Volume 175 Number 1 THE BIOLOGICAL BULLETIN Marine Biological Laboratory LIBRARY AUGUST, 1988 Published by the Marine Biological Laboratory AUG 241988 Woods Hole, Mass. THE BIOLOGICAL BULLETIN PUBLISHED BY THE MARINE BIOLOGICAL LABORATORY Editorial Board GEORGE J. AUGUSTINE, University of Southern California RUSSELL F. DOOLITTLE, University of California at San Diego WILLIAM R. ECKBERG, Howard University ROBERT D. GOLDMAN, Northwestern University EVERETT PETER GREENBERG, Cornell University MICHAEL J. GREENBERG, C. V. Whitney Marine Laboratory, University of Florida JOHN E. HOBBIE, Marine Biological Laboratory LIONEL JAFFE, Marine Biological Laboratory HOLGER W. JANNASCH, Woods Hole Oceanographic Institution WILLIAM R. JEFFERY, University of Texas at Austin GEORGE M. LANGFORD, University of North Carolina at Chapel Hill Louis LEIBOVITZ, Marine Biological Laboratory GEORGE D. PAPPAS, University of Illinois at Chicago SIDNEY K.. PIERCE, University of Maryland RUDOLF A. RAFF, Indiana University HERBERT SCHUEL, State University of New York at Buffalo VIRGINIA L. SCOFTELD, University of California at Los Angeles School of Medicine LAWRENCE B. SLOBODKIN, State University of New York at Stony Brook KENSAL VAN HOLDE, Oregon State University DONALD P. WOLF, Oregon Regional Primate Center Editor: CHARLES B. METZ, University of Miami Associate Editor: PAMELA L. CLAPP, Marine Biological Laboratory AUGUST, 1988 Printed and Issued by LANCASTER PRESS, Inc. PRINCE & LEMON STS. LANCASTER, PA Marine Biological Laboratory LIBRARY AUG241988 Woods Hole, Mass. THE BIOLOGICAL BULLETIN THE BIOLOGICAL BULLETIN is published six limes 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 Bio- logical Laboratory, Woods Hole, Massachusetts. Single numbers, $20.00. Subscription per volume (three issues), $55.00 ($110.00 per year for six issues). Communications relative to manuscripts should be sent to Dr. Charles B. Metz. Editor, or Pamela Clapp, Associate Editor, at the Marine Biological Laboratory. Woods Hole, Massachusetts 02543. POSTMASTER: Send address changes to THE BIOLOGICAL BULLETIN, Marine Biological Laboratory, Woods Hole, MA 02543. Copyright '£) 1988, by the Marine Biological Laboratory Second-class postage paid at Woods Hole, MA, and additional mailing offices. ISSN 0006-3 185 INSTRUCTIONS TO AUTHORS The Biological Bulletin accepts outstanding original re- search reports of general interest to biologists throughout the world. Papers are usually of intermediate length (10-40 manu- script 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 accep- tance. 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Personal communications and mate- rial 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 LIT- ERATURE CITED, and must be typed double spaced on sepa- rate pages, conforming in punctuation and arrangement to the style of recent issues of The Biological Bulletin. Citations should include complete titles and inclusive pagination. Jour- nal abbreviations should normally follow those of the U. S. A. Standards Institute (USASI), as adopted by BIOLOGICAL AB- STRACTS and CHEMICAL ABSTRACTS, with the minor differ- ences set out below. The most generally useful list of biological journal titles is that published each year by BIOLOGICAL AB- STRACTS (BIOSIS List of Serials; the most recent issue). Foreign authors, and others who are accustomed to using THE WORLD LIST OF SCIENTIFIC PERIODICALS, may find a booklet pub- lished by the Biological Council of the U.K. (obtainable from the Institute of Biology, 41 Queen's Gate, London, S.W.7, En- gland. U.K.) useful, since it sets out the WORLD LIST abbrevi- ations for most biological journals with notes of the USASI ab- breviations where these differ. CHEMICAL ABSTRACTS pub- lishes quarterly supplements of additional abbreviations. The following points of reference style for THE BIOLOGICAL BULLE- TIN 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. A //abbreviated components must be followed by a pe- riod, whole word components must no! (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 Kit Visindafjelags Islendinga without abbreviation. F. All single word journal titles in full (e.g. Veliger, Ecol- ogy, 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 pre- ferred forms rather than WORLD LIST or USASI usage (e.g. Na- ture. Science. Evolution NOT Nature. Land., Science, N.Y.; Evolution, Lancaster, Pa.) 6. Reprints, charges. Authors will be charged the excess over $100 of the total of (a) $30 for each printed page beyond 1 5, (b) $30 for each table, (c) $ 1 5 for each formula more com- plex than a single line with simple subscripts or superscripts, and (d) $15 for each figure, with figures on a single plate all considered one figure and parts of a single figure on separate sheets considered separate figures. 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 for foreign authors) will receive page proofs of articles shortly be- fore publication. They will be charged the current cost of print- ers' time for corrections to these (other than corrections of printers' or editors' errors). Reference: Bio Bull. 175: 1-64. (August, 1988) THE MARINE BIOLOGICAL LABORATORY NINETIETH REPORT, FOR THE YEAR 1987 — ONE-HUNDREDTH YEAR I. II III. IV. V. VI. VII. VIII. IX. XI XII XIII Trustees and Standing Committees 1 Members of the Corporation 3 1 . Life Members 3 2. Regular Members 5 3. Associate Members 20 Certificate of Organization 23 Articles of Amendment 24 Bylaws 24 Report of the Director 28 Report of the Treasurer 30 Report of the Librarian 39 Educational Programs 40 1 . Summer 40 2. Spring 46 3. Short Courses 46 X. Research and Training Programs 48 1 . Summer 48 2. Year-Round 53 Honors 58 Institutions Represented 60 Laboratory Support Staff 63 I. Trustees Including Action of the 1987 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, 200 Ocean Lane, Key Biscayne, FL 33 149 Robert Manz, Treasurer, Helmer and Associates, Wes- ton, MA 02 193 Harlyn O. Halvorson, President of the Corporation and Director, Marine Biological Laboratory, Woods Hole, MA 02543 David D. Potter, Clerk, Harvard Medical School, Cam- bridge, MA 02 1 38 Emeriti John B. Buck, National Institutes of Health Aurin Chase, Princeton University George H. A. Clowes Jr., The Cancer Research Institute Seymour S. Cohen, Woods Hole, MA Arthur L. Colwin, Key Biscayne, FL Laura Hunter Colwin, Key Biscayne, FL D. Eugene Copeland, Marine Biological Laboratory Sears Crowell, Indiana University Alexander T. Daignault, Boston, MA Teru Hayashi, Miami, FL Hope Hibbard, Oberlin College (deceased 5/1 1/88) Lewis Kleinholz, Reed College Maurice Krahl, Tucson, AZ Charles B. Metz, University of Miami Keith Porter, University of Maryland C. Ladd Prosser, University of Illinois John S. Rankin, Ashford, CT (deceased 12/12/87) S. Meryl Rose, Waquoit, MA John Saunders Jr., Waquoit, MA George T. Scott, Woods Hole, MA (deceased 9/18/87) Mary Sears, Woods Hole, MA Homer P. Smith, Woods Hole, MA Carl C. Speidel, University of Virginia (deceased 1982) W. Randolph Taylor, University of Michigan George Wald, Woods Hole, MA Class of 1991 Robert B. Barlow Jr., Syracuse University James M. Clark, Edna McConnell Clark Foundation Laszlo Lorand, Northwestern University Lionel I. Rebhun, University of Virginia Carol L. Reinisch, Tufts University School of Veterinary Medicine Brian M. Salzberg, University of Pennsylvania Howard A. Schneiderman, Monsanto Company Sheldon J. Segal, The Rockefeller Foundation Class of 1990 John E. Dowling, Harvard University Gerald D. Fischbach, Washington University School of Medicine Robert D. Goldman, Northwestern University John E. Hobbie, Marine Biological Laboratory Richard E. Kendall, Massachusetts Governor's Office Irving W. Rabb, Boston, Massachusetts 1 \1\R1NE BIOLOGICAL LABORATORY Joan V. Ruderman, Duke Um\ersit\ Ann K. Stuart, University of North Carolina I). Thomas Trigg, Wellesley. MA Class of 1989 Garland E. Allen, Washington University Peter B. Armstrong, University of California, Davis Robert \V. Ashton, Gaston Snow Beekman and Bogue Jt-lle Atema, Marine Biological Laboratory John G. Hildebrand, University of Arizona Thomas J. Hynes Jr., Meredith and Grew, Inc. Robert Mainer, The Boston Company Birgit Rose, University of Miami Gerald Weissmann, New York University Class of 1988 Clay M. Armstrong, University of Pennsylvania Joel P. Davis, Seapuit, Inc. Ellen R. Grass, The Grass Foundation Judith P. Grassle, Marine Biological Laboratory Holger \V. Jannasch, Woods Hole Oceanographic Institution George M. Langford, University of North Carolina Andrew Szent-Gyorgyi, Brandeis University Kensal E. Van Holde, Oregon State University Stanley \V. Watson, Woods Hole Oceanographic Institution Standing Committees Executive Committee of the Board of Trustees Prosser Clifford* Harlyn O. Halvorson* Ray L. Epstein* Robert Man/* John E. Dowling. 1990 Gerald D. Fischbach. 1989 John G. Hildebrand, 1988 Sheldon J. Segal, 1989 Andrew Szent-Gyorgyi, 1988 D. Thomas Trigg, 1990 Animal Care Commitee ( 'anil I . Keinisch. Chairman Ray L. Epstein* I i tula Huffer* Edward Jaskm Andrew H. Mattox* Roxanna Smolowitz Felix Strumwasser J. Richard Whittaker Buildings and Grounds Committee Kenyon S. Twcedell. ( 'hairman Lawrence B ( olu-n * ex-officio Richard D. Cutler* Alan Fein Daniel L. Gilherl Cifford V.Harding Jr. Ferenc I. Harosi Donald B. Leln* Thomas H. Meedel Philip Person Lionel I. Rebhun Thomas S. Reese Evelyn Spiegel Employee Relations Committee John V. K. Helfrich, Chairman Judith Ashmore Florence Dwane Edward Enos William A. Evans John B. MacLeod Fellowships Committee Thoru Pederson. Chairman Judith P. Grassle John Ci. Hildebrand George M. Langford Eduardo Macagno Carol L. Reinisch Housing, Food Service and Day Care Committee Jelle Atema, Chairman Robert B. Barlow Jr. Gail D. Burd LouAnn King* Thomas S. Reese Joan V. Ruderman Ann E. Stuart Institutional Biosafety Raymond E. Stephens. Chairman Paul J. DeWeer Paul T. England Harlyn O. Halvorson* Paul Lee Donald B. Lehy* Joseph Martyna Andrew H. Mattox* Al Senft Instruction Committee Judith P. Grassle, Chairman Ray L. Epstein* Brian Fry John G. Hildebrand Hans Laufer Joan V. Ruderman Brian M. Salzberg Roger D. Sloboda Andrew Szent-Gyorgyi Investment Committee D. Thomas Trigg, ( 'hairman Prosser Gifford* William I. Golden Maurice Lazarus Robert Manz* John W. Speer* W. Nicholas I'horndike Library Joint Management Committee Harlyn O. Halvorson, Chairman* Gurland E. Allen George D. Grice John W. Speer* John II. Steele Library Joint Users Committee Garland L. Allen. ( 'hairman Wilfred B. Bryan A. Farmanfarmaian TRUSTEES AND STANDING COMMITTEES Jane Fessenden* Lionel F. Jafte Laurence P. Madin John Schlee Frederic Serchuk Oliver C. Zafiriou Investment Committee Marine Resources Committee Robert D. Goldman, Chairman William D. Cohen Richard D. Cutler* Louis Leibovitz Toshio Narahashi George D. Pappas Rober D. Sloboda Melvin Spiegel Antoinette Steinacher John Valois* Radiation Safety Committee PaulJ. DeWeer, Chairman Richard L. Chappell Sherwin J. Cooperstein Daniel S. Grosch Louis M. Kerr* Andrew H. Mattox* Harris Ripps WalterS. Vincent Research Services Committee Birgit Rose, Chairman Peter B. Armstrong Robert B. Barlow Jr. Richard D. Cutler* Ray L. Epstein* Barbara Ehrlich John G. Hildebrand Laurinda Jaffe Samuel S. Koide Andrew H. Mattox* Bryan Noe Joel Rosenbaum Rudi Strickler Research Space Committee Joseph Sanger, Chairman Clay M. Armstrong David Landowne Hans Laufer Laszlo Lorand Eduardo Macagno Jerry Melillo Roger D. Sloboda Evelyn Spiegel Steven Treistman Ivan Valiela Safety Committee John E. Hobbie, Chairman Daniel L. Alkon D. Eugene Copeland Richard D. Cutler* Edward Enos* Alan Fein Louis M. Kerr* Alan M. Kuzirian Donald B. Lehy* Andrew H. Mattox* Edward A. Sadowski* Paul A. Steudler Trustees' Committees Audit Committee Robert Mainer, Chairman Robert Manz* * ex-officio Sheldon J. Segal D. Thomas Trigg Kensal E. Van Holde D. Thomas Trigg, Chairman William T. Golden Maurice Lazarus Robert Manz* W. Nicholas Thorndike Compensation Committee Thomas J. Hynes Jr., Chairman James M. Clark John E. Dowling Irving W. Rabb Committee on Laboratory Goals Gerald D. Fischbach, Chairman Michael V. L. Bennett Harlyn O. Halvorson John G. Hildebrand John E. Hobbie David D. Potter Joan V. Ruderman J. Richard Whittaker Centennial Committee James D. Ebert, Chairman Pamela Clapp, Coordinator* Garland E. Allen Robert B. Barlow, Jr. Paul R. Gross Harlyn O. Halvorson" Olivann Hobbie Richard E. Kendall John Pfeiffer Keith Porter Frank Press C. Ladd Prosser John S. Reed D. Thomas Trigg John Valois II. Members of the Corporation Including Action of the 1987 Annual Meeting Life Members Abbott, Marie, c/o Vaughn Abbott, Flyer Rd., East Hart- land, CT 06027 Adolph, Edward F., University of Rochester, School of Medicine and Dentistry, Rochester, NY 14642 Beams, Harold W., Department of Biology, University of Iowa, Iowa City, IA 53342 Behre, Ellinor, Black Mountain, NC 287 1 1 Bernheimer, Alan W., Department of Microbiology, New York University Medical Center, 550 First Ave., New York, NY 10016 Bertholf, Lloyd M., Westminster Village #2114, 2025 E. Lincoln St., Bloomington, 1L 6 1701 M -\RINF BIOLOGICAL LABORATORY Bishop, David \V., Department of Physiology, Medical College of Ohio. C. S. 10008. Toledo, OH 43699 Bold, Harold C., Department of Botany, University of Texas, Austin. TX 787 12 Bridgman, A. Josephine, 715 Kirk Rd., Decatur, GA 30030 Buck, John B., NIH, Laboratory of Physical Biology, Room 1 1 2, Building 6 Bethesda. MD 20892 Burbanck, Madeline P., Box 1 5 1 34. Atlanta, GA 30333 Burbanck, William D., Box 1 5 1 34, Atlanta, GA 30333 Carpenter, Russell, L., 60-H Lake St., Winchester, MA 01890 Chase, Aurin, Professor of Biology Emeritus, Princeton University, Princeton, NJ 08544 Clark, Arnold M., 48 Wilson Rd., Woods Hole, MA 02543 Clarke, George L., Address unknown (deceased 8/23/ 87) Cohen, Seymour S., 10 Carrot Hill Rd., Woods Hole, MA 02543 Colwin, Arthur, 320 Woodcrest Rd.. Key Biscayne, FL 33149 Colwin, Laura Hunter, 320 Woodcrest, Key Biscayne, FL 33149 Copeland, D. E., 41 Fern Lane, Woods Hole, MA 02543 Costello, Helen M., Carolina Meadows, Villa 137, Chapel Hill, NC 275 14 C'rouse, Helen, Institute of Molecular Biophysics, Flor- ida State University, Tallahassee, FL 32306 Diller, Irene C., Rydal Park, Apartment 660, Rydal, PA 19046 (deceased 2/88) Elliott, Alfred M., 428 Lely Palm Ext., Naples, FL 33962-8903 (deceased 1/20/88) Failla, Patricia M., 2149 Loblolly Lane, Johns Island. SC 29455 Ferguson, Frederick P., National Institute of General Medical Science. NIH, Bethesda, MD 20892 Ferguson, James K. W., 56 Clarkehaven St., Thornhill. Ontario L4J 2B4 CANADA Fries, Erik F. B., 4 1 High Street, Woods Hole, Ma 02543 (iilman, Lauren C., Department of Biology, University of Miami, PO Box 249 1 8, Coral Gables, FL 33 1 24 (de- ceased 12/87) Graham, Herbert, 36 Wilson Rd., Woods Hole, MA 02543 Green, James VV., 409 Grand Ave., Highland Park, NJ 08904 Grosch, Daniel S., Department of Genetics, Gardner Hall, North Carolina State University. Raleigh, NC 27607 Hamburger, Viktor, Professor Emeritus. Washington University, St. Louis, MO 63 1 30 Hamilton, Howard I.., Department of Biology, Univer- sity of Virginia, Charlottesville. VA 22901 Ilibbard, Hope, c/o Jeanne Stephens. 374 Morgan St.. Oberlin. OH 44074 (deceased 5/1 1/88) Hisaw, F. L., 5925 SW Plymouth Drive, Corvallis. OR 97330 Hollaender, Alexander, Council for Research Planning, 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 \V., Royal Megansett Nursing Home. Room 205, P. O. Box 408, N. Falmouth, MA 02556 Karush, Fred, Department of Microbiology, University of Pennsylvania School of Medicine. Philadelphia, PA 19104 Kille, Frank R., 1111 S. Lakemont Ave. #444. Winter Park, FL 32792 Kingsbury, John M., Department of Botany, Cornell University, Ithaca, NY 14853 Kleinholz, Lewis, Department of Biology, Reed College, Portland, OR 97202 Lauffer, Max A., Department of Biophysics, University of Pittsburgh, Pittsburgh, PA 15260 LeFevre, Paul G., 15 Agassiz Road, Woods Hole, MA 02543 Levine, Rachmiel, 2024 Canyon Rd., Arcadia, CA 9 1006 Lochhead, John H., 49 Woodlawn Rd., London SW6 6PS, England, U. K. Lynn, VV. Gardner, Department of Biology, Catholic University of America, Washington. DC 200 1 7 Magruder, Samuel R., 270 Cedar Lane. Paducah, KY 42001 Manwell, Reginald, D., Syracuse University. Lyman Hall. Syracuse, NY 13210 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 California, Riverside, CA 9252 1 Moul, E. T., 43 F. R. Lillie Rd., Woods Hole, MA 02543 Nace, Paul F., 5 Bowditch Road, Woods Hole, MA 02543 Page, Irving H., Box 516, Hyannisport, MA 02647 Pollister, A. W., 313 Broad Street, Harleysville, PA 19438 Prosser, C. Ladd, Department of Physiology and Bio- physics, Burrill Hall 524, University of Illinois, Ur- bana. IL61801 Provasoli, Luigi, Haskins Laboratories, 165 Prospect Street, New Haven, CT 065 10 Pryt/., Margaret McDonald, 21 McCouns Lane, Oyster Bay, NY 11771 MEMBERS OF THE CORPORATION Rankin, John S., Jr., Box 97, Ashford, CT 06278 (de- ceased 12/12/87) Renn, Charles E., Route 2, Hempstead, MD 2 1074 Richards, A. Glenn, 942 Cromwell Ave., St. Paul, MN 55114 Richards, Oscar W., Pacific University, Forest Grove, OR 97462 Rockstein, Morris, 8045 SW 107 Ave., #201, Miami, FL 33173 Ronkin, Raphael R., 3212 McKinley St.. NW, Washing- ton, DC 200 15 Sanders, Howard, Woods Hole Oceanographic Institu- tion, Woods Hole, MA 02543 Scharrer, Berta, Department of Anatomy, Albert Ein- stein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461 Schlesinger, R. Walter, University of Medicine and Dentistry of New Jersey, Department of Microbiol- ogy, Rutgers Medical School, P. O. Box 101, Piscata- way, NJ 08854 Schmitt, F. O., Room 16-5 1 2, Massachusetts Institute of Technology, Cambridge, MA 02 1 39 Scott, Allan C, 1 Nudd St., Waterville, ME 04901 Scott, George T., 10 Orchard St., Woods Hole, MA 02543 (deceased 9/1 7/87) slii-miii. David, 33 Lawrence Farm Rd., Woods Hole, MA 02543 Smith, Homer P., 8 Quissett Ave., Woods Hole, MA 02543 Smith, Paul F., P. O. Box 264, Woods Hole, MA 02543 Sonnenblick, B. P., 91 Chestnut St., Millburn, NJ 07041 Speidel, Carl C., 1873 Field Rd., Charlottesville, VA 22903 (deceased 1982) 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, Robert E., 20 Harbor Hill Rd., Woods Hole, MA 02543 Taylor, W. Randolph, Department of Biology, Univer- sity of Michigan, Ann Arbor, MI 48109 Taylor, W. Rowland, 152 Cedar Park Road, Annapolis, MD21401 TeWinkel, Lois E., 4 Sanderson Ave., Northampton, MA 01060 Trager, William, The Rockefeller University, 1 230 York Ave., New York, NY 10021 Wainio, Walter W., 331 State Rd., Princeton, NJ 08540 (deceased 12/87) Wald, George, 21 Lakeview Ave., Cambridge, MA 02138 Waterman, T. H., Yale University, Biology Department, Box 6666, 610 Kline Biology Tower, New Haven, CT 06510 Weiss, Paul A., Address unknown Wichterman, Ralph, 31 Buzzards Bay Ave., Woods Hole, MA 02543 Wiercinski, Floyd J., Department of Biology, North- western Illinois University, Chicago, IL 60625 Wilber, Charles G., Department of Zoology, Colorado State University. Fort Collins, CO 80523 Young, D. B., 1 137 Main St., N. Hanover, MA 02339 Zinn, Donald J., P. O. Box 589, Falmouth, MA 02541 Zorzoli, Anita, 18 Wilbur Blvd., Poughkeepsie, NY 12603 Zweifach, Benjamin W., c/o Ames, University of Califor- nia. La Jolla, CA 92037 Regular Members Ache, Barry W., Whitney Marine Laboratory, Univer- sity of Florida, Rt. 1 Box 1 21, St. Augustine, FL 32086 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 065 10 Adelman, William J., Jr., NIH, Bldg. 9. Rm. IE- 127, Bethesda, MD 20892 Afzelius, Bjorn, Wenner-Gren Institute, University of Stockholm, Stockholm, Sweden Alberte, Randall S., Oceanic Biology Program, Code 1 122B, Office of Naval Research, 800 North Quincy St., Arlington, VA 22217-5000 Alkon, Daniel, Laboratory of Cellular and Molecular Neurobiology, NINDDS/NIH, Bldg. 5, Rm. 435, Bethesda, MD 20892 Allen, Garland E., Department of Biology, Washington University, St. Louis, MO 63130 Allen, Nina S., Department of Biology, Wake Forest University, Box 7325, Reynolds Station, Winston- Salem,NC27109 Allen, Suzanne T., Department of Medicine, Worcester Memorial Hospital 1 19 Belmont St., Worcester, MA 01605 Amatniek, Ernest, 4797 Boston Post Rd., Pelham Manor, NY 10803 Anderson, Everett, Department of Anatomy, LHRBB, Harvard Medical School, 45 Shattuck St., Boston, MA 02115 Anderson, J. M., 1 10 Roat St., Ithaca, NY 14850 Armet-Kibel, Christine, Biology Department, University of Massachusetts- Boston, Boston, MA 02 125 MARINE BIOLOGICAL LABORATORY Armstrong, Clay M.. Department of Physiology, Medi- cal School. University of Pennsylvania. Philadelphia. PA 19174 Armstrong. Peter B., Department of Zoology, Univer- sity of California. Davis. CA 95616 Arnold, John M., Pacific Biomedical Research Center. 209 Snyder Hall. 2538 The Mall. Honolulu. HI 96822 Arnold. William A., 102 Balsam Rd.. Oak Ridge. TN 37830 Ashton, Robert \\ ., Gaston Snow Beekman and Bogue, 14 Wall St.. Suite 1600 New York, NY 10005 Atema, Jelle, Marine Biological Laboratory, Woods Hole. MA 02543 Atwood. Kimball C, P. O. Box 673, Woods Hole. MA 02543 Augustine, George J., Section of Neurobiology, Depart- ment of Biological Sciences, University of Southern California, Los Angeles, CA 90089-0371 Austin, Mary L., 506 1/2 N. Indiana Ave., Bloomington. IN 47401 Ayers, Donald E., Marine Biological Laboratory, Woods "Hole. MA 02543 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 Bang, Betsy, 76 F. R. Lillie Rd.. Woods Hole, MA 02543 Barlow, Robert B., Jr., Institute for Sensory Research, Syracuse University, Merrill Lane, Syracuse, NY 13210 Barry, Daniel T., Department of Physical Medicine and Rehabilitation, ID204, University of Michigan Hospi- tal. Ann Arbor. MI 48109-0042 Barry, Susan R., Department of Physical Medicine and Rehabilitation, ID204, University of Michigan Hospi- tal, Ann Arbor, MI 48 109-0042 Bartell, Clelmer K., 2000 Lake Shore Drive, New Or- leans, LA 70122 Bartlett, James H., Department of Physics, Box 1921, University of Alabama, Tuscaloosa, AL 35489 Bass, Andrew H., Seely Mudd Hall, Department of Neu- robiology and Behavior, Cornell University, Ithaca. NY 14853 Battelle, Barbara-Anne, Whitney Laboratory, Rt. 1, Box 1 21. St. Augustine, FL 32086 Bauer, G. Kric, Department of Anatomy, University of Minnesota, Minneapolis, MN 55455 Beauge, Luis Alberto, Institute de Investigacion Medica, Casilla de Correo 389, 5000 Cordoba, Argentina Beck, L. V., School of Experimental Medicine, Depart- ment of Pharmacology, Indiana University, Bloom- ington, IN 47401 Begenisich, Ted, Department of Physiology. University of Rochester, Rochester, NY 14642 Begg, David A., LHRRB. Harvard Medical School. 45 Shattuck St.. Boston, MA 02 1 1 5 Bell, Eugene, Organogenesis, Inc.. 83 Rogers St., Cam- bridge, MA 02 142 Benjamin, Thomas L., Department of Pathology, Har- vard Medical School, 25 Shattuck St., Boston, MA 02115 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 Col- lege, Waterville, ME 04901 Berg, Carl J., Jr., Bureau of Marine Research, 13365 Overseas Highway, Marathon, FL 33050 Berne, Robert M., University of Virginia, School of Medicine, Charlottesville, VA 22908 Bezanilla, Francisco, Department of Physiology. Uni- versity of California, Los Angeles, CA 90052 Biggers, John D., Department of Physiology, Harvard Medical School, Boston, MA 02 1 1 5 Bishop, Stephen H., Department of Zoology, Iowa State University, Ames, IA 50010 Blaustein, Mordecai P., Department of Physiology, School of Medicine, University of Maryland, 655 W. Baltimore Street, Baltimore, MD 21201 Bloom, Kerry S., Department of Biology, University of North Carolina, Chapel Hill, NC 275 14 Bodian, David, Address unknown Bodznick, David A., Department of Biology, Wesleyan University, Middletown, CT 06457 Boettiger, Edward G., 29 Juniper Point. Woods Hole, MA 02543 Boolootian, Richard A., Science Software Systems, Inc., 3576 WoodclirTRd.. Sherman Oaks, CA 9 1403 Borei, Hans G., Long Cove, Stanley Point Road, Min- turn, ME 04659 Borgese, Thomas A., Department of Biology, Lehman College, CUNY, Bronx, NY 10468 Borisy, Gary G., Laboratory of Molecular Biology, Uni- versity of Wisconsin, Madison, WI 53706 Borst, David W., Jr., Department of Biological Sciences, Illinois State University, Normal, IL 61761 Bosch, Herman F., 17 Damon Drive, Falmouth, MA 02540 Bowles, Francis P., P. O. Box 674, Woods Hole, MA 02543 Boyer, Barbara C., Department of Biology, Union Col- lege, Schenectady, NY 12308 Brandhorst, Bruce P., Biology Department, McGill Uni- versity, 1205 Avenue Dr. Pcnfield, Montreal, P. Q., CANADA H3A 1B1 MEMBERS OF THE CORPORATION Brehm, Paul, Department of Physiology, Tufts Medical School, Boston, MA 02 1 1 1 Brinley, F. J., Neurological Disorders Program, NIN- CDS, 812 Federal Building, Bethesda, MD 20892 Brown, Joel E., Department of Ophthalmology, Box 8096 Sciences Center, Washington University, 660 S. Euclid Ave., St. Louis, MO 631 10 Brown, Stephen C., Department of Biological Sciences, SUNY, Albany, NY 12222 Burd, Gail Deerin, Department of Molecular and Cellu- lar Biology, Biosciences West, Room 305, University of Arizona, Tucson, AZ 85721 Burdick, Carolyn J., Department of Biology, Brooklyn College, Brooklyn, NY 1 1210 Burger, Max, Department of Biochemistry, Biocenter, Klingelbergstrasse 70, CH-4056 Basel, Switzerland Burky, Albert, Department of Biology, University of Dayton, Dayton, OH 45469 Burstyn, Harold Lewis, 216 Bradford Parkway, Syra- cuse, NY 13224 Bursztajn, Sherry, Neurology Department — Program in Neuroscience, Baylor College of Medicine, Houston, TX 77030 Bush, Louise, 7 Snapper Lane, Falmouth, MA 02540 Calabrese, Ronald L., Department of Biology, Emory University, 1555 Pierce Drive, Atlanta, GA 30322 Candelas, Graciela C., Department of Biology, Univer- sity of Puerto Rico, Rio Piedras, PR 0093 1 Carew, Thomas J., Department of Psychology, Yale University, P. O. Box 1 1 A, Yale Station, New Haven, CT06520 Cariello, Lucio, Stazione Zoologica, Villa Comunale, Naples, ITALY Carlson, Francis D., Department of Biophysics, Johns Hopkins University, Baltimore, MD 21218 Carriere, Rita M., Department of Anatomy, Box 5, SUNY, Brooklyn, 450 Clarkson Ave., Brooklyn, NY 11203 Case, James, Department of Biological Sciences, Uni- versity of California, Santa Barbara, CA 93106 Cassidy, Rev. J. D., St. Rose Priory, Springfield, KY 40069 Cebra, John J., Department of Biology, Leidy Labs, G- 6, University of Pennsylvania, Philadelphia, Pa 19174 Chaet, Alfred B., University of West Florida, Pensacola, FL 32504 Chambers, Fdward L., Department of Physiology and Biophysics, University of Miami, School of Medicine, P. O. Box 016430, Miami, FL 33101 Chang, Donald C., Department of Physiology and Mo- lecular Biophysics, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030 Chappell, Richard L., Department of Biological Sci- ences, Hunter College, Box 67, 695 Park Ave., New York, NY 10021 Chauncey, Howard H., 30 Falmouth St., Wellesley Hills, MA 02181 Charlton, Milton P., Physiology Department MSB, Uni- versity of Toronto, Toronto, Ontario, Canada M5S 1A8 Child, Frank M., Department of Biology, Trinity Col- lege, Hartford, CT 06106 Chisholm, Rex L., Department of Cell Biology and Anatomy, Northwestern University Medical School, 303 E. Chicago Avenue, Chicago, IL 606 1 1 Citkowitz, Elena, 410 Livingston St., New Haven, CT 06511 Clark, Eloise E., Vice President for Academic Affairs, Bowling Green State University, Bowling Green, OH 43403 Clark, Hays, Property Management Ltd., 125 Mason St.. Greenwich, CT 06830 Clark, James M., Shearson Lehman Brothers Inc., 14 Wall St., 9th Floor, New York, NY 10005 Clark, Wallis H., Jr., Bodega Marine Lab, P. O. Box 247, Bodega Bay, CA 94923 Claude, Philippa, Primate Center, Capitol Court, Madi- son, WI 53706 Clay, John R., Laboratory of Biophysics, NIH, Building 9, room 1 E- 1 27, Bethesda. MD 20892 Clowes, George H. A., Jr., The Cancer Research Insti- tute, 1 94 Pilgrim Rd., Boston, MA 022 1 5 Clutter, Mary, Office of the Director, Room 5 1 8, Na- tional Science Foundation, Washington, DC 20550 Cobb, Jewel Plummer, California State University, Ful- lerton, CA 92634 Cohen, Adolph I., Department of Ophthalmology, School of Medicine, Washington University, 660 S. Euclid Ave., St. Louis, MO 631 10 Cohen, Carolyn, Roisenstiel Basic Medical Sciences Re- search Center, Brandeis University, Waltham, MA 02254 Cohen, Lawrence B., Department of Physiology, Yale University School of Medicine, B-106 SHM, P. O. Box 3333, New Haven, CT 065 10-8026 Cohen, Maynard, Department of Neurological Sciences, Rush Medical College, 600 South Paulina, Chicago, IL60612 Cohen, Rochelle S., Department of Anatomy, Univer- sity of Illinois at Chicago, 808 S. Wood Street, Chi- cago, IL 606 12 Cohen, William D., Department of Biological Sciences, Hunter College, 695 Park Ave., New York, NY 1002 1 Cole, Jonathan J., Institute for Ecosystems Studies, Cary Arboretum, Millbrook, NY 12545 (resigned 3/7/88) Coleman, Annette W., Division of Biology and Medi- cine, Brown University, Providence, RI 02912 MARINE BIOLOGICAL LABORATORY Collier. Jack R.. Department of Biology, Brooklyn Col- lege. Brooklyn. NY 11210 Collier. Marjorie McCann, Biology Department. Saint Peter's College. Kennedy Boulevard. Jersey City. NJ 07306 Cook. Joseph A.. The Edna McConnell Clark Founda- tion. 250 Park Ave.. New York. NY 10017 Cooperstein. S. J., University of Connecticut, School of Medicine. Farmington Ave.. Farmington. CT 06032 Corliss. John O.. Department of Zoology. University of Mary land. College Park. MD 20742 Cornell. Neal \V., 6428 Bannockburn Drive. Bethesda. MD20817 Cornwall. Melvin C.. Jr.. Department of Physiology L714. Boston University School of Medicine. 80 E. Concord St.. Boston. MA 02 1 1 8 Corson. Da>id Wesley, Jr., 1034 Plantation Lane, Mt. Pleasant, SC 29464 Corwin. Jeffrey T., Bekesy Lab of Neurobiology, 1993 East- West Road. University of Hawaii, Honolulu. HI 96822 Costello. Walter J.. College of Medicine. Ohio Univer- sity. Athens. OH 45701 Couch, Ernest F., Department of Biology. Texas Chris- tian University. Fort Worth. TX 76 1 29 Cremer-Bartels, Gertrud, Universitats Augenklinik, 44 Munster. West Germany Crow. Terry J.. Department of Physiology. University of Pittsburgh. School of Medicine. Pittsburgh, PA 15261 Crowell, Sears, Department of Biology. Indiana Univer- sity. Bloomington. IN 47405 Crowther, Robert. Marine Biological Laboratory. Woods Hole. MA 02543 Currier. David L., P. O. Box 2476. Vineyard Haven. MA 02568 Daignault. Alexander T., 280 Beacon St.. Boston. MA 02116 Dan. Katsuma, Tokyo Metropolitan University. Meg- uro-ku. Tokyo. Japan D'Avanzo, Charlene, School of Natural Science. Hamp- shire College. Amherst, MA 0 1 002 Daud, John R.. Seeley G. Mudd Building. Room 504, Harvard Medical School, 250 Longwood Ave., Bos- ton, MA 021 15 Da\idson, Eric H., Division of Biology. California Insti- tute of Technology Pasadena. CA 91 125 Davis. Bernard D., 23 Clairemont Road. Belmont. MA 02178 Davis, Joel P., Seapuit. Inc.. P. O. Box G. Osterville, MA 02655 Daw, Nigel W., 78 Aberdeen Place. Clayton, MO 63 105 DeGroof, Robert C., Squibb Mark, 105 Carnegie Center, Princeton. NJ 08543 DeHaan. Robert L.. Department of Anatomy, Emory University. Atlanta. GA 30322 DeLanney, Louis E., Institute for MedicaJ Research. 2260 Clove Drive. San Jose. CA 95128 DePhillips, Henry A., Jr., Department of Chemistry, Tnnity College. Hartford, CT 06106 DeTerra, Noel, 2 1 5 East 1 5th St.. New York, NY 10003 Dettbarn. Wolf-Dietrich. Department of Pharmacology. School of Medicine. Vanderbilt University. Nashville. TN37127 DeWeer. Paul J., Department of Physiology. School of Medicine. Washington University. St. Louis, MO 63110 Dixon, Keith E., School of Biological Sciences, Flinders University. Bedford Park. South Australia Donelson, John E., Department of Biochemistry, Uni- versity of Iowa. Iowa City IA 52242 Dowdall. Michael J., Department of Zoology. School of Biological Sciences. University of Nottingham, Uni- versity Park. Nottingham N672 UH. England. UK Dow ling, John E., The Biological Laboratories, Harvard University. 16 Divinity St.. Cambridge. MA 02 1 38 DuBois. Arthur Brooks, John B. Pierce Foundation Lab- oratory. 290 Congress Ave., New Haven, CT 065 19 Dudley. Patricia L., Department of Biological Sciences. Barnard College. Columbia University, New York, NY 10027 Duncan. Thomas K., P. O. Box 662. Woods Hole, MA 02543 Dunham, Philip B., Department of Biology, Syracuse University. Syracuse, NY 13244 Dunlap, Kathleen. Department of Psychology. Tufts Medical School. Boston, MA 02 1 1 1 Ebert, James D., Office of the Director, Chesapeake Bay Institute. The Johns Hopkins University, Suite 340. The Rotunda. 771 West 40th St.. Baltimore, MD 21211 Eckberg, William R., Department of Zoology, Howard University, Washington, DC 20059 Edds, Kenneth T., Department of Anatomical Sciences. SUNY, Buffalo, NY 14214 Eder, Howard A., Albert Einstein College of Medicine. 1300 Morris Park Ave., Bronx. NY 10461 Edstrom, Joan E., 2515 Milton Hills Drive, Charlottes- ville.VA 22901 Edwards, Charles, Rm. 403, Bldg. 10, NIADDK/NIH. Bethesda, MD 20892 Egyud, Laszlo G., 18 Skyview. Newton, MA 02150 Ehrenstein, Gerald, NIH. Bethesda, MD 20892 Khrlich, Barbara E., Division of Cardiology, University of Connecticut Health Center, Farmington, CT 06032 Eisen, Arthur Z., Division of Dermatology. Washington University, St. Louis. MO 63 1 10 MEMBERS OF THE CORPORATION Eisenman, George, Department of Physiology, Univer- sity of California Medical School. Los Angeles. CA 90024 Elder, Hugh Young, Institute of Physiology. University of Glasgow. Glasgow. Scotland. U. K. Elliott, Gerald F., The Open University Research Unit. Foxcombe Hall. Berkeley Rd.. Boars Hill. Oxford. En- gland. UK Englund, Paul T., Department of Biological Chemistry. Johns Hopkins School of Medicine. Baltimore. MD 21205 Epel, David, Hopkins Marine Station. Pacific Grove. CA 93950 Epstein, Herman T., Department of Biology. Brandeis University. Waltham, MA 02254 Epstein, Ray L., Marine Biological Laboratory. Woods Hole. MA 02543 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 Farb, David H., SUNY Health Science Center. Brook- lyn. NY 11203 Farmanfarmaian. A., Department of Biological Sciences. Nelson Biological Laboratory. Rutgers University. P. O. Box 1059. Piscataway. NJ 08854 Fein, Alan, Physiology Department. University of Con- necticut Health Center. Farmington. CT 06032 Feinman, Richard D., Box 8. Department of Biochemis- try. SUNY Health Science Center. Brooklyn, NY 11203 Feldman, Susan C.. Department of Anatomy. University of Medicine and Dentistry of New Jersey, New Jersey Medical School. 100 Bergen St.. Newark, NJ 07103 Fessenden, Jane. Marine Biological Laboratory. W:oods Hole. MA 02543 Festoff. Barry \V., Neurology Service (127), Veterans Administration Medical Center. 4801 Linwood Blvd.. Kansas City. MO 64 128 Fink, Rachel D., Clapp Biology Laboratory. Mount Holyoke College. South Hadley. MA 01075 Finkelstein, Alan. Albert Einstein College of Medicine. 1 300 Morris Park Ave.. Bronx. NY 1046 1 Fischbach, Gerald, Department of Anatomy and Neuro- biology. Washington University School of Medicine. St. Louis. MO 63 110 Fischman, Donald A., Department of Cell Biology and Anatomy. Cornell University Medical College. 1300 York Ave., New York, NY 1002 1 Fishman, Harvey M., Department of Physiology. Uni- versity of Texas Medical Branch. Galveston. TX 77550 Flanagan, Dennis, 12 Gay St.. New York. NY 10014 Fox, Maurice S.. Department of Biology. Massachusetts Institute of Technology. Cambridge. MA 02138 Frank, Peter \V.. Department of Biology. University of Oregon. Eugene. OR 97403 Franzini, Clara. Department of Biology G-5. School of Medicine. University of Pennsylvania. Philadelphia. PA 19174 Frazier. Donald T.. Department of Physiology and Bio- physics. University of Kentucky Medical Center. Lex- ington. KY 40536 Freeman, Gary L., Department of Zoology. University of Texas. Austin. TX 78172 (resigned 3/31/88) Freinkel. Norbert. Center for Endocrinology. Metabo- lism & Nutrition. Northwestern University Medical School. 303 E. Chicago Avenue. Chicago. IL 6061 1 French, Robert J.. Department of Medical Physiology. University of Calgary. 3330 Hospital Dr.. NW. Cal- gary. Alberta. T2N 4N 1 Canada Freygang. Walter J., Jr., 6247 29th St.. NW. Washing- ton. DC 200 15 Fry, Brian, Marine Biological Laboratory. Woods Hole. MA 02543 Fukui, Yoshio, Department of Cell Biology and Anat- omy. Northwestern University Medical School. Chi- cago. IL 60201 Fulton, Chandler M., Department of Biology. Brandeis University. Waltham. MA 02154 Furshpan. Edwin J.. Department of Neurophysiology. Harvard Medical School. Boston. MA 021 15 Fuseler, John \V., Department of Biology. University of Southwestern Louisiana. Lafayette. LA 70504 Futrelle. Robert P., College of Computer Science. North- eastern University. 360 Huntington Avenue. Boston. MA 021 15 Fye, Paul. P. O. Box 309. Woods Hole. MA 02543 (de- ceased 3/1 1/88) Gabriel, Mordecai. Department of Biology. Brooklyn College. Brooklyn. NY 1 1210 Gadsby. David C.. Laboratory of Cardiac Physiology. The Rockefeller University. 1230 York Avenue. New York. NY 10021 Gainer. Harold. Section of Functional Neurochemistrv. NIH. Bldg. 36 Room 4D-20. Bethesda. MD 20892 Galatzer-Levy. Robert M., 180 N. Michigan Avenue. Chicago. IL 60601 Gall. Joseph G.. Carnegie Institution. 1 15 West Univer- sity Parkway. Baltimore. MD 21210 Gallant. Paul E., Laboratory of Preclinical Studies. Bldg. 36. NIAAA/NTH. 1250 Washington Ave.. Rockville. MD 20892 Gascoyne. Peter. Department of Experimental Pathol- ogy. Box 85E. University of Texas System Cancer Center. M. D. Anderson Hospital and Tumor Insti- 10 MARINE BIOLOGICAL LABORATORY lute. Texas Medical Center, 6723 Bertner Avenue, Houston, TX 77030 Gelfant, Seymour, Department of Dermatology. Medi- cal College of Georgia. Augusta, GA 30904 Gelperin, Alan, Department of Biology, Princeton Uni- versity, 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, Waltham. MA 02254 Giblin, Anne E., Ecosystems Center, Marine Biological Laboratory. Woods Hole, MA 02543 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, Bldg. 9, Room IE- 124, Bethesda, MD 20892 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., Room 420 1 , 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 606 11 Goldsmith, Paul K., NIH, Bldg. 10, Room 9C-101, Bethesda, MD 20892 Goldsmith, Timothy H., Department of Biology, Yale University, New Haven, CT 065 1 0 Goldstein, Moise H., Jr., ECE Department, Borten Hall, Johns Hopkins University, Baltimore, MD 21218 Goodman, Lesley Jean, Department of Biological Sci- ences, Queen Mary College, Mile End Road, London, El 4NS, England, U. K. 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 02 1 38 Govind, C. K., Zoology Department — Scarborough, University of Toronto, 1265 Military Trail, West Hill, Ontario, Canada, MIC 1A4 Graf, Werner, Rockefeller University, New York, NY 10021 Grant, Philip, Department of Biology, University of Ore- gon, Eugene, OR 97403 Grass, Albert, The Grass Foundation, 77 Reservoir Rd., Quincy, MA 02 170 Grass, Ellen R., The Grass Foundation. 77 Reservoir Rd., Quincy, MA 02 170 Grassle, Judith, Marine Biological Laboratory. Woods Hole, MA 02543 Green, Jonathan P., Department of Biology, Roosevelt University, 430 S. Michigan Avenue. Chicago. IL 60605 (resigned 2/5/88) Greenberg, Everett Peter, Department of Microbiology. Stocking Hall. Cornell University, Ithaca, NY 14853 Greenberg, Michael J., C. V. Whitney Lab, Rt. 1, Box 1 2 1 , St. Augustine, FL 32086 Greif, Roger L., Department of Physiology. Cornell Uni- versity, Medical College, New York, NY 10021 (re- signed 10/87) Griffin, Donald R., The Rockefeller University, 1230 York Ave., New York, NY 1002 1 Gross, Paul R., Office of the Vice President and Provost, University of Virginia, Charlottesville, VA 22906- 9014 Grossman, Albert, New York University, Medical School, New York, NY 10016 Gruner, John, Department of Neurosurgery, New York University Medical Center, 550 First Ave., 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 1300 Morris Park Ave., Bronx, NY 10461 Hall, Zack W., Department of Physiology, University of California, San Francisco, CA 94143 Halvorson, Harlyn O., Marine Biological Laboratory, Woods Hole, MA 02543 I l:nn kit. Nancy Virginia, Department of Biology, Swarthmore College, Swarthmore, PA 1908 1 Hanna, Robert B., College of Environmental Science and Forestry, SUNY, Syracuse, NY 13210 Harding, Clifford V., Jr., P. O. Box 452, Woods Hole, MA 02543 Harosi, Ferenc I., Laboratory of Sensory Physiology, Marine Biological Laboratory, Woods Hole, MA 02543 Harrigan, June F., 7415 Makaa Place, Honolulu, HI 96825 Harrington, Glenn W., Department of Microbiology, School of Dentistry, University of Missouri, 650 E. 25th St., Kansas City, MO 64108 Harris, Andrew L., Department of Biophysics. Johns Hopkins University, 34th & Charles Sts., Baltimore, MD21218 Haschemeyer, Audrey E. V., Department of Biological MEMBERS OF THE CORPORATION 11 Sciences, Hunter College, 695 Park Ave., New York, NY 10021 Hastings, J. W., Harvard University, 16 Divinity Street, Cambndge, MA02138 Hauschka, Theodore S., RD1, Box 781, Damariscotta, ME 04543 Hayashi, Teru, 7 105 SW 1 12 Place, Miami, FL 33 1 73 Hayes, Raymond L., Jr., Department of Anatomy, How- ard University, College of Medicine, 520 W St.. NW, Washington, DC 20059 Henley, Catherine, 5225 Pooks Hill Rd., #1 127 North, Bethesda, MD 20034 Hepler, Peter K., Department of Botany, University of Massachusetts, Amherst, MA 01003 Herndon, Walter R., University of Tennessee, Depart- ment of Botany, Knoxville, TN 37996-1 100 Hessler, Anita Y., 5795 Waverly Ave., La Jolla, CA 92037 Heuser, John, Department of Biophysics, Washington University, School of Medicine, St. Louis, MO 631 10 Hiatt, Howard H., Brigham and Women's Hospital, 75 Francis Street, Boston, MA 02 1 1 5 Highstein, Stephen M., Department of Otolaryngology, Washington University, St. Louis, MO 63 1 10 Hildebrand, John G., Arizona Research Laboratories, Division of Neurobiology, 603 Gould-Simpson Sci- ence Building, University of Arizona, Tucson, AZ 85721 Hill, Susan D., Department of Zoology, Michigan State University, E. Lansing, MI 48824 Hillis-Colinvaux, Llewellya, Department of Zoology, The Ohio State University, 484 W. 12th Ave., Colum- bus, OH 432 10 Hillman, Peter, Department of Biology, Hebrew Univer- sity, Jerusalem, ISRAEL Hinegardner, Ralph T., Division of Natural Sciences, University of California, Santa Cruz, CA 95064 Hinsch, Gertrude, W., Department of Biology, Univer- sity 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 Hoffman, Joseph, Department of Physiology, School of Medicine, Yale University, New Haven, CT 065 10 Hollyfield, Joe G., Baylor School of Medicine, Texas Medical Center, Houston, TX 77030 Holtzman, Eric, Department of Biological Sciences, Co- lumbia 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 606 16 Houghton, Richard A., Ill, Woods Hole Research Cen- ter, P. O. Box 296, Woods Hole, MA 02543 Houston, Howard E., 2500 Virginia Ave., NW, Wash- ington, DC 20037 Howarth, Robert, Section of Ecology & Systematics, Corson Hall, Cornell University, Ithaca, NY 14853 Hoy, Ronald R., Section of Neurobiology and Behavior, Cornell University, Ithaca, NY 14850 Hubbard, Ruth, 67 Gardner Road, Woods Hole, MA 02543 Hufnagel, Linda A., Department of Microbiology, Uni- versity of Rhode Island, Kingston, RI 02881 Hummon, William D., Department of Zoology, Ohio University, Athens, OH 45701 Humphreys, Susie H., 7 10 Waukegan Rd., Glenview, IL 60025 Humphreys, Tom D., University of Hawaii, PBRC, 41 Ahui St., Honolulu, HI 968 1 3 Hunter, Robert D., Department of Biological Sciences, Oakland University, Rochester, NY 48063 Hunter, W. Bruce, Box 321, Lincoln Center, MA 01773 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, Memorial Sloan Kettering Institute, 1275 York Avenue, New York, NY 11021 Huxley, Hugh E., Department of Biology, Rosenstiel Ba- sic Medical Sciences Research Center, Brandeis Uni- versity, Waltham, MA 02254 Hynes, Thomas J., Jr., Meredith and Grew, Inc., 160 Federal Street, Boston, MA 021 10 Ilan, Joseph, Department of Anatomy, Case Western Reserve University, Cleveland, OH 44106 Ingoglia, Nicholas, Department of Physiology, New Jer- sey Medical School, 100 Bergen St., Newark, NJ 07103 Inoue, Saduyki, McGill University Cancer Centre, De- partment of Anatomy, 3640 University St., Montreal, PQ, Canada, H3A 2B2 Inoue, Shinya, Marine Biological Laboratory, Woods Hole, MA 02543 Issadorides, Marietta, R., Department of Psychiatry, University of Athens, Monis Petraki 8, Athens, 140 Greece Isselbacher, Kurt J., Massachusetts General Hospital, 32 Fruit Street, Boston, MA 021 14 Izzard, Colin S., Department of Biological Sciences, SUNY, Albany, Albany, NY 12222 Jacobson, Antone G., Department of Zoology, Univer- sity of Texas, Austin, TX 78712 Jaffe, Lionel, Marine Biological Laboratory, Woods Hole, MA 02543 12 MARINE BIOLOGICAL LABORATORY Jahan-Parwar, Behrus, Center for Laboratories & Re- search. New York State Department of Health, Em- pire State Plaza. Albany. NY 12201 Jannasch, Holger W., Department of Biology, Woods Hole Oceanographic Institution, Woods Hole, MA 02543 Jeffery, William R., Department of Zoology, University of Texas. Austin, TX 787 1 2 Jenner, Charles E., Department of Zoology. University of North Carolina. Chapel Hill. NC 27514 (resigned 8/87) Jones, Meredith L., Division of Worms, Museum of Natural History, Smithsonian Institution, Washing- ton. 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., Department of Zoology, Arizona State University, Tempe, AZ 85281 Kane, Robert E., University of Hawaii, PBRC, 41 Ahui St.. Honolulu, HI 968 13 Kaneshiro, Edna S., Department of Biological Sciences, University of Cincinnati, Cincinnati, OH 45221 Kao, Chien-yuan, Department of Pharmacology (Box 29), State University of New York, Downstate Medi- cal Center, 450 Clarkson Avenue, Brooklyn, NY 1 1 203 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 Karlin, Arthur, Department of Biochemistry and Neu- rologv Columbia University, 630 West 168th St., New York, NY 10032 Kat/., George M., Fundamental and Experimental Re- search. Merck Sharpe and Dohme, Rahway, NJ 07065 Kean, Edward L., Department of Ophthalmology and Biochemistry, Case Western Reserve University, Cleveland, OH 44 10 1 Kelley, Darcy Brisbane, Department of Biological Sci- ences, 1018 Fairchild, Columbia University, New York, NY 10032 Kelly, Robert E., Department of Anatomy, College of Medicine. University of Illinois. P. O. Box 6998, Chi- cago, IL 60680 Kemp, Norman E., Department of Biology. University of Michigan, Ann Arbor, MI 48104 Kendall, John P., Faneuil Hall Associated, One Boston Place, Boston, MA 02 108 Kendall, Richard E., 26 Green Harbor Rd.. East Fal- mouth. MA 02536 Keynan, Alexander, Hebrew University, Jerusalem. IS- RAEL Kiehart, Daniel P., Department of Cellular and Develop- mental Biology, Harvard University, 16 Divinity Ave- nue. Cambridge. MA 02 1 38 Klein, Morton, Department of Microbiology. Temple University, Philadelphia, PA 19103 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, Charlottesville, VA 22903 Kornberg, Sir Hans, The Master's Lodge, Christ's Col- lege, Cambridge CB2 3BU England, UK Kosower, Edward M., Ramat-Aviv, Tel Aviv, 69978 IS- RAEL Krahl, M. E., 2783 W. Casas Circle, Tucson, AZ 8574 1 Krane, Stephen M., Massachusetts General Hospital. Boston, MA 02 114 Krassner, Stuart M., Department of Developmental and Cell Biology, University of California. Irvine, CA 92717 Krauss, Robert, FASEB, 9650 Rockville Pike, Bethesda. MD 20814 Kravitz, Edward A., Department of Neurobiology, Har- vard 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 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, De- partment of Biology, 3300 South Federal St., Chicago, IL60616 Kuzirian, Alan M., Marine Biological Laboratory, Woods Hole, MA 02543 Laderman, Aimlee, P. O. Box 689, Woods Hole, MA 02543 MEMBERS OF THE CORPORATION 13 LaMarche, Paul H., Eastern Maine Medical Center, 489 State St., Bangor, ME 04401 Landis, Dennis M. D., Department of Developmental Genetics and Anatomy, Case Western Reserve Medi- cal School, 2119 Abington Road, Cleveland, OH 44106 Landis, Story C, Department of Pharmacology, Case Western Reserve University Medical School, 2119 Abington Road, Cleveland, OH 44106 Landowne, David, Department of Physiology, Yale Uni- versity School of Medicine, 333 Cedar St., New Ha- ven, CT 065 10 Langford, George M., Department of Physiology, Medi- cal Sciences Research Wing 206H, University of North Carolina, Chapel Hill, NC 27514 Lasek, 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 Science, Molecular and Cell Bi- ology, Group U-125, University of Connecticut, St- orrs, CT 06268 Lazarow, Paul B., The Rockefeller University, 1230 York Avenue, New York, NY 1002 1 Lazarus, Maurice, Federated Department Stores, Inc., 50 Cornhill, Boston, MA 02 108 Leadbetter, Edward R., Department of Molecular and Cell Biology, U-131, University of Connecticut, Storrs, CT 06268 Lederberg, Joshua, President, The Rockefeller Univer- sity, 1230 York Ave., New York, NY 10021 Lederhendler, Izja I., Laboratory of Cellular and Molec- ular Neurobiology, NINCDS/NIH, Park 5 Building, Room 435, Bethesda, MD 20892 Lee, John J., Department of Biology, City College of CUNY, Convent Ave. and 138th St., New York, NY 10031 Lehy, Donald B., Marine Biological Laboratory, Woods Hole, MA 02543 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 20892 Leinwand, Leslie Ann, Department of Microbiology and Immunology, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461 Lerman, Sidney, Laboratory for Ophthalmic Research, Emory University, Atlanta, GA 30322 Lerner, Aaron B., Yale University, School of Medicine, New Haven, CT 065 10 Lester, Henry A., 1 56-29 California Institute of Tech- nology, Pasadena, CA 91 125 Levin, Jack, Clinical Pathology Service, VA Hospital — 1 1 3 A, 4 1 50 Clement St., San Francisco, CA 94 1 2 1 Levinthal, Cyrus, Department of Biological Sciences, Columbia University, 435 Riverside Drive, New York, NY 10025 Levitan, Herbert, Department of Zoology, University of Maryland, College Park, MD 20742 Linck, Richard W., Department of Anatomy, Jackson Hall, University of Minnesota, 321 Church Street, S. E., Minneapolis, MN 55455 Lipicky, Raymond J., Department of Cardio-Renal/ HFD 1 10, FDA Bureau of Drugs, Rm. 16B-45, 5600 Fishers Lane, Rockville, MD 20857 Lisman, John E., Department of Biology, Brandeis Uni- versity, Waltham, MA 02254 Liuzzi, Anthony, 320 Beacon St., Boston, MA 021 16 Llinas, Rodolfo R., Department of Physiology and Bio- physics, 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 33 101 Loewus, Frank A., Institute of Biological Chemistry, Washington State University, Pullman, WA 99164 Loftfield, Robert B., Department of Biochemistry, School of Medicine, University of New Mexico, 900 Stanford, NE, Albuquerque, NM 87131 London, Irving M., Massachusetts Institute of Technol- ogy, Cambridge, MA 02 1 39 Longo, Frank J., Department of Anatomy, University of Iowa, Iowa City, IA 52442 Lorand, Laszlo, Department of Biochemistry and Mo- lecular Biology, Northwestern University, Evanston, IL 60208 Luckenbill-Edds, Louise, 155 Columbia Ave., Athens, OH 45701 Luria, Salvador E., 48 Peacock Farm Rd., Lexington, MA 02 173 Macagno, Eduardo R., 1003B Fairchild, Columbia Uni- versity, New York, NY 10022 MacNichol, E. F., Jr., 45 Brewster Street, Cambridge, MA 02 138 Maglott-Duffield, Donna R. S., 1014 Baltimore Road, Rockville, MD 20851 Maienschein, Jane Ann, Department of Philosophy, Ari- zona State University, Tempe, AZ 8528 1 Mainer, Robert, The Boston Company, One Boston Place, MA 02 108 Malbon, Craig Curtis, Department of Pharmacological Sciences, Health Sciences Center, SUNY, Stony Brook, Stony Brook, NY 1 1 794-865 1 14 MARINE BIOLOGICAL LABORATORY Malkiel, Saul, Allergic Diseases, Inc., 130 Lincoln St.. Worcester, MA 01605 Manalis, Richard S., Department of Biological Sciences, Indiana University — Purdue University at Fort Wayne. Fort Wayne. IN 46805 Mangum. Charlotte P., Department of Biology, College of William and Man.. Williamsburg. VA 23185 Margulis, Lynn, Department of Biology. Boston Univer- sity. 2 Cummington St.. Boston. MA 02215 Marinucci, Andrew C., 102 Nancy Drive, Mercerville. NJ 08619 Marsh, Julian B., Department of Biochemistry and Physiology. Medical College of Pennsylvania, 3300 Henry Ave.. Philadelphia. PA 19129 Martin, Lowell V., Marine Biological Laboratory, Woods Hole, MA 02543 Martinez-Palomo, Adolfo, Seccion de Patologia Experi- mental, Cinvesav-ipn, 17000 Mexico. D.F. A. P., 14- 740, Mexico Maser, Morton, P. O. Box EM, Woods Hole, MA 02543 Mastroianni, Luigi, Jr., Department of Obstetrics and Gynecology, University of Pennsylvania, Philadel- phia, PA 19174 Mathews, Rita W., Department of Medicine, 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, Phil- adelphia. PA 19104 Mautner, Henry G., Department of Biochemistry, Tufts University, 136 Harrison Ave., Boston, MA 021 1 1 Mauzerall, David, The Rockefeller University, 1230 York Ave., New York. NY 1002 1 Mazia, Daniel, Hopkins Marine Station, Pacific Grove, CA 93950 Mazzella, Lucia, Laboratorio di Ecologia del Benthos, Stazione Zoologica di Napoli, P.ta S. Pietro 80077, Is- chia Porto (NA), Italy McCann, Frances, Department of Physiology, Dart- mouth Medical School, Hanover, NH 03755 McCloskey, Lawrence R., Department of Biology, Walla Walla College, College Place, WA 99324 (resigned 10/ 87) McLaughlin, Jane A., P. O. Box 187, Woods Hole, MA 02543 McMahon, Robert F., Department of Biology, Box 19498, University of Texas, Arlington, TX 76019 Meedcl, Thomas, Marine Biological Laboratory, Woods Hole, MA 02543 Meinert/hagen, Ian A., Department of Psychology, Life Sciences Center, Dalhousie University, Halifax, Nova Scotia, Canada B3H 451 Meinkoth, Norman A., 43 1W Woodland Avenue, Springfield, PA 19064 (deceased 4/87) Meiss, Dennis E., 462 Solano Avenue. Hayward. CA 94541 Melillo, Jerry A., Ecosystems Center. Marine Biological Laboratory, Woods Hole, MA 02543 Mellon, Richard P., P. O. Box 187, Laughlintown, PA 15655 Mellon, DeForest, Jr., Department of Biology. Univer- sity of Virginia, Charlottesville. VA 22903 Menzel, Randolf, Institut fir Tierphysiologie. Free Uni- versitat of Berlin, 1000 Berlin 41 , FEDERAL REPUB- LIC OF GERMANY (resigned 10/87) Metuzals, Janis, Department of Anatomy, Faculty of Medicine, University of Ottawa. Ottawa, Ontario, CANADA K1N9A9 Metz, Charles B., 7220 Southwest 124th St., Miami, FL 33156 Milkman, Roger, Department of Zoology, University of Iowa, Iowa City. IA 52242 Mills, Eric L., Oceanography Dept., Dalhousie Univer- sity, Halifax, Nova Scotia B3H 4J 1. Canada Mills, Robert, 10315 44th Avenue, W' 12 H Street, Bra- denton, FL 33507-1 535 Mitchell, Ralph, Pierce Hall, Harvard University, Cam- bridge, MA 02 1 38 Miyamoto, David M., Department of Biology, Drew University. Madison, NJ 07940 Mizell, Merle, Department of Biology. Tulane Univer- sity, New Orleans, LA 70 1 1 8 Moore, John VV., Department of Physiology, Duke Uni- versity Medical Center, Durham, NC 27710 Moore, Lee E., Department of Physiology and Biophys- ics, University of Texas, Medical Branch, Galveston. TX 77550 Morin, James G., Department of Biology, University of California, Los Angeles, CA 90024 Morrell, Frank, Department of Neurological Sciences, Rush Medical Center, 1753 W. Congress Parkway, Chicago, I L 606 12 Morrill, John B., Jr., Division of National Sciences, New College, Sarasota, FL 33580 (resigned 12/87) Morse, M. Patricia, Marine Science Center, Northeast- ern University, Nahant MA 01908 Morse, Richard S., 193 Winding River Rd., Wellesley, MA 02181 Morse, Robert W., Box 574, N. Falmouth, MA 02556 Morse, Stephen Scott, The Rockefeller University, 1 230 York Ave., Box 2, New York, NY 10021-6399 Moscona, A. A., Department of Molecular Genetics and Cell Biology, University of Chicago, 920 East 58th St., Chicago, IL 60637 Mote, Michael I., Department of Biology, Temple Uni- versity, Philadelphia. PA 19122 Mountain, Isabel, Vinson Hall #1 12, 6251 Old Domin- ion Drive, McLean, V A 22101 MEMBERS OF THE CORPORATION 15 Mullins, Lorin J., University of Maryland, School of Medicine, Baltimore MD 2 1 20 1 Musacchia, Xavier J., Graduate School, University of Louisville, Louisville, K.Y 40292 Nabrit, S. M., 686 Beckwith St., SW, Atlanta, GA 303 14 Nadelhoffer, Knute, Marine Biological Laboratory, Woods Hole, MA 02543 Naka, Ken-ichi, National Institute for Basic Biolgy, Oka- zaki, Japan 444 Nakajima, Shigehiro, Department of Biological Sci- ences, Purdue University, West Lafayette, IN 47907 Nakajima, Yasuko, Department of Biological Sciences, Purdue University, West Lafayette, IN 47907 Narahashi, Toshio, Department of Pharmacology, Med- ical Center, Northwestern University, 303 East Chi- cago Ave., Chicago, IL 606 1 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., 119 Forest Home Drive, Ithaca, NY 14850 Nicholls, John G., Biocenter, KJingelbergstr 70, Basel 4056, Switzerland Nicosia, Santo V., Department of Pathology, University of South Florida, College of Medicine, Box 1 1, 12901 North 30th St., Tampa, FL 33612 Nielsen, Jennifer B. K., Merck Sharp & Dohme Labora- tories, Bldg. 50-G, Room 226, Rahway, NJ 07065 Noe, Bryan D., Department of Anatomy, Emory Uni- versity, Atlanta, GA 30345 Obaid, Ana Lia, Department of Physiology and Phar- macy, University of Pennsylvania, 4001 Spruce St., Philadelphia, PA 19104 Ochoa, Severe, 530 East 72nd St., New York, NY 1002 1 (resigned 3/87) Odum, Eugene, Department of Zoology, University of Georgia, Athens, GA 30701 (resigned 1/87) Oertel, Donata, Department of Neurophysiology, Uni- versity of Wisconsin, 283 Medical Science Bldg., Mad- ison, 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, Biology Di- vision ORNL, P. O. Box Y, Oak Ridge, TN 37830 Olins, Donald E., University of Tennessee-Oak Ridge, Graduate School of Biomedical Sciences, Biology Di- vision ORNL, P. O. Box Y, Oak Ridge, TN 37830 O'Melia, Anne F., 16 Evergreen Lane, Chappaqua, New York 105 14 Oschman, James L., 9 George Street, Woods Hole 02543 Palmer, John D., Department of Zoology, University of Massachusetts, Amherst, MA 01002 Palti, Yoram, Department of Physiology and Biophysics, Israel Institute of Technology, 12 Haaliya St., Bat- Galim, POB 9649, Haifa, Israel Pant, Harish C., NINCDS/NIH, Bldg. 36. Room 4D-20, Bethesda, MD 20892 Pappas, George D., Department of Anatomy, College of Medicine, University of Illinois, 808 South Wood St., Chicago, IL 606 12 Pardee, Arthur B., Department of Pharmacology, Har- vard Medical School, Boston, MA 021 15 Pardy, Rosevelt L., School of Life Sciences, University of Nebraska, Lincoln, NE 68588 Parmentier, James L., Becton Dickinson, P. O. Box 12016, Research Triangle Park, NC 27709 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, St. Louis, MO 63110 Pederson, Thoru, Worcester Foundation for Experimen- tal Biology, Shrewsbury, MA 0 1 545 Perkins, C. D., 400 Hilltop Terrace, Alexandria, VA 22301 Person, Philip, Oral Health Director, Research Testing Labs, Inc., 167 E. 2nd St., Huntington Station, NY 11746 Peterson, Bruce J., 82 Hillcrest Dr., Falmouth, MA 02540 Pethig, Ronald, School of Electronic Engineering Sci- ence, University College of N. Wales, Dean St., Ban- gor, Gwynedd, LL57 IUT, UK Pettibone, Marian H., Division of Worms, W-213, Smithsonian Institution, Washington, DC 20560 (re- signed 1 1/87) Pfohl, Ronald J., Department of Zoology, Miami Uni- versity, Oxford, OH 45056 Pierce, Sidney K., Jr., Department of Zoology, Univer- sity of Maryland, College Park, MD 20740 Poindexter, Jeanne S., Science Division, Long Island University, Brooklyn Campus, Brooklyn, NY 1 1201 Pollard, Harvey B., NIH, F Building 10, Room 10B17, Bethesda, MD 20892 Pollard, Thomas D., Department of Cell Biology and Anatomy, Johns Hopkins University, 725 North Wolfe St., Baltimore, MD 2 1 205 Pollock, Leland W., Department of Zoology, Drew Uni- versity, Madison, NJ 07940 Poole, Alan F., 1 14 Metoxit Road, Waquoit, MA 02536 Porter, Beverly H., 13617 Glenoble Drive, Rockville, MD 20853 Porter, Keith R., Department of Biology, University of Maryland, Catonsville, MD 2 1 228 Porter, Mary E., Department MCD Biology, Campus Box 347, University of Colorado, Boulder, CO 80309 16 M\RIM BIOIOG1CAL I -\BORA TORY Potter, David, Department of Neurohiology. Harvard Medical School, Boston, MA 02 1 1 5 Potts, William T., Department of Biology, University of Lancaster. Lancaster. England. UK Poussart, Denis, Department of Electrical Engineering. Universite Laval. Quebec. Canada Pratt, Melanie M., Department of Anatomy and Cell Bi- ology. 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, Balti- more. MD 2 1205 Presley, Phillip H., Carl Zeiss, Inc., 1 Zeiss Drive, Thorn wood, NY 10594 Price, Carl A., Waksman Institute of Microbiology, Rutgers University, P. O. Box 759, Piscataway, NJ 08854 Price, Christopher H., Biological Science Center, Boston University, 2 Cummington St.. Boston, MA 02215 (resigned 11/87) Prior, David J., Department of Biological Sciences, Uni- versity of Kentucky. Lexington, KY 40506 Prusch, Robert D., Department of Life Sciences, Gon- zaga University. Spokane, WA 99258 Przyby Iski, Ronald J., Case Western Reserve University, Department of Anatomy, Cleveland, OH 44104 Purves, Dale, Department of Anatomy, Washington University School of Medicine, 660 S. Euclid Ave., St. Louis. MO 63 110 Quigley, James, Department of Microbiology and Im- munology Box 44, SUNY Downstate Medical Center. 450 Clarkson Ave., Brooklyn, NY 1 1203 Rabin, Harvey, DuPont Biomed. Prod.-BRL-2, 331 Treble Cove Road, No. Billerica, MA 0 1 862 Raff, Rudolf A., Department of Biology, Indiana Univer- sity. Bloomington, IN 47405 Rakowski, Robert F., Department of Physiology and Biophysics, UHS/The Chicago Medical School, 3333 Greenbay Rd.. N. Chicago, IL 60064 Ramon, Fidel, Dept. de Fisiologia y Biofisca, Central de Investigacion y de, Estudius Avanzados del Ipn, Apur- tado Postal 14-740, Mexico, D.F. 07000 Ranzi, Silvio, Sez Zoologia Sc Nat, Via Coloria 26, 12013. Milano, Italy Rastetter, V ^ard B., Ecosystems Center, Marine Bio- logical L; -Tory, Woods Hole, MA 02543 Hatner, Sara. -partment of Biochemistry, Public- Health Reseai litute, 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, Barbara F., Marine Biological Laboratory. Woods Hole. MA 02543 Reese, Thomas S., Marine Biological Laboratory. Woods Hole. MA 02543 Reiner, John M., 2150 Grand Boulevard. Schenectady, NY 12309 Reinisch, Carol L., Tufts University School of Veteri- nary Medicine. 203 Harrison Avenue, Boston. MA 02 11 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., 30 Burnham Dr., Falmouth. MA 02540 Rich, Alexander, Department of Biology, Massachusetts Institute of Technology. Cambridge, MA 02139 Rickles, Frederick R., University of Connecticut, School of Medicine, VA Hospital, Newington, CT 06 1 1 1 Ripps, Harris, Department of Ophthalmology, Univer- sity of Illinois College of Medicine. 1855 W. Taylor Street, Chicago, IL 606 11 Roberts, John L., Department of Zoology, University of Massachusetts, Amherst, MA 01002 (resigned 10/87) Robinson, Denis M., 200 Ocean Lane Drive, Key Bis- cayne, FL33149 Rose, Birgit, Department of Physiology R-430, Univer- sity of Miami School of Medicine. P. O. Box 016430, Miami, FL 33 149 Rose, S. Meryl, Box 309W, Waquoit. MA 02536 Rosenbaum, Joel L., Department of Biology. Kline Biol- ogy Tower, Yale University, New Haven, CT 06520 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., 18 Millneld Street, Woods Hole, MA 02543 Rowland, Lewis P., Neurological Institute, 710 West 168th St., New York, NY 10032 Ruderman, Joan V., Department of Zoology, Duke Uni- versity, Durham, NC 27706 Rushforth, Norman B., Case Western Reserve Univer- sity, Department of Biology, Cleveland, OH 44106 MEMBERS OF THE CORPORATION 17 Russell-Hunter, W. D., Department of Biology, Lyman Hall 029, Syracuse University, Syracuse, NY 13210 Saffo, Mary Beth, Institute of Marine Sciences, 272 Ap- plied Sciences, University of California, Santa Cruz, CA 95064 Sager, Ruth, Dana Farber Cancer Institute, 44 Binney St., Boston, MA 021 15 Salama, Guy, Department of Physiology, University of Pittsburgh, Pittsburth, PA 15261 Salmon, Edward D., Department of Zoology, University of North Carolina, Chapel Hill, NC 275 14 Salzberg, Brian M., Department of Physiology, Univer- sity of Pennsylvania, 4010 Locust St., Philadelphia, PA 19174 Sanborn, Richard C., 5862 North Olney St., Indianapo- lis, IN 46220 Sanger, Jean M., Department of Anatomy, School of Medicine, University of Pennsylvania, 36th and Ham- ilton Walk, Philadelphia, PA 19174 Sanger, Joseph, Department of Anatomy, School of Medicine, University of Pennsylvania, 36th and Ham- ilton Walk, Philadelphia, PA 19174 Sato, Eimei, Department of Animal Science, Faculty of Agriculture, Kyoto University, Kyoto 606, Japan Sato, Hidemi, Sugashima Marine Biological Laboratory, Nagoya University, Sugashima-cho, Toba-chi, Mie- Ken 517, Japan Sattelle, David B., AFRC Unit-Department of Zoology, University of Cambridge, Downing St., Cambridge CB2 3EJ, England, UK Saunders, John W., Jr., P. O. Box 381 W, Waquoit, MA 02536 Saz, Arthur K., Medical and Dental Schools, George- town University, 3900 Reservoir Rd., NW, Washing- ton, DC 2005 1 Schachman, Howard K., Department of Molecular Biol- ogy, University of California, Berkeley, CA 94720 Schatten, Gerald P., Integrated Microscopy Facility for Biomedical Research, University of Wisconsin, 1117 W. Johnson St., Madison, WI 53706 Schatten, Heide, Department of Zoology, University of Wisconsin, Madison, WI 53706 Schiff, Jerome A., Institute for Photobiology of Cells and Organelles, Brandeis University, Waltham, MA 02 1 54 Schmeer, Arline C., Mercenene Cancer Research Insti- tute, Hospital of Saint Raphael. New Haven, CT 06511 Schnapp, Bruce J., Marine Biological Laboratory, Woods Hole, MA 02543 Schneider, E. Gayle, Department of Obstetrics and Gy- necology, Yale University School of Medicine, 333 Cedar St., New Haven, CT 065 10 Schneiderman, Howard A., Monsanto Company, 800 North Lindberg Blvd., D1W, St. Louis, MO 63166 Schotte, Oscar E., Department of Biology, Amherst Col- lege, Amherst, MA 01002 (deceased 4/12/88) Schuel, Herbert, Department of Anatomical Sciences, SUNY, Buffalo, Buffalo, NY 14214 Schuetz, Allen W., School of Hygiene and Public Health, Johns Hopkins University, Baltimore, MD 21205 Schwartz, James H., Center for Neurobiology and Be- havior, New York State Psychiatric Institute — Re- search Annex, 722 W. 1 68th St., 7th Floor, New York, NY 10032 Scofield, Virginia Lee, Department of Microbiology and Immunology, UCLA School of Medicine, Los Ange- les, CA 90024 Sears, Mary, P. O. Box 152, Woods Hole, MA 02543 Segal, Sheldon J., Population Division, The Rockefeller Foundation, 1133 Avenue of the Americas, New York, NY 10036 Seliger, Howard H., Johns Hopkins University, McCol- lum-Pratt Institute, Baltimore, MD 2 12 18 (resigned I/ 31/88) Selman, Kelly, Department of Anatomy, College of Medicine, University of Florida, Gainesville, FL 32601 Senft, Joseph, English Village Apartments, Bldg. 24, C- 1 , Lower State Rd., North Wales, PA 19454 Shanklin, Douglas R., 1 34 Grove Park Circle, Memphis, TN38117 Shapiro, Herbert, 6025 North 1 3th St., Philadelphia, PA 19141 Shaver, Gaius R., Ecosystems Center, Marine Biological Laboratory, Woods Hole, MA 02543 Shaver, John R., 6 1 5 Jones St., Lansing, MI 489 1 2- 1 7 1 8 Sheetz, Michael P., Department of Cell Biology and Physiology, Washington University Medical School, 606 S. Euclid Ave., St. Louis, MO 63110 Shepard, David C., P. O. Box 44, Woods Hole, MA 02543 Shepro, David, Department of Biology. Boston Univer- sity, 2 Cummington St., Boston, MA 022 1 5 Sher, F. Alan, Immunology and Cell Biology Section, Laboratory of Parasitic Disease, NIAID, Building 5, Room 1 14, NIH, Bethesda, MD 20892 Sheridan, William F., Biology Department, University of North Dakota, Grand Forks, ND 58202 Sherman, I. W., Division of Life Sciences, University of California, Riverside, CA 92502 Shilo, Moshe, Department of Microbiological Chemis- try, Hebrew University, Jerusalem, ISRAEL Shoukimas, Jonathan J., 45 Dillingham Avenue, Fal- mouth, MA 02540 Siegel, Irwin M., Department of Ophthalmology, New 18 MARINK BIOLOGICAL LABORATORY York University Medical Center. 550 First Avenue. New York, NY 10016 Siegelman, Harold \\ '.. Department of Biology. Brook- haven National Laboratory, Upton. NY 1 1973 Silver, Robert B., Laboratory of Molecular Biology. Uni- versity of Wisconsin. 15^ 5 Linden Drive, Madison. \VI 53706 Sjodin, Raymond A., Department of Biophysics, Univer- sity of Maryland, Baltimore. MD21201 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 Sluder, Greenfield, Cell Biology Group, Worcester Foundation for Experimental Biology, 22 Maple Ave., Shrewsbury. MA 01 545 Smith, Michael A., J 1 Sinabung, Buntu #7, Semarang, Java. Indonesia Smith, Ralph I., Department of Zoology, University of California, Berkeley, CA 94720 Sorenson, Martha M., Depto de Bioquimica-RFRJ, Centro de Ciencias da Saude-I. C. B.. Cidade Universi- taria-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, Colum- bia 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 03755 Spiegel, Melvin, Department of Biological Sciences, Dartmouth College, Hanover, NH 03755 Spray, David C., Albert Einstein College of Medicine, Department of Neurosciences, 1 300 Morris Park Ave- nue. Bronx, NY 10461 Steele, John Hyslop, Woods Hole Oceanographic Insti- tution, Woods Hole. MA 02543 Steinacher, Antoinette, Dept. of Otolaryngology, Wash- ington University, School of Medicine, 491 1 Barnes Hospital St. Louis, MO 63 110 Steinberg, M:1 olm, Department of Biology, Princeton Universiu . eton, NJ 08540 Stephens, Gro^ Department of Developmental and Cell Biologv Tsity of California, Irvine, CA 92717 Stephens, Raymond L.rine Biological Laboratory, Woods Hole, MA 025. Stetten, DeWitt, Jr., Senior Scientific Advisor, NIH, Bldg. 16. Room 118. Bethesda. MD 20892 Stetten, Jane Lazarow, 2 W Drive, Bethesda, MD20814 Steadier, Paul A., Ecosystems Center. Marine Biological Laboratory Woods Hole. MA 02543 Stokes, Darrell R., Department of Biology. Emory Uni- versity. Atlanta. GA 30322 Stommel, Elijah W., 766 Palmer Avenue, Falmouth. MA 02540 Stracher, Alfred, Downstate Medical Center, SUNY, 450 Clarkson Ave., Brooklyn, NY 1 1203 Strehler, Bernard L., 2235 25th St., #2 1 7, San Pedro, CA 90732 Strumwasser, Felix, Marine Biological Laboratory, Woods Hole, MA 02543 Stuart, Ann E., Department of Physiology, Medical Sci- ences Research Wing 206H. University of North Caro- lina. Chapel Hill. NC 275 14 Sugimori, Mutsuyuki, Department of Physiology and Biophysics, New York University Medical Center, 550 First Avenue, New York, NY 10016 Summers, William C., Huxley College, Western Wash- ington University. Bellingham, WA 98225 Sussman, Maurice, 72 Carey Lane. Falmouth. MA 02540 Szabo, George, Harvard School of Dental Medicine, 188 Longwood Avenue, Boston, MA 02 1 1 5 Szent-Gyorgyi, Andrew, Department of Biology, Bran- deis University, Waltham, MA 02254 Szent-Gyorgyi, Eva Szentkiraly, Department of Biology, Brandeis University, Waltham, MA 02254 Szuts, Etc Z., Laboratory of Sensory Physiology, Marine Biological Laboratory, Woods Hole, MA 02543 Tamm, Sidney L., Boston University Marine Program. Marine Biological Laboratory, Woods Hole, MA 02543 Tanzer, Marvin L., Department of Oral Biology, Medi- cal School, University of Connecticut, Farmington, CT 06032 Tasaki, Ichiji, Laboratory of Neurobiology, Bldg. 36, Rm. 2B-16, NIMH/NIH, Bethesda, MD 20892 Taylor, Douglass L., Biological Sciences, Mellon Insti- tute, 440 Fifth Avenue, Pittsburgh, PA 15213 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 Trager, William, Rockefeller University, 1230 York Ave., New York, NY 10021 Travis, D. M., Veterans Administration Medical Center, Fargo, ND 58 102 Treistman, Steven N., Worcester Foundation for Experi- mental Biology. Shrewsbury, MA 01545 Trigg, D. Thomas, 1 25 Grove St., Wellesley, MA 02 1 8 1 1 1 ink. ins, J. Philip, Department of Biology, Box 6666, Yale University, New Haven, CT 065 1 0 Troll, Walter, Department of Environmental Medicine, MEMBERS OF THE CORPORATION 19 College of Medicine, New York University, New York, NY 100 16 Troxler, Robert F., Department of Biochemistry, School of Medicine, Boston University, 80 East Concord St., Boston, MA 021 18 Tucker, Edward B., The City University of New York, Baruch College, Box 502, 17 Lexington Ave., New York, NY 100 10 Turner, Ruth D., Mollusk Department, Museum of Comparative Zoology, Harvard University, Cam- bridge, 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 Ueno, Hiroshi, Laboratory of Biochemistry, The Rocke- feller University, 1230 York Ave., New York, NY 10021 Uretz, Robert B., Division of Biological Sciences, Uni- versity of Chicago, 950 East 59th St., Chicago, IL 60637 Valiela, Ivan, Boston University Marine Program, Ma- rine Biological Laboratory, Woods Hole, MA 02543 Vallee, Richard, Cell Biology Group, Worcester Founda- tion for Experimental Biology, Shrewsbury, MA 01545 Valois, John, Marine Biological Laboratory, Woods Hole, MA 02543 Van Holde, Kensal, Department of Biochemistry and Biophysics, Oregon State University, Corvallis, OR 97331 Villee, Claude A., Parcel B, Room 122, Harvard Medical School, 25 Shattuck St., Boston, MA 02 1 1 5 Vincent, Walter S., School of Life and Health Sciences, University of Delaware, Newark, DE 1971 1 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., Whitney Marine Lab, 9505 A1A South, St. Augustine, FL 32086 Wang, An, Wang Laboratories, Inc., One Industrial Ave., Lowell, MA 01 851 Wang, Ching Chung, Department of Pharmaceutical Chemistry, University of California, San Francisco, CA94143 Warner, Robert C., Department of Molecular Biology and Biochemistry, University of California, Irvine, CA 92717 Warren, Kenneth S., The Rockefeller Foundation, 1 133 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 Watson, Stanley, Department of Biology, Woods Hole Oceanographic Institution, Woods Hole, MA 02543 Webb, H. Marguerite, Marine Biological Laboratory, Woods Hole, MA 02543 Weber, Amu-marie, Department of Biochemistry and Biophysics, School of Medicine, University of Penn- sylvania, Philadelphia, PA 19104 Webster, Ferris, Box 765, Lewes, DE 19958 Weidner, Earl, Department of Zoology and Physiology, Louisiana State University, Baton Rouge, LA 70803 Weiss, Dieter, G., Institut fur Zoologie, Technische Un- iversitat Munchen, 8046 Garching, Federal Republic ofGermany Weiss, Leon P., Department of Animal Biology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA 19104 Weissmann, Gerald, New York University, 550 First Av- enue, New York, NY 1 00 1 6 Werman, Robert, Neurobiology Unit, The Hebrew Uni- versity, Jerusalem, ISRAEL Westerfield, R. Monte, The Institute of Neuroscience, University of Oregon, Eugene, OR 37403 Wexler, Nancy Sabin, 1 5 Claremont Avenue, Apt. 92, New York, NY 10027 White, Roy L., Department of Neuroscience, Albert Ein- stein College, 1 300 Morris Park Avenue, Bronx, NY 10461 Whittaker, J. Richard, Marine Biological Laboratory, Woods Hole, MA 02543 Wigley, Roland L., 35 Wilson Road, Woods Hole, MA 02543 Wilson, Darcy B., Medical Biology Institute, 1 1077 North Torrey Pines Road, La Jolla, CA 92037 Wilson, Edward, O., Museum, Comparative Zoology, Harvard University, Cambridge, MA 02 1 38 (resigned 10/87) Wilson, T. Hastings, Department of Physiology, Har- vard Medical School, Boston, MA 02 1 1 5 Wilson, Walter L., 743 Cambridge Drive, Rochester Hills, 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 Wolfe, Ralph, Department of Microbiology, 131 Burrill Hall, University of Illinois, Urbana, IL 61801 Wolken, Jerome J., Department of Biological Sciences, Carnegie Mellon University, 440 Fifth Ave., Pitts- burgh, PA 15213 Worgul, Basil V., Department of Ophthalmology, Co- lumbia University, 630 West 168th St., New York, NY 10032 20 M \R1\I BIOLOGICAL LABORATORY Wu, Chau Hsiung, Depanment of Pharmacology, Northwestern University Medical School, 203 E. Chi- cago Ave.. Chicago. IL 606 1 1 \Vyttenbach, Charles R., Department of Physiology and Cell Biology. University of Kansas, Lawrence, KS 66045 Veh, Jay Z., Department of Pharmacology, Northwes- tern University Medical School, 303 E. Chicago Ave., Chicago, IL 606 1 1 Young, Richard \V.. Mentor O & O. Inc.. 3000 Long- water Dr.. Nonvell. MA 0206 1-16 10 Zackroff, Robert, 66 White Horn Drive, Kingston, RI 02881 Zigman, Seymour, School of Medicine and Dentistry, University of Rochester, 260 Crittenden Blvd., Roch- ester. NY 14620 Zigmond, Richard E., Department of Pharmacology, Harvard Medical School, 250 Longwood Ave., Bos- ton, MA 021 15 Zimmerberg, Joshua J., Bldg. 12A, Room 2007, NIH, Bethesda, MD 20892 Zottoli, Steven J., Department of Biology, Williams Col- lege, Williamstown, MA 01267 Zucker, Robert S., Department of Physiology, Univer- sity of California. Berkeley, CA 94720 Associate Members Ackroyd, Dr. Frederick W. Adams, Dr. Paul Adelherg, Dr. and Mrs. Edward A. Ahearn, Mr. and Mrs. David Alden, Mr. John M. Allen, Miss Camilla K. Allen. Dr. Nina S. Amon, Mr. Carl H. Jr Anderson, Mr. J. Gregory Anderson, Drs. James L. and Helen-. Armstrong, Dr ' Mrs. Samuel C. Arnold. Mrs. Lor, Atwood, Dr. and KimballC., Ill Ayers, Mr. and Mrs. Donald Baker, Mrs. C. L. Ball, Mrs. EricG. Ballantine. Dr. and Mrs. H.T.Jr. Bang. Mrs. Frederik B. Bang, Miss Molly- Banks, Mr. and Mrs. William L. Barkin, Mr. and Mrs. Mel A. Barrows, Mrs. Albert W. Baum. Mr. Richard T. Baylor, Drs. Edward and Martha Beers, Dr. and Mrs. Yardley Belesir, Mr. Tasos Bennett, Dr. and Mrs. Michael V. L. Berg. Mr. and Mrs. C. John Bernheimer, Dr. Alan W. Bernstein, Mr. and Mrs. •.orman 3erwind, Mr. David McM. Bicker, Mr. Alvin Bigelow, Mrs. Robert O. Bird, Mr. William R. Bleck, Dr. Thomas B. Boche, Mr. David Bodeen. Mr. and Mrs. George H. Boettiger. Dr. and Mrs. Edward G. Boettiger, Mrs. Julie Bolton, Mr. and Mrs. Thomas C. Bonn, Mr. and Mrs. Theodore H. Borgese, Dr. and Mrs. Thomas Bowles, Dr. and Mrs. Francis P. Bradley, Dr. and Mrs. Charles C. Bradley, Mr. Richard Brown, Mrs. Frank A., Jr. Brown, Mr. and Mrs. Henry Brown, Mr. and Mrs. James Brown, Mrs. Neil Brown, Dr. and Mrs. Thornton Broyles, Dr. Robert H. Buck. Dr. and Mrs. John B. Buckley, Mr. George D. Bunts, Mr. and Mrs. Frank E. Burt, Mrs. Charles E. Bush, Dr. Louise Buxton, Mr. and Mrs. Bruce E. Buxton, Mr. E. Brewster Calkins, Mr. and Mrs. G. N.,Jr. Campbell, Dr. and Mrs. David G. Carlson, Dr. and Mrs. Francis Carlton, Mr. and Mrs. WinslowG. Case, Dr. and Mrs. James Chandler, Mr. Robert Chase, Mr. Tom H. Child, Dr. and Mrs. Frank M. Church, Dr. Weslev Claff. Mr. and Mrs. Mark Clark, Dr. and Mrs. Arnold Clark, Mr. and Mrs. Hays Clark, Mr. and Mrs. James McC. Clark, Mrs. Leonard B. Clark. Mr. and Mrs. Leroy. Jr. Clarke. Dr. Barbara J. Clement. Mrs. Anthony Clowes Fund. Inc. Clowes, Dr. and Mrs. Alexander W. Clowes, Mr. Allen W. Clowes, Dr. and Mrs. G. H. A. .Jr. Coburn. Mr. and Mrs. Lawrence Cohen, Mrs. Seymour S. Coleman. Dr. and Mrs. John Connell, Mr. and Mrs. W. J. Cook, Dr. and Mrs. Paul W., Jr. Copeland, Dr. and Mrs. D. Eugene Copeland, Mr. Frederick C. Copeland, Mr. and Mrs. Preston S. Costello, Mrs. Donald P. Crabb. Mr. and Mrs. David L. Crain, Mr. and Mrs. Melvin C. Cramer, Mr. and Mrs. Ian D. W. Crane, Mrs. John O. Crane, Josephine B., Foundation Crane, Mr. Thomas S. Cross, Mr. and Mrs. Norman C. Crossley, Miss Dorothy Crossley, Miss Helen Crowell, Dr. and Mrs. Sears Currier, Mr. and Mrs. David L. MARINE BIOLOGICAL LABORATORY 21 Daignault, Mr. and Mrs. Alexander T. Daniels, Mr. and Mrs. Bruce G. Davidson. Dr. Morton Davis, Mr. and Mrs. Joel P. Day, Mr. and Mrs. Pomeroy Decker, Dr. Raymond F. DeMello, Mr. John DiBerardino, Dr. Marie A. Dickson, Dr. Willim A. Dierolf, Dr. Shirley H. Drummey, Mr. and Mrs. Charles E. Drummey, Mr. Todd A. DuBois, Dr. and Mrs. Arthur B. Dudley, Dr. Patricia DuPont, Mr. A. Felix, Jr. Dutton, Mr. and Mrs. Roderick L. Ebert, Dr. and Mrs. James D. Egloff, Dr. and Mrs. F. R. L. Elliott, Mrs. Alfred M. Enos, Mr. Edward, Jr. Eppel, Mr. and Mrs. Dudley Estabrook, Mr. Gordon C. Evans, Mr. and Mrs. Dudley Farley, Miss Joan Farmer, Miss Mary Faull, Mr. J. Horace, Jr. Ferguson, Dr. and Mrs. James J., Jr. Fisher, Mrs. B. C. Fisher, Mr. Frederick S., Ill Fisher, Dr. and Mrs. Saul H. Folino, Mr. John W., Jr. Forbes, Mr. John M. Ford, Mr. John H. Fowlkes, Mr. Aaron Francis, Mr. and Mrs. Lewis W., Jr. Frenkel, Dr. Krystina Fribourgh, Dr. James H. Friendship Fund Fries, Dr. and Mrs. E. F. B. Frosch, Dr. and Mrs. Robert A. Fye, Mrs. Paul M. Gabriel, Dr. and Mrs. Mordecai L. Gagnon, Mr. Michael Gaiser, Mrs. David W. Gallagher, Mr. Robert O. Garfield. Miss Eleanor Garrey, Dr. Walter E. Gellis, Dr. and Mrs. Sydney Gephard, Mr. Stephen German, Dr. and Mrs. James L., Ill Gewecke, Mr. and Mrs. Thomas H. Gifford Mr. and Mrs. Cameron Gifford, Mr. John A. Gifford, Dr. and Mrs. Prosser Gilbert, Drs. Daniel L. and Claire Gilbert, Mrs. Carl J. Gildea. Dr. Margaret C. L. Gillette, Mr. and Mrs. Robert S. Glad, Mr. Robert Glass, Dr. and Mrs. H. Bentley Glazebrook, Mr. James Glazebrook, Mrs. James R. Goldman, Mrs. Mary Goldring, Mr. Michael Goldstein, Dr. and Mrs. Moise H., Jr. Goodwin, Mr. and Mrs. Charles Gould, Miss Edith Grace, Miss Priscilla B. Grant, Dr. and Mrs. Philip Grassle, Mrs. J. F. Green, Mrs. Davis Crane Greer, Mr. and Mrs. W. H.,Jr. Griffin, Mrs. Robert W. Griffith, Dr. and Mrs. B. Herold Grosch, Dr. and Mrs. Daniel S. Gross, Mrs. Mona Gunning, Mr. and Mrs. Robert Haakonsen, Dr. Harry O. Haigh, Mr. and Mrs. Richard H. Hall, Mr. and Mrs. Peter A. Hall, Mr. Warren C. Halvorson, Dr. and Mrs. Harlyn O. Hamstrom, Miss Mary Elizabeth Harrington, Mr. Robert D.,Jr. Harvey, Dr. and Mrs. Richard B. Hassett, Mr. and Mrs. Charles Hastings, Dr. and Mrs. J. Woodland Haubrich, Mr. Robert R. Hay, Mr. John Hays, Dr. David S. Hedberg, Mrs. Frances Hedberg, Dr. Mary Hersey, Mrs. George L. Hiatt, Dr. and Mrs. Howard Hichar, Mrs. Barbara Hill, Mrs. Samuel E. Hirschfeld, Mrs. Nathan B. Hobbie, Dr. and Mrs. John Hocker, Mr. and Mrs. Lon Hodge, Mrs. Stuart Hokin, Mr. Richard Hornor, Mr. Townsend Horwitz, Dr. and Mrs. Norman H. Hoskin, Dr. and Mrs. Francis C. G. Houston, Mr. and Mrs. Howard E. Howard, Mr. and Mrs. L. L. Hoyle, Dr. Merrill C. Huettner, Dr. and Mrs. Robert J. Hutchison, Mr. Alan D. Hynes, Mr. and Mrs. Thomas J., Jr. Inoue, Dr. and Mrs. Shinya Issokson, Mr. and Mrs. Israel Jackson, Miss Elizabeth B. Jaffe, Dr. and Mrs. Ernst R. Janney, Mrs. F. Wistar Jewett, G. F., Foundation Jewett, Mr. and Mrs. G. F.,Jr. Jones, Mr. and Mrs. DeWitt C., Ill Jones Mr. and Mrs. Frederick, II Jones, Mr. Frederick S., Ill Jordan, Dr. and Mrs. Edwin P. Kaan, Dr. Helen W. Kahler, Mrs. Robert W. Kaminer, Dr. and Mrs. Benjamin Karplus, Mrs. Alan K. Karush, Dr. and Mrs. Fred Kelleher, Mr. and Mrs. Paul R. Kendall, Mr. and Mrs. Richard E. Keosian, Mrs. Jessie Keoughan, Miss Patricia Ketchum, Mrs. Paul Kien, Mr. and Mrs. Pieter Kinnard, Mrs. L. Richard Kirschenbaum, Mrs. Donald Kissam, Mr. and Mrs. William M. MARINE BIOLOGICAL LABORATORY Kiv>. Dr. and Mrs. Peter Roller, Dr. Lewis R. Korgen. Dr. Ben J. Kuffler, Mrs. Stephen W. Laderman. Mr. and Mrs. Ezra Lafferty. Miss Nancy Larmon, Mr. Jay Laster, Dr. and Mrs. Leonard Laufer. Dr. and Mrs. Hans Laufer. Jessica, and Weiss, Malcolm LaVigne. Mrs. Richard J. Lawrence, Mr. Frederick V. Lawrence, Mr. and Mrs. William Leatherbee, Mrs. John H. LeBlond, Mr. and Mrs. Arthur Leeson, Mr. and Mrs. A. Dix LeFevre, Dr. Marian E. Lehman, Miss Robin Lemann. Mrs. Lucy B. Lenher, Dr. and Mrs. Samuel Leprohon, Mr. Joseph Levine, Mr. Joseph Levine, Dr. and Mrs. Rachmiel Levitz, Dr. Mortimer Levy, Mr. Stephen R. Lindner, Mr. Timothy P. Little, Mrs. Elbert LiMngstone, Mr. and Mrs. Robert Loeb, Mrs. Robert F. Lovell, Mr a. d Mrs. HollisR. Lovering, Mr. Richard C Low. Miss Dons Lowe, Dr. and Mrs Charles V. 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 Anne Camille Mahler, Mrs. Henry Mahler, Mrs. Suzanne Mansworth. Miss Marie Marsh. Dr. and Mrs. Julian Martyna, Mr. and Mrs. Joseph C. Mason, Mr. Appleton Mastroianni, Dr. and Mrs. Luigi. Jr. Mather, Mr. and Mrs. Frank J., Ill Matherly, Mr. and Mrs. Walter Matthiessen, Dr. and Mrs. G. C. McCusker, Mr. and Mrs. Paul T. McElroy, Mrs. Nella W. Mcllwain, Dr. Susan G. 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 Metz, Dr. and Mrs. Charles B. Meyers, Mr. and Mrs. Richard Milhury. Mr. Edward Van R. Miller, Dr. Daniel A. Miller, Mr. and Mrs. Paul Mixter, Mr. and Mrs. William J., Jr. lizell. Dr. and Mrs. Merle Mniiroy. Mrs. Alberto Montgomery, Dr. and Mrs. Charles H. Montgomery, Dr. and Mrs. Raymond B. Moore, Drs. John and Betty Morgan, Miss Amy Morse, Mrs. Charles L..Jr. Morse, Dr. M. Patricia Moul, Dr. and Mrs. Edwin T. Mountain. Dr. Isabel M. Murray, Dr. David M. Myles-Tochko, Dr. Christina J. Nace, Dr. and Mrs. Paul Nace, Mr. Paul F., Jr. Neall, Mr. William G. Nelson, Dr. and Mrs. Leonard Nelson, Dr. Pamela Newton, Mr. William F. Nickerson, Mr. and Mrs. Frank L. Norman, Mr. and Mrs. Andrew E. Norman Foundation Norris, Mr. and Mrs. Barry Norris, Mr. and Mrs. John A. Norris, Mr. William O'Herron, Mr. and Mrs. Jonathan Olszowka, Miss Janice S. O'Neil, Mr. and Mrs. Barry T. O'Rand. Mr. and Mrs. Michael Ortins, Mr. and Mrs. Armand O'Sullivan, Dr. Renee Bennett Pappas, Dr. and Mrs. George D. Park. Mrs. Franklin A. Park, Mr. and Mrs. Malcolm S. Parmenter, Dr. Charles Parmenter, Miss Carolyn L. Peltz, Mr. and Mrs. William L. Pendergast, Mrs. Claudia Pendleton, Dr. and Mrs. Murray E. 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. Petty. Mr. Richard F. Petty, Mr. William Pfeiffer, Mr. and Mrs. John Plough. Mr. and Mrs. George H. Plough, Mrs. Harold H. Pointe. Mr. Albert Pointe, Mr. Charles Porter. Dr. and Mrs. Keith R. Pothier, Dr. and Mrs. Aubrey- Press, Drs. Frank and Billie Proskauer, Mr. Joseph H. Proskauer, Mr. Richard Prosser, Dr. and Mrs. C. Ladd Psaledakis, Mr. Nicholas Psychoyos, Dr. Alexandre Putnam, Mr. Allan Ray Putnam. Mr. and Mrs. William A., Ill Raymond, Dr. and Mrs. Samuel Reese, Miss Bonnie Reingold, Mr. Stephen C. Reynolds, Dr. and Mrs. George Reynolds, Mr. Robert M. Reznikoff, Mrs. Paul Ricca, Dr. and Mrs. Renato A. Righter, Mr. Harold Riina, Mr. and Mrs. John R. Robh, Mrs. Alison A. MEMBERS OF THE CORPORATION 23 Roberts, Miss Jean Roberts, Mrs. Mervin F. Robertson, Mrs. C. W. Robinson, Dr. Denis M. Root, Mrs. Walter S. Rosenthal, Miss Hilde Roslansky, Drs. John and Priscilla Ross, Dr. and Mrs. Donald Ross, Dr. Robert Ross, Dr. Virginia Roth, Dr. and Mrs. Stephen Rowe, Mr. Don Rowe, Mr. and Mrs. William S. Rubin, Dr. Joseph Rugh, Mrs. Roberts Ryder, Mr. and Mrs. Francis C. Sager, Dr. Ruth Sardinha, Mr. George H. 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, Mrs. George T. Scott, Mr. and Mrs. Norman E. Sears, Mr. Clayton C. Sears, Mr. and Mrs. Harold B. Sears, Mr. Harold H. Seaver, Mr. George Segal, Dr. and Mrs. Sheldon J. Senft, Dr. and Mrs. Alfred Shapiro, Mr. and Mrs. Howard Shapley, Dr. Robert Shemin, Dr. and Mrs. David Shepro, Dr. and Mrs. David Siegel, Mr. and Mrs. Alvin Simmons, Mr. Tim Singer, Mr. and Mrs. Daniel M. Smith, Drs. Frederick E. and Marguerite A. Smith, Mrs. Homer P. Smith, Mr. Van Dorn C. Snyder, Mr. Robert M. Solomon, Dr. and Mrs. A. K. Speck, Dr. William T. Specht, Mr. and Mrs. Heinz Spiegel, Dr. and Mrs. Melvin Spotte, Mr. Stephen Steele, Mrs. John H. Stein, Mr. Ronald Steinbach, Mrs. H. Bun- Stetson, Mrs. Thomas J. Stetten, Dr. Gail Stetten, Dr. and Mrs. H. DeWitt, Jr. Stewart, Mr. and Mrs. Peter Strehler, Dr. and Mrs. Bernard Stunkard, Dr. Horace Sudduth, Dr. William Swanson, Dr. and Mrs. Carl P. Swope, Mrs. Gerard, Jr. Swope, Mr. and Mrs. Gerard L. Szent-Gyorgyi, Dr. Andrew Tabor, Mr. George H. Taylor, Mr. James K. Taylor, Dr. and Mrs. W. Randolph Tietje, Mr. and Mrs. Emil D., Jr. Timmins. Mrs. William Todd, Mr. and Mrs. Gordon F. Tolkan, Mr. and Mrs. Norman N. Trager, Mrs. William Trigg, Mr. and Mrs. D. Thomas Troll, Dr. and Mrs. Walter Tucker, Miss Ruth Tully, Mr. and Mrs. Gordon F. Ulbrich, Mr. and Mrs. Volker Valois, Mr. and Mrs. John Van Buren, Mrs. Harold Van Holde, Mrs. Kensal E. Veeder, Mrs. Ronald A. Vincent, Mr. and Mrs. Samuel W. Vincent, Dr. Walter S. Wagner, Mr. Mark Waksman, Dr. and Mrs. Byron H. Ward, Dr. Robert T. Ware, Mr. and Mrs. J. Lindsay Warren, Dr. Henry B. Warren, Dr. and Mrs. Leonard Watt, Mr. and Mrs. John B. Weeks, Mr. and Mrs. John T. Weinstein, Miss Nancy B. Weisberg, Mr. and Mrs. Alfred M. Wheeler, Dr. and Mrs. Paul S. Whitehead, Mr. and Mrs. Fred Whitney, Mr. and Mrs. Geoffrey G., Jr. Wichterman, Dr. and Mrs. Ralph Wickersham, Mr. and Mrs. A. A. Tilney Wiese, Dr. Konrad Wilhelm, Dr. Hazel S. Wilson, Mr. and Mrs. T. Hastings Winn, Dr. William M. Winsten, Dr. Jay A. Witting, Miss Joyce Wonnsohn, Mrs. Wolfe Woodwell, Dr. and Mrs. George M. Yntema, Mrs. Chester L. Young- Wallace, Miss Nina L. Zinn, Dr. and Mrs. Donald J. Zipf, Dr. Elizabeth III. Certificate 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, Su- san 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 hun- dred 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 Corpora- tion according to the provisions of the one hundred and fif- teenth chapter of the Public Statutes of the Commonwealth of Massachusetts, and the Acts in amendment thereof and in ad- dition thereto. The name by which the Corporation shall be known is THE MARINE BIOLOGICAL LABORATORY. 24 MARINH BIOLOGICAL I NBORUORY The purpose for which the Corporation is constituted is to es- tablish 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 lo- cated is the cit> 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, Wil- liam 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 hun- dred 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 Ste- vens, Treasurer, Edward G. Gardiner, William T. Sedgwick, Susan Mims, Charles Sedgwick Minot. (Approved on March 20, 1988 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 sec- tions 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 t President, and David Shepro, Clerk of the Marine Biolo; .ratory, located at Woods Hole, Massa- chusetts 0254 certify that the following amend- ment to the Article /ation of the Corporation was duly adopted at a nu eld on August 15, 1975, as ad- journed to August 2(>, 19 >f 444 members, being at least two-thirds of its m legalK qualified to vote in the meeting of the corporation: Voted: That the Certificate of Orgam/ution of this corporation be and it hereby is amended by the addition of the fol- lowing provisions: "No Officer. Trustee or Corporate Member of the cor- poration shall be personally liable for the payment or satisfaction of any obligation or liabilities incurred as a result of. or otherwise in connection with. an> commit- ments, agreements, activities or affairs of the corpora- tion. "Except as otherwise specifically provided by the By- laws 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 corpo- ration, 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 U 'itness 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: 1 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) V. Bylaws of the Corporation of the Marine Biological Laboratory (Revised August 16, 1985) I. (A) The name of the Corporation shall be The Marine Biological Laboratory. The Corporation'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 discrimi- nate on the basis of race, color, sex, national and ethnic origin in employment, administration or its educational policies, ad- missions policies, scholarship and other programs. BYLAWS 25 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 deter- mined 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 resigna- tion 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 mem- bership. 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 or- ganized and operated under the general supervision and au- thority of the Trustees. III. The officers of the Corporation shall consist of a Chair- man 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 meet- ing the Members shall choose by ballot six Trustees to serve four years, and shall transact such other business as may prop- erly 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 Meet- ing of Members are fixed by these Bylaws, no notice of the An- nual Meeting need be given. Notice of any special meeting of Members, however, shall be given by the Clerk by mailing no- tice 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 ad- dress 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 Mem- bers present or represented at the meeting, whether or not such Members constitute a quorum. It shall not be necessary to no- tify any Members of any adjournment. 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 Mem- bers according to such procedures, not inconsistent with these Bylaws, as the Trustees shall have determined. Except as pro- vided 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 ("Trustees-at-large") approved by members ac- cording to such procedures, not inconsistent with these Bylaws, as the Trustees shall have determined. Except as provided be- low, such Trustees-at-large shall be divided into four classes of four, 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 other- wise determined by the Trustees for good reason, Trustees-at- large, shall be individuals who have not been considered for election as Corporate Trustees. ( 3 ) Trustees ex officio. who shall be the Chairman, the Presi- dent, 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 qual- ifies 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 Trust- ees 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 Trus- tees-at-large elected in any year (excluding Trustees elected to fill vacancies which do not result from expiration of a term) shall not exceed ten. The number of Trustees-at-large so elected shall not exceed four and unless otherwise determined by vote of the Trustees, the number of Corporate Trustees so elected shall not exceed six. Corporate Trustees shall always constitute a majority on the Board of those elected or approved by the Corporation. (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 26 \I\RIM BIDKHilCM I \HOR \IORY cntiiled 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 \acanc> m the number of Trustees, however aris- ing, may be filled by the Trustees then in office unless and until tilled b> the Members at the next Annual Meeting. (F) A Corporate Trustee or a Trustee-at-large who has served an initial term of at least two 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. IX. (A) The Trustees shall have the control and manage- ment of the affairs of the Corporation. They shall elect a Chair- man 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 four 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 ex- ceed 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. I he> 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 number as provided in Article X, and to delegate to such Committee such of their own powers as they may deem expedient in addi- tion 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 incon- sistent 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 Invest- ment 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 com- mittee shall have tenure and duties as the Trustees shall deter- mine; 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 re- quired for not less than two-thirds of the Investment Commit- tee 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 ot its business; but. unless oth- erwise 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, Di- rector 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. Beginning with the members elected for terms ending in 1990, one of the Trustees elected to serve on the Executive Committee should be a Trustee-at-large. This procedure will be repeated in the class of 199 1 . and hence- forth the Trustees will elect to the Executive Committee Trust- ees to ensure that the composition of the Committee is four Corporate Trustees and two Trust ees-at-large. (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 subcommit- tees as the Committee shall determine. (C) The Executive Committee shall have and may exercise all the powers of the Board during the intervals between meet- ings 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 mat- ters to be acted upon by the Executive Committee or the Board ofTrustees. (D) The Executive Committee shall keep appropriate min- utes of its meetings and its action shall be reported to the Board ofTrustees. (E) The elected Members of the Executive Committee shall constitute a standing "Committee for the Nomination of Officers," responsible for making nominations, at each Annual Meeting of the Corporation, and of the Board ofTrustees, 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 BYLAWS 27 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 tele- phone, 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 dis- solution of the Marine Biological Laboratory. In case of disso- lution, 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 govern- ing (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 enti- tled to vote on amending the Bylaws. Any Bylaw adopted by the Trustees may be amended or re- pealed by the Members entitled to vote on amending the By- laws. XV. The account of the Treasurer shall be audited annually by a certified public accountant. XVI. Except as otherwise provided below, the Corporation shall, to the extent legally permissible, indemnify each person who is, or shall have been, a Trustee, director or officer of the Corporation or who is serving, or shall have served, at the re- quest of the Corporation as a Trustee, director or officer of an- other organization in which the Corporation directly or indi- rectly has any interest, as a shareholder, creditor or otherwise, against all liabilities and expenses (including judgments, fines, penalties and reasonable attorneys' fees and all amounts paid, other than to the Corporation or such other organization, in compromise or settlement) imposed upon or incurred by any such person in connection with, or arising out of, the defense or disposition of any action, suit or other proceeding, whether civil or criminal, in which he or she may be a defendant or with which he or she may be threatened or otherwise involved, directly or indirectly, by reason of his or her being or having been such a Trustee, director or officer. The Corporation shall provide no indemnification with re- spect to any matter as to which any such Trustee, director or officer shall be finally adjudicated in such action, suit or pro- ceeding not to have acted in good faith in the reasonable belief that his or her action was in the best interests of the Corpora- tion. The Corporation shall provide no indemnification with respect to any matter settled or compromised, pursuant to a consent decree or otherwise, unless such settlement or compro- mise shall have been approved as in the best interests of the Corporation, after notice that indemnification is involved, by (i) a disinterested majority of the Board of Trustees or of the Executive Committee or, (ii) a majority of the Corporation's Members. Indemnification may include payment by the Corporation of expenses in defending a civil or criminal action or proceed- ing in advance of the final disposition of such action or pro- ceeding upon receipt of an undertaking by the person indemni- fied to repay such payment if it is ultimately determined that such person is not entitled to indemnification under the provi- sions of this Article XVI, or under any applicable law. As used in this Article, the terms "Trustee," "director" and "officer" include their respective heirs, executors, administra- tors and legal representatives, and an "interested" Trustee, di- rector or officer is one against whom in such capacity the pro- ceeding in question or another proceeding on the same or sim- ilar grounds is then pending. To assure indemnification under this Article of all persons who are determined by the Corporation or otherwise to be or to have been "fiduciaries" of any employee benefit plan of the Corporation which may exist from time to time, this Article shall be interpreted as follows: (i) "another organization" shall be deemed to include such an employee benefit plan, including without limitation, any plan of the Corporation which is gov- erned by the Act of Congress entitled "Employee Retirement Income Security Act of 1974," as amended from time to time ("ERISA"); (ii) "Trustee" shall be deemed to include any per- son requested by the Corporation to serve as such for an em- ployee benefit plan where the performance by such person of his or her duties to the Corporation also imposes duties on, or otherwise involves services by, such person to the plan or participants or beneficiaries of the plan; (iii) "fines" shall be deemed to include any excise taxes assessed on a person with respect to an employee benefit plan pursuant to ERISA; and (iv) actions taken or omitted by a person with respect to an employee benefit plan in the performance of such person's du- ties for a purpose reasonably believed by such person to be in the interest of the participants and beneficiaries of the plan shall be deemed to be for a purpose which is in the best interests of the Corporation. The right of indemnification provided in this Article shall not be exclusive of or affect any other rights to which any Trustee, director or officer may be entitled under any agree- ment, statute, vote of members or otherwise. The Corpora- MARINE BIOLOGICAL LABORATORY lion's obligation to provide indemnification under this Article shall be offset to the extent of any other source of indemnifica- tion or any otherwise applicable insurance coverage under a policy maintained by the Corporation or any other person. Nothing contained in this Article shall affect any rights to which employees and corporate personnel other than Trustees, directors or officers may be entitled by contract, by vote of the Board of Trustees or of the Executive Committee or otherwise. VI. Report of the Director Standing the Test of Experience As the development of an organism can only he understood from the standpoint of genetic continuity. . . . so the de- velopment of the Laboratory, if it is to be fairly understood, must be examined, not in the light of what may transpire in a week or even a whole session, but in the light of its continuous history. Each session may be regarded as a de- velopmental phase, as the promise of the germinal in- ception unfolded to the extent determined by all the coop- erating factors. But each phase or stage is the fulfilment of all that have gone before and the prophecy of all that are tofollow. In i irder to know whether we have fulfilled well or ill, we must go back to the germ in its first stage, and see what promise it contained and what general policy and means were proposed for the fulfilment. If we have pursued one purpose and one method from the beginning, we ought now to be in a position to see whether theory has stood the lest of experience. — C. O. Whitman. Eighth Annual Report. 1895. p. 18 The centennial anniversary of an institution is an ap- propriate occasion for looking back — and an equally ap- propriate occasion for looking around and looking for- ward. In the years leading up to the Marine Biological Labo- ratory's centennial, historians have examined this em- bryonic organism — its origins and its early developmen- tal phases. Historians and philosophers, within the cor- poration and without, have asked what has happened at the MBL, and what role the laboratory has played in spurring the remarkable growth of American biology. (Some of the best of those studies are available in a spe- cial issue of The Biological Bulletin, vol 1 68, no 3.) While the historians have been looking back, other members of the MBL community have been taking a careful look around, asking what the laboratory is today, and forward, asking what role it will play in the next de- cades of American biology — and enterprise several or- ders of magnitude larger and vastly more complex than it was in C. O. Whitman's time. An ad hoc Committee on Long Range Goals spent two years taking a hard look forward, and submitted its report on the laboratory's fu- ture to the corporation in June of 1987. In the second half of the laboratory's one-hundredth year, we have been working to draw together into one workable plan all the ideas inspired by the approach of the centennial year. Well-acquainted now with the labo- ratory's illustrious and unusual history, we are confident that the experiences of the past 100 years show us the way forward. Thus, although the centennial celebration has just begun, the centennial self-examination has al- ready shown us where the laboratory must go in the next decade — if it is to continue to make important contribu- tions to the national biological research effort. Clearly, the MBL's unparalleled summer programs of teaching and research must remain the laboratory's rai- son d'etre. Just as clearly, we must redouble our efforts to apply the most modern biological approaches in our summer courses and in our research programs. We must continue to provide tutorial laboratory courses at the cutting edge of science. For nearly a cen- tury' now, MBL summer courses have attracted faculty and students from the best institutions in the world. Taken together, the MBL summer courses represent a collection of scientific talent that cannot be duplicated at any one university. To ensure the continued health of these one-of-a-kind courses, we must seek over the next decade an educational endowment fund that will cover course expenses not covered by tuition or grants. To sustain and improve our present research technolo- gies and to provide a stable base for summer programs, we will have to encourage new year-round research pro- grams in areas of traditional MBL strength. The new pro- grams will supplement the important existing programs and create a critical mass of year-round investigators in neurobiology, cell biology, and developmental biology. We should build strong year-round programs in microbi- ology and in molecular genetics, molecular evolution, and other areas of research that employ the tools and techniques of modern molecular biology. And of course we must maintain the strong and still-expanding year- round Ecosystems Center. We must continue to capitalize on marine organisms as models for the study of human diseases. As we've known for some time now, we will have to develop new facilities and expertise for cultivating, rearing, and study- ing the many marine animals that are so critical to bio- medical research. And to complement modern molecu- lar approaches, we will need an updated and modernized facility for warm-blooded animals. While we look to expand our year-round programs and modernize our research facilities, we must continue to nourish our traditional programs and resources. We will maintain our relationship with the Boston Univer- sity Marine Program, an association that supports tradi- tional areas of MBL research, provides us with another window on environmental science, and ensures a contin- uous tie to academia. And having completed a $2.5 mil- REPORT OF THE DIRECTOR 29 lion library endowment campaign in 1 987, we've already begun to look for ways to modernize and strengthen this renowned biological science resource. The demands of modern biological research and teaching are great — and accelerating. To meet those de- mands, to take the laboratory where it must go in the exciting decade of biology that lies immediately ahead, we have accelerated greatly our development efforts in 1987. We have asked those who have supported us in the past to maintain their support and to increase it where possible, and we have worked very hard to identify sources of support beyond the core of foundations and friends who have historically been so generous and loyal to the MBL. To support the new development efforts, we have made an effort to improve communications — with the trustees who run the laboratory, with the individuals and foundations who support the laboratory, and with the many individuals and institutions who are potential sup- porters. The emphasis on communications has inspired two new publications: a monthly newsletter (MBL Up- date) from the director to the trustees and an annual de- velopment report (this year titled MBL 87) to acknowl- edge and thank everyone who has supported the labora- tory in the past year. While we have tried to improve communications with our far-flung network of friends, we have tried simulta- neously to improve routine communications within the laboratory. Associate Director Ray Epstein has instituted weekly meetings of the top management staff and monthly meetings of other staff members. The year- round scientists have begun meeting monthly to identify common problems and to search for solutions; those meetings, now formalized as the Forum for Year-Round Scientists, provide a way for the scientific staff to bring their concerns directly to the administrative staffwho are responsible for the area of operation that needs attention. Also within the laboratory, the Employee Relations Committee has begun to publish a staff newsletter (ERC News). If the years leading up to the Centennial have been a valuable period of self-examination for the MBL com- munity, the centennial year of 1988 brings the opportu- nity to share our history and our aspirations with the world at large. Mindful of the many ways the centennial can contribute to the health of the laboratory, we have tried to shape a centennial that will be both celebratory and useful. We have encouraged activities that contrib- ute to one or more of our centennial goals: to search our past for guidance about our future; to bring the story of our past and of our future before a new audience; to give our neighbors, on the Cape and across the country, a glimpse into a modern research laboratory, to raise pub- lic understanding of science by fostering sophisticated yet accessible discussions of science, science education, science communication, and science and public policy; and to mount scientific symposia and other scientific events that make an immediate contribution to the cen- tral activities of the laboratory — teaching and research in basic biology. Having noted some of the directions the laboratory must move in the near future. I'd like to close this 100th director's report with an observation about continuity. We know the MBL as the summer home of neurobiol- ogy, embryology, and cell biology; as the place where ideas are brought together, fertilized, and allowed to de- velop (with occasional interesting mutations); as an insti- tution where whole areas of inquiry can take root quickly; as the training ground of the next generation of American biologists; and as the only major biological in- stitution that recreates itself each year. These fond vi- sions of the laboratory are reconfirmed every year by the scientists, students, scholars, writers, and others who come to the laboratory for the first time. But until you look closely at the historical record, (until you've viewed the laboratory in light of its continuous history, as Whit- man suggested it must be viewed) you're apt to miss one of the outstanding features of this institution: throughout its one hundred seasons, through twelve directorships and many more institutional changes, the MBL has re- mained true to the course set by its original director, Charles Otis Whitman. In the First Annual Report of 1 888, Whitman outlined the essential design of the MBL in a few brief paragraphs: The new Laboratory at Wood's Holl is nothing more. and. I trust, nothing less, than a first step towards the establish- ment of an ideal biological station, organized on a basis broad enough to represent all important features of the sev- eral types of laboratories hitherto known in Europe and America . . . The research department should furnish just the ele- ments required for the organization of a thoroughly effi- cient department of instruction. Other tilings being equal, the investigator is always the best instructor. The highest grade of instruction in any science can only be furnished by one who is thoroughly imbued with the scientific spirit, and who is actually engaged in original work. Hence the propriety — and. I may say. the necessity — of linking the function of instruction with that of investigation . . . To limit the work of the Laboratory to teaching would be a most serious mistake; and to exclude teaching would shut out the possibilities of the highest development. The com- bination of the two functions in mutually stimulating re- lations is a feature of the Laboratory to be strongly com- mended. — C. O. Whitman, First Annual Report, 1888, pp. 16-17 Reflecting on this statement seven years later. Whit- man wrote, "Here [in the 1888 Report] we see sketched •'• M \RI\1 HUH < H ,K \| 1 \HOR Mom the elemental basis of our germ-organization . . . The aim was a permanent biological station; the function was to be instruction and investigation; the formative princi- ple relied upon was co-operation." (Eighth Annual Re- port. 1895. p. 19) Taken together these two passages provide a nearly complete index oi features and functions (in the double sense of rules and roles) that shaped the early develop- ment of the laboratory, still guide its growth today, and will continue to do so into the future. The essential principles of the laboratory are: 1. The MBL is equally devoted to investigation and in- struction; and these are interdependent functions of the institution. 2. The MBL is independent, democratic, and national in character. 3. The MBL is cooperative in spirit and design. 4. The MBL is an institution for teaching and research in basic biology. 5. The MBL is unrestricted in biological scope. 6. The MBL fosters collaboration, interaction, and the exchange of ideas. 7. The MBL is equally dedicated to attracting top-flight investigators and training young scientists. 8. The organization and administration of the MBL is flexible and responsive to the need for change. Throughout its first century, the laboratory has man- aged— sometimes against great odds — to remain true to this ambitious set of principles. Our plans for the first decade of the laboratory's sec- ond century grow out of the recommendations of the forward-looking Committee on Long Range Goals. At the same time, our plans are perfectly in keeping with the principles that have served the MBL — and American biology — so well for 100 years. The close of our first century finds us in a fortunate position. We have a proud and successful history. We have a good base on which to build. And we can look forward with a vision that was elucidated clearly in the laboratory's first decade and adhered to steadfastly ever after. I'm confident that over the next few years that vi- sion will guide us into a future that is relentlessly bright. VII. Report of the Treasurer I want first t<. M vour attention to the strength of the Laboratory's balance sheet at the end of 1987. Current assets exceeded current liabilities by almost $2.000.000 compared to about $760. unn at the end of 1986. This increase is due mainly to the generous five-year grant of $750.000 by the MacArthur Foundation in support of the Parasitology course, and a multi-year grant by the Mellon Foundation to the Ecosystems Center. Such long-term funding considerably improves the financial stability of the MBL and allows the Director to plan and optimize the scientific program of the Laboratory. Another factor contributing to the increase in net cur- rent assets is the net growth of the repairs and replace- ment reserve bv $61.401. even after acquisition of fixed assets and repayment of debt principal of $173. 572. We are continuing to make progress towards our goal of funding depreciation expense from current operations. The need for such reserves is real, and it is underscored by the fact that in 1988 we have expended the balance available for housing repairs and replacements on reno- vations. Endowed fund balances increased by approximately $475,000 in 1987 while quasi-endow ment balances were down slightly. The former is due principally to the gener- ous support of the library endowment by the Bay Foun- dation, which enabled us to meet the challenge of the Mellon Match Grant. In this roller-coaster year in the financial markets we have reason to be satisfied with our portfolio management. Net realized and unrealized losses amounted to approximately $60,000 on portfolios in excess of $10,000.000. We had lightened up on equi- ties in advance of the crash, but I will admit to some con- cern about such a market decline in the year following my recommendation that we show investments at mar- ket value on the balance sheet. I hope you will agree that it is better to see clearly on the balance sheet "how we did" than having to dive into the footnotes to find out. The statement of Support. Revenue, Expenses and Changes in Fund Balances shows how the Laboratory's operations fared this year. The Total Current Unre- stricted Funds (Housing Enterprises) has an excess of revenue over expenses of $290.894, which is gratifying. I reiterate my statement of last year that this cannot be considered a "surplus" because it is before taking ac- count of depreciation expenses. We were only able to contribute $32,274 to Repairs and Replacements from the Housing Enterprise Fund, which is of concern to the Executive Committee. A program is in place to restore the Housing budget to more robust financial health. We were able to set aside $145,701 for the Repairs and Re- placement Reserve this year out of the current unre- stricted fund, a great improvement over 1986. The overall operating results of the Laboratory in 1 987 were quite good, but there are some trends that merit attention and concern. For the third straight year total expenditures on research declined — from $4.048,000 in 1985 to $3.864,000 in ll>87. That actually understates the amount bv which year-round research at the Labora- tory has deceased since a significant portion of the ex- penses of the departing NINCDS program have not been administered by the l.aboratorv and are therefore not in our accounts. I he major financial effect of this shrinking TREASURER'S REPORT 31 of the research base is to cause an increase in our over- head rates. While our proposed rate for 1988 is not high in comparison to other research institutions, the size of the increase in one year has placed a potential burden on our year-round scientists. Recovery of indirect costs for the summer program de- clined slightly, from $525,000 in 1986 to $521,000 in 1987. While this is not a major amount, and is still sig- nificantly above 1985's total of $468,000, any decrease in revenues from the core of the Laboratory's program should alert us for causes and to look carefully at future prospects. Operating expenses of support activities were rela- tively stable in 1987, but this stability masks the fact that during the transition between full-time directors some positions were not staffed, and under the new adminis- tration, some necessary positions have been added. In 1988 we expect expenses to rise more rapidly than revenues, and your Executive Committee has approved a budget for 1988 with a deficit of revenues to expenses of $88,000. This budget received the most careful review by the Executive Committee — a substantial portion of its meetings from September through January were de- voted to it. The decision to incur a deficit was based on the following factors: the need to maintain excellent sup- port of science at the MBL, the soundness of the Labora- tory's financial condition, and reasonable prospects of improved grant funding in the near term. We also felt that the longer term financial prospects of the Labora- tory were bright and that any major retrenchment of ex- penditures might balance the budget in the short term but jeapordize the resources on which future growth de- pends. The Executive Committee's deliberations on the bud- get were an exemplary exercise in financial review. Out of them came a commitment to continued funding of excellence in science and a renewed awareness that that is a difficult challenge. All of us enter this Second Cen- tury dedicated to the greatness of the Laboratory and committed to the effort of sustaining that distinction. M \RINF BIOLOGICAL LABORATORY Coopers &Lybrand certified public accountants To the Trustees of Marine Biological Laboratory Woods Hole, Massachusetts We have examined the balance sheet of Marine Biological Laboratory as of December 31, 1987 and the related statement of support, revenues, expenses and changes In fund balances for the year then ended. Our examination was made In accordance with generally accepted auditing standards and, accordingly, Included such tests of the accounting records and such other auditing procedures as we considered necessary In the circumstances. We previously examined and reported upon the financial statements of the Laboratory for the year ended December 31, 1986, which condensed statements are presented for comparative purposes only. In our opinion, the financial statements referred to above present fairly the financial position of Marine Biological Laboratory at December 31, 1987 and Its support, revenues, expenses and 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. norna Boston, Massachusetts April 15, 1988 TREASURER'S REPORT 33 ON ri — OO CN O § ri o 00 Ov — OO O ON OO j8 S K (N ON ON ^0 oo NO vi <-J Tf Vi oo ON ri vj t — I — r- (•*") — I—- (N VI ON >n i H £ O v-i oo i? oo ) £! = ON — OO s — m .£ ^ u "3 S g 5 -S E g -2 §• ^ £ £ i p. s ,|. & -S M 8 '£ c "c C c S E -^ •o • e 3 f| S 1 j ^ C f y C u a. < K oo p ,£ ^ 15 c I ^ 1 " J D » 0 < Q O H Si; J t J 03 v) •o <: *- e "_3 w ea i— 1 UJ J X OO O O O v~t r*"! OO m ^o J-i WLt rj <; c/3 ON ^ s ^ oo o >/->' (^i oo' ON OO 5 S )5 o' r- oo' 8z rY- ^ rl TJ- i/-i o — — o Tf t*«* — — ~~ — r-j O CQ e 5 o u Z n «r-i X. OO *T 1 — NO O O ON *O ON' 2 M fN O O 1^ - 2 % a§ ? o * *• e- s-g a j.gs £ -c d b .53 = 4J V E E o o Quasi-endowment fund balance Unrestncted Restricted es: eplaceme s 73 lant fund balanc Unrestncted Repairs and r Restricted 2 O - -o c CU nee for uncollectibl on grants and cont E *a c lotes B and C) S2 a 73 rt ^. (A O J S CQ c c C Z "r5 -_ " 3 o c u 0 z U E. c 2 '55 .1 ^ tn 5 U O c C O CL CL c O U 73 & i _0 r: 4J cd cd £ 73 C C3 •o •8 C >j • — 3 c td 00 ^ ~ CQ M _*: 1 j2 "O c K i2 =J ^i qj U 3 s 1 "3 E 7^ i Efl O JO £ o c '3 3 Cash an c J Accoun y > « « -c fc 0 JS o> ~ fti o o Investm "2 r: Less ace 2 o M \KI\1 Hli >1 ' >< .li M I \B< >K \h >K"> - s i o O fi — rj, — i/-, ••» Sw-, o — — r^ v> ^ a ^ III iTi — r-— r«lr*-. «-, OOOC t^«oo^*^w^ -T— «*-»r- > IN FUND BALANCI £ - o 0 X a> 00 2 0 , \ 2 O ffl Q Z < 1 u _) in UJ E 13 1 C/3 u ^^ H Z 4J ^ U a. C > 8 X UJ C/l UJ •o •u I C P t. E 02 UJ Z UJ > I 0 ^ Z UJ Oi £ '5 ' * H Q ^ a: '•—• S 2 a. oo u. O H Z Ul S UJ H f- oo i =e 1 Q ! 5 1 I 111 c^^ III] £ .= $ £. •5 ? 2 — r-~ r- CM oo oo oo oo -» TT r^ — r- — r*% r"> 04 o r~~ ""» "O r- ^i ^o fN — T — OO Q — TREASURER'S REPORT 35 o o •o -o — O oo o - O O I I I I I I wi r-i o ON OO O i r- oo ON ^- ON w*. O " C r i oo T O T (*> "* oo* r-' ir> \o oo in r~~ oo sO p — ' (=fi -T oo (~) in -3- C °\ •rr -t r--' x: w, *T o- sD sO ri r] r~i <^- T\ sO 0 T SO 0 ON' SO sD rn ri &O O § >/"] 3" f~\ sO D- r*i <^i CO ON 'X 0 W"i ON m t"~ in 0 c' b« ~ sO W1 m rj ON t*i OS wl sO -c. pn sD 00 P"; t/^ r- iy-i oo "/"i r*1 tr T t/"i r*i so' r-j r-] ON oo r- t*% r 1 o so r-j ON sD -c r-i ON n-i i/"i <* O *N rn ««9 *• i p* o^ •n sO v") O-; w~l <- i oo' _' *O r- sO -C rvf r4* bO r i (~i -* ^ ON ON 1^1 t ? T r^ »n sO^ &> r i --«C ? n -C oo v-t OO Vi rf (N «-) 00 c u E t/» w 3 "rt '0 c « •^ o> r i O f^-t "S f. -C ON OO r- o ex; ; a feO a 1 I 2 c u 00 c a u E 1 "S OO cd &x c v •(penses: Instruction Research Scholarships and sti Support activities: Dormitory Dining hall t 2 .c .j 5 * "§ B. $ Research services Marine resources Administration Sponsored projec administration en c 0 H E O c T e_ Depreciation Disposal Other Total expenses Excess (deficit) of and revenues o expenses •ansfers among fund Acquisition of fixed Repayment of debl Mellon match trans a -3 C. OJ 1 « C^ To support ecosyste operations Other transfers Total transfers an funds u c c 0 c 1 irealized gains (loss] on investment 0 o c "B o H investments rt change in fund ba! nd balances, beginn year md balances, end of hj £ £ D z ^ 3 U. U- MARINE BIOLOGICAL LABORATORY Notes to I inandal Statements A . I'lirpt >>t' ul ihc Labcirt.il: T\ I he purpov of Marine Biol. • I iboratorj it he "I aboralory") is to establish and maintain a laboratory or station for scientific study and investigations, and a school for instruction in biology and nature history. B. Signi/icunl . l< ' —1-uiiJ lucim/im.' In order to ensure observance of limitations and restrictions placed on the use of resources available to the Laboratory, the accounts of the Laboratory are maintained in accordance with the principles of "fund accounting." This is the procedure by which resources are classified into separate funds in accordance w ith specified activities or objectives. I xtcrnallv restricted funds may onlv be utili/cd in accordance with the purposes established bv the donor or grantor of such funds. However, the I aboratory retains full control over the ulili/alion of unrestricted funds. Restricted gifts, grants, and other restricted resources are accounted for in the appropriate restricted funds. Restricted current funds are reported as revenue when received and as related costs are incurred. Endowment funds ate subiect to restrictions requiring that the principal he invested with income available for use for restricted or unrestricted purposes bv the I aboratory. Ouasi-endovvment funds have been established by the Laboratory for the same purposes as endowment funds; however, the principal of these funds may be expended for various restricted and unrestricted purposes. Fixed Assets Fixed assets are recorded at cost. Depreciation is computed using the straight-line method over estimated useful lives of fixed assets. Reclassifications The financial statements for 1987 reflect certain changes in classification of revenue and expenses. Similar ^classifications have been made to amounts previously reported in order to provide consistency of the financial statements. ( 'ontracts and Grants Revenues associated with contracts and grants are recogni/ed in the statement of support, revenues, expenses and changes in fund balances when received and as related costs are incurred. The Laboratory reimbursement of indirect costs relating to government contracts and grants is based on negotiated indirect cost rates with adjustments for actual indirect costs in future years. Any over or underrecovery of indirect costs is recognued through future adjustments of indirect cost rates. Investments Investments purchased by the Laboratory are carried at market value. Money market securities are carried at cost which approximates market value. Investments donated to the Laboratory are earned at fair market value at the date of the gift. For determination of gain or loss upon disposal of investments, cost is determined based on the average cost method. The Laboratory is the beneficiary of certain endowment investments which are held in trust by others. These investments are reflected in the financial statements. ( ver\ ten vears the Laboratory's status as beneficiary is reviewed to determine that the Laboratory's use of these funds is in accordance with the intent of the funds. The market value of these investments are $3.334.500 and $3.333.054 at December 31. 1987 and 1986. Investment Income and l>i\trihuiii>n I he Laboratory follows the accrual basis of accounting except that investment income is recorded on a cash basis. The difference between such basis and the accrual basis does not have a material effect on the determination of investment income earned on a year-to-year basis. Investment income includes income from the investments of specific funds and from the pooled investment account. Income from the pooled investment account is distributed to the participating funds on the basis of their proportionate share at market value adjusted for any addition or disposals to pooled funds. < I iinJ Buildings, and Equipment The following is a summary of the unrestricted plant fund assets: 1987 1986 Land $ 689,660 $ 689.660 Buildings 16,385,099 IMVV^S Equipment 2.229.651 2.170.878 19.304.410 19.193.896 Less accumulated depreciation (7.707.689) (7.143.565) $11.596.721 $12.050.331 Rcliri'mcnl I'lind I he I aboralory has a noncontrihutory defined benefit pension plan loi substantially all employees. Contributions are intended to provide for benefits attributed to the service date, but also those expected to he earned in the future. TREASURER'S REPORT 37 Actuarial present value of accumulated benefit obligation including vested benefits of $ 1 ,5 14,493 as of January 1 , 1987 $1.566.992 Projected benefit obligation 2,328, 1 90 Plan assets at fair value 2,371,245 Projected benefit obligation less than plan assets 43,055 Unrecognized net (gain) or loss 68.948 Prior service cost not yet recognized in net periodic pension cost Unrecognized net obligation at December 31, 1987 (293.525) Prepaid pension cost (pension liability) recognized in the statement of financial position $ (181,522) Net pension cost for fiscal year ending December 31, 1987: Service cost — benefits earned during the period 1 72,293 Interest cost on projected benefit obligation 1 57,845 Actual return on plan assets 600 Net amortization and deferral (231.898) Net periodic pension cost $ 98.840 The actuarial present value of the projected benefit obligation was determined using a discount rate of 6.5% and rates of increase in compensation levels of 6%. The expected long-term rate of return on assets was 8%. In addition, the Laboratory participates in the defined contribution pension program of the Teachers Insurance and Annuity Association. Expenses amounted to $103,386 in 1987 and $106.535 in 1986. E. Pledges and Grants: As of December 31, 1987 and 1986, the following amounts remain to be received on gifts and grants for specific research and instruction programs, and are expected to be received as follows: December 31, 1987 December 31. 1986 Unrestricted Restricted Unrestricted Restricted 1988 $10,000 $ 951,400 $10,000 $40.764 1989 596,000 5,764 1990 250,000 — $10,000 $1.797.400 $10.000 $46.528 F. Interfund Borrowings: Intertund balances at December 3 1 are as follows: Current funds 1987 1986 Due from plant funds $ 3.240 $ 76,275 Due to restricted endowment fund (120,875) (115.909) Due to restricted quasi-endowment funds (3.000) (200,750) Due from current restricted fund 64.318 64.318 S (56.317) $(176.066) G. Mortgages and Notes Payable: The mortgage note payable with a term of 26 years is in the amount of $1.3 million bearing interest based on the bank's prime rate plus three quarters percent (.75%) on a floating basis for the initial five year period with a floor of 7.50% and a ceiling of 13.00%. The interest rate at December 31, 1987 was 9.75%. The mortgage loan is collateralized by a first mortgage on the land and properties known as Memorial Circle, with recourse in the event of default limited to this land and property and the related revenue. Monthly principal and interest payments of $15,000 commenced January 19, 1987. Other notes payable consist of the following: Unsecured note with interest at 7.90% with monthly principal and interest payments of $221.20 $ 7,048 Unsecured note with interest at 6. 90%> with monthly principal and interest payments of $394.71 8.901 $15,949 At December 31. 1987. these mortgages and notes payable had aggregate future annual principal payments as follows: Amount 1988 $ 78,654 1989 83-899 1990 86-492 1991 94,127 199i 102.957 Thereafter 817,840 1,263,969 Less current portion 78,654 $1.185.315 - M \RI\1 BIOLOGICAL LABORATORY H. The following is a summary of the cost and market value of investments at December 31, 1987 and 1986 and the related investment and distribution of investment income lor the \carsended December 31, 1987 and 1986. Endowment and Quasi-endou mem U.S. Government set unties Corporate fixed income Common stocks Preferred stock Money market securities Real estate Total Less custodian fees I 'nri">lricti'd Current Fund Money market securities Total investments 1987 $1.299.763 :. 136.500 4.08S.042 9,611 1.292,097 15.749 8,841.762 850.000 $9.691,762 1986 $2.278.843 947,287 4.069.893 9,611 639,155 15.749 7.960.538 50.000 $8.010.538 1987 $1,270.857 2.134.363 5,545,523 14,473 1.287,520 15.749 10.268.985 850.000 $11.118.985 1986 $ 2.384.842 933.749 6,101.973 17,975 639.155 15.749 10,093.443 50.000 $10.143.443 ln\c\tmenl Income 1987 $218.919 1(14.583 204,776 1.019 42.91 1 572.208 (54.240) 517,918 40.360 $558,278 1986 $240,056 85.871 178.837 3.214 29.667 537,645 (51.646) 485,999 35.824 $521.823 I. Gift Support for Instructions: Unrestricted Gifts includes $314,309 of gifts for the support of the Laboratory's instruction program available for indirect costs attnbutable to the instruction program. REPORT OF THE LIBRARIAN 39 VIII. Report of the Librarian "A man will turn over half a library to write one book" — if so, scientists throughout the world, including 34 Nobel Prize winners, have "turned over" the MBL Library an amazing number of times in the past 100 years. When established in 1 888. Dr. Whitman, the labo- ratory's first Director, stated in his first report: A library is a necessity in such a laboratory. Boston librar- ies are near enough to be of great service, but we cannot depend on them alone. In addition to textbooks and stan- dard works, we MUST have, at a minimum to begin with, all the important journals now printed in the four principal languages. It is most earnestly to be hoped that adequate means may be found to meet this all-important requisite. Today, one hundred years later, the library has 5000 sep- arate titles of scientific journals, printed in 30 languages, bound in 165,000 volumes. Somehow, over the years "adequate means" have been found to expand the collec- tion to its present size and to keep it available to the sci- entific community on a 24-hour basis. In 1888 an "in- valuable addition" of a $1,000 gift gave the journal col- lection its beginning ("owing to Mrs. Glendower Evans liberality"); in 1988 the completion of an endowment gift of $2.5 million ensures its future. Until 1902 the library depended entirely on donations for books and subscription costs. The 1902 treasurer's report lists the first library expense — $9.29. The Labora- tory did not commit real funds until 1 905 when Cornelia Clapp served as librarian during the summer months. In 1913 it was recommended that a trained assistant be hired to carry on throughout the year "a systematic cam- paign to develop the library since the peculiarly free methods used in this library with free access to the shelves demand constant study to take account of losses, repairs and disarrangements." In 1919 Priscilla B. Montgomery was hired as a year- round Assistant Librarian. The following year the Dewey Decimal Classification system was introduced "after careful consideration of the various schemes in general use — it seems to be the one most familiar to biologists." The main part of the Lillie building was completed in 1924, and a major portion of it was devoted to the Li- brary, which consisted of five floors of stacks and a large reading room with serial racks along the walls. Three hundred and seventy-eight titles were received at the time. Deborah Lawrence was hired as secretary, and the three staff salaries totaled $5,650 for the year. In 1929 The General Education Fund gave $50,000 for the purchase of back sets, serials, and books, and the following year the Director of the newly founded Woods Hole Oceanographic Institution donated $5,000 for the purchase of oceanographic books and journals. Ten years later a gift from the Rockefeller Foundation of $1 10,000 made the addition of the library wing possible. MBL was asked to raise from other sources an additional $25.000 to fill gaps in the journal collection (back sets). The addition cost less than planned, and the extra money was spent "to rebuild the Eel Pond wall, install a book- lift in the stacks, pave the parking lot, and re-grade the lawn around Lillie." The basement floor of the new li- brary wing would house "temporarily, apparatus for ster- ilizing glassware and distilling water." A $25.000 gift was received in 1941 from Carnegie for purchase of foreign journals whenever the market opened again in Europe. Library purchases were limited to the United States and England throughout the war years, although subscriptions were continued for Ger- man. French, and Russian publications. They were stored in a Scandinavian country, and by 1947 most had been received by the MBL Library. Mrs. Montgomery retired in 1947, and Deborah Law- rence was named Librarian. In 1952 WHOI contributed $1,200 annually for their books and journals. In 1956 the reprint collection passed the 200,000 mark, and the first Xerox machine appeared in 1963. The first major physical changes since 1940 were made in 1964, the year Jane Fessenden was named librarian. Tile flooring was placed on all five stacks, and fluorescent lighting was added in the stacks, reading rooms, and offices. Reserve desks received locked cabinets and new lights, and four labs were renovated into library offices. A xerox room and a Rare Book Room were also added to the office area. In 1966 the book collection was recataloged from the Dewey system to Library of Congress. Fifteen thou- sand books changed numbers and positions on the shelves, confusing most of the users. Twelve private car- rels were added on the third floor. By 1970, WHOI was contributing $15,000 annually and in 1971 the reprints were moved to the basement stack and further collection of reprints ended. The collec- tion numbered 250,000 when moved. At the same time, the entire journal collection was moved to cover four stacks instead of three and, we changed the arrangement of titles by dropping the articles "the, les, des," etc. The book collection expanded in 1971 when the MBL Associates gave their annual gift to the Library — $10,500. The following year they donated another $6.000 which was also added to the growth of the book collection. At the same time WHOI added $25,000 spe- cifically for books. Space was not available in the existing book section, therefore Room 306 on the third floor (the equivalent of three laboratories) was renovated for li- brary stacks. Books on ocean engineering, physics, math- ematics, and marine policy were shelved there. In 1 975 the first annual meeting of the East Coast Ma- rine Science Librarians was held in Woods Hole; 49 li- brarians representing 25 institutions met to discuss areas of mutual cooperation. Today membership numbers 140, representing 120 institutions. That same year H. M. 40 MARINE BIOLOGICAL LABORATORY Hirohito. Emperor of Japan, came to Woods Hole and spent an hour in the library catalog room. All offices, reading rooms, and hallways were painted for the occa- sion. The library budget was increasing rapidly, and a num- ber of proposals were made to further involve the Woods Hole Oceanographic. One proposal, submitted to the members of the Corporation in 1976. recommended the creation of a separate corporation for the library, sup- ported by both MBL and WHOI and governed by a sepa- rate Board of Trustees. This met with such opposition from the members that the annual meeting was re-con- vened three times before final adjournment. Not until 1 979 was an agreement finally reached. It was a cost shar- ing plan in which both institutions share certain activities of the library on a 50-50 basis. That agreement is still in effect today. That same year the R. K. Mellon Founda- tion gave $450.000 for the renovation of the Lillie build- ing. The library was included in that renovation. Demo- lition and construction began by 1982. The library now covers about one-half of the I.illie building; eleven labo- ratories were torn down and an area of the third floor, over the present library space, was converted to stack space for the entire book collection. All five floors of ex- isting stacks now house the journal collection, and the Rare Books and Archives occupy three rooms on the first floor. In 1983 we initiated a User Survey. This involved re- cording the use of every journal for a nine month period. We found, in brief, that 53% of the 4765 titles were used during this period and 76c'r involved the years 1980- 1983. In 1985, after years of discussions, the National Marine Fisheries Service in Woods Hole placed their li- brary collection in the MBL Library, thus making the MBL Library the main library for all four institutions in Woods Hole. The scientists at the Fisheries have been using the library since the founding of the laboratory. The minutes of 1889 record that the Fisheries contrib- uted both books and pamphlets to the original collection compiled that first summer of 1 888. The uniqueness of this library lies in the fact that for 100 years scientists — members of the MBL Corpora- tion— have / the Marine Biological Laboratory at II n,>/c Where else would an idiosyncratic worker like me linit ti hhrarv open all the lime, free from the rules and bureaucracy that si i fie scholarship and 'protect' books only by guarding them from use. It is an anomaly in it suspn nn<\ iindummvmous age. May it survive as it is. despite all the improbabilities. " \\. Educational Programs Summer BIOLOGY OK PARASITISM ( 'i>ur\c directors K.nglund, Paul I ., Johns Hopkins University School of Medicine Sher, Alan, NIAID/NIH Other faculty, siatl. ami lecturers Beverley, Stephen. Harvard University School of Medicine Bloom, Barry, Albert Einstein College of Medicine of Yashiva University Boothroyd, John, Stanford University Medical School Burakoff, Steven J., Harvard University School of Medicine Burns, James M., Jr., Hahnemann Medical College & Hospital Butterworth, Anthony, University of Cambridge. UK Cerami, Anthony, Rockefeller University Coffman, Robert, DN'AX, Research Institute of Molecular & Cellular Biology Doering, Tamara L., Johns Hopkins University School of Medicine Donelson, John, University of Iowa Dvorak, James, NIAID/NIH Dwyer, Dennis, NIAID/NIH Karley, Patrick J., Hamilton College Germain, Ronald, NIAID/NIH Glaven, Judy, George Washington University I lart, Gerald \V., Johns Hopkins University School of Medicine Ilenkle, Kim, University of Iowa Hereld, Dale, Johns Hopkins University School of Medicine Joiner, Keith, NIAID/NIH Kumar, Nirbhay, Johns Hopkins University School of Hygiene & Public Health Long, Carole A., Hahnemann Medical College & Hospital Mai/els, Richard, Imperial College of Science and Technology. London. UK Martimv-Palomo, Adolfo, National Polytechnical Institute. Mexico Miller, Louis, NIAID/NIH Modi, < ,m mil, Yale University School of Public Health Nelson, George, Liverpool School of Tropical Medicine, UK Nusseimvcig, Victor, New York University Medical Center Nutiiian. Thomas, NIAID/NIH Pearce, Kdward J., NIAID/NIH Pedersen, Peter, Johns Hopkins University School of Medicine EDUCATIONAL PROGRAMS 41 Pereira, Miercio, Tufts University School of Medicine Pfefferkorn, Elmer, Dartmouth College Medical School Quinn, Thomas, NIH/Johns Hopkins University Sacks, David, NIAID/NIH Scott, Phillip, NIAID/NIH Shapiro, Terry, Johns Hopkins University School of Medicine Shevach, Ethan, NIAID/NIH Smith, Cassandra, Columbia University Sollner-VVebb, Barbara, Johns Hopkins University School of Medicine Spielman, Andrew, Harvard School of Public Health Turner, Mervyn J., Merck, Sharp & Dohme Research Laboratory Walters, Laurel, Yale University School of Public Health Wang, Ching C, University of California, School of Pharmacy, San Francisco Wang, Charlotte, University of California, Berkeley Ward, Samuel, Carnegie Institution of Washington Warren, Kenneth S., The Rockefeller Foundation Wassom, Donald L., University of Wisconsin School of Veterinary Medicine Wellems, Thomas E., NIAID/NIH Young, Richard, Massachusetts Institute of Technology Students Acosta-Gio, Enrique A., National University of Mexico, Mexico Alvarez, Raquel M., Jewish Hospital of St. Louis Aslund, Lena A., Uppsala University, Sweden Barry, Wendy C., International Lab. for Research on Animal Diseases, Kenya Cerami, Carla J., Columbia University Gordon, Dalia L., University of Washington Herwaldt, Barbara L., Washington University School of Medicine Karam, Marc V., Onchocerciasis Control Programme in W. Africa, W. Africa Klinkert, Mo Q., University of Heidelberg, FRG Mani, Sridhar, City College, CUNY Prioli, Reginaldo P., Tufts University School of Medicine Rossi, Cesare, California Institute of Technology Sherman, David R., Vanderbilt University Sjolander, Anders J., University of Stockholm, Sweden Slatter, Andrew F. G., Oxford University, UK Tendler, Miriam, Oswaldo Cruz Institute, Brazil EMBRYOLOGY: A COURSE IN MODERN DEVELOPMENTAL BIOLOGY Course directors Brandhorst, Bruce, McGill University, Canada Jeffery, William, University of Texas, Austin Other faculty, staff, and lecturers Assman, Sally, University of Connecticut Bates, William R., Kyoto University, Japan Beach, Rebecca, University of Texas, Austin Bloom, Theodora, Cambridge University, UK Burdsal, Carol, Duke University Chambers, Edward L., University of Miami Cheng, Andrew, NIAID/NIH Colman, Alan, University of Warwick, UK Crowther, Robert, Marine Biological Laboratory Elinson, Richard, University of Toronto, Canada Etkin, Larry, M. D. Anderson Hospital and Tumor Institute Ford, Christopher, Sussex University, UK Gimlich, Robert, M. D. Anderson Hospital and Tumor Institute Grainger, Robert, University of Virginia Gurdon, John, Cambridge University, UK Humphreys, Tom, University of Hawaii Hunt, Tim, Cambridge University, UK Iwao, Yasuhiro, Yamaguchi University. Japan Jaffe, Laurinda, University of Connecticut Health Center Jaffe, Lionel, Marine Biological Laboratory Kado, Raymond T., C. N. R. S., France kitajima, Takashi, University of California, Berkeley Kline, Douglas, University of Connecticut Health Center Koenig, Gerd, Max Planck Institute. FRG Krieg, Paul, University of Adelaide, Australia Longo, Frank, LIniversity of Iowa Lorraine, Anne, University of Texas, Austin Mailer, James, University of Colorado Masui, Yoshio, University of Toronto, Canada Maxson, Robert E., University of Southern California School of Medicine Melton, Douglas, Harvard University Olins, Joshua, Earlham College Ouellette, Francis, McGill University, Canada Perreault, Sally, EPA, Division of Developmental Biology Poccia, Dominic, Amherst College Raff, Rudolf A., Indiana University Ruderman, Joan, Duke University Saavedra, Carol, McGill University, Canada Sargent, Thomas, NIH Satoh, Noriyuki, Kyoto University, Japan Shibuya, Ellen, University of Toronto, Canada Sluder, Greenfield, Worcester Foundation for Experimental Biology Solursh, Michael, University of Iowa Specksnyder, Johanna, University of Utrecht, Netherlands Stephens, Laurie, University of California, Berkeley 42 MARIM BIOLOGICAL LABORATORY Swalla, Billie J.. Universitv of I c\as. Austin Irinkaus, John IMiilip, Yale Universitv Turner. Paul. Universitv ofCalifornia. Berkeley Vacquier. Victor, Umvi "sii\ ot California, San Diego Velleca. Mark A.. \\ ashmgton University School of Medicine Venuti. Jud>. University of Texas, -\ustin \\augh. I.arr>, Brandeis University \\ hitc. Mar\ E., Universitv of Texas. Austin \N hittakiT, .1. Richard, Marine Biological Laboratory Wilt. Fred II. University ofCalifornia. Berkeley \\ inkier. Matthew M., University of Texas. Austin /ucker. Robert S.. University ofCalifornia. Berkeley Students Beanan, Maureen.]., Indiana University Beer. Donna M.. University of Massachusetts. Amherst Bloom. Theodora I..**, University of Cambridge, UK Coffin, .1. Douglas, SUNY Health Science Center at Syracuse Cox, Cher\l A., Indiana Universitv Dnsdale. Thomas A., I 'niversitv of Toronto, Canada Foster, Barbara A., University ofCalifornia. San Francisco Gewalt. Sally L., University of North Carolina Govind, Shubha. Princeton Universiu Man, ,Iin K., I'niversity ofCalifornia. Davis Jongejan-Zivkovic, Danica I)., University of Utrecht, Netherlands Kdl\, dregon M.. I 'niversitv of Manitoba. Canada Koenig, (ierd**, Max-PIanck-lnstitut. FRG Mandlev. Fli/.aheth N., University of California. Riverside Miranda, Louis M., I 'diversity of Texas Health Science Center. Dallas Monpetit, Isabelle, McGill University. Canada Molla. Chiara Maria, University of Naples. ItaK Niemever. Christina ('., Baylor College of Medici ne Nuelle, .Ion R.. I diversity of Texas, Austin SatU-rwhilc, Lisa I... Johns I lopkins I 'nivcrsity Shillinu, Traser M., University of Southern California Simoneini, I in iana. I inveiMlN ol Washington Sturm. Karin S.. I inns Pauling Institute of Science & Medicine Vond. Bruce I . Rui^i-rs I 'mversitv \ o(-el, .laeahn, Illinois Stale I 'mversitv /.hanu, \\i-i \\ /., I mversitv of Texas Health Science Center. Houston MVRIM I ( Ol.OGY Course director Trank, Peter \V., University of ( )ie>:on * Advanced Research Training Program I'artu ipanis Other faculty, staff, and lecturers Allt-r. Robert, SUNY. Stony Brook Buss, Leo \\ '., Yale University Caraco, Nina, Institute of Ecosystem Studies. Can. Arboretum Carlton, James, \\ illiams College Caron, T)a\id A., Woods Hole Oceanographic Institution Caswell. Hal. Woods Hole Oceanographic Institution Ca^anaugh, Colleen, Harvard College C'ole, Jonathan J., Institute of Ecosystem Studies. Cary Arboretum Da\is, Cabell S., Woods Hole Oceanographic Institution Harrington, John \V., Woods Hole Oceanographic Institution Foreman, Kenneth, BUMP/Marine Biological Laboratory Freadman, Marvin, BUMP/Marine Biological Laboratory Fry, Brian, Marine Biological Laboratory Gallagher, Eugene P., University of Massachusetts, Boston (iiblin. Anne. Marine Biological Laboratory llartman. Jean Marie, Harvard Universitv Jenkins. \Yilliam J., Woods Hole Oceanographic Institution Mann, Kenneth, Bedford Institute of Oceanography. Canada Marey, Maribel, Vassar College Myers, Phillip F... University of South Carolina Nixon. Scott. I 'niversity of Rhode Island Osman, Richard \\ ., Philadelphia Academy of Natural Sciences Peterson, Bruce J., Marine Biological Laboratory Petraitis, Peter S., University of Pennsylvania Plait, Trevor, Bedford Institute of Oceanography. Canada Porter, James, University of Georgia Porter Karen, Universitv of ( ieorgia Pregnall, Marshall A., Vassar College Reiniseh, Carol 1,., I lifts University School of Veterinary Medicine Rhoads, Donald ('., Science Applications International Corp. Rice. Donald, Chesapeake Biological Laboratory. LJniversityof Maryland Roth, Nina, Vassar ( 'ollege Sarda. Raphael, BUMP/Marine Biological 1 aboralon Striekler, Rudi, BUMP/Marine Biological Laboratory \ aliela, I., BUMP/Marine Biological Laboratory Students Bisbal. (iiislavo A., Instituto Nacional de Investigacion v Desarrollo, Argentina EDUCATIONAL PROGRAMS 43 Birne, Patricia P. B., University College, Ireland Charrier Melillan, Maria Elena, Universidad Nacional de Mar del Plata. Argentina DelPArciprete, Olga Patricia, Institute Nacional de Investigation y Desarrollo. Argentina Fernandez, Miriam E., Institute de Biologica Marina y Pesquera "A. Storni," Argentina krishnan, Thankavel, Annamalai University. India Roberts, Michael S., Wesleyan University Sadovsky, Sebastian, Universidade Federal do Espirito, Brazil Shierwater, Bernd, Braunschweig University, FRG Varela, Diana E., Centro Nacional Patagonico, Argentina MICROBIOLOGY: MOLECULAR ASPECTS OF CELLULAR DIVERSITY Course directors Wolfe, Ralph, University of Illinois Greenberg, Peter, Cornell University Other faculty, staff, and lecturers Berg, Howard C., Harvard University Blakemore, Richard, University of New Hampshire Cordts, Marcia L., Cornell University Dimarco, Anthony A., University of Illinois Dore, Joel, University of Illinois Dworkin, Martin, University of Minnesota Frankel, Richard, Massachusetts Institute of Technology Gertman, Eva, Queen's University, Canada Gest, Howard, Indiana University Gibson, Jane, Cornell University Kashket, Eva, Boston University School of Medicine Konisky, Jordan, University of Illinois Kropinski, Andrew M., Queen's University, Canada Krulwich, Terry, Mt. Sinai School of Medicine Marrs, Barry, E. I. DuPont De Nemours & Co. Michel, Tomas A., University of California, Davis Mulligan, Martin E., University of Chicago Olson, Karl, University of Illinois Saulnier, Michelle, Queen's University. Canada Scolnick, Pablo, E. I. DuPont De Nemours & Co. Shapiro, Lucy, Columbia University Spormann, Alfred M., Philipps Universitat, FRG Stetter, Karl, University of Regensberg, FRG Thayer, Rudolf, Phillips Universitat. FRG Widdel, Friedrich W., University of Illinois Students Albertson, Nan H., University of Goteborg, Sweden Arnold, Robert G., University of Arizona Boehme, Susan E., North Carolina State University Conway, Noellette M., Woods Hole Oceanographic Institution Garcia-Pichel, Ferran, University of Oregon Henry, Elizabeth A., Harvard University Holden, Eric G., University of Massachusetts, Amherst Hughes, Robert E., Yale University Kolibachuk, Dana, Cornell University Leisinger, Thomas, Mikrobiologisches Institut, Switzerland Mack, E. Erin, University of Puget Sound Moran, Mary Ann, University of Georgia Parales Rebecca E., Cornell University Roberts, A. Lynn, Massachusetts Institute of Technology Robins, Jeff P., University of Massachusetts Rood, Brian E., University of Florida Schauder, Rolf, University of Ulm, FRG Seeler, Jacob S., Boston University Sment, Karen A., University of Illinois Teiser, Markolf L. O., University of Oregon NEURAL SYSTEMS AND BEHAVIOR Course directors Carew, Tom, Yale University Kelley, Darcy, Columbia University Other faculty, staff, and lecturers Bate, Michael, University of Cambridge. UK Borst, Axel, Max-Planck Institute, FRG Bottjer, Sarah, University of Southern California, Los Angeles Byrne, John H., University of Texas Medical School Calabrese, Ronald, Emory University Cleary, Leonard, University of Texas Medical School Constantine-Paton, M., Yale University Dodd, Frank, Cornell University Eisner, Thomas, Cornell University Fernald, Russell D., University of Oregon Gorlick, Dennis L., Columbia University Hoskins, Sally, Columbia University Jacobs, Gwen, University of California, Berkeley Kent, Karla, University of Arizona Lasansky, Richard, Hebron Academy Levine, Richard B., University of Arizona Macagno, Eduardo, Columbia University Marcus, Emilie, Yale University Menzel, Randolf, University of Berlin, FRG Nusbaum, Michael, Brandeis University Smith, Brian H., University of California, Berkeley Stevens, Charles F., Yale University School of Medicine Streichert, Laura, University of California, Riverside Tompkins, Laurie, Temple University Van Essen, David C., California Institute of Technology Walsh, John P., University of California. Los Angeles Weeks, Janis C., University of California, Berkeley MARINE BIOLOGICAL 1 ABORAlORY \\ enning-Frxleben, Angela. L'niversity of Konstanz, FRG \\ illiams. Heather. Rock Idler University \\ > man, Robert . I.. v ttiversitj Students Born. Richard I .. Harvard Medical School Brainard. Michael S., Stanford University Braun. Got/. Institut fur Tierphysiologie. FRG Casagrand. Janet L., Case Western Reserve University Comfort. Nathaniel, Cornell University Corfas, Gabriel, Wei/mann Institute of Science. Israel F>ans. Bruce I)., Emory University Gallman, Eve A., University of North Carolina Gilbert. C'ole. Indiana University Gruner. Wendy, SUNY. Stony Brook Harty, I. Patrick. University of Pittsburgh Ito. Minami, Osaka University. Japan I.iberstat. Frederic. The Hebrew University of Jerusalem. Israel I.ubischer, .Jane I,., University of California, Los Angeles Matsumoto. Rae R.. Brown University Mendonca, Mary I., University of Texas, Austin Orchinik. Miles, Oregon State University Rinaman. I.inda M., University of Pennsylvania Wright, William G., Yale Universit\ Wutenbach. Robert Alan, Cornell University. NEUROBIOLOGY Course director Karlin. Arthur, Columbia University Other faculty, stall', anil lecturers Andrews, S. B., NINCDS. NIH Armstrong, Katie, Rice University Catterall. William, University of Washington School of Medicine Cepko, C'onnie, Harvard University (hark. Amitabh, Columbia University Cheng, loni, NINCDS/MH C'lapham. Da\id, Brigham and Women's Hospital C'laudio. I oni, Yale University C'orreira. l-rederick F., Albert Einstein College of Medium- : .1 i '-shiva Univcrsitv C /ajkowski, Cynthia. C'olumbia University Dale. Nicholas. ,mbia University Fhrlich. liarbara. n\ of Connecticut Fischbach, Gerald I)., ••> • hmgton University School of Medicine I- rank, F.ric, University of Pittsburgh Gadsh>. David ('., Rockefellci I nnersitv Hall. I.inda M., Albert I instcin ( ollegc of Medicine of Yeshiva University Hess. Peter. Harvard University Inoue. lomo, McGill University. Canada Jessell. Thomas. C'olumbia University Jones. Stephen W., Case Western Reserve University Kacsmarek. I... Yale University Kao. Peter, Columbia University I aiulis. Dennis, Case \\'estern Reserve University I.andis, Story. Case Western Reserve Universitv I.linas, Rudolfo, New York University Medical Center Mac kinnon, Roderick. Brandeis University Majerus. Phil. Washington Universitv School of Medicine Mandel, Gail, Tufts L'niversity School of Medicine Marder, Eve, Brandeis University Matsumoto, Steven, Harvard University Maue, Robert Alan, Tufts University School of Medicine Me Nab, Robert M., Yale University Miller. Christopher, Brandeis University Moosekar, Mark S., Yale University Rauola, Elio, Harvard University Reese, Thomas S., NINCDS/NIH. MBL Rosen, Ora, Memorial Sloan Kettering Cancer Center Schnapp, Bruce, NINCDS/NIH. MBL Schuetze, Stephen M., Columbia L'niversity Sheetz, Michael. Washington University Siegel, Ruth F., Case Western Reserve University Siegelbaum, Steven A., Columbia University Silman, Israel, Weizmann Institute of Science, Israel Smith, Carolyn, University of Pittsburgh Smith, Steve, Yale University Spudich, John, Albert Einstein College of Medicine of Yashiva University Stermveis, Paul C., University of Texas Health Science C "enter Teyler, T., Northeastern Ohio University Vallee, Richard, Worcester Foundation for Experimental Biology Van Wagoner, David R., Case Western Reserve University Vicini, Stefano, Georgetown University Students Aoki, Chiye, Cornell L'niversity Medical College Banin, Eyal, Hebrew University of Jerusalem, Israel Freed. Michael, NINCDS/NIH Gilford. Andrew N., St. Andrews University. Scotland Hsu. llsiao-l.an, Johns Hopkins University kernan. Maurice.!., University of Wisconsin l.armet, Yves, Centre National de la Recherche Scientific) ue. France Mer/dorf, Christa S., Harvard University Mul> III, FmilC., Duke University Medical Center Sands, Steu-n B., University of California. Irvine Van Vactor, l)a>id I... Jr., University of California, Los Angeles Vogel. Steven S., C'olumbia University EDUCATIONAL PROGRAMS 45 PHYSIOLOGY: CELL AND MOLECULAR BIOLOGY Course director Goldman, Robert, Northwestern University Medical School Other faculty, staff, and lecturers Albrecht-Buehler, Guenter, Northwestern University Medical School Asai, David J., Purdue University Bartles, James, Northwestern University Medical School Bloom, Kerry, University of North Carolina Broschat, Kay O., University of Miami School of Medicine Burgess, David, University of Miami School of Medicine Chisholm, Rex L., Northwestern University Medical School Chou, Ying-Hao, Northwestern University Medical School Collins, Christine, Worcester Foundation for Experimental Biology Dahl, Stephen, Wesleyan University Desrosier, David, Brandeis University Dessev, George N., Northwestern University Medical School Earnshaw, William, Johns Hopkins University Foltz, Kathy, Purdue University Fuchs, Elaine, University of Chicago Fukui, Yoshio, Northwestern University Medical School Giroux, Craig, NIEMS Goldman, Anne, Northwestern University Medical School Hammarback, James, Worcester Foundation for Experimental Biology Han, Peter S., Earlham College Helfman, David, Cold Spring Harbor Laboratories Hinds, Kristin, Showa University Research Institute Hinds, Lael, Colorado College Hughes-Fulford, Millie, NASA, University of California Medical Center, S. F. Jamieson, James, Yale University Kenna, Margaret, University of North Carolina Lindberg, Uno, University of Stockholm, Sweden Litman, Gary W., Showa University Research Institute Mayrand, Sandra, Worcester Foundation for Experimental Biology Me Knight, Steven, Carnegie Institution of Washington Morris, N. Ronald, Rutgers University Obar, Robert, Worcester Foundation for Experimental Biology Pederson, Thoru, Worcester Foundation for Experimental Biology Penman, Sheldon, Massachusetts Institute of Technology Petes, Tom, University of Chicago Pollard, Thomas, Johns Hopkins University Medical School Reinisch, Carol, Tufts University School of Veterinary Medicine Rich, Alexander, Massachusetts Institute of Technology Ruderman, Joan, Duke University Shamblott, Mike J., Showa University Research Institute Sloboda, Roger D., Dartmouth College Sluder, Greenfield, Worcester Foundation for Experimental Biology Smith, Allison M., University of Strathclyde, Scotland Spudich, James, Stanford University Steinert, Peter, National Cancer Institute Steinhardt, Richard, University of California, Berkeley Tlsty, Thea, University of North Carolina Vallee, Richard, Worcester Foundation for Experimental Biology Vikstrom, Karen L., Northwestern University Wieben, Eric D., Mayo Clinic, Rochester, Minnesota Wilson, Darcy B., Medical Biological Institute Yeh, Elaine, University of North Carolina Yin, Helen, Harvard Medical School Zeev, Avri Ben, Weizmann Institute of Science, Israel Students Barton, Nelson, R., University of Miami School of Medicine Berryman, Mark A., University of Virginia Carlos, Ruben, University of Hawaii Cowles, Elizabeth A., Michigan State University Curry, Alice M., Yale University Cyr, Janet L., University of Texas Health Science Center, Dallas Dohrmann, Cord E., Duke University Dolan, Liam, University of Pennsylvania Dong, Feng, Oregon State University Feng, Sunlian, Wesleyan University Ferber, Daniel M., Johns Hopkins University Hagstrom, James E., Mayo Graduate School of Medicine Harding, Fiona, University of Rochester Harding, Susan M., University of Alabama Healy, Aileen M., Tufts University Hughes-Fulford, Millie, NASA/University of California Medical Center Kronidou, Nafsika, Dartmouth College Kuppe, Andreas, University of Oregon Lee, Youngsook, University of Connecticut Liang, Bruce T., Harvard Medical School Mackey, Harris M., Columbia Medical School Miller, Rita K., Northwestern University 46 \I\RI\! BIOLOGICAL LABORATORY Hopper, George E., Harvard I niversity Racoosin, Esther I... I lurvurd Graduate School of Arts and Sciences Roberts. Denise M . .,TsUy of Virginia Roy. l.inda M.. V Ellis, Gordon \\ .. I 'niversity of Pennsylvania l.anni. Frederick, Carnegie-Mellon University I.ubv-Phelps. Katherine, Carnegie-Mellon University I.utz, Douglas A., Harvard University Salmon. Edward D., University of North Carolina Taylor, I). Lansing, Carnegie-Mellon University Commercial faculty Aikens, Richard. Photometries. Ltd. Brenner, Mel, Nikon. Inc. Chaisson. Richard. Olympus Corporation of America ( lav pool, David .]