. a. 1979. Central control of swimming in the cubomedusan jellyfish Carybdea ra.stonii. J. Comp. Physiol. 133: 357-367. Satterlie, R. A., and A. N. Spencer. 1979. Swimming control in a cubomedusan jellyfish. Nature 281: 141-142. SCHWARZMANN, G., H. WiEGAND, B. ROSE, A. ZIMMERMAN, D. BEN-HaIM, AND W. R. LOEWENSTEIN. 198 1 . Diameter of the cell-to-cell junctional membrane channels as probed with neutral molecules. Science m-. 551-553. Shelton, G. A. B. 1975. The transmission of impulses in the ectodermal slow conduction system of the sea anemone Calliactis parasitica (Couch). / Exp. Biol. 62: 421-432. SiNGLA, C. L. 1978. Locomotion and neuromuscular system o( Aglantha digitale. Cell Tissue Res. 188: 317-327. Spencer, A. N. 1981. The parameters and properties of a group of electrically coupled neurons in the central nervous system of a hydrozoan jellyfish. / Exp. Biol. 93: 33-50. Spencer, A. N., and R. A. Satterlie. 1980. Electrical and dye coupling in an identified group of neurons in a coelenterate. J. Neurobiol. 11: 13-19. Unwin, p. N. T., and G. Zampighi. 1980. Structure of the junction between communicating cells. Nature 283: 545-549. Werner, B. 1975. Bau and Lebengeschichte des Polypen von Tripedalia cystophora (Cubozoa, class, nov., Carybdeidae) und seine Bedeutung fiir die Evolution der Cnidaria. Helgol. Wiss. Meeresunters. 27:461-504. Reference: Biol Bull. 167: 124-138. (August, 1984) IONIC CONTROL OF SETTLEMENT AND METAMORPHOSIS IN LARVAL MALI OTIS RUFESCENS (GASTROPODA) ANDREA J. BALOUN AND DANIEL E. MORSE Department of Biological Sciences and the Marine Science Institute, University of California, Santa Barbara, California 93106 Abstract An increase in the concentration of K^ in defined sea water medium is dem- onstrated to induce settlement and metamorphosis in larvae of the marine gastropod mollusc, Haliotis rufescens. A decrease in external K^ ion concentration can inhibit the larval response to 7-aminobutyric acid (GABA), a stereochemically specific inducer of metamorphosis of//, rufescens. Stimulation of the metamorphic response by GABA or by increased K^ may depend on transmembrane movement of ions, since induction is sensitive to neuropharmacological blockers of ion conductance. Sulfonyl isothio- cyanostilbene (SITS, an anion exchange blocker) inhibits the larval response to GABA, but does not affect induction by increased external potassium. In contrast, the larval response to potassium is inhibited by tetraethylammonium (TEA, a potassium channel blocker), while induction of metamorphosis by GABA is independent of the presence of TEA. Most manipulations of the concentrations of the other predominant cation components of sea water are not in themselves inductive or inhibitory. However, the actions of GABA and increased K^ as inducers are sensitive to changes in external Ca^^. Potassium may act by directly depolarizing excitable cells involved in the larval perception of inductive stimuli. Activation of metamorphosis by GABA may depend similarly on a depolarizing ion movement at GABA-sensitive cells. Depolarization by manipulation of the ionic environment may offer a general technique for inducing metamorphosis in various marine invertebrate larvae. Introduction Larval metamorphosis, an essential process in the development of most marine molluscs, is a cascade of complex changes initiated in many cases by specific envi- ronmental stimuli (Crisp, 1974; Chia and Rice, 1978). The induction of metamorphosis in larvae of the red abalone, Haliotis rufescens, normally depends on the larval encounter of crustose red algae (Morse et al, 1979; 1980c; Morse and Morse, 1984). This inductive action can be mimicked effectively by micromolar concentrations of 7-aminobutyric acid (GABA). When reared at 15°C the planktonic abalone larvae become competent by seven days post-fertilization to respond to the intact alga, algal homogenate, or to micromolar GABA with rapid metamorphosis (Morse et al., 1979, 1980a, b, c). In the continuous presence of an inducer, the larvae cease swimming and attach by the foot to the substrate; this distinct behavioral transition is followed by the characteristic metamorphic sequence described previously (Morse et al., 1980a). Marine larvae can sense inductive stimuli in the environment, and respond with a coordinated set of behavioral, anatomical, and physiological changes, in a complex process that is likely to involve the larval nervous system (Bonar, 1976; Hadfield, 1978; Burke, 1983a, b). With Haliotis rufescens, the direct electrophysiological analysis Received 9 January 1984; accepted 31 May 1984. 124 IONIC CONTROL OF METAMORPHOSIS 125 of nervous system involvement is handicapped by the small size of the larvae; we have investigated the function of excitable cells instead by manipulation of ion con- centrations and the use of neuropharmacological probes. Evidence presented here demonstrates that the induction of metamorphosis in H. rufescens is directly affected by changes in the external concentration of potassium, a physiologically important ion capable of driving both hyperpolarizing and depolarizing shifts in cell membrane potential. The pattern of dose-dependent mimicry or inhibition of GABA action by K^ is predictable by analogy with the observed influence of K^ on membrane potential in other excitable cell systems. The sensitivity of induction by GABA to changes in external ion concentration, and to specific neuropharmacological probes, suggests that GABA acts similarly as an excitatory agent, producing depolarization of cells capable of activating metamorphosis. Results obtained with neuropharmacological probes suggest that transmembrane movement of specific ions is required for the activation of metamorphosis by increased K^ or by GABA. These results are consistent with the hypothesis that the depolarization of externally accessible excitable cells alone is sufficient to initiate behavioral and developmental metamorphosis. Materials and Methods Larval culture Fertilization was controlled by the mixing of washed gametes, spawned by female and male gravid adult Haliotis rufescens after a brief exposure to dilute hydrogen peroxide (Morse et al, 1977). Clean healthy cultures of the veliger larvae, maintained in flowing 5 /im-filtered ultraviolet-irradiated sea water at 15.0 ± 1.0°C, synchronously developed to a stage of competence to respond to inducers of metamorphosis by seven days post-fertilization (Morse et al, 1980a). Artificial sea water media All experiments were conducted in defined sea water media based on the Woods Hole Marine Biological Laboratory (MBL) recipe (Cavanaugh, 1956). Salt and ion concentrations of this medium are summarized for reference in Table I. Ion con- centrations were manipulated by modification of the MBL formula in two ways: (a) ion excess, in which addition of a salt to MBL sea water increased concentrations of the selected anionic and cationic species without reducing the concentrations of MBL sea water components; and (b) ion replacement, in which a single ion species was partially or completely replaced with a molar equivalent of ionic charge by another species (without compensation for differences in dissociation constants). Artificial sea water media were made with reagent grade salts volumetrically diluted in glass-distilled and microfiltered (Bamstead Nanopure) water. The final pH values of all normal and modified MBL media ranged from 7.8 to 8.1 without adjustment. Just prior to use, media were innoculated with the antibiotics potassium penicillin G and dihy- drostreptomycin sulfate at 150 ppm each, and equilibrated to 15 ± 1°C. Assays of induction All assays were begun with competent veliger larvae (0.2 mm maximum diameter) at 8-10 days post-fertilization. Approximately 200 to 300 larvae were pipetted in a drop of sea water into each 10-ml aliquot of experimental medium, contained in a glass vial (2.4 cm diameter, American Scientific Products). Larvae were incubated in duplicate samples, at 15.0±1.0°C. Induction of plantigrade attachment, assayed as 126 A. J. BALOUN AND D. E. MORSE Table I The salt composition of MBL sea water medium, taken from Cavanaugh (1956) (A), and the calculated maximum free ion concentrations (B) Component Concentration (vaM) A. Salt NaCl 423.0 KCl 9.00 CaCh 9.27 MgClj 22.94 MgS04 25.50 NaHCOj 2.15 B. Ion Na* 425.2 K+ 9.00 Ca^-" 9.27 Mg^"' 48.44 Cr 496.4 S04^- 25.50 the percentage of larvae firmly attached by the foot, provided a quantitative measure of the larval metamorphic response as a function of time. Completion of metamor- phosis was verified by the abcission of the velum (the larval swimming organ) and the initiation of adult shell growth. Modified sea waters found to produce toxic effects were disqualified from further analysis. Moderate toxicity was recognized in non-induced or pre-metamorphic larvae by absence of the normal swimming behavior: many larvae remained withdrawn in their shells; ciliary activity was decreased; the few swimming larvae moved feebly through the lower water column or spun slowly in circles against the bottom. Larvae introduced into highly toxic conditions remained withdrawn; the rapid paralysis of ciliary and muscular activity was followed by death. Neuropharmacological agents tested in conjunction with modified sea water media were added to vials and agitated (Vortex mixer) before temperature equilibration and addition of larvae. 7-Aminobutyric acid (GABA), from Sigma Chemical Company, was used at 4 X 10"^ M, a threshold concentration with which facilitation and inhibition are readily detected. SITS (4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonate) was obtained from ICN Nutritional Biochemicals, and tetraethylammonium chloride (TEA) from Eastman Kodak Company. Mallinckrodt Chemical Works analytical reagent grade salts were used in the construction of artificial sea water media, with the exception of the highly hygroscopic salt MgCb • 6H2O which was purchased as a 4.9 M stock solution from Sigma Chemical Company. Results The concentration- and time-dependent responses of larvae to GABA in MBL sea water (Fig. 1 ) are comparable to the responses of larvae in natural sea water, as defined previously (Morse et ai, 1979; 1980a). Typically, 40-60% of the larvae display an attachment response to 4 X 10"^ M GABA by 40 h. Although this value ranges between extremes of 30-90% for different cultures, larval responses within a healthy culture are consistent; variation between duplicate vials remains small. IONIC CONTROL OF METAMORPHOSIS 127 Figure 1. Larval attachment in response to GABA in MBL sea water. GABA was added at 10"^ A/ (A), 10"* M (9). 4 X 10^ A/ (A), and A/(0). Data are averages of duplicates, with standard deviations indicated by vertical bars. Ion excess effects Increased external potassium effectively induced larval attachment, whether added as sulfate or chloride salts to MBL sea water, or used as a replacement for either Na* or Mg^^ (Fig. 2). In the paired response curves, K^ added with CI" was slightly more efficient as an inducer than when added with S04^ . Similarly, the limiting concen- tration of 20 mM KCl was more rapidly toxic than 10 mM K2SO4 (data not included). Both the inductive and toxic effects of K^ were slightly reduced in medium with sulfate present as the paired anion, instead of chloride. Increases in the external concentrations of other sea water cations, added in excess to MBL sea water as CI" and S04^~ salts, were not inductive (Table II). With the exception of increased Ca^^, the presence of the various excess salts did not inhibit larval attachment in response to 4 X 10"' M GABA, indicating that an increase in osmotic pressure alone neither induces nor inhibits induction of metamorphosis. The inhibitory effect of increased Ca^^ on induction by GABA corroborates the results obtained from media in which Ca^^ concentration was increased by the replacement of Mg^^ (as reported below); these results single out Ca'^^, rather than concomitant alterations in the substitute or paired salt ion concentration, as the cause of the inhibitory effect. We have observed that the induction of metamorphosis by excess K^ is comparable in several respects to that observed with GABA. The efficiency of induction is a dose- dependent function, limited at high concentrations by toxicity. The process of induction involves a temporal component; an optimal concentration of the stimulus (either GABA or increased K^) must be provided continuously for at least 20 h in order for complete metamorphosis to occur. Premature withdrawal or application of subthresh- old levels of the stimulus either fails to induce, or results in only a temporary attachment 128 A. J. BALOUN AND D. E. MORSE 100 80 ^60- c E o r 40 - 20 ] \ IItt - /^^ w / y p5 4-rt 4lli::fe 4 1 A 1 20 40 Time (h) 60 80 Figure 2. Induction of larval attachment by increased external potassium. Potassium was added in excess to MBL sea water as KCl or K2SO4 or was used as a replacement for Mg^^ or Na* in modified MEL sea waters. Excess K* was added to MBL sea water as: 2.0 xnM K2SO4 (A), 4.0 vaM KCl (A), 6.0 mM K2SO4 (O), and 12.0 vxM KCl (•). The effects of increased K"^ concentrations resulting from replacement were tested with media in which: 5.0 mM Mg^^ was replaced with 10.0 mM K^ (D), and 9.0 mM Na"^ was replaced with 9.0 mM K* (■). Data are averages of duplicates, with standard deviations indicated as vertical bars. response that is not followed by completion of metamorphosis. Larvae in media with optimal concentrations of increased K^ or GABA remain active and responsive; premetamorphic larvae swim normally, while attached larvae in the process of meta- morphosis crawl actively on the glass substrate, shed velar lobes, and proceed with growth of new adult shell. Ion replacement effects Most cations tested as potential substitutes for sea water cations had toxic effects on larvae and were not used. These ions, replacing either Na^ or K^ at concentrations of <9 mAf, included Cs^, Li^, choline"^, Tris^ [tris(hydroxymethyl)amino-methane], and TEA (tetraethylammonium). However, partial replacements of cations with other MBL sea water cations were tolerated well by the larvae, and were used in tests representing a matrix of exchanges (Table III). Data from two consecutive experiments, which used larvae from two separate hatches, were compiled by normalizing the responses to those obtained with GABA (4 X 10 '' M) in unaltered MBL sea water. Each of the four cations present in MBL sea water (Na^, K^, Ca'^, Mg^^) was singly replaced by each of the three other species, and the effect of each replacement assayed as a function of time in the presence and absence of 4 X 10"^ MGABA. Response groups presented in Table III show consistent patterns of the effects of the cation exchanges: (Group A) normal induction of metamorphosis by 4 X 10"^ M GABA in unaltered MBL sea water; (Group B) rapid induction of attachment, with or without GABA present, by increased external potassium (except when replacing calcium); (Group C) inhibition of the attachment response to GABA by reduced external po- IONIC CONTROL OF METAMORPHOSIS 129 Table II Larval attachment (other than potassh responses um) in MBL sea water media modified by the addition of excess salts Ion excess Larval attachment (% -GABA , ± S.D.) Salt Cone. (mM) +GABA' None (MBL sea water) 0±0 72 ± 5 NaCl 10 20 40 ±0 1 ±0 1 ± 1 52 ± 10 60 ± 6 65 ± 6 Na2S04 10 20 40 1 ± 1 2 ± 1 8 ± 3 54 ± 1 66 ± 10 64 ± 3 ^CaSO^ 10 20 2 ±0 2± 1 45 ± 21 35 ± 3 ^CaCb 10 20 ±0 1 ± 1 69 ± 11 28 ± 7 MgCl2 10 20 40 o o o 1+ 1+ 1+ o o o 69 ± 13 66 ± 8 40 ± 13 ' Attachment responses are shown at 47 h exposure; GABA was used at 4 X 10"' M. ^ Calcium salts at 40 mM were toxic (as defined in text); larval attachment in these media +GABA was <1%. tassium (except when replaced by magnesium); (Group D) induction, without GABA present, by medium in which sodium was used to replace magnesium, and conversely, inhibition of GABA-induced attachment by medium in which magnesium was replaced by sodium; (Group E) inhibition of the attachment response to GABA by increased external calcium; (Group F) absence of inductive, facilitative, or inhibitory effects of media, without GABA present, in which external calcium concentration was reduced. The absence of inductive ability of increased external K^ when replacing Ca^^ is unique to that exchange condition. In contrast, decreased potassium (Table III, Group C) was not inductive in any exchange condition. When K^ was partially replaced by 5.0 mM Na^ or 2.5 mM Ca^^, inhibition of GABA action was observed, although increased Ca^^ itself also produced inhibition (Group E). The result with substitution by Na^, however, indicates that decreased external K^ can inhibit the larval response to GABA. The only cation replacement capable of inducing attachment of larvae without GABA present, other than replacements resulting in an increase of external potassium, was that in which external Mg^^ was replaced with Na^. The substitution of 23.0 mM Mg^^ with 46.0 mM Na^ (which permitted the concentration of the paired anion to remain unchanged) was inductive at a level comparable to that of 4 X 10^^ M GABA (Table III, Group C). A comparison with the other substitution conditions in Table III in which Mg^^ was decreased, or in which Na^ was increased, indicates that neither ion shift alone can be credited with the inductive action. Apparently, it is the specific replacement of Mg^^ with Na^ that effects larval attachment. The reverse replacement of 46.0 mM Na^ with 23.0 mM Mg^^ strongly inhibited induction by 4 X 10^ M GABA. Again, a comparison of the results obtained with other media in which external Na^ was decreased, or Mg^^ was increased, shows that neither cation change alone is consistently inhibitory. 130 A. J. BALOUN AND D. E. MORSE Table III Larval attachment responses in MBL sea water media modified by cation replacement Replacement media Larval response' -GABA +GABA Cation Cone. Cation Cone. Relative attachment Relative attachment Group replaced (mM) substituted (mM) (% ± S.D.)^ Effect' (% ± S.D.)^ Effect' A (MBL sea water control; no replacement) 0±0.0 N 100 ± 8.4 N B Na^ 9 K* 9 143 ± 5.6 I 148 ± 2.8 F Na* 18 K* 18 152 ± 7.0 I 143 ± 7.0 F Mg^^ 5 K* 10 159 ±4.2 I 158 ± 1.4 F Ca^^ 5 K^ 10 0±0.0 N 101 ± 1.4 N C K* 5 Na* 5 0±0.0 N 61 ± 13 X K-' 5 Mg^^ 2.5 0±0.0 N 92 ± 21 N K* 5 Ca^* 2.5 0±0.0 N 59 ± 4.2 X D Mg^^ 23 Na* 46 97 ± 9.8 I 117 ± 1.4 N Na* 46 Mg^* 23 0±0.0 N 10 ± 2.8 X E Mg^* 23 Ca^^ 23 4 ± 1.4 N 62 ± 2.8 X Na-" 23 Ca^* 11.5 0±0.0 N 46 ± 7.0 X "K* 5 Ca^* 2.5 ±0.0 N 59 ± 4.2 X F Ca^^ 5 Na* 10 ±0.0 N 92 ± 11 N Ca^* 5 Mg^^ 5 0±0.0 N 89 ± 1.4 N ^Ca^* 5 K* 10 0±0.0 N 101 ± 1.4 N ' Attachment responses are shown at 45 h exposure; GABA was used at 4 X 10"' M. ^ Normalized to attachment observed in MBL sea water + GABA, as explained in Results; the absolute value of attachment in MBL seawater + GABA was 47%. S.D. is the absolute standard deviation. ' Effects: (N) no facilitating or inhibitory effect compared with response in unmodified MBL sea water; (1) induction without GABA; (F) facilitation of induction by GABA; (X) inhibition of induction by GABA. * Media listed twice for comparison in separate groups. Table IV The effects of altered concentrations of external Co'* on larval attachment responses to GABA and to increased K* icentrations 1 Larval attachment (% ± S.D.; 1 Altered ion cor -GABA +GABA^ K* Na-' Ca^^ Mg^* 22 h 49 h 72 h 22 h 49 h 72 h None (MBL sea water) ± 1 ± 1 ±0 60+13 80 ± 21 89 ± 4 +4 -4 0± ±0 ±0 36 ± 2 88 ± 6 94 ± +9 -9 0± 1 ± 1 ± 11 ± 6 41+3 52 ± 13 +12 -12 62 ± 12 57 ± 3 74 ± 1 79 ± 3 90 ± 3 94 ± 1 +12 -12 +4 -4 63 ± 3 80 ± 4 93 ± 3 75 ± 95 ± 1 96 ± 3 +12 -12 +9 -9 57 ± 12 81 ± 1 96 ± 3 64 ± 16 83 ± 9 95 ± 4 -4 +4 0± 1 ± 1 ±0 41 ± 17 69 ± 20 95 ± 2 -9 +9 0± 0±0 ±0 0± 1 ± 0± + 12 -12 -4 +4 5 ± 2 4± 3 2±2 72 ± 2 82 ± 87 ± 2 +12 -12 -9 +9 2 ± 1 ± ±0 9 ± 1 43 ± 1 3 44 ± 10 ' Changes in cation concentration .(mA/) with reference to standard MBL sea water. ^ GABA was used at 4 X 10"' M. IONIC CONTROL OF METAMORPHOSIS 131 The actions of GABA and increased external K^ as inducers of metamorphosis both were inhibited by changes in external Ca^^, although the directions of the net change in Ca^^ to which they were sensitive were opposite (Table IV). Without an inducer present, the changes in external Ca^^ (imposed in combination with reciprocal equimolar changes in external Mg-^^) had no effect on exposed larvae. Larval attachment in response to 4 X 10"^ A/ GABA was inhibited by a 9.0 mM increase in Ca^^. Larval responses to increased external K^ (introduced as an equimolar replacement for Na^, without GABA present) were not affected by increased external Ca^\ indicating that inhibition of the response to GABA was not caused by toxicity. In contrast, the larval response to GABA in medium with Ca^^ decreased by 4.0 mM remained comparable to that in MBL sea water with GABA. However, the larval response to increased K^ was strongly inhibited by the 4.0 mM reduction in Ca^^. Despite this strong inhibition, the normal response of larvae to GABA (present in addition to the increased K^ and decreased Ca^^) was retained, again negating the possibility that toxicity was the cause of inhibition. Virtually complete replacement of Ca^^ (—9.0 mA/) inhibited attachment in all conditions, suggesting that this extreme reduction in external Ca^^ was detrimental to the larvae. Neuropharmacological analyses Neuropharmacological probes were used to analyze the effects of external ion changes in the initiation of metamorphosis. Induction by GABA is sensitive specifically to the presence of SITS, an isothiocyanate derivative known to inhibit anion exchange (Cabantchik and Rothstein, 1972). Addition of 1 X 10"^ M SITS to MBL sea water inhibited larval attachment in response to GABA, without altering larval behavior in the absence of GABA (Table V). In contrast, the induction of metamorphosis by increased potassium was not affected by SITS. The presence of SITS did not sub- stantially reduce the increase in larval attachment contributed by GABA, when present in addition to 12 mM excess K^. The effectiveness of SITS as an inhibitor of GABA action depends on its con- centration relative to that of GABA (Fig. 3). SITS at lO""* M fully blocked the inductive effect of 10""* M GABA. A concentration of SITS lower by one order of magnitude (10"^ M) did not block induction by 10""* MGABA but did affect the rate of attachment induced by lower concentrations of GABA. SITS at 10"^ M was relatively ineffective, Table V The effects of a K* -channel blocker (TEA) and an anion exchange blocker (SITS) on larval attachment responses to increased K^ and GABA GABA Larval attachment (% ± S.D.)- > KCl Excess' Alone +TEA +SITS 12 mM 4 X 10"' M 4 X 10-' M 0± 43 ± 10 54 ± 10 82 ± 6 0± 47 ± 18 24 ± 11 43 ± 3 0±0 16 ± 1 57 ±7 75 ±0 ' MBL sea water media prepared as described in text. ^ Absolute percentage of larvae attached after 24 h exposure; S.D. is standard deviation. Concentrations of additions: TEA (5 X 10"' M); SITS (1 X 10"' M). 132 A. J. BALOUN AND D. E. MORSE 100 c 0) E o TO 4x10"'^ 10-6 ;gaba] (M) Figure 3. Inhibition by SITS, as a function of the relative concentrations of SITS and GABA. GABA concentrations are indicated on the horizontal axis. Larval attachment at 28 h in response to GABA is shown for media in which SITS is present at concentrations of: 10"'' M (A), 10"^ M (A), 10"* M (•), and M (O). Data are averages of duplicates, with standard deviations indicated as vertical bars. except when present with the threshold concentration of 4 X 10"^ MGABA. SITS at concentrations lower than those of GABA seemed to have little inhibitory influence. At the erythrocyte membrane, the covalent binding of isothiocyanate groups to the anion transporter protein occurs at specific amino groups (Passow et ai, 1982). The possibility that SITS might inhibit the larval response to GABA by binding the 7-amino group of GABA and thus decreasing the effective concentration, rather than by acting at larval membrane sites, was tested using glycine, a non-inductive and non-facilitating structural analog. Glycine, added with SITS for a one hour prein- cubation prior to the addition of GABA and competent larvae, remained continuously present during the subsequent assays of induction. No competitive protection of induction by GABA from inhibition by SITS was evident in the presence of glycine; SITS fully retained its ability to inhibit GABA action (Fig. 4). Glycine alone had no effect on induction by GABA. The possibility that SITS might act to bind GABA, but not glycine, because of steric hindrance of the amino group in the shorter molecule, was tested by repeating the protocol with e-aminocaproic acid (a longer homolog of GABA) instead of glycine; the identical result further shows that SITS does not act by binding nonspecifically to the amino groups of amino acids in solution. The inhibitory action of SITS appears to be relatively specific. Other potential blockers of ion conductance that were found to have no effect on the normal larval response to GABA include: (a) tetrodotoxin, a blocker of voltage-regulated sodium channels in axonal membranes (review by Armstrong, 1974); (b) picrotoxin, a blocker of GABA-regulated increases in CI" permeability in some systems (Takeuchi, 1976; Gallagher ct ai, 1978; Yarowsky and Carpenter, 1978); and (c) furosemide, an inhibitor of mediated cotransport (Geek et ai, 1980). The action of potassium in the induction of metamorphosis was analyzed using IONIC CONTROL OF METAMORPHOSIS 133 100 - c a> E o TO Figure 4. The inhibitory action of SITS on the larval response to GABA, with or without preincubation of SITS with glycine. SITS and/or glycine, where indicated, were added 1 h before initiation of the experimental assay by addition of GABA, where indicated, and subsequent introduction of competent larvae. Larval responses are shown for MBL sea water with: no addition (O); glycine (•); GABA (A); GABA and glycine (A); SITS (0); SITS and GABA (D); SITS and GABA with glycine (■). Concentrations were: GABA 4 X 10 ' M; glycine, 10"' M; and SITS, 10"' M. Data are averages of duplicates, with standard deviations indicated as vertical bars. tetraethylammonium chloride (TEA), an impermeant blocker of K^ channels in nerve and muscle cells (review by Armstrong, 1974); both intracellular and extracellular applications of TEA block the Ca^^-activated K^ current in molluscan neurons (Her- mann and Gorman, 1981). At concentrations less than lO'* M, TEA specifically inhibits induction of H. rufescens by increased K^; higher concentrations of TEA are toxic, and cause non-specific inhibition of larval responses to all inducers. Concen- trations of TEA less than 1 0"^ M have no apparent inhibitory effect. The presence of 5 X 10^ MTEA reduced the inductive action of 12 mM excess KCl and negated the additive effect of increased K^ when present in combination with 4 X 10^^ M GABA, reducing the attachment to a level equivalent to that of GABA in MBL sea water alone (Table V). Induction by GABA in MBL sea water was unaffected by the presence of TEA at 5 X 10"^, indicating that inhibition by TEA does not result from toxicity. Function of the TEA-sensitive sites thus is required for the induction of metamorphosis by increased K^, but apparently is not essential for the pathway activated by GABA. Discussion The complete process of metamorphosis is induced in Haliotis rufescens larvae by an increase in the concentration of K^ in sea water. Changes in external K"^ concentration can drive electrogenic movements of K^ that directly affect the mem- brane potential; the depolarization of membrane potential as a function of increasing extracellular K^ has been used to demonstrate that the excitable membrane can behave in a classical sense as a K^ electrode (Hodgkin and Horowicz, 1959). The 134 A. J. BALOUN AND D. E. MORSE inductive action of increased K^ suggests that metamorphosis in H. rufescens can be initiated solely by the depolarization of externally accessible excitable cells. Depolarizing electrical stimuli, delivered by suction electrode to the region of the oral ganglion or apical neuropile, have been shown by Burke (1983a) to ehcit immediate metamorphosis in competent larvae of the Pacific sand dollar Dendraster excentricus. This site-specific efficacy suggests that the metamorphic response to an appropriate environmental stimulus is activated in this species by the neural communication of sensory receptors with the larval nervous system. The induction of metamorphosis of H. rufescens by GAB A may depend similarly on the depolarization of GABA-sensitive cells. The initial larval response to GABA in the presence of increased external K^ is greater than that observed with either GABA or increased K^ alone (Table V). In contrast to this combined effect of increased K^ with GABA, a decrease in external K^ can inhibit induction by GABA. Hyper- polarization resulting from the decrease in external potassium, as demonstrated for GABA-regulated postsynaptic cells (Motokizawa et al, 1969), could antagonize a GABA-mediated depolarization. It is unlikely that GABA acts directly by altering membrane permeability to K^ at the same sites utilized during induction by increased K^, since the actions of these inducers are pharmacologically separable. Induction by GABA is sensitive to SITS, and insensitive to TEA; induction by increased K^ is inhibited by TEA but not by SITS. These reciprocal sensitivities also indicate that the inducers operate through pathways that initially are separate; that is, neither follows the other in an obligatory sequence in the process of induction. The separateness of the inductive actions of GABA and increased K+ also is evident in their entirely different sensitivities to alterations in external Ca^^. Induction by GABA is inhibited specifically by increased Ca^^, while induction by increased K"^ is sensitive only to a reduced external concentration of Ca^^. A simple model can be proposed, analogous to other systems, which invokes a single mechanism to explain the opposite sensitivities of these inducers. Increased cytoplasmic concentra- tions of Ca^^ have been shown to activate K^ conductance through Ca^^-regulated K^ channels in diverse cell types (review by Schwarz and Passow, 1983). At physi- ological concentrations of internal and external K^, a Ca^^-activated increase in K^ conductance permits a net K^ efflux that can hyperpolarize a sensory receptor cell, thus decreasing the rate of afferent discharge (review by Edwards, 1984). If we postulate the existence, in larval H. rufescens, of Ca-^-regulated K"" channels in cells that are capable of responding to GABA and to K"", then the effects of Ca^"^ can be explained by a comparable mechanism. In medium with a standard sea water concentration of K^, an increase in calcium (suggested to produce a parallel increase in cytoplasmic calcium) may inhibit the effect of GABA by activating a hyperpolarizing net K+ efflux. With an inductive increase in external K^, however, membrane depolarization rather than hyperpolarization would be expected in response to increased Ca"^; this prediction is supported by the observed absence of an inhibitory effect of increased Ca^^ on induction by K^. In contrast, a decrease in external Ca^^ (suggested to produce a decrease in cytoplasmic Ca^^) may block induction by K^ by antagonizing the necessary electrogenic influx of the cation through Ca'^-regulated membrane channels. The reduced efficiency of K^ as an inducer, when added with sulfate rather than chloride to MBL sea water, may result from a decrease in the sea water con- centration of free Ca'^, since CaS04 has a higher association constant than CaCb- The induction of metamorphosis by GABA is not sensitive to decreased Ca"^, suggesting that its action is not impaired by an increased membrane resistance to K^; this idea is supported by our demonstration that induction by GABA is insensitive to the presence of the K^-channel blocker, TEA. Although a heterogeneous population of larval cells is exposed during the test of an altered sea water medium, the resulting IONIC CONTROL OF METAMORPHOSIS 135 effects on larval metamorphosis are consistent with this model based on the exclusive function of a single group of accessible excitable cells. The selective movement of ions across specialized membranes is a fundamental mechanism in the function of excitable cells. At invertebrate chemoreceptors, a stim- ulus-dependent increase in ion permeability can transduce chemical stimuli into electrical impulses, allowing nervous system analysis of environmental information (Morita, 1972; Thurm and Wessel, 1979; Kaissling and Thorson, 1980). Postsynaptic cells mediate the effect of a chemical neurotransmitter similarly by altering membrane permeability to ions capable of influencing the membrane potential (Takeuchi and Takeuchi, 1960). GABA, as an inhibitory neurotransmitter in both vertebrate and invertebrate systems, acts at postsynaptic sites to increase membrane permeability to chloride (Krnjevic and Schwartz, 1967; Takeuchi et ai, 1978; review by Takeuchi, 1976). While the inhibitory effect of GABA commonly depends on a hyperpolarizing C\ influx, GABA also has been shown to activate a depolarizing efflux of CI in the presynaptic inhibition of vertebrate spinal ganglia (Nishi et ai, 1974; Gallagher et al, 1978). GABA can hyperpolarize or depolarize different cells within the same ganglion in invertebrates such as Helix (Walker et ai, 1975) and Cancer (Marder and Paupardin-Tritsch, 1978). The site of action of exogenous GABA as an inducer of metamorphosis of //. rufescens larvae remains unknown. If acting through the larval nervous system, GABA is likely to function either as a Hgand mimicking the active component of the inductive algae at larval chemoreceptors, or as a neurotransmitter at synapses between neurons regulating the initiation of metamorphosis. We have shown that the larval response to GABA depends on the function of a SITS-sensitive process. This requirement appears to be specific, since induction by increased K^ is not inhibited by SITS, and potential blockers of other ion conductances fail to inhibit the larval response to GABA. It is possible that the larval response to GABA may be directly dependent on a GABA-controlled alteration of SITS-sensitive anion exchange. Although anion exchange processes generally are considered to be electrically neutral, GABA could generate the depolarizing net efflux of an anion such as C\ by promoting "slippage," an exchanger-mediated process in which the unidirectional transport of an anion occurs without an associated anion countertransport (Knauf ^/ al, 1977; Frohlich et al, 1983). Alternatively, the SITS-sensitive system may not be directly controlled by GABA, but may be capable of influencing a state or process on which GABA action does depend. Other data support the suggestion that there may be a functional relationship between GABA as an inducer of metamorphosis, and the transmembrane movement of anions. We have found that the induction of metamorphosis of//, rufescens larvae by GABA is sensitive to changes in CI" concentration in artificial sea water. Fur- thermore, without GABA present, the replacement of 25-75% of CI in sea water with substitute anions (Br", S04^", NO3", acetate, isethionate, or propionate) induces attachment of competent larvae; this inductive action is inhibited by SITS, but not by TEA. The macrocyclic lactone ivermectin, a compound demonstrated to increase Cr conductance at a GABA-regulated synapse in lobster (Fritz et al, 1979), is inductive alone and facilitates induction by media in which Cr is replaced with a substitute anion. An increase in external CI", added in excess with Mg^^, blocks induction by GABA. These results, suggesting that CI efflux may play a role in transduction of the GABA signal, will be presented in more detail elsewhere (Baloun and Morse, in prep.). The data presented here support the idea that GABA and increased K^ work similarly by causing depolarization, but require the function of different ion-conductive processes in the induction of metamorphosis. Additional work will be required to 136 A. J. BALOUN AND D. E. MORSE determine how the specific exchanges of Mg^"^ and Na^ either induce attachment of larvae or block the response to GABA. The induction of larval attachment by medium in which Mg^^ is replaced with Na^ is insensitive both to SITS and TEA, suggesting a mechanism of action separate from those of GABA and increased K^. The culture of marine invertebrates, for research in ontogeny and neurobiology, and for production of food and other resources, would benefit from the development of a general technique for initiating larval metamorphosis. The nature of the specific inducing signals naturally required for metamorphosis is likely to vary among species recruited to different specialized microenvironments. In contrast, the transduction of chemical or other stimuli by receptor cell depolarization may be a more general mechanism in initiating the metamorphic response. The depolarizing effect of small increases in external K^ thus may provide a simple and economical method for the induction of metamorphosis in a variety of marine invertebrates. This idea is supported by our recent finding that larvae of the gastropod mollusc Astraea undosa, for which the natural inducer is not yet identified, efficiently are induced to settle and meta- morphose in a dose-dependent response to increased external potassium (Markell, Baloun, and Morse, unpubl. obs.). The optimal concentration of excess potassium required for the metamorphic response ot A. undosa is close to that described here for Haliotis. While the specific physical and chemical characteristics of substrates influencing settlement of marine invertebrate larvae have been extensively reviewed (Crisp, 1974; Scheltema, 1974; Hadfield, 1978), few studies are available on the role of neuro- physiologically important ions in larval induction. Early work by Lynch (1947) sug- gested that influx of Na^ during brief exposure to greater than normal concentrations of sodium salts could accelerate metamorphosis oiBugula larvae. Spindler and Miifler (1972) demonstrated an inductive response to LiCl in planula larvae of Hydractinia echinata. Subsequent work by Miiller and Buchal (1973) defined a range of inductive responses to Cs+, Rb^, Li^, and K+. Succinyl choline chloride was shown to induce metamorphosis in Phestilla larvae (Bonar, 1976); the active component choline is inductive alone, although less efficient than the natural inducer of metamorphosis (Hadfield, 1978). In these and related studies, the mechanisms of action of the inductive ion changes have remained hypothetical (Muller and Buchal, 1973), or were considered to be unrelated to the normal physiological mechanism (Crisp, 1974, 1984; Hadfield, 1978, 1984). Our resuhs with larvae of Haliotis rufescens suggest that these results obtained in other systems, once considered to be artifactual, may in retrospect be recognized as clues to the integral role of ions in transducing the environmental stimuh required for metamorphosis. Acknowledgments We wish to express our appreciation to Dr. Eugene Roberts and Dr. Philip Laris for their interest and helpful discussions. This research was supported in part by grants to D.E.M. from the NOAA National Sea Grant College Program, Department of Commerce (grant #NA80AA-D-00120, project #R/A-43) through the California Sea Grant College Program; the California State Resources Agency (project #R/A- 43); the U. S. Navy Office of Naval Research (contract #N00014-80-C-03 10); Chevron USA, Inc. and the Atlantic Richfield Foundation (grant #AGR-83 1 519); and to A.J.B. from the International Women's Fishing Association, the Association for Women in Science, the McNaughton Scholarship in Oceanography sponsored by the Women's Home and Garden Association, and the American College Scholarship Program. This assistance is most gratefully acknowledged. IONIC CONTROL OF METAMORPHOSIS 137 LITERATURE CITED Armstrong, C. M. 1974. Ionic pores, gates, and gating currents. Q. Rev. Biophys. 7: 179-210. BoNAR, D. B. 1976. Molluscan metamorphosis: A study in tissue transformation. Am. Zooi 16: 573-591. Burke, R. D. 1983a. Neural control of metamorphosis in Dendraster excentricus. Biol. Bull. 164: 176- 188. Burke, R. D. 1983b. The induction of marine invertebrate larvae: stimulus and response. Can. J. Zool. 61: 1701-1719. Cabantchik, Z. I., AND A. ROTHSTEIN. 1972. 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(August. 1984) BIOLOGY OF HYDRACTINIID HYDROIDS. 2. HISTOCOMPATIBILITY EFFECTOR SYSTEM/COMPETITIVE MECHANISM MEDIATED BY NEMATOCYST DISCHARGE LEO W. BUSS, CATHERINE S. MCFADDEN', AND DOUGLAS R. KEENE^ Department of Biology, Yale University, New Haven. Connecticut 06511 Abstract Intraspecific encounters between colonies of the athecate, colonial hydroid Hydractinia echinata result in contact between mat or stolonal tissues. We have monitored colony ontogeny in five clones of H. echinata and initiated experimental encounters between the two tissue types in both isogeneic and allogeneic combinations. All isogeneic interactions result in fusion, all allogeneic interactions in rejection. Transmission electron microscopy shows that fusion results in the establishment of a common gastrovascular system, whereas rejection is characterized by an electron- dense, fibrous layer separating the two colonies. Rejection involves either the passive cessation of growth along the contact zone or the development of hypertrophied stolons. These hyperplastic stolons destroy foreign tissues and can develop only from existing stolons. Scanning and transmission electron microscopy demonstrates that stolons become hyperplastic through the differentiation of interstitial cells into ne- matocytes and that the destruction of foreign tissue is effected by nematocyst discharge. Experimental elimination of interstitial cells removes the capacity of a colony to produce hyperplastic stolons, but does not affect historecognition. A comparison between these results and similar studies in anthozoans suggests the need to distinguish between the evolution of historecognition and the evolution of mechanisms of in- terference competition. Introduction Cnidarians have evolved a striking array of behavioral repertoires and morpho- logical structures to defend their living space and expand into the space occupied by others. Scleractinian corals contacting other scleractinians extrude mesenterial filaments and actively digest their neighbors (Lang, 1971, 1973; Glynn, 1976; Sheppard, 1979). Scleractinians may also differentiate sweeper tentacles along zones of contact. These modified tentacles are armed with a specialized nematocyst population (den Hartog, 1977; Wellington, 1980) and inflict damage on neighboring colonies (Richardson et al, 1979; Sheppard, 1979; Wellington, 1980; Chomesky, 1983). Certain acontiarian sea anemones display an analogous phenomenon. Following tentacle contact between adjacent anemones, one or both individuals will differentiate catch (or 'killer') tentacles. Like sweeper tentacles, these are elongate, are heavily armed with a specialized ne- matocyst population (Calgren, 1929; Hand, 1955; Williams, 1975; Purcell, 1977; Watson and Mariscal, 1983), and are used to injure neighbors (Williams, 1975, 1980; Purcell, 1977; Purcell and Kitting, 1982; Watson and Mariscal, 1983). Certain en- domyarian sea anemones possess acrorhagia. These structures can inflate and, upon Received 29 March 1984; accepted 24 May 1984. ' Present address: Department of Zoology, University of Washington, Seattle, Washington 98195. ^Present address: Portland Shrine Research Unit, Shriners Hospital for Crippled Children, 3101 S. W. Sam Jackson Park Road, Portland, Oregon 97201. 139 140 BUSS ET AL. contact with the adversary, discharge nematocysts (Abel, 1954; Bonnin, 1964; Frances, 1973b; Bigger, 1976, 1980; Williams, 1978; Ottaway, 1978; Brace and Pavey, 1978; Brace, et al, 1979; Brace, 1981). The evolution of this diverse array of structures is necessarily predicated on the existence of some underlying system of historecognition. The ability to distinguish between isogeneic, allogeneic, and xenogeneic tissues has been demonstrated in certain scleractinians (Lang, 1971, 1973; Hildeman ^/ a/., 1975, 1977a, b, 1980), actiniarians (Frances, 1973a, b, 1976; Purcell, 1977; Bigger, 1980; Brace, 1981), gorgonians(Theo- dor, 1970, 1976;TheodorandSenelar, 1975; Bigger and Runyan, 1979), and hydroids (Teisser, 1929; Schijfsma, 1939; Crowell, 1950; Hauenschild, 1954, 1956; Muller, 1964, 1967; Toth, 1967; Ivker, 1972; Gallien and Gouere, 1974; Tardent and Buhrer, 1982; Muller et al., 1983). It is widely assumed that histocompatibility and deployment of the various effector systems are genetically based alternatives. This assumption is supported by the common observation that aggressive devices are deployed against allogeneic tissue, but not in response to isogeneic tissue (Schijfsma, 1939; Muller, 1964, 1967; Lang, 1971, 1973; Ivker, 1972; Francis, 1973a, b, 1976; Theodor, 1976; Purcell, 1977; Bigger, 1980; Brace, 1981; Tardent and Buhrer, 1982). Genetic data, however, are available for only one cnidarian, the hydractiniid hydroid Hydractinia echinata (Hauenschild, 1954, 1956; Ivker, 1972). Unlike anthozoans, for which there exist substantial data on the manner in which destruction of foreign tissue is effected, there is little comparable information for hydrozoans. Although instances of interspecific and intraspecific competition are known in several hydroid species {e.g., Kato et al., 1962, 1963, 1967; Chiba and Kato, 1966; Muller et al., 1983), structures specialized for competition have been described only for members of the family Hydractinidae. In H. echinata, fusion was first noted by Teisser (1929) between planulae derived from the same cross. Ten years later, Schijfsma (1939) noted that fusion was not the only outcome of intraspecific encounters, noting that "it looks as if the growing borders of two colonies, in striking together and checking each others progress, are stimulated by very active growth and ramifications; resulting in the formation of a dense fringe of intertwined stolons." Subsequent studies by Crowell (1950), Hauenschild (1954, 1956), and Toth (1967) discussed the lack of compatibility between colonies but did not record the behavior of tissues in contact. Muller (1964), however, reported the presence of regions of "wild" stolonal growth in contact with incompatible tissues, observing that such growth may be initiated by both of the colonies in contact. He further observed that these modified stolons were associated with the regression and subsequent demise of one of the interacting colonies and suggested that this regression is due to a toxin released by the modified stolons. Ivker (1972) expanded on Muller's observations, introducing the term "hyperplastic stolon" to describe the modification of normal stolonal growth upon contact with foreign tissue. She likewise found that hyperplastic stolons destroy foreign tissue and hypothesizes that this destruction is the result of an enzymatic secretion from hyperplastic tissue. Subsequent studies of another hy- dractiniid, Podocoryne carnea, have documented a similar hyperplastic response to allogeneic (Tardent and Buhrer, 1982) and xenogeneic tissues (Gallien and Gouere, 1974). In attempt to elucidate the mechanism by which hydractiniid hydroids effect the destruction of foreign tissues, we initiated a study of the fusion-rejection interaction in H. echinata. We find (a) that mat and stolonal tissues differ in their capacity to mount a hyperplastic response, (b) that production of hyperplastic tissue is dependent on differentiation of interstitial cells, and (c) that hyperplastic tissues effect their destruction of foreign tissue by nematocyst discharge. Comparison of anthozoan and HYDROID COMPETITION AND HISTOCOMPATIBILITY 141 hydrozoan responses to foreign tissues suggests the need to distinguish between the selective forces responsible for the evolution of mechanisms of interference competition and those responsible for the evolution of historecognition. Materials and Methods Animal collection, maintenance and propagation We report on a series of laboratory investigations on the phenomenology, ultra- structure, and mechanism of the histocompatibility response in Hydractinia echinata. Methods for each topic considered here are described in separate sections below. Common to all studies, however, are the source of experimental animals and our methods of cultivation and asexual propagation. Hydractinia echinata grows as an encrustation on the surface of gastropod shells occupied by pagurid hermit crabs (Fig. 1). The colonies of//, echinata used in this study were collected on a shallow subtidal (<5 m) gravel-mud bottom at Harrison Point, Long Island Sound, from shells occupied by Pagurns longicarpus. Colonies collected from these shells are assumed to be isogeneic. This assumption is justified because asexual propagation from one shell to another is unknown and several different attempts to detect naturally occurring chimeras have failed (McFadden et ai, 1984). Field-collected colonies were propagated by removing with a scalpel an explant of basal mat containing 1-3 feeding polyps from a shell and gently holding it to a plexiglass slide with a loop of suture thread. After 1-3 days explants attached and threads were removed. Stock colonies established in this manner were maintained in laboratory culture for a period of 2-14 months prior to this study. Colonies were maintained in a recirculating sea water system at room temperature and were fed with one-day-old brine shrimp nauplii for two hours daily. Explants from isogeneic stock colonies were attached to various experimental substrata (detailed below) for observations of colony ontogeny and histocompatibility interactions. Techniques of explantation and laboratory cultivation have been described in further detail elsewhere (Ivker, 1972; McFadden et al, 1984). Colony ontogeny, potential tissue interactions, and histocompatibility Colonies of H. echinata vary considerably in gross morphology during early on- togeny (Schijfsma, 1939; Hauenschild, 1954; Ivker, 1972; McFadden et ai, 1984). The relative rates of production of mat, stolon, and polyps throughout ontogeny differ among colonies, producing a characteristic pattern in gross morphology for a given colony. Mat tissue is composed of a close network of entodermal gastro vascular canals surrounded by interstitial cells and covered by a uniform layer of ectoderm. Stolons are individual periderm-covered canals, composed of a layer of endoderm and a layer of ectoderm, which branch and anastomose to form a highly complex network criss- crossing the substratum. Feeding polyps arise from the mat (Fig. 1), and in some genotypes, from the stolons. Depending on the morphology of colonies and/or the time in ontogeny at which contact is made, there are three possible classes of interactions between isogeneic or allogeneic colonies: (1) mat contacting mat, (2) mat contacting stolon, and (3) stolon contacting stolon. To insure observation of all possible tissue interactions, five genotypes of //. echinata were chosen. The ontogeny of each colony was quantified by observing the number of polyps, the area of mat, and the area of enclosed stolon through time by the methods of McFadden et al. (1984). No replicates were made of these observations, as explants from a given clone produce nearly identical patterns of colony ontogeny 142 BUSS ET AL. Figure 1 . Life cycle of Hydractinia echinata. Fertilized egg develops into crawling planuloid larvae (A) which attaches to a substratum and metamorphoses into a primary polyp (B). By asexual iteration, this polyp develops into a mature colony (C, D) which will produce either male or female reproductive polyps (E). (from McFadden et ai, 1984). (Buss and Grosberg, unpub.). Knowledge of the ontogenetic patterns allowed pairing of colonies at points in ontogeny such that all possible tissue interactions were observed in both isogeneic and allogeneic combinations. Each pairwise combination was rep- licated at least five times. Observations were made on the sequence of events following contact between colonies at SOX using a dissecting microscope. Ultrastructure of the fusion-rejection interaction Three categories of response to contact between colonies were noted using light microscopy: fusion, rejection with hyperplastic stolon formation, and rejection without hyperplastic stolon formation. The development of each of these three outcomes was examined using transmission electron microscopy. Explants of the appropriate colonies were attached to Lux petri dishes and fixed at various times after the initial contact between colonies. Colonies were fixed in modified Kamovsky's fixative (Kamovsky, HYDROID COMPETITION AND HISTOCOMPATIBILITY 143 1965) containing 2% paraformaldehyde, 2.5% gluteraldehyde, 1.5 M CaCh in 0.1 M final concentration sodium cacodylate buffer, pH 7.4, for two hours on ice, rinsed in buffer, then postfixed in 1% OSO4 on 0. 1 M sodium cacodylate buffer for one hour on ice. Colonies were then rinsed in buffer, dehydrated through a graded series of ethanol dilutions, treated with propylene oxide, infiltrated, and flat-embedded in their original Lux permanox petri dishes in Polybed 8 1 2 polymerized at 60°C overnight. Colonies were separated from the dishes, cut out with a jewelers saw, and either ( 1 ) mounted onto a blank for face-on sectioning across histocompatibility interactions or (2) clamped directly into a LKB Huxley ultramicrotome for cross-sectioning. Areas of isogeneic and allogeneic tissue interactions were located via light microscopy by examining 1 ^ thick sections stained in 0.25% Azure I and 0.25% Azure II in 0.25% Sodium Borate. Once located, ultrathin sections from silver to light gold interference color were cut with a diamond knife and mounted on formvar coated 1 X 2 mm slot grids, allowing direct correlation of both the thick section via light microscopy and the entire thin section via transmission electron microscopy. Following staining in 2% Uranyl acetate in 50% Ethanol for 15 minutes and Reynold's lead citrate for 60 seconds, sections were examined and photographed using either a Philips E.M. 200 or Philips E.M. 300 operated at 60 kV. The development of hyperplastic stolons was also observed in scanning electron microscopy, to help correlate transmission microscopy results with observations made with the dissecting microscope. Colonies were grown on glass cover slips and fixed by the same protocol as those prepared for transmission electron microscopy. Following dehydration through a graded series of ethanol, samples were taken through critical point in liquid CO2 in a Sorvall critical point drying apparatus, and sputter coated with 60% Au, 40% Pd. Samples on coverslips were examined and photographed using an ETEC autoscan scanning electron microscope operated at 5-10 kV. Interstitial cells and the development of hyperplastic stolons Colonies were experimentally deprived of interstitial cells (I-cells) to assess the potential influence of the induced differentiation of nematocytes in histocompatibility interactions. The I-cells of hydroids appear to be a multipotent stem cell line, capable of differentiating into any of the various somatic cell types (Lentz, 1966; Muller, 1967, 1968). In the growing colony, however, I-cells only replace those cells incapable of mitotic activity: the nematocytes, the sensory-motor-intemeurons, and the gametes (Diehl and Burnett, 1964, 1965a, b; Muller, 1964, 1967, 1968; Campbell and David, 1974; David and Murphy, 1977; Marcum and Campbell, 1978). In H. echinata, interstitial cells (I-cells) are located between gastrovascular canals within the mat and occur only rarely in the stolons (Muller, 1964). Muller (1967, 1968) has demonstrated that application of mitomycin-C leads to the selective lysis of interstitial cells in H. echinata. Mitomycin-C acts primarily by attacking RNA synthesis and may secondarily lead to structural damage in DNA (Muller, 1967). Application of mitomycin-C leaves cnidoblasts, nerve cells, and ep- ithehomuscular cells intact and thus is preferable to the irradiation or nitrogen mustard techniques typically used with Hydra (Muller, 1967). Colonies exposed to mitomycin retain the ability to regenerate, produce new polyps, and elongate stolons. Treated colonies, however, can no longer differentiate nematocytes and will eventually die unless fed manually. We experimentally eliminated the I-cell population of colonies to determine the capacity of I-cell-depleted organisms to recognize incompatible tissues and to mount a hyperplastic response. Three large colonies were exposed for 14 hours to 0.06 M 144 BUSS ET AL. mitomycin-C. Immediately following the mitomycin exposure, four explants from an isogeneic, but unexposed colony were placed into contact with one of the exposed colonies to determine whether the I-cell-depleted colony retained its fusibility char- acteristics and, if so, to repopulate the depleted colony with I-cells. After two weeks, four explants from this exposed-replenished colony were placed in contact with al- logeneic tissue as controls for the exposure process. The second exposed colony was used to test the capacity of an I-cell-depleted colony to mount a hyperplastic response. Eleven explants of allogeneic tissues were placed in contact with the exposed colony and observations made on the behavior of stolons in contact. The third colony was left unmanipulated and died within three weeks, indicating that the I-cell population of the colony had been effectively eliminated. Results Colony ontogeny and histocompatibility The growth of polyps, mat, and stolon throughout ontogeny for the five genotypes are presented in Figure 2. The five strains differ significantly in the rate of growth of mat (ANOVA, F = 4.49, P < 0.01), polyps (ANOVA, F = 3.03, P < 0.05), and stolonal tissues (ANOVA, F = 5.58, P < 0.005). Log-transformed regressions of mat, polyp, and stolon tissues through time are presented in Table I. Inspection of Figure 2 illustrates that the five strains fall into three distinct groups. Strains 1 and 2 produce no stolons at any point in ontogeny, 4 and 5 produce stolons throughout ontogeny, and strain 3 only produces stolons late in ontogeny. The histocompatibility responses of H. echinata were assessed in all paired com- binations of the five strains (Fig. 3). In addition, strain 3 was paired with all other strains during both its early stolonless stage and late stoloniferous stage of ontogeny. Intraspecific contacts resulted in one of three unambiguous results: fusion, rejection without hyperplastic tissue formation, and rejection with hyperplastic tissue formation (Table II). Fusion is recognized by the disappearance of a discrete margin between tissues and the formation of a shared gastrovascular canal system (Fig. 4A). Rejection without hyperplasticity is recognized as the persistence of a discrete margin separating tissues in contact, with no evidence of shared gastrovascular systems (Fig. 4B). Rejection with hyperplasticity is recognized as the presence of swollen, erect stoloniferous tissues differentiating along, and extending atop, the contact zone (Fig. AC, D). Three relationships emerge from the results of paired histocompatibility inter- actions. First, all isogeneic combinations fuse and all allogeneic combinations reject (Table II). Second, fusion occurs in isogeneic crosses irrespective of the tissues which contact; whereas the pattern in rejection is dependent on the types of tissue which contact (Table II). Finally, mat and stolon tissue differ in their morphogenetic potential; only stolon can produce hyperplastic tissue. In allogeneic crosses, hyperplastic stolon is induced whenever stolons contact either foreign mat or stolon. Rejection without induction of hyperplasticity occurs only when foreign mats contact (Table II). It is important to note that strain 3 produced hyperplastic stolons in late ontogenetic encounters {i.e., stolon-mat contacts) and failed to do so in early ontogenetic encounters {i.e., mat-mat contacts), indicating that the different behavior of mat and stolonal tissues in histocompatibility interactions is purely a difference in the morphogenetic potential of the two tissue types. Ultrastructure of fusion and rejection response Contact between isogeneic tissues results in clear and unambiguous fusion between colonies of//, echinata. Fusion is recognized as the narrowing and rapid disappearance HYDROID COMPETITION AND HISTOCOMPATIBILITY 145 m l> 3 "D O O 2.5—1 2.0 1.5 1.0 0.5 0— ' 5- 4 3 2 I 0- STOLON in H O > m > 3 5- 4 3 2 I 0- 1 2 3 4 5 TIME (wks) Figure 2. Colony ontogeny of the five genotypes of Hydraclinia echinata used in studies of histo- compatibility. Each row represents the growth for one genotype of mat area in cm', of the number of polyps, and the enclosed area of stolons in cm^ versus time. Data for strains 1-5 appear sequentially in row order from top to bottom. Scales are the same for each plot. of the periderm coat in the region of contact immediately following contact between colonies. Ultrastructural observations show no evidence of any boundary between cells of the two colonies as early as 1.5 hours following the initial contact (Fig. 5a). Within four hours of the initial contact a shared gastrovascular system has become 146 BUSS ET AL. Table I Colony ontogeny Slope- Slope- Slope- Strain Mat' R^ Signif.2 Polyp' R^ Signif.' Stolon' R^ Signif.^ 1 0.398 .957 P<0.00\ 0.424 .987 P < 0.001 2 0.568 .988 /'< 0.001 0.397 .999 P< 0.001 — — — 3 0.512 .982 F< 0.001 0.587 .993 P< 0.001 .227 .836 P < 0.05 4 0.549 .990 P <0.00\ 0.734 .948 P < 0.001 .387 .963 P < 0.001 5 0.402 .993 P < 0.001 0.481 .936 P< 0.001 .301 .969 P < 0.001 ' Log (mat, stolon, polyp) versus log (time). ^ F-test. established, as evidenced in live observations by the movement of granular material from one colony into the other. Rejection between allogeneic tissues is characterized by a distinct fibrous boundary separating the two colonies (Fig. 5b, c). This fibrous boundary appears distinct from the periderm coat, is secreted by both colonies, and occurs in both types of rejection responses. At no point have we seen any direct cell-to-cell contact between colonies, nor any evidence of either cells or vesicles crossing this boundary. It is important, 1 2 3E0 3L0 4 5 MM MM MM MS MS MS 2 MM MM MS MS MS 3E0 MM MS MS MS 3L0 MS SS SS 4 SS SS 5 SS Figure 3. Matrix of the tissue interactions resulting from combinations of the five genotypes. Columns and rows represent strain numbers. Note that strain 3 was tested at two different times during ontogeny, during its early ontogenetic (3E0) stolonless phase and its late ontogeny (3LO) stoloniferous stage. Bold face cells represent isogeneic combinations, all other cells represent allogeneic interactions. Five replicates were made for each cell in this matrix. MM-mat versus mat interactions, MS-mat versus stolon interactions, and SS-stolon versus stolon interactions. HYDROID COMPETITION AND HISTOCOMPATIBILITY 147 « • 1 • ■ lElSkT ' 1. "'^^^^4!S|^^^^^^^| M • I\ ' '*-I?lKr^' ••.•»♦% Figure 4. (A) Fusion between two colonies of Hydractinia echinata. Note the continuous gastrovascular canals traversing the margin between colonies. (B) Rejection between mats of two incompatible colonies. Note the failure to fuse along shared colony margin. (C) Rejection between a stolon producing colony and a colony which produces no stolons. Note development of hyperplastic stolons where stolons contact the mat of the foreign colony. (D) Rejection between two stolon producing strains, showing hyperplastic stolon development where stolons of the two colonies contact. however, to recognize that microvillar extensions of ectodermal cells frequently per- forate the mucous layer, hence direct cell surface communication is not ruled out by our observations. 148 BUSS ET AL. Table II Histocompatibility interactions - Rejection* No Hyperplastic Hyperplastic Tissues in Contact n Fusion Response Response A. Isogeneic Interactions Mat versus Mat 15 15 Mat versus Stolon 10 10 Stolon versus Stolon 10 10 B. Allogeneic Interactions Mat versus Mat 15 15-15 0-0 Mat versus Stolon 40 40-0 0-40 Stolon versus Stolon 15 0-0 15-15 * First figure represents behavior of first tissue type listed. Rejection by mat and stolonal tissues differs fundamentally in that stolonal tissues undergo a complex series of morphogenetic transitions following contact with foreign tissue. Within 24 hours of the original contact, stolons become markedly swollen and begin to lose their periderm coat. These swollen or hyperplastic stolons lift up off the substratum and begin to redirect growth toward the foreign colony (Fig. 7a). Upon contacting the foreign tissue, the tissues underlying the stolon lyse. At the ultrastructural level, this series of events is recognized as the movement of numerous cnidoblasts and interstitial cells into the stolon, the development of a distinctive cnidom on the surface of the hyperplastic stolon coming into contact with the foreign tissue (Fig. 6a, b), and the discharge of numerous nematocysts of the basotrichious isorhizal type (Fig. 7c; Mariscal, 1974) into the foreign tissues and the associated lysis of cells in the region of contact (Fig. 6c, d, 7b). Rejection in I-cell-depleted colonies I-cell-depleted colonies retain their fusibility characteristics, fusing with isogeneic colonies (n = 4) and failing to fuse with allogeneic tissues (n = 11). I-cell-depleted colonies, however, failed to display a typical hyperplastic response. Upon contacting foreign tissue, stolons of I-cell-depleted colonies swelled very slightly. These stolons, however, failed to continue to swell in the typical fashion or to lift off the substratum and redirect growth toward the foreign colony. Exposed colonies with their I-cell population replenished (n = 4) displayed a wholly typical hyperplastic response to allogeneic tissues. These experiments demonstrate that the induction of hyperplasticity is dependent upon I-cells, but that the recognition of foreign tissue upon initial contact between colonies is not. DISCUSSION The hyperplastic response of H. echinata to allogeneic tissue bears a number of similarities to anthozoan responses to neighbors. Both hydrozoan and anthozoan responses (1) require contact for induction; (2) are capable of discriminating between HYDROID COMPETITION AND HISTOCOMPATIBILITY 149 A* ■»"■ ^x- -^ 't.