Volume 181 THE Number 1 BIOLOGICAL BULLETIN ne Biological LIBRA AUG22 1991 Woods Hole, Mass. AUGUST, 1991 Published by the Marine Biological Laboratory THE Marine Biological Laboratory ' LIBRARY AUG 2 2 1991 Woods Hole, Mass. BIOLOGICAL BULLETIN PUBLISHED BY THE MARINE BIOLOGICAL LABORATORY Associate Editors PETER A. V. ANDERSON, The Whitney Laboratory, LIniversity of Florida DAVID EPEL, Hopkins Marine Station, Stanford University J MALCOLM SHICK, University of Maine, Orono Editorial Board GEORGE J. AUGUSTINE, University of Southern RUDOLF A. RAFF, Indiana University California KENSAL VAN HOLDE, Oregon State University Louis LEIBOVITZ, Marine Biological Laboratory STEVEN VOGEL, Duke University Editor: MICHAEL J. GREENBERG, The Whitney Laboratory, University of Florida Managing Editor: PAMELA L. CLAPP, Marine Biological Laboratory AUGUST, 1991 Printed and Issued by LANCASTER PRESS, Inc. PRINCE & LEMON STS. LANCASTER, PA THE BIOLOGICAL BULLETIN THE BIOLOGICAL BULLETIN is published six times a year by the Marine Biological Laboratory, MBL Street, Woods Hole, Massachusetts 02543. Subscriptions and similar matter should be addressed to Subscription Manager, THE BIOLOGICAL BUL- LETIN, Marine Biological Laboratory, Woods Hole, Massachusetts 02543. Single numbers, $25.00. Sub- scription per volume (three issues), $72.50 ($145.00 per year for six issues). Communications relative to manuscripts should be sent to Michael J. Greenberg, Editor-in-Chief, or Pamela L. Clapp, Managing Editor, at the Marine Biological Laboratory, Woods Hole, Massachusetts 02543. Telephone: (508) 548-3705, ext. 428. FAX: 508-540-6902. POSTMASTER: Send address changes to THE BIOLOGICAL BULLETIN, Marine Biological Laboratory, Woods Hole, MA 02543. Copyright (E) 1991, 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 Bulk-tin accepts outstanding original research reports of general interest to biologists throughout the world. Papers are usually of intermediate length (10-40 manuscript pages). A limited number of solicited review papers may be ac- cepted after formal review. A paper will usually appear within four months after its acceptance. Very short, especially topical papers (less than 9 manuscript pages including tables, figures, and bibliography) will be pub- lished in a separate section entitled "Research Notes." A Re- search Note in The Biological Bulletin follows the format of similar notes in Nature It should open with a summary para- graph of 150 to 200 words comprising the introduction and the conclusions. The rest of the text should continue on without subheadings, and there should be no more than 30 references. 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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 Ril I 'isindafjelags Islendinga with- out abbreviation. F. All single word journal titles in full (e.g. I'eliger, Ecology, Brain). G. The order of abbreviated components should be the same as the word order of the complete title (i.e. Proc. and Trans. placed where they appear, not transposed as in some BIOLOGICAL ABSTRACTS listings). H. A few well-known international journals in their pre- ferred forms rather than WORLD LIST or USASI usage (e.g. Na- ture, Science, Evolution NOT Nature, Loud., Science, N.Y.; Evolution. Lancaster, Pa.) 6. Reprints, page proofs, and charges. Authors receive their first 100 reprints (without covers) free of charge. Additional re- prints 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 before publication. They will be charged the current cost of printers' time for corrections to these (other than corrections of printers' or editors' errors). Other than these charges for authors' alterations. The Biological Bulletin does not have page charges. The Marine Biological Laboratory Ninety-Third Report for the Year 1990 One-Hundred and Third Year Officers of the Corporation Denis M. Robinson, Honorary Chairman of the Board of Trustees Prosser Gifford, Chairman of the Board of Trustees Harlyn O. Halvorson, President of the Corporation and Director Robert D. Manz, Treasurer Kathleen Dunlap, Clerk of the Corporation Contents . Report of the President and Director Report of the Treasurer . Financial Statements Report of the Librarian 17 Educational Programs Summer Courses . . 19 Short Courses . Summer Research Programs Principal Investigators .... Other Research Personnel ... 30 Library Readers Institutions Represented 34 Year-Round Research Programs Honors Board of Trustees and Committees Laboratory Support Staff Members of the Corporation Life Members Regular Members 54 Associate Members 64 Certificate of Organization 68 Articles of Amendment 68 Bylaws 68 Report of the President and Director I began last year's report by discussing the Laboratory's needs and reminding the Corporation of the pressing need for a new Marine Resources Center capable of providing a reliable, healthy, and genetically denned supply of marine organisms. I closed that discussion of the long-awaited, many-times-planned MRC with the hope that this important facility would become a reality "before too many more director's reports are filed." I begin this director's report, my fourth, the Laboratory's ninety-third (covering its one-hundred third year) with the very good news that the new MRC will indeed be a reality very soon. As I write this report (in the spring of 1991), the building is going up outside my window. Where we used to have a carpenters' shop and a parking lot, we now have pilings on top of which the walls of a state-of-the-art facility for holding marine animals are rising. The key step toward the MRC in 1990 was the passage, in October, of a Federal appropriation bill that included $4.75 million toward the MRC. Coupled with earlier Federal support, this brought funding for the MRC and related projects to a total of $8. 95 million. Part of the planning for the new MRC involved making alternate plans for parking and a re-location of the carpenters' shop. The plan at the close of 1990 is to build a new carpenters' shop on the MBL campus next to the Broderick House and to put a new, off-site parking lot in the MBL Woods off Oyster Pond Road near Memorial Circle. In the next director's report, I fully expect to be able to report that these auxiliary projects are completed and that the new MRC is up and very close to going on-line. While preparations for construction of the MRC occupied much of our attention in 1990, we simultaneously continued to plan for an Advanced Studies Laboratory (ASL), which together with the MRC will constitute the Marine Biomedical Institute for Advanced Studies. At the close of 1990, the new ASL was in the second of three design phases. The construction of the new Marine Resources Center and the progress toward a new Advanced Studies Laboratory are important steps toward ensuring a bright future for the Laboratory, but other long- standing and well-documented needs remain. To address these additional needs, the Trustees at their June 22 meeting initiated a long-range development program. The Executive Committee approved the use of a development consultant to help us review our readiness for mounting a major fund raising campaign. The consultant. Browning Associates, presented a cogent analysis of the Laboratory, which will help us greatly expand our development operation in 1991 and beyond. Research At the urging of the External Scientific Advisory Committee, we established in 1990 a Scientific Council to function in an advisory capacity to the Executive Committee. The Council is charged with guiding the Laboratory in: the development of scientific and educational programs the use of scientific resources the evaluation and promotion of scientific staff the recruitment of new scientific positions the initiation of institutional grant proposals. The council is to work in conjunction with existing committees, such as the Research Space Committee and the Instruction Committee. Council members are appointed by the director, with the approval of the Executive Committee. The council is to include up to three members of the year-round 2 Annual Report Aerial view of the MBL's Marine Resources Center. Photo by Robert Colder. scientific community, up to three members of the summer community, two non-MBL scientists, the MBL director who serves e.\ officio. and an c.\ officio executive secretary. The first council is composed of: year-round MBL scientists Drs. John Hobbie (vice- chairman), Mitchell Sogin, and Felix Strumwasser; summer MBL scientists Drs. Barbara Ehrlich, Gerald Fischbach, and Joseph Sanger, Dr. Holger Jannasch. Woods Hole Oceanographic Institution, and Dr. Howard Hiatt, Brigham and Women's Hospital. Dr. Leslie Garrick serves as ex officio executive secretary, and, as director of the MBL, I serve as ex officio chairman of the council. The council met throughout 1990 to evaluate scientists for appointment and promotion and to set directions for future scientific development and expansion. They completed a draft of one position paper on Laboratory directions in cellular and developmental biology. The council plans to prepare additional position papers on environmental sciences/ ecology, microbiology, molecular evolution, neurobiology, and plant sciences. These documents are targeted for completion in 1991 and 1992. Instruction The instruction program continued in 1990 with its unanimously acclaimed courses. One new short course was added: Pathogenesis of Neuroimmunologic Diseases, co-directed b\ J. Murdoch Ritchie, Yale LJniversity, and Byron H. Waksman, Harvard University and New York University. At the urging of the External Scientific Advisory Committee, we have been taking a careful look at the cost of the courses. The Instruction Committee looked at cost containment in 1990, and, while there is as yet no consensus on how to proceed, it is clear that we will have to do something about spiraling cost increases in this age of decreasing federal support for advanced training. New fellowships in 1990 included the Nikon Fellowship: a Bernard Davis Fellowship for studies in microbiology or molecular evolution; the Daniel S. Grosch Scholarship Fund for studies in environmental toxicology; and the Porter Fellowships for Minority Students/Investigators for work in physiology. A list of fellowship recipients appears later in this issue. Library The Library made continued progress toward applying computing technology to library services. As described in the Report of the Librarian, Jane Fessenden, the MBL Librarian of 29 years, retired in 1990. In December, Dr. David Stonehill accepted the position of Director of the MBL/WHOI Library and Scientific Information Research Center. Dr. Stonehill has been a national leader in the development of modern information services, having worked for Report of the President and Director 3 Jelle Atema, the new director of the Boston University Marine Program. Photo by Judith Anderson. NASA, a number of leading universities, and most recently the President of the United States. Governance In August, Dr. Prosser GifTord announced his intention to step down from the chairmanship of the MBL Board of Trustees. Dr. Gifford led the MBL for 14 years, through a period of unprecedented change and growth. In a little short of a decade and a half, he worked with 4 of the 1 1 directors the Laboratory has had in its 103 year existence. Under his leadership, the Laboratory's facilities were significantly upgraded, the year-round science program grew substantially, the educational program maintained its character, the library prospered and began an exciting modernization program, and the administrative and development staffs were strengthened. Neurobiologist Dr. Jelle Atema assumed the directorship of the Boston University Marine Program in August. He replaces Dr. Rudi Strickler. who had directed BUMP since 1987. Dr. Atema has energetic plans for BUMP's graduate and undergraduate programs, and his long acquaintance with the MBL bodes well for our partnership with Boston University. In their February meeting, the Trustees approved a new Long Range Financial Planning Committee charged with reporting to the Trustees and/or the Executive Committee on the existence and appropriateness of long-range financial planning mechanisms of the laboratory. Responsibility for planning remains with the administration of the laboratory, while the new committee is to assure that the financial risks of growth have been properly anticipated and planned for. The committee is to include two at-large trustees, two corporate trustees, and the treasurer, e.\ officio. Treasurer Robert Manz chaired the new committee, which began by making a comprehensive review of the new MRC. In August, the Long Range Financial Planning Committee gave the Trustees a favorable report on the financial planning for the MRC. That favorable report was an important part of the briefing that led to the Trustees' decision to proceed toward construction of the new MRC. Personnel A group of MBL employees, including service, maintenance, and clerical workers, voted in February to affiliate with the Hospital Workers Union, Local 767. Collective bargaining negotiations began almost immediately after the election, and with the union and management both negotiating in a spirit of cooperation and mutual respect, we were able to negotiate our first contract in an impressively short time. A fair, reasonable, and workable contract was signed in July, and the work of the Laboratory proceeded without disruption. Long-time manager of marine resources John Valois retired in November after 40 years at the MBL. In addition to running the collecting operation, Mr. Valois has served for years as a spokesman for marine biology and has appeared regularly in newspapers and on television extolling the virtues of marine animals as models for research. On his departure, Edward Enos was promoted to Superintendent of Marine Resources. Mr. Enos has been a collector for many years and was eminently well-prepared to take the reins of the department. MBI, Science Writing Fellows in the Ilands-On Laboratory Course. Photo by James Hrynyshyn. 4 Annual Report The Biological Bulletin Under the editorship of Michael J. Greenberg, The Biological Bulletin continued to publish well-presented reports of outstanding research that is of general interest to biologists throughout the world. In 1990, Dr. Greenberg announced that all page charges would be dropped, and that authors would be offered 100 free reprints for publishing in the journal. Most importantly, he noted in his 1990 report to the corporation, a carefully prepared manuscript can now appear in print as soon as 3.6 months after its submission. In fact, some manuscripts meeting the criteria of the new Research Notes section which features brief communications of high quality and currency may appear in print even sooner. All of these initiatives have been highlighted in the journal's newsletter. The Biological Board. The newsletter, which is published "occasionally" by the editorial staff, was created to highlight and promote the articles appearing in. and the policies of, the journal. Dr. Greenberg has also been attempting to adjust the mix of papers appearing in the Bulletin so that the journal more closely reflects research here at the MBL. To assist in this effort. Dr. Greenberg has recruited three Associate Editors to aid in the review and solicitation of manuscripts. Drs. J. Malcolm Shick (University of Maine, Orono), Peter A. V. Anderson (The Whitney Laboratory), and David Epel (Hopkins Marine Station) will serve four-year terms as Associate Editors in their respective fields of physiology and metabolism, neurobiology, and developmental biology. Science Writing Fellowships In its fifth year, the MBL Science Writing Fellowships Program evolved a one-week, hands-on course in cellular and molecular biology for science writers. Co-directed by Dr. Robert Goldman, Northwestern University Medical School, and Boyce Rensberger, The Washington Post, the course began with an introduction to cells and a microscopy demonstration, and ended five days later with the writers cloning and sequencing DNA. The course, which will be expanded to include a neurobiology component in 1991, is open to all science writers and serves as an orientation for the MBL Science Writing Fellows. Directorship of the Science Writing Program has passed from founding director James Shreeve to Dr. Byron Waksman. Mr. Shreeve will continue to serve on the program's advisory board. The Tokyo String Quartet. Pholo by Christian Steiner. Public programs We continued to offer a few modest programs for our non-scientist neighbors on the Upper Cape. In July we held a public symposium on Science and Public Policy. The keynote address was delivered by Massachusetts Senator John Kerry, who urged the science community to become more involved in the very political process of forging a coherent national science policy. The symposium was followed by the second annual MBL Chamber Music Concert, featuring the Tokyo String Quartet. The first annual Falmouth Forum concluded in early 1990 with presentations by Dr. Allen Counter on Black Arctic explorer Matthew Henson, Anne Hawley on the government as patron of the arts, a musical portrait of Harry Truman by David McCullough, and a panel discussion on energy and the environment. The series was well enough received by local audiences that the MBL Associates brought it back for the winter of 1990- 9 1 . Marshall Goldman began the second Falmouth Forum series in November with a presentation on Perestroika, and in December actress Julie Harris read from Lucifer's Child, her new one-woman play. Harlyn O. Halvorson Report of the Treasurer The year 1990 was one of building for the Laboratory. Total support and revenues increased from $16.2 to $17.1 million due primarily to the initiation of construction of the Marine Resources Center, supported by a grant from the federal government; $1.4 million was received and expended on this project in 1990. Gifts received decreased approximately $1.6 million; 1989 was the last year of the receipt of significant funds from the Howard Hughes Foundation multi-year grant for the Library and the Education programs. Dining hall revenues rose $170,000 due to increases in food service functions, attendees, and meal card prices. Investment income grew $200,000 from 1989 to 1990 due to increases of long- and short-term investments and the length of time that they were available to the Laboratory during the respective years. Recovery of indirect costs related to research and instruction grew by approximately $150,000, all of which was attributable to the growth of MBL sponsored research. Other operating revenues were essentially unchanged from 1989. Total expenses grew approximately $700,000, from $15 million in 1989 to $15.7 million in 1990. The most significant increases were in Research Services and Plant Operations; the former is due primarily to the expansion of the operations of the Mass Spectrometry Laboratory, the Instrument Development Laboratory, and the Protein and Nucleic Acid Chemistry Center operated jointly with the Woods Hole Oceanographic Institution; the latter is due primarily to increased utility, insurance, and labor costs, as well as significant expenses involving the removal of asbestos and other repairs and renovations of our housing. It is also worth noting that the Laboratory was able to offer approximately $100,000 more scholarships, fellowships, and stipends in 1990 than in 1989, a 35% increase. While our aim is to provide much more, the trend is very heartening, as we have almost doubled the amount of scholarships, fellowships, and stipends from 1988. The Current Unrestricted Fund ended the year with an excess of support and revenues over expenses of $93,407. This combines the results of the Housing and Dining Auxiliary with all other Operations. The Housing and Dining Auxiliary had an excess of support and revenues over expenses of $267,761, of which $65,000 was used for scheduled repayment of debt owed on the Memorial Circle cottages and $199,863 was transferred to the Repairs and Replacements Reserve for housing. More sobering and troublesome, however, is that the operations of the Laboratory without the Housing and Dining operation experienced a deficiency of revenues to expenses of $174,354. The single greatest reason for this was our inability to meet budgeted goals for unrestricted current gifts. The balance sheet shows that we ended 1990 with a significant degree of liquidity as cash and short-term investments accounted for approximately two-thirds of current assets. Investments at market grew by approximately $400,000. Essentially all of that was attributable to gifts for endowment, as the net impact of the market on the valuation of our investments was a decline of less than $100,000. Land, buildings and equipment, net of accumulated depreciation, grew by almost $1 million due primarily to the beginning of construction of the Marine Resources Center. When completed, the cost of this project is expected to total $9 million. The operating budget for 1991 reflects a determination to maintain the current level of services at the Laboratory in the most cost-effective manner possible, while we redouble our efforts to generate more funds to support operations. Our greatest single challenge is still the need to find a cost-effective way to provide services that are primarily 6 Annual Report used during the summer months. The strategy of the Laboratory for the last 20 years has been to increase the size of the year-round program to provide a more stable financial base for the Laboratory. The Ecosystems Center has been the most notable success to date, and we trust that this will be a model for other MBL sponsored programs. The principal financial benefit of this strategy is the potential to achieve efficiencies in the cost of administration and services by spreading them across a broader base. We recognize that these will only be achieved by focusing on ways to be as efficient as possible. The principal financial cost of this strategy is the capital cost of the laboratory building and equipment to house the scientists and their science, as well as the reserve or endowment funds necessary to attract scientists. Through the federal funding to date of the MBIAS project, we are well on our way towards funding the necessary buildings. The next significant challenge will be generating the necessary program support funds. This strategy presumes the continued excellence of the summer research and education programs. Our education program shows many signs of health and vitality. Courses are oversubscribed; we have had renewed success in obtaining grants, and funding remains in place to support the overhead costs of the program for the next few years. Our single greatest challenge at the moment is the renewal of this support either through endowment or renewed medium-term funding. Our summer research program shows signs of financial stress. Applications, occupancy, and revenues have been flat to marginally declining for the last couple of years. It is clear that reductions in federal funding have placed a greater budgetary burden on scientists seeking to come to the MBL in the summer, and some have had to withdraw. The Laboratory has long subsidized the cost of a summer stay for all scientists from its discretionary resources the summer laboratory fee falls significantly short of the fully allocated cost of the space but our ability to do so is limited. We can selectively support a few scientists, principally young investigators, and we have had some success recently with the establishment of two Nikon fellowships for young summer scientists, but we need to do much more. It is clear that increased attention needs to be paid to the summer research program to continue its financial vitality. We need to attract more applicants; we need to find more funds to allow young scientists to become familiar with the MBL; and we need to be as efficient as possible in offering and delivering the services that make a stay at the MBL the most productive scientific experience possible. Financially, your Laboratory lives on the edge. On the positive side, we have continued to demonstrate our ability to attract funds from the federal government, from foundations, and from individuals. We are in the process of significantly upgrading our physical plant, to make the Laboratory an even more attractive place to do science. Our housing budget is currently generating the funds necessary to assure that we will be able to continue to maintain and upgrade those facilities. Our endowment has continued to grow. On the negative side, our endowment is not large enough to provide a financial cushion for any of our major programs with the significant exception of the Ecosystems Center. We have limited funds to recruit scientists whether for summer or year-round programs. Our support services do not cover their costs through charges for their services, and operations aside from Housing and Dining are in deficit. To move off the edge we will need to do three things: More clearly define, articulate, and communicate the MBL's special contribution to science and society Significantly improve our generation of capital and operating support through fund raising Redefine for ourselves what constitutes the most effective delivery of services in the most efficient manner. Robert D. Manz Financial Statements Coopers &Lybrand certified public accountants REPORT OF INDEPENDENT ACCOUNTANTS To the Trustees of Marine Biological Laboratory Woods Hole. Massachusetts We have audited the accompanying balance sheet of Marine Biological Laboratory as of December 3 1 , 1990 and the related statement of support, revenues, expenses and changes in fund balances for the year then ended. We previously examined and reported upon the financial statements of the Laboratory for the year ended December 31, 1989, which condensed statements are presented for comparative purposes only. These financial statements are the responsibility of the Laboratory's management. Our responsibility is to express an opinion on these financial statements based on our audit. We conducted our audit in accordance with generally accepted auditing standards. Those standards require that we plan and perform the audit to obtain reasonable assurance about whether the financial statements are free of material misstatement. An audit includes examining, on a test basis, evidence supporting the amounts and disclosures in the financial statements. An audit also includes assessing the accounting principles used and significant estimates made by management, as well as evaluating the overall financial statement presentation. We believe that our audit provides a reasonable basis for our opinion. In our opinion, the financial statements referred to above present fairly, in all material respects, the financial position of Marine Biological Laboratory at December 31, 1990, and its support, revenues, expenses and changes in fund balances for the year then ended in conformity with generally accepted accounting principles. Our audit was conducted for the purpose of forming an opinion on the basic financial statements taken as a whole. The supplemental schedules of support, revenues, expense and changes in fund balances for current funds (Schedule I), endowment funds (Schedule II) and plant funds (Schedule III) as of December 31, 1990 are presented for purposes of additional analysis and are not a required part of the basic financial statements. Such information has been subjected to the auditing procedures applied in the audit of the basic financial statements and, in our opinion, is fairly stated, in all material respects, in relation to the basic financial statements taken as a whole. Boston, Massachusetts April 19. 1991 LjQOOtno t oCu O ^~ r~~- _^ fN iO r^ OO O r^j oo >o r~- __ rO t O n VO r-~. o <^ oo q P! S r-- S 3 oo r- o oo ON OO O OO r- p- P^ ON OO rn S rn >/-> r-- o -o C-N] ri u-, O ^ ^O iy~i O <^i NO ^r TT o r\ m O oo 2 VO ri oo O I ^T O^ (N d f*"i <^| Tj- ^- oo ^^ TJ- r^i oo ^ *o ON ri r- (N r*-i " ^" -"V u-i r^ mrr -* r-i (N O "* rn O O ON OO O oo ON ^ 00 ON m oo TJ- ^ u-, ui m m fN O <*"> ^^f OO . oo ON 00 ^H r-- o ^t P-- /% VD O oo o^ SO rn ON ON iO f^J */^ Tf ^O VO ON so /"> 1 oo r-l I/-, ^J- QV DC O 'T r~- _J ON P- Cl Vi ON VO 00 O ON 00 sO \o r- P- w~ O r~ ON __< r*^ ON O) ^O p- -< O "0 SO (N r~^ I 'O >o O ^O n r-i vi ON fN ON ^ ON cn ' ^^ ' 1-' vO p^T ro U-, TT <^l 2 ON ' - t a & Z. g 8 -ic 5 " "S < r^ W5 ABORATORY ;TS 990 Is for 1989) LIABILITIES AND FUND B Current portion of long-term debt Accounts payable and accrued ex] Deferred income Total current liabilities Mortgage and notes payable (Not( Deferred support (Note K) Q h I? i >, U ^ ' G 2 - - - = ( Total liabilities Current unrestricted fund balance Endowment funds: Quasi-endowment unrestricted Endowment, income for unrest Endowment, income for restric Quasi-endowment restricted - 1 UJ " $ X ' O -t o ^T oo oo * ON O l~~ -r ~ -. ^^ ^D so "/" tO rn it O OO O r- -5 U 1J . 0^7 ,C ^^ *! c 03 O < c ta i -J ^ S -J <- O CL io NO "/"i O O "^ ;> ON VO O O rn rn rn Os OO ON 00_ OO "X O Oo' ri ^-~ 1 Oa. < oa /< c > i U o &0 CQ rj O VO Tf- rn O ON fNJ n so O oo rN UJ -C Z~ oo ON Tf oo oo r- _ ON r~ O ON OO so vo OO O /"! ""' O g "^ Os ^f "/"i "^ O */"> r> O ^ /"> Tf OO rn NO S ? S ?, ^' CM ^^ -~, rn i/"t so o> 3 c3 "O C J^ > 3 > 8 = b 2-0 A U U C 09 O urren I |H a "S E ^ g 3 23 o t> '5 3 c -* o v,j ^ H t o 'C a 5 1 "| *- a V) t/5 O ^ ' ' -C IsJJ 8 cement res 5 o 1 o i/i 73 C/3 c U ^ C C. E C t/i r3 "^ JZ "3 J U C T3 C a C/5 T3 U c _ 2 C '- 5 V) OJ 5 c. c S 2 "n C 75 c D u Oi U a: c c o C ca a c c o 00 c c' a * I vi O % b 73 ? ^ ci o -c h- t- r oo rn oo oo rn x o __ ^ sO c/l _ TD - S ^ ^^dso^-S i C SO Os O O"> oo 2 U. O^f ooo ^ r~i C?N oo oo cj oo [^- OO Os s a r I 2 ^ iX r I - "* C so' in SO so 3 r-ici T T'^-f O^r^ooOu/^Vi VI C Tt Os v! so oo * Tf 9 a- 2 o u- r-ri r*-ioc oor-r-n^^Tj- O" O CJ ol O so rn p < ^ r i ri' r-' o ri r i fN r 1 ri rl rn o oC o\ Os' U i^ CN! rsi r4 (A (U Tf ^ ^ < $ _i c DC S ^^ ~ "^ z 2 P < UJ Q ^ | o w fe > o u IT* "S u 'C 2 I w fc-J flj O- 4J O 3 u S g S s C D LU > ' J^ 7- w r: 5 a: > 0:1 H o o ^ ri -^t so ri rj ^-' so' Tt r-- p Tf Os Os' T' i' O V) Os_ r- oo r*^ r- r^ r 4 s i i OJ BC oo ^- ri so S OS rn so Cs| oo' on c | 1 o O C"1I W~i/"1d | Y~i o-i r~ j Tf ^-t 1 C 1 ^O ""> o^ so OO ri OO r-4 SO sO_ Z ^ u 1 2 lipiE!ill DC o- r*-, SO sf SO -f T S c ^ sC' r-' LU D W o "O " ^ C o O Si "5 ~ o-2 o g c '-5 i! ' ~ "S '> 3 C | a tU OJ 3 3 ^ C/l C T3 \ C t ~ a C __ QJ 4J t/5 3 s.sz O OJ c D. 3 CA "7 "*" "" _2 C3 Ofi il "3 8 H ou 'S C/l *O SS ^ P-'O;?;^-^^ ss=ss (N Tt ri !n 5 ON oo OX c r3-f~- sO f^ir~-sor'--"/~i/^Ov i/~i r**j \o O O oo in ^> t 00 oo ON 5 o^ csr~- so r^-rj-^Ov^Tr-ov - ioooor~- r*~)O r-j r-- o r- oo r^-i oo mTfi/->i/^ Sv o' oo TI-' NO OX in ox m' in \f\ -t ON ^f i/~i . ^~ i r | ^ -i (N r\ ^D O ox n ^ Ox 1^ i^ -^ c ^1 ON' n o> ri ON' r- o' Ov ON NO ON < T3 *** -J c tf> *C 3 Q c o ri - 0. ro ~ ON O Z -2 ^J ri O O r i r CC m j^^ Tf P Q- ON (N NO **H c ' oo' ' ^ ^ Ox' c/3 O fN r I ^ u D 60 Qi G o O g; ~ ri ? m ^ r- vo r- oo Ox r] >j tj _' ^ +-> ON m o oo_ I 5s D rr "' *~ -rl \ T^-' ' ON' f^ ^ Ox ON^ Ox' O ~7 *~ e \ ~ oo ri w~) ON in . CQ ^ o <2 3 8 m "* 3- '* 'O Ox_ **- c/) "c ^~ a: fxj r i J m c c3 r~- r- &i ' Z H 2 ~ C Q .5 u 00 r?: C ~ rN Ox O U "2 '1 I 1 o oo in NO ON 1^ O 00 Ox o , "O c X t c g ^ J H4 (U Q. QQ U 1 ON' ' ~ t^ oo ON oo NO r ri O D u E c <*) 5 g 8 <*> 3 w > L"J= u 7 w > ~ * i fy* * ^ -rt f" 1 ^D *^~i OO sO ~" 'T s . OO i ^ Ox OO r- ^ 5 w M> So S ?! SS^o o >c P * NO NO OO >/"i OV fS S ^ A3 'C O* oo' so' r [ r-* so' trT ' r [ O r- ON' ' ON to s s 1 (/I I-n r! ^ S 5 Ox r i rxi 1 CL, ^ c v5 cd c fe 1 | ^ nrestricted Oo' O' Ov oo' rn Ov oo' Tf i^i c tn Ox r~l C' C (N oo ON' ** O cd a. H _j 60 T3 c CO 5> o u n w o t -> 21 II M Is llill lllilF 2<^ o-QO O c c l oi 'a -o J< C ! HZ a E U.H 10 Notes (o Financial Statements 1 1 Marine Biological Laboratory Notes to Financial Statements A. Pnrpi isc ( if the Labi >ruti iry The purpose of Marine Biological Laboratory (the "Laboratory") is to establish and maintain a laboratory or station for scientific study and investigations, and a school for instruction in biology and natural history. B. Significant accounting policies: Basis of presentation -fund accounting 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 with specified activities or objectives. Separate accounts are maintained for each fund; however, in the accompanying financial statements, funds that have similar characteristics have been combined into fund groups. Accordingly, all financial transactions have been recorded and reported by fund group. Externally restricted funds may only be utilized in accordance with the purposes established by the donor or grantor of such funds. However, the Laboratory retains full control over the utilization 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 as the related costs are incurred (see Note K). Endowment funds are subject to restrictions requiring that the principal be invested in perpetuity with income available for use for restricted or unrestricted purposes by the Laboratory. Quasi-endowment 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. Contracts and grants Revenues associated with contracts and grants are recognized in the statement of support, revenues, expenses and changes in fund balances as the related costs are incurred (see Note K). 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 recognized through future adjustments of indirect cost rates. Investments purchased by the Laboratory are carried at market value. Money market securities are earned at cost which approximates market value. Investments donated to the Laboratory' are carried 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 reported in the financial statements which are held in trust by others. Every ten years the Laboratory's status as beneficiary of these funds is reviewed to determine that the Laboratory's use of these funds is in accordance with the intent of the funds. The market values of these investments are $4, 125.093 and $4.039,803 at December 31. 1990, and 1989, respectively. Investment income ami distribution The 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 market value unit basis (Note L). Annuities payable Amounts due to donors in connection with gift annuities is determined based on remainder value calculations which generally assure a rate of return at 10%, maximum payout terms of eighteen years, and interest payout rate of 8%. C. Land, buildings and equipment: The following is a summary of the unrestricted plant fund assets: 1990 1989 Land $ 689,660 $ 689,660 Buildings 16,955,015 16,926,715 Equipment 2,819.202 2,672,838 Construction in progress 1.969.713 580.598 22.433,590 20,869,811 Less accumulated depreciation (9.459.548) (8.858.047) $12,974,042 $12,011,764 12 Annual Report D. Retirement plan The Laboratory participates in the defined contribution pension program of the Teachers Insurance and Annuity Association College Retirement Equities Fund. Contributions amounted to $451,665 in 1990 and $393,422 in 1989. E. Restricted pledges and grants. As of December 31. 1990. the Laboratory reported active pledge and grant commitments outstanding of $492,939 (unaudited) to be received. The restricted pledges are not included in the financial statements since it is not practicable to estimate the net realizable value of such pledges. Restricted pledges of $467,337, and $14,151 and $1 1,451 are scheduled to be paid in 1991, 1992, and 1993, respectively. F. Intcrfiind borrowings: Current fund interfund balances at December 31 are as follows: Due to restricted endowment fund Due to restricted quasi-endowment funds 1990 $(4,750) (1.650) $(6.4001 1 989 $(2,190) (200) $(2,390) G. Mortgage and notes payable: Long-term debt at December 31, 1990 amounted to $1,265,000. The aggregate amount of redemption due for each of the next five fiscal years is as follows: 1991 1992 1993 1994 1995 Thereafter Less current portion Amount $ 65.000 60.000 60.000 60,000 60,000 960.000 1,265,000 65.000 $1.200.000 In 1989. the Laboratory issued $1,330,000 Massachusetts Industrial Finance Authority (MIFA) Series 1989 Bonds, which pay varying annual interest rates and mature on October 31, 201 1. The Series 1989 bonds are collateralized by a first mortgage on certain Laboratory property. The interest rate is adjustable and was 7.25% and 6.5% at December 31, 1990 and 1989. In compliance with the Laboratory's MIFA bond indenture, deposits with Shawmut Bank N.A., as trustee, represent investments in the debt service reserve fund of $143.006 in 1990 and $133.000 in 1989. H. Investments: The following is a summary of the cost and market value of investments at December 31. 1990 and 1989 and the related investment income and distribution of investment income for the years ended December 31. 1990 and 1989. Cost Market Investment Income Endowment ami Quasi-Endowmenl U.S. Government securities Corporate fixed income Common stocks Money market securities Real estate Total Less custodian and management fees Total Restricted Current Fund Certificates of deposits Money market securities Total Total investments 1990 1989 $ 1.934,834 6.663,376 4,284,165 590,735 343.247 $ 2.595.407 5.900,736 3,392,001 595,467 345,749 13,816.357 12,829,360 13.816,357 12,829.360 502,360 1.500.000 490,263 965.000 2,002,360 1.455.263 $15,818,717 $14.284.623 1990 1989 $ 1.957.512 6,809,264 6,346,778 590.735 343,247 $ 2.607.537 6.032.642 5,901,724 593.544 345.749 16,047.536 15,481.196 16.047,536 15,481,196 502,360 1.500.000 490,263 965,000 2.002.360 1,455,263 $18.049.896 $16.936.459 1990 $ 214.300 506,490 234,690 121,787 1,077,267 (55.592) 1.021.675 20.312 99.367 119.679 $1.141,354 1989 $ 1 34,394 363,439 196,452 87,994 782,279 (49.318) 732.961 34,785 I 75.205 209,990 $942,951 Notes to Financial Statements 13 I. Gift support for instruction: Current year unrestricted gifts includes $500,000 of gifts for the support of the Laboratory's instruction program available for indirect costs attributable to the instruction program. J. Tax-exempt status: The Laboratory is exempt from federal income tax under Section 501(c)3 of the Internal Revenue Code. K. Restricted current funds deterred support: The Laboratory defers recognition of revenue on current restricted funds until the related costs are incurred. Amounts received in excess of expenses are recorded as deferred support. The following summanzes the activity in the deferred support account for 1990 and 1989, respectively: 1990 Balance at beginning of year Additions: Gifts, endowment income and grants received Unrealized gains Realized gain Deductions: Funds expended under gifts and grants Transfers Balance at end of vear $4,450,222 8,499,360 19,868 1,649 7,977,187 219.294 $4,774.618 $2.951,662 9,382,212 30,672 7,542.909 371.415 $4,450.222 L. Accounting for pooled investments: The major portion of investment assets is pooled for investment purposes with each participating fund subscribing to, or disposing of. units at market value at the beginning of the current quarter. The unit participation of the funds at December 31, 1990 and 1989, respectively, is as follows: Endowment and similar funds: Quasi-unrestricted Quasi-restricted Restricted endowment 1990 3,909 7,436 39.401 50,746 1989 3.887 7.416 36.488 47.791 Pooled investment activity on a per-unit basis was as follows: Unit value at beginning of year Unit value at end of year Increase (decrease) in realized and unreal- ized appreciation Net income earned on pooled investments Total return on pooled investments $109.57 108.90 (.67) 5.99 $ 5.32 $100.00 109.57 9.57 5.73 $ 15.30 Investment income is distributed to individual funds as earned. SCHEDULE I MARINE BIOLOGICAL LABORATORY STATEMENT OF SUPPORT, REVENUES, EXPENSES AND CHANGES IN FUND BALANCES CURRENT FUNDS for the year ended December 31,1 990 SUPPORT AND REVENUES: Grant reimbursement of direct costs Recovery of indirect costs related to research and instruction programs Tuition Support activities: Dormitories Dining hall Library Biological Bulletin Research services Marine resources Investment income Gifts Change in deferred support Miscellaneous revenue Total support and revenues EXPENSES: Instruction Research Scholarships, fellowships, and stipends Support activities: Dormitories Dining hall Library Biological Bulletin Research services Marine resources Administration Sponsored projects administration Plant operations Other Total expenses Excess (deficit) of support and revenues over expenses Unrealized gain on investments Realized gain on investments Total gain on investments TRANSFERS AMONG FUNDS: Debt service Acquisition of fixed assets To unrestricted plant fund Housing transfer To support operations Instruction Capitalize ecosystems income Other Total transfers among funds Net change in fund balance Fund balances, beginning of year Fund balances, end of year Operating Fund $3.136.240 273.475 172,085 491,642 149,555 466,931 4.689.928 634,428 634.428 114.516 5.438,872 Auxiliary Enterprises Fund 579,898 141.118 658.115 443.676 1.867.738 278,221 1 .644.460 5.613.226 (174.3541 (21,653) 2.898 1 90,000 (5,045) 6.642 172.842 (1.512) 21.030 19,518 906,292 802,447 1,708,739 1,708,739 638,531 690,644 111,803 1.440.978 267.761 (65,000) (199.863) (2.898) (267,761) Total Current I 'nres-tricted Fund $3.136.240 906.292 802,447 273.475 172,085 491,642 149,555 466.931 6.398.667 634,428 634,428 114.516 7.147,611 638,531 690,644 579,898 141.118 658.1 15 443,676 1.979.541 278,221 1.644.460 7.054.204 93,407 (65,000) (21,653) (196,965) 190.000 (5.045) 3.744 (94.919) 11.512) 21.030 $ 19.518 Current Restricted Fund $4,872,529 484,749 236.366 674.423 6.268.067 1,979,547 (324.396) 1.655.151 224.740 8.147,958 1,370,282 5,228,030 356,253 172,398 347.996 6.791 35,014 1,009 432.408 7.950.181 197.777 19,868 1.649 21,517 (144,881) 135.209 5,045 (215,764) 1.097 (219.294) Total $4,872,529 3,136,240 4S4.749 906,292 802.447 273.475 172.085 728,008 149,555 1.141.354 12.666.734 2.613,975 (324,396) 339.256 15,295,569 1,370.282 5,228.030 356,253 638,531 690,644 752,296 141,118 1.006.1 1 1 450.467 2,014,555 278.221 1.645,469 432.408 15.004.385 291.184 19,868 1.649 21,517 (65,000) (144,881) (21,653) (196,965) 325.209 (215.764) 4.841 (314.213) (1.512) 21.030 $ 19,518 MARINE BIOLOGICAL LABORATORY STATEMENT OF SUPPORT, REVENUES, EXPENSES AND CHANGES IN FUND BALANCES ENDOWMENT FUNDS for the year ended December 31,1 990 SCHEDULE II Restricted Unrestricted Qiiasi- Endowmenl Endowment. Income for Unrestricted Purposes Endowment. Income for Restricted Purposes Quasi- Endowment Total Restricted SUPPORT AND REVENUES: Gifts Total support and revenues Excess of support and revenues over expenses Realized gains on investments Unrealized gain (lossl on investments Total gain (loss) on investments TRANSFERS AMONG FUNDS: Capitalize ecosystems income Endowment transfers Other transfers Total transfers among funds Net change in fund balances Fund balances, beginning of year Fund balances, end of vear $ 312.522 $ 2,450 $ 114,972 $ 314.972 312.522 2.450 314.972 314,972 312.522 2.450 314,972 314,972 9,488 $ 165,441 121,543 194.377 481,361 490,849 (11.451) (110.737) (113,465) (204.872) (429.074) (440,525) (1.963) 54.704 8.078 (10,495) 52,287 50.324 215.764 215.764 215.764 (190,000) (135,209) (135.209) (325.209) 2.260 1.921 (2.158) 12.277 12.040 14.300 (187,740) 1.921 (2.158) 92.832 92.595 (95.145) (189.703) 56.625 318.442 84,787 459,854 270.151 426.982 3.234.878 4.804.006 4.480.887 12.519.771 12.946.753 $237.279 $3.291.503 $5.122.448 $4,565.674 $12,979,625 $13,216,904 15 SCHEDULE III MARINE BIOLOGICAL LABORATORY STATEMENT OF SUPPORT, REVENUES, EXPENSES AND CHANGES IN FUND BALANCES PLANT FUNDS for the year ended December 31, 1990 SUPPORT AND REVENUES: Grant for capital additions Total support and revenues EXPENSES: Depreciation Plant operations Other Total expenses Excess (deficit) of support and revenues over expenses TRANSFERS AMONG FUNDS: Debt service Acquisition of fixed assets Transfers to unrestricted plant fund Housing transfers Other transfers Total transfers among funds Net change in fund balances Fund balances, beginning ol year Fund balances, end of year 601,501 8.500 610.001 (610.001) 65.000 180,662 245.662 (364.339) 10.272.954 $ 9,908,615 Unrestricted Repairs and Replacement Reserve $111,922 111.922 (II 1,922) (14.128) 199,863 227,892 413.627 301,705 148,820 Total Unrestricted Restricted Total SI. 429.322 $ 1.429,322 1,429.322 1,429,322 601.501 601,501 111.922 111.922 8.500 8.500 721.923 721,923 (721,923) 1,429,322 707,399 65,000 65.000 180,662 180,662 (14.128) (14.128) 199.863 199.863 227.892 (249.931) (22.039) 659.289 (249,931) 409,358 (62,634) 1.179,391 1.1 16.757 10.421.774 790.322 11.212.096 $450.525 $10,359,140 $1.969,713 $12,328.853 16 LI L LIE Report of the Librarian Library directors In 1990. Jane Fessenden, who had been the librarian and worked at the MBL for 29 years, retired. Jane made the Library a productive home for its users, improved the collection whenever financially possible, and lead the Library through innumerable changes. During her tenure, the Library was converted from the Dewey Decimal System to Library of Congress, the first "stack move" took place (after three years of planning), the reading rooms were redone, the Rare Books Room was created, the Copy Service Center was developed in its present location, staff was increased, and new technology was initiated. She will be best remembered, however, for her unique ability to provide the services and support most desired by our users. In December. Dr. David Stonehill accepted the position of Director of the MBL/WHOI Library and Scientific Information Research Center. Dr. Stonehill managed computer facilities on various NASA projects and directed computing services at academic institutions. From 1988 until his move to MBL, he was Director of Information Resources Managements for the Executive offices of the President of the United States. Under Dr. Stonehill, the Library plans to launch a Scientific Information Research Center, and we look forward to his leadership as we meld the traditional with the new methods of scientific information management. New age information delivery Using the report created by the Hughes Committee, the Library immediately began to implement some of its recommendations. A network manager was hired, and the network design was completed. What followed was a frenzy of equipment ordering and the installation of cable and wires to extend the network out of the library and into classrooms and labs in the Lillie and Loeb buildings and the Ecosystems Center. Because of the ease of communicating over the network, some of the traditional methods of the reference librarians moved into a new era during 1990. One advance. Current Contents loaded onto the librarians' computers, allowed us to send weekly up-dated bibliographies to requesters over the MBLnet. The Library's catalog has been converted to electronic records, and all the books in the library were barcoded as part of the automation of library systems. The CLAMS network of libraries came on-line in 1990, and scientists in their laboratories, who are on the Woods Hole network, can connect to the catalog and see whether the library holds the book they are seeking. Journal rates increase Our Library has studied the effect of rising book and subscriptions costs beginning with the comprehensive Rockefeller Journal Use study. In 1990, the close to 30% projected journal price increase reduced our options and resulted in the cancellation of 338 current subscriptions. Our User Panel worked throughout the summer with lists of journals targeted for cancellation, using criteria established by the Joint Users Committee, to insure the best possible preservation of our collection and services. This was not the only journal crisis of 1990 the space allocation for housing the journals had reached saturation. For over a year, the creation of a design to preserve our stack space has been developed and refined and the periodical collection will be moved during the beginning of 1991. The logical placement of the 17 18 Annual Report collections according to the alphabet will be maintained, albeit into two places. The A-Z arrangement will be in the front part of the stacks for currently received journals and also in the back wing for the pre-1976 journals. In an attempt to recover some of the escalating costs incurred in the Library, we have tried to create a model for users who are not directly associated with the contributing Woods Hole institutions. We have also changed our interlibrary loan policies and have increased resource sharing with a number of institutions, i.e.. Brandeis, University of Massachusetts Medical School, Wesleyan, University of Rhode Island and the Pell Laboratory. Escalating costs and U. S. postage rates drove the increase in interlibrary loan charges, and at the International Association of Marine Science Libraries and Information Centers' conference in Seattle, Washington, we were challenged, without ill will, but with some concern from the international community, about our rate increases. A solution to this problem may involve sharing the responsibility for serials collection development at the national and international level during the 1990s. Preservation Two documents that have been of historic and sentimental value to the scientists, staff, and visitors of the MBL were appraised and declared endangered. Study Nature Not Books the black charcoal, handwritten sign of Louis Agassiz had adhered to its backing and tears, holes, stains, rust, scratches, and smudges were evident everywhere. The same was true ot the two-page Last one to Go message to American soldiers by Katsuma Dan. Both of these documents were sent to the Northeast Document Conservation Center for treatment and have been returned to the library for display. Cathy Norton Acting Librarian Educational Programs Summer Courses Biology of Parasitism (June 10 to August 10) Co-Directors John Donelson, University of Iowa College of Medicine Carole Long, Hahnemann Medical College Faculty Steven Anderson, University of Iowa John Boothroyd. Stanford University Ted Bianco. Imperial College of Science & Technology, UK Patrick Farley, Hahnemann Medical College Steven Hajduk, University of Alabama, Birmingham Peter Ham, Liverpool School of Tropical Medicine, UK Michael E. Harris, University of Alabama, Birmingham Mary Alice Hartman, University of Kentucky Kwang S. Kim, University of Iowa Peter Kima, Hahnemann Medical College Yien Ming Kuo, Imperial College, UK Rick Martin. University of Iowa David Moser, University of Iowa Elonne Petrin, University of Cincinnati David Russell, New York University David Sachs, NIH Judy Sakanari, University of California, San Francisco Sam Turco. University of Kentucky Lecturers Nina Agabian, University of California, San Francisco Steven Beverley, Harvard Medical School Kent Campbell, Centers for Disease Control Dickson Despommier, Columbia University Paul Englund, Johns Hopkins School of Medicine Don Harn, Harvard University Stephanie James, NIH Keith Joiner, Yale University School of Medicine Patricia J. Johnson, UCLA Don Krogstad, Washington University, St. Louis Ira Mellman, Yale University School of Medicine George Nelson, Liverpool School, UK Ruth Nussenzweig, New York University Victor Nussenzweig, New York University Richard Olds, Brown University Bill Petri, University of Virginia Robert Sauer, Massachusetts Institute of Technology Alan Sher. NIH Irwin Sherman, University of California, Riverside Larry Simpson, UCLA Mitch Sogin, MBL Rick Tarleton. University of Georgia Merv Turner, Merck Sharp & Dohme Research Laboratory C. C. Wang, University of California, San Francisco Leon Weiss, University of Pennsylvania Don Wiley, Harvard University Dyann Wirth, Harvard University Students Wanida Asawanahasakda, Mahidol University, Thailand Fernanda R. Gadelha, University of Illinois Eileen S. Gruszynski, University of California, Los Angeles Michael J. Howard, Vanderbilt University Christopher A. Hunter, University of Glasgow, Scotland Gregory J. Jennings, Tulane University Christopher L. Leptak, Yale University Congjun Li, Worcester Foundation for Experimental Biology 19 20 Annual Report Leo X. Liu, Beth Israel Hospital/Harvard University James J. McCoy, University of Virginia Gloria I. Palma, Univ. del Valle. Colombia Laura J. Rocco, Johns Hopkins University Nicola N. Schweitzer, Imperial College of Science, UK Frank Seeber, Institiit fur Tropenhygeine, Germany Philippe G. Vandekerckhove, University of Leuven, Belgium Gayl Wall, University of Dundee, Scotland Embryology: Cell Differentiation and Gene Expression in Early Development (June 21 to July 30) Directors Eric Davidson, California Institute of Technology J. Richard Whitaker, MBL (Assistant Director) Faculty Michael Akam, University of Cambridge, UK Amy Bejsovic, University of Cambridge, UK Marianne Bronner-Fraser, University of California, Irvine Scott Fraser, University of California, Irvine Katherine Harding, Columbia University Janet Heasman, University of Cambridge, UK Linda Huffer, MBL Wendy Katz, California Institute of Technology Robert Kingsbury, Carnegie Institution, Baltimore Thomas Lallier, University of California, Irvine Robert Leclerc, University of Maryland Michael Levine, Columbia University David McClay, Duke University Steven McKnight. Carnegie Institution of Washington Robert Nickells, California Institute of Technology Jerome Regier, University of Maryland John Shuman, Carnegie Institution. Baltimore Paul Sternberg, California Institute of Technology Nicholas Torpey, University of Cambridge, UK Kellie Whittaker, California Institute of Technology Christopher Wylie, University of Cambridge, UK Students Kamran Ahmad, University of Utah Michael J. Bank, Columbia University Peter B. Bokor, Rockefeller University James B. Castelli-Gair, University of Madrid, Spain Robert A. Cornell. University of Washington Maria G. Di Bernardo, Italian National Research Council, Italv Win J. Dictus, University of Utrecht, The Netherlands Anne D. Donaldson, MRC Laboratory, UK Bruce W. Draper, University of Washington Silvia B. Frenk, King's College, Cambridge, UK Kareen M. Guida, University of Paris, France Nan Ho, University of California, Berkeley Jon F. Kayyem, California Institute of Technology Dangeruta Kersulyte, Acadamy of Sciences of Lithuania, USSR Daniel S. Kessler, Rockefeller University Mary Ellen Lane, Columbia University Thierry Lepage, University of Nice, France Donal T. Manahan. University of Southern California, Los Angeles Jeffrey R. Miller, Duke University Anne Marie Murphy, Johns Hopkins University Christof Niehrs, European Molecular Biology Lab., Germany Mary E. Pownall, University of Virginia Inge J. Van Wijk, Max-Planck Institute, Germany Tongweng Wang, University of Florida Marine Ecology: Concepts, Techniques and Applications of Molecular Probes (June 17 to July 28) Director J. Woodland Hastings. Harvard University Faculty Cheryl Booth, Falmouth, MA Ann Bucklin, Marine Biological Laboratory Thomas T. Chen, Center of Marine Biotechnology, University of Maryland Clara Cheng, University of Maryland Toby Cole, Hopkins Marine Station, Stanford University Lynna Hereford, Hopkins Marine Station, Stanford University Chun-Mean Lin, University of Maryland Kenneth Nealson, Great Lakes Research Center, LIniversity of Wisconsin, Milwaukee Dennis Powers, Hopkins Marine Station, Stanford University T. Roenneberg, University of Munich, Germany Simona Sorger, Hopkins Marine Station, Stanford University Keno Truper, Bonn, Germany Barbara Wimpee, Great Lakes Research Center, University of Wisconsin, Milwaukee Charles Wimpee, University of Wisconsin, Milwaukee Educational Programs 21 Lecturers David Caron Colleen Cavanaugh Penny Chisholm Ed De Long Paul Dunlap Brian Fry Linda Goff John Kessler Lynn Margulis James McCarthy Dan Morse Rob Olsen Hans Paerl Jack Palmer Ned Ruby G. Savior Ann Sesholz Bob Simon Mitch Sogin Felix Strumwasser John Waterbury Students Abdiel J. Alvarez, University of Puerto Rico Brian J. Binder, Massachusetts Institute of Technology Alice F. Brown, Brown University Ka Hou Chu, Chinese University of Hong Kong, Hong Kong Peter J. Edmunds, Northeastern University Oivind Enger, University of Bergen, Norway Jonathan B. Geller, University of Oregon Gregory J. Hinkle, University of Massachusetts, Amherst Robert E. Hodson, University of Georgia Eric R. Holm, Duke University Jerilyn Jewett-Smith, Whitman College Lisa M. Kann, University of Rhode Island James S. Maki, Harvard University Kirk D. Malloy, University of Delaware Adam G. Marsh. University of New Hampshire Tracie-Lynn Nadeau, University of Wisconsin Martin Polz, University of Vienna, Austria Michael C. Schmale, University of Miami Robin M. Schneider, University of Southern Louisiana Lynda P. Shapiro, Bigelow Laboratory for Ocean Sciences Robert L. Sinsabaugh, Clarkson University Erika Stephens. Harvard University Stephen C. Tsoi, University of Hong Kong, Hong Kong Karl E. Wommack, University of Maryland Microbiology: Molecular Aspects of Cellular Diversity (June 10 to July 26) Co-Directors Martin Dworkin, University of Minnesota John Breznak, Michigan State University Faculty Richard Behmlander, University of Minnesota Pamela Contag, University of Minnesota Christiane Dahl, University of Bonn, Germany Deborah Eastman. University of Minnesota Andrew M. Kropinski, Queen's University, Canada Hans Truper, University of Bonn, Germany Stefan Wagener, Michigan State University Lecturers Paul Dunlap, Woods Hole Oceanographic Institution Holger Jannasch, Woods Hole Oceanographic Institution J. Waterbury. Woods Hole Oceanographic Institution Students Diane K. Arwood, University of Southern Mississippi Joanna S. Brooke, University of Western Ontario, Canada Joseph P. Calabrese, West Virginia University Neena Din, University of British Columbia, Canada Amis Druka, Latvian State University, USSR Olivia T. Harriott, University of Connecticut Robert Huber, University of Regensburg, Germany Jennifer B. Klenz, University of Saskatchewan, Canada Judith A. Koskella, New York University Bridget E. Laue, University of Colorado Jared R. Leadbetter, Goucher College Timothy C. Lilburn, University of British Columbia, Canada Shi Liu, University of Oklahoma Lynn V. Mendelman, Harvard Medical School Elizabeth J. Orle, Colorado State University Mechthild Pohlschroder, University of Massachusetts, Amherst Frank J. Slack, Tufts University Barth F. Smets, University of Illinois Claire S. Ting, Cornell University Mary A. Wyka, Merck & Co., Inc. Neural Systems & Behavior (June 10 to August 1) Co-Directors Ronald Calabrese, Emory University Martha Constantine-Paton, Yale University 22 Annual Report Faculty Arthur Arnold, University of California, Los Angeles Alexander Borst, Max-Planck-Institut filr Biologisch Kybernetick, Germany John Byrne, University of Texas Medical School Thomas Carew, Yale University Leonard deary. University of Texas Medical School at Houston Robin Cloues, Harvard University Michael Davis, Yale University Robert M. Douglas, University of British Columbia, Canada Lise Eliot, Center for Neurobiology & Behavior Russell Fernald, University of Oregon Leslie Fischer, Columbia University William Frost, University of Texas Medical School Cole Gilbert, University of Arizona Dennis Gorlick, Columbia University Jon Hayashi, Arizona Research Laboratory Sally Hoskins, City College of CUNY John Koester, New York State Psychiatric Institute Richard B. Levine, University of Arizona Yurin Levy, Brandeis University Margaret Livingstone, Harvard Medical School Anne Lohoff, Columbia University Eduardo R. Macagno, Columbia University Emilie Marcus, Yale University Eve Marder, Brandeis University Michael P. Nusbaum, University of Alabama, Birmingham Mu-Ming Poo, Columbia University David J. Sandstrom, University of California Patricia Steen, Yale University Nacita Tabti, Columbia University Janis C. Weeks, University of Oregon Angela Wenning, Universitat Konstanz, Germany Michael Nitabach, Columbia University Lecturers Catherine Carr, University of Rochester Joe Martinez, Jr., University of California Students Robert A. Berkowitz, Washington LJniversity James P. Burke, University of Alabama, Birmingham Belinda S. Chang, Harvard University Miles G. Cunningham, Massachusetts Institute of Technology Graeme W. Davis, University of Massachusetts, Amherst Mayra Garcia-Ruiz, University of Nat. Autonoma, Mexico John F. Hamilton, Meharry Medical College, Nashville Tamara L. Harris, University of Ottawa, Canada Valerie L. Kilman, University of Illinois Barlett W. Mel, California Institute of Technology Brett D. Mensch, Baylor College of Medicine Alison R. Mercer, University of Otago, New Zealand Edward P. Monaghan, University of California, Berkeley Tanja Quenzer, Max-Planck-Institut, Germany Adina L. Roskies, University of California, San Diego Hyunjune S. Seung, Harvard University Deana L. Shackelford, University of Oklahoma Petra Skiebe, Universitat Hamburg, Germany John E. Spiro, University of California, San Diego Douglas Syme, University of California, Irvine Neurobiology (June 10 to August 11) Co-Directors Leonard Kaczmarek, Yale University Irwin Levitan, Brandeis University Christopher Miller, Brandeis University Faculty Cecilia Armstrong, University of Pennsylvania Gary Banker, LIniversity of Virginia Synnove Beckh, Max-Planck-Institiit fur Biophysikalische Chemie, Germany Andrew Czernik, Rockefeller University Jan De Weer, Duke University Judith Drazba, NINDS/NIH Keith Elmslie. Case Western Reserve University Stuart Firestein, Yale University School of Medicine Paul Forscher, Yale University Robert French, University of Calgary, Canada Sara Garber, University of Alabama, Birmingham Allison Hall, Case Western Reserve University Richard Horn. Roche Institute for Molecular Biology Richard Huganir, HHMI, Johns Hopkins Medical School Stephen Jones, Case Western Reserve University Richard Kramer, Columbia University Kyu-Ho Lee, Johns Hopkins Medical School Andrew Matus, Friedrich Meischer Institute, Switzerland Robert Miller, Case Western Reserve University Angus Nairn, Rockefeller University Randall Reed, HHMI, Johns Hopkins Medical School Thomas Reese, NINDS/NIH Talvinder Sihra, Rockefeller University Carolyn Smith, NINDS/NIH Walter Stuhmer, Max-Planck-Institiit fur Biophysikolische Chemie, Germany Educational Programs 23 Students Ricardo C. Araneda, Albert Einstein Medical School Sylwester Chyb, Wesleyan University Dan H. Cox, Tufts University Stuart D. Critz, University of Texas Medical School Peter F. Drain, MIT Kathryn J. Edson, University of Minnesota Julie A. Haack, University of Utah Lise R. Heginbotham, Harvard University Marc A. Post, University of Michigan Haohua Qian, University of Illinois Hanno M. Roder. Massachusetts Institute of Technology Maria A. Sosa, University of Florida Physiology: Cell and Molecular Biology (June 10 to July 21) Director Thomas D. Pollard. Johns Hopkins University Faculty Steven Almo, Johns Hopkins Medical School Kerry Bloom, University of North Carolina William Busa, Johns Hopkins University Antony Galione, Johns Hopkins University Neal R. Gliksman, University of North Carolina Robert Jensen. Johns Hopkins School of Medicine Margaret Kenna, University of North Carolina John Maslanski. Johns Hopkins University Jonathan McMenamin-Balano, University of Massachusetts, Boston Robert Palazzo, Marine Biological Laboratory Katherine Pollard, Johns Hopkins Medical School Ted Salmon, University of North Carolina Sue Schmidt, Glyndon, MD John Simon, University of North Carolina John Sinard, Johns Hopkins Medical School Tammy Smith, University of North Carolina Cynthia V. Stauffacher, Purdue University Murray Stewart, Medical Research Council, UK Elaine Yeh, University of North Carolina Students Robert L. Bacallao, University of California, Los Angeles Sandra A. Brockman, Carnegie Mellon University Thomas O. Carpenter, Yale University Isabelle A. Carre, SUNY, Stony Brook Joseph A. Cerro, Columbia University Marc D. Coltrera, University of Washington Tod A. Critchlow, Scripps Institute of Oceanography Spencer J. Danto, Cornell Medical College Michele I. Flatters, Tufts University Holly V. Goodson, Stanford University Supriya Jayadev, Duke University John R. Jordan, University of Utah David L. Keefe, Yale University Karen L. King, Florida State University Qingwen Li, University of Kansas Helen McNeill, University of Pennsylvania Michael E. Mendelsohn, Harvard/Brigham & Women's Hospital Christa S. Merzdorf, Harvard University Robert Mirro, University of Tennessee Karen M. Page, Dartmouth College Alice P. Pentland, Washington University, St. Louis Zhican Qu, Johns Hopkins University Joe W. Ramos, University of Virginia Jean F. Regal, University of Minnesota Eric A. Shelden, University of Massachusetts, Amherst Charles B. Shuster, Tufts University Thomas W. Smith, Brigham & Women's/Harvard Medical School Robin L. Stears, SUNY, Stony Brook Salme Taagepera, University of Virginia Charlotte M. Vines, Harvard Medical School Yingjian Wang, University of Miami Christiane Wiese, Johns Hopkins Medical School Elizabeth L. Winter, City College of New York Vicki L. Wolff, Brandeis University Qi Yang, University of Connecticut Guangwen Zhou, Oregon State University Short Courses Analytical and Quantitative Light Microscopy in Biology, Medicine, and Materials Science (May 10 to 18) Co-Directors Edward D. Salmon, University of North Carolina Greenfield Sluder, Worcester Foundation for Experimental Biology David E. Wolf, Worcester Foundation for Experimental Biology Faculty and Course Assistants Brad Amos, MRC, Cambridge, UK Orit Baha, Worcester Foundation for Experimental Biology Steven M. Block, Rowland Institute for Science Richard Cardullo, Worcester Foundation for Experimental Biology Walter Carrington, University of Massachusetts Medical School 24 Annual Report Gordon Ellis, University of Pennsylvania Fred Fay, University of Massachusetts Medical School JeffGelles, Brandeis University Richard Haugland, Molecular Probes, Eugene, OR Linda Huffer. MBL Shinya Inoue. MBL Anthony Moss, Worcester Foundation for Experimental Biology Rudolf Oldenbourg, MBL Stephen Parsons, University of North Carolina Robert V. Skibbens, University of North Carolina Kenneth R. Spring, NIH D. Lansing Taylor, Carnegie-Mellon University Richard Walker, University of North Carolina Students Julia Barsony, NIDDK/NIH Harold G. Bohlen, Indiana University Medical School Daniel J. Brat, Mayo Graduate School Barry J. Burbach, SUNY, Stony Brook John P. Caufield, Harvard Medical School Wendy Cheng, International Paper Company, Tuxedo, NY Matthew H. Chestnut, The Procter and Gamble Co., Cincinnati, OH Diana M. Cordova, Alcon Laboratories, Inc., Ft. Worth. TX Sascha Dho, The Hospital for Sick Children, Toronto, Canada Ulrich Dirnagl, Forschungslabor, Germany Cynthia J. Forehand, LIniversity of Vermont John J. Freeman. Monsanto Co., St. Louis, MO Jill Gemmill, University of Alabama, Birmingham John A. Hammer. Ill, NHLBI/NIH Ray S. Hartman, Children's Hospital of Los Angeles Donald T. Haynie, The Johns Hopkins University Walter J. Koroshetz, Massachusetts General Hospital Dan Green (left) demonstrates a ratio imaging package from Universal Imaging during an AQLM course session. Julia M. Lash, Indiana University School of Medicine Michael I. Lethem, University of North Carolina Steve Paddock, University of Wisconsin Shoshana Paglin, Boston University School of Medicine James R. Sellers. NHLBI/NIH Anna Spudich, Stanford University Fei Wang, Syracuse University Measurement and Control of Chemical Stimuli (April 25 to 30) Director Greg A. Gerhardt, University of Colorado Faculty and Course Assistants Barry W. Ache, C.V. Whitney Laboratory, University of Florida Jelle Atema, Boston University Marine Program, MBL Scott Brock, University of Colorado Marilyne Friedemann, University of Colorado John S. Kauer, Tufts New England Medical Center Stuart Firestein, Yale University School of Medicine Paul Moore. Boston University Marine Program, MBL Mike Palmer, University of Colorado Michael Parrish, University of Colorado Wayne L. Silver, Wake Forest University Students Carol E. Diebel, SUNY Health Science Center Richard L. Doty, Hospital of the University of Pennsylvania Heather L. Eisthen, Indiana University Tim Granata, Southeastern Massachusetts University Kristina-Viveka Hillegaart, Astra Research Centre, Sweden Herman K. Lehman, University of Arizona Celia Marrase. Boston University Marine Program, MBL Pricilla E. Purnick, Columbia University John R. Welborn, University of Southern California Methods in Computational Neuroscience (August 5 to September 1) Co-Directors James M. Bower, California Institute of Technology Christof Koch. California Institute of Technology Educational Programs 25 Lecturers and Instructors Paul Adams, SUNY, Stony Brook Edward Adelson, Massachusetts Institute of Technology Daniel Alkon, N1H Richard Anderson, Massachusetts Institute of Technology David Beeman, University of Colorado, Boulder Avis H. Cohen, Cornell University Norberto Grzywacz, Massachusetts Institute of Technology Nancy Kopell, Boston University Rudolfo Llinas, NYU Medical Center Kevan Martin, MRC, Oxford, UK Michael Mascagni, Washington, DC Kenneth Miller, University of California, San Francisco Mark Nelson, California Institute of Technology John Rinzel, NIH David Rumelhart, Stanford University Sylvia Ryckebusch, California Institute of Technology Terrence Sejnowski, Salk Institute Allen I. Selverston, University of California, San Diego John Uhley, California Institute of Technology David Van Essen, California Institute of Technology Lucia Vaina, Boston University Matthew Wilson, California Institute of Technology Students Aric Agmon, University of California, Irvine Hagai Agmon, Hebrew University of Jerusalem, Israel Dale Anders, University of California, San Diego Evyatar Av-Ron, The Weizmann Institute of Science, Israel Ellen Barton, University of Pennsylvania Eyal Bartfeld, Rockefeller University David Berkowicz, Yale University School of Medicine Neil J. Berman, Oxford University, UK Peter J. Braam, University of Utah Dennis Bray, Colorado State University Trevor Darrell, Massachusetts Institute of Technology Gyongyi Gaal, University of Pennsylvania Kurt Haas, Albert Einstein College of Medicine Dirk Kautz, University of Oregon Markus Lappe, NIH Sean Marrett, Montreal Neurological Institute, Canada Douglas Morton, Case Western Reserve University Dietmar Rapf, MPI fur biologische Kybernetik, Germany Walter Schneider, University of Pittsburgh Nelson Spruston, Baylor College of Medicine Christoph Staub, Brain Research Institute, Switzerland Fan-Gang Zeng, Syracuse University Molecular Evolution (August 19 to 31) Director Mitchell L. Sogin, MBL Faculty Michael Clegg, University of California, Riverside Daniel B. Davison, University of Houston W. Ford Doolittle, Dalhousie University, Canada Robert Dorit, Harvard University John W. Drake, National Institute of Environmental Health Sciences Joseph Felsenstein, University of Washington Walter M. Fitch, University of California, Irvine Barry G. Hall, University of Rochester Andrew Knoll, Botanical Museum of Harvard University David R. Maddison, Harvard University Peter Maloney, The Johns Hopkins Medical School Roger Milkman, The University of Iowa Gary Olsen, University of Illinois Norman R. Pace, Indiana University David Joseph Patterson, University of Bristol, UK Margaret Riley, University of Massachusetts, Amherst David A. Shub, SUNY, Albany Steve Smith, Harvard University Temple F. Smith, Dana Farber Cancer Institute Mark Wheelis, University of California, Davis Allan C. Wilson, University of California, Berkeley Elizabeth A. Zimmer, Smithsonian Institution Students Alexandra L. Basolo, University of Texas, Austin Heidi G. Blair, SUNY, Albany Judith Anne Blake, Smithsonian Institution Laura Blinderman, San Diego State University David J. Bogler, University of Texas, Austin Diane M. Bridge, Yale University Henner Brinkmann, Technische Universitat, Germany James R. Brown, Simon Eraser University, Canada Bruce E. Byers, University of Massachusetts, Amherst Robert E. Calhoon, Queens College 26 Annual Report Bruce J. Cochrane, University of South Florida Rafael O. De Sa, University of Texas, Austin Marcela Descalzi, University of Houston Philippe Djian, Harvard Medical School James W. Edwards, Salem College Bruce A. Eichhorst, University of North Dakota William Fischer, Los Alamos National Laboratory Dean A. Glawe, University of Illinois Robert M. Gould, New York State Institute for Basic Research Sharon P. Gowan, Harvard University Herbaria Lawrence I. Grossman, Wayne State University Radhey S. Gupta, McMaster University, Canada Raymond W. Holton, University of Tennessee Bradford E. James, Dartmouth College Matthew D. Kane, University of Illinois Jessica C. Kissinger, Indiana University Paul J. Kores, Tulane University Maja Kricker, National Institute of Environmental Health Sciences Uri Ladror, Chicago Medical School Laura Landweber, Harvard University Bang-Ning Lee, University of Texas, Houston Heikki Lehvaslaiho, University of Helsinki, Finland Wayne S. Leibel, Lafayette College Enrique P. Lessa, University of California, Berkeley Louise Ann Lewis, Ohio State University Paul O. Lewis, Ohio State University John M. Logsdon, Jr., Indiana University Kenneth L. McNally, University of California Linda K. Medlin, University of Bristol, UK Thomas Mills, University of Houston Judith Mongold, University of Massachusetts, Amherst Barbara Moore, Massachusetts Institute of Technology Rana Muzaffar. Florida State University Lena M. Nielsen, University of Lund, Sweden Elizabeth A. Oakenfull, MRC Haematology Unit, Oxford, UK Yves P. Quentin, Los Alamos National Laboratory James Ramsay, University of Utah Judith M. Rhymer, Smithsonian Institution Kenneth A. Rice, Harvard University Bernardo Rudy, NYU Medical Center Frank G. Salinas, University of Houston Leo C. Schalkwyk, Dalhousie University, Canada Michael Schloemann, Yale University Jeffrey D. Silberman, University of Miami Rama S. Singh, McMaster University, Canada Elizabeth M. Snella, Indiana University Elizabeth T. Snow, NYU Medical Center Peter L. Starkweather, University of Nevada Jeffrey L. Stein, University of California, San Diego Youngbae Suh, Louisiana State University Thomas R. Sutler, CUT, Research Triangle Park, NC Cheryl L. Tarr, University of North Dakota Richard N. Williams, Boise State University Charles G. Wray, Yale University Optical Microscopy & Imaging in the Biomedical Sciences (October 6 to 12) Co-Directors Nina Stromgren Allen, Wake Forest University Colin S. Izzard, SUNY, Albany Faculty and Course Associates Steven M. Block, Rowland Institute for Science Joseph De Pasquale, SUNY, Albany Alec Harootunian, HHMI, University of California, San Diego Fredric S. Fay, University of Massachusetts Medical Center Kenneth A. Jacobson, University of North Carolina John M. Murray, University of Pennsylvania Kenneth Orndorff, Dartmouth College Julia S. Sizemore, Wake Forest University Stephen J. Smith, Stanford University School of Medicine Kenneth R. Spring, NHLBI/NIH Anna Spudich. Stanford University School of Medicine Roger Tsien, HHMI, University of California, San Diego Lecturers Jan Hinsch, Leica, Inc., Rockleigh, NJ Shinya Inoue, MBL Ernst Keller, Carl Zeiss, Inc., Thornwood, NY Rudolf Oldenbourg, MBL Students Lawrence W. Argenbright, Boehringer Ingelheim Pharmaceuticals, Inc., Ridgefield, CT Elikplimi K. Asem, Purdue University Thomas G. Burke, City of Hope National Medical Center Dean Cole, Los Alamos National Laboratory R. Ford Denison, USDA Camille DiLullo, University of Pennsylvania Harold F. Dvorak, Beth Israel Hospital David H. Eidelman, McGill University, Canada T. R. Gowrishankar, University of Chicago John W. Hanrahan, McGill University, Canada Christopher M. Kenyon, Centre Hospitalier Thoracique de Montreal, Canada Educational Programs 27 Jeff Lansman, University of California, San Francisco Stephen Lin, Harvard Medical School William W. Mantulin, University of Illinois, Urbana Champaign Jose A. Mari Mutt, University of Puerto Rico Jane E. Minturn, Yale University School of Medicine Spering A. Scott, Purdue University Jacqueline Sterner. University of Rochester Scott J. Sternberg, Colorado State University Xiao-Ying Tien, Case Western Reserve University Susan M. Wall, NIH David C. Zawieja, Texas A&M University Pathogenesis of Neuroimmunologic Diseases (August 19 to 31) Co-Directors J. Murdoch Ritchie, Yale University School of Medicine Byron H. Waksman, Foundation for Microbiology Faculty Vahi E. Amassian, SUNY Health Science Center Barry G. W. Arnason, University of Chicago Joel A. Black, Yale University Pietro DeCamilli, Yale University Medical School Judah A. Denburg, McMaster Medical Center, Canada Marc A. Dichter, University of Pennsylvania Charles A. Dinarello, Tufts University Medical School Diane Griffin, The Johns Hopkins University Stephen L. Hauser, Massachusetts General Hospital Henry Khachaturian, NIMH/NIH Norman Latov, College of Physicians and Surgeons of Columbia University Carl M. Leventhal, NINDS/NIH W. Ian Lipkin, University of California. Irvine Cathy G. McAllister, University of Pittsburgh Dale E. McFarlin, NINDS/NIH John Newsom-Davis, Oxford University, UK Robert B. Nussenblatt, NEI/NIH Nathanial G. Pitts, National Science Foundation Jerome B. Posner, Memorial Sloan-Kettering Cancer Center Donald L. Price, Johns Hopkins Hospital Richard W. Price, University of Minnesota Health Center James W. Prichard, Yale University Medical School Cedric S. Raine, Albert Einstein College of Medicine Anthony T. Reder, University of Chicago David M. Regan, York University, Canada Stephen C. Reingold, National Multiple Sclerosis Society Benjamin F. Roy, Georgetown University School of Medicine Clifford B. Saper, University of Chicago Randolph B. Schiffer, Strong Memorial Hospital Eli E. Sercarz, University of California, Los Angeles Moon L. Shin, University of Maryland, Baltimore Michael E. Shy, Thomas Jefferson University Hospital Lawrence Steinman, Stanford Medical Center Stephen G. Waxman, Yale University Medical School Howard L. Weiner, Brigham and Women's Hospital Jerry Wolinsky, University of Texas Health Science Center at Houston Students Anat Achiron. Beilinson Medical Center, Israel Peter-Brian Andersson. Sir William Dunn School of Pathology, UK Jody L. Baron, Yale University School of Medicine Bruce F. Bebo, Jr., Texas A&M University John R. Bethea, University of Alabama, Birmingham Helen Clare Bodmer, Institut de Chimie Biologique, France Arlene R. Collins, SUNY, Buffalo Steven W. Dow, Colorado State University Lorise C. Gahring, Research Institute of Scripps Clinic Maureen N. Gannon, Rockefeller University Claude Paul Genain, University of Kentucky Koenraad Gijbels, University of Leuven, Belgium Jonathan D. Glass, Johns Hopkins Hospital John J. Hemperly, Becton Dickinson Research Center Nancy A. Johnson, Washington University Medical School Abraham Kessler, Weizmann Institute of Science, Israel Judith Luber-Narod, University of Massachusetts Medical School Mary Ann McKee, Columbia Presbyterian Hospital Rune Midgard, Molde County Hospital, Norway Nicholas T. Potter, University of Connecticut School of Medicine Reijo Salonen, University of Turku, Finland Jun-ichi Satoh, University of British Columbia, Canada Tiziana Savio, Institute Superiore di Sanita. Italy Daryth D. Stallone, University of Pennsylvania School of Medicine Sharon A. Stranford. Hahnemann University Ursula I. Wesselmann, Northwestern University Jurgen Zielasek, Diabetes Research Institute, Germany Summer Research Programs Principal Investigators Alkon, Daniel, NIH, LMCN Armstrong, Clay M., University of Pennsylvania Armstrong, Peter, University of California Augustine, George J., University of Southern California Baker, Robert, New York University Medical Center Barlow, Robert, Syracuse University Barry, Daniel, University of Michigan Medical Center Barry, Susan R., University of Michigan Bass, Andrew H., Cornell University Bates, William R., Carleton University, Canada Bearer, Elaine L., University of California Beauge, Luis. Institute M. y M. Ferreyra, Argentina Begenisich, Ted, University of Rochester Medical Center Bennett, Michael V. L., Albert Einstein College of Medicine Bezanilla, Francisco, University of California, Los Angeles Bloom, George S., University of Texas Southwestern Medical Center Bodznick, David, Wesleyan University Borgese, Thomas A., Lehman College, CUNY Boron, Walter F., Yale University School of Medicine Borst, David W., Illinois State University Boyer, Barbara C, Union College Brady, Scott, University of Texas Southwestern Medical Center Brehm, Paul, SUNY, Stony Brook Brown, Joel E., Washington University School of Medicine Bryant, Shirley, University of Cincinnati Bug, William J., Columbia University Burdick, Carolyn J., Brooklyn College Burger, Max M., Friedrich Miescher-Institute, Switzerland Callaway, Joseph C., University of North Carolina Cariello, Lucio, Stazione Zoologica, Italy Chang, Donald C., Baylor College of Medicine Chappell, Richard L., Hunter College, CUNY Charlton. Milton, University of Toronto, Canada Charmantier, Guy, Universite des Sciences et Techniq. du Languedoc, France Charmantier-Daures, Mireille, Universite des Sciences et Techniq. du Languedoc, France Clay, John, NIH, NINDS Cohen, Lawrence B., Yale University School of Medicine Cohen, William D., Hunter College, CUNY Collin, Shaun P., University of California, San Diego Cooperstein, Sherwin J., University of Connecticut Health Center Cuppoletti, John, University of Cincinnati D'Alessio, Giuseppe, University of Naples, Italy D'Avanzo, Charlotte, Hampshire College De Weer, Paul, Washington University School of Medicine Deitmer, Joachim W., University of Kaiserslautern, Germany Dunlap, Kathleen, Tufts Medical School Eckberg, William R., Howard University Ehrlich, Barbara E., University of Connecticut Health Center Feinman, Richard, SUNY Health Science Center Ferguson, Donald, University of Cincinnati Fieber, Lynne A., University of Miami Fink. Rachel, Mount Holyoke College Fishman, Harvey M., University of Texas Medical Branch Gadsby, David C., Rockefeller University Gainer, Harold, NIH, NINDS Garrick, Rita Anne, New Jersey Medical School and Fordham University Giuditta. Antonio, University of Naples, Italy 28 Principal Investigators 29 Glynn, Paul, Hunter College Goldman, Robert D., Northwestern University Medical School Gonzalez-Serratos, Hugo, University of Maryland School of Medicine Griff, Edwin, University of Cincinnati Haimo, Leah T., University of California Hardin, John A., Yale University Heiny, Judith, University of Cincinnati Hernandez-Cruz, Arturo, Roche Institute of Molecular Biology Highstein, Stephen M., Washington University Holman, Molly A., The Whitney Laboratory, University of Florida Hoskin, Francis C.G.. Illinois Institute of Technology Hoy, Ronald R., Cornell University Hummon. William, Ohio University Ilan. Joseph, Case Western Reserve University Ilan, Judith, Case Western Reserve University Ip. Wallace, University of Cincinnati Johnston, Daniel, Baylor College of Medicine Josephson, Robert K., University of California Kaminer, Benjamin, Boston University School of Medicine Kaneshiro, Edna S., University of Cincinnati Kaplan, Ehud, Rockefeller University Kaplan, Ilene M., Union College Karp, Richard, University of Cincinnati Kremer, James N., University of Southern California Kriebel, Mahlon, SUNY Health Science Center, Syracuse Langford, George, University of North Carolina, Chapel Hill Laufer, Hans, University of Connecticut Lee, Youngsook, Harvard University Lehman, Michael, University of Cincinnati Levin, Jack, University of California School of Medicine, San Francisco Levine, Robert Paul, Washington University School of Medicine Lian, Jane, University of Massachusetts Medical School Linck, Richard W., University of Minnesota Lipicky, Raymond John, U. S. Food and Drug Administration Lisman, John, Brandeis University Llinas, Rodolfo R., New York University Medical Center Loewenstein, Werner R.. University of Miami School of Medicine Lohmann, Kenneth J., University of Washington Malchow. Robert Paul, University of Illinois College of Medicine Malinowska, D. H., University of Cincinnati Martinez, Joe L., Jr., University of California Matteson, Donald R., University of Maryland Meinertzhagen, Ian A., Dalhousie University, Canada Metuzals, Janis, University of Ottawa, Canada Mitchison, Timothy, University of California, San Francisco Moreno, Alonso P., Albert Einstein College of Medicine Nasi, Enrico, Boston University School of Medicine Nelson, Leonard, Medical College of Ohio Noe, Bryan D., Emory University School of Medicine Northcutt, R. Glenn, University of California, San Diego Obaid, Ana Lia, University of Pennsylvania School of Medicine Pant, Harish, NIH, NINDS Pappas. George D., University of Illinois, Chicago Parysek. Linda, University of Cincinnati Peckol, Paulette, Smith College Pierce, Sidney K., University of Maryland, College Park Pierson, Beverly K., University of Puget Sound RarTerty. Nancy S., Northwestern University Rakowski, Robert F., UHS/The Chicago Medical School Render, JoAnn, Hamilton College Ripps, Harris, University of Illinois College of Medicine Rose, Birgit, University of Miami School of Medicine Ross. William N., New York Medical College Ruderman, Joan V., Harvard Medical School Russell, John M.. University of Texas Medical Branch, Galveston Ryan, Una S., Washington University Salzberg, Brian M., University of Pennsylvania School of Medicine Segal, Sheldon, Rockefeller Foundation 30 Annual Report Sheetz, Michael P., Washington University Medical School Silver, Robert B., Cornell University Sloboda, Roger D., Dartmouth College Speksnijder, Johanna E., Utrecht University, The Netherlands Sperelakis, Nicholas, University of Cincinnati Spiegel, Evelyn, Dartmouth College Spiegel, Melvin, Dartmouth College Spray, David C, Albert Einstein College of Medicine Stein, Gary, University of Massachusetts Medical School Steinacker, Antoinette, Washington University Stracher, Alfred, SUNY Health Science Center, Brooklyn Stuart, Ann E., University of North Carolina Swandulla, Dieter, Max-Planck-Institut, Germany Swenson, {Catherine I., Harvard Medical School Tanguy, Joelle, Northwestern University Telzer, Bruce R., Pomona College Terada, Hirotoshi, Hamamatsu Photonics, K. K., Japan Treistman, Steven N., Worcester Foundation for Experimental Biology Trinkaus, John Philip, Yale University Troll, Walter, New York University Medical Center Tucker, Edward B., Baruch College Tykocinski. Mark L., Case Western Reserve University Tytell, Michael, Bowman Gray School of Medicine of Wake Forest University Vaisberg, Eugeni A., Protein Research Institute, USSR Vallee, Richard, Worcester Foundation for Experimental Biology Van Egeraat, Jan M., Vanderbilt University Vogel, Steven S., NIH, NIDDK Weiss, Dieter G., Zoology Institute, Germany Welsford, Jan G.. University of Pennsylvania School of Medicine Wolszon, Laura R., Columbia University Worden, Mary Kate, Harvard Medical School Yeh, Jay Z., Northwestern University Zigman, Seymour, University of Rochester School of Medicine and Dentistry Zottoli, Steven J., Williams College Zukin, R. Suzanne, Albert Einstein College of Medicine Summer investigators (left to right) David Borst, Guy Charmantier, Mireille Charmantier-Daures, Hans Laufer, and Brian Tsukimura Other Research Personnel Akeson, R. A., University of Cincinnati Altamirano, Anibal A.. University of Texas Medical Branch Andrews. S. Brian, NIH. NINDS Arnold, John, University of Hawaii Bamrungphol, Wattana. University of Pennsylvania Bargeron. Mary, Syracuse University Batten, Bruce E., Dublin, OH Bechtold-Imhof, Ruth, Boston University Medical Center Belmont, Lisa, University of California, San Francisco Bernal-Martinez, Juan, University of Connecticut Health Center Bersoff, Rochelle. Washington University School of Medicine Bezprozvanny, Ilya, University of Connecticut Health Center Blanchard, Charles, Cambridge, MA