MBL Volume 1 79 THE Number 1 BIOLOGICAL BULLETIN AUGUST, 1990 Published by the Marine Biological Laboratory THE BIOLOGICAL BULLETIN PUBLISHED BY THE MARINE BIOLOGICAL LABORATORY Editorial Board GEORGE J. AUGUSTINE, University of Southern JOHN E. HOBBIE, Marine Biological Laboratory California GEORGE M. LANGFORD. LIniversity of RUSSELL F. DOOLITTLE, University of California North Carolina at Chapel Hill at San Diego Louis LEIBOVITZ. Marine Biological Laboratory WILLIAM R. ECKBERG. Howard University ROBERT D. GOLDMAN, Northwestern University RUDOLF A. RAFF, Indiana University EVERETT PETER GREENBERG, Cornell University K.ENSAL VAN HOLDE, Oregon State University EJitur: MICHAEL J. GREENBERG, The Whitney Laboratory. University of Florida Managing Editor: PAMELA L. CLAPP. Marine Biological Laboratory AUGUST, 1990 Printed and Issued by LANCASTER PRESS, Inc. PRINCE &. LEMON STS. LANCASTER, PA BIOI.OCICAI. 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 BULLETIN, Marine Biological Laboratory, Woods Hole. Massachusetts 02543. Single numbers, $25.00. Subscription per volume (three issues), $57.50 ($ 1 15.00 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 *"> O O '0 s ~~ ^i Tf U~i ON OO K s a- o u-i ri oo i OO r>l r) > r i Tf r', r*-, rt c O 4 8~T r~~- O r-j) O 1^- OO ' ~i r*~t oo O oo r^ [^ m -t r i C 1 ir. i^, ri r i r~" ON <^"i OO C' \O *f <~ 1 r-l \O 'Vi O T 2; O ri ri Tf c r-l ri o oo fj Tf oo Tf ri u*, >O ri o oo -f r- ON Tf a- r i r- ' Tf r Tf Tf rr-j r| r | O s u^ O 5s 9- UJ O p. 3 3 <^ ( , , a 5 - 1 g o -o _o ffl & S Q ^^ z Z c y 1 -si i 1 -Hill i _l z- -^ t? ^ ^ w o y o ' o c "7 O ' u e 'So 00 w S* V IS ~ C -rj U S X t^ C j. n, QJ 2 E ^ E E OJ ~ o c -r - -2 Z ? coco -' J wi '-Z o C O C - ^- TD 3j "O 1> S a C Jf ||| r; T3 E ( i 00 2 S O ^ J 0Q.C O rt~^^ S 2- ^ '.5 1 | .8 J < | || | ||| ^ ! i ? I x i - e > ^ O ^ O i 1 S 1 1 1 "S 5 - ^ Ouj aw ails -2 i 1 1 *S c 5J C 3 ai &. ~ ?2 oo "*- d ^8 * O ^ O ri 'OO a z || r> <; c J 1 ?' ? ^ pfj J^ ! oo -^ -t ri oo ON -J < o D. O m X E te oa S uj .e Z " -T ^r oo oo Tf a-. O ~ o -t oo O r- yn O Tf -f ~. o\ ^ ^; ^ ' < ^ Tf S -t r'l ' a; O ri ri 00 Ov 2 I I -a o Z U c CQ O u o Z "O C "rt t'-] "P O o 1/5 C LU U a c O 5 Z PJ ^ E o UJ Z Lu Ji Di - 1 Qi o ^ O T3 c ti c_ a. U. t/3 C U. h 3 O i u Z to LU C S c LU ' H - \o r I r I l ON oo' ^1- * r-' ON" -c m t oo oo so ri o o o u s *J D. c/i UJ 0> "1 C O 6 o Z o o 3 LU _ ^ ^ *^ c LU c u Oi Q S -5 c o G3 o W5 Z j-^ ' J^ .i UPPORTA E "S pd Recovery o researc Tuition Support acl 1 00 '= I a a II) * -r' rl" O r\ r~~ "^ ' ~T <~ 1 O "T O r ] O >T1 Tf oo v~> f -f Ch oo - OO O *"*"' ""O <"*"i "-" f 1 f*"' *"*"' *~~~ '*& " s "/~> f i r~- <"*~i r** t *^ ^o o^ "^ r~- s ~~ oo oo o in r- o oo o * >/", rn r^i O r i r- i-r, r- oo r*-, oo ^ -t l /"i ^O -t - - r i * o o <*-( ^ r i /~i o ^-' r-i ri r- O C a c a o O. a P C D. C ^ ^_ ra 1 s 1 esources c o o a o c Q c 0. .C ?* tn c. XPENSES Instructio Research Scholarsh Support a Dormit 00 c c 5 ',"] Cq f" Marine Admin c a C/3 o c a. Depreciat Other _ 11 oi 1_ rt accountin] ids ( Note 1 fl W PI-. c ,5 4J >i ances ^_ O 00 C a OJ I OJ 00 c 73 | U O OO c rt u >-, S c uJ T3 t*~ CA 'c c u- ~3 '5b -^' o ^ 'oh -^ J ^ 3 1 C - " C o> o l> O C J3 c rt ^ ,'J M c cd C3 ^r u 73 o C cd 3 E T3 C ^ o c 3 a D 3 H z U- O u. U. a & o c cm c a E o H 12 Annual Report Marine Biological Laboratory Notes to Financial Statements A. Purpose of the Laboratory: 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 at 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 accompany- ing 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 L). 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 endow- ment 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 L). 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 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 $4,039,803 and $3,551,482 at December 31, 1989, and 1988. respectively. Investment income and 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 M). 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 nineteen years, and interest payout rate of 8%. C. Lund, buildings, and euiiipmcnl: The following is a summary of the unrestricted plant fund assets: 1989 1988 Land $ 840,594 $ 689,660 Buildings 16,926.715 16,694.233 Equipment 2,521.904 2,299,094 Construction in progress 580.598 20,869.811 19.682,987 Less accumulated depreciation (8.858.047) (8.268.927) $12.011.764 $11.414,060 Notes to Financial Statements 13 D. Rclircnu-nl luiui On May 23. 1489, the Laboratory terminated its noncontributory defined benefit pension plan, which covered substantially all employees. Benefits earned by employees under the terminated plan became fully vested and were distributed to plan participants. Net pension cost for fiscal year ending December 31.1 989 was: Service cost $ 27,041 Interest cost 173,227 Actual return on plan assets (296.846) Net amortizations and deferrals 82.291 Net periodic pension income $ (14.287) On February 17. 1989, in anticipation of the plan termination, effective May 23, 1989, the Laboratory froze future benefit accruals. The accumulated benefit obligation of $2.569.454 was settled through the purchase ot non-participating annuity contracts and distribution ol lump sum settlements. Because all excess assets were allocated among the participants, the Laboratory recognized no curtailment gain or loss. There was a settlement gain of $289.650 attributable to amounts previously accrued by the Laboratory and the plan ceased to exist as an entity. A portion of the settlement gain of $192.285 was used to account for separation agreements with certain current employees. The balance of the settlement gain. $97,365 was returned to grants and contracts. The Laboratory participates in the defined contribution pension program of the Teachers Insurance and Annuity Association. Expenses amounted to $393.422 in 1989 and $130.677 in 1988. E. Rcstriclctl /'/ctto'.v uinl grants: As of December 3 1 . 1989. the Laboratory reported active pledge and grant commitments outstanding of $ 1 ,073,986 (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 $978,786 and $95.200 are scheduled to be paid in 1 990 and 1 99 1 . respectively. F. line: luiul hi immings: Current fund interfund balances at December 3 1 are as follows: 19SV Due to restricted endowment fund $(2.190) $(31.600) Due to restricted quasi-endowment funds (200) $(2,340) $(31,600) G. Long-term debt at December 31.1 989 amounted to $ 1 .330,000. The aggregate amount of redemption due for each of the next five fiscal years is as follows: Aniintnl 1990 $ 65.000 1991 65,000 1992 60.000 1993 60,000 1994 60,000 Thereafter 1 .020.000 1.330.000 Less current portion 65.000 $1.265,000 During 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 3 1 , 20 1 I The bonds are payable annually with the first payment of $65,000 due October 1 , 1 990. The interest rate is adjustable and was 6.5% at December 31. 1989. In compliance with the Laboratory's MIFA bond indenture, deposits with Shawmut Bank, as trustee, represent investments in the debt service reserve fund of $ 1 33.000. The Series 1 989 bonds are collateralized by a first mortgage on certain Laboratory property. H. The following is a summary of the cost and market value of investments at December 31,1 989 and 1 988 and the related investment income and distribution of investment income for the years ended December 31. 1989 and 1988. 14 Annual Report Investment Incumc Endowment and mid^i- U.S. Government securities Corporate tixed income Common slocks Preferred stock Money market securities Real estate Total Less custodian and management fees Total Restricted current fund Certificates of deposits Money market securities Total Total investments , 2.^95,407 5.900.736 3.392.001 595.467 345.749 12.829.360 490.263 965.000 1.455.263 $14.284.623 1988 1.328.927 3.124,493 3.717,850 916.280 15.749 9.103.294 638,603 1.750.000 2.388.603 $1 1.491,902 1989 b 2.607,537 6,032,642 5.901.724 593,544 345.749 15.481.196 490,263 965.000 1.455.263 $16.936.459 1988 i 1.323.105 3.131.404 5.375.980 916,280 15.749 10.762,518 638,603 1.750.000 2.388.603 $13,151,121 19K9 1988 $134,394 $ 95.234 363.439 257,692 196.452 211.319 250 87,994 49,995 782,279 (49,318) 614.490 (44,073) 732.961 570,417 34.785 175,205 38.603 66.248 209,990 104,851 $942.951 $675.268 I. Gill rl /or in\tn/fln>n Unrestricted gifts includes $406.524 of gifts for the support of the Laboratory's instruction program available for indirect costs attributable to the instruction program. LiliKcilion: The Laboratory is involved in litigation on several matters and is subject to the possibility of certain claims arising in the normal course of business, none of which, in the opinion of management are expected to have a materially adverse effect on the Laboratory's financial position. K. The Laboratory is exempt from federal income tax under Section 501(c)3 of the Internal Revenue Code. L. Change in McouniniK methttd tr current rcsinclcJ /iimlx Effective January 1. 1989, the Laboratory adopted the accounting policy of deferring recognition of revenue on current restricted funds until the related costs are incurred. Amounts received in excess of expenses are recorded as deferred support. This change has been retroactively applied to the fund balances as of January 1. 1988. The cumulative decrease in the fund balances was $4.450.222 and $2,951,662 at December 31, 1989 and 1988, respectively. The following summarizes the activity in the deferred support account for 1 989 and 1988, respectively: Balance at beginning of year Additions: Gifts, endowment income and grants received Unrealized gains Deductions: Funds expended under gifts and grants Transfers Balance at end of year 1989 $2.951.662 9.382.212 30.672 7.542.909 371.415 $4,450.222 1988 $1.653,334 7,298,318 6,047.936 (47.946) $2.951,662 M. .tccounlini; /or pooled invcMmcnlx: The major portion ot 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. 1989 is as follows: Endowment and similar funds: Quasi-unrestricted Quasi-restricted Restricted endowment 19S9 3.975 7,551 37.220 48.746 Pooled investment activity on a per-unit basis was as follows: Unit value at beginning of year Unit value at end of year Increase in realized and unrealized appreciation Net income earned on pooled investments Total return on pooled investments $100.00 107.42 7.42 5.61 $ 13.03 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 vear ended December 31,1 989 SUPPORT AND REVENUES: Grant reimbursement of direct costs Change in deferred support Recover. 1 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 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 TRANSFERS AMONG FUNDS: Debt service Acquisition of fixed assets Net transfer to restricted plant fund Transfers to unrestricted plant fund Housing transfer Endowment transfers Instruction transfer Capitalize ecosystems income Other transfers Total transfers among funds Net change in fund balances Fund balances, beginning of year Fund balances, end of vear Fund $2.948.4() George Grice, WHOI John W. Speer* Gary Walker, WHOI Library Joint Users Garland Allen, Chairman Henry Dick, WHOI A. Farmanfarmaian Lionel Jaffe Catherine Norton* John Teal, WHOI Geoff Thomson, WHOI Page Valentine, USGS Carole Winn*. WHOI Marine Resources Robert Goldman, Chairman 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 Paul De Weer Louis M. Kerr* Andrew Mattox* Walter Vincent Research Services Birgit Rose. Chairman Peter Armstrong Robert B. Barlow Jr. Richard Cutler* Barbara Ehrlich Ehud Kaplan Samuel S. Koide Aimlee Laderman Andrew Mattox* Bryan Noe Bruce J. Peterson Rudi Strickler 52 Annual Report Research Space Steven Treistman Ivan Valiela Joseph Sanger, C hairman Clay Armstrong Ray L. Epstein* Safety Leslie D. Garrick* John Hobhie, Chairman David Landowne ^ c D. Eugene Copeland Hans Lauler D . , , , Richard Cutler* Laszlo Lorand c . , c * Edward Enos* Eduardo Macagno . ., ..... Louis kerr* Jerry M.MeIiUo Alan Kuzinan o an c, K f man Donald B. Lehv* Roger Sloboda . ,, ^ Andrew Mattox* Paul Steudler Tom Fisher Laboratory Support StafT Biological Bulletin Clapp. Pamela L.. Managing Editor Puckctt. Kathryn C'oniroller's Office Speer, John W., Controller Accounting Services Binda, Ellen F. Campbell, Ruth B. Davis, Doris C. Gilmore, Mary F. Godin, Frances T. Goldsmith, Ruth E. Hohhs, Roger W., Jr. Hough. Rose A. Oliver. Elizabeth Poravas. Maria Chem Room Chisholm, Caroline G. Miller, Lisa A. Sadowski. Edward A. Computer Services Tollios. Constantine Purchasing Evans. William A. Hall, Lionel E., Jr. * Including persons who joined or left the stafl'during 1989. Copy Service C'cnler Gibson. Caroline F. Jackson, Jacquelyn F. Mountford. Rebecca J. Ridley, Sherie Devcloi'iiicin Office Ayers. Donald E., Director Berthel. Dorothy Lessard. Kelley J. O'Hara. Aqua Thimas, Lisa M. Director's O/fice Halvorson, Harlyn O., President and Director Epstein, Ray L. Kinneally, Kathleen R. Watkins, Joan E. Gray Museum Bush, Louise, Curator Armstrong, Ellen P. Montiero. Eva Housing King. LouAnn D.. Conference Center and Housing Manager Crocker, Susan Farrell, Bernice R. Gomes, Susan A. Gross, Laura F. Johnson, Frances N. Klopfer, (Catherine Krajewski, Viola I. Kuil, Elisabeth Lewis, Shirley A. Mancevice, Denise M. McNamara, Noreen Potter, Maryellen Sadovsky. Sebastian Telephone Office Baker, Ida M. Geggatt, Agnes L. Ridley, Alberta W. Human Resources Goux, Susan P., Manager Donovan, Marcia H. Lihrtiry Fessenden, Jane, Librarian Norton, Catherine N., Acting Librarian Ashmore. Judith A. Costa, Marguerite E. Mirra. Anthony J. Mountford, Rebecca J. Nelson, Heidi Page, Joel Page, Kristin Pratson, Patricia G. Tamm. Ingrid deVeer, Joseph M. 53 54 Annual Report MRL Associates Liaison Scanlon. Deborah Puhlic Inl'imnaiiim Office Liles, George W.. Jr.. Director Anderson, Judith L. Stone, Beth R. Radiation Safer]' 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 Sen-ices. Projects, and Facilities Cutler, Richard D.. Manager Buildings and Grounds Lehy. Donald B., Superintendent Allen, Wayne D. Anderson, Lewis B. Baldic, David P. Blunt, Hugh F. Boucher, Richard L. Bourgoin, Lee E. Carini, Robert J. Carr, Edward T., Jr. Collins, Paul J. Conlin, Henry P. Conlin, Mary E. Dunne, James Fish, David L., Jr. Fuglister, Charles K. Gibbons, Roberto G. Gonsalves, Walter W., Jr. Hathaway, Peter Jones. Leeland Justason, C. Scott Klinger. Michael Krajewski, Chester J. Lochhead, William M. Lunn, Alan G. Lynch. Henry L. MacLeod, John B. McAdams, Herbert M.. Ill Mills. Stephen A. Rattacasa, Frank D. Rossetti, Michael F.. Jr. Schoepf, Claude deVeer, Robert L. Ward. Frederick Weeks, Gordon W. Wilson, Mitchell J. Electron Microscopy Lab Kerr, Louis M. Machine Shop Sylvia. Frank E. Marine Resource* Center Valois, John J.. Manager Enos, Edward G., Jr. Enos, Joyce B. Fisher. Harry T.. Jr. Hanley, Janice S. Moniz. PriscillaC. Revellese, Christopher Sullivan. Daniel A. Tassinari, Eugene Torres, Sophie J. Photolab Colder, Linda M. Colder, Robert J. Rugh, Douglas E. Sponsored Programs Gamck, Leslie D.. Assistant Administrator Dwane, Florence Hurler. Linda Lynch. Kathleen F. Tilghman, Alison Animal Care Facility Hanley. Janice S. Povio, Sandra C. Shephard. Jennifer 7 V.S'9 Summer Supiwrt Staff Albrecht, Helen Allen. Tania L. Amon, Tyler C. Ashmore, Lynne E. Avery, Deirdre Ayers, Andrew D. Beetlestone, Linda Bolton, Hugh Buckley, Joseph Burgess. Kristin Burke. Sean Capobianco. James A. Chen, Chong Child. Malcolm S. Cishek. Dawn Costa, Christopher Cullen, Timothy DeGiorgis, Joseph Dickerson, Catherine Dickerson, Veronica Dodge. Michael F. Dodge, Susan A. Donovan, Jason P. Dooley, Kimberly A. Frye. Jennifer Grassle, John T. Grimes, Jeffrey Grossman, Howard Hallock. David Hamilton. Elizabeth R. Hill. Evan S. Horowitz. Rachel Illgen, Robert C. Jones, Leeland A. Kinneally. Kara J. Langton. Lori Loebach, Mary Marini, Michael F. Milliken, Sally T. Northern, Marc D. Peal, Richard W. Percy. Mary R. Raab. Michael M. Redlich, Sanford Remsen, Andrew S. Romeo, Denise E. Rook. Kellyann Schauer, Caroline L. Shaw, Trevor P. Snyder, Rebecca Sohn, Marcus J. Strout. Matthew P. Swope, John G. Swope. Nathaniel I. Teixeira, Anita Ulbrich. Ciona Valois, Francis X. Varao, John Villalobos, Maria T. Wetzel, Ernest D. Whitehead, Glen C. Winspear, David A. Members of the Corporation* Life Members Abbott, Marie, c/o Vaughn Abbott. Flyer Rd.. East Hartland. CT 06027 Beams, Harold \\ '., 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. IL61701 Bishop, David \V., Department of Physiology, Medical College of Ohio, C. S. 10008. Toledo. OH 43699 (deceased) Bodian, David, 4100 North Charles St., #9 1 3. Baltimore. MD 21218 Bold, Harold C., Department of Botany, University of Texas, Austin. TX 78712 (deceased) Bridgman. A. Josephine, 7 1 5 Kirk Rd., Decatur. GA 30030 Buck, John B., N1H. Laboratory of Physical Biology. Room 1 12, Building 6 Bethesda, MD 20892 Burbanck, Madeline P., Box 1 5 I 34, Atlanta, GA 30333 Burbanck, William D., Box 1 5 1 34, Atlanta. GA 30333 Carpenter, Russell L., 60- H Lake St.. Winchester. MA 01890 Chase, Aurin, Department of Biology, Guyot Hall. Princeton University, Princeton, NJ 08544 Clark, Arnold M., 53 Wilson Rd.. Woods Hole, MA 02543 Cohen, Seymour S., 10 Carrot Hill Rd., Woods Hole, MA 02543-1206 Colvvin, Arthur, 320 Woodcrest Rd.. Key Biscayne. FL 33 149 Colvvin, Laura Hunter, 320 Woodcrest, Key Biscayne, FL 33149 Copeland, D. E., 41 Fern Lane, Woods Hole, MA 02543 Costello, I lelen M., Carolina Meadows, Villa 1 37, Chapel Hill, NC 275 1 4 Crouse, Helen, Institute of Molecular Biophysics, Florida State University, Tallahassee, FL 32306 Failla, Patricia M., 2149 Loblolly Lane, Johns Island, SC 29455 Ferguson, James K. W., 56 Clarkehaven St., Thornhill, Ontario L4J 2B4 CANADA Fries, Erik F., 4 1 High Street, Woods Hole, MA 02543 Goldman, David, 63 Loop Rd., Falmouth, MA 02540 * Including action of the 1989 Annual Meeting. 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 University. St. Louis. MO 63 1 30 Hamilton, Hovuird L., Department of Biology, University of Virginia, 238 Gilmer Hall, Charlottesville. VA 22901 I larding, Clifford V., Jr., Wayne State University School of Medicine. Department of Ophthalmology, Detroit, MI 4820 1 Haschemeyer, Audrey E. V., 21 Glendon Road, Woods Hole. MA 02543 I luuschka, Theodore S., FD 1 , Box 78 1 , Damariscotta. ME 04543 Ilisaw, F. L., 5925 SW Plymouth Drive. Corvallis, OR 97330 Hollaender. Alexander, Council for Research Planning, 1717 Massachusetts Ave. NW. Washington, DC 20036 Hubbard, Ruth, 2 1 Lakeview Avenue, Cambridge, MA 02 1 38 Humes, Arthur G., Marine Biological Laboratory, Woods Hole, MA 02543 Johnson, Frank H., Department of Biology, Princeton University. Princeton. NJ 08540 Kaan, Helen \V., Royal Megansett Nursing Home. Room 205, P. O. Box 408, N. Falmouth, MA 02556 Karush, Fred, Department of Microbiology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104- 6076 Kille, Frank R., I 1 1 1 S. Lakemont Ave. #444, Winter Park. FL 32782 Kingsbury, John M., Department of Plant Biology, Cornell University, Ithaca, NY 14853 Kleinholz, Lewis, Department of Biology. Reed College, 3203 SE Woodstock Blvd., Portland, OR 97202 Laderman, Ezra, P. O. Box 689. 18 Agassiz Road. Woods Hole. MA 02 543 Eauffer, Max A., 1273 Folkstone Drive, Pittsburgh, PA 15243 LeFevre, Paul G., 1 5 Agassiz Road, Woods Hole, MA 02543 Levine, Rachmiel, 2024 Canyon Rd., Arcadia, CA 91006 Lochhead, John H., 49 Woodlawn Rd., London SW6 6PS, England, UK Loevvus, Frank A., Washington State University, Institute of Biological Chemistry. Pullman, WA 99 1 64 Lynn, W. Gardner, Department of Biology. Catholic LIniversity of America, Washington, DC 200 17 56 Annual Report Magruder, Samuel R., 270 Cedar Lane, Paducah, KY 42001 Manwell, Reginald D., Syracuse University, Lyman Hall. Syracuse. NY 13210 Mathews, Rita \\ ., Box 131, Southfield, MA 1259 Miller, James A., 307 Shorewood Drive, E. Falmouth. MA 02536 Moore, John A., Department of Biology, University of California. Riverside. CA 9252 1 Moscona, Arthur A., University of Chicago. Department of Molecular Genetics and Cell Biology. 920 East 58th Street. Chicago, IL 60637 Moul, E. T., Woodbriar, 339 Gifford St., Falmouth, MA 02540 (deceased) Mullins, LorinJ., University of Maryland School of Medicine, Department of Biophysics, Baltimore. MD 21201 Nace, Paul F., P. O. Box 529, Cutchogue. NY 1 1935 Page, Irving H., Box 516. Hyannisport. MA 02647 Pollister, A. W., 8 Euclid Ave.. Belle Mead, NJ 08502 Prosser, C. Ladd, Department of Physiology and Biophysics, Burrill Hall 524. University of Illinois. Urbana. IL 6 1 80 1 Provasoli, Luigi, Via Stazione 43, 2 1025 Comerio (VA), Italy Prytz, Margaret McDonald, Address unknown Renn, Charles E., Route 2, Hempstead, MD 2 1074 Richards, A. Glenn, 942 Cromwell Ave., St. Paul, MN 55 1 1 4 Richards, Oscar W., Route 1 , Box 79F, Oakland, OR 97462 (deceased) Rockstein, Morris, 600 Biltmore Way, Apt. 805, Coral Gables, FL 33 1 34 Ronkin, Raphael R., 3212 McKinley St., NW, Washington. DC 200 15 Sanders, Howard, Woods Hole Oceanographic Institution. Woods Hole, MA 02543 Scharrer, Berta, Department of Anatomy. Albert Einstein College of Medicine. 1300 Morns Park Avenue, Bronx, NY 10461 Schlesinger, R. Walter, University of Medicine and Dentistry of New Jersey. Department of Molecular Genetics and Microbiology. Robert Wood Johnson Medical School, Piscataway, NJ 08854-5635 Schmitt, F. O., Room 16-512, Massachusetts Institute of Technology, Cambridge, MA 02 1 39 Scott, Allan C., 1 Nudd St., Waterville, ME 04901 Shemin, David, 33 Lawrence Farm Rd., Woods Hole, MA 02543 Shilo, Moshe, The Hebrew University, Division of Microbial and Molecular Biology, 91904 Jerusalem, Israel (deceased) Silverstein, Arthur M., The Johns Hopkins Hospital Wilmer Institute. Baltimore, MD21205 Smith, Homer P., 8 Quissett Ave., Woods Hole. mA 02543 Smith, Paul F., P. O. Box 264, Woods Hole, MA 02543 Sonnenblick, B. P., 5 1 5A Heritage Hill, Southberg. CT 06488 Steinhardt, Jacinto, 1 508 Spruce St., Berkeley, CA 94709 Stunkard, Horace W., American Museum of Natural History. Central Park West at 79th St., New York, NY 10024 (deceased) Taylor, Rohert F.., 20 Harbor Hill Rd., Woods Hole, MA 02543 Taylor, W. Randolph, The Herbarium, North University Bldg., University of Michigan. Ann Arbor, MI 48109 Taylor, \V. Rowland, 152 Cedar Park Road. Annapolis. MD 21401 (deceased) TeVVinkel, Lois E., Rockridge. 25 Coles Meadow Road, Northampton, MA 01060 Trager, William, The Rockefeller University, 1230 York Ave.. New York, NY 10021 Villee, Claude A., Harvard Medical School. Parcel B/Room 122. 25 Shattuck Street. Boston. MA 02 1 1 5 Wald, George, 2 1 Lakeview Ave.. Cambridge, MA 02 1 38 Waterman, T. H., Yale University, Biology Department. Box 6666. New Haven. CT 065 1 1 Weiss, Paul A., Address unknown Wichterman, Ralph, 3 1 Buzzards Bay Ave., Woods Hole, MA 02543 VV'iercinski, Floyd J., Department of Biology, Northeastern Illinois University, Chicago, IL 60625 Wilber, Charles G., Department of Zoology, Colorado State University, Fort Collins, CO 80523 Young, D. B., 1 137 Main St.. N. Hanover. MA 02339 (deceased) /.inn, Donald J., P. O. Box 589. Falmouth, MA 0254 1 Zorzoli, Anita, 18 Wilbur Blvd.. Poughkeepsie, NY 12603 /weifach, Benjamin W., 88 1 1 Nottingham Place, La Jolla, CA 92037 Regular Members Abt, Donald A., University of Pennsylvania School of Veterinary Medicine, 3800 Spruce Street, Philadelphia, PA 19104-6044 Acheson, George H., 25 Quissett Ave., Woods Hole, MA 02543 Adams, James A., Department of Natural Sciences, University of Maryland, Princess Anne, MD 21853 Adelberg, Edward A., Provost's Office, 1 1 5 Hall of Graduate Studies, Yale University, New Haven. CT 06520 Adelman, William J., Jr., NIH, Bldg. 9, Rm. IE- 127, Bethesda, MD 20892 Afzelius, Bjorn, Wenner-Gren Institute, University of Stockholm. Stockholm, Sweden Alberte, Randall S., Department of Molecular Genetics and Cell Biology. University of Chicago, 1 103 E. 57th Street, Chicago. IL 60637 Alkon, Daniel, Laboratory of Cellular and Molecular Neurobiology, NINDS/NIH, Bldg. 5, Rm. 417, Bethesda. MD 20892 Allen, Garland E., Department of Biology, Washington University, St. Louis, MO 63 104 Allen, Nina S., Department of Biology, Wake Forest University, Box 7325, Winston-Salem, NC 27 109 Regular Members 57 Allen, Suzanne T., Department of Medical Oncology, Boston University Medical Center, 75 E. Newton Street, Boston, MA 02 11 8-2393 Amatniek, Ernest, 4797 Boston Post Rd., Pelham Manor, NY 10803 Anderson, Kverett, Department of Anatomy & Cell Biology, LHRBB. Harvard Medical School, 45 Shattuck St., Boston, MA 021 15 Anderson, J. M., 1 10 Roat St., Ithaca, NY 14850 Armet-Kibel, Christine, Biology Department, University of Massachusetts-Boston, Boston. MA 02125 Armstrong, Clay M., Department of Physiology. Medical School. University of Pennsylvania. Philadelphia. PA 19104 Armstrong, Peter B., Department of Zoology, University of California, Davis, CA 956 1 6 Arnold, John M., Pacific Biomedical Research Center, 209 Snyder Hall, University of Hawaii. Honolulu, HI 96822 Arnold, William A., 102 Balsam Rd.. Oak Ridge, TN 37830 Ashton, Robert W., Gaston Snow Beekman and Bogue. 666 5th Avenue, 3 1st Floor, New York, NY 10005 Atema, Jelle, Marine Biological Laboratory, Woods Hole, MA 02543 Atwood, Kimball C., Ill, P. O. Box 673, Woods Hole, MA 02543 Augustine, George J., Section of Neurobiology, Department of Biological Sciences, University of Southern California. Los Angeles, CA 90089-037 1 Austin, Mary L., 506 1/2 N. Indiana Ave.. Bloomington, IN 47401 Ayers, Donald E., Marine Biological Laboratory, Woods Hole, MA 02543 Baker, Robert G., Department of Physiology and Biophysics, New York University Medical Center. 550 First Ave., New York, NY 10016 Baldwin, Thomas O., Department of Biochemistry and Biophysics. Texas A&M University, College Station, TX 77843 Bang, Betsy, 76 F. R. Lillie Rd.. Woods Hole, MA 02543 Barlow, Robert B., Jr., Institute for Sensory' Research. Syracuse LIniversity, Merrill Lane. Syracuse. NY 13244- 5290 Barry, Daniel T., Department of Physical Medicine and Rehabilitation, ID204, University of Michigan Hospital. Ann Arbor, MI 48 109-0042 Barry, Susan R., Department of Physical Medicine and Rehabilitation, ID204, University of Michigan Hospital. Ann Arbor. MI 48109-0042 Bartell, Clelmer K., 2000 Lake Shore Drive, New Orleans, LA 70 1 22 Bartlett, James H., Department of Physics, Box 870324, LIniversity of Alabama. Tuscaloosa, AL 35487-0324 Bass, Andrew H., Seely Mudd Hall. Department of Neurobiology, Cornell University. Ithaca. NY 14853 Battelle, Barbara-Anne, Whitney Laboratory, 9505 Ocean Shore Blvd., St. Augustine. FL 32086 Bauer, G. Eric, Department of Anatomy, University of Minnesota. Minneapolis, MN 55455 Beauge, Luis Alberto, Department of Biophysics, Institute M.Y.M. Ferreyra. Casilla de Correo 389, 5000 Cordoba, ARGENTINA Beck, L. V., School of Experimental Medicine, Department of Pharmacology. Indiana University, Bloomington, IN 47401 Begenisich, Ted, Department of Physiology, University of Rochester, Medical Center, Box 642. 601 Elmwood Ave., Rochester. NY 14642 Begg, David A., LHRRB, Harvard Medical School, 45 Shattuck St., Boston, MA 02 1 1 5 Bell, Eugene, Organogenesis. Inc.. 83 Rogers St., Cambridge. MA 02 142 Benacerraf, Baruj, Dana-Farber Cancer Institute, 44 Binney Street, Boston. MA 021 15 Benjamin, Thomas L., Department of Pathology, Harvard Medical School, 25 Shattuck St., Boston, MA 02 1 1 5 Bennett, M. V. L., Albert Einstein College of Medicine, Department of Neuroscience, 1300 Morris Park Ave., Bronx. NY 10461 Bennett, Miriam F., Department of Biology, Colby College, Waterville, ME 04901 Berg, Carl J., Jr., Bureau of Marine Research. 1 3365 Overseas Highway, Marathon. FL 33050 Berne, Robert M., Department of Physiology, University of Virginia. School of Medicine, Charlottesville, VA 22908 Bezanilla, Francisco, Department of Physiology, University of California, Los Angeles, CA 90024 Biggers, John D., Department of Physiology, Harvard Medical School, Boston. MA 02 1 1 5 Bishop, Stephen H., Department of Zoology, Iowa State University, Ames, IA 500 10 Blaustein, Mordecai P., Department of Physiology, School of Medicine, LIniversity of Maryland, 655 W. Baltimore Street, Baltimore, MD 2 1 20 1 Bloom, Kerry S., Department of Biology, University of North Carolina, Wilson Hall, CB #3280, Chapel Hill, NC 275 14 Bodznick, David A., Department of Biology, Wesleyan University, Lawn Avenue, Middletown. CT 06457 Boettiger, Edward G., 29 Juniper Point, Woods Hole, MA 02543 Boolootian, Richard A., Science Software Systems, Inc.. 3576 WoodchfTRd., Sherman Oaks, CA 91403 Borgese, Thomas A., Department of Biology, Lehman College. CUNY. Bronx, NY 10468 Borisy, Gary G., Laboratory of Molecular Biology, University of Wisconsin, Madison, Wl 53706 Borst, David W., Jr., Department of Biological Sciences, Illinois State University, Normal, IL 6 1 76 1 -690 1 Bosch, Herman F., 43 Windward Way, No. Falmouth, MA 02556 Bowles, Francis P., P. O. Box 674. Woods Hole, MA 02543 Boyer, Barbara C., Department of Biology, Union College, Schenectady, NY 12308 Brandhorst, Bruce P., Department of Biological Sciences, Simon Fraser University, Barnaby, BC V5A 156 Canada Brehm, Paul, Department of Neurobiology and Behavior. SUNY at Stony Brook. Stony Brook, NY 1 1794 Brinley, F. J., Neurological Disorders Program, NINCDS. 812 Federal Building, Bethesda. MD 10892 58 Annual Report Brown, Joel E., Department of Ophthalmology. Box 809c Sciences Center, Washington University, 660 S. Euclid Ave.. St. Louis. MO 63 110 Brown, Stephen C., Department of Biological Sciences, SUNY, Albany, NY 12222 Burd, Gail Deerin, Department of Molecular and Cell Biology, University of Arizona, Tucson, AZ 8572 1 Burdick, Carolyn J., Department of Biology, Brooklyn College, Bedford Avenue & Avenue H, Brooklyn, NY 11210 Burger, Max, Freidrich Miesner Institut Bau 1060 Postfach 2543. Basel 4002, Switzerland Burky, Albert, Department of Biology, University of Dayton, Dayton. OH 45469 Burstyn, Harold Lewis, Melvin and Melvin. 