^ * . '*'6 • ♦ # i 4 • it ^' • ' • *■ ^^ * M »« i^m C M ^) i Figure 5. (A) Fusion of mat and stolonal tissues 1.5 hours after initial contact between colonies (1390X). Arrow points to region of initial contact. Note the lack of any distinct boundary separating cells of the two colonies. (B) Rejection between mats of two allogeneic colonies (1200X). Lying between the two colonies, along the entire length of boundary, is an electron-dense fibrous material. This fibrous layer, shown at higher magnification (8040X) in (C), is not in contact with the tissues of either colony. M = mat, S = stolon. isogeneic and allogeneic tissues; (3) respond by site-specific cellular differentiation; and (4) involve the discharge of nematocysts to effect destruction of foreign tissues. Recognition elements Anthozoan responses to foreign tissues are apparently elicited by contact with either the tentacles, coenosarcs, or mesenterial filaments of other cnidarians. In H. echinata, the response is elicited following contact with either mat or stolonal tissue. Cnidarians are typically covered with a copious mucous layer, perforated with mi- crovillar extensions of ectodermal cells. Tardent and Buhrer (1982) suggest that rec- 150 BUSS ET AL. \ M • «. » • d m^ 9 . «^® •o'^aflr5®* • ■ '*■',:•(♦. ^•' • ® «;"«• «^ «i HP 6«« •-• gif •yiS ■■fA ^, id HYDROID COMPETITION AND HISTOCOMPATIBILITY 151 ognition elements lie within the mucous layer of Podocoryne carnea, but they do not consider the possible influence of cell-surface markers on ectodermal villi. Bigger (1976), however, tested the capacity of allogeneic mucus to elicit an acrorhagial response in Anthopleura krebsi and found no such effect. Lubbock ( 1 979) demonstrated that mucous extractions of various sea anemone and coral species have markedly different antigenic determinants. He failed, however, to detect differing antigenic determinants in mucus within a given species. The localization and eventual char- acterization of recognition elements remains a central, unresolved issue. Historecognition A hallmark of anthozoan responses to neighboring cnidarians is the capacity to distinguish between isogeneic and allogeneic tissues and to selectively deploy effector systems against allogeneic forms. To my knowledge, all substratum-bound cnidarians investigated are able to distinguish between isogeneic and allogeneic tissues (Table III). In contrast to the apparent uniformity of allorecognition, cnidarian recognition of xenogeneic tissues is quite variable. Several anemones fail to display acrorhagial responses upon interspecific encounters with other anemones (Francis, 1973; Bigger, 1976, 1980; Williams, 1978), despite the ability of at least one anemone to recognize tissues as different as that of a scyphozoan medusae (Bigger, 1976, 1980). Similarly, catch tentacle development in Metridium senile may vary greatly in both occurrence and effect on other anemones (Purcell and Kitting, 1982). Among scleractinians, sweeper tentacles in Agaricia agaricities may develop in response to encounters with the encrusting gorgonian Erythropodium caribaeoreum and the zooanthid Palythoa caribbea (Chornesky, 1983). Similarly, the hydrocoral Millepora dichotoma displays varying degrees of interspecific aggression in response to xenogeneic neighbors (Muller et ai, 1983). The apparent ubiquity of allorecognition may reflect a primitive capability of cnidarians and the variability in deployment of effector systems in xenogeneic en- counters may be a relatively recent adaptation to local circumstances. If this hypothesis is correct, xenogeneic effector systems should be found most frequently between species in which the frequency and potential severity of interspecific encounters is great. This suggestion is tentatively supported by observations of the interactions among hydractiniid hydroids in Long Island Sound. Hydractinia echinata is the most common hydractiniid and interactions are primarily intraspecific contacts, whereas Podocoryne carnea is relatively rare and makes frequent interspecific encounters (Buss and Yund, unpub.). As expected, P. carnea is capable of mounting a sustained hy- perplastic response to H. echinata, whereas H. echinata is incapable of maintaining a similar response to P. carnea (McFadden, unpub.). Further study of the relationship between the occurrence of xenogeneic effector systems and the relative frequency of intraspecific and interspecific competition is warranted. Figure 6. (A) Section across the tip of a hyperplastic stolon in contact with foreign mat (800X). Note high density of nematocysts in hyperplastic stolon. 96 hours after initial contact between colonies. (B) Inset of this cross-section in higher magnification (3273X), shows that each cell harbors a nematocyst. (C) Section across hyperplastic stolon in contact with foreign mat (800x). Note the concentration of capsules of discharged nematocysts along the margin of the hyperplastic stolon where it is in contact with foreign tissue and the zone of destruction directly underlying this region. These discharged capsules are eventually sloughed off, a new set of nematocytes are differentiated, and the interaction repeated until the foreign tissue is completely eliminated. (D) Inset shows the contact zone at greater magnification (3273x). showing shafts of the nematocysts embedded in the foreign tissue. HP = hyperplastic stolon, M = mat, NC = nematocyst capsule. 152 BUSS ET AL. %^ r .3k HYDROID COMPETITION AND HISTOCOMPATIBILITY 153 Table III Cnidarian hislocompatibility and competition Taxon Effector System References Hydrozoa Hydroida Hydractinia echinata Podocaryne carnea Milleporina Millepora dichotoma Hyperplastic stolons Hyperplastic stolons Unknown Schijfsma. 1939; Muller, 1964; Ivker, 1972 Tardent and Buhrer. 1982 Muller et ai, 1983 Anthozoa Gorgonacea Lophogorgia sarmentosa Eunicella stricta Leptogorgia virgidata Psuedopterogorgia elisahethae Plexaura flexuosa Actiniaria Actinea equina Anthopleura artemisia A. ballot i A. elegantissima A. Krebsi Anemonia sargassensis Bunodosoma cauernata Phymactis clematis Cereus pendunculatus Diadumene cincta Halipanella luciae Metridium senile Sargartia elegans S. troglodytes Scleractinia Agaricia agaricites Montastrea cavernosa Montipora verrucosa Pocillopora damicornis P. robusta Unknown Unknown Unknown Unknown Unknown Acrorhagi Acrorhagi Acrorhagi Acrorhagi Acrorhagi Acrorhagi Acrorhagi Acrorhagi Catch Tentacles Catch Tentacles Catch Tentacles Catch Tentacles Catch Tentacles Catch Tentacles Sweeper Tentacles Sweeper Tentacles Unknown Sweeper Tentacles Sweeper Tentacles Theodor, 1970 Theodor, 1976 Bigger and Runyan, 1979 Bigger and Runyan, 1979 Bigger and Runyan, 1979 Francis, 1973b, Brace and Pavey, 1978 Bigger, 1980 Williams, 1978 Francis, 1973b Bigger, 1976, 1980 Bigger, 1980 Bigger, 1980 Brace, 1981 Williams, 1975 Williams, 1975 Williams, 1975; Watson and Mariscal, 1983 Purcell, 1977 Williams, 1975 Williams, 1975 Chomesky, 1983 Richardson et al.. 1979 Hildeman et al. 1975, 1 Wellington, 1980 Wellington, 1980 980 Site-specific differentiation The occurrence of such a diverse array of responses to foreign tissues testifies to the chronic occurrence of intra- and interspecific competition in cnidarians. Contacts between cnidarians are typically site-specific; interactions among scleractinians and Figure 7. (A) Scanning electron micrograph showing a hyperplastic stolon arching off the substratum toward a polyp of an allogeneic colony (120x). (B) Contact between a hyperplastic stolon (arrow) and a foreign polyp (190X). Note the concentration of nematocysts threads where hyperplastic stolon contacts the foreign polyp. (C) Artificially discharged nematocysts from a hyperplastic stolon (440X), showing these nematocysts to be basotrichious isorhizas. 1 54 BUSS ET AL. hydrozoans are typically made only along colony margins and interactions between anemones are often limited to only a portion of a clonal patch. Several cnidarian responses to foreign tissues {e.g., sweeper tentacles, catch tentacles, hyperplastic stolons) share a common feature: the capacity for site-specific differentiation of specialized tissues and morphologies. The capacity for site-specific differentiation is enormously important as it allows a colony to divert energies to aggression only in those tissues where they may be most effective. Site-specific differentiation, however, can only occur if the group is capable of transporting multipotent stem cells (or their products) to the zone of combat. This trait is limited in phyletic distribution; only sponges, cnidarians, platyhelminthes, echinoderms, and chordates have been found to possess a mitotically active multipotent stem line throughout ontogeny (Nieuwkoop and Sutasurya, 1981; Buss, 1983a, b). The dependence of several effector systems on site-specific differentiation under- scores the need for caution in the interpretation of immunologic "memory" in in- vertebrates. The repeated reports of memory in invertebrates involve systems in which the effector mechanisms are unknown (Hildeman, 1975, 1977a, b, 1980; Manning, 1980; Bigger et ai, 1982). However, if these responses require differentiation of mul- tipotent stem cells the observation of memory may simply reflect the deployment of specialized cells or cell products the differentiation of which had been previously induced. Although this will result in an accelerated second-set response, this observation does not imply that (a) the putative memory will be retained over ecologically relevant time scales or that (b) the accelerated second-set response will be observed to display any specificity whatsoever with respect to antigenic determinants. In the absence of a detailed knowledge of the nature of the effector system and appropriate third party experiments, the observation of an accelerated second-set response cannot be con- sidered evidence of existence of a memory component homologous to that of vertebrate immune systems. Effector systems Perhaps the most striking similarity between the various groups of cnidarian responses to foreign tissues is the evolution of a nematocyst-based effector system. Nematocyst function is remarkable in its evolutionary lability; various specialized nematocysts are used for attachment, prey immobilization, prey capture, and clone defense (Mariscal, 1974). Nematocysts appear in structures as different and as limited in phyletic distribution as scleractinian sweeper tentacles and mesenterial filaments (den Hartog, 1977; WeUington, 1980), actinarian catch tentacles (Calgren, 1929; Hand, 1955; Williams, 1975; Purcell, 1977; Watson and Mariscal, 1982) and acrorhagia (Calgren, 1949; Abel, 1954; Bonnin, 1964; Francis, 1973b), and hydroid hyperplastic stolons (Figs. 6, 7). The use of nematocysts in histocompatibility and competition is likely a convergence in function. Evolution of histocompatibility The similarity of anthozoan and hydrozoan responses to foreign tissue suggests the need to distinguish between selection for histocompatibility and selection for competitive ability. Several authors have suggested that competition between indi- viduals (or species) was the primitive selective agent shaping the evolution of allo- recognition (e.g., Kaye and Ortiz, 1981). This hypothesis seems unlikely for two reasons. It is difficult to understand how a diversity of different competitive behaviors and structures could have evolved if there were not a pre-existing system allowing for the recognition of those individuals and species against which they might be effective. In addition, cnidariians are uniformly capable of recognizing allogeneic tissues. HYDROID COMPETITION AND HISTOCOMPATIBILITY 155 even in forms in which competition between conspecifics seems highly unlikely. A more parsimonious explanation is that genes for historecognition and totipotent cells capable of differentiating into nematocysts were ancestral features of cnidarians which became linked into certain groups. The diversity of cnidarian responses to competition may ultimately reflect the co-occurrence in this group of (1) a primitive system of historecognition, (2) a mitotically active multipotent stem cell lineage, and (3) an effective device, the nematocyst, which might be coopted to defensive functions. If this is the case, selective forces other than competition between individuals must account for the evolution of historecognition. A frequently cited alternative explanation for the evolution of histocompatibility is that of defense against microbial and viral infections, cancer, and pathogen mimicry ("surveillence theory," e.g., Burnet, 1970). Although microbial infections are un- doubtedly of considerable importance, there is little data upon which to assess this theory in cnidarians. Allorecognition might, for example, be interpreted as a defense against the potential of fusion acting as a vector for pathogens. However, nematocyst- based effector systems are clearly unsuitable for employment against pathogens. Ne- matocysts are an order of magnitude larger than microbes and their unique method of deployment is clearly unrelated to any microbial clearance function. Cnidarians, however, may not be limited to nematocyst-based effector systems. For example, certain classes of cellular (Hildemann, 1975, 1977a, b) or allelochemic (Sammarco et ai, 1983) interactions have been suggested. Adequate assessment of the relevance of the "surveillence theory" to allorecognition in cnidarians must await further in- formation on their mechanisms of microbial detection and clearance. An alternative, but complementary, explanation for the evolution of histocom- patibility is the somatic cell parasitism hypothesis (Buss, 1982). This hypothesis is based on the fact that the primordial germ cells of certain simple metazoans are not sequestered in early ontogeny. Fusion between conspecifics results in the passage of totipotent cells {i.e., competent to produce gametes) from one individual into another. If the totipotent ceUs of one individual prove more effective in differentiating into gametes than do those of the other component of the chimera, then one individual has effectively parasitized the other (Buss, 1982). Fusion between individuals with an active totipotent cell lineage produces a chimera in which the fitness of the com- ponents of the chimera is determined not only by the fitness of the chimeric individual relative to other individuals in the population, but also by competition between components of the chimera for representation in the gametes. Systems of allorecog- nition serve to prevent fusion and the subsequent invasion of the totipotent cell line of one individual into another, hence acting to defend an organism from somatic cell parasitism. If this scenario is correct, the totipotency of cell lines provides both the raison d'etre for the evolution of historecognition and the mechanism permitting the subsequent evolution of specialized competitive mechanisms in the Cnidaria. Acknowledgments We thank K. Carle, E. Chornesky, D. Green, R. Grosberg, B. Keller, C. Wahle, and P. Yund for comments on the manuscript, and R. Lerner and T. Jenkin for technical assistance. Support was provided by the National Science Foundation (OCE- 8117695 and PCM-83 10704). LITERATURE CITED Abel, E. F. 1954. Ein Beitrag zur Giftwirkung der Actinien und Function der Randsackchen. Zooi Anz. 153: 259-268. 156 BUSS ET AL. Bigger, C. H. 1976. The acrorhagial response in Anthopleura krebsi: intraspecific and interspecific recognition. 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Fecal material of these predators consisted primarily of zooxanthellae, which were shown to be photosynthetically active and capable of re-establishing symbioses with aposymbiotic A. pulchella. Introduction Symbiotic dinoflagellates (zooxanthellae = Symbiodinium microadriaticum) are major primary producers on coral reefs (Muscatine, 1980). They occur in high densities in corals and other cnidarian hosts but have yet to be found in abundance in the water column. Since zooxanthellae must be acquired de novo with each sexual gen- eration in many symbioses (Trench, 1979), and since cnidarians show specificity for different strains of zooxanthellae (Schoenberg and Trench, 1980), the question often arises: how are zooxanthellae dispersed over coral reefs? There are several ways in which zooxanthellae are freed from animal tissue: by extrusion, either spontaneous (Steele, 1977) or as a result of osmotic (Goreau, 1964) or temperature (Buchsbaum, 1968) stress, or as a result of predation on the host. Motile stages also can arise from zooxanthellae in decaying host nudibranch tissues (Kempf, 1984). The potential sig- nificance of predator feces as an agent for the dispersal of viable zooxanthellae has not been previously reported. Many predators on corals and other symbiotic cnidarians have been described. For example, fishes of the Marshall Islands include browsers on coral polyps (Families: Chaetodontidae, Monacanthidae), grazers on coral heads (F.: Chaetodontidae, Scaridae, Balistidae, Monacanthidae), and feeders on branching coral tips (F.: Balistidae, Mon- acanthidae, Tetraodontidae, Canthigasteridae) (Hiatt and Strasburg, 1960; see also Hobson, 1974; Randall, 1974). Robertson (1970) reviewed invertebrate feeders on corals, particularly gastropods. However, none of these authors considered whether zooxanthellae were digested by the predators. The liberation of zooxanthellae from host tissue has two important ecological consequences: zooxanthellae are dispersed and may reinfect other hosts, and they become available as a food source for herbivores. The Hawaiian sea anemone Aiptasia pulchella is found in shallow (<2 m) reef areas and in protected lagoons. The puflfer Arothron meleagris, two species of but- terflyfish, Chaetodon auriga and C. unimaculatus, and the nudibranch Berghia major were found to feed on A. pulchella. As each sea anemone contained from 1.0 to 1.5 X 10^ zooxanthellae, or about 3.0 X 10^ zooxanthellae per mg animal protein (Parker, in prep.), large numbers of zooxanthellae were consumed. This paper presents data which show that fecal pellets from these predators contained stages which were pho- Received 26 April 1984; accepted 29 May 1984. * Current address: School of Life Sciences, University of Nebraska, Lincoln, Nebraska 68588. 159 160 G. M. PARKER tosynthetically active and gave rise to motile zooxanthellae. Fecal zooxanthellae were also capable of re-establishing symbioses with aposymbiotic A. pulchella; this indicates that predator feces may be important as a mode for the dispersal of symbionts and reinfection of symbiotic hosts. Materials and Methods Feeding experiments Experiments with Arothron meleagris were conducted at the University of Cali- fornia, Los Angeles, using fish and Aiptasia pulchella collected in Hawaii. Those with butterflyfishes and nudibranchs were conducted at the Hawaii Institute of Marine Biology (HIMB), Coconut Island, Kaneohe Bay, Oahu, Hawaii, using freshly collected organisms. Butterflyfishes were collected on the reefs near HIMB with baited live traps. The nudibranch was found among populations of ^. pulchella near the docks at HIMB. In controlled experiments I allowed predators which had been starved for 24 hours to feed on sea anemones, then isolated them in tanks of clean sea water. Fecal material was collected with a pipette in some cases within minutes, but usually within several hours after defecation. Photosynthetic ability of fecal zooxanthellae The photosynthetic ability of fecal zooxanthellae from Arothron meleagris was tested by fixation of '''C-bicarbonate. Feces were suspended in sea water, briefly homogenized with a glass tissue homogenizer, and filtered through coarse "nitex" screen to remove large clumps. The filtrate was centrifuged at 300 X ^ in an lEC HN-S table centrifuge for two minutes to separate zooxanthellae from debris. The algal pellet was resuspended in filtered sea water, assayed for cell number, and diluted to a concentration of 2.90 X 10^ zooxanthellae- ml"'. Cells were incubated with NaH'^'COs (0.5 tiC\ • ml"') in duplicate test tubes at an irradiance of 100 ^E • m"^ • s"' at 25 °C for one hour. Replicate tubes wrapped in foil were also incubated to correct for any heterotrophic fixation of ''^C-bicarbonate. Tubes were inverted every 1 5 minutes to resuspend settled cells. Photosynthesis by fecal zooxanthellae was compared with that of freshly isolated zooxanthellae. The latter were obtained from sea anemones maintained in the dark for periods corresponding to the residence times of consumed zooxanthellae in fish guts. Zooxanthellae were isolated as follows. Sea anemones were homogenized using a glass tissue homogenizer. The homogenate was centrifuged at 550 X ^ to separate animal and algal fractions and the resulting algal pellet was washed in filtered sea water several times. Final suspensions were diluted to the same cell concentration used for fecal zooxanthellae, and cells were incubated with '"^C-bicarbonate under the same conditions used for the fecal zooxanthellae and at the same time. At the end of the incubations, tubes of fecal and freshly isolated zooxantheUae were centrifuged at 550 X g, and the resulting algal pellets were rinsed three times in filtered sea water. The supernatant and final algal volumes were recorded, and three replicate 100 n\ aliquots were taken from each fraction for liquid scintillation counting. The aliquots were acidified with an equal volume of 1 A^ HCl and placed under a heat lamp for three hours to drive off" inorganic ''*C02, then neutralized by addition of 1 A^NaOH. Scintillation fluor (10 ml) was added and samples were counted on a Beckman LS lOOC scintillation counter. CPM were converted to DPM by the external standard ratio method. ZOOXANTHELLAE IN PREDATOR FECES 161 Reinfection experiments with aposymbiotic A. pulchella To determine if fecal zooxanthellae from ail four predators could re-establish symbioses with aposymbiotic sea anemones, two containers of aposymbiotic A. pul- chella (with four sea anemones per container) were set up for each source of feces. Feces were added directly to one container. The other container served as a control for spontaneous reinfection. Both containers in each of the four sets were aerated and sea anemones were maintained under the same irradiance and fed every other day with freshly hatched Anemia nauplii. After four days the sea water was changed in both containers and feces were removed from the experimental container. Containers were then rinsed with fresh sea water every two days. One or two tentacles were removed every two days from all sea anemones, squashed, and examined micro- scopically for the presence of zooxanthellae. The number of zooxanthellae and the day of first appearance were recorded. To determine the extent of reinfection after a long period of time, the photosynthetic abilities of experimental and control sea anemones were compared after 50 days by measuring oxygen flux and fixation of ''*C-bicarbonate. Oxygen flux measurements were made in a rectangular plexiglas chamber (volume: 49.5 ml) with a Clark YSI 4004 oxygen electrode connected to a chart recorder. The chamber was surrounded by a water jacket maintained at 25 °C. Irradiance sufficient for light-saturated pho- tosynthesis by zooxanthellae in these sea anemones, 2000 yiE • m~'^ • s"', was provided by a 250 watt tungsten-halogen lamp. Several measurements in the light and dark were made for each sea anemone. Sea anemones were then incubated with 0.25 /iCi NaH''*C03 per ml for one hour at 2000 ^E-m'^'S"' irradiance and 25°C. At the end of the incubations they were rinsed in non-radioactive sea water, homogenized, and the homogenate sampled for liquid scintillation counting, zooxanthellae cell numbers, and total protein. Aliquots for liquid scintillation counting were treated as previously described. To determine the density of zooxanthellae in experimental and control sea anemones, the homogenates were centrifuged to separate animal and algal fractions and the resulting algal pellet was washed three times with filtered sea water. Cell counts were made on the final algal suspensions. Supematants were combined and aliquots analyzed for animal protein. Protein analysis was done by the method of Lowry et al. (1951). The density of zooxanthellae was expressed as numbers of zooxanthellae per mg animal protein. Statistics To determine whether the results of the reinfection experiments were significantly different for control and experimental groups of sea anemones, I used the Mann- Whitney U test (Sokal and Rohlf, 1969). Nonparametric statistics were necessary as data sets were found to be heteroscedastic (F^ax-test; Sokal and Rohlf, 1969). Results Feeding experiments A. pulchella was fed to puffers (Arothron meleagris) previously maintained on fresh mussel meat and occasionally sea anemones. Puffers curled back their lips and used their fused beak-like teeth to chop off" the crown of tentacles from individual sea anemones. After the tentacles were consumed the puffer would eat the rest of the body column before proceeding to the next sea anemone. A puffer ate 1 5 to 45 sea anemones at one feeding. Defecation of sea anemone remains occurred 1 2 to 46 hours afterwards. 162 G. M. PARKER Individuals of^A. pulchella from natural populations living on dead coral skeletons and rocks were offered to many different reef fishes in Hawaii. The butterflyfishes Chaetodon auriga and C. unimaculatus readily ate these sea anemones, with each fish consuming about five sea anemones at one feeding. The tentacle crowns and upper parts of the body column of sea anemones were preferred, and defecation occurred within 12 to 24 hours after feeding. The nudibranch Berghia major was found in association with natural populations of yl. pulchella in Hawaii. It may be that A. pulchella is a significant prey item for B. major, as nudibranchs laid egg strings close to these sea anemones. The nudibranch limited its feeding to sea anemone tentacles. One nudibranch consumed the tentacles of three sea anemones (approx. 120 tentacles) daily. Tentacles were clipped off near the oral disk. Nudibranchs defecated within 24 hours after feeding. B. major differs from the fish in that it stores zooxanthellae and nematocysts in its cerata. Zooxanthellae stored in the cerata are presumed to be photosynthetically active to some extent, as nudibranchs consumed less oxygen in the light than in the dark (Parker, unpubl.). Fecal material from the three predatory fish and the nudibranch consisted mostly of zooxanthellae. Light microscopic examination of feces showed that fecal zoox- anthellae appeared intact and that many of the cells were in the process of dividing (Fig. 1 ). Motile zooxanthellae arose within a few hours from defecated zooxanthellae when feces were placed under bright light. Photosynthetic ability of fecal zooxanthellae Photosynthetic rates of fecal zooxanthellae from the puffer .4. meleagris are shown in Table I. Assimilation numbers, corrected for dark heterotrophic fixation, of fecal zooxanthellae are similar to those obtained for the freshly isolated zooxanthellae. Figure 1. Light microscopic preparation of a fecal pellet from the nudibranch Berghia major which fed on the sea anemone Aiptasia pulchella. Arrow points to a dividing zooxanthella cell. Scale bar = 35 Min. ZOOXANTHELLAE IN PREDATOR FECES 163 Table I Assimilation numbers for zooxanthellae from Arothron meleagris y^'a'i and freshly isolated from Aiptasia pulchella Fecal zooxanthellae Freshly isolated zooxanthellae Time in Assimilation number* Time sea anemones Assimilation number fish gut (mg C-h"'-zoox. ceir') kept in dark (mg C-h"' -zoox. cell"') Sample (hours) (XlO"') (hours) (XIO"') At 12-24 3.30 2.32 A 24-36 0.02 36 0.77 B 24-36 2.65 46 1.55 C 46 0.94 .... , r/li&ht '"C fixation - dark '"C fixation\ /0.09 mg COjX * Assimilation number = I I • I ) • L\ added activity / \ ml / /total volume\ / 12C \ ^_, , , ^ r . .. i1 I incubation j-liT^j-^ -(total number of zooxanthellae)- 'J = mg C • zooxanthella cell ' • h"'. t Letters refer to different fish individuals used. These results indicate that fecal zooxanthellae are photosynthetically active after passage through the predator's gut. Reinfection experiments with aposymbiotic A. pulchella Within six to ten days after the initial addition of feces to containers of aposymbiotic A. pulchella, zooxanthellae, including some in division stages, appeared in all ex- perimental sea anemone tentacles. Fecal zooxanthellae from all four predators rein- fected aposymbiotic sea anemones. Control sea anemone tentacles remained initially free of zooxanthellae, although some contained zooxanthellae after 50 days in the light. To determine the extent of reinfection after a long period of time, experimental and control sea anemones were compared at 50 days. The data in Table II show that after 50 days experimental sea anemones contained high densities of zooxanthellae. Significantly fewer zooxanthellae per mg animal protein were found in control sea anemones than in experimental ones which had been exposed to predator feces [Mann- Whitney U test: Us = 16, P < .05 (for C. unimaculatus experiment) Us = 12, P < AO (for B. major experiment)]. The photosynthetic performance of control and experimental sea anemones after 50 days was evaluated from '''C-bicarbonate fixation and oxygen production and consumption data (Table III). Significantly more carbon (DPM-mg sea anemone protein"') was fixed in experimental sea anemones than in control sea anemones [Mann- Whitney U test: Us = 16, P < .05 (for C. uminaculatus experiment) Us = 12, P < .10 (for B. major experiment)]. Experimental sea anemones showed net oxygen production in the light whereas control sea anemones consumed oxygen under the same conditions. Rates of oxygen consumption in the dark for both groups are included for comparison. In separate experiments, fecal pellets were placed onto the oral disks of sea ane- mones to determine whether these were ingested. In most trials, feces were readily ingested and retained by both symbiotic and aposymbiotic sea anemones. 164 G. M. PARKER Table II Zooxanthellae in experimental and control sea anemones 50 days after initial challenge Donor predator (source of feces) Recipient sea anemones Total number of zooxanthellae per sea anemone (XIO*) Number of zooxanthellae per mg animal protein (XIO*) Chaetodon unimaculatus Experimental (+ feces) 5.78 (±.28)t n = 4 2.88 (±.33) n = 4 Control 0.08 (±.04) n = 4 0.05 (±.02) n = 4 Berghia major Experimental (+ feces) 8.76 (±.78) n = 3 2.70 (±.02) n = 3 Control 1.09* (±.84) n = 4 0.50 (±.38) n = 4 * One control sea anemone became densely packed with zooxanthellae. If it is excluded the mean number of zooxanthellae per sea anemone is 0.268 X 10*. t ±S.E. Discussion Many symbiotic associations rely on a renewed establishment of the symbiosis after sexual reproduction (Trench, 1979). A few examples include the gorgonian Pseudopterogorgia bipinnata (Kinzie, 1974), the coral Astrangia danae (Szmant-Froe- lich et al, 1980), the sea anemones Anthopleura elegantissima and A. xanthogrammica (Siebert, 1974), and the clam Tridacna squamosa (Fitt and Trench, 1981). The eggs and planula larvae of Aiptasia pulchella do not contain zooxanthellae (Parker, unpubl. obs.). As symbionts must be obtained from the environment, predator feces may be an important source of zooxanthellae for reinfection. Table III Productivity in experimental and control sea anemones 50 days after initial challenge O: produced (+) or consumed (-)* Donor predator (source of feces) Recipient sea anemones '"C fixedt Light (XIO-^) Dark (XlO-=) Chaetodon unimaculatus Experimental (+ feces) 53,302 (±8568)tt n = 4 +2.381 (±.538) n = 4 -1.003 (±.159) n = 4 Control 2126 (±1057) n = 4 -0.411 (±.067) n = 3 -0.434 (±.143) n = 3 Berghia major Experimental (+ feces) 131,600 (±33,533) n = 3 +2.327 (±.288) n = 3 -0.779 (±.221) n = 3 Control 16,119 (±12,863) n = 4 -0.140 (±.253) n = 4 -0.809 (±.202) n = 4 * (mg 02)- (mg sea anemone protein) ' -h '. t DPM'(mg sea anemone protein) '. tt ±S.E. ZOOXANTHELLAE IN PREDATOR FECES 165 Although zooxanthellae are believed to be a single species, some strain specificity has been demonstrated in certain hosts (Schoenberg and Trench, 1980). Kinzie and Chee (1979) showed that aposymbiotic A. pulchella reinfected with zooxanthellae isolated from different hosts had different growth rates; sea anemones reinfected with zooxanthellae from A. pulchella and the scyphozoan Cassiopea xamachana grew as well as normal A. pulchella whereas those infected with zooxanthellae isolated from the gastropod Melibe pilosa and the clam Tridacna maxima grew no better than control aposymbiotic sea anemones. Therefore mechanisms which increase zoox- anthellae dispersal, and thus contribute to the probability of host contact with the "correct" strain, are important. Mobile predators such as reef fish which release viable zooxanthellae in their feces may be significant in the dispersal of zooxanthellae over long distances. Aposymbiotic sea anemones which had been exposed to feces contained high densities of zooxanthellae after 50 days (Table II). These densities were similar to those of symbiotic A. pulchella, which have algal densities ranging from 1.5 to 3X10^ zooxanthellae per mg animal protein (Parker, in prep.). Although sea anemones exposed to fecal zooxanthellae had significantly more zooxanthellae, some of the control sea anemones became repopulated with zooxanthellae. Zooxanthellae in control sea anemones were probably residual cells, occasionally found in aposymbiotic A. pulchella, which multiplied under the favorable culture conditions. Predation on symbiotic cnidarians may increase the chances of zooxanthellae coming into contact with other host organisms, as fecal pellets were found to be readily ingested by A. pulchella. Feces contain varying quantities of semi-digested animal remains which may stimulate ingestion in potential host organisms. It is not yet known if motile zooxanthellae released from the fecal material, direct ingestion of feces, or both processes, are responsible for reinfection of aposymbiotic hosts. Observations with cultured zooxanthellae indicate that the motile forms are more readily ingested by potential hosts than the non-motile zooxanthellae (Kinzie, 1974; Fitt and Trench, 1981), but predator feces consist of freshly isolated zooxanthellae associated with animal remains and hence may not be directly compared with cultured zooxanthellae. It is likely that both motile zooxanthellae from fecal pellets and direct ingestion of fecal pellets are modes for acquisition of symbionts. Many zooxanthellae in the process of cell division were found in predator feces (Fig. 1 ). It is possible that passage through the predator gut may expose zooxanthellae to higher nutrient levels than are found in sea water. This may actually stimulate growth and the survival of fecal zooxanthellae, as has been shown for algae in the guts of freshwater Daphnia magna (Porter, 1976). This study shows that zooxanthellae defecated by some predators on cnidarians are viable. The different assimilation numbers for fecal and freshly isolated zoox- anthellae cannot be directly attributed to factors such as residence time in the fishes or dark preconditioning of host sea anemones (Table I). Variability in the photosyn- thetic performance of fecal zooxanthellae may result from differences in the physi- ological environment encountered by zooxanthellae during passage through the fish guts. There is a possibility that some of the consumed zooxanthellae were digested. Herbivorous reef fishes may break down plant material by mechanical grinding or lysis by gastric acidity (Lobel, 1981). All zooxanthellae in the feces appeared intact and healthy (for example, Fig. 1 ), suggesting that mechanical breakdown is negligible. Harmehn- Vivien and Bouchon-Navaro (1983) studied the diets of butterflyfishes in Moorea. They measured the ratio of the weight of the alimentary tract to the weight of the fish (defined as a repletion index) and found that diet was correlated to the repletion index. They found that chaetodontids feeding primarily on corals had a 166 G. M. PARKER greater proportion of their body weight as ahmentary tract, and from this concluded that corals represent more of a vegetable food {i.e., zooxanthellae) than an animal food for butterflyfishes. However, they did not examine the fecal material of these butterflyfishes, nor did they present any physiological evidence for this conclusion. Although some herbivorous reef fishes have acidic gastric fluids and plant material is degraded by these (Lobel, 1981), no data on the acidity of gastric fluids of butter- flyfishes and puffers are available. There is no information on cellulase activity in the butterflyfishes and puffer used in this study, however two species of estuarine puflfers from Georgia showed no cellulase activity (Stickney and Shumway, 1974). The results of this study indicate that at least a significant proportion of consumed zooxanthellae are not digested by the butterflyfishes and puffers. The nudibranch diflfers from the fish in that zooxanthellae are selected and stored in the cerata. Since these were photosynthetically active in the nudibranch, zoox- anthellae must have a process whereby digestion in B. major is avoided. It has been suggested that the nutritional status of nudibranchs may influence the relative pro- portion of degenerate and healthy zooxanthellae in fecal material (Kempf, 1984). Fish feces have been shown to be a food source for other reef fish (Robertson, 1982), but the importance of zooxanthellae released from feces as a food source for coral reef filter-feeding herbivores is unknown. Large numbers of zooxanthellae are released in predator feces. As an example, a puffer weighing 270 g wet weight readily consumes 30 sea anemones at one feeding. One feeding may thus liberate up to 330 million zooxanthellae. The relative contribution of fecal zooxanthellae to reef food sources will depend on the density of predators and the amount of symbiotic tissue consumed and assimilated. Zooxanthellae in fish feces may be an important source of energy for the reef community as well as a source of zooxanthellae for the reinfection of nonsymbiotic larvae and juveniles of host species. Acknowledgments I thank Dr. P. Helfrich and the staff" at HIMB, particularly Mr. L. Zukaran for collection of fish, Dr. M. S. Gordon for use of some puffers and sea water facilities at UCLA, and Dr. L. Muscatine for advice and support. Dr. S. Kempf helped identify the nudibranch. Drs. E. Gladfelter, W. Hamner, S. Kempf, L. Muscatine, and F. Wilkerson reviewed drafts of this manuscript. Funded in part by a research grant from the UCLA Academic Senate Research Committee. LITERATURE CITED BuCHSBAUM, V. 1968. Behavioral and physiological responses to light by the sea anemone Anthopleura elegantissima as related to its algal symbionts. Ph.D. Dissertation, Stanford University, Palo Alto, California. FiTT, W. K., AND R. K. Trench. 1981. Spawning, development, and acquisition of zooxanthellae by Tridacna squamosa (Mollusca, Bivalvia). Biol. Bull. 161: 213-235. GOREAU, T. F. 1964. Mass expulsion of zooxanthellae from Jamaican reef communities after Hurricane Flora. Science 145: 383-386. Harmelin-Vivien, M. L., andY. Bouchon-Navaro. 1983. Feeding diets and significance of coral feeding among chaetodontid fishes in Moorea (French Polynesia). Coral Reefs 2: 1 19-127. HlATT, R. W., AND D. W. Strasburg. 1960. Ecological relationships of the fish fauna on coral reefs of the Marshall Islands. Ecol. Monogr. 30: 65-127. HoBSON, E. S. 1974. Feeding relationships of teleostean fishes on coral reefs in Kona, Hawaii. Fish. Bull. 72:915-1031. Kempf, S. C. 1984. Symbiosis between the zooxanthella Symbiodinium (= Gymnodinium) microadrialicum (Freudenthal) and four species of nudibranchs. Biol. Bull. 166: 110-126. Kjnzie, R. a. III. 1974. Experimental infection of aposymbiotic gorgonian polyps with zooxanthellae. / Exp. Mar. Biol. Ecol. 15: 335-345. ZOOXANTHELLAE IN PREDATOR FECES 167 KiNZiE, R. A. Ill, AND G. S. Chee. 1979. The effect of different zooxanthellae on the growth of experimentally reinfected hosts. Biol. Bull. 156: 315-327. LOBEL, P. S. 1981. Trophic biology of herbivorous reef fishes: alimentary pH and digestive capabilities. J. Fish Biol. 19: 365-397. LowRY, O. H., N. J. ROSEBROUGH, A. L. Farr, AND R. J. RANDALL. 1951. Protein measurement with the Folin phenol reagent. / Biol. Chem. 193: 265-275. Muscatine, L. 1980. Productivity of zooxanthellae. Pp. 381-402 in Primary Froduclivily m the Sea. P. G. Falkowski, ed. Plenum Publishing Co., New York. Porter, K. G. 1976. Enhancement of algal growth and productivity by grazing zooplankton. Science 192: 1332-1334. Randall, J. E. 1974. The effect of fishes on coral reefs. Proc. Second Int. Coral Reef Symp. 1: 159-166. Robertson, D. R. 1982. Fish feces as fish food on a Pacific coral reef Mar. Ecol. Prog. Ser. 7: 253-262. Robertson, R. 1970. Review of the predators and parasites of stony corals, with special reference to symbiotic prosobranch gastropods. Pac. Sci. 24: 43-54. Schoenberg, D. a., and R. K. Trench. 1980. Genetic variation in Symbiodinium (= Gymnodinium) microadiaticum Freudenthal, and specificity in its symbiosis with marine invertebrates. III. Specificity and infectivity of Symbiodinium microadriaticum. Proc. R. Soc. Lond. B. 207: 445-460. Siebert, a. E., Jr. 1974. A description of the embryology, larval development, and feeding of the sea anemones Anthopleura elegantissima and ^. xanthogrammica. Can. J. Zool. 52: 1383-1388. Steele, R. D. 1977. The significance of zooxanthella-containing pellets extruded by sea anemones. Bull. Mar. Sci. 27: 591-594. Stickney, R. R., and S. E. Shumway. 1974. Occurrence of cellulase activity in the stomachs of fishes. / Fish Biol. 6: 779-790. SOKAL, R. R., and F. J. Rohlf. 1969. Biometry. W. H. Freeman and Co., San Francisco. 776 pp. Szmant-Froelich, a., P. Yevich, and M. E. Q. Pilson. 1980. Gametogenesis and early development of the temperate coral Astrangia danae (Anthozoa: Scleractinia). Biol. Bull. 158: 257-269. Trench, R. K. 1979. The cell biology of plant-animal symbiosis. Ann. Rev. Plant Physiol. 30: 485-531. Reference: Biol Bull. 167: 168-175. (August, 1984) MORPHOLOGICAL AND BEHAVIORAL DEFENSES OF TROCHOPHORE LARVAE OF SABELLARIA CEMENTARIUM (POLYCHAETA) AGAINST FOUR PLANKTONIC PREDATORS J. TIMOTHY PENNINGTON AND FU-SHIANG CHIA Department of Zoology, University of Alberta. Edmonton, Alberta, Canada T6G 2E9 Abstract Controlled experiments were conducted by offering eggs, pre-setal trochophores, and setose trochophores of the polychaete Sabellaria cementarium to four planktonic predators, Pleurobrachia bachei (Ctenophora), Aequorea victoria (Hydrozoa), brach- yuran megalopa (Crustacea), and juvenile Sebastes spp. (Pisces). Each predator species captures prey with different mechanisms and the prey, while similar in size, differ in motility and presence or absence of setae. Consumption of non-motile eggs was greater by megalopa but less by A. victoria than consumption of pre-setal trochophores; it is suggested that differences in predator feeding mechanisms account for these differences. Setose trochophores were always consumed at lower rates than the younger stages. The evidence suggests that setae can function in larval defense against an array of predators with different feeding mechanisms, but that swimming may increase, decrease, or have no effect upon rate of predation, depending upon predator species. Introduction Thorson (1946), Young and Chia (in press), and others have suggested that the major source of larval mortality for benthic marine invertebrates is predation. While this conjecture may be true, little empirical information supports it. Predation upon invertebrate larvae is generally documented during gut content analyses of predators; larvae usually constitute a minor portion of the diet (reviewed by Young and Chia, in press), and larvae thus observed are often partially digested and therefore difficult to identify. However, Cowden et al. (1984) provide data on differential predation upon several pelagic larvae by two benthic filter-feeders. Models of reproductive strategies of benthic invertebrates have generally assumed that rates of predation upon larvae are constant throughout ontogeny (Vance, 1973; Pechenik, 1979; Jackson and Strathmann, 1981), though Christiansen and Fenchel (1979) did consider large, late- stage larvae less susceptible to predation than small, early larvae. Motility is a factor which may alter rates of predation upon developing larvae. Gerritsen and Strickler (1977) have predicted on the basis of encounter rates that prey could minimize predation by minimizing movement. However, it remains unclear whether diversity of planktivores and feeding mechanisms will render this hypothesis relatively unimportant in marine environments, especially for slow-swimming in- vertebrate larvae. A second factor which may alter rates of larval predation is the development of structures such as larval setae (Fig. Id). A wide variety of planktonic organisms develop setae or spines, including larvae of many benthic polychaetes (Bhaud and Received 16 March 1984; accepted 11 May 1984. 168 DEFENSES OF A POLYCHAETE LARVA 169 Cazaux, 1982; review by Schroeder and Hermans, 1975) and articulate brachiopods (Long, 1964). These larval setae project posteriorly during normal swimming, but are erected to spread out radially when larvae encounter objects or are otherwise disturbed (Fig. Ib-c). Since larval setae are typically lost during metamorphosis, they are presumed to be adaptations to pelagic existence. Setae and spines have been postulated to function both as "parachutes" which slow sinking rates and as defense mechanisms (Wilson, 1929, 1932; Hardy, 1956; Blake, 1969;Fauchald, 1974; Schroeder and Hermans, 1975). In defense, setae are presumed to function both by increasing a larva's effective size and by making it difficult to swallow. Spines of freshwater rotifers and cladocerans are known to be effective defenses against small plantivorous invertebrates, but are apparently not effective against fish predation (Gilbert, 1966; Dodson, 1974; Kerfoot, 1975, 1978, 1980). The only observations regarding the function of setae or spines for marine organisms are those of Lebour (1919) and Wilson (1929). Lebour (1919) observed a megalopa's dorsal spine lodging the larva into the esophagus of a small fish; the fish was neither able to expel or ingest it and eventually died. Wilson (1929) described small fish ejecting Sabellaria aheolata trochophores from their mouths and suggested that erected setae rendered the trocho- phores offensive. This study was designed to examine whether motility and setae of trochophores of the polychaete Sabellaria cementahum Moore are effective defenses against pre- dation by four planktonic predators. S. cementarium was used as prey because its embryos and larvae were readily available, and because of the prominent setae that its trochophores develop (Fig. Ib-d). Materials and Methods Adult Sabellaria cementarium were dredged in the vicinity of San Juan Island, Washington. Gametes were obtained and embryos and larvae were cultured as in Smith (1981). Non-motile eggs, 2 day-old pre-setal trochophores and 5 day-old setose trochophores were used as prey (Fig. 1 a-c). Body size and shape was relatively constant during the first five days of development (70-90 ^m), though eggs were disk-shaped and somewhat broader when freshly spawned. Predator species from four phyla, Pleurobrachia bachei (Ctenophora), a medusa Aequorea victoria (Hydrozoa), unidentified brachyuran megalopa (Crustacea), and juvenile Sebastes sp. (Pisces), were chosen because they were common near Friday Harbor during summertime, and because of their different feeding mechanisms. Al- though in some cases predators were kept in the laboratory for several days before experiments and fed Artemia salina nauplii or goldfish food, they appeared to be in good condition at the time of experiments. For each experiment fifty eggs or larvae were placed into each of 16 1.0 1 jars which contained 960 ml of 3 /im filtered sea water. Twelve of the jars were divided into four sets of three replicates. Each set received a different predator species: ( 1 ) one 10 mm diameter P. bachei per jar; (2) one 30 mm diameter A. victoria per jar; (3) five 3 mm long megalopa per jar; or (4) two 15 mm long Sebastes sp. per jar. The four remaining jars served as controls, measuring background mortality and handling errors. All jars were capped and strapped horizontally around the horizontal axis of a "grazing wheel" which rotated at 1.6 rpm, gently stirring the water and keeping the prey evenly distributed within the jars. Experiments were run for 24 hours in a 12:12 light:dark, 14°C coldroom. At the end of each experiment, predators were removed and water was siphoned from the jars through 41 ^m Nitex mesh, concentrating the 170 J. T. PENNINGTON AND F. S. CHIA Figure 1 . Selected developmental stages of Sabellaria cementarium; A, B, and C slightly compressed and to same scale. A: unhatched embryo of the same size and shape as eggs and pre-setal trochophores; B: five day-old setose trochophore swimming with unerected setae; C: five day-old trochophore with erected setae; D: seta of 5. cementarium trochophore. PT, prototroch; PS, provisional setae. remaining prey in a small volume of residual water. The prey were then washed into vials and preserved in 2% formalin. The preserved prey were later counted in a Bogorov Tray under a dissecting microscope. Data analysis was performed according to the methods of Zar (1974). DEFENSES OF A POLYCHAETE LARVA 171 Results Predation rate upon the three developmental stages of Sabellaria cementarium by each of the four predators is presented in Figures 2a-d. All control values were averaged because loss from control jars was stage-independent; the slope of a least- squares regression of number of larvae missing from controls upon prey stage did not differ significantly from zero (F-test; P < .05). A one-way analysis of variance (ANOVA) was calculated from the data for each predator species to determine if there were significant differences between the number of prey missing in the four treatments (controls, eggs, pre-setal trochophores, and setose trochophores). The anal- yses were done with untransformed data since Bartlett's Test indicated that the data was sufficiently homoskedastic for ANOVA. For all ANOVA's there were significant overall differences between treatments {P < .02 or less), indicating that all predators ate some prey. A posteriori Student-Newman-Keuls Range Tests (SNK Tests) were then calculated which compared all possible combinations of treatments and grouped treatment subsets that were not significantly different {P < .05). The different predator species exhibited different rates and patterns of predation upon eggs and pre-setal trochophores, but in all cases setose trochophores were eaten at low rates, not significantly different than control values (Fig. 2). For Pleurohrachia bachei, the SNK Test grouped values for the controls and setose trochophores as not different or homogeneous, indicating non-significant predation upon setose trocho- phores while eggs and pre-setal trochophores were eaten significantly more often. For Aequorea victoria the SNK Test grouped values for the controls, eggs, and setose trochophores as homogeneous, indicating uniformly low rates of loss from 20i 1 PST ST B) A. victoria a ab b 1 1 ab C D) S a E PST ST ebastes b b ab PST ST Figure 2. Histograms showing mean number of Sabellaria cementarium eggs and larvae missing from treatments for each of the four predator species, ± 1 standard deviation. Treatments are C, controls (n = 12); E, eggs (n = 3); PST, pre-setal trochophores (n = 3); ST, setose trochophores (n = 3). Letters over each bar denote the results of a Student-Newman-Keuls Multiple Range Test, where the same letter occurs over two or more bars the bars were grouped as not significantly different (P < .05). 172 J. T. PENNINGTON AND F. S. CHIA these groups. Thus, pre-setal trochophores appear to be vulnerable to predation by A. victoria, but eggs and setose trochophores are either neglected or avoided. The SNK Test indicated that brachyuran megalopa ate significantly more eggs than the other prey stages, but that insignificant numbers of setose trochophores were eaten. For juvenile Sebastes the SNK Test again grouped the controls and setose trocho- phores as homogeneous, indicating that eggs and pre-setal trochophores combined were eaten significantly more often than setose trochophores. Discussion The effect of motility on predation rate varied among predators, probably a result of the predators' different feeding mechanisms. Predation by medusae involves re- sponses to individual prey in the sense that nematocysts must be stimulated to fire, and prey motion is an important cue in this response (Pantin, 1942). Non-motility may explain the lack of consumption of eggs by Aequorea victoria. Prey motion is presumably an important cue for ctenophores and fishes as well, since coUoblasts must be stimulated to release adhesive substance in ctenophores (Franc, 1978) and most fish locate prey visually (Kislalioglu and Gibson, 1976; Hyatt, 1979). However, Pleurobrachia bachei and Sebastes sp. did not eat more motile than nonmotile prey. Megalopa ate significantly more eggs than all other stages of prey. It thus appears that swimming helped trochophores to escape or avoid these predators. The mech- anisms by which most megalopa feed on small prey are not known, but many crus- taceans both filter small particles and feed raptorially upon larger prey (Marshall and Orr, 1960; McLaughlin, 1982). If the megalopa did filter-feed, prey capture was probably not dependent upon recognition of individual eggs or trochophores. If so, non-motile eggs would be encountered and captured nearly as often as swimming trochophores, but if swimming enabled some trochophores to escape, the rate of predation upon eggs would be higher, as was observed. Predation upon setose trochophores was insignificant while oocytes and pre-setal trochophores were eaten more often by all predators (except A. victoria, which did not eat eggs). The methods by which setae function defensively have not been in- vestigated, but the radial splay of setae could create at least three potential defenses: (1) the effective size of a larva increases; (2) a buffer zone of setae and water around a larva's tissues is formed; (3) the barbed setae become oriented so that they may pierce objects impinging upon a larva. The possible roles of these mechanisms are discussed below. Erection of setae increases the overall diameter of a larva, possibly deterring predation by small-mouthed predators as has been shown for freshwater rotifers (Gilbert, 1966). However, the predators used in the present experiments all eat prey much larger than trochophores. Reeve et al. (1978) fed Pseudocalanus minutus (<650 fiuv long) to P. bachei during production experiments, and Lebour ( 1 924) observed P. bachei eating larval fish. A. victoria has been commonly observed eating large prey, including fish and other jellyfish (Lebour, 1924; Hyman, 1940; Arai and Jacobs, 1980). The juvenile Sebastes sp. fed successfully on Anemia salina nauplii {ca. 600 ^m) as well as upon pieces (> 1 mm) of goldfish food. Many species of crab larvae are also cultured successfully on A. salina nauplii (Rice and Wilhamson, 1971) and the megalopa used in these experiments fed on goldfish food as well. It thus seems unlikely that the size increase created by setal erection prevents predation by any of the predator species used here. However, megalopa have far smaller mouths than the other predators tested; erected setae may substantially increase handling difficulty if DEFENSES OF A POLYCHAETE LARVA 173 megalopa cannot swallow larvae whole but must manipulate and dismember them. Similarly, spines of cyclomorphic cladocerans and rotifers have been shown to reduce predation by freshwater predators with small mouths (reviewed by Zaret, 1980). It thus seems probable that setae function defensively against small-mouthed predators such as megalopa by increasing handling time. In contrast, the other predator species used here could easily swallow whole setose S. cementarium trochophores. The buffer zone of sea water surrounding a trochophore with erected setae may be important as a defense against medusae and tentaculate ctenophores. As described above, both P. bachei and A. victoria must sense and capture indivdual prey. If a predator's tentacles touch only the erected setae of a trochophore, the tactile or chemical cues necessary to elicit a response may not be perceived. Further, even if a larva is recognized as food, nematocysts and colloblasts may work inefficiently upon setae or across the buffer zone {ca. 1 50 ^m) of water created by the setae. If trochophores are first trapped by nematocysts or colloblasts, then ingested and finally expelled, their chances of surviving are probably slim. The numerous trochophores surviving experiments appeared to be in good shape; few were deformed or entangled in mucus. It seems unlikely the surviving trochophores were captured at all by these predators, but that setae prevented recognition or prey capture. Setae may also deter predation by irritating mouthparts as originally implied by Wilson (1929), whose suggestion seems intuitively reasonable because fish capture prey within the buccal cavity where setae could easily pierce oral tissues as trochophores are bitten or swallowed. Predatory fish are also deterred by the spines of sticklebacks (Hoogland et al, 1957), but the spines of some cyclomorphic rotifers and cladocerans are not considered to be effective against fish (Greene, 1983). Other work on predation upon marine larvae has found patterns of predation comparable to those presented here. For a predator who senses individual prey at a distance, Landry (1978) found that weakly motile early copepod nauplii were poorly detected by the copepod Labidocera trispinosa, and were thus eaten at low rates. Large active nauplii were eaten at the highest rates, while copepodids developed an escape response and were eaten rarely. Also, work with marine fish larvae as prey for various crustaceans has generally found that non-motile eggs are not detected by predators and eaten rarely while motile yolk-sac larvae are eaten at high rates. Feeding larvae develop an escape response and are captured and eaten much less often (Lil- lielund and Lasker, 1971, Theilaker and Lasker, 1974; Bailey and Yen, 1983). The low rates of predation upon later stages in all cases are due to the development of fundamentally new structures or behaviors during ontogeny, processes not observed for freshwater prey (Greene, 1983). At present it is not possible to assess the potential impact these predators have on pelagic larval populations of S. cementarium. No quantitative estimates of the densities of any of the predator or prey species have been made in the Puget Sound area, though all are common in the plankton during summer. Similarly, except for P. bachei (see Reeve and Walter, 1978), quantitative observations of the predation rates of the predators upon other prey types have not been made. However, for the predators used we have shown that rates of predation upon setose trochophores are low. Susceptibility to predation is a ubiquitous and important problem for embryos and larvae of benthic invertebrates (Thorson, 1946; Young and Chia, in press) which should generate strong selective pressures for larval defense. If effective defenses have evolved, larval forms, behaviors, chemicals, and ultimately reproductive strategies should reflect such selection. Reproduction in many benthic invertebrates with pelagic larvae is characterized by a short period of rapid embryogenesis followed by a prolonged 174 J. T. PENNINGTON AND F. S. CHIA period of larval feeding and growth. This pattern may be faciUtated by the development of efficient larval defenses. ACKNOWLEEXjMENTS We are grateful to George Shinn and Richard Strathmann, who contributed both to the conceptual and technical elements of this study. We thank A. O. D. Willows, director of Friday Harbor Laboratories, for providing facilities. This work was supported by a University of Alberta Graduate Assistantship in Zoology to J.T.P. and by an NCERC grant to F.S.C. LITERATURE CITED Arm, M. N., and J. R. Jacobs. 1980. Interspecific predation of common Strait of Georgia planktonic coelenterates: laboratory evidence. Can. J. Fish. Aquat. Sci. 37: 120-123. Bailey, K. M., and J. Yen. 1983. Predation by a carnivorous marine copepod, Euchaeta elongata Esterly, on eggs and larvae of the Pacific Hake, Merluccius produclus. J. Plankton Res. 5: 71-82. Bhaud, M., and C. Cazaux. 1982. Les larves de polychetes des cotes de France. Oceanis 8: 57-160. Blake, J. A. 1969. Reproduction and larval development of Pol ydora from northern New England (Poly- chaeta: Spionidae). Ophelia 7: 1-63. Christiansen, F. B., andT. M. Fenchel. 1979. Evolution of marine invertebrate reproductive patterns. Theor. Pop. Biol. 16: 267-282. Cowden, C, C. M. Young, and F. S. Chia. 1984. 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Pearse, eds. Academic Press, New York and London. Zar, J. H. 1974. Biostatistical Analysis. Prentice-Hall, Inc., New Jersey. 620 pp. Zaret, T. M. 1980. Predation and Freshwater Communities. Yale Univ. Press, New Haven. 187 pp. Reference: Biol Bull. 167: 176-185. (August, 1984) SUN AND SHADE MEDIATE COMPETITION IN THE BARNACLES CHTHAMALUS AND SEMIBALANUS: A FIELD EXPERIMENT DAVID S. WETHEY Department of Biology and Marine Science Program, University of South Carolina, Columbia, South Carolina, 29208 Abstract The barnacles Chthamalus fragilis and Semibalanus balanoides compete for space in the high intertidal zone in southern New England. Chthamalus settles throughout the intertidal and persists in the absence of competition with Semibalanus. Semibalanus also settles throughout the intertidal but is usually eliminated from the high intertidal zone by heat and/or desiccation. In a field experiment in the high intertidal zone, Semibalanus survived the high summer temperatures and overgrew Chthamalus under an opaque roof. Under a transparent roof and in control areas with no roof, Semi- balanus died in mid summer, and Chthamalus persisted. Hence the intensity of interspecific competition is mediated by physical stress which primarily affects the dominant competitor. Introduction A paradigm of intertidal zonation is that local upper shore limits are set by physical stress (heat, desiccation) and local lower shore limits are set by biotic interactions (predation, competition) {e.g., Connell, 1961, 1972, but see also Underwood and Denley, 1984). A corollary is that the intensity of interspecific interactions should decrease as one approaches a local upper shore limit, because the lower shore species should increasingly suffer from physical stress. A second corollary is that in the absence of the physical stress, a low shore species should be able to exclude a high shore species on the high shore. There are several possible tests of these hypotheses. Connell (1961) transplanted a high shore species below its lower shore hmit and showed that it survived in the absence of competitors, but died in the presence of competitors. The reverse experiment, reducing the physical stress and determining the outcome of competition, has not been done. An earlier paper (Wethey, 1983) gave evidence from field transects that the outcome of competition between the barnacles Semibalanus and Chthamalus was mediated by heat and/or desiccation stress acting primarily on Semibalanus. On transects in Connecticut, Semibalanus was found at higher levels on the shore in shaded locations than in sunny areas. The upper shore limit of Semibalanus distribution was higher in damp areas than in adjacent dry locations (Wethey, 1 983). Chthamalus was abundant in areas where Semibalanus was absent (Wethey, 1983). In low shore areas from which I removed Semibalanus, Chthamalus survived for over a year well below its normal lower intertidal distribution limit (Wethey, 1983 and unpub.). There is one field experiment that implicated direct sun (rather than emersion) as a limiting factor on Semibalanus. Hatton (1938, p. 274) fixed a sun shade several centimeters above a south facing rock surface and noted after 13 months that Received 15 February 1984; accepted 11 May 1984. 176 SHADE AND COMPETITION IN BARNACLES 177 Semibalanus in the experimental shade area had grown bigger than those in direct sun. The growth rates on shaded south-facing sites were indistinguishable from those on north-facing surfaces that were naturally shaded. The effects of heating by the sun were probably more important than drying of the surface, because the experimentally shaded sites dried out at low tide whereas the north facing rocks did not (Hatton, 1938, p. 275). This experiment was unreplicated and had no controls for the effects of the structure of the shade. Hatton did not report on the effect of the experiment on competition between Semibalanus and Chthamalus. In this paper I provide experimental evidence for the influence of physical stress on the intensity of competition between two rocky intertidal barnacles. I show that, as I had previously suggested (Wethey, 1983), the intensity of competition is determined on the high shore by physical stress, which primarily affects the dominant competitor. Materials and Methods The study was carried out at the Yale University Peabody Museum Field Station, Guilford, Connecticut. Field experiments were established on the south shore of Horse Island, Long Island Sound (41° 16?