Bloom, Jonathan, Northwestern University Bloom, Theodora, Harvard Medical School Blubaugh, Diane, University of Puget Sound Bollner, Tomas, Dalhousie University, Canada Braithwaite, Scott, SUNY, Stony Brook Breitweiser, Gerda E., Johns Hopkins University School of Medicine Brezina, Vladimir, University of Connecticut Health Center Brient, Heather L., Ohio University Brodwick, M., University of Texas Medical Branch Brooks, Brian P., University of Pennsylvania Other Research Personnel 31 Buelow, Neal, Syracuse University Buerkett, Christopher G.. Illinois State University Burkart. Werner, Paul Scherrer Institut, Switzerland Campos de Carvalho, Antonio, Federal University of Rio De Janeiro, Brazil Capano. Carla Perrone. Stazione Zoologica, Italy Caputo, Carlo, IVIC-Inst. Venezolano de Inv. Ciento, Venezuela Carey, John P., University of Washington Caviedes. Pablo, NIH, NIDDK Chow, Robert H., University of Pennsylvania Cohen, Avrum, Yale University School of Medicine Collin, Carlos, NIH, LMCN Correa, Ana Maria, University of California, Los Angeles Cota, Gabriel, Centre de Investigacion del IPN, Mexico Couch, Ernest, Texas Christian University Davis. Marion, Yale University School of Medicine Demers. David, University of California, Riverside Dermietzel. Rolf. University of Regensburg, Germany Derrick, Brian, University of California, Berkeley Dessev, George N., Northwestern University Medical School DiPolo, Reinaldo, IVIC, Venezuela Diebel, Carol, SUNY Health Science Center at Brooklyn Dohrmann, Cord, Harvard Medical School Drazba, Judith, NIH, NINDS Evans, Wayne A., Ohio University Faeta, Hillary H.. Ohio University Falk, Chun Xiao, Yale University Medical School Floyd, Carl C., NIH, NINDS Flucher, Bernhard E., NIH, NINDS Forman, Robin, Medical College of Virginia Franzini-Armstrong, Clara, University of Pennsylvania Fujiki, Hirota, National Cancer Center Research Institute, Japan Gao, Qian, University of Pennsylvania School of Medicine Gavi, Benny, SUNY, Binghamton Genao. Ivan, Lehman College Gerosa, Daniela, Friedrich Miescher Institut, Switzerland Gill-Kumar, Pritam, U. S. Food and Drug Administration Goldman, Anne E., Northwestern University Medical School Gomez, Maria, Boston University Gomez-Laganas, Froylan M., Centra de Investigacion del IPN, Mexico Goodwill, Ken Gould, Robert, New York State Institute for Basic Research Grant, Philip, NIH, NINDS Grassi. Daniel. Ft. Lauderdale. FL Hammar. Kasia, NIH Hamosh, Leora Y., Johns Hopkins School of Medicine Haneji, Tatsuji, Chiba University School of Medicine, Japan Hernandez, Michael R., University of Texas Medical Branch Herzog, Eric, Syracuse University Hessinger. David, Loma Linda University Hogan, Emilia, Yale University School of Medicine Homola, Ellen. University of Connecticut Huang, Jack, NIH, NIDDK Huddie, Patrick L., NIH, NINDS Huerta. Patricio, Brandeis University Jaffe, David, Baylor College of Medicine Johnston, Jennifer, Dartmouth College Jordan, John R., University of Utah Kadam, P. A., The Population Council Kahana, Alon, Brandeis University Kenner. Glenda S., Ohio University Key, Brian, Children's Hospital Medical Center, Cincinnati Khanna, Anu, Lehman College Kim, Nam Hew, Albert Einstein College of Medicine Kingston, Samuel, New York KJein, Kathryn, Emory University School of Medicine Knudsen, Knud D.. U. S. Food and Drug Administration Koide, Samuel S., Population Council Komura, Hitoshi, University of Pennsylvania School of Medicine Konnerth, Arthur, Max-Planck-Institiit fur Biophysikalische Chemie, Germany Kowtha, Vijayanand C., NIH Kozlowski, David, University of Connecticut Krishna, Gobal, NIH Kronidou, Nafsika, Dartmouth College Kuhns, William, Hospital for Sick Children, Canada Kumar, Sanjay S., University of Pennsylvania Landau, Matthew, Stockton State College Larsen, Mark, University of Puget Sound Lasser-Ross, Nechama, New York Medical College Latorre, Ramon, Universidad de Chile, Chile Lederhendler, I. Izja, NIH, NINDS 32 Annual Report Leidigh. Christopher, Brown University Leonard II, Edward E., University of Pittsburgh Levitan, Herbert, University of Maryland Lin, Jen-Wei, New York University Medical Center Locke, Rachel, Washington University Lowe, Kris, New College Luca, Frank, Harvard Medical School Martin, Melissa, Illinois State University McDonald, John K., Emory University School of Medicine Menichini, Enrico, University of Naples, Italy Miledi, Ricardo, University of California Milgram, Sharon L., Emory University School of Medicine Mimori, Tsuneyo, Keio University. Japan Misevic, Gradimir. University Hospital of Basel, Switzerland Moogan, Teresa, Hunter College Moreira, Jorge E., NIH, NINDS Moshiach, Simon, NIH Murray, Sandra A., University of Pittsburgh Niclas, Joshua, University of California, San Francisco Nitabach, Michael, Massachusetts Institute of Technology Norgren. Robert, University of Cincinnati Olds, James, NIH, LMCN Sagi, Amir, Hebrew University, Israel Sakakibara, Manabu, Toyohashi, Japan Sala, Salvador, University of Maryland, Baltimore Sanchez, Ivelisse, Hunter College Sanchez-Andres, Juan V., NIH Sato, Eimei, Kyoto University, Japan Schiminovich, Samuel, Englewood, NJ Seemes, Eliana, University of Sao Paulo, Brazil Sheetz, Jennifer, Woods Hole, MA Sheller, Rebecca, University of Texas, Austin Shibuya, Ellen, Harvard Medical School Sivaramakrishnan, Shobhana, University of Southern California Sosnicki, Andrzej A., University of Pennsylvania Spires, Sherrill, University of Rochester Steffen, Walter, University of Minnesota Stewart, Patricia, University of Rochester Stokes, Darrell R., Emory University Stout, Matthew P., SUNY, BufTalo Sugimori, Mutsuyuki, New York University Medical Center Sweet, Hyla C, Union College Syme, Douglas, University of California Tewari, Kirti, University of Texas Medical Branch Todaro, M. Antonio D., University of Modena, Italy Tsukimura. Brian, Illinois State University Turner, Robert, Lehman College Papaconstandinov, Eleni, University and Cantonal Hospital, Switzerland Pardo, Alex, Hampshire College Parsey, Ramin. University of Maryland Parsons, Stephen, University of North Carolina, Chapel Hill Perozo, Eduardo, University of California, Los Angeles Piccoli, Renata, University of Naples, Italy Plant. Charles P., Tufts University Powers, Maureen K., Vanderbilt University Pumplin, David W., University of Maryland, Baltimore Quigley, James P., SUNY, Stony Brook Rafferty, Keen A., University of Illinois Rasgado-Flores, Hector, University of Health Sciences/ Chicago Medical School Reese, Thomas, NIH, NINDS Rodriguez, Katrin, University of Illinois Romero, Adarli, Washington University Roth, William W., Emory University School of Medicine Russell, Joshua C., University of Texas Medical Branch Ueno, Hiroshi, Rockefeller University Vargas, Fernando, U. S. Food and Drug Administration Vogel, Jackie M., Illinois State University Wache, Susanne C., University of Connecticut Wadsworth, Patricia, University of Massachusetts Watson, Win, University of New Hampshire Wells, Dan, Lexington, MA Wiercinski, Floyd J., Northeastern Illinois University Wu, Jian-Young, Yale University School of Medicine Zakevicius, Jane M., University of Illinois College of Medicine Zecevic, Dejan, University of Belgrade, Yugoslavia Zheng, Qiang, Baylor College of Medicine Zhi-quo, Liang, Population Council Zigman, Bunnie Rose, University of Rochester School of Medicine and Dentistry Zipser, David, E. Lansing, MI Library Readers 33 Library Readers: General Adler, Elizabeth, MBL Apter, Nathanial, Nova University Baccetti, Baccio, University of Siena, Italy Barrett, Dennis, University of Denver Bartolucci, Simonetta, Naples, Italy Burdick, Jonathan, MBL Bursztajn, S., Baylor College of Medicine Carriere, Rita M., Downstate Medical Center Child, Frank M., Trinity College Chinard, Francis P., New Jersey Medical School Cobb, Jewel Plummer, California State University, Fullerton Cohen, Leonard, American Health Foundation DeSimone, Douglas W., University of Virginia Health Science Center DeToledo-Morrell, Leyla, Rush Medical Center Dowling, John, Harvard University Duncan, Thomas K., Nichols College Edds, Kenneth T., SUNY, Buffalo Eisen, Herman, Massachusetts Institute of Technology Farmanfarmaian, A., Rutgers University Fox, Tom, Harvard Medical School Frenkel, Krystyna, NYU Medical Center Friedler, Gladys, Boston University School of Medicine German, James L., The New York Blood Center Gilbert, Daniel L., NIH Goldstein, Moise H., John Hopkins University Goodman. Dewitt S.. Columbia University Gormley, Gerard, MBL Guttenplan, Joseph, NYU Dental Center Hill, Richard, Michigan State University Humphreys, Tom, University of Hawaii International Wildlife, MBL Kaltenbach, Jane, Mount Holyoke College Karlin, Arthur, Columbia University Kelly, Robert. U.I.C. College of Medicine Kisten & Babitsky, MBL Klemow, Kenneth M.. Wilkes University Kremer, James N., University of South California Laderman, Aimlee D., Yale University Lee, John J., City College of CUNY Levitz, Mortimer, NYU Medical Center Marine Research, MBL May, Ronald, MBL Mitchell, Ralph, Harvard University Mooseker, Mark S., Yale University Musacchia, X. J., University of Louisville Olins, Ada L., University of Tennessee, Oak Ridge Olins, Donald, University of Tennessee, Oak Ridge Oschman, James L., MBL Passano, Leonard, University of Wisconsin Peisach, Jack, Albert Einstein College of Medicine Pollen, Daniel, University of Massachusetts Medical School Prosser, C. Ladd. University of Illinois Prusch, Robert, Gonzaga University Robinson, Denis, MBL Romagnani, Sergio, Universita di Firenze, Italy Rossi, Mose, Naples, Italy Rourke, John, MBL Russell-Hunter, W. D., Syracuse University Sanger, Jean, University of Pennsylvania Sanger, Joseph W., University of Pennsylvania Schippers. Jay M., WAFRA, New York, NY Shriftman, Mollie-Starr, North Nassau Health Center Singh, Ajai Pratap, Bareilly College Solomon, Dennis, MBL Stein, Leonard, Health Sciences Center SUNY Stephens, Philip J., Villanova University Stephenson, William K., Earlham College Sweet, Frederick, Washington University School of Medicine Szent-Gyorgyi, Andrew, Brandeis University Szulman, Aron, MaGee Womens Hospital Trager. William, The Rockefeller University Van Holde, Kensal E., Oregon State University Vaina, Lucia, Boston University Wagner, Robert R., University of Virginia Wallace, Robert W.. MBL Warren, Leonard, Wistar Institute Wilber, Charles G., Colorado State University Wimpee. Charles, MBL Wittenberg, Jonathan, Albert Einstein College of Medicine Wolken, Jerome, Carnegie Mellon University Worgul, Basil, Columbia University Worgul, Kathleen, MBL Young, Wise, NYU Medical Center Young, Lily, NYU Medical Center 34 Annual Report Library Readers: Desks Anderson, Everett, Harvard Medical School Avioli, Louis V., Jewish Hospital, St. Louis Boyer, John F., Union College Candelas, Graciela C., University of Puerto Rico Chaet, Alfred B., University of W. Florida Clark, Arnold, MBL Cohen, Seymour, MBL Collier, Marjorie M., St. Peters College Copeland, Eugene, MBL Corliss, Bruce, Duke University Crews, David, University of Texas, Austin Czinn, Steven J., RB&C Hospital, Cleveland Festoff, Barry, VA Medical Center, Kansas City Fussell, Catherine, University of Pennsylvania Gibson, Kevin, University of Pittsburgh Gross, Paul R., University of Virginia Grossman, Albert, NYU Gruner, John, NYU Medical Center Haubrich, Robert, Denison University Herskovitz, Theodore, Fordham University Inoue, Sadayuki, McGill University, Canada Katz, George M., Merck, Sharp & Dohme King, Kenneth, Childrens Hospital Krane, Stephen, Mass. General Kravitz, Edward, Harvard Medical School Leighton, Joseph, Peralta Cancer Research Lorand, Laszlo, Northwestern University Malbon, Craig C., SUNY Mauzarall, David, Rockefeller University Mizell, Merle, Tulane University Morrell, Frank, St. Lukes Medical Center, Chicago Narahashi, Toshio, Northwestern University Medical School Nickerson, Peter A., SUNY, Buffalo Paton, David, MBL Person, Philip, VA Medical Center, Brooklyn Robinson, Denis, MBL Roth, Jay, University of Connecticut Roth, Lorraine, MBL Shanklin, Douglas, University of Tennessee Shepard, Frank, Woods Hole Database Shepro, David, Boston University Sonnenblick, B. P.. Rutgers University Spector, Abraham. Columbia University Speck, William, Case Western Reserve Stuart, Ann, University of North Carolina Sundquist, Eric, USGS, Woods Hole Sydlik, Mary Anne, Eastern Michigan University Tweedel, Kenyon, University of Notre Dame Webb, Marguerite, MBL Wittenberg, Beatrice, Albert Einstein College of Medicine Yow, Frank, Kenyon College Library Readers: Rooms Hines, Michael, Duke University Medical School Moore, John W., Duke University Medical School Rabinowitz, Michael. Harvard Medical School Reynolds, George, Princeton University Weidner, Earl, Louisiana State University Weissman, Gerald, NYU Medical Center Zweig, Ronald, MBL Domestic Institutions Represented Alabama, University of Birmingham Albert Einstein College of Medicine American Bionetics, Inc. American Psychological Association Ames Laboratory Analytical Luminescence Laboratory Applied Biosystems Arizona Research Laboratory Arizona, University of Arizona, University of. School of Medicine Aspen Research Institute Atlantex & Zieler Instrument Corporation Axon Instruments, Inc. Bareilly College Baruch College of CUNY Baylor College of Medicine Beckman Instruments, Inc. Bethesda Research Labs Bio-Rad Laboratories Bodega Marine Station Boston University Boston University School of Medicine Bowling Green State University Bowman Gray School of Medicine Brandeis University Brigham & Women's Hospital Brinkmann Instruments. Inc.. Brooklyn College of CUNY Brown University Institutions Represented Bryn Mawr College Bunion Instrument Company, Inc. California Institute of Technology California, University of, Berkeley California, University of, Davis California, University of, Irvine California, University of, Los Angeles California, University of. Riverside California, University of. San Diego California, University of, San Francisco California, University of, Santa Cruz Cambridge Instruments Cambridge Technology Carnegie Institute of Washington Carnegie-Mellon University Case Western Reserve University Center for Agricultural Biotechnology Center for Marine Biotechnology Center for Microbial Ecology, Michigan State University Chicago, University of Children's Hospital Medical Center Ciba Corning Diagnostics Corp. Cincinnati, University of City College of New York City University of New York (CUNY) Clark University Colgate University Colorado College Colorado, University of. Boulder Columbia University Columbia University College of Physicians and Surgeons Connecticut, University of Connecticut, University of. Health Center Cornell University Coy Laboratory Products Crimson Camera Technical Sales CSPI, Inc. Dage MTI. Inc. 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School of Medicine Michigan State University Michigan, University of, Ann Arbor Minnesota, University of Molecular Probes Monsanto Company Mount Holyoke College Murray State University National Institutes of Health/NINDS National Institutes of Health/NIEHS National Institutes of Health/NIDDK National Jewish Center for Immunology & Respiratory Medicine National Science Foundation Naval Medical Research Institute Neslab Instruments, Inc. New Brunswick Scientific Company, Inc. New College of the University of South Florida New England Medical Center New Hampshire, University of New Jersey Medical School New York Medical College New York University Medical Center 36 Annual Report Nikon, Inc. North Carolina, University of. 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Williams College Wisconsin, University of Wistar Institute Woods Hole Oceanographic Institution Worcester Foundation for Experimental Biology Yale University Yale University School of Medicine Zeiss, Carl, Inc. Foreign Institutions Represented Academy of Sciences, USSR Basel, University of, Switzerland Belgrade, University of, Yugoslavia Bergen, University of, Norway Bielefeld, University of, Germany Biozentrum, Basel. Switzerland Bonn, University of, Germany British Museum of Natural History, UK Calgary, University of, Canada Cambridge, University of, UK Carlton LIniversity, Canada Catania. University of, Italy Centro de Investigacion del IPN, Mexico Chiba University, Japan Chiba University School of Medicine, Japan Chile, University of. C.E.C.S., Chile Institutions Represented 37 CINEVESTAV-IPN, Mexico Cologne, University of, Germany Dalhousie University, Canada Dalhousie University Medical School, Canada Ecole normale superieure, France Federal University of Rio de Janeiro, Brazil Freie Universitat Berlin, Germany Friedrich Miesener Institut. Switzerland Gottingen, University of, Germany Hamamatsu Photonics, K. K.. Japan Hebrew University, Israel Hong Kong, University of. Hong Kong Imperial College of Science & Technology, UK Institute for Biological Research. Yugoslavia Instituto di Biologia dello Sviluppo, CNS, Italy Instituto M. y. M. Ferreyra, Argentina I.V.I.C., Venezuela Kaiserslautern, University of, Germany Katholieke Universiteit Leuven, Belgium Keio University, Japan Kobe University, Japan Koln, University of, Germany Konstanz, University of, Germany Kyoto University, Japan Life Sciences Institute, Israel London School of Hygiene & Tropical Medicine, UK London, University of, Egham, UK Max-Planck-Institut fur Biophysikalische Chemie, Germany Max-Planck-Institiit, Gottingen, Germany McGill University, Canada Medical Research Council, UK Milan, University of, Italy Modena, University of, Italy Montreal. University of, Canada Naples, University of, Italy Nice, University of, France Nottingham, University of, UK Osnabriick, University of, Germany Ottawa, University of, Canada Paris, University of, France Paul Scherrer Institute, Switzerland Philipps-Universitat, Marburg, Germany Philipps-Universitat, Germany Protein Research Institute, LISSR Queens College, UK Queens University, Canada Queensland, University of, Australia Regensburg, University of, Germany Sao Paulo, University of, Brazil Saskatchewan. University of, Canada St. Andrews University, Scotland, UK Seoul National University, Korea Siena, University of. Italy Simon Bolivar, University of, Venezuela State University of Utrecht, The Netherlands Stazione Zoologica, Italy Stockholm, University of, Sweden Swiss Federal Institute, Switzerland Swiss Federal Institute of Technology, Switzerland Toronto, University of, Canada Tubingen, University of, Germany Universita di Firenze, Italy Universita di Palermo, Italy Universite des Sciences et Techniq. du Languedoc, France University College, UK University Hospital of Basel, Switzerland Utrecht University, The Netherlands Vienna, University of, Austria Vrije Universiteit Brussel, Belgium Wolfson College, UK Z.L.F. Kantonsspital, Basel, Switzerland Zoologie Institut, Germany Year-Round Research Programs Boston University Marine Program Atema, Jelle (Professor of Biology, Program Director) Humes. Arthur G. (Professor of Biology Emeritus) Strickler, J. Rudi (Professor of Biology, Program Director) Tamm, Sidney L. (Professor of Biology) Valiela, Ivan (Professor of Biology) I '/siting tacultv and investigators Ache. Barry (C. V. Whitney Lab, St. Augustine. Florida) Bamhach, Richard (Virginia Polytech and State University) Bloomer, Sherman (Boston University) Borisy, Gary (University ot Wisconsin, Madison) Caballero. Pascual (University of Las Palmas. Gran Canaria) Chang, Donald (Baylor College of Medicine. Texas) D'Avanzo. Charlene (Hampshire College) Dionne. Vincent (University of California) Gerhardt, Greg (University of Colorado) Good. Michael (Lltrecht University, The Netherlands) Hinga. Kenneth (LJniversity of Rhode Island) Josephson, Robert (University of California, Irvine) Kauer. John (Tufts University) Kaufman, Les (New England Aquarium) Kremer, James (University of Southern California) Linck, Richard W. (University of Minnesota. St. Paul) Marrase, Celia (University of Barcelona, Spain) Meinertzhagen, Ian (Dalhousie University, Canada) Nakamura. Shogo (Toyama University, Japan) Patterson. David (University of Bristol. UK) Peckol, Paulette (Smith College) Perez Castillo. Fernando (CIQRO. Mexico) Rhoads, Donald (Adjunct Professor of Geology) Rietsma. Carol (SUNY, New Paltz) Rosenbaum. Joel (Yale University) Sardet. Christian (Villefranche-sur-Mer. France) Sardu. Rafael (Centre d'Estudio Avancats de Blanes, Spain) Singarajah, K. V. (University of Brazil. Brazil) Steffan, Walter (University of Minnesota, St. Paul) Speksnijder. Annelies (Utrecht University, The Netherlands) Sulak. Ken (Huntsman Marine Science Centre, Canada) Terasaki, Mark (NIH) Research stall Foreman, Kenneth (Research Associate) Gerardo. Hortense (Research Associate) Tamm, Signhild (Senior Research Associate) Voigt. Rainer (Research Associate) Teaching assistants and stall Alber, Merryl (Course Assistant) Coughlin, David (Course Assistant) Cowan, Diane (Course Assistant) Gomez, George (Course Coordinator) Hahn, Dorothy (Senior Administrative Secretary) Hersh. Douglas (Course Assistant) LaMontagne. Michael (Course Assistant) Mosiach. Simon (Course Assistant) Mulsow. Sandor (Course Assistant) Paullin. Susanne (Assistant to the Director) Scholz, Nathaniel (Course Assistant) Sunlcy, Madeline (Administrative Manager) Varela, Diana (Course Assistant) Graduate students Alber, Merryl Banta. Gary Brewer, Matthew Bohachevsky, Boris Bryden, Cynthia Coughlin. David Cowan, Diane Elskus. Adria Gallager. Scott Gomez. George Hersh. Douglas Hwang, Jiang-shiou Karavanich. Christy Katz, Andrea Year-Round Research Programs 39 Kennedy, Blain LaMontagne, Michael Lavalli. Kan Lindstrom, Daniel Mazel. Charles Merrill, Carl Moore. Paul Mosiach, Simon Mulsow, Sandor Portnoy. John Scholz. Nathaniel Strubel, liana Tamse Armando Trager, Geoffrey Usup, Gires Varela, Diana Weinstein, Diana White, David Undergraduate student* I-'all 1990 Annis, Eric Boulanger, Lisa Brand. Michelle (University of California. San Diego) Carrama. Carolyn Christine. Stephanie Collumb, Chris (Trinity University) Christman, Laurie Cruse, Jennifer Deiner, Michael (Tulane) DeSantis, Michael Dougall, David (University of Pittsburg, Johnstown) Fernandez, Cecilia Forrest. Davina Fox, Pamela Gibson. Mark (Wesleyan) Goodbred, Steven Guertin, Laura (Bucknell) Harris. Matthew Haven. Michael Hover, Eric (Lawrence University) Kinkade, Christopher Kozlowski, Wendy MacDonald, Robin McKee, Nancy (Middlebury) McNeil. Sean Polsky, Matthew Russell, John (Franklin & Marshall) Seton-Haris, Genevieve Shaz, David Smith. Douglas Smith. Kirsten Spradlin, Trevor Staunton, Edward Steenburgh. Eric Stousland. Brett (Lawrence University) Tello. Christine Valdes. Hugo Warner, Jacqueline BUMP graduate student Diane Cowan explains the finer points of the lobster to BUMP undergrads. Youngberg, Jill Ziemba, Robert Visiting graduate .stndentx Fall 1991) ll'HMS Burner Michael (Lawrence University) Jones, Fynn (Vanderbilt) Klaper. Rebecca (University of Illinois) Maselli. Andrew (Knox College) Naessig, Tricia (Augusta College) Theis, Lisa (Lawrence University) Summer undergraduate interns Altes, Hester Bergles, Dwight Butler, Nina (Westown School. PA) Casterline, Jennifer Call, Christopher Hoagland. Todd (Bucknell) Lacomis, Lynne (SUNY. Binghamton) Pfeifer, Shaili Pardo. Alex (Five Colleges) Reischauer, Alyssa (USC) Sanders, Sophie (Dalton School) Short, Graham Szulgit, Greg Fall undergraduate intern Waggett, Caryl (Brown) 40 Annual Report Laboratory ofJelle Atema Organisms use chemical signals as their main channel of information about the environment. These signals are transported in the marine environment by turbulent currents, viscous flow, and molecular diffusion. Receptor cells extract signals 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 animal and its narrowly tuned receptor cells. Research focuses on amino acids (food signals) and pheromones (courtship and dominance) neurophysiology of receptor cells, behavior guided or modulated by chemical signals, and computational models of odor plumes and neural filters. Laboratory of Arthur G. Humes Research interests include systematics, development, host specificity, and geographical distribution of copepods associated with marine invertebrates. Current research is on taxonomic studies of copepods from invertebrates in the tropical Indo-Pacific area, and poecilostomatoid and siphonostomatoid copepods from deep-sea hydrothermal vents and cold seeps. Laboratory of Ivan Valiela Our major research activity involves the Waquoit Bay Land Margin Ecosystems Research Project. This work examines how human activity in coastal watersheds (including landscape use and urbanization) increases nutrient loading to groundwater and streams. Nutrients in groundwater are transported to the sea, and, after biogeochemical transformation, enter coastal waters. There, increased nutrients bring about a series of changes. The Waquoit Bay LMER is designed to help us to understand and model the coupling of land use and consequences to receiving waters, and to study the processes involved. A second long-term research topic is the structure and function of salt marsh ecosystems, including the processes of predation, herbivory. decomposition, and nutrient cycles. The Ecosystems Center The Center was established in 1975 to promote research and education in ecosystems ecology. Eleven senior scientific staff and forty research assistants and support staff study the terrestrial 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 controls of tundra, lake, and stream biota) to the Harvard Forest (long-term studies of the effects of disturbance in forest ecosystems) 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 nutrients and make use of the Center's mass spectrometry laboratory (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 dioxide-caused climate change. Opportunities are available for postdoctoral fellows. Staff and consultants Hobbie, John E., Co-Director Melillo, Jerry M., Co-Director Banta, Gary Bauman, Carolyn Berger, Laurel Bowles, Francis Bowles, Margaret Castro, Nancy Cochran, Wendy Davis, Sarah Deegan. Linda Dornblaser. Mark Downs, Martha Fafinski, Stephen Fry. Brian Garritt, Robert Giblin, Anne Gregg, Tim Griffin, Elisabeth Helfrich, John Hopkinson, Charles Hullar, Meredith Jesse. Martha Jordan, Marilyn Kicklighter, David Kracko, Karen Laundre, James Michmerhuizen, Catherine Miliefsky, Michelle Moller, Bernard Nadelhoffer, Knute Padien. Daniel Pallant. Julie Parmentier. Nancy Peterson. Bruce Raich, James Rastetter, Edward Regan, Kathleen Ricca, Andrea Russell, Ann Saupe. Susan Schwamb, Carol Schwarzman, Elisabeth Semino. Suzanne Shaver, Gaius Shulman, Laura Steudler, Paul Tholke, Kristin Tucker. Jane Bowden. Richard Peterjohn. William Castro. Mark Ryan. Michael Klmg, George Wainright. Sam McKane, Robert I 'iMiinx scholars Joyce, Linda, U.S.D.A. Forest Service McGuire. David, U.S.D.A. Forest Service Neill. Christopher. University of Massachusetts, Amherst Laboratory for Marine Animal Health 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 investigated. Staff Abt, Donald A., Director and The Robert R. Marshak Term Professor of Aquatic Animal Medicine and Pathology, School of Veterinary Medicine, University of Pennsylvania Year-Round Research Programs 41 A view of Alaska's Brooks Range form the Ecosystems Center's Toolik Lake research station. Photo by Mark Dornblaser. Bullis, Robert A., Research Associate in Microbiology, University of Pennsylvania Leibovitz, Louis, Director Emeritus McCafferty, Michelle, Histology Technician, University of Pennsylvania Moniz, Priscilla C., Secretary Smolowitz, Roxanna. Research Associate in Pathology, University of Pennsylvania Wadman. Elizabeth A., Microbiology Technician, University of Pennsylvania Laboratory of Aquatic Biomedicine This laboratory investigates leukemias of soft shell clams. Monoclonal antibodies developed by this laboratory and techniques in molecular biology are used to investigate the differences between normal and leukemic cells and their ontogeny. The impact of pollutants on leukemogenesis is currently being studied with an emphasis on regional superfund sites. Staff Reinisch, Carol L., Investigator, MBL, and Chairperson Department of Comparative Medicine, Tufts University School of Veterinary Medicine Miosky, Donna, Laboratory Technician White. Marja. Postdoctoral Fellow Laboratory of Cell Biochemistry This laboratory studies developmental, metabolic, and environmental influences on the genetic regulation of cellular enzymes. Current emphasis is on the gene products involved in hepatic heme biosynthesis and utilization in marine fish. These processes are responsive to hormonal and nutritional signals as well as to environmental pollutants and carcinogens. This work is being conducted with fish liver in vivti, with primary cultures of normal hepatocytes. and with cultured hepatoma cells isolated from a fish tumor. Gene activity is quantitated with cDNA probes, and the relevant genes are being cloned in bacteria to define better the actions of chemical inducers. Other research is concerned with translocation of proteins between various subcellular compartments both in fish hepatocytes and in invertebrate eggs before and after fertilization. Staff Cornell, Neal W., Senior Scientist Bruning, Grace, Research Assistant Foley. Kathleen, Research Assistant I'tviting Fox, T. O., Harvard Medical Center 42 Annual Report Laboratory of Developmental Genetics This research group studies the early gene control of cellular differentiation pathways (cell lineage determination) in the embryos of tunicates and other marine invertebrate species. Staff Whittaker, J. Richard, Senior Scientist Crowther, Robert, Research Assistant Loescher, Jane L., Research Assistant Meedel, Thomas H.. Assistant Scientist I 'isitint( investigators Collier, J. R., Brooklyn College Lee, James J., Columbia University, College of Physicians & Surgeons Laboratory* of Judith P. Grassle Studies on the population genetics and ecology of marine invertebrates living in disturbed environments, especially of sibling species in the genus Capilclla (Polychaeta). Staff Grassle, Judith P., Senior Scientist Feinsilber, Sigalit, Research Assistant Mills, Susan W.. Research Assistant Laboratory ofHaryln O. Halvorson This laboratory is interested in the molecular process of sporulation and germination in members of the genus Bacillus. Our earlier work has involved characterization of the ger.l gene in Bacillus subtilis, and determination of the germination requirements of marine endospore-forming bacteria. Over the past year, we have isolated a large number of sporeformers from various marine environments like deep sea cores and sediments. Our intention is to characterize these bacteria at the molecular level, with emphasis on genes associated with sporulation and germination. Protocols based on DNA fingerprinting and quantitative hybridizations have been developed to differentiate these bacteria from one another, as well from terrestrial sporeformers. The hybridization data has shown that the bacterial isolates are not closely related to one another. Numerical taxonomic methods are also being used to cluster the various isolates. The physiologically interesting sporeformers will also be characterized by physical mapping using rare-cutting restriction endonucleases. Staff Halvorson. Harlyn O., Principal Investigator Chikarmane, Hemant, Assistant Scientist Glick, Beatriz. Research Assistant Pratt. Sara. Research Assistant VanLoov. Lori. Research Assistant I isiting investigators Anderson, Porter, University of Rochester Keynan, Alex, Hebrew University, Jerusalem, Israel Kornberg, Hans, Christ's College, Cambridge. UK. Vincent, Walter, University of Delaware Yashphe. Jacob, Hebrew University, Jerusalem, Israel Laboratory of Shiny a Inoue Mechanism of mitosis and related motility. Development of high resolution 3-D video microscope systems. High resolution polarized light microscopy of muscle fibrils. Physical origin of edge birefringence and image formation in the polarized light microscope. Staff Inoue, Shinya, Distinguished Scientist Knudson, Robert. Instrument Development Engineer Oldenbourg, Rudolf. Visiting Assistant Scientist Stukey, Jetly, Research Assistant Szent-Gyorgyi, David. Research Assistant Taracka, Richard. Instrument Maker Woodward. Bertha M., Laboratory Manager I 'isiting investigators Inoue, Theodore, Universal Imaging Corporation, West Chester, Pennsylvania Silver, Robert B., Cornell University. Ithaca, New York Stemmer, Andreas, Lhiiversity of Basel, Switzerland Laboratory of Alan M. Ku^irian The research explores the functional morphology and ultrastructure of various organ systems in opisthobranch mollusks. The program includes mariculture of the nudibranch, Hermissenda crassicornis, with emphasis on developing reliable culture methods for rearing and maintaining the animal as a research resource. Studies include optimization of adult and larval nutrition, control of facultative pathogens and disease, and development of morphologic criteria for staging larvae and juveniles. These morphologic studies stress the ontogeny of neural and sensory structures. Concurrent with these studies is the development of a new technique to obtain and reconstruct serial block face images (SBFI) of epoxy embedded tissue actually sectioned inside an SEM by an in situ miniature ultramicrotome. Additional collaborative research includes histochemical investigations on strontium's role in initiating calcification in molluscan embryos (shell and statoliths). as well as immunocytochemical labelling of cell-surface and secretory product antigens of neurosecretory neurons in the eye of . l/'/r.v/fl. Staff Alan M. Kuzirian. Assistant Scientist Catherine T. Tamse, Research Assistant Year-Round Research Programs 43 Dr. Alan Kuzirian and Catherine Tamse in the laboratory. Photo by George Liles. Laboratory of Molecular Evolution The major research effort of this laboratory is the structure analysis of ribosomal RNA. Similarities between small subunit ribosomal RNA sequences are used to infer the evolutionary history of eukaryotic microorganisms and to design molecular probes for studies in marine ecology. Staff Sogin, Mitchell L., Director Bhattacharya. Debashish, Postdoctoral Fellow Bibeau, Claude, Research Technician Bucklin, Ann, Visiting Scientist Stickel, Shawn, Research Technician Wainwright, Patricia, Postdoctoral Fellow Laboratory of Neuroendocrinology This laboratory studies the molecular and cellular bases of two neural programs that regulate different important behaviors in the model mollusk Aplysia. Research is conducted on the mechanisms of the neuronal circadian oscillators located in the eyes. These circadian oscillators drive the circadian activity rhythm ot 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 associated 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. In another project, the laboratory has discovered and is continuing research on a novel second messenger enzyme, an NADase, in the oocytes of Aplysia. that generates cyclic ADPR, a Ca :+ -mobilizing product. Staff Strumwasser, Felix, Director Cox, Rachel L., Senior Research Assistant Glick, David, Senior Postdoctoral Fellow Hellmich, Mark, Postdoctoral Fellow Rainville, Carol, Laboratory Assistant Vogel, Jackie, Research Assistant Laboratory of Robert E. Palazzo This laboratory studies the biochemical regulation of cellular events during meiosis and mitosis. An integral part of the research effort is the design of reconstitution systems that faithfully execute cell cycle dependent events under defined conditions. Current cell biological, immunochemical, biochemical, and microscopic methodologies are employed. Using marine eggs as a material source, assays have been developed that allow the study of germinal vesicle breakdown (GVBD), aster formation, and reactivation of isolated mitotic apparatus in vitro. Current focus of the laboratory is on the identification of cell cycle dependent regulatory events with major emphasis on protein phosphorylation and other post- translational modifications. The ultimate goal is the identification of key enzymes and target substrates that are involved in the regulation of cell division and are highly conserved during evolution. Laboratory of Monica Riley Research in this laboratory focuses on the molecular evolution and gene expression in the bacterium Escherichia coli. In a collaborative effort, a database containing information on the intermediary metabolism and biochemical pathways of E. coli is being developed. When completed, this database is expected to contain information on each metabolic reaction, the enzyme, the reactants, products, cofactors, activators, inhibitors, kinetics, equilibrium constants, binding constants, etc. Related research is on the evolution of the K. coli DNA and organization of the genes in the chromosome. Comparative nucleotide and amino acid sequence data provide information on the evolutionary relationships of E. coli genes to homologous genes in related bacteria. Laboratory of Sensory Physiology Since 1973, the laboratory has conducted research on various aspects of vision. Current studies focus on photoreceptor cells, on their light-absorbing pigments, and on their biochemical reactions initiated by light stimulation. Microspectrophotometric and biochemical techniques are used to study the receptors of both vertebrates (amphibia, fish, and mammals) and invertebrates (horseshoe crab and squid). 44 Annual Report Staff Harosi, Ferenc, Director, Associate Scientist, MBL. and Boston University School of Medicine Szuts, Ete, Associate Scientist, MBL, and Boston University School of Medicine I 'isiting investigators Evans, Barbara I., University of Oregon, Eugene Hawryshyn, Craig W., University of Victoria, Canada Lall, Abner B., Johns Hopkins University Laboratory ofOsamu Shimomura Biochemical studies of the various types of bioluminescent systems. Preparation of the improved forms of aequorin for measuring intracellular free calcium. Staff Shimomura, Osamu, Senior Scientist, MBL, and Boston University School of Medicine Shimomura, Akemi, Research Assistant I'isiting investigator Nakamura, Hideshi, Harvard University Laboratory of Raquel Sussman We investigate the molecular mechanism of DNA damage- inducible functions in E culi. Present studies deal with novel genes that affect radiation-induced mutagenesis and analysis of RecA functions. Staff Sussman, Raquel, Associate Scientist Hemant Chikarmane, Postdoctoral Research Associate Dudley. Karen, Research Assistant National Vibrating Probe Facility We are exploring the roles of ionic currents, gradients, and waves in controlling development. We focus on controls of pattern and controls by calcium ions. Staff Jaffe, Lionel, Senior Scientist and Facility Director Kuhtreiber, Willem, Physiologist McLaughlin, Jane, Research Assistant Miller, Andrew, Research Associate Sanger, Richard, Technician Shipley, Alan, Technician I 'isiling investigators Allen, Nina, Wake Forest College Bates, William, Carleton University, Canada Buonano, Mark. Massachusetts Institute of Technology Chrystal, Jane, University of Sidney, Australia Cornwall, Carter, Boston University Danilchik. Michael, Wesleyan University Gillot, Isabelle. Station Zoologique. Villefranche-sur-mer, France Isaacs, Hugh, Brookhaven National Laboratories Kinnamon, Sue, Colorado State University Koshian, Leon, Cornell University Lucas, William, University of California, Davis McConnaughey, Ted, Marine Biological Laboratory O'Donnell, Michael, McMaster University, Canada Ripps, Harris, University of Illinois College of Medicine Rubinacci, Allessandro, University of Milan, Italy Sardet, Christian, Station Zoologique, Villefranche-sur-mer, France Saunders. Mary Jane, University of South Florida, Tampa Schiefelbein, John, University of Michigan Shapiro. James, University of Chicago Smith, Peter, Cambridge University, UK Speksnijder, Johanna, University of Utrecht. The Netherlands Other Year-Round Investigators and Staff Stephens, Raymond E., Principal Investigator Szent-Gyorgy, Gwen, Research Assistant Warren, Lisa, Research Assistant Honors Friday Evening Lectures Fellowships Dennis Powers, Stanford University. Hopkins Marine Station, 29 June "Adapting to a Changing Environment: Genetic and Physiological Mechanisms" Joan Ruderman, Harvard Medical School, 6 July "Controlling Cell Division" James Spudich, Stanford University School of Medicine, 13 July "Manifestation of a Molecular Motor: From Muscle to Amoebae" Ricardo Miledi, University of California, 19, 20 August (Forbes Lectures) "How to Study the Brain Using Frog Oocytes" James Tiedje, Michigan State University, 27 July "Destruction ofPCBs and Other Pollutants by a New Class of Anaerobic Sediment Microorganisms" Ruth Sager, Dana-Farber Cancer Institute, 3 August "Tumor Represser Genes" A. James Hudspeth, University of Texas Southwestern Medical Center, 10 August "How the Ear's Works IVork: Mechanoelectrical Transduction, Frequency Tuning, and Synoptic Transmission by Hair Cells of the Internal Ear" Phil Leder, Harvard Medical School, 17 August "Limb Deformity: A Pleiotropic Mutation Governing Embryonic Pattern Formation in the Mouse" Brian Fry, MBL Ecosystems Center, 24 August "Consumers and Carbon Isotopes: Good Chemistry in the Sea" Thoru Pederson, Worcester Foundation for Experimental Biology, 31 August "Between Nucleus and Cytoplasm: The Baroque Process of Making mRNA as Studied from the Perspective of Cell Biology" Robert Day Allen Fellowship Eugeni A. Vaisberg, Protein Research Institute, USSR Frederick B. Bang Fellowship Fund Elaine Bearer, University of California, San Francisco Jean and Ratsuma Dan Fellowship Douglas E. Koshland (financial support by Japanese Dan Fellowship), Carnegie Institution of Washington M. G. F. Fuortes Fellowship Joseph Charles Callaway, University of North Carolina, Chapel Hill Stephen W. Kuffler Fellowship Joachim W. Deitmer, University of Kaiserslauten, Zoologie, Germany Dieter Swandulla, Max-Planck-Institiit, Germany Hayden-Baille Fellowship Neena Din, University of British Columbia, Canada Anne Donaldson, MRC Laboratory of Molecular Biology, UK Faith Miller Fellowship Elaine L. Bearer, University of California, San Francisco Youngsook Lee, Harvard University 45 46 Annual Report Nikon Fellowship Timothy Mitchison. University of California, San Francisco Science Writing Fellowships Robin Henig. Freelance Bruce Jacobs. Freelance Bill Krasean, Kalamaioo Gazelle June Kinoshita, Freelance Kerning Kuo, Voice of America Beth Livermore, Freelance Michael Skoler, National Public Radio Pamela Weintraub, Omni Rick Weiss, Science News H. Burr Steinbach Memorial Fellowship Youngsook Lee, Harvard University Steps Toward Independence MBL Summer Fellowship Elaine Bearer, University of California, San Francisco Robert Paul Malchow, University of Illinois JoAnn Render, Hamilton College Katherine Swenson, Harvard Medical School Johanna E. Speksnijder, Utrecht University, The Netherlands Eugeni Vaisberg, Protein Research Institute, USSR Scholarships APA Fellow Maria A. Sosa, University of Florida John F. Hamilton, Meharry Medical College ARCS Foundation Fellow Daniel S. Kessler, Rockefeller University ASCB Fellows Abdiel J. Alvarez, University of Puerto Rico Robert L. Bacallao, University of California, Los Angeles Miles G. Cunningham. Massachusetts Institute of Technology Charlotte M. Vines, Harvard Medical School Biology Club of CUNY Elizabeth L. Winter, City College of New York Father Arsenius Boyer Scholarship Fund Haohua Qian. University of Illinois C. Lalor Burdick Scholarship Ann Marie Murphy, Johns Hopkins University Gary N. Calkins Memorial Scholarship Hanno M. Roder, Massachusetts Institute of Technology Frances S. Claff Memorial Scholarship Ka Hou Chu, Chinese University of Hong Kong, Hong Kong Edwin Grant Conklin Memorial Scholarship Sylwester Chyb, Wesleyan University Lucretia Crocker Endowment Fund Silvia E. Frenk, King's College, Cambridge. UK Bernard Davis Scholarship Diane K. Arwood, University of Southern Mississippi Joanna S. Brooke, University of Western Ontario, Canada Joseph P. Calabrese, West Virginia University Judith A. Koskella, New York University Bridget E. Laue, University of Colorado Jared R. Leadbetter, Goucher College Shi Liu, University of Oklahoma Lynn V. Mendelman, Harvard Medical School Elizabeth J. Ode, Colorado State University Mechthild Pohlschroder, University of Massachusetts, Amherst William F. and Irene Diller Scholarship Fund Sylwester Chyb, Wesleyan University Caswell Grave Scholarship Ricardo Araneda. Albert Einstein College of Medicine Sylwester Chyb, Wesleyan University Kareen M. Guida. University of Paris, France Aline D. Gross Scholarship Anne Marie Murphy. Johns Hopkins University Arthur Klorfein Fund Scholarship Dangeruta Kersulyte, Academy of Science, Lithuania, USSR Honors 47 Thierry Lepage, University of Nice, France Kareen M. Guida. University of Paris, France Edwin S. Linton Memorial Scholarship Michael C. Schmale, University of Miami S. O. Mast Founders Scholarship Peter J. Edmunds. Northeastern University Ka Hou Chu, Chinese University of Hong Kong, Hong Kong Center for Microbial Ecology, Michigan State Scholarship Olivia T. Harriott, University of Connecticut Alberto Monroy Fellow Maria G. DiBernardo, Institute di Biologia dello Sviluppo, CNS, Italy James S. Mountain Memorial Fund Scholarship Karen M. Page, Dartmouth College Eric A. Shelden, University of Massachusetts, Amherst Salme Taagepera, University of Virginia Sandra A. Brockman, Carnegie-Mellon University Tod A. Critchlow, Scripps Institute of Oceanography Planetary Biology Internship Jennifer E. Klenz, University of Saskatchewan. Canada William Townsend Porter Foundation Fellowship Abdiel J. Alvarez, University of Puerto Rico Robert L. Bacallao, University of California, Los Angeles Miles G. Cunningham, Massachusetts Institute of Technology Domingo T. Rivera, Worcester Foundation for Experimental Biology Charlotte M. Vines, Harvard Medical School Ebenezer Yamoah, University of Alberta, Canada Herbert \V. Rand Scholarship Brian J. Binder, Massachusetts Institute of Technology Sandra A. Brockman, Carnegie-Mellon University Isabelle Carre, SUNY, Stony Brook Joseph Cerro, Columbia University Ka Hou Chu, Chinese University of Hong Kong, Hong Kong Tod A. Critchlow, Scripps Institute of Oceanography Michele I. Flatters, Tufts University Holly Y. Goodson, Stanford University Jerilyn Jewett-Smith, Whitman College John R. Jordan, University of Utah David L. Keefe, Yale University Karen L. King, Florida State University Qingwen Li, University of Kansas Helen McNeill, University of Pennsylvania Christa S. Merzdorf. Harvard University Robert Mirro, University of Tennessee Qi Yang, University of Connecticut Society for General Physiologists Scholarships Robert A. Berkowitz. Washington University Daniel H. Cox, Tufts University Holly Y. Goodson, Stanford University Daniel S. Kessler, Rockefeller University Marjorie W. Stetten Fund Haohua Qian. University of Illinois Surdna Foundation Scholarship Peter J. Edmunds, Northeastern University Oivind Enger, University of Bergen, Norway Robert E. Hodson, University of Georgia James S. Maki, Harvard University Martin Polz. University of Vienna. Austria Stephen C. Tsoi, University of Hong Kong, Hong Kong William Morton Wheeler Family Founders' Scholarship Hanno M. Roder, Massachusetts Institute of Technology Awards Lewis Thomas Award Natalie Angier, The New York Times MBL Tour Guides Award for Outstanding Science Presentation to the General Public Nancy Rafferty, Northwestern University Seymour Zigman, University of Rochester School of Medicine Board of Trustees and Committees Corporation Officers and Trustees Ex officio Honorary Chairman of the Board of Trustees, Denis M. Robinson. Key Biscayne. FL Chairman of the Board of Trustees, Prosser Gifford. Washington. DC President of the Corporation and Director. Harlyn O. Halvorson. Marine Biological Laboratory. Woods Hole, MA Treasurer. Robert D. Manz. Helmer & Associates. Waltham. MA Clerk of the Corporation, Kathleen Dunlap, Tufts University School of Medicine. Boston, MA Class of 1994 Trustees Robert D. Goldman, Northwestern University, Chicago, IL Rodolfo R. Llinas. New York University Medical Center, New York. NY Thomas D. Pollard, Johns Hopkins University, Baltimore. MD Joan V. Ruderman, Harvard University School of Medicine, Boston, MA Joseph Sanger, University of Pennsylvania School of Medicine. Philadelphia, PA Ann Stuart, Marine Biological Laboratory. Woods Hole, MA Class of 1994 Trustees-at- Large Frederick Bay. Gaston Snow Beekman & Bogue, New York. NY Mary-Ellen Cunningham. Grosse Pointe Farms, MI Robert W. Pierce, Boca Grande, FL Irving Rabb, University Place at Harvard Square, Cambridge. MA Class of 1993 Garland E. Allen, Washington University, St. Louis. MO Jelle Atema, Manne Biological Laboratory, Woods Hole, MA William L. Brown, Weston, MA Alexander W. Clowes, University of Washington School of Medicine. Seattle, WA Barbara Ehrlich, University of Connecticut. Farmington, CT Edward A. Kravitz, Harvard Medical School. Boston. MA Robert E. Mainer, The Boston Company, Boston, MA Jerry M. Melillo. Marine Biological Laboratory, Woods Hole, MA Roger D. Sloboda. Dartmouth College, Hanover, NH Class of 1992 Norman Bernstein, Bernstein Management, Inc., Washington, DC Ellen R. Grass, Grass Foundation, Quincy, MA Neil Jacobs, Hale & Dorr, Boston. MA Sir Hans Kornberg, Christ's College, Cambridge, UK George Langford, University of North Carolina, Chapel Hill. NC Jack Levin, V.A. Medical Center. San Francisco, CA Evelyn Spiegel, Dartmouth College, Hanover, NH Andrew G. Szent-Gyorgyi. Brandeis University, Waltham, MA Kensal E. VanHolde, Oregon State University, Corvallis. OR Stanley W. Watson. Associates of Cape Cod. Inc.. Falmouth, MA Class of 1991 Robert B. Barlow Jr.. Syracuse University, Syracuse, NY Dieter Blennemann, Carl Zeiss, Inc., Thornwood, NY James M. Clark, Palm Beach, FL Wensley G. Haydon-Baillie, Porton, Int., London. UK Laszlo Lorand, Northwestern University, Evanston. IL Lionel I. Rebhun. University of Virginia, Charlottesville, VA Carol L. Reinisch, Tufts University School of Veterinary Medicine. Boston, MA Brian M. Salzberg, University of Pennsylvania. Philadelphia, PA Sheldon J. Segal, The Rockefeller Foundation. New York, NY Emeriti John B. Buck, NIH, Bethesda. MD Seymour S. Cohen. Woods Hole, MA Arthur L. Colwin, Key Biscayne, FL Laura Hunter Colwin, Key Biscayne. FL D. Eugene Copeland. MBL, Woods Hole, MA Sears Crowell. Indiana University, Bloomington, IN Alexander T. Daignault, Boston, MA William T. Golden, New York, NY Teru Hayashi, Woods Hole, MA Ruth Hubbard. Cambridge, MA Lewis Kleinholz, Reed College. Portland. OR Maurice E. Krahl, Tucson, AZ Charles B. Metz, Miami, FL Keith R. Porter, University of Pennsylvania, Philadelphia, PA C. Ladd Prosser, University of Illinois. Urbana. IL S. Meryl Rose. E. Falmouth, MA W. D. Russell-Hunter, Syracuse University, Syracuse, NY John Saunders, Jr., Waquoit, MA Mary Sears, Woods Hole. MA Homer P. Smith, Woods Hole, MA W. Randolph Taylor, Ann Arbor, MI D. Thomas Tngg, Wellesley, MA Walter S. Vincent, Woods Hole, MA George Wald, Cambridge, MA 48 Trustees and Committees 49 Executive Committee of the Board of Trustees Prosser Gifford*. Chairman Robert B. Barlow Jr.. 1991 Ray L. Epstein* Robert D. Goldman, 1994 Harlyn O. Halvorson* Robert E. Mainer. 1993 Robert D. Manz* Jerry M. Melillo. 1992 Irving W. Rabb, 1991 Sheldon J. Segal. 1992 D. Thomas Tngg, 1990 Trustee Committees 1990 Audit Robert Mainer. Chairman Ray L. Epstein* Robert D. Manz* Sheldon J. Segal Andrew G. Szent-Gyorgyi D. Thomas Tngg Kensal E. Van Holde Stanley W. Watson Compensation Prosser Gifford, Chairman Robert E. Mainer Robert D. Manz Irving W. Rabb D. Thomas Tngg Investment D. Thomas Trigg, Chairman William L. Brown Ray L. Epstein* William T. Golden Maunce Lazarus Werner R. Loewenstein Robert D. Manz* Irving W. Rabb W. Nicholas Thorndike Standing Committees for the Year 1990 Buildings & Grounds Institutional Animal Care and Use Craig Dorman. WHOI Ray Epstein Kenyon Tweedell. Chairman Leslie D. Garrick*. Chairman George Grice, WHOI Alfred Chaet Robert B. Bullis John W. Speer* Lawrence B. Cohen Alfred Chaet Gary Walker. WHOI Richard Cutler* Ray L. Epstein* Alan Fein Ferenc Harosi Edward Jaskun Andrew Mattox* Library Joint Users Donald B. Lehy* Thomas Meedel Philip Person Evelyn Spiegel Fellowships Thoru Pederson, Chairman Martha Constantme-Paton Ray L. Epstein* Leslie D. Garrick* Ann Giblin George M. Langford Eduardo Macagno Carol L. Reinisch J. Richard Whittaker Housing, Food Service, and Child Care Thomas Reese. Chairman Jelle Atema Andrew Bass Susan Barry Donald Chang Richard Cutler* Stephen Highstein LouAnn King* ex officio Institutional Biosafety Raquel Sussman. Chairman Paul De Weer Paul Englund Harlyn O. Halvorson* Paul Lee Donald B. Lehy* Joseph Martyna Andrew Mattox* Alfred W. Senft Instruction Roger Sloboda. Chairman Ray L. Epstein* Rachel Fink Leslie D. Garrick* Leah Haimo Ron Hoy Hans Laufer Joan Ruderman Robert Silver Ray Stephens Library Joint Management Harlyn O. Halvorson*, Chairman Garland Allen Garland Allen. Chairman Henry J. B. Dick. WHOI A. Farmanfarmaian Lionel Jafle Catherine Norton* John Teal. WHOI Geoff Thomson, WHOI Page Valentine, USGS Carole Winn*. WHOI Marine Resources Robert Goldman. Chairman Donald Abt William Cohen Richard Cutler* Toshio Narahashi George Pappas Roger Sloboda Melvin Spiegel Antoinette Steinacher John Valois* Radiation Safety Ete Z. Szutz, Chairman David W. Borst Richard L. Chappell Sherwin J. Cooperstein Louis M. Ken- Andrew Mattox* Robert Rakowski Walter Vincent 50 Annual Report Research Services Robert Palazzo Robert Silver Paul Steadier Steven Treistman Brian Noe, Chairman , van Valie|a Peter Armstrong Richard Va ,, ee Robert B. Barlow jr. Research Space Richard Cutler* Safety Barbara Ehrhch Jose P h San 8 er - Chairman Ken Forman ' 3au ' ^ e Wecr J nn Hobbie, Chairman Ehud Kaplan Ra y L - Epstein* D. Eugene Copeland Samuel S Koide Leslie D. Garnck* Richard Cutler* Aimlee Laderman John Hildebrand Edward Enos* Jack Levin David Landowne Louis Kerr Andrew Mattox* Hans Laufer Alan Kuzirian Eduardo Macagno Donald B. Lehy* Jerry M. Melillo Andrew Mattox* *c\v afficio Joan V. Ruderman Paul Steudler Laboratory Support Staff* Biological Bulletin Clapp. Pamela L., Managing Editor Puckett. Kathryn Stone, Beth Ready Computer Services Tollios, Constantine D.. Manager Schmidt. Valerie Controller's Office Speer. John W.. Controller Accounting Services Binda. Ellen F. Campbell. Ruth B. Davis. Doris C. Ghetti. Pamela M, Gilmore. Mary F. Hobbs. Roger W.. Jr. Hough, Rose A. Oliver. Elizabeth Poravas. Maria Riley, Janis E. Chein Room Chisholm. Caroline G. Miller. Lisa A. Sadowski. Edward A. Purchasing Hall. Lionel E.. Jr. Schorer. Timothy M. Copy Service Center Mountlbrd. Rebecca J.. Supervisor Jackson. Jacquelyn F. Ridley, Sherie Including persons who joined or left I he staff during 1990. Development Office Avers, Donald E.. Director Berthel, Dorothy Lessard, Kelley J. Thimas, Lisa M. Director 's Office Halvorson. Harlyn O., President and Director Burrhus, I. Elaine Catania. Didia Epstein, Ray L. Kinneally. Kathleen R. Power. Linda M. Watkins, Joan E. Gray Museum Backus, Richard H., Curator Armstrong, Ellen P. Montiero. Eva Housing King. LouAnn D.. Conference Center and Housing Manager Barnes. Susan M. Farrell, Bermee R. Johnson, Frances N. Krajewski. Viola I. Kuil, Elisabeth Mancevice, Denise M. McNamara, Noreen Sadovsky, Sebastian Telephone Office Baker, Ida M. Geggatt. Agnes L. Ridley. Alberta W. Human Resources Goux, Susan P., Manager Donovan. Murcia H. Library Norton, Catherine N., Acting Librarian Ashmore, Judith A. Costa, Marguerite E. Fessenden. Jane Fisher, Susan Goux, Randal Huguenin. Sanders Keenan, Patrick M. Mirra, Anthon> J. Mountford. Rebecca J. Nelson. Heidi Pratson, Patricia G. Showalter, Christine M. Tamm, Ingrid deVeer. Joseph M. Wnght. Rosemary MBL Associates Liaison Scanlon, Deborah Public Information Office Liles, George W., Jr., Director Anderson, Judith L. Kaye-Peterson, Amy Stone, Beth Ready Safety Services Mattox, Andrew H.. Safety Officer Apparatus Barnes. Franklin D. Haskins, William A. Martin. Lowell V. Nichols, Francis H., Jr. Shipping and Receiving Geggatt. Richard E. Illgen, Robert F. Monteiro, Dana 52 Annual Report Services, Projects, and Facilities Cutler. Richard D.. Manager Enos, Joyce B. Kurland. Charles J. Buildings and Grounds Lehy, Donald B., Superintendent Baldic. David P. Blumsack. Jeffrey J. Blunt, Hugh F. Bourgoin, Lee E. Carini, Robert J. Fish, David L., Jr. Fuglister, Charles K. Gonsalves, Walter W., Jr. Hathaway, Peter Jones, Leeland Justason. C. Scott Lochhead, William M. Lunn. Alan G. MacLeod. John B Me Adams. Herbert M., Ill Mills, Stephen A. Olive, Charles W.. Jr. Rattacasa. Frank D. Schoepf. Claude deVeer, Robert L. Weeks, Gordon W. Electron Microscopy Lab Kerr. Louis M. Housekeeping Services Jerry Phillabaum, General Supervisor Allen, Wayne D. Anderson, Lewis B. Boucher, Richard L. Collins, Paul J Conlin, Henry P. Gibbons. Roberto G. Krajewski, Chester J. Lynch, Henry L. Instrument Development I. til' Robert Knudson Machine Shop Sylvia, Frank E. Marine Resources Center Valois, John J.. Manager Enos. Edward G.. Jr.. Superintendent Fisher, Harry T.. Jr. Hanley. Janice S. Moniz, Pnscilla C. Monteiro. Dana Revellese, Christopher Sullivan, Daniel A. Tassinari, Eugene Torres, Sophie J. Photolab Colder, Linda M. Colder, Robert J. Rugh, Douglas E. Sponsored Programs Garnck, Leslie D.. Administrator Dwane. Florence Huffer. Linda Lynch, Kathleen F. Animal Care Facility Hanley. Janice S. Shephard, Jennifer Summer Support Stall' Albrecht, Helen Amon, Tyler C. Anderson, Penny Ashmore. Lynne E. Avers, Andrew D. Bolton. Hugh Burke, Sean Cadwalader. George, Jr. Campbell, Robert E. Capobianco, James A. Child, Malcolm S. Cishek. Dawn Clinard, Nathan Cullen, Timothy Cutler, Laura Demir, Oktay Dias, John. Ill Donovan, Erin Donovan, Jason P. Ford, John. Ill Grassle, J. Thomas Halpm. Michelle Hamilton, Elizabeth R. Hibhitt, Catherine Hrynyshyn, James Hullum, Rebekah Illgen, Robert C. Kaplan. Liat Kinneally, Kara J. Langton, Lori Leatherbee. Amy Marini. Michael F. Montroll, Charles Northern, Marc D. Peal, Jennifer Peal, Richard W. Philbm, Linda M. Phillips, Daniel Piazza. Lucia Remsen, Andrew S. Roderick, Paul Rosenkranz, Margalil St. Jean, Jeannette Scherer, Aimee Sheetz, Jonathan Sheffield. James Shepherd. Jennifer Shock, Duane Snyder. Matthew Snyder, Rebecca Sofferman. Rebecca Swope. John G. Tilghman. Alison Varao. John Vardac. Michael Wetzel, Ernest D. Wilkes, Jennifer Members of the Corporation' Life Members Abbott, Marie, c/o Vaughn Abbott. Flyer Rd., East Hartland, CT 06027 Beams, Harold W., Department of Biology, University of Iowa, Iowa City, IA 52242 Bernheimer, Alan \V., 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. IL 61701 Bodian, David, 4100 North Charles St., #913, Baltimore, MD 21218 Bridgman, A. Josephine, 715 Kirk Rd., Decatur, GA 30030 Buck, John B., NIH, Laboratory of Physical Biology. Room 1 12. Building 6 Bethesda, MD 20892 Burbanck, Madeline P., Box 15134, Atlanta, GA 30333 Burbanck, William D., Box 15134. Atlanta. GA 30333 Carpenter, Russell L., 60-H Lake St., Winchester, MA 01890 Chase, Aurin, Department of Biology, Guyot Hall, Princeton University, Princeton, NJ 08544 (resigned) Clark, Arnold M., 53 Wilson Rd.. Woods Hole, MA 02543 Cohen, Seymour S., 10 Carrot Hill Rd., Woods Hole, MA 02543-1206 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 27514 Crouse, Helen, Address unknown Kailla, Patricia M., 2149 Loblolly Lane, Johns Island. SC 29455 Ferguson, James K. \V.. 56 Clarkehaven St., Thornhill, Ontario L4J 2B4 Canada Fries, Erik F., 41 High Street, Woods Hole. MA 02543 (deceased) Goldman, David, 63 Loop Rd., Falmouth, MA 02540 Graham, Herbert, 36 Wilson Rd.. Woods Hole, MA 02543 Green, James W., 409 Grant Ave., Highland Park, NJ 08904 Grosch, Daniel S., 1222 Duplin Road. Raleigh, NC 27607 Hamburger, Viktor, Department of Biology, Washington LIniversity. St. Louis, MO 63 130 Hamilton, Howard L., Department of Biology, University of Virginia. 238 Gilmer Hall. Charlottesville, VA 22901 Harding, Clifford V., Jr., Wayne State University School of Medicine. Department of Ophthalmology, Detroit, MI 48201 Haschemeyer, Audrey E. V., 2 1 Glendon Road, Woods Hole, MA 02543 Hauschka, Theodore S., FD1, Box 781, Damariscotta. ME 04543 Hisaw, F. L., 5925 SW Plymouth Drive, Corvallis, OR 97330 * Including action of the 1990 Annual Meeting. Hollaender, Alexander, Council for Research Planning, 1717 Massachusetts Ave. NW. Washington, DC 20036 (deceased) 1 1 ul>l>:irn. William D., Department of Zoology. Ohio University, Athens. OH 45701 Humphreys, Susie H., Research & Development. Kraft. Inc., 801 Waukegan Rd., Glenview, IL 60025 Humphreys, Tom D., L'niversity of Hawaii. PBRC, 41 Ahui St., Honolulu, HI 96813 Hunter, Robert D., Department of Biological Sciences, Oakland University, Rochester. MI 48309-4401 Hunter, W. Bruce, Box 321. Lincoln Center, MA 01773 Hurwitz, Charles, Basic Science Research Lab, Veterans Administration Hospital, Albany, NY 12208 Hurwitz, Jerard, Sloan Kettenng Institute for Cancer Research, 1275 York Avenue. New York. NY 1 1021 Huxley, Hugh E., Department of Biology, Rosenstiel Center, Brandeis University, Waltham, MA 02154 Hynes, Thomas J., Jr., Meredith and Grew, Inc., 160 Federal Street, Boston. MA 021 10-1701 Han, Joseph, Department of Developmental Genetics and Anatomy, Case Western Reserve University School of Medicine, Cleveland, OH 44 106 Ingoglia, Nicholas, Department of Physiology, New Jersey Medical School, 100 Bergen St., Newark, NJ 07103 Inoue, Saduyki, Department of Anatomy, McGill University Cancer Centre, 3640 University St.. Montreal, PQ, H3A 2B2, Canada Inoue, Shinya, Marine Biological Laboratory, Woods Hole, MA 02543 Isselbacher, Kurt J., Massachusetts General Hospital, 32 Fruit Street, Boston, MA 02114 Issidorides, Marietta, R., Department of Psychiatry, University of Athens, Moms Petraki 8, Athens, 140 Greece Izzard, Colin S., Department of Biological Sciences, SUNY, 1400 Washington Ave., Albany, NY 12222 Jacobs, Neil, Hale & Dorr, 60 State St.. Boston. MA 02019 Jacobson, Antone G., Department of Zoology, University of Texas, Austin, TX 78712 Jaffe, Lionel, Marine Biological Laboratory, Woods Hole. MA 02543 Jannasch, Holger W., Department of Biology, Woods Hole Oceanographic Institution, Woods Hole. MA 02543 Jeffery, William R., Bodega Marine Laboratory. Box 247, Bodega Bay, CA 94923 Jones, Meredith L., Division of Worms, Museum of Natural History, Smithsonian Institution. Washington, DC 20560 Josephson, Robert K., Department of Biological Sciences, University of California, Irvine, CA 92717 Members of the Corporation 59 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 IS Kane, Robert E., PBRC. University of Hawaii. 41 Ahui St., Honolulu. HI 96813 kaneshiro, Edna S., Department of Biological Sciences. University of Cincinnati. Cincinnati. OH 45221 kao, Chien-yuan, Department of Pharmacology, Box 29, SUNY, Downstate Medical Center. 450 Clarkson Avenue. Brooklyn. NY 11203 kaplan, Ehud, Department of Biophysics. The Rockefeller University, 1230 York Ave., New York, NY 10024 karakashian, Stephen J., Apt. 16-F. 165 West 91st St., New York. NY 10024 km INI. Arthur, Department of Biochemistry and Neurology, Columbia University. 630 West 168th St.. New York, NY 10032 Katz, George M., Fundamental and Experimental Research Labs. Merck Sharp and Dohme. P. O. Box 2000. Rahway. NJ 07065 kelly, Robert E., Department of Anatomy. College of Medicine. University of Illinois. P. O. Box 6998. Chicago, IL 60680 kemp, Norman E., Department of Biology, University of Michigan, Ann Arbor, MI 48109 kendall, John P., Faneuil Hall Associates, 176 Federal Street, 2nd Floor, Boston, MA 02110 kendall, Richard E., Commissioner of Environmental Management, 100 Cambridge Street, Room 1905, Boston, MA 02202 kerr, Louis M., Marine Biological Laboratory, Woods Hole, MA 02543 keynan, Alexander, Hebrew University. Jerusalem, Israel kiehart, Daniel P., Department of Cellular and Developmental Biology, Harvard University. 16 Divinity Street, Cambridge, MA 02138 kirk, Mark D., Division of Biological Sciences. University of Missouri, Columbia, MO 652 I 1 klein, Morton, Department of Microbiology, Temple University Medical School, Philadelphia, PA 19103 klotz, Irving M., Department of Chemistry, Northwestern University, Evanston. IL 60201 knudson, Robert A., Marine Biological Laboratory. Woods Hole, MA 02543 koide, Samuel S., Population Council, The Rockefeller University, 1230 York Avenue, New York, NY 10021 kornberg. Sir Hans, The Master's Lodge, Christ's College. Cambridge CB2 3BU. England. UK kosower, Edward M., Ramat-Aviv. Tel Aviv, 69978 Israel krahl, M. E., 2783 W. Casas Circle. Tucson, AZ 85741 krane, Stephen M., Arthritis Unit, Massachusetts General Hospital, Fruit Street, Boston, MA 021 14 krauss, Robert, FASEB, 9650 Rockville Pike, Bethesda. MD 20814 kravitz, Edward A., Department of Neurobiology. Harvard Medical School, 220 Longwood Ave., Boston, MA 02 1 1 5 kriebel, Mahlon E., Department of Physiology, SUNY Health Science Center, Syracuse, NY 13210 kristan, William B., Jr., Department of Biology B-022, University of California San Diego, La Jolla, CA 92093 kropinski, Andrew M. B., Department of Microbiology/Immunology, Queen's University. Kingston. Ontario K7L 3N6, Canada kuhns, William ,1., Hospital for Sick Children, Department of Biochemistry Research, Toronto, Ontario M5G 1X8, Canada kuhtruiber, Willem M., Marine Biological Laboratory, Woods Hole. MA 02543 kusano, kiyoshi, NIH. Bldg. 36. Room 4D-20, Bethesda. MD 20892 kuzirian, Alan M., Marine Biological Laboratory. Woods Hole. MA 02543 Laderman. Aimlee, Yale University, New Haven, CT 06520 LaMarche, Paul H., Eastern Maine Medical Center. 489 State St., Bangor. ME 04401 I ainlis. Dennis M. D., Department of Developmental Genetics and Anatomy, Case Western Reserve University School of Medicine, Cleveland. OH 44106 Landowne, David, Department of Physiology. P. O. Box 016430, University of Miami School ot Medicine. Miami. FL 33101 Langford, George M., Department of Physiology. CB7545 University of North Carolina School of Medicine. Chapel Hill. NC 27599-7545 Laster, Leonard, University of Massachusetts Medical School. 55 Lake Avenue. North, Worcester. MA 01655 Laufer, Hans. Department of Biological Science, Molecular and Cell Biology, Group U-125, University of Connecticut, Storrs, CT 06268 Lazarow, Paul B., Department of Cell Biology and Anatomy, Mount Sinai Medical School. Box 1007, 5th Avenue & 100th Street, New York, NY 10021 Lazarus, Maurice, Federated Department Stores, Inc.. Sears Cresent, City Hall Plaza. Boston. MA 02108 Leadbetter, Edward R., Department of Molecular and Cell Biology, U-131, University of Connecticut, Storrs, CT 06268 Lederbcrg, Joshua, The Rockefeller LIniversity, 1230 York Ave.. New York. NY 10021 Lee, John J., Department of Biology, City College of CLINY, Convent Ave. and 138th St.. New York, NY 10031 Lehy, Donald B., Marine Biological Laboratory, Woods Hole, MA 02543 Leibovitz, Louis, 3 Kettle Hole Road, Woods Hole, MA 02543 Leighton, Joseph, 2324 Lakeshore Avenue. #2, Oakland, CA 94606 Leighton, Stephen, NIH, Bldg. 13 3WI3, Bethesda. MD 20892 Leinwand, Leslie Ann, Department of Microbiology and Immunology. Albert Einstein College of Medicine, 1 300 Morris Park Ave., Bronx, NY 10461 Lerman, Sidney, Eye Research Lab. Room 41, New York Medical College, 100 Grasslands Ave., Valhalla. NY 10595 Lerner, Aaron B., Yale University, School of Medicine, New Haven, CT06510 Lester, Henry A., 156-29 California Institute of Technology, 156-29, Pasadena, CA 91 125 Levin, Jack, Veterans Administration Medical Center, I 13 A. 4150 Clement St., San Francisco, CA 94 1 2 1 Levinthal, Cyrus, Department of Biological Sciences, Columbia University. Broadway and 1 16th Street. New York. NY 10026 (deceased) Levitan, Herbert, Department of Zoology, University of Maryland, College Park, MD 20742 Levitan, Irwin B., Department of Biochemistry, Brandeis University, Waltham, MA 02254 Linck, Richard \\ '., Department of Anatomy, Jackson Hall, University of Minnesota, 321 Church Street. S. E., Minneapolis. MN 55455 Lipicky, Raymond J., Department of Cardio-Renal/Drug Prod. Div., FDA, Rm. 16B-45, 5600 Fishers Lane, Rockville, MD 20857 Lisman, John E., Department of Biology, Brandeis LIniversity, Waltham, MA 02254 Liuzzi, Anthony, 55 Fay Rd., Box 184. Woods Hole. MA 02543 60 Annual Report Llinas. Rodolfo R., Department of Physiology and Biophysics, New York University Medical Center. 550 First Ave., New York, NY 10016 Loew, Franklin M., Tufts University School of Veterinary Medicine, 200 Westboro Rd.. N. Grafton. MA 01536 Loewenstein, Birgit R., Department of Physiology and Biophysics, R- 430. University of Miami School of Medicine, Miami, FL 33101 Loewenstein, Werner R., Department of Physiology and Biophysics, University of Miami. P. O. Box 016430. Miami, FL 33101 London, Irving M., Massachusetts Institute of Technology, E-25-551. Cambridge, MA 02 139 Longo, Frank J., Department of Anatomy, University of Iowa, Iowa City. IA 52442 Lorand, Laszlo, Department of Biochemistry and Molecular Biology, Northwestern University, 2153 Sheridan Road. Evanston, IL 60208 Luckenbill-Edds, Louise, 155 Columbia Ave., Athens, OH 45701 Luria, Salvador E., 48 Peacock Farm Rd., Lexington, MA 02173 (deceased) Macagno, Lduardo R., 1003B Fairchild, Department of Biosciences, Columbia University. New York. NY 10027 MacNichol, E. F., Jr., Department of Physiology. Boston University School of Medicine, 80 E. Concord St., Boston, MA 021 18 Maglott-Duffield, Donna R., American Type Culture Collection, 12301 Parklawn Drive, Rockville, MD 20852-1776 Maienschein, Jane Ann, Department of Philosophy. Arizona State University, Tempe, AZ 85287-2004 Mainer, Robert, The Boston Company, One Boston Place. OBP-15-D, Boston. MA 02 168 Malbon, Craig Curtis, Department of Pharmacology. Health Sciences Center, SUNY. Stony Brook, NY 1 1744-8651 Manalis, Richard S., Department of Biological Sciences, Indiana University Purdue University at Fort Wayne, 2101 Coliseum Blvd.. E. Fort Wayne. IN 46805 Mangum, Charlotte P., Department of Biology, College of William and Mary. Williamsburg, VA 23185 Manz, Robert D., Helmer and Associates, Suite 1310. 950 Winter St., Walthan, MA 02 154 Margulis, Lynn, Botany Department, University of Massachusetts. Mornll Science Center. Amherst. MA 01003 Marinucci, Andrew C, 102 Nancy Dnve, 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 Experimental, Cinvesav-ipn, 07000 Mexico. D.F. A. P.. 140740, Mexico Maser, Morton, Woods Hole Education Assoc., P. O. Box EM. Woods Hole. MA 02543 Mastroianni, Luigi, Jr., Department of Obstetrics and Gynecology, Hospital of the University of Pennsylvania, 106 Dulles, 3400 Spruce Street, Philadelphia. PA 19104-4283 Matteson. Donald R., Department of Biophysics, University of Man-land School of Medicine, 660 Redwood Street, Baltimore, MD 21201 Mautner, Henry G., Department of Biochemistry, Tufts University School of Medicine, 136 Harrison Ave., Boston, MA 021 1 1 Mauzerall. David. The Rockefeller University, 1230 York Ave., New York, NY 10021 McCann, Frances, Department of Physiology, Dartmouth Medical School, Hanover, NH 03755 McLaughlin, Jane A., Marine Biological Laboratory, Woods Hole, MA 02543 McMahon, Robert F., Department of Biology. Box 19498. University of Texas. Arlington. TX 76019 Meedel, Thomas, Marine Biological Laboratory. Woods Hole, MA 02543 Meinert/hagen, Ian A., Department of Psychology, Life Sciences Center. Dalhousie University, Halifax, Nova Scotia B3H 451. Canada Meiss, Dennis E., 462 Soland Avenue, Hayward, CA 94541 Melillo, Jerry A., Ecosystems Center, Marine Biological Laboratory, Woods Hole, MA 02543 Mellon, DeForest, Jr., Department of Biology. Gilmer Hall. University of Virginia, Charlottesville, VA 22903 Mellon, Richard P., P. O. Box 187. Laughlintown. PA 15655 Metu/.als, Janis, Department of Pathology. University of Ottawa, Ottawa, Ontario, K1H 8M5 Canada Metz, Charles B., 7220 SW 124th St.. Miami. FL 33156 Milkman, Roger, Department of Biology. University of Iowa, Iowa City, IA 52242 Mills, Robert, 10315 44th Avenue. W 12 H Street. Bradenton. FL 33507-1535 Mitchell. Ralph, DAS. Harvard University, 29 Oxford Street. Cambridge. MA 02 1 38 Miyamoto, David M., Department of Biology. Drew University, Madison, NJ 07940 Mizell, Merle, Laboratory of Tumor Cell Biology, Tulane University, New Orleans, LA 70118 Moore, John W., Department of Neurobiology, Box 3209, Duke University Medical Center, Durham, NC 27710 Moore, Lee E., Department of Physiology and Biophysics, University of Texas Medical Branch, Galveston, TX 77550 Morin, James G., Department of Biology, University of California, Los Angeles, CA 90024 Murrell, Frank, Department of Neurological Science. Rush Medical Center. 1753 W. Congress Parkway, Chicago, IL 60612 Morse, M. Patricia, Marine Science Center, Northeastern University, Nahant, MA 01 908 Morse, Robert W., Box 574. N. Falmouth. MA 02556 Morse, Stephen Scott, The Rockefeller University, 1230 York Ave., Box 2. New York, NY 10021-6399 Mote, Michael I., Department of Biology. Temple University. Philadelphia. PA 19122 Mountain, Isabel, Vinson Hall #112, 6251 Old Dominion Drive, McLean, VA 22101 Muller, Kenneth J., Department of Physiology and Biophysics. University of Miami School of Medicine. Miami. FL 33101 Murray, Sandra Ann, Department of Neurology, Anatomy and Cell Science, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261 Musacchia, Xavier J., Department of Physiology and Biophysics, University of Louisville School of Medicine, Louisville, KY 40292 Nabrit, S. M., 686 Beckwith St., SW, Atlanta. GA 30314 Nadelhoffer, knute. Marine Biological Laboratory. Woods Hole. MA 02543 Naka, Ken-ichi, 2-9-2 Tatsumi Higashi. Okazaki, Japan 444 Nakajima, Shigehiro, Department of Pharmacology and Cell Biology, University of Illinois College of Medicine at Chicago. 808 S. Wolcott Street, Chicago. IL 60612 Nakajima, Yasuko, Department of Anatomy and Cell Biology. University of Illinois College of Medicine at Chicago. M/C 512, Chicago, IL60612 Narahashi, Toshio, Department of Pharmacology, Northwestern University Medical School, 303 East Chicago Ave., Chicago. IL 60611 Members of the Corporation 61 Nasatir, Maimon, Department of Biology. University of Toledo, Toledo. OH 43606 Nelson, Leonard, Department of Physiology. CS10008, Medical College of Ohio. Toledo, OH 43699 Nelson, Margaret C., Section of Neurohiology and Behavior. Cornell University. Ithaca. NY 14850 Nicholls, John G., Biocenter. Klingelbergstrasse 70. Basel 4056. Switzerland Nickerson, Peter A., Department of Pathology, SUNY, Buffalo. NY 14214 Nicosia, Santo V., Department of Pathology. University of South Florida. College of Medicine. Box 11. 12901 North 30th St., Tampa. FL 33612 Noe, Bryan D., Department of Anatomy and Cell Biology, Emory University School of Medicine, Atlanta, GA 30322 Northcutt, R. Glenn, Department of Neuroscience, A-001. Scripps Institution of Oceanography, La Jolla, CA 92093-0201 Norton, Catherine N., Marine Biological Laboratory. Woods Hole. MA 02543 Obaid, Ana Lia, Department of Physiology and Pharmacy, University of Pennsylvania School of Medicine, B-400 Richards Bldg. Philadelphia. PA 19104-6085 Oertel, Donata, Department of Neurophysiology. University of Wisconsin. 281 Medical Science Bldg.. Madison. WI 53706 O'Herron, Jonathan, 45 Swifts Lane. Darien. CT 06820 Ohki, Shinpei, Department of Biophysical Sciences, SUNY at Buffalo, 224 Can Hall, Buffalo, NY 14214 Olins, Ada L., University of Tennessee-Oak Ridge. Graduate School of Biomedical Sciences, Biology Division ORNL, P. O. Box 2009, Oak Ridge. TN 37831-8077 Olins, Donald E., University of Tennessee-Oak Ridge, Graduate School of Biomedical Sciences. Biology Division ORNL, P. O. Box 2009, Oak Ridge, TN 37831-8077 O'Melia. Anne F., 16 Evergreen Lane, Chappaqua, New York 10514 Oschman, James L., 31 Whittier Street, Dover, NH 03820 Palazzo, Robert E., Marine Biological Laboratory. Woods Hole. MA 02543 Palmer, John D., Department of Zoology, University of Massachusetts. Amherst, MA 01002 Palti, Yoram, Rappaport Institution, Technion, POB 9697, Haifa, 31096 Israel Pant, Harish C, NINCDS/NIH, Laboratory of Neurochemistry. Bldg. 36. Room 4D-20. Bethesda. MD 20892 Pappas, George D., Department of Anatomy, College of Medicine, University of Illinois, 808 South Wolcott St.. Chicago, IL 60612 Pardee, Arthur B., Department of Pharmacology. Harvard Medical School. Boston. MA 02 1 1 5 Pardy, Roosevelt L., School of Life Sciences. University of Nebraska. Lincoln, NE 68588 Parmentier, James L., Becton Dickinson Research Center. 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 631 10 Pederson, Thoru, Worcester Foundation for Experimental Biology, Shrewsbury, MA 01 545 Perkins, C. D., 400 Hilltop Terrace, Alexandria. VA 22301 Person, Philip, Research Testing Labs. Inc., 167 E. 2nd St.. Huntington Station, NY 1 1746 Peterson, Bruce J., Ecosystems Center, Marine Biological Laboratory, Woods Hole, MA 02543 Pethig, Ronald, School of Electronic Engineering Science, University College of N. Wales. Dean St., Bangor, Gwynedd. LL57 IUT, UK Pfohl. Ronald J., Department of Zoology. Miami University, Oxford. OH 45056 Pierce, Robert \V., 4851 Shore Lane. P. O. Box 1404, Boca Grande, FL 33921 Pierce, Sidney K., Jr., Department of Zoology, University of Maryland, College Park, MD 20742 Poindexter, Jeanne S., Science Division, Long Island University. Brooklyn Campus, Brooklyn, NY 1 1201 Pollard. Harvey B., NIH, NIDDK.D. Bldg. 8, Rm. 401, Bethesda, MD 20892 Pollard, Thomas D., Department ot Cell Biology and Anatomy. Johns Hopkins University, 725 North Wolfe St., Baltimore, MD 21205 Poole, Alan F., P. O. Box 533, Woods Hole. MA 02543 Porter, Beverly H., 13617 Glenoble Drive, Rockville. MD 20853 Porter, Keith R., Department of Biology, Leidy Laboratories, Rm. 303, University of Pennsylvania. Philadelphia. PA 19104-6018 Porter, Mary E., Department of Cell Biology and Neurology, Llniversity of Minnesota, 4-147 Jackson Hall, Minneapolis, MN 55455 Potter. David, Department of Neurobiology, Harvard Medical School, Longwood Avenue, Boston, MA 021 15 Potts, William T., Department of Biology, University of Lancaster. Lancaster. England. UK Powers, Maureen K., Department of Psychology, Vanderbilt Llniversity. Nashville. TN 37240 Pratt. Melanie M., Department of Anatomy and Cell Biology, University of Miami School of Medicine (R124), P. O. Box 016960, Miami, FL 33101 Prendergast, Robert A., Wilmer Institute, Johns Hopkins Hospital, 601 N. Broadway, Baltimore, MD 21205 Presley, Phillip H., Carl Zeiss. Inc., I Zeiss Drive. Thornwood. NY 10594 Price. Carl A., Waksman Institute of Microbiology, Rutgers University, P. O. Box 759, Piscataway, NJ 08854 Prior. David J., Department of Biological Sciences. NAU Box 5640, Northern Arizona University, Flagstaff, AZ 8601 1 Prusch, Robert D., Department of Life Sciences, Gonzaga University, Spokane, WA 99258 Purves, Dale, Department of Neurobiology. Duke University Medical School, Box 3209, Durham, NC 27710 Quigley, James, Department of Pathology. SUNY Health Science Center. BHS Tower 9, Rm. 140. Stony Brook, NY 1 1794 Rabb, Irving V\ ., 1010 Memonal Drive. Cambridge, MA 02138 Rabin, Harvey, DuPont Co., CRD. Exp. Station 328/358, Wilmington. DE 19880 Rabinowitz, Michael B., Marine Biological Laboratory. Woods Hole. MA 02543 Rafferty, Nancy S., Department of Anatomy. Northwestern University Medical School, 303 E. Chicago Avenue, Chicago, IL 60611 Rakowski, Robert K., Department of Physiology and Biophysics. UHS/The Chicago Medical School, 3333 Greenbay Rd.. N. Chicago, IL 60064 Ramon, Fidel, Dept. de Fisiologia y Bionsca, Centre de Investigacion y de Estudius Avanzados del ipn, Apurtado Postal 14-740. D.F. 07000, Mexico 62 Annual Report Rabb, Irving VV., 1010 Memorial Drive. Cambridge, MA 02138 Rabin, Harvey, DuPont Co., CRD, Exp. Station 328/358, Wilmington, DE 19880 Rabinowitz, Michael B., Marine Biological Laboratory. Woods Hole. MA 02543 Rafferty, Nancy S., Department of Anatomy, Northwestern University Medical School, 303 E. Chicago Avenue. Chicago, IL 60611 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 Bionsca. Centra de Investigacion y de Estudius Avanzados del ipn. Apurtado Postal 14-740, D.F. 07000, Mexico Ranzi, Silvio, Sez Zoologia Sc Nat, Via Coloria 26, 120133. Milano. Italy Rastetter, Edward B., Ecosystems Center. Marine Biological Laboratory, Woods Hole, MA 02543 Ratner, Sarah, Department of Biochemistry, Public Health Research Institute. 455 First Ave., New York. NY 100 Id Rebhun, Lionel I., Department of Biology, Gilmer Hall. L'niversity of Virginia, Charlottesville, VA 22901 Reddan, John R., Department of Biological Sciences, Oakland University, Rochester, MI 48309-4401 Reese, Barbara F., NINCDS/NIH, Bldg. 36, Room 3B26, 9000 Rockville Pike. Bethesda, MD 20892 Reese, Thomas S., NINCDS/NIH, Bldg. 36. Room 2A27. 9000 Rockville Pike. Bethesda. MD 20892 Reiner, John M., 1 1 1 Emerson St.. Apt. 623. Denver, CO 80218 Reinisch, Carol L., Tufts University School of Veterinary Medicine, 136 Harrison Avenue, Boston, MA 021 I I Reynolds, George T., Department of Physics, Jadwin Hall, Princeton University. Princeton, NJ 08544 Rice, Robert V.. 30 Burnham Dr., Falmouth. MA 02540 Rich, Alexander, Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02 1 39 Rickles. Frederick R., Department of Medicine. Division of Hematology-Oncology, University of Connecticut Health Center, Farmmgton, CT 06032 Ripps, Harris, Department of Ophthalmology, University of Illinois College of Medicine, 1855 W. Taylor Street, Chicago, IL 6061 1 Robinson, Denis M., 200 Ocean Lane Drive #908. Key Biscayne, FL 33149 Rosenbaum, Joel L., Department of Biology, Kline Biology 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, Department of Biological Sciences, Simon Fraser University. Burnaby, BC, V5A 1S6, Canada 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, P. O. Box 285, Woods Hole. MA 02543 Rowland. Lewis P., Neurological Institute. 710 West 168th St., New York, NY 10032 Ruderman, Joan V'., Department of Anatomy and Cell Biology, Harvard University School of Medicine, Boston. MA 021 15 Rushforth, Norman B., Department of Biology, Case Western Reserve University, Cleveland, OH 44106 Russell-Hunter, \V. D., Department of Biology. Lyman Hall 012, Syracuse University, Syracuse. NY 13244 Saffo, Mary Beth, Institute of Marine Sciences. 272 Applied 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, Pittsburgh. PA 15261 Salmon, Edward D., Department of Biology. Wilson Hall, CB3280, University of North Carolina, Chapel Hill, NC 27599 Salzberg, Brian M., Department of Physiology, University of Pennsylvania. B-400 Richards Bldg.. Philadelphia. PA 19104-6085 Sanborn, Richard C., 1 1 Oak Ridge Road. Teaticket, MA 02536 Sanger, Jean M., Department of Anatomy. School of Medicine. University of Pennsylvania, 36th and Hamilton Walk, Philadelphia. PA 19174 Sanger, Joseph, Department of Anatomy, School of Medicine, University of Pennsylvania. 36th and Hamilton Walk, Philadelphia, PA 19174 Sato, Hidemi, Faculty of Social Science, Nagano University, Shiminogo, Ueda, Nagano 386- 1 2, Japan Sattelle, David B., AFRC Llnit-Department of Zoology, University of Cambridge. Downing St.. Cambridge CB2 3EJ, England, UK Saunders, John \\ ., Jr.. P. O. Box 381. Waquoit Station. Waquoit. MA 02536 Saz, Arthur K., Department of Immunology. Georgetown University Medical School, Washington. DC 20007 Schachman, Howard K., Department of Molecular Biology, University of California, Berkeley. CA 94720 Schalten, Gerald P., Integrated Microscopy Facility for Biomedical Research. University of Wisconsin. 1 1 17 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 02254 Schmeer, Arline C., Mercenene Cancer Research Institute, Hospital of Saint Raphael, New Haven. CT 0651 1 Schmidek, Henry H., Henry Ford Neurosurgical Institute. Henry Ford Hospital. Detroit. MI 48202 Schnapp, Bruce J., Department of Physiology, Boston University Medical School, 80 East Concord Street. Boston. MA 02 1 1 8 Schneider, E. Gayle, Department of Obstetrics and Gynecology. Yale University School of Medicine. 333 Cedar St.. New Haven, CT 06510 Schneiderman, Howard A., Monsanto Company. 800 North Lindbergh Blvd., D1W. St. Louis, MO 63166 (deceased) 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 Behavior. New York State Psychiatric Institute Research Annex. 722 W. 168th St., 7th Floor. New York, NY 10032 Scofield, Virginia Lee, Department of Microbiology and Immunology, UCLA School of Medicine, Los Angeles, CA 90024 Scott, Allan C., 1 Nudd St., Waterville, ME 04901 Sears, Mary, P. O. Box 152. Woods Hole, MA 02543 Segal, Sheldon J., Population Division, The Rockefeller Foundation, 1 133 Avenue of the Americas, New York. NY 10036 Selman, Kelly, Department of Anatomy. College of Medicine. University of Honda. Gainesville. FL 32601 Members of the Corporation 63 Shanklin, Douglas R., Department of Pathology, Room 584, University of Tennessee College of Medicine, 800 Madison Avenue, Memphis, TN 38163 Shapiro, Herbert, 6025 North 13th St., Philadelphia, PA 19141 Shaver, Gaius R., Ecosystems Center, Marine Biological Laboratory, Woods Hole, MA 02543 Shaver, John R., 18 Las Parras, Cayey, PR 00633 Sheetz, Michael P., Department of Cell Biology and Physiology. Washington LIniversity Medical School. 606 S. Euclid Ave., St. Louis, MO 63 110 Shepard, David C, P. O. Box 44, Woods Hole, MA 02543 Shepro, David, Department of Microvascular Research, Boston University, 5 Cummington St.. Boston. MA 02215 Sher, F. Alan, Immunology and Cell Biology Section. NIAID/NIH. Laboratory of Parasitic Disease, Building 5, Room 1 1 4, Bethesda, MD 20892 Sheridan, William F., Biology Department, LIniversity of North Dakota, Box 8238, University Station, Grand Forks, ND 58202- 8238 Sherman, I. W., Department of Biology, LIniversity of California. Riverside, CA 92521 Shimomura, Osamu, Marine Biological Laboratory, Woods Hole, MA 02543 Siegel, Irwin M., Department of Ophthalmology, New York University Medical Center, 550 First Avenue, New York. NY 10016 Siegelman, Harold W., Department of Biology. Brookhaven National Laboratory, Upton, NY 1 1973 Silver, Robert B., Department of Physiology. Cornell University, 822 Veterinary Research Tower, Ithaca, NY 14853-6401 Sjodin, Raymond A., Department of Biophysics, University of Maryland, Baltimore. MD 21201 Skinner, Dorothy M., Oak Ridge National Laboratory, P. O. Box 2009, Biology Division, Oak Ridge. TN 37831 Sloboda, Roger D., Department of Biological Sciences. Dartmouth College. Hanover, NH 03755 Sluder, Greenfield, Worcester Foundation for Experimental Biology, 222 Maple Ave.. Shrewsbury. MA 1 545 Smith, Michael A., J 1 Sinabung, Buntu #7. Semarang, Java, Indonesia Smith, Ralph I., Department of Zoology. LJniversity of California, Berkeley. CA 94720 Sogin, Mitchell, Marine Biological Laboratory. Woods Hole, MA 02543 Sorenson, Martha M., Depto de Bioquimica-RFRJ. Centro de Ciencias da Saude-I. C. B., Cidade Universitaria-Fundad, Rio de Janeiro, Brasil 21.910 Speck, William T., Department of Pediatrics. Case Western Reserve University, Cleveland, OH 44106 Spector, Abraham, College of Physicians and Surgeons, Columbia University, 630 West 168th Street, New York, NY 10032 Speer, John W., Marine Biological Laboratory, Woods Hole, MA 02543 Sperelakis, Nicholas, Department of Physiology & Biophysics, University of Cincinnati, Cincinnati, OH 45267-0576 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. 1300 Morris Park Avenue, Bronx, NY 10461 Steele, John Hyslop, Woods Hole Oceanographic Institution, Woods Hole, MA 02543 Steinacker, Antoinette, Dept. of Otolaryngology, Washington University. School of Medicine, Box 8115. 