700 Merchants Bank Bldg.. Syracuse, NY 13202-1686 Bursztajn, Sherry, Neurology Department, Program in Neuroscience. Baylor College of Medicine, Houston, TX 77030 Bush, Louise, 7 Snapper Lane. Falmouth. MA 02540 Calabrese, Ronald L., Department of Biology. Emory University. 1555 Pierce Drive. Atlanta, GA 30322 Candelas, Graciela C., Department of Biology, University of Puerto Rico. Rio Piedras. PR 0093 1 Carew, Thomas J., Department of Psychology, Yale University, P. O. Box I 1 A, Yale Station, New Haven. CT 06520 Cariello, Luciu, Biochemistry Department. Stazione Zoologica, Villa Comunale. 80120 Naples, ITALY Carlson, Francis D., 2302 W. Rogers Avenue, Baltimore, MD 21209 Carriere, Rita M., Department of Anatomy and Cell Biology, Box 5. SUNY Health Science Center, 450 Clarkson Ave.. Brooklyn, NY 11203 Case, James, Office of Research Development, Cheadle Hall, University of California, Santa Barbara, CA 93 1 1 1 Cassidy, Rev. J. D., Pope John Center, 1 86 Forbes Rd., Braintree, MA02184 Cebra, John J., Department of Biology, Leidy Labs, G-6, University of Pennsylvania. Philadelphia. PA 19174 Chaet, Alfred B., University of West Florida, Pensacola, FL 32504 Chambers, Edward L., Department of Physiology and Biophysics, University of Miami, School of Medicine, P. O. Box 016430, Miami, FL 33 101 Chang, Donald C., Department of Physiology and Molecular Biophysics. Baylor College of Medicine, One Baylor Plaza. Houston, TX 77030 Chappell, Richard L., Department of Biological Sciences, Hunter College, Box 210, 695 Park Ave., New York, NY 10021 Charlton, Milton P., Physiology Department MSB, University of Toronto, Toronto, Ontario. M5S 1 A8 Canada Chauncey, Howard H., 30 Falmouth St.. Wellesley Hills, MA 02 1 8 1 Child, Frank M., Ill, Department of Biology. Trinity College. Hartford. CT 06 106 Chisholm, Rex L., Department of Cell Biology and Anatomy. Northwestern University Medical School. 303 E. Chicago Avenue. Chicago. IL 606 I 1 Citkowitz, Elena, 410 Livingston St.. New Haven. CT 0651 1 Clark, Eloise E., Vice President for Academic Affairs, Bowling Green State University, Bowling Green. OH 43403 Clark, Hays, 26 Deer Park Drive. Greenwich. CT 06830 Clark, James M., Shearson Lehman Brothers Inc.. 14 Wall St., 9th Floor, New York. NY 10005 Clark, VVallis H., Jr., Bodega Marine Laboratory. P. O. Box 247. Bodega Bay. CA 94923 Claude, Philippa, Primate Center, Capitol Court, Madison, WI 53706 Clay, John R., Laboratory of Biophysics. N1H. Building 9. Room IE- 127. Bethesda. MD 20892 Clutter, Mary, Office of the Director, Room 518. National Science Foundation. Washington. DC 20550 Cobb, Jewel Plummer, California State University. 800 State College Boulevard, Fullerton, CA 92634 Cohen. Adolph L, Department of Ophthalmology. School of Medicine, Washington LIniversity, 660 S. Euclid Ave., St. Louis, MO 63 110 Cohen, Avis H., Section of Neurobiology and Behavior. Mudd Hall. Cornell University. Ithaca, NY 14853-2702 Cohen, Carolyn, Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, MA 02254 Cohen, Lawrence B., Department of Physiology. Yale University School of Medicine, 333 Cedar Street, New Haven, CT 065 10-8026 Cohen, Leonard A., 279 King St.. Chappaqua. NY 105 14 Cohen, Maynard, Department of Neurological Sciences, Rush Medical College. 600 South Paulina. Chicago. IL 60612 Cohen, Rochelle S., Department of Anatomy, University of Illinois, 808 W. Wood Street, Chicago, IL 60612 Cohen, William D., Department of Biological Sciences, Hunter College. 695 Park Ave.. Box 79, New York. NY 10021 Coleman, Annette W., Division of Biology and Medicine, Brown University, Providence. RI 1 9 1 2 Collier, Jack R., Department of Biology, Brooklyn College, Bedford & Avenue H, Brooklyn. NY 1 1210 Collier, Marjorie McCann, Biology Department. Saint Peter's College. 2641 Kennedy Boulevard, Jersey City. NJ 07306 Cook, Joseph A., The Edna McConnell Clark Foundation. 250 Park Ave.. New York. NY 1001 7 Cooperstein, S. J., University of Connecticut, School of Medicine, Farmington Ave.. Farmington. CT 06032 Corliss, John O., P. O. Box 53008, Albuquerque. NM 87 1 53 Cornell, Neal W., Marine Biological Laboratory, Woods Hole. MA 02543 Cornwall, Melvin C., Jr., Department of Physiology L7 14, Boston LIniversity School of Medicine, 80 E. Concord St., Boston. MA 02 1 1 8 Corson, David Wesley, Jr., 1034 Plantation Lane. Mt. Pleasant. SC 29464 Corwin, Jeffrey T., Department of Otolaryngology. University of Virginia Medical Center, Box 430, Charlottesville, VA 22908 Regular Members 59 Costello, Walter J., Department of Zoology, College of Medicine, Ohio University, Athens, OH 45701 Couch, Ernest F., Department of Biology, Texas Christian University. Fort Worth, T\ 76 1 29 Cremer-Bartels, Gertrud, Universitats Augenklimk. 44 Munster. FRG Crow, Terry J., Department of Neurobiology and Anatomy, University of Texas Medical School, Houston, TX 77225 Crowell, Sears, Department of Biology, Indiana University, Bloomington, IN 47405 Crowther, Robert, Marine Biological Laboratory. Woods Hole. MA 02543 Currier, David L., P. O. Box 2476. Vineyard Haven, MA 02568 Daignault, Alexander T., 280 Beacon St., Boston, MA 02 1 16 Han, Katsuma, Tokyo Metropolitan Union, Meguro-ku, Tokyo, Japan D'Avanzo, Charlene, School of Natural Science. Hampshire College. Amherst, MA 01002 David, John R., Seeley G. Mudd Building. Room 504. Harvard Medical School. 250 Longwood Ave.. Boston. MA 02 1 1 5 Davidson, Eric H., Division of Biology. 1 56-29, California Institute ot Technology. Pasadena. CA 9 1 1 25 Davis, Bernard D., Bacterial Physiology Unit. Harvard Medical School. Boston, MA 02 1 1 5 Davis, Joel P., Seapuit. Inc., P. O. Box G. Osterville, MA 02655 Daw, Nigel \\ '., 78 Aberdeen Place. Clayton, MO 63105 DeGroof, Robert C., E. R. Squibb & Sons, P. O. Box 4000, Princeton. NJ 08543-4000 I HI la. in, Robert L., Department of Anatomy and Cell Biology, Emory University School of Medicine. Atlanta. GA 30322 DeLanney, Louis E., Institute for Medical Research. 2260 Clove Drive, San Jose, CA 95 1 28 DePhillips, Henry A., Jr., Department of Chemistry, Trinity College, 300 Summit Street, Hartford. CT 06 106 DeTerra, Noel, 2 1 5 East 1 5th St.. New York. NY 1 0003 Dettbarn, Wolf-Dietrich, Department of Pharmacology. School of Medicine, Vanderbilt University, Nashville, TN 37127 De Weer, Paul J., Department of Cell Biology and Physiology, School of Medicine, Washington University, St. Louis, MO 63 110 Dixon, Keith E., School of Biological Sciences, Flinders University, Bedford Park, 5042, South Australia, Australia Donelson, John E., Department of Biochemistry, University of Iowa. Iowa City, IA 52242 (resigned 8/14/89) Dowdall, Michael J., Department of Zoology. School of Biological Sciences, University of Nottingham, University Park. Nottingham N67 2RD. England, UK Dowling, John E., The Biological Laboratories. Harvard University. 1 6 Divinity St.. Cambridge, MA 02 1 38 DuBois, Arthur Brooks, John B. Pierce Foundation Laboratory, 290 Congress Ave.. New Haven, CT 065 19 Dudley, Patricia L., Department of Biological Sciences, Barnard College, Columbia University, 3009 Broadway. New York, NY 10027 Duncan, Thomas K., Department of Environmental Sciences, Nichols College. Dudley. MA 01 570 Dunham, Philip B., Department of Biology, Syracuse University. Syracuse, NY 13244 Dunlap, Kathleen, Department of Physiology, Tufts University Medical School. Boston. MA 02 1 1 1 Ebert, James D., Office of the Director, Chesapeake Bay Institute. The Johns Hopkins University, Suite 340, The Rotunda, 77 1 West 40th St., Baltimore, MD 2 1 2 1 1 Eckberg, William R., Department of Zoology, Howard University, Washington, DC 20059 Edds, Kenneth T., Department of Anatomical Sciences, SUNY, Buffalo. NY 14214 Eder, Howard A., Albert Einstein College of Medicine, 1300 Morris Park Ave.. Bronx, NY 10461 Edwards, Charles, University of Southern Florida College of Medicine. MDC Box 40. 12901 Bruce B. Downs Blvd.. Tampa, FL 336 12 Egyud, Laszlo G., 1 8 Skyview. Newton. MA 02 1 50 Ehrenstein, Gerald, NIH. Bethesda. MD 20892 (resigned) Ehrlich, Barbara E., Division of Cardiology. University of Connecticut Health Center, 263 Farmington Avenue. Farmington, CT 06032 Eisen, Arthur Z., Division of Dermatology, Washington University. St. Louis, MO 631 10 Eisenman, George, Department of Physiology, University of California Medical School, Los Angeles, CA 90024 Elder, Hugh Young, Institute of Physiology, LIniversity of Glasgow, Glasgow, Scotland G 12 8QQ Elliott, Gerald E., The Open University Research Unit, Foxcombe Hall, Berkeley Rd., Boars Hill, Oxford, England OX 1 5HR Englund, Paul T., Department of Biological Chemistry, Johns Hopkins School of Medicine, Baltimore, MD 21205 Epel, David, Hopkins Marine Station, Pacific Grove, CA 93950 Epstein, Herman T., 1 8 Lawrence Farm Road, Woods Hole, MA 02543 Epstein, Ray L., Marine Biological Laboratory, Woods Hole, MA 02543 Erulkar, Solomon D., 3 1 8 Kent Rd., Bala Cynwyd, PA 19004 Essner, Edward S., Kresge Eye Institute, Wayne State University, 540 E. Canfield Ave.. Detroit, MI 48201 Farb, David H., SUNY Health Science Center. Brooklyn. NY ! 1 203 Farmanfarmaian, A., Department of Biological Sciences, Nelson Biological Laboratory', Rutgers University, Piscataway. NJ 08855 Fein, Alan, Physiology Department, University of Connecticut Health Center, Farmington. CT 06032 Feinman, Richard D., Box 8. Department of Biochemistry. SUNY Health Science Center. 450 Clarkson Avenue. Brooklyn. NY 1 1 203 60 Annual Report Feldman, Susan C., Department of Anatomy. University of Medicine and Dentistry of New Jersey, New Jersey Medical School. 100 Bergen St., Newark, NJ 07103 Fessenden, Jane, Marine Biological Laboratory, Woods Hole, MA 02 543 Festoff, Barry W., Neurology Service (127), Veterans Administration Medical Center, 4801 Linwood Blvd.. Kansas City, MO 64 128 Fink, Rachel D., Department of Biological Sciences. Clapp Laboratory, Mount Holyoke College, South Hadley, MA 01075 Finkelstein, Alan, Albert Einstein College of Medicine, 1300 Morris Park Ave.. Bronx. NY 10461 Fischbach, Gerald, Department of Anatomy and Neurobiology. Washington University School of Medicine, St. Louis, MO 63 110 Fishman, Harvey M., Department of Physiology and Biophysics, University of Texas Medical Branch, Galveston.TX 77550 Flanagan, Dennis, 12 Gay St.. New York. NY 10014 Fox, Maurice S., Department of Biology. Massachusetts Institute of Technology. Cambridge. MA 02 1 39 Frank, Peter W., Department of Biology, University of Oregon, Eugene, OR 97403 (resigned 1 1/15/89) Franzini- Armstrong, Clara, Department of Biology G-3, School of Medicine, University of Pennsylvania. Philadelphia, PA 19104 Frazier, Donald T., Department of Physiology, University of Kentucky Medical Center, Lexington. KY 40536 Friedler, Gladys, Boston University School of Medicine. 80 East Concord Street. Boston, MA 02 1 1 8 Freinkel, Norbert, Center for Endocrinology, Metabolism & Nutrition, Northwestern LJniversity Medical School, 303 E. Chicago Avenue, Chicago, IL 606 1 1 (deceased) French, Robert J., Health Sciences Center, University of Calgary. Calgary, Alberta, T2N 4N 1 . Canada Freygang, Walter J., Jr., 6247 29th St., NW, Washington, DC 20065 Fry, Brian, Marine Biological Laboratory, Woods Hole, MA 02543 Fukui, Yoshio, Department of Cell Biology and Anatomy, Northwestern University Medical School, Chicago. IL 60201 Fulton, Chandler M., Department of Biology, Brandeis University, Waltham, MA 02254 Furshpan, Edwin J., Department of Neurophysiology, Harvard Medical School, Boston, MA 02 1 1 5 Fuseler, John W., Department of Biology, University of Southwestern Louisiana, Lafayette, LA 70504 Futrelle, Robert P., College of Computer Science, Northeastern University, 360 Huntington Avenue, Boston, MA 02 1 1 5 Gabriel, Mordecai, Department of Biology, Brooklyn College, Brooklyn. NY 1 1210 Gadsby, David C., Laboratory of Cardiac Physiology, The Rockefeller University, 1230 York Avenue, New York, NY 10021 Gainer, Harold, Section of Functional Neurochemistry, NIH, Bldg. 36. Room 4D-20, Bethesda, MD 20892 Galatzer-Levy, Robert M., 180 N. Michigan Avenue. Chicago, IL 60601 Gall, Joseph G., Carnegie Institution, 1 1 5 West University Parkway. Baltimore, MD 2 12 1(1 Gallant, Paul F., Laboratory of Preclinical Studies. Bldg. 36. NIAAA/NIH. 1250 Washington Ave.. Rockville, MD 20892 Gascoyne, Peter, Department of Experimental Pathology, Box 85E, University of Texas System Cancer Center, M. D. Anderson Hospital and Tumor Institute. Texas Medical Center. 6723 Bertner Avenue. Houston. TX 77030 Gelfant, Seymour, Department of Dermatology. Medical College of Georgia. Augusta, GA 30904 (deceased 6/1/89) Gelperin, Alan, Department of Biophysics, AT&T Bell Labs, Room 7C305, 600 Mountain Avenue. Murray Hill, NJ 07974 German, James L., Ill, Lab of Human Genetics. The New York Blood Center, 310 East 67th St.. New York. NY 10021 Gibbs, Martin, Institute for Photobiology of Cells and Organelles, Brandeis University, Waltham, MA 02254 Giblin, Anne E., Ecosystems Center, Marine Biological Laboratory. Woods Hole, MA 02543 Gibson, A. Jane, Wing Hall, Cornell University, Ithaca, NY 14850 Gifford, Prosser, 560 N Street. SW, S-903. Washington. DC 20024 Gilbert, Daniel L., Laboratory of Biophysics, NIH/NINDS. Bldg. 9, Room IE- 124. Bethesda, MD 20892 Giudice, Giovanni, Dipartimento di Biologia e Dello Sviluppo. 1-90123, Via Archirafi 22, Universita di Palermo. Palermo. Italy Glusman, Murray, 50 East 72nd Street, New York, NY 10021 Golden, William T., Golden Family Foundation, 40 Wall St., Room 4201. New York. NY 10005 Goldman, David E., 63 Loop Rd.. Falmouth, MA 02540 Goldman, Robert D., Department of Cell Biology and Anatomy, Northwestern L'niversity. 303 E. Chicago Ave.. Chicago, IL 606 11 Goldsmith, Paul K., NIH, Bldg. 10. Room 9C- 101. Bethesda, MD 20892 Goldsmith, Timothy H., Department of Biology, Yale University, New Haven, CT 065 10 Goldstein, Moise H., Jr., ECE Department, Barton Hall. Johns Hopkins University, Baltimore. MD 2 1 2 1 8 Goodman, Lesley Jean, Department of Biological Sciences, Queen Mary College, Mile End Road, London, El 4NS, England, UK Goudsmit, Esther, M., Department of Biology. Oakland University, Rochester. MI 48309 Gould, Robert Michael, Institute for Basic Research in Developmental Disabilities, 1050 Forest Hill Rd.. Staten Island, NY 10314 Gould, Stephen J., Museum of Comparative Zoology, Harvard University, Cambridge, MA 02138 Govind, C. K., Life Sciences Division, University of Toronto, 1 265 Military Trail. West Hill, Ontario, M 1C 1 A4, Canada Regular Members 61 Graf, Werner, Rockefeller University, 1230 York Ave., New York. NY 10021 Grant, Philip, 2939 Van Ness Street. N.W., Apt. 302. Washington, DC 20008 Grass, Albert M., The Grass Foundation, 77 Reservoir Rd., Quincy, MA 02 170 Grass, Ellen R., The Grass Foundation. 77 Reservoir Rd.. Quincy. MA 02 1 70 Grassle, Judith, Marine Biological Laboratory. Woods Hole. MA 02543 Graubard, Katherine, Department of Zoology, NJ-15, University of Washington, Seattle. WA 98195 Greenberg, Everett Peter, Department of Microbiology. Stocking Hall, Cornell University, Ithaca, NY 14853 Greenberg, Michael J., Whitney Laboratory, 9505 Ocean Shore Blvd., St. Augustine. FL 32086 Griffin, Donald R., Concord Field Station, Harvard University, Old Causeway Road, Bedford, MA 1 730 Gross, Joan E., 25 1 5 Milton Hills Drive. Charlottesville, VA 22901 Gross, Paul R., Center for Advanced Studies. University of Virginia, 444 Cabell Hall. Charlottesville. VA 22903 Grossman, Albert, New York University Medical Center, 550 First Ave., New York. NY 10016 Grossman, Lawrence, Department of Biochemistry. Johns Hopkins LJniversity, 615 North Wolfe Street, Baltimore, MD 21205 Gruner, John, Department of Neurosurgery, New York University Medical Center, 550 First Ave.. New York, NY 10016 Gunning, A. Robert, P. O. Box 165, Falmouth, MA 0254 I Gwilliam, G. P., Department of Biology, Reed College. Portland, OR 97202 I hi i mi i. Leah, Department of Biology, University of California, Riverside, Riverside, CA 92521 Hall, Linda M., Department of Biochemistry and Pharmacology, SUNY, 3 1 7 Hochstetter, Buffalo. NY 14260 Hall, /ack W., Department of Physiology, Llniversity of California, San Francisco. CA 94143 Halvorson, Harlyn O., Marine Biological Laboratory. Woods Hole. MA 02543 Hamlett, Nancy Virginia, Department of Biology. Swarthmore College. Swarthmore, PA 19081 Hanna, Robert B., College of Environmental Science and Forestry, SUNY. Syracuse, NY 13210 Harding, Clifford V., Jr., Wayne State University School of Medicine. Department of Ophthalmology, Detroit, MI 48201 Harosi, Eerenc L, Laboratory of Sensory Physiology, Marine Biological Laboratory, Woods Hole, MA 02543 Harrigan, June F., 741 5 Makaa Place, Honolulu. HI 96825 Harrington, Glenn \V., Division of Cell Biology and Biophysics, 403 Biological Sciences Building, University of Missouri, Kansas City, MO 641 10 Harris, Andrew L., Department of Biophysics. Johns Hopkins University, 34th & Charles Sts., Baltimore, MD 2 1 2 1 8 Hastings, J. W., The Biological Laboratories, Harvard University, 1 6 Divinity Street, Cambridge, MA 02 1 38 Hathaway, Warren, Hathaway Publishing, 780 County Street, Somerset, MA 02726 Hayashi, Teru, 7105 SW I 12 Place. Miami, FL 33173 Haydon-Baillie, Wensley G., Porton Int., 2 Lowndes Place, London, SW I X 8DD, England. UK Hayes, Raymond L., Jr., Department of Anatomy, Howard University. College of Medicine, 520 W St.. NW. Washington, DC 20059 Henley, Catherine, 5225 Pooks Hill Rd., #1 127 North, Bethesda, MD 20034 (resigned 8/14/89) Hepler, Peter K., Department of Botany. University of Massachusetts, Amherst, MA 01003 Merndon, W alter R., LJniversity of Tennessee, Department of Botany, Knoxville, TN 37996-1 100 Heuser, John, Department of Biophysics. Washington LJniversity. School of Medicine, St. Louis, MO 63 1 10 (resigned 8/14/89) Hiatt, Howard II., Department of Medicine, Brigham and Women's Hospital, 75 Francis Street, Boston, MA 02 1 1 5 Highstein, Stephen M., Department ot Otolaryngology, Washington Llniversity, St. Louis, MO 63 1 10 Hildebrand, John G., Arizona Research Laboratories, Division of Neurobiology, 61 1 Gould-Simpson Science Building, Llniversity of Arizona. Tucson. AZ 8572 1 Hill, Richard W., Department of Zoology, Michigan State University, E. Lansing, MI 48824 Hill, Susan D., Department of Zoology, Michigan State University, E. Lansing, MI 48824 Hillis-Colinvaux, Llewellya, Department of Zoology, Ohio State University. 484 W. 1 2th Ave., Columbus, OH 432 1 Hillman, Peter, Department of Biology, Life Sciences & Neurobiology, Hebrew University, Jerusalem 91904, Israel Hinegardner, Ralph T., Division of Natural Sciences, LJniversity of California, Santa Cruz, CA 95064 Hinsch, Gertrude, W ., Department of Biology, University of South Florida, Tampa, FL 33620 I lobbie, John E., Ecosystems Center, Marine Biological Laboratory. Woods Hole, MA 02543 Hodge, Alan J., 3843 Ml. Blackburn Ave., San Diego, CA 92111 I 1 i.ll 111:1 M. Joseph, Department of Physiology, School of Medicine. Yale Llniversity, New Haven, CT 065 1 5 I loll> field. JoeG., Baylor School of Medicine, Texas Medical Center, Houston, TX 77030 I lull /man. Eric, Department of Biological Sciences, Columbia University, New York, NY 1001 7 Holz, George G., Jr., Department of Microbiology, SLJNY, Syracuse, NY 13210 (deceased 9/ 1 7/89) I 1 cskin, Francis C. G., Department of Biology, Illinois Institute of Technology, Chicago, IL 60616 Houghton, Richard A., Ill, Woods Hole Research Center, P. O. Box 296, Woods Hole. MA 02543 Houston, Howard E., 2500 Virginia Ave., NW, Washington, DC 2003 7 (resigned) Hoy, Ronald R., Section of Neurobiology and Behavior, Cornell University, Ithaca, NY 14853 Hufnagel, Linda A., Department of Microbiology, University of Rhode Island, Kingston, RI 02881 62 Annual Report Itummon, William D., Department of Zoology, Ohio University, Athens, OH 45701 Humphreys, Susk' II., Research & Development, Kraft, Inc., 801 Waukegan Rd., Glenview, IL 60025 Humphreys, Torn D., University of Hawaii, PBRC. 41 Ahui St., Honolulu, HI 968 13 Hunter, Robert D., Department of Biological Sciences, Oakland University, Rochester, MI 48309-4401 Hunter, VV. Bruce, Box 321, Lincoln Center, MA 01773 Hurwitz, Charles, Basic Science Research Lab, Veterans Administration Hospital, Albany, NY 122U1S Hurwitz, Jerard, Sloan Kettering Institute for Cancer Research, 1275 York Avenue, New York, NY 1 1021 Huxley, Hugh E., Department of Biology, Rosenstiel Center, Brandeis University, Waltham. MA 02 1 54 Hynes, Thomas J., Jr., Meredith and Grew, Inc., 160 Federal Street, Boston, MA 02 I 10-1701 Man, Joseph, Department of Developmental Genetics and Anatomy. Case Western Reserve University School of Medicine, Cleveland, OH 44106 Ingoglia, Nicholas, Department of Physiology, New Jersey Medical School, 100 Bergen St.. Newark, NJ 07103 Inoue, Saduyki, Department of Anatomy, McGill L'niversity 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, Monis Petraki 8, Athens, 140 Greece Izzard, Colin S., Department of Biological Sciences, SUNY, 1400 Washington Ave., Albany, NY 12222 Jacobson, Antone G., Department of Zoology. University of Texas, Austin, TX 787 12 Jaffe, Lionel, Marine Biological Laboratory, Woods Hole, MA 02543 Jahan-Parwar, Behrus, Center for Laboratories & Research. New York State Department of Health, Empire State Plaza, Albany, NY 12201 Jannasch, Holger W., Department of Biology, Woods Hole Oceanographic Institution, Woods Hole, MA 02543 Jeffery, William R., Department of Zoology. University of Texas. Austin, TX 787 12 Jones, Meredith L., Division of Worms, Museum of Natural History, Smithsonian Institution, Washington, DC 20560 Josephson, Robert K., Deparment of Psychobiology, University of California, Irvine, CA 92664 Rabat, 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 02118 Kammer, Ann E., Department of Zoology. Arizona State University, Tempe, AZ 85281 (resigned 8/14/89) Kane, Robert K., PBRC. University of Hawaii, 41 Ahui St., Honolulu. HI 968 13 Kaneshiro, Edna S., Department of Biological Sciences, University of Cincinnati, Cincinnati, OH 4522 1 Kao, C'hien-yuan, Department of Pharmacology, Box 29, SUNY, Downstate Medical Center, 450 Clarkson Avenue. Brooklyn. NY 11203 Kaplan, Ehud, The Rockefeller University. 1230 York Ave., New York, NY 10021 Karakashian, Stephen,]., Apt. 16-F, 165 West 91st St., New York, NY 10024 Karlin, Arthur, Department of Biochemistry and Neurology, Columbia University, 630 West 168th St., New York, NY 10032 Katz, George M., Fundamental and Experimental Research Lab, Merck Sharpe and Dohme. P.O. Box 2000, Rahway, NJ 07065 Kelley, Darcy Brisbane, Department of Biological Sciences. 1018 Fairchild, Columbia University, New York, NY 10032 Kelly, Robert E., Department of Anatomy, College of Medicine, University of 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 021 10 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 02 1 38 Klein, Morton, Department of Microbiology, Temple University, Philadelphia, PA 19103 Klotz, Irving M., Department of Chemistry. Northwestern Llniversity, 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 Konigsberg, Irwin R., Department of Biology, Gilmer Hall, University of Virginia, Charlottesville, VA 22903 Kornberg, Sir I lans. 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 208 1 4 Regular Members 63 Kravit/, Edward A., Department of Neurohiology, Harvard Medical School, 25 Shattuck St., Boston, MA 02 1 1 5 Kriehel, Mahlon E., Department of Physiology, SUNY Health Science Center, Syracuse. NY 1 32 10 Kristan, William B., Jr., Department of Biology B-022, University of California San Diego, La Jolla, CA 92043 Kropinski, Andrew M. B., Department of Microbiology/ Immunology. Queen's University. Kingston. Ontario K7L 3N6. Canada Kuhns, NMIIiam J., Hospital for Sick Children, Department of Biochemistry Research. Toronto, Ontario M5G 1X8, Canada Kusano, Kiyoshi, N1H, Bldg. 36, Room 4D-20, Bethesda, MD 20892 Kuhtreiher, NVillem M., Marine Biological Laboratory, Woods Hole, MA 02543 Kuzirian, Alan M., Marine Biological Laboratory, Woods Hole, MA 02543 Laderman, Aimlee, P. O. Box 689, 18 Agassiz Road, Woods Hole, MA 02543 LaMarche, Paul H., Eastern Maine Medical Center, 489 State St.. Bangor, ME 04401 I iimlis, Dennis M. D., Department of Developmental Genetics and Anatomy, Case Western Reserve School of Medicine, Cleveland, OH 44106 I .mil is. Story C., Center for Neurosciences, Case Western Reserve LIniversity School of Medicine, Cleveland. OH 44106 Landowne, David, Department of Physiology, P. O. Box 016430, LIniversity of Miami School of Medicine, Miami, FL 33101 I .angford, George M., Department of Physiology, CB7545 University of North Carolina School of Medicine, Chapel Hill, NC 27599-7545 Lasek, Raymond J., Case Western Reserve University, Department of Anatomy. Cleveland, OH 44106 (resigned 6/1/89) Laster, Leonard, University of Massachusetts Medical School. 55 Lake Avenue, North, Worcester, MA 01655 Laufer, Hans, 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 1002 1 Lazarus, Maurice, Federated Department Stores, Inc., 50 Cornhill, Boston, MA 02 108 Leadhetter, Edward R., Department of Molecular and Cell Biology, U- 1 3 1 , University of Connecticut, Storrs, CT 06268 Lederberg, Joshua, The Rockefeller University, 1230 York Ave., New York. NY 10021 Lederhendler, Izja I., Laboratory of Cellular and Molecular Neurobiology. N1NCDS/NIH, Park 5 Building. Room 435, Bethesda, MD 20892 (resigned 8/14/89) Lee, John J., Department of Biology, City College of CUNY, Convent Ave. and 138th St., New York, NY 10031 Lehy, Donald B., Marine Biological Laboratory, Woods Hole, MA 02543 Leibovitz, Louis, 3 Kettle Hole Road, Woods Hole, MA 02543 Leighton, Joseph, 2324 Lakeshore Avenue, #2, Oakland, CA 94606 Leighton, Stephen, N1H. Bldg. 1 3 3W 1 3, Bethesda, MD 20892 Ltinwand, Leslie Ann, Department of Microbiology and Immunology, Albert Einstein College of Medicine, 1300 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. CT 065 10 Lester, Henry A., 1 56-29 California Institute of Technology, Pasadena, CA 9 1 1 25 Levin, Jack, Clinical Pathology Service, VA Medical Center, 1 1 3A, 4 1 50 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 Levitan, Herbert, Department of Zoology, University of Maryland. College Park, MD 20742 Levitan, Irwin B., Department of Biochemistry, Brandeis University, Waltham, MA 02254 I i MI- k. Richard W., Department of Anatomy, Jackson Hall, University of Minnesota. 32 1 Church Street, S. E., Minneapolis. MN 55455 Lipicky, Raymond J., Department of Cardio-Renal/HFD 1 10, FDA Bureau of Drugs, Rm. 16B-45, 5600 Fishers Lane, Rockville, MD 20857 Lisman, John E., Department of Biology, Brandeis University, Waltham, MA 02254 Liuzzi, Anthony, 55 Fay Rd., Box 184, Woods Hole, MA 02543 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 1 536 Loewenstein, Birgit R., Department of Physiology and Biophysics, R-430, University of Miami School of Medicine. Miami. FL 33 101 Loewenstein, Werner R., Department of Physiology and Biophysics, University of Miami. P. O. Box 01 6430, Miami, FL33101 Loftfield, Robert B., Department of Chemistry, School of Medicine, University of New Mexico, 900 Stanford, NE, Albuquerque, NM 87131 London, Irving M., Massachusetts Institute of Technology, E-25-55 1 , Cambridge, MA 02 1 39 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, Irvine Hall. 1 55 Columbia Ave., Athens, OH 45701 Luria, Salvador E., 48 Peacock Farm Rd., Lexington, MA 02173 64 Annual Report Macagno, Eduardo R., 1003B Fairchild, Department of Biosciences. Columbia University, New York, NY 10027 MacNichol, K. F., Jr., Department of Physiology, Boston University School of Medicine, 80 E. Concord St.. Boston, MA 02 118 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. 5-D. Boston, MA 02 106 Malbon, Craig Curtis, Department of Pharmacology. Health Sciences Center. SUNY. Stony Brook. NY 1 1794-8651 Malkiel, Saul, Allergic Diseases. Inc., 130 Lincoln St., Worcester, MA 01 609 M. malls, 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. Williamshurg. VA 23185 Margulis, Lynn, Botany Department, University of Massachusetts. Morrill Science Center, Amherst, MA 01003 Marinucci, Andrew C., 102 Nancy Drive, Mercerville, NJ 08619 Marsh, Julian B., Department of Biochemistry and Physiology, Medical College of Pennsylvania, 3300 Henry A ve.. 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, P. O. Box EM, Woods Hole Education Assoc.. 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 19174 Mathews, Rita W., Box 131, Southfield, MA 01259 Matteson, Donald R., Department of Biophysics, University of Maryland School of Medicine, 660 Redwood Street, Baltimore, MD21201 Mautner, Henry G., Department of Biochemistry, Tufts University School of Medicine, 136 Harrison Ave., Boston, MA 021 11 Mauzerall, David, The Rockefeller University, 1230 York Ave.. New York, NY 1002 1 Mazia, Daniel, Hopkins Marine Station, Pacific Grove, CA 93950 (resigned 8/1 4/89) Mazzella, Lucia, Laboratorio di Ecologia del Benthos. Stazione Zoologica di Napoli, P.ta S. Pietro 80077. Ischia Porto (NA), ITALY (resigned 8/14/89) McCann, F ranees. 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 Meinertzhagen, Ian A., Department of Psychology, Life Sciences Center, Dalhousie University, Halifax, Nova Scotia B3H 45 I.