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.) The original manuscript must be typed in double spacing (including figure legends, footnotes, bibliography, etc.) on one side of 16- or 20-lb. bond paper, 8'/2 by 1 1 inches. Manuscripts should be proofread carefully and errors corrected legibly in black ink. Pages should be numbered consecutively. Margins on all sides should be at least 1 inch (2.5 cm). Manuscripts should conform to the Council of Biology Editors Style Manual, 4th Edition (Council of Biology Editors, 1978) and to American spelling. Unusual abbreviations should be kept to a minimum and should be spelled out on first reference as well as defined in a footnote on the title page. Manuscripts should be divided into the following components: Title page, Abstract (of no more than 200 words). Introduction, Materials and Methods, Results, Discussion, Acknowledgments, Literature Cited, Tables, and Figure Legends. In addition, authors should supply a list of words and phrases under which the article should be indexed. 2. Figures. Figures should be no larger than 8'/2 by 1 1 inches. The dimensions of the printed page, 5 by 7% inches, should be kept in mind in preparing figures for publication. We recommend that figures be about Vh times the linear dimensions of the final printing desired, and that the ratio of the largest to the smallest letter or number and of the thickest to the thinnest line not exceed 1:1.5. Explanatory matter generally should be included in legends, although axes should always be identified on the illustration itself Figures should be prepared for reproduction as either line cuts or halftones. Figures to be reproduced as line cuts should be unmounted glossy photographic reproductions or drawn in black ink on white paper, good-quality tracing cloth or plastic, or blue-lined coordinate paper. Those to be reproduced as halftones should be mounted on board, with both designating numbers or letters and scale bars affixed directly to the figures. All figures should be numbered in consecutive order, with no distinction between text and plate figures. The author's name and an arrow indicating orientation should appear on the reverse side of all figures. 3. Tables, footnotes, figure legends, etc. Authors should follow the style in a recent issue of The Biological Bulletin in preparing table headings, figure legends, and the like. Because of the high cost of setting tabular material in type, authors are asked to limit such material as much as possible. Tables, with their headings and footnotes, should be typed on separate sheets, numbered with consecutive Roman numerals, and placed after the Literature Cited. Figure legends should contain enough information to make the figure intelligible separate from the text. Legends should be typed double spaced, with consecutive Arabic numbers, on a separate sheet at the end of the paper. Footnotes should be limited to authors' current addresses, acknowledgments or contribution numbers, and explanation of unusual abbreviations. All such footnotes should appear on the title page. Footnotes are not normally permitted in the body of the text. 4. A condensed title or running head of no more than 35 letters and spaces should appear at the top of the title page. 5. Literature cited. In the text, literature should be cited by the Harvard system, with papers by more than two authors cited as Jones et al, 1980. Personal communications and material in preparation or in press should be cited in the text only, with author's initials and institutions, unless the material has been formally accepted and a volume number can be supplied. The list of references following the text should be headed LITERATURE CITED, and must be typed double spaced on separate pages, conforming in punctuation and arrangement to the style of recent issues of The Biological Bulletin. 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. D., 400 Hilhop Terrace, Alexandria, VA 22301 Person, Philip, Special Dental Research Program, Veterans Administration Hospital, Brooklyn, NY 11219 Peterson, Bruce J., Ecosystems Center, Marine Biological Laboratory, Woods Hole, MA 02543 Pethig, Ronald, School of Electronic Engineering Science, University College of N. Wales, Dean St., Bangor, Gwynedd, LL57 lUT, U. K. 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. Quebec, Canada Pratt, Melanie M., Department of Anatomy and Cell Biology, University of Miami School of Medicine (R124), P.O. Box 016960, Miami, FL 33101 Prendergast, Robert A., Department of Pathology and Ophthalmology, Johns Hopkins University, Baltimore, MD 21205 Price, Carl A., Waksman Institute of Microbiology, Rutgers University, P.O. Box 759, Pis- cataway, NJ 08854 Price, Christopher H., Biological Science Center, 2 Cummington St., Boston, MA 02215 Prior, David J., Department of Biological Sciences, University of Kentucky, Lexington, KY 40506 Provasoli, Luigi, 474 Whitney Ave., New Haven, CT 0651 1 (Life Member 12/83) Prusch, Robert D., Department of Life Sciences, Gonzaga University, Spokane, WA 99258 20 MARINE BIOLOGICAL LABORATORY Przybylski, Ronald J., Case Western Reserve University, Department of Anatomy, Cleveland, OH 44104 PURVES, Dale, Department of Physiology, Washington University School of Medicine, 660 S. Euclid Ave., St. Louis, MO 631 10 QuiGLEY James, Department of Microbiology and Immunology Box 44, SUNY Downstate Medical Center, 450 Clarkson Ave., Brooklyn, NY 1 1203 Rabin, Harvey, P.O. Box 239, Braddock Heights, MD 21714 Raff, Rudolf A., Department of Biology, Indiana University, Bloomington, IN 47405 Rakowski, Robert F., Department of Physiology and Biophysics, Washington University, School of Medicine, 660 S. Euclid Ave., St. Louis, MO 631 10 Ramon, Fidel, Dept. de Fisiologia y Biofisca, Centrol de Investigacion y de, Estudius Avanzados del Ipn, Apurtado Postal 14-740, Mexico, D.F. 07000 Ranzi, Silvio, Department of Zoology, University of Milan, Milan, Italy Ratner, Sarah, Department of Biochemistry, Public Health Research Institute, 455 First Ave., New York, NY 10016 Rebhun, Lionel I., Department of Biology, Gilmer Hall, University of Virginia, Charlottesville, VA 22901 Reddan, John R., Department of Biological Sciences, Oakland University, Rochester, MI 48063 Reese, Thomas S., Marine Biological Laboratory, Woods Hole, MA 02543 Reiner, John M., Albany Medical College of Union University, Department of Biochemistry, Albany, NY 12208 Reinisch, Carol L., Tufts University School of Veterinary Medicine, 203 Harrison Avenue, Boston, MA 02 1 1 5 Reuben, John P., Department of Biochemistry, Merck Sharp and Dohme, P.O. 