^, 72°45'W). The smooth granite shore has a 15° slope to the southwest. The tidal range is approximately 1.9 meters, and experiments were established at +1.5 m. Seven treatments were used to test the effects of shade on the distribution and abundance of Chthamalus and Semibalamis. The treatments were: 1. Unmanipulated site. 2. Two-sided cage (roofless except for mesh, upper and lower shore sides open). 3. Full cage (roofless except for mesh). 4. Two-sided cage with clear plastic roof (upper and lower shore sides open). 5. Full cage with clear plastic roof. 6. Two-sided cage with opaque plastic roof (upper and lower shore sides open). 7. Full cage with opaque plastic roof. Galvanized steel hardware cloth cages (10 cm 1 X 10 cm w X 3 cm h, 1.5 cm mesh) were attached with stainless steel screws set in plastic wall anchors in holes drilled in the rock. Roofs of clear Plexiglas were held onto the tops of the cages with the attachment screws. In the shade treatments, the roofs were wrapped with duct tape, making them opaque. Clear roof treatments, roofless treatments, and unmanipulated areas were used as controls for testing the effects of shade and the effects of the presence of a roof. The design was also used to test for the effects of a cage and cage structure. Three replicates of each treatment were established. Photographs of each of the sites were taken on 14 June 1983 at initiation of the experiment and on 17 July, 10 August, 19 October, and 1 December, 1983. A focal framer on a 3:1 closeup ring provided registration of camera position (Nikonos, 35 mm lens, flash-lit, Panatomic-X film). The number of individuals of each species in each site was counted on enlargements of the photographs. Percent cover was estimated by placing a transparent sheet with 49 uniformly spaced dots over the enlargements. The percent of the dots touching Semibalanus, or Chthamalus is an estimate of the percent cover of each species {e.g., Menge, 1976; Wethey, 1983). All Semibalanus were counted in the percent cover and census measures. Only the Chthamalus that were present at the beginning of the experiment were counted. Semibalanus settlement had finished before the start of the experiment, but Chthamalus larvae settled during the period August to October. 178 D. S. WETHEY The tests for the effects of shade, and the various controls were made as follows: 1 . The control for the presence of an opaque roof is a clear roof The effects of a roof in the absence of shade were determined by comparing the roofless treatments to the clear roof treatments. 2. The effects of shade in the presence of a roof were tested by comparing the clear roof treatments to the opaque roof treatments. 3. The the control for the presence of a cage is a two-sided cage. The effect of a cage was tested by comparison of the roofless full cages to roofless two-sided cages. 4. The control for the presence of the support structure (wire mesh) was the unmanipulated area. The comparison of unmanipulated areas with two-sided cage treatments is a test of the effect of the presence of the structure. These tests were all pre-planned contrasts, which were carried out as part of an analysis of variance. Two parameters were tested: change in percent cover of both Semibalanus and Chthamalus, and percent survival of the two species. Data were transformed by the arcsin transformation prior to analysis to normalize the distributions. Because all shade and roof treatments had both full cage and two-sided cage supports, it was necessary to test for the effect of the cage before proceeding with the rest of the analysis. If the cage effect is not significant then the remaining pre-planned contrasts can be used. Despite the fact that sums of squares for caged and two-sided treatments are pooled in the shade and roof contrasts, the tests are considered a priori because they were all planned in advance (were not suggested by the data), and only a specific limited subset of all possible comparisons was made. The confidence level (i'-value) only applies to each particular test, not to the whole series of tests, and is only appropriate when the test is pre-planned {e.g., Neter and Wasserman, 1974, p. 472). If the only important effects are those of sun versus shade, then only the shade test should be significant. Percent cover data were used only to calculate the change in occupation of space between initiation and termination of the experiment. The uniformly spaced dots tended to fall on the same locations on the two samples, making the estimate of absolute change in percent cover more accurate than would be possible with truly spatially independent samples. Because only a difference in percent cover is calculated, the lack of independence does not compromise the analysis. The test of the effect of treatments on changes in percent cover is equivalent to a test of the effect of treatments on survival of a cohort of individuals. Results All sites were equivalent in terms of percent cover at the initiation of the experiment (Table I). This means that any differences detected at the end of the experiment are the results of the treatments, not historical effects carried through from initiation. Population densities of Semibalanus were 10.9 (S.D. 3.4) individuals/cm^ at ini- tiation of the experiment, which was approximately 60 days after settlement (Table I). Densities at settlement were likely to have been much higher than this, since mortality is high in the period soon after settlement (e.g., ConneU, 1961; Wethey, 1984). There were no effects of caging on the survival of Chthamalus or Semibalanus (Tables II, III). The tests for effect of the structure of the support (two-sided cages versus unmanipulated sites) indicated no structure effect (Tables II, III). The exper- iments were set up at a tidal level above the local upper foraging limit of the primary SHADE AND COMPETITION IN BARNACLES 179 Table I Percent cover o/Semibalanus and Chthamalus at initiation on 14 June. 1983 Semibalanus Treatment Roofless Clear Roof Opaque Roof Unmanip Contrast Roof Shade Cage Structure MSE = 1490, df Mean 57 62 57 58 DF SE 5 5 5 7 F P 1.77 0.2047 0.47 0.5042 0.99 0.3356 1.10 •0.3111 14 Chthamalus Treatment Roofless Clear Roof Opaque Roof Unmanip Contrast Roof Shade Cage Structure MSE = 310, df 14 Mean 22 15 20 14 DF SE 5 5 5 7 F P 0.50 0.4927 0.19 0.6723 0.65 0.4352 0.09 0.7685 Analysis of variance carried out on arcsin-square root transformed percentages. The mean values are transformed back to percents. Abbreviations: DF = degrees of freedom, MSE = mean square error, F = variance ratio, P = probability of Type I error. Roof contrast is a priori comparison of roofless and clear roof treatments. Shade contrast is a priori comparison of clear roof and opaque roof treatments. Cage contrast is a priori comparison of roofless cages to roofless two-sided cage treatments. Structure contrast is a priori comparison of roofless two-sided cages to unmanipulated treatments. Population densities at initiation: Semibalanus 10.9/cm^ (S.D. 3.43), Chthamalus 1.3/cm^ (S.D. 1.06). predator {Urosalpinx cinerea). The cages served primarily to exclude the herbivorous gastropod Littorina littorea (mean number per quadrat in July, August, and October samples: cage 0.3, two side 2.1). Littorina saxatilis were small enough to enter the cages and were found in abundance at each survey (for example, mean number per quadrat in July samples; cage 27.9, two side 26.9). The primary effect of the roofless and clear roof cages was the growth of a canopy of green filamentous algae from June to October (canopy appeared in 3 of the 6 unshaded cages, percent cover in August 44%, S.D. 33%), and the appearance of a few Fucus vesiculosus plants thereafter (Fucus appeared in 2 roofless cages, 4 plants in one and 1 plant in the other). The canopy of filamentous green algae evidently did not provide enough shade or water retention to mitigate the effects of sun on Semibalanus. Because there was no cage effect on survival of Semibalanus or Chthamalus. the pre-planned pooling of data from full cages and two sided cages was retained from the tests of the shade and roof eflTects. 180 D. S. WETHEY Table II Percent survival o/Semibalanusyrow June to December Treatment Mean Roofless 7 Clear Roof 7 Opaque Roof 21 Unmanip Contrast DF Roof 1 Shade 1 Cage 1 Structure 1 MSE = 305, df = 14 SE 3 3 3 4 F P 0.01 0.906 7.64 0.015 0.21 0.657 1.11 0.309 Analysis of variance carried out on arcsin-square roof transformed percentages. The mean values are transformed back to percents. Abbreviations and contrasts are as in Table I. Percent survival of Semibalamis was higher in the shade treatments than in the treatments exposed to direct sun (roofless and clear rooO (Table II). Survival was significantly less under the clear roof (7%) than under the opaque roof (21%) (shade effect, Table II). There was no effect of the presence of the roof alone, based on comparison of the clear roof (7% survival) and roofless (7% survival) treatments (roof effect, Table II). Therefore the shade effect is the result of the shade alone and is not confounded by the presence of the structure of the roof Percent survival of Chthamalus was lower in the shade treatments than in the treatments exposed to direct sun (Table III). Survival under the clear roof (76%) was significantly higher than under the opaque roof (36%) (shade effect, Table III). There was no effect of the presence of the roof alone, based on comparison of the clear roof (76% survival) and roofless (67% survival) treatments (roof eflfect. Table III). Therefore the shade effect is the result of the shade alone, and is not confounded by the presence of the roof Table III Percent survival o/ Chthamalus yrow June to December Treatment Mean SE Roofless 67 12 Clear Roof 76 12 Opaque Roof 36 12 Unmanip 95 17 Contrast DF F P Roof 1 0.32 0.5805 Shade 1 5.64 0.0324 Cage 1 0.03 0.8611 Structure 1 2.62 0.1278 MSE= 1330, df= 14 Analysis of variance carried out on arcsin-square root transformed percentages. The mean values are transformed back to percents. Abbreviations and contrasts are as in Table I. SHADE AND COMPETITION IN BARNACLES 181 Occupation of space was dramatically affected by the experimental treatments. In the shade treatments Semihalanus increased its occupation of space (Table IV) and formed 2.5 cm tall hummocks under the roofs. In the clear roof and roofless treatments, occupation of space by Semihalanus decreased (Table IV); any surviving Semihalanus were relatively small and no hummocks formed. Chthamalus slightly decreased (not statistically significantly) in occupation of space in the shade treatments and remained constant in the clear roof and roofless treatments (Table IV). The majority of the deaths o^ Chthamalus in the shade treatments were the result of direct interactions with Semihalanus. Chthamalus individuals were overgrown, crushed, and undercut by Semihalanus (Table V). Individuals that were far enough away from Semihalanus that they did not experience direct interference, survived in the shade treatments. Few Chthamalus individuals in the sun treatments (roofless and clear roof) were close enough to surviving Semihalanus to sustain damage. The Chthamalus that were close to Semihalanus died as a result of the interaction. There were proportionally very few deaths of Chthamalus as a result of unknown causes or of interactions with conspecifics (Table V). Table IV Changes in space occupation from June to December Semihalanus Treatment Mean SE Roofless -34.7 15.4 Clear Roof -46.4 15.4 Opaque Roof 30.4 15.4 Unmanip -86.4 21.7 Contrast DF F P Roof 1 0.08 0.7833 Shade 1 11.38 0.0046 Cage 1 0.35 0.5618 Structure 1 1.21 0.2908 MSE = 2215, df= 14 Chthamalus Treatment Mean SE Roofless 3.4 10.4 Clear Roof 0.01 10.4 Opaque Roof -25.2 10.4 Unmanip 26.1 14.7 Contrast DF F P Roof I 0.10 0.7623 Shade 1 2.12 0.1674 Cage 1 0.22 0.6468 Structure 1 0.46 0.5075 MSE = 2689, df= 14 Analysis of variance carried out on arcsine-square root transformed percentages. Mean values transformed back to percents. Values are changes in percent cover from the beginning to the end of the study (positive values indicate an increase in percent cover, negative values indicate a decrease). Abbreviations and contrasts are as in Table I. 182 D. S. WETHEY Table V Causes of death o/Chthamalus Cause Shade trtmts. percent Sun trtmts. percent Killed by Semibalanus Overgrown 73 6 Crushed 13 34 Undercut 7 21 Killed by Semibalanus and Chthamalus Crushed 2 Killed by Chthamalus Crushed 4 Unknown 6 34 Values are percents of total deaths from particular identifiable causes in the shade and sun treatments. Shade treatments = opaque roof Sun treatments = clear roof and roofless. Shade treatments: number of individuals = 130, sun treatments: number of individuals = 53. Discussion This study examined the influence of physical factors (heat and/or desiccation) on the intensity of interspecific competition between the barnacles Semibalanus and Chthamalus near the northern geographic limit of Chthamalus in New England. In sunny locations the upper shore limit of Semibalanus distribution is lower than in more shaded locations and Chthamalus survives in areas where Semibalanus dies (Wethey, 1983). I argued that the intensity of the competitive interactions between Chthamalus and Semibalanus were mediated by intolerance of heat and/or desiccation by Semibalanus (Wethey, 1983). The experiment described here is a field test of this hypothesis. The results of the experiment are consistent with the shade/competition hypothesis. In the shade treatments Semibalanus survived (Table II) and grew to form hummocks 2.5 cm high. It increased occupation of space at the expense oi Chthamalus (Tables III, IV, V). In the clear roof and the roofless treatments Semibalanus died (Table II) and its occupation of space decreased during the experiment (Table IV). Chthamalus remained unchanged in the sun treatments (Tables II, III, IV). No hummocks formed in any of the sun treatments. The results were striking enough that at termination the shade treatments were recognizable from a distance of several meters away on the shore after the hardware was removed. The controls for the effect of the roof alone and the support structure alone are essential to allow the results of the experiment to be applied to the real world. These controls allow one to separate the effect of shade from the effect of the structure holding the shade above the experimental plots. The clear roof controls were indis- tinguishable from the roofless treatments, indicating that shade alone was the important factor affecting survival in the shade treatments (Table II). Therefore the results of the experiment can be applied to any locations where shade occurs naturally. The survival of Semibalanus is therefore strongly influenced by shade in southern New England. SHADE AND COMPETITION IN BARNACLES 183 These results are consistent with the hypothesis that the upper shore limit of Semibalamis is set by intolerance of heat and/or desiccation rather than intolerance of emersion. The effect of the shade treatment was similar to that reported by Hatton (1938). He concluded that shade alone strongly influenced growth. Other experiments could not distinguish between the effect of emersion and the effect of heat/desiccation. Hatton (1938) reduced the importance of desiccation and raised the upper shore limit o{ Semibalamis by means of the drips from a slowly draining basin fixed in the high intertidal. Hatton's (1938) drip treatment also may have added food to the experimental individuals (Underwood and Denley, 1984). Foster (1969, 1971a, b) concluded that heat and desiccation were important, based on field and laboratory experiments and observations of changes in the upper shore limit of Semibalamis in mid summer (see also Bowman, 1982). Connell (1961b) found that mortality rates measured during hot dry weather were greater than those measured in cooler periods and concluded that desiccation was important. In northern Scotland, Chthamalus is more abundant on surfaces that dry out at low tide and Semibalamis is more abundant on surfaces that remain wet (Lewis, 1964). Semibalamis is more common on north-facing than south-facing shores in the south of England (Crisp and Southward, 1958). As one approaches the southern limit of Semibalamis. it become progressively more restricted to shaded locations (Barnes, 1958). The present study provides the first controlled experimental demonstration that shade alone can determine the local upper shore limit. Underwood and Denley (1984) state that one cannot make the generalization that local upper limits are set by physical stress operating after larval settlement. They erect several alternative hypotheses: animals on the high shore may starve during calm weather because they are not submerged long enough to feed. Alternatively larvae may actively avoid settlement on the high shore. In addition transplant "... experiments only reveal sources of mortality of organisms moved outside their normal zone and do not teU us anything definite about reasons for the absence of organisms from such areas" (Underwood and Denley, 1984). The present experiments falsify all of these alternatives posed by Underwood and Denley (1984) and demonstrate that physical stress operating after larval settlement directly limits the upper shore distribution limit of Semibalanus in southern New England. On a local scale the distribution of sun and shade will likely correlate strongly with the distribution of Semibalamis and Chthamalus, with the latter being prevalent in sunny sites on the high shore {e.g., Wethey, 1983). Semibalamis is the dominant competitor and exerts a strong influence on the distribution and abundance of Chthamalus. In the treatments where Semibalanus survived, it killed neighboring Chthamalus individuals by overgrowing, crushing, or undercutting them (Table V). In the absence of Semibalamis, Chthamalus survived (Tables II, III, V). Hence the survival of Semibalamis to a large extent determines the fate of Chthamalus as a result of competition for space. The intensity of competition is in turn determined by the action of heat and desiccation on Semibalanus. In sunny sites on the high shore, Semibalanus dies and Chthamalus experiences little competition (Tables II, III, V). In shaded sites Semibalanus survives and Chthamalus loses in competition (Tables II, III, V). Thus local zonation is likely the result of postsettlement mortality from interspecific competition in Chthamalus rather than the result of any requirements for dry conditions on the part of Chthamalus adults or juveniles. These results are consistent with those of Connell (1961) who showed that interspecific competition with Semibalanus had a much greater influence on the distribution of Chthamalus than did intraspecific competition, or larval settlement pattern. 134 D. S. WETHEY On a geographic scale, Chthamalus is likely to persist only in areas where Semibalanus predictably dies on the high shore, providing a refuge from competition (Wethey, 1983). In Massachusetts north of Cape Cod, Semibalanus does not die on the high shore (Wethey, 1983), and as a result its upper shore limit does not change during the year. At this location, Semibalanus does not settle above the upper shore limit of adults. At Nahant, Massachusetts (north of Cape Cod), I have monitored settlement in sites 10 cm above the upper shore limit oi Semibalanus and have not seen more than 1 cyprid larva/cm^ (unpub.). This is in striking contrast to the pattern in Connecticut, where I measured densities of Semibalanus metamorphosed spat of 10/cm^ in June (6 weeks after the end of settlement) at shore levels 20 cm above the upper shore limit of adult distribution (Table I). Chthamalus is absent at Nahant and is abundant on the high shore in Connecticut. On a temporal scale, after a period of hot summers, Chthamalus would be expected to increase in abundance, as a result of the lessening of the intensity of competition with Semibalanus. After a period of cold summers, Chthamalus should decrease as a result of the greater intensity of competition with Semibalanus. Such a temporal pattern has been documented by Southward and Crisp (1956), Southward (1967) and Crisp et al. (1981) in England. The abundance of Chthamalus in southern New England may have increased since the climatic minimum in the early 1800's. The species was noted by Darwin (1854) in collections from Charleston, South Carolina, but not in collections from Delaware Bay or Massachusetts. It was first noted at Woods Hole in 1898 by M. A. Bigelow (Sumner et al, 1913, pp. 191, 646). The first published report was by Sumner (1909). Sumner et al. (1913) note "It is hard to believe that this species has been habitually confused with [Semi]balanus balanoides by the long succession of field naturalists and systematic zoologists who have exploited the shores of New England for over a century. These men erred rather in the direction of discovering too many new species than in ignoring well established ones." Pilsbry (1916) in his monograph on the North American barnacles was equally puzzled by this. Perhaps Chthamalus really was rare in the mid 1 800's and reinvaded from the south as a result of release from competition with Semibalanus brought about by the climatic warming. The physical environment and biotic interactions combine to determine the dy- namics of this high intertidal barnacle assemblage. The intensity of interspecific com- petition is mediated by physical stress, which primarily affects the dominant competitor. Semibalanus can significantly reduce the population densities of Chthamalus, even on the high shore, if Semibalanus is not killed by physical stress. An understanding of the interplay between physical stress and biotic interactions may allow us to un- derstand not only local zonation, but also geographic limits of species and patterns of long term temporal variation in relation to climatic change. Acknowledgments This study was supported by the National Science Foundation (OCE 8208 1 76) and the University of South Carolina. L. W. Buss provided access to the field sites at the Yale University Peabody Museum Field Station Horse Island property and provided lab space, housing, and transportation. M. W. Reed and T. Jenkin assisted in the field and took photographs when I was in South Carolina. D. Felkel and R. Williams assisted in the darkroom. S. A. Woodin provided advice on the experimental design and critically read the manuscript. Reviewer C. H. Peterson made numerous helpful suggestions. SHADE AND COMPETITION IN BARNACLES 185 LITERATURE CITED Barnes, H. 1958. Regarding the southern limits of Balanu.s halanoides (L.). Oikos 9: 139-157. Bowman, R. S. 1982. The role of stochastic events in Balamis/Chthamalus interactions on Scottish shores. Abstr. 17th Eur. Mar. Biol. Symp. CONNELL, J. H. 1961. The influence of interspecific competition and other factors on the distribution of the barnacle Chthamalus stellatus. Ecology 42: 710-723. CoNNELL, J. H. 1972. Community interactions on marine rocky intertidal shores. Artn. Rev. Ecol. Syst. 3: 169-172. Crisp, D. J., and A. J. Southward. 1958. The distribution of intertidal organisms along the coasts of the English Channel. / Mar. Biol. Assoc. V. K. 37: 157-208. Crisp, D. J., A. J. Southward, and E. C. Southward. 1981. On the distribution of the intertidal barnacles Chthamalus stellatus, Chthamalus montagui and Euraphia depressa. J. Mar. Biol. Assoc. U. K. 61: 359-380. Darwin, C. 1854. A Monograph on the Sub-class Cirripedia. Ray Society, London. 684 pp. Foster, B. a. 1969. Tolerance of high temperature by some intertidal barnacles. Mar. Biol. 4: 326-332. Foster, B. a. 197 la. Desiccation as a factor in the intertidal zonation of barnacles. Mar. Biol. 8: 12-29. Foster, B. A. 197 lb. On the determinants of the upper limit of intertidal distribution of barnacles (Crustacea: Cirripedia). / Anim. Ecol. 40: 33-48. Hatton, H. 1938. Essais de bionomie explicative sur quelques especes intercotidales d'algues et d'animaux. Ann. Inst. Oceanogr. 17: 241-348. Lewis, J. R. 1964. The Ecology of Rocky Shores. Hodder and Stoughton, London. 323 pp. Menge, B. a. 1976. Organization of the New England rocky intertidal community: role of predation. competition and environmental heterogeneity. Ecol. Monogr. 46: 355-393. Neter, J., AND W. Wasserman. 1974. Applied Linear Statistical Models. Irwin, Homewood Illinois. 842 pp. PiLSBRY, H. A. 1916. The sessile barnacles (Cirripedia) contained in the collections of the U. S. National Museum; including a monograph of the American species. Bull. U. S. Nat. Mus. 93: 1-366. Southward, A. J. 1967. Recent changes in the abundance of intertidal barnacles in south west England: a possible effect of climatic deterioration. J. Mar. Biol. Assoc. U. K. 47: 81-95. Southward, A. J., and D. J. Crisp. 1956. Fluctuations in the distribution and abundance of intertidal barnacles. / Mar. Biol. Assoc. U. K. 35: 211-229. Sumner, F. B. 1909. On the occurrence of the littoral barnacle Chthamalus stellatus (Poli) at Woods Hole, Mass. Science 30: 373-374. Sumner, F. B., R. C. Osburn, and L. J. Cole. 1913. A biological survey of the waters of Woods Hole and vicinity. Bull. (J. S. Bureau Fisheries 31: 1-794. Underwood, A. J., and E. J. Denley. 1984. Paradigms, explanations and generalizations in models for the structure of intertidal communities on rocky shores. Pp. 151-180 in Ecological Communities: Conceptual Issues and the Evidence, D. R. Strong, Jr., D. Simberloff, L. G. Abele, and A. B. Thistle, eds. Princeton University Press, Princeton. Wethey, D. S. 1983. Geographic limits and local zonation: the barnacles Semibalanus (Balanus) and Chthamalus in New England. Biol. Bull. 165: 330-341. Wethey, D. S. 1984. Spatial pattern in barnacle settlement: day to day changes during the settlement season. J. Mar. Biol. Assoc. U. K. 64: (in press). Reference: Biol Bull. 167: 186-199. (August, 1984) GROWTH RATES OF THE SEA SCALLOP, PLACOPECTEN MAGELLANICUS, DETERMINED FROM THE '^O/'^O RECORD IN SHELL CALCITE DAVID E. KRANTZ', DOUGLAS S. JONESl AND DOUGLAS F. WILLIAMS' ^Marine Science Program, University of South Carolina, Columbia, South Carolina 29208, ^Department of Geology, University of Florida, Gainesville, Florida 32611, and ^Department of Geology and Belle W. Baruch Institute, University of South Carolina, Columbia, South Carolina 29208 ABSTRACT Present age determination techniques for the sea scallop, Placopecten magellanicus (Gmelin), rely on the subjective interpretation of lines on the shell exterior as rep- resenting periods of annual growth. This study compares scallop age and growth estimates from the external line method with a stable isotope technique. The oxygen isotopic records from serially sampled carbonate powders taken from two scallop specimens collected alive off the Virginia coast show annual cycles which closely approximate the isotopic composition predicted as a function of observed salinity and temperature. Since these annual isotopic cycles are controlled by physical-chemical processes, they provide an independent time scale for age and growth rate determi- nation. Growth rates determined from the isotopic records are roughly twice those estimated from the external line method and from a published average growth curve for Placopecten magellanicus. Introduction The sea scallop, Placopecten magellanicus (Gmelin), is an important economic resource for the New England and Atlantic Canada fisheries, providing the fourth highest income from landings (Bourne, 1964; Serchuk et al, 1979). Average annual production from combined U. S. and Canadian catches is on the order of 13,000 metric tons of meats (Serchuk et al, 1979). Harvestable populations of sea scallops occur from St. Lawrence Bay to the Virginia-North Carolina continental shelf just north of Cape Hatteras (Posgay, 1957; Merrill, 1962). Sea scallops in the northern portion of the range inhabit water depths from approximately one meter below mean low tide to just deeper than 100 meters on the continental shelf (Dickie, 1955). The more southerly populations inhabit increasingly deeper waters. In the Virginia Bight, the southern extreme of their range, sea scallops are confined to 40-100 meter water depths (Merrill, 1971), presumably in response to lethal summer temperatures above 20-23°C (Dickie, 1958) in shallower waters. The considerable economic importance and heav>' fishing of sea scallop stocks underscore the need for accurate age and growth rate estimates for this species. Such information is vital if the sea scallop fishery is to be managed effectively. The techniques which are presently employed for age determination of the sea scallop rely on the interpretation of lines visible on the exterior of the shell or on the hinge ligament (Stevenson and Dickie, 1954; Merrill et al., 1965). Based primarily on mark and recovery studies, several pectinids, including Placopecten magellanicus, deposit annual rings or growth lines in late winter or early spring (Stevenson and Dickie, 1954; Received 10 April 1984; accepted 30 May 1984. 186 SEA SCALLOP GROWTH RATES 187 Taylor and Venn, 1978; Serchuk et al., 1979; Paul, 1981). Scallops deposit calcium carbonate in the form of calcite to the shell margin in concentric increments. During the warmer months of the year, while the scallops are growing rapidly, the distance between consecutive increments is relatively wide (Stevenson and Dickie, 1954; Mason, 1957; Taylor and Venn, 1978). As growth slows during the winter and early spring, the growth increments are crowded together, forming what appears to be a concentric line or ring on the shell (Fig. 1 ). The interpretation of external lines as representing years of growth for an individual specimen is often complicated by numerous disturbance or shock rings. Sea scallops are notably sensitive to physical disturbances and sudden changes in environmental conditions such as sharp temperature or salinity changes, or storm-related turbulence (Merrill et al., 1965). In response to strong stimuli, scallops retract the mantle and cease calcification along the shell margin. This action leaves a noticeable line after shell growth resumes. Distinguishing annual lines from disturbance lines macro- scopically is frequently difficult and often very subjective (Stevenson and Dickie, 1954; Merrill et al., 1965). Other shell characteristics, such as seasonal variation in shell color, convexity of the shell profile, and activity of boring organisms, may provide some information for the interpretation of external lines (Merrill et al., 1965). However, these additional methods are still very subjective and are not conclusive. In view of the uncertainties involved in the accurate interpretation of growth rings, additional methods of establishing the periodicity of these features have been pursued in other species of molluscs. Principal among these efforts has been the investigation of stable isotope variations across shell increments {e.g., Yavnov and Ignat'ev, 1979; Jones et al., 1983). Figure 1. Two specimens of Placopecten magellanicus. PMIO (left) and PM26 (right), used for isotopic analyses. Years of growth estimated from external lines are marked on the shell with corresponding year number. Position of shell margin for each summer of growth as determined from the isotopic profiles are indicated by white arrows. Grooves drilled to collect carbonate powder samples are visible on the shell exterior. 188 KRANTZ ET AL. Application of stable isotopic methods to mollusc studies This study utilizes the ratio between the stable isotopes of oxygen, '^O and '^O, in scallop shell carbonate to monitor ambient water conditions during shell deposition, and ultimately to make interpretations about shell growth. Because of the thermo- dynamic behavior of the oxygen isotopes in chemical reactions, the ratio of '^O/'^O in the product is a function of the '^O/'^O ratio of the reactants and the temperature at which the reaction occurred (Urey, 1947). During the deposition of shell carbonate by a mollusc, this isotopic fractionation is controlled by the '^O/'^O ratio of the water in which the animal is living and the ambient temperature during shell deposition. With a constant '^O/'^O ratio for water, relatively fewer '^O atoms are incorporated into shell carbonate in the warmer summer months ("lighter" isotopic values) and proportionately more '^O atoms are incorporated in the cooler winter months ("heavier" isotopic values). The temperature control on the fractionation of oxygen isotopes is a function of the reaction kinetics and in molluscs is, for the most part, independent of physiological processes (Epstein and Lowenstam, 1953; Epstein et al, 1953; Jones ^/ a/., 1983). The isotopic composition of shell carbonate is also a function of the '^O/'^O ratio of the water. The water '^O/'^O is a conservative property and may be related to salinity in that sea water is isotopically heavier (relatively more '^O) than freshwater (Epstein and Mayeda, 1953; Fairbanks, 1982). Shell carbonate deposited by molluscs living in marine conditions will be isotopically heavier than shell carbonate deposited under freshwater conditions (Epstein et al, 1953; Keith et al, 1964; Eisma et ai, 1976). The controlling factors of water '^O/'^O and temperature have been quantified and related to shell carbonate isotopic composition in the calcite paleotemperature equation (Epstein et ai, 1951; Epstein et al, 1953). The principal application of this equation is to calculate temperature of formation from a known carbonate isotopic value when water isotopic composition is known or can be reasonably estimated. Epstein et al. (1953) emphasize that seasonal temperature cycles produce significant variations in isotopic composition within the shell. By sequentially sampHng a mollusc shell at closely spaced intervals (approximately 1 mm), it is possible to check for seasonal changes in the '^O/'^O ratio and compare these cycles with the shell growth record. Information obtained by sequential sampling of mollusc shells has allowed the interpretation of annual growth patterns (Wefer and Killingley, 1980; Cochran et ai, 1981), periodicity of growth increment formation (Horibe and Oba, 1972; Williams et ai, 1982; Jones et ai, 1983), and correlation of the shell isotopic record with upwelling events (Killingley and Berger, 1979), seasonal productivity changes, and thermocline development (Arthur et ai, 1983). Hydrographic conditions of the study area Hydrographic data obtained from 40 to 60 meter water depths in the shelf area near the collection site of specimens PMIO and PM26 were averaged by month for the years 1975 to 1979, which represent the growth period of the specimens. The area of the mid-shelf from which the scallop specimens were collected is essentially full marine with mean salinity of 33.7 %o and an average annual salinity range from 32.7 to 34.7%o (unpubhshed data from NOAA-NODC; Nickerson and Mountain, 1983). A certain degree of seasonality is associated with salinity in that highest salinity values occur in the spring and fall, while lowest salinity values occur during mid- summer (Fig. 2). Occasional short-term deviations occur from this average trend, most orobably in response to extreme precipitation or local hydrographic fluctuations. SEA SCALLOP GROWTH RATES 189 35.5 35.0 ^ 34.5 H 34.0 33.5 33.0 32.5 < CO M — r- A M J J MONTH — r- S o p18 -16 H m - 14 S -0 m -12 D > - 10 H (_ J3 - 8 m ^^ o - 6 o ^^ - 4 - 2 Figure 2. Mean monthly bottom water salinity (solid line) and temperature (dashed line) for areas of the Virginia Bight continental shelf with water depths between 40 and 60 meters. Data obtained from NOAA-NODC and Nickerson and Mountain (1983) are summarized for the years 1975-1979. Average monthly bottom water temperatures for the same area range from ap- proximately 6.0°C to a maximum of approximately 13.0°C. Extreme temperatures may be 2 or 3°C on either side of the average range, dipping to 4°C or rising to 16°C. Throughout most of the winter, spring, and early summer, average bottom water temperatures remain below 8.0°C (Fig. 2). Beginning in late summer and continuing through the fall, bottom water temperatures steadily increase to an annual maximum. An abrupt temperature drop with the onset of winter is followed by a fairly constant low temperature. As in the case of yearly salinity variations, short- term excursions in temperature also occur throughout the year, probably caused by local hydrographic events. Predicted isotopic composition of shell calcite The isotopic composition of shell calcite was predicted using: (1) an unpublished regression equation relating the water 6'^0 to salinity for the Virginia Bight (R. G. Fairbanks, pers. comm.), (2) the average bottom-water salinity and temperature ob- servations, and (3) the calcite paleotemperature equation (Epstein et al., 1953). The predicted 6'^0 of calcite can be estimated by solving the paleotemperature equation (Epstein et al., 1953) using the quadratic formula, such that: 5'«0 (calcite) = 5'^0 (water) + [(4.38 - Vl9.18 -0.4(16.9 - T)]/0.20 where T = temperature in degrees C. Temperature observations and water ^'''O estimates from salinity observations were substituted into this derivation of the pa- leotemperature equation to predict the average oxygen isotopic composition of shell calcite deposited during each month of the year. For areas of the Virginia Bight continental shelf with water depths between 40 and 60 meters, the predicted annual isotopic record from scallop shell calcite is as shown in Figure 3. A distinct seasonality is evident with the lightest isotopic values of approximately 0.5%o occurring in the late summer and early fall, and the heaviest values of approximately 2.5%o occurring in the winter. Since the curve is based on average hydrographic conditions, any given year of actual conditions may deviate 190 KRANTZ ET AL. -0.5 0.0 o ^ 5 -.^ m Q Q. O 1 CD 1.5 2.0 2.5 M M J J A MONTH Figure 3. Predicted average monthly 5'*0 values for scallop shell calcite. Shell carbonate isotopic composition was estimated using average monthly salinity and temperature values (Fig. 2) and the calcite paleotemperature equation (Epstein et ai. 1953). A distinct seasonality is evident, with lightest 6'^0 values in the late summer and heaviest values in the winter and early spring. somewhat from this predicted curve, and actual seasonal extremes may be somewhat greater. The short-term temperature and salinity excursions, which are not uncommon on the shelf, would be expected to show up in the shell isotopic record as deviations from the annual trend. However, the seasonal cycle should still be the major signal recorded in the shell carbonate of the scallop, as has been demonstrated for other mollusc species (Wefer and Killingley, 1980; Williams ^/ a/., 1982; Jones ('Z a/., 1983). Materials and Methods Living specimens of Placopecten magellanicus used in this study were collected by National Marine Fisheries Service (NMFS) personnel during the 1 979 yearly shellfish assessment survey. Isotopic analyses were performed on two specimens which were collected alive on 2 1 May 1979 from a station at 57 meters water depth approximately 90 km off the coast of Virginia (37°15'N, 74°45'W). These two specimens (PMIO, PM26) were chosen from a group of thirty because the exterior of both shells showed no evidence of boring or extensive erosion. Estimation of the yearly growth for each individual was made by NMFS personnel using lines on the shell exterior in a method described by Merrill et al. (1965). Indelible marks representing estimated years of growth were placed on each shell by NMFS personnel, and were later used for com- parison with the growth interpretation based on the stable isotope record. Both PMIO and PM26 specimens were prepared for isotopic analysis by first lightly grinding the exterior of the shell to remove the periostracum and any foreign debris. Discrete samples of carbonate powder were then drilled from the outer shell layer using a 0.5-mm dental drill bit. The calcium carbonate powders were collected in a series by drilling consecutive grooves parallel to shell growth increments from the umbo to the ventral margin along the axis of maximum growth. Samples were taken only from the outer prismatic shell layer which is deposited sequentially along the shell margin during the growth of the scallop. Care was taken to avoid the inner shell layer which is deposited in thin sheets over existing calcite. Any given point of SEA SCALLOP GROWTH RATES 191 the inner shell layer will be composed of calcium carbonate deposited over an extended period of time instead of the single time slice represented by the overlying prismatic layer. The stable isotopic composition of the shell powders was determined using stan- dardized techniques (Williams et ai, 1977; Jones et ai, 1983). Approximately 0.5 mg of each carbonate sample was first roasted in vacuo at 380°C for one hour to remove any remnant of the organic matrix. Each roasted sample was then reacted in purified phosphoric acid at 50°C usmg a technique modified from McCrea (1950). The oxygen and carbon isotopic compositions of the evolved carbon dioxide gas were determined on a VG Micromass 602-D mass spectrometer. By convention, the isotopic values are recorded relative to the carbon dioxide gas derived from the Pee Dee Belemnite (PDB) standard carbonate powder (Epstein et al, 1953) in conventional delta notation (6, %o). Analytical precision was ±0.1 0%o for prepared samples. Results Specimen PM 10, with a shell height of 75 mm, was estimated by NMFS personnel to have completed three years of growth and begun shell deposition in a fourth year. This age determination was based on an interpretation of the external growth lines represented diagrammatically in Figure 4. The millimeter scale on the horizontal axis relates the position of these external lines to shell height (as measured from the umbo to the margin) and the position of each carbonate powder sample drilled from the shell. Isotopic determinations were made on 46 discrete powders secured from the shell of specimen PMIO. The oxygen isotope data (see Krantz, 1983 for data) are plotted in Figure 4 with the 5'^0 scale reversed so that lower 5 values, representing "warm" isotopic temperatures, are at the top of the vertical scale. The oxygen isotope record from specimen PMIO exhibits two major cycles with approximately 2%o variation between minimum and maximum 5'*^0 values (Fig. 4). The heaviest (most positive) 5'^0 value in the record is +2.46%o and the lightest (most negative) is +0.29%o. The 5'^0 curve is roughly sinusoidal although occasional deviations of a few tenths of a per mil are observed from the trend. As previously discussed, ambient temperatures during shell formation may be estimated from the calcite paleotemperature equation (Epstein et ai, 1953) using the 6'^0 values of the individual carbonate samples and the 6'^0 values of the water. As a first approximation, if one makes the simplifying assumption that changes in water 5'^0 at the collection site are negligible, an average water 5'^0 value of — 0.25%o (R. G. Fairbanks, pers. comm.) can then be used to estimate temperature during shell deposition. In this manner, the shell carbonate 6'^0 values can be converted to temperature minima and maxima. Using this approximation, the minimum isotopic temperature recorded in the shell of PMIO is 6.5°C, the maximum is 14.6°C, and the average range is 7.5°C. Specimen PM26, with a shell height of 126 mm was collected on the same day and from the same station as specimen PMIO. NMFS personnel estimated the age of specimen PM26 to be seven years with the beginning of an eighth year of growth, again relying on the interpretation of external growth lines (Fig. 5). The 6'**0 profile of PM26 shows four large amplitude cycles (Fig. 5) with approximately 2%o variations occurring between minimum and maximum 6'^0 values. Superimposed on these cycles are several smaller amplitude excursions of approximately 0.2 to 0.5%o. As with the oxygen isotope data from specimen PMIO, the data from PM26 may be converted to isotopic temperature estimates using an average water 6'**0 of -0.25%o. The minimum and maximum calculated temperature values vary from 5.7°C to 16.5°C, with an average range of 8.4°C. 192 KRANTZ ET AL. 0.0 1.0 - m a a. o •O 2.0 3.0 — I — 10 — I — 20 30 — I — 40 — I — 50 — I — 60 — I — 70 SHELL HEIGHT (mm) Figure 4. Position of external lines and shell carbonate oxygen isotopic record for the shell of specimen PMIO. The external lines illustrated in the diagram are those interpreted by NMFS personnel as representing years of growth. These are numbered in reverse chronological order (line 1 is the most recently formed) to facilitate comparison with those on specimen PM26. The isotopic values of discrete sample powders are plotted with the 6'*0 scale reversed so that lower b values, which represent "warm" isotopic temperatures, are at the top of the vertical scale. The millimeter scale on the horizontal axis relates the position of the external lines and the position of individual carbonate powder samples to shell height. Discussion Interpretation of observed oxygen isotope records With the predictive model outlined previously, the oxygen isotope records obtained from the scallop shells may be interpreted as yearly cycles controlled by seasonal hydrographic conditions. The isotopic record from specimen PM 1 is interpreted as showing two full years of growth with the beginning of a third year (Fig. 4). Specimen PMIO completed one year of growth at a shell height of 25 mm as determined by one full cycle in the isotopic record. Shell deposited from to 10 mm gradually becomes isotopically lighter (more negative), representing late winter to late spring, and reaches an inferred late summer maximum temperature at approximately 15 mm shell height (Fig. 4). The seasonal trend continues into the late fall and early v/inter as represented by shell deposited from 1 5 to 25 mm. A second annual cycle SEA SCALLOP GROWTH RATES 193 0,0 - CD a a. o 00 2.0 - 3.0 20 40 60 80 SHELL HEIGHT (mm) 100 I 120 Figure 5. Position of external lines and shell carbonate oxygen isotopic record for specimen PM26. The external lines illustrated in the diagram are those which were interpreted by NMFS personnel as representing years of growth. As in Figure 4 for specimen PM 1 0, the Hnes are numbered in reverse chronological order, and the oxygen isotopic values for shell carbonate samples are plotted with a reversed d'^O scale. begins at 30 mm, reaches a late summer temperature maximum at approximately 55 mm, and proceeds to a yearly minimum at 65 mm shell height. The beginning of a third annual cycle represented by the last 10 mm of shell and terminating at the ventral margin coincides well with the spring collection date (Fig. 4). Not only does the beginning of the isotopic cycle coincide with the seasonal hydrographic cycle, but the 6'^0 values of samples taken from the shell margin are very close to the \J%o value predicted by the model for May (Fig. 3). This agreement between isotopic "season" and collection date supports the validity of the isotopic record interpretation. These results indicate a discrepancy between the three years of growth interpreted from the external lines and the two years of growth inferred from the 6"*0 record. Further, the external lines, which are presumed to be deposited annually in the spring, appear to occur in various seasons according to the inferred seasonality of the isotopic profile. For example, external line number 3 was deposited in the fall of the first growth year, line 2 during summer of the second year, and line 1 the following spring (Fig. 4). In this particular specimen, there does not appear to be a consistent season 194 KRANTZ ET AL. for external line formation, therefore, the assumption that these external lines represent true annual events does not appear to be valid. As in the smaller specimen, the isotopic record from the shell of PM26 demonstrates what appear to be distinct, hydrographically controlled, annual 5'^0 cycles (Fig. 5). Again, the four years of growth are recorded in PM26 by the cycles in the isotope curve. This age estimate is at variance with the seven years of growth interpreted from the external lines. The occurrence of external lines on the shell of PM26 appears to have a more consistent relationship to the seasonal 6'^0 cycles than in PMIO. Counting backwards from the ventral shell margin, and hence backwards chrono- logically, external lines 2, 4, 6, and 7 of specimen PM26 were deposited during late summer maximum temperatures (Fig. 5). A little more in line with the presumed deposition of annual lines in the spring by Placopecten, external lines 1 , 3, and 5 appear to have been deposited in middle spring to possibly early summer. As in specimen PM 10, the termination of the isotopic record at the shell margin is consistent with the spring collection date. Comparison of the records from the two specimens Since both scallop specimens were collected alive in 1979, calendar years may be assigned to years of growth by counting backwards from the shell margin (which represents the collection date). By assigning calendar years in this manner, the in- terpretation of the isotopic records and the external growth lines from the two specimens may be compared directly. In Figure 6 the shell records of specimens PM 1 and PM26 are overlaid based on distance from the shell margin and on an interpretation of the isotopic record. The cycles in the isotopic record of specimen PM26 which we interpret as rep- resenting the calendar years 1978 and 1979 do not include the isotopically heavy carbonate which the model predicts should be deposited during the winter. In com- parison, the isotopic record for these same years from specimen PMIO includes carbonate isotopic values as heavy as those predicted by the model. Since other bivalves have been documented to slow or cease calcification during the winter (Taylor and Venn, 1978; Clark, 1979; Jones, 1980), it is reasonable to assume that the values "missing" from the isotopic record of specimen PM26 represent cessation of shell deposition. Similar interpretations have been proposed for the attenuation of cycles in the isotopic record for conchs (Epstein and Lowenstam, 1953; Wefer and Killingley, 1980) and surf clams (Jones et ai, 1983). The isotopic record of specimen PMIO is used in Figure 6 to represent the complete record for the years 1978 and 1979, while the isotopic record of specimen PM26 has been separated to illustrate periods of cessation of shell deposition. The first two years of growth in specimen PM26 appear to be relatively complete. However, the isotopic values representing the winter months of 1975-76 are slightly lighter than expected which may indicate lack of shell deposition for a short period. Alternatively, the winter of 1975-76 may have been slightly warmer than 1976-77, but this possibility can be confirmed without more complete hydro- graphic information. Overall, the two major cycles in the isotopic record for specimen PMIO coincide well with the cycles from specimen PM26. The records from both shells have very similar amplitudes and absolute isotopic values. As previously discussed, the predicted 6"^0 values demonstrate a distinct seasonality in varying from 2.5%o in the winter to 0.5%o in the late summer. The cycles in the isotopic records of both specimens fall almost exactly within the predicted range estimated from the hydrographic data. The isotopic temperature range of 5.7°C to 16.5°C calculated from shell calcite 6'^0 SEA SCALLOP GROWTH RATES 195 -0.5 PM10 H J °^^ o CO 'jO 15- 2 2 5 PM26 IL H IL IL A. •^T-TT 1975 1976 1977 1978 YEARS OF GROWTH lTZ] 1979 Figure 6. Comparison of the 5'*0 records of specimens PMIO and PM26. The isotopic profile of specimen PMIO is represented by the dashed line, that of specimen PM26 by the solid line. Calendar years assigned to each annual cycle in the 6'*0 records of each specimen comprise the horizontal axis. External lines interpreted as annual marks are represented for each specimen at the top of the diagram. Breaks in the record of specimen PM26 correspond to presumed curtailment of calcification by the scallop. values corresponds well to the observed water temperatures for the area. The few excursions outside of the predicted range represent minimum and maximum tem- peratures which were probably dampened in the model by using hydrographic data which were averaged by month. These calculated temperatures also seem reasonable in that maximum temperatures fall below the reported lethal temperature of ap- proximately 20°C (Dickie, 1958). Seasons and years determined from the isotopic record allow correlation of growth lines between the two specimens and aid in evaluating possible mechanisms for line formation. External line 1 on the shell of specimen PMIO was formed in the spring before the collection date in May, 1979. Line 2 on specimen PMIO appears to cor- respond with line 2 on specimen PM26, both having been deposited in summer 1978 just prior to the annual temperature maximum. Similarly, growth lines 6 and 7 on specimen PM26 were deposited in middle to late summer of years 1976 and 1975 respectively. The previously mentioned external lines appear to have been formed with little cessation of growth as evidenced by loss of "time" from the isotopic record. In contrast, external lines 1 and 4 in specimen PM26, and line 3 in specimen PMIO appear to coincide with periods of missing isotopic values. As was pointed out pre- viously, two gaps in the isotopic record of specimen PM26 corresponding to the winters of 1978 and 1979 are interpreted as cessation of shell growth. Each of these gaps is marked by a line on the shell. On specimen PMIO, line 3 coincides with a sudden shift in isotopic values, which suggests that it may be analogous to line 4 in specimen PM26 and was probably formed at approximately the same time. In each case, the specimens appear to have stopped or drastically slowed calcification in the fall and added no shell material during the winter, hence there are no isotopic values representing those periods. After this cessation of growth, specimen PMIO appears to have resumed calcification much sooner than PM26, which explains the difference in the two isotopic records for the early spring of 1978. Specimen PMIO apparently 196 KRANTZ ET AL. did not curtail calcification in the winter of 1978-79, as evidenced by a fairly complete isotopic record for the period. Sea scallops are sensitive to disturbance or abnormal changes in environmental conditions (Merrill et al, 1965). External line 5 on the shell of specimen PM26 seems to coincide with anomalously light isotopic values during the early spring of 1977. The lighter than expected isotopic values were possibly the result of a low salinity event which may have eventually caused mantle retraction, shell closure, and a tem- porary halt to calcification. Growth rate determinations Since the isotopic composition oi Placopecten appears to be controlled by seasonal hydrographic factors, the isotopic record from a shell should provide a useful time scale for determining rate of growth. The number of months from the beginning of shell deposition may be estimated for obvious seasonal peaks such as summer maxima and winter minima. This time scale may then be related directly to the positions on the shell of the respective samples. Constructed in this manner, a standard graph of shell height to years of growth (Fig. 7) illustrates that specimens PMIO and PM26 show an average rate of growth of approximately 35 mm per year (as interpreted from the isotopic record). The first two years of growth in both specimens show a very similar, approximately linear, growth rate even though they represent different calendar years. This supports the growth rate interpretation by suggesting a similar trend in ontogenetic development. For years two through four in specimen PM26, the rate of growth gradually slows, which is typical for many species of molluscs (Mason, 1957; Hallam, 1967; Jones et al, 1978; Serchuk et al., 1979). The growth rates inferred from the isotope profiles of PMIO and PM26 can also be compared to the rates as determined using the external line aging technique (Fig. 8). Growth rate estimates based on the external lines would suggest that the two specimens grew approximately 20 mm per year for the first three years of growth. The larger specimen, PM26, shows a significant decrease in growth rate with age. 140 -\ 1 20 - £ 100 E X 80 g ^ 60 LU I 40 - 20 - P/ 4' a;- ,^' 'p PM 10 D PM26 ■ YEARS OF GROWTH Figure 7. Growth rates of specimens PMIO and PM26 interpreted from the isotopic profiles. Points on each curve correspond to shell height at obvious seasonal peaks (summer maximum and winter minimum). Open squares represent shell heights for specimen PMIO; closed squares represent specimen PM26. SEA SCALLOP GROWTH RATES 197 140 f MERRILL el al ( 1965) YEARS OF GROWTH Figure 8. Comparison of growth rates determined from the external line technique and from the isotopic record. Growth rates for specimens PMIO and PM26 interpreted from the isotopic profiles are represented by dashed lines. Growth rates for the same two specimens determined from the external lines are represented by solid lines. Open squares indicate shell heights for specimen PMIO; closed squares indicate specimen PM26. Dotted line illustrates an average growth curve for 351 Placopcclen specimens aged using the external line method published by Merrill et al. (1965). The growth rate estimates for both specimens fall very close to a curve constructed from average age/height data that Merrill et al. (1965) generated by estimating the age of 35 1 scallops using the external hne technique. Although the growth rate estimates based on the external lines are internally consistent, they differ considerably from those interpreted from the isotopic record (Fig. 8). The growth rate determined from the isotopic record is roughly twice that from the external line method. This would be expected considering the discrepancy between the age (years of growth) estimates. Migration Results from previous tag-release studies suggest that sea scallops do not show any widespread or directed seasonal migration (Dickie, 1955; Posgay, 1963, 1981). The shell isotopic records presented here support this idea of limited movement. If the scallop specimens had migrated from either deeper or shallower water areas, the movement should have appeared as a significant change in the amplitude of the annual 6'^0 cycles. In general on the continental shelf of the Virginia Bight, seasonal bottom water temperature fluctuations are greater in shallower water, and become progressively more dampened with increasing depth (Nickerson and Mountain, 1 983). The isotopic record contained in shell carbonate, being controlled largely by ambient temperature, should also have a greater amplitude for a scallop living in shallower water. Taking into account the interruptions during calcification noted previously, specimens PMIO and PM26 have very similar isotopic amplitudes. This similarity in isotopic amplitude is seen in comparing individual cycles within the record of a single specimen, and in comparing the records of the two specimens. Therefore, it appears that the two scallop specimens, PMIO and PM26, remained in essentially the same depth habitat during their lives. 198 KRANTZ ET AL. Implications for shellfisheries management Methods presently used for the determination of age and growth rate in sea scallops rely on the interpretation of lines or rings on the shell exterior. Although some mark and recovery studies have shown that sea scallops produce an external line in the spring, lines are also produced during other times of the year, presumably in response to physical disturbance. This study demonstrates that the ratio of '^O/ '^O incorporated in the shell carbonate can be used to avoid the subjectivity of interpreting external lines as annual marks. One particular advantage of using the isotopic record as an age determination tool in molluscs is the fact that the 6'^O-derived time scale is primarily controlled by the seasonal changes in hydrographic conditions. Because of this independent control, the interpretation is less subjective than distinguishing between shock lines and true annual lines on a scallop shell. From the evaluations performed in this study, external lines do not always form concurrently or consistently for all individuals in a scallop population. On the other hand, the shell isotopic composition should record the same annual cycles for the entire population. It should, however, be noted that the stable isotope method does not lend itself to use with large numbers of specimens, primarily because of the time and expense involved in the isotopic analyses. However, the technique does allow accurate age determination for small groups of specimens used for modeling periodic external line formation, growth rates, and other processes related to biomineralization. Acknowledgments We thank Dr. Richard Fairbanks of Lamont-Doherty Geological Observatory for generously sharing his unpublished water isotope data, and Dr. Norimitsu Watabe of the University of South Carolina and two anonymous reviewers for helpful com- ments about the manuscript. The members of the Stable Isotope Laboratory of the University of South Carolina provided invaluable laboratory assistance. This research was supported in part by National Science Foundation grants (ATM-8204525, OCE80- 08239, OCE82-089 1 1 ). Further financial assistance was provided by a Sigma Xi Grant in Aid of Research, and by the Marine Science Program and the Office of the Graduate School at the University of South Carolina. This is contribution number 540 of the Belle W. Baruch Institute of Marine Biology and Coastal Research. LITERATURE CITED Arthur, M. A., D. F. Williams, and D. S. Jones. 1983. Seasonal temperature-salinity changes and thermocline development in the mid-Atlantic Bight as recorded by the isotopic composition of bivalves. Geology 11: 655-659. Bourne, N. 1964. Scallops and the offshore fishery of the Maritimes. Bull. Fish. Res. Board Can. 145: I- 59. Clark, G. R. 1979. Seasonal growth variations in the shells of recent and prehistoric specimens of Mercenaria mercenaria from St. Catherines Island, Georgia. Anthropological Papers, Am. Mus. Nat. Hist. 56: 161-179. Cochran, J. K., S. M. Rye, and N. H. Landman. 1981. Growth rate and habitat of Nautilus pompilius inferred from radioactive and stable isotope studies. Paleobiology 7: 469-480. Dickie, L. M. 1955. Fluctuations in the abundance of the giant scallop Placopecten magellanicus (Gmelin), in the Digby Area of the Bay of Fundy. / Fish. Res. Board Can. 12: 798-856. Dickie, L. M. 1958. Effects of high temperatures on survival of the giant scallop. / Fish. Res. Board Can. 15: 1189-1211. Eisma, D., W. G. Mook, and H. a. Das. 1976. Shell characteristics, isotopic composition and trace- element contents of some euryhaline molluscs as indicators of saUnity. Palaeogeogr. Palaeoclimatol. Palaeoecol. 19: 39-62. Epstein, S., and H. A. Lowenstam. 1953. Temperature-shell-growth relations of recent and interglacial Pleistocene shoal water biota from Bermuda. J. Geol. 61: 424-438. Epstein, S., R. Buchsbaum, H. A. Lowenstam, and H. C. Urey. 1951. Carbonate-water isotopic temperature scale. Geol. Soc.,Am. Bull. 62: 417-426. SEA SCALLOP GROWTH RATES 199 Epstein, S., R. Buchsbaum, H. A. Lowenstam, and H. C. Urey. 1953. Revised carbonate-water isotopic temperature scale. Geol. Soc. Am. Bull. 64: 1315-1326. Epstein, S., and T. Mayeda. 1953. Variation of '"O content of waters from natural sources. Geochim. Cosmochim. Acta 4: 2 1 3-224. Fairbanks, R. G. 1982. The origin of continental shelf and slope water in the New York Bight: Evidence from '^O/""© ratio measurements. / Geophys. Res. 87: 5796-5808. Hallam, a. 1967. The interpretation of size-frequency distributions in molluscan death assemblages. Paleontology 10: 25-42. HoRiBE, Y., AND T. Oba. 1972. Temperature scales of argonite-water and calcite-water systems. Fossils 23: 69-79. Jones, D. S. 1980. Annual cycle of shell growth increment formation in two continental shelf bivalves and its paleoecologic significance. Paleobiology 6: 331-340. Jones, D. S., D. F. Williams, and M. A. Arthur. 1983. Growth history and ecology of the Atlantic surf clam (Spisula solidissima) as revealed by stable isotopes and annual shell increments. / lixp. Mar. Biol. Ecol. 13: 225-242. Jones, D. S., I. Thompson, and W. G. Ambrose. 1978. Age and growth rate determinations for the Atlantic surf clam Spisula solidissima, based on internal growth lines in shell cross-sections. Mar. Biol. 47: 63-70. Keith, M. L.. G. M. Anderson, and R. Eichler. 1964. Carbon and oxygen isotopic composition of mollusk shells from marine and fresh-water environments. Geochim. Cosmochim. Acta 28: 1757- 1786. KiLLiNGLEY, J. S., AND W. H. Berger. 1979. Stable isotopes in a mollusk shell: detection of upwelling events. Science 205: 186-188. Krantz, D. E. 1983. Environmental and growth information from stable isotope records in the sea scallop, Placopecten magellanicus. M.S. Thesis, University of South Carolina, Columbia. Mason, J. 1957. The age and growth of the scallop, Pecten maximus (L.), in Manx waters. J. Mar. Biol. Assoc. U. K. 36: 473-492. McCrea, J. M. 1950. On the isotopic chemistry of carbonates and a paleotemperature scale. / Chem. Phys. 18: 849-857. Merrill, A. S. 1962. Abundance and distribution of sea scallops off the Middle Atlantic Coast. Proc. Nad. Shellfish. Assoc. 51: 74-80. Merrill, A. S. 1971. The sea scallop. Am. Malacol. Union Inc. Ann. Rep. 1970: 24-27. Merrill, A. S., J. A. Posgay, and F. E. Nichy. 1965. Annual marks on the shell and ligament of the sea scallop {Placopecten magellanicus). Fish. Bull. 65: 299-3 1 1 . Nickerson, S. R., and D. G. Mountain. 