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 Metuzals, Janis, Department of Pathology, University of Ottawa, Ottawa, Ontario, K1H 8M5 Canada Metz, Charles B., 7220 SW 1 24th St., Miami, FL 33 1 56 Milkman, Roger, Department ot Biology, University of Iowa, Iowa City, IA 52242 Mills, Eric L., Oceanography Dept., Dalhousie University, Halifax, Nova Scotia B3H 4J 1 , Canada Mills, Robert, 103 15 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 70 1 1 8 Moore, John VV., Department of Neurobiology, Box 3209, Duke University Medical Center. Durham, NC 27710 Moore, Lee E., Department of Physiology and Biophysics. LIniversity of Texas Medical Branch, Galveston, TX 77550 Morin, James G., Department ot Biology, LIniversity of California. Los Angeles, CA 90024 Morrell, 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 01908 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 (resigned 8/14/89) Mote, Michael I., Department of Biology. Temple University. Philadelphia, PA 19122 Mountain, Isabel, Vinson Hall #112, 625 I Old Dominion Drive, McLean. VA 22 101 Muller, Kenneth J., Department of Physiology and Biophysics. University of Miami School of Medicine, Miami, FL 33 101 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 Nadelliotier, Knute, Marine Biological Laboratory, Woods Hole. MA 02543 Regular Members 65 Naka, Ken-ichi, PHL 821, Department of Ophthalmology, NYU Medical Center. 550 First Avenue. New York, NY 10016 Nakajima, Shigehiro, Department of Anatomy and Cell Biology, University of Illinois College of Medicine at Chicago, 808 S. Wood Street, Chicago, IL 606 1 2 Nakajima, Yasuko, University of Illinois College of Medicine at Chicago, Department of Anatomy and Cell Biology, M/C 512, Chicago, IL 606 12 Narahashi, Toshio, Department of Pharmacology, Northwestern University Medical Center, 303 East Chicago A ve., Chicago, IL 606 11 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 Neurobiology and Behavior. Cornell University. Ithaca. NY 14850 Nicholls, John G., Biocenter. Klingelbergstr. 70. Basel 4056, Switzerland Nicosia, Santo V., Department of Pathology, University of South Florida. College of Medicine. Box 1 1, 12901 North 30th St., Tampa. FL 336 12 Noe, Bryan D., Department of Anatomy and Cell Biology. Emory University. Atlanta, GA 30322 Norton, Catherine N., Marine Biological Laboratory. Woods Hole, MA 02543 Obaid, Ana Lia, Department of Physiology and Pharmacy, University of Pennsylvania, 4001 Spruce St., Philadelphia, PA 19104-6003 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 Cary 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 37830 Olins, Donald E., University of Tennessee-Oak Ridge, Graduate School of Biomedical Sciences. Biology Division ORnL. P. O. Box 2009, Oak Ridge. TN 37830 O'Melia, Anne F., 16 Evergreen Lane. Chappaqua. New York 10514 Oschman, James L., 3 1 Whittier Street, Dover, NH 03820 Page, Irving II., Box 516. Hyannisport. MA 02647 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. 3 1096 Israel Pant, HarishC, 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 Wood St., Chicago, IL 606 12 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 I.., Becton Dickinson Research Center, P. O. Box 12016. Research Triangle Park, NC 27709 Passano, Leonard M., Department of Zoology, Birge Hall. Llniversity of Wisconsin, Madison. WI 53706 Pearlman, Alan L., Department of Physiology. School of Medicine, Washington University, St. Louis, MO 63 I 10 Pederson, Thoru, Worcester Foundation for Experimental Biology, Shrewsbury, MA 01 545 Perkins, C. D., 400 Hilltop Terrace. Alexandria. VA 2230 1 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 Plohl, Ronald J., Department of Zoology, Miami University, Oxford. OH 45056 Pierce, Sidney K., Jr., Department of Zoology. University of Maryland, College Park, MD 20742 Poindexter, Jeanne S., Science Division, Long Island Llniversity, Brooklyn Campus, Brooklyn, NY 1 1201 Pollard, Harvey B., NIH. NIDDKD. Bldg. 8. Rm. 401. Bethesda, MD 20892 Pollard, Thomas D., Department of Cell Biology and Anatomy, Johns Hopkins University, 725 North Wolfe St., Baltimore, MD21205 Pollister, A. W., 8 Euclid Avenue, Belle Mead, NJ 08502 Poole, Alan F., P. O. Box 533, Woods Hole. MA 02543 Porter, Beverly II., 1 3617 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 02 1 1 5 Potts, William T., Department of Biology, University of Lancaster, Lancaster, England, UK 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 1 1., Carl Zeiss, Inc., 1 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, NALI Box 5640, Northern Arizona University, Flagstaff, AZ 8601 1 66 Annual Report Prusch, Robert D., Department of Life Sciences, Gonzaga University, Spokane, WA 99258 Przybylski, Ronald J., Case Western Reserve University, Departmer ol \natomy. Cleveland, OH 44104 (resigned 8/14/89) Purves, Dale, Department of Anatomy, Washington University School of Medicine, 660 S. Euclid Ave.. St. Louis. MO 63 110 Quigley, James, Department of Pathology, SUNY Health Science Center, 450 Clarkson Avenue, Stony Brook, NY 11794 Rabb, Irving VV., 1010 Memorial Drive, Cambridge, MA 02138 Rabin, Harvey, DuPont Biomedical Products, BRL-2, 500-2, 33 Treble Cove Road, No. Billerica, MA 01862 Rabinowitz, Michael B., Marine Biological Laboratory, Woods Hole, MA 02543 Raff, Rudolf A., Department of Biology, Indiana University, Bloomington, IN 47405 Rafferty, Nancy S., Department of Anatomy, Northwestern University Medical School, 303 E. Chicago Avenue, Chicago IL 606 11 Rakowski, Robert F., Department of Physiology and Biophysics. UHS/The Chicago Medical School, 3333 Greenbay Rd., N. Chicago, IL 60064 Ramon, Fidel, Dept. de Fisiologia y Biofisca, Centra de Investigacion y de Estudius Avanzadosdel ipn, Apurtado Postal 14-740, D.F. 07000. Mexico Ranzi, Silvio, Sez Zoologia Sc Nat, Via Coloria 26, 1 20 1 33, 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 10016 Rebhun, Lionel I., Department of Biology, Gilmer Hall, University of Virginia, Charlottesville, VA 22901 Reddan, John R., Department of Biological Sciences, Oakland University, Rochester. MI 48063 Reese, Barbara F., 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., Department of Biochemistry, Albany Medical College of Union University, Albany. NY 12208 Reinisch, Carol L., Tufts University School of Veterinary Medicine, 1 36 Harrison Avenue, Boston, MA 02 1 1 5 Reuben, John P., Department of Biochemistry, Merck Sharp and Dohme, P. O. Box 2000, Rahway, NJ 07065 (resigned 8/14/89) Renn, Charles E., Address unknown 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 Richards, A. Glen, 942 Cromwell Avenue. St. Paul, MN 55114 Rickles, Frederick R., Department of Medicine, Division of Hematology-Oncology, University of Connecticut Health Center, Farmington, CT 06032 Ripps, Harris, Department of Ophthalmology, University of Illinois College of Medicine, 1855 W. Taylor Street, Chicago, IL 606 11 Robinson, Denis M., 200 Ocean Lane Drive #908. Key Biscayne, FL33149 Rose, S. Meryl, 32 Crosby Ln.. E. Falmouth, MA 02536 Rosenbaum, Joel I,., 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 Llniversity, Burnaby, BC, V5A186, 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., 1 8 Millneld Street, P. O. Box 285, Woods Hole. MA 02543 Rowland, Lewis P., Neurological Institute, 7 10 West 168th St., New York, NY 10032 Ruderman, Joan V., Department of Anatomy and Cell Biology, Harvard University School of Medicine, Boston, MA 02 1 1 5 Rushforth, Norman B., Department of Biology, Case Western Reserve University, Cleveland, OH 44106 Russell-Hunter, \\. D., Department of Biology, Lyman Hall 029, 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 02 1 1 5 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 27514 Salzberg, Brian M., Department of Physiology. University of Pennsylvania, 4010 Locust St., 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, Nagoya University School of Science. Sugashima-cho. Toba-shi, Mieken 517, Japan Sattelle, David B., AFRC Unit-Department of Zoology. Llniversity of Cambridge. Downing St.. Cambridge CB2 3EJ. England. UK Regular Members 67 Saunders, John \V., Jr., P. O. Box 38 1 . Waquoit Station. Waquoit. MA 02536 Saz, Arthur K., Department ot'Immunology. Georgetown University Medical School, Washington, DC 20007 Schachman, Howard K., Department of Molecular Biology. University of California. Berkeley, CA 94720 Schatten, Gerald P., Integrated Microscopy Facility for Biomedical Research. University of Wisconsin. I 1 1 7 W. Johnson St.. Madison. WI 53706 Schatten, Heide, Department of Zoology. University of Wisconsin. Madison, WI 53706 SchirF, Jerome A., Institute for Photohiology of Cells and Organelles, Brandeis University. Waltham. MA 02 1 54 Schmeer, Arline C., Mercenene Cancer Research Institute. Hospital of Saint Raphael, New Haven, CT 065 1 1 Schnapp, Bruce J., Department of Physiology. Boston University Medical School, 80 East Concord Street. Boston, MA 021 18 Schneider, E. Gayle, Department of Obstetrics and Gynecology, Yale University School of Medicine, 333 Cedar St., New Haven, CT 065 10 Schneiderman, Howard A., Monsanto Company, 800 North Lindbergh Blvd.. D1W. St. Louis. MO 63 166 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, LICLA School of Medicine, Los Angeles, CA 90024 Sears, Mary, P. O. Box 1 52. Woods Hole, MA 02543 Segal, Sheldon J., Population Division. The Rockefeller Foundation. 1 1 33 Avenue of the Americas, New York. NY 10036 Selman, Kelly, Department of Anatomy, College of Medicine, University of Florida, Gainesville, FL 32601 Senft, Joseph, Biology Department, Juniata College, Huntingdon, PA 16652 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., 1 8 Las Parras, Cayey, PR 00633 Sheetz, Michael P., Department of Cell Biology and Physiology, Washington University Medical School, 606 S. Euclid Ave.. St. Louis. MO 63 1 10 Shepard, David C., P. O. Box 44. Woods Hole. MA 02543 Shepro, David, Department of Biology. Boston University. 2 Cummington St.. Boston, MA 022 1 5 Slier, F. Alan, Immunology and Cell Biology Section, Laboratory of Parasitic Disease, NIAID, Building 5, Room 1 14. NIH, Bethesda, MD 20892 Sheridan, William F., Biology Department, University of North Dakota, Box 8238, University Station, Grand Forks. ND 58202-8238 Sherman, I. \V., Department of Biology, University of California, Riverside, CA 92502 Shimomura, Osamu, Marine Biological Laboratory, Woods Hole, MA 02543 Shoukimas, Jonathan J., 45 Dillingham Avenue. Falmouth, MA 02540 (resigned 8/14/89) 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, MD21201 Skinner, Dorothy M., Oak Ridge National Laboratory, P. O. Box 2009, Biology Division, Oak Ridge, TN 37830 Sloboda, Roger D., Department of Biological Sciences, Dartmouth College, Hanover, NH 03755 Sluder, Greenfield. Cell Biology Group, Worcester Foundation for Experimental Biology, 222 Maple Ave.. Shrewsbury'- MA 1 545 Smith, Michael A., J 1 Sinabung, Buntu #7, Semarang. Java, Indonesia Smith, Ralph I., Department of Zoology, University of California, Berkeley, CA 94720 Sorenson, Martha M., Depto de Bioquimica-RFRJ, Centro de Ciencias da Saude-I. C. B.. Cidade Universitaria-Fundad. Rio de Janeiro, Brasil 21.910 Speck, William T., Case Western Reserve University, Department of Pediatrics. Cleveland, OH 44 1 06 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 Spiegel, Evelyn, Department of Biological Sciences, Dartmouth College, Hanover, NH 03755 Spiegel, Melvin, Department of Biological Sciences, Dartmouth College, Hanover, NH 03755 Spray, David C., Albert Einstein College of Medicine, Department of Neurosciences, 1 300 Morris Park 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 81 15, 4566 Scott Avenue, St. Louis, MO 63110 Steinberg, Malcolm, Department of Biology, Princeton University, Princeton, NJ 08540 Stephens, Grover C., Department of Developmental and Cell Biology, University of California, Irvine, CA 927 1 7 Stephens, Raymond E., Marine Biological Laboratory, Woods Hole, MA 02543 68 Annual Report Stetten, DeVVitt, Jr., NIH. Bklg. 16, Room 118, Bethesda, MD 20892 Stetten, Jane Lazarow, 2 W Drive, Bethesda. MD 20814 Steadier, Paul A., Lcosystems 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.. #2 1 7. San Pedro. CA 90732 Strunwasser, 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 1 00 1 6 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 Sydlik, Mary Anne, Department of Biology, Eastern Michigan University, Ypsilanti, MI 48197 Szabo, George, Harvard School of Dental Medicine, 188 Longwood Avenue, Boston, MA 021 15 (resigned 8/14/89) Szent-Gyorgyi, Andrew, Department of Biology, Brandeis University, Bassine 244, 4 1 5 South Street, Waltham, MA 02254 Szuts, Ete Z., Laboratory of Sensory Physiology, Marine Biological Laboratory, Woods Hole, MA 02543 Tamm, Sidney L., Boston University Marine Program, Marine Biological Laboratory, Woods Hole, MA 02543 Tanzer, Marvin L., Department of Oral Biology, Medical School, University of Connecticut, Farmington, CT 06032 Tasaki, lehiji. Laboratory of Neurobiology, Bldg. 36, Rm. 2B- 16, NIMH/NIH, 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, William H., Department of Biology, University of Pennsylvania, Philadelphia, PA 19104 Telzer, Bruce, Department of Biology, Pomona College. Claremont. CA9171I Thorndike, W. Nicholas, Wellington Management Company, 28 State St., Boston, MA 02109 Trager, William, Rockefeller University, 1230 York Ave., New York, NY 10021 Travis, D. M., Veterans Administration Medical Center, 2101 Elm Street, Fargo, ND 58 102 Treistman, Steven N., Worcester Foundation for Experimental Biology, 222 Maple Avenue, Shrewsbury. MA 01 545 Trigg, D. Thomas, 1 25 Grove St.. Wellesley. MA 02 1 8 1 I rinkaus, .). Philip, Department of Biology. Box 6666, Yale University. New Haven, CT 06510 Troll, Walter, Department of Environmental Medicine, College of Medicine, New York University, New York, NY 10016 Troxler, Robert F., Department of Biochemistry, School of Medicine, Boston University, 80 East Concord St., Boston. MA 021 18 Tucker, Edward B., 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 02 138 Tweedell, Kenyon S., Department of Biology, 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 01 545 Valois, John, Marine Biological Laboratory. Woods Hole, MA 02543 Van Molde, Kensal, Department of Biochemistry and Biophysics, Oregon State University, Corvallis, OR 9733 1- 6503 Vincent, Walter S., 16 F. R. Lillie Road, Woods Hole. MA 02543 Waksman. Byron, National Multiple Sclerosis Society. 205 East 42nd St.. New York. NY 10017 Wall, Betty, 9 George St., Woods Hole, MA 02543 Wallace, Robin A., Whitney Laboratory, 9505 Ocean Shore Blvd.. St. Augustine. FL 32086 Wang, An, Wang Laboratories. Inc.. One Industrial Ave., Lowell, MA 01851 (deceased) Wang, Ching Chung, Department of Pharmaceutical Chemistry. LIniversity of California, San Francisco, CA 94143 Warner, Robert C., Department of Molecular Biology and Biochemistry, University of California, Irvine, CA 927 1 7 Warren, Kenneth S., Maxwell Communications Corp., 866 Third Avenue. New York, NY 10022 Warren, Leonard, Wistar Institute, 36th and Spruce Streets. Philadelphia, PA 19104 Waterbury, John B., Department of Biology, Woods Hole Oceanographic Institution, Woods Hole, MA 02543 Associate Members 69 Watson, Stanley, Associates of Cape Cod, Inc., P. O. Box 224, Woods Hole, MA 02543 Waxman, Stephen G., Department of Neurology, LCI 708, Yale School of Medicine, 333 Cedar Street, New Haven, CT 06510 \Yebb, II. Marguerite, Marine Biological Laboratory, Woods Hole, MA 02543 Weber, Annemarie, Department ot Biochemistry and Biophysics, School of Medicine. University of Pennsylvania, Philadelphia, PA 19104 \Yebster, Ferris, Box 765, Lewes, DE 19958 (resigned 8/14/ 89) Weidner, Karl, Department of Zoology and Physiology, Louisiana State University, Baton Rouge, LA 70803 Weiss, Dieter, G., Institut fur Zoologie, Technische LIniversitat Munchen. 8046 Garching, FRG Weiss, Leon P., Department of Animal Biology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA 19104 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 White, Roy I... Department of Neuroscience, Albert Einstein College, 1300 Morris Park Avenue, Bronx. NY 10461 (resigned 8/1 4/89) Whittaker, J. Richard, Marine Biological Laboratory. Woods Hole. MA 02543 Wigley, Roland L., 35 Wilson Road, Woods Hole. MA 02543 Wilson, Darcy B., Medical Biology Institute, 1 1077 North Torrey Pines Road, La Jolla. CA 92037 Wilson, Kdward O., Museum of Comparative Zoology, Harvard University. Cambridge. MA 02 1 38 Wilson, I. Hastings, Department of Physiology, Harvard Medical School, Boston, MA 02 1 1 5 Witkovsky, Paul, Department of Ophthalmology, New York LIniversity Medical Center, 550 First Ave., New York, NY 10016 Wittenberg, Jonathan B., Department of Physiology and Biochemistry. Albert Einstein College, 1300 Morris Park Ave.. Bronx, NY 01 461 Wolfe, Ralph, Department of Microbiology, 131 Burrill Hall, University of Illinois, Urbana. IL 61801 Wolken, Jerome J., Department of Biological Sciences, Carnegie Mellon University, 440 Fifth Ave., Pittsburgh. PA 15213 Worgul, Basil V., Department of Ophthalmology, Columbia University. 630 West 168th St.. New York. NY 10032 Wu, Chau Hsiung, Department of Pharmacology, Northwestern LIniversity Medical School, Chicago, IL 606 1 1 Wyttenbach, Charles R., Department of Physiology and Cell Biology, University of Kansas, Lawrence, KS 66045 Y'eh, Jay Z., Department of Pharmacology. Northwestern University Medical School, Chicago, IL 6061 1 Young, Richard, Mentor O & O, Inc.. 3000 Longwater Dr.. Norwell. MA 0206 1 (resigned 10/5/89) Zackroff, Robert, 80 Kersey Rd.. Peacedale, RI 02883 (resigned 8/1 4/89) Zigman, Seymour, School of Medicine and Dentistry. University of Rochester. 260 Crittenden Blvd., Rochester, NY' 14620 Zigmond, Richard E., Center for Neurosciences, School of Medicine. Case Western Reserve University. Cleveland, OH 44106 Zimmerberg, Joshua J., Bldg. 12A, Room 2007, NIH, Bethesda, MD 20892 Zottoli, Steven J., Department of Biology, Williams College, Williamstown, MA01267 Zucker, Robert S., Neurobiology Division. Department of Molecular and Cellular Biology, University of California, Berkelev, CA 94720 Associate Members Ackroyd. Dr. Frederick W. Adams. Dr. Paul Adelherg, Dr. and Mrs. Edward A. Ahearn, Mr. and Mrs. David Alden, Mr. John M. Allard, Dr. and Mrs. Dean C, Jr. Allen, Miss Camilla K.. Allen, Dr. Nina S. Amon, Mr. Carl H. Jr Anderson, Mr. J. Gregory Anderson, Drs. James L. and Helene M. Antonucci. Dr. Robert V. Armstrong, Dr. and Mrs. Samuel C. Arnold. Mrs. Lois An/in, Ms. Kara L. Aspinwall. Mr. and Mrs. Duncan Atwood. Dr. and Mrs. Kimhall C. Ill Avers, Mrs. Donald Backus, Mrs. Nell Baker, Mrs. C. L. Ball, Mrs. Eric G. Ballantine. Dr. and Mrs. H. T., Jr. Bang, Mrs. Frederik B. Bang, Miss Molly Banks, Mr. and Mrs. William L. Barkan. Mr. and Mrs. Mel A. Barrows, Mrs. Albert W. Baum. Mr. Richard T. Baylor, Drs. Edward and Martha Beers. Dr. and Mrs. Yardley Belesir. Mr. Tasos Bennett, Drs. Michael and Ruth Berg, Mr. and Mrs. C. John Bernheimer, Dr. Alan W. Bernstein, Mr. and Mrs. Norman Bicker, Mr. Alvin Bigelow, Mr. and Mrs. Robert O. Bird. Mr. William R. Bishop, Mrs. John Bleck, Dr. Thomas B. Boche, Mr. Robert Bodeen, Mr. and Mrs. George H. Boettiger, Dr. and Mrs. Edward G. Boettiger, Mrs. Julie Bolton, Mr. and Mrs. Thomas C. Bonn. Mr. and Mrs. Theodore H. Borg, Dr. and Mrs. Alfred F. Borgese, Dr. and Mrs. Thomas Bowles, Dr. and Mrs. Francis P. Bradley, Dr. and Mrs. Charles C. Bradley, Mr. Richard Brown, Mrs. Frank A.. Jr. Brown, Mr. and Mrs. Henry Brown, Mr. James Brown. Mrs. Neil Brown. Mr. and Mrs. T. A. Brown, Dr. and Mrs. Thornton Broyles. Dr. Robert H. Buck. Dr. and Mrs. John B. Buckley, Mr. George D. Bunts. Mr. and Mrs. Frank E. 70 Annual Report Bun. Mrs. Charles E. Burwell, Dr. and Mrs. E. Langdon Bush, Dr. Louise Buxton, Mr. and Mrs ! Buxton. Mr. E. Brew Cadualader. Mr. ( Calkins, Mr. and \lrs.G.N.Jr. Campbell. Dr. an '. Mrs. David G. Carlson. Dr and Mrs. Francis Carlton, Mr. and Mrs. Winslow G. Case, Dr. and Mrs. James Chandler. Mr. Robert Chase. Mr. Thomas H. Child, Dr. and Mrs. Frank M.. Ill Chisholm, Dr. Sallie W. Church. Dr. Wesley Claff. Mr. and Mrs. Mark Clark, Dr. and Mrs. Arnold Clark, Mr. and Mrs. Hays Clark, Mr. James McC. Clark. Mr. and Mrs. Leroy. Jr. Clark, Dr. Peter L. Clarke. Dr. Barbara J. Clement, Mrs. Anthony C. Cloud. Dr. Laurence P. Clowes Fund. Inc. Clowes. Dr. and Mrs. Alexander W. Clowes. Mr. Allen W. Clowes. Mrs. G. H. A., Jr. Cobb, Dr. Jewel P. Collier, Mr. Christopher Coburn. Mr. and Mrs. Lawrence Cohen, Mrs. Seymour S. Coleman, Drs. John and Annette Collum. Mrs. Peter Colt. Dr. LeBaronC.Jr. Connell, Mr. and Mrs. W. J. Cook, Dr. and Mrs. Joseph Cook, Dr. and Mrs. Paul W., Jr. Copel. Mrs. Marcia N. Copeland. Dr. and Mrs. D. Eugene Copeland, Mr. Frederick C. Copeland, Mr. and Mrs. Preston S. Costello. Mrs. Donald P. Cowan. Mr. and Mrs. James F., Ill Crabb. Mr. and Mrs. David L. Crain, Mr. and Mrs. Melvin C. Cramer, Mr. and Mrs. Ian D. W. Crane. Mrs. John O. Crane, Josephine B.. Foundation Crane, Mr. Thomas S. Crosby. Miss Carol Cross, Mr. and Mrs. Norman C. Crossley, Miss Dorothy Crossley. Miss Helen Crowell, Dr. and Mrs. Sears Currier. Mr. and Mrs. David L. Daignault. Mr. and Mrs. Alexander T. Daniels. Mr. and Mrs. Bruce G. Davidson, Dr. Morton Davis, Mr. and Mrs. Joel P. Day, Mr. and Mrs. Pomerov Decker. Dr. Raymond F. DeMello. Mr. John DiBerardino. Dr. Marie A. DiCecca. Dr. and Mrs. Charles Dickson, Dr. William A. Dierolf, Dr. Shirle> H Donovan. Mr. David L. Dorman. Dr. and Mrs. Craig Dreyer, Mrs. Frank Drummey. Mr. and Mrs. Charles E. Drummey, Mr. Todd A. DuBois. Dr. and Mrs. Arthur B Dudley, Dr. Patricia DuPont, Mr. A Felix. Jr. Dutton, Mr. and Mrs. Roderick L. Ebert, Dr. and Mrs. James D. Egloft". Dr. and Mrs. F. R. 1 .. Elliott. Mrs. Alfred M. Enos. Mr. Edward. Jr. Eppel. Mr. and Mrs. Dudley Epstein. Mr. and Mrs. Ray L. Estabrooks, Mr. Gordon C. Evans. Mr. and Mrs. Dudley Farley, Miss Joan Farmer, Miss Mary Faull. Mr. J. Horace, Jr. Ferguson, Mrs. James J., Jr. Fisher, Mrs. B. C. Fisher, Mr. Frederick S.. Ill Fisher. Dr. and Mrs. Saul H. Fluck, Mr. Richard A. Folino, Mr. John W., Jr. Forbes, Mr. John M. Ford, Mr. John H. Fowlkes. Mr. Aaron Francis. Mr. and Mrs. Lewis W.. Jr. Frenkel. Dr. Rrystina Fribourgh. Dr. James H Friendship Fund Fries. Dr. and Mrs. E. F. B. Frosch, Dr. and Mrs. Robert A. Fye. Mrs. Paul M. Gabriel, Dr. and Mrs. Mordecai L. Gagnon. Mr. Michael Gaiser. Mrs. David W. Gallagher. Mr. Robert O. Garcia. Dr. Ignacio Garneld. Miss Eleanor Gellis, Dr. and Mrs. Sydney Gephard. Mr. Stephen German. Dr. and Mrs. James L.. Ill Gewecke, Mr. and Mrs. Thomas H. Giflbrd. Mr. and Mrs. Cameron Giftord, Mr. John A. Gilford. Dr. and Mrs. Prosser Gilbert. Drs. Daniel L. and Claire Ciildea. Dr. Margaret C. L. Gillette. Mr. and Mrs. Robert S. Glad. Mr. Robert Glass. Dr. and Mrs. H. Bentley Glazebrook, Mr. James G. Glazebrook, Mrs. James R. Goldman. Mrs. Mary Goldring, Mr. Michael Goldstein, Dr. and Mrs. Moise ll.Jr. Goodgal. Dr. Sol H Goodwin, Mr. and Mrs. Charles Gould. Miss Edith Grace, Miss Priscilla B. Grant. Dr. and Mrs. Philip Grassle, Mrs.J.K. Green. Mrs. Davis Crane Greenberg. Noah and Mosher. Diane Greer. Mr. and Mrs. W. H.. Jr. Griffin, Mrs. Robert W. Griffith. Dr. and Mrs. B. Herold Grosch, Dr. and Mrs. Daniel S. Gross. Mrs. Mona Gunning, Mr. and Mrs. Robert Haakonsen, Dr. Harry O. Haigh, Mr. and Mrs. Richard H. Hall. Mr. and Mrs. Peter A. Hall. Mr. Warren C. Halvorson, Dr. and Mrs. Harlyn O. Hamstrom. Miss Mary Elizabeth Harrington. Mr. Robert D.. Jr. Harrington, Mr. Robert B. Harvey. Dr. and Mrs. Richard B. Hassett, Mr. and Mrs. Charles Hastings. Dr. and Mrs. J. Woodland Haubrich. Mr. Robert R. Hay. Mr. John Hays, Dr. David S. Heaney. Mr. John D. Hedberg, Mrs. Frances Hedberg. Dr. Man.' Hersey, Mrs. George L. Hiatt. Dr. and Mrs. Howard Hichar, Mrs. Barbara Hill. Mrs. Samuel E. Hirschfeld. Mrs. Nathan B. Hobbie. Dr. and Mrs. John Hocker. Mr. and Mrs. Lon Hodge. Mr. and Mrs. Stuart Hokin. Mr. Richard Hornor, Mr. Townsend Horwitz. Dr. and Mrs. Norman H. Hoskin. Dr. and Mrs. Francis C.G. Houston, Mr. and Mrs. Howard E. Howard, Mrs. L. L. Hoyle, Dr. Merrill C. Huckle. Mrs. Eleanor L. Huettner. Dr. and Mrs. Robert .1. Hutchison. Mr. Man D. Hyde. Mr. and Mrs. Robinson Hynes. Mr. and Mrs. Thomas J.. Jr. Inoue. Dr. and Mrs. Shinya Issokson. Mr. and Mrs. Israel Jackson. Miss Elizabeth B. Jafle. Dr. and Mrs. Ernst R. Janney. Mrs. F. Wistar Jewell. G. F.. Foundation Jewell. Mr, and Mrs. G. F., Jr. Jewell. Mr. and Mrs. Raymond L. Jones. Mr. and Mrs. DeWilt C.. Ill Jones, Mr. and Mrs. Frederick. II Jones, Mr. Frederick S.. Ill Jordan. Dr. and Mrs. Edwin P. Raan. Dr. Helen W. Kahler, Mrs. Robert W. Rammer. Dr. and Mrs. Benjamin Karplus. Mrs. Alan K. Karush, Dr. and Mrs. Fred Kelleher. Mr. and Mrs. Paul R. Kendall, Mr. and Mrs. Richard E. Keosian. Mrs. Jessie Keoughan, Miss Patricia Retchum. Mrs. Paul Kinnard. Mrs. L. Richard Kirschenbaum. Mrs. Donald Kissam, Mr. and Mrs. William M. Ki\ y. Dr. and Mrs. Peler Roller. Dr. Lewis R. Rorgen, Dr. Ben J. Rravilz. Dr. and Mrs. Edward Ruffler. Mrs. Slephen W. Laderman. Mr. and Mrs. Ezra Latterly. Miss Nancy Lahner, Mrs. Alia S. Larmon, Mr. Jay Lasler. Dr. and Mrs. Leonard Latham. Miss Eunice Lauter, Dr. and Mrs. Hans Laufer, Jessica, and Weiss, Malcolm LaVigne. Mrs. Richard .1. Lawrence. Mr. Frederick V. Lawrence, Mr. and Mrs. William Leach. Dr. Berton J. Lealherbee. Mrs. John H. LeBlond, Mr. and Mrs. Arthur Leeson. Mr. and Mrs. A. Di.x Associate Members 71 LeFevre. Dr. Marian E. Lehman, Miss Robin Lenher. Dr. and Mrs. Samuel Leprohon. Mr. Joseph Levine, Mr. Joseph Levine. Dr. and Mrs. Rachmiel Levitz. Dr. Mortimer Levy, Mr. and Mrs. Stephen R. Lindner, Mr. Timothy P. Little, Mrs. Elbert Livingstone, Mr. and Mrs. Robert Lloyd. Mr. and Mrs. James Loeb, Mrs. Robert F. Loessel, Mrs. Edward Lovell. Mr. and Mrs. Hollis R. Levering. Mr. Richard C. Low, Miss Dons Lowe, Dr. and Mrs. Charles U. Lowengard, Mrs. Joseph Mackey, Mr. and Mrs. William K. MacLeish, Mrs. Margaret MacNary, Mr. and Mrs. B. Glenn MacNichol, Dr. and Mrs. Edward F.. Jr. Maddigan. Mrs. Thomas Maher. Miss Anne Camille Mahler. Mrs. Henry Mahler. Mrs. Suzanne Mansworth, Miss Marie Maples. Dr. Philip B Marsh. Dr. and Mrs. Julian Martyna, Mr. and Mrs. Joseph C. Mason. Mr. Appleton Mastroianni, Dr. and Mrs. Luigi, Jr. Mather, Mr. and Mrs. Frank J., Ill Matherly. Mr. and Mrs. Walter Matthiessen, Dr. and Mrs. G. C. McCusker, Mr. and Mrs. Paul T. McElroy, Mrs. NellaW. Mcllwain. Dr. Susan G. McMurtrie. Mrs. Cornelia Hanna Meigs. Mr. and Mrs. Arthur Meigs, Dr. and Mrs. J. Wister Melillo, Dr. and Mrs. Jerry M. Mellon, Richard Ring, Trust Mellon, Mr. and Mrs. Richard P. Mendelson, Dr. Martin Metz, Dr. and Mrs. Charles B. Meyers, Mr. and Mrs. Richard Miller. Dr. Daniel A. Miller, Mr. and Mrs. Paul Mills, Mrs. Margaret A. 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. Murray, Mr. David M. Myles-Tochko, Drs. Christina J. and John Nace, Dr. and Mrs. Paul Nace. Mr. Paul F., Jr. Neall. Mr. William G. Nelson. Dr. and Mrs. Leonard Nelson, Dr. Pamela Newton. Mr. William F. Nickerson, Mr. and Mrs. Frank L. Norman. Mr. and Mrs. Andrew E. Norman Foundation Norris, Mr. and Mrs. Barry Norris, Mr. and Mrs. John A. Norris, Mr. William Norton, Mrs. Thomas J. 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 Pappas, Dr. and Mrs. George D. Park. Mr and Mrs. Malcolm S. Parmenter. Dr. Charles Parmenter. Miss Carolyn L. Pearee. Dr. John B. Pearson. Mrs. Oscar H. Peltz, Mr. and Mrs. William L. Pendergast. Mrs. Claudia Pendleton. Dr. and Mrs. Murray E. Pen, Mr. and Mrs. John B. Perkins. Mr. and Mrs. Courtland D Person, Dr. and Mrs. Philip Peterson. Mr. and Mrs. E. Gunnar Peterson, Mr. and Mrs. E. Joel Peterson, Mr. Raymond W. Petty, Mr. Richard F. Pfeiffer. Mr. and Mrs. John Plough. Mr. and Mrs. George H. Plough, Mrs. Harold H. Pointe, Mr. Albert Pointe. Mr. Charles Porter. Dr. and Mrs. Keith R. Pothier. Dr. and Mrs. Aubrey Press, Drs. Frank and Billie Proskauer. Mr. Joseph H. Proskauer, Mr. Richard Prosser, Dr. and Mrs. C. Ladd Psaledakis, Mr. Nicholas Psychoyos, Dr. Alexandre Putnam, Mr. and Mrs. Allan Ray Putnam, Mr. and Mrs. William A.. Ill Rankin, Mrs. John Raphael, Ms. Ellen S. Raymond, Dr. and Mrs. Samuel Reese. Miss Bonnie Regis, Ms. A. Kathy Remgold, Mr. Stephen C. Reynolds. Dr. and Mrs. George Reynolds, Dr. John 1 Reynolds. Mr. and Mrs. Robert M. Reznikoff, Mrs. Paul Ricca, Dr. and Mrs. Renato A. Righter. Mr. and Mrs. Harold Riley, Dr. Monica Rnna, Mr. John R. Robb, Mrs. Alison A. Roberts. Miss Jean Roberts, Mr. Mervin F. Robertson. Mrs. C. W. Rohmson, Dr. Denis M. Robinson. Mr. Marius A. Root, Mrs. Walter S. Rosenthal, Miss Hilde Roslansky. Drs. John and Pnscilla Ross. Dr. and Mrs. Donald Ross. Dr. Robert Ross, Dr. Virginia Roth. Dr. and Mrs. Stephen Rowan. Mr. Edward Rowe, Dr. Don Rowe, Mrs. William S. Rugh, Mrs. Roberts Ryder, Mr. and Mrs. Francis C. Sager. Dr. Ruth 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 Schuamb. 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 Segal, Dr. and Mrs. Sheldon J. Selby. Dr. Cecily Senft. Dr. and Mrs. Alfred Shanklin, Mr. D. R. Shapiro. Mr. and Mrs. Howard Shapley. Dr. Robert Shemin, Dr. and Mrs. David Shepro. Dr. and Mrs. David Sherblom. Dr. James P. Siehel. Dr. Enid Siegel, Mr. and Mrs. Alvin Simmons, Mr. Tim Singer, Mr. and Mrs. Daniel M. Smith. Drs. Fredenck E. and Marguerite A. Smith, Mrs. Homer P. Smith. Mr. Van Dorn C. Snyder. Mr. Robert M. Solomon. Dr. and Mrs. A. K. Sonnenblick. Mrs. Perle Speck, Dr. William T. Specht, Mr. and Mrs. Heinz Spiegel. Dr. and Mrs. Melvin Spotte, Mr. Stephen Steele. Mrs. John H. Steele, Dr. Robert E. Stein, Mr. Ronald Steinbach. Mrs. H. Bun- Stetson. Mrs. Thomas J. Stetten, Dr. Gail Stetten, Dr. and Mrs. H. DeWitt. Jr. Stunkard. Dr. Horace Sudduth, Dr. William Swanson. Mrs. Carl P. Swope. Mrs. Gerard. Jr. Swope, Mr. and Mrs. Gerard L. Szent-Gyorgyi, Dr. Andrew Taber, Mr. George H. Taylor. Mr. James K. Taylor, Dr. and Mrs. W. Randolph Tietje, Mr. and Mrs. Emil D.. Jr. Timmins. Mrs. William Todd, Mr. and Mrs. Gordon F. Tolkan. Mr. and Mrs. Norman N. Trager. Mrs. William Trigg, Mr. and Mrs. D. Thomas Troll. Dr. and Mrs. Walter Trousof. Miss Natalie Tucker. Miss Ruth Tully. Mr. and Mrs. Gordon F. Ulbrich. 