4566 Scott Avenue, St. Louis, MO 63 110 Steinberg, Malcolm. Department of Biology, Princeton University, Princeton, NJ 08540 Stephens, Grover C., Department of Ecol. and Evol. Biology, School of Biological Sciences. University of California. Irvine, CA 92717 Stephens, Raymond E., Marine Biological Laboratory. Woods Hole, MA 02543 Stetten, DeWitt, Jr., NIH, Bldg. 16, Room 1 18, Bethesda, MD 20892 (deceased) Stetten, Jane Lazarow, 4701 Willard Ave., Chevy Chase, MD 20815 Steudler, Paul A., Ecosystems Center, Marine Biological Laboratory. Woods Hole, MA 02543 Stokes, Darrell R., Department of Biology. Emory University, Atlanta, GA 30322 Stommel, Elijah W., Marine Biological Laboratory, Woods Hole, MA 02543 Stracher, Alfred, Department of Biochemistry. SUNY Health Science Center, 450 Clarkson Ave.. Brooklyn, NY 1 1203 Strehler, Bernard L., 2235 25th St., #217. San Pedro. CA 90732 Strickler, J. Rudi, Center for Great Lakes Studies. 600 East Greenfield Ave., Milwaukee, WI 53204 Strumasser, Felix, Marine Biological Laboratory, Woods Hole, MA 02543 Stuart, Ann E., Department of Physiology, Medical Sciences Research Wing 206H. University of North Carolina, Chapel Hill, NC 27599- 7545 Sugimori, Mutsuyuki, Department of Physiology and Biophysics, New York University Medical Center, 550 First Avenue, New York, NY 10016 Summers, William C., Huxley College of Environmental Studies, Western Washington University, Bellingham, WA 98225 Suprenant, Kathy A., Department of Physiology and Cell Biology, 4010 Haworth Hall. University of Kansas, Lawrence, KS 66045 Sussman, Maurice, 72 Carey Lane, Falmouth. MA 02540 Sussman, Raquel B., Marine Biological Laboratory, Woods Hole, MA 02543 Sweet, Frederick, Box 8064, Washington University School of Medicine. 499 South Euclid, St. Louis. MO 631 10 Sydlik, Mary Anne, Department of Biology, Eastern Michigan University, Ypsilanti, MI 48197 Szent-Gyorgyi, Andrew, Department of Biology. Brandeis University, Bassine 244. 415 South Street. Waltham, MA 02254 Szuts, Ete Z., Laboratory of Sensory Physiology, Marine Biological Laboratory, Woods Hole. MA 02543 Tabares, Lucia, AVDA. Department of Physiology, Sanchez, Pizjuan 4, 411009 Seville, Spain Tamm, Sidney L., Boston University Marine Program. Marine Biological Laboratory, Woods Hole. MA 02543 Tanzer, Marvin L., Department of Oral Biology, Medical School, University of Connecticut, Farmington, CT 06032 Tasaki, Ichiji, Laboratory of Neurobiology, NIMH/NIH, Bldg. 36, Rm. 2B-16. Bethesda, MD 20892 Taylor, Douglass L., Center for Fluorescence Research, Carnegie Mellon University, 440 Fifth Avenue. Pittsburgh, PA 15213 Teal, John M., Department of Biology, Woods Hole Oceanographic Institution, Woods Hole, MA 02543 Telfer, \\ illiam H., Department of Biology, University of Pennsylvania, Philadelphia. PA 19104 Telzer, Bruce, Department of Biology, Pomona College, Claremont, CA 9 1 7 1 I 64 Annual Report Thorndike, W. Nicholas, Wellington Management Company. 28 State St., Boston. MA 02 109 Trager, William, Rockefeller University, 1230 York Ave., New York, NY 10021 Travis, D. M., Veterans Administration Medical Center, 2101 Elm Street, Fargo, ND 58102 Treistman, Steven N., Worcester Foundation for Experimental Biology, 222 Maple Avenue, Shrewsbury, MA 01545 Trigg, D. Thomas, 1 25 Grove St.. Wellesley, MA 02 1 8 1 Trinkaus, J. Philip, Department of Biology. Box 6666, Yale University, New Haven, CT 065 1 1 Troll, Walter, Department of Environmental Medicine, College of Medicine. New York University. New York, NY 10016 Troxler, Robert F., Department of Biochemistry, School of Medicine, Boston University. 80 East Concord St., Boston. MA 02 1 1 8 Tucker, Edward B., Department of Natural Sciences. Baruch College, CUNY, 17 Lexington Ave., New York, NY 10010 Turner, Ruth D., Mollusk Department, Museum of Comparative Zoology. Harvard University. Cambridge. MA 02138 Tvteedell, Kenyon S., Department of Biological Sciences, University of Notre Dame, Notre Dame, IN 46656 Tytell. Michael. Department of Anatomy, Bowman Gray School of Medicine, Wake Forest University. Winston-Salem. NC 27103 Ueno, Hiroshi, Department of Biochemistry. The Rockefeller University. 1230 York Ave.. New York. NY 10021 Valiela, Ivan, Boston University Marine Program, Marine Biological Laboratory, Woods Hole, MA 02543 Vallee, Richard, Cell Biology Group. Worcester Foundation 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 97331-6503 Vincent, Walter S., 16 F. R. Lillie Road, Woods Hole, MA 02543 Vogel, Steven S., LBM, NIDDK/NIH, Bldg. 10, Rm. 9B04. Bethesda. MD 20894 Waksman, Byron, Foundation for Microbiology. 300 East 54th St.. New York, NY 10022 Wall, Betty, 9 George St.. Woods Hole. MA 02543 Wallace, Robin A., Whitney Laboratory, 9505 Ocean Shore Blvd., St. Augustine, FL 32086 Wang, Ching Chung, Department of Pharmaceutical Chemistry, University of California, San Francisco, CA 94143 Warner, Robert C., Department of Molecular Biology and Biochemistry. University of California, Irvine, CA 92717 Warren, Kenneth S., Maxwell Communications Corp., 866 Third Avenue. New York. NY 10022 Warren, Leonard, Wistar Institute, 36th and Spruce Streets, Philadelphia, PA 19104 \\aterbury, John B., Department of Biology. Woods Hole Oceanographtc Institution, Woods Hole, MA 02543 Watson, Stanley, Associates of Cape Cod, Inc., P. O. Box 224, Woods Hole, MA 02543 Associate Members Waxman, Stephen G., Department of Neurology, LCI 708. Yale School of Medicine. 333 Cedar Street. New Haven. CT 06510 Webb, H. Marguerite, Marine Biological Laboratory. Woods Hole. MA 02543 Weber, Annemarie, Department of Biochemistry and Biophysics, School of Medicine. University of Pennsylvania. Philadelphia. PA 19066 Weidner, Earl, Department of Zoology and Physiology, Louisiana State University, Baton Rouge, LA 70803 Weiss, Dieter G., Institut fur Zoologie. Technische Universitat Munchen. 8046 Garching, FRG Weiss, Leon P., Department of Animal Biology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA 19104 Weiss, Paul A., Address unknown Weissmann, Gerald, New York University Medical Center. 550 First Avenue. New York, NY 10016 Werman, Robert, Neurobiology Unit, The Hebrew University, Jerusalem, Israel Westerfield, R. Monte, The Institute of Neuroscience. LIniversity of Oregon, Eugene, OR 97403 V\ hittaker, 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. I 1077 North Torrey Pines Road, La Jolla. CA 92037 Wilson, T. Hastings. Department of Physiology, Harvard Medical School, Boston, MA 02 1 1 5 \\ilkovsky, Paul, Department of Ophthalmology, New York University Medical Center, 550 First Ave., New York, NY 10016 Wittenberg, Jonathan B., Department of Physiology and Biophysics, Albert Einstein College, 1300 Morris Park Ave.. Bronx, NY 01461 Wolfe, Ralph, Department of Microbiology, 131 Burrill Hall, University of Illinois, Urbana, IL 61801 \\olken, Jerome J.. Department of Biological Sciences. Carnegie Mellon University, 440 Fifth Ave.. Pittsburgh, PA 15213 \\orgul, Basil V., Department of Ophthalmology, Columbia University, 630 West 168th St., New York, NY 10032 W'u, Chau Hsiung, Department of Pharmacology. Northwestern LIniversity Medical School, Chicago, IL 6061 1 Wyttenbach, Charles R., Department of Physiology and Cell Biology. University of Kansas, Lawrence, KS 66045 Yeh, Jay Z., Department of Pharmacology. Northwestern University Medical School. Chicago. IL 6061 1 Zigman, Seymour, School of Medicine and Dentistry. LIniversity of Rochester. 260 Cnttenden Blvd., Rochester, NY 14620 Zigmond, Richard E., Center for Neurosciences, School of Medicine, Case Western Reserve University, Cleveland. OH 44106 Zimmerberg, Joshua J., NIH, Bldg. 12A. Room 2007, Bethesda, MD 20892 Zottoli, Steven J., Department of Biology. Williams College, Williamstown, MA 01267 Zucker, Robert S., Neurobiology Division, Department of Molecular and Cellular Biology, University of California. Berkeley, CA 94720 Zukin, Ruth Suzanne, Department of Neuroscience, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461 Ackroyd, Dr. Frederick W. Adams, Dr. Paul Adelberg, Mrs. Edward A. Ahearn. 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Mizell, Dr. and Mrs. Merle Monroy. Mrs. Alberto Montgomery, Dr. and Mrs. Charles H. Montgomery, Mrs. Raymond B. Moore, Drs. John and Betty Morgan, Miss Amy Morse, Mrs. Charles L.. Jr. Morse, Dr. M. Patricia Moul. Mrs. Edwin T. Munson, Mr. William Murchelano, Dr. Robert Murray, Mr. David M. Myles-Tochko, Drs. Christina J. and John Nace, Dr. and Mrs. Paul Nace, Mr. Paul F., Jr. Naugle, Mr. John E. Neall, Mr. William G. Nelson. Dr. and Mrs. Leonard Nelson, Dr. Pamela Newton. Mr. William E. 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 Norton. Mrs. Thomas J. O'Connell, Dr. and Mrs. Clifford Offenback, Dr. Jack A. O'Herron, Mr. and Mrs. Jonathan Olszowka, Dr. Janice S. O'Neil, Mr. and Mrs. Barry T. O'Rand, Mr. and Mrs. Michael O'Sullivan, Dr. Renee Bennett Ott, Drs. Philip and Karen Palmer, Mr. and Mrs. David Pappas, Dr. and Mrs. George D. Park, Mr. and Mrs. Malcolm S. Parmenter, Dr. Charles Parmenter, Miss Carolyn L. Pearce, Dr. John B. Pearson, Mrs. Oscar H. Peltz. Mr. and Mrs. William L. Pendergast, Mrs. Claudia Pendleton, Dr. and Mrs. Murray E. Peri, Mr. and Mrs. John B Perkins, Mr. and Mrs. Courtland D. Person, Dr. and Mrs. Philip Peters, Mr. and Mrs. Frederick S. Peterson, Mr. and Mrs. E. Gunnar Peterson, Mr. and Mrs. E. Joel Peterson, Mr. Raymond W. Petty, Mr. Richard F. Pteiffer, Mr. and Mrs. John Plough. Ms. Frances 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, Dr. Frank Proskauer, Mr. Joseph H. Proskauer, Mr. Richard Prosser, Dr. and Mrs. C. Ladd Psaledakis, Mr. Nicholas Psychoyos, Dr. Alexandre Putnam, Mr. and Mrs. Allan Ray Putnam, Mr. and Mrs. William A., Ill Rankin, Mrs. John Raphael, Ms. Ellen S. Raymond, Dr. Samuel Reese. Miss Bonnie Regis. Ms. A. Kathy Reingold, Mr. Stephen C. Reynolds, Mrs. George Reynolds, Dr. John L. Reynolds. Mr. and Mrs. Robert M. Rezmkoff, Mrs. Paul Ricca. Dr. and Mrs. Renato A. Righter, Mr. and Mrs. Harold Riina, Mr. John R. Riley. Dr. Monica Robb, Mrs. Alison A. Roberts, Miss Jean Roberts, Mr. Mervin F. Robertson, Mrs. C. W. Robinson. Dr. Denis M. Robinson, Mr. John G. Robinson, Mr. and Mrs. Marius A. Root. Mrs. Walter S. Rosenthal. Miss Hilde Members of the Corporation 67 Roslansky, Drs. John and Priscilla Ross, Dr. and Mrs. Donald Ross, Dr. Robert Ross. Dr. Virginia Roth, Dr. Stephen Rowan. Mr. Edward Rowe, Dr. Don Rowe, Mrs. William S. Rugh, Mrs. Roberts Ryder, Mr. Francis C. Sager. Dr. Ruth Sanidas. Dr. and Mrs. Dennis J. Sardinha, Mr. George J. 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 Schwamb. Mr. and Mrs. Peter Schwartz, Dr. Lawrence 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 Seder, Mr. John Segal, Dr. and Mrs. Sheldon J. Selby, Dr. Cecily Senft. Dr. and Mrs. Alfred Shanklm. Dr. D. R. Shapiro, Mr. and Mrs. Howard Shapley, Dr. Robert Sharp. Mr. and Mrs. Robert W. Shemin, Dr. and Mrs. David Shepro, Dr. and Mrs. David Sherblom. Dr. James P. Sichel, Dr. Enid Siegel, Mr. and Mrs. Alvin Simmons. Mr. Tim Simon, Mr. and Mrs. Stephen Singer, Mr. and Mrs. Daniel M. Smith, Drs. Frederick E. and Marguerite A. Smith. Mr. and Mrs. Homer P. Smith. Mr. Van Dorn C. Snyder, Mr. Robert M. Solomon, Dr. and Mrs. A. K. Sonnenblick, Mrs. Perle Specht, Mr. and Mrs. Heinz Speck, Dr. William T. Spiegel. Drs. and Mrs. Melvin and Evelyn Spotte, Mr. Stephen Steele, Mrs. John H. Steele, Dr. Robert E. Stein, Mr. Ronald Steinbach. Mrs. H. Burr Stetson. Mrs. Thomas J. Stetten, Dr. Gail Stetten. Dr. and Mrs. H. DeWitt. Jr. Stracher. Dr. Dorothy Sudduth. Dr. William Swain, Mr. Albert H. Sussman. Dr. and Mrs. Maurice Swanson. Mrs. Carl P. Swift, Mr. and Mrs. E. Kent Swope. Mrs. Gerard, Jr. Swope, Mr. and Mrs. Gerard L Szent-Gyo'rgyi, Dr. Andrew Taber, Mr. George H. Talamas-Rohana, Dr. Eduardo Taylor, Mr. James K. Taylor. Dr. and Mrs. W. Randolph Tietje, Mr. Emil D., Jr. Timmins. Mrs. William Tochko, Dr. John S. 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 Trousof. Miss Natalie Tucker, Miss Ruth Tully. Mr. and Mrs. Gordon F. Ulbnch, Mr. and Mrs. Volker Valois, Mr. and Mrs. John Vancouver Public Aquarium Van Buren, Mrs. Harold Van Holde, Mrs. Kensal E. Veeder, Mrs. Ronald A. Veeder, Ms. Susan Vincent. Mr. and Mrs. Samuel W. Vincent, Dr. Walter S. Vonderhaar, Dr. William 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. Weift'enbach, Dr. and Mrs. George Weinstein. Miss Nancy B. Weisberg, Mr. and Mrs. Alfred M. Weissmann, Dr. and Mrs. Gerald Wheeler, Dr. and Mrs. Paul S. Wheeler, Dr. William M. Whitehead. Mrs. Fred Whitney. Mr. and Mrs. Geoffrey G., Jr. Wichterman, Dr. and Mrs. Ralph Wickersham, Mr. and Mrs. A. A. Tilney Wiese, Dr. Konrad Wilber. Mrs. Clare M. Wilhelm. Dr. Hazel S. Willis. Mr. Herbert F. Wilson, Mr. and Mrs. Leslie J. Wilson, Mr. and Mrs. T. Hastings Winn. Dr. William M. Winsten, Dr. Jay A. Witting, Miss Joyce Woitkoski, Miss Nancy Wolfinsohn, Mrs. Wolfe Woodwell, Dr. and Mrs. George M. Wrigley, Mrs. Roland Yntema, Mrs. Chester L. Young, Miss Nina L. Zinn, Dr. and Mrs. Donald J. Zipf. Dr. Elizabeth Gift Shop Volunteers Marian Adelberg Michael Goldring Bertha Person Louise Atkins Rose Grant Julia Rankin Barbara Atwood Martha Griffin Virginia Reynolds Gloria Borgese Edith Grosch Lilyan Saunders Jennie Brown Jean Halvorson Elsie Scott Elisabeth Buck Helen Hodosh Deborah Senft Patricia Case Adele Hoskins Charlotte Shemin Summers Case Pauline Hyde Manlyn Shepro Shirley Chaet Sona Jones Cynthia Smith Julia Child Sally Karush Marguerite Smith Vera Clark Ruth Ann Laster Louise Specht Margaret Clowes Barbara Little Judith Stetson Villa Crowell Sarah Loessel Dorothy Stracher Elizabeth Daignault Winnie Mackey Mary Ulbrich Janet Daniels Constance Martyna Barbara van Holde Alma Ebert Florence Mixer Barbara Whitehead Margaret German Lorraine Mizell Clare Wilber Rebeckah Glazebrook Phyllis Meyers MEL Tour Guides Teni Hayashi Betsy Bang Sally Loessel Lola Robinson John Buck Isabel Mountain Donald Zinn Sears Crowell Julie Rankin Margery Zinn Certificate of Organization Articles of Amendment Bylaws of the MBL 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. Susan Mims and Charles Sedgwick Mmot being a majority of the Trustees of the Marine Biological Laboratory in compliance with the requirements of the fourth section of chapter one hundred and fifteen of the Public Statutes do hereby certify that the following is a true copy of the agreement of association to constitute said Corporation, with the names of the subscribers thereto: We. whose names are hereto subscribed, do, by this agreement, associate ourselves with the intention to constitute a Corporation according to the provisions of the one hundred and fifteenth chapter of the Public Statutes of the Commonwealth of Massachusetts, and the Acts in amendment thereof and in addition thereto. The name by which the Corporation shall be known is THE MARINE BIOLOGICAL LABORATORY The purpose for which the Corporation is constituted is to establish and maintain a laboratory or station for scientific study and investigations, and a school for instruction in biology and natural history. The place within which the Corporation is established or located is the city of Boston within said Commonwealth. The amount of its capital stock is none. In Witness Whereof, we have hereunto set our hands, this twenty seventh day of February in the year eighteen hundred and eighty-eight, Alpheus Hyatt, Samuel Mills. William T. Sedgwick, Edward G. Gardiner. Charles Sedgwick Minot. William G. Farlow, William Stanford Stevens, Anna D. Phillips, Susan Mims, B. H. Van Vleck. That the first meeting of the subscribers to said agreement was held on the thirteenth day of March in the year eighteen hundred and eighty-eight. In Witness Whereof, we have hereunto signed our names, this thirteenth day of March in the year eighteen hundred and eighty-eight. Alpheus Hyatt. President. William Stanford Stevens, Treasurer, Edward G. Gardiner. William T. Sedgwick. Susan Mims. Charles Sedgwick Minot. (Approved on March 20. 1888 as follows: / hereby a-mfr that it appears upon an examination of the within written certificate and the records of the corporation duly submitted to my inspection, that the re- quirements of sections one, two and three of chapter one hundred and fifteen, and sections eighteen, twenty and twenty-one of chapter one hundred and six, of the Public Statutes, have been complied with and 1 hereby approve said certificate this twentieth day of March A.D. eighteen hundred and eighty-eight. Charles Endicott Commissioner of Corporations) Articles of Amendment (On File in the Office of the Secretary of the Commonweallh) We, James D. Ebert. President, and David Shepro. Clerk of the Marine Biological Laboratory, located at Woods Hole. Massachusetts 02543, do hereby certify that the following amendment to the Articles of Organization of the Corporation was duly adopted at a meeting held on August 15, 1975, as adjourned to August 29. 1975, by vote of 444 members, being at least two-thirds of its members legally qualified to vote in the meeting of the corporation: Voted: That the Certificate of Organization of this corporation be and it hereby is amended by the addition of the following provisions: "No Officer, Trustee or Corporate Member of the corporation shall be personally liable for the payment or satisfaction of any obligation or liabilities incurred as a result of, or otherwise in connection with, any commitments, agree- ments, activities or affairs of the corporation. "Except as otherwise specifically provided by the Bylaws of the corporation, meetings of the Corporate Members of the corporation may be held anywhere in the United States. "The Trustees of the corporation may make, amend or repeal the Bylaws of the corporation in whole or in part, except with respect to any provisions thereof which shall by law, this Certificate or the bylaws of the corporation, require action by the Corporate Members." The foregoing amendment will become effective when these articles of amendment are filed in accordance with Chapter 180. 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 amend- ment will become effective on such later date. In Witness whereof ami Under the Penalties of Perjury, we have hereto signed our names this 2nd day of September, in the year 1975. James D. Ebert, President: David Shepro, Clerk. (Approved on October 24, 1975, as follows: I hereby approve the within articles of amendment and. the filing fee in the amount of $10 having been paid, said articles are deemed to have been filed with me this 24th day of October. 1975. Paul Guzzi Secrelan of the Commonwealth) Bylaws of the Corporation of the Marine Biological Laboratory (Revised August 17, 1990) (These Brians hem' been e.\lensirely amended by the Board ol Trustees and are \ubieel lo approval br the Corporation II these amendments are not approved by the Corporation, the existing Bylaws mil then remain in place.) 68 Bylaws of the Corporation 69 ARTICLE I THE CORPORATION A Name ami Purpose. The name of the Corporation shall be The Marine Bio- logical Laboratory The Corporation's purpose shall be to establish and maintain i laboratory or station for scientific study and investigation and a school for in- struction in biology and natural history. B Sondtscntninalion The Corporation shall not discriminate on the basis of age, religion, color, race, national or ethnic origin, sex or sexual preference in its policies on employment and administration or in its educational and other programs. ARTICLE II MEMBERSHIP I Members. The Members of the Corporation ("Members") shall consist of persons elected by the Board of Trustees (the "Board"), upon such terms and conditions and in accordance with such procedures, not inconsistent with law or these Bylaws, as may be determined by the Board- At any regular or special meeting of the Board, the Board may elect new Members. Any Member may vote at any meeting of the Members either in person or by proxy executed no more than three months prior to the date of such meeting. Except as otherwise limited therein, proxies shall entitle the persons named therein to vote at any adjournment of such meeting, but shall not he valid after final adjournment of such meeting. Proxies need not be sealed or attested and proxy purported to be executed by or on behalf of a Member entitled to vote shall be deemed valid unless challenged at or poor to its exercise. Members shall serve until their death or resignation unless earlier removed with or without cause by the affirmative vote of two-thirds ot the Trustees then in office. Any Member who has retired from his or her home institution may. on written request to the Corporation, be designated a Life Member. Life Members shall not have the right to vote and shall not be assessed for dues. 6 Meetings The annual meeting of the Members shall be held on the Friday following the second Tuesday in August of each year, at the Laboratory of the Corporation in Woods Hole. Massachusetts, at 9:30 a.m. If no annual meeting is held in accordance with the foregoing provision, a special meeting may be held in lieu thereof with the same effect as the annual meeting, and in such case all references in these Bylaws, except in this Article II. B., to the annual meeting of the Members shall be deemed to refer to such special meeting. Members shall transact business as may properly come before the meeting. Special meetings of the Members may be called by the Chairman or the Trustees, and shall be called by the Clerk, or in the case of the death, absence, incapacity or refusal by the Clerk, by any other officer, upon written application of Members representing at least ten percent of the smallest quorum of Members required for a vote upon any matter at the annual meeting of the Members, to be held at such time and place as may be designated. The Chairman of the Board shall preside at all meetings of the Corporation. C Notice of Meetings. Notice of any annual meeting or special meeting of Members, if necessary, shall be given by the Clerk by mailing notice of the time and place and purpose of such meeting at least 1 5 days before such meeting to each Member at his or her address as shown on the records of the Corporation. D ll'u/ivr ol Notice Whenever notice of a meeting is required to be given a Member under any provision of the Articles or Organization or Bylaws of the Corporation, a written waiver thereof, executed before of after the Meeting by such Member, or his or her duly authorized attorney, shall be deemed equivalent to such notice. E Adjournments Any meeting ot the Members may be adjourned to any other time and place by the vote of a majority of those Members present or represented at the meeting, whether or not such Members constitute a quorum, or by any officer entitled to preside at or to act as Clerk of such meeting, if no Member is present or represented. It shall not be necessary to notify any Members of any adjournment unless no Member is present or represented at the meeting which is adjourned, in which case, notice of the adjournment shall be given in accordance with Article II E. Any business which could have been transacted at any meeting of the Members as originally called may be transacted at an adjournment thereof. ARTICLE III ASSOCIATES OF THE CORPORATION l\\oiwies ol the Corporation. The Associates of the Marine Biological Laboratory shall be an unincorporated group of persons (including associations and corporations) interested in the Laboratory and shall be organized and operated under the general supervision and authority of the Trustees. The Associates of the Marine Biological Laboratory shall have no voting nghts. ARTICLE IV BOARD OF TRUSTEES A Pon-ers The Board of Trustees shall have the control and management of the affairs of the Corporation. The Trustees shall annually elect a Chairman ot the Board who shall serve until his or her successor is selected and qualified. They shall annually elect a President of the Corporation who shall also be the Vice Chairman of the Board and Vice Chairman of meetings of the Corporation. They shall annually elect a Treasurer. They shall elect a Clerk, who shall be a resident of Massachusetts and shall serve a term of four years. Eligibility for re-election of the Clerk shall be in accordance with the content of this Article IV as applied to Corporate or Board Trustees. They shall elect Trustees-at-Large as specified in this Article IV. They shall appoint a Director of the Laboratory for a term not to exceed five years, provided the term shall not exceed one year, if the candidate has attained the age of 65 years prior to the dale of the appointment. They may choose such other officers and agents as they shall think best. They may fix the compensation of all officers and agents of the Corporation and may remove them at any time. They may fill vacancies occurring in any of the offices. The Board shall have the power to choose an Executive Committee from their own number as provided in Article V. and to delegate to such Committee such of their own powers as they may deem expedient in addition to those powers conferred by Article V. They shall, from time to time, elect Members to the Corporation upon such terms and conditions as they shall have determined, not inconsistent with law or these Bylaws. B Composition and Election. There shall be four groups of Trustees: ( 1 1 Trustees (the "Corporate Trustees") elected by the Members according to such procedures, not inconsistent with these Bylaws, as the Trustees shall have determined. Except as provided below, such Trustees shall be divided into four classes of six, one class to be elected each year to serve for a term of four years. Such classes shall be designated by the year of expiration of their respective terms. (2) Trustees ("Trustees-at-Large"). Nominees for Trustees-at-Large shall be introduced at the annual meeting of the Corporation for subsequent election by the Board according to such procedures, not inconsistent with these Bylaws, as the Trustees shall have determined. Such Trustees-at-Large shall be divided into four classes of four Trustees, one class to be elected each year to serve for a term ol lour years. Such classes shall be designated by the year of expiration of their respective terms. It is contemplated that, unless otherwise determined by the Trustees for good reason. Trustees-at-Large. shall be individuals who have not been considered for elections as Corporate Trustees. (3) Trustees e\ offiao shall be the Chairman, the President, the Treasurer, the Clerk and the Director of the Laboratory. (4) Trustees emeriti shall include any Member who has retired from his or her home institution and has requested to serve as a Trustee emeritus provided he or she has served at least two terms as a Trustee. A Trustee ex officio is eligible to serve as a Trustee ementus provided he or she has served as Trustee e\ officio for at least eight years. Trustees ex ofticio and emeriti shall have all the nghts of the Trustees, except that Trustees emeriti shall not have the right to vote. (5) The total number of Corporate Trustees and Trustees-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 Members. (6) Newly elected Trustees shall take office at the February meeting of the Board, but may participate in discussions at intervening meetings following their election, without voting rights. (7) The Trustees and officers shall hold their respective offices until their suc- cessors are chosen and quahfied. C. Eligibility 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/she has last served as a Trustee. D Removal. Any Trustee may be removed from office at any time without cause, by vote of a majority of the Members entitled to vote in the election of Trustees; or for cause, by vote of two-thirds of the Trustees then in office. A Trustee may be removed for cause only if notice of such action shall have been given to all of the Trustees or Members entitled to vote, as the case may be. prior to the meeting at which such action is to be taken and if the Trustee to be so removed shall have been given reasonable notice and opportunity to be heard before the body proposing to remove him or her. E I 'ticuncies Any vacancy in the Board, unless and until filled by the Members at any annual or special meeting of the Members, may be filled by vote of a majority of the remaining Trustees present at a meeting of Trustees at which a quorum is present or by appointment of all of the Trustees if less than a quorum shall remain in office. F. Meetings. The annual meeting of the Trustees shall be held promptly after the annual meeting of the Members at the Laboratory in Woods Hole, Massachusetts. Special meetings of the Trustees may be called by the Chairman, the President or 70 Annual Report by any seven Trustees to be held at such time and place as may be designated. The Chairman of the Board, when present, shall preside overall meetings of the Trustees. Wntten notice shall be sent to a Trustee's usual or last known place of residence at least two weeks before the meeting. Notice of a meeting need not be given to any Trustee if a written waiver of notice executed by such Trustee before or after the meeting is hied with the records of the meeting, or if such Trustee shall attend the meeting without protesting prior thereto or at its commencement the lack of notice given to him or her. G Quorum Twenty-five Members shall constitute a quorum at any meeting. Except as otherwise required by law or these Bylaws, the affirmative vole 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 consitute action on behalf of the Members. // Transfers of Interests in Land. There shall be no transfer of title or long-term lease of real property held by the Corporation without pnor approval of not less than two-thirds of the Trustees. Such real property transactions shall be finally acted upon at a meeting of the Board only if presented and discussed at a pnor meeting of the Board. Either meeting may be a special meeting and no less than four weeks shall elapse between the two meetings. Any property acquired by the Corporation after December I, 1989 may be sold with the pnor approval of not less than two-thirds of the Trustees (other than any Trustee or Trustees with a direct or indirect financial interest in the transaction being considered for approval) who are present at a regular or special meeting of the Board at which there is a quorum. ARTICLE V COMMITTEES A Executive Committee The Executive Committee is hereby designated to consist of not more than ten Trustees, including the t'.v n/licio Trustees (Chairman of the Board. President. Treasurer, and Director of the Laboratory); and six additional Trustees, two of whom shall be elected by the Board 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 shall be a Trustee-at-Large. Beginning with the Members elected for terms ending in 1991. the Trustees will elect, to the Executive Committee. Trustees to ensure that the Committee includes four Cor- porate Trustees and two Trustees-at-Large. The Chairman of the Board shall act as Chairman of the Executive Committee and the President as Vice Chairman. The Executive Committee shall meet at such times and places and upon such notice and appoint such subcommittees as the Committee shall determine. The Executive Committee shall ha\e and may exercise all the powers of the Board dunng the mter\als between meetings of the Board except those powers specifically withheld, from time to time, by vote of the Board or by law. The Executive Committee may also appoint such committees, including persons who are not Trustees, as it may. from time to time, approve to make recommendations with respect to matters to be acted upon by the Executive Committee or the Board. The Executive Committee shall keep appropnate minutes of its meetings, which shall be reported to the Board. Any actions taken by the Executive Committee shall also he reported to the Board. 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 Members and of the Board for candidates to rill each office as the respective terms of office expire (Chairman of the Board. President. Treasurer, Clerk and Director of the Laboratory). B Board Committee-. Henerallv The Board shall have the power, by vote of a majonty of the Trustees then in office, to elect an Investment Committee, a Nom- inating Committee and any other committee and, by like vote, to delegate thereto some or all of the powers of the Board except those which by law. the Articles of Organization or these Bylaws they are prohibited from delegating. The members of any such committee shall have such tenure and duties as the Trustees shall determine. The Investment Committee, which shall oversee the management of the Corporation's endowment funds and marketable securities shall include as t'.v n/fieio members, the Chairman of the Board, the Treasurer and the Chairman of the Audit Committee, together with such Trustees as may be required for not less than two-thirds of the Investment Committee to consist of Trustees. Except as otherwise provided by these Bylaws or determined by the Trustees, any such com- mittee may make rules for the conduct of its business, but, unless otherwise provided by the Trustees or in such rules, its business shall be conducted as nearly as possible in the same manner as is provided by these Bylaws lor the Trustees ( Iciion', Without a Meeting Any action required or permitted to be taken at any meeting of the Executive Committee or any other committee elected by the Trustees may be taken without a meeting if all Members of such committees consent to the action in wnting and such wntten consents are filed with the records of meetings. Members of the Executive Committee or any other committee elected by the Trustees may also participate in any meeting by means of a telephone con- ference call, or otherwise take action in such a manner as may. from time to time, be permitted by law. ARTICLE VI OFFICERS A Enumeraiiiin The officers of the Corporation shall consist of a President, a Treasurer and a Clerk, and such other officers having the powers of President. Treasurer and Clerk as the Board may determine, and a Director of the Laboratory. The Corporation may have such other officers and assistant officers as the Board may determine, including (without limitation) a Chairman of the Board, and one or more Vice-Presidents, Assistant Treasurers or Assistant Clerks. Any two or more offices may be held by the same person. An officer need not be a Member or Trustee of the Corporation. If required by the Trustees, any officer shall give the Corporation a bond lor the faithful performance of his or her duties in such amount and with such surety or sureties as shall be satisfactory to the Trustees. B Tenure Except as otherwise provided by law. by the Articles of Organization or by these Bylaws, the President. Treasurer, and all other officers shall hold office until the first meeting of the Board following the annual meeting of Members and thereafter, until his or her successor is chosen and qualified. C. Resignation Any officer may resign by delivenng his or her wntten resignation to the Corporation at its principal office or to the President or Clerk and such resignation shall be effective upon receipt unless it is specified to be effective at some other time or upon the happening of some other event. D Removal The Board may remove any officer with or without cause by a vote of a majonty of the entire number of Trustees then in office, at a meeting of the Board called for that purpose and for which notice of the purpose thereof has been given, provided that an officer may be removed for cause only after having an opportunity to be heard by the Board at a meeting of the Board at which a quorum is personally present and voting. E lummy A vacancy in any office may be filled for the unexpired balance of the term by vote of a majonty of the Trustees present at any meeting of Trustees at which a quorum is present or by written consent of all of the Trustees if less than a quorum of Trustees shall remain in office. F. Dinrlor T he Director shall be the chief operating officer and. unless otherwise voted by the Trustees, the chief executive officer of the Corporation. The Director shall, subject to the direction of the Trustees, have general supervision of the Lab- oratory and control of the business of the Corporation. At the annual meeting, the Director shall submit a report of the operations of the Corporation for such year and a statement of its affairs, and shall, from time to time, report to the Board all matters within his or her knowledge which the interests of the Corporation may require to be brought to its notice. G. Deputy Direetor. The Deputy Director, if any. or if there shall be more than one. the Deputy Directors in the order determined by the Trustees, shall, in the absence or disability of the Director, perform the duties and exercise the powers of the Director and shall perform such other duties and shall have such other powers as the Trustees may. from time to time, prescribe. // r<0.005]. This bar graph is based on 15 no- stimulus control experiments and 30 single-arm experiments, 15 each for "nonlayer. no eggs" and "egg layer, eggs"; in the single-arm experi- ments, animals were choosing between a stimulus in one arm and no stimulus in the other. without egg cordons [X : (2) = 34. 12; P < 0.005], dem- onstrating that egg-laying animals are attractive. Because the "egg-laying animal" stimulus has two components the egg layer and its egg cordon subse- quent experiments focused on their relative contributions to pheromonal attraction. More animals were attracted to egg layers without egg cordons than had been attracted to nonlayers without egg cordons, and fewer made no choice (Fig. 3); the difference in response patterns was statistically significant [X 2 (2) = 15.38; P < 0.005], dem- onstrating that the egg layer is a source of aggregation pheromones. Identical results were obtained when a non- layer with a recently deposited egg cordon was used as the stimulus (Fig. 3), indicating that the egg cordon is also SEXUAL PHEROMONES IN APLYSIA 85 0} a co n 3 15 10 Positive No choice Negative Nonlayer Egg layer Nonlayer Egg layer No eggs No eggs Eggs Eggs Stimulus Figure 3. Egg layers are attractive to Aplysia braxiliana: a larger number of animals was attracted to an egg layer without eggs than was attracted to a nonlayer without eggs, and the patterns of responses to the two stimuli are significantly different [X 2 (2) = 15.38; P < 0.005]. Egg cordons are also attractive to A. brasiliana: a larger number of animals was attracted to a nonlayer with eggs than was attracted to a nonlayer without eggs, and the response patterns are significantly different [X : (2) = 15.38; P < 0.005]. The effects of egg layers and egg cordons are not additive at the concentrations tested: the response patterns for a nonlayer with eggs and an egg layer without eggs are identical, and do not differ significantly from the pattern obtained for an egg layer with eggs [X 2 (2) = 2.83; 0.25 < P < 0.50]. This bar graph is based on 60 single-arm experiments, 1 5 per stimulus; in each experiment, animals were choosing between a stimulus in one arm and no stimulus in the other. a source of pheromonal activity. Neither pattern differed significantly from that obtained for an egg-laying animal with an egg cordon [X 2 (2) = 2.83; 0. 10 < P < 0.25], dem- onstrating that the effects of the layer-derived and cordon- derived factors are not additive at the concentrations tested. Subsequent experiments examined whether animal- derived factors are required for the attractiveness of the egg cordon and whether the attraction is visually mediated. Two series of experiments were performed. In the first, an egg cordon without any animal served as the stimulus. The level of attraction and pattern of responses were identical to those obtained using an egg layer and its cor- don as the stimulus (Figs. 3, 4), demonstrating that egg cordons are sufficient to attract conspecifics. In the second series of experiments, a "sham" cordon (a tangled mass of silastic tubing; vol = 2 ml) served as the stimulus. The level of attraction and pattern of responses differed sig- nificantly from those observed with the egg cordon [X 2 (2) = 1 5.27; P < 0.005], but did not differ from those observed in no-stimulus control experiments [Fig. 4; X 2 (2) = 1.70; 0.25 < P < 0.50]. The differential responses to egg cordons and sham cordons suggests that the attraction is chemi- cally rather than visually mediated. The results of these two series of experiments, in conjunction with those re- ported above, demonstrate that both egg layers and egg cordons are sufficient to attract conspecifics, but that nei- ther is uniquely required. As a first step toward identifying tissue sources of the cordon-derived aggregation pheromones, acidic extracts of the atrial gland (equivalent to 50% of the material in one gland) were assayed for the ability to increase the attractiveness of a nonlaying animal when placed in the surrounding ASW. A higher level of attraction to nonlay- ing animals and lower level of no-choice responses was observed when the extract was present (Fig. 5); the dif- 0) a E c ffl 4) n 15 10 Positive No choice Negative No animal No animal No animal No eggs Sham eggs Eggs Stimulus Figure 4. Animals are not required for an egg cordon to be attractive to Aplysia brasiliana: a larger number of animals was attracted to an egg cordon without an animal than was attracted to a sham cordon without an animal, and the response pattern was significantly different [x : (2) = 15.27; P < 0.005]. The sham cordon was a tangled mass of silastic tubing, 2 ml in volume. The attractiveness of an egg cordon is not visually mediated: the pattern of responses to a sham cordon without an animal did not differ significantly from the no-stimulus control (no animals, no eggs) (X-(2) = 1.70; 0.25 < P < 0.50]. (Note: for the no- stimulus control, movement to the left was defined as a positive response and movement to the right a negative response.) This bar graph is based on 15 no-stimulus control experiments and 30 single-arm experiments, 1 5 each for "no animal, sham eggs" and "no animal, eggs"; in the single- arm experiments, animals were choosing between a stimulus in one arm and no stimulus in the other. 86 S. D. PAINTER ET AL CO 'c a E z 15 10 Positive No choice Negative Nonlayer Nonlayer Nonlayer No eggs AQE Eggs Stimulus Figure 5. Secretory products of the Aply.iia californica atrial gland (or A. brasiliana AG-LE) may contribute to the attractiveness of an egg cordon. The number of animals attracted to a nonlayer was increased when an extract of the A. californica atrial gland (AGE) was placed in the surrounding ASW. The pattern of responses differed significantly from that observed for a nonlaying animal without eggs [X 2 (2) = 10.58: F < 0.01], but did not differ significantly from that for a nonlaying animal with eggs [ V(2) = 0.44; 0.90 < P < 0.95]. This bar graph is based on 45 single-arm experiments. 1 5 per stimulus: in each experiment, an- imals were choosing between a stimulus in one arm and no stimulus in the other. Terence in response patterns was significant [X 2 (2) = 10.58; P < 0.0 1 ]. Interestingly, the level of attraction and pattern of responses did not differ significantly from those ob- tained when a recently deposited conspecific egg cordon was placed in the same location [Fig. 5; X 2 (2) = 0.44; 0.75 < P < 0.90], suggesting that products of the A. californica atrial gland (or A. brasiliana AG-LE) might significantly contribute to the attractiveness of an egg cordon. Induction of mating activity Animals. A pool of 205 sexually mature Aplysia brasil- iana individuals was used in these studies. Small plastic fish tags ( 1 1 mm in diameter; Howitt Plastics, Molalla, Oregon) were sutured to the caudal region of the right parapodium so that individuals could be identified. Four criteria were used to select animals for each experiment: ( 1 ) the animal must not have laid eggs during the preced- ing 24 h; (2) the animal must not have participated in a behavioral experiment during the preceding 24 h; (3) the animal must not have been tested with the stimulus; and (4) all of the animals in an experiment must have been housed in the same aquarium. Once selected, the animals were randomly assigned to treatments. All experiments were begun between 9 and 10 am, because there is evi- dence of a circadian rhythm in Aplysia mating behavior (A.J'asciata. Susswein et al.. 1983, 1984). Relative contributions of the egg layer and egg cordon: experimental protocol and statistical analyses. Eight an- imals were used in each experiment (Fig. 6). One animal was injected with 0. 1 ml of atrial gland extract and placed in a 4-1 plastic beaker containing 3 1 of aerated non-con- ditioned ASW; this treatment induced egg laying, usually within 30 min, and the egg layer conditioned the ASW in the beaker. A second animal was handled and placed in an identical beaker; this treatment did not induce egg laying, but the nonlayer conditioned the ASW. When the injected animal finished laying eggs (70.3 4.1 min after injection; mean S.E.M.), it was removed, rinsed in fresh non-conditioned ASW and transferred to a third beaker; the handled animal was treated in the same way and transferred to a fourth beaker. Nontreated animals were then distributed among the four beakers so that each con- tained two animals. The resulting experimental conditions are: ( 1 ) two nonlayers in animal-conditioned ASW with an egg cordon; (2) two nonlayers in animal-conditioned ASW without an egg cordon; (3) one egg layer and one nonlayer in non-conditioned ASW without an egg cordon; and (4) two nonlayers in non-conditioned ASW without an egg cordon. The reproductive activity of each individual was as- sessed at 10-min intervals for 270 min. Three categories of mating activity were recognized (male, female, and si- multaneous hermaphrodite); egg-laying activity was also recorded, but courtship was not. For calculation purposes, animals that did not mate or lay eggs during the obser- vation period were assigned a 280-min latency for that activity. Although this approach underestimates the dif- ference in mean latency to mating between strongly pos- itive and control conditions, the effect is relatively small because at least 85% of the animals mated in every ex- perimental condition tested. Statistical significance was assessed by a one-way analysis of variance, followed by Duncan's multiple range test for pairwise comparisons. When time courses of mating activity were compared, statistical significance was assessed by X 2 analysis of in- dividual time points. Egg cordon volume was measured at the end of each experiment by ASW displacement in a graduated cylinder and averaged 1.7 ml (1.7 0.2 ml; mean S.E.M.). Twenty experiments were performed in this series. Results. Animal-conditioning the ASW with a nonlay- ing animal resulted in an increase in the percentage of SEXUAL PHEROMONES IN APLYSIA 87 Handle animal; inject atrial gland extract I Conditions ASW; lays eggs Transfer egg layer to Conditioned ASW Non-conditioned ASW with eggs without eggs I I Add 2 nonlayers Add 1 nonlayer Handle animal; no injection i Conditions ASW; does not lay eggs Transfer nonlayer to Conditioned ASW Non-conditioned ASW without eggs without eggs I Add 2 nonlayers Add 1 nonlayer 2 nonlayers in Egg layer, nonlayer in conditioned ASW non-conditioned ASW with eggs without eggs 2 nonlayers in conditioned ASW without eggs 2 nonlayers in non-conditioned ASW without eggs Figure 6. Flow diagram of the protocol used in the first series of mating experiments. One animal was injected with an extract of the atrial gland to induce egg laying and placed in a beaker containing aerated non-conditioned ASW; a second animal was handled and placed in a second beaker containing aerated non- conditioned ASW. When egg deposition was complete, the egg layer was transferred to a third beaker and the handled nonlayer to a fourth beaker. Additional nontreated animals were then distributed among the four beakers so that each contained a pair of Aplysia: the resulting experimental conditions are indicated at the bottom of the diagram. Mating and egg-laying behaviors were scored at 10-min intervals for 270 min: animals failing to exhibit a behavior during the observation period were assigned a 280-min latency for calculations. animals mating at early time periods relative to non-con- ditioned ASW controls (Fig. 7A); the difference was sta- tistically significant at three time periods 40, 50, and 60 min [X'( 1 ) > 3.84 for each; P < 0.05]. The mean latency to mating was also reduced (Fig. 7B), but did not differ significantly from that of the non-conditioned ASW con- trols (P = 0.29; one-way analysis of variance). It is im- portant to note that these effects, although small, were consistently observed when the ASW was animal-condi- tioned. Comparable results were obtained in an indepen- dent series of experiments performed in our laboratory (A. R. Gustavson, unpubl. data). The studies used a dif- ferent pool of A. brasiliana and animal-conditioned the ASW for 60 rather than 70 min, but produced quantita- tively similar responses. A higher percentage of animals mated at early time periods relative to the non-conditioned ASW controls [X 2 (l) > 3.84 at 30, 40, 70, 80. 90, 100, and 1 10 min; P < 0.05 for each]; the mean latency to mating was reduced, but the change was not statistically significant (P = 0.28; one-way analysis of variance). The consistency of these two sets of results suggests that ani- mal-derived factors induce mating, but that their activity or concentration is relatively low under the conditions tested. The idea that animal-derived factors induce mating in Aplysia is consistent with a recent report in the literature that the amount of time that A. fasciata spend mating is a function of the number of animals available as copu- latory partners (Ziv el al, 1989) and thus, presumably, a function of the concentration of animal-derived factors in the ASW. Similar, but quantitatively larger, effects were observed when the ASW was animal-conditioned by an egg layer and contained an egg cordon (Fig. 7 A. B). The percentage of animals mating at early time periods was increased relative to non-conditioned ASW controls, and the difference was statistically significant for every observation period from 10 through 1 10 min [X 2 (l) > 3.84 for each; P < 0.05]. The mean latency to mating was significantly reduced (P = 0.002; one-way analysis of variance). Assuming that animal-con- ditioning the ASW with an egg layer is comparable to ani- mal-conditioning with a nonlayer (see below), these results demonstrate that cordon-derived factors induce mating, and suggest that the effects of the animal-derived and cordon- derived factors may be additive. S. D. PAINTER ET AL E c a o 01 5 Q. 100 - 80 40 20 Nonlayer, No eggs Conditioned Nonlayer, No eggs Non-conditioned 40 80 120 160 200 240 280 Time (mini 150 _ 125 1 CD 100 75 50 25 Nonlayer Nonlayer Nonlayer No eggs No eggs Eggs Non-cond. Conditioned Conditioned Stimulus Figure 7. Both animal-derived and cordon-derived factors induce mating activity in Aplysia brasiliana. (A) The percentage of animals mating at early time periods was increased by animal-conditioning the ASW with a nonlaying animal; the difference was significant at 40, 50, and 60 mm [X 2 ( 1 ) > 3.84; P < 0.05]. The percentage was further increased by animal-conditioning the ASW with an egg-laying animal and leaving the egg cordon in the ASW; the percentages were significantly higher than those obtained in non-conditioned ASW without an egg cordon at every observation period from 10 through 1 10 min (X'U) > 3.84 for each; P < 0.05]. The experimental protocol is shown in Figure 6 (n = 20 experiments). Because Aplysia tend to mate in bouts lasting approximately 60 min and the bouts are often separated by periods during which no mating occurs (Leonard and Lukowiak, 1987), it is not possible to read a mean latency to mating directly from this graph. (B) The latency to mating (mean S.E.M.) was reduced by animal-conditioning the ASW with a nonlaying animal, but the difference was not statistically significant (P = 0.29; one-way analysis of variance). The latency was further reduced in animal-conditioned ASW with an egg cordon, and differed significantly from that obtained for non-conditioned ASW without an egg cordon (P < 0.002; one-way analysis of variance). Animals did not distinguish between recent egg layers and nonlayers in non-conditioned ASW without an egg cordon. There were no significant differences in the time courses of mating activity (Fig. 8A), in the latencies to mating (Fig. 8B), or in the sexual role first assumed by the animals (Table I). These results suggest that there is not a prolonged change in the motivational state of the egg layer (i.e.. an increase in receptivity to courtship) that persists in the absence of an egg cordon. More importantly, they suggest that the egg cordon, rather than the egg layer, may be primarily responsible for the relatively short la- tencies to mating observed when animals are actively lay- ing eggs. The experiments did not address the question of whether specific layer-derived or animal-derived factors are required for the induction of mating by egg cordons, however, and this issue was examined in the next series of experiments. Induction of mating by egg cordons in non-conditioned ASW ': experimental protocol and statistical analyses. Five animals, selected as described above, were used in each experiment (Fig. 9). One was injected with atrial gland extract and placed in a beaker to lay eggs. When deposition was complete, the egg cordon was removed, quickly rinsed, and transferred to a second beaker containing non- conditioned ASW; a pair of animals was then placed in this beaker and another pair placed in a third beaker that contained only non-conditioned ASW. The resulting ex- perimental conditions are: (1) two nonlayers in non-con- ditioned ASW with an egg cordon; and (2) two nonlayers in non-conditioned ASW without an egg cordon. Repro- ductive behavior was assessed for each animal at 10-min intervals for 270 min and analyzed as in the preceding experiments. Egg volume was measured after each ex- periment and averaged 2.0 ml (2.0 0.3 ml; mean S.E.M. ). Fifteen experiments were performed. Results. Placing a recently deposited egg cordon in the non-conditioned ASW surrounding two nonlaying ani- mals significantly increased the percentage of animals mating in 9 of the first 14 observation periods [Fig. 10A; X : ( 1 ) > 3.84 at 20-40 min, 70 min, and 100-140 min: P < 0.05 for each], and significantly reduced their mean latency to mating relative to the control group (Fig. 10B; P < 0.01; one-way analysis of variance). These results demonstrate that cordon-derived factors alone are suffi- cient to induce mating. Induction of egg-laying activity Egg deposition was also monitored in the experiments described above. Neither animal-derived nor cordon-de- rived factors significantly affected the percentage of ani- mals laying eggs (Table II), and the low percentages made calculations of mean latency to deposition meaningless. Because Aplysia brasiliana lays eggs more frequently when SEXUAL PHEROMONES IN APLYSIA 89 100 Egg layer, No eggs Non-conditioned Nonlayer, No eggs Non-conditioned 40 80 120 160 200 240 280 Time (min) 150 125 c E o> 100 75 50 25 B Nonlayer No eggs Non-cond. Egg layer No eggs Non-cond. Stimulus Figure 8. Aplysia brasiliana does not distinguish between recent egg layers and nonlayers in non-conditioned ASW without an egg cordon. (A) Recent egg layers and nonlayers mated at the same frequency in non-conditioned ASW without an egg cordon. The experimental protocol is shown in Figure 6 (n = 20 experiments). (B) There is no difference in mean latency to mating between recent egg layers and nonlayers in non- conditioned ASW without an egg cordon. caged alone rather than in pairs (Blankenship et a!., 1983), the experiments were repeated with one animal in each beaker rather than two. Single-animal experiments: protocol. Four test animals, selected as described in the mating studies, were used in each experiment. One was placed in each of four beakers and egg-laying activity assessed at 10-min intervals for 270 min. The ASW in each beaker contained a different combination of animal-derived and cordon-derived fac- tors: ( 1 ) non-conditioned ASW without an egg cordon (negative control); (2) non-conditioned ASW with an egg cordon (cordon-derived factors only); (3) animal-condi- tioned ASW without an egg cordon (animal-derived fac- tors only); and (4) animal-conditioned ASW with an egg cordon (both animal-derived and cordon-derived factors). The conditions in each beaker were established as de- scribed in the section on mating (see Figs. 6 and 9). The volumes of the stimulus egg cordons were measured at the end of every experiment and averaged 3.2 ml (3.2 0.6 ml; mean S.E.M.). In five experiments, all animals that were not induced to lay eggs were injected with atrial gland extract to verify that they were physiologically com- petent to do so; all laid egg cordons in response to the injection, demonstrating that the experimental conditions were not interfering with the activity. Results. Neither animal-derived nor cordon-derived factors had a significant effect on egg deposition (Ta- ble II). Discussion Pheromonal attraction These studies have shown that Aplysia brasiliana is not attracted to nonlaying animals in the absence of an egg cordon. The results contrast with those of Lederhendler and colleagues (1977), which showed that Aplysia dac- tylomela is attracted to nonlaying conspecifics and that the magnitude of the attraction increases as the number of stimulus animals increases. We do not know whether the difference reflects species differences or whether it re- sults from differences in experimental design (e.g., from differences in concentration produced by using 5-min rather than 60-min conditioning periods), and we have not examined the possibility that a group of nonlaying A. brasiliana would be attractive. We have, however, tested A. californica in T-maze experiments and have found that A. californica, like A. brasiliana, is not attracted to non- laying conspecifics under these conditions (S. D. Painter, unpubl. data). These results are consistent with earlier studies by Audesirk (1977), which showed that A. cali- fornica is not attracted to nonlaying animals in Y-maze experiments. Although there is electrophysiological evi- dence that A. californica detects the odors of nonlaying conspecifics (Audesirk and Audesirk, 1977; Chase, 1979), there is no evidence to date that the electrophysiological response is to species-specific odors (Chase, 1979) and no behavioral evidence that the odors are attractive. Aplysia brasiliana is attracted to egg-laying animals with egg cordons. The ability to distinguish egg-laying animals with egg cordons from nonlaying animals without egg cordons was previously described in burrowing studies in this species (Aspey and Blankenship, 1976). In those studies, when a nonlaying animal was introduced into an aquarium containing a burrowed conspecific, the intro- duced animal burrowed; when an egg-laying animal was 90 S. D. PAINTER ET AL Table I The effects of recent egg deposition on sexual role in Aplysia brasiliana Conditions t & of animals first mating as % Not mating Animal Female Male Hermaphrodite Conditioned ASW. egg cordon present" Non-conditioned Egg layer 90 10 ASW, no egg cordon* Non-conditioned Egg layer 45 30 1 5 10 ASW, no egg cordon* Nonlayer 40 40 10 10 " From Painter et al ( 1989). Some egg layers were actively laying eggs when the second animal was introduced into the chamber. The ASW was animal-conditioned by the egg layer, n = 20. * In each case, the experimental animal was introduced into a new chamber at the beginning of the observation period and the ASW was not animal-conditioned. Egg layers were animals that had just completed egg deposition; nonlayers were those that had not laid within the preceding 24 h. The activity pattern of an egg layer in non-conditioned ASW without an egg cordon differed significantly from that of an egg layer in animal- conditioned ASW with an egg cordon [X 2 (3) = 16.67; P < 0.005], but did not differ from that of a nonlayer in non-conditioned ASW without an egg cordon (X : = 1.11; 0.75 < P < 0.90). n = 20 for each. introduced, however, the burrowed animal emerged to mate with it. The distinction is also evident in mating experiments ( Painter t'//., 1989), which showed that egg- laying animals have significantly shorter latencies to mat- ing than do sexually mature but nonlaying animals. Other species of Aplysia also make the distinction. Copulatory Handle animal; inject atrial gland extract Conditions ASW; lays eggs Conditioned ASW with egg layer Transfer eggs to Non-conditioned ASW without eggs Non-conditioned ASW without eggs Add 2 nonlayers Add 2 nonlayers 2 nonlayers in non-conditioned ASW with eggs 2 nonlayers in non-conditioned ASW without eggs Figure 9. Flow diagram of the protocol followed in the second series of mating experiments. One animal was injected with an extract of the atnal gland to induce egg laying and placed in a beaker containing aerated non-conditioned ASW. When egg deposition was complete, the egg cordon was removed from the container, quickly rinsed, and transferred to a second beaker containing non-conditioned ASW. Two nontreated animals were then added to the beaker containing the egg cordon, and two others added to a third beaker containing only non-conditioned ASW. The resulting experimental conditions are indicated at the bottom of the diagram. Reproductive behaviors were scored at 10-min intervals for 270 min. SEXUAL PHEROMONES IN APLYSIA 91 100 * 80 OB c 60 ra CL 40 20 Nonlayer, Eggs Non-conditioned Nonlayer, No eggs Non- conditioned 40 80 120 160 200 240 280 Time (min) 200 150 100 50 Nonlayer No eggs Non-cond. Nonlayer Eggs Non-cond. Stimulus Figure 10. High concentrations of animal-denved factors are not required for the induction of mating activity by recently deposited egg cordons. (A) A higher percentage of nonlaying .l/Vrau brasiliana mated at early time periods when an egg cordon was placed in the surrounding non-conditioned ASW; the increase was statistically significant at 20, 30,40. 70, and 100-140 min [X 2 (l)> 3.84 in each case; P < 0.05]. The experimental protocol is shown in Figure 9 (n = 15 experiments). (B) Placing an egg cordon in the non-conditioned ASW surrounding two nonlaying animals significantly reduced their latency to mating relative to nonlaying animals without an egg cordon (P < 0.0 1 ; one-way analysis of variance). The experimental protocol is shown in Figure 9 (n = 15 expenmentsl. chains of A. californica, for example, are both more stable and more attractive when the first animal in the chain is laying eggs (Audesirk, 1977). The present studies have shown that both egg-layer- derived and cordon-derived factors contribute to the at- tractiveness of an egg-laying animal. Although there are two apparent sources of this pheromonal activity, it re- mains to be demonstrated whether the two sets of factors differ biochemically. There is extensive and prolonged contact between the egg layer and its cordon, both during and following oviposition, which would facilitate a transfer of activity between the two. The attractiveness of a nonlaying animal was increased by placing a recently deposited egg cordon in the same arm of the T-maze, providing the basis for a simple bioas- say system in which to identify potential tissue sources of the "cordon-derived" pheromonal activity. Extracts of the A. californica atrial gland were assayed in this system and also increased the attractiveness of a nonlaying animal, suggesting that secretory products of the A. californica atrial gland (or A. brasiliana AG-LE) may contribute to the cordon-derived activity. The extracts each contained 50% of the material in a single atrial gland and were as attractive as A. brasiliana egg cordons, suggesting that the A. brasiliana and A. californica aggregation pheromones may be chemically similar or identical. This issue is cur- rently being examined by HPLC analyses of egg cordon eluates, and by compositional and microsequence analyses of active fractions. It is worth noting that extracts of the A. californica atrial gland also induce copulatory activity in A. brasiliana (Painter et ai. 1989), and that field studies suggest that Aplysia aggregation pheromones may not be entirely species-specific (A. vaccaria, for example, has been observed in association with A. californica aggregations: Kupfermann and Carew, 1974). Mating activity The experiments showed that both animal-derived and cordon-derived factors reduce the latency to mating in Table II Neither animals nor egg cordon', stimulated egg-laying activity in Aplysia brasiliana % Animals laying eggs Conditions 2 Animals in chamber 1 Animal in chamber Non-conditioned ASW No egg cordon 0" 0" Egg cordon 6.7 6.7" Animal-conditioned ASW No egg cordon 7.5' O d Egg cordon 12.5' I0 d " n = 1 5; 30 possible egg-laying episodes. Not significantly different [X-(D = 2.14:0.10 < /><0.25]. * n = 15; 15 possible egg-laying episodes. Not significantly different [X 2 (D = 1.07:0.25 < P < 0.50]. ' n = 20; 40 possible egg-laying episodes. Not significantly different [X 2 (l) = 1.44:0.10 - Enzyme-Treated No Addition ' / i I 6 12 18 24 48 Duration Larval Exposure (hr) Figure 4. Inaetivation of the cell wall-associated inducer by treatment with digestive enzymes from abalone. Equivalent amounts of inducer were incubated in parallel ( I h, 28C) with or without exposure to en- zymes. Samples then were washed by ultrafiltration. Assays with the chromogenic substrate for sulfatase. p-nitrophenylsulfate. confirmed that all of the enzyme was removed from the paniculate samples by this washing procedure. The washed samples then were assayed in quadru- plicate for remaining morphogenic activity (in parallel with assays with no addition). After 24 h of exposure of the larvae to each of the three assay conditions, fresh samples of the inductive crustose coralline red alga Hydmlithon boergesenii (CCA) were added to duplicate samples of each assay type (open symbols), to assess the remaining responsiveness of the larvae. pet. Patella vulgata (Table II). A significant reduction in the dose-dependent activity of the cell wall-associated morphogen resulted from this enzyme treatment. When Table II Effect of sulfatase purified from Patella vulgata on cell wall-associated morphogen Inducer (equivalents)' Untreated Enzyme-treated Metamorphosis- 0+0 1 70 10 6 100 1 6 20 1 1 70 10 1 The decalcified morphogen was incubated with or without enzyme for 1 h at 28C. and subsequently washed free of enzyme, as described in Materials and Methods. One equivalent corresponds to 0.25 ml of the original decalcified morphogen. Portions of the two samples then were assayed, either singly or in pre-mixed combination, in the relative amounts shown. 2 Assays were conducted with 5 larvae/trial, and scored for metamor- phosis after 24 h: results = mean S.D.; n = 2 trials for each condition. 10 D. E. MORSE AND A. N. C. MORSE 100 ,- _ 80 - None Trps. Papn |3-Gal. c-Gal 0-Glu Hyal Bactri Haiiotis Patella Wu JOOu 200u 200u Sulfatase Figure 5. Effects of purified enzymes used as probes for structural determinants of the cell wall-associated inducer of larval metamorphosis. Trps. = trypsin; Papn. = papain; /J-Gal. = /i-galactosidase; -Gal. = - galactosidase; fi-Glu. = /j-glucuronidase; Hyal. = hyaluromdase; sulfatases purified from the bacterium Acrohuctcr acrnt;cne.t, the limpet Piilclln vitlgiild. and the abalone Haliolis craclierndii. were used in the amounts (enzyme units) indicated. Assays, after treatment and washing of the paniculate samples, were performed in duplicate; all other details as in Figure 4 and Materials and Methods. equivalent amounts of the enzyme-treated and the un- treated morphogen were mixed and assayed together, no reduction in the activity of the untreated morphogen was observed, thus confirming the conclusion that the mol- luscan enzymes inactivated the inducer, and not the larvae. Larvae exposed to molluscan digestive enzyme- treated inducer, and either subsequently (Fig. 4) or si- multaneously (Table II) exposed to untreated inducer, re- sponded normally and completed metamorphosis. The digestive enzyme preparation from Haiiotis that inactivated the coral morphogen (Fig. 4) is a relatively crude mixture containing high quantities of a sulfatase (Spaulding and Morse, 1991) and lower quantities of /3- glucuronidase and several other enzymes. When purified molluscan sulfatase and /i-glucuronidase were tested sep- arately, the sulfatase was a potent inactivator of the cell wall-associated morphogen, whereas jtf-glucuronidase had little if any significant activity (Fig. 5). The data show that sulfatases purified from Haliolis, Patella, and from a bac- terium all inactivate the morphogen, and that the effect of the Haiiotis sulfatase is concentration-dependent. (The slight inactivation caused by treatment with the /3-gluc- uronidase preparation is likely to reflect the activity of the small amount of sulfatase known to still contaminate this preparation.) In similar tests, five other purified enzymes were used to probe for essential features of the morphogen structure. Of these, only hyaluronidase, which cleaves sulfated poly- saccharide chains, reduced the activity of the insoluble, cell wall-associated morphogen (Fig. 5). The proteolytic enzymes trypsin and papain, and the exosaccharidases a- galactosidase and 0-galactosidase, were completely with- out effect. These results, and those presented above. strongly suggest that a sulfated polysaccharide is an es- sential component of the morphogen recognized by the Agaricia humilis larvae. Enzymes release and degrade soluble morphogen Four purified endopolysaccharidases, including agarase, endo-/i-galactosidase, lysozyme, and hyaluronidase, re- leased a soluble morphogen from the insoluble cell wall preparation (Fig. 6). The subsequent time-dependent de- cline in soluble morphogen activity seen with prolonged exposure to agarase suggests that this enzyme may con- tinue to attack the solubilized inducer; this suggestion is confirmed by experiments presented below. None of the other enzymes tested in the experiment shown in Figure 5 released any detectable soluble activity. The enzyme-solubilized morphogen was a potent in- ducer, causing the Agaricia humilis larvae to quickly at- tach either to the sides or bottoms of the clean polystyrene assay beakers, and to undergo rapid and normal meta- morphosis and post-metamorphic growth (cf. Fig. 10). These results, and those with the alkali-solubilized ma- terial discussed below, prove that the larvae do not require any specific tactile stimulus from the morphogen, but re- spond solely to its chemical structure. Experiments in which aliquots of the cell wall-associated morphogen were incubated in the presence and absence of purified enzymes, inside dialysis tubing, confirmed that agarase and other endosaccharidases continue to attack and subsequently degrade the soluble morphogenic mol- ecules they first release (as suggested by the data in Fig. 100 - Digestion (hr) Figure 6. Solubilization of morphogen by treatment with purified endopolysaccharidases. Agarase (squares): endo-/3-galactosidase (filled circles); hyaluronidase (upward triangles); lysozyme (downward triangles); no enzyme controls (open circles). Other procedures similar to experiment shown in Figure 5. Details are in Materials and Methods. CHARACTERIZATION OF CORAL MORPHOGEN 111 100 - _ 80 - + Agarase tn O 9- o E 3 QJ 60 - 0246 + Agarase, 1 sample) No Enzyme! No Inducer J "j 10 12 14 16 18 20 22 Digestion (hr) Figure 7. Enzymatic solubilization and further purification of small morphogen. The insoluble, cell wall-associated inducer of larval meta- morphosis was digested inside a dialysis tubing ( retention limit = 1 4,000 Da) with purified agarase. Small morphogens released from the insoluble inducer were allowed to accumulate in the external dialysate, which was removed and assayed, and replaced with fresh external medium, every two hours. An otherwise identical sample was digested in parallel with no change of dialysate for 22 h, after which the external medium was removed and assayed (dotted line). Other details as described in the text; all assays were performed in duplicate under standard conditions. Con- trols conducted in parallel included assays of dialysates changed every 2 h from a sample incubated with no enzyme, and larvae incubated with no additions; these gave 0% metamorphosis. Results show meta- morphosis induced by the dialysates. 6). The external dialysates (including the permeant small morphogens released) were allowed to diffuse and accu- mulate outside of the membranes for a fixed interval, after which they were removed for assay, and replaced with fresh external liquid for the next interval of incubation. With purified agarase, and 2-h intervals for accumulation, removal, and replacement of the external dialysates. we observed a time-dependent increase and subsequent de- cline in the rate of appearance of dialyzable morphogenic activity with M < 14,000 Da (Fig. 7). However, far less of the total morphogenic activity could be detected in an otherwise identical incubation, in which only one sample of dialysate was collected and assayed after prolonged in- cubation (24 h). All of the dialysates were held at 28C until the end of the experiment (24 h) and assayed si- multaneously, to control for any time-dependent decay of activity. Because the difference between the two parallel incubations thus was in the limitation (2 h interval) or prolongation (24 h interval) of the opportunity for the small dialyzable morphogen to diffuse back and forth be- tween the outside and inside of the dialysis tubing, we conclude that prolonged exposure of released, dialyzable morphogen to the agarase resulted in degradation of the active morphogen. We therefore conclude that agarase, which cleaves sulfated polysaccharides, particularly at 0- 1,4-linked galactose residues, not only releases the active morphogen from a sulfated polysaccharide parent mole- cule, but degrades the morphogen itself, as well. Therefore, the structure of the morphogen would seem to contain a sulfated polysaccharide with galactose residues. In similar experiments, we compared dialysis mem- branes with calibrated porosites ofca. 10,000-14,000 Da and ca. 6.000-8,000 Da, and two other endosaccharidases in addition to agarase (Fig. 8). When lysozyme was used we again saw a time-dependent increase and subsequent decline in the rate of accumulation of dialyzable mor- phogen with M < 14,000 Da. There was a similar, but displaced, rise and fall in the accumulation of smaller morphogen (M < 8000 Da), suggesting that these smaller molecules may be derived, in part, by enzymatic cleavage of the larger dialyzable inducers. With the 8-h intervals 100 80 60 40 20 M< 14,000 Agarase M < 8,000 Lysozyme 0-8 8-16 16-24 Digestion (hr) Figure 8. Enzymatic solubilization and further purification of small morphogen through dialysis membranes of two different porosities. Ex- periment performed as in Figure 7, except that: agarase, lysozyme and endo-i-galactosidase were compared; the digestions were performed in parallel in dialysis tubings with retention limits of 14.000 Da (hatched bars) and 8,000 Da (solid bars); the external dialysates were changed and saved for assays every 8 h. 12 D. E. MORSE AND A. N. C. MORSE Table III Specificities and effects <>l'en:yinc probes ;<csenii was incubated with vigorous stirring at pH 12.0. At the times indicated, aliquots were withdrawn, neutralized to pH 8.2. filtered through nitrocellulose filters (0.2 ^m. 47 mm diam.), and the soluble fractions assayed (in duplicate 10 ml samples) under the standard conditions. Details are in Materials and Methods. Results shown are the average percentages of larvae induced to attach and metamorphose by the soluble fractions, S.D. Results of a parallel and otherwise identical incubation held continuously at pH 8.2 are included for comparison. polysaccharide (cleaved by hyaluronidase, agarase, and sulfatase), with multiple, substituted residues of N-ace- tylglucosamine (linkages cleaved by lysozyme and endo- itf-galactosidase) and multiple residues of galactose (link- ages cleaved by agarase and endo-/3-galactosidase). Protein (or peptide) does not appear to be an essential component, nor do several specific free or terminal sugars or saccharide units. Hydrolysis with dilute alkali releases small anionic morphogen containing sulfate The finding that specific endopolysaccharidases can re- lease a soluble morphogen from the insoluble cell wall- associated fraction suggested that non-enzymatic, partial hydrolysis under mild conditions might yield similar re- sults. While some desulfation also would occur, hydrolysis with sufficiently dilute alkali would be expected to yield oligosaccharide fragments with retention of at least some of the original sulfate groups (Percival and McDowell, 1967). As predicted, exposure of the decalcified, partic- ulate cell wall fraction to dilute alkali (pH 11-12) released a soluble, sulfate-containing morphogen. The increase in yield of soluble morphogenic activity as a function of the time of incubation at pH 12 is shown in Figure 9. The apparent decline in yield after 6 h suggests that the mor- phogen itself is slowly inactivated at pH 12. Exposure to strong acid (4 N HC1) or strong base (4 TV NaOH) causes more rapid inactivation. The morphogen released by mild alkaline hydrolysis is of relatively low molecular weight, strongly anionic, sul- fate-containing, and unstable. Estimates of the apparent molecular weight were made by three independent meth- ods: ultrafiltration through a calibrated membrane, di- alysis through a calibrated dialysis membrane, and Seph- adex gel filtration (Table IV). In ultrafiltration, all of the applied activity was recovered in the YM-5 membrane ultrafiltrates, consistent with M < 5000 Da. In dialysis through a calibrated membrane, all of the recovered ac- tivity (equal to that in the non-dialyzed control) was found in the first external dialysate, (see Materials and Methods), consistent with M < 2000 Da. These results were con- firmed by gel-filtration through Sephadex G-10, in which all of the applied activity was eluted at the end of the included volume, indicating M < 2000 Da. These findings all indicate, therefore, that the morphogen solubilized by alkaline hydrolysis may be as small as 2000 Da or less (Table IV). This low molecular weight material is strongly anionic; it binds tightly to DEAE Sephadex and to DEAE-nitro- cellulose (Table V). Because the material does not bind to Sephadex or nitrocellulose alone, without DEAE sub- stitution, the binding is most likely dependent on ionic interaction. This binding proves to be very strong; little activity could be removed from the DEAE Sephadex by elution with 0.4 M NaCl, and 75% of the initially applied activity was found still adsorbed to the resin (Table V). Similarly, 63% 13% of the activity applied to a DEAE- nitrocellulose filter was found still adsorbed to the filter, even after elution with 0.4 N HC1. That the morphogen was not eluted from DEAE by 0.4 TV HC1 indicates that its ionic binding was dependent on more strongly anionic groups than uronic acids or other simple carboxylates (pK ca. 2). These results are consistent with the conclusion, based on sulfatase sensitivity (cf. Figs. 4, 5; Table II). that the morphogen contains sulfate groups (see Discussion). The presence of sulfate esters in this solubilized mor- phogen was confirmed independently by turbidimetric analysis with barium after strong acid hydrolysis (method according to Beeley, 1985). The yields obtained indicate Table IV Estimation of molecular weight of the small morphogen solubilized by partial alkaline hydrolysis Method M Estimate (Da) Ultrafiltration Dialysis Sephadex gel filtration <5000 <2000 <2000 Details are as described in Materials and Methods. Independent ex- periments verified that there was no significant adsorption of the mor- phogen to the gel-filtration matrix or to the ultrafiltration or dialysis membranes. 114 D. E. MORSE AND A. N. C. MORSE Table V Adsorption of the small morphoxen. xolubilizeil by partial alkaline hydrolysis, to DEAE Recovery Adsorbant Eluant or fraction Units DEAE Sephadex Application 1,000 1 m.U Tris 1 mM Tns + 0.4 A/ NaCl 50 5 Remaining on DEAE 753 75.3 (Total) (80.3) DEAE Nitrocellulose Application 640 1 mM Tris 1 mM Tns + 0.4 A/ NaCl 0.4 N HCI Remaining on DEAE 400 80 63 13 (Total) (63 13) Details are as described in Materials and Methods. Morphogen re- covered in the high-salt eluate. and that remaining on the DEAE, induced larvae to attach firmly to the polystyrene assay beakers and metamorphose normally. Control experiments demonstrated that the DEAE adsorhants without the applied morphogen had no such activity. Additional controls showed that there is no binding of the soluble morphogen either to Seph- adex or nitrocellulose alone, without the DEAE ion-exchange groups present. the presence ofca. 8-14% (w/w) sulfate in the alkali-sol- ubilized morphogenic fraction. Agaricia humilis larvae exposed to the alkali-solubilized low molecular weight morphogen, either free in solution or bound to DEAE (Sephadex or nitrocellulose) showed rapid, normal, and complete metamorphosis. The activity of both the soluble and DEAE-bound morphogen was concentration- or dose-dependent. Activity, specificity, and stability oj the small soluble morphogen The small, dialyzable morphogens obtained in the ex- periments shown in Figures 6-9 induce rapid and normal larval settlement, attachment, complete metamorphosis, and normal post-metamorphic growth (Fig. 10). These processes induced by the small morphogenic molecules are indistinguishable from those induced by the native, intact nongeniculate coralline alga. The low molecular weight morphogens produced by enzymatic or alkaline hydrolysis of the paniculate cell wall fraction from Hydrolithon boergesenii (or associated microbial symbionts) induce metamorphosis of at least one other agariciid coral, in addition to Agaricia humilis. Larvae of the sympatric A. lenuifolia. which also are in- duced to settle and metamorphose by the intact Hydro- lithon boergesenii (A. Morse et ai, in prep.), are induced to metamorphose by the dialyzable morphogen released by agarase digestion of the cell wall fraction from this alga, with an efficiency comparable to that exhibited for the A. humilis larvae (Table VI). In both of these species, the coral larvae are induced to attach to the walls or bot- tom of the polystyrene containers, metamorphose com- pletely, and begin normal post-metamorphic growth in response only to the low molecular weight chemical mor- phogen. In contrast, larvae of the sympatric ahermatypic coral, Tubastraea aurea, which are not induced to me- tamorphose by Hydrolithon boergesenii. are not induced by the low molecular weight morphogen enzymatically released from the algal cell wall fraction (Table VI). This demonstrates that the morphogenic activity of this chem- ical is biologically specific for those larvae that respond to the intact alga. Whereas the decalcified, cell wall-associated, insoluble morphogen is relatively stable when frozen at - 10C, the small, dialyzable morphogen (M < 8000 Da) released from this parent material by endo-0-galactosidase is stable for only a few days at -10C. The lower molecular weight morphogen released by mild alkaline hydrolysis (M < 2000) proved to be even more unstable. Even when frozen at -10C, this material lost activity with a half- life that varied in different preparations between 24 and 72 h. Activities of known compounds We have tested a wide variety of sulfated and non-sul- fated polysaccharides and related polymers, including several substrates for the enzymes used in this study. These (all pre-adjusted to the pH of ambient seawater prior to testing) have included: agar; agarose; agaric acid; alginic acid; ascophyllan; X-, (-, and K-carrageenans; fucoidan, furcellaran; laminarin; chitin; di-N-acetylchitobiose; tri- N-acetylchitotriose; keratan sulfate; chondroitin sulfates A, B, and C; heparin; heparan sulfate; hyaluronic acid; asialofetuin; dextran sulfate; pentosan sulfate; polyvinyl sulfate; polyanethol sulfate; pectin; amylopectin; cellulose; cellulose sulfate; sulfoethylcellulose; and sulfopropyl- sepharose. Most of these proved inactive with Agaricia humilis larvae. Only the /,-carrageenan (a sulfated polymer rich in galactose), fucoidan (a sulfated polymer rich in fucose), and keratan sulfate (a sulfated glycosaminoglycan) induced metamorphosis, although this activity was weak and evident only at very high concentrations (>5 mg/ml). Discussion Recent field and laboratory studies have shown that recruitment of the shallow- water agariciid corals, Agaricia humilis and A. tenmfolia, is determined in part by larval recognition of a chemical inducer of substratum-specific settlement and metamorphosis (Morse et ai. 1988). This inducer is associated with specific nongeniculate coralline red algae. In the case of A. humilis, only certain coralline CHARACTERIZATION OF CORAL MORPHOGEN 115 Figure 10. Normal attachment and metamorphosis of Agaricia humilis larva induced by the soluble morphogen. Scanning electron micrographs of the endoskeleton of a post-metamorphic corallite attached to polystyrene, dorsolateral (a) and dorsal (b) views, and for comparison, a pre-metamorphic planula larva (c). The pronounced calcified septa and the attachment plaque are clearly visible in the 2-day post-metamorphic corallite. Diameter of the corallite ca. 1.5 0.1 mm; length of the planula ca. 0.7 0.1 mm. The soluble morphogen was obtained by digestion with endo-ff-galactosidase. followed by dialysis, as in Figure 8. Tissue- digestion, fixation, dehydration, critical-point drying, and electron microscopy were by standard procedures. red algae contain the inducing morphogen (Fig. 1: Table I: cf. Morse et ai. 1988; A. Morse and R. Steneck, in prep.). We have extended our previous finding that Agaricia humilis larvae maintain both the stringency of their de- pendence upon the alga-associated morphogen, and the specificity of this requirement, for at least 30 days follow- ing their release (Fig. 1 ). While most larvae probably settle and metamorphose in less than 30 days in the natural environment, this capacity to delay metamorphosis in the absence of the required morphogen can enhance both the dispersal of the larvae, and the substratum-specificity of the final distribution of recruits. Although much of scler- actinian recruitment may be locally seeded (Bak and En- gle, 1979; Rylaarsdam, 1983; Baggett and Bright, 1985; Sammarco and Andrews, 1988), the larvae of A. humilis (cf.Fig. 1 ; also Morse etal., 1988) and certain other species (cf. Harrigan, 1972; Richmond, 1981, 1985, 1987; Har- rison et ai. 1984; Scheltema, 1986; Morse et ai. 1988; Richmond and Hunter, 1990) are capable of distant dis- persal as well. Indeed, temporal pulses of scleractinian recruitment at certain sites on the Great Barrier Reef are dependent on the settlement of larvae produced at distant locations (Wallace, 1985; Babcock, 1988). In addition to the importance of hydrodynamic, topographic, and geo- graphic features for the control of larval dispersal and consequent recruitment (e.g.. Frith et ai. 1986; Rough- garden et ai, 1988; Sammarco and Andrews, 1988; Black, 1988), and the effects of predation, nutrition, competition, and other biotic factors, studies of A. humilis (Morse et ai. 1988; and those reported here) indicate that for some coral species, the stringency and specificity of the larval requirements for the induction of settlement and meta- morphosis also can be important in controlling the spatial distribution of recruits. A similar larval requirement for a substratum-specific biochemical inducer of settlement Table VI Induction oj metamorphosis of Agaricia humilis and A. tenuifolia /an'ae. and absence of induction o/'Tubastraea aurea, by the low molecular weight (LA/H'J morphogen Treatment Metamorphosis at 24 h (X S.D.) LMW morphogen Seawater control Agaricia humilis Agaricia tenuifolia Tubastraea aurea 95% 10% (n = 4) 0% 0% (n = 5) 87% 23% (n = 3) 0% 0% (n = 5) 0% 0% (n = 3) 0% 0% (n = 3) Soluble morphogen was prepared by agarase digestion of the particulate cell wall preparation of Hydrolithon boergesenii (agarase. 