1983. Surface and bottom temperature and bottom salinity distributions on the continental shelf. Cape Hatteras to Cape Sable from MARMAP cruises, 1977- 1982. MARMAP Contrib. MED/NEFC 83-16. Paul, J. D. 1981. Natural settlement and early growth of spat of the queen scallop, Chlamys opercularis (L.), with reference to the formation of the first growth ring. J. Molluscan Stud. 47: 53-58. Posgay, J. A. 1957. The range of the sea scallop. Nautilus 71: 55-57. Posgay, J. A. 1963. Tagging as a technique in population studies of the sea scallop. ICNAF Spec. Pub. 4:268-271. Posgay, J. a. 1981. Movement of tagged sea scallops on George's Bank. Mar. Fish. Rev. 43: 19-25. Serchuk, F. M., P. W. Wood, J. A. Posgay, and B. E. Brown. 1979. Assessment and status of sea scallop (Placopecten magellanicus) populations off the northeast coast of the United States. Proc. Natl. Shellfish. As.soc. 69: 161-191. Stevenson, J. A., and L. M. Dickie. 1954. Annual growth rings and rate of growth of the giant scallop, Placopecten magellanicus (Gmelin) in the Digby Area of the Bay of Fundy. / Fish. Res. Board Can. 11:660-671. Taylor, A. C, and T. J. Venn. 1978. Growth of the queen scallop, Chlamys opercularis. from the Clyde Sea area. / Mar. Biol. Assoc. U. K. 58: 687-700. Urey, H. C. 1947. The thermodynamic properties of isotopic substances. / Chem Soc. 1947: 562-581. Wefer, G., and J. S. KiLLiNGLEY. 1980. Growth histories of strombid snails from Bermuda recorded in their O- 18 and C-13 profiles. Mar. Biol. 60: 129-135. Williams, D. F., M. A. Arthur, D. S. Jones, and N. Healy-Williams. 1982. Seasonality and mean annual sea surface temperatures from isotopic and sclerochronologic records. Nature 296: 432- 434. Williams, D. F., M. A. Sommer, II, and M. L. Bender. 1977. Carbon isotopic compositions of recent planktonic foraminifera of the Indian Ocean. Earth Plan. Sci. Lett. 36: 391-403. Yavnov, S. v., and a. V. Ignat'ev. 1979. Shell structure and growth temperature of molluscs, family Mactridae. Sov. J. Mar. Biol. 5: 409-414. Reference: Biol Bull. 167: 200-209. (August, 1984) SELF-GENERATED VERSUS ENVIRONMENTALLY PRODUCED FEEDING CURRENTS: A COMPARISON FOR THE SABELLID POLYCHAETE EUDISTYLIA VANCOUVERI RACHEL ANN MERZ* Department of Anatomy, The University of Chicago, Chicago, Illinois 60637 Abstract The feeding currents produced by the branchial crown of the tube-dweUing sabellid polychaete Eudistylia vancouveri are compared with ambient currents experienced by the in situ worm. The speeds of the branchial currents range between 0.025 and 0.080 cm/s and are similar to the patterns mapped by Nicol (1930) for Sabella pavonina. The ambient currents in contact with the branchial crown of a worm in the field are up to three orders of magnitude higher (33.6 cm/s). When these worms are clustered together in the field, their tubes form hemispherical mounds that aflfect the pattern of ambient currents. Flow over the surface of the cluster is augmented in comparison to pre-cluster velocities. Thus animals within a cluster experience higher feeding currents than do solitary worms. This increase in feeding current velocity is not without potential competition for food from cluster- mates. Depletion of natural particles during the passage of a single wave through a cluster ranges from 45 to 65%. Introduction The mechanisms by which suspension feeding animals remove particles from the surrounding fluid is a topic of current and historical interest. Ruid movement de- termines to a large degree the mechanical forces impinging on an organism {e.g.. Wainwright and Koehl, 1976; Merz, 1984), the rates of respiration and excretion (LaBarbera, 1982), and the feeding mode employed by some organisms {e.g., Lewis, 1968; Warner, 1977; Tagon et al, 1980; LaBarbera, 1984). Therefore, to fully and accurately understand the feeding processes and behavior of aquatic organisms, the natural flow regime of the animal in question must be taken into account (Reidl, 1971; Vogel, 1981). Nicol (1930) describes the morphology, ciliary tracts, and feeding currents of the sabellid polychaete Sabella pavonina. This very detailed work is one of the most complete descriptions of the feeding mechanisms of a polychaete (Fauchald and Jumars, 1979) and has been used as a model for other studies of sabellid polychaetes (Fitzsimmons, 1965; Lewis, 1968; Bonar, 1972). It has also been incorporated into the literature as a general model for feeding in the Sabellidae (Jorgenson, 1956, 1966; Dales, 1970; Ba:rnes, 1980). Nicol suggests that all water movement through the branchial crown of sabellids is due to ciliary activity. However, Nicol did not account for possible effects that ambient flow may have in this process. Her observations on whole worms were carried out in small closed containers of still water; finer details were ascertained by examining excised portions of branchiae. Received 29 March 1984; accepted 29 May 1984. * Present address: Marine Science and Maritime Studies Center, Northeastern University, East Point, Nahant, Massachusetts 01908. 200 FEEDING CURRENTS COMPARISON 201 Dales (1957) estimated the filtration rate (volume of material strained per unit fresh worm weight) of a variety of fan worms (sabellids and serpulids) in still water by measuring changes in the optical density of graphite particles and unicellular algal suspensions. He concluded that fan worms are "clearly . . . less efficient than other suspension-feeding invertebrates, both in the volume of water they are capable of straining, and in the kind of particles which can be retained" (p. 315). However, Warner (1977) suggested that sabellids are among the suspension feeders which can use ambient water movement to augment their own self-produced currents. Because the effect of the environmental regime has never been addressed in any study of sabellids, an important aspect of the feeding mechanisms and ecology of these animals has been neglected. Many species within Sabellidae aggregate into densely packed nonclonal mounds (Hartman, 1969). Aggregations of tube-dwellers have been shown to affect the pattern of ambient currents (Eckman, 1979, 1983; Nowell and Church, 1979). The amount of suspended material in the water may be augmented by resuspension of particles due to the presence of tubes (Eckman et ai, 1981; Carey, 1983) or may be depleted by the biological activity of the tube dwellers (Eager, 1964; Woodin, 1978, 1981; Levin, 1982). None of these studies has examined these phenomena for epifaunal tubes on hard substrates. This work examines three aspects of suspension feeding in the sabellid polychaete, Eudistylia vancouveri. First is a comparison of the water velocities produced by the cilia of the branchial crown with the velocities of ambient currents. Second, the effect of the dense hemispherical aggregates of worms on the water flow near the feeding crown is described. Third, removal of natural particles during a single passage of water across the surface of a worm cluster in the field is quantified. Materials and Methods Field site and animal collection Cattle Point, San Juan Island, Washington (48°27'N, 122°57'W) was the site for all in situ flow measurements, worm collection, and particle sampling. This rocky point extends into the Strait of Juan De Euca and is one of the most exposed points in the San Juan Islands. Specimens of E. vancouveri were collected by carefully peeling intact tubes away from the rock substrate during low tides. The animals were held in sea water tables with continuous circulation of fresh sea water. Only whole, undamaged worms were used for flow observation and measurement. Flow observation and measurement To measure and observe currents produced by cilia of the branchial crown, in- dividual worms were supported upright in their natural tubes in a 15 X 10 X 20 cm clear plastic container and fresh sea water was circulated through the space between this inner container and an outer chamber to keep the inner box at ambient sea water temperature (10°-12°C). Flow patterns produced by the branchial crown were visualized by releasing flu- orescein sodium (uranine) dye (dissolved in sea water) at various locations around the worm. This dye was injected through PE-50 catheter tubing, the end of which had been drawn into a fine point (~300 ^lm diameter). The flow rate of the dye was controlled with a micrometer buret. The dye injection apparatus was mounted on a micromanipulator (for further details, see LaBarbera, 1981). 202 R. A. MERZ I observed the positions adopted by the worms at different ambient velocities using a recirculating flow tank with a variable speed motor (Vogel and LaBarbera, 1978). Worms in their natural tubes were arranged with the tubes extending into the center of the flow tank. The velocity of ciliary currents in still water was measured with a thermistor flowmeter (LaBarbera and Vogel, 1976) modified to improve spatial resolution and to record very low velocities (see LaBarbera, 1981, for specific modifications and calibration procedure). Precision of velocity measurements was ±0.03 cm/s; accuracy was approximately the same. The probe was 500 (xm in diameter and its spatial resolution was 0.5 mm. Current velocities within and around in situ clusters were measured with a portable electromagnetic water current meter (Model 511, Marsh-McBimey, Inc.). Precision of this instrument was ±2% of reading. The probe was 2.5 cm in diameter. An adjustable aluminum scaffolding was used to hold the flow probe securely in the desired location while readings were taken. All in situ velocities were recorded on a Linear Model 142 portable chart recorder. All velocities reported here are the mean (±S.D.) of the peak velocities of a series of waves moving through a surge channel at a particular location. Measurement of particle depletion To measure removal of particles from the water by a cluster of E. vancouveri, water samples were taken from a single wave before and after it washed through a worm cluster. To insure that the same water mass was sampled on both sides of a cluster, fluorescein dye was released upstream from the worm. A water sample was taken immediately before this marked water moved through the worm cluster, and again as it emerged from the downstream side. During sampling the water surface was never more than 3 cm above the worm tubes. Thus, these samples represent water moving through the worm cluster at crown height. The water samples (30-80 ml) were taken with a large bore (5 mm) suction device. Each water sample was transferred to a sterile glass bottle, sealed, and stored on ice in the dark for transport to the laboratory. Elapsed time from sample collection to particle counting was less than four hours. Three different worm clusters were sampled in this manner. To estimate the repeatability of sampling, one cluster was sampled twice (two separate waves, 20 minutes apart). At another cluster, two downstream samples were taken from a single wave. In the laboratory, each water sample was gravity filtered through a 1 02 ^m Nitex filter. This filtered sample was then gently inverted several times and a 2 ml subsample was removed for measurement. The precision of counts from multiple subsamples is better than 5% (B. Best, pers. comm.). The frequency distribution for 128 particle size classes was tallied and recorded by an Elzone 80XY particle counter (Particle Data, Inc.). Particle size was measured as displacement volume and is reported as the diameter of a sphere of equivalent volume. For a description of this technique, see Haven and Morales-Alamo (1970). Results Pattern and velocities of worm-generated currents The pattern of water movement around a fully expanded branchial crown of Eudistylia vancouveri in still water agrees with Nicol's (1930) description (Fig. IB). FEEDING CURRENTS COMPARISON 203 t 0.070 Figure 1. Posture of the branchial crown of Eudistylia vancouveri in still and moving water. (A) Enlargement of a filament and its associated pinnules. (B) Position of the filaments in still water, arrows indicate the dye streams produced by ciliary currents, the speeds of which are reported as cm/s. (C) Position of the filaments in flowing water (20 cm/s), heavy arrow shows the direction of water movement, the position of filaments and pinnules were traced from photographs. The cilia-driven currents flow from under the branchial crown through the network of filaments. This flow is completely laminar. There is no evidence of the pulsatile flow that would result if this current were produced by the peristaltic pumping of the body within the tube. The streamlines do not mix within the branchial crown, but converge towards the midpoint above it. Current speed at the periphery of the crown (0.5 cm above the distal tips of the filaments) ranged between 0.025 and 0.045 cm/s (X = 0.035 ± 0.004 S.E.). The current speed above the center of the crown (no more than 0.5 cm from the midpoint) ranged between 0.056 and 0.080 cm/s (X = 0.070 ± 0.006 S.E.). Thus the speed at the center of the crown is about twice that at the periphery. This higher speed cor- responds to the larger number of cilia-covered filaments in this center region. Ad- ditionally, this central stream of higher speed may act to entrain the peripheral stream- lines and result in their convergence above the branchial crown. Crown posture in ambient currents The filaments of the branchial crown of £. vancouveri are arranged in two lateral spirals of equal size (Banse, 1979). When the worm emerges from its tube these paired whorls of filaments unfurl and fill a volume above the tube that is like a rounded cone or pointed hemisphere in shape. Each filament describes an arc, with the pinnules on the upper or oral surface (Fig. 1 A). In still water, the branchial crown is positioned symmetrically over the tube, with the plane of the base of the crown perpendicular to the long axis of the tube (Fig. 1 B). 204 R. A. MERZ In flowing water, 10 to 40 cm/s, the apex of the crown is angled downstream. In this position the leading edge of the crown is raised and the plane of the base of the crown is no longer perpendicular to the long axis of the tube (Fig. IC). The spiral tiers of the crown intersect the ambient flow at approximately 30° to 40°. This orientation of suspension feeding structures is known for a wide variety of animals (Warner, 1977). The paths of natural particles and fluorescein dye streams around the branchial crown in this orientation indicate that there is a downstream eddy into which the pinnules of the filaments project. At higher speeds (above 40 cm/s), the worms partially withdraw into their tubes, drawing the filaments of the crown together. Flow patterns in the field The distribution of flow velocities around a cluster indicates that the cluster acts as a semi-porous barrier or breakwater, causing the bulk of water in a passing wave to flow over the surfaces of the cluster rather than through the mass of tubes (Table 1, Fig. 2). The flow speed is highest at the surface of the cluster where the branchial crowns are positioned. Lower values occur within the cluster, below the surface of the branchial crowns. There is no appreciable change in flow speeds around solitary animals in the same habitat. Thus, animals in clusters experience higher flow at crown level than do solitary animals when both are located in the same habitat. Particle removal The total particle depletion during a single wave passage through a cluster ranges from 45 to 65%. Figure 3 gives the size-frequency distribution of particles in a wave before and after passing a worm cluster; Figure 2 illustrates the water sampling locations. There is less than a 9% difference in this value for two waves passing over the same cluster; and less than a 7% difference for duplicate samples taken from the same wave downstream from a cluster. The fraction of particles removed was not constant over all sizes {a = 0.05, Kolmogorov-Smimov; Siegel, 1956). In all cases the lowest percent particle removal was at the large end of the size distribution, above 7 ^m. The highest percent removal was between 3 and 6 /^m. Discussion Two factors have led to potentially erroneous views about the method and degree of success of suspension feeding in the sabellid polychaetes. In the first case, previous Table I Current speeds around clustered and solitary worms^ Mean maximum Mean maximum Mean maximum Cluster speed ±S.D. speed ±S.D. speed ±S.D. Worm length (N) (cm s"') (N) (cm s ') (N) (cm s^') density parallel to pre-ciuster crown level below crowns (tubes m^) flow (cm) Cluster 1 11.2 ±2.1 (13) 12.7 ± 1.0(12) 8.0 ±2.2 (13) 1243 50 Cluster 2 23.6 ±2.1 (11) 31.8 ± 1.8(11) 3.7 ±0.8(13) 2576 150 Cluster 3 27.7 ±2.9(16) 33.6 ± 2.3 (23) 16.6 ±2.7(17) 2448 50 Solitary worm 8.5 ±0.7(15) 8.5 ± 1.3(15) — 1 — The placement of probes around clusters is diagrammed in Figure 2. FEEDING CURRENTS COMPARISON 205 lOcm Figure 2. Flow and water sampling sites through a section of a cluster of Eudistylia vancouveri. (A) Site of pre-cluster water sample removed. (B) Site of post-cluster water sample removed. (C) Site of upstream dye release and velocity measurements. (D) Site of velocity measurements at crown height. (E) Site of below crown velocity measurements. Arrow indicates the direction of flow during water sampling and traces the path of the dye marker during one wave passing through the cluster. Note that the sampling site is in a surge channel and flow is bidirectional. Cluster drawn to scale. workers (Nicol, 1930; Dales, 1957; Fitzsimmons, 1965; Lewis, 1968; Bonar, 1972; Sorokin, 1973) have not taken into account the importance of ambient water movement in suspension feeding. They have all studied the process in still water, a fluid regime the worms rarely, if ever, experience. However, if the natural flow conditions are considered, it is clear that the velocity of water in contact with and surrounding the branchial crown is two to three orders of magnitude greater than that produced by the cilia alone. Thus, any estimate of feeding that is based on only ciliary currents grossly underestimates the rate at which water and food are processed by the worm. Second, it is important to realize that flow rates per se are not the sole criterion by which filtration efficiency should be evaluated. If a suspension feeder can acquire all the food it needs by generating low filtration velocities, it may well be more efficient than suspension feeders that generate high flow rates at greater energetic cost (this latter point is addressed in LaBarbera, 1984). In ambient flow, the majority of the feeding surface of the crown (the pinnules) is positioned on the downstream side of the worm. The wake of tubes is characterized as an area of relatively slow moving fluid, forming erratic spirals or eddies (Carey, 1983). Warner (1977) suggests that the advantage of positioning particle capture surfaces in this area is that reduced flow speed and chaotic or recirculating particle movement may enhance capture of the particles (also see Meyer, 1973; Rubenstein and Koehl, 1976). Clusters of E. vancouveri remove up to 65% of all particles in a wave washing over them (more than 70% of particles 3 to 6 ^m in diameter. Fig. 3). Sabellids are known to filter bacterial cells 0.5 nm in diameter from a suspension of single cells. Only sponges were shown to be as successful at this small size range (Sorokin, 1973). Other suspension feeders (ascidians, bivalves, and calanoid copepods) take larger particles (above 3 to 7 ^m in diameter) (Haven and Morales-Alamo, 1970; Sorokin, 1973; Vahl, 1973; Wright et al, 1982). Thus it may be that sabellids are concentrating on the smaller range of plankton composed of bacterio-, myco-, and small phyto- plankton, which are readily assimilated (Sorokin, 1973) and constitute the greatest proportion of the planktonic biomass (Sieburth et al, 1978). The densely packed mounds of tubes of E. vancouveri alter the velocity profile of an incoming wave. The thicket of tubes restricts water flow and forces the bulk of the water to travel over the surface of the mound. This flow pattern has two advantages for the worms. The tubes are subjected to lower drag forces (Merz, 1984) u 1200 / \ Cluster 1 900 /\ 600 300 f 1200 4) 900 Cluster 2 600 4) -Q E z 300 600 400 200 600 400 200 4 6 8 10 20 Particle Diameter (microns) Figure 3. Size-frequency distributions for natural suspended particles before and after passing through a worm cluster. In each case line U represents the upstream pre-cluster sample, line D or !> represent the post-clus';er sample (see Fig. 2). The results from three clusters are shown. In cluster 2, lines D and U represent two post-cluster samples taken from the same wave. Cluster 3A, B are measurements of the same cluster for two different waves, 20 minutes apart. (These clusters are not those shown in Table I). 206 FEEDING CURRENTS COMPARISON 207 and the feeding crowns are positioned in the fastest flowing water at the surface of the cluster (Fig. 2, Table I) where they are exposed to more water and food per unit time than is a solitary animal (Table I). Koehl (1977) suggests that sessile suspension feeders should minimize drag on their support structure while maximizing flow through their feeding structure. The hemispherical aggregates of E. vancouveri accomplish both of these objectives. Many suspension feeders live with non-clonal conspecifics in hemispherical mounds. For example, sabellid genera contain species that aggregate in this way (O'Donoghue, 1924; Chapman, 1955; Hartman, 1969; Koechlin, 1977), as do some chaetopterid polychaetes (Bailey-Brock, 1979), phoronids (Johnson, 1959; Ronan, 1975), and goose barnacles (Kozloff", 1973). All these animals are either passive sus- pension feeders or are facultatively active suspension feeders {sensu LaBarbera, 1977), and as such depend to some extent on ambient flow for feeding. These animals cluster together into potentially competitive aggregates for several reasons. One may be that domes increase flow through the feeding structures and simultaneously reduce drag on supportive structures. The mound itself forces the bulk of water to flow over the surface. Thus each animal is acting in its own interests, but in so doing contributes to the formation that benefits others in the mound. ACKNOWLEEXjMENTS I wish to thank the director and staff" of the Friday Harbor Laboratories for their help and the use of their facilities. In addition, the Department of Anatomy at the University of Chicago made it possible for me to live and work in Washington. Special thanks go to M. Landry and B. Best for generously loaning equipment and supplying technical assistance. R. Aller, B. Clark, M. Feder, M. Telford, S. Woodin, and an anonymous reviewer all added valuable ideas and criticism. I am particularly grateful to M. LaBarbera for his expertise, support, and enthusiasm. The research was supported by a Harper Fellowship from the University of Chicago to the author and NSF grant DEB-7823292 to M. LaBarbera. LITERATURE CITED Bailey-Brock, J. H. 1979. Sediment trapping by chaetopterid polychaetes on a Hawaiian fringing reef. / Mar. Res. 31: 643-656. Banse, K. 1979. Sabellidae (Polychaeta) principally from the Northeast Pacific Ocean. / Fish. Res. Board Can. 36: 869-882. Barnes, R. 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(August, 1984) GIANT SMOOTH MUSCLE nBERS OF THE CTENOPHORE MNEMIOPSIS LEYDII: ULTRASTRUCTURAL STUDY OF IN SITU AND ISOLATED CELLS MARI-LUZ HERNANDEZ-NICAISE, GHISLAIN NICAISE, AND LUC MALAVAL Histologie et Biologie Tissulaire, Universite Claude Bernard, 43 Boulevard du 11 Novembre, 69622 — Villeurbanne, France^ Abstract The lobate ctenophore Mnemiopsis leydii possesses giant smooth muscle fibers grouped in two sagittal bundles. Functional isolated cells were obtained by an enzymatic digestion of mesoglea and epithelia. Each bundle is made of 30 to 50 multinucleated cylindrical cells which may reach 35 ^m. in diameter and 4 cm in length. The nuclei and non-contractile organelles (mitochondria, golgi, rough endoplasmic reticulum) are contained in a discontinuous axial core, surrounded by a thick sheath of myofilaments. Thin (actin) filaments, 5.9 nm in diameter, form irregular rosettes around the thick (myosin) filaments, 16.1 nm in diameter. An actin:myosin filament ratio of 7:2 and a myosin density of 249 filaments per ftm^ were found in cross-sections of relaxed in situ cells. No dense bodies nor attachment plates were observed. From the coiled shape of contracted single cells and from the rearrangement of organelles in such coiled cells, we propose that myofilaments are organized in thin long myofibrils attached to the cell membrane at both ends, and that the attachment sites follow two (sets of) enantiomorphic helices. The sarcoplasmic reticulum is a longitudinally oriented 3-dimensional network of narrow tubules among the myofilaments. Its relative volume, estimated from cross sections, amounts to 0.9% of the contractile cytoplasm. No peripheral couplings have been observed, nor any tubular or vesicular invagination of the sarcolemma. Introduction The first giant smooth muscle fiber so far known has been described in a planktonic invertebrate, the ctenophore Beroe (Hemandez-Nicaise and Amsellem, 1980; Her- nandez-Nicaise et al, 1980). These multinucleated cells can reach several centimeters in length and 50 nm in diameter; they have no striation, dense bodies or attachment plates, at least when observed after conventional processing for electron microscopy. A simple technique using hyaluronidase and trypsin has been performed to isolate functional muscle cells of Beroe. Their ultrastructure and the electrical properties of their membrane are similar to those of /« situ fibers (Hemandez-Nicaise et al, 1982). Such a single giant smooth muscle fiber provides a multi-purpose model for cell biological studies of smooth muscle. Recent studies using a single cell preparation from a lower vertebrate (Bagby et al, 1 97 1 ) have already lead to a better understanding of smooth muscle cell organization and function (Fay, 1976; Walsh and Singer, 1981; Fay et al, 1983). These possibilities are hindered by the limited availability o^ Beroe. To our knowl- edge, this species is available along the French Mediterranean coast (Station Zoologique at Villefranche-sur-Mer) only from March to May, and as the animals feed upon Received 20 March 1984; accepted 29 May 1984. ' Present address: Cytologic experimentale, Universite de Nice, Pare Valrose, 06034 Nice. France. 210 MNEMIOPSIS GIANT SMOOTH MUSCLE RBERS 211 other fragile planktonic species they cannot be kept in good condition for more than one or two weeks in the laboratory. We thus tried to identify another species which could (1) yield giant smooth muscle fibers, (2) be available over long periods at low cost, and (3) be kept in a marine laboratory without excessive care. We found that the lobate species Mnemiopsis leydii (Fig. 1 ) meets all these re- quirements: it is possible to isolate functional giant smooth muscle fibers from the two sagittal muscle bundles; it is an ubiquitous neritic species of the southern shores of North America and is, for example, common throughout the summer in Woods Hole, Massachusetts; and the animals can be kept several days in still sea water, renewed every day. The possibility of rearing closely related species through long periods has been demonstrated (Baker and Reeve, 1974; Ward, 1974). The present report describes the method used to obtain functional single muscle cells and gives a detailed electron microscopic description of the in situ and isolated cell together with a stereological analysis of the sarcoplasmic reticulum (SR). The ultrastructural features oi Mnemiopsis giant smooth muscle cells are compared with those of Beroe. A preliminary electro-physiological investigation has been conducted prior to this morphological study to ascertain the physiological integrity of freshly isolated cells Figure 1 . Photograph of a living Mnemiopsis leydii. viewed from the tentacular side. The animal is swimming downward with mouth (m) and oral lobes (lo) below and aboral organ (arrow) uppermost. The pharynx (p) is visible through the transparent mesoglea. 1.5X. 212 HERNANDEZ-NICAISE £r .4L. (Hernandez-Nicaise et al. 1981) and has shown that the resting potentials (average: —56 mV), membrane impedances, and action potentials of isolated and in situ fibers do not differ significantly. Further electrophysiological studies are in progress (An- derson, in press). Materials and Methods Specimens oi Mnemiopsis leydii were collected with plastic beakers along the jetty of the N. M. Fisheries Service, Woods Hole, Massachusetts. The two sagittal muscle bundles (Fig. 2) were mechanically removed from the animals. Care was taken to eliminate as much mesoglea as possible, and to preserve the attachment of the muscle cells at both ends (endodermic lining of aboral canal and epidermis of the lips). The dissected bundles were kept in cooled artificial sea water (ASW) prepared according to Cavanaugh (1956) (NaCl, 423 mM; KCl, 9 mM; CaCb, 9 mM; MgCb, 23 roM; MgS04, 26 mM; NaHCOj, 2 mM). Cell preparation The procedure devised for Beroe muscles (Hernandez-Nicaise et al, 1982) appeared poorly adapted for Mnemiopsis muscular bundles, and was modified as follows: after a brief rinse in Ca-free ASW, the dissected bundles were pre-incubated at 30°C for 75-90 min in 0.3% hyaluronidase (type III, Sigma) in nominally Ca-free ASW. After this incubation the mesoglea surrounding the muscle cells was considerably softened and could be removed by dissection. The tissues were then incubated in 0.05% trypsin (type III, Sigma) and 0.3% hyaluronidase at 30°C for 20-30 min in Ca-free ASW. The bundles were then trans- ferred to Ca-free ASW. They were gently agitated by blowing them in and out of a large bore siliconized Pasteur pipette, until the individual cells were freed from the bundle. The released muscle cells were transferred to cold Ca-containing ASW. From this stage on, siliconized glassware was used. Electron microscopy Freshly dissected bundles could not be fixed by immersion into a fixative. Ob- servation of the bundles under the microscope during the onset of such fixation showed that the contact of the extremities of muscle fibers with the fixative triggers such a violent contraction that the muscles break into small fragments and disintegrate. To prevent this contraction two alternative protocols were devised: (1) the dissected bundles were incubated for 30 min in 0.3% hyaluronidase in Ca-free ASW prior to fixation; the cells remained relaxed when immersed in fixative, or (2) the bundles were incubated for one hour in Ca-free ASW prior to fixation; in this case the cells had an undulated appearance probably due to an interaction between the remaining intact mesoglea and the fixative. All the figures of in situ fibers presented in this study were obtained from bundles pretreated with hyaluronidase. Single cells were transferred by pipette into fixative without any damage. Bundles and single cells were fixed with 5% glutaraldehyde in cacodylate-buffered ASW (pH 7.7) at room temperature. Following a brief rinse in this buffered isotonic saline, the cells or the whole bundles were post-fixed in 2% osmium tetroxide in the same saline. Some specimens were block-stained with 1% tannic acid (Mallinckrodt) in 0.1 M sodium cacodylate (pH 6.5). The specimens were subsequently dehydrated in a graded series of ethyl alcohol followed by three changes in propylene oxide and embedded in durcupan. Silver grey sections were stained with uranyl acetate followed by lead citrate. MNEMIOPSIS GIANT SMOOTH MUSCLE FIBERS 213 Morphometry Six bundles from different individuals were cut transversally. An electron micro- graph at an initial magnification of 10,000X was taken from sections of seven fibers randomly chosen in each bundle. The magnification of the electron microscope was repeatedly calibrated against an optical grating replica (1260 lines/mm). Measurements were performed on prints at an exact magnification of 30,0(X)X, with an image analyzer Videoplan (Kontron, Germany). The total surface of each muscle fiber section and the surface occupied by the axial column and the mitochondria were measured. The difference between these two areas was defined as the contractile cytoplasm area ("Ace"). The area, "Asr," and membrane length, "Bsr," of sarcoplasmic reticulum (SR) were also measured and the number of SR profiles, "Nsr," was counted. Our hypothesis was that the morphology of the SR (see Results) is consistent with a stereological model of continuous tubules parallel to the main axis of the fiber, as defined by Weibel (1972) for the SR of striated muscle fibers. Under these conditions, the relative area, Asr/ Ace, and the relative membrane length, Bsr/Acc, of the SR on a given section are representative of the relative volume, Vv; and if the section is transverse, they are representative of the relative surface, Sv, of the SR in the contractile cytoplasm (Weibel, 1972). These stereological parameters were estimated, after a preliminary statistical study, by averaging the calculated ratios of each section (estimator 1 Asr 1 Rsr 1 of case II in Cruz-Orive, 1980), thus: Vv = - Z , Sv = - Z , n being the n Ace n Ace number of sections of measured fibers. In the case of Sv, the value obtained by . . ^ 4 Bsr assummg an isotropic organization Sv = — z , has also been calculated. The Trn Ace numerical density of the SR tubules in sections was estimated in the same way by 1 ^ Nsr NA = - Z . n Ace Quantitative study of myofilaments The densities and diameters of myofilaments were estimated from micrographs taken from the same sample as above. Measurements were performed on prints at a final magnification of 100,000X. The number of myosin filaments per ^m^ was counted on cross sections of 14 cells from 4 bundles. A one way variance analysis showed that there was no significant differences between two fields within a same section, between two cells of the same bundle, nor between two cells of different animals. Therefore a smaller sample was used to determine actin density (4 cells) and filaments diameters (4 cells, 50 filaments of each category per cell). Freezefracturing For freeze-fracturing studies whole bundles were fixed at Woods Hole in 5% glutaraldehyde in cacodylate buffered, Ca-free ASW. They were kept in ice-cold fixative for several days (3 to 7) until they were processed in our laboratory in France. The tissue samples were thoroughly rinsed in several baths of buffered saline and dissected into small pieces. The specimens were infiltrated with 30% glycerol in the same buffer for 3 hours. Tissue blocks were then mounted between two copper discs, rapidly frozen at -210°C in nitrogen slush, and placed into the double-fracture device of a "Reichert Jung CF 250" unit. They were then fractured at a stage temperature of 214 HERNANDEZ-NICAISE ET AL. — 1 50°C, under a vacuum of 1 . 10"^ Torr without etching. The exposed surfaces were shadowed with carbon/platinum at an angle of 45°. Replicas were cleaned with sodium hypochlorite, repeatedly rinsed in distilled water, and mounted on copper grids. Results Light microscope observations on living bundles The mesoglea of Mnemiopsis leydii is crossed by numerous muscle fibers. Most of them are too widely separated from each other and too small in diameter to be interesting either for in situ studies or as single cell preparations as is the case for Beroe muscle cells. Two sagittal bundles of thick long muscle fibers are known to occur in Mnemiopsis (Fig. 2) as well as in other lobates (Chun, 1 880), but have been paid little attention; they have even been reinterpretated as connective tissue. From our own observations on living animals, we are now able to give a more precise description of these muscular tracts. Each bundle consists of 30 to 50 muscle fibers, tightly packed into a flat ribbon contained in the sagittal plane; the bundles follow one edge of the pharynx wall along its entire length. At their aboral extremities, all the muscle fibers in each bundle are attached to the walls of the axial aboral canal which is a continuation of the pharynx cavity immediately below the apical sensory organ (see Hyman, 1 940). Muscle fibers c.r. au Figure 2. Diagram oi Mnemiopsis drawn after Figure 1 showing the course of the sagittal muscle bundles (mu) along the pharynx (dotted). At their oral extremities, the muscle fibers may either enter the base of the auricle (au) or lips; same legends as in Figure 1. On the left part of the drawing the auricle has been cut away (b.au; cut base of the auricle), and the paratransversal comb row is not represented. MNEMIOPSIS GIANT SMOOTH MUSCLE HBERS 215 progressively spread out from the bundle and insert on the body wall at various levels from the bases of the auricles down to the lips (Fig. 2). Each muscle fiber appears as a transparent cylinder running through the entire length of the bundle. The length of the bundle — and thus of the fibers — is directly related to the size (and age) of the animal. The longest fibers are also the thickest. Our electron microscope studies were conducted on a sample of bundles ranging from 1 to 2.5 cm in length, whereas in experiments and fixations on single cells we used the longest bundles, up to 3.5-4 cm. Light microscope observations on living cells Undisturbed isolated cells, or cells kept in Ca-free ASW, appear as smooth surfaced cylinders, as in the intact animal. In our experiments, the extremities of single cells either taper into thread-like endings, or swell into a bulbous stump suggestive of resealing which has been demonstrated in Beroe muscle cells (Hernandez-Nicaise et al, 1980) (Figs. 3, 4). Upon weak mechanical or electrical stimulation, the cells contract and become coiled (Fig. 4). The coiling is either clockwise, counter-clockwise, or both, along a given cell. Usually a weak stimulus initiates a localized thickened coil which may spread slowly along the cell, so that part of the whole length of the cell will take this spring-like profile. Such coiled fibers are never observed in an intact animal nor in bundles. Further stimulation or excess external K^ ( 100 mM) induces a strong contraction leading to a full shortening of the fiber. Usually it undergoes a brief coiled phase, then shortens dramatically into a thick straight cylinder with a transversally plicated surface (Fig. 5). Such an extreme shortening cannot be obtained in situ: in the intact mesoglea a strongly stimulated fiber will break into a row of bead-like fragments. In most cases an isolated cell will not stand such extreme shortening: the membrane usually bursts apart at one end, or the contractile cytoplasm detaches from the cell membrane and recoils at one extremity of the cell. Under Nomarski optics, the fibers exhibit an axial row of nuclei with no intervening septa. The muscle fibers of Mnemiopsis thus appear as long multinucleated cells. The contractile cytoplasm shows no transverse or oblique striation. It displays a fibrillar organization: fibrils run along the entire length of the cell and are entwined in crossed helices (Fig. 6). Electron microscope study The ultrastructure of cells in situ and of single cells appears similar. No modi- fications suggestive of damage caused by the enzymatic dissociation have been found in isolated cells. The following description is thus largely based on a survey of several bundles, with additional data gained from the study of sections of isolated cells. Examination of cross sections of various bundles confirms that the diameter of muscle cells is correlated with their length, and shows that the cells of one bundle are fairly homogenous in size. The shortest bundle examined has the smallest fibers, from 3 to 4.3 nm in diameter (average: 3.5 nm, 30 fibers), and the longest bundle has the largest fibers, with diameters up to 36 nm (Figs. 7, 8). The muscle cells of a bundle are attached to their neighbors by thin strands of dense mesoglea, usually less than 1 nm in width (Fig. 7). Each cell is surrounded by a basal lamina, probably modified by the hyaluronidase treatment, but nevertheless densely stained by tannic acid (Figs. 7, 8, 10). 216 HERNANDEZ-NICAISE ET AL. V \^ © © Figures 3-6. Micrographs of living muscle fibers isolated from the longitudinal sagittal bundles of Mnemiopsis; Nomarski interference contrast. Figure 3. Relaxed muscle fiber. The upper end has probably been cut during isolation and is slightly bulbous. Note the smooth sarcolemmal surface and the axial row of organelles. Scale bar: 100 fim (lOOX). Figure 4. Coiled muscle fiber. Parts of the fiber coil clockwise (dark triangle) while others coil counterclockwise (open triangle). Note again the bulbous stump at one extremity. Scale bar: 100 ^m ( lOOX). Figure 5. Fully contracted fiber. The cell has the appearance of a tightly compressed spring. The contraction has been obtained by exposing the cell to 100 mA/ external K"^. Scale bar: 100 ^m (150x). Figure 6. Relaxed cell, slightly compressed to show the double spiral pattern of fibrils. Scale bar: 50 ^m (350X). MNEMIOPSIS GIANT SMOOTH MUSCLE HBERS 217 y Figure 7. Part of a sagittal muscle bundle seen in cross section from a small Mnemiopsis. The diameter of the cells appears uniform. The cells are mechanically coupled by their longitudinal ridges (r) and/or strands of thickened mesoglea (arrows). Scale bar: 2 /xm (7000X). As observed with light microscopy the axial core contains a row of nuclei. The nuclei are euchromatic with a single large nucleolus, and have a smooth oval outline in relaxed cells (Figs. 7, 8, 11). They are surrounded by various organelles: sacs and tubules of rough and smooth endoplasmic reticulum, small Golgi bodies with their associated vesicles, and mitochondria. Between these organelles the axial cytoplasm may be filled by polysomes and free ribosomes, or by bundles of myofilaments. Mitochondria are restricted to the central core. Their number increases with the size of the muscle fiber. In our preparations they display a clear matrix which sometimes seems swollen, a feature known to occur in other ctenophores (Hemandez-Nicaise and Amsellem, 1980). If the cells have been allowed to remain in ASW containing Ca^^ (10 mM) the mitochondrial matrix contains several dark granules similar to those described in Beroe muscles (Nicaise and Hemandez-Nicaise, 1980). A peculiar feature of these organelles is the continuity of their external membrane with the outer nuclear envelope. Such contacts have been regularly observed in various sections from different animals. Myofilaments. In all cells examined, two types of filaments have been found, thick and thin (Figs. 8, 9, 10). Their size and distribution have been studied in cross sections of cells from bundles incubated in Ca-free ASW prior to fixation, these cells are thus presumed to be in a relaxed state. The thick, myosin-Hke filaments have a minimum diameter of 15.30 nm ± 1.79 (S.D.). Their density is 249 ± 17 (S.D.) filaments per ^m^ of organelle-free contractile cytoplasm in cross-section, and is remarkably constant in the sample studied, varying within a small range from cell to cell (231-264 filaments/^m') and within each cell (less than 10% variation). These filaments are regularly distributed in a nearly hexagonal r /_ /f^r f mi *«y> t»* A^^ ^^?S ¥.; K if- ^..■v..>- u ^(r- © «5^^v -^ .- Jr J ^C'-^'iBSB Figure 8. Cross section of an in situ muscle fiber from a medium sized Mnemiopsis. The axial nucleus is surrounded by several mitochondria (mi). The myofilaments are cut at various angles, the overall pattern suggesting an helicoidal arrangement. Note the numerous small SR tubules (arrowheads), nearly all sectioned transversally, and the clear "vesicles" tightly apposed to the cell membrane. Scale bar: 1 nm (14,500X). 218 MNEMIOPSIS GIANT SMOOTH MUSCLE HBERS 219 pattern (centered hexagons), with a center-to-center spacing varying from 70 to 95 nm (Fig. 9). The thin, actin-hke filaments have a diameter of 5.9 ± 0.84 nm. If the bundles are incubated in a solution containing the SI fragment of myosin (from chick smooth muscle), the thin filaments exhibit the classical arrowhead configuration. On this basis Figure 9. Transverse section of thick and thin myofilaments. The thick, myosin-Hke filaments form a nearly hexagonal lattice. The thin actin filaments form irregular rosettes around the thick myofilaments and are linked by finer microfibrils. Scale bar: 0.1 nm (140,000x). Figure 10. Periphery of a muscle fiber in cross section. Microfibrils link the myofilaments and attach on the plasmalema (open arrows). At the same sites fibrils originating from the basal lamina (b.l.) insert on the external side of the cell membrane (arrowheads). Scale bar: 0.1 /xm (140,000X). 220 HERNANDEZ-NICAISE ET AL. they will be referred to here as actin filaments. The ratio of thin-to-thick filaments is very stable, varying from 7.2 to 7.3, with a mean value of 7.22. In some areas of cross sections the thick and thin filaments are linked by radial cross-bridges stemming from the thick filaments. There is no definite orbital pattern, the thin filaments being arranged in irregular rosettes around the thick filaments (Figs. 9, 10). In restricted patches between thick filaments, adjacent actin filaments are linked by strands of an amorphous electron-dense material building an irregular network. No striation pattern or dense anchoring structures such as intracytoplasmic dense bodies or peripheral attachment plates have been found in these muscle fibers. However, following tannic acid block staining, discrete patches of fuzzy material can be seen underlying the inner leaflet of the cell membrane. This internal coat is continuous with the material linking the actin filaments. At these same localizations, microfil- aments, originating from the basal lamina, attach to the external leaflet of the cell membrane (Fig. 10). In longitudinal sections of relaxed isolated cells, the myofilaments run nearly parallel to the axis of the fiber and there is no evidence of existing myofibrils. In cross or longitudinal sections of coiled fibers, the overall aspect of the contractile cytoplasm is a zig-zag pattern. The myofilaments appear grouped in bundles cut at various angles, and delineated by a narrow clear space containing a tubule of the SR or one or several microtubules. In a given section the width of such bundles varies from 0.6 to 2 ^m (a more accurate estimate requires serial sectioning). We believe that these bundles are the myofibrils observed in living cells. Surface of muscle cells. Neither cells observed in sections from bundles nor isolated cells exhibit any permanent system of sarcolemmal invaginations. Some occasional infoldings may occur and are easily recognized in tannic acid-contrasted tissues. In the bundles so far examined, the surface of muscle cells shows longitudinal ridges which appear in cross sections as short (less than 1 urn) finger-like evagina- tions. The distribution of these ridges very nearly follows an axial symmetry (see Figs. 7, 8). Single cells do not display these ordered ridges, but instead show numerous slender processes, which are mobile in living cells (Fig. 1 1 ). Sarcoplasmic reticulum. In all the sections observed — from in situ and isolated cells — the SR appears distributed among the whole contractile cytoplasm. It is distinctly impregnated by tannic acid in isolated cells which have been fixed immediately upon enzymatic dissociation with no further recovery in normal ASW. From such prep- arations, the SR appears as a set of longitudinal tubules running along the myofilaments; they may be linked by lateral branches (Figs. 11, 12) or merge into larger vesicles. In longitudinal sections intersecting the nuclei, the tubules are in continuity with the nuclear envelope. The SR is not particularly abundant in the immediate vicinity of the plasma membrane. No peripheral coupling has been found so far in Mnemiopsis fibers, although some vesicles are very close to the cell membrane. On cross sections the SR profiles are irregularly distributed among the contractile cytoplasm, but their distribution is not random: in most cases the SR sections are aligned on imaginary spirals originating in the axial core toward the periphery of the cell (see Figs. 7, 8). From our sample of bundles the following quantitative data have been obtained: The SR profile in cross-section has an average area of 7.6 X 10"^ ^m' (±0.3 X 10"^ SEM) and an average perimeter of 0.36 /um (±8.5 X 10"^). The SR accounts for 0.91% (±0.035) of the volume of the contractile cytoplasm, and its relative surface MNEMIOPSIS GIANT SMOOTH MUSCLE RBERS 221 * I \ s.r. ■^■$igj4 "^ irr •® s.r. / / V ' -ii^sS?^-*^*''^'-' t ® >-ij2:^5-^-^3«®^ Figure 11. Longitudinal section of a relaxed isolated muscle fiber through the axial core. The elongated nucleus (N) is flanked by tightly packed mitochondria (mi). The SR (s.r.) is densely stained by tannic acid. A branching of a tubule is visible at the upper left (open arrow). Scale bar: 1 nm (9000x). Figure 12. Detail of the SR tubules from another section of the same cell as in Figure 1 1. The arrow points to a "mesh" of the sarcotubular network. Scale bar: 0.5 ^lm (30,000X). is 0.45 ^im^ltxvsY' (±0.018) if we assume a complete consistency with Weibel's parallel cylinders model (1972). It may amount to 0.57 jim^lnvcv' (±0.023) if we assume that the organization of the SR elements is completely isotropic amongst the myofilaments (see Discussion). The numerical density of SR profiles in the contractile cytoplasm is 1 .26 tubules per )um' (±0.06 1 ). These values are homogeneous for the whole sample despite the large variability in size of the muscle cells (one way variance analysis, F = 1.40). 222 HERNANDEZ-NICAISE ET AL. There is a slight but significant correlation between the value of the ratio Asr/Acc (or Bsr/Acc) and the area of contractile cytoplasm of the section if they are calculated on each section (r = 0.35 in both cases, P < 0.5%). Junctions. We looked for intermuscular junctions in all bundles studied. Few were found with most of them located between fibers of the periphery of the bundles. At these junctions the plasma membranes are apposed along variable lengths (0.3-4 )um), and may form close contacts at some restricted patches. At these close contacts, tannic acid post osmium stains the intercellular space — 4 to 5 nm wide — ?>^nN^ ■.0 ,■ >''»<■, ■■>■ > '® ->' .1*^%^.; :;1*-., H 14 ^ Figure 13. Junction between two cells of the bundle. Gap is stained giving rise to a pentalayered structure (tannic acid 1% post osmium). Note the thin bridges across the gap. Scale bar: 0. 1 Mm ( 1 50,000X). Figures 14-15a, b. Freeze-fracture replicas of a muscle fiber. Figure 14. The cleavage plan exposes the P face of the cell membrane and runs through a longitudinal ridge (r) thus exposing a band of cytoplasm (c). The replica shows a large array of polygonal gap junctional plaques, intercalated with particle-free aisles. Scale bar: 0.5 ^m (44,000X). Figures 1 5a, b. Complementary freeze-fracture replicas of part of the junction of Figure 14. E-face particles (Fig. 15a) correspond to a honeycomb array of pits on the P-face (Fig. 15b) (framed area in Fig. 14). Scale bar: 0.1 ^m (100,000X). MNEMIOPSIS GIANT SMOOTH MUSCLE RBERS 223 thus revealing a pentalayered structure (Fig. 13). The intercellular space contains bridges repeating every 16-20 nm. In freeze-fracture replicas, features characteristic of gap junctions were found on some muscles (Figs. 14, 15a, b). The E-face, or ectoplasmic fracture, of the muscle membrane exhibits polygonal clusters of 1 1-14 nm particles (Fig. 15a) and a com- plementary ordered lattice of pits — with a 17 nm center-to-center spacing — on the P, or protoplasmic fracture, face (Figs. 14, 15b). The clusters may be isolated or appear in large arrays (Fig. 14). In any cross section of a bundle, nearly each muscle profile is in contact with one or two clear circular profiles (0.3-1 ^m in diameter) encased between two pro- jections of the muscle membrane. These vesicles do not contain any structure except for an occasional network of microfilaments. At some discrete points the muscle cell and the "clear vesicle" come into close contact, with an intercellular space of 20-40 nm. Survey of the mesoglea surrounding the bundle has not given evidence for a special category of cell as the origin of these structures. We have found that muscle cells of the bundle and of the neighboring mesoglea can emit "blisters" of variable sizes which are apparently empty. We have not observed such evaginations in contact with a neighboring muscle cell. In some cases these "blisters" make reflexive contact with the mother cell. Some of the gap junctions observed in our freeze-fracture replicas may thus fall into this category of junctions. Discussion Viability of isolated cells Our light and electron microscope observations together with the electrophysi- ological data reported previously (Hemandez-Nicaise et al, 1981) demonstrate that enzymatically isolated muscle cells of Mnemiopsis leydii are functional cells: ( 1 ) the membrane appears ultrastructurally and physiologically intact, (2) the cells contract if stimulated or in the presence of excess of K^ ions in the bathing fluid, and (3) the ultrastructural features appear unchanged. Myofilaments We consider that the thick and thin filaments are myosin and actin filaments, respectively. As already mentioned the thin filaments are decorated by the S 1 subfrag- ment of heavy meromyosin, and preliminary data from gel electrophoresis indicate that Beroe muscle cells contain myosin but not paramyosin (A. V. Somlyo, pers. comm.). In ultrastructural cross sections the overall pattern of actin and myosin is very similar to the distribution observed in Beroe muscle cells (Hemandez-Nicaise and Amsellem, 1980). However, the quantitative data for myosin density and actin-to- myosin ratio differ significantly between the two species as reviewed in Table I. The actin-to-myosin ratio is distinctly lower than the values given for vertebrate smooth muscle (see review in Somlyo, 1980) and is closer to the figures published for several invertebrate phyla (see review in Hemandez-Nicaise and Amsellem, 1980; Plesch, 1977). Organization of contractile units One of the most striking features of Mnemiopsis muscle cells, apart from their length, is the absence of peripheral and intracytoplasmic dense bodies. 224 HERNANDEZ-NICAISE ET AL. Table I Comparison between morphological parameters ofMnemiopsis leydii" and Beroe ovata'' muscle filaments Beroe ovata in Beroe ovata Beroe ovata Mnemiopsis leydii situ cells state isolated cells isolated cells in situ cells relaxed unknown relaxed coiled Thick filament diameter 15.30 ± 1.79 nm 16.4 ± 0.3 nm 16.11 ± 2.12 nm 16.11 ± 2.12 nm Thick filament density 249 ± 17 fiL/^m^ 320-450 fil.//im2 457 ± 15 fil./Mm' 350-660 fil./Mm^ Thick filaments spacing 70-95 nm 40-60 nm Thin filament diameter 5.9 ± 0.84 nm 6.2 ± 1.6 nm 6.3 ± 0.6 nm 6.3 ± 0.6 nm Thin/thick filament ratio 7.22 2.5-7.5 5.22 ±0.21 4.88 ± 0.29 ' Means ± S.D. •"Data from Hemandez-Nicaise and Amsellem (1980) and Hemandez-Nicaise et al. (1982). In vertebrate smooth muscles the dense bodies are the morphological and functional correlates of fragmented Z Hnes (Bond and Somlyo, 1982). Their spatial distribution in oblique strands across the cell points to an organization of the myofilaments into small contractile units (Bond and Somlyo, 1982; Fay et al., 1983). A similar type of organization is found in invertebrate smooth muscles like the byssus retractor of Mytilus (Sobieszek, 1973). From our observations reported in the present paper, we propose that, as in Beroe (Hernandez-Nicaise et al., 1982), the myofilaments may be organized into two sets of thin myofibrils attached at their extremities on the muscle membrane along two (or two sets oO enantiomorphic helices. The small patches of sarcolemma coated internally by a filamentous coat are likely candidates as mini-attachment plates. The microfilaments linking those patches to the basal membrane are suggestive of fibronectin microfilaments known in other cell types to "cooperate" with actin bundles (Singer, 1979; and review by Hynes, 1981). Such bonds between the muscle membrane and the mesoglea may explain the fragmentation of muscle fibers under the action of fixatives. Each of the oblique myofibrils may be constituted of serial units, i.e., pseudo- sarcomeres. The diameter of the muscle fibers and the low angle between the myo- filaments and the fiber axis at rest, call for very long fibrils; but the possibility of a 75% shortening of single fibers implies that a fibril is made of a series of units with an important overlapping of myosin and actin filaments, rather than a single pseudo- sarcomere with one set of myosin filaments. The staggering of myosin and actin filaments in such small myofibrils accentuated by an unavoidable shearing, may thus account for the fact that in cross sections actin and myosin filaments are distributed as in A-bands of obliquely striated muscles fixed in a contracted state (see review in Rosenbluth, 1972). The structural and biochemical equivalents of intracytoplasmic dense bodies therefore have to be demonstrated in order to establish the existence of serial pseudo- sarcomeres. Indications of such attachment structures have already been obtained from electron microscopy of highly stretched Beroe muscles (unpub. obs.). Z elements with little or no contrast have been shown (if not specifically reported) in other primitive metazoans, namely Cnidaria. In figures published by various authors, we have noted that in cnidarian epitheliomuscular cells, the smooth muscle fibrils are devoid of dense bodies, and that striated myofibrils display a thin, wavy strand of fuzzy material in place of Z lines (Chapman, 1974; Amerongen and Peteya, 1976; Keough and Summers, 1976; Anderson and Schwab, 1981). Hoyle (1983) reports a striated muscle in a primitive Crustacean with "barely discernible Z regions." MNEMIOPSIS GIANT SMOOTH MUSCLE RBERS 225 Mnemiopsis muscles (and all other ctenophore muscles we have observed so far) also lack the so-called intermediate filaments, or desmin filaments. From published figures these filaments are equally absent in Cnidaria muscles. Desmin filaments occur in both smooth and striated muscles, and are associated with dense bodies (Small and Sobieszek, 1977; Bond and Somlyo, 1982) and the Z line (Behrend, 1977; La- zarides, 1980), respectively. The protein a-actinin is one of the main components of the Z line and dense bodies (Ebashi et ai, 1966; Schollmayer et ai, 1976; Endo and Nasaki, 1982) and is responsible for most of the electron-opacity of these structures together with desmin and vinculin (see review in Lazarides, 1980). Ctenophores and Cnidaria may lack a-actinin and desmin and may use other proteins (see review in Weeds, 1982), with a weak affinity for electron microscope stains, as bonds between the actin filaments and anchoring structures. Sarcoplasmic reticulum The SR of the giant muscle cells o^ Mnemiopsis is very similar in its morphology and distribution to that of 5^roe giant muscle cells (Hemandez-Nicaise and Amsellem, 1980; Malaval et ai, 1981; Malaval, 1982). In both species the heterogeneous dis- tribution of SR in the myoplasm and the alignment of SR profiles in cross sections along spirals, suggest an ordered three-dimensional pattern of this organelle along the fiber, which may reflect to some extent the organization of the myofibrils (Her- nandez-Nicaise et ai, 1982; Malaval, 1982). Our estimates of SR relative volume and correlated parameters have been calculated on the assumption that the SR distribution in relaxed Mnemiopsis muscles follows Weibel's model of parallel tubules (1972). Table II gives the values obtained for Mnemiopsis and Beroe muscles. It is theoretically possible to assess the relevance of this model for a given specimen by comparing the values obtained for Sv in different planes of section (Eisenberg et al, 1974). However, such a control appears only feasible on striated fibers, for which an estimate of the angle between the plane of section and the axis of the cell can be calculated from the length of sarcomeres. With Mnemiopsis fibers we can only consider the extreme values of Sv, the "real" value probably being closer to the figure obtained with Weibel's anisotropic model. To summarize, the relative volumes of SR in Mnemiopsis and Beroe muscles are closely similar in value but in Mnemiopsis the tubules are smaller and more numerous per cross sectional unit. Table II Comparison between morphological parameters of SR in muscle fibers o/Mnemiopsis leydii" and Beroe ovata^ Average area of SR tubule in cross section Average Relative Surface Average diameter of volume of SR density of Numcncal circumference of SR SR tubule in % of SR. density of SR tubule in cross in cross contractile expressed in tubules in section section c>1oplasm >im^/>im' cross sections Mnemiopsis 7.6- IQ-Vm^ ± 0.3 • 10"' 0.36 ^m ± 8.5 ■ 10" O.II4>im 0.91% ±0.035 0.45 + 0.018 1.26 ±0026 Beroe 1 15- 10"' >im^ ± 1 • 10"' 2 19- 10"' Mm^ ± 2- 10"' 0.48 >im ± 20- 10"' 0.59 *im ± 30- 10"' 0. 1 52 >im 1 .24% ± 0.070 0.40 ± 0.020 0.90 ± 0.070 0.186 ^m 0.70% ±0.50 0.22 ± 0.020 0.38 ± 0.0.30 * Means ± S.D. ""Data from Malaval (1982). The values were obtained from two samples from two animals and the two sets of figures differed significantly and are thus given separately (see Schmalbruch, 1979. for a similar problem with muscle parameter variability in a vertebrate). 226 HERNANDEZ-NICAISE ET AL. The wide range of sizes in our sample of sagittal bundles of muscle cells in Mnemiopsis has enabled us to find a positive correlation between fiber size and the SR volume. The relative volume of SR of ctenophore giant smooth muscles is thus low if compared with the figures generally given as reference for vertebrate smooth muscle, namely 5.75% in vascular smooth muscle and 2% in phasic smooth muscle (Somlyo, 1980). However very similar figures have been reported for mammalian smooth and striated muscles (Schmalbruch, 1979; McGuffee et ai, 1981). Furthermore, these studies show the unsuspected influence in such quantitative studies of various factors extrinsic to the preparation such as the fixation procedure (McGuffee et ai, 1981), the buffer used in fixing and rinsing solutions (Moriya and Miyazaki, 1979) and the physiological condition of the animal (Schmalbruch, 1979). A relative volume of 1% is estimated by Somlyo (1978) as the minimum amount of SR to allow a release of Ca^^ sufficient for activation of the contractile proteins. Assuming that this statement is true for ctenophores whose internal fluid and tem- perature differ widely from those of mammals, the value of 0.9 1% found in Mnemiopsis muscle cells is not too low to rule out that this organelle is involved in the onset of contraction. Another important indication for the role of SR in contraction is the presence of peripheral couplings (or dyads) between sarcoplasmic cistemae and the plasma membrane which presumably are crucial in excitation-contraction coupling. No cou- plings have been observed in Mnemiopsis, and very few cistemae are found close to the membrane. In Beroe muscles most of these couplings are found at neuromuscular and intermuscular junctions. We may have missed them in Mnemiopsis as we were not specifically looking for neuromuscular junctions. This observation does not nec- essarily exclude the intervention of the SR in ctenophore muscles in excitation- contraction coupling and certainly does not rule out a role for this organelle in the relaxation of these giant fibers (Malaval et ai, 1981; Malaval, 1982). If we adopt the criteria of classification proposed by Josephson (1975) and Plesch ( 1 977), the giant muscles oi Mnemiopsis and Beroe clearly fall into the same category. The muscle cells of the sagittal bundles of Mnemiopsis show fewer cross-bridges available per actin filament and fewer mitochondria than Beroe muscles. They are thus likely to be less powerful, less sustained, and slower than Beroe muscle cells. This is consistent with the behavioral data (Swanberg, 1974; Harbison et al, 1978). Junctions The few intermuscular junctions observed in the sagittal bundles o{ Mnemiopsis present the features of gap junctions, and more precisely the ultrastructural charac- teristics of E-gap junctions which are predominantly found in invertebrates (see reviews in Flower, 1977; Peracchia, 1981; Larsen, 1983). The precise distribution of these junctions among the muscles of a bundle remains to be evaluated. In vertebrates, gap junctions are widely accepted as the ultrastructural correlate of electrical coupling (Peracchia, 1981). They occur between smooth muscle cells (Fry et ai, 1977) which work in a coordinated fashion; for example, in a pregnant uterus the number of gap junctions between myometrial cells increases significantly at the onset of labor (Garfield et al, 1977). The contacts described in this study may link together the fibers of the sagittal bundles. To date however, an electrical coupling between muscles of a bundle has not been found (Anderson, in press). The empty "blisters" point toward another possibility: they may be broken parts of radial muscle fibers destroyed during dissection. Such a possibility is also suggested by Anderson (in press). With the isolation technique reported in this study, it is possible to use the giant MNEMIOPSIS GIANT SMOOTH MUSCLE FIBERS 227 smooth muscle fibers of Mnemiopsis for a variety of studies while controlling them individually under a simple dissecting microscope. In addition, Mnemiopsis sagittal bundle of parallel fibers can be used as multicellular homogeneous sample for quan- titative studies. Acknowledgments The authors thank Dr. S. Tamm, and Dr. T. S. Reese for their help during their stay at the M.B.L., Woods Hole; Drs. A. P. and A. V. Somlyo for the gift of SI fragment and many helpful comments; Dr. T. Simpson for critical reading of the manuscript; and A. 