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. WalterS. 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. 72 Annual Report Weiffenbach. Dr. and Mrs. George Weinstein, Miss Nancy B. Weisberg. Mr. and Mrs. Alfred M Weissmann. Dr. and Mrs, i iKI Wheeler. Dr. and Mrs Paul S. Wheeler. Dr. Will; Whitehead. Mr. and Mrs. Fred Whitney. Mr. and Mrs. GeorTrey G.,Jr. Wichterman, Dr. and Mrs. Ralph Wickersham, Mr. and Mrs. A. A. Tilney Wiese. Dr. Konrad Wilbur. Mrs. Claire M. Wilhelm. Dr. Hazel S. Wilson. Mr. and Mrs. Leslie J. Wilson, Mr. and Mrs. T. Hastings Wmti. Dr. William M. Winsten, Dr. Jay A. Witting. Miss Joyce Woltinsohn, Mrs. Wolfe Woitkoski. Miss Nancy Woodwell. Dr. and Mrs. George M. Yntema, Mrs. Chester L. Young. Miss Nina L. Zinn. Dr. and Mrs. Donald J. Zipf. Dr. Elizabeth Gift Shop Volunteers Marian Adelberg Margaret German Lorraine Mizell Louise Atkins Rebeckah William Neall Barbara Atwood Glazebrook Bertha Person Patricia Barlow Michael Goldring Julia Rankin Gloria Borgese Rose Grant Lilyan Saunders Jennie Brown Martha Griffin John Saunders Kitty Brown Edith Grosch Elsie Scott Elisabeth Buck Jean Halvorson Deborah Sen ft Patricia Case Adele Hoskins Charlotte Shemin Summers Case Pauline Hyde Marilyn Shepro Julia Child Sona Jones Cynthia Smith Vera Clark Sally Karush Marguerite Smith Margaret Clowes Barbara Little Judith Stetson Elizabeth Daignault Sarah Loessel Barbara van Holde Janet Daniels Winnie Mackey Barbara Alma Ebert Constance Martyna Whitehead Elinor Gabriel Nella McElroy Clare Wilber MBL Tour Guides Betsy Bang Teru Hayashi 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 Minot being a majority of the Trustees of the Marine Biological Laboratory 1 in compli- ance 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 H'HHf.vv Whereof, we have hereunto set our hands, this twenty seventh day of February in the year eighteen hundred and eighty-eight, Alpheus Hyatt, Samuel Mills. William T. Sedgwick, Edward G. Gardiner. Charles Sedgwick Minot, Wil- liam G. Farlow. William Stanford Stevens. Anna D. Phillips. Susan Mims, B. H. Van Vleck. That the first meeting of the subscribers to said agreement was held on the thir- teenth day of March in the year eighteen hundred and eighty-eight. In H'Hi'\\ H'/UTCC/, 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. 1 888 as follows: / hereby certify that it appears upon an examination of the within written certifi- cate and the records of the corporation duly submitted to my inspection, that the requirements of sections one. two and three of chapter one hundred and fifteen, and sections eighteen, twenty and twenty-one of chapter one hundred and six. of the Public Statutes, have been complied with and I hereby approve said certificate this twentieth day of March A.D. eighteen hundred and eighty-eight. Charles Endicott (_ 'on r ol Corpt>>'Lini>tt\} A rticles of A mend men t (On File in the Office of the Secretary of the Commonwealth) We, James D. Ebert. President, and David Shepro, Clerk of the Manne 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. 1175. as adjourned to August 24, 1175. 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 liabil- ities incurred as a result of. or otherwise in connection with, any commit- ments, agreements, activities or affairs of the corporation. "Except as otherwise specifically provided by the Bylaws of the corpora- tion, meetings of the Corporate Members of the corporation may be held anywhere in the L'nited 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 provi- sions thereof which shall by law, this Certificate or the bylaws of the corpo- ration, require action by the Corporate Members." The foregoing amendment will become effective when these articles of amend- ment are filed in accordance with Chapter 1 80, Section 7 of the General Laws unless these articles specify, in accordance with the vote adopting the amend- ment, a later effective date not more than thirty days after such filing, in which event the amendment will become effective on such later date. //; ir//wv,v iv/ic/vo/ ami I /; shall have determined, not inconsistent with law or these By- laws. (B) The Board of Trustees shall also have the power, by vote of a majority of the Trustees then in Office, to elect an Investment Committee and any other committee and. by like vote, to delegate thereto some or all of their powers except those which by law. the Articles of Organization or these Bylaws they are prohib- ited from delegating. The members of any such committee shall have tenure and duties as the Trustees shall determine; provided that the Investment Committee, which shall oversee the management of the Corporation's endowment funds and marketable securities, shall include the Chairman of the Board of Trustees, the Treasurer of the Corporation, and the Chairman of the Corporation's Budget Committee, asex officio members, together with such Trustees as may be required for not less than two-thirds of the Investment Committee to consist of Trustees. Except as otherwise provided by these Bylaws or determined by the Trustees, any such committee may make rules for the conduct of its business; but. unless otherwise provided by the Trustees or in such rules, its business shall be conducted as nearly as possible in the same manner as is provided by these Bylaws for the Trustees. X. (A) The Executive Committee is hereby designated to consist of nol more than ten members, including the ex officio Members (Chairman of the Board of Trustees, President, Director, and Treasurer); and six additional Trustees, two of whom shall be elected by the Board of Trustees each year, to serve for a three- year term. Beginning with the members elected for terms ending in 1 WO, one of the Trustees elected to serve on the Executive Committee should be a Trustee-at- large. This procedure will be repeated in the class of I'M!, and henceforth the Trustees will elect to the Executive Committee Trustees to ensure that the compo- sition of the Committee is four Corporate Trustees and two Trustees-at-large. (B) The Chairman of the Board of Trustees shall act as Chairman of the Execu- tive Committee, and the President as Vice Chairman. A majonty of the members of the Executive Committee shall constitute a quorum and the affirmative vote of a majority of those voting at any meeting at which a quorum is present shall constitute action on behalf of the Executive Committee. The Executive Commit- tee shall meet at such times and places and upon such notice and appoint such subcommittees as the Committee shall determme. (C) The Executive Committee shall have and may exercise all the powers of the Board during the intervals between meetings of the Board of Trustees except those powers specifically withheld from time to time by vote of the Board or by law. The Executive Committee may also appoint such committees, including per- sons who are not Trustees, as it may from time to time approve to make recom- mendations with respect to matters to be acted upon by the Executive Committee or the Board of Trustees. (D) The Executive Committee shall keep appropnate minutes of its meetings and its action shall be reported to the Board of Trustees. (E) The elected Members of the Executive Committee shall constitute a stand- ing "Committee for the Nomination of Officers," responsible for making nomina- tions, at each Annual Meeting of the Corporation, and of the Board of Trustees, for candidates to fill each office as the respective terms of office expire (Chairman of the Board. President, Director, Treasurer, and Clerk). XI. A majority of the Trustees, the Executive Committee, or any other com- mittee elected by the Trustees shall constitute a quorum; and a lesser number than a quorum may adjourn any meeting from time to time without further notice. At any meeting of the Trustees, the Executive Committee, or any other committee elected by the Trustees, the vote of a majority of those present, or such different vote as may be specified by law, the Articles of Organization or these Bylaws, shall be sufficient to take any action. XII. Any action required or permitted to be taken at any meeting of the Trust- ees, the Executive Committee or anv other committee elected by the Trustees as referred to under Article IX may be taken without a meeting if all of the Trustees or members of such committee, as the case may be, consent to the action in writ- ing and such wntten consents arc tiled with the records of meetings. The Trustees or members of the Executive Committee or any other committee appointed by the Trustees may also participate in meeting by means of conference telephone, or otherwise take action in such a manner as may from time to time be permitted h> la XIII. The consent of every Trustee shall he necessary to dissolution of the Ma- nne Biological Laboratory. In case of dissolution, the property shall be disposed of in such a manner and upon such terms as shall be determined by the affirmative vote of two-thirds of the Board of Trustees then in office. XIV. These Bylaws may be amended by the affirmative vote of the Members at any meeting, provided that notice of the substance of the proposed amendment is stated in the notice of such meeting. As authorized by the Articles of Organiza- tion, the Trustees, by a majority of their number then in office, may also make, amend, or repeal these Bylaws, in whole or in part, except with respect to (a) the provisions of these Bylaws governing (i) the removal of Trustees and (ii) the amendment of these Bylaws and (b) any provisions of these Bylaws which by law, the Articles ot Organization or these Bylaws, requires action by the Members. No later than the time of giving notice of the meeting of Members next follow- ing the making, amending or repealing by the Trustees of any Bylaw, notice thereof stating the substance of such change shall be given to all Corporation Members entitled to \ote on amending the Bylaws. Any Bylaw adopted by the Trustees may be amended or repealed by the Mem- bers entitled to \otc on amending the Bylaws. XV. The account of the Treasurer shall be audited annually by a certified pub- lic accountant. XVI Except as otherwise provided below, the Corporation shall, to the extent legally permissible, indemnify each person who is. or shall have been, a Trustee, director or officer of the Corporation or who is serving, or shall have served, at the request of the Corporation as a Trustee, director or officer of another organiza- tion in which the Corporation directly or indirectly has any interest, as a share- holder, creditor or otherwise, against all liabilities and expenses (including judg- ments, fines, penalties and reasonable attorneys' fees and all amounts paid, other than to the Corporation or such other organization, in compromise or settlement) imposed upon or incurred by any such person in connection with, or arising out of. the defense or disposition ol any action, suit or other proceeding, whether civil or criminal, in which he or she may be a defendant or with which he or she may be threatened or otherwise involved, directly or indirectly, by reason of his or her being or having been such a Trustee, director or officer. The Corporation shall provide no indemnification with respect to any matter as to which any such Trustee, director or officer shall be finally adjudicated in such action, suit or proceeding not to have acted in good faith in the reasonable belief that his or her action was in the best interests of the Corporation. The Cor- poration shall provide no indemnification with respect to any matter settled or compromised, pursuant to a consent decree or otherwise, unless such settlement or compromise shall have been approved as in the best interests of the Corpora- tion, after notice that indemnification is involved, by (i) a disinterested majonty of the Board of Trustees or of the Executive Committee or, (ii) a majority of the Corporation's Members. Indemnification may include payment by the Corporation of expenses in de- fending a civil or criminal action or proceeding in advance of the final disposition of such action or proceeding upon receipt of an undertaking by the person indem- nified to repay such payment if it is ultimately determined that such person is not entitled to indemnification under the provisions of this Article XVI, or under any applicable law. As used in this Article, the terms "Trustee," "director" and "officer" include their respective heirs, executors, administrators and legal representatives, and an "interested" Trustee, director or officer is one against whom in such capacity the proceeding in question or another proceeding on the same or similar grounds is then pending. To assure indemnification under this Article of all persons who are determined by the Corporation or otherwise to be or to have been "fiduciaries" ol any em- ployee benefit plan of the Corporation which may exist from time to time, this 76 Annual Report Article shall be interpreted as follows: 1 1 1 "another organization" shall be deemed to include such an employee benefit plan, including without limitation, any plan of the Corporation which is govcrtu-il b, the Act of Congress entitled "Employee Retirement Income Secuni 974." as amended from time to time ("ER- ISA");(ii) "Trustee" shall I i.\l to include any person requested by the Cor- poration to serve as si iplmoe benefit plan where the performance by such person of his . luties to the Corporation also imposes duties on. or otherwise m\ol\c>. Mich person to the plan or participants or benefi- ciaries of the plan: (iii) ' lines" shall be deemed to include any excise taxes assessed on a person with respect to an employee benefit plan pursuant to ERISA; and (iv) actions taken IT omitted by a person with respect to an employee benefit plan in the pertormaiv eol such person's duties fora purpose reasonably believed by such person to he in the interest of the participants and beneficiaries of the plan shall be deemed to he for a purpose which is in the best interests of the Corporation. The nghl of indemnification provided in this Article shall not be exclusive of or ailed any other nghts to which any Trustee, director or officer may be entitled under any agreement, statute, vote of members or otherwise. The Corporation's obligation to provide indemnification under this Article shall be offset to the ex- tent of any other source ofindemmfication or any otherwise applicable insurance coverage under a policy maintained by the Corporation or any other person. Nothing contained in this Article shall affect any nghts to which employees and corporate personnel other than Trustees, directors or officers may be entitled by contract, by vote of the Board ofTrustees or of the Executive Committee or other- wise. XVII. There shall be no transfer of title or long-term lease of real property held by the MBL Corporation without pnor approval of two-thirds of the full Board of Trustees. Such real property transactions shall be presented and discussed at one meeting of the Board and finally acted upon at a subsequent meeting of the Board. Either meeting could be a special meeting and no less than four weeks should elapse between these meetings. Reference: Biol. Bull 179: 77-86. (August, 1990) Hsr-omega, A Novel Gene Encoded by a Drosophila Heat Shock Puff* M. L. PARDUE, W. G. BENDENA 1 , M. E. FINI : , J. C. GARBE 3 , N. C. HOGAN, AND K. L. TRAVERSE Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139 Abstract. Although originally identified because of its abundant transcription in heat shock, the hsr-omega gene is active, at generally lower levels, in non-stressed cells. The locus produces an unusual set of three tran- scripts. Evidence from a variety of experiments suggests that one of these transcripts acts in the nucleus, possibly to regulate the activity of a nuclear protein. Another of the transcripts appears to act in the cytoplasm, possibly monitoring or regulating some aspect of translation. The two transcripts together could have a role in coordinating nuclear and cytoplasmic activity. A number of processes occur in eukaryotic cells in which nuclear and cytoplas- mic activities need to be coordinated; we suggest that hsr- omega plays a role in such coordination. Introduction Biologists have often found that a biological peculiar- ity in one organism can be exploited to study general questions that apply to many organisms. The polytene chromosomes that are found in some cells in Drosophila and some other organisms are examples of such a biolog- ical peculiarity. Studies of these chromosomes have con- tributed significantly to our understanding of how gene activity changes during development or in response to various agents (see Beermann, 1972; Hennig, 1987). Polytene chromosomes are giant chromosomes, made up of many chromatids lying side by side in precise align- ment. Although polytene chromosomes are condensed * This paper is based on a Friday Evening Lecture delivered at the Marine Biological Laboratory. Woods Hole, MA on 30 June 1989. Present addresses: 'Department of Biology. Queen's University, Kingston, Ontario, Canada K7L 3N6. 2 Eye Research Institute of the Retina Foundation, Boston, MA 02 1 14, and 'Department of Genetics, University of California, Berkeley, CA 94720. enough to allow cytological mapping, they are interphase chromosomes and thus allow us to actually see, in situ, chromatin structures involved in transcription and DNA replication. When a gene is being very actively tran- scribed, the site of that gene frequently undergoes a local- ized puffing of the many DNA strands that make up the chromosomes. Many transcribed regions do not make detectable puffs, but when a puff is seen, it is always a sign of very active transcription. A new puff indicates that transcription at the puff site has either been turned on, or turned up very sharply (Bonner and Pardue, 1977). About 25 years ago, F. Ritossa ( 1 962, 1 964) found that when Drosophila larvae were placed at 37C for a short period, nine new puffs were induced, suggesting that this heat shock induced nine new genes (Fig. 1). The genes were scattered over the chromosomes but still seemed to be controlled coordinated. Further, the same set of genes could be induced by other kinds of stresses, including a wide variety of chemicals. This induction was reversible; the puffs regressed as soon as the stress was removed. This was clearly a fascinating set of genes, but a good many years were to pass before it became technically pos- sible to find out what the genes coded for. When that happened, the puffs were found to encode a small set of proteins now called the heat shock proteins, or hsps (see Ashburner and Bonner, 1979). The name "heat shock" is really a historical one because the proteins were first identified after a 37C heat shock. We know now that these proteins are also induced by a variety of stresses and they are sometimes called the stress proteins. More recently it has become apparent that all of the hsps also have roles in non-stressed cells (for recent reviews on heat shock see Lindquist, 1986; Lindquist and Craig, 1988; Pardue el ai, 1988). In some cases, the hsps in non-stressed cells are encoded by the same genes that 77 78 M. L. PARDUE ET AL show increased activity in response to heat shock. In other cases, the hsps may be encoded by closely related genes, some activated by heat shock and some regulated in other ways. After hsps had been identified in Drosophila, it be- came evident th.^ 11 organisms make very similar sets of stress protein--. Animals, plants, and bacteria all show this heat shock , csponse, so presumably the response has been around almost as long as cells have. The conserva- tion of the major hsps is striking. For example, the hsp70 of Drosophila has 48% amino acid identity with the equivalent lisp of E. coli (Bardwell and Craig. 1984). Al- though the major proteins produced in the heat shock response are strongly conserved, the stimuli that induce the response vary from organism to organism and reflect the conditions under which the organism lives. For in- stance, the temperatures that induce heat shock in Dro- sophila cells are well below those that heat shock mam- malian cells. This is not surprising because Drosophila cells usually live 10-20 degrees below the temperature of mammalian cells. Evolutionary conservation argues that the heat shock response is very important, yet we know only a little about how it helps the organism. Clearly the response helps cells endure, for a short time, temperatures slightly above what they normally tolerate. If cells are subjected to a mild heat shock and make a low level of heat shock proteins and RNA, then they can survive temperatures that would kill them if they were moved directly to those temperatures. We do not know how any of the hsps protect the cell from the heat shock, but studies of these proteins in non- stressed cells are showing that the hsps have very interest- ing roles in normal cells in addition to their roles in stressed cells. Three major families of hsps are now known to be conserved in plants, animals, and bacteria. For each of these families there is evidence suggesting that members act as "molecular chaperones." That is, these proteins appear to regulate the association of pro- teins with other macromolecules. The hsp70 family has many members; one of them has been implicated in translocation of secretory proteins into the endoplasmic reticulum and another appears to translocate proteins into mitochondria (Chirico el a/.. 1988; Deshaies et ai, 1988). The hsp 90 family (which includes the Drosophila hsp 82) appears to chaperone steroid hormone receptors and has also been found in association with some protein kinases (Catelli et ai, 1985). The bacterial hsp 70 is dna K, which is involved in protein-protein interactions in DNA replication (Georgopoulos et al.. 1989). A major bacterial hsp is groEL, which participates in bacterio- phage assembly, although it probably has other roles (Herendeen et ai, 1979). Recently groEL has been shown to be related to an animal mitochondria! protein B Figure 1. Drosophila melanogaster salivary gland chromosomes showing heat shock-induced puffing. (A) Part of chromosome 3 show- ing lour of the major heat shock purls (arrows, solid arrow indicates the 93D puff), x 900. (B) Higher magnification view of a 93D heat shock puff (arrows), xl 100. (C) The 93D region from a non-heat shocked larva. The region that puffs in heat shock is indicated by arrows, x 1 100. The chromosomes in A and B are from larvae that had been heat shocked at 36C for 30 min. that is increased on heat shock (McMullin and Hallberg, 1988) and to a chloroplast protein that is involved in as- sembling the oligomeric enzyme Rubisco (Hemmingsen etaL 1988). Thus the polytene puffs have revealed the existence of perhaps the most basic cellular response to stress and led to extensive study of this homeostatic mechanism. In turn, the studies of heat shock have accelerated our un- derstanding of a new class of proteins, the chaperonins. The polytene puffs have still more to tell us. For instance, all of the known Drosophila heat shock protein genes have been found. As expected, the genes are in the heat shock puffs, but there are still a few heat shock puffs with no known function. One that has intrigued us especially is in region 93D in D. melanogaster. HSR-OMl-XiA. A NOVEL HEAT SHOCK GENE 7') 3 O Figure 2. The transcripts of hsr-omega. The autoradiogram shows total RNA from control (lane C) and heat shocked (lane H) cultured Dro.tophila cells. The RNA has been gel fractionated, transferred to a filter, and hybridized with 3: P-labeled probe containing the sequence of transcript omega 3 (and therefore complementary to a portion of the sequences of omega 1 and omega 2. also). Longer exposures of the autoradiogram show that all three transcripts are present in the control cells (Garbe el ai. 1896). The top diagram shows the transcribed region of the gene. The shaded areas indicate the "unique portion" of the gene, much of which is incorporated in the cytoplasmic omega 3 transcript. The white area represents the intron that is spliced out in the processing of omega 3. The striped area represents the region of small tandem direct repeats. The two arrows mark the polyadenylation signals used in the processing of omega 2 (left arrow) and omega I (right arrow). The three transcripts are diagrammed below the transcription unit. The Unusual Puff at 93D The puff at 93D is one of the very largest puffs, suggest- ing that it is actively transcribed during heat shock. Cyto- logical studies have shown that 93D is unusual in a num- ber of ways. ( 1 ) 93D is a bona fide member of the heat shock set, but it can also be induced by a number of agents that do not induce the other members of the fam- ily. (2) 93D contains large RNP granules never seen in other puffs. (3) 93D binds antibodies to several nuclear antigens not found in the other heat shock puffs. Every species ofDrosophila studied has one (and only one) heat shock puff that has all these strange features (see Lak- hotia, 1987). The cytological studies strongly suggested that the un- usual member of the heat shock puff set in each Drosoph- ila species is homologous to the unusual member in each of the other species. We now have evidence that this is true and have named this puff the lisr-omega locus. (The name reflects its original identification as the locus en- coding /feat shock /?NA omega, although we now think that the locus is important in almost all cells, whether or not they are heat-shocked.) Although the homology story turned out happily in the end, it seemed for awhile that there was no sequence homology between these puffs. Berendes and his col- leagues in The Netherlands isolated a cDNA clone from the D. hydci 2-48B puff (i.e., the D. liydei heat shock puff with unusual features). The cDNA consisted of tandem 1 1 5 nt repeats (Peters et ai, 1984). The clone was used to select a cosmid clone of D. liydei DNA that covered the entire puff region. Neither the cosmid nor the cDNA showed any cross-hybridization with D. melanogaster DNA. Thus the 2-48B gene appeared to have no homo- logue in D. mi'lanogasterDNA. However, we now know that 2-48B is the D. liydei homolog of D. melanogaster 93D (Garbe et ai, 1986). Apparently the sequence at this locus has evolved faster than the sequences of the other heat shock genes, whereas the phenotype, as deduced from cytological puffs, has been conserved. As discussed below, our studies of the 93D gene substantiate, and help to explain, this somewhat paradoxical conclusion. We began our study of the 93D puff by using a cloned gene to determine the structure of the locus and its tran- scripts. The structure is quite different from that of the other heat shock genes or any other known gene (Fig. 2). 