1 mg. 1000 units), and dialysis through a membrane retaining molecules with M > 14,000 Da, as described in Materials and Methods. Larvae of each of the three coral species were assayed for metamorphosis in response to the dialyzable morphogen and seawater controls in parallel: 5 larvae/trial: n = number of trials. 116 D. E. MORSE AND A. N. C. MORSE and metamorphosis, and larval recognition of the chem- ical inducer in the ocean environment, recently were shown to determine the fine-scale spatial distribution of recruitment of the polychaete, Phragmatopoma califomica (Jensen and Morse, 1990). Chemical nature ofihe inducer The inducer of Agaricia humilis settlement and meta- morphosis that is associated with specific nongeniculate coralline red algae is chemical in nature (Table I; cf. Morse el a/.. 1988). This morphogenic substance is insoluble in a wide range of solvents, apparently because it is associated with cell wall polysaccharides (with which the morphogen is partially purified; cf. Fig. 2). Solubilized fractions of the inducer can be generated, however, by hydrolysis with enzymes or mild alkali. The fact that these solubilized fractions are sufficient to trigger normal attachment and metamorphosis of A. humilis larvae on clean polystyrene surfaces (Fig. 10) proves that the requirement of the larvae for a morphogenetic inducer is satisfied by a chemical substance from the inductive algal substratum. Non-re- cruiting algal substrata do not yield this activity (Table I). These results thus confirm and extend the finding that larval recognition of the inductive but insoluble paniculate fraction, partially purified from the settlement-inducing algal surface, is dependent upon the integrity of a perio- date-sensitive (Fig. 3) and sulfatase-sensitive (Fig. 5; Table II) chemical structure. These sensitivities, the insolubility of the crude inducer, and its insensitivity to proteolytic enzymes, suggested that the inducer is associated with a sulfated polysaccharide. Consistent with this suggestion, we have found that the inducer can be solubilized by cleavage with purified en- doglycosidases that can act on sulfated polysaccharides (Figs. 6-8). As summarized in Table III. the cleavage by agarase and by endo-0-galactosidase indicates that the substrate polymer contains galactose units; cleavage by lysozyme and by endo-0-galactosidase indicates that this polymer also contains 0-1,4 linked N-acetylglucosamine or N-acetylglucosamine sulfate units (cf. Li el a/.. 1982; Kitamikadocfa/., 1982; Scudder et at.. 1983, 1984; Mor- rice et at.. 1983a, b; Usov and Ivanova, 1987). Because continued hydrolysis by each of these enzymes leads first to solubilization. and then to progressive inactivation of the inducer, the inductive moiety itself probably contains the above-mentioned sites of hydrolysis. The morpho- genetic inducer is thus associated with, and may itself contain, a sulfated glycosaminoglycan, i.e., a sulfated polysaccharide that includes multiple N-acetylglucos- amine and galactose units. The finding that partial alkaline hydrolysis liberates a sulfate-containing, strongly anionic, small morphogen is consistent with this conclusion. Re- cently, hydrolysis, ion-exchange HPLC, and sensitive de- tection of the resolved monosaccharides by pulsed am- perometry have independently confirmed that glucos- amine (derived from N-acetylglucosamine) and galactose are indeed principal components of this solubilized small morphogen (M. Hardy, Dionex Corp., pers. comm.). Sul- fated polysaccharides that contain amino sugars and are rich in galactose, similar to those in foliose red algae (cf, Percival and McDowell, 1967; Percival, 1978; Mc- Candless, 1981; Yaphe, 1984), previously were found in other coralline red algae (Turvey and Simpson, 1965). Keratan sulfate, which proved to be slightly active as a morphogen, has three characteristics that make it unique among the known sulfated glycosaminoglycans tested. In these same features, it also, to some degree, resembles the morphogen recognized by Agaricia humilis larvae: ( 1 ) it contains multiple galactose-/3-l,4-N-acetylglucosamine units (whereas heparin, heparan sulfate, and the three dif- ferent chondroitin sulfates all contain glucuronic acid or other uronic acid units in place of galactose); (2) it is readily cleaved by endo-0-galactosidase (Scudder el at.. 1983), whereas the other known compounds tested are not; and (3) the keratan sulfates from some sources cannot be eluted from anion exchangers at salt concentrations below 3-4 M (Roden et til., 1972; cf. our results with DEAF, Table V), whereas the other sulfated glycosami- noglycans are readily eluted at significantly lower con- centrations (Roden et at.. 1972). Very highly sulfated gal- actans from red algae also are not eluted from DEAF, or else are eluted only at high concentrations of urea or at high temperature (Yaphe, 1984). However, the weak morphogenetic activity evident at only high concentra- tions of keratan sulfate, carrageenan, and fucoidan indi- cates that although these substances may be somewhat similar to the natural inducer, they differ in structural features (possibly including positions of the sulfate groups or other linkages) from those of the natural morphogen. The morphogenetic activity of the natural inducer sol- ubilized from the cell wall fraction obtained from Hy- drolithon boergesenii or its associated microflora is bio- logically specific (Table VI). This molecule induces normal attachment and metamorphosis in larvae of two species of Agaricia that are also induced by the intact alga from which the morphogen was obtained. However, the mol- ecule fails to induce these reactions in larvae of the sym- patric Tubastraea aurea. which are not induced by the intact alga. The high specificity of the Agaricia humilis larvae for morphogens associated with only certain nongeniculate coralline red algae (Fig. 1; Table I; Morse et a/.. 1988; A. Morse and R. Steneck, in prep.) is apparently a reflection of the chemical specificity of the larval receptors for only some unique sulfated glycosaminoglycans. This suggestion is supported by the finding that a wide variety of synthetic natural sulfated polysaccharides. glycosaminoglycans, and CHARACTERIZATION OF CORAL MORPHOGEN 117 structurally related polymers have either little or no mor- phogenetic activity. A better understanding of the chem- ical basis for the high specificity of the larval receptors awaits further information about the stereochemistry and structure of the organic sulfate esters and other substitu- ents in the natural morphogen. Relation to other systems Sulfated glycosaminoglycans and other sulfated poly- saccharides have been widely implicated in other highly specific cell recognition phenomena that control differ- entiation, including inter-phyletic symbiosis, fertilization, aggregation of sponge cells, the "homing" of circulating mammalian lymphocytes into the lymph nodes, pattern formation in the developing nervous system, and the me- tastasis and invasiveness of tumors. The number of struc- tural permutations of such molecules, and hence the complexity of coding of cell recognition ideotypes, far ex- ceed those of the proteins, which are more fully under- stood (Drickamer, 1988; Sharon and Lis, 1989). Recent evidence has shown that the host specificity for nodule induction in leguminous plants by the nitrogen- fixing symbiotic bacterium, Rliiiobium meliloti, is deter- mined by recognition of a unique sulfated glycosamino- glycan signal (a tetrasaccharide containing one sulfate and one acylamino group) produced by the bacterium (Ler- ouge el al.. 1990). Recognition, by the host plant, of this inducing bacterial signal, and the resulting specificity of the inter-phyletic symbiosis, has long been thought to be governed by a class of receptors known as lectins, present on the plant root hair surfaces (Kijne et ai. 1989). Re- cently, this suggestion was strongly confirmed when the intergeneric transfer between plants of the DNA coding for one such root lectin resulted in the transfer of host specificity for nodulation induced by the bacterium (Diaz et ai. 1989). Recognition of specific sulfated polysaccha- rides at the surface of Strongylocentrotus pwpiiratus sea urchin eggs by conspecific sperm has been demonstrated to be essential for fertilization (Rossignol et al., 1984; DeAngelis and Glabe, 1987). In this reaction (DeAngelis and Glabe, 1987), and in the binding of heparin to anti- thrombin (Atha et ai, 1985), the locations of the sulfate esters on the sugars have been found to be of critical im- portance; in the latter case, removal of one specific sulfate reduced the affinity of specific binding by as much as 10,000-fold (Atha et ai, 1985). The lengths of the sulfated polysaccharides are also critical determinants of the strength (and hence, the specificity) of these binding re- actions (Hoylaerts et ai, 1984; DeAngelis and Glabe, 1987). Evidence also suggests that lectin-like recognition of cell surface sulfated glycosaminoglycans controls an essential phase in the species-specific reaggregation and subsequent differentiation of sponge cells (Henkart et ai. 1973; Turner and Burger, 1973; Jumblatt et ai, 1980; Conrad et ai, 1984; Diehl-Seifert et ai, 1985, 1989; Mar- goliash et ai, 1965; Coombe et ai, 1987; Coombe and Parish, 1988; Schroder et ai, 1988;Gramzowe/a/.. 1989). Recognition and binding of sulfated glycosaminoglycan moieties of cell surface proteoglycans also controls adhe- sion, tissue-specific differentiation and growth in a wide variety of mammalian and other higher systems (Edelman, 1985; Fransson, 1987; Ruoslahti, 1989). The structures and positions of the sulfate esters can be critically im- portant in the control of these functions as well (Fransson, 1987). Sulfated glycosaminoglycans and other sulfated polysaccharides bind to lectin-like receptors on the sur- faces of lymphocytes (Parish et ai, 1984; Parish and Snowden, 1985; Chong and Parish, 1985, 1986; Thurn and Underbill, 1986; Brenan and Parish, 1986; Brandley et ai. 1987). Recently, the genes coding for two distinct "homing receptors" from the surfaces of mammalian lymphocytes have been cloned and sequenced (Gallatin et ai, 1986; Yednock et ai, 1987a; Goldstein et ai, 1989; Holzmann et ai, 1989; Siegelman et ai. 1989; Stamen- kovic et ai, 1989; Stoolman, 1989). Lectin-like recogni- tion of specific sulfated or other anionic carbohydrates in the target lymphoid tissues, mediated by these receptors, is thought to direct the homing of specific subsets of lym- phocytes from the circulation to adhere to the blood vessel endothelia of their target lymphoid organs (lymph nodes. Peyer's patches, etc.), where the recruited lymphocytes then differentiate to produce antibodies (Brenan and Par- ish, 1986; Gallatin et ai, 1986; Yednock et ai, 1987a, b; Jalkanenc/a/.. 1988; Stoolman, 1989; Coombe and Rider, 1989). The parallels between this lymphocyte homing reaction and the settlement and metamorphosis ofAgaricia humilis larvae are potentially interesting. In both, substratum- specific "recruitment," attachment, and differentiation are apparently induced by recognition of a non-diffusing, substratum-specific sulfated polysaccharide. Our further observation that the partially purified sulfated polysac- charide that induces A. humilis larvae to attach and me- tamorphose also induces murine lymphocytes to undergo mitosis (Morse and Eardley, unpub. obs.), may therefore be worth further investigation. The specificity of this latter reaction is unclear, however, as a wide variety of sulfated polysaccharides and related polymers, including several carrageenans, fucoidan, and ascophylan, induce lympho- cyte mitosis. Whether the same subset of lymphocytes responds to each of these compounds has not yet been determined. In contrast, the A. humilis larvae respond only slightly and incompletely to carrageenan and fucoi- dan, and only at very high concentrations. Cell-surface recognition of polysaccharides and other complex carbohydrates in such non-immune systems is generally considered to be mediated by lectins, a broad 18 D. E. MORSE AND A. N. C. MORSE class of ubiquitous, carbohydrate-specific receptors that recently has been redefined (Barondes, 1988; Drickamer, 1988; Sharon and Lis, 1989). Mitchell and his colleagues first demonstrated that the settlement and metamorphosis of larvae of the polychaete, Janua brasiliensis, are me- diated by a lectin-like recognition of inductive exopoly- saccharides produced by specific bacteria (Kirchman el a/., 1982a, b; Mitchell and Kirchman, 1984; Maki and Mitchell, 1985, 1986). These authors first pointed out the similarities between this larval settlement reaction and other lectin-mediated recognition phenomena, including the root nodule-bacteria symbiosis discussed above. Wei- ner et al. (1985) and Bonar el al. ( 1986) also have shown that specific bacterial exopolysaccharides may play a role in the induction of settlement and metamorphosis of Crassostrea virginica and C. gigas oyster larvae, although induction in those systems is complex, and more than one class of compound is known to be involved (Coon et al., 1985; Coon and Bonar, 1987; Fitt el al., 1989; Bonar et al., 1990). The results reported here, demonstrating sensitivity of the Agaricia humilis morphogen to cleavage by endo-/3- galactosidase and agarase, (and resistance to the exogly- cosidic /3-galactosidase), indicate that the morphogen contains essential internal /3-galactoside units. This finding may be of particular interest in view of the suggested im- portance of j3-galactoside-specific lectins in controlling differentiation in higher animal systems (Barondes et al., 1988; Sharon and Lis. 1989). Remaining problems Two problems remaining are the determination of the complete chemical structure of the inducing molecule recognized by the Agaricia humilis larvae, and the un- equivocal identification of the biological source of this inducer. A number of compounds that induce metamor- phosis of various marine invertebrate larvae have been partially purified from the respective inductive substrata, and the structures of these compounds have been partially characterized. But we are aware of only two natural in- ductive molecules that have been completely character- ized. These include the algal molecules that induce meta- morphosis of the scallop, Pecten ma.\imits (Yvin et al., 1985), and the hydrozoan, Corync uchidai (Kato et al., 1975). Significantly, both of these are small molecules soluble in organic solvents, and thus amenable to gas- chromatography and mass spectroscopy. In a large num- ber of the other cases investigated, however, the native inducers have proved to be either water soluble (e.g., Highsmith, 1982; Hadfield and Scheuer, 1985; Burke, 1 986; Hadfield and Pennington, 1 990), or polymeric and insoluble (e.g., Jensen and Morse, 1984. 1990; Morse et al.. 1988). [We are not including in this discussion such molecules as potassium or calcium salts, fatty acids, cyclic nucleotides, or other widely active effectors of depolar- ization, protein phosphorylation, or signal transduction pathways: these all have been shown to induce meta- morphosis of larvae without species- or substratum-spec- ificity (eg., Baloun and Morse, 1984; Morse, 1985, 1990; Yool et al.. 1986; Pechenik and Heyman, 1987; Jensen and Morse, 1990; Jensen et al., 1990)]. The inducer of Agaricia humilis metamorphosis described here is in its native form associated with a substratum-specific insol- uble polymer. Partial hydrolysis with either enzymes or dilute alkali releases a smaller, water-soluble and strongly anionic inducer which is markedly unstable. This insta- bility has hindered analyses of the active morphogen. The use of highly purified enzymes, and employment of their specificities for selective cleavage, solubilization, and as probes of the structural determinants of morphogenetic activity, may prove widely useful in further studies of such otherwise intractable molecules. The inductive molecule that we have described is ob- tained from homogenates of the nongeniculate coralline red alga, Hydrolithon boergesenii. Larvae of Agaricia hu- milis are induced to metamorphose by contact with intact specimens of that alga, and recruits of the coral are found preferentially on that alga in the field (Morse et al., 1988; A. Morse and R. Steneck, in prep.). Three other species of anthozoan, including the scleractinian Agaricia ten- iiifo/ia (Morse et al., 1988), the temperate octocoral Al- cyonnim sidcriiim (Sebens, 1983a. b), and a tropical gor- gonian, Plexanra sp. (Lasker, 1990), also have been found to settle and metamorphose in response to crustose red algal surfaces. But in each of these cases, the inductive molecule could have been produced by bacteria or other microorganisms associated with the algal surfaces. Bacteria or bacterial films have been implicated in the control of larval settlement and metamorphosis in a few other cni- darians in which these processes are chemically induced. Larvae of the hydroid, Hydractinia echinata. settle and metamorphose in response to films of the bacterium, Al- teromonas sp., on shells inhabited by hermit crabs (Spin- dler and Miiller, 1972). Cassiopea andromeda (scypho- zoan) larvae also are induced to settle and metamorphose by bacteria, apparently in response to soluble peptides produced by the action of bacterial degradative enzymes (Fitt and Hofmann, 1985; Fitt et a/., 1987; Hofmann and Brand, 1987). Bacterial films have been widely implicated in the control of larval settlement and metamorphosis in many other kinds of invertebrates as well (Wilson, 1955; Cameron and Hinegardner, 1974; Brancato and Woola- cott, 1982; Kirchman et at.. 1982; Mitchell and Kirchman, 1984; Bonar et al. 1986; Maki and Mitchell. 1986; Fitt et al.. 1989; Maki et al., 1990). Moreover, as Maki et al. ( 1990) have suggested in the case of barnacle larvae, the larval response may depend in a complex way on the CHARACTERIZATION OF CORAL MORPHOGEN 119 interaction between bacteria and the surface on which they are attached. The structure of the morphogen recognized by Agaricia hunulis larvae, suggested by the results reported here to be a sulfated glycosaminoglycan, would be equally con- sistent with a molecule of the red algal cell wall and of a cell wall or other exopolymer produced by an associated bacterium (Percival and McDowell, 1967; Mackie and Preston, 1974; Sanford et a/., 1977; McCandless, 1981; Drews and Weckesser, 1982; Boyle and Reed, 1983). We have found, however, that the activity of crude homog- enates appears markedly enhanced following decalcifi- cation, consistent with the unmasking of constituents of the algal cell wall. Attempts to culture the algal cells ax- enically from isolated protoplasts, by the methods of Polne-Fuller and Gibor (1984) and Kloareg et al. (1989), and attempts to culture the alga-associated microbial symbionts, may help further resolve the source of the in- ducer. 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The isolated copepods were maintained in 2 ml clear plastic culture chambers in an incubator at 20C and at a 16:8 light:dark schedule. They were fed a mixture of Tetramin flaked fish food and nutritional yeast. I performed three types of crosses to estimate outbreed- ing depression and inbreeding depression. The crosses were female X non-poolmate, female x poolmate, and female X sibling. Poolmate matings represent the normal breeding situation for non-dispersing Tigriopus. Non- poolmate matings should represent the breeding situation for dispersing copepods; any reduction in fitness in the non-poolmate matings reflects outbreeding depression. Similarly, sibling matings should represent the worst-case result of not dispersing; any reduction in fitness in sibling matings reflects inbreeding depression. However, if pools are full of close relatives, and therefore close inbreeding represents the normal breeding situation, then sibling matings and poolmate matings should have similar results. Furthermore, inbreeding depression is expected to be minimized in this case because many of the deleterious alleles will have been eliminated from the pool population. After mating occurred in the lab test pairs, I removed the males and placed each female in a large (50 ml) vial with excess food to minimize cannibalism following hatching of the juveniles. From the single mating, females then produced continuous broods. At the end of three weeks (approximately one generation), I counted the total number of individuals (nauplii, copepidites, and adults) in each vial and used this number as a measure of fitness. The rationale for using this number as an index of fitness is that Tigriopus probably maximize their fitness most effectively by producing the greatest possible number of offspring. The high fecundity of Tigriopus may support this hypothesis. The number of offspring produced varies with type of cross (Fig. 1). Females mated with random poolmates produced significantly more offspring than did either fe- males mated with non-poolmates or females mated with siblings (Ei 2 = 0.061, P = 0.02). Number of offspring was compared by isotonic regression (see Gaines and Rice, Table I Characteristics <>/ PI>i>i which lab populations were taken Salinity (ppl) Pool mean (range) Temperature (C) mean (range) Pool size (cm) Substratum type 1 5.7(0-151 14.9(9-25) 100 X 100 X 17 upright algae, sand 2 9.2 (0-20) 15.9(9-24) 63 x 28 x 3 algal mat 3 10.0(0-20) 13.9(9-20) 45 x 25 X 3 rock, sand 4 17.1(0-40) 15.6(9-28) 100 x 50 x 4 algal mat 5 10.0(0-20) 14.8(8-27) 100 x 53 X 8 sand 6 9.2(5-15) 15.2 (9-23) 100 x 100 x 28 upright algae, sand Mean salinity and mean temperature over a four month sampling period are shown. Approximate pool size is given for maximum values of three dimensions: length ' width depth. OUTBREEDING DEPRESSION IN TIGRIOPUS 125 (3 z .50 3.60 P > .05 2. Sibling and Non-poolmate S P stained unstained y 15 13 28 18 33 I 1 24 29 57 0.02 P> .90 1.41 P > .05 3. Poolmate and Non-poolmate P N stained unstained 12 10 11 23 7 17 18 40 0.40 P> .50 1.40 P>.05 In each test, the female was given a choice of two males sibling + poolmate, sibling + non-poolmate, or poolmate + non-poolmate. Sample sizes were 40, 57, and 40, respectively. "Stained" males were dyed red with carmine powder to facilitate individual recognition tor testing. S = sibling, P = poolmate, and N = non-poolmate. 126 A. F. BROWN suits suggest that frequent dispersal may not always be the most adaptive reproductive strategy. Acknowledgments I wish to thank T. Farrell. J. Lubchenco, B. Menge, and the rest of the Zoology gang for their advice and pa- tience. C. King graciously provided the lab space. The manuscript was improved greatly due to comments from R. Burton, S. Gaines. P. Yund. an anonymous reviewer, and the attendees of the Brown Bag seminars. Support came through a ZoRF grant from Oregon State University. Literature Cited Brown, A. F. 1985. An experimental test of optimal outbreeding in the harpacticoid eopepod Tigriopus califomicus. MS Thesis, Oregon State University. Burton, R. S. 1986. Evolutionary consequences of restricted gene flow among natural populations of the eopepod Tigriopus califomicus. Bull- Mar. Sa. 39: 526-535. Burton, R. S. 1990. Hybrid breakdown in developmental time in the eopepod Tigriopus califomicus. Evolution 44: 1814-1822. Burton, R. S., and M. W. Feldman. 1981. Population genetics of Ti- griopus califomicus. II. Differentiation among neighboring popula- tions. Evolution 35: 1 192-1205. Burton, R. S., and S. G. Swisher. 1984. Population structure of the intertidal eopepod Tigriopus califomicus as revealed by field manip- ulation of allele frequencies. Oecologia 65: 108-1 1 1. Cohen, D., and U. Motro. 1989. More on optimal rate of dispersal: taking into account the cost of dispersal mechanisms. Am. Nat. 134: 659-663. Egloff, D. A. 1966. Ecological aspects of sex ratio and reproduction in experimental and field populations of the marine eopepod Tigriopus califomicus (Baker). ./ Exp. Mar. Biol Ecol 42: 99-1 1 1. Gaines, S. D., and VV. R. Rice. 1990. Analysis of biological data when there are ordered expectations. Am. Nat. 135: 310-317. Greenwood, P. J., P. H. Harvey, and C. M. Perrins. 1978. Inbreeding and dispersal in the great tit. Nature 271: 52-54. Holekamp, K. E., and P. W. Sherman. 1989. Why male ground squirrels disperse. Am. Sci 77: 232-239. Hoogland, J. L. 1982. Prairie dogs avoid extreme inbreeding. Science 215: 1639-1641. Howe, H. F., and J. Smallwood. 1982. Ecology of seed dispersal. Anna. Rev. Ecol. Syst 13: 201-228. Johnson, M. L., and M. S. Gaines. 1990. Evolution of dispersal: theo- retical models and empirical tests using birds and mammals. Annu. Rev. Ecol. Syst. 21: 449-480. Packer, C. 1979. Inter-troop transfer and inbreeding avoidance in Papio anuhis. Annn. licliav 27: 1-36. Palmer, A. R., and R. R. Strathmann. 1981. Scale of dispersal in varying environments and its implications for life histories of marine inver- tebrates. Oecologia 48: 408-418. Price, M. V., and N. M. VVaser. 1979. Pollen dispersal and optimal outcrossing in Delphinium nelsoni. Nature 277: 294-297. Shields, \V. M. 1982. Philopatry. Inbreeding, and the Evolution oj Sex. SUNY Press, Albany. Strathmann, R. R., E. S. Branscomb, and K. Vedder. 1981 . Fatal errors as a cost of dispersal and the influence of intertidal flora on set of barnacles. Oecologia 48: 13-18. Vittor, B. A. 1971. Effects of the environment on fitness-related life history characters in Tigriopus califomicus Ph. D. Thesis. University of Oregon. Waldman, B. 1988. The ecology of kin recognition. Annu. Rev Ecol. Svst 19: 543-571. Reference: Biol. Hull 181: 127-134. (August, Alloimmunity in the Gorgonian Coral Swiftia exserta LUISA SALTER-CID AND CHARLES H. BIGGER Department of Biological Sciences, Florida International University. Miami, Florida 33199 Abstract. This study of histocompatibility demonstrates that the gorgonian Swiftia exserta (Coelenterata, Antho- zoan) fulfills the minimal functional criteria of cytotox- icity, specificity, and altered secondary response (memory) that characterize an adaptive immune response. All au- tografts (self grafts) fused, and all allografts (intraspecific grafts) underwent rejection, which is characterized by rapid and progressive blanching, necrosis, and loss of tissue in the immediate contact area. Initial reactions required 7-9 days to produce 1 mm of necrosis, but after a resting period, a second contact at a new tissue area yielded the same reaction in 3-4 days. After primary sensitization, intervals of up to eight weeks still produced a significantly accelerated secondary response. Significant differences between the reaction times of second set and third party allografts demonstrated recognition specificity in these responses. Thus, this is the first report of an adaptive al- loimmune response in gorgonians. Introduction Most immunologists would agree that specificity and memory are the hallmarks of adaptive immune reactivity. According to some authors (Hildemann el ai, 1979), only the following functional criteria are necessary to define such immunological competence: ( 1 ) antagonistic or cy- totoxic reaction after sensitization; (2) selective or specific reactivity, and (3) inducible memory, i.e.. selectively al- tered (positive or negative) reactivity on secondary con- tact. Possession of a specific adaptive immune system, in- cluding immunorecognition leading to selectively induc- ible responses with a memory component, was, until re- cently, considered to be restricted to vertebrates (Klein, 1989). Invertebrate defense mechanisms, often associated Received 2 May 1990; accepted 1 April 1991. Abbreviations: RT reaction time, MRT mean reaction time. 1- first (primary) graft, 2 secondary graft, 3P third party graft. with phagocytosis and encapsulation, were thought to lack sharp specificity. We now know that at least some me- tazoan invertebrates, ranging from sponges to protochor- dates, possess a well-developed capacity for allogeneic recognition followed by incompatibility reactions (see Bigger. 1988). Some definitive studies have been carried out on in- vertebrates. Relatively short-term memories were found in the sponge Callyspongia dijfusa (4 weeks; Bigger et al., 1982) and the coral Montipora verrucosa (8 weeks; Hil- demann et ai, 1980a), but a longer term memory was reported for allograft rejection in the sea urchin, Lyte- chinus pictus (6 months; Coffaro, 1980). Specific immune memory, however, has not been found in all invertebrates even within the same class or phylum. For example, im- mune memory has been reported to be absent in some sponges (e.g.. Van de Vyver, 1980; Smith and Hildemann, 1984) and earthworms (Parry, 1978). Therefore, without testing, we cannot assume that the details of an immune response in one species are transferable to other members of the phylum. The gorgonians (Anthozoa; Alcyonaria) are an Order of soft corals that includes sea fans and sea whips. Theo- dor's extensive studies (Theodor, 1966, 1969, 1970, 1976; Serre and Theodor, 1967; Theodor and Carriere, 1975; Theodor and Senelar, 1975) of Eunicella stricta and Lo- phogorgia sarmentosa, and the preliminary work of Bigger and Runyan ( 1979) on Leptogorgia virgulata, Pseudopte- rogorgia elisabethae and Plexuraflexulosa, have identified some of the major characteristics of histocompatibility in the Order. Naturally occurring grafts within the same col- ony (autografts) occur in field, and all experimentally in- duced autografts rapidly fuse within 24 h. (Theodor, 1970; Bigger and Runyan, 1979). Rapid xenogeneic reactions (between two individuals of different species) were the focus of Theodor' s study of gorgonian histocompatibility; he hypothesized a system of "induced suicide" underlying graft rejection. The delay in the cytotoxic responses of 127 128 L. SALTER-CID AND C, H. BIGGER allografts (between two individuals of the same species) suggested, to Theodor, fundamental differences in the mechanisms operative in xenogeneic versus allogeneic cytotoxic incompatibilities. Gorgonian histoincompati- bility mechanisms have not been characterized at the cel- lular and molecular levels. As for immunocompetence, the gorgonians certainly fulfilled the first criterion of cy- totoxic alloincompatibility; but prior to this study, the parameters of rejection and other aspects, such as those of memory and specificity, had not been tested or were not found. The purpose of this project was to study tissue incom- patibility reactions between different colonies of the gor- gonian coral Swift ia exserta (Fig. 1). We asked whether the corals possess attributes of an adaptive immune system. Materials and Methods Animal colled ion and maintenance Swiftia exserta individuals were purchased from various sources and were collected in the waters off Southeast Florida, at depths of approximately 30 m. The animals were transported to Florida International University, where they were maintained in 25 or 100 gallon seawater aquaria with sub-gravel filters, and fed newly hatched Ar- lemia. All aquaria were subjected to alternating periods of 12 hours of light and dark. The water temperature was gen- erally maintained at 20-22C with aquarium heaters, al- though during the summer, the temperatures occasionally rose to 25C. Individual experiments were confined to a range of about 1 C or less. Salinity values ranged from 31 to 34 0/00. Because all treatments in an experiment were run concurrently in the same aquarium, all were subjected to the same general conditions. Although there were several colonies in each aquarium, they were not in contact. The animals were acclimated for at least 4 days before being placed in experimental contact. As opposed to many other gorgonians, Swiftia exserta lives well under laboratory conditions. Also, as we report in this study, its rapid allogenic reactions make it especially attractive for immunological studies. Techniques of grafting and scoring Small branches, 2-3 cm, were clipped from a gorgonian colony with surgical scissors and immediately placed in either allogeneic (intraspecific) or isogeneic (= autograft) combinations. To eliminate any influence of size on the reactions, all pairings involved tissue pieces of about the same size. The two pieces were gently placed in close con- tact without traumatizing the cell surfaces. We call this grafting. Unless otherwise stated, secondary grafts (2) were performed after a sensitization period of three days; i.e., the tissues were put in contact for three days and then separated for some given length of time before regrafting. A three-day period of sensitization was chosen to assure full maturation of the immune response; i.e., at this time cytotoxic reactions were underway in all graft pairs but not completed. A system of third-party grafting (3P) was used to test for specificity in the allograft response. It was performed like the secondary graft except that tissue from an allo- geneic animal not used in the sensitization was separately grafted with each of the test tissues for the second inter- action. A lack of specificity should result in all allogeneic tissue being treated the same, and that would be reflected in similar second set and third-party rejection times. Specificity would be demonstrated if the third party grafts were treated in a naive fashion. The coral branches were immobilized in small holders, each consisting of a 4 mm diameter X 5-6 cm length of glass tube bent into a "v" shape. The ends of the glass tube were covered with Nalgene 8000 plastic tubing (1/8"IDX I/ 16" wall) 1-1. 5 cm long. Small cuts (2 mm) were made in the plastic into which the coral branches were inserted (Fig. 2). A 2-3 mm long section of tissue was removed from the axial skeleton at the cut end of the branch that was inserted into the tubing; this operation precluded the necrosis that is induced when the tissue is pinched. Contact was between intact surfaces rather than the cut-ends of the branch. Tip to side, side to side, and tip to tip cytotoxic reactions were similar in direction and appearance, and paired /- tests between reaction times showed no significant differ- ences between the rates (data not shown). Therefore, the reaction is independent of the site chosen for pairing. In tip to tip, and some tip to side grafts, the support was unstable, and the individual pieces separated before the reaction endpoint was reached. No response would occur in such cases, because rejection seems to depend on un- broken physical contact. Because no significant influence of tissue locality was observed, side to side grafting was used in all the subsequent experiments and technical problems were minimized. After grafting, the individuals were examined daily un- der a dissecting microscope at 40X magnification. One o'' two responses occurred: either fusion complete gr together of the tissues of the two branches (Fi^ _\\ or rejection tissue necrosis of one or both individuals 2* the contact zone between the two colonies (Fig. 4). Cytoxic reactions were arbitrarily scored as definitive when soft tissue destruction extended 1 .0 mm or more to either side of an interface. The time required, in days, to reach that point was called the reaction time (RT). The reaction is easy to score for this species because the white necrotic tissue stands out in marked contrast to the orange GORGONIAN ALLOIMMUNITY 129 Figure 1. Colony of Swift ia exseria. Figure 2. Technique of pair grafting. Coral pieces cut from the same or separate colonies of S exseria are immobilized by the holders with their contact surfaces intimately opposed (between arrows). Figure 3. Intracolony autografts of 5. exseria showing compatible fusion at interface (arrows). Figure 4. Intracolony allografts of .5 exseria showing cytotoxic incompatibility restricted to the immediate contact zone. Note blanching and soft tissue death at interface (arrows) and exposed axial skeleton. color of the living soft tissues. Because as little as 0. 1 mm of necrosis can usually be discriminated, end points can be determined quantitatively. Data analysis Mean reaction times (MRT) and their standard devia- tions in days were determined. Paired /-tests were used to determine the significance of the differences between the experimental and control data because the animals, in both cases, were genetically identical. Results Pathology Grafts between parts of the same colony were always compatible. Soft tissues fused in the area of contact as early as one day after grafting. Neither tissue bleaching nor necrosis accompanied fusion in any of the ten pairs tested (Fig. 3). Compatible fusion persisted indefinitely. The fused branches could not be pulled apart easily; the tissues always tore before separating. Rejection of initial allografts was preceded by a lag of 3-5 days; during this time intimate tissue contact was required. Progressive allograft rejection occurred in the following sequence: (1) tissue blanching caused by the disappearance of pigmentation in the immediate contact zone; (2) loss of spicules from the tissues; and (3) tissue death eventually leading to exposure of the hard axial skeleton (Fig. 4). Intensified reactivity (more rapid and acute necrosis) was accompanied by secretion of mucus at the interface. On two occasions, overgrowth of one col- 130 L. SALTER-CID AND C. H. BIGGER Table I Timing of initial allosensitization or immune induction in Swiftia exserta Difference between Duration of initial No. of coral Range of indiv. 1st and 2nd allogeneic contact pairs tested Graft Mean reaction react, times reaction times (a) (days) (n) (b) time (days) (c) (days) (d) 1 45 ctl. test 8.3 0.5 8. 2 0.7 7-9 7-9 N.S. P > 0.4 2 45 ctl. 8.0 + 0.4 7-9 SlG />< 0.001 test 3.3 0.5 3-4 3 45 ctl. test 8.3 0.6 3.7 0.5 7-9 3-4 SlG P < 0.001 (a) Sensitization by first-set allografting was allowed for 1, 2, or 3 days at 22-24C; coral pairs were then each regrafted at a new interface 3 days later. (b) Concurrent controls (primary grafts) were performed with the same combinations as the test grafts. (c) Mean standard deviation of the mean. (d) N.S. not significant. SlG significant at the 0.1% level. Significance was determined by the paired /-test. ony by the other at one or both sides of the interface was observed; this overgrowth was concurrent with the usual necrosis. Parameters of graft response Reproducibility of reaction times. The first experimental set consisted of grafting 10 replicates of each of 10 allo- geneic combinations and 8 replicates of each of 10 iso- geneic combinations. The very similar reaction times (RTs), and standard deviations that were less than one day for each combination, justified our use of a single graft of each combination in all subsequent treatments (data not shown). Alloimnnme memory. In a series of 45 test pairs, control primary allografts yielded a mean reaction time of 8.3 0.6 days. Secondary allograft tests, established by re- setting the same coral pairs at new interfaces away from the original contact area following a three-day separation, showed accelerated and intensified cytotoxic reactions with a MRT of 3.7 0.5 days (Table I). The MRT dif- ference, with an interval of three days between first-set and second-set grafting, was highly significant (P < 0.001 ), indicative of immune memory. Intensified second-set reactivity was characterized by an earlier onset and stron- ger necrosis and secretion of mucus at the interfaces. Three days of presensitization were successful in elic- iting an accelerated second-set reaction. Possible earlier induction of memory was tested by disjoining initial al- lografts after one or two days of contact and regrafting the same pairs at new interfaces three days later (Ta- ble I). Coral pairs regrafted after only 1 day of allogeneic con- tact yielded a MRT of 8.2 days with a range of individual cytotoxic reaction times of 7-9 days (Table I). Thus, no significant difference was observed between the RTs of the control and test pairs. As shown in Table I, after 2 days of presensitization, all second-set grafts already dis- played accelerated activity with a MRT of 3.3 0.5 days. Paired /-tests showed that the difference between the pri- mary and secondary RTs was significant (P < 0.001). Two days of tissue contact were sufficient to elicit ac- celerated allograft responses. Longer sensitization periods did not improve the response time, and the sensitization appeared to be "all or nothing" rather than a gradual transition. To test for specificity, 90 pairs of third-party grafts were performed. The protocol is shown in Figure 5. Rather than responding in a purely naive or sensitized manner, these third-party grafts yielded a broad range of cytotoxic reaction times reflecting a distribution of accelerated to non-accelerated responses as is shown by the range of individual reaction times of 4-9 days (Table II). The re- action time of third-party grafts (mean 5.9 days) was sig- nificantly different from both first and second-set graft reaction times (8.2 days and 3.5 days respectively. Ta- ble II). To test for long-term memory, groups of grafts were reset at new interfaces at 2, 4, 6, and 8 weeks after sepa- ration from primary contact. The resulting MRTs, stan- dard deviations, and the range of individual cytotoxic re- action times, are given in Table III. Strong alloimmune memory was present in the second-set group grafted 2 weeks after first-set separation, as shown by an early MRT of 4.1 days 0.5 days and a significant difference (P < 0.001) between the primary and secondary RTs. This sensitized state was still present after 4 weeks, with an MRT of 3.6 0.6 days. At 6 weeks after first-set sepa- GORGONIAN ALLOIMMUNITY 131 ration, the accelerated reaction started to fade, as is shown by the MRT of 6.4 and the individual reaction times of 5-8 days, but is still significant (P < 0.001 ). This fading of alloimmune memory was still more accentuated at the 8-weeks period, with a MRT of 6.9 0.9 and an individual range of 6-9 days. Although there is some degree of over- lapping in the values of the control and test grafts from the 8-week group, the difference between the primary and secondary RTs is still significant (P < 0.00 1 ); the response gets progressively weaker in all combinations approaching the timing of a naive response. Discussion This study demonstrates that the anthozoan gorgonian Swiftia exserta displays: (a) cytotoxic reactivity accom- panying allogeneic incompatibility, (b) early and vigorous primary allograft rejection, and (c) selective alloimmunity with a specific memory component. Thus, S 1 . exserta ful- fills the three criteria for an adaptive immunological re- sponse, described by Hildemann ct al. ( 1979). Among other coelenterates, allogeneic incompatibilities have been documented for hydrozoans (e.g., Ivker, 1972; Buss et a!.. 1984) and anthozoans, including gorgonians (e.g., Theodor, 1970), sea anemones (e.g.. Bigger, 1980), and scleractinian corals (e.g., Hildemann et al., 1977). Except for the coral Montipora vcmicosa (Hildemann et ul.. 1980a), however, no substantial data bearing on the existence or persistence of immune memory in this phy- lum has heretofore been available. In the gorgonian coral, Swiftia exserta, autografts fuse compatibly, but allografts were invariably incompatible. The pattern of necrosis (Fig. 4) suggests that the response is triggered by a cellular process rather than a diffusible factor; i.e., because tissue destruction was limited to the graft interface, either cell contact or a very short-range cytotoxic molecule was responsible for the cytotoxic re- sponse. Swiftia exserta is an attractive model for tissue Grafting Protocol First set Second set together for 3 days graft on day six 00 @ separated for 3 days regraft at new interface with naive partner Third Party Third Party Controls B together for 3 days separated for 3 days graft on day six regraft at new interface with third party Figure 5. Experimental protocols used in the specificity study (Table II). A, B, and C stand for naive tissue from colonies (clones). A, B, and C. A' and B' denote presensitized individuals from the same respective colonies. 1 "first-set; 2 second-set; 3P third party; ctl third-party controls. 132 L. SALTER-CID AND C. H. BIGGER Table II Specificity oj allograft reaction limes in Swiftia exserta Range of No. of coral individual pairs tested Mean reaction reaction times Grafts (a) (n) time (b) (days) (days) First set 45 8. 2 0.6 7-9 (c) Second set 90 3.5 0.4 3-4 (c) Third party 90 5.9 1.5 4-9 (c) Control 90 8.3 0.6 7-9 (c) (a) Concurrent controls were performed with the same combinations as the third party grafts. (b) Mean standard deviations of the mean. (c) The difference between second-set and third-party (3P) graft re- sponses and the difference between 3P and control graft responses are both highly significant (P < 0.001. paired I test). Experimental temperature range: 21-24C. grafting, because the responses are easily scored and the rapid rates of rejection (primary MRTs of about 7 to 10 days) are closer to those observed in mammalian trans- plantation than in the coral M. verrucosa (MRTs of about 18 to 22 days, Hildemann el a/., 1980a). As Burnet (1969) pointed out, recognition of self/not- self is the cornerstone of immunological recognition. It has been suggested that while vertebrates recognize not- self, invertebrate responses are based on recognition of self (e.g., Burnet, 197 1 ). Although S. exserta shows a range of reaction times and severity, the results from a given combination of colonies were highly reproducible. This pattern of differing responses between different allogeneic combinations and the specificity associated with memory, i.e., that different allogeneic "not-self" elicits different re- sponses, supports the opposite hypothesis, that allogeneic rejection is based on a recognition of "not-self" (see Big- ger, 1988; Neigel, 1988). Among 1479 alloparabiotic combinations of the gorgonian Eunicella stricta collected in the Banyuls-sur-mer (France) area, Theodor (1976) ob- served only 1 1 or 0.7% compatible fusions, whereas the remaining 99.3% exhibited rejection reactions. Even the small number of compatible gorgonians could have orig- inated from the same clone, because all the specimens were collected from the same area. That impressive vari- ation of histocompatibility markers, as well as that found between the 10 colonies employed in the present study, suggest that each separate clone or colony has a unique array of alloimmunorecognition molecules, as predicted by the concept of the "uniqueness of the individual" de- veloped by Medawar (e.g., Jokiel el ai, 1983). The early acquisition of immune memory in other cor- als (Hildemann et a/.. 1980a) seemed to be a gradual or quantitative process, with accelerated reactivity present after 2 to 4 days of contact and maximal presensitization developed after 4 to 8 days; that activation or positive memory then persisted or diminished only slightly after prolonged primary contact. In S. exserta, because there is apparently no significant difference between the MRTs of the secondary responses elicited after 2 or 3 days of primary contact (3.3 and 3.7 d respectively. Table I), it seems to be an all or none process occurring at approxi- mately 2 days. The present study demonstrates that significant short- term alloimmune memory persists for at least 8 weeks. Table III Diiralinn i>l Swiftia exserta alloimmune memory Time interval between 1st sensitization and 2nd graft (weeks) No. of coral pairs tested (n) Graft (a) Mean reaction time (b) (days) Range of indiv. react, times (days) Significance of the difference between the primary and secondary reaction times (c) 3 43 ctl. test 8.1 1.0 4. 1 0.5 7-10 3-4 SIG 4 45 ctl. test 7.9 0.8 3.6 0.6 7-9 3-5 SIG 6 42 ctl. test 7.8 0.8 6.4 1.0 7-9 5-8 SIG 8 39 ctl. test 7.9 0.8 6.9 0.9 7-9 6-9 SIG (a) Concurrent controls (primary grafts) were performed with the same combination as the test grafts. (b) Mean standard deviation ot the mean. (c) Significance was determined by the paired /-test and in all cases represents P < 0.001. GORGONIAN ALLOIMMUNITY 133 but starts to fade at 6 weeks in the gorgonian coral 5". exserta (Table III). As with similar short-term immune memory in the sponge C diffma (Bigger ct a/.. 1982), annelids (Dales, 1978), and echinoderms (Coffaro, 1980), this appears to constitute a major difference from the long- term alloimmune memory found in mammals (Hilde- mann, 1984). In mice and rats, immune memory de- monstratable by accelerated rejection of test skin allografts resides in lymphocytes and persists more than one year after sensitization (Billingham et ai, 1963). It is important to note however, that in this study, no tests were performed after prolonged sensitization (> 3 days) and that there was no investigation of environmental factors such as temperature, light, and salinity, which may affect the ef- fectiveness of sensitization and the cytotoxic reaction times (e.g., Johnston et ai. 1981). Further investigation of conditions that might favor long-term memory is de- sirable, because the absence of this characteristic could be a major distinguishing feature of invertebrates. Memory that lasts only weeks (Bigger ct ai, 1982), rather than months or years (Billingham ct ai. 1963), could have sev- eral causes. This immunologic feature in sponges and corals may be the result of shorter-lived memory residing in immunocytes with commensurate life spans (Hilde- mann et ai. 1980b). For example, the sensitized immu- nocytes may be replaced by naive cells. Alternatively, the molecules in which memory is imprinted may become inactive with time or with immunocyte division. Killer cells or molecules involved with memory have yet to be identified as a special subset in any coelenterate (see Bigger and Hildemann, 1982). Thus, these studies of memory must await further progress. Only 27% of the third-party individuals reacted as fast as the slowest of the second-set allografts, while the re- mainder responded in a similar fashion to primary grafts or at intermediate times, demonstrating a specificity in- volved in the secondary response. These results contrast with the ones obtained by Hildemann et nl. (1980a) in M. verntcosa. A bimodal distribution of third-party re- action times such that approximately 66% of the third- party individuals reacted as fast as the second-sets led them to suggest the occurrence of cross reactivity. The present study presents a different situation; third-party reaction times were not bimodal and, while there is a demonstrable specificity, an allogeneicly non-specific effect appears to be manifested in the third-party allografts. Alternatively, another explanation is that the accelerated responses of some third-party individuals could be the result of the sharing of a limited repertoire at minor histocompatibility loci (locus), which might exist together with a not-shared, highly polymorphic MHC. However, there is no genetic data that pertains. Why third-party grafts reacted in a sig- nificantly different way from primary as well as secondary grafts must await further investigation. This study supports the hypothesis (e.g., Burnet, 1976) that invertebrates may be capable of anticipatory immune responses, as opposed to suggestions (Klein, 1989) that, whereas vertebrate immune responses are based on rec- ognition of not-self, invertebrate responses are not antic- ipatory and are non-specific. Unfortunately, the study of immunity in invertebrates has been restricted so far to very few animals, and most of these have been investigated in little detail; therefore, many questions remain unan- swered. Swift ia exserta provides a very good model in which to study these invertebrate defense mechanisms, which need clarification. Acknowledgments Thanks are due to Ms. Cecile Olano for providing pho- tographic assistance, Ms. Lois Bigger for editorial assis- tance, and anonymous reviewers for their helpful com- ments. Support was provided by NIH grant RR08205. Literature Cited Bigger, C. H. 1980. Interspecific and intraspecific acrorhagial aggressive behavior among sea anemones, a recognition of self and not-self. Biol. Bull 159: 117-134. Bigger, C. H. 1988. Historecognition and immunocompetence in se- lected marine invertebrates. Pp. 55-65 in Invertebrate Historecog- nition. R. Grosberg. D. Hedgecock, and K. Nelson, eds. Plenum Press, New York. Bigger, C. H., and VV. H. Hildemann. 1982. Cellular defense systems of the coelenterata. Pp. 59-87 in The Reticuloendothelial System. N. Cohen and M. M. Sigel. eds. Plenum Publishing Corp.. New York. Bigger, C. H., P. L. Jokiel, VV. H. Hildemann, and I. S. Johnston. 1982. Characterization of alloimmune memory in a sponge. J Im- inunoi 129: 1570-1572. Bigger, C. H., and R. Runyan. 1979. An in situ demonstration of self- recognition in gorgonians. Dev. Comp. Immunol. 3: 591-594. Billingham, R. E., VV. K. Silvers, and D. B. Wilson. 1963. Further studies on adaptive transfer of sensitivity to skin homografts. J. Exp. .\fecl 118: 397-401. Burnet, F. M. 1969. Self and Not-Self: Cellular Immunology, Book L Melbourne and Cambridge University Presses. Carlton, Victoria, and London. Burnet, F. M. 1971. "Self-recognition" in colonial marine forms and flowering plants in relation to the evolution of immunity. Suture 232: 230-235. Burnet, F. M. 1976. The evolution of receptors and recognition in the immune system. Pp. 35-58 in Receptors and Recognition I. P. Cua- trecasa and M. F. Greaves, eds. Chapman and Hall, London. Buss, L. W., C. S. McFadden, and D. R. Keene. 1984. Biology of hy- dractmid hydroids. 2. Histocompatibility effector system/competitive mechanism mediated by nematocyst discharge. Biol. Bull. 157: 139- 158. Coffaro, K. A. 1980. Memory and specificity in the sea urchin l.yte- chimts pictus. Pp. 77-88 in Phytogeny of Immunological Memory. M. J. Manning, ed. Elsevier/North Holland, New York. Dales, R. P. 1978. The basis of graft rejection in the earthworms Lum- hrictis terrestris and Eisema foetida. J. Inverlebr. Pathol. 32: 264- 268. Hildemann, \V. H. 1984. A question of memory. De\. Comp. Immunol. 8: 747-756. 134 L. SALTER-CID AND C. H. BIGGER Hildemann, VV. H., C. H. Bigger, and I. S. Johnston. 1979. Histoincompatibility reactions and allogeneic polymorphism among invertebrates. Transplant Proc 11:11 36- 1141. Hildemann, VV . H., P. L. Jokiel. C. H. Bigger, and I. S. Johnston. 1980a. Allogeneic polymorphism and alloimmune memory in the coral Mimtipora verrucosa. Transplantation 30: 277-301. Mildemann, VV. H., C. H. Bigger. P. L. Jokiel, and I. S. Johnston. 1980b. Characteristics of immune memory in invertebrates. Pp. 9- 14 in Phytogeny oflmmunological Memory, M. J. Manning, ed. El- sevier/North Holland, New York. Hildemann, W. H., R. L. Raison, G. Cheung, C. J. Hull, C. Akabe, and J. Okanoto. 1977. Immunological specificity and memory in a scleractinian coral. Nature 270: 219-223. Ivker, F. B. 1972. A hierarchy of histoincompatibility in Hydractinia cchiuaui Biol. Bull 143: 162-174. Johnston, I. S., P. L. Jokiel, C. H. Bigger, and VV. H. Hildemann. 1981. The influence of temperature on the kinetics of allogralt re- actions in a tropical sponge and a reef coral. Biol. Bull 160: 280- 291. Jokiel, P. L., W. H. Hildemann, and C. H. Bigger. 1983. Clonal pop- ulation structure of two sympatric species of the reef coral Monlipora Bull Mar. Sci. 33: 181-187. Klein, J. 1989. Are invertebrates capable of anticipatory immune re- sponses? Scand. J Inununol 29: 499-505. Neigel, J. E. 1988. Recognition of self or non-self? Theoretical impli- cations and an imperical test. Pp. 127-142 in Invertebrate Histore- cognition, R. Grosberg, D. Hedgecock. and K. Nelson, eds. Plenum Press. New York. Parry, M. J. 1978. Survival of body wall autografts. allografts and xenografts in the earthworm Eisenia foetida. J. Invertebr. Pathol. 31: 383-392. Serre, A., and J. I heodor. 1967. Mise en evidence d'une reconnaissence immunologique de tisus chez un invertebre. G R Acad. Sc. Paris. 264: 513-514. Smith, L. C., and VV. II. Hildemann. 1984. Alloimmune memory is absent in Hymeniacidon sinapium, a marine sponge. J. Immunol. 133: 2351-2355. Theodor, J. 1966. Contribution a I'etude des Gorgones (V) les greffs chez les gorgones: etude d'un systeme de reconnaissence du tissus. Bull. In.il. Oceanogr. Monaco. 66: 1-7. Theodor, J. 1969. Histotoxicite in vivo el in vitro entre tissus xeno- geniqus et entre tissus allogenipus chez un invertebre. C R Acad. Sc. Pans 268: 2534-2535. Theodor, J. 1970. Distinction between "self" and "not-self" in lower invertebrates. Nature. 227: 690-692. Theodor, J. 1976. Histoincompatibility in a natural population ofgor- gonians. Zoo/. J. Linnean Soc 58: 173-176. Theodor, J., and J. Carriere. 1975. Direct evidence of heterolysis of gorgonian target cells. Pp. 101-104 in Immunologic Phytogeny, W. H. Hildemann and A. A. Benedict, eds. Plenum Press, New York. Theodor, J., and R. Senelar. 1975. Cytotoxic interaction between gor- gonian explants: mode of action. Cell. Immunol. 19: 194-200. Van de Vyver, G. 1980. Second-set allogralt rejection in two sponge species and the problem of an alloimmune memory. Pp. 15-25 in PliylKCuy ol Immunological Memory, M. J. Manning, ed. Elsevier/ North Holland. New York. Reference: Bwl Bull 181: 135-143. (August, 1991) Restriction Fragment Length Polymorphism Analysis Reveals High Levels of Genetic Divergence Among the Light Organ Symbionts of Flashlight Fish CONNIE J. WOLFE AND MARGO G. HAYGOOD Scripps Institution of Oceanography, University of California, San Diego. California 92093 Abstract. Restriction fragment length polymorphisms within the lu.\ and 1 6S ribosomal RNA gene regions were used to compare unculturable bacterial light organ sym- bionts of several anomalopid fish species. The method of Nei and Li (1979) was used to calculate phylogenetic dis- tance from the patterns of restriction fragment lengths of the lux A. and 16S rRNA regions. Phylogenetic trees con- structed from each distance matrix (luxA. and 16S rDNA data) have similar branching orders. The levels of diver- gence among the symbionts, relative to other culturable luminous bacteria, suggests that the symbionts differ at the level of species among host fish genera. Symbiont re- latedness and host geographic location do not seem to be correlated, and the symbionts do not appear to be strains of common, free-living, luminous bacteria. In addition, the small number of hybridizing fragments within the 16S rRNA region of the symbionts, compared with that of the free-living species, suggests a decrease in copy number of rRNA operons relative to free-living species. At this level of investigation, the symbiont phylogeny is consistent with the proposed phylogeny of the host fish family and suggests that each symbiont strain coevolved with its host fish spe- cies. Introduction Members of the teleost fish family Anomalopidae har- bor luminous bacterial symbionts in specialized light or- gans, located under each eye. Light is produced contin- uously by the bacteria through the action of bacterial lu- ciferase; but depending on the species, the light may be occluded, either by rotation of the entire light organ, or by a shutter mechanism (Kessel, 1977; Leisman el a!., 1980; Johnson and Rosenblatt, 1988). The four genera of Received 20 November 1990: accepted 18 March 1991. anomalopid fishes contain five species: Anomalops katop- tron, Photoblepharon palpebratus, Photoblepharon stein- il:i, Kryptophanaron alfredi, and Phthanophaneron har- vevi. Evidence from comparative morphology and bio- geography suggests the following familial phylogeny (Johnson and Rosenblatt, 1988). The anomalopids appear to have branched off of the Trachithyid lineage during the Cretaceous period. The genus Anomalops probably diverged later in the Cretaceous and is radically different from the other genera. Phthanophaneron diverged in the Miocene with Kryptophanaron. and Photoblepharon di- verged most recently, in the Pliocene. Luminous bacterial symbionts in several other families of fishes have been cultured. In these instances, a single species of symbiont is associated with an entire family of host fish. These bacteria belong to species that have also been isolated directly from seawater, and the juvenile host may acquire its symbionts from the surrounding water at each generation (Ruby and Nealson, 1976; Fitzgerald, 1977; Reichelt el al. 1977; Leis and Bullock, 1986; Wei and Young, 1989). Other light organ symbioses, in cer- atioid fishes and pyrosomes, contain unculturable sym- bionts, and the identity of the symbionts is unknown in those cases. The anomalopid symbionts have, as yet, defied all of our attempts to culture them in laboratory media. Our inability to culture the symbionts, combined with the extensive morphological adaptations of the host fish, suggest an obligate symbiosis in which the bacteria may have coevolved with the fish host. We have compared the symbionts in four species of anomalopid fish by analyzing restriction fragment length polymorphisms (RFLPs) of fragments containing either the luciferase structural genes (/j/.vAB), or the 16S ribo- somal RNA genes. RFLPs of other structural genes have frequently been used to compare species and populations of bacteria (Stanley el al.. 1985; Franche and Cohen-Ba- 135 136 C. J. WOLFE AND M. G. HAYGOOD zire, 1987; Denny el a!., 1988; Lindblad el at., 1989). Ribosomal RNA genes are very conserved, and compar- isons of rRNA gene restriction patterns had previously been used to differentiate between species, as well as sub- species, of bacteria (Gottlieb and Rudner, 1985; Grimont and Grimont, 1986; Saunders et a/., 1988; Stull el a/., 1988; Buyser cl al, 1989). Our goal was to analyze both gene regions to determine: ( 1 ) whether the symbionts are the same species in all of the anomalopids or are specific to fish species, genus, or geographic location; (2) the in- terrelationships of the symbionts compared with the phy- logeny of the host fish, to support or refute the coevolution hypothesis; and (3) the broader phylogenetic affiliations of the symbionts within the luminous bacteria. Materials and Methods Bacterial strains Bacterial strains used in this study are described in Table I. The free-living luminescent strains were grown in com- plex seawater broth (SWC: Ruby and Nealson, 1978) at 25C. Fish collection Fish were hand-collected by SCUBA divers in the lo- cations shown in Table I. Samples of four species of fish were obtained, including two species (.-1. katoptron and P. palpebratus) from two genera collected at the same location in Papua New Guinea. Both specimens of Pho- toblepharon from Papua New Guinea would be identified as P. palpebratus on the criteria given by McCosker and Rosenblatt (1986). The two specimens are similar in mer- istics and overall morphology, but differ in coloration. P. palpebratus # 1 is dark, has the white area on the opercle characteristic of the species, and the white reflecting scales and fin edgings shared with P. steinitii. P. palpebratus #2 is grey, and lacks contrasted white areas on the body and fins (R. H. Rosenblatt, pers. comm.). The P. steinitzi specimen was obtained at Eilat, Israel, and is thus virtually certainly P. steinitii, although the specimen is no longer available for positive identification. DNA preparation Samples of DNA were prepared from single, entire, fish light organs as in Haygood and Cohn (1986). Total DNA yield per light organ ranged from 5 to 63 ^g of DNA. DNA from the other luminous bacterial strains was iso- lated by the method of Ditta et al. (1980). DNA concen- tration was determined spectrophotometrically by absor- bance at 260 nm. DNA digestion, electrophoresis, and hybridization DNA samples were digested with the following en- zymes: Hpal Seal Bglll, Kpnl, Pst\, C/al, Hindi and Sphl for lu\ RFLP analysis; and EcoRl, Bglll, Hindlll and Neil for the 16S rRNA gene comparison. Each digest consisted of about 200 ng of DNA. The digests were sep- arated by 0.8% agarose gel electrophoresis in 40 mA/ Tris acetate/ 1 mA/ EDTA pH 8.0. The agarose gels were dried at 60C, denatured, neutralized (Tsao et al., 1983), and prehybridized at 60C in 6 X SSC (sodium chloride/so- dium citrate; Maniatis et al.. 1982), 2.5 mA/ EDTA. 200 yug/ml denatured salmon sperm DNA and 0.5 mg/ml dried milk. In previous work, the A', alfredi symbiont lux region was cloned into pDR720 (Haygood and Cohn, 1986). A 1.73 kb Hpa\/Kpn\ fragment containing ln.\A and half of Table I Origins ol D.\.l .si/m/>/c.v Bacterial species Strain Host Collection site Source or reference 'ibrio harvcyi B392 NA Unknown Reichelt and Baumann. 1973 'ibno han-evi BB7 NA Sargasso Sea Belasrt /.. 1984 'ibrio (irieittalix #1 ATCC #33933 NA Yellow Sea Yang ei al.. 1983 'ihrin iirientalt\ #2 ATCC #33934 NA Yellow Sea Yangrtu/., 1983 'ibnn lichen B6I NA 2030' latitude, 15730' longitude Baumann et al., 1971 'ihrin /Lichen MJ1 Monocentris japonicus 50 miles southeast of Tokyo, Japan Ruby and Nealson. 1976 "hotobacterium phosphoreum NZ1 ID Ne:nmiu iiei/ualix 2842'N, 138'W: 906 to 936 m depth Ruby and Morin, 1978 MA NA Kyptophanaron allmli #1 La Parguera. Puerto Rico Haygood and Cohn, 1986 MA NA Kryptophanaron alfredi #16 Roatan, Honduras This study MA NA t>it>inu/i>i>\ katoptnm #1 Port Moresby, Papua New Guinea This study MA NA Inomalops kaptoptron #2 Madang, Papua New Guinea This study MA NA Photoblepharon palpebratus #1 Port Moresby. Papua New Guinea This study MA NA Photoblepharon palpchiaius #2 Port Moresby, Papua New Guinea This study SIA NA Photoblepharon MCI nil :i Eilat. Israel This study NA, not applicable. FLASHLIGHT FISH SYMBIONT RFLPS 137 Table II Pairwise comparisons of the percentage conserved fragments and the genetic distances (see Materials and Methods) within the lux gene regions <>/ Vibrio harveyi and the anomalopid symbionts Strain K han-cvi 1 kill apt mn A', altredi P. palpebratus P steimtzi Strain B392 #1 #\ #2 I 'ibrio liarvevi B392 16.0 >17.8 >16.0 >16.8 Anomalops ka/optron #1 8.3 >16.8 >14.7 >15.7 Kyplophanaron alfredi #1 <6.5 <7.4 12.0 17.6 Photoblepharon palpebratus #2 <8.3 <10.0 14.8 10.9 P steinii-i <7.4 <8.7 6.7 17.4 The lower values are the percentage of conserved fragments and the upper values the estimated genetic distances (multiplied by 100). In cases where no fragments were conserved, values are listed as being less than the minimum conservation which can be calculated from the total number of observed fragments or greater than the minimum calculable genetic distance. lu.\E was isolated from the plasmid by 0.8% low melting point agarose electrophoresis. Plasmid pNOl 30 1 (Course el al. 1982; Jinks-Robertson el al, 1983) carrying rRNA genes of Esclie richia colt was digested with Hindlll, and a 568 bp fragment within the 16S rRNA gene was isolated on a 0.8% low melting point agarose gel. The fragments were cut out of the gel, the gel slices were melted, and this material was used directly with the Pharmacia random priming kit to obtain [a- 32 P]dCTP labeled probe. Probes were added to each prehybridized gel and incubated for 16 h at 60C. After hybridization, the gels were washed in 1 X SSC, 0.1% SDS at 60C, and exposed to X-ray film. Each gel was exposed for several different intervals to ensure the detection of all hybridization fragments. Control experiments indicated that there was no hybrid- ization of the lu.\ probe to E. colt genomic DNA, or of the E. co/i 16S probe to flashlight fish genomic DNA under these conditions. Estimation of sequence divergence An estimation of sequence divergence (number of nu- cleotide substitutions per site) was calculated from the fraction of conserved hybridizing fragments for each pair of DNAs (Nei and Li, 1979; Nei, 1987). This estimation is applicable to closely related DNA sequences and is based on the assumption that fragment changes occur due to base substitutions within the restriction sites, rather than to major deletions or rearrangements. The sequence di- vergence within and surrounding the lu.\A. gene was es- timated for the four symbiont species and Vibrio harveyi by comparing the hybridization patterns obtained from restriction digests hybridized to the A", alfredi symbiont lux probe. All of the restriction enzymes used in the lux analysis recognized 6 bp; thus the data from all digests were additive and analyzed together (Nei and Li, 1979; Nei, 1987). Divergence within and around the 16S rRNA genes of the symbionts and several strains of free-living luminous bacteria was determined by analysis of common hybridizing fragments to the 568 bp probe. The 1 6S rRNA data from Hindlll, Bg/ll, and EcoRl enzyme digestions were additive and were analyzed together; the data from Neil digestion were analyzed separately because Neil rec- ognizes 5 bp rather than 6 (Nei and Li, 1979; Nei, 1987). Data for each enzyme within both analyses were also an- alyzed individually and compared with the additive data set. When no common hybridizing fragments were ob- served between a pair, the fraction of conserved hybrid- izing fragments was given as less than the smallest cal- culable fraction, and the genetic distance between the pair was given as greater than the maximum calculable dis- tance. All resulting distance matrices were analyzed using the Fitch-Margoliash method to form trees [program FITCH, from the Phylogenetic Inference Package (PHY- LIP) version 3.2, by Dr. J. Felsenstein]. Each group of tree files was read by the CONSENSE program (also from PHYLIP), and consensus trees were generated and com- pared. Results Comparison of RFLPs within the ItixAB gene region The percentage of conserved fragments among I ". har- veyi and the various anomalopid symbionts was calculated from pair-wise comparisons of fractions of conserved hy- bridizing fragments (Table II, lower left). The A", alfredi symbiont lu.\ probe hybridized to restriction fragments of each of the symbionts, but the hybridization patterns showed little conservation (Fig. 1). In fact, no enzyme of the eight tested produced a hybridization fragment con- served among all of the symbionts. Three of the enzymes (Bglll, Hindi and Hpal) revealed no conserved hybrid- izing fragments between the symbionts, or between any symbiont and I', harveyi B392. The Photoblepharon pal- pebratus and P. steinitii symbionts ( P. p. # 1 and P. s. # 1 ) 138 C. J. WOLFE AND M. G. HAYGOOD Kpn\ 12345 Hpa\ 12345 Bgl\\ 12345 kb 23.13 9.42 6.68 4.36- Kigure I. Autoradiogram obtained from hybridization of the 1! P- labeled 1.73 kb /H.V probe to DNA extracted from I'ihrio harveyi B392 ( 1 ) and the light organs of Anomafaps katoptnm #1 (2), Kryptophanaron alfrcdi #1 (3). Pholohlcpharon palpehratus #2 (4), and PholMepharon xlcinil:i (5). DNA was digested with Kpn\. Hpa\ and B,?/II. are most similar, with a distance of 0.109. The A. katop- tron specimen has no conserved hybridizing fragments with any of the other anomalopid symbionts; thus the calculated distances are underestimates of an unknown distance. The divergence (d) among symbionts and be- tween symbionts and I". han>eyi ranges from 0.109 to >(). 1 78 when calculated with Nei and Li's method (Table II, upper right). Values for (/have been multiplied by 100 in Table II, to facilitate easy visual comparison. The con- sensus tree is shown in Figure 2. Comparison ofRFLPs detected by the 16S rRNA gene probe In DNA digests of every bacterial strain tested, the 568 bp 16S rRNA gene probe hybridized with between 1 and 12 restriction fragments generated by enzymes with six- base-pair recognition sequences (Fig. 3). Digestion of the 1 '. liarveyi strains generated the highest number of hy- bridizing fragments (~9/digest), while digestion of the symbiont DNAs, particularly from Anomalops and Kryp- loplumaron, resulted in fewer hybridizing fragments (~2/ digest). In both the lux and 16S comparisons, the number of hybridizing fragments indicates that a single symbiont inhabits each light organ or that any additional symbionts are present at undetectable levels. The number of hybrid- izing fragments in the lux comparisons are consistent with the presence of a single copy of the lux genes, in both the symbionts and the culturable strain. In the 16S compar- isons, the low number of symbiont fragments indicates that a mixed symbiont culture is unlikely. As shown by the fractions of conserved hybridizing fragments (Table III. lower left), the 16S probe generally yielded a higher number of conserved fragments than the lux probe. Dis- tances calculated from hybridization patterns among cul- turable species range from 0.062 to 0.216, with a mean of 0.1 13 0.040 (the standard deviation was calculated from values in Table III). As in Table II, the d values shown in Table III have been multiplied by 100 to ease visual comparison. The symbionts differ from the cultur- able species with distances ranging from 0.099 to >0. 1 80. Distances among symbionts from fish of different genera range from >0. 1 17 to >0. 151, contrasting strongly with the level of difference seen between strains of culturable species (0.029 0.02 1 ). Thus, the symbionts in this family do not appear to be members of a single species. In two instances, a strain-level relationship was seen between symbionts. The two specimens of A. katoptron were indistinguishable, and distance values for symbiont DNAs from P. steinitzi and P. palpebratus #1 are also close, with a calculated distance of 0.023. Thus, because these values are within the range that we have seen when comparing strains of the same species of Vibrio (Table III), these two symbiont strains should be considered as ^Vibrio harveyi B392 Figure 2. Phylogenetic tree for anomalopid symbiont strains and I 'ibrio harveyi constructed from the lux region genetic distance data in Table II. The tree is a majority rule consensus tree derived from the CONSENSE program of PHYLIP (see Materials and Methods). Branch lengths were obtained from the data in Table II using the FITCH program of PHYLIP. The arrow indicates that the following branch lengths are minimum estimates: there were few or no conserved hybridizing frag- ments between this group of strains and the others compared. FLASHLIGHT FISH SYMBIONT RFLPS EcoR! Sg/ll H/ndlll 12345678910111213 12345678910111213 12345678910111213 139 21 .23- kb -21.23 Figure 3. Autoradiogram obtained from hybridization of the 32 P-labeled 16S rRNA gene probe to DNA extracted from the light organs of Krypti iphanaron allrcdi #16(1). Anomalops kaloplron #2(2). Anomalops katoptron #1 (3), Photoblepharon palpebratus #2 (4), Photoblepharon palpebratus #\ (5). Photoblepharon steinit:i (6), and from laboratory cultures of I 'ibrio tischcri B61 (7), Vibrio fischeri MJ1 (8), I 'ibrio oricnhili\ #2 (9). I 'ibrio orienialis #\ ( 10). I 'ibrio Imn-eyi BB7 ( 1 1 ), and I 'ibrio han-eyi B392 (12). DNA was digested with f<)RI. %/II and Hind\\\. Molecular weight markers are the same for both EcoR] and fi.tf/II. belonging to the same species. All of the Photoblephanm symbionts(P. p. #1, P. p. #2, and P. s. #1) are more similar to each other than to A", alfredi and A. katoptron. However, two samples from different individuals of P. palpebratus (P. p. #1 and P. p. #2) collected in the same geographic location showed considerable differences in their restric- tion patterns. Moreover, one P. palpebratus sample (P. p. #1) is more closely related to the sample of P. steinitzi than to the other P. palpebratus sample (P. p. #2). The degree of difference between the symbionts of the P. pal- pebratus specimens is even more anomalous when com- pared to the total conservation of restriction fragments from the DNA of two Anomalops specimens from differ- ent geographic locations. The possibility of inadvertent exchange of samples was investigated and it does not ap- pear to have occurred; the experiments were repeated with 140 C. J. WOLFE AND M. G. HAVGOOD <,-- VO "5 % '< ON p p OO od \C * v-> in p ON ON O v v o o V V V r ! *"""": "! ~T ~ . 'J "^ *-? "!""' v~> ~ O O o-' rn -d r-' o O O V V V ON o od od r-j N so" r-' r--' od < ": ""; P "* P "O P P ^ , r*- ( . _ ^ C il , si .- a Ef o o^ 2 i! s J i 3 F i 00 13 = e ^ -c s r 1 S! S \f: Si S '-< t/i -o identical results. The consensus tree generated is shown in Figure 4. Discussion Our data indicate that the bacterial symbionts differ dramatically within the family Anomalopidae. Based on RFLP analysis within the /.v gene region, the symbionts from different genera are almost as different from one another as the Kryptophanaron symbiont is from I', har- veyi. Comparison of the symbiont restriction patterns with those of known species of luminous bacteria within the 16S rRNA gene region strongly supports the conclusion, drawn from the lux data, that the symbionts differ at greater than strain level between genera within the family Anomalopidae. In other light organ symbioses, in which the symbionts have been cultured, all members of the fish family contain the same bacterial species. RFLP analysis supports this assertion for Photobacterium phosphoreum, a bacterial species that inhabits light organs of several families of fishes. The genetic distances between three strains of P. phosphort'iim from two species of opisthoproctid fishes (Opisthoproctus gnnuildii and Opisthoproctus soleatus) and P. phosphoreum NZ 1 1 D, a symbiont from a macro- urid fish, were determined using the same 16S RFLP analysis method, and ranged from 0.002 to 0.006 (data not shown). The small genetic distances observed for the opisthoproctid symbionts contrast greatly with the large differences observed among the anomalopid symbionts. Clearly, the anomalopids do not appear to host a single species. Symbionts from different anomalopid genera dif- fer at least as much as species of culturable luminous bac- teria. In the 16S rRNA analysis, the observed distances between the anomalopid symbionts are considerably greater than the distance between I '. harveyi and ( '. orien- talis, approaching the distance between I '. harveyi and I ". fischeri, two highly divergent members of the genus I 'ibrio (Baumann el a/.. 1980). In contrast, there is a high level of conservation among strains of characterized Vibrio species, even among the free-living and monocentrid symbiont strains of I '. fischeri from different locations (strains B61 and MJ1, Table I and Fig. 4). Within the anomalopid family, equivalent strain levels of similarity are found only within genera, in two of the three samples from the genus Photoblepharon (P. p. #1 and P. s. #1) and the samples from different specimens of A. katoptmn. In addition, we recently collected several specimens of Kryptophanaron alfredi from Roatan, Hon- duras, and compared five light organ samples, using the 16S probe and the same restriction enzymes previously used in this study. The specimens were indistinguishable, with all fragments conserved. Thus, although we do not have additional samples of each fish species within the anomalopid family, it seems likely that symbionts within FLASHLIGHT FISH SYMBIONT RFLPS 141 P f^lfrbrolul fl 2 Figure 4. Phylogenetic tree for anomalopid symbionts and several free-living luminous bacteria constructed from the 16S rRNA gene region genetic distance data in Table III. The tree is a majority rule consensus tree derived from theCONSENSE program of PHYLIP. Branch lengths were obtained from the data in Table III using the FITCH program of PHYLIP. Arrows on two of the branches indicate that the branch lengths are minimum estimates; there were few or no conserved hybridizing fragments between these strains and the others compared. each host species comprise a single species. The anomalous result with P. palpebratus #2 is discussed below. The observed diversity among symbionts within the family Anomalopidae is not associated with geographical location, and this might be expected if the symbionts en- gage in genetic exchange with bacterial populations in the environment. DNA from specimens of P. palpebratus and A. katoptron collected from the same location in Papua New Guinea are no more similar to each other than each is to the K. alfredi symbiont from the Caribbean. In fact, they are considerably less similar than the symbionts from Photoblepharon specimens from the Red Sea and Papua New Guinea (P. p. #1 and P. s. #1 ). In addition, symbionts from two samples of A. katoptron, collected from different locations in Papua New Guinea (~800 miles apart) ap- pear identical within the 16S rRNA gene region. Thus, the genetic similarity of symbionts seems to correlate more with host phylogeny than geographic location. Precise comparisons of the symbiont branching order to the proposed evolutionary history of the host fish are difficult because the branch lengths are very rough esti- mates. However, the branching order, is similar in both analyses and is consistent with that of the fish host (John- son and Rosenblatt, 1988). These highly conservative es- timates of distance indicate that the Anomalops symbiont differs from the other symbionts at the specific, and pos- sibly the generic, level. Anomalops is very different from the other genera in the family Anomalopidae and is thought to have diverged from the ancestral lineage at least 60 million years before the divergence of Krypto- plmnawn and Photoblepharon (<5 million years ago). The Kryptophanaron symbiont also differs from the Photo- blepharon symbionts, but does share some conserved hy- bridizing fragments with Photoblepharon symbionts in both the lux and rRNA analyses. These data suggest that the symbionts may have coevolved with the host fish. However, the divergence between the two P. palpebratus samples raises the possibility that the apparent corre- spondence between symbiont and host trees could dis- appear when more samples are examined; i.e., that ex- treme divergence may occur in symbionts within host species as well as between host genera. However, this hy- pothesis is not supported by the identity observed between the two Anomalops samples from different locations, the similarity of P. palpebratus #1 and P. steinitzi, and our comparison of five K. alfredi samples. Equally likely al- ternatives are that ( 1 ) the genus Photoblepharon is more complex than current systematics would suggest, and that more than one type of Photoblepharon may occur at our collection site (see Materials and Methods) or (2) a gene rearrangement in the P. palpebratus #2 symbiont relative to the other anomalopid symbionts could give an over- estimate of genetic distance. Unfortunately, the additional specimens needed to resolve this anomaly are not cur- rently available and will be difficult to obtain. Further study of both the systematics of Photoblepharon and the genetic relatedness of the symbionts is required. Another interesting aspect of the 16S rRNA RFLP analysis is the variation in number of hybridizing restric- tion fragments between symbiont and free-living bacterial DNA. In all cases, the symbionts show fewer hybridizing fragments to the 16S probe than the free-living strains. The low number of hybridizing fragments revealed in this study suggests the presence of a bacterial monoculture in the light organ. In addition, these data could indicate a difference in copy number of the rRNA genes; i.e., the symbionts have a lower number of operons than the free- living luminescent strains. Similar studies on pea-aphid endosymbionts revealed that the endosymbionts have only a single copy of the rRNA operon, while related free-living organisms have several (Unterman and Baumann, 1990). Restriction fragment analyses of mycobacteria 1 DNA have shown that the number of rRNA copies correlates with growth rate, i.e.. slow growing species have fewer copies of the rRNA operon than those growing more rap- idly (Bercovier et a!., 1986; Suzuki et al, 1987). Previous studies of anomalopids suggested that symbionts in the light organ have slow growth rates [doubling times of 8- 23 hours: (Haygood et al., 1984)]. A reduced rRNA op- eron copy number in the anomalopid symbionts may represent a permanent adaptation to light organ condi- tions, such that the growth rate is inherently low in the anomalopid symbionts. 142 C. J. WOLFE AND M. G. HAYGOOD The taxonomic identity of the anomalopid symbionts remains unknown. None of the luminous species included in the 16S rRNA region analysis appear to be the same species as the symbionts, which fall somewhere between I '. orientalis and F iisi-lieri. The 1 6S rRNA region analysis suggests that the symbionts are more closely related to V. orientalis than to I harveyi. Ln.\A nucleotide sequence data for I '. harveyi, I '. fischeri, and the Kryptophanaron aljredi symbiont reveal a closer relationship between V. harveyi and the A", aljredi symbiont (75% identity) than between the symbiont and I', fischeri [63% (Haygood, 1990)]. This relationship is not observed in the 16S data, which indicate that the symbionts are equally related to both r. harveyi and ('. fischeri. This discrepancy could indicate that the similarity of the symbiont with I ' harveyi is found only within the lux region and is not distinguish- able at the rRNA level. The RFLP approach has some limitations, due to errors in the estimation of distance. These errors result from small, undetected fragments, or fragments of slightly dif- ferent lengths, that are indistinguishable at the level of gel electrophoresis. The major virtue of the RFLP approach is that many samples can be analyzed directly and si- multaneously. The calculations of genetic distance are based on assumptions about the substitution rate and fre- quency of the four nucleotides within a sequence (Nei, 1987). The errors in these assumptions are negligible when the estimated nucleotide substitution (d) is small [>0. 1; (Nei, 1987)], but have an increasingly large effect as d increases, resulting in an underestimation of the distances and a corresponding decrease in accuracy. The distances between the symbionts in this study are large, approach- ing, and in some cases exceeding, the upper limit of ac- curacy for this method. Differences in copy number are also a problem in the 16S RFLP analysis, because these differences represent differences in genome organization rather than actual se- quence divergence. Including all fragments in an analysis between two samples with different copy numbers will overestimate the calculated divergence by inflating the denominator in the fraction of conserved hybridizing fragments. There is no valid way to normalize the data, but to test the robustness of our conclusions, we attempted a conservative compensation for copy number discrep- ancies by recalculating divergence with the denominator normalized to the smallest number of fragments in each sample pair. The values obtained by these calculations should be the lowest possible estimates of divergence within each pair. Using this method of estimation, the intergeneric divergences among the symbionts are not significantly changed [ranging from 0. 1 1 2 to >0. 1 22 (cor- rected) versus >0. 1 1 7 to >0. 