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. Meryl, Box 309W, Waquoit, MA 02536 ROSENBAUM, Joel L., Department of Biology, Kline Biology Tower, Yale University, New Haven, CT 05610 Rosenberg, Philip, School of Pharmacy, Division of Pharmacology, University of Connecticut, Storrs, CT 06268 Rosenbluth, Jack, Department of Physiology, New York University School of Medicine, 550 First Ave., New York, NY 10016 Rosenbluth, Raja, 3380 West 5th Ave., Vancouver 8 BC, Canada V6R 1R7 Roslansky, John, Box 208, Woods Hole, MA 02543 Roslansky, Priscilla F., Box 208, Woods Hole, MA 02543 Ross, William N., Department of Physiology, New York Medical College, Valhalla, NY 10595 Roth, Jay S., Division of Biological Sciences, Section of Biochemistry and Biophysics, University of Connecticut, Storrs, CT 06268 Rowland, Lewis P., Neurological Institute, 710 West 168th St., New York, NY 10032 Ruderman, Joan V., Department of Anatomy, Harvard Medical School, Boston, MA 02 1 1 5 MEMBERS OF THE CORPORATION 21 RUSHFORTH, Norman B., Case Western Reserve University, Department of Biology, Cleveland, OH 44106 Russell-Hunter, W. D., Department of Biology, 110 Lyman Hall, Syracuse University, Syracuse, NY 13210 Saffo, Mary Beth, Department of Biology, Swarthmore College, Swarthmore, PA 1 908 1 Sager, Ruth, Sidney Farber Cancer Institute, 44 Binney St., Boston, MA 02 11 5 Salama, Guy, Department of Physiology, University of Pittsburgh, Pittsburgh, PA 15261 Salmon, Edward D., Department of Zoology, University of North Carolina, Chapel Hill, NC 27514 Salzberg, Brian M., Department of Physiology, University of Pennsylvania, 4010 Locust St., Philadelphia, PA 19174 Sanders, Howard, Woods Hole Oceanographic Institution, Woods Hole, MA 02543 Sanger, Jean M., Department of Anatomy, School of Medicine, University of Pennsylvania, 36th and Hamilton Walk, Philadelphia, PA 19174 Sanger, Joseph, Department of Anatomy, School of Medicine, University of Pennsylvania, 36th and Hamihon Walk, Philadelphia, PA 19174 Sato, Hidemi, Sugashima Marine Biological Laboratory, Nagoya University, Sugashima-cho, Toba-chi, Mie-Ken 517, Japan Saunders, John, Jr., Department of Biological Sciences, SUNY, Albany, NY 12222 Saz, Arthur K., Medical and Dental Schools, Georgetown University, 3900 Reservoir Rd., NW, Washington, DC 2005 1 SCHACHMAN, HOWARD K., Department of Molecular Biology, University of California, Berkeley, CA 94720 SCHIFF, Jerome A., Institute for Photobiology of Cells and Organelles, Brandeis University, Waltham, MA 02154 Schlesinger, R. Walter, University of Medicine and Dentistry of New Jersey, Department of Microbiology, Rutgers Medical School, P.O. Box 101 Piscataway, NJ 08854 (Life Member 11/83) SCHMEER, Arlene C, O.P., Mercene Cancer Research Hospital of Saint Raphael, New Haven, CT 065 1 1 Schneider, E. Gayle, University of Nebraska Medical Center, Department of Biochemistry, 42nd and Dewey Ave., Omaha, NE 68105 SCHNEIDERMAN, HOWARD A., Monsanto Company, 800 North Lindberg Blvd., DIW, St. Louis, MO 63166 ScHOPF, Thomas J. M., Department of Geophysical Sciences, University of Chicago, 5734 South Ellis Ave., Chicago, IL 60637 (deceased 3/84) SCHOTTE, Oscar E., Department of Biology, Amherst College, Amherst, MA 01002 SCHUEL, Herbert, Department of Anatomical Sciences, SUNY, Buffalo, NY 14214 SCHUETZ, Allen W., School of Hygiene and Public Health, Johns Hopkins University, Baltimore, MD 21205 Schwab, Walter E., Virginia Polytechnical Institute and State University, Department of Biology, Blacksburg, VA 24601 Schwartz, James H., Center for Neurobiology and Behavior, New York State Psychiatric Institute— Research Annex, 722 W. 168th St., 7th Roor, New York, NY 10032 Schwartz, Martin, Department of Biological Sciences, University of Maryland Baltimore County, Catonsville, MD 21228 ScoRELD, Virginia Lee, Department of Microbiology and Immunology, UCLA School of Medicine, Los Angeles, CA 90024 Scott, Allan C, 1 Nudd St., Waterville, ME 04901 Scott, George T., 10 Orchard St., Woods Hole, MA 02543 (Life Member 10/83) Sears, Mary, P.O. 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. Bruce, Harvard Medical School, Berman-Gund Laboratory, Massachusetts Eye and Ear Infirmary, 243 Charles St., Boston, MA 021 14 Szent-Gyorgyi, Albert, Marine Biological Laboratory, Woods Hole, MA 02543 Szent-Gyorgyi, Andrew, Department of Biology, Brandeis University, Waltham, MA 02 1 54 Szent-Gyorgyi, Eva Szentkiraly, Department of Biology, Brandeis University, Waltham, MA 02154 Szuts, EteZ., Laboratory of Sensory Physiology, Marine Biological Laboratory, Woods Hole, MA 02543 Takashima, Shiro, Department of Bioengineering, University of Pennsylvania, Philadelphia, PA 19174 Tamm, Sidney L., Boston University Marine Program, Marine Biological Laboratory, Woods Hole, MA 02543 Tanzer, Marvin, L., Department of Biochemistry, Box G, Medical School, University of Connecticut, Farmington, CT 06032 Tasaki, Ichiji, Laboratory of Neurobiology, National Institute of Mental Health, NIH, Bethesda, MD 20205 Taylor, Douglass L., Biological Sciences, Mellon Institute, 440 Fifth Avenue, Pittsburgh, PA 15213 Taylor, Robert E., Laboratory of Biophysics, NINCDS, NIH, Bethesda, MD 20205 Taylor, W. Rowland, 4800 Atwell Rd., Shady Side, MD 20764 Teal, John M., Department of Biology, Woods Hole Oceanographic Institution, Woods Hole, MA 02543 Telfer, William H., Department of Biology, University of Pennsylvania, Philadelphia, PA 19174 Thorndike, W. Nicholas, Wellington Management Company, 28 State St., Boston, MA 02109 Travis, D. M., Veterans Administration Medical Center, Fargo, ND 58102 Treistman, Steven N., Worcester Foundation for Experimental Biology, Shrewsbury, MA 01545 Trigg, D. Thomas. 125 Grove St., Wellesley, MA 02181 Trinkaus, J. Philip, Osbom Zoological Laboratories, Department of Zoology. Yale University, New Haven, CT 06510 Troll, Walter, Department of Environmental Medicine, College of Medicine, New York University, New York, NY 10016 Troxler, Robert F., Department of Biochemistry, School of Medicine, Boston University, 80 East Concord St., Boston, MA 021 18 Tucker, Edward B., Biology Department, Vassar College, Poughkeepsie, NY 12601 Turner, Ruth D., MoUusk Department, Museum of Comparative Zoology, Harvard University, Cambridge, MA 02 1 38 Tweedell, Kenyon S., Department of Biology, University of Notre Dame, Notre Dame, IN 46656 Tytell, Michael, Department of Anatomy, Bowman Gray School of Medicine, Winston- Salem, NC 27103 Uretz, Robert B., Division of Biological Sciences, University of Chicago, 950 East 59th St., Chicago, IL 60637 Valiela, Ivan, Boston University Marine Program, Marine Biological Laboratory, Woods Hole, MA 02543 Valois, John, Marine Biological Laboratory, Woods Hole, MA 02543 24 MARINE BIOLOGICAL LABORATORY Van Holde, Kensal, Department of Biochemistry and Biophysics, Oregon State University, CorvalUs, OR 