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ENCAPSULATION AND TISSUE INJURY NICOLO PARRINELLO, ELEONORA PATRICOLO, AND CALOGERO CANICATTI Institute of Zoology, Palermo University. Via Archirafi, 18 90123 Palermo. Italy ABSTRACT Particulate (sheep erythrocytes, ascidian oocytes, stromata, colloidal carbon) or soluble agents (bovine serum albumin or hemoglobin, hemocyanin) were injected in varying doses into the tunic oiCiona intestinalis. This ascidian reacted by producing a capsule and/or tissue injury. Statistical analysis suggests that the two phenomena are independent, probably related to the nature and dose of the irritant. Light histological observations showed granulocyte degranulation in the damaged tissue, suggesting that an acute inflammatory-like process is involved in the tunic reaction. Introduction To maintain body integrity, tunicates have evolved mechanisms which destroy and eject foreign materials. The defense responses include both humoral and cellular components. They could constitute a surveillance system ancestral to the vertebrate immune system (Parrinello and Patricolo, 1975, 1984; Parrinelloe/a/., 1977;Parrinello and Canicatti, 1982, 1983; Wright and Ermak, 1982). Attempts to demonstrate immunological capabilities in the ascidian Ciona in- testinalis have shown that it possesses natural (non-inducible) bacteriocidins (Johnson and Chapman, 1970) and hemagglutinins (Parrinello and Patricolo, 1975; Wright and Cooper, 1975) and that it reacts by phagocytosis and encapsulation to foreign materials inserted into the tunic (Parrinello et ai, 1977), and rejects a first set of tunic allografts (Reddy et al, 1975). However the source of the natural defense responses needs further examination, while tunic graft rejection involves some persistent non-specific inflammatory responses. C intestinalis non-specifically reacts toward large concentrations of erythrocytes injected into the tunic and produces a capsule around them (Parrinello et ai, 1977). Cells infiltrate the area and release substances enveloping the foreign material. This response is often strong enough to produce large capsules visible through the tunic. Also in a variable number of treated specimens, the tunic matrix over the injected erythrocytes lysed, and a tunic wound was produced. Animals with the injured tunic survived for a period of time dependent on the seriousness of the trauma. In specimens which showed a slight reaction the wound healed. Preliminary light microscopic histological observations did not clarify the nature of such a reaction. Moreover, the data did not establish a relationship between the capsule and the injury. In this study the C. intestinalis tunic injury and encapsulation produced by various doses of particulate or soluble agents were investigated by examining their external Received 6 March 1984; accepted 29 May 1984. Abbreviations: SE = sheep erythrocytes; PBS = phosphate-buffered saline (0.01 A/ pH 7.4 phosphate buffer containing 0.15 M NaCl); BSA = bovine serum albumin; Hb = bovine hemoglobin; He = Octopus vulgaris hemocyanin. 229 230 PARRINELLO ET AL. appearance. It is shown that they are two un-related processes which most frequently appear as a resuh of high doses of either particulate material or soluble proteins. The capsule structure is described elsewhere (Parrinello and Patricolo, 1984). We now report some observations on the injured tissue. Histological studies relate the gran- ulocyte degranulation to the injury process. Materials and Methods Adult Ciona intestinalis L. specimens (about 1 0- 1 2 cm in length) were collected from the harbors of Palermo and Porticello, Italy. Animals showing a tunic free of external marine matter were selected; they were maintained in aerated and frequently renewed sea water at 15-18°C. After several washings sheep erythrocytes (SE) (5 X 10^ 5 X 10^, 5 X 10^ cells/ml) and colloidal carbon (G. Wagner, Lot C 11/1431 A) (2 and 20 mg/ml), were suspended in phosphate-buffered saline pH 7.4 (PBS). Hemoglobin-free red cell membranes were prepared using the method of Davis and Bakerman (1972); packed ghosts were suspended at final concentrations (v/v) of 1, 3, and 10% in PBS. The response to a more complex cellular system was investigated by injecting C. intestinalis oocytes collected from several specimens, washed and suspended in PBS (1-1.2 X 10^ oocytes/ml). Various concentrations (0.2, 2.0, and 20.0 mg/ml) of bovine serum albumin (BSA) (Sigma), bovine hemoglobin (Hb) (Sigma), and Octopus vulgaris he- mocyanin (He) (kindly supplied by Dr. G. Nardi, Zoological Station, Naples), were prepared in PBS. 0.2 ml volumes of these preparations were injected into the tunic under the cuticle in the region of the sigmoid intestine with a syringe equipped with a 27-gauge needle. Control specimens were injected with 0.2 ml PBS in each experiment. The animals were inspected daily for signs of tissue reaction. Statistical analyses were performed using analysis of variance; the acceptable level of significance was P < 0.05. For light optical studies a large fragment (about 1 cm wide) of the injured tunic was fixed in 70% ethanol and embedded in paraplast, 5 Mm sections were stained with hematoxylin-eosin and Mallory's stains (Beccari and Mazzi, 1966). Hemagglutinating activity was assayed as previously described (Parrinello and Patricolo, 1975). Blood was collected from the heart. Hemagglutination titers are expressed as reciprocal of the last dilution giving agglutination. Results The tunic reaction Table I gives the external observations of the injected area during the tunic reaction. They show that, while PBS-injections never induce reaction in the control animals, particulate agents or soluble proteins can elicit two distinct types of tunic response. (1) SE, ascidian oocytes, BSA, Hb, and He induce a capsule which includes the injected materials. This appears 2 to 8 days after the injection as a whitish circular or elliptical disc ( 1 .5-3.0 cm wide) included in the tunic tissue (Fig. 1 ). (2) SE, oocytes, the highest doses of stromata and colloidal carbon, BSA, and Hb induce a drastic response which produces a local injury of the tunic: a blister forms in the treated area, and the overlaying cuticle becomes thin and finally ruptures (Fig. 2a). When the foreign material is particulate the debris can disappear. In some specimens the two responses against SE, BSA, Hb, and He can occur together. In this case the injury can appear before or after the encapsulation. Both types of response show some degree ASCIDIAN INFLAMMATORY-LIKE REACTIONS 231 Table I Reaction o/Ciona intestinalis to intratunic injection of particulate or soluble materials Dose'" Injected specimens Number of reacting specimens showing Irritant agent Capsule A Capsule plus injury B Injury C Days to produce 50% reacting specimens SE 1 X 10' 1 X 10* 1 X 10' 270 422 937 49(18.1) 176(41.7)" 332 (35.4)'' 13(4.8) 24 (5.7) 266 (28.4)^ 10(3.7) 40 (9.5) 33 (3.5) 6-8 4-8 3-6 Stromata from SE 1% 3% 10% 134 111 135 — — 40 (29.6) 5 Oocytes 1-1.2 X 10' 50 36 (72.0) — 12 (24.5) 5 Colloidal carbon 0.2 mg 2.0 mg 113 106 — 7 (6.6) 9 BSA 0.04 mg 0.4 mg 4.0 mg 100 100 100 1 (1.0) 16 (16.0) 19(19.0) 40 (40.0) 8 (8.0)" 92 (92.0) 8 4 1 Hb 0.04 mg 0.4 mg 4.0 mg 130 124 126 4(3.1) 48 (38.7) 14(11.1)'' 15(12.1) 68 (53.9)" 2(1.6) 14(11.1) 4 2 2 He 0.2 mg 112 19(16.9) 17(15.2) — 4 PBS 0.2 ml 627 — — — SE = sheep erythrocytes; BSA = bovine serum albumin; Hb vulgaris hemocyanin; PBS = phosphate-buffered saline. '" In 0.2 ml. Percentage is reported in parentheses. 'P < 0.01 in comparison to B and C. ^ P < 0.05 in comparison to C, f < 0.01 in comparison to C. " Z' < 0.01 in comparison to C. '' F < 0.01 in comparison to B. bovine hemoglobin; He = Octopus of modulation depending on the injurious agent. The capsule visible through the tunic appears in some of the specimens injected with erythrocytes (1 X 10"'-1 X 10^), ascidian oocytes, BSA, Hb (0.04-4.0 mg), or He (0.4 mg). It is invisible when colloidal carbon or stromata is used even in high concentrations. The injury is produced by either particulate or soluble materials. Each agent, if injected in high doses, can elicit tunic damage as the only visible phenomenon (Table I). The results in Table I suggest that encapsulation is not related to the injury formation. Significant differences (P < 0.01) were found between the frequencies of the capsule and injury responses produced by 1 X 10^, 1 X 10^ erythrocytes, or 4.0 mg BSA. Significant differences also resulted when "capsule plus injury" response was compared with each of the other two reactions produced when 1 X 10^-1 X 10' SE, 4.0 mg BSA, or Hb were injected. The dose of the irritant affects the response type and frequency, the largest dose being more effective in producing the tunic reaction. Different doses were examined statistically for each response type (Table II). Higher SE and protein concentrations 232 PARRINELLO ET AL. Figure 1 . Capsule seven days after 0.4 mg bovine serum albumin injection into the tunic of Ciona intestinalis (arrowhead). produced capsules in a significantly {P < 0.01) larger number of specimens. The highest doses also elicited an increased injury frequency. There is variability in the time required for the appearance of the tunic reaction in about 50% of the reacting specimens in each treatment (Table I). The appearance time is inversely proportional to the dose and depends on the nature of the foreign material. Fast reactions (1-2 days) of either capsule, injury, or both were observed when concentrated protein solutions were used (4.0 mg BSA, 0.4-4.0 mg Hb) whereas slow reactions appearing in 4-8 days resulted at lower concentrations (0.04, 0.4 mg BSA, and 0.04 mg Hb). The same pattern characterized the effects of the various SE doses (see below) while only the highest concentration of stromata produced this response. The reaction time is also influenced by animal variability: in 10 different C. intestinalis groups (937 specimens) injected with the highest erythrocyte dose, about 50% of 631 specimens reacted within 3-6 days depending on each animal lot, the remainder reacted after 7-8 days. Variability in response frequency was also observed between the lots. The greatest variability characterized the mean frequency values of the injury responses. The mean values calculated from all experiments are indicated in Figure 3. Hemagglutinating activity of the serum was tested with sheep or rabbit (RE) erythrocytes. To perform the assays the blood of five specimens was collected and pooled daily. Sera from PBS-injected animals showed no activity against SE, but agglutination titers of 2-4 were observed against RE. No changes were found in the hemagglutinating titers throughout the reaction period following SE injection. The response toward particulate materials Encapsulation is the most frequent response to erythrocytes and ascidian oocytes (Table I). Sheep erythrocytes can produce either a capsule or, less frequently, a tunic injury. Both responses are dose-dependent. The frequency of the tunic injury, even ASCIDIAN INn.AMMATORY-LIKE REACTIONS 233 •^^^.iJ A ,^|B ;^--— ^^Tmjf-^ . \: tm 0^ \ Figure 2. Tissue damage seven days after erythrocyte injection into the tunic of Ciona iniesiinalis. (a): Injured tunic (arrowhead); (b-f): transverse sections of the injured tunic (Mallory's stain); (b): lesion of the tunic, 80X; (c): hole in the tunic matrix (tm), 380X; (d): edge of the injured tunic, 380X; (e): eosinophil granulocyte, 1420X; (f): degranulation by cell membrane dissolution, 1420X. g = granulocyte; h = hole; arrows indicate granules. when appearing with the capsule, increased with increased dosage (Fig. 3). The results in Figure 3 also show a pronounced variability in the responsiveness of the various animal groups to each dose. The stromata at the highest dose and oocytes at the dose used also frequently elicited tunic injury, both being not very injurious agents. In fact, several days after the injection (5-8 days), these irritants produced tiny blisters (5-6 mm wide) containing gelatinous materials. Tissue damage rarely occurred when stromata were the irritants and the tunic subsequently healed. Colloidal carbon was the least effective injurious agent even if injected in high quantities (2.0 mg/specimens). Only 6.6% of the 106 treated specimens showed a small blister like that described above. 234 PARRINELLO ET AL. Table II Tunic reaction o/'Ciona intestinalis: comparison of the dose response by analysis of variance /'-values*" Irritant Capsule and agent Dose compared Capsule injury Injury SE 1 X 10' vs. 1 X 10' P<0.0\ N.S. P<0.0\ 1 X 10' vs. 1 X 10' P<0.01 P<0.0\ P<0.0\ 1 X 10' vs. 1 X 10' P<0.01 p • •??/,.: ?.-->> >;:.:fei. ■.^im^'.mi Figure 1 . Encapsulation of ascidian oocytes injected into Ciona intestinalis tunic, (a) Capsule seven days after injection (arrowhead), (b-e) Transverse sections of the tunic containing capsule (Mallory's stain); (b) arrows indicate the lined up cells which envelop the foreign material, 40X; (c) closer view of the capsule 470X; (d) amoeboid vesicular cells (v) forming the capsular outline, 630X; (e) vesicular cells which release vacuolar content to form an encapsulating strip, 1420X. c, cuticle; e, epithelium; gp, granule-packed cells; od, oocyte debris; tw, tunic wound. originating from the tissue under the epithelium to enclose the injured tunic up to the cuticle (Fig. 2). Histological observations of tunic fragments from five specimens injected with 1 X 10^ SE and two specimens injected with oocytes, showed that cells accumulated around the wound in large bands in the tunic, from the epithelium to the foreign 242 PARRINELLO AND PATRICOLO Figure 2. Capsule transverse sections seven days after sheep erythrocyte injection into Ciona intestinalis tunic (Mallory's stain), (a) Tunic inner zone Hned by epithelium (e), 95X. (b) Closer view showing granule- packed cells (gp), released granular material (rg) and vesicular cells (v), 570X. material. They are called "granule-packed cells" because their cytoplasm is completely filled with strongly aniline blue (Mallory's trichrome) stained fine granules which mask the nucleus (if present); their diameters range from 2.1 to 5.6 ^m (Fig. 3a-c). The granules are positive to Millon, PAS, and alcian blue reactions. In some cells they are less packed and more easily distinguished, in other cells the granular material apparently dissolves while cells are elongated and stretch out releasing their contents into the tunic matrix. In three specimens injected with erythrocytes, granule-packed cells were found in close contact with the epithelium and up to the inner border of the wound. In longitudinal sections they were arranged in 2-3 concentric layers around the wound. ENCAPSULATION IN ASCIDIAN TUNIC 243 Figure 3. Capsular components seven days after erythrocyte injection into Ciona inieslinalis tunic, 1420X (Mallory's stain), (a-c) Various features of granule-packed cells, (d) Vesicular cells (v) and granule- packed cells (gp) in close contact, (e) Lymphocyte (arrow) among vesicular cells and elongated granule- packed cells, (f) Transitional cell (arrow), (g) Immature vesicular cell (arrow), (h) Large vesicular cells releasing amorphous material, (i) End stages of granule-packed cells and vesicular cells, n, nucleus. 244 PARRINELLO AND PATRICOLO Vesicular cells filled the reacting tunic particularly at the inner edge of the wound. They range in size from 3.5 to 7.1 ^m and vary in appearance according to their stage of development. These cells can assume an amoeboid shape and fall into line to form the capsular outline and encapsulating strip, some release their amorphous contents by dissolving a portion of their membrane (Fig. Id, e). The vacuolar substance was negative with Mallory's stain, PAS, Millon, and alcian blue reactions. Granule-packed cells and vesicular cells can be in close contact; their membranes apparently dissolve and their contents mix (Fig. 3d, i). Both cell types appear to contribute to the production of capsular substance. In some zones, the granular material has a streaked appearance due to ghosts of the large vesicular cells. Small lymphocytes (2. 1-3.4 ^m) with an aniline blue slightly cytoplasm (Fig. 3e), are present in the reacting tunic. Spherical cells, 3.2-4.5 ^m in diameter, with an eccentric or central nucleus and aniline blue staining cytoplasm are considered transitional cells. They are frequent among the granule-packed cells and are numerous near the epithelium as far as the inner edge of the wound. Figure 3 (e-g) shows the presumptive stages in the vesicular cell differentiation. The cells of the mantle epithelium (9.2-1 1.3 nm, in surface view), show vacuoles 3.5-7.4 nm wide containing an amorphous substance which is negative to Mallory's trichrome, PAS, Millon, and alcian blue histochemical reactions. Large vacuoles can release their contents into the tunic matrix by dissolving a portion of their membranes. Multi-layered epithelium was observed in some capsule transverse sections (Fig. 4). Granulocytes (6.2-7.6 nm) are distributed along the edges and inside the wound; they contain eosinophil granules 0.6-0.8 ^m in diameter and can degranulate, releasing the latter by cell membrane dissolution. In wounds produced by erythrocyte injection, the intensely stained material distributed along the edges makes it difficult to identify the granulocytes. Large round cells (7.5-8.5 ^m), probably phagocytes, containing vacuoles full of aniline blue-staining material, are frequently found at the edges of the wound. These vacuoles occupy the cytoplasm and contain substances which are PAS and Millon positive but alcian blue negative. The nucleus is indistinguishable (Fig. 5a). Large refringent granule cells (4.5-6.4 fxm) are numerous in the wound. A single large orange-stained granule occupies almost all of the cell (Fig. 5b). Cells with a smaller refringent granule show more evident semilunar cytoplasm. Other cells release their granule by dissolving a portion of the cell membrane. These last two cell types are not frequent in the tunic wound containing oocytes. Compartment cells (6.3-7.9 ^m) are characterized by 2-4 vacuoles separated from each other by cytoplasmic partitions (Fig. 5c); they are similar to those described in the blood of other ascidians (cfr. Wright, 1981). The vacuolar contents can be slightly stained by aniline blue or appear as yellow stained refringent inclusions; in some cells a prominent refringent inclusion can occupy a large part of the cytoplasm (Fig. 5d). The nucleus is not visible and the cell outlines are often obscure (Fig. 5e, f). Free yellow refringent granules (2. 1-2.8 ^^m) can be found (Fig. 5g). Large numbers of compartment cells and free granules populate the wound. The specimens injected with stromata (1% or 10%) or colloidal carbon (2 or 20 mg/ml) never showed an evident capsule. However histological investigations revealed some capsular components in the injected area. Within 6-7 days vesicular cells in various differentiation stages were distributed in the tunic matrix between the epi- thelium and the inner edge of the wound. Round phagocytes full of deeply aniline blue-stained material or carbon masses, and large refringent granule cells are scattered ENCAPSULATION IN ASCIDIAN TUNIC 245 '^■f\ I i .^kX^ ' } I Figure 4. Epithelium and inner zone of Ciona intestinalis tunic after sheep erythrocyte injection (Mallory's stain), (a) Transverse sections showing single layered epithelium (e) and granule-packed cells (gp), 570x. (b, c) Active epitheUum releasing vacuolar content, 1420X. (d) Surface view of the epithelial cells, 1420X. V, vesicular cell; n, nucleus; tm, tunic matrix. among the foreign materials. When the highest doses were used these cells were more numerous. Capsule induced by soluble proteins. Histological transverse sections of capsules obtained 6 days after 0.4 mg protein (BSA, He, Hb) injection, showed that the tunic around the wound is populated with vesicular cells varying in differentiation stages. Inside the wound, aniline blue-stained large round cells and large refringent granule cells are frequent; granulocytes are settled on the inner edge, where they can degranulate. Fine granular materials (PAS, Millon, and alcian blue positive) were layered on 246 PARRINELLO AND PATRICOLO Figure 5. Cells inside the tunic wound seven days after erythrocyte injection; erythrocyte debris is indistinguishable, 1420X (Mallory's stain), (a) Large round phagocyte, (b) Large refringent granule cell, (c) Compartment cell, (d-f) Compartment cell with refringent inclusions, (g) Free refringent globules (arrow) inside the wound. the epithelium forming a band which gradually decreased in thickness toward the capsule margin. Early stages in the tunic reaction Histological study of the tunic showed that rapid cellular responses follow eryth- rocyte injection. After 2-4 hours vesicular cells in various differentiation stages pre- dominate near the epithelium. Aniline blue-stained large round phagocytes were scattered among the erythrocytes; the latter formed large masses at the inner edges of the wound. In tunics fixed after 12-16 hours (Fig. 6a, c-e) many hyaline and granular amoe- bocytes (7.1-11.2 X 2.3-3.5 /xm) formed a band along the inner wound edge and ENCAPSULATION IN ASCIDIAN TUNIC 247 If li-mrmiiftfiiiraiiM tw « J^ I i#i' b . /- i Figure 6. Early response in the tunic reaction of Ciona intestinalis after erythrocyte injection, (a, b) Transverse sections showing tunic wound (tw) and amoebocyte (a) infiltration, (a) Twelve hours after injection, 160X. (b) Twenty-four hours after injection, 390X. (c) Benzidine-positive amoebocytes, 980X. (d) Hyaline amoebocyte (arrow), 980X. (e) Granular amoebocyte (arrow), 980X. g, granule; v, vacuole, a, b, d, e: Mallory stained. showed lobose pseudopodia. Some vesicular cells lined up forming an encapsulating strip while others filled the tunic matrix and may be am.oeboid in shape. Large refringent granule cells are numerous inside the wound and are also present in the inner tunic layer below the wound. Nodules of transitional cells are observed near the epithelium. In tunics fixed after 24 hours, the above cells are more numerous. On the wound inner edge, the cells are full of aniline blue-stained material (Fig. 6b) while the eryth- rocyte masses are not easily distinguished. After 48 hours many compartment cells, with vacuoles containing yellow refringent material alone were found inside the wound. Granule-packed cells were also present in the tunic while large vacuoles characterized the epithelial cells. At 12 and 24 hours the large round cells inside the wound and the amoebocytes were positive to the benzidine histochemical reaction for hemoglobin (Fig. 6c). Tests carried out at 48 hours and 6 days were negative. The tunics from specimens which 6-7 days after SE injection did not show an apparent capsule, presented in the injection site the same components found in the early stages (12-48 hours) of the capsule development. Low general cell frequency and few granule-packed cells characterize these tunic areas. The capsule increases in thickness and 12-20 days after 5 X 10^ SE injection, forms a protuberance of the tunic and a decreased number of cells. The wound was reduced, probably by coalescence of the edges which appear partially free of cells and material. Encapsulating strips, when present, contained few vesicular cells. A quick response followed stromata or colloidal carbon injection. Vesicular cells 248 PARRINELLO AND PATRICOLO formed thin encapsulating strips in the tunic fixed after 24-48 hours. Small carbon masses were in the round phagocytes inside the wound. Hyaline and granular amoe- bocytes with carbon inclusions, formed a band along the inner wound edge. Granule- packed cells or free granular material were never observed in the tunic. Discussion Ciona intestinalis is capable of recognizing non-self materials and reacts by cellular responses. Foreign agents inserted into the tunic, including tunic tissue allografts, are removed from the body (Reddy et al, 1975; Wright and Cooper, 1975; Parrinello et al, 1977). These reactions are considered to form part of the internal defense system. They show characteristics of inflammatory-like processes including phagocytosis, en- capsulation, and tissue damage. Encapsulation is a response elicited by both corpuscular material and soluble proteins (Parrinello et al, 1984). The nature and dose of the foreign agent determine the strength of the response and influence the structure of the capsule which is visible in the most reactive specimens. Early stages of encapsulation also occur in the tunic of apparently non-reactive animals; the absence of granule-packed cells or granular material lining the active epithelium could account for the non-appearance of the capsule. The removal of the foreign material and tunic debris may be rapidly effected by phagocytes. These cells correspond reasonably well to the blood hyaline and granular amoebocytes described by Rowley (1981). They may begin the tunic reaction by performing non-self recognition. The nature of the cell contents in the responses to various agents suggests that the large round cells inside the wound could also be phagocytes. The removal of large masses may be facilitated by a lysosomal mechanism dependent on the eosinophil granulocyte degranulation. Large numbers of these cells were found in the wound containing oocytes, where the resultant degraded material may induce further inflammation {cfr. Hirschhom, 1974; Gleisner, 1979). Such activity may also lead to tunic injury (Parrinello et al., 1984). Lymphocytes can infiltrate the inflamed tunic, however their quantity is not important to capsule composition. The transitional cells are numerous. They are a first stage in which stem cells differentiate to produce capsule cells. From morphological evidence the presumptive stages in vesicular cell differentiation are as described in the blood by Millar (1953). Lymphocytes and transitional cells as well as granular and hyaline amoebocytes may originate from the blood or from the nodules of hemopoietic tissue situated in the pharingeal wall and around the gut loop (Ermak, 1976). An unidentified amorphous substance contributes to the manufacture of the capsule matrix. It is contained in large vacuoles of mantle epithelium cells and vesicular cells. It is released by vacuole and cell membrane dissolution. Vesicular cells rapidly fill the inflamed area and line up in a continous layer, isolating the injured tissue. In this respect encapsulation may involve the mechanisms that construct or regenerate the tunic. Enlargement and vacuolization of epithelial cells and cell types ("cell with a small acidophil vacuole" and "phagocyte univacuolated"), probably corresponding to differentiation stages of vesicular cells, have been described by Peres (1948) in the tunic regeneration of C intestinalis. Moreover, in the ascidian tunic, epithelial cells take up the glucose used in ground substance production. In C. intestinalis this monosaccharide is used in the manufacture of acid mucopolysaccharides, and may be incorporated into the tunic fibers (Robinson et al, 1 983). Evidence for the cellulose nature of this capsular material is not available, but histochemical evidence suggests ENCAPSULATION IN ASCIDIAN TUNIC 249 that it collaborates with mucopolysaccharides in tunic matrix formation. This was particularly evident when the fate of the vesicular cells and the granule-packed cells was observed. The packed fine granules consisting of mucopolysaccharides gradually dissolve and apparently mix with the vesicular cell products. The origin of the granule packed cells and fine granular material is unknown. They might have an epithelial origin because they frequently appear in close contact with the epithelium. Large refringent granule cells have been described in the regeneration of the tunic as occasionally occurring across the epithelium (Peres, 1948) and usually present just below the cuticle. They may be involved in the production of this glycoprotein layer (De Leo et ai, 1981) and may produce the glycoproteins inside the wound and contribute to the healing by cuticular material formation. Compartment cells could be stages in morula cell development as proposed for the corresponding blood cells in other ascidian species (Endean, 1960; Kalk, 1963; Smith, 1970). The absence of mature morula cells in the inflamed area could depend on the activity of lysosomal enzymes which interrupt cell development and induce granule release. It is known that morula cells congregate and break down at the edge of wounds produced by extirpating fragments of Halocynthia aurantium tunic (Smith, 1970). Moreover, they form the capsule which surrounds glass fragments inserted into the tunic of Molgula manhattensis (Anderson, 1971). Differences in the cell composition of the capsules apparently depend on the nature and size of the irritant. Apart from the granule-packed cells and round phago- cytes, the other cells are also found in the normal or PBS-injected tunic. They are variably distributed and definitely less frequent. As yet the origin of the capsule cells is unknown. The contribution of naturally occurring humoral factors in the ascidian non-self recognition mechanisms is unclear. However, hemagglutinins are probably not involved in the erythrocyte tunic encapsulation of C intestinalis (ParrineUo et ai, 1984). In summary, the tunic reaction of C intestinalis is a response consisting of several processes: (1) non-self recognition by phagocytes which can intervene in early stages of the reaction; (2) degranulation of eosinophil granulocytes which can disrupt large foreign material, probably by lysosomal mechanisms which in some cases may cause tissue damage (ParrineUo et al, 1984); (3) tunic matrix substance production with formation of a thick capsule to isolate the inflamed area; and (4) cuticular glycoprotein production to heal the tunic wound. Acknowledgments This work was supported by an M.P.I, grant. We would like to thank Mr. G. Miceli for his assistance in printing the photographs. LITERATURE CITED Anderson, R. S. 1971. Cellular responses to foreign bodies in the tunicate Molgula manhattensis (De Cai). Biol. Bull. 141: 91-98. Beccari, N., and V. Mazzi. 1966. Manuale di Tecnica Microscopica. See. Ed. Libraria. 366 pp. Bresciana, J., and J. LCtzen. 1960. Gonophysema gullmarensis (Copepoda Parasitica). An anatomical and biological study of an endoparasite living in the Ascidian Ascidiella aspersa. I. Anatomy. Cac. Biol. Mar. 1: 157-184. Davis, R. F., and S. Bakerman. 1972. Pp. 151-153 in Cellular Antigens. A. Nowotny ed., Springer- Verlag, New York. De Leo, G., E. Patricolo, and G. D'Ancona Lunetta. 1977. Studies on the fibrous components of the test of Ciona intestinalis Linnaeus. I. Cellulose-like polysaccharide. Acta Zool. (Stockh.) 58: 135-141. De Leo, G, E. Patricolo, and G. Frittitta. 1981. Fine structure of the tunic oi Ciona intestinalis L. 250 PARRINELLO AND PATRICOLO II. Tunic morphology, cell distribution and their functional importance. Acta Zool. (Stockh.) 62: 259-271. Dudley, T. L. 1968. A light and electron microscopic study of tissue interactions between a parasitic copepod, Scoledocus hunlsmani (Henderson), and its host ascidian Styela gibbsi (Stimpson). / Morphol. 124: 263-281. Endean, R. 1960. The blood cells of the ascidian Phallusia mammillata. Q. J. Microsc. Sci. 101: 177- 197. Ermak, T. H. 1976. The hematogeneic tissues of tunicates. Pp. 45-56 in Phylogeny of Thymus and Bone Marrow-Bursa Cells, R. K. Wright and L. C. Cooper, eds. Elsevier/North-Holland, Amsterdam. Ganter, p., and G. Joll^s. 1970. Histochemie Normale et Pathologique. Vol. 2. Gauthier-Villars, Paris. Gleisner, J. M. 1979. Lysosomal factors in inflammation. Pp. 229-260 in Chemical Messengers of the Inflammatory Process, J. C. Houck, ed. Elsevier/North-Holland, Amsterdam. Hirschhorn, R. 1974. Lysosomal mechanisms in the inflammatory process. Pp. 259-285 in The Inflam- matory Process, Vol. I, B. W. Zweifach, L. Grant, and R. T. Mc Cluskey, eds. Academic Press, New York. Kalk, M. 1963. Intracellular sites of activity in the histogenesis of tunicate vanadocytes. Q. J. Microsc. Sci. 104: 483-493. Luna, L. G. 1968. Manual of Histologic Staining Methods, of the Armed Forces Institute of Pathology. McGraw-Hill, New York. 258 pp. Millar, R. H. 1953. Ciona. L.M.B.C Memoirs on Typical British Marine Plants and Animals. 35: 1-123. MONNIOT, C. 1963. Kystodelphvs drachi n.g.n. sp., copepode enkyste dans une branchie d'Ascidie. Vie Milieu 14: 263-273. Parrinello, N., and E. Patricolo. 1975. Erythrocyte agglutinins in the blood of certain ascidians. Experiential: 1092-1093. Parrinello, N., G. De Leo, and E. Patricolo. 1976. Evolution of the immune response. Tunic reaction of Ciona intestinalis L. to erythrocyte injection. Some ultrastructural aspects. Boll. Zool. 43: 390. Parrinello, N., E. Patricolo, and C. Canicatti. 1977. Tunicate immunobiology. I. Tunic reaction 0^ Ciona intestinalis L. to erythrocyte injection. Boll. Zool. 44: 373-381. Parrinello, N., E. Patricolo, and C. Canicatti. 1984. Inflammatory-like reaction in the tunic of Ciona intestinalis (Tunicata). I. Encapsulation and tissue injury. Biol. Bull. 167: 229-237. Patricolo, E., and G. De Leo. 1979. Studies on the fibrous components of the test oi Ciona intestinalis Linnaeus. II. Collagen-elastin-like protein. Acta Zool. (Stockh.) 60: 259-269. P^Rfes, J. M. 1948. Recherches sur la genese et la regeneration de la tunique chez Ciona intestinalis L. Bull. Inst. Oceanogr. (Monaco) 936: 1-12. Reddy, a. L., B. Bryan, and W. H. Hildemann. 1975. Integumentary allografts versus autografts reactions in Ciona intestinalis: A protochordate species of solitary Tunicate. Immunogenetics 1: 584-590. Robinson, W. E., K. Kustin, and G. C. McLeod. 1983. Incorporation of '''C glucose into the tunic of the ascidian Ciona intestinalis (Linnaeus). J. Exp. Zool. 225: 187-195. Rowley, A. F. 1981. The blood cells of the sea squirt Ciona intestinalis: Morphology, differential counts and in vitro phagocytic activity. / Invert. Pathol. 37: 91-100. Smith, M. J. 1970. The blood cells and tunic of the ascidian Halocynthia aurantium (Pallas). I. Hematology, tunic morphology, and partition of cells between blood and tunic. Biol. Bull. 138: 354-378. Thomas, J. A. 1931. Sur les reations de la tunique d'Ascidia mentula Miill., a I'inoculation de Bacterium tumefaciens Sm. C R. Soc. Biol. 108: 694-696. Wright, R. K. 1981. Urochordates. Pp. 565-626 in Invertebrate Blood Cells. Vol. 2, N. A. Ratcliffe and A. F. Rowley, eds. Academic Press, London. Wright, R. K., and E. L. Cooper. 1975. Immunological maturation in the tunicate Ciona intestinalis. Am. Zool. 15: 21-27. Wright, R. K., and T. H. Ermak. 1982. Cellular defense systems of the Protochordata. Pp. 283-320 in The Reticuloendothelial System, a Comprehensive Treatise. Phylogeny and Ontogeny, Vol. 3, N. Cohen and M. M. Sigel, eds. Plenum Press, New York. Reference: Biol. Bull. 167: 251-263 (August. 1984) INFLUENCE OF CHEMICAL COMPOSITION OF ALGAL FOOD SOURCES ON GROWTH OF JUVENILE OYSTERS, CRASSOSTREA VIRGINICA GARY H. WIKFORS, JOSEPH W. TWAROG, JR., AND RAVENNA UKELES National Marine Fisheries Service, Northeast Fisheries Center, Milford Laboratory, Milford, Connecticut 06460 ABSTRACT Two algal flagellates, Diinaliella tertiolecta Butcher and Tetraselmis maculata Butcher, harvested in the stationary phase from a semi-continuous carboy culture apparatus, were analyzed for dry weight, total carbohydrate, total protein, and total lipid. Each species was cultured in three different growth media. The growth response of D. tertiolecta was similar in all three formulations but populations of T. maculata were considerably limited in the reduced-nutrient medium, Xi. Both algal species cultured in the Xi medium had significantly greater dry weights and contained more carbohydrate and less protein than cells cultured in the standard formulation (E). A third formulation (N/P), in which all medium components were reduced except nitrate and phosphate, produced algae with reduced carbohydrate and increased protein as compared with E medium. The total lipid content of Z). tertiolecta was significantly less than that of T. maculata regardless of the culture medium. Algae cultured in the three formulations were fed to juvenile oysters, Crassostrea virginica. T. maculata was a consistently better food source than D. tertiolecta, in- dicating a probable causal relationship between algal lipid content and oyster growth. Growth of oysters fed algae cultured in Xi medium was increased as compared with oysters fed algae cultured in E or N/P medium, suggesting a nutritional requirement for relatively more carbohydrate than protein as well. Results indicate that differences in growth media affect the gross chemical composition of algal food sources which alone can account for differences in algal nutritional value to C virginica. Introduction The laboratory or hatchery method of rearing molluscs in which brood stocks of adults are conditioned for spawning and induced to release gametes, and whose fertilized eggs are reared through larval stages to metamorphosis, requires that a suitable source of nutrition be available throughout the life cycle of the species that is being reared. This nutrition is derived most often from the culture of selected species of unicellular algae (see review, Walne, 1964; Ukeles, 1971, 1980; Epifanio, 1982), which are introduced into trays and tanks holding the molluscs. Other methods of obtaining algae have also been used (Glancy, 1965; Castagna, 1975), different sources of nutrition have been explored (Epifanio, 1979), and the development of artificial diets is in progress (Gabbott et al, 1976; Langdon, 1983). From the earliest work in which it was recognized that unicellular algae are food sources for molluscs (Cole, 1936; Bruce et al, 1939) investigators have queried why some species of algae are better food sources for molluscs than others. Certain factors related to the algal Received 18 April 1984; accepted May 1984. • -' Use of trade names does not imply endorsement by the National Marine Fisheries Service. 251 252 WIKFORS ET AL. cell have been suggested as explanations, e.g., cell size, cell wall composition, di- gestibility, the presence or absence of toxic metabolites, and chemical composition of the algal cell. None of these characteristics alone has offered an entirely satisfactory explanation. Although it seems that gross chemical composition (protein, carbohydrate, hpid) of different algae should be highly significant in affecting molluscan growth, research on this subject has yielded inconclusive results (see review, Webb and Chu, 1982). The research reported here describes the manner in which the gross chemical composition of two algal species of close taxonomic standing can be induced to vary and how the chemistry of the algal food source influences the growth response of juvenile oysters, Crassostrea virginica. Materials and Methods Algal culture and population determination Dunaliella tertiolecta Butcher and Tetraselmis maculata Butcher, obtained from the Milford algal culture collection, were examined. These species, cultured axenically in an enriched sea water growth medium, E, have been in the collection for many years, and strains of these phytoplankters have also been maintained in a reduced- nutrient enriched sea water medium, X i , for several years. Strains cultured in a third formulation in which all medium components except nitrate and phosphate were reduced, N/P, were developed during the course of these experiments. Inocula for the growth curves reported in this study had been subcultured routinely in N/P for over one year. Medium formulations are shown in Table I. Algae were cultured in the following types of Pyrex glassware: 20 X 150-mm screw-capped test tubes (with the liners removed) that were matched and calibrated for use as cuvettes in growth experiments; 125- and 500-ml Erlenmeyer flasks; and Fembach flasks fitted with siphons and filling bell attachments to inoculate semi- continuous 18-liter carboy cultures (Ukeles, 1973). Table I Formulations for algal growth media; final concentrations for 1 liter Medium Component X, Sea Water* 500 ml NaNOj 77.5 mg KH2PO4 5.0 mg NaFe Sequestrene 0.5 mg tham** 0.5 gm Vitamin 8,2 0.8 Mg Thiamin HCl 0.08 mg CuS04-5H20 4.9 ng ZnS04 • 7H2O 11 ng CoCl2-6H20 6.5 ng MnCl2-4H20 90 ng NaMo04-2H20 3.1 ng N/P 500 ml 500 ml 300 mg 300 mg 20 mg 20 mg 0.5 mg 5 mg 0.5 gm 1.0 gm 0.8 Mg 3.0 Mg 0.08 mg 0.3 mg 4.9 ng 9.8 ng 1 1 ng 22 ng 6.5 ng 13 ng 90 ng 180 ng 3.1 ng 6.2 ng Adjust pH to 8.0 and bring to volume of 1 liter with double glass-distilled water. ♦ Salinity 27-28%o. ** Tris(hydroxymethyl)aminomethane. ALGAL CHEMICAL COMPOSITION 253 Media were steam-sterilized in a Castle autoclave for 20 minutes (40 minutes for carboy media) at 15 psi pressure. Test tube and flask cultures were incubated in a GPI Model RI Incubator/Growth Chamber at 20°C, illuminated to about 300 ftC with cool-white fluorescent lights on a 12:12-h light/dark cycle. Carboys received constant iHumination (300 ftC) at 20°C ± 1°C. Culture densities for constructing growth curves were determined turbidimetrically with a Bausch & Lomb Spectronic 20 Spectrophotometer-colorimeter. Algal popu- lations were also determined by microscopic counts in an Improved Neubauer He- macytometer (Bright Line). Carboy cultures which had been harvested daily and replenished weekly with sterilized media for four weeks were selected for analyses. Samples of algae taken five days after the most recent addition of medium were subjected to dry weight determinations according to the method of Epifanio and Ewart (1977) and chemical analyses, as described in the next section. Analyses of algal chemical constituents Analyses of total protein were conducted using a heated biuret-Folin (HBF) pro- cedure modified from Dorsey et al. (1977, 1978), a method which provided increased sensitivity and a more stable end point as compared with the well-known method of Lowry et al. (1951). Cells were collected on a 25-mm glass fiber filter, and protein was extracted with appropriate reagents at 100°C for 100 minutes. Modifications from the published procedure primarily involved the concentrations and final volumes of reagents in the reaction tube (Table II). Sample reactions were read in a Beckman DB GT Grating Spectrophotometer. Protein nitrogen values were determined by interpolation from a standard curve obtained with prepared solutions of bovine serum albumin. Total protein was then calculated using a conversion factor of 6.25 generally accepted for most marine algal species (Dorsey et al., 1978). Carbohydrate determinations were made using a phenol-sulfuric acid method for analysis of algae reported by Kochert (1978) based upon procedures developed by Dubois et al. (1956). Algal culture samples were collected and washed with sterile isotonic NaCl by repeated cold centrifugation in an lEC Model Pr-2 centrifuge. Cells to be assayed were then homogenized in ethanol as in Myklestad and Haug (1972); this method was modified by using a Sonicor 50W ultrasonic bath. Prepared glucose solutions were assayed to construct a standard curve as recommended by Marshall and Orr (1962). Table II Preparation of solutions for heated biuret-Folin protein assay w/v concentration Concentration of ml for intermediate ml for 100 ml upon addition to Reagent stock solution working solution final solution* reaction tube* NaoCO, 20 g/100 ml _ 10 ml 2% NaOH 40 g/ 1000 ml — 10 ml 0.4% NaKTartrate 20 g/100 ml 2 ml/10 ml combine' •■* 1 ml 0.04% CuS04-5H:0 5 g/100 ml 2 ml/10 ml 0.01% * This corresponds with "reagent f" of Dorsey et al. (1977). ** To keep CuSOj -51420 from precipitating, it is necessary to dilute NaKTartrate to about 7 ml before adding Cu solution, mix thoroughly, and then bring to final volume of 10 ml with twice -distilled water. 254 WIKFORS ET AL. Determinations of total lipid were conducted using the method of Mukerjee ( 1 956) as adapted by Strickland and Parsons (1968). Cells for lipid analyses were collected, washed, and homogenized as in the carbohydrate procedure described above. A pre- cipitate of algal material obtained by evaporating the ethanol from the homogenate was assayed, and extinctions were corrected with appropriate stearic acid standard and blank determinations. Oyster feeding experiments Juvenile oysters, Crassostrea virginica, were grown in moUuscan rearing chambers, described in Ukeles and Wikfors (1982), which provided a constant flow of filtered, UV-irradiated sea water with temperature adjusted to 26°C. Each day, sea water flow was interrupted for four hours during which oysters were permitted to feed upon algal food suspensions introduced into the chambers. Daily rations of algal food cultures harvested from carboys were equilibrated to the same cytoplasmic volume of 0.6 ml packed cells per chamber as determined by centrifugation in modified Hopkins tubes (Ukeles, 1973). Resulting daily nutritional inputs in terms of algal cell number, dry weight, protein, carbohydrate, and lipid were calculated for each algal food source. Differences between means of replicate chemical analyses and daily nutritional inputs were tested with a Z test (a = 0.05). Each molluscan rearing chamber contained 50 juvenile oysters of similar initial size. Brood stocks of adult oysters obtained from a commercial landing at New Haven, Connecticut, were conditioned in the laboratory for spawning and induced to release gametes by warm-water stimulation (Loosanoff'and Davis, 1963). Fertilized eggs were reared through setting in filtered sea water on a diet of cultured algae. Young juvenile oysters thus obtained were utilized in these experiments. Growth of oysters was de- termined weekly by weighing pooled groups of 50 live oysters from each chamber on a Sartorius top loading balance. Mean live weight per oyster was calculated for each population and plotted versus time in weeks. Results Algal growth populations Population growth of D. tertiolecta cultured in E, N/P, and Xi media was nearly identical (Fig. 1). The effects of various NO3/PO4 ratios were investigated in a factorial design experiment where 16 combinations of NaNOs (77.5, 150, 300, and 350 mg/1) and ICH2PO4 (5, 10, 20, and 25 mg/1) were included in the Xi basal medium. Population growth of D. tertiolecta was similar over this range of concentrations; representative growth curves in nitrate/phosphate ratios of 77.5/5, 300/5, 77.5/20, and 300/20 mg/1 are shown in Figure 2. Increased concentrations of chelated iron (up to 1.5 mg/1) and vitamins (B12 up to 6.4 /lig/l and thiamine -HCl up to 0.64 mg/1) were also tested in an X 1 base formulation for possible growth-promoting effects upon D. tertiolecta. Again, no differences in the growth of/), tertiolecta were detected. Clearly, population grov^h of D. tertiolecta is not affected by the reduction of major medium components from E to Xi levels. In contrast, Tetraselmis maculata demonstrated a considerable reduction of growth in the Xi formulation (Fig. 3). This difference did not appear as a diminished rate of logarithmic growth but, rather, as a decrease in the maximum population as compared with E medium. The addition of nitrate and phosphate to Xj medium in concentrations equivalent to those in the E medium (= N/P) produced a population density equivalent to that obtained in E medium (Fig. 3). Increasing the vitamin ALGAL CHEMICAL COMPOSITION 255 6 .15 .1 .08 .06- .04- .02- .01 - .005- ^\^i .^y ^'^' .^' A- / f /J D. tertiolecta c L. N/P X. • .// — I 1 1 1 1 — 10 15 20 25 30 Days Figure 1 . Growth curves of Dunaliella tertiolecta populations in three enriched sea water media: E, N/P, and X,. d .06- .04- .02- .01- • I .' •'/ ■' / • Y . . • ^1 / / / / • / .005-^ / • — 1 1 1 i — 1 10 15 20 25 30 Days Figure 3. Growth curves of Tetraselmis maculata populations in three enriched sea water media: E, N/P, and X,. concentrations in Xi medium up to 6.4 ^^g/l for B12 and 0.64 mg/1 for thiamine • HCl did not affect any increase in T. maculata growth. Dry weights of 10^ cells of D. tertiolecta and T. maculata differed significantly (a = 0.05) when algae were cultured in the three formulations. D. tertiolecta cells cultured in Xi medium had dry weights that were larger than those from E or N/P; the dry weights of cells from the latter two were not different statistically (Table III). Similarly, T. maculata cells had greater dry weights when cultured in Xi medium than in E or N/P (Table III). Table III Gross chemical composition of algal cells Species Medium Dry weight Mg/10* cells % Total protein % Total carbohydrate % Total lipid Dunaliella tertiolecta Tetraselmis maculata X, 89.93 17.2 56.0 2.4 E 78.26 39.4 31.1 2.0 N/P 77.69 57.4 23.2 1.5 X, 119.5 15.6 36.5 15.3 E 110.9 31.0 23.9 8.3 N/P 114.7 37.9 13.5 9.2 ALGAL CHEMICAL COMPOSITION 257 Algal chemical composition Although population growth of D. tertiolecta was not affected by the reduction of nutrients to concentrations contained in X, or N/P formulations, the chemical compositions of cells cultured in E, N/P, and X, media differed statistically. Differences in percent total carbohydrate between D. tertiolecta cells cultured in the three media were significant {a = 0.05) X, > E > N/P, and the same observation was made for T. maculata (Table III). Conversely percent total protein for each species cultured in the three media showed the opposite relationship (a = 0.05) N/P > E > X, (Table III). The percentage of the dry weight accounted for by the lipid fraction was signif- icantly (a = 0.05) larger in T. maculata than in D. tertiolecta regardless of growth medium. Lipid content of both algae seems to be somewhat increased in X, over E and N/P media, but these differences could not be tested statistically. Oyster feeding experiments Daily rations of algal food sources were adjusted to provide identical cytoplasmic volumes as previously described. Because algal cells cultured in different media were shown to have different dry weights and chemical compositions, it was necessary to compare daily rations for all algal food cultures in terms of number of cells, dry weight, and the chemical components in mg fed per day (Table IV). The outstanding difference in daily ration between D. tertiolecta and T. maculata involved the lipid component of the diet: oysters fed T. maculata received significantly more lipid than those fed D. tertiolecta. Daily rations of protein varied from 24.2 mg in X, to 68.2 mg in N/P for D. tertiolecta and from 23.9 mg in X, to 81.8 mg in N/P for T. maculata. Total carbohydrate in the algal diets ranged from 27.6 mg in N/P to 78.6 mg in X, for D. tertiolecta and from 33.4 mg in N/P to 55.9 mg in X| for T. maculata. Dry weights of 0.6 ml packed cells were higher for T. maculata than for D. tertiolecta with the T. maculata N/P diet having the largest dry weight, 248 mg, and D. tertiolecta in E medium having the smallest, 1 1 3 mg. Numbers of cells contained in the daily rations were very similar for the three D. tertiolecta diets ranging only from 1 .44- 1.56 X 10^ cells. The T. maculata diets showed more variation with a range of 1.28- 2.16 X 10^ cells (in X, and N/P, respectively). Growth responses of juvenile oysters to D. tertiolecta or T. maculata cultured in E, N/P, or Xi medium are shown in Figure 4. Oysters exhibited increased growth when fed T. maculata as compared with D. tertiolecta regardless of the medium in which the cells were cultured (a = 0.05). After six weeks of observation, oysters fed either alga cultured in Xi medium were larger than oysters fed algae cultured in E Table IV Composition of 0.6 ml packed cells for six algal diets fed to oyster popidations daily Culture Number of Dry Total Total Total Algal species medium cells weight carbohydrate protein lipid Dunaliella tertiolecta X, 1.56 X 10-^ 140 mg 78.6 mg 24.2 mg 3.34 mg E 1.44 X 10" 113 mg 35.1 mg 44.4 mg 2.22 mg N/P 1.53 X 10" 119 mg 27.6 mg 68.2 mg 1.74 mg Tetraselmis maculata X, 1.28 X 10' 153 mg 55.9 mg 23.9 mg 23.4 mg E 1.94 X 10" 215 mg 51.4 mg 66.8 mg 18.0 mg N/P 2.16 X 10" 248 mg 33.4 mg 81.8 mg 22.8 mg 258 WIKFORS ET AL. .? S3 > w -I e 2 Figure 4. Growth of juvenile oysters, Crassostrea virginica. fed Dunaliella tertiolecta (dashed Hne), and Tetraselmis maculata (solid line), each cuUured in three different medium formulations: E, Xj, and N/P. medium which, in turn, had grown more than oysters fed algae cultured in the N/P formulation. This relationship was consistent for both T. maculata and D. ter- tiolecta; however, statistical evaluation was difficult. Although data points shown in Figure 4 represent an average weight for 50 oysters from one chamber, these 50 were weighed as a pooled group; the small size and large numbers of the oysters rendered weighing individuals impractical. Over six weeks, calculated differences between mean weekly growth rates of oysters fed Xi, E, or N/P cultures were not significant {a = 0.05) with the exception of T. maculata in which Xi > N/P. This is not ALGAL CHEMICAL COMPOSITION 259 surprising considering the somewhat hyperbolic shapes of the growth curves. Nev- ertheless, the consistency of the growth curves suggests that differences in growth of oysters fed either alga cultured in Xi , E, or N/P represent a genuine response to the different diets. Discussion In the present investigation, T. maculata and D. tertiolecta were compared as food sources for juvenile C virginica. These algae are similar in that both are chlo- rophyte flagellates, ovoid or elliptical in shape with yellow-green chromatophores, and are of similar size (cell length of Z). tertiolecta ranges from 6.4-1 1.7 /im and T. maculata from 6.2-13.8 nm)\ the main difference is the presence of a medium rigid cell wall in T. maculata and a thin hyaline periplast in D. tertiolecta (Butcher, 1959). The algae were cuhured in three different media formulations which resulted in differences in cell chemistry. These findings afforded a unique opportunity for eval- uation of the significance of algal chemical composition in the nutrition of the filter- feeding bivalve, Crassostrea virginica. The highly controlled conditions provided by the use of axenic cultures and the chambers designed for rearing the bivalves served to eliminate many of the unknown variables that can affect data collected in feeding studies conducted under less rigorous experimental conditions. D. tertiolecta tolerated a wide variation in nutrient enrichment of its growth medium; population growth showed little variation between the standard E formu- lation, the much reduced X|, and the N/P formulation. In an earlier study it was also observed that two algal species used as molluscan food sources, Monochrysis lutheri Droop (= Pavlova lutheri comb. nov. Green) and Phaeodactylum tricormitum Bohlin can tolerate a substantial (50%) reduction of phosphate and nitrate concen- trations in the growth medium (Ukeles, 1977). T. maculata, in contrast, responded to the low NO3 and PO4 concentrations of Xi medium with a reduced population; its needs for N and P were evidently higher than for D. tertiolecta. Although the growth of D. tertiolecta populations was unaffected by variations in the three nutrient formulations of the growth medium, the chemical composition varied considerably. Stationary phase cells cultured in E medium and, more so, in X| medium accumulated more carbohydrate than in the N/P medium. The com- position of cells in N/P medium was higher in protein. The low carbohydrate and high protein contents found in N/P medium were similar to those reported for log phase D. salina cultured in an artificial sea water medium (Parsons et al., 1961). The protein concentration of both flagellates is lowest in the Xi medium and, in fact, the concentrations of protein in the three growth media are directly reversed to those of carbohydrates. Algae seem to have exhausted nitrogen for protein synthesis in the Xi medium and shunted fixed carbon to carbohydrate synthesis without suffering a reduction in final population as measured by optical density. This observation agrees with previous studies reporting that stationary phase algal cells, which accumulate carbohydrates (Handa, 1969; Myklestad and Haug, 1972; Chu et al., 1982a; Loos and Meindl, 1982), are actually doing so in response to nitrogen depletion in the medium (Antia et al., 1963; Werner, 1970; Hobson and Pariser, 1971). Stationary phase cultures of T. maculata from N/P medium, similar to D. tertiolecta, contained a high protein, low carbohydrate composition, agreeing with the results reported for analysis of log phase cultures of T. maculata by Parsons et al. ( 1 96 1 ). D. tertiolecta, as an oyster food, did not increase in nutritional value relative to T. maculata when cellular protein or carbohydrate was increased. Similarly, T. ma- culata did not diminish in value relative to D. tertiolecta when protein or carbohydrate 260 WIKFORS ET AL. was decreased in the different growth media. Additional evidence that the amount of protein or carbohydrate fed per day does not limit the growth of juvenile C. virginica lies in the observation that the D. tertiolecta X| diet contained more car- bohydrate than any T. maculata diet, and the D. tertiolecta N/P diet contained more protein than the T. maculata Xi diet (Table IV). In no case was growth of oysters fed D. tertiolecta greater than that of oysters fed T. maculata. However, the lipid contents of T. maculata in all three diets were greater than those of D. tertiolecta by a factor of 1 (Table IV). Thus, there is strong evidence that total lipid, or a component of the lipid fraction, in T. maculata cells accounts for this alga's greater food value as compared with D. tertiolecta. Evidence that lipid content of a food source is critical in determining the growth response of oysters has been suggested in feeding studies with juvenile C. virginica offered different artificial diets (Castell and Trider, 1974), and the importance of lipids in the diet for larval bivalves has been well-documented (Millar and Scott, 1967; Helm et al, 1973; Holland and Spencer, 1973; Holland, 1978; Chu and Dupuy, 1980; Chu et al, 1982b). Components found in lipids extracted from algal cells showed that certain fatty acids, particularly those of the 6 w 3 group, are required by oysters (Langdon and Waldock, 1981; Langdon, 1982). The former study showed that a diet of cultured D. tertiolecta cells, fortified with encapsulated 22:6 w 3 fatty acid, produced better growth of C. ^/ga^ juveniles than did D. tertiolecta alone. It is possible, then, that T. maculata contains a required lipid component, perhaps a fatty acid, in large amounts as compared with D. tertiolecta. The greater nutritional value of T. maculata than D. tertiolecta is clearly correlated with the greater lipid concentrations in T. maculata. It is noteworthy that both species cultured in the Xi formulation in which the carbohydrate content was higher than the other two growth media offered the best oyster nutrition of the three media tested. Several investigators have implicated car- bohydrates, particularly glucose, as being important in oyster nutrition (Gillespie et al, 1964; Haven, 1965; Dunathan et al, 1969). Flaak and Epifanio (1978), using Thalassiosira pseudonana as a food source, concluded that the growth of the oyster, C. virginica, was more rapid when algae were richer in carbohydrate than protein. It has been known for some time that the chemical composition of freshwater algae can be affected by variation in environmental factors (Spoehr and Milner, 1948; Taub and Dollar, 1965; Saddler and Taub, 1972). Marine species, particularly diatoms, also have been shown to undergo changes in composition with alterations of envi- ronmental conditions. Observations of a phytoplankton bloom induced in a plastic sphere showed that with nitrogen depletion in the medium a significant change in the carbohydrate and protein composition of cells occurred (Antia et al., 1963). A decrease in N/C ratio as a result of nitrogen deficiency has been observed in Skele- tonema costatum (Holm-Hansen et al, 1968), Cyclotella nana, and Thalassiosira fluviatilis (Hobson and Pariser, 1971). Myklestad and Haug (1972) found that with a depletion in nitrate there was a change in the protein/carbohydrate ratio in Chae- toceros affinis. Protein, carbohydrate, and lipid composition of two strains of Phaeo- dactylum tricornutum were found to vary in light and dark periods (Terry et al, 1983) and it was also observed that temperature and light conditions will vary the composition of Thalassiosira allenii (Redalje and Laws, 1983). The present study has shown that the chemical composition of two closely related flagellates can depend upon the availability of nutrients in the growth medium. The extreme variation in chemical composition of these algal species that was shown to be dependent upon nutrient availability poses a number of questions pertinent to ecological monitoring, as well as to the practical considerations of providing a source of nutrition for artificially reared filter-feeding species. High primary productivity ALGAL CHEMICAL COMPOSITION 261 of marine and estuarine waters, as measured by algal cell numbers, chlorophyll content of sea water, dry weight of biomass, fixed carbon, or other methods currently employed by environmental biologists and oceanographers, may not necessarily reflect accurately the ability of these waters to support large populations in higher trophic levels. Pre- dictions of harvests of commercially valuable species based upon "primary produc- tivity" measurements alone could be overestimated if phytoplankton populations are deficient in some nutrient essential to the grazing species. Similariy, the nutritional values of cultured algae used to rear moUuscan or other species in the laboratory or commercial hatchery could vary considerably depending upon algal species, medium formulations, growth phase of algae at harvest, or temperature and light conditions of culture. Acknowledgments The authors are indebted to Elizabeth N. Wikfors who, during volunteer service in our laboratory, developed the modified HBF protein procedure used in these studies. LITERATURE CITED Antia, N. J.. C. D. McAllister, T. R. Parsons, K. Stephens, and J. D. H. Strickland. 1963. Further measurements of primary production using a large-volume plastic sphere. Limnol. Oceanogr. 8: 166-183. Bruce, J. R., M. Knight, and M. W. Parke. 1939. The rearing of oyster larvae on an algal diet. / Mar. Biol. Assoc. U. K. 24: 337-374. Butcher, R. W. 1959. An introductory account of the smaller algae of British coastal waters. Part I: Introduction and chlorophyceae. Min. Agric. Fish. Food Fishery Invest. Ser. IV: 1-74. Castagna, M. 1975. Culture of the bay scallop, Argopecten irradians. in Virginia. Mar. Fish. Rev. 37: 19-24. Castell, J. D., AND D. J. Trider. 1974. Preliminary feeding trials using artificial diets to study the nutritional requirements of oysters (Crassostrea virginica). J. Fish. Res. Board Can. 31: 95-99. Chu, F.-L. E., AND J. L. DuPUY. 1980. The fatty acid composition of three unicellular algal species used as food sources for larvae of the American oyster (Crassostrea virginica). Lipids 15: 356-364. Chu, F.-L. E., J. L. Dupuy, and K. L. Webb. 1982a. Polysaccharide composition of five algal species used as food for larvae of the American oyster, Crassostrea virginica. .Aguaculture 29: 241-252. Chu, F.-L. E., K. L. Webb, D. Hepworth, and M. Roberts. 1982b. The acceptability and digestibility of microcapsules by larvae of Crassostrea virginica. J. Shellfish Res. 2: 29-34. Cole, H. A. 1936. Experiments in the breeding of oysters (Ostrea edulis) in tanks, with special reference to the food of the larva and spat. Min. Agric. Fish. Fishery Invest. Ser. II. Vol. XV: 1-24. DORSEY, T. E., P. W. McDonald, and O. A. Roels. 1977. A heated biuret-Folin protein assay which gives equal absorbance with different proteins. Anal. Biochem. 78: 156-164. DoRSEY, T. E., P. McDonald, and O. a. Roels. 1978. 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Epifanio, C. E., and J. Ewart. 1977. Maximum ration of four algal diets for the oyster Crassostrea virginica Gmelin. Aquaculture 11: 13-29. Flaak, a. R., and C. E. Epifanio. 1978. Dietary protein levels and growth of the oyster Crassostrea virginica. Mar. Biol. 45: 157-163. Gabbott, p. a., D. a. Jones, and D. H. Nichols. 1976. Studies on the design and acceptability of micro- 262 WIKFORS ET AL. encapsulated diets for marine particle feeders. II. Bivalve molluscs. Pp. 127-141 in Tenth European Symposium on Marine Biology (Ostend, Belgium), G. Persoone and E. Jaspers, eds. Universa Press, Wetteren. Gillespie, L., R. M. Ingle, and W. K. Havens. 1964. Glucose nutrition and longevity in oysters. Q. J. Florida Acad. Sci. 27: 279-288. Glancy, J. B. 1965. Method of raising shellfish seed in a simulated habitat. U. S. Patent Office. Patent No. 3,196,833. Handa, N. 1969. Carbohydrate metabolism in the marine diatom, Skeletonema costatum. Mar. Biol. 4: 208-214. Haven, D. S. 1965. Supplemental feeding of oysters with starch. Chesapeake Sci. 6: 43-51. Helm, M. M., D. L. Holland, and R. R. Stephenson. 1973. The effect of supplementary algal feeding of a hatchery breeding stock of Ostrea edulis L. on larval vigour. / Mar. Biol. Assoc. V. K. 53: 673-684. HOBSON, L. A., and R. J. Pariser. 1971. The effect of inorganic nitrogen on macromolecular synthesis by Thalassiosira fluviatilis Hustedt and Cyclotella nana Hustedt grown in batch culture. J. Exp. Mar. Biol. Ecol. 6: 71-78. Holland, D. L. 1978. Lipid reserves and energy metabolism in larvae of benthic marine invertebrates. Pp. 85-123 in Biochemical and Biophysical Perspectives in Marine Biology, 4, D. C. Malins and J. R. Sargent, eds. Academic Press, London. Holland, D. L., and B. E. Spencer. 1973. Biochemical changes in fed and starved oysters, Ostrea edidis L. during larval development, metamorphosis and early spat growth. / Mar. Biol. Assoc. U. K. 53: 287-298. Holm-Hansen, O.. W. H. Sutcliffe, Jr., and J. Sharp. 1968. Measurement of deoxyribonucleic acid in the ocean and its ecological significance. Limnol. Oceanogr. 13: 507-514. KoCHERT, G. 1978. Carbohydrate determination by the phenol-sulfuric acid method. Pp. 95-97 in Handbook of Phycological Methods — Physiological and Biochemical Methods. J. A. Hellebust and J. S. Craigie, eds. Cambridge University Press, New York. Langdon, C. J. 1982. New techniques and their application to studies of bivalve nutrition. Pp. 305-320 in Proc. Second International Conference on Aquacullure Nutrition: Biochemical and Physiological Approaches to Shellfish Nutrition, G. D. Pruder, C. Langdon, and D. Conklin, eds. Louisiana State University, Baton Rouge. Langdon, C. J. 1983. Growth studies with bacteria-free oyster {Crassostrea gigas) larvae fed on semi- defined artificial diets. Biol. Bull. 164: 227-235. Langdon, C. J., and M. J. Waldock. 