80 M. L. PARDUE ET .11. I I Figure 3. Autoradiograms showing the localization of omega 1 to the nucleus in both diploid cells (A) and polytene cells (B). In both experiments the cells were fixed, permeablized, and hybridized with a 'H-labeled probe complementary to the hsr-omega repeats (Bendena el at.. I989a). The cells in (A) are cultured cells, xi 100. (B) shows the nucleus and part of the cytoplasm from a larval salivary gland cell. X700. The cells in (A) have been heat shocked for I h at 36C. The cell in (B) has not been heat shocked. Heat shock increases the amount of omega 1 in the cell but does not affect the nuclear location. The transcribed region is greater than 10 kb and can be divided into two parts: a 5' unique region (approximately 3 kb) and a region of tandem repeats (8-25 kb, depend- ing on the allele). These repeats are found nowhere else in the genome. Typical heat shock transcription signals can be found upstream from the start of transcription. Because the transcript is regulated in multiple ways, there must be other control signals also, but these have not yet been dissected (Garbe el at.. 1989). In both normal and stressed cells, the hsr-omega locus produces a distinctive set of three major transcripts, all starting from the same nucleotide (Fig. 2). The largest transcript, omega 1, contains all of the sequence between the start and the second termination site (which is marked by a poly-adenylation signal). Omega 1 is not a precursor for the other transcripts. It remains in the nucleus, and its turnover is controlled differently from the other two transcripts (Fig. 3). Its most remarkable feature is the segment of over 8 kb of tandem repeats. Each repeat is only 284 bp long, and the repeats differ from each other by less than 10% (Garbe el ai. 1986). The second 93D transcript, omega 2. appears to be made by an alternative termination near the first polyadenyla- tion signal. Omega 2 is also nuclear but it seems to be a more-or-less typical precursor that is spliced to make the cytoplasmic transcript, omega 3. These three transcripts have typical RNA processing sequences for splicing, polyadenylation, etc., but show no other similarities to known RNAs that might provide clues to function (Garbe el ai. 1989). We have, therefore, used a number of techniques biochemical, cytological, and genetic (both classical and reversed) to search for function (Bendena ei til.. 1989). The results of these experiments will be discussed below. Our studies to date suggest a working hypothesis about the function of the hsr-omega locus. Although not the only possible hypothesis, this seems to provide the sim- plest explaination of all the observations. Briefly, we sug- gest that omega 1 serves to bind some component in the nucleus, thereby affecting either the activity or the level of this component in the nucleus. Because levels of omega I vary quickly in response to cellular conditions, this binding would make the activity or the nuclear level of the bound component respond rapidly to those condi- tions and could thus serve a regulatory role. In the case of the cytoplasmic transcript, omega 3. we propose that the RNA, not a protein product, is important. One possi- bility is that the level of omega 3 RNA reflects the rate of protein synthesis at any given time and provides a way to link another cell process to this rate. Finally, we won- der why both the nuclear and the cytoplasmic transcripts of hsr-omega come from the same start site. Does this in some way coordinate the initial levels of the nuclear and cytoplasmic transcript? A number of processes in eu- karyotic cells have nuclear and cytoplasmic activities that must be coordinated. Perhaps this gene plays a role in such coordination. Evolutionary Comparisons of Hsr-omega Genes When we found that 93D had short tandem repeats, we were struck by its resemblance to the D. hydei 2-48 B gene. Although the D. melanogaster repeats (284 bp) are larger than those in D. hydei (115 bp), such long stretches of repeats in a transcribed region were too unusual to dismiss as unrelated, notwithstanding the previous evi- dence that the genes shared no sequence homology. We used small pieces of the D. melanogaster DNA sequence to probe DNA from the cosmid clone of the D. hydei 2- 48B locus. Our hybridization results confirmed those of Berendes and his colleagues, with one significant excep- tion (Garbe and Pardue, 1986). Most fragments showed no cross-hybridization at any stringency. The small re- I1SR-OMEGA. A NOVEL HEAT SHOCK GENE 81 gion that did show cross-hybridization, however, encour- aged us to clone and sequence the rest of the D. hydei gene. The sequence analysis showed that the cross-hy- bridization was due to 60 nt of perfect homology and, surprisingly, 40 of the conserved nucleotides were in the intron, while the rest extended beyond the 3' splice site. In spite of the differences in sequence, the D. hydci gene had the same structure as the D. melanogaster gene, and the location of the conserved region was the same in both genes. The same conserved sequence has now been found in the hsr-omega loci of all of the other Drosophila species that have been studied. The homology can be de- tected by in situ hybridization to polytene chromosomes. The sequence homology has been used to clone the lisr- omega gene from D. pseudoobscura. The three cloned genes enable us to compare hsr-omega sequences from distantly related Drosophila: D. melanogaster and D. pseudoobscura are separated by 46 million years, and both are separated from D. hydei by 60 million years (Beverley and Wilson, 1984). Our studies of the D. hydei and D. pseudoobscura genes have shown that the structure of the hsr-omega lo- cus is conserved, although the sequences have diverged (GarbetVt;/.. 1988). In each Drosoptiilu species, the locus has a unique region followed by a long string of tandem repeats. In each species, there are three transcripts of ap- proximately the sizes found in D. melanogaster. In each species the 60 nt conserved region is in the intron and overlaps the 3' splice site. In spite of the strong evolutionary divergence, the se- quence comparisons give useful clues about possible functions. The clues are strong because the evidence that so much of the sequence can change increases the sig- nificance of the parts that have not changed. The unique portion oj the gene In any pairwise alignment of the sequences from D. melanogaster, D. hydei, or D. pseudoobscura, the longest conserved sequence is the 60 nt around the 3' splice site (Garbe et al., 1988). (The conserved region rises to 62 nt when the D. melanogaster sequence is compared with that of D. pseudoobscura.) This conserved sequence might be necessary for splicing in heat shock; however that seems unlikely if one considers a similar sequence comparison of the hsp83 gene. The hsp83 transcript is also spliced in heat shock and it shows very little se- quence conservation in the intron ( Blackman and Mesel- son, 1986). The hsp83 exon shows significant sequence conservation, but because the exon codes for protein, it is probably the protein sequence that is conserved, an explanation that does not hold for the 93D exon, which does not encode a protein. At this point, the reason for this conserved sequence is a puzzle. With the exception of the 60-62 bp conserved region, pairwise alignments of the rest of the unique region of hsr-omega show few stretches of conserved sequences longer than 10-20 bp. Interestingly, some of the longest regions of homology surround sites that our RNA studies indicate to be important for RNA processing and func- tion. These include 14-16 bp (depending on the species compared) at the 5' splice site, and 1 5-2 1 bp at the poly- adenylation signal. In addition, the transcription start site shows conservation between the species and also has five of the six specific nucleotides that are conserved in all Drosophila heat shock mRNAs, except the hsp83 mRNA. The sequence alignments also show why there is so little cross-hybridization between hsr-omega genes. There have been many short insertions and deletions that eliminate runs of sequence long enough to hold a hybrid. The deletions and insertions tend to balance out, so the sizes of the exons and introns are conserved. Another conserved feature of the hsr-omega genes is the lack of long open reading frames (Garbe et ai, 1 986). The only open reading frames (ORFs) are very short (shorter than those found on the opposite, non-tran- scribed strand). A comparison of the transcripts from the three Drosophila species shows only one ORF that is at all conserved in location or sequence (Garbe et a!., 1989). The location is interesting because, in each species, this ORF is the first one that is in a sequence context thought to be favorable for translation. The sequence conserva- tion is not very strong; only the first four amino acids, plus a few other scattered amino acids, would be the same in all three translation products. Even the size of the ORF varies; the translation product would contain 23, 24, and 27 amino acids, depending on the species. In spite of the low level of conservation, other studies (discussed below) strongly suggest that this ORF is im- portant in the function of the omega 3 transcript. The general conclusion from these sequence studies on the unique part of the gene seems to be that the cell is conserving the ability to make, splice, and polyadenylate an RNA of this size. This conclusion is based on the small conserved regions, the significance of which we al- ready know. There are other small conserved segments that we cannot now decode; these regions probably also have functional importance. The tandemly repeated segment of the gene Restriction enzyme mapping of DNA from hsr-omega loci indicates that, in all Drosophila species, this locus has >8 kb of short tandem repeats. Repeats from both D. melanogaste r and D. hydei have been sequenced. Within each Drosophila species the repeats show <10% diver- gence, but between species the repeats differ in both size and sequence. There is, however, a conserved 9 nt seg- 82 M L. PARDLIE ET AL. ment, AUAGGUAGG, that is found once in the 1 1 5 nt repeat of D. hydci and twice in the 284 nt repeat of D. melanogaster (Garbe et a/., 1 986). The 9 nt segment thus occurs at about the same frequency along the transcripts. D. pseudoohsciira repeats have not yet been sequenced, but preliminary evidence suggests that they will also have the 9 nt sequence. The sequence, AUAGGUAGG, is in the size range of sequences that have been shown to serve as binding sites for proteins and RNAs. The sequence may serve as a binding site in the omega 1 RNA. If so, the repeats may be a device to maintain a certain number and spacing of copies of the binding site. The evolution of the hsr-omega repeats appears much like that of satellite DNA. That is, the repeats are very homogeneous within each species, but diverge rapidly between species. The D. melanogaster repeal will hybrid- ize only with DNA from the sibling species, D. simulans, and, even in this case hybrid stability is reduced, and re- striction site differences indicate some sequence change. The Cytoplasmic Transcript, Omega 3 The cytoplasmic RNA, omega 3, is spliced and poly- adenylated, two characteristics usually associated with mRNAs, yet the only open reading frames (ORFs) are small and show very little conservation. Surprisingly, omega 3 is found on polysomes (monosomes and di- somes)by all known criteria ( Fini t'/t//.. 1989). The local- ization in control cells does not change when the cells are heat shocked. In both cases almost all of the transcript was loaded on the polysomes; rarely is any of the tran- script free. In control cells, omega 3 turns over rapidly but is stabilized by all inhibitors of protein synthesis (Fig. 4) (Bendena et a/., 1989). There is now evidence that turnover of certain mRNAs is linked to their presence on polysomes (Hunt, 1988). Although the studies described below suggest that omega 3 is not an mRNA, omega 3 shows a specific and rapid turnover when it is associated with active polysomes, as do these mRNAs (Bendena et ai. 1989b). These evidences of an association between omega 3 and protein synthesis have led us to search very hard for an hsr-omega translation product (Fini et al, 1989). That search has been unsuccessful; but we have obtained indirect evidence that the small, partially conserved, ORF in omega 3 is translated. The indirect evidence comes from experiments with recombinant DNA mole- cules in which a bacterial chloramphenicol acetyltrans- ferase (CAT) gene was joined to the 5' part of the hsr- omega sequence (Fig. 5). The constructs were stably transformed into Drosophila cultured cells and tested for their ability to direct synthesis of mRNA and CAT pro- tein. The experiments showed that omega 3 ORF blocks translation of a CAT gene placed just 3' to it, as long as ABC D E F G H I 10 t 1.2 - Figure 4. Autoradiograms showing differential regulation of the nuclear and cytoplasmic transcripts of hsr-omega. Inhibitors of protein synthesis lead to preferential accumulation of the cytoplasmic tran- script, omega 3 Drugs that induce putting of 93D without inducing other heat shock loci (e.g . colchicine, henzamide) lead to preferential accumulation of the nuclear transcript, omega I. To make the autora- diogram, RNA from cultured Drosophila cells has been fractionated and probed with the omega 3 sequence, as in Figure 2. RNA from con- trol cells (A. D, G), cells treated for 2 h with I0~" A/cycloheximide (B). cells treated for 2 h with ICT 7 A/ pactamycin (C), cells treated with It) mAl benzamide for 12 h (E) or 24 h (F). cells treated with 100 Mg/ml colchicine for 12 h (H) or 24 h (I). The 10 kb omega 1 and the 1.2 kb omega 3 transcripts are indicated. the omega 3 ORF has a termination codon so that the ribosome must reinitiate in order to translate CAT. In contrast, when the omega 3 ORF is fused in frame to CAT, a CAT protein of appropriately larger size is pro- duced. These transformation experiments give evidence that the omega 3 ORF is translated in vivo, although no product can be detected. Thus the product of the ORF must either be degraded or be sequestered very rapidly. We think the first alternative is most likely because we have been unable to find any evidence of the product, even in the transformed cells where the excess product might be expected to saturate or slow a sequestration mechanism (Fini et al., 1989). Taken together, the studies on omega 3 have suggested the following working hypothesis: the translation of the omega 3 ORF functions to allow a polysome-associated turnover of omega 3 and this turnover in some way serves to monitor, or regulate, some aspect of protein synthesis in the cell. Possibly, the omega 3 RNA, or a degradation product of this RNA, has a function with a rate determined by the rate at which omega 3 turns over on the polysomes. Admittedly this is highly speculative, but polysome-associated turnover may be common for RNAs that can be turned over rapidly (Hunt, 1 988). The turnover of omega 3 would reflect the level of protein HSR-OMEGA, A NOVEL HEAT SHOCK GENE 83 67 base 93D leader hydei leader Figure 5. Recombinant DNA constructs used to analyze the trans- lation of the most conserved small open reading frame (ORF) in hxr- onu'Kti- (A) The 93Xho construct was made by joining a bacterial chloramphenicol acetyltransferase (CAT) gene to DNA from the D mclanoKU.iier hsr-nmega gene. The junction was 30 nucleotides past the termination of the small ORF. The 93XR construct was made by deleting the small ORF from 93Xho. If the small ORF is translated in vm>. the ribosomes would not be expected to reinitiate on the CAT gene. Therefore the 93Xho construct would not be expected to direct the synthesis of CAT while the 93XR construct, where the CAT transla- tion start is not blocked, should. When the constructs were stably trans- formed into Dmsophila cultured cells, both constructs yielded abun- dant RNA but only the 93XR CAT gene was translated. (B) The S3- 1 28 construct was made using the D. hydei hsr-omega gene. In this case the bacterial CAT gene was joined in frame to the conserved ORF so that ribosomes translating the ORF should continue translating into the CAT gene and yield CAT protein that is larger by the size of the ORF peptide. Cells carrying this construct do produce the predicted larger CAT. All of the experiments give strong evidence that the hsr- oiiH'xii ORF is translated in wvo(Fini el at-, 1989). synthesis at any particular time and could link some other cellular process to this level. The Nuclear Transcript, Omega 1 One of the questions that we had about hsr-omega was whether all of the agents that induced the puff were act- ing on the hsr-omega sequences. Puffs usually involve more DNA than is actually transcribed, so a puffin the 93D region might indicate activation of a transcription unit that is not hsr-omega. The question had interesting implications. If the agents that induce the hsr-omega puff, but not the rest of the heat shock puffs, were induc- ing the transcripts that we are studying, it would suggest that hsr-omega is more sensitive to its environment than the other heat shock loci. Other experiments had shown that treatments inducing a puff at 93D blocked induction by a second agent, if the two inducers are applied in a relatively short time (Lakhotia, 1987). If all the inducers were acting on the hsr-omega gene, the observation that one inducer blocks activation by a second agent suggests that the hsr-omega locus can autoregulate, with products of the first induction inhibiting later induction. We have tested several inducers (Bendena ci ai, 1989) and have found that all of the inducers do act on the hsr-omega locus (Fig. 4). These results suggest a responsiveness to external agents and an autoregulation that could charac- terize a regulatory locus. This sensitivity to the environ- ment is consistent with the rapid response seen in heat shock in which the 93D region puffs slightly before the other loci and also returns to control levels more rapidly when cells are returned to normal temperature (Fig. 6). These experiments with the other inducers gave an un- expected result. The level of omega 1, but not omega 2 or omega 3, is increased by the agents that induce the 93D puff without inducing the rest of the heat shock loci (Fig. 4). The level rises rapidly in response to the agent, remains high so long as the agent is present, and drops rapidly when the agent is removed. Thus, the level of omega I at any time reflects something that the cell per- ceives in its environment. The omega 1 transcript accu- 36 25 36 25 .5' .5 1.0 1.5 2 3 4 .5 5 1.0 1.5 2 34 93D hsp83 Figure 6. Autoradiogram showing that hsr-omega transcripts re- turn to control levels more rapidly than the hsp83 transcripts upon recovery from heat shock. Cultured Drosophila cells were heat shocked for 30 min at 36C and then returned to 25C. Aliquots were taken at times indicated above each lane and the RNA was analyzed as in Figure 2. The panel marked 93D shows the hsr-omega transcripts as detected by the probe for the omega 3 sequence. The panel marked hsp83 shows the same samples probed for the sequences encoding the D. melanogas- ter hsp83. The hsr-omega transcripts return to control levels at 1-1.5 h while the hsp83 transcripts do not return to control levels until 4 h after return to 25C. (The autoradiogram shows only samples taken up to the time at which hsp83 returned to control levels.) 84 M. L PARDUE A/ !/ 1 Ul(JK)e-Up4 | 1 -- Df(3R)GC14 --1 Df(3R)epl 1 " (erl) (er8 erl 1 er9) (er3) (er!9 er!3 er!6) GC8 EC 13 EC36 GC23 GC31 EC17 EC38 EC15 EC 19 EC28 EC20 EC32 DC3 EC37 Ier7 er4| (erg) Ier5) EC3 GC15 GC20 GC18 DC1 GC11 EC4 GC16 EC7 GC19 EC21 ECS GC17 EC 11 GC21 EC24 EC9 EC2 EC1 EC10 EC16 EC18 EC30 EC25 EC26 EC27 EC33 Figure 7. Diagram of the chromosomal deletions that bracket the hsr-omega locus and of the lethal complementation groups in that region in D. melanogaster. The lines indicate the relative locations and sizes of the three deletions. The brackets indicate the region of overlap of Df(3R)e Gp4 and Df(3R)GC14. which contains the hsr-umega locus. The complementation groups are listed below in order. In those cases where the order has not been established, the complementation groups are enclosed in parentheses. Below the name of each complementation group are given the names of the individual mutations that have been recovered in that group. The region of overlap of Df( 3R)e cip4 and Df( 3R )GC 1 4 is designated complementa- tion group er3; it appears to contain only the hsr-omega locus but this has not been formally proven. Animals carrying both deficiencies and, therefore completely lacking er3, grow very poorly and do not survive to adulthood. Thus er3 appears to be an essential locus, but no point mutations in er3 have been recovered. Complementation group e is the ebony locus: several mutations in this group were recovered but were not scored. Because multiple mutants have been recovered in most of the complementation groups, it appears that er3 is unusually resistant to mutagenesis. Df(3R)epl removes the heat shock control elements, but not the constitutive activity of hsr-omega. Animals carrying this deletion and Dt(3R)GC14 are fully viable at optimum temperatures but are much more sensitive to slightly increased temperatures than wild type flies. GC30 EC40 GC27 EC43 EC44 mulates around the nucleus, apparently just under the nuclear membrane (Fig. 3). The striking feature of the omega 1 sequence is the array of tandem repeats containing an evolutionarily conserved potential binding site for a protein or RNA. The idea that the hsr-omega transcript binds protein is attractive because one of the distinctive features of the locus is the presence of 300 nm RNP granules not seen at any other locus. These granules could be formed by omega I and its bound protein. This structural evidence, coupled with the evidence that levels of this RNA can change rapidly, suggests that omega I might sequester variable amounts of a protein or RNA in response to variable cell conditions. The binding might affect the ac- tivity of the bound agent, either positively or negatively, or it might simply change the concentration that is pres- ent in the nucleus. In any case, such binding could allow omega 1 to play a regulatory role for some aspect of cell metabolism. One way to test this hypothesis is to try to find a molecule that specifically binds to the omega 1 RNA. We have recently found that an //; vitro RNA tran- script containing only the repeats from omega 1 binds specifically to three bands on a blot of gel-fractionated Drosophila proteins: one band migrates at about 1 70 kD and a doublet migrates at 53-55 kD. The bands could represent a set of related proteins; alternatively, the smaller doublet could represent monomers or degrada- tion products of the 170 kD band. These studies are pre- liminary, but the characterization of the proteins repre- sented by these gel bands should give us useful informa- tion about the function of omega 1. Genetic Analyses of Hsr-omega Our genetic studies (Mohler and Pardue, 1984) are consistent with the hypothesis that hsr-omega has some sort of regulatory role. We have identified two chromo- somal deletions, e Gp4 and GC14, that completely remove the hsr-omega sequences. e p4 removes 1 1 complemen- tation groups proximal to hsr-omega, and GC14 re- moves 3 complementation groups distal to hsr-omega, but the 2 deficiencies appear to overlap only for hsr- omega (Fig. 7). Extensive attempts to recover point mu- tations in the region of overlap gave no lethal, visible, or temperature-sensitive mutations. What we know about the sequence conservation of hsr-omega suggests that it HSR-OMI-X1A. A NOVEL HEAT SHOCK GENE 85 would be almost impossible to inactivate it by a point mutation (or even by a tiny deletion), so we are not sur- prised to get no hsr-omega mutants. The genetic evi- dence provides a strong argument that no other genes (of conventional mutability) are located in the region of overlap. Ife p4 /GC14 heterozygotes are indeed homozy- gous deficient only for hsr-omega, then their phenotype indicates that hsr-omega is important in non-stress situa- tions. These e Gp4 /GC14 heterozygotes hatch as well as normal sibs (but mothers had one hsr-omega gene) but grow very slowly, dying at all stages of development. None survive as adults. More recently we have recovered another deletion, epl, that removes six complementation groups proximal to hsr-omega and also deletes the heat shock transcrip- tion signals, but not the constitutive transcription signals or the transcribed sequence of hsr-omega (Garbe, 1988). Heterozygous epl/GC14 flies are viable and fertile at normal growth temperatures (25C) but cannot grow at 3 1 C, a temperature at which wild type flies grow ( but do not make sperm). The simplest explanation of the analy- sis of these two heterozygotes is that e c ' p4 /GC14 flies die because they lack any hsr-omega gene, and the epl/ GC14 flies die at 3 1C because their only hsr-omega gene has no heat shock control. But it is formally possible (considering the two results together) that the overlap of e Gp4 and GC14 also includes a lethal gene distal to hsr- omega, while the overlap of epl and GCI4 includes a temperature-sensitive gene proximal to hsr-omega. (The other possibility, that either phenotype is simply due to the sum of the hemizygous loci, was excluded by testing animals carrying larger deficiencies for this region.) We have mapped the breakpoint of the epl deficiency and find that it is just downstream of the closest heat shock transcription signal of hsr-omega. The breakpoints of the other two deficiencies lie outside the region that we have cloned (about 5 kb 5' and about 1 kb 3' ). The ep 1 deletion shows that there is no undiscovered lethal gene 5' to hsr- omega that could cause death of the eGp4/GC 1 4 hetero- zygotes. (If there were, epl/GC14 flies would also die.) A highly repeated element just past the 3' end of hsr-omega has made walking in that direction difficult, and we have not pursued it because breakpoint mapping cannot de- finitively reveal whether the phenotype we observe is due to the hsr-omega gene or to some other gene. The proof must be genetic. Our mutagenesis experiments were ex- tensive, and they detected no mutable gene in this region. Thus, the genetic evidence argues that hsr-omega is the only gene in the overlap region; however, the definitive test must be rescue of the mutants by P-element transfor- mation with the cloned gene. In the meantime, we sug- gest that, if the two transheterozygote phenotypes are due to hsr-omega, then they are consistent with a regulatory role for this locus. One possible type of regulatory role would be to link two processes in a way that increases metabolic efficiency. Mutants lacking the regulator would have the two processes running freely, at consider- able expense of energy. Such mutants might grow slowly and have trouble making it past crucial developmental points or surviving in sub-optimal environments, as do the putative hsr-omega deletions. Because hsr-omega produces both nuclear and cytoplasmic transcripts, we speculate that the locus acts to link a nuclear with a cy- toplasmic process. Conclusion We now have several kinds of information about the hsr-omega locus. It is clearly different from other genes, and some of those differences might explain why the lo- cus has not attracted attention before. It seems to be rela- tively insensitive to mutagenesis and so would not be eas- ily picked up in genetic studies. (Non-protein coding genes may be generally less sensitive to mutagenesis be- cause, while any frame-shift or stop codon can destroy the function of an mRNA, RNAs with other functions are not so strongly polar as tnRNAs; many small dele- tions or base changes in a non-coding RNA may make small local perturbations that do not affect function.) Like the heat shock response, which was identified from polytene puffs, the hsr-omega locus may be found in many organisms. Because the sequence is evolving so rapidly, it may not be possible to use hybridization to detect the DNA in other genera, and the search for the gene in other organisms must wait until the function is better understood. Nevertheless, we think that this locus, like the loci encoding hsps, is common to many organ- isms. Acknowledgments This work has been supported by a grant from the Na- tional Institutes of Health to M. L. Pardue. M. E. Fini was the recipient of a postdoctoral fellowship from the American Cancer Society. J. C. Garbe was a predoctoral trainee of the National Institutes of Health. Literature Cited Ashburner, M., and J. J. Bonner. 1979. The induction of gene activ- ity in Drosophila by heat shock. Cell 17: 241-254. Bardwell, J. C. A., and E. A. Craig. 1984. Major heat shock gene of Drosophila and the Escherichia cult heat-inducihle dnaK gene are homologous. Proc. Neil. Acacl. Sci. USA 81: 848-852. Beermann, VV., ed. 1972. Developmental Studies of Giant Chromn- somes. Results ami Problems in Cell Differentiation. 1 'ol. 4. Spring- er- Verlag, New York. 277 pp. Bendena, W. G., M. E. Fini, J. C. Garbe, G. M. Kidder, S. C. Lakhotia, and M. L. Pardue. 1989a. hsr-omega: a different sort of heat shock locus. Pp. 3-14 in Stress-Induced Proteins. ICN-UCLA Symposia on Molecular and Cellular Biology. }'ol. 96. M. L. Pardue, J. R. Feramisco and S. Lindquist, eds. Alan R. Liss. Inc., New York. 86 M. L. PARDUE ET AL Bcndcna, W. G., J. C. Garbe, K. L. Traverse, S. C. Lakhotia, and M. L. Pardue, 1989b. Multiple inducers of the Drosophila heat shock locus 93D( ii . ,;): inducer-specific patterns of the three transcripts. ./ Cel 108: 201 7-202S. Beverley, S. M., and A.C.Wilson. 1984. Molecular evolution in Dro- sophila and the higher Diptera. II. A time scale for fly evolution. ./ M, 2 mm strip of thick photographic film resting on glass bead. Pronotal end of strip tapered. Other end with wax bead tare. Not to scale. I IRFR.Y UNISEX FLASH CONTROLS 89 sessed by Student's /-test. Mean response delays are given with standard deviations (s), not standard errors. In mak- ing comparisons, variance is indicated by V, the coeffi- cient of variation (s/M). Results Flight On both suspensions, about two thirds of the hundred- odd males mounted flew reliably; about two thirds of those flying responded to LED stimulation. Flight vari- ability was possibly due to the difficulty in mounting the animals so that the suspension did not touch the spread elytra or antennae; response variability was perhaps due to the body not being in exactly the normal flight attitude [head extended forward from under the nearly horizon- tal pronotum, thorax inclined downward toward the rear about 30, abdomen hanging down almost vertically, di- recting the light from the ventral lanterns in the 6th and 7th segments downward and forward (Fig. 1 )]. The flight reflex is very compelling; males will even fly upside- down. With both suspensions, many continuous flights of up to 30 min were observed, and some animals flew on more than one day if demounted between flights. Wing- beat frequency of STR animals was found stroboscopi- cally to be about 80 Hz at about 25C. Flying STR ani- mals sometimes writhed the abdomen, a behavior that P. pyralis does not exhibit in normal flight. STR males that flashed spontaneously (i.e., more than 4 s after an LED flash; see Discussion) showed no flight change cor- responding to the dip and hover behaviors that are nor- mally associated with flashing during field display. When given an LED answer about 2 s after flashing, males sometimes turned the head immediately toward the sig- nal (video observations with J. F. Case). Presumably this was the functional equivalent of the normal response in which the body turns as a whole. Many CIR records showed an artifact due to ambient light reflected off or interrupted by the rotating arm ( Figs. 7a, 16, arrows). From the typical, indicated 2-Hz rate of rotation, and the 1 1-cm radius of rotation, the velocity of linear flight was calculated to be about 3 mph. Rate of rotation was constant. The results reported below suggest that a normally hid- den 2-s flash-control circuit of the P. pyralis male was sometimes revealed by forcing the animal to fly in a cir- cle. As a control background for this hypothesis, I there- fore first present full ranges of both the spontaneous flashing and the short-delay photic responses of both STR and CIR males. Spontaneous flushing Flashing in the absence of stimulation was usually spo- radic, but, on both suspensions, some males flashed spontaneously and consecutively for a number of cycles in approximately the normal rhythm. These spontane- ous flashes were sometimes quite uniform in intensity and were emitted as regularly as by males flashing in the field. The STR series of Figure 3, for example, shows uni- form flashes and regular rhythm. In contrast, the Figure 5 STR series illustrates flashes that were highly variable in form (perhaps because of abdominal twisting), though emitted with respectable regularity (see figure legends). With the CIR suspension, apparent flash intensity, con- tour, and duration varied widely as the animal rotated and the ventral abdominal lantern was alternately partly occluded and then exposed to the photometer, but rhythmic flashing was nonetheless observed (Fig. 7 and legend). Short-delay photic response (SD) The effects of exposing flying males to LED flashes were quite variable in mode, between individuals, and between suspensions, and often the firefly did not flash for many seconds after a signal. Two types of consistent response were observed. In the more frequent, seen in animals on both suspensions, the male flashed from 0.2 to 0.7 s after the LED stimulus. Many hundreds of these "triggerings" were recorded from the dozens of males studied. Figures 4 and 9-14 illustrate variations in flash form and delay observed in six STR males, and Figures 8 and 16-18 do the same for two CIR animals. Mean response delays for individual males ranged from 0.26 0.02 s to 0.46 0.08 s, and some differed significantly from each other. Mean delay ranges for individual males were typically less than 0. 