97331 ViLLEE, Claude A., Department of Biological Chemistry, Harvard Medical School, Boston, MA 02115 Vincent, Walter S., School of Life and Health Sciences, University of Delaware, Newark, DE 19711 Wainio, Walter W., Box 1059 Nelson Labs, Rutgers Biochemistry, Piscataway, NJ 08854 Waksman, Byron, National Multiple Sclerosis Society, 205 East 42nd St., New York, NY 10017 Wall, Betty, 9 George St., Woods Hole, MA 02543 Wallace, Robin, A., Department of Anatomy, College of Medicine, University of Florida, Gainesville, FL 32610 Wang, An, Wang Laboratories, Inc., Bedford Road, Lincoln, MA 01773 Warner, Robert C, Department of Molecular Biology and Biochemistry, University of California, Irvine, CA 92717 Warren, Kenneth S., The Rockefeller Foundation, 1133 Avenue of the Americas, New York, NY 10036 Warren, Leonard, Department of Therapeutic Research, School of Medicine, Anatomy- Chemistry Building, University of Pennsylvania, Philadelphia, PA 19174 Waterman, T. H., Yale University, Biology Department, Box 6666, 610 Kline Biology Tower, New Haven, CT 06510 Watson, Stanley, Woods Hole Oceanographic Institution, Woods Hole, MA 02543 Webb, H. Marguerite, Marine Biological Laboratory, Woods Hole, MA 02543 Weber, Annemarie, Department of Biochemistry, School of Medicine, University of Penn- sylvania, Philadelphia, PA 19174 Webster, Ferris, Box 765, Lewes, DE 19958 Weidner, Earl, Department of Zoology and Physiology, Louisiana State University, Baton Rouge, LA 70803 Weiss, Leon P., Department of Animal Biology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA 19174 Weissmann, Gerald, New York University, 550 First Avenue, New York, NY 10016 Werman, Robert, Neurobiology Unit, The Hebrew University, Jerusalem, Israel Westerreld, R. Monte, The Institute of Neuroscience, University of Oregon, Eugene, OR 37403 Wexler, Nancy Sabin, 15 Claremont Avenue, Apt. 92, New York, NY 10027 Whittaicer, J. 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. Beers, Dr. and Mrs. Yardley Bennett, Dr. and Mrs. Michael V. L. Bernheimer, Dr. Alan W. Bernstein, Mr. and Mrs. Norman Berwind, Mr. David BiGELOw, Mrs. Robert O. BOCHE, Mr. David Bodeen, Mr. and Mrs. George H. BoETTiGER, Dr. and Mrs. Edward G. Bolton, Mr. and Mrs. Thomas C. BoTKiN, Dr. and Mrs. Daniel B. Bowles, Dr. and Mrs. Francis P. Bradley, Dr. and Mrs. Charles C. Bronson, Mrs. Samuel C. Brown, Mrs. Dugald E. S. Brown, Dr. and Mrs. Frank A., Jr. Brown, Mr. and Mrs. Henry Brown, Mrs. Neil Brown, Dr. and Mrs. Thornton Buck, Mrs. John B. BuFPiNGTON, Mrs. Alice H. Burrough, Mrs. Arnold H. Burt, Mrs. Charles E. Butler, Mr. and Mrs. Rhett W. Buxton, Mr. and Mrs. Bruce Calkins, Mr. and Mrs. G. N. Jr. Campbell, Dr. and Mrs. David G. Capobianco, Mr. and Mrs. Pat J. Members Carlson, Dr. and Mrs. Francis Carlton, Mr. and Mrs. Winslow G. Cheney, Mrs. Ralph H. Claff, Mr. and Mrs. Mark Clark, Mr. and Mrs. Hays Clark, Mr. and Mrs. James McC. Clark, Dr. and Mrs. Leonard B. Clark, Mrs. W. 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. Marlboro College Maryland, University of Maryland, University of, Baltimore County Massachusetts Eye and Ear Infirmary Massachusetts General Hospital Massachusetts Institute of Technology Massachusetts State Lobster Hatchery Massachusetts, University of, Boston Merck, Sharp and Dome Research Labora- tories Mercy College Miami, University of Miami, University of, School of Medicine Michigan Cancer Foundation Michigan State University Minnesota, University of Miriam Hospital Moravian College Mount Allison University Mount Holyoke College Mount Sinai School of Medicine Mystic Marinelife Aquarium NINCDS National Heart, Lung and Blood Institute National Institute of Mental Health National Institutes of Health National Institutes on Alcohol Abuse and Alcoholism National Marine Fisheries Service Nebraska, University of Nebraska, University of. Medical Center New College New England Medical Hospital New Jersey Medical School New Mexico, University of New York Aquarium New York Blood Center New York, City College of New York, City University of, Brooklyn College New York, City University of. Hunter College New York, City University of, Herbert Lehman College New York Institute of Basic Research for Mental Retardation New York Medical College New York, Neurological Institute New York State College of Veterinary Medicine New York, State University of, Albany New York, State University of Binghamton New York, State University of, Buffalo New York, State University of. College of Medicine New York, State University of, Downstate Medical Center New York, State University of. New Paltz New York, State University, Stony Brook New York University College of Dentistry New York University Medical Center New York University School of Medicine New York, Veterans Administration Hospital Nikon, Inc. North Carolina State University North Carolina, University of. Chapel Hill North Carolina, University of. School of Medicine Northeast Fisheries Center Northeastern University Northwestern University Northwestern University Medical School Norwich University Notre Dame, University of Oberlin College Ohio, Medical College of Ohio State University College of Medicine Ohio, University of Oklahoma, University of. Health Sciences Center Olympus Corporation of America Oregon, University of Oxford Marine Research Laboratory Pacific Biomedical Research Center Pennsylvania, Medical College of Pennsylvania, University of Pennsylvania, University of. School of Dental Medicine Pennsylvania, University of. School of Med- icine Pittsburgh, University of PMI-Strang Clinic Princeton University Puerto Rico, University of Puget Sound, University of Purdue University Rainbow Babies and Children's Hospital Rensselaer Polytechnic Institute Research Triangle Institute A. O. Reichert Scientific Rhode Island, University of Rochester, University of Medical Center Rochester, University of. School of Medicine and Dentistry Rockefeller Foundation 90 MARINE BIOLOGICAL LABORATORY Rockefeller University, The Rowland Institute of Science Rush Medical College Rutgers University Saint Andrew's Presbyterian College Saint Peter's College Scarborough College Seton Hall University Smith College Smithsonian Institution South Carolina, University of South Florida, University of Southhampton College Standard Oil of Indiana Stanford University Stanford University School of Medicine Stonehill College Swarthmore College Syracuse University Temple University Tennessee State University Tennessee, University of Texas, University of, Health Science Center Texas, University of. Medical Branch Texas A and M University Texas A and M University College of Medicine Texas Christian University Trinity College Tufts Medical School Tufts University Tufts University School of Medicine Tufts University School of Veterinary Medicine Tulane University Tulane University School of Medicine Upjohn Company United States Department of Agriculture, Plant Protection Institute United States Department of Health and Human Services, Food and Drug Administration Utah, University of, School of Medicine Fran M. Valenti, Inc. Vanderbilt University Vanderbilt University Medical Center Vassar College Venus Scientific Vermont, University of. School of Medicine Veterans Administration Hospital Virginia Institute of Marine Science Virginia Medical Center Wake Forest University Wallace and Company Washington, University of Washington, University of. 