., Atlantex & Zieler Instrument Corp. Cohen, Daud, I 'niversal Imaging Corporation Esser. Hermann .L, Optical Elements Corp Goldherg, Michael, Research Imaging Systems, Inc. Hannanav, Wyndham, G. W. Hannaway Associates Ilinsch. .Jan. F:,. Lcitz. Inc. Howard, Michael, Quanta Systems, Inc. Keller, Ernst, irl Zeiss, Inc. klotsche. Rich., < < OHU, Inc. Km kiiliv K.I. , Spcx Industries, Inc. Mengers, Paul,Quante nrporation Olwell, Patricia, I I at/, liu . Ota, Boh. I riilenl 1 lectronics Presley. Phillip II., Carl Zeiss. Inc. Taylor, Richard, C'olorado Video Ihomas, Paul, I)A(II -M II \N ick, Rohert, Photonic Microscopy, Inc. Students Blumenleld. Hal. Howard Hughes Medical Institute (hen. Nong-Ruay, Cornell University Cheng, Foni. Marine Biological Laboratory /N I H Collin, Carlos E., NINCDS/NIH. Marine Biological Laboratory Dissing, Steen, University of Copenhagen. Denmark Faltermeier, Bernd, Carl Zeiss. Inc. Fink, Rachel D., Mount Holyoke College Frostig. Ron D., Rockefeller University Gibson, Sarah Frisken, Carnegie-Mellon University Holmes, Tim, University of Missouri Hutchison, Nancy .L, Fred Hutchinson Cancer Research Center Jamieson, James D., Yale University School of Medicine Lechleiter, James D., Tufts University School of Medicine Lowy, Robert Joel, National Institutes of Health Martin, James C., University of Alabama Pratt, Melanie, M., University of Miami School of Medicine Salzman, Gary C., Los Alamos National Laboratory Sardet, Christian, Station Zoologique. France Stump, Robert F., University of New Mexico School of Medicine Telzer, Bruce R., Pomona College I eragawa, Carolyn K., University of California. Irvine \Veiss, Dieter G., Technische Universitat Munchen. FRG Short Courses CELL AND MOLECULAR BIOLOGY OE PLANTS August 3- 15, 1987 Directors Dure, Leon S., University of Georgia Key, Joe L., University of Georgia Lecturers Binns, Andrew, University of Pennsylvania Chua, Nam-Hai, Rockefeller University Crouch, Martha, Indiana University Darvill, Alan, U niversity of Georgia Fraley, Rob, Monsanto Company Guilfoyle, Tom, University of Missouri I lallick, Richard, University of Arizona llaselkorn, Robert, University of Chicago Levings III, C'. S., North Carolina State University Long, Sharon, Stanford University Meagher, Richard, I inivcrsity of Georgia Meverowit/., Elliott, California Institute of Technology Quail, Peter, University of Wisconsin Ryan, Clarence A., Washington State University Siltlow, Carolyn, University of Minnesota I imberlake, \N illiam. University of Georgia EDUCATIONAL PROGRAMS 47 Verma, Desh Pal S., McGill University Wessler, Susan, University of Georgia Yoder, Olin, Cornell University Students App, Alva, A., Rockefeller Foundation Armbrust, Ginger, WHOI/MIT Basson, Bruce R., University of North Carolina Baumgarten, Miriam, Columbia University Becker, David W., Pomona College Brady, Kevin P., Indiana University Bruemmer, Joseph H., USDA Couch, Jennifer, Pennsylvania State University Diebold, Ronald, Marquette University Heeyong, Tai, Pennsylvania State University Jayne, Susan M., Ciba-Geigy Corp. Lahners, Kristine, Ciba-Geigy Corp. Palenik, Brian, WHOI/MIT Robertson, Borre, University of Tromso, Norway Rusnak, Suzanne, Parma City School. OH Sasavage, Nancy, Bethesda Research Laboratories Toenniessen, Gary H., Rockefeller Foundation Waddle, James A., University of California Ward, Michael R., University of California MOLECULAR AND CELLULAR IMMUNOLOGY August 3- 15, 1987 Course directors Reinisch, Carol, Tufts University School of Veterinary Medicine Wilson, Darcy, Medical Biological Institute Oilier faculty, staff and lecturers Bevan, Michael J., Scripps Clinic and Research Foundation Broedeur, Peter, Tufts University Hogg, Nancy, Imperial Cancer Research Fund, England Janeway, Charles A., Jr., Yale University School of Medicine Rabat, Elvin A., Columbia University Leskowitz, Sidney, Tufts University School of Medicine Morse, Herbert C, HI, NIH Mosier, Donald E., Medical Biological Institute Prendergast, Robert A., Johns Hopkins Hospital Rosenwasser, Larry J., Tufts New England Medical Center Springer, Timothy, Dana-Farber Cancer Institute Strominger, J., Harvard University Sunshine, Geoffrey, Tufts University School of Veterinary Medicine Valentine, Fred T., New York University Medical Center VVeissmann, Gerald, New York University Medical Center Winchester, Robert, New York, NY Wortis, Henry H., Tufts University School of Medicine Students Allen, Suzanne T., Worcester Memorial Hospital Carlson, David L., University of California, Davis Chang, Yueh-jong, Indiana State University Fitzgerald, Kathleen A., Bristol-Myers Harshan, K. V., All India Institute of Medical Sciences, India Hayflick, Joel S., Oregon Health Sciences University Marx, James J., Jr., Marshfield Medical Research Foundation Miller, Lynn, Hampshire College Pender, Daniel J., Columbia University Petty, Richard F., Brick Township School, Brielle, NJ Read, Dorothy L., Southeastern Massachusetts University Saugstad, Julie A., University of Oklahoma Tomlinson, Gail E., Children's Hospital National Medical Center Zaroogian, Gerald E., United States Environmental Protection Agency CELLULAR NEUROBIOLOGY IN THE LEECH August 5-25, 1987 Course director Nicholls, John G., University of Basel, Switzerland Oilier /acuity, staff, and lecturers Blackshaw. Susanna, University of Glasgow, Scotland Calabrese, Ronald, Harvard University Cohen, Lawrence G., Yale University School of Medicine Friesen, W. Otto, University of Virginia Kristan, William B., Jr., University of California, San Diego Macagno, Eduardo, Columbia University Muller, Kenneth J., University of Miami School of Medicine Payton, Brian W., Memorial University of Newfoundland, Canada Ross, William, New York Medical College Salzberg, Brian M., University of Pennsylvania Stent, Gunther S., University of California, Berkeley Weisblat, David, University of California, Berkeley Zipser, Birgit, Michigan State University Students Clarke, William P., Mount Sinai School of Medicine Gleizer, Lidia, University of California, Berkeley Gu, Xiao-nan, University of Miami School of Medicine Karrer, Tracy A., Yale University Nakagawa, Liria, University of Basel, Switzerland Passani, M. Beatrice, Columbia University Szczupak de Rodgers, Institute Nacional de Investigacion, Argentina Venable, Nancy L., University of Basel, Switzerland 48 \1\RINF BIOLOGICAL LABORATORY Wallace. Mark I .. Temple I 'imersitv \\cdcen. Cath> J.. Univcrvu »t ( aliforma. Berkeley U ittenberg. George F., I imcrsiu of California. San Diego Xiao. C'hun. Yale ' crsiiy School of Medicine HISTOm mOIOGYrllFRFDITY AND DEVELOPMENT August 2-15. 1987 Course directors Garland, Allen E., Washington University Fantini. Bernardino. University of Rome, Italy Maienschein, Jane, Arizona State University Other faculty, staff, anclkriwct •<, C'hurehill. Frederick, Indiana University Gilbert. Scott. S\\arthmore College Groeben, Christiane, Naples Zoological Station, Italy Grmek. Mirko, L'Ecole des Hautes Etudes, Le Sorhonne. France ()lb>. Robert, University of Leeds. UK Roe, Shirley A., Harvard University Students Aseuitto, James, Mahwah High School. NJ Bogin, Mar>, COrnell University Burian. Richard M., Virginia Polytechnic Institute and State University Cadwallader, Joyce V., Saint Mary -of-the- Woods College Cronin. Funice A., Belmont Abbey College De Jonghe-Mnrphy, Viviane, Stamford, CT Doering. Grant R., College of the Academy of the New Church Fausto-Sterling. Anne, Brown University Gariepy. Thomas P., Stonehill College Hammonds, Ftelynn M., Harvard University Howard, Heidi, Harvard University Jungck, John R., Beloit College Karustis, Marlene, Ml. St. Mary's Academy I .each, Berton J., Rockville. MD Lcwin. Susan ()., Shodair Children's Hospital I.)ons, SluTrie 1.., University of (Chicago Miles. Sara .Juan, Wheaton College Mvlott, Anne. ma I 'niversity Opit/, John M . >ntana State University Paraccr. Surindai <-ster State C'ollege Richardson, Robert t 'niversity of Cincinnati Sawin. Clark I ., I till-, ! * Mt\ Shaver, John R., 1 'niveisn Ho Rico Sloan, Jan Bntin, Kansas < n . \n institute Swetlit/, Marc, University of C'hicago \\ allev, \\ illis Wayne, Delta State University Weidman, Nadine M., Bryn Mawr College \\ inisatt, \\ illiam. University of C'hicago \. Research and Training Fronrams Summer Principal Intcstigators Alford, Simon, St. Georges Hospital Medical School, U. K. Anderson, Winston A., Howard University Angstadt, James D., Emory University Vrmstrony. Clay M., University of Pennsylvania Armstrong, Peter B., University of California. Davis Arnold, John, University of Hawaii Atwood, Kim ha 1 1. Woods Hole, MA Augustine, George, University of Southern California Barlow, Robert B., Jr., Syracuse University Barry, Daniel, University of Michigan Barry, Michael A., Albert Einstein College of Medicine of Yeshiva University Barry, Susan R., University of Michigan Bass, Andrew, Cornell University Beauge, Luis A., Institute de Investigacion Medica, Argentina Begenisich, Ted, University of Rochester Bennett, Michael V. L., Albert Einstein College of Medicine of Yeshiva University Be/anilla, Francisco, University of California, Los Angeles Bloom, George S., University of Texas Health Science Center. Dallas Bod/nick, David, Wesleyan L' niversity Borgese, Thomas A., Lehman College Boron, Walter F., Yale University School of Medicine Borst, David \\ ., Illinois State University Brady, Scott T., University of Texas Health Science Center. Dallas Brehm, Paul, Tufts University School of Medicine Bre/ina, Vladimir, University of California, Los Angeles Brown, Joel F., Washington University School of Medicine Burdick, Carolyn J., Brooklyn C'ollege Burger, Max M., University of Basel, Switzerland Burgos, Mario, I 'niversity ofCuyo, Mendoza, Argentina Butt, Arthur M., East Carolina University School of Medicine (hang, Donald ('., Baylor College of Medicine C happell, Richard I.., Hunter College Charlton. Milton P., University of Toronto, Canada Cinelli, Angel R., University of Pennsylvania School of Dental Medicine Clark, John M., Universitv of Massachusetts. Amherst Cohen, Avis II., Cornell University Cohen, Lawrence B., Yale Universitv School of Medicine Cohen, \> illiam I)., Hunter College ( oopcrslein, Sherwin J., University ofConnecticut Health Center RESEARCH AND TRAINING PROGRAMS 49 De \Veer, Paul, Washington University School of Medicine Delaney, Kerry, Princeton University Dunlap, Kathleen, Tufts University School of Medicine Eckberg, \YiIliam E., Howard University Ehrlich, Barbara E., University of Connecticut Health Center Feinman, Richard D., SUNY Health Science Center, Brooklyn Eishman, Harvey M., University of Texas Medical Branch Gadsby, David C., Rockefeller University Gainer, Harold, NINCDS/NIH Gilbert, Daniel L., NINCDS/NIH Gilbert, Susan P., Pennsylvania State University Giuditta, Antonio, University of Naples, Italy Gonzalez-Serratos, Hugo, University of Maryland School of Medicine Gould, Robert M., New York State Institute for Basic Research Govind, C. K., University of Toronto, Canada Graf, Werner, Rockefeller University Greengard, Paul, Rockefeller University Haimo, Leah, University of California, Riverside Halvorson, Harlyn O., Brandeis University Hess, Stephen D., University of Southern California, Los Angeles Highstein, Stephen M., Washington University School of Medicine Hill, Susan D., Michigan State University Hoskin, Francis C. G., Illinois Institute of Technology Humphreys, Tom, University of Hawaii Ingoglia, Nicholas, New Jersey Medical School Josephson, Robert K., University of California, Irvine Kaminer, Benjamin, Boston University School of Medicine Keynan, Alexander, Hebrew University of Jerusalem, Israel Kornberg, Hans, University of Cambridge, UK Kriebel, Mahlon E., SUNY Health Science Center, Syracuse Landowne, David, University of Miami School of Medicine Langford, George M., University of North Carolina School of Medicine Lasek, Raymond J., Case Western Reserve University Laufer, Hans, University of Connecticut Lechleiter, James D., Tufts University School of Medicine Levin, Jack, University of California School of Medicine Levis, Richard A., Rush Medical Center Lewbart, Gregory A., University of Pennsylvania Veterinary School Lichtman, Jeff W., Washington University School of Medicine Lipicky, Raymond J., Food and Drug Administration Lisman, John, Brandeis University Llinas, Rudolfo, New York University Medical Center Loewenstein, Werner R., University of Miami School of Medicine Lorand, Laszlo, Northwestern University Malbon, Craig C., SUNY, Stony Brook Marcum, James, Harvard Medical School Matsumura, Fumio, Michigan State University Matteson, Donald R., University of Maryland School of Medicine McMahon, Douglas G., Harvard University Mendelson, Bruce, University of Pittsburgh School of Medicine Metuzals, J., University of Ottawa, Canada Moore, John W., Duke University Medical Center Miillins, Lorin J., University of Maryland School of Medicine Narahashi Toshio, Northwestern University Medical School Nelson, Leonard, Medical College of Ohio Noe, Bryan D., Emory University School of Medicine Pappas, George D., University of Illinois College of Medicine Pierson, Beverly K., University of Puget Sound Pumplin, David W., University of Maryland School of Medicine Purves, Dale, Washington University School of Medicine Quigley, James P., SUNY Health Science Center, Brooklyn Rakowski, Robert F., University of Health Sciences/ The Chicago Medical School Rebhun, Lionel I., University of Virginia Rickles, Frederick R., University of Connecticut Health Center Ripps, Harris, University of Illinois College of Medicine Rose, Birgit, University of Miami School of Medicine Ruderman, Joan, Duke University Russell, John M., University of Texas Medical Branch, Galveston Saez, Juan C., Albert Einstein College of Medicine of Yeshiva University Salzberg, Brian M., University of Pennsylvania Sanger, Jean M., University of Pennsylvania School of Medicine Sanger, Joseph W., University of Pennsylvania School of Medicine Segal, Sheldon J., Rockefeller Foundation Silver, Robert B., University of Wisconsin Sloboda, Roger D., Dartmouth College Smith, Stephen J., Yale University School of Medicine Speck, William T., Case Western Reserve University/ University Hospitals of Cleveland Steinacker, Antoinette, Washington University Medical School 50 \1\RIM BIOICXiK \l 1 \BOR\IORV Sluart. Ann V.., I 'mvcrsiu oi North Carolina Suprenant. kath> V.. I imcrsiU of Kansas lablin. Fern, Unncrvr ol California. Davis Telzer, Bruce R.. Pomona College lilnev. I.fwis(... rsit\ of Pennsylvania Treistman, Stt-u- V, Worcester Foundation for Experin, 'logy I rinkaus. John Philip. Yale University Troll. Walter, New York University Medical Center Tucker, Kdward B., Baruch College Tytell. Michael, Wake Forest University. Bowman Gray School of Medicine Vincent, Walter. University of Delaware Weiss, Dieter G., Technical University of Munich at Garching. FRG Weissmann, Gerald, New York University Medical Center Wiens, T. J., University of Manitoba. Canada W u. Jian->ouns, Yale University School of Medicine Yen. .Ja> /.., Northwestern University Zigman, Seymour, University of Rochester Medical Center /.ukin. R. Suzanne, Albert Einstein College of Medicine of Yeshiva University Other Research Personnel Abramson, Charles, Downstate Medical Center Adler, Elizabeth, University of Toronto. Canada Adra, Chaker N., University of Ottawa. Canada Alberghina, Mario, University of Catania. Italy Albrecht, Kenneth, University of Connecticut Altamirano, Anibal, University of Texas Medical Branch. Galveston Armstrong. Margaret, University of California. Davis Baccetti, Baccio, Howard University Baker, Margaret, Cornell University Baker, Robert G., New York University Medical Center Bamrungphol. Wattana, University of Pennsylvania Bern, Dwight, Howard University Blumer, Jeffrey L., Case Western Reserve University Breitwieser, Gerdna F., University of Texas Medical Branch, Galveston Brew ton, Kevin, Howard University Brosius, "..iimi. Albert Einstein College of Medicine of Yeshiva ' Diversity Brown. Vim. • ase Western Reserve University Browne, C'aro. • Forest University Bro/en. Reed, Yale rsity Buchanan, JoAnn, Y; I niversity School of Medicine Callawa>, Joseph C'harles, 1 'niversity of Washington ( aputo. Carlo, Instituto \Viii 'ol.i node Investigaciones Cientifican. Venezuela Cariello, l.ucio. /oological Station. Italv Chen, F.ric, Northwestern University Medical School C'how, Robert II., I Inivcrsity of Pennsylvania Cohen, Avrum, University of Chicago Cohen, Sarah R., Yale University Cole, Neil M., I 'niversity of Michigan C ontanche, Douglas A., Medical University of South Carolina C'ota-Penuelas, Gabriel, Universiu of Pennsylvania Couch, Krnest F., Texas Christian University Cruise, F.nid, Howard Universiu Dailey, Jessica, Northwestern Urmersit\ Da>idson, Sarah. New York University Medical Center Deak, Peter. University of Connecticut DiFranco, Marino, Central University of Venezuela I)i Polo, Reinaldo, Institute Venezolanode I in estigaciones Cientifican. Venezuela Dome, Jeffrey S., University of Pennsylvania School of Medicine Dowling, John F., Harvard Universiu Duffy, Steven, University of Toronto. Canada Duran, Carlos L., Lehman College Eatock. Ruth Anne, University of Rochester Ferkowic, Michael, Michigan State University Fink, Rachel D., Mount Holyoke College Flacker. Jonathan, University of Chicago School of Medicine Flores, Roberto, University of Pennsylvania School of Dental Medicine Forman, Robin, Medical College of Virginia Fox, Geoffrey Q., Max Planck Institut fur Biophysikalische Chemie. FRG Frank, Dorothy M., Case Western Reserve University School of Medicine Frederick. Judith I.., University of Puget Sound Freepong-Buodo, Anthoin. Howard University (iao, Pei-qing, Baylor College of Medicine Garcia, Richard, Baruch College George, Fdwin B., Case Western Reserve University Gershfeld, Norman, NIAMS/NIH Gill-Kumar. Pritam, Food and Drug Administration Grassi, Daniel, Ft. I auderdale Graubard, Catherine, Albert Einstein College of Medicine of Yeshiva University Greif, Peter C'., Food and Drug Administration Greiner, Francine, Emory University School of Medicine Grob, Marianne, Friedrich Miescher-lnstitut, Switzerland Gruner, John A., New York University Medical Center llamosh. I.eora Y., University of Michigan Ilaneji, Tatsuji, Population Council. Rockefeller I 'niversity Hernandez, Michael R., I muTsit\ of Texas Medical Bianch. Galveston I lerring, Alex McNcely, University of North Carolina Hill, Christine, Mount Holyoke College Ilines, Michael, Duke University Medical Center RESEARCH AND TRAINING PROGRAMS 51 Hogan, Emilia, Yale University School of Medicine Holmstrom, Diane, University of Puget Sound Homola, Kllen, University of Connecticut Hopp, Hans, Yale University School of Medicine Hollis, Vincent VV., Jr., Howard University Hunt, John R., Baylor College of Medicine Jumblatt, James, Tufts University kadam, Arjun L., Population Council, Rockefeller University Kaplan, Ehud, Rockefeller University Keller, Franz, Technical University of Munich at Garching, FRG Kissee, Linda, Illinois State University Khier, J., Albert Einstein College of Medicine of Yeshiva University Knudsen, Knud, Food and Drug Administration Koide, Samuel S., Population Council, Rockefeller University Kosik, Kenneth S., Brigham & Women's Hospital Landau, Matthew, University of Connecticut Lehman, Herman K., Syracuse University Leidigh, Christopher, Brown University Leopold, Philip L., University of Texas Health Science Center, Dallas Leuchtag, H. Richard, Texas Southern University Levitan, Herbert, W/L. Cohen Li, Hui, Illinois State University Lintl«ren, Clark A., Duke University Medical Center Llinas, Rafael, Washington University School of Medicine Lowe, Kris, New College of University of South Florida Luca, Frank, Duke University Luthi, Theres, University of Basel, Switzerland Maldonado, Pedro E., University of Miami School of Medicine Matsumura, Fumihiko, Michigan State University Mauney, Donald, University of North Carolina School of Medicine McGuinness, Teresa, Rockefeller University Mcllveen, Anita, University of Connecticut McKee, Juliet M., University of Kansas Menichini, Enrico, Institute di Biologia Cellulare, Italy Meyer, Monica A., Thomas Jefferson University Milgram, Sharon L., Emory University School of Medicine Misevic, Gradimir, Friedrich Miescher-Institut, Switzerland Murray, Sandra, University of Pittsburgh Mushynski, Walter E., McGill University, Canada Nealey, Tara A., University of Rochester Nicholas, Craig J., Syracuse University Nishio, Matomo, Northwestern University Medical School Obaid, Ana Lia, University of Pennsylvania School of Dental Medicine Oberhauser, Andres, University of Pennsylvania Osses, Luis R., University of Southern California, Los Angeles Palazzo, Robert E., University of Virginia Pant, Harish, NINDCDS/NIH Parsons, Thomas D., University of Pennsylvania Pearce, Joanne, University of Toronto, Canada Perozo, Eduardo, University of California, Los Angeles Porter, Charles VV., Rockefeller University Powers, Maureen K., Vanderbilt University Rasgado-Flores, Hector, University of Maryland School of Medicine Repucci, Anthony, Case Western Reserve University Requena, J., Institute Internacional de Estudios Avanzados, Venezuela Riesen, William, Yale University Robinson, Phyllis R., Brandeis University Robitaille, Yves, Montreal Neurological Institute, Canada Rodriguez, Richard, Baruch College Rooks, Arthur, University of North Carolina School of Medicine Rudolph, Rebecca E., University of Puget Sound Salvati, Serafina, N. Y. S. U. for Basic Research in Developmental Disabilities Sanchez, Ivelisse, Hunter College Sands, Peter J., New York University Medical Center Sanger, Jean M., University of Pennsylvania School of Medicine Schiminovich, David, Yale University Schneider, Eric J., Wesleyan University Schneider, Melissa R., Hamilton College Seitz-Tutter, Dieter, Technical University of Munich at Garching, FRG Shockley, Ronald, University of California, Irvine Spires, Sherrill, University of Rochester Spray, David C, Albert Einstein College of Medicine of Yeshiva University Stadler, Herbert, Max Planck Institut fur Biophysikalische Chemie, FRG Stokes, Darrell R., Emory University Sugimori, Mutsuyuki, New York University Medical Center Swandulla, Dieter, University of Pennsylvania Swenson, Katherine, Harvard Medical School Sydlik, Mary Anne, Syracuse University Tanguy, Joelle, Ecole Normale Superieure, France Tricas, Timothy C., Washington University School of Medicine Ueno, Hiroshi, Rockefeller University Vandenberg, Carol A., University of California, Los Angeles Vaysse, Pierre, Albert Einstein College of Medicine of Yeshiva University MARINE BIOLOGICAL LABORATORY Verselis, V\tautas, Albert I mstem College of Medicine of Yeshiva University Vilijn, Marie-Helene. Albert Einstein College of Medicine of Y^ nvcrsiu \ogel. Jacal>n M iern Illinois University Waxman. Stephen G., Yale University School of Medicine Webb, ( hi Ktina Kae, University of California. Los Angeles Weiss. .Jerry S., Northwestern University Medical School Weiss, Leo, Venice. CA YA estendorf. Joanne M., Duke L'niversity \\ hite, Roy L., Albert Einstein College of Medicine of Yeshiva University \\ hittembury. Jose, Case Western Reserve University- Xiao, C'hun, Yale University School of Medicine /.akevicius, Jane M., University of Illinois College of Medicine 7,a\ilowitz, Joseph, Albert Einstein College of Medicine of Yeshiva University /ecevic, Dejan, Institute of Biological Research. Yugoslavia /hang. Lan, Syracuse University- Library Readers Adelberg, L'dward A., Yale Medical School Alkon. Dan, NIH Allen, Garland, Washington University Anderson, Kverett, Harvard Medical School Apter. Nathaniel S., Nova University Bang, Betsy, MBL Bauer, G. Kric, University of Minnesota Medical School Boettiger, Julie, Temple University Buck, John, Nil I C'apeC'od Planning & Kconomic Development ( andelas, Graciela, University of Puerto Rico Carriere, Rita, Downstate Medical Center, SUNY Cathcart, lorn, Mississippi State University Child, Frank, I rimty College Chambers, Kdward L., University of Miami C'hinard. Francis P., New Jersey Medical School Churchill. I red. History of Biology Course Clark, \r. I, MBL Cobb, Jewel Hummer, California State University Cohen, Leonard \mericanHealthFoundation C'ohen. Se>mout Corliss. Brute I L, Dui- I niversity Dancis, Joseph. Neu YOT\ S. hool of Medicine Deloledo-Morrell, Leyla, Rush IVcslntcrian St. Lukes Medical (enter Dodge, Frederick A., IBM Research Duncan, I homas. Nichols College Kbert, James, Carnegie Institute of Washington Kder, Howard A., Albert Einstein College of Medicine Farb, David, SUNY Farmanfarmaian, A., Rutgers University Feingold, David S., New England Medical Center Fisher, Saul H., NYU Medical Center Frenkel, Krystyna, New York University Medical Center Friedler, Gladys, Boston University School of Medicine Fussell. Catharine P., The Pennsylvania State University Galatzer-Levy, Robert, University of Chicago German, James L., The New York Blood Center Gibbs, Terrell T., SUNY Health Science Center at Stony Brook Goldstein, Moise H., Jr., Johns Hopkins University. EECS Department Goodgal, Sol H., Pennsylvania School of Medicine Grant, Philip, University of Oregon Greelish, Stephen J., Liberty Mutual Research Center Grossman, Albert, NYU Guttenplan, Joseph B., New York University Dental Center Harding, Clifford V., Kresge Eye Institute. Wayne State University llaubrich, Robert, Denison University Herskovitz, Theodore T., Fordham University Hildehrand, John G., University of Arizona Hill, Richard W., Michigan State University Hoffman, Peter R. Hosteller, Karl Y., University of California Hughes-Fulford, Millie, Johnson Space Center Ilan, Joseph, Case Western Reserve University Man, Judith, Case Western Reserve University Inoue, Sadayuki, McGill University Jarvik, Murry F... U.C.L.A. Kaltenbach, Jane C., Mount Holyoke College Kaplan, Ilene M., Union College Karush, Fred, University of Pennsylvania Kelly, Robert, University of Illinois, College of Medicine Kemlo>v, Kenneth M., Wilkes College King, Kenneth, Jr., Children's Hospital Kisten & Babitsky, Private Lawyers Klein, David I.., University of California San Francisco Koulish, Sasha, College Staten Island, CUNY Krane, Stephen M., Massachusetts General Hospital Kravit/, F.dward A., Harvard Medical School .aderman. Aimlee, MBL .a/arow. Normand II., Mayo Clinic .each, Berton .1. «e, John J., City College NY .eighton, Joseph, Medical College of Pennsylvania .evine, Rachmiel, City of Hope Medical Center ,evit/., Mortimer, New York University Medical Center ,ewis, Larry, Millersville University RESEARCH AND TRAINING PROGRAMS 53 Luckenbill-Edds, Louise, Ohio University Maienschein, Jane, Arizona State University Marine Research Inc., Marine Research Inc. Mautner, Henry G., Tufts University School of Medicine McCann-Collier, Marjorie, Saint Peter's College McNabb, F. M. Anne, Virginia Polytechnic Institute & State University Mecurio, Arthur M., Harvard Medical School Mecurio, Kimberly, MBL Meinertzhagen, I. A., Dalhousie University Mitchell, Ralph, Harvard University Mizell, Merle, Tulane University Morrell, Frank, Rush Presbyterian St. Lukes Medical Center Moyer, Carolyn F., E. G. & G. Mason Research Institute Musacchia, X. J., University of Louisville Nagel, Ronald L., Albert Einstein College of Medicine Nickerson, Peter A., SUNY at Buffalo Nowotny, Alois H., University of Pennsylvania Olby, Robert, History of Biology Course Oschmann, James, MBL Ott, Karen J., University of Evansville Paton, David Person, Philip, VA Medical Center, Brooklyn, NY Pierce, Sidney K., University of Maryland Pollen, Don, University of Massachusetts Medical Center Prusch, Robert D., Gonzaga University Reiner, John M., Albany Medical College Reynolds, George T., Princeton University Ringer, Steven, Childrens Hospital Rosenkranz, Herbert S., Case Western Reserve University Roth, Jay S., University of Connecticut Rowland, Lewis P., Neurological Institute Rudmann, Daniel G., Kenyon College Russell-Hunter, W. D., Syracuse University SMU Library, Southeastern Massachusetts University Schippers, Jay, WAFRA Advisory Seaver, George, Seaver Engineering Shapley, Robert, Rockefeller University Shemin, David, Northwestern University School of Medicine Shepard, Frank, Woods Hole Data Base Shepro, David, Boston University Shriftrnan, Mollie Starr, North Nassau Mental Health Center Sluder, Greenfield, Worcester Foundation for Experimental Biology Spector, Abraham, Columbia University Spiegel, Evelyn, Dartmouth College Spiegel, Melvin, Dartmouth College Stephens, Philip J., Villanova University Stephenson, William K., Earlham College Stracher, Alfred, State University of New York Szent-Gyorgyi, Andrew G., Brandeis University Szentkiralyi, Eva M., Brandeis University Tashiro, Jay Shiro, Kenyon College Tellez, Alexander, Harvard University Jessie, Richard, University of Montreal Tilney, Lewis, University of Pennsylvania Trager, William, The Rockefeller University True, Merrill, MBL Tweedell, Kenyon S., University of Notre Dame Vaida, Akhil B., Hahneman University Van Holde, Kensal E., Oregon State University Wagenbach, Gary, Carleton College VVainio, Walter, Rutgers University Warren, Leonard, Wistar Institute W ebb, H. Marguerite, MBL Weidner, Earl H., Louisiana State University Wheeler, George, Brooklyn College Whittaker, J. Richard, MBL W ichterman, Ralph, MBL Wilber, Charles G., Colorado State University Williams, Wendy, Cape Cod Times Wirth, Dyann, HSPH/TPH Wittenberg, Jonathan, Albert Einstein College of Medicine Wittenberg, Beatrice, Albert Einstein College of Medicine Wolken, Jerome J., Carnegie Mellon University Yow, F. W., Kenyon College Zacks, Sumner I., The Miriam Hospital/Brown University Zigmond, Richard E., Harvard Medical School Zottoli, Steven J., Williams College Year-Round Programs Adelman, William J., Jr., Director, and Chief, Section on Neural Membranes, Laboratory of Biophysics, Ma- rine Biological Laboratory, NINCDS/NIH The Section on Neural Membranes concentrates on the bio- physical mechanisms underlying the conduction of the nerve impulse, synaptic transmission, and muscular transmission. The primary research animal is the Woods Hole squid, Loligo pealei. Staff Clay, John Fohlmeister, Jorgen Goldman, David Hodge, Alan Muller, Ruthanne Rice, Robert Ross, Darci Vick, Sherry Alkon, Daniel L., Chief, Section on Neural Systems, Laboratory of Biophysics, Marine Biological Labora- tory, NINCDS/NIH 54 M \RINI BIOLOGICAL LABOR \TORY The Section on Neural Systems studies the cellular basis of learning and memory InvestiiMtois relate changes in behavior caused by associative learning to biochemical and biophysical transformation of spo-itu nerve cells using a California nudi- branch. Hcrmi^, >rnis. Ku/irian. Alan l.ederhendler, Izju I.oturco, Joe Nelson, 1'om Tvndal, Cl\de Staff Bank. Bam Chen. Chong Collin. Carlos Garner. Lisa ilnpp. Hans Ikeno. Hide Boston I'niversity Marine Program Staff Halm. Dorothy. Administrative Secretary Sunle>, Madeline, Administrative Consultant Graduate students Alber. Merryl Banta, Gary Barshaw, Diana Borroni, Paola Brooks, Cydney Bnden, Cindy Chen, ('hong Corroto, Frank Costa, Joseph Coughlin, David Cowan, Diane Ellis, Sarah Elskus, Adria Foreman, Kenneth Gallager, Scott Click, Stephen Undergraduates Bower, Patricia Carlon, David Coburn, Cara C'orker, Amy Cromarty. Stuart Dawson, Steffany Dolun. ; Caspar, katli Mill. Chrisin. Howard, Katherine Hoffman, .Jennifer Hahn.Jill Hersh, Douglas Krieger, Yutta Lavalli, K.iri Mercurio, Kim Merrill, Carl Moore, Paul Mulsow, Sandor Scott, Marsha lamse, Armando Trager, Geoffrey Trott, Thomas Webb, .Jacqueline White, David Wood, Susan Kaska, David Kelly, Jennifer Marsich, Victor Me Walters, Brian Sammon, Leslie Santa Ana, Jeff Schad, Andrea Souders, Donna Taft, Natalie Weisman, Kitty /filer, Robert Visiting investigators D'Avanso, C'harlene, I lampshirc ( 'ollege Poole, Alan, Boston University Kietsma, Carol, SUNY. New Pall/ ' Note: All staff of Boston I 'mversiiv unless otherwise indicated. Atema. Jelle. Professor of Biology. Boston University Organisms use chemical signals as their main channel of in- formation about the environment. These signals are trans- ported in the marine environment by turbulent currents, vis- cous flow, and molecular diffusion. Receptor cells extract sig- nals through various filtering processes. Currently, the lobster with its exquisite sense of taste and smell is our major model to study the signal filtering capabilities of the whole animals and its narrowly tuned receptor cells. Research focuses on amino acids (food signals) and pheromones (courtship), neuro- physiology of receptor cells, and computer modeling of odor plumes. Staff Voigt, Rainer, Research Associate Freadman, Marvin, Visiting Assistant Professor of Biol- ogy, Boston University Principal research interests: comparative animal physiology, respiratory and circulatory physiology, physiology and hydro- mechanics of animal locomotion. Current research: ( 1 ) mecha- nisms of thrust production in marine fishes, drag relations in swimming fishes: (2) influence of hydrodynamic phenomena on circulatory and respiratory function in fishes: (3) determi- nants of circulatory system function in horseshoe crab. Linni- lus polyphemus; (4) locomotor muscle function at varying tem- peratures and levels of performance. Humes, Arthur G., Professor of Biology Emeritus, Bos- ton University Research interests include systematic^, development, host specificity, and geographical distribution of copepods associ- ated with marine invertebrates. Current research is on taxo- nomic studies of copepods from invertebrates in the tropical Indo-Pacific area, and poecilostomatoid and siphonostoma- toid copepods from deep-sea hydrothermal vents and cold seeps. Strickler, Rudi J., Professor of Biology. Boston Univer- sity Use high-speed cinematography and special laser light opti- cal systems with target tracking devices to observe zooplank- ton-algae. carnivorous-herbivorous /ooplankton. and fish-zoo- plankton interactions. Lab and field results show the degree to which abiotic forces influence the evolution of species, feeding guilds, and predator-prey interactions. Additional topics in the feeding ecology of crinoids. bryo/.oans and other suspension feeding invertebrates enhance perception of the first consumer level in the aquatic food chain. Staff ( 'ostello, John, Research Associate I .mini. Sidney I.., Associate Professor of Biology, Boston University We investigate the mechanism and control of diverse types of motility and behavior on the organismal, cellular, and sub- cellular levels. Techniques include clectrophysiology. microin- jection, laser microsurgery, cytochemistry and histochemistry, video-enhanced phase contrast and interference contrast mi- RESEARCH AND TRAINING PROGRAMS 55 croscopy, biochemistry, transmission and scanning EM, and freezer-etch EM. Other studies include the role of massive actin filament bundles, biology of prokaryotic-eukaryotic cell associ- ations, fluid dynamics and biomechanical coordination of cilia, and predator-prey interactions of herbivorous-carnivorous macroplankton. Staff Tamm, Signhild, Research Associate Valiela, Ivan, Professor of Biology, Boston University Research emphasis is on structure and function of salt marsh ecosystems and coastal embayments, including the processes of predation, herbivory, decomposition, and nutrient cycles. A parallel line of work, with more applied aspects, is eutrophica- tion in coastal marine communities and interactions between watersheds and coastal waters. Staff Dzierzewsky, Michelle, Research Assistant Lohmann, Denah, Research Assistant Surda, Rafael, Research Associate Taylor, Margery, Research Assistant \ 'isiting Investigators Woodward, Helen, Undergraduate Researcher Copeland, D. Eugene, Investigator, Marine Biological Laboratory Electron microscopy of luminescent organs (photophores) in deep sea fish; gas secretion in swimbladders of deep sea fish; and osmoregulatory tissue in Limiilus. The Ecosystems Center The Center was established in 1975 to promote research and education in ecosystems ecology. Nine scientists study the ter- restrial and aquatic ecology of a wide variety of ecosystems ranging from northern Europe (trace gas emission from acid- rain affected forests) to the Alaskan Arctic (long-term studies of the control tundra, lake, and stream biota) to Buzzards Bay (controls of anaerobic decomposition). Many projects, such as those dealing with sulfur transformations in lakes and nitrogen cycling in the forest floor, investigate the movements of nutri- ents and make use of the Center's mass spectrometry labora- tory (directed by Brian Fry) to measure the stable isotopes of carbon, nitrogen, and sulfur. The research results are applied wherever possible to questions of the successful management of the natural resources of the earth. In addition, the ecological expertise of the staff is made available to public affairs groups and government agencies who deal with such problems as acid rain, ground water contamination, and possible carbon diox- ide-caused climate change. There are opportunities for post- doctoral fellows. Administrative staff Hobble, John E., Director Helfrich, John V. K. Semino, Suzanne Griffin, Elisabeth A. Scientific staff Hobbie, John E., Senior Scientist Melillo, Jerry M., Associate Scientist Peterson, Bruce J., Associate Scientist Shaver, Gaius R., Associate Scientist Fry, Brian D., Assistant Scientist Giblin, Anne E., Assistant Scientist Nadelhoffer, Knute J., Assistant Scientist Rastetter, Edward B., Assistant Scientist Steadier, Paul A., Research Specialist Consultants Bowles, Francis P. Jordan, Marilyn J. Education staff appointments Bowden, Richard D., Postdoctoral Fellow Kling, George W., Postdoctoral Fellow Me Ivor, Carole, Postdoctoral Fellow Raich, James, Postdoctoral Fellow Technical staff Banta, Gary, Research Assistant Dornblaser, Mark, Research Assistant Downs, Margaret, Research Assistant Hooper, David, Research Assistant Kicklighter, David, Research Assistant Laundre, Jim, Research Assistant Me Kerrow, Alexa, Research Assistant Michener, Bob, Research Assistant O'Brien, Margaret, Research Assistant Regan, Kathleen, Research Assistant Tucker, Jane, Research Assistant Turner, Andrea R., Research Assistant White, David, Research Assistant Fein, Alan, Investigator, Laboratory of Sensory Physiol- ogy, Marine Biological Laboratory, and Department of Physiology, Boston University School of Medicine Physiology and biochemistry of invertebrate photoreceptors. Research into the inositol polyphosphate pathway in transduc- tion in Limiilus ventral photoreceptors in response to light and squid photoreceptors. Recent work: studying oxygen con- sumption in Limiilus ventral photoreceptors in response to light. Staff Payne, Richard, Assistant Scientist McBride, Jim, Technician Wood, Susan, Boston University Marine Program Graduate Student Grassle, Judith P., Senior Scientist, Marine Biological Laboratory Studies on the population genetics and ecology of marine invertebrates living in disturbed environments, especially of sibling species in the genus Capital/a (Polychaeta). Staff Mills, Susan W., Research Assistant 56 \I\RINE BIOLOGICAL 1 \BOR\IORY Hahtirson. Harlvn ().. President and Director. Marine Biological Laboratory Research focuses on the regulation of phosphate metabolism in Acineitihacier /u, i< ular. sv nthesis and utilization of polymerized inor-.i ic phosphate (polyphosphate), and the process of spori .non in Bacillus .tuhtilis. Staff Chikarmuiu. ilcrnant. Research Associate Pratt. Sara. Research Assistant I'isilin.u in\r*iis:aii>r\ Atwood. Kim. C'olumhia University kornbcrg. Huns. I 'niversity of Cambridge Ke\nan. Alev Memorial Sloan Kettering Cancer Center N incent. Walter, Universitv of Delaware Dole, Stephen, Science Writing Fellow. The Miami Herald llarosi. Ferenc I., Investigator. Associate Scientist. Ma- rine Biological Lahoraton,. Boston University School of Medicine Native and analogue visual pigment studies in situ. The ma- jor technique is microspeetrophotometry. Retinal photorecep- tors are obtained from amphibian, lizard, fish and primate eyes. Staff /.ahajs/k). Tiber, Research Associate Visiting //m'W;t,-<;;.'M Cornwall. Carter, Boston I 'niversity School of Medicine llawrvshvn. Craig \V., McMaster University. Hamilton. Ontario. Canada Retry, Heywood M., SUNY, Stony Brook Inoue. Shinya. Distinguished Scientist. Marine Biologi- cal Laboratory, and the University of Pennsylvania Mechanism of mitosis and related motility. Development of high resolution 3-D v ideo microscope systems. Staff \nniballi, D\on. Programming Engineer. Cornell University College of Engineering Bo\d. Steu-n, Programming Engineer. Cornell University College of Engineering Green. Daniel. Programming Engineer. University of Illinois, School of Engineering Inouc. I heodore. Programming Engineer. Cornell University College of Engineering Kiibinuu. .1 Programming Engineer. Cornell University College ol ring Shimomura. Sat... .iivh Assistant, Stanford University Taracka, Robert, 1 i \ssistant Woodward, Bertha M '.iiory Manager I 'isiting investigating Baji-r. Andrew S., University of Oregon Buruos, Mario H., University ol ( uyo, Mendo/a. Argentina l-ukiii, Yoshio, Northwestern University Medical School kit/hurt, Daniel P., Harvard Umversitv Salmon. Fdward I)., University ol North Carolina Sardt-l. Christian. Biol. Cell. Marine. Ville France-Sur-Mer Siher. Robert B., University of Wisconsin .Jatfe, Lionel, Senior Scientist. Marine Biological Labo- ratory. and Director. National Vibrating Probe Facility We are exploring the roles of ionic currents, gradients, and waves in controlling development. We focus on controls <)/ pat- tern and /T calcium ions. Staff Kuhtreiher, \Viel, Physiologist Shipley, Alan. I'echnician \\ illiams, Phillip C"., Engineer Speksnijder. Annelies. Postdoctoral Fellow. Royal Veterinary University. Denmark Asman, Sally, Harvard University H:iimi:mn. Steve, EPA. Research Triangle Park. NC Biggers, John, Harvard Medical School Bowdan, Fli/abeih, University of Massachusetts Chen, I sung-llsien, Academia Sinica. Taiwan C'ruwford, Karen, Swarthmore College ( ullander, Chris, University of California. S. F. Devlin, Leah, University of Rhode Island Diekc>, Joe. Clemson College Diehl-.Iones, \V. I.., University of Manitoba. Canada Fink, Rachael, Mount Holyoke College HoltuK, Klars, Royal Veterinary University. Denmark Kunkel, Joseph, University of Massachusetts Mladeno\, Phillip. Mount Allison University. New- Brunswick. Canada Payne, Richard. University of Maryland Pethin, Ron, University College of North Wales. U. K. Sardet. Christian. Station Marine Villelfranche sur mer. France I rinkaus, John, Yale University Trovell. Cindy, University of Colorado V'er Aehtert. Barend, Universitv ol Leuven. Belgium \\ vinan. Robert, ^'ale Llniversity I.eibovit/., Louis, Director. Laboratory for Marine Ani- mal Health. Marine Biological Laboratory, and Profes- sor, Department of Avian & Aquatic Animal Medicine. New York State College of Veterinary Medicine The laboratory provides diagnostic, consultative, research and educational services to the institutions and scientists of the Woods Hole community concerned with marine animal health. Diseases of wild, captive, and cultured animals are in- vestigated. Staff \bt, Donald A., Co-Investigator. University of Pennsylvania Bullis, Robert A., Senior Research Associate. Cornell University llansen, Sandra B., Secretary. Cornell University Mi-('atfert), Michelle, Histological Technician. Cornell University Moni/, I'riseilla ('., Administrative Secretary \\adnian. Fli/abeth A., Microbiological technician. Cornell Universitv RESEARCH AND TRAINING PROGRAMS 57 I 'isiiing investigators Koulish, Sasha, College of Staten Island, CUNY Lewbart, Gregory, University of Pennsylvania Rabinowitz, Michael, Investigator, Marine Biological Laboratory, and Instructor in Neurology, Harvard Med- ical School Measurement of lead in baby teeth to see if lead exposure at different ages, recorded at different sites within teeth, are re- lated to child development. Staff Lewandowski, Ann, Research Assistant, Harvard Medical School Reese, Thomas S., Chief, Laboratory of Neurobiology, Marine Biological Laboratory, NINCDS/NIH The Laboratory of Neurobiology is concerned with the secre- tory mechanism underlying synaptic transmission, the mecha- nism of organelle movement underlying axoplasmic transport, and the organization of neural cytoplasm. Staff Andrews, S. Brian, Research Associate Bechtold-Imhof, Ruth, Research Assistant Cheng, Toni, Research Associate Chludzinski, John, Research Technician Coyle, Jo-Anne, Secretary Gallant, Paul, Research Associate Garbus-Gooch, Cynthia, Research Assistant Hammar, Katherine, Research Assistant khan, Shahid, Visiting Research Associate Reese, Barbara F., Research Technician Sheetz, Michael P., Visiting Research Associate Schnapp, Bruce J., Research Associate Tatsuoka, Hozumi, Research Associate Terasaki, Mark, Research Associate Reinisch, Carol L., Investigator, Marine Biological Lab- oratory, and Chairperson. Department of Comparative Medicine, Tufts University School of Medicine Our laboratory is studying hematopoietic neoplasia, a leu- kemia-like disease of soft shell clams. Monoclonal antibodies developed by this laboratory and techniques in molecular biol- ogy are used to investigate the differences between normal and leukemic cells and their ontogeny. Staff Miosky, Donna, Laboratory Technician Smolowitz, Roxanna, Postdoctoral Fellow Shimomura, Osamu, Senior Scientist, Marine Biological Laboratory, and Boston University School of Medicine Biochemical studies of the various types of bioluminescent systems. Preparation of the improved forms of aequorin for measuring intracellular free calcium Staff Shimomura, Akemi, Research Assistant I 'isiting investigators Musicki, Branislav, Harvard University Nakamura, Hideshi, Harvard University Stephens, Raymond E., Investigator, Marine Biological Laboratory, and Boston University School of Medicine Biochemistry of microtubules in cilia, flagella, and the cyto- plasm; mechanosensitivity and the control of ciliary move- ment. Staff Good, Michael J., Research Assistant Oleszko-Szuts, Susan, Research Associate Stommel, Elijah W., Research Associate, St. Elizabeth's Hospital ! 'isiting investigator Holz, George G., Tufts University School of Medicine Strumwasser, Felix, Director, Laboratory of Neuroen- docrinology, Boston University School of Medicine, and Marine Biological Laboratory This laboratory studies the molecular and cellular bases of two neural programs that regulate different important behav- iors in the model mollusc Aplysia. Research is conducted on the mechanisms of the neuronal circadian oscillators located in the eyes. These circadian oscillators drive the circadian activity rhythm of the animal, which is concerned with the daily timing of food gathering and of prolonged rest. Additional research is conducted on a group of neuroendocrine cells that produce a peptide, "egg-laying hormone," that initiates egg laying and as- sociated behaviors. The laboratory is interested in how the three-dimensional shape of this peptide hormone allows a highly specific interaction with its receptor and the intracellular processes that are triggered by it. Staff Eason, Barbara, Laboratory Assistant Click, David, Senior Postdoctoral Fellow Hellmich, Mark, Graduate Student Viele, Daniel P., Senior Research Assistant Sussman, Raquel, Associate Scientist, Marine Biological Laboratory Investigation of the molecular mechanism of DNA damage- inducible functions. Present studies deal with the structure- function relationships of A represser analyzed by immunologi- cal techniques. Staff McLaughlin, Jane, Research Assistant Cornuel, Catherine, Research Assistant Szuts, Etc Z., Investigator/Assistant Scientist, Marine Biological Laboratory. Laboratory of Sensory Physiol- ogy, and Department of Physiology, Boston University School of Medicine Biochemical reactions (phosphorylation, inositide metabo- lism) that mediate the light-induced response of retinal photo- 58 MARINE BIOLOGICAL LABORATORY receptors in both vertebrates ( trot: and cattle) and invertebrates (squid and Limuliis). Staff Irapp. Susan ( \ssisiant Visiting invei Cla>. John K.. ' ial l'ho\phoprotem\ \\\a< uilnc Memorv \\hal the Snail \ l-.\e I . ", Judith P., I he At .nli.i Institute. 6 August. Hiro- shima Day Lecture, "liui I I line l'i/mine^ in f\eef Reflect ions on the Moral Lite oi Si iei Ka>en, Peter, Missouri Botanical (iarden. 7 August, Monsanto Biotechnology Lecture. "Population, Pov- erty and Politics in the Tropics" Kandi. James, Conjurer, Lecturer and MacArthur Fel- low, 14 August. "Search for the Chimera" Capu//o. Judith McDowell, Woods Hole Oceano- graphic Institution, 21 August. "Cross Ecosystem Companion < >t II 'n\ie Disposal Impacts: The Scientific Hci\/\ tor Decision Makingot Environmental Issues" (ierbi, Susan, Brown University. 28 August, "Evolution ol u Molecular Machine 1 lie Rihosome"' Frank A. Brown, ,)r. Memorial Readership Hill. Richard \V., Michigan State University Robert l)a> Allen Fellowship Suprenant, Kathy A., University of Kansas Frederik B. 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Laboratory Support Staff Including Persons Who Joined or Left the Staff During 1987 Biological Bulletin Anderson, Lewis B. Metz, Charles, B., Editor Baldic, David P. Bauer, Diane Blunt, Hugh F. Clapp, Pamela L. Bourgoin, Lee E. Mountford, Rebecca J. Brunette, Clifford J. Buildings and Grounds Carini, Robert J. Cutler, Richard D., Collins, Paul J. Services, Projects, and ConJin, Henry P. Facilities Manager Finegan, Timothy B. Lehy, Donald B., Fuglister, Charles K. Superintendent Gibbons, Roberto G. 64 MARINE BIOLOGICAL LABORATORY Gonsalves. Walter W., Jr. Illgen. Rohcn }•'. Jennings. Da\ul \ Lewis. Ralph H. Lochhead. William M. Lunn. Alan ( i MacLeod. John B. McAdams. Hcibert. Ill Mills. Stephen A. Rattacasa, Frank Rossetti. Michael Schoepf. Claude Schwamb. Peter J. Varao, John deVeer. Robert L. Ward. Frederick Weeks. Gordon W. \Vhittaker. William \V indie. Irvin Controller's OH ice Speer. John W.. Controller Binda. Ellen F. Campbell. Ruth B Davis. Doris C. Gilmore. Man, F. ( lodin. I ranees I . Hobbs. Roger \V.. Jr. Hough. Rose A. Mahan. Joan M. O'Brien-Sibson, Patricia J. Oliver. Eli/ubcth Tollios, Constantine Copy Service Center Gibson, Caroline F. Jackson. .laci|iiel\n F. Mountford, Rebecca J. l)f\c/nf»ncnl Olln e A\ers. Donald I , 1 1 Lyons. Elaine 1). Thimas I l)in ; !• '/ ( Halvorson. I l.n President/Dili \\ hiltaker. J. Richard. Acting Director Berthel. Dorotln ( lark. C'athanne I . Fpstein. Ra\ 1 KmncalK. Kathleen R. Gray Museum Armstrong. Ellen P. \lontiero. Eva Housing King. LouAnn D.. Conference Center and Housing Manager Adolf. Bozena Andrews, Loretta Baptiste. Winnie Chamberlain. Bonn> Ldd\. Knstine A. Ellsworth. Lynne M. Hamilton. Heather A. Heins. Christine Johnson, Frances N. Kuil. Elisabeth Leach, Adele Lewis, Sheryl I. Lewis, Shirley A. McNamara. Noreen Palmer. Doreen P. Price. Dale L. Rutf. Corinne M. Library Fessenden, Jane. Librarian Ashmore. Judith A. Corbett. Marguerite Mirra. Anthoin .1. Mountford, Rebecca J. Munson. Robin Nelson. 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Linda O'Brien-Sibson. Patricia .1 Telephone Otlicc Baker. Ida M. Geggatt. Agnes L. ( Irace. Patricia A. 1V8^ Siiinnh'i Support Staff Allen. I ama L. Armstrong, Nicholas B. Armstrong. Timothy C. Ash more. Lynne E. Becker. Sharron A. Beetlestone. Linda Belheau. Christine A. Burdick. Jonathan R. Burr. Michelle Burr, Su/anne Butler. KelK A. Cadwalader, George, Jr. Callagy. Kristen ( 'amphell. Andrew Carrier, Michelle A. Child. Malcolm S. Collins. Jessica J. Dickman. Michael C. Dino. Victor H. Donovan. Christine B. Donovan. Jason P. Felcyn. George D. Hahn. Heidi Hines. Kristen Hodapp. John M. [llingworth, Dawn Ingersoll. Da\ id Ireland. Lisa Mackey, William T. Magcnnis, Colleen A. Magennis, Maureen P. Manheim. Francesca Marini. Michael F. Martyna, Jonathan W. McMenamin-Balano. Jonathan Montroll. Charles Nelson. Christen L. Pachter. Jane E. Parsons. Marc L. Peal. Richard W. Philbin. Linda M. Remsen, Andrew W. Richardson. Keith W. Rickles. Andrew H. Rose. Christine M. Sadovsky. Sebastian Shaw. Trevor P. Shay. Pamela I . Sheet/. Jonathan P. Shiplc>. Michael Sirrico, Jennifer M. Sohn. Marcus Stesens. Lisa D. Sun. Ya-ping Syhia. James A. Valois, Francis X. \\ el/el. Ernest D. Woodward, Helen Reference: Bio/ Bull. 175: 65-78. (August. 1988) Development of Nerve Cells in Hydrozoan Planulae: II. Examination of Sensory Cell Differentiation Using Electron Microscopy and Immunocytochemistry VICKI J. MARTIN Department of Biological Sciences, University of Noire Dame. Notre Dame. Indiana 46556 Abstract. The development of sensory cells in hydro- zoan planulae of Halocordyle disticha was examined us- ing transmission electron microscopy and light immuno- cytochemistry. Sensory cells arise in the anterior end of the planula in the ectoderm at 24 hours postfertilization. These cells extend from the free surface of the planula to a ganglionic plexus located just above the mesoglea. The cytomorphosis of sensory cells is characterized by the ap- pearance of a single apical cilium, microtubules, mito- chondria, one to several Golgi complexes, electron-dense droplets, dense-cored vesicles, and neurites. The basal end of the sensory cell forms one to several processes (neurites) which contribute to the ganglionic plexus. Api- cal specialization of the sensory cell precedes basal differentiation. Sensory cells increase in number as plan- ulae develop and many become organized into clusters of 3-6 cells distributed along the entire length of the planula. Within some of these clusters, two morphologi- cal types of sensory cells are discernible: light sensory cells and dark sensory cells. Light sensory cells outnum- ber the dark sensory cells and are the first sensory cells to appear at 24 hours postfertilization. Use of immunocyto- chemical techniques on wholemounts and paraffin-em- bedded sections of planulae demonstrates the presence of FMRFamide-like immunoactivity associated with some of the sensory cells. Such FMRFamide-like expres- sion is first detected at 24 hours postfertilization in the anterior ectoderm of the planula. By 96 hours postfertil- ization, the spatial distribution of FMRFamide-like posi- tive sensory cells is such that many are found in clusters along the entire anterior-posterior axis of the planula. There is, however, an abundance of FMRFamide-like positive cells in the anterior region of the planula just prior to metamorphosis. The apices and cell bodies of the Received 26 October 1987; accepted 31 May 1988. sensory cells exhibit intense immunostaining, whereas the basal processes stain faintly. This study identifies neuropeptide-like substances in nerve cells of cnidarian larvae and demonstrates a developmental correlation be- tween the time of appearance of the synthetic machinery of sensory cells with the pattern of expression of the FMRFamide-like peptide. Introduction Early light microscopists defined two types of nerve cells in cnidarians: sensory cells and ganglionic cells ( Burnett and Diehl, 1 964). Sensory cells are oriented per- pendicular to the mesoglea with their apical ends con- tacting the outer free surface of the animal and their basal ends drawn out into processes. Ganglionic cells exhibit round perikarya and lie in the basal part of the ectoderm with their axes oriented parallel to the mesoglea. Further- more, Westfall and associates have demonstrated that, in hydra, many types of sensory cells and ganglionic cells exist which can be classified as unipolar, bipolar, or mul- tipolar depending on the number of processes extending from the perikaryon (Yu el al., 1985). Chemical synapses with electron-dense and dense- cored vesicles have been observed in the nervous systems of adult hydrozoans, scyphozoans, and anthozoans (Horridge and Mackay, 1962; Lentz and Barrnett, 1965; Jha and Mackie, 1967; Davis el al.. 1968; Westfall, 1970, 1973; Westfall et al.. 1971; Stokes, 1974; Peteya, 1975; Yamasu and Yoshida, 1976; Singla, 1978; Spencer, 1 979). However, only recently has a specific peptide been identified in adult cnidarians that might be acting as a neurotransmitter (Grimmelikhuijzen and Graff, 1986; Grimmelikhuijzen and Groeger, 1987). Electron-dense droplets and dense-cored vesicles also have been identi- fied in planular nervous systems (Martin, 1988), and 65 66 V. J. MARTIN SENSORY CELL DIFFERENTIATION 67 Kolberg and Martin (1988) have demonstrated catechol- amines in association with planular nerves. Further- more, they provide evidence that such catecholamines may be functioning as neurotransmitters, neurohor- mones, or neuromodulators during embryogenesis (Kolberg and Martin, 1988). A reagent (anti-FMRFamide) is available which will stain cells containing peptides ending in -Arg-Phe-NH2. The work of Grimmelikhuijzen and associates suggests that, when this antiserum is applied to cnidarians, the peptides bound to it are likely to be related to pGlu-Gly- Arg-Phe-amide (PQGRFa) which is present in large amounts in nervous systems of adult anthozoans and probably also in scyphozoans and hydrozoans (Grim- melikhuijzen and Graff, 1986; Grimmelikhuijzen and Groeger, 1987). The question is: how early in develop- ment, and in what cells, is the gene for this peptide (or peptide family) expressed? The planula larva is a good system in which to examine this problem because the number of cell types in the larva is small, their arrange- ment is simple, and neither the variety nor the arrange- ment are very far from those of the adult (Martin and Thomas, 1980; Martin et ai, 1983: Thomas el ai. 1987; Martin, 1988). In this study, the development of the planula of the marine hydrozoan Halocordyle disticha was followed with transmission electron microscopy to determine when sensory nerve cells appeared and when the syn- thetic machinery of these cells appeared. Different aged planulae were exposed to FMRFamide antiserum, and the pattern of expression of the FMRFamide-like pep- tide was correlated with the electron microscopic find- ings. Materials and Methods Mature colonies of Halocordyle disticha were col- lected from wharf pilings in Morehead City, North Caro- lina. Fronds from male and female colonies were placed together in large finger bowls of filtered seawater. The bowls were placed in the dark at 6:00 pm, and at 9:00 pm, early cleavage embryos were collected, placed in small finger bowls of seawater, and reared at 23°C. Eight-hour embryos, as well as 10-, 16-, 24-, 48-, 72-, 96-, and 1 20-hour planulae, were prepared for transmis- sion electron microscopy. The animals were fixed for 1 hour in 2.5%glutaraldehyde, pH 7.4, in 0. 2 M phosphate buffer. They were postfixed for 1 hour in 2% osmium tetroxide, pH 7.2, in 1.25% sodium bicarbonate. The specimens were dehydrated in an ethanol series, infil- trated, and embedded in Spurt's embedding medium. Serial thin-sections were cut with a Porter-Blum MT-2B ultramicrotome, placed on 150-mesh copper grids, and stained with 3.5% uranyl acetate in ethanol followed by lead hydroxide. The grids were examined and photo- graphed with a Hitachi H-600 transmission electron mi- croscope. Thick plastic sections were also cut, placed on subbed glass slides, and stained with methylene blue- azure II. To better visualize the basal processes of sensory cells, early cleavage embryos were cultured in seawater con- taining 0.01 M hydroxyurea until they reached the ma- ture planula stage (Martin, 1985, 1986). These treated larvae were then prepared for transmission electron mi- croscopy. Embryos reared continuously in hydroxyurea contain reduced numbers of ganglionic cells and slightly fewer ganglionic neurites, yet possess the same number of sensory cells as do comparable controls (Martin, 1 985, 1986, pers. obs.). The sensory cells of hydroxyurea- grown planulae are morphologically identical to those of comparable controls, and their basal processes are more easily traceable due to the reduced size of the ganglionic plexus (Martin, 1985, 1986, pers. obs.). Planulae of eight different ages, wholemounts and par- affin sections, were tested for their ability to bind a rabbit antiserum raised to FMRFamide (Immuno Nuclear Corporation). The planular ages included 10-, 16-, 24-, 36-, 48-, 72-, 96-, and 120-hour planulae. To visualize Figure 1. Single light sensory cell (S) in the anterior ectodermal region of a 24 hour planula. The cytoplasm is filled with microtubules, Golgi cisternae. some rough endoplasmic reticulum. and a few mito- chondria. EM. epitheliomuscle cell. X8400. Figure 2. Cluster of three light sensory cells (S) in the mid ectodermal region of a 72 hour planula. Each cell possesses an apical cilium and a cytoplasm nch in microtubules, Golgi cisternae, electron-dense droplets (arrow), and dense cored vesicles, x 1 1 ,200. Figure 3. Ganglionic nerve cell (G) and ganglionic plexus (GP) at the base of the ectoderm in a 72 hour planula. Basal neurite extensions (P) of light sensory cells (arrow) and dark sensory cells (not visible here) project into and help constitute the ganglionic plexus. Neurites of the plexus contain microtubules, mito- chondria, electron-dense droplets, and dense-cored vesicles. M, mesoglea. x 10,000. Figure 4. Cluster of light (S) and dark (D) sensory cells in the ectoderm of a mature hydroxyurea-grown planula. From the early cleavage stage, animals were continuously cultured in 0.01 M hydroxyurea in seawater. All cells in treated planulae are morphologically identical to those in control planulae. Photo- graphs of treated embryos (Figs. 4, 6, 9) were included because excellent planes of section illustrating clustering of sensory cells and sensory cell processes were obtained from these embryos. X7000. JV '• SENSORY CELL DIFFERENTIATION 69 the binding of FMRFamide antiserum on wholemounts of planulae, the procedure presented by Koizumi and Bode (1986) was followed with some modifications. Planulae were fixed for 1 hour in 10% formalin in seawa- ter. After fixation, the animals were washed 3 times, for 15 minutes each, in 10 mM phosphate-buffered saline (PBS, pH 7.2). Incubation with the FMRFamide antise- rum was for 1 8 hours, with the primary antibody diluted 1:200 with 10 mM PBS, pH 7.2, containing 2% neonatal calf serum (Irvine Scientific), 0.3% Triton X-100, and 0. 1% sodium azide. The incubation was carried out with the planulae in lid-covered 96 well tissue culture plates that were resting on a rotating shaker platform set at 60 rpm. After the first incubation period, the primary anti- body was pulled off with a pipette, and the animals were washed for three 1 5-minute changes in 1 0 mA/ PBS, pH 7.2. Incubation with the second antibody was for 1 hour in fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit immunoglobins (U. S. Biochemical Corpora- tion) diluted 1:120 in 10 mM PBS, pH 7.2, containing 10% fetal calf serum, 0.3% Triton X-100, and 0.1% so- dium azide. The second incubation was also in 96 well plates rotated at 60 rpm. After the second incubation, the animals were washed 3 times, for 15 minutes each, in fresh 10 mM PBS, pH 7.2. Wholemount preparations were examined for fluorescently labelled cells with a Zeiss microscope equipped with epifluorescence. To visualize binding of FMRFamide antiserum to par- affin sections of planulae, the following procedure was followed. Samples fixed in formalin were dehydrated through an alcohol series, infiltrated and embedded in paraffin, and serially sectioned at 8 /urn. Approximately nine sections were mounted in the center of a single glass slide, three rows one above the other, and each row con- taining three sections. The slides were rehydrated to dis- tilled water, and the sections were surrounded by an outer ring of vacuum grease (the grease ring was just to the outside of the sections). The grease was applied in a moist chamber to prevent the sections from drying. The protocol for indirect immunofluorescence for par- affin sections was identical to that described for whole- mounts. The FMRFamide antiserum was placed in the grease-created well thus immersing the sections. Such slides were placed in a lid-covered moist chamber and rotated at 40-60 rpm for 1 8 hours. PBS rinses and incu- bation in the second antibody were also carried out in the moist chamber. After incubation, the grease was carefully removed from the slides, and the sections were covered with mineral oil and examined for fluorescently labeled cells. Some of the paraffin sections were subse- quently stained with azure B after their initial examina- tion for immunofluorescence. For wholemounts and paraffin sections, the binding specificity of the FMRFamide antiserum was deter- mined by preincubating a 1:200 dilution of the antise- rum with either 1 or 10 Mg/ml synthetic FMRFamide (Peninsula Lab) for 24 hours at 4°C before using it to stain the samples. Results Sensory cells begin to arise in the ectoderm of the plan- ula at 24 hours postfertilization (Fig. 1 ). They first appear as single cells scattered in the anterior region of the plan- ula. As development progresses, these cells increase in number and become distributed along the entire length of the planula, many arranged in clusters of 3-6 cells (Fig. 2). Sensory cells are columnar and extend from the free surface of the planula to a ganglionic plexus located just above the mesoglea (Fig. 3). Sensory cells are charac- terized by an apical cilium, a medially to basally located nucleus, and small basal neurite extensions which pro- ject into and help constitute the ganglionic plexus. Two morphological types of sensory cells are identifi- able at the fine-structural level: a light sensory cell and a dark sensory cell (Fig. 4): the light sensory cell has a more electron-lucent cytoplasm than does the dark sensory cell. The light and dark sensory cells can be distinguished on the basis of their distribution and time of appearance, their cytology, and their neurite processes. First, the light cells are in the vast majority, and they appear first in planulae that are only 24 hours old (Fig. 1 ). I have, as yet, only seen dark sensory cells in the most Figure 5. A cluster of sensory cells containing one dark sensory cell (D) and several light sensory cells (S) in the anterior ectoderm of a 72 hour planula. The dark sensory cell has a cytoplasm rich in microtu- bules, mitochondria aligned in rows between the microtubules. electron-dense droplets, and dense-cored vesicles. The nucleus (N) of the dark sensory cell is mid to basally located, and the basal extension is bipolar (arrows). xSOOO. Figure 6. Medially located nucleus of a dark sensory cell. A single Golgi complex (arrow) is found in close association with the nucleus, as are numerous granules and vesicles, x 1 1 ,900. Figure 7. Cytoplasm of a dark sensory cell in a maturing planula. Electron-dense droplets (single arrow) and dense-cored vesicles (double arrows) are abundant in the Golgi region (GO) of the cell. X4 1.000. Figure 8. Cytoplasm of a light sensory cell in a mature planula. Multiple Golgi complexes (GO) appear throughout the apical cytoplasm, as do electron-dense droplets (arrow) and dense-cored vesicles (double arrows). X4 1,000. I- inure 9. Basal region ol .1 ,l.nk sensory cell (D) in a mature hydroxv urea-grown planula. The base of the cell forms two processes il'i which contribute to the ganglionic plexus (GP). Mitochondria, rmcrotu- hules. electron-dense droplets (single arrow), and dense-cored vesicles (double arrows) till the sensory neu- rites and arc abundant in the other neurites of the ganglionic plexus. E.endoderm: M. mesoglea. x 1 9, 200. SENSORY CELL DIFFERENTIATION 71 Figure 10. Basal regions of light sensory cells in a maturing planula. Basal processes (P) from two light sensory cells extend into the ganglionic plexus (GP). Numerous microtubules occupy the cytoplasm of these processes, however, electron-dense droplets and dense-cored vesicles have not yet appeared. The appearance of these droplets and vesicles in the basal extensions constitutes the last phase of sensory cell differentiation. E, endoderm; EC. ectoderm; M. mesoglea. x 19.000. mature planulae (36-96 hours postfertilization depend- ing on temperature: just prior to attachment). Further- more, the dark sensory cells do not appear in all sensory cell clusters (Fig. 2), and when they are present, they gen- erally occur singly or in pairs (Fig. 5). Light sensory cells may occur singly along the length of the mature planula, but dark sensory cells have only been seen among the clusters. The cytoplasm of the dark sensory cells contains nu- merous bundles of microtubules, rows of mitochondria dispersed in between the microtubule bundles, generally a single Golgi complex in close proximity to the nucleus, and electron-dense, non-membrane bound droplets and dense-cored, membrane-bound vesicles (Figs. 4-7). The cytoplasm of light sensory cells also contains numerous bundles of microtubules, many Golgi complexes in the 72 v. j. MAR i IN SENSORY CELL DIFFERENTIATION 73 upper apical regions, and electron-dense, non-mem- brane bound droplets and dense-cored, membrane- bound vesicles (Figs. 1, 2, 4, and 8). However, there are fewer mitochondria than in dark sensory cells, and the mitochondria are not arranged in the distinct rows that characterize the dark cells (Fig. 4). The basal extensions of dark sensory cells contain nu- merous mitochondria, microtubules, electron-dense droplets and dense-cored vesicles and often bifurcate to form two neurites that project into the ganglionic plexus (Fig. 9). In contrast, the basal processes of light sensory cells have not been seen to bifurcate, and they appear to contain fewer droplets and vesicles than do the processes of dark sensory cells (Fig. 10). Thus, the dark sensory cells appear to be bipolar, whereas the light cells may be considered as unipolar. During the development of both types of sensory cells, the apical region of each cell becomes specialized before the basal region (Figs. 7, 8, 11-13). One or several Golgi complexes, depending on the type of sensory cell, form early in close association with the nucleus. Droplets and vesicles soon appear within the region of the Golgi (Figs. 7, 8, 13). Concurrent with the appearance of the Golgi, mitochondria and microtubules fill the apical cytoplasm. Next, the basal regions of the cells become specialized to form neurites. Mitochondria, microtubules, droplets, and vesicles appear within the forming basal neurites (Figs. 9, 14). FMRFamide-like immunoactivity is observed in par- affin sections and wholemounts of planulae of Halocor- dyle disticha (Figs. 15, 17-26). Such immunoactivity is first detected in single cells in the ectoderm at 24 hours postfertilization in the anterior region of the planula (Fig. 1 5). Before 24 hours animals lack immunostaining (Fig. 16). As the planulae mature, the immunostaining in- creases and appears scattered along the planular ante- rior-posterior axis (Figs. 17-22). The appearance and distribution of the FMRFamide-like positive cells corre- sponds to the appearance and distribution of some of the sensory cells as viewed by transmission electron micros- copy. As planulae age, some of the positively staining cells appear in clusters (Figs. 1 8-22) and display the char- acteristic morphology of sensory cells: columnar cells in the ectoderm with tiny tortuous processes that project toward the mesoglea. An examination of the FMRF- amide-like positive cells in paraffin sections confirms that they are sensory cells (Fig. 28). When such sections are subsequently stained with azure B, the immunoposi- tive cells stain faintly as they lack apical granules. The only other columnar cells in the ectoderm, glandular and epitheliomuscle cells, possess numerous large apical granules; such granules stain darkly. Thus, epitheliomus- cle cells and glandular cells stain darker with azure B than do the sensory cells. Furthermore, light azure B- staining sensory cells are first detected at 24 hours post- fertilization, whereas dark azure B-staining cells are visi- ble shortly after gastrulation (10-12 hours postfertiliza- tion). No distinction between dark and light sensory cells, as seen via transmission electron microscopy, is possible at the light microscopic level. Examination of paraffin sections from different aged planulae and from different axial regions of the planula illustrates the abundance and distribution of immuno- positive cells with respect to axial location and develop- mental time. All FMRFamide-like expression, with the exception of a few scattered fluorescent dots in the endo- derm, is confined to the planular ectoderm (Figs. 15, 17- 23). Developmental expression of the FMRFamide-like peptide by sensory cells is such that it is first detected in the upper apical two-thirds of the cell and last, if at all, in the basal region (Figs. 15, 17-22). The upper portion of the cell exhibits brilliant immunostaining while the basal processes stain weakly. Hence, the first immuno- positive cells to appear at 24 hours exhibit intense fluo- rescence in their apical regions and little or no staining in their basal ends (Fig. 15). Examination of different axial regions of mature planulae indicates that FMRFamide- like immunopositive cells are found along the entire planular axis by 48-72 hours postfertilization (Figs. 18- 22). However, there does appear to be more immuno- positive cells at the anterior end of the planula than at the posterior end (Figs. 20-22). Just prior to metamor- Figure 11. Apical region of a differentiating light sensory cell in a 72 hour planula. Apical differentia- tion of sensory cells precedes basal differentiation as Golgi complexes, microtubules. electron-dense drop- lets (single arrow), and dense-cored vesicles (double arrows) fill the apical cytoplasm. Electron-dense drop- lets, dense-cored vesicles, microtubules, and mitochondria form later in the basal processes. X32.000. Figure 12. Immature basal extensions of light sensory cells. The processes (P) are not yet filled with electron-dense droplets, dense-cored vesicles, or mitochondria. These neurites extend into a well-formed ganglionic plexus (GP). M. mesoglea. x 15.000. Figure 13. Golgi region in the apex of a light sensory cell. Electron-dense droplets and dense-cored vesicles (arrows) are found in the area of the Golgi. X63.000. Figure 14. Differentiating basal processes (P) of light sensory cells. These processes become filled with mitochondria, electron-dense droplets (arrows), and dense-cored vesicles ( not visible). Compare this micro- graph with Figure 12 illustrating immature basal processes of light sensory cells. x3 1,000. 74 V. J. MARTIN SENSORY CELL DIFFERENTIATION 75 Figure 21. Slightly oblique paraffin section through the anterior region of a young 96 hour planula. Basal processes (arrow) of some sensory cells exhibit weak FMRFamide-like activity. E, endoderm. X150. Figure 22. Longitudinal paraffin section of a young 96 hour planula. Immunopositive cells are abun- dant in the anterior region (A) and a few are visible along the mid-sides (MI) of the animal. The arrow denotes the location of the mesoglea. E, endoderm. X220. Figure 23. Longitudinal paraffin section of a mature 120 hour planula just prior to metamorphosis. Immunopositive cells are present in the anterior (A) region of the planula, however, they are absent from the more posterior (P) regions of the animal. E, endoderm. xlOO. Figure 15. Paraffin section through the anterior region of a 24 hour planula. A few FMRFamide- positive cells first appear in the anterior region of the planula at this stage of development. These cells probably correspond to some of the first sensory cells seen via transmission electron microscopy. The FMRFamide-like peptide is first expressed in the apices of these cells (as indicated here) and only later in the mid to basal regions of the cells. Expression of the peptide-like material at this time is confined to a few ectodermal cells, as the endoderm (E) lacks immunostaining. The single arrow denotes the outer edge of the ectoderm, whereas the double arrows indicate the mesoglea. X230. Figure 16. Paraffin section through the anterior region of a 16 hour planula. These embryos do not express the FMRFamide-like peptide. as indicated by their lack of immunostaining. The arrow indicates the outer margin of the ectoderm. E, endoderm. x250. Figure 1 7. Longitudinal paraffin section of a 36 hour planula. As development proceeds, the number of cells expressing the FMRFamide-like peptide increases, as demonstrated by the larger number of positive- staining cells at 36 hours compared to 24 hours (Fig. 15). By 36 hours, immunopositive cells are visible in the anterior region (A) of the planula and also along the sides of the planula. For the most part, the immunostaining at this stage is strongest in the apical regions of cells. The arrow indicates the location of the mesoglea. E, endoderm; P, posterior. X 150. Figure 18. Paraffin cross section through the mid region of a 72 hour planula. The number of immuno- positive cells has increased by 72 hours, and many of these cells associate to form intense immunopositive clusters (arrows) along the length of the planula. E, endoderm. X200. Figure 19. Oblique paraffin section of a 72 hour planula. Single immunopositive cells and clusters of immunopositive cells are visible in the planular ectoderm. By 72 hours, in those cells that express the FMRFamide-like peptide, the immunostaining is not confined solely to the apices of the cells but has extended to include the mid regions of the cells, and in some cases the basal regions of the cells (arrow). Faint staining of basal processes is seen in the anterior region of the planula and decreases towards the posterior end of the planula. E, endoderm. X220. Figure 20. Oblique paraffin section of a 72 hour planula. Immunopositive sensory cells are found in the ectoderm along the entire anterior-posterior axis of the planula. There appears to be more immunoposi- tive cells in the anterior region (A) of the planula than in the more posterior regions (P). A few immunoposi- tive small cells are visible in the anterior endoderm (E) at this stage, and these cells represent a subpopula- tion of interstitial cells differentiating along the ganglionic cell line. X200. 76 \ J M \RIIN H(jure 24. Distribution (if clusters of itnmunopositi ve cells in a wholemount of a 96 hour planula. These cells are found in the anterior (A), middle, and posterior (P) regions of the planula. X80. Figure 25. Wholemount of the mid to posterior region of a 96 hour planula showing the distribution ofFMRFamide-like positive clusters of cells. X90. I- inure 26. Wholemount of a mature planula showing the anterior region. Processes (arrows) stain faintly and are located in the ectodermal region of the ganglionic plexus and also just above the plexus. E, endoderm: EC. ectoderm. X250. h inure 27. Wholemount of a mature planula showing the posterior region. There are no positive stain- ing processes detected in this area. K, endoderm; !•"(', ectoderm. X250. phosis, a large '^her of immunopositive cells are de- tected in the extan. interior region of the planula (Fig. 23). Basal processes ol vjnsory cells located in the ante- rior region of the planula slain more intensely than do those of sensory cells distributed in the mid to posterior region of the animal (Figs. 21-23). The spatial distribution of cells expressing the FMRFamidc-like pcptide is easily visualized using wholemounts of planulae (Figs. 24-27). In mature plan- ulae (96 h) clusters of immunopositive cells are visible as large dots scattered along the length of the animal. A few of these FMRFamide-like positive clusters first appear in the anterior region of the late 24 hour planula and later in the mid to posterior regions of the mature planula. In wholemounts, the basal processes of the sensory cells arc very difficult to visuali/e as thev are tinv and stain only SENSORY CELL DIFFERENTIATION 77 28 Figure 28. Paraffin section through the anterior region of a matur- ing planula stained with azure B. Light staining sensory cells (arrows) are found among darker staining epitheliomuscle cells and glandular cells. Dark staining cells outnumber the light staining cells. When pro- cessed for immunocytochemistry the light cells exhibit a positive re- sponse for FMRFamide-like activity. E. endoderm; M, mesoglea. X250. faintly (Fig. 26). Visualization of weak immunopositive basal processes is more obvious in paraffin sections (Fig. 21). Wholemounts and paraffin sections of planulae stained with FMRFamide antiserum preabsorbed with 10 Mg/ml synthetic FMRFamide do not exhibit any im- munopositive staining. Furthermore, treatment of sam- ples with antiserum preabsorbed with 1 Mg/m' FMRFamide results in very dim staining of cells. Discussion Using immunocytochemistry and radioimmunoas- says, Grimmelikhuijzen and associates identified sub- stances resembling vertebrate or invertebrate neuropep- tides in the nervous systems of adult cnidarians (Grim- melikhuijzen and Graff, 1985, 1986; Grimmelikhuijzen and Groeger, 1987). The most common neuropeptides seen were those related to the molluscan neuropeptide Phe-Met-Arg-Phe-amide (FMRFamide). When anti- FMRFamide was applied to adult cnidarians, cells con- taining peptides ending in -Arg-Phe-NH; bound the antiserum. Recently, Grimmelikhuijzen and co-workers isolated and sequenced a specific neuropeptide, pGlu- Gly-Arg-Phe-amide (antho-RFamide), from sea anemo- nes and pennatulids (Grimmelikhuijzen and Graff, 1985, 1986; Grimmelikhuijzen and Groeger, 1987). In this study hydrozoan planulae of different develop- mental ages were tested for their ability to bind a rabbit antiserum raised to FMRFamide to determine if pep- tides ending in -Arg-Phe-NHi were present in larval cnidarians and, if so, to determine when in development the gene for such a peptide (or peptide family) was ex- pressed. Planulae of Halocordyle disticha exhibited a positive staining response when exposed to anti- FMRFamide, indicating that peptides ending in -Arg- Phe-NH: are present in cnidarian larvae. The expression of the FMRFamide-like peptide was first observed at 24 hours postfertilization in the ectoderm in association with some of the sensory cells. As planulae matured, the number of immunopositive sensory cells increased, and such cells were seen along the entire length of the plan- ula. Just prior to metamorphosis a large number of FMRFamide-positive cells appeared in the anterior ecto- derm (attachment end), suggesting the involvement of the FMRFamide-like peptide in planular attachment or metamorphosis. The time of appearance of the synthetic machinery of planular sensory cells, and the pattern of appearance of FMRFamide-like immunofluorescence, appear to be correlated. Ultrastructural examination of planulae re- veals that the planular nervous system begins to form in the ectoderm at 24 hours in development. A few sensory cells are found in the anterior end of the planula at this time, and as planulae age, the number of sensory cells increase. Furthermore, as sensory cells differentiate, their apical regions become specialized before their basal re- gions. One of the first differentiative events detected in the sensory cell is the formation of one to several Golgi complexes in close association with the nucleus. Shortly after the formation of the Golgi complexes, electron- dense droplets and dense-cored vesicles appear in the apical cytoplasm, and only later in the forming basal neurites. Individual immunopositive cells first appear at 24 hours in the anterior ectoderm of the planula and, most probably, correspond to the first sensory cells seen via transmission electron microscopy. As development progresses, many of these immunopositive cells become organized into clusters along the length of the planula. Furthermore, such immunopositive cells exhibit bril- liant immunofluorescence first in their apical regions, and only later do they show a dim staining in their basal regions. Such a spatial and temporal staining response in the immunopositive cells is expected in view of the Ultrastructural findings concerning time of appearance and location of the Golgi complexes, droplets, and vesi- cles. The presence of peptides in the nervous systems of planulae suggest that: ( 1 ) peptides may play crucial roles in the development of these larvae; and (2) peptides may be important for metamorphosis. In either case, the sim- V. J. MARTIN pie nervous system of the planula provides an excellent system with which to anal> ze neuropeptide action on de- velopmental processes. .' Iso. because the planula can he easily visualized, rrnintained, and acquired throughout the entire ITK ic process, it offers a unique devel- opmental mo examining the temporal appearance of new neu ,'iidesand for analyzing possible switches that may • ur in neuropeptide phenotype (i.e.. plastic- ity of neuropeptide expression) as an animal passes from the embryonic condition to the adult state. Acknowledgments This research was supported by National Science Foundation Grants DCB-8702212 and DCB-871 1245. Literature Cited Burnett, A., and .V Diehl. 1964. The nervous system of Hydra. I. Types, distribution and origin of nerve elements. / Exp. Zoo/ 157: 217-226. Davis, I,., A. Bunn it. and J. Havnts. 1968. Histological and ultrastruc- tural study ol the muscular and nervous systems in hydra. J A. y Zool 167: 295-332. Grimnu'likhuijzen, C.. and D. Graff. 1985. Arg-Phe-amide-like pep- tides in the primitive nervous systems of coelenterates. Pcptides 6: 477-483. Grimmelikhuijzen. C., and D. Graff. 1986. Isolation of pGlu-Gly-Arg- Phe-NH: (Antho-RFamide). a neuropeptide from sea anemones. Proc. Natl. Acad. Sci. I'SA 83: 98 1 7-982 1 . Grimniflikhuij/cn. ('.. and A. Groencr. 1987. Isolation of the neuro- peptide pGlu-Gly-Arg-Phe-amide from the pennatulid Renilla kol- likt-ri FEBSLctt 211: 105-108. Horridge. G., and B. Mackay. 1962. Naked axons and symmetrical synapses in coelenterates. Q J Microsc. Sci. 103: 53 1 -54 1 . Jha, R., and G. Mackie. 1967. The recognition, distribution and ultra- structure of hydrozoan nerve elements. J. Morphol. 123: 43-62. Km/iiim. O., and H. Bode. 1986. Plasticity in the nervous system of adult Hydra I. The position-dependent expression of FMRFamide- like immunoreactivity. Dev. Biol. 1 16: 407-42 1 K. k.. and \ . Martin. 1988. Morphological, cylochemical and neuropharmacological evidence for the presence of catecholamines in hydro/oan planulae. Development 103: 1-10. I.entz, I., and R. Barrnett. 1965. Fine structure of the nervous system ofhvdra .Am /.ool. 5: 341-356. Martin. V. 1985. Interstitial cellsare required for polyp morphogenesis during metamorphosis of planulae. Am. Zool. 25:6IA. Martin. V. 1986. Development of nerve cells in hydrozoan planulae ofPennuria narella. Am. Zool. 26: 53. Martin. V. 1988. Development of nerve cells in hydrozoan planulae: I. Differentiation ofpanglionic cells. Biol. Bull 174: 319-329. Martin, \ .. and M. Thomas. 1980. Nerve elements in the planula of the hydrozoan Pennaria narella. J Morphol. 166: 27-36. Martin. \ ., F. Chia. and R. Koss. 1983. A fine structural study of meta- morphosis of the hydrozoan Mnrocomella polydiademala. J Mor- phol 176:261-287. Peteya, D. 1975. The ciliary-cone sensory cell of anemones and cerian- tids. Tissue Cell!: 243-2*2. Singla. C. 1978. Fine structure of the neuromuscular system of/'. as recently reported (Smiley and Cloney, 1985). "I uc s are the structural unit in holo- thunan gonads, and each consists of three tissues. Outer- most is a complex pernnm-iim composed of peritoneal epithelial cells, nerves, and muscle cells. Innermost is the inner epithelium composed of oocytes and somatic inner epithelial cells of two types. Lying between the basal lam- inae of the peritoneum and the inner epithelium is the (onnective tissue compartment composed of libers, the i FECUND TUBUUS Figure I. Diagrammatic depiction of a left lateral view of the Sn- I'lin/'ii'i califomicus ovary. This ovary is bilaterally symmetric about the dorsal mesenten, . mesenchymal cells, and the genital hemal sinus. The gen- ital hemal sinus is not lined by cells but is a lacuna within the connective tissue compartment. The oocyte basal lamina (oolamina) limits direct connection of the oocyte and the jelly space with the fluid in the genital hemal sinus. No outer sac of tissues surrounds holothurian go- nads. Spent tubules. Spawning evacuates post-vitellogenic oocytes from the fecund tubules (Smiley and Cloney, 1985). Just after spawning, spent tubules are a maximum of 4 cm long, and their pigmentation has decreased markedly. A week later, they have shrunken and become a rust color owing to phagocytosis (Fig. 9). This phago- cytic action reduces the ovarian inner epithelium to few identifiable structures; the most obvious are the oocyte oolaminae (Fig. 9). The densely staining material in peri- toneal cells and coelomocytes are birefringent residual bodies, products of phagocytic activity. A month after spawning, only pigmented plaques on the posterior of the gonad basis remain, testifying to the former presence of spent fecund tubules (Fig. 1). Since these tubules are lost, subsequent generations of oocytes must be recruited from other ovarian tubules. Secondary tubules. The secondary tubules are just an- terior to fecund tubules on the gonad basis (Fig. I ). They average 2.5 cm in length and 0.35 mm in diameter. They branch once or twice along their length, and each branched portion is slightly elongate. Their histology is similar to the fecund tubules, but the peritoneal epithe- lial cells are more columnar in secondary tubules (Fig. 2). and the genital hemal sinus is less extensive, giving the connective tissue compartment a more fibrous ap- pearance. Fibroblasts, morula cells, and especially petal- HOLOTHURIAN OOGENES1S AND REPRODUCTIVE CYCLE 81 oid amoebocytes are more common in the connective tissue compartment of secondary tubules. Secondary tu- bules can be divided into two categories. Late secondary tubules are more posterior on the basis; early ones are more anterior. In late secondary tubules, coincident with the onset of vitellogenesis in the fall, somatic inner epithelial cells form the inner wall of the ovary, and surround the oo- cytes (Fig. 10). Together, the inner epithelium and con- nective tissue compartment of these tubules form stubby longitudinal folds (Fig. 2) that run short distances along the length of the tubule. These develop into the long deep longitudinal folds in fecund tubules (Smiley and Cloney, 1985). Some inner epithelial cells form a follicle around the oocytes; these adhere to the oocyte surface, and a jelly space is not present. The lumen of late secondary tubules is much less occluded by oocytes than that of fecund tu- bules. Sexes can be reliably identified from late second- ary tubules, after oocytes have formed germinal vesicles. The peritoneum of early secondary tubules is thinner than in late secondary tubules, but is otherwise similar. The inner epithelium in early secondary tubules is sim- ple in spite of its stratified appearance (Fig. 3). Here, lon- gitudinal folds are reduced in size compared to late sec- ondary tubules. The monociliated somatic cells are smaller than oocytes and have nuclei with denser periph- eral heterochromatin. Even by electron microscopy, it is not possible to distinguish between oocytes and sperma- tocytes in early secondary tubules, or between oogonia and spermatogonia in primary tubules. The ovarian lu- men of early secondary tubules is not seriously occluded by oocytes or longitudinal folds (Fig. 2) and is more ex- pansive than in late secondary tubules. Within it is weakly staining hemal fluid containing morula cells and petaloid amoebocytes. Primary tubules. The primary tubules, the smallest and most anterior on the gonad basis, are often difficult to discern even with magnification (Fig. 1 ). The most an- terior are less than 2 mm in length, 0.25 mm in diameter, and do not bifurcate along their length. Those primary tubules closest to secondary tubules are slightly larger than those more anterior, and they may branch, but the tips of their branches are globose rather than elongate. The distinction between primary and secondary tubules is made on size, position, and cytology. There are no lon- gitudinal folds of the inner epithelium and the connec- tive tissue compartment in primary tubules (Fig. 4). The histological organization of primary tubules is similar to the other tubules (Smiley and Cloney, 1985), but the connective tissue compartment is substantially smaller, particularly the genital hemal sinus. The lumen of primary tubules also contains hemal fluid which ap- pears to be identical with that found in the genital hemal sinus of late secondary and fecund tubules. In spite of the adhering junctions between cells of the inner epithelium. fluids and mesenchymal cells within the lumen appear to mix freely with those in the genital hemal sinus. I con- clude that the lumen of primary and early secondary tu- bules is in direct connection with, if not a part of, the ovarian connective tissue compartment. Gonad basis. The gonad basis in S. californicus is a 5 to 7 mm long saddle-shaped thickening (Fig. 1) of the dorsal mesenteric connective tissue compartment (Smi- ley and Cloney, 1985; Cameron and Fankboner, 1986). It is totally enclosed by the dorsal mesenteric peri visceral peritonea (Fig. 6), which are structurally identical to, and continuous with, the investing peritonea of the ovarian tubules (Smiley and Cloney, 1985). The dorsal mesen- tery results from the lateral fusion of the left and right somatocoels during metamorphosis (Smiley, 1986), and the peritonea of the gonad basis and tubules have the same ontogeny. The gonoduct inserts into the dorsal anterior aspect of the gonad basis and ascends within the connective tissue compartment of the dorsal mesentery to the gonopore located anteriorly in interambulacrum CD (Hyman, 1955). In all specimens sectioned (Fig. 8), the thinner wall of the gonoduct faces one side of the dorsal mesen- tery, and the long axis of its elliptical lumen is parallel with it. The duct is lined with a simple epithelium com- posed of columnar and exaggeratedly columnar mono- ciliated cells. No genital cord (Theel, 1901; Haanen, 1914) is present adjacent to the gonoduct in Stichopus californicus. nor is there any genital rachis (Smiley, 1 988b). Entering the basis at a ventral anterior aspect is a channel connecting the genital hemal sinus of the gonad with the dorsal hemal structures of the gut (Fig. 1 ). The composition and structure of the connective tis- sue compartment in the gonad basis is complex (Figs. 6, 7). Within it, muscle cells run between the fibers of the connective tissue matrix. Lacunae are found in the con- nective tissue of the gonad basis, but based on evidence from serial semithin sections, neither the genital hemal sinus of late secondary and fecund tubules, nor the cen- tral lumina of primary and early secondary tubules are in direct connection with these lacunae. The gonad basis contains a reduced central lumen continuous with lu- mina from more advanced tubules (Fig. 6). Columnar somatic inner epithelial cells of the less advanced tubules partially occlude the opening near the point of their in- sertion onto the gonad basis. At the most anterior lateral aspect of the gonad basis, the connective tissue compart- ment contains small cell nests, about 50 /im in diameter (Fig. 7), similar to those described for Holothuriaparvula (Kille, 1942). In S. californicus, each cell nest has a re- duced central lumen isolated from the lumen of the go- nad basis. The nests are separated from the connective tissue compartment by a basal lamina (Fig. 13). Cytology of the inner epithelial cells Primordial germ cells in the gonad basis. The cells comprising the cell nests are easily differentiated into two S. SMILEY ••^iin- 2. Light micrograph of a cross section of an early secondary tubule. C. connective tissue com- I 1 iiu-ipient longitudinal folds. O. oocyte. P. peritoneum. The arrows demark a longitudinal fold 4 KiKurt- .V Light micrograph of the inner epithelium in an early secondary tubule. MC. mitochondria! cloud in a spue me stage oocyte. O. oocyte, SC. inner epithelial somatic cell. 9 1 Ox. HKUIT 4 I i in micrograph of a cross section of a primary tubule. C. connective tissue compartment. IE. inner epithelium. P. peritoneum, PA. petaloid amoebocytes within the lumen. I75X. HKurvfi. Light micrograph of details on the inner epithelium of a primary tubule. C. connective tissue compartment. MO. mitotic oogonium, OL. ovarian lumen. P. peritoneum, SC. somatic cell of the inner epithelium. I050X. HOLOTHURIAN OOGENESIS AND REPRODUCTIVE CYCLE 83 types found in approximately equal abundance (Fig. 13). The smaller type, averaging about 6 ^m in diameter, have more electron-dense cytoplasm, nuclei with distinct peripheral heterochromatin, prominent centrioles and a cilium; these are interpreted as somatic cells. The larger cells (Fig. 13), averaging about 10 ^m in diameter, have more electron-lucent cytoplasm, nuclei with reduced pe- ripheral heterochromatin, prominent centrioles (Fig. 18), and appear to bear a cilium. The cytoplasm in the larger cells (Fig. 13) contains aggregations of mitochon- dria and associated electron-dense bodies (Fig. 19), here called type I nuage. Nuage is germ line specific electron- dense granules, unbound by membranes, and has been described for germ line cells from other species (Eddy, 1975; Kessel, 1983). These criteria indicate that the larger cells (Figs. 7, 1 3) are primordial germ cells (PGCs), the progenitors of the mitotic gonial cells, themselves precursors of spermatocytes or oocytes. PGCs lack yolk, are bound to neighboring cells with adhering junctions ( Fig. 19), and appear attached to the thickened basal lam- ina (Fig. 13). Oogonial mitoses in primary tubules. The inner epi- thelium of primary tubules contains two distinct cell types, somatic cells and oogonia (Fig. 5). The smaller cells, with more basophilic cytoplasm and nuclei with more distinct peripheral heterochromatin are somatic cells, homologous with the smaller cells in cell nests within the gonad basis. These monociliated cells are not organized into a simple epithelium typical of late second- ary and fecund tubules. Their jumbled arrangement makes the organization difficult to classify (Fig. 5). TEM reveals that all cells have adhering junctions attaching them to their neighbors, and the basal most cells rest on a basal lamina, therefore the inner cells of primary tubules form a true epithelium. Within primary tubules, germ line cells of the inner epithelium are less electron dense, and their nuclei show substantially less peripheral heterochromatin than so- matic cells. They average about 10 jum in diameter, lack yolk, and contain type I nuage. Like the somatic cells, these are bound to neighbors by adhering junctions, and appear attached to the thickened basal lamina. Germ line mitoses are frequently encountered, and are re- stricted to the primary tubules (Fig. 5) in 51. californicus. Mitoses indicate that these germ line cells are oogonia, which correspond to PGCs in cell nests. The criteria sup- porting this conclusion include: ( 1 ) they are part of the inner epithelium of the tubules, (2) they are larger and are morphologically similar to other germ line cells, (3) their staining pattern is more like germ cells than somatic cells, and (4) because TEM reveals that they contain type I nuage. The frequency of mitotic figures among the oogonia of primary tubules is higher than that found in any other ovarian tissues of S. californicus. The plane of the divi- sion spindles in oogonia is parallel to the inner epithe- lium, preventing daughter cells from being pushed into the tubule lumen at mitosis. Oogonia occur throughout the length of the primary tubule; indicating there is no discrete zone of mitotic proliferation in this tissue. Peri- toneal epithelial cells in primary tubules also divide, al- though not as frequently as oogonia. Prophase ofmeiosis I in early secondary tubules. In the inner epithelium of secondary tubules, the somatic cells cannot be distinguished as parietal or follicular. These cells are bound to other inner epithelial cells, somatic and germ line, by adhering junctions; or they may be closely applied to the surface of adjacent cells but show- ing no junctions. The somatic cells are smaller than germ line cells and have nuclei with peripheral heterochro- matin. The germ line cells here are quite small relative to those in late secondary tubules, and their chromosomes are in configurations characteristic of early prophase stages ofmeiosis (compare Fig. 21). These cells average about 15 ^m in diameter, lack yolk, have adhering junc- tions with adjacent cells, contain nuage, and appear to be associated with a special basal lamina, the oolamina. Because of the small oocyte size, oolaminae in early sec- ondary tubules extend over a greater area at the basal surface of the oocyte than in the fecund tubules (Smiley and Cloney, 1985), but it has the same staining proper- ties and fine structure. The differences in relative size of the oolamina in secondary and fecund tubules are proba- bly a result of oocyte growth. An aggregation of densely staining particles lies adja- Figure6. Light micrograph of the gonad basis. C. connective tissue compartment, IE. inner epithelium, L. lumen, P. peritoneum. 375X. Figure 7. Light micrograph of a cell nest within the connective tissue compartment of the gonad basis. BL. basal lamina limiting the nest, CF. connective tissue fibers, GC. germ line cell. SC. somatic cell. 1025X. Figure 8. Light micrograph of the gonoduct. C. connective tissue compartment of the dorsal mesen- tery. GL. gonoduct lumen, P. peritoneum. Note the absence of a genital cord. 255X. Figure 9. Light micrograph of a cross section of a resorbing spent tubule. M . muscle cells of the complex peritoneum. L. ovarian lumen. Oo. oolamina, P. peritoneum, PC. petaloid amoebocytes containing lipo- fuscin granules. 525X. Figure 10. Light micrograph of a cross section of a late secondary tubule. CF. connective tissue fibers, Fc. follicular inner epithelial cells, L. ovarian lumen, Oo. oolamina. P. peritoneum. 250X. :- - ." -i. V • 'vvr>- h iijurv 1 1 . Tf M of a germ line cell 1mm a late primary luhule. N. nuclcolus, Nu. nucleus. PB. pericen- triolar body (satellite structures). 6980X. Kigurc 12. TEM at higher magnification of the centriolar satellite structure in Figure 1 1. G. Golgi licxly. M. mitochondrion. I'H periccntriolar body (satellite structures). Arrowhead points to microtubules • ing from the ccntriole. 2 1 .700X. 13. TEM of cell nest within the gonad basis. BI .. basal lamina separating cell nest from. C. • e tissue compartment. M. mitochondria, Nul. nucleus of a germ line cell. Nu2. nucleus of a sorn.. (I Hjjim M II M showing nuagc in a spireme stage oocytc from an early secondary tubule. G. Golgi body. M.rn: n.lnon. I. type I nuage. II. type II nuage. I3.330X. KiKurv 15. II M showing mitochondria! cloud from a previtellogemc ooc>te in a secondary tubule. MC. mitochondria! cloud. Nu. nucleus of oocyte, 1. type I nuage. Type 11 nuage is located between the mitochondria of the cloud. 8200X. KiKurc 16. TI-M \egetal mu m\ Hli in an oocyte from a late secondary tubule just beginning vitellogen- esis. Many of the paniculate densities are precipitated lead stain, an artifact. JS. jelly space, M V. microvilli. O. oocyte. 20,000> HOLOTHURIAN OOGENESIS AND REPRODUCTIVE CYCLE 85 111 .Ilit'. iiiiil . ;. ??m: - ^ ; , • ' I '\J ' r "' ' • ' • * ' f'f- • - 1 - » • • " " ::*%&*';V M ra;'"--; - • V. 1 " ^j8w:-^.^w.-' •*. '.•/•••'-•• j . v • ; -' , . } , : -' ' >•' . L§A ' :. ' "' '- ;- •:'>.••;" /*•<• Figure 17. TEM showing unusual spherical aggregations of mitochondria. M. spherical aggregations of mitochondria, NE. nuclear envelope, I. type I nuage, II. type II nuage. 13.060X. Figure 18. TEM of cilia found on somatic and germ line cells within the cell nests. C. cilium, GC. germ line cell nucleus. Arrowhead marks an apical centriole in this germ line cell. 11,1 50X. Figure 19. TEM showing intercellular junctions between adjacent inner epithelial cells in the cell nests. AJ. adhering junctions, GC. germ line cell, SC. somatic cell, I. type I nuage. 8360X. cent to the nucleus, in the vegetal region of prediplotene oocytes of the secondary tubules (Fig. 3). These are not yolk, but a cloud of mitochondria (Fig. 1 5). As oogenesis progresses these mitochondria become arranged in strik- ing spherical aggregations which enclose granular elec- tron-dense material (Figs. 14, 17), here called typell mi- age, which is ultrastructurally indistinguishable from similar nuage found in other echinoderm oocytes (Mil- lonig et at., 1968; Eddy, 1975). Type I nuage (Fig. 14) is also present in these oocytes. Oocytes of secondary tu- bules have a centriole located in the peripheral cyto- plasm (Figs. 11, 12). The centriole has a full compliment of satellite structures (Fig. 12) as would be expected in a cilium producing centriole, yet holothurian oocytes are not known to bear a cilium and none were found in these sections. The centriole and the mitochondria! cloud de- fine the future egg axis, judged by their relationship to the oolamina. It is possible that the centriole acts to fash- ion the oocyte protuberance, which contains dense ar- rays of microtubules (Smiley and Cloney, 1985). Diplotene oocytes in late secondary tubules. The term previtellogensis is used here to include all phases of oo- cyte development beginning with the production of oo- cytes in the mitotic proliferation of oogonia, and extend- ing to the inception of active vitellogenesis. Cytologi- cally, the earlier portions of oocyte previtellogenesis can be referred to as spireme stages (Wilson, 1925), describ- ing the characteristic chromosome morphologies. The spireme stages of leptotene, pachytene, and zygotene are evidently accomplished rapidly in S. californicus since they are only rarely encountered in sections. The chro- mosomes decondense at diplotene; the nucleus enlarges and assumes an expanded germinal vesicle configura- tion, giving the oocyte its most recognizable morphol- 86 S. SMILEY 4 . * **^l^ rf^ *» *. >A Figure 20. Light micrograph ol'prcvitellogcnic oocytes in a late secondan, tubule. MS. spherical aggre- i-'ations of mitochondria (see Fig. 17). 1. type I nuage. 1I30X. ! icure 21. Light micrograph of diplotenc configuration to chromosomes of secondary tubule ooc>1es. diplotenc chromosomes. N. nucleolus. Sc. somatic cells. 940X. turc 22. Light micrograph of early vitellogenic oocyte in the late secondary or early fecund tubule. II genital hemal sinus of the connective tissue compartment. Oo. oolamina. 375X. KiRun- .' i micrograph of frontal section through the developing gonad of a juvenile .V californi- cus.CC.cc tissue compartment, Gd. goncxluct, IE. inner epithelium, P. peritoneum, T. tubules. 75x. Figure 24. Light micrograph of a cross section of a vitellogenic tubule. PC. follicle. H. genital hemal sinus. LF. longitudinal fold. P. peritoneum. Pvo. previtellogenic oocytes, Vo. vilellogenic oocytes. I SOX. HOLOTHURIAN OOGENESIS AND REPRODUCTIVE CYCLE 87 ogy. The diplotene stage extends into the vitellogenic pe- riod, and is terminated when the chromosomes enter diakinesis. Diplotene has the longest duration of any of these stages in 5. californicus (Fig. 20) and most oocytes in late secondary tubules have their chromosomes in this configuration. The morphology of oocytes and somatic cells in sec- ondary tubules changes greatly during early oocyte growth and differentiation. When tubules have advanced to where their oocytes are in the diplotene stage, somatic inner epithelial cells adhere to the surface of the oocyte (Fig. 10). During the transition from early to late second- ary tubules, these somatic cells form junctional com- plexes between themselves, creating a true follicle around the oocyte, but it is only in late secondary tubules that the parietal inner epithelium is clearly identifiable (Fig. 24). When a follicle is present around secondary tu- bule oocytes, the jelly space is absent. Oocytes adhere to the connective tissue layer of the ovary (Fig. 10) by their oolamina in all but the most advanced secondary tu- bules (Smiley and Cloney, 1985), and attempts to man- ually dislodge oocytes with fine needles were unsuccess- ful. Oocytes here average about 40 ^m in diameter, con- tain type I and II nuage, and for the most part lack yolk. The large diplotene germinal vesicle nucleus is probably active in synthesizing messages required for further de- velopment, but no specific information on this point is available. Vitellogenesis in smaller fecund tubules. Stichopus cal- ifornicus oocytes start to accumulate yolk late in the fall, beginning at about the end of October in the San Juan Archipelago population. The onset of vitellogenesis oc- curs only in late secondary tubules, and is heralded by a substantial increase in cellular debris found in the genital hemal sinus of these tubules (Fig. 22), during the resorp- tive period and torpor that follows spawning (Fank- boner and Cameron, 1985; Cameron and Fankboner, 1986). Vitellogenesis initially proceeds at a leisurely pace, determined by the slow increase in dense baso- philic granules in oocytes of animals fixed during No- vember. By late November, there is a marked increase in the number of granules per oocyte section, indicating that the pace of vitellogenesis has increased. The pace continues to increase until mid January when it appears to level off. The accumulation of cellular debris within the genital hemal sinus is paralleled by elaboration of mi- crovilli at the vegetal pole of the oocyte adjacent to the oolamina (Fig. 16). TEM reveals that the majority of these granules are yolk platelets (Smiley and Cloney, 1985; Fig. 23), but some are mitochondria, and others type I nuage. During vitellogenesis the spherical aggregations of mitochondria that surrounded nuage in early secondary tubules break up. The mitochondria and nuage disperse within the ooplasm and are not localized to the perinuclear vegetal region. Vitellogenesis is detectable earlier in more cen- trally located oocytes (Fig. 24). Oocytes near the periph- ery of tubules are the last to show an increase in size and in accumulation of the dense basophilic yolk granules diagnostic of vitellogenesis. The average diameter of oo- cytes increases during the late fall and through winter and early spring in S. californicus. The juvenile gonad. In juvenile Stichopus californicus, the gonad has started to develop by three to four months after metamorphosis (Fig. 23). The initial structures to form include an expanded central connective tissue component of the gonad basis, a developing gonoduct, and two to four pairs of tubules, similar to the conditions reported for Holothuria parvula (Kille, 1942). The tu- bules are small and unbranched. The most posterior tu- bules are slightly larger than those more anterior, but the germ line cells within their inner epithelia are all in the proliferative oogonial stage, as determined by the pres- ence of mitotic figures in serially sectioned gonads. Juve- niles are difficult to raise, and consequently are rare. I took serial 1 ^m sections of the gonads from the four specimens I raised, and found no genital rachis (Hyman, 1955; Smiley, 1988b) present in any of these specimens. Discussion Structural aspects ofoogenesis in Stichopus californicus This data shows that the gametogenic holothurian ovary consists of three classes of ovarian tubules, which can be defined by their size, their location along the go- nad basis, and the cytological stage of the germ line cells within them. These results are harmonious with and ex- pand upon those of Theel ( 1 90 1 ), Mitsukuri ( 1 903 ), and Kille (1942) concerning the function of the smaller ovar- ian tubules anterior on the basis in other holothurians. However, my results are in conflict with the arguments of Delage and Herouard (1903) who suggested that the function of the smaller tubules was to provide a fluid that augmented the spawn. The tubules represent a contin- uum between the smallest most anterior primary tu- bules, and the largest most posterior fecund tubules. This continuum is broken by the annual episodic cytological changes in the development of the oocytes. All primary tubules, whether larger or smaller, contain only oogonia. All secondary tubules, whether early or late, contain pre- vitellogenic oocytes. The onset of vitellogenesis in sec- ondary tubules during the fall is a convenient marker for determining the initial stage of the fecund tubules. This description of the resorption of spent tubules, the progressive increase in size of the secondary tubules dur- ing autumn, and the localization of separate functions to particular classes of tubules supports the hypothesis that tubules are progressively recruited to a more posterior position along the flanks of the gonad basis as they de- velop. Direct testing of this hypothesis is not easy. Vital S.S s SMII I > dye markers disappear long before a year has passed. 5. califomicus eviscerates when tagged, and animals kept in aquaria for more than a few months shrink in size, and fail to develop OOC\'.-N properly (Smiley, pers. obs.). In the field, indr ange freely and readily change depth (Counnc 17). The most direct test of this hy- pothesis o to mark mitotic oogonial cells with radioactive nidine. A sizable number of animals would ha\e to be tagged to insure significant recovery, and doses of the marker would have to be initially high to be detected reliably. Because the morphological evi- dence is so compelling, use of this much radioisotope is probably not warranted. Oocyte polarity and attachment Holothurian oocytes are among the most visibly polar- ized in the animal kingdom (Smiley and Cloney. 1985). In fully formed oocytes. this polarization is referred to as the egg axis, which passes from the oocyle protuberance at the animal pole, through the eccentric germinal vesi- cle, to the oolamina at the vegetal pole. This axial polar- ization develops gradually as oocytes increase in size and is continuously congruent with an epithelial cell polar- ization of the germ line cells that is evident from the time they are first identifiable in the cell nests. This epithelial character of the polarization is defined by the presence of a luminal apical surface, the apical centriole, junctional complexes with adjacent cells, and a basal lamina. The centriole of 5. califomicus oocytes is apical (ani- mal quadrant), and similar in position to those in oocytes of the asteroid Pinaster ochraceus (Schroeder and Otto. 1984: Schroeder, 1985). This is in contrast to reports in Xenopux oocytes of a basal centriole (in the vegetal quad- rant) which is associated with the mitochondria! cloud (Al-Mukhtar and Webb. 1971; Coggins, 1973). a posi- tion not homologous to that in any other epithelial cell. I contend that these workers have mistakenly assigned a basal position to this centriole because they assumed a vegetal location for the aggregated mitochondria of the Balbiani body. But. the terms 'Balbiani body' and 'yolk nucleus' actually mean any basophilic zone near the ger- minal vesicle (NoYrevang, 1968), and neither aggregated mitochondria norGolgi bodies are restricted to the vege- tal quadrant or to the mitochondria! cloud according to other investigators (Heasman et a!., 1984; Wylie el a/., 1985). Wilson i ) suggested promorphological homology for the vegetal loci ion of the oocyte centrioles through his comparison ol < \\ ith spermatozoa which have a centriole basal to the niu k-us. However, his depiction of spermatozoa is inverted compared to their true pro- morphological architecture. The trailing flagellum is ac- tually an apical cilium; and the centrioles of spermatozoa are apical to the nucleus. In other words, sperm swim backwards. Consequently, if the centriole is indeed vege- tal in Xenopus oocytes, one would expect to identify cen- trioles in two different axial positions in premetamorphic tadpole gonocytes. Al-Mukhtar and Webb did not report this observation. Recent analyses using immunocyto- chemical methods did not identify a vegetally located centriole in Xenopus (Palacek el a/.. 1985: Dent and Klymkowsky, 1988). Given these arguments, it seems prudent to reinvestigate the axial pattern in Xenopus pre- diplotene oocytes using these techniques. In some previous descriptions of other holothurians (Inaba. 1930) and other echinoderms(Boveri, 1901). oo- cytes were often thought to be attached to the somatic ovary by their vegetal surfaces. Other investigators hold that oocytes are attached by the animal surface ( Lindahl, 1932; Monne, 1946, Holland et al., 1975). In Stichopus califomicus, both views are correct depending on the stage of oogenesis. In early stages, oocytes are connected to the somatic ovary by the oolamina at their vegetal pole. In more advanced stages, the protuberance, an ani- mal pole elaboration, connects oocyles to the cells of the somatic ovary. Sections through less fully developed ova- ries might lead one to erroneously conclude that holo- thurian oocytes are always attached by their oolaminae. Information derived from thicker sections of poorly fixed and paraffin embedded specimens might also lead to erroneous conclusions. Origin of the germ cells If we accept type I nuage as a more critical marker of germ line cells than either alkaline phosphatase or dense RNA accumulation (Eddy, 1975), then PGCs can be reli- ably identified only with TEM or immunocytochemistry (Strome and Wood. 1982, 1983). Between the somato- coels in newly metamorphosed S. califomicus there is set of mesenchymal cells which contain dense RNA accu- mulations characteristic of germ line cells, but these do not have unambiguous type I nuage as determined by TEM (Smiley, 1986). In the present study. I show that when Stichopus califomicus is six months old. gonado- genesis has begun, and unambiguous PGCs are located in the connective tissue compartment of the dorsal mes- entery. The location and timing of the appearance of these germ line cells is consistent with previous reports of the onset of holothurian gonadogenesis (Cuenot. 1948; Wootton, 1949). These results support the views of Nieuwkoop and Sutasurya (1981) who concluded that echinoderm germ cells first become visible some months after settlement. However, primordial germ cells have been described in newly metamorphosed echinoids (I li.uk and Hinegardner. 1980). These arguments should not be interpreted to mean either that germ line cells of holothurians or other cchi- noderms show an irrevokable lineage restriction, or that HOLOTHURIAN OOGENESIS AND REPRODUCTIVE CYCLE 89 there is an inviolable restriction barring other cell types from becoming germ line cells. The results of Kille (1942), on posterior half regenerates of Holothuria par- vula following binary fission, suggest that at least perito- neal epithelial cells can transform into germ line cells un- der some conditions. Theel ( 1 90 1 ) described cell aggrega- tions lying against the ascending gonoduct in the dorsal mesentery of adult Mesothuria intestinalis, as a genital cord, which contained cells that he interpreted to be PGCs. The histological description of the genital cord in M. intestinalis does not match that of the cell nests in the gonad basis of S. californicus, but the location and description of M. intestinalis PGCs themselves are quite similar to those of S. californicus reported here. Pre- sumptive PGCs occur in cell nests lodged within the dor- sal mesentery of the aspidochirote Holothuria parvula (Kille, 1942). Until PGCs have been described in holo- thurians from different taxa, we must assume that the differences in location and structure of the germ line bearing tissues reflect intraordinal variations. Cytological aspects ofoogenesis Nuage. This is the first specific identification of nuage in a holothurian oocyte. Nuage is electron-dense mate- rial, lacking a unit membrane, which is found in the cyto- plasm of germ line cells where it commonly occurs in two forms (Eddy. 1975, Kessel, 1983). Type I nuage is about 1 nm in diameter and is a granular electron-dense material (Fig. 17) forming rough spheres. Type II nuage consists of minute paniculate electron-dense granules surrounded by a homogeneous matrix which is slightly more electron dense than ordinary cytoplasm and which excludes ribosomes (Figs. 14, 17). Both types of nuage are often associated with mitochondria, especially in fe- male germ line cells (Fig. 17; and Millonig el al, 1968). The relationship between nuage and the mitochondrial yolk cloud is likely to be a fundamental one judging from the association between these elements in the oocytes of species from numerous phyla (Eddy, 1975). To date there is no detailed explanation for this association. In echinoderms, a number of different names have been applied to nuage including 'dense lumps' in oocytes of the crinoid Nemaster rubignosa (Holland, 1971) and probably the extruded nucleoli in oocytes of the holothu- rian Thyone briareus (Kessel, 1966). Type I and type II nuage have been reported in oocytes of adult echinoids (Millonig el al.. 1968). Houk and Hinegardner (1980) found structures similar to type I nuage, which they called goniosomes, in cells presumed to be PGCs in newly metamorphosed Lytechinus pictus, and types I and II nuage were found in oocytes of Xenopus laevis (Al-Mukhtar and Webb, 1971;Coggins, 1973). Available reports suggest that all animals probably have some form of germ line specific granule, whether it be called nuage (Eddy, 1975), polar granules as in Drosophila melano- gaster, or P granules as in Caenorhabditis elegans (Wolf et a!.. 1983). Nuage appears to be germ line specific, be- cause somatic cells have not been shown to contain it (Eddy, 1975). Determination of the origin of germ line cells in other echinoderms may be facilitated by using nuage as a marker. Nuage is distinct from the heavy (dense) bodies, large granular inclusions surrounded by annulate lamellae often encountered in a variety of echi- noderm oocytes (Afzelius, 1957; Eddy, 1975; Kessel, 1966; see Smiley and Cloney, 1985. Fig. 22). Nuage is no longer aggregated in S. californicus oo- cytes after the onset of vitellogenesis. The spherical ag- gregations of mitochondria which surround type II nu- age disperse, presumably carrying nuage along with them. A similar dispersal was noted in echinoids (Mil- lonig et al., 1968). If nuage is a germ line determinant, then its dispersal may be expected in those animals, such as echinoderms, where regulative development is the rule. If the nuage were not to disperse it would be absent from isolated blastomeres which then would lack the germ line determinant, a condition incompatible with numerous surgical studies on echinoderm embryos (Hor- stadius, 1973). In support of this assessment is a recent report by Wylie et al. (1985), which suggests that the mi- tochondrial cloud of Xenopus laevis disperses through- out the vegetal ooplasm when the oocyte becomes fully formed. In this case mitochondria and nuage remain somewhat localized to the vegetal quadrant. Mitochondria! cloud. This structure (Fig. 15) has been referred to as the yolk nucleus, the Balbiani body, and the Balbiani vitelline body in other oocytes (Nerrevang. 1968;Guraya, 1979), as well as the mitochondrial cloud (Heasman et al., 1984; Wylie et al., 1985). The defini- tions for these terms appear to be capricious; they refer to a number of structures having only a juxtanuclear po- sition and basophilic staining characteristics in common (Norrevang, 1968;Guraya, 1979). The term 'mitochon- drial cloud' is at least accurately descriptive and is used here. In S. californicus, the mitochondrial cloud is always found in the vegetal hemisphere of developing oocytes, close to the nuclear envelope. In sections through sec- ondary tubules (Figs. 2, 3), adjacent oocytes appear to have their mitochondrial clouds in opposing orientation. But the complex topology of the inner epithelium shows that this is not the case (Smiley and Cloney, 1985). In these oocytes, as well as in those of Xenopus laevis, the vegetal mitochondrial cloud appears to be the site for mi- tochondrial proliferation (Heasman £? al., 1984), judging by the frequency with which dumbell-shaped mitochon- dria are encountered. However, the identity of the bio- 90 S SMII IV chemical pathways controlling this proliferation are not known. Among other echinoderms. a mitochondrial cloud is found in crinoid oocytes (Holland. 1976). The report that echinoid c jytes contain yolk nuclei ( Verhey and Moyer, 1 96~ viust be reinterpreted, because the mi- crographs tl' > \estigators present as documentation of their c tow only annulate lamellae (their Fig. 20). I 'lit . \ Intense vitellogenesis begins in the late seconds: > tubules after the autumnal resorption of the viscera in Slichopus californicus. At this time, the genital hemal sinus contains cellular debris and unidentifiable paniculate matter, and the number of yolk granules in oocytes increases. Vitellogenesis begins in oocytes at the center of the 5. californicus ovary, but an explanation for this is not readily apparent. It appears likely that oocyte growth constricts passage of hemal fluid through the gen- ital hemal sinus toward the interior of the tubule (Smiley and Cloney. 1985). But rapid growth of central oocytes, occluding only the most central parts of the ovary, would still allow the peripheral oocytes access to an unrestricted hemal sinus. Yolk proteins appear to be preferentially taken up at the vegetal pole of the S. californicus oocyte. based on fine structural information alone. Elongate microvilli are found at the vegetal surface of 5. californicus oocytes during the post resorptive period of vitellogenesis (Fig. 16). Similar structures have been described at the pre- sumptive vegetal pole of an asteroid oocyte (Beijnink a ell., 1984). Some elasipod holothurians have very large eggs, and uptake occurs over the entire egg surface (Eckelbarger. pers. comm.). Recent studies of echinoid vitellogenesis indicate that cells within the echinoid ovary, either accessory cells or oocytes or both, may synthesize some of the yolk glyco- proteins (Ozaki rttf/., 1986; Shy u et ai. 1986). Cells ex- plicitly homologous to the accessory cells do not occur either in any holothurian yet described (Smiley and Clo- ney, 1985) or in crinoids (Holland et ai, 1975). Conse- quently, these classes require a different mode of nutrient replenishment, which may be provided by the coelomo- cytes of the perivisceral coelomic fluid. The most abun- dant protein found in echinoid coelomic fluid shows im- munocytochemical cross reactivity with the egg 23S yolk glycoprotein (Giga and Ikai, 1985a, b). Coelomocytes in the coelomic fluid may be the largest single source for all yolk gl ik-ins. at least in Dendraster cxceniricus (Harrington and Ozaki, 1986), but ovary (Ozaki et a/.. 1986) and gut (Sh el ai, 1986) also produce yolk pre- cursor proteins. k>. Its from the experiments of Shyu i't ai ( 1986), while do. uncnting that the coelomic fluid contains large amounts of the yolk precursor proteins in Strongylocentrotus purpuratus, appear to contradict the conclusion that coelomocytes arc a major source. Shyu et ai ( 1 986) did not include a divalent cation chelator in their coelomocyte culture medium, and they only la- beled for about one quarter the time that Harrington and Ozaki did. In the absence of 10-50 m.\/ EDTA, echinoid petaloid amoebocytes undergo an irreversible clotting re- action (Otto et ai, 1979: Edds, 1980). This suggests that the failure of Shyu el ai to record radiolabeled amino acid incorporation into vitellogenin in the coelomocytes might be due to differences in the duration of labeling or to the clotting response. Future research into holothu- rian vitellogenesis should include an examination of coe- lomocytes to see if they are a rich source of vitellogenins. If they prove to be, then the absence of a discrete nutrient storage organ in this class might be explained, and the pathway of nutrient replenishment proposed by Smiley and Cloney ( 1985) supported. The annual cycle of the S. californicus ovary The results of this study demonstrate that holothurian oogenesis follows the same cytological course that has been described for a number of other animals in different phyla (Wilson, 1925). In all non parthenogenetic ani- mals, the PGCs, proliferating oogonia, spireme stages of meiosis I prophase, diplotene, and diakinesis sequence is followed in exact order. Repetition of this order, coupled with the localization of these clearly interpretable cyto- logical stages into discrete and linearly ordered structures within the 5. californicus ovary, offers a simple explana- tion of the annual ovarian cycle in holothurians. The cytology of the germ line cells of cell nests, pri- mary and secondary tubules, and the complete resorp- tion of spent fecund tubules support the idea that more anterior tubules are progressively recruited to a more posterior position on the gonad basis with the same tim- ing as that shown by changes in the cytological stages in the oocyte nuclei. This suggestion was also made in other aspidochirotes by Mitsukuri ( 1 903) for Stichopus japoni- cus, Kille (1942) for Holothuria pan'ula. and Deich- mann ( 1948) for Neostichopus grammatus, and for the dendrochirote Thyone briarcus by Kille ( 1 939). This no- tion of progressive recruitment is attractive in S califor- nicus because it is a large animal that doubtless lives for many more than six years, and probably spawns in all but the first three (Fankboner and Cameron, 1985: Cam- eron and Fankboner, 1986). If progressive recruitment of new tubules did not occur in .S. californicus, then after its sixth year the animal would be without spawn. The Model My results provide the information necessary to pro- pose a model of how the annual cycle of the S. calijorni- c//.v ovary progresses. The annual cycle begins with the resorption of spent tubules in year N, and can be summa- ri/ed as follows. Some of the nutrients derived from HOLOTHURIAN OOGENESIS AND REPRODUCTIVE CYCLE 91 phagocytosis of the spent tubules are taken up into the genital hemal sinus of the secondary tubules, as indicated in Figure 22. Concurrent with the increase of material in the genital hemal sinus, vitellogenesis begins in these oocytes. Therefore, from late fall to early spring, second- ary tubules become the fecund tubules of year N + 1 and are located at the posterior of the gonad basis. Immedi- ately after resorption of the spent tubules, the genital he- mal sinus and lumina of primary tubules and hemal la- cunae in the connective tissue compartment also become occluded with debris and nutrients. The primary tubules, having completed the oogonial proliferative divisions, become the secondary tubules of year N + 1 and are now located further back on the basis. During the fall, winter, and early spring, oocytes in these new secondary tubules (yearN + 1 ) undergo the early prophase stages of meiosis I, culminating in diplotene. Nests of cells within the go- nad basis emerge from the connective tissue compart- ment during the early fall. Surrounded by the perivis- ceral peritoneum, they become the primary tubules of year N + 1 and are found in the anterior location charac- teristic of primary tubules. General applicability of the model Hyman (1955) suggested that resorption of spawned tubules would prove to be the rule among holothurians. If spent tubules are resorbed after spawning, it is likely that progressive recruitment of tubules also occurs, with anterior less advanced tubules moving to a more poste- rior location concomitant with advancement in the cyto- logical development of the germ line cells. Progressive recruitment of ovarian tubules has been strongly sug- gested in several aspidochirotes; Stichopus califomicus (this study), Mesothuria intestinalis (Theel, 1901), as well as in the dendrochirote Thyone briareus (Kille, 1939). However, there is too little comparative informa- tion on oogenesis in apodan, molpadian, dactylochirote, and elasipod holothurians to assess the applicability of this model to them. The best information supporting this model are the surgical experiments of Kille (1939) with the dendrochirote Thyone briareus. These showed that of all the ovarian tissues, the gonad basis alone can regen- erate oocytes. When only fecund tubules were ablated, new oocyte-bearing tubules regenerated from the basis. When the entire gonad including the basis was removed, regeneration failed. Kille's (1942) study of the gonad in Holothnria pannila, an animal which also reproduces asexually through binary fission, provides additional support for this model. The data and analysis presented in this paper, while considerably more precise in determining cell and tissue level changes, is limited by this precision, and cannot supplant population level surveys. Information derived by the gonad index method may be applicable in assess- ing this model's generality. From the data presented here, it is clear that these gonad index studies measure vitellogenesis and not the process of oogenesis. A num- ber of species have been investigated in this way, includ- ing Stichopus califomicus (Cameron and Fankboner, 1986), Stichopus japonicus (Tanaka, 1958), Thelenota ananas and Microthele nobilis (Conand, 1983), and the data they present are entirely consistent with the pattern described here (see Smiley et al., for a more complete review). Consistency, not confirmation or refutation, is all that can be expected from such studies, because gonad index assessments only measure average changes in pop- ulations. Analysis of reproductive cycle data derived from gonad indices of populations of many different ho- lothurians shows that most species have the general fea- tures predicted by this model. This includes marked di- minishment in gonad index after spawning, the subse- quent further reduction in index corresponding to phagocytosis of spent tubules, the measurable lowest size corresponding to the basis and more anterior tubules, and the gradual build up in index as vitellogenesis for the next season commences (Smiley et al.. 1988). This model may not account for the gonad index data of male holothurians of any order, nor of small and her- maphroditic holothurians, such as Cucumaria curata (Rutherford, 1973), or Rhabdomolgus ruber (Menker, 1 970). Because many Arctic, Antarctic, and deep sea spe- cies are thought to produce eggs continuously (Feral and Magniez, 1985; Tyler et al., 1985) they may also pose problems for this model. Comparisons with other echinoderm classes The anatomical and histological simplicity of this ho- lothurian ovary offers unique information that is appli- cable to understanding the dynamics of oogenesis in other echinoderm ovaries (Smiley, 1984, 1986, 1988b; Smiley and Cloney, 1985; Smiley et al., 1988). It is difficult to study microscopic morphological changes among a synchronously developing population of oo- cytes in other echinoderms. Primordial germ cells, mi- totic oogonia, and early meiotic prophase staged oocytes are considerably smaller than previtellogenic or vitello- genic oocytes; they can be quite difficult to locate within the inner epithelium. This difficulty is reflected in the limited reports on the earliest stages of oogenesis in other echinoderms (Holland etai, 1975; Walker, 1982). These same structural problems have prevented biochemical analysis of oogenesis in any echinoderm. Given the iden- tifiable and discrete localization of the major cytological stages in oogenesis in the holothurian ovary, such bio- chemical analyses may now be possible. 92 S. SMILEY Unanswered questions This study offers a number of important observations and analyses which require more detailed investigation. Among these are t'u ble signalling role of the re- sorptive pha*. onset of torpor in driving subse- quent cogent"; c is no clear description of the ear- liest stages 'opment in the PGCs of any holothu- rian. nor o ir ultimate source. If cells other than those containing :iuageare capable of transforming into PGCs (Kille. 942), then the conditions under which this trans- formation can occur are important to know. This knowl- edge would allow a more detailed analysis of the mecha- nisms of coordinated control over the regulatory path- ways for differentiation in an echinoderm. an elusive problem made more difficult by the extreme regulative development of these animals. The mechanisms of con- trol over the proliferate divisions of the mitotic oo- gonia are not known, nor is the control of entry into the prophase stages of meiosis I or the biochemical details of these changes. Even if the onset of vitellogenesis is con- trolled by resorption of the viscera, a mechanism that could explain how the interior oocytes of late secondary tubules are directed to begin vitellogenesis prior to the peripheral oocytes is elusive. We have very little informa- tion on the relative contribution of the oocyte itself on yolk formation in any holothurian species. Finally, the reasons for the general resorption of the viscera of this holothurian each fall, even in those individuals that have not yet reached sexual maturity (Fankboner and Cam- eron. 1985). remain a mystery. Acknowledgments Much of this work was done at the Friday Harbor Lab- oratories. 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ANDERSON1, AND MONIQUE DURAND-CLEMENT2 1 Woods Hole Oceanographic Institution, Woods Hole, Massachusetts 02543, and - Unite ISSERM 303. Marine Station. 06250 I'illejranche Sur Mer. France Abstract. Preliminary assessment is made of two key components in ciguatera ecology. First, we examined the numerical variability ofGambierdiscus toxicus as an epi- phyte on the macroalgae, Diclyota and Galaxaura. Vari- ability was examined by a statistical bootstrap technique to determine the minimum number of samples required to adequately estimate the abundance of G. toxicus at one station and to test for statistically significant differ- ences between two stations. A minimum of 10 replicates were needed at the relatively low G. toxicus abundance found at our study site. Second, we demonstrated the fea- sibility of conducting controlled laboratory experiments to assess the short-term colonization behavior of G. toxi- cus on selected macroalgae offered in varying mass and surface area ratios. To assess G. toxicus abundance and distribution, the number of cells per unit of host alga must be standardized. We show that contradictory con- clusions can be reached depending on whether the num- ber of dinoflagellate cells is normalized to algal biomass or surface area. Introduction The continuing enigma of ciguatera, namely the sto- chastic v;m:ihj|jty offish toxicity in time and space, must have its ba^: in the ecology and population dynamics of the benthic di , flagellates that produce ciguatoxin and maitotoxin. Si 'loflagellate species have been im- plicated as produci mical compounds that likely result in fish toxicity jmoto el ai, 1987). One of these Gamhierdiscus /< u ppears to be most signifi- cant in ciguatera ecology. Despite the identification over a decade ago of the most likely progenitors of the toxins Received !9Octobcr I987;acceptcd 31 May 1988. and numerous field and laboratory studies throughout the tropics (reviewed in Withers, 1982; Ragelis. 1984; Salvat, 1985; Anderson et ai, 1985; Anderson and Lo- bel. 1987), the state of knowledge about ciguatera and G. toxicus physiology and ecology remains "very primitive indeed" (Scheuer and Bagnis, 1985). Clearly, one reason for this situation is the complex and gradual manner in which small quantities of the highly potent ciguatoxin and related toxins move through the food chain to the higher predators. We be- lieve that a contributing factor has been the lack of a standardized, statistically rigorous methodology for ex- amining the distribution and abundance of the ciguatera dinoflagellates in time and space. In this preliminary study, we addressed the question: "How many samples of a macroalgal species must be collected at one time and at one site and depth to adequately quantify the abun- dance of G'. toxicus'.'" Strictly speaking, the results of our data analysis are valid only for one study site at one point in time and for the two macroalgal hosts we examined, but these data and methodologies document the large er- rors associated with inadequate sample size and provide a means to determine the minimum number of samples needed to test for statistically significant differences be- tween stations. In addition, we tested the feasibility of conducting laboratory studies of the preference of G. tox- icus for a particular macroalgal host. Analysis of these data demonstrate the misleading nature of dinoflagellate abundance expressed per gram of host alga and provide a good argument for yet another change to common enu- meration methodologies — namely the normalization of dinoflagellate abundance to host surface area rather than biomass (Bomber el al.. 1985). Ciguatera is a complex ecological phenomenon that is poorly understood. It seems appropriate to re-evaluate commonly accepted 94 CIGUATERA ECOLOGY 95 methodologies and assumptions and to test the feasibility of addressing certain fundamentally important behav- ioral and physiological issues in the laboratory. Materials and Methods Smdv site This field study was conducted at St. Barthelemy (17°54'N, 62°50'W) in the Caribbean (French West In- dies) from 8 to 20 August 1986. Extensive collections were made at the "Pointe de Negre" site located on the south side of the island, about 3 km from the port of Gustavia. Laboratory facilities and logistics on the island were provided by the New England Biolabs Foundation. Macroalgal collection and dinoflagellate enumeration Two macroalgal species were selected for intensive study, Dictyota sp. (probably D bartayresii and maybe mixed with D. dichotoma and D. divaricata) and Galax- aura sp. (probably G. corneum). Both species were epi- phytized by G. toxicus. Dictyota is a brown alga and Ga- laxaura is a red articulated coralline alga. The growth form of each is a complex 3-D structure of branches. Both algae were growing abundantly in close proximity. All specimens were collected between 5-6.5 m depth at the Pointe de Negre study site on 17 and 19 August 1986. Specimens were picked, placed in individual plas- tic bags that were sealed underwater, and transported to the laboratory in the dark at ambient water temperature. The bags were then vigorously shaken and kneaded to dislodge G, toxicus. The 20-250 ^m size fraction was col- lected by pouring the contents through Nitex sieves. The adequacy of this method was confirmed by successive shaking and washing, which produced few additional di- noflagellates. The collected material was backwashed into a vial with approximately 10 ml of filtered seawater. G. toxicus indi- viduals were enumerated with a compound microscope at 100X total magnification. A minimum of one ml of the concentrated suspension was scanned for each sam- ple. Macroalgae were blot-dried and weighed. Masses ranged between 1 and 20 grams. Dinoflagellate abun- dance was initially expressed as cells per gram blot dry weight of host algae. Subsequent measurements of algal surface area per gram blot dry weight allow the data to be normalized to surface area as well. Preference studies Two laboratory experiments were conducted. One used equal biomass amounts of macroalgae and the other combined two algae in different proportions. In all treatments, algae were rinsed several times to remove most epiphytes (determined by visual inspection of algae under a dissecting microscope) and placed in pairs in 125-ml beakers with filtered seawater. A known number of G. toxicus cells were then added (final concentration approximately 80 mr'). The samples were maintained for 24 hours in an incubator at 26°C on a 1 2: 1 2 L:D cycle at approximately 50 ^Einst m~2s~'. Beakers were mildly swirled every 4 h (except overnight) to evenly distribute those dinoflagellates which had not yet settled. When the experiments were terminated, the number of G. toxicus cells on each alga and those remaining unattached in the beaker were counted separately. Because of the differ- ences in algal morphology, the results are expressed as the percentage of all G. toxicus attached to the algae, as cells per unit mass and as cells per surface area. Two G. toxicus isolates were used-strain T3 isolated from Gam- bier Islands by R. Bagnis and strain SB01 isolated from Lorient, St. Barthelemy, by M. Durand-Clement in July 1986. They were grown in modified ES medium (Dur- and, 1984). Macroalgae measurements Surface area, wet weight, and displacement volume were assessed for the macroalgae Galaxaura and Dicty- ota. Surface area is the most difficult measurement to ob- tain accurately. We used three methods for surface area calculations. One technique consisted of dipping a dried macroalgal sample in a detergent solution that is sup- posed to adhere to the algal surface in a layer of consis- tent thickness (Harrod and Hall, 1962). By knowing the weight of solution that coats a standard area of plastic sheeting, the surface area of an alga can be calculated. We encountered numerous problems with this method and did not obtain consistent or reliable data. Clearly, this concept has potential, and modifications made by Bomber (1985, pers. comm.) using wet specimens and full strength detergent might be necessary, but problems exist with how the solution coats and is adsorbed by different algae. Other techniques involved morphologi- cal measurements of surface area. Enlarged silhouettes of Dictyota were analyzed using a computer and a digitizer graphics unit since that species' shape is essentially two- dimensional. Galaxaura was measured in pieces under a microscope with a micrometer. Galaxaura 's shape is tubular, and painstaking measurements were made of all individual pieces in a sample. Surface area by these latter two methods provided consistant data. Results Algal mass measurement The primary problem we encountered was the mea- surement of algal characteristics for valid interspecific comparison of host-alga selection by G. toxicus. The P. S. LOBEL ET Al. 10- -...-. A 8- i*mfc 6- 104% c o 69% 69/0 ~ 0 10 20 30 40 5O 6 Number ol Cells gm Diclyota -, , 70% 1 6- B Z 4- 3- 2- 20% 10% 1- n- 0 10 20 30 40 50 60 Number ol Cells gm Galaxaura Figure 1. Frequency distribution of the number of (iamhierili\ni\ toxicus cells on (A) Diclyota and (B) (itilti.Miiiru. Percentage relative frequency is specified above each column common measure of 'cells per gram blot-dry weight' is suitable for m/raspecific comparison of G. toxicus on a host alga. This measurement had the advantage of being easy and rapid in the field. However, the best measure- ment for interspec'\f\c host algal comparisons is 'cells per surface area.' The surface area ofDictyota was 105 ± 31 (range 67- 151) crrr gm' ' (n = 1 3). The surface area of Galaxaura was 31 ± 8 (range 25-42) cm2 gm"' (n = 4). Conversion of the number of G. toxicus cells per gm alga to cells per cm: was obtained by dividing by 1 05 for Dictyola and 3 1 for Galaxaura. The variance in surface areas is probably due in large part to measurement error, but the possibil- ity of allometric variation in these algae also needs to be examined. i'u'ld abundance Gambierdiscus toxicus was present at the study site, but in relatively low abundance. Every Dictyota sample (n = 29) hosted at least 5 cells g ' blot-dry weight. Eighty- six percent of the collection had greater than 10 cells g ' (Fig. 1A). The mean (±S.D.) number of G. toxicus on Dictyola wa> 24 ± 14 (range 5-56) cells g"' or 23 cells per 100 cm half of the Galaxaura samples (n = 10) were e] -ed by G. toxicus and another 20% had fewer than 1 < ' (Fig. 1 B). The mean (±S.D. ) abundance of G toJ on Galaxaura was 6 ± 10 (range 0-30) cells g ' or 11K>,- net 100cm2. The number of O' toxicus per gram of host alga was not a function of the size of individual Dtctyolti ( Fig. 2) or Gain \ O 60 n 50- 40- 30- 20- 10- z " Dictyota Sample Weight (blot-dry), grams !• iyure 2. Relationship between the number ofGombierdiscus toxi- i u\ cells per gm of Dictyola as a function of the sample weight of indi- vidual I)icl\'iiia masses (n = 29). No statistical correlation was found. CIGUATERA ECOLOGY 9' 50 50 A A '• 40 40 E 1 TO ! = . • ' : • . : : • • O 30 3 30 • • i : i • ' . ! i i : - i t 01 a at 20 ' . i i '•• s i ! • 1 i i i ! I i j MlMMI a ; : • : ) : ; ; j j ; \ \ \ \ \\\\\\\\\\ * 2° "3 : ' : • ! ! : • O O 1 i . ' ' • 10 • • •! ' ' t ' ; i t • ; ; : 10 Q : j ! \\ !: ji o- 10 e B 10 c TO c a Q) o S8 / : 1 / \ i "o \ T • "5 »- 6 , o 6 s "4it;;:- s \ UJ •0 4 "imliiiii ' 4 ,i i 1 rr~kJ T i i . ' i . \ N * • TO :"-:'-!-::1!mmtH|4444 * I s~--- C , • • . • • ' * t ! TTn c 2 TO - ' * • 2 55 CO 0' :•'«... o • 100-j c C re II c re .£ * 80 T .£ Jj so | n 0) U °)>o j go A re >- 60 ' re >- 60 £ O ' o 2 u t LU , — UJ i "H -Q 40 c -n 40 / 01 t_ u re v' o re a"? Q- re 20 , \' T . : • ^ re 2° ^^/ 55 ^_ , , , ... 55 Y 0 • 5 10 15 20 25 30 15 20 25 30 Number of Samples Figure 3. Dictyola. (A) Variations in means for given sample sizes drawn from the total pool of 29 samples calculated by the "bootstrap" method. The mean was calculated 25 times for each sample size. At sample sizes less than n = 5, some points exceed the limit of the ordi- nate. (B) Standard error of the means in Figure 5 calculated 25 times with the overall average connected by a solid line across all sample sizes. This indicates the variance associated with a mean calculated for a given sample size. As sample size increases, this variance decreases. (C) Percent change in the standard error of the means from n to n + I calculated 25 times for each n. The overall average is connected by a solid line across all n's. In this case, as sample size increased, the per- centage change in possible means decreased substantially up to about n = 15 after which additional samples did not significantly affect the mean. 10 Number of Samples Figure 4. Galaxaura. (A) Variations in means. (B) Standard error of the means calculated in A. (C) Percent change in the standard error of the means. In this case, n = 1 0 was insufficient to determine the point at which additional samples would not significantly affect the results. See Figure 3 for details. pies would be statistically adequate using parametric analyses to estimate the abundance of G. toxicus (Fig. 3). For comparison to other collections, these analyses can be used to determine the variance values that would be significantly different for specified sample sizes. Given the means and standard errors calculated above, the val- 98 P. S. LOBEL ET At. ues of sample means, \\hich \\ould be signiticanth different at P< .05 and I' < .01 assuming the same degree of standard error, can \ ply specified by a /-test. It would be necessary generate a new set of plots in other studies ur < .'av/n/.v abundance and distribu- tion. Several experiments were designed to evaluate the fea- sibility of determining if the G. toxicus abundance on different macroalgae is the result of stochastic processes or a demonstration of active substrate preference and se- lection. Preference was evaluated by counting the num- ber of dinoflagellate cells on each of the macroalgal choices (which were offered alone or in pairs in several different mass ratios) after 24 hours. Although in some cases the mass of the algal choices were the same, com- parisons are complicated by the different morphologies. For example, Dictyota is flat and Galaxaura is round, but both have equivalent mass to volume ratios { /. e. , 1 .05 and 0.96. respectively), although Dictyota has more than triple the surface area compared to the same mass ofGa- laxaura. In control trials where only one macroalga was available, approximately the same fraction of the intro- duced G. toxicus population chose to settle on Dictyota and Galaxaura (55 and 51%, respectively) after 24 h. A detailed breakdown of the Dictyota:Galaxaura se- lectivity is given in Table I, which shows the effects of differential availability of the competing host algae. The data are expressed in three ways. When the number of attached cells on each host species was expressed as a per- centage of the total attached cells, the Dictyota portion varied systematically between 24 and 74% as the Dicty- oia:Galaxaura mass ratio changed from 0.25 to 2.0 be- tween treatments. Stated differently, when Dictyota only represented 20% of the macroalgal biomass, 24%. of the cells selected it as a host. At the other extreme, when Dic- i villa represented 67% of the biomass, 74% of the cells selected it. Alternatively, if the G. toxicus abundance was normalized to the mass of the host species, the number on Diet \-itta was between 1 .3 and 2.0 times greater than that on a/ilaxuura for the four treatments. Comparative analysis of the number of G. toxicus cells per unit , area on different algae suggests a differ- ent pic 1 1 nta has approximately three-times greater sin a per gram wet weight than does Ga- laxaura (ratio D. 'm is 3.4). On a per unit surface area basis, the m. >f cells on Dictyota was between 1.7 and 2.7 times less than on Galaxaura. The number of G. toxicus per gram and per cm2 of Dictyota or Gala\- aura remained remarkably consistent even when the ra- tio of the macroalgal masses varied eight-fold. At Pointede Negre, the number of (/ ii>\icu.\ cells per gram of Dictyota was significantly greater (a ratio of 4: 1 ) than on Galuxaura (/-test. t == 3.6449, DF = 37. P < 0.01). The two macroalgal species were common and present in approximately equal abundance at the study site. Discussion Numerous investigators world-wide have conducted field surveys to elucidate the in .situ population biology of G. toxicus and the other ciguatera dinoflagellates. To date, reports on the distribution and abundance of these dinoflagellates have been uniformly based on small sam- ple sizes (n < 10), with the dinoflagellate numbers being variously described in terms of: (A) the maximum num- ber of cells per gram of host algae (genera or species not always specified; Carlson et ai, 1984: Ballantine el al., 1985; Carlson and Tindall, 1985; Taylor, 1985; Taylor and Gustavson, 1983); (B) cells per gram of a specified alga (Shimizu et al., 1982; Bagnis el al.. 1985a; Ballan- tine et al.. 1985; Taylor, 1 985); (C) cells per gram of mul- tiple unspecified algae (Bagnis et al.. 1985b; Caire et al.. 1985; Gillespie et al.. 1985a, b; Taylor and Gustavson, 1983); and (D) cell counts per algal surface area (Bomber et al.. 1985). The purposes of these surveys varied, as did the macroalgal species distributions, but the lack of a co- herent picture of G. toxicus ecology (Scheuerand Bagnis, 1985; Anderson and Lobel, 1987) nevertheless argues that a re-examination of commonly used methodologies and assumptions is warranted. A desirable initial goal should be the standardization of sampling and enumera- tion procedures. One of the first questions facing any field survey is the number of macroalgal species to sample and the number of replicates of each species to include. The major logisti- cal constraints of time, money, and distance to cover of- ten have led researchers to sample multiple species of macroalgae, each collected with few if any replicates at a given site. The statistical analysis of our Pointe de Negre data makes it clear that small sample sizes are inherently misleading, and that only when the Dictyota sample size exceeded n = 10 did the variance reach acceptable levels. Of course, a sample size of 10 is minimal for any para- metric statistical test. Given the high variance that we observed for small sample sizes, non-parametric tests would have been of dubious merit. The issue of sample variance is further complicated by the level of absolute numerical abundance of cells per sample. Phytoplank- ton ecologists recogni/ed early that the accuracy of a count varies as a function of the square root of the num- ber counted (Lund et al.. 1958). To obtain twice the ac- curacy, four times the number of organisms must be counted. Consequently, there is a critical population level below which field survey data will not resolve sub- strate preference or hiogeography with certainty. CIGUATERA ECOLOGY Table I Gambierdiscus toxicus substrate preference with differing amounts of macroalgal hosts 99 Dictyota ( = D) Galaxaura Ratio D.G Ratio D.G Ratio G:D ( = G) # cells g-' #cells/cm2 #cells/cm2 Treatment 1 : Mass ratio 0.25 D:G blot weight, g 0.2 0.8 % of total macroalgal biomass 20% 80% % of total macroalgal surface area 46% 54% % attached cells* 24% 76% # cells per g 2750 2167 1.3 # cells per cm2 26 69 0.25 2.7 Treatment 2: Mass ratio 0.5 D.G blot weight, g 0.4 0.8 % total macroalgal biomass 33% 67% % of total macroalgal surface area 63% 37% % attached cells* 49% 51% # cells per gram 3168 1625 2.0 # cells per cm2 30 52 0.58 1.7 Treatment 3: Mass ratio 1.0 D:G blot weight, g 0.4 0.4 % of total macroalgal biomass 50% 50% % of total macroalgal surface area 75% 25% % attached cells* 60% 40% # cells per gram 3250 2168 1.5 # cells per cm2 31 70 0.44 2.3 Treatment 4: Mass ratio 2.0 D G blot weight, g 0.8 0.4 % of total macroalgal biomass 67% 33% % of total macroalgal surface area 87% 13% % attached cells* 74% 26% # cells per g 2188 1500 1.5 # cells per cm2 21 48 0.44 2.3 * Attached cells on each species as a percent of the total cells attached to macroalgae. Another problem encountered in attempts to compare field distributional data for G. toxicus is that the species of host macroalgae collected for the various surveys differ so dramatically. This is not only a reflection of the difficulty in finding one or two macroalgal species dis- tributed throughout all coastal marine habitats, but it also indicates that little is known about the differential abundance of G. toxicus and other benthic dinoflagel- lates on particular macroalgal species. If researchers could go to a site and know that they could obtain mean- ingful data on G. toxicus abundance by sampling only one or two algal species, sampling statistics could be im- proved and ecological issues more easily resolved. Given that it seems desirable to designate key macroalgae that are significantly associated with ciguatera dinoflagellates, we argue that an experimentally derived hierarchy of host species is needed. A laboratory procedure to deter- mine this hierarchy is described here. Algae to be exam- ined in the laboratory should include representatives of the flora found in each of the coastal marine habitats (e.g., tidepool, forereef, backreef, lagoon, etc.). Adhering to our belief that more can be learned from a statistically relevant number of replicates of one or two key host species rather than an equal number of samples split between the various macroalgae present in a study area, we focused our attention on Dictyota and Galax- aura during our field study. We chose these species be- cause of their circumtropical distribution, because they are abundant at most shallow and deep reef environ- ments, and because they have been cited as supporting high G. toxicus populations (especially Dictyota; Carl- son, 1984; Carlson et a/., 1984; Ballantine el ai, 1985; Carlson and Tindall, 1985; Gillespie et ai, 1985a; Tay- lor, 1985; Taylor and Gustavson, 1983). Ballantine et at. (1985) studied the seasonal abundance of G. toxicus on Dictyota at Puerto Rico and found typical densities rang- ing between 1 00-300 cells g~ ' with a maximum of 8000. They recognized the huge variability in cell counts among samples collected close to one another, and noted that Dictyota appeared to be preferred as a substrate over the seagrass Thalassia testudinum. Elsewhere in the Car- ibbean, G. toxicus was also found to be significantly more abundant in association with Dictyota spp. than sympatric Spyridea ftlamenlosa and Cladophora hetero- 100 P. S. LOBtl hi II nema (Carlson, 1 984; Carlson and Tindall. 