1 5 s. CIR animals tended to respond after somewhat longer delays, and flash more dimly, than STR animals, al- though there were substantial overlaps. I found that males walking on a smooth horizontal surface did not respond to flashed answers to their signals if the answer was delivered from directly behind or from the rear within 30 to either side of the longitudinal body axis. This means that CIR males might have been unable to see the LED signals for up to 1/6 revolution (ca. 0.083 s). When apparent CIR delays were each reduced by an average blind-spot correction (0.04 s), the overall mean delay duration was not significantly greater than that of STR animals at the same temperature. Ninety-five per- cent of both STR and CIR measurements fell between 0.25 and 0.65 s. For the present, accordingly, the most parsimonious conclusion is that STR and CIR males give the same short-delay photic response. The narrow fre- quency distribution peaking at 0.4 s in Figure 19 is the averaged delay for STR and CIR males. The mean delay for the combined group was 0.38 s. 90 J. BUCK 4.9 1 a b 1 1 ; \ s \ , K 1 *? i i sec*- 6.6 Figure 3. Four successive spontaneous flashes ofSTR male 126. a-d, flashes 2-5 in a rhythmic series of 8 (mean period 4.92 0.16 s; V = 3). Numbers are flash-to-flash intervals. T = 25. Note: in the chart records, the rapidly rising and falling limbs of some flashes have been reinforced. Jagged or sawtooth traces are instrumental AC noise revealed by high amplification. Decline of traces below baseline after some flashes is an instrumental artifact. Rash intensity is arbitrary. Time scale for all records indicated on Figure 7a. Figure 4. SD response of STR male 126 to 0.4 s LED stimulus. Delay 0.35 s, showing stimulation at the LED "on" phase (arrow). Figure 5. Rhythmic spontaneous flashing of STR male 124. a-f, flashes 1, 4, 5, 6. 8, and 15 in a series of 27, showing variability of flash intensity and form. Record also showed a 0.4-s difference in mean delay between two sections of same run; mean of first 13 cycles. 6.06 0.41 s(V = 6); mean of last 14 cycles, 6.46 1.04 s (V = 16). Figure 6. Spontaneous flash of STR male 132. T = 25. Figure 7. Four consecutive spontaneous flashes of CIR male 94. a-d, flashes 9-12 in rhythmic series of 13, showing 0.55 s rotation artifacts (arrows in a) and effects of rotation on flash form delineation by photometer. Numbers, interflash intervals. Mean period for series, 7.2 0.93 (V = 13). T = 22. Figure 8. SD response of CIR male 94. Delay 0.46s. Figure 9. SD response of STR male I. Delay 0.36s. Figure 10. SD response of STR male 98. Delay 0.28 s. Figure 11. First of two consecutive SD responses of STR male 105 that were 5 s apart. Peak clipped by over-amplification. Delay 0.60s. Figure 12. NextSD response of STR male 105. Delay 0.3 s. Figure 13. SD response of STR male 282, showing 0.8 s flash duration. Delay 0.3 s. T = 27. Figure 14. SD response of STR male 123 to 0. 1 s LED flash, showing slow light accretion but typical 0.37 s delay. Figure 15. Spontaneous flash of CIR male 107. T = 23. Figure 16. Two consecutive SD responses of CIR male 107, 4.4 s apart. First delay, 0.37 s; second delay, 0.58 s. Arrows, rotation artifact. Figure 17. SD response of CIR male 107 showing delay of 0.35 s. close to that of first flash in Figure 16, but of lower intensity. F'igure 18. SD response of CIR male 107, showing delay of 0.56 s, close to that of second flash in Figure 16, but of lower intensity, and distorted by rotation of suspension. i IRI:FLY UNISEX FLASH CONTROLS 91 Long-delay plunk- response (LD) A second, less frequent type of photic response (Figs. 20, 22-24; filled columns in the 1.5 to 3 s range in Fig. 19) was given only by CIR males. In 15 of 31 individuals, from 1 to 23 such responses, with delays averaging 2.3 s. were recorded, sometimes several in succession. No flashes in the 2-3 s post-stimulus range were seen in any of 20 different males flying from the STR suspension. In some instances, the CIR firefly's response flash was followed by a second, spontaneous flash at about the same interval (Fig. 24). On several occasions, two spon- taneous flashes about 2.3 s apart were emitted (Figs. 21, 24; unfilled column caps in Fig. 19). In sum, LED stimuli evoked two photic responses: a short-delay (SD) variety delayed an average of 0.38 s (Fig. 19, first distribution frequency peak); and a long- delay (LD) one delayed an average of 2.3 s (Fig. 19, 1.5 to 3 s concentration). Both distributions were shown to be significant (/>> 95%) by Wallenstein's ( 1 980) scan sta- tistic, using an 0.5-s window. With one exception among hundreds of records (Fig. 23). LED stimulation evoked one response or the other, not both. Discussion Spontaneous flashing In experiments on intact fireflies, there is no direct way of ascertaining which flashes are initiated endogenously and which are responsive. In the present work, absence of significant clumping of flashes later than 3 s after LED stimulation (Fig. 19) indicates that the SDand LD distri- bution peaks reflect true photic responses, and con- versely that firefly flashes that occurred more than 3 s after exogenous input were spontaneous (endogenous). This conclusion is supported by both field and laboratory observations. Of 378 display-flashing periods recorded in 18 series, each from a different male of Phot inns pyralis flying free in the field at 23 (ave. 6.15 s), only one was shorter than 4 s. 2 Similar results were obtained by Ed- munds (1963) and Maurer (1968). Though spontaneous STR and CIR flashing tended to be less regular than in the field, due to frequent flash- skipping, series of consecutive flashes were well within the reported range of field rhythm variability 2 (legends : The many hundred raw measurements in Buck (1936) that were given antique statistical treatment by Buck (1937) were reexamined to assess the range of individual period variation in free-flying males in nature. Using 38 series of 8 to 71 consecutive Hashes, it was found: that a typical V value is 10, with values as low as 3 and as high as 20 occur- ring occasionally; that mean period decreases about 0.4 s for each de- gree (C) nse in temperature: and that statistically significantly different individual means occasionally occur even between two individuals at the same temperature. Nonetheless, as shown by Buck and Buck (1968, footnotes 42-47), the P. pyralis rhythm, though interior in regularity of Figs. 3, 5, and 7). The special case of 2-3-s pairs of apparently spontaneous flashes (Fig. 2 1 ; unfilled column caps in Fig. 19), is discussed below. The SD ("reflex"'.') response Aside from irregularities in recorded flash form due to the motion of the CIR males, the SD photic responses observed in STR and CIR males differ in no essential respect from each other, or from those seen normally in free-flying animals. 3 Short-delay photic interactions between males of P. pvralis had been observed in synchronized flashing among males courting the same female (Buck, 1935; Maurer, 1 968 ) and between males flying indoors in dark- ness (Buck, 1938b), but it was not until the response was measured and studied intensively that it was recognized as part of the male's normal repertory (Buck et al, un- pub.). By programmed stimulation, the triggering was confined to the latter half of the flashing cycle (i.e., more than 3 s after a male's flash) an interval dubbed the "late window" to distinguish it from the 1.5-2.5-s post- flash "early window," which is tuned to the female's re- sponse and mediates orientation (Case, 1984; Buck, 1988). In the field, the SD interaction occurs as a triggering of one flying male by the flash of a close neighbor or of an artificial light. The present laboratory-triggered delays are consistent with those measured in video records from free flying males ( Buck et al. , unpub.). When male A thus triggers the flash of B, neither animal pays any attention to the other, but B's flashing rhythm is reset so that there- after he flashes in synchrony with A (Case, 1984; Buck. to those of some tropical synchronizing species, is quite in line with many other biological periodicities, including human heartbeat during sleep. ' The variations were not necessarily due to the experimental condi- tions. Even in free field flashing, the magnitudes of all flash parameters show centrally peaked frequency distributions, and all vary with tem- perature. In considering photic effects on flash liming, flash initiation (with which this study is principally concerned) must be distinguished from flash modulation. Initial excitation depends on neuronal volleys from the brain (Case and Buck, 1963; Buonamici and Magni. 1967; Brunelli el al.. 1977) but there is some evidence that flash form (inten- sity, duration, time-course) may be affected also by activity of the final cord ganglia (Christensen and Carlson, 1981). The number, firing se- quence, and areal distribution of the individual flashing units may also vary (Buck, 1955, 1966; Hanson el al.. 1969). Thus there are many potential sources of variation. Some STR records show flash form vary- ing independently of the spontaneous flashing rhythm (Fig. 5). and be- tween individuals given comparable stimulus flashes ( Figs. 9-12). Aside from effects of changing firefly-photometer geometry, the same conclu- sions hold for the flashes of CIR males (Figs. 7, 8, 15-18. 20-24). The important point is that both the SD and LD photic responses maintain their characteristic and exclusive delay ranges independently of varia- tions in flash form between individuals and between runs. 92 J. BUCK (\) j n \ ; \ V r \ (2 3) 1 , -/ . I / v , - 2345678 LED FLASH TO FIREFLY FLASH (SECONDS) Figure 19. Frequency distributions of SD and LD responses. First peak (0.2-0.8 s) is the average of 180SL responses of 6 STR males and 180 SD response of 6 CIR males (30 consecutive responses for each individual). Second peak ( 1 .3-3 s) is 72 LD responses of the 10 CIR males that emitted more than one LD Hash (filled columns), plus 1 1 corresponding spontaneous flash-to-flash intervals from the same males (unfilled caps). Figure 20. LD response of CIR male 107. Delay 1.96 s. Figure 21. Pair of spontaneous flashes 2.2 s apart. First flash was 1 1 s after previous flash. CIR male 107. Figure 22. LD response of CIR male 250. Delay 1.94 s. T = 22. Figure 23. Rare apparent SD and LD responses to same LED flash. SD delay 0.44 s; LD delay 2.2 s. CIR male 250. Figure 24. LD response (delay 2.0 s). followed by two sponta- neous flashes, the first 2.3 s later, the second 2.3 s after the preceding. CIR male 250. Figure 25. Normal response of freely perched female to LED flash. Delay 2.3 s. Female 1. Figure 26. Same as Figure 25. Delay 1 .92 s. Female 2. 1988). The figures in the present paper are intended only to illustrate the variation range of the response. Its de- tailed aspects will be taken up in another paper. Its puta- tive functions are discussed by Buck (1988). The SD male-male triggering behavior in P. pyralis is also of interest because its delay is often not greatly different from that for flashes elicited by electrical stimu- lation in the head (Case and Buck, 1963). Similarly, the 0.3-s delay of the L. lusitanica male-male response (Papi. 1969) corresponds to the electrical brain delay in that species (Brunelli el at.. 1977). A 0.3-s photic response, distinct from the 0.6 male-female interval, has been found also in P. concisits and shown to correspond to the head-lantern electrical delay in that species (Hanson and Buck, unpub.). [It may also be the inter-male interval involved in the synchronized field flashing observed by Otte and Smiley (1977).] Papi used the term "reflex" for the SD male-male in- FIRHFLY UNISEX FLASH CONTROLS 93 teraction interval in L. lusilanica. perhaps implying that it involves the sort of minimum brain-lantern delay ex- pected in a nonspecific reflex that is, a fixed response inherent in the way the flash-control system is con- structed rather than one evolved specifically in a com- municative context. (The human knee-jerk, a response incidental to the presence of stretch-receptors that func- tion normally in locomotion, is a case in point.) In this vein, and because of the lack of interaction between free P. pyralis males after such triggering. I use "reflex" provi- sionally to suggest a possible qualitative distinction be- tween the 0.38-s SD and 2.3-s LD photic responses. The LD response (female-type circuit in mule) Because males of L. lusitanica, I', concisus, and P. pyr- alis respond (by orienting) to the characteristically de- layed response flashes of their conspecific females, each must have a response-timing circuit that corresponds to the emission-timing circuit of the female. Males of L. Im- iianica and P. concisus also flash in response to other, conspecific males, and after the same delay used by their respective females. This suggests that the timing process initiated by seeing a flash of light may. potentially, termi- nate by mediating either orientation or flashing. Whether this lability involves bifunctional or parallel cir- cuits, and what determines whether female simulation occurs normally (L. lusilanica, P. concisus) or not (P. pyra/is), are less important in the present context than the apparent presence in both sexes of the same emis- sion-timing. The 2.3-s (LD) signal-male delay induced in CIR P. pyralis males (23) should presumably be shortened about 8% as a rotational correction, but is, in any case, close to the average 2. 1-s response delay of P. pyralis fe- males answering flashlight flashes at about 23 (Buck. 1937) or LED signals (Figs. 25 and 26). Thus, this labora- tory finding appears to parallel Papi's SD finding and to strengthen the idea that emission-timing circuitry of the female type is also present in the male. Why female-simulating behavior is overt in L. lusitan- ica and P. concisus and latent in P. pyralis is unknown. P. pyralis is the most abundant and widespread Ameri- can photinid firefly, occurring in at least 23 states (Lloyd, 1966), whereas its sibling species, P. concisus, is limited to a small area of central Texas. These distributions are consistent with the expectation that a signal that identi- fies the female unambiguously would have selective ad- vantage over one that does not. Possibly P. pyralis has evolved a step beyond P. concisus. There were not enough spontaneous intervals of 2-3 s duration (Figs. 21, 24; unfilled column caps in Fig. 19) to assert that they derive from endogenous excitation of the same LD flash-timing circuit that is sometimes ex- cited by LED flashes (Figs. 20, 22, 24). However, the con- centration of such intervals strongly suggests that the stress of flying on the CIR suspension does induce spon- taneous flashing at 2-3-s intervals in addition to the also atypical 2.3-s LD photic response to LED stimulation. Use of male circuitry by female In the three fireflies discussed above, the male recog- nizes the female's emission pattern specifically, but there is no evidence that the female recognizes the rhythmic spontaneous interflash interval of the male. However, in certain species in which the male's emission signal is a pair of flashes rather than a single flash, the female does recognize the male specifically. She responds only after being presented with a pair of flashes timed in the charac- teristic pattern of her conspecific male, and thus must have a circuit tuned to that interval. In P. greeni, for ex- ample, the male emits a pair of flashes 1.5 s apart every 5 or 6 s, and the female responds about 0.8 s after the second flash of the 1.5-s pair (Lloyd. 1969; Buck and Buck, 1972). No instance has been reported of a female of a pair- flashing species mimicking her male's flash pattern in the field, but P. greeni females have been induced to flash in pairs 1.5 s apart by strong repetitive photic stimulation (Buck and Case, 1986). Thus, as with P. pyralis males forced to fly in tight circles, it appears that abnormal stimulation sometimes uncovers latent flash-timing ca- pacity. Significance and genesis of unisex flash-controls In a cricket in which females cannot call, Huber(1962) found that females nevertheless ". . . possessed a ner- vous organization sufficient for primitive stridulatory movements in spite of the absence of stridulatory struc- tures." Alexander (1962) suggested that if both sexes were at least potentially able to call ". . . it would repre- sent an interesting simplification of evolutionary change in a communicative system something of an assurance that the . . . song of the male and the ability of the fe- male to respond to it . . . will evolve as a unit." The pres- ent evidence that male and female dialog fireflies share specific, quantitatively matched, flash-timing controls, overt or latent, may implement Alexander's insight. Because males and females of dialog fireflies are al- most identical in lantern structure and control mecha- nisms (Buck, 1948), the shortest photic delay circuit, if it is indeed a reflex, would be expected to be present in both sexes. It would be understandable, then, that this circuit could have been co-opted during evolution to mediate both the female's response delay and the matching recog- nition interval in the male (L. lusitanica and P. concisus), and to confer supplementary reproductive advantage via 94 J. BUCK male flash synchronization (Buck, 1988) in P. pyralis and P. concisus. It is less obvious how and why, in some species, this potentially unerring clue to female identification has evolved (or retained) the ambiguity of being used by males as well as females. The surmise that dialog ques- tions and answers ought to evolve as a unit seems not readily compatible with paradoxical behaviors like those in L. luaitanica and P. concisus in which males normally decoy other males as well as seek females. The existence of female-signal simulation seems to argue that the dupli- cate timing circuits owe their evolutionary fixation to that behavior, but it also seems obvious that dialog in which males can identify females unequivocally (as in P. pyralis) should be more strongly selected than dialog in which males are also attracted by males. Among suggested functions of female-simulation, "improving the female's chances of fertilization" (Papi, 1969) implies altruistic group selection. "Giving a re- jected male an opportunity to see and approach the fe- male's flashed answers to another male, and thus another chance to mate with her . . ." (Lloyd, 1979) seemingly has the rejectee and the female synchronizing with each other as both flash in response to the primary male. This would require the rejectee to recognize a flash that not only did not occur at the proper female-recognition in- terval after his flash but was in a phase relation (simulta- neity) that has been found, in other species, to be the point of minimum sensitivity to photic input (Buck et at, 1981; Buck, 1988). It is also not at all clear that males giving the female-simulating response have, in fact, been rejected previously. A third possible function of female-signal simula- tion distracting the deceived male from courting the real female, and so boosting the decoy's statistical chances of finding a mate (E. Arbas and S. Lewis, pers. comm.) may have more promise, particularly, as Dr. Lewis has pointed out to me, with the strongly male-bi- ased operational sex ratio that is usual in dialog popula- tions early in the season. Summary 1 . In timing her flashed answer to the male's signal, a female dialog firefly uses the same delay interval that the male uses in timing the interval between his own flash and her answer. 2. In three species, males answer the flashes of other males after the same specific response-delay interval that is characteristic of their conspecific females. 3. Experimentally, the male of a fourth species has been shown to be capable of flashing responsively after the same delay interval as the female. In a fifth species, the female can be induced to emit flashes with the same timing as one element of the male's spontaneous display. 4. The above data are compatible with the hypotheses that male and female firefly share some of the same courtship flash-timing circuits in overt or latent forms, and that a particular control circuit may, on occasion, time either detection or emission. The overall neuro- physiological picture is of a pool of timing circuits that can connect in various input/output combinations to mediate a variety of behavioral patterns. The data are consistent with Alexander's ( 1962) sur- mise that courtship questions and answers should evolve together. All present-day circuitry must, of course, derive by selection from ancestral flash-controls. In another communication I plan to compare firefly unisex re- sponses with possible analogs in other animals, and to examine the speculation that duplicate circuits in con- specific male and female fireflies hark back to a stage in dialog evolution in which both sexes flashed alike. Acknowledgments Many colleagues have enhanced my understanding of control physiology and evolution theory, but Drs. Ed- mund Arbas, Albert Carlson, Joseph Cicero, Sara Lewis, and Stephen Shaw helped particularly with the present communication. Jon Copeland suggested trying tethered flight. Richard Raubertas recommended the Wallenstein statistical analysis. I am also grateful to Elisabeth Buck, James Case, and Frank Hanson (the "et at" cited in the text) for allowing reference to unpublished joint work, to Dr. Hanson for firefly shipments, and to Jeff Carpenter for field assistance. Charlette Lancaster made the draw- ings. Literature Cited Alexander, R. D. 1962. Evolutionary changes in cricket acoustical communication. Evolution 16: 443-467. Hi nut Mi. M., F. Magni, and M. Pcllegrino. 1977. Excitatory and in- hibitory events elicited by brief photic stimuli on flashing of the firefly Lticiola lusiianica (Charp.). J. Comp. Physiol. (A) 119: 15- 35. Buck, J. B. 1935. Synchronous flashing of fireflies experimentally in- duced. Science^: 339-340. Buck,J.B. 1936. Studies on the firefly. Ph.D. thesis. The Johns Hop- kins University, Baltimore. Buck, J. B. 1937. Studies on the firefly. II. The signal system and color vision in Photinus pyralis. Physiol. Zoo/. 10:412-419. Buck, J. B. 1938a. A device for orienting and embedding minute ob- jects. Slain Tcchnol. 13:65-68. Buck, J. B. 1938b. Synchronous rhythmic flashing of fireflies. Q Rev. Biol. 13:301-314. Buck, J. B. 1948. The anatomy and physiology of the light organ in fireflies. Ann. N. Y. Acatl. Sci. 49: 397-482. Buck, J. 1955. Some reflections on the control of bioluminescence. Pp. 323-333 in The Luminescence of Biological Systems, Frank H. Johnson, ed. AAAS, Washington, DC. Buck, J. 1966. Unit activity in the firefly lantern. Pp. 459-474 in Bio- FIREFLY UNISEX FLASH CONTROLS 95 liiinnu"-cence in Pnyresx. F. H. Johnson and V. Hanoda, eds. Princeton Llniversity Press, Princeton, N.I. Buck, .1. 1988. Synchronous rhythmic flashing of fireflies. 11. Q Rev Hail 63: 265-289. Buck, J., and E. Buck. 1968. Mechanism of synchronous flashing of fireflies. Science 159: 1319-1 327. Buck, J., and E. Buck. 1972. Photic signaling in the firefly Plioimu\ greem. Biol Bull. 142: 195-205. Buck, J., E. Buck, J. F. Case, and F. E. Hanson. 1981. Control of flashing in fireflies. V. Pacemaker synchronization in Ptcroptyx cri- hellahi .1 Comp . Pliysiol (A) 144: 287-298. Buck, J., and J. F. Case. 1986. Flash control and female dialog reper- tory in the firefly Photinusgreeni. Biol Bull. 170: 176-197. Buonamici. M., and F. Magni. 1967. Nervous control of flashing in the firefly Ludola ilaliea L. Arch. Ital. Biol. 105: 323-338. Case, J. F. 1984. Vision in the mating behavior of fireflies. Pp. 195- 222 in Inxcct Communication. Trevor Lewis, ed. Royal Entomolog- ical Society of London. Academic Press, London. Case, J. F., and J. Buck. 1963. Control of flashing in fireflies. II. Role of central nervous system. Btol. Bull. 125: 234-250. Christensen, T. A., and A. D. Carlson. 1981. Symmetrically orga- nized dorsal unpaired median (DUM) neurones and flash control in the male firefly, Photnris wrsicolor ./ /-.'A/I liiol.93: 133-147. Edmunds, L. N., Jr. 1963. The relation between temperature and flashing intervals in adult male fireflies, Photinu* pyralis Ann. Ent. Soc.Am. 56:716-718. Hanson, F. E., J. Miller, and G. T. Reynolds. 1969. Subunit coordi- nation in the firefly light organ. Biol. Bull. 137: 447-464. Huber, F. 1962. Central nervous system control of sound production in crickets and some speculations on its evolution. Evolution 16: 429-442. Lloyd, J. E. 1966. Studies on the flash communication system in Pho- tinii.i fireflies. Ali.\c I'uhl ,\/HY /<>/ L'niv. Michigan. No. 130, pp. 1-95. Lloyd, J. E. 1968. A new Phnnmix firefly, with notes on mating be- havior and a possible case of character displacement. Coleopiermls ' Bull 22: 1-10. Lloyd, J. E. 1969. Flashes, behavior and additional species of nearctic Photintix fireflies (Coleoptera: Lampyridae). Coleopterists' Bull 23: 29-40. Lloyd, J. E. 1979. Sexual selection in luminescent beetles. Pp. 239- 342 in Se.\ncil Selection mill Reproductive Competition in /mw/.v. M. S. Blum and N. A. Blum, eds. Academic Press, New York. Maurer, Ll.M. 1968. Some parameters of photic signalling important to sexual and species recognition in the firefly Photmus pyralis. M. S. Thesis. State University of New York Stony Brook, NY. Olte, D., and J. Smiley. 1977. Synchrony in Texas fireflies with a consideration of male interaction models. Biol. Behav. 2: 143-158. Fapi, F. 1969. Light emission, sex attraction and male flash dialogue in a firefly, l.ttcioUi litvUinica (Charp.). Monilore Zool. Ital. (N. S.) 3: 135-184. \\allenstein, S. 1980. A test for detection of clustering over time. Am J Epiilemiol. 111:367-372. Reference: Bitil. Bull 179: 96-104. (August, 1990) The Structure of Sweeper Tentacles in the Black Coral Antipathes fiordensis WALTER M. GOLDBERG 1 , KEN R. GRANGE 2 , GEORGE T. TAYLOR'. AND ALICIA L. ZUNIGA 1 * Department of Biological Sciences, Florida International University, University Park, Miami, Florida 33 1 99 and 2 Department of Scientific and Industrial Research, Division of Water Sciences, Wellington, New Zealand Abstract. Normal tentacles on polyps of the black coral Antipathes fiordens ware less than 2 mm long and display well-defined, wart-like structures, the centers of which are marked by both flagella and microvilli. Both of these microappendages are characteristic of spirocytes, the dominant type of cnidocyte in normal tentacles. Sweeper tentacles, up to 15 mm long, form in apparent response to an alcyonacean epibiont. The external surface of the sweeper tentacle lacks the well-defined, wart-like batter- ies of the normal tentacle, and exhibits a general reduc- tion in the appearance of surface microappendages. Nonetheless, there is a greater number of cnidae per unit area. No spirocysts are found in these sweeper tentacles. Instead, the cnidom is composed entirely of microbasic b-mastigophores (MbMs). More than 99% of these are of a single type that are structurally different from the MbMs found as a minority of the cnidae in normal tenta- cles. Changes in sweeper tentacle cnidae are compared with those occurring in modified tentacles of other an- thozoans. Introduction Aggressive behavior by anthozoan coelenterates is effected by a variety of specialized structures containing nematocysts (see review by Bigger, 1988). Extrusion of septal filaments (preferred to "rnesenterial" filaments; see Bayer and Owre, 1968) onto a competitor is a com- mon strategy among scleractinian corals (Lang, 1973; Glynn, 1974; Loya, 1976; Cope 1981; Bak et ai, 1982; Logan. 1984), and has been described in a corallimor- pharian as well (Chadwick, 1987). Some anemones pos- Received 22 January 1 990; accepted 1 6 May 1 990. sess specialized marginal vesicles the acrorhagi that are used for this purpose (Francis, 1973; Ottaway, 1978; Bigger. 