1981. The effect of algal and artificial diets on the growth and fatty acid composition of Crassostrea gigas spat. J. Mar. Biol. Assoc. U. K. 61: 431-448. Loos, E., AND D. Meindl. 1982. Composition of the cell wall of Chlorella fusca. Planta 156: 270-273. LoosANOFF, V. L., AND H. C. Davis. 1963. Rearing of bivalve mollusks. Pp. 1-136 in Advances in Marine Biology, I, F. S. Russell, ed. Academic Press, London. LowRY, O. H., N. J. ROSEBROUGH, A. L. Farr, AND R. J. RANDALL. 1951. Protein measurement with the Folin phenol reagent. / Biol. Chem. 193: 265-275. Marshall, S. M., and A. P. Orr. 1962. Carbohydrate as a measure of phytoplankton. / Mar. Biol. Assoc. U. K. 42: 511-519. Millar, R. H., and J. M. Scott. 1967. The larvae of the oyster Ostrea edulis during starvation. / Mar. Biol. Assoc. U. K. 47: 475-484. MUKERJEE, P. 1956. Use of ionic dyes in the analysis of ionic surfactants and other ionic organic compounds. Anal. Chem. 28: 870-873. Myklestad, S., and a. Haug. 1972. Production of carbohydrates by the marine diatom Chaetoceros affinis var. willei (Gran) Hustedt. I. Effect of the concentration of nutrients in the culture medium. J. Exp. Mar. Biol. Ecol. 9: 125-136. Parsons, T. R., K. Stephens, and J. D. H. Strickland. 1961. On the chemical composition of eleven species of marine phytoplankters. / Fish. Res. Board Can. 18: 1001-1016. Redaue, D. G., and E. a. Laws. 1983. The effects of environmental factors on growth and the chemical and biochemical composition of marine diatoms. I. Light and temperature effects. J. Exp. Mar. Biol. Ecol. 68: 59-79. Saddler, J. B., and F. B. Taub. 1972. Chemical variability of algal shellfish feeds. Proc. Natl. Shellfish. Assoc. 62: 6-7. Spoehr, H. a., and H. W. Milner. 1948. The chemical composition of Chlorella; effect of environmental conditions. Plant Physiol. 24: 120-149. Strickland, J. D. H., and T. R. Parsons. 1968. A practical handbook of seawater analysis. Bull. Fish. Res. Board Can. 167: 1-310. Taub, F. B., and A. M. Dollar. 1965. Control of protein level of algae, Chlorella. J. Food Sci. 30: 359- 364. ALGAL CHEMICAL COMPOSITION 263 Terry, K. L.. J. Hirata, and E. A. Laws. 1983. Light-limited growth of two strains of the marine diatom Phaeodactylum tricornulum Bohlin: Chemical composition, carbon partitioning and the die! pe- riodicity of physiological processes. / Exp. Mar. Biol. Ecol. 68: 209-227. Ukeles, R. 1971. Nutritional requirements in shellfish culture. Pp. 43-64 in Proc. Conference on Artificial Propagation of Commercially Valuable Shellfish, K. S. Price, Jr. and D. L. Maurer, eds. University of Delaware, Newark. Ukeles, R. 1973. Continuous culture — a method for the production of unicellular algal foods. Pp. 233- 254 in Handbook of Phycological Methods — Culture Methods and Growth Measurements. J. Stein, ed. Cambridge University Press, London. Ukeles, R. 1977. Culture of algae for feeding larval and juvenile molluscs in controlled aquaculture. Third Meeting of the I.C.E.S. Working Group on Mariculture, Brest, France, May 10-13. Actes de Collogues du C.N.E.X.O. 4: 361-370. Ukeles. R. 1 980. American experience in the mass culture of micro-algae for feeding larvae of the American oyster, Crassostrea virginica. Pp. 287-306 in Algae Biomass. Production and Use, G. Shelef and C. J. Soeder, eds. Elsevier/North-Holland Biomedical Press. Amsterdam. Ukeles, R., and G. H. Wikfors. 1982. Design, construction, and operation of a rearing chamber for spat of Crassostrea virginica (Gmelin). J. Shellfish Res. 2: 35-39. Walne, p. R. 1964. The culture of marine bivalve larvae. Pp. 197-210 in Physiology of Mollusca. I. K. M. Wilbur and C. M. Yonge, eds. Academic Press, New York. Webb, K. L., and F.-L. E. Chu. 1982. Phytoplankton as a food source for bivalve larvae. Pp. 272-291 in Proc. Second International Conference on Aquaculture Nutrition: Biochemical and Physiological Approaches to Shellfish Nutrition, G. D. Pruder, C. Langdon, and D. Conklin, eds. Louisiana State University, Baton Rouge. Werner, D. 1970. Productivity studies on diatom cultures. Helgol. Wiss. Meeresunters. 20: 97-103. Reference: Biol. Bull. 167: 264-269. (August, 1984) SIDE-SCAN SONAR RECORDS AND DIVER OBSERVATIONS OF THE GRAY WHALE (ESCHRICHTIUS ROBUSTUS) FEEDING GROUNDS JOHN S. OLIVER AND RIKK G. KVITEK Moss Landing Marine Laboratories, Moss Landing, California 95039 Abstract Gray whales (Eschrichtius robustus) excavate infaunal invertebrates and sediment by suction, producing many large depressions in the sea floor. Diver observations indicate that side-scan sonar provides accurate estimates of the size of feeding ex- cavations and the area of bottom covered by excavations (>30% of the bottom). Although side scan does not detect some excavations because of smaU size (particularly <3 m^) or their orientation with respect to the side-scan track, it gives a quantitative impression of the relative intensity of bottom disturbance by whales. This disturbance is directly related to habitat and prey utilization by whales. Introduction Gray whales {Eschrichtius robustus) extensively excavate the sea floor while feeding on benthic invertebrates (Oliver et al, 1983b, 1984). The major prey are amphipod crustaceans living in bottom sediments (Rice and Wolman, 1971; Zimushko and Ivashin, 1980; Bogoslovskaya et al, 198 1). Field observations show that both infaunal prey and sediments are extracted by suction (Oliver et al., 1983b, 1984). (See Ray and ScheviU, 1974 for "laboratory" observations of suction.) Sediment is expelled through the baleen. Excavation size and shape are highly variable. Gray whales rework single or multiple feeding excavations into much larger and complex features. Distinct excavations range in diameter from less than 1 m to over 20 m. While some feeding excavations are shallow surface sucks (3-10 cm deep), many excavations are 15-30 cm deep, and some are over 40 cm deep (Oliver et al., 1983b, 1984). The feeding excavations of gray whales are detected by side-scan sonar (Johnson et al., 1983) and are easily distinguished from other depressions in the sea floor made by walrus (Ohver et al., 1983a), ice gouging (Reimnitz et al., 1977; Thor and Nelson, 1981), and gas craters (Nelson et al., 1979). However, does side-scan sonar accurately represent feeding excavations and provide a useful relative impression of feeding disturbance? We answer this question by comparing side-scan records and diver observations of a highly accessible feeding ground along Vancouver Island, where prey communities and feeding records are remarkably similar to the primary feeding ground in the Bering Sea (Oliver et al., 1984). Materials and Methods The major study area was in Pachena Bay on the west coast of Vancouver Island, British Columbia. Gray whales fed in the bay during the spring and summer on a dense community of tube-dwelling Ampelisca amphipods (Oliver et al., 1984). Two permanent underwater stations were established in Pachena Bay to compare side- scan records with diver observations from 16 July to 1 5 August 1983. The two stations represented areas with relatively few and many excavations, and were designated the Received 2 March 1984; accepted 29 May 1984. 264 GRAY WHALE FEEDING GROUNDS 265 sparse and dense stations, respectively. A 50-m line (marked every 5 m) was staked to the bottom at both sites. The Hnes were perpendicular to the general direction of sand ripple marks on the sea floor. The ends of the lines were marked with surface buoys and a large metal barrel or beam that gave a distinct trace on record. As a result, each 50-m line could be located on a record and placed within a known pattern of excavations. The side-scan sonar was a 500 kHz system (KJien 521 dual channel side-scan). Recordings were made on wet paper at 60 lines/cm. All records were made on the 50-m range scale, giving a record with a 50-m width on both sides of the tow fish (a hydrodynamically designed body containing the underwater transducers). The record was uncorrected for ship speed and depth of tow (see below). The fish was towed at a depth of 5-6 m above the bottom (45-60° wire angle), at constant rpm, and at a constant compass direction. We used two boats with deep V-huUs: a 21 -foot Lucas (Hurricane 600) and a 40-foot converted Bristol Bay fisheries boat (R/V ALT A). While the side-scan was towed under a variety of sea conditions, quantitative mea- surements were taken only from records made in seas with <0.5-m swell and no wind chop. Divers placed sea floor targets 50 meters apart within the study area. These were visible on the side-scan displays, allowing the records to be corrected for ship speed. This was done by digitizing the records and redrawing them to the correct scale using a computer. Diver estimates of percent bottom covered by excavations, mean excavation size, and size distribution of excavations all came from diver maps of the dense and sparse station areas. Parallel estimates were made in several ways from side-scan records. Excavation patterns usually were measured only from a single run over a diver station. If estimates came from a single run, the run is numbered (run 1 or run 2). A composite sample was taken by examining all the single runs over a diver station and locating as many of the diver-observed excavations as possible. Finally, several single runs over the diver stations and over nearby areas were sampled to make regional estimates of excavation patterns. The regional areas were larger than the station area, but still represented the relatively sparse or dense feeding records. All the underwater observations were done by divers using SCUBA. Divers located and examined all feeding excavations at least 20 m on both sides and at the ends of the 50-m lines. They measured to the nearest 0.5 m the relative position, shape, major dimensions, and depth (to nearest cm) and noted edge conditions (steep or gently sloping) for each excavation. These observations were facilitated by good water clarity (5-8 m). AU excavations were less than one month old (Oliver et ai, 1984). Results The dense and sparse areas were easily distinguished by side scan. The percentage of the sea floor covered by feeding excavations was significantly greater at the dense compared to the sparse station {P < 0.05, Mann Whitney U-test). The mean size of excavations was significantly larger {P < 0.05, Mann Whitney U-test) at the dense station (Table II). There was also a significantly greater proportion of large excavations at the dense station {P < 0.05, Kolmogorov-Smimov test). In all estimates from the same station, the three samples from side-scan records (single runs, comp>osites, regional samples) were not significantly different from each other {P > 0.05, same tests). Finally, the dense study site was not located in the most intensely disturbed feeding area, where we found over 30% of the sea floor covered with feeding excavations. Quantitative measurements of the feeding record made by divers were similar to quantitative estimates from side-scan records. Diver and side-scan measures of the 266 J. S. OLIVER AND R. G. KVITEK Table I Percent area of the sea floor covered with gray whale feeding excavations at the relatively sparse and dense feeding areas in Pachena Bay Side Scan* Diving observations Run 1 Run 2 Composite Dense feeding station (6450 m^) 11.7% 9.3% 9.8% 10.8% Number of excavations 42 31 34 40 Sparse feeding station (2680 m^) 4.5% 3.4% 4.9% 3.6% Number of excavations 14 8 11 12 * See Methods section for explanation of single run versus composite. Diver and side-scan estimates are similar. percentage of bottom covered by feeding excavations were remarkably similar (Table I). There was no significant difference {P > 0.05, Wilcoxon's signed-ranks test) between the mean size of excavations estimated by divers and by side scan at either the dense or sparse stations (Table II). At the dense station there was no significant difference {P > 0.05, Kolmogorov-Smimov test) between diver and side-scan estimates of the size distribution of excavations (Fig. 1 ). However, at the sparse station, there was a significant difference {P < 0.05, Kolmogorov-Smimov test) between diver and side-scan estimates of the size distribution of excavations (Fig. 2). This difference could only be detected when the side-scan records were examined from the entire region around the sparse station. This region was qualitatively similar to the station site, but contained more features. Since our sample size was small at the sparse station (Tables I, II), the regional sample increased the power of the statistical test (Sokal and Rohlf, 1981). Furthermore, this difference could only be established by dividing the excavation area into 1 m^ size classes rather than 5 m^ (Fig. 2). Smaller excavations were more difficult to detect on side-scan records (Table III). At the dense station, only two excavations were never located on the records, both were <3 m^. Eighteen excavations between 3 and 1 3 m^ were missed on at least one record at the dense station. No excavations larger than 13 m^ were missed on records from either station (Table III). Table II Mean size (m^) of excavations estimated by divers and side scan in the relatively dense and sparse feeding areas in Pachena Bay (± standard errors) Diving observations Side scan* Run 1 Run 2 Composite Dense feeding station (6450 m^) 18.0 ± 2.6 18.1 ±3.4 18.6 ± 2.6 17.4 ± 2.4 Number of excavations 42 31 34 40 Sparse feeding station (2680 m^) 8.6 ± 2.8 11.5 ±3.2 12.0 ± 3.3 8.0 ± 3.2 Number of excavations 14 8 11 12 * See Methods section for explanation of single run versus composite. GRAY WHALE FEEDING GROUNDS 267 u c u DENSE SITE DIVING OBSERVATIONS N 42 2- SIDE SCAN SONAR 8 ' — ( Composite ) N 40 6- 4 2 H — 1 — ' 1 T— ^-1 ^-T ) , n , 10 20 30 40 50 60 Excavation size ( m ^ ) Figure 1. Size distribution of feeding excavations measured by divers and from side-scan records (a composite sample) at the dense station. o c o 16 -1 14 12 ^ 10 8 6 - 4 - 2 - 12 -, 10 - 8 - 6 - 4 2 H SPARSE SITE DIVING OBS. N 21 SIDE SCAN SONAR ( Regional Sample ) N 31 -1 1 1 1 1 r- 10 20 30 Excavation size ( m^ ) Figure 2. Size distribution of feeding excavations measured by divers and from side-scan records (regional sample) at the sparse station. 268 J. S. OLIVER AND R. G. KVITEK Table III The ability of side scan to detect relatively small (13 trr) <13 m^ > 13 m^ Never seen Missed at least once Never seen Missed at least once Dense feeding area Number of excavations Sparse feeding area Number of excavations 9% 27% 23 9 43% 78% 0% 0% 19 5 0% 0% Only a few excavations were never located by side scan; more were missed on at least one run over the station areas. Discussion Side-scan sonar can give an excellent impression of the relative intensity of gray whale feeding in soft-bottom habitats. Some feeding excavations are undetected because of their small size or orientation to the sonar signal. Nevertheless, side-scan and diver estimates of excavation sizes and the percent area disturbed show the same relative differences between a dense and a sparse feeding record, even when the dense and sparse records were relatively similar. The feeding record is quantified more precisely and easily, and for large areas more effectively, from records than from diver obser- vations. Side-scan sonar has considerable potential as a tool for documenting feeding patterns of gray whales. Spatial and temporal variations in the relative intensity of feeding can be documented. Ideas about large-scale patterns of habitat and prey utilization can be tested. Side scan also has applications in future management of bottom-feeding marine mammals such as the gray whale, and perhaps the walrus (Oliver et al, 1983a). Just as the browse patterns of deer and other ungulates are used by terrestrial biologists (de Voos and Mosby, 1971), excavation patterns doc- umented by side scan can help to assess the interactions between large marine grazers and their benthic food. The most important contribution of side-scan sonar to future management may be in evaluating long-term ecological questions involving the ex- ploitation of peripheral and central feeding grounds as the gray whale population grows, stabilizes, or declines in size. Acknowledgments We are grateful to Jon Jolly, Kirk Johnson, and Hans Nelson for their help in the field and in side-scan art. Our coworkers Peter Slattery and Ed O'Connor helped with all aspects of the study. Comments from Kirk Johnson, Lloyd Lowry, Jim Oakden, and Mark Silberstein improved the presentation. The support of the Bamfield Marine Station was essential, thanks to Sabina Leader and Ron Foremen. The work was funded by the World Wildlife Fund and the National Science Foundation (DPP- 8121722). LITERATURE CITED BOGOSLOVSKAYA, L. S., L. M. VoTROGOV, AND T. N. Semenova. 1981. Feeding habits of the gray whale off Chukotka. Rep. Int. Whaling Comm. 31: 507-510. GRAY WHALE FEEDING GROUNDS 269 Johnson, K. J., C. H. Nelson, and H. L. Mitchell. 1983. Assessment of gray whale feeding grounds and sea floor interaction. USGS Open-File Report 87-727. Nelson, C. H., D. R. Thor, M. W. Sandstrom, and K. a. Kvenvolden. 1979. Modem biogenic gas- generated craters (sea-floor "pockmarks") on the Bering Shelf, Alaska. Geol. Soc. Am. Bull. 90: 1144-1152. Oliver, J. S., P. N. Slattery, E. F. O'Connor, and L. F. Lowry. 1983a. Walrus, Odobenus rosmarus. feeding in the Bering sea: a benthic perspective. Fish. Bull. 81: 501-512. Oliver, J. S., P. N. Slattery, M. A. Silberstein, and E. F. O'Connor. 1983b. A comparison of gray whale, Eschrichtius robustus, feeding in the Bering Sea and Baja California. Fish. Bull. 81: 513- 522. Oliver, J. S., P. N. Slattery, M. A. Silberstein, and E. F. O'Connor. 1984. Gray whale feeding on dense ampeliscid amphipod communities near Bamfield, British Columbia. Can. J. Zool. 62( 1 ): 41-49. Ray, G. C, and W. E. Schevill. 1974. Feeding of a captive gray whale, Eschrichtius robustus. Mar. Fish. Rev. 36: 31-38. Reimnitz, E., p. W. Barnes, L. J. Toimil, and J. Melchior. 1977. Ice gouge recurrence and rates of sediment reworking, Beaufort Sea, Alaska. Geology 5: 405-408. Rice, D. W., and A. A. Wolman. 1971. The life history and ecology of the gray whale {Eschrichtius robustus). Am. Soc. Mammal. Spec. Publ. 3: 1-142. SOK.AL, R. R., and F. J. ROHLF. 1981. Biometry. W. H. Freeman and Company, San Francisco. 859 pp. Thor, D. R., and C. H. Nelson. 1981. Ice gouging on the subarctic Bering shelf Pp. 279-292 in The Eastern Bering Sea Shelf: Oceanography and Resources, Vol. 1. D. W. Hood and J. A. Calder, eds. Vol. 1, Univ. of Washington Press, Seattle. de Voos, a., and H. S. Mossy. 1971. Habitat analysis and evaluation. Pp. 1 35- 1 72 in Wildlife Management Techniques (3rd ed.), R. H. Giles, ed. The Wildlife Society. ZiMUSHKO, V. v., and M. v. Ivashin. 1980. Some results of Soviet investigations and whaling of gray whales {Eschrichtius robustus Lilljeborg, 1 96 1 [sic, for 1 86 1 ]). Rep. Int. Whaling Comm. 30: 237- 246. Parrinello, Nicolo, and Eleonora Patricolo Inflammatory-like reaction in the tunic of Ciona intestinalis (Tunicata). II. Capsule components 238 WiKFORS, Gary H., Joseph W. Twarog, Jr., and Ravenna Ukeles Influence of chemical composition of algal food sources on growth of juvenile oysters, Crassostrea virginica 251 SHORT REPORT Oliver, John S., and Rikk G. Kvitek Side-scan sonar records and diver observations of the gray whale (£"s- chrichtius robustus) feeding grounds 264 CONTENTS Annual Report of the Marine Biological Laboratory 1 INVITED REVIEWS GOVIND, C. K. Development of asymmetry in neuromuscular system of lobster claws 94 «' Mackie, G. O., p. a. V. Anderson, and C. L. Singla Apparent absence of gap junctions in two classes of Cnidaria 120 DEVELOPMENT AND REPRODUCTION Baloun, Andrea J., and Daniel E. Morse Ionic control of settlement and metamorphosis in larval Haliotis rufescens (Gastropoda) 124- ECOLOGY AND EVOLUTION Buss, Leo W., Catherine S. McFadden, and Douglas R. Keene Biology of hydractiniid hydroids. 2. Histocompatibility effector system/ competitive mechanism mediated by nematocyst discharge 139 Parker, GiSELE MULLER .^^ Dispersal of zooxanthellae on coral reefs by predators on cnidarians 159 Pennington, J. Timothy, and Fu-Shiang Chia Morphological and behavioral defenses of trochophore larvae of Sa- bellaria cementanum (Polychaeta) against four planktonic predators 168 Wethey, David S. Sun and shade mediate competition in the barnacles Chthamalus and Semibalanus: a field experiment 476^ GENERAL BIOLOGY Krantz, David E., Douglas S. Jones, and Douglas F. Williams Growth rates of the sea scallop, Placopecten magellanicus, determined from the '*0/^*0 record in shell calcite 186 Merz, Rachel Ann Self-generated versus environmentally produced feeding currents: a com- parison for the sabellid polychaete Eudistylia vancouveri 200 PHYSIOLOGY Hernandez-Nicaise, Mari-Luz, Ghislain Nicaise, and Luc Malaval Giant smooth muscle fibers of the ctenophore Mnemiopsis leydii: ul- trastructural study of in situ and isolated cells 210 Parrinello, Nicolo, Eleonora Patricolo, and Calogero Canicatti Inflammatory-like reaction in the tunic of Ciona intestinalis (Tunicata). I. Encapsulation and tissue injury 229 Continued on Cover Three Volume 167 Number 2 THE •"'t7^''^i: BIOLOGICAL BULLETIN IS ise4 PUBLISHED BY THE MARINE BIOLOGICAL LABORATORY Editorial Board Robert B. Barlow, Jr., Syracuse University Wallis H. Clark, Jr., University of California at Davis David H. Evans, University of Florida C. K. GoviND, Scarborough Campus, University of Toronto Judith P. Grassle, Marine Biological Laboratory - Harlyn O. Halvorson, Brandeis University Maureen R. Hanson, University of Virginia Ronald R. Hoy, Cornell University xSamuel S. Koide, The Population Council, Rockefeller University Frank J. Longo, University of Iowa Charlotte P. Mangum, The College of William and Mary Michael G. O'Rand, Laboratories for Cell Biology, University of North Carolina at Chapel Hill Ralph S. Quatrano, Oregon State University at Corvallis Lionel I. Rebhun, University of Virginia Dorothy M. Skjnner, Oak Ridge National Laboratory John D. Strandberg, Johns Hopkins University John M. Teal, Woods Hole Oceanographic Institution J. Richard Whittaker, Boston University Marine Program and Marine Biological Laboratory George M. 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IV ERRATUM THE BIOLOGICAL BULLETIN, Volume 166, Number 3, Page 529 The following correction should be made in the paper by Chizuko Obata and Shin-Ichi Nemoto entitled, Artificial parthenogenesis in starfish eggs: production of parthenogenetic development through suppression of polar body formation by me- thlxanthines (1984, Biol. Bull. 166: 525-536): the scale bar in A of Figure 4 should read 150 ^m. Thus the last sentence of the legend should read: Bar in A representing 150 ^m is common to A through F. Reference: Biol. Bull. 167: 271-309. (October, 1984) THE PREVENTION OF POLYSPERMIC FERTILIZATION IN SEA URCHINS' HERBERT SCHUEL Department of Anatomical Sciences. University at Buffalo. SUNY. Buffalo. New York 14214 and Marine Biological Laboratory. Woods Hole. Massachusetts 02543 Introduction During fertilization a single haploid sperm nucleus fuses with the haploid egg nucleus to establish the normal diploid chromosomal complement of the new in- dividual. This is a critical event in the initiation of development because polyspermy, the fusion of more than one sperm pronucleus with the female pronucleus, results in abnormal development and the eventual death of the embryo (Lillie, 1919; Roths- child, 1956). Various strategies have evolved to assure monospermic fertilization. Several sperm normally may enter large yolky eggs (certain insects, mollusks, sharks, reptiles, birds, urodels, etc.) but only one fuses with the egg's pronucleus (Lillie, 1919; Rothschild, 1956). Supernumerary sperm nuclei within the cytoplasm of these eggs undergo de- generation. The mechanism responsible for this process is poorly understood (Jaffe and Gould, 1984). In sea urchins and most other animals including mammals poly- spermy is prevented by blocks that operate at the egg surface to prevent supernumerary sperm from entering the egg's cytoplasm (reviewed by Lillie, 1919; Allen, 1957; Rothschild, 1956; Runnstrom, ^?a/., 1959; Runnstrom, 1966; Ginzburg, 1972; Gwat- kin, 1977; Epel, 1978; Schuel, 1978, 1984; Dale and Monroy, 1981; Wolf, 1981; Whitaker and Steinhardt, 1982; Schmelle/a/.. 1983; Jaffe and Gould, 1984; Nuccitelli and Grey, 1984). The purpose of this review is to examine all processes that are currently known to contribute to the prevention of polyspermy in sea urchins. Sea urchin gametes are an ideal model system to study polyspermy preventing mechanisms as well as other aspects of fertilization and development. Fertilization in echinoderms normally takes place externally in sea water which facilitates culturing and experimental manipulation of gametes in the laboratory. The sexes are separate and large quantities of gametes can be collected from adult animals (Harvey, 1956). For example, a single female Arbacia yields 5 ml of eggs containing approximately 4 X 10^ ova, while a male of this species yields 5 ml of semen containing about 10" sperm. The eggs undergo synchronous development following fertilization under lab- oratory conditions. These features make it possible to study polyspermy-preventing mechanisms by biochemical, physiological, and morphological techniques that are difficult to apply to other types of eggs. This is especially true with respect to mammalian eggs which share many functional aspects of fertilization in common with sea urchins (Gwatkin, 1977; Schuel, 1978; Lopo, 1983; Schmell et ai, 1983). Polyspermy can be quantitated by counting the number of sperm pronuclei within the cytoplasm of sea urchin eggs fixed prior to the time of pronuclear fusion (Presley and Baker, 1970; Byrd and ColHns, 1975; Schuel and Schuel, 1981), or by counting the incidence of multipolar divisions at the time of first cleavage (Wilson, 1900; Clark, Received 16 June; accepted 24 July 1984. ' Dedicated to the late Professor E. E. Just, in commemoration of the hundredth anniversary of his birth. 271 272 A H. SCHUEL POLYSPERMY BLOCKS 273 1936; Schuel et ai, 1973). Monospermic eggs divide into two blastomeres at first cleavage, while polyspermic eggs either divide into more than two cells or can be seen to contain multiple asters. The Egg Surface and Investing Coats Since polyspermy in sea urchins is prevented by processes that operate at the egg surface, the morphological and biochemical properties of the egg surface and investing coats must be considered, as well as changes that take place in these structures during fertilization. These surface structures include the jelly coat, vitelline layer, plasma membrane (oolemma), and subjacent cortical granules (Fig. 1 ). The mature sea urchin egg is surrounded by a jelly coat that hydrates and expands upon contact with sea water. The jelly slowly dissolves, charging the surrounding sea water with its biochemical components. The jelly coat can be removed from the eggs by treating them briefly with acidified (pH 5.0) sea water, by straining through bolting cloth, and by other treatments (Harvey, 1956). The jelly coat is transparent because its refractive index is the same as sea water, but its presence is indicated by the spacing between individual eggs. If the eggs are contiguous, there is no jelly coat. However, some jelly coat material remains attached to the surfaces of acid-dejellied eggs under such circumstances (Vacquier et a!., 1979). The jelly coat is known to contain glyco (sialo)-proteins, fucose sulfate polysaccharides, and small peptides (reviewed by: Runnstrom et ai, 1959; Tyler and Tyler, 1966a; Metz, 1967; Lopo, 1983). Sperm must swim through the jelly coat and its dissolved components to reach the surface of the egg. As they do, they are activated by molecular constituents of the jelly as evidenced by increased motility, increased respiration, a transient isoag- glutination, and induction of the acrosome reaction (Lillie, 1919; Tyler and Tyler, 1966a, b; Metz, 1967; Summers et ai, 1975; SeGall and Lennarz, 1979; Lopo, 1983; Monroy and Rosati, 1983). Increased sperm motility near eggs is an obvious advantage in increasing the probabihty of fertilization. The transient isoagglutination may perform a similar role by keeping sperm in the vicinity of eggs, especially in the animals' native marine habitat. The acrosomal filament that is formed at the apical end of the sperm by the acrosome reaction binds the sperm to the egg surface (vitelline layer) and fuses with the egg's plasma membrane to initiate sperm penetration (Dan, 1967; Colwin and Colwin, 1967; Summers et al, 1975; Lopo, 1983). Acrosomal enzymes such as phospholipase A2 (Conway and Metz, 1976), proteases (Levine and Walsh, 1979; Yamada and Aketa, 1981; Green and Summers, 1982), and arylsulfatase (Hoshi and Moriya, 1980) have been implicated in facilitating sperm penetration. The vitelline layer is a thin extracellular coat attached to the external surface of the egg's plasma membrane (Fig. la). The vitelline layer is composed of glycoproteins and contains species specific sperm receptors (Summers et ai, 1975; Glabe and Vac- quier, 1977; Schmell et al, 1977; Glabe and Lennarz, 1981; Kinsey and Lennarz, 1981; Rossignol et al, 1981; Lopo, 1983; Niman et ai, 1984). Monoclonal antibodies against vitelline layer proteins prevent sperm binding and fertilization (Gache et ai. Figure 1. Transmission electron micrographs showing surface morphology of sea urchin, Slron- gylocentrotus droebachensis, egg. Hylander (1984, unpubl. data). A: Unfertilized egg. The vitelline layer (VL) is attached to the outer surface of the egg's plasma membrane (PM). Numerous short microvilli (MV) are located at the egg surface. Cortical granules (CG) showing the spiral lamellae structure characteristic of this genus are located immediately subjacent to the plasma membrane. Jelly coat (J). 22,500X. B: Surface of fertilized egg fixed 60 min after insemination. The cortical granules have secreted their contents. The fertilization envelope (FE) is separated from the egg surface by the perivitelline space (PV). The hyaline layer (HL) invests the egg surface. Yolk platelet (Y). 20,300X. 274 H. SCHUEL 1983). Cross species fertilization is facilitated by removal of the vitelline layer (Longo, 1977). The egg's plasma membrane (oolemma) exhibits many short microvilli. As is true for all living cells, the sea urchin egg maintains an unequal distribution of ions across its plasma membrane which is reflected in a resting potential of —60 to —70 mV. The fertilizing sperm triggers a rapid electrical depolarization of the oolemma which acts as a rapid block to polyspermy (reviewed by: Hagiwara and Jaffe, 1979; Whitaker and Steinhardt, 1982; Shen, 1983; Gould-Somero and Jaffe, 1984;NucciteUi and Grey, 1984; also see Electrical block section, below). Cortical granules, Golgi-derived secretory organelles, are located immediately sub- jacent to the plasma membrane (Fig. la). The morphology and biochemical com- position of sea urchin cortical granules have been studied intensively (reviewed by: Runnstrom, 1966; Epel, 1978; Schuel, 1978, 1984; Shapiro and Eddy, 1980). The cortical granules form an irregular monolayer under the plasma membrane, with areas of tight packing interspersed with numerous patches of oolemma that are devoid of subjacent cortical granules. This packing arrangement is directly related to the sites where sperm penetration can take place and to the prevention of polyspermy (Schuel, 1978 and 1984, also see Cortical granule protease section, below). The surface of the egg is drastically altered as a result of cortical granule secretion during fertilization (Fig. lb). Cortical granule exocytosis begins at the site where the fertilizing sperm fuses with the oolemma, rapidly propagates around the entire surface of the sea urchin egg, and is completed within 1 -2 min depending upon species and temperature (reviewed by: Schuel, 1978, 1984). Secretion of the cortical granules is triggered by an increased concentration of free calcium ions within the egg's cytoplasm (reviewed by: Epel, 1978; Schuel, 1978, 1984; Jaffe, 1980). A similar calcium transient triggers exocytosis in stimulated somatic secretory cells (Poste and Allison, 1973; Rubin, 1982). Released cortical granule contents promote the detachment of the vitelline layer from the egg's plasma membrane and its elevation from the egg surface to become the fertilization envelope (reviewed by: Epel, 1978; Schuel, 1978, 1984; Shapiro and Eddy, 1980). This process is known as the cortical reaction. The fertil- ization envelope (membrane) acts as a mechanical barrier to penetration by super- numerary sperm (see Fertilization envelope section, below). The hyaline layer is formed by secreted cortical granule product(s) as well. Its primary function is to act as an extracellular cement to maintain blastomere adhesion during cleavage (reviewed by: Schuel, 1978, 1984), but it can also assist in preventing polyspermy under appropriate conditions (see Hyaline layer section, below). Cortical granule secretion is an important aspect of polyspermy preventing mech- anisms in sea urchins. Immature oocytes at the germinal vesicle stage of development do not have cortical granules in association with their plasma membrane and therefore can not undergo a cortical reaction in response to stimulation by sperm. These immature oocytes are extremely vulnerable to polyspermy (Harvey, 1956; Longo, 1978; De Felice and Dale, 1979). Furthermore, treatments that inhibit cortical granule discharge promote polyspermy. These include: application of hydrostatic pressure (Chase, 1967); colchicine (Hagstrom, 1956a); narcotics such as urethane (Longo and Anderson, 1970b) and chloral hydrate (Hagstrom, 1956a; Lonning, 1967); nicotine (Hagstrom and Allen, 1956); enzymatic inhibitors of the cortical granule protease (Hagstrom, 1956a; Lonning, 1967; Longo and Schuel, 1973; Schuel et al, 1973; Longo et al., 1974; Alliegro and Schuel, 1984); and the phospholipase A2 inhibitor quinacrine (Ferguson and Shen, 1984). Finally, premature discharge of the cortical granules induced by activating eggs with saponin or calcium ionophore A23187 pre- vents the penetration of subsequently added sperm (Ginzburg, 1964; Schuel et al., 1976b). Sperm can penetrate eggs after parthenogenetic activation by ammonia, a POLYSPERMY BLOCKS 275 treatment that does not trigger premature secretion of the cortical granules (Schatten, 1978; Longo, 1983). Reproduction in Nature The properties of fertilization studied with sea urchin gametes in the laboratory reflects reproductive processes of the animals in their natural habitat. Prior to spawning, adult urchins migrate together to form very dense local populations. All the members of the local population spawn at the same time (Harvey, 1956; Boolootian, 1966; Reese, 1966). Other marine invertebrates exhibit similar spawning behavior (Harrison et al, 1984). The synchronous release of large quantities of gametes is clearly ad- vantageous for animals that reproduce externally. Since sperm must swim to the egg to fertilize it, the production of an excess number of sperm (approximately 10" to lOVegg) also favors fertilization (Harvey, 1956). The probability that an individual egg will be fertilized depends upon the number of sperm in its immediate vicinity, which in turn depends upon the proximity of an adult male to the spawning female and the direction of current flow. Given this set of circumstances, there must have been strong evolutionary pressures to select for fertilization mechanisms likely to be successful over a wide range of sperm/egg ratios. Thus, to increase the chances of a successful hit under conditions of a low sperm density, the unfertilized egg should be highly receptive to sperm with numerous functional receptors at its surface. To prevent polyspermy under conditions of a very high sperm density, the fertilized (activated) egg must be able to effectively exclude supernumerary sperm on its surface. These expected features have been documented in laboratory studies of sea urchin fertilization (see below). Concepts of Fast and Permanent Polyspermy Blocks Our conceptual framework for analysis of polyspermy prevention in sea urchins is based upon the responses of eggs to insemination with excess sperm (Fig. 2). Large numbers of sperm rapidly attach to the egg surface within seconds after insemination (Fig. 2a). The fertilization envelope lifts off' the surface of the egg starting at the site of activation by the fertilizing sperm (Moser, 1939; Anderson, 1968; Green and Summers, 1980), spreads around the surface of the egg, and is completely elevated within about a minute (Fig. 2b-2d). Most of the sperm initially attached to the egg surface are detached as the fertilization envelope elevates. The fertilization envelope formed as a result of the cortical reaction functions as an absolute mechanical barrier to penetration by supernumerary sperm (Rothschild, 1956; Schuel, 1978, 1984). Even under the kind of conditions illustrated in Figure 2, polyspermy is a rare event since 85 to 95% of eggs in such cultures will admit only a single sperm (Ginzburg, 1964; Schuel and Schuel, 1981; Nuccitelli and Grey, 1984; Schuel et al, 1984). Eariy students of fertilization had made similar observations and concluded that the fer- tilization envelope (permanent polyspermy block) did not elevate rapidly enough from the entire surface of the egg to be exclusively responsible for the prevention of polyspermy in the presence of excess sperm (Just, 1919; Lillie, 1919; Just, 1939). Hence, the belief arose that a more rapid block to polyspermy also must function to protect the egg until the permanent block is established by completion of the cortical reaction. The existence of the putative rapid (pre-cortical) block to polyspermy was dem- onstrated by measuring the rates of initial fertilization and re-fertilization (polyspermy) in egg cultures upon insemination with excess sperm (Rothschild and Swann, 1952; 276 H. SCHUEL Figure 2. Fertilization reaction of Lytechinus pictus eggs upon insemination with excess sperm. From Vacquier and Payne (1973), reprinted with permission of Academic Press, Inc. A: Egg fixed at 10 s after insemination showing numerous sperm bound to the entire surface of the egg. B: Egg fixed at 25 s POLYSPERMY BLOCKS 277 Rothschild, 1956; Presley and Baker, 1979). In these studies the sperm were either removed by dilution or killed by addition of spermicides at various times after in- semination. The percent of eggs in the cultures that were unfertilized, fertilized, monospermic, and polyspermic was determined. The first successful sperm-egg reaction occurs within seconds after insemination, while re-fertilization (polyspermy) occurs at a much slower rate and stops entirely at about 60 s when the cortical reaction has been completed (Rothschild and Swann, 1952). The receptivity of the fertilized egg to refertilization is reduced about 20 fold during this interval. The conduction time for the establishment of this rapid but incomplete block to polyspermy was estimated to be less than 1 s. Subsequent studies by Presley and Baker (1970) confirmed that the observed incidence of monospermic fertilization under such conditions is far greater than predicted if receptivity of the egg were not reduced after the first sperm- egg fusion. Together these findings show that some mechanism must operate prior to completion of the cortical reaction in sea urchins to reduce the probability of polyspermy (reviewed by: Whitaker and Steinhardt, 1984; Nuccitelli and Grey, 1984). Nevertheless the existence of a rapid pre-cortical block to polyspermy has been questioned (Hagstrom and Allen, 1956; Kille, 1959; Byrd and Collins, 1975; Epel, 1978; DeFelice and Dale, 1979; Dale and Monroy, 1981; Dale et ai, 1982). The validity of this point of view must be examined in light of currently known properties of fertilization in sea urchins, and a consideration of how quickly a rapid block to polyspermy would have to be established in order to be effective. The half time for eggs to be fertilized upon insemination with excess sperm is about 0.5 to 1.0 s (Rothschild and Swann, 1952; Byrd and Collins, 1975; Schuel and Schuel, 1981). Since a portion of this period must represent the time required for the sperm to reach to the egg surface, the reaction of the fertilizing sperm with its receptor must be extremely rapid. Sperm binding to receptors in the vitelline layer is a prerequisite for fertilization (Summers et ai, 1975; Glabe and Vacquier, 1978). There are 1 500 to 6000 sperm binding sites on the surface, presumably the vitelline layer of the unfertilized sea urchin egg (Vacquier and Payne, 1973; Decker and Lennarz, 1979; Green, 1983). How many of these correspond to potential penetration sites is unknown. However, the penetration sites must be numerous since eggs can be fertilized upon insemination with as few as 10 sperm/egg (Byrd and Collins, 1975; Schmell et al, 1977). Another important factor is the interval between activation of the egg by the fertilizing sperm and completion of the cortical reaction. Cortical granule exocytosis begins within 3-40 s of activation and is completed within 1-2 min (reviewed by: Schuel, 1978, 1984; Nuccitelli and Grey, 1984). The range of values reported in the literature probably reflects species diflferences, temperature differences, and factors associated with the experimental criteria used to quantitatively evaluate exocytosis. In any case these observations show that a significant portion of the egg's surface remains unprotected by an absolute mechanical block to polyspermy (fertilization envelope) for an appreciable time until the cortical reaction is completed. Under these circumstances polyspermy would be the rule rather than the exception even at low or moderate sperm densities in the absence of a rapid block. To be effective the rapid block would have to be established within a fraction of a second after insemination showing early stage in elevation of the fertilization envelope (FE) and development of the perivitelline space (PV). Sperm detachment from the elevating fertilization envelope is evident. C: Egg fixed at 35 s. The cortical reaction has spread around more of the egg surface. Note that a few sperm (SP) remain bound to the partially elevated fertilization envelope. D: Egg fixed at 50 s. The fertilization envelope is elevated over the entire surface of the egg and detachment of bound sperm has been completed. 400x. 278 H. SCHUEL after the first successful sperm-egg reaction to prevent supernumerary sperm from entering (see Electrical block section, below). Thus, the rapid block to polyspermy should no longer be regarded as a controversial issue. Rather it should be recognized as a physiological necessity for normal fertilization in sea urchins, and in many other organisms as well. Our attention now should be directed toward identifying the processes that act to limit sperm penetration prior to completion of the cortical reaction and to the elucidation of their mechanisms of operation. Multiple Polyspermy Blocks Although it is conceptually convenient to think of polyspermy prevention in terms of separate rapid and permanent blocks, it is now evident that several processes act together to reduce the probability of polyspermic fertilization in sea urchins. The role of the elevated fertilization envelope as a mechanical barrier to polyspermy has been recognized for over a century (Fol, 1 877; Hertwig and Hertwig, 1 887). Other processes involving ion-dependent changes in membrane potential (Jaffe, 1976, 1980; Schuel and Schuel, 1981), various secretory products released by the egg's cortical granules (reviewed by: Epel, 1978; Schuel, 1978, 1984), release of peroxide to inactivate sperm (Boldt et al, 1981), production of arachidonic acid oxidation products (Moss et al, 1983; Schuel et al, 1984), and the protective (?) role of the jelly coat (Hagstrom, 1956b) have been discovered during the past 30 years. The list is probably still in- complete. These multiple defenses tend to limit sperm penetration by supernumerary sperm even when any one of the polyspermy blocking systems is inhibited experi- mentally. Even in the presence of an effective polyspermy promoting agent, the induction of polyspermy also depends upon the sperm density in the cultures since sufficient numbers of sperm must be present to overwhelm the egg's remaining defenses (Boldt et al, 1981; Schuel and Schuel, 1981; Schuel et al, 1973; Schuel et al, 1984). The existence of these multiple defenses also can be inferred from the wide variety of agents that are known to cause polyspermy in sea urchins (Hertwig and Hertwig, 1887; Clark, 1936; Harvey, 1956; Schuel, 1978, 1984; Dunham et al, 1982; AUiegro and Schuel, 1984). Jelly coat The jelly coat that invests the surface of the sea urchin egg may act as a filter to regulate the arrival of sperm at the egg surface (vitelline layer) and thereby reduce the probability of polyspermic fertilization. Evidence for such a role is provided by observations that eggs fertilize more rapidly and are more vulnerable to polyspermy after the jelly coat is removed (Hagstrom, 1956b, 1959; Runnstrom et al, 1959; Schuel et al, 1974; Vacquier et al, 1979; Schuel and Schuel, 1981). However, there is considerable variability in the reported susceptibility of eggs to polyspermy after removal of the jelly coat, with some studies showing a large increase (Hagstrom, 1956b) and others a much smaller but still detectable increase (Schuel et al, 1974; Schuel and Schuel, 1981). This may reflect species differences in the importance of the intact jelly coat in preventing polyspermy. Alternatively, it may not be the absence of the jelly coat per se, but rather damage produced by the treatment used to remove the jelly coat from the eggs that is responsible for the increased polyspermy (Hagstrom, 1959). This possibility also is suggested by abnormalities in the electrical properties of the egg's plasma membrane and the high levels of polyspermy sometimes seen in acid de-jellied eggs (DeFelice and Dale, 1979; also see Electrical block section, below). Furthermore, arylsulfatases isolated from sea urchin sperm or from hmpets can dissolve POLYSPERMY BLOCKS 279 the jelly coat but do not cause polyspermy (Hoshi and Moriya, 1980), even when treated eggs are inseminated with excess sperm (Schuel and Schuel, unpubl. data). Additional work is required to resolve these questions. The ambient sea water near the eggs contains dissolved jelly coat components which affect sperm function in a variety of ways (see The Egg Surface and Investing Coats section, above). Of these, the transient isoagglutination and induction of the acrosome reaction also may be contributing factors in preventing polyspermy. Since agglutinated sperm are not free to react with eggs until they disperse, this process may tend to reduce temporarily the frequency of sperm-egg collisions. The infertility of sperm that are permanently agglutinated by antibodies is consistent with this notion (Metz et ai, 1964). Induction of the acrosome reaction is followed by a rapid decline in the fertilizing capacity of sperm (Tyler and Tyler, 1966b; Kinsey et ai, 1979; Vacquier, 1979; Sano and Kanatani, 1980; Christen et ai, 1983; Nakano et ai, 1984). Such a reduction in the number of potent sperm near already fertilized eggs would be expected to have a salutary effect in reducing the chances of re-fertilization (poly- spermy). Quantitative studies are required to determine whether these processes actually have a significant physiological role in preventing polyspermy. Electrical block Electrical depolarization of the plasma membrane is a universal response of cells including eggs to stimulation (Tyler et ai, 1956a). Jaffe (1976) discovered that sea urchin eggs use this process to establish a rapid but transient block to polyspermy (reviewed by: Hagiwara and Jaffe, 1979; Dale and Monroy, 1981; Whitaker and Steinhardt, 1982; Shen, 1983; Nuccitelli and Grey, 1984; Gould-Somero and Jaffe, 1 984; Jaffe and Gould, 1 984; Schuel, 1 984). Jaffe's electrophysiological studies showed that: (1) the resting membrane potential of unfertilized eggs is -70 mV; (2) the oolemma depolarizes to + 10 to +20 mV with 3 s of insemination; (3) the depolarized state is maintained for about 60 s until the cortical reaction is completed; (4) thereafter the oolemma repolarizes to about —70 mV during the next 5-10 min; and (5) eggs which fail to depolarize to above —10 mV become polyspermic, while those that depolarize to +10 to +20 mV are typically monospermic. Finally, fertilization is prevented if the membrane potential is set at +5 mV but occurs at —10 mV. The electrical block is not absolute since some of the unfertilized eggs voltage clamped at +10 to +20 mV do fertilize at high sperm densities while none of these clamped eggs fertilize at low to moderate sperm densities (Jaffe, 1976; Jaffe et ai, 1982; Lynn and Chambers, 1984). Nicotine can cause polyspermy by inhibiting the electrical block (Jaffe, 1980). The kinetics and ionic basis for the fertilization (activation) potential in sea urchin eggs has been studied intensively (reviewed by: Hagiwara and Jaffe, 1979; Whitaker and Steinhardt, 1982; Shen, 1983; Nuccitelli and Grey, 1984). Its major features are illustrated (Figs. 3-6) from the work of Chambers and DeArmendi (1979). Normal unfertilized eggs show a resting potential of -70 to -80 mV (Fig. 3a). The membrane is electrically excitable and responds to stimulation with a positive going action potential of brief (3.5-14.6 s) duration. Sperm triggers a long lasting biphasic (segments a and b) fertilization potential which remains positive until fertilization envelope elevation is completed. The initial phase of segment a of the fertilization potential corresponds to an action potential that is triggered by the fertilizing sperm (Fig. 3b). Segment b begins at the end of segment a (about 15 s) and reaches a maximum at 33 s. De- polarization begins when the sperm reacts with its receptor at the egg surface (DeFelice and Dale, 1979; Hulser and Schatten, 1982), and attains levels (+5 to +20 mV) 280 H. SCHUEL + 50, -75 I sec Figures 3-6. Electrical recordings of fertilization potentials in Lytechinus variegatus eggs. Abscissa: time (see scales); ordinate: {upper) current in pA; (lower) membrane potential in mV. Vertical arrow: start of depolarization in inseminated egg; horizontal bracket: interval during which fertilization membrane (FM) elevation occurred. Temp. = 22°C. From Chambers and DeArmendi (1979), reprinted with permission of Academic Press, Inc. Figure 3. Action potential and fertilization potential recorded in an egg cultured in sea water. A: Oscillograph record of unfertilized egg {left) injected with pulses of depolarizing current. An action potential was elicited by 20 pA pulse of 2 s duration. The pulse was cut off during the rising phase of the action potential. The same egg (right) after insemination. For segments (a) and (/?), see text. Break in curve represents 10 min period when membrane potential repolarized steadily to -70 mV. B: Same oscillograph records as A. wath time scale expanded 8X. The steep rising limb of the action potential ( ) and the fertilization potential ( ) are superimposed. C Same record as B. but time scale expanded 4X (32x scale of A). capable of excluding sperm (Jaffe, 1976) within 100 m s (Fig. 3c). Repolarization of the fertilized egg is completed in 10 min, and is associated with increased potassium conductance (Steinhardt et al, 1971). Damaged eggs have leaky membranes and show a resting potential of - 10 to -20 mV (Fig. 4). They can not be excited electrically. Upon insemination, the sharp early peak of the fertilization potential (segment a) is missing. The membrane potential slowly rises to positive levels (segment b) within about 1 5 s. Such eggs lack a voltage sensitive mechanism to generate a rapid spike depolarization, which can be triggered only if the egg's membrane potential is more negative than -40 mV. Eggs with leaky membranes can be sealed and the resting potential restored to -70 mV by the transient injection of negative current (Chambers and DeArmendi, 1979). The rapid influx of calcium (Azamia and Chambers, 1976; Paul and Johnston, 1978) and sodium (Payan et al., 1981; Sardet et al, 1982) ions POLYSPERMY BLOCKS 281 +25 -25 -50 -75 a— - + 13.5 mV 10 min h. JO ^ec ^ _70 mV Figure 4. Fertilization potential of an egg in sea water whose resting potential was -15 mV. at fertilization appears to be the ionic basis for the fertilization potential. This hypothesis is supported by recordings of fertilization potentials generated by eggs inseminated in sodium and calcium-depleted sea water. In low (4.8 mA/) sodium (choline-sub- stituted sea water), the membrane of the unfertilized egg is electrically excitable and can generate action potentials of briefer than normal duration upon stimulation (Fig. 5). After insemination these eggs fire a very brief action potential, and the fertilization potential (both segments a and h) is suppressed greatly. In low calcium sea water, the first component (segment a) of the fertilization potential is suppressed (Fig. 6). These findings show that both calcium and sodium influx generate the first response phase (segment a) of the fertilization potential, while sodium influx generates the second response phase (segment h). These electrophysiological studies make it possible to predict key properties of the rapid electrical block to polyspermy: (1) Unfertilized eggs that are not electrically excitable and/or that have low (-10 to —20 mV) resting potentials are abnormal or damaged, and should be susceptible +100 :r^JlJL__ mV mV -t-l mV UNFERTILIZED FERTILIZED 10 tec Figure 5. Action potential and fertilization potential recorded in an egg cultured in choline substituted sea water containing 4.8 mAf Na^. Action potentials of briefer than normal duration (compare with Fig. 3 A, above) were elicited in the unfertilized egg (left) by pulses of 65 pA 1 .2 s and 100 pA 100 ms duration. The same egg (right) after insemination. 282 H. SCHUEL ACTIVATION POTENTIAL OF EGG IN 10"' M Cq'^-SW AND IN SW +23 mV +24 mV 10~''M Ca^*-SW Figure 6. Recording of fertilization potential of egg inseminated in sea water containing 0.1 mM Ca^^ ( ), compared with egg fertilized in normal (10 mM Ca*^) sea water ( ). to polyspermy. This expectation has been realized since such eggs are highly vulnerable to polyspermy (Jaffe, 1976; DeFelice and Dale, 1979; Whitaker and Steinhardt, 1983). (2) Eggs fertilized in low sodium sea water, which suppresses the fertilization potential (Fig. 5), should be highly susceptible to polyspermy. This expectation has been realized since polyspermy results when eggs are fertilized in sodium depleted (choline-substituted) sea water (Nishioka and Cross, 1978; Jaffe, 1980; Schuel and Schuel, 1981). However, ahhough a high incidence of polyspermy is obtained, the eggs do not fill up with supernumerary sperm when heavily inseminated in low sodium sea water (Schuel and Schuel, 1981). Two factors tend to limit sperm pen- etration under these conditions: the normal completion of the cortical reaction (Schuel and Schuel, 1981), and the adverse effects of the low sodium environment on sperm fertility (Schuel and Schuel, 1981; Bibring et al, 1984). (3) Eggs fertilized in calcium-depleted sea water, which suppresses the first com- ponent (segment a) of the fertilization potential (Fig. 6), should be susceptible to polyspermy. Such a phenomenon might account, in part, for the elevated polyspermy seen in 80% isotonic magnesium chloride/20% sea water (Clark, 1936). Magnesium is a potent ionic antagonist of calcium (Heilbrunn, 1956), and could suppress the calcium component of the fertilization potential (Jaffe, 1980). Increased polyspermy was not observed when eggs were fertilized with acrosome reacted sperm in zero calcium sea water (Schmidt et al., 1982). However, sperm fertility is reduced 20X in zero calcium sea water, and these authors may not have used sufficient numbers of sperm to challenge the egg's remaining defenses. The experiments with low sodium and zero calcium sea water illustrate an im- portant point. The results of polyspermy inducing treatments can be complicated if the treatment also injures the sperm and thereby reduces its fertilizing capacity. Normal sperm function can not be assumed. It must be evaluated. The cortical reaction normally propagates around the egg surface while the rapid electrical block is believed to be operational. In order to measure the duration and efficacy of the rapid block in Arbacia, advantage was taken of the fact that the cortical POLYSPERMY BLOCKS 283 granule secretion mediated block can be suppressed by soybean trypsin inhibitor (Schuel and Schuel, 198 1 ). The rationale for these experiments was based on previous observations that a soybean trypsin inhibitor (SBTl) sensitive protease is secreted by the egg's cortical granules at fertilization (Schuel et al, 1973; Vacquier et ai, 1973), that SBTI inhibits the cortical reaction and promotes polyspermy (Hagstrom, 1956a; Lonning, 1967; Longo and Schuel, 1973), that SBTl-treated eggs are monospermic initially and gradually become polyspeimic with time (Hagstrom, 1956a; Longo and Schuel, 1973; Longo et ai, 1974; Schuel et ai, 1976a), and that all potential sperm penetration sites remain available at the surface of SBTI-treated Arhacia eggs for 3 min post insemination (Schuel et ai, 1976a; also see Cortical granule protease section, below). Under these conditions the time at which the SBTl-treated eggs first became polyspermic (Schuel and Schuel, 1981) was used to titrate the decay of the rapid block (Fig. 7). The half time for the first supernumerary sperm fusion event in natural (425 mM Na^) sea water is 89.9 ± 4.7 s, compared to 15.8 ± 1.6 s in 26 mM Na^ {P < 0.01). Furthermore, the decay in the rapid block corresponded to the period iOOr o o c o o *^ Q. ^ O Q. • SBTI in SW (425mMNaM o SBTI in SW (26 mM No") 120 180 Time (sec) 300 Figure 7. Kinetics of sodium-dependent block to polyspermy in Arhacia punctulata eggs as determined by the rate at which eggs become polyspermic in 2.5 ml SBTI (0.5 mg/ml) dissolved in natural (425 mM Na"^) and choline-substituted (26 mM Na"^) sea water. Eggs inseminated with excess (0.1 ml of 8%) sperm. Unfused sperm were killed at indicated times by addition of equal volume of sodium laur\l sulfonate (Hagstrom and Hagstrom, 1954), final concentration 0.0007%. Incidence of polyspermy estimated by counting multipolar divisions at first cleavage. Data normalized and expressed as percentage of all SBTI- treated eggs that were polyspermic in the absence of spermicide (91.3 ± 5.9 in 425 m.\/ Na* and 71.7 ± 3.8 in 26 mM Na"^), minus the percentage of control eggs that were polyspermic in natural sea water alone (5.3 ±2.1 in 425 mM Na^ series and 7.0 ± 4.0 in 26 mM Na"^ series), n = 3. From Schuel and Schuel (1981), reprinted with permission of Academic Press, Inc. 284 H. SCHUEL when the oolemma that had initially depolarized to +20 mV is gradually repolarizing to the level (-70 mV) characteristic of the fertilized egg (Fig. 8). During normal fertilization in Arbacia elevation of the fertilization envelope (cortical reaction) is completed by 60 s after insemination, long before the rapid electrical (sodium de- pendent) block decays to 50% of its effectiveness at 90 s. Thus sea urchin eggs have adapted two typical cellular responses to stimulation, electrical depolarization of the plasma membrane and exocytotic release of stored secretory products, to operate in tandem to assure monospermic fertilization (Schuel and Schuel, 1981). Presently available data suggests that the egg's fertilization potential regulates sperm-egg membrane fusion by blocking insertion/translocation into the oolemma of potential-sensitive components of the sperm's plasma membrane (Gould-Somero and Jaffe, 1984). Studies of cross species fertilization using voltage clamped sea urchin {Strongylocentrotus purpuratus) and Urechis caupo (a echiuroid worm) eggs showed that the blocking voltage for fertilization in these crosses is determined by the sperm species (Jaffe et ai, 1982). The electrical block to fertilization in Lytechinus eggs clamped at +15 mV can be overcome by brief repolarization to —60 mV for 35-45 ms (Shen and Steinhardt, 1984). These findings show that a sperm attached to the surface of an unfertilized egg can complete its electrically sensitive insertion/transfer process into the egg membrane within milliseconds. Finally, sperm can activate but will not penetrate Lytechinus eggs whose membranes are clamped at potentials more negative than —30 mV (Lynn and Chambers, 1984). The activating sperm is detached from the surface of these eggs as the fertilization envelope elevates. Hence, the oolemma T>20 100 r- O O X E ^ »- o o p CL f- O CL c 0) c o E -90 -240 Time (sec) Figure 8. Decay of rapid block to polyspermy (normalized curve for polyspermy rate in SBTI- treated eggs in 425 xnM Na* from Fig. 7) versus electrophysiological recording (provided by Jaffe and Tilney) of changes in membrane potential in an Arbacia egg during fertilization. From Schuel and Schuel (1981), reprinted with permission of Academic Press, Inc. POLYSPERMY BLOCICS 285 must partially depolarize after the sperm reacts with its receptor at the egg surface for membrane fusion to take place. These activated but unpenetrated eggs do not divide. They lack the sperm centriole required to organize the mitotic apparatus, and are comparable to parthenogenetically activated eggs that fail to cleave for the same reason (Lillie, 1919; Mazia, 1961; Schuel et al., 1976b; Schuel, 1978). Together these findings indicate that the electric field across the egg's plasma membrane regulates sperm fusion and entry. The existence of a rapid electrical block to polyspermy in sea urchins has been questioned (DeFelice and Dale, 1979; Dale and DeSantis, 1981; Dale el al., 1982). This has generated considerable controversy in the literature (reviewed by: Dale and Monroy, 1981; Whitaker and Steinhardt, 1982; Shen, 1983; Nuccitelli and Grey, 1984; Schuel, 1984). According to Dale and his colleagues, the resting potential of physiologically normal (optimal) Psammechimis and Paracentrotus eggs varies from —8 to —16 mV. In their hands immature or aged eggs were reported to have resting potentials to -60 to -80 mV. Upon insemination of "optimal" eggs, the fertilizing sperm triggers a 1 to 2 mV step depolarization which precedes the main positive going depolarization (presumed to reflect propagation of the cortical reaction) by \?>- 15 s. Polyspermic eggs typically show additional step depolarizations (reflecting other successful sperm-egg fusion events) during this 13-15 s lag period. However, it should be noted that the fertilization potential changes recorded by Dale and his colleagues in what they consider to be "optimal" (physiologically normal) eggs are identical to what is seen in damaged eggs with leaky membranes (Chambers and DeArmendi, 1979). Such eggs lack the initial spike depolarization phase of the positive going fertilization potential (see Fig. 4, above). Technical problems rather than species differences in polyspermy prevention probably account for the differences in experimental results reported by Jaffe (1976 and 1980) and by DeFelice and Dale (1979). The membrane of the sea urchin egg has a very high input resistance (Jaffe and Robinson, 1978; Chambers and DeArmendi, 1979) so that even minor leaks in the seal between the oolemma and a recording electrode can cause spuriously low readings in resting potential in the range of -10 mV (Nuccitelli and Grey, 1984). Independent data from studies which do not involve the potentially damaging effects of micro-electrode impalement of individual eggs are helpful in resolving the issue. Ion flux data confirm that the true resting potential of normal unfertilized eggs is about -70 mV (Jaffe and Robinson, 1978; Chambers and DeArmendi, 1979). The rapid spike depolarization seen in electrode-impaled eggs at fertilization (Jaffe, 1976; Chambers and DeArmendi, 1979; see Fig. 3, above) also can be recorded by the non-invasive measurement of action currents on the egg surface (Whitaker and Steinhardt, 1983). Suppression of the fertilization potential by low sodium sea water (Steinhardt et al., 1971; Ito and Yoshioka, 1973; Chambers and DeArmendi, 1979) induces polyspermy in large populations of sea urchin eggs (Nishioka and Cross, 1978; Jaffe, 1980; Schuel and Schuel, 1981). Finally, the quality of the eggs used by DeFelice and Dale (1978) is questionable in view of the high incidence of polyspermy (70.1% avg., sperm density 2.4 to 21 X lOVml) observed in the non-impaled eggs in their cultures. This may reflect damage to the eggs resulting from the acid sea water treatment used to remove the jelly coats (Hagstrom. 