1985). Spyri- dea in turn was considered a preferred host for G. toxicus based on field distributions in Hawaii (Shimizu el ai, 1982). These d. assed here because they emphasize the value in .omparable macroalgal species (or an estab! Hierarchy of species) as substrates to be colic .eld surveys, but they are also examples of how pic'.icnce has been inferred without suitable back- group..., .nformation. The relatively obvious source of er- ror that, with one exception (Bomber el a/.. 1985). has largely been ignored in field studies to date is that cell counts normalized to mass are only comparable between macroalgal hosts if these hosts have the same surface area per unit mass. The value of this concept was first recog- nized by Bomber et al, (1985) who saw no correlation between the field abundance of another epiphytic dino- flagellate. Prorocentrum lima, and the mass of macroal- gal species. The dinoflagellate distribution was best ex- plained on the basis of available surface area. On first inspection, our laboratory data might be seen as evidence for active preference of G. toxicus for Dictyota. since the dinoflagellate abundance per gram of host alga was al- ways higher on Dictyota than on Galaxaura. In fact, when the dinoflagellate abundance is normalized to host surface area, the opposite conclusion is reached — namely that the preference is for Galaxaura. As seen in Table I, the percentage of all attached cells that selected Galaxaura was always higher than the percentage of available macroalgal surface area represented by Galax- aura. typically 1.5-2 times higher. If attachment were simply surface area dependent, a 1:1 correspondence would be expected. This apparent preference is also seen in the ratio of cells cm~2 on Galaxaura versus Dictyota, which varies between 1.7 and 2.7. A simple surface area dependence with no preference would again be evi- denced by values closer to 1 .0. In this context, it is noteworthy that we typically saw four times as many G. toxicus cells (per gram) on Dicty- ota than on Galaxaura at Pointe de Negre (Figs. 3 A, 4A). This corresponds to a nearly equal dinoflagellate abun- dance per cm2 (i.e., a ratio near 1 .0 as discussed above), so active preference seems unlikely. However, these data might be the end result of an initial colonization based on prellu-'u-c. as seen in our short-term laboratory ex- periments, iwed by differential growth or mortality of the din. • on each macroalga. The separation andquantifu se two processes clearly requires further study thai ,ond the scope of this paper. Our intent is to emphasi/r the difficulties associated with comparisons between dilk-i nt macroalgal hosts and the ease with which incorrect interpretations can be made if data are expressed in commonly accepted units of cells g'1 of host algae. Bomber (1985) reports that macroalgal species can be divided into three general groups on the basis of surface areag ', with differences spanning a fac- tor of four between species. Until surface area mass ' re- lationships are determined for other important macroal- gae, we argue that G. toxicus abundance data cannot be interpreted either in terms of substrate preference or geo- graphic distribution patterns. Only data for the same host species would be comparable, and then only if the number of replicate samples is sufficient. Another fascinating and unexpected result from the preference experiments is that a relatively constant num- ber of G. toxicus cells attached to each gram or cnr of our host algae, even when each host species' fractional biomass varied eight-fold. One possible interpretation is that there is a "carry ing capacity" for each species. Given reports of much higher numbers of G. toxicus per gram of Dictyota by Ballantine et al. (1985), it seems more likely that we are seeing colonization that was still in progress when the experiment was terminated after 24 h. This consistency is reassuring and argues that the studies of the dynamics of G. toxicus substrate attachment and preference are feasible in the laboratory. We have shown that valuable information can be obtained by comparing the short-term colonization behavior of G. toxicus when offered different macroalgal hosts. These results suggest that there is preference expressed in the early stages of colonization. Our next step is to extend these experi- ments in time so as to evaluate other factors that will affect the final abundance of dinoflagellates, namely host chemistry, dinoflagellate growth, water turbulence effects. light effects, and so forth. In summary, we have initiated a ciguatera research program that we hope will generate field data that are not only statistically sound but that also will allow compari- sons to be drawn with results from other researchers throughout the world. Central to this approach is a focus on one or two key macroalgal host species, as well as the collection of sufficient replicates for our abundance esti- mates to be a valid representation of the real G. toxicus distribution. Normalization of these data to host surface area would be more informative and less subject to mis- interpretation than the more common units of cells g" '. Finally, we have demonstrated the ease with which sub- strate preference studies can be conducted in the labora- tory. We recognize that the natural abundance and dis- tribution of G. toxicus in the field is a reflection of both substrate attachment and the resulting growth and mor- tality of the established dinoflagellate population. This complex phenomenon must first be separated into dis- crete components, however, each to be studied in isola- tion if we are ever to fully comprehend the spatial and temporal dynamics of ciguatera. Acknow lodgments We gratefully acknowledge the assistance of J. Aubin, B. Keafer, D. Kulis, and the support of D. Combs and CIGUATERA ECOLOGY 101 M. Kellett. We thank D. Smith for the computer pro- gramming and J. Weinberg for discussion about statisti- cal bootstrap methods. This research was supported in part by the New England Biolabs Foundation, by the Office of Sea Grant in the National Oceanic and Atmo- spheric Administration through grants NA84AA-D- 00033 (R/B 56 and R/B 86 and NA86AA-D-SG090 (R- V76), and by the National Science Foundation (OCE- 8614210). Contribution No. 6614 from Woods Hole Oceanographic Institution. Literature Cited Anderson, D. M., and P. S. Lobel. 1987. The continuing enigma of ciguatera. Bio. Bull. 172: 89-107. Anderson, D. M., A. W. White, and D. G. Baden, eds. 1985. Toxic Dinojlagettates. Elsevier, New York. 56 1 pp. Bagnis, R., J. Bennett, C. Prieur, and A. M. Legrand. 1985a. The dy- namics of three benthic dinoflagellates and the toxicity of ciguateric surgeonfish in French Polynesia. Pp. 177-182 in Toxic Dinoflagel- lates. D. M. Anderson, A. W. White, and D. G. Baden, eds. Elsevier. New York. Bagnis, R.. J. Bennett, M. Barsinas, M. Chebret, G. Jacquet, I. Lechat, Y. Mitermite, Ph. Perolat, and S. Rongeras. 1985b. Epidemiology of ciguatera in French Polynesia from 1960 to 1984. Pp. 475-482 in Proceedings of the 5th International Coral Reef Congress, B. Sal- vat, ed. Antenne Museum-EPHE. Vol. 4. Ballantine, D. L., A. T. Bardales, T. R. Tosteson, and H. Dupont-Durst. 1 985. Seasonal abundance ofGambierdiscus toxicus and Ostreopsis sp. in coastal waters of southwest Puerto Rico. Pp. 417-422 in Pro- ceedings of the 5th International Coral Reef Congress, B. Salvat, ed. Antenne Museum — EPHE. Vol. 4. Bomber, J. \V. 1985. Ecological studies of benthic dinoflagellates asso- ciated with ciguatera from the Florida Keys. M. S. Thesis, Florida Inst. Tech. 104pp. Bomber, J. \V., D. R. Norris, and L. E. Mitchell. 1985. 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(August, 1988) Ontogeny of Osmoregulation and Salinity Tolerance in Two Decapod Crustaceans: Homarus americanus and Penaeusjaponicus G. CHARMANTIER', M. CHARMANTIER-DAURES', N. BOUARICHA1. P. THUET', D. E. AIKEN2, AND J.-P. TRILLES1 [Laboratoire de Physiologie des Invertebres, Universite des Sciences el Techniques du Languedoc. PL E. Baiaillon. 34060 Montpellier Cedex, France, and2Department of Fisheries and Oceans, Invertebrate Biology and Aquacidture, Biological Station, St. Andrews. \c\\ Hrunswick EOC 2X0, Canada Abstract. Osmoregulation and salinity tolerance were studied in larvae and post-larvae of two species of crusta- ceans. Homarus americanus and Penaeus japonicus, that have different types of embryonic development. In both species, salinity tolerance decreased through the larval stages, was at a minimum at metamorphosis, and increased in post-larval stages. In H. americanus, the lethal salinity for 50% of the animals (24 h LS50) at 20°C was about 1 7%o at metamorphosis, and about 10.5-12%o in stages IV and V. In P. japonicus, the 24 h LS50 at 25°C was about 25%o at metamorphosis, and about 7-10%« from the sixth post-larval stage onwards. In both species, larvae were hyper-osmoconformers and the osmoregulatory pattern changed after metamor- phosis to the juvenile/adult type. In //. americanus, stages IV and V slightly hyper-osmoregulated in low sa- linities. In P. japonicus, post-larvae hyper-hypo-regu- lated, and their regulatory capacity increased up to the fifth post-larval stage. In young stages of H. americanus and P. japonicus, Osmoregulation and salinity tolerance appear correlated, and are modified at metamorphosis. These results are discussed • iih regard to their ecological and physiologi- cal implic i md to previous studies on other species. Introduction Most studies on crusui "an Osmoregulation deal with adult forms (review in M mtel and Farmer, 1983), and only a few data are a\ n larval and post-larval Received 8 February 1988; accepted 29 April 1988. Osmoregulation. These are summarized in Table I. De- pending on species, the osmoregulatory abilities can vary among the successive larval stages or remain unchanged. The adult type of regulation is also established at variable stages in different species. Numerous studies have been concerned with larval and post-larval salinity tolerance, but few have attempted to correlate the salinity tolerance of different developmental stages with their correspond- ing osmoregulatory capabilities in a given species. The objective of this study conducted with the Ameri- can lobster Homarus americanus H. Milne Edwards. 1 837, and the shrimp Penaeusjaponicus Bate, 1 888, was to determine the salinity tolerance of larval and post-lar- val stages of these species, to define the ontogeny of their Osmoregulation. and to attempt to correlate osmoregula- tory abilities and salinity tolerance. //. americanus and P. japonicus are both economically important, and any knowledge of their larval and post-larval environmental tolerance and physiology can be valuable for their man- agement and potential culture. Moreover, their patterns of post-embryonic development are different, offering the opportunity for comparisons of larval physiology. In the genus Homarus, larval development comprises one prelarva and three zoea or mysis larvae — stages I to III — before a metamorphosis leading to post-larvae — stage IV or megalopa — and then to juvenile stages. In //. americanus, Osmoregulation has been studied in adults (Dall, 1970) and juveniles (Charmantier el al.. 1981. 1984a); information on ionic regulation (Charmantier el a/.. 1984b) and preliminary data on Osmoregulation (Charmantier el a/.. 1 984c) are also available for the early post-embryonic stages of this species. Osmotic and ionic 102 ONTOGENY OF DECAPOD OSMOREGULATION 1U3 Table I Summan1 oj studies on larval/post-lar\'al crustacean osmoregulation Species Larval/ post-larval development Type(s) of osmoregulation dunng post- embryonic development Type of osmoregulation in adults Rhithropanopeus hanisii ( 1 ) Cardisoma guanhumi (3) Callinectes sapidus (6) Hepalus epheliticus (6) Libinia emarginala (6) Sesanna reticulatum (9) Clibanariiix villains (10) Macrobrachium pelersi (12) Uca subcvlmdnca ( 1 3) Callianassa Jamaica louisianensis ( 14) 4 zoeae 5 zoeae 7 zoeae several megalopae 3 zoeae 1 megalopa 3 zoeae 1 megalopa 5 zoeae 1 megalopa 9 larval stages 1 megalopa Hyper-osmoconform from 10 to 40 %o Slight hyper-regulation in lowest salinities. Except osmoconform in diecdysial stage V Hyper-osmoconform <20 %a Tendency to hyper-regulate in 30- 40 %o in 3rd, 4th, 5th zoeae Hyper-isoregulation in 1st, 2nd zoeae and late megalopa. Osmoconform in 7th zoea. Hyper-osmoconform in other stages Hyper-isoregulation in 1st zoea. Slightly hypo-regulate in 40 %o in following stages. Larvae approaching megalopa and settling crab stages gradually osmoconform 1st zoea and late megaloga osmoconform. Slight hypo- regulation in 40 %o in 2nd zoea and early megalopa Hyper-regulate from 10-35 %o. Slight hyper-regulation in 40 %o Hyper-osmoconform from 200 to 1 200 mosm-kg"' Hyper-hypo-regulation Stronger ability in 1st stage and in post-larvae Slight hyper-regulation in zoeae I-I1 Slight hyper-hypo-regulation in megalopa Slight hyper-regulation in media <800-900mosm.kg-' Hyper-hypo-regulation (2) Hyper-hypo-regulation (4) (5) Hyper-hypo-regulation (7) Supposedly osmoconform (6) Osmoconform (6) (8) Hyper-hypo-regulation (9) Hyper-regulation Isosmotic regulation (11) Hyper-hypo-regulation (12) Hyper-hypo-regulation (13) Hyper-hypo-regulation (15) ( 1 ) Kalber and Costlow ( 1 966) (2) Smith (1967) ( 3 ) Kalber and Costlow ( 1 968 ) (4)Pearse(1932) (5) Quinn and Lane (1966) (6) Kalber (1970) (7) Ballard and Abbott (1969) (8)Gilles(1970) (9)Foskett(1977) (10)Young(l979a) (11) Young (1979b) (12) Read (1984) ( 1 3) Rabalais and Cameron (1985) (14)Felderrta/. (1986) (15)Felder(1978) regulation of//, gammants has been studied in juveniles (Charmantier eta/., 1984d). In contrast to the simple post-embryonic development of homarid lobsters, the penaeid shrimps have numerous larval instars beginning with nauplii stages, which is an exception among decapod crustaceans. The post-embry- onic development ofPenaeusjaponicus goes through six nauplius stages, three zoeae, and three mysis. The third mysis stage ends with a metamorphosis: the succeeding post-larval stages can be named either by their stage (e.g., PL5, fifth post-larva) or by the time elapsed since meta- morphosis (e.g., P5, 5 days after metamorphosis). Al- though osmoregulation has been studied extensively in penaeid shrimps (review in Charmantier, 1987b), only ionic regulation is known in P. japonicus (Exbrayat and Bourguet, 1982). Preliminary data have also been gath- ered on osmoregulation of their larvae (Charmantier, 1986). Materials and Methods This study was conducted at two different locations: St. Andrews (New Brunswick, Canada) for Homams americanus and Montpellier (Herault, France) for Pen- aeus japonicus. Standardized methods were used for both species and two of us were members of both the 104 CHARMANT1BR ET AL Canadian and French groups who performed the experi- ments. Animals Homan lU larvae were obtained during the summer ftvi -lers captured in Passamaquoddy Bay and held i: . lobster culture facility at the Biological Statii Andrews. After hatching, larvae were trans- ferred to 40-1 planktonkreisels (Hughes et at., 1974) sup- plied with flow-through seawater at a salinity of 30-3 1 %o, a temperature of 20°C under natural photoperiod. Lar- <. ac \v ere fed three times a das w ith frozen adult Anemia. As each larval stage lasts several days, molting stages were obtained according to the time elapsed from the preceeding molt, and three groups of animals, postmolt stage A. stage C and premolt stage D. were selected. Larvae of P. japonicus were obtained in early spring from the Ifremer Station (Deva-Sud) at Palavas (Her- ault). They were reared in semi-recirculated systems us- ing a clear water technique at a salinity of 35-36%o, a temperature of 25-27°C and under an artificial photope- riod (12L/12D) (Aquacop, 1983;Laubier, 1986). Larvae were fed with algae or Anemia nauplii according to their stage. Each larval stage lasted 24 h or less, so it was not possible to select animals according to molting stages. Preparation of media Dilute media were prepared by addition of tap water to seawater and high salinity media were prepared by adding "Instant Ocean Synthetic Sea Salts" (Aquarium Systems, Inc.) to seawater. All experiments were con- ducted at 20°C (Homarus) or 25°C (Penaeus). Salinities were expressed according to the osmotic pressure in mosm-kg ' and to the salt content of the medium in %». Osmotic pressure was measured on an Advanced Instru- ments 31 LA or Roebling osmometer, and salinity on a YSI 33 salinometer. A value of 3.4 %o is equivalent to 1 00 mosm-kg '. Sun-ival bioassays To determine salinity tolerance, acute static bioassays were conducted with animals held in test media ranging from tiv h water (~ 10 mosm-kg"1) to seawater (~ 900- 1100 H kg"1) and differing by increments of 100 mosm-kg '%o). Penaeid shrimp larvae were held communally , containers holding 0.5 1 of me- dium; due to agnnis -havior, lobster larvae were held in individual comp ents partly immersed in 3.5-1 containers. All media ated. Animals were not fed during the bioassays. The 'luration of lobster experi- ments was 48-96 h. The shrimp experiments lasted 24- 96 h due to the shorter duration of the stages. Each bioassay was run on a group of 10 animals and 'i/; U ' 4 l Figure 1. SaliniU tolerance in larval (I. II. Ill) and posi-lar\al (IV, V) Homarus americanus at 20°C. Variations in LS50 in %o and mosm • kg ' according to larval (L)/post-larval (PL) and molt stages and todays of development. Each point represents the mean value of at least two determinations from 10 animals, with 95'7 confidence interval. Closed circles: 24 h LS50: open circles: 48 h LS50; open triangles: 96 h LS50. replicated. Animals were counted and dead animals re- moved at 0.5, 1 . 2. 3. 6. 1 2. 24. and 48 or 96 h according to the prescriptions of Sprague (1969) in toxicity studies. The criteria for death were total lack of movement, im- mobility of the scaphognathite (lobster) and of the heart (both species), and lack of response after repeated touches with a probe. Median times to 50r; mortality (LT50) and their 95°r confidence limits were determined from a computer program (Lieberman. 1983) based on the probit technique of Lichtfield and Wilcoxon (1949) and Finney (1962). Median lethal salinities (LS50) and 95% confidence intervals were calculated by standard techniques of probit analysis (Finney. 1962; Davies, 1971) computerized on the Letcur program (Zitko, 1982). LS50 were calculated at 24 and 48 h. and at 96 h in some longer lasting post-larval stages. Osmoregulation Animals were reared at different selected salinities in recirculated planktonkreisels (lobster) or 0.5-1 plastic containers (shrimp). Individuals were dried on filter pa- per and hemolymph was sampled by inserting a micropi- pette into the heart. This operation was conducted under mineral oil in the smallest stages in order to avoid rapid evaporation and desiccation. In shrimp, reproducible data were obtained only from stage zoea 2, i.e.. more than 1 .3 mm long. Osmotic pressure of hemolymph was measured on a Kalber-Clifton micro-osmometer requir- ing 30-50 nl, with reference to the osmotic pressure of the medium. Results Homarus americanus: xalinity tolerance The ability of// americanus to tolerate low salinities varied with post-embryonic development (Fig. 1). After ONTOGENY OF DECAPOD OSMOREGULATION 105 a slight decrease early in stage I, the 24 h LS50 increased through stages I, II, and III from about 410 mosm-kg~' (14%o) to a maximum (corresponding to a minimum tol- erance) of 500 mosm- kg'1 (17%o) in stages III D and IV A-B preceding and following metamorphosis. In post- larval stages IV and V (in molting stage C), salinity toler- ance reached its maximum, with 24 h LS50 values down to 340 mosm-kg"1 (11.6%o) and 310 mosm-kg"1 ( 10.5%o); however, LS50 increased during the molt from stage IV to stage V. The 48-h LS50, although higher by 10-40 mosm-kg'1 (0.3-1.4%o), followed the same pat- tern of variation. Molting between larval stages was pos- sible in respectively 0%, about 50%, and more than 80% of the larvae in media of <400, 500-600 and >700 mosm- kg'1 (13.6, 17-20.4, 23.8%o). H. americanus: osmoregulatory ability Adaptation time. After a rapid transfer from seawater at 850 mosm-kg'1 (29%o) to a dilute medium of 500 mosm-kg"1 (17%o), the hemolymph osmotic pressure stabilized within 1 h in stages I and II, 2 h in stage III and 3-6 h in stage IV (Fig. 2). Osmotic adaptation to concentrated media of 1 100 and 1300 mosm-kg'1 (37 and 44%o) was completed in 1-3 h. In all subsequent ex- periments we kept the animals 6-24 h in each medium before sampling. Osrnoregulation. The types of osmoregulation were 900i- 800 600 500 - 6 18 20 Time (h) 24 Figure 2. Change in hemolymph osmotic pressure (HL) in stages I-IV of Homarus americanus after rapid transfer from seawater (850 mosm • kg~ ' , 29%o) to a dilute medium ( 500 mosm • kg" ' . 1 7%o ) at 20°C. Each point represents the mean value of determinations from 12-15 animals, with 95% confidence interval. IV MO (moam kg-') Figure 3. Variations in the difference between the osmotic pres- sures of hemolymph and medium (HL-MD) according to the osmotic pressure of the medium (MD)in stages I-V of Homarus americanus a.\ 20°C. Each point represents the mean value of determinations from 12-20 animals (except in extreme salinities: 5-16 animals) with 95% confidence interval. O O: post-molt; • •: stage Ci A A: premolt. similar in larval stages I, II, and III. In molting stage C, these larvae hyper-osmoconformed over the whole range of salinity, the osmotic pressures of hemolymph and me- dium differing by about 1 0-20 mosm • kg" ' . Their regula- tion was slightly more hyper-osmotic in premolt and nearly isosmotic in post-molt (Fig. 3). The pattern of osmoregulation changed after meta- morphosis. Stage IV and V post-larvae in molting stage C hyper-osmoconformed in high salinities and seawater and their regulation was slightly hyper-osmotic in dilute media (hemolymph-medium difference of about 80 mosm-kg'1 in a 500 mosm -kg'1 or 17%o medium). No significant difference was found in the regulation of pre- molt post-larvae, but the ability to hyper-regulate in di- lute media significantly decreased in post-molt stages IV and V (hemolymph-medium difference of about 30-35 mosm-kg'1 in a 500 mosm-kg'1 or 17%o medium) (Fig. 3). Penaeus japonicus: salinity tolerance Tolerance of low salinities varied with the develop- mental stages of P. japonicus (Fig. 4). The 24 h LS50 in- creased during larval development from about 460-600 mosm-kg'1 (16-20%») in nauplii and zoeae 1 up to 730 mosm • kg"1 (25%») just prior to and after metamorphosis in mysis 3 and first post-larval stages (PL1). Salinity tol- erance increased progressively thereafter up to stage PL6, P12, i.e.. 12 days after metamorphosis (24 h LS50 about 300 mosm-kg"1 or 10%o) and more slowly up to stage 106 CHARMANTIER ET AL. Figure 4. Salinity tolerance in lar\al il ) and post-Ian al (PL) Pen- aeus japonicus at 25°C. Variations in LS50 in %o and mosm-kg"1 ac- cording to larval/post-larval stages and days of development. Each point represents the mean value of at least two determinations from 10 animals, with 95^ confidence interval. N. nauplius: Z. zoea: M. mysis; PI. post-larval stage. Closed circles: 24 h LS50; open circles: 48 h LS50; open triangles: 96 h LS50. PL10. P20 (24 h LS50 about 200 mosm-kg'1 or ?%„). The 48 h LS50. higher than the 24 h LS50 by 20-150 mosm-kg"1 (0.7-5. l%o), followed the same pattern of variation. Molting between larval stages was possible in 0%, about 50%, and 60-100% of the larvae in media of <400. 600-700. and ;>800 mosm - kg" ' ( 1 3.6, 20.4-23.8, 27.2%o), respectively. P. japonicus: osmoregulatory ability Adaptation time. After a rapid transfer from seawater (1050 mosm -kg"' or 35.5%o) to a dilute medium (500 mosm-kg"' or 17%o), the hemolymph osmotic pressure stabilized in 1 h in stage zoea 3, 3 h in fourth post-larval stage (PL4), and 6 h in stage PL 10 (Fig. 5). In all subse- quent experiments, we kept the animals 6-24 h in each medium before sampling. Osmoregulation. Larval stages (zoea 2 and 3, mysis 1- 1) h\per-osmoconformed over the whole range of tested salinities: the osmotic pressures of hemolymph and me- dium differed by about 10-40 mosm-kg '. In stage mysis 3, which lasted 2 days, regulation was slightly more hy- per-osmotic towards the end of the stage, i.e., in prcmolt animals ' . 6). At the end of the larval period, most of the animal lied in the dilute media and the few survi- vors teni1 iismoconform. The pattern o smoregulation changed after meta- morphosis. Starting I n the end of first post-larval stage (PL1) which lasted 1 the regulation shifted to slightly hyper-osmotic ii •lute media and hypo-osmotic in seawater. Hyper- and i po-osmoticity increased pro- gressively thereafter up to PL5-PL6, P10-P12. From these stages onwards, the difference between the osmotic pressures of hemolymph and media reached about 300, 200. and -150 mosm-kg ' in 300. 500, and 1000 mosm-kg"' media (corresponding salinities: 10. 17. 34%o), respectively. The isosmoticity medium changed progressively from 900 mosm-kg ' (30.5%o) in PL1 to 800 mosm-kg"1 (27%o) in PL5-PL6 (Fig. 6). Discussion Salinity tolerance In Homarus americanus. salinity tolerance at 20°C ex- pressed by the 24 h LS50 varies from 14-17%o in larvae down to 10.5-12%o in post-larvae and is minimum at metamorphosis — about 1 7%o. These results are in agree- ment with previous data: in //. gammarus and H. ameri- canus, respectively, Gompel and Legendre (1927) and Templeman (1936) found that the larval period could progress to metamorphosis and stage IV only at 1 5- 17.5°C in salinities above 17V Sastry and Vargo (1977) observed that larvae developed to stage V in salinities above 20%o at 15°C and 15%o at 20"C. Thus, from these studies, the minimum salinity compatible with larval de- velopment and metamorphosis can be estimated to be 1100r- 1000 900 800 700 • PL10 PL4 600 5001- I inure 5. Change in hemolymph osmotic pressure (HL) in stages /DIM t and post-larvae 4 and 10 ofPenaeusjaponicus after rapid trans- fer from seawater (1050 mosm • kg ', 35.5%o) to a dilute medium (500 mosm-kg"'. I7%o) at 25°C. Each point represents the mean value of determinations from 10- I 5 animals, with 95'1; confidence interval. ONTOGENY OF DECAPOD OSMOREGULATION 107 300 -, 250 - 40 20 !z2 !-!- -H P3 PL2 \^ " 50 20oU 20 -Z3b I-h -H •• _ k \ " P4 PL 3 \ 150J- 40 -Z3e j i- -H \- XT! '00 E 100 i ~ N\] •T 40 T 1 — — •T"""~~~ 1 1 -50 _ e JE 20 E n P6 PL4 - 50 3 50 - 0 J 20 _ M2 J, -!— I - I X 1 20 _M3 IT —I 1 ,00 _ \ - — - | \ {" -50 -50- 40 - Pi PLi b [ Pe PL4 6 \ 20 ^H 5° " 40 - Pz PLi e I \ 20 N \ . k -150- -20 -40 _ '\{ ..o I 1 - , XI 300 500 700 900 300 500 700 900 MD (moam Kg "') MD (mosrn kg'1 } Figure 6. Variations in the difference between the osmotic pressures of hemolymph and medium (HL- MD) according to the osmotic pressure of the medium (MD) in larval and post-larval stages of Penaeus japonicus al 25°C. Each point represents the mean value of determinations from 10-15 animals (except in some low salinities, 5), with 95% confidence interval. Z, zoea; M, mysis; P, number of days after metamor- phosis; PL, post-larval stage; b, beginning; e, end. about 17%o at 20°C. After metamorphosis, the values of LS50 found in post-larvae are similar to those observed in one-year-old juveniles (10%o: Charmantier. unpub. data) and compatible with the lethal limits known in adults (8%«: McLeese, 1956). The salinity tolerance of post-larvae decreases at the time of molt, which is a fre- quent observation among crustaceans. In P. japonicus, the 24-h LS50 at 25°C varies from 1 6- 20%o to 25%o in larval stages with a maximum value at metamorphosis. It decreases progressively to 10%« from post-larval stage PL1 to PL6, and to 7%o in stage PL 10. In P. japonicus (Hudinaga, 1942), P. duorarum (Ewald, 1965), P. marginatus (Gopalakrishnan, 1976), and Met- apenaeus bennettae (Preston, 1985), zoea larvae are less resistant to low salinity than mysis, which is not the case in our study. There are two possible explanations of this. The different feeding conditions could interfere with the effect of salinity. Preston (1985) observed that "starva- tion was a more potent factor than the effects of tempera- ture and salinity in determining survival through the protozoeal larval stages." On the other hand, most of these studies were conducted for long periods to com- plete the larval development, which is not the case in our stage-by-stage study. After metamorphosis, the values of LS50 from PL6-PL8 were similar to those found in juve- niles of the same species (8%»: Dalla Via, 1986; 6%o: Thuet et al., unpub. data). In H. americanus and P. japonicus, the 48 or 96 h val- ues of LS50 are higher than those at 24 h. The difference is highest in the stages that last less than 48 h (Penaeus larval stages, young Homarus in molting stage D). High mortality occurs in the lowest salinities when these ani- mals attempt to molt. Adaptation time In H. americanus and P. japonicus the time required for osmotic equilibration to a dilute medium is about 1- 2 h in larvae, and 3-6 h in early post-larvae. It is between 12 and 24 h in juvenile lobster (Charmantier et al., 1984a) and adult shrimp (Charmantier, unpub. data), and about 75 h in adult lobster (Dall, 1970). Therefore, adaptation time is size dependent. In larvae of other spe- cies, adaptation time is similar to that of young stages of Homarus and Penaeus (Ka\beT and Cosllow, 1966, 1968; Kalber, 1970;Foskett, 1977; Felder^a/., 1986). Foskett (1977) stressed the physiological and ecological impor- tance of rapid adaptation of hemolymph osmotic pres- sure to changes in the salinity of the medium. This may be particularly true in the case of larval Homarus and Penaeus which are planktonic and thus exposed to sud- 108 CHARMANTIER ET AL den changes in saliniu following heavy rainfall. These rapid changes also require the existence of intracellular osmotic adaptation. eV'rvuilh in the hyper-osmocon- forming larval stages two species. Osmoregulai In // at is, larval stages I-III hyper-osmocon- form and pv al stages IV and V slighth hyper-regu- late in dik - media. The adult type of osmoregulation (Dall. 19 which is identical to juvenile regulation ( Charmantier tY a/., 1981, 1984a). is acquired at stage IV. following metamorphosis. We found the same pattern of changes in the osmotic regulation of larvae and post- larvae of//, gammarus (Thuet ct a/.. 1988). confirming the physiological likeness of the two species. The molt cycle affects osmotic regulation in //. ameri- canits in two ways. In premolt larvae in stages I-III, the osmotic pressure of the hemolymph increases in all me- dia. Similar variations have been shown in the larvae of Rhithropanopeus harrisii (Kalber and Costlow. 1966) and Cardisoma guanhumi (Kalber and Costlow. 1968). These authors suggested that a hyperosmotic internal medium would favor uptake of water at molt. In H. am- ericanus. the tendency toward increased hyper-regula- tion before ecdysis is lost after metamorphosis, but postmolt post-larvae demonstrate a lower ability to hyper-regulate than do stage C animals. The decrease in hemolymph osmotic pressure or ion concentration is well known in postmolt adult crustaceans, where it is re- lated to the water intake at molt, as exemplified by different lobsters (Travis, 1955; Glynn. 1968). Thus the effect of molting on osmotic regulation is different before and after metamorphosis. This could be related to varia- tions in the permeability of the cuticle in larvae and post- larvae and to changes in the mechanisms involved, as discussed later. In P. japonicus, zoeal and mysis larval stages hyper- osmoconform and the type of osmoregulation changes to hyper-hypo-regulation after metamorphosis. In post- larvae, the ability to regulate increases progressively up to stages PL5-PL6. In P. japonicus, the adult type of regu- lation is hyper-hypo-osmotic as in most species of pen- aeid shrimps (see review in Charmantier, 1987b); thus this type > .f regulation appears soon after metamorphosis butitselt: IA is only gradually established over 10-12 days at 25"( A few studi addressed the evolution of osmo- regulatory al n? the post-embryonic develop- ment of decapod (Table I). In several species larvae are hyper-osmu< mers, for example Rhithro- pannpeus harrisii (Kalba ..nd Costlow. 1966), Calli- nectes sapidus (Kalbcr, 1970), Scsarma reticulaiiim (Foskett, \911),Clibananu\ \ -ituttus (Young, I979a). //. americanus and P.japonicus larvae exhibit a similar type of osmoregulation. In other species such as C'urJn<>tn War. BioI.Ecol. 76: \9\-l99. I>all. \V. 1970. OST ilation in the lobster Homants americanus. J.Fish A' 27:1123-1130. Dall. V\ l*»x i .latory ability and juvenile habitat preference in som prawns. J. Exp. Mar Biol. Ecol. 55: 219-232. UallaMj.' .^86. Salinity responsesof the juvenile penaeid shrimp fen... ../V/WIM. I. Oxygen consumption and estimations of pro- duc'.n iu . Aquaculture 55: 297-306. DUMI-.. R. G. 1971. Computer Programming in Quantitative Biology. VjJemic Press, London. Fwald. J.J. 1965. The laboratory rearing of the pink shrimp. Penaeus rfuoftvwn (Burkenroad). Bull Mar Sci 15:436-449. Exbrayat.J.-M.. andJ.-P. Bourguct. 1982. Variations du milieu inter- ieur de Penaeus japonicus en fonction de certaines conditions na- turelles (cycle de mue et age I et de certaines conditions experimenta- les (epedonculation et ablation des organes Y). Bull. Soc. Zoo/, t'r 107:33-51. Felder. D. L. 1978. Osmotic and ionic regulation in several western Atlantic Callianassidae (Crustacea. Decapoda, Thalassinidea). Biol. Bull 54: 409-429. Felder.J.M., D. L. Felder. and S.C. Hand. 1986. Ontogeny of osmo- regulation in the estuanne ghost shrimp Cal/ianassa jamaicense louisianensis Schmitt (Decapoda, Thalassinidea). J Exp Mar Biol. Ecol 99:91-105. Finney, D. J. 1962. Prohn Analysis. • a Statistical Treatment of the Sig- moid Response Curve. Second Edition. Cambridge Univ. Press. London. Foskett, J. K. 1977. Osmoregulation in the larvae and adults of the grapsid crab Sesarma relicutalum Say. Biol Bull. 153: 505-526. Gilles, R. 1970. Osmoregulation in the stenohaline crab Libinia eniar- ginaia Leach. Arch. Int. Physiol. Biochim. 78: 9 1 -99. Glynn, J. P. 1968. Studies on the ionic, protein and phosphate changes associated with the moult cycle of Honiara* vulgaris. Comp. Bio- chem Physiol 26: 937-946. Gompel, M., and R. I.egendre. 1927. Effets de la temperature, de la salure et du pH sur les lanes des Homards. C. R. Seanc Soc. Biol. 97: 1058-1060. Gopalakrishnan. K. 1976. Larval rearing of red shrimp Penaeus margi- natus Aquaculture 9: 145-154. Hudinaga. M. 1942. Reproduction, development and rearing of Pen- aeus japonicus Bate. Japn J /.not. 10: 305-393, PI. 16-46. Hughes. J. T., R. A. Shleser, and G. Tchobanoglnus. 1974. A rearing tank for lobster larvae and other aquatic species. Prog. Fish Cult. 36: 129-132. Kalbcr. F. A. 1970. Osmoregulation in decapod larvae as a consider- ation in culture techniques. Helgol H'iss. Meeresunlers. 20: 697- 706. Kalber. F. ' and J. D. Costlow. Jr. 1966. I he ontogeny of osmoregu- latmi neurosecretory control in the decapod crustacean Rhi- ihra/' \>n /< ml 6: 22 1-229. Kalbcr, F. A., an.. ",il(i», ,Ir. 1968. Osmoregulation in larvae of the land crab < ma guaflhumi t^jtreille. Am /.ool. 8: 4 1 I - 416. Ijiubitr. \.I986. IX-SCTCM it-ides. Pp. 459-491 in I.'Auuacul- ture. G. Barnabe. coordin. I . Pan 3. Technique et Documentation, Lavoisier. l.ieberman. H. R. 1983. Estimating LD50 using the probit technique: a basic computer program. Drug Chem Toxicol 6: 1 1 1-1 16. I iu-htield. J. I., and F. \Vilco\on. 1949. The reliability of graphic esti- mates of relative potency from dose-percent effect curves. J. Phar- macol. Exp Ther 108: 18-25. Mantel. L. H., and I.. I.. Farmer. 1983. Osmotic and ionic regulation. Pp. 53-161 in I lii • liiii/i>v\ ,'lCn^uicca. I.-/ .* Internal Anatomy and Physiological Regulation, L. H. Mantel, ed. Academic Press. NY. Mcl.i'cse, 1). \\ . 1956. Effects of temperature, salinity and oxygen on the survival of the American lobster. / Fish. Res Board Can 13: 247-272. Pearse, A. S. 1932. Freezing-points of blood of certain littoral and estuanne animals. Carnegie Inm. Hash. Puhl. n° 43? 28:93-102. Preston, !Y 1985. The combined effect of temperature and salinity on hatching success and the survival, growth, and development of the larval stages of Meiapenaeus bennettae (Racek and Dall). / Exp. Mar. Biol. Ecol. 85: 57-74. Quinn. D. J., and C. E. Lane. 1966. Ionic regulation and Na-K stimu- lated ATPase activity in the land crab, Cardisoma giianhumi Comp Biocftem Physiol. 19: 533-543. Rabalais, N. N'., and J. N. C'ameron. 1985. The effects of factors impor- tant in semi-arid environments on the early development of L'ca sithcylindnca. Biol. Bull 168: 147-160. Read, G. H. L. 1984. Intraspecific variation in the osmoregulatorv capacity of larval, post-larval, juvenile and adult Macrobrachium /><7crw(Hilgendorf). Comp Biochem Physiol 78A: 501-506. Sastry, A. N., and S. I.. Vargo. 1977. Variation in the physiological responses of crustacean larvae to temperature. Pp. 40 1 -423 in Phys- iological responses ol marine biola to pollutants, F. J. Vernberg. A. Calabrese. F. P. Thorberg, and W. G. Vernberg, eds. Academic Press, NY. Smith, R. 1. 1967. Osmotic regulation and adaptive reduction of water- permeability in a brackish-water crab, Rhithropanopeus harrisii (Brachyura, Xanthidae). Biol. Bull 133: 643-658. Sprague. J. B. 1969. Measurement of pollutant toxicity to fish. I. Bio- assay methods for acute toxicity. H 'ater Res 3: 793-82 1 . Templcnian. \\ . 1936. The influence of temperature. saliniU. light and food conditions on the survival and growth of the larvae of the \ob- ster (Homarus americanus). J Biol Board Can 2:485-497. Thuet, P., M. CharmantiiT-Daures, and G. Charmantier. 1988. Rela- tion cutre Osmoregulation et activites d'ATPase Na*-K* et d' anhy- drasc carbonique chez Panes et postlanes de Homarus gainmarus ( L.) (Crustacea: Decapoda). ./ A.'\/> Mar Hi«l /.,.>/ 115:249-261. Travis, D. F. 1955. The molting cycle of the spiny lobster Panulirus art;tis ( Latreille). III. Physiological changes which occur in the blood and urine during the normal molting cycle. Biol. Bull 109: 484- 503. Young, A. M. 1979a. Osmoregulation in larvae of the striped hermit crab Clihananux villains (Bosc). (Decapoda:Anomura:Diogem- J.ie) KM. Coast I/,;- Set 9:595-601. Young, A. M. 1979b. Osmoregulation in three hermit crab species, Clihanariux villains (Bosc). I'agurus longicarpus Say and P. polli- caris Say (Crustacea:Decapoda:Anomura). Comp Biochem Phys- iol 63A: 377-382. /.itko, N'. 1982. l.etcur. the lethalitv curve program. Can Tech. Rep. l-ish -t^iiat. Set. 1134: I Opp. Reference: Bio! Bull 175: 1 1 1-121. (August, 1988) Swimming Speed and Oxygen Consumption in the Bathypelagic Mysid Gnathophausia ingens DAVID L. COWLES1 AND JAMES J. CHILDRESS2 1 Department of 'Biology, Loma Linda University, Riverside, California 92515-8247 and 2 Marine Science Institute, University of California, Santa Barbara, California 93106 Abstract. The energetic costs of swimming were deter- mined for the bathypelagic mysid Gnathophausia in- gens. Individuals over a large size range spontaneously swam at speeds from 5 to 6.5 cm/s. To maintain this speed, smaller animals swam at much higher relative swimming speeds than did larger animals. Routine rates of oxygen consumption were thus considerably higher in the smaller instars. The relationship between standard rates of oxygen consumption and animal size was slightly less than the standard log-log allometric slope of 0.75. Within the speed range of 0-8 cm/s, oxygen consump- tion appeared to increase as a linear function of speed. Cost of transport was very high at low speeds. At 5.5 cm/ s, cost of transport was lower than that measured for other crustaceans, but higher than that of fish. Swim- ming efficiency increased with speed. While the lower cost of transport and higher swimming efficiency may contribute to G. ingens ' reduced rates of oxygen con- sumption as compared to those of shallower-living crus- taceans, the major factor appears to be G. ingens' lower level of swimming activity. Introduction It is well known that deep-living pelagic fish and crus- taceans have metabolic rates considerably lower than those of shallower-living pelagic species (Childress, 1 969, 1971b, 1975, 1977; Smith and Hessler. 1974; Torres et ai, 1979; Smith, 1978; Smith and Laver, 1981; Cowles, 1987). This reduction is of an order of magnitude or more, and can be only partially accounted for by changes in temperature, pressure, and animal protein content with depth (Childress, 1975; Mickel and Childress, 1982). Lower metabolic rates at depth have generally been attributed to selection for energy conservation due Received 30 October 1987; accepted 31 May 1988. to food limitation at depth (Childress and Nygaard, 1973, 1974; Bailey and Robison, 1986). A more recent hypothesis contends that, for deeper-living animals such as fish and crustaceans which rely on vision for detection of predators or prey, the reduction in metabolic rate is related to a decrease in activity and the capacity for activ- ity, which is allowed by the shorter reactive distances at depth and consequent relaxation of selection for capaci- ties for rapid swimming (Childress et ai, 1980; Childress and Mickel. 1985). For any active pelagic species, locomotory activity may be expected to play a prominent role in determining the overall metabolic rate. Activity may be critical for feeding, escape from predation, vertical migrations, finding a mate, and maintaining station in the water col- umn. However, little is known of the normal activity lev- els of deep-living pelagic crustaceans nor of the amount of metabolic energy such activity requires. A few studies have been made on swimming speeds and rates of aero- bic metabolism of shallow-living pelagic crustaceans. Torres et al. (1982) and Torres and Childress (1983) used an annular chamber to measure swimming activity and rate of oxygen consumption in the shallow-living Eu- phausia pacifica. Kils (1979a, b) measured mean rates of oxygen consumption and recorded average swimming speeds for the Antarctic krill, Euphausia superba. No comparable data, however, are available for deep-living crustaceans. Mickel and Childress (1978, 1982) and Quetin and Childress (1980) measured pleopod beat rates and oxygen consumption in the mysid Gnatho- phausia ingens strapped to a fixed underwater frame. It is not clear, however, what correlation exists between pleopod beat rates and swimming speeds in swimming crustaceans. Gnathophausia ingens Dohrn ( 1 870) is a large cosmo- politan bathypelagic mysid from the family Lophogastri- 111 112 D. L. COW I I- S AM) J J CIII1DKISS dae. The species is negatively buoyant in seawater and appears to be an active swimmer. Most of the population of this species off California live at depths of 400 to 900 meters (ChiKv . Since the species can be main- tained in the 1; tory for long periods of time, more has been learr. xmt its physiology than that of nearly am other ! . pelagic crustacean (Childress, 1968, 1969. I*' . Fuzessery and Childress. 1975: Belman and Childress. 1976; Childress and Price, 1978. 1983: Mickel and Childress. 1978. 1 982: Quetin and Childress. 1980: Hiller-Adams and Childress. 1983a. b. c: Cowles, 1987). Like many deep-sea animals, its rate of aerobic metabolism is significantly lower than that of compara- ble epipelagic animals. In this study we measured the relationship between routine swimming speeds and rates of oxygen consump- tion in G. ingens. These data were used to characterize energetic costs of swimming for this species. Swimming energetics of G. ingens were then compared to those of shallower-living crustaceans and offish to evaluate G. in- gens' relative swimming abilities and costs in compari- son to those of other active pelagic swimmers. Materials and Methods Seventy-two Gnathophausia ingens of instars 5- 1 1 (Childress and Price, 1978, 1983) were obtained from depths of 450-750 meters from San Clemente, Catalina, and Santa Cruz basins off Southern California, using a 1 0-foot square Tucker Trawl fitted with a thermally insu- lated cod end (Childress el ai, 1978). The mysids were maintained in the laboratory in 5.5°C seawater in 1 -liter plastic containers and were fed once a week to satiation with an alternating diet of salmon and shrimp. Individu- als kept in the laboratory were starved at least twenty- four hours before being used for an experiment. Length of stay in the laboratory before use in an experiment ranged from one hour to six months, with the majority being used within twenty days. Swimming speed and rate of oxygen consumption were measured in a recirculating swim tunnel similar to that described by Cowles et at. (1986). Modifications in- cluded an increase in diameter to 10.16 cm, the total en- closure of the tunnel for respiration measurements, and the conn of the tunnel to a computer-based data acquisition ai >ntrol system for continuous data log- ging. Ultraviolet /ed, 0.2 ^m filtered seawater con- taining 25 mg/l eaL .streptomycin and penicillin was used during oxygen nion experiments to mini- mize background micro, ial respiration. A dark cover was placed over the chamber to minimize disturbance to the animal during the experiment. Experimental animals were sealed individually in the swim tunnel and allowed to swim at spontaneous speeds while speed and oxygen consumption were measured continuously. Individual experiments varied in length from four to thirty hours. An experiment was terminated when it had proceeded long enough so that at least sev- eral hours of steady oxygen consumption data had been obtained, preferably at a range of speeds. The animal was then removed, weighed wet, and returned to a holding tank of chilled seawater. Later the live, anesthetized ani- mal's underwater weight was determined in 5.5°C seawa- ter, and the animal was dried to determine dry mass. Data obtained from the swim tunnel were used to cal- culate rate of oxygen consumption [MO:, micromoles O:/ (mg wet wt X h)] as a function of absolute (Sa. cm/ s) and relative (Sr, lengths/s) for each mysid. The mean absolute (Smar) and relative (SmrT) swimming speeds and rate of oxygen consumption (MO;mr) for the entire ses- sion in the swim tunnel were calculated for each mysid. with the assumption that these speeds, swum spontane- ously by the mysid, represent routine swimming speeds. In addition, the maximum swimming speed maintained for at least one minute, identified as the maximum short- term swimming speed, was noted for each individual. Data for all animals from each instar were grouped to- gether, and the mean Smar. Smrr, MO:mr, and wet, dry, and underwater weights were calculated for each instar. Best fit mean-square linear and power regressions for the relationship between Smar, Smrr and MO;mr were calcu- lated for each instar and for all the instars combined. Swimming speeds and rates of oxygen consumption for each instar were compared by analysis of variance and by a regression of these variables against carapace length. The best-fit equation relating rate of oxygen consump- tion (MO:) to swimming speed (Sa) and body mass (grams wet weight) was determined by least squares mul- tiple regression. All references to statistical significance in these experiments were based on the 95r< confidence level. References to rates of oxygen consumption con- form to the terminology conventions of Piiper et al. (1971). Results Swimming s/>m/.s ami rute\ of oxygen consumption Upon first being placed in the swim tunnel, many of the animals swam rapidly and erratically for some time. Maximum short-term speeds were usually recorded dur- ing this early period (Table I). Maximum short-term speeds varied from 10.3 to 18.2 centimeters per second ( 1 length per second for the larger instars, 2 lengths per second for the smaller). After this initial acclimation period of one to three hours, most individuals swam at a characteristic speed that varied little throughout the rest of the experiment. G. INGENS SWIMMING ACTIVITY 113 Table I Swimming speeds and rates of oxygen consumption of Gnathophausia ingens maintained in the laboratory for less than 30 days, by instar Swimming speed* Mean weight (g) Mean length Mean Rate of oxygen Maximum cm/s consumption Instar n Wet Dry Under water Carapace mm Total cm Smar cm/s s.d. length/s s.d. Short Sustained M02nuT# s.d. 5 7 0.718 0.114 0.015 15.9 5.2 3.5 2.6 0.67 0.49 10.3 7.5 0.0125 0.00943 6 13 1.27 0.260 0.021 20.2 6.2 5.8 1.7 0.93 0.27 12.8 8.6 0.0116 0.00811 7 14 2.22 0.382 0.030 24.3 7.2 6.2 2.3 0.86 0.32 15.4 10.3 0.00575 0.00307 8 13 4.07 0.772 0.047 30.0 8.5 6.3 2 2 0.74 0.26 18.2 11.6 0.00453 0.00229 9 10 7.12 1.321 0.086 35.9 9.9 5.1 1.1 0.51 0.10 18.2 12.2 0.00271 0.00172 5-9 72 3.10 0.472 0.040 25.6 7.5 5.6 2.1 0.76 0.32 18.2 12.2 0.00725 0.00641 * Mean speeds are the average swimming speeds of all individuals of the instar combined. Maximum "short" speeds are the highest speeds maintained by any individual of the instar for 1 minute; maximum sustained speeds are the highest speeds sustained by any individual in the instar for at least 20 minutes. # MO; units = micromoles O;/(mg wet wt x h). No significant differences in swimming speed between day and night were observed. Swimming speed and the accompanying rates of oxygen consumption were sig- nificantly lower for animals that had been maintained in the laboratory for thirty days or longer (P < .005 and .0002, respectively). Mean routine swimming speeds (Smar, cm/s) and rates of oxygen consumption [MO^mr, micromoles O;/ (mg wet wt X h)] for each instar are sum- marized in Table I. This table contains data for only those animals that had been in the laboratory for less than thirty days. Except for the smallest individuals (in- star 5), routine absolute swimming speed averaged around 5 to 6.5 cm/s for all animals, regardless of instar. Instar 5 animals swam significantly more slowly than the other instars (P < .05), but there was no significant difference in absolute swimming speeds among instars 6 through 9 (Fig. 1 A). The slope of a regression of Smar ver- sus animal length for instars 6 through 9 was not signifi- cantly different from zero (P = .52). Significant trends in mean relative routine swimming speeds (SmrT) were found among the instars. The relative routine swimming speeds of each of the instars 6 through 8 were significantly higher than those of all larger instars. A regression of Smrr versus animal length for instars 6 through 9 had a highly significant downward trend (P < .001), indicating the slower relative swimming speeds of the larger instars. Most instar 5 animals swam at a slower relative speed than predicted by this regression, but faster than the largest instars (Fig. IB). The body angle of the swimming mysids changed with swimming speed. At low speeds the body angled upward anteriorly, becoming more horizontal as speed in- creased. For any given absolute swimming speed, body angle was greater for the smaller instars. By 8 cm/s the body was essentially horizontal. This trend is similar to that reported by Kils ( 1979b) for swimming Euphausia superba. and that reported by Cowles et al. (1986) for passive body movements of dead G. ingens. In the animals for which pleopod beat rate was re- corded, no significant relationship was found between pleopod beat rate and swimming speed within the nar- row range of swimming speeds observed. The pleopods beat at around 1 50-230 strokes per minute, regardless of swimming speed. The relationship between pleopod beat rate, swimming speed, and body angle for one individual is shown in Figure 2. Significant differences in rates of oxygen consumption were measured between instars. With the exception of instar 5, routine mass-specific metabolic rates (MO2mr) were significantly higher for the smaller instars than for larger ones (Fig. 1C). Most individual animals swam at a typical speed for that individual, with little deviation throughout the ex- periment. The maximum sustained speed, defined as the highest swimming speed maintained for at least 20 min- utes, recorded for any animal of a particular instar was typically less than twice the average swimming speed of individuals from that instar. Few animals swam at a large enough range of speeds to determine the relationship be- tween swimming speed and rate of oxygen consumption. In those that did, the relationship was approximately lin- ear (Fig. 3). The best-fit equation relating rate of oxygen consump- tion (MO2) to swimming speed (Sa) and body mass (g wet wt) was: MO: = 0.00289 - 0.00216 log,0g + 0.00156 Sa -0.001 67 (log,0g)XSa (1) 14 I) 1 ( y 'te swimming speed, MO;,,,, of the smaller instars w;i- , ,< than that of the larger instars (Fig. 4). This effect v. -duced but not eliminated by converting swimming spe Is to lengths per second for each instar. The smaller am ;,i;iK thus had higher rates of oxygen consumption than did the larger animals at any given swimming speed. £• 0.006 ~ X — : S 0.005 - 5 a» 0.004 s ^ D) E, CM O 0.003 0.002 - TgDHODO « 0.001 JL O E =1 0.000 C — i r~ i i i i » 1 234 56 Swimming Speed cm/s Htjure.l. Relationship of swimming speed (X,cm/s) to rate of oxy- gen consumption (Y, micromoles O:/ (mg wet wt x h) for one tinatho- lilniiHia int;en*. Error bars are standard deviation. G. 1NGENS SWIMMING ACTIVITY 115 02468 Swimming speed cm/s Figure 4. A comparison of the best-fit linear regressions of mass- specific rate of oxygen consumption [micromoles O?/ (mg wet wt X h)] as a function of absolute swimming speed (cm/s) for Gnathophausia ingens instars 5-9, based on equation 1 . Discussion Swimming speeds Comparison of G. ingens' routine swimming speed of 5 to 6.5 centimeters per second (0.5 to 1 body length per second) to that of other, shallower-living pelagic crusta- ceans is difficult, since few comparable measurements have been reported (Table II). All shallow-living species cited are smaller than the deep-living G. ingens but swim at faster relative speeds. G. ingens is also capable of swim- ming much faster than its routine speeds, as demon- strated by several animals that swam at over 18 cm/s (over 2 lengths/s) for several minutes when first intro- duced to the swim chamber, and by the maximum sus- tained speeds of 7.5-12.2 centimeters per second (1-1.5 lengths/s) (Table I). However, these relative speeds for G. ingens are as little as one tenth that of the shallower-liv- ing species when the difference in body length is taken into account. If the highest speeds cited above for each species are taken as an estimate of the animals' burst speeds, G. ingens' burst speeds are also markedly lower than those of shallower-living pelagic crustaceans. To attain the routine swimming speeds of 5 to 6.5 cm/ s, the swimming speed of most G. ingens instars, the smaller instars had to swim at relative speeds of nearly twice as many body lengths per second as compared to the larger instars. The routine swimming speeds of the smaller instars thus approached more closely to their maximum sustained speeds (Table I). This may be why the absolute swimming speeds of instar 5 animals were somewhat lower than those of most of the larger instars. If instar 5 animals were to swim at 5 to 6.5 centimeters per second, as the larger instars did, they would be swim- ming at 1 to 1 .25 lengths/s, or nearly the maximum sus- tained relative speed (1.4 lengths/s) attained by any in- star. Swimming speeds and rates of aerobic metabolism Due to the relatively restricted range of routine swim- ming speeds selected by G. ingens in this experiment, it was not possible to determine conclusively the shape of the relationship between swimming velocity and rate of oxygen consumption. However, several lines of evidence indicate that the relationship is linear over the limited range of speeds studied. In the few animals that swam at a wide range of speeds, the relationship between velocity and rate of oxygen consumption appeared to be approxi- mately linear, as shown in Figure 3. When data from all individuals of a given instar were pooled, the best-fit rela- tionship in each instar measured was linear over the range of speeds tested. Stepwise linear regression indi- cated that rate of oxygen consumption was related di- rectly to swimming speed, and not to the logarithm nor Table II Comparison of crustacean swimming speeds reported in the literature Species Length (cm) cm/s Lengths/s Reference Gnathophausia ingens 5.2-9.9 5.0-6.5 0.5-1 This paper Euphausia superba 4-5 5.6 1.25 Kils(1979a) Mysis relicta 3 5-10 2-3 Robertson et al. (1968) Neomysis americana 11.4 6-8 5-7 Hargreaves(1981) 0.6-1.7 3-10 5-6 Hargreaves(1981) Acanthomysis sp. 0.7 9-10 10-14 Allen (1978) Mysidium columbiae 20 Steven (1961) 116 D. L. COWLES AND J. J CH1I DKI SS to the square of speed. In addition, Cowles et al. (1986) showed that drag on a dead mysid's body increases lin- early with velocity c ver the speed range at which these animals were swi nmmg. Since thrust in a steadily swim- ming animal is ualtodrag(Wu, 1977), thrust and met- abolic ei' 'nsumption would also increase linearly with swimming speed in these animals if drag on a dead mysid : opresentative of drag on a live, swimming mysid. A linear relationship between swimming velocity and rate of oxygen consumption has also been reported for several other crustacean species. Halcrow and Boyd, (1967) found a linear relationship for the amphipod Gammarus oceanicus, as did Torres and Childress. ( \()%l)forEuphausiapaciftca. A number of other crusta- ceans, however, have been found to have nonlinear rela- tionships between swimming velocity and rate of oxygen consumption. The basis for these differences is not clear. However, it appears likely that at higher speeds the rela- tionship between velocity and oxygen consumption in G. ingens would begin to conform more closely to an exponential relationship (Hargreaves. 1981; Webb, 1 97 5a; Cowles etal.. 1986). Size dependency of oxygen consumption It has been shown for numerous organisms that the slope of the allometric equation of the logarithm of total oxygen consumed (Y) versus the logarithm of the ani- mal's mass (X) generally falls in the range of 0.67 to 1, usually being about 0.75 (Kleiber, 1947; Wolvekamp and Waterman, 1960; Wu, 1977; Schmidt-Nielsen, 1979). This relationship holds for standard or basal me- tabolism (Winberg. 1956, 1961; Hemmingsen. 1960; Brett. 1965; Brett and Glass, 1973;Wilkie, 1977; Peters, 1983). for routine metabolism (Job. 1957), and for active metabolism (Brett, 1965, Brett and Glass, 1973; Taylor ci al.. 1981; Prothero, 1979). Childress (197 la) and Miller-Adams and Childress ( 1 983c) found a similar rela- tionship between animal size and routine oxygen con- sumption in G. ingens. The standard rates of aerobic me- tabolism determined in this study were generally lower than these rates (Fig. 5). The routine rates of oxygen con- sumptu'i reported by Childress (1971a) and Miller- Adams.:, H ">ildress(1983c) were similar to rates asso- ciated with nming speeds of 0.25 lengths/s in this study. Though the slop.. <~ the allometric relationship be- tween size and standard iietabolic rate (equation 2) was lower than the 0.75 generally found for such relation- ships, the difference was barely significant. On the other hand, the slope of the allometric regression of the ani- mals' routine metabolic rates IITS//A wet mass was highly significantly less than 0.75 (equation 3). and for the 1 235 Wet weight (g) Figure 5. A comparison of the best-tit least-squares linear regres- sions of the allometric relationship between total rate of oxygen con- sumption ( Y. micromoles Oi/h) vwsi/.v wet mass(X. grams), from sev- eral studies of(inathophausia ingens'oxyg/sn consumption rate. Lines: A: from Childress (1971). B1-B3: from Hiller-Adams and Childress ( 1 983c). C to E: rates of oxygen consumption as measured in this study. C: standard rate (0 cm/s). D: oxygen consumption rate at 0.25 lengths per second swimming speed. E: routine oxygen consumption rates. smaller instars the rates of oxygen consumption were sig- nificantly higher than those previously reported for G. ingens (Fig. 5). This trend reflects the higher relative swimming speeds and rates of oxygen consumption of the smaller instars under the conditions of the swim tun- nel. In this experiment, the animals swam freely in the tunnel at a speed they set themselves, restrained only by connection to a movable force transducer. In previous reports (Childress, 1971a; Hiller-Adams and Childress, 1983c), oxygen consumption was measured within a small enclosed respiration chamber in which the animals lay, beating their pleopods. Since these animals were free to set their own pleopod beat rates within the confines of the chamber, it may be assumed that the rates of oxygen consumption measured under these conditions were routine rates. The fact that the slopes of the allometric relationships obtained under these conditions were sim- ilar to the expected slopes of 0.75 supports this assump- tion. However, in light of the data obtained in the present study, it appears that the definition of routine activity as applied to active, negatively buoyant crustaceans such as G. ingens needs to be refined. It appears that the animals in the enclosed chambers, which were not free to swim about, assumed a uniform level of activity that was sim- ilar in all instars and equivalent to a swimming speed of approximately 0.25 lengths per second. The present experiment shows that when free to swim through the water, however, the smaller instars assume a much higher level of spontaneous activity than the larger in- stars do. "Routine" activity levels are markedly different for the different instars if the animal is in a free-swim- G INGENS SWIMMING ACTIVITY 117 ming state, as in this experiment, but not if the animal is not free to swim about, as in the Childress ( 197 la) and Hiller-Adams and Childress ( 1983c) experiments. If one is interested in comparing rates of oxygen consumption at some standard level of activity, then the rates mea- sured by Childress (197 la) and Hiller-Adams and Childress ( 1 983c) will do. However, this study shows that if one is estimating actual energy expenditures as may occur under routine conditions in the field, one must ac- count for the different levels of routine activity the different instars assume when left to swim freely. Cost of transport The energy expenditure of an actively moving animal can be described in terms of cost of transport, or the amount of energy required to move a given distance through the medium. Cost of transport is influenced by a number of variables including speed, mode of transport, animal size and shape, and medium. For calculating cost of transport, the linear relationship between swimming speed and rate of oxygen consumption was recalculated in terms of energy expended per unit distance [calories/ (g X km)]. A respiratory quotient (RQ) of 0.79 was used, reflecting metabolism of a mixture of carbohydrate, pro- tein, and fat (Bartholomew, 1977). When this RQ is used, 1 micromole of oxygen is equivalent to 0. 1075 cal- ories. Instar 8 was selected as an average animal for cost of transport estimation. Instar 8 animals weighed an av- erage of 4.07 grams, and the relationship between speed and rate of oxygen consumption is given in equation 1 . Using these data, this animal's energy expenditure per unit distance (CT, calories per gram-kilometer) [cal/ (g X km)] while swimming is: CT= (4) This relationship is shown graphically in Figure 6. As can be seen, the energy required per unit distance is very high for low swimming speeds, dropping rapidly with in- creasing speed at low speeds and then much more grad- ually at speeds above 3 cm/s. This relationship makes it clear that, for G. ingens, lowest costs of transport per unit distance are incurred at speeds above 3 cm/s. This fits well with the empirical observation that these mysids swim at a characteristic speed of 5 to 6.5 cm/s. Slower swimming speeds would be energetically expensive, en- tailing a high cost per unit distance. On the other hand, equation 4 predicts that swimming faster than 5 or 6 cm/s would at best result in only minimal reduction in cost of transport. In reality, higher swimming speeds are likely to result in even higher costs than predicted, due to increasing turbulence and to the exponential increase in drag with speed predicted by hydrodynamic equations 10 - 8 - ra ra K O) "o^f 4 ~ „ o; °£ ° S 2 - 02468 Swimming Speed (cm/s) Figure 6. Cost of transport (CT, calories per gram-kilometer) as a function of swimming speed (Sa, cm/s) for Gnalhophausia ingens of instar 8. Equation: CT = 1.63 [(3.87/SJ + 1] (Webb, 1975a). Over the 0 to 8 cm/s range of swimming speeds measured in this experiment, change in body an- gle with speed appears to mask these effects, but at higher speeds they can be expected to become more prominent, resulting in an increase in cost of transport at higher speeds. In terms of energy expenditure per unit time, higher swimming speeds also have higher costs due to the in- crease in metabolic rate with speed. G. ingens ' routine swimming speeds thus appear to be intermediate be- tween the very low speeds, with their high costs of trans- port per unit distance, and very high speeds, with their high metabolic costs per unit time and distance. At a speed of 5.5 cm/s, a 4.07 gram G. ingens would have a cost of transport of 2.78 cal/(g X km), or 1 1.3 cal/ km. This value is slightly higher than values estimated from the regression lines shown in Schmidt-Nielsen, (1972) Tucker, (1975), Beamish, (1978), and Har- greaves, (1981), all of which are for swimming fish. None of these authors state the equation for their regression lines; however, Schmidt-Nielsen's data are calculated from data given by Brett (1965) for swimming sockeye salmon of 3.38 to 1432 grams. A regression of Brett's data (for salmon in 15°C water), converted to the units of equation 4, is: CT = 2.05 WWT -0.254±0.054 (5) A 4.07 gram G. ingens swimming at 5.5 cm/s would have a cost of transport of 2.78 cal/(g X km), while the above equation predicts that cost of transport for a fish of the same size would be 1 .43 cal/(g X km). Torres (1984), using data from Brett and Glass (1973) for a size range of sockeye salmon, calculated net cost of transport for swimming fish. Torres' equation is: 118 D. L. COWLES AND J. J. CHII OKI SS CTn = 1.416\V\\ I (6) (CTn in this equatio s net cost of transport [cal/(g x km)], therefore standard metabolic rate must be sub- tracted from th • e metabolic rate before using this equation.) \v ras equation is based on salmon data. Torres has < mt the cost of transport of a number of other hsh species falls near this line as well. For an instar 8 /'. ingens swimming at 5.5 cm/s. the net cost of transport would be 1.82 cal/(g X km), while the value Torres' equation predicts for a fish of similar size is 0.997. G. ingens' cost of transport thus appears to be twice as high as that offish of similar size. This trend has been noted for other crustaceans as well. Torres (1984) compiled net cost of transport data for a number of crus- tacean species and calculated an equation for crustacean net cost of transport analogous to equation 6. For crusta- ceans Torres' best-fit regression is: CTn = 6.26 WWT~028 (7) This equation predicts a net cost of transport for a 4.07 gram crustacean swimming at 5.5 cm/s of 4.23 cal/ (g X km), twice as high as the 1.82 calculated for G. in- gens. Thus. Gnathophausia ingens ' net cost of transport appears to be relatively low for crustaceans, which use paddle propulsion, but is higher than that for fish, which use an undulatory propulsion mode. Swimming efficiency Swimming efficiency, the ratio of the mechanical power required to overcome the drag an animal experi- ences while swimming to the metabolic power the ani- mal uses for swimming, is a useful way to compare the efficiency of different propulsive mechanisms. Swim- ming efficiency has been determined for a number of fish, including salmon (Osborne, 1961; Webb, 1973, 1975a) and trout (Webb. I971a, b), which swim in the subcarangiform mode, and Cymatogaster aggregaia (Webb, 1975b) and goldfish (Smit, 1965, Smit el at.. 1971). which use pectoral fin propulsion. Calculated swimming efficiencies for the subcarangiform swimmers were low at low speeds, increasing to 15.8% at critical swimming speeds for trout and to 26% for salmon. Effi- cien< matogaster aggregata at critical swimming speed 2 to 14%. Efficiency tends to increase with fish size • imming speed (Webb, 1975a), and ap- pears to be h ir the subcarangiform mode than for pectoral fin pi Previous studies mming efficiency in hard-bod- ied organisms such as crustaceans have mainly been esti- mates based on what is kn iwn about muscle efficicnc\ and efficiency in fish. Klyashtorin and Yarzhombek ( 1973) used various efficiencies cited in the literature for ATP conversion, muscle efficiency, and paddle propul- sion efficiency to arrive at an estimate of 5% for crusta- cean swimming efficiency. Hargreaves (1981) used sim- ilar calculations, along with the fish swimming effi- ciencies cited above, to estimate crustacean swimming efficiency at 10%. Nachtigall (1977) used a swimming efficiency of 10% for swimming water beetles, based in part on a calculation of 30% efficiency for the rowing ap- paratus. Torres ( 1 984) made a more direct calculation of swim- ming efficiency in the euphausiid E. pacifica by measur- ing rate of oxygen consumption at various swimming speeds and comparing these values to estimated drag based on hydrodynamic formulas. On the basis of this partly empirical data, he calculated swimming efficiency to vary from 0.014%, at 1 cm/s to 2.85% at 20 cm/s. If the animal's drag were higher than his estimates based on hydrodynamic formulas, then the animal's swim- ming efficiency would be correspondingly higher. Swimming efficiency in G. ingens may be calculated based on oxygen consumption data from this experi- ment (equation 1 ) and on drag data from Cowles el al. (1986). For an instar 8 individual (carapace length 3.0 cm ). swimming efficiency Es is described by the equation: ES = 2.96X 10~3(Sa) (8) At a swimming speed of 5.5 cm/s, swimming efficiency- would be 1.6 percent. This efficiency is higher than the 0.097 to 0. 1 33% reported by Torres ( 1 984) for E. pacifica swimming at this speed, but is below that reported for fish. Equation 8 indicates that (/. ingens' swimming effi- ciency increases linearly with speed. At 8 cm/s, efficiency for an instar 8 animal would be 2.4%. Swimming effi- ciency also increases with speed for fish (Webb, 1975a), and for E. pacifica (Torres, 1984), though not linearly. In fish, the increase in efficiency with speed is thought to be linked to changes in propeller and muscle efficiency. It is not known whether this is also true for G. ingens. Efficiency and the changes in efficiency with speed of the multiple-paddle mode of propulsion used by G. ingens and many other pelagic crustaceans have not been ade- quately studied. Kils (1979b) found that Euphausia \\ipcrhu changes many aspects of the pleopod stroke with increases in speed over the range of 0-1 5 cm/s, including increasing abduction of the protopodites, increasing de- gree of spreading of propulsive setae, holding pleopods closer to the body on the return stroke, directing the pro- pulsive stroke more directly to the posterior, and bring- ing the whole body into a more nearly horizontal orien- tation. These adjustments result in changes in the flow direction and size of the wake and in increased swim- ming velocity. Change in pleopod beat rate is small over this entire range of speeds, increasing from 150 to 180 beats per minute. Increase in swimming speed is accom- G. INGENS SWIMMING ACTIVITY 119 plished by a linear increase in the transport distance per beat rather than an increase in pleopod beat rate. E. sitperba thus appears to control swimming speed by modifying stroke efficiency at speeds up to 15 cm/s. At this speed maximum stroke efficiency appears to have been reached, and further increases in speed are brought about by changes in stroke rate. Mickel and Childress (1978) and Quetin and Childress (1980) observed that the pleopod beat rate of G. ingens strapped to a frame is remarkably constant, remaining at an average of be- tween 140 and 210 beats per minute or stopping com- pletely. G. ingens in the swim tunnel also maintained a similarly high, stable rate of pleopod beats, even with changes of swimming speed of at least a factor of two. It thus appears likely that G. ingens also adjusts swimming speed largely by changes in stroke characteristics, as does E. superba. Which parameters of the stroke are varied and how these changes contribute to stroke efficiency re- main to be determined. One likely factor influencing the increase in swimming efficiency in G. ingens with increasing speed is the change in body attitude (Fig. 2). At low speeds the animal swims with its body angled upward, directing a larger propor- tion of its thrust downward and thereby increasing lift. As speed increases, the body assumes a more horizontal orientation, so that a larger vector percentage of thrust is directed directly backward. This trend is likely to result in increasing efficiency in the generation of forward thrust with increasing speed, as observed. Eventually, however, the animal reaches a speed at which it assumes a nearly horizontal orientation in the water. The speed varies between instars, but by 8 cm/s most animals are nearly horizontal. This speed, at which increases in effi- ciency due to changes in body angle would be maxi- mized, would correspond to an efficiency of 1.8% for G. ingens of instar 8. Calculations for other instars, such as those carried out above for instar 8, indicate that swimming efficiency also increases with size in G. ingens. At 5.5 cm/s, efficiency for an instar 5 individual would be 0.8%, while that of an instar 9 individual would be 5.36%. Such a trend has also been noted for fish (Webb, 1975a). Gnathophausia ingens as a bathypelagic crustacean As an active pelagic crustacean, G. ingens appears to be more efficient and has lower costs of transport than shallower species, such as E. pacifica. However, the order of magnitude reduction in rate of oxygen consumption of the bathypelagic G. ingens can only be partially ac- counted for by these relatively small increases in swim- ming efficiency or reductions in cost of transport. The most obvious factor contributing to G. ingens' low rate of oxygen consumption is its reduced swimming speed relative to surface-living crustaceans. G. ingens' routine relative swimming speeds are as low as one tenth those measured for shallower-living crustaceans, and its maxi- mum speeds appear to be lower by the same factor. On the other hand, G. ingens is not inactive. The mysid swims constantly and shows no tendency for hanging motionless in the water. These observations are consis- tent with present hypotheses regarding the selective fac- tors responsible for the low metabolic rates of deep-living pelagic species, and provide experimental evidence of re- duced activity levels in deep-living animals. Acknowledgments We thank the crews of the research vessels New Hori- zon and Velero for their help in gathering G. ingens for this research, and George Hilton for his help in statistical analysis. Our thanks also to A. Alldredge, A. Ebeling, M. S. 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(August. 1988) Energy Metabolism During Anoxia and Recovery in Shell Adductor and Foot Muscle of the Gastropod Mollusc Haliotis lamellosa: Formation of the Novel Anaerobic End Product Tauropine GERD GADE Instituijiir Zoologie IV, Universitdt Diisseldorf, Universitdtsstr. I, D-4000 Diisseldorf I , Federal Republic of Germany Abstract. Metabolic responses to experimental anoxia and subsequent recovery, and to exercise were investi- gated in two different muscular tissues of the ormer, Ha- liotis lamellosa. The tissues are employed for different tasks by the animal. The foot is mainly responsible for slow gliding movements. The shell adductor muscle pulls down the shell for protection and in righting an ani- mal which has been dislodged from the rocks. Tissue- specific differences in anaerobic energy metabolism oc- cur. During 6 h of experimental anoxia, energy for both muscles was provided by arginine phosphate and co-fer- mentation of glycogen and aspartate. Glycolysis in the shell adductor muscle led mainly to the formation of the novel end product tauropine; D-lactate production pre- dominated in the foot. This pattern is consistent with ob- served enzymatic profiles in the two muscles and with the equilibrium constants of the respective enzymes, tauropine and D-lactate dehydrogenase. Recovery from anaerobiosis was characterized by a rapid return of the phosphagen pool and the energy charge to the aerobic state. A protracted time-course was observed for the clearance of glycolytic end products. Exercise, primarily powered by the shell adductor muscle, was mainly fueled by glycolysis resulting mostly in the accumulation of tauropine. Introduction In recent years different enzymes that terminate anaer- obic glycolysis, so-called opine dehydrogenases, have Received 15 March 1988; accepted 31 May 1988. been identified in the tissues of many marine inverte- brates (for a review, see Gade and Grieshaber. 1 986). The products formed (octopine, strombine, or alanopine) via the reductive condensation of pyruvate with the respec- tive amino acid (arginine, glycine or alanine) are collec- tively known as opines: arginine pyruvate + glycine + NADH + H+ oclopmc dehydrogcnase stromhine dchydrogcnase alanine alanopine dchydrogcnase octopine strombine + NAD* + H:O. alanopine Recently, a unique compound was detected in muscle extracts of the prosobranch gastropod, the abalone Hali- otis discus hannai (Sato ct ai, 1985). It was identified as D-rhodoic acid (now termed tauropine), previously iso- lated from some red algae (Kuriyama, 1961). The re- sponsible enzyme, rhodoic acid dehydrogenase or tauro- pine dehydrogenase, catalyzing the reaction: pyruvate + taurine + NADH + H" ^ tauropine -I- NAD* + H:O, has been found in muscle tissue of the Japanese abalone as well as in the European ormer, Haliotis lamellosa (Sato and Gade, 1986), and subsequently purified and characterized in detail in the latter species (Gade, 1986). Certain features of this enzyme suggest a role in main- taining cytoplasmic redox balance during hypoxic condi- tions in the ormer (Gade, 1986). Various invertebrates can withstand hypoxic condi- tions in their habitats. Their metabolism during such a period of environmental anoxia is characterized by co- fermentation of aspartate and glycogen leading to the ac- 122 ANAEROBIC ENERGY METABOLISM OF HALIOT1S 123 cumulation of the end products succinate, alanine and (in some cases) propionate and acetate. The rate of en- ergy production is low, but the yield of ATP increased (reviewed by de Zwaan, 1977; Schottler, 1980; Living- stone, 1982; Gade, 1983a; Storey, 1985). During exces- sive locomotory activity, the capacities of muscle tissue to synthesize ATP rapidly by aerobic means are limited and energy provisions are met during functional anoxia via anaerobic glycolysis resulting in the accumulation of lactate or the opines. The rate of energy production is high, but the efficiency is low (references as above and Gade, 1980; de Zwaan and van den Thillart, 1985; Gade and Meinardus, 1986; Gade and Grieshaber, 1986; Gade, 1987a). The present study concerns the anaerobic energy me- tabolism of muscle tissue in the ormer Haliotis lamel- losa. The strategies used to provide energy during envi- ronmental and functional anoxia are of paramount im- portance for the survival of this species. The ormer is epifaunal in the littoral zone, attached by its foot to wave-swept rocks. The broad shell acts as a protective shield. It is pulled down tightly by the large shell adduc- tor muscle (the right retractor or columella muscle) dur- ing low tide or vigorous wave action. When dislodged, ormers are extremely vulnerable to predators, especially since they often lie upside down. Therefore, these gastro- pods typically right themselves as fast as possible. This, again, is achieved mainly by relatively active movements of the large shell adductor muscle and with less input of the foot (unpub. obs.). Previous studies revealed interesting patterns of dehy- drogenase distribution in muscle tissue of the ormer (Gade, 1986). Most tauropine dehydrogenase activity is found in the shell adductor muscle, which contains only minute levels of D-lactate dehydrogenase activity. In the foot muscle D-lactate dehydrogenase displays the highest activity. This almost mutually exclusive distribution of the dehydrogenases, combined with the different in- volvement of the muscle tissues during environmental (both tissues) and functional (mainly shell adductor) an- oxia, led us to compare the energetics of the two muscle tissues. Furthermore, we investigated the metabolic events during recovery in well-aerated seawater, immedi- ately following experimental anoxia, since data on the fate of the accumulated end products and re-charging of the depleted high energy phosphates are rather scarce (see review by Ellington, 1983). This paper shows unequivocally that the fermentation of glycogen to the novel end product tauropine main- tains cytoplasmic redox balance during experimental as well as functional anoxia in the shell adductor muscle. D-lactate is the main fermentation product in the foot during 6 h of anoxia. Both glycolytic end products are apparently oxidized very slowly in situ. Comparison of w.wt. LDH TDH 69 157 Figure 1. The sites of tissue-sampling (shell adductor muscle and foot) in the ormer, Haliotis lametlosa. The boxes identify the sites. Ad- ditionally, the enzyme activities for D-lactate dehydrogenase (LDH) and tauropine dehydrogenase (TDH ) for each tissue are given as means ± 1 S.D. (n = 4) in units per gram wet weight (U g~' wt wgt). the glycolytic rates during experimental and functional anoxia reveals a 10-fold increase in the shell adductor, but only an enhancement by a factor of 2 in the foot muscle. Materials and Methods Animals and tissues Specimens of the ormer Haliotis lamel/osa (5-7 cm maximal shell length) were collected by local fishermen from the Bay of Naples, Italy, during October 1986. Ani- mals were maintained in flowing seawater (22-24°C) at the Stazione Zoologica. Animals were used in experi- ments four to six days after collection. Due to different profiles in the enzyme activities for pyruvate reductases (see Introduction), muscle tissue from two different organs were used in this study (Fig. 1): we compared the metabolic changes occurring in the shell adductor muscle to those in the foot. The dorsal part of the shell adductor (near the attachment of this muscle to the shell), and the anterior edge of the foot just below the head, were always excised for study (Fig. 1 ). Biochemicals Biochemicals were from Sigma Chemical Company (Deisenhofen, FRG) and Boehringer GmbH (Mann- heim, FRG). All other chemicals were of reagent grade quality and came from Merck (Darmstadt, FRG). Tau- ropine dehydrogenase (EC 1.5.1.?), used to determine taurine and tauropine, was purified from the shell adduc- tor muscle of//, lamellosa as outlined previously (Gade, 1 987b). D-lactate dehydrogenase (EC 1.1.1 .28) and octo- pine dehydrogenase (EC 1.5.1.1 1), used to assay for D- lactate, arginine phosphate, and arginine, respectively, 124 G. CADE * 100 5090 50.80^ r Time (h) 12 16 Figure 2. Alterations in the adenylate energy charge (E.G.) in the shell adductor (SA; solid circles) and foot (F; open circles) muscle of Halunis lamellosa during environmental anoxia and recovery (onset murki/d by arrow). Each value is a mean ± I S.D. (for n, see Materials and Methods). The asterisks denotes a significant change compared to controls. were purified from muscle tissue of the horseshoe crab, Limulus polyphemus (Carlsson and Gade, 1 985), and from the adductor muscles of the scallop. Pecten jaco- baeits (Gade and Carlsson. 1984), respectively. h '\[H'ri mental procedure Metabolic responses to environmental hypoxia and re- covery. Twenty specimens of H. lamellosa were incu- bated in wash bottles (10 animals each) filled with about 2 1 of seawater (22-24°C) that had been gassed with pure nitrogen until P0. (monitored with an oxygen electrode) reached almost zero mm Hg. After the animals were in- serted, the wash bottles were flushed with a constant, slow stream of nitrogen gas. After 6 h of anoxic incuba- tion, seven animals were removed, and their shell adduc- tor and foot muscles excised, blotted, and frozen in liq- uid nitrogen. The remaining ormers were returned to well-aerated seawater, and subsets of four animals were removed at various intervals (1,3, and 1 3 h) and treated as above. Furthermore, a zero time group of seven gas- tropods were chosen for the controls. The frozen tissues were subsequently transported from Naples to Diissel- dorf on dry ice and stored at -35°C. Metabolic responses to functional hypoxia. Four ani- mals were exercised for 1 2 to 1 5 min in a large aquarium with flowing-seawater system. To induce exercise, the animals were placed upside-down on their shells; their righting movements involved, primarily, relatively vig- orous contractions of the shell adductor muscle. When the animals had regained their normal posture, they were immediately inverted again. This work was continued for up to 15 min. when movements became much slower and the animals appeared to be exhausted. Shell adduc- tor and foot muscles were then removed and treated as above. Metabolite assays Neutralized perchloric acid extracts were prepared from the frozen tissues of H. lamellosa according to pre- viously published methods (Gade el a!., 1978: Carlsson and Gade, 1986). The levels of ATP. ADP. AMP, argi- nine, and arginine phosphate were determined spectro- photometrically by the methods of Lamprecht and Trautschold (1974), Jaworeck et a/. (1974). and Gade ( 1985a); the determinations were made immediately af- ter neutralization of the extracts to eliminate sample losses. Other metabolites were quantitated spectrophotomet- rically after storage of the extracts at -25°C. The meth- ods used were those of Gawehn and Bergmeyer (1974) for D-lactate. Gra/31 (1974a, b) for L- and D-alanine, Bergmeyer et al. (1974) for aspartate, Williamson (1974) for succinate, and Gade ( 1987b) for taurine. Tauropine was determined enzymatically using tauropine dehydro- genase in an assay system almost identical to that used for octopine quantification (Gade, 1985b). All metabolite data were analysed for significant Table I Alteralil'adcn\-ltitc\ crimcnttil ani <\ia ami \ uh\c 3 O; O E 2 1- 0 L-AIonine D-Alanme U 8 Time (h) 12 16 Figure 5. Time-course of the levels of L-alanine (upper panel) and D-alanine (lower panel) in the shell adductor (SA; solid circles) and foot (F; open circles) muscle ofHaliotis lamellosa during experimental anoxia and recovery. For further details, see Figure 2. 126 G. G\I>I 6). Anoxic incubation resulted in a significant increase in both tissues. In the loot muscle, initial values were rap- idly achieved after 1 h > ,\. In contrast, succinate levels in the shell adductor declined more slowly, and took 3 h to reach pre-ano\ic levels. Taurine levels were about twice as high in the shell ad- ductor as in the foot of control abalones. No significant changes occurred in the foot. There was a significant de- cline in the shell adductor muscle during anoxia without any restoration in the recovery period (Figs. 7, 8). Sub- stantial accumulations of tauropine were evident in the shell adductor muscle during anoxia, while a small, but significant, formation occurred in the foot muscle. In the latter tissue, the main anaerobic end product was D-lac- tate. which also accumulated in the adductor, but to a much lesser extent (50% ) than tauropine (Figs. 7. 8). D- lactate levels were rapidly cleared to 50% of the anoxic level during the first hour of recovery in the foot. Both tauropine and D-lactate levels remained high in the shell adductor after 3 h of recovery. Even after 13 h of recov- ery these levels were still higher than the initial concen- trations before anoxia. No significant changes in the levels of glucose-6-phos- phate (0.50 and 0.15 /zmoles/g w. wi. in shell adductor and foot muscle, respectively) were observed during an- oxic incubation and recovery (results not shown). Metabolic responses to functional hypoxia The levels of the adenylates, arginine-containing com- pounds, and various other metabolites in the shell ad- ductor muscle and the foot during exercise are listed in Table II. There was no significant change in the energy charge or in the levels of arginine phosphate in either tis- sue. Aspartate levels were marginally, but significantly, diminished in the shell adductor, whereas a small, but significant rise in the levels of L-alanine was observed in the foot. As main glycolytic end products, levels of D- lactate (marginally) and tauropine (primarily) were ele- -: - . r 1 Succinate 12 Time(h) 16 I inure 6. Time-course of the levels of succinate in the shell adduc- tor (SA; solid circles) and foot (I : open > m Irsi muscle of lliilu 150- 100- 50- 0 9- V I, 0 T E ~ =L2 1 0 SA TQ u r op i n e D-Lactate L 8 Time (h) 12 16 Figure 7. Time-course of the levels of taurine (upper panel) and (lower panel ) D-lactate ( solid circles) and tauropine (open circles) in the shell adductor muscle (SA) ofHaliotis tamellosa during experimental anoxia and recovery. For further details, see Figure 2. vated significantly in the shell adductor muscle, whereas the D-lactate levels were doubled in the foot without any significant rise in the tauropine levels. Discussion This study in the ormer is a good example of the prin- ciple that anaerobic energy metabolism in a muscle is specifically matched to the function of the muscle. It re- flects adaptation to specific metabolic need of the tissue. The two investigated tissues, the foot and the shell ad- ductor muscle, are employed by the animal for different tasks. The foot is mainly responsible for slow gliding movements that very likely are supported by aerobic me- tabolism. In contrast, the shell adductor muscle pulls the ormer's shield-like shell tightly to the substratum to pre- clude dessication during low tide and to prevent dislodg- ing by wave action. The shell adductor also rights an ani- mal that has been detached from the rocks. Thus, the shell adductor muscle is mctaholically more active than the foot and performs burst contractions which, in gen- eral, rely on anaerobic metabolism. However, when the whole animal has to cope with hypoxic or even anoxic conditions, both tissues need to have the capacity for maintaining metabolism anaerobically. ANAEROBIC ENERGY METABOLISM OF HAL1OT1S 127 16 Figure 8. Time-course of the levels of taurine (upper panel) and (lower panel) D-lactate (solid circles) and tauropine (open circles) in the foot (F) of Ha/iotis lamellosa during experimental anoxia and re- covery. For further details, see Figure 2. This partitioning in function is also reflected in the metabolism of the two tissues. Compared to foot muscle, the shell adductor contains 4-fold higher levels of high- energy phosphates, ATP (and, in fact, the total adenylate pool), and arginine phosphate, suggesting a higher meta- bolic rate. This is confirmed when the energy demand for both tissues is calculated from the decreased levels of the phosphagens and the increased levels of glycolytic products occurring during experimental anoxia (Table III): the ATP production rate (^moles g~' wt wgt min~') for the shell adductor muscle is about twice as high as for the foot. In both tissues the bulk of the energy is provided by anaerobic glycolysis (between 70 and 80%), the re- mainder by the phosphagen (Table III). Again, the glyco- lytic flux (calculated in nmoles glycosyl units g~' wt wgt min'1; Table III) is also 1.5-fold higher in the shell ad- ductor. The main qualitative difference between the two tis- sues is the involvement of two different glycolytic end products in anaerobic metabolism. Whereas glycogen breakdown in foot results in the production of D-lactate, glycolysis in the shell adductor is terminated with the for- mation of the novel end product tauropine. Thus, the tauropine/tauropine dehydrogenase system — function- ally analogous to the lactate/lactate dehydrogenase sys- tem— is active in the shell adductor muscle to maintain cytoplasmic redox balance. This pattern of end product formation in the different tissues is in agreement with the enzymatic complement of the respective tissue: lactate dehydrogenase is the predominant pyruvate reductase present in the foot; tauropine dehydrogenase is almost exclusively present in the shell adductor (Gade, 1986). We now ask why tauropine dehydrogenase turns up in this mollusc, and why taurine is used as a substrate for a dehydrogenase. Opine dehydrogenases which use the amino acids L-arginine, glycine, and L-alanine for the condensation reaction with pyruvate are already known (see review by Gade and Grieshaber, 1986). Obviously, the opine dehydrogenases that have evolved are those that would make use of the most abundant amino acids in the species. The same is true for the amino acid tau- rine. From the amino acids used for opine production (arginine, glycine, alanine, taurine) it is the one with the highest concentration in the shell adductor muscle of the ormer (Gade, 1986 and this study for arginine). There seems a sort of "co-evolution" of the most abundant amino acid and the corresponding opine dehydrogenase; thus, the specificity of the enzyme for the amino acid may be evolutionarily altered as a consequence of the "makeup" of the pool of amino acids in the different tis- sues. The mechanism of this is not understood yet, how- ever another example is in the literature. In the poly- chaete worm Aphrodite aculeata, strombine dehydroge- nase was found in pharynx muscle containing extremely high levels of glycine, whereas alanopine dehydrogenase was present in body wall musculature (Storey, 1983). Siegmund (1986; also cited in Grieshaber and Kreutzer, 1 986) compared the concentrations of those amino acids involved in the action of octopin-, strombine-, and ala- nopine dehydrogenase from various marine inverte- brates (coelenterates, molluscs, and annelids) to the amounts of octopine, strombine, and alanopine actually formed during environmental anoxia. He showed that in the species investigated the most abundant of these amino acids was used as a substrate for opine formation. This was the case for alanine/alanopine in Littorina litlo- ralis, L. littorea, Nucella lapillus, and Glycera convolula, and forglycine/strombine in Halichondrapanicea, Myti- lus edulis, Crassostrea angulata, Pharus legumen, Solen marginal us, Ensis siligua, Lutraria lutraria, Arenicola marina, Nephtys hombergi, Pherusa plumosa, and Pecti- naria koreni. However, tauropine production was not analyzed, but in many of the species taurine concentra- tions are higher than those of the other amino acids de- termined. Thus, with the present small data base avail- able— tauropine dehydrogenase has additionally only re- ported from muscle tissue of the brachiopod Glotlidea pyramidata (Doumen and Ellington, 1987) — it is not possible to conclude from a high taurine concentration to the presence of tauropine dehydrogenase and/or pro- duction of tauropine. Another question concerning H. lamellosa is: what is the significance of tissue-specific differences in pyruvate metabolism? We speculate that the driving force that led to the appearance of tauropine dehydrogenase in the shell adductor muscle is the requirement for burst activ- 128 G. CADE Table II Levels of adenvlaies. w ' ' wt- wgt) and adenyfate energy charge in ••hell atlihictor and fool muscle I-! H.I ring exercise (15 min) Shell adductor muscle Foot muscle MOM-, ; te Control (n = 7) Exercise (n = 4) Control (n = 7) Exercise (n = 4) ATP 3.25 - 0.99' 2.65 - 0.11 0.73 ± 0.31 1.00 • 0.59 ADP 0.96 - 0.20 1.02 • 0.18 0.14 + 0.06 0.20 + 0.07 A Mi- 0.23 - 0.16 0.14 i 0.07 0.03 ± 0.01 0.05 + 0.02 Sum 4.44 • 1.23 3.81 • 0.28 0.90 ± 0.32 1.25 • 0.64 Energy charge 0.85 ± 0.04 0.83 ' 0.03 0.88 ± 0.03 0.86 * 0.06 Arginine 19.63 -t- 4.38 21.25 • 4.00 3.56 ± 1.23 5.54 • 2.46 Argmine phosphate 11.77 - 2.92 9.88 * 3.37 3.42 ± 1.18 4.41 • 3.91 Sum 31.40 + 2.31 31.13 • 4.65 6.98 ± 2.27 9.95 ' 6.17 D-lactate 0.50 • 0.10 0.86 * 0.16" 0.33 ± 0.19 0.77 * 0.15** Tauropine 1.22 - 0.57 5.83 - 2.09** 0.23 ± 0.10 0.40 + 0.26 Taurine 243.2 - 39.50 168.90 • 22.0** 91.6 ± 50.5 84.0 • 23.9 L-alanine 1.72 i 0.46 2.83 + 1.48 0.56 ± 0.28 1.26 ' 0.57** D-alanine 1.60 + 0.45 1.09 • 0.74 0.37 ± 0.18 0.18 • 0.15 Aspartate 1.61 - 0.47 0.60 ' 0.48** 1.77 ± 0.80 1.05 • 0.66 Succinate 0.36 + 0.14 0.40 * 0.25 0.11 ± 0.03 0.10 + 0.09 * All values given as mean ± SD. ** Significant to control. ity creating functional anoxia during the righting move- ments. Such active muscle work needs a rapid activation of glycolytic energy production, which eventually leads to an increased redox status (NADH/NAD* ratio). Based on theoretical considerations of thermodynamic properties of opine and lactate dehydrogenases, it was proposed that the large amino acid pool used for opine production is decisive for maintaining the NADH/ NAD* ratio lower than using the lactate pathway (see review by Fields, 1983). Most other arguments for the possible advantages of opine synthesis versus lactate for- mation (e.g., lack of disturbance of internal osmolarity and less acidification) have been dismissed, as discussed recently by Grieshaber and Kreutzer ( 1 986). Besides glycogen and arginine phosphate breakdown, the amino acid aspartate provides energy during anoxia. The simultaneous depletion of aspartate and accumula- tion of succinate and alanine in both tissues indicates cofermentation of glycogen and aspartate as known to occur in other invertebrates during lack of oxygen (see, for example. Cade, 1983a, 1987a; Kreutzer el al.. 1985). The observation of only very small amounts of succinate Table III ( 'nmi\in\nn • and glycolytic flux (nmoles 1 wgt min 'im ihell adductor and loni nut^lc <>i Hal ions lamellosa during experimental anoxia and exercise* ATP equivalents (jimnlcs g ' wt wgt) from Glycolysis** Phosph.igen Rate of consumption of ATP (/imolesg ' wt- wgt min~') * For calculations sec Mcinardus- 1 ! ,,ide, 1987. '* The small increase of succinate was assumed to derive by aspartate breakdown and is included in this calculation. *•* ( nntnhutinn of gjycolysisas percentage ol total equivalents of ATP is given in brackets. ( il\i oKlu' !lu\ (n moles glycosyl unit g ' wt-wgt mm ') Six-hour expert rm- shell adductor 18.9(70<7,)*** 8.2 0.08 16.3 foot 12.2(79%) 3.2 0.04 10.2 Exercise shell adductor Hl%) 2.2 0.77 206 !.,.,! 'i' i — 0.10 22.7 ANAEROBIC ENERGY METABOLISM OF HAL/OTIS 129 formed in both tissues of the ormer during anoxia and no production of propionate makes it highly unlikely that succinate is derived from glycogen by the so-called phos- phoenolpyruvate carboxykinase route. This pathway is apparently only operative in "good anaerobes" tolerat- ing prolonged hours of anoxia (see Introduction). The lack of propionate (and acetate) formation in the ormer is then indicative that this species can tolerate anoxic conditions for only a fraction of the time compared to species such as certain blue mussels, oysters, and many annelids. Indeed, preliminary experiments with speci- mens of H. lamellosa showed that these animals were unable to survive experimental anoxia longer than eight hours. Recovery in both muscles of H. lamellosa was quite similar. Levels of the high-energy phosphates and succi- nate were rapidly restored, but asparate levels increased slowly (and only) in the foot. Similar changes have been reported during recovery in the foot of the cockles, Car- dium edule (Gade and Meinardus, 1981) and C. tubercu- lalum (Meinardus-Hager and Gade, 1987), and the ad- ductor muscle of the file shell, Lima hians (Gade, 1983b). Levels of the respective glycolytic end products in the tissues of the ormer, D-lactate and tauropine, were not cleared during the first h (foot) or 3 h (shell adductor) of recovery, but also did not significantly increase during this time period. Thus, it is highly unlikely that an anaer- obic initial phase of recovery occurs as observed in tis- sues of the bivalves Mytilus edulis (de Zwaan el al., 1983), M. squamosus (Nicchitta and Ellington. 1983), and Crassostrea gigas (Eberlee el al, 1983). The power output during exercise in the ormer is low relative to the jet propulsion of cephalopods and scallops or the jumps of the cockle, C. tubercidatum. The present study shows that energy demand increases for both mus- cles in comparison to experimental anoxia: about 10- fold for the shell adductor and about 2-fold in the foot (Table III). Since the energy is mainly or exclusively (foot) derived from glycolysis, the same increases are cal- culated for the glycolytic fluxes (Table III). These in- creases are small when compared to C. tuberculatum, where during jumping the glycolytic rate is enhanced 100-fold above the resting rate (Gade and Meinardus- Hager, 1986). The calculations in Table III also show that the shell adductor muscle is particularly involved in exercise of the ormer; its rate of energy consumption and its glycolytic flux are about 8- to 9-fold higher than those of the foot. The bulk of the energy during exercise came from glycolytic tauropine formation in the shell adduc- tor muscle, although there was also some D-lactate pro- duction. Thus, anaerobic breakdown of glycogen to tau- ropine also supports exercise metabolism, as it did me- tabolism during experimental anoxia. The formation, during both experimental and func- tional anoxia, of both glycolytic end products — tauro- pine and D-lactate — in the shell adductor muscle may be explained by theoretical considerations of the equilib- rium constants of the respective reactions. Since the de- gree of product formation is determined by the equilib- rium constant (K<,q) of a reaction, we measured the K<,q constants for the reactions catalysed by tauropine (TDH) and D-lactate dehydrogenase (D-LDH). These were K^q(TDH) = ([pyruvate] X [taurine] X [NADH] X [H+]) X ([tauropine] X [NAD+])~' = 7.15 X 1 0" ' 3 M (Gade, 1986) and K«,(D-LDH) == ([pyruvate] X [NADH] X [H+])X ([D-lactate] x [NAD+]) ' = 1.3 x l(Tl2(Gade and Meinardus-Hager, 1986). We can use these values to assess whether this theoreti- cal equilibrium is reached in vivo by both enzymes. The actual equilibrium is given by the mass action ratio, e.g., MARLDH = ([pyruvate] X [NADH] X [H+]) X ([D-lac- tate] X [NAD+])~' and MARTDH = ([pyruvate] X [tau- rine] X [NADH] X [H+]) X ([tauropine] X [NAD+])"'. These ratios, however, were not calculated because data for neither the internal proton concentration, nor the NAD+/NADH ratio, are available for the shell ad- ductor muscle. However, assuming that the reaction catalysed by LDH is at or near equilibrium in most bio- logical systems, we can obtain indirect information on the equilibrium situation of the TDH reaction using the K<,q values (see above). The ratio K^TDHyK^D- LDH) has the value of 0.55 M and corresponds to the quotient ([D-lactate] X [taurine]) X ([tauropine])"1, since TDH and LDH share common substrates such as pyruvate, NADH, H+, and NAD+ (see above). Thus, if we use the concentrations of D-lactate, taurine, and taur- opine measured in the shell adductor, the calculated ra- tio should be close to the theoretical value, 550 mM, if both reactions are near equilibrium (our assumption). Table IV shows that the reactions are not exactly at the theoretical equilibrium, but the "close equilibrium" of neither reaction changed much during anoxia and subse- quent recovery. Thus, the formation of primarily tauro- pine and a little D-lactate is according to the equilibrium constants of the reactions. In contrast, after exercise the quotient was 10-fold lower than the theoretical value in- dicating that the reactions of D-LDH and/or TDH are in "disequilibrium." Our data do not indicate which reac- tion that is, but a recent paper on a similar phenomenon in foot muscle of C. tuberculatum argues for a "disequi- librium" of D-LDH because of its low activity and the enhanced production of pyruvate and NADH due to the increased glycolytic flux (see details in Gade and Meinar- dus-Hager, 1986; pages 197 and 198). The same argu- ments can be applied to the ormer during exercise: D- LDH activity in the shell adductor is much lower than TDH activity and glycolytic flux is enhanced. 130 G. CADE Table IV -hell adductor muscle of Haliotis lamellosa during experimental anoxia, recown: and exercise* pine] 6-h anoxia I -h recovers' 3-h recovery I 3-h recovery Exercise Quotient (m .'.' 199 190 133 217 133 50 • Tissue c nirations calculated from data presented in Table II and Figures 7 and 8 under the assumption that the water content of the tissues In conclusion, the energy metabolism in both the shell adductor and foot muscle is powered by co-fermentation of ghcogen and aspartate and transphosphorylation dur- ing experimental anoxia. According to differences in tis- sue activities, as well as therm odynamic properties of the two pyruvate reductases (TDH, LDH), tauropine is the preferred end product in the shell adductor, while D-lac- tate accumulation occurs in the foot. Recovery in well- aerated seawater reverses most of the metabolic changes seen during anoxia, but the time courses for high-energy phosphates (fast) and glycolytic end products (slow) are quite different, as in other molluscs. Enhanced glycolysis and maintenance of redox balance by the reaction of tauropine dehydrogenase are the main features of exer- cise in the shell adductor, and the foot is only minimally involved during this behavior. Acknowledgments The author thanks Dr. Georg Meinardus-Hager for help with the experiments, Dr. W. Ross Ellington (The Florida State University, Tallahassee) for commenting on and correcting the manuscript, and the staff of the StazioneZoologicadi Napoli for their hospitality. Finan- cial support was provided in part by grants from the Deutsche Forschungsgemeinschaft (Ga 241/4-3 and Gr 456/ 12-1) and by a Heisenberg Fellowship awarded from the Deutsche Forschungsgemeinschaft (Ga 241/5-2). Literature Cited BiTKmi > i r. 1 1 I .. K. Bcrnt, H. Mollerinu, and G. Pfleiderer. 1974. 1 Aspartat und .-Asparagin. Pp. 1741-1745 in Methoden dcr En:y- e. Vol. 2, H. U. Bergmeyer. ed. Verlag Chemic. Weinheim. (arlsson, K.-H., arid i di-. 1985. Isolation and characterisation of tissue-specific isot Lictate dehydrogenase from muscle and hcpatopancreas > imulus polyphonic J Comp. Ph\-\iol B 155: 72 3-731. (arlsson, K.-ll.. and <;. Gade. I9K6. Metabolic adaptation of the horseshoe crab. l.tmulu\ /« during exercise and environ- mental hypoxia and subsequent recovery. Rial Bull 171:217-235. Doumen, ('., and \\ . R. Kllington. 1987. Isolation and charactm/a- lion of a taurine-spccific opine dehydrogenase from the pedicles of the brachiopod. (ilottidea pyramiduiu ' / •/' /""/ 243:25-31. Kberlee, J. C., J. M. Storey, and K. B. Storey. 1983. Anaerobiosis. recovery from anoxia and the role of strombine and alanopine in the oyster, Crassostrea virginica. Can. J. Zoo/. 61: 2682-2689. Ellington. \V. R. 1983. The recovery from anaerobic energy metabo- lism in invertebrates. J Exp. Zoo/. 228: 43 1 -444. Fields, J. H. A. 1983. Alternatives to lactic acid: possible advantages. J. Exp. 7.001 228: 445-457. Gade, G. 1980. Biological role of octopine formation in marine mol- luscs. Mar. Biol Lea. 1: 121-135. Gade. G. 1983a. Energy metabolism of arthropods and mollusks dur- ing environmental and functional anaerobiosis. J. Exp. Zool 228: 415-429. Gade, G. 1983b. Energy production during anoxia and recovery in the adductor muscle o! the tile shell, Lima hians. Comp Biochem Physiol 76B: 73-77. Gade, G. 1985a. Argmine and arginine phosphate. Pp. 424-431 in Methods of Enzymatic Analysis. Vol. VIII, H. U. Bergmeyer. ed. Verlag Chemie, Weinheim. Gade, G. 1985b. Octopine. Pp. 419-423 in Methods of Enzymatic Analysis, Vol. VIII, H. U. Bergmeyer. ed. Verlag Chemie. Wein- heim. Gade, G. 1986. Purification and properties ol tauropine dehydroge- nase from the shell adductor muscle of the ormer. Haliotis lamel- losa. Eur J Biochem 160: 3 11-31 8. Gade, G. 1987a. Leben ohne Sauerstoff: Die Rolle der anaeroben Glykolyse bei aquatischen wirbellosen Tieren. I 'IT/I Dtsch Zool. Ges. 80:93-110. Gade, G. 1987b. A specific enzymatic method for the determination oftaurine. Biol (.'hem //<>/>/><•- .SVv/cr 368: 1519-1523. Gade, G., and K.-H., C'arlsson. 1984. Purification and characterisa- tion of octopine dehydrogenase from the marine nemertean Cere- hralit/u.\ lacteus (Anopla: Heteronemertea): comparison with scal- lop octopine dehydrogenase. Mar. Biol 79: 39-45. (•ade, G., and M. K. Grieshaber. 1986. Pyruvate reductases catalyze the formation of lactate and opines in anaerobic invertebrates. Comp Biochem Physiol 83B: 255-272. Gade. G.. anil G. Meinardus. 1981. Anaerobic metabolism in the common cockle ( 'ardium ediile V. Changes in the levels ol metabo- lites in the foot during aerobic recovery after anoxia. Mar Biol 65: 113-116. (Jade, G., and G. Meinardus-l lager. 1986. Anaerobic energy metabo- lism in Crustacea. Xiphosura and Mollusca: lactate fermentation versus multiple fermentation products. Zool. Beilr A' F. 30: 187- 203. Gade, G., K. Wceda, and I'. A. Gabbotl. 1978. Changes in the level of octopine during the escape responses of the scallop Pecten ma\i- musL.J Comp Physiol 124: 121-127. GaHehn, K., and II. t1. Bergmcyer. 1974. D-Laktat. Pp. 1538-1541 [n Methoden der Enzymatischen Analyse, Vol. 2. H n. Bergmeyer. ed. Verlag Chemie, Weinheim GralJI, M. 1974a. I.-Alanm. Pp. 1727-1730 in Methoden der Enzy- ANAEROBIC ENERGY METABOLISM OF HALIOT1S 131 matischen Analyse, Vol. 2, H. U. Bergmeyer, ed. Verlag Chemie, Weinheim. GraBl, M. 1974b. D-Alanm. Pp. 1731-1734 in Methoden der En:y- matischen Analyse, Vol. 2, H. U. Bergmeyer, ed. Verlag Chemie, Weinheim. Grieshaber, M. K., and U. Kreutzer. 1986. Opine formation in ma- rine invertebrates. Zoo/. Beitr. N. F 30: 205-229. Jaworeck, D., \V. Gruber, and H. U. Bergmeyer. 1974. Adenosin- 5'-diphosphat und Adenosin-5'-monophosphat. Pp. 2179-2181 in Methoden der Enzymatischen Analyse, Vol. 2. H. U. Bergmeyer, ed. Verlag Chemie, Weinheim. Kreutzer, U., B. Siegmund, and M. K. Grieshaber. 1985. Role of cou- pled substrates and alternative end products during hypoxia toler- ance in marine invertebrates. Mol. Physwl. 8: 37 1-392. Rumania. M. 1961. Ninhydrin reactive substances in marine al- gae— III. On the chemical structure of "unknown A" isolated from red algae. Bull. Jpn. Soc. Sci. Fish. 27: 699-702. Lamprecht, W., and I. Trautschold. 1974. ATP. Bestimmung mil Hexokinase und Glucose-6-phosphat-Dehydrogenase. Pp. 2151- 2159 in Me/hoden der Enzymatischen Analyse, Vol. 2, H. U. Berg- meyer, ed. Verlag Chemie, Weinheim. Livingstone, D. R. 1982. Energy production in the muscle tissue of different kinds of molluscs. Pp. 257-274 in Exogenous and Endoge- nous Influences on Metabolic and Neural Control, Vol. 1, A. D. F. Addink and N. Spronk, eds. Pergamon Press, Oxford. Meinardus-Hager, G., and G. ( .ado. 1987. Recovery from environ- mental anaerobiosis and muscular work in the cockle, Cardium lu- berculatum: oxygen debt and metabolic responses. J. Exp. Zoo/. 242:291-301. Nicchitta, C. V., and W. R. Ellington. 1983. Energy metabolism dur- ing air exposure and recovery in the high intertidal bivalve mollusc Geitkensia demissa granosissima and the subtidal bivalve molluscs Modiolus squarnosus. Biol. Bull. 165: 708-722. Sato, M., and G. Gade. 1986. Rhodoic acid dehydrogenase: a novel amino acid-linked dehydrogenase from muscle tissue of Haliotis species. Naturwissenschaften 73: 207-209. Sato, M., N. Kanno, and Y. Sato. 1985. Isolation of D-rhodoic acid from theabalone muscle. Bull Jpn. Soc. Sci. Fish. 51: 1681-1683. Schottler, LI. 1980. Der Energiestoffwechsel bei biotopbedingter An- aerobiose: Untersuchungen an Anneliden. Verh. Dtsch. Zoo/. Ges. 73: 228-240. Siegmund, B. 1986. Funktionsbedingte und biotopbedingte Hypox- ien: Zur Bedeutung der Opine im anaeroben Energiestoffwechsel. Ph. D. Thesis. Universitat Dusseldorf. Storey, K. B. 1983. Tissue-specific alanopine dehydrogenase and strombine dehydrogenase from the sea mouse. Aphrodite aculeata (Polychaeta). J. Exp. Zoo/. 225: 369-378. Storey, K. B. 1985. A re-evaluation of the Pasteur effect: New mecha- nisms in anaerobic metabolism. Mol. Physwl. 8: 439-46 1 . Williamson, J. R. 1974. Succinat. Pp. 1661-1666 in Methoden der Enzymatischen Analyse. Vol. 2, H. U. Bergmeyer. ed. Verlag Chemie, Weinheim. de Zwaan, A. 1977. Anaerobic energy metabolism in bivalve mol- luscs. Oceanogr. Mar. Biol. 15: 103-187. de Zwaan, A., and G. van den I hi Hart 1985. Low and high power output modes of anaerobic metabolism: Invertebrate and vertebrate strategies. In Circulation. Respiration, and Metabolism, R. Gilles, ed. Springer- Verlag, Berlin, Heidelberg. de Zwaan, A., A. M. T. de Bont, W. Zurburg, B. L. Bayne, and D. R. Livingstone. 1983. On the role of strombine formation in the energy metabolism of adductor muscle of a sessile bivalve. J. Comp. Physiol. 149: 557-563. Reference: Biol Bull 175: 132- 1 36. (August. 1988) Control of Cnida Discharge: II. Microbasic p-Mastigophore Nematocysts are Regulated by Two Classes of Chemoreceptors GAIL E. MUIR GIEBEL, GLYNE U. THORINGTON, RENEE Y. LIM, AND DAVID A. HESSINGER' Department of Physiology and Pharmacology. School of Medicine, Loma Linda University. Lonui Linda. California 92350 Abstract. Using tentacles of the sea anemone. Aiptasia pallida. Thorington and Hessinger (1984. 1988a, b) re- cently identified two classes of chemoreceptors involved in sensitizing cnidocytes to discharge cnidae in response to mechanical stimuli. Discharge of cnidae was quanti- fied by measuring adhesive force between the tentacles and a test object. This measurement is assumed to reflect the contribution of the three types of cnidae present in the tentacles of A. pallida: the adherent spirocysts and two types of penetrant nematocysts, the predominant microbasic p-mastigophores and the basotrichous isorhi- zas. In the present paper we directly measure the dis- charge of the microbasic p-mastigophores and show that mastigophore-containing cnidocytes are sensitized by representative agonists for these two classes of chemore- ceptors. We also show that under certain conditions the number of discharged microbasic p-mastigophores cor- relates linearly to a major component of the measured adhesive force. This enables us to calculate the contribu- tion to adhesive force made by individual mastigo- phores. Introduction Nearly one hundred years ago, Nagel ( 1 892) suggested that chemicals d t from prey elicit feeding in cnid- arians. Recently, tu. groups of prey-derived chemicals, including N-acetylated sugars and a variety of amino compounds, were identified as being involved in prey capture. In the tentacles ofthc sea anemone Air>ia\ia /> 'w Figure 1. The effects of glycine on adhesive force (mgf) and on the number of discharged mastigophores adhering to test probes. Values for adhesive force (triangles) and for the number of discharged masti- gophores (circles) are expressed as means of the daily means of separate measurements with vertical bars representing standard errors. differences are apparent. First, the measurement of adhe- sive force initially decreases at 1CT9 M glycine before in- creasing to the maximum effect, while the number of dis- charged mastigophores shows no such decrease at 10 9 A/. Second, the magnitude of the maximum increase in adhesive force at Ema, versus naive controls is by 15%, whereas the magnitude of the maximum increase in dis- charged mastigophores is by more than 100% (Fig. 1). Third, the concentrations of glycine which yield the half- maximum effect (i.e.. KO}) are somewhat different for adhesive force measurements at 5.0 X 10~8 M than for the number of discharged mastigophores at 1.6 / io-7A/. NANA as a representative N-acetylated sugar sensitizer The effects of different concentrations of NANA on mean adhesive force (Fig. 2, triangles) and on the mean number of discharged mastigophores (Fig. 2, circles) are also biphasic and essentially coincidental. Each dose-re- sponse curve has regions of sensitization at low concen- trations of NANA, Emax values occurring at 10"5 M NANA, and regions of apparent desensitization at still higher concentrations. On the other hand, the magni- tude of the increase of adhesive force is by 25%. whereas the magnitude of the increase in the number of dis- charged mastigophores is by nearly 200%. In addition, the concentration to give the half-Emax (i.e.. the Ko5 value) for adhesive force at 3.2 X 10~7 A/ is about four times as much as that for the discharged mastigophores at 7.8 X 1(T* A/. Proportionality ofnematocysts discharged to adhesive force The number of discharged mastigophores is directly proportional to the measured adhesive force for sensitiz- ing doses of agonists up to 10 6 A/ glycine and 10 ' A/ NANA (Fig. 3). The calculated line for these data, when extrapolated, intercepts the abscissa to the right of the origin. This indicates that the measured adhesive force consists of at least two components, one that is indepen- dent of mastigophores and one that is dependent upon mastigophores. Thus, under these experimentally con- trolled conditions tentacles exhibit an adhesiveness of 160 120 o c. Q. O 01 0) n E 3 •z. HI) Mastigophores Adhesive Force SEM. I 40 ."• 10-9 10-' 10-5 NANA Cone., M 10-3 O> E 65 o>" o o LL CD > 60 S 55 50 45 !• inure 2. The effects ofN-acetylnetlraminic acid (NAN A) on adhe- sive force (mgf land on the mimlx-i u ('discharged mastigophores adher- ing to tcsl probes. Data expressed as in l-'igure 1 . CHEMORECEPTORS SENSITIZE CNIDOCYTES TO DISCHARGE NEMATOCYSTS 135 C/} 0> O 120 .c a. o 01 t5 5 80 I 40 0 20 40 60 80 Adhesive Force, mgf Figure 3. Correlation of discharged mastigophores to measured ad- hesive force (mgf). Horizontal and vertical bars represent standard er- rors of the mean (95% confidence limits) for adhesive force and for the number of discharged mastigophores, respectively. Plotted values represent all data measured at sensitizing concentrations of glycine (empty circles) and NANA (filled in circles) from Figures 1 and 2 (R = 0.92). Insert: data points obtained by subtracting 43. 3 mgf from each mean measurement of adhesive force and dividing by the number of discharged mastigophores and then plotting these values as a function of the number of mastigophores discharged. Ordinate-intercept of cal- culated line (dashed line) is 0.2 1 7 ± 0.028 mgf. approximately 43.3 mgf that is unrelated to the discharge of mastigophores (Fig. 3). This mastigophore-indepen- dent component of adhesiveness can be subtracted from each mean measurement of adhesive force. The resulting value can be expressed as the mean, corrected adhesive force per mastigophore. We find that the contribution of each mastigophore to the adhesive force measurement is slightly dependent on the number of mastigophores discharged (Fig. 3, insert). On the other hand, at desensi- tizing doses of sensitizers the number of discharged mas- tigophores does not correlate with measured adhesive force (data not shown). Discussion Discharge of mastigophores is influenced by two chernoreceptor classes The adhesion of tentacles to test objects has been used by researchers to detect in situ cnida discharge (Williams, 1968; Lubbock, 1979). More recently, using a novel and sensitive technique to quantify adhesion, Thorington and Hessinger (1984, 1988a, b) identified two classes of chemoreceptors that sensitize cnidocytes to discharge cnidae in response to mechanical stimuli. There are possible limitations, however, associated with using adhesive force to study responsiveness of cni- docytes. One such possible limitation is that measured adhesive force is an aggregate indicator of the discharge of several types of cnidae and, therefore, cannot distin- guish between the different types of responding cnido- cytes. In the present paper we have shown that a specific type of cnidocyte — those bearing the predominant ne- matocyst in the tentacles of A. pallida, the microbasic p-mastigophore — respond in a biphasic, dose-dependent manner to the chemosensitizers glycine and NANA (Figs. 1, 2, circles). Similar dose-response curves are ob- tained by measuring adhesive force under identical con- ditions (Figs. 1, 2, triangles; Thorington and Hessinger, 1988a). Thus, we conclude that the discharge of masti- gophores in this anemone is influenced by the two classes of sensitizing chemoreceptors that detect N-acetylated sugars and a variety of amino compounds, including gly- cine. Williams (1968), using the sea anemone Haliplanella luciae, concluded that the discharge of spirocysts, an ad- herent and non-penetrating type of cnida, but not that of mastigophores, was sensitized by food extracts. In con- trast, using A. pallida. we find that the discharge of masti- gophores is sensitized by optimum concentrations of both glycine and NANA (Figs. 1, 2, circles), each likely to be constituents of their natural diet. Similar findings for the effects of NANA on the discharge of mastigoph- ores of H. luciae have been found by Watson and Hes- singer (in press). In sea anemones the cnidome of the tentacle consists of the adherent spirocysts and the penetrant nemato- cysts. In acontiate sea anemones such as A. pallida and H. luciae, the cnidome of the tentacles is made up of spi- rocysts, microbasic p-mastigophores, and basitrichous isorhizas(Hand, 1955). Of these three types of cnidae the basitrichous isorhizas comprise a comparatively small portion of the total cnidae in the tentacles of A. pallida (Giebel, unpub. obs.) and H. luciae (Watson and Maris- cal, 1983) and are likely to contribute relatively little to the adhesive force measurements. Dose responses for nematocyst discharge and adhesive force compared The major difference between the dose-response curves for adhesive force and for the number of dis- charged mastigophores (Figs. 1, 2) is a proportionally greater increase in the number of discharged mastigoph- ores at Emax values than in adhesive force. For example, the maximum increase in number of discharged masti- gophores is 100% and 200% for glycine and NANA, re- spectively, as compared to only 1 5% and 25% increases in adhesive force, respectively. These differences in max- 136 G. E MUIR GIEBEL ET AL imum effects are not surprising since adhesive force is a composite measure of several contributing factors, in- cluding cnida-mediated and non-cnida-mediated (i.e.. "stickiness") factors, o A hich the discharged mastigoph- ores is only one. Contribui: • arious tentacle factors to adhesive force The daia in this paper show that within the range of sensitizing doses of glycine and NANA. the measure- ments of adhesive force correlate linearly with the num- ber of discharged mastigophores (Fig. 3). By extrapolat- ing this plot to the abscissa we estimate the adhesive force expected in the absence of discharged mastigophores to be approximately 43 mgf (Fig. 3). Therefore, contribu- tions to adhesive force up to 43 mgf are independent of mastigophores and due to, most probably, a combina- tion of factors, including discharged spirocysts and any inherent "stickiness" of the surface mucus. Recently we obtained measurements for the mucus. The mucus on the tentacle surface contributes approximately 30 mgf to the measure of tentacle adhesive force (Thorington and Hessinger. in prep.) in the absence of cnida discharge. The difference between 43 mgf (due to surface mucus plus discharged spirocysts) and 30 mgf (due to surface mucus alone) is approximately 13 mgf, possibly ac- counted for by the sum of all discharged spirocysts. How- ever, this is not to say that the contribution of spirocysts is constant at different concentrations of sensitizer. At sensitizing doses of glycine and NANA, contributory in- crements in excess of 43 mgf are due to discharged masti- gophores. From the slope of the linear correlation be- tween the number of discharged nematocysts and the measured adhesive force we calculate that the contribu- tion of each discharged mastigophore to adhesive force is approximately 0.17 mgf. A comparable value, which is somewhat dependent upon the number of discharged mastigophores. is obtained as the ordinate intercept of a plot when 43 mgf is subtracted from the adhesive force measurements and then plotted as mgf/mastigophore irniM the number of discharged mastigophores (Fig. 3, insert). The slight dependence of the calculated adhesive force per mastigophore upon the number of discharged mastigophores (Fig. 3, insert) is possibly due to a soften- ing effect of discharged mastigophores on the gelatin coating of the probe. The correlation between number of discharged masti- gophores and adhesive force occurs only at stimulatory doses of the tested sensitizing agents. At higher than opti- mum doses of sensiti/er the measurement of adhesive force does not correlate very well with the number of ad- hering nematocysts, possibK indicating that dramatic changes in other contributions to adhesive force, such as from discharged spirocysts, may also occur. ( 'onclusions In summary, we have shown that the discharge of mi- crobasic p-mastigophore nematocysts is under the con- trolling influence of at least two classes of chemoreceptor systems, one that is responsive to amino compounds as represented by glycine, and another that is responsive to N-acetylated sugars as represented by NANA. Further- more, the majority of the increase in adhesive force in response to these chemosensitizers is due to the discharge of the microbasic p-mastigophores. In addition, we have shown that under defined conditions the number of dis- charged nematocysts is proportional to the measured ad- hesive force. Thus, measurements of adhesive force can be used to quantify the extent of total cnidae discharged. Acknowledgments We thank Drs. G. Watson, P. McMillan, and C. Clausen for their valuable advice in relation to this proj- ect. Funded in part by BRSG grant RR 05352-24 and NSF grant DCB-8609859 to D.A.H. Literature Cited Blanquet, R., and H. M. I^nhoff. 1966. A disulfide-linked collage- nous protein of nematocysts capsules Science 154: 152-153. Dorgan, I... and D. A. Hessinger. 1984. GRAFPAC. a graphics and formal package for the Apple 1 1 and (1 le) computer. Copxright 1984. Hand, ('. 1955. The sea anemones of central California part III. The acontianan anemones. H'usmannJ Biol. 13: 189-202. Hessinger, I). A., and J. A. Hessinger. 19SI . Methods tor rearing sea anemones in the laboratory. Pp. 153-179 in Marine Invertebrates. Committee on Marine Invertebrates. National Academy Press. Washington. D. C. I.ubbock. R. 1979. Chemical recognition and nematocyte excitation in a sea anemone ./ /-.\/> BfO/. 83: 283-292. Nagel, \V. 1892. Das geschmacksinn der actinien. Zoo/. An:. 15: 334-338. Thorington. (.',. I ., and I). A. Hessinger. I9S4. Identification and par- tial characteri/ation ol cnidocyte chemoreceptors on the sea anem- one. .-I iptaxia pallida .1 Cell Biol 99(4): 221a. Thorington. (.',. t ., and I). A. Hessinger. 1988a. Control of cnida dis- charge: I. Evidence for two classes of chemoreceptor. Bio. Bull 174: 163-171. Thorington, (i. I ., and I). A. Hcssinger. 1988H. Chemical control of cnida discharge In Biology of \eniutovyxlx, D. A. Hcssinger and H. M. Lenhoff. eds. Academic Press. Orlando. (In press.) Watson, G. M., and I). A. Hessinger. 19KKa. Receptor-mediated en- docytosis of a chemoreceptor involved in triggering the discharge of cnidae in a sea anemone tentacle. Tissue Cell 19: 747-755. Watson, (i. M.. and I). A. Hessinger. 1988b. Locali/ation of a pur- ported chemoreceptor involved in triggering cnida discharge in sea anemones. In Biology of Nematocysts, D. A. Hessinger and H M. I enhotl. eds. Academic Press. Orlando. (In press.) Watson, <;. M., and R. N. Mariscal. 1983. Comparative ultrastruc- ture of catch tentacles and feeding tentacles in the sea anemone llaliplanella. Tissue (.'ell 15: 939-953. \\illiams. R. B. 1968. Contiol of the discharge of cnidae in Diadu- meneluaae(Vcm\\). Nanire2t9: 959. Reference: Biol. Bull. 175: 137-143. (August. 1988) Aspects of Entrainment of CHH Cell Activity and Hemolymph Glucose Levels in Crayfish* JANINE L. KALLEN, N. R. RIGIANI, AND H. J. A. J. TROMPENAARS Zoologiscli Laboratorium, Faculteit der Wiskunde en Natuurwetenschappen, Katholieke Universiteit, Toernooiveld, 6525 ED Nijmegen, The Netherlands Abstract. We investigated the effects of several experi- mental conditions, such as constant darkness, light/dark phase-shift, covered eyes, eyestalks and rostral regions, and optic tract sectioning, on the entrainment of daily blood glucose rhythmicity in the crayfish. Hemolymph glucose determination over a 24 h period and a morpho- metrical study on the secretory activity of the Crustacean Hyperglycemic Hormone (CHH)-producing cells in the eyestalk using immunocytochemistry were carried out. Our results indicate that Astacus leptodactylus exhibits an endogenous circadian blood glucose rhythm en- trained by the light/dark schedule. The light stimuli that control the rhythm are not de- tected by the compound eyes nor by the caudal photore- ceptor but most probably by a photoreceptor located elsewhere in the eyestalk. After disruption of the neural connection between the optic lobes and the cerebral gan- glion, blood glucose rhythmicity persists, which indicates that the biological clock of the blood glucose rhythm is located within the optic lobes. Introduction The Crustacean Hyperglycemic Hormone (CHH)- producing system of the crayfish Astacus leptodactylus consists of a number of neurosecretory perikarya located on the rostral latero-ventral side of the medulla termi- nalis ganglionic X-organ (MTGX). The axons of these cells pass through the medulla terminalis (MT) and ter- minate in the sinus gland. The location and morphology of this cell system has been described in detail (for Asta- cus leptodactylus see Van Herp and Van Buggenum, Received 3 March 1988; accepted 31 May 1988. * Results were presented at the 'Reunion des Carcinologistes de Langue Francaise.' Concameau. France (6-9 June 1987). 1979; Gorgels-Kallen and Van Herp, 198 1 ; Gorgels-Kal- len and Voorter, 1984; for other decapod species see Jaros and Keller, 1979; Gorgels-Kallen et al. 1982; Van Herp et al.. 1984). Under constant light/dark conditions, the CHH cell system of Astacus leptodactylus reveals a daily rhythmic- ity in the synthetic activity of the perikarya, transport of CHH-material to the sinus gland, and release of CHH into the hemolymph which results in a 24 h rhythm of blood glucose level (Gorgels-Kallen and Voorter, 1985). Similar results are described for the prawn Palaemon ser- ratus (Van Herp et al., 1984). Diurnal rhythmicity of hemolymph glucose content is also described for the crayfish Orconectes limosus (Hamann, 1 974) and for the freshwater field crab Oziotelphusa senex senex (Reddy et al., 1981). Hamann (1974) examined the day/night rhythmicity of blood glucose content of Orconectes limo- sus under various conditions. His findings signified the importance of light signals as entraining stimuli to main- tain blood glucose rhythmicity and indicated the pres- ence of an endogenous pacemaker. Furthermore, we re- cently described the presence of synaptic input on the ramifications of CHH axons in the MT neuropileum (Gorgels-Kallen, 1985). All the above mentioned find- ings point to the presence of a system controlling the en- trainment of CHH metabolism. The present work was undertaken in an effort to ob- tain additional information on the role of the prevailing environmental light/dark conditions in the entrainment of daily rhythmicity of CHH cell activity and, as a conse- quence, the hemolymph glucose level in Astacus lepto- dactylus. Based on Hamann's (1974) experiments, the daily blood glucose rhythm was examined under various conditions in order to study its exogenous or endogenous character and to learn more about the location of photo- 137 138 J L KALLEN ET AL receptor(s) and oscillator(s) involved in modulation. Furthermore, since \\o are immunocytochemically able to determine the secretory activity of individual CHH cells (Gorgels-K ..nd Voorter, 1984, 1985), we in- vestigateil ect of the experimental conditions on the cellular . ity of the CHH cell system. Materials and Methods Animals and blood sampling Crayfish of the species Astacus leplodactylns (Nord- mann), were imported from Turkey via a commercial dealer and kept in the laboratory in running tap water (13-15°C), and fed weekly with pieces of meat or fish. Except when otherwise stated, animals were kept under light/dark conditions (LD 12:12. light on 8.00 am). Ex- periments were performed with adult female and male crayfish of equal size and in intermolt stage. Blood sam- ples, obtained from 5 to 10 animals per sample time, were collected over a 24 h period at regular intervals. 100 ^1 of hemolymph was drawn into a calibrated capillary pipet which was inserted between the coxa and the basis of the left cheliped. Samples were taken in duplicate from each animal and frozen immediately. The blood glucose level was determined using the Gluco-Quant Test Combination (Boehringer Mannheim GmbH). Experimental conditions Constant darkness. Crayfish were kept in constant darkness (code DD). Blood samples were taken at the start of the experiment and 6. 1 1. and 35 days after the onset of DD conditions. Phase-shift. Crayfish kept under normal LD condi- tions were exposed to a 1 2 h phase-shift (code PS) accom- plished by lengthening the light period with 12 hours at the onset of the experiment. Hemolymph samples were taken at the start of the experiment and 6. 12, and 18 days after introduction of the phase-shift. Covered retinas and rostral regions. Several experi- ments were performed covering different regions of the eyestalk and the rostrum. Either both retinas (code RR), both eyestalks (code EE), or the rostral cephalic region including the eyestalks (code CR) were painted using a black textile dye (Silka; Talens, Apeldoorn, The Nether- lands) which formed a completely opaque, water-resis- tant coverall- Before painting blood samples were taken. The experimental conditions were maintained for 35 days, then hem- »lj mph was again sampled. Optic tract operation. Between the hard cxoskeleton of the eyestalk and rostrum there is a softer region enabling articulation of the eyestalk. In order to disrupt the neural connection between the optic ganglia and CHH cell sys- tem with the cerebral ganglion, a fine pointed cauteriza- tion needle or a microdissection scalpel was pushed through the soft region to section the optic tract locally (code NO). Blood samples were taken before the start of the experiment and 35 days after operation. At the end of the experiment, crayfish were sacrificed and the eyestalks were microscopically examined to check the success of the optic tract section, the condition of the isolated gan- glia and the normal course of the blood circulation. Light microscopy From animals kept under the above described experi- mental and control conditions, eyestalks were ablated at 12.00 am and fixed in Bouin Hollande fluid (24 h) con- taining 10% of a saturated aqueous solution of subli- mate. The fixed material was embedded in Paraplast (57°C). The immunocytochemical staining was based on the peroxidase-antiperoxidase (PAP)-method (Stern- berger, 1 974) and was performed on 7 ^m sections with an overnight incubation at 4°C for the anti-CHH. The primary antiserum was raised in rabbits against a puri- fied hyperglycemic fraction derived from eyestalks of As- tacus leptodactylus (for further details of the purification of the antigen and the production of the antiserum see Gorgels-Kallen and Van Herp, 1981 ). The procedure of the immunostaining followed Van Herp and Van Bug- genum (1979). with the primary antiserum applied in a dilution series. For each experiment the optimal dilution of the primary antiserum proved to be 1/1 50. which con- forms to the optimal dilution used for eyestalks from ani- mals kept under normal LD and laboratory conditions. The specificity of the immunostaining was tested as described previously (Gorgels-Kallen and Van Herp, 1981). The secretory cell stages of individual CHH cells were determined on the basis of the observed differences in staining intensity which enables the arbitrary division of the immunoreactive cells into three categories: +, ++, and +++, representing the cells with least intense, mod- erately intense, and most intense immunostaining, re- spectively. In a previous study, morphometric analyses of the cells at both the light and electron microscopic lev- els led to the following characterization of the three cell stages: + cells show the weakest immunoreaction which is correlated to the low content of CHH granules in their cytoplasm. The cells possess a large nucleus which points to a high level of mRNA production. ++ Cells show an intermediate immunoreaction. In their cytoplasm the content of granules is increased. These cells possess the largest nuclear and cytoplasmic volumes, which indicate high synthetic activity. + + + Cells show an intense im- munoreaction which is correlated with the largest nu- merical density of CHH granules in their cytoplasm. The synthetic level of these cells is low as documented by ENTRAINMENT OF CHH-SYSTEM IN CRAYFISH 139 their small cytoplasmic and nuclear volumes. Moreover, the CHH granules show a lower electron density and an increased diameter as compared with those of + and + + cells. These facts point to the occurrence of a maturation process, indicating that the secretory granules are youn- gest in the + cells and oldest in the +++ cells, which indicates different degrees of synthetic activity of the three distinguished cell stages. For a detailed description of the morphology and the results of a morphometric study of the CHH cell stages we refer to our previous study (Gorgels-Kallen and Voorter, 1984) and to our study on the secretory dynamics of the CHH cells in the course of the day/night cycle (Gorgels-Kallen and Voorter, 1985). The numbers of +, ++, and +++ cells were counted in the left eyestalks from three specimens per experimental or control condition. In addition, the cytoplasmic and nuclear volumes of five of each of the +, ++, and +++ cells, per eyestalk, were calculated according to Weibel's (1979) method and as described in detail previously (for illustration see Fig. 3; Gorgels- Kallen and Voorter, 1984, 1985). Results Hemolymph glucose rhythmicity Control experiments. Figure 1 a represents the results of the glucose determination in the hemolymph for all specimens used in the DD and PS experiment, deter- mined during normal 12 h light and 12 h dark condi- tions. The blood glucose values show the usual rhythmic- ity: around 4 hours after the onset of the dark period the glucose level doubles compared to the level found in the daytime. Constant darkness. Figures Ib-d show the blood glu- cose levels over a 24 h period, measured after 6 (Fig. Ib), 1 1 (Fig. Ic), and 35 days (Fig. Id) under constant dark- ness conditions. After 6 days absence of the light stimu- lus, the circadian hemolymph glucose pattern still per- sists, although the maximum decreases around mid- night. After 1 1 days the normal rhythm is tempered: yet, at midnight an increased variation of the mean glucose level can still be seen. Constant darkness for 35 days re- sults in total absence of any daily rhythm. Furthermore, the data show that removing the periodic changes in light intensity leads to consistently low glucose levels during the whole 24 h period. Phase-shift. Figures le-g show the hemolymph glu- cose values over a 24 h period measured 6 (Fig. le), 12 (Fig. If), and 18 days (Fig. Ig) after the introduction of a 1 2 h shift in the normal LD pattern, by lengthening one light period by 12 hours. Six days after the phase-shift blood glucose rhythmicity is disturbed. The graph shows three zones with an increase followed by a decrease in blood glucose content and increased variation in the 10 -/->! ® 1 1 . .[ ^ /\^ 2 - 8 12 16 20 24 4 8 10 "(ib) 10 I '© 6 Ll i lT\ 6 t i 1 ml hemolymph O NJ ^\l YK-* 2 n ' K! i\ /hi A •/ f \ \ ' • • • -^ , / • '~td 8 12 16 20 24 4 8 ;© 8 12 16 20 24 4 8 8 A ! ^ 6 6 - J\ /M i m O u o> 2 g , -/ iV^'l -r • v, 8 12 16 20 24 4 8 8 16 20 24 4 8 10 '(id) 10 - ,-. A — / \ 6 6 AU , 2 - ^f A/^i 2 -/' ; [^h 8 12 16 20 24 4 8 8 12 16 20 24 U 8 t (hours) Figure 1. (a-g) Daily hemolymph glucose pattern: (a) under nor- mal light/dark conditions (LD 12:12;n = 10); (b) after 6 days constant darkness (n = 5); (c) after 1 1 days constant darkness (n = 5); (d) after 35 days constant darkness (n = 5); (e) six days after a 12 h phase shift (n = 5 );(0 twelve days after a 12 h phase-shift (n = 5); (g) eighteen days after a 12 h phase-shift (n = 5). Means ± SEM. mean glucose values for most sample points. After 12 days the normal circadian blood glucose pattern reap- pears; the maximum in glucose level is found at 12 a.m., 4 hours after the onset of the "new" dark period. How- ever, during the light period at 12 pm a second smaller peak is seen. This glucose pattern persists and even be- comes more pronounced 1 8 days after the start of the experiment. At 12 am, the new midnight, the mean glu- cose level increases firmly whereas at 12 pm, the former midnight, a second smaller, although distinct rise in blood glucose content is found. Painted eyestalks and rostral regions. Prevention of light perception via the ommatidia (light percepting units forming the compound eye) does not abolish circa- dian rhythmicity in the hemolymph glucose content (Fig. 2b). During the dark period the glucose level still doubles. The amplitude and mean glucose levels during the total 24 h period are closely comparable to the con- 140 J. [.. K.ALLEN ET AL X) 2a / /r^-f 10 I 2 o> ? fi 12 16 20 24 4 8 10 8 12 16 20 24 4 8 8 12 16 20 24 4 8 ® '° 8 12 16 20 24 8 8 12 16 20 24 4 8 t ( hours ) Figure 2. (a-e) Daily hemolymph glucose pattern after several ex- perimental conditions: (a) under normal light/dark conditions before the start of the experiment (LD 12:12;n = 5): (b) thirty-five days after covering of the compound eyes (code RR; n = 5); (c) thirty-five days after covering of the whole eyestalks (code EE; n = 5): (d) thirty-five days after covering of the whole eyestalks and the rostral region of the cephalothorax (code CR; n = 5); (e) thirty-five days after section of the optic tract (code NO: n = 5). Means ± SEM. trol values obtained from these specimens before starting the experiments (Fig. 2a). Yet, some influence must be noticed. The nocturnal rise in glucose is found immedi- ateh at the start of the dark period and the mean values show increased variation. After painting the whole eye- stalks, an increased glucose level during the dark period is still seen, although the amplitude is very reduced (Fig. 2c). Covering the whole eyestalks including the rostral part of the cephalothorax leads to a complete absence of circadian blood glucose rhythmicity and during the whole 24 h period the glucose levels consistently stay very low (Fig. 2d). Optic trad operation. Disruption of the connection be- tween the eyestalk and the cerebral ganglion by section- ing the optic nerve does not interfere with the persistancc of a daily rhythmicity in hemolymph glucose content (Fig. 2c). .Set retory activity of the CHH cells We investigated the effect of a change in environmen- tal conditions on the secretory activity of the CHH cells. Animals that exhibit both a change (DD experiment). and no change (RR experiment) in hemolymph glucose rtnthmicity were used. The results of this morphomctric investigation are presented in Table I. Animals kept in constant darkness have a ratio between the number of +, ++, and + + + cells which differs from the ratio found for crayfish kept under control conditions. The number of + + + cells increases accompanied by a decrease in the number of ++ cells. The morphometric data reveal an increase in the cellular volume of the + + + cells. Immu- nocytochemical staining of the eyestalks of the RR ani- mals reveals that both the proportion of + , ++, and ++ + cells, and their cellular and nuclear volumes, are similar to those of the control animals. Disruption of the optic nerve produces a ratio of the +, ++, and + + + cells different to the ratio found for control animals. The number of + cells increases firmly, accompanied by a decrease in ++ cells. The morphometric results reveal increased cellular and nuclear volumes of the -I- cells. Furthermore, the overall conditions of these operated eyestalks were normal: blood circulation was not affected and no signs of degenerating eyestalk tissues were ob- served, nor regeneration of the sectioned optic nerve. Discussion Blood glucose levels found in Astacus leptodactylus af- ter exposing the animals to constant dark conditions re- veal that the normal 24 h rhythm persists for many cy- cles. The rise in blood glucose content around midnight remains clearly distinguishable after 6 days of constant darkness; after 1 1 days the rhythm is tempered, but the high variation in the mean glucose content at 12 pm still indicates a masked presence of the nocturnal peak. Intro- duction of a 1 2 h phase-shift results in a very slow adap- tation of the 24 h rhythm to the newly imposed light schedule. Six days after the onset of the phase-shift, the normal blood glucose rhythm is still disturbed. This dis- turbance is expressed as: (a) a high variation in the mean glucose levels of most sample points, and (b) three peri- ods with an increase followed by a decrease. This result corresponds to the description of the so-called transient phase, i.e., a temporary loss of synchrony among the var- ious units involved in a particular circadian rhythm, which results in frequency beats modulating the free- running period (Pavlidis, 1473). Twelve days after intro- duction of the phase-shift, the normal circadian blood glucose rhythm is restored, adapted to the new light/dark scheme, i.e., around 4 hours after the onset of the dark period a firm blood glucose peak is detected. However, even 1 8 days after the phase-shift the "old" glucose peak still can be seen. In his introduction of the symposium "Biological Clocks" (Cold Spring Harbor, 1960) Aschott" underlines the importance of the establishment of the free-running ENTRAINMENT OF CHH-SYSTEM IN CRAYFISH 141 0> E _ . 3 "a rju^^l'^iro^— >q " a. +i ii +i ii +1^ ii 3 ii 3 — ^ C C "^ C ^ C "" 2. z i c E - o tft +| — +| (N +| — +| S1 j£ 7 — ^- — • - • ^-^ & X 0 •o 4J *s ^ E - « "*• c ft ® ii +j ii +!_ u +j u £ § (U 0 •o j; § 's N-"' «N ^T^ r-^'^4 ^^^ c •£ 3 ;_ ^ E z ^£^S^ S^ * a° E o o •& u a T3 g (U flj 5h E > "^ I + 1"| + > a- +1 - +1 ^ +1 — +1 ^ •— " II '—'II ""^ II ""^ II o u ^. o ii fNl ,- c — •£ * 5 So I E ro T]- (N r| • (N * *N • fS • fN J^c^c^c^c , — , « Q «3 "° u " j > "^ u S "O •5 ^ o c <— . — 1 i .0 ^ ^^|T§7§7 If S| *£.§| 3 ^ Ch c ~ C ^_ C ^^ C •rt wi *" Z8 *Z? % ^* ~£ U i "3 •5."!' E ~ H E ^ ™ 0 3"| "*. ^2 "! f] ""! 2 "*. S i 2 •« 2 E F o > ;i u* ir> +1 II JH II +1^ II +[ II fe o I o _t Q rt 0 •o o 2 c ^3 4j — "o X r^i c"^ c ^ c^ c a u >- fc ^ y, V, 3 v Z a "c "g Ts E £^ •c o. s a t/1 QJ "* rMrnTr"' -O 2*. +1 II +1 II +1 II +1 II c <*- ^ **— 3 3 4J E -~. 3 J£ CTs=r£2i.?;i. 1 "8 "9 "8 ^ c 5i c e3 J3 ,U u = (e Z | o .— £• •— E o *5 cA «3 "c3 «3 3-§l^ 55 c« (« "o "o £ " " a 1.9 "o ll|l C C ao II § Q oi O Q Q ai Z g a i 6 u a £* z C C M x ° W period of individual organisms in order to establish a pe- riodicity as an endogenous one. However, establishment of the free-running period of the blood glucose rhythm in crayfish in this way is not possible, since repeated sam- pling of hemolymph from the same specimen leads, within several hours, to hyperglycemia caused by stress. Hamann's (1974) method of measuring the circadian hemolymph glucose levels from individual crayfish via an extracorporeal circulatory system only succeeded for a few cycles and cannot be performed with large numbers of animals. As such, blood glucose rhythmicity can only be depicted by sampling different groups of crayfish as described in this paper. However, despite the fact that we were not able to determine one of the major characteris- tics of circadian rhythm, we believe that the above men- tioned results, i.e., (a) the persistance of the rhythm for many cycles without external periodic light stimuli, and (b) the slow adaptation of the rhythm to changed light/ dark conditions, allow us to postulate that the daily blood glucose level in Astacus leptodactylus is generated within the organism and therefore may be called endoge- nous or circadian. Furthermore, since prolonged exposure of the animals to constant darkness eliminates hemolymph glucose rhythmicity and changing the external light stimuli pro- duces entrainment, our data show the final indispens- ability of the prevailing light/dark cycle as a Zeitgeber in entrainment of the daily blood glucose rhythm. The importance of an external light stimulus can also be seen from the results on the determination of the secretory activity of the CHH cells after 35 days DD conditions: an increased number of enlarged ++ + cells and a decreased number of actively producing ++ cells is found. These data point to a reduced production of CHH and in- creased storage in the perikarya. Covering both retinas does not abolish reception of a light entraining signal, as blood glucose rhythmicity still persists. This is also supported by the results of the deter- mination of secretory activity of the CHH cells: the resul- tant data are closely comparable to those of control ani- mals. Painting both eyestalks results in a very faint nocturnal blood glucose increase and covering both eye- stalks and the rostral region of the cephalothorax leads to a complete disappearance of rhythmicity and a consis- tently low glucose level during the whole 24 h period: the resulting hemolymph glucose graph is closely compara- ble to the graph obtained after constant DD conditions. Comparable experiments with the crayfish Orconectes li- mosus, performed by Hamann (1974), show the same blood glucose patterns. Therefore it appears that the eye- stalks (optic lobes) have a dominant role in perception of light, but the presence of an involved photoreceptor in the rostral region of the cephalothorax cannot be ex- cluded. The effect of disruption of the optic tract further 142 J L K.ALLEN ET AL o .. Figure 3. PAP-staining of CHH-producing cells in the MTGX. il- lustrating +, ++, and + + + cells. Bar represents 50 nm. + cells: mean cell volume 32.5 x 103 nm\ mean nuclear volume 5.5 x 10' nm3; + + cells: mean cell volume 38.8 x 103 nm}, mean nuclear volume 5.6 X 103/im3; +++ cells: mean cell volume 27.8 X 103/im3. mean nuclear volume 4.1 x I03^m3. (Morphometrical data from Gorgels-Kallen and Voorter. 1984). supports the importance of the entraining function of the eyestalk: daily rhythmicity of the hemolymph glucose level is not affected after disturbance of the neural con- nection between the cerebral and optic ganglia. This re- sult points to the presence of a pacemaker or biological clock of the glucose rhythm located in the optic lobes, although hormonal modulation from a pacemaker lo- cated elsewhere cannot be ruled out. Indeed, such hor- monal influence is not disturbed by the optic tract opera- tion, since microscopic investigation of these eyestalks did not reveal any irregularities concerning blood circu- lation or the condition of eyestalk structures. However, the secretory activity of the CHH cells of these NO ani- mals does not show the same picture as found in control animals. Yet, as the blood glucose rhythmicity is compa- rable to control animals, it could be that CHH release is modulated by an oscillator located in the eyestalk. How- ever, that regulation of CHH synthesis is more complex and (also) affected by a pacemaker located elsewhere. Another possibility might be that the CHH cells not only produce a hyperglycemic factor but also other hormon- allv active substances. Synthesis of hyperglycemic hor- mone might be affected by optic nerve section, while the synthesis of other hormones might go on undisturbed. Such an effect can be visualized immunocytochemiculh . Our results exclude any regulatory effect caused by the caudal photorcceptor described for the first time in cray- fish by Prosser ( 1934). Our data are supported by the work of Page and Lari- mer (1972), who studied the entrainment of the circa- dian locomotor activity in the crayfish Procamhants clarkii. They found that removal of the caudal photore- ceptor, removal of the ommatidia of both eyes, or re- moval of both the ommatidia and the lamina ganglion- aris, did not effect entrainment of rhythmicity in loco- motion. Comparable results were also obtained by Pollard and Larimer (1977) regarding circadian rhyth- micity of the heart rate in Procambarus clarkii. Page and Larimer ( 1976) demonstrated the existence of a photore- ceptor in the brain in the same species. In contrast with these findings are data by Glantz el a/. (1983). who intra- cellularly recorded the electrical activity of neurosecre- tory cells in the eyestalk induced by illumination of reti- nal fields. Fuentes-Pardo and Inclan-Rubio (1987) recently described the participation of the caudal photo- receptor in synchronizing the circadian locomotor rhythm in Procambarus hinivieri. Williams (1985) pro- posed, after his evaluation of the impact of optic tract section on the locomotor activity of the shore crab C'arci- nus maenas, the presence of a presumptive neural clock in the cerebral ganglion involved in regulation of loco- motor rhythm. In conclusion, we postulate that Aslacus leptoductylus exhibits an endogenous circadian hemolymph glucose rhythm entrained by the prevailing light/dark schedule. Neither the retinas nor the caudal photoreceptor repre- sent the main modulating receptors for blood glucose level and synthetic activity of the CHH cells. The present results indicate that the eyestalks possess the major re- ceptors for the entraining light stimulus and also contain the oscillatory center for the blood glucose rhythm. Acknowledgments The authors are grateful to Prof. Dr. J. M. Dcnuce and Dr. F. van Herp for discussing the manuscript. We thank the Illustration Services of the Faculty of Sciences for preparing the figures, and Mrs. E. Derksen for typing the manuscript. Literature C'ited Aschotf, .1. I960. l-xogcnous and endogenous components in circa- dian rhythms. In Hitiltwal Clocks Cold Spring Harb Symp. Quuni Hint 25: 1 I -28 ENTRAINMENT OF CHH-SYSTEM IN CRAYFISH 143 Fuentes-Pardo, B., and I. Inclan-Rubio. 1987. Caudal photoreceptors synchronize the circadian rhythms in crayfish. I. Synchronization of ERG and locomotor circadian rhythm. Camp. Biochem. Physiol. 86A: 523-527. Glantz, R. M., M. D. Kirk, and H. Arechiga. 1983. Light input to crustacean neurosecretory cells. Brain Res 265: 307-3 1 1 . Gorgels-Kallen, J. L. 1985. Appearance and mnervation of CHH- producing cells in the eyestalk of the crayfish Asiacus leptodactylus examined after tracing with Lucifer Yellow. Cell Tissue Res 240: 385-391. Gorgels-Kallen, J. L., and F. Van Herp. 1981. Localization of crusta- cean hyperglycemic hormone (CHH) in the X-organ sinus gland complex in the eyestalk of the crayfish Astacus leptodactylus (Nord- mann, 1842). 7. Morphol. 170: 347-355. Gorgels-Kallen, J. L., and C. E. M. Voorter. 1984. Secretory stages of individual CHH-producing cells in the eyestalk of the crayfish Astacus leptodactylus determined by means of immunocytochemis- try. Cell Tissue Res. 273: 291-298. Gorgels-Kallen, J. L., and C. E. M. Voorter. 1985. The secretory dy- namics of the CHH-producing cells in the eyestalk of the crayfish Astacus leplodactylus, in the course of the day/night cycle. Cell Tis- sueRes. 241:361-366. Gorgels-Kallen, J. L., F. Van Herp, and R. S. E. W. Leuven. 1982. A comparative immunocytochemical investigation of the crustacean hyperglycemic hormone (CHH) in the eyestalks of some decapod Crustacea. J. Morphol. 174: 161-168. Hamann, A. 1974. Die neuroendoknneSteuerungtagesrhythmischer Blutzuckerschwankungen durch die Smusdriise beim Flusskrebs. J. Com/7. Physiol. 89: 197-214. Jaros, P. P., and R. Keller. 1979. Immunocytochemical identifica- tion of hyperglycemic hormone-producing cells in the eyestalk of Carcinus maenas. Cell Tissue Res. 204: 379-385. Page, T. L., and J. L. Larimer. 1972. Entrainment of the circadian locomotor activity rhythms in crayfish. J. Comp. Physiol 78: 107- 120. Page, T. L., and J. L. Larimer. 1976. Extraretinal photoreception in entrainment of crustacean rhythms. Photochem. Photobiol. 23: 245-251. Pavlidis, T. 1973. Biological phenomena attributable to populations of oscillators. Pp. 159-186 in Biological Oscillators: Their Mathe- matical Analysis, T. Pavlidis, ed. Academic Press. New York. Pollard, T. G., and J. L. Larimer. 1977. Circadian rhythmicity of heart rate in the crayfish, Procambarus clarkii. J. Comp. Physiol. 57:221-226. Prosser, C. L. 1934. Action potential in the nervous system in the crayfish. II. Responses to illumination of the eye and caudal gan- glion. / Cell. Comp. Physiol. 