1 5 1 (presented in Table III)] and still correspond to species level differences among the characterized specimens. The number of symbiont samples examined in this study is sub-optimal, due to the difficulty and expense involved in obtaining specimens, but we have attempted to compensate for the small sample size. The large inter- generic differences among the anomalopid symbionts is supported by 17 independent comparisons, i.e., each symbiont sample compared to each sample from a dif- ferent fish genus. Even iff. palpebratus #2 were excluded due to a possible gene rearrangement, there are still 15 independent comparisons in support of our conclusion. Despite the limitations of the analysis, several conclu- sions can be drawn. First, the symbionts are obviously not the same species in all members of the family Anom- alopidae. The differences are so extreme that even the largest possible estimates of variance cannot obscure them. This result stands in distinct contrast to all those symbioses in other fish light organs in which symbiont identity has been studied. In addition, no geographical connection be- tween host location and symbiont species is apparent, im- plying that no significant genetic exchange occurs between symbionts from different hosts via free-living populations. Furthermore, the symbiont branching order is consistent with the proposed sequence of evolution within the family Anomalopidae. Thus, at this level of analysis, the sym- bionts appear to have coevolved with the host fish. The large difference between P. pa/pehratus #1 and #2 could be due to a rearrangement or unrecognized difference in the host fishes. Finally, the apparent low copy number of the 16S rRNA operon in the symbionts may represent genetic adaptation to the light organ environment. In any case, the symbiont divergence observed in the association between these luminescent bacteria and the anomalopid fishes appears to be novel among previously characterized light organ symbioses. Acknowledgments We thank A. C. Arneson and the Department of Marine Sciences of the University of Puerto Rico for assistance in collecting A', aljredi, A. C. Arneson and P. Colin for the A. katoptron and P. palpebratus specimens, and the staff of the H. Steinitz Marine Biological Laboratory, Eilat, Israel, for the P. sleinitzi sample. We are grateful to Dr. G. Wilson for help in obtaining and running the com- puter programs, and Dr. R. Rosson and B. Tebo for their helpful suggestions. We also thank Dr. R. Rosenblatt for his identification of the Photoblepharon specimens and critical reading of the manuscript. Thanks are also due to M. P. Quaranta for help in writing the distance esti- mation program. This research was funded by the Office of Naval Research, grant no. N00014-89-J-1742. Literature Cited Baumann, P., I Baumann, S. S. Bang, and M. J. Woolkalis. 1980. Reevaluation of the taxonomy of I'ihrio. 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Characterization of marine luminous bacteria isolated off the coast of China and description of I 'ibrio orientalis sp. nov. Curr. Microbiol. 8: 95-100. Reference: Biol. Bull. 181: 144-157. (August, 1991) Fine Structure of Photophores in Gonostoma elongatum: Detail of a Dual Gland Complex D. EUGENE COPELAND Marine Biological Laboratory, Woods Hole, Massachusetts 02543 Abstract. Gonostoma elongatum Gunther 1 878 is a me- sopelagic fish that has numerous light-bearing organs (the photophores). Some of the photophores are unusual in that they incorporate two different glands: glands D and V. These dual gland photophores are found: (1) in the upper serial row on the body, (2) among the suborbital photophores, and (3) in the caudal photophores. In a ven- tral serial row of photophores, each organ possesses one gland only: gland D. Glands D and V in the upper serial photophores have secretory ducts that fuse to form a common duct that empties to the surface. Gland D, which produces a bluish light, has dioptric material that could provide both spectral reflection and filter transmission. Gland V also has dioptric material associated with it. The entire glandular complex is covered by a shield of alter- nately layered collagen, which has a lens-like bulge over gland D. Immediately beneath the shield are slanted, overlapping rows of dioptric material. The detailed mor- phology of the dual gland photophore in the upper serial row is presented and briefly compared to some of the other photophores on the fish. Noteworthy are the various arrangements of materials that can efficiently guide the light from gland D and, possibly, from gland V. The result would be a diffuse glow suitable for counter illumination. Introduction Throughout the oceans there is a large population of mesopelagic fishes, the so-called midwater fishes. The term "midwater" is somewhat misleading in that, even in the deepest of oceans, the average habitat depth is within the range of 300 to 1500 meters, and at night some of the fish migrate to the surface. The primary concentration is usu- ally associated with a thermocline marking the junction Received 24 April 1990; accepted 8 April 1991. of warm, actively mixed surface water and the colder, less active deeper waters. The specific gravity difference at that interface permits trapping of material that provides an ecological niche. One very interesting adaptation of mesopelagic fishes to the darkness of their habitat is the presence of photo- phores (bioluminescent organs). The photophores are usually distributed in a characteristic order over the body, so much so that the pattern can be used for taxonomic identification (Grey, 1964). The anatomical morphology of the photophores ranges from the huge, complex organs in Argyropelecus to the simple skin photophores ofCliau- liodus (Bassott, 1966; Herring and Morin. 1978). Gonostoma elongatum is a relatively large mesopelagic fish that possesses many of the various types of photo- phores, such as lateral serial, orbital, opercular, branchio- stegal, and caudal (sternchase). Representative photo- phores from the upper lateral serial row were chosen for study. Attention was drawn to the upper serial photophores because each has two parts, a dorsal spherical unit and a closely associated ventral unit, plate-like, silvery, and re- flective. The upper unit is accepted as a glandular pho- tophore in the usual sense and used for taxonomic pur- poses (Grey, 1964; Badcock, 1984), but there is no general agreement as to the nature of the lower unit. One of the first to describe it was Brauer ( 1908) who called it a "sack formigen Organ." In the more recent literature, it is re- ferred to as "glandular" (Grey, 1964, and Badcock, 1984). I find no cytological descriptions of the lower gland or estimates as to its function. Buck (1978) and Young (1983) provide extensive dis- cussion of bioluminescence as linked to counterillumi- nation, species and sex recognition, searchlights, lures, flashes or clouds to distract predators, and so forth. Clarke (1963) observed that most photophores in fish provide a 144 PHOTOPHORES OF GONOSTOMA 145 light nearly matching the blue spectral value (478 nm) of residual down-dwelling light from the ocean surface. Thus, the light from the photophores could "camouflage" the fish from the sight of predators, and especially from below. This possibility is described in some detail by McAllister (1967), Herring (1982), and Denton el at. (1985). Details as to the spectral values of photophore light, as compared to those of the environment at oceanic depths, are pro- vided by Denton and Herring (1978) and Herring (1984). Evidence that fish can adjust the strength of their pho- tophore emissions to match the intensity of down-dwelling light is provided by Case el al. (1977), Lawry (1974), and Young and Roper (1977). As an additional comment about spectral values and their ecological import, it should be noted that the eyes of deep sea fishes have a chrysopin pigment that absorbs maximally at about 480 nm, vir- tually the same as the down-dwelling light and that of the photophores (see discussion by Nicol, 1989). Gonostoma might be an exceptional fish, in that Swift ct al. (1977) have reported a photophore spectral value of 503 nm for it, but this observation of Gonostoma was based on a single photophore on a single specimen that was so badly damaged the species could not be identified with certainty. More significantly. Swift ct al. used an early spectrometric system requiring slow point by point measurement, which could lead to uncertainty. The ob- servation need not be discarded, but it should always be qualified. Widder ct al. (1983) used the more sophisticated optical multichannel analyzer (OMA) and surveyed spec- tral values emitted by some 70 marine species; the values mostly ranged from 470 to 480 nm. This article describes in detail the histological and cy- tological morphology of the two units in the upper serial row of photophores in G. elongatum and the connection between the two. Also of note is the associated array of dioptric tissues that may guide light emission. Brief com- parisons are made between the upper serial photophore and three others in the same fish: lower serial, suborbital. and caudal photophores. Materials and Methods The largest collections of Gonostoma elongatum were made on two cruises: the Woods Hole Oceanographic In- stitute (WHOI) RV Knorr cruise #118-4 on a transect from San Juan, Puerto Rico, to Woods Hole, Massachu- setts; and the WHOI RV Oceanus cruise #183. On both trips, a 10-ft. 2 MocNess midwater trawl was used, and the best collections were made near the outer edge of the con- tinental shelf in the general area of the Hudson Canyon or slightly south thereof. On other trips, few random G. elongatum individuals were otherwise collected by using less efficient meter nets. Estimated collection depth ranged from 350 to 900 meters. The specimens were between 150 and 225 mm in length. The retrieved fish were moribund by the time they reached the decks (hearts fibrillating and weak reflexes), but showed little trauma compared to others, such as Benthosema. Air emboli in the circulatory systems was a probable cause of morbidity. Whole fish were fixed im- mediately after retrieval by total immersion in cold 3% glutaraldehyde buffered to 7.4 pH with 0.1 M sodium cacodylate at refrigerator temperature (ca. 4.0C). Spec- imens were stored in a refrigerator, and the fixative was replaced with fresh fixative after about 12 h. After 2-3 days, the fish were finally stored in 1% glutaraldehyde in 0. 1 M cacodylate. Dissection of photophores, post os- mication, and embedment in epoxy resin of tissues for future study were made shortly after returning to port. Thick ( 1 micron) epoxy sections for light microscopy were stained with methylene blue-azure II. The light mi- crographs in Figures 3. 11, 15, and 17 were made with a Zeiss Axiophot microscope. Thin sections for electron microscopy were stained with uranyl acetate, followed by Reynolds lead citrate, and were studied with a Zeiss 10 electron microscope. Figure 1. Diagram of an entire photophore complex [from the upper serial row (OA)] as viewed through the surface of the fish. (1) Joint se- cretory duct orifice to external surface. (2) Secretory duct of gland D with attached clusters of secretory cells. (3) Lateral hemispheric canopy of gland D. (4) Gland D. (5) Transparent collagen covering shield. (6) Secretory duct of gland V. ( 7) Gland V. Dashed lines indicate the pattern of overlapping rows of dioptric material (see Fig. 1 7) that He immediately under the shield. 146 Figure 2. Diagram of gland D, same orientation as in Figure 1 . ( 1 ) Canopy with outer connective tissue layer, a middle layer of indosomes and an inner layer of thin platelets composed of laminations of dioptric material. (2) Radiating spindle shaped cluster of secretory cells arranged in packets of five or six cells. (3) Thin layer of connective tissue. (4) Layer of thin dioptric platelets similar to the layer in the canopy. (5) Beaded layers of oriented iridosomes (see Fig. 7a). (6) Layer of connective tissue that encompasses both gland D and its canopy. (7) Blood vessel. (8) Thick laminated dioptric facet (see Figs. 3. 10, 1 1). (9) Location of a continuation of the platelet layer that drops down in hack of gland D forming a cup-like shelf that terminates near the plane of section. / < . the free edge faces the surface of the fish (see 4 in Fig. 3). (10) Oriented layers of sparse, flattened connective tissue cells (see Fig. 8) embedded in finely granular lucent material that extends from under the canopy to form a lenticular bulge. (11) Cluster of secretory cells similar to those within the gland proper but on a smaller scale. (12) Small clusters of cells containing irregularly arranged iridosomes. ( 1 3) Epithelial layer of secretory duct. (14) Connective tissue sheath of secretory duct. Figure 3. Light micrograph of gland D. Plane of section is at right angle to the diagram in Figure 1, i.e.. at right angle to the surface of the fish. ( I ) Thin laminated dioptric platelets. (2) Sheets of beaded melanin- like iridosomes. (3) Thick dioptric plates or facets. (4) Shelf of thin dioptric platelets continuing from the posterior-ventral edge of layer 1 ; its out of plane position is indicated in Figure 2 b\ dashed line. (5) Overlapping dioptric material, closely packed. (6) Dioptric material, more dense, and oriented at right angle to the lens of the shield. (7) Lens-like thickening of the shield. (8) Cross section of overlapping dioptric ribbons that range on down to the level of gland V (see dashed lines in Fig. 1 ). (9) The main bulge of lenticular material. The curving layers of connective tissue help to maintain the symmetry of the bulge. Collecting channels of the secretory cell packets are indicated by arrows (also see Figs. 5. 6). Scale bar = 20 ^m. PHOTOPHORES OF GONOSTOMA 147 148 D. E. COPELAND Light microscope studies of whole mounts were made by two methods. First, reasonable detail could be seen when the flat, embedded epoxy blocks were placed on a slide and observed through the upper (shiny) surface with the 10X and 40X objectives of a compound microscope. The osmium tetroxide in the post osmication process acted as a stain. However, the best whole mount prepa- rations were obtained after the melanin pigment had been bleached away (1% chromic acid in 1% calcium chloride, 6 to 12 h at room temperature). The tissues were then stained in Lynch's precipitated borax carmine, followed by dehydration and mounting on slides with Permount. An additional effort was made to reveal the internal fine structure of the iridosomes, which, although "mela- nin-like," resisted bleaching with chromic acid. Treatment with the more potent peracetic acid (Barka and Anderson, 1963) to the point of tissue maceration produced no marked bleaching. Some visualization of internal ultra- structure was obtained only by use of very thin (grey) sections. Several attempts were made to demonstrate nerves with Protargol (Winthrop Chemical Co.), but the efforts were not successful. Because silver stains for nerves are notoriously capricious, the failure does not indicate that nerves are absent. Results The serial photophores of Gonostoma elongatum are arranged in two rows on the ventro-lateral side of the body. The upper serial row (OA) runs from the operculum to the anal level, and each photophore includes two glan- dular structures. The lower row (IV, VAV, AC) runs the full length of the body and each has only one structure. (For diagrams and keys to the photophores of Gonostoma see pp. 284 and 300 in Badcock, 1984.) Each of the upper serial photophores has two distinct glands (Fig. 1). Each gland has a duct that joins the other, and the common duct exits by an orifice to the surface of the fish. The whole glandular complex is covered by a transparent shield of multilayered collagen. The common secretory duct empties to the external surface close to the posterior edge of the shield. For convenience, the dorsal gland is re- ferred to as "gland D," and the ventral gland is called "gland V." A faint, bluish light was observed along the ventro- lateral aspects of several G. elongatum in a darkened room aboard ship, but I did not have equipment to localize or enhance the light source. Also, admittedly, my primary concern was to initiate fixation as quickly as possible. Gland D Gland D is spherical and composed of long secretory cells radiating from a central collecting cavity (Fig. 2). The cavity is lined with epithelial cells that continue on to form the lining of the secretory duct. The radiating cells are grouped in clusters of 5 or 6 in such a way that the central adjoining membranes of the cluster form a channel (Figs. 5, 6) that connects to the central collecting cavity. (The term "channel" is preferred over "duct" be- cause there is no epithelial lining present.) The lateral surfaces of each spindle-like cluster of cells borders on areas filled with connective tissue and blood vessels (Fig. 4). Each glandular cell is filled tightly, in an oriented fash- ion, with secretory granules and multiple layers of rough endoplasmic reticulum (RER). The RER is layered par- allel to that part of the cell adjacent to the connective tissue space (Fig. 4) and fills the outer, more broad end of the cell. The secretory granules are found in that part of the cells adjacent to the common channels (Fig. 5), and that association persists to the point where the chan- Figure 4. Longitudinal section along a radius of gland D, including the surfaces of two cells from adjoining packets of cells, and together with intervening connective tissue and vascular supply (C). Rough endoplasmic reticulum (R) lies parallel to the cell surfaces facing the connective tissue. Large secretory granules (S) are located away from the connective tissue layers and are found adjacent to the secretory channels in the middle of the packets (see Fig. 5). Scale bar = 1 fim Figure 5. Cross section near the midpoint of a radiating packet of secretory cells in gland D. Secretory granules (S) are packed about a central collecting channel (C). There is no tubular epithelium but the glandular cells do have tight junctions (twin arrows) and desmosomes (single arrow). Scale bar = I ^m Figure 6. Section closely parallel to the central collecting cavity of gland D cutting through the epithelium lining which has dense nuclei (Nu) and dense cytoplasm. Each packet ot radiating glandular cells (G) narrows and penetrates the epithelium so that the collecting channels (C) connect to the central cavity. At this level, secretory granules are predominant in the secretory cells. Scale bar = 1 fim Figure 7a. Flattened, oval-shaped indosomes held in orderly fashion by parallel membranes having cross bridges. They are quite dense, and both chromic acid and peracetic acid treatment failed to bleach the melanm-appeanng granules. Scale bar = 0.27 ^m. Figure 7b. Higher magnification of Figure 7a (which is an extremely thin section) with enhanced contrast to reveal the faint laminations that are registered parallel to the bounding membranes of the indosomes. PHOTOPHORES OF GONOSTOMA 149 150 D. E. COPELAND nels empty into the central secretory collecting cavity after penetrating the epithelial lining (Fig. 6). The secretory duct immediately adjacent to gland D has small clusters of cells attached to it. The cells are se- cretory and similar to those in the main part of the gland in having plentiful RER and secretory granules. They dif- fer only in being more rounded and having very short collecting channels (Fig. 2). The base of the secretory duct and its attached cluster cells is covered by a cup-like hemispheric "canopy" extending laterally from the spherical gland (Fig. 2). The dorsal three quarters of gland D is covered by a double layer, the innermost being com- posed of a thin layer of laminated, dioptric platelets (Figs. 2, 3); [structures in this article that respond to polarized light are called "dioptric" (see Fig. 12)]. The outer layer is composed of beaded rows of melanin-like iridosomes (Figs. 2. 3, 7). A similar double layer also extends over the inner surface of the canopy. Irregular cellular clusters of iridosomes are found at the free edge of the canopy (Fig. 2). The dioptric platelet layer covering gland D extends down on all sides, slightly more so on the ventro-medial side where it forms a narrow shelf or band (out of plane of section in Fig. 2, but seen in Fig. 3). The lower surface of the gland D lacks iridosomes and has a circular pattern of dioptric material forming thickened, multilayered plates or facets. The facets slightly overlap each other inward to the central dorso- ventral axis of the gland (Figs. 2, 3). The facets, when viewed with transmitted light in epoxy embedded material, can be seen to form a brilliant blue pattern resembling that of mosaic tile. The facets are composed of orderly, multiple layers of membrane-bound material (Figs. 10, 11), which presumably accounts for the response to polarized light. All of the other dioptric materials mentioned in this article have similar layering. The iridosomes are small, flattened, lozenge-shaped particles with their long axes held in register by mem- branes. The result is a sheet of iridosomes that in cross section looks like a beaded necklace (Fig. 7). The outer layer covering gland D is composed of many sheets of such material. The pigment of the bead-like iridosomes is quite resistant to chromatic acid digestion and responds only slightly to the more corrosive peracetic acid. It prob- ably is not representative of the usual melanin. A multiple layering of material can be seen within the matrix of the ovoid iridosome particle. The layers are parallel to the free surface of the beaded sheets of iridosomes and can be visualized only in very thin (grey in color) ultratome sections. The space covered by the canopy is filled with an orderly arrangement of loose connective tissue (Fig. 2). Sparse, oriented, flattened cells (Figs. 8, 9), supported by some random collagen, fill the cavity and extend ventrally. The ventral extension expands into a bulge underneath gland D (Figs. 2, 3). Because of its spherical shape, and because a better designation is lacking, I call it a "lenticula." It has a fine glandular matrix that is completely transparent in preserved material. Where the lenticula adjoins the covering shield the concentration of collagen in the matrix markedly increases (Fig. 9). The secretory cavity of gland D is emptied by a duct that also connects to a similar duct from gland V (Fig. 1). The common duct then exits through the epithelial surface of the fish (Fig. 12). The cavity and the ducts are lined with a distinct epithelium characterized by dense nuclei (Fig. 6). A layer of connective tissue surrounds both ducts, and the lumen of each duct contains a glandular non- descript material. Gland V The plate-shaped gland V is anatomically and cytolog- ically different from gland D. The secretory cells form a single layer adherent to a network of thin-walled inter- connecting capillaries. The labyrinthic space between the capillaries (with their adherent cells) is packed with se- cretory material (Fig. 1 3). There is no collecting chamber, and the duct for gland V is attached directly to one of the upper (dorsal) arms of the sprawling labyrinth that con- tains secreted material. The cells of gland V have obvious Figure 8. Cross section of flattened connective tissue cells in the transparent bulge (lenticula) located below gland D. Note oriented parallel array. Small clusters of collagen fibers (arrow) are barely visible at this magnification. Matrix is of fine amorphous material. Scale bar = 20 ^m. Figure 9. Higher magnification of the lenticula as it adjoins the covering shield. Collagen fibers (asterisk) are much more numerous and the connective tissue cells are larger and more irregular. Arrow points in the direction of the attachment to the covering shield. Scale bar = 5.0 iim. Figure 10. Cross section of a multilayered dioptric facet of gland D. Note the regular periodicity of the layer being sequestered from a parent cell (arrow). Scale bar = I j ^^H 57 152 D. E. COPELAND secretory elements such as granules and rough endo- plasmic reticulum (Fig. 14). The number of secretory granules and the number of cells having the granules is quite variable. Cells possessing granules are usually dis- tributed in a gradient, the cells having the highest con- centration of granules being found at the upper (dorsal) end of the gland. Gland V has dioptric material in a layer immediately under that organ (Fig. 15). It is in the form of thin rec- tangular ribbons overlapping and randomly disposed with no prevailing orientation when viewed in embedded ma- terial by light microscopy. Each ribbon is about 20 X 100 Mm and has four or five layers of laminated material sim- ilar to that in Figure 10. The ribbons probably provide the whiteish, silvery reflection seen in the living, though moribund, animal. Sometimes the reflection has a faint yellow color in fixed material. Shield The entire dual gland complex is covered by a trans- parent shield (Fig. 1 ) of collagen orientated in alternating layers that are at right angles to each other (Fig. 16). Fifteen or so of the layers form a lens-like thickening at the level of gland D (Fig. 3). The number of layers drops off in all directions until there is only one layer of amorphous ma- terial at the edge of the shield. That periphery is embedded in a rod-like rim of cellular material (Fig. 18). The free surface of the shield is covered by a thin layer of epidermal epithelium that is frequently lost during collection and preparation. Immediately beneath the shield is oriented dioptric material arranged in rows parallel to each other (dotted lines in Fig. 1). The rows of ribbons overlap in a steeply pitched, shingle-like fashion (Fig. 17). The angle of the pitch is downward, i.e.. laterally and ventrally to the body. The region of layered shingles reaches from about the median level of gland D (Fig. 3), down to the upper edge of gland V (i.e., it does not cover gland V). The parallel rows of dioptric material extend straight across the shield just above gland V level, but as the gland D level is ap- proached, the rows increasingly bulge upward (Fig. 1). There is a regional change in the orientation of the shingles where the connective tissue of the lenticula adheres to the shield. In that region, the dioptric material is more coarse and oriented at right angles to the shield (Fig. 3). The modified shingles lie between the lens of the shield and the lenticula of gland D. Above that region, the dioptric material again slants downward, is more compact, and ends. Although the light microscope cross sections of the shingles responded to polarized light, at no time was color or reflection evident in the intact animal, living or fixed. The dioptric material is transparent and can easily be overlooked at the light level unless differential interference contrast (DIG) lighting of embedded material is used. Comparison with some other types ofphotophore in G. elongatum The lower serial row, the suborbital, and the caudal photophores each have a gland that, in size and cytology, is similar to gland D. However, the lower serial photo- phores have no gland V; the singular gland D connects directly to the surface via a single duct. Both the suborbital and caudal photophores have gland V type tissue, but noteworthy in each case, the glands V and D are connected directly to each other by a duct that does not approach the surface. Discussion Gland D and its dioptric material Herring and Morin ( 1978, p. 306) have provided (af- ter Bassott) a line drawing of gland D in Gonostoma at the light histology level. They diagram the radiating arrangement of secretory cells focussed on a central collecting cavity and say that a duct connects to the surface of the fish, but do not show a clearly organized duct or associated cluster cells. Also, items such as can- opy, facets, and lenticula are not depicted, and gland V is not mentioned. Gland D of Gonostoma is better illustrated in a micrograph by Nicol (1969, p. 365). The Figure 13. Cross section of several thin-walled capillaries (arrows) in gland V. The capillaries form a flattened irregularly branching, network of vessels and are covered by a single layer of glandular cells (G). The labyrinthic space between the capillaries is filled with secretory material (S). The capillaries are distended, and the lumen (L) is empty of red blood corpuscles because of ernboli located somewhere else in the system. Scale bar = 10 ^m. Figure 14. Detail of a secretory cell in gland V. Note usual glandular characteristics such as rough endoplasmic reticulum (R). Golgi apparatus (G), and secretory 1 granules (S) and secreted material (asterisk). Also, note the irregular protrusions of the cell surface that may aid in secretory release. Scale bar = I ^m. Figure 15. Light micrograph of a section through the layer of dioptric ribbons that lay beneath gland V. The ribbons are thin (4 or 5 laminations), rectangular (about 20 X 100 ^m), and randomly disposed within the layer. Cross section of a ribbon (single arrow) and longitudinal section (twin arrows). Scale bar = 20 Mm. PHOTOPHORES OF GONOSTOMA 153 , sxf . I ^^P ^ 15 154 D. E. COPELAND photophore is from the "lower trunk." If trunk is de- nned as post-anal, then the photophore is one of the lower serial row that has no gland V (the upper serial row of dual gland photophores is on the abdomen an- terior to the anus). However, because gland D in the upper and lower serial rows are quite similar, the pho- tograph by Nicol is comparable to the gland D described here (but not in as much detail). The secretory cells of gland D are equivalent in all respects to the A photocytes of Bassott ( 1966). However, I found no cells equivalent to his type B photocytes. The lack of type B cells in the photophores of gonostomids has been noted by Bassott (1966) and Nicol (1969). Gland D is capped with a layer of dioptric platelets backed by a layer of iridosomes in such a manner that light can be reflected ventrally and laterally toward the external surface, a common phenomenon in photophores located laterally on the body. These layers may selectively reflect spectral light (Denton el til., 1985). The canopy, as an auxiliary structure, may have a light concentrating role. The layer of dioptric platelets, plus iridosomes, could have a reflective and light concentrating effect. It is in a position suited to reflect some light from gland D and much of the light from the cluster cells at the base of the collecting duct. The slightly overlapping, concentric ar- rangement of the dioptric facets on the ventral side of the sphere could have a concentrating effect as well as a pos- sible light filter effect similar to that described by Denton et at. (1985). Furthermore, the shelf of dioptric platelets (Figs. 2, 3) that slopes toward the shield could provide a deflecting effect, guiding more light through the lenticula into the lens. Lenticula as a light collector and anchor The lenticular tissue is in the shape of an inverted comma with the tail under the canopy and the spherical bulge positioned below the gland proper (Fig. 2). The highly oriented loose connective tissue and amorphous matrix could allow for an optically clear structure with an index of refraction suitable for concentrating light. The tissue might also act as a light filter (Denton et a!.. 1985). Bassott (1960) describes a photophore in the closely related gonostomatid Maurolicus that has a body that is gelati- nous, transparent, homogeneous, and refractive; and sug- gests that it may act as a lens. The oriented collagenous attachment of the lenticular apparatus to the shield could serve two purposes. The most obvious would be to pass light to the lens of the shield in a registered fashion. The other purpose, in lack of other connective tissue, would be to serve as an anchor for gland D, keeping it oriented and in place. Gland I ' and its dioptric material Although gland D in Gonostoma has been repeatedly identified in the literature as a photophore and considered as furnishing a taxonomic pattern (Grey, 1964), the as- sociated gland V has received little attention. One of the first to identify it was Brauer (1908), who referred to it as a "sack formigen Organ." It has been referred to as being glandular and having a direct connection to gland D, but there is no description of the two secretory ducts fusing into a common duct to empty to the surface. Also, I find no cytological descriptions of the gland V. The secretory cells of gland V have little or nothing in common with the secretory cells in gland D, either ana- tomically or cytologically. The type cell of gland V has a well-developed Golgi apparatus and associated granules that indicate the usual merocrine type of secretion. How- ever, granules do not approach the dense population seen in gland D. There is no evidence in the literature suggesting that gland V produces luminescence. Most certainly it has no category "A" photocytes as found by Bassott (1966) in a broad spectrum of photophore types in teleosts. The dioptric ribbon-like material beneath gland V must serve as a reflector, and account for the silvery appearance. The shield and its dioptric material The shield is composed of multiple layers of collagen, each arranged at right angles to the neighboring layers. Thus, maximum stiffening is obtained. The shield is fur- ther strengthened at the tapering circumferences by a rod- like rim of cells. The thin overlying cutaneous epithelium is easily displaced in dissection and probably does not contribute much strength to the system. The shield may be involved in two aspects of light re- lease. First, the shield itself is thickened to form a lens, albeit it may be weak. That lens is positioned directly opposite and attached to the lenticula of gland D, so that light passing from the lenticula may be collected by it. Second, the oriented rows of dioptric material associated with the shield may have indices of refraction that would help to guide light downward from gland D (with a pos- sible exception at the level of the lenticula). The rather complicated orientation of the rows in relation to gland D, straight rows transitioning to curved, supports this in- terpretation. Moreover, there is no such dioptric material accompanying the shield over gland V. Whatever the function of the dioptric material associated with the shield, it must be in reference to gland D. Iridosomes Bassott (1966) describes the photophores found throughout the stomiatoidei (which includes Gonostoma) V c 5j&r , <_- :-~^ij5r Figure 16. Cross section of the shield, median between its free edge and the lens thickening. Note that the alternating layers of parallel collagen fibers are at 90 angles to each other, thus adding strength to the shield. Collagen (C). Skin epithelium (E). Scale bar = 1 urn. Figure 17. Light micrograph of a cross section of the dioptric ribbons that lay in overlapping fashion immediately under the shield. (See Fig. 1 for their distribution). Ventral direction (V). Collagen shield (S). Dioptric material (arrow). External medium (asterisk). Scale bar = 10 ^m. Figure 18. Cross section of the edge of the collagen shield. The shield proper tapers peripherally to a narrow flange of dense material (D), almost devoid of collagen fibers, and is finally bordered by a rod of supporting cells that may have participated in the growth of the shield. Scale bar = 1 t^m. 18 155 156 D. E. COPELAND as "urn, sphere or retort shaped." The wall of the sphere is always formed of reflector cells which, in turn, are coated with a "layer of iridocytes of melanine" (Bassott, 1966, p. 566). The particles in Figure 7 could be called "mela- nosomes" because they look melanin-like and have no proven iridescence. However, the particles, by reason of their laminated substructure, their rigid orientation within sheets, and their resistance to chromic acid and peracetic acid digestion, may not be usual melanin granules. For example, Nicol ( 1989) describes two kinds of "melanoid substances" to be found as chemical components in the reflectors of fishes. He uses the term "melanoid" because the two are chemically derived in part from tyrosine by tryosinase catalysis and exhibit certain parallels to melanin synthesis. By precedent, the term "iridosome" is chosen in this article as representing the particles within the "ir- idocyte" of Bassott. The sheets of iridosomes are ideally located about gland D and under the ribbons of gland V for a possible light modifying function. The hitherto unreported laminations in the particle, plus their orientation to the long axis of the sheets, suggests that such could be true. (On the other hand, they may be exceptionally efficient light suppres- sors.) Functional aspects I approach the discussion of possible functions with trepidation, and I quote Buck (1978, p. 420): "At least 20 functions of bioluminescence in one animal or another have been proposed Though often quite plausible, most of these proposals were originally more a reflection of the ingenuity of observers than of the solidity of the obser- vations, and there has been an unfortunate tendency for later authors who are casting about for functions of lu- minescence in their own material simply to list possibilities without adducing any additional support." This comment is particularly appropriate when considering the meso- pelagic fish, which are difficult to observe or to test in any way. Doubtless, type D gland photophore luminesces. Swift c/ al. (1977) recorded luminescence in a Gonostoma pho- tophore, "about 2/3 of the distance from the head to the tail." That distance would locate it in the area of the body that has only lower serial photophores with gland D (see illustration. Badcock, 1984. p. 300). Furthermore, Anctil (1972) has stimulated luminescence in the photophores of the closely related gonostomid, Maurolicus. I find no record where gland V is given the status of a photophore. However, the enlarged type V gland tissue in the caudal gland of G. elongatum has been referred to as "luminous tissue" by Grey ( 1964), and it has associated with it, at a tangent and relatively inconspicuous, type D gland that is cytologically akin to the lower serial pho- tophore that has been proven to luminesce (as stated above). Thus, both glands D and V may produce light. Keeping Buck's admonition in mind, one would add that proof for a luminescent function for gland V is faint. The possibility that luminescence may occur other than in gland D or gland V (i.e.. in surface slime) cannot be discounted. Bassott (1966) says of Gonostoma that the absence of clearly characterized B cells in association with the A cells could be explained by the possibility that the A cell secretion is secreted to the outside and there mixed with other agents. Also, Swift et al. (1977, p. 822) quote Brauer ( 1908) and say "the light organs in G. elongatum have ducts leading externally which suggests along with the large value of FWHM (Table 1) that its spectra show little effect from the masking of overlying tissue." The implication is that the ducts bring the luminescence to the surface, where it is more easily seen. My own obser- vations of the faint bluish luminescence in G. elongatum were not sufficient for a specific localization of source. I hazard no thoughts regarding the function of a direct duc- tal connection between glands D and V in the suborbital and caudal photophores. It is interesting that, at least in G. e/ongatum. gland V is always associated with a gland D. However, gland D is not necessarily associated with gland V. One of the noteworthy features of this article is the detailed analysis of a whole array of dioptric materials that can efficiently handle the luminescent light in such a way as to produce a downward, evenly diffused lighting for the fish, that could function as counterillumination. Acknowledgments This work was supported by grant NSF-PCM-80-23 1 66 to D.E.C. and grant NSF-OCE-84- 16206 to Holger Jan- nasch. I am grateful to J. A.C. Nicol for his comments and encouragement and to Margaret McFall-Ngai for the loan of a midwater collecting net. Not least, the cooperative crews and excellent ship facilities of the RSV Oceanus and RSV Knorr (Woods Hole Oceanographic Institution) must be acknowledged. Literature Cited Anctil, M. 1972. Stimulation of bioluminescence in lantern-fishes (Mycotphidae). 11. Can. J /<><>/. 50: 233-237. Badcock, J. 1984. Gonostomatidae. Pp. 284-301 in l-'ixlif.i of Norlh- ciiMcrn Atlantic and Mediterranean., Vol. 1. P. J. Whitehead. M.-L. Bauchot. J.-C. Hureau. .1. Nielsen, and E. Tortonese, eds. Unesco Publishers. Paris. Barka. T.. and P. J. Anderson. 1963. Histochemistry. Theory. Practice and Hihlmxrupln: Harper and Row, New York, Evanston, and Lon- don. PHOTOPHORES OF GONOSTOMA 157 Bassott, J.-M. 1960. Donnees histochimiques et cytologiques sur les phophores due teleosteen Maurolicus pennanti. Arch. Ana/, Morphol. E.\y. 49: 23-71. Bassott, J.-M. 1966. On the comparative morphology of some lumi- nous organs. Pp. 557-610 in Bioluminescence in Progress, F. H. Johnson and Y. Haneda. eds. Princeton University Press, New Jersey. Brauer, A. 1908. Die Tiefsee-Fische. H'iss. Ergebn. deut Tiefsee- E.\pecl.. "laldma" 15: 1-432. Buck, J. 1978. Functions and evolutions of hioluminescence. Pp. 419- 460 in Bioluminescence in Action. P. J. Herring, ed. Academic Press. New York. Case, J. F., J. Warner, A. T. Barnes, and M. Lowenstein. 1977. Bioluminescence of lantern fish Myctophidae in response to changes in light intensity. Nature 265: 179-181. Clarke, VV. D. 1963. Function of hioluminescence in mesopelagic or- ganisms. Nature 198: 1244-1246. Denton, E. J., and P. J. Herring. 1978. On the filters in the ventral photophores of mesopelagic animals. J. Physio/. (Loud.) 284: 42 P. Denton, E. J., P. J. Herring, E. A. Widder, M. A. Latz, and J. F. Case. 1985. The roles of filters in the photophores of oceanic animals and their relation to vision in the oceanic environment. Prix: R. Six: Loud B 225: 63-97. Grey, M. 1964. Genus Gonostoma. Pp. 163-180 in Fishes of Western North Atlantic. Part 4, H. B. Bigelow, ed. Sears Foundation for Marine Research, Yale University, New Haven, CT. Herring, P. J. 1982. Aspects of hioluminescence of fishes. Oceanogr. Mar Biol. Ann. Rev. 20: 415-570. Herring. P. J. 1984. The spectral characteristics of luminous marine organisms. Proc. R Sue. Land. B 220: 183-217. Herring, P. J., and J. G. Morin. 1978. Bioluminescence in fishes. Pp. 273-329 in Bioluminescence in Action, P. J. Herring, ed. Academic Press. Lawry, J. V. 1974. Lanternfish compare down dwelling light and bio- luminescence. Nature 247: 155-157. McAllister, J. V. 1967. The significance of ventral hioluminescence in fishes. J. Fish Res. Bd. Can. 24: 537-554. Nicol, J. A. C. 1969. Bioluminescence. Pp. 355-400 in Fish Physiology. IW. ///. W. S. Hoar and D. J. Randall, eds. Academic Press, New York. Nicol, J. A. C. 1989. Eyes of Fishes. Clarendon Press of Oxford Uni- versity, Oxford. Swift, E., \V. H. Biggley, and T. A. Napora. 1977. The bioluminescence emission spectra of Pyrosoma atlanticum. P sriinosum (Titnicata). Euphausia lenera (Crustacea) and Gonostoma sp. (Pisces). J. Mar Biol. Assoc. U.K. 57:817-823. \\ idder, E. A., M. L. Latz, and J. F. Case. 1983. Marine biolumines- cence spectra measured with an optical multichannel detection sys- tem. Biol. Bull 165: 791-801. Young, R. E. 1983. Oceanic bioluminescence: an overview of general functions. Bull Alar. Sci. 33: 829-845. Young, R. E., and C. F. E. Roper. 1977. Intensity regulation of bio- luminescence during countershadmg in living midwater animals. Fish. Bull. 75: 239-252. Reference: Biol. Bull. 181: 158-168. (August, 1991) Morphology and Behavior of an Unusually Flexible Thoracic Limb in the Snapping Shrimp, Alpheus heterochelis A. T. READ, J. A. McTEAGUE, AND C. K. GOVIND Life Sciences Division, Scarborough College, University of Toronto, 1265 Military Trail, Scarborough. Ontario MIC 1A4 Abstract. The second thoracic limb in the snapping shrimp. Alpheus heterochelis. is much thinner, more elongated and flexible, and has a larger ganglion than its serial homologs. The greater length and flexibility is largely due to one of the limb segments viz., the carpus which consists of five separate segments, rather than the single segment typical of the other limbs. Externally, the multi- segmented carpus is relatively free of cuticular projections except for scattered simple setae. The adjoining seg- ments the merus and the propus are also smooth ex- cept for clusters of long simple setae on the pollex and dactyl. Internally, each of the carpal segments has three muscles a bender, stretcher and rotator all restricted to the distal half of the segment. In keeping with the sen- sillum-free exterior of the multisegmented carpus, only about 1000 axon profiles originate in the carpus out of a total of 6000 counted at the base of the ganglion. This total number is roughly half that found in the first thoracic limbs. Conversely, the number of axon profiles in the lon- gitudinal connectives to the second thoracic ganglion is about 25% greater than that to the first thoracic ganglion and may partly account for the size difference between these two ganglia. In terms of their behavior, the second thoracic limbs are almost constantly active, mostly probing the substrate, and occasionally grooming various body parts. Part of the probing behavior consists of food foraging and retrieval, especially from concealed and hard-to-reach locations. Because of their flexibility, these limbs are particularly adept at such movements. Received 22 January 1 991; accepted 8 April 1991. Introduction Shrimps of the family Alpheidae are commonly referred to as snapping shrimp, or pistol shrimp, because of the audible popping sound they make when closing their ma- jor cheliped. The major or snapper cheliped is highly spe- cialized both morphologically and physiologically, to produce this defensive response (Prizbram, 1901; Ritz- mann, 1974). The opposite cheliped or claw, referred to as the minor or pincer claw, is smaller, less elaborate, and used primarily in burrowing behaviors. Although these paired claws have attracted much attention, because of their asymmetry and their capacity to reverse this asym- metry (Prizbram, 1901; Wilson, 1903), the next pair of thoracic limbs, which are also highly specialized in form and function, have been somewhat neglected. The second thoracic limbs are bilaterally symmetrical and very thin compared to the chelipeds. The most striking morphological specialization is, however, the multiseg- mented carpus, which is made up of five separate segments rather than the single segment characteristic of all the other thoracic limbs in the shrimp and of crustacean limbs in general. The multisegmented carpus gives the second tho- racic limb a high degree of flexibility. Indeed, in another snapping shrimp, Alpheus pachychirus, these second tho- racic limbs are used as needles with which to stitch algal mats into a temporary retreat (Schmitt, 1975). Although this type of behavior is not seen in Alpheus heterochelis. the second thoracic limbs are, nevertheless, strikingly flexible in their movements. Moreover, these movements are not associated with walking, which is performed by the remaining three pairs of thoracic limbs, but with ex- ploring the environment, feeding, and grooming. Thus 158 SHRIMP FLEXIBLE LIMB 159 the second thoracic limbs make almost constant searching movements, by which their distal ends delicately explore the surrounding substrate. Another interesting point about these highly flexible second thoracic limbs is that their ganglion appears to be larger than that of any of the other thoracic ganglia. This would imply that the volume of neural tissue concerned with the behavior of the second thoracic limb is greater than that of any other thoracic limb. We have therefore investigated the morphology and behavior of these limbs. Materials and Methods Adult snapping shrimps, Alpheus heterochelis (Say), were collected from tidal pools around Beaufort, North Carolina, and shipped to our laboratory in Scarborough, Ontario. In the laboratory, the animals were held in 25 1 glass aquaria equipped with a bottom gravel filter and partitioned into 12 compartments with fiberglass screens. The aquaria were filled with artificial seawater that was kept at room temperature (22 C). A specially prepared diet a blended mash of chicken livers and hearts and commercial dog food was fed to the animals daily. The shrimps were sexed on arrival in the laboratory, and their molt history during captivity was recorded. Behavioral and morphological observations were made on adult an- imals obtained from the wild, and a few juveniles reared in our laboratory were used to supplement the mor- phology. Morphology Scanning electron microscopy. The second thoracic limbs of adult shrimps were removed by a gentle pinch, which induces the animal to autotomize the limb at its base. These isolated limbs (or in the case of juvenile shrimps, whole animals) were fixed for 1-3 h in a 0.15 M sodium cacodylate buffer (pH 7.4) containing 2.5% glu- taraldehyde, 0.2% formaldehyde, 2 mAI CaCl 2 , 0.06 AI NaCl, and 0.3 M sucrose. Next, the tissue was washed in 0.15 M cacodylate buffer, containing 2 mAf CaCl 2 , 0.06 A/ NaCl, and 0.3 M sucrose for 1 h, with several changes. Following dehydration in a graded ethanol series, the tis- sue was transferred into acetone, before being critical point dried, and mounted with silver paste on Cambridge SEM stubs. The tissue was sputter-coated with gold-palladium and examined with a Hitachi S-530 scanning electron mi- croscope. Transmission electron microscopy. The focus of this study was the muscles and nerves within the 2nd thoracic limb. Isolated limbs were pinned out in a dish and su- perfused with the fixative described previously. When the nerves at the base of the ganglion were to be studied, the thoracic nervous system was exposed on its ventral side in situ and superfused with fixative. Following an initial fixation of 1 h, the tissues were further dissected, and se- lected pieces were removed and allowed to fix for an ad- ditional hour. A rinse in buffer solution for 0.5 h followed, and the pieces of tissue were then post fixed in 2% O V O 4 for 1 h. Next, the tissue was briefly rinsed in buffer so- lution, dehydrated in a graded ethanol series, cleared in propylene oxide, and embedded in Epon-Araldite (Pearce etai. 1986). Thin ( 75- 1 00 nm) cross-sections of the nerve were taken close to the ganglion to capture all the axons between the limb and its hemiganglion. These sections were mounted on Formvar-coated single-slot grids, stained with uranyl acetate and lead citrate, and examined with a Zeiss 9S and a Siemens 102 electron microscope. Cross-sections of the ganglionic nerves were photo- graphed in their entirety via a series of overlapping ex- posures at 1800X. The resulting negatives were printed to 6000X and assembled into a montage in which the smallest, usually unmyelinated, axons (0.2 ^m diameter) were distinct, and could be easily counted. A similar pro- cedure was followed for the ventral nerve cord connec- tives, except that the initial exposures were at 500X and the final prints were at 3500X. At this magnification, in- dividual axon profiles were easily recognized, as the over- whelming majority were myelinated and therefore rela- tively large. Behavior Observations were carried out on animals acclimated to conditions in the laboratory (room temperature, 10: 14 L:D photoperiod) for at least one month. Two small ob- servation tanks (32 cm X 9 cm X 16 cm) were set up as follows: the sides and back were covered with opaque pa- per, and the inside was partially filled with sloped gravel or sand, and half-filled with artificial seawater. The ani- mals to be observed were placed in the observation tanks and allowed to acclimate overnight. Observations were carried out in the morning and afternoon, under artificial lighting. Time budget. The focus of our reconnaissance obser- vation was the flexible limb, and our ethogram was con- structed with this in mind. We then determined the per- centage of time occupied by the cataloged behavioral states using instantaneous sampling (Lehner, 1979). Behavioral states were scored at 20-s interval points throughout an 1 1-min observation period. Foraging experiments. An experiment was designed to test the importance of the flexible limb in foraging, spe- cifically for concealed food; i.e., the foraging behavior of shrimp with intact flexible limbs (control) was compared to that of shrimp with missing flexible limbs. Males rang- ing in length (rostrum to telson) from 28 to 34 mm were used. The flexible limbs were autotomized and the shrimp 160 A. T. READ ET AL allowed at least a week to recover. The procedure appeared to cause little stress, as expected, because autotomy is a defensive adaptation and. indeed, shrimp are frequently found with missing limbs. Their general behavior ap- peared normal. The trials for observing the foraging behavior were set up as follows: the individual to be tested was confined to one end of the tank with a piece of screen and, at the other end, a small piece of coral was partially buried. Into one of the corralites (approximately 5 mm deep X 2 mm wide) was placed a previously frozen brine shrimp, along with a generous squirt of "brine-shrimp water." The shrimp was released from confinement by removal of the dividing screen, and the time taken to locate the food and actually retrieve it were both recorded. In order for the trial to be valid, the animal had to demonstrate foraging behavior; i.e., frantic crawling around with intense sub- strate probing. If the food was not located or found within 10 min, the trial was terminated. Each animal was tested twice daily, in the morning and afternoon. Results External morphology Alp/ieiis heterochelis bears five pairs of thoracic limbs: the first two pairs are chelated, and the remaining three pairs are not (Fig. 1A). The first pair of limbs is elaborated into chelipeds; they are bilaterally asymmetrical, one bearing a major (snapper) chela, and the other a minor (pincer) chela, both held in front of the animal. The re- maining four pairs of thoracic limbs are not as elaborate, are much smaller, and are held to the side of the animal. The second pair of thoracic limbs differ from the others in that they are much thinner and have a multisegmented carpus. There are five carpal segments; the proximal two are much longer than the distal three, but the joint between each of the segments is in the same orientation as that between the most proximal segment and the merus (Fig. IB). Consequently, at each joint the distal segment can lie in line with its proximal partner, or it can be bent to almost touch its proximal partner through an angle of 130-140. This degree of bending at each of the four in- tercarpal joints makes this limb extremely flexible, allow- ing its chelated propus-dactyl segment to reach parts of the body otherwise inaccessible to a limb with a single segmented carpus. The multisegmented nature of the carpus also makes it the longest segment of this limb, much longer than its counterpart in the other three posterior limbs. In contrast, the propus is much shorter in the second limb than in the remaining limbs. Each of the carpal segments, as well as the more prox- imal merus. is smooth and bare (Fig. 1 B) except for a few scattered short simple setae (Fig. 1C). The apical end of these simple setae are specialized into a sheath (Fig. 1 D). The propus is also relatively free of cuticular projections (Fig. IB) except for its most distal parts, the pollex and the dactyl (Fig. IE). Situated at the distal end of the pollex and dactyl, on each of the medial and lateral aspects, are prominent clusters of long setae. Scattered more proxi- mally are one or two smaller clusters. These long setae are serrulate in form and have an apical pore (Fig. IF). Others have their tip elaborated into a sheath (Fig. 1G). A row of short setae occur along the closing edge of the dactyl (Fig. IE) and these are serrulate and have an apical pore (Fig. 1H). An opportunity to examine juvenile shrimps arose in our laboratory when a berried female hatched its eggs and we were able to rear these developing shrimps into juvenile forms. In the early juvenile stages, when the paired che- lipeds have not yet differentiated into snapper and pincer types, the carpus in the second thoracic limb was fully differentiated with five segments on the one side but only four on the other (Fig. 2A). Of these four segments, the distal three were matched in shape, size, and location to their counterparts on the opposite limb, while the fourth, most proximal segment had not yet subdivided into two. Indeed, a short, shallow furrow on its ventral side (Fig. 2B, C) seemed to mark the formation of a new segment. This observation suggests that the segmentation of the carpus may occur in a distal to proximal direction; pos- sibly the carpus arises as a single unit initially and sub- sequently becomes segmented. No sensilla occur on the carpal segments at this juvenile stage; only a single cluster of long setae was present at the distal tip of the pollex and dactyl (Fig. 2A). Internal morphology Muscle elements. The musculature in the propus and carpus was examined in thick and thin cross-sections of these segments. The propus typically has two muscles, a small opener and a large closer muscle. Both originate on the exoskeleton and insert via apodemes to the dactyl, which opens and closes in response to contraction of the respective muscles. The fine structure of these muscles is typical of other crustacean striated muscles (Govind and Atwood, 1982) and of the snapping shrimp claw closer muscle (Mellon and Stephens, 1980); vi:.. myofibrils composed of serially repeating sarcomeres, in which thick filaments are surrounded by thin filaments. We did not further characterize these muscles into fiber types. Mi- tochondria were found typically around the periphery of the fiber, where they formed a relatively thick rind. They also occurred occasionally interspersed within the fiber, usually in a single row separating myofibrils. Both muscles also occasionally displayed nerve ter- minals that resembled those previously described in the SHRIMP FLEXIBLE LIMB 161 Figure 1 . External morphology of the second thoracic limb of the snapping shrimp viewed with scanning electron microscopy. (A) Line drawing of the intact animal showing the five pairs of thoracic limbs, of which the first pair is the enlarged, bilaterally asymmetric chelipeds. and the second pair is the thin, elongated, chelated, and highly flexible limb. (B) The second thoracic limb with dactyl (d) and propus (p), 5-segmented (1 to 5) carpus and merus (m). Morphology and distribution of setae on this limb are shown in the accom- panying figures. (C) A typical carpal segment with few sensilla which are of the setal type (D). (E) Distal end of the propus and dactyl with several clusters of long setae and a single row of short setae along the apposing edge of the dactyl. (F) Typical long setae showing serrulate nature and an apical pore. (G) Long setae with a sheath-like tip. (H) Typical short setae with serrulate form and apical pore. Scale bars: B. C. 500 pm; E, 250 M m; D, F, G, H, 2 M m. 162 A. T. READ ET AL Figure 2. (A) External morphology of the paired second thoracic limbs in a juvenile shrimp. The carpus of the right limb is differentiated into five segments (1 to 5), while the left limb shows the most distal three segments (3 to 5) differentiated but not the most proximal two. (B) High power views of the most proximal segments (1.2) of the right carpus while the corresponding region of the left carpus (C) is a single segment with a furrow (arrow) marking the beginning of segmentation into two. Scale bars: A, 200 pm; B, C. 50 jjm. closer muscle of the first thoracic limb (Phillips el al, 1982). The nerve terminals were characterized by a pop- ulation of clear synaptic vesicles that were spherical in most cases; occasionally, terminals with more irregularly shaped vesicles were encountered. The shape of these syn- aptic vesicles with aldehyde fixation i.e.. spherical or irregular effectively denotes excitatory or inhibitory nerve terminals (Atwood el al., 1972). The most distal carpal segment contains three muscles, a large stretcher muscle, a small bender muscle, and an even smaller rotator muscle (Fig. 3). The latter two mus- cles are closely juxtaposed and are situated in one com- partment while the stretcher muscle lies by itself in the other compartment. The apodemes of these muscles at- tach to the next distal segment, although the muscles themselves do not traverse the full length of the segment but are restricted to the distal half. In fine structure these carpal muscles are similar to those in the propus, and they also display neuromuscular terminals indicative of in- nervation by both excitatory and inhibitory axons. The musculature in each of the other segments of the carpus resembles that found in the most distal segment. Neural elements. The second thoracic ganglion is con- sistently larger than the first (Fig. 4). In freshly dissected preparations from three adult shrimps, the surface area of the second ganglion was 25 to 30% larger than the first ganglion. Differences in the input to the ganglia might account for the size differences between them. Hence we examined the nerves and connectives belonging to these ganglia. A. Nerve. Each of the thoracic limbs is served by two separate nerves, the first and second nerve (Fig. 5A), which originate from the hemiganglion. Each nerve is mixed, composed of sensory and motor axons. The majority of axons within crustacean nerves are sensory, with their cell bodies located at the periphery (Bullock and Horridge, 1964). These sensory axons enter the ganglion and ramify in the neuropil, the integrative region of the ganglion. In crustaceans, relatively few (<60) motor axons innervate the limb musculature (Wiersma, 1961; Govind and At- wood, 1982). The nerves of the snapping shrimp are unusual in that they have myelinated axons (Ritzmann, 1974), although peneid shrimp also have this feature (Heuser and Dog- genweiler, 1966). Usually, however, crustacean nerves have only unmyelinated axons (Bullock and Horridge, 1964). Consequently in cross-sections, snapping shrimp nerves prominently display numerous, large Schwann cell nuclei characteristic of myelinated axons (Fig. 5A). At a higher magnification, myelinated axons are readily dis- tinguishable from their unmyelinated counterparts, be- cause the myelin forms a dense sheath around the axon (Fig. 5B). The unmyelinated axons are wrapped by glial sheaths to different degrees, depending on their size. The SHRIMP FLEXIBLE LIMB 163 Figure 3. Cross-section through the first carpal segment with an exoskeleton (e) boundary and the interior separated into two compartments (asterisks) by a thin septum (arrow). One compartment has the stretcher (s) muscle and a large branch of the limb nerve (n) while the other compartment has the bender (b) and rotator (r) muscles and two smaller nerve (n) branches. Scale bar: 50 ^m. smaller axons are naked, whereas the larger axons have several layers of glial covering. Because most of the axons in the nerves are sensory, we can estimate the sensory innervation from the limbs by counting the total number of axons close to the gan- glion. Such counts were made in two animals, and the results were similar in both cases (Table I). The total numbers of axons on the right and left sides are almost equal. There is also an equal distribution between my- elinated and unmyelinated axons on the right and left sides in each of the two animals. To determine how the numbers of axons in the second limbs compare to those of the highly specialized and asymmetrical first thoracic limbs or chelipeds, counts were also made of the nerves to the pincer and snapper che- lipeds (Table I). The present counts of numbers of axons to the snapper and pincer nerves confirms previous find- ings; the snapper side has more axons than the pincer side (Govind and Pearce, 1988). Comparison between the first and second limbs reveals that the second limb has a much smaller number of axons than either the pincer or the snapper (Table I). In shrimp # 1 , for example, the second thoracic limb has 6000 axons, whereas the snapper on the first limb has over 13,000 axons and the pincer has 10,000 axons. In terms of total numbers of axons, the snapper has the largest number, followed by the pincer, and then the second thoracic limbs, which have the smallest number. The distribution of un- myelinated and myelinated axons is interesting; the first thoracic limbs (both the pincer and the snapper) have more (60-70%) unmyelinated than myelinated axons, whereas the second limbs have an equal number of my- elinated and unmyelinated axons. The above counts of axons taken close to the ganglion represented the total number to the limb, including those to the thorax at the base of the limb. Because we were interested largely in the multisegmented carpus, we counted the axons in the nerve running through the distal segments of a second thoracic limb. In all cases, counts were made from the most distal end of each segment. As anticipated, the axon number increased progressively, be- ginning at 1 500 in the propus, to 1 700 in the fourth carpal segment, to 2000 in the second carpal segment, to 2400 in the merus. The difference in number between the pro- 164 A. T. READ ET AL. Figure 4. Photomicrograph of the thoracic nervous system in a freshly dissected shnmp, showing the paired hemiganglia to the first ( 1 ), second (2), third (3), and fourth (4) thoracic limbs, the nerves (arrows) from these hemiganglia and the opening (asterisk) for the dorsal artery in the connective between the third and fourth ganglia. Note the larger size of the second ganglia compared to the first or third. Scale bar: 250 /im. pus and merus of 1000 represents the number of sensory axons originating in the multisegmented carpus. B. Connectives. Apart from the nerves, the only other external source of neural input to the ganglia is via the connectives (Fig. 4). Therefore the number of axons that enter the ganglia via the connectives may be estimated by counting axon profiles in both the anterior and pos- terior connectives to the ganglion, as this would encom- pass both ascending and descending inputs. Consequently, counts were made in two animals, of the connectives an- terior to the first, second, and third thoracic ganglia to estimate the anterior and posterior inputs to the first and second thoracic ganglia (Table II). The number of axons in the paired, left and right connectives at each of the three sampling stations were highly symmetrical. But the number in each of the three sampling stations was distinct, indicative of the relative degree of neural traffic to and from each of the first and second thoracic ganglia. Because the direction of the neural traffic i.e.. whether it is as- cending, descending or through-going cannot be distin- guished in these cross-sections of the connectives, the numbers from the anterior and posterior connectives were simply added as an estimate of the input into each of the first and second thoracic ganglia. The second thoracic ganglion had a higher number than the first, approxi- mately 4000 versus 3000 axons, suggesting that the neural input is greater to the second thoracic ganglion than to the first. Behavior Both casual and formal observations of adult snapping shrimps in glass aquaria show the second thoracic limbs to be almost constantly active, primarily in probing the environment and, to a lesser degree, in grooming body parts. Probing is the rapid, jerky touching of the substrate or other objects by the chelae of the flexible limb. This is greatly facilitated by the multi-segmented carpus, which gives the limb enhanced flexibility, allowing it to probe deeply into the benthic crevices. Probing occurs when the shrimp are crawling (back- wards and forwards), burrowing (shovelling substrate with pereiopods, pleopod beating of substrate), or just standing still (including pleopod beating of the water). Grooming involves picking at various parts of the body with the rapidly opening and closing chelae of the flexible limbs. Although virtually every part of the body is acces- sible, the most frequently groomed areas include the ros- trum, gills, ventral thorax and abdomen (including pleo- pods), and the large chelae. The flexible limb also retrieves and brings food to the mouth. Occasionally these limbs lifted and carried rela- tively heavy objects, such as large pebbles. Time budget. As mentioned previously, one of the most striking aspects of the flexible limbs was their almost con- stant activity. This is reflected in the time budget analysis (Table III), which shows these limbs as being active 95% of the time and inactive only 5%. The majority (77%) of this activity was devoted to probing the substrate, be it sand or gravel. Moreover, immobilizing the carpal seg- ments, by gluing them, had little effect on the probing activity. Probing occurred whether the shrimp was sta- tionary, crawling, or burrowing, and the time devoted to this activity was approximately similar for each of the three states. Grooming was the only other activity that occupied a significant amount of the time of the flexible limbs, al- though the time occupied (13%) was considerably smaller than that spent in probing. Grooming was confined prin- cipally to the head region. Foraging experiments. The flexible limbs were actively engaged in foraging for food, the introduction of which caused very intense probing activity. When found, food was retrieved and brought to the mouthparts exclusively by the flexible limbs. Sometimes, the food was held by the pincer while bits were torn off by the flexible limb chelae, and the pincer was occasionally used to push the food into the sand. Rarely, jets of water produced by clo- sure of the snapper were used to uncover food. SHRIMP FLEXIBLE LIMB 165 2 ^:fMp. .. ' t* * / *. :' M "> -I ^% ^%- - *H^* K^'lldk ** * "" ;r* v ^- : . # ^ J, Jifs. ;; ^Pi^^ ft i 1 ^* ^ B Figure 5. (A) Cross-section of the small first ( 1 ) and large second (2) nerves at the base of the hemiganglion to a second thoracic limb: each nerve has tightly packed axons and prominent nuclei of Schwann cells. (B) Representative area of the nerve cross-section showing clusters of small unmyelinated axons which are naked while myelinated axons have a densely stained sheath and scattered dense staining nuclei. Scale bars: A. 50 Mm; B. 5 ^m. 166 A. T. READ ET AL Table I Number of axon profiles in the paired (right and left) nerves to the first and second thoracic limbs in two snapping shrimps Table II Number of axon profiles in the paired (left and right) connectives to the first and second thoracic ganglia in two snapping shrimps Thoracic limbs Myelinated Unmyelinated Both Connectives Total Shrimp #1 i noracic loiai unienor ana ganglia left right (right and left) posterior) First thoracic limb Shrimp #1 right (pincer) left (snapper) Second thoracic limb 4071 4286 6441 8722 10512 1 3008 First thoracic 403 443 846 Second thoracic 1187 1260 2447 Third thoracic 876 846 1722 3293 to first ganglion 4169 to second ganglion right 3197 3072 6269 left 2885 3117 6002 Shrimp #2 First thoracic limb Shrimp #2 First thoracic 486 454 940 Second thoracic 1087 II 10 2197 Third thoracic 1120 1031 2151 3 1 37 to first ganglion 4348 to second ganglion right (pincer) 3247 6214 9461 left (snapper) 4637 9232 13869 Second thoracic limb right 3451 3880 7331 Discussion left 3691 3791 7482 To evaluate the role of the second thoracic limbs in locating and retrieving food, these functions were com- pared in two groups of 10 shrimp each. The flexible limbs of the first group were intact; they were autotomized in the second group. In 2 1 separate trials, the intact shrimps were able to locate and retrieve the concealed food in all cases. The time taken to locate the food was 72 18s (SEM), and to retrieve it with the second thoracic limbs added only a few more seconds (80 18 s SEM). Un- expectedly, the autotomized shrimps, in 1 7 out of 1 9 sep- arate trials, successfully located the concealed food and the time taken was 1 15 24 s (SEM) which was not significantly different (Students /-test) from the time for the intact group. The food location was done by frequent and direct contact of the substrate with the mouthpart region, i.e.. substrate pressing. However, once the food was located via such substrate pressing, the shrimps were unable to retrieve it because the second thoracic limbs were missing. Only in exceptional cases, and with the per- sistent use of other appendages, were the autotomized shrimps able to retrieve the food placed in the coral. Clearly, the second thoracic limbs were not required in locating concealed food, but were indispensable for re- trieving it. Conspeciftc interact ions. To determine the role of the second thoracic limbs in conspecific interactions, obser- vations were made of male/female and male/male pairs. The flexible limbs appeared to play little part in hetero- or homosexual interactions, and, in fact, during such en- counters, the limbs were held reflexed posteriorly in a locked position, completely out of the way. The chitinized exoskeleton of crustaceans restricts flex- ibility within a limb to its segmental joints. Hence the unusual occurrence of a multisegmented carpus in the second thoracic limb of the snapping shrimp, Alpheus heterochelix, makes this limb particularly adept at ex- ploratory behavior, food gathering, and grooming. Such behaviors occur constantly and are subserved by a gan- glion which is larger than its neighbors. This size difference is related to the greater number of axon profiles in the longitudinal connectives to this ganglion than to the neighboring ganglia. The significance of these findings concerning the external and internal morphology of the second thoracic limbs and their behavior are discussed below. Table III Time budget for activities related to the second thoracic limbs in snapping shrimps % of time Sand Gravel Overall Probing: total 79.5 75.1 77.0 burrowing 16.8 32.3 24.6 crawling 34.1 20.5 27.3 stationary 28.6 ->i "> 25.2 Grooming: total 16.3 10.4 13.3 head 12.1 7.4 9.8 chelae 2.5 1.0 1.7 abdomen 1.7 2.0 1.9 Miscellaneous 3.0 6.7 4.8 Inactive 1.2 8.4 4.8 Number animals/observations 6/405 6/405 12/810 SHRIMP FLEXIBLE LIMB 167 Morphology The features of the second thoracic limb in the snapping shrimp. Alpheus heterochelis. that prompted this study were: its multisegmented carpus, its unusual flexibility, and its ganglion, which is larger than that of its neighbors. The first two are directly related, in that division of the carpus into five segments makes the limb highly flexible. The extent to which this specialization in the structure and function of the second thoracic limb regulates the size of its ganglion is somewhat more difficult to resolve. A simple possibility was that the input to the ganglion would regulate its size, and we tested this possibility by counting the number of axon in the limb nerve and the longitudinal connectives to this ganglion. Numbers of axons in nerves. Cross-sections of nerves to the thoracic ganglia in several crustaceans reveal thou- sands of axon profiles of varying diameters, with the ma- jority being small (<5 M) (Sutherland and Nunnemacher, 1968; Govind and Pearce, 1985). Some of the large profiles are presumably motor axons. and these are comparatively few because crustacean limb muscles are innervated by only a few ( 1 -5 ) motor axons (Govind and Atwood, 1982). For each of the distal segments of the limb the merus, carpus, and propus there are typically two antagonistic muscles, and each muscle is innervated by 2-5 axons: thus there are altogether about 30 axons. Another 30 mo- tor axons would account for the more proximal muscles of the limb, making a total of 60 motor axons. This num- ber is a very small percentage of the thousands of axons counted in the nerves. The vast majority of axons in these nerves are presum- ably sensory. Consequently, counting axon profiles in these nerves provides an index of the sensory input to the ganglion. Furthermore, because the size of the ganglion will, in part, be governed by the sensory input, differences in the axonal counts between the first and second thoracic limbs may underlie the differences in size of the respective ganglia. The correlation we find between axon numbers and size in the snapping shrimp hemiganglia is a negative one, however. The chelipeds, both of which have a substantially larger number of axons (9,000 and 13,000), are each as- sociated with a smaller ganglion than that of the second thoracic limbs. Therefore, as a first approximation, the peripheral input to the ganglia cannot explain the size differences between the first and second ganglia. Alternatively, the size differences between the ganglia might be attributed to the extra carpal segments in the second thoracic limb; there are five of these, compared to only one carpal segment in the chelipeds. Each of the five carpal segments in the second thoracic limb has at least three muscles, and they may receive their own motor axons. If this were the case, and assuming that each muscle receives a minimum of two axons, the extra segments would add an additional 24 motor axons. Because each motor axon extends a dendritic tree within the neuropil, it is likely that the addition of 24 motor axons would increase the size of the neuropil. Numbers of axom in the connectives. Apart from pe- ripheral nerves, the only other source of nerve input to the ganglia is via the connectives. The total input to each of the ganglia was estimated by simply adding the numbers of axons in the connectives anterior and posterior to each ganglion; ascending and descending input could not be distinguished. A positive correlation was seen between axon numbers and the size of the ganglia, as there were 4000 axons to the second thoracic ganglion, and 3000 axons to the first ganglion. This 25% difference is close to the 25-30% difference in the size of the hemiganglia. In summary, the greater size of the second thoracic ganglion, relative to its first thoracic counterpart, is not simply due to differences in the number of sensory axons from the periphery to the ganglion. Consequently, it must be due to central factors. Finding a greater number of axons in the ventral connectives to the second thoracic ganglion, compared to the first, is consistent with this conclusion. The exact nature of these central features is unknown, but they may be related to the specialized na- ture of the second thoracic limb as a highly flexible me- chanosensory "arm" capable of delicate movements. Consequently, we may anticipate additional motor axons, as in the extra carpal segments, a higher concentration of proprioceptors and, perhaps, a higher concentration of interneurons emanating from the ganglion. All of these features would contribute to increasing the size of the ganglion. Behavior Nolan and Salmon (1970) observed that A. heterochelis spent most of its active time grooming. In contrast, we found that the overwhelming majority of time was spent probing. Undoubtedly though, the flexible limbs are vitally important in both functions and are in almost constant motion, whether the shrimp is burrowing, crawling (walking), cleaning, feeding, or stationary. The ability to effectively "put them away" during agonistic encounters perhaps underscores their importance. Food foraging. Animals that had autotomized their flexible limbs were at a great disadvantage in foraging for food. The ability to find concealed and hard-to-get-at food would obviously be beneficial in the coral reefs, oyster beds, and sponges frequently inhabited by A. heterochelis (Brooks and Herrick, 1892). Since alpheid shrimps spend much time actively browsing across the substrate, using their flexible limb chelae as probes and micromanipula- tors, this is likely an important means of foraging. In this 168 A. T. READ ET AL. respect they are similar to prawns (Hindley and Alexander, 1978). However, alpheid shrimps are reported to stun prey such as other shrimps (McLaughlin, 1982) or to crack open clam shells by means of their snap. This would be another means of foraging, although the availability of such large prey would likely be insufficient to support a dense population of shrimps (Dahl, 1968). Autotomized shrimp easily located concealed food, suggesting that the limbs are not important for olfaction. Indeed, the flexible limb has relatively few external sen- silla, even taking into account their small size. Those seen were similar to sensilla observed on the chelipeds ( Read and Govind, 1990). Functions of the flexible limb sensilla, probably related to foraging, may include contact che- moreception (i.e.. taste as opposed to olfaction), contact mechanoreception, and perhaps texture sensitivity. Grooming. Grooming reduces the incidence of epizoic and sediment fouling, which could seriously affect the health and sensory and locomotory abilities of individuals and increase the mortality of brooding embryos (Bauer. 1975, 1979). A. helemchelis, in particular, is parasitized by a conspicuous epicaridean isopod. Seen in about 2% of captured specimens, this parasite lodges in the ventral surface of the abdomen (pers. obs.), undoubtedly impair- ing the reproduction, if not the health and the molt cycle of affected animals. Obviously, grooming would be im- portant in discouraging this parasite. In addition, A. het- erochelis hosts another parasite which was a sac-like or- ganism filled with eggs and which lodges beneath the car- apace in the thoracic region, an area which is subject to very frequent grooming. Molting, which occurs every 18- 25 days, does not rid the shrimp of either the abdominal or thoracic parasites, thereby underscoring the importance of grooming. Acknowledgments We thank Blair Feltmate for advice on the behavior experiments. Bill Kirby-Smith for collecting snapping shrimps, and Christine Gee and Joanne Pearce for animal rearing and maintenance and for criticism of the study. Financial support was provided by the Natural Sciences and Engineering Research Council of Canada. Literature Cited Atwood, H. L., F. Lang, and W. A. Morin. 1972. Synaptic vesicles: selective depletion in crayfish excitatory and inhibitory axons. Science 176: 1353-1355. Bauer, R. T. 1975. Grooming heha\ lor and morphology of the candean shrimp Panda/us danae Stimpson (Decopoda: Natantia: Pandalidae). /.ool ./ Linn. Site. 56:45-71. Bauer, R. T. 1979. Anti fouling adaptations of marine shrimp (Deca- poda: Candea): gill cleaning mechanisms and grooming of brooded embryos. Zoo/. J. Linn. Sue. 65: 281-303. Brooks, \V. K., and F. H. Herrick. 1892. The embryology and meta- morphosis of the Macrura. Mem. Nat. Acad. Sci. 5: 322-576. Bullock, T. H., and G. A. Horridge. 1964. Structure and Function in the Nervous System of Invertebrates. W. H. Freeman & Co., San Francisco. Dahl, W. 1968. Food and feeding of some Australian peneid shnmp. F. A O. Fish Rep. 57: 251-258. Govind, C. K., and H. L. Atwood. 1982. Organization of Neuromuscular Systems. Pp. 63-103 in The Biology of Crustacea, Vol. 3. Neuro- hmloxy Structure and Function. H. L. Atwood and D. C. Sandeman. eds. Academic Press, New York. Govind, C. K.. and J. Pearce. 1985. Laterahzation in the number and size of sensory axons to the dimorphic chelipeds of crustaceans. ./. Nei/robiol 16: I 11-125. Govind, C. K., and J. Pearce. 1988. Remodelling of nerves during claw reversal in adult snapping shrimps. J Comp. Neurol 268: 121-130. Heuser, J. E., and C. F. Doggemveiler. 1966. The fine structural or- ganization of nerve fibers, sheaths, and glial cells in the prawn Pa- lacinoneles vulgaris. J. Cell Bio/. 30: 381-403. Hindley, J. P. R., and C. G. Alexander. 1978. Structure and function of the chelate pereiopods of the banana prawn Penaeus merguiensis. Mar, Bio/. 48: 153-160. U'hner, P. N. 1979. Handhook of Ethological Methods. Garland STPM Press, New York. McLaughlin, P. A. 1982. Comparative morphology of crustacean ap- pendages. Pp. 197-256 in The Biology of Crustacea, Vol. 2, Em- /inv>/f't,'r. Morphology and Genetics, D. E. Bliss, ed. Academic Press, New York. Mellon, DeF., Jr., and P. J. Stephens. 1980. Modification in the ar- rangement of thick and thin filaments in transforming shrimp muscle. J. E\p Zoo/. 213: 173-179. Nolan, B. A., and M. Salmon. 1970. The behaviour and ecology of snapping shrimp (Crustacea: Alp/wits heterochelis and Alpheiis nor- 1111111111). Forma Funclio 2: 289-335. Pearce, J., C. K. Govind, and R. R. Shivers. 1986. Intramembranous organization of lobster excitatory neuromuscular synapses. / Neu- rocytol 15: 241-252. Prizbram, II. 1901. Experimentell Studien liber Regeneration. Arch. EnlH-iek. Mech. 11: 321-345. Phillips, C. E., J. A. Wilson, and DeF. Mellon, Jr. 1982. A comparative study by serial section electron microscopy of neuromuscular junc- tions in the dimorphic claws of the snapping shrimp, Alpheiis het- ernchelis. J Neurohiol 13: 495-505. Read, A. T., and C. K. Govind. 1990. Composition of external setae during regeneration and transformation of the bilaterally asymmetric claws of the snapping shnmp. Alpheu.i heterochelis. J Morphol 207: 1-9. Ritzmann, R. 1974. Mechanisms for the snapping behavior of two Al- pheid shnmps, Alpheiis calitormensis and Alpheiis heterochelis. J. Comp Physiol 114: 91-101. Schmitt, W. L. 1975. Crustaceans. University of Michigan Press. Ann Arbor. Sutherland, R. M., and R. F. Nunnemacher. 1968. Microanatomy of crayfish thoracic cord and roots. J. Comp. Neurol. 132: 499-518. Wiersma, C. A. G. 1961. The Neuromuscular System. Pp. 191-240 in The Physiology of Crustacea. T. H. Waterman, ed. Academic Press, New York. Wilson, E. B. 1903. Notes on the reversal of asymmetry in the regen- eration of chelae in Alpheiis hclerochelis. Bio/ Bull. 4: 197-210. Reference: Bin/. Bull. 181: 169-174. (August. 1991) Ecdysteroid Treatment Delays Ecdysis in the Lobster, Homarus americanus JIN-HUA CHENG 1 AND ERNEST S. CHANG 2 Bodega Marine Laboratory, University of California, P. O Box 247, Bodega Bay. California 94923 Abstract. Premolt stage D 3 of juvenile lobsters, Homarus americanus. was further divided into five substages ac- cording to the degree of cuticle digestion along the dorsal midline of the carapace and on the dorsal surface of the merus of the chelipeds. The mean times to ecdysis for intact lobsters (5.5 1.1 g) at each substage were 71.4, 57.7, 30.0, 16.1, and 6.6 h, respectively. The level of ec- dysteroids dropped continuously during stage D,, from 0.5 Mg/rnl at substage 1 , to less than 0. 1 Mg/ml at substage 5. Injections of 20-hydroxyecdysone (20-HE) (1.0 or 5.0 Mg/g) delayed ecdysis in animals receiving an injection at substages 3 or 4, but not 1, 2, or 5. The staging method can be applied to eyestalk-ablated (ESX) lobsters as well; but those animals complete stage D, and molt much more rapidly. In addition to the time of ecdysis, the rate of development (based on the degree of cuticle digestion) in both intact and ESX lobsters was decreased by injections of 20-HE. We conclude that decreased ecdysteroid tilers in the hemolymph of lobsters is a prerequisite to the ini- tiation of ecdysis, and that rates of development during stage D, are regulated negatively by ecdysteroids. We sug- gest that the time of ecdysis is controlled in lobsters through the regulation of the rate of decline of ecdysteroid liters. Introduction Like most olher crustaceans, lobsters (Homarus amer- icanus) increase iheir body size by moiling. Before each moll, Ihe inner layers of Ihe old culicle are reabsorbed, and Ihe ouler layers of the new cuticle are synthesized underneath the old one (Skinner. 1985, for review). Shed- Received 20 December 1990; accepted 8 April 1991. 1 Present address: Tungkang Marine Laboratory, Tungkang, Pingtung, Taiwan 928, Republic of China. 2 To whom all correspondence should be addressed. ding Ihe culicle al ecdysis is accompanied by a sudden increase in body weighl because of rapid waler uplake (Mykles, 1980). This waler occupies Ihe space made available by the larger and more pliable new cuticle syn- thesized before molt. The enlarged space is needed for subsequenl lissue growth prior to Ihe nexl moll. This cyclic event which is repeated throughoul Ihe life of many crus- taceans, is under the control of ecdysteroids (moiling hor- mones) (Chang, 1989, for review). In lobslers, as in many olher cruslaceans, Ihe ecdyste- roid level rises during early premolt and reaches its peak at lale stage D : , or early D,, then drops lo a low level immediately before ecdysis (Chang and Bruce, 1 980, 1981; Chang and O'Connor, 1988). A similar ecdysteroid profile has been found in insects (Steel and Vafopoulou, 1989, for review). In bolh insecls and cruslaceans, Ihe rising liters of ecdysteroids initiate Ihe cascade of evenls lhal culminate in ecdysis (Skinner, 1985; Chang, 1989). In addilion, decreasing tilers of ecdysteroids in lale premolt are necessary for Ihe inilialion of ecdysial behavior in some insecls (Slama, 1 980; Truman eta/.. 