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. Ellis, Nancy L. Flynn, Susan HoBBS, Roger W. Hough, Rose A. Messer, Jill Development Office Salguero, Carol G., Development Officer Bravo, Luz Scarborough, Bonnie M. Weiss, Nan Director's Office Gross, Paul R., President and Director Thimas, Lisa Marie General Manager's Office Smith, Homer P., General Manager CoLBURN, Karen H. Geggatt, Agnes L. Johnson, Frances N. Plummer, Cynthia C. Wagner, Carol Ann Grants and Educational Services Howard, Joan E., Coordinator Ferzoco, Susan J. Foley, Joanne A. Vasquez, Eileen Gray Museum TiFFNEY, Wesley N., Curator Bush, Louise, Assistant Curator BoRETOS, C. Diane MoNTiERO, Eva S. Library Fessenden, Jane, Librarian AsHMORE, Judith A. Berrios, Jose R. Gibbons, Roberto G. Hanley, Janice S. Hough, Nancy L. Joseph, E. Lenora Margolin, Jill Moss, Ann H. MouNTFORD, Rebecca J. Norton, Catherine N. Swain, Laurel E. deVeer, Joseph M. Ward, Mary LABORATORY SUPPORT STAFF 93 Marine Resources Valois, John J., Manager BoRETOS, C. 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? Finally, a significant component missing from the present consideration of the claw neuromuscular system is the inhibitory (CI) motoneuron. There is a clear need to examine both its central and peripheral mechanisms in order to establish its role in claw asymmetry and to follow its development. Acknowledgments It is a pleasure to thank my colleagues who have contributed to the research reported here and the Natural Science and Engineering Research Council of Canada, the Muscular Dystrophy Association of Canada and the Grass Foundation of the U.S.A. for financial support. LITERATURE CITED Atwood, H. L. 1967. Crustacean neuromuscular mechanisms. Am. Zool. 7: 527-551. Atwood, H. L. 1973. An attempt to account for the diversity of crustacean muscles. Am. Zool. 13: 357- 378. Atwood, H. L. 1976. Organization and synaptic physiology of crustacean neuromuscular systems. Prog. Neurobiol. 7: 291-391. Atwood. H. L., andG. D. Bittner. 1971. Matching of excitatory and inhibitory inputs to crustacean muscle fibers. / Neurophysiol. 34: 157-170. Atwood, H. L., and L Kwan. 1976. Development of synapses in crayfish opener muscle. / Neurobiol. 7:289-312. Atwood, H. L., F. Lang, and W. a. Morin. 1972. Synaptic vesicles: selective depletion in crayfish excitatory and inhibitory axons. Science 176: 1353-1355. Bittner, G. D., 1968. The differentiation of crayfish muscle fibers during development. / Exp. Zool. 167: 439-456. Bittner, G. D., and D. L. Traut, 1978. Growth of crustacean muscle and muscle fibers. / Comp. Physiol. 124: 277-285. Burke, R. E. 1981. Motor units: anatomy, physiology and functional organization. Pp. 345-422 in Handbook of Physiology. The Nen'ous System, Vol. II, J. M. Brookhart and V. B Mountcastle, eds. Williams & Williams Co., Baltimore. Corballis, M. C, and M. J. Morgan. 1978. On the biological basis of human laterality: evidence for a maturational left-right gradient. Behav. Brain Sci. 2: 261-336. Costello, W. J., and C. K. Govind. 1983a. Contractile responses of single fibers in lobster claw closer muscles: correlation with structure, histochemistry and innervation. J. Exp. Zool. 227: 381-393. Costello, W. J., and C. K. Govind. 1983b. Correlation of motoneuron activity, muscle proteins and claw dimorphism in the lobster Homarus americanus. Am. Zool. 23: 902. Costello, W. J., and C. K. Govind. 1 984. Contractile proteins of fast and slow fibers during differentiation of lobster claw closer muscles. Dev. Biol. (In Press) Costello, W. J., and F. Lang. 1979. Development of the dimorphic claw closer muscles of the lobster, Homarus americanus. IV. Changes in functional morphology during growth. Biol Bull. 156: 1 79- 195. Costello, W. J., R. Hill, and F. Lang. 1981. Innervation patterns of fast and slow motor neurones during development of a lobster neuromuscular system. / Exp. Biol. 91: 271-284. Costello, W. J., R. Hill, andF. Lang, 1984. Firing patterns ofcloser motoneurons during reflex activity in the dimorphic claws of the lobster. / Exp. Zool. (In Press) Crane, J., 1977. Fiddler crabs of the world. Ocypodidae: Genus. L'ca. Princeton University Press. Princeton, New Jersey. 736 pp. El-Haj, a. J., C. K. Govind, and D. F. Houlihan. 1984. Growth of lobster leg muscle fibers over intermolt and molt. / Crust. Biol. 4: 536-545 Emmel, V. E. 1908. The experimental control of asymmetry at different stages in the development of the lobster. / Exp. Zool. 5: 471-484. 118 C. K. GOVIND GOUDEY, L. R., andF. Lang. 1974. Growth of crustacean muscle: asymmetric development of the claw closer muscles in the lobster, Homarus americanus. J. Exp. Zool. 189: 421-427. GoviND, C. K., 198 1 . Does exercise influence the differentiation of lobster muscles. F*p. 2 1 5-253 in Locomotion and Energetics in Arthropods. C. F. Herreid II and C. R. Fourtner, eds., Plenum Publ. Corp., New York. GoviND, C. K., AND H. L. Atwood. 1982. Organization of neuromuscular systems. Pp. 63-103 in The Biology of Crustacea, Vol. 3, Neurobiology: Structure and Function, D. E. Bliss, H. L. Atwood, and D. C. Sandeman, eds. Academic Press, New York. GoviND, C. K., AND R. H. Hill. 1982. Paired impulses in lobster claw motoneurons: in vitro and in vivo production. Can. J. Zool. 60: 1096-1099. GoviND, C. K., AND K. S. Kent. 1982. Transformation of fast fibers to slow prevented by lack of activity in developing lobster muscle. Nature 298: 755-757. GoviND, C. K., AND F. Lang. 1974. Neuromuscular analysis of closing in the dimorphic claws of the \obs\,ex Homarus americanus. J. Exp. Zool. 190: 281-288. GoviND, C. K., AND F. Lang. 1978. Development of the dimorphic claw closer muscles of the lobster, Homarus americanus. III. Transformation to dimorphic muscles in juveniles. Biol. Bull. 154: 55- 67. GoviND, C. K., AND F. Lang. 1979. Physiological asymmetry in the bilateral crusher claws of a lobster. J. Exp. Zool. 297: 27-32. GoviND, C. K., AND F. Lang. 1981. Physiological identification and asymmetry of lobster claw closer motoneurones. / Exp. Biol. 94: 329-339. GoviND, C. K., AND J. Pearce. 