4: 363-377. Reddy, C. S. D., M. Raghupathi, V. R. Pursushotham, and B. P. Naidu. 1981. Daily rhythms in levels of blood glucose and hepatopan- creatic glycogen in the freshwater field crab Oziotelphusa senex senex (Fabricius). Indian J. Exp. Biol. 19: 403-404. Sternberger, L. A. 1974. Immunocytochemistry. Prentice Hall, Inc., Englewood Cliffs, New Jersey. Van Herp, F., and H. J. M. Van Buggenum. 1979. Immunocyto- chemical localization of hyperglycemic hormone (HGH) in the neurosecretory system in the eyestalk of the crayfish Astacus lepto- dactylus. Experientia 35: 1 527- 1 528. Van Herp, F., A. Van Wormhoudt, W. A. J. Van Venrooy, and C. Bellon-Humbert. 1984. Immunocytochemical study of crusta- cean hyperglycemic hormone (CHH) in the eyestalks of the prawn Palaemon serranis (Pennant) and some other Palaemonidae, in re- lation to variations in the blood glucose level. J. Morphol. 182: 85- 94. Weibel, E. R. 1979. Stereological Methods. Practical Methods for Bi- ological Morphometry, Vol. I. Academic Press, London. 415 pp. Williams, J. A. 1985. Evaluation of optic tract section on the locomo- tor activity rhythm of the shore crab Carcinus maenas. Comp. Bio- chem. Physiol. 82A: 447-453. Reference: a/o/. Bull. 175: 144-153. (August, 1988) Electrophysiological and Histological Observations on the Eye of Adult, Female Diastylis rathkei (Crustacea, Malacostraca, Cumacea) V. B. MEYER-ROCHOW AND M. LINDSTROM Department of Biological Sciences, University of Waikato, Hamilton. \ewZealand, and Tvdrminne Zoological Station, University of Helsinki, SF-IOVOO Hanko, Finland Abstract. The approximately 200 ^m wide eye of Dias- tylis rathkei consists of two closely apposed eye halves with four lenticular complexes measuring 40 nm in di- ameter in each. Each lenticular complex consists of a lens rich in 30 nm electron-opaque glycogen-like particles made up of smaller (5-6 nm) subunits. and a rhahdom comprising regularly aligned microvilli. The retinula cell somata. which are in a proximal location, are linked with the distally placed rhabdom via approximately 10 ^m thick, cellular strands. The strands are surrounded by cells crowded with reflecting organelles of ca. 0.8 nm in diameter. Dark/light adaptational changes affect the position of uniformly spherical organelles measuring 0.4-0.5 Mm 'n diameter and presumed to contain carotenoids, the over- all size of the rhabdom. and the diameter of individual microvilli. The latter measure 75 nm in the light-adapted state and 90-120 nm in the dark-adapted state. There is ultrastructural evidence (swollen and abundant endo- plasmic reticulum and widely distributed glycogcn-like particles) that, under light-adapted conditions, the retin- ula cells are in a phase of intense metabolic activity. A multilamellar structure, similar in appearance to that found in the organ of Bellonci of other crustaceans, but also resembling a trophospongium, was noticed in close proximity to the eye within the optic lobe. Extra- cellular ek-' 'rophysiological recordings obtained with Nad-filled glass electrodes consisted of a cornea-nega- tive potential change and reached a maximum ampli- tude of nearly 400 ^V to 300 ms flashes of white light. Superimposed spectral response curves from eight different animals, based on a criterion amplitude of 50 Kcmved 2 February 1988; accepted 31 May 1988. /iV, were nearly congruent in shape and displayed one single sensitivity peak to light of 512-549 nm in wave- length. Intensity/response curves obtained to light of 472, 549, and 628 nm wavelengths and the single spectral sensitivity peak strongly suggest that only one type of ex- citatory visual pigment is involved in the visual process of D. rathkei. It is concluded that in spite of its tiny size, the eye of D. rathkei could be useful in the coordination of repro- duction and synchronization of vertical migrations. Introduction Cumaccans are an order of peracaridan crustaceans traditionally placed near the Isopoda (Siewing, 1956; Fryer, 1967). Certain species of Cumacea, including Di- astylis rathkei, regularly occur in the plankton, some- times hundreds of meters above the seabottom (Fricke, 1931; Forsman, 1 938 ). It is thought that the males, which have considerably larger eyes than the females (Zimmer. 1941 ). seek the latter in the open water at night during the mating season (Forsman, 1938). At certain times of the year, these small crustaceans can be very abundant (1214/m:: Kaestner, 1959) and represent an important component of the diet of various species of fishes; yet virtually all we know about the cu- maccan photorcceptor goes back almost 60 years to a study of D rathkei by Fricke ( 193 1 ), who asserted that ihc cumacean eye was a degenerated compound eye. Though in some species of cumaceans, e.g., NannastdCUS eii.\inicus, two clearly separated, lateral eyes are present (Bacescu, 1951), this view was challenged by Mayrat ( 1 9X I ), who claimed the cumacean eye was a dorsal ocel- lus. Arguments for and against either opinion were sum- 144 OBSERVATIONS ON D. R.4THKEI EYE 145 marized by Meyer-Rochow (1988) and supplemented with ultrastructural observations on the larval eye of D. rathkei. However, a detailed examination of the struc- ture and function of the eyes of the adults is still lacking. Since male and female D. rathkei differ in their behavior to a light source (Forsman, 1938; Zimmer, 1941) and the short-lived males are much less common than the fe- males, this paper is concerned with one form only: the female sex. Materials and Methods Collection and maintenance of organisms In early April, live, adult specimens of the cumacean Diastylis rathkei were dredged from the sandy bottom of the Baltic Sea southwest off the Danish island of Lange- land in approximately 40 m depth. The animals, all fe- males, were taken to the Zoological Institute of the Uni- versity of Kiel and kept in brackish water aquaria. Within a week, during which time histological prepara- tions of the D, rathkei eye were being made, 1 0 individu- als in a 2 liter thermos bottle were taken by one of us (V. B. Meyer-Rochow) to Finland in an airplane and subsequently housed at Tvarminne Zoological Station at 6°C in total darkness. Individuals were picked at random for both anatomical and physiological observations. Histology Eyes of daytime specimens as well as night specimens were carefully extirpated under a dissecting microscope at 10:00 h and 24:00 h, respectively. A paraformalde- hyde/glutaraldehyde mixture of 7.4 pH, buffered in Mil- lonig's phosphate and adjusted with 3 g d-glucose/100 ml for reasons of osmolarity matching Baltic Sea water, served as the initial fixative, in which the eyes stayed for about 40 h. They were then washed in buffer and post- fixed for 1.5 h in 2% osmiumtetroxide using the same buffer. The specimens were then embedded in Spurr's medium. One jum sections were stained with toluidine-blue for light microscopy. Gold or silver sections were stained with uranyl acetate and lead citrate for electron micros- copy. Four light-adapted and two dark-adapted speci- mens were examined. Electrophysiology Experimental procedures closely followed those re- ported by Lindstrom and Meyer-Rochow ( 1 987). During preparation, using infra-red image converters mounted on a Wild-5 stereo-microscope, each animal was illumi- nated by light that had passed through two Kodak Wrat- ten 87 gelatin filters and a heat filter. These were inserted in the ray path of white light coming from a 1 5 W micro- scope lamp. The incident light leaving the 3 mm wide tip of a light guide perpendicular to the eye, was centered around the hole through which the recording electrode was lowered some 40-50 ^m into the eye. The light spot made by the stimulating light flash covered the entire eye. The light output had been calculated in absolute units (qu -cirrus"1) by an Airam UVM-8LX luxmeter calibrated by Airam Laboratories for a wavelength of 564 nm(Donnerand Lindstrom, 1980). Based on these read- ings, we estimate the quantal amount of the flashes of light on the eye to elicit a response to have been 3.4 X 10'°qu-cm 2-s '. Measurements of light levels in the field at the site of capture are unavailable, but Lindstrom and Nilsson (1983) mention summer and autumn fig- ures of 1012 and 109 qu-cnT2-s~', respectively, for a depth of 100 m at "Norwegian northern latitudes." The stimulus time was usually set at 300 ms. Tips of glass electrodes drawn with an Ealing micro- electrode puller were cut off to an outer diameter of ap- prox. 10 ^m. The measuring electrode was filled with a 1 MNaCl solution and connected to a Tektronix 5031 dual beam storage oscilloscope. Setting-up procedures averaged no longer than 5-10 minutes. Thereafter the test animals were given 30 minutes of total darkness to recuperate from the operation. In the spectral sensitivity studies a criterion response of 50 uV was employed. All recordings were made in the AC-setting. Results Histology Gross anatomy. The eye consists of two symmetrical eye halves on a common forward-projecting ocellar lobe, separated from each other along the midline by a 1 ^m wide gap (Fig. 1 ). There are four bright red retinal com- plexes in each eye half, but facets or corneal lenses are not developed. The integument covering the eye is trans- parent and uniformly 5 ^m thick. Distally, each retinal complex possesses a lenticular structure (diameter ap- prox. 40 jim) and is in intimate contact with the micro- villi of a rhabdom that is produced by retinula cells. The large nuclei (Fig. 2) of these cells are located some 100- 1 50 fim more proximally near the center of the dome- shaped eye. Cytoplasmic strands of approx. 10 ^m diameter swerve in an arc through a massively developed layer of reflecting material and connect retinula cell bodies and rhabdom (Fig. 1 ). The rhabdom/lens interface is charac- terized by unclear cell boundaries that give the impres- sion that the two form a functional unit (Figs. 3, 4). The lens component of each such complex, contrary to Fricke (1931), appears to contain more than one nucleus (Fig. 3). Apart from the large, chromatin-rich nuclei on the proximal or lateral sides of the lens, each lens is made 146 \ B Ml VI R-R--•»'.••- - ••:5»;.,.; r- •>.-v*: ••••£.. v. '•;*-• •/.*••*'. |igv;l Figure I. Light micrograph of horizontal section through the eye of an adult female. The symmetrical eye halves joined along the midline are apparent and lenticular complexes (I ). retinulacell strands (S). and nuclei (N) are clearly visible. Scale = 20 /im. Figure 2. Electron micrograph ofproximally located retinulacell nucleus with surrounding clusters of electron-opaque glycogen particles and presumed camlinoid bodies (arrows). Scale 2 fjm. up of a dense aggregation of electron-opaque particles, measuring 30 nm in diameter (Figs. 3,4). These particles are composed of 40-50 smaller suhunits of approx. ft nm in si/e (Fig. 5). Cumacean retinula cells contain uniformly shaped 0.4-0.5 ^m large, spherical organelles (Fig. 3). These are identical in appearance and location with bodies identi- fied as carotinoid grains in the crustacean chromato- phore (J-lofsson and Hallherg. 1973). They are thought to he present in the photorcccptive cells of other peraca- OBSERVATIONS ON D. R.4THKEI EYE 147 ~ ' ., ^ * V~ l"U> li-'Kff-\f.r •*.r part of the organ of Bellonci' (Renaud-Mor- i . 1977). However, because of its proximity to glial '.vlls. it probably represents a trophospongium (Scharrer. 1964: Eguchi and Meyer-Rochow. 1983). No further observations on it or the higher visual centers were made. Dark/light adaptational changes. Fricke (1931) ob- scrv ed that the yellow pigment inside the retinula cells of Cuma rathkei was not stationary but had the ability to migrate towards or away (= up and down) from the len- ticular apparatus. A dark-adapted eye. in which most of the carotenoid pigment is withdrawn and only the whit- ish reflecting organelles remain, changes within 4 min- utes into the light-adapted condition upon exposure to daylight (Fricke, 1931). Ultrastructurally, at night the eye (Fig. 9) had considerably more voluminous rhab- doms than during the day (Fig. 8) and the diameters of the individual microvilli in the "night eye" were signifi- cantly enlarged over those of the "day eye" (i.e.. 0.09- 0. 1 25 ^m versus 0.075 j/m). A centrally placed cytoskele- tal rod was also more obvious within the dark-adapted microvilli. An actual change in shape of the microvillar transverse profile upon dark and light adaptation, as claimed by Yoshida and Kaga ( 1 983), was not seen. The border between rhabdom and cytoplasm in the light adapted eye, however, gave the impression of greater jaggedness in comparison to the dark adapted condition. Also there were consistently more tiny ribo- some-like dark particles and Golgi bodies in the cyto- plasm of the light adapted eye (Fig. 8), and the endoplas- mic reticulum was more obvious and more widely dis- tributed. Longitudinally arranged microtubules, along u Inch glycogen-like particles from storage areas near the proximally located retinula cell nuclei could possibly be transported and carotenoid pigment bodies could slide, were identified in both the dark and light adapted eyes (see discussion on transport mechanisms in the crusta- cean cell: Frixione el ai. 1979; Rao and Fingerman, 19X3). However, large, empty cisternae and a lack of multi-M.-sicular and Golgi bodies were predominantly confined to the dark adapted cells. No obvious difference with regard to rise and abundance of mitochondria be- tween dark and light adapted states was seen, but occa- sionally tinv vesicles (50 nm diameter) budded off the cristae in light adapted mitochondria. Electrophysiology Results were obtained from eight often animals tested. Responses were generally small and rarely reached the maximum amplitude of 360 pV, recorded in one animal to the highest available flash of white light. The ERGs were typical cornea-negative responses of the slow type with little or no positive component (Fig. 10) similar to the responses of the isopod Porcellio loen ;s (Benguerrah and Carricaburu. 1976). The spectral response curves (Fig. 1 1 ) had but one smooth sensitivity peak in the vicinity of 512-549 nm. Responses to long ultraviolet radiation were down by one sensitivity log unit from the maximum, whereas to light of wavelengths greater than 600 nm the drop was even steeper. The curves of the 8 successfully tested ani- mals were remarkably congruent and fitted a Dartnell nomogramme of a rhodopsin visual pigment with Xma, at 530 nm. Intensity/response curves to light flashes of 472 nm, 549 nm, and 628 nm were, of course, horizontally shifted and had different maximal amplitudes. They shared more or less the same slope over the linear part of the curve with only the 628 nm curve being slightly less steep (Fig. 12). The fact that there was not even an obvious difference to the V/logI curve obtained by using flashes of white light is interpreted as evidence that there is only a single excitatory visual pigment present in the photore- ceptor of D. rathkei and that the ERG-recordings cor- rectly reflect the eyes' spectral capacity in the scotopic state. Discussion There is nothing about the overall anatomy or ultra- structure of the eye of D rathkei that would preclude it from being functional photoreceptors. Absolute sensitiv- ity is high and spectral sensitivity appears to match the downwelling spectrum of the prevalent light. In fact, the presence of internal lenses of a diameter close to 40 ^m and a rhabdom only 20 ^m away from the center of the lens would result in an F-number of only 0.5, indicative of considerable light-gathering power in each lenticular complex. We know little about the optical quality or properties of the lens, or whether a radial gradient of re- fractive index exists as, for example, in the eye of the aquatic beetle Cyhisler (Meyer-Rochow, 1973). that could produce a focus in the distal end of the rhabdom. However, even if the lenticular refractive index only "lii'gl el was uher tiein des Seewassers" (Fricke, 1931), each retinular complex in the eye of/), rathkei obtains a further boost in photon-capture from the all-abundant reflecting organelles. Similarities in lenticular ultrastructure between D nii/ikci and other arthropods exist. The tiny, compact clusters of electron-opaque particles making up the bulk of the lens or crystalline cones have repeatedly been claimed to represent glycogcn material (Perrelct, 1970; Eakin and Kuda, 1971; Elofsson and Odselius, 1975). OBSERVATIONS ON D R.4THKEI EYE 149 KlL - • Figure 6. Inward-projecting narrow elongations of hypodermal cells (N), under the cuticle (C), separate the two eye halves, whose major components are cells that are crowded with circular vesicles which are thought to be reflecting in nature, to isolate individual retinular (Rh) complexes, and thus to improve photon capture. Scale = 2 ^m. Figure 7. A short distance behind the eye, this multi-tubular structure, at first glance resembling a rhabdom and probably identical to what Stahl (1938) interpreted as the "X-organ" in the eye-bladder of D ralhkei, was located. Scale = 2 nm. The granular fine structure in the center of the crystalline croscope sections is obviously made of material that is cone in the eye of the mantis Ciulfina resembles that of richer in protein than the surrounding tissue." Be this as the D. rathkei lens, but Horridge and Duelli ( 1 979) state it may, there seems little controversy about the chemical that "the crystalline cone in histological and electron mi- nature of the intracellular 'islands' of granular inclusions 150 V B MEVER-ROCHOW AND M LINDSTROM " * •'* V fe ' - " *flw/ - .* W? > • ' ' -'fcr •.,.,•••* - A ilK * ' X • 3' <> *•*. • • -f • • • •».•* .. - • v. v .-. y >•'-,*; -w*-- • " f f .^ '• n, • • '^' * **- • . • .' * • . ...'.-> • • - - .*«*, •;•*•» • - >" ' "I-. -'ii / U "* -*^ ' v '"IV -L . f*y /s « , ' to . ^L. •-•^•"~*- ~^^— i *- » Figure H. In the lighl adapted condition the total rhahdom volume is reduced, carotcnoid organelles migrate towards the rhahdom edge, and microvilli are 75 nm in diameter. Scale I ^m. Figure 9. In the dark adapted e\e the rhahdom volume is enlarged, electron-empty cisternae instead of carotenoid bodies are more numerous around the edge of the rhahdom. and microvillus diameters have become noticeably wider and more variable. Scale • 1 nm. near the retinula cell nuclei, for they are identical to those of several species of isopods, in which specific glycogcn reported from the eye of the tick Ainhlyonuna cinuri- tests revealed their true character (Martin, 1976). The canum (111 and Cromroy, 1977) and intracerebral ocelli great abundance of tiny black particles in virtually the OBSERVATIONS ON D R.1THKE1 EYE 151 1 to o 3 a * w at « a a a n\ and /' lemorala (Crust. i ..I. n ( dinnti'tiiai Hiol 41: 1-17. Donner. k. O ' mdstrom. 198(1. Sensitivity to light and circa- dian activn;. t,-i,i al/inii (Crustacea, Amphipoda). Ami /out h'enn 17: Kakin, R. M., and A K ., 1972. Glycogen in lens of tunicate tad- pole. J Kxp /.mil l«d ihi. K.. and \. B. M.-K-, ,,,». 198.1. I rophospongium-likc structures in an insi-tt iscol rctmula cells of l'ili<> \u r/iu.s(Lcpidoptcra) to irradiatmn with ultr;m..k-t h|>ht ( ell I i\\m- Res 231:519-526. Hofssnn. R., and K. llallhtTK. 1973. Correlation of ultrastructureand chemical composition of crustacean chromatophore pigment. J. I'l- trastruc. Res. 44: 42 1-429. Klofsson. R., and R. Odselius. 1975. The anostracan rhahdom and the basement membrane — an ultrastructural study of the Anemia compound eye. Ada J.ool 56: 141-153. Ktequoz, V., A. Stadelmann. and M. Tsacopoulos. 1983. The effect of light on glycogen turnover in the retina of the intact honeybee drone (Apis mellifera). J. Comp. Physiol 150: 69-75. Korsman, B. 1938. Untersuchungen liber die Cumaceeen des Skager- aks. /Mil. Bid L'pp 18: 1-162. Forward. R. B., and I. \V. C'ronin. 1979. Spectral sensitivity of larvae from intertidal crustaceans. J Comp .Physio!. 133:311-315. Kricke. M. 1931. Die Komplexaugen von Diastylis rathkei. Zoo/. Jahrh Ahi Anal Onlogenie Tien- 53: 701-724. Krixione, K., II. Arcchiga, and V. Tsutsumi. 1979. Photomechanical migrations of pigment granules along the retmula cells of the cray- fish. J. Neurohioi 10: 573-590. Kryer, (j. 1967. IV — Studies on the functional morphology and feed- ing mechanism of Monndclla urgentum Stella (Crustacea: Ther- mosbaenaceae). Trans. R Sue. Edmh 66: 49-90. (ioldsmith, T. H. 1978. The spectral absorption of crayfish rhab- doms: pigment, photo-product, and pH-sensitivity. I 'is/on Res. 18: 463-473. Hallberg, K. 1977. The fine structure of the compound eyes of mysids (Crustacea: Mysidacea). Cell TI.\MIC Res 184: 45-65. I l.illlu iii. K., M. Andersson, and D.-K. Nilsson. 1980. Responses of the screening pigments in the compound eye of ,\i'i>mysis integer (Crustacea: Mysidacea). J K\p /.<><>1 212:397-402. Hallbcrg, E., H. L. Nilsson, and R. Klofsson. 1980. Classification of amphipod compound eyes — the fine structure of the ommatidial units (Crustacea. 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OBSERVATIONS ON D R.4THKEI EYE 153 Meyer- Rochow, V. B. 1973. The dioptric system of the eye ofCybis- ter (Dytiscidae: Coleoptera). Proc. R Soc. Land. 183B: 159-178. Meyer-Rochow, V. B. 1985. A study of unusual intracellular organ- elles and ultrastructural organisation of the eye ofGammarits ocea- wn«(Segerstrale 1 947) fixed in the midnight sun of the Spitsbergen (Svalbard) summer. Bwmed. Res. 6: 353-365. Meyer-Rochow, V. B., and E. Eguchi. 1984. The effects of tempera- ture and light on particles associated with crayfish visual mem- brane: a freeze-fracture analysis and electrophysiological study. J. Neurocytol. 13: 935-959. Meyer-Rochow, V. B., and G. A. Horridge. 1975. The eye ofAnoplog- nathus( Coleoptera, Scarabaeidae). Proc. R Soc. Loud 188B: 1-30. Michel, A., and F. Anders. 1954. Uber die Pigmente in Auge von Gammarus pule.\ Naturwissenschqften 41:72. Nassel, D. R., and T. H. Waterman. 1979. 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G. 1970. Light control of the reproductive cycle of Pontoporeia affims Lindstrom (Crustacea, Amphipoda). / Exp Mar. Biol. Ecol. 5: 272-275. Slewing, R. 1956. Morphologische Untersuchungen an Cumaceen (Cumopsis goodsiri v. Beneden). Zool. Jahrb. Anal. Onlogeme Tiere 72: 522-559. Stahl, F. 1938. Preliminary report on the colour changes and the incretory organs in the heads of some crustaceans. Ark. Zool. 30B: 1-3. Straiten, W. P., and T. E. Ogden. 1971. Spectral sensitivity of the barnacle eye, Balanus amphilrite. J Gen Physiol 57: 435-447. Yoshida, M., and K.-I. Kaga. 1983. Photoinduced ultrastructural changes in microvilli of a cumacea Dimorphostylis asiatica. Zool. Mag. 92: 529. Zimrner, C. 1941. Cumacea. Pp. 1-222 in Bronn's Klassen & Ord- nungen des Tierreichs, Vol. 5(4). Akad. Verlagsges, Leipzig. Reference: Bio! Bull 175: 154-166 i August, 1988) Inorganic Aspects of the Blood Chemistry of Ascidians. Ionic Composition, and Ti, V, and Fe in the Blood Plasma of Pyura chilensis and Ascidia dispar DOMINGO A. ROMAN, JUSTA MOLINA, AND LIDIA RIVERA Departamento de Quimica, Facultad de Ciencias Bdsicas, Campus Coloso, Universidad de Antofagasta, Casilla 1240 Antofagasta, Chile Abstract. Iron, titanium, and vanadium analysis were performed on the tunicates Pyura chilensis Molina. 1 782. and Ascidia dispar, and the inorganic chemistry of blood was investigated. The major ionic characterization of the blood plasma and cytosolic solutions were deter- mined. Gel chromatography was used to secure informa- tion on the possible existence of metal organic com- plexes. I'vura chilensis accumulates Fe and Ti, and Ascidia dispar accumulates Fe, Ti, and V in blood cells in this quantitative order. Significant levels of metals are associ- ated with cell residues (membrane cells), although this may be, to some extent, dependent on the cell lysis tech- nique. The elution behavior of plasma in Sephadex G-75 and LH-20 gels and the respective absorption spectra of the fractions showed evidence of organic metal complexes in the plasma of both tunicate species. Introduction For years tunicates have piqued the curiosity of biolo- gists because of their unusual physiological peculiarities and because they may have given rise to the vertebrates (Bcrril. I v^5). Among the physiological peculiarities that distinguish these organisms from others are the follow- ing: (i) They need a low tension of oxygen (Goodbody, 1 974). To date, no reversible binding of oxygen has been detected nor the unequivocal existence of a proteic O: transport compound that transports O: through the blood (Macara el al.. !979a; Agudelo el a/., 1982). (ii) They are entirely ammonotelic in their protein metabo- Reccivcd 6 July 1987; accepted 20 May 1988. lism, but are uricotelic with respect to nucleic acid me- tabolism. Therefore, they differ from most invertebrates that are wholly ammonotelic, accumulating uric acid and purines in nephrocyte vacuoles (Goodbody, 1974; Wright, 1 98 1 ). The functional importance of this storage remains obscure, (iii) They are capable of humoral and cellular immunological responses (Wright. 1 98 1 ) and are rich in bio-active substances (Roman. 1986). (iv) They accumulate metal ions. With respect to metal ions, tunicates are known for the uptake of selected metals from seawater and for accumu- lating them in their blood (Carlisle, 1968; Swinehart el a/., 1974; Senozan, 1974; Biggs and Swinehart, 1976). Members of the order Enterogona can accumulate vana- dium (Kustin et at.. 1975; Kustin and McLeod, 1977; Macara el a!.. 1979b; Biggs and Swinehart. 1979; Botte el al.. 1979; Dingley el a/., 1981; Hori and Michibata. 1981;Rowley. 1982; Dingley elal.. 1982). However, the type of coordination compound(s) in which the metal is involved in the blood is unknown (Carlson, 1975; Tul- lius el al., 1980; Dingley et al., 1982; Hawkins el al.. 1983a; Bruening et al.. 1985; Frank el al.. 1986). Mem- bers of the order Pleurogona. sub-order Stolidobranchi- ata, accumulate iron (Endean. 1955a, b, c; Agudelo et al.. 1982; Agudelo et al.. 1983a. b: Agudelo et al.. 1985). but nothing is known about its function in blood cells (Hawkins et al.. 1983H). In plasma, iron is associated with transferrin-like metalloproteins (Martin el al.. 1984; Finch and Huebers, 1986). Hawkins el al. ( I983c) proposed that ascidian taxon- omy reflects a separation into vanadium- and iron-con- taining species. Tunicates accumulate other metals be- sides vanadium and iron (Monniot. 1978; Macara et al.. 1979c; Agudelo et al.. 1981; Rowley, 1982), which may 154 INORGANIC ASPECTS OF THE BLOOD CHEMISTRY OF ASCIDIANS 155 not be essential elements subjected to selective accumu- lation mechanisms. Sessile filter feeding animals are very sensitive to their immediate environment, and signifi- cant amounts of contaminating metallic elements could be taken up by ascidians (Papadopoulou and Kanias, 1977). In processes in which metals are accumulated in blood cells, it is logical that metals make a transient or perma- nent appearance in blood plasma. Once metals gain ac- cess to the body interior, they must be appropriately dis- tributed, but because of its hydrolysis property some of these metals cannot be held in solution, in the interior media, without some mechanism to prevent its precipi- tation. No metalloproteins such as hemocyanin have been re- ported in ascidian blood plasma. However, Hawkins et al (1980a) and Webb and Chrystal (1981) studied the metal binding properties — including spectral character- ization and metal contents — of some tunicates ( Hawkins et al.. 1980b). They found preliminary evidence of metal complexing. This was confirmed by Martin et al. (1984) in the plasma of Pyura stolonifera, by demonstrating an iron-binding protein of about 40,000 daltons molecular weight with one iron-binding site considered as one Py- ura transferrin (Finch and Huebers, 1986). In this work, the Ti, V, and Fe contents were deter- mined in several tissues. Also, the major characterization and chromatographic elutive behavior on Sephadex G- 75 and LH-20 gels of Pyura chilensis Molina, 1782, and Ascidia dispar blood plasma were examined. These are two phylogenetically diverse ascidians. Materials and Methods Chemicals were from Merck. 3,3'-dimethylnaphthi- dine was from Eastman organic chemicals and ophenan- throline hydrochloride was from Riedel-De Haen. Seph- adex G-75, LH-20 gels and blue dextran 2000 were from Pharmacia Fine Chemicals. Deionized water was pre- pared from distilled water passed through a disposable demineralizer cartridge (Corning 3508-B). Specimens of P. chilensis and A. dispar were collected at Bahia Mejillones del Sur (Antofagasta-Chile) from marine pools, in which they were found as encrusting fouling organisms. P. chilensis afixes itself to ropes while A. dispar attaches itself to painted floating metallic bar- rels where they coexist with hydrozoans and bryozoans. Before drawing blood, specimens were maintained for some time in seawater at room temperature, and then were gently squeezed to remove most of the seawater. Blood samples of both species were obtained by cut- ting the base of the body. Blood cells were removed from the plasma by centrifuging (2500 rpm; 10 min). Plasma was kept at 4-5°C while carried to the laboratory and was used as soon as possible. Cellular residues presumably consisted of cell mem- branes. No distinction was made between cell surface and intracellular membranes. Cell samples were rinsed with seawater and then subjected to two different cell ly- sis processes. In the first procedure, cells were subjected to three freeze-thaw cycles in deionized water media ( 1 .4 parts of triturated ice + 2 parts of CaCl2 X 6H2O freeze/ room temperature), gently squeezed with a cell teflon ho- mogenizer, and then centrifugated at 8000 rpm. In the second procedure cells were subjected twofold to an ex- cess of methanolic solution of 0.75% HC1 (Hawkins, pers. comm.) and centrifuged at 8000 rpm. In both cases the cytosolic solution and methanolic extract were made up to the original volume from which the cells were ob- tained. Whole blood samples of P. chilensis were sub- jected to the first cell lysis procedure, but without deion- ized water. A Sorvall refrigerated centrifuge was used. Metal analysis Prior to the Ti, V, and Fe determinations in specimens and tissues, a qualitative analysis was performed on di- gested blood cells. Cells rinsed with microfiltered seawa- ter were digested with binary HNO3/HC1O4 acid system (Jones et al.. 1982), performing assays for Cu, Mn, Fe, Ni, Co, Ti, V, and Nb (Feigl and Anger, 1972). Mn and Fe were also subjected to semi-quantitative assays with Merkoquant sticks. Pyura chilensis and Ascidia dispar were analyzed indi- vidually. Tissues including blood were obtained from 10-20 specimens of P. chilensis and 30-50 specimens of A. dispar. Bodies were separated from tunics and rinsed with filtered seawater. Tunics were gently scrubbed with a plastic brush to remove dirt and rinsed in a similar manner. Siphons and tunics were cut off with a hard acrylic knife. Specimens and tissues, including some samples of plasma, cells, and cellular residues, were then dried at 1 10°C to constant weight, digested with a binary acid procedure (Jones et al.. 1982), and then treated ac- cording to the respective metal analysis. In tissues, iron was determined with 1 , 10-phenanthro- line (Sandell, 1959; Fries, 1972), and Ti and V were sepa- rated (Korkisch, 1969; Fukasawa and Yamane, 1977) prior to their determinations. Titanium was determined according to Qureshi et al. (1968), and vanadium using the methods of Bannard and Burton (1968) and Fuka- sawa and Yamane (1977). In the fractions, iron was de- termined using 2,4,6-tri-2 pyridyl-l,3,5-triazine (Collins et al., 1959; Box, 1981), and vanadium and titanium as above, without separating them after digestion of the fractions with a binary HNOj/HClCX acid system (Jones etai, 1982). Blank controls were used in every metal analysis, and except in the fractions, all the determinations were per- formed in triplicate. 156 D A. ROMAN ET AL Determination of the major ionic composition and relative reduced feature of the fluids Chlorinity and salinity \\ere determined conductimet- rically with respect to standard seawater at 25°C (con- ductimeter RaJi. >uieter COM 2e. with a standard cell CDC 104) Chloride was determined by Mohr titration and sulphate '\v direct titration with barium perchlorate using Thonn as indicator. Subsequently, cations were re- moved by passing the sample through a strong acid cat- ion exchange resin column (Fritz and Yamamura, 1955 ). except in seawater in which case sulphate was de- termined gravimetrically as BaSO4. Successive determi- nation of calcium and magnesium were made by poten- tiometric titration with a calcium ion selective electrode (Roman el al, 1982); Na, K, and Li analysis were per- formed by flame emission spectrophotometry on a Radi- ometer FLM-3; pH measurements were made potentio- matrically on a Radiometer pH Meter 26 with glass membrane electrode. All major component determina- tions were made in triplicate. The relative reduced feature of the plasma and cyto- solic solutions were tested by two redox potentiometric titrations (non-standard biochemical methods). In the first, aliquot samples (10-20 ml) in polypropylene vessel were put into a Radiometer TTA-80 titration assembly, acidified with 0.75% HC1. and then titrated with a stan- dard solution 0.1 A'K.MnO4. In the second, aliquot sam- ples (10-20 ml) were acidified with 2 ml of concentrated HC1O4, treated with 5 ml of a standard solution 0.1 N K:Cr:O-, and then titrated with a standard solution of " Chromatographicfractionation of blood plasma Fractionations were performed on Sephadex G-75 and LH-20 gels, in thermostated chromatography columns (Pharmacia Fine Chemicals K.26/40) loaded with 4 g of Sephadex G-75 and 13 g of Sephadex LH-20, respec- tively. The column temperature was 20°C, but all sam- ples and eluants were cooled at 4-5°C. Plasma samples were concentrated by freeze-dry (Freezer-dryer-5 Lab- conco), five-fold for P. chilensis and two-fold for .4. dis- par before running the chromatography procedures. The void volume of the column (V()) was determined using blue dextran-2000, and the bed volume (V,) was calcu- lated according to the height and diameter of the gel column. In G-75 chromatography the sample volumes were 6 and 10 ml for .1 . narand P. chilensis; the eluants were 0.01 A/NaClam; icetic acid, respectively, cooled and dcaereated, collecting fractions of 10 ml (plasma of A. dispar) and 6 ml (plasma of P. chilensis). The absor- bance at 278, 288, 310, 37!, 454, and 675 nm (plasma ot'/J chilensis). 265 and 322 nm (plasma of .1 dispar), and the metal analysis in all fractions were monitored with respect to eluant solutions, previously passed through the respective column, as reference or blank, re- specti\d\ In LH-20 chromatography, to minimize inhomogene- ities in the column the gel was packed after swelling in deaereated methanol. One bed volume column of each of the following eluants: water, 25, 50 and 75% methanol in water (v/v), were then passed through the column, fol- lowed by 99.8% methanol, collecting two 10 ml fractions per eluant for use as reference or blank solutions. The column was then loaded with the sample ( 10 ml). Chro- matography was performed using 1 .5 bed volume of each cooled deaereated methanol/water gradient from 0 to 99.8% methanol according to Macara et al. ( 1979b), col- lecting 6 ml (plasma chromatography of P. chilensis) and 10 ml (plasma chromatography of A. dispar) fractions. Absorbance at 272, 288, 3 1 0. 320, 375, 454, and 675 nm (plasma of P. chilensis), 266, 280, 326 and 660 nm (plasma of A. dispar), and metal analysis in all fractions were monitored. Ultra-violet and visible spectra were re- corded for whole plasma and the peak-fractions from the eluting patterns, employing a Beckman 35 spectropho- tometer. All other absorciometric measurements were also made using this instrument. Results Metal analysis Metal concentrations found on specimens and various tissues of tunicates are listed as mg/Kg dry weight (Table I). Concentrations for plasma are given in mg/1. Ni. Co. Mn, and Nb were not detected in blood cells. Higher con- centrations of iron and titanium, and iron, titanium, and vanadium were found in /'. chilensixandA. dispar blood cells, respectively. Although V was not detected in P. chi- lensis blood cells and was found in A. dispar blood cells, only trace levels of it were found in both blood plasmas. Ti was not detected in P. chilensis blood plasma. Results of the metal analysis in cell lysate (cytosolic solution), calculated by the difference between the metal contents in whole blood cells and in blood cell residues, are tabulated as percentage of metals in Tables Ha, b, c, respectively. Here, the cellular residues were not washed with acid prior to analysis. Aqueous and 0.75% HC1/ methanol cell lysis procedures were considered (P. chi- lensis). These results show that iron content in cell resi- dues from aqueous and HCl/methanol cell lysis proce- dures are low and comparable, but the titanium content, surprisingly, was higher and greater in the cellular resi- dues than in the cytosolic solution for both cell lysis pro- cedures, but higher in cell residues from HCl/methanol lysis method. Therefore, it is possible that metallic pre- cipitation by extensive hydrolysis (Agudeloc/ at.. 1983b. INORGANIC ASPECTS OF THE BLOOD CHEMISTRY OF ASCIDIANS Table I Relative distribution ofFe. Ti, and I' contents (mg/kg dry) in Pyura chilensis and Ascidia dispar 157 P. chilensis A. dispar Fe Ti V Fe Ti V Specimen 191.8 n.d. n.d. 94.1 107.2 25.7 Body (without tunic) 84.3 n.d. n.d. 717.4 53.1 25.4 Siphons 70.7 n.d. n.d. 86.4 16.2 34.8 Tunic 243.9 n.d. 1.5 74.3 125.6 0.4 Blood plasma 45.0 n.d. 1.9 93.7 61.4 22.0 a(1.5 n.d. 0.06 2.9 2.1 0.8) Blood cells 1,105.4 277.8 n.d. 2.181.5 1,552.5 692.9 Blood cell residues6 7.4 132.8 n.d. 586.4 784.1 163.7 Blood cell residues' 17.3 258.9 n.d. d d d J mg/l; bfrom lysed cell preparations produced by subjecting the cell samples to several freeze-thaw cycles in deionized water media and then centrifuging them at 8000 rpm; cfrom lysed cell preparations produced by subjecting the cell samples to treatment with methanolic solution of 0.75% HC1 centnfugmg them at 8000 rpm; dnot determined; n.d. = not detected. 1985) may have been minimized during water cell lysis in the conditions of this work. Thus, it appears that more attention should be focused on tunicate blood cell lysis procedures. Table Ha Relative iron distribution in blood cells as determined in pooled samples Cytosolicd-e Cell Species solution residues P. chilensis 99.3% 0.7% P. chilensis 98.4%f 1.6%' A. dispar 73.1% 26.9% Table lib Relative titanium distribution in blood cells as determined in pooled samples P chilensis P chilensis A, dispar 52.2% 6.8%r 49.5% 47.8% 93.2%f 50.5% Table He Relative vanadium distribution in blood cells as determined in pooled samples A. dispar 23.6°. d Calculated by difference between contents in whole blood cells and in blood cells residues (Table I); cin respect to lysed cells preparations produced by subjecting the cell samples to several freeze-thaw cycles in deionized water media/centrifuging them at 8000 rpm; fwith respect to lysed cell preparations produced by subjecting the cell samples to treatment with a methanolic solution of 0.75% HCl/centrifuging them at 8000 rpm. Ion composition and reduced tendency of blood fluids The pH and ionic composition of plasma, lysed whole blood, and cytosolic solutions, for both species, are shown in Tables III and IV. The sulphate content in A. dispar plasma was greater than in P. chilensis, but both contents were lower than in seawater. In the cytosolic solutions, e.g.. aqueous intracellular media, the concen- trations of sulphate were low with respect to the plasma. Calcium and magnesium contents in P. chilensis plasma are higher than in A. dispar. In P. chilensis some enrich- ment occurred with respect to seawater, which also oc- curs for sodium and potassium. Calcium, magnesium, sodium, and potassium contents also were lower in whole lysed blood than in plasma (P. chilensis). In P. chi- lensis blood cells, the sodium concentration in the cyto- solic solution is only 50% of the A. dispar cytosolic solu- tion. However, potassium concentration is very low. The pH of the whole lysed blood (P. chilensis) was nearly alkaline, the salinity almost equal to the seawater from which the specimens were obtained. The sulphate concentration was only 62% of its concentration in blood plasma. Plasmas, 0.75% HCl/methanolic extracts from blood cells, and cytosolic solutions had reducing tendency in both species in respect to dichromate and permanganate, respectively. Spectral-separative chromatographic behavior of iron, titanium, and vanadium in plasma P chilensis plasma is pink-orange and A. dispar plasma is greenish-yellow. Figure 1 shows the UV-visible spectra of both species' plasma. The bands 265-290, 300-330, and 675 nm regions were common to both 158 D. A. ROMAN / / i/ l.il.K III Ionic composing'. l'\ ura chilensis and Ascidia dispar Plasma P. chilensis Plasma A. dispar Surface coastal seawater' Chlonnit\ 19.38 18.64 19.44 Salinity ":- 32.35 33.67 35.13 19.86 19.05 19.51 so; (g/i) 0.60 0.79 2.60 Ca:'u 0.51 0.34 0.44 Mg:' (g/l) 1.54 1.14 1.33 B/l) 15.68 9.34 1 1.16 K*(g/l) 0.78 0.42 0.40 LJ- (mg/1) 0.87 0.84 1.32 pH 6.77 6.48 8.03 Na/K 0.33 0.30 0.23 Ca/Mg 20.10 22.20 27.90 ' Surface coastal seawater of Bahia Mejillones del Sur. spectra, with a light bathochromic effect in the U V bands of A. dispar plasma with respect to the P. chilensis UV spectrum of plasma, which also shows a shoulder in the 280-290 nm zone. The elution patterns detected at 265 nm for. 4. dispar and at 310 nm for P. chilensis are given in Figure 2a. None of the P. chilensis fractions were colored, but frac- tions 9-1 1 were yellowish in A. dispar plasma chroma- tography. The elution profiles of iron and vanadium for Table IV limiL'ciinipnsiiiim <>t l\\i'd whole blood and cytosolic solutions nt Pyura chilensis and Ascidia dispar" Lysed whole blood of P. chilensis* Cytosolic solution of P. chilensis' Cytosolic solution of .•I. dispar' Chlorinity %c 19.74 n.m. n.m. Salinity %o 35.66 n.m. n.m. f 1 (g/l) 19.57 n.m. n.m. so; (g/D 0.37 III! '0 0.051 ' a ig/1) 0.30 n.d. n.m. Mg2" (g/l) 1.03 o.d. n.m. Na Ig/l) 9.59 0.051 0.104 K'lmg/l) 4X4.90 11.20 0.30 1 i img/l) 0.94 n.d. 0.02 pH 7.82 7.01 7.36 Na K 0.29 4.6 346.7 Ca/Mg 19.8 — — ' Analysis on lysed whole blood of A. dispar were not made due to lack of samples; hfrom subjecting the samples of blood to several Imvc thav. cycles and then centrifuging at 8000 rpm; 'from subjecting the cell samples to several freeze-thaw cycles w ith deiomzcd water and then centrifuging them at 8000 rpm; n.m. = not measured: n.d. = not de- tected. 200 350 240 450 280 550 WAVELEMOTH IN fll 320 650 360 750 Figure 1 . Ultraviolet-visible spectra of blood plasma ot'.-l \cidia dis- par (concentrated twofold by freeze-dry and acidified at pH 3 with ace- tic acid, that also was the reference solution AL v • • •; fresh, water as reference AUV.VIS )• ar>d I'yura chilensis (fresh, water as reference B, \ Ms solid line). Cell pathlength 1 cm. Dilution shown were appli- cable. P. chilensis, and iron, titanium, and vanadium for A. dis- par are also presented in Figure 2b. In both species pat- terns, two peaks were obtained with respect to absor- bance, each one in fractions 3, 4; 6-9 (A. dispar). and 5. 6; 10-12 (P. chilensis). The first band eluted was in the void volume of the column (V0 = 30 ml) and should have contained compounds with greater molecular weight or at least comparable to the upper exclusion limit of the G- 75 column bed. The second band eluted was at a greater volume than V,(VC = 6 1 and 67 ml for P. chilensis and A. dispar plasmas, respectively) and should have contained compounds with less molecular weight or comparable to the lowest exclusion limit of the G-75 column bed. This also should be valid for the yellow fractions (9- 1 1 ) from A. dispar plasma chromatography. The absorbance pro- file at 322 nm showed equal characteristics for. 4. dispar, and the same occurred with the profiles at 278, 288, 375. 454, and 675 nm for I' chilensis. Four peaks were obtained for P. chilensis with respect to the iron content in fractions, whose elution volumes ( Vc) were 1 3, 25, 55, and 67 ml. The second peak had the same values of the chromatographic behavior parameter (VC/V0, Vr/V,, KaJ of the first band in function of absor- bance at 3 10 nm, and so on. These fractions (5, 6) should have contained iron compounds of high molecular weight, found for the first iron band. The other peaks should correspond to iron compounds of low molecular weight. Vanadium was also eluted after the bed volume. INORGANIC ASPECTS OF THE BLOOD CHEMISTRY OF ASCIDIANS 159 3.6 7121 61 FRACTION Figure 2a. Elution patterns of blood plasma of Ascidia dispar from Sephadex G-75 chromatography at 270 nm (6 ml concentrated twofold by freeze-drying, 6 ml fractions. A dash line), and Pyura chilensis at 310 nm (10 ml concentrated fivefold by freeze-drying, 6 ml fractions, B solid line). 10 FRACTION 15 20 Figure 2b. Elution patterns of metal contents per fraction from Sephadex G-75 chromatography: in plasmas of Pyitra chilensis (iron E solid line: vanadium F~), and Ascidia dispar (iron G open circles; titanium H : vanadium I closed circles). Conditions, samples, and fraction volumes are of Figure 2a. Four bands were also obtained for A. dispar with re- spect to iron content in fractions (Vc = 57, 87, 107, and 137 ml). None had the same values of the chromato- graphic parameters of the bands in function of absor- bance at 265 nm. The four Ve values are greater than the V,, therefore they should not contain iron compounds of high molecular weight. However, for titanium (three bands, Ve = 37, 87, and 1 17 ml) the first peak is super- posed and similar in the profile at 265 nm, which should mean that it corresponds to titanium compounds with a high molecular weight. The other bands are after the bed volume. The eluted vanadium show increasing contents after fraction 1 0, for which only two bands were consid- ered (Ve = 67 and 87 ml), both after the bed volume, where the first is superposed with the second peak at 265 nm. The elution profiles for P. chilensis and A. dispar blood plasma chromatography on Sephadex LH-20, em- ploying methanol/water gradient as eluants are given in Figures 3 and 4. None of the fractions were colored. At 272 nm, two major bands and one shoulder were ob- tained for/3, chilensis, each in fractions 6-9, 11-13, and 14-15. At 310 nm, three bands and two shoulders were obtained, each in fractions 6-9, 1 1-12. 14-15, and 18- 19, respectively. Profiles were also detected at 288 nm (which is superposed with the profile at 272 nm), 320, 375 nm (which were superposed with the profile at 310 nm), and at 454, and 675 nm, which were superposed between them (no bands were obtained in fractions 5-6, 7-8, 10-11, and 15-16). In A. dispar, 6 peaks and 1 shoulder were obtained at 266 nm, each in fractions 4-6, 9, 15-16 (shoulder), 21- 22, 25, 29, and 34 (small). At 326 nm one major band was obtained (fractions 3-6), although two small peaks were also observed at fractions 29 and 34, respectively. In addition, patterns were detected at: 288 nm (that was not superposed with the profile at 266 nm, only for the shoulder, fraction 1 1 ) and at 660 nm (no bands were ob- tained in fractions 2-3 and 33-34). Iron was eluted in all LH-20 chromatography of P. chi- lensis plasma. The Ve of the main bands were at 49, 61, 85, 97, 1 1 5, 1 33, and 235 ml. The first three bands were superposed with the respective eluting peaks at 272 nm, and also with three eluting bands of the profile at 310, and with two peaks of eluting profile at 675 nm. Most of the main iron bands in the profiles were observed at a greater volume than V, of the bed column, and after frac- tion number 20, appeared not to have association with the patterns at 272, 288, 310, 375, 454, and 675 nm. Va- nadium was not considered in this opportunity. Iron was also found in all LH-20 chromatography of A. dispar. and the Ve of the main peaks were obtained at 35, 55 (shoulder), 115, 145, 165, 195, 215, 265, 295, and 330 ml, in which the chromatographic parameters of any of them correlates with the eluting peaks with respect to absorbance eluting patterns. Titanium was not found in fractions 7-14, and the Vc of the main bands were ob- tained at 45, 155, 185, 205, 265, and 305 ml. The second titanium eluting band correlates with the respective peaks in the profile at 266 nm, and the fourth is super- posed with the patterns at 266 and 280 nm. Vanadium was found in all the chromatography, but most was 160 D. A. ROMAN KT AL. 22.5! 3.8 6 ml FRACTIONS Figure 3. Elution patterns of blood plasma of I'ytiru chiU-nsis from Sephadex LH-20 chromatography at 272 nm (C solid line) and 310 nm (D X X X), and elution profile of iron (circles). 10 ml concentrated fivefold by freeze-drying. 6 ml fractions. el uted from fractions l-!7(Vc = 5.25,75,95, 1 15, 145, 265, 285, and 335 ml). Fractions 5 and 1 1 absorption spectra from Sephadex G-75 chromatography of P. c/ulen.\is blood plasma are shown in Figure 5a. Fraction 5, that also corresponds to the second iron-band in the respective eluting profile (Fig. 2b), had an absorption band at 276 nm with one shoulder at 400-425 nm. Fraction 1 1 shows absorption maxima at 270, 3 1 0, and 460 nm with a shoulder at 360- 375 nm, and is not in the area of an iron-band, although it is between the third and fourth iron-band, in the re- spective eluting pattern (Fig. 2b). r110r55 flO 2 > a c 2 *c Cfl 15 25 FRACTION 35 F.lution patterns of blood plasma ol Iv ului i/n/tar from Sephadex I.H-20 chromatography at 266 nm (I dntsi and ^2li nm (FOOO), and dution profiles of iron (X x .< ), titanium (dash line), and vanadium (solid line). Id nil lomi-nlruk-d Iwnlold In lu-c/i- ilrvinj;, Id ml fractions. INORGANIC ASPECTS OF THE BLOOD CHEMISTRY OF ASCIDIANS 161 2.0,1.0 21 200 350 240 280 450 550 WAVELENGTH IN flfTl 320 650 360 750 Figure 5a. Absorption spectra of fractions 5 (Auv solid line, AVIS dots) and 1 1 (Buv dash line, BVIS circles) from Sephadex G-75 chroma- tography of blood plasma ofPyura chilensis. Ultraviolet spectra of fractions 4, 7 and 1 1 from Seph- adex G-75 chromatography of A. dispar blood plasma are shown in Figure 5b. Fraction 4 had an absorption shoulder at 265-285 nm and also corresponds to the first titanium-band in the respective profile (Fig. 2b). Frac- tion 7 shows absorption bands at 2 10, 260-280, and 326 nm, and it corresponds to the first vanadium band (Fig. 2b). Fraction 1 1 (yellowish) had two absorption maxima, at 266 and 326 nm, respectively, and corresponds to the third iron band (Fig. 2b). Ultraviolet spectra of fractions 5, 8, 17, 21,25, 29, and 36 from Sephadex LH-20 chromatography of A. dispar blood plasma are shown in Figure 6a. Fractions 3-5 had absorption maxima at 266-270 and 322-324 nm, corre- sponding moreover to the border-line zone between the first iron band and the respective iron shoulder, and to the first titanium band (Fig. 4). Fraction 8 also had two absorption bands, at 262 and 320 nm, which only appear to be associated with the third vanadium peak (Fig. 4). Fraction 17 had ultraviolet bands at 232 nm and in the zone of 280 nm, corresponding to the fourth iron peak in Figure 4. Fraction 2 1 had one absorption band at 280 nm and two small shoulders at 274-276 nm and 286- 288 nm, respectively. This fraction also corresponds to the first titanium band (Fig. 4). Fraction 25 had ultravio- let bands at 230 and 270 nm, and one shoulder at 292- 294 nm. This fraction appears not to be associated with 0.5 200 240 280 320 WAVELENGTH IN 111 Ml 360 Figure 5b. Ultraviolet spectra of fractions 4 (C circles), 7 (D, D' dots), and 1 1 (E solid line) from Sephadex G-75 chromatography of blood plasma ofAscidia dispar. Dilution shown were applicable. any metal. Fraction 29 had the following absorption maxima: at 210, 232 (shoulder), 270, and 292-294 nm (shoulder), and should correspond to the same group of compounds as fraction 25 (have similar UV spectra). Fraction 36 had three ultraviolet maxima, at 218 (not shown), 296, and 328 nm, and it corresponds to the last iron band (Fig. 4). Fraction 34 had a spectrum similar to fraction 36, except for the band at 296 nm, which in fraction 34 appears as a shoulder in the zone of 280 nm. Also, fraction 34 correspond to the penultimate iron peak (Fig. 4). The visible spectra of fractions only showed absorption increasing monotonically with a decreasing wavelength. Ultraviolet spectra of fractions 6, 8, and 1 1 from Seph- adex LH-20 chromatography of P. chilensis blood plasma are shown in Figure 6b. Fraction 6 had absorp- tion maxima at 280 nm and in the 3 1 0-320 nm zone. It appeared not to be associated with any principal iron band (Fig. 3) although it is in the borderline of a minor iron peak (fraction 5). Fraction 8-9 also had two ultravi- olet bands, at 270 and 302-306 nm, but are in the first principal iron peak zone (Fig. 3). Fraction 1 1 had an ab- sorption shoulder band at 260-280 nm and another that tends to disappear at 286-288 nm. This fraction is in the second principal iron peak zone (Fig. 3). In the 12-24 fraction range, the absorption spectra showed no bands. From fractions 25 to 29, the ultraviolet spectra only showed one light band at 266 nm. The visible spectra of fractions also consisted in absorptions increasing mono- tonically with decreasing wavelength. 162 D. A. ROMAN ET AL 2 0.5 0.2 2.0 200 240 280 WAVELENGTH IN Mill 320 36C Figure 6a. Ultraviolet spectra of fractions 5 (F - •-), 8 (G dash line). 17(H x x x), 21 (I OoO). 25 (J solid line), 29 (K dots), and 36 (L circles) from Sephadex LH-20 chromatography of blood plasma of Ascidia dispar. Discussion The analysis reported here should support the conclu- sion that P. chilensis is an iron and titanium accumula- tor, and that A. dispar is an iron, titanium, and vana- dium accumulator. In both species the predominant metal was iron, which in the case of P. chilensis is consis- tent with ascidian phylogeny with respect to vanadium- and iron-containing species (Hawkins el ai, 1983c). In the order Pleurogona, all of its family species are iron accumulators (Swinehart el a/., 1974; Agudelo et al. 1 982). However, A. dispar appears to he an iron-predom- inant species, although, it also accumulates titanium and vanadium at greater levels than considered non-biologi- cal (Saxby. 1969; Hawkins el ai. 1983c) with respect to metal contents in blood cells. Results from the whole body (specimens) are not reliable because when the ani- mal is removed it immediately begins to lose blood. In the sub-orders Aplousobranchia and Phlebobranchia. the majority contain vanadium in their blood (Hawkins el al., 1983c: Michibata et al.. 1986). Titanium has been reported in ('tuna inteslinalis (Noddack and Noddack, 1939) and Eudt* nmaritleri (\jt\\nc. 1961. 1962a,b), but according to Goodbody ( 1974), there is no concrete evi- dence that titanium would be concentrated in blood cells. In the present work evidence is presented of this metal in the blood cells ol/' chilensis and . I dispar. However, some of these results could be only apparent from the biochemical point of view, because they may be influenced bv the ascidians immediate environment 200 240 280 320 WAVELENGTH IN tl 111 360 Figure 6b. Ultraviolet spectra of fractions 6 (M dots). 8 (N dash line), and 1 1 (O solid line) from Sephadex LH-20 chromatography of blood plasma of Pyura e.g.. the floating metallic barrels of marine pools where fixation occurs. TiCK and FejOj are frequently used as pigments in many paints (Orna, 1980). Because of their ability to accumulate metallic trace elements from sea- water, tunicates also have been suggested to serve as ma- rine pollution indicators (Papadopoulou and Kanias, 1977). Therefore, the Ti in P. chilensis, and the higher concentrations of Fe and Ti in A. dispar. may also be associated with this aspect, rather than being considered essential elements subjected to selective accumulation mechanisms. The accumulation of uncommon metals by ascidians in significant concentrations is still an open question. For instance, something similar to what hap- pens to Ti, occurs to Nb (Rayner-Canham, 1984). Iron is the predominant metal in P. chilensis cyto- plasm, but in .1 dispar 26.9% could be in cell mem- branes. Titanium is almost distributed likewise in both species' cytoplasm and cell membranes. Vanadium is predominant in .1 dispar cytoplasm cells, although 23.6% could be bound to membrane cells. Therefore, variable fraction of metals, which may depend on the species, are associated with blood cell membranes of tu- nicates. Blood plasma of both species were nearly neutral, with a lower salinity than the habitat seawater and with low concentrations of sulphate ions. Besides, the Ca/Mg con- centration ratios were greater (0.33 for P. chilensis and INORGANIC ASPECTS OF THE BLOOD CHEMISTRY OF ASCIDIANS 163 0.30 for A. dispar) compared with the seawater (0.23). The Na/K concentration ratios were lower (20. 1 for P. chilensis and 22.2 for A. dispar) than in seawater (27.9). Calcium and magnesium were not detected in the cy- tosolic solutions, and the Na/K concentration ratios were very different (4.6 for P. chilensis and 346.7 for A dispar). Nevertheless, both were nearly neutral and their sulphate ion contents were low, reaching 11.7% and 6.5% of their contents in plasmas of P. chilensis and A. dispar. respectively. This implies that the low concentration of sulphate in plasmas (in respect to the concentration of sulphate in seawater), is not the result of the accumula- tion into cytoplasmic blood cell solutions. Considerable controversy still exists on the intracellular pH and con- centration of sulphate in the intact blood cells of tuni- cates(Dingley elal.. 1982; Hawkins?/ al, 1983a; Frank etal.. 1986). To obtain more knowledge about the behavior of some major tunicate blood components, plasma-cell in- teraction was abruptly induced in the blood itself (due to lack of A. nigra blood, this experiment was carried out only with P. chilensis blood). Blood cells apparently were not lysed under whole blood lysis procedures, according to microscope observations and to differential UV-spec- tra of plasma, cytosolic solution and lysed whole blood samples. The results (Table IV), are consistent with the fact that the blood cells of P. chilensis are not acidic and it seems that interactions could occur between plasma and cellular compounds, that could account for the de- crease of sulphate, calcium, magnesium, sodium, and potassium concentrations in whole lysed blood solution, in respect to their concentrations in blood plasma. Part of these components could be taken up by some com- pound(s) of the cellular membranes. It is also possible that sulphate, calcium, and magnesium in particular, in- teract with some intracellular compounds, which would mean, for instance, that sulphate is consumed by intra- cellular compounds of cytosolic solutions. Due to the complexometric titration method by means of which cal- cium and magnesium were determined (Roman et al., 1982), it is feasible that intracellular strong metal ligands take up part of the calcium and magnesium of the plasma. Therefore, this could be the first evidence of sul- phate consumption by blood cell components of tuni- cates, as hypothesized by Hawkins et al. (1983b). It should explain its low concentration in ascidian blood plasma as compared to the blood plasma of other marine animals (Burton. 1973). Both plasmas and cytosolic solutions were reducing with respect to permanganate and dichromate, respec- tively. However, deproteinization prior to the titration were not made. However, in the case of the back titration of dicromate method, the sample was acidified with con- centrated perchloric acid, a deproteinizant (Carr et al.. 1983). In the pioneering studies of Endean ( 1 985a) sim- ilar assays were tested, and Muzzarelli (1973) used back titration of dicromate forchitin determination. Hawkins el al. (1980a) have detected N-acetylaminosugar com- pounds in the blood plasma of tunicates. Other reducing components that have been reported in ascidian blood include some reduced form of metals, the tunichrome like compounds and the so called apoferreacids (Macara et al.. 1979a, b, c; Agudelo et al.. 1982, 1983b, 1985; Hawkins et al., 1983b; Bruening et al.. 1985; Frank et al.. 1986). Maintaining iron and vanadium in reduced forms in specialized blood cells, and also in some extension in the plasma in the case of iron (Agudelo et al.. 1983b; Ro- man, unpub. results from P. praeputialis). required more investigation in adequately controlled artificial condi- tions. The plasma spectra (Fig. 1 ) are similar for P. chilensis and A. dispar. The main differences are the presence of a shoulder at 375-385 nm, and the existence of pink- orange compound(s) having an absorption band at 450- 475 nm in the plasma spectrum of P. chilensis. P. stoloni- fera pink compound(s) had a visible band at 497 nm (Hawkins et al., 1980a). The plasmas UV- spectra of A. nigra (Kustin et al., 1976), A. ceratodes (Hawkins et al., 1980a), Podoclavella moluccensis, Polycarpa peduncu- lata (Hawkins et al., 1 980b), and P. stolonifera (Hawkins et al., 1980a) also have bands at 260-275 nm and 300- 330 nm ranges. A band at 335 nm (Agudelo et al.. 1982) was only detected in plasma of B. ovifera. The main simi- larity of the visible spectra of P. chilensis and A. dispar plasma is the band at the 675 nm zone. Anion exclusion, cation retardation, and other prob- lems occur in the chromotography of metal-containing substances on Sephadex G and LH types. This is due to the small amounts of donor groups present in the mate- rial (Pharmacia Fine Chemicals, l977;Kuraetal.. 1977; Johnson and Evans, 1980; Lonnerdall and Hoffman, 1 98 1 ). To minimize this problem, 0.0 1 M NaCl and 0.06 M acetic acid solutions were used as eluents with Sepha- dex G-75, and methanol/water gradient with Sephadex LH-20 chromatography, respectively. Some level of methanol was always maintained in the separative pro- cess and prior to the sample run, the column was condi- tioned with methanol p.a. As Sephadex LH-20 was used with a mixture of polar solvents, adsorption and parti- tion effects must be considered to play major role in the separation. Gel filtration effects can be disregarded. The elution behavior of plasmas of P. chilensis and A. dispar from Sephadex G-75, were similar in respect to absorbance versus fraction collected (Fig. 2b), but the patterns for metal contents versus fraction collected (Fig. 2b), were not similar in function to the same metal con- sidered. In P. chilensis plasma, evidence of iron com- 164 D. A. ROMAN ET AL pounds with a high molecular weight was found (fraction 5-6). in addition to i mds corresponding to low mo- lecular weight • pounds. However, these might correspo! i pounds of high molecular weight that show e ater affinity for the gel phase than for theaquc.'..s ,.hase. In A dispar, no evidence of high mo- lecular \ ight iron compounds was found. However, these were found in the case of titanium (fraction 4). Low molecular weight compounds of iron and titanium, or metal compounds that showed greater affinity for the sta- tionary phase were also detected. In both plasmas vana- dium appears to exist as low molecular weight com- pounds, unless the high molecular weight compounds were retarded by adsorption phenomena. The absorption spectra of the fractions associated with high molecular weight iron compounds (Fig. 5a, fraction 5), cannot correspond to an Fe (III) hydrolytic polymer, which only showed a shoulder at 470 nm (Flynn. 1984). The absorption spectrum of fraction 1 1 (Fig. 5a) appears to correspond to G-75 low molecular weight organic pig- ment that could be a tunichrome-like compound(s). The absorption spectra of fractions 5 and 1 1 account for the spectrum plasma of P. chilensis. so these results appear not to be "artifacts." The ultraviolet spectra of the fractions associated with apparently high molecular weight titanium compounds, from A. dispar plasma chromatography on Sephadex G- 75 (Fig. 5b, fraction 4), only shows a shoulder at 270- 286 nm. This absorption zone was also checked for the indication of a high molecular weight iron compound(s), but no visible bands were observed. The ultraviolet spec- tra of fractions 7 and 1 1 (Fig. 5b) appeared to correspond to closely related compounds, apparently of low molecu- lar weight, associated with vanadium and iron, respec- tively. Their UV spectral features suggest that tuni- chrome-like compounds may also be involved in these fractions (Brueningrt al., 1985). In comparison with the absorption spectrum of the whole blood plasma of A. dix- par (Fig. 1). in the chromatographic fractions, the ab- sorption peak at 675 nm zone was not observed. The elution behavior of plasma of P. chilensis and A. dispar on Sephadex LH-20 with methanol/water gradi- ent, showed similar patterns for absorbance, and iron contents versus fraction collected (Fig. 3, 4). For P. chi- lensis plasma, chromatographic evidence of iron-com- pounds were i >htained, and the same occurs for iron, tita- nium, and vanadium compounds in A. dispar plasma, respectively, which appear not to be inorganic hydrolytic products of metal ions. In A. dispar. the absorption spectra of fractions 3-5 (Fig. 6a) appear to be associated with iron and titanium- compounds, but according to the spectra of fractions 4, 7, and 1 1 from Sephadex G-75 (Fig. 5b), the titanium compound(s) should tend to absorb at 260-290 nm /one. Iron, vanadium-compounds and tunichrome like substances also absorb at 320-330 nm. The ultraviolet spectrum of fraction 8 (Fig. 6a), should correspond then to vanadium compound(s). The ultraviolet spectrum of fraction 17 may correspond to iron compounds of pro- teinaceous nature, due to the band at 280 nm zone, and the same seems to occur in fraction 2 1 for titanium com- pound(s). Fractions 25-29 (Fig. 4) were not associated to any metal ions, and by their spectra appear to correspond to closely related compounds. Fractions 34-36 are re- lated to iron, and by their spectral features should corre- spond to iron compound(s) similar to those obtained from the interaction between iron and fractions 8-13 G- 75 chromatography of A. ceratodes plasma (Hawkins el a/., 1980a). Therefore, compounds of fractions 3-5 should be closely related to iron compound(s) of frac- tions 34-36. In P. chilensis plasma chromatography on Sephadex LH-20 gel. fraction 6 (Fig. 3) appear not to be associated with iron, and their spectrum (Fig. 6b) could correspond to tunichrome-like substances similar to spectrum of fraction 1 1 from Sephadex G-75 (Fig. 5a). However, fractions 8, 9 (Fig. 3) are related to a main iron peak, then those should contain iron compound(s), whose ab- sorption peaks show (Fig. 6b) hipsochromic shifts in re- spect to the spectrum of fraction 6. Hiper- and hipo- chromic effects in the bands can also be observed. Frac- tion 11 is in the zone of the second iron peak (Fig. 3), and by their ultraviolet spectra (Fig. 5a), may correspond to iron compound(s) of proteinaceous nature. It is likely that by dilution the visible absorption max- ima were not observed in the spectra of fractions coming from LH-20 chromatography of blood plasmas. The complicated hydrolytic processes of iron (Flynn, 1984), titanium (Pascal, 1963, Ciavatta el ai. 1985) and vanadium (Kustin and Macara, 1982) in a pH media close to neutrality, such as the blood plasma of tunicatcs. suggests that these elements could be found as coordina- tion compounds with protcic or non proteic organic li- gands. The ligands that have been associated with metals, in tunicate plasma, are proteins (Hawkins ct a/.. 1980a: Webb and Chrystal, 1981; Agudelof/a/., 1983b)andN- acetylaminosugar compounds (Hawkins el al., 198()a, b). However, Agudelo el al. 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Studies on the structure, reproduction, develop- ment and accumulation of metals in the colonial ascidian Eudis- toma ritteri Van Name, 1 945.7 Morphoi 111: 105-137. I.onnerdal. B., and B. Hoffman. 1981. Alkaline reduction of dextran gels and crosslinked agarose to overcome non specific binding of trace elements. Biol. Trace Element Res. 3: 301-307. Macara, I. G., G. C. McLeod, and K. Kustin. 1979a. Vanadium in tunicates: oxygen-binding studies, Comp Biochem. Physiol 62A: 821-826. Macara, I. G., G. C. McLeod, and K. Kustin. 1979b. Isolation, prop- erties and structural studies on a compound from tunicate blood cells that may be involved in vanadium accumulation. Biochem. J. 181:457-465. Macara, I. G., G. C. McLeod, and K. Kustin. 1979c. Tunichromes and metal ion accumulation in tunicate blood cells. Comp Bio- " Physiol 638:299-302. Martin. V \\ .. K. Iluebers, H. lluebers, J. Webb. C. A. Finch. 1984. A monosited transferrin from a representative deuteros- tome. «/«/W64: 1047-1052. Miccra, (i.. S. ; •••i:ma, A. Dessi. P. Decock, B. Dubois, and II. Ko/lon- ski. 1985. Coppertll) compexation by D-glucosamme. Spectro- scopic and potentiometnc studies. Inorg. Chim Ada 107: 45-48. Michibata. 11., T. Tcrada. N. Anada, K. Viin.ik.m.i. and 1. Numaku- nai. 1986. The accumulation and distnbution of vanadium, iron, and manganese in some solitary ascidians. Biol. Bull 171:672-681. Monniot. F. 1978. Connaissances actuelles sur les ions metalliques chez les ascidies. Actualites de Biochimie Marine Colloque GABIN, la Rochelle. Pp. 185-194. Muzzarelli, R. A. A. 1973. Natural Chelatirtg Polymer International series of monographs in analytical chemistry. Pergamon Press. Hungary. Vol. 55. Pp. 95-96. Noddak, 1.. and \V. Noddak. 1939. Die haufigkeiten der schwermet- alle in meerestieren. Ark /.no! 32: 1-35. Orna, M. V. 1980. Chemistry and artist' colors, part I, II. III. 7 Chem. Educ 57: 256-258; 264-267; 267-269. Papadopoulou. ( .. and G. D. Kanias. 1977. 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The blood cells afCiona mtestmalis: an electron probe X-ray microanalylical study. 7 Mar Biol Assoc, U. A 62: 607-620. Sandell. K. B. 1959. C 'nlonmeinc Determination of Trace* of Metals. Interscience Publishers. Inc., New York, third ed. Pp. 537-542. Saxby, J. D. 1969. Metal-organic chemistry of the geochemical cycle. Rev PureAppl. Chem 19: 131-150. Senozan, N. M. 1974. Vanadium in the living world. 7 Chem Educ 51:503-505. Sephadex 1,11-20. 1977. Technical report. Pharmacia Fine Chemi- cals. Uppsala, Sweden. Pp. 1-23. Swinchart, J. 11., W. R. Biggs. 1). .1. llalko, and N. C. Schroeder. 1974. The vanadium and selected metal contents of some ascidi- ans. Biol. Bull 146: 302-312. I ullius, I U., W . O. < .ilium. R. M. K. Carlson, and K. U. Hodgson. 1980. Structural study ol the vanadium complex in living ascidian blood cells by X-ray absorption spectroscopy. 7. Am. Chem. Soc. 102: 5670-5676. Webb, J., and P. Chrystal. 1981. Protein binding of iron in blood plasma of the ascidian Hermania momus Mar Biol 63: 107-112. Wright. K. K. 1981. I'rochordates. Pp. 565-626 in Invertebrate Blood Cells, N. A. RatcliH'e and A. F. Rowley, eds. Academic Press. London. Reference: Bio/. Bull 175: 167-174. (August, 1988) The Behavioral Response of Spiny Lobsters to ATP: Evidence for Mediation by P2-like Chemosensory Receptors RICHARD K. ZIMMER-FAUST1, RICHARD A. GLEESON2 AND WILLIAM E. S. CARR2 1 Marine Science Institute, University of California, Santa Barbara. California 93106 and 2 The H 'hitney Marine Laboratory and Department ofZoologv. University of Florida. St. Augustine, Florida 3 2086 Abstract. The results of both behavioral and electro- physiological studies with the California spiny lobster, Panulirus interntptus, support the hypothesis that a loco- motory response evoked by ATP in seawater may be me- diated by chemoreceptors akin to P2-type purinoceptors. Behavioral results consistent with this hypothesis are: ( 1 ) an activity sequence of ATP > ADP > AMP or adeno- sine; (2) the behavior is also evoked by ATP analogs with modifications in both the adenine and ribose moieties; and (3) the slowly degradable analogs, /3,7-methylene ATP and 0,7-imido ATP (AMPPNP) are active. Extra- cellular recordings from single chemosensory cells show that ATP-sensitive cells are present in the antennule of P. interruptus and exhibit marked similarities to the P2- like chemoreceptors identified earlier in P. argus. Al- though the ranked order of behavioral activity to ATP and eight analogs parallels that measured physiologi- cally, important differences include: ( 1 ) AMPPNP is rel- atively more active physiologically; and (2) the behav- ioral sensitivity to ATP, ADP, and AMP is greater than that measured physiologically. Since degradation of ATP proceeds rapidly in animal flesh and in seawater, it is pro- posed that ATP may represent a particularly appropriate signal molecule for foraging by the lobster as it is indica- tive of recently injured or freshly killed organisms. Introduction Receptors for purine nucleotides, referred to as puri- nergic receptors or purinoceptors, occur in various inter- Received 29 February 1988; accepted 31 May 1988. nal tissues of mammals (see Burnstock, 1978). One type of purinergic receptor, the P2-type, is most sensitive to the nucleotide adenosine triphosphate (ATP) (Burn- stock, 1978). Interestingly, chemoreceptors stimulated by ATP, which exhibit properties similar to P2-type re- ceptors, have been demonstrated electrophysiologically in the olfactory organ of the Florida spiny lobster, Panul- irus argus (Carr et ai. 1986). However, their role in the chemically mediated behavior of P. argus has not been explored. Recently, in the California spiny lobster, P. interrup- tus, ATP was shown to be a potent chemoattractant, evoking a locomotory response associated with the rec- ognition and finding of food (Zimmer-Faust, 1 987; Zim- mer-Faust and Dyson-Hudson, in prep.). When the effectiveness of adenosine and adenine nucleotides was compared, the potency sequence for the locomotory re- sponse was: ATP > ADP > AMP or adenosine. This se- quence was identical to that found by Carr et al. (1986) in the physiological studies with P. argus. Collectively, the above findings provided the impetus for initiating an integrated behavioral and physiological study to ascertain if the behavioral response of P. inter- ntptus to ATP might be mediated by P2-like chemore- ceptors. In this study, we have extended the behavioral data for P. interruptus using a series of ATP analogs to permit comparison with the physiological results from P. argus. We also demonstrate physiologically that P. inter- ruptus has ATP-sensitive chemoreceptors which exhibit major similarities to those characterized in P. argus. To- gether, our findings reveal that P. interruptus does have P2-like chemoreceptors which may mediate its behav- ioral response to ATP. 167 168 R. K. ZI.MMER-FAUST ET AL Materials and Methods Collection and maintenance of animals Specimen^ »i .". interruptus were captured by hand on reefs near Sania Barbara, California. Before use in behav- ioral studies, lobsters were held for 7 to 14 days in large circular tanks. A continuous flow of seawater main- tained the temperature at 15 to 17°C. and a 12:12 D:L cycle was imposed. Only hard-shelled animals (n = 108) of 60 to 72 mm carapace length were used; each was tat- too marked on the ventral sternites for individual recog- nition (Kuris. 1971). Lobsters were fed live mussels (Mytilus californianus and M. edulis) and sea urchins (Strongylocentrotiis purpuratus) ad libitum; all food was removed 24 h before tests. Specimens of P. interruptus used for electrophysiologi- cal studies were shipped by overnight courier from Santa Barbara to the Whitney Laboratory (St. Augustine, Flor- ida) where they were held together in a tank with a flow- through seawater supply maintained at 14 to 16°C. Spec- imens of P. argus were collected in the Florida Keys and held at ambient seawater temperature in flow-through tanks at the Whitney Laboratory. All animals were fed a diet offish, squid, and shrimp. Behavioral assays Individual lobsters were assayed for locomotory re- sponses to chemical solutions in rectangular aquaria, 30 X 30 X 13 cm. a size shown previously to permit both careful control of stimulus flow and rapid testing, with- out inhibiting behavior (Zimmer-Faust and Case. 1 983). Earlier studies revealed excellent agreement between the locomotory responses evoked by chemostimulants in natural habitats and in the small aquaria used for assays in the current study (e.g.. Zimmer-Faust and Case, 1 982: Zimmer-Faust el ai, 1984). Seawater entered each aquarium at a flow rate of 2 ml/s from a head-tank main- tained under constant hydrostatic pressure. A three-way valve was used to introduce a 25-ml volume of each test solution into the seawater flow. Dilution associated with stimulus delivery was determined in 18 trials by intro- ducing a fluorescent dye (sodium fluorescein) and con- tinuously monitoring fluorescence using optical fiber probes attached to the antennules of unrestrained ani- mals (see Zimmer-Faust and Stantill. 1986: Zimmer- Faust c/ ai. in press). Maximum concentrations contact- ing the ank-nnuK-s were determined to be 7.57 X 10~3 (±3.62 X 10 ' S.D.) times the injected concentration, with dye peaks attained 29.8 s (±5.8 s S.D.) after initial dye input. Concentrations presented herein are corrected for this dilution. A locomotory response by /'. interruptus is defined as forward ambulatory movement to a distance greater than one carapace length. Previously, ATP was found to stimulate other behaviors associated with appetitive feeding (Zimmer-Faust. 1987); however, these other be- haviors were more variable than forward ambulatory motion. Individual lobsters were tested only once every 72 to 96 h for a maximum of 5 tests during a 20-day period. Animals were put into experimental aquaria 45 to 60 min prior to testing and usually settled within 30 to 40 min. Observations were initiated 1 min before in- troduction of a chemical solution and continued for 4 min afterwards; lobsters were tested only if they were in- active during the first minute of observation. Each assay consisted of a randomized presentation of a test or con- trol stimulus with the exception that identical solutions were never repetitively presented to the same animal. All trials were conducted according to a double-blind proto- col. At least 20 different lobsters were assayed with each test solution; seawater alone was the control. Electrophysiological recordings Extracellular recordings were made from ATP-sensi- tive sensory cells in the olfactory organ (lateral filament of the antennule) of both P. interrupt us and P. argus. The preparation was similar to that used by Carr el al. ( 1986) and has been described in detail (e.g.. Gleeson and Ache, 1985). The essential features of the preparation were as follows. An excised, lateral filament was placed in an ol- factometer and maintained via arterial perfusion with Pamtlirus saline. Selected volumes of chemical stimuli were injected into a carrier stream of artificial seawater ( ASW) which continuously flowed through the tuft of ol- factory sensilla on the filament at a rate of 3 ml/min. Suction electrodes were used to obtain action potential recordings from the axons of individual sensory cells in- nervating the sensilla. These recordings were made from the antennular nerve which was exposed at the proximal end of the filament and separated into small bundles within a bath of Pamilirus saline. ATP-sensitivc cells were identified by their initiation of action potentials (impulses) following the introduc- tion of a search stimulus of ATP (10 ^M. ca. 20 n\) into the carrier flow of ASW; ASW alone was presented in an identical manner as the control. The response of a cell to defined chemicals was determined by injecting a 190 j/l volume of each test substance into the carrier flow. Con- ductivity measurements showed that this volume gener- ated a stimulus profile in the olfactometer that reached the injected concentration within one second and re- mained constant for approximately two seconds before beginning to wash out. [Note: due to an error in conduc- tivity measurements, the stimulus profile reported pre- viously (Carr el al.. 1986) overestimated the time a stim- ulus remains at the injected concentration.] Intervals of BEHAVIOR MEDIATED BY P, RECEPTORS 169 4 min were maintained between stimulus presentations, during which time ASW continuously flowed over the filament. In each trial, stimuli were presented in a ran- dom sequence except in dose-response determinations where an ascending concentration series was used. Single cell responses were recorded using an amplitude/time window discriminator, the output of which was moni- tored with a microprocessor to measure and store the time intervals between impulses for on-line or subse- quent display and analysis of the response. In this report, cell responses are expressed as the total number of evoked impulses. Relevant physiological results obtained for P. argus (Carr el al, 1986) are included in some figures and sum- marized in the text to facilitate their comparison with the results obtained with P. interruptus. Chemical solutions All chemicals were from Sigma Chemical Company. Structural formulae of ATP and the analogs included in the study are shown in Figure 1 . For behavioral assays, solutions were prepared immediately before tests in membrane-filtered (0.45 nm) seawater and adjusted to pH 7.8. Aliquots were stored on dry ice and warmed to ambient seawater temperature just prior to use. Solu- tions for physiological studies were prepared as stocks in ASW, adjusted to pH 7.8, stored at -70°C, and aliquots were brought to room temperature just prior to use. Results Behavior Figure 2 shows the results of the behavioral assays comparing the stimulatory activity of ATP and nine structurally related substances. Although ATP was the most stimulatory substance, all of the analogs, except for 8-bromo-ATP and adenosine, were significantly more effective than seawater alone (G-test for Independence with Williams' correction: G > 7.60, d.f. = \,P< 0.01, all comparisons). We observed no responses to seawater in 66 trials. The rank order of behavioral activity for these analogs parallels that previously measured physio- logically in ATP-sensitive cells of P. argus (Fig. 2). A Kendall's Tau analysis of these ranks revealed a highly significant association (T = 0.721, n = 10, P < 0.008). The most notable exception to this ranking concerns the stable analog, 0,7-imido ATP (AMPPNP), which in the physiological tests proved to be more stimulatory than ATP itself. Behaviorally, the relative activity of ADP and AMP was greater than that measured physiologically; however, the behavioral activity induced by these ana- logs was significantly less than that for ATP (G-Test with Williams' correction: G > 4.31, d.f. = 1, P < 0.05, both comparisons). To characterize the dose-response (D-R) relationships of selected analogs, behavioral assays were conducted over a range of concentrations with ATP and with three analogs having structural modifications in either the ni- trogenous base, the ribose, or the triphosphate moiety. The results revealed that the slopes of the D-R curves for ATP and the analogs were not significantly different (Fig. 3). This finding is consistent with the notion that the ac- tions of these substances are mediated by a common population of receptors. The rank order of potencies found in this analysis was ATP > dATP > CTP ^ AMP- PNP. This ranking confirmed the order of activities ob- served earlier with the single-dose determinations (see Fig. 2). Physiology Physiological studies revealed that P. interruptus, like P. argus, has a distinct population of olfactory cells that are selectively activated by ATP. In both species, the re- sponse of these cells is characterized by a short burst of impulses, the duration of which is generally only a few hundred milliseconds in spite of the fact that the stimu- lus is present for several seconds (Fig. 4A). A comparison of the D-R relationships for these cells reveals similar sensitivities in the two species. In P. interruptus, how- ever, the D-R function exhibits a significantly steeper slope [Test for Parallelism (Tallarida and Murray, 198 1 ): P < 0.05], with a response maximum of approximately 17 impulses (Fig. 4B); the cells in P. argus show a maxi- mum response of about 1 1 impulses. Comparisons of the stimulatory capacities of the ade- nine nucleotides and adenosine on the ATP-best cells of both species show marked similarities. In both species the activity sequence is ATP ?> ADP > AMP and adeno- sine (Fig. 5A). In both species ADP is a very poor stimu- lant and, like AMP and adenosine, is virtually inactive. Moreover, the ATP-best cells in both species show greater responses to the slowly degradable analog, AMP- PNP, than to ATP itself (Fig. 5B). A similar specificity for ATP in the cells of both species was also indicated in trials in which 10 \iM glutamate, taurine, betaine, and glycine were each individually tested. None of these sub- stances elicited responses from any of the nine cells ex- amined in P. interruptus. and only glycine evoked a re- sponse (a single impulse) in one of the seven cells tested in P. argus. Discussion This study shows that the behavioral response to ATP exhibited by the California spiny lobster, P. interruptus, may be mediated by chemoreceptors related to the P2- 170 R. K. ZIMMER-FAUST ET AL Adenlne Alterations Ribose-P-P-P ATP HN Ribose-P-P-P GUANOSINE-5'-TP (GTP) NH2 r-N L >- - Br Ribose-P-P-P 8-BROMO-ATP (BRATP) NH, VN I Ribose -P-P-P CYTIDINE-5'-TP (CTP) Ribose Triphosphate Alterations ooo HO-P-0-P-0-P-0-CH2 Adenine 1 I I I . I OH OH OH 1 3' 2'i OH OH ATP 0 0 ii II HO-P-0-P-O-CH, 1 I I - OH OH I --0 Adenine OH OH ADP OH 0=P-0-CH OH Adeninp OH OH AMP HO-CH2 Adenine OH OH ADENOSINE (ADO) 0 O 0 n n ii HO-P-0-P-0-P-0-CH2 Adenine OH OH OH OH 2'-DEOXY-ATP (DATP) 0 00 HO-P-NH-P-0-P-0-CH2 Adenine III OH OH OH v & a OH OH P, y-IMIDO ATP (AMPPNP) 0 00 II ii ii HO-P-CH^-P-O-P-O-CH? 1 I I OH OH OH Adenine OH OH },><- METHYLENE ATP (AMPPCP) Figure 1 . Structural formulae of ATP and analogs tested physiologically and/or behaviorally in I'unitli- n/.v interrupts. type purinoccptMrs described by Burnstock ( 1978). Data supporting this i- ,v>thesis include: ( 1 ) structure activity relationships (SAR) lor the behavior which show congru- ence with the SAR I'm P -like chemoreceptors previously described in P argus (Carr el al, 1986); and (2) the elec- trophysiological identification of ATP-sensitive chemo- receptors in P. interrupts that are virtually identical in their sensitivity, specificity, and temporal response char- acteristics to those described in P art;u\. The stimulation of the oriented locomotory response in /'. inlerruptus by adenine nucleotides and adenosine shows a potency sequence of ATP > ADP > AMP or adenosine. This behavior is also evoked by ATP analogs with modifications in the adenine moiety (e.g., GTP, CTP), the ribose moiety (2'-deoxy ATP), and the triphos- phate moiety (AMPPNP, AMPPCP) (see Fig. 2). How- ever the analog 8-Bromo-ATP is only a weak behavioral stimulant. These SAR are consistent with the hypothesis BEHAVIOR MEDIATED „ 60 Figure 2. Relative activities of ATP and analogs as determined by behavioral assays in Panulirus interruptus (solid bars) and by physiolog- ical recordings from ATP-best cells in P. argus (hatched bars). In behav- ioral experiments each compound was tested on at least 20 lobsters at a concentration of 2.3 nAf. In physiological studies each compound was tested at 100 \iM on at least six cells; bars represent mean responses ± SEM. The physiological data are derived in part from Carr el al., ( 1 986). Abbreviations as in Figure I . that the locomotory behavior is mediated by chemore- ceptors akin to the P:-type purinoceptors that have been found by various workers to exhibit the following SAR: (1) potency sequence of ATP > ADP > AMP or adeno- sine (Burnstock, 1978); (2) broad sensitivity to nucleo- tide triphosphates including those with modifications in both the adenine and the ribose moieties (Maguire and Satchell, 1981; Phillis and Wu, 1981; Lukacsko and Krell, 1982); (3) tolerates modifications in the triphos- BY P2 RECEPTORS 171 A P. interruptus . ATP(IO)jM) B c o CL cr> lobster. Panuhnts argus: studies of taurinest ors. Biol Hull 154:226-240. Galun. R.. N ' .: M. Zecharia. 1984. Effect of plasma compo- nents on i: .-'.nng response of the mosquito Aedes aegypti L. to adenme nuc1. oiides. Physioi Enlomol. 9: 403-408. Galun. R., L. C. Koontz, R. \V. Gwadz, and J. M. C. Ribeiro. 1>)S5 * -u , i of ATP analogues on the gorging response of Aedes l-.momol. 10:275-281. Gleeson. R. A., and B. \\ . Ache. 1985. Amino acid suppression of taunne-sensitivechemosensory neurons. Brain Res 335:99-107. Ho\le.C. H. \.,and M. J. Greenberg. 1988. Actions of adenyl com- pounds in invertebrates from several phyla: evidence for internal punnoceptors. Comp Binchcm Phv\iol 90C: 113-122. kuris. A. M. 1971. Population interactions between a shore crab and twosymbionts. Ph.D. Dissertation. University of California, Berke- le\ 345 pp. l.ukacsko, P., and R. D. Krell. 1982. Responses of the guinea-pig uri- nar> bladder to purine and pyrimidme nucleotides. Eur. J Pharma- c<>/80:401-406. Maguirt'. M. H.. and D. G. Satchell. 1979. Specificity of adenine nu- cleotide receptor sites: inhibition of the guinea pig taenia coli by adenine nucleotide analogs. Pp. 33-43 m Physiological and Regula- tory Functions of Adenosine and Adenine Nucleotides, H. P Baer, and G. I. Drummond. eds. Raven Press, New York. Maguire. M. H., and D. G. Satchell. 1981. Purinergic receptors in visceral smooth muscle. Pp. 49-92 in Purinergic Receptors, G. Burnstock. ed. Chapman and Hall. London. Mankin, R. \V., and M. S. Mayer. 1983. Stimulus-response relation- ships of insect olfaction: correlations among neurophysiological and behavioral measures of response. / Theor liiol. 100:613-630. Mato, J. M., B. Jastorff, M. Morr, and T. M. Konijn. 1978. A model for cyclic AMP-chemoreceptor interaction in Dictyostelium dis- coideum. Biochem. Biophys Ada 544: 309-314. Mitchell, B. K. 1976. Physiology of an ATP receptor in labellar sen- silla of the tsetse fly Glossina morsilans morsitans Westw. (Diptera: Glossinidae). / E\p Biol 65: 259-271. Olberg, R. M. 1983. Inlcrneurons sensitive to female pheromone in the deutocerehrum of the male silkworm moth. Bi>mhy\ mori. I'hyvol l-:m»m«l 8:419-428. PhillN, J. \V., and P. H. \Vu. 1981. The role of adenosine and its nucleolides in central synaptic transmission. Prog Neurobiol. 16: 187-239. Smith, J. J. B. 1979. The feeding response of Rhodnius prolixus to blood fractions, and the role of ATP. 7 Exp Bwl 78: 225-232. Spencer. M. 1986. The mnervation and chemical sensitivity of single aesthetasc hairs. J Comp. Physioi. A Sens. Keiiral Behav Phy^inl 158:59-68. Sutclitfe. J. K., and S. B. Mcher. 1979. Experiments on biting and gorging behaviour in the black fly. Simulium n'nuslim. Physioi. En- tomol. 4: 393-400. lallarida. R. J., and R. B. Murra>. 1981. Manual of Pharmacologic Calculations with Computer Programs Springer, New York. Pp. 11-12. Yatani, A., Y. Tsuda, N. Akaike, and A. M. Brown. 1982. Nanomolar concentrations of extracellular ATP activate Ca channels in snail neurones, \alitre 296: 1 69- 171. Zimmer-Faust. R. K. 1987. Crustacean chemical perception: towards a theory on optimal chemoreception. Biol. Bull 172: 10-29. /.immer-Faust, R. K., and J. F. Case. 1982. Odors influencing forag- ing behavior of the California spiny lobster. Paniilirus inierrupius. and other decapod Crustacea. Mar. Behav Physioi. 9: 35-58. Zimmer-Faust, R. K., and J. F. Case. 1983. A proposed dual role of odor in foraging by the California spiny lobster, Panulints interrup- iin ( Randall). Biol Bull 164:341-353. Zimmer-Faust, R. K., \V. C. Michel, J. E. Tyre, and J. F. Case. 1984. Chemical induction of feeding in California spiny lobster. Panulirus interrtiptus ( Randall): responses to molecular weight frac- tions of abalone. / Chcm. Ecol. 10:957-971. Zimmer-Faust. R. K., and M. F. StanhTI. 1986. What do aquatic ani- mals smell'.'. -tin. Zool 26: 9A. Zimmer-Faust, R. K., .1. M. Stanh'll. and S. B. Collard III. In press. A fast, multichannel lluorometer for investigating aquatic chemore- ception and odor trails. I.nunol Oceanogr. 33. Reference: Biol. Bull. 175: 175-179. (August. 1988) Growth Rate of Jamaican Coral Reef Sponges After Hurricane Allen* CLIVE R. WILKINSON AND ANTHONY C. CHESHIRE Australian Institute of Marine Science, P.M.B. No. 3. Townsville M.C. 4810, Queensland, Australia Abstract. Growth rate estimates for five coral reef sponges on the Discovery Bay fore-reef are presented. These were determined from the size of individual sponges growing on the coral rubble that was deposited when Hurricane Allen struck the north coast of Jamaica in August 1980. Sponges collected in February 1986 were weighed and their growth rates determined using the MIX program, originally developed to analyze size- frequency data in fish populations. Sponge doubling times were between 232 and 304 days, with evidence that early exponential growth may be slowing down after four years. The fastest growing sponges were those with small populations of symbiotic cyanobacteria, indicating that there may be a selective advantage for those sponges with photosynthetic symbionts. Introduction Hurricane Allen passed within 50 km of the north coast of Jamaica on 6 August 1980. Large seas generated by winds in excess of 250 km per hour caused extensive damage to the coral reefs in the vicinity of the Discovery Bay Marine Laboratory (Woodley, 1980; Woodley et al, 1981). The damage, however, was patchy; it was exten- sive in some areas and minor in those areas where local topogiaphy resulted in the attenuation or deflection of the waves (Woodley et a!., 1981). Prior to the hurricane, the fore-reef slope contained extensive, dense thickets of Acropora cervicornis between 10 m and 25 m (Goreau and Goreau, 1973; Pang, 1973). Some of these thickets were destroyed, resulting in the deposition of coral rub- ble which buried other sessile invertebrates. Received 26 August 1987; accepted 20 May 1988. 'Contribution No. 410 from the Australian Institute of Marine Science and No. 425 from the Discovery Bay Marine Laboratory, Jamaica. Prior to Hurricane Allen, sponge populations on the fore-reef slope of Discovery Bay were large (Reiswig, 1973) and considered to be ecologically significant as they could filter a volume of water equivalent to the en- tire water column to a depth of 40 m each day (Reiswig, 1974). The only evidence of these populations in Febru- ary 1986 was below 30 m depth where some of the exten- sive populations reported by Reiswig (1973) remained. Reliable estimates of the growth rates of marine sponges generally are not available. Reiswig ( 1 973), Day- ton et al. (1974), and Wilkinson (1978) all attempted to determine growth rates by measuring sponges underwa- ter, however their estimates were not particularly suc- cessful. More accurate estimates were obtained by Wil- kinson and Vacelet (1979) who transplanted Mediterra- nean species onto plastic plaques for subsequent periodic measurement. These techniques, however, involved trauma to the specimens and were time consuming. Sponge populations were surveyed on the north coast of Jamaica as part of a larger study (Wilkinson, 1987). The site chosen off the Discovery Bay Marine Labora- tory was covered by a dense bed of Acropora rubble in excess of half a meter thick, which had accumulated dur- ing Hurricane Allen. This bed covered large areas with no evidence of the previous coral or sponge fauna. The site, however, did contain numerous small sponges and coral colonies growing on pieces of the rubble. This study reports the estimated growth rate of one sponge species from 20 m on the fore-reef slope off Discovery Bay. Four other species were collected, and speculative doubling rates are presented based on the rate estimated for the first species, Pseudoceratina crassa. Materials and Methods Specimens of five massive sponge species were col- lected in February 1 986 from 20 m depth, approximately 175 176 C R \\IIKl\so\ \\[) V C CTHSHIRI 1 km to the west of the Discover. Ba> Marine Labora- tory (see Goreau and Goreau, 1973, for site description). Care was taken to select only regular-shaped animals (presumably derived from a single larva) which were at- tached to rubble and without obvious signs of predation damage. The sponges were weighed after draining for ap- proximately 20 seconds and the volume measured by displacement in water. Estimations of dry weight were made on 5 individuals of each species after dry ing for 36 h at 80°C. The size-class structure of the Pseudoceratina crassa population was estimated using the MIX program of Macdonald and Pitcher (1979; Macdonald and Green, 1 986). MIX is an interactive program used to fit distribu- tions to grouped data by maximum likelihood estima- tion. The program has been used effectively in the analy- sis of fisheries size-frequency data where the groups rep- resent successive year classes. For P. crassa. it was assumed that there were five size groupings representing the recruitment from five annual spawning events be- tween Hurricane Allen and the date of collection. To test this assumption, all possible combinations of groups from two to ten were tried, but a significant fit was only obtained for five groups. The mean size of sponges in each size-class was determined from the significant fit ob- tained to the size-frequency data with the MIX program. These mean values for each size-class were analyzed us- ing a least squares regression to provide an exponential growth model ( W, = aeat) from which the relative growth rate « was estimated. Growth rates for the other four sponge species could not be obtained by the same method due to the limited number of specimens. Growth rate approximations for these species were derived using the growth model from P. crassa. It was assumed that the average size at 50 days after spawning was the same for all species. Exponential growth curves were then derived using simultaneous equations starting with a wet weight at 50 days of 2.93 g (as for P. crassa) and the weight of the largest individuals at the date of collection. The age of these individuals was defined as the number of days between the first spawning after Hurricane Allen and the date of collection. Dates for spawning of these species were extracted from the few observations of mass release of sperm reported in the lit- erature (Reiswig, 1983;Hoppe, 1988). Results The most prevalent sponge in this area, Pseudoccra- tina crassa, has an approximate doubling time of 8.5 months (257 days) with individuals after 5 years weigh- ing, on average, 350 g wet weight (Fig. I ; Table I ). Growth rate estimates for the other four sponges sug- gest that the doubling times are between 7.6 anil 10 300 r 200 g> 'o O 100 Hurr cane Allen 1986 Al AT 1985 1984 IT 1983 IT 1982 1981 1980 Year classes Figure I. Pseudoceratina r/u.vvu growth curve prepared from MIX program analysis of sponge wet weights on the collection date in Febru- ary 198ft. The mean size tor the live size-classes with standard errors are positioned at the suggested date of spawning (solid arrows) in May of each sear. Hurricane Allen occurred on 6 August 1980. The expo- nential growth curve (W, = ae"'. where a = 2.567, a = 0.00270. and t = days since spawning) is represented by the curve drawn through the open circles, which are derived "mean si/es" tor time year classes. months (232 and 304 days). These species were less abundant in the area surveyed, hence fewer samples were available for growth rate analysis. Confidence in these estimates is substantially lower than for P. crassa. espe- cially for Age/us dispar (Table I). The full data sets for the five species are listed in Figure 2 with the estimated mean size of each year-class. It was assumed that there were five spawning events after Hur- ricane Allen in August 1980, and that each spawning event contributed individuals to the population. In the case of Ircinia J'cli. v. the position is different in that there are possibly two annual sperm release events, one in Oc- tober and another in February. Six year-classes are repre- sented on Figure 2 assuming an October release, al- though it must be recognized that semi-annual classes could exist. The largest sponges are assumed to have arisen from larvae settling within two months of the hur- ricane, hence, the estimated growth rates are the most conservative. For the other species, there is a presumed GROWTH RATE OF CORAL REEF SPONGES Table 1 Estimated growth rale parameters for sponge species at 20 in on the fore-reef slope of Discovery Bav. Jamaica 111 Sponge Spawning date used Abundance irT2 No. collected Doubling time days Growth const. « Growth const, a Sizeg 5 years Dry Wt. Vol. Wet Wt. Wet Wt. Pseudocerat ina 28 May 0.57 135 257 0.00270 2.567 354 0.175 ±.021 0.946 ± .023 crassa Irciniafelix 14 February 0.12 54 235 0.00295 2.535 552 0. 1 54 ± .032 n 1 October F 'erongula. 14 March 0.05 39 232 0.00294 2.531 587 0.108 ±.023 0.927 ± .034 aniis Smenospongia 14 March 0.02 48 304 0.00228 2.621 168 O.I 55 ±.01 3 .930 ± .050 aurea Agelas dispar 20 July 0.05 17 250 0.00277 2.557 404 0.150±.013 n Spawning dates used in the exponential equations are from Reiswig (1983) and Hoppe (1988). Abundance reports incidence irT2 in 120 m2 surveys reported in Wilkinson ( 1987). The doubling times and growth constants a and a are from the exponential equation W, = ae"' which was used to estimate size (wet weight) after 5 years. Dry weight to wet weight and volume to wet weight ratios plus standard deviations are included for comparison with other studies; n. no data available. lag of 6.3 to 1 1.5 months between the date of the hurri- cane and the first spawning event. Discussion Hurricane Allen presented the opportunity to estimate the growth rates of some Caribbean reef sponges. These estimates, although approximate, represent rare exam- ples of sponge growth rate statistics at the early stages of growth. A similar opportunity was used by Scoffin and Hendry (1984) to assess the effects of Hurricane Allen on the recruitment of sclerosponges to the Discovery Bay reef. The estimated growth rates of the five species indicate doubling times in the range 232-304 days, i.e., all in- crease by more than 100% per annum. These exceed the rates reported during studies of larger specimens of coral reef sponges (Reiswig, 1973; Wilkinson, 1978). These rates are only applicable to young sponges in the early exponential stage of growth, and cannot be extrapolated to larger sponges where proportional growth rates are considerably less (Reiswig, 1973). There is evidence in Figure 1 that the rate of growth of Pseudoceratina crassa is slowing down after four years with the fifth year-class sponges being 40 g (15%) smaller than sponges predicted from unrestricted exponential growth. A reduction in growth rates after the early, exponential phases of growth was observed by Reiswig (1973) for three large sponge species on the Discovery Bay reef. These estimated growth rates are considered more use- ful than those recorded previously for coral reef sponges. The estimates are conservative as it is assumed that the larger sponges resulted from larvae settling out within the first year of Hurricane Allen. If the larger sponges re- sulted from later settlings, then more rapid growth rates would apply. The estimates were based on measure- ments of differences in biomass (both wet and dry weight measurements), whereas in previous studies growth rates were based on estimated changes in the size of large, ir- regular specimens underwater. The assertion that these estimates may be more reli- able depends on several assumptions: (i) All sponges in the region surveyed were destroyed by the hurricane. The specimens collected origi- nated from newly settled, individual larvae pro- duced by undamaged populations from deeper water; (ii) The individuals collected, particularly the large ones, originated from single larvae; (iii) The sponge species examined produce recognizable size classes which can be related to annual or semi- annual spawning events; (iv) The largest sponges resulted from larvae which set- tled out within the first year after Hurricane Allen. With respect to assumption (i), destruction in the re- gion surveyed in 1 986 was extensive; no large corals (e.g., Montastrea annularis) or sponges survived. The area is seaward of site B in Woodley el al. (1981) where there was extensive damage to gorgonian colonies. This site (similar in profile to E in Fig. 1 of Woodley et al.. 1981) was fully exposed to wave action because it slopes gently, thereby acting as the final repository for considerable amounts of Acropora rubble from shallower depths. Hence, it is unlikely that any sponge fragments survived in this habitat. All sponges appeared to be individuals that had settled on the rubble. Of the five sponges present in sufficient numbers to warrant collection for this study, three form distinct. C R. WILKINSON AND A. C. CHFSH1RI 800 600 400 200 100 § I HI-IH M| osmoregulalion and s.ilinity tolerance in UMI ilei.ipod crustaceans: Hiiutit\ inm-riii*f. and .\\inlui ili\/xir . . 154 Zimmer-Faust, Richard K .. Richard A. Gleeson, and William E. S. Carr The behavioral response of spiny lobsters to ATP: evidence for mediation by P2-like chemosensory re- ceptors 167 SHORT REPORT Wilkinson, Clive R., and Anthony C. Cheshire Grov>ith rate of Jamaican coral reel sponges after Hurricane Allen 175 Volume 175 THE Number 2 BIOLOGICAL BULLETIN Marine Biological Laboratory LIBRARY DEC 9 1988 Woods Hole, Mass. OCTOBER, 1988 Published by the Marine Biological Laboratory THE Marine Biological Laboratory LIBRARY DEC 9 1988 Woods Hole, Mass. BIOLOGICAL BULLETIN PUBLISHED BY THE MARINE BIOLOGICAL LABORATORY Editorial Board GEORGE J. AUGUSTINE, University of Southern California RUSSELL F. DOOLITTLE, University of California at San Diego WILLIAM R. ECKBERG, Howard University ROBERT D. GOLDMAN, Northwestern University EVERETT PETER GREENBERG, Cornell University MICHAEL J. GREENBERG, C. V. Whitney Marine Laboratory, University of Florida JOHN E. HOBBIE. Marine Biological Laboratory LIONEL JAFFE, Marine Biological Laboratory HOLGER W. JANNASCH, Woods Hole Oceanographic Institution WILLIAM R. JEFFERV, University of Texas at Austin GEORGE M. LANGFORD. 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Authors will be charged the excess over $100 of the total of (a) $30 for each printed page beyond 1 5, (b) $30 for each table, (c) $ 1 5 for each formula more com- plex than a single line with simple subscripts or superscripts, and (d) $15 for each figure, with figures on a single plate all considered one figure and parts of a single figure on separate sheets considered separate figures. 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 for foreign authors) will receive page proofs of articles shortly be- fore publication. They will be charged the current cost of print- ers' time for corrections to these (other than corrections of printers" or editors' errors). Reference: Biol. Bull. 175: 181-184. (October, 1988) Morning Release of Larvae Controlled by the Light in an Intertidal Sponge, Callyspongia ramosa SHIGETOYO AMANO Cancer Research Institute, Kanaiawa University. Kanazawa. Ishikawu 920, Japan Abstract. The intertidal sponge Callyspongia ramosa releases larvae in the morning under natural light. The photic control of this morning release was studied under experimental light-dark (LD) cycles. Under LD 12:12h cycles (light period, from 6:00 to 18:00), release peaked about 6:00. But the release was not stimulated by the illu- mination in the morning because the sponge colonies re- leased larvae even in the darkness. The experiments un- der various light regimes showed that the photic stimulus is not the onset of darkness but. unexpectedly, the onset of light the day before. In fact, C. ramosa colonies invari- ably released larvae about 24 hours after the onset of light under all illumination regimes tested. The tidal cycle and the daily cycle of the seawater temperature did not in- fluence the time of release. Therefore, in nature, the dawning light most likely stimulates larval release on the next day. This photoadaptation in the larval release of C. ramosa suggests that the morning release is advanta- geous for their free-swimming larvae to seek out and set- tle on the suitable substratum in the intertidal region within their short dispersive period. Introduction Many sponges are phototrophic and some derive at least 50% of their energy requirements from large popu- lations of photosynthetic symbionts, usually blue green algae (Wilkinson, 1983, 1987). Obviously these sponges must occupy a substratum of full sunlight. The free- swimming larvae of such species are probably capable of locating themselves on a habitat exposed to the sun in this dispersive period. On the other hand, the free-swim- ming larvae of the sponges adapted to the shady habitat must selectively settle themselves on a shaded substra- tum. The free-swimming larvae of many sponges re- Received 23 May 1988; accepted 25 July 1988. spond to light (Warburton, 1966: Bergquist and Sinclair, 1968: Uriz, 1982a, b). However, it has not been shown experimentally that the larvae of phototrophic and non- phototrophic sponges show different patterns of behav- ior in habitat selection (Bergquist etai. 1970; Fell, 1974). Morning releases of larvae have been reported in sev- eral sponge species (Levi, 1951, 1956). Halichondria panicea (order Halichondrida) also releases larvae in the morning (Amano, 1986). It was shown that the stimulus for larval release in this sponge is the onset of darkness the evening prior to release, which occurs 1 5 hours later. In this report I show that Callyspongia ramosa (order Haploschlerida) releases larvae in the morning, but its stimulus for release is the onset of light the day before. This is quite different from //. panicea. The ecological significance of these photic controls of larval release and of the photoadaptations in the habitat selection of sponge larvae are discussed. Materials and Methods Callyspongia ramosa colonies were collected in June from rafts at the Breeding Center of Aomori Prefecture, in northern Japan, about three kilometers from the Asa- mushi Marine Biological Laboratory where all the exper- iments were performed. Sponge colonies were collected carefully to minimize damage. They were placed in water-tight containers under water, brought to the labo- ratory, and transferred into running seawater. Sponges were maintained in clean running seawater to ensure their health throughout the experiments. In the early morning following collection, colonies re- leasing many larvae were selected. Larvae filled the me- sohyle of such colonies. Usually colonies released larvae for more than ten days in the laboratory. The larvae re- leased from a colony under experimental LD cycles were counted using the method described previously (Amano, 181 182 s \\l\\0 Ijj 1 3UU CL < 1000 u. 0 UJ 50° 1 . 3000 2000 1000 0 3 6 9 12 15 18 21 0 TIME OF DAY Figure 1. Typical lanal release pattern off \ill\ \fnnviti ramo\a un- der LD 12:12h e>cles (light period, from 6:00 to 18:00). The colony released largest number oflarvae between 6:00 and 9:00. and the num- bers decreased thereafter. The arithmetical mean of the release time (MR I) is 6:44. At the top. illumination schedule is shown: the black- ened bar indicates the dark period and the white portion, the light pe- riod. 1986). Briefly, the colony was fixed with a cotton thread in a photographic developing tank and supplied continu- ously with running seawater. Thus it could be illumi- nated or shielded from light at will without interrupting the seawater supply. The released larvae were washed with the outflow, caught by a piece of nylon mesh ap- plied to a plastic vessel, and counted every three hours. Results Although the exact reproductive period off. ramo^i in nature is unknown, it released parenchymula larvae in June in the Asamushi Marine Biological Laboratory. The larvae ejected from a osculum swam just below the water surface. The dimensions of a typical larva were about 250 X 150 /urn. The larvae were thickly covered with flagella except for a bare posterior pole, and the bare pole was encircled with a band of long flagella. They were dull yellow but contained reddish brown pigments in the posterior pole. Under natural light. (' riiini>\ci released larvae around dawn in the laboratory. This apparent diurnal periodic- ity was confirmed under artificial LD 1 2: 1 2h cycles. Fig- ure I shows a typical larval release off. rcimusa during a 24-hour-period under these conditions. Many larvae were released before 6:00. but most were released be- tween 6:00 ami (':00: the numbers decreased graduallv after 9:00. "I he mean release time (MRT) was arithmeti- cally calculated with these data, the MR I of the larval release shown in I igurc I is 6:44. The results in Figure I suggest that the onset of light on that morning could not be a photic stimulus to the larval release because main larvae had been released be- fore 6:00 while still dark. I his suggestion is shown to be true in Figure 2. A sponge colonv that had been under 0 3 6 9 12 15 18 21 0 3 S 9 12 15 18 21 0 3 6 9 12 15 18 21 0 TIME OF DAY Figure 2. Inhibition of larval release brought about bv continuous darkness on the da> before. ( 'allyspongia raitwiu released larvae in the dark on the first day. but did not release on the second day despite illumination from 6:00 to 18:00. MRTs of the 1st and 3rd days are 7: 1 4 and 7:26. respectively LD 12:12h cycles was kept in the dark on the first day. On the second and third days it was illuminated under LD 12:12h cycles again. On the first day. the colony released larvae in the dark as if it were still under a LD 12:12h cycle. It did not release on the second day, al- though it was illuminated from 6:00 to 1 8:00. This result was not brought about by the loss of release ability of the colony because it released larvae as usual on the third day. These results show that the time of release had been determined by the illumination on the day before. I his conclusion is consistent with that of Halichondria pani- < r<; (Amano. 1986). Figures 3 and 4 show the results of the experiments designed to determine which photic stimulus, the onset of light or the onset of dark on the day before, triggers larv al release. In Figure 3. a colony was illuminated from 12:00 to 18:00: that is putting off of the onset of light bv six hours. This putting off of the onset of light delayed the next day's larval release for about 4.5 hours. In Figure 4. a colony was illuminated from 6:00 to 12:00. that is advancing the onset of darkness by six hours. This ad- vance, however, did not significantly advance the time of release on the next dav. The delav of the larval release uj 4000 < 3000 0 2000 or ID § 1000 z ~h 0 3 6 9 12 15 18 21 0 3 6 9 12 15 18 21 0 TIME OF DAY 1-iKiirc.V Delay of larval release brought about by six hours' putting nil nl the ousel of light on the first day. The release on the second day was dclavcd loi .ihout •) ^ hours. MRTs of the 1st and 2nd days are 6:47 and 1 1 : 1 8, respective!) LARVAL RELEASE IN SPONGE 183 uj 3000 -1 2000 fe — CE £ 1000 z •z. r — i . . . . ~L . . . 0 3 6 9 12 15 18 21 0 3 6 9 12 15 18 21 0 TIME OF DAY Figure 4. Although the onset of darkness was advanced for six hours, the colony released larvae about the same time on the second day as that under LD 12:12 h cycles. MRTs of the 1st and 2nd days are 7:26 and 5:32, respectively. in Figure 3 was not a result of the shortening oflight pe- riod because the colony in Figure 4 was also illuminated for six hours. Thus it is clear from these results that larval release was stimulated by the onset of light on the day before. This conclusion is distinctively different from that of H. pauicca where the onset of darkness was the stimulus (Amano, 1986). Results of Figure 5 confirm the above conclusion. If the onset oflight is truely a stimulus, the time between photic stimulus and larval release must be about 24 hours because C. ramosa released larvae at about 6:00 under LD 12:12h cycles (light period, from 6:00 to 18:00). Instead, if the onset of dark were the stimulus, the duration should be about 12 hours. Preliminary ex- periments showed that a dark-adapted C. ramosa colony reacts to illumination for one hour. Figure 5 shows the result of one such experiment where a colony was illumi- nated from 17:00 to 18:00. Under these conditions it re- leased larvae around 17:00 on the next day, that is, after about 24 hours. Table I summarizes the results of the experiments pre- sented in this paper and shows the temporal relationships 2000 UJ cr 1500 O 1000 cr LU CD 5 500 0 3 6 9 12 15 18 21 0 3 6 9 12 15 18 21 0 TIME OF DAY Figure 5. Larval release brought about by one-hour illumination. The colony released larvae about 24 hours after the illumination. MRTs of the 1st and 2nd days are 6:59 and 17:04, respectively. Table I Constant temporal relationship of larval release to onset oflight on the day before under various illumination regimes in callyspongia ramosa Hours from onset oflight* Hours from onset of dark* Reference 24:44 12:44 Fig. 1 23:18 17:18 Fig. 3 23:32 17:32 Fig. 4 24:02 23:04 Fig. 5 * Duration between onset time of light or dark and arithmetical mean of release time. between larval release and the two possible photic sig- nals. Durations between the onset oflight on the day be- fore and larval release are constantly about 24 hours un- der all illumination regimes tested. On the other hand, the onset of dark has variable temporal relationships to larval release. Thus, at least under these experimental conditions, the larval release of C. ramosa is brought about by a photic stimulus, that is, the onset oflight on the day before. Discussion Under natural illumination, C. ramosa releases larvae in the early morning with apparent diurnal periodicity. It is obvious from the results shown in this paper that the larval release is controlled by the light and does not depend on other possible environmental factors. Larval release is independent of the tidal cycle and the daily change of seawater temperature because it showed no temporal relationship to such factors during this study. Besides, it is not controlled by a circadian rhythm be- cause a release peak occurred only once in the continu- ous darkness. It must be noted, however, that all of the results shown in this paper are based on laboratory ex- periments. I have yet to study larval release of a sponge in the field; it might be possible in a particular species only. The morning releases of larvae have been reported in several sponge species: Halichondriapanicea (Amano, 1986), Ha/iclonapermolis(A.mano, unpub. data), Oscar- ella lobnlaris, Hymeniacidon sanguinea. and Halisarca metsclmikovi (Levi, 1951, 1956). Thus the morning re- lease of larvae is likely to be ubiquitous in viviparous sponges. I have already shown that the onset oflight on the day before is the photic stimulus to the larval release of C ramosa (order Haploschlerida) (see Results). In fact, col- onies of C. ramosa released larvae about 24 hours after the onset of light under all illumination regimes tested (Table I). In nature, the dawning light is probably a stim- ulus to the larval release of the next day. Therefore C. ramosa may release larvae a little earlier even on a dark, cloudy morning if the previous morning was clear. In 1X4 s \M\NO Halichondria panicea (order Halichondrida), however, the photic stimulus '•>! its larval release is the onset of darkness on the prcv ions da> ( Amano. 1986). Thus, the onset of light be a photic stimulus in the order Haplosch! i the onset of darkness ma\ he a stim- ulus in UK Halichondrida. although more studies are required to verify this presumption. Which receives the photic stimulus, a mother sponge or the lar\ae?The sponge has no ovary: larvae are eiue- loped by a layer of follicle cells and embedded in the mesohyle. The structure of the follicle and that of the mcsohyle seem too simple to receive the photic stimulus and to perform complicated functions associated with lar\al release. It is more likely that the larvae receive the stimulus directly. Light can reach the larvae through the mesohyle because ( >ani<<\a colonies are small and pale in color. When stimulated by the onset of light, mature larvae ready to be released may migrate toward an exhal- ant canal. Although almost nothing is know n about their migration, the larvae must rupture the follicle and move toward an exhalant canal through the mesohyle (Brien and Meewis. 1938: Fell. 1464). ( nunoMi larvae max require about twenty-four hours to reach the exhalant canal, that is to say. this is probably the lag time before release. To test this hypothesis, the migration of larvae within the mesohyle must he studied in detail during the twenty-four hours following the photic stimulation. Although sessile animals of acquatic habitat belong to taxa which are very ditlerent phylogenetically. they max be grouped together ecologically as producers of disper- sive propagules such as planktonic larvae (Sara. 1984). This has been an evolutionary trend, as dispersion and substrate occupation are obviously essential for the suc- cess of any sessile animal (Meadows and Campbell. 1972). In the sponge, all viviparous species produce free- swimming larvae as dispersive propagules. It is notewor- thy that most intertidal sponges are viviparous and most oviparous sponges inhabit the deeper sea-tloor ( Reiswig. 1976: Watanahe. 1978: Simpson. 1980). Viviparity ap- pears to be advantageous lor the intertidal sponges to oc- cupy a suitable substratum (Bergquist and Sinclair. 196S: Ayling. 1980). If they were oviparous, their eggs and de- veloping embryos might he swept away from the inter- tidal region by strong currents and waxes during a long embryonic period. Free-swimming larvae seem to he able to find and settle promptly on a suitable substratum within the dan. ;(>6; Bergquist eta!.. 1970; I riz, i H !a hi. I he photic controls ol larval release shown in this and in a previous paper (Amano. 1986) arc probably two instances of the adaptation for efficient dispersion of the swimming larvae of these ses- sile sponges in the interlidal region. Acknowledgments Sincere thanks are expressed to Dr. Numakunai and other members of the Asamushi Marine Biological Lab- oratory for their hospitality and help during my stay. I am grateful to T. Mayama. S. Tamura. and M. W'ashio who collected the sponges. Literature Cited Amano, S. 1986. Larval release in response to a light signal bv the intertidal sponge Halichondria panicea liml Hull 171:371-378. Ayliny. \. I . 1980. Patterns of sexuality, asexual reproduction and recrmlment in some subtidal marine demospongiae. Biol. Bull 158: 271-2X2. Beruquist, P. R., and M. F. Sinclair. 1968. The morphology and be- haviour of larvae ol some intertidal sponges. .\ '. / ./ Mar /-Vcs/i- ual K«-v 2:426-437. Bergquisl. P. R., M. K. Sinclair, and J. .1. Hogg. 1970. Adaptation to intertidal existance: reproductive cycles and larval behaviour in demospongiae. .S r»i/i /<><>/ St>c /.nm/. 25: 247-271. Brien. P.. and II. Meow is. 1938. Contribution a I'etudcde 1'embrvo- genesedesSpongillidac. Arch. Hint. 49: 177-25(1. Fell. P. F. 1969. The involvement ol nurse cells in oogenesis and em- bryonic development in the marine sponge. Haliclona echasis. J. Morpliol. 127: 133-150. Fell, P. K. 1974. Pont'era. Pp. 51-132 in Rci'iodiulion ,>l Marine In- nvichratc"*. Vol. I. A. C. Giesc and J. S. Pearse. eds. Academic Press. NY Levi, C. 1951. I 'ovip.mtechez les spongiaires. ( R \t,iti Sci Paris 233: 272-274. l.o\i, ( . 1956. F.tude des llalisarca de Roscoff. Embryologie et sys- tematique des demosponges. inli /m>l Exp. Gen.93: 1-181. Meadows, P. S.. and .1. I. Campbell. 1972. Habitat selection b\ acqualic invertebrates. lnni;i- liiology. \ . W. I larrison and R. R. Cowden. eds. Academic Press. NY. Sara, M. 1984. Reproductive strategies in sessile macrofauna. Boll /MO!. 51:243-248. Simpson, T. I.. 1980. Reproductive piocesses in sponges: a critical evaluation ol current dala and \ievvv //;/ ./ Inwru-hr. Rcfirnd. 2: 251-269. tlriz, M. .1. I982a. Reproduccion en Hymeniacidon sanguined « ii a nt. 1X26): hiologiadela larva v primeros estadios postlarvarios Im- 1'c^i -46: 29-39. I n/, M. .1. 1982b. Morfologia v comportamiento de la larva paren- quimula de Scu/'iilma lophyropoda Schmidt 19S2 (Demospongia, Halichondrida) v fonmacion del rhagun. Inv. I'esti 46: 313-322. \\arhiirt(in. !•. V.. 1966. I he behavioui ol" sponge larvae. / m/. \\ilkinson. (. R. 1983. Net primary productivitv in coral reef sponf.es S,(,w, '219:410-412. \\ iikiiisnii. ( . R. i')87. Interocean differences in size and nutrition of i oi. il ieel sponge populations S. ii-ih r 236: 1654-1657. Reference: Biol. Bull. 175: 185-192. (October, 1988) Characterization of the Molt Stages in Penaeus vannamei: Setogenesis and Hemolymph Levels of Total Protein, Ecdysteroids, and Glucose SIU-MING CHAN*. SUSAN M. RANKIN**, AND LARRY L. KEELEY Laboratories for Invertebrate Neuroendocrine Research. Department of Entomology, Texas A&M University, Texas Agricultural Experimental Station, College Station, Texas 77843 Abstract. The molting cycle of Penaeus vannamei ju- veniles was characterized by distinct and predictable changes in the setae of pleopods. The molt pattern was diecdysic with a relatively short intermolt period (40%) and a long proecdysial period (>53%). The levels of both total protein and ecdysteroids increased in the hemo- lymph during proecdysis, whereas the level of hemo- lymph glucose was low at metecdysis and proecdysis and maximal during anecdysis. As revealed by SDS-PAGE, the relative concentrations of two polypeptides (32 kD; 175 kD) changed during the molting cycle. Introduction Molting in arthropods includes not only the act of ec- dysis, but also new cuticle formation, apolysis, the imme- diate postecdysis, and tissue growth (Passano. 1960). This dynamic cycle has been divided into four phases in crustaceans: (1) metecdysis (stages A, B), the period immediately following ecdysis; (2) anecdysis (stage C). a period of tissue growth and accumulation of food re- serves; (3) proecdysis (stage D). a period of active mor- phological and physiological changes in preparation of the next molt; and (4) ecdysis (stage E), the shedding of the old cuticle (Drach, 1939). Several methods are used to determine the molt stages of crustaceans. These methods include histological ex- amination of the integument, measurement of the size of the gastroliths or the regenerating pereiopods. and deter- Received 10 March 1988; accepted 28 July 1988. * Department of Wildlife and Fisheries Sciences. Texas A&M Uni- versity. ** To whom correspondence should be addressed. mination of setal development on the appendages. De- termination of molt stages by the state of setogenesis on the appendages is rapid and inflicts little harm to the ani- mals, even after repeated sampling. Setogenesis is used as a criterion to stage a number of decapods, including the natantians (Scheer, 1960; Kamiguchi. 1968). an- omurans (Kurup. 1964), and macrurans (Aiken, 1973). Among the penaeids, criteria for assessing molt stages are described for Penaeus duorarum (Schafer, 1968), Pen- aeus merguiensis (Longmuir, 1983), Penaeus escidentus (Smith and Dall, 1 985), Penaeus stylirostris (Huner and Colvin, 1979; Robertson eta/., 1987), and Penaeus set if- enis (Robertson et ai, 1987). Molting is stimulated in Crustacea by one or more of a group of closely related steroid hormones, the ecdyste- roids (Skinner, 1985). Hemolymph ecdysteroids during the molting cycle have been measured in only a few deca- pods by use of radioimmunoassay (RIA) (Andrieux et ul.. 1976; Chang et a/.. 1976; McCarthy and Skinner, 1977; Keller and Schmid, 1979; Stevenson et a/., 1979; Chang and Bruce, 1980; Charmantier-Daures and De- Reggi. 1980: Hopkins, 1983; Jegla et a/.. 1983; Soumoif and Skinner. 1983). No determinations are reported for penaeid shrimp. In most cases, hemolymph ecdysteroid liters increase rapidly during proecdysis; however, pre- cise patterns are species-specific (e.g.. Stevenson, et al.. 1979; Chang and Bruce, 1980). Other hemolymph pa- rameters, such as the levels of glucose (Telford, 1968) and protein (Dall, 1974) also undergo cyclic changes that correlate with the molt stage. This paper describes the molting cycle of Penaeus van- namei, an economically important shrimp in the mari- culture industry of the southern United States. We have characterized molt stages based on setogenesis of the 185 186 S. M. CHAN KT AL plcopods. determined hemolvmph levels of protein, glu- cose, and ecdysu . and analv/ed the hemolymph proteins b> poK n le gel electrophoresis (PAGE). i.ils and Methods Animals r \annamei juveniles were purchased from Ocean Venture Inc. in Port Lavaca. Texas. Thev were held for two weeks in 250 gallon circular tanks for acclimation at 20-22°Candatasalinitv of28-30%o.Shrimp(l 1.5-13.0 cm total length) \\ere maintained individually in plastic hoses (23 cm X 12.5 cm X 12 cm) and fed once daily with commercial maturation diet at a rate of 4'"< total hod> weight (Rangen Inc.. Buhl. Idaho) v .'genesis To measure setogenesis. the distal third of the pleopod was excised, floated in saline on a microscope slide, and observed at 100X with a compound microscope. Photo- micrographs were prepared from pleopods mounted in saline. Setal development was observed at 6-h intervals for 2 days after ecdysis and then twice daily until the next molt. Hemolymph measurements Forty n\ of hemolymph were withdrawn through the arthropodial membrane of the fifth pereiopod using a chilled microsyringc. HemoKmph sampling was per- formed with minimal handling of animals and took <20 s. This brief period of handling precluded induction of an endocrine-dependent stress response that would in- fluence the levels of hemolymph metabolites during the time of sampling. All sampling was performed 2-5 h af- ter the onset of the photophase. Sampling of pleopods and of hemolymph at four-day intervals had no apparent effects on the animals. Results from both sexes were pooled since preliminary studies had indicated no sex- related differences in hemolymph metabolite levels. I cdysteroid liters were determined in the hemolymph by RIA. Ecdysone antiserum was a gift from Dr. W. E. Bollenhacher (University of North Carolina. Chapel Hill). 23.24 !H2(N) ecdysone (specific activity 58-60 Ci/ m.U) was purchased from New England Nuclear (Bos- ton. MA): the ecdysone standard was purchased from Sigma ' In mi al Co. (St. Louis, MO). Ten pi of hemo- lymph were added to 150 pi methanol. After precipita- tion of protein lor l> minutes at 4°( . the sample was centrifuged for 5 minutes at 12.000 ' .t,' The supernatant was collected and cva|"i'.iu-d under nitrogen, and ecdy- steroids were measured I ( hani1 <•/<;/, 1976). Hemolymph total protein was measured using the Coomassie brilliant blue test i hiadtord. 1976). Hemolymph glucose was determined by the glucose oxidase procedure ( Mark, 1 959) using a commercial re- agent kit (Sigma ( 'hemical Co.). Polyacrylamide gel eU\ tro^lim-esis (PAGE) HemoKmph proteins were analy/ed b> slab gel elec- trophoresis (Laemmli. 1970). Native PAGE (6<7< ). was performed with hemolymph mixed with a sample buffer (0.125 M Tris-HCI. pH 6.8) containing 0.01'" brom- phenol blue as the tracking dye. SDS-PAGE (10-15% linear gradient) was performed with hemolymph incu- bated in sample buffer containing 2r; SDS and 1% 2- mercaptoethanol for 15 minutes in a boiling water bath. Electrophoresis was run at 50 V in the stacking gel and 1 00 V at the resolv ing phase. Gels were stained with Coo- massie blue R for total protein or w ith dithiooxamide for copper (Whittaker. 1959). Results 1 he molting cycle: setogenesis Setae are external outgrowths from appendages such as uropods. pleopods. and antennule scales. In P. vanna- mei. the degree of setal development was not identical on different appendages and in different regions of the same appendage. For example, if pleopod setae were in stage D0. most of the setae on the antennule scales were still in late anecdysis (C3). Similarly, when the setae on the proximal portion of the pleopod entered proecdysis. the setae on the distal half of the pleopods were still at anecdysis. To standardize the criteria for determining molt stages, setogenesis was based on changes in the setae on the distal third of the pleopods. Singe A (metecdysis). Stage A lasted about one day (Table I), and the newly molted animals were inactive and did not feed. The exoskeleton was soft, parchment- like, and uncalcihed. The epidermis was transparent with little pigmentation. Setal lumens were filled with translucent fiber-like matrices (Fig. 1 ). and the epidermis near the setal base was less granular than that of later stages. Stage n Imetectlysisi. The exoskeleton hardened, pre- sumably due to deposition of calcium, and epidermal pigmentation increased. At this time, setal matrices ap- peared granular and began to retract from the setal lu- mens towards the bases of the setae. An internal cone (conical base) began to form in each seta during later Stage B. Sttige ( (anecclysis). Calcification of the exoskeleton was completed. This stage occupied 10 to 15 days (35- 40'. ) of the mtermolt period (Table I). Animals were maximally active at this stage and resumed feeding. Most of the setal lumens were clear ol'setal matrix at this time. MOLT STAGING IN PENAEVS VANNAME1 Table I Characteristics ofl/ie molting slages of juvenile Penaeus vannamei 187 Stage Duration Feeding/activity Exoskeleton Epidermis Setal development A B 1 8-28 h< 1-2%) 2 3-40 h( 3-4%) none/weak none/restored soft hardened transparent granular granular matrix fills lumen granular matrix retracts; internal 1.5davs(5%) restored/maximal hard granular cone formation begins; setal organs become visible granular matrix retraction completed, internal cone formation completed C2 6-8 days (20%) maximal/maximal hard granular c, 4-7 days (15%) maximal/maximal hard granules very dense D0 3-6 days (15%) decreasing/maximal No new cuticle yet retracts (apolysis) D, 8- 10 days (28%) decreasing/maximal New cuticle appears invaginates new setae begin to develop D2 2-3 days (6-7%) Space forms mvaginates new setae form barbules between old and new cuticle D, 1-2 days (3-4%) no feeding, water is old skeleton soft old setal organs disappear, new setae absorbed fold E 1-2 min no feeding, body old cuticle is shed expands Formation of internal cones was completed in the C, stage (Fig. 3). Setal organs, cylindrical structures that give rise to the setae (see Aiken, 1973), became clearly visible in the C2 stage (Fig. 4). The C3 stage differed from the C2 stage (Fig. 4) in that during C, the setal bases were more dense and the setal organs more distinctive (Fig. 5), pre- sumably due to mobilization of granules in the epidermis just before proecdysis. Stage D (proecdysis). Proecdysis lasted 15 to 19 days and could be divided into stages D0-D3 . Early proecdysis began with apolysis (Jenkin. 1966), the separation of the endocuticle from the epidermis. In P. vannamei, the pro- cess began first in the posterior region in the endopodites (Fig. 6) and was accompanied by the resorption and pre- sumed decalcification of the exoskeleton. New cuticle was not present at the D0 stage. As the epidermis re- tracted, it invaginated (D, ) (Fig. 7) at the setal bases as new cuticle was deposited (D, ). At the late Dr stage, the new epidermis continued to invaginate and new setae be- gan to develop and protrude from the new cuticle (Fig. 8). At the D2 stage (Fig. 9), new setae formed barbules and the setal spines extended into the base of the former seta. The epidermal retraction continued and resulted in large empty spaces between the old and new exoskele- tons(D3). Setal organs were no longer evident as discrete organs. Presumably as a result of muscular contraction, the new setae folded, and disrupted the regular pattern of setal arrangement (Fig. 10). Immediately before ecdysis, late proecdysis was characterized by absorption of water, expansion of the body, muscular contraction/relaxation. and breakage of the intercalary sclerites in the abdom- inal dip. Stage E (ecdysis). As the animal shed the exuvium, the invaginated setae everted. Ecdysis lasted for less than 2 minutes (<1%). Early post-molt animals usually did not feed, although some newly molted animals consumed the old exuviae, possibly to recover calcium and other cuticular components. The molting cycle: hemolymph ecdysteroids and metabolites We measured levels of circulating ecdysteroids and metabolites during different stages of the molting cycle as determined by the criteria of setal formation and mor- phology described above. Figure 1 1 shows the ecdysteroid levels in the hemo- lymph during each molt stage. Ecdysteroid titers were approximately 30 pg/pl during metecdysis (A and B). The titers dropped to a minimum of about 16 pg/^1 at stage C:, began to increase at C3 and reached a maxi- mum of approximately 200 pg/n\ at D, . The increases in ecdysteroid liter correlated with the onset of proecdy- sis and the events related to new cuticle formation. The pattern of hemolymph protein levels was similar to that of the ecdysteroids. Hemolymph protein levels were low during metecdysis (20 mg/ml) and anecdysis (C,) and increased to a plateau (85-95 mg/ml) at the pro- ecdysis (D3)( Fig. 12). The changes in levels of hemolymph glucose (Fig. 13) during the molting cycle did not resemble the S-pattern 188 S. M. CHAN KT AL 3 . I ;irl> metecdssis. stage A. Most of the setal lumens arc filled with setal matrix (sm). The bases of setae (sb) are agranular and setal articulations (sa) are opaque. (100X). scale bar = 45 ^m. Figure 2. Mctecdysis. stage B. The setal matrix (smi lias begun to retract towards the base of the seta (sb). revealing the setal lumen (si). ( IOOX). scale bar = 45 pm. Huurv .V \nccd\sis. stage CV Retraction ot'selal matrix (sm) has ne.iied completion and the setal lumens (si) are almost empty . Forma- tion of internal cones (ic) has been completed in most setae (IOOX). scale bar = 45 nm. Hcurt- 4. \needysis. stage CV Setal organs (so), which gi\e rise to the internal cones (ic). and setal articulations (sa) arc evident. ( Kid • i. scale bar = 45 pm. observed for the protein and ccdvsteroid tilers. The glu- cose levels were lowest immediately before and alter the molt and increased significantly (I' < O.O.v /-test) during anecdysis (€3). Thirteen proteins were detected in stained native gels: the dominant protein. #6. stained positively for copper with dithionxamide (data not shown) and is therefore likely to be the respiratory protein. hemoc\anin. No dis- tinctive changes in proteins during the molting c\cle were revealed by native I sl)S-l'\(il- ofhemolymph proteins of the molt cycle revealed more than 2(1 pol\- peptidcs (Fig. 14). No changes were observed in the ma- jor polypeptidcs: however, ol the less abundant polypep- tides. one small (32 kD) and one high molecular weight polypeptide (175 kD) changed in relative abundance during the molting cycle. The relative concentrations of these two polypeptidcs were low during late metecdysis (B) and anecdysis (C). and increased during proecdy- sis(D). Discussion The molting cycle in crustaceans is characterized by distinct morphological, physiological, and biochemical events. We have identified and characterized several of these parameters for the South American white shrimp. /' \itnnamci. -so I 8 os- I1 iyurc 5. Anccdvsis. stage ( ',. Setal bases (sb) ha\e become denser anil seial OIJMMS (so) moie distinct. Well-defined internal cones (ic) are evident. ( Kid- ). scale bar 45 »m. I'iuiiri- 6. l'ioecd\sis. stage D0. Apolysis. The separation of the cpi- deunisiepi hum the cuticle is evident. ( IOOX). scale bar =- 55 /jm. Hniin- 7. 1'ioecdysis, stage D, . Invagmalion u\ ) ol the epidermis (ep) has left a clear space between the old cuticle and the epidermis. I he new setae (ns) and cuticle have begun to form. ( lOOx), scale bar = 45 ftm. I inure S. Proeedysis. stage D,-. The epidermis (ep) has continued to invaginate (iv); new barbules (h) have formed on some new setae (ns).(K)Ox). scale bar - MOLT STAGING IN PENAEUS VANNAME1 189 Molt stages ns »- 10 Figure 9. Proecdysis. stage D:. New barbules (b) are present on all new setae (ns). ( lOOx), scale bar = 30 ^m. Figure 10. Proecdysis, stage D, . The pattern of new setae (ns) is interrupted by folds of the epidermis (ep). The animal is ready to molt in this stage. ( 120x) scale bar = 40 nm. The crustacean molting cycle: setogenesis Although setogenesis has been used as a criterion for molt staging for many years (see Drach, 1939). species variations in setal morphology and development result in differences among crustaceans in both staging criteria and in easily-defined subdivision of the molt stages. We have established criteria for the molt stages and substages in P. vannanu'i. These criteria include the discernment in the pleopods of the epidermis, setal lumens, internal cones, and setal organs (see Figs. 1-10). Similar criteria have been used to determine stages for the penaeidae shrimp P. ciiliforniensis, P. stylirostris (Huner and Col- E 1 c . Q. _>. O 12 for each point). vin. 1979). P. mcrguiensis (Longmuir. 1983). P. duor- arum (Schafer, 1968) and P. esculentns (Smith and Dall, 1985). Setogenesis in these shrimp species differs primar- ily in the degree of pigmentation in the appendages and in the duration of the molt stages. Furthermore, the com- plete retraction of the setal matrices was observed in other penaeids during anecdysis, whereas in some indi- viduals of P. vannanu'i retention of setal matrices was observed. Deviations in setogenesis are even more pro- nounced in other decapods. For example, in the lobster Panulims nuirginulm. internal cones are lacking; thus. AB c Molt stages C O) Q. c > I V c o (A •D O 4> .c Q. "5 o 250 - 200 - 150 - 100 • 50 - • i D D D D D 0 — 1' — 1" • 1"' 2,3 • 10 — i — — I — 20 Time (days) 30 Figure 11. Ecdysteroid liters in the hemolymph of juvenile I3 van- namei during Ihe molting cycle. Each point represents the mean ± stan- dard error of the mean (S.E.M.) (n > 12 for each point). 0) in o o 3 ABC 55 T !• 45- 35- 25 - 15 Molt stages C D D D D D 3 0 — V 1" - 1" 2,3 . 10 20 Time (days) 30 Figure 13. Total glucose levels in the hemolymph of juvenile P. vannamei during the molting cycle. Values represent mean ± S.E.M. (n > 12 for each point). 190 S M CHAN £7 .-It. 17 205- 115- 97- r 66 -175 45- 29- *. -32 cean molting cycle: (1) anecdysic that has a relatively long intermolt and (2) diecdysic that has a long premolt (Knoweles and Carlisle. 1956). In general, crustaceans with an anecdysic molting cycle usually enter a terminal anecdysic (Cjj) stage (Skinner. 1985). Whether the molt- ing cycle of adult P. vanminici (which continue to molt throughout their life) is diecdysic or anaccdy sic remains to be determined, but in juvenile /' vunnaniei. the pre- molt period occupied 50-55% of the molting cycle. Thus, the molting cycle of juvenile P. vannamci is diec- dysic (Table I). Our observations on durations of molt stages in juvenile P. vannaniei are similar to those re- ported for juvenile P. esciilentus (Smith and Dall. 1985); however, in juvenile P. iner^iiiensis (Longmuir. 1983). juvenile P. califomiensis, and juvenile P. xty/irostris (Huner and Colvin. 1979) the D(, stage is proportionally much longer. C3 DO D1 D2 D3 Figure 14. 1 0-1 5' linear gradient SDS-PAGE of hernolymph pro- teins from juvenile P rumiamt'i during the molting cycle \ alues in the tar left column indicate molecular weight determinations (kDl. Arrows indicate polvpeptides which increase in relative quantities as the molt- ing cycle progresses. Letters (bottom (indicate the molt stage. the distinction between stages B and C depends mainly on the thin and hollow appearance of the setal lumen in anecdysisfLyle and MacDonald. 1983). These examples emphasi/e that molt staging must rely on a combination of setal characters. Furthermore, substaging varies ac- cording to the investigators. We found that the molting cycle in I' vannaniei was readily divided into stages: A. B. C, ,. and D0 i. In the crayfish Asiacm leptodactylus, the molting cycle was divided into AI_:. B, :. C,_4. and I ),, ; (Van Herp and Bellon-Humbert. 1978). C4 and D4 stages were not described in P. vannamei because those putative stages were of extremely short duration. Molt staging may he accomplished using setogenesis in a variety ol appendages. These appendages include the pk-opods, as demonstrated in /'. nnir^inuiiis (Lyle and MacDonald. 1983). .1 lcpt<>iliictylu\ (Van Herp and Bellon-Humbert, 1978) and (hchc^tiu ciivnuinti (Ciraf. 1972): the maxillae in (.'hionoeeeie^ npilin (Moriysau and Mallet. 1986); and the uropods in Petroli\ilu^ cin- neclines (Kurup. I9(>4). /' \n-///vn//vs (Huncr and Col- \m. 1979: Robertson eta/.. 1987) and/' v<-///mM Rob- ertson el nl.. 1 987 ). We have used the pleopods for delei - mmation of molt stages in /' Minihiinci because removal of other appendages results in trauma or death, and be- cause the relatively thin t. uncle of the pleopods facilitates observations on setal development. Two molting patterns fui\c beeiulefined for the crusta- I'lie crustacean molting cycle: heniolympli ccd\'\ien>icl\ ami meiaholites The titer of hemoly mph glucose was low during stages A and B. rose gradually during stage C. reached a maxi- mal concentration in early proecdysis (Do. D, ). then de- clined in late proecdysis (Fig. 1 1 ). A similar situation was reported for C 'arcinas wtfivw.v (Spindler-Barth. 1 976), al- though liters of circulating glucose in C niaenas were more than twice those reported here for P. rannamei. In contrast to this pattern. Telford (1968) demonstrated that hernolymph glucose liters increased shortly before ecdysis in three species of crabs. Maximal levels of glu- cose in /'. vannaniei during the intermolt probably re- sulted from an accumulation of food reserves during ihis period of active feeding. Likewise, ihe gradual decline in glucose liters during late proecdysis corresponded with reduced feeding. We believe that the glucose lilers of P. rannanici correlate principally with ihe feeding pattern and do nol reflecl concurrent changes in metabolism. Since glucose levels were lowest just before and after ec- dysis, it is unlikely thai the glucose was essential for ei- ther chitin synthesis for ihe new cuticle or as a source of energy during molting. Gwinn and Slevenson (1973) have speculated thai in Orconectes limosus. the major energy source is chilin because the chitin resorbed by the epidermis before molting provides sufficient material for both new chilin sy nthesis and energy for molting. The use of the RIA to measure hernolymph ecdyste- roid liters in decapods has been limited to only a few spe- cies. including the crayfish. Orconectes sanbomi (Steven- son el nl.. 1979) and (). liniosns (Keller and Schmid, 1979; Jegla ct al . 1983). the lobster. Hninnni\ ameri- ciinii\ (Chang and Bruce. 1980). and the crabs C. niaenas (Andrieux el al.. 1976). (.'ullineetes sapidiis (Soumotf and Skinner. 1983). (iec Mar. Biot. Ecol. 16: 167 IM> Draeh. P. 1939. el cvcle d'mtermue ehe/ les crustace's deca- podes..!' Oceanogr. Paris \ -f. 19: 103-391. Fielder. D. R . K. K. Rao. and M. FinRerman. 1971. A female-limited lipoprolem and the diversity of hcmocyunin components in the di- morphic variants of the hdder crab. Lea fiiixilator. as revealed bv disc dccirophoresis.Cow/> Kxuhcm Plivuol 398:291-299. Gurell. I . A., and I.. I. Gilbert. 1971. Protein and RNA synthesis in the premolt crayfish Orconectes virilis. Z. I'vl l'h\-\u>l. 73: 345- (iraf. F. 1972. Etude comparative de I'actiondeguset le talitride eige Orchestia (Crustaces. Amphipodesl. t R .Uciii .S'-35(v I Inn. i I \ „ and I . B.( ohin. 1979. Observations on the molt cvclcs of two species of juvenile shrimp, Peniicm califoriensis and 1'ciuicin \lvlirn\trn (Decapoda: Crustacea). I'ruc .\iill Shell/nil l\v>< 69: 77-84. Jegla. I. C., C. Ruland. G. KcRel. and R. Keller. 1983. I he role of ihe Y-organ and cephalic gland in ecdv steroid production and the conlrol of molting in the cravlish. (M "//<•( /r\ lini»\u\ ./ ( 'mii/i Physiol. 152:91-95. .li-nkin. P. M. 1966. \polvsis in Arthopoda molting ode. \iiniii- 221:871. KamiKiichi. V 1968. A new method for the determination of inlcr- ninlt stage in the fresh water prawn I'ulacinon ihnui<>l Man 77: 326-329. Keller, R.. and F. Schmid. 1979. /;; vitro secretion of ecd> steroids b> Y-organ and lack ol secretion hs mandihular organs of crayfish fol- lowing molt induction. ./ Cnint> /'/irs/fi/ 130:347-353. Keer, M. S. 1969. The hemohmpli proteins of the blue crab ( 'til/i- rttr/i'\ \u/>i(lii\ II. A lipoprotein serologically identical to oocstc li- povitellin. I1c\' Biol 20: 1-17. KnoHt-les. t- . (,. \\ .. and I). B. Carlisle. 1956. I ndocrme control in the crusiacca. Hint Rev. 31: .396-473. hiirup. V (,. 1964. 1 he mtermoll cycle of an anomuran. /'l•/^(>//^//;wl 33: 278-292. Morivsau. M.. and P. Mallei. 1986. Molt stages ol the snow crab ( 7//<'//.'r, r/('v i'i'i/ic by observation of morphogenesis of setae on the maxilla. ./ ( 'ru\unriin Iliol 6: 709-718. Passano, I,. M. 1960. Molting and its control. Pp. 473-536 in The Physiology of Crustacea, Vol. 1. T. H. Waterman, ed. Academic Press. New York. Robertson. L., \V. Bra>. J. Leung-'! rujillo. and A. L. Lawrence. 1987. Practical molt staging of /V>;,;i'i/\ MY/VivM.v and I'cinicin v/\-- lirastrix.J. H'orld .•laiiaailinif S.'< 18: 180-185. Schafer. II.. I. 1968. The determination of some stages of the molting cycle of I'l'titicm din>i\irwn. by microscopic examination of the se- tae of the endopodites of pleopods. /•' .1 O. h'uli Kcr 57: 38 1 -39 1 . Seheer. 8. I. I960. Aspects of the intermolt evcle in natantians. Coin/t HitifliciH I'liviiol 1:3-18. Senkhell, F. (;., and J. ( . \\ riston. 1980. Catabolism of hemocyanin in the American lobster. Homarus americamis. Ctunp. Biochem. l'h\M»l. 698:781-790. Skinner, I). M. 1985. Molting and regeneration. Pp. 43-146 in The li/nli>i;\ ni ( "nnr<;n'i;. Vol. 9. D. E. Bliss and L. H. Mantel, eds. Academic I'icss. New "1 ork Smith, t). M., and \\ . Dull. 1985. Moult staging the tiger prawn Pe- naeu.\ c\citU'ntn\ Pp. S5-95 in Second Australian National Prawn Si-miinir. P C. Rothlisberg. B. J. Hill, and D. J. Staple, eds NPS2. Cleveland. Australia. Smirnoff. ('., and I). M. Skinner. 1983. I cdysteroid liters during the molt cvcle ot the blue crab resembles those of other crustaceans. liml. Hull 165:321-329. Spindler-Barlh. M. 1976. Changes in the chemical composition of the common shore crab, Carc/nus maenos ./ C»;n/> l'/n\ml 105: 197-205. Sleu-nson, .1. R.. P. N. Armslronu. F. S. Chang, and .1. D. O'Connor. 1979. I cdvsteroul tiler during the molt cvcle of crayfish Oreo- nct'tc\ Minhi'iin den CII/H/I I.ndivnnol 39:20-25. I > llorcl. M. 1968. I he idenlilication of sugars in the blood of three species of Atlantic crabs. Hml. Hull 135:674-584. I rat is. I). F. 1955. The molting cvcle ol the spun lobster. Panulirus iiri;ii\ 1 atreille. l\ . 1'ost-ecdvsial luslological and histochemical changes in the hepatopancreas and integumental tissue, liml Hull 113:451-479. \ an llerp. F., and C. Bellon-l liniihert. 1978. Setal development and molt prediction in the larvae and adults of crayfish. Asiacux /<'/>/»- ilui-n-lin \tinu, nil inr\4: 289-301. \\ hitlaker. .1. R. 1959. I ocah/ation of hemocvanin on starch gel elec- trophorelic pattern, \niiirc 184: 194. Reference: Biol. Bull 175: 193-201. (October, 1988) The Effect of Host Feeding on the Contribution of Endosymbiotic Algae to the Growth of Green Hydra KENNETH W. DUNN* Department of Ecology and Evolution, State University of New York, Stony Brook, New York 11794 Abstract. Previous work has shown that the advantage conferred by endosymbiotic algae on the growth of green hydra is most evident during periods of food shortage. This advantage disappears when hydra are amply fed. However, evidence is presented here which suggests that endosymbiotic algae are not sensitive to the nutritional needs of the host. In controlled feeding studies, green hy- dra produced more bud tissue than did aposymbionts at all feeding levels. Per capita algal contribution to host growth was independent of host feeding rate. Starvation had little effect on rates of algal photosynthesis. The algae of unfed hydra translocated a larger proportion of photo- synthetically fixed 14C to the host than did the algae in recently fed hydra. The differences in algal translocation were small, however, and unlikely to significantly affect hydra growth rates. Evidence is presented suggesting that the decrease in the rate of algal translocation in fed hydra may result from an increased algal demand for photosyn- thate to support the rapid algal growth that follows host feeding. Introduction Studies comparing the budding rates of green and apo- symbiotic (algae-free) hydra show that possession of en- dosymbiotic algae increases the rate of hydra bud pro- duction. The advantage conferred by endosymbiotic al- gae was greatest when hydra received little food. There was no difference in budding rate between amply fed green and aposymbiotic hydra (Muscatine and Lenhoff, 1965a, 1965b, Stiven. 1965). A similar phenomenon oc- curs in the symbiosis between Chlorella and Parame- cinm bursaria. Endosymbiotic algae augment the cili- Received 12 February 1988; accepted 29 July 1988. * Present address: Department of Pathology. College of Physicians and Surgeons. Columbia University, New York, New York 10032. ate's growth at low but not high concentrations of bacte- rial food (Karakashian, 1963). It is generally believed that the algae augment hydra growth by providing or- ganic materials to the host (Muscatine and Lenhoff, 1965b, Smith el ai. 1969. Muscatine, 1971, Thorington and Margulis, 1981). The question then arises whether the endosymbiotic algae may increase the rate of photo- synthate translocation during periods when the host is without food (Smith et a/., 1969, Mews, 1980). Regulation of algal translocation according to host need suggests a high degree of coevolution between the symbionts. On the other hand, feeding the host stimu- lates algal growth (McAuley, 1981, 1982, 1985a, 1985b, 1986a, Bossert and Dunn, 1986. Dunn. 1987). If algae need much of their photosynthate to grow, they may nec- essarily translocate less carbon when growing rapidly (Pardy and White, 1977: Mews, 1980). Mews (1980) showed diminished translocation by the rapidly growing algae repopulating hydra that had been artificially de- pleted of algae. Thus the rate of algal carbon transloca- tion may increase when the host is without food through a mechanism which involves no algal response to host need per se. To determine if the contribution of algae to host growth is influenced by host nutritional state directly, rather than by algal growth rate, one would like to vary hydra food income while holding specific algal growth rate constant. It may be possible to accomplish this by studying hydra at "steady state" with their food incomes (Otto and Campbell, 1977, Gurkewitz et al., 1980). Otto and Campbell (1977) showed that after ten days on a fixed feeding regime, hydra come to a "steady state" in which the specific growth rate of hydra cells is constant across feeding rates (see also Bosch and David, 1984). In the studies described here, bud production is measured in hydra at steady state with their feeding rates. Under 193 194 K. \V. DUNN these conditions, the contribution of algae to host hud production was either independent of. or an increasing function of host feeding rate. One might expert iluit starving hydra, because of their depressed growth rates, would receive more translocated carbon from their algae than do well fed India. Previous examinati.'iis of the distribution of photosyntheticall) fixed I4C in green hydra have not supported this expecta- tion. Similar percentages of fixed carbon were found to be translocated in hydra starved one and three days (Eisenstadt. 1971), and one and nine days (Mews. 1980). Although the percentage of fixed carbon translocated was unaffected by starvation, it algae increase their rate of photosynthesis during host starvation, the actual mass of carbon translocated may have varied with the rate of algal photosynthesis. This was not measured. In studies presented here, measurements of the percentage of pho- tosynthetically fixed I4C translocated from algae to host were taken alongside measurements of the rate of algal photosynthesis, measured with a polarographic oxygen electrode. Algal photosynthesis was unaffected by starva- tion. However, feeding induced small, brief declines in the percentage of I4C translocated from algae to host to levels below the baseline characteristic of unfed hydra. Thus: a. the contribution of algae to hydra growth, b. the rates of algal photosynthesis and. c. the percentages of fixed carbon translocated from algae to host, provide no evidence to support the idea that endosy mbiotic algae respond according to host nutritional need. Materials and Met hods Experimental organisms The Carolina strain of Ilydm viridissima was obtained from the C'arolina Biological Supply Co. Aposymbiotic clones were derived from individuals whose algae were removed by the method of Pardy ( 1976). Stock culture conditions Hydra were maintained in M solution ( Muscatine and I.enhotf. 19foa) minus Iris butler at I 7 degrees C' under continuous illumination of 15 to 25 ^F.m : s '. Stocks were fed to repletion with freshly hatched \rtemia nauplii even, Monday. Wednesday and I rulav tor a pe- riod of several months before the start of any study I he culture dishes were rinsed 2 hours and 10 hours alter feeding and were scrubbed once a week. Maintenance of hydra mi //m/ /<•<•/ hydra bud production The number of detached buds was recorded daily for each hydra prior to feeding. On Monday. Wednesday, and Friday detached buds were removed, rinsed in dis- tilled water, and lyophilized. Buds were later weighed in- dividually on a Cahn G-2 microbalance. Estimation of parentiil tissue A positive relationship between parental si/.e and feed- ing rate is characteristic of hydra at equilibrium with then feeding rates (Otto and Campbell. 1977. Gurkewit? c/ ul.. 1980). To verity that this relationship existed in hydra whose bud production was to be measured, paren- tal si/e had to be measured without harming the experi- mental hydra. Consequently, parental si/e was measured photographically. Hydra were photographed at the be- ginning, middle and end of each feeding experiment. Pa- rental hydra volumes (exclusive of bud tissue) were com- puted from these photographs as previously described (Slobodkin and Dunn. 1983). Calibration curves were constructed to convert photo- graphic estimates of volume into masses. Budless adult hydra were each photographed twice, then lyophili/ed and weighed. Geometric mean regressions (Sokal and Rohlf. 