1980; Ayre, 1982;Sebens, 1984). A fewanthozo- ans are capable of forming specialized tentacles during aggressive interaction, den Hartog ( 1977) described the formation of specialized bulbous tentacle tips in coralli- morphs after contact with stony coral competitors. In other species, the entire tentacle structure can be modi- fied under such conditions. Some acontiate anemones develop short, opaque, blunt-tipped "catch" tentacles (e.g., Williams, 1975), while some scleractinian corals can form elongated "sweeper" tentacles (Lewis and Price, 1975; Bak and Elgerschuizen, 1976; den Hartog, 1977; Richardson et ai, 1979; Wellington. 1980;Chorn- esky, 1983). Sweeper tentacles have also been described in a few gorgonian corals (Sebens and Miles, 1989 and references therein). In all these cases, the transformation occurs within 4-9 weeks, when the normal or feeding tentacle converts to a sweeper or catch tentacle as the result of contact with other coelenterate species (Welling- ton, 1980; Bak et ai, 1982; Bigger, 1982; Chornesky, 1983). Intraspecific competition (Purcell and Kitting, 1982; Kaplan, 1983; Hidaka and Yamazato. 1984; Hi- daka, 1985; Fukui, 1 986) and competition with non-coe- lenterates (Hidaka and Miyazaki, 1984) also appear to be associated with changes in tentacular morphology. The induction of sweeper and catch tentacles is ac- companied by changes in the relative proportions of ne- matocysts and spirocysts, as well as by shifts in the type of nematocyst (den Hartog, 1977; Purcell. 1977; Wel- lington, 1980; Watson and Mariscal, 1983a, b; Hidaka and Yamazato, 1984; Fukui. 1986; Hidaka et a/., 1987). However, these earlier observations were made with op- tical or scanning electron microscopes and the intracap- 96 BLACK CORAL SWEEPER TENTACLE 97 sular details of cnidae before and after conversion to the sweeper tentacle were not compared. Thus, only changes between classes of nematocysts could be detected, while more subtle changes within-class were not. Similarly, though some of the external differences between tentacle types have been documented (den Hartog, 1977; Hidaka and Miyazaki, 1984; Fukui, 1986; Sebens and Miles, 1989), little attention has been given to histological and cytological distinctions. Watson and Mariscal ( 1983a, b) examined catch tentacles from this perspective, while Doumenc ( 1972) and Bigger ( 1982) documented the ul- trastructural differences between acrorhagi and tenta- cles. We know of no corresponding work for sweeper ten- tacles. Antipatharian corals are little known, largely due to their typical occurrence in deeper water. However, on the southwest coast of New Zealand's south island, large, virtually monotypic black coral populations occur in depths of 5-30 m (Grange el ai. 1981; Grange, 1985), and are thereby accessible to study. The structure of the polyp, including the tentacle and gastrodermis, has been examined by Goldberg and Taylor (1989a, b) who re- ferred to this species as Anlipathes apcria Totton. How- ever, further work has resulted in its re-description as the endemic A. fiordensis Grange, 1990. We now report on the occurrence of sweeper tentacles in these animals, and compare their morphological and cellular structure with unmodified tentacles. Materials and Methods The site for this study was Doubtful Sound, Fiordland, New Zealand (45 20.95' S, 167 02.83' E). Living mate- rial was photographed /;; situ at 10-20 m to document the incidence of sweeper tentacles. Color transparencies were examined microscopically to estimate tentacular size. Branchlets with normal polyps and tentacles, as well as areas of the colony where sweeper tentacles had formed, were collected and immediately fixed for 4 h in a solution of 3% glutaraldehyde and 1% paraformaldehyde in sea- water containing 0. 1 M cacodylate at pH 7.4. The fixed tissues were then transferred to cacodylate-buffered sea- water and express-shipped to Miami for secondary fixa- tion in osmium tetroxide, alcohol dehydration, and em- bedment in Spurr resin. Sections of tentacle 1 ^m thick were normally stained for 30 s at 55C with borax-buffered 0.05% Toluidine blue, but sweeper tentacles required much shorter stain- ing times (see Results). A Philips EM 200 operated at 60 kV was used for transmission electron microscopy; scanning electron microscope observations were made with an ISI Super 3A SEM, using material critical-point dried from liquid CO 2 . Proportional counts of cnidae types were made by examining randomly chosen 1 //m sections taken from 8 normal and 8 sweeper tentacles until at least 1000 cnidae were counted. Results The appearance of the normal polyp of A. fiordensis is shown in Figure 1 . Each polyp has six tentacles (five are shown) that surround the mouth and pharynx. The pha- ryngeal region also projects from the polyp as an oral cone (see Goldberg and Taylor, 1989a, for details). The living tentacles are 1.2 0.6 mm long and taper to a blunt tip. There is no distinct acrosphere. The tentacle surface is organized into a series of raised, wart-like bat- teries, with flagella projecting from their centers (Figs. 2, 3). The warts in the apical regions are flagellated, but are better defined proximally. Ciliary cones (stereocilia sur- rounding a longer central cilium) are scattered and are not a prominent feature of the tentacular epidermis. Spirocysts comprise 88.7% of the tentacular cnidae. Their capsules are clear and do not stain with Toluidine blue (Fig. 3 inset). The hirsute appearance of the wart center is largely a function of the mature spirocyte (Fig. 3), the apical end of which bears a circlet of microvilli and a single, acentrically placed flagellum 1 jum or more in length (Goldberg and Taylor, 1989a). Cells at the wart periphery and base tend to stain deeply with Toluidine blue. Part of this pattern is ac- counted for by the staining of the mucus cells, and part by the staining of nematocysts. Mucus cells stain a meta- chromatic pink, especially around the vesicles. The vesi- cles themselves develop a much lighter metachromasia. Most of the nematocysts stain a deep purple. The cap- sules average 2.5 ^m wide by 18.0 ^m long, and contain a triply pleated tubule in a poorly infiltrated, generally electron-opaque matrix (Fig. 3). The shaft is cylindrical, nearly the length of the capsule, and 0.5 0.2 /urn wide. It is approximately uniform in diameter, tapers toward the base as it meets the tubule, and ends apically in a distinctive cap. These characters are consistent with the description of microbasic b-mastigophores (MbMs) given by Mariscal (1974), and are the nematocysts re- ferred to by Goldberg and Taylor (1989a) as type A MbMs. They comprise about 8.7% of the tentacular cni- dae. A second type is larger (up to 35 urn long and 10 ^m wide), and displays a weak orthochromasia with Tolu- idine blue. The matrix has a granular appearance in the electron microscope. Conversely, the shaft, and to some extent the tubule, are electron-opaque. The shaft is 1.8 0.3 nm wide and about a third the capsule length. De- spite these distinctions, this second type falls into the same nematocyst category as those described above, re- ferred to as type B MbMs by Goldberg and Taylor (1989a). These large, granular nematocysts comprise 98 W. M. GOLDBERG ET AL. Figure 1. SEM preparation of Anlipathex Iwrdenxix polyp. Five of six tentacles are shown surrounding the mouth (M) in a central oral cone. Scale bar = 20 fim. Figure 2. Normal tentacle showing wart-like structures (W). The rim of the wart is composed of mucus cells: the depressed wart centers contain clusters BLACK CORAL SWEEPER TENTACLE 99 Figure 4. Sweeper tentacles (SW) adjacent to a contracted alcyonacean colony (A). Polyps with normal tentacles (NT) are located on branchlets (BR), while sweeper tentacles are usually confined to larger branches. Scale bar = 5 mm. Figure 5. //; xitn photograph showing development of sweeper tentacles (SW) in association with expanded polyps of the alcyonacean coral Alcyoiiiunt mtrantiaciim (A). Note apparent gradation of tentacular length toward the epibiont. There are no normal A. fwrdensis tentacles in this photograph. Scale bar = 5 mm. about 2.6% of the cnidae in the normal tentacle, but are the dominant cnida in the gastrodermis (Goldberg and Taylor, 1989b). Sweeper tentacles in A. fiordensis are thread-like and extend up to 1 5 mm long (Fig. 4). A gradation of tentacle length may occur, either through contraction, distance from the competitor, or both, so that some tentacles may be as short as 2 mm. We have examined all tentacle sizes histologically (see below), and have not found intermedi- ates between normal and sweeper tentacles. There is no distinct acrosphere, in contrast to their formation or en- largement in other corals with sweeper tentacles (den Hartog, 1977; Chornesky and Williams, 1983; Hidaka and Miyazaki, 1984; Sebens and Miles, 1989), and their agonistic analogues in corallimorphs (den Hartog, 1 977). A. fiordensis sweeper tentacles tend to form on thicker (older) branches, generally 4 mm or more in diameter and are especially common in the presence of the alcyo- nacean Alcyonium aurantiacum Quoy and Gaimard (Fig. 5). We have also noted them near red algae (Epy- menia sp. and Lithothamnion sp.). However, in these in- stances we have not ruled out the presence of foreign of flagella, representing the apical ends of cnidocytes. Scale bar = 20 ^m. Figure 3. TEM preparation through a wart center perpendicular to the tentacular axis. Flagella and prominent microvilli (MV) are associated with the surface of mature spirocytes (SP). An arrow connects the flagellum and rootlet in this spirocyte. Two type A microbasic b-mastigophores (mbm) are shown. Upper left: a mature capsule with characteristic poorly infiltrated matrix. Lower right: an immature capsule with typical electron-opaque matrix. The raised edges of the wart are formed by mucus cells (MU). Scale bar = 5 jim. Inset: tentacular cross-section showing arrangement of stained mucus cells (arrows) surrounding unstained spirocyte clus- ters. Light microscopy; Scale bar = 20 100 W. M. GOLDBERG /:/ I/ t ^ \SRw/ Figure 6. SEM preparation of sweeper tentacles. Note apparent absence of wart and microappendage structure. Surface cracks are artifacts of preparation. Scale bar = 50 nm. Figure 7. Higher magnification shows that surface filiation occurs, but is greatly reduced. The center of a single wan (W) is indicated by BLACK CORAL SWEEPER TENTACLE 101 coelenterate material, or the possibility that algal coloni- zation was secondary. Sweeper tentacles in these black corals are extremely fragile, especially after fixation and dehydration. Except for artifactual cracks, their surfaces appear smooth, with- out the wart-like structure of normal tentacles (Fig. 6). At higher magnification, low mound-like warts with hirsute projections at their centers can be distinguished (Figs. 7, 8). The cells in the wart center are primarily cnidocytes, the surfaces of which form short microvilli and have a central cilium usually 3 nm or less in length (Fig. 9). The microvilli often form a collar-like arrangement at the tentacular surface (Fig. 8 inset), but ciliary cones, as such, apparently are absent (see Discussion). Histologically, sweeper tentacles are completely different from their normal counterparts. The wart mar- gins are still defined by mucus cells, but in sweeper tenta- cles these cells tend to be longer, narrower, and less nu- merous than those in normal tentacles (compare Figs. 3 inset and 9 inset). In addition, mucus cells stain more uniformly and produce a more intense pink metachro- masia compared to their normal counterparts. More im- portantly, spirocysts are absent. All of the cnidae in the warts are nematocysts, and nearly all of them stain rap- idly and deeply with Toluidine blue, giving the sweeper tentacle a comparatively uniform structure (Fig. 9 inset). Staining time must be held to less than 5 s at room tem- perature to avoid overstaining these cnidae. The capsules are 3.5 0.5 jum wide and 22.5 2.5 j/m long. This is 40% wider and 25% longer than the dominant nemato- cyst found in normal tentacles. Warts generally contain 20-30 mature cnidae each, half or less of the 50-60 cni- dae (mostly spirocysts) found in normal tentacles (Gold- berg and Taylor, 1898a). Conversely, there is a greater number of warts per unit surface area in sweeper tenta- cles. In the boxed area of Figure 7, for example, 1 1 to 12 warts can be distinguished, whereas a comparable area from normal tentacle contains little more than two warts. Thus, sweeper tentacles contain approximately twice the number of cnidae per unit surface area. Transmission electron microscopy shows that the structure of the shaft and capsule in the sweeper tentacle nematocysts are very similar to the type A MbMs de- scribed above from normal tentacle. However, differ- ences in these cnidae become evident as they mature. The matrix becomes electron-lucent, tends to infiltrate poorly, and often shatters during sectioning. The shaft, matrix, and capsule wall usually lack adequate contrast (Fig. 9) even after overstaining. Occasionally, large ne- matocysts with a granular matrix can be found in or near final position (Fig. 9). These appear to be the same gas- trodermal-type mastigophores that constitute 2.6% of the cnidae in the normal tentacle, but account for less than 1 % of the sweeper tentacle nematocysts. These large cnidae also occur in the body wall epidermis of both nor- mal and sweeper polyps. The latter appear to possess more of the gastrodermal cnidae than the sweeper tenta- cle, but proportionately no more than normal tentacle or body wall. Unlike normal polyps, however, the body wall is devoid of spirocysts. This condition indicates that the cnidae in the epidermis surrounding the sweeper ten- tacle are modified along with the tentacle itself. Discussion A few studies of sweeper or catch tentacle formation have included quantitative observations of the cnidae (Table I). In all but one of the specialized tentacles stud- ied to date, spirocyst production is completely or almost completely suppressed in favor of nematocysts. In Ga- laxea fascicularis there is a 50% reduction in the spiro- cyst population of the sweeper tentacle acrosphere (Hi- daka and Yamazato, 1984). Because spirocysts function in adhesion during food capture (Mariscal, 1974; Maris- cal et al.. 1 977 ). a change in favor of nematocysts is con- sistent with a change in tentacular function. However, a reduction in the development of surface microappen- dages in sweeper tentacle might not be predicted with the change from spirocysts to nematocysts. Mariscal et al. ( 1976) found microvilli, but no ciliary structures associ- ated with spirocytes in several anthozoan species, and suggested that this may be an additional distinction be- tween spirocytes and nematocytes. To the contrary, the normal tentacles ofA.fiordensix are dominated by spiro- the arrow. Scale bar = 20 ^m. Figure 8. Triple magnification of boxed area in Figure 7 shows reduced wart-like structure. Scale bar = 5 jim. Inset: surface detail showing part of a single wart. Clustered microvilli suggest ciliary cones, but a prominent central cilium is not apparent. Scale bar = 5 ^m. Figure 9. TEM preparation showing typical appearance of sweeper tentacle epidermis. The only cnidae are microbasic b- mastigophores. The two at left are the type that constitute >99% of the nematocysts. accompanied by an occasional large, gastrodermal-type microbasic b-mastigophore at right. The dominant cnida has an apical cap (ac), a triply pleated tubule (t), and distinct shaft (sh) in a poorly infiltrated, electron-lucent matrix. The rim of the sweeper tentacle wart is formed by mucus cells (MU). The surface of each cnidocyte is drawn into a short collar of microvilli (arrows) that surround a single cilium. Scale bar = 1 ^m. Inset: mucus cells in sweeper tentacles tend to be elongated (arrows), and stain intensely with Toluidine blue, as do the dominant nematocysts, giving the sweeper wart structure a totally different appearance in the light microscope. Scale bar = 20 ^m. 102 W. M. GOLDBERG ET AL. TABLE I Quantitative distribution of cnidae types in normal and modified tentacle Taxon Normal tentacle cnidae Sweeper or catch tentacle cnidae Scleractinia: 10% Sp 1 , 1 5% MpM 2 in acrosphere; 50%. MbM 3 , 35% Sp in acrosphere; ( ialcLwa lascicularis* HI J in tentacle and column with some MpM & Sp 4 MhM in tentacle with some HI and Sp Scleractinia: 83% Sp. 8% MbM in acrosphere 63% HI, 30% interm 5 in acrosphere Mi Hitastrea cavemosa b Actiniaria: 82% Sp. 17% MbM 58% HI, 38% AI 6 Mclriilium senile' Actiniaria: 57% Sp. 26% MpM 99+ % HI Huliplanella hicuic' i Antipatharia: 89% Sp, 9% MbM all MbM Antipathesfiordensis' a Hidaka and Yamazato, 1984. "denHartog, 1977. 'Purcell, 1977. d Watson and Mariscal, 1983a. ' This paper ' Spirocyst = Sp. : Microbasic p-mastigophore = MpM. 3 Microbasic b-mastigophore = MbM. 4 Holotrichous isorhiza = HI. 5 Cnidae intermediate in form between HI and MbM. 6 Atrichous isorhiza = AI. cytes that not only possess well-developed microvilli, but are clearly ciliated (flagellated) as well (Goldberg and Taylor, 1989a). The distinct reduction in the hirsute ap- pearance of its sweeper tentacles is therefore consistent with the absence of spirocytes. Further reduction in sur- face microappendage formation might result from a gen- eral decrease in epidermal microvilli. Because microvilli may serve as a reserve pool of membrane (Erickson and Trinkhaus, 1976), they might assist in the formation of elongated structures in coelenterates (Eppard et at. 1 989) including sweeper tentacles. Similarly, the absence of typical ciliary cones may be due to the shortness of the central cilium in the MbMs. Therefore, the absence of these putative receptor structures (cf. Mariscal, 1974) may be more apparent than real. Indeed, Hidaka and Miyazaki (1984) have suggested that ciliary cones may be typical of microbasic-b or p-mastigophores, as op- posed to other nematocyst types. The presence of occa- sional ciliary cone structures in normal A.fwrdensis ten- tacles may correspond to the lower relative abundance of these cnidae compared to sweeper tentacles. The change in nematocyst type on conversion to sweeper tentacle appears to be inconsistent across taxa. Some species replace MpM (microbasic p-mastigo- phores) or HI (holotrichous isorhizas) in feeding tenta- cles with MbMs (microbasic b-mastigophores), whereas others replace MbM or MpMs with His, among others (Table I). In the case of A. ftordensis, the change in the nematocyst population involves differences in mean cap- sule size, appearance of the mature matrix, and staining characteristics. Other characters, especially the appear- ance of the shaft, suggest that the sweeper tentacle cnidae remain as MbMs. However, this designation is tentative due to the relatively small population of mature cnidae that remain undamaged after sectioning. Changes in the size, shape, and other details of nematocyst structure have been documented during the conversion to sweeper tentacles in other species (den Hartog, 1977; Hidaka et at. 1987), as well as between metagenetic and ontoge- netic stages of the same species (Fautin, 1988, and refer- ences therein). Some of these distinctions may be a func- tion of taxonomy. Hidaka et at (1987) discuss the prob- lems of classification when attempting to distinguish populations of MbMs, basitrichs, and holotrichous ne- matocysts. They agree with Schmidt (1974), who con- cluded that intergrades exist between MbMs and basi- trichs, and refer to both as b-rhabdoids. The inconsisten- cies listed in Table I may be resolved by lumping morphological characters of the shaft and tubule, but this may not address important functional changes that could be represented by increased capsule size (Watson and Mariscal, 1983a; Hidaka and Yamazato, 1984; Hi- daka el at. 1987; this paper) and changes in the physical properties of the nematocyst matrix (this paper) that ac- company the conversion of normal to sweeper or catch tentacle. Several authors have correlated the morphogenetic pattern of catch tentacle development with the forma- BLACK CORAL SWEEPER TENTACLE 103 tion of its cnidae (Purcell, 1977; Watson and Mariscal, 1983a; Hidaka and Yamazato, 1984; Hidaka ct ill.. 1 987). In our study, sweeper tentacles irrespective of size, had already formed at the time of sampling. Thus, we have not had an opportunity to observe changes presum- ably occurring in the cnidom over time. Watson and Mariscal (1983b) showed that in Haliplanellu Indue. catch tentacles not only exhibited a change in the type of cnidae, but displayed a maturational gradient in cnidae development as well. The catch tentacle base contained 96.3% cnidoblasts (cells with immature cnidae). Con- versely, the catch tentacle tip contained mature cnidae almost exclusively. The opposite pattern was seen in feeding tentacles, where the gradient in maturity oc- curred cross-sectionally, i.e.. cnidoblasts were distributed along the base of the epidermis, while mature cnidae were restricted to the outer epidermis. These opposing patterns were not present in the sweeper tentacles of .1. fiordensis. In both the normal tentacle (Goldberg and Taylor, 1989a) and the sweeper tentacle, nematocyst de- velopment took place in a cross-sectional gradient, not a longitudinal one. 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An- thozoa). Biol. Bull 164: 506-517. Watson, G. M., and R. N. Mariscal. 1983b. Comparative ultrastruc- ture of catch tentacles and feeding tentacles in the sea anemone Haliplanella. Tissue Cell 15: 939-953. Wellington, G. M. 1980. Reversal of digestive interactions between Pacific reef corals: mediation by sweeper tentacles. Oecologia 47: 340-343. W illiams, R. B. 1975. Catch tentacles in sea anemones: occurrence in Haliplanella luciae (Verrill) and a review of current knowledge. J.Nat. Hist 9:241-248. Reference: Bin/ Bull 179: 105-1 12. (August, 1990) Parasitism and the Movements of Intertidal Gastropod Individuals LAWRENCE A. CURTIS* University Parallel, Ecology Program School of Life Sciences, and College of Marine Studies, University of Delaware, Newark, Delaware 19716 Abstract. Movements of marked individuals of Ilya- nassa obsoleta (n = 500) were charted in an intertidal environment for about one week. At the end of observa- tions, 260 marked individuals, which had been sighted 1017 times collectively, were recollected and examined for trematode infections. Six trematode species were found in 19 infection combinations including unin- fected, singly, doubly, and triply infected snails. We know that most snails found high on beaches and on sandbars carry Gynaecotyla adunca infections. It has been hypothesized that this host behavior modification is a parasite adaptation to enhance cercarial transmission to a semi-terrestrial next host. Observations reported here support this hypothesis and reveal some of the com- plexity in the behavior imposed on /. obsoleta by G. ad- unca. Individuals that were uninfected or infected with other parasites demonstrated no unique movement pat- terns, but individuals infected with G. adunca made re- peated excursions into the upper shore habitat. These ex- cursions were timed so that host-parasites were left emerged at high elevations primarily during nighttime low tides. Because many snails were multiply infected, data presented support the idea that gastropod popula- tions have the potential to be used as systems for the study of the nature of ecological and evolutionary inter- actions among parasite species. Introduction Spatial and temporal patterns associated with inter- tidal animals have been studied by many authors. A re- cent review (Foster el al., 1988) discusses the ecological Received 28 March 1990; accepted 29 May 1990. * Mailing address: Cape Henlopen Laboratory, College of Marine Studies. University of Delaware, Lewes, DE 19958. factors thought to cause these patterns. However, point- ing to a shortage of specific studies, these authors ex- pressly (p. 1 3) did not include the effects of parasitism in their discussion, although they noted that the effects of parasites on their hosts can be substantial. Several studies have examined the movements of intertidal gastropods (e.g., Underwood, 1977; Borowsky, 1979; Hazlett, 1984), but parasitism has not often been considered an important factor in these studies. Gastropods are an im- portant component of marine and estuarine benthic sys- tems. That parasites should not be ignored in these sys- tems has been shown by previous studies with the mud snail Ilyanassa obsoleta and its trematode parasites (Sindermann, 1960; Stambaugh and McDermott, 1969; Curtis and Hurd, 1983; Curtis, 1985, 1987). The signifi- cance of parasitism has also been shown for at least one other abundant gastropod, Littorina littorea (e.g.. Sindermann and Farrin. 1962; Lambert and Farley, 1968; Williams and Ellis, 1975). In a previous observation (Curtis, 1987) I noted a rela- tionship between trematode parasitism and vertical zo- nation of the mud snail Ilyanassa obsoleta on the shore. Snails found high on beaches and on sandbars were usu- ally parasitized by Gynaecotyla adunca. Snails unparasit- ized or parasitized with other species remained at lower levels. Data were interpreted to mean that the parasite alters the normal behavior of the host snail to enhance transmission of cercariae to semi-terrestrial (beach- dwelling) crustacean second hosts. Many parasites alter host behavior to enhance host-to-host transmission by means of predation [see Dobson (1987) for references], but this is apparently the first observation suggesting al- tered behavior with the adaptive advantage of enhancing cercarial transmission. Thus far, inferences concerning the behavior-altering 105 106 L. A. CURTIS capacity of Gynaecotyla ailnnca have been drawn from correlative population-level data with no attempt to de- termine the behavior of individual snails. Observations of individual host behavior are necessary because one cannot determine the exact composition of the altered behavior by comparing parasitism in populations at different vertical levels on the shore (Curtis, 1 987). Many questions are left unanswered by this approach. Does the same host repeatedly move onto the same area of beach or sandbar? If repeated visits occur, is there a schedule? Do hosts always move to a similar vertical position? Does this mean that G. aditnca-'mfecled snails move around in the habitat significantly more than other snails? In gen- eral, just how simple or complex is the altered behavior imposed on Ilyanassa obsoleta by this parasite? Data presented in this paper help address these ques- tions. Positional histories of individually marked snails with various infections (including uninfected) were charted in a natural sandflat environment for approxi- mately one week. Data indicate that Gynaecotyla adun- ra-infected snails, despite the presence or absence of co- occurring trematodes, have a complex, patterned behav- ior that is much different from the behavior of snails when unparasitized or parasitized by other trematodes. In a broader sense, the results illustrate the depth to which parasites become insinuated into ecological sys- tems and their contribution to the complexity of those systems. Additionally, the high frequencies of double and triple infections revealed by this (and other) work support the idea that host gastropod populations could be more useful as systems for the study of parasite species interactions than is currently appreciated. Materials and Methods The experiment took place during July 1985 on the Cape Henlopen sandfiat in Delaware Bay (described in Curtis and Hurd, 1983). The same sandbar area that had been used in 1984 (Curtis, 1987) was used again. The configuration and position of the sandbar had changed somewhat over the winter, so on 2 July sandbar topogra- phy was again mapped (Fig. 1 ) using the methods de- scribed in Curtis (1987). A 3 X 5 m plot was marked out at the peak of the sandbar and a pipe was driven deep into the center of the plot as a vertical (zero elevation) and horizontal reference point for measurements made during the experiment (see Fig. 1 ). Cape Henlopen has two low and two high tides per day that are roughly equal in range (ca. 1-1.5 m). The sandbar peak was emerged for approximately 3 h on ei- ther side of low tide and submerged for approximately 3 h on either side of high tide. In 1984 there was a clear correlation between propor- tion of snails parasitized by Gynaecotyla aclunca and ele- Figure 1. A sketch map of the Cape Henlopen sandbar used for this study. Zones of elevation and position of the 3x5 meter plot are shown. vation. To determine whether this was also true in 1985, snails were collected from the sandbar peak area (n = 185) and, for comparison, from 11 sites on the sandflat peripheral to the sandbar (n = 125). Sandflat snails were collected from areas at least 28 cm down from the sand- bar plot (Fig. 1 ). These were examined for size, sex, and parasitism as described in Curtis (1985). Generally, the design of the experiment involved col- lecting two sets of snails: sandbar/beach snails (which were likely to be infected with Gynaecotyla adunca): and sandflat snails (which were unlikely to be so infected). Snails in both sets were marked individually and released on the sandbar. The sandbar, adjacent sandflat, and beach were searched for marked individuals during each subsequent low tide for about one week. As marked indi- viduals were located, their positions relative to plot cen- ter were recorded using polar coordinates. More specifically, snails were marked with numbered insect tags glued onto a dried and roughened shell area with clear fingernail polish. Sandflat snails (n = 250) were collected on 1 5 July during the 1 309 h low tide. (All times are Eastern daylight savings time.) They were measured for size, marked, and retained in the labora- tory until release. Sandbar/beach snails (n = 250) were collected during the 0216 h low tide on 16 July. They were measured, marked, and retained in the laboratory. On the next low tide (16 July, 1354 h) the 500 marked snails were released on the sandbar in 20 groups of 25 (13 sandbar/beach snails plus 12 sandflat snails, or the reverse) placed at equal intervals along a circumference PARASITISM AND SNAIL MOVEMENTS Table 1 Nearby physical conditions and numbers of snails at lite sandbar peak 1 3 5 in plot) during the lh-?4 July 1 985 Cape llenlopcn experiment n'llli individually marked Ilyanassa obsoleta 107 Low tide # Time EDST Lighting Ta Tw Sal Snails in plot % marked 1354 L 25 24 29 (marked snails released) 1 0257 D 24 23 28 42 12 2 1437 L 27 28 7 4 25 3 0339 D 21 21 9 63 40 4 1522 L 29 32 28 15 27 5 04 IS D 18 19 18 102 37 6 1608 L 24 28 29 3 33 7 0500 D 23 21 27 70 36 8 1655 L 28 30 30 7 9 0543 D 25 23 24 27 48 10 1745 1 26 26 26 3 67 11 0627 D 24 24 26 45 40 12 1X40 1 26 26 29 15 47 13 0713 D 20 21 -> 39 36 14 1938 D 23 23 30 14 27 15 0803 1 16 2040 D Abbreviations: Ta and Tw = air and water temperature (C): sal = salinity (g/kg): and lighting conditions (L = light. D = dark). A tide is labeled "dark" if it occurred between sunset minus one hour! 1900 h)and sunrise plus two hours (0800 h). at a 10-m radius from the plot center (Fig. 1). On this and all subsequent low tides during the experiment, all snails inside the plot were evicted (scattered just outside the plot). Thus on any given low tide, counts of plot snails reflected those that had moved in during the previ- ous high water and remained. The procedure for locating marked snails involved searching the sandbar and its periphery. Whenever a marked snail was sighted, mark number, time, meters from plot center, and number of degrees clockwise from magnetic north were noted. The sandbar and adjacent sandflat were searched in ever wider circles until water became too deep (about 15 cm) to observe snails. De- pending on tidal range, the sandbar and adjacent areas could be effectively searched for up to three hours on one low tide. If the release tide is tide zero, searches were per- formed and snail positions noted through tide 16 (24 July, 2040 h). Most marked snails were recollected on tides 14-16. Many marked snails were recollected after tide 16 (through 28 August). Sightings of these snails dur- ing the experimental week were used, but positions re- corded at recollection were not. By transferring position of a snail at sighting to the contour map of the sandbar (Fig. 1 ) elevation of the snail at that sighting and net distance moved since the previ- ous sighting were determined. Snail elevations were as- signed to discrete elevation zones (0, 2, 4. 