1984. Lateralization in number and size of sensory axons to the dimorphic chelipeds of crustaceans. (Submitted) GoviND, C. K., H. L. Atwood, and F. Lang. 1974. Sarcomere length increases in developing crustacean muscle. J. Exp. Zool. 189: 395-400. GoviND, C. K., J. She, and F. Lang. 1977. Lengthening of lobster muscle fibers by two age-dependent mechanisms. Experienlia 33: 35-36. GoviND, C. K., P. J. Stephens, and V. Trinkaus-Randall. 1981. Differences in motor output and fiber composition of the opener muscle in lobster dimorphic claws. J. Exp. Zool. 218: 363-370. GUTH, L. 1968. Trophic influences of nerve on muscle. Physiol. Rev. 48: 645-687. GuTMANN, E., 1976. Neurotrophic relations. Ann. Rev. Physiol. 38: 177-216. Hajek., I., N. Chari, a. Bass, and E. Gutmann. 1973. Differences in contractile and some biochemical properties between fast and slow abdominal muscles of the crayfish (Astaais leplodactylus). Physiol. Bohemslov. 22: 603-612. Harris, A. J. 1974. Inductive functions of the nervous system. Ann. Rev. Physiol. 36: 251-305. Herrick, F. H. 1895. The American lobster: a study of its habits and development. Fish Bull. V. S. 15: 1-252. Herrick, F. H. 191 1. Natural history of the American lobster. V. S. Bur. Fish. 29: 149-408. Hill, R. H., and C. K. Govind. 1981. Comparison of fast and slow synaptic termials in lobster muscle. CellTiss. Res. 221: 303-310. Hill, R. H., andC. K. Govind. 1982. Functional subdivision within a lobster motor unit. Experientia 38: 362-363. Hill, R. H., andC. K. Govind. 1983. Fast and slow motoneurons with unique forms and activity patterns in lobster claws. / Comp. Neurol. 218: 327-333. Hill, R. H., and C. K. Govind. 1984. Larval innervation of lobster claw closer muscle. J. Exp. Zool. 229: 393-399. Hill, R. H., and F. Lang. 1979. A revision of the inhibitory innervation pattern of the thoracic limbs of crayfish and lobster. J. Exp. Zool. 208: 129-135. Hughes, J. T., J. J. Sullivan, and R. Shleser. 1972. Enhancement of lobster growth. Science 111: 1110-1111. Hughes, J. T., R. A. Shleser, and G. Tchobanoglous. 1974. A rearing tank for lobster larvae and other aquatic species. Prog. Fish. Cult. 36: 129-132. Jahromi, S. S., and H. L. Atwood. 197 1 . Structural and contractile properties of lobster leg muscle fibers. J. Exp. Zool. 176: 475-486. Jahromi, S. S., andH. L. Atwood. 1974. Three dimensional ultrastructure of the crayfish neuromuscular apparatus. / Cell Biol. 63: 599-613. Jahromi, S. S., andM. P. Charlton, 1978. Transverse sarcomere splitting: a possible means of longitudinal growth in crab muscle. / Cell Biol. 80: 736-742. JOLESZ, F., and F. a. Sreter. 1981. Development, innervation, and activity-pattern induced changes in skeletal muscle. Ann. Rev. Physiol. 43: 531-552. Kent, K. S., and C. K. Govind. 1981. Two types of tonic fibers in lobster muscle based on enzyme histochemistry. / Exp. Zool. 215: 113-116. DEVELOPMENT OF ASYMMETRY 119 King, J. J., andC. K. Govind. 1980. Development of excitatory innervation in lobster claw closer muscle. / Comp. Neurol. 194: 57-70. Lang, F. 1975. A simple culture system for juvenile lobsters. Aquaculture 6: 389-393. Lang, F., W. J. Costello, andC. K. Govind. 1977a. Development of the dimorphic claw closer muscles of the lobster, Homarus americanus. I. Distribution of fiber tvpes in adults. Biol. Bull. 152: 75- 83. Lang, F., C. K. Govind, and J. She. 1977b. Development of the dimorphic claw closer muscles of the lobster, Homarus americanus. \\. Distribution of muscle fiber types in larval forms. Biol. Bull. 152: 382-391. Lang, F., C. K. Govind, W. J. Costello, and S. L Greene. 1977c. Developmental neuroethology: changes in escape and defensive behavior during growth of the lobster. Science 197: 682-685. Lang, F., C. K. Govind, and W. J. Costello. 1978. Experimental transformation of muscle fiber properties in lobster. Science 201: 1037-1039. Lang, F., M. M. Ogonowski, W. J. Costello, R. Hill, B. Roehrig, K. Kent, and J. Sellers. 1980. Neurotrophic influence on lobster skeletal muscle. Science 207: 325-327. Lehman, W., and A. G. Szent-GyOrgyi. 1975. Regulation of muscular contraction: distribution ofactin control and myosin control in the animal kingdom. / Gen. Physiol. 66: 1-30. Mellon, DeF., Jr. 1981. Nerves and the transformation of claw type in snapping shrimp. Trends Neurosci. 4: 245-248. Mellon, DeF., Jr., and P. J. Stephens. 1978. Limb morphology and function are transformed by contralateral nerve section in snapping shrimps. Nature 272: 246-248. Mellon, DeF., Jr., J. A.Wilson, andC. E. Phillips. 1981. Modification ofmotoneuron size and position in the central nervous system of adult snapping shrimp. Brain Res. 223: 134-140. Morgan, T. H. 1923. The development of asymmetry in the fiddler crab. Am. Nat. 57: 269-273. Morgan, T. H. 1924. The artificial induction of symmetrical claws in male fiddler crabs. Am. Nat. 58: 289-295. Neal, D. M., D. L. MacMillan, R. M. Robertson, and M. S. Laverack. 1976. The structure and function of the thoracic exopodites in the larvae of the lobster, Homarus gammarus L. Phil. Trans. R. Soc. Lond. B. 274: 53-68. Nottebohm, F. 1977. Asymmetries in neural control of vocalization in the canary. Pp. 23-44 in Lateralization in the Nervous System. S. Harnard, R. W. Doty, L. Goldstein, J. Jaynes and G. Krauthamer, eds. Academic Press, New York. Ogonowski, M. M., and F. Lang. 1979. Histochemical evidence for enzyme differences in crustacean fast and slow muscle. J. Exp. Zool. 207: 143-151. Ogonowski, M. M., F. Lang, andC. K. Govind. 1980. Histochemistry of lobster claw-closer muscles during development. / Exp. Zool. 213: 359-367. Przibram, H. 1901. Experimentelle studien iiber Regeneration. Arch. Entm. Mech. Org. 11: 321-345. Rhodes, W. R., Jr. 1977. Anatomical and physiological correlates of asymmetry and courtship display by male fiddler crabs. Ph.D. Thesis, University of Wisconsin, Madison. Ripley, S. H., andC. A. G. Wiersma. 1953. The effect of spaced stimulation of excitatory and inhibitory axons of the crayfish. Physiol. Comp. Oecol. 3: 1-17. Solon, M. H., and J. S. Cobb. 1980. The external morphology and distribution of cuticular hair organs on the claws of the American lobster, Homarus americanus (Milne-Edwards). J. Exp. Mar. Biol. Ecol.4S: 205-215. Stephens, P. J., and C. K. Govind. 1981. Peripheral innervation fields of single lobster motoneurons defined by synapse elimination during development. Brain Res. 212: 476-480. Stephens, P. J., and DeF. Mellon, Jr. 1979. Modification of structure and synaptic physiology in transformed shrimp muscle. J. Comp. Physiol. 132: 97-108. Wiersma, C. A. G. 1955. 