1981) computed from these data explained 90', and 57'"; of the variation in the masses of green hydra and aposy mbionts, respectively . These regressions were then used as calibration curves to estimate the mass of parental tissue of each experimental hydra from its pho- tographic volume measurements. llgal photosynthesis and translocation lo measure the cllect of feeding on translocation of pholosy nthetically fixed carbon from algae to host, the FEEDING AND GROWTH OF GREEN HYDRA 195 rates of photosynthesis and the distribution of photosyn- thetically fixed I4C between algae and host were mea- sured before and at various times after feeding. Photosynthetic oxygen production was measured us- ing a polarographic oxygen electrode chamber (volume = 6.4 ml) fitted with a YSI model 5331 oxygen probe (Dunn. 1986). Illumination from a Bausch and Lomb fluorescent lamp was passed through a Kodak 301 A in- frared cutoff filter to minimize heating of the incubation chamber. Photon flux at the experimental hydra was ap- proximately 30 jjEirT2 s~', an illumination level that caused no chamber heating. All incubations were done in 0.45 n filtered M solution maintained at 1 7°C by circu- lating water from a constant temperature bath through the chamber water jacket. For each experiment, 5 groups of 30 hydra each were assembled randomly from a pool of standard hydra (hy- dra with one fully formed bud) which had been unfed for 11 hours. One of these five groups was set aside for measurements on unfed hydra. The remaining 4 groups were fed and sequentially selected for measurement at the time points 12, 24, 36, and 48 hours after feeding. In one experiment hydra were fed one Anemia nauplius apiece and in a second, hydra were fed two nauplii apiece. At each time point, hydra were placed in the respirom- eter, and all light was excluded from the chamber. Upon equilibration of the chamber temperature a constant rate of oxygen depletion was produced, reflecting respiration. Upon illumination a slower rate of oxygen depletion was immediately established and its slope, reflecting net pho- tosynthesis (the combined rates of respiration and photo- synthesis), was recorded for 15 minutes. At the end of this period, 50 n\ of Na2'4CO3 (approximately 1 ^Ci per n\) was injected through a side port. Hydra were incu- bated in light in this solution for 45 minutes. At the end of the incubation, the hydra were rinsed and then homogenized in a glass tissue homogenizer at 0°C. Algae were separated from host tissue by three rounds of centrifugation in M solution (at 600 g). Host and algal fractions were then frozen for later analyses. An average of 1.3% (±.15%, standard error) of the algae were found to be included in the host fraction. At the end of each incubation, after hydra were re- moved from the chamber, the oxygen electrode was cali- brated by means of measurements taken of air saturated distilled water at 17°C and of the deoxygenated solution after addition of a mixture of sodium dithionite and CoCl;,. Additional I4C partitioning data were collected in three more experiments in which hydra were otherwise treated as described above, but no respirometry data were collected. In one experiment, hydra were starved for 72 hours, then fed a single nauplius apiece. In a sec- ond experiment, hydra were starved for 72 hours, then fed ad lib. In the third experiment, hydra were starved for 120 hours, then fed ad lib. Triplicate samples of both animal and algal fractions for each time point were prepared for liquid scintillation counting. 0.2 ml of 6 TV acetic acid was added to 0.2 ml aliquots from each fraction. The mixture was then placed in a warm air stream and shaken intermittently over a period of 30 minutes to allow unfixed 14CO2 to escape. A stable transparent mixture was obtained after addition of 10 ml of scintillation fluor (8 grams Om- nifluor, 2 liters toluene, 1 liter Triton X- 100) and 1.2ml deionized water. Samples were counted with a Beckman LS-100C liquid scintillation counter. Counts per minute were corrected to disintegrations per minute by the exter- nal standards channel ratio method. Of the radioactivity found in the host fraction, 84% was assumed to have been translocated from the algae, since the host fractions of three samples of green hydra incubated in darkness contained an average of 16% (±2%, standard error) of the radioactivity present in the host fraction of identically treated but illuminated green hydra. Likewise, a sample of aposymbionts incubated with radioactive sodium carbonate accumulated 16% of the radioactivity present in the host fraction of illumi- nated green hydra (after correcting for differences in pro- tein content between the two). Measurement of algal growth Algal growth was estimated from four to eight hemacy- tometer counts of both the host and the algal fractions of a particular sample under 400X epifluorescence. To avoid bias, samples were analyzed without knowing their identity. The data in Figure 7 on algal growth are also presented as part of another report (Dunn, 1987). Protein determination Triplicate protein determinations were made of both host and algal fractions by the method of Lowry et al. (1951) using bovine serum albumin as a standard. Sam- ple absorption values were read at 750 nm to avoid inter- ference from chlorophyll. The difference between the protein content of hydra before and 1 2 hours after feeding (thus following regurgi- tation) was used as a measure of food intake for hydra fed ad lib. In the two experiments described above in which each hydra was fed one nauplius, the protein con- tent of an individual Anemia nauplius was calculated ac- cording to this procedure to be 1.6 and 1.8 /ng of protein 1% K. \V. Dl \\ - E c c re CL 13 £ eo E 130 110 90 70- 50 30- 10 (A) 3 meals/week D o - 130 110- 90- 70 50- 30- tc (B) 6 meals/week 1234 nauplii consumed per meal Figure 1. I stimatcd masses of given (D) and aposymhiolic (O) hy- dra at various steads state feeding rates for hydra fed three times weekly (A) and six times weekly IB). Masses were derived from photographic size estimates of parental tissue only (exclusive of bud tissue) by the method described in Slobodkinand Dunn( 1983). Plotted values repre- sent the means of determinations made at the beginning, middle, and end of the 21-day experiment. Least squares regressions (shown) yielded R; \alues of 0.5 1 and 0.82 for green hydra fed three times and six times weekly, respectively, and 0.69 and 0.33 for aposymbionts fed three times and si\ limes weekly, respectively. In this and following figures, plotted values for green hydra and aposymbionts have been slightly offset hon/ontally for clarity per nauplius. These values agree well with a direct deter- mination of 1 .8 jig of protein per nauplius. Results The ('//(•< / c/ t Parental body si/e of both green and aposymbiotic //i - draviridissimawasan increasing function of steady slate feeding rate (I ig. I ). The expei un. m.il Imlra produced more buds when provided with more food. For any food income, ru-cn hydra appeared to pmdm e more buds than did apos\ m- biotic hydra (Fig. 2). (Because some of the cells of these meristic data have zero variance, the statistical sii'inli- cancc of these regressions or the dillerenccs belueen them cannot be tested parametricalh ) Mud si/e is like- \\ isc an increasing function of feeding rate for both forms of hydra ( I-'ig. 3 ). The green hydra made larger buds than did the apos\mbiont (ANC'OVA. P < .025 in the low frequency feeding study. P < .001 in the high frequency feeding study). To assess the contribution of algae to host growth, esti- mates of host tissue production \\ere derived from mea- surements of the mass of buds produced. An average of M' - of the Carolina strain's protein was found in the host traction when algae were removed by centrifugation. McAuley (14,X6b) showed that another 17P; of hydra protein is lost by the host fraction to the algal fraction by this procedure. Accordingly. I assumed that 80' ; of the total protein produced by the Carolina strain is of host origin. Assuming that this protein ratio is proportional to the biomass ratio of host tissue to total hydra tissue, rough estimates of host tissue production can be made. Note that these estimates could be confounded by sys- tematic variation in the biomass ratio with feeding rate (see Discussion). When hydra were fed daily, the beneficial effect of al- gae on host tissue production was evident at all food lev- ra •o 13 0) o 3 •o o 0) J3 E 0.6- 0.5- 0.4 0.3- 0.2- 0.1 - 0.0 (A) 3 meals/week -o o Q. i/l 0.6- 0.5 0.4 0.3- 02- 0.1 ' 0.0 1 2 (B) 6 meals/week 23 nauplii consumed per meal l'i|>iiri> 2. I he numbers ol "buds produced by green (I I) and aposym- biotic (O) hydra during 2 I days at various steady state feeding levels lor hydra fed three limes (A) and si\ tunes weekly (H). I east squares regressions (shown) yielded R \.ilucs of 0.75 and 0.90 for green hydra fed three times a nil si\ times weekh . respectively, and 0.74 for aposym- bionls led thiee limes or si\ limes weekly . respectivelv FEEDING AND GROWTH OF GREEN HYDRA 197 13- 12- 11 - lo- g- s' 7- 6- 5- 4 (A) 3 meals/week (A) 3 meals/week 13- 12- 11 - lo- g- s' 7- 6- 5 4 (B) 6 meals/week 012345 nauplii consumed per meal Figure 3. Mean masses of buds produced by green (D) and aposym- biotic (O) hydra during 21 days at various steady state feeding levels for hydra fed three times (A) and six times weekly (B). Least squares regressions (shown) yielded R2 values of 0.70 and 0.90 for green hydra fed three times and six times weekly, respectively, and 0.65 and 0.91 for aposymbionts fed three times and six times weekly, respectively. els (Fig. 4b), and the benefit increased as food level in- creased (slopes differ significantly. P < .05, ANCOVA). For hydra fed every other day (Fig. 4a), green hydra pro- duced more animal tissue than did aposymbionts (P < .001, ANCOVA). but the increment of difference was independent of food level (slopes do not differ signifi- cantly. P > .25, ANCOVA). There is no evidence at ei- ther feeding frequency that the contribution of algae to host tissue production increases as host food level de- creases. The effect of host feeding on algal photosynthesis The rates of photosynthesis and of respiration were measured as a function of time after feeding for hydra consuming either one or two Anemia nauplii apiece. For each sample, the rate of gross photosynthesis was derived by subtracting the rate of oxygen depletion in the dark (due to respiration) from the rate of oxygen depletion in the light (due to the combination of respiration and pho- tosynthesis). As shown in Figure 5, feeding appeared to have little effect on either the photosynthetic rates or the o O) u> o ro T3 01234 nauplii consumed per meal Figure 4. Estimated total amount of host tissue produced by green (D) and aposymbiotic (O) hydra during 2 1 days at various steady state feeding levels for hydra fed three times (A) and six times weekly (B). Least squares regressions (shown) yielded R: values of 0.85 and 0.94 for green hydra fed three times and six times weekly, respectively, and R: of 0.82 and 0.90 for aposymbionts fed three times and six times weekly, respectively. respiratory rates of starved hydra, at least at the level of resolution of twelve hours. In all cases, hydra consumed oxygen, indicating that 1.0- 0.0 -1.0- -2.0- -3.0 12 24 36 hours after feeding 48 Figure 5. Gross photosynthetic oxygen production (hatched bars) and respiratory oxygen consumption (open bars) of samples of 30 hydra apiece at various times after feeding. Data shown are from two experi- ments. In one. hydra were fed a single Artenua nauplius apiece, in the other, hydra consumed an average of two nauplii apiece. Since no differences between the two feeding conditions were apparent, the data were pooled to yield the means and standard errors shown. 198 k. \V. DUNN . - c o a o c . each consuming two nauplii (O), or an average of three nauplii (A), or four nauplii (D). Hydra were starved for three days prior to experimental feeding in all cases except for the hulru w hich ate an average of three nauplii. which had been starved for five days. 30 nEm~2 s ' is below this association's light compensa- tion point. Phipps and Pardy ( 1 982) report a compensa- tion point of I 75 viridissima : s ' for the Florida strain of // The did i ni iccilim; mi photosynthate partitioning between /MS/ and algae The percentage of fixed I4C translocated from the algae to the host in a 45 minute incubation is plotted against time afler feeding lor five experiments (Figs. (ui. b). Ini- tial values reflect translocution in hydra starved for three days (four experiments) or lue davs (one experiment). The act lil< lo-ilmg experiments \\ere charactcri/cd In a decline in the |>< i' village of '''(' translocated during the interval from 12 to M, hours following a meal (Fig. 6b). I he mean percentage translocated during that interval varied inversely with both the average meal si/e ol the hydra (Fig. 7a) and with the net algal growth rate during I he interval (Fig. 7b). However, the mean pcivcniagc translocated was not related to the algal mitotic index (data not shown ). As in the studies of Eisenstadt (1971) and Mews (1980). extending the period of starvation beyond two days had little or no etlect on translocation rates in h\dra studied here: within 48 hours after feed ing ad lib. translo- cation appears to stabili/e near 50'i (51.0 ± 2.1c; at 4S hours, 48.7 ± 1.1% at 72 hours, and 49.4', at 120 hours, one sample). Discussion T/ii'c/fcci <>l lecilinx rate /nt\ in tin1 xnwth ot yeen hydra Previous studies showed that the contribution of endo- symbiotic algae to hydra budding increased at low food intakes. In the steady state feeding studies presented here, the difference in budding rate between green and aposymbiotic forms of the Carolina strain was either in- dependent of. or an increasing function of. feeding rate. It ma\ be that well-fed Imlra enjoyed a greater benefit o 01 o .0 ra o o 50 i 48- 46- 44- 42 (A) 50 i 48- 46- 44 - 3456 ug protein consumed (B) 42- -0.2 0 1 0.0 01 algal growth rate (per day) 0.2 Mean percentage ol photosvntheticallv lived IJ(' translo- cated lo the host during the period from \2 lo .75. ANCOVA). but lowers aposymbiont budding efficiency from 43% to 35% (nearly significant, .05 < P <.l. ANCOVA). For quantifying the beneficial effect of algae on the growth of hydra, the rate of host tissue production is an arguably better criterion than is the rate of bud produc- tion, which includes both host and algal components. However, the conclusion remains the same regardless of whether hydra growth is quantified in terms of number of buds, mass of buds, or mass of host tissue produced per day; the augmentation of hydra growth caused by algae is either independent of, or an increasing function of. hydra feeding rate. However, it should be noted that an implicit assump- tion in the correction applied to convert the mass of buds produced into mass of host tissue produced is that the ratio of algal to host biomass is independent of hydra feeding rate. Large systematic variation in this ratio could seriously confound this correction. While no evi- dence exists that directly pertains to this, it is generally noted that starvation increases the ratio of algal to host biomass (Muscatine and Pool, 1979; Douglas and Smith; 1984, McAuley, 1985a;Muller-Parkerand Pardy, 1987). If the same sort of variation occurs in steady state hydra such that the amount of algal tissue increases relative to host tissue at low feeding rates, it would only serve to accentuate the results already found, that is, the benefi- cial effect of algae increases with hydra feeding rate. Many of the experimental hydra developed testes dur- ing the daily feeding experiments. This sexuality may have resulted from the constant feeding regime imposed on the hydra (Rutherford, et ai, 1983) since non-sexual animals were chosen for the experiment. Sexuality did not measurably affect asexual reproduction by these hy- dra. The proportion of the experimental interval in which hvdra were observed to exhibit testes was not cor- related to the efficiency of bud production of either form of H. viridissima (correlation coefficients were not sig- nificantly different from zero, r = .04 for each form, arc- sine-square-root transformed proportions). The results suggest an increased contribution of endo- symbionts to bud production seen at low host food levels (Muscatine and Lenhoff, 1965a, b; Stiven. 1965) may pertain only to hydra that are not at steady state with their feeding rates. The cell-specific growth rate of steady state hydra is independent of host feeding rate (Otto and Campbell, 1 977), but the cell-specific growth rate of non- steady state hydra may vary with feeding rate. If this is true, it is possible that the beneficial effect of algae on host budding depends less on feeding rate per se than on the specific cellular growth rate of one or both symbionts. The effect of host feeding on the rate of alga/ carbon translocation The translocation data presented here qualitatively fit the model presented earlier in which algae translocate more carbon during periods of slow growth than during periods of rapid growth; the algae of unfed or poorly fed hydra translocated a larger proportion of their fixed car- bon than algae in recently fed and well-fed hydra, respec- tively. The simplest interpretation of these data is that the algae in well-fed hydra retain more of their photosyn- thate to support their own rapid growth. Since the carbon fixation rates of endosymbionts are relatively constant regardless of host nutritional condition, slowly growing algae in poorly fed hydra may translocate more of their photosynthate. Using 14C as a tracer to estimate the rates of carbon translocation from algae to host incurs certain errors (see Mews, 1980; Muscatine et ai. 1984 for discussion). In particular, the specific activity of newly fixed carbon or of translocated carbon may both vary (e.g., when hydra respiratory |:CO: production changes or if translocated carbon contains some variable fraction of previously fixed algal i:C). Consequently, the amount of 14C trans- located may not be strictly proportional to the total amount of carbon translocated. Translocated carbon is quantified here as the percentage of fixed 14C translo- cated from algae to host, a variable that is independent of variations in the specific activity of newly fixed carbon. Mews (1980) showed that the specific activity of translo- cated maltose varied with time of incubation as well as with intensity of illumination. These variables were care- fully controlled in my studies, but comparisons of per- cent translocation rates in different incubations may still be compromised if the specific activity of translocated carbon varied despite these precautions. Muscatine et al. (1984) suggest that while the I4C tracer technique may 200 K W. DUNN be adequate for "short term relatne comparisons." their "growth rate method" is to be preferred for estimation of absolute amounts il nanslocution integrated over pro- tracted period^ Considered quantitatively, the changes in percent translocation rates with host nutritional state were small. It seems unlikely that they explain the dramatic changes in the algal contribution to budding seen in the non- stead> state feeding studies of Muscatine and Lenhotf ( 1965a, b)and Stiven (1965). During the 36 hour decline in percent translocation following feeding, the algae of hydra ingesting an average of four nauplii apiece translo- cated approximately 44r; of their photosynthate. while those in hydra eating only one nauplius translocated ap- proximately 49%. Assuming equivalent carbon fixation rates at these two feeding levels, this represents an I I' . increase in the amount of photosynthate translocated. From Stiven's (1965) regressions, green hydra and apo- symbionts produced buds at essentially the same rate when fed four nauplii a day. Green hydra receiving one nauplius a day produced buds 2.5 times as fast as did comparably fed aposymbionts. It seems unlikely that an 1 l'< difference in carbon translocation could alone ac- count for such a difference in host growth. Muscatine and Lenhoff (1965b) found that popula- tions of aposymbionts and green hydra grow identically when fed daily, but green hydra grow twice as fast as apo- symbionts when fed every two days. We assume that hy- dra fed every other day receive translocated carbon as in the ad lib feeding experiments shown in Figure 6. As- suming that daily feeding maintains translocation rates at the levels observed from 12 to 24 hours after feeding. daily fed hydra receive only 5C* less translocated carbon than hydra fed every 2 days. Again, it seems unlikely that a 5% difference in translocation rate could have such a dramatic effect on hydra growth rate. Even considering possible differences in culture condi- tions between this and previous studies, these calcula- tions suggest that the enhanced beneficial effect of algae on host budding (Muscatine and I.cnhoff, 1965a, b: Sti- ven. 1965) at low feeding frequencies ma> depend on an algal factor other than carbon translocation. I he impor- tance of algal carbon translocation to the growth of regu- larly fed green hydra has been questioned by Mews and Smith (19X2) and Muller-Parker and Pardy (1987). Mews and Smith ( 19X2) found no relation between the rates of translocation and host budding in several artifi- cial green hydra associations. In studies of an artificial association between aposymbiotic // viriilistinui and al- gae isolated from symbiotic I'liranicciuni hur\nnn. Miil- ler-Parkcr and Pardy (1987) found that hydra raised at 30 pEm : s ' fixed almost four times as much carbon as those raised at 5 jjEm : s '. but grew only 10''; faster. They concluded that "the growth rates of fed hydra are regulated by factors other than light-dependent carbon fixation." The light intensity in the studies reported here was be- low the association's compensation light intcnsit) where photosynthetic oxygen production matches respiratory oxygen consumption. Therefore, hydra satisfied a certain fraction of their respiratory carbon requirement through feeding. If the experiments had been conducted at a higher illumination, algal photosynthesis and transloca- tion may have satisfied the association's respirator} needs so that heterotrophy would not be required for maintenance. However, as discussed above, increased carbon translocation might not result in increased hydra growth. The amount of algal disintegration in green h>dra can be substantial and may increase when hydra are under- fed (Dunn. 1987). Disintegration of algae may provide the host with a variety of nutrients, some of which may be more limiting to bud production than reduced car- bon. Insofar as disintegrating algae are incapable of fix- ing carbon, this form of nutrient translocation will not be detectable in short-term radioactive carbon partitioning assays. Acknowledgments This research was supported by research grants from the National Science Foundation, the Mellon Founda- tion, and the Hudson River Foundation to L. B. Slobod- kin and a grant from Sigma Xi to Kenneth Dunn. I am grateful to Leonard Muscatine for his encouragement and guidance during a nine-month stay in his laboratory at UCLA. I thank Heidi C'hapnick, Jay Fader, and John LeGuyadcr for technical assistance. I also thank John McDonald for suggested improvements in the manu- script. I i In. il NIC Cited Bosch, T.C.G.. and C'.N. I)a»id. 198-4. Growth regulation in Hydra: relationship between epithelial cell cycle length and growth rate. I>CY Hi,lvr. 1988) Egg Capsule Catechol Oxidase from the Little Skate Raja ennacea Mitchill, 1825* THOMAS J. KOOB1 AND DAVID L. COX1 - * Mount Desert l\luiul liit>loxicul l.nhomtory. Sn/\/nirr C'orc. Maine and2 Department <> i 'niversity of Oregon, Eugene. Oregon \hstract. A phenoloxidase was demonstrated in ox- tracts of egg capsules tanning in ittcroand ofnidamental glands from spawning little skate. Raja ennacea. The en- zyme was identified as a catechol oxidase based on its ability to oxidize the <>r//;r>-diphcnols pyrocatechol. 4-methylcatechol. 3.4-dihydroxyphenylalanine, 3-hy- droxytyramine and /V-acetyldopamine to their corre- sponding orf/zoquinones and its relative inactivity against monophenols. 4-methylcatechol was oxidized at the greatest rate, while 3.4-dihydroxyphenylalanine. 3- hydro.xytyramine and . V-acetyldopamine were oxidized at slower rates. The nidamental gland enzyme was inhib- ited by cyanide, nitrogen, and diethyldithiocarhamate. Oxidase activity in crude extracts from nidamental glands was enhanced by addition of a-chymotrypsin. suggesting that the en/yme is produced in a latent form. Ammonium sullate fractionation of nidamental gland and capsule extracts resulted in a fifteen-fold purification of the en/yme. This partially purified catechol oxidase from the nidamental gland exhibited optimal rates of ox- idation at 0.5 M NaCI and pH 7.0. The en/yme, how- ever. showed a wide tolerance for elevated salinity and alkaline pH. These observations indicate that the oxidase acts principally //; utero. but may remain active in seawa- ter following oviposition of the capsule. This cn/yme plays a pivotal role during the formation ol skate egg cap- sules by cataly/ing the oxidation of capsular catechols to highly iv, i> live quinones forming dark pigments which tan the capsul.n matrix. Introduction Oviparous elasmohranchs encapsulate eggs in curi- ously shaped, leathery capsules produced bv speciali/ed d l'< May I987;aox-pta1 2X JuK I'JXS * Portions nl Ihis \\ork hau- appr.iird in ahsli.nl lorn). siv Kooh \ ( OX, I'>X4. I 'IKS. and 1 nidamental glands in the upper oviduct (for review of the structure and composition of these capsules see Hunt. 1985). These glands are highly developed during spawn- ing and in some species are the predominant organs in the reproductive tract. The early structural studies of Perrevex ( 1 884). Henneguv ( 1 893). Borcea (1904, 1905), and Widakowich ( 1906) established that the nidamental glands of Scyliorhinus canicula and several other ovipa- rous species have distinct glandular regions each with an extensive tubular system leading to lamellae at the lume- nal surface. Typically, three regions were discriminated: an albumen-secreting zone, a mucous-secreting zone, and a shell-secreting zone. The tubules in the shell-secret- ing zone were bordered by epithelial cells which con- tained abundant cytoplasmic granules filled with the pre- cursors of capsules. During capsule formation, these granules are secreted from the epithelial cell into the lu- men where they coalesce and then are transported to the lamellae bv ciliated tubule cells (Filhol and Garrault. 193S). Borcea (1905) and Widakowich (1906) showed how these lamellae mould the newly secreted capsular material and produce the layered organization of the capsular wall. This basic structure appears common to nidamental glands from both oviparous sharks and skates, having now been described in Scyliorhinus cani- cula (Perrovex. ISM: Henneguv. 1893: Borcea. 1904. 1905: Widakowich. 1906: Filhol and Garrault, 1938: Metten. I1H'»: I hrcadgold. 1957; Krishnan. 1959; Ru- saouen. 1978). (.'/n/o\cr/liiiin (;ri.\euni (Nalini, 1940). Rant />45h. 1948). Rusaouen ( l'» 'Si .uul Rusamien el ill ( ll)76) proxulcd 202 SKATE EGG CAPSULE CATECHOL OXIDASE 203 ultrastructural and histochemical evidence that six zones of secretory activity containing five types of secretory granules could be distinguished in the nidamental glands of Scyliorliinus canicula. Histochemical tests identified neutral and sulfated mucopolysaccharides, sulfated gly- coproteins rich in tyrosine. a fibrillar collagenous pro- tein, sulfhydryl groups, indole radicals, peroxidase, and phenoloxidase activities, each localized in granules of specific regions and cell types within the glands. In the shell secreting zone alone. Rusaouen (1978) found all of these components except the mucopolysaccharides. Her studies provide convincing evidence that the nidamental gland is an extremely complex organ which synthesizes a variety of secretory products and that capsule forma- tion and composition are equally complex. Formation of skate egg capsules begins in the nida- mental gland with the secretion and assembly of capsular precursors. These materials are white when assembled but then gradually develop color with time /'/; ntcro, eventually producing the deep greenish brown character- istic of skate capsules at oviposition. In Raja erinacea, the tanning of capsules in idem is coincident with the introduction of catechols into the capsular matrix (Koob and Cox, 1986b). An enzymic activity able to oxidize catechols to quinones has been demonstrated histo- chemically in tanning capsules and nidamental glands from several oviparous elasmobranchs. Brown (1955) re- ported that sections of newly formed Raja egg capsules turned brown upon incubation with tyrosine and that this reaction could be blocked by potassium cyanide. She believed that these results demonstrated a polyphenol oxidase which would oxidize the polyphenol present in the capsule to quinone which, in turn, would tan the cap- sule. A polyphenoloxidase was demonstrated histochem- ically in shell glands of Scyliorhinus canicula by incubat- ing sections of fixed glands with catechol (Threadgold. 1957). Krishnan ( 1 959) showed that both capsular mate- rial and sections of frozen glands from Chiloscyllium gri- seum oxidized catechol and that the capsule had chemi- cal properties like other quinone tanned matrices. He suggested that capsule formation involved a form of qui- none autotanning (sensu Smyth. 1954) catalyzed by a phenoloxidase. In Scyliorhinus canicula this enzyme is localized both to a narrow zone in the upper region of the nidamental gland and to a broad band in the caudal region (Rusaouen, 1978). Further information regarding the nature of this oxidase activity is lacking. Therefore we set out to characterize the biochemical properties of the oxidase involved in forming egg cap- sules of the little skate. Raja erinacea. We were especially interested in defining the substrate specificity of the oxi- dative activity and in determining the sensitivity of this activity to inhibitors, salinity, pH. and urea to gain in- sight into the conditions within the tanning capsular ma- trix. Materials and Methods Selection of animals Females of Raja erinacea were selected from otter trawl catches on the basis of ovarian size and color as viewed through the translucent ventral body wall. Bum- pus (1898) showed that ripe females can be discrimi- nated in this way. We found that females so selected will produce egg capsules during short term captivity (Koob el a/., 1986). Females landed with capsules in utero were also selected. Skates were maintained in 2400 1 aquaria supplied with fresh circulating seawater and were fed Maine Gulf shrimp. Every twelve hours females were palpated for egg capsules in the uterus. Only females that produced eggs were used for collection of nidamental glands. Egg capsule preparation A female which had just completed secretion of egg capsules was sacrificed and the oviducts containing newly formed capsules were excised in low. Later exami- nation revealed that the egg capsules were fully formed but untanned at the anterior, more recently secreted end. The oviducts were ligated at the cervix and just cephalad to the nidamental gland to isolate the tanning capsule within the uterine portion of the oviduct. Ten ml of 1.0 M NaCl, 0.05 M NaH2PO4, pH 7.5 chilled to 4°C were introduced into each uterine lumen with a syringe via the cervical canal. After manipulating the buffer to thor- oughly wash the uterine contents, it was collected through the opened upper oviduct. This uterine flush contained much particulate which was removed by cen- trifugation at 3,000 rpm and 4°C for 10 minutes. The capsules were then removed from the oviduct and placed into 60 ml of the same salt buffer at 4°C with occasional stirring for 15 minutes. This capsular wash was centri- fuged as above to remove particulates. The attachment fibers from these capsules were then removed from the lateral seams and homogenized on ice in 30 ml of the 1 .0 M NaCl buffer using a glass homogenizer. The homoge- nate was centrifuged at 37,000 X g for 1 5 minutes at 4°C. The supernatants from the uterine flush, capsular wash, and attachment fiber extract were assayed directly for ox- idase activity. A second, partially tanned capsule was removed from the uterus and extracted directly with 1.0 M NaCl, 0.05 Af NaH2PO4 , pH 7.0 by homogenization with a polytron (Brinkmann Instruments Inc., Westbury, New York). Following centrifugation at 37,000 X g and 4°C for 30 204 T. J. KOOB \ND D. L. ( o\ minutes, the extract was fractionated bv ditlcrcntial am- monium sulfute precipitation as described below. Nidamental gland • ci'uraiion Nidamental -Is from spawning females were ex- cised fro i oviduct, minced over ice and disrupted with homogenizer in 1.0 M NaC'l. 0.05 M NaH '.. pll ".0. fhe homogenate, which appeared ge- latinous and slighth pink, was cent rifuged at 37.000 Xg and 4°C forthirtv minutes. The pinkish supernatant was collected and analyzed directly for oxidase activity. This 1 .0 M NaCl extract was subsequently fractionated bv se- quential precipitation at 5. 10. 20. 30. and 40r; ammo- nium sulfate at neutral pH. Precipitates were collected by centrifugation at 4°C and 37.000 X g for thirty min- utes, and redissolved in 1.0 M NaCl. 0.0? M NuH;PO4. pH 7.0. Not all the precipitate formed at low ammonium sulfate concentrations dissolved in the butler, therefore it was necessary to clarity these solutions bv centrifuging at 25.000 X gand 4°C for 1 5 minutes. Protein determina- tions were performed on diluted aliquots of the original extract and on the redissolved ammonium sulfate frac- tions (Low r\ ct ill.. 1^51 ). Enzyme assay Oxidase activity was measured in the various cn/vme preparations by incubating diluted aliquots with I ml/ substrate in 0.5 M NaCl. 0.025 M NaH2PO4, pH 7.5 at ambient temperature and spectrophotometrically moni- toring for increases in absorbance at product specific wavelengths. Substrates generally employed for oxidase assavs were 3.4-dihydro\yphenylalanine (1-dopa) or 4- methylcatechol: other substrates tested were /7-cresol. 3- hvdroxytyramine. A-acetyldopamine and tyrosine (all obtained from Sigma Chemical Co., St. Louis, Missouri). Extinction coefficients for the substrates were from Waite (1976). For each assay the en/yme solution and diluent were mixed in 1 ml cuvettes. The reaction was initiated by adding substrate in a 0.0 1 \l 1 1( 'I stock solu- tion and mixing. The change in absorbance at product specific wavelengths was recorded for periods up to 1 20 minutes. All assays were performed at room temperature in a reaction volume of 1 ml. The change in absorhancc in the en/vme solutions was compared to that in control incubates which contained boiled en/vmc. substrate. and butler. The kinetic parameters Km and V,,,,, were estimated by direct linear plm 'I ivnthal and Cormsh-Bowdcn. 1974) using only the initial, bnells linear icaction veloci- ties from assays performed asdcscnbed above. I his lim- ited the usable portion of such kinetic assa\s to about 30 seconds. Inhibitors of other catechol oxidases were tested tor cllecl on skate enzyme preparations. KCN or dicthyldi- thiocarbamatc was added to a final concentration of 50 M.V/and incubated 1 5 minutesat room temperature prior to the addition of 4-methylcaiechol. In all other respects the assays were performed as above. Results from these assavs were used to determine the type of inhibition ob- served and to estimate K,. both bv means of direct linear plots (Eisenthal and Cornish-Bowden. 1974). The effect of nitrogen on oxidase activitv was estimated by exten- sive purging of reaction solutions prior to 4-melhylcate- chol addition and bv assaying the reactions under ni- trogen. To examine the effects of salinity, pH. and urea on oxi- dase acti\ ity. the desired concentrations were effected by diluting the enzyme solution with concentrated stock buffers. NaCl concentration was varied from 0.25 M to 1 .0 M in 0.05 M NaH:PO4. pH 7.0. pH was varied from 4.5 to 9.0 using three buffers: pH 4.5 to 6.0 in 0.05 M sodium acetate: pH 6.0 to 7.5 in 0.05 M sodium phos- phate: and pH 7.5 to 9.0 in 0.05 M Tris. Urea concentra- tions were varied from 0 to 4.8 M in 0.05 M sodium phosphate. pH 7.0. Gel electrophoresis Discontinuous gel electrophoresis was performed by a modification of the method of Laemmli (1970) either with or without sodium dodecyl sulfate. Acr\ lamide and N,N'-methylene-bisacrylamide concentrations for the separating gel were 5'< and 0.1 y, (w/v). respectively, those for the stacking gel were 3r; and 0.08% (w/v). Elec- trode buffer was 0.025 M Tris. 0. 192 M glycine, pH 8.3. To estimate protein molecular weights, gels and reservoir butler included ().\'"<- SDS. Prior to such electrophoresis. samples and molecular weight standards (Pharmacia Inc.. Piscataway, New Jersey) were heated for three min- utes at 100°C in 2'"< SDS and 5'i /tf-mercaptoethanol. In- soluble material was removed by centrifugation at 1 2.000 X i> for three minutes. Electrophoresis was carried out at 1 5 m A per slab for 30 minutes after which current was doubled for three hours. During electrophoresis the apparatus was maintained at ambient seawater tempera- ture (approximately I5°C). Proteins were visualized by fixing and staining gels overnight at ambient tempera- ture in 0.5'- (w/v) Coomassie brilliant blue G-250 dis- sol\cd in methanol:acelicacid:water (40:15:45, v/v), and were subsequently destained first in mclhanol:acetic aeid:water (45:10:45. v/\ ) and then in 5% acetic acid. Catechol oxidase activity was localized bv immersing un- fixed native gels for one hour in a solution of I ml/ 4- methylcatechol. 0.5 M NaCl. 0.05 M NaH:PO4. pH 7.0. Results The uterine Hush, capsular wash and attachment fiber extract catalv/ed the conversion of 3,4-dihydroxyphe- SKATE EGG CAPSULE CATECHOL OXIDASE 205 nylalanine to dopaquinone. The oxidase in the uterine flush oxidized 4-methylcatechol, 1-dopa and yV-acetyldo- pamine. All preparations showed an initial low rate of oxidation which eventually increased and became linear at the later time points. Boiling the extracts for one min- ute destroyed this activity indicating the enzymic nature of the oxidizing principle. These measurements showed that an oxidase was associated with tanning capsules in utero. Since this enzyme could be flushed from the uter- ine lumen without mechanical disruption of the capsular material, the enzyme obtained must have been on the capsule surface, on the surface of the uterine epithelium, or free in the uterine lumen. Extracts of nidamental glands and tanning capsules from spawning females oxidized a variety of catechols (Fig. 1 ). Ammonium sulfate fractionation of 1 .0 A/ NaCl extracts resulted in a significant enrichment in enzyme specific activity (Table I for data on shell gland extract). Catechol oxidase activity was found predominantly in precipitates formed at 5 and 10% (NH4):SO4. Most of the protein precipitated at higher concentrations. Based on specific activity, the enzyme was purified 1 5-20 fold with respect to the initial homogenate. Since both the 5% and 10% ammonium sulfate precipitates contained active catechol oxidase, they were combined for further characterization of the enzyme. Discontinuous gel electrophoresis (Fig. 2) showed that ammonium sulfate fractionation produced a substantial purification of the shell gland catechol oxidase. The pre- cipitate formed in 5% ammonium sulfate consisted mostly of material aggregated at the bottom of the sam- ple well and several proteins having an apparent molecu- lar weight around 63.000 daltons (Lane 2, Fig. 2). Mate- rial precipitated by 10% ammonium sulfate also con- tained aggregates in the sample well (Lane 3. Fig. 2), in addition to a predominant band with an estimated mo- lecular weight of 85,000 daltons. Using 4-methylcatechol as substrate, catechol oxidase activity in this 10% ammo- nium sulfate fraction was localized not to the major pro- tein, but in a slower migrating diffuse band between 440,000 and 230,000 daltons. This band was not visible when stained with Coomassie blue (Fig. 2). This indi- cated that the active enzyme preparation contains rela- tively little protein having catechol oxidase activity and that the predominant protein could be a contaminant or a reduced or inactive form of the enzyme. Most of the protein in the original extract precipitated at ammonium sulfate concentrations higher than 10%, appearing pre- dominantly in the 20% fraction (Lane 4, Fig. 2). These data confirm the substantial purification of the enzyme by ammonium sulfate fractionation. Partially purified oxidases in 1 .0 M NaCl extracts of tanning capsules and nidamental glands were compared with respect to substrate specificities. The two enzymes 20 10 A. 4OO nm Uj CO § CO 00 20 « 20 10 - C. 465 nm capsule extract gland extract Figure 1. Oxidation of (A) 4-methylcatechol. (B) 3,4-dihydroxy- phenylalanine. (C) 3-hydroxylyramine by partially purified extracts of tanning capsule and nidamental glands. Aliquots of redissolved 10% (NH.jhSO., precipitates of these extracts were incubated with 1 mAl substrate in 0.5 M NaCl, 0.05 A/NaH:PO4 . pH 7.5 at ambient tempera- ture. Absorbance was monitored at the indicated wavelengths. Values shown are means of triplicate analyses. showed similar oxidative activities against 4-methylcate- chol, 3-hydroxytyramine. and 3,4-dihydroxyphenylala- nine (Fig. 1). Tyrosine was little affected by these en- zymes. The substrate specificity of the nidamental gland ex- tract was examined in greater detail by determining its Km and Vmax for selected catecholic and phenolic com- pounds (Table II). Using the ratio Vmax/Km as an index of substrate preference (Segal, 1976), 4-methylcatechol was clearly the most favored substrate followed distantly by jV-acetyldopamine and 3-hydroxytyramine. These data indicate that the nidamental gland enzyme has a strong preference for a methyl group substituted in para orientation to the first aromatic hydroxyl group. Chemi- cal modification of the «-carbon by charged moieties 206 ' -' K.OOB AND I) I i ' >\ I abk- I Catechol oxidase activity in ammonium <.iilhiu- iraciiian , iiilu- 1 "M \tii'l c\trtni , (n U/min/ml) Protein (mg/ml) Specific actmt\ (n.l//min/mg) Purification factor (Fold) 1.0 > -.tract 26.4 ±0.8 12.5 2.1 ±0.1 1 (Ml v. i4 tractions: - 152.4 3.8 39.7 ± 1.9 18.91 10% 160.4 • 5.1 5.1 U .4 ± 0.2 14.45 20% 40.4 ± 1.2 19.0 2.1 ±0.1 — 30% trace 9.0 — — 40% — 5.5 — — such as the carho.\\ls or amines of dopa and 3-hydroxy- tyramine or even hy the A'-acetylethyl side chain of N- acetyldopamine markedly diminished the en/yme's cat- al\tic efficiency. Monophenols such as/J-cresol and tyro- sine were little a flee ted h\ the shell gland enzyme within the assa\ period. Table III shows effects of inhibitors on the nidamental gland eatechol oxidase. Like other phenoloxidases. the nidamental gland en/.yme was inhibited by oxygen com- petitors such as cyanide and nitrogen. Diethyldithiocar- 1 234567 1 2 3 4 5 6 / ir 1 r-< n -669K — -440K -232K 140K 67K COOMASSIE BLUE 4 METHYICATECHOI - 2. Pol yacrylanmlc gel electropboresis of ammonium suliau- fractions from nidamenlal gland extract A. ( oomassu- blue R-25II stained SDS r,cl i-K't irophori-sis of nu'ivaptoctlianol reduced nidamcn- tal gland s;un. 'inws Lane ( 1 ) original extract; (2) 591 ammo- nium sulfate prei i, in ammonium sulfate precipitate MI 20% ammonium sulfate precipitate ivi *n ammonium sulfate precip itatc;(6)40'/, ammonium sull.ii pn -, i pi tale: and (7| moleculai wcii'.hl standards as indicated ilnun hi^lu-st to lowest: thyroglohulin. fenitm. lactute dehydrogenase. anil IIOMIIC si-rum alhuminl H I mcthylcatechol (I m I/I stain i in1 Ol a I without delei^cnt was < trri •! out foi i h at pH 7.5 and room temperatun sample lam- order is tin- same as lor < oomassie blue stained gel. hamate, a metal chelator especially effective against cop- per-containing en/\ mes. also inhibited the enzyme. Oxidase activity was sensitive to the pH of the reaction mixture (Fig. 3). Both partialK purified extracts from ni- damental gland and tanning capsules exhibited maximal oxidation rates at pH 7.0-7.5. Little oxidation occurred at or below pH 5.0. Enzyme in the capsule extract was also sensitive to alkaline pH. retaining only a small por- tion of its activity towards 4-methylcatechol and 1-dopa at pH 8.0-8.5 (Fig. 3). The nidamental gland enzyme was apparently less sensitive to alkaline pH. Oxidation rates of 4-methylcatechol by the partially purified nida- mental gland enzyme were substantially above the natu- ral oxidation rate of this substrate (Fig. 3). Even at pM 9.0 the enzyme retained some of its activity. At alkaline pH these catechols rapidly oxidize, so measurement of enzymic activity at pH X.O to pH 9.5 is only an estimate. While it is clear that oxidase activity from both capsule and nidamental gland declines above pH 7.5. some oxi- dative activity is retained at the pH of seawater (8.0-8.5 ). The concentration of sodium chloride in the eatechol oxidase assay was varied from 0.25 M to 1 .0 M in 0.05 M \aH:PO4. pH 7.0. Enzymatic activity at NaCI con- centrations below 0.25 M could not be accurately mea- sured because of the substantial increase in turhiditx re- la bit- 1 1 S»/' \f /, /ii- /a i Vi 'it-Hi i' i 'i \ln-ll xlcinil fiiii'i //I'/.'Ui/.Mi'/r. 'in Kaia ennacea Substrate Km(m,U) V * * max • mm/K-m p 1 K'SOl 1 1 ', msinc 0 (1 0 (1 0 (i Pyrocatechol VI » .05 1. 'I ' 24 2 2 1 Mrtlnli-atcchol I.I ' l'> 45. s ± .46 41.4 1 -Dopa [ Jopaminc \.Aivt\ldop.imme 1.37 • II ' O.I 1 ±.01 0.60 ± .22 VI ' .20 0.7 ± .10 4.3 ± .48 2.3 6.1 7.2 * /;inoles oxidized/min/mg pmiein M V SKATE EGG CAPSULE CATECHOL OXIDASE 207 Table III Inhibitors of shell gland catecholoxidase. 4-methylcatechol was used as substrate in all assavs Inhibitor Type n = 3. Cyanide Diethyldithiocarbamate N: Noncompetitive Noncompetitive Probably n.c. 4.02 ± .03 166± .04 not measured for catechol oxidases from mussel byssus (Waite, 1985) and periostracum (Waite and Wilbur, 1976). While the native substrate for the catechol oxidase in the egg cap- sule has not been characterized, we have recently de- tected 3,4-dihydroxyphenylalanine in hydrolyzates of freshly oviposited capsules of Raja erinacea (Cox et a/.. 1987). In addition. Hunt (1985) has reported identifica- tion of three catechols. including 3,4-dihydroxyphenyl- alanine, in hydrolyzates of egg capsules of Scyliorhinus canicula. It is uncertain whether these catechols are in- sulting from protein precipitation. NaCl concentrations above 0.25 M had little effect on the rate of oxidation of 4-methylcatechol by the nidamental gland extract (Fig. 4). A slight increase in the oxidation rate was observed at 0.5 M and this was statistically different from the rate at 0.4 M and 1.0 A/. Urea inhibited catechol oxidase in the partially puri- fied nidamental gland extract in a concentration-depen- dent manner (Fig. 5). At the lowest concentration exam- ined, 0.15 M. a slight reduction in oxidase activity was detected. Fifty percent inhibition occurred at approxi- mately 4.0 .17 urea. At the concentration of urea gener- ally maintained in elasmobranch tissues the oxidation rate of 4-methylcatechol was reduced by about 10%. Typically oxidation did not commence immediately upon addition of the substrate, but rather occurred only after a brief delay. The duration of this delay was reduced by incubating the extract with «-chymotrypsin prior to adding substrate to initiate the reaction (Fig. 6). When crude nidamental gland extracts were stored at 4°C for several hours, their oxidative activity increased. These results suggest that the enzyme is produced in a latent form and that some endogenous factor in the crude ex- tract is able to activate the latent enzyme. Discussion These observations confirm the presence of a catechol oxidase in tanning capsules and mature nidamental glands of the little skate. Raja erinacea, and thus support previous reports that this type of enzyme might be in- volved in the formation of elasmobranch egg capsules. We biochemically identified this enzyme as a catechol oxidase on the basis of its ability to catalyze the conver- sion of ort/zo-diphenols to their corresponding quinones. The nidamental gland enzyme is markedly inhibited by both cyanide and nitrogen, as expected of any oxidase. The enzyme is also inhibited by diethyldithiocarbamate which suggests that like other phenoloxidases it may con- tain copper. The enzyme prefers catechols bearing a methyl side chain which lacks exposed charged groups. These substrate prejudices are similar to those reported CAPSULE -•» 6 \ a A A / NIDAMENTAL GLAND Q O 56789 Figure 3. Effects of pH on oxidation rate of 4-methylcatechol by the capsule extract and nidamental gland extract. pH was varied from 5.0 to 8.5 using the three buffers: pH 4.5-6.0 in 0.05 At sodium acetate; pH 6-7.5 in 0.05 M sodium phosphate; pH 7.5-8.5 in 0.05 M Tris- HC1. Values shown are means of triplicate analyses of experimentals and boiled controls, and bars show the S.E.M. 208 1 I KOOB AND D. L. COX 2 4 O 02 0.6 1.0 NaCI CONCENTRATION M Figure 4. Effects of NaCI on the oxidation rate of 4-methylcatechol bs the partial!) punned nidamental gland extract. NaC'l was \aned in 0.05 \l \aH.-POj. pH 7.0. Values are means ± S.E.M. of triplicate analyses. troduced as free amino acids or occur covalentlv bound to capsular proteins. Spectral analyses of intact capsular material suggested that the catechol in Rani cnnaccd capsules at oviposition is peptide bound (Koob, DX7). We have also shown that catcchols are introduced into the capsular matrix following secretion and assembly of capsule precursors, while the formed capsules move into and reside in the uterine lumen. This accumulation of catechol is coincident with color development (Koob X. O 12345 UREA CONCENTRATION . M I inure 5. Effects of urea on the oxidation late ol 4-mctlnlcatcchol In the partially purified nidamental gland extract. I !ica was \.nu-il in '..,( I 0(1* I/ NaH:PO4.pH 7.0. Values arc means 'SI M ..I triplicate analyses 120 80 40 CHfMOTRYPSIN yX *x* cowrwoi ..» • * .,"T TIME I m i n> Figure 6. Initial rates of oxidation of 4-methylcatechol h\ extracts of nidamental glands with and without 40 ^g of u-chymotrypsm. Ali- quots of the 1 .0 \l NaCI extract were premcuhated for 20 minutes at room temperature with 40 mg ot'a-ehymotrypsin. Controls were incu- bated in parallel. Final assax conditions were 0.5 M NaCI. 0.05 A/ NaH:PO4. pH 7.5. and values presented are means ± S.E.M. and Cox. ll)S6b). The presence of catechol oxidase in tanning capsules indicates that once catechols are intro- duced, they are susceptible to oxidation. These observa- tions support Brown's (1955) original contention that this enzyme plays a pivotal role during the formation of skate egg capsules by catalyzing oxidation of catechols to highly reactive quinones forming dark pigments which tan the capsular matrix. These experiments also establish the optimal condi- tions for assay of catechol oxidase from Raja crinacea nidamental glands. The partially purified en/yme exhib- ited maximal activity at 0.5 M NaCI and pH 7.0. Whether these conditions obtain in capsular material during tanning is not known, however, they closely re- semble the osmolality and pH generally maintained in elasmobranch tissues. The sensiti\it\ of the enzyme to urea is expected since renal and branchial enzymes from other elasmobranchs show identical inhibition by urea (Malyusz and Thicmann. ll)76). We do not know whether urea is present in fluid bathing the tanning cap- sule or in the capsular material itself. The chemical con- ditions within the capsular matrix could be established during secretion of capsule precursors or alternatively could result from regulation of the intrauterine milieu. Ihe wide tolerance of nidamental gland catechol oxi- dase to alkaline pi I and elevated salt concentrations pro- \ides evidence thai the en/vme might remain active in seavvater following oviposition of the capsule. I gg cap- sules of the little skate continue to tan during incubation by a process which mav involve catechol oxidation (Koob. 1 987). While it appears from the data presented here that catechol oxidase operates principally during capsular tanning /'« utcn>. it could also play a role in post- ovipositional tanning of the capsule. SKATE EGG CAPSULE CATECHOL OXIDASE 209 One well characterized quinone tanning system that operates in seawater is the tanning of the attachment disc and byssus ofMytilus edulis. The byssal catechol oxidase displays optimum activity in salinity and pH near those of seawater (Waite, 1985). This enzyme's pH optimum (8.0) is slightly above that of the skate egg capsule en- zyme, suggesting that the byssal enzyme may be more effective in seawater. Further study will be necessary to determine whether the egg capsule catechol oxidase in fact retains activity in seawater following oviposition. Such experiments will also furnish additional evidence regarding the role of this enzyme in the incubation-re- lated tanning of the capsule. The ability of «-chymotrypsin to shorten the delay in commencement of catechol oxidation by extracts of ni- damental glands suggests that the egg capsule catechol oxidase is produced in a latent form which can be acti- vated by proteolytic cleavage. Since this enzyme appears to auto-activate during storage as well as in the presence of substrate, these results also suggest that the gland pro- duces a native activator. Whether the native activator re- sembles bovine «-chymotrypsin is not yet known. These observations are consistent with reports of latent phenol- oxidases from both invertebrate and other vertebrate tanning systems. For example, during sclerotization of the silkworm (Bomhyx ninri) cuticle, a latent phenoloxidase is acti- vated by a serine protease (Dohke, 1973a, 1973b; Ashida and Dohke, 1980). A similar protease activates the pro- phenoloxidase of the arthropod immune response to in- vasive parasites (Ashida, 1971; Soderhall, 1982; Ashida and Soderhall, 1984;DularayandLackie, 1985;Yoshida and Ashida, 1986; Saul and Sugumaran, 1987. 1988; for a review see Gotz and Boman, 1985). In addition, Alyti- lus cditlis produces a catechol oxidase that is latent to- wards catecholic substrates without prior activation by a-chymotrypsin (Waite, 1985). Among vertebrate tan- ning systems, the tyrosinase of amphibian skin is known to be produced in a latent form. Wittenberg and Triplet! ( 1985a, b) have shown that detergents activate the latent tyrosinase from Xenopus laevis. This evidence is consis- tent with the preliminary data presented here. Together they implicate an activation process involving a zymo- gen of catechol oxidase during the formation of skate egg capsules. Catechol oxidases have been detected in materials in- vesting germ cells from many species and widely diver- gent taxa. Among fungi increases in phenoloxidase activ- ity coincident with the development of fruiting-bodies have been noted for many species and have been investi- gated particularly in Neurospora crassa (Hirsch, 1954; Horowitz el at., 1961), Hypomyces solani (Wilson, 1968), Schizophylhim commune (Phillips and Leonard, 1976, 1977; Leslie and Leonard, 1979), and Aguricn.* bispoms (Lindeberg, 1950; Turner, 1974; Rast el al.. 1981). Latent catechol oxidase has been identified as the predominant form of the enzyme in fruiting-bodies of A. bispoms (Yamaguchi ct al.. 1970), though the mecha- nism of its activation has not been determined. The fruit- body phenoloxidase initiates melanization of propagule walls which is thought to confer protection from desicca- tion (reviewed by Sussman, 1968), lysis by other micro- organisms (Potgieter and Alexander, 1966; Kuo and Al- exander, 1967), and damage from ultraviolet light and other radiations (Sussman, 1968). Seed coats of several wild legumes contain catechol ox- idase. In Pisitm elatins, for example, catechol oxidase ac- tivity in the seed coat rises sharply during the later devel- opmental stages, especially during dehydration of the seed coat (Marbach and Mayer, 1975). The enzyme is believed to catalyze the generation of specific physico- chemical properties important for dormancy and subse- quent germination. Catechol oxidases have also been detected in the egg- shells and reproductive tracts of various invertebrates. Phenoloxidase activity has been histochemically demon- strated in both eggshells and vitellaria of many monoge- netic and digenetic trematodes, and in certain cestodes (for a review see Clegg and Smyth, 1 968; and Smyth and Halton, 1983). Recently, a dopa-rich eggshell precursor protein from the vitellaria ofFasciola hepatica has been purified and characterized ( Waite and Rice-Ficht, 1987). Catechol oxidase activity has also been detected in the Faxciola vitellaria, and this enzyme has been partially purified (Cox and Waite, unpub. results). Like the elas- mobranch egg capsule, the eggshells of many trematodes and cestodes appear to be stabilized by a form of scleroti- zation involving phenoloxidase catalyzed quinone tan- ning. Egg cocoons of the leech Erpobdella octoculata un- dergo post-ovipositional hardening and darkening sug- gestive of some form of tanning. Knight and Hunt( 1974) found the cocoons to be insoluble in the solutions used by Brown (1950a) with the exception of sodium hypo- chlorite. They also reported that the cocoons contain 3,4- dihydroxyphenylalanine and a catechol oxidase. Qui- none tanning may occur in the egg cocoons of other an- nelids, but the evidence is as yet circumstantial (Brown, 1950b). Among the Arthropoda, oothecae and left colleterial glands of the cockroaches Blatta orientalis (Pryor, 1940) and Periplaneta americana (Brunei, 1952) contain cate- chol oxidase which is believed to catalyze oxidation of 3,4-dihydroxybenzoic acid during the sclerotization of the ootheca. Likewise, eggshells of the house cricket, Acheta domesticus, reportedly contain a catechol oxi- dase (MacFarlane, 1960), while presence of the enzyme in shells of the mosquito, Aedes aegypli, has been in- 210 T. J. KOOB \ND D L. COX ferred (NValker and Men/or. 1969). Although several <>- and />-diphenols ha\c boon extracted from eggshells and reproductive tract^ >>t other insects, relatively little is known aboir .-orresponding synthetic enzymes (for review of and eggshell proteins see Hinton. 1981). To our knowledge, this is the first report of a catechol • :n egg capsules and oviduct of a vertebrate. The identification of catechol oxidase in egg capsules of Raja erinacea broadens the view that the products of this path- wax possess particular properties adaptable to the needs of developing germ cells of fungi, plants, and animals. Note added in proof Inclusion of proteinase inhibitors (EDTA, benzami- dine, N-ethylmaleimideand PMSF) during extraction of nidamental glands virtually eliminated catechol oxidase activity. The crude extract of one gland containing pro- teinase inhibitors contained less than 10f; of the catechol oxidase activity found in the paired control gland ex- tracted without proteinase inhibitors. These results sup- port our suggestion that the egg capsule catechol oxidase is produced in a latent form requiring proteolytic activa- tion. Acknow lodgments These studies were supported in part by the Lucille P. Markey Charitable Trust and Mount Desert Island Bio- logical Laboratory. Literature C ited Ashida, M. 1971. Purification and characteri/ation of prephenoloxi- dase from hemoKmph of the silkworm Bomliyx mon. Arch. Bio- chem Ui,ii>h\\ 144: 749-762. \shida. M.. and K. Dohke. 1980. Activation of prophenoloxidase by the activating enzyme of the silkworm HI»>I/>\-\ m«n Inject lln>- chem 10: 37-47. Ashida, M., and k. Soderhall. 1984. Ihc prophenoloxidase activat- ing system in Crayfish. C0mp Hintlifiii r/ivsinl 77B:2l-26. Hurn-a, I. 1904. Sur la glande nidumcntaire de I'oviductedesF.lasmo- hranches. (.' K llchil Scans I,,;,/ Sci 138:94-1(11. Hun en. I. 1905. Recherches sur la systeme uro-genital des lilasmo- hranchcs. .It, h /,«,! I \f> <„•/; 4: 199-484. Brown. ('. II. I95/ 107:71-74. Dohke. k. I973a. Studies on prophenoloxidase-activating enzyme from cuticle of the silkworm Ht'»ih\'\ nmri I. Activation reaction by the enzyme. Arch. Biochcin Hinphys. 157: 203-209. Duhkc. k. I973b. Studies on prophenoloxidase-activating enzyme from cuticle of the silkworm B»inhy\ nwri II. Purification and characterization of the enzyme. .1/v/i Binchi'in Hmphvs 157:210- 221. l)tilara>. B.. and A. M. Laekit. 1985. Haemocytic encapsulation and the prophenoloxidase-acth ation pathway in the locust Schistocerca urcxurui I orsk. In vet Biochem 15: 827-834. Eisenthal. R..and A.C'ornish-Bunden. 1974. The direct linear plot. A new graphical procedure for estimating en/yme kinetic paranicu-Tv Biochfm .1 139: 7|S-^2tl. l-ilhul, .1., and II. Garrault. 1938. La secretion de la prokeratine et la formation de la capsule ovulaire chez les Selaciens. Arch Anal Micr,i\L 34: 105-145. Gotz, P. and H. G. Boman. 1985. Insect immunity. Pp. 453-485 in Comprehensive Insect r/iy\i<>luKy. Biochemistry and Pharmticul- (>K\: Vol. 3. G. A. Kerkut and L. E. Gilbert, eds. Pergamon Press, Oxford. lU-nnrguy, I.. F. 1893. Sur la structure de la glande nidamentaire de I'oviducte des Selaciens. C R Sue. PhilomatigueSthsN.,5. Ilintun. II. !•:. 1981. Hi,il,<\;\ oflruecl I wv Vol. I. Pp. 201-210. Per- gamon Press. Oxford. Hirseh, H. M. 1954. Environmental factors influencing the differen- tiation of protoperithecia and their relation to nrosinase and mela- nin formation in .Vcm«\/>n;(/ cru\.\a I'hv\n>logia Plantarwn 1: 72- 97. Horowitz, N. H., M. Kliny, II. I.. Maeleod. and Y. \\atanabc. 1 96 1 . Structural and regulative genes controlling tyrosine synthe- sis in Neurn\i«>ru Cold Spring Harbor Symp Quant liiol. 26: 233- 238. Hunt, S. 1985. 1 'he selachian egg case collagen. Pp. 409-434 in Biol- i>Ky "/ ln\-enelf'Turin. 10123 Torino. Italy Abstract. Ophryotrocha diadema. a simultaneous her- maphroditic polychaete worm, forms pairs in which both partners regularly alternate sex roles and trade eggs. Since O. diadema has a protandrous phase, safeguards against cheating by a non-reciprocating partner, either male or hermaphrodite, have evolved. Results of a mate choice experiment indicate that protandrous males are generally discarded as mates because they are unable to reciprocate with eggs. Reproductive success (measured by estimating the mean number of egg masses per indi- vidual per day) of hermaphrodites paired with males was significantly lower than the reproductive success of her- maphrodites paired with hermaphrodites. This indicates that O. diadema is able to time spawning activity accord- ing to the sexual condition of its partner. On the other hand, oogenesis and the production of multiple batches of mature oocytes is independent of the presence of a partner. Worms did not discard mates with substantially fewer eggs. The small size of clutches and the short inter- val between successive spawnings could he considered a form of egg parcelling, which would prevent exploitation of hermaphroditic individuals by partners unable to re- ciprocate. Introduction Ophryotro ha i/ii/dcuui \kesson. 1976. is a small (4 mm long) simultaneous hermaphrodite \\hich was dis- covered in 197(1 h. Reish and Akesson among the foul- ing fauna in l.os Angeles Harbor. I arvac are released from the egg case at a body length of 4 setigerous seg- ments. Its sexual life begins with a protaiulrous phase. which lasts two to three weeks. I he simultaneous her- Recenc.1 12 Auj-usl I 'W7; accepted !2July 1988. maphroditic phase begins at the fourth week of life, at a body length of 14 setigers. Sperm are produced through- out life in the third and fourth setigers only, and eggs are produced from the fifth setiger onwards. Fecundity is age-dependent (Akesson, 1976). In the first week of re- production, cocoons contain 15-17 eggs. Maximum re- productive output (30-40 eggs/day) occurs four weeks after spawning begins and then slowly declines (Akesson, 1982). Cocoons are released at intervals of about three days. The main features of the mating system of 0. diadema were described by Sella ( 1985) and can be summarized as follows: ( 1 ) Pairs are formed preferentially between simulta- neous hermaphrodites. Spawning synchronization is achieved by means of close mutual contact during a courtship lasting several hours. (2) Partners repeatedly alternate sex roles v\nh the same partner at intervals of about 30 hours. As one part- ner releases its eggs, the other fertilizes them. This alter- nate egg laving has been denned as egg trading by Fischer (1980) and Mavnard Smith (1982. p. 160). ( 3 ) Both partners care for the developing embryos. This paper addresses the following question: does the mating system ofY/ diadema represent an evolutionarily stable strategy (I-SS). \enxn Mavnard Smith ( 1982. p. 12) and Parker (1978). i.e.. a strategy such thai, if all the members of a population adopt it, no mutant slrategy is likely to invade it under the influence of natural selec- tion'.' In the case of O diadema. the mutant strategy unulil be reproducing only as a male. O. diadema eggs require greater nutritive resources than sperm (Sella, in prep I. so reproducing only as a female would probably be selected ai-amst. 212 RECIPROCATION IN A POLYCHAETE WORM 213 A mating system involving reciprocity would not be stable if safeguards against non-reciprocating individuals had not evolved. Two partners have a common interest in reciprocating but there is a potential advantage in non- reciprocation. Since eggs require greater nutritive re- sources than sperm, a hermaphrodite could increase its fitness by fertilizing the eggs of other individuals on days when it has no mature eggs to give. For reciprocal spawn- ing to be critical to reproductive success, there should be strong constraints on reproducing only as a male in both the protandrous and hermaphroditic phases. Therefore, reproductive success is expected to be significantly lower than average for those individuals that fail to reciprocate egg exchange. In O. diadema at each spawning, ovaries release all their mature eggs. Therefore, in order not to be a loser in egg trading, a hermaphrodite is expected to have evolved an ability to perceive how many eggs its partner has and, eventually, to discard a partner with substantially fewer eggs than it has. To assess the evolutionary stability of the mating sys- tem of O. (/iuilcnui I estimated the probability that sub- adults will succeed in mating with hermaphrodites, com- pared the reproductive success of a hermaphrodite mated to a male with that of a hermaphrodite paired to another hermaphrodite, and investigated whether worms can perceive egg loads of their partners and dis- card partners having fewer stored eggs. Materials and Methods Animals used in experiments were taken from labora- tory populations originating from specimens of O. dia- dema collected in Los Angeles Harbor (Akesson, 1976). Populations were reared according to the methods de- scribed by Akesson (1976) and Sella (1985). They were placed in filtered sea water with a salinity of 34%» at a constant temperature of 20°C and fed parboiled spinach. A genetically determined yellow or white coloration of eggs permitted identification of the partners in a pair since both eggs and embryos are colored according to the mother's phenotype (Sella and Marzona, 1983). There- fore, color of eggs was used to infer which partner spawned eggs or fertilized them in a white and yellow worm pair. No difference in the mean number of eggs per cocoon was observed between yellow and white indi- viduals (Akesson, 1976; Sella, unpub.). In all experiments, only virgin individuals of the same age were used, since both fecundity and fertility are age dependent in O. diadema (Akesson. 1982). To obtain virgin individuals, individual males were reared in sepa- rate containers until they reached the length (14-15 set- igers) at which oocyte production begins. Only when their first oocyte batch was mature, were they used in experiments. Therefore, the term "virgin worm" refers to a simultaneous hermaphrodite which is ready to spawn its first batch of eggs. Since body walls are trans- parent and ripe oocytes measure approximately 180 X 200 nm, oocyte growth (as well as embryo develop- ment) could be easily observed with a stereomicroscope at low magnification. Although "reproductive success" generally is defined as the number of progeny that survive to reproduce, this number is often impossible to measure. When pairs are formed between two simultaneous hermaphrodites, ap- proximately 95% of the eggs are fertilized and develop (Sella, in prep.). The mean number of eggs per cocoon per week has a standard error never greater than 1 (Akes- son, 1976). Therefore, if individuals of the same age are paired, reproductive success can be indirectly estimated by counting the cocoons released in a given time interval. The term "young male" refers to subadults 7-8 setigers long which have not yet reached the simultaneous her- maphroditic phase and release only sperm. The following three experiments were conducted. Mate selection To study the intensity of sexual selection against males, 44 bowls each containing a yellow virgin her- maphrodite, a white virgin hermaphrodite, and a male six setigers long, were set up. Each hermaphrodite could choose to pair either with the male or with the other her- maphrodite. Animals were observed until a pair was formed in each bowl and courtship terminated with egg spawning by one of the two partners. Reproductive success and failure to reciprocate If reciprocal spawning is critical to mating success, re- productive success of a hermaphrodite mated to a young male is expected to be significantly lower than that of a simultaneous hermaphrodite paired with another simul- taneous hermaphrodite. The reproductive success (num- ber of cocoons spawned in 10 days) of a set of 72 pairs of yellow and white hermaphrodites was compared to a set of 72 pairs of young males and either yellow or white simultaneous hermaphrodites. In the latter situation, the simultaneous hermaphrodites can act only as females, since alternation of sex roles and reciprocation of fertil- ization is precluded. Each pair was kept in a separate container. Ability to perceive partner's eggs load There are two ovaries per setiger in O. diadema. Each ovary produces one or two mature oocytes at a time. Eggs are spawned only if a partner able to fertilize them is present. Isolated individuals accumulate ripe eggs in the coelomic cavity because they do not spawn. The experi- 214 G. SELLA TahU I Reproductive success (mean mm; ; vr individual) in 10-day interval I .n/enalhelHeen f>u> s span-nings (Bn>< • ' > ' mai'lirodiies paired »;/// u simiiltaneci, • «r \\iih a \vnnt; male (A) (B) K pairs -V egg masses/ \ individual x days between NB successive spaw m ngs #X# 144 : 1 • 0.02 54 2.97 ± 0.20 72 1.8 ±0.07 34 5 .24 ± 0.26 (A) One-way ANOVA. F, 142 = 7.58:0.005 <0.01 (B) One-way ANOVA, F,.86 = 6.26:0.02 < f< 0.01 NA = number of sampled hermaphrodites: NB = number of indn id u- als from sample NA which spawned twice during the experiment ment takes advantage of these characteristics to deter- mine if an individual is able to perceive the number of eggs held by its partner and whether it can modulate its spawning behavior accordingly. Worms are expected to discard potential partners with fewer stored eggs. Three equal groups of hermaphrodites with a different number of eggs in their coelomic cavity were allowed to choose their partner according to the number of mature eggs it had. The experiment lasted 124 hours. To obtain virgin individuals with different numbers of ripe oocytes, 3 groups of 100 virgin worms were isolated in separate containers for: (A) two weeks, (B) one week, and (C) one day. respectively, from the day the first ripe oocytes were visible through their body walls. The experimental set up for matching individuals from groups A. B, and C was the following: in each of 50 Petri dishes filled with 30 ml of seawater. 6 individuals (2 from each group) were put together. A cellulose triace- tate disc of the same size as the dish, printed with a milli- meter grid, was placed beneath each dish to record the location of each pair upon its formation. Results Pans between males and hermaphrodites formed in 14 bowls (32' i :md pairs between hermaphrodites formed in 30 bowls (6Xf; ) out of 44. These values are signifi- cantly different than those expected (29.2 and 14.6) it mating choice had been random, (d test 39.54; /' < 0.001). When hermaphrodites could choose between a male and another hermaphrodite. the> prefer red to mate with the hermaphrodite, i.e.. with a partner able to reciprocate egg Reproductive MICH'S \ and failure to reciprocate The mean number of egg masses each partner spanned in the set of simultaneous hermaphrodite pairs was significantly greater than the mean number each her- maphrodite spawned in the set of pairs between her- maphrodites and young males (Table I). Moreover, the mean number of days between successive spawnings of a simultaneous hermaphrodite mated to another her- maphrodite is significantly fewer than the mean interval between successive spawnings of an adult mated with young males (one-way ANOVA on data transformed in a Vx + 0.5 scale) (Table I ). Therefore, the reproductive success of a simultaneous hermaphrodite is significantly influenced by the sexual phenotype (either male or hermaphrodite) of its partner. The number of spawned cocoons by a simultaneous her- maphrodite decreases when it cannot reciprocate be- cause it has a young male as a partner. Ability to perceive partner's cw load Isolation treatment led to a significantly different number of ripe eggs accumulated in the coelomic cavity of worms from groups A. B. and C. (Table II). A posteri- ori comparisons among mean numbers of eggs (SS-STP test, Sokal and Rohlf. 1981. p. 245) from individuals which spawned eggs in groups A. B. and C are highly significant (Table II). thus indicating that egg accumula- tion in the coelomic cavity is significantly affected by the length of the isolation period. The first pairs formed at the end of the first day after the beginning of the experiment and the first egg spawn- ings were observed during the second day. Although the aim of this experiment was not to study reciprocation, it is worth observing that at the end of the experiment 67 of I 10 pairs had reciprocated and that the I \ HI i ii \lean number of eggs i"'* < ofoon laid by individuals isolated foi 2 weeks (A), 1 »<- the first maturation of oot vies A B C .v±S.E. 32.35 « 1.51 24.46 ± 1.42 17.54± 1.03 N 60 53 46 Onc-wa> ANOVA, F2. ,4, = 25.34; /> • (Midi t I'i'sii-iion comparisons arm nit: means. (SS-STP tot I ininpaiisons A versus H u versusC C versus \ i nlical SS ss I3h2.264 significant 1227.5642 significant 4986.718 significant 591.43 RECIPROCATION IN A POLYCHAETE WORM 215 Table III Frequence of pairs formed between individuals belonging to groups A. B. and C. in 6 days Rjnds of pairs Ax A AXC BxC Bx B CXC total observed 9 33 33 30 expected 12.2 24.4 24.4 24.4 G-test, G = 3 1 . 1 2. P < 0.0 1 Numbers of individuals, out of 100. involved in pair formation either as males or as females from group A = 84 group B = 69 group C = 67 Heterogeneity G-test, G2 = 9.3. P = 0.0 1 . 3 12.2 12.2 110 110 proportions of individuals acting only as fertilizers was not significantly different in all three groups (heterogene- ity G test, G: = 1.19). Most fertilizers of pairs formed in the last two days of the experiment did not have the opportunity to reciprocate by spawning. Expectations about pair formation were not fulfilled by the experimental data (Table III). Animals of group A, with the greatest number of oocytes, became involved in pair formation in a significantly greater number than animals of groups B and C, but they did not pair more frequently among themselves than with worms with a lower number of oocytes belonging to groups B and C. During the experiment no individual paired twice. Pairing was not random. An excess of pairs were formed by individuals belonging to different groups and a deficiency of pairs were formed by individuals belong- ing to the same group compared with frequencies ex- pected under the hypothesis of random mating. Worms released all the eggs they currently had without parcelling them, irrespective of how many eggs their partners had, thus indicating that O. diadema does not select its mate by number of eggs. Discussion Simultaneous hermaphroditism with juvenile protan- dry would be an evolutionarily unstable reproductive strategy if males, competing for fertilization with her- maphrodites, had greater reproductive success than si- multaneous hermaphrodites. This does not seem to hap- pen in the mating system of O. diadema because there are strong constraints limiting reproductive success ei- ther as a non-reciprocating hermaphrodite or as a male. Hermaphrodites, which did not reciprocate because they mated with young males, laid fewer cocoons than recip- rocating hermaphrodites. Males experience the follow- ing three selective constraints: ( 1 ) they are generally dis- carded as mates because they are unable to reciprocate; (2) if they are paired to a hermaphrodite, the number of egg masses laid by the hermaphrodite per day is less than the number of egg masses laid by a hermaphrodite paired with another hermaphrodite; and (3) they do not fertilize eggs as efficiently as hermaphrodites (Sella, in prep.). Therefore, one would expect a very brief protandrous phase, or for such a phase to be completely absent in the early part of the life cycle ofO. diadema, because of the selective constraints against it. Indeed, the protandrous phase lasts about 'A of the most fertile period of the life ofO. diadema. Testes are comparable in size to ovaries, but, since they are present only in the third and fourth setigers throughout life, investment in testicular tissue may be much less. The return of this investment in juveniles is small, because they do not have many oppor- tunities to pair, compared to the return of the same investment during the hermaphroditic phase, where op- portunities to fertilize eggs are regularly offered to recip- rocating individuals. Despite the constraints on reproducing only as a male, 32% of young males engaged in mating competition with hermaphrodites succeeded in pairing with hermaphro- dites. In another mate choice experiment described by Sella (1985), males succeeded in being the first to pair with hermaphrodites in three out of sixteen bowls. The difference between frequencies of males paired with her- maphrodites in both experiments is not statistically sig- nificant (heterogeneity G test, G, = 1.04). The only ap- parent competitive advantage that young males might have over hermaphrodites is their greater mobility, which could help them in searching for and courting a partner if population density were low. Ghiselin (1974, p. 194) advanced a similar hypothesis to explain the exis- tence of small males associated with hermaphrodites in many animal groups; when adults have a mode of life in which motility is restricted, small males, able to capital- ize on their motility, tend to evolve. Hermaphrodites of O. diadema and adults of other Ophryotrocha species have a restricted motility since they spend much of their time in egg brooding. Ghiselin (1974, p. 210), Gould (1977, p. 330), and Charnov (1982, p. 250) observed that 216 c, sn i \ selective pressures favoring progcncMs in males concom- ituntly increase as population density decreases. A her- maphrodite living in a low density population would in- cur sonic UK ' ..ost in searching for a better mate. and. at a ce age of oogencsis. might be hormonally program n. mate with the first available mate. In general, interstitial fauna are present at low densities (S> 'uuk. 19M). I 'nfortunately. nothing is known about the population structure of O diailcnui in the held, apart from the original observation in L.os Angeles Harbor that only 2 individuals o\' (> duu/cniti were col- lected with more than 30(1 individuals of O lahnniica I'lhitun Since then, no additional specimens have been found in the same locality (D. Reish. pers. comm.l. It is difficult not to expect some form of egg parcelling in O. duulcnui. since it would prevent exploitation of hermaphroditic individuals by partners with no eggs. Al- though the excess of matings between individuals with a different number of oocy tes observed in the third experi- ment merits further investigation, the results indicate that in O diadcnia each partner releases all of its ripe eggs. The higher frequency of pairing by group A individ- uals may be explained by the greater number of accumu- lated eggs compressing coelomic walls and causing re- lease of all the eggs. Results of the second experiment indicate that there is. at least, a sort of temporal parcel- ling of cocoon production: (I) time intervals between acts of egg laying by a worm are briefer if immediately reciprocated by an egg laying partner: (2) conversely, longer intervals between acts of egg-laying occur in mat- ings with partners that do not lay eggs. The small si/e of the clutches and the short intervals between them can be considered a form of egg parcelling. It is interesting to observe that in other simultaneous hermaphroditic spe- cies of Qp/jryo/roc/KJ \i c o hartmanni, O maculata, and O. /'(>). this form of egg parcel- ling can be regarded as a game of incomplete informa- tion, since each fish knows whether it h.is eggs to trade, but not if its partner has eggs. In () 'i/i<>/ \/. Scr. 2: 145- 155. Akesson. B. 1976. Morphologv and life cvclc of O/'lirvmrnt'liti Jui- ili'iihi. a new polvchaclc species from California. Ophelia 15: 23- 35. Akesson. B. 1982. \ hie table studs on Ihree genetic strains of Of/iry- olnieha tliiiilemu (I'olychaeta. Domlleidae). Int. J. Invert Reprint S: S9-69. Axelrod. R.. and \\ . I). Hamilton. 19X1. I he evolution of coopera- tion. .V, «•/;«• 21 1: I3W-I3"<> ( liarncn. K. I . 19X2. The Theory of Sex l/A'n inon \l«nt<\>iiiil 29: 53-72. l-iseher. I-:. A. 19X11. I he iclalionship between matin;; system and si- multaneous hei m.i|ihiodiliMii in the coral reel lish Hypoplectrus ni- gricans (Semnidae). \>um f!clin\ 28:d2()-633. «.in-i IIM xi i. |97J. The Economy of Nature and the Evolution of Sc\ I'niveisiiv ol ( 'alilomia. Hei kelev. and l.os Angeles. 343 pp. RECIPROCATION IN A POLYCHAETE WORM 217 Gould, S. J. 1977. Ontogeny and Phylogeny. Belknap Press of Har- vard University Press, Cambridge, Massachusetts and London U.K. 501 pp. Grolhe, C. and II. D. Pfannenstiel. 1986. Cytophysiologieal study of neurosecretory and pheromonal influences on sexual development in Ophryotrochapuerilis. Ini. J Invert Reprod. Dev 10: 227-239. Marchionni, V. and A. Rolando. 1981. Sex reversal in Ophryolrocha puerili.s induced by ethereal extracts of female phase individuals. Boll. Zooi 48:91-96. Maynard Smith, J. 1982. Evolution and I lie Theory of Games. Cam- bridge University Press. U.K. 224 pp. Parker, G. A. 1978. Selfish genes, evolutionary games, and the adap- tiveness of behaviour. Nature 274: 849-855. Pfannenstiel, H. D. 1977. Experimental analysis of the "paarkultur- effect" in the protandric polychaete Ophryotrocha puerilis Clap. Mecz. J. Exp. Mar. Biol. Ecol. 28: 3 1 -40. Pfannenstiel, H. D. 1984. Sex determination and intersexuality in polychaetes. Forlschr. Zoo/. 29: 81-98. Rolando, A. 1984. The sex induction hypothesis and reproductive be- haviour in four gonochoristic species of the genus Ophryotrnehti (Annelida, Polychaeta). MonitoreZool. Ital. (N.S.) 18: 287-299. Sella, G. 1985. Reciprocal egg trading and brood care in a hermaph- roditic polychaete worm. Anim. Be/iav. 33: 938-944. Sella, G. and M. Marzona. 1983. Inheritance, maternal influence and biochemical analysis of an egg color polymorphism in Ophryotro- cha diadema. Expenenlui 39: 97-98. Sokal, R. R. and F. J. Rholf. 1981. Biomeiry. second edition. W. H. Freeman and Co., San Francisco. 859 pp. Svedmark, B. 1964. The interstitial fauna of marine sand. Biol. Rev. 39: 1-42. Westheide, VV. 1984. The concept of reproduction in polychaetes with small body size: adaptation to interstitial environment. Forl- schr. Zool. 29: 265-287. Reference: B»W Bull. 175:21- Geographically Widespread, Non-hybridizing, Sympatric Strains of the Hermaphroditic, Brooding Clam Lasaea in the Northeastern Pacific Ocean DIARMAID 6 FOIGHIL* AND DOUGLAS J. EERNISSE** l-'ritJav Harhur Lahoniii v/cs. / nn'crMiy »/ \\'u.\liin^t<>n. l-'nday Harhor. \\'u\liiiiifti>n V8250 Abstract. We hase studied phenotypic variation in six en/ymes of Lasaea. a taxonomically complex genus of small brooding clams, from nine northeastern Pacific sites. Each of the individuals examined produced one of five combinations of electromorph patterns. /, pcs could be differentiated into two main types, one containing two and the other three phenotype combina- tions. Samples from each population contained from one to three phenols pe combinations and there was no e\ idence for crossbreeding among phcnotypes. These re- sults are strongly at variance with random mating expec- tations and indicate that the phenotype combinations represent reproductively isolated strains. This is substan- tiated hv a more detailed studs of the McNeill Bay. Brit- ish Columbia, population where both main strains co- exist. Electrophoretie characterization of l.tisacu from individual 100 cnr samples of barnacle cover revealed that strains are not spatially segregated. Progeny of ( 1 ) pair mating experiments. (2) brooding field individuals, and (3) specimens that reproduced in isolation, all per- petuated the maternal electromorphs. Data from prcsi- nus studies of reproduction in northeastern Pacific / l a planktonic larval stage. Separation of the main strains in Received 24 I-ehru:ir> I1'- d !9 Inl-. I9i ' I 'u-xent Address: Biolm--. I >> •|Miinu-nl. I 'IIIUTSIU "I S u IOM.I. 1' < > "i> s KI.MI.I. British f nlumi-u < anada s ss\ 2V2. ** Present Address: Museum of Zoology and IVp.iiliiH-iii ol Hu>loi'\. University ofMichigan. Ann Arhoi. Mu IHIJ.IM 4S Id1) Victoria. British Columbia, populations on the basis of shell color phenotype is to some degree possible, but is often equivocal. 1 lectrophoretic analysis (especially of reddish specimens) is necessary for their reliable identi- fication. Introduction Members of the taxonomically complex bivalve ge- nus, Ld\ih'ii. are reproduces els speciali/ed. minute, m- tertidal. crevice dwellers (Keen. 1938: Oldfield. 1964: Glynn, 1965: Ponder. 1971) ssith a near-cosmopolitan distribution (Cliavan. 1969). A prominent reproductive/ developmental dichotomy exists within the genus. La- sdi'ii juseniles ( Pelseneer. 1903: Oldfield. 1964: (ilvnn, 1965;Rosessatcr. 1 975: Booth, 1979;Kay, 1979). I here is as set no evidence for cross-fertilization in La- saea that have crass l-ass as iiiveniles (Crisp ct ai. 1983: 6 Foighil. 19S6a. Crisp and Standen. 1988), which to- gether form a complex assembledge of nominal species and subspecies of unknoss n phv logenetic alhmts . Molluscan systematists have consentionally relied heasils on shell morphologs to distinguish between spe- cies. There is great indisidual variation in Lasaea valves (l)all. IS')'); Ponder. 1971: Roberts. 1984: 6 Foighil. ll'S6a). even among those collected from any particular siir. and this poses a difficult taxonomic dilemma. Keen (1'MS) lists >4() species o! / ,/s 120) in Kerguelen Lasaea and pro- posed that this resulted from an apomictic mode of re- production. However, re-examination of their histologi- cal material has revealed that Kerguelen Lasaea are simultaneous hermaphrodites with disproportionately tiny testicular versus ovarian tissue (6 Foighil, unpub.). The presence of sperm in European and Kerguelen La- saea leaves open the possibility of reproductive modes besides apomixis, including self-fertilization. Considerable data are available on the reproduction of northeastern Pacific Lasaea. They brood their young to a crawl-away juvenile developmental stage (Keen, 1938; Glynn, 1965; Beauchamp, 1986; 6 Foighil, 1986b), are simultaneous hermaphrodites (Glynn, 1965; O Foighil, 1985a; Beauchamp, 1986), and can reproduce in isola- tion (6 Foighil, 1986b, 1987). Isolated individuals simul- taneously spawn sperm and eggs into the suprabranchial chamber, sperm attach to eggs by an acrosomal reaction, the male pronucleus is incorporated into the egg cyto- plasm and the oocyte produces two polar bodies before first cleavage (6 Foighil. 1987). A relatively tiny amount of testis is produced in the ovotestis, sperm occupy ap- proximately 5% of gonadal volume, the rest being de- voted to oogenesis (6 Foighil, 1985a). This pattern of reduced sperm production is theoretically consistent with the hypothesis that self-fertilization is common (Heath, 1979; Fischer, 198 1 ; Charlesworth and Charles- worth, 198 1 ). Indeed, sperm production in the randomly mating L. austra/is is an order of magnitude greater (ap- proximately 50% of gonadal volume) than in northeast- ern Pacific congeners (6 Foighil, 1988). Several observa- tions imply that cross-fertilization might be a relatively rare phenomenon in northeastern Pacific Lasaea popu- lations. Sperm are present only in small numbers and have reduced motility (6 Foighil, 1985a), and there is an apparent absence of specialized sperm transfer mecha- nisms typically found in cross-fertilizing brooding bi- valves, such as spermatophores/spermatozeugmata (Coe, 1931; 6 Foighil, 1985b), dwarf/complemental males (Turner and Yakovlev, 1983; 6 Foighil, 1985c), and pseudocopulation (Townsley el al.. 1965). In this study, we examined isozyme variation of four British Columbia (B. C), Canada, and five California, U. S. A. populations of Lasaea. The F, progeny of adults from a B. C. site, either resulting from pairs or single indi- viduals that reproduced when placed in isolation, or from broods collected from field individuals, were also characterized electrophoretically. We used isozyme data from adults and juveniles ( 1 ) to help identify the breed- ing system ofLasaea by comparing observed phenotype frequencies to random mating expectations and (2) to assess the systematic status of Lasaea populations using shell and protein phenotypes. Materials and Methods After collecting, adult nonbrooding animals were ei- ther analyzed electrophoretically within 2 days following storage in seawater tables or placed at — 70°C until pro- cessed. Electrophoresis was performed at Friday Harbor Laboratories using 1 3% starch (Sigma hydrolyzed potato starch) gels, standard power supplies, and horizontal electrophoretic apparatus. Whole animals were individ- ually homogenized with glass rods in an approximately equal volume of gel buffer and gels were run, not exceed- ing 200 volts, until the front had reached a preset "des- tiny" 80 mm from the origin. A single discontinuous Tris-citrate buffer system (electrode: 18.55 g boric acid/1 and 2.4 g sodium hydroxide/1, pH 8.2; gel: 9.21 g tris/1 and 1.05 g monohydrate citric acid/1, pH 8.7) was used for the following enzymes: esterase with a-naphtyl ace- tate substrate (EST; nonspecific), (leucine) aminopepti- dase (LAP; E.C. 3.4. 11.1), peptidase with glycyl-leucine substrate (PEP-GL). peptidase with leucyl-glycyl-glycine substrate (PEP-LGG). and peptidase with leucyl-valine and leucyl-tyrosine substrate (PEP-LVLT). In addition, phosphoglucomutase (PGM; E.C. 2.7.5.1) and glucose- phosphate isomerase (GPI; E.C. 5.3.1.9) were investi- gated using Crisp el o/.'s (1983) discontinuous Tris-ci- trate buffer system. Most individuals showed no activity for PGM and the results for this enzyme are not pre- sented. Enzyme staining assays for EST, LAP. and PGM 220 D. O HW.IIII \ND D. J FFRMSSE-: HALF MOON BAY CAYUCOS CYPRESS ?T-*2 MONTEREY figure I. Sampling sites for northeastern Pacific / . were as described by Ayala ct aL ( 1972) and for GPI by Tracey ct ul. (1975). The PEP-GL, PEP-PP and PEP- LVLT staining assays consisted of 30 ml of a 2'V agar solution (60°C) added to 20 mg of peptide substrate. 2000 units of peroxidase. 10 mg of O-Dianisidine. 5 mg of ( '<>rt>ui/n\ iulainuiitcu\ toxin. 0.5 ml of 0.1 M Mn('l: and 20 ml ot'0. 1 M Na:HPO4 butler. At least one McNeill Bay individual of known protein phenotype was included per gel to provide a standard electromorph. and specimens from all nine populations were repeatedly run together on the same gels to verify electromorph scoring. The right valves of 73 McNeill Bay specimens were retained and analy/ed for possible correlations of shell morphology and color to protein phenotype. Subsequent to the initial electrophoretic survey of l.a- Micii from the nine study sites (Fig. 1 ). a more detailed investigation of the McNeill Bay population was per- formed. This involved elcctrophoietic characterization of the proj.'1-nv off I ) pair mating experiments. (2) speci- mens that lepi 'liiavd in isolation. (3) individuals that reproduced in the held. The degree of spatial overlap be- tween strains in tlu \1. \c-ill Bay study site was also elec- trophoretically assessed In February 1987. non-brooding I.n^acd adults were sampled from McNeill Bav and specimens were soiled according to shell color phenotype. Thirty pairs, each containing one white and one red shelled individual, were placed in separate culture vials containing 20 nils of 1 ^m tillered seawater. l.usaca are positively thigmo- tactic (Morton. I960) and each pair was positioned in v lose physical contact to ensure mutual attachment with byssal threads to facilitate potential cross-fertilization. The culture vials were maintained at 18°Cand the clams were fed on cultured Thalassiosira pseudonana (strain 3H)and had salt water changes twice weekly. All individ- uals were checked weekly for brooding activity using a dissecting microscope (broods are visable through the translucent shells). Once a brooding individual was de- tected, the non-brooding partner was preserved at -70°C. as was the brooding parent when all the F, juve- niles had been released from the brood chamber. The F, juveniles were cultured under the same conditions until September 1987. when parents, non-brooding partners, and the F, progenies of pairs containing adults of differ- ent enzyme phenotypes were electrophoretically ana- lyzed. Originally, it was hoped to type the offspring (0.7- 1.2 mm in valve lengths) using both LAP and PEP-GL enzyme assays, however, results were obtained only for the latter. Ten red-shelled non-brooding adults collected from McNeill Bay in February 1987 were placed individually in culture vials and maintained in the laboratory, as pre- viously described, until they released juveniles. In May 1987. 30 Lasaca adults brooding early embryos were se- lected from pooled McNeill Bay samples. They were also individually raised in laboratory conditions until juve- nile release. Both sets of parents were then fro/en at -70°C and electrophoretically characterized together with their offspring in September 1987 for the PEP-GL enzyme phenotypes. Twenty five random 100 cnr samples of barnacle cover containing 7. 1 strain. Assuming that these electro- morph patterns have a similar genetic basis to that ob- served in other organisms, this is markedly at variance with random mating expectations. The observed and D. O FOIGMI1 AND D. J. EERNISSE E E t— I O u_ I LU > . O GO O O O GDC O O O O O O O I O D 2.0 VALVE LENGTH mm » Huurc 3. Plot of valve height against valve length for 73 McNeil] Bay Lasiiea characteri/ed according to protein phenou pe. Squares rep- resent strain 1 animals(n = 27). solid squares are double scores, correla- tion coefficient (r) = 0.976. Circles represent strain 3 animals (n = 46), solid circles are double scores, r = 0.921 (0.01 < P < 0.02). When the large strain I individual (3.6 mm in length) is excluded, strain I r = 0.967 is not significant^ different from 0.92 1 (0.05 0.50) among while and predominantly red-shelled in- dividuals (0.942 and (1.953 respect i\ el \ Ml ig. 4). Anterio- dorsal shell margins saricd considciabK in shape within each strain and shell coin. iMoup. (>.(/., see specimens 2. 4. 5 and 31. 37. 38 (Fig. 5i. 'I here was no consistent difference in the maximum sizes attained by either pro- tein phenotype or shell color groupings. The relationship between shell color and protein phe- notype was also investigated for Lasaea from Victoria's McNeill Ba\ population. Figures 5 and 6 show the right valves and the respective PEP-GL electromorphs for forty specimens (20 white: 20 predominantly red). The 20 white-shelled individuals, sorted before electropho- retic analysis, all exhibited the PEP-GL 3 electromorph. Four hundred and forty-one (44 1 ) adult specimens from McNeill Bay were typed for the PEP-GL 1 enzyme dur- ing the initial electrophoretic survey, and the subsequent investigation of spatial overlap between the two main strains. Of these. 272 and 169. respectively, expressed PEP-GL 3 and PEP-GL 1 phenotypes. If shell color is independent of PEP-GL protein phenotype. a ratio of 12. 34 PEP-GL 3 to 7.66 PEP-GL 1 for the 20 white speci- mens in Figure 5 is expected (when analyzed by Chi square test: P < 0.001). The 19 predominantly red- shelled animals for whom electromorphs were obtained, contained both PEP-GL 1(11 specimens) and PEP-GL 3 (8 specimens) patterns (expected ratios: 1 1.72 PEP-GL 3. 7.27 PEP-GL 1 : Chi square test: 0.25 < P < 0.50). Of these 19 predominantly red animals. 1 1 were uniform in color and 8 had some white patches. Both of these two color subgroupings. however, were heterogenous in pro- tein phenotype expression. Eight of the totally red speci- mens had the PEP-GL 1 pattern and four were type PEP- GL 3. Four of the eight mixed color individuals were PEP-GL 1 and the rest PEP-GL 3. Similar results were obtained when shell coloration and protein phenotypes of the Ten Mile Pt. population were investigated: white E 2.1 E £ •" O » o o a o o o o a o • on o a o o a a o o o Z.6 2.1 10 U 3.4 3.6 3.8 VALVE LENGTH mm » 4. Plot of valve height aguinst valve length for 73 McNeill Bay l.tiMicu characteri/cil accordinp to shell color. Squares represent predominentlj red individuals (n = 48). solid squares are double scores. correlation coefficient MI 0.453. Circles represent while shells (n = 25). solulaiclesaie.liiulilc scores. r = 0.942. (P> 0.50). NON-HYBRIDIZING, SYMPATRIC LASAEA STRAINS 223 • * • »**»!»** ® * * • "• ft* Figure 5. Right valves of 40 Lasaea individuals from McNeill Bay. Victoria. Specimens 1-20 (top two rows) have white shells and specimens 21-40 (bottom two rows) are predominantly or totally red. Scale = 5 mm. individuals all expressed the PEP-GL 3 phenotype (15 specimens) and totally red or mixed color specimens pro- duced either the PEP-GL 1 or PEP-GL 3 phenotypes (64 specimens). No mortality of adult McNeill Bay Lasaea occurred during laboratory culture. Less than 5% of F, progeny died in culture, mainly as a result of premature release from the brood chamber before the development of valve opposition. Four of the 30 Lasaea pairs used in the pair mating experiments did not spawn while in the labora- tory. One member of all remaining pairs released F! progeny, however, in eight of these pairs both adult speci- mens expressed the PEP-GL 3 electromorph. The re- maining 18 pairs were composed of individuals exhibit- ing different PEP-GL phenotypes and in 17 of these cases the individual that initiated brooding expressed a PEP- GL 1 electromorph. When analyzed by Chi square test, this result is significantly different (0.005 < P < 0.01) from an expected ratio of 9 PEP-GL 1 to 9 PEP-GL 3, if precedence in the onset of brooding were independent of PEP-GL phenotype. Mean brood size was 17.8 ± 6.4 S.E. and a total of 330 F, progeny were typed for the PEP-GL enzyme. In all cases, the F, PEP-GL phenotypes were identical to those of the confirmed parents (brooding in- dividuals) and did not reveal any evidence of cross-fertil- ization by the potential sperm donors (non-brooding partners) (see Fig. 7). The 10 adult McNeill Bay Lasaea maintained in isola- tion during laboratory culture reproduced successfully. Eight and 2 individuals, respectively, expressed the PEP- GL 1 and PEP-GL 3 electromorphs. All 146 F, progeny (mean brood size of 14.6 ± 5.0 S.E.) perpetuated the pa- rental PEP-GL phenotypes, including the 26 progeny of the 2 PEP-GL 3 parents. Assuming that the isolated PEP- GL 3 phenotype parents reproduced by self-fertilization, as previous cytological evidence would suggest (6 Foighil. 1987). the result for the 26 F, progeny is signifi- cantly different from the 1:2:1 phenotype ratios expected if the PEP-GL 3 electromorph represented a heterozy- gous dimer protein, or if the 2 slow bands represented a heterozygous monomer protein (Chi square test, P < 0.001). Eleven of the 30 brooding adults sampled in McNeill Bay in May 1987 expressed a PEP-GL 3 phenotype and the remainder exhibited the PEP-GL 1 electromorph. Altogether, 435 F, progeny (mean brood size = 14.5 ± 8.4 S.E.) were typed and all perpetuated the parental phenotypes, with a single exception. This exceptional in- dividual occurred in a brood of 2 1 juveniles, 20 of which expressed the maternal PEP-GL 3 electromorph, the other produced the PEP-GL 1 phenotype. It is more likely that this individual resulted from inadvertent transfer between cultures, rather than from cross-fertil- ization between the strains, because it totally lacked the maternal phenotype for this brood. Three hundred and one (30 1 ) individuals were charac- terized for the PEP-GL enzyme from the 25 samples of barnacle cover (1002 cm2) taken from McNeill Bay in October 1987. Both main Lasaea strains co-occurred in 18 of these samples (Figs. 8, 9) and there was no signifi- 224 D. O FOIGHIt \ND D. J 1 1 RMSSL • P NB 7 " 8 Figure6. PEP-GL electromorphs of the same 40 l.n^ticn individuals in Figure 5. Specimens 1-20, 27. 28. 30. 33. 34. 36. 39. and 40 produced a PEP-GL 3 electromorph pattern. Individuals 21-23. 25. 26. 2s). 3 1 . 32. 35, 37. and 38 gave a PEP-GL 1 electromorph pattern. Specimen 24 ga\ e no detectable result and specimen 25 was too taint to photograph well. Figure 7. PEP-GL phenoupcs of McNcill Ba\ / in<; / 0.5). It appears that the 2 main Lasaeci strains are not segregated in McNeill Bav on this spatial scale and show a high degree of overlap. i24 o "> 20 . . ~ 12 z o No 8 12 16 PEP-GL3 Phenotypes Per Sample 20 I inure 9. Rclah il'll'dl I .m.l I'l l> < ,1 i phcno types in 25 100 Cm im| - I • HM i Imm McNeill Ba\. \'ic- toria, B. C. Large circle doubli or < orrelation . •« -tin lent (r) oovci. /• 0 Discussion The absence of putative intermediate protein pheno- t\pcs and the consequent de\'iations from random mat- ing expectations in spatially overlapping, sympatric field populations and in the progeny of pair mating experi- ments suggest that ( I ) several to many strains of l.asaca coexist and are widespread along the west coast of North America, and (2) if mating occurs between the various strains, it must be very rare. Population genetic structure of our study populations resemble those described by Crisp <•/ and each hand in the complex multi-banded GL, PEP-LGG and LAP elect ro- morph par rded in this study should represent a separate ous locus. Each "B" strain typicallv yielded a .r number of protein phenotypes (i.e.. bands) than did each "A" strain in our study. One inter- esting possibility is that the strains ditl'cr in ploidy and perhaps one or more ploidy duplication events has been responsible, in part, for the reproductive isolation(s) of Lii\ucii strains (C. Moritz. pers. comm.). The only- published work to date on ].a\mg to a ciaul-a\\a\ juvcnilcsi.ii:e in many marine iiueitehrate laxa. Strathmann ct al. sug- gest that reduced dispersal promotes inbreeding winch lowers heterozygosity. exposing deleterious homozygous combinations to selection and eventual elimination. Thus prolonged inbreeding dilutes the genetic penalty of inbreeding depression caused by self-fertilization. If a self-fertile individual happens to arise in an already in- bred population, and it additionally produces relatively few sperm, it would then be at a reproductive advantage, because of the knvered cost of spermatogenesis and at a genetic advantage due to the reduced "cost of meiosis" (Williams. 1975: Maynard Smith. 1978: Bell, 1982). In- deed, the genetic advantage of a reduced "cost of meio- sis" implies that once developed, the evolution of com- pletely self-fertilizing lineages may be an irreversible step (Bull and" Charnov, 1985). Strathmann ci al. (1984) were concerned primarily with explaining hermaphroditism in groups that are normally gonochoric (e.g.. echino- derms. anemones, chitons, sipunculans. etc.), but they also point out that the basic model should apply to ex- plaining exceptional self-fertilizers in hermaphroditic ta.xa that normally have effective blocks to self-fertiliza- tion (e.g.. tunicates. nudibranchs. certain bivalve taxa in- cluding Lasaca). The lack of a planktonic dispersive stage, simultaneous hermaphroditism. and apparent high level of self-fertilization of northeastern Pacific La- saca are consistent with the proposal (Strathmann et al.. 1984; Eernisse. 1988) that having crawl-away offspring can lead to departure from cross-fertilization. Cross-fertilizing marine invertebrate species that are typically sedentary as adults and lack a planktonic larval stage show significant interpopulational genetic diver- gence on a relatively small geographic scale (Berger. 1973. 1977;SnyderandGooch, 1973; Campbell. 1978: Ward and Warwick. 1980; Bulnheim and Scholl. 1981: Burton. 1983; Janson and Ward. 1984: Palmer. 1984; Grant and Utter. 1988). Interpopulational genetic drift in these cases may originate from a founder effect (Hoi- gate. 1966: Nei cl al.. 1975) during an initial coloniza- tion, or a later genetic bottleneck event, that may be maintained and enhanced by infrequent genetic ex- change with other populations over time. In contrast, our results for northeastern Pacific Lasaca show that some strains ( 1 . 4) are present in geographically distant sites. A number of potential factors may have contributed to this, including alternative dispersal techniques such as byssus drifting (Lane ct al.. 1985) and rafting (High- smith. 1985) (short and long distances respectively), low rates of mutation, and a predominantly self-compatible reproductive mode. Self-fertilization is genetically con- servative because new alleles formed by mutation are rapidh expressed in homozygous combinations and are thus dirccllv exposed to selection (Bell. 1982). Newly formed populations \\ill not experience a founder effect if the initial colonizers previously existed as reproduc- tively isolated strains at the source site. Taxonomic interpretations based on a small number NON-HYBRIDIZING. SVMPATRIC L.4SAEA STRAINS 227 of loci can lead to a potentially serious bias (Nei, 1972). Even though we examined relatively few loci, it is clear that northeastern Pacific Lasaea populations are com- posed of a variety of reproductively isolated strains as Crisp et a/. ( 1983) found for British populations. These strains are readily distinguishable by electrophoretic analysis. Diagnostic separation of strains from Victoria, B. C. populations on the basis of shell phenotype appears un- reliable, except for some apparent color differences be- tween the strains. We can predict with a high degree of confidence that white-shelled Lasaea from Victoria will express particular protein electromorphs. However, specimens with the same protein phenotypes may also have mixed shell coloration (red/white) or totally red shells. Shell pigmentation is known to be light-induced in juvenile mussels (Trevelyan and Chang, 1987), and, in Lasaea rubra hinemoa, the extent of red coloration in the valves is thought to be related to the degree of expo- sure to sunlight (Ponder, 1971 ). This may also be the case for at least strain 3 of northeastern Pacific Lasaea be- cause specimens recovered from deep crevices had the whitest shells. There may be a partial habitat difference between the two main strains in Victoria, with strain 3 occurring in both deep and shallow crevices and strain 1 found only in shallow crevices. Data from the laboratory pair mating experiments hint at physiological or spawn- ing differences between the two main strains: 17 of the first individuals to spawn from the 18 heterogeneous pairs expressed a PEP-GL 1 phenotype. Criteria used by Keen (1938) to separate two nominal Lasaea species in Californian populations are inade- quate when applied to Victoria Lasaea of known protein phenotype. Keen ( 1938) distinguished L. cislu/a from L. subviridis by its smaller size, less oblique outline, darker color, higher umbones and by its more abrupt slope from the umbone to the anterior margin. However, Ponder (1971) and Beauchamp (1985) could not distinguish on morphological grounds two different forms of Lasaea in Californian populations. As in this present study. Ponder (1971 ) found Keen's ( 1938) distinguishing morphologi- cal characteristics to be highly variable among individual shells. For the present, it would appear that electropho- retic analysis is necessary for the reliable identification of northeastern Pacific Lasaea strains. Whether the two main strains detected in this study correspond to L. sub- viridis and/or L. cistula will require careful comparisons of any diagnostic morphological distinctions, should they be found, with existing type material for northeast- ern Pacific Lasaea. There is as yet no evidence for cross-fertilization be- tween the strains found in British (Crisp et a/., 1983) and northeastern Pacific Lasaea populations. Therefore the biological species concept (Mayr, 1957, 1963), which ap- plies best to randomly mating organisms, would seem inappropriate for these populations. Ponder ( 197 1 ) con- cluded that most nominal species of Lasaea are merely regional subspecies, or ecotypes of the type species L. nt- bra. Alternatively, one could conclude that every distinct electrophoretic strain is a distinct historical entity or, per- haps, species. Efforts to determine the level at which the category "species" best applies would best wait for repro- ductive investigations of other Lasaea populations and a better understanding of Lasaea historical relationships, which are undoubtedly complex among Lasaea that lack a dispersive larval stage (Ponder, 1971; Crisps/ a/.. 1983; OFoighil, 1986a; Thiriot-Quievreux t>/ #/., 1988). Reso- lution of these relationships will require a multidisciplin- ary approach applied to a variety of populations of this near-cosmopolitan genus. Acknowledgments Funding was provided by a University of Victoria graduate student fellowship and a Friday Harbor Labo- ratories post-doctorate fellowship to D. 6 Foighil; and NSF grant OCE-841 5258 to R. R. Strathmann and D. J. Eernisse. We are indebted to D. J. Crisp for sending us an unpublished manuscript and to C. Thiriot-Quievreux for letting us examine her histological material. A. R. Palmer, R. Grosberg, C. Moritz, F. Sly, R. R. Strath- mann, and anonymous reviewers gave valuable com- ments on earlier drafts. We thank A. O. D. Willows, di- rector of FHL, for the use of facilities. Literature Cited Antonovics, J. 1968. Evolution in closely adjacent plant populations. V. Evolution of self-fertility. Heredity 23: 219-238. Ayala, F. J., J. R. Powell, M. L. Tracey, C. A. Murao, and S. Perez- Salas. 1972. Enzyme variability in the Drosophila willisloni group. IV. Genie variation in natural populations of Drosophila willisloni. Genetics 70: 1 13-139. Barnard, K. H. 1964. 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Evolution 40: 388-404. Uzzell, T. M. 1964. Relations of the diploid and triploid species of the Ambystoma le/fersonium complex (Amphibia, Caudata). Copeia 2: 257-300. Vrijenhoek, R. C. 1985. Homozygosity and interstrain variation in the self-fertilizing hermaphroditic fish, Rivulus marmoratus. J Hered. 76: 82-84. Ward, R. D., and T. Warwick. 1980. Genetic differentiation in the molluscan species Littorina rudis and Littorina arcana (Prosobran- chia: Littorinidae). Biol. J. Linn. Soc. 14:417-428. White, M. J. D. 1973. Animal Cytology and Evolution. Cambridge University Press. 961 pp. Williams, G. C. 1975. Sex and Evolution. Princeton University Press, Princeton, N. J. 255 pp. Reference: Bn>l Bull r- -..'her. 1988) Allelochemical Interactions Between Sponges and Corals JAMES W. PORTER' AND NANCY M. TARGETT2 /< " >l»g\ Depdrinieni. I 'niversity of Georgia, . ti/ien.\. (.ieor^iu 30602, and ( 'allege «t Marine University of Delaware, /raw Delaware /niln>ule\. the liver sponge, and Agaricia lamareki. the sheet coral. This sponge/coral as- sociation was studied //; .\ini both at points of natural contact and following manipulations that artificially brought the sponge and coral together. Plakorti\ kills \vaneiu upon direct contact and upon indirect contact (i.e., waterborne metabolites only). Plakonis creates a dead zone of coral around its base as it overgrows the coral. The effect of either direct or indirect contact by Plak<>ni\ is to reduce: ( 1 ) the number of zooxanthellae in K'("''i -in. (2) the weight of chlorophyll a per unit area of the coral, and (3) the weight of tissue nitrogen per unit area of the coral. The necrotic effect also evidences itself as changes in o.xvgcn flux characteristics such as significant increases in the compensation point anil the nocturnal respiration rate, and significant reductions in the maximum net and gross photosy nthctic rates. As a consequence, the diet in- tegrated production to respiration ratio falls below units t>ii t'.'drifid colonies in contact with Plakortia; this does not occur lor coral without neighboring sponges. Because direct contact between l'/iil\<>rii\ and .-Ixiineiti is not IK-' ' • .11 -in ellect stress in the coral, the presence nl aitivc chemical metabolites from l'lakorn\ is sug- gested. Ihus in •vhanical abrasion is excluded as the sole met I i.in i si 11 ol di H nuance by the sponge. Introduction Sessile and scdcn il reef organisms frei]ucnlh compete lor space adopting mechanisms to Received H October. 1 987; accepted ' Ink 1'iss minimi/e fouling or overgrowth by epihionts and maxi- mi/e their own space-capture abilities. Several biological mechanisms that mediate ecologically significant inter- actions among coral reef organisms have been described. For example, scleractinian corals effect extracoelenteric damage to neighbors via extended mesenterial filaments and long sweeper tentacles (Francis. 1973: Lang. 1973: Richardson el til.. 1979; Wellington. 1980: Sheppard. 1982). Likewise, hydrocorals and octocorals can move onto and spread across scleractinians. and thereby com- pete successfully for space with reef-building corals (Wahle. 1980; La Barre and Coll. 1982: Tursch and Tursch. 1982). Bryozoans employ sweeper appendages which are effective in competition and in prevention of fouling (Jackson. 1977). These structures are used in spe- cific behaviors that involve the recognition of potential competitors and the direction of interference mecha- nisms against them. Chemical defense mechanisms have also been sug- gested. These mechanisms have demonstrable effects on species distributions anil individual survivorship in ter- restrial plant communities (Fraenkel. 1969; Whittaker and Feenv. 1971; Rosenthal and Jan/en. 1979; Mein- wald, 1982: Target! and Isman. 1986). and have recently been rev icwcd in ecological contexts for marine commu- nities (Barbier. 1981: Bak el at.. 1982: Fenical. 1982; Norris and 1 emcal. 1982; Palumbi and Jackson. 1982; ( olwell. 1983; I aulkner and Ghisclin. 1983; Steinberg. 1984;Scheuer. 1985; Bakus el nl.. 1986). Unusual sccondarv metabolites have been isolated from numerous sessile solitarv and colonial coral reef or- ganisms (Tursch el a/.. 1978: Cimino el a/.. 1983; Sulli- van el til . 1 983; ( 'oval el nl., 1 984; Bandurraga and Feni- cal. 1985; Kashman el til . 1985: and Coll el /fl/.. 1981;Carmely et al.. 1983; Cimino ct al.. 1983; Gonzalez etal., 1984;Nakatsut'/a/.. 1983, 1984; Manes et al.. 1985; Braekman et al., 1985; Walker etal., 1985; Nakamura etal., 1986; Mayol et al , 1986) and also identifies several ecological contexts in which the metabolites might function (Thompson et al., 1982; Cimino el al.. 1982). Sullivan el al. (1983) demonstrate that the burrowing sponge Siphonodictyon sp. secretes a guanidine-contain- ing sesquiterpene. siphonodictidine, in its mucus. This compound kills adjacent coral tissue, thereby preventing the coral from overgrowing the sponge's oscular chim- neys. The compound stimulates coral respiration and it has been speculated that increased respiration rate and decreased photosynthetic rate would probably result in death for the coral. However, Sullivan el al. (1983) point out that because so many factors can affect hard corals under aquarium conditions, one must be cautious in ex- trapolating to the long-term effects of siphonodictidine at subacute concentrations. Environmental information on the effects of these kinds of compounds //; silu is needed. In this paper, we characterize the consequences of chemical-biological interactions in interspecific space competition between the coral Agaricia lamarcki (Milne Edwards & Haime, 1 848) and the sponge Plakortis hali- condroides (Wilson. 1902). Interactions between Pla- Figure 1 . Natural associations of the sponge Plakortis halichon- droides and the coral Agaricia lamarcki show a bleached area at the zone of contact. The necrotic area is evident in this photograph on the left side of the coral along the region adjacent to the sponge and above the 1 .0 cm scale bar in the center. (Salt River Canyon. St. Croix, 25 m depth; photograph by R. S. Smith). kortis and Agaricia are common in St. Croix, U. S. Vir- gin Islands, and in Jamaica where Plakortis overgrows living coral, utilizing the newly dead coral skeletons as a primary point of attachment and growth (N. Targett, J. Neigel, J. Porter, unpub. data). Upon physical contact or proximity of less than five centimeters to the sponge, Agaricia bleaches and shows a marked necrosis in the region of contact (Fig. 1 ). Using the Plakortis /Agaricia interaction as a basis for study, we: ( 1 ) describe the in situ effect of Plakortis hali- condroides on Agaricia lamarcki both at points of natu- ral contact and following manipulations that artificially bring together the sponge and coral, (2) quantify the //; silu effects of direct sponge contact and indirect sponge contact (i.e., exposure to whole sponge exudates only) 232 J. W. PORIT.R AND N M. 1 \Rdl I I on photosynthetic and respiratory oxygen fluxes in the coral, and (3) suggest the long-term effects on tissue bio- mass, growth rate, and survival of Agaricia colonies growing in a-^ : ion with Plakorlis sponges. Materials and Methods Site description This research was conducted utilizing the NOAA un- derwater habitat. Hydrolab. located at a depth of 17 m on the north coast of St. Croix. U. S. Virgin Islands. Ex- perimental material and line transect data were collected from the east coral reef slope of the Salt River Canyon within the research area available from the habitat. Fit-Id studies Transects ten meters in length and two meters wide (for a total of 20 m: in each transect area) were laved parallel to depth contours at 20 and 30 m. The number of Plakorlis halichondroides colonies within one meter of the line were counted. The number of times P. hali- chondroides grew within five centimeters of living tissue of Agaricia lamarcki was also noted. Additionally, a swimming census covering 1000 m: was conducted be- tween the 20 and 30 m depth contours to record all spe- cies of scleractinian corals adjacent to /'. halichondroides colonies. Biomass determinations Coral tissue was removed from the surface of the coral skeleton using a Water Pik (Johannes and Wiebe, 1970) with tillered seawater. The tissue slurry was homogc- ni/ed for one minute in a blender and triplicate aliquots were removed for analysis of nitrogen content, /ooxan- thellae cell density, and chlorophyll u concentration. Standard protocols for sample preparation and analysis were used for fluorometric determination of chlorophyll pliyllui Icmx Wells, 1973 als are more similar morphologically than taxonomi- cally; they are all horizontally flattened, and as such, provide a level substratum on which Plakortis can grow. The only exception to this pattern is the finger coral, Afa- dracis mirabilis, which is killed at the base of its branches in contact with the sponge. Approximately one-third (34.0 ± 6.8%) of all Plakortis colonies occur on or directly adjacent to living coral (Ta- ble II). The remaining members of the population grow on stable substrata, many of which are recently dead coral plates. Almost half of the corals with sponges on or near them show signs of bleaching and necrosis (40.8 ± 3.4%, Table II), but all coral specimens show tissue death in the area directly underneath the sponge. Biomass Our results clearly demonstrate a marked effect on al- gal densities, algal pigment concentration, and coral tis- sue mass for all of the experimental conditions relative to the control condition (Table III). The number of zoo- xanthellae per unit area, and as a consequence, the chlo- rophyll a per unit area of coral, decreases by a factor of three after contact with injured sponges, uninjured sponges, and sponge water (Table III). The amount of chlorophyll a per algal cell does not follow a consistent pattern under the experimental treatments; only after ex- posure to sponge water does the mass of chlorophyll a per algal cell decline significantly (Table III). 234 j w PORII R AND v M i \KI,I i i i .,1,1, ii Plakortis halichondroki. ,1,'iniiy iiml inteniclitms MI//; xclcraftinian a>ral\ I \ ± one S.D.; n = 4) Total number of \umberofcoral lumber of sponge colonies Number of coral colonies T of contacted ge colonies willun 5 cm of c'r of sponge population colonies within showing visible corals showing Station name per 20 nv coral in contact with coral 5 cm of sponges signs of stress signs of stress 20m Right 62 18 29 16 6 38 30m Left 39 13 33 13 5 38 30m Right 105 32 30 33 15 45 20m Left 90 40 44 36 15 42 74 i 29 26 ± 12 34.0 ± 6.9 24± 12 10±5 40.8 ± 3.4 Nitrogen mass per unit area of coral tissue decreases b> a factor of two (Table III). This effect cannot be ex- plained solely by the loss of zooxanthellae since they con- stitute only 7 ' ', of the coral tissue biomass (Porter and Muscatine. in prep.). Coral tissue lysis also occurs (Fig. 2). The effect of these combined plant and animal tissue responses is to create a bleached /one. or necrotic halo, on the coral in the vicinity of the sponge that is visually obvious from a meter awa> (Fig. 1 ). u\ Contact with Plakorm effects both o\\gen production and oxygen consumption (Fig. 3). Oxygen consumption more than doubles for corals in direct contact with in- jured or uninjured sponges, rising from approximately 6 to 12 ^gO:cm : h ' (Table IV). Coral respiration rate stays the same during injection of sponge water in the short-term indirect contact experiments. However, the maximum net photosynthetic rate drops by almost half for both direct contact with injured or uninjured sponges and for indirect contact with sponge water. These low- ered photosynthetic rates occur within eight minutes of injecting sponge water, suggesting that sponge metabo- lites rapidh diminish this coral species' photosynthetic capacity. Rising respiration rates and diminishing pro- duction rates tend to offset one another mathematically. And hence, only for indirect contact with sponge water, where production falls but respiration does not rise con- comitantly. does the maximum gross production show a significant decline. The compensation light intensity is significantly higher under all experimental treatments relative to the control (Fig. 3: Table IV). This demonstrates that more light is needed for corals near or adjacent to sponges to meet their basal metabolic demands through photosyn- thesis than for corals at a distance from sponges. Further, it suggests that coral production balances coral respira- tion later in the morning and stops earlier in the after- noon for corals near sponges (Fig. 3). Given the lowered production rates and increased res- piration rates observed under the experimental treat- ments, it is not surprising that coral P/R ratios are also substantially lower among corals in contact w ith sponges (Table IV). The overall effect is that while the integrated P/R ratio is always at or above unity in control corals, it is always below 1 .0 in corals exposed to injured or unin- jured sponges under field conditions (Table IV). P/R ra- tios below 1 .0 are never found for this species in Jamaica, Table III ltn'nili-.\ \tindi ii in i \ » oneS D.;h v, /,„ , olonies *>t ilw \hivi-cural, Agaricia lamarcki under different exposures imhcliw v'""i.'''. Plakortis halichondroules (see I /i,'v I & 2) 1 xnerimental treatment Characteristic 1 nils No contact K ..nirol) Contact with sponge water ( 'untact with injured sponge Contact with uninjured sponge Zooxanthellae Pigments Pigments Nitrogen 10 llS( m 1 H jig (111 ,; mgTKNcm 2 I.I 14 ,s 1(102 • 0.1(1 10.39 ±0.86 0.57 ± 0.06 0.36 ± 0.08* 5.82 ±0.93* 2.08 ±0.13* 0.25 ± 0.03* (1 IS • 0.06* 9.81 ±4.05 3.79 ± 1.61* 0.25 ±0.16 0.40 ±0.1 4* 10.57+ I.SS 2 S4± 1.00* 0.25 + 0.10* M- Miissi^nihcaiilK dilleienl from the Control (/J 0.05 \NOVA). SPONGE-CORAL ALLELOCHEMISTRY 235 Figure 2. After two hours of experimentally induced contact be- tween Agaricia lamarcki and Plakortis halichondroides (a, top), a bleached, necrotic area appears on the coral (b, bottom). (Salt River Canyon, St. Croix, 25 m depth; photograph by N. M. Targett). even over greater depth ranges ( Porter and M uscatine, in prep.), and therefore these values for coral colonies in contact with sponges are indicative of unsustainable car- bon deficits. In all cases, the differences observed between control and experimental coral oxygen flux patterns demon- strate significantly reduced photosynthetic capacity in coral colonies in contact with sponges or their metabo- lites. Therefore, the null hypotheses (no discernable effect) must be rejected for hypotheses (3) compensation point, (4) net production, (5) gross production, (6) noc- turnal respiration rate, and (7) integrated P/R ratio un- der either field conditions or idealized "cloudless day" illumination. Hypothesis (8) P/R ratio > 1 .0 is also falsi- fied. Other species of sponges (Agelas conij'era, Haliclona rubens, and 1'emngnla sp.) were also placed in contact with Agaricia lamarcki. They did not bleach the coral, thus indicating that mechanical irritation and pressure are not responsible for the effect observed with P. chondroides. Crude organic extracts were isolated from Plakortis halichondroides and coated onto synthetic cellulose pads, "tuffy sponges." When tied to living coral, these extract-soaked pads caused bleaching within 24 hours. Control pads (uncoated or coated with ether solvent only) produce no effect. While the exact nature of these organic compounds is still unknown, comparative thin layer chromatography of crude extracts from whole P. halichondroides and compounds isolated from waters surrounding uninjured Plakortis suggests that com- pounds in the surrounding water are the same as those produced naturally by the sponge. Discussion The sponge Plakortis halichondroides actively inhibits the metabolism and tissue survival of adjacent corals. In- hibition results from both direct and indirect contact (waterborne metabolites only) suggest that Plakortis uses allelochemicals as one means to secure and occupy space on the reef. Plakortis halichondroides bleaches Agaricia lamarcki and causes marked tissue necrosis. The effects on coral biomass and coloration are sufficiently dramatic that it is possible to survey these interactions visually from some distance above the reef surface. The loss of zooxanthellae from corals following exposure to sponges or sponge exu- dates parallels the loss of symbiotic algae during other natural stresses such as abnormal temperatures, salinity fluctuations, or high rates of sedimentation (Porter, 1987). These coral biomass reductions contribute to the pro- found effects that sponges have on coral oxygen metabo- lism. Parallelling the loss of zooxanthellae is a significant decline in primary production. Although the algae that remain appear to have normal concentrations of photo- synthetic pigments, the few remaining zooxanthellae cannot compensate for the overall loss of algae. Further, despite the fact that there is significantly less coral tissue per unit area on corals adjacent to sponges than on corals without sponge contact, the respiration rate is still sig- nificantly higher. This respiratory increase suggests a stress or repair-metabolism response to the active sponge metabolites. The overall effect on the P/R formula of decreasing the numerator and increasing the denominator is to lower the ratio below one for Agaricia lamarcki. These subopti- mal values are not found in this photoautotrophic coral species. For example, even to depths of 50 m, Agaricia has an annual integrated P/R ratio of 1.13 (Porter and Muscatine, in prep.). The ecological role of specific secondary metabolites 236 J W PORTER AND N. M. TARGETT I able IV I 'ariaiion (x± 9.5' " in i>ln>i<>\ynilu">ii-liKln utilization characteristics for in situ colonies oj Agaricia lamarcki under different c\r MUIIS halichondrmdcs teeFig 3) Experimental treatment Ch.ir.KU" Units No con tact (Control) Contact with sponge water Contact with injured sponge Contact with uninjured sponge MgO: cm : h ' nE ' m s 0.222 ± 0.046 O.I40± 0.058 0.266 ± 0.141 0.303 0.196 1, nEm s 137.82 ±9.16 129.64 ±27.93 109.40 ±25.10 101.32 36.12 l,(cm:) jiEm s 27.69 ± 1.70 60.54 ± 15.58* 76.90 ±21.00* 85.84 19.68* p net nia\ /igO: cm : h ' 24.88 ± 1.95 10.64 ± 2.22* 17.90 ± 4.19* 15.05 7.12* pc gross ma\ ^gO: cm • h ' 30.66 ± 1.95 14.78 ± 2.22* 29.16 ± 4.19 id 'IS 7.12 •\\erage r. night »/gO:cm h 5.77 ±0.21 4.14 ± (i 29 11.26 ± 0.80* 15.93 1.09* P. gross/Re 24 h Ratio (held) 1.98 ±0.51 1.37 ± 0.31 0.83 ± 0.34* 0.73 0.23* ' Means sign i In. anth di tiercm from the Control i/' ' 0 OM in marine organisms is known in only a feu cases (Webb ami ("oil. 1983: Sullivan ct a/.. 1983: Morse and Morse. 1984: Target! ct a/.. 1986; Pawlik. 19X6). In the boring sponge Siphonoiliciyon. mucus-borne metabolites kill corals (Sullivan ct a/.. 1983). Our study demonstrates that chemical-biological interactions are important in non-boring sponge species and in over-growth, not just anti-fouling. processes. Several toxic exudates from soft corals appear to be <£ — l/> -7 c >< in O £ O 30 r -to NO CONTACT 400 Irrodiance -30 L I miirr V Net photOSyntheSJS-irradiance cm\cs are graphed I'm .\Karu-iu liiiHiinki colonies In 1) from 20 m depth under dillcrcnl levels of contact unh l'/ii/..n>/i\ lit/In lninitnni/i'\ I he i'iii\es are lilted to a hyperhi lurutuin (see I able l\ Im the sl.ilisln.il com- parisons hetv. i I ..|vnniental ( oiuliliuii I : control, no con- tact with I'liii 01 les). 1 xperinu'nl.il Cunditnin 2: indirect contact with water cor mdate from coarselj mashed Plakortii (solid squares). Cxpcrim -idilidti V direct c|" •niin-iil.il ( imdilimi •) iliu-il contact with uninjured I'lukm 'solid dianioiuKl (see I ij1, I I. I \|ien- mental C'ondilion 4 most clusel-. mimics condiiums ol natural i CMH.H i on the reef. responsible for causing localized mortality in hard corals, since extensive mortality of hard corals can occur even when direct contact is not established with the soft coral (Sammarco ct til.. 1985). Coll ct al. ( 1982b) isolated sec- ondary metabolites from the water surrounding two spe- cies of octocorals that were identical to those isolated from the octocorals themselves. Flexibilide. dihydro- flexibilide. sarcophine. and sarcophytoxide were isolated from crude extracts ofSinularia /Icxihilis and Surcopliy- lon crassocaula, respectively, and from their surround- ing water when sampled /».v////(Coll el al.. 1982b). Flexi- bilide and sarcophytoxide cause death in scleractinian corals at concentrations > 10 ppm (Webb and Coll, 1983). These compounds are also structurally related to sinulariolide. a potent algal growth inhibitor (Tursch. 1976). The sponge Plakonis halichondroides contains nu- merous unusual secondary metabolites such as cyclic peroxides and aromatic lactones (Faulkner ct al.. 1979; Stierlc and Faulkner. 1980). It is possible that the cyclic peroxides cause the bleaching response described above and that the phenolic compounds contribute to tissue ly- sis. The closely related congener. Plukortis :yx«nip/hi. contains (Z)-7-meth\ l-4-octen-3-one and several deriva- tives of 3-hydroxy-4-hydroxymethyl-4-pentenoic acid (Faulkner and Ravi. 1980). These may be involved in both the coral biomass changes and the oxygen flux mod- ifications observed for the Plakortis/Agaricia associa- tion. Between 30 and 40 percent of all Flakortis colonies on the reef are contiguous with scleractinian corals. Of these contacts, half show bleaching and necrosis on the coral tissue near the sponge: all show death of the coral tissue mulct ncaih the sponge. More importantly, based on the results of our /// Min manipulations, the same elfect is observable prior to the establishment of direct sponge- SPONGE-CORAL ALLELOCHEMISTRY 237 coral contact. This implies that the sponge exudes water- borne metabolites which have a detrimental effect on coral respiration and photosynthesis. If even naturally low concentrations of these waterborne metabolites are effective at suppressing coral photosynthesis, then there is the distinct possibility that sponge exudates may in- fluence ecosystem productivity in the deeper zones of the reef. Plakortis kills and colonizes the dead skeletons of at least 14 scleractinian coral species. These species are dis- tributed in all 8 Caribbean coral families located in the 20-30 m survey area. Our demonstration that toxins from Plakortis are so effective against corals, the other- wise most successful order of benthic invertebrates on the reef, suggests that further investigation of this interac- tion is warranted. Future research should focus on the mechanisms of action and chemical structure of the compounds that inhibit photosynthesis, stimulate respi- ration, and reduce coral colony biomass. The range of potency and the half-life of these allelochemicals once released into the water column is also of critical interest. Finally, information on the long-term survival of corals at varying distances from sponges, and the sources of mortality for Plakortis would be of great value to our un- derstanding of the chemical ecology of this association. Allelochemistry gives Plakortis a measurable advantage in space competition with scleractinian corals, but is only one factor among many other biotic and abiotic factors which determine its population structure and dynamics. Acknowledgments We thank the Hydrolab staff for their support during Missions 84-10 and 85-4, and particularly thank aqua- nauts George Schmahl, Joe Schubauer, Paul Preston from 84-10, and Nick Vrolijk, Ray Jakubczak. and Rob- bie Smith from 85-4. We are also grateful to topside sci- entists Hany Salam (84-10 and 85-4) and Susan Sennett (85-4). Dr. Willard D. Hartman identified our Plakortis specimens. This work is supported by grants from the Hydrolab Program of NOAA and Office of Naval Re- search Grant Number N-000-14-84-K-057 1 . Literature Cited Bak, R. P. M., R. M. Termaat, and R. Dekker. 1982. The complexity of coral interactions: influence of time, location of interaction, and epifauna. Mar. Biol. 69: 2 1 5-222. Baker, J. I., and V. Murphy. 1976. Compounds from Marine Organ- isms. \ 'olwnc I CRC Press, Cleveland. 226 pp. Bakus, G. J. 1981. Chemical defence mechanisms and fish feeding behavior on the Great Barrier Reef, Australia. Science 211: 497- 499. Bakus, G. J., N. M. 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Effects of alcyonarian coral terpenes on scleractinian coral photosynthesis and respiration. Toxicon Sitppl. 3: 485-488. Wellington, G. M. 1980. Reversal of digestive interactions between Pacific reef corals: mediation by sweeper tentacles. Oecologia 47: 340-343. \\hittaker, R. H., and P. P. Feeny. 1971. Allelochemics: chemical interactions between species. Science 171: 757-770. Reference: Bu>l Bull 175: :4i>-:-i> A New Type of the Manifestation of Colony Specificity in the Compound Ascidian, Botrylloides violaceus Oka1 EUICHI HIROSE. VASUNOR1 SAITO. AND HIROSH1 WATANABE Shiinoda Marine Research (.'enter. University of Tsukuba, Slummla 5-10-1. Sltiiuoka 415. .Japan Abstract. A new t\ pe of colony specificity ( = allogeneic recognition) is shown for Botrylloides violaceus. All bo- tr\llid ascidians previoush studied for colony specificity sho\\ allo-recognition reactions, manifested as fusion or nonfusion (rejection), both at the colonial margin (= growing edge) and at the cut surface. By contrast allo- recognition in Bolrylloiile\ violaceiis is absent at the cut surface, but present at the growing edge. Juxtaposition of cut surfaces resulted in fusion of the colonies regardless of origin, while juxtaposition of natural growing edges resulted in fusion or rejection, according to the genetic combination of colonies. Similar results occurred among the sibling colonies derived from the same mother col- ons, in which pairs tunic necrosis was observed in areas where the two colonies partially fused. These features of allogeneic rejection in B. vinliici'ii\ were very similar to those of "nonfusion" in Boiryl/oiih"* s/w/" resulted in "nonfusion" without a particular reaction. Introduction >n\ spcciticiu represented b> allograft rejection has been demonstrated for man> colonial forms of ani- mals, from sponges to ascidians. Colony specificity in some compound aseidians is manifested b> fusibiliu be- tween colonies, two colonies cither form a single mass (fusion) or do not ' ncicction). when thc\ come into con UK! Some .1 -.peciesdo not show coloin spcci- Rcccived 7 Januars I98f ' !6 luh l'»ss 'Contribution number 4K4 from the Slnmo,l.i M.IMIK- KI-MMH.II Center. ficity. while others exhibit it (Koyama and Watanabe. 1 982). In those that do. isogeneic colonies are always fus- ible at their natural growing edges, and allogeneic colo- nies are either fusible or not fusible. Colony specificity in compound ascidians has been studied primarily in species of the family Botr\llidae(bo- tryllid ascidians). These species form sheet-like colonies in which /ooids are buried in a gelatinous tunic. Zooids are arranged in rosettes or ladder-like systems with com- mon cloacal apertures, and are connected to one another by a ramif) ing network of blood vessels which terminate in sausage-shaped ampullae at the pcriphen of the col- ony. All botryllid ascidians that have been studied so far exhibit colon> specificity. In some of them, genetic con- trol of their fusibiliu has been demonstrated (Oka and Watanabe. 1957. 1960. 1967; Sabbadin. 1962. 1982; Scofield ciu/.. 1982). The morphology and cell biology of fusion and nonfu- sion (rejection) in botryllids have been studied in detail in four species: Botryl/u\ va;A/m (Saito and Watanabe. 1982). B. /»•/»;/!,'<•"»'> (Oka and Watanabe. 1%7; Tanaka and Watanabe. 1973: Tanaka. 1973: Katow and Wata- nabe, 1980: Taneda and Watanabe. 1982). B. sehlosseri (Milanesi cl nl.. 1978: Scofield and Nagashima. 1983). and lioiryl/oii/i"* s//;;r«/cv;\M (Mukai and Watanabe. 1974: Saito. [976; cj Sailor/,//, 1981). I he course of fusion is essentially the same in all these species. By con- trast, the rejection reaction is initiated at distinctly dilferent stages of fusion in different species. These facts impK that comparative studies of the processes of fusion and nonfusion in various species might be useful forana- ly/ing the mechanism of allo-recognition, as well as for considerations of the evolution of colony specificity in botryllid aseidians. Here we ha\e iiuesligaled the pro- cesses of lusion and nonfusion in the Japanese species Boirvlloiilcs vinlaeeus. anil found that this species shows COLONY SPECIFICITY IN BOTRYLLO1DES 241 Table I Fusion experiments in Botrylloides violaceus at the cut surface Combination No. combination Fusion Nonfusion Between colonies of the Shimoda population 112 112 0 Between colonies of the Asamushi population 27 27 0 Between the two populations 33 33 0 a new and instructive type of colony specificity pre- viously undescribed for compound ascidians. Materials and Methods Colonies of B. violaceus -were collected in the Shimoda Floating Aquarium (Shizuoka Prefecture) and in the vi- cinity of the Asamushi Marine Biological Station (Ao- mori Prefecture). The two collecting sites are about 1 100 km apart in linear distance. B. simodensis colonies were also collected in the vicinity of the Shimoda Marine Re- search Center (Shizuoka Prefecture). To facilitate han- dling of the colonies, they were fixed on glass plates and reared in the culture boxes floated in the bay near the Shimoda Marine Research Center. The fusibility of colonies was tested by fusion experi- ments. Procedures for fusion experiments were as fol- lows: a piece was cut out of each of the two colonies be- tween which fusibility was to be tested. The two similarly sized pieces (about 15 zooids) were placed in juxtaposi- tion on a glass slide, and they were brought into contact with each other either at the cut surfaces or at the grow- ing edges. The colonies on the glass slide were then kept in a moisture chamber for 30 minutes, so that the colo- nies might attach to the glass slides prior to their place- ment in running seawater. The experimental animals were observed under a binocular stereomicroscope each day. The paired colonies showed clear fusion or nonfu- sion reactions. Fusion here means the establishment of a common vascular system between the two colonies, and nonfusion the absence of it. In the latter case, several tri- als with the same combination of colonies were carried out to avoid the possibility of accidental failure of fusion. Oozooids of B violaceus were obtained from some colonies from the Shimoda Floating Aquarium popula- tion. In the fusion experiments of sibling colonies, a pair of oozooids derived from the same mother colony were placed on a glass slide so that they came into contact by their growing edges. Further experimental methods were the same as those just mentioned. For histological studies, specimens in the process of fusion or nonfusion were fixed in a solution containing 2.5% glutaraldehyde and 0.45 M sucrose buffered with 0.1 M cacodylate at pH 7.4. The fixed specimens were then dehydrated through a butanol series, embedded in paraplast. sectioned at 5 pm and stained with Congo red, Delafield's hematoxylin, and eosin-orange G. Results Fusion experiment* at the cut surface When colonies of Botrylloides violaceus were apposed by their cut surfaces, we were surprised to note that all colonies fused (Table I). This was true both in intrapopu- lational and in interpopulational combinations. How- ever, separation of such fused colonies between which the common vascular system had been established for a few days or more was occasionally observed. This sug- gests the presence of a kind of long term (or induced) allo-recognition. It should be noted, however, that in these experiments it was difficult to determine whether separation was caused by allo-recognition or by unsuit- able conditions for long-term observation. By contrast, a rapid, necrotic xeno-graft rejection was observed be- tween similarly juxtaposed pieces of B. violaceus and B. simodensis colonies. Blood cells from the blood vessels clustered in the tunic at the boundary between the colo- nies. The clusters of cells were observed as a clear black line along the boundary (Fig. 1). The area with the cell infiltration eventually degenerated and was apparently cleared from the area. Fusion experiments at the growing edge By contrast to the results with cut colonies, apposition of colonies by their naturally growing edges resulted ei- ther in fusion or nonfusion, depending on the particular combination of colonies employed. In the case of fusion. Figure 1. Xeno-rejection at the cut surface. Upper colony is Botryl- loides violaceus, and lower colony is B. simodensis. Necrosis is ob- served along the boundary between the two colonies, z. zooid. Scale bar = 1 mm. 242 I / ; it 11 and nontusion at the growing edge between sibling colon i :!>ndes \iolaceus. a: Fusion, four days after settlement of the i . •. ooids. Common vascular system has been established, b: Nonfusion, a week after settlement, am. ampullae; o. oozooid. Scale Kirs « *• mm. disappeared and was filled with continuous test matrix containing normally distributed tunic cells (Fig. 4a-c). Fragments of cuticle were sometimes observed in the original boundary /one (Fig. 4b). In the case of nonfu- sion, the tunic layers sometimes fused, but only in small areas along the boundary. In the fused areas, tunic cells \\ere considerably more abundant than usual (Fig. 4d). In addition, blood cells (particularly morula cells) infil- trated the tunic from the blood vessels in the contact ar- eas ( Fig. 4e). These cells were found clustered and disin- tegrated in the rejection zone. The disintegrated cells subsequently were released from the tunic in the form of massive aggregations (Fig. 4f). tip to side contacts occurred between ampullae of the two colonies, and then fusion took place at those sites to produce a common vascular system. In the case of non- fusion, ampullae of both colonies pushed against each other, but never penetrated the tunic of the facing col- ony. Although signs of rejection were not clearly visible in the contact area under the binocular stereomicro- scope, the necrotic rejections were evident in subsequent histological sections of the rejection zone (below •). Similar experiments between sibling oozooids gave similar results (Fig. 2). Out of the 32 combinations stud- ied. 14 resulted in fusion and 10 resulted in nonfusion. The results of the remaining eight combinations could not be assessed because of the degeneration or mechani- cal separation of the colonies. All xenogeneic combinations between B. violaceus and B. simodensis resulted in nonfusion reactions sim- ilar to those in incompatible intraspecies pairs. //; \ti>h >gical tihscmitions A frontal section of normal ampullae at the periphery of a colony is shown in Figure 3. The ampullae are buried in the tunic. Many blood cells are seen in the ampullar lumen, and are not usually seen in the tunic. Tunic (or "test") cells are dispersed throughout the tunic, and can be distinguished from blood cells by their morphology and staining characteristics. A cuticular layer is differen- tiated at the external surface of the tunic. The histological features of fusion and nonfusion at the growing edge of B. violaceus were very similar to those"! I. --'ilcnsis as reported by Saito( 1976). In the case of fir ' boundary between the two colonies Discussion In Botrylltiiilf* rio/thcuy short term allo-recognition appears to be absent at the cut surface and present at the growing edge. This type of colony specificity has not been described previously. In compound ascidians previously studied, mechanisms of allo-recognition have been stud- ied in Didemnum in<>st'/cyi (Mukai and Watanahe. 1974). I\-n>pln>ru juponica (Koyama and Watanabe. Figure 3. Frontal section through the periphery of a colony of Bo- n \-llmtlcs violaceus The substratum is to the bottom and the colony is growing to the right. Some of the tunic cells arc pointed by arrow heads, c. cuticle: e, epithelium of ampulla; lu. lumen of ampulla with blood cells. Scale bar Ml vm. Histological aspects of fusion and nonfusion at the growing edge between sibling colonies of i'1'ilaceus. a-c: Fusion, (a) Contact of colonies. Dissolution ol cuticular layers occurs at the i colonies (arrow ). (bi Tiision ol colonies. I' ragments of cuticle are still observed (arrow head) (c)( I ••. luscd tunic, d-f: Nonlusion. kll Partially fused tunic in which there are many tunic cells (ei In iiiliratc into the tunic and form a mass of cells (arrow heads), (f) The mass of de- structed idls is r to the outside of the tunic (arrow lic.uli Scale bars •*->L2S 243 244 E. HIROSE ET AL. 1981). P. sagamiensis (K .ma and Watanabe. 1982). Symplegma replm and Watanabe. 1974). Bo- iryllus scalari\ v -;d Watanabe. 1982), B. primi- genus (Oka -ibe. 1957). B. schlosseri (Ban- croft. 190' am. 1962; Scoficld and Nagashima. 1983). ami ales simodensis (Mukai and Wata- nabe \lthough allo-recognition at the cut surface is uncertain in the two species of Perophora and B. . all the other ascidians listed clearly have the : . tor allo-recognition, both at the cut surface and at the growing edge (reviewed by Watanabe and Taneda. I1'*:: Taneda et at.. 1985). On the other hand, in the species we and others found lacking colom speciticitv. c i.' Polycitor proliferus (Oka and Usui. 1944: ct. Toki- oka. 1953) and Perophora mitlticlalhrata (Mukai and Watanabe. 1974: cf. Nishikawa. 1984). two colonies never fuse naturally at their growing edges, but invari- ably fuse at their cut surfaces, regardless of their origin. In B. violaceus, a colony invariably fuses with am other member of the same population or of another popula- tion at the cut surface. At the growing edge, however. colonies show fusion or nonfusion depending on the combination of colonies. From this we conclude that B violaceus has a new type of colony specificity. The nonfusion reaction observed at the growing edges of B. violaceus is similar to that of B. .simoa'ciisi\. which sometimes has been called "indifference." This term means nonfusion without particular reaction, such as the nonfusion of the ascidians lacking colony specificity. e.g., Perophora multiclathrata (Mukai and Watanabe. 1974). In the nonfusion of both B. simoilensis and B. violaceus. the necrotic reaction between two colonies is barely observable under a binocular stereomicroscope. but is clearlv observed histologically. Consequently, the nonfusion of these species should be placed in a category other than indifference. We propose, therefore, to use the term "sub-cuticular rejection" to describe nonfusion re- actions in these two species. In sub-cuticular rejection, the necrotic reaction with blood cell infiltration is limited to the tunic along the boundary between colonies, and never occurs in the ampullae and blood vessels. In sub-cuticular rejection, allo-recognition appears to occur in the suh-cuticular region of the colonial margin because the reaction is limited to that area. In fact. H. viulai'cu*. may conduct allo-recognition only in the sub- cuticulai region, since allo-recognition is absent at the cut surface in this species. The sub-cuticular region of tunic consists of tunicin libers and tunic cells. Therefore. the tunic cells i> • 'In play an important role for allo-rec- ognition. All bolryllid ascidians studied with regard to colom specificity arc capable < tural allo-recognilion at the growing edge. The fusion n.m isessentiall> thcs.mic in all species studied, but the nonfusion reaction is initi- lahlc II The stage at which the nonfusion reaction is initiated at the growing t'l/t'i Species Stage Reference B. scalaris ampullar fusion Saito and Watanabe. 1982 II iirunigenus ampullar penetration Taneda and Watanabe. 1982 /; \imodensis partial fusion of tunic Saito. 1976 H ui>/ijtvii>/di;\: I /;/Y (eh. ' lul ponents of the male reproductive system and. to a lesser extent, the presence or absence of muscular prostatic or- gans also known as adenodactyls. The distribution of Polyet'li.\ covers a large portion of the Northern Hemisphere (Kenk. 1953: Ball. 1975). In North America, the known species are restricted to the western third of the continent. Kenk (1953) proposed that the North American distribution of Pol reel is is a re- sult of pre-glacial dispersal from Asia across the Bering Strait when a land bridge existed during periods of lower sea stands. According to Kenk ( 1953). subsequent glacia- tions have controlled or adjusted the distribution of cer- tain species. Kenk's ( 1953) synthesis of Polyeelis distri- bution has been adopted by Ball ( 1975). Recent investigations of springs in western Massachu- setts, in the northeastern United States, have revealed the existence of an undescribed species of Polyeelis. The new species possesses, among other distinctive morphological features, a speciali/ed gland situated near the reproduc- tive structures, termed the ventral gland, which is unlike that of any other described species of /WroY/.s or any other known North American triclad species. This paper provides a description of the new species and attempts to explain the disjunct occurrence of the new species in New Fngland. Materials and Methods The spring in which the new species was found is in Sunderland. Franklin County, Massachusetts and repre- sents the i\ pe locality. The spring is the principal source of water for a state-owned fish hatchery. Specimens were collected on 25 August, 22 September, 30 October, 1987, l< NEW SPECIES OF FLATWORM FROM NEW ENGLAND 247 and 13 January, 1988. One hundred and fifty specimens were collected and examined. Of the series, 34 animals had fully formed reproductive organs. Most specimens were killed in 2% nitric acid and fixed in FAA. A few specimens were maintained in laboratory conditions for behavioral observations. Twelve speci- mens were serially sectioned using conventional tech- niques and stained with Delaneld's or Ehrlich's hema- toxylin and eosin. Though most sections were of the sag- ittal plane, cross sections were prepared in two cases. An additional three specimens were dissected. The Holotype and the Paratypes (all slides) and a series of whole speci- mens have been deposited into the collections of the Mu- seum of Comparative Zoology, Harvard University, Cambridge, Massachusetts. The remaining specimens and prepared slides have been deposited into the Mu- seum of Zoology, University of Massachusetts. Amherst, Massachusetts. Systematic Account and Results Family Planariidae Stimpson, 1857 Genus Polycelis Ehrenberg, 1 83 1 Polycelis remota, new species Diagnosis Polycelis remota is a medium sized species (maximum length, 17 mm) demonstrating characteristics represen- tative of the genus, including the presence of an arc of eyespots following the anterior contour of the body of the animal, distinct cephalic auricles produced anterolat- erally. testes situated ventrally, and pre-pharyngeal and paired oviducts uniting posteriorly of the penis bulb to form a common oviduct which descends to the genital atrium. Otherwise, it is distinct from all known species of Polycelis by the possession of a large, transverse mus- cular gland with a single exterior sucker-like organ situ- ated always on the right ventral side of the animal, ante- rior and separate from the gonopore and its associated atrial cavities. The new species is further distinguished from all other known species of Polycelis by the peculiar seminal vesicle, by having a greatly elongated penis bulb, and by the position of the copulatory bursa which ex- tends anterior of the posterior margin of the pharyngeal cavity. Description General characteristics of Holotype (living animal, J 1.0 mm) (Fig. 1A. B). The anterior margin of the head forms a low inverted "V," and continues laterally with sub-triangular auricles. The neck region is posterior to auricles with slight constriction. The body widens poste- riorly, reaching its greatest width in the region of phar- Figure 1 . External characteristics of Polycelis remota: A, dorsal view of living animal; B. anterior end of living animal; C, anterior end of preserved animal; D, ventral view of posterior portion of preserved animal. Scale line equals 1 mm; G = gonopore, M = mouth pore, R = region of ventral gland. S = "sucker." ynx. The posterior part of the animal tapers to a rounded point. Eyes are present and numerous, and extend across the margin of the head and along the lateral margins, ex- clusive of auricles, some distance posterior to auricles. The arc is interrupted at the midpoint of the anterior margin. The dorsal surface anterior to the pharyngeal re- gion with the low median keel extending anteriorly to the apex of the anterior margin. The pharynx is single, medially placed, and occupies about one third of the length of the animal. The inner muscle layers of pharynx are composed of an outer, thin longitudinal layer and an inner, thick circular layer. Rhabdites are present along the margins of the animal. Both mouth and genital pores open ventrally on the midline. The color of the dorsal surface of animal, exclusive of the area of digestive caeca, is olive, and the digestive caeca are brown to yellowish- brown. The pharyngeal region is a lighter whitish-yellow. Ventrally, the animal is grayish-white, and the region of the atrium and gland is lighter. The pharynx is unpig- mented. Anatomy of the Holotype (Figs. ID, 2, 3a-d). In the 248 D. G. SMIIH CO . Sagittal MOW nl posterior male and female reproduclne structures of l'«l\ccli\ rcm<>ta, semi-diagrammatic: -\ atrium. B hursj. BD = bursa duet. CO = common oviduct. LD ejaculalon duct. GP = gonopore, M mouth pore. PB = penis bulb. PH = pharvnx, PP = penis papilla. PV = papilla of \esicle. SV = seminal \esicle. VD \as deferens (narrowed spermiductal \esicle), VG - \entral gland: stipple indicates presence of muscle tissue. female reproductive system, the ovaries are situated ven- trally and just posterior to first lateral digestive caecum and slightly to side of midline. Oviducts pass posteriorly and ventrally. medially to nerve cords, to a point just posterior of the ventral gland, under which they pass, and ascend on either side of the penis hulh. The right oviduct passes hetween the hursa duct and penis hulh. The two ov iducts join posterior to the penis bulb to form a com- mon oviduct which proceeds to female portion of the atrium. The copulatory hursa is a lohatc. somewhat flat- tened sac placed dorsully and to right of the pharyngeal cavity. The hursa stalk or duct proceeds posteriorly from the bursa to the right of the penis hulh and joins the atrium ventrally along side the penis papilla. The histol- ogy ofthc hursa duct changes during its course. The duct wall is anteriorly similar to the bursa wall and contains a thick cellular lining composed of a tall, spacious, and densely staining columnar epithelium without a detect- able muscle coat. The posterior portion of ventral gland is surrounded by a coat of connective tissue fibers; the inner epithelium is characteri/ed by thin columnar cells which stain densely only at their bases. The male reproductive system contains separate testes extending ventrally from between second and third di- gestive caeca posteriorly to near base of the pharynx. Up ; ht testicular masses are evident at one time. The two vasa dcfercntia pass ventrally and posteriorly, and medially to the nerve cords near the distal third of the pharynx the ducts enlarge to form spermiductal vesicles. I osicles narrow somewhat and separately enter the & -side at approximately opposite lat- eral points on Ir I he seminal vesicle is lativ oval, and situ -diately posterior to the phai v n- geal cavity and IKI I:1'! ll\man. I- II- 1^51. '.:u-\ Playhelminthes and Rhyn- chocoela. Vol. II -.-Hill Book Co . New York. 550pp. kenk. R. 193(1. i L- zum system der Probursalier (Tncladida pa- ludicola) / 89:145-162 ki-nk. R. I1'- -h-watermcladsl I urbcllarialof Alaska / t'.V '. 103: 163-186. kenk. R. l"~ I leshwater triclads (Turbellaria) of North America. V':tl >Polycelii .s/m//iM'»i <.'I'/IIH/< /<« (;V;i 78: 44- RfMinldsDii. I. B. 1966. The distribution and abundance of lake- dwelling triclads-towardsa hypothesis, li/i i.« 52 Smith. 1). (,. 19S3. A new species ol Ircsh-water gammaroidean am- phipod (Crangonyctidae) from southeastern New England. Trans In, U« 102:355-365. Smith. 1). (.. 1986. The occurrence of the troglobitic amphipod. Slv- VI'/TO/HI/A ft'Hi/M /ivm/s (Smith) (Crangonyctidae) in the Taconic Mountains of southwestern Massachusetts (USA): a case for the ex- istence of a subterranean relugium in a glaciated region. Inl. J Spel- eol 14(19X4-19X5): 31-37. \ andi'l, A. 1921. \oteshiologiquessurlesPlanaircsdesenvironsde Montpellier. Hull Hi, 'I I-'r . licl.n. 55: 239-259. \\ insor. I.. 1981 . I he taxonomy, zoogeograpln and biologx afliipul- nini /u'luvnr Moseley, 1878 (Tncladida. Temcola). llnJriihi,>lni;ui 84: 17. Reference: Biol. Bull 175: 253-260. (October. 1988) Temperature and Relative Humidity Effects on Aerial Exposure Tolerance in the Freshwater Bivalve Corbicula fluminea ROGER A. BYRNE1, ROBERT F. McMAHON2, AND THOMAS H. DIETZ' 1 Department of Zoology and Physiology, Louisiana State University. Baton Rouge. Louisiana 70803. and2Section of Comparative Physiology, Department of Biology, Box 19498, The University of Texas at Arlington, Arlington, Texas 76019 Abstract. The exposure tolerance, aerial respiratory be- haviors, and the rates of water loss of the Asian freshwa- ter clam, Corbicula fluminea, were assessed under three temperature conditions ( 1 5°, 25° and 35°C) and five rela- tive humidity (RH) treatments (5%. 