1956b, 1959). Consistent with this view, acid dejellied eggs have resting potentials initially of -8 to -15 mV which gradually change with standing in sea water to -60 to -80 mV (DeFelice and Dale, 1979; Taglietti, 1979). This might reflect recovery of the eggs from acid-induced damage. It would be of interest to test the capacity of these eggs to resist polyspermy. Approximately 85-95% monospermic fertilization should be obtained with good batches of eggs inseminated with 10^ to 10** sperm/ml (Ginzburg, 286 H. SCHUEL 1964; Boldt et ai, 1981; Schuel and Schuel, 1981; Alliegro and Schuel, 1984; Schuel etai, 1984). The weight of available evidence thus supports the operation of an ion-dependent electrical block to polyspermy in sea urchins as proposed by Jaffe (1976). It is probably functionally equivalent to the rapid block postulated by Rothschild and Swann (1952) in the sense that it is quickly established after the first successful sperm-egg reaction, is transiently active until the cortical reaction is completed, and reduces the probabihty that supernumerary sperm can penetrate the egg. Electrical blocks to polyspermy have been described in a variety of invertebrate and vertebrate eggs (reviewed by: Gould-Somero and Jaffe, 1984; Jaffe and Gould, 1984; Schuel, 1984). Fertilization envelope Observations of living sea urchin eggs during fertilization (see Fig. 2, above) led to the belief that the fertilization envelope acted as an absolute mechanical barrier to further sperm penetration (Fol, 1877; Hertwig and Hertwig, 1887). For the egg confronted by large numbers of excess sperm seeking entry, it provides an impregnable defense — a complete and continuous Maginot line. Sperm penetration ceases when elevation of the fertilization envelope is completed (Rothschild and Swann, 1952; Ginzburg, 1964; Schuel et al, 1976b). If the elevated fertilization envelope is removed, subsequently added sperm can penetrate (re-fertilize) the egg to produce polyspermy (Sugiyama, 1951; Tyler ^/ a/.. 1956b; Longo, 1980, 1984). The fertilization envelope is derived from the vitelline layer, a thin extracellular coat or glycocalyx, that is attached to the plasma membrane of the unfertilized egg. It is detached from the oolemma and transformed into the elevated and hardened fertilization envelope by the actions of products secreted by the cortical granules at fertilization (reviewed by: Epel, 1978; Schuel, 1978, 1984). This process is begun by the secreted cortical granule protease which cleaves peptide bonds linking the vitelline layer to the egg's plasma membrane (see Cortical granule protease section, below). The subsequent lifting of this detached investment (nascent fertilization envelope) is promoted by hydration and/or osmotic effects resulting from the secretion of sulfated acid mucopolysaccharides and other hydrophylic colloids into the developing peri- vitelline space by the discharging cortical granules (Schuel et al, 1974; Schuel, 1978; Green and Summers, 1980). Immediately after its elevation (1-2 min post insemi- nation) the fertilization envelope is easily removed by various mechanical or chemical treatments, but it gradually hardens over the next 10-30 min and becomes extremely difficult to remove or disperse (Runnstrom, 1966; Veron et al, 1977; Schuel et al, 1982b). Hardening is a complex process involving covalent cross-linking mediated by peroxidatic (Foerder and Shapiro, 1977; Hall, 1978) and possibly transpeptidation (Lallier, 1970) reactions, as well as structuralization by cortical granule derived struc- tural proteins (reviewed by: Shapiro and Eddy, 1980; Schuel, 1978, 1984). Structur- alization and cross-linking normally are completed by 10-15 min after insemination. During the next 15-20 min the permeability of the fertilization envelope to proteins is reduced greatly (Veron et al, 1977). Collectively, these transformations act to protect the developing embryo from predation and other environmental hazards (Schuel, 1984). As the fertilization envelope elevates, most of the sperm that initially were bound to the vitelline layer at the egg surface are detached (Vacquier and Payne, 1973). However, some sperm remain attached to the elevating or fully elevated fertilization envelope for an extended period of time, yet they can not penetrate it (Summers et al, 1975; Summers and Hylander, 1976; Schuel and Schuel, 1981; also see Fig. POLYSPERMY BLOCKS 287 2, above). How are they excluded? One possibility is that hardening of the fertilization envelope makes it resistant to sperm penetration. However, sperm are unable to penetrate the soft fertilization envelope immediately after its elevation (Rothschild and Swann, 1952; Schuel et al. 1976b) while structuralization and cross linking require 10-15 min for completion (Veron et al., 1977; Schuel et al., 1982b). Nicotine accelerates structuralization of the fertilization envelope (Longo and Anderson, 1970a). However, it has been reported that sperm can continue to enter nicotine-treated eggs to produce polyspermy after fertilization envelope is completely elevated (Hagstrom and Allen, 1957; Dale et al.. 1982). In addition, 3-amino-l,2,4-triazole inhibits the ovoperoxidase-catalyzed cross linking of the fertilization envelope during hardening (Foerder and Shapiro, 1977) but does not promote polyspermy (Boldt et al., 1981; Coburn et al., 1981). These findings suggest that either hardening is not involved in preventing polyspermy, or that sperm are excluded by a manifestation of hardening that cannot be detected by presently available experimental probes. Alternatively sperm may be inactivated by H2O2 that is produced during the cortical reaction (Boldt et al., 1981; Coburn et al., 1981) until some aspect of the hardening process renders the fertilization envelope completely resistant to sperm penetration. Future work should resolve these questions. Cortical granule protease A soybean trypsin inhibitor (SBTI) sensitive protease(s) is localized in cortical granules of unfertilized sea urchin eggs (Schuel et al., 1973; Decker and Kinsey, 1 983; Kopf e/ al., 1983), and is secreted during the cortical reaction at fertilization or upon parthenogenetic activation (Vacquier et al., 1973; Fodor et al., 1975; Carroll, 1976; Schuel et al., 1976b). This protease has been purified from unfertilized eggs and fertilization product (cortical granule exudate) by SBTI-affinity chromatography and characterized biochemically (Fodor et al., 1975; Alliegro and Schuel, 1983, 1984; Sawada et al., 1984). It is a serine protease that is trypsin like in its enzymatic properties. The biological functions of the cortical granule protease in fertilization have been studied using SBTI and other serine protease inhibitors, and by the direct application of the purified protease to fertilization cultures (reviewed by: Epel, 1978; Schuel, 1978, 1984). The protease helps prevent polyspermy by promoting cortical granule exocytosis (Hagstrom, 1956a; Lonning, 1967; Longo and Schuel, 1973), by detaching the vitelline layer from the egg's plasma membrane to initiate elevation of the fer- tilization envelope (Longo and Schuel, 1973; Longo et al., 1974; Carroll and Epel, 1975; Schuel et al., 1976a), by proteolytic removal of sperm receptors in the vitelline layer (Aketa et al., 1972; Vacquier et al., 1973; Carroll, 1976; Glabe and Vacquier, 1978; Carroll et al., 1982; Acevedo-Duncan and Carroll, 1983), and by promoting the generation of H2O2 by fertilized eggs (Coburn et al., 1981). Limited proteolysis of egg surface proteins in the plasma membrane and/or vitelline layer takes place during fertilization (Shapiro, 1975; Ribot et al., 1983). These processes may reflect multiple direct functions of the protease, a cascade reaction involving several proteases as in blood coagulation, and/or the activation of other enzymatic systems. Zymogen activation is a ubiquitous property of serine proteases in a wide variety of cellular systems, and produces a prompt and irreversible response to physiological stimuli to initiate new functions (Neurath and Walsh, 1976; Neurath, 1984), as occurs during fertilization (Schuel, 1978, 1984). Eggs become polyspermic in SBTI because the cortical reaction is inhibited (Longo and Schuel, 1983; Vacquier et al., 1973; Longo et al., 1974; Schuel et al., 1976). The 288 H. SCHUEL addition of equivalent amounts of bovine serum albumen, an inert protein, to the cultures does not affect the cortical reaction and does not cause polyspermy (Schuel and Schuel, 1981). SBTI is the most potent enzymatic inhibitor of the cortical granule protease known at present (Alliegro and Schuel, 1984). Furthermore, acid or alkali inactivation of SBTI (Kunitz, 1947) abolishes its capacity to inhibit this sea urchin egg protease and to cause polyspermy (AUiegro and Schuel, 1984). Other serine protease inhibitors (ovomucoid, limabean trypsin inhibitor, antipain, leupeptin, tosyl lysine chloromethyl ketone, benzamidine, etc.) mimic the effects of SBTI in producing polyspermy by inhibiting the cortical reaction (Hagstrom, 1956a; Runnstrom, 1966; Vacquier et al, 1972a, b; Schuel et al, 1973, 1976a, b; Hoshi et al, 1979; Alliegro and Schuel, 1984; Sawada et al., 1984). The biological effectiveness of trypsin inhibitors in promoting polyspermy shows a high correlation with their relative potencies as enzymatic inhibitors of the purified cortical granule protease (Table I). In view of these findings the claim by Dunham et al. ( 1 982) that SBTI causes polyspermy because of a non-specific protein effect is unlikely to be correct. SBTI-treated sea urchin eggs are monospermic initially (Hagstrom, 1956a) and polyspermy arises gradually by refertilization extending for a 1 5-20 min period after the initial insemination (Hagstrom, 1956a; Longo and Schuel, 1973). This process was elucidated in SBTI-treated Arbacia eggs by: (1) determining the receptivity of monospermic eggs (obtained by minimal initial insemination) to refertilization upon subsequent re-insemination with excess sperm; combined with (2) a morphometric analysis (transmission electron micrographs) of the cortical reaction (Longo et al, 1973; Schuel et al, 1976a). The results of these studies are depicted schematically in Figure 9. In control eggs the cortical reaction (transition from Fig. 9A-C) normally is completed by 60 s after insemination but requires 15-20 min for completion in SBTI-treated eggs. Cortical granule exocytosis is completed in SBTI-treated eggs by 3 min post insemination (Fig. 9B). At this time the vitelline layer has detached only over regions formerly occupied by the discharged cortical granules, but remains attached to the plasma membrane at numerous sites (cortical projections) that were devoid of subjacent cortical granules prior to fertilization. These regions correspond to the functional sperm penetration sites because receptivity of the SBTI-treated egg to polyspermy (refertilization) at 3 min is the same as that of the unfertilized egg. Receptivity to refertilization only declines coincidentally with the gradual detachment Table I Comparison of the potencies of serine protease inhibitors in inhibiting the purified cortical granule protease and promoting polyspermy in sea urchins Inhibitor ID50 (M) P50 (M) Soybean trypsin inhibitor 6.01 X 10"' 4.94 X 10"* Ovomucoid 1.06 X 10"* 1.21 X IG"" Limabean trypsin inhibitor 3.47 X 10~* 1.42 X 10"'' Leupeptin 4.22 X 10"* 8.48 X 10"" Antipain 3.16 X 10"' 3.69 X 10"" ID50: Inhibitor cone, to inhibit 50% of enzyme activity using benzoyl- 1 -arginine ethyl ester as substrate. Protease purified from extract of unfertilized Stronglyocentrotus purpuratus eggs by SBTl-affinity chro- matography. P50: Inhibitor cone, to promote polyspermy in 50% oi Arbacia punctulata eggs inseminated with excess sperm. According to Spearman's rank correlation, r^ = 0.90. From Alliegro and Schuel (1984), reprinted with permission oi Biol. Bull. POLYSPERMY BLOCKS 289 VL -^ PM _, CG ^'^^^ // // /////. igUUV/ Figure 9. Schematic diagram depicting the effects of SBTI in causing polyspermy and inhibiting the cortical reaction in sea urchin eggs. The transition from A to C is normally completed in control Arbacia punclulata eggs within 60 s after insemination. From Schuel (1978), reprinted with permission of Alan R. Liss. Inc. A: Surface of an unfertilized egg showing regions where cortical granules (CG) are tightly packed under the plasma membrane (PM) interspersed with randomly distributed areas devoid of subjacent cortical granules. The vitelline layer (VL) is attached to the outer surface of the egg's plasma membrane (PM). Transition from A to B is sensitive to inhibition of SBTI. B: Surface of SBTI-treated egg at 3 min after insemination. The fertilization envelope has elevated over regions previously occupied by the discharged cortical granules. The vitelline layer remains attached to the egg's plasma membrane at the apex of the cortical projections. These sites correspond to regions where the plasma membrane was devoid of subjacent cortical granules before fertilization. Sperm can continue to fuse with and penetrate the egg at these sites as long as they are available. Transition from B to C sensitive to inhibition by SBTI. C: Surface of SBTI- treated egg at 20 min after insemination. The fertilization envelope has elevated from the entire surface of the egg and prevents the entrance of additional sperm. of the vitelline layer from the cortical projections over the next 15-20 min (Fig. 9C). Sperm penetration can continue at these sites in SBTI-treated eggs as long as they are available (Fig. 10). Detachment of the vitelline layer from these potential penetration sites is required to complete the cortical block to polyspermy. Refertilization (poly- spermy) also is facilitated because SBTI-treated eggs can not produce H2O2 to inactivate sperm at their surfaces (Cobum et al, 1981). However, SBTI-treated eggs do not fill up with supernumerary sperm under these conditions because the electrical (sodium dependent) block is still operative and tends to limit sperm penetration (Schuel and Schuel, 1981). Large numbers of sperm rapidly bind to the vitelline layer at insemination, and most of them subsequently detach from the elevating fertilization envelope (Vacquier and Payne, 1973; Summers and Hylander, 1976). Sperm detachment is prevented by SBTI (Fig. 1 1 ). Application of the cortical granule protease (Carroll and Epel, 1975; Carroll, 1976) or bovine trypsin (Aketae/fl/., 1972) to unfertilized eggs prevents the binding of subsequently added sperm. These findings suggest that the cortical granule protease removes sperm receptors from the vitelline layer (Glabe and Vacquier, 290 H. SCHUEL FE / t ^ ^v ?.»- ' -"^i n? . -vV r "^ . V ■ . ** J i!*&i .35I. '-■ '"» » (tT^jp^* i i t ^f^ Ik 2r"- '^' ' FE tt^tlsK^f^Uih.^i Figure 10. Spermatozoon associated with apical surface of a cortical projection (CP) via its acrosomal process (AF) in SBTI-treated Abracia zygote seven minutes after initial minimal insemination, fixed 2 min after subsequent re-insemination with excess sperm. Fertilization envelope (FE), periviteUine space (PV), sperm nucleus (SN). 37,800x. Inset: Photomicrograph of zygote re-inseminated 12 min after initial insem- ination. Zygote nucleus (ZN), refertilizing spermatozoon (RS). 450X. From Longo et al. (1974), reprinted with permission of Academic Press, Inc. POLYSPERMY BLOCKS 291 9 UJ a. c O CD CO \ SBTI \ \ \ \ •v Seconds After Insemination Figure 1 1 . Effect of SBTI on sperm binding and detachment during fertilization in Strongylocentrolus purpuratus. Eggs were inseminated in sea water (• •) or 1 mg/ml SBTI (• •), and the number of sperm bound per egg was determined at indicated times after insemination. Sperm rapidly bind to the egg surfaces and then begin to detach as the cortical reaction propagates around the surface of the eggs (compare with Fig. 2, above). This process is completed in control eggs within 50 s. SBTI-treated eggs do not show the normal detachment phase. From Vacquier et al. (1973), reprinted with permission of Academic Press. 1978; Yoshida and Aketa, 1978). This process tends to reduce the probability of polyspermy. Detachment of the vitelline layer and destruction of sperm receptors in sea urchins may be promoted by two distinct SBTI-sensitive proteases that are secreted by the cortical granules (Carroll and Epel, 1975; Carroll, 1976; Carroll et al.. 1982). Similar phenomena apparently operate in mammalian eggs where the secretion of a "trypsin-like" cortical granule protease has been implicated in altering the zona pel- lucida to prevent polyspermy (Gwatkin et al., 1973; Bleil et al., 1981; Wolf 1981). Peroxide mediated block Phagocytic leukocytes employ peroxidatic reactions involving secreted myelo- peroxidase and H2O2 to kill bacteria and other microorganisms (Klebanoff, 1980). The peroxidatic system activated in sea urchin eggs at fertilization, secreted ovope- roxidase and H2O2 which promotes cross-linking of the fertilization envelope, was 292 H. SCHUEL postulated to also act as a spermicidal agent to help prevent polyspermy (Foerder and Shapiro, 1977; Klebanoff ^/ al, 1979; Shapiro and Eddy, 1980), since H2O2 is toxic to sperm (Evans, 1947). Evidence for a peroxide-mediated block to polyspermy in sea urchins has been obtained (Boldt et al, 1981; Cobum et al, 1981). Sperm are rapidly inactivated by egg-derived H2O2 . However, a peroxidase endogenous to the sperm appears to be responsible for their inactivation (Boldt, 1982; Boldt et al, 1981, 1984). Sea urchin eggs release H2O2 into the ambient sea water during the cortical reaction at fertilization (Fig. 12). The fertilizing capacity of sperm is rapidly reduced by treatment with equivalent concentrations of H2O2 (Boldt et al, 1981; Cobum et al, 1981). For example, with minimal sperm densities, 300 tiM H2O2 produces an instantaneous 35% reduction in sperm fertility which is further suppressed to a 90% reduction over five minutes (Boldt et al, 1981). The fertilizing capacity of control sperm in sea water remains constant during this period. Removal of egg-derived H2O2 by adding catalase to fertilization cultures of Arbacia produces a concentration dependent induction of polyspermy (Cobum et al, 1981; Dunham et al, 1982). Catalase does not impair elevation of the fertilization envelope. It must be added prior to completion of the cortical reaction to cause polyspermy (Fig. 13). The addition of equivalent amounts of bovine serum albumin, a non-enzymatic protein, does not cause polyspermy (Cobum et al, 1981; Schuel, 1984). Furthermore, inactivation of catalase by heating or addition of 3-amino-l,2,4-triazole, an inhibitor which binds irreversibly with the catalase-H202 300i- //-• TIME(min.) iH Figure 1 2. Release of H2O2 by Stwngylocentrotus purpuratus eggs during fertilization. Eggs inseminated with a sperm density just sufficient to obtain 100% fertilization. Gametes were removed from the cultures at indicated times after insemination. The concentration of H2O2 was determined in the supematants by the horseradish peroxidase mediated oxidation of pyrogallol; - O - O -, 0.2 ml packed eggs/ 10 ml culture; -•-•-, 0.1 ml packed eggs/ 10 ml culture. From Boldt et al. (1981), reprinted with permission of Alan R. Liss, Inc. POLYSPERMY BLOCKS 293 o 100 80 r*x, I 60h- Q. I/) 40 20 J L 15 30 Time (sec) Figure 13. Timing of catalase induced polyspermy in Arbacia punctulata eggs. Catalase (2 mg/ml final cone.) added to cultures at indicated times after insemination with excess sperm. When catalase is added after the cortical reaction is completed at 60 s post insemination, the incidence of polyspermy is the same as in control eggs fertilized in sea water. From Cobum et al. (1981), reprinted with permission of Academic Press. complex (Margoliash and Novogrodsky, 1960), abolishes its capacity to promote polyspermy (Cobum et al.. 1981). Together these findings show that: (1) sea urchin eggs produce H2O2 to help prevent polyspermy until the cortical reaction is completed; and (2) catalase promotes polyspermy by the enzymatic removal of egg-derived H2O2. The claim that catalase causes polyspermy because of a non-specific protein effect (Dunham et al., 1982) is unlikely to be correct. The putative role of ovoperoxidase in preventing polyspermy was examined using phenylhydrazine and 3-amino-l,2,4-triazole (Boldt et al., 1981). These are the two most potent inhibitors of ovoperoxidase in Strongylocentrotus (Foerder and Shapiro, 1977), and both would be expected to cause polyspermy if ovoperoxidase were involved. Phenylhydrazine promotes polyspermy while 3-amino-l,2,4-triazole does not (Boldt et al, 1981). These results suggested that the egg's ovoperoxidase does not have a functional role in preventing polyspermy, and implied that a peroxidase endogenous to the sperm might be involved. Evidence for such a sperm peroxidase has been obtained (Boldt et al., 1981, 1984). Phenylhydrazine protects sperm from inactivation by H2O2, while 3-amino- 1,2,4- triazole potentiates the adverse effect of H2O2 on sperm fertility (Boldt et al.. 1981). Phenylhydrazine is a well known peroxidase inhibitor (Hidaka and Udenfriend, 1970), while 3-amino- 1,2,4-triazole is a classic catalase inhibitor (Margoliash and Novo- grodsky, 1960). Biochemical studies have shown that sea urchin sperm actually contain a peroxidase that is preferentially inhibited by phenylhydrazine and a 3-amino- 1,2,4- triazole sensitive catalase (Boldt, 1982; Boldt and Schuel, 1982; Boldt et al.. 1984). These data suggest that sperm fertility is modulated by reactions of their endogenous catalase and peroxidase with egg-derived H2O2 during fertilization. Hydrogen peroxide 294 H. SCHUEL is a potent cytotoxic substance (Klebanoff, 1980). Catalase is a ubiquitous enzyme in aerobic cells and protects them by removing H2O2 generated during normal oxidative metabolism (Chance et ai, 1979). The sperm catalase probably performs a similar function (Rothschild, 1950). It also appears to protect sperm from H2O2 produced by fertiUzed eggs (Boldt et al, 1981; Cobum et ai, 1981). Peroxidases use H2O2 to oxidize other substances. The products of such reactions can be cytotoxic (Klebanoff, 1980). The sperm peroxidase may perform an analogous function by utilizing H2O2 released by fertilized eggs to reduce sperm fertility, and thus assist in preventing polyspermy. As in phagocytic cells, peroxide production by eggs during fertilization is associated with a burst in cyanide insensitive oxygen consumption (Foerder et al, 1978; Klebanoff et ai, 1 979; Klebanoff, 1 980; Perry and Epel, 1981). Two thirds of the oxygen consumed by Strongylocentrotus eggs at fertilization is converted into H2O2 (Foerder et ai, 1978). Superoxide anion is produced along with H2O2 during the respiratory burst by phago- cytes. Together these oxygen species can generate other extremely cytotoxic oxygen radicals via the Haber-Weiss reaction (Klebanoff, 1980). Chemiluminescence detected during fertilization (Foerder et ai, 1978) may reflect the formation of such oxygen radicals. The cytotoxic effects of phagocytes on microorganisms and tumor cells can be reduced by addition of catalase and/or superoxide dismutase to the cultures (Kuehl and Egan, 1980; Fridovich, 1982; HalliweU, 1982; Nathan, 1982). Similar processes may operate in sea urchin gametes at fertilization since added catalase or superoxide dismutase promote polyspermy (Cobum et ai, 1981; Dunham et ai, 1982). The respiratory burst and H2O2 production may be linked to other early events in eggs at fertilization such as the release of calcium from intracellular stores, the influx of external sodium ions, and cortical granule secretion (Steinhardt and Epel, 1974; Foerder et ai, 1978; Whitaker and Steinhardt, 1982). The respiratory burst in sea urchin eggs is blocked in sodium-free sea water (Whitaker and Steinhardt, 1982). This finding suggests that there may be a functional link between the sodium dependent (Jaffe, 1980; Schuel and Schuel, 1981) and peroxide mediated (Boldt et ai, 1981; Cobum et ai, 1981) blocks to polyspermy. A similar situation may apply to the functions of the cortical granule protease. SBTI is a potent enzymatic inhibitor of the cortical granule protease (see Cortical granule protease section, above), and it also blocks the production of H2O2 by fertilized eggs (Cobum et ai, 1981). This may account, at least in part, for the high levels of polyspermy seen in SBTI-treated sea urchin eggs. SBTI and other serine protease inhibitors prevent the production of oxygen radicals by phagocytes (Janoff and Carp, 1982; Troll et ai, 1982). This again is suggestive of the many biochemical similarities between sea urchin gametes and somatic phagocytes. Does a peroxide mediated block to polyspermy operate in mammals? Studies intended to answer this question have yet to be conducted, but there are several highly suggestive similarities between sea urchins and mammals. Cortical granule derived ovoperoxidase is involved in cross-linking the fertilization envelope in sea urchins (Foerder and Shapiro, 1977; Hall, 1978) and the zona peflucida in mammalian eggs (Gulyas and Schmell, 1980; Schmell and Gulyas, 1980). Given this situation, the fertilized mammalian egg would be expected to produce H2O2 required for cross- linking as well. Furthermore, H2O2 and other active oxygen metabolites are very toxic to mammalian sperm (Smith and Klebanoff, 1970; Jones and Mann, 1973; Holland et ai, 1982; Alvarez and Storey, 1982, 1983), just as they are to sea urchin sperm (Evans, 1947; Boldt et ai, 1981). These phenomena in mammalian gametes should be studied in relation to polyspermy prevention. POLYSPERMY BLOCKS 295 Arachidonic acid cascade Changes in phospholipid metaboHsm occur in sea urchin eggs during fertilization (Schmell and Lennarz, 1974; Barber and Mead, 1975; Byrd, 1975; Kozhina et ai, 1978; Barber, 1979; Turner et ai, 1983). Membrane fusion between secretory granules and the plasma membrane is promoted by the action of a calcium-activated phos- pholipase A2 in both somatic secretory cells (Laychock and Putney, 1982; Rubin, 1982) as well as sea urchin eggs (Ferguson and Shen, 1984) and sperm (Conway and Metz, 1976; SeGall and Lennarz, 1981). Phospholipase A2 releases a free fatty acid (usually arachidonic acid) and lysophosphoglycerides from membrane phosphoglycer- ides (Fig. 14). These substances may be re-incorporated into membrane phospholipids via reacylation reactions in sea urchin eggs during fertilization (Schmell and Lennarz, 1974; Turner et ai, 1983) or in secreting somatic cells (Laychock and Putney. 1982; Rubin, 1982). Lysophosphoglycerides can initiate exocytosis by promoting membrane fusion (Lucy, 1970; Laychock and Putney, 1982). Free arachidonic acid can be oxidized by two major pathways (Kuehl and Egan, 1980). The cyclooxygenase pathway leads to the production of prostaglandins and thromboxanes. Non-steroidal anti-inflam- matory drugs (NSAID) act by inhibiting cyclooxygenase (Flower, 1974; Kuehl and Egan, 1980). The lipoxygenase pathway leads to the production of the leukotrienes and hydroxy fatty acids (HETE's). Cells may contain different lipoxygenases which oxidize the carbon atom at positions 5, 11, 12, or 15 of arachidonic acid (Nelson et ai, 1982; Samuelsson, 1983). Leukotrienes are products of the 5-lipoxygenase pathway. The slow reacting substance of anaphylaxis (SRS-A) consists of leukotrienes LTC4, MEMBRANE PHOSPHOLIPIDS PHOSPHOLIPASE A2 CA-^+ACTIVATED QUINACRINE INHIBITS REACYLATION ARACHIDONIC ACID +LYSOP HO SPHOGYICERIDE S ►REACVIATION CYCLOOXYGENASE NSAID NHIBIT LIPOXYGENASES PROSTAGLANDINS + THROMBOXANES HPETE'S LEUKOTRIENES HETE'S MEMBRANE FUSION EXOCYTOSIS REGULATORS OF CELLULAR FUNCTIONS Figure 14. Schematic diagram depicting role of phospholipase Aj in the initiation of the arachidonic acid cascade and the membrane fusion reaction in exocytosis. as discussed in text. Non-steroidal anti- inflammatory drugs (NSAID), hydroperoxy eicosatetraenoic acids (HPETE'S), hydroxy eicosatetraenoic acids (HETE'S). 296 H. SCHUEL LTD4, and LTE4 (Samuelsson, 1983). Products derived from the enzymatic oxidation of arachidonic acid by cyclooxygenase and lipoxygenases are widely distributed in invertebrates and vertebrates (Flower, 1974; Nomura and Ogata, 1976; Morse et al, 1977), are extremely potent mediators of inflammatory reactions and also are mod- ulators of a wide variety of normal cellular functions in somatic tissues (Kuehl and Egan, 1980; Laychock and Putney, 1982; Nelson et al, 1982; Samuelsson, 1983). Several lines of evidence suggest a role for phospholipase A2 in triggering cortical granule discharge in sea urchin eggs at fertilization. The cortical reaction can be induced by treating unfertihzed eggs with meUttin which is a phospholipase A2 activator from bee venom (Ohman, 1945; Shimada et al., 1982). Similar results can be obtained by the application of lysolecithin (Ohman, 1945; Schuel, 1978), a potent fusigen liberated from membrane lipids by phospholipase (Lucy, 1970). Quinacrine inhibits phospholipase A2 in blood platelets (Lapetina et al, 1981). Unfertilized sea urchin eggs contain a calcium activated phospholipase A2 that is sensitive to inhibition by quinacrine (Ferguson and Shen, 1984). Furthermore, quinacrine inhibits the cortical reaction and promotes polyspermy in fertilization cultures (Ferguson and Shen, 1984). Evidence has been obtained implicating arachidonic acid oxidation in preventing polyspermy (Schuel et al, 1983; Schuel et al, 1984). Arachidonic acid is located at the second acyl position of phosphoglycerides and is released by the action of phos- pholipase A2 (Hill and Lands, 1970; Lapetina et al, 1981; Bach, 1982; Laychock and Putney, 1982). Membrane phosphoHpids of cortical granules and plasma membrane in unfertilized sea urchin eggs contain abundant (about 1 7% of total fatty acids) arachidonic acid (Decker and Kisney, 1983). Arachidonic acid is liberated from phos- pholipids in eggs at fertilization by a calcium activated phospholipase (Perry, 1979), presumably phospholipase A2 (Ferguson and Shen, 1984). The synthesis of arachidonic acid oxidation products of the cyclooxygenase (prostaglandins) and cyclooxygenase (12-HETE-like molecule) pathways at fertiUzation has been reported (Perry, 1979). Enzyme inhibitors and specific product antagonists have been used as probes to show a role for these reactions in the block to polyspermy. Indomethacin, a NSAID that is a potent cyclooxygenase inhibitor in somatic tissues (Flower, 1974; Kuehl and Egan, 1980), causes a dose and sperm density dependent induction of polyspermy if added before the egg completes the cortical reaction (Schuel et al, 1984). Indomethacin does not retard the cortical reaction (Elhai, 1981; Schuel et al, 1984), and does not promote polyspermy by protecting sperm from peroxidatic inactivation by egg-derived H2O2 (Schuel et al, 1984). Other potent cyclooxygenase inhibitors (flufenamate and meclofenamate) also cause polyspermy (Table II). FPL-55712, an antagonist for leu- kotrienes LTC4 and LTD4 (Sheard et al, 1982), causes a dose and sperm density dependent induction of polyspermy (P50 = 2.5 ± 0.8 nM in Arbacia at 4.0 ± 2.2 sperm/ml, n = 6) if added before the eggs complete the cortical reaction (Moss et al, 1983). To determine which gamete is affected by FPL-55712, eggs and sperm were pretreated with the drug which was removed by dilution at fertilization. Sperm pretreated with FPL-55712 do not cause polyspermy in control eggs. However, eggs pretreated with FPL-55712 become heavily polyspermic upon insemination with control sperm. These findings suggest that reaction of leukotrienes with receptors on the egg's surface modulate its receptivity to sperm during the cortical reaction. BW755C, a potent inhibitor of the 5-lipoxygenase involved in leukotriene synthesis during inflammatory reactions (Higgs and Mugridge, 1983), also causes polyspermy in sea urchins (Schuel and Schuel, unpubl. data). Hence products derived from both the cyclooxygenase and 5-lipoxygenase pathways for oxidation of arachidonic acid appear to modulate gamete interaction and help prevent polyspermy in sea urchins. POLYSPERMY BLOCKS 297 Table II Relative potency ofNSAID in promoting polyspermy in Stronglyocentrotus Drug P50* ifiM) Indomethacin (n = 8) 38.1 ± 18.4 Rufenamic Acid (n = 8) 20.6 ± 10.5 Meclofenamate (n = 4) 16.9 ± 9.4 * P50: Concentration of drug at which 50% of the eggs were polyspermic. Incidence of polyspermy in control eggs; 14.7 ± 1 1.8%. Sperm density: 9.8 ± 4.5 X lOVml. n = 13. Aspirin, a weak cyclooxygenase inhibitor, did not cause polyspermy at cone, up to 5 mM (data not shown). From Schuel et al. (1984), reprinted with permission of Alan R. Liss, Inc. The metabolism of arachidonic acid in other systems is regulated by the amount of substrate (free arachidonic acid) available for oxidation (Vogt, 1978; Racowsky and Biggers, 1983). If this were true for sea urchin eggs as well, then addition of exogenous arachidonic acid to unfertilized eggs should result in the formation of products that inhibit fertilization. Added arachidonic acid has been reported to reduce the fertility of sea urchin eggs (Elhai and Scandella, 1983). Additional work is required to identify directly the arachidonic acid products that are produced during fertilization and to determine how they act to prevent polyspermy. Similar processes may operate in other animals as well. For example, the mechanism by which molluscan eggs prevent polyspermy has been an enigma because there is no overt structural change at the egg surface comparable to the cortical reaction in sea urchins (Longo, 1973; Alliegro and Wright, 1983). However, molluscan eggs are a rich source of cyclooxygenase (Morse et al., 1977). Do they use cyclooxygenase metabolites to prevent polyspermy? Arachidonic acid derived products also may regulate cortical granule secretion (Elhai, 1981) and other aspects of fertilization. An ionophore-like role for lipoxygenase products in releasing calcium from internal stores during egg activation has been proposed (Epel et al., 1982). Both cyclooxygenase and lipoxygenase products are known to exhibit properties of calcium ionophores in somatic tissues (Laychock and Putney, 1982; Weissmann et al., 1982). The acrosome reaction in human sperm involves a membrane associated phospholipase Ai (Thakkar et al., 1984) and appears to be regulated by arachidonic acid oxidation products (Meizel and Turner, 1983). In view of these observations it is tempting to speculate that arachidonic acid derived metabolites also may help prevent polyspermy in mammals. There may be a functional link between the arachidonic acid cascade and the peroxidatic block to polyspermy in sea urchins (see Peroxide mediated block section, above) since: ( 1 ) cytotoxic oxidizing radicals are produced during peroxidatic reactions in the arachidonic acid cascade (Kuehl and Egan, 1980; Kuehl et al., 1982); (2) both H2O2 and superoxide anion are required to initiate the arachidonic acid cascade (Kuehl and Egan, 1980; Fridovich, 1982); and (3) H2O2 can have a role in the metabolism of prostaglandins (Paredes and Weiss, 1982). There also may be a link between the functions of the cortical granule protease and the arachidonic acid cascade since thrombin treatment activates phospholipases and arachidonic acid oxidation in platelets (Lapetina et al., 1981). In addition, serine protease inhibitors block the stimulus-induced release of arachidonic acid by platelets (Feinstein ('/ al.. 1980). 298 H. SCHUEL Hyaline layer A role in polyspermy prevention has been attributed to the hyaUne layer (reviewed by: Rothschild, 1956; Allen, 1957; Runnstrom et al, 1959; Tyler and Tyler, 1966b; Epel, 1978; Schuel, 1978, 1984). This concept is based upon findings that the hyaline layer restricts sperm penetration into eggs that are experimentally denuded of their fertilization envelopes (Ishida and Nakano, 1950; Sugiyama, 1951; Hagstrom and Hagstrom, 1954b; Nakano, 1956; Tyler et al., 1956b; Longo, 1980). Such demem- branated eggs can be refertilized, but the frequency of polyspermy declines with time (Tyler et al., 1956b; Schatten, 1978; Longo, 1980). The reduced receptivity of these eggs to refertilization is caused by the formation of the hyaline layer which gradually covers up sperm receptors in the egg's plasma membrane (Tyler and Tyler, 1956b). Removal of this barrier by dispersing the hyaline layer with calcium-magnesium free sea water makes it possible to refertilize zygotes or embryos through the 8-cell stage of development (Sugiyama, 1951; Runnstrom et al., 1959; Longo, 1980 and 1984). Demembranated eggs gradually become refractory to sperm penetration even after the hyaline layer is removed (Schatten, 1978; Longo, 1980). This effect was attributed to the establishment of a late permanent block to polyspermy at the level of the egg's plasma membrane (Schatten, 1978). This interpretation may not be correct. Hyalin, the major structural protein of the hyaline layer, is stored in cortical granules of unfertilized eggs (reviewed by: Schuel, 1978, 1984). The hyahne layer that is formed during fertilization is derived from this source. However, hyalin also is stored in small cytoplasmic vesicles of unfertilized eggs that do not secrete during fertilization (Hy- lander and Summers, 1982) and which normally discharge during subsequent em- bryonic development (Kane, 1973). This secondary reservoir is probably the source of hyalin protein that can regenerate the hyaline layer after its experimental removal (Citkowitz, 1971; Kane, 1973; Schuel et al., 1982a). Such regeneration of the hyaline layer probably accounts for the gradual reduction in receptivity of demembranated eggs to refertilization (polyspermy) after the initially formed hyaline layer is removed (Longo, 1980). The hyaline layer is unlikely to be a significant factor in preventing polyspermy during normal fertilization because sperm can not penetrate either the elevating or fully elevated fertilization envelope (Rothschild and Swann, 1952; Rothschild, 1956; Schuel, 1978, 1984; also see Fertilization envelope section, above). Another possibility is that the protein hyalin also might impregnate the fertilization envelope and help render it resistant to sperm penetration. However, immuno-electron cytochemical studies using mono-specific antibodies against pure hyalin did not detect hyalin in the fertilization envelope (Hylander and Summers, 1982). Nevertheless, the hyaline layer may serve as a final line of defense against polyspermy for an egg that has lost its fertilization envelope because of an environmental insult in nature, or experimental manipulation in the laboratory. Sulfated acid mucopolysaccharides Cortical granules of unfertilized sea urchin eggs contain sulfated acid mucopoly- saccharides (SAMP) that are secreted during the cortical reaction at fertilization (Is- hihara, 1968; Bal, 1970; Schuel et al., 1974). These acid polyanions are thought to participate in the lifting of the fertilization envelope by promoting the influx of water into the developing perivitelline space (Schuel et al., 1974; Schuel, 1978). The cytochemical localization of SAMP in the cortical granules was facilitated by using quaternary ammonium salts to prevent loss of SAMP during fixation (Schuel et al., 1974). The natural ionic environment of sea urchin eggs in sea water favors POLYSPERMY BLOCKS 299 the formation of stable complexes between SAMP and quaternary ammonium salts (Kelly et al, 1963). Brief exposure of unfertilized eggs to several quaternary ammonium salts (cetyltrimethyl ammonium bromide, cetylpyridinium chloride and bromide, tetraethylammonium chloride, etc.) produces residual adverse effects on subsequent fertilization in sea water in terms of reduced fertility and greatly increased vulnerability to polyspermy in eggs that do fertilize (Schuel et al., 1974). Eggs cannot be fertilized in the presence of the quaternary ammonium salts because these substances inactivate and/or kill the sperm. These findings suggest that cortical granule derived SAMP may have a role in preventing polyspermy (Schuel et al., 1974). However, the qua- ternary ammonium salts could produce polyspermy by different mechanisms since some of them may alter the egg's plasma membrane by acting as detergents or otherwise modifying its electrical properties (Schuel et al., 1974; Schuel, 1978; Shen. 1983). This phenomenon should be studied further. The Nicotine Controversy Nicotine has been recognized to be a polyspermy promoting agent for almost a century (Hertwig and Hertwig, 1 887; Clark, 1 936). However, the mechanism by which nicotine causes polyspermy remains controversial because the drug appears to produce multiple effects on gamete interactions, and because observations made by various investigators are not consistent with each other. This may reflect species differences in response to nicotine, and/or differences in experimental procedures used by various investigators. Binding of sperm to the egg surface is greatly enhanced in nicotine-treated eggs, especially if the jelly coat had been removed prior to insemination (Hagstrom and Allen, 1956; Longo and Anderson, 1970a). Sperm remain bound to the elevated fertilization envelope in nicotine-treated eggs. Thus nicotine promotes a more stable adhesion of the gametes which could allow more sperm to fuse with the egg than normal (Longo and Anderson, 1970a). Nicotine inhibits the rapid block to polyspermy (Rothschild, 1956). Nicotine- treated eggs may fertilize more rapidly than controls (Rothschild and Swann, 1950; Rothschild, 1953; Dale et al., 1982), although contrary results have been reported (Hagstrom and Allen, 1956). Sperm do not swim more rapidly in nicotine so sperm motility can not account for the higher fertilization rate (Rothschild and Swann, 1950; Rothschild, 1953). Nicotine also affects the electrical properties of the egg's plasma membrane as reflected by decreased membrane resistance, altered current voltage relation such that outward current produces a smaller shift in potential, and reduced amplitude of the fertilization potential (Jaffe, 1980; Dale et al., 1982). These findings show that nicotine inhibits the electrical block to polyspermy (Jaflfe, 1980). Dale's group (1982) claim that these electrical phenomena are not related to polyspermy prevention (see Electrical block section, above). Another view holds that nicotine produces polyspermy by inhibiting some aspect of the cortical reaction (Hagstrom and Allen, 1956; Dale et al.. 1982). Elevation of the fertilization envelope in nicotine-treated eggs usually is completed at the same time as in controls (Rothschild, 1953; Hagstrom and Allen, 1956; Longo and Anderson, 1970a). In some cases it elevates more rapidly in nicotine because cortical granule discharge is initiated at multiple loci which correspond to the sites where supernumerary sperm fuse with the egg (Rothschild and Swann, 1950; Longo and Anderson, 1970a). Sperm may continue to enter nicotine-treated eggs after elevation of the fertilization envelope has been completed (Hagstrom and Allen, 1956; Dale et al.. 1982). If correct, this finding suggests that nicotine may inhibit the transformation in the fertilization 300 H. SCHUEL envelope that is responsible for excluding sperm. Impaired structuralization of the fertilization envelope can not be responsible for continued sperm penetration because nicotine accelerates structuralization (Longo and Anderson, 1970a). However, other workers found that sperm incorporation ceases in nicotine-treated eggs when elevation of the fertilization envelope is completed (Baker and Presley, 1966). Cortical granule secretion occasionally is arrested by nicotine so that the fertilization envelope elevates only over a restricted portion of the egg surface (Hagstrom and Allen, 1956; Hulser and Schatten, 1982). Another possibility is that nicotine may act by inhibiting the cortical granule protease (Carroll, 1976). How nicotine produces polyspermy remains enigmatic. Multiple actions of the drug on gamete binding, fertilization potential, cortical granule secretion, and the fertilization envelope may be involved. Additional work under standardized conditions is required to clarify this question. Conclusions Sea urchin eggs show an extraordinary capacity to resist polyspermy upon insem- ination with excess sperm. Research summarized above has revealed that this is due to the redundancy of defense mechanisms all of which act together to help assure monospermic fertilization. Factors associated with external reproduction in the ocean must have exerted tremendous selective pressures for the development of these multiple polyspermy preventing mechanisms because a great excess of sperm is required to insure that fertilization would take place. Several of these polyspermy preventing mechanisms have been conserved in mammals (Gwatkin, 1977; Schuel, 1978, 1984; Wolf, 1981; Schmell et al, 1983) where an excess of sperm may arrive at the site of fertilization within the female's reproductive tract. Heretofore it has been assumed that the responsibility for polyspermy prevention resided exclusively with the fertilized egg. The sole function attributed to the sperm during fertilization was to try to enter the egg. However, functional cooperation by both interacting gametes appears to be involved in preventing polyspermy. Sperm are active participants in both the electrical (Jaffe et al, 1982) and peroxide mediated (Boldt et al, 1981, 1984) blocks. They are pre-programmed to reduce their fertility in response to electrical and chemical signals produced by the fertilized egg. Natural selection and evolution have fashioned an exquisite set of cellular responses and regulatory processes that are elicited during gamete interaction to increase the prob- ability of normal monospermic fertilization. Within this context, it is not an individual spermatozoon but rather the production of a diploid zygote that is important for successful reproduction and continuation of the species. Fertilization is a cellular process. Typical responses to stimulation that are common to all cells (electrical depolarization of the plasma membrane, calcium-triggered release of secretory products via exocytosis, limited proteolysis, respiratory burst, release of H2O2, and generation of arachidonic acid oxidation products, etc.) have been adapted by the interacting gametes to help prevent polyspermy. For somatic secretory cells such as those involved in inflammatory reactions, these responses are initiated by the reaction of a specific ligand with its receptor in the cell's plasma membrane. The equivalent function is subserved during fertilization by the reaction of complementary molecular determinants on the surface of the egg and the sperm at the site of gamete fusion. In this sense, fertilization and polyspermy prevention can be considered to be akin biochemically to an inflammatory reaction in somatic tissues. Multiple polyspermy preventing processes have been identified in sea urchins. 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YOSHIDA, M., AND K. Aketa. 1978. Localization of species-specific sperm-binding factor in sea urchin eggs with immunofluorescent probe. Acta Embryol. Exp. 3: 269-278. Reference: Biol. Bull. 167: 310-321. (October, 1984) HNE STRUCTURE AND VITAL STAINING OF OSPHRADIUM OF THE SOUTHERN OYSTER DRILL, THAIS HAEMASTOMA CANALICULATA (GRAY) (PROSOBRANCHIA: MURICIDAE) DAVID W. GARTON', RICHARD A. ROLLER, AND JOHN CAPRIO Department of Zoology and Physiology, Louisiana State University, Baton Rouge, Louisiana 70803 Abstract The morphology of the osphradium of Thais haemastoma canaliculata (Gray) was examined using light microscopy, SEM, and TEM. The osphradium is composed of approximately 150-200 lamellae, each of which is divided into two distinct regions by a groove situated parallel to the dorsal edge of the organ. The dorsal one-fourth of each lamella is covered by dense cilia that are assumed to generate water currents about the osphradium. Ciliary tufts, located in small depressions, and numerous secretory cells are distributed uniformly on the ventral three-fourths of the lamellae. A thin tract of cilia borders the ventral edge of each lamella. The overall cellular organization is less complex than has been reported previously in other marine pro- sobranchs. Selective staining of putative chemoreceptors was performed using Procion Brilliant Yellow. Individual cells in the ventral region and the ventral edge of each lamella were Procion-positive. Results of this study suggest that ventral interlamellar regions and the ventral edge of each lamella are chemosensory regions, while the dorsal portion of each lamella is indifferent epithelium. INTRODUCTION The chemosensory function of the osphradium in prosobranch gastropods is well established (Brown and Noble, 1960; Bailey and Laverack, 1966; Bailey and Benjamin, 1968). Ultrastructural studies on several prosobranch species reveal a similar overall pattern of cellular organization, although there is little agreement on the functional interpretation of individual cell types (Welsch and Storch, 1969; Alexander, 1970; Crisp, 1973; Alexander and Weldon, 1975; Newell and Brown, 1977). Five different types of presumed sensory cells ("Sinneszelle") were identified from transmission electron microscopy of the osphradium of Buccinium undatum (Welsch and Storch, 1969). These five cell types, dubbed Sil-Si5 (Crisp, 1973), were thought to be either chemoreceptors or proprioreceptors. Crisp (1973) found cell types Sil-Si4 in the osphradia of five prosobranch species, but she concluded that cell types Si 1 and Si2 were not sensory receptors because neither axons nor intracellular vesicles were ob- served. The presumed sensory receptors, Si3 (similar to a free nerve ending) and Si4 (similar to a primary receptor) were located primarily in a specialized region of the interlamellar surface (Crisp, 1973). Regional specialization of lamellar epithelium has been observed in other marine prosobranchs (Alexander and Weldon, 1975; Newell and Brown, 1977). Newell and Brown (1977), on the basis of "presynaptic vesicles," reported that some ciliated cells in the osphradium of Bullia digitalis might be sec- ondary receptors. As yet ultrastructural observations have not elucidated the primary or secondary nature of receptors and no electrophysiological data have confirmed the conclusions drawn from these ultrastructural observations. Received 23 March 1984; accepted 2 July 1984. ' Present address: Department of Ecology and Evolution, State University of New York at Stony Brook, Stony Brook, New York 1 1 794. 310 HNE STRUCTURE OF THAIS OSPHRADIUM 311 The present investigation was initiated to study the gross morphology and ultra- structure of the osphradial epithelium of Thais hacmastoma canaliculata (Gray) using light microscopy, SEM, and TEM and to identify putative chemoreceptor cells by vital staining with Procion Brilliant Yellow. Materials and Methods Adult oyster drills, Thais haemastoma canaliculata (Gray), were collected from the vicinity of Caminada Pass near Grand Isle, Louisiana. Drills were transported to the laboratory and placed in a 38 1 aquarium at room temperature (23-25 °C) and 20 %oS (Instant Ocean® Seawater Mix). Snails were removed from their shells by gently cracking open the shell at the base of the body whorl and severing the columellar muscle. The dorsal mantle was dissected open and folded back to expose the osphradium at the base of the ctenidium. The osphradium was dissected from the ctenidium and preserved for histological examination. For scanning electron microscopy (SEM), osphradia were fixed overnight with 2.5% gluteraldehyde in 0.2 M sodium cacodylate-sucrose buifer (585 mOsm; pH = 8.0). Specimens were rinsed in distilled water to remove all buffer salts, dehydrated in acidified 2,2-dimethoxypropane, critical-point dried in CO2 and coated with 200A of Au/Pd. Osphradia were examined at 25kV with a Hitachi S-500 SEM. For transmission electron microscopy (TEM), osphradia were fixed overnight with 2.5% gluteraldehyde in 0.2 M sodium cacodylate-sucrose buffer (585 mOsm; pH = 8.0) and post-fixed for 2 h in 1% OSO4 buffered in 0.2 M sodium cacodylate (pH = 8.0). Specimens were dehydrated in ethanol and embedded in Epon. Ultrathin sections were placed on copper grids and contrasted with uranyl acetate-lead citrate. Sections were examined with a JEOL 100-CX TEM at 80 kV. Vital staining of epithelial receptor cells in the osphradium was accomplished by a modification of the method of Holl (1981). Live drills acclimated to 7.5 %oS were anesthetized by chilling on ice. The shell of each snail was gently cracked at the body whorl, immediately over the osphradium. The mantle edge was cut and reflected and the exposed osphradium was periodically irrigated for 30 minutes with a 4% solution of Procion Brilliant Yellow in 0. 1 M KCl. For four species of freshwater fish, Holl (1981) used a 0.01 M KCl staining solution, a concentration we considered too low for an estuarine gastropod. Thais haemastoma canaliculata can be readily acclimated to low sahnity (Garton and Stickle, 1980), therefore a 0.1 A/ KCl solution (200 mOsm/kg) was used so that osmotic differences between the staining solution and the hemolymph would be minimized in low salinity acclimated snails (7.5 %oS equals 223 mOsm/kg). Excess stain was flushed away with 7.5 %oS sea water. The osphradium was post incubated at room temperature (24°C) for 30 minutes, removed from the snail and fixed in buffered formalin (pH = 6.5). The specimen was dehydrated in alcohol, cleared in xylene and embedded in paraffin. Sections, 7 ^m, were mounted on slides and the paraffin removed with xylene. Sections were coated with immersion oil and viewed at 390-490 nm (mirror — 510 nm; suppression — 515 nm) with an epifluorescence light microscope equipped with a Leitz H2 filter block. Non-stained osphradia were examined as controls. Results SEM and TEM observations The general morphology of the osphradium in Thais haemastoma canaliculata is typical of prosobranch gastropods, being bipectinate and containing approximately 312 D. W. GARTON ET AL. 150-200 lamellae (Fig. 1). The pseudostratified epithelium is separated from the central region of the lamella by a distinct basal lamina. Connective tissue, muscle fibers, nerves, and blood spaces are present in the central region of each lamella (Fig. 2). In the live snail the lamellae are active and capable of independent movement. In Thais haemastoma canaliculata a groove 10-15 ^m wide and 5 ^m deep runs parallel to the dorsal edge of each lamellae and separates the lamellar epithelium into two distinct regions (Figs. 3, 4). A thin tract of cilia is present along the ventro-lateral edge of each lamella (Figs. 3, 5). The epithelium dorsal to the groove is composed entirely of a single layer of ciliated cells. There are relatively few mitochondria in these cells, and the mitochondrial cristae are diffuse, indicating a relatively low met- abolic rate (Munn, 1974). Ciliary rootlets are short and many microvilli cover the cell surface. The epithelium ventral to the lamellar groove, occupying approximately mm Figure 1. SEM of the anterior portion of the osphradium (Os) showing its relation to the ctenidium (Ct) and incurrent siphon (S). Me-mantle epithehum; R-raphe. FINE STRUCTURE OF THAIS OSPH RADIUM 313 VT ^9^ mm <% f ' . i» ■T\ lOTHm' Figure 2. Longitudinal thin section (1 ^m) of a single osphradial lamella stained with Paragon. BL-basal lamina; CT-ciliary tuft; CR-central region; M-mucus layer; NB-nerve bundle; VT-ventral ciliary tract (between arrows). three-fourths of the surface area of each lamella, contains uniformly distributed, tufted ciliated cells (Figs. 6, 7). These cilia originate from cells lying depressed between neighboring cells. Numerous mitochondria with well developed cristae are concentrated 314 D. W. GARTON ET AL. Figure 3a. SEM of an inverted osphradium showing directional references for the lamella. Note the unequal extension of individual lamellae. D-dorsal edge of lamella; G-lamellar groove; L-lamella; R-raphe; V-ventral edge of lamella, b. Lateral view of single lamella (SEM), orientation reversed from a. D-dorsal edge; G-lamellar groove; VT- ventral tract of cilia. at the apical end of the ciliated cell (Fig. 8). Long ciliary rootlets extend basally through the aggregate of mitochondria. Ciliary tufts project laterally into the inter- lamellar space. The interlamellar space is filled with mucus and membrane-bound HNE STRUCTURE OF THAIS OSPHRADIUM 315 Figure 4. Transitional region on the dorsal edge of a single lamella (SEM). CT-cilia tuft; G-lameliar groove; NSC-non-sensory cilia. Figure 5. Transitional region (SEM) on the ventral edge of a single lamella. CT-cilia tuft: VT-ventral tract of ciha. 316 D. W. GARTON ET AL. Figure 6. SEM of ciliary tufts (CT) on the interlamellar surface. Figure 7. SEM of individual ciliary tuft in Figure 6. Figure 8. TEM of ciliary tuft (CT) projecting into mucus layer (M). Magnification = 4125X. FINE STRUCTURE OF THAIS OSPHRADIUM 317 secretory vesicles produced by the surrounding non-ciliated cells present in the ventral region of the lamellae (Fig. 8). All cilia observed by TEM, regardless of location, were of the 9X2 + 2 fibril arrangement. Cilia were traced distally in many sections and no deviation from the 9X2 + 2 arrangement was observed. Nerve tracts were located in the central region of the lamellae but no axons or synapses bridging the basal lamina were observed. Vital staining Selective staining of individual receptor cells is shown in Figures 9-12. Epithelial cells present in the region ventral to the groove and along the ventro-lateral tract of Figures 9-12 Ruorescent light micrographs of Procion Brilliant Yellow stained material. Figure 9 Low magnification of osphradial leafiets. D dorsal; V ventral. Figure 10. Procion-positive cells (RC) at ventral tip of lameUa. CR^entral region; Nt-nerve tract. Figure 1 1 . Procion-positive cell (RC) m mterlamellar region FIGURE 12. Example of a Procion-positive cell (RC) at ventral or distal region of lamella. 318 D. W. GARTON ET AL. cilia stained as well as nerve tracts in the central region (Figs. 9-12). Although the individual cells that were stained by Procion Brilliant Yellow could not be identified as specific cell types observed in the SEM and TEM material, their location suggests that tufted ciliated cells along the interlamellar surface and ciliated cells of the ventro- lateral tract were stained. Discussion The osphradial lamella in Thais haemastoma canaliculata (Gray) is divided into two morphologically distinct regions. This division has been observed in other proso- branchs (Crisp, 1973; Newell and Brown, 1977; Altner and PriUinger, 1980). Welsch and Storch (1969) and Crisp (1973) reported that the presumed sensory receptors are concentrated in the epithelium adjacent to the "transition zone" or groove on the lamella. In Conus flavidus the arrangement is more complex. Alexander (1970) and Alexander and Weldon (1975) observed presumptive receptor cells along interdigiform grooves on the interlamellar surfaces of the osphradial lamellae in Conns flavidus. These cells were also located in small depressions, similar to those in T. haemastoma canaliculata. Hackney et al. (1983) identified tufted, ciliated cells as putative che- moreceptors on the paUial tentacles of the limpet Patella vulgata. The gross morphology of these tufted cells is similar to that of the tufted ciliated cells observed in this study. Our observations imply that rather than being restricted to a specialized "transition zone," chemosensory cells are distributed over a major part of the osphradial lamella in T. haemastoma canaliculata. Results of the vital staining in Thais haemastoma canaliculata also indicate that sensory cells (possibly chemosensory) are uniformly distributed across the interlamellar surface and ventro-lateral edge of the lamella. Procion-positive cells are denser along the ventral edge than across the interlamellar surface. Ventral edges of lamellae project into the inhalant water stream (the osphradium is located on the dorsal wall of the mantle cavity adjacent to the ctenidium), providing maximal exposure to environ- mental stimuli. After contacting ventral edges, water currents pass through mucus- filled interlamellar spaces, then along the dorsal edge of lamellae, adjacent to the mantle epithelium. Any chemosensory cells present in the dorsal region would come into contact with odorant molecules last. Deviations from the 9X2 + 2 fibril arrangement in osphradial cilia have been observed in prosobranchs (Crisp, 1973). Crisp (1973) reported (8 + 1), (8 + 0), (7 + 1), (7 + 0), and (6+1) arrangements. Other ciliary modifications for a putative chemoreceptor cell have been observed in an opistobranch (Davis and Matera, 1982; Matera and Davis, 1982). "Discocilia" were observed only in chemosensitive regions of the body oi Pleurobranchaea californica (Davis and Matera, 1982). SEM and TEM observations in our study revealed no specialized modifications of cilia in diflferent regions of the osphradial lamellae that might suggest chemosensory function. All cilia observed were of the 9X2 + 2 fibril arrangement. Both primary and secondary receptors have been reported in osphradial receptors (Crisp, 1973; Newell and Brown, 1977). Crisp (1973) speculated that the Si3 and Si4 cell types are primary receptors, although no axons were observed. Given the volume of epithelial cells compared to axon sizes (tenths of microns), the probability that we could observe axonal connections in TEM material is quite low. Crisp ( 1 973) reported no evidence for secondary receptors (synaptic vesicles); however, Newell and Brown (1977) reported chemical synapses in the osphradial epithelium o^ Bullia associated with tufted ciliated cells, although no high magnification figure of the vesicles was provided. In invertebrates, the great majority of sensory cells are primary receptors; FINE STRUCTURE OF THAIS OSPHRADIUM 319 Figure 13. Scanning electron micrograph (SEM) of the ventral surface of the osphradium of the southern oyster drill, Thais haemasloma canaliculata (Gray). The osphradium is suspended from the roof of the mantle cavity and the lamellae project ventrallv into the incurrent water stream. Anterior is to the left. Figure 14. SEM of the dorsal surface of the olfactory rosette of the channel catfish. Icuilurus punctatus. The rosette is attached to the floor of the olfactory capsule and the lamellae project dorsally into the olfactory chamber. Anterior is to the right. 320 D. W. GARTON ET AL. development of secondary receptors is considered a vertebrate characteristic (Bullock and Horridge, 1965). Nerve tracts associated with putative (chemo?)-receptors were observed in the Procion Brilliant Yellow stained material, although none were observed in TEM or fractured SEM specimens. Transport of the stain along the nerve tracts suggests that the selectively stained epithelial cells are primary receptor cells. Also, no synapses or intracellular vesicles indicative of secondary receptors were observed. Cellular organization of the osphradial epithelium in Thais haemastoma cana- liculata is less complex than has been reported for other prosobranchs (Crisp, 1973; Newell and Brown, 1977; Altner and Prillinger, 1980). The results of this study suggest a functional separation of different epithelial regions of each lamella (sensory versus non-sensory) similar to that found in many teleosts (Yamamoto, 1982). The gross morphology of the prosobranch osphradium (Fig. 13) and the teleost olfactory organ (Fig. 14) are strikingly similar, suggesting convergent evolution. Both organs are composed of numerous, laterally radiating lamellae organized into specialized sensory and indifferent epithelial regions (Caprio and Raderman-Little, 1978; Yamamato, 1982). Because definitive axonal connections or synapses between ciliated cells and nerve fibers have not yet been identified, the assignment of sensory function to cells in the prosobranch osphradium is currently based solely on cytological observations and selective vital staining. Although the results of our and other studies have identified putative chemoreceptors, electrophysiological investigations will be necessary to con- firm and elucidate structure-function relationships of receptor cells and specialized lamellar regions of the prosobranch osphradium. Acknowledgments The authors would like to acknowledge the assistance of Dr. W. L. Steffens with the transmission electron microscopy and the fluorescent photomicrography. We are also indebted to Mr. Russell Goddard for his invaluable advice and assistance and to Mr. Jay Erickson for the SEM photomicrograph of the catfish olfactory organ. This research was partially supported by the Petroleum Refiners Environmental Council of Louisiana and NSF Grant DEB-7921825 to Dr. William B. Stickle, Jr., who kindly provided laboratory facilities. LITERATURE CITED Alexander, C. G. 1970. The osphradium of Conus flavidus. Mar. Biol. 6: 236-240. Alexander, C. G., and M. W. Weldon. 1975. The fine structure of the osphradial leaflets in Conus flavidus. Mar. Biol. 33: 247-254. Altner, H., and L. Prillinger. 1980. Ultrastructure of invertebrate chemoreceptors, thermoreceptors and hygroreceptors and its functional significance. Pp. 69-140 In International Review of Cytology, Vol. 67, G. H. Bourne, and J. F. Danielli, eds. Academic Press, New York. Bailey, D. P., and M. S. Laverack. 1966. Aspects of the neurophysiology of Buccinium undatum. I. Central responses to stimulation of the osphradium. J. Exp. Biol. 44: 131-148. Bailey, D. F., and P. R. Benjamin. 1968. Anatomical and electrophysiological studies on the gastropod osphradium. Symp. Zool. Soc. Lond. 23: 263-268. Brown, A. C, and R. G. Noble. 1960. Function of the osphradium in Bullia. Nature 188: 1045. Bullock, T. H., and G. A. Horridge. 1965. Structure and Function in the Nervous System of Invertebrates. W. H. Freeman and Co., San Francisco and London. 1610 pp. Caprio, J., and R. Raderman-Little. 1978. Scanning electron microscopy of the channel catfish olfactory lamellae. Tissue Cell 10: 1-9. Crisp, M. 1973. Fine structure of some prosobranch osphradia. Mar. Biol. 22: 231-240. Davis, W. J., and E. M. Matera. 1982. Chemoreception in gastropod molluscs: Electron microscopy of putative receptor cells. J. Neurobiol. 13: 79-84. HNE STRUCTURE OF THAIS OSPHRADIUM 321 Garton, D., and W. B. Stickle. 1980. Effects of salinity and temperature on the predation rate of Thais haemasloma on Crassostrea virginica spat. Biol. Bull. 158: 49-57. Hackney, C. M., C. R. McCrohan, and S. J. Hawkins. 1983. Putative sense organs on the pallia! tentacles of the limpet. Patella vulgala (L.). Cell Tissue Res. 231: 663-674. HOLL, A. 1981. Marking of olfactory axons of fishes by intravital staining with Procion Brilliant Yellow. Slain Technol. 56: 67-70. Matera, E. M., and W. J. Davis. 1982. Paddle cilia (discocilia) in chemosensitive structures of the gastropod mollusk Pleurobranchaea californica. Cell Tissue Res. 222: 25-40. MUNN, E. a. 1974. The Structure of Mitochondria. Academic Press, New York. 465 pp. Newell, P. P., and A. C. Brown. 