1983; Reynolds, 1986; Zdarek and Denlinger, 1987). Injections of ecdysteroids during late premolt inhibit ecdysis in some amphipods (Graf. 1972a, b). Similar re- sulls. Ihough, have not been oblained in olher cruslacean species (Skinner. 1985, for review). One possible expla- nation for these negalive resulls is lhat injections were not made al Ihe right time. In an insect, Manduca sexta. injeclions made before or after Ihe crilical period have no effecl on Ihe lime of ecdysis (Truman el a/.. 1983). The lack of a precise slaging melhod for cruslaceans in lale premoll may have prevented Ihe discovery of an analogous crilical period. In insecls, decreasing liters of ecdysteroids prior to molt not only regulate Ihe time of ecdysis itself, but also mod- ulate Ihe rate of premolt developmenl (Schwartz and Truman, 1983). Although crustaceans can regulate the 169 170 J.-H. CHENG AND E. S. CHANG time of ecdysis to varying degrees (Skinner, 1985), a study comparable to that in insects has not yet been conducted. During the last few days before ecdysis, certain regions of the cuticle are digested to a greater extent than others, enabling the lobster to shed its old exoskeleton. In our study, we developed precise staging criteria for stage D, lobsters. The time of ecdysis during the last three days of the molt cycle could be predicted on the basis of these criteria. In addition, the effect of injecting 20-hydroxyec- dysone on the rate of cuticle digestion and the time of ecdysis was investigated. Materials and Methods Maintenance of animals Two families of full-sibling juvenile Hoinams ameri- canns(5.5 1.1 and 3.2 0.7 g wet weight; mean S.D.) were used for these experiments. The lobsters were raised in a semi-recirculating system at 20.0 0.5 C on a diet of live adult brine shrimp (Chang and Conklin, 1983; Conklin and Chang, 1983). The photoperiod was 16L:8D. Molt prediction Before stage D,, intact and eyestalk-ablated (ESX) lob- sters (5.5 g) were staged based on the setal development of the pleopods (Aiken, 1973). Stage D, lobsters, about three days before molt, were further subdivided into five substages, based on the degree of cuticle breakdown along the dorsal midline of the carapace, and on the dorsal sur- face of the merus of the chelipeds (Table I). These are areas where extensive cuticle digestion occurs prior to ec- dysis. For staging, the lobster's dorsal carapace was wiped dry and evaluated with a dissecting microscope. Exogenous ecdysteroid injection Intact lobsters (5.5 g) at various substages of stage D 3 received a single injection (0.2, 1 .0, or 5.0 ^g/g wet weight) of 20-hydroxyecdysone (20-HE; Rohto Pharmaceutical; purity checked by high-performance liquid chromatog- raphy before use). Control animals received vehicle only (lobster saline; Mykles, 1981). ESX lobsters (3.2 g) were used for one injection study. Both eyestalks were removed from stage B-C animals, with fine scissors, about 5-10 days after molting. When animals were approaching the first postoperative molt, they were staged by the same method described above and injected with lobster saline, either containing 20-HE or not. Only one dose of 20-HE (1.0 ^g/g wet wt.) was used per animal in these experiments. The time between injection and ecdysis was recorded on time-lapse video with a time stamp. Endogenous ecdysteroid determination Before injection, a hemolymph sample (25 n\) was taken from each animal and the content of its endogenous ec- dysteroids determined quantitatively by radioimmunoas- say (Chang and O'Connor, 1979); the IB-4 antiserum from Dr. W. E. Bollenbacher was used (University of North Carolina, Chapel Hill). Rate of development The effect of ecdysteroid liters on the rate of progression through the D, substages was investigated. Lobsters (5.5 g for both intact and ESX animals) received an injection of 20-HE ( 1 .0 Mg/g). once per day, starting from substage 1 of stage D, . The rates of development were recorded twice daily based on criteria described in detail below. Results Stage D, was divided into five substages according to the degree of the cuticular digestion in the regions shown in Figure 1. At substage 1, only the posterior region of the dorsal midline of the carapace shows signs of digestion. This is manifested as a narrow crack that starts at the posterior end of the midline and proceeds anteriorly about one-third of its total length. At substage 2, another narrow crack appears in the dor- sal midline of the carapace. It starts from the anterior of the carapace and proceeds posteriorly to about one-third of the total length of the dorsal line. At substage 3, the narrow crack forms along the entire dorsal midline. At substage 4, the crack widens to occupy the entire width of the dorsal midline along the posterior two-thirds of its length. At substage 5, the crack widens along the entire length of the midline. Also, the dorsal surface of the merus of each cheliped becomes soft (Ta- ble I). The times to ecdysis for animals in substages 1 to 5 are given in Table I. Although the molt-staging technique can be applied to ESX lobsters, these animals spend much less time in each substage (Table I). The levels of the ec- dysteroids in the hemolymph of intact lobsters drop con- tinuously during stage D,, from 0.55 /ug/ml at substage 1. to less than 0.1 /jg/ml at substage 5 (Fig. 2). The role of ecdysteroids in regulating the time of ecdysis was examined by injecting stage D, lobsters with various doses of 20-HE. Lobsters were divided into the five sub- stages as described. Each animal received a single injection of lobster saline, with or without 20-HE. A low dose (0.2 Mg/g) of 20-HE did not significantly delay ecdysis in an- imals receiving the 20-HE injection at any substage (Fig. 3). High doses ( 1 .0 and 5.0 ng/g) delayed ecdysis in ani- mals receiving an injection at substages 3 and 4, but not 1, 2. or 5 of stage D,. LOBSTER MOLT STAGING AND REGULATION 171 Figure 1. Carapace of juvenile lobsters, Homanis americanus. showing the progress of cuticular digestion along the dorsal midhne. Single arrow indicates slight digestion. Double arrows indicate heavy digestion (see Table I). The numbers indicate the substages of D, . Field of view is approximately 7 x 20 mm. Ecdysis of ESX lobsters receiving an injection of 20- HE was also delayed (Fig. 4). Ecdysis was delayed in all lobsters receiving a 20-HE injection at substages 1 or 2, and in seven of nine lobsters at substage 3, but in none of the lobsters at substages 4 and 5. The unresponsiveness of the two lobsters (at substage 3) to the 20-HE injections might have been due to lower ecdysteroid liters (0. 1 8 and 0.28 Mg/ml) than the other seven animals (range of 0.35 to 0.66 Mg/ml). These two animals may have been ap- proaching substage 4. Injection of 20-HE not only delayed ecdysis, but also depressed the rate of development. This was assayed by the degree of cuticle digestion over time in both intact and ESX lobsters (Fig. 5). Table I Siibsiugn ofDj based upon cuticle digestion in both intact and eyestalk-ablated juvenile Homarus americanus (5.5 g) at 20C Cuticle digestion at Dorsal midline of carapace Hours before ecdysis (n) SE ) Substage Anterior Central Posterior Dorsal surface of ofD, region region region merus of cheliped Intact Ablated 1 + hard 71.4 9.5 (9) 32.4 8.2 (8) 2 + + hard 57.7 4.0(9) 21.8 5.4(6) 3 + + + hard 30.0 6.0(6) 10.8 5.0(6) 4 + ++ ++ hard 16.1 2.0(7) 6.0 1 .0 (4) 5 ++ + + ++ soft 6.6 0.7(7) 4.2 2.3 (5) - Indicates digestion absent, + indicates slight digestion (width of the crack is less than the width of the dorsal line). ++ indicates heavy digestion (width of the crack is wider than the width of the dorsal line). 172 J.-H. CHENG AND E. S. CHANG 0.8 E 0.6 5" tr UJ 0.2 Q O UJ 0.0 12345 SUBSTAGE OF D 3 Figure 2. Ecdystcroid tilers in the hemolymph of intact 5.5 g lobsters, plotted against substage of D, (means 1 S.D.). Each datum point rep- resents 10-19 animals. Discussion Intensive cuticle digestion along the dorsal midline of the carapace starts about three days before ecdysis and permits lateral expansion of the carapace at ecdysis. The intensive cuticle digestion on the dorsal surface of the merus of the chelipeds permits lobsters to withdraw their large chelipeds through the narrow basiischial joints. Cheliped withdrawal is also facilitated by the differential degeneration of cheliped muscle tissue (Mykles and Skin- 50 <> 40 Q a 'o to 20 rr I 10 D control | 1.0ug/g 1 12345 SUBSTAGE OF D 3 Figure 4. The effect of 20-HE injection (1.0 Mg/g) on time of ecdysis in eyestalk-ablated 3.2 g lobsters (means 1 S.D.). Animals received a single injection of saline with or without 20-HE at the stage indicated. The time of ecdysis was recorded using time-lapse video. Asterisks (** and ***) indicate significant differences (P < 0.0 1 and 0.001 , respectively). Each group contained 6-12 animals. ner, 198 1 ) and active water uptake (Mykles, 1980; Cheng, 1990). Our molt-staging technique, based on cuticle digestion, reliably predicted the time of ecdysis, which was critical for this study. This technique should also be applicable to other species, for research and for the commercial pro- duction of soft-shell crustaceans. 1 C.U D control V2 100 H / en fcj .z ug/g 8 80 UJ ii i * 1-OM9/9 | 5.0 ug/g * 60 h- A * ^ 40 1 20 H fis! n U i ^Ai u 1 : 3 4 5 SUBSTAGE OF D 3 Figure 3. The effect of injection of various concentrations of 20-HE (0, 0.2, 1.0, or 5.0 Mg/g) on time of ecdysis in intact 5.5 g lobsters (means 1 S.D.I. Animals received a single injection of saline with or without 20-HE at the stage indicated. The time of ecdysis was recorded using time-lapse video. Asterisks (* and **) indicate significant differences (P < 0.05 and 0.01, respectively). Each group contained 7-9 animals. Q b_ O UJ o CO CD CO A ablated control A A ablated treated o o intact control intact treated 24 48 72 96 120 TIME SINCE D, (hours) Figure 5. The effect of 20-HE injection on the rate of cuticle digestion in intact and eyestalk-ablated 5.5 g lobsters. Animals received an injection of saline with (treatedl or without (control) 20-HE (1.0 Mg/g) every 24 h. starting from substage I . The stage of cuticle digestion was subsequently checked every 12 h. Each group contained 7-12 animals. E indicates ecdysis. LOBSTER MOLT STAGING AND REGULATION 173 We observed that ecdysteroid tilers continued to decline during the last three days of the molt cycle. Similar results have been reported for juvenile lobsters in our laboratory (Chang and Bruce, 1980, 1981). The mechanisms re- sponsible for the decline are not clear, but a decrease in the rate of production of ecdysteroid by the Y-organ may be the predominant means of controlling the declining hormone liters. Evidence for this is Ihe observalion lhat Ihe secrelory rale of ecdysleroids by Ihe Y-organs of Pachygrapsus crassipes in vitro was correlaled wilh cir- culating ecdysteroid concenlralions (Chang and O'Con- nor, 1978). Both negalive and posilive short-loop feedback mechanisms have been demonslraled in Ihe prolhoracic glands of Manduca sexta (Sakurai and Williams, 1989). and similar mechanisms may operale in Y-organs of cruslaceans as well. In addilion, increased rales of deg- radalion of ecdysleroids in lale premoll may play an im- portanl role in regulaling Ihe declining lilers of ecdysle- roids (Snyder and Chang, 1991 ). High doses of 20-HE ( 1 .0 or 5.0 Mg/g) delayed moll in inlacl lobslers receiving injeclions al subslages 3 or 4. This observalion is consislenl with Ihe observalion in in- secls lhal Ihe decline in circulating ecdysteroids musl pre- cede Ihe occurrence of ecdysis (Slama, 1980; Truman et a!.. 1983; Reynolds, 1986; Zdarek and Denlinger, 1987). In addition, the delay of ecdysis by exogenous ecdysteroid in lobslers is similar lo observalions in insecls, where Ihere is a crilical period when animals are mosl sensilive lo hormonal Irealmenl (Truman et al., 1983; Zdarek and Denlinger, 1987). Inlacl lobslers become insensitive to exogenous 20-HE when Ihey reach substage 5, as developmenl nears com- plelion (within 7 h before ecdysis), and as the circulaling level of Ihe hormone declines lo ils low level (less than 0.1 Mg/ml). This insensilivity suggesls lhal Ihe ecdysial programs have already been Iriggered. In M. sexta, Ihis Irigger includes Ihe acquisilion of Ihe sensitivily lo and release of eclosion hormone (EH), which coordinale ec- dysial behavior and related physiological evenls (Truman, 1985). Allhough Truman et al. (1981) found EH activity in insects from five non-lepidopleran orders, and quan- tified il wilh a Manduca bioassay, direcl demonslralion of Ihe existence of an EH-like factor capable of initialing ecdysis has nol been reported in any non-lepidopteran. In crustaceans, an exuviation factor analogous lo EH has been proposed, based on Ihe observalion lhal ecdy- steroid injection blocked ecdysis in some amphipods (Graf, 1972a, b; Charmanlier and Trilles, 1976). There is no direcl evidence, however, for EH in cruslaceans. Pep- lide exlracls from brains and Ihoracic ganglions of pre- and poslmoll crabs were assayed for EH activily, all wilh negalive resulls (Cameron, 1989). Cruslaceans and non- lepidopleran insecls may nol use an EH-like factor lo initiate ecdysis, but instead direclly rely on Ihe declining liters of ecdysteroids lo Irigger ecdysial behavior. The declining ecdysteroid lilers al lale premoll influence olher aspecls of developmenl, in addilion lo the lime of ecdysis. As shown in Figure 5, culicular digestion was delayed by multiple injeclions of 20-HE. These resulls suggest lhal Ihe events in late premolt are negatively mod- ulated by ecdysteroids. This is consistenl wilh observalions in insecls (Schwartz and Truman, 1983). In M. sexta, however, morphological developmenl was completely suspended by continuous ecdysteroid infusion. The neg- alive effecl of ecdysleroids on lale premoll evenls is in conlrasl lo Iheir posilive effecl on developmenl al olher moll slages in bolh insecls and cruslaceans. Acknowledgments We lhank D. K. Aronstein, M. J. Bruce, and W. A. Hertz for technical assistance and Dr. M. Snyder for help- ful discussions. The gifts of lobslers from M. Syslo (Mas- sachusetts State Lobster Halchery and Research Slalion) and ecdysteroid anlisera from Dr. W. E. Bollenbacher (Universily of North Carolina, Chapel Hill) are gratefully acknowledged. The Aquacullure and Fisheries Program of Ihe Universily of California, Davis, is acknowledged for financial support (lo J.-H.C.). This work is a resull of research sponsored in part by NOAA, National Sea Granl College Program, Departmenl of Commerce, under Granl NA85AA-D-SG140, Projecl R/A-80, Ihrough Ihe Cali- fornia Sea Granl College Program (lo E.S.C.). The U.S. Governmenl is authorized lo reproduce and dislribute copies for governmental purposes. Literature Cited Aiken, D. E. 1973. Proecdysis, setal development, and molt prediction in the American lobster (Homarus americanus). J. Fish. Res. Board Can 30: 1337-1344. Cameron, J. N. 1989. Post-moult calcification in the blue crab, Cal- linecles sapidus: timing and mechanism. J Exp. Biol. 143: 285-304. Chang, E. S. 1989. Endocrine regulation of molting in Crustacea. Rev. Aquatic Sci. 1: 131-157. Chang, E. S., and M. J. Bruce. 1980. 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Taghert. P. F. Copenhaver, N. J. Tublitz, and L. M. Schwartz. 1981. Eclosion hormone may control all ecdyses in insects. Nature 291: 70-71. Truman, J. W., D. B. Rountree, S. E. Reiss, and I.. M. Schwartz. 1983. Ecdysteroids regulate the release and action of eclosion hor- mone in the tobacco hornworm Manduca sexta (L.). J. Insect P/irsiol. 29: 895-900. Zdarek, J., and D. L. Denlinger. 1987. Pupal ecdysis in flies: the role of ecdysteroids in its regulation. / Insect Physiol. 33: 123-128. Reference: Bint. Bull 181: 175-180. (August, 1991) Long-Term Culture of Freshwater Mussel Gill Strips: Use of Serotonin to Affect Aseptic Conditions D. B. GARDINER, F. S. TURNER. J. M. MYERS, T. H. DIETZ, AND H. SILVERMAN Department of Zoology and Physiology. Louisiana Slate University. Baton Rouge. Louisiana 70803 Abstract. Serotonin relaxes the musculature and in- creases epithelial ciliary activity in freshwater mussel gills. This results in greater than normal water flow through the labyrinth of water canals and channels of the gill. These water spaces harbor significant microbial populations that make aseptic culture of freshwater mussel gills difficult. High concentrations of antibiotics can maintain short- term cultures, but are toxic to the tissue and reduce the lifespan of the culture. Moderate levels of antibiotics used in combination with 0. 1 mA/ serotonin during a single, short pretreatment produces aseptic cultures. These cul- tures can now be established routinely and are viable for over a month as assayed by gill structural integrity, trypan blue exclusion, leucine incorporation into TCA precipi- table protein, and normal physiological responsiveness to serotonin re-exposure. Introduction Attempts at establishing molluscan organ cultures have been made since the mid-1920's. Zweibaum (1925) tried to culture freshwater mussel gills in various diluted ver- tebrate Ringer's solutions enriched with peptone. Mantle tissue explants, from the land snail Helix aspersa (Ga- tenby, 1 93 l;Gatenby etai, 1934) and the marine bivalve Pinctata (Bevelander and Martin. 1949), were cultured. Cultured explants of the heart, lungs, and foot of various Helix species have also been reported (Konicek, 1933). Unfortunately, these organ cultures remained functional for only a few days. More recently, certain cells and tissues, particularly of pulmonate gastropods and some marine organisms, have been successfully cultured. The cell lines cultured were Received 12 December 1990; accepted 1 April 1991. Abbreviations: Penicillin (Pen); streptomycin (Strep); amphotericin B (Am-B); Artificial mussel hemolymph (AMH). derived from several tissues: ganglia, gills, heart, digestive tract, foot, mantle, oviduct, connective tissue, and gonad (BurchandCuadros, 1965; Cheng and Arndt, 1973; Han- sen, 1975; Kaczmarek et al.. 1979; Sengal, 1961a,b; Ste- phens and Hetrick, 1979; Vago and Chastang, 1958). Un- fortunately, attempts at culturing freshwater mussel tissue have failed due to fungal and bacterial contamination (Sengel, 1964), and the lack of well-defined maintenance media. The gills of freshwater mussels are complex organs that function in ion transport, respiration, food capture and sorting, storage and maintenance of embryos during re- production, calcium storage, and water movement. Sev- eral different cell types are associated with the gill, but culturing gill explants or sub-culturing specific cell lines allows the study of tissue- and cell-specific responses of the gill in isolation from the rest of the organism. Previ- ously we have studied gills excised from the animal im- mediately before use (Dietz and Findley, 1980; Dietz et al., 1982; Dietz and Hagar, 1990; Silverman etai, 1991). Having viable gill explants functioning in culture expands the range and duration of experiments that can be accom- plished. Here we report the successful long-term culture of freshwater mussel gills under aseptic conditions. A suitable medium, methods for reducing antibiotic exposure, and culture viability (as judged by normal appearance and physiological responses in gill tissue) are documented. Materials and Methods Animals and maintenance Freshwater mussels Anodonta grandis and Ligumia subrostrata were collected from shallow ponds near Baton Rouge, Louisiana. The animals were maintained in aer- ated artificial pond water (Table I) at 22-25C, and were 175 176 D. B. GARDINER ET AL. allowed to acclimate to laboratory conditions for a week before use. Initial short-term culture methods Gills were washed (3 X 20 min) in sterile pondwater or 30 mA/ Tns-HCl, pH 7.8. containing up to 1500 U ml"' penicillin G (Pen) and 1.5 mg ml" 1 streptomycin (Strep). The preliminary media we tested contained mul- tiple components chosen for their presence in diluted ver- tebrate culture media, or in mussel hemolymph (Table I). As the major source of the denned nutrients, we selected either a medium based on vertebrate Ham's F- 1 2 (Gibco) (dilution I), or artificial mussel hemolymph (AMH). All cultures contained 1 mg 1 ' phenol red, which allowed us to monitor pH. Cultures in each medium appeared normal and were viable for 1-4 days, but microbial and fungal contamination remained a problem. Antibiotic treatments To reduce fungal contamination, we exposed gill strips to amphotericin B (Am-B) in concentrations ranging from 25 to 500 Mg ml ' and for exposure times of from 1 to 24 h. We also pretreated intact animals in solutions of an- tibiotics at concentrations patterned after those used by Stephens and Hetrick (1979) to decontaminate oyster tis- sues. For such whole animal treatment, the following an- tibiotics were added to mussel Ringer's or pondwater (Table I): 1000 U ml" 1 penicillin G and 1 mg ml ' strep- tomycin, 100 ^/g ml" 1 neomycin, 50 Mg ml ' chlortetra- cycline, 1 25 Mg nil" ' gentamicin, 500 Mg ml ' kanamycin sulfate, 500 Mg ml"' polymyxin B sulfate, 100 Mg ml" 1 erythromycin, 25 Mg m ' ' amphotericin B, and 250 U ml" 1 nystatin. The mussels were immersed in this solution for 3-4 days. Gills were removed from both pretreated and non-pretreated clams, cut into 3-5 mm strips, and cultured as described above. Serotonin application Serotonin enhances the ciliary activity and relaxes the muscles of the gills in freshwater mussels (Gardiner et al., 1991), so we used it as an aid in decontamination. Gills were isolated and placed in sterile tubes containing 10~ 4 M serotonin in freshwater mussel Ringer's, pH 7.8. The solution was changed every 5 min for 20 min. The gills were removed and placed in sterile tubes containing freshwater mussel Ringer's with 500 U ml"' penicillin G, 0.5 mg ml" 1 streptomycin, 500 Mg ml" 1 colistin, 10 /ug ml" 1 chlortetracycline, 250 U ml ' nystatin, and 10 4 A/ serotonin, pH 7.8. This solution was replaced every 15 min for a total incubation period of 45 min. Gills were cut into 3-5 mm strips that were distributed in 24-well Corning culture plates containing an enriched Ham's F- 12 medium (Table I, Ham's F-12 dilution II) or artificial mussel hemolymph (AMH, Table I). Both media were fortified with 500 U ml" 1 penicillin G, 0.5 mg ml ' strep- tomycin, and 5 Mg ml ' chlortetracycline, pH 7.8. To some of these cultures, 250 U ml ' nystatin also was added. The pH was adjusted to 7.8, and the osmolality adjusted to 60 mosm (Precision System Micro Osmometer), as needed. The explants were maintained at 20-23C, and the explant was placed in a new well with fresh medium every two days. Assessment of tissue viability The initial assessment of viability was by observation, with an inverted microscope, of explant integrity, periodic muscular contractions, and ciliary activity. Trypan blue exclusion was used to determine cellular viability (Fresh- ney, 1987). Cells detached from the explant were placed in a 0.2% trypan blue in mussel Ringer's for 5 min. Cells were viewed under a light microscope, and dying ones were identified by their uptake of trypan blue. Serotonin ( 10 5 A/) increases the gill ciliary activity and relaxes gill musculature. We used these responses to assay for the proper physiological response of the explants. The explants were placed into fresh medium containing se- rotonin and observed for 5 min for muscular reflex activity and changes in the pattern of ciliary motion (Gardiner et al., 1991). Several cultures were assayed for their ability to incor- porate 'H-leucine into a trichloroacetic acid (TCA) pre- cipitable protein fraction. Approximately 1 nCi ml"' 3 H- leucine (specific activity 1 mCi MA/"' leucine) was added to AMH culture medium in which the leucine concen- tration had been reduced to 1 nAI. After a 1-h exposure, the gills were denatured with 10% TCA, rinsed in pond- water, blotted, and their wet tissue weights recorded. The gills were homogenized in 1 ml 10% TCA and centrifuged at 5000 X g for 5 min. The pellet was twice resuspended and centrifuged in 10%. TCA. The pellet was dissolved in 1 A/ NaOH, and the radioactivity was determined in a liquid scintillation counter. Protein concentration was determined by the method of Bradford (1976). Analysis of microbial contaminants Fungi growing in the cultures were identified only by the appearance of structural hyphae; no attempt was made to identify the species. The bacterial contaminants sur- viving antibiotic treatment were cultured in antibiotic- free Ham's F-12 dilution II (Table I) and sent to Louisiana State Veterinary Medical Diagnostic Laboratory for iden- tification. Results Our initial attempts to establish sterile gill explants in culture, using a variety of antibiotics, were unsuccessful. MUSSEL GILL CULTURE 177 Table I Composition of media used for culturing freshwater mussel gills Pond water FW mussel Ringer's Ham's F- 12 dilution I Ham's F-12 dilution II Art. mussel hemolymph FW mussel hemolymph Inorganic salts (m.U) CaCl, 0.40 5.0 0.04 0.03 4.79 a. b, c CuSO 4 -5H,O 1.4 x 10"' 1.0 x 10-' FeS0 4 -7H,0 4.2 x \Q-> 2.99 x 10~ 4 0.028 d KC1 0.05 0.5 0.438 0.313 a, b. c K.,HPO 4 0.197 a, b, c MgCI,-6H,O 0.09 0.061 0.187 b MnCl 2 -4H 2 O 0.12 d NaCl 0.50 5.0 18.2 13.0 16.18 a, b, c NaHCO 3 0.20 5.0 4.59 a. b, c Na,HPO 4 0.190 0.142 a, b, c Na,SO 4 5.0 b ZnSO 4 -7H,O 4.2 X 10~ 4 3.0 x 10 4 0.0048 d Amino acids (i*M) L-Alanine 14.0 10.0 55.0 55 7 (4) e L-Arginine 140.0 100.0 14.0 144(4)e L-Asparagine (I 14.0 10.0 L-Aspartate 14.0 10.0 27.0 27 10 (4) e L-Cysteine (1 28.0 20.0 L-Glutamate (I 14.0 10.0 54.0 54 16 (4) e L-Glutamine (1 140.0 100.0 Glycine 14.0 10.0 12.0 124(4)e L-Histidine 14.0 10.0 26.0 26 4 (4) e L-Isoleucine 4.2 3.0 8.0 8 1 (4)e L-Leucine 14.0 10.0 13.0 1 3 1 (4) e L-Lysine (1 28.0 20.0 15.0 15 4(4)e L-Methionine 4.2 3.0 L-Phenylalanine (1 4.2 3.0 8.0 8 1 (3)e L-Proline 42.0 30.0 14.0 14 1 (4)e L-Serine 14.0 10.0 83.0 837(4)e L-Threonine 14.0 10.0 134.0 59; I34e L-Tryptophan 1.4 1.0 L-Tyrosine 4.2 3.0 3.0 3.0 e L-Valine 14.0 10.0 14.0 14 2 (4)e Vitamins (mM) Biotin 4.2 X lO" 6 3.0 x I0' 6 4.09 x 10-' Choline chloride 0.02 0.01 7.16 X 10-' Folic acid 4.2 X 10- 4 3.0 X 10 4 2.26 x 10-' myo-inositol 0.014 0.01 0.011 Niacinamide 4.2 x 10~ 5 3.0 X ID' 5 8.19 x 10~' Pantothenate (Ca) 1.4 X \Q-< 1.0 X 10~ 4 2.09 x 10-' Pyridoxine 4.2 X 10~ 5 3.0 x 10~ 5 4.86 X 10~' Riboflavin 1.4 X 10~ 5 1.0 x 10~ s 2.70 x 10~ 4 Thiamine 1.4 x 10 4 1.0 x 10-" 2.96 x 10-' Vitamin B-12 1.4 x 10~ 4 i.o x 10-" Other (mA/) Glucose 1.4 5.55 5.55 Fetal serum (v/v) 8.7% 5% 5% Hemolymph (v/v) 8.7% 100% Hypoxanthine 0.0042 0.003 Insulin (U ml'') 0.25 Linoleic acid 4.2 x 10-' 3.0 X 10~ 5 Lipoic acid 1.4 x 10~ 4 pH 7.0 7.8 7.8 7.8 7.8 7.8 Phenol red (mgl"') 1.0 1.0 1.0 1.0 Putrescine 1.4 X 10~ 4 1.0 X 10~ 4 Pyruvic acid 0.15 0.1 TES buffer 2.0 Thioctic acid 1.0 x 10~ 4 Thymidine 1.4 x 10" 4 3.0 x 1Q-" Refer to (a) Dietz (1979), (b) Potts (1954), (c) Silverman el al . ( 1983), (d) Silverman el at.. ( 1987) for ionic composition and (e) Hanson and Dietz (1976) for amino acids in freshwater mussel hemolymph. Art. = artificial, FW = freshwater. 178 a D B. GARDINER ET AL Figure 1. (a) Light micrograph of Ligiimin .tiihro.slrala gill explant. in culture less than 24 h, showing normal filament (F) organization, (b) All of the explants remained functional for several days, but were contaminated by predominantly gram-negative bacteria of the genus Leukothrix, commonly associated with freshwater mussels and shrimp (Louisiana State Vet- erinary Medical Diagnostic Laboratory). In addition, the gill cultures treated with Am-B at concentrations up to 500 us, ml"' for 12 h had fungi within a few days. High doses of Am-B caused copious mucus secretion and in- hibition of ciliary activity. With the growth of fungi and bacteria, the culture medium rapidly acidified in spite of frequent (1-2 days) medium changes. Treatment with high concentrations of antibiotics was ineffective; 75% of the cultures still contained fungal con- tamination, and all contained 2-3 strains of bacteria. Ex- plants not contaminated with fungus remained viable for 10-14 days, but all had bacterial contamination. Pre- treatment of the intact animal with antibiotics did not reduce microbial contamination. The microorganisms were unlikely to be resistant to the wide spectrum of an- tibiotics we used; we conclude rather that the gill was harboring the microorganisms in the extensive branchial canals and channels, isolating them from the antibiotics in the medium. Serotonin prctrcatmenl oj gill explants Explants treated with serotonin rarely exhibited bac- terial or fungal contamination. When nystatin was only present in the initial wash, 15-25% of the explants had fungi. If nystatin were present continuously in the me- dium, only 4-8% of the cultures were infected. The se- rotonin pretreatment, in combination with moderate lev- els of antimicrobial agents, resulted in cultures that were visibly free of bacterial and fungal contamination. After 38 days in culture, 96% of the explants remained free of bacteria. Once pretreated with serotonin, gill explants remained viable and free of bacteria for over a month. Neither the cells in the explant nor detached cells showed any uptake of trypan blue. The gill explant is lined with a ciliated epithelium organized into filaments. Filament organiza- tion remained unchanged (Fig. 1), and ciliary and mus- cular activity continued throughout the life of the explant. Both muscular and ciliary activity were altered by re-in- Higher magnification micrograph of (a) showing filaments (F) and active cilia (arrows) indicated by the retractile halo pattern, (c) Light micrograph of Anodonta grandis gill explant cultured for 30 days, showing filaments (F) and active cilia (arrows) as indicated by the halo pattern, (d) Light micrograph of Anodonla grandis gill explant maintained in culture for 40 days. Gill integrity has been compromised and cells have detached from explant. This detachment is mostly observable at the filaments (F). Bars (a) = 137 /xm; (b) = 3? ^m; (c) = 37 M m; (d) = 77 jim. MUSSEL GILL CULTURE 179 traducing serotonin into the culture medium, and the ef- fects were the same as those seen in freshly isolated gills. That is, ciliary activity increased and was more coordi- nated, and the gill musculature was relaxed by serotonin treatment. Such treatment dilates the openings to the wa- ter canals in the gills (Gardiner et a/., 1991). Thus, phys- iological responses to a known effector of gill activity still occur in long-term explant culture. Media development Various media and supplements to those media have been examined for their ability to improve short-term gill culture (Table I). Osmolarity, mussel blood, vitamin so- lutions, and insulin are important factors in such consid- erations. For maintenance of gill explants for a week or less, both diluted Ham's F-12 and AMH were acceptable. At 10 days, some explants in Ham's F-12 showed in- creased cellular detachment, and at two weeks, ciliary ac- tivity was reduced or non-existent. AMH medium main- tained explants for over 30 days in culture, and explants exhibited coordinated ciliary activity and periodic mus- cular contractions. In addition, few cells detached from the explants cultured in AMH. Those explants continu- ously cultured with nystatin in the AMH could not be distinguished from those exposed only initially to nystatin. After 40 days in AMH culture, some explants lost their integrity; epithelial cells began to detach from explants (Fig. 1). Metabolic viability of the cultured explants The ability of the cultures to incorporate 3 H-leucine into TCA-precipitable protein was maintained at stable levels for 30 days in culture. In a representative series of A. grandis gill tissues cultured for 10, 24, or 31 days, in- corporation of ^H leucine was 5.73 0.41 (n = 17) CPM (^g protein h)~ ', and there was no significant difference between the three groups of gills (ANOVA, P > 0.05). Beyond 30 days, the viability of the gill explants declined in both A. grandis and L. subrostrata, as indicated by reduced leucine incorporation. In another study of A. grandis gills, leucine incorporation remained essentially constant through 44 days of culture, but declined 50% in gills cultured for 54 days. Discussion Explants of freshwater mussel gill can be cultured under aseptic conditions, with minimal exposure to antimicro- bial agents, by treating the gill in the initial decontami- nation stage with serotonin in combination with antibiotic and antifungal agents. Serotonin relaxes musculature in a number of molluscan systems (Twarog, 1954; Cam- bridge et ai. 1959; Kobayashi and Hasimoto, 1982). When applied to freshwater mussel gill, exogenous sero- tonin relaxes the branchial musculature, dilating the water canals leading into the central water channels. This effect, combined with an increased and more synchronous ciliary beat, maximizes fluid flow through the gill. These physi- ological effects of serotonin on gill explants occur within seconds (Gardiner et al, 1991). Incubating gills in a com- bination of serotonin and moderate concentrations of an antibiotic mixture for 45 min virtually eliminates all mi- crobial and fungal contamination. This suggests that many microbes are normally sheltered within the water spaces of the gill. One of the advantages of the serotonin treat- ment is that aseptic cultures can be established at lower concentrations of antibiotic and antifungal agents. These cultures can be maintained in an aseptic state with low levels of antibiotics, or even none. Freedom from the con- founding results of antibiotics makes gill cultures far more suitable for physiological study. Our initial attempts to achieve aseptic cultures of fresh- water mussel gills were mainly directed at elimination of the microorganisms by increasing the variety of antibiotics and elevating their concentrations. The assumption in this approach is that microorganisms are "resistant" to the antibiotics being used. This is not the case. As observed in this study, our initial failure to establish sterile cultures was due to the tissue harboring microorganisms and fungal spores in compartments that were partially isolated from the antibiotics. Perhaps a principal reason for difficulty in establishing any aseptic organ culture is the complex architecture of the organ, which provides microenviron- ments relatively free of antibiotics. Elevating the concentration of antibiotics may actually be detrimental to the tissue explant. Amphotericin B proved ineffective in controlling fungal contamination, and higher concentrations were toxic to the gill explants. High concentrations of Am-B resulted in abnormally high mucous secretion and cessation of ciliary activity. Thus, while fungal contamination is an important consideration in establishing long-term explants of freshwater mussel gill, Am-B should be avoided. We can now routinely culture viable gills for over a month. Freshwater mussel gills will survive in a variety of media for short periods. Several functions, including explant structural integrity, ciliary beat, muscular reflex activity, and a continued ability to respond to re-addition of serotonin, all indicate that the explants are functioning in organ culture. Trypan blue is excluded from the ma- jority of cells associated with the explant whether in place or detached. However, longer-term gill culture is appar- ently affected by medium composition and, as yet, un- known deficiencies. The established explant cultures are useful for the study of many events associated with gill 180 D. B. GARDINER ET AL. cellular activity (e.g.. calcium concretion synthesis, cell membrane ion channel characteristics). Acknowledgments The Cell and Tissue Culture Facilities, Department of Veterinary Microbiology and Parasitology, Louisiana State University, offered facilities and helpful suggestions throughout this study. 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CASE Department of Biological Sciences and Marine Science Institute, University of California, Santa Barbara, California 93106 Abstract. Bioluminescence in the midshipman fish. Porichthys notatus from the Santa Barbara coastal region, was quantified from onset through the first two years of life. Maximum light emission was 2.5 X 10 9 photons s~' upon leaving the nest and reached 2.0 X 10' photons s~" within the first year. These intensities may be sufficient for counterillumination in moon or starlight over most of the depth range of the fish. The bioluminescence of juveniles recently detached from the nest was depleted by multiple topical applications of a dilute noradrenalin so- lution. A luciferin-free diet also exhausted luminescence in 10-18 months. Bioluminescence was restored within 24 h after feeding depleted fish with dried specimens of the bioluminescent marine ostracod I 'argula hilgendorfti. and light emission capacity was correlated with the amount consumed. Predation by second year fish (18 months) upon juvenile P. notatus (3 months) or upon live I ". tsujii also restored luminescence. After restoration, lu- minescence gradually disappeared within several months. Consumption of luciferin-containing organisms by al- ready competent fish did not increase light intensity. Ju- venile P. notatus from the Santa Barbara coastal region require exogenous luciferin to remain luminescent. Introduction The midshipman fish, Porichthys notatus. has been the subject of many investigations since Greene ( 1899) first noted its bioluminescence originating from hundreds of dermal photophores, primarily confined to the ventral surface. Its natural history has been reviewed by Hubbs (1920), Arora (1948) and Ibara (1967). A nocturnal pred- ator, it remains buried in the sand during the day and ascends at night to feed. The habitat is moderately deep Received 17 December 1990; accepted 21 March 1991. waters to 400 m on the coastal shelf except during the breeding season. Then, during late spring and early sum- mer, sexually mature adults migrate inshore to spawn in the intertidal to 80 m depths (Feder et al. 1974). Males establish sheltered nesting sites and acoustically attract females. Females deposit up to 400 eggs, and multiple matings may result in the male guarding nests containing more than 1 000 eggs. P. notatus ranges from southeastern Alaskan waters to Baja California (Wilimovski, 1954) with a discontinuity along the coast of Oregon dividing the group into northern and southern divisions (Warner and Case, 1980). Al- though the photophores of both groups are ultrastructur- ally indistinguishable, the northern fish are non-lumines- cent (Strum, 1969). Both populations contain luciferase, but the northern population lacks luciferin (Tsuji et al., 1972; Barnes et al.. 1973). Administration of the biolu- minescent marine ostracod I 'argula hilgendorfti, induces luminescence in Puget Sound fish (Tsuji et al.. 1972; Barnes et al.. 1973). Fish of the southern division, south of Monterey Bay, are uniformly luminescent, while the central population, north of Monterey to Cape Mendocino, includes lumi- nescent and non-luminescent fish (Warner and Case, 1980; Thompson and Tsuji, 1989). The analog of V. hil- gendorfii along the western North American coast is I '. tsujii, whose distribution coincides with the southern midshipman population (Kornickerand Baker, 1977). Its absence from northern waters led to speculation that I '. tsujii is the luciferin source for the southern population (Warner and Case, 1980). The inability to experimentally deplete luminescence in luminous adults despite long periods of captivity and repeated challenges with noradrenalin suggested that adult P. notatus have already acquired large luciferin reserves from the diet or that there is a luciferin recycling or syn- 181 182 A. F. MENSINGER AND J. F. CASE thesis mechanism (Barnes el a/., 1973). Ingestion of small numbers of luminescent ostracods rendered northern adults capable of luminescence for up to two years, with light yield greater than was considered theoretically pos- sible for the amount of luciferin consumed, suggesting that de novo synthesis is triggered by exogenous luciferin or that there is a recycling mechanism (Thompson el al, 1988a). A preferential mechanism for rapid uptake of lu- ciferin from the digestive system has also been reported in P. notatus (Thompson el al., 1988b). The purpose of our study was to observe the effects of a luciferin-free diet on the development of biolumines- cence in P. notatus juveniles of the southern population and to ascertain whether the fish of this population need exogenous sources of luciferin to remain luminescent. To this end, light emission was quantified from its onset in larval fish through the first two years in laboratory-reared and in locally collected individuals of P. notatus. The ef- fects of multiple challenges with noradrenalin on the de- pletion of luminescence in the fish are described as are experiments that contributed to the evaluation of the lu- ciferin recycling hypothesis. Materials, Methods and Results Collection Breeding pairs of P. notatus were collected intertidally along the Pacific Coast, just north of Santa Barbara, Cal- ifornia. They were maintained in large aquaria with sand filtered, running seawater ( 1 6-20C). Masonry and large, inverted abalone shells provided nesting sites. Mating usually ensued during the first night in captivity and pro- duced between 1 00-400 eggs. Females were removed after spawning and males were retained to maintain the nests. Additional nests with guardian males were obtained by divers. After larval detachment (35-50 days post-fertil- ization), juveniles were placed in aquaria with sand-cov- ered bottoms sufficient for burrowing. Free-living juveniles were collected with a twenty-five foot, semi-balloon otter trawl from October to May. Min- imum depth of capture of first year fish ranged from 30 m in the fall to 80 m in late spring. All fish were main- tained on a luciferin-free diet consisting of \\v\ngArteniia and kelp mysids and frozen squid. Laboratory reared and trawled fish of the same year class had similar growth rates (Fig. 1A). Photophore di- ameter increased directly with standard length (Fig. IB). Light meaxnrenicni Bioluminescence was quantified with an integrating sphere quantum counting photometer (Latz et al.. 1987). Small fish, less than 4 cm si (standard length) were placed individually in a head down position in a clear plastic test 1 W.V - A 1 8.0 I .I'-' S=J 6.0 UJ | J _i g 4,0 i A < Q Z 5 ^ 2.0 5 0) n n . i i i < i i 50 100 150 200 250 300 350 400 TIME (DAYS) 15 O I 10 I o LJJ Q 5 5 Q B 0.0 0.2 04 0.6 0.8 PHOTOPHORE DIAMETER (MM) 1.0 Figure 1. Porichthys nolutus growth and photophore development. (A) Standard length (cm) versus time (days) post-fertilization. For each group tested, mean values are shown; error bars represent one SD of mean. Data represent laboratory raised (circles) and captured (squares) fish. For wild fish, age was determined from average time when fish left nest at capture site. Sample size ranged from 20 to 150. (B) Standard length (cm) irrvjw photophore diameter (mm). The fifth anteriormost photophore of each series from the right side of the fish was measured. One specimen contributed one point for each series. Data for lateral (circles), mandibular (squares), and gular (triangles) series are presented together with calculated least-squares linear regressions of length on time according to the equations: y = 0.31 + 17x, r : = 0.93 (solid) for lateral; y = -1.38 + 21.8x, r : = 0.90 (dashed) for mandibular; y = -1.08 + 27.8x, H = 0.88 (dotted) for gular. tube (10 X 75 mm or 15 X 75 mm) inside the 0.25 m diameter integrating sphere. Approximately 6 ml of free volume remained in the test tube after the introduction of the fish. A translucent stopper, containing delivery and output intramedic tubing, sealed the tube. Twelve milli- liters of freshly prepared 0.005 M ()-noradrenalin (Sigma) in filtered seawater was then administered by means of a syringe attached to the input tubing externally to the sphere. The fish was completely immersed in the solution. Two minutes later, a second dose of 6 ml was administered and the overflow collected outside the BIOLUMINESCENCE IN P. NOTATVS 183 1E1 1 o o 1E09 1E08 III!, *r V 1 100 200 300 400 500 600 TIME (DAYS) Figure 2. Bioluminescence induction and depletion. Maximum in- tensity (photons s~') versus time (days) after fertilization. Data represent laboratory raised (circles) and captured (squares) fish. Laboratory raised fish received no exogenous luciferin in the diet. Trawled fish were tested two weeks after capture. For each group tested, mean values are shown; error bars represent one SD of mean. Sample size ranged from 10 to 25. Dotted line represents noise background of integrating sphere and points on line are to be considered nonluminescent. sphere. Larger fish were placed in a 7.0 cm diameter cir- cular plexiglass chamber, 3.5 cm high, containing 40 ml of 0.005 M noradrenalin. Bioluminescence was recorded for 5 min with a RCA 8850 photomultiplier tube operated at -1700 V and baf- fled so that only light reflected from the sphere internal surface was measured. This procedure is essential to ac- curately measure total emission from large organisms containing many non-isotrophically radiating photo- phores. The detector signal was processed through a dis- criminator calibrated at -0.315 V and the resulting fre- quency signal was counted with an Ortec No. 776 counter/ timer and displayed on a Norland No. 5400 multichannel analyzer (MCA). Radiometric calibration of the detection system involved determination of the combined spectral responsitivity of the integrating sphere and photomulti- plier tube using an Optronic Laboratory Model 310 cal- ibration source. Light absorbance by fish within the sphere was checked against a C 14 activated phosphore and found to be negligible. The MCA trace was stored on film or floppy disk. Immersion in noradrenalin solution elicited biolumi- nescence within 20 s by transcutaneous absorption as shown by the effectiveness of topical application in air, thus avoiding mouth and gills. Luminescence was not observed prior noradrenalin application. Maximum light emission was usually attained within 5 min and gradually declined until exhaustion approxi- mately 40 min later. Exposure to fresh noradrenalin so- lutions during the trial did not appreciably raise intensity levels. A 365 nm emitting ultraviolet (UV) lamp (UVSL 25, Ultraviolet Products Inc.) was used to qualitatively test for luciferin in photophores by observing fluorescence vi- sually (Barnes et a/.. 1973). Bioluminescence depletion and induction Luminescence was initially detected about 30 days after spawning in 1.7 cm si larval fish still attached to the nest. Bioluminescence capacity in the laboratory population maintained on a luciferin-free diet increased for 6 months, before gradually declining (Fig. 2). Loss of luminescence capacity occurred from 10 to 18 months after spawning. Laboratory reared and trawled fish showed indistinguish- able luminescence levels up to approximately 300 days. Recently detached juvenile fish (2.5-3.5 cm si) were exhausted of luminescence capacity by immersing them in 0.005 M noradrenalin for approximately 5 min every other day until no luminescent response was elicited (Fig. 3). Depleted animals remained nonluminescent unless their diet was supplemented with luciferin as described below. The remaining untreated population was tested bimonthly for luminescence with noradrenalin. For each sample of the latter group, the tests represented the first exposure to noradrenalin. Noradrenalin-depleted fish invariably could be restored to luminescence competence by the administration of 1E-HO p- o LU cn O 1E+09 o , SV N \ ;"*-* % o '' ', CONTROL PN 1 PN 2 ---- PN 3 10 20 30 TIME (DAYS) Figure 3. Noradrenalin induced depletion of bioluminescence. Maximum intensity (photons s ') versus time (days) after first challenge with noradrenalin. For control group tested (squares), mean values are shown; error bars represent one SD ot mean. Data points represent time of first challenge with noradrenalin. Control fish received only one chal- lenge. For the three experimental fish (circles and triangles), each point represents a challenge. Sample size of each control group was 10. Solid line represents calculated least-squares logarithmic regression of intensity on time according to equation y = 2.3 X 10 9 + 0.02x, r- = 0.61. 184 A. F. MENSINGER AND J. F. CASE dried Vargula hilgendorfii (0.7 to 4.0 mg). Depleted fish were anesthetized in tricaine methane sulfonate (MS 222, ICN-K&K Laboratories), 100 mg/1 filtered seawater, and fed 6- 1 2 whole, dried I 'argula hilgendorfii via intramedic tubing (PE 190) slipped into the anterior gut. Controls were anesthetized but not fed. To guard against regurgi- tation, fish were monitored for 15 min after regaining their ability to swim. The few ostracods expelled were dried and their weight subtracted from the total. Restored luminescence neared but never exceeded pre- vious maximum output. Restoration was evident within 24 h, but maximal light output was usually not attained for several weeks. Total light emission was related to the amount of ]'argitla administered (Fig. 4). Naturally depleted, second year P. notatus (9.0 cm si) also had luminescence capacity restored by ingestion of dried Vargitla hilgendorfii (5.0-9.0 mg), live I', tsujii (n = 3) and recently detached juvenile P. notatus (n = 3 to 5) (Fig. 5). For the live feedings, fish were placed in small aquaria containing either three live J r . tsujii. or five re- cently detached juvenile P. notatus (2.5 cm si) and mon- itored until prey were consumed. Restored luminescence neared but never exceeded previous maximum output. Living I 'argula produced the most rapid onset and greatest intensities. Reinduction was not permanent, and lumi- nescence capacity gradually disappeared with time (Fig. 5). The rise indicated after day 90 in Figure 5 for juvenile P. notatus feeding on other P. notatus is an aberration representing one fish that had been gradually losing its capacity for luminescence. The other three fish were de- pleted, but died before testing on day 120. Ingestion of live I '. tsujii by already competent juveniles did not result in increased light output. LIVE PORICHTHYS FREEZE DRIED CONTROL VAHGULA JUVENILES VAHGULA O 2E-I-08 I MAXIMUM ffi + O ~g 1 1 i 1 20 30 VARGULA (MG) 40 Figure 4. Maximum intensity (photons s ' ) ivrvi/.v weight of I 'argula (mg) fed to depleted fish. Points represent individual fish tested 48 h after administration. Line represents calculated least-squares linear regression of light on weight of I 'argula according to equation y = 0.43x + 5.9 x 10 7 , T- = 0.62. CO CO O CO z LU t; 1E-KJ8 30 60 90 120 150 TIME (DAYS) Figure 5. Maximum intensity (photons s ') versus time (days) after feeding with luciferin sources at day 0. Prey included live I 'argula isu/ii (squares), freeze dried ('. hilgendorfii (triangles), and recently detached P. notatus (circles, dotted line). Controls are represented by circles, dashed line. Sample size ranged from 4 to 8 poor to day 90, and I to 4 for the rest of the experiment. Dashed line represents background of integrating sphere and points on line are to be considered nonlummescent. Trawling also reduced or depleted luminescence. The effect was more pronounced in younger (4.2 cm si) fish captured in November than in older fish of the same year class (6.9 cm si) trawled the following spring (Fig. 6). Two weeks were needed for captured animals to attain maxi- mum light levels. Individual photophore responses Light from individual photophores was also quantified. Thirty microliters of 0.001 M noradrenalin was injected under the branchiostegal photophores of a fish anesthe- tized with tricaine methane sulfonate MS 222 as outlined by Thompson et al. ( 1 987 ). After a 1 0-min delay to insure that the fish was luminescing. 5-10 photophores of a spe- cific anatomical series were removed surgically as a strip, and immediately placed in filtered seawater in a small, clear plexiglass chamber (4 X 2 X 0.3 cm) in the integrating sphere. Luminescence was measured for 5 min. To insure that preparation time did not bias the measurements, the test order was reversed for every other fish in the test series. All photophore series were tested within 1 h of injection. The average light intensity of single photophores varied with location on the fish. Photophores from the gular series were significantly brighter (ANOVA: P < 0.01) than all other photophores except those from the branchiostegal series (Table I). Discussion Larval P. notatus from the southern population are ini- tially luminescent. However, these experiments demon- BIOLUMINESCENCE IN P. NOTATUS 185 o o O =J 1E + 09 11/85 3/86 5 10 15 20 25 30 35 TIME (DAYS) Figure 6. Bioluminescence recovery after collection by trawling. Maximum intensity (photons s~') versus time (days) after trawl. For each group tested, mean values are shown; error bars represent one SD of mean. All fish are from first year class. Points indicate average intensity from Porichthys nolalus captured off Santa Barbara during 1 1/85 (circles) or 4/86 (squares). Average standard lengths were 4.2 cm (November) and 6.9 cm (April). The initial two points in each graph represent fish receiving first challenge with noradrenalm. Remaining points represent fish with at least one previous challenge. Sample size ranged from 1 2 to 25. strate that exogenous sources of luciferin must be acquired during at least the first two years of life if luminescent capacity is to be retained. Bioluminescence was detected in smaller (1.7 cm si) fish than has been previously re- ported, owing perhaps to the increased sensitivity of the integrating sphere photometer over previously used de- tectors, and to the fact that this photometer allows accurate quantification of luminescence for entire animals rather than only for groups of photophores as in previous studies (Tsuji rt a/., 1972;Anctil, 1977). The increase in bioluminescence capacity during the initial six months in laboratory animals shows the im- portance of maternally provided luciferin reserves. Lu- minescent capacity did not increase with added exogenous luciferin during this period, indicating emission capacity was not initially substrate limited. The rise in light output during juvenile life is positively correlated with increasing photophore area. Other contributing factors might be in- creased emission per unit area or increased efficiency of use of luciferin reserves. A comparison of light emission and standard length of laboratory and trawled fish of the same age indicated that our laboratory maintenance pro- cedures had no detrimental effect on bioluminescence ca- pacity or growth during the first half year. Trawling resulted in an average 60-90% temporary re- duction of light capacity with a much greater effect noted in smaller than in larger fish of the same year class. Ap- proximately 25% of trawled fish tested negative for both fluorescence and bioluminescence within 24 h of retrieval. As even dip netting can stimulate luminescence in captive fish, it is highly likely that the stress of trawling invokes luminescence. Although trawl stress may have depressed neurological pathways controlling luminescence, the ab- sence of fluorescence and subsequent return of lumines- cence indicates that the cause is luciferin depletion in the light organs and that time is required to replenish the substrate from reserves. Larger fish exhibited less reduc- tion perhaps due to greater luciferin concentration in their larger photophores and reserves or better resistance to stress. These results strongly indicate that care should be taken in interpreting luminescence capacity of recently trawled fish, especially smaller ones, because our tests with UV and noradrenalin indicate that 25-50% of a particular trawl would be non-luminescent immediately after cap- ture, even though all regained luminescence within a week. Our results indicate that Thompson and Tsuji (1989) may have overestimated the number of nonluminescent fish in their trawls as fish were not given time to recover. The adaptive significance of bioluminescence in the midshipman has long been obscure. It has been speculated that the photophores attract prey by mimicking euphau- siid swarms (Tsuji et ai. 1971). Displays have been ob- served during mating and in response to predators (Crane, 1965; Lane, 1967). A role in mating is unclear because females are acoustically attracted to mate and the nesting environment does not optimize the effect of biolumines- cent displays. We have observed numerous successful matings in laboratory tanks and only once was lumines- cence briefly noted. Furthermore, the northern population reproduces without luminescence capability. The dominantly ventral photophore position and the intensity range of light emission seems ideal for counter- Table l Average intensity (photons s~') of individual photophores from various anatomical series Individual Photophore Response Photophore Average intensity 1 S.D. location' n photons s~' per photophore Mandibular 36 1.7 X 10 s 1.1 X 10 6 Branchiostegal 32 4.3 x 10 7 1.6 x 10 7 Gular 29 7.6 x 10 7 4.5 x I0 7 Ventral 36 2.7 x 10 6 2.6 x 10" Pleural 33 4.0 x 10 6 2.9 X 10 6 Scapular 23 4.4 x 10 5 8.1 x 10 4 1 According to Greene (1899). Light was quantified for five minutes. Five to ten photophores were tested in each fish and n represents total number of photophores tested. Photophores were taken from four adult fish. 186 A. F. MENSINGER AND J. F. CASE illumination. Although the P. notatus emission spectrum (Tsuji el al.. 1975) does not exactly match nocturnal as- tronomic light (Munz and MacFarland, 1977). this may be unimportant because many of the California nearshore fishes that feed primarily at night lack the visual pigments necessary to detect hue differences (Hobson el al.. 198 1 ). Additionally, the variability in water quality of nearshore waters can alter the apparent emission spectrum. Lumi- nescence intensity is sufficient to counterilluminate moon or starlight throughout most of the normal depth range. Upon leaving the nest, fish emit at 2.0 X 10 4 photons s~' cirT : , which is sufficient to counterilluminate starlight at approximately 30 m depth and full moonlight at 70 m depth in Class 1A oceanic water (Jerlov, 1976) on cloud- less nights. The fish probably could match intensities at somewhat shallower depths because the nests are found in water usually less clear than class IA and the effects of kelp canopies or clouds are considerable. Although counterillumination in fish has not been demonstrated in captivity, its presumed importance may explain the early onset of luminescence in larval fish that become competent to luminesce weeks before detach- ment. Given the inverted position of the larvae, low re- flectivity of the substrate and presence of a guardian male, it is doubtful that luminescence has much functional sig- nificance on the nest. However, it may be vital to counter- illuminate immediately after detachment. This study shows, for the first time, depletion of lu- minescence in previously competent fish. Other investi- gators had been unable to deplete naturally luminous adult fish or ostracod-induced northern fish despite long periods of captivity or multiple challenges with noradrenalin (Tsuji et til.. 1972; Barnes ct al.. 1973; Thompson et al., 1987). In our study, luminescence was depleted by fre- quent challenges with topically applied noradrenalin or by maintenance on a luciferin-free diet. Noradrenalin treatment depleted luminescence within three weeks in recently detached fish, thus leading to the conclusions that frequent stimulation expends maternally acquired reserves and that a regeneration process is not present. In juvenile southern fish, lack of exogenous luciferin also depleted luciferin reserves within 10-18 months. The depletion may have been the result of spontaneous luminescence or metabolic elimination of luciferin. Loss of luminescent capacity by whatever means was not detrimental to the photophores as luminescence could be restored by lucif- erin administration. The three luciferin sources used in our experiments all induced luminescence between 20 and 48 h, more than twice as quickly as reported in previous studies (Tsuji et al.. 1972; Barnes ct al.. 1973). This is attributed to the smaller body size of the fish, which presumably allowed quicker substrate transport to the photophores, and im- proved instrumentation. Although it has long been speculated that I '. tsujii is the source of P. notatus luminescence, this is the first study in which fish were found to actively prey on (". tsujii, albeit in laboratory tanks. Cannibalism was also observed for the first time in P. notatus. First year fish are easy prey of older fish, and small conspecifics have been noted in the stomach of the closely related species, P. myriaster (Allen, 1982). Previously it had been thought that young fish must accumulate adequate life-time luciferin reserves before reaching maturity (Warner and Case, 1980), be- cause it seemed unlikely that larger fish would consume small ostracods and would, at any rate, occupy deeper water outside the range of 1'argula. Clearly, such is not the case and the overlapping ranges of first and second year fish off Santa Barbara make it possible for younger fish to be an additional exogenous source of luciferin for older conspecifics. thus establishing a link which would allow the larger fish effective indirect access to I 'argula luciferin. It was calculated using the methods of Thompson et al.. ( 1987) that the fish should theoretically yield approx- imately 2 X 10' 5 photons s~' for the amount of dried I'argiila ingested. Although light emission was only quantified for 5 min, none of the induced fish emitted light for over 40 min and many displays lasted less than 20 min towards the end of the study. Even using the ex- tremely liberal calculation of multiplying the maximum intensity attained during the run by 40 min to derive pho- ton yield, and then summing the trials for each fish, the brightest fish only produced 2 X 10 13 photons s ' (range 2 X 10 i: to 2 X 10" photons s '), a value two orders of magnitude below the theoretical yield. Thompson et al. ( 1988) report that the fish retain approximately 1% of the luciferin ingested. Even after estimating the luciferin re- tention rate at two orders of magnitude less than actually ingested, the majority of the induced fish fall short of at- taining theoretical values. These experiments do not support either a luciferin synthesis or recycling mechanism for juveniles of the southern population. Luminescence could be depleted by continual challenges with noradrenalin or maintenance on a luciferin-free diet. Although luminescence could be restored with exogenous sources of luciferin, the effect was not prolonged and emission values fell short of theo- retical values. These results contrast with the reports of Thompson et al. (1987; 1988a,b) who found that the persistence of T. hilgenclorfii [ l4 C]luciferin in the photophores and long lasting luminescence after feeding small amounts of lu- ciferin suggested an active recycling mechanism in Puget Sound adults (Thompson et al.. 1988b). Furthermore, they reported I'argula feeding to previously non-lumi- nescent fish, yielded more light than was theoretically possible for amount of luciferin ingested. B1OLUMINESCENCE IN P. NOTATVS 187 A major difference between our investigations and those of Thompson el al. (1987; 1988a,b) was our use of south- ern juveniles instead of northern adults. These populations may have evolved different luciferin pathways in response to availability of exogenous luciferin in the diet of adults. The southern habitat includes two luciferin sources, V. tsujii and young P. notatus. Perhaps owing to this, they have lost or have never evolved an alternative mechanism, or possibly it does not function until their later, deep water stage when they are out of range of exogenous sources. If \ 'argula disappeared gradually from the northern range, the northern fish might have evolved mechanisms to maximize the effects of increasingly rare encounters with luciferin sources. Another possibility is that the pathways require minimal amounts of luciferin to function. Once levels drop below a critical concentration, enzymes needed for synthesis or recycling are no longer produced. A second difference in these investigations is that the use of the integrating sphere photometer allowed us to quantify more accurately the total light emission per fish and avoid the assumptions concerning total light emitted, average intensity, and differential photophore emission made in previous reports (Thompson el al., 1987). For example, Thompson el al. (1987) multiplied the directly measured intensity of 127 photophores mostly from the branchiostegal and gular series (Greene, 1899) by 4.3 to determine light emission from the whole fish, with the underlying assumption that all photophores emit equal intensities. However, we find that the branchiostegal and gular series contain the most intensely emitting photo- phores of the entire fish (Table I). We calculated emission based on photophore average intensity by dividing the fish into three areas: ventral photophores inside the light capture geometry of the Thompson et al. (1987) photo- multiplier (branchiostegal and gular series; n = 127), re- maining ventral and lateral photophores (mandibular, ventral, and lateral series; n = 397) and head and dorsal photophores (scapular series; n = 150). We determined total light output by taking the average intensity of the photophores contained within representative series (listed above for each area) and multiplying the average photo- phore intensity by the approximate number of photo- phores contained within each area. Our values indicate that quantification of light intensity for the whole animal by extrapolating from only the branchiostegal and gular photophores overestimates light emission by at least a factor of 3.5. Another factor not considered by Thompson et al.. (1987) in arguing for luciferin recycling is that, in the course of losing luminescence capability, fish initially lose luminescence capacity in posterior photophores. Fish low in luminescence capacity were noted by dark adapted ob- servers to have many posterior photophores unresponsive to noradrenalin application (Mensinger and Case, un- pub.). Whether this is due to smaller reserves in posterior photophores or a transport mechanism that gives the an- terior sites higher priority remains unknown. A mecha- nism favoring supply of luciferin to anterior ventral pho- tophores during luciferin limitation would have adaptive value in preserving counterillumination capability for the larger and therefore more conspicuous, anterior regions. If the latter is true, however, the localized anterior nor- adrenalin injection sites used in Thompson et al. (1987) may have disproportionately depleted luciferin reserves from the anterior series, thus resulting in the transport of luciferin from the more distal sites. Whatever the mech- anism, extrapolating intensities from anterior photophores would, therefore, result in erroneously higher emission estimates and thereby induce error into calculations of luciferin use. We found no evidence of a mechanism for long-term maintenance of luminescence capacity in southern fish. Eventual loss of luminescence in animals without previous noradrenalin exposure rules out the possible deleterious effects of repeated noradrenalin challenges in the fish used in our investigation. The loss of luminescence capacity after induction with luciferin showed that there was no synthesis mechanism dependent on priming with non- maternal sources of luciferin. The loss of maternally or naturally acquired reserves through spontaneous lumi- nescence, diffusion, or autoxidation may have decreased luciferin below recyclable levels. However, the relatively short reinduction periods (3-6 months) and low photon yields cast doubt on the presence of a recycling mechanism in second year southern fish. We conclude that fish of the southern population must continually acquire exogenous sources of luciferin, at least during their early life history, to remain luminescent. Acknowledgments J. Warner contributed to the early phases of this study. J. G. Morin and A. Huvard kindly provided living Var- gii/u. J. G. Morin also most helpfully reviewed the manu- script and provided other useful comments. A. Uriu and L. Trebasky helped with animal maintenance. Research was supported in part by Office of Naval Research Con- tracts N00014-84-K-0314 and N00014-89-J-1736. Literature Cited Allen, M. J. 1982. Functional structure of soft-bottom communities of the southern California shelf. Ph. D. thesis. University of California, San Diego, La Jolla, California. Anctil, M. 1977. Development of bioluminescence and photophores in the Midshipman fish, Porichlhvs notatus. J Morptwl. 151: 363- 396. Arora, H. L. 1948. Observations on the habits and early life history of the batrachoid fish. Porn-hthys notatus Girard. Co/via 1948: 89-93. Barnes, A. T., J. F. Case, and F. I. Tsuji. 1973. Induction of biolu- minescence in a luciferin deficient form of the marine teleost Poncli- 188 A. F. MENSINGER AND J. F. CASE ilivs. in response to exogenous lucifenn. Com/). Biochem. Physiol. 46A: 709-723. Crane, J. M. 1965. Bioluminescent courtship display in the teleost Porichlhys notalus Copeta 1965: 239-241. Feder, H. M., C. H. Turner, and C. Limbaugh. 197-1. Observations on fishes associated with kelp beds in Southern California. Calif. Dept. Fish and Game, Fish Bull 160. 144 pp. Greene, C. W. 1899. The phosphorescent organs in the toadfish, Por- ichthvs nolalus Girard. J. Morphol. 15: 667-6%. Hobson, E. S., VV. N. McFarland, and J. R. Chess. 1981. Crepuscular and nocturnal activities of Californian nearshore fishes, with consid- eration of their scotopic visual pigments and the photic environment. Fish. Bull. 79: 1-30. Hubbs, C. L. 1920. The bionomics ofPorichthys notatus Girard. Am. Nat. 54: 380-384. Ibara, R. M. 1967. Biology of the midshipman fish, Porichlhys nolatux Girard. M. A. thesis. University of California, Santa Barbara, Cali- fornia. Jerlov, N. G. 1976. Marine Optics, Elsevier, Amsterdam, 231 pp. Kornicker, L. S., and J. II. Baker. 1977. I 'argula txuiii. a new species of luminescent ostracoda from lower and southern California (My- odocopa: Cypridininae). Proc Biol. Soc. Wash. 90: 218-231. Lane, E. D. 1967. A study of the Atlantic midshipman Porichthyx po- rosixximux in the vicinity of Port Aransas. Texas. Conlri. Mar Sci. 12: 1-53. Latz, M. I., T. M. Frank, M. R. Bowlby, E. A. Widder, and J. F. Case. 1987. Variability in Hash characteristics of a bioluminescent co- pepod. Biol. Bull 173:489-503. MUM/, F. \V., and W. N. McFarland. 1977. Evolutionary adaptation of fishes to the photic environment. Pp. 193-274 in Handbook ol Sensory Physiology. Vol. VII/5, F. Crescitelli, ed. Springer. Berlin, Heidelberg, New York. Strum, J. 1969. Fine structure of the dermal luminescent organs, pho- tophores, in the fish Porichlhyx notalus. Anal. Rec. 164: 433-46 1 . Thompson, E. M., B. G. Nafpaktitis, and F. 1. Tsuji. 1987. Induction of bioluminescence in the marine fish Porichlhys by I 'argula (crus- tacean) lucifenn. Evidence for de nmo synthesis or recycling of lu- ciferin. Pholochcm. Photobiol 45: 529-533. Thompson, E. M., V. Toya, B. G. Nafpaktitis, T. Goto, and F. I. Tsuji. I988a. Induction of bioluminescence capability in the marine fish. Porichthyx nolalux. by }' argula (crustacean) [ l4 C]luciferin and un- labeled analogues. J. Exp. Biol. 137: 39-51. Thompson, E. M., B. G. Nafpaktitis, and F. I. Tsuji. 1988b. Dietary uptake and blood transport of I'argula (crustacean) lucifenn in the bioluminescent fish, Porichlhys notalus. Comp. Biochem. Physiol. 89A: 203-209. Thompson, E. M., and F. I. Tsuji. 1989. Two populations of the marine fish Porichthyx notatux, one lacking in luciferin essential for biolu- minescence. Mar. Biol 102: 161-165. Tsuji, F. I., V. Haneda, R. V. Lynch III, and N. Sugiyama. 1971. Luminescence cross-reactions of Porichlhys luciferin and theories on the origin of luciferin in some shallow-water fishes. Comp. Biochem. Physiol. 40A: 163-179. Tsuji, F. L, A. T. Barnes, and J. F. Case. 1972. Bioluminescence in the marine teleost, Porichlhys nolatux. and its induction in a non- luminous form by Cypridma (ostracod) luciferin. Nature 237: 515- 516. Tsuji, F. I., B. G. Nafpaktitis, T. Goto, M. J. Cormier, J. E. Wamplcr, and J. M. Anderson. 1975. Spectral characteristics of the biolu- minescence induced in the marine fish, Ponchthys no/a/us, by Cy- pndina (ostracod) lucifenn. Molec. Cell. Biochem. 9: 3-8. Warner, J. A., and J. F. Case. 1980. The zoogeography and dietary induction of bioluminescence in the midshipman fish, Porichthys nolalus Biol Bull. 159: 231-246. \\ilimovski, N. J. 1954. List of the fishes of Alaska. Stanford Ichlhyol. Bull 4: 279-294. Reference: Biol Bull 181: 189-194. (August, 1991) Vanadobin, a Vanadium-Binding Substance, Extracted from the Blood Cells of an Ascidian, Can Reduce Vanadate(V) to Vanadyl(IV) HITOSHI MICHIBATA 1 *, AKEMI MORITA 1 , AND KAN KANAMORI 2 ^Biological Institute and ^Department of Chemistry, Facultv of Science, Toyaina University, Gofiiku 3190, Toyama 930 Japan Abstract. Ascidians specifically accumulate high levels of vanadium from seawater in their blood cells. Almost all of the vanadium is present in a reduced form in the blood cells, although the metal exists in a +5 oxidation state in seawater. It has, therefore, been assumed that agents that cause the reduction of vanadate(V) to vana- dyl(IV) must be present within ascidian blood cells. In this regard, we have extracted a vanadium-binding sub- stance, which we have called vanadobin, from the vana- docytes of ascidians. We examined whether vanadobin is involved in the reduction of vanadate(V) accumulated from seawater. Data obtained by spectrophotometry and ESR spectrometry revealed that not only a crude homog- enate of vanadium-rich blood cells but also a purer form of vanadobin eluted from a column of Sephadex G-15 could reduce vanadate(V). Our experiments demonstrate that vanadobin, a vanadium-binding substance extracted from ascidian blood cells, can reduce vanadate( V) to van- adyl(IV) and maintain it in the reduced form. Introduction Vanadium, a multivalent metal, is generally present in the biosphere in the +5, +4, and +3 oxidation states (Chasteen, 1983; Kustin et a!., 1983). Among all organ- isms examined, ascidians appear to be the only ones that contain high levels of vanadium. High levels of specifically accumulated vanadium in the blood cells of ascidians are reduced predominantly to the +3 oxidation state, with a small amount of vanadium also present in the +4 oxi- dation state (Tullius el al., 1980; Dingley et a/., 1981; Received 16 January 1 99 1 ; accepted 18 March 1991. * Present address: Mukaishima Marine Biological Laboratory, Hiro- shima University, Mukaishima-cho. Hiroshima 122, Japan. Frank et al.. 1986; Lee etui., 1988; Hirata and Michibata, 1990), even though the vanadium dissolved in seawater seems to be present as vanadate(V) anions in the +5 ox- idation state ( McLeod et al., 1975). Therefore, it has been assumed that some agent that causes the reduction of vanadate(V) to vanadyl(IV) must be present in ascidian blood cells because they accumulate the metal from sea- water. We have already reported the extraction of a vanadium- binding substance, which we have called vanadobin, from the vanadocytes of ascidians (Michibata et al., 1986; Mi- chibata and Uyama, 1990; Michibata et al.. 1990a). Va- nadium incorporated into vanadobin is maintained in the reduced form and, therefore, vanadobin seems to be able to reduce vanadate(V) to vanadyl(IV). In the present ex- periments, the metal-reducing ability of vanadobin was examined by spectrophotometry and ESR (electron spin resonance) spectrometry, after we demonstrated that the supernatant of a homogenate of the blood cells could re- duce the vanadate(V). Materials and Methods Homogenates of blood cells Specimens of Ascidia gemmata were collected at the Asamushi Marine Biological Station of Tohoku University in Asamushi, Aomori, Japan. The animals were trans- ported to our laboratory and maintained in an aerated aquarium at 12C until use. Blood was collected by car- diac puncture under an anaerobic atmosphere of nitrogen gas to preclude oxidation by air; subsequent manipula- tions were also carried out under the same conditions. The blood cells were separated from the blood plasma by centrifugation at 3000 X g for 10 min at 4C. About 189 190 H. MICHIBATA ET AL. 10 g wet weight of the pellet of blood cells were resus- pended in 3 ml of acidified, deionized, and distilled water (acidic DDW) that had been degassed, bubbled with ni- trogen gas, and adjusted to pH 2.3 with 2 A/ HC1. We feared that the buffer solution, such as HCl-glycine buffer, might interfere with ESR spectrometry; therefore, no buffer solution was used in these experiments. The sus- pension was then ground in a glass-Teflon homogenizer at 4C. After adding 1 2 ml of acidic DDW, the homog- enate obtained was centrifuged at 1 1,500 X g for 10 min, to remove the cell debris. An aliquot of the supernatant of the homogenate was examined for its ability to reduce vanadate(V) to vanadyl(IV). Extraction ofvanadobin (vanadium-binding substance) The extraction of vanadobin was carried out as de- scribed previously using 7 ml of supernatant obtained as described above (Michibata el ai, 1 990a). The supernatant was loaded onto a column (3.6 cm X 56 cm long) of Sephadex G-15 (Pharmacia Fine Chemicals, Uppsala, Sweden) and eluted with acidic DDW in 5 ml. The peak fractions, monitored at 254 nm, were sepa- rately pooled and lyophilized, and then were redissolved in 10 ml of acidic DDW and kept in anaerobic atmosphere before use. Spectrophotometric measurements of the reduction ofvanadate(V) Aqueous solutions of vanadium(III) sulfate [V : (SO 4 h] and vanadium(IV) oxide sulfate (VOSO 4 ) at low pH ex- hibit absorption maxima at 420 nm and 620 nm, and at 760 nm with a shoulder around 625 nm, respectively, whereas an aqueous solution of sodium vanadium(V) (Na,VO 4 ) exhibits no absorption maximum in the visible range. We have already demonstrated that the ratio of vanadium ions in the +3 state to those in the +4 oxidation state, which are associated with vanadobin. can be cal- culated from the respective molar absorption coefficients (t) (Michibata el ai, 1990a), using the following formulae: D 620 = A[M] X t D 760 = A[M] X " B[M] X 760 ,iv 760 Here, D 620 and D 76I) are the observed absorbance of vanadobin at 620 nm and 760 nm, respectively and e'" 6 2o- t m 1M , e IV 62r> and c IV 76 o are the molar absorption coeffi- cients of inorganic vanadium(lll) and vanadium(lV) in water at each wavelength. Molar concentrations of va- nadium(III) and vanadium(IV) associated with vana- dobin, A[M] and B[M], can be calculated from the ob- served absorbance at 620 nm and 760 nm. Electron spin resonance (ESR) measurements of the reduction ofvanadate(V) ESR spectrometry was carried out as described previ- ously (Hirata and Michibata, 1990). Briefly, 100 n\ of a mixture of two volumes of sample and one volume of 4 M H ; SO 4 were put into a quartz tube. We used a JES- RE1X ESR spectrometer (JEOL Ltd., Tokyo) for ESR spectrometry. The instrument conditions were adjusted as follows: microwave frequency, 9.2 GHz; magnetic field, 330 100 mT; microwave power, 5 mW; field modula- tion frequency, 100 kHz; field modulation width, 0.63 mT; and sweep time for recording, 4 min. Chemicals All chemicals used were obtained from commercial sources and were of special grade, except for V 2 (SO 4 ),, which was prepared according to the literature (Claunch and Jones, 1963). Results Reduction ofvanadate(l') by a homogenate of blood cells As shown in Figure 1, the supernatant of the homog- enate of blood cells exhibits two absorption maxima at 08-i 0.4 0.8i B 400 500 600 700 800 900 Wavelength (nm) 400 500 600 700 800 900 Wavelength (nm) Figure 1. Reduction of vanadate(V) by the supernatant of a ho- mogenate of blood cells from Ascidia gemmata, as monitored by spec- trophotometry. A. Immediately after the start of the reaction. B. Thirty- one hours after the start of the reaction. Absorption spectrum of the supernatant of the homogenate of blood cells (a), of 8 m.M vanadate(V) (b), and of 8 roA/ vanadyl(IV) (c). When an 8 mA/ solution of vanadate( V) was added to the supernatant, absorbance in the vicinity of 760 nm due to vanadyl(IV) increased (d). Thirty-one hours after the reaction, absor- bance at the shorter wavelength than 530 nm due to vanadate(V) de- creased and that at 760 nm became clear (d), suggesting that the added vanadate(V) was reduced to vanadyl(IV) by the supernatant. By contrast, addition of an 8 m.A/ solution of vanadyK IV ) to the supernatant resulted in little change in the absorption spectrum (e) both immediately after and 31 hours after the start of the reaction. The higher base line in the absorption spectrum of (d) than the others caused turbidity appeared when vanadate(V) solution was added to the sample. VANADATE REDUCTION BY VANADOBIN 191 Peak 2 1.50 Peak 5 CJ.2 mg v = 40 ~~60 80 Fraction number 100 120 140 Figure 2. Elution profile of the supernatant of a homogenate of blood cells from Ascidia gemmata from a column of Sephadex G-15. The supernatant (7 ml) was loaded onto a column (3.6 cm X 56 cm long) of Sephadex G-15 (Pharmacia Fine Chemicals) and eluted with DDW (see text) at pH 2.3 in 5 ml. Fractions composing each peak were sepa- rately pooled, and amounts of vanadium in individual aliquots were measured by ESR spectrometry. Then, the material under each peak was lyophilized and kept under anaerobic conditions. The material in the second peak contained the highest amount of vanadium. V,: 570 ml, V : 226 ml, and V c : 280ml. 620 nm and 760 nm, which are assignable to vana- dium(III) and vanadyl(IV), respectively. Comparing the corresponding spectra observed in inorganic vanadium complexes, as demonstrated previously (Michibata et ai, 1990a), we calculated that the supernatant of the homog- enate of the blood cells intrinsically contained 25 mAf vanadium in the +3 and +4 oxidation states at a ratio of 30:70. After adding 1 .2 ml of an 8 mAf solution of vanadate( V) to an equal volume of the supernatant, spectral changes were recorded. Figures 1A and IB show the spectra im- mediately after mixing and after 3 1 h. The absorbance at 760 nm due to vanadyl(IV) became conspicuous, accom- panying the decrease of the absorbance at the wavelength shorter than 530 nm due to vanadate(V) with time, sug- gesting that the added vanadate(V) was reduced to van- adyl(IV) by the supernatant. By contrast, the addition of the same amount of an 8 mAf solution of vanadyl(IV) resulted in little change. These observations indicate clearly that some reducing agent is present in the super- natant of homogenate of blood cells from Ascidia gem- mat a, which can reduce vanadate(V) to vanadyl(IV). As shown in Figure 1 B, the spectral change with time reveals that reduction of vanadate(V) to vanadyl(IV) occurred very slowly over the course of 3 1 h after the onset of the reaction. Reduction ofvanadate(V) hy vunadobin; observations bv spectrophotometry When the supernatant of the homogenate of blood cells was eluted from Sephadex G-15, five peaks were obtained (Fig. 2). Amounts of vanadium contained in each peak of material are illustrated as shaded squares in Figure 2. Reduction experiments were performed with samples of elutant that had been lyophilized and redissolved in 10 ml of acidic DDW. From the ratio of absorbance at 620 nm to that at 760 nm, it was calculated that the material in peak 2 contained vanadium in the +3 and +4 oxidation states at a ratio of 1:25. When 0.8 ml of a solution of vanadyl(IV) or vanadate(V) at concentrations from 4 mM to 16 mA/was added to each peak fraction, reduction of vanadate(V) to vanadyl(IV) was significant in the case of the material in peak 2, when monitored by spectropho- 0.2 400 500 600 700 800 900 Wavelength (nm) Figure 3. Reduction of vanadate(V) by vanadobin from Ascidia Kcmmata as monitored by spectrophotometry. The samples of lyophilized material (described in the legend to Fig. 2) were redissolved in 10 ml of acidic DDW and reacted with an equal volume of either vanadate(IV) or vanadylf V), both solutions were at 8 mA/ to examine their ability to reduce vanadium. Absorption spectra of: (a) vanadobin; (b) an 8 mA/ solution of vanadate(V); and (c) an 8 mA/ solution of vanadyl(IV). (d) When vanadate(V) (8 mA/) was reacted with vanadobin, a marked in- creased in absorbance at about 760 nm due to vanadyl(IV) was observed, indicating that vanadate(V) was reduced to vanadyl(IV) by vanadobin. (e) By contrast, addition of vanadyl(IV) (8 mA/) to vanadobin resulted in little change in the absorption spectrum, indicating that no further reduction of vanadyl(IV) to vanadium(lll) had occurred. Unlike the re- sults obtained with the supernatant of the homogenate of blood cells depicted in Figure 1, the above changes in absorbance were observed immediately after the start of the reaction, and the reduction was main- tained for at least 24 h. 192 H. M1CHIBATA ET AL 250 410 Figure -4. ESR spectra of vanadobin and of a mixture of vanadobin with an 8 m.U solution of vanadate(V) at 77 K under anaerobic atmo- sphere of nitrogen gas. (a) Vanadobin, (b) a mixture of vanadobin and 8 mM vanadate(V). Oxovanadium [VO 2+ (IV)J that was intrinsically present in the vanadobin gave typical ESR signals (a). The addition of an 8 m.U solution of vanadate(V), which alone gave no ESR signal, to an equal volume of vanadobin increased the signal intensity to about 1. 2 times that generated by vanadobin alone. tometry. Figure 3 shows the rapid reduction of vana- date(V) to vanadyl(IV) that followed the addition of the material in peak 2 (vanadobin). Although vanadate(V) in solution exhibits no absorption in the vicinity of 760 nm, the addition of vanadobin induced a drastic increase in absorbance at 760 nm, which indicates the reduction of vanadate(V) to vanadyl(IV). This spectral change was ob- served immediately after the start of the reaction, and the reduction was maintained for at least 24 h. Amounts of reduced vanadium lot Amounts of non-reduced vanadium for given amounts of added vanadium (mM) given amounts of added vanadium (mM) 5.0 5.0 10.0 Peak 2 4 mM VI VI 4 00 mM (100'/.| Peak 2 . 8 mM V(V> 5.24 mM (65.5V.) Peak 2 16 mM V(V) .................... ....... 522 mM (326''.) 276 mM (345*.) 