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. The structural organization of the septate and gap junctions oi Hydra. J. Cell. Biol. 52: 397-408. Hyman, L. H. 1940. The Invertebrates: Protozoa through Ctenophora. McGraw Hill, New York. 726 pp. JOSEPHSON, R. K. 1974. Cnidarian neurobiology. Pp. 245-280 in Coelenterale Biology, L. Muscatine and H. M. Lenhoff, eds. Academic Press, New York. JosEPHSON, R. K., AND W. E. ScHWAB. 1979. Electrical properties of an excitable epithelium. / Gen. Physiol. 74: 213-236. King, M. G., and A. N. Spencer. 1979. Gap and septate junctions in the excitable endoderm of Polyorchis penicillatus (Hydrozoa, Anthomedusae). / Cell Sci. 36: 391-400. LOEWENSTEIN, W. R. 1981. Junctional intercellular communication: the cell-to-cell membrane channel. Physiol. Rev. 61: 829-913. LoEWENSTElN, W. R. 1984. Cell individuality and connectivity, an evolutionary compromise. Pp. 77-87 in Individuality and Determinism. S. W. Fox, ed. Plenum Publ. Corp., New York. Mackie, G. O. 1976. 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. The nature of membrane sites controlling anion permeability of human red blood cells as determined by studies with disulfonic stilbene derivatives. / Memb. Biol. 10:311-330. Cavanaugh, G. M. 1956. Pp. 62-69 in Formulae and Methods IV of the Marine Biological Laboratory Chemical Room. Woods Hole, Massachusetts. Chia, F. S., and M. E. Rice, eds. 1978. Settlement and Metamorphosis of Marine Invertebrate Larvae. Elsevier, New York. Crisp, D. J. 1974. Factors influencing settlement of marine invertebrate larvae. Pp. 1 77-265 in Chemoreception in Marine Organisms, P. T. Grant and A. M. Mackie, eds. Academic Press, New York. Crisp, D. J. 1984. Overview of research on marine invertebrate larvae, 1940-1980. Pp. 103-126 in Marine Biodeterioration, J. D. Costlow and R. C. Tipper, eds. Naval Institute Press, Annapolis, Maryland. Edwards, C. 1983. The ionic mechanisms underlaying the receptor potential in mechanoreceptors. Pp. 497-503 in The Physiology of Excitable Cells, A. D. Grinnell and W. J. Moody Jr., eds. Alan R. Liss, Inc., New York. Fritz, L. C, C. C. Wang, and A. Gorio. 1979. Avermectin B|a irreversibly blocks postsynaptic potentials at the lobster neuromuscular junction by reducing muscle membrane resistance. Proc. Natl. Acad. Sci. USA 76(4): 2062-2066. Frohlich, O., C. Leibson, and R. B. Gunn. 1983. Chloride net efflux from intact erythrocytes under slippage conditions: Evidence for a positive charge on the anion binding/transport site. / Gen. Physiol. 81: 127-152. Gallagher, J. P., H. Higashi, and S. Nishi. 1978. Characterization and ionic basis of GABA-induced depolarizations recorded "in vitro" from cat primary afferent neurones. J. Physiol. 275: 263-282. Geck, p., C. Pietrtyk, B. C. Burckiiardt, B. Pfeiffer, and E. Heinz. 1980. Electrically silent cotransport of Na"^, K"^ and CI" in Ehrlich cells. Biochim. Biophys. Acta 600: 432-447. Hadfield, M. G. 1978. Metamorphosis in marine molluscan larvae: an analysis of stimulus and response. Pp. 165-175 in Settlement and Metamorphosis of Marine Invertebrate Larvae. F. S. Chia and M. E. Rice, eds. Elsevier, New York. Hadreld, M. G. 1984. Settlement requirements of molluscan larvae: New data on chemical and genetic roles. In Recent Innovations in Cultivation of Pacific Molluscs, D. Morse, K. Chew, and R. Mann, eds. Elsevier, New York. (In press.) Hermann, A., and A. L. F. Gorman. 1981. Effects of tetraethylammonium on potassium currents in a molluscan neuron. J. Gen. Physiol. 78: 87-1 10. HODGKIN, A. L., and p. Horowicz. 1959. The influence of potassium and chloride ions on the membrane potential of single muscle fibres. / Physiol. 148: 127-160. Kaissling, K. E., and J. Thorson. 1980. Insect olfactory sensilla: structural, chemical and electrical aspects of the functional organization. Pp. 261-282 in Receptors for Neurotransmitters. Hormones and Pheromones in Insects, D. B. Satelle et ai, eds. Elsevier/North-Holland Biomedical Press, New York. Knauf, p. a., G. F. Fuhrmann, S. Rothstein, and a. Rothstein. (1977). The relationship between anion exchange and net anion flow across the human red blood cell membrane. / Gen. Physiol. 69: 363-386. Krnjevic, K., and S. Schwartz. 1967. The action of 7-aminobutyric acid on cortical neurones. Exp. Brain Res. 3: 320-336. Lynch, W. F. 1 947. The behavior and metamorphosis of the larva of Bugula neritina (Linnaeus): Experimental modification of the length of the free-swimming period and the responses of the larvae to light and gravity. Biol. Bull. 92: 1 15-150. Marder, E., and D. Paupardin-Tritsch. 1978. The pharmacological properties of some crustacean neuronal acetylcholine, 7-aminobutyric acid, and L-glutamate responses. J. Physiol. 280: 213- 236. Morita, H. 1972. Primary processes of insect chemoreception. Adv. Biophys. 3: 161-198. Morse, a., and D. E. Morse. 1984. Recruitment and metamorphosis of Haliotis larvae are induced by molecules uniquely available at the surfaces of crustose red algae. J. Exp. Mar. Biol. Ecol. 75: 191-215. 138 A. J. BALOUN AND D. E. MORSE Morse, D. E., H. Duncan, N. Hooker, and a. Morse. 1977. Hydrogen peroxide induces spawning in molluscs, with activation of prostaglandin endoperoxide synthetase. Science 196: 298-300. Morse, D. E., N. Hooker, H. Duncan, and L. Jensen. 1979. 7-Aminobutyric acid, a neurotransmitter, induces planktonic abalone larvae to settle and begin metamorphosis. Science 204: 407-410. Morse, D. E., H. Duncan, N. Hooker, A. Baloun, and G. Young. 1980a. GABA induces behavioral and developmental metamorphosis in planktonic molluscan larvae. Fed. Proc. 39: 3237-3241. Morse, D. E., N. Hooker, and H. Duncan. 1980b. GABA induces metamorphosis in Haliotis, V: Stereochemical specificity. Brain Res. Bull. 5, Suppl. 2: 381-387. Morse, D. E., M. Tegner, H. Duncan, N. Hooker, G. Trevelyan, and A. Cameron. 1980c. Induction of settling and metamorphosis of planktonic molluscan larvae III: Signaling by metabolites of intact algae is dependent on contact. Pp. 67-86 in Chemical Signaling in Vertebrate and Aquatic Animals, D. Miiller-Schwarze and R. M. Silverstein, eds. Plenum Press, New York. MoTOKiZAWA, P., J. P. Reuben, and H. Gru>jdfest. 1969. Ionic permeability of the inhibitory postsynaptic membrane of lobster muscle fibers. J. Gen. Physiol. 54: 437-46 1 . Muller, W. a., and G. Buchal. 1973. Metamorphose-induktion bel Planulalarven. II. Induktion durch monovalente Kationen: Die Bedeutung des Gibbs-Donnan-Verhaltnisses und der Na*/K*-ATPase. Wilhelm Roux' Arch. 174: 122-135. NiSHi, S., S. MiNOTA, AND A. G. Karczmar. 1974. Primary afferent neurones: The ionic mechanism of GABA-mediated depolarization. Neuropharmacology 13: 215-219. Passow, H., H. Fasold, M. L. Jennings, and S. Lepke. 