1977. The fine structure of the osphradium of Bullia digitalis Meuschen (Gastropoda: Prosobranchia). Malacologia 16: 197-205. Welsch, U., and V. Storch. 1969. Uber das Osphradium der prosobranchen Schnecken Buccinium imdatum und Neptunea antiqua. Z. Zellforsch. 95: 317-330. Yamamoto, M. 1982. Comparative morphology of the peripheral olfactory organ in teleosts. Pp. 39-59 In Chemoreception in Fishes: Development in Aquaculature and Fisheries Science, Vol. 8, T. J. Hara, ed. Elsevier, New York. Reference: Biol. Bull. 167: 322-330. (October, 1984) CHEMOSENSORY RESPONSES TO AMINO ACIDS AND CERTAIN AMINES BY THE CILIATE TETRAHYMENA: A FLAT CAPILLARY ASSAY M. LEVANDOWSKY, T. CHENG, A. KEHR, J. KIM, L. GARDNER, L. SILVERN, L. TSANG, G. LAI, C. CHUNG, AND E. PRAKASH Haskins Laboratories of Pace University, 41 Park Row, New York, New York 10038 Abstract An assay for chemosensory responses by the ciliate Tetrahymena thermophila is described that uses glass capillaries with a rectangular cross-section (inner dimensions, 20 X 2 X 0.2 mm). These have optical and geometrical properties permitting convenient observation of cell behavior within the capillaries. Washed cells, starved for 12 h, accumulated preferentially in capillaries containing L-methionine, L-leucine, L-cysteine, L-histidine, L-histamine, cimetidine, agmatine, and berenil at concentrations of 10"^ M or less. They avoided capillaries containing tripelennamine, diphenhydramine, and pentamidine at these concentrations. It is argued that the actual response thresholds are much lower than the concentrations put into the capillaries, since cells respond to the gradient of the diffusing chemical. L-Isoleucine, itself inert, blocked the response to L-leucine but not to L-methionine, L-cysteine, or L-histidine. L-Ethionine and 1 -homocysteine caused accumulation but not L-cysteine or DL-cystathionine. L-Cystine did not block the response to L-cysteine. Cells accelerated when entering a capillary where accumulation occurred. On reaching the interior they swam more slowly and uniformly, and with fewer turns or stops than in control capillaries lacking the chemical signal, or when outside of the capillaries. Cells were inhibited from leaving both control and test capillaries, possibly because of accumulated wastes or secretions in the surrounding medium. Introduction Chemical senses have been analyzed extensively in bacteria (Adler, 1975; Koshland, 1980) and the cellular slime molds (Mato and Konijn, 1979), and are probably ubiquitous among motile microorganisms (Levandowsky and Hauser, 1978). Among ciliate protozoa, chemosensory responses have been much studied in Paramecium (Van Houten et ai, 1981) and Blepharisma (Miyake, 1980). The genus Tetrahymena, easily grown in chemically defined media and the subject of numerous genetic and biochemical studies, appears particularly promising for the analysis of the mechanisms of these responses. Csaba and Lantos (1973, 1975) demonstrated enhanced phagocytosis by starved Tetrahymena cells in the presence of low levels of serotonin and other vertebrate neurohormones. Ueda, Kobatake, and their colleagues (Ueda and Kobatake, 1977; Ataka et ai, 1978; Tanabe et al, 1980) described dispersion or accumulation in response to a number of chemicals, particularly those producing a bitter taste in humans, and related these responses to interactions with membrane lipids and mem- brane potential as measured with fluorescent dyes. Almagor et al (1981) adapted capillary methods which have been used with bacteria (Adler, 1975) and flagellates (Sjoblad et al, 1978; Spero, 1984). They demonstrated responses to methionine and leucine, and related these to the influence of those chemicals on swimming behavior. Received 7 May 1984; accepted 24 July 1984. 322 CHEMOSENSORY RESPONSES OF TETRAHYMENA 323 We describe another assay method based on capillaries with a rectangular cross section. These have optical and geometrical properties which permitted detailed ob- servations of behavior within them. We also extend the results of Almager et a/, to include other amino acids and certain amines. Materials and Methods Organisms and culture methods Tetrahymena thermophila (CU strain 307, mating type BIV) was obtained from Dr. Lea Bleyman and cultured at room temperature in autoclaved HTM medium: (gm/100 ml H2O) dextrin 0.8; yeast autolysate (Difco) 0.5; liver concentrate (Sigma) 0.06; casein hydrolysate (Hycase, Sheffield) 0.6; morpholinopropane sulfonic acid (MOPS) 0.1; pH adjusted to 7.0 with NaOH. Starvation pretreatment Three to six day old cuhures (late log phase, approximately 2 X 10' cells/ml) were harvested and washed twice by gentle centrifugation (2 min in a clinical centrifuge followed by resuspension in distilled H2O), then recentrifuged and resuspended in starvation medium (S) (mg/100 ml H2O): CaCb 2H2O 15.0; Na2EDTA 60.0; N-tris (hydroxymethyl) methyl-glycine (TRICINE) 18.0; adjusted to pH 7.0 with NaOH. (A somewhat more complex medium, containing also magnesium, sodium, and po- tassium salts was used in early experiments, but later work showed these were not required for the chemosensory response). After starvation in S for at least 6 h, cells were washed once in distilled H2O as before and resuspended in S and allowed to stand at least an hour before use in an experiment. During the starvation period the cells underwent metamorphosis to the faster-swimming dispersal form (Nelson and DeBault, 1978). In this assay significant chemosensory responses were seen only with the starved dispersal form, and never with well-fed cells. The behavior of starved, washed cells appeared to be quite sensitive to the presence of trace contaminants. In preliminary studies it was found that the presence of Na2EDTA or other organic ligands was desirable for normal swimming behavior. Salt solutions lacking this, made up with samples of glass-distilled water from six different laboratories of the New York-New Haven area, collected and transported in clean glass or plastic containers, resulted in abnormal swimming behavior such as swimming backward or in circles; this problem could be cured by addition of ap- propriate amounts of Na2EDTA and CaCl2 H2O (see Discussion section). Capillaries, glassware, and plasticware Borosilicate glass capillaries ("microslides," Vitro Dynamics Inc., P. O. Box 285, Rockaway, NJ 07866) with rectangular cross-section and inner dimensions (mm) 20. X 2. X 0.2 were used. Before use, both new and used capillaries were cleaned by soaking overnight in concentrated H2SO4. Before and after soaking they were boiled in several changes of distilled H2O. Capillaries cleaned in this manner yielded best results. Glassware and plasticware used for these experiments were kept segregated and washed separately from other labware, using Alconox detergent and many rinses in hot water, followed by soaking in distilled H2O. New or only slightly used plastic dishes tended to inhibit cell motility (see also Wolfe and Colby, 1981); this problem could be eliminated by soaking the dishes overnight in concentrated H2SO4, but concentrated HCI, NaOH 5 A^or 10% Na2EDTA were not effective (see Discussion). 324 M. LEVANDOWSKY ET AL. Assay method Capillaries were filled with S medium (control capillaries) or with S and a solute to be tested (test capillaries), by touching one end to the fluid, allowing the latter to be rapidly drawn in by capillarity. Properly cleaned capillaries fill very quickly. Cap- illaries were handled with clean fi^rceps. Filled capillaries were then placed in a plastic dish with 40 mm inner diameter (the top half of a Coming 35-mm tissue culture dish) containing 3 ml of a cell suspension, diluted to approximately 25,000 cells/ml. Two capillaries, test and control, were placed in each dish and gently submerged to lie flat and parallel on the dish bottom, with ends separated by several mm from each other and from the sides of the dish. It should be noted that both ends of the capillary are open in this assay, in contrast to the capillary assays commonly used with bacteria (Adler, 1975). Experiments were done at 28 °C. Below 25 °C the chemosensory response was never detected. Results of replicate experiments done in the dark and in various light regimes appeared to be the same. After 5 min, capillaries were removed with forceps, gently blotted on the outside with tissue, and placed in a dry plastic dish. Behavior of organisms swimming inside the capillaries was observed with the microscope. Then the dish with the two capillaries was floated for 1 min on a hot water bath (65-75 °C). The heat-killed cells in each capillary were then counted. Six replicate assays of each concentration of the test chemical were done in each experiment. Experiments were then repeated several times with different cell prep- arations. Cells were also checked in a control experiment for response to 10"^ M L- methionine or L-histamine before being used to test other compounds. Purest available reagent grade commercial chemicals were used. Results Statistical aspects. To establish a statistical base line we did experiments in which no chemical cue was tested, but rather both capillaries in each dish contained only the salts solution S. Table I shows data for ten replicate experiments with the same cell preparation. These data appear inconsistent with a simple statistical model in which each cell "decides" independently of others whether to enter one or the other of the two identical capillaries. Such a model would predict a binomial distribution in which the great differences between the two capillaries in trials 1, 2, 3, 6, and 9 would be very improbable. This great variability may perhaps be due to cell interactions, in which case the assumption of statistical independence of the cells is not valid. Thus, standard parametric tests for significance are probably inappropriate. Table II shows the effect of introducing 10~^ M L-methionine into one of the capillaries in each dish. Though there is great variability in both absolute numbers Table I Numbers of cells entering pairs of identical capillaries in replicate trials Trial number 1 2 3 4 5 6 7 8 9 10 Capillary 1 Capillary 2 228 92 34 199 164 91 78 82 75 96 238 157 120 76 158 172 135 240 212 200 CHEMOSENSORY RESPONSES OF TETRAHYMENA 325 Table II Response to L-methionine in six replicate trials Trial number 1 2 3 4 5 6 Test capillary: S medium + 10'^ M Methionine Control capillary: S medium 225 211 568 385 637 227 852 132 468 273 347 179 and ratios of cells in test and control capillaries, the former always had more cells than the latter in a given dish, and it is clear that they accumulated preferentially in the presence of 10^ M methionine. By the nonparametric sign test, the probability of the data in Table II occurring by chance is less than .0 1 6. Because of high variability, of unknown origin, we adopted the sign test as a conservative measure of statistical significance, and each experiment was therefore done in six or more replicates. This was repeated with several different cell preparations before we accepted a response as significant. Amino acid responses Of 23 common amino acids tested, only four elicited significant responses by the above criterion (Table III). The L forms of methionine, leucine, histidine, and cysteine were active at 10"^, 10"", and sometimes lower molar levels. Others tested and found inert were the L forms of alanine, arginine, asparagine, aspartic acid, cystine, glutamic acid, glutamine, glycine, isoleucine, lysine, ornithine, phenylalanine, proline, serine, taurine, threonine, tryptophan, tyrosine, and valine. In an abstract of preliminary results (Levandowsky et al, 1982) we had reported responses to several of the latter also, but in subsequent experiments these were not significant by the above criterion. Table III Response to amino acids, amines, and their antagonists Molar concentration 10-^ 10-" 10-5 Amino acids L-methionine + + +* L-histidine + + +* L-leucine + + +• L-cysteine + Amines L-histamine 2HCi + + +* Agmatine SO4 + + +* Histamine and diamine antagonists Cimetidine HCl + + Berenil - + + Tripelenamine HCl - Diphenhydramine HCl - - Pentamidine isethionate - - + = accumulation (more ceils in test capillary). - = dispersion (fewer cells in test capillary). * = effect true only in some experiments. = response not significant (see text). 326 M. LEVANDOWSKY ET AL. Specificity of amino acid responses Isoleucine was usually inert (in a few preparations slight accumulation occurred at 10"^ M). When added to the background, so that 10"^ M L-isoleucine was present in both control and test capillaries, and in the surrounding fluids, it blocked the response to leucine but not to methionine, cysteine, or histidine. Several methionine analogs were tested. DL-Ethionine elicited a strong response in the same range as methionine, but not seleno-DL-methionine. DL-Homocysteine, a cysteine analog, was active in the same range as L-cysteine, but L-cystine and DL- cystathionine were inert. L-Cystine added to the background at 10^^ M did not block the response to either cysteine or methionine. Histamine and the diamines L-Histamine HCl (Table III) elicited a somewhat stronger aggregation response than histidine, over the same concentration range. Because of certain biochemical similarities, discussed below, we also tested a group of diamines. Of these, only agmatine proved active, causing aggregation in the same range as histamine and the four amino acids. Diaminopropane, putrescine, spermidine, spermine, and cadaverine were in- active. Antihistamines and diamine antagonists We examined several antihistamines, as well as anti-parasite drugs which are antagonists to both histamine and polyamines (see Discussion). Of these, the H-2 antihistamine cimetidine and the trypanocide Berenil (diminazene aceturate) eUcited aggregation at 10"^ M. Dispersion (fewer cells in the test capillary) occurred with the H-1 antihistamines tripelennamine and diphenhydramine, and with the polyamine antagonist pentamidine. Behavioral basis of the responses Using an ocular micrometer and a stopwatch we measured swimming speed and turning frequency of individual cells selected arbitrarily by following the first cell to swim past a line on the ocular micrometer grid in a given time. Tables IV and V show differences in swimming behavior in the test capillary and elsewhere. A basic element in the repertoire of swimming behavior of starved T. thermophila cells, and the most important one for getting cells into the capillary during a five minute experimental period, consists simply of straight or nearly straight "runs" punctuated by turns. Unlike Paramecium, T. thermophila does not usually stop, but often slows down during the turn. On entering a test capillary containing an attractant, cells immediately accelerated and swam rapidly without turns for 4-8 mm. On reaching the midregion of the Table IV Average swimming speed (mm/s, ±2 a) in a typical experiment 1. Midregion of test capillary .44 ± 0.3 (n = 10) 2. Entering the test capillary .72 ± 0.4 (n = 10) 3. Midregion of control capillary .58 ± 0.4 (n = 10) 4. Entering control capillary .50 ± .03 (n = 10) 5. Outside capillaries .49 ± .05 (n = 10) CHEMOSENSORY RESPONSES OF TETRAHYMENA 321 Table V Average time between turns or stops (s. ± 2a) in a typical experiment Middle of test capillary 8.2 ± 1.2 (n = 20) Middle of control capillary 4.8 ± 1.1 (n = 20) Outside of capillaries 4.2 ± 1.0 (n = 20) capillary they swam more slowly and uniformly than elsewhere, with fewer turns than in the control capillary or outside of the capillaries (Tables IV, V). On attempting to leave, cells in both the test and control capillaries tended to stop and turn more frequently in a zone near the end of the capillary, and in some cell preparations appeared virtually unable to leave once they had entered either the test or the control capillary. When cells inside the capillary swam into the wall they usually just "reflected" like billiard balls, at an equal angle, without slowing down. These were not counted as turns in the observations. In summary: outside the test capillary, where there was no added chemical cue, the cells moved in a series of small, relatively fast "runs." There was a large variability in individual speed and turning frequency, and the overall impression was of a jerky, erratic searching behavior. At the entrance to the test capillary, where there is a gradient of diffusing chemical signals, they suddenly and dramatically accelerated and swam up the gradient to the midregion of the capillary. This appears to be the main factor causing greater accumulation in the test capillary than in the control during the five minute duration of the experiment. In the midregion, where the chemical concentration was presumably constant, they slowed down and turned less frequently than outside, and the overall impression was of a very uniform, even motion, with few turns or stops. On attempting to swim out of either test or control capillary, their motion became jerky and irregular, with many stops and turns. In some cell preparations they seemed to be unable to leave. Since this occurred also in the control capillary, this was probably the effect of wastes or cell secretions in the fluid outside, which had cells in it for at least an hour before the exper- iment began. Discussion Responses to methionine and leucine were also studied by Almagor et al. (1981), using the WH-52 strain of T. thermophila and a somewhat different capillary assay method, as well as microscopic observation to record motility in various attractant concentrations. The latter method detected responses at much lower levels than the capillary method. As they note, organisms responding to a gradient of a diffusing chemical would initially encounter a much lower concentration than that originally placed in the capillary. Thus capillary assays, though convenient and meaningful, are relatively insensitive and overestimate the response threshold by at least an order of magnitude. Similar conclusions have been drawn regarding capillary assays of bacterial chemosensory assays (Hazelbauer and Adler, 1971). Our capillary assays and those of Almagor et al. appear to be similar in sensitivity. Ordinary capillaries with a circular cross section present difficulties however, when they are submerged in the cell suspension it is difficult to determine whether cells swimming at a given focal depth are inside the capillaries. Furthermore, when they are removed from the medium, fluid-filled capillaries act as lenses, and cells swimming 328 M. LEVANDOWSKY ET AL. inside them are extremely difficult to watch with the microscope. These problems do not arise with the rectangular capillaries used in this assay. The abnormal behavior in the absence of organic ligands such as Na2EDTA, noted in the materials and methods section, may be due to heavy metals leaching from glass or plastic containers (Bemhard, 1977). Inhibition of cell motility (sticking to the bottom) in new, untreated plastic dishes probably stems from surface charge- related hydrophobic interactions between cells and the plastic surface (Kitamura, 1982; D. Rittshoff, pers. comm.). It would be interesting to know whether the WH-52 strain used by Almagor et al. also responds to cysteine, histidine, and the amines listed in Table III. There is a great deal of biochemical variation among morphologically indistinguishable members of this group (Nanney, 1980), and one would like to know whether sensory responses are a conservative or a variable feature. As noted above, we had indications in early experiments of responses to other amino acids, notably tyrosine, phenylalanine, ar- ginine, and serine, but these did not appear consistently in subsequent experiments. Such inconsistency could have various explanations, one of which is a latent sensitivity to these compounds that is not always expressed. This deserves further study. Amines and amino acids may serve as useful ecological signals for food. Thus, Fuzessery et al. (1978) suggested a correlation between the spiny lobster's chemo- sensitivity to very low levels of taurine and the latter's particular value as a potential food indicator in the marine environment. In the case of Tetrahymena, a freshwater phagotroph, there are numerous laboratory studies of feeding behavior (Nilsson, 1979), but virtually no field studies of its natural history — its natural diet is, strictly speaking, unknown. In the lab it is usually grown on living or dead bacteria or yeast, or in defined or undefined liquid media containing precipitated particles. Histamine is a common waste product of bacterial decomposition, particularly of plant tissues (Gug- genheim, 1951), and agmatine is an amine restricted to certain plants and bacteria. Beyond these hints, we have no clues yet regarding the adaptive value of responses to this particular set of amino acids and amines. From a comparative, phylogenetic point of view the histamine response is of particular interest. Csaba and Lantos (1975) found that low levels of histamine and two histamine antagonists also stimulated phagotrophy in their GL strain. The positive (accumulation) response to the drug cimetidine, used to block the H-2 histamine receptor in treating ulcers, probably reflects its structural similarity to histamine (the presence of an imidazole group). Tripelennamine and diphenhydramine on the other hand, drugs used to block histamine H-1 receptors in treating allergy and cold symptoms, gave rise to a negative (dispersion) response. Though they are antihistamines, these do not resemble histamine structurally and lack the imidazole group, but do have pharmacological and chemical affinities to local anesthetics of the cocaine family. The mode of action of the latter is thought to involve, among other possibilities, alteration of the physical properties of the cell membrane. We think it likely that some chemosensory responses of Tet- rahymena will prove to be due to binding of the signal molecule to specific membrane- bound or intracellular receptor molecules, probably proteins, but some responses may be simply due to non-specific changes in physicochemical properties of the membrane. This was indicated by studies in which negative (repulsion) responses to various hydrophobic chemicals and to bitter substances were correlated with changes in membrane fluidity (Ataka et al, 1978; Tanabe et al, 1980). Berenil (diminazene aceturate) and pentamidine are known mainly as anti-try- panosomal drugs. It appears that these and a number of other anti-trypanosomal and anti-malarial drugs are inhibitors of both histamine N-methyl transferase and also CHEMOSENSORY RESPONSES OF TETRAHYMENA 329 diamine oxidase (Duch et ciL, 1984). This link between histamine and the diamines is not yet understood, but appears promising as a pharmacological principle. Further analysis of the Tetrahymena response to such compounds would be desirable and might yield useful information on clinically important questions. Chemosensory responses to amino acids are phylogenetically widespread, being found in bacteria (Adler, 1975; Koshland, 1980), algae (Hauser et ai. 1975; Sjoblad et ai, 1978), many invertebrates {e.g., Ache, 1972) and vertebrates {e.g., Caprio, 1978). In catfish, L-cysteine was the most effective olfactory stimulus tested (Caprio, 1978). Amino acids also act as excitatory transmitters in the brain and may be involved in some forms of epilepsy (Croucher et ai, 1982). In particular, L-cysteine may function as a transmitter in the brain (Watkins and Evans, 1981). We have also found responses by T. thermophila to several other chemical groups, including neurotransmitters and hormones. Preliminary accounts of this work and of studies of the ionic requirements of the chemosensory response have appeared (abstracts: Levandowsky et ai, 1982; Gardner and Levandowsky, 1983; Tsang and Levandowsky, 1983). The possibility of homologies between Tetrahymena responses to amino acids and amines, and those in higher organisms is intriguing. In addition to its evolutionary interest, this would suggest the practical possibility of using this organism as a model system to investigate chemosensory mechanisms, profiting from the ease with which it can be grown in mass culture, and the possibilities of genetic analysis. Acknowledgments We thank N. Sauter, and especially I. R. Lapidus for help in the early stages of development of the assay method, and J. Van Houten, D. Lynn, L. Bleyman, C. J. Bacchi, K. Foster, A. Ron, and an anonymous reviewer for useful suggestions and comments on the manuscript. LITERATURE CITED Ache, B. W. 1972. Amino acid receptors in the antennules of Homarus americanus. Comp. Biochem. Physiol. 42: 807-811. Adler, J. 1975. Chemotaxis in bacteria. Ann. Rev. Biochem. 44: 341-366. Almagor, M., a. Ron, and J. Bar-Tana. 1981. Chemotaxis in Tetrahymena thermophila. Cell Motility 1: 261-268. Ataka, M., a. Tsuchii, T. Ueda, K. Kurihara, and Y. Kobatake. 1978. Comparative studies on the reception of bitter stimuli in the frog, Tetrahymena. slime mold and Nitella. Comp. Biochem. Physiol. 61 A: 109-115. Bernhard, M. 1977. Chemical contamination of culture media: assessment, avoidance and control. Pp. 1459-1499 in Marine Ecology. Vol. Ill, part 3, O. Kinne, ed. Wiley & Sons, New York. Caprio, J. 1978. Olfaction and taste in the channel catfish: an electrophysiological study of the responses to amino acids derivatives. / Comp. Physiol. 123: 357-371. Croucher, M. J., J. F. Collins, and B. S. Meldrom. 1982. Anticonvulsant action of excitatory amino acids. Science 216: 899-901. CSABA, G., and T. Lantos. 1973. Effect of hormones on protozoa. Studies on the phagocytotic effect of histamine, 5-hydroxytryptamine and indolacetic acid in Tetrahymena. Cylohiologie 7: 311-315. CSABA, G., AND T. Lantos. 1975. Specificity of hormone receptors in Tetrahymena. Experiments with serotonin and histamine antagonists. Cytohiologie 11: 44-49. DucH, D. S., C. J. Bacchi, M. P. Edelstein, and C. A. Nichol. 1984. Inhibitors of histamine metabolism In vivo and In vitro: correlations with anti-trypanosomal activity. Biochem. Pharmacol. 33: 1 549- 1553. Fuzessery, Z. M., W. E. S. Carr, and B. W. Ache. 1978. Antennulary chemosensitivity of the spiny lobster, Panulirus argus: studies of taurine sensitive receptors. Biol. Bull 154: 226-240. Gardner, L., and M. Levandowsky. 1983. Ca^* dependence of chemosensory response and swimming speed in Tetrahymena thermophila. J. Protozool. 30: 13A (Abstr.). 330 M. LEVANDOWSKY ET AL. Guggenheim, M. 1951. Die biogenen Amine. S. Karger, Basel. 619 pp. HaUSER, D. C. R., M. LEVANDOWSKY, S. H. HUTNER, L. CHUNOSOFF, AND J. S. HOLLWITZ. 1975. Chemosensory responses by the heterotrophic marine dinoflagellate Crypthecodinium cohnii. Microb. Ecol. 1: 246-254. Hazelbauer, G. L., and J. Adler. 1971. Role of the galactose binding protein in chemotaxis oi Escherichia coli toward galactose. Nature 230: 101-104. Kjtamura, a. 1982. Attachment oi Paramecium to polystyrene surfaces: a model system for the analysis of sexual cell recognition and nuclear activation. / Cell Sci. 58: 185-199. Koshland, Jr., D. E. 1980. Bacterial Chemotaxis as a Model Behavioral System. Raven Press, New York. 193 pp. LEVANDOWSKY, M., T. CHANG, A. KEHR, J. KlM, L. GARDNER, L. SiLVERN, AND L R. LaPIDUS. 1982. Chemosensory responses of Tetrahymena: a flat capillary assay. J. Protozoal. 29: 498 (Abstr.). LEVANDOWSKY, M., AND D. C. R. Hauser. 1978. Chemosensory responses of swimming algae and protozoa. Int. Rev. Cytol. 53: 145-210. Mato, J. M., AND T. M. KONUN. 1979. Chemosensory transduction in Dictyostelium discoideum. Pp. 181-221 in Biochemistry and Physiology of Protozoa. 2nd Ed., Vol. 2, M. Levandowsky and S. H. Hutner, eds. Academic Press, NY. MiYAKE, A. 1981. Physiology and biochemistry of conjugation in ciliates. Pp. 125-198 in Biochemistry and Physiology of Protozoa, 2nd Ed., Vol. 4, M. Levandowsky and S. H. Hutner, eds. Academic Press, NY. Nanney, D. L. 1980. Experimental Ciliatology. Wiley and Sons, New York. 304 pp. Nelsen, E. M., AND L. DeBault. 1978. Transformation in Tetrahymena pyriformis: description of an inducible phenotype. J. Protozoal. 23: 113-119. NiLSSON, J. R. 1979. Phagotrophy in Tetrahymena. Pp. 339-380 in Biochemistry and Physiology of Protozoa. 2nd Ed., Vol. 2, M. Levandowsky and S. H. Hutner, eds. Academic Press, NY. Sjoblad, E. D., I. Chet, and R. Mitchell. 1978. Chemoreception in the green alga Dunaliella tertiolecta. Curr. Microbiol. 1: 303-307. Spero, H. J. 1984. Chemosensory behavior in the phagotrophic dinoflagellate Gymnodium fungiforme. J. Phycol. (In press.) Tanabe, R., K. Kurihara, and Y. Kobatake. 1980. Changes in membrane potential and membrane fluidity in Tetrahymena pyriformis in association with chemoreception of hydrophobic stimuli: fluorescence studies. Biochemistry 19: 5339-5343. Tsang, L., and M. Levandowsky. 1983. Cholinergic chemosensory responses of Tetrahymena. J. Protozoal. 30: 13A (Abstr.). Ueda, T., and Y. Kobatake. 1977. Hydrophobicity of biosurfaces as shown by chemoreceptive thresholds in Tetrahymena. Physarum and Nitella. J. Membrane Biol. 34: 351-368. Van Houten, J., b. C. R. Hauser, and M. Levanexdwsky. 1981. Chemosensory behavior in protozoa. Pp. 67-124 in Biochemistry and Physiology of Protozoa, 2nd Ed., Vol. 4, M. Levandowsky and S. H. Hutner, eds. Academic Press, NY. Watkins, J. C, and R. H. Evans. 1981. Excitatory amino acid transmitters. Ann. Rev. Pharmacol. Toxicol. 21: 165-204. Wolfe, J., and R. H. Colby. 1981. A method for immobilization of living Tetrahymena. Exp. Cell Res. 134: 313-317. Reference: Biol. Bull. 167: 331-338. (October, 1984) TRANSFER OF NEMERTEAN EGG PREDATORS DURING HOST MOLTING AND COPULATION DANIEL E. WICKHAM', PAMELA ROE^ AND ARMAND M. KURIS' ^Bodega Marine Laboratory, Bodega Bay, California 94923, ^Deparlmenl of Biology, California State College Stanislaus, Turlock, California 95380, and ^Marine Science Institute and the Department of Biological Sciences, University of California, Santa Barbara, California 93106 Abstract Juvenile nemertean egg predators were able to efficiently transfer from the premolt cuticle to the postmolt cuticle of male and female crabs when the host molted. These worms also efficiently transferred from male to female hosts at copulation. The syn- chronized responses of the nemertean worms to host physiology and behavior dra- matically concentrate the nemertean population on the sole food source required for worm reproduction: crab eggs. The efficient location of reproductive crabs by juvenile worms increased the likelihood that these worms can have significant effects on crab fisheries when worm population density is high. Introduction Worms of the nemertean genus Carcinonemertes are ectosymbionts that feed on the eggs of decapod crustaceans. Infestation of hosts follows a planktonic larval period. The juvenile worms then ensheath in various protected spots on the host exoskeleton (Humes, 1942; Kuris, 1978; Wickham, 1980). These quiescent worms apparently subsist on dissolved organic matter leaked from crab arthrodial membranes and absorbed through the tegument of the worms (Roe et ai, 1981; Crowe et ai. 1982). Carcinonemertes errans infests the dungeness crab. Cancer magister, and will settle on crabs of any age or sex (Wickham, 1980). This crab is long lived and females produce their first egg clutch when two or three years old (Butler, 1961). Mating occurs only at the female molt. Males sequester premolt females and copulation occurs immediately following ecdysis of the female (Snow and Nielson, 1966). They reproduce once a year, brood eggs for about 90 days in central California, and must molt before producing subsequent broods. Worms infesting this crab species must feed on crab eggs in order to reproduce themselves. In 1974 the average burden of nemerteans on ovigerous female dungeness crabs in central California was 29,000 C. errans per crab causing the mortality of over 50% of the crab eggs produced in the San Francisco region (Wickham, 1979). This level of infestation still persists and has shown no evidence of decline even though host abundance has been dramatically reduced relative to historical levels for this entire time period. Nemertean abundance on dungeness crabs from northern California, Oregon, and Washington has increased from 1000-2000 in 1974 and 1975 to levels comparable to those found in central California with a concomitant increase in crab egg mortality, possibly in response to record high populations of host crabs followed by a drastic cyclic decline (Wickham, 1980; unpubl.) Nemerteans have been observed on many species of decapod crustaceans, oc- casionally with large numbers of worms on individual hosts (Humes, 1942; Aiken et Received 17 May 1984; accepted 25 July 1984. 331 332 D. E. WICKHAM ET AL. al, 1983) but never to the extent found on Cancer magister. The only other instance of widespread infestations in epidemic proportions by nemerteans is on the Alaskan king crab, Paralithodes camtschatica. Populations over much of the range of this crab are currently suffering dramatic brood wastage apparently related to a marked increase in the abundance of nemerteans which are either new to this host, or, more likely were not observed previously on this heavily studied host because of typically low numbers (Wickham and Kuris, unpubl.) Factors which allow the maintenance of such large densities of ectosymbionts on hosts which periodically molt and have been thought to shed their symbionts at the molt (Humes, 1942) must be identified to understand the role of these organisms in their hosts' dynamics. Materials and Methods Specimens of juvenile and mature Cancer magister with resident populations of Carcinonemertes errans were collected from the waters near Bodega Bay, California. Mature male and female crabs which showed evidence of approaching molt were held separately in laboratory tanks and monitored through the molt. Worm distri- butions over the host exoskeleton were assayed by enumerating worms at a repre- sentative selection of sites normally occupied by worms. These included walking legs, axillae, chelipeds, thorax, abdomen, pleopods (female), or copulatory appendages (male). Photographs were taken to display worm disposition at the time of ecdysis. Worms were counted on the exuviae after molt. Redistribution of the worms on the new exoskeleton was followed over the first few days following molting by sampling the original sites. An exhaustive census of worms on the new exoskeleton was then conducted to determine the proportion of the original population which transferred at molt. Intermolt transfer was also observed on juvenile crabs held in cubicles in plastic parts boxes. These crabs had low numbers of worms and, in 7 of 1 2 instances, worm populations on them were augmented by the addition of worms to the exoskeleton prior to ecdysis. The additional worms were obtained from adult male crabs. Worms crawled onto juvenile crabs placed with the worms in finger bowls. Observations of worm behavior at host ecdysis were conducted only after an acclimatization period of approximately a week. Transfer of worms to females at mating was observed during two mating instances in two fashions. In the first instance the exoskeleton of a female was cleaned by sparging with 95% ethanol until all worms were killed and removed. She was then mated with an infested male and the number of worms on her exoskeleton was counted the following day and compared with the number remaining on the male to determine the proportion transferred. In the second instance an infested female was mated with a male whose worms had been vitally stained with neutral red dye. The distribution of these stained worms was then noted on the female the day after mating and the number was again compared with the number remaining on the male. Observations were also made with C epialti worms on the crab Hemigrapsus oregonensis. The proportion of worms transferring at molt was measured in the same fashion as with C magister. Results When mature female Cancer magister molt, an average of 88% of the resident worms migrate to the new exoskeleton (Table I). The manner of migration is active, directed and strikingly illustrated in Figure 1 . Several days prior to host ecdysis worms become more concentrated near the base of the abdomen where they have greater INTERHOST TRANSFER BY CARCINONEMERTES 333 Table I Numbers o/Carcinonemertes errans on adult female crabs and exuviae after molting Proportion Crab No. worms No. worms Total no. of worms No. on exuvium on new crab worms transferring 1 134 1891 2025 .934 2 111 899 1166 .762 3 89 1612 1701 .948 4 827 5536 6363 .870 X = .88 S.D. = .09 access to the epimeral suture. Worms crawl toward the epimeral suture when de- calcification begins. By the time the suture opens most of the worms are massed against the new exoskeleton of the crab so they can remain with the crab as the exuvium is shed. Shortly after ecdysis worms can be observed migrating forward and outward until they assume a typical distribution pattern (see Table IV). During intermolt periods worms tend to be contracted and clustered in groups. On the early post-ecdysial crab, worms are elongated and can often be seen moving across calcified portions of the exoskeleton where they normally are not found. At this time worms can often be found head to tail in a single file along suture lines in the axils. Transfer at ecdysis on male crabs appears to be more variable than on females. Figure 1. of carapace. Worms actively crawling toward decalcifying epimeral suture on postero-latcral surface 334 D. E. WICKHAM ET AL. Table II Numbers o/Carcinonemertes errans on adult male crabs and exuviae after molting Proportion Crab No . worms No . worms Total no. of worms no. on 1 new crab on ( exuvium worms transferring Spring 1 22 3 25 .12 2 14 5 19 .26 3 24 3 11 .27 4 145 27 145 .16 5 124 22 146 .15 X = .19 S.D. = .06 Late summer 1 25 377 402 .94 During spring when male crabs carried lower worm burdens most worms were lost with the molt. Worms on the male crab which molted in late summer transferred with an efficiency comparable to those on female crabs (Table II). On juvenile crabs worms transferred on both sexes (Table III). Worms disappeared from the system in these experiments. An average of only 22% of the premolt worms were found on both the post molt and exuviae with male crabs (n = 6) and 73% of the premolt worms found with female (n = 9) post molt and exuviae. Comparable figures for loss to the system on adult crabs are not available given the difficulty of quantifying total worm abundance on pre-molt mature female crabs without first sacrificing the crab. During the two mating instances approximately 90% and 85% respectively of the worms from the male transferred to the female (400 of approximately 5000, and 600 of approximately 3200 worms were left on the males after mating). In the observations done on worms dyed with neutral red the worms from males were distributed in a different pattern than the resident worms (G-test, P < .01). They tended to be slightly more concentrated under the abdomen (thoracic sutures plus bases of pleopods) than the preexisting population of worms on the female (Table IV). The typical distribution of juvenile worms on intermolt males was much more concentrated in sites under the abdominal flap than on intermolt females (Table V). Worms on the host Hemigrapsus oregonensis transferred in a fashion similar to that found with worms on Cancer magister but with somewhat less efficiency. Worms transferred more efficiently on male crabs (Table VI). Discussion The striking behavior exhibited by Carcinonemertes errans on the crab Cancer magister at the molt represents an important adaptation for increasing the likelihood Table III Average number o/ Carcinonemertes errans on premolt and postmolt juvenile Cancer magister and their exuviae (±S.D.) Crab sex Average no. worms on exuviae Average no. worms on new crab M F (n = 7) (n = 5) .57 (±.8) .60 (±1.3) 7.43 (±4.8) 4.40 (±2.1) Average proportion transferring .93 (: .94 (: :.l) :.l) INTERHOST TRANSFER BY CARCINONEMERTES 335 Table IV Distribution of worms dyed with neutral red compared to undyed worms on adult female crab after mating No. dyed No. undyed Proportion Proportion Site on crab worms worms of total dyed of total undyed Chela 2 16 .01 .03 Coxal-basis joint on walking leg 35 101 .13 .17 Merus-carpus joint on walking leg 9 37 .03 .06 Thoracic sutures under abdomen 155 235 .59 .40 Bases of pleopod 60 192 .23 .33 of eventual arrival on a crab egg clutch where worms can complete their life cycle. The manner of movement by worms prior to actual ecdysis suggests that worms receive some type of preliminary information which causes them to begin to move toward areas with more direct access to the epimeral suture. The normal location of worms on males suggests that there is some recognition system involved in site selection. Juvenile worms on females can be found in virtually any protected crevice on the ventral surface of the body and on the limbs. On males most juvenile worms are under the folded abdomen near the copulatory appendages. This obviously facilitates transfer to females at mating but it also suggests that worms can distinguish male from female hosts and modify their site of infestation accordingly. The nature of the signal for nemertean concentration near the abdomen just prior to ecdysis is unknown. Juvenile C. errans occur on or near arthrodial membranes which are known to be "leaky" (Crowe et ai, 1982). Worms actively absorb dissolved organic matter being leaked by the host at these sites (Roe et ai, 198 1 ) so it is possible that alteration of the chemical nature of worm infestation sites provides a cue. One event which occurs at the time of molting is the separation of the epicuticle from the newly forming cuticle below, which could possibly alter the rate of leakage through Table V Distribution of juvenile worms on intermolt host exoskeletons (±S.D.) Male (n = 6) Female (n = 3) Proportion Proportion of total of total Site No. worms worm pop. No. worms worm pop. Axillae 24.0 (±13.7) .06 167.3 (±16.7) .12 Walking legs 10.3 (±4.4) .02 891.3 (±276.3) .61 Chelipeds 0.0 .00 6.7 (±5.8) .01 Thorax 141.3 (±21.2) .33 54.3 (±28.2) .04 Abdomen 183.8 (±33.1) .43 133.7 (±73.5) .09 Pleopods or copulatory apjxndages 65.0 (±32.5) .15 200.0 (±130.0) .13 Proportion under abdomen (.92) G test, P< .01 (.26) 336 D. E. WICKHAM ET AL. Table VI Proportion of worms transferring to the new molt on male and female Hemingrapsus oregonensis (±S.D.) No. worms No. worms Average proportion Sex on exuvium on new crab transferring M(n = 7) 0.14 (±.38) 1.71 (±.76) .95 (±.12) F (n = 12) 2.25 (±1.5) 3.00 (±4.2) .46 (±.35) the membranes or worms could sense molting fluid present between old and new cuticle. The juvenile nemerteans on a pre-molt adult crab appear to aggregate where they have access to the epimeral suture but it is not until the suture actually begins to open that they migrate out across open calcified carapace to reach the suture. Again the chemicals which might be the actual attractants are unknown. Clearly the de- calcification occurring there is a unique chemical event on the host and any number of compounds such as enzymes or breakdown products could be utilized as a cue fiDr worm migration. The process of transfer on molting female crabs appears to be a multi-stage process involving recognition of diverse agents and specific action patterns. In contrast the shift from male to females at mating may be a one-stage process cued by the ecdysis of the female crab. Males mate with females just after the female has shed her exoskeleton and it is then that transfer from the male to female by worms occurs. In the transfer of worms marked with neutral red, worms from the male were more concentrated under the female abdomen relative to the distribution of the worms already on the female. It is not known if the male-derived worms would eventually have spread out similar to the worms already there. The different behavior of worms on male crabs that molt before and after the spring mating season is consistent with our other observations and reinforces the adaptive behavior for C errans. The one example of efficient transfer occurred on a male in late summer, several months after the mating season which is usually in February or March in central California. Worms on males transferring at this time become available for venereal transmission the next breeding season. Male crabs in early spring had a mean burden of 129.4 worms (n = 23). In contrast in the fall male crabs carried 1603.5 worms each (n = 29). The lower burden just after the mating season is consistent with the observation of venereal transfer. The inability of a high proportion of worms to transfer at ecdysis on male crabs that molt immediately after the mating season is intriguing. Perhaps most of the remaining worms represent remnant population that were either unresponsive to previous transfer stimuli or are located in sites relatively inaccessible to such stimuli. In the experiment involving transferral of worms on juvenile Cancer magister a large proportion of the worms disappeared and were found on neither the new crab cuticle nor on the exuvium. Most of this loss to the system occurred on male juvenile hosts but these were the hosts which had worms placed on them because of low original worm burdens. Perhaps acclimatization to the new host was insufficient resulting in worm departure. Worm transfer at molt was highly efficient in the worms which stayed in the experimental chambers. The ability to transfer and concentrate on mature female crabs is highly adaptive for C. errans. It is only after feeding on host eggs that the worm can complete its own life cycle (Wickham, 1980). This transfer ability coupled with the fact that C errans appears to be able to survive on amino acids leaked by the host for an indefinite INTERHOST TRANSFER BY CARCINONEMERTES 337 period (Roe et al, 1981) means that once a larval worm finds a host it has a relatively high probability of eventually feeding on crab eggs. The fact that Carcinonemertes epialti on the host Hemigrapsus orcgonensis also is able to transfer at host ecdysis suggests that this behavior is general for Carcino- nemertes. One other worm has been observed to transfer at host ecdysis on the host Pinnixia tubicula (J. McDermott, pers. comm.). Humes (1942) noted that C. carci- nophila was shed with the exuvium when the host Callinectes sapidus molted. The location of worms on non-ovigerous hosts in the Atlantic differs in one important respect, however. Carcinonemertes carcinonphila is generally found sheathed between the gill lamellae of its host when not on an ovigerous crab (Humes, 1942; Hopkins, 1947). Pseudocarcinonernertes homari also lives in the branchial chamber and on the gills of the lobster Homarus americanus (Fleming and Gibson, 1981). On the Pacific coast, nemerteans have never been found in any number on the host gills (Wickham, 1978; unpubl.). It is possible that the location of the juvenile worms prevented transfer at molt, but in the case of the host Pinnixa tubicola the worms which transferred also lived in the host branchial chamber (J. McDermott, pers. comm.). Our observations of transfer by C. errans occurred because of the unusually high densities of worms involved. This behavior only became obvious when vast numbers of worms were observable. Further studies on transfer at molt by Carcinonemertes carcinophila are warranted. The most significant consequence of these findings is C. errans has a heretofore unsuspected ability to accumulate on ovigerous crabs. This worm has been an enigma in that in central California crabs can carry as many as 100,000 worms. Yet C errans has a fecundity on the order of 1000 eggs per year (Wickham, 1980) which is quite low when compared to other planktonically dispersed marine organisms. Efficiency in finding and remaining with hosts is critical to their success. Thus, the predatory impact of C. errans on crab reproduction is maximized. Since the commercial season (for male dungeness crabs only) follows the channelization of the worm population to the reproductive female crabs, only a very small fraction of the worm population is lost to the principal source of crab mortality: the fishery. While crab populations may be temporarily depressed due to fishing, the worm pop- ulation is buffered by the relative longevity of their unfished female hosts. The long- term consequences of the complex but efficient life cycle of C. errans for the dungeness crab fishery must now be explored. Acknowledgments We would like to thank the director of the U.C. Bodega Marine Laboratory, Cadet Hand, for assistance in conducting this study. We also thank Lab Manager Paul Siri for the loan of photographic equipment. Thanks also go to L. DeKeyser for manuscript preparation. This work was supported by the California Office of Sea Grant Project No. RF-75A, Grant No. NA80AAD00120. LITERATURE CITED Aiken, D. E., S. L. Waddy, L. S. Uhazy, and A. Campbell. 1983. A nemertean destructive to the eggs of the Lobster, Homarus americanus. Rapp. P. -v. Reun. Cons. Int. E.xplor. Mer 182: 130-133. Butler, T. H. 1961. Growth and age determination of the Pacific edible crab. Cancer magisier Dana. J. Fish. Res. Board Can. 18: 873-891. Crowe, J. H., L. M. Crowe, P. Roe, and D. E. Wickham. 1982. Uptake of DOM by nemertean worms: association of worms with arthrodial membranes. Am. Zool. 22: 671-682. 338 D. E. WICKHAM ET AL. Fleming, L. C, and R. Gibson. 1981. A new genus and species of monostiliferons hoplonemertean, ectohabitant on lobsters. J. Exp. Mar. Biol. Ecol. 52: 79-93. Hopkins, S. 1947. The nemertean Carcinonemertes as an indicator of the spawning history of the host, Callinectes sapidus. J. Parasitol. 33: 1 46- 1 50. Humes, A. G. 1942. The morphology, taxonomy and binomics of the nemertean genus Carcinonemertes. III. Biol. Monogr. 18: 1-105. KuRiS, A. M. 1978. Life cycle, distribution, and abundance of Carcinonemertes epialti. or nemertean egg predator of the shore crab, Hemigrapsus oregonensis in relation to heart size, reproduction, and molt cycle. Biol. Bull. 159: 247-257. Roe, p. 1979. Aspects of development and occurrence of Carcinonemertes epialti (Nemertea) from shore crabs in Monterey Bay, California. Biol. Bull. 156: 130-140. Roe, p., J. H. Crowe, L. M. Crowe, and D. E. Wickham. 1981. Uptake of amino acids by juveniles of Carcinonemertes errans (Nemertea). Comp. Biochem. Physiol. 69: 423-427. Snow, C. D., and J. R. Nielson. 1966. Premating and mating behavior of the Dungeness crab (Cancer magister Dana). J. Fish. Res. Board Can. 23: 1319-1323. Wickham, D. E. 1978. A new species of Carcinonemertes (Nemertea: Carcinonemertidae) with notes on the genus from the Pacific Coast. Proc. Biol. Soc. Wash. 91: 197-202. Wickham, D. E. 1979. Predation by the nemertean Carcinonemertes errans on eggs of the Dungeness crab. Cancer magister. Mar. Biol. 55: 45-53. Wickham, D. E. 1980. Aspects of the life history of Carcinonemertes errans (Nemertea: Carcinonemertidae) an egg predator of the crab Cancer magister. Biol. Bull. 159: 247-257. Reference: Biol. Bull. 167: 339-353. (October, 1984) CHEMICAL MEDIATION OF APPETITIVE FEEDING IN A MARINE DECAPOD CRUSTACEAN: THE IMPORTANCE OF SUPPRESSION AND SYNERGISM RICHARD K. ZIMMER-FAUST,* ' JEFFREY E. TYRE,t WILLIAM C. MICHEL.t AND JAMES F. CASEf * Department of Zoology, University of Queensland, St. Lucia, Brisbane 4067, Queensland. Australia and ^Department of Biological Sciences and the Marine Science Institute University of California Santa Barbara. California, 93106 Abstract The California spiny lobster, Panulirus interruptus, failed to exhibit appetitive feeding or locomotion in response to a low molecular weight fraction (< 1000 daltons) prepared from a sea water extract of muscle from abalone, a natural prey. This lack of response was caused by chemical suppressants, rather than by lack of stimulatory compounds. Excitatory responses were induced by single, low molecular weight com- pounds, but these responses were inhibited by suppressants which occur naturally in the muscle fraction. Amino and organic acids were found highly stimulatory to lobsters, but nucleotides and sugars were not. A mixture of monocarboxylic amino acids and dicarboxylic organiic acids was much more effective in elliciting behavior than either of the constituents tested alone, at the same overall concentration. Mixtures which combined either ammonium or urea with amino or organic acids significantly reduced behavioral activity caused by these latter substances. Results indicate that tests of single chemicals cannot always reliably predict the stimulatory properties of solutions, combining even as few as two or more compounds. The stimulatory properties of complex odorants, including prey extracts, are best assessed by fractionating and then combining and testing the fractions in bioassays of factorial design. Introduction Behavioral investigations of feeding and electrophysiological studies of chemo- sensory afference have usually shown that decapod crustaceans are sensitive to low molecular weight compounds. Of these, organic nitrogenous substances and organic acids are the most stimulatory (Laverack, 1963; Case, 1964; McLeese, 1970; Kay. 1971;Shepheard, 1974; Allen ^/ a/., 1975; Hindley, 1975; Ache t'/ a/.. 1978; Johnson and Ache 1978; Mackie et al, 1980; Derby and Atema, 1982a, b). Carbohydrates (Ashby and Larimer, 1965; Hartman and Hartman, 1977; Zimmer et al., 1979; Robertson et al., 198 1) and nucleotides (Shelton and Mackie, 197 1 ; Carr and Thomp- son, 1983) also evoke responses. It is generally assumed that low molecular weight substances are the dominant natural feeding attractants for marine decapods since they stimulate receptors, cause behavioral responses, and are highly soluble and dif- fusable in sea water (Ache et al., 1976). These latter properties are thought to result in their rapid release from tissues of prey and from carrion (Rittschof, 1980; Zimmer- Faust and Case, 1982a). Because low molecular weight compounds may provide the earliest chemosensory clues to distant food sources, it is generally assumed that decapod predators emphasize their detection in food search and feeding. Received 30 March 1984; accepted 25 July 1984. ' Present address: Marine Science Institute, University of California, Santa Barbara. California. 93106. 339 340 R. K. ZIMMER-FAUST ET AL Recent electrophysiological investigations have demonstrated that certain low molecular weight substances, abundant in animal flesh, suppress the neural responses of lobster and crab antennule chemoreceptors to stimulatory compounds (Gleeson and Ache, 1983; Johnson and Atema, 1983). While this finding is interesting, there are as yet no clear behavioral correlates for these physiological observations. Sup- pressants may be found to serve a vital role in the control of foraging and feeding by these animals. For example, suppressants might reduce ingestion of harmful sub- stances, reduce the search for food of low caloric value, or reduce locomotory activity of an animal in the vicinity of a valuable food item, increasing the likelihood of food discovery. Most previous behavioral studies of chemoreception in feeding and food search by decapod Crustacea have focused on the role of low molecular weight sub- stances as excitants, typically with tests performed of single chemicals and simple mixtures (McLeese, 1970; Kay, 1971; Allen et al, 1975; Fuzessery and Childress, 1975; Hindley, 1975; Hamner and Hamner, 1977; Hartman and Hartman, 1977; Ache et al, 1978; Robertson et al, 1981; Carter and Steele, 1982; Zimmer-Faust and Case, 1982b). These experiments assumed that summative chemosensory inputs di- rectly control behavior, and little attention has been given to CNS processing. However, in the few studies in which interactions among stimuli were considered synergy was observed {e.g., Shelton and Mackie, 1971; Mackie and Shelton, 1972; Carr, 1978; Robertson et a/., 1981). Under field conditions, we have found the California spiny lobster, Panulirus interruptus (Randall), arriving in greatest numbers at abalone muscle {Haliotis spp.) baits, after 24-48 h (Zimmer-Faust and Case, 1982a). This occurs even though small molecules (primary amines) are released from the baits predominantly over the first 3 h. Moreover, laboratory experiments demonstrate that P. interruptus responds sig- nificantly to a high molecular weight fraction (>1000 daltons), but not to a low molecular weight fraction (<1000 daltons) prepared from abalone muscle (Zimmer- Faust et al, 1984). Though our field and laboratory observations are in agreement, they differ from what is commonly believed true for decapods, namely, that low molecular weight substances control foraging and feeding. Our results are further at odds with previous electrophysiological investigations which show that P. interruptus possesses chemoreceptors sensitive to low molecular weight compounds, occurring abundantly in the flesh of abalone (Fuzessery and Childress, 1975; Lindsey, 1976). These considerations prompted the present investigation into the behavioral re- sponses o^ Panulirus to low molecular weight substances. Compounds were individually assayed for their ability to stimulate early arousal and appetitive phases of feeding. Some were effective, thus demonstrating specifically for Panulirus that inability to respond to the low molecular weight fraction of abalone does not arise from behavioral insensitivity to low molecular weight substances. It was further experimentally dem- onstrated that this inability was caused by chemical suppressants, and that both synergistic and suppressant interactions occur among substances naturally residing in the tissues of prey and carrion of lobsters. Our results demonstrate that the con- tributions made by specific chemical agents to the stimulatory capacity of a complex prey extract, cannot be properly specified without considering the entire chemosensory integrative capacity of the responding organism. Materials and Methods General procedures General procedures and apparatus were identical to those previously described (Zimmer-Faust and Case, 1983; Zimmer-Faust et al, 1984). Animals captured in CHEMICAL MEDIATION OF FEEDING IN LOBSTERS 341 traps or by hand (SCUBA) at More Mesa reef, 4 km east of the UCSB campus, were brought immediately to our laboratory and held in 3000 1 aquaria for 14 days before experiments were initiated. Incoming animals were tattoo marked for individual recognition (Kuris, 1971), and only hard-shelled animals of 60-68 mm carapace length were used. Animals were fed abalone muscle, mackerel muscle (Scomber ja- ponicus), and opened mussels (Mytilus califomianus) and were deprived of food for 24 h before testing. Lobsters were individually tested for responses to chemical solutions in rectangular aquaria, 30 X 30 X 13 cm, a size permitting the control of stimulus flow, without inhibiting behavior. Sea water (980 ml/min) entered each aquarium at a velocity of ~50 cm/s, from a head-tank maintained under constant hydrostatic pressure. Stim- ulants were introduced from a reservoir (10 ml/7 s) by opening a three-way valve. Dilution associated with stimulus delivery was 1.02 X 10 ^(±0.13 X 10^^ S.D.) times original concentrations, as previously determined by fluorometric measurement of fluorescein dye dilution (Zimmer-Faust and Case, 1983). Concentrations reported are not corrected for this dilution. Lobsters were tested once in 48 h for a maximum of 6 times during a 14-day period. They were put in experimental aquaria 90- 1 20 min prior to testing and usually settled within 30-40 min. Observations of behavior were initiated 1 min before in- troduction of a chemical solution and continued for 3 min afterwards. All trials were conducted according to a double-blind protocol, in which the observer was unaware of the composition of test or control solutions being tested. Order of stimulus pre- sentation did not influence the behavior of animals, since for each substance the proportion responding was unrelated to the test sequence. All solutions were prepared from analytical grade reagents and 5 ixm filtered sea water, adjusted to pH 7.8 before testing. Antennule flicking and wiping, pereiopod probing, and locomotion were moni- tored, since these are behaviors commonly exhibited by Panulirus and other decapods in response to chemicals associated with food {e.g., Maynard and Dingle, 1963; McLeese, 1970; Kay, 1971; Mackie and Shelton, 1972; Snow, 1973; Allen et ai, 1975; Fuzessery and Childress, 1975; Hindley, 1975; Pearson and OUa, 1977; Pearson et ai, 1979; Zimmer-Faust and Case, 1982b). Appetitive feeding was defined as the occurrence of increased flicking (>1.0 flick/s), wiping and probing each within a 3 min trial period. Further justification for the emphasis on these behavioral acts appears elsewhere (Zimmer-Faust et al, 1984), and their definitions are given in Table I. Chemicals were considered stimulatory when proportions of responding animals dif- fered significantly from the proportion responding to sea water {P < 0.05). A G-Test for Independence was used with Williams' correction for 2 X 2 contingency tables, in analyzing data from experiments presenting test solutions to differing groups of animals. A binomial test was used (Sokal and Rohlf, 1981, p. 774; F = q = 0.5), for experiments presenting test solutions to the same group of animals, with changes in individual responsivities compared. Experiment 1: responses to single compounds Previous investigations showed that lobsters are unresponsive to a low molecular weight fraction (< 1000 daltons) of an extract prepared from abalone muscle (Zimmer- Faust et ai, 1984). For this reason, tests were conducted to determine if lack of response is caused by behavioral insensitivity to low molecular weight substances. Thirty-two compounds were individually assayed at 10'^ A/, and each chemical was tested on 20 different animals in conjunction with 40 sea water controls. 342 R. K. ZIMMER-FAUST ET AL. Table I Definitions of behavioral elements in appetitive feeding and locomotion by Panulirus Act Definition Feeding Antennule flicking Vertical deflection of a lateral antennular flagellum to a position nearly contacting the medial flagellum. A response was defined as > 1 .0 flick per second. Leg probing Any non-locomotor movement of a pereiopod, either raking a dactyl across the substratum, or elevating a dactyl to a position no longer in contact with the substratum. Antennule wiping A downward and vertical deflection of an antennule, resulting in simultaneous contact of both antennular flagella with the third maxillipeds. Locomotion A laterally or anteriorly directed movement of the body to a distance, >l/2 carapace length. Experiment 2: interaction between glycine and the <1000 dalton fraction of freeze-dried abalone muscle extract (FDAME) In the first experiment, glycine was found to be the most stimulatory of all tested substances (see Results, Table II). Because a low-molecular weight fraction of FDAME is ineffective in stimulating feeding in Panulirus, yet contains a high concentration of glycine (4.5 X 10""* M) (Zimmer-Faust et al, 1984), this finding suggested the existence of suppressants within the fraction. An experiment was conducted to test for this possibility. A low molecular weight fraction of FDAME was prepared from a standard extract (6.00 g/1) of lyophilized abalone muscle and filtered sea water, by the procedures of Zimmer-Faust et al. (1984). Ultrafiltration of the extract was performed using an Amicon model 402 pressure ultrafiltration vessel and UM-2 membrane, with ultra- filtrate (<1000 dahons) collected undiluted, and stored frozen (-20°C) in aliquots. Aliquots of 10" M glycine, <1000 fraction with 1 0""* M glycine added, and sea water were each presented to the same 27 animals. The application of glycine, by itself, served to control for the possibility that lack of response might be caused by factors other than chemical composition. It was expected that the glycine-enhanced low molecular weight fraction would be ineffective, if suppressants for glycine existed. Experiment 3: interactions between glycine and other defined compounds Experiments employing the <1000 dalton fraction could not be used to identify the mechanism(s) of feeding suppression, because its constituents might have two types of effects. They might bind to and thereby limit the action of stimulatory molecules, or they might act directly to influence chemosensory processes. Therefore, to approach this question we were constrained to examine the simplest of interactions in this system, namely, those between glycine and other defined compounds. Such tests could demonstrate if suppression directly involves either primary chemosensory processes or CNS mechanisms, by eliminating the possibility of chemical binding or chelating among assayed substances. Jests with glycine, urea, and ammonium. We first explored the interaction between glycine and urea. Urea was selected because it is highly abundant in an extract known CHEMICAL MEDIATION OF FEEDING IN LOBSTERS 343 to be noxious to lobsters (J. E. Tyre, upubl. data), prepared from the muscle of angel shark (Squat ina califomica), and because it cannot bind glycine under our present test conditions. Aliquots of 10' M glycine, 10^^ M glycine plus 10 - A/ urea, 10"^ M urea, and sea water were each presented to the same 52 animals. Experiments were then performed to investigate the possible interaction between glycine and ammonium. Ammonium was selected because of its close similarity to the major molecular subcomponent of urea, and because of its abundance in the low molecular weight fraction of FDAME (1.0 X 10~^ M). Thus, ammonium might serve as a natural suppressant in FDAME to glycine-induced feeding responses. Like urea, it does not bind to glycine under present test conditions. Tests were conducted injecting aliquots of 10' M glycine, 10"' M glycine plus 10^ M ammonium, 10 ' M am- monium, and sea water, each to the same 32 animals. Additional tests were performed to further examine the interaction between glycine and ammonium. These tests were conducted by injectin