10.78 mM (67 4V Figure 5. Reduction of vanadate(V) at different concentrations by material from peak 2 contained vanadobin, as measured by ESR spec- trometry. All vanadate(V) was reduced to vanadyl(lV) at a concentration of 4 mM vanadate(V). However, at 8 mM and 16 mM. a part of the vanadate(V) added was reduced by vanadobin. Thus, 0.8 ml of solution of vanadobin could completely reduce about 5.2 mM of vanadate. In 0.8 ml of the solution of vanadobin, the concentration of vanadium was intrinsically 25 mM. If I mole of vanadobin contains I mole of vanadium ions, then 5 moles of vanadobin readily reduce 1 mole of vanadate(V) to vanadvl(IV). results obtained are summarized in Figure 5: all of the vanadium in 4 mM vanadate(V), 65.5% of that in 8 mM vanadate(V) and 32.6% of that in 16 mM vanadate(V) was reduced, respectively, after adding vanadobin. It is clear that 0.8 ml of vanadobin prepared by us can reduce 5.2 mM vanadate(V) to vanadyl(IV). An aliquot of 0.8 ml of vanadobin contained 25 mAf intrinsic vanadium. If 1 mole of vanadobin contains 1 mole of vanadium, 5 moles of vanadobin readily reduce 1 mole of vanadate(V) to vanadyl(IV). This assumption, however, needs further investigation. When the same experiments were carried out with the material in peak 1 , a decrease in reducing ability was de- tected, as shown in Figure 6. The material in peak 1 re- Reduction ofvanadote(V) by vanadobin: observation by ESR spectrometry ESR spectrometry was used to confirm the above re- sults. The oxovanadyl chemical species [VO 2+ (IV)] is the only species of vanadium that is detectable with an ESR spectrometer. Because the intensity of the ESR signal due to VO 24 (IV) depends on the pH, the pH of samples was adjusted to 0.21 (the pH at which the highest intensity of signals was obtained) by the adding one half volume of 4 M H : SO 4 . Figure 4 shows the ESR spectra derived from (a) vanadobin and (b) from a mixture of vanadobin and 8 mM vanadate(V). The signal intensity of the latter was about 1.2 times as strong as that of the former solution, suggesting that a portion of the added vanadate(V) was reduced to the vanadyl(IV) species by vanadobin. The Amounts ol reduced vanadium (Of Amounts ot non-reduced vanadium for given amounts of added vanadium (mM) given amounts of added vanadium (mM) 50 50 10.0 150 1.4 mM V( V) 069 mM (173V.) Peak I 8 mM VI VI 049mM(61V.) Peak I 16 mM V(VI 052 mM (33V. 3 31 mM (82 7V.) 751 mM (939'.) 1548 mM (967V.) Figure 6. Reduction of vanadate(V) at different concentrations by material in peak I which contained no vanadobin, measured by ESR spectrometry as described in the legend to Figure 5. Less reducing ability was observed in this case, unlike that shown in Figure 5. The material in peak I reduced a solution of about 0.5 mM vanadate(V) which cor- responded to between 17.3% and 3.3% of the added metal. VANADATE REDUCTION BY VANADOBIN 193 duced about 0.5 mM vanadate(V), which corresponded to 17.3% to 3.3% of the added metal. The material in the other peaks eluted from the column showed little reducing ability. Among the rest, the material in peak 3 did not show a reducing ability, although it contained the second highest level of vanadium. Discussion Because almost all the vanadium contained in vana- dobin is kept in a reduced chemical form (Michibata et a!.. 1990a), it seems clear that vanadobin cannot only reduce the metal but also maintain in the reduced form. The present results have demonstrated that vanadobin, extracted from the vanadium-rich blood cells of Ascidia gemmala by elution from a column of Sephadex G-15, can reduce vanadate(V) to vanadyl(IV), as shown both by spectrophotometry and ESR spectrometry. We have confirmed that no reduction of vanadate(V) occurred when inorganic vanadate(V) and vanadyl(IV) (8 mM) were mixed together under the same conditions as those used in the experiments described here (data not shown). It is therefore clear that vanadobin reduces vanadate(V) to vanadyl(IV). Except for the vanadobin eluted in peak 2 (Fig. 2), the materials in the other peaks showed little ability to reduce the metal. Unexpectedly, the material in peak 3, which contained the second highest amount of vanadium (Fig. 2), did not show a reducing ability. Although its absorption spectrum was different from that of peak 2, which was the typical spectrum of vanadobin, it closely resembled that of the inorganic vanadium complex in the +4 oxi- dation state, as reported previously (Michibata et ai, 1990a). In other words, it may be that peak 3 did not contain vanadobin but inorganic vanadium released from vanadobin; therefore, no reducing ability would be ob- served. A crude supernatant, obtained from a homogenate of the blood cells of A. gemmata, was also able to reduce vanadatef V). This result is not surprising. In living blood cells, various reducing agents, such as ascorbic acid, glu- tathione, and cysteine, are generally present. In fact, the supernatant reduced vanadate(V) slowly for over 31 h after mixing of the supernatant with vanadate(V) while the extracted vanadobin rapidly reduced the metal after mixing vanadobin with vanadate(V). These phenomena suggest that the crude supernatant contains not only sev- eral reducing agents but also several oxidizing agents and, therefore, the reduction of vanadate(V) to vanadyl(IV) may compete against a re-oxidation of vanadium(IV) by some intrinsic oxidizing agents. This may require much more time than that required for purer vanadobin. Nakanishi's group isolated a tunichrome, composed of three pyrogallol subunits, from ascidian blood cells, and they proposed that it was involved in both the accumu- lation and the reduction of vanadium in the blood cells (Macara et ai, 1979a, b: Bruening et ai. 1985). However, in addition to the fact that no fluorescence due to the tunichrome was observed from the vanadocytes (Michi- bata et ai, 1988; 1990b), Bulls et ai (1990) pointed out that analogues of the tunichrome were barely able to re- duce vanadium(V) to vanadium(IV). Moreover, specific binding of vanadium has not yet been observed within the tunichrome. Therefore, it is noteworthy that the agent that combines with vanadium can reduce vanadate(V), as shown in the present experiments. We have already demonstrated that the vanadium- containing blood cells the vanadocytes are the signet ring cells, and not the morula cells as previously thought (Michibata et ai, 1987); we have also shown that vana- dobin is contained in the signet ring cells (Michibata and Uyama, 1990) and we have demonstrated that vanadobin may well be a universal complex in ascidians, playing a prominent role in the accumulation of vanadium in blood cells and in the maintenance of its concentration (Mi- chibata et ai, 1990a). Therefore, we conclude that van- adate(V) is reduced by vanadobin in the vanadocytes of vanadium-containing ascidians, even though details of the mechanism remain unresolved. The physiological ability, observed uniquely in ascidian blood cells, to reduce and maintain vanadium in the low-oxidation state attracts many investigators in a variety of fields. Acknowledgments We would like to express our heartfelt thanks to Dr. T. Numakunai and all other members of the staff of the As- amushi Marine Biological Station of Tohoku University in Asamushi, Aomori, Japan, for supplying the materials and facilitating parts of our work. Thanks are also due to Dr. A. Takeuchi of Toyama University for kindly making his ESR spectrometer available to us. This work was sup- ported in part by Grants-in-Aid from the Ministry of Ed- ucation, Science and Culture, Japan (#01480026 and #01304007) and was also supported financially from the Institute for Marine Biotechnology. Literature Cited Bruening, R. ( . E. M Oltz, J. Furukawa. K. Nakanishi, and K. Kustin. 1985. Isolation and structure of tunichrome B-l, a reducing blood pigment from the tunicate Ascidia nigra L. J Am. Chern. Soc. 107: 5298-5300. Bulls, A. R., C. G. Pippin, F. E. Hahn, and K. N. Raymond. 1990. Synthesis and characterization of a series of vanadium-tun- ichrome B 1 analogues. Crystal structure of a tris(catecholamide) complex of vanadium. J. Am. Chem. Soc. 112: 2627-2632. Chasteen, N. D. 1983. The biochemistry of vanadium. Structure ami Bonding 53: 105-138. ('launch, R. T., and M. M. Jones. 1963. Vanadium(III) sulfate. Inarg. Synthesis 1: 92-94. 194 H. MICH1BATA ET AL. Dingley, A. L., K. Kustin. I. G. Macara, and G. C. McLeod. 1981. Accumulation of vanadium by tunicate blood cells occurs via a specific anion transport system. Biochim, Biophys, Ada 649: 493-502. Frank, P., R. M. K. Carlson, and K. O. Hodgson. 1986. Vanadyl ion EPR as a noninvasive probe of pH in intact vanadocytes from Ascidia ceralodes. Inorg. Chem. 25: 470-478. Ilirata. J., and H. Michibata. 1990. Valency of vanadium in the van- adocytes of Ascidia gcmmata separated by density-gradient centrif- ugation. / Exp. Zoo/. 257: 160-165. Kustin, K., G. C. McLeod, T. R. Gilbert, and I,. B. R. Briggs. 1983. Vanadium and other metal ions in the physiological ecology of marine organisms. Structure and Bonding 53: 139-160. Lee, S., K. Kustin, VV. E. Robinson, R. B. Frankel, and K. Spartalian. 1988. Magnetic properties of tunicate blood cells. I. Ascidia nigra. J Inorg. Biochem 33: 183-192. Macara, I. G., G. C. McLeod, and K. Kustin. I979a. Isolation, properties and structural studies on a compound from tunicate blood cells that may be involved in vanadium accumulation. Biochem ./. 181: 457- 465. Macara, I. G., G. C. McLeod, and K. Kustin. 1979b. Tumchromes and metal ion accumulation in tunicate blood cells. Ctmip. Biochem. Physiol 63B: 299-302. McLeod, G. C., K. V. Ladd, K. Kustin. and D. L. Tnppen. 1975. Extraction of vanadium(V) from seawater by tunicates: a revision of concepts. Limnol. Oceanogr 20: 491-493. Michibata, H., T. Miyamoto, and H. Sakurai. 1986. Purification of vanadium binding substance from the blood cells of the tunicate, Ascidia sydneiensis samea. Biochem. Biophys. Res Commim 141: 251-257. Michibata, H., J. Hirata, M. (Jesaka, T. Numakunai, and H. Sakurai. 1987. Separation of vanadocytes: determination and characteriza- tion of vanadium ion in the separated blood cells of the ascidian. Ascidia ahodon. J Exp. /Mol. 244: 33-38. Michibata, H., J. Hirata, T. Terada, and H. Sakurai. 1988. Autonomous fluorescence of ascidian blood cells with special reference to identification of vanadocytes. Expenentia 44: 906-907. Michibata, H., H. Hirose, K. Sugiyama, V. Ookubo, and K. Kanamori. 1990a. Extraction of a vanadium-binding substance (vanadobin) from the blood cells of several ascidian species. Bio/ Bull. 179: 140- 147. Michibata, H., and T. Uyama. 1990. Extraction of vanadium-binding substance (vanadobin) from a subpopulation of signet ring cells newly identified as vanadocytes in ascidians. J. Exp. Zool. 254: 132- 137. Michibata, H., T. fyama, and J. Ilirata. 1990b. Vanadium-containing blood cells (vanadocytes) show no fluorescence due to the tuni- chrome in the ascidian. Ascidia sydneiensis samea. Zool. Sci. 1; 55-61. Tullius, T. D., \V. O. Gillum, R. M. K. Carlson, and K. O. Hodgson. 1980. Structural study of the vanadium complex in living ascidian blood cells by X-ray absorption spectrometry. J. Am. Chem. Soc. 102: 5670-5676. Reference: Biol. Bull. 181: 195-198. (August, 1991) A Breeding Population of the Western Pacific Crab Hemigrapsus sanguineus (Crustacea: Decapoda: Grapsidae) Established on the Atlantic Coast of North America JOHN J. McDERMOTT Department of Biology, Franklin and Marshall College. Lancaster, Pennsylvania 17604 The west Pacific grapsid crab Hemigrapsus sanguineus was found in the United States for the first time in 198S. Additional crabs were recovered in 1990 from Townsends Inlet and Cape May Harbor, New Jersey (22 males, 16 females), and four of the females collected from June through September were ovigerous. Thus, H. sanguineus has now established itself in southern New Jersey, the first well-documented case of an exotic brachyuran becoming established along the east coast of the United States. The marine grapsid crab Hemigrapsus sanguineus (de Haan, 1853), native to the western Pacific Ocean, was first recovered in the eastern United States in September 1988; a single ovigerous female was found in a rocky in- tertidal zone under a bridge at Townsends Inlet, Cape May County, New Jersey (1) (Fig. 1). The site was not reinvestigated until 20 months later (28 May 1990), and at that time an immature female H. sanguineus was re- covered [carapace width (CW) X carapace length (CL) = 12.8 X 10.8 mm]. This second finding suggested that the original record of the crab in New Jersey was not simply fortuitous, but that this Pacific brachyuran was established in Atlantic waters. The discovery also provided a rare opportunity to document a potentially major in- troduction. Subsequently, 36 additional mature and immature crabs (22 males and 14 females) (Fig. 2) were collected (26 June, 3 and 6 July, 22 August, 2 1 September, 1 5 and 28 October 1990) at the same site. Crabs were usually located in the mid to upper intertidal zone under rocks covered with Fucus vesiculosus, but at low tide some moved below the mid intertidal. As with many intertidal Received 15 November 1990; accepted 30 April 1991. grapsid crabs that live among rocks, individuals of H. sanguineus are secretive and swift, and one must turn rocks over rapidly and snatch them quickly in order to catch them; otherwise, they retreat among the lower in- accessible rocks. All crabs were transported to the Franklin and Marshall laboratory where they were easily main- tained individually in 2 cm of unaerated seawater (tem- perature 19C, salinity ~30%o); they subsisted on pieces of the macroscopic algae Enteromorpha sp. and Ulva lac- tuca. and otherwise required minimal care, except for a daily water change. Crabs were measured with a dial cal- ipers to the nearest 0.1 mm. The 22 males ranged from 8.3 to 24.1 mm CW (mean 17.2 4.6), and from 7.3 to 21.0 mm CL (mean 15.2 4.1); 15 females ranged from 3.6 to 24.4 mm CW (mean 17.6 5.1), and from 3.3 to 21.3 mm CL (mean 15.3 4.4). The greater range in females was due to one juvenile (3.6 mm), resulting from the 1990 spawning season, collected 15 October. The next smallest female was the 12.8 mm specimen collected 28 May; the smallest male (8.3 mm) was collected 26 June. These and other juveniles were probably from the 1989 spawning season. Ovigerous females (n == 4) were found from June through September (not in July collections). One oviger- ous crab, obtained on 26 June (CW X CL = 24.4 X 21.3 mm), carried embryos half filled with yolk but with no apparent pigment or eye development. The crab was pre- served three days later, and the embryos were counted (n = 28,702). Two ovigerous crabs (CW X CL = 1 5.2 X 1 3.2 mm, 23.2 X 19.8 mm) were collected on 22 August. The smaller of them released its zoeae (n = 6328) two days later, and on 27 August it had another brood. Although the crab aborted many undeveloped eggs from this brood. 195 196 J. J. McDERMOTT -CMH CAPE MAY 7C50' Figure 1. Sites on the Atlantic coast of the Cape May peninsula, New Jersey, where Hfmigrupsus sanguineus has been discovered. TI = Townsends Inlet (the main study location; CMH = Cape May Harbor, which is inside of Cape May Inlet (indicated). the zoeae (n = 3360) hatched from the remaining eggs 25 days later. The larger of the two crabs had nearly the full complement of yolk in its embryos, and released its zoeae after 1 7 days. Many zoeae were eaten by the crab, so the brood was not counted. One of the two females collected on 21 September (CW X CL = 19.3 X 16.8 mm) was ovigerous, and it released its zoeae the next day (n = 13,090). The first female H. sanguineus collected 24 September 1988 (1) was also ovigerous. Thus the egg- bearing period extends from at least June through Sep- tember. A non-ovigerous female H. sanguineus (CW X CL = 18. 5 X 16.2 mm) was collected by Lisa Wargo sometime during January or February 1990, in Cape May Harbor (38 57' 12" N, 74 53' 20" W), 23.4 km south of the Townsends Inlet site (Fig. 1 ). This crab was one of at least three specimens, of apparently the same species; they were recovered from small clumps of mussels (Mylilus editlis L.) obtained in the Harbor and were maintained in an aquarium, with fish and other invertebrates, at the Cape May Point State Park. The crabs must have been very small when collected, because they were only noticed later when they were ~ 10 mm CW. The crab delivered to me was maintained in the aquarium until it was preserved on 18 June 1990. H. sanguineus has become established on the ocean side of Cape May County, New Jersey, and likely will be found in environmentally suitable locations elsewhere in the state (i.e., in the upper intertidal levels of rocky areas or possibly on artificial substrates suspended near the wa- ter line). This crab was probably introduced into the United States before 1988, because its presence could have been easily overlooked. New Jersey may not be the focal point or the only focal point of its introduction, and per- haps it is already established in adjoining mid-Atlantic states. H. sanguineus is now the only other member of the genus found in the Atlantic Ocean (2,3,4), except for H. affinis Dana, 1851, which ranges from Cape St. Roque, Brazil, to the Gulf of San Matias, Patagonia (5). The genus is represented in the northern hemisphere of the eastern Pacific by H. niulus (Dana, 1851) and H. oregonensis (Dana, 1851) (5,6), and by //. creniilatus (H. Milne Ed- wards, 1837) in the southern hemisphere (5). A brief resume of its life history characteristics may give insight into the potential impact that this grapsid might have on the intertidal environment along the At- lantic seaboard. In Japan, at about the same latitude as New Jersey, H. sanguineus is one of the largest and most common grapsid crabs living in rocky intertidal habitats (7). It is sympatric there with at least one other abundant grapsid, H. penicillatus (de Haan, 1835). In the rocky in- tertidal of New Jersey, it may be competitive with the native brachyurans found in the same habitat; i.e.. three xanthids, Eurypanopeus depressus (Smith, 1869), Neo- panope sayi (Smith, 1869), and Panopeus herbstii H. Milne Edwards, 1 834; juveniles of the portunid, Carcinus maenas (Linnaeus, 1758); and juveniles of the cancrid, Cancer irroratus Say, 1817. Fukui's studies (8) on the fecundity of H. sanguineus in Japan, along with my observations in New Jersey, in- dicate the potential for its rapid increase in Atlantic waters. Fukui determined that females may live for at least three years, the largest capable of producing more than 5 broods/year, with as many as 56,000 eggs/brood. Using Fukui's regression equation for the correlation between the number of eggs/brood and carapace width, the 35.8 mm ovigerous crab collected in 1988 ( 1 ) may have carried more than 52,000 embryos. My preliminary data, how- ever, on brood size versus carapace width from the other New Jersey specimens give values somewhat greater than those predicted by Fukui's equation. In Japan, the crab's breeding season is from March to October, with the main peak in May-June (8), a period longer than the four- month season suggested by my preliminary information from New Jersey. The 25-day developmental period at 19C, recorded for the second brood of the 15.2 mm H. EXOTIC CRAB ESTABLISHED IN U.S. 197 B Figure 2. Three living Hemigrapsus sanguineus collected 26 June 1990 at Townsends Inlet, New Jersey: A. mature female. 18.5 mm CW. scale = 20 mm; B. mature male, 14.2 mm CW and immature female. 8.3 mm CW. sanguineus mentioned above, is predicted by Fukui's regression equation for the relationship between incuba- tion period and water temperature for embryos raised in Japan. Water temperatures in New Jersey from June through September would be conducive to the production of about four broods during this period. Fukui found that females reach maturity at 14.0 mm CW, and have a max- imum CW of 39.0 mm. Thus, the 35.8 mm ovigerous female collected in New Jersey in 1988 is near the max- imum size recorded for Japan, and the smallest mature crab found at Townsends Inlet (CW = 15.2 mm) is close to Fukui's value. H. sanguineus has not been recorded from the west coast of the United States (6,9,10,1 1), where the only ex- otic brachyuran is Rhithropanopeus harrisii (Gould, 1841). a xanthid introduced from the Atlantic east coast. There may have been numerous attempts at colonization of the Atlantic coast of the United States and Canada by species of exotic brachyurans, but most seem to have failed prior to H. sanguineus. Several authors, cited by Williams (4). agree that the green crab, C. maenas, was probably introduced into North America from the eastern Atlantic; it now ranges from Nova Scotia to Virginia (greatest abundance in New England). The timing of such an in- troduction is not known, and because it could have been prehistoric, its establishment has never been documented. Carcinus has had considerable impact in the United States because it prefers bivalves (some commercial) as prey (12.13). The notorious Chinese mitten crab, Eriocheirsi- nesis H. Milne Edwards, 1 853, has been reported in Lake Erie, but seems never to have established a breeding pop- ulation in the Great Lakes ( 14). A single specimen of Er- loc/icir was reported from Louisiana's Mississippi River Delta region in 1987(15,16), but has not been found again since. H. sanguineus could have a great impact on the normal rocky intertidal environment. Therefore, the latitudinal extent of the species must be determined, its spread mon- itored, and its community interactions studied. Research on its further distribution in New Jersey and other mid- Atlantic states is now underway. Acknowledgments 1 thank L. Wargo, formerly of the Cape May Point State Park, New Jersey, for sending me a specimen of//. sanguineus from Cape May Harbor and supplying per- tinent information on its collection. I am grateful to J. N. Kraeuter and R. Wargo of the Rutgers University Shellfish Research Laboratory for their assistance, to J. L. Rich- ardson of Franklin and Marshall College and A. B. Wil- liams of the NMFS Systematics Laboratory. Smithsonian Institution, for reviewing the manuscript, and to C. R. Shearer, Jr. and R. A. Fluck of Franklin and Marshall College for providing photographs and for help in the maintenance of crabs, respectively. Literature Cited I. Williams, A. B., and J. J. McDermott. 1990. An eastern United States record for the western Indo-Pacific crab. HcmiKfapsus sail- 198 J. J. McDERMOTT .tfii/wii.v (Crustacea: Decapoda: Grapsidae). Proc. Dial. Soc. Wash. 103: 108-109. 2. Monod, Th. 1956. Hippideael Brachyuraouest-africains. Memoires ill' I'liislilW Franc.(iis tl'Ajriuue Noire. No. 45. 674 pp. 3. Manning, R. B., and L. B. I lollhuis. 1981. West African brachyuran crabs (Crustacea: Decapoda). Smithson. Contrib. Zoo/. No. 306, 379 pp. 4. Williams, A. B. 1984. Shrimps, Lobsters, and Crabs of the Atlantic Coast of the Eastern United States. Maine to Florida. Smithsonian Institution Press, Washington, DC. 550 pp. 5 Kathbun, VI. J. 1918. The grapsoid crabs of America. U. S. Natl. Mux. Bull No. 97: 461 pp., 161 plates. 6. Morris, R. H., D. P. Abbott, and E. C. Haderlie. 1980. Inlenidal Invertebrates ofCalifornia. Stanford University Press. Stanford, CA. 690 pp. 7. Sakai, T. 1976. Crabs oj Japan and the Adjacent Seas. Kodansha Ltd. Tokyo, 773 pp. (English text), 251 plates (many colored), 461 pp. (Japanese text), as 3 separate volumes. 8. Fukui, V. 1988. Comparative studies on the life history of the grapsid crabs (Crustacea, Brachyura) inhabiting intertidal cobble and boulder shores. Pub. Seto Mar. Biol. Lab. 33: 121-162. 9. Carlton, J. T. 1979. Introduced invertebrates of San Francisco Bav. Pp. 427-444 in San Francisco Bay the Urbanized Estuary. T. J. Conomos, ed. Pacific Division, American Association for the Ad- vancement of Science, San Francisco, CA. 10. Carlton, J. T. 1985. Transoceanic and interoceamc dispersal of coastal marine organisms: the biology of ballast water. Oceanog. Mar Biol. Aim. Rev. 23: 313-371. 1 1. Carlton, J. T. 1989. Man's role in changing the face of the ocean: biological invasions and implications for conservation of near-shore environments. Consen: Biol. 3: 265-273. 12. Glude, J. B. 1955. The effects of temperature and predators on the abundance of the soft-shell clam, Mya arenaria. in New England. Trans. Am. Fish. Soc. 84: 13-26. 1 3. Ropes, J. W. 1968. The feeding habits of the green crab, Carcinus niucnux (L.). Fish. Bull. 67: 183-203. 14. Nepszy, S. J., and J. H. Leach. 1973. First records of the Chinese mitten crab, Erioeheir sinensis, (Crustacea: Brachyura) from North America. J. Fish. Res Bd Can. 30: 1909-1910. 15. Horwath, J. L. 1988. Injurious wildlife: mitten crabs. Proposed rule. Federal Register 53(219): 45784-45788. 16. Horwath, J. L. 1989. Importation or shipment of injurious wildlife: mitten crabs. Rules and regulations. Federal Register 54(96): 22286- 22289. CONTENTS Annual Report of the Marine Biological Laboratory 1 Lenhoff, Howard M. Ethel Browne, Hans Spemann, and the discovery of the Organizer Phenomenon 72 BEHAVIOR Painter, Sherry D., Maria G. Chong, Mary A. Wong, Ann Gray, Jeffry G. Cormier, and Gregg T. Nagle Relative contributions of the egg layer and egg cor- don to pheromonal attraction and the induction of mating and egg-laying behavior in Aplwia 81 GENERAL BIOLOGY Copeland, D. Eugene Fine structure of photophores in Gonostoma elonga- titin: detail of a dual gland complex 144 Read, A. T., J. A. McTeague, and C. K. Govind Morphology and behavior of an unusually flexible thoracic limb in the snapping shrimp, Alpheus het- erochelis 158 PHYSIOLOGY DEVELOPMENT AND REPRODUCTION Ikegami, Susumu, Tomoko Mitsuno, Masanori Ka- taoka, Satoshi Yajima, and Mieko Komatsu Immunological survey of planktonic embryos and larvae of the starfish Astrrina pectinifera. obtained from the sea, using a monoclonal antibody directed against egg polypeptides 95 Morse, Daniel E., and Aileen N. C. Morse Enzymatic characterization of the morphogen rec- ognized by Agaricia hiimilis (scleractinian coral) lar- vae . 104 ECOLOGY AND EVOLUTION Brown, Alice F. Outbreeding depression as a cost of dispersal in the harpacticoid copepod, Tigriopu.60mm sex removals sex changes 9A 3 2 6 21 6 4 2 1 9B 1 4 4 14 4 3 2 2 10 1 1 23 5 T ) -) B. TP and INT remain at outset, subsequently removed: No. males Remaining Removed No. females remaining No. No. No. changing subsequent subsequent Reef TP INT IP <60mm >60mm sex removals sex changes 12 4 5 7 38 5 9 6 14 3 4 24 1 3 2 TP = Terminal Phase coloration; INT = Intermediate coloration (early Terminal Phase); IP = Initial Phase coloration. In the first set (A), all males (TP, INT, and IP) were removed at the outset. In the second set (B), only IP males were removed. The seventh column shows the number of individuals having changed sex two weeks after the experiment began. At that point, in both sets of experiments, some TP and INT individuals were subsequently removed, and the responses of the remaining individuals were noted, again after a two-week interval. 202 R. R. WARNER AND S. E. SWEARER 16 14 - O 12 - 10 < O 8 cc LJJ CO 6 2 - 4 6 8 10 NUMBER REMOVED 12 16 Figure 2. Responses of remaining individuals to removals of INT and TP males of Thalassoma bifasciatum on patch reefs in Tague Bay, St. Croix. Shown are the results from the removals from reefs shown in Table II (initial and subsequent removals shown separately) and four larger reefs. On the vertical axis are the total number of individuals changing from IP coloration to INT coloration during a two-week period. The dotted line represents a one-for-one response. The solid line is a regression of the number changing (NC) against the number removed (NR). The regression equation is NC = .894NR - 0.049; r = .91, P < 0.001. response. It should be noted, however, that not all of the responses on larger reefs were necessarily sex changes, be- cause IP males were not removed along with the TP and INT males. Changes in coloration As early as the first day of the manipulation, IP indi- viduals sometimes slightly darkened their heads while courting and mating. These coloration differences were not detectable outside of the mating period. By day 3, however, the largest individuals had begun the transition to TP coloration. Over the course of the observations (a maximum of 28 days), only the two or three largest in- dividuals on each reef proceeded to develop full INT col- oration, and this was apparent within five days. In the rest of the changing individuals, the head region had a permanent bluish tinge, and the two anterior lateral blotches had darkened and enlarged, but the body was not yet green. No individuals attained full TP status over the course of the experiment. None of the IP fishes on the control reefs initiated color change during the initial experimental period. Sex change verified No individual identified as a potential sex-changer had functional ovaries. All 10 gonads examined histologically were testes engaged in active sperm production (Table II). Furthermore, all of these testes were secondary (derived from previously functional ovaries), with varying amounts of degenerating oocytic material still in the gonadal la- mellae. Sex change apparently can proceed quite rapidly. Even individual 10A3, observed spawning as a female before the manipulation and captured 8 days later, was already producing tailed sperm. Of the 10 individuals investigated histologically, in only three had spermatogenesis not yet proceeded to the production of mature sperm. All of these had been first observed to display early intermediate col- oration 8 to 9 days before capture. Discussion It is clear that female bluehead wrasses change sex when larger brightly colored males are removed from the local population. Social control of sex change in reef fishes was first documented two decades ago (Fishelson, 1970; Rob- Table II Si:e, coloration, two estimates of time since initiation of sex change, and gonadal state for ten individuals obserfed to change jnini initial phase coloration and female behavior to male coloration and behavior over the course of the experiments Days since Days since individual began Histological Fish Length at capture beginning of to display male condition of ID (mm S.L.) Coloration experiment coloration gonad 9A2 67.0 INT 19 19 Act 2, t.s. 9A6 65.8 early INT 28 9 Act 2, t.s. 9B1 65.2 INT 28 28 Act 2, t.s. 9BX 63.9 early INT 28 8 Recent 2, t.s. 9BY 55.0 early INT 28 8 Act 2. t.s. 9BZ 55.2 early INT 28 8 Recent 2, no t.s. IOA1 64.6 early INT 17 9 Act 2, no t.s. 10A3 69.3 early INT 8 8 Act 2, t.s. 10A4 65.3 early INT 17 9 Act 2. no t.s. T41 64.5 early INT 17 9 Act 2, t.s. INT = Intermediate coloration; Act 2 = active secondary male, with most of the gonad occupied by spermatogenic crypts; Recent 2 = transitional gonad. with much of the gonad still containing degenerating oocytic material, some spermatogenic crypts present; t.s. = tailed sperm present. SEX CHANGE IN THE BLUEHEAD WRASSE 203 ertson, 1972), and its precision has been described on sev- eral occasions (e.g., Fricke and Fricke, 1977; Moyer and Nakazono, 1978; Shapiro, 1979, 1980; Ross el at.. 1983, 1990; comprehensively reviewed by Ross, 1990). Within the genus Thalassoma, social control has been shown in captive groups of T. lucasanum (Warner, 1982), and T. diiperrey(Rossetal.. 1983, 1990), but never before under field conditions. In T. duperrey it was further shown that it was the relative size of the individual within the local social group that was the cue for sex change; neither the sex nor the coloration of the other individuals in the group appeared to have any additional stimulatory or inhibitive effect. While this is likely to be true for Thalassoma bi- fasciatum as well, it cannot be conclusively shown, given our experimental design. Shapiro (1980) obtained a remarkably precise response to multiple removals of males in the serranid Anthias squamipinnis, and also observed that not all individuals responded immediately to the removal. Instead, sex changes (as indicated by coloration changes) tended to be separated by an interval of about two days. Shapiro hy- pothesized that the subsequent development of sex change in subordinate females was slowed by the initial devel- opment of male characters in higher-ranked females. While our monitoring was not conducted often enough to detect the exact sequence of successive sex changes, some individuals did not initiate the process immediately. Of the 22 individuals that changed sex on the small reefs, 4 were known to have delayed changing at least 7 days after the removal of the males. The aggression directed by large sex-changing females toward other large individ- uals may have contributed to this delay. While the removal of TP males in Thalassoma bifas- ciatitm results in a precise sex-change response (Fig. 2), it is unclear how this precision is attained. Particularly on bigger reefs with large populations, it seems unlikely that the mechanism is a direct response by one individual to the loss of a particular male. On the basis of their ex- periments with small groups of fishes, Ross et al. (1990) suggested that sex change in T. duperrey was mediated by a critical size-ratio mechanism: individuals would change sex when there were sufficiently few larger indi- viduals as well as enough smaller individuals present in the local population. In our study, it is difficult to dis- tinguish direct responses to removal from reactions to changes in the size, sex, or color ratios because removals changed these ratios as well. The proportions of color types in the pre-manipulation populations in our study was variable: from 8 1 to 96% of the total population was in the IP color phase (Table I). The mean percentage of the total population within the IP size range was 88%, a ratio of IP to TP of 7. 3:1. Of the 147 females remaining on all reefs after the initial removals, the 17 largest (11.6%) changed sex, resulting in a color ratio of 7.6:1. One can also investigate the possibility of a critical ratio by mon- itoring the minimum sex- or size-ratio at which individuals were seen to change sex. In other words, what percent of the population was smaller than the smallest individual seen changing sex on each reef? This varied from 83 to 94%, with a mean of 88.2%. (n = 5), suggesting a critical ratio of 7.5 smaller individuals to larger. However, we stress that these various ratio measures would be very similar even if a simple one-for-one replacement sex- change mechanism was in operation. To resolve this issue, one would need to vary the number of smaller individuals left on the reef after the male removals, similar to the experiments of Ross et al. (1990) for captive populations of T. duperry. In any event, there is certainly no fixed critical ratio for T. bifasciatum as a whole, because the sex and col- oration ratios change radically with reef size (Warner and Hoffman, 1980). It may be, however, that individuals re- spond to a ratio appropriate to the reef on which they find themselves (termed the behavioral-scaling model in Ross, 1990). Sex change of a number of large females also reduces the pool of potential mates for the newly domi- nant males. If the critical-ratio hypothesis is correct, there should be slightly fewer individuals changing sex than were removed on the experimental reefs. There does exist such a trend in our removal experiments (Fig. 2), but it will take much larger sample sizes to distinguish this trend from random variation. The behavioral, coloration, and gonadal responses to male removals among the females on the experimental reefs were very rapid, as were seen in other sex-changing species (Robertson, 1972; Shapiro, 1979, 1985). Behav- ioral responses occurred almost instantaneously, suggest- ing that the initiation of mating and aggressive behaviors characteristic of males in this species are not dependent on hormone levels (see Crews and Moore, 1986, for a discussion of the various possible relationships between hormones and mating behavior). While we did not collect gametes from matings, it is extremely unlikely that sex- changing individuals could produce sperm in the first few days after the manipulations. Thus these individuals ap- pear capable of spawning in the male role without release of sperm. Ross (1990) suggested that gametocytes of the heter- ologous sex are maintained in the gonads of some species to facilitate rapid sex change. While T. bifasciatum shows no sign of such heterologous gametocytes in the ovary, this does not appear to inhibit the ability of females to rapidly assume functional male status. In this study, we could not determine the relative timing of changes in coloration and gonadal condition; all in- dividuals that were collected had initiated changes toward TP coloration, and all were well advanced in sperm pro- duction. Stoll (1955) induced both color change and sex change in 1 3 females with injections of methyl testoster- one; the color change began four days after treatment, 204 R R WARNER AND S. E. SWEARER and all individuals were producing sperm when they were examined 20 days after the injection. However, other studies on this species have shown that under normal cir- cumstances, sex change can precede color change, because larger IP individuals can be secondary males (Warner and Robertson, 1978). Individuals should change sex when the reproductive prospects of functioning as the opposite sex exceed the expectations of the current sex (the size-advantage model; seeGhiselin, 1969; Warner el a/., 1975; Charnov, 1982a; Warner, 1988b). If mate monopolization depends on the ability to dominate other individuals, and if dominance depends on relative size, then it is the largest individuals in a local population that should change sex in response to large male removals. Relative size does convey domi- nance in the bluehead wrasse (Warner and Schultz, in prep.), and the largest males are most reproductively suc- cessful (Warner, 1984). Accordingly, it is only the largest females that change sex when the TP and INT males are removed (Fig. 1), as predicted by the size-advantage model. Acknowledgments We thank J. Caselle, C. Gabor, and B. L. Rogers for assistance in the field, and E. T. Schultz and two anon- ymous reviewers for helpful comments. This research was supported by the National Science Foundation (BSR 8704351 toR.R.W.). Literature Cited Charnov, E. 1982a. The Theory of Sex Allocation. Monographs in Pop. Biol. 18. Princeton University Press, Princeton, NJ. Charnov, E. 1982b. Alternative life-histories in protogynous fishes: a general evolutionary theory. Mar. Ecol. Prog. Ser 9: 305-307. Crews, D., and M. C. Moore. 1986. Evolution of mechanisms con- trolling mating behavior. Science 231: 121-125. Feddern, H. A. 1965. The spawning, growth, and general behavior of the bluehead wrasse. Thalassoma bifasciatum (Pisces: Labndae). Bull. Mar. Sci. 15:896-941. Fishelson, L. 1970. Protogynous sex reversal in the fish Anthias squamipinnis (Teleostei. Anthiidae) regulated by the presence or ab- sence of male fish. Nature 221: 90-9 1 . Fricke, H. W., and S. Fricke. 1977. Monogamy and sex change by aggressive dominance in a coral reef fish. Nature 266: 830-832. Ghiselin, M. T. 1969. The evolution of hermaphroditism among an- imals. Q Rev. Biol 44: 1 89-208. Hoffman, S. G., M. P. Schildhauer, and R. R. Warner. 1 985. The costs of changing sex and the ontogeny of males under contest competition for mates. Evolution 39: 915-927. Moyer, J. T., and A. Nakazono. 1978. Protandrous hermaphroditism in six species of the anemonefish genus Amphiprion in Japan. Japan J Ichthyol. 25: 101-106. Reinboth, R. 1967. Biandric teleost species. Gen. Coin/) Endocrinol.. Abstracts 9: 46. Reinboth, R. 1973. Dualistic reproductive behavior in the protogynous wrasse Thalassoma bifasciatum and some observations on its day- night changeover. Helgol. H'iss. Meeresuiilers. 24: 174-191. Robertson, D. R. 1972. Social control of sex reversal in a coral reef fish. Science 177: 1007-1009. Roede, M. J. 1972. Color as related to size, sex, and behavior in seven labrid fish species (Genera Thalassoma. Halichoeres, and Hemipter- inotus). Studies on the fauna of Curacao and other Caribbean islands 138: 1-264. Ross, R. M. 1990. The evolution of sex-change mechanisms in fishes. Environ. Biol. Fish. 29: 81-93. Ross, R. M., T. F. Hourigan, M. M. F. Lutnesky, and 1. Singh. 1990. Multiple simultaneous sex changes in social groups of a coral- reef fish. Copeia 1990: 427-433. Ross, R. M., G. S. Losey, and M. Diamond. 1983. Sex change in a coral reef fish: dependence on stimulation and inhibition on relative size. Science 221: 574-575. Sadovy, Y., and D. V. Shapiro. 1987. Criteria for the diagnosis of her- maphroditism in fishes. Copeia 1987: 136-156. Schultz, E. T., and R. R. Warner. 1989. Phenotypic plasticity in life- history traits of female Thalassoma bifasciatum (Pisces: Labridae). 1 . Manipulations of social structure in tests for adaptive shifts of life- history allocations. Evolution 43: 1497-1506. Shapiro, D. Y. 1979. Social behavior, group structure, and the control of sex reversal in hermaphroditic fish. Adv. Studies Behav. 10: 43- 102. Shapiro, D. V. 1980. Serial female sex changes after simultaneous re- moval of males from social groups of a coral reef fish. Science 209: 1136-1137. Shapiro, D. V. 1985. Behavioral influences on the initiation of adult sex change in coral reef fishes. Pp. 583-585 in Current Trends in Comparative Endocrinology. B. Lofts and W. N. Holmes, eds. Uni- versity of Hong Kong Press, Hong Kong. Shapiro. D. Y. 1989. Sex change as an alternative life-history style. Pp. 177-195 in Alternative Life-History Styles in Animals M. N. Burton, ed. Kluwer Publishers, Dordrecht, Netherlands. Stoll, I.. M. 1955. Hormonal control of the sexually dimorphic pig- mentation of Thalassoma bifasciatum. Zoologica 40: 125-132. Tee-Van, J. 1932. Color changes in the bluehead wrasse, Thalassoma bifasciatum (Bloch). Bull. N. Y. Zool. Soc. 35: 43-47. Victor, B. C. 1986. Larval settlement and juvenile mortality in a re- cruitment-limited coral reef fish population. Ecol. Monogr. 56: 145- 166. Warner, R. R. 1975. The reproductive biology of the protogynous her- maphrodite Pimelometopon pulchrum (Pisces: Labridae). Fish. Bull. 73: 262-283. Warner, R. R. 1982. Mating systems, sex change, and sexual demog- raphy in the rainbow wrasse, Thalassoma htcasanum. Copeia 1982: 653-661. Warner, R. R. 1984. Deferred reproduction as a response to sexual selection in a coral reef fish: a test of the life historical consequences. Evolution 38: 148-162. Warner, R. R. 1985. Alternative mating behaviors in a coral reef fish: a life-history analysis. Proc. Fifth International Coral Reef Congress, Tahiti 4: 145-150. Warner, R. R. 1987. Female choice of sites versus males in a coral reef fish. Thalassoma bifasciatum. Anim. Behav 35: 1470-1478. Warner, R. R. 1988a. Traditionally of mating-site preferences in a coral-reef fish. Nature 335: 719-721. Warner, R. R. 1988b. Sex change in fishes: hypotheses, evidence, and objections. Environ Biol. Fishes 22: 81-90. Warner, R. R. 1990. Male versus female influences on mating-site de- termination in a coral reef fish. Anim. Behav 39: 540-548. Warner, R. R., and S. G. Hoffman. 1980. Local population size as a determinant of mating system and sexual composition in two tropical reef fishes (Thalassoma spp.). Evolution 34: 508-518. Warner, R. R., and D. R. Robertson. 1978. Sexual patterns in the labroid fishes of the western Caribbean, I: The wrasses (Labridae). Smith- sonian Contrib. Zool. 254: 1-27. Warner, R. R., D. R. Robertson, and E. G. Leigh, Jr. 1975. Sex change and sexual selection. Science 190: 633-638. Reference: Biol Bull. 181: 205-215. (October, 1991) Ontogeny Versus Phylogeny in Determining Patterns of Chemoreception: Initial Studies with Fiddler Crabs MARC J. WEISSBURG'* AND RICHARD K. ZIMMER-FAUST 12 * 1 Marine Environmental Sciences Consortium and ~ Department of Biology. University of Alabama, P.O. Box 369-370. Dauphin Island. Alabama, 36528 Abstract. Fiddler crab ( L'ca longisignalis) first stage zoea and adults were assayed for behavioral responses to 16 amino acids and sugars. Larval chemosensitivity was ex- amined using computer-video motion analysis of swim- ming behavior. Adult sensitivity was assayed by deter- mining the substances that elicit feeding. The pattern of chemoreception expressed by V. longisignalis adults is strongly correlated with that measured previously in adult sand fiddler crabs, Uca pugilator. This concordance among abilities probably reflects shared trophic ecologies of the two species. In contrast, a quantitative analysis shows no significant correlation between the sets of com- pounds inducing chemoreceptive behavior by larval and adult U. longisignalis. The strongest responses (by both stages) are elicited by substances found in potential prey, and differences in prey types among larvae and adults appear responsible for the lack of correlation. Larvae do, however, respond to substances abundant in prey con- sumed by adults, even though these substances are absent, or occur at low levels, in larval prey. Adults, on the other hand, appear insensitive to compounds that cue only lar- val food, but which are maximally stimulatory to larvae. Consequently, our results indicate that the abilities of one life-history stage may be constrained, through develop- ment, by the requirements of later stages. The patterns of correlation among adults of different species, and among life-history stages within a species, indicate that both eco- logical context and developmental factors influence pat- terns of chemosensitivity. Introduction Studies on marine invertebrates have engendered a large body of literature on chemosensory properties and their Received 26 April 1991; accepted 23 July 1991. * Present address: Department of Biology and Marine Sciences Pro- gram. University of South Carolina. Columbia. South Carolina, 29208. ecological implications. Several broad scale patterns are apparent, based on current work. In general, the particular spectrum of substances eliciting a response is correlated with diet. Flesh eaters tend to respond strongly to amino acids, sometimes to organic acids and nucleotides, and are generally unresponsive to sugars (see reviews of Ache, 1982; Carr, 1988; Laverack, 1988; Zimmer-Faust, 1989). The particular substances generating large responses are often dominant organic components in prey tissues. In contrast to flesh eaters, herbivorous organisms are highly responsive to carbohydrates (Zimmer et al.. 1979; Rob- ertson el al, 1981; Trott and Robertson, 1984; Rittschof and Buswell, 1989), which occur as algal storage products and components of extracellular mucous sheaths (Fogg, 1966; Fogg #a/.. 1973;Craigie, 1974;Darley, 1977; Paul- sen et al., 1978) and sometimes to amino acids that are characteristic of algal material. In spite of broad scale associations between diet and the response spectrum of organisms, related or ecologically similar species can display quite dissimilar fine scale pat- terns of chemosensitivity. The spiny lobsters Panulirus argus and P. interrupt us each respond to a wide array of amino and organic acids, and exhibit a broad overlap in the substances eliciting a response. However, within the overlapping subset of stimulatory substances, these two species are differentially responsive to the same com- pounds. For example, P. argus is most responsive to citric acid, ascorbic acid, succinic acid, taurine, and glycine (Ache et al., 1978), whereas P. interruptus responds mostly to glycine, alanine, serine, succinic acid, oxalic acid, and adenosine triphosphate (Zimmer-Faust et al., 1984; Zim- mer-Faust, 1987). Similarly, the coexisting copepods Acartia hudsonica and Eurytenwra herdmani both re- spond to amino acids, although Acartia is stimulated mostly by aliphatic amino acids (e.g., leucine and valine) whereas Eurytemora is most responsive to the dicarboxylic 205 206 M. J. WEISSBURG ET AL amino acids (i.e., glutamic and aspartic acid). When re- sponses to the same compounds are observed, each species reacts to them in different ways (Poulet and Ouellet, 1982). Acartia, for instance, is repelled by aspartic acid, while Eurytemora is attracted to it. To date, explanations of chemosensory abilities have focussed largely on adaptive hypotheses. It is implicitly assumed that the response magnitude varies directly with the relative concentration of substances in prey, thus al- lowing animals to locate potential food items more effi- ciently. Other explanations exist, especially where organ- isms progress through a series of life-history stages re- quiring different chemosensory abilities. At any one stage, abilities (1) may be functionally important, (2) may rep- resent incipient abilities required at a later stage, or (3) may be relictual abilities that were required by previous life-history stages. Developmental explanations for pat- terns of chemosensitivity may be especially important for marine organisms where differences in the habitats oc- cupied by larval and adult stages can be large. Fiddler crabs (genus Uca) provide an excellent system with which to quantitatively investigate correlations of abilities among species, and life-history stages. All adult fiddler crabs are deposit feeders, foraging on intertidal sediments (Crane, 1975). Trophic habits are quite uniform (Teal, 1958; Miller, 1961; Crane, 1975; Robertson et al, 1980; Weissburg, 1990), and several species often coexist within a limited geographic area (Crane, 1975). Further, adult and larval habitats are markedly different in terms of abundance levels, and specific components of the chemical environment (e.g., Degensetal., 1964; Andrews etui., 1 971; Clark f/fl/.. 1972; Mopper et al., 1980; Mop- perand Lindroth, 1982). The investigation reported here was designed to explore the potential influence of both ecological and develop- mental processes on adult and larval chemosensitivity. The logical starting point for detecting developmental constraints on chemoreception is to determine patterns of chemosensitivity at the endpoints of the ontogenetic series. Similarly, the concordance of chemosensory abil- ities in at least one pair of species with similar trophic habits should be established before a detailed survey of ecologically and taxonomically related species is at- tempted. Therefore, we have examined, quantitatively, the chemosensory abilities of first stage zoea and adult stages of the crab, Uca longisignalis, and have compared the properties of different life-history stages in the same species, and among adults of U. longisignalis and U. pug- ilator. Materials and Methods Preparation of test solutions Metabolites released by Artemia salina in culture. An initial experiment was performed to explore larval swim- ming behavior in response to substances released by a documented prey species. The solution was prepared from a culture of young brine shrimp (Artemia salina) a stan- dard food source used in rearing carnivorous larvae of marine invertebrates, including fiddler crabs (e.g., Herrnkind, 1968; Rabalais and Cameron, 1983; Christy, 1989). Anemia nauplii were hatched from cultures in- cubated in 20 ppt artificial seawater media (ASW) (Forty Fathoms Marinemix) prepared with HPLC grade deion- ized water. When nauplii were 1-3 h old, 100 ml of the batch culture were spun down in a Beckman (Model J2- 21) centrifuge at 10,400 X g and 2C for 30 min. The resulting supernatant was decanted, filtered through a 0.45 ^m membrane filter, diluted to a concentration of 1 5 ppt, then stored at -87C. Amino acids and sugars. Chemicals were prepared in ASW media at a concentration of 15.0 ppt, using HPLC grade deionized water. The following amino acids were used as test compounds: /i-alanine (ALA), arginine (ARG), cysteine (CYS), glutamic acid (GAD), glycine (GLY), proline (PRO), taurine (TAU), and valine (VAL). The following sugars were used: galactose (GAL), glucose (GLU), maltose (MAL), sorbose (SOR), sucrose (SUC), and trehalose (TRE). These compounds were selected be- cause they are important chemical constituents in either plant or animal components of the diet of marine zoo- plankters (e.g., Jeffries, 1969), or have been found stim- ulatory to other deposit feeding crustaceans (e.g., Rob- ertson et al., 1981; Rittschof and Buswell, 1989). Except where noted, all amino acids were L-isomers, and all sug- ars D-isomers. Chemicals were all reagent grade, obtained from the Sigma Chemical Company. Test solutions were prepared in single batches at a concentration of 10~ 4 M for experiments performed using larvae, or 10"' M for experiments performed using adults, divided into 20 ml aliquots, and stored at 87C until needed. Where nec- essary, solutions were adjusted to pH 8.0 using 1 A/NaOH or HC1. Experiments testing larval chemosensory abilities Gravid Uca longisignalis females, collected from Air- port Marsh, Dauphin Island, Alabama, were held until their broods hatched. Females were placed in finger bowls containing a small amount of filtered 1 5 ppt seawater, and kept in incubators at 26 C under a 14:10 light-dark cycle. Females were checked for brood release twice per day, and water was changed daily. Usually several females simultaneously released larvae, and these broods were combined, and held in filtered seawater at densities of about 10 larvae/ml prior to assaying chemosensory be- havior. Experiments on larval chemosensory abilities began 24- 36 h after hatching and ran for 24 h. We specifically re- CHEMORECEPTION IN FIDDLER CRABS 207 stricted our experiments to these early stages because lar- vae are only competent to settle on substrates and me- tamorphose to the adult form later in the developmental series, after they have reached the megalopa stage (e.g., Epifanio el a/., 1988). Therefore, behavior in response to test compounds cannot be ascribed to substrate selection and settlement processes. Twenty larvae were transferred in 5 ml of ASW into plexiglass microcosms (3 cm long X 3 cm wide X 5 cm high), and 20 ml of the 10 4 A/ test solution was gently added. After about 30 s of gentle stir- ring to disperse the test compound, the microcosm was placed into a darkened chamber, and larval swimming behavior was recorded. Each trial was video-recorded for a 10-min period. Order of the test solutions was deter- mined by a random numbers table, subject to the con- straints that: a specific compound was not presented twice in succession; and that seawater treatments were per- formed after every block of five test solutions. Five to eight replicate trials of each test compound were per- formed. Individual larvae were tested in only one trial and then discarded. Larval swimming behavior in the horizontal plane was filmed using a Sony infrared-sensitive CCD camera (model H VM-200) equipped with a Tamron 1 80 mm lens, mounted beneath the microcosm, and focused 1.0 cm (25 to 35 larval body lengths) above the bottom. Similarly sized chambers and techniques have been successfully used for video recording locomotory behavior of small zooplankters, including copepods (Buskey, 1984), tintin- nids (Buskey and Stoecker, 1989), and polychaete (But- man et ai, 1988) and molluscan larvae (Zimmer-Faust and Tamburri, in prep.). The chamber was illuminated with an infrared light source (>830 nm) oriented 90 to the axis of the video camera. The field of view was 6.9 X 6.8 mm, about 6.4% of the total cross-sectional area of the microcosm. Depth of field was determined by mount- ing a pipette (tip diameter = 300 ^m) on a micromanip- ulator calibrated in 10 ^m increments. Using the com- puter-video motion analyzer (see below) with gray scale threshold set for discriminating larvae, the pipette tip was moved vertically to determine the limits at which the an- alyzer would 'recognize' the tip as an object. The recog- nition distance was 1.5 mm in the vertical dimension. Quantification of swimming speed and turning behav- ior was accomplished by replaying each video tape through a computer video motion analysis system (Motion Analysis Corp. Model VP 1 10 and Expert Vision software package) interfaced with an Amdek 386 microcomputer. The system constructs a digitized record of the raw video data, using a gray scale detector to enhance the contrast between objects in the video field and background. The outlines of the objects are defined, and the x,y coordinates of the centroid (geometric center) are calculated. The path of an object is reconstructed, on a frame-by-frame basis. as the translational movement through space of an object's centroid (see Buskey and Stoecker, 1989; Zimmer-Faust and Tamburri, in prep.). The video sampling rate of the motion analysis hardware was set at 10 frames/s, and, based on preliminary results, only the first 5 min of each trial were used in the analysis. Analysis of larval behavior consisted of determining swimming speed and net-to-gross displacement ratio (NGDR) for each larval swimming path. The NGDR is the ratio of the linear distance between the starting and ending points (net distance) and the total distance tra- versed by the path (gross distance). The NGDR measures the tendency of paths to be circular or twisted, and reaches a maximum value of 1.0 for paths that are completely straight. An NGDR of zero implies a looped or circular path, with the origin and endpoint occurring at the same spatial coordinates. For each test substance, data for all paths were pooled across trials, and means compared to swimming behavior observed in seawater using a Student's /-test. Experiments exposing larvae to Artemia culture water and test compounds indicated mean speeds, and NGDRs did not differ significantly among paths during the 5-min analysis period. Based on initial findings, glucose, valine, and trehalose were selected for dose-response trials because they were among the compounds producing the largest changes in larval swimming behavior. Using the above protocol, lar- val swimming behavior was determined for larvae exposed to a series of half log-step dilutions from 10~ 4 to 10~ 6 M. Three replicate trials were run at each concentration, and within each dilution series the order of presentation was randomly determined. Seawater controls were run every fifth trial, as before. Solutions were prepared by serial di- lutions of the primary stock used in the initial assays. Individual larvae were tested once, then discarded. Swim- ming speed and NGDR were regressed on concentration using SAS regression procedures (SAS Institute, Carey, North Carolina). Experiments testing adult chemosensory abilities Adult Uca longisignalis, also collected from Airport Marsh, were held in a 1 X 2 m sea table equipped with a continuous flow of filtered, UV sterilized seawater, and a sandy substrate allowing animals to construct burrows. Animals had continuous access to both shallow water (5 cm depth) and exposed sandy regions. Air temperature varied between 27-32C, water temperature was 24-27C at a salinity of 18-20 ppt. Animals were fed a diet of commercial fish food pellets, and the benthic diatom Cv- lindrotheca closteriiim. Forty-eight hours prior to exper- imentation, animals were removed from sea tables, and in small groups, isolated in bowls containing clean azoic substrate and 15 ppt ASW held at 26C. 208 M. J. WEISSBURG ET AL Chemosensory assays were conducted on individual adult crabs (mean carapace width: 18.4 1.8 mm SD) exposed to one of the 16 amino acids or sugars used pre- viously in tests with larvae. A 40-1 aquarium was subdi- vided into three sections using opaque partitions, and the bottom lined with a layer of clean beach sand. The sand was sloped so that when approximately 200 ml of ASW was added, the back third of each section contained about 1 cm of standing water. This arrangement allows crabs access to water required in feeding (Miller, 1961), and crabs forage readily in this type of arena (Weissburg, 1990). A glass cover maintained humidity. Each crab was presented with a food patch embedded in the sand at the front of the test section. The patch was a 2.3 cm diameter culture dish, containing 4.0 g substrate mixed with 3 ml of the test solution. Based on the work of Robertson el al. (1981), test solutions were prepared at 10"' A/, because this concentration proved maximally stimulatory to adult U. pugilator. The parallel method- ology employed by Robertson el al. (1981) and by us fa- cilitates a quantitative comparison between the two stud- ies. The substrate was mud, collected from a location at Airport Marsh where crabs were observed feeding. Sedi- ment was washed through a 1 mm sieve, ashed overnight at 600C, treated in hot 10 M HC1 for 8 h, then washed repeatedly with HPLC-grade distilled water to remove all traces of acid. Feeding experiments took place at 26C. Crabs were allowed to feed for 2 h while their behavior was recorded on video tape, using a NEC black and white CCD video camera (Model Ti 22AII) equipped with a Tokina 35-70 mm macrofocal lens and 2X multiplier, connected to a JVC (Model BR 3200U) tape recorder and a Panasonic TR- 1 24-MA video monitor. All three animals in the aquarium were visible, and at the front of the tank (where the patches were embedded), the image of the an- imals was magnified by approximately 25%, facilitating accurate analysis of feeding behavior. Each chemical was tested on 15 individuals of each sex. The tapes were reviewed and the frequency of feeding induction was determined, where feeding was defined as more than three successive lifts of the chelae to the mouth in any 5-s interval. We chose this criterion to be conser- vative in distinguishing a feeding response from patch sampling behavior. We observed that 75-80% of the an- imals, including those tested on seawater controls, lifted a chelae to their mouth at least once during a trial, al- though none of the animals lifted its chelae more than three consecutive times in sediments containing only sea- water. Fiddler crabs possess chemosensitive organs on the minor chelae (Robertson el al., 1981). and probing is a mechanism to allow animals to determine the contents of each patch. Three consecutive lifts seems to discrimi- nate feeding from sampling. Any animal that was not scored as sampling the patch was excluded from analysis. Approximately 75% of the animals met the criterion of exhibiting sampling behavior, and additional animals were tested as needed to produce sample sizes of 1 5 animals of each sex per compound. Thus, results cannot be con- sidered artifacts of differences in sampling frequency among compounds. The total time spent feeding was also measured to indicate the intensity of the response, and was recorded to the nearest 1 s with the time counter on the video tape recorder. Preliminary results indicated that males and females were similar in their responses, so the data from both sexes were pooled. A William's corrected G-test was used to determine whether the percentage of animals respond- ing to a substance was greater than that of seawater con- trols. Time spent feeding was analyzed using a Student's /-test. Animals did not feed on the seawater controls, giv- ing feeding times of 0.00 (see Results). Because feeding times on other compounds cannot be less than zero, this was a one-tailed /-test to determine whether feeding times were significantly greater than zero. Comparing lan'al and adult chemosensory responses Responses of larval and adult Uca longisignalis were quantitatively compared using a non-parametric corre- lation analysis. Kendal's Tau was computed based on the rankings of the significance tests of larvae and adults. Each stimulatory compound (for either life history stage) was ranked by the /-value (larvae) or G-value (adults). For larvae, the highest /-value from measurement of either NGDR or swimming speed was used (see Results). We ranked the G-scores for the adults, because, unlike the /- test, they are less affected by variation in the time at which an animal commenced feeding. If, for instance, an animal began feeding near the end of a trial, feeding times could be truncated as an artifact of the experimental trial length, whereas estimates of the percentage of animals feeding are immune to the timing of the response. In any case, results obtained using /-test scores differ little from an analysis done using the G-test scores (see Results). Because we are interested in comparing the subsets of chemicals stimulatory to each life-history stage, compounds pro- ducing no response in both larvae and adults were ex- cluded from the correlation analysis. Responses of adult Uca longisignalis and adult Uca pugilator were also quantitatively compared using Ken- dal's Tau. Rankings for Uca pugilator were taken from Robertson et al. (1981), giving the number of foodballs/ animal produced by crabs foraging on a particular sub- strate and compound. The rankings for the stimulus in- tensity of each compound on U. pugilator were computed by averaging results from all trials performed presenting a particular compound. Several compounds were tested in only one experiment, and the confidence limits for the CHEMORECEPTION IN FIDDLER CRABS 209 Table I Sample sizes and t-va! lies for Uca longisignalis larval swimming responses to chemical test solutions, relative to seawater controls Compound Number of paths analyzed 7-value Speed NGDR Anemia culture water 66 2.48" 2.97" Amino acids 0-aIanine 108 2.86" 0.76 arginine 65 1.18 0.58 asparagine 120 1.14 1.77 cysteine 141 0.30 0.72 glutamic acid 40 0.77 0.76 glycine 145 0.68 1.19 proline 119 0.25 0.16 serine 109 0.41 0.23 taurine 150 0.49 1.77 valine 98 7.60"* 3.76*" Sugars galactose 124 2.99" 0.37 glucose 114 3.72** 0.01 maltose 106 0.71 2.18* sorbose 109 3.36" 0.16 sucrose 134 2.54** 0.61 trehalose 140 0.01 4.37*** Seawater 259 = P<0.05, ** = P<0.01.' 0.001. number of foodballs produced in response to these com- pounds overlapped. In this case, the rankings were con- sidered ties. Results Experiments testing larval chemosensory abilities Responses to metabolites released by Artemia in culture. Larvae responded to solutions derived from Artemia cul- tures by changing both swimming speed and turning be- havior. Larval swimming speeds and NGDR's were sig- nificantly higher, relative to behavior in seawater (Table I; Figs. 1, 2). We measured ammonia-N and dissolved organic carbon (DOC) in ASW and in culture water to assay for the presence of Artemia metabolites. The con- centration of ammonia in culture water was 8.5 pAf, and in ASW was 0.8 nAl, as determined with an Alpkem nu- trient autoanalyzer (Model RFA/2). Dissolved organic carbon occurred at 3.2 1 mg/1 in culture water and at 0.75 mg/1 in ASW, determined with a Shimadzu organic car- bon analyzer (Model TOC-5000). The values obtained in culture water were typical for estuarine waters (Dame et ai. 1989; J. R. Pennock, pers. comm.) and clearly indi- cated the presence of Artemia metabolites. Responses to amino acids and sugars. All of the sugars tested, as well as the amino acids valine and /i-alanine. CD CU Q_ 1.7y 1.6 1.5- 1.4" 1.3- 1.2" 1.1 -- 1 VAL GAL GLU ART SOR ALA SUC SW Figure 1. Mean swimming speeds (1 SEM) of larvae exposed to test compounds at 10~ 4 M. metabolites released by Anemia salma (ART), and seawater. Data appear only for those compounds producing a sig- nificant change in larval swimming speed relative to seawater controls (P < 0.05; see Table I). were stimulatory. Each substance, except for trehalose, caused changes in larval swimming behavior consistent with larval responses to Anemia culture water. Swimming speeds were increased in the presence of most sugars, and the two amino acids (Table I; Fig. 1). The response to valine was the most dramatic, while responses to the other compounds were weaker. Galactose, glucose, sucrose, and 0-alanine constituted a group producing roughly equal increases in swimming speed. Path trajectories were also changed in response to valine, maltose, and trehalose, as shown by the changes in NGDR (Table I; Figs. 2, 3). Valine and maltose resulted in straighter paths, while lar- vae in trehalose solutions produced paths that were more circuitous (Fig. 3). For each of the other compounds listed in Table I, but not appearing in Figures 1 or 2, mean swimming speeds were within 0.02 mm/s of that in 0.95 T O O 0.85 0.75 0.65 VAL MALT ART SW TRE Figure 2. Mean NGDR ( 1 SEM) of larvae exposed to test com- pounds at 10~ 4 M, metabolites released by Anemia salina (ART), and seawater. Data appear only for those compounds producing a significant change in larval turning behavior relative to seawater controls (P < 0.05; see Table I). 210 M. J. WEISSBURG ET AL. 240 240 D 256 x coordinate (pixels) 256 < coordinate (pixels) Figure 3. Typical swimming paths in the horizontal plane by larvae exposed to seawater and to test substances causing maximal responses. The x,y coordinates are in units of picture elements (pixels), where 1 pixel = 0.028 mm. All substances were tested at 10 4 M: A. seawater, B. glucose, C. valine. and D. trehalose. Relative to seawater, larvae swim faster in glucose, faster and straighter in valine, and turn more frequently in trehalose. seawater, and /-tests yielded values of 0.0022-1.183 (P > 0.20, Table I). Mean NGDR values for non-stimulatory compounds were all within 0.02 of the NGDR in sea- water, and /-values ranged from 0.020 to 1 .77 (P > 0.05, Table I). The dose-response curves for glucose, valine, and tre- halose generally display a log-linear form (Fig. 4), allowing analysis by linear regression. There is a significant rela- tionship between solution strength and response magni- tude for all compounds. Responses to valine display the most sensitivity to dosage (slope = 0.097), followed by glucose (slope = 0.062), with responses to trehalose being least sensitive to dosage (slope = -0.048). The y-intercept of each curve is significantly different from seawater con- trols, according to a /-test (Fig. 4, / > 8.94, d.f. > 229, P < 0.01, all comparisons). Larvae therefore respond to mi- cromolar concentrations, and can probably respond to sub-micromolar stimulus concentrations as well. None of the regressions explain a great deal of the observed vari- ance (r < 0.05 in all cases), either due to the small slopes, or because additional factors may be important in deter- mining larval swimming responses. Experiments testing adult chemnsensory abilities Adult Uca longisignalis were induced to feed by a lim- ited subset of the amino acids and sugars (Table II). The monosaccharide glucose, and the glucose-containing di- saccharides, maltose and sucrose, induced the greatest percentage of animals to feed. Feeding was also elicited by the sugars, trehalose and sorbose, but to a much smaller degree, and the amino acids serine, glutamic acid, and /3- alanine also provoked minor responses. Except for valine, which generated a non-significant increase in feeding, none of the other compounds elicited any behavioral re- sponse at all G- values were 0.00 (Table II). The feeding intensity elicited by each of the test com- pounds is also given in Table II, which lists foraging time for each treatment. All of the compounds that caused crabs to feed generated feeding times significantly greater than zero (one-tailed Mest) and, in general, the magnitude of response initiation and intensity was correlated. A Kendal's Tau, based on the rankings of feeding percent CO 1.6-j A. Valine T "* 1.5- T E 1 ,, 1.4- T 1.3- J J lu 1.2- , CL 1.1 - | J 1 CO 1.0- 1.6- 1 1 B. Glucose * 1.5- E T ^ 1.4- ,1 i $ 1.3- I " (D 1.2 . 0) CL 1.1 1 n ! 0.95-1 0.90- QL g 0.85 0.80- 0.75 C. Trehalose SW 10~ 6 10~ 5 Concentration (M) 10 -4 Figure -4. Dose-response curves for larvae exposed to test substances causing maximal responses. Each point gives the mean 1 SEM. Values for seawater controls (SW) at extreme left are shown for comparison, n for seawater = 79. A. Swimming speed in valine as a function of con- centration, FI.^ = 5.932, P < 0.025; /-score testing for intercept different from seawater = 8.937, n = 131, P < 0.001. B. Speed in glucose as a function of concentration, f ,. 2 , 5 = 3.65, P < 0.05; /-score testing for intercept different than seawater = 45.88 1 , n = 1 55, P < 0.001 . C. NGDR in trehalose as a function of concentration, /", 5 j 8 = 22.897, P < 0.001; /-score testing for intercept different than seawater = 12.204, n = 181, P< 0.001. CHEMORECEPTION IN FIDDLER CRABS 211 Table II Feeding responses by adult Uca longisignalis to chemical test solutions and seawater controls Proportion feeding Feeding time Compound Proportion G-value Time (s) /-value Atnino acids /5-alanine 0.17 7.33** 11 2 4.59*** arginine 0.00 0.00 asparagine 0.00 0.00 cysteine 0.00 0.00 glutamic acid 0.13 5.98* 28 5 4 92*** glycme 0.00 0.00 proline 0.00 0.00 serine 0.17 7.33* 57 23 2.51* taurine 0.00 0.00 valine 0.07 2.83 Sugars galactose 0.00 0.00 glucose 0.60 36.61*** 841 164 5.12*** maltose 0.80 43.71*** 367 63 6.91*** sorbose 0.20 8.22** 269 108 2.48* sucrose 0.60 36.61*** 437 86 5.91*** trehalose 0.33 10.97** 535 81 6.63*** Seawater 0.00 Thirty different crabs were tested with each compound and with sea- water. Mean feeding time is given I SEM. When there was no significant feeding as judged by a G-test, /-tests of feeding times were not performed. * = P 0.50). Behavior of larvae in trehalose is quite different than responses to the other compounds, making the functional significance of the response to trehalose unclear. Therefore, the correlation analysis was repeated with the larval response to trehalose ranked last, and also with this compound deleted from the analysis entirely. In both cases, the results are unchanged; Kendall's Tau, Table III Analysis of chemosensory responses between life-history stages in Uca longisignalis, and between adults of U. longisignalis and U. pugilator Larvae Adults U. longisignalis U. longisignalis U. pugilator Compound Score Rank Score Rank No. Rank Valine 7.60 1 2.84 9 a _ Trehalose 4.31 2 10.97 4 20.3 5 Glucose 3.75 3 36.61 2.5 60.0 3 Sorbose 3.36 4 8.24 5 a Galactose 2.99 5 0.00 10" fJ-alanine 2.86 6 7.33 6.5 b 10.0 7 Sucrose 2.54 7 36.61 2.5 133.7 1 Maltose 2.14 8 43.71 1 85.5 2 Serine 0.79 9 7.33 6.5" 51.1 4 Glutamic acid 0.77 10 5.98 8 C 10.0 7 Asparagine 0.00 8" 10.0 7 Score refers to the results of a /-test (larvae) or G-test (adults) for significant behavioral responses to the test compound. For U. pugilator. No. refers to the mean number of foodballs produced by individual crabs foraging on the test compound, with the data taken from Robertson el al. (1981). a Ranking for adult-larval comparison only, not used in adult-adult comparison. b Ranking for adult-larval comparison only, rank for adult- adult comparison is 5.5: c Ranking for adult-larval comparison only, ranking for adult-adult comparison is 7; d Ranking for adult-adult comparison only, compound not used in adult-larval comparison. See text for details. 212 M. J. WEISSBURG ET AL. computed with trehalose ranked last, is -0.3423, and is -0.2319 with trehalose removed (P> 0.25 in both cases). The difference in line versus coarse scale patterns re- flects changes in the stimulatory capacity of substances excitatory to both life history stages. Only four compounds were significantly stimulatory exclusively to larvae or to adults (valine and galactose were highly excitatory solely to larvae, while glutamic acid and serine induced only adults to feed); the majority of the remaining compounds tended to be more highly excitatory to one stage or the other. Maltose and sucrose, for instance, were highly stimulatory to adults, but less stimulatory to larvae, while trehalose was more stimulatory to larvae than to adults. In contrast to the within species comparison, the re- sponses of adult U. pugilator and U. longisignalis are highly correlated at both coarse and fine scales. A binomial test indicates that adults from both species mutually re- spond either positively or negatively to the same com- pounds more frequently than expected by chance (bino- mial test: n= 14. .v = 1 3, P < 0.00 1 ). Similarly, a Kendall's Tau, computed on the rankings in Table III, gives a value of 0.78 (P < 0.03), indicating a quantitatively similar re- sponse pattern to individual compounds among adults of each species. Although we present our tabulation using rankings of the proportion feeding for Uca longisignalis, the results using feeding time are not substantially different (Tau = 0.57, 0.03 < P < 0.06). The monosaccharide glu- cose, its disaccharide isomers, and the amino acid serine were the most stimulatory, while the remaining amino acids were weakly stimulatory to both species. Discussion This is the first examination of patterns of chemore- ception in zoea stage larvae. We have shown that brach- yuran crab larvae possess chemosensory abilities, and that these abilities are present, although perhaps not fully de- veloped, at a very early stage. Larvae respond to various amino acids and sugars with a change in swimming be- havior. Such changes have previously been demonstrated as reliable indicators of chemosensitivity in a variety of zooplankters, such as copepods (Poulet and Ouellet, 1982; Buskey, 1984). tintinnids (Buskey and Stoecker, 1989), oyster larvae (Zimmer-Faust and Tamburri. in prep.), and ciliates (Levandowsky et a/.. 1984). Workers have interpreted the changes in swimming be- havior as indicating ' > >i waterborne substances act to cue resource acquisition behavior, since changes often involve decreases in swimming speed and in NGDR, and an in- creased rate of turning (Buskey, 1984; Buskey and Stoecker, 1989; Zimmer-Faust and Tamburri, in prep.). Together, these changes have been viewed as 'adaptive' responses, allowing predators to remain in aggregations of potential prey, or enabling larval settlers to conduct site-restricted searches for microhabitats favorable to sub- strate colonization. Clearly, the increased turning behavior displayed by fiddler crab larvae in trehalose solutions might facilitate location of prey within patches, because the turning causes animals to loop back, focusing loco- motory activity within a small area (e.g.. Koopman, 1980). Paradoxically, increases in swimming speed and NGDR also seem to be associated with prey detection. In this study, Uca zoea responded to metabolites from Anemia. a known zooplankton prey species, with an increase in both speed and NGDR. Buskey (1984) determined that, when copepods are given diatom suspensions, swimming speed and NGDR decrease, whereas copepods exposed to diatom exudates alone, increase both speed and NGDR. In the absence of mechanical stimuli, these behaviors might allow animals to cover large areas in search of prey patches, whereupon encountering prey, paired mechanical and chemical stimuli would result in site-restricted be- havior. Additional experiments are necessary to determine the functional roles of chemoreception and mechanore- ception in mediating prey search by Uca zoea. To some degree, substances stimulatory to larvae are those that signal the presence of potential food items. Zooplankton are the primary prey species of Uca larvae, which have been successfully reared only using various motile zooplankton (e.g., Herrnkind, 1968; Christiansen and Yang, 1976: Rabalais and Cameron, 1983; Christy, 1989). The larvae lack setae necessary for capturing phy- toplankton, instead pinning prey between the telson and anteroventral spine (Herrnkind, 1968). Uca longisignalis larvae respond to the amino acids valine and /^-alanine. Although the response spectrum is narrower than that observed in other (adult) crustaceans (see reviews of Ache, 1 982; Carr, 1988; Laverack. 1988), these two amino acids strongly signal the presence of fleshy prey. Beta-alanine constitutes as much as 12% of the free-amino acids re- leased into the water by zooplankton, with both valine and /3-alanine being consistently among the most abun- dant amino acids released into the surrounding environ- ment by potential zooplankton prey (Webb and Johannes, 1 967). Beta-alanine is generally one of the most abundant free amino acids present in flesh, and valine is quite often abundant, averaging over 5% of the free-amino acid pool (Cowey and Corner, 1964; Srinivasagam et at.. 1971). Zooplankton assemblages have 2-3 times the free amino acid concentration of valine and /3-alanine as phyto- plankton-dominated communities (Jeffries, 1969). Some of the stimulatory sugars may also signal the presence of prey. Glucose is the major blood sugar present in animals, while trehalose is found in the hemolymph of some crustaceans (Telford, 1968). Both of these com- pounds evoke filter feeding responses in the planktivorous porcelain crab Petrolisthes cinctipes (Hartman and Hart- CHEMORECEPTION IN FIDDLER CRABS 213 man, 1977). There is little work done on the distributions of other sugars in seawater or in tissues of marine animals, although galactose and galactose derivatives found in sea- water may originate from flagellates (Sakugawa el a/.. 1985). Adult U. longisignalis respond to a rather limited suite of the compounds tested, primarily carbohydrates, and stimuli that are more effective at inducing feeding also produce more intense feeding responses (Table II). Glu- cose and glucose dissacharide moieties are particularly potent stimuli. Contrary to what has been found in other studies, there is little evidence that disaccharides are more stimulatory than constituent monosaccharides (e.g.. Zimmer el ai. 1979; Robertson el al. 1981; Trott and Robertson. 1984). Judging by both the induction as well as the intensity of the response, crabs respond more highly to glucose compounds than to other substances. Some additional sugars produce appreciable responses of one type or the other (i.e.. induction or intensity), but the joint function of the two components is not as great as for glucose and its dissacharides. Although a few amino acids, particularly serine, provoke responses, they have small effects both as inducers, and in terms of the response intensity. The pattern of chemosensitivity suggests adult U. lon- gisignalis are well constructed to allow efficient foraging in their natural habitat. Diatoms, bacteria, and, occasion- 1 ally, blue-green algae are dominant flora of the mud-flats preferred by crabs. The primary storage product in dia- toms is a glucan, and carbohydrates are primary constit- uents of the extracellular mucose sheaths of diatoms and blue-green algae (e.g.. Fogg, 1966). These sugars are often released into the environment by algal species (Fogg, 1 966; Foggt'/a/.. 1973;Craigie, 1974; Darley, 1977; Paulsen el al. 1978). Serine is the dominant amino acid of the free amino acid pool of diatoms (Parsons el al, 1961), and it appears that adult fiddler crabs are unique in responding primarily to this amino acid (i.e.. Robertson el al, 1981). Strong responses to carbohydrates and serine may be ex- pected in light of their specificity as a cue to microalgal food resources. The methodology of this study and that of Robertson el al (1981) on the sand fiddler crab, Uca pugilator, are parallel enough to quantify the correlation among che- mosensory abilities of the two species. The analysis in- dicates that, in at least this species pair, similar trophic ecologies have produced a broad concordance among patterns of adult chemosensitivity, even on a fine scale. The high correlation among the abilities of Uca species may be a function of the habitat occupied by adult fiddler crabs. For the semi-terrestrial Uca. molecules present in sediments where crabs forage are derived from a variety of sources, including microbial breakdown processes (Rittschof, 1980), and leakage from dead cells and decay- ing material (Kennish, 1986). High background levels of substances in sediments, and the large number of potential non-prey sources of stimulatory molecules, may make intertidal mudflats particularly "noisy" environments. Under these conditions, only a few substances most closely associated with the presence of food (i.e.. serine and car- bohydrates) may be good signal carriers, forcing conver- gence among the properties of organisms occupying these habitats. Future studies on animals in subtidal, intertidal, and semi-terrestrial habitats are needed to fully discern the influence of habitat type on patterns of correlation among chemosensory abilities of related crustacean spe- cies. In contrast to the between-species comparison, larval and adult Uca longisignalis express different patterns of chemosensory ability at the fine scale. It appears that dif- ferences in the chemical environment of the pelagic and benthic habitats, or differences in diet, may require that varying life-history stages respond disparately to similar suites of chemicals. A number of compounds stimulatory to one life-history stage of U. longisignalis are either non- stimulatory or only slightly stimulatory to the other stage. For instance, valine and galactose are excitatory only to larvae; serine and glutamic acid only to adults. Valine and serine are cues that are well-defined markers for prey types favored by a particular form, so it is perhaps not surprising that each substance is strongly stimulatory to only one life-history stage. The significance of glutamic acid and galactose to certain stages is less clear. Glutamic acid is not readily released into seawater by zooplankton (Webb and Johannes, 1967), but is an abundant constit- uent of the amino acids found in diatoms (Cheucas and Riley, 1969). Glucose appears to be unique in its ability to function as a cue for both larval and adult food sources, and is highly stimulatory to both life-history stages. Patterns of larval and adult abilities must be cautiously interpreted, because it appears that some stimulants may produce responses that do not enhance the ability of the organism to detect prey. There is no evidence that the sugars sorbose, maltose, and sucrose are present in zoo- plankton, but they are intimately associated with adult food sources. It may be that detection of these substances by larvae has some function. However, in view of the close association of these substances with adult food, we tentatively hypothesize that the chemosensory responses of larvae to these sugars are incipient abilities. That is, sensitivity to these compounds by larvae may be the result of developmental processes required in providing sensi- tivity by adults. While larval abilities appear partially a consequence of adult requirements, adult abilities do not seem to reflect larval needs. Substances which act as potential cues to larval, but not adult prey items (e.g.. valine), do not elicit adult feeding behaviors, while still evoking strong larval 214 M. J. WEISSBURG ET AL. responses. Central neural processing is known to attenuate chemical signals, thereby reducing or eliminating behav- ioral responses, even though substances may evoke strong electrophysiological activity from receptor cells (Dethier, 1980; Derby et ai, 1985). It is sometimes observed that substances that produce action potentials from sensory cells do not always have detectable effects on behavior. Shepheard (1974) compared results of his electrophysio- logical study on responses by olfactory cells of Homarus americanus, to behavioral studies of McLeese (1970), and concluded that a variety of compounds with strong elec- trophysiological activity elicited little or no behavioral re- actions. Working with U. pugilator, Vermeer (1981) has documented that valine and /i-alanine are both extremely potent substances in electrophysiological assays, although valine is not an adult feeding stimulant, and /3-alanine is only weakly so (Robertson et ai, 1981). We suggest this type of discordance between electrophysiological and be- havioral responses in adults may be adaptive, serving to reduce or eliminate adult feeding reactions to substances that cue only larval food resources. The conclusions reached as a result of our study must be regarded as working hypotheses, primarily because we did not assay the changes in chemosensory-mediated be- havior continuously through ontogeny from zoea and megalopa stages, to the adult stage. Our aim is to call attention to the patterns we have observed, to help in- vestigators interpret chemosensory abilities of other or- ganisms. Additional studies on the development of che- moreceptive behavior in fiddler crabs and in other organ- isms will be needed to confirm the patterns we have detected. Further investigations also will be required to determine the conditions under which chemosensory abilities of early life-history stages are constrained by de- velopmental processes and ecological requirements of later forms. 