1982. The study of the anion transport protein ("band 3 protein") in the red blood cell membrane by means of tritiated 4,4'-diisothiocyano- dihydrostilbene-2,2'-disulfonic acid (^H2-DIDS). Pp. 1-31 in Chloride Transport in Biological Membranes, J. A. Zadunaisky, ed. Academic Press, New York. Scheltema, R. S. 1974. Biological interactions determining larval settlement of marine invertebrates. Thallasia Jugoslav. 10: 263-296. ScHWARZ, W., AND H. Passow. 1983. Ca^^-activated K^ channels in erythrocytes and excitable cells. Ann. Rev. Physiol. 45: 359-374. Spindler, K-D., AND W. A. Muller. 1972. Induction of metamorphosis by bacteria and by a lithium- pulse in the larvae of Hydractinia echinata (Hydrozoa). Wilhelm Roux' Arch. 169: 271-280. Takeuchi, a. 1976. Studies of inhibitory effects of GABA in invertebrate nervous systems. Pp. 255-267 in GABA in Nervous System Function. E. Roberts, T. N. Chase, D. B. Tower, eds. Raven Press, New York. Takeuchi, A., and N. Takeuchl 1960. On the permeability of the end-plate membrane during the action of the transmitter. / Physiol. Land. 154: 52-67. Takeuchi, H., K. Watanabe, and H. Tamura. 1978. Penetrable and impenetrable anions into the GABA-activated chloride channel on the postsynaptic neuromembrane of an identifiable giant neurone of an African giant snail {Achat inafulica Ferussac). Comp. Biochem. Physiol. 61C: 309- 315. Thurm, U., and G. Wessel. 1979. Metabolism-dependent transepithelial potential differences at epidermal receptors of arthropods. J. Comp. Physiol. 134A: 119-130. Walker, R. J., M. J. Azanza, G. A. Kerkut, and G. N. Woodruff. 1975. The action of 7-aminobutyric acid (GABA) and related compounds on two identifiable neurones in the brain of the snail Helix aspera. Comp. Biochem. Physiol. 50C: 147-154. Yarowsky, p. J., AND D. O. Carpenter. 1978. A comparison of similar ionic responses to 7-aminobutyric acid and acetylcholine. / Neurophysiol. 41: 531-541. Reference: Biol Bull. 167: 139-158. (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. Pp. 127-136 in Coelenterate Ecology and Behavior, G. O. Mackie, ed. Plenum Press, New York. Bigger, C. H. 1980. Interspecific and intraspecific acrorhagial aggressive behavior among sea anemones: a recognition of self and not-self Biol. Bull. 159: 117-134. Bigger, C. H., and Runyan, R. 1979. An in situ demonstration of self-recognition in gorgonians. Dev. Comp. Immnol. 3: 591-597. Bigger, C. H., P. L. Jokiel, W. H. Hildeman, and I. S. Johnston. 1982. Characterization of alloimmune memory in a sponge. J. Immunol. 129: 1570-1572. BONNIN, J. P. 1964. Recherches sur la 'reaction d'aggression' et sur le fonctionnement des acorhages d' Actinia equina L. Bull. Biol. Fr. Belg. 1: 225-250. Brace, R. C. 198 1 . Intraspecific aggression in the colour morphs of the anemone Phymactis clematis from Chili. Mar. Biol. 64: 85-93. Brace, R. C, and J. Pavey. 1978. Size-dependent dominance hierarchy in the anemone Actinia equina. Nature 273: 752-753. Brace, R. C, J. Pavey, and D. L. J. Quickie. 1979. Intraspecific aggression in the color morphs of the anemone Actinia equina: the convention governing dominance ranking. Anim. Behav. 27: 553- 561. Burnet, M. 1970. Self and Not-Self. Cambridge Univ. Press, Cambridge. 318 pp. Buss, L. W. 1982. Somatic cell parasitism and the evolution of somatic tissue compatibility. Proc. Natl. Acad. Sci. U.S.A. 79: 5337-5341. Buss, L. W. 1983a. Evolution, development and the units of selection. Proc. Natl. Acad. Sci. U.S.A. 80: 1387-1391. Buss, L. W. 1983b. Somatic variation and evolution. Paleobiology. 9: 12-16. Calgren, O. 1929. Uber eine Actiniariengattung mit besonderen Fangtentakeln. Zool. Anz. 81: 109-1 13. Campbell, R. D., and David, C. N. 1974. Cell cycle kinetics and development oi Hydra attenuata. II. Interstitial cells. / Cell. Sci. 16: 349-358. Chiba, Y., and M. Kato. 1966. Interspecific relation in the colony formation among Bougainvillia sp. and Cladonema radiatum (Hydrozoa, Coelenterata). Sci. Rep. Tohoku Univ. Ser. IV (Biol.) 32: 201-206. Chornesky, E. a. 1983. Induced development of sweeper tentacles on the reef coral Agaricia agaricites: a response to direct competition. Biol. Bull. 165: 569-581. Crowell, S. 1950. Individual specificity in the fusion of hydroid stolons and the relationship between stolonic growth and colony growth. Anat. Rec. 108: 560-56 1 . David, C. N., and S. Murphy. 1977. Characteristics of interstitial stem cells in Hydra. Dev. Biol. 58: 372-383. DiEHL, F. A., AND A. Burnett. 1964. The role of interstitial cells in the maintenance of hydra. I. Specific destruction of interstitial cells in normal, asexual, non-budding animals. J. Exp. Zool. 155: 253- 260. DiEHL, F. A., AND A. Burnett. 1965a. The role of interstitial cells in the maintenance of hydra. II. Budding. J. Exp. Zool. 158: 283-298. DiEHL, F. A., AND A. Burnett. 1965b. The role of interstitial cells in the maintenance of hydra. III. Regeneration of hypostome and tentacles. J. Exp. Zool. 158: 299-318. Francis, L. 1973a. Clone specific segregation in the sea anemone Anthopleura elegantissima. Biol. Bull. 144: 64-72. Francis, L. 1973b. Interspecific aggression and its effects on the distribution oi Anthopleura elegantissima and some related sea anemones. Biol. Bull. 144: 73-92. Francis, L. 1976. Social organization within clones of the sea anemone Anthopleura elegantissima. Biol. Bull. 150: 361-376. Gallien, L., and M. C. Gouere. 1974. Incompatibilite entre cultures inergeneriques d'explants chez hydraires Hvdractinia echinata Fleming et Podocoryne carnea Sars. Comptes. r. hebd. Seane. Acad. Paris' Ser. D. 11%: 107-110. Glynn, P. W. 1976. Some physical and biological determinants of coral community structure in the eastern Pacific. Ecol. Mongr. 46: 431-456. Hand, C. 1955. The sea anemones of Central California, Part III. The acontiarian anemones. Wasmann J. Biol. 13: 189-251. den Hartog, J. C. 1977. The marginal tentacles of Rhodactis sanctithomae (Corallimorpharia) and the sweeper tentacles of Montastrea cavernosa (Scleractinia); their cnidom and possible function. Pp. 463-469 in Proc. Third. Int. Coral Reef Symp., D. L. Taylor, ed. Univ. of Miami Press. Coral Gables. Hauenschild, V. C. 1954. Genetische und entwichlungphysiologische Untersuchungen uber Intersexualitat und Gewebevertraglichkeit bei Hydractinia echinata Flem. Wilhelm RouxArch. Entwicklungsmech. Org. 147: 1-41. HYDROID COMPETITION AND HISTOCOMPATIBILITY 157 Hauenschild, v. C. 1956. Uber die Vererbung einer Gewebevertraglichkeits-Eigenschaft bei dem Hy- droidpolypen Hydractinia echinala. Z. Naturforsch. lib: 132-138. HiLDEMAN, W. H., D. S. LiNTHicuM, AND D. C. Vann. 1975. Transplantation and immunoincompatibility reactions among reef-building corals. Immunogenetics 2: 269-284. HiLDEMAN, W. H., R. L. Raison, G. Cheung, C. J. Hull, L. Akaka, and J. Okamoto. 