33%, 53%, 75% and 95%). C. fluminea displayed low tolerance to aerial exposure (range of median tolerance times: 23.8-24.9 h at 35°C, 7 1 .4-78.2 h at 25°C, and 248.5-34 1 .6 at 1 5°C). Relative humidity had no effect on median tolerance time except at 15°C. Body size was reciprocally related to water loss rate at all temperatures, and on longevity at 25° and 35°C. Cumulative rates of water loss at 95%. and 75% RH were lower than the other humidities at 15°C, but no differences were found at 25° or 35°C. Mantle edge expo- sure behavior was inhibited by low humidity and high temperature. Exposing mantle tissues did not increase rate of water loss except at humidities below which the behavior was very rare. The occurrence or extent of the behavior did not affect individual clam longevity. The results suggest that C. fluminea can detect rates of desic- cation and make behavioral adjustments. Introduction The Asian clam, Corbiculafluminea ( Miiller) is a com- mon inhabitant of freshwater habitats in the southern United States (McMahon, 1982, 1983a). Its migration to freshwater is relatively recent and evidence for its estua- rine past exists in its higher blood osmolality and differ- Received 20 February 1988; accepted 27 July 1988. Abbreviations: RH: relative humidity. LSL: log,0 shell length, ME: proportion of time spent with mantle exposed. ent ion ratios compared to other freshwater bivalves (Dietz, 1979). The adaptations displayed by C. fluminea for survival in freshwater must be recently evolved and may have arisen from estuarine/marine adaptations (Gainey, 1978). Furthermore, the adaptations of corbi- culids are derived independently of those of another ma- jor freshwater bivalve family, the unionids. Freshwater bivalve molluscs inhabiting shallow lentic or lotic habitats are subject to periodic emersion as water level drops. Typically, reduced water levels commonly occur during summer months when rainfall is lower and temperatures are higher. Emersion periods are not pre- dictable in their duration or timing. As an adaptation to this stress, some freshwater bivalve species are capable of surviving up to a year out of water (Hiscock, 1953). McMahon (1979) showed that survivability of C. flumi- nea in air is affected by temperature and relative humid- ity. Associated with emersion are an array of behaviors which include gaping or mantle exposure. This appears to be associated with an aerial respiratory function (Mc- Mahon, 1979; McMahon and Williams, 1984). There is evidence that other freshwater bivalves can obtain oxy- gen directly across the valves (Collins, 1967; Dietz. 1 974). Many intertidal marine bivalves maintain aerobic metabolism by continually gaping valves and allowing direct exchange of gasses with the atmosphere (Bayne et ill.. 1976:BrinkhoffiYtf/., 1983; Snick et a/., 1986; fora review see McMahon, 1988). Exposure of large soft tis- sue surfaces to air should lead to water loss. Under pro- longed aerial exposure conditions, C. fluminea must bal- ance the advantages of maintaining an aerobic metabolic mode against the requirement to conserve water. The aims of this study are to investigate the interrela- tionships of temperature and relative humidity on sur- 253 254 K \ BYRNI /•/ I/ vivability ofC.Jlumimu in air. \Ve examined the ellccts of body size, and the ex '.nu-nce and frequency of aerial respirator. beha\ 101 . ••• .ner loss rates under these envi- ronmental conditii • \\e are particularly interested in the interplay bet\v ^-n the requirement of preventing de- hydration ss Inch necessitates valve closure and the meta- bolic ads :• >f maintaining contacts with the exter- nal environment. Materials and Methods Specimens of the Asian clam. ('. tlununca. for all ex- periments were collected in June. 1 985. from an outflow of Lake Benbrook. Tarrant County, Texas. Animals were maintained in filtered, aerated tapwater at labora- tory temperatures (20°-23"C) for at least one week prior to use. A sample of 300 animals was used for the experi- ments. 20 per relative humidity/temperature combina- tion. Each animal was identified by a lightly etched num- ber on the left valve. The etchings removed the outer per- iostracal layer to reveal the light colored mineral underneath. It is unlikely that the etchings svould have affected gas permeability. Specimens were blotted dry. weighed to the nearest 0. 1 mg. and placed onto a desicca- tor plate in desiccators maintained at the appropriate rel- ative humidity and temperature. Five relative humidities were chosen, <5%, 33%, 53%, 75%, and >95%. These humidities were maintained in closed desiccators (plate diameter 190 mm) by using sil- ica gel (<5%), supersaturated solutions of MgCl:-6H:O (339 ). Mg(NO,):-6H:O (53%). NaCl (75%), and water (>95%). Salt solutions used to establish the various hu- midity values are from Wexler and Hasegawa (1954). Relative humidity was measured using a hygrometer (Airguide) and remained ±10''; of the starting value dur- ing the experiments. Experiments were performed at 15°, 25°. and 35°C (±0.5°C). temperatures chosen to repre- sent a range comparable with those experienced in the environment. Field observations show that individuals of this population when aerially exposed lie just beneath the surface of the sandy substratum, exposing little of the shell. Around 70-80% of the ventral aspect of the valve junction may be exposed. Under these conditions the effects of wind on esaporatise loss are reduced and our design. s\hich does not involve air movement, is appro- priate. Animals were reweighed at approximately 3, 6, or 12 hour intervals for 15". 25°, and 15°C treatments, respec- tively. Prior to amoving animals from the desiccators, the number of individuals exposing the mantle edge, a respiratory behavior, was noted, as were occasions when obvious quantities ol Quid had been expelled. At death, the soft tissue was excised di ied to constant weight (>48 h. 90°C) and weighed. I he shell was blotted dry anil weighed. The experiment continued until all animals were dead. The total available water [total weight at beginning of the experiment - (wet shell weight + dry tissue weight)]. s\as calculated for each individual. Water loss was ex- pressed as cumulative percent water lost ( 100 x weight lost since the start of the experiment/total available wa- ter). The points on the curses of the time course of cumu- latise ssater loss (Figs IB. 2B. and 3B) represent values for a standard (grand mean sized) animal of 20.3 mm shell length. Values svere derived by performing regres- sion analyses of log,,, shell length versus cumulative wa- ter loss at each sampling interval, and deriving an esti- mate fora 20.3 mm animal from the regression equation. The number of animals used for the regression analyses (the survivor number) ranged from 20 at the beginning to 3 near the end of the experiments. Of the approxi- mately 150 separate regressions performed. 66'; ssere significant (1J < 0.05). A significant si/e relationship to cumulative svater loss was more prevalent at 15°C and 25°C (85 and 93r; of regressions performed, respectively) than at 35°C (33%). If no significant relationship was in- dicated (I>> 0.05) then the mean water loss svas utilized. Hourly rates of water loss were calculated as the per- cent water lost/sampling interval duration and were cate- gorized as to whether the animal svas exposing mantle edge tissues, had closed valves, or had expelled fluid dur- ing the sampling interval. Analyses of variance using a repeated measures design on logm transformed rates were performed, followed by Duncan's multiple range tests to determine differences between temperatures. Analyses of covariance were run ssith log,,, shell length as the covariate and Duncan's multiple range tests were performed to detect relative humidity effects within each temperature. Student's Mests were performed on paired comparisons of water loss rates when valves ssere closed. when exposing mantle tissues, and when fluid was ex- pelled for each animal at each temperature/relative hu- midity combination. An index of individual clam behav- ior is the proportion of time a clam spends with mantle exposed (ME), exclusive of periods during which mantle fluid svas expelled. A multiple regression relating clam longevity to relative humidity . log,,, shell length, and ME was performed. Median tolerance times (TLm or 1TM), the elapsed time to 50% mortality) were calculated from the mortal- ity data using the SAS PROBIT procedure (SAS. Cary. N("). Probit analysis transforms the sigmoid time/mor- tality curve to a linear shape and, by means of linear re- gression, a mid-point or median value can be dcris-ed. Significant differences in median tolerance times were detected by non-overlap of 95^ fiducial limits. Other sta- tistical tests also utilized SAS procedures. Results I here was a significant (/' < 0.05) temperature effect on median tolerance time ( 1 I m) ( I able I) resulting in CORBICULA AERIAL EXPOSURE 255 Table I Median tolerance limes, lime elapsed (hours) to 50% mortality as determined by prohit analysis. /orCorbicula fluminea under various conditions of temperature and relative humidity Median tolerance times (hours) relative humidity Temp. 33? 53? >95% 15°C 259.7 A 248. 5 A 253.0 A 305.4 B 341.6C 25°C 72. 2 A 7 1.4 A 73.4 A 78.2 A 75. 6 A 35°C 23.8 A 24.0 A 24.0 A 24.4 A 24.9 A Different letters within a temperature treatment (rows) indicate non- overlap of 95% fiducial limits between those relative humidity treat- ments. an approximately threefold increase in TLm with every 10°C decline in temperature. At 15°C, theTLm'sat 75% and at 95% RH were significantly different (P < 0.05) from one another and both were higher than those at the other relative humidities. No such relationship of rela- tive humidity to TLm was found at either of the other temperature treatments. During emersion, C. fluminea displayed three catego- ries of behavior. The first was simply the closed condition with valves tightly shut, displaying no tissue directly to the environment. The second behavior was that de- scribed by McMahon ( 1979) where the leading edge of the mantle tissue is protruded past slightly gaping valves so that moist tissue is exposed. This mantle edge expo- sure behavior has been linked to possible aerial oxygen uptake (McMahon and Williams, 1984). Under condi- tions of lower relative humidity the exposed tissues may appear dry, or a hardened mucus may form between the parted valves. Clams displaying these conditions also were scored as exposing mantle tissues. The third cate- gory of behavior was a complete or partial emptying of the mantle cavity water store. This was evidenced at higher humidities by a pool of fluid around the animal. At lower humidities the fluid may have dried, but evi- dence in the form of dried matter around the animal was an indication that fluid had been expelled. Mantle edge exposure was influenced both by temper- ature and relative humidity (Figs 1 A. 2A and 3A). High temperature (35°C) inhibited exposure (Fig. 3A), whereas at 25°C and 1 5°C mantle edge exposure was con- trolled primarily by relative humidity (Figs 2A and 1 A). At 1 5°C (Fig. 1 A) the behavior was observed at every rel- ative humidity treatment. However, the incidence of mantle edge exposure was inhibited at humidities of 33% or lower. At 95% RH, approximately 40% of individuals were exposing tissue at any time. The initial incidence of mantle edge exposure behavior at 75% RH and 1 5°C was similar to that at 95%- RH (Fig. 1 A). At 53%. RH mantle edge exposure behavior was reduced and occurred in two bouts, the first during the initial 100 hours of emersion and a second commenced after 200 hours. During these periods few (<20%) individuals were displaying the be- havior. The incidence of the behavior was reduced even more at 33% and 5% RH. At 25°C mantle edge exposure behavior, with one ex- ception, was confined to relative humidities of 53% or greater (Fig. 2A). A distinct correlation existed between the frequency of mantle edge exposure behavior and rel- ative humidity. Fifty to eighty percent of individuals at 95% RH were displaying the behavior between 24 and 54 hours of emersion. During the same period, individu- als at 75% RH were exposing mantle 1 0-40% of the time, while 5-20%. of those at 53% RH were exposing tissues. At 35°C mantle edge exposure behavior was curtailed (Fig. 3A). The occurrences were limited to 53%. RH and O 25- SLMrrT I • • i A^ >st .^>y ,J//D I ,>• ^\/T .^>- ./ Vl ^^A- 6-flCI 480 Exposure Duration (hours) Figure 1. A. Time course of occurrence of mantle edge exposure behavior in Corbicula fluminea under five relative humidity treatments at 15°C. Mantle edge exposure behavior is quantified as the percent of individuals observed with mantle protruding at the close of a sampling interval. The abscissal scale is the same as for Figure 1 B. Symbols repre- sent A— >95% RH; •— 75% RH;D— 53% RH; O— 33% RH; A— <5% RH. B. Time course of cumulative percent body water lost for a standard sized (20.3 mm shell length) Corbicula fluminea under the same five relative humidity treatments at 15°C as in Figure 1 A (symbols the same as Fig. IA). The points represent estimations of the cumulative water lost for this standard sized animal based on linear regressions of log,0 shell length on cumulative percent water lost where these regressions were significant (P < 0.05, 85% were significant). Where regressions proved not significant mean cumulative water loss is recorded. The number of animals ranged from 20 at the beginning of the experiment to 3 at the close. The vertical bars are standard errors of the estimate. For clarity, error bars were included at intervals. Declines in cumula- tive water lost were due to mortalitv. 256 R. A. BYRNE ET AL _ 75- *f /° ^ ^ s 1 24 1 T 1 1 48 72 9 Exposure Duration (hours) Figure 2. A. Time course of occurrence of mantle edge exposure behaviorin Cnrhiailariiiinincu under five relative humidity treatments at 25°C. Mantle edge exposure behavior is quantified as in Figure I A. The abscissa! scale is the same as for Figure 2B Sy mbols represent RH: •— 75'-. RH:D— 53r; RH:C— .W RH:A— <5^ RH. B. Time course of cumulative percent body water lost for a standard si/ed (20.3 mm shell length) Curhunki lliiniim-u under the same five relative humidity treatments at 25°( ' as in Figure 2 A ( sv mbols the same as Figure 2 \) Methods lor calculating curves were the same as in Fig- ure IB. Approximately 9391 of the si/e-walei loss regressions were sig- nificant (P < 0.05). Declines in cumulative water lost were due to mor- talitv. above, with only single individuals recorded at 53' < . The maximum occurrence was onh MY < atl>5'< RH and less at the lower humidities. The incidences of the beha\ior were reduced during the period of accelerated water loss (sec Fig. 3B). and ceased after 2 1 hours emersion. At 15°C the pattern of cumulative water loss showed a distinct relative humidity effect (Fig. IB). Cumulative water loss values for 5. 33, and 53' < Rl I cluster together and display no differences at any interval. The curves for these treatments are approximately linear throughout the experiment. The curves for 75 and l>5' - RH are lower than the other relative humidity treatments. The 75% curse was linear whereas the ''5' ! curve showed an initial slow increase |. Jlnwcd b> an acceleration to a higher loss rate later in the experiment. At 25T le\o iier loss (Fig. 2B) were 2-6 times those at I > < h >ss levels for 5-75' , R II were ap- proximately linear and lustered together. At no time during the experiment • m\ significant difference (P > 0.05 (found in water loss Ivtwcen the relative humidiu treatments. Cumulative water loss lmm clams at 35°C (Fig. 3B) was 2-3 limes the 25"( values. \\ alei loss was similar for all rclatne humidities and no significant differences (P > 0.05) were found between treatments. The curves of cumulative water loss were non-linear, showing an in- crease after 15-18 hours exposure, leveling off at 24 hours as mortality increased. Overall hourly rates of water loss showed a threefold increase from 15°C to 25°C and a doubling from 25°C to 35°C (Fig. 4). The magnitude of these differences was reduced w hen rates of water loss during periods of valve closure were considered. When examining rates during both mantle exposure and fluid expulsion, in most cases temperature differences were more pronounced. There were significant differences (P < 0.05) between tempera- ture treatments for all relative humidities and behavioral categories except for rates when fluid was expelled be- iween 25° and 35°C at 33% and 53r; RH. No significant differences (P> 0.05) in overall rates of water loss existed between 5% and 53% RH treatments at 1 5°C (Fig. 4). The water loss rates at 75% and 95''. RH were significantly less (P < 0.05) than the rates at the lower humidities. At 25°C and 35°C no differences in overall mean hourly water loss rates were found between the relative humidity treatments. When rates of water loss after periods of valve closure I inure .V V I ime course of occurrence of mantle edge exposure behavior in ( n//>;i uld tluminca under five relative humidity treatments at 35°C. Mantle cdi'c exposure behavior is quantified as in Figuic I \ I he absciss.il scale is ihe same as for Figure 3B. Symbols represent A .'is.RH.» 75 RH;O— 53%RH;C— 33%RH;A— <5%RH. B. Time course ol cumulative percent body water lost for a standard si/ed (2(1 1 nun shell length) <. I'tl'iailii Ihimiiicn uiulei the same five relativehumidilv treatments at 35°Casin Figure 1 \ ( sv mbols the same .is I IT \\\ Methods loi calcul.Hinr curves vveie the s.nne .is in I igure I H ( )ulv s v; of size-water loss regressions were significant. Declines in cumulative water lost were due to mortality CORBICULA AERIAL EXPOSURE 257 0.0 Overall Closed Mantle Fluid exposed expelled Figure 4. Mean rates of water loss for Corhicula Iliiniineii at five relative humidity treatments (5%, 33%, 53%, 75%.. and 95%) and three temperatures (I5°C, 25°C, and 35°O. Rates of water loss are divided into four behavioral categories (overall average, with closed valves, with mantle exposed, and after fluid expulsion from the mantle cavity; see text). The letters A. B. and C refer to results of Duncan's multiple range tests comparing rates between temperatures, within a relative humidiu and behavioral category. Categories marked with dissimilar letters de- note significant differences (P < 0.05). The letters W, X, Y, and Z refer to results of Duncan's multiple range tests comparing rates between relative humidity treatments, within a temperature and behavioral cat- egory. Categories with dissimilar letters denote significant differences (P < 0.05). The asterisks denote significant differences (P < 0.05; paired 't' test) in mean rates between both the "mantle exposed" and "fluid expelled" behavioral categories and the "closed" category, within each temperature and relative humidity treatment. (Fig. 4) were examined there were distinct relative hu- midity effects especially at 15°C. At this temperature the mean rate of water loss at 95% RH was only 40% of the 5% or 33% RH rate. These differences were less apparent at 25°C where the rate at 95% RH was significantly less than the other rates, but represented at most only a 30% reduction. At 35°C there was no consistent pattern of rel- ative humidity effects on mean rates of water loss when valves were closed. Rates of water loss after periods of mantle edge expo- sure (Fig. 4) showed no significant differences between relative humidity treatments at 25°C or 35°C, and no consistent relative humidity effect was found at 15°C. Similarly, rates when fluid was expelled showed no sig- nificant (P> 0.05 ) relative humidity effect at any temper- ature. No significant differences were found between rates of water loss when valves were closed and rates when man- tle edge tissue was exposed at 95% RH at any of the tem- peratures (Fig. 4). At 1 5°C when the mantle edge was ex- posed, the water loss rate was significantly higher than the rate at 53% RH when valves were closed, whereas at 25°C the loss rate when mantle was exposed was signifi- cantly higher at 75% RH. Rates of water loss when closed and when exposing mantle tissues were not significantly different at 35°C. at which temperature the behavior of exposing mantle tissues is non-existent at relative hu- midities below 53%, and is uncommon even at higher humidities. In most cases rates when fluid was expelled were significantly (P < 0.05) higher than when closed (Fig. 4). Only the rates at 1 5°C and 5% RH were not sig- nificantly different. The multiple regression equation relating longevity to relative humidity, logu> shell length, and ME (mantle edge exposure behavior) is shown in Table IIA. Relative humidity had a significant (P < 0.05) effect on longevity at 15°C only, as would be expected from the median tol- erance time statistics. Animal size had a significant nega- tive effect at both 25°C and 35°C. No significant effect of Table II Multiple regression variable estimates relating Corbicala fluminea aerial longevity l.-t I and mean Hater loss rate while closed (B), to relative humidity (RH), logn, shell length (LSL). and average proportion oj time spent with mantle exposed (ME) (longevity only) at 15°. 2?°. and 35°C Intercept RH LSL ME R: A. Longevity (hours) I5°C 209.3* 0.897* -2.6 21.7 0.22* (99.3) (0.2941 (75.6) (46.7) 25°C 122.1* 0.069 -47.1* -2.0 0.08 (26.1) (0.060) (20.1) (6.7) 35°C 41.2* 0.006 -17.0* 1.5 0.12* (6.4) (0.012) (4.8) (3.1) B. Mean water loss rate (valves closed) (% body water- hour ') 15°C 0.959* -0.002 1 * -0.462* 0.52* (0.126) (0.0003) (0.097) 25°C 3.40* -0.0017* -2.061* 0.36* (0.39) (0.0007) (0.304) 35°C 4.18* -0.0024 -2.077* 0.06 (1.22) (0.0023) (0.927) Values in parentheses are standard errors of estimates. Asterisks indi- cate estimates significantly different from zero (P < 0.05). 258 K \ UN KM / / I/ mantle edge exposure behav ior on clam longevity was noted at any of the temjvratui Larger individual- lose water at a slower rate than smaller clams .1- was a consistently significant (P < 0.05) negati t of bodv si/e. as measured bv the logic of shell length, on mean water loss rate when \ahes were d -ill temperatures ( 1 able IIB). There also was a sigmricant effect of relative humidity at 1 5°( and 25 ' -is would be expected from the results in Figure 4. Relative humidity had a significant effect on water loss alter mantle edge exposure at 15°Cand 25°C (P < ()(>•>) Discussion Temperature is the major factor affecting water loss rate, exposure tolerance, and mantle edge exposure be- hav ior in ( tluniim-u under conditions of prolonged ae- rial exposure. The effects of relative humiditv are less pervasive but significant. Whether water loss is deter- mined as cumulative loss or mean hourly loss rates, rela- tive humidity has little or no effect at 25 or35°C. At 15°C a distinct mediating effect of higher relative humidities is detected. The major influence of relative humidity is on the incidence of mantle edge exposure behavior. Al- though temperature is dominant in determining the ex- tent of this response, within a temperature treatment, in- creases in relative humidity increase the frequency of mantle edge exposure behavior. This indicates that speci- mens of C. tluininca can perceive desiccation and com- pensate behav iorally. This ability is further evidenced by examining rates of water loss in individuals with closed valves and after bouts of mantle edge exposure. Under conditions of high relative humidity there is no differ- ence in rates of water loss within a temperature group. Significant increases in water loss rate occur only at rela- tive humidities where mantle edge exposure behavior is transitional between being commonplace and being rare. Thus, under conditions where there are no adverse con- sequences of exposing tissues with regard to losing water, then the behavior is common. When water loss rates ex- ceed rates when the animal has closed valves, then the behavior is inhibited. The occurrence of a second period of mantle edge ex- posure behavior at the lower humidities suggests addi- tional factors become important. When clams had lost 60-70 water, which corresponds to the total mantle i water store plus some hcmolymph water (McMahon. I i second bout of mantle edge expo- sure behavior i In all cases this behavior isdis- played in individi in dealh and might represent a loss of muscle ton d with a weakened condi- tion. Another possibility is that it represents a final gas exchange event prioi to complete shutdown to conserve water. Clams that expose tissues extensivelv do not survive longer than those that remain closed for extended peri- ods. Furthermore, there is no relative humidity effect on median tolerance time at 25°C even though mantle edge exposure is significantly enhanced by higher relative hu- midity. 1 hus the adaptive significance to mantle edge ex- posure is obscure. McMahon and Williams (1984) showed a direct relationship between duration of mantle edge exposure behavior and aerial oxygen uptake. Other experiments (Bvrne. unpuh.) show that clams prevented from gaping their valves in air have a lower median toler- ance time than those permitted to expose tissues (at 20°C and 95' ; RH the TLm is 1 54 hours when allowed to open valves, and is onlv 96 hours with valves prevented from opening. P < 0.05). There are several adaptive explanations of mantle edge exposure behavior. First, it is possible that gas exchange necessary for the maintenance of aerobic metabolism may be accomplished with little exposure of tissues. The measurement of mantle edge exposure utilized here is essentially a "snapshot" of behavior. There is evidence (Byrne, unpub.) that clams, in addition to the more con- stant mantle edge exposure behav ior also perform valve movements which ventilate the mantle cavity. Such be- haviors are relatively short in duration and could be missed. Second, being out of water, there would be a buildup of metabolic waste products including dissolved ("O: and concomitant acid-base problems as a result of reduction in gas exchange capability. Intermittent man- tle edge exposure may aid gas exchange but may not sig- nificantly alter hemolymph solute concentrations, which might be the cause of the reduced survival time. Under aerial exposure conditions, blood osmolality increases threefold and calcium levels rise fourfold indicating mo- bilization of calcium carbonate from the shell (Bvrne. unpuh.). Third, the behavior may conserve energy stores, liven the most energv "efficient" anaerobic pathwavs are far less efficient than aerobic metabolism (Hochachka and Somero, 1984). Although the extent of the mantle edge exposure did not increase the survival time in air. the animal might have been conserving limited energy stores by maintaining aerobic metabolism. Whereas ae- rial survival would not be influenced by mantle edge ex- posure, subsequent abilities to resume aquatic existence would be enhanced in individuals that conserved sub- strates (McMahon, 1988). The effect of body size on the ability to survive aerial exposure is two sided. There is a distinct reciprocal rela- tionship between si/e and water loss rale. However, at 25 and 35°(' there also is a negative relationship between individual clam longevity, and si/e I he temperature ellect is most severe at 35°C whereas size-dependent ellecls on water loss rates are significant at all tempera- tures I hus. uiulei short term conditions of aerial expo- sine, larger clams would have an adaptive advantage over smaller clams because of the larger clam's slower CORBICULA AERIAL EXPOSURE 259 rate of water loss. The effect of body size is not significant after periods of mantle edge exposure suggesting that evaporative water loss under these conditions is a simple function of the linear size of the exposed mantle, an al- most two-dimensional surface, rather than being related to a surface area of the whole clam while valves are closed. When compared to other freshwater bivalves, C. Jhtminea displays a low tolerance of aerial exposure. Mc- Mahon (1979) showed a somewhat longer aerial expo- sure tolerance for C fluminea than reported here, but also found that relative humidity had an effect on me- dian tolerance time at the lower temperature (20°C). Mantle edge exposure behavior was inhibited completely at the lower humidity (approximately 5% RH), but was common at the higher humidity (ca. 95% RH). The unio- nid Ligumia submstrata could survive up to 40 days ex- posure under conditions of high relative humidity at temperatures of 22°-25°C (Dietz, 1974). An Australian unionid, Hyridella aiistralis, apparently is capable of withstanding at least 60 days of exposure at room tem- peratures (Hiscock, 1953). Sphaerium occidental, the fingernail clam, can survive aerial exposure for up to a month; the survival time is related to relative humidity (Collins, 1967). This species undergoes a period of aesti- vation during which the tolerance to aerial exposure is increased (McKee and Mackie, 1980). Non-aestivating 5. occidentals has a median tolerance time at 20°C of 1- 3 days, and aestivating clams 8-34 days, the range con- trolled by relative humidity. The record for exposure en- durance is held by the African unionid, Aspatharia pet- ersi, which has been reported to survive over a year out of water (Dance, 1958). The lower tolerance of C. fluminea to exposure is sim- ilar to that of more marine/estuarine species. Median tol- erance times for Cerastroderma. ednleal high relative hu- midity range from 129 h at 15°C to 9.5 h at 35°C (Boy- den, 1972). These values are lower than those of C. fluminea, but represent an extended tolerance when compared to less-often exposed marine bivalves. Ceras- troderma glaucum, found subtidally, has TLm's of 86.7, 42, and 9.7 h at 15, 25, and 35°C, respectively (Boyden, 1972). Cerastroderma editle gapes almost continuously when emersed and can survive 42.9% of tissue water loss, whereas C. glaucum remains closed on exposure and can withstand only 33% tissue water loss (Boyden, 1972). However, the high intertidal bivalve Modiolus modiolus air gapes while emersed and has a TLm of 9 days in air at 24.5°C (Lent, 1968). Survival time is associated with the volume of oxygen present in the atmosphere. Desic- cation is an unavoidable side effect of an aerial respira- tory adaptation ( Lent, 1968). Pulmonate snails represent a group of molluscs that have adapted to a range of habitats from completely ter- restrial to obligate aquatic (McMahon, 1983b). Length of survival in air depends on the rate of water loss from the snail (Machin. 1975). Tolerance of a high loss of body water results in increased tolerance times. Biomphalaria glabrata can withstand 70% reduction in total body wa- ter and has a TLm of 64 days at 27°C at 96% RH (von Brand et al, 1957). There are sharp distinctions of TLm between relative humidity treatments ranging down to 2.8 days at 15% RH (von Brand et al, 1957). Melampus bidentatns can lose up to 78.5% of its body water (Price, 1980) and there is a major effect of body size in that larger snails can withstand in excess of 14 days exposure at 20°C and 97%. RH whereas juvenile snails cannot sur- vive 12 hours emersion without free water. C. fluminea when aerially exposed under conditions of low humidity may form a hardened mucus between slightly parted valves. This is superficially similar to the epiphragm sometimes produced by estivating stylomato- phoran snails which is thought to function in retarding evaporative water loss by reducing convective move- ment of air over moist tissues (Machin, 1975). The hard- ened mucus may perform a similar function in C. Jhtm- inea while also allowing a diffusive contact with the envi- ronment for gas exchange purposes. It has been noted previously (McMahon, 1979, 1983a; McMahon and Williams, 1984) that C. fluminea. be- cause of its relatively recent history in freshwater, dis- plays physiological and behavioral adaptations that seem to be intermediate between those of more ancient fresh- water forms such as the unionids and sphaeriids, and its estuarine and marine relatives. Its blood osmolality is higher, and the major hemolymph ions differ from those of unionids (Dietz, 1979). McMahon and Williams (1984) suggested that the respiratory adaptation to aerial exposure is intermediate between the continual gaping of some intertidal bivalves (e.g., Cerastroderma edule) and the closed valve response of unionids, or the trans- valvular gas exchange capabilities of some sphaeriids. This intermediate nature also is evidenced in this study by the lower exposure tolerances found for this species when compared to most other freshwater bivalves, and the higher tolerance than that reported for most of estua- rine/intertidal lamellibranchs. There are two major problems facing a clam that has been exposed in air. First, there is the problem of main- taining metabolism and homeostasis without the normal modes of waste excretion, gas exchange, and ion regula- tion. Second, there is the loss of fluid either by passive surface evaporation or by active expulsion. Loss of nutri- tional opportunities frequently is regarded as not very significant owing to the overriding importance and im- mediacy of the other factors. However, in C. fluminea, carbohydrate stores are limited (Williams and McMa- hon, 1985) and prolonged emersion might cause severe carbohydrate depletion. Preserving water stores necessi- tates maintaining closed valves and preventing contact 260 R. A. BYRNE ET AL with the external environment. Ameliorating internal conditions in the f.i. lining o.xxgen tensions, in- creasing carho1 ns. and increasing concen- trations of met. quires some gas exchange with the environn- J the consei|iient exposure of living tissues to . or less arid atmosphere. Corf './ possesses adaptations which en- able it to surv tv c periods of emersion longer than its cstu- arine relan \pparenth ('. thiminca can detect the loss of water and modify its gas exchange heha\ior ac- cordingly.. It can withstand a loss of up to 60r"< of its avail- able water: ho\\e\er. it does not display the capabilities »f some unionid species to withstand verv lone periods of aerial emersion. Unlike the marine and estuarine cnv i- ronments. freshwater animals may experience periods of emersion that are unpredictable either in timing or dura- tion. Some unionid clams, with their long history in freshwater, appear to ha\e evolved adaptations enabling them to withstand protracted exposure. C.thiminca in its own adaptations shows interesting modes of prolonging existence out of water. These adaptations, including the modification of the "gaping" behavior in response to temperature and relative humidity may be regarded as intermediate in nature between those of more ancient freshwater and estuarine forms, but might also be looked upon as novel adaptations to a new environment by a relatively recent invader. Acknowledgments We thank Edith Bvrtie. David Long, and Bruce White- head for assistance in data collection. Dr. Harold Silver- man tor critically reviewing the manuscript, and Vicki Lancaster for advice on statistics. This studs forms part of a dissertation submitted bv R \B to the (iraduate School of 1 ouisiana State I 'niversity and A&M College in partial fulfillment of the Ph.D. degree. Supported by research grant from the University of Texas at Arlington to Rl M and \SI grants DCB 83-03789 and DCB 87- (115(14 to HID. Literature CiU-d Bavin. lil< .1. Ba\m-. I.C. Can-foot, and R. .}. Thompson. r>~<). fhe physiological ecolog) »i \4ytiluscalifornianusConnd. - gen tensions and air exposure (>m>li>via (Bcri :i Ho\di-n. ( . K I1' i.-s|niatnin ol ilu- cockle < \-iii\liinl,-rni,i • -mperaturc. Co»i/> Hun hem /'/;rw.'/ 43\: 697-712 ton Brand. I.. I'. XI MO. Nolan. 1957. I'livsiol-.n al obscrsatK .limn nl "the snail \\(\it,ilnrln\ Klahralux. ISixl Hull I I Brinkhotf. V. K. Sim km.i . irshahc-r. 198.1. Naluialoi- currence ofanaerobiosis in moll .nicmdal habitat-- login ST. 1 5 1- I < nllins. I. \\.I967. ( )\ ./en-uptake. •,'• ind desicca- lion of the fingernail clam. Sphaerium occidemale Prime. Ph.D. Ilu-sis t mvcrsiiv of Minnesota. 166pp. (Diss. Abslr. 28B: 5238). Daiu-i-. v I'. 1958. Drought resistance in an African freshwater bi- valve../ Conchol 24::si-:s:. Dii-i/. I. II. 1974. Bixlv lluid composition and aerial o\vgen con- sumption in the freshwater mussel, Ligiunia subrostrata (Say): ctlccts ol ili-lmli. linn and anoxie stress. Biitl. Hull 1-47: 560- - Did/. 1. 11.1979. Uptake of sodium and chloride b) freshwater mus- sels ( 'an ./ Zool 57: 156- iwi. (•ainc-v. I.. K., Jr. 1978. 1 he respunso nf the Corhiculklae I Mullusca: Bi\alvia) to osmotic stress: the cellular response. Ph\-\ml /<»>/ 51: 79-91. Ilisou-k. I. D. 195.1. Osmoregulatum in Australian freshwater mus- sels (Lamellibranchiata). 1. Water and chloride ion exchange in 1 1 \ridflla aiMi\iii^ (I am I. Ins/. J. ,\far. Freshwater Rex, I: 317- $29 llochachka. P. \\ ., and (i. N. Sonu-ro. 1984. limclu'inu-til Adapta- tion Princeton I niscrsiu Press. Princeton. New Jersey. 537 pp. I riil. ( '.. M. 1968. Air-gaping by the ribbed mussel. I/HI/M/KS ii,'nn.\- s»s(|)illw\ni: elKvtsanil adaptive siiiniticaiKv. Hint Hull 134:60- 73. Xlachin, ,J. 1975. \\ater relationships. In Pulmonates vol. I. l-nnc- lidiml Anntil Ccrhicnln S\mpi>\iwn. J. C. Britlon. ed. I'exas Christian I 'niversitv Rest-arch Foundation. Fort Worth. XlcMahon, R. F. 1982. The occurrence and spread of the introduced Asiatic freshwater clam. (<//i/s, ,/ \nl. (v Ecolog) VA I). Russell-Hunter, ed. Academic Press. San Di.-rn Mi-Miilion. R. F. 1988. Respirators response to periodic enio m micrtida! molluscs in Physiological Compensation in IntertidaJ \nntnil\ .Sr/u/'"1""" K. lillington and B. R. McMahon eds. Am. /<«u»a. 1954. Relative humidity-temperature lelatiunships ol some saturated salt solutions in the temperature rangi 0 to iO°( .1. Res. ,\'at. Bur Stand 53:1" 16 \\illianis. C. .1.. and R. F. MeMahon. 1985. Seasonal vanalion in owi'.en consumption rates, nitrogen excretion rates and tissue or- r.iinc - .nbon: nitrogen ratios in the introduced \sian freshwater bi- valve. C'orhiciila /Iiiininca (Muller) (l.amellibranchia: Corbicula- eea) tm \l,i/,n-»l. Hull. 3:267-268. Reference: Biol. Bull 175: 261-273. (October, 1988) Visual Spectral Sensitivities of Bioluminescent Deep-sea Crustaceans TAMARA M. FRANK1 AND JAMES F. CASE Department of Biological Sciences and Marine Science Institute, University of California, Santa Barbara, California 93106 Abstract. The spectral sensitivities of eight species of deep-sea decapod shrimps (Family Oplophoridae) were determined from shipboard measurements of electroreti- nograms of dark-captured specimens. I\'oti>\f<>iin<\ xih- bosus and N. elegans are maximally sensitive at 490 nm, and chromatic adaptation experiments indicate that a single visual pigment is present. Peak sensitivities of Acuni/iep/iyni smithi and A. curtirosiris are at 5 10 nm. a longer wavelength than expected for such deep-sea dwellers. The four photophore-bearing species, Systel- laspis debilis, Janicella spinacauda, Oplophorus spino- sus. and O. gracilirostris have sensitivity maxima at 400 and 500 nm, and chromatic adaptation experiments in- dicate the presence of two visual pigments. This unusual short wavelength sensitivity may provide the basis for congener recognition based on the spectral bandwidth of luminescence. Introduction The light field in the deep-sea consists of essentially monochromatic light from two sources: (1) dim down- welling light with a chromatic spectrum centering on 475 nm(Jerlov, 1968; Dartnall, 1974;Cronin, 1986); and (2) bioluminescence, with spectra characteristically peaking at 460-490 nm( Herring, 1983; Widder <>/<;/., 1983;Latz et a!.. 1988). It has long been assumed that the visual systems of deep-sea organisms would also have mono- chromatic sensitivity, with visual pigment absorption maxima blue-shifted (as compared with shallow water species) for maximum sensitivity to the existing light re- gime (Bayliss et ai. 1936; Clarke, 1936; Goldsmith, Received 24 December 1987: accepted 28 July 1988. 1 Current Address: Department of Physiology. University of Con- necticut Health Center. Farmington, CT 06032. 1972; Shaw and Stowe, 1982; Cronin, 1986). Almost all studies on deep-sea fish (Denton and Warren, 1957; Munz, 1957; WaldcYrt/.. 1957; Denton and Shaw, 1963; Fernandez, 1978; Crescitelli et a/., 1985), cephalopods (Hara and Hara, 1979) and crustaceans (Fisher and Gol- die, 1958, I960; Denys and Brown, 1982) support this hypothesis, reporting single visual pigment systems with absorption maxima between 470 and 490 nm, in con- trast with maxima of 490-550 for shallow water species (reviewed by Goldsmith, 1972; Lythgoe, 1972). How- ever, these studies were performed on visual pigment ex- tracts or via microspectrophotometry (MSP), which pro- vide excellent information on the absorption characteris- tics of the visual pigments (see Menzel, 1979, for review of problems associated with extracts of invertebrate vi- sual pigments), but may not necessarily reflect the physi- ological spectral sensitivity. For example, in the crayfish, Procambarus, the spectral sensitivity of the dark-adapted eye, measured electrophysiologically, peaks at about 570 nm (Wald, 1968). while the Xmax of the visual pigment is 530 nm; (Cummins and Goldsmith, 1981). Similar, though smaller, red shifts in spectral sensitivity are also found in the lobster. Homarus, (Wald and Hubbard, 1957; Wald, 1968) and the shrimp, Palaemonetes, (Fer- nandez, 1965; Wald and Seldin, 1968; Goldsmith and Fernandez, 1968). Goldsmith (1978) found that these shifts could be attributed to the filtering effects of red- leaky screening pigments. It is difficult, from pigment ex- tract and MSP data, to accurately assess the degree of pre-retinal filtering and its effect on spectral sensitivity, but this must be taken into account before making any assessment of an organism's visual capacity. Recording the electroretinogram, the mass response of a large number of photoreceptor cells to a flash of light, is a simple way to obtain physiologically relevant infor- mation about an organism's visual capabilities. For our 261 262 T. M. FRANK AND J. F. CASE purposes, it is superior . llular recording meth- ods because of th >i working on a vibrating, unstable ship. Altho Horn this method cannot definitive! vther one or several visual pig- ments are prc^ e is excellent e\ idence that ERG- determined ; correspond to spectral cell t\pcs (Goldsmith . :nandcz. 1968: Stieve et at.. 1978: Laughlin c . 1980: Cummins and Goldsmith. 1981: Goldsmith. 1986) and this method is often the method of chmce for comparative studies, particularly on pre- \iouslv untested organisms (Kobayashi and Ali. 1971: Eguchi eiul.. 1982). The first evidence fora short wavelength receptor in a deep-sea organism comes from Wald and Rayport's (1977) electrophysiological study of the alciopid worm I'anadis. Its dark-adapted spectral sensitivity curve ex- hibits a violet shoulder in addition to a blue-green peak, and response waveforms to 380 nm light are different from those to 480 nm light, arguing for the presence of two spectral classes of photoreceptor cells. The fact that this is the only example of enhanced short wavelength sensitivity in deep-sea organisms mu\ be because so few animals from this environment have been studied electrophysiologically. While both pigment extracts and MSP have proven successful in identifying dual red-shifted pigments in deep-sea fish (Denton a al .. 1970; O'Day and Fernandez, 1974: Partridge et ul.. 1987), neither method has led to conclusive identifica- tion of the violet visual pigment (whose presence was ver- ified with intracellular recordings) of some shallow water crustaceans (Goldsmith ci a/.. 1968; Goldsmith and Bruno, 1973; Cummins and Goldsmith. 1981: Martin and Mote. 1 982; Cummins e? a/., 1984). This may be due to the small quantity of pigment present, which would be swamped by the dominant pigment or its photoproducts during absorption measurements on extracts, or due to its location in small cells which may be inaccessible to. or overlooked by. MSP measurements. I )eep-sea crustaceans are useful subjects for explor- atory electrophysiological studies of receptor systems of deep-sea animals, because they can be retrieved in good conditinii. anil remain viable for many days under the i maintenance conditions. Soft-bodied fish and in- vertebrates are g lu-rallv dead upon retrieval or die within hours .ipture. However, electrophysiological technique > lorn been used with deep-sea crusta- ceans because trely survive transport to land- based labs in go< lition. I or this reason, we have- developed a i siological apparatus en- abling shipboard nis of spectral sensitivities of freshly caught sjn. Members of the family Oplophoridae were cho ludj because! 1 ) this family contains both phoinphmv ng and non-pho- tophore bearing species. (2) then depth rani-es are lairlv well known, and (3) viable specimens could be obtained in sufficient numbers fora comprehensive study. An un- expected result of this \vork was the discovery of en- hanced sensitivitv to violet light in the four photophore bearing species examined. Preliminary reports have been presented in abstract form (l-'rank. 1986). Materials and Methods Specimen collect ion anil maintenance Specimens of the eight species of deep-sea shrimp used in this studv (Table 1) were collected during two cruises on the R.V. AVn Horizon off the southwest coast of Oahu. Hawaii, with an opening/closing 3.1 m Tucker Trawl, fitted with a thermally protected, light-tight col- lecting container (Childress el ai, 1977; Childress and Price. 1978). This container was closed at depth, ensur- ing recoverv of healthv organisms whose eyes had not been irreparably damaged by surface light levels, a well known concern in working with deep-sea species (Loew, 1976; Nilsson and I indstrom. 1983: Shelton et ai. 1985). The container was opened in a light tight room, and animals were sorted under dim red light. Experi- mental animals were maintained in chilled seawater (5°C) in light proof containers and studied within 24 hours of capture. Dim red illumination was also used while setting up for experiments. Animals were mounted in a holder sus- pended in a 5°C seawater bath, allowing enough pleopod movement to maintain respiratory water currents across the gills. Temperature was maintained by pumping -PC antifreeze from a I .auda cooler through cooling coils sub- merged in the seawater bath. The eyes were stabilized by gluing (Superglue) to small posts attached to the holding chamber on either side of the head. Electrical recording ERGs were recorded with 5 ^m tip. glass insulated, metal microelectrodes (F. Haer & Co.). placed subcor- neally with the aid of a dissecting microscope equipped with an infrared light source ( W ratten Filter 89C) and an infrared image converter (FJW Industries). A reference electrode was placed in the other eye, and a silver-chlo- ride electrode grounded the water bath. The electrodes were used with a Grass high impedance probe (Model I IIP5 I 1 . l()7 M ohms impedance) to eliminate electrode polarization artifacts (Kugel. 1977). Signals were ampli- fied with a Grass AC Pre-amplifier (Model H1P51 I.I). with the low frequency filter set for minimal filtering (0.1-0.3 Hz) to minimize distortion due to AC-amplili- cation. VISION IN DEEP-SEA CRUSTACEANS 263 Optical apparatus Monochromatic test flashes were provided by an American ISA Monochromator(full width at half maxi- mum intensity [FWHM] = 2 nm) with a tungsten - halo- gen light source powered by a Weston regulated power supply (Model 752 1 ). Flash duration of 100 ms was con- trolled by a Uniblitz Shutter (Model 100-2) triggered by a Grass S44 Stimulator. Light intensity was controlled with a neutral density wedge and neutral density niters, and was calibrated at each wavelength with a UDT Op- tometer( United Detector Technology Model 61 )and ra- diometric probe, with point calibrations referenced to NBS provided by UDT. Test flashes were presented to the eye through one end of a branched quartz fiber optic light guide (Welch-Al- len). The 2 mm output diameter of the light guide was large enough to illuminate the whole eye, and experi- ments showed that this light did not reach the refer- ence eye. The adapting light source for chromatic adaptation ex- periments was an incandescent light filtered by a 400 nm broadband filter (Melles Griot BG 1 2. FWHM = 1 1 0) for violet adaptation, and a 520 nm broadband filter (M. G. VG6, FWHM = 90 nm) for green adaptation. The adapt- ing light was delivered to the eye through one branch of the light guide, and test flashes were superimposed on this background light through the other branch. This en- sured that both the adapting light and the stimulus light acted upon the same group of photoreceptor cells. Experimental procedure The eye was stimulated with 100 ms test flashes of monochromatic light adjusted for intensity until a de- fined criterion response was attained at each wavelength tested. The criterion was usually set 20 pV above baseline noise, ensuring that the intensity of the light flashes used was very near the threshold of sensitivity, so as not to light-adapt the eye. Signals were instantaneously an- alysed for peak to peak response height after digital con- version by an LSI/PDF 1 1 computer, and stored on mag- netic tape (Lockheed Store 4 Recorder) for later wave- form analysis. The order of the flashes was random, and the response to a standard flash of set wavelength and intensity was tested periodically throughout the experi- ment, to ensure the stability of the eye and to monitor the state of dark-adaptation. Spectral sensitivity mea- surements were started when the response to the stan- dard flash was stable, for both dark-adapted and chro- matically adapted eyes. Spectral sensitivity curves were generated as the reciprocal quanta needed to produce the criterion response at each wavelength. Absorptance spec- tra were constructed from Dartnall nomograms (Dart- nail, 1953), using the analysis provided by Cornwall et al. (1984). The inefficiency of the monochromator at shorter wavelengths limited the intensity of the adapting light that could be used. To ensure that a full spectral sensitiv- ity curve could be measured, the intensity of the adapting light was adjusted so that a criterion response to 370 nm test flashes could still be elicited. Due to the varying sen- sitivity of some species to short wavelength light, the in- tensity of the adapting light necessarily varied between experiments. Results Notostomus gibbosus and N. elegans The results for N. gibbosus and TV. elegans were identi- cal and will therefore be described together with no dis- tinction made between species. The mean dark-adapted spectral sensitivity curve for Notostomus (Fig. 1 A) shows that the sensitivity maximum occurred at about 490 nm. The absorptance spectrum for a 490 nm pigment with an optical density (O.D.) of .5 provides an excellent fit to the spectral sensitivity curve. Green chromatic adapta- tion uniformly depressed sensitivity across the spectrum (Fig. 1 B), indicating that only one visual pigment is pres- ent in both species. The dark-adapted response waveforms of the ERGs were identical at all wavelengths (Fig. 1C). Chromatic adaptation of the eyes with green light produced no dis- cernible effects on the waveforms, supporting the conclu- sion that both species possess a single visual pigment. Acanthephyra smithi and A., curtirostris Although these two species have different depth distri- butions (Table I), their spectral sensitivities were identi- cal, and will again be described with no distinction be- tween species. Maximum sensitivity in the dark-adapted eye was at 510 nm (Fig. 2A). An absorptance spectrum, constructed based on the known absorption maximum (490 nm) and O.D. (.6) of A. smithi visual pigment (Hil- ler-Adams et al., 1988), was offset from the spectral sensi- tivity curve by 20 nm. Chromatic adaptation experiments indicate that only one visual pigment is present, as there were no selective effects of green and violet adaptation on the shape of the spectral sensitivity function; spectral sensitivity de- creased uniformly across the spectrum (Fig. 2B, C). The ERG response waveforms in dark-adapted eyes were identical at all wavelengths for individual speci- mens and adaptation with violet and green lights had no discernible effects on the shape of the response wave- forms (Fig. 3). Lack of wavelength-specific effects of B T M. FRANK AND J. F. CASE 1 able I Ih'i'ili distribution and bioluminescence mode 420 500 Wavelength (nro) 420 500 580 Wavelength (nm) c 370 %w *- 570 Figure 1. Spectral sensitivity nl \i>i<"nni\ (A) Standardised mean spectral sensitivity curse (solid line I I'oi \ cj/'/u'vin and \ .7, gans (n = 10). Criterion responses ranged from 20-50 /jV. Standard errors are represented by vertical bars. Sensitivity is defined as the recip- rocal of the quantum flux (photons/cnr/s) required to elicit the crite- rion response at each wavelength. Maximum sensitivity centers on 490 nm. Dashed line represents the absorptance curve tor a hypothetical rhodopsm with .1 AI]14, of 490 nm. and an O.D. of .5. (B) Green chro- matic adaptation had DO effect on the spectral sensitivity of.Yn/900 >900: spew gibbosus Votostomw >900 >9002 spew elegaru Systellaspii 620-900 100-300 spew debilis pholophorcs Janicella 500-600 30-250 spew spinacauda photophorcs Oplophortu 490-750 140-375 spew spinosiu photophores Opti >;'/!( in/\ 440-650 60-750 spew gracilirostris pholophores 'Zieman. 1975. 2 J. Childress. pers. comm. ever, the location of the two sensitivity maxima at 400 and 500 nm. when present, was very consistent, as seen in the average curve for the eight specimens in which two maxima were present (Fig. 5 A). Spectral sensitivity did B -7 4 Wavelength (nm) Wavelength (nm) Wavelength (nm) 2. Speclial sensitiviiv ol l< ,iinlii'pli\ra. (A) Standardized mean spectral sensitivity curve for I curtirostristaidA.smithi(n • 14). Criterion responses i.uif.ed from 20-60 nV . Peak sensitivity centered on 5IOnm. Absoipi.incespeciium (dashed line), was constructed based (in the known absorption maximum (490 nm) and O.I) I M ol I \mithi visual pigment (Hiller-Adams « a/., 1988). (B) Green chromatic adaptation did not diminish long wavelength sensitivity with respect to shun wavelength sensiiiviiy (data from one specimen). Results from three othei specimens aie the same. Intensity ol adapling light was 3.2 in (A\ iin.s M | Similarly, violet chromatic adaptation did not enhance lone, wavelength sensitivity iclative lo shoit wavelength sensi- livitv (data liom one specimen). Intensitv of adapting light was 2.4 in '(/W/cnv/s. VISION IN DEEP-SEA CRUSTACEANS 265 f\ DARK B DARK ADAPTED VIOLET ADAPTED 370 390 590 Figure 3. ERG response waveforms matched for equal amplitude (50 /iV) in dark-adapted and chromatically adapted Acanlhcphyra. (A) Waveforms were identical across the spectrum in the dark -adapted eye. Upon green-adaptation, the responses were different from those in the dark-adapted eye, but were identical to each other. (B) Response wave- forms from another dark-adapted specimen were also identical across the spectrum and adaptation with violet light did not affect their shape. not appear to depend on the size of the criterion response for the range of criterion responses used (20-100 ^V), as curves generated for an animal using two different crite- rion response levels were the same (Fig. 5B, C). B -8 8 - 340 4OO 460 520 580 340 400 460 520 580 Wavelength (nm) 340 400 460 520 580 Wavelength {nm) Figure 4. Dark-adapted spectral sensitivity curves from four speci- mens of Systellaspis dehilis. demonstrating the variability in their spec- tral sensitivity. Criterion response = 50 n V. Wavelength (nm) Figure 5. (A) Average standardized spectral sensitivity curve from only those dark-adapted 5. ilchilis that possessed bimodal spectral sen- sitivity curves (n = 8). The two sensitivity maxima were consistently at 400 and 500 nm. (B. C) Dark-adapted spectral sensitivity curves for one preparation at two different criterion response levels were identical. Results of chromatic adaptation experiments indicate that two visual pigments may be present. Under green adaptation, the spectral sensitivity curve was markedly depressed in the long wavelength part of the spectrum (Fig. 6A). Green adaptation also brought out the violet peaks in two specimens where there was no evidence of a short wavelength peak in the dark-adapted eye (Fig. 6B). The effect of violet adaptation was to depress the short wavelength peak with respect to the long wave- length peak, although the effects were not equally distinct in all experiments. The strongest effects were seen in those specimens that had large 400 nm peaks in the dark- adapted spectral sensitivity curves (Fig. 6C). Differences in waveform responses to short versus long wavelength light suggest that the two putative pigments are in separate cells. Again, because of variability in elec- trode placement, the dark-adapted waveforms were not identical from animal to animal. In one specimen, the short wavelength response waveforms were distinctly 266 I M I K \\K \ND J. F. CASE 34O 430 600 MO Wavelength |rm) J*D 420 BOD sao Wavelength (nm) 310 «0 500 580 Wavelength (r¥n) I- igure 6. Effects ofchromatic adaptation on the spectral sensiti\ it> of S Jchilis. (A) Green chromatic adaptation had a greater effect on thesensitmtv ol'thc blue-green receptors than the violet receptors, lead- ing toa relative enhancement of the uolet peak. (B) The dark-adapted spectral sensitivity curve lor another preparation showed no distinct violet peak. Green adaptation depressed the sensitivity of the blue- green receptors, exposing the violet receptors and thereby producing a distinct violet peak in the spectral sensitivity function. (C) Blue adapta- tion had a greater effect on short wavelength sensitivity, depressing the sensitivity of the violet receptors to such an extent that only the blue- green peak is now visible. Results of four other green adaptation and four other blue adaptation experiments were consistent with the results shown. Intensities of adapting lights were (A) 1.2X 10 4.(B) I.I x 10"'. and (C) 1 .2 x I0~ different from the long wa\ clength responses in the dark- adapted c\e (Fig. 7A). For another specimen, the wave- forms were identical in the dark-adapted eye. but upon green chromatic adaptation, the short wavelength re- sponses became markedly different from the long wave- length responses (Fig. 7B). Blue adaptation had either no effect when waveforms were identical in the dark- adapted eye. or actually diminished differences that were initially present in the dark adapted eye (Fig. 7C). All of these results support the conclusion that Systcl- A/v/'/s possesses two spectral classes of receptor cells with different response characteristics: one with maximal blue-green sensitivity and the other maximally sensitive in the \iolet. Janicella spinacauda The dark-adapted spectral sensitivity curves of the four specimens tested displayed a consistent maximum atSOOnm in the blue-green, but the position of the short wavelength peak varied from 350 to 420 nm (F'ig. 8A. B). No correlation (.mild be found between these results and time of capture or lime of experimentation. The results of t\v< . chromatic adaptation experiments indicate that two \isual pigments are present. I he effect of green adaptation was in depress ihe blue-green peak with respect to the \mlet peak, as well as shift the short wavelength maximum lmm ^0 to 410 nm (1 if X( ). \ iolet adaptation selectively depressed the violet peak relative to the blue-green peak (Fig. 8D). As in .Sivr//<;s/'/v. the response waveforms were either distinctly ditlerent in the dark-adapted eye (Fig. 9A). or were identical in the dark-adapted state, and changed dramatically at the shorter wavelengths upon green ad- aptation (Fig. 9B). Conversely, violet chromatic adapta- tion had no selective effects on response waveforms that were identical in the dark-adapted eye (Fig. 9C). These results indicate that .lanicclla also possesses two spectral classes of receptor cells. B DARK ADAPTED DARK GREEN ADAPTED ADAPTED DARK VIOLET ADAPTED ADAPTED 370 410 430 440 370 430 "~lN- **v- 370 430 450 470 4 JL " 450 J/-' 530 "/** 550 6IOB»^* 610— V" 810 Jf»iV) in another piep.nalion were identical at all wavelengths in the dark-adapted eye. with simple, monophasic. corneal negative waveforms. Green adapta- tion pioduced distinct wavelength specific eM'ects. The waveforms Irom ls(l to 4^(1 nm weie reversed in pol.intv. while the responses from 470 to 610 nm remained unchanged, with the transition occurring at 460 nm. (C'l Daik-adapted response waveforms (50 i*V) demonstrate the same differences as described in ( \ ). with waveforms between 370 and 4 Id n m exhibiting one i h.iiacteristie waveform, and responses between 470 and MO nm exhibiting another. The transition from one type to iln- .'ther occurred at 450 nm. Blue adaptation markedly altered the response wavelnims between 370-450 nm; these waveforms became identical to the long wavelength responses, which were unalfected by Mill ,l.l.l|)t.llloM VISION IN DEEP-SEA CRUSTACEANS 267 B 320 400 480 560 640 340 420 500 580 Wavelength (nm) Wavelength (nm) Daik-8dapt»d \ Dartk-edaptsd . n -.-^ -. 1 320 400 4SO 560 Wavelength (nm) 420 500 580 Wavelength (nm) Figure 8. Dark-adapted spectral sensitivity curves for individual specimens of Janice/la spinacauda. (A) The short wavelength sensitiv- ity peaks at about 350 nm, while the long wavelength sensitivity peaks at 500 nm. (B) In another specimen, the short wavelength peak was at 420 nm. while the long wavelength sensitivity maximum was a shoul- der rather than a distinct peak. (C) Green chromatic adaptation en- hanced the relative size of the violet peak, as well as shifting the sensitiv- ity maximum from 350 to 410 nm. Data from one specimen. Intensity of adapting light was 1.56 x 10~6 nW/cnr/s. (D) Violet chromatic ad- aptation had a greater effect on short wavelength sensitivity, resulting in a relative enhancement of the blue-green peak. Data from one speci- men. Intensity of adapting light was 1.2 x 10 3 >tW/cm2/s. Oplophorus spinosus andO. gracilirostris The results for O. spinosus and O. gracilirostris were the same, and will be discussed together with no distinc- tion between species. Representative examples of dark- adapted spectral sensitivity curves for two specimens are shown in Figure 10 (A, B). The variability in these curves is similar to that seen in the previous two species. Chromatic adaptation experiments again provide evi- dence that more than one visual pigment is present. Vio- let adaptation resulted in a small depression in the violet shoulder (Fig. IOC). The only specimen that had a dis- tinct violet peak in its dark-adapted spectral sensitivity curve (see Fig. 10B) died during the chromatic adapta- tion experiment; therefore, the effects of violet adapta- tion are not as apparent as in Systellaspis or Janicella. The effects of green adaptation were much more dra- matic. The sensitivity to long wavelength light was greatly diminished with respect to the short wavelength sensitivity, resulting in either two peaks, or, with more intense adaptation, a distinct peak at 400-410 nm, and a plateau centering at 500 nm (Fig. 10D). The shapes of the response waveforms in dark-adapted and chromatically adapted eyes were the same as those described for Systellaspis and Janicella (Fig. 1 1C), again pointing to the presence of two spectral classes of recep- tor cells. Oplophorus spinosus proved to be unusually robust, and in two instances we were able to record responses after the eye had recovered from chromatic adaptation. Green chromatic adaptation distinctly altered the shape of the spectral sensitivity curve as well as the response waveforms (Fig. 11 A. C). Both the spectral sensitivity curve and the response waveforms, measured two hours after extinguishing the adapting light, were the same as those measured before chromatic adaptation (Fig. 1 IB, C). This indicates that waveform changes were due to the effects of the adapting light, and not to changes in electrode position or to degenerative changes in the eye during the course of an experiment. B DARK ADAPTED -^ >v 470 — \ v* Figure 9. ERG response waveforms matched for equal amplitude in J. spinacauda. (A) The response waveforms (amplitude = 30 nV) in this dark-adapted preparation were markedly different between re- sponses to short versus long wavelength light. The major component of the short wavelength responses (350-450 nm) was comeal positive (shown by the downward deflection), while the major component of the longer wavelength responses was negative. (B) In another prepara- tion, the dark-adapted response waveforms (40 MV) were virtually iden- tical, and were all corneal positive. Upon adaptation with green light, the waveforms between 370-450 nm were reversed in polarity, while the waveforms from 470-570 nm remained unchanged. (C) Dark- adapted waveforms in another preparation were virtually identical, and remain unchanged under a blue adapting light. Polarity differences in long wavelength response waveforms between different specimens are probably due to differences in the depth of the recording electrode (see Discussion). 268 T. M. FRANK AND J. F. CASE B -7 . • -8.8 -7.5 -9.5 340 400 460 520 580 Wavelength (nm) 320 400 480 560 Wavelength (nm) D -7 Cr««n- adoplto 340 420 500 580 Wovelength (nm) 340 420 500 580 Wavelength (nm) I i i;u re 10. Dark-adapted spectral sensitivitj curves f or Oplophorus. (A) Six of the seven specimens tested possessed broad spectral sensitiv- ity curves similar to the one shown, with small variations in sensitmts at the shorter wavelengths. (Hi Only one distinctly bimodal spectral sensitivity curve was measured, with peaks at 350 and 500 nm. (C) Selective etl'ects of \iolet adaptation are small hut discernible: sensitiv- ity was slightly more depressed at the shorter wav clengths. diminishing the small violet peak seen in the dark-adapted eve Intensity of adapting light was 2.4 • 10 ' jiW/cnv/s. (D) Green adaptation selectively de- pressed sensitivity at the longer wavelengths, producing a much larger violet peak relative to the blue-green peak, lender a higher intensitv adapting light, the blue-green peak was completely depressed in the same specimen. Results from four other specimens arc compatible w nh those shown Discussion In ck-ar oceanic waters, the \\a\eleiigth of maximum light iransmittance is 510 nm in the surface laxers. with the FVVHM . nvcring a spectral range from 440 to (-00 nm. At 100 m depth, selcctne absorption and scattering have shifted the transmission maximum to 475 nm and narrowed the spectral distribution to a I \\ I IM cmctint' 440-500 nm (JerKn . |9l - D.ninall, 1474; ,li-.K.\. 1976: Cronin, 1986). I hi lit) that deep-sea organisms may have blue-shifted \istial pigments as an adaptation for maximum scnsitiviu to this light ur.ime (the Sensi- tivity Hypothesis) was first suggested b\ Clarke (1936) and Bayliss ct al. (1936). and this idea of sensitivity peaks matching ambient light distribution has since been ex- teiuled to other en\ ironments. Although Lythgocf 1968) has shown that the Scnsitiviu Hypothesis may not neces- sarih hold true for all shallow water species, which live in a \cr> "complex" \isual environment, it has been strongly supported b\ studies on organisms living in the B t-7 > t in o o -8.8 -9.6 380 +60 540 620 380 620 WAVELENGTH (NM) WAVELENGTH (NM) D»nK oneeN POST AOA 390 430 440 450 510 550 610 A. V" A- - 400 ms (•inure II. I Meets of chromatic adaptation on spectral sensitivity a nil response waveforms in (.»/>/< >/>/;, 'MM ( M (iieen adaptation selec- tivclv ilepresseil the lone wavelength sensitivity, leading to a bimodal s|iei'iial sensitmu eur\e «nli mavima at 400 and 500 nm. (B) Two hunts altet the adapting liyhl «as turned oil', the eye had recovered completely from the elicits ol'lhc adapting light, and the spectral sensi- tivity function uas identical to that of the dark-adapted eye. (O Daik- ailai>teil response wa\etonns toi the same preparation shown above ueic shiihtls iliilru in .it the shortest wavelengths. Circcn adaptation alteied the i espouse waveloi ins at the shorter wavelengths as previously descnheil I wo h.uiis altei the adapting light was turned oil. the re- sponse wau-lonns were again identical to those in the dark-adapted e vlapimr hi'ht intensity = 2.5 X 10 ' ^W/cm2/s.: criterion re- sponse -10 i/^ VISION IN DEEP-SEA CRUSTACEANS 269 "simpler" deep-sea visual environment. The visual pig- ments of most deep-sea species studied to date have peak absorption maxima clustered between 470 and 490 nm. which are about 10-20 nm shorter than those of their shallow water counterparts, thus supporting the Sensitiv- ity Hypothesis (reviewed by Goldsmith, 1972; Cronin, 1986)'. Single visual pigment systems The results from two species in this study. N. gihhusus and TV. elegans. support the Sensitivity Hypothesis. The maximum sensitivity of these species (490 nm) is at shorter wavelengths than those of shallow water crusta- ceans (510-550 nm), and fall into the same range as those of the deep-sea fish (Denton and Warren, 1957; Munz, 1 957; Wald ?/«/.. 1957; Denton and Shaw, 1963; Fernandez, 1978: Crescitelli el ai. 1985). The spectral sensitivities of A. curtirosiris and A. smithi peak at 510 nm, seemingly more appropriate for shallow water crustaceans than for species that maintain daytime depths of greater than 500 m. The absorptance spectrum matches the shape of the spectral sensitivity curve, but is offset by 20 nm. This suggests that these species may possess some type of non-moving distal pig- ment screen, as found in A. purpurea (Welsh and Chace, 1937), that would shift the sensitivity maximum away from the visual pigment absorption maximum. In cray- fish and lobsters, this pigment screen is believed to be responsible for the 10-30 nm difference between the vi- sual pigment absorption and the spectral sensitivity func- tion (Goldsmith, 1978). Why such a screening pigment shield would be needed, particularly in A. curtiroMn*. which never migrates to shallower waters, remains ob- scure. It is unlikely that self-screening by metarhodopsin contributed significantly to the long wavelength shift in spectral sensitivity. Although both species possess meta- rhodopsins with Xmax at shorter wavelengths (A. cwtiros- trjs — 481 nm; A. stuil/ii — 483 nm; Hiller-Adams et ai, 1988) than those of their rhodopsins, so that self-screen- ing by metarhodopsin would shift the spectral sensitivity to longer wavelengths, our experimental protocol en- sured that the eye was fully dark-adapted before starting an experiment. According to Goldsmith (1978), self- screening by metarhodopsin should be negligible if: ( 1 ) the eye is dark-adapted. (2) near-threshold flashes are used to stimulate the eye (preventing isomerization of a sizable fraction of rhodopsin to metarhodopsin), and (3) the organism has other mechanisms than photo-regener- ation for restoring a full liter of rhodopsin. The first two conditions were met by our experimental protocol, and while these two species have not been studied with re- spect to dark-regeneration, such a system was found in another oplophorid occupying the same depth range (Hiller-Adams et ai, 1988.) Additionally, specimens tested within three hours of capture demonstrated the same spectral sensitivity as those that were maintained in the dark for 24 hours before testing. Therefore, self- screening by metarhodopsin is not a reasonable explana- tion for the observed results. The difference in the polarities of the representative response waveforms shown for Notostomus (Fig. 1 ) and Acanthep/iyra (Fig. 3) may be due to differences in the electrode depths from preparation to preparation. In both genera, preparations were found in which the re- sponse waveforms were of the opposite polarity to those shown in the figures, so these polarity differences are not species specific, but probably depend on the recording parameters. Konishi (1955), working with the lobster eye, showed that an electrode just beneath the corneal surface recorded a corneal negative response. With deeper insertion into the eye, the recorded response re- versed in polarity to a corneal positive response. Since the depth of electrode penetration varied between prepa- rations in our study, electrode position may explain the ERG polarity differences. Dual visual pigment m/cms The most interesting visual systems are found in the remaining four species, S. debilis. J. spinacauda, O. spinosus. and O. gracilirostris. which appear to possess a violet sensitive pigment in addition to one with maximal sensitivity in the blue-green. The variation in the shapes of the dark-adapted spectral sensitivity curves is much greater than in those of the single pigment species, and this may be due in part to the location of the electrode in the eye, particularly if different parts of the eye have different spectral sensitivities. Regional differences in spectral sensitivity have been found in the eyes of several species of insects (Walther, 1958: Ruck, 1965; Bennett and Ruck, 1970) and results of experiments on these in- sects are similar to ours. Goldsmith (1960) also reports that the relative contribution of the UV and green recep- tor systems to the ERG in the honeybee eye could be altered somewhat by moving the electrode to another part of the eye. Due to our experimental protocol in testing the eye with a standard flash throughout the experiment, in addi- tion to the fact that those crustaceans which had a dis- tinct violet peak exhibited the same overall sensitivity as those which did not (see Fig. 4). we are confident that the differences in shapes of dark-adapted spectral sensitivity curves were not due to differences in the degree of dark- adaptation. The conclusion that two visual pigments are present in these four species is strongly supported by the differential 270 T. M. FRANK AND J. F l \M effects of the different adapting lights on the shape ot 'the spectral sensitivitv 1C selective cllccts of violet adaptation were gc- not as great as those of green adaptation, a- he attributed to the fact that all visual pigmer s a .i'-band that absorbs in the shorter igths. meaning that \iolet adaptation would affei voth receptor s> stems. However, the fact that \ioiei adaptation had a stronger effect on the short wavelength svstem, so that differential effects in the shape ot'the spectral sensitivity functions could be seen. indicates that the violet peak is not due to the ^-absorp- tion band on the blue-green pigment as in the \\oodlouse .'7/0 (Goldsmith and Fernande/. 1968). If this \\ere the case, the sizes of the two peaks relative to each other u ould remain the same during all adaptation conditions. Waveform differences between responses to short \'cr- M(S long wavelength light indicate that the two visual pig- ments are housed in different receptor cells. Single cells ha\e never been shown to respond dilferentiallv to different wavelengths of light (Graham and Hartline. 1935; Naka and Rushton. 1966: Stark and Wasserman. 1974). and if several spectral mechanisms with different time courses contribute to the ERG. equal amplitude re- sponses at all wavelengths can never be matched (Chap- man and Lall. 1967). In several species of muscid flies, waveform differences between short and long wave- length responses were initially attributed to the presence of a red sensitive receptor in addition to short wavelength receptors (Autrum and Burkhardt, 1961; Burkhardt. 1962: Ma/okhm-Porshnyakov. I960). However. Gold- smith (1965) found that these differences were due to differences in the sizes of ganglionic on-off effects in the ERG. rather than the presence of several spectral classes of receptor cells. Crustacean ERGS do not exhibit these ganglionic on/off effects, since the ERG is a more purely retinal response (Naka and Kuwahara. 1956; Chapman and Lall, 1967; Goldsmith and Fernandez. 1968). and there is no experimental evidence for any contribution by the optic ganglion to the ERG (Ruck and Jahn, 1954; Konishi. 1955). Therefore, differences in waveform re- sponses to short and long wavelength light in crusta- ceans, based on current knowledge, can onlv be attrib- uted to two different populations of receptor cells with dillerent membrane properties (Wald. 1968). Further support for two spectral classes of receptor cells comes from the wavelength-specific effects of different adapting lights on response waveforms. These results can tx :ied bv assuming that the visual pig- ments are housed i, litlerent receptor cells, and that the numeric distnU.i : the two receptor classes is not equal. This situation is mimd in cravlish ami lobsters. where the long wavelength pirmcnt occupies seven of the eight retinula cells present in i: "imnaiidium. and the violet pigment occupies nisi one (Cummins and Gold- smith. 1 981: Cummins i-l al.. 1984). A similar unequal distribution also appears to be present in our deep-sea species. The ERG responses in the dark-adapted eyes were generally characteristic of those attributed to the blue-green receptors. Upon green adaptation, the rela- tive contribution of the violet receptors was enhanced. and distinct differences in response waveforms at the shorter wavelengths were observed. Conversely, violet adaptation had no affect on response waveforms, or di- minished differences present at the shorter wavelengths in the dark-adapted eyes, as expected when the minor contribution of the violet receptors was further dimin- ished. The location of the violet receptor cells in an accessory eve would provide an explanation for the unusual "hy- perpolarizing" responses seen to short wavelength light. All known microvillar photoreceptors. which are the type possessed by all crustaceans (Eakin. 1972). depolar- ize in response to light (reviewed by Jarvilehto. 1979), but an unusual orientation of the short wavelength re- ceptors to the electrode could produce an apparently "hyperpolarizing" response. In the alciopid worm Tor- ri'ti. which also has microvillar photoreceptors. responses from the main retina are depolari/ing, while responses contributed by an accessory retina are hyperpolarizing. and this has been attributed to the reversed arrangement of the receptors of the accessory retina relative to the electrode position (Wald and Rayporl. 1977). <>/ h\'<> visual pigments in organisms Many other crustaceans, such as shallow water crabs (Wald. 1968; Hyatt. 1975; Martin and Mote. 1982), lob- sters (Cummins cl al.. 1984), estuarine shrimp (Wald andSeldin. 1968; Goldsmith and Fernandez. 1968). and crayfish (Goldsmith and Fernandez. 1968; Wald. 1968: Waterman and Fernandez. 1970; Cummins and Gold- smith. 1 98 1 ) appear to possess a short wavelength visual pigment. The purpose of this pigment is not clear, al- though Hyatt ( 1975) feels that it may be a mechanism for hue discrimination in the fiddler crab. Even though the rationale for the pigment is not known, all of these species live in shallow or near-surface waters where UV light is abundant and may play some role in their visual environment. In insects, the presence of a UV peak has been closely correlated with some behavioral patterns. Behavioral studies on I 'V-sensitive pierid butterflies in- dicate that they visit violet and blue (lowers more often than butterflies without the short wavelength sensitivity (Use. 1928: Eguchi cl al.. 1982). Obara and Hidaka ( 1968) also report that male pierid butterflies approach females after identifying the U V patterns on their wings. However, the reason for a UV visual pigment among some nocturnal moths (Eguchi cl al.. 1982; Mikkola. VISION IN DEEP-SEA CRUSTACEANS 271 1972) remains obscure, since U V light is absent in moon- light as well as in background galactic light at night (MunzandMcFarland, 1973, 1977). There is a similar absence of UV and near-UV light in the deep-sea. Although UV light may penetrate signifi- cantly in the surface layers (Jerlov, 1968; Dartnall, 1974; Jerlov, 1976). it is virtually absent by 500 meters— .09% of the 500 nm light present at the surface remains, while only .00007% of the 400 nm light is still present (Type 1 water. Table XXVI— Jerlov, 1976). Bioluminescence, the other source of light in the deep- sea, is considered by some to be the major visual stimulus present in this environment (Beebe, 1935; Clarke and Hubbard, 1959; Jerlov, 1968). It is also the logical candi- date to provide an explanation for the violet visual pig- ment, since the four species with the enhanced violet sen- sitivity possess photophores, while the four species with- out photophores are not violet sensitive. Examples of unusual visual systems correlated with bioluminescence are found in three species of malacosteid fish, which pos- sess red-shifted visual pigments as an apparent adapta- tion for enhanced sensitivity to their own red biolumi- nescence (O'Day and Fernandez, 1974; Denton el ai. 1970; Bowmaker and Herring, unpub.). While the vast majority of bioluminescence emission maxima in the deep-sea, including those from the photophores of 5. de- bilis and O. spinosiis, are clustered around the same wavelengths as the downwelling illumination (460-490 nm), with no emission maxima below 430 nm (Herring, 1976; Herring, 1983; Widder el ai. 1983; Latz et ai, 1988), bioluminescence may still provide the explana- tion for the unusual violet pigment. It has been suggested that the presence of two blue- green visual pigments in some species of deep-sea fish may serve as a system for discriminating between differ- ent bioluminescent organisms by using the spectral bandwidth as the basis for discrimination (Partridge et ai. 1988). The putative violet visual pigment may be serving the same purpose in these oplophorids. Spectra with broader spectral bandwidths would be more effi- cient in stimulating the violet-receptor, and in this man- ner could be distinguished from spectra with narrower bandwidths. The spectral emissions from most of the species with similar depth distributions as these oplopho- rids, including other crustaceans (except euphausiids), si- phonophores, fish, and cephalopods. are remarkably similar, with the peaks lying between 465 and 485 nm, and FWHM covering 65-90 nm (Widder et ai, 1983; Herring, 1983; Latz etai. 1988). However, the emissions from the photophores ofS. debilis and O. spinosm, while peaking in the same range, have FWHMs of 48-58 nm (Herring, 1983; Latz et ai, 1988), and perhaps this difference in spectral bandwidth is enough to facilitate congener recognition. Additionally, the FWHM of their luminous secretion, which is thought to be used during escape responses, is between 65 and 75 nm, and could potentially be distinguished from the photophore emis- sion to serve as warning signs to congeners. Acknowledgments We thank Dr. James Childress. his laboratory associ- ates, and the captains and crews of the RV New Horizon for assistance with animal collection. We also thank Dr. Childress for generously providing shipboard laboratory space; Dr. Steve Bernstein for writing the digitizing pro- gram; and Mark Lowenstine, Robert Fletcher, and Joel Dal Pozzo for help in designing and building the portable ERG apparatus. Drs. 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Pp. 16-20. Reference: Biol. Bull 175: 274-283. (October, 1988) Visual Spectral Sensitivity of the Bioluminescent Deep-sea Mysid, Gnathophausia ingens TAMARA M. FRANK1 AND JAMES F. CASE Ih'f>arinu'ni <>i Biological Sciences and Marine Science Institute, University of California, Santa Barbara. Ca/iloniia 93106 Abstract. The spectral sensitivity of the deep-sea my- sid. Gnathophausia ingens (Family Lophogastridae), was measured by electroretinography on intact specimens. High sensitivity to orange light was found. This was an unexpected result for a species whose adult members are never found above 400 m. Results of chromatic adapta- tion and silent substitution experiments were not com- patible with either a one pigment or dual pigment visual system, making this one of the more unusual visual sys- tems ever described. Introduction In this, the second of two papers on the spectral sensi- tivities of deep-sea crustaceans, we report on the unusual visual system of the deep-sea m\sid, Gnathophausia 111- gens, a species whose adult members are found below 400 m. This robust animal survives under laboratory conditions for over two years (Childress and Price, 1 978). making it an ideal candidate for electrophysiological studies. Its visual system proved to be unlike any pre- viously described crustacean visual system, including those of the deep-sea crustaceans described in our previ- ous paper (Frank and Case. 1988). Prolonged laboratory maintenance was not responsible for the unusual results. since specimens tested three months after capture pos- sessed identical threshold and spectral sensitivities to those tested within 24 hours of capture. Materials and Methods Animal collection and maintenance Specimens of if Con- necticut Health Center. Farming!" >n < I ni.nP Electroretinograms (ERGs) were recorded using the experimental set-up described in our previous study (I i;ink ;nul Case. I^SS). Chromatic adaptation experi- ments were conducted as previously described, with a 400 nm broadband tiller (Melles Griot BG12. FWHM 274 VISION IN A DEEP-SEA MYSID 275 = 110 nm) for violet adaptation, a 520 nm broadband filter (M.G. VG6, FWHM = 90 nm) for green adapta- tion, and a 570 nm short wavelength cut-ofF filter (M.G. OG570) for orange adaptation. Silent substitution experiments were conducted using a modification of the methods of Forbes el al. (1955) and Donner and Rushton ( 1959). The light output from two monochromators was controlled by two electromagnetic shutters (Uniblitz) connected in such a way that when one shutter opened, the other simultaneously closed. Light from the two monochromators was conducted to the eye through the two branches of a bifurcated light guide, ensuring that upon shifting illumination from one monochromator to the other, the same receptor field was illuminated. Thus, when the two light sources are matched for equal intensity and wavelength, switching from one monochromatic source to the other should produce no visual response; i.e., the substitution should be "silent." Light intensity was controlled with glass neu- tral density filters and a neutral density wedge. Experimental procedure Test flashes of 100-ms duration were used, and were repeated at one minute intervals. The response to a stan- dard flash of set wavelength and intensity was tested ev- ery five flashes to monitor the stability of the preparation. The reciprocal of the quantum flux (/L I he ellecis of orange adaptation were very small (Fig. 2C): in individual ani- mals, the curves measured under orange adaptation were identical to those measured in the dark-adapted eyes (see I ii:. 5C). The chromatic adaptation results are presented on a log scale to be consistent with prcv iouslv published spectral sensitivity curves on other crustaceans. How- ever. when presented on a linear scale, the dramatic difference between the effectiveness of green and violet adaptation on altering the shape of the spectral sensitiv- itv curve as compared with orange adaptation is much more visible (Fig. 3). Results of the linear regression analysis on the regres- sion lines calculated for the mean difference values are shown in Figure 4. The fit to a single linear regression was poor for the data set obtained by subtracting values measured under violet adaptation from those measured in the dark-adapted eve. so these data were divided into two groups. The cut-off point for each group w^as chosen so that the regressions shown provided the best fit ac- cording to the F-test. The mean difference values for green adaptation were also best fit by two linear regres- sions. while a single regression provided the best fit to the mean difference data for orange adaptation. Both the long wavelength and short wavelength regression lines were significantly different from zero under violet adap- tation (P < .001 ). Although the curve under green adap- tation appears similar to that obtained under violet adap- tation (Fig. 2 A. B). only the long wavelength regression line (for points past 510 nm) was significantly different from zero. (P < .001 ). This indicates that green light did not have a statistically significant effect on spectral sensi- 340 400 460 520 580 640 340 400 460 320 580 640 340 400 460 520 5BO 640 Wavelength (nm) Wavelength (nm) Wavelength (nm) Figure 3. I he effects of chromatic adaptation displayed on a linear scale, to accentuate the dillcicnccs in the shapes of the chromatically adapted spectral sensitivity curves. Data are the same as those displayed in Figure 2. Bars indicate standard errors. I he effects of violet and green adaptation were visibly greater and different from those of orange adap- tation. A small shift in maximum sensitivity from 510 to 470 under mange adaptation is now visible. VISION IN A DEEP-SEA MYS1D 277 1.500-r 0.500 0.000 300 350 400 450 500 550 600 650 700 1.500-r c 1.000 0.000 B 300 350 400 450 500 550 600 650 700 1.500-r 0.000 300 350 400 450 500 550 600 650 700 Wavelength (nm) Figure 4. Statistical analysis of chromatic adaptation effects on spectral sensitivity. Each point is the mean of the difference values ob- tained by subtracting log light intensities required to elicit the criterion response during chromatic adaptation from those required in the dark- adapted eyes: difference values from individual animals were normal- ized before grouping into color classes. Standard errors are shown where they are larger than the point markers (.030). Slopes (m) of the regressions appear on each graph. (A) The mean difference values un- der violet chromatic adaptation (n = 11) were best tit by two regression lines. The regressions were generated using an iterative procedure in which data points were sequentially added or subtracted at 490, 510, 530, and 550 nm until the best fit was attained using the F-test. Both slopes are significantly different from zero (t = 5.89. d.f. = 7; t = 20, d.f. = 5; P < 0.001. both comparisons). (B) The mean difference data set for green adaptation was also best fit by two regressions. The slope of the regression from 350-530 nm is not significantly different from zero(t = .371. d.f. = 8); however, the slope of the line fitting data from 5 10-650 nm is(t = 1 1.2. d.f. = 6: P< 0.001). (C) The mean difference data for orange adaptation (n = 9) was best fit by one linear regression, the slope of which is not significantly different from zero (t = . 1 10. d.f. tivity at the shorter wavelengths, but did significantly al- ter the shape of the spectral sensitivity curve at the longer wavelengths. The regression for the mean difference data under orange adaptation was not significantly different from zero, indicating that the shape of the spectral sensi- tivity curve under orange adaptation was the same as that of the dark-adapted eye. The selective effects of the different colors were not due to intensity differences in the adapting lights. The effects were visible at the lowest intensities, and higher intensities only slightly enhanced these differences (Fig. 5). Selective effects were also not due to animal variabil- ity, as demonstrated by the results from three different chromatic adaptation experiments on one specimen (Fig. 6). This experiment also demonstrates that the re- sults seen were not due to degenerative changes in the eye overtime; complete recovery to the pre-adapted level after a sufficient dark interval can be seen (Figs. 6B, C). Response waveforms Analysis of the response waveforms indicates that a dual receptor system may be present, although again, the evidence is inconclusive. The ERGs were generally sim- ple, monophasic, corneal-negative signals, characteristic of crustacean visual systems. Waveforms matched for equal amplitude in the dark-adapted eye were either sim- ilar (Fig. 7F), or, more commonly, were noticeably different only at the longest wavelengths (Fig. 7D). ERGS at the shorter wavelengths were simple in form, while at 550 to 570 nm, additional small waves appeared prior to the large wave. Occasional small positive shoul- ders preceding the larger negative waves were found at shorter wavelengths (Fig. 7 A). Violet, green, and orange adaptation had the same ' , __^ > J40 400 460 520 580 640 J40 4QO 460 S Wavelength (nm) Wavelength (nm) 400 460 520 580 640 Wavelength (nm) Figure 5. The effects of varying intensities of adapting lights on the spectral sensitivity function. Each graph represents data from one ani- mal. The numbers below the plots indicate the quantum flux of the adapting light in jjW/cm:/s. (A) Violet adaptation at two different in- tensities produced essentially the same results. (B) The selective effects of green adaptation were more evident at the higher intensity, but were still visibly different from violet or orange adaptation at the lower inten- sity. (C) Selective effects of orange adaptation were not visible at either intensity, although the threshold was depressed by the same amount (1-2 log units) as under violet adaptation in A. 278 T. M. FRANK AND J. F. CASE S40 400 4M MO MO I Wavelength (nm) 340 400 4*0 520 MO 640 Wavelength (nm) 140 400 460 b20 i50 640 Wavelength (nm) Figure 6. Selective effects of chromatic adaptation on the spectral sensitivity of one specimen. I ach chromatically adapted curve is shown with the dark-adapted cur\e measured just prior to that chromatic ad- aptation experiment. The effect of the adapting light was to lower the sensitivity by approximately 4-5 log units in each experiment. The se- lective effects of the adapting lights are consistent with the results seen in the grouped data. (A) Blue adaptation produced two peaks at 450 and 550 nm. (B) Green adaptation similarly produced a relatively bimodal curve, with peak sensitivities at 430 and 550 nm. (C) Orange adaptation had no apparent effect on the shape of the spectral sensitivity function. effects on the response waveforms. Under chromatic ad- aptation, all waveforms became hiphasic. with a very small first wave (or cusp at the shortest wavelengths) fol- lowed by a second larger wave (Fig. 7B, E. G). The size of the first wave increased with increasing wavelength af- ter 570 nm. Upon extinction of the adapting light, the response waveforms recovered to the pre-adapted state, indicating that the alteration in waveform was due to the adapting light (Fig. 7C). Silent Mih\niuiion Substituting light from one monochromator to an- other did not produce a discernible dark period for G. ingens, since light of the same color could always be sub- stituted without eliciting a response as long as the intensi- ties were matched (Fig. 8A). When intensities were not properly matched, a distinct electrical signal was seen. the polarity of which depended on whether the substitut- ing light was of a lower or a higher intensity. Silent substi- tution .if >no nm light for 400 nm light was always possi- ble (Fig. SB), indicating that the same receptor system was operating at these wavelengths. I lowever. silent sub- stitution was never possible between 500 and 600 nm (Fig. 8C), or 40(i and 600 nm (Fig. HD). An "on" re- sponse never completely disappeared before the "off" re- sponse became apparent. Increasing or decreasing the in- tensity only increased the si/c of the electrical signal. These results support the premise of two spectral classes of receptor cells, one dominating the responses in the blue-green, and the other operating primarily in the or- ange. Discussion Gnaihophausia ingens is a deep-sea mysid whoso depth distribution depends on its life history stage. The members of the size classes studied here (Instars 6-10) have a da\ time depth range of 650-750 m (Childress and Price, 1978). They do not undergo a typical vertical mi- gration, but instead disperse at night to depths between 400 and 900 m. This species possesses the typical crusta- 50 uV ABC 80 uV 40 uV D E F G \r \r Figure 7. Response waveforms matched for equal intensity within one preparation. Data from three individuals are shown. I he criterion response is shown at the top of each data set. Time bar designates 400 ms. (A) Dark-adapted waveforms were monophasic corneal-negative responses, indicated b> the downward deflection, with a small corneal positive wave preceding the larger negative component at the shorter wavelengths, and a small corneal negative wave present at the longer w.m-lcngths (arrows). (B) Violet adaptation changed all the response w.iv.-lorms such that a small negative wa\e was present at all wave- lengths (arrow). with the si/c of the wave increasing at the longer wave- lengths. (C) Waveforms recorded two hours after extinction of the adapting light were identical to those in the dark-adapted eye. (D) The response waveforms recorded in the dark-adapted eye of another speci- men were distinctly different at the longer wavelengths (arrow). The simple monophasic waveforms seen at the shorter wavelengths devel- oped a small cusp by 540 nm. and two distinct waves were present at 610 and MO nm (1)1 he effects of a green adapting light were identical in i hose of the violet adapting light; the large corneal negative waves were preceded In .1 small corneal negative wave (arrow). The size of the small wave again increased with increasing wavelength. (F) The w av dorms in this dark-adapted eye were virtually identical at all wave- lengths. (Ci) Orange adaptation also produced the small corneal nega- 1 1 v c w ave (arrow) at all wavelengths, and the small wave again increased in si/e at the longer wavelengths. VISION IN A DEEP-SEA MYSID 279 A 400/400 BCD 400/500 400/600 500/600 Figure 8. Results of silent substitution experiments for one speci- men. The first number at the top of the graphs is the wavelength setting for Monochromator 1 (M 1 ); the second number is for Monochromator 2 ( M2 ). The square waves designate which monochromator was illumi- nating the eye for the responses seen. (A) Silent substitution was possi- ble when M 1 and M2 were set for the same wavelength, demonstrating that no discernible dark penod was present during the substitution. The ( 1 ) under the square wave indicates that M 1 was on at this time: when the square wave reverses polarity, light from M2 was substituted. (B) Silent substitution was also possible between 500 and 400 nm. The top figure shows the response when the intensity of the light from Ml was too high. There was a discernible "on" response at the start of the sub- stitution, and an "off' response at the end. Responses were corneal positive in this specimen, as they were in several others, which can be attributed to the depth of the recording electrode (Konishi, 1955). The response was not a maintained "on" response for the duration of the stimulus due to the amplifier time constant. The intensity of the light from M 1 is shown below the square wave pulse in photons/cnv/s. With decreasing intensity of light, responses diminished until an intensity was reached at which no response was discernible (7.8 x 10" photons). For comparison, the last figure shows the off response when the inten- sity from M 1 was too low and the on response upon substituting in light from M2. (C) Silent substitution was not possible between 400 and 600 nm light. Distinct on and off responses are visible in the first figure. With decreasing intensity, the on and off responses became smaller, but never disappeared. The smallest response was seen at 3.66 x 109 photons. Increasing the intensity increased the size of the on response, while decreasing the intensity produced a discernible oft" response. (D) Silent substitution was also not possible between 500 and 600 nm. The smallest response was at 2.73 x 109 photons, and increasing or decreas- ing the intensity from this value increased the size of the electrical signal. cean spherical compound eye, but its visual physiology appears to be very unusual, and the results of this study are not entirely compatible with either a single or dual visual pigment system. Spectral sensitivity The absorptance curves in Figure 1 suggest two possi- ble explanations for the unusually broad dark-adapted spectral sensitivity curve. A single pigment present in very high concentrations could give rise to such a broad spectral sensitivity curve, due to self-screening by rho- dopsin. Such high concentrations of visual pigment have been found in several species of deep-sea fish (Denton and Warren, 1957) and crustaceans (Hiller-Adams el al., 1988), and may increase sensitivity to light, a useful ad- aptation in the dimly lit deep-sea environment. How- ever, two pigments with fairly close absorption maxima (490 and 520 nm) could also give rise to a broad spectral sensitivity curve, as demonstrated by the absorptance curve in Figure IB. The effects of chromatic adaptation do not support the single pigment/self-screening hypothesis. Only the re- sponse to orange adaptation is compatible with this hy- pothesis. The shift in maximal sensitivity is not signifi- cant, and the shape of the curve under orange adaptation is identical to the curve recorded from dark-adapted eyes in individual specimens. Under this hypothesis, the effects of green and violet adaptation should be the same as those of orange adaptation — decreasing sensitivity but not changing the position of peak sensitivity. However, violet and green light produced visible changes in the shape of the spectral sensitivity curve, and these changes were significant. Even when the adapting lights de- creased the overall sensitivity by the same amount (see Fig. 6), the different colors had different effects on the spectral sensitivity. Therefore, a single visual pigment with a high optical density cannot alone explain the spec- tral sensitivity of G. ingens. The spectral sensitivities of some shallow water lob- sters, shrimp and crayfish are markedly shifted from the absorption maxima of their visual pigments (De Bruin el al. 1957: Wald and Hubbard, 1957; Kennedy and Bruno, 1961; Goldsmith and Fernandez. 1968; Wald, 1968; Bruno et al.. 1977) and this has been attributed to selective filtering by screening pigments (Goldsmith, 1978; Cummins et al.. 1984). Although the eye ofGna- thophausia ingens has not been investigated histologi- cally, Elofsson and Hallberg (1977) and Hallberg (1977) have studied seven other species of deep-sea and shallow living mysids, and found several common characteris- tics. All possessed superposition eyes with a layer of red pigment cells around the proximal part of the rhabdom and below the basement membrane. In the deep-living species, the red pigment cells appeared to replace the darker retinular pigment found in other species. Our gross histological examination of unfixed G. in- gens eyes also revealed an abundance of red pigment cells. The eye also had a large eyeglow area, probably due to the presence of a tapetum or reflecting pigments. The eyeglow area did not change upon light adaptation, as it does in many insects and shallow living crustaceans with mobile proximal screening pigments (reviewed by Sta- 280 T. M. FRANK AND I I i \sl venga. 1979). Tim sug vsts that a dark proximal screcn- ing pigment is probablv .ilso absent in this species. Due to the apparent lack of a proximal screening pig- ment in ti -wi/ ini;c>i\. the following discussion is based on >'• s assumption that the red screening pig- ment ; .iiound the proximal ends of the rhah- doms. as it is in the other deep-sea mysids lacking the proxim.:: screening pigment (Elofsson and Mallbcrg. 1977: Hallberg. 1977). In this configuration, the red pig- menl would be in a position to filter light before it reached the visual pigment. MSP on a similar red pig- ment in the crab /<•/>/( >i,'/-<;/>s(/\ showed that maximal ab- sorbance was at 500 nm. with low absorbance in both the UV and red (Stowe, 1980): i.e., UV and red light are not blocked by this pigment. Stowe has estimated that under strong light adaptation, when the pigment extends one third to one halfway up the rhabdom. (as it does in the deep-sea mysid Krytlin>ps — Hallberg. 1977). a "kink" would be seen in the spectral sensitivity curve at 380 nm. This is not the case in (.i. inxen\. neither the dark-adapted or chromatically adapted eyes ever showed a \ lolet sensi- ti\it> peak. Additionally, the longwave-length peak in 6'. ingens under violet and green adaptation always oc- curred between 530 and 550 nm. If a red-leaky screening pigment is responsible for the chromatically adapted spectral sensitivity (as it is in se\eral species of muscid flies— Goldsmith. 1965). a red peak should be visible be- tween 600 and 650 nm. Therefore, based on what is known about mysid screening pigments, the leakv screening pigment hypothesis also cannot provide an ex- planation for the spectral sensitivity of(/. ini>cns. In other species where there is good evidence for dual visual pigment mechanisms, the selective effects of chro- matic adaptation are dramatic and undeniable under adapting lights that depressed sensitivity by 1 -2 log units (Chapman and I.all. 1967: Goldsmith and Fernande/. 1968; VVald. I9ILX: I rank and Case. I9XS). 1 he effects of chromatic adaptation on (V //W/M were not as distinct. Green and violet adaptation significantly changed the shape of the spectral sensitivitv cur\e compared to or- ange adaptation (Tig. 4). but adapting lights that de- pressed sensitivity up to five log units never produced ellects of the magnitude seen in other crustaceans. Addi- tionalh. we were ne\er able to completely depress the short wavelength peak, which should be possible if a short wavelength pigment was present I all and Cronin ( I987)descnlir a similar situation in the purple land crab (Gecardnus lateral i\ I. I he spectral sensitiv itv of this spe- cies was much I ilian the absorption maximum of a single visual pigment, but chromatic adaptation with different colors did not have pronounced selective effects. They suggest that this alone does not preclude the possibility of two rcccptoi i lasses. Receptors containing visual pigments adjacent in the spectrum (such as blue versus green) would be difficult to isolate with ERGs. which are gross responses from the whole eve. and this problem would be compounded if one receptor class were numcricallv significantly inferior to the other class. as in the blue crab ( 'allincctc^ ( Martin and Mote. 1982). The shapes of the ERG response waveforms in the dark-adapted eve and under chromatic adaptation point towards the presence of a dual receptor mechanism, but there are inconsistencies. Under the dual receptor system hypothesis, at the shorter wavelengths, the 490 nm recep- tor cells would dominate the ERG. At longer wave- lengths. the contribution of these shorter wavelength re- ceptors would be diminished, and the contribution of the 520 nm receptors would become evident. The differ- ences in response waveforms to long wavelength light (570-630 nm) and shorter wavelengths in the dark- adapted eye of (/'. ingen* are consistent with this hypothe- sis (Fig. 7). Responses to shorter wavelengths were sim- ple. monophasic corneal negative (downward) wave- forms. At the longer wavelengths, the waveforms became more complex, with a vcrv small negative wave preced- ing a much larger one. The fact that these small waves were only visible at the longest wavelengths in the dark- adapted eve. and that they became larger with increasing wavelength (Fig. 7 A. D). supports the premise that they are due to the contribution of the long wavelength recep- tor system to the ERG. The effects of green and violet chromatic adaptation on response waveforms are also compatible with this hy- pothesis. Green and violet adapting lights should and did selectively diminish the contribution of short wavelength receptors to the ERG. unmasking the contribution of the long wavelength receptors. Under these adapting lights. all waveforms were composed of a small wave preceding a larger wave, and the si/e of the small wave increased with increasing wavelength (Fig. 7B. E). Conversely, or- ange adaptation should diminish the contribution of the long wavelength receptors, and eliminate the small first waves that may have been present in the dark-adapted eve. However, this is not what occurred. The effects of orange adaptation were identical to those of violet and 1'ieen adaptation: a small first wave was visible at all wavelengths and increased in si/eat longer wavelengths (Fig. 7G). This indicates that the small first waves are not 1'ioiluccd In long wavelength receptor cells. These effects of mange adaptation are not consistent with the dual re- ceptor svstem hvpothesis. but there is currently no other plnsiological explanation for waveform differences in cmsiaccan ERGs. In insects, wavelength-specific waveform differences have been attributed to differences in the si/e of the gan- VISION IN A DEEP-SEA MVSID 281 glionic on/off effects in the ERG due to a leaky screening pigment. If a red leaky screening pigment is present, as in several species of muscid flies, then red stimulation would stimulate more receptors than expected, produc- ing a larger on/off effect (Goldsmith. 1965). However, the ERG recorded in crustaceans is a more purely retinal response, with no evidence for any ganglionic compo- nent (Naka and Kawabara, 1956; Burkhardt. 1962; Chapman and Lall, 1967; Goldsmith and Fernandez, 1968). The small waves also occur only at the beginning of the response, indicating that they are not due to gangli- onic contributions, since the "off" response is missing. Currently, the only explanation for waveform differences in dark-adapted crustacean eyes is that two different classes of receptor cells with different response character- istics are contributing to the ERG (Chapman and Lall, 1967; Fernandez and Goldsmith, 1968; Wald, 1968). Si/em substitution The best evidence for a dual receptor mechanism in this species comes from the silent substitution experi- ments. The idea behind silent substitution (as described by Forbes el ai. 1955, and Donner and Rushton. 1959) is that if an eye is adapted to a steady monochromatic light, and this is replaced by a light of the same color from another source, a response will be seen if the intensity difference is within a detectable range for the eye. The substitution will only be silent when the eye can no longer detect an intensity difference, provided that the act of substitution does not produce a detectable dark period. If the lights are of different colors, the result will depend on the type of receptors contributing to the visual response. If only one type of receptor cell is present, then any two lights equally absorbed by the pigment can be silently substituted. Hence, in a single visual pigment sys- tem, an intensity can be found at each wavelength at which substitution will be silent. If more than one recep- tor type is contributing to the response, each with its own response characteristics, then in principle, substitution cannot be silent at all wavelengths. A response will be seen due to cessation of excitation of cells already re- sponding, and the excitation of cells (with different mem- brane characteristics) previously not responding. With our experimental design, we demonstrated that silent substitution was possible if monochromatic lights of the same colors were matched for equal intensities, indicating that an instrumental dark period was not dis- cernible during the switch. Silent substitution was also possible between 400 and 500 nm, indicating that re- sponses to these wavelengths are dominated by the same receptor cell class. However, 400 nm and 600 nm light could not be "silently" substituted at any intensity. Sim- ilarly, the substitution of 500 for 600 nm light always produced a discernible response. These results support the hypothesis that two receptor systems are present. Autrum and Stumpf ( 1953) described the presence of a red receptor in Museu. basing their hypothesis partly on their heterochromatic flicker results that red light al- ways elicited a response when substituted for blue or green light, while blue and green light intensities could be adjusted to achieve silent substitutions. However, this wavelength dependence was later attributed to stimula- tion of different numbers of ommatidia by red versus green light, due to the presence of a red leaky screening pigment (Goldsmith, 1 965). Goldsmith found that it was possible to produce receptor components of equal size to green and red stimulation, but the on/off components, which are ganglionic in origin and depend on the num- ber of receptors stimulated, could never be matched. This interpretation cannot explain our results, however, because we were working with a crustacean. As stated above, the ERG in crustaceans is a more purely retinal response, with no on/off component. Without the com- plicating on/off component, if a red-leaky accessory pig- ment was present, light intensities could be found at which weak stimulation of many receptors under red light would produce the same size response as stronger stimulation of fewer receptors under green light. Unusual effects of adapting lights on portunid crabs (Wald, 1968; Leggett, 1979) have been attributed to the presence of a single visual pigment, whose absorption is modified by different colored filters abutting different rhabdoms upon light adaptation. Our results argue against this mechanism, as the difference in waveform responses and the inability to silently substitute between 400-500 nm versus 600 nm cannot be explained by a single photoreceptor class with colored filters. We are confident that these remarkable results are not consequences of the experimental procedure. The identi- cal apparatus was used to measure the spectral sensitivity of deep-sea oplophorids (Frank and Case, 1988) and pro- vided clear evidence for either single or double visual pig- ment systems in those species, comparable to published results for shallow water crustaceans. The capture and maintenance of Gnathophausia ingens was identical to that of the oplophorids. The only difference is that some G. ingens specimens had been maintained in the labora- tory for up to five months. However, both the spectral sensitivity and threshold sensitivity of specimens main- tained for months in the laboratory were identical to those of specimens tested within twenty-four hours of capture, eliminating laboratory maintenance as an ex- planation for our unusual results. The presence of a single highly concentrated visual pigment can be readily correlated with the deep-sea habi- tat of this organism, as mentioned above. However, the explanation for the long wavelength shift of peak sensi- 282 I \1 I K \\k \\|) J I CASE tivity to 510 nni. and therefore enhanced scnsiti\it> to orange light, remains unknown. The lite history ot ' dna- (hophausia pit mswer. Fhe size classes used in this study are .-mid above 400 m. and although smaller - are found ai shallower depths, they are alway i than lot) m. \\here the spectral distri- butio' lias already significantly narrowed to- wards the bluer wavelengths (Jerlov. 1968: Dartnall. 1974;( ,onin. 1986). The rational for the presence of two visual pigments, if the> arc indeed present, is even more difficult to con- cei\e. 1 nexpected dual visual pigment systems have been found in several species of deep-sea fish (Denton el >70;< >'Day and I ernande/. 1474; Bow maker. Dart- nall and Herring, unpub.: Partridge el ui. 1988). and max be present in some deep-sea crustaceans as well (Frank and Case. 19SS). All these species possess photo- phores. and the cited authors have suggested that posses- sion of dual visual pigments may play a role in congener recognition. However. G ;;;.t,v/;.v does not possess any photophores. It does emit a bioluminescent spew from the oral region with a peak spectral emission at 485 nm (Illig. 1905: Frank el al.. 1984). This is close to the peak sensitivity of one of its proposed visual pigments, but is also the same as most of the bioluminescence that has been measured from organisms obtained from these depths (Herring. 1976: Widder <•//;<>/<; / I i;/ /'/MAM/ .15: 71-82. Beebe. \V. 1935. Hull Mile l>,mn John Lane, London. Bruno. M. S., S. V Barnes, and T. II. Goldsmith. 1977. The visual pigment and visual cycle of the lobster, /fcwianis ./ < ,>w/> rh\\i«l 120: 123-142. Burkhardt. I). 1962. Spectral scnsitivitv ami other response charac- teristics of single visual cells in the arthropod eve. S\i>i/> \n< /-.'i/> liiol 16:86-109. Chapman. R. M., and A. B. I.all. 1967. Llectroretinogram character- istics and the spectral mechanisms ot the median ocellus and lateral eye'mLimuIuspolyphemus .1 (,<_'n /'/aw,./ 50:2267-2287. Childress. J. J., and M. H. Price. 1978. Growth rate of the hatln pe- lagic crustacean Gnathophausia mirivi.v 1. Dimensional growth and population structure. Mar Ki«l 50:47-62. Cornwall. M. (., E. F. MaoNichol Jr., and A. Fein. 1984. Ahsorptance and spectral scnsitiv itv measurements of rod photore- ceptors of the tiger salamander. l/nh\ Miami nxnnwii I 'i\inn AY* 24(1 1): !65l-lt.-"i C'ronin, T. \V. 1986. Photoreeeption in marine invertebrates. Am. /««!. 26:403-415. Cummins. I). K.. I'M (hen. and I II. Goldsmith. 1984. Spectral sensitivity of the spiny lobster. Panulint\ an;ii\ liin/ Hit// 166: 269-276. Dartnall. II. .1. A. 1953. Hie interpretation of spectral sensitmu curves. /(/ \lcil Hull 9: 24-30. I)e Bruin, (i. II. P., and I). .J. Crisp. 1957. The influence of pigment migration on vision of higher Crustacea. J /-.'\/> Hiol 34:447-463. Denton, F. .1.. .1. B. Gilpin-Brown. and P. G. \\riKht. 1970. On the "niters" in the photophores of mesopelagic fish and on a tish emit- ting red light and especialK sensitive to red light. ./ /Virvif/ 208: 72-73. Denton. !•'.. .1.. and F. .1. XVarren. 1957. Photosensitive pigments in the retinae of deep-sea fish. J \iin Hi<>/ i \ MIC, r, A'. 36: 651-662. Donner, K. ()., and \\ . A. II. Rushlon. 1959. Retinal stimulation by light substitution . ./ I'hvMol 149: 288- 'o:. Flofsson, R., and F. llallheru. 1977. Compound eves ol some deep- sea and hord imsid crustaceans I, iii /Hi'/ i.Sini A/i i 58: ldl)-177. Forbes, A.. S. Biirleiuh. M. \e\land. 1955. I leetrie responses to color shift in frog and turtle retina ./ Vmv>/>/M wo/ 18: 517-535. Frank, I. M., F. A. \Mdder, M. I. Fat/, and. I. F.Case. 1984. Dietary maintenance of bioluminescence in a deep-sea mysid ./ / */> liml 109: 385- ^S'i (•rank. I. M., and .1. F. Case. 1988. Visual specti.il sensitivities ol bioluminescent deep-sea crustaceans, liiol Hull 175:255-267. Goldsmith. 1 . 1 1. 1965. I )o Hies have a red receptor? ./ Gen. Physiol. 49: 265-287. VISION IN A DEEP-SEA MVSID 2X3 Goldsmith, T. H. 1978. The effects of screening pigments on the spec- tral sensitivity of some Crustacea with scotopic (superposition) eyes. risianRex. 18:475-482. Goldsmith, T. H., and H. R. Fernandez. 1968. Comparative studies of crustacean spectral sensitivity. Z I'gl. Physiol. 60: 156-175. Hallberg, E. 1977. The tine structure of the compound eyes of mysids (Crustacea: Mysidacea). Cell Tissue Res. 184: 45-65. Herring, P. J. 1976. Biolummescence in decapod Crustacea. ./ Miir Biol.Assoc. U.K. 56: 1029-1047. Hiller-Adams, P., E. A. VVidder, and J. F. Case. 1988. A microspec- trophotometric study of visual pigments in deep-sea crustaceans. J. Camp. Physio/. 163: 63-72. Illig, G. 1905. Das leuchten der Gnatliophausen. '/.vol. An:. 28: 662. Jerlov, N. G. 1968. Optical Oceanography. Elsevier. Amsterdam. Kennedy, D., and M. S. Bruno. 1961. The spectral sensitivity of cray- fish and lobster vision. / (Jen. Physiol 44: 1089-1 102. Konishi, J. 1955. Retinal and optic nerve response of the compound eye of the spiny lobster, Panulims juponicits von Siebold. Rep. Fac. Fish. Vmv MIC 2(1): 138-144. Lall, A. B., and T. W. Cronin. 1987. Spectral sensitivity of the com- pound eyes of the purple land crab Gecardnus laieralis Bio! Bull 173:398-406. Leggett, L. M. W. 1979. A retinal substrate for color discrimination in crabs. J. Comp. Physiol. 133: 159-166. Martin, F. G., and M. I. Mote. 1982. Color receptors in marine crus- taceans: a second class of retinula cells in the compound eyes of Ca/linectesand Cardans. J. Comp. Physiol. 145: 549-554. Naka, K., and M. Kunabara. 1956. The component analysis of the ERG from the compound eyeofCambarus. Mem. Fac Sci Kyushu Univ. Series E 2(2): 75-86. O'Day, VV. T., and H. R. Fernandez. 1974. Aristoslomias sdnlillans (Malacosteidae): a deep-sea fish with visual pigments apparently adapted to its own bioluminescence. ( 'isinn Res. 14: 545-550. Partridge, J. C., S. N. Archer, and J. N. Lythgoe. 1988. Visual pig- ments in the individual rods of deep-sea fishes. J. Comp. Physiol. 162A: 543-550. Stavenga, D. G. 1979. Pseudopupils of compound eyes. Pp. 358-435 in Handbook of Sensory Physiology, \'nl. Vll/bA. Springer-Verlag. Berlin. Stowe, S. 1980. Spectral sensitivity and retinal pigment movement in the crab, Leptograpsus variegatus (Fahridus). J. E.\p. Bio/. 87: 73- 98. Wald. G. 1968. Single and multiple visual systems in arthropods. J. Gen. Physiol. 51(2): 125-156. \\ald, G., and R. Hubbard. 1957. Visual pigment of a decapod crusta- cean: the lobster, feature 180: 278-280. Widder, E. A., M. I. Latz, and J. F. Case. 1983. Marine biolumines- cence spectra measured with an optical multichannel detection sys- tem. Biol. Bull 165: 791-810. Zar, J. H. 1974. Biostatislical Analysis. W. D. McElroy and C. P. Swanson, eds. Prentice-Hall, Englewood Cliffs. Reference: Buil Bull I'- :..her. 1988) Fiber Types in the Limb Bender Muscle of a Crab (Pachygrapsus crassipes) MICHAEL P. McDERMOTT AND PHILIP J. STEPHENS* I'illanvva L'niver\iiy. Department of Biology, \'illano\-a. Pennsylvania 1908? Abstract. The bender muscle in the walking limb of the Pacific shore crab (Pachygrapsus mnw/vo is composed of fibers with different structural (sarcomere length) and histoehemical (NADH diaphorase and myonbrillar ATPase) propenies. Slow fibers are located along the dorsal margin of the muscle and along the ventral margin in the distal portion of the muscle. The remaining bender muscle is composed of intermediate-type fibers, which can be differentiated into two groups based upon the pH sensitivity of the my otibrillar ATPase activity and the polysaccharide content of the fibers. Introduction Vertebrate motor neurons are considered to have a trophic influence on their target muscle fibers, since their physiological characteristics have a profound effect on the physiological properties and structural integrity of the muscle (Guth. 1968: Gutmann. 1976). Altering the firing activ ity or changing the motor supply can alter the contraction speed (Bullcr r/ al.. I960: Buller and Lewis, iw-x Lomo el al.. 1974: Luff. 1975). calcium uptake (Sreter <•/c.s) were obtained from the Pacific Bioniaimc laboratories. Venice. California, and were CRAB BENDER MUSCLE FIBER TYPES 285 kept in the laboratory at 24°C. The animals were kept individually, and feeding (Purina rabbit chow) and sub- sequent seawater changes were performed every 2 to 3 days. Observations were made using autotomized, sec- ond and third walking limbs removed from crabs that had been acclimated to laboratory conditions for at least 4 weeks. Certain histochemical properties were examined in frozen sections of the bender muscle. The cuticle of the carpus was reduced in thickness with a dental drill. The stretcher muscle was removed, and the bender muscle plus the remaining thin cuticle were mounted on a chuck in Histo Prep (Fisher Scientific) and immersed in liquid nitrogen; the tissue was allowed to equilibrate to — 25°C in the cryostat. Sections (20 ^m thick) were mounted on glass slides and air dried for 1 5 to 60 min. Sections were stained for activity of the mitochondria! enzyme nicotinamide dehydrogenase (NADH) diapho- rase using the method of Ogonowski and Lang (1979), and for calcium-activated myofibrillar adenosine tri- phosphatase ( ATPase) activity with acidic or basic prein- cubation, using a modified procedure of Padykula and Herman (1955). Acidic preincubation was performed in a solution of 100 mAI KC1 and 100 mM Na-acetate at pH 5.0 for 10 min at room temperature. Preliminary ex- periments using pH 4.6 pre-incubation (Maier ct ai. 1984) did not produce differentiation of muscle fibers in sections of the bender muscle. Alkaline preincubation was carried out in a solution of 1 8 mM CaCl2 and 25 mM Na barbitol at pH 9.4 for 5 min, then transferred to the same solution containing 0.05% mercaptoethanol for 20 s. For both preincubation regimes, myofibrillar ATPase activity was determined as described by Maier ct a/. ( 1984). Sections were dehydrated in a graded series of ethanol. cleared in xylene, and mounted in Permount. The polysaccharide content of bender muscle fibers was determined in unfixed, frozen sections using the method of Lillie and Fullman (1976); no counterstain was used. Sarcomere length measurements were made from sin- gle bender muscle fibers fixed at resting length (O'Con- nor el a/., 1982). The cuticle over the stretcher muscle was removed. The underlying stretcher muscle, limb nerve, and connective tissue layer were carefully re- moved to expose the bender muscle. The preparation was bathed in a calcium-free, high magnesium crab sa- line for 1 h in an attempt to reduce muscle contraction during fixation. The carpus-propus joint was positioned so that the bender muscle was stretched and the prepara- tion was immersed in Bouin's fixative for 36 h. The ben- der muscle was removed from the carpus and stored in 70% ethanol. Sarcomere length measurements were made from sin- gle muscle fibers that had been teased into myofibrils in a drop of 70% alcohol on a glass slide. The myofibrils I = 12.2 pm X = 10.4 pm X = 9.9 pm LL X = 9.0 tim SARCOMERE LENGTH (,,") Figure 1. Sarcomere length measurements of 25 fibers removed from each quadrant of the bender muscle (inset diagram). were examined under Nomarski optics and the length of five successive sarcomeres was measured using a cali- brated ocular micrometer. Measurements were made from five different myofibrils and an average sarcomere length value was calculated for the muscle fiber. Mea- surements were made for 100 fibers removed from each bender muscle. Results The bender muscle is located in the carpopodite seg- ment of the limb. In P. crassipes, the walking legs are compressed in the anterior-posterior direction, but the degree of compression of any one segment is not always constant. In the carpopodite, for example, the proximal region exhibits little compression and is essentially cylin- drical (see Fig. 2). By contrast, the distal portion of the carpopodite is compressed to form flat anterior and pos- terior surfaces (see Figs. 3D and 4D). The inset of Figure 1 shows the orientation of the carpopodite. The limb is viewed from the anterior surface and is connected proxi- mally to the meropodite and distally to the propodite. The other two surface are called dorsal and ventral. Measurements made from 100 fibers removed from different areas in the bender muscle revealed an average sarcomere length of about 10.5 ^m, with a range of 4 to 16 nm. In one preparation, the bender muscle was di- vided into four regions and measurements were made from each quadrant (Fig. 1 — inset). The proximal-dorsal quadrant had fibers with the longest mean sarcomere 286 M. P. McDFRMOTT AND P. J. STFPIII \S A d B . \ 'mure 2. Histochcmical properties of 'tin- bender muscle. Semi xec- lions stained for NADU diaphorasc activity (A), myofihrillar A I Paxc activity with alkilme (Bl and acid (C) preincuhation, and for polysac- charide content (D). a: anterior: d: dorsal: p: posterior: \ : ventral. Cali- bration: 500 ^m (A-C) and 4 1 5 ^m 1 1) i length (12.2 ^m: range 8 to 16 /jin). The distal-dorsal quadrant contained a similar population of long-sarco- mere fibers but also had some fibers with shorter (4 to 5 Mm) sarcomeres. Fibers located on the ventral surface of the bender muscle had sarcomeres between 4 and 16 ^m; the distal-ventral quadrant had a higher proportion of shorter-sarcomere (5 to 8 pm) fibers (Fig. 1 ). Sections of the dissected carpopoditc revealed that the bender muscle extends along the entire anterior surface and along some of the posterior surface of the segment (Fig. 2). The smaller stretcher muscle (which was re- moved prior to sectioning) is located along the posterior surface of the carpi ipodite and is confined to the ventral and central portion ol'the segment. Bender muscle fibers can he differentiated In their contrasting histochcmical properties. I his can be seen in Figure 2. which shows four serial sections ol'the bender muscle taken at a level about one-third from the proxi- mal end of the carpopodite. There is a population of muscle fibers that displays high NADH diaphorase activ- ity, as seen by dark rings around the margin of individual fibers (Fig. 2A). In all preparations (n = 16). these types of fibers were located in the dorsal half of the muscle, on either side of the apodeme. With regard to myofibrillar ATPase activity, these same libers stained poorly with alkaline preincubation (Fig. 2B) but well with acidic pre- incubation (Fig. 2C). Finally, these fibers had a high polysaccharide content (Fig. 2D). Most of the other fibers in the bender muscle showed poor NADH diaphorase activity (Fig. 2A) and high alka- line myotibrillar ATPase activity (Fig. 2B). In all prepa- rations (n = 16). staining for myotibrillar ATPase activ- ity with acidic preincubation revealed that these fibers are not a homogeneous group. In Figure 2C there is one group of lighter staining fibers located towards the center of the muscle, and a second group of darker staining fi- bers around the peripheral margins of the muscle. A sim- ilar difference was also seen in sections stained for poly- saccharide content (Fig. 2D). Fibers in the central por- tion of the muscle had a level of staining that was intermediate between fibers located more dorsally (dark staining) and fibers on the ventral margin of the muscle (light staining). Finally, in all preparations there were al- ways some fibers that exhibited poor myofibrillar ATPase (at both pH levels) and NADH diaphorase activ- ities (Fig. 2 — arrows). It is apparent that polysaccharide content may be cor- related with the histochemical properties of most fibers in the bender muscle (Fig. 2). In all preparations (n = 16) fibers with high polysaccharide content (Fig. 2D) stained well for NADH diaphorase (Fig. 2 A) and had high acid myofibrillar ATPase activity (Fig. 2C). Fibers with lower polysaccharide content (Fig. 2D) stained well for alkaline myofibrillar ATPase activity (Fig. 2B) and poorly for NADH diaphorase (Fig. 2 A). We have used this correla- tion between fiber-type and polysaccharide content to differentiate liber-types in the different regions of the bender muscle (Fig. 3); these observations were con- firmed by examination of adjacent sections stained for NADH diaphorase activity (Fig. 4) and for myofibrillar ATPase activity with acid and alkaline pre-incubation ( micrographs not shown). Figures 1 and 4 each show four sections taken at different levels of the bender muscle; the inset numbers represent the level of the section of the carpopodite, with proximal as 0 and distal as 100. In the proximal portion of the muscle, fibers with high polysaccharide content and high NADH diaphorase activity form a "V-shaped wedge" around the apodeme in the dorsal portion of the muscle (Figs. 3A, B. and 4A. B). Intermediate staining libers are located on either side ol'the "wedge," while poorly staining fibers arc located mostly ventrally in the bender muscle (Fig. 3A. B). CRAB BENDER MUSCLE FIBER TYPES 287 8 B 32 -% \ : 68 \ I \ v Figure 3. Polysaccharide content of bender muscle fibers. The inset numbers represent the level of sectioning in the muscle. 0 represents proximal. 100 represents distal, a: anterior, d: dorsal; p: posterior; \: ventral. Calibration: 500 fim. In the distal half of the bender muscle, fibers with high polysaccharide content and high NADH diaphorase ac- tivity are located on the dorsal and ventral margins of the bender muscle (Figs. 3C, D, and 4C, D). Intermediate staining fibers are located mostly anteriorly, while poorly staining fibers are found mostly in the posterior-central portion of the bender muscle (Fig. 3C, D). The above staining profiles were consistent in all preparations (n= 16). Discussion The present study shows that the bender muscle in the walking limbs of P. crassipes is not composed of a uni- form population of fibers. In other crustacean muscles a correlation has been found between fiber type, sarco- mere length, NADH diaphorase activity and myofibrillar ATPase activity (Atwood, 1973; Ogonowski and Lang: 1979; Tseetul.. 1983; Maierrf at., 1984; Stephens?? ai. 1985). Slow muscle fibers tend to have long sarcomeres (>10 Mm), high NADH diaphorase activity, and high acid myofibrillar ATPase activity. In the proximal por- tion of the bender, fibers with long sarcomere lengths were found predominantly in the dorsal half of the mus- cle. The presumption that these fibers are slow is sup- ported by the histochemical data, since there is a distinct population of muscle fibers with high NADH diaphorase activity and acid myofibrillar ATPase activity in this re- gion (Fig. 2A, C). In the distal region of the muscle, how- ever, presumptive slow fibers were concentrated in the dorsal, ventral and anterior portions of the muscle (Fig. 4C, D). It is interesting that NADH diaphorase activity in individual slow muscle fibers is confined to the periph- eral margin (Figs. 2A and 4). A similar observation has been made in the tails of lobster (Ogonowski and Lang, 1979) and crab (Stephens el a/., 1985) and indicates that B 32 /' L i t m ' Figure 4. NADH diaphorase activity of bender muscle fibers. The inset numbers represent the level of sectioning in the muscle. 0 repre- sents proximal, 100 represents distal, a: anterior, d: dorsal; p: posterior, v: ventral. Calibration: 500 ^m. 288 M P. McDERMOTT AND P. J. STEPHENS the mitochondria containing this enzyme are concen- trated in the peripheral margins of the slow fibers. In man> crustac. an muscles, last fibers have short sar- comeres (<4 < \ \l >1 1 diaphorase activity and high alkali hrillar ATPase activity. Ourobsena- tions revealed no sarcomcres less than 4 ^m in length (Fig. I), indicating that the bender muscle contains no fast fibers The remaining fibers in the bender muscle are therefore assumed to be intermediate-type fibers. The in- termediate-type fibers appear to be concentrated Neu- trally in the proximal portion of the muscle (Fig. 2\. and posteriorly in the distal portion of the muscle (Figs. 3 and 4). In the present study we have found that the pohsac- charide content is highest in slow fibers(Fig. 2). A similar correlation has been made between polysaccharide con- tent and fiber-type in crab swimming muscles (Tse ci a/.. 1983). Furthermore, based on polysaccharide content there appear to be t\\ o t\ pes of intermediate fibers. More- o\er. the distribution of the fibers \\ith different polysac- charide content appears to be in line with those that exhibit contrasting acid ATPase activity (Figs. 2C. D). Fibers adjacent to the slow fibers have higher polysaccha- ride content and have a lighter staining profile for ATPase activity. Paradoxically, in some sections there were one or more fibers with intermediate polysaccha- ride content, and low NADH diaphorase and myotibril- lar ATPase activities (Fig. 2 — arrows). We have not been able to typify these fibers. It may be argued that the polysaccharide content of the different fibers was influenced by the dissection pro- cedure prior to freezing of the tissue. The dissection may have selectively stimulated one axon so that the inner- vated fibers could have been actuated and thus eould have decreased or depleted their polysaccharide stores. To examine this possibility, we froze several limbs imme- diately after autotomy. Although the presence of cuticle decreased the i|iialit> of the sections, no differences be- tween the staining profiles of these sections and those from dissected preparations were observed ( McDermott. unpub. observations). We conclude thai the difference in the polysaccharide content of the various muscle libers is not artifact. Acknowledgments This wniL \\as funded by a grant from the Research Corpoiaii. I'vhnical assistance of Ms. I omse di- Cola isgratclui I nowledged. I ilcraturc (lied \(«(xid. II. I.. 1973. An .iii' n:;'i 1. 1. mount for the diversity oU msta- ccan muscle. Am. /.mil 1.1: MHIMM), II. I... and.l. M. \\ojlim kv I')S6. Short-term and long-term plasticity and physiological differentiation of crustacean motor syn- apses. Int. «ev \eunthiol. 28: 275-36 1 . H.n.uiv M.. and R. I. Close. 1971. I lie transformation of myosin in cross-in nervated rat muscles. J. Physiol. 213: 455-474. Buller. A.. I ..and I). M. Lewis. 1965. I urther observations on mam- malian cross-innervau\l skeletal muscle. J. Physiol. 178: 343-358. Buller. A. .).. J. C. Kccles, and R. M. Eccles. I960. Interaction be- tween motor neurons and muscles in respect to the characteristic speed of their response. ./. Phyuol 150:417-439. Buller. A. .1., \V. F. II. M. Mninmaerts. and K. Seraydarian. 1969. l:n/ymic properties of myosin in fast and slow twitch mus- cle of the cat following cross-reinnervation. / Phvsiol 205: 581- 597. (•nth. 1.. 1968. Trophic influences of nerve on muscle. Physiol Rev 48: 645 i (.nun. HIM. P. V. 1976. Neurotrophic relations, .-inn. Rev. 1'hynnl 38: 177-216 Lang, I-.. M. M. Ogunnwski, \V. J. Costello. R. Mill, B. Roehrig, K. Kent, and .). J. Sellars. 1980. \eurotrophic influence on lobster skeletal muscle. .S'< vivirr 207: 325-327. l.nenieka. (,. A., and II. I.. AtHood. 1985. Age-dependent long-term adaptation of crayfish phasic motor axon synapses to altered activ- its../ Armv-v/ 5:459-467. l.nenieka, (.,. \., II. I.. AlHood, and I . Marin. 1986. Morphological transformation ol synaptic terminals of a phasic motoneuron by long-term tonic stimulation . ./ V'/I/HM; 6:2252-2258. l.illie, R. 1)., and II. M. Pullman. 1976. lli\t«i\uh\;if Technic and I'rtiftu al III v/i'i hcnii\ir\4i\\ Ed. McGraw-Hill. New York. Lomo. I '., R. 1 1. \\ fsigaard, and II. A. Dahl. 1974. Contractile prop- erties of muscle: control by pattern of muscle activity in the rat. Proi K Soi /<"/kiila.ll. \..and \.Mermann.l955. Factors affecting the activity of adenosine tnpliosphate and other pliosphotascs as measured by histochemical techniques. J llniinlii'm Cytochem.3: 161-169. Pahapill. P. A.. (;. A. l.nenieka, and II. I.. Atwood. 1986. Neuronal experience modifies s\ naptic loni'.-teim tacihtation. ( 'tin .1 I'hysiol. l'htirmtit-,'1 64: 1052-1(154. IVlli-. I). 1984. ActiMty-iiuluced last to slow transitions in mamma- lian muscle. Mctl .Si / S/>i'i/\ / \ci\ i\c 16: 5I7-52S. Sreler. K. A., A. K. l.ulf, and .(.(iergely. 1975. I llect ol cross-reinner- \ at ion un physiological parameters and on properties of myosin and sarcoplasmic feticiilnni nl last and slow muscles of the rabbit. ./ Gen Physiol 66: si i 821 Stephens, I*. .1., .1. M. l.efennieh, and P. Klainer. 1985. \ciiionnisiiil.ip u-l.iiionships in the abdomen of the Californian shoie ciab /',/, :n grapsiu c rtixxi/wx. ./. Newobiol. 16: 127-136. 1st'. I . \\ ,,C. K.(; ama. 1984b) to document that insects can actively and selecti\el> control the bulk How of gases through the spiracles. The homeostatic control of hemo- coehc pressure on tracheal ventilation appeared to be elfccted by a novel, brain-independent, cholinergic cir- cuitry with the centers located in thoracic ganglia of the ventral nerve cord (Slama el ul , 1979; Slama. 1986). Such an autonomic. parasympathetic-like nervous sys- tem has recently been found in various species and devel- opmental stages of insects. It has been called the coelo- pulse system (Slama. 1988b). Encouraged by these find- ings I have worked since 1979 on a method that could displa> the dynamics of respiration through the spiracles. This paper describes some initial results obtained with a microancmometric network that can monitor over pro- longed periods the gas flow through one or more spira- cles. Material and Methods The pupae of all the investigated species were obtained from our laboratory cultures. Larvae of Act/as xek'nc were fed fresh Rhododendron v/>. leaves. Eggs were pur- chased from dealers, and the pupae were kept and mea- sured at 25°C. Spliin.\ lixiiMn larvae were fed fresh leaves of I.i.uiiMnon vulture outdoors in September. Diapaus- ing pupae were stored at 5°C: measurements were made at room temperature (24-25°C). Ilyaltiplioru cecropia larvae were reared on willow (\ are caused bv contraction-- ol ihe intcrsegmcntal ahdi>minal muscles. the hear; perates independently on two different frequeni:c^ and causes li)()-times smaller changes in hemocoelic pressure. Figures 2A and B slum \entilatory functions of three selected spiracles during the terminal pan of one extra- cardiac pulsation. The Kmcst trace is an auxiliary record obtained from the isotonic transducer attached to the tip of the abdomen. This record shows the frequency of the extracardiac hemocoelic pulsation to varv trom 26 to 18 strokes per min. and the amplitude of the associated ab- dominal movements from 5 to 1 5 /*m. Another instruc- tive feature of the record is the intervals when all spira- cles are hermetically sealed (indicated by hori/ontal lines at the bottom of Fig. 2A and B). During this time, hemo- coelic pressure decreases and the abdomen contracts with constant velocity of 84-90 ftm • min '.which is pro- portional to the O: consumption rate. A further charac- teristic of the record is large inspirations of air (indicated by triangles). These are connected with a sudden increase of internal body \olunic. elevation of the subatmo- spheric hemocoelic pressure and. finally, with sudden elongation of the abdomen. This has been recorded bv the transducer in Figures 2A and B. The above relationships suggest that the record from the isotonic transducer can re\eal main details related to the respiration dynamics of the investigated pupa. However, it does not show which of the spiracles was functioning. This information is partly provided by the anemometric records in Figures 2A and B. The spiracles on the third abdominal segment are most frequently used in this species. Kadi of the spiracles measured can function independently. Thus, the left thoracic spiracle (TL) was hermetically closed at the beginning of record- ing, it was slightly opened but held considerably con- Strii led between the third and fifth min. and it opened after both 3A spiracles were lightly closet! (Fig. 2B). This experiment does not determine how much the thoracic spiracular vahcs were opened during the maximum am- plitude of the anemometric responses. It is also unknown whether som of the remaining intact spiracles would flutter at the same time. Nevertheless, the amplitudes showing the movement of up to +0.5 n\ of air across the spiracular valv erj stroke of the intersegmental muscles (I it's 2A, B) provide clear experimental evi- dence that hcni' • ils.itions may indeed cause a very efficient trachea I ventilation. The comparison of anemometric records Irom the right and left 3A spiracles documents that each spiracle Fit-lire 2 A. l,-im\ v'/cvic. midwav during the pupal-adult transfor- malion. Example of the selective recording of inspirations (I) and expi- rations (!•) I'rom three spiracles CM. -left prothoracic. 3 AI -left and 3 AR-right 'ill abdominal I during the second half-period of an extracar- duc pulsation. Each of ihe three anemometric channels had one "ac- tive" thermistor connected with spiracle while the other was free. Lower trace shows relative changes of internal volume and hemocoelic pressure, recorded indirect!) v la an isotonic transducer attached to the tip of the abdomen. The heav > black lines on the bottom indicate peri- ods when all spiracles arc hernieticallv closed; triangles show larger in- spirations of air can instantly open or close in full synchronization with individual strokes of the hemocoelic "hydraulic bel- lows." I he frequency of 0.3 to 0.5 H/ (one stroke in 2-3 s) is sufficiently low to allow such synchronization. Al- though the 3AI. and 3AR spiracles function in concert for some time, certain strokes are missing on one or an- other trace. Moreover, larger inspirations of 1 .0 to 1 .5 ^' of air were reali/ed selectively In a sudden 300ms flutter of the 3AR valve, while the contralateral valve remained silent I hese results show that functioning of the spiracu- lar valves is controlled by a nervous system whose func- tions are precisely coordinated with nervous control of the exlracardiac pulsations i ofdiapausing cccrojiia pupae llvn/i>[>ln>r(i ('<•< •!•< i/iid invariably enters a prolonged pu- pal diapause which persists for at least d months at room INSECT RESPIRATION 293 Figure 2B. Continuation of the recording from Figure 2A showing the terminal part of the extracardiac pulsation including ventilation of the left prothoracic spiracle. temperature. In this case, anemometric recordings were preceded by one or more days of continuous monitoring of respiratory dynamics using the isotonic transducer alone (at 25°C). This was necessary to recognize possible abnormalities in respiratory functions resulting from the attachment of spiracles to anemometric tubing. The ane- mometric technique permits unrestrained movement of air across the spiracles. However, the records occasion- ally signaled suffocation or incomplete CO2 ventilation after prolonged anemometric recordings. Usually this was recognized by supernumerary, out-of-schedule pul- sations. In this case the connectors were dismantled for some time. In the majority of diapausing Cecropia pupae (15 specimens; some of them measured several times) there were regular bursts of CO2 release at 5 to 7 h inter- vals. In addition, there were brief expiratory outbursts of intratracheal gases associated with abdominal rotation once per 12-16 h. Figure 3 shows a typical sample of the combined ten- sio-anemometric recording during the interburst period. The lower trace from the isotonic transducer reveals rela- tive changes of internal body volume. It shows that the spiracles were hermetically sealed most of the time (inter- nal pressure was subatmospheric throughout). The clo- sure is indicated by the periods when the abdomen re- tracts due to decreasing pressure with a constant speed of 3 ^m per min. Volumetric calibration of this pupa under water revealed that 1 ^m of abdominal contraction was equivalent to 240 nl of internal volume. Thus, isotonic transducer can be used as a rapid and simple detector of O: consumption rate. The constant rate of 3 /urn • min~' of abdominal retraction in Figure 3 corresponded to O2 consumption of 720 nl-min~' (43.2 ^il O2-h '). The diapausing Cecropia pupae maintain subatmo- spheric hemocoelic pressure during the whole interburst period. Except for the CO2 burst, they perform mechani- cal expiration only during a brief rotational response once per 12-16 h. In such prolonged inspirations, which is very common in all diapausing lepidopteran pupae, the anemometric network can be used for simultaneous recording from eight spiracles, as shown in Figure 3. In the upper part we find four anemometric traces corre- sponding to four pairs of eight reciprocal gates. Each trace is thus common to two spiracles whose inspirations are displayed in the opposite directions (see arrows in Figure 3. Hyalnphoru cccropia. diapausing male pupa during the interburst period (25°C). Recording of inspirations from eight spiracles (T-thoracic. AL-left AR-right abdominal spiracles). Note that in con- trast to single gate operation as shown in Figure 2, the two reciprocal gates of each channel record here inspirations in the opposite direction from the midline (see arrows). Lower trace from the isotonic transducer reveals intervals and magnitude of all inspirations, i.e., sudden abdomi- nal elongation. Hermetical closure of spiracles between inspirations is manifested by a steady upward movement of the pen driver (abdominal contraction due to decrease of hemocoelic pressure). 294 K s| \\| \ Fig. 3). The intcr\als anJ magnitudes of all inspirations in the body can K- i on lower trace from the iso- tonic transducer. plained aho\e. \ccordmgly. any sudden expirati> >i mtratracheal gas. if present, should be recogm by instantaneous abdominal con- traction. « ;\issi\e inspirations are caused by ab- dominal c!i :IL .ition (for more details see Slama. 19,x4a). ire 3 shows that the pupa used only two spiracles tor periodic inspirations during the interburst period. , i second and fourth left abdominal. Their function was coordinated with an accuracy of a few ms. With val- ues of internal pressure ranging from —300 Pa to —2 kPa, the 2 \L spiracle showed larger inspirations up to 200 nl of air. while the 4 AL gave smaller and more variable responses of 20 to 50 nl at a time. This suggests that the aperture of each spiracle can be individually controlled. The general respiratory pattern of this pupa was that air was taken in discontinuous!) in sudden gulps lasting only 100-200 ms at more or less regular intervals of 3 to 4 per min. The intervals between inspirations could be prolonged by decreasing ambient temperature. For ex- ample. at 1 5°C the intervals were approximately twice as long as at 25°C. Near the CO; burst period, the passive respiratory movements often disappeared from the rec- ords though hemocoelic pressure remained slightly be- low barometric level. Gentle touching of the surface (causing small volumetric changes within the pupal body) always evoked an immediate anemometric re- sponse in one or both thoracic spiracles. This suggests that some spiracles can he maintained constricted, allow- ing a constant inflow of 720 nl-min ' of air into the bod. \hinwment d ( (>• IT ii pupae Figure 4 shows the anemometric responses during the whole period of CO: burst in the same pupa used in Fig- ure 3. The bottom trace from the isotonic transducer shows rather delicate cxtracardiac hemocoelic pulsation with an amplitude of only about I ^m of abdominal movement and a frequency of 21-23 strokes per min. In principle, the movement of flexible abdominal segments acts bellows-like on the tracheae and produces the inflow or outflow of gas through any open spiracle. The ampli- tudes D| the anemometric responses are directly propor- tional to the aperture of the spiracular valve. For exam- ple, a completely closed spiracle gives no response, a partly constricted in • imcs an intermediate response. and a fully op> u le should give the maximum response. In additim plitudcs of the individual ane- mometric responses are -ncdby an increasing num- ber of spiracles that open smmiiancously . Some of the ahme outlined relationships are illus- trated by the uppci traces in I igmc I I he "nonselec- tive" variant of the anemometric recording and slow- chart speed do not show which of the two spiracle mates on each channel have actually responded. The arrange- ment of the pairs of spiracles shown in Figures 3 and 4 was made after the foregoing finding that the two spira- cles of a pair did not function at the same time. Figure 4 shows that some spiracles, such as 7 AR and 4 AR re- mained closed throughout almost the entire period of the CO: burst. It also shows that the "master spiracles" from Figure 3 (2 AL and 4 AL) were probably functional dur- ing the initial half-period of the burst, whereas a larger thoracic spiracle (TR) opened at the end of the burst. Different amplitudes of the anemometric responses and permanent closure of some spiracles suggest that the spi- racles cannot he maintained widely opened by high CO: concentration during the burst, as has been generally be- lieved. In reality, some spiracles can be selectively venti- lated at different sites and at determined periods of the CO; burst. The records of other burst periods in this and other pupae indicated that the pattern in Figure 4 is vari- able. This suggests that alteration of the spiracle opening sequence is under non-stereotyped physiological control. Pupae of Cecropia and some other saturniids usually close all spiracles for 10 to 20 min after termination of a ("O: burst, when internal pressure is close to atmospheric level. During this period, abdominal segments retract with the velocity of 5 to 12 /nin-min ' and hemocoelic pressure declines to —5 kPa or less. Such a large vacuum inside the body becomes sequentially reduced to the usual values by large inspirations of air. most frequently through thoracic or last abdominal plus thoracic spira- cles. Sometimes more than 20 n\ of air are taken in a single surge and sometimes more than 0.5 ml of air is taken in during one min. The process continues until hemocoelic pressure becomes adjusted to about -500 Pa. This is followed by the type of respiration as shown in Figure 3. Re.\i>iniii<»i ofdiapausing Sphinx ligustri Sphingid pupae typically show regular periods of in- spirations that give the records of hemocoelic pressure a saw-tooth appearance. The records from isotonic trans- ducers are mirror images of changes in hemocoelic pres- sure (see Slama. !9X4a). i.e.. the abdomen contracts slowly when hemocoelic pressure passively decreases. The bottom trace in Figure 5 shows the saw-tooth pat- tern in S[>/iin\ HitiiMri. The teeth indicate intervals and size of the inspirations. A further peculiarity of sphingid pupae is the slowly expanding internal volume (visible as the slow decline tendency of the lower trace in Fig. 5). This is terminated once per several hours by a large expi- ration associated w nh abdominal rotation or. eventually . bv a CO, burst. I Kelul information from this record are INSECT RESPIRATION 295 10 12 MINUTES Figure 4. Hyalophora cecmpia. the same preparation as in Figure 3. recording from eight spiracles during the 20-min period of CO: burst associated with an extracardiac pulsation (25°C). Each of the four anemometric traces gives unresolved responses from four pairs of spiracles (arrows indicate inspirations in the particular spiracles, but they may be expirations in the respective counterparts). The amplitudes of the anemometric responses are proportional to the degree of opening of the spiracular valves. Lower trace shows the bellows-like ventilatory movements of the distal abdominal segment. more or less regular intervals of inspirations at 30 s, and the velocity of the constant abdominal contraction of 2.5 Mm • min ' . Calibration of the pupa under water revealed that 1 /urn of abdominal contraction was equivalent to 1 10 nl of air transported through the spiracle or 1 10 nl of O: consumed (O: consumption of 16.5 ^1-rT1). 296 K. SLAMA 200 200- 2AL TR 2AR 7AR 4AR 7AL 4AL , z g 5< ± • §2 £ a. OL 10 12 16 18 20 22 MINUTES 1-inure 5. Spliiin livimri. diapausmg pupa. A sample from prolonged recordings between the CO: bursts during the period of discontinuous inspirations of air. Lower trace shows the characteristic "saw- tooth" pattern ol hcmocoelic pressure changes revealed indirectly by an isolonic transducer Irom the tip nl the abdomen. Inspirations are indicated b\ sudden abdominal prolongation (sudden decrease of the internal vacuum) I he anemometric traces slum inspirations in eight spiracles indicated h> the arrows Note that only 4 Al and 2 \R spiracles were used for inspirations, the I R spiracle was used only twice. while the rest of spiiailcs were hermetically closed all time. The anemometric traces (Fig. 5) document that this pupa also used in: .'nations through selected abdominal spiracles. Inspirali. m red only in 4 AL assisted b\ 2 AR. A sudden mspn.ition throm'h the right thoracic spiracle occurred as UK two abdominal spiracles go silent (around 13:00 min record in;1 nmci Such suilt interplay between close or more distant spiracles is quite common. Direct evidence that it \\as not expiration through the paired 2 AR spiracle is provided by the tensiometric rec- ord below, which shows abdominal elongation due to volumetric increase, not contraction. The pattern ot inspirations in Figure 5 shows that the "master" and assisting spiracles taking part in discontin- uous air intake are not onlv located on dillerent body INSECT RESPIRATION 297 segments, but can occur on contralateral sides. More- over, the two functioning spiracles opened for a total of only 7 s of 22 min, while all other spiracles were hermeti- cally closed. This pattern when all spiracles are closed while only two of them would flutter for no more than 0.53 per cent of time seems to be a common feature in diapausing pupae of Lepidoptera. It provides a serious argument against the belief that the pupa could breathe by simple diffusion of respiratory gases through spiracles. The situation in Fig. 5 cannot be taken as a stereotypic model for all pupae of a species. Other pupae ofS. ligus- tri did not use 4 AL as the most active spiracle. Some used preferentially TR, 3 AL, or 7 AL. The tobacco hornwonn Manduca sexta Large sphingid pupae (Acheronlia atropos. Herse con- volvuli. Manduca sex/a) show very special respiratory scenarios during diapause. They maintain an internal vacuum and tend to use a single "master" spiracle for prolonged discontinuous inspirations, while all other spi- racles are tightly closed. This often continues unaltered for periods of more than 1 5 h at room temperature or for several days at 5°C. Figure 6 shows a 24-min segment taken from an uninterrupted 48-h recording in diapaus- ing Manduca. The bottom trace comes from the isotonic transducer. It shows regular inspirations at 1 .5 to 2 min intervals. The inspirations have been associated with sudden increases of internal body volume which is mani- fested on the record by sudden elongations of the abdo- men. After termination of the anemometric measure- ments, the hemocoel cavity of this pupa was connected with the hydraulic transducer for calibration of the sys- tem. The bottom trace in Figure 5 appeared as a mirror image of changes in hemocoelic pressure (for more de- tails see Slama. 1984a). The records in Fig. 6 show that, of eight spiracles, the pupa inspired only through the left thoracic spiracle. All others were kept hermetically closed. Each inspiration lasted approximately 200 ms, the anemometer detected a rapid flow of 600 nl of air. Further measurements on this and other diapausing Manduca pupae revealed im- portant biophysical data that can be summarized as fol- lows: (a) the velocity of the steady abdominal contraction is 2 nm • min '; (b) 1 ^m of abdominal movement corre- sponds to 2.9 Pa change in hemocoelic pressure; (c) 1 urn of abdominal movement is equivalent to 150 nl of air inspired or O2 consumed, and (d) the baseline hemo- coelic pressure is 0.8 kPa under atmospheric level. The above data reveal a hitherto unknown homeo- static mechanism. This mechanism regulates a constant body length within the limits of ±5 ^m (i.e., l/10000th of pupal length), maintains more or less constant body volume within the limits of ±750 nl (i.e., 1/1 3000th of body volume), or regulates hemocoelic pressure within the limits of ±14. 5 Pa (i.e.. 1.4mm hydrostatic pressure). This illustrates remarkable accuracy in the underlying sensory and neurophysiological mechanisms. Extensive anemometric studies with diapausing pupae of various lepidoptera consistently revealed subatmo- spheric hemocoelic pressures and predominantly closed tracheal systems. Air was mechanically sucked into the body whenever some spiracle opened while mechanical expiration was unusually rare. In certain cases, as in the pupa of Manduca. body volume remained constant for many hours in spite of a well-documented net inflow of air into the otherwise hermetically sealed pupal body. This effective nitrogen concentration within the closed pupal case has been studied intensively. Details will be described elsewhere. Discussion The mechanics of insect respiration and tracheal ven- tilation were studied some 80 years ago (for review see Babak, 1912). About 30 years ago, the discontinuous res- piration of insects became a favorite subject of insect physiology (Punt, 1950; Schneiderman and Williams, 1955; Buck, 1962; Keister and Buck, 1964). Though it still is a favorite subject of more recent reviews (Miller, 1981; Kestler, 1985), few additional insights have been added since Schneiderman and his co-workers denned changes in intratracheal pressure, described the micro- cycles of suction respiration, and explained the basic mechanisms of spiracular functions (Levy and Schnei- derman, 1966; Brockway and Schneiderman, 1967; Burkett and Schneiderman, 1966). The present study stems directly from these publications and confirms most, though not all, of the conclusions. Information is available on neuromuscular control of spiracular functions and ventilatory movements (review by Miller, 1981). Anemometric techniques, combined with the direct or indirect detectors of hemocoelic pres- sure, now permit monitoring the actual passage of respi- ratory gases through individual spiracles. These tech- niques, in combination with very sensitive microrespiro- graphic methods (see Slama. 