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Chemical mediation of appetitive feeding in a marine decapod crus- tacean: the importance of suppression and synergism. Biol. Bull 167: 339-353. Reference: Biol Bull 181: 2 16-221. (October, 1991) Genetic Variation in the Timing of Gonadal Maturation and Spawning of the Eastern Oyster, Crassostrea virginica (Gmelin) BRUCE J. BARBER 1 , SUSAN E. FORD 2 , AND ROBERT N. WARGO 2 * 1 Virginia Institute of Marine Science. College of William and Mary, Gloucester Point, I 'irginia 23062, and -Shellfish Research Laboratory, Rutgers University, New Jersey Agricultural Experiment Station, Institute of Marine and Coastal Sciences, Port Norris, New Jersey 08349 Abstract. The gonadal cycles of four groups of eastern oysters, Crassostrea virginica (Gmelin), including native stocks collected that year and inbred strains (reared in Delaware Bay for 5-6 generations) from both Long Island Sound and Delaware Bay, were compared in Delaware Bay in 1987. Inbred strains resembled their respective na- tive stocks; both Long Island groups initiated gonadal de- velopment and spawning about one month earlier and spawned over a shorter duration than both Delaware Bay groups. Analysis of covariance revealed that the effect of time on gonadal development was statistically different (P < 0.05) for all between-location group comparisons, but not for the two within-location comparisons. Thus, after six generations of inbreeding in Delaware Bay, Long Island oysters maintained their characteristic pattern of gonadal development and spawning, indicating the exis- tence of genetically different environmental requirements for gonadal maturation between the two locations. Introduction Intraspecific variation in various aspects of reproduc- tion has been noted for several species of marine bivalves, and can be either genetic or non-genetic (adaptive) in na- ture (see Sastry, 1979; Barber and Blake, 1991). The sug- gestion that there are genetically distinct populations or "races" of eastern oysters, Crassostrea virginica (Gmelin), was first made by Loosanoff and Engle (1942). Stauber (1950) concluded that there were three physiological races of oysters, based on differences in temperatures at which Received 25 February 1991; accepted 24 May 1991. Present address: AT&T, 4 1 1 Mt. Kemble Ave., Morristown, New Jer- sey 07960. spawning was initiated in Long Island, New Jersey, and Texas populations. Because transplant experiments were not conducted, the relative contribution of genetic versus non-genetic factors to the observed differences was not determined. Loosanoff and Nomejko (1951) and Loos- anoff (1969) transplanted stocks from various locations along the eastern and Gulf coasts of the U.S. to Milford, Connecticut, and histologically compared gonadal devel- opment. Differences in the timing and extent of gonadal development and spawning between some of these stocks in the common environment suggested the existence of ge- netic differences in the environmental factors regulating gonadal maturation. The possibility of acclimatization after transfer to the new environment, however, was not precluded. As stated by Loosanoff ( 1969), the best way to separate the genetic and environmental aspects of oyster reproduction would be to use "successive generations of laboratory-reared oysters originating from parents of dif- ferent geographical areas." The oyster (C. virginica) selective breeding program of the Rutgers University Shellfish Research Laboratory in- cludes a variety of wild (imported) stocks and laboratory- reared strains that originated from several locations (Has- kin and Ford, 1979; Ford and Haskin, 1987). Initial ex- amination of some of these strains (originating from Long Island Sound, Delaware Bay, and James River, Virginia, stocks) indicated that after several generations of inbreed- ing and maintenance in Delaware Bay, the timing of spe- cific reproductive events remained characteristic of the site of geographic origin (Ford et a!., 1990). The object of the present study was to establish whether these ob- served differences are attributible to intraspecific genetic variation. This was accomplished by simultaneously 216 VARIATION IN GONAD MATURATION 217 comparing cycles of gonadal maturation and spawning in both wild stocks and inbred strains originating from two locations: Long Island Sound and Delaware Bay. Materials and Methods Native oysters from Long Island Sound (LIN), native oysters from Delaware Bay (DBN), a 6th generation, 1985 year class, inbred strain originally imported from Long Island Sound to Delaware Bay in 1964 (LII), and a 5th generation, 1985 year class, inbred strain originating from Delaware Bay (DBI) were obtained in March 1987. Be- cause the endoparasite Haplosporidium nelsoni (MSX) alters gonad development and relative fecundity (Barber el al., 1988; Ford and Figueras, 1988), all oysters used in this study were first "purged" of MSX (Ford and Haskin, 1988a, b) by being suspended in wire trays from the lab- oratory dock (Bivalve, New Jersey) in the Maurice River (salinity < 5 ppt) for several weeks. On 1 May 1987, all experimental groups were moved to the tidal flats of lower Delaware Bay, Cape May County (salinity 18-24 ppt), for the remainder of the study. Each group of oysters was sampled periodically between 17 March and 5 October 1987; ten individuals were shucked and fixed in Davidson's solution. A standard transverse (anterior) section across gill, stomach, intestine, and digestive diverticula was dehydrated, cleared, and embedded in paraplast. Six-^m sections were stained with iron hematoxylin, acid fuchsin, and aniline blue. Slides were examined for MSX prevalence (% of sample infected) and intensity (Ford, 1985; Barber el al., 1988). Gonadal development was assessed from the histological sections using the Bioquant Image Analysis System (R&M Bio- metrics, Inc., Nashville, Tennessee) to obtain a Gonadal Area Index, defined as the ratio of gonadal area to the entire visceral mass area X 100. The gonadal area tech- nique is a sensitive indicator of gametogenic events in oysters (Mori, 1979; Barber el al., 1988) and is more pre- cise than measuring the thickness of the gonadal layer alone, as performed by Loosanoff and Nomejko ( 195 1 ). Cycles of gonadal development of the four groups of oysters, based on mean gonadal area indices, were com- pared using analysis of covariance after correcting for het- eroscedasticity with the least squares method of White (1980). Analysis of covariance was accomplished using the dummy variable regression procedure of Wonnacott and Wonnacott (1970). Gonadal area index was regressed on a quadratic function of time (elapsed days from the date of the first sample) as: Gonadal Area Index = , Days + ajDj Days + ft Days 2 + ftDj Days 2 , using the Tobit estimation to overcome the problem of zeros in the data set (Goldberger, 1964). The t statistic was then used to test the null hypothesis that , = a, and ft = ft, for each pair-wise group comparison. Conven- tional analysis of variance was not applied because of the presence of zero-valued observations, which invalidated the required normality assumption. Moreover, conven- tional analysis of variance compares only means, while the procedure used here allowed a global comparison. Water temperature data from the experimental site were collected only sporadically during 1987. Weekly averages of "calculated" daily water temperatures were therefore obtained from the regression of daily air temperatures (Cape May, New Jersey) on actual water temperatures collected at the experimental site from 1978-80 and 1985- 88 (n = 358; r = 0.77). These are presented in Figure 1. Results The haplosporidan parasite H. nelsoni had little effect on gonadal development in any of the groups in this study. Prevalence and intensity of the parasite remained rela- tively low throughout the first four months of the study period because of the exposure of oysters to low salinity prior to initial sampling (Table I). Prevalence of//, nelsoni did not exceed 50% until 1 5 July, and systemic infections exceeded 50% only in the LIN group, after 15 July and the completion of spawning in that group. Gonadal de- velopment in C. virginica is most affected in individuals having systemic H. nelsoni infections (Barber et al., 1988; Ford and Figueras, 1988; Ford et al.. 1990). Cycles of gonadal maturation and spawning for the four groups of oysters are represented as mean gonadal area indices in Figure 2. Both Long Island groups (LIN and LII) initiated gonadal development in April and had maximal gonadal areas in late May (Figs. 2A, B). Some spawning occurred in both Long Island groups in late May and June, but greatest spawning activity occurred in July. Long Island oysters (both LIN and LII) were spent by August. Although some gonadal development was ap- parent in the DBI group in late April, most gonadal growth in both Delaware Bay groups (DBN and DBI) occurred in May (Figs. 2C, D). Maximal gonadal areas were found for both Delaware Bay groups in June, which was followed by a period (from June to August) of what appeared to be partial spawning and redevelopment. Final spawning and the cessation of gonadal activity in the Delaware Bay groups occurred in September. Thus there were similarities between inbred strains and native stocks within each site of origin but differences between sites of origin. The null hypothesis of similarity of coefficients was re- jected in all four pair-wise comparisons between strains having different geographic origins (Table II), indicating that the timing of gonadal growth and spawning of both native stocks and inbred strains from Delaware Bay were statistically different (P < 0.05) from those of oysters (both 218 B. J. BARBER KT AL T E M P E R A T U R E 35 30 25 20 15 10 M A R A P R M A Y J U N J U L 1987 A U G S E P O C T N O V Figure 1. Weekly mean water temperature at the experimental site, lower Delaware Bay, as calculated from the regression ot daily air temperature at Cape May, New Jersey, on water temperature readings from 1978 to 1980 and 1985 to 1988. native and inbred) from Long Island Sound. On the other hand, the null hypothesis was accepted in the two com- parisons involving inbred and native groups having the same site of origin (LIN-L1I and DBN-DBI), indicating that the timing of gonadal maturation cycles was similar for native stocks and inbred strains within each site of origin. Discussion There were distinct differences in the timing of gonadal development and spawning of both native stocks and inbred strains between the Long Island and Delaware Bay sites of origin. Oysters in the Long Island groups initiated gonadal growth, achieved maximal gonadal development, and began spawning about one month earlier than oysters in the Delaware Bay groups. Additionally, both Delaware Bay groups exhibited a protracted period (about three months) of partial spawning and redevelopment. The sta- tistical comparison of gonadal area indices reinforced the observations reported in this study and those of Ford el al. (1990). Time (elapsed days) had the same effect on gonadal area index for the LIN and LII groups and for the DBN and DBI groups, but not for any between-lo- cation comparison. Thus, the timing of gonadal devel- opment differed significantly between the Long Island and Delaware Bay locations. These differences were main- tained in the Long Island inbred strain, even after six generations (23 years) in Delaware Bay, demonstrating that there are genetically determined differences in en- vironmental criteria necessary for the initiation or com- pletion of a particular gametogenic event at these two locations. Several potential environmental regulators of bivalve reproduction have been identified, with the foremost of these being temperature (Sastry, 1979; Barber and Blake, 199 1 ). Scallops (Argopecten irradians) from Woods Hole, Massachusetts, and Beaufort, North Carolina, acclimated to similar laboratory conditions prior to the initiation of gonadal development, exhibited maximal gonadal growth at 15 and 23C, respectively, indicating a non-adaptive (genetic) difference in temperature requirements for go- nadal development between these two populations (Sastry, 1966). One of the original criteria upon which the deter- mination of physiological races of oysters was based was the temperature at which spawning began. According to VARIATION IN GONAD MATURATION 219 Table I Prevalence (P. %) and intensity (I. expressed as % systemic injections) o] Haplospondium nelsoni in experimental groups of oysters, Crassostrea virginica, on the various sampling dates LIN LII DBN DBI Date (1987) 17 March _ 20/0 50/20 0/0 29 April 0/0 10/10 20/0 0/0 7 May 0/0 0/0 13 May 0/0 0/0 0/0 0/0 21 May 0/0 0/0 0/0 0/0 27 May 0/0 20/10 0/0 0/0 3 June 0/0 20/10 20/10 10/10 12 June 0/0 40/20 40/10 0/0 18 June 0/0 20/20 20/20 30/10 24 June 40/10 10/0 40/20 1 0/0 2 July 20/0 0/0 15 July 90/0 60/10 50/0 0/0 29 July 100/60 10/0 60/20 30/0 12 August 70/20 10/0 26 August 90/70 0/0 40/0 20/0 19 September 40/30 10/0 5 October 0/0 10/0 60/20 40/30 LIN = Long Island Native. LII = Long Island Inbred. DBN = Delaware Bay Native. DBI = Delaware Bav Inbred. Stauber (1950), the Long Island Sound population was distinct because it began spawning when temperature reached 16.4C, while other populations spawned at higher temperatures. In this study, both Long Island groups began spawning when water temperature was be- tween 15 and 20C, while spawning in the Delaware Bay groups began when water temperature was about 25-28C (Fig. 1 ). Thus there is little difference in the temperature at which Long Island oysters spawn in their native setting or in Delaware Bay, even after six generations of inbreed- ing in Delaware Bay. Gonadal development is an energy demanding process, with necessary nutrients coming either from stored re- serves, recently ingested food, or both (Sastry, 1979; Bar- ber and Blake, 1991). Site-specific variation in gonadal development of Mytilus edulis. A. inadians. and Placo- pecten magellanicus has been reported to be due to adap- tion (non-genetic) to local variations in environmental factors, most notably food availability (Newell et a!.. 1982; Bricelj et ai. 1987; MacDonald and Thompson, 1988). The fact that at the same site in Delaware Bay, both inbred and native Long Island oysters have distinctly different gonadal cycles than Delaware Bay oysters, effectively eliminates food abundance as the sole regulator of gonadal development in C virginica. Perhaps gonadal develop- ment in oysters is initiated in the presence of adequate food supplies, at a genetically determined temperature. The results of this study and previous studies establish the existence of populations of C. virginica along the east coast of the United States that are genetically distinct based on differences in the timing of cycles of gonadal devel- opment and spawning. These intraspecific differences oc- cur between populations located north and south of Long Island, New York. Loosanoff and Nomejko ( 195 1 ) found that oysters from Massachusetts and Long Island Sound spawned successfully in Milford, Connecticut, while oys- ters from New Jersey and Virginia did not spawn or only partially spawned. Similarly, Loosanoff ( 1969) saw a dif- ference in the temperature at which gonadal development was initiated between oysters from north of Long Island and south of Long Island. Oysters transplanted from Chesapeake Bay (Maryland) to Florida conformed to local spawning conditions after one year (Butler, 1955), indi- cating that observed differences in spawning temperatures between these locations were non-genetic. Genetic discontinuities along the distributional range of C. virginica have been reported at Brownsville, Texas, based on electrophoretic variation in proteins encoded by nuclear genes (Buroker, 1983) and along the mid-Atlantic coast of Florida based on restriction site variation in mi- tochondrial DNA (Reeb and Avise, 1990). It is noteworthy that populations of oysters exist that have genetically dis- tinct gonadal maturation cycles and yet are indistinguish- able based on both allozyme frequencies and mtDNA re- striction site variation. The fact that discrepancies exist between allozyme frequencies, mtDNA patterns, and physiological processes such as reproduction, point out that there is no simple means of delineating genetic boundaries among oyster populations (see review by Gaffney, 1991). The existence of stocks of oysters having genetically distinct reproductive cycles has implications for the oyster fishery. Oysters transplanted from a northern location (north of Long Island) to a southern location (south of Long Island), requiring lower temperatures to initiate go- nadal development, would spawn earlier than the local population. In the case of Long Island oysters in Delaware Bay, there is a potential one month overlap in spawning periods during which inter-breeding could occur. For the most part, however, the spawning periods would remain distinct. Oysters transplanted from southern to northern locations might not experience temperatures high enough to initiate gonadal development or spawning. In this case, oysters with unspawned or partially spawned and resorb- ing gametes would have little reproductive value and would be of poorer quality and lower commercial value. Hatchery production of oysters would be enhanced by the ability to obtain naturally conditioned broodstock over a wider time interval than what is available with local stocks alone. 220 B. J. BARBER ET AL LONG ISLAND NATIVE B LONG ISLAND INBRED z o Q z z o DELAWARE BAY NATIVE D DELAWARE BAi INBRED I 987 o z z o I 987 Figure 2. Mean gonadal area index (1 SD) for four groups of Crassostrea virginica: A = Long Island Native (LIN); B = Long Island Inbred (LII); C = Delaware Bay Native (DBN); D = Delaware Bay Inbred (DBI). Fitted lines are spline interpolations. Table II Statistical comparison (t-tfsl) oj coefficients a and /3 from the quadraliL model for each possible combination of oyster, Crassostrea virginica. groups H : a, = a, and 0, = J JP when 1 i and I are different groups of oysters Groups i J t (, = ,) t (0, = ?,) P* LIN DBN 6.17 7.72 P < 0.002 LIN LII 1.39 0.44 LIN DBI 4.01 6.33 P&Q.02 DBN LII 5.50 6.66 P < 0.005 DBN DBI 2.62 1.59 LII DBI 2.89 4.36 P < 0.05 * If comparisons of both a and fJ are rejected. LIN = Long Island Native. LII = Long Island Inbred. DBN = Delaware Bay Native. DBI = Delaware Bay Inbred. Acknowledgments We thank J. Kirkley for generous assistance with the statistical analysis and D. O'Connor and R. Barber for histological preparation and disease analysis. Comments by R. Mann, J. Graves, and P. Gaffney improved the manuscript. This work is the result of research sponsored by NOAA, Office of Sea Grant, Department of Com- merce, under grant No. NA89AA-D-SG057 (Project No. R/F-23). The U.S. Government is authorized to produce and distribute reprints for governmental purpose not- withstanding any copyright notation that may appear hereon. This is New Jersey Sea Grant Publication No. NJSG-90-238 and New Jersey Agricultural Experiment Station Publication No. D 32405-3-90, supported by state funds. This is contribution No. 1673 from the Virginia Institute of Marine Science, College of William and Mary, and No. 91-22 from the Institute of Marine and Coastal Sciences, Rutgers University. VARIATION IN GONAD MATURATION 221 Literature Cited Barber, B. J., S. E. Ford, and H. H. Haskin. 1988. Effects of the parasite MSX (Haplosporidiwn nelsoni) on oyster (Crassostrcu virginica) en- ergy metabolism. 1. Condition index and relative fecundity. ./ Shellfish Res 7:25-31. Barber, B. J., and N. J. Blake. 1991. Reproductive physiology. Pp. 377_428 in Scallops: Biology. Ecology and Aauacultwe, S. Shumway, ed. Elsevier Science Publishers B. V., Amsterdam. Brk-elj, V. M., J. Epp, and R. E. Malouf. 1987. Intraspecific variation in reproductive and somatic growth cycles of bay scallops Argopeclen inadians. Mar. Ecol. Prog. Set: 36: 123-137. Buroker, N. E. 1983. Population genetics of the American oyster Cras- sostreu virginica along the Atlantic coast and the Gulf of Mexico. Mar. Biol. 75:99-112. Butler, P. A. 1955. Reproductive cycle in native and transplanted oys- ters. Proc. Nail. Shellfish Assoc. 46: 75. Ford, S. E. 1985. Chronic infections of Haplosporidiwn iie/soni (MSX) in the oyster Crassostrea virginica. J Invertehr. Palhol 45: 94-107. Ford, S. E., and A. J. Figueras. 1988. Effects of sublethal infection by the parasite Haplosporidiwn nclsoni (MSX) on gametogenesis, spawning, and sex ratios of oysters in Delaware Bay, USA. Diseases At/mil. Org. 4: 121-133. Ford, S. E., and H. H. Haskin. 1987. Infection and mortality patterns in strains of oysters Crassoslrea virginica selected for resistance to the parasite Haplosporidiwn nelsoni (MSX). ./. Parasitol 73: 368- 376. Ford, S. E., and H. H. Haskin. 1988a. Management strategies for MSX (Haplosporidiwn nelsoni) disease in Eastern oysters. Am. Fish. Soc. Spec. Puhl. 18: 249-256. Ford, S. E., and H. H. Haskin. 1988b. Comparison of//; vitro salinity tolerance of the oyster parasite Haplosporidiwn nelsoni (MSX) and hemocytes from the host, Crassostrea virginica. Coinp. Biochem. Phy.siol.90A.: 183-187. Ford, S. E., A. J. Figueras, and H. H. Haskin. 1990. Influence of selective breeding, geographic origin, and disease on gametogenesis and sex ratios of oysters, Crassostrea virginica. exposed to the parasite Haplosporidiwn nelsoni (MSX). Aquaculture 87: 285-301. Gaffney, P. M. 1991. Biochemical and population genetics. In Biology. Culture and Management of the American Oyster. V. L. Kennedy, A. F. Eble. and A. Rosenfield, eds. Maryland Sea Grant, College Park. In Press. Goldbcrger, A. S. 1964. Econometric Theory. John Wiley and Sons. Inc., New York. Haskin, H. H., and S. E. 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Econometrica 48: 817-838. Wonnacott, R. J., and T. H. Wonnacott. 1970. Econometrics. John Wiley and Sons, Inc., New York. Reference: Bml Bull 181: 222-231. (October, 1991) Ploidy and Pronuclear Interaction in Northeastern Pacific Lasaea Clones (Mollusca: Bivalvia) DIARMAID 6 FOIGHIL 1 AND CATHERINE THIRIOT-QUIEVREUX 2 1 Department of Biological Sciences, Simon Fraser University. Bwnaby. British Columbia, Canada \'5A 1S6 and 2 Universite P. el M. Curie, Observatoire Oceanologique, B. P. 28. 06230 I 'illetranche-sur-mer. France Abstract. A natural population of the bivalve genus La- saea from Victoria, British Columbia, Canada was kary- ologically characterized, and pronuclear interaction was studied in newly spawned eggs. Mitotic metaphases from 95 cells were enumerated, and chromosome numbers ranged from 58 to 108, with 90 to 100 being most frequent. Ten well-spread metaphases were karyotyped, and the chromosomes were classified into 32 triplet subgroupings on the basis of shared morphology and size, together with a variable number of supernumerary chromosomes. Northeastern Pacific Lasaea clones share broad karyo- logical features with direct developing congeners, but de- tailed comparisons reveal that they have experienced dif- ferent evolutionary mechanisms of polyploidy. The pron- uclear interaction study generated two key pieces of evidence that establish that northeastern Pacific Lasaea clones do not reproduce by self-fertilization, but that par- thenogenetic development is triggered by autosperm. The incorporated sperm nucleus disintegrates in the egg cortex and does not fuse with the egg "pronucleus," i.e., syngamy does not occur. Both polar bodies have a diploid chro- mosome number, a result inconsistent with meiosis, im- plying that they are products of mitotic divisions. Non- hybridizing lineages of northeastern Pacific Lasaea there- fore represent true asexual clones, not inbred lines. Lasaea is the first bivalve genus in which asexual reproduction has been confirmed and is also the first molluscan genus in which pseudogamy (gynogenesis) has been detected. Introduction Lasaea is a near-cosmopolitan genus of minute, her- maphroditic, brooding clams that inhabit crevices in rocky Received 5 March 1991; accepted 8 July 1991. intertidal shores (Keen, 1938; Ponder, 1971; Beauchamp, 1986). We are interested in these organisms because they allow valuable insights into the genetic and evolutionary consequences of alternate life history trait combinations in marine benthic invertebrates. A major reproductive and developmental dichotomy exists within Lasaea. One species, L. australis (Lamarck, 1818), reproduces by cross- fertilization, releases its progeny as planktotrophic larvae (6 Foighil, 1988; Tyler- Walters and Crisp, 1989) and is restricted in its distribution to the western Pacific (6 Foighil, 1989). In contrast, the congeners of L. australis all release their young as crawl-away juveniles and form a complex grouping of poorly defined systematic status (6 Foighil, 1989). Although lacking pelagic larvae, this group of organisms ha^e attained possibly by rafting a remarkably exten- sive collective geographic range, which includes all con- tinents apart from Antartica and a large number of oceanic islands (6 Foighil, 1989). Population genetic studies of Lasaea with this developmental mode have revealed that natural populations are composed of non-hybridizing, frequently sympatric, genetic strains (Crisp et a/., 1983; Crisp and Standen, 1988; 6 Foighil and Eernisse, 1988; Tyler- Walters and Crisp, 1989; Tyler- Walters and Dav- enport, 1990). There is currently no evidence for cross- fertilization in this grouping. Individuals can reproduce in isolation for at least two generations (Crisp et a/., 1983), and progeny from pair mating experiments (6 Foighil and Eernisse, 1988) and from field brooding specimens (6 Foighil and Eernisse, 1988; Tyler- Walters and Dav- enport, 1990) preserve maternal protein phenotypes. La- saea strains studied to date in Europe and Kerguelen Is- land are highly, and variably, polyploid; indeed, they pro- duce a record number of chromosomes for the Class Bivalvia (Thiriot-Quievreux et a!., 1988, 1989). It is not PLOIDY AND PRONUCLEAR INTERACTION IN Lasaea CLONES 223 clear, however, if polyploidy is linked to this develop- mental mode, and the evolutionary mechanism of poly- ploidy in these organisms is obscure. The general biology of Lasaea is known in considerable detail, but one important question still remains to be re- solved: what is the reproductive mode of Lasaea strains that lack pelagic larvae? This has proven to be surprisingly difficult to answer, and there are lines of evidence for both self-fertile (6 Foighil, 1987) and asexual reproductive modes (Crisp and Standen. 1988; Thiriot-Quievreux et ai, 1988, 1989; Tyler- Walters and Crisp, 1989). All stud- ied populations of Lasaea that lack pelagic larvae are now known to be simultaneous hermaphrodites with a minute male allocation (6 Foighil and Eernisse, 1988). Prelimi- nary data on sperm-egg interaction are consistent with self-fertilization: both gamete types are spawned simul- taneously into the brood chamber: sperm bind to eggs via an acrosomal reaction, penetrate the egg, and two polar bodies are extruded prior to first cleavage (O Foighil, 1987). A variety of other data, however, suggests an asex- ual reproductive mode in which the sperm merely trigger parthenogenetic development (pseudogamy). Individual Lasaea frequently express putative heterozygote electro- morphs at multiple loci (Crisp and Standen, 1988; O Foighil and Eernisse, 1988; Tyler- Walters and Crisp, 1989). These electromorphs, if real, are inconsistent with a self-fertile reproductive mode. Karyological analyses of Lasaea strains have failed to find meiotic metaphases, and the presence of odd ploidy numbers and of super- numary chromosomes may render accurate meiosis im- possible (Thiriot-Quievreux et ai, 1988, 1989). The aims of this study were twofold. First, we karyo- logically characterized northeastern Pacific populations, an approach to understanding how the exceptionally high chromosome assemblages in Lasaea strains evolved. Sec- ond, we studied pronuclear interaction in newly spawned eggs to distinguish between the two competing hypotheses about the reproductive mode in Lasaea strains. Materials and Methods Karyology Specimens of Lasaea were sampled at McNeill Bay, Victoria, British Columbia, Canada in October 1989 and air-mailed live to Villefranche-sur-mer for karyotyping. The McNeill Bay population is composed of at least five non-hybridizing genetic strains that can be reliably dis- tinguished only by electrophoretic analyses (6 Foighil and Eernisse, 1988). Air-mailed specimens were maintained on arrival in aquaria for 10 days and fed cultured mi- croalgae (Isochrysis galbana) to stimulate cell divisions. Specimens were incubated for 12 h in seawater con- taining 0.005% colchicine, following which the valves of each clam were gently half-opened to allow effective hy- potonic treatment (45 min in 0.9% sodium citrate). Sub- sequent processing involved fixation in freshly mixed ab- solute alcohol and acetic acid (3:1) with three changes of 20 min duration. During the second fixation step, the bodies were dissected from the valves. Slide preparations were made from 1 to 4 bodies using an air-drying tech- nique (detailed in Thiriot-Quievreux and Ayraud, 1982). The preparations were stained for 10 min with 4%. Giemsa (pH 6.8), and photographs of well-spread metaphases were taken with a Zeiss III photomicroscope. For karyotyping, chromosomes were cut out of the photomicrographs and were paired on the basis of size and centromere position. Measurement of chromosomes from the best karyotypes were made with a Digitizer (BIT PAD 10, Summa Graphic) interfaced with a microcom- puter (ATV 286). Statistical interpretations were made using a CHROMOS program (Thiriot-Quievreux, 1984; Thiriot-Quievreux et a/., 1988). Terminology relating to centromere position follows that of Levan et al. (1964). When a centromere position was borderline between two categories, the mean was calculated with 95%. confidence limits, and both categories were listed. Pronuclear interaction Additional animals were sampled from the McNeill Bay in February 1990 for the pronuclear interaction study. Gravid individuals were cultured in petri dishes containing seawater at room temperature and checked daily using a dissecting stereomicroscope for evidence of spawning activity. Newly spawned individuals were detected by the presence of eggs in the brood chamber, an event visible through the semi-transparent valves of most adults. Fifty-four early broods (uncleaved eggs 4 cell stage) were dissected from the parents and individ- ' . >.* * *^ * * * * , V*** ,*^u * * * * Figure I. Mitotic metaphase with 100 chromosomes in northeastern Pacific Lasaea- Scale bar = 10 ^m. 224 D. 6 FOIGHIL AND C. THIR1OT-QUIEVREUX l v' * 1 19 22 25 2 8 31 1 1 < 16 17 .K- 20 21 23 24 26 27 2 9 30 32 Figure 2. Raryotype taken from a northeastern Pacific Laxaea metaphase with 97 chromosomes. Scale bar = 5 ^m. ually fixed in three changes of freshly made Carney's fixitive (3:1 methanol, glacial acetic acid). Fixed broods were cleared in 45% glacial acetic acid and were gently pipetted into Vaseline-sealed wells on microscope slides. The cleared cells were viewed under phase-contrast or darkneld optics using a compound Zeiss photomicro- scope and photographed with Kodak Tech Pan film. Cy- tological events following spawning were reconstructed PLOIDY AND PRONUCLEAR INTERACTION IN Lasaea CLONES Table I Chromosome measurements and classification in seven metaphases of northeastern Pacific Lasaea 225 Chromosome pair no. Relative length Arm ratio Centromeric index Classification Mean SD Mean SD Mean SD 1 5.75 0.16 0.864 0.037 46.18 1.06 m 2 4.69 0.28 0.757 0.066 42.85 2.05 m 3 3.98 0.21 0.733 0.056 42.07 1.77 m 4 3.51 0.33 0.777 0.078 43.92 2.42 m 5 2.63 0.33 0.811 0.075 44.43 2.30 m 6 2.38 0.31 0.842 0.069 45.46 1.97 m 7 2.11 0.39 0.873 0.056 46.37 1.54 m 8 1.92 0.33 0.821 0.087 44.75 2.64 m 9 1.62 0.26 0.842 0.082 45.45 2.52 m 10 5.89 0.55 0.436 0.063 29.91 3.00 m 1 1 4.50 0.37 0.423 0.056 29.42 2.86 sm 12 2.66 0.15 0.355 0.039 26.02 2.15 sm-st 13 2.47 0.20 0.353 0.079 25.70 4.14 sm-st 14 2.27 0.12 0.352 0.063 25.68 3.56 sm-st 15 2.16 0.22 0.366 0.067 26.41 3.43 sm-st 16 2.03 0.21 0.366 0.056 26.46 2.99 sm-st 17 1.80 0.22 0.346 0.075 25.27 4.05 sm-st 18 3.67 0.31 0.306 0.094 22.87 5.36 st-sm 19 3.28 0.22 0.328 0.038 24.32 1.98 st-sm 20 3.01 0. 1 5 0.306 0.023 23.28 1.33 st 21 2.84 0.12 0.314 0.046 23.68 2.66 st 22 5.33 0.42 0.089 0.022 8.05 1.75 t 23 4.84 0.30 0.071 0.013 6.58 1.10 t 24 4.50 0.14 0.088 0.022 8.04 1.82 t 25 4.03 0.22 0.081 0.014 7.46 1.22 t 26 3.32 0.26 0.099 0.013 8.96 1.02 t 27 2.84 0.23 0.108 0.036 9.61 2.91 t 28 2.68 0.14 0.099 0.017 8.93 1.34 t 29 2.31 0.20 0.116 0.044 10.23 3.44 t 30 1.87 0.28 0.137 0.029 11.88 2.22 t-st 31 1.66 0.24 0.115 0.010 10.26 0.75 t 32 1.31 0.16 0.134 0.038 11.57 2.94 t-st by examining broods that were fixed at different devel- opmental stages. Results Karyology The 85 slide preparations examined in this study con- tained large interphase nuclei (approximately 35 nm in diameter) and mitotic metaphase spreads. Meiotic divi- sions were not encountered. The mitotic metaphases were remarkable due to their large sizes and their high chro- mosome numbers (Fig. 1). Ninety-five metaphase spreads were photographed, and chromosome counts were scored, although the latter process was frequently complicated by the presence of overlapping chromosomes. Chromosome numbers ranged from 58 to 108; the majority of meta- phases (76 out of 95) contained more than 80 chromo- somes, and 32 spreads had 90-100 chromosomes. Ten well-spread metaphases were karyotyped. Figure 2 shows the karyotype taken from one metaphase spread containing 97 chromosomes. The chromosomes have been arranged into 32 triplet subgroupings on the basis of shared morphology and size. Triplets can be further classified into groups of similar morphology: metacentrics, submetacentrics, subtelocentrics, and telocentrics. The identification of homologous chromosomes is relatively unambiguous for the largest chromosomes. But the smallest chromosomes have less distinctive morphologies, so ambiguities remain. One minichromosome within this metaphase (Fig. 2) could not be classified. The chromo- somes of other karyotyped metaphases could similarly be arranged into triplets. Variation in chromosome number between metaphases resulted from the absence of single chromosomes from individual triplet subgroupings or from the occurrence of supernumerary chromosomes among the smaller members of each morphological group. 226 D. 6 FOIGHIL AND C. THIRIOT-QUIEVREUX Relative length 6.00 - 5.00 - 4.00 - 3,00 - 2,00 - 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 ly 2(1 21 22 23 24 25 26 27 28 29 30 31 32 Chromosome pairs B m D sm D sm-st D st D st-sm [3 t t-st Figure 3. Ideogram of northeastern Pacific Lasaea chromosomes constructed from relative length and centromeric index values. Lengths are in ^m. m, metacentric; sm, submetacentric; sm-st, submetacentric- subtelocentric; st, subtelocentric; st-sm, subtelocentric-submetacentric; t, telocentric; t-st, telocentric-sub- telocentric. Chromosomes were measured from seven well-spread metaphases (3 with 95 chromosomes, 3 with 97, and 1 with 100). Table I gives the mean and standard deviations of the relative lengths, arm ratios, and centromere indices of 32 chromosome triplets and their classification. An ideogram (Fig. 3) was constructed from the relative length and centromere index values so that the different mor- phological types of chromosomes could be better visual- ized. The karyotype of northeastern Pacific Lasaea consists of 8 metacentrics, 8 submetacentrics (including 6 sub- metacentric-subtelocentrics), 4 subtelocentrics (including 2 subtelocentric-submetacentrics), and 1 1 telocentrics (including 2 telocentric-subtelocentrics). Within each morphological grouping of chromosomes, some triplets were difficult to distinguish because of their similar lengths: e.g.. metacentrics no. 7-8, submetacentric-sub- telocentrics no. 12-13 and 15-16, subtelocentrics no. 20-21, and telocentrics no. 27-28 and 30-31. In these cases, the loss of individual chromosomes or the occur- rence of supernumary chromosomes could not be as- signed with accuracy. Pronnclear interaction A single sperm penetrates the cell membrane of each egg shortly after spawning. The egg germinal vesicle breaks down, and the egg chromosomes condense and become aligned for first metaphase (Fig. 4a). The incorporated sperm nucleus remains in the egg cortex in a condensed Figure 4a. Phase contrast light micrograph (PCLMI of an uncleaved northeastern Pacific Lasaea egg showing the incorporated, condensed sperm nucleus (arrowl in the cortex and the egg chromosomes arranged for first metaphase (on left). Scale bar = 20 urn. Figure 4b. PCLM showing detail of sperm nucleus from Figure 4a. Scale bar = 6 nm. Figure 5. PCLM of decondensing sperm nucleus (N) located in the cortex of an northeastern Pacific l.iiMicu egg. Scale bar = 10 ^m. Figure 6. Darkfield light micrograph (DFLM) of an uncleaved northeastern Pacific Lasaea egg with a decondensing sperm nucleus situated in its cortex (large arrow). This egg has extruded two polar bodies and the egg chromosomes are arranged for first cleavage metaphase (small arrow). Scale bar = 50 fim. Figure 7. DFLM of a northeastern Pacific La.iaea first cleavage telophase. Note presence of aggregated sperm chromatin (n) in one of the daughter cells and of daughter chromosomes (large arrows) in both daughter cells. The cleavage furrow (c) is also apparent, as are the first (lower small arrow) and second (upper small arrow) polar bodies. Scale bar = 40 jim. PLOIDY AND PRONUCLEAR INTERACTION IN Lasaea CLONES 227 4a 228 D. 6 FOIGHIL AND C. THIRIOT-QUIEVREUX state (Figs. 4a, b), until after the production of the two polar bodies. The sperm nucleus then slowly decondenses and can be distinctly visualized in the egg cortex with phase contrast. ^Fig. 5) and, especially, darkfield optics (Fig. 6). However, the sperm nucleus does not recondense to form chromosomes or to associate with the egg chro- mosomes. During first cleavage, the sperm chromatin re- mains aggregated and typically ends up in the cortex of one of the two daughter cells (Fig. 7). Prior to first cleavage, the egg extrudes two polar bodies, each involving the formation of a spindle at the animal pole and the division of chromosomes. The first division (Fig. 8) results in the formation of a polar body containing an average of 48 (5.2 S.E.; n = 17) well defined chro- mosomes (Fig. 9). After formation of an additional spin- dle, a second polar body is extruded adjacent to the first (Fig. 10). Unlike those of the first polar body, the chro- mosomes of the second are tightly aggregated and are very difficult to distinguish microscopically (Fig. 10). In a pre- liminary study, 6 Foighil (1987) assumed that the second polar body of northeastern Pacific Lasaea strains is hap- loid. During the present work, however, the second polar body chromatin was sufficiently dispersed in a few cases, and we could determine that each chromosome is com- posed of two homologous interphase chromatids (Fig. 1 1 ), i.e., is diploid. In 2 eggs out of the 123 examined, the number of chromosomes in the second polar body could be established accurately and were in each case (49, 45) equal to that of the adjoining first polar body. The egg chromosomes that remain within the egg (female "pro- nucleus") migrate to the center of the cell and become arranged in homologous pairs for first cleavage (Fig. 12), without associating with the sperm nucleus. A mean of 95.9 (12 S.E.; n = 9) chromosomes were arranged at the first cleavage metaphase. This is consistent with the results obtained in metaphase spreads of adult tissue. During first cleavage, the homologous chromosome pairs separate (Fig. 13) and migrate into the forming daughter cells. Discussion Northeastern Pacific Lasaea strains have high chro- mosome numbers, ploidy grouping of the chromosomes by three, and variable numbers of supernumerary chro- mosomes. Congeners that lack pelagic larvae have also been characterized karyologically from European (Thiriot- Quievreux el ai, 1989) and from Kerguelen Island pop- ulations (Thiriot-Quievreux et ai, 1988). These congeners share with northeastern Pacific strains the karyological features of high chromosome numbers and the presence of supernumerary chromosomes, but show varying de- grees of polyploidy. In Kerguelen strains, a chromosome number of 100-120 was found, but ploidy levels could not be determined (Thiriot-Quievreux et al., 1988). Chro- mosome numbers in European strains range greatly (63- 340) and can be grouped into different ploidy levels of 3, 5, and 6, in addition to variable supernumeraries (Thiriot- Quievreux et al., 1989). A meaningful comparison of karyotypes among these different Lasaea populations is complicated because we cannot precisely distinguish homologous chromosome sets from the large and variable number of supernu- maries. However, we can compare the first 17 chro- mosome pairs identified in the karyotype of Kerguelen strains (Thiriot-Quievreux et al., 1988) with the first 17 sets of homologous chromosomes ordered in decreasing size in the karyotypes of European (Thiriot-Quievreux et n/.. 1989) and of northeastern Pacific strains (this study). The karyotypes of the three populations differ in their relative numbers of metacentric, submetacen- tric, subtelocentric, and telocentric chromosome sets (8m, 3sm, 2st and 4t in Kerguelen strains; 6m, Ism, 6st and 4t in European strains; 4m, 2sm, 4st and 7t in northeastern Pacific strains). These karyological dis- tinctions not only imply that the three geographically isolated populations are reproductively incompatible, but also that they have experienced different evolution- ary mechanisms of polyploidy. Polyploidy in mollusks has been reported in several hermaphroditic pulmonate snails that are capable of self- fertilization or asexual reproduction (Jacob, 1957; Burch and Huber, 1966; Patterson and Burch, 1978; Goldman et al., 1984). But among the Bivalvia, this phenomenon is exceptional, and beside the polyploid Lasaea strains, only one species, Corbicula leana, with a triploid chro- Figure 8. PCLM showing a lateral view of the first metaphase spindle in a northeastern Pacific Lasaea egg. Scale bar = 8 ^m. Figure 9. PCLM of the first polar body extruded by a northeastern Pacific Lasaea egg. Scale bar = 10 jum. Figure 10. PCLM of both first (P) and second (arrow) northeastern Pacific Lasaea polar bodies. Scale bar = 20 pm. Figure II. PCLM showing details of northeastern Pacific Lasaea second polar body chromosomes. Arrows point to homologous chromatids. Scale bar = 7 ^m. Figure 12. PCLM of northeastern Pacific Lasaea egg chromosomes arranged at first cleavage metaphase. Arrows point to pairs of homologous chromosomes. Scale bar = 6 pm. Figure 13. PCLM of first cleavage anaphase. Arrows indicate daughter chromosomes. Scale bar = 35 PLOIDY AND PRONUCLEAR INTERACTION IN Lasaea CLONES 229 230 D. 6 FOIGHIL AND C. TH1R1OT-QUIEVREUX mosome number of 54, has been recorded (Okamoto and Arimoto, 1986). The genus Ltisaea contains one sexual species, L. aus- tralis, that undergoes a planktotrophic larval development (6 Foighil. 1988; Tyler- Walters and Crisp, 1989). 6 Foighil (1988) proposed that this reproductive and de- velopmental combination represented the primitive con- dition in the genus. Tyler- Walters and Crisp (1989) pro- vided a preliminary estimate of chromosome number in two L. australis eggs (one with n = 2 1-22, the other with 2n = 42-44). A more detailed karyological analysis cur- rently underway has yielded a diploid number of 2n = 36 for this species (C. Thiriot-Quievreux, unpubl.). Work in progress on the karyotype of L. australis may help to clar- ify chromosomal evolutionary mechanisms within the genus Lasaea. Karyological analyses of Lasaea strains in both present and previous studies (Thiriot-Quievreux eta!., 1988, 1989) have failed to find meiotic metaphases and the presence of odd ploidy numbers and of supernumary chromosomes presumably renders accurate meiosis impossible. Al- though not all aspects of egg maturation have been re- vealed in this present study, two key pieces of evidence establish that northeastern Pacific Lasaea strains are par- thenogens, not self-fertilizers. The incorporated sperm nucleus disintegrates in the egg cortex and does not fuse with the egg "pronucleus"; i.e.. syngamy does not occur. Both polar bodies have a diploid chromosome number, a result inconsistent with meiosis, implying that they are products of mitotic divisions. Our data confirm Crisp and Standen's (1988) proposal that parthenogenetic devel- opment is triggered by auto sperm in Lasaea strains lack- ing dispersive larvae. Non-hybridizing lineages of north- eastern Pacific Lasaea therefore represent true asexual clones, not inbred lines. Lasaea is the first bivalve genus in which asexual reproduction has been confirmed and is also the first molluscan genus in which pseudogamy (gyn- ogenesis) has been detected. Egg maturation in northeastern Pacific Lasaea differs from that of the great majority of other apomictic (ameiotic) organisms in that two polar bodies are extruded rather than one (Hughes, 1989). Two polar bodies are also mitotically produced in the gastropods Thiara (Afe- lunoides) tuberculatus and T. lineatns. which avoid a re- duction in chromosome number by arresting an oogonial division (Jacob, 1957). More detailed analyses of Lasaea egg maturation divisions are needed to determine if these organisms restitute chromosome numbers in a similar manner prior to first cleavage. We have no direct data on the chromosome comple- ment of the northeastern Pacific Lasaea sperm cells, but meiotic metaphases have not been discovered in Lasaea clones despite intensive karyological study of populations from British Columbia, Kerguelen Island, and Europe (present study, Thiriot-Quievreux et al.. 1988, 1989). Re- liance on sperm activation leaves open the possibility of occasional leakage of sperm chromosomes and may be the origin of the supernumary chromosomes that are characteristic of asexual Lasaea karyotypes. Pseudogamy (gynogenesis) occurs in a wide variety of taxa (Kiester et al.. 1981; Stenseth et al.. 1985; Hughes, 1989) and pseudogamous individuals are typically sexual parasites of closely related cross-fertilizing species. Lasaea clones, however, are exceptional in that each individual is reproductively independent, using its own sperm to trigger asexual development. Lasaea clones, therefore, have much greater evolutionary potential than most gyn- ogenetic forms. Gynogens typically originate from hybridization events between related species, e.g., the Ambyostomajef- Jersoniainun complex (Uzzell, 1964), Rana escnlenta (Uzzel and Berger, 1975; Turner and Nopp, 1979), and possibly the freshwater bivalve Corbicitla leana (Oka- moto and Arimoto, 1986). Hybrid, gynogenetic Fl prog- eny are frequently triploid, incapable of meiosis, and reproduce by sexually parasitizing males of the parental species (Hughes, 1989). Northeastern Pacific Lasaea clones are triploid and may have arisen from rare hy- bridization events between ancestral lineages. Hybrid, triploid Lasaea Fl progeny that produced a small amount of phenotypically normal sperm cells (6 Foighil, 1985, 1987) may then have reproduced by autogyno- genesis. This evolutionary scenario is, however, specu- lative and needs to be verified by independent phylo- genetic reconstruction. It is now apparent that the genus Lasaea includes some very unusual marine mollusks. Direct developing members are collectively much more successful than sexual congeners, which retain pelagic larval develop- ment (6 Foighil, 1989). In the present study, we have confirmed that direct development in this genus is linked to polyploidy and to the presence of supernumary chromosomes (Thiriot-Quievreux et al.. 1988, 1989). Direct development is also linked to minute male al- location (Pelseneer, 1903; Oldfield, 1964; 6 Foighil, 1985; McGrath and 6 Foighil, 1986; 6 Foighil and Eernisse, 1988) and to a clonal population genetic structure (Crisp et al.. 1983: Crisp and Standen, 1988; 6 Foighil and Eernisse, 1988; Tyler- Walters and Crisp, 1989). Autogynogenesis, which we have documented in northeastern Pacific Lasaea populations, may also be a persistent theme in other members of the genus that share this developmental mode, but see Tyler-Wal- ters and Davenport (1990) for a potential exception. The genus Lasaea promises to become a valuable model system for exploring the long-term evolutionary and genetic consequences of contrasting reproductive modes in benthic marine metazoans. PL.OIDY AND PRONUCLEAR INTERACTION IN Lasaea CLONES 231 Acknowledgments Thanks are due to A. Insua and G. Quelart for assistance in karyotyping. This work was supported by an NSERC postdoctoral fellowship to D. 6 Foighil. 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Naturwissenschaften 66: 268-269. LIzzell, T. M. 1964. Relations of the diploid and tnploid species of the Ambyostoma jeffersonium complex (Amphibia, Caudata). Copeia 2: 257-300. Uzzell, T. M., and L. Berger. 1975. Electrophoretic phenotypes of Rana ridibunda. Rana lessonae and their hybridogenetic associate Rana escitlenta. Proc. Nat. Acad. Sci. 127: 13-24. Reference: Biol Bull 181: 232-237. (October. 1991) Variation in Fertilization Rate in the Tropical Reef Fish, Halichoeres bivattatus: Correlates and Implications CHRISTOPHER W. PETERSEN Smithsonian Tropical Research Institute. Box 2072, Balboa, Panama Abstract. Fertilization rates were estimated for natural spawns in the tropical wrasse, Halichoeres bivattatus. Fertilization rate averaged 88%, but varied with both sea conditions and with the addition of males (streakers) to pair spawns. As sea conditions became rougher, the mean fertilization rate for a day decreased. This effect was due to the two days with the roughest sea conditions and the lowest mean fertilization rates; there was no obvious trend when these two days were excluded from the analysis. On a given day, pair spawns, with a single male and female, had fertilization rates approximately 7% lower than spawns where 1-2 streakers joined the pair spawn. These results suggest that variation in fertilization rate must be considered as a potential selective force in shaping repro- ductive behavior in fishes with external fertilization of pelagic eggs. Introduction The social and mating systems of tropical reef fishes, especially the more colorful and abundant species like the wrasses (Pisces: Labridae), have been the focus of a large body of research (Robertson, 1972, 1981; Robertson and Choat, 1974; Robertson and Hoffman, 1977; Warner and Robertson, 1978; Thresher, 1979; Warner and Hoffman, 1980a, b; Moyer and Yogo, 1982; Tribble, 1982; Kuwa- mura, 1984; Warner, 1984a, b, 1987, 1988; Nemtzov, 1985; Hoffman, 1985; Ross, 1986; Lejeune, 1987; Victor, 1987; Baird, 1988). Much of this work has involved doc- umenting spawning behavior, quantifying the reproduc- tive success of various mating tactics, and speculating on Received 1 Apnl 1991; accepted 24 May 1991. Present address: College of the Atlantic, 105 Eden St., Bar Harbor, Maine 04609- 11 98. the adaptive significance of morphological and behavioral differences between and within the sexes. The wrasses have reproductive behaviors typical of free- spawning tropical reef fishes, with spawning occurring at downcurrent sites of reefs. Spawning consists of one to over ten males and a female rushing upward 0.2 to several meters toward the surface and releasing gametes into the water column at the apex of the spawning rush. Although past workers have proposed many alternative mechanisms for the evolution of reproductive behaviors in tropical reef fishes, one fundamental factor has been almost com- pletely ignored: the effect of fertilization rate on individual reproductive success and spawning behavior (for excep- tions see Shapiro, 1989; Petersen, 1991). In this paper, I provide measurements for fertilization rate in the tropical wrasse, Halichoeres bivattatus, examine patterns and the potential causes of variation in fertilization rate, and dis- cuss the role that fertilization rate may play in the evo- lution of reproductive behavior in this species. Selection on males has been generally thought to act such that individuals produce enough sperm to fertilize all of the eggs of a female, implying that investigations of variation in fertilization rate would be uninteresting. However, as the amount of sperm released in a spawn increases, the number of additional fertilizations for a given number of sperm released should decrease, reducing the benefit for producing more sperm (Petersen, 1991). There is no reason to expect that the benefits to males for producing additional sperm will outweigh the costs of sperm production until all eggs are fertilized. Under these circumstances, the selected level of sperm production may not be enough to fertilize all available eggs, and the fer- tilization rate should be less than 100% (Petersen, 1991). The extent to which fertilization rate is important in discussions of mating-system evolution depends on how 232 FERTILIZATION RATE IN HALICHOERES 233 much variance exists in fertilization rate and how much of that variance is predictable. Given that fertilization rate may not always be 100%, it is still not clear whether fer- tilization rate plays a significant role in determining male and female mating strategies in organisms that have ex- ternal fertilization of pelagic eggs. In many tropical reef fishes, there is more than one male mating behavior within a population (e.g.. Warner et a/.. 1975; Robertson and Warner, 1978; Warner and Robertson, 1978; Thresher, 1984; Petersen, 1990). Spawning sites may be occupied by either a large territorial male who mates singly with the female (pair spawning) or by a group of smaller males who mate together with the female (group spawning). In addition, small non-ter- ritorial males are known to rush in and join pair spawns at the moment of gamete release, a mating behavior called streaking (Warner el al.. 1975; Thresher, 1984). In many species, there may be several active spawning locations within the range of an individual female, providing fe- males with a choice of conditions for spawning. The extent that males choose or compete for spawning locations and females choose either spawning locations or the identity of the male-role spawners based on fertilization rate is unknown. Reproductive Biology of Halichoeres bivattatus The slippery dick, Halichoeres bivattatus, is the most common Caribbean wrasse found over shallow reefs and seagrass communities. Smaller males and females have an initial-phase (IP) drab coloration; large terminal-phase (TP) individuals are more brightly colored and are in- variably male (Warner and Robertson, 1978). TP males may be the result of sex change, or merely a coloration change from IP males (Warner and Robertson, 1978). Sex ratio is only slightly biased towards females (1:1.2), and IP males outnumber TP males approximately 2.3:1 (Warner and Robertson. 1978). Spawning occurs daily in the mid-afternoon over the lee edges of reefs and over turtle grass (Warner and Rob- ertson, 1978). In pair spawning, gravid females enter the territories of TP males, are approached by the TP male, and after one to several rushes by the male toward the female both rise quickly 0.3 to over 1 m in the water column in a fast spawning rush. Gametes are released at the apex of this spawning rush, after which both fish dart back to their normal positions closer to the bottom. The eggs are fertilized externally as they begin to float away from the location of the spawn. The TP male continues to court additional females, and may spawn over 20 times in a day (pers. obs.). Streaking occurs when an IP male joins the spawning pair at the apex of their spawning rush and presumably releases sperm. Only TP males were ob- served pair spawning in this study; IP males were observed streaking pair spawns and, on one occasion, approxi- mately 10 IP males took part in a group spawn. Both pair and group spawning appear to be common in this species (Warner and Robertson, 1978). All data reported here are from pair spawns with or without streakers. Materials and Methods Data were collected during February 1990 along the northeast coast of St. Croix, Virgin Islands, and during March 1990 in the San Bias Islands of Panama. At St. Croix, collections of eggs were made in shallow water ( 1- 4 m depth) while snorkeling behind the barrier reef ap- proximately 1 km west of Tague Bay. In the San Bias, collections were made while snorkeling at 1-3 m depth at Smithsoniantupo reef adjacent to the Smithsonian Tropical Research Institute San Bias Field Station. To collect eggs from natural spawnings, observers po- sitioned themselves within 2-4 m of a spawning site near the beginning of the daily spawning period. When a spawn was observed, the gamete cloud was marked by a small amount of fluorescein dye released by the observer near the spawn. After waiting for at least 30 s, the observer swept the area around and including the expanded fluo- rescein cloud for 30 s with a 6-inch brine-shrimp net (maximum mesh size approximately 100 X 300 nm}. Collections of eggs from another wrasse ( Thalassoma bi- fasciatum. Petersen et al.. 1992) indicated that harsher- textured nylon nets, such as plankton netting, severly re- duced the fertilization rates while the brine-shrimp net had no effect. At the end of the sweep, the net was drawn away from the fluorescein cloud, the contents of the net were transferred to a small plastic bag, and the bag was sealed. For each spawn, the location of the spawn was recorded, and the number of streakers that joined the spawn (0-2) was noted. The next morning the contents of each bag were filtered through 100 nm nylon mesh to collect the eggs, which were then examined under a dissecting microscope and scored as fertilized or unfertilized. By counting eggs 16- 20 h post-spawning, fertilized, developing eggs could be unambiguously distinguished from undeveloped eggs. Developing eggs contain nearly fully developed larvae, because hatching is completed within 24 h. Undeveloped eggs were scored as unfertilized. Estimates of fertilization rate for spawns relied on collections with at least 20 eggs. To test whether the 30-s delay before collection was adequate to allow fertilization to occur, on three days col- lections were alternated between 30-s delays and 60-s de- lays. A significant increase in fertilization rate in the 60- s sample would suggest that the 30-s samples underesti- mate fertilization rate. In addition, collections were made at spawning locations during the spawning period but not immediately after a spawn to verify that eggs collected 234 C. W. PETERSEN C 03 Q. (/D 0) .0 E 50 i 40- 30- 20- 10- \/A 20 40 60 Fertilization rate 80 Figure 1. Frequency distribution of fertilization rates for all spawns with >20 eggs. The lower hound for the 10% categories is given, the last category included two spawns with 100% fertilization of collected eggs. after spawns were not in the water column from spawns elsewhere. On each date, a qualitative score of sea conditions was made during the spawning period. This score varied from 1 for the calmest conditions to 5 for the roughest condi- tions experienced during the study. These data were used to test for decreased fertilization rate on days with higher water velocities and increased water mixing. Based on theoretical studies of water turbulence and fertilization rate, increased water mixing was predicted to reduce sperm concentrations and reduce fertilization rates (Denny and Shibata, 1989). The possibility that sperm depletion in males during the daily spawning period causes lower fertilization rates was examined by testing for a negative correlation between fertilization rate and the order of collection of spawns for a specific male during a spawning period. Only sequences with at least seven samples were used for this purpose. For statistical analysis, fertilization rate data were an- gularly transformed before applying parametric statistical techniques. In cases where the sea-condition ranking or the spawning-order ranking was used, non-parametric statistics were employed. Results A total of 180 collections of eggs were taken over 19 days. Of the 180 collections, 102 (57%) had the mini- mum of 20 eggs and were used in the analysis of fertil- ization rate. The mean fertilization rate of these collections was 88.1% (median = 87.3%) (Fig. 1 ). The number of eggs collected from a spawn varied from to 536 (median = 26.5; for collections with at least 20 eggs, median = 65.5). The three control collections had 0-4 eggs, im- plying that eggs in collections do come largely from the spawn that was observed immediately prior to sampling. There was no evidence that collecting eggs after 30 s detrimentally affected the estimate of fertilization rate. Eggs collected from spawns after 60 s had virtually iden- tical fertilization rates to spawns collected after 30 s (F, , 5 = 0.001, P= 0.98). Spawning date had a significant effect on fertilization rate. Over the 1 8 dates with at least two collections with 20 eggs, fertilization rate varied significantly among dates (ANOVA, F I784 = 8.3 !,/>< 0.001). One of the causes of variation in fertilization rate among days appeared to be sea conditions. There was a negative correlation between the qualitative score of sea condition and the mean fertilization rate for a day, with rougher days having lower fertilization rates (Fig. 2, i\ = -0.44, /V.aiied] < 0.05, n = 18 days). This analysis used only spawns without streaking; an identical trend existed for the spawns with streakers (r s = -0.48, / 3 [i. ta ,icd] < 0.05, n = 16). Much of the variation in fertilization rates among dates was due to the two dates that had the roughest sea conditions and also had the lowest mean fertilization rates. Excluding these two days, there was no significant effect of sea conditions on fertilization rate (both types of spawns, P| Mj ,i,d] > 0.25). Spawns with streakers had significantly higher fertil- ization rates than pair spawns without streakers collected on the same day. To test for changes in fertilization rate with the presence of streakers, date of collection and the presence of streakers were examined simultaneously in a two-way ANOVA for the subset of days with collections from pair spawns both with and without streakers (n = 16 dates). Using this database, both the presence of a streaker joining the pair spawn and the day a spawn was collected 100- w 5 80 C o ' 60- N oS 40- 20 o 8 o e o o o o 12345 Sea conditions (rank) Figure 2. The relationship between sea conditions (rank score) and mean fertilization rate on a day for all spawns without streakers with >20 eggs. FERTILIZATION RATE IN H.4LIC1IOERES 235 had a significant effect on fertilization rate (Table I). Spawns with streakers had an average of 6.6% higher fer- tilization rate on a day (range = -4 to 18%) compared with pair spawns without streakers. The identity of the male did not appear to affect fer- tilization rate. On two days collections were made si- multaneously from three males on the same back reef area within 30 meters of one another. There was a sig- nificant effect of date (F ug = 6.41, P = 0.02), but no significant effect of male identity (F 2 .it = 0.56, P = 0.58). There was also no evidence that fertilization rate for spawns by a male changed in any systematic way during the spawning period. Over the course of the study, there were three sequences of 7-10 spawns successfully collected from a male. These sequences occurred over a period of 33-48 min. The order in the spawning sequence was not significantly correlated with fertilization rate (r s = -0.25 to 0.64, Quailed] > 0.1 in all cases). Thus, the fertilization rate data does not provide evidence for sperm depletion by TP male H. bivattatus. Discussion Fertilization rate has been a previously ignored aspect of the reproductive biology of tropical reef fishes. The result that lower fertilization rates occur on days of rougher water conditions and higher current velocity is consistent with a theoretical treatment of the effect of turbulence on fertilization rate (Denny and Shibata. 1989) and with em- pirical data for marine invertebrates (Pennington, 1985; Levitan, 1989) and another marine fish (Petersen el a/., 1992). A difference in fertilization rate was also observed be- tween pair spawns and pair spawns joined by streaking males. The most likely cause of the increase in fertilization rate in spawns with streakers is a higher total amount of sperm released with the addition of another male. How- ever, this result could also be caused by some other factor associated with streaking. For example, a slower spawning ascent or a lower spawning rush height might be associated both with increased fertilization rates and with a higher probability of additional males joining the spawn. Al- though these alternatives are possible, 1 will assume that the most straightforward interpretation is correct, that streakers directly increase fertilization rate in Halichoeres bivattatus. If so, the result that streaking increases fertil- ization rate indicates that some of the assumptions that have been made about reproduction in reef fishes have been violated and that fertilization rate must be considered as a potential selective force in shaping spawning behavior. If females attempt to maximize fertilization rates in their spawns, irrespective of the identity of the male that fertilizes the eggs, then these data provide evidence for a conflict of interests between the TP territorial male and Table I Two-way mixed-model ANOl'A of the effect of presence of streakers and collection day on fertilization rale Dependent variable Fertilization rate (angular transformation) Independent variable df MS F P Day of collection 15 0.10 6.62 <0.001 Presence of streakers 1 0.12 6.77 <0.05 Interaction 15 0.02 1.1 >0.25 Error 65 0.016 R- = 0.74. the spawning female. Females gain fertilizations by having a streaking male join the spawn, while the TP male will almost certainly lose fertilizations due to sperm compe- tition with the streaking IP male. Territorial males chase IP individuals away from the spawning site during the spawning period, and many, if not all, of these are pre- sumably males and potential streakers. In addition, im- mediately before spawning, males sometimes exhibit a behavior called "looping" (see Thresher, 1984, for a de- scription and drawing). Looping consists of the TP male going through a series of quick rushes upward near the spawning site. Looping has traditionally been thought of as a courtship behavior, but appears to have a second function of exposing potential streakers in H. bivattatus. In several instances, IP individuals streaked while the male was looping, and were immediately chased by the male. Thus, looping appears to be a tactic by territorial males to expose potential streakers before spawns. Although there is convincing evidence that males at- tempt to exclude streakers, there is no convincing evidence that females alter their behavior to increase their fertil- ization rate by increasing the probability of successful streaking. In the Mediterranean wrasse, Sytnphodus tinea. females avoid nesting males with large numbers of pe- ripheral males (van den Berge et ai, 1989). Fertilization rates of the demersal eggs were near 1 00% and did not differ for nests with and without frequent spawning by peripheral males, so spawning site selection did not appear to be based on differences in fertilization rate (van den Berge et ai. 1989). In the seabass, Serranus fasciatus, Petersen (1987) in- terpreted hesitation by female-role spawners before be- ginning the spawning rush as a behavioral tactic to reduce streaking. When the spawning partner hesitated, males often broke off spawning and patrolled the spawning area, chasing any individuals that were close enough to the spawning location potentially to streak. However, the in- creased fertilization rate for spawns with streakers in //. bivattatus suggests a different interpretation for this be- 236 C. W. PETERSEN havior. Hesitations by females before beginning the spawning rush in tropical reef fishes could be a female mating tactic to increase streaking rate, and not a female response to imperfect positioning by the TP male prior to spawning or a tactic to reduce streaking rate. This hy- pothesis needs to be tested with comparative data, in- cluding species with and without streaking males. Females in H. hivattatus often hesitate several times before ulti- mately spawning with a TP male (pers. obs.). Despite the possible advantages of female spawning be- havior that increases fertilization rate, these modifications may be highly constrained by predation pressure during spawning. Behaviors that may increase the chance of a nearby male seeing and joining the spawn, such as slowing down the spawning rush, may also increase the vulnera- bility of the female to predators. In two instances during this study, a lizardfish (Synodus sp.) struck at the fish in a pair spawn at the apex of the spawning rush. Although neither attempt was successful, predators may limit the advantages of female behaviors that could increase their visibility to streakers but at the same time make them more vulnerable to predators. Thresher (1984) has noted that many of the observations of reef-fish being preyed upon have occurred during spawning. In another Caribbean wrasse, Thalassoma bifasciatum, fertilization rates do not differ between pair spawns and group spawns (Petersen ct a!., 1992), despite an estimated 80-fold increase in sperm released in group spawns relative to pair spawns. Group spawns typically involve at least five males, and may produce higher levels of turbulence and water mixing, reducing the concentration of gametes faster than in pair spawns. Group spawns may also lack the close juxtaposition of a male and female during gamete release that exists between the pair-spawning male and female with or without streaking (Petersen et ai, 1992). Thus, fertilization rate comparisons between different types of spawns may depend both on the number of males in the spawn and the type of spawn. This difference for spawns with streaking and group spawns compared with pair spawns should be tested for other species to determine its generality. In transforming male mating success into reproductive success, researchers have had to rely on approximations of how fertilizations are divided among males in cases of sperm competition. The approach has varied from as- signing all of the reproductive success to the male using the alternative reproductive pathway due to his higher sperm production and proximity to the female (Gross and Charnov, 1 980) to dividing the fertilization evenly among all males in the spawn (Warner and Hoffman 1980a, b; Petersen 1987, 1990). In all of these studies, fertilization rate was assumed constant. This study shows that addi- tional males alter the fertilization rate, and, although it is not clear how fertilizations are divided among the two types of males, it is clear that our assumptions of how mating success is translated into reproductive success in these fishes needs to be reappraised. These results suggest two avenues for future research in the reproductive biology of tropical reef fishes. First, variation in female mate choice, spawning site selection, and temporal patterns of spawning may be at least in part a response to variation in fertilization rate. Second, the spawning behavior of both males and females needs to be reconsidered as a potential tactic to increase individual fertilization rates. Acknowledgments This study was supported by NOAA grant NURC 90-1 and a Smithsonian Tropical Research Institute post- doctoral fellowship to the author. I would like to thank Sarah Cohen, Helen Hess, and Amy Sewell for help with data collection and Helen Hess and two anonymous re- viewers for making helpful comments on the manuscript. Literature Cited Baird, T. A. 1988. Abdominal windows in straight-tailed razorfish, .\Y- richlhvs maninicensis: an unusual female sex character in a poly- gynous fish. Copeia 1988: 496-499. van den Berge, E. P., F. Wernerus, and R. R. Warner. 1989. Female choice and the mating costs of peripheral males. Anim. Behav. 38: 875-884. Denny, M. \\ ., and M. F. Shibata. 1989. Consequences of surf-zone turbulence for settlement and external fertilization. Am. Nat. 134: 859-889. Gross, M. R., and E. L. Charnov. 1980. Alternative male life histories in bluegill sunfishes. Proc. Nat. Acad. Set. U.S.A. 76: 6937-6940. Hoffman, S. G. 1985. Effects of size and sex on the social organization of reef-associated hogfishes, Bodianus sp. Environ. Bio/ Fish. 14: 185-197. Kuwamura, T. 1984. Social structure of the protoynous fish Labroides dimtdialus Publ. Sew Mar. Biol Lab 29: 117-177. Lejeune, P. 1987. The effect of local stock density on social behavior and sex change in the Mediterranean labrid Com julis. Environ Bid Fish. 18: 135-141. Levitan, D. R. 1989. Life history and population consequences of body size regulation in the sea urchin Diadema antillarnm Phillipi. Ph.D. Dissertation University of Delaware, Newark. Moyer, J. T., and V. Yogo. 1982. The lek-like mating system of Hal- ichivrex melanochir (Pisces: Labridae) at Miyake-jima, Japan. Z. Ticrpsychol. 60: 209-226. Nemtzov, S. C. 1985. Social control of sex change in the Red Sea ra- zorfish Xyrichtys pentadactylus (Teleostei, Labridae). Environ. Biol. f-'uh. 14: 199-211. Pennington, J. T. 1985. The ecology of fertilization of echinoid eggs: the consequences of sperm dilution, adult aggregation, and synchro- nous spawning. Biol. Bull. 169: 417-430. Petersen, C. W. 1987. Reproductive behaviour and gender allocation in Serranus fasciatus, a hermaphroditic reef fish. Anim. Behav. 35: 1601-1614. Petersen, C. \V. 1990. The relationships among population density. individual size, mating tactics, and reproductive success in a her- maphroditic fish, Serranus fasciatus. Behaviour 113: 57-80. FERTILIZATION RATE IN HALICHOERES 237 Petersen, C. W. 1991. Sex allocation in hermaphroditic seahasses. Am. Nat. (in press). Petersen, C. W., R. R. Warner, S. Cohen, H. C. Hess, and A. T. Sewell. 1992. Variation in pelagic fertilization success: implications for production estimates, mate choice, and the spatial and temporal dis- tribution of mating. Ecology (in press). Robertson, D. R. 1972. Social control of sex reversal in coral-reef fish. Science 111: 1007-1009. Robertson, D. R. 1981. The social and mating systems of two lahrid fishes. Halichoeres maculipinna and H. garnoti. off the Caribbean coast of Panama. Mar. Biol. 64: 327-340. Robertson, D. R., and J. H. Choat. 1974. Protogynous hermaphroditism and social systems in labrid fishes. Proceedings Second International Symposium Coral Reefs 1: 217-225. Robertson, D. R., and S. G. Hoffman. 1977. The roles of female mate choice and predation in the mating systems of some tropical labroid fishes. Z Tierpsychol. 45: 298-320. Robertson, D. R., and R. R. Warner. 1978. Sexual patterns in the labroid fishes of the western Caribbean. II: The parrotfishes (Scaridae). Smith. Contrih. Zoo/. 255: 1-26. Ross, R. M. 1986. Social organization and mating system of the Ha- waiian reef fish Thalassoma dupcrrey (Labridae). Pp. 794-802 in Indo-Padfic Fish Biology: Proceedings of the Second International Conference on Indo-Pacific Fishes. T. Uyeno, R. Arai. T. Taniuchi. and K. Matsuura, eds. Ichthyological Society of Japan. Tokyo. Shapiro, D. V. 1989. Sex change as an alternative life-history style. Pp. 177-195 m Alternative Life-history Styles in Animals, M. N. Burton, ed. Kluwer Publishers, Dordrecht, The Netherlands. Thresher, R. E. 1979. Social behavior and ecology of two sympatric wrasses (Labridae: Halichoeres spp.) off the coast of Florida. Mar. Biol. 53: 161-172. Thresher, R. E. 1984. Reproduction in Reef Fishes. T.F.H. Publications. Neptune City. NJ. Tribble, G. W. 1982. Social organization, patterns of sexuality, and behavior of the wrasse Coris dorsomacu/ala at Miyake-jima, Japan. Environ. Biol. Fish. 1: 29-38. Victor, B. C. 1987. The mating system of the Caribbean rosy razorfish, Xyrichthys martinicensis. Bull. Mar. Sci. 40: 152-160. Warner. R. R. 1984a. Mating behavior and hermaphroditism in coral reef fishes. Am. Sci. 72: 128-136. Warner, R. R. 1984b. Deferred reproduction as a response to sexual selection in a coral reef fish: a test of the life historical consequences. Evolution 38: 148-162. Warner, R. R. 1987. Female choice of sites versus mates in a coral reef fish, Thalassoma bifasciatiim. Anim. Behav. 35: 1470-1478. Warner, R. R. 1988. Traditionally of mating-site preferences in a coral- reef fish, \atiire 335: 719-721. Warner, R. R., and S. G. Hoffman. 1980a. Local population size as a determinant of mating system and sexual composition in two tropical reef fishes (Thalassoma spp.) Evolution 34: 508-518. Warner, R. R., and S. G. Hoffman. 1980b. Population density and the economics of territorial defense in a coral reef fish. Ecology 61: 772- 780. Warner, R. R., and D. R. Robertson. 1978. Sexual patterns in the labroid fishes of the western Caribbean, I: The wrasses (Labridae). Smith. Contrih. Zool. 254: 1-27. Warner, R. R., D. R. Robertson, and E. G. Leigh, Jr. 1975. Sex change and sexual selection. Science 190: 633-638. Reference: Biol Hull 181: 238-247. (October. 1991) Spermatophore Diversity Within and Among the Hermit Crab Families, Coenobitidae, Diogenidae, and Paguridae (Paguroidea, Anomura, Decapoda) C. C. TUDGE Zoology Department, University of Queensland, Queensland, 4072. Australia Abstract. The spermatophore morphology of 1 3 species of hermit crab from the families Coenobitidae, Diogenidae and Paguridae is described and illustrated, and compar- isons are made with existing descriptions to show that spermatophore form, at the light microscope level, can be used to separate three families of the Paguroidea. Sper- matophores from members of the family Coenobitidae are robust in nature with large, ovoid-spherical ampullae mounted on short, thick stalks. Members of the family Diogenidae have more fragile spermatophores with small spherical ampullae mounted on long, slender stalks. The spermatophores of members of the family Paguridae are distinctive in possessing large, elongate, ampullae, an ac- cessory ampulla at the base of the main ampulla and a pseudo-stalk analogous with the true stalk of the Coe- nobitidae and Diogenidae. The occurrence of double- headed spermatophores (two ampullae on a single stalk) is recorded for the first time, in a Dardanus species. The ultrastructure of the lateral ridge, which divides the am- pulla of the paguroidean spermatophore into two halves, is described using both scanning and transmission electron microscopy. A simple, branching key for classifying the investigated hermit crabs (from the families Coenobitidae, Diogenidae and Paguridae only) into their respective family, based on the gross morphology of their spermato- phore, is presented. Introduction The infraorder Anomura consists of 13 families of which only 6 have been investigated for spermatophore morphology. Representatives of three hermit crab families in the superfamily Paguroidea (sensit McLaughlin, 1983) Received 12 April 1 99 1 ; accepted 17 July 1991. have been investigated for spermatophore morphology. These are listed below. Coenobitidae: Birgus latro (Lin- naeus), Matthews, 1956; Tudge and Jamieson, 1991. Coenohita rugosus H. Milne Edwards, Matthews, 1956. Diogenidae: Clibanarius misanthropus(R\sso), Mouchet, 1930, 1931. Dardanus arrosor (Herbst), Mouchet, 1931 (as Pagurus arrosor). Dardanus asper (De Haan), Mat- thews, 1953. Dardanus punctulatus (Olivier), Matthews, 1956. Diogenes pugilator (Roux), Mouchet, 1930, 1931. Pagnrisles oculatiis (Fabricius), Mouchet, 193 1 . Aniculus inaxiiniis Edmondson, Triiopagurus maxinuis (Herbst) and T. slrigatits (Herbst), Matthews, 1957 (as Aniculus strigalnx). Paguridae: Anapagurus hyndmanni (Thomp- son), Mouchet, 1930, 1931. Anapagurus brevicarpus A. Milne Edwards and BouvierandA /cms (Bell), Mouchet, 1931. Cestopagurus timidus (Roux), Pagurus anachorelus Risso, P. cuanensis (Thompson), P. excavatus (Herbst) and P. sculptimanus (Lucas), Mouchet, 1931 (all as Eu- pagurus). Pagurus prideaux Leach, Mouchet, 1931; Ha- mon, 1937 (both as Eupagurus prideauxi ). Pagurus bern- hardus (Linnaeus), Mouchet, 1931; Chevaillier, 1970 (both as Eupagurus bernhardus). Pagurus novizealandiae (Dana), Greenwood, 1972 (as P. novae-:ealandiae). Pa- gurus excavatus (Herbst), Schaller, 1979 (as P. meticu- /osus). All of these previous studies of spermatophore structure have been at the light microscope level. Except for Triiopagurus maximus and T. strigatus (Matthews 1957), the hermit crabs previously studied have a typically pedunculate spermatophore that can be divided into three major regions: a sperm-filled ampulla, a colum- nar stalk of variable length, and a foot or pedestal. The ampulla has a partition or line of division that runs around the lateral edge and separates the ampulla into two halves. This suture line is the point of weakness where the ampulla breaks to release the spermatozoa prior to fertilization. 238 HERMIT CRAB SPERMATOPHORE DIVERSITY 239 Earlier pioneers in hermit crab spermatophore mor- phology include Mouchet (1930, 1931) and Matthews ( 1953, 1956, 1957), whose important contributions to this field also covered other decapods. These references, along with many other papers on decapod spermatophores, are adequately tabulated in Dudenhausen and Talbot (1983). Some additional, more recent, references to anomuran and brachyuran spermatophore studies can be found in Mann's (1984) book on spermatophores. Studies on her- mit crab spermatophores have been neglected for the past 10 years, except for a recent review by Hinsch (1991); although some important works on brachyuran (Jeyalec- tumie and Subramoniam, 1989; Subramoniam, 1991) and caridean shrimp (Chow et a/.. 1989) spermatophores have been published. Comparisons of the functional mor- phology of genitalia and subsequent sperm transfer and storage mechanisms among taxa can provide useful in- formation on phylogenetic relationships and evolutionary divergence; especially in the Decapoda (Bauer, 1986, 1991). The present paper describes and illustrates the spermatophore structure of 1 3 species of hermit crab, from 3 families in the Paguroidea, and introduces a simple key to classify these hermit crabs to family, based on light microscope observations of their spermatophores. Materials and Methods Collection sites and dates for the species in this paper are as follows: Family Coenobitidae Birgits latro (Linnaeus 1767), Malaita Island. Solomon Islands, SW Pacific, October, 1988; Coenobita brevimanm Dana 1852, Coenobita perlatus H. Milne Edwards 1837 and Coenobita ntgosus H. Milne Edwards 1837, Suwarrow Atoll National Park, Cook Islands, SW Pacific, August, 1990; Coenobita spinosus H. Milne Edwards 1837, Dar- win, Northern Territory, Australia, May, 1990. Family Diogenidae Calcium latens (Randall 1839), Calcinus mimitus Bui- tendijk 1937 and Clibanarius corallinus (H. Milne Ed- wards 1848), Heron Island, Queensland, Australia, De- cember, 1990; Clibanarius virescens(Krauss 1843). Dun- wich. North Stradbroke Island, Queensland, Australia, April, 1990; Dardanus lagopodes (Forskal 1775) and Dardanus megistos (Herbst 1804), Heron Island, Queensland, Australia, December, 1990; Diogenes gar- dineri Alcock 1905, Mooloolaba, Queensland, Australia, October, 1990. Family Paguridae Pagtints hirtimanus Miers 1880, Heron Island, Queensland. Australia, December, 1990. The male reproductive system was dissected from fresh specimens, 10% buffered formalin-fixed or 3% glutaral- dehyde-fixed specimens. The spermatophores were teased out of the distal part of the vas deferens onto microscope slides. Spermatophores were viewed and photographed with an Olympus BH2 microscope equipped with No- marski interference contrast optics. After the initial glu- taraldehyde fixation and first phosphate buffer wash, the fixation procedure for transmission electron microscopy was carried out in a Lynx-el. Microscopy Tissue Processor. Portions of the vas deferens were fixed in 3% glutaralde- hyde in 0.2 M phosphate buffer (pH 7.2) for 1 h at 4C. They were washed in phosphate buffer (3 washes in 15 min), postfixed in phosphate buffered 1% osmium tetrox- ide for 80 min; similarly washed in buffer and dehydrated through ascending concentrations of ethanol (40-100%). After being infiltrated and embedded in Spurr's epoxy resin (Spurr, 1969), thin sections (50-80 nm thick) were cut on a LKB 2128 UM IV microtome with a diamond knife. Sections were placed on carbon-stabilized colloidin- coated 200 ^m mesh copper grids and stained in 6% aqueous uranyl acetate for 40 min; rinsed in distilled wa- ter; stained with Reynold's lead citrate (Reynolds, 1963) for 20 min; and further rinsed in distilled water. Micro- graphs were taken on a Hitachi 300 transmission electron microscope at 80 kV. For scanning electron microscopy, 3% glutaraldehyde- fixed spermatophores were dehydrated through a graded series of ethanol (80-100%) at 10 min intervals. They were then dried after being placed in a drop of ether and sputter coated with gold; micrographs were taken on a Philips 505 scanning electron microscope at 20-30kV. Results Family Coenobitidae The tripartite, pedunculate spermatophores of Birgits latro are approximately 650 nm in length from pedestal to apex of the ampulla. The sperm-filled ampulla is 440 fj.m long and 450 ^m wide and forms an inverted heart- shape (Fig. 1A). The stalk of the spermatophore is ap- proximately 100 /urn in length. The spermatophore is lat- erally wider than deep, and therefore is slightly spatulate and is composed of two halves, which meet at the lateral edge as a raised ridge (Fig. 3 A). This ridge is 20 ^m thick while the remaining spermatophore wall is only 10 nm thick. Under a double Mallory's staining procedure (Mai- lory, 1936) the spermatophore wall is shown to be com- posed of two layers: an inner, darker staining layer, which is discontinuous at the ridge, and an outer, lighter staining layer, which covers the entire ampulla and increases in thickness at the ridge. The pedunculate spermatophores of Coenobita spinosus are similar in shape to those of Birgus latro but much 240 C. C. TUDGE Coenobita rugosus ^'^--' Coenobita spinosus Coenobita J brevimanus Coenobita perlatus Clibanarius corallinus Clibanarius virescens Diogenes gardineri urus hirtimanus M HERMIT CRAB SPERMATOPHORE DIVERSITY 241 smaller (Fig. IB). Each individual spermatophore is ap- proximately 170 jum in length from base of the pedestal to the top of the ampulla. The ampulla, which is approx- imately 1 10 ^m long, is composed of two halves that join at a conspicuous lateral ridge. The stalk of the spermato- phore is only 20 j/m long. The width of the ampulla from ridge to ridge is approximately 1 10 nm, while the depth of the ampulla is only 100 ^m. This gives the ampulla a slightly spatulate shape, with the ridge running around the lateral edge. The ampulla is surrounded by a sper- matophore wall 2 ^m in thickness, which increases to 3 nm at the ridge, and is filled with many closely packed spermatozoa, separated by an extracellular matrix. Ultra- structural studies show the ridge to be a break in the fi- brillar structure of this wall (Fig. 3C). The spermatophores of Coenobita nigosus are larger than those of Coenobita spinosus. being 190 ^m long, with an ampullar length and width of 150 and 100 ^m. respectively. The ampulla, which is slightly more elongate than either Birgus latro or Coenobita spinosus, sits on a stalk 32 urn high (Fig. 1C). Coenobita brevimamis has a spermatophore 290 /urn in length which is composed of a stalk that is 90 nm long and an ampulla that is 180 ^m long and 1 10 jum wide. Although the ampulla is larger than that of Coenobita rugosus, its shape is very similar (Figs. ID, 2 A). The spermatophore of Coenobita perlatus is the largest of the four species of Coenobita studied. At 600 ^m in length, it is only slightly smaller than the spermatophore of Birgus latro (Fig. 1A). The ampulla, 400 nm long and 230 ^m wide, is similar in shape to those of Coenobi