1977a. Im- munological specificity and memory in a scleractinian coral. Nature 270: 219-223. HiLDEMAN, W. H., R. L. Raison, C. J. Hull, L. Akaka, J. Okumoto, and G. Cheung. 1977b. Tissue transplantation immunity in corals. Pp. 537-543 in Proc. Third Int. Coral Reef Symp.. D. L. Taylor, ed. Univ. of Miami Press, Coral Gables. HiLDEMAN, W. H., p. L. Jokiel, C. H. Bigger, AND I. S. JOHNSTON. 1980. Allogeneic polymorphism and alloimmune memory in the coral, Moniipora verrucosa. Transplantation 30: 297-301. IVKER, F. B. 1972. A hierarchy of histo-incompatibility in Hydractinia echinata. Biol. Bull. 143: 162-174. Karnovsky, M. J. 1965. A formaldehyde-gluteraldehyde fixative of high osmolality for use in electron microscopy. / Cell. Biol. 27: 137A-138A. Kato, M., K. Nakamura, E. Hirai, and Y. Kakinuma. 1962. Interspecific relation in the colony formation among some hydrozoan species. Bull. Mar. Bio. St. Asamushi. Tohoku Univ. 11: 31-36. Kato, M., E. Hirai, and Y. Kakinuma. 1963. Further experiments on the interspecific relation in the colony formation among some hydrozoan species. Sci. Rep. Tohoku Univ. Ser. IV (Biol.) 29: 317-325. Kato, M., E. Hirai, and Y. Kakinuma. 1967. Experiments on the coaction among hydrozoan species in the colony formation. Sci. Rep. Tohoku Univ. Ser. IV (Biol.) 33: 359-373. Kaye, H., and T. Ortiz. 1981. Strain specificity in a tropical marine sponge. Mar. Biol. 63: 165-173. Lang, J. C. 1971. Interspecific aggression by scleractinian corals I. the rediscovery of Scolymia cubensis (Milne Edwards and Haime). Bull. Mar. Sci. 21: 952-959. Lang, J. C. 1973. Interspecific aggression by scleractinian corals II. Why the race is not always to the swift. Bull. Mar. Sci. 23: 260-279. Lentz, T. L. 1966. The Cell Biology of Hydra. North-Holland Publ. Co., Amsterdam. 199 pp. Lubbock, R. 1979. Mucous antigenicity in sea anemones and corals. Hydrobiologia. 66: 3-6. Manning, M. 1980. Phylogeny of Immunological Memory. Elsevier, New York. 359 pp. McFadden, C. S., M. McFarland, and L. W. Buss. 1984. Biology of hydractiniid hydroids. I. Colony ontogeny in Hydractinia echinata. Bio. Bull. 166: 54-67. Mariscal, R. N. 1974. Nematocysts. Pp. 129-178 in Coelenterate Biology: Reviews and Perspectives, L. Muscatine and H. M. Lenhoff, eds. Academic Press, New York. Marcum, B. a., and R. D. Campbell. 1978. Development oi Hydra lacking nerve and interstitial cells. / Cell Sci. 29: 17-33. Muller, W. E. G. 1964. Experimentelle Untersuchungen uber Stockentwicklung, Polypendifferenzierung und sexual Chimaren bei Hvdractina echinata. Wilhelm Roux' Arch. Entwicklungsmech. Org. 155: 181-268. Muller, W. E. G. 1967. Differenzierungspotenzen und Geschlechtstabilitat der I-Zellen von Hydractinia echinala. Wilhelm Roux Arch. Entwicklungsmech. Org. 159:412-432. Muller, W. E. G. 1968. Elimination der I-Zellen durch alkylierende Cytostatika und deren Effekte auf die Embryonalentwicklung bei Hydractinia echinata. Exp. Cell. Res. 49: 448-458. Muller, W. E. G., A. Maidhof, R. K. Zahn, and I. Muller. 1983. Histocompatibility reactions in the hydrocoral Millepora dichotoma. Coral Reefs 1: 237-241. NiEUWKOOP, p. D., AND L. A. SuTASURYA. 1981. Primordial Germ Cells in the Invertebrates. Cambridge Univ. Press, Cambridge. Ottaway, J. R. 1978. Population ecology of the intertidal anemone Actinia tenebrosa. I. Pedal locomotion and intraspecific aggression. / Mar. Freshwater Res. 29: 787-802. PURCELL, J. E. 1977. Aggressive function and induced development of catch tentacles in the sea anemone Metridium senile (Coelenterata, Actiniaria). Biol. Bull. 153: 355-368. PURCELL, J. E., AND C. L. KITTING. 1982. Intraspecific aggression and population distribution of the sea anemone Metridium senile. Biol. Bull. 162: 345-359. Reynolds, E. S. 1963. The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. / Cell. Biol. 17:208-212. Richardson, C. A., P. Dustan, and J. C. Lang. 1979. Maintenance of the living space by sweeper tentacles of Montastrea cavernosa. Mar. Biol. 55: 181-186. Sammarco, p. W., J. C. Coll, S. LaBarre, and B. Willis. 1983. Competitive strategies of soft corals (Coelenterata:Octocorallia): allelopathic effects on selected scleractinian corals. Coral Reefs 1: 173-178. Schijfsma, K. 1939. Preliminary notes on early stages in the growth of colonies of Hydractinia echinata (Flem.). Arch. Neerland. Zool. 4: 93-102. 158 BUSS ET AL. Sheppard, C. R. C. 1979. Interspecific aggression between reef corals with reference to their distribution. Mar. Ecol. Prog. Ser. 1: 237-247. Tardent, p., and M. Buhrer. 1982. Intraspecific tissue incompatibilities in the metagenetical Podocoryne carnea M. Sars (Cnidaria:Hydrozoa). Pp. 295-303 in Embryonic Development, Part B. Cellular Aspects. M. M. Burger and R. Weber, eds. A. R. Liss, New York. Teissier, G. 1929. L'Origine multiple de certaines colonies d'Hydractinia echinata (Flem.) et ses consequences possibles. Bull. Soc. Zool. Fr. 54: 645-647. Theodor, J. L. 1970. Distinction between 'self and 'not-self in lower invertebrates. Nature 111: 690- 692. Theodor, J. L. 1976. Histocompatibility in a natural population of gorgonians. Zool. J. Linn. Soc. 58: 173-176. Theodor, J. L., and R. Senelar. 1975. Cytotoxic interaction between gorgonian explants: mode of action. Cell. Immunol. 19: 194-200. TOTH, S. E. 1967. Tissue compatibility in regenerating explant from the colonial marine hydroid Hydractinia echinata. J. Cell Physiol. 69: 125-131. Watson, G. M., and R. N. Mariscal. 1983. The development of a sea anemone tentacle specialized for aggression: morphogenesis and regression of the catch tentacle of Haliplanella luciae (Cnidaria, Anthozoa). Biol. Bull. 164: 506-517. Wellington, G. M. 1980. Reversal of digestive interactions between Pacific reef corals: mediation by sweeper tentacles. Oecologia 47: 340-343. Williams, R. B. 1975. Catch-tentacles in anemones: occurrence in Haliplanella luciae (Verill) and a review of current knowledge. / Nat. Hist. 9: 241-248. Williams, R. B. 1978. Some recent observations on the acrorhagi of sea anemones. J. Mar. Biol. Assoc. U.K. 58: 787-788. Williams, R. B. 1980. A further note on catch tentacles in anemones. Trans. Norfolk Norwich Nat. Soc. 25: 84-86. U) Reference: Biol Bull. 167: 159-167. (August, 1984) DISPERSAL OF ZOOXANTHELLAE ON CORAL REEFS BY PREDATORS ON CNIDARIANS GISELE MULLER PARKER* Department of Biology, University of California, Los Angeles. California 90024 Abstract Fish and nudibranchs prey on cnidarians that contain high densities of symbiotic dinoflagellates (zooxanthellae). Several fish {Arothron meleagris, Chaetodon auriga. and Chaetodon unimaculatus) and one nudibranch (Berghia major) feed on the Ha- waiian symbiotic sea anemone Aiptasia pulchella. 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 calcul