1984b). help to obtain simultaneous monitoring of the course of O2 consump- tion, internal volume, and hemocoelic pressure changes and, most importantly, monitoring of the functioning spiracles (for more technical details see Slama, 1984a, 1984b, 1988a). August Krogh's pioneering work led to a theory of purely diffusive gas transfer within the tracheal system (Krogh, 1920). This model was subsequently corrobo- rated by Weiss-Fogh (1964) who concluded that there were no reasons to assume mechanisms for insect respi- ration other than simple gaseous diffusion. The model 298 K SI \\l \ 822 411 2 o- 411 - c 822- TR 2AL Tl u < Z o o 7AL 4AL 7AR 4AR 2AR 50 j 10 12 M I N 14 16 18 20 22 24 Figure 6. Maiuhua \c\la, diapausing t'emale pupa. Recording of regular inspirations from eight spira- cles during the interhurst period. Onl> left prothoracic spiracle was functional while all others were tightly sealed. The bottom trace from the isotonic transducer displays the associated pressoric or volumetric changes from movement of the tip of the abdomen. was thoroughly analyzed by Buck (1962) and recently updated by Kestler (1985). The most important chal- lenge to the diffusion theory has hitherto been the results of Hazelhoff. The latter's work is known mainly from the description of F'rof. H. Jordan (see Jordan. 1 927). Hazel- hoff found that insect spiracles were completely closed most of the time — a finding that few have recognized but which is fully confirmed in the present study. I he ditlusion theory of insecl respiration was ques- tioned in various review articles (Chauvin. 1 949: Kuz- netzoff, 195.V. Buck. 1962: Miller. 1981: Kestler, 1985). but the critiques lacked experimental data. The original reasonings of Kmgh ( 1920) supporting the purely diffu- sive made ot insect respiration were based on the premise that immobile sta;,'< •, nl insects did not show ventilators movements. H". we see (I igs. 1A. B and 3) that immobile pupae do e-.lnbit minute hemocoelic pulsa- tions. These result in active inn. heal \entilation. Buck (1962) postulated that a mtuective stream of intratra- cheal gas can be achieved In extrcmelv small changes in internal pressure. Indeed, the changes in mechanical pressure associated with the described pulsations are so small (sometimes less than 5 Pa or less than 1 ^m ofin- tegumental movement) that they can be visualized only via recently available electronic devices. This may ex- plain wh\ the existence of extracardiac pulsations in hemocoelic pressure remained unknown until 1976 (Slama, 1976). Recent investigations show that the pul- sations are present everywhere. They occur, for example, in immobile prepupae and in pupae of all major endo- pterygote groups, including Colcoptera. I.epidoptera. Hymenoptera. and Diptera (Slama. 1984a). The wide- spread occurrence of these pulsations (ventilaton, move- ments) in the immobile stages with low metabolic rates provides strong circumstantial evidence that simple diffusion principles are not satisfactory for the transport nl r.ises through spiracles. However, the diffusion princi- ples formulated bv Krogh ( 1920) may find practical use for the internal transport of O: between tracheae and tis- sues. I Ins view is consistent with the calculations of Buck INSECT RESPIRATION 299 (1962), and of Kestler (1985), as well as with the recent views concerning respiratory functions of insect tra- cheolesfWigglesworth. 1984). To illustrate some arguments against the role of diffu- sion in the exchange of O2, N2, and CO2 between the pupae and the environment, we may discuss again the case of Mandnca in Figure 6. Here the unidirectional suction stream of air first passes through a narrow spirac- ular sieve into a cavity above the spiracular valve. The valve flutters only in the left thoracic spiracle for 100- 200 ms about once per minute. Thus it opens only for approximately 0.5% of the time. During its opening, air is propelled inside vigorously by a 0.5 to 0.8 kPa pressure difference (according to preliminary calculations, the speed of the air stream is close to 20 m-s~'). All other spiracular valves are permanently and hermetically sealed for several hours, and at lower temperatures (5- 10°C) they may be closed for several days. For obvious reasons, it is unrealistic to expect diffusion of the respira- tory gases through just one spiracle that is sealed 99.5% of time and whenever it opens there is a fast stream of air. The respiration pattern of.Munditai is not restricted to the large-sized sphingid pupae. It is quite common among diapausing pupae in a number of lepidopteran families, including miniature pupae of Geometridae. where diffusion principles in respiration would be most likely. Reasons why lepidopteran pupae must live with closed spiracles, are still unknown. According to the liter- ature (Buck, 1962; Kestler, 1985), the principal reason is water conservation. The foregoing facts strongly argue that separate zones of the tracheal system can be ventilated by selective opening of the determined spiracles. Actual ventilation is brought about by genuine pulsations in hemocoelic pressure. These are generated in the majority of insect groups by contractions of the intersegmental muscles of the abdomen. Usually utilization of O2, fixation of CO;, in buffers, and hermetical closure of the spiracles, create subatmospheric pressures which are automatically con- veyed to the gas-filled tracheae. An instantaneous inflow of fresh air occurs whenever a spiracle opens. This brief recapitulation of the observed respiratory relationships suggests that insects possess a neuromuscular mecha- nism for controlling inspirations and expirations through individual spiracles. The nervous system con- trolling opening or closing the spiracles has apparent mo- tor outflow via a nerve system regulating the interseg- mental muscles of the abdomen and, thereby, the hemo- coelic pressure. In Tenebrio, Galleria. and some other insects an auto- nomic (brain independent), parasympathetic-like ner- vous system regulating hemocoelic pressure has been de- scribed (Slamat^fl/.. 1979; Slama. 1986). More recently. this mechanism has been termed the coelopulse system (from the Greek koiloma or Latin coelom for cavity and piilsus for beating or striking). It regulates certain ho- meostatic functions in reproducing adults of various in- sect groups (Slama, 1988b). There is increasing evidence that insect respiration is regulated by the same coelopulse system that regulates hemocoelic pressure. The mecha- nism mutually determines the duration of the pressure pulsations, controls the level of the baseline hemocoelic pressure, and regulates the intervals of inspirations (see Slama, 1984a). The present anemometric data show that it may also control the function of individual spiracles. Thus, the coelopulse mechanism of insect respiration is composed of two elements: (a) neuromuscular system regulating the opening or fluttering of spiracular valves (metameric system of unpaired central and transverse nerves innervating the spiracles, with the adjacent peri- sympathetic neurohaemal organs), and (b) what may be termed "hydraulic bellows" driven by the intersegmental muscles of the abdomen with nerve impulses coming from the thoracic ganglia (generating changes in hemo- coel pressure that force the air in or out through the se- lected spiracle). These complex physiological functions can be compared to playing an accordion. There are two interconnected nerve functions: one is responsible for pulling the bellows and the other for pressing the right keys on the keyboard. We know the instrument but we must now learn to listen to the melody of different in- sects. Acknowledgments I am greatly indebted to Prof. C. M. Williams of Har- vard University, Cambridge, Massachusetts, for valuable help in preparation of this manuscript: Prof. J. B. Buck of National Institutes of Health, Bethesda, Maryland, for helpful criticisms, and Dr. J. H. Willis of The University of Illinois, Urbana, Illinois, for suggesting the title. Literature Cited Babak, E. 1912. Die Mechanik und Innervation der Atmung. Win- lerslein 's Handh. ! 'ergl. Physiol. I: 265-640. Brockway, A. F., and H. A. Schneiderman. 1967. Strain-gauge trans- ducer studies on intratracheal pressure and pupal length during dis- continuous respiration in diapausing silkworm pupae. J. Insecl Physiol. 13: 1413-1451. Buck, J. 1962. Some physical aspects of insect respiration. Annu. Rev. Enlomol. 7: 27-56. Burkett, B. N., and H. A. Schneiderman. 1974. Discontinuous respi- ration in insects at low temperatures: intratracheal pressure changes and spiracular valve behavior. Biol Bull. 147:249-310. Chauvin, R. 1949. Physiologic dcl'Insecte. 2nd ed. Paris 1958. Jordan, H. 1927. Die Regulierung der Atmung bei Insekten und Spinnen. Z \\-gl. Physiol. 5: 179-190. kaars, C. 1981. Insects — spiracle control. Pp. 337-366 in Locomo- 300 K si \\l \ lion and Energciu^ m ' • Herreid and C. R. Fourtncr, eds. Plenum. New x .1:- ndon. keislcr. M., and J. Buck. »<-4. Respiration: some exogenous and endogenous ell. piration. Pp. 617-658in ///r/'/ns/ ologyofh: Kstein.ed. kestler, P. 1985. .nul icspiratorx water loss. Pp. 137-183 in Emir. iologj ..".'\-y ti:n>li>i;ii mm'komycli (Principles n!' Insect Phvsiologx I l/d. Acad. Sei. Moscow. 402 pp. (In Russian.) 1 t'\>. R. I., and H. A. Schneidrrman. 1966. Discontinuous respira- tion in insects. IV. Changes in intratraeheal pressure during the res- pirators cycle of silkworm pupae. ./ lim\:i Physiol. 12: 465-492. Mill. P. J. 1974. Respiration: aquatic insects. Pp. 403-467 in The Plivsii>fox\ q) Insei ta, 2nd ed.. vol. 4. M. Rockstein. ed. Academic Press. New York. London. Miller. P. I.. 1974. Respiration — aerial gas transport. Pp. 345-402 in I he /Vn w.'/.ifr <>t In\cita. 2nd ed.. vol. 4. M. Rockstein. ed. Aca- demic Press. New N ork. I ondon. Miller. P. I.. 1981. Ventilation in active and inactive insects. Pp. 367-390in Locomotion and Energetics in lnlir,ii>ntl\. C. F. Herreid andC. R. Fourtner. eds. Plenum Press. New York. London. Protansul. \.. N. Baudry-Partiaoelou, and K. Slama. 1977. llacmolunph pressure pulses in the metamorphosis of l>-nchni< inuliit'i I. .'.: / Htiininl flo/itviiHv/nv 74:362-374. Punl. A. 195(1. 1 he respiration in insects. Phyvnl ( 'amp. 2: 59-74. Schiu-iderman. II. A., and ( . M. Williams. 1955. An experimental anahsis olthe discontinuous respiration of the cecropia silkworm. Ilii-l Hull 1(19: 123-143. Slama, K. 1976. Insect haemohmph pressure and its determination. .\cldi.nii\\ 73:65-75. Slama. k. !9H4a. Recording of haemolymph pressure pulsations from the insect body surface. / Cunip !'/i\v.hcitn>\lnv (in press). Slama, k. I988h. Role of the autonomic nervous system (coelopulse) in insect reproduction. 1'rm Ini Synip Inject Reproduction, Zin- kovy 1487 (in press). Slama. K., N. Baudr>-Partiaoglou. and A. Proiansal-Baude/. 1979. Control of extracardiac haemolymph pressure pulses in / , 'nebrit > m, >hi, »- 1 . ./ Inwl Pliytiol. 25: 825-83 1 . \\'eiss-Kogh, 1 . 1964. Diffusion in insect wing muscle, the most ac- tive tissue known. ./ /.' \/> liml. 41: 229-256. \\ iKHlesHiirlh, \ . B. 1984. The physiology of insect tracheoles l,l\ Insect Physiol 17:85-148. Reference: Biol. Bull. 175: 301-319. (October. 1988) Abstracts of Papers Presented at the General Scientific Meetings of the Marine Biological Laboratory August 22-24, 1988 Abstracts are arranged alphabetically hy first author within the following categories: cell biology, comparative and general physiology, developmental biology ami tenil- i-ation. ecology, ami neurobiology. Author and subject references will be found in I lie regular volume index in the December issue. Cell Biology The packing density of cytoskeletal polymers in a.\o- I'/i/sm affects the resistance to polymer sliding. AN- THONY BROWN AND RAYMOND J. LASEK (Bio-archi- tectonics Center, School of Medicine, Case Western Reserve University, Cleveland, OH 44106). In the polymer sliding hypothesis of slow axonal transport, the rate of cytoskeletal polymer sliding is partly a function of the resistive drag that the polymers encounter as they slide past adjacent structures within the axon ( Lasek 1 986. / Cell Sci. [Suppl.] 5: 1 6 1 - 1 79). A recent study of two side-by-side populations of axons that have different neu- rofilament transport rates has shown that the neurofilament density is higher in those axons with the slower rate (Price el al. 1 988. / Neurocy- lol. 17: 55-62). This suggests that an increase in polymer packing den- sity may slow the rate of polymer sliding in axons. To test this, polymer sliding was mechanically induced /;; vitro by slowly stretching axoplasm extruded from squid giant axons (George and Lasek 1986, Biol. Bull. 171: 469). The resistance to stretch was measured at various polymer packing densities. Cytoskeletal polymer density was increased by os- motically compressing axons with hypertonic sucrose solutions prior to extrusion of the axoplasm. With 1 M sucrose in seawater the mean axon volumes decreased by 63% (n = 9. minimum = 53%, maximum = 71%). Electron microscopy showed that the polymers were more densely packed. Axoplasm from these compressed axons showed a much greater resistance to polymer sliding. The mean modulus of elas- ticity, which is a measure of this resistance, was 1 3 dyn/min (minimum = 2 dyn/min, maximum = 30 dyn/min, n = 16) for untreated axoplasm and 60 dyn/min (minimum = 44 dyn/min, maximum = 77 dyn/min, n = 6) for axoplasm compressed with 1 M sucrose solutions. This indi- cates that compressing the axonal polymers more tightly together in- creases the resistive drag thai they encounter during sliding. In this way. the external compressive forces on axons that pack axonal polymers /;/ situ may increase the resistance to polymer sliding, thereby decreasing the rate of slow axonal transport. Cell fusion and cell poration using a radiofrequency elec- tric field. D. C. CHANG AND P. Q. GAO( Baylor College of Medicine). Cell fusion and cell poration are important biological techniques that have a variety of applications in cell biology, molecular biology, and immunology. Recently we have developed a new method that induces cell fusion and cell poration using a pulsed radiofrequency (RF ) electric field. This field is of high strength, typically 3-5 kV/cm. The pulse width is of the order of 100 microseconds, and the oscillating frequency varies from 50 kHz to 1 MHz. We have applied this new method to fuse and porate a number of cell types, including human red blood cells (RBC). Light microscopy using DIC optics and fluorescence micros- copy were used to study the process of cell fusion induced by the RF field. Two types of fusion were observed in RBC. The first type is a membrane fusion in which the fusing cells retain their individual shapes, but a membrane-labelling dye can pass from one cell to another. The second type is a cytoplasmic fusion in which the fusing cells merge to become a single larger cell. The yield of cytoplasmic fusion is strongly dependent on the oscillating frequency of the applied RF field, with the highest yield at 100 kHz. Analysis by video microscopy of the fusion process shows that RBC first shrink in size following the RF pulses and then partially swell before fusion takes place. This observation suggests that the RF pulses may create large membrane pores which allow rapid exchange of intra- and extracellular substances. Using freeze-fracture electron microscopy, we have observed such pores. The pore size is large (up to 0.3 ^m in diameter) so that large macromolecules such as coiled DNA can readily pass through the membrane of the porated cells. Our study suggests that the RF poration method will be highly useful for gene transfection or microinjection of biologically active sub- stances (e.g.. antibodies or molecular markers). Supported by the Advanced Research/Technology Program of Texas. Ca:+ -dependent catec/iolamine modulation of sperm movement in Arbacia punctulata. LEONARD NELSON (Medical College of Ohio Toledo, OH 43699) AND Lucio CARIELLO. Sperm motility is a quantifiable phenomenon providing a readily ac- cessible model for the analysis of complex regulatory mechanisms of cellular function. Bovine spermatozoa show saturable binding of radio- labeled norepinephrine with an affinity constant of about 0.5 nA/. Coin- cubation of sperm cells with non-labeled compounds and blocking agents causes displacement of the bound isotope. Isoproterenol. dopa- mine. and epmephrine reduced the binding to a degree which suggested 301 302 CELL BIOLOGY thai the challenging agents would cttc.-i the rate of sperm cell progres- sion. Sea urchin sperm assas adapted to lest the physiological responses revealed that Arhacia sperm •>•< ihited dose-, time-, and C 'a "'-depen- dent action. Norepinephni :crenol. and dopamine increased the motile rate h> 7! the control sperm alter 10 min of exposure, while ep: *->s without etl'ect. Receptor Mockers also induced im- ,-SMVC movement. 1 he pranolol. another ff-blocker. moderately increased the motiht> while the mildly acti\e o-adrenergic compound quinidine had noeffeci except when administered along with propranolol. Thus when d-adrenergic agonists occupy binding sites on the sperm cell surface, lhe> prevent the stimulation due to agents which act as /^-antagonists in neuromuscular systems. The o-blocker phentolamine. however, po- tentiates the action of the fJ-agonist isoproterenol. I he absence of Ca:' or the presence of( 'a '-channel blockers reduce the stimulator, effects of the catecholammes which appear to be in- volved in ion channel regulation. The catecholamines appear to inter- act with cell surface receptors that activate the adenylate cyclase/cyclic AMP second messenger system. Caffeine (an inhibitor of cAMP phos- phodiesterase) and 8-Br-cyclic AMP reduce the stimulation caused by norepinephrine and atenolol. Epinephrine also depresses the stimula- tory action of 8-Br-cyclic AMP. Support by the Sage Foundation. Initial studies of marine vertebrate lens eytoskeleton. NANCY RAFFERTV. KRIS LOWE, KEEN RAFFERTY. AND SEYMOUR ZIGMAN (University of Rochester Medical Center). The anatomical nature ol the lenses of marine vertebrates does not support a lens accommodative process that alters their shapes. Unlike the lenses of many terrestrial mammals. they are not elastic. However, accommodation is accomplished b> translocation of the lens by retrac- tor or protractor muscles in the eye I'hese lenses also resist swelling both in hypotonic media and when their Na/K ATPase is inhibited, which suggests cytoskeletal rigidity I lerein. we identify the cytoskeletal proteins in these lenses, and describe the structures of their cytoskele- tons. The lenses ol'teleosts and elasmohranchs were sunned for actin using rhodammephalloidin reagents. Morphological studies of the cytoskele- ton were aided by electron microscopic examinations by using gold labelled antibodies. Lens extracts were examined b> polyacrylamide gel electrophoresis (PAGE) with special attention to the cytoskeletal proteins, actin and vimentin. Immunoblotting was done with purified antibodies to actin and vimentin (Western blots). Exposure of the lenses in \itro to ncar-UV radiation (365 ± 30 nm: .5 mW/cnv) was used to study the induction of light-associated alter- ations of cytoskeletal elements Extracts of both dogfish and sea robin lenses were shown to contain both actin and vimentin I I'M ,1 and immunohlotting. The antibod- ies against actin and vimentin were made in rabbits and mice, respec- tively; common antigenic determinants are thus present in the cy toskel- etal elements d! marine lenses. Actin filamentsv.ru ;issouatcd with the epithelial cell plasma mem- branes and in the pcrmuclcar region. U V -exposure in dogfish appeared to cause depoly men/a i K in ol the filaments. Immunogold E.M. showed actin to be associated with the basal plasma membrane (in the dogfish! Tclcost vimentin was found to be associated with the pcrinuclear net- work. PAGE plus immuno-hl.itting resulted in bands proving that actin and vimentin were present in these lenses. Preliminary immunoblots i that some cytoskeletal element1, may comigrate with the lens crystallins. especially in the cortex Che structure and chemistry of m.i- rine lens cy toskeletons appear to be similar to those of mammals. Thus, an altered cytoskelcton cannot explain the lack of swelling and accom- modation in marine lenses. Support: MH and RPB. Inc. Isolation ol the doltish erythroeyte marginal hand using liciewnls. IVELISSE SANCHEZ AND WILLIAM D. CO- HEN (Hunter College, NY). Isolation of the marginal band (MB) of microtubules from the eryth- roc\ les of non-mammalian vertebrates is a useful approach to the mo- lecular composition and the structural and mechanical properties of the nucleated erythrocyte cytoskeletal system. In our prev lous methods for MB isolation, proteases have been used to digest the cell surface- associated cytoskeletal network (SAC), containing actin and spectrin, fodrin-like proteins, that encloses the MB. In the present work, ey- toskeletons were prepared from erythrocytes of the smooth dogfish ( l/in?i'/!n «;/m)hy lysis with Brij-58 in the presence of protease inhibi- tors, washed in microtubule stabilizing medium, and stored at -20°C in 50% (v/v) glycerol containing taxol to help stabilize the MB. This ser\ ed as the standard starting material for testing potentially selective, non-proteolytic SAC solubilizing agents systematically. Various phos- phates and reducing agents suggested by literature on the mammalian actin-spectnn network proved ineffective, as did numerous detergents used individually. However. MB release from cytoskeletons was ob- tained readily in Triton X-100 (0.1-0. 4'T I containing low concentra- tions of SDS (0.025-0. \ glycerol. I herclbre. these re- agents can be used to stop the release reaction during quantitative time- course studies. Added standard proteins are not proteolwed during MB release, nor is release blocked by a protease inhibitor "cocktail." indi- cating that activation of endogenous proteases is not involved. As deter- mined by SDS-PAGE. tubulin ( four gel bands) is the dominant compo- nent of the isolated dogfish MB preparations. Results to date with am- phibian and avian erythrocytes indicate that the method may be widely applicable to other species, and therefore useful for comparative studies. Supported by MBRS-NIH S06RR08 176-07 and NSFDCB871 1810. \s\cinhly and changes in the fibrous substructure of the cleavage Jurrow in living cells. J. M. SANGER, J. S. DOME, B. MnTAi . AND J. W. SANGER (University of Pennsylvania). We have microinjected tracer amounts of fluoreseently labeled myo- sm light chains, monomei acini, and phallotoxms into PtK; cells to observe the assembly of myosm and actin into the cleavage furrows of these cells. We selected large cells that often formed multipolar mitotic spindles and subsequently, multiple cleavage furrows that could he vis- ualized with the fluorescent probes lor myosin and acini Mitotic cells that had been injected with fluorescent myosin light chains or fluores- cent monomer actin had spindles whose fluorescence w.is brighter than that of the surrounding cytoplasm I he same was true of cells that had been iniecled with bovine serum albumin In contrast, the fluorescence m spindles of cells that had been injected with F-actin probes, fluores- cent phallotoxins. was approximately the same as in the cytoplasm ad- lacent to the spindle We interpret this to mean that soluble proteins bee, line concentrated in the milotic spindle, whereas the actin that re- mains m the I -form during mitosis is present in the spindle at the same level .is ii is in the extra-spindle cytoplasm. In late anaphase, a band of ABSTRACTS FROM MBL GENERAL MEETINGS 303 fluorescence was visible in the forming cleavage furrow of all cells that had been injected with one of the three probes for myosin and actin. but not in cells injected with bovine serum albumin. Fluorescent bands of actin and myosin formed not only between each group of separating chromosomes, but also midway between adjacent asters of multi-polar spindles. In one cell in which two mitotic spindles were oriented parallel to one another, four fluorescent bands formed: one between each of the two groups of separating chromosomes and one between each of the two pairs of adjacent asters. In a majority of exceptionally large cells, fibers were discernible in the cleavage furrows. In a few cases, the fibers appeared striated or beaded, in a manner similar to stress fibers in inter- phase cells. The fibers shortened and disassembled during cytokinesis and the tluorescently labeled proteins became localized on either side of the midbody and eventually were redistributed in the stress fibers of the daughter cells. Cytoplasmic dynein is the motor for retrograde vesicle transport in squid axons. BRUCE J. SCHNAPP (Marine Biological Laboratory, Woods Hole, MA 02543). Intracellular vesicles in axons move, without reversing, along uni- formly oriented microtubules. A soluble fraction (S2) from extruded axoplasm was previously found to promote the bidirectional move- ment of plastic beads on purified microtubules in vitro. Two microtu- bule-based translocators were identified: an anterograde (+ end di- rected) motor, kinesin. and a retrograde motor whose purification and role in vesicle transport is described here. The retrograde motor in squid axoplasm is a microtubule-associated protein previously termed HMWI (Vale el ai 1985. Cell 42: 39-50). Like cytoplasmic dynein purified from bovine brain ( Paschal etal.J. Cell Biol. 105: 1273-1282), HMWI promotes microtubule sliding on glass, shows nucleotide-de- pendent binding to microtubules, is a 22s particle, and has a heavy chain of M, > 400,000 that co-electrophoreses with axonemal dynein heavy chains. Like other dyneins, the heavy chain of HMWI is specifi- cally cleaved into two >200 kd fragments when irradiated with 254 nm light in the presence of 20 fiM vanadate and 2 m.l/ ATP, leading to inactivation of both motility and ATPase activity. UV-vanadate treat- ment of axoplasmic S2 completely blocks retrograde bead movement, indicating cytoplasmic dynein is the retrograde microtubule motor pre- viously identified in this system. Using Kl-extracted vesicles whose movement is dependent on the addition of S2, vesicle movement was 63% retrograde when assayed on centrosome microtubules in control S2 (irradiated in the absence of vanadate) and 7% retrograde in the presence of S2 exposed to 20 /iA/ vanadate during UV irradiation. In assays that quantified the absolute numbers of moving vesicles, the numbers of vesicles moving in the anterograde direction were unchanged by the UV-vanadate treatment. This indicates dynein operates specifically with a population of vesicles programmed to move toward the minus end of microtubules. Purified dynein. either alone or together with kinesin. did not promote vesicle movement, consistent with our previous finding (Schroer el ai 1988. / Cell Biol . in press) that soluble proteins, in addition to kinesin, are required for vesicle transport. Comparative and General Physiology The Limulus amebocyte contains a:-macroglobulin. PE- TER B. ARMSTRONG (U. of California, Davis), JAMES P. QUIGLEY, AND FREDERICK R. RlCKLES. Alpha;-macroglobuhn, a protease-bmding protein that is reactive with almost all endopeptidases, is present in high concentrations in the plasma of the horseshoe crab. Limulus (Quigley and Armstrong 1985, J. Biol. Chem 260: 12715). Alpha-2-macroglobulin was demonstrated by its ability to protect the active site of trypsin from inactivation by the macromolecular active site inhibitor, soybean trypsin inhibitor (Armstrong el al. 1985, J. E\p Zool. 236: 1). and by reaction with an antiserum prepared against purified Limulus «2-macroglobulin. The blood cells also contain «2-macroglobulin in a form that is released when washed cells are stimulated to undergo exocytosis by treatment with the lonophore, A23 1 87. Alpha-2-macroglobulm is detected in the materials released from the cells during degranulation both by activity in the soybean trypsin inhibitor-protection assay and by immunochem- ical staining of Western blots. The subunit molecular weight of the cell- associated c*2-macroglobulin, 185 kd, is identical to that of the plasma form. Although cells contain large quantities of the cytoplasmic marker enzyme (lactate dehydrogenase). none is released during ionophore- stimulated degranulation, indicating that cell lysis does not occur and is not responsible for the release of a2-macroglobulin. The penultimate wash buffer lacks «2-macroglobulin, demonstrating that the cells have been washed free of plasma proteins. We were unable to detect a;-mac- roglobulin in Western blots of degranulated cells, indicating that most or all of the cell-associated a2-macroglobulin is released during degran- ulation. The amount of «2-macroglobulin contained within the cells of a given volume of blood is 2-5% of the quantity free in that volume of plasma. The distilled water lysates of N-ethylmaleimide-treated amebocytes used to detect endotoxin (e.g.. Limulus amebocyte lysate or LAL) contain relatively large quantities of active a2-macroglobulm. These preparations are essentially free of the principal plasma protein, hemocyanin, indicating that the cells have been well washed prior to lysis. Supported by NIH Grant No. GM 35185. Suppression of common mode signals within the electro- sensory system of the little skate. DAVID BODZNICK (Wesleyan University) AND JOHN MONTGOMERY. Elasmobranch fishes possess an acutely sensitive electrosensory sys- tem which can detect bioelectric fields produced by other animals. However, they produce bioelectric fields themselves (e.g.. during venti- lation) which result in unwanted self-stimulation of the electrorecep- tors. Recordings from the brain show that central neurons are respon- sive to extrinsic fields, but manage to reduce the ventilatory modulation occurring in their afferent input. The likely basis of this suppression is that ventilatory stimulation is common mode to the receptors whereas extrinsic fields are differential (Montgomery 1984, J. Comp. Physiol. 155A: 103-111; New and Bodznick \981.Neurosci.Abstr. 13:399). To test the hypothesis of common mode suppression, a stimulus was introduced via a gut electrode. The distribution of recorded electric fields, and recordings from primary afferents, show that this method provides a good common mode stimulus to all of the receptors (with the exception of the dorso-medial hyoid group which is above the water level in the tank). A 1 Hz sinusoid delivered through the gut electrode, was adjusted to give a 20 //V potential between the bath and a Ag/ AgCl electrode placed in the interior of the body in the region of the electroreceptor clusters. This electrode also measures fluctuations in potential during normal ventilation which range from 0-60 ^V . Defin- ing the modulation produced by the gut electrode as noise, and the response to a 2 ^V/cm uniform field as signal, the signal to noise ratio (S/N) in primary afferents ranged from 0.3 to 1 .8 (mean 0.79. S.D. 0.32, n = 24). In the output neurons of the medullary nucleus the range of S/N was 0.7-65.8 (mean 4.8. S.D. 1 1.9. n = 29). The high values of S/N found in some central neurons is evidence that a common mode suppression mechanism functions to suppress CNS responses to venti- latory self stimulation. 304 ( i All1 \R\TIVE AND GENERAL PIIYSIOKXiY Bacteriologic investigati II disease in the deep sea red crab. Gervon qtiinquedens. ROBFRT BULLIS (Marine Biological 1 , rator\). Lot 'is LEIBOVITZ, LARRYSWV K\Mn YOUNG. We studied the • ctlects of pollution from sewage sludge dumping jt i >'l shell disease in crustaceans. Deep sea red crabs. ' iftu chosen -is a representative species, were collected .'ntincntal shell' c.in\ons which may have re- ceived d rom Dump sue mi.. iwcmv crabs were examined representing populations that were close (Hudson canyon-8). interme- diate (Block canyon-6). and relati\el> tar ( \tlantrs canyon-6) from the dump sue. Ml crabs examined exhibited darkly pigmented lesions of the exoskeleton which suggested crustacean shell disease. The lesions had a himodal distribution pattern. A random unilateral hy perpigmen- tation was associated with apparent abrasions and scratches. A bilateral lesion symmetry was also observed. These bilateral lesions appeared t.i evolve as hyperpigmentation of the uniformly spaced microscopic sensory organelles located on the surface of the carapace. These pig- mented areas became enlarged, confluent, and occasionally resulted in shell defects. Crabs with amputated appendages had lesions on the ex- posed proximal remaining articular surfaces. Cultures were taken from lesion and non-lesion areas on the crab on a variety of selective and non-selective media and incubated aerobicallv. Bacteria isolated pre- sumptively included Vibrio alginolylicus, \'ihrn> cumphclln. 1'ihnn jlwialiy Flavobacter meningosepticutn. 1-UiM'hacicr hrc\-c. and /.M/K-- richia co/i. Several as yet unidentified fungi have been isolated. This study suggests that the shell disease lesions found in (jeryon spp. are distinct from other previously described shell diseases, including "cigarette-burn disease." This study is supported in part hy a grant from the Division of Re- search Resources. National Institutes of \ lealth ( P40-RR 1 333-08). Computer model of light-induced voltage response of the Hermissenda type K plioiorecepior hased on seven light- ami voltage-gated ionic conductances. CHONG CHEN (Computation & Neural Systems. 216-76 Cal- tech. Pasadena. CA 91125). CHRISTOF KOCH. AND DANIEL L. ALKON. type B pholoreceptors ofa sea snail. lli-mn^i'iii/Li. have been dem- onstrated as primary convergent neurons during associative condition- ing. To understand its iole in mloimation processing and the underly- ing mechanism ol ionic channel modulation, we implemented a math- ematical model of the B-photoreceptor on a 3/160 SUN workstation. Hodgkin-Huxley-like equations are used to describe the kinetics of seven ionic conductances. The somata of the photoreceptor isapptoxi- mated as a sphere of 30 /im in diameter 1 hree of the conductances are gated by light-induced chemical changes. gNa(L. V. t). gK88. Bwl Bull.. 175: 302). was used to con- struct an affinity chromatography gel. Squid optic lobe homogenate was soluhili/ed. reacted batchvvise with the gel and the bound protein eluted. and reconstituted into lipid vesicles by sonication/dialysis. I hese vesicles were preloaded the with Quin-2. Addition of Ca" to the external medium produced a rapid, sustained increase in the fluores- cence not seen in control vesicles. Influx was blocked by 50 n\l Cd'*. I I \ blocked in a dose-dependent and competitive manner with exter- nal Ca' When a Nernst potential was established by valinomycin. the ( a influx into the vesicles was voltage dependent. Vesicles were also fused with lipid hi layers formed across i he tip of a patch-clamp micropi- pette. Two types of channel-like activ ity were found. The first was char- acten/ed bv voltage-dependent openings of 1-3 ms duration (mean). I he opening probability, which was also voltage dependent, reached a maximum of 0.35 at a potential of 0 mV. The conductance was 15-20 pS in si l m \l Ba" and 5-8 pS in 100 m.U Ca". Comparison of the macroscopic currents obtained by summing multiple pulses repro- duced closely the macroscopic lea in squid terminal (LlinastVu/. 1981. /)'i,>/'/Ms ./ . 3.3: 28')). When the cvtoplasmic face of the protein was exposed to high concentrations of Ba". extremely long mean open times (300 ms) were observed having a similar conductance. In sym- metric Ba" solutions, replacement of the cvtoplasmic solution with Cs' resulted in conversion of long openings to short openings. High internal calcium ( 100 m W Hhd not change the opening-time mode. We conclude that using an alhnity gel based on FTX. it has been possible to isolate and partially purity a calcium channel with the properties expected lor the press naptic channel. Ilix/i ammonium hackxround does not alleel response function of narrowly tuned chemoreceptor cells. ( ' \R \ M. COBURN, RAINLR VOIGT, AND JELLE ATEMA (Boston University Marine Program. Marine Biologi- cal Laboratory). We tested the effects of elevated ammonium (NH4) background on the stimulus-response functions (S-R) ol hydroxyprolme- (Hyp), argi- nine- (Argl. and taunne-scnsitive ( I an) cells of the lobster (Hninarn.\ iinii'i'iciinit*) medial antennule. Nil., is a biologically relevant stimulus found in high levels in coastal waters Action potentials ol single eel Is were recouled extracellularly using a suction electrode. Chemotcceplor cells were identified with a search stimulus (equimolar mixture of Hyp. Arg. and I'au; each at an applied peak concentration of 7 • 10 ' I/) injected into a carrier flow of artifi- cial seawatei i \sv\ | which supervised the excised appendage. Individ- ual responses to Hyp. Arg. and Lau (7 • 10 * I/) were recorded to ulenlify (he best-stimulus. I hen. S-R functions were determined for best -stimulus and Ml, in \S\\ (7 • 10 " to 7 • 10 ' I/). Aflei a 1 mm adapl.ilion to a 10 ' U N114 background, the S-R functions were rcdelermmed I mallv. as a conlrol lor viability and NH4 effects. S-R functions weie repeated in \S\\ Ten Hvp cells were narrowly tuned (except one cell responded equal I v to \rgl i >nlv one i ell responded to high concent i at ions of NIL, in ASW. None of the 10 cells tested altered S-R functions in the NH4 kn kri.uind ( )l two narrowly tuned Arg cells, one responded slightly io\uv ingh concentrations ol Ml, m \s\\ Neither cell was affected bv the Ml, backgiound. One of two narrowly tuned I au cells was re- sponsive to NH4 in ASW. However, both cells showed a suppressed response in the NH4 background ABSTRACTS FROM MBL GENERAL MEETINGS 305 We conclude that Hyp and Arg cells on the medial antennules are narrowly tuned and unaffected by even abnormally high NH4 back- grounds. This characteristic makes them suitable for detection of chem- ical contrast. Similar effects have been described for chemoreceptor cells in other lobster chemoreceptor organs. Supported by NSF (BNS-85 1 2585) to JA. Circulatory responses of bluefish to epinephrine, phentol- amine. and atropine. STEPHEN H. Fox, CHRISTOPHER S. OGILVY, AND ARTHUR B. DuBois (John B. Pierce Foundation Laboratory, New Haven, CT 065 19). The cardiovascular responses to epinephrine, phentolamine. and at- ropine were studied in resting bluelish (Pomatomits sallairix). Blood pressure and heart rate were monitored through a 1-mm tube placed in the ventral aorta. Change in mean blood pressure in mmHgin response to different doses of epinephrine chloride reached a maximum of 60 mmHg and had an ED50 of 5 Mg/kg body weight. Following competi- tive blockade of the alpha adrenergic sympathetic system with phentol- amine mesylate, 400 Mg/kg intraarterially. the ED50 for epinephnne chloride increased to 15^g/kg(n = 5). The phentolamine at this dose level dropped the resting blood pressure in fish lightly anesthetized with tricaine from a control of 83/54 to 64/45 (P < 0.001 ), and the heart rate remained unchanged (n = 9). But a dose of 200 Mg/kg did not change the resting blood pressure or heart rate significantly (n = 4, P = 0.2). Atropine sulfatc. 10 ng/kg. increased the heart rate from 48/ min SE 4 to 87/min SE 12. Blood pressure increased from 98/61 to 112/81 (n = 5,P<0.02). At 80 Mg/kg, the heart rate increased from 42/ min to 1 1 2/min and the blood pressure changed from 82/52, control, to 96/78. This tachycardia caused a significant increase in diastolic pres- sure (n = 5. P< 0.01). These results show that blood pressure and heart rate of bluefish are under tonic vagal and alpha adrenergic control, as in some other teleosts. This suggests the presence of well-developed parasympathetic and sympathetic regulation of the cardiovascular sys- tem in bluefish. Calcium-dependent incorporation of serine into phospha- tidylserine in the squid giant axon: physiological role in excitable membranes'.' P. G. HOLBROOK (Labora- tory of Bioorganic Chemistry, National Institute of Di- abetes, Digestive and Kidney Diseases, National Insti- tutes of Health, Bethesda. Maryland 20892) AND R. M. GOULD. Studies of the calcium-dependent, energy-independent incorpora- tions of 14C-labeled bases (choline, ethanolamine. and senne) into their respective phospholipids, phosphatidylcholine(PC). phosphatidyletha- nolamine (PE). and phosphatidylserine (PS) in subcellular fractions of whole rat brain demonstrated that these phospholipase-D type activi- ties, known as the base-exchange enzymes, were enriched in both the microsomal and synaptosomal plasma membrane fractions and sug- gested that they might play a specific role in excitable membranes re- lated to calcium-signaling (Holbrook and Wurtman 1988, J. Neuro- chcm 50: 151-161). To obtain evidence that this type of activity is transported to nene-endings. studies were initiated to establish whether serine-exchange activity was present in pure axoplasm ex- truded from the giant axon of the squid (l.iiligo peali) (Holhrook and Gould 1986, Biol. Bull 171: 494). We report here that axoplasm incor- porates 'H-serine into PS at a rate which, when expressed on the basis of lipid phosphorous, is comparable to that observed in squid stellate ganglion and optic lobe. This incorporation meets accepted criteria for mediation via the base-exchange pathway: ( 1 ) it is stimulated by cal- cium and blocked by EGTA. (2) It has an alkaline pH optimum peak- ing at 8.6. The activity did not appear to saturate at the highest concen- tration of calcium tested (25 mAf ). .-I hi V able pigment system in Hermissenda type A photo- receptors. H.-P. HOPP AND D. L. ALKON (Sect./Neural Systems, Lab. of Biophysics, NINCDS-NIH, Marine Biological Laboratory, Woods Hole, MA 02543). On the basis of anatomy, electrophysiologic properties, and synaptic interaction, the five photoreceptors in each Hermissenda eye were pre- viously distinguished (Alkon and Fuortes. 1972 J. Gen. Physiol 60: 63 1-649) as type A (2) and type B (3). White light elicits depolarizing generator potentials (with early transient and later sustained responses) from both types of cells. Following the offset of such light stimuli, how- ever, type B cells show a long-lasting depolarization ( LLD) while type A cells frequently exhibit long-lasting hyperpolanzations. In the present study, generator potentials of photoreceptors in isolated eyes. i.e.. with all synaptic interactions and impulse activity eliminated by axotomy. were exposed to a variety of light stimuli with different wavelength compositions. After previous adaptation to while or orange light, both medial and lateral type A photoreceptors responded to blue light stimuli (blue edge filters, Xc = 530 nm. 3.5 x 10" W/m:) with a markedly reduced sus- tained depolarization. Additional stimulation resulted in a strong de- crease or even complete disappearance of the early transient. The oppo- site effects, i.e.. increased sustained depolarization and reappearance or increase of the early transient, were elicited from blue adapted photore- ceptors with white or orange light stimuli (orange edge filters, Xc = 540 nm, 3.5 x 10: W/m:). Typically, the offset of the blue light stimuli was followed by a prolonged depolarizing afterpotential (PDA) of up to 50 mV which could last for up to 3 hours and showed immediate restora- tion after exposure of the cell to white or orange light. In contrast to these color-specific changes in light responses from type A cells, the responses of the three type B photoreceptors exhibited no comparable bistable changes over the wavelength range of 400 nm to 700 nm. Using a double-pulse and a single-pulse paradigm, monochromatic light stimuli of equal duration and quantum flux ( 10 nm FWHM inter- ference filters. .9 x 10: W/rrr) were used to measure the spectral depen- dency of the decrease and increase in size of the early transient. The decrease was maximal at 470 nm while the increase was maximal at 570 nm. The existence of the two different wavelength maxima suggests two distinct visual pigment states with different absorption peaks. Membrane conductance was reduced during the PDA evoked by blue light, suggesting the shutdown of an outward directed conductance as its underlying ionic mechanism. Furthermore, the strong increase in membrane conductance accompanying the early depolarizing transient was reduced and eventually eliminated as the early transient was re- duced by the repetitive application of blue light. Conversely the same reduction in conductance reappeared as the early transient was restored with repetitive yellow light stimulation. A thermostable rhodopsin-metarhodopsin conversion is thought to underly the color-specific regulation of the membrane potential and the conductances. Supported by DFG Forschungsstipendium HO-989/1-1. Organophosphorus acid (OP A) anhydrase from squid: a calcium-dependent P-h'-splitting enzyme. FRANCIS C. G. HOSKIN, K. S. RAJAN, AND K. E. STEINMANN (Illinois Institute of Technology Center, Chicago. IL 60616). The enzymes that hydrolyze the P-F bond of certain cholinesterase inhibitors (e.g., diisopropylphosphorofluoridate. DFP: 1.2.2-trimethyl- 306 COMPARATIVE AND GENERAL PHYSIOLOGY propyl methylphosphonofluoridate. Soman ) are known as organophos- phorus acid anhydrascs [OP \ anhydrase; Ibrmcrly DFPascl. The\ are categorized as "Ma/n- biquitous; Mn- stimulated: Soman/ DFP hydrolysis ratios 1 60-90.000) and "squid tvpc" (limited to ceph. . hepatopancreas. and saliva: Mn:* in- dirterent or slightly < vman/DFP = 0.25; mol. wt. 30.000: Hoskir< 1/7'. /.>"/ 4: SI65-S172). We now find another rr ! that distinguishes these two OPA anhydrasesand may boar or ,iu>n of a physiological role for these unusual en- zymes. The ^iuid cnz.yme is non-competitively inhibited by LDTA (K, a 10~? M i- I lie inhibition is resolvable into last and slow components (tv, '= 1 h. reversible: t... = 16 h. irreversible). I he puntied squid en- /\ me is inhibited 90'" -t- by EDTA. about M > In I ( ITA. and not at all b> the transition metal ehelators 8-hydroxyquinoline-S-sulfonate and 1 . 1 0-phenanthroline. all at 1 0 J M EGT A followed by C'a" ' causes 80% recovery of activ it>. whereas EGTA and then Mg- ' causes 10% recov- ery. Identical results are obtained with freshly extruded axoplasm. In sharp contrast. Mazur type OPA anhydrase (various sources) is inhib- ited about 30' • bs all four ehelators at 10 4.U. and is stimulated (mam- fold) by Mn:* and sometimes Mg:* at 10 ' M. We conclude that squid type OPA anhvd rase isa Ca:" en/vme. We suggest that the Mazur type OPA anhydrases. while Mn:* stimulated, are not divalent cation de- pendent. In \iew of a C'a:' involvement in many cellular processes, these results imply a physiological role for squid type OPA anhydrase. The actual role, and the nature of the Ca:* regulatory site, remain un- known. Supported by a grant from Army Research Office. . I \-ihraiinf! calcium-selective electrode tor detecting ex- tra-cellular calcium gradients. WIEL M. KUHTREIBER, PHILIP C. WILLIAMS. AND LIONEL F. JAFFE (Marine Biological Laboratory. Woods Hole. MA 02543). We have developed a vibrating, extracellular, calcium-selective mi- croelectrode for measuring calcium gradients caused by localized cal- cium currents in or out of cells or tissues. The sensor is of the neutral carrier type and the electrode has a resistance of a few hundred Meg- Ohms. Gradients are measured by slowly vibrating the electrode and feeding its output into a phase-sensitive detector. In this way it is possi- ble to measure calcium-fluxes smaller than 1 pmol/cnr/s in a large background of other ions. Preliminary measurements with this new device on Dui\n\iflniin and Polysphondilium show that these slime molds have large inward calcium currents ranging from about 1 to 3- 30 pmol/cnr/s at aggregation stages and at migrating slug stages, re- spectively. The influx just behind the front of the slug is two to three times the influx at more posterior locations. This area of reduced influx probably corresponds to the expected prestalk area of the slug. We have also detected calcium-fluxes in preliminary experiments on developing eggs of the fucoid seaweed /'flvclia. and in the growing pollcntube of the tobacco plant. These feasibility studies show that the vibrating cal- cium-selective electrode produces biologically meaningful data. This device should provide a novel way to study the role of calcium fluxes in biological systems. Supported hv N I H-grants to I I I llentaio/tniciii i lilainvdiosi\ ol the rock crah (Cancer ir- roratus) and the ionali crah (Cancer horealis). Louis IIIUIAM/ ( I .ihoralory tor Marine Animal Health. Marine Biological Laboratory). I he results nl an approximate sivvcai study ( I983-I98X) of a pre- viously unrcported highly fatal li.nr.missiblc disease of laboratory- maintained populations of i< uih nabs were reported. Hc- matopoietic tissues and circulating blood cells were primarily affected. I he disease incidence was low m newly harvested wild crabs, which became infected rapidly when introduced into cannibalistic laboratory crab colonies. The percentage mortalities increased in direct proportion to the length of time crabs were maintained in the laboratory. 1 he disease was easilv diagnosed by light microscopic examinations of fresh and fixed-stained blood smears and tissues demonstrating the pathognomonic. greatlv swollen, immature and mature, infected circu- lating hemocytes. Such infected hemocvtes were tilled with line baso- philic (.'hlamydm that compressed and displaced the nuclei and cy- toplasmic organelles ot the host cells outwardly . As the disease contin- ued, the crab's hematopoietic tissues were destroyed, and swollen cells frequently produced microscopic emboli and vascular occlusion. In- fected cells frequently ruptured, releasing Clilamyiiia into the blood and tissue spaces. Ultrastructural studies demonstrated oval or round developmental stages afCh/ainyiJui sp. within infected host cells. Reticulate stages ranged from a mean length of 560 nm and 475 nm in width, intermediate and condensing stages had a mean length of 422 nm and 354 nm in width: and elementary bodies had an approximate mean diameter of 214 nm This is the first report of a hematopoietic chlamydiosis. and chla- mydiosis in Crustacea. The specific features of the chlamydial agent, the importance of the disease, its epizootiology. and its comparative pathology were discussed. This study is supported in part by a grant from the Division of Re- search Resources. National Institutes of Health (P40-RR 1 333-08). The analysis of miniature synoptic potentials in the squid giant synapse. J.-W. LIN, M. SUGIMORI, AND R. LLI- NAS(Dept. of Physiology and Biophysics, NYU Medi- cal Center, New York, NY 10016). The miniature synaptic potentials recorded from the squid giant syn- apse have a near-symetrical waveform (Joyner ci al 1975. liinphys J. 15: 37). Typically, it reaches peak amplitude in about 1 ms and decays in 5-6 ms. The time course can be approximated by a difference be- tween two exponential functions, with a rising and a decay time con- stant. / i- . F(t) = a[e 1/ld - e '"]. Synaptic noise simulated on the basis of this observation was then used to test the resolution ofspectral analy- sis. Briefly, a fast Fourier transform was performed and a best fit of the spectra was located by searching through many time constant combina- tions. The results of the simulation show that the calculated time con- stants recreated the original miniature potential waveform satisfacto- rily as long as the standard deviation of the background noise was less than twice the miniature amplitude. Thus, only those experiments where a background noise level lower than 200 ^V: were selected for spectral analysis. ; c . with a standard deviation slightly larger than the mmipotentia! amplitudes that has been estimated to be about 10 ^A (Miledi I967..7. /Vin/o/ 192: .179: Augustine and Fckcrt I'»S4. ./ I'/n * ml 346: 257). This procedure was applied to experimental data, where dillerent levels of noise wen- evoked bv presvnaptic depolari/ations, and the frequency characteristics of these spectra provided consistent time constants for each sv napsc. In one example, rise time constants of 0.2-0.3 msand decay time constants of 1 .5-1.6 ms were obtained from recordings where there was a 7-fold dillerence in transmitter release. Similar spectral consistency was obtained before and alter LI \ appli- cation, .mil picn ules an additional support for the presv naptic calcium conductance blockage mle of this toxin (Sugimon ft ill IVJvS. Hint. Hull 175: S02) / //.'I / ol sr//(//'wi / tu/d ( '. I.I/ l\i/iti\e II I'll cvokcil and spontaneous transmitter release in the squid giant svn- ABSTRACTS FROM MBL GENERAL MEETINGS 307 apse. R. LLINAS, M. SUGIMORI, J.-W. LIN, T. L. Mc- GUINNESS*. AND P. GREENGARD* (NYU Medical Center and *Rockefeller University). Synapsin I. in its dephospho- and its phospho- form, and CAM ki- nase II were injected into the presynaptic terminal digits of the squid giant synapse. The location of these proteins was monitored with fluo- rescent microscopy. The effects of these injections on spontaneous transmitter release were studied using the technique described in an accompanying abstract (Lin el ai 1988, Biol. Bull 175: 300). The re- sults indicated that dephosphosynapsin I reduced spontaneous and evoked quantal release in a manner which correlated temporally with its diffusion into the preterminal. Phosphosynapsin I showed no effect. By contrast, CAM kinase II did not modify spontaneous release if the presynaptic potential was negative to -70 mV. However, the low level of transmitter release produced by one second depolarizing pulses of the preterminal was increased by as much as 300% after the CAM ki- nase II injection. This indicated that a calcium entry, beyond that pro- duced by the resting calcium conductance, is required for the injected CAM kinase II to increase transmitter release. The above results are consistent with the previous suggestion (Llinas el a/. 1985, PNASK: 3035) that these proteins control the availability ot releasable vesicles by regulating the amount of vesicular caging by synapsin I whose bind- ing to vesicles and to actin is phosphorylation-dependent (De Camilli and Greengard 1986. Biocliem. Pharmacol 35: 4349). Learning in the green crab. Elect romyograms reveal that movement of the eye is not required for classical condi- tioning of the eye withdrawal reflex. RAFAEL H. LLI- NAS, RICHARD D. FEINMAN, ROBIN R. FORMAN, AND CHARLES I. ABRAMSON (SUNY Health Science Cen- ter at Brooklyn, Brooklyn, NY 1 1203). The eye withdrawal reflex of the green crab, Cari'imt.t inucnii\. can be conditioned according to a classical (Pavlovian) procedure by pairing a mild vibration to the carapace as a conditioned stimulus (CS) with an air puff to the eye as an unconditioned stimulus (US). (Abramson and Feinman 1 988,7. Ncurosci. 8: 2908-2912). Eye retraction results from the activity of several muscles, the largest of which is the main abductor muscle. 19a. Stereotyped electrical activity in this muscle accompanies the eye retraction and can be recorded as electromyograms ( EMG). The EMG record was used as an indicator of behavior to determine if the pattern of acquisition of conditioned responses was the same in an im- mobilized eye as in one that was freely moving. The results indicate that acquisition is the same in most cases and suggest that, as in many cases of classical conditioning, integration of the two sensory stimuli (CS and US) is sufficient for learning. Animals were assigned to either an experimental group with EMG leads implanted and the eye re- strained, or to one of two control groups with freely moving eyes: either with EMG leads or unoperated. The pattern of acquisition for 5 of 8 experimental animals was the same as that for controls with moving eyes. Animals subjected to unpaired stimuli (fixed or moving eyes, with or without electrodes) showed few conditioned responses. After the ac- quisition trials, the eyes were freed, the wires were cut, and animals were returned to home tanks and later tested for behavioral responses in a sequence consisting of CS alone after 4 h and 24 h and a second period of paired training. Animals showed substantial retention and the second training period produced enhanced performance compared to training on the first day. The profile of behavior of all eight paired ani- mals with fixed eyes was the same as that for animals with freely moving eyes. The unpaired animals that had been trained with eyes restrained were likewise indistinguishable from unpaired subjects with moving eyes. This work was supported, in part, by funds from Margaret H. Mor- gan and from the Research Foundation of SUNY. A lustamine-gated union channel suppresses lobster ol- factory receptor cell activity. TIMOTHY S. McCLiN- TOCK (The Whitney Laboratory). Application of histamine to the soma of American lobster receptor cells suppressed both spontaneous and odor-evoked spiking as pre- viously discovered in spiny lobster olfactory receptor cells (T. Bayer and B. W. Ache, unpub. data). In intracellular recordings, histamine caused an increase in conductance, a chloride-dependent change in membrane potential, and reductions in odor-evoked depolarizations. In isolated, voltage-clamped soma, pressure-applied histamine acti- vated a largely chloride-dependent current. This current was not affected by external cobalt and cadmium (n = 2) or internal perfusion with 1 mA/GTP--y-S(n = 18) orGDP-0-S (n = 3). Applying histamine outside the patch pipette activated channels in outside-out patches but not in cell-attached patches (n = 19). Placing 1 mM GTP-i-S in the pipette did not alter the activation of this channel in outside-out patches (n = 3). The mean slope conductance calculated from current- voltage relationships was 44 ± 6 pS. Extrapolated reversal potentials suggested that chloride was the major permeant ion present, but that some cation permeability also exists. In steady state concentrations of histamine, the probability of the channel being open began to rise rap- idly between . 1 pM and I //A/, and appeared to saturate between 10 /iA/ and 100nA/(n = 2). Channel opening evoked by 10 //A/ histamine was blocked by 500 pM cimetidine but not by 500 pM pyrilamine. These results show that the modulatory action of histamine upon lobster ol- factory receptor cells is mediated by a histamine-gated anion channel. I thank the Grass Foundation and the NIMH for supporting this work, the members the 1988 Grass Fellow program, and B. W. Ache for advice. Modification ot the veslibulo-ocular reflex (} 'OR) after 6- OHDA induced catecholamine depletion in the CNS of goldfishes. JAMES G. MC£LLIGOTT (Temple Univ. School of Medicine), MICHAEL WEISER, AND ROBERT BAKER. The vestibulo-ocular reflex ( VOR) helps to stabilize visual images on the retina during movements of the head. This compensatory reflex moves the eyes in a direction opposite to that of the head. Modification of the VOR provides an experimental paradigm for examining hypoth- eses about plasticity in the central nervous system (CNS). These experi- ments were designed to test the effects of depleting CNS catechol- amines, especially norepinephrine (NE), on modifiability of the VOR in the goldfish. Past experimentation in various mammalian systems had shown that depletion of NE affects developmental plasticity in the visual system as well as VOR modifiability in cats. CNS catecholammes in the goldfish were depleted by small multiple injections of 6-OHDA into the brains of anesthetized goldfishes. Experimentation com- menced two weeks after injection in order for CNS catecholamine de- pletion to occur. Eye movements were measured in chronically re- strained alert animals. After initial measurements of the VOR gain (eye velocity/head velocity = 0.82). each fish underwent 4.5 hours of VOR gain modification. Gain was increased by presentation of visual stimuli in the presence of vestibular stimulation ('/g Hz ± 20 degrees). Gain increased towards 2x and then towards 3x over a 4.5 hour period. In all cases, goldfishes (n = 4) produced robust VOR postmodification gain increases that averaged 2.6. When the brains of these animals were analyzed for catecholamine content by High Pressure Liquid Chroma- tography with Electrochemical Detection, there was a severe depletion - - T \K\IIVI-: AND GENERAL PHYSIOLOGY of brain NE to less than 4'" for all brain rep. TIN measured as compared to controls. Thus. unhV manr MI -ms. d-OHD \ deple- tion ofCNSNE in the K1 oticeablj inlluenee VORgain modifiability. Physiologic ;;;. • n-eniiiii; ihe COmpci l //; hliu'lisll. CHRISTOPHER S. OCILVY. Si MM II N H. FOX. \\D \KIIII R B. Dl'BOIS (John B. Pierce Foundation Laboratory. New Haven, CT 06519). Bluetish maintain ventral aortic blood pressure (BP) and develop a tachycardia during passive head-up tilting in air. This is true for un- anestheti/ed or anestheli/ed fish despite a lower BP in anestheti/ed ani- mals. Prior to tilting, we gave atropine sulfate (80^g/kg) to block para- sympathetic innervation to the heart, or phentolamine mesylate (400 fig/kg) to block alpha-adrenergic vasoconstriction. Or. we gave the neu- romuscular junction blocking agent pancuronium bromide (100 ^g/ kg) to prevent body movements during tilting. In liverish, we transected the spinal cord 3 mm caudal to the obe\ before tilting. In others, the \agi were cut as well. After phentolamine. control BP was 73/47 mm Hg. and fell to 54/40 5 min into a 30° tilt, remaining low for the 30 mm of tilting. Atropine increased the heart rate (HR) to I 1 1 per min from a control of 42. and BP to 96/78 from a control of 82/52. HR remained unchanged during tilting, while BP decreased to 74/58 (P < 0.05) and remained low during the 30-min tilt. Flexion of the lower body muscu- lature during tilting transiently increased the BP. Pancuronim pre- vented this and lowered BP during tilting. Cord transection without vagotomy lowered BP during the tilt despite an increase in HR. Cord section plus vagotomy produced a fall in BP and increase in HR. When these fish were tilted to 10° (2 min), 20° (2 min). or 30° (5 mm), blood pressure fell precipitously and heart rate was unchanged. These results indicate that the cardiovascular reflex system in the hluefish is well de- veloped and similar to that in land vertebrates. I'pon tilting, there is inhibition of the vagus, with tachycardia, an apparent alpha adrencrgic mediated vasoconstriction. and lower body muscular contractions. These aid venous return and maintain blood pressure to help perfuse the brain and other vital organs. Further contributions in odor contrast ({election in hermit crahs I i si IE SAMMON, SOPHIE SANDERS, AND JELLE ATE MA (Boston University Marine Program, Marine Biological Lab. Woods Hole, MA 02543). Hermit crabs (Paguru\ longicarpus) luce, in part, scavengers searching for food by smell in marsh environments with high organic hack- grounds We expect that the detection of chemical contrast is important tor these animals. We tested their ability to locate upstream odor sources in the laboratory counting the fraction of individuals attracted through a slightly avcrsive dark corridor. We chose three odors that were highly attractive .it different dilutions: shrimp juice (SJ) was most attractive! MID', at 10 .Million I lollowcd In lish iincell I: at Id 'land mussel juice i M.I 10 i Response functions were essential!) parallel: for all tin ' 0 ittractioi ..... urred at inn- further dilution. Rc'.p nnMili (scawaici (averaged In self-adapting I o ' dilution), all three i espouse func- tions were suppr. ..... n,, i values) for MI "and 10 4 stimulus dilutions, and ! '). A toxin fraction isolated from American funnel web spider venoms (FTX) blocks calcium currents in central neurons (Sugimori and Llinas 1987. .VCK/VIVC; l/iv;r ). This fraction has been recently shown to have a low molecular weight (200-300 dalton) (Cherksey ft at. 1988. Biol Hull 175: 298). When tested on transmission at the squid giant synapse, the toxin blocked synaptic transmission without affecting either so- dium or potassium voltage-dependent conductances. The blockage oc- curred without affecting the pre- or post-fiber action potentials. Volt- age-clamp experiments of the presy naptic terminal show that the in- ward calcium current is blocked at submicromolar concentrations by l"l \ within 5 to 10 minutes after direct application to the hath. The toxin was very slowly reversible, and Us blockage was related in a com- petitive way to [Ca:']. Finally, pressure injection of glutamate in the area of the postsynaptic fiber demonstrated that FTX had no effect on the post synaptic potential generated, thereby indicating no effect of FTX on glutamate dependent post-synaptic channels. liye movement repertoire of the t'lihuion sculpin, Scor- paenichthys marmoratus. MICHAEL WEISLR. AN- DREW BASS, JAMES G. McFi i IGOTI. AND ROBERT BAKER (NYU Medical Center. New York. New York). ( Observation ol eve movements in the Caba/on sculpin. a teleost. sug- gested goal-directed changes in ga/c to attend small targets much like the oculomotor performance described for many foveated mammals, lo test tins hypothesis, spontaneous, oplokinetic. and veslibular in- duced eve movements were quantitatively investigated employing the magnetic search coil technique. Saccades and fixation in the light were found in both the horizontal and vertical directions llori/ontal sac- cades occurred simultaneously in both eves but often with dispaiale amplitude, direction, and velocitv \ ertical saccades were independent of hon/ontal eve movements. I ew spontaneous eye movements were observed in the dark. Visual following was elicited using either full field 01 subtended 5° targets deliveied simisoidally over a frequency range of 0.25 ll/ lo 1.0 1 1/ at 8-40°/s. Gain, measured as eve velocity/target velocitv. was o d and o 4. lespectively . throughout the frequency range. Smooth Hacking was nevei elicited in the vertical direction. V estibular evoked eve movements had .1 gain (eve vclocitv 'head velocity) of 0.8 throughout the same frequency range. ( 'ombined visual and \estibular inteiaclion produced eve movements at a gam of 1 .0 from O.Od \ 1 1/ 'oil/ Adaptive rain control of the vestibulo-ocular reflex towards a gain of 2.0 at O.S Hz (±10' am pi itude I was examined after 3 h of train- ing with .1 lull held visual stimulus of v1 s at a he. id velocity of I6°/S. ABSTRACTS FROM MBL GENERAL MEETINGS 309 Vestibular evoked eye movements measured in the dark were increased from 0.8 to 1.8. We conclude that the oculomotor system of the Caba- zon sculpin is remarkably robust, exhibiting more of the features de- scribed in foveated mammals than those reported in other teleosts as exemplified by the goldfish. Stimulus/response coupling in sponge cell aggregation- differential effects of cocaine derivatives and non-ste- roidal antiinflammatory agents. GERALD WEISSMAN, REED BROZEN, GIULIA CELLI, AND PETER SANDS (Marine Biological Laboratory). Dissociated marine sponge cells (.Microcmna proli/eru) re-aggregate when protein kinase C and Ca fluxes elicit secretion of a species-specific aggregation factor. Aggregation is regulated by a guanine-nucleotide binding protein (G-protein) and is inhibited by pertussis toxin (Weiss- mann el al . 1986 PNAS 83: 2914-2918). Using quantitative aggreg- ometry. we now show that agents which mimic binding of GTP to the G-prolem ( vanadate, > 1 mA/. fluoroaluminate. >5 m A/) elicit aggrega- tion of sponge cells exposed to sub-optimal amounts of Ca( 5 m.U)and ionomycin ( 1 n\D. Arachidonic acid (20:4) had a similar effect. Cells treated with local anesthetics, i.e.. tetracaine(IOO jjA/). dibucame(150 n.\l ). procaine ( I mA/ ). or lidocaine (3.5 mA/) failed to undergo normal aggregation in response to vanadate or arachidonate. Non-steroidal antunflammatory drugs, i.e., indomethacin ( 100 MA/), piroxicam (100 ii.M). sodium salicylatel 5 mA/). or aspirin (5 mA/) also inhibited aggre- gation. Neither class of drug inhibited aggregation induced by the direct activator of C-kinase. phorbol myristate acetate. The two groups of in- hibitors differed in their mode of action with respect to the twin signals of protein kinase C and Ca movements (measured as 45Ca efflux and appearance of 'H-arachidonate in diacylglycerol on thin-layer chroma- tography in hexane:ether:aceticacid, 50:50: 1). Whereas cocaine deriva- tives provoked enhanced 45Ca fluxes from sponge cells exposed to Ca and ionomycin. aspirin-like drugs inhibited 45Ca efflux. Moreover, whereas local anesthetics had no effect on diacylglycerol synthesis, aspirin-like drugs inhibited 3H-arachidonate incorporation into diacyl- glycerol. The experiments not only suggest that cocaine derivatives ex- en their membrane effects at sites distal to C-kinase and that aspinn- like drugs work at the level of the G-protein. but that these primitive cells resemble human blood cells in their response to products of the coca plant or the willow. Artificial diets of fish food are potentially useful for Her- missenda marieiilture. EBENEZER YAMOAH, ALAN M. KUZIRIAN (Marine Biological Laboratory), DONNA McPHiE, AND Louis MATZEL. An advantage of using marine invertebrates as biomedical research models is their ability to be kept in laboratory culture. With the increas- ing dependence on closed-recirculating systems using natural or artifi- cial seawater by non-coastal research laboratories, effective and efficient maintenance conditions are important. The question of feeding density and growth rates for juveniles was addressed. Ten-day, post-metamorphic animals were kept in individual 5-ml flow-through test tubes in a l-l container and fed 2-3 stalks of the coelenterate hydroid, Tithularia sp. (n = 10). Concurrently, animals were pooled in a 250-ml crystalizing dish with 10-12 hydroid stalks. All animals were kept in a 14°C incubator, their length measured, water changed, and food replenished every 3 days for 15 days. Instantaneous growth rates (K-values) for individually cultured and fed animals were compared with the multiple cultured animals. The data revealed no significant (Student /-test) differences between the two conditions (K- values: 13.4, 16.5, respectively). Artificial fish food pellets were used by the Laboratory of Cellular and Molecular Neurobiology. NINCDS-NIH. Bethesda, MD. for maintenance of adult Herinisaemhi. Although artificial diets (microen- capsulated food and amino acid suppliments) have been used for filter feeding larval and adult molluscs, this represented a unique opportu- nity to evaluate its use on a totally carnivorous species. Hard and soft fish food pellets, containing fish and shrimp meal, yeast, wheat-germ, seaweed meal, sucrose, vitamins, and trace elements were compared with a proven maintenance diet of crab viscera to test their ability to promote growth of healthy adult animals. Eight animals per feeding paradigm were fed either hard or soft fish pellets, or crab viscera. All animals were starved for five days, weighed, assigned to individual 50- ml flow-through test tubes, and fed (2 pellets/tube; viscera from 3 crabs split between 8 Henniwmltn. Thereafter they were weighed, and fed every 3 days for 15 days. The following K-values based on weight gained (mg) were obtained: hard pellets, 2.18; soft pellets, 3.21; crab viscera. 4.30. Based on Student Mests, the soft pellets were significantly better (P < .05) than hard, but not significantly less than the crab diet (P> . 1 ). The hard pellets provided a significantly less effective diet than the crab (P< .01). The results of the juvenile growth and culture conditions indicate that pooling of reasonable numbers of individuals and their food pro- vides conditions which promote good growth. This is extremely impor- tant for ease of maintenance under manculture conditions. The artifi- cial diet study indicates that the soft fish food pellets provide adult growth rates comparable to that of crab viscera. They are somewhat better because the pellets foul the water much less than does crab vis- cera. The hard pellets which contain more crude protein (hard. 40%; soft, 35%) do not support comparably high growth rates. They probably are less palatable. This finding is important because it means there is a standard, extremely low maintenance diet potentially available that will provide consistant dietary conditions for Hermissenda maricul- ture. E. V. gratefully acknowledges the support provided by the American Society for Cell Biology, Minorities Student Program, and the Labora- tory of Cellular and Molecular Neurobiology. NINCDS-NIH. Some morphological and physiological properties ol the sea robin Maul liner cell. STEVEN J. ZOTTOLI (Wil- liams College), GRAEME W. DAVIS, AND SUSAN C. NORTHEN. Comparative morphological studies of teleost fish have revealed differences in Mauthner cell (M-cell) size; somata can be categorized as either large or small. In addition. M -cells have not been found in certain fish. Most M-cell studies have been conducted on cyprinids which have M -cells in the large size class. We have analyzed the M-cell of the sea robin. Prionolut curnlinus. to learn more about the properties of a cell in the small size category. M-axons were identified as described previously (Zottoli and Agos- tini 1984. Biol. Bull 167: 535) using intracellular recordings with KC1 electrodes in lightly anesthetized (0.007% MS-222) fish. M-axons could be activated by antidromic stimulation about halfway down the spinal cord but could not be activated further caudally. Morphological studies of the M-axons revealed a steady decrease in axonal size from the re- cording site in the medulla (40 /im) to halfway down the spinal cord (20 Mm ). Further caudally, it became increasingly difficult to distinguish the M-axons from other axons in the cord, suggesting that the M-axon does not extend the full length of the spinal cord. Antidromic activation frequently resulted in a second component on the falling phase of the action potential. This later component may represent the spike generated at the initial segment which has traveled back passively to the axonal recording site. Double antidromic stimula- tion at 1/s resulted in blockage of the later component of the second 310 DEVEL< )I'NU N TAL BIOLOGY AND FF.RTILIZA I K )\ spike when the second stimui I approximatel) less after the first. Thief- lenl returned after increasing the frequency of stimu '.ite that this blockage is the result ofa M-cell collateral in: • similar to that described in the goldfish and that this nei common to M-cells independent of their size. These results pi , m the analysis of a M-cell in the small si/ecatcgoiy and are a necessary prelude for com parati\e investi- gations of M-cell mediated beha\ior. Supported b\ \l\( US Grant *NS 2('032. Sue Northen was sup- ported by the Ford-Mellon Research Scholar Program. Developmental Biology and Fertili/ation N 1 1 I ai .1 concentration of 5.0 p.\l induced G VBD in more than 90"'! oi oocytes while 5-methoxytryptamine (5-MT) and 5-hydroxyindole acetic acid (5-HTAA) at the same dose did not induce GVBD. Mian- serin, a 5-HT, antagonist, at 5.0 n\l dose totally blocked 5-HT-induced ( A HI) Kelanserini 5-HT:) and metergoline (5-HT(( )at the same dose failed to block 5-H I -induced GVBD. I he 5-11 I agonist. 8-OH-DPAT (5-HT, o induced GVBD in SO' . of the oocytes-. whereas RU-24969 (5- HT1B)and 2-meth>l-serotonin( 5-HT,) did not induce GVBD. The present findings suggest that \/i/w//<; oocy ics possess 5-HT,A re- ceptor sites. This sludy was supported by grant no. INT 82 1 1 350 and NSF and GA PS 87123 from the Rockefeller Foundation. The 5-HT antagonists and agonists were obtained as gifts from Glaxo. Sandoz. Roussel L'CI.AF. and Farmitaha Protein phosphorylation t/urini; oocylc mat unit ion in Spisula tfw/Asterias. T. HAM n \\oS. S. KoiDE(The Population Council, New York, NY 1002 1 ). Spisula oocytes were preincubated for 2 h with |':P]phosphate at a concentration of 200 ^Ci/ml to radiolabel the adenosine tnphosphate (ATP) pool. Maturation was induced with 5-hydroxytryptamine (5- HT) at a final concentration of 10 n\l After 10 and 30 min of treat- ment, oocytes were homogeni/ed and the paniculate fraction prepared. The phosphorylated proteins were separated b\ sodium dodecyl sul- fale-polyacrylamide gel electrophoresis (SDS-PAGE) and the radiola- belled proteins analyzed by autoradiographv. -\ marked increase in the radiolabelling of a protein with an estimated Mr of 30,000 occurred with the 5-HT-treated oocytcs. A similar result was obtained with the paniculate fraction of oocyles inseminated with sperm and incubated with [7-3:P]ATP. M. duration was induced in A\icna'> oocytes by treatment with 1- methyladenine for 10 and 30 min. Cytosol fraction was prepared and incubated with both [>-3:P]A I P and [v':P]GTP 1 'he radiolabelled proteins were analyzed by SDS-PAGE and autoradiography. With [-,- l:P]ATP a marked increase in the incorporation of radioactivity in- curred with proteins of estimated molecular weights of 70.000 and 62,000; whereas a marked increase in radiolabelling occurred with a protein of estimated Mr of 56.000 using h-':P]G I P. The present findings suggest that protein phosphorylation is an earls biochemical event during the resumption of meiosism S/>i \itlu and.-l.v- lerias oocytes. This study was supported by a grant No. Int 82 1 1 350 from NSF and GS PS 87 1 2 from The Rockefeller Foundation. Evidence /»r \crotonin O // / , ,/ rac/ttoi \iic on Spisula »<>t vie. A. L. KADAM, S. J. SFGAI., AND S. S. KOIDH (Population Council. New York). Serotonin MIHIMC. Ml I I. a ncurotransmitter. in- duces spawning when administered to Spisula and germinal vesicle breakdown (GVBD) of Spisula oootcs when added in vitro (Hirai i-t :4^:MSi I he present study was undertaken to determine thi n >r sites present on Sp/sw/a oocytes. Oocytc maturation i adding one drop containing ;n to i " ml of artificial seawaler (ASW) containing 5 n '. IMHI of lest samples. After 30-40 min at ambient tempcralun ou-d loi ( A BD by light microscopy. Serotonin rin (! HI,), ketansenn (5- HT:), metergoline (5-H 1 i -s h\diox\-2-(di-n-piop\l animol-tetralm (X-OII \ >]• \ \ • I'li/M^-ll I ,„). and 2- Diethylserotonin were used to i ol HI " • • -pioi site on Spisula oocytes. Stimulation ol Spisula s/>cr»i motility by serotonin (5-hy- droxytryptamini'). A. L. KADAM, S. J. SEGAL, AND S. S. KOIDE (Population Council. New York). Hirai cml (./ Av/> Zoo/. 245: 318. 1988 (demonstrated that seroto- nin (5-HT) induces spawning when administered to male surf clam. Spi\uhi i for 5 min. The supernatant was recentrifuged at 1000 x g for 10 min at 4°C. The pellet of sperm was suspended in ASW to the original \olume. refrigerated overnight, and used the next da\ for motilitv assay Appropriate concentrations of test substances were added to 0.5 ml of sperm suspension. After 5 min of treatment, the percent motile sperm were estimated using the "cell soft" semen analyzer (Cryoresources). MotiliU was also deter- mined using an axunert microscope with video-enhancement (Zeiss). The oxygen consumption of treated and control sperm was measured by using the YSI ox\gen meter (Clark t\pe 02 electrode. Yellowstone Instrument Co.). Linear consumption of o\\gen was recorded for 1 h. The fertilizing ability was assayed with washed Spisulu oocytes and scored for germinal vesicle breakdown (GVBD) and embryo develop- ment by visual examination using an Axiovert microscope (Zeiss). 5-HT stimulated Spixula sperm motility. The effect was dose-depen- dent (I p\l H5%\2fiM 7791 : and 5 MA/ = 92'-;). S-H\drox\-2(di-n- propylaminol-tetralin. (8-OH-DPAT) (5-HT,A agonist), and 5-meth- oxytr\plamme(5-M I (also stimulated sperm molility b\ Si) ' and 70'";. respectively. RU-24969 (5-HT|B) and 5-hydroxyindoleacetic acid (5- 1 1 1 \ \ i had no effect on motility. Serotonin antagonists, mianserin (5- HT,)and ketansenn (5-HT:). at 5. OpM concentration did not block 5- 11 1 -induced sperm motility. 5-HT at 5.0 n.M concentration did not stimulate sperm motility in sea urchin (Arhaciti). parchment worm (( V;i;iYi>/>/iT//\i. and so,uid (/ c/wi). The oxygen consumption of 5-HT- stimulated .S/'M/i/i/ sperm increased by 40'. of the control value in 40 min. 5-HT-stimulated motile speim retained then capacity to fertilize V/w//<; oocMcs that developed normally. In conclusion. 5-H I . S-OII-DPAT. and 5-MT stimulate sperm mo- tility in .S'/xvii/d I Ins si ml \ was supported by grant no. INT 82 1 1350 from NSF and GA PS 8712 from the Rockefeller Foundation. The 5-11 I agonists and antagonists weie gilts from Sandoz. Glaxo. Roussel. and Farmitalia. '/'//<• exx conical endop/iiMnic rcrii'iilnni. C. SARDFT, M. I 1 K \S\KI. .1. I . SIM KS\M)| K. AND L. F. J A FFE (Sta- tion Zoologiquc. Villefranche-S-Mer, Laboratory of Ncurohiology. N1NCDS. Nil! at Woods Hole, and Marine Biological Laboratory. Woods Hole). ABSTRACTS FROM MBL GENERAL MEETINGS 311 Eggs of echinoderms, molluscs, amphibians, and mammals display numerous strands of endoplasmic reticulum (ERI immediately be- neath the plasma membrane (Sardet 1984, Dc\: Biol. 105: 196: Char- bonneau and Grey 1984, De\: Biol. 102: 90: Luttmer and Longo 1985. Dev. Growth Differ. 27: 349; Speksnyder el al. 1986, Progress in Devel- opmental Biology, part B. pp. 353-356). We have shown that this corti- cal ER can be retained in isolated cortices of sea urchin eggs as a hones- comb lattice encircling cortical granules ( 1 ). Using fluorescent dyes (Terasaki el al 1986. J. Cell Biol. 103: 1557), we have confirmed and extended these observations to several species of sea urchin eggs. A tu- bulovesicular network of rough ER adheres to the plasma membrane and is continuous with the cytoplasmic ER on which many organelles remain attached. We have furthered our observations on cortical ER in eggs of the tunicate Phallusia mammillata. which do not have cortical granules. Cortices from these eggs are characterized by an extensive network of ER tubes and sheets, whose dynamics can be observed by fluorescence or DIC microscopy. Perfusion with hypotonic solution indicates that there are discrete anchorage points of the ER network to the plasma membrane. We could also localize microfilaments along the internal side of the plasma membrane using rhodamine-labeled phalloidin, and microtubules coursing along ER strands in the isolated cortex using immunofluorescence. The cortical ER network and microtubules were also observed by electron microscopy of replicas of fixed, rotary -shad- owed whole mount preparations. In view of the probable role of cortical ER in calcium regulation, its anchorage to the plasma membrane and its continuity throughout the cytoplasm, we must consider that the egg's ER may act as a scaffolding or framework able to control assembly-disassembly of associated cy- toskeletal elements. These cytoskeletal elements as well as organelles may. in turn, move with respect to the ER. .1 cell-free preparation of endoplasmic reticulum derived from eggs. M. TERASAKI, C. SARDET*, AND T. REESE (Laboratory of Neurobiology, NINCDS, NIH at Woods Hole, and *Station Zoologique. Villefranche- S-Mer). The classic method for producing cell-free preparations of ER mem- branes is homogenization followed by differential centrifugation. This method has been useful, but ( 1 ) the continuity of the ER. which distin- guishes it from other membranes, is not preserved. (2) other mem- branes may contaminate this fraction, and (3) compartmentation in the ER is difficult to study. A cell-free preparation of variably sized networks of tubular membranes was produced from sea urchin eggs (Arhacia piiiiclnlula) and tunicate eggs (Phallusia mammillata) by shearing attached eggs or by squashing eggs between two cover slips. The membranes which remain on the cover slip not associated with the cortices after washing were observed by fluorescent dye staining [DiOC6(3)]. by video enhanced DIC. and by whole mount electron microscopy of glutaraldehyde-osmium-fixed and critical point dried samples. Ribosomes on many of the membranes were observed by EM and by staining with Hoechst dye 33258 at pH 2 (Hilwig and Gropp, ECR 91: 457). Since some of the disadvantages of differential centrifu- gation can be avoided, this preparation makes possible new kinds of studies on activities of the ER. such as calcium regulation, protein or lipid synthesis, or protein filament binding and motility. We used a different dye, DilC16(3) (8 /ug/ml for 1 min diluted just before use from 2.5 mg/ml ethanol stock), to examine the organization of the cortical ER in these cortices. This dye stains cortical ER but not cortical granules of sea urchin egg cortices. Comparison with phase contrast images shows that the ER often encircles cortical granules on the cortex, verifying the original observations of Sardet (1984, DB 105: 196). Diamino acids hlock plutei formation in developing Ar- bacia mimicking other chemopreventive anticancer agents. WALTER TROLL (NYU Medical Center, New York. NY) AND JESSICA A. FEINMAN. Protease inhibitors and retinoids interfere with plutei development in Arbacia punclulata when added up to six hours after fertilization. The development of plutei is specifically modified without any effect on earlier differentiation. The protease inhibitor f-amino caproic acid (EAC) was effective at 0. 1 M concentration while leupeptin, antipain. and a,-antitrypsin. as well as retinoids. blocked plutei formation at mM concentrations. Similar concentrations of chemopreventive agents sup- pressed H-ras oncogene transformation of NIH 3T3 cells (Garte et al. 1987. Cancer Res. 47: 3159-3162). We investigated lysine, the amino acid analog of EAC. and noted that it blocked plutei development at 1 mM. 1 00-fold lower concentration than the effective dose of EAC. The closely related amino acids hydroxylysine and ornithine also blocked plutei formation at mM concentrations. Other amino acids, including argimne. histidine. glutamic acid, and aspartic acid, were inactive. Thus, the diamino acids mimic the action of chemopreventive agents in blocking plutei development in Arhacia. It will be of interest to study these diamino acids further in their anticarcinogenic action in NIH 3T3 cells and in animal and carcinogenic model systems. They have the advantage that they are non-toxic and can be readily added to media or diets. Supported by Center Grant ES-00260 from the National Institute of Environmental Sciences. Ecology Effect of tail regeneration on early fecundity in Capitella sp. I and sp. II (Po/yc/iaeta). SUSAN D. HILL (Michi- gan State University), MICHAEL J. FERKOWICZ, AND JUDITH P. GRASSLE. Capilella sp. I and sp. II are sibling species which co-occur in the vicinity of Woods Hole. Both species have comparatively large yolky eggs, and lecithotrophic larvae that colonize disturbed, organically rich habitats. Consequently, reduction in size of first brood or delay in first spawning may be significant in determining their rates of population increase in the field. We investigated the effect of posterior segment amputation and sub- sequent regeneration on size of the first two broods and on time of emergence of the metatrochophore larvae. Capitella spp. I and II late juveniles and young females were paired according to size and stage of vitellogenesis. Animals in which oocytes were macroscopically unde- tectable were classified as juveniles; animals with visible oocytes were categorized by oocyte development. Tails were amputated at the 25th abdominal setiger from one of each pair. Each worm was mated with a male. Embryos in brood tubes produced by the first and second spawn- ings of each female were counted. Date of hatching of metatrocho- phores from the first brood tube was determined. No significant difference was found in brood size in Capitella sp. I: regenerating worms produced as many eggs in their first and second broods as intact worms. Capitella sp. II worms that had their tails amputated as juveniles produced significantly fewer eggs in their first and second broods than did intact worms. Capitella sp. II females, in which oocytes were detectable at amputation, showed no reduction in either brood. It appears that, following amputation prior to vitellogen- esis, Capitella sp. I produces its usual complement of eggs while Capi- lella sp. II reduces the number. 312 I C 01 (HA In both species, tail loss in juvenile delay s the production and hatch- ing of the tirs- : >pliore lar\ae b> several days, an important dela> in sp, orl ivncrauon timov Support tor this icscarch was Pro\ided h> NSF OCE-8509169 (S.D.H.). Mann, implications related to the commercial vu/i,. :entilic collect/Hi; ol the \\lielk Bus\con. h i \i \l. K MM AN. BARBARA C. BOYLR. AND KRIS- TEN \ S \NIOS (Union College). This stud> examines socio-economic and ecological trends associ- ated with fluctuations in the commercial value of the whelk MIM. on, the basis of the New 1 ngland conch fishery, and evaluates relevant ma- rine policies. Interviews of fishermen, seafood buyers, and marine pol- icy oHieials were conducted: information was also collected by setting our own conch traps for monitoring and research purposes. Results indicate a small but active conch industry in New England that has undergone significant price fluctuations and technological diversifica- tion. Specifically, within the last few years the price of conch has almost doubled and competition between conch pot fishermen and draggers now lishing for conch has increased. Depletion of foreign conch fisher- ies and overtishing of other domestic fishes have contributed to the in- creased activity in the New England conch industry. Ecological prob- lems involving the possible depletion of the New England fishery and scientific collecting problems associated with scarcity and rising costs as well as changes and confusions regarding recent marine policies regu- lating the New England conch fishery are also examined. 1 he authors gratefully acknowledge the support of the Woods Hole i Kvanographic Institution, the Marine Biological Laboratory, and Union College/Dana I ellowships. (iniy seal imps cstahlisli critical marine liahitat in \an- tuckei Sound. I. S.I DAVID PA ION (Fayston. Ver- mont). Halichoerus grypw hirthmgsat Muskeget Island. Nantucket Sound. during the winter of 1987/1988 occurred during the last week of Janu- ary. Surveys of the Nantucket and Martha's Vineyard Sounds, the His- abeth Islands, and Nomans Island were begun in early November. Soli- tary males were seen at the North Shore of Muskeget in early Decem- ber. Common seals arrived in numbers at this time and posted themselvesat their usual haulinggrounds. Aerial reconnaissance at ele- vations of 241 I and 1000 meters were made with the Hasselhlad 250 and 80 mm Zeiss lenses and Kodochrome ASA 6-4 film. 1 he photographic transparencies were magnified and enhanced with digital techniques and flat plane microscopic objectives. On 28 January. 10 males were grouped at the north shore of the south hook of the island upwind from 7 females and 2 white-coated pups Sea birds flocked to tin- edge of the nursery grounds. Two males attended the shore near them. On 29 January, two placenta! remains showed at the middle of the bar. Two more white-ci laled pups were photographed On the 30th. the pi » enlas hail been lemoved except for red slams in the sand I JIIPI, these three days, a small seal that had the pattern and shape of a molter was included in the nursery group. A high altitude reconnaissance was made of the area No other white-pelt pups wcic detected 'in 'lit swimming animals were seen near the island. Twenty-si--. I were munted at the nursery; one .'I ihc five pups was re | -i>eitv owners. Molting pods chose Wasque Shoal and Muskcp-: Khnd north bar during April and Mav Andrew-sand M.n: i - ibhslicd llu- continuous chau. lei of this species' use of these water II 1 -Junes I here is irrefutable evi- dence in these recent surveys that the lit. -I, n ofapodofgraj seals is begun here. The habitat includes pupping grounds, fishing, and molting hauloutslBoyd. 1961). I wish to acknowledge Joe Costa, the Dickenson I" rust. Ovide Fon- lier. the llolgate Family, and the V1PIRG C orpmation for their sup- port. I'reliminary report: composition of communities resident on Limulus carapaces. LINDA L. TURNER, CARRIE KAMMIRE. AND MARY ANNE SYDLIK (Eastern Michi- gan University). Horseshoe erabl/ inni/in /Wr/'/ji-wnv) carapaces act as moving sub- strates for simple to complex assemblages of small marine organisms. ( arapace communities are unique in two ways. First, maximum com- munity age is constrained by horseshoe crab moult patterns. Second, horseshoe crabs spend most ol'the year buried offshore, then repeatedly move into subtidal eelgrass communities and harsher intertidal /ones during the breeding season. These movements probably have a signifi- cant impact on carapace community composition and complexity . Taxon frequency and diversity were determined by sampling 41 cara- paces (intraocular distance range 21-41 cm) at Mashnee Dike. Cape Cod. Neither horseshoe crab size nor sex significantly influenced com- munity structure. Barnacles (Chlhamalus and Balanus) and slipper limpets (Crcpulula) were the most common of the >10 faunal genera. More than 16 algal genera were present. Green blades, tubes, and branched filaments made up more than 50' ; of all algae. Brown and red filaments were intermediate and rare, respectively. Pnnclana (brown blades) and Clumilms (red erect algae) w-ere found only as germlmgs. Algal crusts (all orders) were relatively rare. The subtidal eelgrass com- munity may have contributed up to eight faunal and twelve algal genera. No significant changes in relative frequencies of algal orders were seen between 1-23 June 1988. There were shifts in the relative fre- quencies of algal morphologies over this period: blades and filaments alternately dominated, while ( 'hontlms. ( '< ulnini. and crusts exchanged subdommant positions. The preliminary study suggests a possible optimum in taxon diver- sity. Eighty-three percent of samples bore between four and nine ta\a per carapace (mean 6.3; range 1-13). Increases in taxa beyond five pel carapace were due mainly to the addition of like organisms: two genera of barnacles instead of one; three species of branched filament rather than one. Supported by NSF Grant BNS-8719325 to M.A. Sydhk Neurobiology i'.tlccts ot intracellular alien calcium clicliitors on trans- miller re/ease at the sc/uid xiant srn qumacnnc. a compound known to inhibit sodium channels in meta/oan cells 1 hesedata show that quinacnne is an additional tool for the stud> of the voltage-sensitive calcium channel in paramecium. I his work was supported by NIH grant CiM- 3W92 and NSF grant HNS S5-06778. BEE is a PEW Scholar in the Biomedical Sciences. Neurophysiological corn-lute* <>t ' *e.\ clillerence* in the \<>nic minor system of the midshipman, Porichthys no- tatus. ANDREW BASS (Cornell University) AND ROB- i R r BARER. In the sound-producing fish. I'/mchtkvs mnnlus. there are two classes of sexually mature males. A large size class (Type I) generates several types of "vocalizations" during the breeding season, unlike the smaller size class of males (Type II) and females which have not yet been found to be sonic. Previous studies have shown that the morphology of the central (sonic motor nucleus) and peripheral (swimbladder "drum- ming" muscles) components of the sonic motor system are similar in lype II males and females. In these studies all animals were acclimated to a temperature of 1 5°C. Surprisingly, sonic activity could be evoked in ((//classes of animals following midbrain stimulation and intracranial recording from exiting sonic motor nerves. A synchronous response was always evoked bilaterally in females and both classes of males. In- tracellular recordings of synaptic and action potentials from every sonic motoneuron in Type II males and females, as in Type I males, exhibited a direct 1:1 correspondence with each evoked response. Temperature over a range of 6-24°C was then used as a physiological variable to further compare sex differences in functional organization. At the same temperature, the fundamental frequency in Type II males and females was similar but significantly lower than in Type I males. These data indicate that the sonic motor system of females and Type II males are qualitatively similar to that of Type I males, but there is a detectable dilleience in discharge frequency . We conclude that the central neuro- nal circuitry in Type II males and females can generate a rhythmic sonic output despite dramatic sex differences in the si/cs of motoneu- rons. total muscle mass, and individual muscle diameter. (r-/>rnieiii\ modulate the calcium action potential inPaia- mecium calkinsi. JI\N BERNAI AND BARBARA EHR- 1 K lid 'imersity of Connecticut, Furmington, CT). Activation of voltage-dependent calcium channels initates backward swimming in I'tirumcciiim The duration of backward swimming was modulated by ( i-proteins in the marine paramecium /'urtiinci ntni «//- AmwlMcIlveen <•/S. anon- hvdroly /able analogue of GTP which activates G-proteins. was injected into the cell, the duration of calcium action potential was increased by '(id1 , . This effect was maintained over 20 min. GDPJS. a non-hydro- 1\ /able analogue of GTP which inhibits (i-proleins. decreased the dura- tion and amplitude of the calcium action potential. Five minutes after injection of GDP^S. the voltage response to a current pulse was re- duced to the passive properties of the cell. These results suggest that G- proteins are involved in calcium action potential modulation. We are in the process of doing voltage clamp experiments to clarify the mecha- nism of the modulation of calcium action potential by G-proteins. This work was supported by NIH grant CMS 39092. BEE is a PEW Scholar in the Biomedical Sciences. .-1 biologically inotivaieil artificial neural network. DAN- IEL ALK.ON (Laboratory for Molecular and Cellular Neurobiology, NINCDS, NIH), KIM TIPLITZ BLACK- WELL*. TOM P. VOGL*. VASSILIOS KOUNTOURIS*. Most artificial neural networks utilize neuronal elements whose pre- synaptic strengths are modulated by the output, and are set by a num- ber of different iterative, non-linear, or stochastic algorithms. The net- work described here, a dynamically stable associative learning (DYS- TAL) network, is based on a biological neural network: the convergent visual and vestibular pathways which mediate associative learning of Hermissenda iriMwi'ivmv. The response of these pathways to their pre- ferred stimuli are of fixed synaptic strength. In addition, there are loci of convergence which are modified by repeated presentation of tempo- rally related patterns. By virtue of unique learning rules, the DVSTAL network displays a number of desireable features some of which may be found in other models but which have not previously been combined. The network is self-adapting and the strength (weight) associated with each synapse is adjusted by a rule that only requires information regarding the pre- and post-synaptic neurons involved. A consequence of self-adaptation is that the network can he scaled for the solution of large problems with only a linear increase in computation time, even on a serial machine. Finally, the network can associate different patterns which are pre- sented sequentially. The network was trained by presenting to the 3 by 3 input array either "T" or "E". The network was tested and was able to properly classify (distinguish) incomplete "T"s and "L~"s. In addition, when presented with an ambiguous pattern (one which could be either a "T" or a "C") the network responds with the union of the patterns (T.) when trained to associate "T"s and "C's. and with the intersection of the two ( P) when no association had been formed o1 presynaptic Ca channel* at active zone* <>/ ///c squid giant \r//sr /.'I/ analv*i* <»/ active :one (liMrihitiion in a tiiru-2 imaging *pecimen. JoANN BU- CHANAN (Yale Univ. Medical School), GEORGE J. AUGUSTINE, MII.TON P. CII\KI ION. Luis OSSES, AND STEPHEN J SMI in. We have pel formed cunelalive light and electron microscopical studies of the distribution of active /ones in the presynaptic terminal of the giant synapse I lie goal of tins study was to test the hypothesis that ( 'a channels are clustered at piesv naptic activ e /ones by comparing * I nviionmcnlal Research Institute of Michigan. Arlington. VA 22209. ABSTRACTS FROM MBL GENERAL MEETINGS 315 the distribution of active zones to sites of Ca influx. This was accom- plished by examining a squid giant synapse preparation which had been injected with the Ca indicator dye, fura-2, to study the pattern of Ca influx following presynaptic stimulation (see Smith cl al. 1988, Biol. Bull. 175: 311). Following physiological studies, the preparation was immersed in 2"< glutaraldehyde in 0.1 A/cacodylate butter containing 0.8 M sucrose and further processed for electron microscopy. Thick sections (2 ^m thickness) were cut through the 800 /jm-long synapse, digitized, and reassembled with computer-aided reconstruction meth- ods. At this level of resolution it was possible to visualize sites of synap- tic contact along the synapse; the distribution of contacts was closely correlated with the longitudinal distribution of Ca influx sites detected with fura-2 in the living synapse. In addition, thin sections were col- lected at 50 ;jm intervals through the synapse and photographic mon- tages were made to examine the distribution of active zones around the perimeter of the presynaptic terminal. It was found that the number of active zones in the region of the presynaptic terminal nearest the postsynaptic axon was 10-times greater than in the region away from the postsynaptic axon. This closely corresponds to the lateral gradients of Ca influx identified in the fura-2 measurements. These two findings demonstrate a close correlation between active zones and sites of Ca influx and therefore support the hypothesis that Ca channels are clus- tered at active zones. Supported by Howard Hughes Medical Institute funds. NIH grant NS-2 1624, and MRC (Canada) funds. Identification and characterization of peptidergic m/;«,'<> pcalei. The enzyme was readily extract- able from squid tissues using 50 mA/ Tris buffer pH 7.4 containing 0.5% (w/v) Triton X-100 and in optic lobe ODH had Km's (mA/) for pyruvate and L-arginine ofO.2 and 0.9, respectively. Optic lobe activity was 24.6 ± SD 3.5 ^mol/min/g wet wt. (n = 4) at pH 6.6 and 25°C using 4 mA/ each of pyruvate and L-arginine, and 0.09 mA/ NADH. This was 15 times the LDH activity in this tissue using optimal assay conditions. Tentacle muscle and retinal fibers had comparable activit- ies to optic lobe, whereas mantle muscle was approximately four times higher. Tissues with much lower activities included testes, sperm sacs, gill, hepatopancreas. giant axon, and fin nerve. In muscle, optic lobe, retinal fibers, testes, and gill, ODH activity was eight times or greater than that of LDH. This suggests that ODH rather than LDH plays a major role in energy metabolism in many tissues of/., pealei. Subcellular fractionation of optic lobe showed ODH to have a classi- cal cytoplasmic distribution with the synaptosomal fraction containing 20% of the activity in a 100% occluded form. Supported by Travel Grants awarded to MJD from the Royal Society and the Wellcome Trust. 316 NFt'ROBIOLOGY Protein synthesis in the ifiiini ii \on anil in nerve endings of the sQiiul v >ITTA (Dept. General Physiology, i Naples, Ital>). ENRICO MENICHINI. IMIM\ ( -.SIK.II. AND BARRY B. KAPLAN. Axonsarcgener.. Uk the system of protein synthesis. In accord with this view. th.- svmhcsis ol' axoplasmic protein b> the isolated giant axon ol" the squid Kiiuditta <•;,;/ I%X. /'\ '.IS 59: I2.X4- 1287) was attributed to the activity of penaxonal glial cells, from which newly made proteins are transferred to the a\on (Lasek ci ill 1977. ./ Cell i • ~* However, our experiments demonstrate that polysomes earning nascent peptidc chains are present in squid a\o- plasm. Cleaned giant axons. still associated \vith intact giant a\on sys- tems, were incubated in Millipore-liltcrcd seawater containing lOOjiCi/ ml"S-meihionme( I hat20-22°O Radioactive polysomes were puri- fied from extruded axoplasm by sedimentation through a layer of 2 M sucrose and fractionated on 15-45 sucrose gradients I he radioactive polysomal profile was shifted to the top of the gradient by treatment ol the rcsuspendod pellet with RNase or EDI A ( ycloheximide (50 ^g/ ml) strongly inhibited the labelling of axoplasmic polysomes. while chloramphcnicol (50wg mltdid not induce appreciable changes. 1 he occurrence of protein synthesis in a purified fraction of optic lohcsynaptosomesl Hernandez cl at. 1976. Aaa <. icni \ cnci.11: 120- 123) was contirmed by demonstrating "S-methionine incorporation into protein. The incorporation was linear for at least one hour, and dose-dependent in a limited protein concentration range. Cyclohexi- mide was strongly inhibitor,, while chloramphenicol induced only a limited degree of inhibition. Preta-atment of the synaptosomal fraction with RNase (ID Mg/nil) did not affect incorporation, while hypo-os- motic shock completely abolished protein labelling. Strong inhibition was also induced by depolan/mg conditions ( 100 ml/ KC'l). I hese data demonstrate that protein synthesis occurs in the giant axon axoplasm and that a similai process may be present in nerve end- ings. lleiniluhynnilieclomy und selective ololith lesion synip- iom\ in the thnlish \\IK\IK GRAF- AND ROBERT BAKI R (Marine Biological Laboratory. Woods Hole, \1 \ (12 Dunng metamorphosis. lUitlish tilt '»()" to one side or the other to become bottom-adapted adult animals. In this position, the labyrinths are rotated 40" relative to their prcmctamoiphic orientation in space. In this attitude the utncultis is le.ist sensitive to small displacements. and therefore the other otoliths. in particular the sacculus. have been suggested to substitute for the alleged inscnsitmty ol the utriculus. Nev- enheless. the utncular nerve h.is the largest caliber by far of all labyrin- thine nerve branches. In this context, we tested the relative contribution of the two labyrinths and single otolitlucendorgans to maintain posture in the adult winter flounder. r\ciiil«im\!i's iiincru utnn Ilcrni- labyrinthcclorny was either performed by central ncurectomy ol the labyrinthine nerve brain hes m by removal of the entire labyrinth via a lateral appi". i ! B Ided similar results. Lesions of the down-side labyrinth (n :dlv resulted in any abnormal move- ment. In some • ' iralled towards the lesioned side as observed in upright fish. I I ol the up-side labyrinth, in con- trast. produccddram.il .'ilisui 1 4 1. I he animals per- formed several full / fore settling on the tank floor. If "translated" into uprii'l i-nicnt. this response could he described as a rotation about , ntl il axis (/iun from the lesioned side. Thus, two fundamental d I .-observed when comparing the hemilabyrintheetomy involving the up-side labyrinth in the adult flatfish to thai of upnfht lish I MM .. , /ju;a,'<' in tin,', lion of the lesion symptoms from spiralling movements about the rostro-cau- dal axis to rotations about the doiso-ventral axis. Second, a change of laleralilv from bodv mov cnients towards the lesioned side to rotations away from the lesion. Similai obscivalions were made after hemilaby- rintheclomy of the up-side labyrinth in two adult summer flounders, l\nuti, luln N dentalUS, a left-sided flatfish. Selective neurectomy of the utncular nerve pioduced identical body movements as those following hemilabyrintheetomy (up-side: n = 3: down-side: n -- 1 ). Selective sac- culus (up-side: n = 4) and lagena lesions (up-side: n = 2) produced movement patterns inconsistently or not at all. Thus, the utriculus re- tains us original functional role in respect to posture. In general, lesion symptoms were not pronounced but could be exacerbated by neurec- tomy of the optic nerves. We interpret the peculiar bilaterally asymmet- ric labyrinthine orgam/ation ol the adult flatfish to be of fundamental importance for maintaining the flatfish as a flatfish Supported by \lllgrant NS20^S Sliuly ol' the resting conductance ot the squid a.\on niem- hrane. J. R. HUNT AND D. C. CHANG (Baylor College of Medicine). Prev ious studies in this laboratory suggested that the resting conduc- tance which determines the resting potential (\u.,,l in the squid giant axon may he largely controlled by a pathway different than the delayed rectifier K channel. Because the resting conductance is very small, we developed a special voltage clamp procedure using extensive signal av- eraging to measure minute changes in conductance (g). This procedure consists of superimposing a 40 ms rectangular pulse with a series of small square pulses (±3 mV high. 1-mswidel I hus. the slope conduc- tance at a given potential can be determined directly from the current changes in response to the square pulses. Using this method, we mea- sured the membrane conductance over a wide range of test potentials. The Na channel was blocked by externally applied tetrodotoxin. While conductance al depolan/mg potentials was dominated by the delayed rectifier, at V more negative than the resting potential, we found a small component of K conductance (gK:( which had V dependence deviated from the Hodgkm-Huxley equations describing the delayed rectifier. Our preliminary results of the ion selectiv ity measurements ol gK; show that the relative permeabilities of gk: for K'. Rb'. NH4'. Na*. Cs*. and Li' ions are 1.0.0.71. 0.14. 0.13.0.10. and 0.04. respectively. We also determined the ion selectivity ol the resting membrane by studying the effects ol various ions on the \ '„,.., of the axon. The ion selectivity ofgK.: is qualitatively similar to that determined from Vrcsl. On the other hand, the ion selectivity of the resting membrane is known to be dillci- ent than that of the delayed rectifier. I or example. Cs' seems to be readily permeable to the resting membrane but is virtually imperme- able to the delaved rectifier. I his is not the ease for gKJ. Hence, based on pi opei ties of both the V dependence and ion selectivity, it is reason- able to interpret that g^, may he the maior pathway for resting current. Supported in part by NIH grant *NS25S()3-OI. Siereo\cof>y ol'lii.^li resolution video inicroseope images reconstructed from serial optical sections. SIIINYA IN- CH n (Marine Biological Laboratory) AND TED INOUE. We will demonstrate high-resolution stereoscopic video microscope images dvnamicallv. I he stereo-pan video pictures were obtained by reconstruction from through-local, veiv linn optical sections (Inoue I')SS. U,v/;.'[A ( ',•/! Hn'l 3d: in pi ess I stored on an optical memory' disk recoulcr K >MDR> I 01 images stored on theOMDR. and for intensity contours of micioscope images, stereo pans were reconstructed by shearing the image stack (Inoue and Inoue 1486. Ann. NY Acad. Sci. •4H3: 342 404) with an I mage- 1 /AT digital image processor (Universal Imaging Corp.. Media. I'M I he same processor compresses the left ABSTRACTS FROM MBL GENERAL MEETINGS 317 and right images each to half height vertically but not horizontally, and places them in the top and bottom halves of the video field. A Stereo- graphies processor re-expands and projects the left and right images alternately at 120 Hz as left and right circularly polarized images (Ste- reographies Corp., San Rafael, CA). Complementary filters worn by the observer provide striking, pseudocolor or monochrome, flickerless, dynamic stereoscopic images. Details of Golgi-stained neuronal pro- cesses and intensity contours of a human oral epithelial cell were pro- jected at the meeting. Supported by NIH grant GM 31617-07 and NSF grant DCB 8518672. Contractility of the squid stellate ganglion. CLAUDIA M. NUNO (Univ. Southern California). MARIA ELENA SANCHEZ, JOANN BUCHANAN, AND GEORGE J. AU- GUSTINE. Studies of synaptic transmission at the squid stellate ganglion some- times are impeded by endogenous contractions of the ganglion. We characterized these contractions to identify experimental treatments that selectively eliminate movement without altering synaptic trans- mission. Contractions were detected in isolated stellate ganglia of Lol- i,!>o pea/ei by using a fiber optic light collector and photodiode to mea- sure changes in light transmission associated with movement. With such an arrangement rhythmic movements of the ganglion were readily detected. The amplitude, but not the frequency, of these signals de- pended upon the position of the fiber optic collector. Electron micros- copy of the stellate ganglion identified two types of cells containing or- ganized arrays of contractile filaments: smooth muscle cells of blood vessels and unidentified cells dispersed throughout the ganglion. Either of these cells could be involved in producing ganglion contractions. Ion substitution and pharmacological experiments were used to ex- amine the ionic basis of the ganglionic contractions. Removal of Na ions from the extracellular medium had effects that depended upon the nature of the substance used as a substitute for Na. Replacement of Na with Tris rapidly and reversibly abolished movements, while replace- ment with sucrose slowed contractions but did not entirely eliminate them. The Na channel blocker tetrodotoxin ( 1 nM) had no effect on contractions. In contrast, removal of Ca ions (with Mg as a substitutel completely eliminated contractions. Inorganic Ca channel blockers. such as Cd (2 mM) and Mn (12.5-25 m.U). eliminated contractions, as did the dihydropyridine Ca channel blockers nitrendipine (10-20 M.U) or nimodipine (20 ^M). We conclude that Na ions play some role in generating movements, but are less important than Ca ions. Block- ade of contractions by Tns is likely to be due to a secondary pharmaco- logical action of this ion, rather than Na removal. Either Na or Ca could be involved in producing spontaneous electrical activity, while Ca might play some other role in excitation-contraction coupling. Of the several conditions that eliminate ganglion contractions, dihydropyri- dines are the only treatments which do not also affect synaptic trans- mission. Supported by NIH grant NS-2 1 624. Multiple site optical recording from small ensembles of Aplysia californica neurons in culture. A. L. OBAID, T. D. PARSONS, AND B. M. SALZBERG (University of Pennsylvania School of Medicine). Cell culture of 2-dimensional ensembles of identified invertebrate neurons permits one to construct truly simple "nervous systems" in a dish. We have consistently observed a wide variety of synaptic interac- tions in such preparations, using conventional electrophysiological techniques. These networks are uniquely amenable to study by means of multiple site optical recording of transmembrane voltage (MS- ORTV). We have used the potentiometric dye RH-155 and a 12 x 12 photodiode array to record changes in extrinsic absorption, at 700 ± 25 nm, from up to 9 neurons simultaneously. These optical signals provide a faithful representation of voltage changes at 124 loci during trials of 8-s duration. Rhythmic electrical activity is observed in many cells and connecting processes, in the absence of any electrical stimulation. This activity may be endogenous, or it may be light induced. In either case, computer averaging over spike occurrences in individual detector ele- ments reveals complex correlations in the MSORTV records. These experiments suggest that multiple site optical recordings from small ensembles of interconnected neurons may help to unravel the intricate relationships that characterize even the simplest nervous systems. We are grateful to Tom Capo and the Howard Hughes Medical Insti- tute for providing us with juvenile Aplysia, to David Kleinfeld for intro- ducing us to the Aplysia culture system, and to Larry Cohen and his co-workers for their generous provision of software for data acquisition and analysis. Supported by USPHS grants NS 1 6824 and H L 07499. Electrical connections to cultured invertebrate neurons using nnilttelcctrode arrays. WADE REGEHR (Bell Labs, Murray Hill, NJ), YUAN Liu, MICHELLE MISCHK.E, AND B. M. SALZBERG. Recently it has become possible to culture a variety of invertebrate neurons from leech (Hirtulo medicinalis), slug (Aplysia californica), and snail (Hclisoma mvo/iv.v). The ability to form small networks of synaptically connected identified neurons has facilitated the study of synaptogenesis and neuronal development. LIsing conventional elec- trophysiological techniques it is difficult to study more than 2 or 3 neu- rons for longer than several hours. For cultured neurons extracellular recording and stimulation is difficult, since the medium shunts the cur- rent How to ground. Multielectrode arrays (Thomas 1972, Exp. Cell Rc\ 14: 6 1 -66; Pine 1980. ./ \cnnnci Methods 2: 19-3l;Gross 1982, J. Nmrosi'i. Methods 5: 1 3-22) overcome these limitations. These pla- nar arrays consist of 6 1 electrodes embedded in the bottom of a culture dish. Both the conductive leads and the insulation are transparent, making the dishes compatible with voltage-sensitive dyes and inverted microscopy. Large invertebrate neurons from Aplysia. leech, and Hcli- soma, grown on the array, form seals over dish electrodes. Action po- tentials have been recorded simultaneously from many neurons for up to two weeks, with signal-to-noise ratios as large as 500: 1 . The signals recorded from electrodes under cell bodies are essentially derivatives of the intracellular potential, while signals recorded from electrodes sealed under axon stumps are primarily due to active channels. Multielectrode arrays have also been used to stimulate neurons and to record the re- sulting action potential. While it is relatively easy to record action po- tentials, it is difficult to record post-synaptic potentials because of their small size and slow rate of rise. By improving the seal to tens of meg- ohms it should be possible to record fast post-synaptic events. We are grateful to Tom Capo and the HHMI for providing juvenile Apl\-\ui. Supported by USPHS grant NS 16824 and Grass Foundation Fellowships to W.R. and Y.L. Clustering ofpresynaptic Ca channels at active zones of the squid giant synapse: fur a- 2 movies of Ca influx. STEPHEN J. SMITH (Yale Univ. Medical School), GEORGE J. AUGUSTINE, JOANN BUCHANAN, MILTON P. CHARLTON, AND Luis OSSES. Recordings of fluorescence changes from intracellularly injected fura-2, a calcium indicator, were used to test the hypothesis that Ca channels at the giant synapse of Loligo pealei are localized at presynap- tic active zones. A SIT video camera and an analog video disc recorder were used to record fluorescence images, which were subsequently ana- 318 SI 1 R( IBIOI ' ><,1 lyzed using a digital vid ith excitation light of 3.Mi nm wavelength, lluorcv.-- order of 25 were observed at the end 01 I trains. With 350 nm excita- tion, fluoro ) under the same conditions These optical change-, likelv reflect complexation of fura-2 with C'a ions. At earl> tin stimulation, these indicator sig- nals were local i • i -ij'lasmic C'a signals were more diffuse, prohablv reflecting diffusion ot'Ca ions away from Ca channels clustered at active /ones The patterns of early fluorescence change after action potential stimulation were consistent with the hypothesis that Ca channels clus- ter at active zones within the presvnaptic membrane of this giant pre- svnaptic terminal. Supported by Howard Hughes Medical Institute funds. NIH grant NS-2 1624, and MRC (Canada) funds. (i-proieins in the si/uiii Loligo pealeii: characterization, i/iiiiniiitition, and examination ofpossihle role in axo- mil transport. STEVEN S. VoGEL (Columbia Univer- sit\ ). STEPHEN D. HESS, AND THOMAS S. REESE. G-proteins have been implicated in mediating exocytosis. If they are m\ohed in release of transmitter from synapses, we might expect them to be transported to. and enriched in. svnaptic terminals. We used the giant axon and a synaptosome preparation from squid to examine this idea. Using pertussis toxin (PTX) catah/ed [i:P]-ADP-ribosylation fol- lowed by SDS-PAGE. we find that optic lobe synaptosomes contain 2S pmoles ADP-nbose/mg protein in a M, 40.000 protein. Synaptosomal labeling was enriched hv a factor of 1.7 over optic lobe homogenate. Homogenates of giant axon had 0.68 ± 0.07 pmoles ADP-ribose/mg protein (n = 3) while extruded axoplasm had 0.32 ± 0.01 pmoles/mg i n 1 1. It is now possible to calculate that 44' ; of the axonal G-protein is in axoplasm. To determine whether Ci-protems are transported to terminals, we cooled a I mm segment of axon to 4°C for 2 h while maintaining the rest at 16°C. Because transport is blocked at low temperatures, we ex- pected a build-up of antcrograde vesicles and a depletion of retrograde vesicles on the proximal side of the cold block (and the opposite on the distal side). Labeling in homogenates made from 5-mm segments just proximal to the block was consistently greater than in homogenates in comparable segments just distal to the block (6 of 7 experiments): with- out the cold block, labeling in both segments was the same. The build up of G-protems on the proximal side was not correlated with any change in C'oomassie staining including neurofilamenls. actin. and tu- bulm In one experiment, with three 2-mm segments on each side o! the block, there was a build-up of G-protein on the proximal side, and a depletion on the distal side of the block. I ' i squid nervous system contains PTX sensitive G-protcins that are concentrated in synaptosomes anil transported to terminals b\ last axonal transport. S.S.V. i i a 1 988 Grass Fellowship. Channel 'ill muscle \arcopla\nuc relieii/nin an- o/>ene,/ i <\nol 1 ,4,5-trisphosphate and arc 111- J. \\ \ i K \s \\D B. E. F.IIKI ic n (Uni\crsit\ ofO iicut. 1 arminglon. (T). Inositol 1.4.5-tnsiilr: la beer nnphcateil as the link be- tween excitation and conti n smooth muscle However, the mechanism of the stimulation In II'. w.r uiul.-.n \\ e have lound a novel channel in the sarcoplasmic retieulum (SK) ol amtie smooth muscle that is opened b> IP. and inhibited b> hepann. This channel is guile different from the calcium release channel in SR from skeletal and cardiac muscle Channel properties were determined by measuring calcium release from SR vesicles with the calcium indicator antipyry- la/o III and hv measuring calcium currents through single channels that had been incorporated into planar lipid hilavers. IPi actuation of calcium release from aortic SR vesicles occurs at low concentrations of IP, (Ko< = I it.M) and activation is specific. Several other inositol phosphales (inosilol l-phosphaie. inosilol 1.4-bisphosphaie. inositol 1,3,4-trisphosphate, inositol l,3,4,5-tetrakisphosphate;each at 20/j.U) and caffeine (1-50 m.U) did not initiate calcium release from these vesicles. Activation of the channels incorporated into bilayers were ob- served at 0. 1 n\l IP,. In addition. IP, was only effective when added to one side of the bilayer showing that channels inserted preferentially in oneorientalion. These results contrast with those obtained with skeletal SR in which I m \1 caffeine induced calcium release and activated channels incorporated inlo planar hilayers. IP, (20 ^M ) aclivaled nei- ther calcium release nor channel activity in the bilaver with vesicles made from skeletal SR. Rulhenium red (1 p.\l) inhibiled caffeine-in- duced calcium release from skeletal SR but 20 /i.U ruthenium red did not affecl IP,-induced calcium release from aortic SR. Similarly, only skeletal SR channel activity was inhibited by ruthenium red. Heparin ( 10/ig/ml) inhibited both calcium release and channel activity in aortic SR but concentralions 100 limes larger were ineffective in skeletal SR assays. These results support the hypothesis that IP, is the trigger for contraction in smooth muscle. This work was supported by NIH grant HL-33026 and GM-39IW2. BEE is a PEW Scholar in the Biomedical Sciences. I 'oltage sensitive dye recording from the abdominal nerve cord of the American cockroach. JIAN-YOUNG Wu, HANS-PETER HOPP. CHUN XIAO. AND LARRY COHEN (Yale University School of Medicine). Recently Yagodin. Pushkarev. and Slutsky reported that thev were not able to measure voltage-sensilive dye signals from axons in insect nerve cord. We have tried to repeal this experiment using a microscope and 1 24 elements photodiode array to measure absorption signals from the abdominal chord of the American cockroach. Periplaneta anicri- ctina. The cereal nerve was stimulated with a suction electrode and ac- tion potentials were also monitored b> an extracellular electrode. In agreement with Yagodin cl nl . when we used intact (non-desheatlied) preparations, none of the 1 2 different dyes we tried penetrated the sheath and no signals were detected. However, when the nerve cord was partially desheathed and ihen incubated in dye solution for 30 minutes, the axons appeared to be stained. Two oxonol dyes, RH474 and RH4S2. provided by RHUI Hil- deslieim and Amiram Cirinvald. gave absorption signals when the cer- eal nerve was stimulated. These signals could he detected in a single trial. We think these signals are real because il the stimulus inlensily \v.is i educed or the polantv icversed. the signal disappeared. The signal also disappeared if a wavelength which was not absorbed bv the dve was used. When we reduced the stimulus intensity so that there was only one all-or-none event on the electrical recording, we could nol deln I .in optical signal I hus the signal vv.is not large enough to detect a single action potential from a single axon. Wilh the stimulation paradigm and recording arrangement we used, onlv axons which receive excilaton svnaptic connections from the cereal sensory libers in the (ith abdomi- nal ganglion, and ascend acioss the Mil abdominal ganglion would be dele, led furthermore, the stimulus was less than maximal. Thus we llnnk thai onlv a iclativelv small fraction of I he axons in the cord gener- ated the signals w Inch we dele, led Supported In l'IIS('ianI \S(IS437. ABSTRACTS FROM MBL GENERAL MEETINGS 319 Optical recording oj membrane potential changes from single neurons with intracellulur dyes. CHUN XIAO (Yale University) AND DEJAN ZECEVIC. We investigated the absorption and fluorescence optical signals from individual neurons from the suboesophageal ganglion of Helix n\f>cr\u and the segmental ganglion of Hirudo medicinalis selectively stained by intracellular application of voltage sensitive dyes. We looked for op- tical signals that are large enough to allow the analyses of regional prop- erties of neurons in the intact isolated ganglia. The preparation was positioned on the microscope stage and its im- age was formed by 25 • . 0.4 NA long working distance objective. In the plane of the magnified image we positioned the 12 • 12 array of photodiodes for multi-site recording of transmitted or fluorescence light intensity changes that correspond to changes in membrane poten- tial. In absorption measurements, negatively charged oxonol dye (JPW 1034) gave largest signals when applied from outside in both He/i.\and Hirudo, No signal occurred when dye was injected into the neurons. Photodynamic damage with this dye was relatively pronounced in leech neurons where it prevented extensive averaging. Fluorescence optical signals from intracellularly stained Helix neurons were obtained using positively charged styryl dyes RH461 and RH437. With RH437 we recorded absorption signals but the signal-to-noise ratio was not as good as in fluorescence. Improvement in dye structure, staining protocol, and measuring technology is needed to increase the resolution in order to record optically from dissociated neurons in culture and to record from individual cells in intact ganglia. We are grateful to L. B. Cohen, B. Salzberg, and A. L. Obaid for valuable discussion. Supported by NIH grant NS-08437. CONTENTS BEHAVIOR GENERAL BIOLOGY Amano, Shigetoyo Morning release of larvae controlled by the light in an intertidal sponge, Callyspongia ramosa 181 DEVELOPMENT AND REPRODUCTION Chan, Siu-Ming, Susan M. Rankin, and Larry L. Keeley Characterization of the molt stages in Penaeus van- namei: setogenesis and hemolymph levels of total protein, ecdysteroids, and glucose 185 Dunn, Kenneth W. I he effect of host feeding on the contribution of endosymbiotic algae to the growth of green hydra 193 Koob, Thomas J., and David L. Cox Kgg capsule catechol oxidase from the little skate Raja erinacea Mitchill, 1 825 202 Sella, Gabriella Reciprocation, reproductive success, and safe- guards against cheating in a hermaphroditic polv- chaete worm, Ophryotrm hn iliuilrma Akesson, 1976 212 Hirose, Euichi, Yasunori Saito, and Hiroshi Wata- nabe A new type of the manifestation of colony specificity in the compound ascidian, Botrylloides violaceus Oka 240 Smith, Douglas G. A new, disjunct species of triclad flat worm (Turbel- laria: Tricladida) from a spring in southern New England 246 PHYSIOLOGY Byrne, Roger A., Robert F. McMahon, and Thomas H. Dietz Temperature and relative humidity effects on aerial exposure tolerance in the freshwater bivalve Carbi i ii/n /liiiiiniri: ................................. Frank, Tamara M., and James F. Case Visual spectral sensitivities of bioluminescent deep- sea crustaceans ..................... Frank, Tamara M., and James F. Case Visual spectral sensitivity of the bioluminescent deep-sea mysid, Gnathophausia ingens . 274 McDermott, Michael P., and Philip J. Stephens Fiber types in the limb bender muscle of a crab -v) ....................... 284 253 2fil Sl.im.i. Karel A new look at insect respiration ECOLOGY AND EVOLUTION ABSTRACTS 6 Foighil, Diarmaid, and Douglas J. Eernisse ( .I-D^I, ijiliM ,ill\ \\idrsprcad, non-hybridizing, sym- |).in K si i.i i us ni the hermaphroditic, brooding dam fa in the northeastern l*a< ilic Ocean 218 Porter, James W., and Nancy M. Targett .Mli-lniln-mu.il iiiii-i.ii lions Kciuccii sponges and corah 230 Abstracts of papers presented at the General Scien- tific Meetings of the Marine Biological Laboratory 301 CHI biology Comparative and general physiology . Developmental biology and fertili/ation . . 310 I- 1 ology Neurobiology Volume 175 THE Number 3 BIOLOGICAL BULLETIN Marine Biological Laboratory LIBRARY t JAN 19 1989 Woods Hole, Mass. DECEMBER, 1988 Published by the Marine Biological Laboratory Marine Biological Laboratory ; LIBRARY JAN 1 9 1989 Woods Hole, Mass. THE BIOLOGICAL BULLETIN PUBLISHED BY THE MARINE BIOLOGICAL LABORATORY Editorial Board GEORGE J. AUGUSTINE. University of Southern California RUSSELL F. DOOLITTLE. University of California at San Diego WILLIAM R. ECKBERG. Howard University ROBERT D. GOLDMAN, Northwestern University EVERETT PETER GREENBERG, Cornell University MICHAEL J. GREENBERG. C. V. Whitney Marine Laboratory. University of Florida JOHN E. HOBBIE. Marine Biological Laboratory LIONEL JAFFE. Marine Biological Laboratory HOLGER W. JANNASCH, Woods Hole Oceanographic Institution WILLIAM R. JEFFERY. University of Texas at Austin GEORGE M. LANGFORD, University of North Carolina at Chapel Hill LOUIS LEIBOVITZ. Marine Biological Laboratory GEORGE D. PAPPAS. University of Illinois at Chicago SIDNEY K. PIERCE, University of Maryland RUDOLF A. RAFF. Indiana University HERBERT SCHUEL. State University of New York at Buffalo VIRGINIA L. SCOFIELD, University of California at Los Angeles School of Medicine LAWRENCE B. SLOBODK.IN. State University of New York at Stony Brook K.ENSAL VAN HOLDE. Oregon State University DONALD P. WOLF. Oregon Regional Primate Center 1'Jnor CHARLES B. METZ. University of Miami c Editor: PAMELA L. CLAPP. Marine Biological Laboratory DECEMBER, 1988 Printed and Issued by LANCASTER PRESS, Inc. PRINCE & LEMON STS. 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Tahles, footnotes, figure legends, etc. Authors should follow the style in a recent issue of I lie Bi< deiapod i i iisiai cans: llniiuinn inn, 1 1< iiiin\ ynd Penaeus japonicui Cowles, David I... and |ames J. Childress Swimming speed anil <>\\i;en i inisiiiiiplioii in llie bathypelagii m\sid Gnathophausia / No. 1, At i.i M I'.iss I Gade. Gerd I- net n\ met a 1 10 1 ism dm mi; anoxia and t ecoxei \ m shell adduclol and loot muscle ol the gastropod mollusc Haliotii luiiii lln\n lot malion ol (he no\cl anaeroliii end pi odnc I lam opine ] 2'J Giebel, Gail E. Muir. Glyne U. Thorington, Renee Y. Lim, and David A. Hessinger Control of cnida discharge: II Mtc i obastc p-m.isii- Ij- gophoi c ncm.ilcx \sts ate- I ci; iil.H ed li\ I ui i c lassc-s of chemoreceptors I3'_' K. ill. n Janine L., N. R. Rigiani, and H. J. A. J. Trompenaars -(| Aspects of cut I ammcnl ol (Jill cell acli\H\ and hemolymph glucose levels in < i a\ hsh 137 Meyer-Rochow, V. B., and M. I.indstrom Klec It oph\sioloi;ic al and hislole>i;ie al nbsei \, it lolls on tin- c\c- ol aclull . fern. lie Dimhli* ni/lihi i (( a usla- i ea. M.il.icostraca, (aimacea) Ill Roman, Domingo A.. Justa Molina, and I.idia Rivera Inorganic aspcc Is of tin- blood c hcmisti \ ol ascidi- ans. lomi c omposiiion. and I I. \'. and |-c in the blood plasma ol I 'MI in < lull /MM and . Ui nlin ill \jiin . . 1 5 I Zimmer-Kaust, Richard K.. Richard A. Glecson. and William E. S. Carr I he- behavioral response- ol spun lobsters to \ I I' evidence foi mediation b) I '--like- c hemosensory re- c c-plol s H'i7 SHORT RKPORT Wilkinson, Clive R., and Anthony ('.. Cheshire Growth talc- ol |. nil. Mi .ill coial led sponges allei III 1 1 m i u .me \llc-n . 1 7") NO. L1. 0< IOIU-K 1988 BEHAVIOR Amano, Shigetoyo Momingreleaseof larvae controlled by the light in ail intcrlid.il sponge. ( .nlh^inii^in nnii<>\u ......... DEVELOPMENT AND REPRODUCTION (itian. Sin-Mini;. 'Misan M. Rankin. and Larry I S I Dunn, Kenneth W. I In i Hi i i ol host I. c . In r; on the- contribution ol endosymbiotil ali;ae lo ihc- i;ioulli ol i;u-en India HIS Koob, I homas | and David L. Cox I ' i;i; capsule- catechol oxidase liom tbc little skate- R a , rinaren Mm lull. I M''. L'(i'_> Characterization ol ih< moll stages \n Penaeus van litillli'i: sclo^enesis and I n i • ii M ll.i Reciprocation, reproductive SUM ess. and sail guards against i IH-.U mi; ma hermaphroditii pol\ chaete \\ el- laria: I ri< laclida) from a spring in southern New Kngianci .................................... 246 PHYSIOLOGY Byrne, Roger A., Robert F. McMahon, and Thomas H. Dietz Temperature and relative humidity effects on ae- rial exposure tolerance in the freshwater bivalve ('.ni'/in iiln ftwninea 253 Frank, Tamara M., and James F. Case Visual spectral sensitivities of bioluminescent deep- sea crustaceans 26 1 Frank, Tamara M., and James F. Case Visual spectral sensitivity of the bioluminescent deep-sea niysid, Gnathophausia uigi'ii* 274 McDermott, Michael P., and Philip J. Stephens Fiber types in I he limb bender muscle of a crab (Pachfgrapsus < >-i'\) 284 Slama. Karel A new look at insect respiration 289 ABSTRACTS Abstracts of papers presented at the General Scien- tific Meetings of the Marine Biological Laboratory 301 Cell biology 301 Comparative and general physiology 303 Developmental biology and fertilization 310 Ecology 311 Neurobiology 312 No. 3, DECEMBER Hiss 321 DEVELOPMENT AND REPRODUCTION Quackenbush, L. Scott, and L. L. Keeley Regulation of vitellogcnesis m the fiddler crab, I '«i ECOLOGY AND EVOLUTION Best, Barbara A. Passive suspension feeding in a sea pen: effects of ambient flow on volume flow rate and filtering efficiency 332 Campos, Bernardita, and Roger Mann Discocilia and paddle cilia in the- larvae of Muliinn Ititrntlis and Sfii^nlci W«/I.\»;/H« (Mollusca: Bivalvia) 343 Fairweather, Peter G. Consequences of supply-side ecology: manipulating the recruitment of interticlal barnacles affects the intensity of predation upon them 349 Jasnow, Michael, Cynthia L. Crown, Stanley Feldstein, Linda Taylor, Beatrice Beebe, and Joseph Jaffe Coordinated interpersonal timing of Down-syn- drome and nondelayed infants with their mothers: evidence for a buffered mechanism of social inter- action . 355 MacDonald, B. A., and R. J. Thompson liiuaspc-c ific variation in growth and reproduction in latitudmally differentiated populations of the gi- ant s( allop Plti/njii'i ti'ii magellanicus (Gmelin) 361 Miyazaki, Jun-Ichi, Rei Ueshima, and Tamio Hira- bayashi Application of a two-dimensional electrophoresis method to the systematic study of land snails of sub- genus Liuli ii/ilui/'i/n v/ from southwestern Japan is- lands 372 Sebens, Kenneth P., and Julia S. Miles Sweeper tentacles in a gorgonian octocoral: mor- phological modifications for interference competi- tion . 378 PHYSIOLOGY Doeller, Jeannette E., David W. Kraus, James M. Colacino, and Jonathan B. Wittenberg Gill hemoglobin may deliver sulfide to bacterial symbionts of Solemya ivlum (Bivalvia, Mollusca) . . . 388 McFall-Ngai, Margaret, Lin Ding, James Childress, and Joseph Horwitz Biochemical characteristics of the pigmentation of mesopelagic fish lenses 397 Saffo, Mary Beth Nitrogen waste or nitrogen source? Urate degrada- tion in the renal sac of molgulid tunicates 403 VI i oNIENTS Siebenaller. Joseph '• ••n.i'. F. Murray F.Vollltillll.lM tl .ill. ill III .I binding 10 ill |>ioi lid Tablin. Fern. .. I In- tun - icbocyte m ilu- blood <>l - II. I In- .IHU-IMK \li- i Moski-lo lon: .1 inojpl' 30g) male and female l'cmu'ii\ set (ferns were obtained frozen from a commercial shrimp processor (Coastal Freezing. Port Aransas, Texas). One hundred cvestalks (22.25 g) were ground frozen in a mortar and pestle and extracted in distilled water. The extract was gently boiled for 5 min and then centrifuged at 10.000 • t,' for 30 min at 4°C. The super- natant tl evestalk equivalents/ml) was stored fro/en at 4°C. In \i\o am l>i/5', CO: at 4 psi. During a 4-h incubation at 3()°( '. the beakers were gently agitated on an orbital shaker. At the termination of the incubation, tissues were removed from the media, rinsed with chilled saline, and then homogem/ed in ice cold extraction buffer. The tissue homogenates were cen- trifuged at 8000 > i; for 15 mm al 4°C. Proteins in the supernatant were precipitated bv the addition of 3 vol- UCA VG 323 umes of ice cold 100% (NH4)2SO4 and then recentrifuged at 8000 X g for 15 minutes at 4°C. The pellet of protein was resuspended in extraction buffer, and the entire pre- cipitation procedure was repeated twice more. The final pellet of protein was resuspended in PBS. Aliquots ( 100 jtl) of the solubilized proteins were taken for the determi- nation of both total protein and total I4C leucine incor- poration as described above. Differences between treat- ment groups were tested using a Mest (Sokal and Rohlf. 1969). Time course oj in vitro I4C leucine incorporation Tissues were dissected (n = \2;2 replicates) and incu- bated for 2, 4, 8. 16, or 22 hours as described above. At the termination of incubation, the tissues were rinsed with chilled crustacean saline, homogenized in 0.4 A' per- chloric acid and centrifuged at 2000 * g for 10 minutes at 22°C. The resulting pellet of protein was resuspended in 90% ethanol/1% sodium acetate and centrifuged as above. The resulting pellet of protein was retained and extracted in chloroform:methanol (2:1) and centrifuged as above. The final pellet of protein was air dried at 50°C and then dissolved in 1 A'NaOH. Aliquots ( 100^1) of the solubilized proteins were taken for total protein determi- nation and total I4C leucine incorporation as described above. Bovine serum albumin in 1 A' NaOH served as the standard for the protein determinations. Differences between groups were tested with a /-test (Sokal and Rohlf, 1969). I '/; /niri/it'iition and antibody production Uca pugilator vitellogenin was characterized as a large purple lipoprotein with two subunits of 100,600 and 1 25,000 daltons. This protein was about 90% of the total protein in crab ovaries (Eastman-Reks and Fingerman, 1984). We used a slight modification of the Eastman- Reks and Fingerman (1984) method to isolate vitello- genin from L'ca pugilator. Ovaries from crabs (0.10 to 0.25 mm oocyte diameter) were pooled and homoge- nized in ice cold extraction buffer. Phenylmethonyl sul- fonyl flouride (0.001%) was added to the extraction buffer just before use to inhibit general proteases. Ovar- ian proteins were extracted as above using 3 cycles of ex- traction buffer and ice cold 100% (NH4)2SO4 precipita- tion. The final protein pellet was resuspended in PBS (2 mg/ml) and applied to a Sephadex G-200 column (2.5 cm X 20 cm; void volume = 18 ml with blue dextran). The protein extract retained the purple color characteris- tic of the crab ovaries and eggs. This characteristic color aided us in the purification procedure. The column was eluted with PBS (4 ml/h) at 4°C. 1-ml fractions were col- lected. Column fractions were monitored at 280 nm, and fractions with the characteristic purple color of crab ova- ries were pooled and dried on a rotary evaporator (Speed- Vac, Savant Instruments. Hicksville, New York). Sam- ples of this crude ovarian protein extract were character- ized on 7% polyacrylamide gels. Sodium dodecyl sulfate was added to the protein sample and the polyacrylamide to dissassociate the large proteins into subunits (Hames and Rickwood, 1981). The crude extract was dominated by 2 bands of protein in the size range of vitellogenin characterized by Eastman-Reks and Fingerman, 1984. A preparative gel procedure was used to purify enough of the crude material from crab ovaries for rabbit immuni- zation. Ovarian protein samples from the G-200 column were resuspended in a dissassociating buffer with sodium dodecyl sulfate. This was applied to a large preparative gel of 7% polyacrylamide (1.0 X 150 X 130 mm) in a single well which spanned the entire width of the gel (Hames and Rickwood, 1981. Laemmli, 1970). After the tracking dye eluted from the gel. a single vertical slice of the gel was removed and stained for proteins with 0.5%' Coomassie Blue to determine mobilities of the proteins within the preparative gel. The remainder of the prepara- tive gel was sliced horizontally ( 1-mm segments); the re- sulting gel segments were placed in an elution buffer of 0.05 m.U ammonium acetate. Proteins were eluted from the gels into the buffer at 4°C for 24 hours. Proteins that eluted from the gels were freeze dried and stored at -4°C. Samples of the proteins eluted from the gel segments were characterized on another analytical 7% polyacryl- amide gel with sodium dodecyl sulfate buffers. This pro- cedure allowed us to obtain sufficient quantities of par- tially purified Vg for immunization procedures. The mo- lecular weight of the Vg subunits were determined by comparison to standards simultaneously separated in analytical polyacrylamide gels of various percentages. Molecular weight standards were obtained from Bio Rad and used according to the instructions provided. Myosin (200,000), beta-galactosidase (1 16,250), phosphorylase- B (97,400). bovine serum albumin (66,200), and oval- bumin (42,699) were the protein standards used to cali- brate the subunit molecular weight of ovarian proteins. Serum was obtained from rabbits before immuniza- tion via ear vein puncture. Rabbits were immunized with 50 subepidermal injections (20 n\ each) of complete ad- juvant containing 40 jig egg yolk protein/ml as described previously (Quackenbush and Fingerman, 1985). Rab- bits were bled each week via cardiac puncture. Whole blood was allowed to clot then centrifuged at 10,000 X g for 30 minutes at 4°C. The supernatant serum was stored at -72°C. Serum was screened against partially purified egg yolk proteins using Ouchterlony plates (Ouchter- lony, 1949). Serum showing a precipitation line against partially purified egg yolk proteins was further purified. Positive serum was passed through a column of DEAE- Affi-Gel-Blue (Bio-Rad) to separate the IgG fraction 324 I s 01 \( kl \BI SH \\[) I I kl I I M from other serum componc"1 \ column (14 ml hod volume) was pre|- flowing methods de- scribed in Bi 062 (Bio Rad). fractions amtainn aliquoted (0.5 mg/ml). and dried on uitor. The antibody stocks were diluted with PB> containing 0.01% sodium a/ide for use in the assays. Antibody characterization Rabbits were immunixed with either the high molecu- lar weight Yg suhunit (V,) or the low molecular weight suhunit (\ •). Since the hepatopancreas samples con- tamed only \':. onl> the antibodies against V; were char- acteri/ed. Antibody characterization included Ouchter- lony plates, immunoprecipitation. and western blotting. Extracts in PBS of gonad (100 ^g/ml), hepatopancreas llld ^g'ml). and hemolymph (110 j/g/ml) from both male and female crabs were tested against the antibody in Ouchterlony plates ( \'"< agar in PBS). Extracts of go- nad. hepatopancreas. and hernolymph (100 jug/300 ^1) were incubated at 4°C for IX hours with 10 n\ of rabbit serum. The mixtures were then centnfuged at 8,000 X g at 4°C for 10 min and the resulting protein pellets washed w ith ice cold PBS 4 times. Pellets of immunoprecipitated protein were solubili/ed in electrophoresis buffer con- taining sodium dodecyl sulfate and separated on a !'"< polyacnlamide gel. This assay should recover the pro- teins that the antibody recognizes and precipitated from the crude extracts containing several different proteins. Extracts of gonads and hepatopancreas from females in s itellogenesis were separated on a !'"< polyacrylamide gel in sodium dodecyl sulfate. The gel was then electroblot- tedonto nitrocellulose paper (Tow bin ct nl .. 1979; Doug- las and King. 1984). The rabbit serum was incubated with the filter paper blots for 2 h in PBS. A goat anti- rabbit IgG antibody with peioxidase conjugated to the antibody was then incubated with the blots in PBS (Kir- kegaard and Pern, (iailhersburg. Maryland). I he blots were then stained with 4-chloro-l-napthol (Sigma Chemical Co. St. Louis. Missouri) and H:O:. This west- ern blotting procedure was used to determine if the rab- bit serum bound selectively to the V: proteins contained in these extracts, when it was challenged with all the different proteins in the crude extracts. Immunoassay The immunuassav consisted of a 4-h ;// vitro incuba- tion as described ' I issues were then homogenized in extraction butler I ins were extracted with 3 cycles of extraction hullei and K > old 10(1'- i \l I ,) S( ),. The final protein pellet was ^suspended in 600 ^1 of PBS. Miquolsl 100 ^ll were taken hum this solution lor deter- mination ol total protein and ( leucine incorporation. rifty M' of the rabbit serum (25 ^g protein) was added to the remaining 400 n\ of each tissue homogenate and the mixture was incubated at 4°C for 18 hours. Afterincuba- tion. the tissue homogenates were centrifuged at 8000 x g at 4°C for 1 5 minutes. The supernatant was carefully removed, and 100 n\ aliquots were taken for determina- tion of total protein and I4C leucine incorporation. The pellet of immunoprecipitated protein was washed four times with ice cold PBS. The final protein pellet was solu- bili/ed in 500 /j| of 1 Y NaOH. and aliquots of 100 n\ were taken for determination of total protein and I4C leucine incorporation. Differences between treatment groups were tested for significance with a /-test (Sokal andRohlf. 1969). GIH purification Samples of crude /•*. u'//7 Ml values are means ± one standard error. n = 12 for each group. 2 replicates ture of protein svnthesis. A 4-h incubation time was used for all subsequent assays. Crude shrimp eyestalk extracts \\crc tested for their ability to atl'ect /';; vitro protein synthesis in the ovaries from intact and eyestalk-ablated crabs (Fig. 4). The threshold tor statistically significant inhibition of protein svnthesis uas the same regardless of the origin of the ovarian tissues (0.006 FSI . /-test for two percentages. /' OOH Maximum inhibition of the protein synthesis was produced b> 0.05 FSI in ovaries from eyestalk ab- l.ited crabs. l'i it n sv nthcsis in ovaries from intact crabs was maximal: ii , bited by 0.0 1 2 ESE. Ovaries from the intact crabs were more sensitive to the evcstalk extract than ovaries I: .] crabs. \ dose of 0.012 FSF produced a sifmlican .iler inhibition in the ovaries from intact crabs than , d in the ovaries from evestalk ablated crabs (/-test for i '-mages./'- o.(>5). I he highest dose tested. O.os I \i ;.,nduccd less, not more, inhibition of protein synthesis lli.ni a lower dose I Ins suggests that the crude extract probably contains many factors, perhaps even a factor that can increase protein synthesis /// w'Zro (Charniaux-Cotton, 1 1>S5). I 'itellogenin intrittcation and antibody characterization The partially purified extract of ovaries from lator was dominated by the two distinct bands of protein characteristic of the egg yolk proteins (Eastman-Reks and Fingerman. 1984: Fig. 5A. B). The molecular weights of the two groups of egg yolk proteins were 1 03.000 ± lOOOdaltons. V,. and 8 1,000 ± lOOOdaltons. V: (n = 12 measurements. Fig. 5 A, B). These bands of protein in the polyacrylamide gels clearly represent two classes of proteins, which each may contain several yet unresolved distinct polypeptides. The hepatopancreas extracts contained only the V: group of egg yolk proteins (n = 8; Fig. 5 A. B). Both V, and V: are the dominant proteins of the egg yolk, they stain positive for both lipid and sugars, and they contain the purple pigment of the egg yolk. Thus, these proteins, V, and V: fulfill the cri- teria established for vitellogenin (Eastman-Reks and Fin- german, 1984; Wallace end.. 1967). The antiserum (#1790-12-4) to the V: protein group Uca pugilator in vitro 80r 6O c o 20 0 .OOO6 .012 .025 05 Eyestalk Equivalents l-iuiiri- 4. I he /'/ vilrn inhibition of incorporation of IJ( leucme i nil MI\, n i, m proteins In crude shrimp esestalk extract. Ovarian tissues from intact crahs (open circles) or evestalk ablated crahs (tilled circles) were tested All \aluesare means • one standard error, n = 8 for each group. _ replicates. An extract of shrimp tail muscle uas used fora control for evestalk exlr.icls ( ontrol injections were adjusted by dilu- tion with crab saline to ei|ual the total protein concentration of the micctions ,,l evestalk extract. Percent inhibition was calculated as de- scnhed in Materials and Methods. UCA VG 327 reacted with crude extracts from female crab gonads, hepatopancreas. and hemolymph in Ouchterlony plates. The antiserum did not produce any reaction with similar extracts of tissue from male crabs. Therefore the antise- rum was specific for female proteins (data not shown). The antiserum precipitated both V, and V: proteins from ovary extracts, but only V2 proteins from hepato- pancreas extracts (Fig. 5 A). Thus when the antiserum was presented with crude tissue extracts containing many different proteins and some breakdown products of large proteins, the antiserum only precipitated the \^ and V: proteins. The western blot of proteins from crude tissue extracts showed that the antiserum selectively bound to only the V: proteins in the hepatopancreas (Fig. 5C). The antiserum did bind to some low molecular weight proteins other than V; in the ovarian homoge- nate. The antiserum had low affinity for any of the other proteins known to be in these crude extracts (Fig. 5A-C). Thus this antiserum (#1790-12-4) was specific to female proteins, and relatively specific for V, and V: proteins from tissue extracts. The immunoprecipitation of both V, and V: from the ovarian tissue extracts suggests that these two proteins may be linked in the ovary. The antibody was used to measure ;/; vitro Vg synthe- sis in homogenates of ovaries, hepatopancreas and he- molymph (Table I). The ovary had more immunopreci- pitatable protein than either the hepatopancreas or the hemolymph, consistent with the role of the ovary as a yolk storage site. However, the I4C leucine content of the immunoprecipitated Vg was similar in both the ovary and hemolymph samples, suggesting that both ovary and hemolymph can produce new egg yolk proteins. Recov- ery of all the proteins and radioactive labeled amino acid was near 90% for these assays. Some label and protein was lost due to the procedures used. Though the amount of immunoprecipitated Vg was less in the hepatopan- creas than the ovary, the Vg in the hepatopancreas had about three times the I4C leucine incorporated into Vg than the ovary. This is consistent with the hepatopan- creas as a site of Vg production but not Vg storage. Ovary, hepatopancreas, and hemolymph can incorpo- rate 14C leucine into new egg yolk proteins, the hepato- pancreas incorporates much more than the other groups, whereas the ovary seems to retain more Vg than either the hepatopancreas and the hemolymph. Partial purification ofGIH Fractions from crude shrimp eyestalk extract were tested for their ability to inhibit in vitro ovarian protein synthesis. The values for immunoprecipitated Vg are given in Figure 6 and some values for non-specific inhibi- tion are reported in Table II. Eighty-six percent of the inhibitory activity applied to the column was recovered 200 116 97.4 66.2 42.7 7 % SDS PAGE Uca pugilator 12345678 Ov Std Hp Ov Std Hp Ov V P P 2 1234567 200 116 97.4 66.2 42.7 7 ' , SDS PAGE Uca pugilator Western blot 200 116 v 97.4 66.2 42.7 Hp Ov •- Hp Hp Std V V Ov Std P 2 1 Figure 5. Isolation of egg yolk proteins from ovaries (Ov) and hepa- topancreas (Hp) from the tissues of Uca put;ilali>r A. Proteins from crude extracts of ovary and hepatopancreas. Immunoprecipitates from these crude extracts of ovary (Ovp) and hepatopancreas (Hpp) are com- pared to the complete extracts and a sample of partially purified V, (V:). In the sample of ovanan proteins the antibody precipitated both V, and V:. but only V: was precipitated from the sample of hepatopan- creas. The location of the molecular weight standards are labeled on the left side in daltons • !()' B. Isolation of egg yolk proteins. Lane 7 contains a crude ovarian homogenate. Lane 6 contains the fraction with purple color from the G-200 column. Lanes 5 and 4 contain the proteins eluted from the preparative polyacrylamide gels, labeled V, and V, respectively. Lane 2 contains a crude extract of hepatopancreas. while lane 1 is the immunoprecipitated protein from this hepatopan- creas homogenate. C. Western blot of a T"r. polyacrylamide gel. Samples of crude ovarian and hepatopancreas extract were separated in a poly- acrylamide gel. a sample of V: was run in the third lane. The polyclonal antibody to V, bound to a single band in the hepatopancreas sample, and to three bands in the ovarian sample. The antibody did not bind to V, , though it was present in the ovarian sample. in the 35 fractions that were tested (5 replicates). Three peaks of inhibitory activity were consistently resolved (fraction #29, Rf = 0.48; fraction #35, Rf =0.41; and fraction 64. Rf = 0.23, 5 replicates). These fractions had 64% of all the inhibitory activity applied to the column. All three fractions had about the same specific activity (2200 ± 300 units/mg; 5 replicates), fraction 29 at Rf = 0.48 had the most protein (3 j/g ± 0.7 jug; 5 replicates). The size of proteins eluting at fraction #29, Rf = 0.48 was estimated to be 3.300 ± 500 (5 replicates). This is a smaller size estimate for GIH that the GIH previously isolated from the lobster. Panulirus argus, (Quacken- bush and Herrnkind. 1983). Based on the bioassay, the column chromatography produced a 37-fold purifica- 328 L. S. QUACKENBUSH AND L. L. KEI I I N. Ic I vipn^-. • ablati Sample Proteins (mg) Hepatopancreas llemolymph ("rude I Pellet Supernatanl Sample ' ii 04 (1.421 ±0.05 0.55 ±0.06 0.045 ±0.01 0.401 ±0.02 DPM/MG/Hour 0.50 ±0.01 (1.014 ±0.01 (1.390 ±0.01 Ovarj Hepatopancreas Hemolvmph Crude Extract Pellet Supernatant 5.611 ± 1.065 865 - 181 2.973 ± 1.208 8,536 ± 1.425 2.977 ± 755 3.803 ± 567 1.448 ± 123 792 ±71 778 ± 79 Ml values are means ± one standard deviation, n = 12 for all cases, 2 replicates. C'rude extract is the initial tissue homogenate, Pellet is the protein precipitated bv the antihod> to vitellogenin, Supernatant is the protein not precipitated bv the antibod>. Recovers of both label and protein was between SO-901 ; tor these assays. lion from the crude material (traction #29. Rf = 0.48). Fraction -2^ inhibited Vg 14C leucine incorporation. It was specific to Vg proteins. Fraction #29 had no signifi- cant effect on I4C leucine incorporation into proteins other than Vg. represented by the supernatant in the im- munoassay (Table II). Both the crude starting material and material from the column void volume inhibited MC leucine incorporation into both Vg and non-Vg proteins, demonstrating non-specific protein synthesis inhibition (Table II. Fig. 6). Based on the si/e estimate for fraction #29 (3.300 ± 500 daltons) the material in this fraction was biologically active at 1.8 < 10 ".I/ protein. Discussion The primary source of crustacean egg yolk proteins was first suggested to be extra-ovarian (Wallace cl V Ganion and Kessel, 1972; Wolin cl til. 1973: Schade and Si ivers, 1980). Direct demonstration of pioiem synthi o\anan tissue supported the new view that the • ontribution to overall egg yolk protein sv nth mficant. I he relalivclv slow rale M| ovarian prolcm s in isolated ovarian tissue supported the argument >'iat extra-ovarian tissue also contributed to egg voU | i"trin svnlhesis (lui and O'Connor. 1976. 1977: Fasiman-Reks and F'ingerman. 1985). Ovarian egg yolk protein synthesis does not preclude extra-ovarian egg yolk protein synthesis. The unstated assumption in previous work was that egg yolk proteins in crustaceans were produced exclusivelv in a single tis- sue, as in insects and vertebrates. Insects produce vitello- genin exclusively in ihe fat body A similar paltern was expected in arthropod relatives, the crustaceans. In the isopod. Iilotcu haihica /uM/rr;, and Ihe amphipod, Or- c/u'\iui gammarella, egg yolk proleins are produced in a subepidermal adipose lissue. Ihe fat body (Blanchet- Tournier, 1982: Souty and Picaud. 1984). However, vi- tellogenin is also made in the ovaries of the crab Pacliy- ,i,'// labeled leueine in isolated ovarian traxnient.'i from the fiddler crab I Uca pugilator,) (:i Inhibition of protein incorporation of labeled leueine G-25 Fraction # Pellet Supernatant Crude material 64%* 22%* # 15 (void volume) 64%* 22'-- * #22 6% 4% #29 64%* 6% Rf=0.48 #31 48%* 14% #35 58%* 12% #42 7% 10% #52 5% 6% #64 28%* 12% #70 3% 5% All values are means, n = 8 for each fraction from 4 replicates. An * indicates a statistically significant difference between the fraction tested and a muscle extract control (t test for two percentages. P < 0.05). One unitofGIH activity produces a 20'"; inhibition of protein incorporation of labeled leueine, by definition. Crude material is the initial eyestalk extract: all test does were adjusted with saline to a protein concentration of 10 fig/ml. Fraction numbers in this table correspond with fraction numbers in Figure 6. Fractions were selected from a complete data set (35 fractions) as representative of the observations. //; vitro. Comparisons of protein synthesis in the tissues from Uca pugilator to other crustaceans is limited by the important variations in the reported methods, proce- dures, and other variables. Ovarian incorporation of la- beled umino acids into egg yolk proteins from Procamba- nis sp. (477 DPM/mg/h) and Piiiln^rapsiis m;.v.s//>c.s (966 DPM/mg/h) were suggested to be too low to ac- count for complete vitellogenesis (Lui and O'Connor, 1977). Incorporation of labeled amino acids into egg yolk proteins in the hepatopancreas of the fiddler crab (2927 DPM/mg/h) was more than three times the incor- poration of labeled amino acids into egg yolk proteins in either the ovarian tissue or the hemolymph. This signifi- cant difference (/-test, P < 0.05) suggests that the hepato- pancreas in the fiddler crab can contribute to overall egg yolk protein production. The demonstrated role of the crustacean hepatopan- creas is the synthesis and secretion of digestive enzymes (Gibson and Barker, 1979). This tissue is also a major site of lipid storage and carbohydrate metabolism (Chang and O'Connor, 1983: Sedlmeier, 1985). In the lobster, Homarus americanus, the hepatopancreas is the principal tissue source of hemocyanin synthesis (Senk- beil and Wriston, 1981). Eyestalk factors can affect lipid metabolism, protein synthesis, enzyme synthesis, and ri- bonucleic acid synthesis in the crustacean hepatopan- creas (Fingerman et ai. 1967; O'Connor and Gilbert. 1968; Gorell and Gilbert, 1971; Bollenbacher el ul.. 1972: Wormhoudt. 1974: Momin and Rangneker, 1975). In our study, eyestalk ablation significantly in- creased both in vivo and in vitro incorporation of labeled amino acids into proteins of the hepatopancreas. Crude extracts of eyestalks decreased protein synthesis in the hepatopancreas of the crayfish, Orconectes virilis, but in- creased ribonucleic acid synthesis in the hepatopancreas of the crayfish, Procambarus clarkii (Fingerman el ai, 1967; Gorell and Gilbert. 1971). Both protein synthesis and egg yolk protein synthesis in ovaries and hepatopan- creas appear to be affected by the eyestalk endocrine sys- tem in crustaceans. The coordination of egg yolk protein synthesis in several tissues by the eyestalk endocrine sys- tem would be one mechanism to optimize the energy in- vestment required for the production of many yolk- laden eggs. Insects produce egg yolk proteins exclusively in the fat body (Downer and Laufer, 1983). Fiddler crabs can make egg yolk proteins in at least three sites: ovaries, he- patopancreas, and hemolymph. Other crustaceans ap- pear to produce egg yolk proteins from several tissues as well (Blanchet-Tournier, 1982; Charniaux-Cotton, 1985: Fingerman. 1987). One hypothesis for these fun- damental differences among the arthropods may be linked to the differences in life histories. Pterygote insects do not molt as adults, whereas most adult crustaceans continue to molt. Molting is a physiologically demand- ing process requiring extensive protein synthesis and lipid metabolism (Chang and O'Connor. 1983; Skinner, 1 985). The repetitive egg yolk production of adult crusta- ceans which live for several years is an equally demand- ing physiological process. The mature ovary of a fiddler crab is 4-6% of the total body wet weight; the ovary con- tains about 30-40 mg of egg yolk proteins (Webb, 1977). During the reproductive period in the summer, a single female crab will produce two broods of several thousand eggs (Webb, 1977; Christy. 1978). The eyestalk endo- crine system is capable of regulating both molting and ovarian development with inhibitory factors so that these two process do not occur simultaneously (Webb, 1977; Adiyodi, 1985). The established need for the syn- chronization of egg release or egg hatching to systematic environmental variation further constrains the produc- tion of mature oocytes (Hartnoll, 1969; Christy, 1982). Fiddler crabs precisely time the release of larvae to opti- mize their survival (Bergin, 1981: Christy, 1982). Both physiological and physical limitations may require that the massive egg yolk protein synthesis be completed quickly. Therefore, using several sites to synthesize egg yolk proteins may be one strategy to maximize both so- matic growth and reproductive output within these con- straints. The diversity of crustacean life histories and the habitats they exploit make this a testable hypothesis (Christy, 1982; Hartnoll, 1969). 1 s oi \( kl \Bl Ml \ND L. L. KEl I M The partial!}. purified C.I! 1 inhibited egg \olk protein synthesis directK ' varies. Use of antibod) to Vgallo\ isurementofeggyolk protein production cific assay is a significant im- provem, is assays for CilH which were based on tern synthesis or simply ovarian wet weight ch -mirski i-i nl.. 1981;Quackenbush and Herrnkind. I9f I astman-Reks and Fingerman. 1984: C'harniauvCotlon. 1985). The development of a bioas- sa> for CilH or am inhibitory hormone requires ade- quate controls to detect the potential toxic effects of non- specific agents that mav be present in crude extracts (Channing ci i Bliss and L. H. Mantel, eds V i.lrmic Press. New ^ oik Beams. II. \\ .. and R. d. kesscl. 1963. lilectron microscope studies on developing crux fish oocyles with special reference to the origin ofyolk../ Cell Hiol 18:621-649. Herein. M. 1981. Hatching rhythms in t/ca pug/'teor \hii liiol. 63: 151-158. Blanche!- 1 ournicr, M. h. 1982. Quelquc aspects des interactions hor- monales cnlre la mue et al \itellogenese chez le Crustace Amphi- pode, Orcheslia gammarella. Reprod \un /Vi 22:325-344. Bolk-iihacher. \\ . h ., S. M. Hechner, and .1. I). O'Connor. 1972. Regulation of hpid synthesis during earl> premolt in deca- pod crustaceans. Cuiii/) Hii'thcni /'/irwii/ 42B: 157-165. Bomirski. \.. M. Arvndarc/k. K. kanainska, and I,. II. kleinhol/. 1981. Partial characterization of crustacean gonad-inhibhing hor- mone. Int. .1 Invertehr. Re/>roti. 3: 213-219. Bro»n. K. A. Jr., and G. M. Jones. 1948. Ovarian inhibition by a sinus gland principle in the tiddler crah. Rial Hull 96: 22S-232. (hang, K. S., and J. I). O'Connor. 1983. Metabolism and transport of carbohydrates and lipids. Pp 263-287 in The Biol<i s.', l-:\l> «/.-/ \lctl 178:339-361. C harniaux-C'otton. II. 1985. Vitellogenesis and its control in mala- costracan Crustacea. Am. /.on/. 25: 197-2116. ChrisH. J. II. 1982. Adaptive significance of semilunar cycles of lar- val releasein fiddler crabs (Genus L\'u): test of an hypothesis. Biol. Hull 163:251-263. C'hrisu, J. II. 1978. Adaptive significance of reproductive cycles in the fiddler crab. ( 'fa />ni;i Imor. a hypothesis. Sm'/uc 199: 453-455 ( ooke. I. M.. B. llajlctt. and I. \\oathvrh>. 1977. Electncalh elic- ited ncurosecreton and electrical responses of the isolated crab si- nus gland in normal and reduced calcium salines. ./. /-.'.v/> Iliol 7(1: 125-149. Douglas. (;.('., and B. K. king. 1984. \ filter paper sandwich method using small volumes of reagents for the detection of antigens elec- trophoretically transferred onto nitrocellulose. ./ lininunol Mcl/i- oafs 75: 333-338. I)o»ner, R. (;. II.. and 11. I.aufer. 1983. l.ndinrinoli^v ul //;MI/V Alan I.iss Inc.. New York. Kastman-Rcks, S., and M. Kinyerman. 1984. 1 tlects of ncuroendo- crine tissue and cyclic AMP on ovarian growth (// \i\<> and in vitro in the fiddler crab, Uca pugilatoi (.'onip liioclicm lin:\i«l 79 A: 674-6X4. Kastman-Reks, S., and M. l-inctrnian. 1985. In > -tiro sy mhcsis of vi- tellin by theovan of the tiddler crab, lea puxilator .1 /•. \/i /on/ 233: I I 1-1 16. KifldtT. I). R.. k. R. Rao, and M. Kinucrman. 1971. \ female-limited lipoprotein and the diversity of hemocyanin components in the di- morphic variants of the tiddler crah. ( 'ca pugilator, as revealed hv disc electrophoresis. Civn/' Hiochcni /'/MA;,./ 39B: 291-297. hinyerinan, M. 1987. 1 ndocnnc mechanisms in crustaceans./ Crux- nil-can H/ol 7: 1-24. I in".! m.iii M.. I . I >i mi i ii n /.i I. . M. Miya»aki,( . OgurO, and V . ^ ama- molo. 1967. Neuroendocrine control of the hepatopancreas m the crayfish, Procambarus clarkii l'h\^i«l /on/ 40: 2^ 'o (ianion, I . R.. and R.(i. ki-ssi-l. 1972. Intracellular synthesis. Trans- port and packaging ol proleiiiaceous volk in oocvtes nl OrconeCtfS //ii/im/iis. ./ Cell Hn>l 52:42(1-437. (iil>son. R., and I'. I. Barker. 1979. I he decapod hepalopancie.is Oi-ennot; \l,ir Hiol .Inn Her 17: 2X^ 116 (iorell, I. A., and I . (.illierl. I97|. I'loiem and RNA synthesis in premoll cravlish. Oreomrlt's wn/n / I \'l l'h\-\tol 73: 345 ^f> llames. B. I)., and 1). RicUood. 1981. del /.7/vWon oj Pro it-in^ ii I'rtii n, til l/>/>i-oii,-li IRI Press. Washington. I )< UCA VG 331 Hartnoll, R. G. 1969. Mating in the Brachyura. Crustaeeana 16: 161- 181. Kerr, M. S. 1969. The hemolymph proteins of the blue crab. Calli- necles sapuliis II. A hpoprotein serologically identical to oocyte li- povitellin. Dev. Bin/. 20: 1-17. l.aemmli, V. K. 1970. Cleavage of structural proteins during assem- bly of the head of bacteriophage T4. Nature 221: 680-685. Lui, C. \V., and J. D. O'Connor. 1976. Biosynthesis of lipovitcllin b\ the crustacean ovary. II. Characterization of and in vitro incorpora- tion of amino acids into purified subunits. J. E\p. Zool 195: 41- 52. Lui, C. \V., and J. D. O'Connor. 1977. Biosynthesis of lipovitellin. III. The incorporation of labeled amino acids into purified lipovitel- lin of the crab, Pachygrapsus crassipes. J. E.\p. Zoo/ 199: 105-108. Mi. linn. M. A., and P. V. Rangneker. 1975. Histochemical localiza- tion of oxidative enzymes in the hepatopancreas of Scyllu terrain (Forskal)(Brachyura:Decapoda). J. E.\r */"' Bin! Eeol. 20: 249- 264. O'Connor, .J. D., and L. Gilbert. 1968. Aspects of lipid metabolism in crustaceans. Am. Zool 8: 529-539. Ouchterlony, (). 1949. Antigen-antibody reactions in gels. II. Factors determining the site of the precipitate. .Irk kemi 1:43-50. Paulus, .1. K., and II. I.aufer. 1987. Vitellogenocytes in the hepato- pancreas ofCareiniismaenns and Libinia emarginata. Ini ./. Invert Reprod Dev. 11:29-44. Panousc, J. B. 1943. Influence d'ablation dc peduncle oculaire sur la croissance de 1'ovaire chex la crevette. Leander terrains. Compies RenduAcad. Sci. (Pans) 217: 553-555. Quackenhush, L. S. 1986. Crustacean endocrinology: a review. Can ./. Fish Aqua .SV; 43:2271-2282. Quackenbush, 1,. S., and M. Finnerman. 1985. En/yme-linked im- munosorbant assay of black pigment dispersing hormone from the tiddler crab. L'capiixilator lien Comp. Endocrinol. 57: 438-444. Quackenbush, 1.. S., and VV. F. Herrnkind. 1983. Partial characteriza- tion of eyestalk hormones controlling molt and gonadal develop- ment in the spiny lobster. Panulirus argns. ./. Crustacean Biol. 3: 34-44. Redd>. S. R. R., and G. R. \\yatt. 1967. Incorporation of uridine and leucine in vitro by Cercropia silkmoth wing epidermis during diapause and development. ./ Insect Physiol 13: 981-994. Schade, M. L., and R. R. Shivers. 1980. Structural modulation of the surface and cytoplasm of oocytes during vitellogenesis in the lob- ster. Honnirus ainencanns: an electron microscope protein tracer study. J Morp/iol 163: 13-26. Sedlmeier, D. 1985. Mode of action of the crustacean hyperglycemic hormone. Am. Zool. 25: 223-232. Senkbeil, E. G., and J. C. Wriston. 1981. Hemocyanin synthesis in theamencan lobster, Homiirns americanus. Comp. Biocliem. Pliys- /<>/. 68B: 163-171. Skinner, D. M. 1985. Molting and regeneration. Pp 43-146 in The Biology of Crustacea. Vol. 9. D. E. Bliss and L. H. Mantel, eds. Academic Press, Orlando. Sokal. R., and F. J. Rohlf. 1969. Biometry. Freeman Press. San Fran- cisco. Souty, C., and J-I-. Picaud. 1984. EfFet de 1'injection d'une gonado- tropin humaine sur la synthese et la liberation de la vitellogenine par le tissu adipeaux du Crustace Isopod marin, Idotea halluca bas- len Audouin. Gen Comp Endocrinol. 54: 418-42 1. Tom, M., M. Goren, and M. Ovadia. 1987. Localization of the vitel- lin and its possible precursors in various organs of Parapenaeus l<»if;ir<>\tri.s Int J Invenebr Reproii. Dev 12: 1-12. To« bin, II., T. Staehelin, and J.Gordon. 1979. Electrophoretic trans- fer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Set. 76: 4350- 4354. \\ allace, R. A.. S. L. \\ alker, and P. V. Hauschka. 1967. Crustacean lipovitellin. Isolation and characterization of the major high density Hpoprotein from eggs ofdecapoda. Biochemistry 6'. 1582-1590. \\ebb, M. 1977. Eyestalk regulation of molt and vitellogenesis in Uca pugilator. Biol Bull 153:630-642. \\olin, F. M., H. I.aufer. and D. F. Albertini. 1973. Uptake of yolk protein, lipovitellin by developing crustacean oocytes. Dev. Biol. 35: 160-170. \\ourmhoudt, A. 1974. Variations in the level of the digestive en- zymes during the intermolt cycle of Palaemon serratus: influence of the season and effect of the eyestalk ablation. Comp. Biochem. P/irsiol.49\: 707-7 15. Reference: Biol Bull I "5: ;:N ;-i: UVccmbor. l' Passive Suspension Feeding in a Sea Pen: Effects of Ambient Flow on Volume Flow Rate and Filtering Efficiency BARBARA A. BEST1 Depannu'nl <>t /.oology, Duke L'niversity. Durham, \orili ( 'urolina 27706 Abstract. An integrative analysis of passive suspension feeding is developed and tested. It emphasizes the func- tional role of overall organism design in enhancing the hydromechanic conditions necessary for feeding. Feed- ing rate, defined as the total number of particles captured per time, is a function of the ambient flow speed which independently affects both the \olume How rate and the filtering efficiency. In the sea pen I'liloMirits gurneyi. volume flow rate initially increases with increasing ambi- ent flow speed, peaks, and then decreases as the animal deforms with the flow. Filtering efficiency, for a given filter geometry, decreases with increasing velocity. How- ever, due to deformation of the filter with flow, higher filtering efficiencies are maintained as a result of the vari- able porosity tiller. Feeding rate is strongly dependent on volume flow rates. The feeding rate initially increases with increasing ambient flow, but then peaks and de- creases similar to the volume flow rate. Both volume flow rate and filtering efficiency depend upon the size of the organism and the relative position of the organism in the boundary layer. Introduction •Viualii. suspension feeding organisms use an array ol filtering elements to separate particles from the passing fluid nu-<: <\i\\ivc suspension feeders rely solely on the rclatr ni of their filter and the particle- laden watei foi ' .iplurc. In many sessile ami benthic Rccciu-il 24 IKvcmlx-i : .! 1 6 September ' Present address: On i i in li I ahin.ilniA. lil.uk 14-1412. C(ilumhi.i I nr.rrsils. < li.Mli Si . \eu York. York organisms, the relative movement is generated by the ambient flow, leaving them highly dependent on the am- bient flow for feeding. Active suspension feeders use cilia or muscles for pumping water past the filtering surface. This feeding may be augmented by the ambient velocity, which can be an order of magnitude greater than the self- generated currents (Merz. 1984:Okamura. 1984. 1987). Recently a few studies have begun to examine and clarify particle capture mechanisms in biological sys- tems. Particle size selection has been shown to depend on the diameter and spacing of the filtering elements, flow velocity past the elements, diameter of the particles, and surface chemistry of both particles and filtering elements (Rubenstein and Koehl. 1977; LaBarbera. 1978. 1984: Gerritsen and Porter. 1982). However, the most broad- scale aspects of filter feeding are still poorly understood, especially the functional role of overall organismal de- sign in maintaining the organism's exposure to flow and in influencing the hydromechanical conditions which aid in particle capture. In some stony corals the branch- ing pattern prevents the formation of stagnant zones within the colony. This assures adequate flow through- out (Chamberlain and Graus, 1975). In flexible organ- isms, their shape and exposure to flow depend on the flow velocity and the structure's response to drag forces imposed by the flow (Koehl. 1976; Patterson. 1984; Vo- gel. 1984). thereby changing the tnicroenvironment around the filtering elements (Harvell and LaBarbera, 1985). The present study emphasizes the effect of gross mor- phology on feeding performance in a particular suspen- sion feeder to highlight the potential interactions be- tween morphology and flow which may occur in a wide range of suspension feeders. The ecological consequence 132 PASSIVE SUSPENSION FEEDING 333 of functional morphology is equated with "feeding rate" which is denned as the total number of particles caught per unit time. In this analysis, feeding rate is a function of three parameters: ( 1 ) the volume of water processed by the organism per unit time, (2) the proportion of par- ticles removed per volume of water processed, (3) and the density of particles in the ambient water. The first two factors, the "accessibility" to flow and the "filtering efficiency" or proportion of particles retained, may both be influenced by the ambient velocity. For a benthic or- ganism, several factors may affect the ambient velocity it experiences, including its own size and height above the substratum into the boundary layer (Nowell and Jumars, 1984). This paper first describes an approach for examining the effect of structure-flow interactions on feeding. It then describes a series of field and laboratory studies of feeding rate in a passive suspension feeder which address these questions: ( 1 ) how does ambient flow speed inde- pendently affect volume flow rate and filtering efficiency, two components of feeding rate? (2) How are volume flow rates and filtering efficiency affected by organism size, as the organism grows and extends into a different ambient flow environment? (3) How does the structural response of the organism to ambient flow influence parti- cle capture on the level of the filtering elements, or polyps? Integrative analysis of suspension feeding An approach useful for evaluating the effect of mor- phological features on the feeding rate of passive suspen- sion feeders is presented. I first identify parameters (envi- ronmental and morphological) which may influence feeding and then define the relationships of these param- eters. For example, an analysis of feeding mechanics en- compasses several aspects of structural design which em- phasize the interaction between animal structure and ambient flow: ( 1 ) Structural features related to maintaining filter surface area exposed to the //on1. Drag imposed by the flow will tend to deform flexible organisms. In organisms which require flow through the filter for feeding, it is the drag-induced pressure drop across the filter that drives the water through the filter: hence, the organism cannot "hide" from the flow and must have support structures to maintain the filter in the flow. In organisms which uti- lize flow over or around the filter, such as in gravitational deposition feeders, filter surface area or position in flow must be maintained. (2) Structural features which siahilne the tiller in rela- tion to changes in /low direction or amplitude. For short- term fluctuations in current speed or direction, on the order of seconds to hours, the animal may stabilize the filter either by actively or passively re-orienting, and thus ensure proper exposure. For long-term fluctuation, from days to years, re-orientation and stabilization may occur via growth responses (e.g.. Wainwright and Dillon. 1969). (3) I- low-structure interactions related to particle cap- lure- Large scale features may pre-position particles be- fore they contact the filter (Craig and Chance, 1981), or pre-condition the water and its flow characteristics, such as flow speed, through the filter (Harvell and LaBarbera, 1985). The design aspects listed above all influence feeding. The first two aspects influence the volume of water pro- cessed by the organism and the last influences filtering efficiency. The total number of particles extracted per unit time is a function of the volume of water processed per unit time, the number of particles in the ambient wa- ter, and the proportion of particles removed per unit vol- ume of water processed, and can be expressed as the equations given in Figure 1. Structural and flow features can be identified which may affect either "accessibility" or "filtering efficiency" (refer to Fig. 1 ). For example, filtering efficiency depends on the flow pattern and particle paths around the filtering elements, and thus is a function of flow velocity around the elements: the size, spacing, orientation, and surface charge of elements; the diameter, density, motility, and surface charge of the particles; and possibly the fre- quency with which the elements are cleaned (Fuchs, 1964;RubensteinandKoehl, 1977; Spielman, 1977; La- Barbera, 1978, 1984:Gudmundsson, 1981). "Accessibility" can refer to the volume of water pass- ing either through a filter or in close enough proximity that particle extraction is possible. In organisms where the flow is quasi-perpendicular to the filter, as in the sea pen Ptilosarcus gurneyi. access to flow is equated with volume flow rate. It is equal to the surface area of the filter exposed to the flow times the velocity of flow through that area (refer to Fig. 1 ). The surface area of the filter exposed to the flow is a function of the organism's vertical stance and orientation to the flow; the shape, size, and flexibility of the filter; the ambient velocity and the drag imposed by the flow; the position of the organ- ism above the substratum and the presence of other or- ganisms. The influence of most of these parameters on accessibility in the suspension feeding sea pen Ptilosar- cus gurneyi is addressed in Best (1985, in prep.). Having identified both flow characteristics and mor- phological features which may affect feeding rate, I ex- amined the influence of flow velocity on both volume flow rate and filtering efficiency, with particular attention 334 B. A. HI s I | particles in proportion of ambient water * particles reta unit volui»« by filler Feeding Rate = accessrbi I > ty particle concentration » filtering efficiency Fi Itering Accessibi I > tj - surface area of filter * speed of flo> through filter area efficiency exposed to f lo« f(organis*'s vertical stance, filter's orientation to flow) f(drag forces acting on organism; support structures resisting deformation) f(ai»bient flow speed, organism's shape, size) f (relative position in boundary layer, presence of other organisms) proportion of particles retained by filt through the filter's boundary f (particle contact «ith element, retention by element) f (streaml i nes; particle diameter, density, motility and surface charge, surface charge of elements) f (velocity around elements, filter porosity, dependent on size, spacing and geometry of elements) f(ambient velocity; structural response of filter to flow) HUUIT I. Diagram of integral! vc meirnxl. show me relationships among paranu'tcrs. Soe text for discussion. to flow-structure interactions that change with ambient (low speed. bor Laboratories of the rniversits of Washington. Ani- mals were maintained in running seawater. Materials and Methods / \i'crimi'ntal on.w./v The sea pen hili>\urcu\ winicyi (C'nidaria: Pennatula- cea) was used in this study because it has a well-defined morphology which can be easily characterized on the ba- sis of size and age (Birkcland. 1969. 1974). I he upper portion of the organism, the rachis. contains the filtering elements — the polyps — which extend from the semi-cir- cular horizontal plates termed "leaves" (fig. 2). With an increase in ambient selocitv. the flexible rachis bends downstream (Fig. 2). The polyps form a semi-cylindrical filter on the downstream side of the organism, extending the length of the rachis (fig. 2). The height (\ertical stance! of the rachis was the indicator of organism si/e. The rachis behaves as a single unit which orients to the flow (Best. I9XS). ensuring water passage through the fil- ter in the same direction — into the concave side of the semi-cylindrical filter. This sea pen feeds prirnarils on phytoplankton (Birkeland. 1969). Its bright orange color is the result of carotcnoids incorporated from ingested dinoflagellates and can be passed on to the tissues of nu- dibranch predator (S Kemp, pers. comm.). /'///<»v/n ;,\ (,•;,»•//, vi is a common inhabitant of soft- sediment envi: - in Puget Sound and the San Juan Archipel. hington. from an initial length of under I mm. /' wnih TOW toa total length ol o\ci 80cm. extending over 40 <. ove the benlhic surface Sea pens were hand collcu d iiom lope/ Souml bv SC'UBA diving and transpmied back to the I ridas llai- \4orphometrics Photographs of organisms in the lield were compared to photographs of fully expanded organisms in the labo- ratory. Since there were no differences detected in stance or bod> si/e between laboratory and tield photographs, all morphometrics were performed in the laboratory. With a Wild dissecting microscope, polvp si/es in small (3-10 cm rachis height) and large (20-30 cm rachis height) sea pens were measured. For each organism, polyp width, tentacle width and length, and pinnule length and spacing were measured to the nearest 0.01 mm. Polyps from the bottom, middle, and top of the ra- chis from each sea pen were examined. Sea pens were placed in a large recirculating flow tank (working area of 32 by 34 cm: Vogcl and LaBarbera. 1978). continuously supplied with fresh seawater. I o ex- amine spacing between polyps as a function of flow speed, close-up photographs of the sea pens were taken as thev deformed under How speeds up to 25 cm s '. From these photographs, the spacing between the leaves and the dcnsitv ol'polv ps were determined. Pol\ p dcnsitv was calculated as the number of polyps cm in the plane of the filter, approximating a plane perpendicular to the direction of (low. Sea pens were studied within I ope/ Sound where wa- ter depth averaged 10 m and the bottom consists of san- ds-mud, lo characterize the How environment on the PASSIVE SUSPENSION FEEDING 335 Figure 2. Photographs of small sea pens. Pliluxtircit.i gumeyi. Left. View of sea pen showing the array of polyps on the downstream side of the rachis (rachis height = 4.5 cm). "J" -Joint. "P" - Peduncle. "L" - Leaf. Arrow points to internal style visible through body wall. Note the parallel leaves "L" through which water would flow. Right. Side view of sea pen (rachis height = 3.0 cm). "P" - Peduncle. "J" - Joint. "S" - Siphonozooids. "R" - Rachis. Arrow points to the top rigid portion of the style. Row is from left to right. scale of the sea pens, a thermistor flow probe (LaBarbera and Vogel. 1976) was used to record local flow speeds at 18 cm above the substratum, the average height of the sea pens. Flow direction was also continuously moni- tored with a weather vane/potentiometer (Best. 1985). Velocity recordings were made over 6-8 hour periods, including one low and high tide, on three different days. To construct a velocity profile and determine the range of velocities experienced by small, medium, and large sea pens, velocity measurements were taken at different heights above the substratum. Volume/low rates There is a postural change in Ptilosarcus gurneyi with increasing flow speed. The area of the feeding filter ori- ented perpendicular to the current also changes with the flow speed. Therefore, it was necessary to measure di- rectly the volume flow rate through the filter as a func- tion of ambient flow speed. Sea pens acclimated in the recirculating flow tank. An imaginary plane was established upstream of the organ- ism and horizontal transects across this plane were made with a fluorescein dye injector. For each horizontal tran- sect, it was noted for which points the dye, once released, subsequently passed through the filter of the downstream sea pen. Horizontal transects were spaced vertically ev- ery 1 .0 cm for large and medium sea pens and 0.5 cm for small pens. From these transects an area on the imagi- nary plane was determined, representing the source of water actually passing through the filter. The volume flow rate was calculated by multiplying this area by the water flow speed through the plane (area X speed = vol- ume time"1). Flow speeds in the flow tank were mea- sured with a thermistor flowmeter which had been cali- brated with an electromagnetic water current meter 336 » \ m s i (Model 511. Marsh-V.-Birnc . Inc.. with a precision ±2%). For each low rates were determined over a range ! to --s '-•m s ' lo' medium and large - cm s lor small pens. Deter- mination rates were repeated three times for a largi. nd small sea pen: replicated values were within 3' for the small sea pen and less than 6', for the Velocity through nuliis A thermistor How probe measured the flow speed be- tween the leaves and through the mesh-work of polvps as sea pens were exposed to ambient speeds of I to 25 cm s m the How tank. Filtering efficiency In the field, water samples were collected upstream and downstream of sea pens from three si/e classes: a small sea pen with a rachis height of 7 cm. medium sea pen 15 cm high, and large sea pen 25 cm high. For each sea pen three concurrent upstream-downstream water samples were taken by SCUBA divers positioned on ei- ther side of the organism. 1 ach diver held a transparent plastic tube, parallel to the current, either directly in front of the rachis or directly behind the rachis. at the same height. The tubes were held in place long enough to be Hushed with seawater several times. Simulta- neously the ends of both tubes were sealed. Three si/ed tubes were used for the different si/ed sea pens, with 3 cm. 5 cm. and 7 cm diameters, respectively Upon leaving the water, the divers transferred the wa- ter samples into clean bottles, stored them on ice in the dark, and immediatclv took them to the laboratory for analysis. In the laboratory each water sample was first tillered through a 163 ^m N'itex filter. An Fl/onc S()\Y particle counter (Particle Data. Inc.) was used to count the total number ol' particles in a 2 ml subsample. and to con- struct a si/e-licqucncv distribution of the particles into 12X particle si/e classes in the range 0- 100 ^m. 1 ive sub- samples wen- analv/ed from each upstream and down- stream held sample. The precision of counts from multi- ple subsamples is better than 5' To examine the effect of How speed on particle reten- tion, a lahoratoi uncut was conducted involving a medium-si/ed sea pen (rachis height = 12 cm). I he sea pen was allowed male in the recuvulaling llow tank. A filtrate of naln> il particles — containing phvlo- plankton and parliculaies ranriiH: liom I -SO ^m in di- ameter— was added in III ink. A dcnsiiv between 3000 4000 particles nil was maintained thimighoul the experiment. At each of three How speeds. 1.5, 3.0. and 6.0 cm s '. water samples (40-70 ml) were collected concurrently with a suction device (5 mm diameter opening) immediatelv upstream and downstream of the rachis. Three sets of water samples were taken at each velocity. In addition, experiments similar to the above were conducted with a medium sea pen feeding on one si/e range of panicles, bv adding only the chlorophycean phytoplankter Diinaliclla ( 10 /nin diameter) to the flow- tank. 1 he water samples from the laboralorv experi- ments were treated and analv/ed for particle counts in the same manner as in the field experiment, but with 1 ml subsamples. Filtering efnciencv was calculated from the difference in panicle counts from two samples, taken eoncurrentlv up- and downstream, and expressed as a percentage of the upstream count. Results Field velocities Flows at the field site were relatively steady, hydrauli- cally smooth and bidirectional, and changed orientation approximately 180° with the tides. The mean How speed at IS cm above the substratum ranged from 8 to 1 1 cm s '. I he maximum speeds ranged from 13 to 17 cm s"1. Over a 10 minute period, for a mean flow speed of 9.8 ± 0.42 cm s ', the turbulence intensity (standard dev ia- tion of the flow /mean How speed) was 0.043. At 8 cm above the sediment — the approximate height of small sea pens — the mean flow speed ranged from 3 to 7 cm s ' and the maximum speeds ranged from 8 to 10cm s '. Velocity readings taken over a few minutes at 5. 10, 30, 50. and 100 cm above the substratum showed mean flow- speeds of 6. 5. 7.7. X.7. 9.(>. and 10.2 cm s '. respectively Using the standard log layer equations for a geoplnsical boundarv layer and the linear regression of the above means, the boundarv shear velocity (u.) — a measure of shear stress acting on the bed and a useful parameter of the How field— was estimated to be 0.50 cm s '. Worphometrics Polvp width, the distance across the polvp between tentacle lips, is uniform throughout large sea pens and the middle and bottom regions of small sea pens (3.59 mm ' 0.157.H 25). The very top 30-40 polyps on the small sea pens are slightly larger with a mean width of 3.88 mm 1 0.2 I 7 (Student's /-test, i 2.34. a = 0.05, n 30). lentacle length ranged from 1.4 to 1.9 mm. with a mean of 1 .59 mm < 0. 1 3 (n - 30). The tentacle width was approximate^ 0.34 mm at its base and tapers to the PASSIVE SUSPENSION FEEDING 337 io E I io2 O) _5 £ io1 Large 0 2 4 6 8 10 12 14 16 18 2022 24 Ambient Velocity (cm/s) Figure 3. Volume flow rate as a function ot ambient velocity. The volume of water passing through the rachis initially increases as velocity rises, then peaks and declines due to the postural change of a sea pen. tip. Eleven to twelve pairs of pinnules project out along the length of the tentacle, increasing from a length of 0.07 mm at the tentacle base to 0.32 mm at the tentacle tip. These pinnules do not project laterally out from the tentacle, but are angled downstream. Pinnule width ranged from 0.03 to 0.05 mm. Spacing between pinnules ranged from 0.06 to 0. 1 3 mm. The spacing between the leaves, on the downstream side of the animal, was smaller in the small sea pens (3.8 mm mean spacing. 3. 1-4.2 range, n = 6 organisms) than in the large sea pens (5. 7 mm mean. 5. 4-5. 9 range, n = 7) in still water. With increasing flow speeds both small and large sea pens flex with the flow, resulting in a decreased spacing between leaves. At 1 3 cm s~ ', small sea pens had a mean spacing of 2.3 mm, range 2.6-2.8: large sea pens had a mean spacing of 3.9 mm, range 3.5-4.2. At 20 cm s~', large sea pens had a mean of 3.4 mm. range 3.2-3.6. In still water polyp density, in the plane of the filter, is 30.2 ± 1.76 polyps cm : (n = 12) in large sea pens and 29.4 ± 2.35 polyps cm~: (n = 12) in small sea pens. As ambient flow speed increases to 1 3 cm s~ ', polyp density does not change significantly in small sea pens. However, in large sea pens there is a significant increase in polyp density to 33.1 ±0.13 polyps cm~: (Student's /-test, / = 3.58, « = 0.05. n = 24). I 'olumeflow rates The volume of water flowing through the filter per unit time (volume flow rate) is dependent on both the ambi- ent flow speed and the area of filter projected into the flow as determined by the posture of the organism ( Best, 1985). As shown in Figure 3, the volume flow rate for a single organism increases with an increase in ambient flow until the organism becomes so bent back by the flow that more water tends to flow over rather than through the filter. The maximum volume flow rate, y (ml s~'), attained for an organism is best fit by a linear function (stepwise multiple regression analysis) of its rachis height, x (cm), where y = -208.4 + 54.6X (r = 0.93. n = 18). The flow speeds at which these maximum volume flow rates occur are also related to the organism's size. For small sea pens, the maximum volume flow rates occur at flow speeds ranging between 6.5 and 8.5 cm s~', between 12 and 14 cm s~' for medium sea pens, and between 14 and 1 8 cm s~ ' for large sea pens. I 'clocity through rachis and Reynolds number Figure 4 shows the flow speed between the leaves and directly behind the polyps in relation to the ambient flow speed. As the ambient speed increases, the flow speed be- tween the leaves and polyps also increases, but at a lower rate (slope of regression lines less than 1.0: for leaves, b = 0.55. ts = 8.58. t.05[6] = 2.31. P < 0.05; for polyps, b = 0.33. ts = 26.78. t.05I6] = 2.3 1 . P < 0.05). At an ambient speed of 25 cm s~'. the flow speed between the polyps is only 6-8 cm s~ ' , and 11-13 cm s~ ' between the leaves. For an object moving relative to the ambient fluid, the relative importance of inertial and viscous forces acting on the object — and therefore the nature of flow around the object — is represented by the dimensionless parame- 25 20 E o o _o 5 10 15 20 25 Ambient Velocity (cm/s) Figure 4. Flow velocities between leaves and behind the polyps as a function of ambient velocity. Note the much reduced flows occurring by the polyps. $38 B \ BIS I f * S M L 153060 Velocity (ems' I Figure 5. \ 1 ilteringefficienc) as a function of organism size. Fil- tering etticienc) is calculated as the percentage difference in particle counts from upstream and downstream water samples taken in the tield. Means • SH are shown for 15 paired water samples (2 ml) ana- I) A-d for each small "S". medium "M". and large "L" sea pen. Field flow speeds for the small sea pen were approximate!) 5-6 cms ' and 8- 9 cms ' for the medium and large sea pens. Panicle retention in the small sea pen is significant!) lower than the medium and large sea pens (2x2 G-test of independence. G = 56.6. « = 0.05) B. Filtering effi- cienc) as a function of ambient flow speed. Filtering efficient:) lor a single sea pen feeding in the flow tank at three different speeds. Means ± SE are shown for 1 5 paired water samples (I ml I. Particle retention decreases » ith increasing velocity (ANOVA, F2.M = 75.52. P < .00 1 . n 45). ter "Reynolds number" (Re). For medium and large sea pens, assuming the characteristic length to he the length of the rachis parallel to flow, the Reynolds number is around 8 X 10: to 1 X 104 for flows from 2 to 25 cm s ': for small sea pens the Reynolds number is around 2 • 1(): to 3 x 10\ However, due to the small size of the pinnules and tentacles (i.e., small characteristic length) and the reduced flows over these structures, the nature of the flow is very different as reflected in the Reynolds number (Re: approximately 1-10 for tentacles, and 0. 1- 1 .0 for the pinnules). Particle capture is occurring under low Rev nolds number, viscous flow conditions. l-'ilh'int" effli /<•//< i Figure 5 A shows the filtering efficiencies — the percent- age of particles present in the downstream water sample relative to the upstream sample — for the three dillerenl si/ed sea pens in the field. The small sea pen had the low- est retention of particles which was signitieanth different from the highei i trillions of the medium and large sea pens (2 • 2 (I-K ol independence performed on raw particle counts. (. 6,0 0.05). I he relationship of tillering elhciencv I" i mhicnt flow speed for one sea pen is shown in figure 5B • rllicicncv decreased lioin a mean of 42. X', *-. 3.5''< al a velocit) of 1.5 em s ' to a mean of 29.99! ' 5.19i at .1 u-locity of Ml cm s '. An \\()\ \ tailed In show a sii'inliranl difference in the number of particles in the upstream samples throughout the experiment (F..4J 1.62. I' > 0.21. n = 45). but did show a significant interaction between ambient flow speed and the number of particles in the downstream samples (F;44 = 75.52. !' « .001. n = 45). Filtering effi- ciency decreases with increasing velocity. The si/e-frcquencv distribution of particles in one set of upstream-downstream water samples taken in the field is shown in Figure 6; the difference between the area under these two curves represents the number of parti- cles removed during one passage through the rachis. Ev- ery field feeding test failed to show a significant difference in the size-frequency distributions of the upstream- downstream water samples (G-test for goodness of fit), implying that no particle size selection was occurring. However, for the laboratory feeding experiments, there were significant differences (G-test for goodness of fit) in the size-frequency distributions of every upstream- downstream comparison (Table I). The mean particle size in the downstream sample was smaller than that for the upstream sample, implying that larger particles had a higher probability of being retained. The difference be- tween the mean particle size of upstream-downstream samples increased as ambient flow speed increased (Ta- ble 1). suggesting a greater selection for larger particles at higher ambient flow speeds. Feeding performance The total number of particles removed from the ambi- ent water per unit tune, the feeding rate, is equal to the product of volume flow rate and filtering efficiency. Feeding rates, calculated from these two independent!) measured parameters, of a large, medium, and small sea pen are shown in Figure 7. assuming an ambient particle concentration of 1000 particles ml ' which is within the range of concentrations present in the field. Because fil- tering efficiencies were only measured tor flow speeds up — upstream downstream 10 15 20 25 30 40 5060 80 100 Particle size (microns) I- inured. Si/r liaiuciK \ disii i billion ol one sel ol 'upMream-dow n- strcam w.ilet samples from tile field for a 15 cm sea pen in 6 cm s How fhe total number of particles in 1 ml subsamples are shown. Note lh. -.imilarit) in curves: the si/e-fret|uenc\ distributions ,ue not sii'iuli cantly different (G-test for goodness of fit, G 80.5, x3»i >i 93.9, P - o. i ). PASSIVE SUSPENSION FEEDING 339 Table I Laboratory feeding experiments iliflerenees in panicle xi:c distribu- tions between paired upstream-downstream water samples Ambient flow speed Mean particle size (^m) Up Down G-test 1.0 cms ' 9.6 9.6 95.6* 1 0.1 9.7 219.0* 10.3 9.7 168.4* x = 10.0 9.7 3.0cm s ' 10.1 9.6 195.6* 10.3 9.6 231.2* 10.3 9.9 377.8* x= 10.2 9.7 6.0 cms"1 10.4 10.0 146.1* 10.2 9.9 145.1* 10.6 9.9 276.5* x = 10.5 9.9 Means tor each water sample are based on a count of 1 0.000 particles from natural filtrate. G-test for goodness of fit used to compare fre- quency distribution of 75 particle size classes! 1-100 Mm) of upstream and downstream water samples. G-test, XTOITJI = 93.9. * significant at « = 0.05. around the polyps, coupled with polyp density, influence the size range of particles retained by the filter. Size, flexibility, and volumeflow rale In many benthic communities, occupation of space above the substratum is at a premium (Jackson and Buss, 1975; Jackson, 1977; Sutherland and Karlson, 1977). Access to flow for these benthic organisms will depend on their protrusion above the substratum and the pro- jected area of the filtering appendages relative to the flow direction. Volume flow rates are dependent on ambient flow speed and filter size, both of which increase as the organism grows. Volume flow rate refers to the volume of water which passes through the filter "boundary" of an organism per unit time. For a sea pen. the volume flow rates initially increase with increasing flow speed, but then peak and decline due to deformation of the organism. The maximum vol- ume flow rate and the ambient velocity at which it occurs are both dependent on organism size. As the animal bends in the flow, more water passes over it rather than to 6 cm s 'in the laboratory, filtering efficiency values for higher flow speeds were estimated to decrease to 20% in the absence of rachis deformation (closed circles). From the field experiments, efficiencies are known to re- main around 30% (represented by open circles), proba- bly due to a decrease in filter porosity as the rachis bends downstream. Feeding rate is largely determined by vol- ume flow rate (compare Fig. 7 with Fig. 3); with increas- ing flow speed feeding initially increases, peaks, then de- creases. Discussion Structure-flow interactions Structure-flow interactions are important to suspen- sion feeders which depend on relative movement of food particles past a filtering structure. ( 1 ) In flexible organ- isms, the drag forces on the organism will change the shape and vertical stance of the filter. These drag forces are dependent on the filter shape, size, orientation, and flexibility. The absolute (rachis height) and relative (de- gree of bending) vertical stance of the filter determine the volume of water flowing through the filter. (2) Flexibility regulates the speed and pattern of the flow past the filter- ing structures, and thus filtering efficiency. Efficiency de- pends on the packing array and density of polyps, which can change with ambient velocity. (3) Flow patterns Loige 0> O O Q. I io4 I03 / Small • *-*-~""V . 0 2 4 6 8 10 12 14 16 18 2O 22 Ambient Velocity (cm/s) Figure 7. Feeding rate as a function of flow speed and rachis defor- mation. Using the determinations of volume flow rate and filtering efficiency, feeding rates of a large (rachis = 24 cm), medium (rachis = 15 cm), and small (rachis = 8 cm) sea pen are shown. Open circles represent feeding rates calculated using 30% efficiency at the higher flow speeds, as recorded in the field for medium and large sea pens; closed circles beneath the open circles represent feeding rates assuming that efficiency would continue to decrease to 20% at the higher flow speeds in the absence of rachis deformation. 340 » \ HIM through the rachis K..Jns flexibiliiv prevents drag from rising exponential! : p.) inamUiins lower ve- locities through tiu past the polyps, and changes the p« k-nsity ) ol'the filter. Parame- ters influencing —such as niter shape and size. drag, am ^img drag, are discussed in detail in Best : ... : .\mtncni velocity, titter poroviy. and filtering efficiency filtering efficiency, in engineering vernacular, is de- Inied as the mini her of particles contacting a fiber relative to the number of particles which would have passed through the fiber area had the fiber not been there to di- vert streamlines (Fuchs. 1964; Spielman. 1977). Most bi- ological filters are a composite of fibers of various dimen- sions and configurations that can change with organism growth and ambient flow. \ more usable definition of efficiency tor biological tillers is the proportion of parti- cles retained from a given volume of water within an or- ganism's "access" to flow. For filters perpendicular to steady flows, access is the volume of water which passes tin i nigh the filter's boundary. For filters perpendicular to oscillating flows or oriented parallel to flows, access is the volume of water passing sufficiently close to the filter from which particles uniy be extracted. Filtering effi- ciency depends on the position of fluid streamlines around the filtering elements and the particle paths around the filtering elements. Rubenstein and Koehl ( 1977) were the first to com- pare filtering structures of aquatic organisms to man- made aerosol niters that have the potential of capturing particles by a number of non-sieving mechanisms («.'.#., I uchs. 1464). Spielman ( 1477 (extended the engineering analysis of particle capture by filters in aqueous media, leading to predictive models ol'the mechanisms, nature, and si/c of particles captured bv these filters. The pri- mary mechanism of particle capture for main biological filters appears to be by direct interception, aided possibly by electrostatic or London-van der Waals forces (for re- view see 1 aHarhcra. 1984). This mechanism has been used i" < \plain particle capture by brittle stars (I.aBar- bera. 1978) rmoids (Meyer. 1979). aquatic insects (Craig and < hance, 19X1: Silvester. 1983). and cnid- arians (Ko.-lil 19' 7; Patterson. 14X4). As originally de- veloped I' particles, capture bv direct intercep- tion assumes nierofa neutrally buoyant parti- cle follows an ' <-d streamline around a filtering fiber: if the Stre •' the center ol'the particle pass within one particl fiber, loniacl occurs and the particle is retained aterborne particle ap- proaches a liber, it must de z from the undisturbed stu-amlme due to the slow di fluid from tin- IMP between the surfaces as the particle and fiber approach (Spielman. 1977). For contact to occur in aqueous me- dia, the action of attractive forces (electrostatic or van der Waals forces) must be invoked to overcome the slow. viscous drainage. If the center of the particle passes within some critical distance, the attractive forces will be sufficient to insure contact with the fiber (Spielman. 1977). These models apply to single fibers and assume that all particles contacting the fiber remain caught. In a test of Spielman's theories. LaBarbera (1984) found relatively good agreement between the predicted and actual si/e distribution of Sephadex beads (40-360 ^m) caught by the brittle star Ophiopholis uciilcaia. Dis- agreements between observed and predicted distribu- tions may be due to two factors. First, the model assumes a single filtering fiber, but brittle stars use an array of pin- nules, resulting in flow interaction and changes in the direction of streamlines (Fuchs. 1964). Second, it is as- sumed that all particles contacting the fiber remain caught; this assumption has not been tested for a biologi- cal filter, especially when large active particles, such as zooplankton, are caught. Direct interception is inversely dependent on flow ve- locity (Spielman. 1977). Contrary to the volume flow rate, filtering efficiency decreases with an increase in am- bient flow speed, as shown in the laboratory experiment. In this laboratory experiment. I used a maximum flow speed of 6.0 cm s ' . At speeds faster than this, the sea pen in the flow tank would begin to bend back significantly, increasing polyp density, and thus altering the geometry of the filter by compressing the polyps closer together. The laboratory experiment showed that, for a given po- rosity filter, a decrease in particle retention occurs with increasing ambient velocity. The filtering network of I'tiloMrcns xunu-yi is not strictly two-dimensional. Water entering the rachis first passes between the leaves, and a velocity profile is estab- lished with the faster flow down the middle. On the downstream edge of the organism, the polyps extend be- tween the leaves into the faster flow. The polyps, and then tentacles and pinnules, are oriented slightly down- stream in such a way that they continually bisect the ve- locily distribution. In this way. the filtering elements are placed in the regions of "untiltercd" water, increasing the probability of particle contact. In the field experiments, it was found thai the medium and large sea pens hail higher filtration elliciencies than the small sea pen. even though they were exposed to higher ambient flow speeds above the substratum. Under ambient flow, polyp density is higher in the larger sea pens, resulting m a lower "porosity" or spacing between the polyps. \ lower filter porosity would increase the to- tal filtering surface in a given volume, reduce the velocity PASSIVE SUSPENSION FEEDING 341 profile between neighboring filtering elements, and de- crease the distance between particle streamlines and the element's surface. As ambient flow speed increased, sea pens are bent back by the flow, compressing the down- stream array of polyps and decreasing porosity further. Structure-flow interactions therefore result in a "variable porosity filter" which can maintain a higher efficiency at higher ambient flow speeds. Panicle size selection A review of Figure 6 reveals that, overall, the size dis- tribution of particles retained follows closely the size dis- tribution in ambient water. In fact, in the feeding experi- ments conducted in the field, there was no difference be- tween upstream and downstream water samples in size distribution of particles. There was a difference detected in the laboratory experiments, perhaps because of the higher particle counts used to construct the distributions (10.000 particles in laboratory experiments versus 1 500- 3000 particles in field experiments). Although there was a preference towards the retention of larger particles in the laboratory experiments here and in LaBarbera's ( 1984) study with brittle stars, the distribution of parti- cles retained still closely follows the availability of parti- cles. There is no steep demarcation or stepped character in the distribution. Due to the mechanisms of particle capture operating in aqueous media, and the abundance of small particles in natural seawater (McCave. 1984), a high degree of selectivity for large particle sizes, to the exclusion of small particles, appears to be precluded. Whether or not an organism feeds on the small particles of detritus and phytoplankton in the sea, its filter will tend to collect these particles. Feeding performance Feeding performance is dependent on flow velocity in bryozoans (Okamura. 1984, 1985) and octocorals (Le- versee, 1976; Patterson, 1984), and on swimming speed in juvenile fish (Friedland el a!.. 1984). In the flexible bryozoans and octocorals studied, colony shape changed with ambient velocity: bryozoan branches collapsed in- ward and downstream (Okamura, 1984), while in octo- corals individual polyps bent downstream (Patterson, 1984). These structural changes can influence feeding performance by altering both volume flow rate and fil- tering efficiency — two parameters influenced by ambient velocity and resulting structure-flow interactions. In this study I examined these two parameters independently. Feeding rate in the sea pen Ptilosarcus gurneyi is highly dependent on volume flow rate. Both feeding rate and volume flow rate initially increase sharply as flow speed increases, and then decrease as the upright posture de- creases. Small sea pens have a lower feeding rate because of lower volume flow rates, due to smaller filters and lower ambient flow speeds near the substratum in the boundary layer. Filtering efficiency decreases with in- creasing flow speed, which may be compensated for by a change in filter porosity; polyps become more closely packed as flow speed increases. Because feeding rate is dependent on a complex inter- play of volume flow rates, filtering efficiencies, organism size and flexibility, and ambient velocity, competitive feeding interactions between organisms may be due not only to direct reduction of resource supplies, but may also be mediated by altering the ambient velocity. In the arborescent bryozoan Bugu/a stolonifera, Okamura ( 1984) found that feeding success in small colonies was reduced by both fast ambient flows and the presence of a large colony upstream. In the first case, fast ambient flows may have resulted in lower volume flow rates or reduced efficiencies. In the second case, the larger colony upstream may have depleted particles in the ambient wa- ter, or restricted the volume of water passing through the smaller colony. Acknowledgments I thank K.. London. S. Vogel, J. Voltzow, and S. Wain- wright for their stimulating discussions and suggestions. The penultimate draft of the manuscript was improved by the helpful comments of two anonymous reviewers. This work was supported by a Cocos Foundation Train- ingGrant in Morphology Grant and a Lerner-Gray Fund for Marine Research Grant. Literature Cited Best, B. A. 1985. An integrative analysis of passive suspension feed- ing: the sea pen Ptilnsarcus gurneyi as a model organism. Ph.D. Dissertation. Duke University. Durham, NC. Birkeland.C. l%9. Consequences of differing reproductive and feed- ing strategies for the dynamics of an association based on the single prey species, Ptilosarcus gurneyi (Gray). Ph.D. Dissertation. Univ. of Washington, Seattle, WA. Birkeland, C. 1974. Interactions between a sea pen and seven of its predators. /u«/ Monogr. 44: 21 1-232. Chamberlain, J. A., and R. R.Graus. 1975. 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Si/e spectra and aggregation ol suspended parti- cles in the deep ocean. /Vr/'-.S,-,, Ko 3K4A): 329-352. Mcr/, K. V 1984. Self-generated \ersus environmental!) produced feeding currents: a comparison for the Subelhd polvchaele / tuli-.- tyUa\aih<'ii\vn liml Hull 167:200-209. Mevcr. I). I.. 1979. Length and spacing of the tube leet in crinoids (Echmodeimalai and their role in suspension-feeding. Mar. Biol. 51: 3M 169 N»»ell. \. R. M., and P. A. Jumurs. 1984. Mow environments of aquatic benthos. Anmi. Rt'i: Ecol. Sysl 15: 303-32S. Okaniuni. B. 1984. 1 lie cllccls of ambient How velocitv. colon v si/e. and upstream colonies on the feeding success ol ht\o/oa I. liui;ulii Kotow/iva Ryland, an arborescent species. J l:\/< \ta> Hi,': I 83: 1 79- 1 9 5 Okainura. B. 1985. I he etlccts ol ambient How velocitv. colonv si/e. and upstream colonies on the feeding success of hrvo/oa II (.'nn- I'l't'iini rciitiihini (I mnaeus). an encrusting species. ./ / x/> \l,n «/c/ Ecol 89:i.9 MI Okamuru. B. 1987. ['article si/e and How velocitv induce an inferred switch in brvo/oan suspension-feeding behavior. Iliol. Hull 173: 222—229 I'alkTson. M. R. 1984. Patterns ol whole colonv prev capture in the oclocoral, Alcyonium siderium Hml Bull 167:613-6.29. Ruhenstein. 1). I., and M. \. R. kuchl. 1977. 1 he mechanisms of tiller feeding: some theoretical considerations \m \>n 111:981-994. SihfsiiT, N. R. 1983. Some hvdrod\numic aspects of filter-feeding with rectangular-mesh nets. ./ Tlwur. Biol 103: 265-286. Sutherland. .1. P.. and R. II. karlsun. 1977. Development and stabil- n\ of the fouling communit) at Beaufort. N. ('. l-'t-ol. .\ftmogr 47: 425-446 Spielman. I,. A. 1977. Particle capture from low-speed laminar Hows Inini Wei- /• 'Iniit McJi 9:297-319. \oyel.s.i98l. 1 iic in \l< >\'int; iluul\ The Physical Biology ofFlov \\ illard Grunt Press. Boston. 357pp. Viiui'l. S. 1984. Drag and flcxibihtv in sessile organisms. Am. Zoo/. 24: 37-44. \ <>(jel. S.. and M. l.aBurl>eru. 1978. Simple How tanks for research and teaching, liin^u-iiic 28: 638-643. \\ainwriyhl, S. A., and .1. R. Dillon. 1969. On the orientation of sea fans. Binl Hull 136: 130-139. Reference: Bwi Bull. 175: 343-348. (December. 1988) Discocilia and Paddle Cilia in the Larvae of Mulinia late ralis and Spisula solidissima (Mollusca: Bivalvia) BERNARDITA CAMPOS' AND ROGER MANN2* { Institute of Oceanology, University of Valparaiso, P.O. Box 13-D. \'madel Mar. Chile. and-\'irginia Institute of Marine Science. School of Marine Science. The C 'ol/e^c of William and Mary. ( i/oucesier Point. I 'irginia 23062 Abstract. The bivalve larval velum contains four bands of cilia: inner and outer preoral bands, an adoral band, and a postoral band. The preoral bands of compound cilia are generally considered to be used for both locomo- tion and food gathering. The adoral and postoral bands function in concert with the preoral bands in food gath- ering and transfer of food to the mouth. Cilia are usually described as cylindrical structures which taper to a blunt tip. Modified cilia with disc-shaped (discocilia) or pad- dle-shaped ends have been recorded in several inverte- brate species. Here, for the first time, we demonstrate the presence of discocilia in the velum of Mulinia lateralis and paddle cilia in the velum of Spisula solidissinni. Such cilia are restricted to the preoral bands and the cen- tral ciliary tuft. The presence of such cilia does not ap- pear to increase the swimming velocity of these larvae in comparison to that of Rangia enneata larvae of similar size. The possibility that these modified cilia have en- hanced sensory capability remains to be tested. Introduction The larvae of bivalve molluscs are one of the major components of the meroplankton(Thorson, 1950). Most bivalve larvae develop from the fertilized egg to the veli- ger stage in twenty four hours or less. The veliger larva is characterized by a soft body enclosed by laterally com- pressed, semitransparent. paired valves and a protrud- Received 8 August 1988; accepted 29 September 1988. * To whom correspondence should be sent. Contribution number 1481 from the Virginia Institute of Marine Science. The College of William and Man.1. ing, oval, ciliated velum. The velum contains four bands of cilia: inner and outer preoral bands, an adoral band, and a postoral band (Elston, 1980; Waller, 1981). The preoral bands, consisting of compound cilia 20-80 /jm long, are responsible for locomotion and food gathering. The adoral band, of shorter cilia approximately 8 ^m long, transfer food particles to the mouth. The postoral band consists of complex cilia 15-20 ^m in length. The efficiency of food concentration from the water depends on the harmonic beating of the preoral and postoral bands of cilia (Strathmann ct al.. 1972; Strathmann and Leise, 1979). Cilia are generally considered to be cylindrical struc- tures with a constant diameter except for a tapering, blunt distal tip (Sleigh and Blake, 1977). Morphologi- cally different cilia, discocilia, have been described for the polychaete Lanice conehilega (Pallas, 1776) by Heimler (1978). Tamarin et al. (1974) described cilia with "biconcave flattened discs or paddles," 1.33 ^m in diameter in the ventral pedal groove of the juvenile mus- sel Mytilus califomianus (Conrad, 1 837) and ascribed to them a secretory function of adhesive material. Arnold and Williams-Arnold (1980) described discocilia in the embryo of the squid Loligo pealei (Lesueur, 1 82 1 ); Ma- tera and Davis ( 1982) observed paddle cilia in the rhino- phore of the marine gastropod Pleurobranchaea califor- >;/«/( MacFarland, 1 966); and OFoighil ( 1985) described papillae bearing cilia with bulbous tips of approximately 0.25 ^m diameter in the mantle fold of temporary dwarf males of the bivalve Pseudophythina rugifera (Carpen- ter, 1864). In this report we describe, for the first time, the presence of modified cilia in the velum of larvae of two marine bivalves, the mactrids Mulinia lateralis (Say. 1 882) and Spisula solidissima ( Dillwyn. 1817). 343 344 ». ( \\I1H )S \\I) R MANN MaU'-l:iK and Methods Muliniii u ere obtained from a cultured populate m Shore Laboratorv of the Vir- ginia In Marine Science (VIMS). Wacha- preanue. \ > .*"/<' solidissima adults were obtained from a con . u-ial fishing dock at Willis Wharf. VA. For comparison purposes a third mactnd. Rtin^ia cuncala (Cira\. 1.N3I). was collected from the Rappahannock Ri\er. V A. All adult bivahes were maintained at the \ l\ls \\ achapreague laboratory in water of appropriate salimtv : 30 ppt for the marine stenohalme .V \olnlissinui. 25 ppt tor the eury haline M. lutcrn/i\. and 10 ppt for the oligohaline R ciincata. Adults were induced to spawn by thermal stimulation (24. 28. and 32°C for S. solidissima. M latcniliy and R cuncata. respectively). Larvae were cultured using the procedures of Cullinev ci . at which time larvae were fed on mixtures of the phvtoplankters I'uvlovu (Monncliry.^is) lnl/icn (Droop Green) (formcrlv M<»n>c/ir\'\/\ luilicrii Droop). Isochry.M^ xnlhaihi Parke. and Isoclirysis aft", ^alhana (clone T-Iso). General procedures for phytoplankton culture followed the guidelines of Guillard ( 1983). Preparation of larvae for scanning electron micros- copy (SEM) followed the guidelines of Turner and Boyle ( 1975). Umbo stage larvae were siphoned from the cul- ture container, retained on a 63 nm nylon mesh screen, thoroughly rinsed in 0.45 n\n tillered water of the appro- priate salinity, transferred to 10 ml of tillered seawater and relaxed by sequenlial addilions of I ml of 8"i (w/ v) MgC'l: in distilled water. Osmolarity of final relaxing solutions was not measured bul relaxation was typically obtained following addition of 3-4 ml of MgCU solution. Larvae were concentrated by centrifugation. and fixed for 2 hours with 2.5"! chilled glularaldehyde in distilled waler buffered al pH 7.2 with 0. 1 M sodium cacodylate. I he fixative was subsequently pipetted off and the larvae subjected to three rinses, of 30 minutes each, of 3 ml of 0.1 \l sodium cacodvlalc in 0.25 A/ Nad. Larvae were posl fixed tor one hour m 5 ml of I'. OsOj in 0.19 A/ NaCI hullered at pH 7.2 with sodium cacodylate. Larvae were again rinsed, three times, in 0. 1 M sodium cacodyl- ate in 0. 1 5 A/ NaC'l. and stored o\ernight at 4°C. Dchv- dralion was ellened by 20-rnin exposures to a graded al- cohol series (30. 50, 70. 90, 95. and 100'. ) followed h\ three changes in I On1 acetone. Critical point drying was effected using a nn HI, nuo Polaron dryer. 1 arvaewere mounted on stubs using double sided adhesive tape, des- sicated for a further 24 hours, coated with gold-palla- dium, and examined with an \MK model 1 000 scanning electron microscope. Photographs were made with Po- laroid 52 film. Results Figures 1 through 3 illustrate, with increasing magni- ficalion. ihe comparative morphology of the velum of the three species examined. In all species the posloral band consists of "typical" cylindrical cilia with distally tapering, blunted tips (Fig. 1 A-C): however, the preoral bands exhibit species-specific differences. Ranxia cu- nctiia exhibit again, "typical" cilia (Fig. 1C). The distal portions of the preoral cilia of Spisula solidissima and Mitlinin lutcnilis terminate in biconcave paddles (Figs. 1 A. 2A, 3A) or slightly intlaled discs (discocilia, see Figs. 1 B, 2B, 3B), respectively, the terminal structures measur- ing approximately 1-1.3 ^m in diameler. A single pre- oral cilia in A/, luicralis was observed wilh Ihe disc 1-2 ftm dislal to the tip (Fig. 3B). The preoral cilia appear clustered and conform to ihe description of compound cilia as given by Waller ( 198 1 ). The central ciliary tufts of the velum (Fig. 4) further exhibit species-specific cilia morphology: "typical" compound cilia in R. cuncala (Fig. 4B), cilia with terminal paddles in 5. .wlidissima. and a mixture of cilia with terminal discs and discs 1-2 nm distal to the cilia tip in A/ kileralis (Fig. 4A). In all three species the diameter of the ciliary shaft was between 0.2 and 0.4 ^m. Discussion The comparatively rare occurrence of modified cilia in the animal kingdom prompts Ihe queslion as lo whether their presence is the result of artifacts during preparation for examination. Indeed. Lhlers and Fillers (1978). ex- amining flatworms. concluded lhal both paddle cilia and discocilia were absent in untreated tissue, but appeared onl\ as artifacts after exposure to formaldehyde, sodium phosphate, and sodium cacodylate during preparation for SHM. Bergquist ct til. ( 1977) contend that modified cilia obscrsvd in sponge larvae are real structures. Matera and Davis ( 1 982), after a comprehensive study ofPleitro- hranclhica ailifomicti using light and transmission and scanning electron microscopy, rejected the conclusion of Fillers and Fillers ( 1978). noting that the paddle cilia can be seen with light microscopy in isosmotic seawater and made to straighten reversibly by exposure to hypertonic seawater. We also reject the conclusion of Fhlers and Fillers ( 1978). In the present study discocilia or paddle cilia were a consistent characteristic of particular cilia bands within a species but not characteristic of the whole \elum. Umlbrmits of cilia morphology throughout the velum, an observation consistent with the hypothesis ol artifactuial production during SFM preparation, was not MODIFIED CILIA IN BIVALVE LARVAE 345 most instances, simultaneously. The question remains as to whether cilia morphology in these species will change with changing salinity, however, the ecological signifi- Figure 1. Scanning electron micrographs of the velum of (A) Spixula solidtssimu, (B) Mulinia laieralis, and (C) Rangia cuncaia lar- vae, pr, preoral cilia: po. postoral cilia; va, valve. Scale bar = 20 nm observed. The larvae used in this study were cultured at different salinities. The osmolarities of the final relaxing solutions were therefore different; however, larvae were fixed and subsequently processed identically and, in Figure 2. Scanning electron micrographs of the velum of (A) Spisula solidissima. (B) Mulinia laleralis. and (C) Rangia cuneata lar- vae. Details of the ciliary bands, pr, preoral cilia; po, postoral cilia; va, valve. Scale bar = 10 ^m 346 IV ( \\II'OS \\1) K. \1\NN .V Scanning electron micrographs of the prcoral velar cilia tips of(A) Spmilti \iiliiln\tniti. (B) Miilinui liiii-rcili\. and ((') Rangm iiiiH'iila larvae. Note ihe biconcave paddles in V w>//(/m;m,; and the terminal discs in M. lui. \imvv identifies a single disc distal to the cilia tip in U liiii-inii i id tim cancc of this question is pinbablv minimal in lh;it hotli the larvae and adults of these species exhibit distinctly different salmiiv optima « ampos. ll)SS) which are re- flected in the culture conditions used here. In summary, we believe the paddle and discocilia described here to be genuine structures. The function of the modi tied velar cilia is debatable. Matera and Davis ( 1982) concluded that previous litera- ture "collectively indicate that dilations of ciliary mem- branes represent a common morphological specializa- tion subserving chemosensation." The structures de- scribed by OFoighil ( 1985) are also appropriately located for sensory function. However, they are slightly smaller than previously described cilia modifications. Onlv the "secretory" cilia described by Tamarin ct ul. ( 1974) are thought to have a primary function that is other than sensory. As mentioned earlier, the primary function of the preoral bands in the bivalve larval velum is generally considered to be in locomotion and food gathering. The sensory function has received little attention; however, consideration of veliger swimming behavior, wherein larvae progress in a vertically oriented helix with the ve- lum extended in the direction of motion (see Cragg and Gruffydd, 1975; Cragg. 1980: Mann and Wolf. 1983). suggests that such a function is reasonable. The ability to combine locomotion and chemosensation to direct ori- ented movement along a gradient of chemostimulant has yet to be demonstrated in bivalve larvae. Chemosensory responses associated with settlement and metamorphic inducers have been demonstrated in well-mixed labora- tory containers (e.g.. Coon el al.. 1985), but responses to gradients per vc remain untested. Despite the apparent lack of an organized nervous system in velar tissue Elston ( 1980) suggests that "a cell to cell transmission of im- pulses" would fulfill this sensory function. The presence of paddle or discocilia do not appear to enhance rate of movement in the species examined here. In larger animals, paddle structures would generally be considered advantageous in overcoming drag and en- hancing propulsion. Pelagic bivalve veliger larvae gener- ally range in size from 75 to 400 /um maximum dimen- sion and move at absolute velocities of less than 10 mm s '. At this size and velocity, Reynolds numbers are less than or approach 1 . a region where viscous forces pre- dominate in determining maximal velocity (sec Vogcl. 1981). In a complementary study (Campos. 1988) a comparison of rates of vertical displacement (time to as- cend through a unit vertical distance while swimming in a helical pattern) was made for three si/e ranges of larvae for each of the species examined here at the temperature and salinity of culture. The "D" or straight hinge veliger larvae of R cnncalu. M lulcnili\. and .V. so///<; ex- hibited mean (n 25) rates of 0.38. 0.25. and 0.26 mm s '. respectively. Comparable values for umbone larvae were 0.49, 0.49, and 0.40 mm s '. respectivelv. Mean values for pcdiveliger larvae of the three species were 0.45. 0.34. and 0.40 mm s '. respectivelv. Despite the MODIFIED CILIA IN BIVALVE LARVAE 347 Figure 4. Scanning electron micrographs of the central ciliary tuft of (A) Mulinia laterals and (B) Rungui eiineaiu larvae. Note the pres- ence of both terminal discs and discs distal to the cilia tip (see arrows) in M. laleralis. Scale bar = fact that interspecific comparison is confounded by mi- nor differences in morphometry, size, and, we suspect, specific gravity of larval stages, it is evident that the pres- ence of discocilia and paddle cilia in M. lalcralis and 5. solidissima. respectively, does not apparently confer higher rates of vertical displacement when compared with R cituccita. We did not compare absolute velocity (that which describes movement along the helical path rather than just vertical displacement) in the swimming study. Nonetheless, the ecologically meaningful value for vertical displacement (see discussion in Mann. 1986) suggests that the presence of modified cilia is not accom- panied by greater ability to depth regulate in stratified water columns, an arguable advantage to any larvae en- countering estuarine or shallow coastal environments. Examination of previous descriptions of larvae or lar- val velar morphology for Crassostrea virginica Gmelin (Elston. 1980). Ostrca eclnlis Linne (Waller, 1981), and Airiica isltuulica Linne (Lutz cl al., 1982) have failed to demonstrate the presence of velar paddle cilia or discoci- lia— although in fairness only Waller (1981) provides micrographs of sufficient magnification and appropriate content for definitive statements. The taxonomic sig- nificance of these structures is also debatable. A signifi- cant component of bivalve taxonomy has historically fo- cussed on adult shell characteristics and the present focus of larval taxonomy is on valve morphometry and hinge ultrastructure (see comments in Lutz et a/.. 1982). Yet within one family, the Mactridae, we have examined three phylogenetically associated species and demon- strated the presence of three distinct cilia morphologies, each unique to one species. Clearly, determination of the frequency of occurrence, function, and taxonomic sig- nificance of these modified cilia in the bivalve larval ve- lum awaits further investigation. Acknowledgments This work was supported in part by a Fulbright Fel- lowship to Bernardita Campos and funds provided by the Council on the Environment, Commonwealth of Virginia. We thank Prof. Ruth D. Turner for useful dis- cussion on larval morphology. Mr. Michael Castagna for encouragement and use of facilities at the Wachapreague laboratory, Ms. Patrice Mason for assistance in electron microscopy, and an anonymous reviewer for construc- tive criticism of the manuscript. Literature Cited Arnold. J. M.. and I,. D. \\illiams-Arnold. 1980. Development of the ciliature pattern on the embryo of the squid Loligo pealei: a scan- ning electron microscope study. Biol. Bull 159: 102-1 16. Bergquist, P. R., C. R. Green, M. E. Sinclair, and H. S. Roberts. 1977. The morphology of cilia in sponge larvae. Tissue Cell 9: 179-184. Campos. B. 1988. Swimming responses of larvae of three mactnd bi- valves to different salinity gradients. M. A. thesis. College of Wil- liam and Mary. Williamsburg. Virginia. 1 1 5 pp. Chanley, M.H. 1981. Laboratory culture of marine bivalve molluscs. Pp. 233-249 in Marine Invertebrates: Laboratory Animal Manage- ment National Academy Press, Washington. DC. Coon, S. L., D. B. Bonar, and R. M. Weiner. 1985. Induction of settle- ment and metamorphosis of the Pacific oyster. Crassostrea gigas (Thunberg). by L-DOPA and catecholamines. J. Exp. Afar. Biol £«>/.94:211-221. Cragg, S. M. 1980. Swimming behaviour of the larvae ofPecten max- imux(L.) (Bivalvia). J Mar Biol. Assoc. L' A'. 60: 55 1-564. Cragg, S. M., and 1. Id. Gruffydd. 1975. The swimming behaviour and pressure responses of the vehconcha larvae of Ostrea edulis L. Pp. 43-57 in Proceedings Vtli European Marine Biology Symposium. H. Barnes, ed. Aberdeen University Press. Aberdeen. Scotland. Culliney, J. L., P. J. Bo\le, and R. D. Turner. 1975. New approaches and techniques for studying bivalve larvae. Pp. 257-27 1 in Culture nt Marine Invertebrate Animals. W. Smith and M. Chanley. eds. Plenum Press. New York-London. Ehlers. L .. and B. Ehlers. 1978. Paddle cilia and discocilia — genuine structures? Cell Tiss. Res. 192: 489-50 1 . Elston, R. 1980. Functional anatoirn. histolog> and ultrastructure of 348 B r\\IPOS AM) R M \\\ the soft tissues of the lar\al aim- ^^in\i \iri-inica. Proc. Nail. She! Guillard. R. R i 'plankton lor (ceding marine invertehra. i Culture of Marine Invertebrates i I Berg, ed • . Co.. Pennsylvania. llrimk-r. \\ .1 now type of kmocilia in the larvae , il'olyehaeta. Terehelloniorph.il ( .•// 1 1\\ R^ I.H- Lutz.R. \.. K. Mann. J. G. Goodsell. and M. Castat-na. 1982. Larval and carl, post iar\al development of the Ocean Ouahog. .trcticu islamlicti ./ U,;r Hiol Issot I A 62: "4s-769. Mann. R. I486. Sampling of bivalve larvae. 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Reference: Bin! Bull 175: 349-354. (December, 1988) Consequences of Supply-Side Ecology: Manipulating the Recruitment of Intertidal Barnacles Affects the Intensity of Predation Upon Them PETER G. FAIRWEATHER* Institute of Marine Ecology. University of Sydney, NSW 2006. Australia Abstract. An experimental manipulation of barnacles successfully recruiting to two seashores tested the conse- quences of these variations to predation by whelks and the eventual population structure of the barnacles. Very young barnacle spat were removed from some areas with and without predatory whelks. The whelks moved away from areas without the juvenile barnacles, but stayed and ate barnacles present on the treatment with barnacle re- cruitment. Predation resulted in almost complete elimi- nation of the cohort of barnacles. In contrast, barnacles survived and grew to reproductive sizes on areas initially without whelks but with recruits. Because of the move- ments of whelks and their predation on the barnacles, the final abundances of whelks and barnacles in each plot bore little relationship to their initial experimental treat- ments. Thus caution is needed in interpreting the causes of static patterns of abundance in the field, where the processes involved earlier are not definitely known. These results point to a need to incorporate variation in recruitment into models of biological interactions. Introduction An important development in marine ecology within the last five years has been the reappraisal of the inci- dence and implications of variation in larval settlement and recruitment (Lewin, 1986; Young, 1987; Under- wood and Fairweather, 1988). Biological oceanogra- phers and fisheries scientists (e.g.. Thorson, 1950; Coe, 1953; Ricker, 1 954; Beverton and Holt. 1957; Loosanoff, 1966) have long focussed on the settlement from the wa- Received 13 June 1988; accepted 19 August 1988. * Present address: Centre for Environmental & Urban Studies, Mac- quarie University, NSW 2109. Australia. ter column of planktonic larvae of marine invertebrates and fishes, and their subsequent recruitment to adult populations (and hence to exploitable stocks). Field ecol- ogists have recently turned their attention to the magni- tudes of local variation in this recruitment (Victor, 1983; Caffey, 1985; Gaines and Roughgarden, 1985), and the consequences this has for diversity and abundance of marine species (Underwood and Denley, 1984; Connell, 1985; Peterson. 1986). This new emphasis has been termed "supply-side ecology" (Lewin, 1986). Specific theoretical models (Sale, 1982; Roughgarden, 1986; Mengeand Sutherland, 1987) have been proposed incor- porating such variation for the subsequent demography of these "open" populations and whether recruitment variation determines the size of populations. Variation in recruitment is considered an important alternative ex- planation for many ecological patterns (Underwood and Denley, 1984;Lewin, 1986). However, community ecol- ogists have given less consideration to the consequences of this variation for biological interactions (such as pre- dation or competition) among marine organisms (Un- derwood and Fairweather, 1988). It has been shown experimentally that variation in the availability of different potential prey in a rocky inter- tidal area can greatly affect predation on them (Fair- weather, 1985, 1987). One mechanism by which avail- ability of prey might vary is via the settlement of their larvae and their recruitment as juveniles. In such circum- stances, variation in the recruitment of particular prey species might also influence the outcome of predatory interactions and hence the structure of the community (Underwood and Fairweather. 1988). Although observa- tions in other intertidal work suggest these effects (Fi- scher-Piette, 1935; Dayton. 1971;Menge, 1972; Morgan, 1972), no experiments involving manipulations of re- 349 350 P. G 1 \IRV\I Mill R cruitmcm have ' Here I describe an experi- ment that sim "' recruitment bv an im- portant memK •. k\ intertidal community into areas wil predators on two seashores in New v . Australia. Species considered were the barnaele ..' rosea (Krauss) and the muneid uhelk ' narginalba&lainville. r. rosea is the most abundant sessile animal at midtidal levels on shores ex- posed to wave action: M. marginalba is the most com- mon predator in such areas (Denley and Underwood. ! >'"•». i ndenvood. 198la: t 'nderwood ct ul . 1983). Materials and Methods The experiment used simultaneous manipulations of recruitment and predation on small patches of rocky shore to test whether whelks are capable of responding to a simulation of an extreme case of recruitment varia- tion. On some shores around Sydney. NSW, Australia (Underwood ct ul.. 1983). large areas of the midshore were devoid of nearly all animals that, prior to 1983. had supported dense populations of Tc\M'n>pora (from a set- tlement in 1978: Underwood and Denley. 1984) and their associated communities. The two seashores used were Green Point in Broken Bay (33°3()' S. 15T17' E). and Maitland Bay ( 33°28' S. 1 5 1 "22' E) on the open coast. I simulated the failure of barnacles to recruit by remov- ing ncwlv settled individuals surrounding selected pools on each shore. Two 400 nr sites at each of the two shores were chosen within which at least 24 slight depressions, pools, and small crevices could be found. All were sepa- rated by distances of at least 2 m. These crevices were suitable to shelter Morula (Moran. 1985). and a few ani- mals had found refuge there. Around these pools were generally homogeneous bare areas of at least 1 nr with few prey (barnacles and gastropods) at a total density of onl> about 15 per nr. In the first week of March. 1984. I noted a reasonably dense set of Tesscmpora that had occurred about a week before at Maitland Bay (approxi- mately 2000 per nr) and Green Point (1 100 per nr). Spat at Green Point may have settled a week earlier than at Maitland Bay (pers. ohs.). At eaeh site (two on each shore, consisting of 24 pools each), six ),. iols were assigned to each of four treatments (see Fig. 1 ): z < mtrol with whelks and recruits present, a treatment with recruits removed but whelks present, a treatment with .. Iks absent, and a treatment with both whelks and re> •ihsent. Initially, the one- to two- week-old barn. i <> comprised some 9S'. of the po- tential prey on all pi uuigh it is unusual for whelks to eat them so young;! ither. 1985). Barnacles were not seen being eaten bv ill1 hHks piesent at this time and probably very lew were killed by predators during ihc lust month (although morlalilv did occur). Barnacle whelks newly recruited barnacles young adult barnacles whelks recruits + whelks - recruits - whelks + recruits - whelks - recruits Figure I. Pictorial representation ot'lhe initial and hnal conligura- tions of the loin treatments in the experiment. I he whelks .ire shown hiding in the central crevice in each plot and the survival and growth of the barnacles is indicated hv the number and si/es in each treatment ( t whelks/ 1 recruits are the control plots). spat were removed from the appropriate plots by careful scraping with a knife on the 5th and 6th of March (so that 0.05 Degrees of Mean Source of variation freedom square F- ratio P %of variation Main Effects Shores, Sh 1 37 4 NS 0.8 Sites within Shores, Si(Sh) 2 10 1 NS 0.5 Predators. P 1 1204 143 <0.01 27.1 Recruits. R 1 771 1927 <0.001 17.3 Interaction Sh • P 1 88 1 1 NS 2.0 Sh • R 1 43 107 <0.01 1.0 Si(Sh)XP 2 8 1 NS 0.4 Si(Sh) x R 2 (1 0 NS 0.0 PX R 1 141 1 941 <0.001 31.7 Sh X P X R 1 81 54 <0.05 1.8 Si(Sh) • P • R 2 2 0 NS 0.1 Residual 80 10 17.4 Total 95 b) Student-Newman-Keids lest sot mean number \ at barnacles .surviving lur the Sh ^ P \ R interaction. "MB" = Mankind Kay. "GP" = Green Pom/. "+" = present. "-"= absent Treatments Shore Predators Recruits Means MB GP GP MB GP MB GP MB 18. 5 > 12.3 > 2.8= 2.5= 2.1= 2.0= 1.3= 0 Whelks, and Recruits), mixed model analysis of variance (after procedures in Underwood, 1981b) (see design in Table I). Differences among means were revealed by use of Student-Newman-Keuls tests on the means (Under- wood, 198 Ib). Results Differences in the analyses among sites or shores are less relevant to the original hypothesis about recruit- ment, but may represent significant sources of variation in barnacle or whelk numbers. For example, in the final sample. Shores differed (see Tables I, II) in that there were more surviving barnacles in the plots from which whelks had been removed at Maitland Bay than at Green Point (as might be expected from the starting numbers i.e.. Fig. 2). Sites within each shore were not divergent (i.e.. despite different numbers of recruits initially, the two sites at each shore yielded a similar result). The treat- ment with recruits, but without whelks, had more barna- cles than all other treatments, and this treatment at Mait- land Bay had more than the corresponding treatment at Green Point (reflecting the initial numbers, see Table II, Fig. 2). During the four-month experimental period, the num- ber of whelks on plots declined rapidly where there were no recruits available as prey (Fig. 2a), while they did not move away from plots with juvenile barnacles. The whelks that disappeared were found in crevices outside the experimental plots and hence had not died. Thus, whelks responded readily and negatively to experimen- tally induced variation in recruitment of a major prey: the predators avoided the areas that simulated failures of recruitment. Analyses of the number of barnacles present at the start of the experiment revealed significant differences between shores, and between plots where recruits were present and where they had been removed (see Tables I, II: Fig. 2b). In contrast, the final configuration of prey in plots was rather different from initial populations (Fig. 1 ). Numbers of recruits declined on all plots, but were annihilated by whelks (to local extinction at Maitland Bay and nearly so at Green Point; Fig. 2b). The rate of decline was greater at Maitland Bay. Some small recruit- ment (from the plankton) occurred subsequently on each shore, but at different times among the sites. After four months, there were again two statistically distinct groups of "barnacle" and "non-barnacle" plots, but den- sities of young Tesseropora in the controls had declined so much that these plots were indistinguishable from the "non-barnacle" group (Fig. 2b). Discussion The most direct test of whether variation in recruit- ment interacts with predation would include manipula- tion of the number of recruits to populations of prey and/ or modifications of the timing of their arrival. Of course this would be difficult to do, especially regarding the tim- ing of episodes of recruitment (because of uncertainty in the availability of larvae at any time). The plankton represents a "mystery stage" (Spight, 1975) in the life cy- cle of many marine organisms that is currently impossi- ble to predict for these and other species (Underwood, 1979). The experiment used a somewhat less direct ap- proach. By removing recruits as soon as they were ob- served, it was possible to create situations where a species had effectively "failed" to recruit, which could be com- pared to undisturbed areas with the "normal" number of recruits. This methodology is particularly applicable to sessile species such as barnacles, while it is difficult to increase the number of recruits to an area without great disturbance. In this experiment, whelks left plots without recruits; Mont/a individuals migrated from unmanipulated areas without prey (Fairweather, 1988). These data suggest 352 I' G. I AIRWLAIIH R labk II Summur\-i'i*. ' • barnacles in the experiment Significant factors Results !i (initial) Mi, ues MB>GP Recruits +r> — r(= zero) -\pnl Shores MB>GB Whelks • Recruits -w/+r > +w/+r > +w/-r = -w/-r(= zero) MJ\ Sites (undefined spatial \uriation) Whelks +w > — w Recruits +r > — r June Shores x Whelks MB/-w > GP/-w > MB/fvv > GP/+w Shores x Recruits MB/+r > GP/+r > MB/-r > GP/-r July! final i Shores X Whelks x Recruits MB/-w/+r > GP/-w/+r > other treatments «helks. "r" = recruits. 'M " = present, "- absent. "MB" = Maitland Ba>. "GP" = Green Point. Results of Student-Newman-Keuls tests of differences among means are also given. that persistent, dense aggregations of \\helks require the natural equivalent of the treatment with recruitment and \\helks initially. Where whelks remained, the barnacles suffered greater mortality than in areas without \\helks. I hus the four different initial treatments changed into three sorts of communities (Fig. 1 ). Plots from which bar- nacles had been experimentally removed finished with no or feu barnacles or whelks. Control plots finished with whelks but few or no barnacles, while plots which started with only barnacles remained that way (with some thinning). Very few whelks located these plots (Fig. 2a) Although this pattern could change if later migra- tions of whelks located plots with surviving barnacles, the results suggest that persistent and dense populations of barnacles require the natural equivalent of the treat- ment with recruitment in the absence of whelks. This sit- uation docs occur on the coast of New South Wales, but the locutions of such patches vary through time (Fair- weuther. I9XXI. 1 he hnal abundances of predators and prey in the ex- perimental communities, however, gave little indication of the interaction ol prcdation with recruitment (see Fig. 1). Without direct knowledge of these processes during the previous four months, the pattern of these divergent plots could be mistakenly attributed to many combina- tionsot thesn or failure of recruitment, the intensity of predalion. and other factors (such as physical distur- bance) not coiv.it _] here. It would be perilous to inter- pret any extant p -umlancc of prev as being the result ol \ ,hle predatory interactions without experimental a : tornal evidence. Descrip- tive surveys of com mum '. lure cannot tell us much about the cstablishmei ntenance of these pat- ternsfWiens. 1V,X| I h Kerved interactions may constitute mere epiplu i ndenvood cl til.. 1983; Sutherland and Ortega inberg <•/ 85; Underwood and Fainveather. 1988). not just characleri/ed as consistently great or small (as in Roughgarden. ll>X6: Menge and Sutherland. 14X7). In providing a variable input, recruitment has been shown here to inlluence the sorts of small scale interactions that organi/e intcrtidal assemblages. RECRUITMENT AND PREDAT1ON 353 GP MB 60 40- 0J SO- LO- 'S 10- o o-i M A M J J M A M J J Figure 2. Densities of (a) whelks per m: and (b) barnacles per 0.04 m: during the experiment. GP denotes Green Point. MB = Maitland Bay, O = controls, • = predators removed, A = recruits removed, A = predators and recruits removed. Means and their standard errors are shown as points and error bars. ;/ = 6 replicate plots. Data for only one site are shown for each shore because the Sites factor was rarely significant in the analyses (see Table II). Acknow ledgments Thanks to A. J. Underwood. P. D. Steinberg, G. P. Quinn. K.. A. MeGuinness, M. A. O'Donnell, and V. M. Nelson for advice on a draft, and to many others for dis- cussions. Prof. J. Connell and an anonymous reviewer improved the clarity of the manuscript. L. Bragg drew the figures, and was supported by a Macquarie Univer- sity Research Grant. This forms part of a Ph.D. thesis (University of Sydney) supported by a Commonwealth Postgraduate Research Award and a University of Syd- ney Research Grant. Literature Cited Beverton, R. J. H., and S. J. Holt. 1957. On the Dynamics of Ex- ploited Fish Populations. HM Stationery Office. London. Caffey, H. M. 1985. Spatial and temporal variation in the settlement and recruitment of intertidal barnacles. Ecol. Monogr. 55: 3 1 3-335. Coe. \\. R. 1953. Resurgent populations of littoral marine inverte- brates and their dependence on ocean currents and tidal currents. Ecology 34: 225-229. Connell, J. H. 1985. The consequences of variation in initial settle- ment vs. post-settlement mortality in rocky intertidal communities. J. Exp. Mar. Biol. Ecol 93: 1 1-45. Dayton. P. K. 1971. Competition, disturbance and community orga- nization: the provision and subsequent utilization of space in a rocky intertidal community. Ecol. Monogr. 41: 351-389. Denley, K. J., and A. .J. Underwood. 1979. 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D., and J. Roughgarden. 1987. Fish in offshore kelp forests affect recruitment to intertidal barnacle populations. Science 235: 479-48 1 . Gaines, S., S. Brown, and J. Roughgarden. 1985. Spatial variation in larval concentrations as a cause of spatial variation in settlement for the barnacle, Balaam glundula. Oecologia 67: 267-272. Lewin, R. 1986. Supply-side ecology. Science 234: 25-27. Loosanoff, V. L. 1966. Time and intensity of setting of the oyster, Crassasirea virginica. in Long Island Sound. Biol. Bull. 130: 211- 227. Menge, B. A. 1972. Foraging strategy of a starfish in relation to actual prey availability and environmental predictability. Ecol. Monogr. 42: 25-50. Menge. B. A., and J. P. Sutherland. 1987. Community regulation: variation in disturbance, competition, and predation in relation to environmental stress and recruitment. Am Nat. 130: 730-757. Moran, M. J. 1985. 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A longitudinal study of four- and nine- month-old infants indicates that they coordinate the tim- ing of their vocal behavior with that of their mothers and vice versa. Maternal interactions of Down-syndrome and nondelayed infants were analyzed and found not to differ with regard to such temporal coordination, indi- cating that it is independent of level of cognitive func- tioning. The capacity for coordinated timing is proposed as a mechanism for the facilitation of social interaction. Such coordination parallels temporal matching observed in a variety of species along the phylogenetic scale. Introduction Beginning at least with the work of the Gardeners (Gardner and Gardner, 1969; Gardner and Gardner, 1 974), researchers have explored the extent to which ani- mals can communicate as do human beings. Our re- search, on the other hand, has been concerned, in part, with the question of whether human social interaction is made possible, or facilitated by, capacities that are shared with other species and serve the same functions. We re- port here the results of a longitudinal study of the tempo- ral structure of social communication between nonde- layed and Down-syndrome infants in the first year of life and their mothers. The results suggest that coordinated interpersonal timing may serve as a mechanism for the facilitation of social interaction. We conclude that such timing shares features of functionally adaptive social pre- dispositions present in other species. Received 4 January 1988; accepted 27 September 1988. Conversation is the primary mode of conspecific com- munication employed by homo-sapiens. Such exchange is an important mechanism serving the organization and maintenance of human society. In this respect, conversa- tional exchange may be viewed as the functional ana- logue of the bird song and cricket chirp. While the infor- mation encoded in a chirp or a song sequence and a conversation may differ radically, the functional conse- quences of such a signal may be identical: facilitation of mating, bonding between infant and caretaker, guarding against predation, etc.. It is in this functional sense that we are considering a human vocal exchange as equiva- lent to vocal behaviors observed over a wide range of or- ganisms. There is an extensive body of evidence (Feldstein and Welkowitz, 1987) showing that conversa- tional exchange between adult speakers possesses a com- plex statistical temporal structure; a structure not en- tirely subsumed by the syntactic and semantic aspects of such an exchange. Of central interest to our investigation is coordinated interpersonal timing, which refers to an alteration in the temporal patterning of one speaker's be- havior as a function of that of the other speaker. Work with invertebrates, especially insects, and with simple vertebrate models, has begun to delineate a vari- ety of genetic and neurologic factors that are responsible for the temporal organization of social behavior. Thus, for example, investigators (Zerhring et al, 1984; Ham- blen et a/.. 1986) have isolated mutations mapped to a particular region of the X chromosome in Drosophila. Mutations on this locus increase, decrease, or destroy completely the temporal pattern of the male fly's mating song. A unique coding sequence that forms a portion of 355 356 \1 .1 \s\o\\ / / i/ this locus has lecently been identified in several verte- brates (Schilo "'iis of neurons that act as temporal tillers have been identified in crickets (Schildberger. MN4). 1 hese tillers are ••tuned" to the .-onspecific song. Temporal niters sensitive to bi alient stimuli have also been identified in several species of toads (Rose and Ca- pranica. 1^-ii. the cKvirie lish. ci,afniniinniii (Partridge and Heiligenberg. 1981 ). and in rats (Rees and Moller. • J). The importance of a capacity for temporal attunement in terms of the organism's survival is not to be underesti- mated. Zeliek ( 1986) emphasized the crucial ecological function served by the temporal patterning of vocali/a- tion in certain frogs and the electric organ discharge in the weakly electric nsh. Both fish and frogs have adopted similar strategies of signal oscillator timing to avoid sig- nal overlap and jamming between conspecifics. Lam- precht ci nl (1985) detailed the utility of distance-call duets in bar-headed geese (. l/;s 3 4 5 6 78 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 Figure 1. A diagramatic representation of a conversational se- quence. The numbered line at the bottom represents time in 250-ms units. V stands for \-ocali:atwn. P (or pause, and SP tor switching pause (the silence that occurs immediately prior to a change in the speaking turn). The arrows that point down denote the end of the infant's turns; the arrows that point up denote the end of the adult's turns. ISS and NSS stand for intcrrupnvc and naninterruplive simultaneous speech. respectively. (Adapted from Figure II-2 of Jaffe and Feldstein. 1970). any discernible silence. A pause is an interval of joint silence that is initiated and terminated by vocalizations of the same participant. A switching pause is a joint si- lence initiated by the participant who has the turn and terminated by a vocalization of the other participant (Fig. 1). Statistical analyses The dyadic time series was divided into five-second2 segments, or time units, yielding 144 five-second units (over 12 min). A time-series regression (TSR) analysis (Ostrom. 1978) was computed for each parameter to as- sess the occurrence of coordinated interpersonal timing for each mother-infant pair. The TSR was accomplished by a three-step procedure. The series were first subjected to an ARIMA (SPSSX) modeling procedure for the pur- pose of "pre-whitening" the data. The ACF subprogram of SPSSX Trends was used to allow for visual and statisti- cal checks to test which model parameters best fit to the data and met the assumptions made by the model. It was determined that the most useful parameter values were 2, 0, 0. Each series was prewhitened separately. After the selection of the appropriate model, the TSR analyses were computed by the AREG subprogram of SPSSX Trends. It is the temporal coordination that oc- curs in the current 5- or 30-second sampling interval that was used in this report. In other words, we were con- cerned with the degree to which changes in one series are reflected by changes in the other series within the same time frame. This relationship is indexed by the standard- 2 The five-second time unit was used for the TSR analyses of all the parameters but maternal speaking turns. Average values were com- puted for each parameter for every five seconds of interaction and for every 30 seconds in the case of maternal turns. Five seconds was chosen because it is approximately equal to the mean + 1 standard deviation (breach parameter. Maternal speaking turns had a significantly greater mean value and thus 30 seconds was selected as a more appropriate time unit. \i I \s\o\v /;r AL Table I Summary of Chi siiuiir,-. -lie retail* nl lime- wn D>ad i>pe SP Four month-- \ 10 10 7 10 iher-infant R 52 .14 .07 .21 X f.S h 1 35.27 5.78' 126.4? y X 7 6 8 I )S mother-infant R 61 .15 .11 .21 * inn s5 :s - ; 17.48 64.63 Nine months A 10 10 6 10 N: mother-infant R ,63 .14 .14 .24 , 79.96 34.89 36.66 89.49 A 10 9 5 9 DS: mother-infant R .89 .09 .07 .16 x- 86.68 17.39 6.81* 30.33 • /' > .05. Note A is the ill for the Chi square. The R represents the average standardi/ed partial regression coefficient. T stands for Turns. P for Pauses. SP for Switching Pauses. V for Vocali/ations. The "N" stands for "N'ondelaved," the "DS" stands for "Dow n's s> ndrome." ized partial regression coefficient, which is used as a co- efficient of coordinated timing. We wanted two kinds of information. One was whether the group of dyads insolving Down-syndrome infants and the group of dyads miohing nondelayed in- fants each engaged in coordinated interpersonal timing. This information was provided by a meta-analytic ap- proach in which standard normal deviate scores are ob- tained for the probability values associated with the re- gression coefficients. Each of these standard scores is squared to yield a C'hi square with one degree of freedom. The Chi squares are then summed for each group of dy- ads to provide a Chi square test (with (//equal to the number of ( hi squares in the sum) of whether the regres- sion coefficients in each group were significantly different from zero. Another was whether the two groups ditlered in terms of the extent with which they engaged in coordi- nated timing. Differences between the two groups of mother-infant pairs (nondelayed and delayed) and be- tween the two age groups (four and nine months) were assessed b i .phi plot anaKsisul \anaiuv. Results The Chi square malyses of the results indicate that mutual coordination -curred for all but one of the tem- poral parameters, at I>M|I 4 and 9 months, regardless of diagnosis! I able I) (iiven that the meta-anahtic results demonstrate that temporal coordination occurred across all but one of the vocal behaviors, or parameters, the question is whether the two groups can be discriminated on the basis of their magnitudes of coordination. I he analysis of variance of the pauses, switching pauses, and vocalizations yielded a significant main effect for diagnosis (/•'[!, 17] = 4.34, P = .05, t = .40). indicating that the dyads \\ ith the delayed infants seemed to engage in less coordination than their nondelayed counterparts. However, the occurrence of a significant interaction of diagnosis by age (/•'[!. 17] = 4.34. /' .05. » .40) indicates that the apparent gen- eral difference between the two groups is primarily attrib- utable to a significantly lower degree of coordination of the Down-syndrome dyads at four months of age. By the time the delayed infants reach nine months of age. their a\ ei age degree of coordination with their mothers is sim- ilar to that of the nondelayed dyads (Fig. 2). The results of the analysis of speaking turns (done sep- arately because of the larger sampling interval) proxide no evidence of a difference in degree of coordination be- tween the dyads with the delayed infants and those \\ith the nondelayed infants ( /•'[ 1 . 17] = 0.00. P = .959). Nor did the magnitude of coordination of either group of dy- ads change markedly with time (/-'[I. 17] = 0.70. P = .415). Discussion The results offer support for the hypothesis that infants and their mothers coordinate the temporal organization of their vocal behavior both when the infants are four 4 T 0 16 • o a u 4: 014-1 o to Nondtlayed Pown .-y (Hi ip •IIME AGE IN MONTHS I inure 2. I he interaction of the diagnosis h> age. indicating that wheieas the degree of Coordinated interpersonal timing of the dv.nls wiih iioiulcl.ncd infant is similar when the ml. mis aie four and nine months ot air. I hat ol the dyads with I Xmn'ssv ndrome infant increases from four to nine months. A MECHANISM OF SOCIAL INTERACTION 359 months and nine months old. They demonstrate that the temporal phenomenon found to characterize adult con- versation (Partridge and Heiligenberg. 1981; Feldstein and Welkowitz, 1987) is present in adult-infant interac- tions from as early as four months of age and that the results are true not only for nondelayed infants, hut also for infants afflicted with Down syndrome. Thus the study, having used a group about whose cognitive im- pairment there can be no doubt, represents a strong test of the proposition that coordinated interpersonal timing is independent of cognitive ability. Note, however, that although coordination appears to be a general phenomenon detected in both groups at both ages, the two groups could be discriminated on the basis of the lower degree of coordination exhibited by the Down-syndrome infants and their mothers at four months of age. This finding of lower coordination at four months increasing, by nine months, to a level similar to that of the nondelayed dyads, is consistent with the find- ings from a wide array of studies about the social behav- ior of Down-syndrome infants. These studies (Ciccheti and Sroufe, 1976; Serafica and Ciccheti, 1976; Ciccheti and Serafica, 1981; Spiker. 1983) have shown that dys- functional aspects of social behavior of Down-syndrome infants and young children are related to deviations in rate of development and not to deficits in development. The capacity to process and respond to the temporal patterning of human vocalizations may enable the infant to select and "lock onto" a biologically important envi- ronmental stimulus. That this capacity is present in in- fants suffering from severe cognitive impairment sug- gests that it may be buffered against insults to the organ- ism. In other words, it may be that the capacity functions to make social interaction possible. The underlying neu- romechanisms responsible for such temporal sensitivity are not known. Workers such as Rose (1986) have ob- served that many different types of organisms employ the same set of neurons in the midbrain for processing cer- tain varieties of temporal information. Rose speculated that similar mechanisms may be operative in human be- ings. Zelick (1986) pointed out that the behavior strate- gies adopted by certain frogs and the weakly electric fish to avoid signal jamming are quite similar, and suggested that common neuromechanisms may be responsible for the common behavioral strategy. Whether the mecha- nisms underlying the behaviors described in this report are similar to those that operate in nonhuman organisms remains open to investigation. There is no doubt that the temporal patterning of so- cial interaction is a fundamental aspect of behavior in any given ecological setting. Marler and Terrace ( 1984) noted that "The mechanisms that underlie imprinting and song learning cannot be understood without first ac- knowledging the pervasive role of unlearned, function- ally adaptive predispositions to associate particular classes of stimuli" (p. 5). We conjecture that the respon- sivity to the temporal patterning of vocal behavior dem- onstrated by the findings presented here is an instance of such a functionally adaptive predisposition in human beings. Acknowledgments The study was supported by Research Grant No. 1 2- 152 from the March of Dimes, by the Fund for Psycho- analytic Research, and. in part, by Research Grant No. R01-MH41675 from NIMH. The investigators are in- debted to the Academic Computing Center of the Uni- versity of Maryland Baltimore County for its generous contribution of computer time and services. Literature Cited Bayley, N. 1969. Hayley Scales <>l Infant Development. Psychological Corp. Ciccheti, D., and K. Serafica. 1981. Interplay among behavioral sys- tems: illustrations for the study of attachment, affiliation and wari- ness in young children with Down's syndrome. Dev Psychol. 17: 36-49. Ciccheti, D., and A. Sroufe. 1976. The relationship between cognitive development in Down's syndrome infants. Child Dev. 47:920-929. Feldstein, S., and J. \Velkcmitz. 1987. A chronography of conversa- tion: in defense of an objective approach. Pp. 435-499 in Nonverbal Behavior and Communication. A. W. Siegman and S. Feldstein. eds. Erlbaum, Hillsdale, NJ. Gardner, R. A., and B. T. Gardner. 1969. Teaching sign language to a chimpanzee. Science 165: 664-672. Gardner, B. 1 '., and R. A. Gardner. 1974. Two-way communication with an infant chimpanzee. Pp. 1 71-184 in Behavior of Nonhuman Primates, Vol. 4. A. M. Schrier el ai. eds. Academic, New York. Gibson, D. 1978. Down's Syndrome: The Psychology of Mongolism. Cambridge University Press, New York. Hamblen. H., \V. A. Zehring, C. P. Kyriacou, P. Reddy, Q. Yu, D. A. Wheeler, L. J. Zwiebel, R. J. Konopka, M. Rosbash, and J. C. 1 lall. 1986. Germ-line transformation involving DNA from the period locus in Drosophila melangaster: overlapping neural fragments that restore circadian and ultradian rhythmicity to per" and per" mu- tants. J Neuragcn. 3: 249-29 1 . Jaffe, J., and S. Keldstein. 1970. Rhythms of Dialogue. Academic, New York. Jasnow, M. D., and S. Feldstein. 1986. Adult-like temporal charac- teristics of mother-infant vocal interactions. Child Dev. 57: 754- 761. Lamprecht, J., A. Kaiser, A. Peters, and C. Kirchgessner. 1 985. Distance call duets in bar-headed geese (Anser indicits): Co- operation through visual relief of the partner? 7. Tierpsychol. 70: 211-218. Marler, P., and H. Terrace, eds. 1984. Dahlcm Konleranzen Sponger- Verlag. Berlin. Ostrom, C. W. 1978. Time Series Analysis. Regression Techniques. Sage. Beverly Hills. CA. Partridge, B. L., and \V. Heiligenberg. 1981. Pp. 309-319 in Ad- 360 M ,1 \S\0\\ / / I/ vances in I 'cru-i I' I wen .•.',/. cds Plenum. New ^ ork Radlntf. 1 I1'"" Vale \ sell-report dcpicssum Male for .l.iu.ni. I/'/)/ /'M,/;,'/ l/Mjwi',1 I: vx> 401. Rees,A..and \ .oiler. 1983. Responscsof neurons in the inferior It t.i \M .iiul I \I tones, //cur Kcv 10: 301-330. Rose. (, 1986. \ temporal processing mechanism tor all species. Bra . £vo/. 28: 1 U-144 RUM. (... and R. ( apranica. 19N4. \eeessmg amplitude-modulated sounds rn the auditon midhrain ot two species of toads: matched temporal niters. ./ C,<»i/< l'h\'M»I 154: 2 I I -2 1'). Sehildhcrt-iT. k. 198-1. Temporal selectmt> of auditon neurons in the cricket../ Comp I'ln^nl 155:171-185. Si-ratiea. 1 .. and I). ( ieeheli. 1976. Down's s\ndrome children in a strange situation: attachment and exploration behaviors. Merrill- Palmer Q 22: 137-150 Shin. II. S.. I. A. Banjidlo. B. I. (lark. I . R. Jackson, and M. \\ . Young. 1985. An unusual coding sequence from a />M\H/>/IJ/ mtcncnuon for young children with Down's s\ndrome: new diiections lor enhancing parent child s\nchron>. In /),m/; \ Si >/[/(, ii ii/ii/fin'v in Biomedicine and the Behavioral s, fences, S Peuschel and J. R\nders. eds. The \\'are Press. Cam- bridge. MA. /.crhrint;. \\ . V. 1). \. \\hi-eli-r. P. Redd>. R. .1. Konopka.C . P. k>ria- eou. M. Kosbash. and.l.C. Hall. ll)84. P-element transformation of period locus D\ A restores rhuhmicity to mutant, arlnthmic Drosophila melangaster. (V//39: 369-376. /.click, R. 1986. Jamming avoidance in electric fish and frogs: strate- gies of signal oscillator timing. Bruin lieliu\ i'.vnl 28: 60-69. /.ucki'rman. M.. and B. I ubin. 1965. \luniiul l,n the \I IK/ 1 duca- lional and Industrial 1 esting Serx ice. San Diego. ( \ Reference: Biol. Bull 175: 361-371. (December. 1988) Intraspecific Variation in Growth and Reproduction in Latitudinally Differentiated Populations of the Giant Scallop Placopecten magellanicus (Gmelin) B. A. MACDONALD AND R. J. THOMPSON Marine Sciences Research Lahoraion; Memorial University of Newfoundland, St. John's, Newfoundland A1C 5S7, Canada Abstract. The giant scallop, Placopecten magellanicus, exhibits a discrete gametogenic cycle which varies be- tween populations. In our study, spawning occurred later in scallops from New Jersey than in those from New- foundland, but there is no latitudinal trend when data from the literature are considered. Reproduction is prob- ably controlled by local environmental factors. There was high intraspecific variation in shell and so- matic growth rates, and in the production of somatic and germinal tissue. Reproductive output in particular showed great plasticity. Variation in these traits along a depth gradient on a micro-geographical scale was equal to or greater than variation on a latitudinal scale, al- though reproductive output in New Jersey scallops ex- ceeded that of scallops from Newfoundland. Enhanced reproductive output was associated with reduced lon- gevity. Introduction Many species of marine ectotherms are distributed over a wide latitudinal range and often display intraspe- cific variation in physiological characteristics and life- history strategies (Levinton, 1 983). Such species are ideal candidates for determining which environmental fac- tors, such as water temperature, that vary with latitude in a predictable manner may influence the growth and reproduction of individual animals. Causal relationships between water temperature and growth or reproductive output have proved difficult to establish unequivocally, owing to local variations in environmental conditions, such as food availability and temperature (Newell el ill.. Received 21 July 1988: accepted 20 September 1988. 1982; MacDonald and Thompson, 1985a). There is a need for studies in which intraspecific variation on a mi- cro-geographic scale is examined for a number of charac- ters and related to observations on latitudinally sepa- rated populations. With some exceptions, the general consensus in the literature is that bivalve molluscs from low latitudes grow more rapidly at ambient temperature, attain a smaller maximum size, and have a shorter lifespan than do conspecifics from higher latitudes (Newell, 1964). This view is supported by studies on several species of bivalves, including Silic/uci pcilula (Weymouth el ai, 1931) and Mytilits eilnlis (Seed, 1 976), but clear latitudi- nal trends have not been observed in others, e.g., Mya arenaria (Brousseau, 1979) and Placopecten magellani- cus (Posgay, 1979). In Macoma halthica from North America, however, maximum size is greatest in popula- tions from low latitudes, whereas in M. halthica from Eu- rope growth is faster at intermediate latitudes (Gilbert, 1973; Bachelet, 1980; Beukemaand Meehan, 1985). There is an extensive literature on the gametogenic cy- cle and the timing of spawning in many bivalve species (Giese and Pearse, 1974; Sastry, 1979; Newell el al., 1982). For several species in the northern hemisphere, spawning occurs at higher temperatures and later in the year in southern populations than in northern ones (Sas- try, 1970, 1979; Seed, 1976; Barber and Blake, 1983), and is often more synchronized at higher latitudes (Oc- kelmann, 1 95 8; Bricelj . 1° ter, 1974). In previous papers, we have descriK ow local variations in temperature and food supplv can influence shell growth, somatic production. gametogenesis, and reproductive characteristics in popu- lations from Newfoundland (MacDonald and Thomp- son. 19S5a. b. 1986: MacDonald ctal.. 1987). In this pa- per, we integrate this information with data from scallop populations in New Brunswick and New Jersey, to fur- ther our knowledge of intraspecific variation in I3, magel- kinicus. The objectives are to establish whether growth and reproductive parameters show identifiable latitudi- nal trends, to determine which ones should be regarded as plastic or variable on a local scale, and to understand the possible adaptive value of the observed strategies. Materials and Methods Sntily s//t-s and environmental data Scallops were collected from Sunnyside (47°5T N, 53°55' W) in Trinity Bav. Newfoundland, by SCUBA di- vers, from St. Andrews (45°04'N. 67°04'W) in Passama- quoddv Bay. New Brunswick, and from a bed near As- burv Park (4()°1 3'N. 73°47'W). New Jersey, (Fig. 1 ) using a modified Digby dredge. Collections were made approx- imatelv monthlv between July 1982 and November 1983 at Sunnvside and New Jersey for determination of the gametogenic cycle. In 1983 a complete size range of scallops was obtained in the months immediately before and after spawning at Sunnvside (Julv/September). St. Andrews (July/November), and New Jersey (September/ November), to measure the weight loss of the gonad on spawning. Samples were obtained from depths of 10 and 3 1 m in Sunnyside and St. Andrews but were only avail- able from 31 m at the New Jersey site. Seasonal water temperatures were recorded in Sunnv- side using moored 180 d continuous recording thermo- graphs (Ryan Instruments. Seattle. Washington) and in New Jersey by means of a maximum-minimum ther- mometer. Temperature cycles for St. Andrews were ob- tained from Forgeron (1959) and represent mean values for the 1957 and 1958 seasons combined. An approxi- mation for water temperature at 10 m was calculated by averaging the temperature at the surface and the bottom (24m). Growth rutc\ \t'cs ofindivuln.il .illops were estimated by inter- preting external growth mil's on the shell (Stevenson and Dickie. 1954) and growth increments on the calcareous portion of the ligament (Merrill >•! til , |9Mi). Measure - 1. Sites from which scallops. Placn/h\li-/i were collected. A — Avalon Peninsula. Newfoundland (SS Sunny- siclcl. B — Passamaquodih Ba\ |S\ = St. Andrews); C — New Jersey NL— northern limit of distribution. SI. — southern limit of distribu- tion. ments of shell height [maximum distance between the dorsal (hinge) and ventral margin (Seed. 1980)] were re- corded to the nearest 0.1 mm using vernier calipers. Mean shell heights for each age class were estimated us- ing the von Bcrlalanffv equation: H, = H / [ 1 - e Kit- 1. where H, shell height at lime t. M / mean asvmp- totic shell height. K the Brodv growth coefficient and t,, a parameter representing time when shell height equals zero. The von Bertalanfiv functions were fitted by itera- tion, using the Marquardt algorithm available in the NUN procedure of the Statistical Analvsis Svstem (SAS Institute Inc.). Weights of the gonad and remaining (somatic) tissue were determined separatelv for individual scallops after drying at 9()°C for 48 h. The mean somatic weight for INTRASPECIFIC VARIATION IN SCALLOPS 363 each age class was estimated using polynomial regres- sion, which has some advantages over the von Berta- lanffy function for describing somatic growth rates (Mac- Donald and Thompson. 1985a). Polynomial regression, which was computed by the General Linear Model (GLM) procedure of SAS, may be described by the fol- lowing equation: y = • -/3m\ where i30, fi\- • -0m = population parameters, y = the pre- dicted somatic weight for a given value of x (age) and ( = random error at observation x. Linear correlation between regressors(multicollinearity) was reduced by re- placing values of x with (x — x) (Neter ft ai, 1983). For predictive and comparative purposes, relation- ships between shell height and somatic or gonad weight were fitted by SAS (GLM procedure) to the allometric equation y = axh, where y is the predicted weight (g) at a given shell height x (mm), and a and b are fitted parame- ters. A linear form of this equation was obtained by transforming both variates to logarithms and fitting the data to a straight line by least squares regression. Owing to possible seasonal differences in shell and somatic growth rates, only those scallops collected from Sunny- side and New Jersey between July and December were used in comparisons with St. Andrews. Statistical com- parisons between scallops from the three locations were only made on those individuals collected in 1 983 ranging in age from two to eight years because these were the only age classes common to all three populations. Gametogenic cycle and gamete volume fraction To establish the gametogenic cycle, histological sec- tions were prepared from the gonads of six male and six female scallops in each monthly sample from Sunnyside (10 m depth) and New Jersey. The proportions of the gonad occupied by developing gametes and mature ga- metes (the volume fractions) were estimated by a stereo- logical procedure (Lowe el a/.. 1982). The gamete vol- ume fraction (G VF ) was calculated as the sum of the val- ues for developing and mature gametes (for details see MacDonald and Thompson, 1986). Production Somatic tissue production (Pg) was calculated from the increments in dry tissue weight between consecutive year classes, assuming that 1 g dry weight = 24.5 kJ (Thompson. 1977). Since Placopecten nuigellanicns has a discrete reproductive cycle and spawns only once a year, gamete production (Pr) was estimated from the weight loss of the gonad on spawning in scallops of given age (determined from the von Bertalanffy equation de- scribing shell height as a function of age), and then con- verting to units of energy ( 1 g dry gametes = 26.0 kJ; MacDonald and Thompson. 1985b). Data from males and females were combined, because there were no con- sistent differences between the sexes in somatic or gonad growth curves (MacDonald and Thompson, 1985b). Reproductive effort Reproductive effort (RE), defined as the proportion of non-respired assimilation allocated to reproduction, was calculated for each age class: RE = [Pr/(Pg + Pr)]-100 To compensate for differences in growth rate between populations, RE was also expressed as a function of so- matic weight (MacDonald el a/.. 1987). Furthermore, there were differences in longevity between populations (MacDonald and Thompson 1985a; this paper), so we also related RE to the proportion of the lifespan repre- sented by any given age. Results Water temperature Water temperatures were higher off New Jersey than at the more northerly sites, except in the summer, when the water at St. Andrews was warmer than elsewhere (Fig. 2). In New Jersey, the temperature reached 1 7°C in November, but never fell below 5°C during the winter, whereas at St. Andrews and particularly at Sunnyside. winter temperatures were much lower. The form of the temperature cycle was similar at St. Andrews and Sunny- side. Water temperatures in the shallower depths at these two locations generally exceeded those in deeper water, except during the winter (December- April), when the water columns were vertically mixed. Cumulative an- nual day degrees were estimated as 3 1 80 for New Jersey, 2536 (10m) and 2400 (31 m) for St. Andrews, and 1451 (10 m) and 957 (31 m) for Sunnyside. Gametogenic cycle Gametes observed in histological sections were di- vided into two categories: ( 1 ) developing gametes (DG), representing early stages, and (2) mature or ripe gametes (MG), (MacDonald and Thompson. 1986). Since the ga- metogenic cycles of Sunnyside scallops from 10 m and 31 m were similar (MacDonald and Thompson, 1986). only data from 10 m were used in comparisons with New Jersey scallops. In both populations (Sunnyside and New Jersey), the seasonal cycles for GVF (TG; males and females com- bined) were very similar, although in both years Sunny- side scallops spawned two months earlier than those from New Jersey (Fig. 3). According to Dickie (1953) 364 B. A. M\( l)()\\l D AND R. J. THOMPSON 16 . N D Figure 2. Water temperatures. •• • •• Sunnysidc ( It) m): O Sunnyside (3 1 m):B---«St. Andrews ( 10 m); D---D St. Andrews (3 1 m): A---A New Jeisev and Beninger( 1 987). spawning in Placopecten nuigcllan- icns from the St. Andrews area occurs at the same time as it does at Sunnyside, i.e., late August to early September. Whereas gametogenesis began earlier in the year at Sun- nyside than at New Jersey, mature (ripe) gametes did not appear in Sunnyside scallops until April, compared with January for New Jersey scallops in which mature ga- metes were present almost year-round. There was a small decrease in GVF (MG) during June and July in scallops from New Jersey, followed by an increase in August. \\hich may suggest partial or dribble spawning (Newell ctai. 1 982). Shell growth Von Bertalantfy equations were used to relate shell height to age for all age classes represented in each sam- ple ( I able I ). There was a latitudinal gradient in longev- n\ i Sunnyside > St. Andrews > New Jersey: see legend to Table I), although there was no clear trend for asymp- totic heiuhl ( 1 1 / ). Shell height was greatest at Sunnyside ( H) ml and least at New Jerse>. with intermediate values in scallops from St. Andrews, but in deeper water (3 1 m) at Sumnsule II / was relatively small. The Brod> growth coefficient (K.) was lower in the Sunnyside popu- lation (especialh at 3 I m) than in the others, indicating that scallops from Sunmsidc readied asymptotic height relatively slow l\ con i>;ircd with those from more soutli- erl\ locations, but caution must be exercised in compar- ing growth coefficients when II / values are different (see Discussion) For a rigorous comparison ol growth rates, polynomial regressions afford the advantage that they can be handled by linear models (MacDonald and Thompson, I985a). Comparisons were made between scallops from 3 1 m at Sunnyside. St. Andrews, and New Jersey, and also be- tween scallops from the shallowest depths from which they were obtained at each location, using data for indi- viduals two to eight years old (Fig. 4. Table II). Scallops from 3 1 m at New Jersey and St. Andrews grew at similar rates but significantly faster than scallops from 3 1 m at Sunnyside. However, when scallops from the shallowest collection depths were compared, shell growth was sim- ilar at all three sites. Somatic uv/X'/// Polynomial regressions of somatic weight against age were also compared (Fig. 5, Table II). For scallops from 80 60 40 20 -| JASON D'JFMAMJJASON 1982 1983 Figure .V (iamctc \olumc traction for scallops. /'A;. n/><'< icn nm- (,'< 'lltinii /iv from Sunmsidc (Id m depth: •••••) and New Jerse\ (A ---A). Data lor developing gametes (DG) and mature gametes (MG) are for males onl\. whereas dula lor total gametes ( TO) are for males and females combined. Values arc means • >)>'. confidence limits INTRASPECIFIC VARIATION IN SCALLOPS Table I Parameters (± 95% C.LJ of the von Benalanffy equations describing shell height (H, mm) as a function of age (years) in Placopecten magellanicus collected from depths of 10 in ami 31 m in three locations 365 Sunnyside, Newfoundland St. Andrews. New Brunswick New Jersey 1 0 m 31 m 10m 31 m 10m 31m H, 176.5 ±3.0 158.4 ±3.3 166.9 ± 12.5 166.0 ±8.1 155.9 ±9.6 K 0.19 ±0.013 0.16 + 0.015 0.21 ± 0.033 0.21 ±0.032 0.22 + 0.04 to 0.55 0.10 0.51 0.53 0.32 r 0.97 0.97 0.96 0.98 0.95 n 272 243 83 73 — 145 The age classes found were 1-20 years at Sunnyside. 1-12 years at St. Andrews, and 1-10 years at New Jersey, r = coefficient of determination, n = number of observations. 3 1 m, all regressions were significantly different, somatic weight being greatest at St. Andrews and least at Sunny- side. However, at the shallowest depths somatic weight was greater in scallops from St. Andrews and Sunnyside than in those from New Jersey. Significant differences were also observed between linear regressions of somatic weight against shell height (both variates transformed to logarithms), excepting the samples from the shallowest collections at Sunnyside and New Jersey (Tables II, III). Production There was a clear latitudinal trend in gamete produc- tion (Pr) by individual scallops from 31 m depth (New Jersey > St. Andrews > Sunnyside) which was also re- flected in total production (Pg + Pr), but the greatest so- matic production (Pg) was at St. Andrews and the least at Sunnyside (Fig. 6). Scallops from New Jersey also pro- 140 ., 100. o UJ I UJ x to 60. 20. •f •r AGE (yr) Figure 4. Shell growlh in scallops. Placopecten magellanicus, from Sunnyside (• 10 mi O 31 m), St. Andrews (• 10m;D31 m) and New Jersey (A). duced more gametes than those from shallow water (10 m) at the more northerly locations, and older individuals from New Jersey (>5 years) showed greater total produc- tion. For scallops from the shallowest depths at each site, Pg increased at higher latitudes (Sunnyside 10 m > St. Andrews > New Jersey), although the lowest values for Pg were observed in samples from 3 1 m at Sunnyside. All comparisons of gonad dry weight at any given shell height showed significant differences between popula- tions, excepting that between Sunnyside ( 10 m)and New Jersey (Table II). 20, 15. 2 UJ 5 o 10. 5. 2468 AGE (yr) Figure 5. Somatic growth (dry tissue less gonad and shell) in Placo- pecten magellanicus from Sunnyside (•• • •• 10 m; O---O 31 m), St. Andrews (•---• IOm;D— O31 m), and New Jersey (A — A). 366 B \ \! \( DON \l I) \\DR.J. I'HOMI'SON Table I] Summary »1' iai I values lor c«iii{wi/ eoiu/iansoiis nlal/oinetne relationships hctwcen somatu weight, gonad weight, and v/uV/ height for populations ot Placopecien magellanicus 3 1 m vs 3 1 m vs 3 1 m 10m vs3l m" vs 10m M NJ vs NB M \s\B NFvsNJ NJ vsNB M \s\B (a) Polvnomial t values Shell ft 5.05*" (!(,'! 4.1 1** 0.85 0.32 1.59 Height 0, 4.37*** 0.44 4.82*** 1.41 0.58 1.12 vs. 0.70 0.73 0.10 1.13 1.33 i) in age ft 1.84 1.29 0.88 0.87 1.31 0 }•) Somatic 00 3.58*** 4.51*** 7.71*** 4.29*** 7.06*** 3.67"* Weight 0, 3.06** 1.22 5.47*** 2.05 2.71" 0.73 \s age 02 0.99 0.63 0.55 0.57 0.62 0.02 (b) Allometric regression t values Somatic log a 2.71" 1 1.10*** — 8.96**' 10.12** 0.67 Weight vs. Shell h 0.59 1.54 2.48* 1.15 0.12 1.53 Height Gonad log a — 8.00*** — 2.32 6.52*" — Weight vs. Shell b 4.88*** 0.45 5.64*** 0.35 2.22 Height 2.54* Samples were collected from New Jersev I\J:31 m). St. Andrews. New Brunswick (NB; 10and31 ml. and Sunnyside. Newfoundland (NF: 10 and 31 ml. (*P < 0.05. **/>< 0.0 !."*/>< 0.00 1. + indicates NJ sample). The large reproductive output and low body weight of New Jersey scallops resulted in a higher turnover ratio [production: hiomass ratio, (Pr & Pg)/B] than in individ- lahlflll , illiiinetric relationships he/neen I issue weight and shell height in Placopecten magellanicus collected in IVX3 In mi depths <>t in in and 31 in in three locations Sunnyside. St. Andrews. New Newfoundland Brunswick New Jersey 10m 31 m 10m 31 m 10m 31 m Somatic log i -4.67 -3.76 -4.77 -4.55 -4.94 b 2.77 2.28 2.83 2.72 — 2.86 r2 0.97 0.83 0.99 0.99 0.93 n 93 97 83 75 48 Gonad log a -6.29 -5.75 -9.96 8.21 -7.89 b 3.29 2.87 5.05 4.16 4.18 r 0.83 0 0.92 0.93 0.95 n 41 48 43 41 24 Regressions are of the liirni W allh. where W = dry weight (gl of the somatic tissue or of the gonad immedialels before spawning. II shell height (mm), a and ban- luted parameters. uals from the other populations (Fig. 7). At Sunnyside. production per unit weight was greater at 10 m than at 3 1 m. whereas at St. Andrews the turnover ratio for scal- lops less than 6 years old was independent of depth. With the exception of the New Jersey population, turnover ra- tio was a decreasing function of age. Reproductive effort There was considerable variation in RE between sites and between depths (Fig. 8). For all ages and si/es. RE at any given age or somatic weight was greatest in scallops from New Jersey, owing to higher Pr and lower Pg values than in individuals from the other populations. At Sun- nyside. scallops from 10 m had a greater RE than those from 31 m. Reproductive dibit was greater in young scallops (<5 years) from Sunnyside than in those from similar depths at St. Andrews, but lower in scallops older than 5 years. However, when expressed as a function of lifespan, RE was greatest in Sunnyside scallops and least in those from St. Andrews (Sunnyside 10 m > Sunnyside II m > New Jersey > St. Andrews 10 m - St. Andrews 31 m), /.('.. the maximum values observed tor RE were in large, old individuals from the Newfoundland location. z o I- I u Q O a. o. IOO- 5O- INTRASPECIFIC VARIATION IN SCALLOPS b) Pg c) Pr + Pg 367 4'.- 0/ 0— o 2OO- IOO- 2468246 24 68 AGE (yr) Figure 6. Gonad production (Pr). somatic production (PE) and total production (Pg + Pr) by individual scallops, Placopeclen magellaniciis. from Sunnyside (••••• 10m; O---O 31 m). St. Andrews (•---• 10 m; D---D 3 1 m). and New Jersey (A--- A). Discussion In all the populations of Placopecten magellaniciis ex- amined here and in others described elsewhere (Thomp- son, 1977; Robinson et al., 1981; Beninger, 1987) there is a discrete annual reproductive cycle with a well-syn- chronized spawning period. A slight decrease in GVF in New Jersey scallops during June and July may represent a minor spawning of the type described by Naidu ( 1970) for scallops from a bed in western Newfoundland, but we did not observe this phenomenon in Sunnyside scal- lops. In Newfoundland, scallops spawn in August-Sep- tember (Naidu, 1970; this study). Beninger (1987) and Robinson el al. ( 1981 ) report a similar timing in P. ma- gellaniciis from the Bay of Fundy and from Maine, re- spectively. Our observation that the giant scallop spawns later in the year off the coast of New Jersey suggests that this species may be similar to the bay scallop Argopecten irradians. in which spawning occurs later in southern populations than in those further north (Sastry, 1970; Barber and Blake, 1 983). Sastry ( 1 970) attributed this lat- itudinal differentiation in the gametogenic cycle of A. ir- radians to differences in food supply, since the peak in phytoplankton availability occurs later at the southern location than the northern one. However, according to some reports, P. magellaniciis spawns early (July) at the southern limit of its range (MacKenzie, 1979). On the north shore of the Gulf of St. Lawrence, which is close to the northern limit, spawning also takes place in July (Gaudet. pers. comm.). Thus, there are no clearly identi- fiable latitudinal trends in the timing of spawning, al- though the variation appears to be less than in some other bivalves, notably Mytilux eiiulis. in which the ga- metogenic cycle may be highly variable over a small geo- graphic range (Lowe et a!.. 1982; Newell et al., 1982). Borrero ( 1987) found that the temporal variation across the intertidal zone in the reproductive cycle of the ribbed mussel Geukensia demissa may exceed that among lati- tudinally separated populations. As in other species, re- 100, 75. ICQ *v Q. 50. 25 AGE Vyr/ Figure 7. Turnover ratios (production: biomass P/B, where P = Pg + Pr) in scallops, Placopecten magellanicus, from Sunnyside (••••• 10m:O---O3l m), St. Andrews (•—• 10m;D— D31 m), and New Jersey (A---A). 368 H V M\( DONAID AND R .1 IHOMPSON 100, ot O £ 75 VJ 50 O 8 Of. o. 25. // 46 AGEiyr) 5 10 15 SOMATIC WEIGHT (g) 025 050 075 10 LIFESPAN Figure 8. Reproductive effort. 100 X Pr/(PB + P,). as a function of age. somatic weight and lifespan in the scallop Placn/'cilcn niuxcl/unicm •- - • Sunnyside (10 m); O---O Sunnyside (31 ml: •---• St. An- drews! 10 m); D---DSI. Andrews (31 m); A ---A New Jersey. production in P. magellanicus is probably controlled pri- marih h\ local environmental factors, especially food supply, which determines the nutrient reserve and hence the capability to initiate gamete development (Newell cl ul . 1982). Comparisons of shell growth rates from our own study and others show that differences in shell height at any given age in scallops from Newfoundland. New Bruns- wick, and Georges Bank are small (Fig. 9). and that there is as much variation between depths at several sites in eastern Newfoundland (MacDonald and Thompson. 1985a) as there is between populations at different lati- tudes. We have some evidence for an increase in asymp- totic height in PlacopctU'ii magellanicus at higher lati- tudes, which is consistent with studies on some bivalve species, hut not others (see Introduction). However, there is a clear differentiation in longevity, which is greater in northern than in southern populations. Care must be taken when comparing shell growth rates from ditlerent populations, especially when the von Ber- talanlh function is used. It is not appropriate to base comparisons on the parameter K when the asymptotic heights or lengths differ considerably between popula- tions because K is inversely related to H / (Ralph and Maxwell. 1977; Haukioja and Makala. 1979). Further- more. K is a growth coefficient and should not he re- garded as a growth rate />X^}. Main ot the literature values for von Bertalanlly parameters are of limited \alue for comparative purposes, since confidence limits are often not provided. but several statistical packages (including SAS) now in- clude algorithms for handling nonlinear functions of the von Bertalanffy type and provide not only estimates of the parameters but also their variances. We circum- vented the problems inherent in the von Bertalanffy function by also fitting polynomials to the shell growth data, which demonstrated that growth was slower at Sun- nyside (especially at 3 1 m depth) than at more southerly 180 160-1 _ 140 — 120 O u] 100 J 80 x 60 — i — — i — — i — 8 10 12 AGE (yr) 14 16 18 l-'i|>urf 9. Shell gicmth cinu-s tui sv.illops. /'/.Ai'/'iwc'i mii < in. from several locations. I Sunnvsule, Newfoundland. Kim depth; 2 = Sunnvside. 'I m; 3 St. Andrews. New Iliunswick (all from Mac- Donald and Thompson. l"Wa): 4 - (leorges Bank (Brown cl ul.. l>)72); s Mid-Atlantic Bight (Serchuk cl ul.. 1 982); 6 = New Jersey (this paperl. INTRASPECIFIC VARIATION IN SCALLOPS 369 locations, but the problem with this approach is that the coefficients themselves have no biological significance. There is considerable intraspecific variation in the so- matic growth rate of Placopecten magellanicus, and in the production of somatic and germinal tissue, but only in reproductive output is there any evidence of latitudi- nal differentiation: scallops from New Jersey are more fecund than those from locations further north. For the most part, variation along a depth gradient on a micro- geographic scale may be as great or greater than variation on a latitudinal scale (MacDonald and Thompson, 1985a. b; this study). Reproductive output in particular shows great plasticity, viz. the variation between years in shallow water at Sunnyside. Furthermore. MacDonald ( 1986) recorded greater shell growth rates, reproductive output, and somatic production in giant scallops main- tained in suspended culture than in those growing on the bottom nearby. These findings are consistent with observations on other bivalve species. In a comparison of several popula- tions ofArgopecten irradians, Bricelj ct al. ( 1987) found the greatest differences in fecundity, timing of spawning, adductor muscle weight, and shell height to be between locations less than 2 km apart. In the weathervane scal- lop. Patmopecten caiirinus. individuals from offshore beds show slower shell growth, a reduced asymptotic height and lower somatic and germinal production than is found in conspecifics from inshore areas, where food is more plentiful (MacDonald and Bourne, 1987). The growth of shell and somatic tissue is enhanced in the scal- lop Chlamys islamlica by growing the animals in sus- pended culture (Wallace and Reinsnes. 1985). The mus- sel Mytilus edulis also exhibits considerable phenotypic variation in reproductive characteristics over a small geographic range (Bayne ct al.. 1983). Similar observa- tions have been made on fish (Kipling and Frost. 1969; Mann el al.. 1984). According to Calow ( 1979), selection for enhanced re- productive output should result in a reduction in the life- span, and there is a considerable body of evidence to sup- port this position. Thus, in Placopecten magellanicus, the shift in emphasis from growth to reproduction occurs earlier in scallops from New Jersey than in those from more northerly locations, and total production is greater in the former than the latter, whereas longevity is re- duced in these productive, fecund individuals from New Jersey. Further evidence comes from our observations on scallops from various depths at Sunnyside, New- foundland. Since we were unable to resolve accurately growth lines for animals older than twenty years, these individuals were excluded from our analyses. However, we did record that a greater proportion (25%) of the total number of scallops (426) taken from 3 1 m depth fell into this category, compared with only 8.6% (n = 43 1 ) at 10 m. Thus on this extremely small geographic scale, lon- gevity is reduced when food and temperature conditions are favorable and production (somatic and gonadal) by individual scallops is increased. Furthermore, MacDon- ald (1986) has demonstrated that asymptotic height de- creases in Placopecten magellanicus grown in suspended culture, which is associated with greater production; and in a review of data from several species of limpets, Gra- hame and Branch (1985) describe a trend whereby lon- gevity is a decreasing, non-linear function of reproduc- tive output and of the growth coefficient K. The importance of water temperature in determining geographic differentiation in growth rate and fecundity has been emphasized by Levinton ( 1983) and Lonsdale and Levinton ( 1 985). The latitudinal variation in longev- ity which we have recorded in Placopecten magellanicus is certainly correlated with an observed temperature gra- dient, and at the Sunnyside site there is also an increase in longevity (associated with decreased production) in scallops from deeper, colder water. There is little information on the degree to which in- traspecific variation in growth and reproduction is envi- ronmentally induced or genetically determined. Levin- ton (1983) identified a genetic component to somatic growth differences among latitudinally separated sibling species in a genus of dorvilleid polychaete, and Lonsdale and Levinton (1986) described genetically based differ- entiation in both growth rate and fecundity along a lati- tudinal gradient in a harpacticoid copepod. Thompson and Newell (1985) recorded differentiation in the physio- logical response to high temperature in two latitudinally separated populations of the mussel Mytilus edulis, but were unable to determine whether this was genetic or a result of irreversible phenotypic adaptation. In a recipro- cal transplant experiment, Widdows el al. ( 1984) found that most of the physiological variation between two populations of M. edulis was attributable to environ- mental rather than to genotypic factors, although accli- matization was not complete and a genetic component may therefore have been present. This is consistent with other observations on M. edulis demonstrating that most of the variation in tissue growth is accounted for by site rather than by stock (Mallet el al.. 1987). However, in another study in which reciprocal transplants were made between populations of M. edulis, Dickie et al. (1984) found a large environmental influence on growth com- bined with a significant genetic effect. We have no information on genetic control of growth and reproduction in Placopecten magellanicus, but the considerable phenotypic plasticity in this species on a microgeographic scale suggests a strong environmental influence, especially since we have correlated changes in growth and reproduction with temperature and food conditions. This does not necessarily imply that local ge- 370 B \ MACDON SI I) AND R. J. THOMPSON nctic sariabilits is ot "no importance, since genetic differ- entiation can still be significant when phenols pic s aria- lion in growth and reproductis-e traits is greater than the genetic component. The giant scallop exhibits a growth pattern described by Sebens ( 1987) as Type I indetermi- nate grout h (plastic asymptotic gross th). in which the si/eait.un.\i bs an mdisidual is largely governed by envi- ronmental conditions. Such phenols pic sariabilits for a single species merits consideration in any analysis of ihe adaptise value of reproductive tactics, but has receised little atlenlion (Bayne ct ul.. 19X3). In the same sein. Stearns ( 1 976) has cautioned investigators against invok- ing evolutionary explanations to account for reproduc- tise trends ssithout first eliminating genetic and ens iron- mental factors, especially food. We conclude that growth and reproductis-e traits in Placopecten magellanicus are inter-related and highly sariable on a lemporal as svell as aspalial scale, aslhey probably are in many olherbivals'e species: lhat local s'ariability often exceeds sarialion along a latitudinal gradient; and that enhanced repro- ductise oulput is associated svilh reduced longevity. Acknowledgments We lhank Dr. R. W. F.lner. Dr. E. Gould, and Mr. R. Chandler for arranging scallop collections from St. An- drews and from New Jersey. Collections from New- foundland svere made by stall" of the MSRL diving unit. Laboratory assistance was presided by Ms. J. Seneiall. We thank Drs. B. L. Bayne. D. J. Innes. J. S. Levinton, and R. I. E. Newell for reviewing Ihe manuscript. The work was supported by an NSERC operating grant to R.J.T. and by scholarships to B.A.M. from the Marine Sciences Research Laboratory and the Faculty of Gradu- ate Sludies. Memorial University of Newfoundland. This is Publ. No. D-3200 1-3-88 of the New Jersey Agri- cultural Experimental Station, supported by state funds. Marine Sciences Research Laboratory Contribution Number 731. Literature Cited \ P 1 1. I il'».i n R. S. 1983. Variation in I In- grim In rale of \l\a arenann and its relationship lo the environment as analv/cd 111 rough pnnci- p.il , OtnpOnentS analysis and llic ^- parameter nl the \nn Bcrlalanllv itior An /i Hull 81: 75-84. Bachi-lcl. (, 1980. 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Application of a Two-Dimensional Electrophoresis Method to the Systematic Study of Land Snails of Subgenus Luchuphaedusa from Southwestern Japan Islands JUN-ICHI MIYAZAKI, REI UESHIMA, AND TAMIO HIRABAYASHI Institute ot Biological Sciences, The i 'nivcrsity oj l\ukuha. Tsukubu-slri. Ibaraki 305. Japan Abstract. A land snail. Tyrannophaedusa (Litchuphac'- (h/^u nf'/ii Jo/m (family Clausiliidae). is classified into two types, large and small forms, according to shell size. Using a two-dimensional electrophoresis method, we compared the total protein components of the large form with seven members of Luchuphaedusa and three species of different suhgenera of the same genus, and obtained the similarity ranging from 0.989 to 0.667. The similarity between the large form and / (Decolliphaedusa) hilti- hraia was the lowest. Two species of the other subgenus. Nesiophaedusa, were very similar to the large form. The differences were verv small between two specimens of the large form. The small form of 7. iai is \m',ust iiixs. ium is very peculiar and unlike those of any closely re- lated Clausiliid group. l.iicliiiplHiciiiisii comprises eight species (two having one subspecies) and lives mainlv on the Nansei Islands between the Pacific Ocean and the East China Sea (Kur- oda. 1963). Only one species inhabits a small area of the Kyushu mainland: two species live on the western is- lands of Kyushu. Their distribution is distinctive and in- compatible with biogeographical boundaries suggested by distributions of terrestrial vertebrates, avians, spiders, insects, and other land snails ( Tokuda. 1978). Therefore, it is intriguing to investigate how these slow-moving ani- mals have expanded then habitats, established their unii.]ue distribution, and speciated. There is no study so far concerning patterns of their expansion of distribution and speciation and the phylogenetic relationships of spe- cies belonging to this group. / 1 1..) ophidoon among Luchuphaedusa was origi- nally described by Pilsbry ( 1905) as making up a distinc- tive "section Oophaedusa." This species has a peculiar shell shape which does not taper at the summit, while shells of other species are shaped like those of ordinary clausiliids. The species has been classified as the large or small form according to shell si/e (Pilshrv. 1905; Minato. 19X5). Although Minato ( 1 9X5) concluded that these two forms were conspecilic, recent studies show that they are considerabh different judging from some anatomical characters and allo/yme variation (Ueshima. in prep.). Thus, these two forms are very ambiguous in their spe- cies identities. Therefore, it is intriguing to examine their characters tiom ditlerenl viewpoints and to discuss the phylogenetic position of / // .1 <>/>/i/t/<>i>/i in subgenus 372 SYSTEMATIC STUDY OF LAND SNAILS 373 Luchuphaedusa and taxonomic status of the two forms of this species relating to other species. We have established a basis for elucidating the evolu- tionary process of Luchuphaedusa by analyzing protein constituents of whole bodies using two-dimensional elec- trophoresis. The method permits the simultaneous anal- ysis of many characters (Aquadro and Avise, 1981; Mi- yazaki et a/.. 1 987) and avoids the need to consider intra- specific variation. This is in contrast to one-dimensional electrophoresis which analyzes variable proteins, i.e.. en- zymes (Ayala ct al. . 1974: Avise, 1975). We calculate the similarity among species based on two-dimensional elec- trophoretic patterns and discuss a radiation pattern and the relationships between these land snails. Materials and Methods Samples Species used in this study are listed below. Three spe- cies belonging to two different subgenera of Tyranno- phaedusa, including type species of respective subgenera, were also examined. (i) T. (L.) aiunuii aiiinuii and T. (L.) ophidoon (large and small forms) from Shimo Koshiki-jima Is. (ii) T. (L.) nesiothauma and T. (L.) oshimae from Amami O-shima Is. (iii) T. (Nesiophaedusa) okinoerabuensis from Okinoer- abu-shima Is. (iv) T. (L.) callistochilu. T. (L.) inclyta. and T (N.) her- nardi from Okinawa Is. (v) T. (Decolliphaedusa) hilahniia from Sho-o-cho, Okayama. Honshu. The system of classification was according to Kuroda (1963), although some authors treat Luchuphaedusa, Nesiophaedusa, and Decolliphaedusa as full genera. Nc- siophaedusa, consisting of two species, is endemic to Nansei Islands and is similar to Luchuphaedusa in gen- eral shell characteristics ( Pilsbry, 1 90 1 ). Decolliphaedusa is widely distributed in the southwestern area of Japan. T. (L.) ophidoon is classified according to shell and an- atomical characteristics into large and small forms. A re- cent study revealed that the large form was composed of two divergent populations inhabiting northern and southern parts of Shimo Koshiki-jima Island (Ueshima, in prep.). Thus, we used only the large form specimens from the southern part of the island to avoid confusion. A distribution of members of Luchuphaedusa and Nesiophaedusa and localities where specimens were ob- tained are shown on the map in Figure 1 . Electrophoresis Whole bodies of land snails, removed from their shells, were used for electrophoresis. One pair of specimens was used for each comparison. Goto Is KoshikMuna 18. 6 a Pacific Ocean 1 0 East China Sea Nansei Is. Amami Is Okinawa Is Oklno«ribu-slww It Okinawa Is. «) Figure 1. Map of southwestern Japan showing a distribution of members ofLuchuphacilusa and Nesiophaedusa and localities of speci- mens used in this study. White numbers in closed circles represent is- lands where specimens for this study were obtained. Black numbers in open circles represent other areas in which members of Luchuphaedusa inhabit. 1 . The large and small forms of Tyrannophaedusa (Luchuphae- dusa) ophidoon: 2. T (L.) azumai a:umai: 3, T. (L.) azumai una: 4, T (L.) nesiothauma: 5. T. (L.) oshimae; 6, T. (L.) mima mima: 7. T. (L.) mimatokunoshimana;%, T. (L.)degenerata;9, T. (Nesiophaedusa) oki- noerabuensis: 10, T. (N.) bernardi: 11.7". (L.) callistoctula: 12, T. (L.) inclria. T. (Decolliphaedusa) bilabrata is not included. Sample solubilization and two-dimensional electro- phoresis were carried out as described previously (Hira- bayashi, 1981; Hirabayashi et al., 1983; Oh-Ishi and Hi- rabayashi, 1988). In brief, the whole body was homoge- nized thoroughly in 20 volumes of an extraction medium containing 8 yl/guanidine HC1, which prevents protease activity completely. The homogenate was dia- lyzed against 5 M urea and 1 A/thiourea and centrifuged 374 \/\KI at 60.000 X gfor 45 mm. 1 IK- supernatant ISO ^1 or 120 n\) was subjected to the first dimension isoelectnc focus- ing with agarosc gels foi I 2.500 \'-h. The second dimen- sion SDS-polyacr>lamide gel electrophoresis was per- formed as dcsi : iped hv I aemmli ( 1 470). at 30 m A when the bromophenol tracking dye was in the stacking gel of 3% acrylamide and at 60 mA until the dye reached the lower end of the running gel of a concentration gradient of l2-2n acrvlamide. After electrophoresis. proteins were stained b> Coomassie brilliant blue as described by StephanoiYi//. (1986). Analysis A method for comparison between tuo specimens has been described (Miva/aki. 1987). For comparison of two-dimensional electrophoresis patterns, we used a trip- let method in which two different samples to be com- pared (each SO ^1) ami their mixture (60 + 60 jul) were focused and electrophoresed at the same time. One set composed of three patterns was examined on photo- graphs. The overlapping of protein spots was confirmed on the mixture pattern after it was presumed from com- parison between two individual patterns. A close exami- nation of the mixture pattern is indispensable to identify subtle differences in positions of spots and to judge pre- cisely the overlapping of spots, although only individual patterns were used for comparison by some authors (Oh- mshi cl al.. 1983a: Ohnishi ci id.. 1983b: Williams. 1984). In most cases we used the large form as a standard counterpart for comparison, because itstaxonomic posi- tion relative to others is especially intriguing. About two to four hundred protein spots were examined on each electrophoretic pattern and the similarity was calculated as described by Aquadro and Avise ( 1 98 1 ). Results Two typical sets for comparison between two-dimen- sional electrophoresis patterns are represented in Figure 2. Triplet patterns on the left are for investigating varia- tion between different individuals of the large form of Tyrannophaedusa (Luchuphaedusa) ophiilonn (Fig. 2a, h. c). I hose on the right are for comparison between the large form of '/ / • "/i/ii(li><»i and / <\i'\n>plnu'iluMi) hernanli (I i;- 2d. e. I ). 1 he pattern of / i.\ i hcnntnli (f in Fig. 2) r different from that of the large form (d in Fig. 2). The patterns from different specimens of the large form Imm same locality (a. c in Fig. 2) are verj similar, showing aln. i no variation between individu- als. Middle patterns (b. e in Fig. 2) of each set are derived from mixtures of two different samples. The similarity was calculated according to the for- Mciirv 2. 1 wo-dimensional electrophoresis patterns of whole bod- ies ol land snails in genus Tyrannophaedusa Hixtrophoresis was c. li- ned out as described previously ( Ilirabavashi. 1481; Oh-Ishi and Hira- bavashi. 1988). Triplet patterns lor investigating \anation between different individuals of the large form ol / / in /m/'/Mr./imr />/>liitl«»n are represented on the left (a-c). Patterns from two different specimens of the same locality (a and c) and their mixture pattern (b) are shown. I riplet patterns for comparison between the large form of / <>/>/;/- tli«»i and 7" /.\c\ini'/HU'ilini/i hcrmmli are represented on the right (d- II. where d is the pattern of the large form. I' is that of / l.\ > hcrnanti. e is that of their mixture. mula: F = 2Nxy/(Ns 4 NJ. where F is the similarity be- tween specimens x and y. Nx> is the number of spots shared by x and y, and Nx and Ny are the numbers of spots scored for x and v. respectively (Aquadro and Avise. 198 1 ). The result of calculations is shown in Table I. The similarity value between different specimens of the large form was very large (0.989). while the value be- tween the large and small forms was smaller (0.9 1 3). sug- gesting that the large and small forms are definitely different populations. The result supports that T. (Dccol- lifi/uicihiMi) />///dc -TT b T a 0600 l 0 700 i 0800 l 0900 -\ 1 000 A I A k Figure 3. Schematic drawing of a distribution of similarity values among land snails. The similarity values to the large form of Tyranno- phaedusa (Luchuphaedusa) ophidoon. which is a standard counterpart for comparison, are shown on the upper line, and those of T. (L.) oshi- mae and T. (Nesiophaedusa) okinoerabuensis to T. (L.) callistochila on the lower line. Letters correspond with those in Table I. 376 -i-i \in \/\KI Table 1 1 Distribute" be ivi en intrast ' Mouse Fruit llv I and snail Similarit\ Intra Inter Intra Inter Intra Inter D.981 4 1 n.961 i : 0.960-0.941 1 1 3 0.940-'- 6 0.920-0.901 1 1 1* 0.900-0.881 1 6 0.880-0.861 5 1 0.860-0.841 1 6 1 0.840-0.821 8 3 0.820-0.801 1 2 Figures represent the numbers of pairs which show the similarity within the range given in the left column. Intra and Inter indicate mtr.i- specific and interspecific populations. respectively. * Represents the position of the similant> between the large and small forms of / (A.) opliuionn The data on mice is from Aquadro and A vise ( 198 1 ) and that on fruit flies (DroMiphila numnwn species subgroup) is from Ohmshi ctal. ( 19X3b). Intraspecific populations were not found in ranges below 0.800. (L.) m'^iot/hiiinm. of which the similarity to the large form is higher next to the two above mentioned mem- bers, is an inhabitant of Amami O-shima Is. (Fig. 3. d). T. (N.) okiHomihucmn in Okinoerabu-shima Is. is less similar to the large form than T. (L.) ncsiothauma (Fig. 3. e). '/'. (L.) ccillistoclula, T. (L.) inclvui. and "/'. (N.) />cr- nardi. which have the smaller similarity values to the large form, live on Okinawa Is. (Fig. 3, f, g. i). Therefore, the values show that the more remote the island is from Shimo Koshiki-jima Is., the lower similarity the species living on it has. This may be accounted for by sequential colonization of a single ancestor from either Shimo Ko- shiki-jima Is. or Okinawa Is., and by radiation within each island. '/'. (I..) <>\lnnuic is the only exception (Fig. 3, h) in this interpretation of radiation of these species. This species lives on Amami O-shima Is., but has significantly lower similarity than '/'. fl.j nc'\iiilliaiiinu. After the main current of radiation was over, its ancestor may havecome from some other island. The presumptive do- IKII island is possibly Okinawa Is., because the species has the similarity value close to those of other members on this island. It is remarkably similar to / /I..) aillis- tochilain the protein constituent ( I able Hand shell mor- phology. I he last problem concerns the taxonomic positions of thesmallandl.ii fi nt / ,1 i ,>in I heir shell shapes are so peculiar that 1'ilsbry ( I'«)S) ivteited them to "section Oophaedusa" and included them as a single species However, they are dillerent to each other not only in shell si/e but also in anatomical and biochemical characteristics (Minato. ll'S5: Ueshima, in prep.). Our result also shows that they constitute considerably different populations, since there is a large gap (0.076) between the two forms (Fig. 3. the upper line). Therefore, it is questionable whether they should be included in the same species or not. When our result is compared with those reported by others using the two-dimensional elec- trophoresis method (Table II). the relationship between the large and small forms corresponds to that of closely related interspecific or distantly differentiated intraspe- cific populations. Data suggesting reproductive isolation between these two forms is under study of their allo/y me variation (Ueshima. in prep.). Since available data from two-dimensional electrophoresis is insufficient, and since classification systems currently used are probably unique to different ta.xa. we can only assume taxonom im- positions of organisms examined. Collection of data from other organisms by the two-dimensional electro- phoresis method and investigation on other characteris- tics of the two forms are now being carried out to decide more precisely their taxonomic positions. Acknowledgments We express special thanks to Dr. Tetsuo Iwami (Tokyo Kasei Gakuin College) for his advice and encouragement throughout this work. We also thank Mr. Kunitsugu Ka- wabe and Dr. Sen Takenaka (The University of Tsu- kuba) for collecting specimens. Literature Cited Aquadro, C. F., and .1. ( . \\ise. 1981. Genetic divergence between rodent species assessed h> using two-dimensional electrophorcsis. /'/..< .\