6 50 cm be- low plot center). A mean elevation at sighting was calcu- lated for each recovered marked snail. Net distance moved per tide by a snail between sightings was deter- mined by converting polar coordinates from field mea- surements to rectangular coordinates, calculating dis- tance between the two points and dividing by the num- ber of elapsed tides. To reduce any effects of the marking and release procedure, movements between points of re- lease and first sightings were excluded. A mean net movement per tide (rounded to the nearest meter) was calculated for each recovered marked snail that was ob- served at least twice. Recovered snails were examined in the laboratory by dissection so individual snail histories during the experiment could be compared based on par- asitism and other characteristics. Comparisons among snails with different infections in terms of elevation on the sandbar and net distance moved were made using nonparametric statistical methods (Sokal and Rohlf, 1981). Results Table I shows some of the physical conditions prevail- ing on low tides during the experiment as well as num- bers of snails entering the 3 X 5 m plot at the sandbar peak during the previous high water. Temperatures and salinities were in the usual ranges for Cape Henlopen. No information is given for tides 1 5 and 16 because from tide 14 on snails were being collected as sighted. In Table I, tides are classified as occurring under light or dark con- ditions, and it is apparent that more snails were present in the plot on dark low tides (mean = 50.25, S.D. = 27.53) than on light low tides (mean = 7.83, S.D. 108 L. A. CURTIS Table II Si an \iii~, lur shell size and sex ratio, and number of snails in cadi trematotle inh'clion ailcgon; lor groups of Ilyanassa obsoleta cullc and examined in mniicc/ion with the snail movement experiment Unmarked snail samples Parameter Sandbar peak Sandllat Recollected (marked) snails Shell height (mm) mean S.D. ', female: ' r ; male 21.1 1.7 47:53 20.3 2.3 50:50 21.4 1.5 44:56 Infection* Nl 39 64 32 Hq 11) 8 Ls d 4 5 Zr 2 35 19 Av 2 5 Ga 78 106 Dn (I 1 1 HZ 1 1 3 HG d 3 LZ 1 1 4 LG 22 1 31 ZA 1 ZG 25 30 AC 5 6 CD 1 GSt (1 1 LZG 5 1 3 LAG 2 3 ZAG 1 TOTALS 185 125 260 % Ga-infected 75.1 1.6 70.4 * Abbreviations: NI = not infected; Hq = ffimasthlaquissetensis;L& = Lepocreadium setiferoides; Zr = Zoogonus ruhellus: Av = Austrobi- Iharzia variglandis; Ga = Gynaecotyla aduneu; Dn = Diplostomum nasxa; St = Stephanostomum leinie; multiple infections are abbreviated with the generic initials of the species involved (e.g.. ZAG = ZrAvGa triple infection). = 5.74). Table I further shows that on most tides many snails in the plot had marks, which suggests that they made up a substantial proportion of total snails using the sandbar. Table II shows that the groups of snails exam- ined in this study did not differ significantly in terms of age (shell height) or proportions of males and females. However, it is clear that snails visiting the sandbar peak (plot) were largely Gynaecotyla adunca-infecled, whereas snails from the adjacent sandflat were not (Table II, unmarked snails). Table II also shows the variety of multiple infections present and, in particular, the large proportion of G. adunca infections that were combined with other trematode infections: of the 324 observed G. adunca-mkcted snails in Table II, nearly half (43.2%) were multiply infected. Of the 500 snails marked and released, 260 were ulti- mately recovered. Frequencies of trematode infections among recollected marked snails, which were sighted 1017 times collectively over the 16 tides, are shown in Table II. Based on initial collection site, there were two groups of marked snails: a sandbar/beach group (n = 250, mean shell height = 21.5mm, S.D. = 1.5); and a sandflat group (n = 250, mean shell height = 20.8 mm. S.D. = 1.5). The likelihood of sighting and recovering a snail was clearly different for individuals in the two groups: 74.6% of the sandbar/beach snails were recov- ered (93.6% were sighted at least once after release): only 31.2% of the sandflat snails were recovered (68.4%. were sighted after release). Almost all (99.5%) recovered snails from the sandbar/beach group (n = 182) had Gynaeco- tyla adunca infections, while very few (2.6%) recovered snails from the sandflat group (n = 78) had this parasite. Marked snails apparently stayed near the sandbar during the experiment because regular searches of the adjacent beach (Fig. 1) and a neighboring sandbar 30 m SW re- vealed no marked snails. The less frequent observation and greater loss of marked sandflat group snails can be explained by their being dispersed in a larger area (see below). Three marked snails were sighted in the area of the 1985 sandbar during July and August 1986. By this time, the experimental sandbar no longer existed. Snail A was uninfected when examined and had a shell height of 20.0 mm ( 19.9 mm when marked). It was observed twice in 1985 around the periphery of the sandbar. Snails B and C were both double infected with Zoogonus rubellus and Gynaecotyla adunca and had shell heights of 24.9 and 23.8 mm (24.7 and 23.7 mm, respectively, when marked). Both snails had been sighted several times (to- tal = 8) on the peak of the sandbar in 1985. Such data are subject to multiple interpretations, but indications are: ( 1 ) that growth for these large snails was slight during the previous year; and (2) based on behavior, snail A was probably not infected in 1985 and remained so, while snails B and C were both infected with (at least) G. ad- unca in 1985 and had carried their infections through the winter. Sightings of marked snails during the experiment pro- vided information on elevation at sighting and move- ment between sightings for individuals with various sin- gle and multiple trematode infections. Because Gynaeco- tyla adimca-'mfecled snails aggregated near the sandbar peak, probability of observing these snails was enhanced. Snails that did not behave this way were dispersed in a larger area (the outer edge and periphery of the sandbar), and the probability of observing them was reduced. Ac- tivities of G. adtincii-infected snails would be over-repre- sented in the data if all the individual sightings (range = 1-9/snail) were used as datum points. To correct for PARASITISM AND SNAIL MOVEMENTS I a !>!. Ill Relationship between trematode parasitism and elevation at sighting (cm down from bar peak) of marked Ilyanassa obsoleta individuals on a Cape Henlopen sandbar* 109 Parasitism Parameter Nl Hq Ls Zr Av Ga LG ZG AG # marked snails 32 8 5 19 5 106 31 30 6 Total # sightings 78 17 18 53 14 460 151 115 34 Median of mean elevs. (cm) 38 34.7 30 38.7 42 5.5 4.7 4.5 4.5 Range (cm) 3-50 33-48 22-47 25-50 34-51 0-32 0-19 0-30 1-8 Avg. rank 201 205 195 209 219 93 75 88 79 * There was a significant effect of parasitism on the mean elevations of individual snails (Kruskal-Wallis test, H = 139.456, d.o.f. = 8, P < 0.001 ). Higher average ranks denote lower elevations. Abbreviations as in Table II. this, I used mean elevation at sighting and mean net movement per tide between sightings as datum points for each snail. This procedure emphasizes infrequently sighted snails (i.e., not G. adunca-mfected). Neither ten- dency is desirable, but using the mean value for each snail leads to more conservative comparisons because statistical tests are not swamped with the behavior of fre- quently observed G. adunca-mfecied snails. Categories of infection with fewer than five snails were eliminated from Tables III and IV. Table III shows that the elevation of Ilyanassa obsoleta at sighting was influ- enced by parasitism. Individuals infected with Gynaeco- tyla adimca, either singly or in multiple, were sighted at higher elevations on the sandbar (lower average ranks in the table) than uninfected or otherwise infected individu- als. Table IV shows that net movement per tide of indi- vidual snails was also influenced by parasitism. It should be recognized that these measurements record minimum movement. Snails could not have moved less than the distances measured, but undoubtedly moved more. Av- erage ranks and medians (Table IV ) show that there were two groups of snails with respect to net movement per tide; those infected with G. adunca and those not. A sta- tistical comparison of just these two groups confirms that G. adunca-mfected snails moved around more than other snails (Mann-Whitney U Test. U = 5657.5, P < 0.001). A long interval between pairs of sightings could affect the magnitude of calculated net movement per tide. With snails remaining in the same general area, as many did in this study, this would probably lead to an underes- timation of net movement. If time between sightings was routinely greater for snails lacking Gynaecolyla adunca (which were seen less often) compared to those infected, this could introduce a bias and compromise the result (Table IV) that snails with G. adimca moved around more than other snails. The range of elapsed tides be- tween sighting pairs was the same for both groups (1-13). The mean number of tides between pairs of sightings for all recollected G. adunca-inkcted snails (Table II) was 2.5, with 91% of sighting pairs (n = 625) coming four or fewer tides apart; the corresponding mean for recollected Table IV Relationship between trematode parasitism and mean net movement between pairs oj low tides Jor individually marked Ilyanassa obsoleta on a Cape Henlopen sandbar* Parasitism Parameter NI Hq Ls Zr Ga LG ZG AG # marked snails 22 6 5 17 104 30 28 6 Total # net moves obs. 48 9 13 35 351 120 84 27 Median of mean net moves (m) 1.6 1.5 1.7 1.8 2.5 2.6 2.9 2.5 Range (m) 0-15 0-4 1-3 0-7 0-11 0-8 1-8 1-3 Avg. rank 75 83 67 90 114 121 130 113 * There was a significant effect of parasitism on net movement per tide of individual snails (Kruskal-Wallis test, H = 1 6. 987, d.o.f. = 7, P< 0.02). Higher average ranks denote more movement. Abbreviations as in Table II. 10 L. A. CURTIS 100 T 80 - LU 60 o a: LU Q- 40 20 - NON-Ga SNAILS (n=77) ,Ga SNAILS (n-183) _n n 0123456 NO. TIMES OBS. ON PEAK Figure 2. Frequencies of visitation to the sandbar peak ( upper verti- cal 4 cm in Fig. 1 ) by //iwiu.v.va nhsoleta infected with Gynaecotyla mill/tea (Ga snails) and those not infected ( non-Ga snails). snails without G. adunca was 3.4, with 75% of sighting pairs (n = 131) coming within four tides. Therefore, the usual number of tides between sightings was similar ( 1- 4) for both groups of snails. Even if longer periods be- tween sightings tend to reduce calculated net movements per tide, relatively few measurements would have been involved. Moreover, both groups of data would have been affected similarly. A biased underestimation, on this account, is unlikely. Frequency of visitation to the sandbar peak by the 260 recovered snails (Table II) is shown in Figure 2. Snails not infected with Gynaecotyla adunca almost never showed up in the upper 4 cm area of the sandbar. Only a single snail not infected with G. adunca (recorded as uninfected) made multiple visits to the peak. Among G. adunca-vafected snails, however, many individuals vis- ited the peak several times (up to six) during the 16-tide experiment. Visitations could have been more frequent and regular than indicated (Fig. 2) if buried marked snails escaped notice on some tides. In any case, it is clear that many host snails made repeated excursions onto the same sandbar peak. The behavior of Gynaecotyla adunca-mfecied snails has a diurnal component because they are most frequent on beaches and sandbars during night low tides (Curtis. 1987; Table I). The diurnal pattern of visitation to the sandbar peak by host individuals, which results in this observation, is revealed by the following data. The peak is denned here as the area within the 4-cm contour (Fig. 1 ). Among G. adunca-inkcled snails, 1 1 2 were observed on the peak two or more times. Most sightings for these (90%, n = 377) were on night low tides. None of these snails was sighted exclusively on daytime low tides; 72% were sighted exclusively on nighttime low tides and 28% showed up on both day and night low tides. Among the 28% seen on both dark and light tides, 71% of sightings were on dark tides. Therefore, host-parasites were typi- cally emerged during night low tides with daytime emer- gence being relatively infrequent. Among recovered Gynaecotyla adunca-infected snails, 77 were sighted on both light and dark low tides. Were they sighted at similar elevations on both types of tides? In a Wilcoxon Signed-Ranks Test for two groups (paired observations), these snails had a significantly higher ele- vation when sighted on the sandbar at night (mean = 7.0 cm down from peak, range = 0-34) than when sighted during the day (mean = 9.5 cm. range = 0-50) (Wil- coxon T = 869.5, P < 0.002). Therefore, not only did infected snails tend to make night visits to the sandbar, but on those night visits they positioned themselves higher than on day visits. Discussion My results show that Ilyanassa obsolcta infected with Gynaecotyla adunca exhibits a complex behavior unlike that of snails lacking this parasite. These snails make re- peated migrations (Fig. 2) to the higher reaches (Table III; Curtis, 1987) of sandbars and beaches, and these ex- cursions entail a generally greater amount of movement per tide than that demonstrated by other snails (Table IV). These migrations leave hosts emerged primarily (but not exclusively) during low tides that occur at night (Ta- ble I). When a host snail is sighted on both night and day low tides, it is usually found at higher elevations at night. All this suggests that there is adaptive value to the para- site in repeatedly inducing its host to be located high on the shore during nighttime low tides. Ilyanassa obsoleta is the main first intermediate host for Gynaecotyla ad- unca. Definitive hosts include any of a variety of shore birds and certain fish (Hunter, 1952) and mammal spe- cies (Harkema and Miller, 1962). I proposed previously (Curtis, 1 987) that the adaptive value lies in an enhanced probability of cercarial transmission to semi-terrestrial, crustacean second intermediate hosts [the amphipod beach-hoppers, Talorchestia longicornis (Rankin, 1940) and T. megaloptha/mia( Hunter and Vernberg, 1957) or the fiddler crab Ucapugilator( Hunter, 1952)]. The pres- ent results support, and allow refinement of this general hypothesis. Fiddler crabs do not inhabit Cape Henlopen, but beach-hoppers do. It should be noted, however, that these second hosts are beach (not sandbar) dwellers. Be- cause the basic behavior of infected snails is the same on sandbars and beaches (Curtis, 1987), the two habitats are apparently indistinguishable by infected individuals. It is worth noting that, in adaptive terms, migrating up and down Cape Henlopen sandbars appears to be a waste of PARASITISM AND SNAIL MOVEMENTS 111 parasite time and energy because the next host is not found there. Presumably, however, over the geographical range of the parasite, a next host is present often enough that parasite fitness is enhanced by inducing host vertical migrations regardless of whether a second host is present or absent on a particular shore. What cues could be used to control the migration? Based on field observations (Curtis, 1987), I concluded that the day-night difference in numbers of host-parasites on the sandbar is a matter of differential immigration during submergence, not differential emigration during emergence. Thus, when a sandbar or beach is examined at low tide, snails found there had moved into position hours before during the previous high tide. This means that the host-parasite tends not to move up the shore dur- ing a high tide to be followed by a daytime low tide, but tends to do so on a high tide that will be followed by a nighttime low tide (Curtis, 1987; present results). There- fore, a remarkable feature of the host-parasite is its ability to track and respond to high tides associated with appro- priate future low tides. From an adaptive standpoint, why avoid stranding the host on daytime low tides? Curtis (1987) noted that the diurnal difference in migratory behavior could be adap- tive because ( 1 ) it matches diurnal behavior patterns of the next host or (2) it lessens the risk of desiccation to the host or the parasite. Beach-hoppers (Talitridae) tend to be active at night and burrow during the day (Kaestner, 1970: pers. obs.), making ( 1 ) above very probable. In this study, individuals infected with Gynaecotyla achinca were observed on daytime tides and later again on night- time tides, which shows that daytime exposure is not le- thal to the infection. Anyway, parasite stages within the host are probably not the ones at risk. More likely, cer- cariae cannot survive the rigors of exposure outside the host during daytime low tides. Tentatively then, host ex- cursions onto a daytime beach would reduce the para- site's fitness for two reasons: the next host would not be around to infect; and released cercariae would not sur- vive until hosts become active on a subsequent nighttime tide. In studies where the effect of trematode parasitism on intertidal snail movement has been considered (Sinder- mann, 1960; Lambert and Farley, 1968; Stambaugh and McDermott, 1969; Williams and Ellis, 1975), the general conclusion has been that parasitism inhibits movement of the host. Perhaps parasitism does decrease gastropod movement in some circumstances, but in this study (Ta- ble IV) it only increased it. Snails infected with Gynaeco- tyla adunca, singly or in combination with other species, could be distinguished from the rest because, consistent with their migratory behavior, they exhibited greater mean net movement per tide. However, uninfected snails could not be distinguished from infected ones (ex- cept G. adunca-inkcled) based on movement. This re- sult suggests that, during summer and in terms of loco- motion in the field, Ilyanassa obsoleta. is not significantly impaired by trematode parasites. Gynaecotyla adunca is found in many multiple infec- tions(Curtis, 1985, 1987, Table II)and has an overriding influence on the host regardless of the presence or ab- sence of other trematodes (Fig. 2, Tables III, IV). This suggests that interactions with other trematode species are likely. Most accounts of ecological and evolutionary relationships among co-occurring helminths emphasize the interactions of adult parasites in vertebrates (e.g., Price, 1980; Holmes, 1983, 1986; Holmes and Price, 1986). Holmes and Price (1986) and Holmes (1986) de- lineate three hierarchical levels of parasite assemblage or- ganization: infra-, component, and compound commu- nities. Data from this study (Table II) are, in themselves, probably too few for meaningful analysis, but in kind they stand to reveal organization at infra- and compo- nent levels. The development of more host-parasite systems ame- nable to study is essential to progress in the field of para- site community (guild) ecology (Price, 1986). Larval trematodes in marine gastropods have not been much studied in this context because multiple infections are, or are considered to be, too infrequent (e.g., see Vernberg c/ a/.. 1969). Gastropod-trematode systems have been analyzed for parasite species interactions (see Rohde, 1981 and references therein) and co-occurrence interac- tions have been noted, but such systems have probably been underutilized. Field studies (Cort et a/., 1937; Rohde, 1981; Curtis, 1985, 1987) show that multiple in- fections can be commonplace. In this study, 570 snails were examined (Table II): 75.4% were infected (19 different combinations); 26.7% were multiply infected. Ilyanassa obsoleta individuals and populations should be added to the list of systems in which interactions among parasites can be studied. Acknowledgments I thank L. E. Hurd for stimulating comments on the manuscript, T. K. Wood for giving me the insect tags, andH. Hudson for dependable assistance in making field observations under difficult conditions. I also thank anonymous referees for their time and valuable critical contribution. 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JONASDOTTIR Marine Sciences Research Center, State University of New York at Stony Brook, Stonv Brook, New York 11 794-5000 Abstract. Newly hatched nauplii of Scottolana cana- densis ( Willey) collected from two locales in Maine were larger than Maryland nauplii when females were reared under identical conditions (20C and high food concen- tration, 2.5 X 10 5 algal cells ml" 1 ). Under high food con- centration, Maryland nauplii had faster growth rates (logm M m h ') than Maine nauplii, but survivorship was similar. Growth rates were lower under low food concen- tration (0.5 X 10 5 cells ml" 1 ), and were the same for all locales, whereas survivorship of the Maine nauplii through NV was higher than the Maryland nauplii. We hypothesize that size-related differences in naupliar feed- ing efficiency may explain the variation in survival under low food stress. Introduction In many marine organisms with mechanisms for dis- persal and hence the potential for gene flow, genetic differentiation of populations has occurred (e.g., as found in copepods; Bucklin and Marcus, 1985; Burton, 1986; reviewed in Hedgecock, 1986). The potential for genetic differentiation of estuarine populations may be greater than that for coastal populations because of phys- ical barriers to dispersal (e.g., salinity and temperature; KoehruYtf/., 1980; McAlice, 1981) and because selective forces arising from human activities may be more in- tense (e.g., PCB pollution; Cosper et ai, 1984, 1988; see review by Levinton, 1980). Scottolana canadensis (Willey: but see For, 1984) is a widespread, brackish-water harpacticoid copepod (Wil- ley, 1 923; Haertel ?//.. 1969;Coull, 1972). When com- mon-rearing techniques were used in the laboratory, fe- males from Maine (43N) produced larger eggs that took Received 30 August 1989; accepted 23 April 1990. longer to develop at all test temperatures than those from Maryland (38N) and Florida (27N; Lonsdale and Lev- inton, 1 985a). These results were contrary to the expecta- tion that northern-derived populations would demon- strate compensation for low temperature. Furthermore, low food stress (2.5 X 10 4 algal cells ml') produced lo- cale differences in newborn survival, with Maine nauplii surviving in the highest proportion. Although variation in egg size does not necessarily reflect differences in egg organic content (McEdward and Carson, 1987), the cor- respondance between large egg size and increased sur- vival of newborns suggested that the Maine females pro- duced eggs with more yolk than did females from other locales, resulting in differences in maternal reserves for newly hatched nauplii (Lonsdale and Levinton, 1985a). The Saco River, from which the Maine S. canadensis individuals were collected, is characterized by extremely high rates of freshwater flow (B. McAlice, pers. comm.), and Saco Bay receives "sediment input grossly out of proportion to (its) size" ( Kelly et at. , 1986; cited in Jacob- son et ai, 1987). The planktonic nauplii of S. canadensis would probably reach their physiological limits once car- ried into the Gulf of Maine due to rapidly declining tem- peratures (McAlice, 1981) and possibly to increased sa- linities. Laboratory studies have shown that the survivor- ship of S. canadensis is adversely affected by higher salinities (responses to 10, 15, and 20%o were studied; Lonsdale, 1981), but the differences were largely found in the epibenthic copepodites rather than the planktonic nauplii. Thus, the efficacy of salinity as a barrier to dis- persal in the species is moot. In this paper, we present the results of a test of the "nauplius development time restriction" hypothesis; i.e., that "yolkier eggs may enhance the rate of nauplius de- velopment to Copepodite I, at which stage they migrate 113 114 D. J. LONSDALE AND S. H. JONASDOTTIR Table I Location and physical characteristics nl cullcciinn siic\ /nr Scottolana canadensis Collection site Date Temperature Salinity (V) (%) Sheepscott River (SHP). Wiscasset, Maine May 1988 17 12 Saco River (SR). Biddetord, Maine May 1988 13 10 Patuxent River(MD), Lushy, Maryland May 1988 18 10 to the bottom, thereby increasing the probability of S. canadensis remaining within the (Saco River) estuary" (p. 428; Lonsdale and Levinton, 1985a). Thus, the egg and naupliar traits of the Maine copepods may not have reflected a latitudinal trend, but rather a localized evolu- tion to a unique hydrodynamic condition. To test the hypothesis, we compared egg development time, egg size, and naupliar growth and survival rates of S. cana- densis collected from the Saco River (SR ) and Sheepscott River (SHP) estuaries in Maine, and the Patuxent River estuary in Maryland (MD). Materials and Methods Field collections Planktonic nauplii of Scottolana canadensis were ob- tained with a 63 /um-mesh net from three sites on the east coast of North America; pertinent collection informa- tion is listed in Table I. Separate collections consisted of 250 or more nauplii. Specimens of Scottolana collected from Maine and Maryland are interfertile (Lonsdale et ai, 1988). Culture methods In the laboratory, wild-caught copepods were cultured at 20C in 1000-ml Erlenmeyer flasks containing 15%o seawater. Seawater was prepared with water from Stony Brook Harbor, New York (~27 %), adjusted to 15%o with distilled water, filtered through 20-yum mesh, and autoclaved. Algae also were cultured at 20C and 15%o with a 14: 10 hour light-dark cycle in f/2 enrichment me- dium (Guillard, 1975), and maintained in an approxi- mately log phase of growth by harvesting and adding me- dium three times weekly. Generally, algal cultures used both in copepod culturing and experiments ranged in age from 5 to 14 days. Cell densities were assessed with a he- mocytometer. A mixture of two algal species Iso- chrysis galbana (ISO; ~4-6 /um diameter and ~ 1 4. 1 pg C ceir 1 according to the Strathmann equations; Strath- mann, 1967) and Thalassiosira pseudonana (3H; ~4 ^m and ~9.0 pg C ceir') was added to each copepod culture three times weekly to produce a minimum den- sity of 2.5 x 10 5 cells ml '. Copepod batch cultures from each locale (SHP, SR, MD) were maintained for at least two months prior to experimentation. Experimental procedures Egg development times. Seventy-five nauplii (all stages) from each locale were removed from batch cul- ture and reared in 1000-ml beakers using the above methods. These and all other experiments were con- ducted at 20C. At this temperature, copepods from Maine and Maryland exhibited little difference, either in mean growth rate from nauplius I to adult, or in adult female energy budgets (Lonsdale and Levinton, 1985b, 1986, 1989). Several additional cultures of SHP nauplii were set up because the first had produced many non- reproducing females, possibly due to an infectious agent (see Lonsdale and Levinton, 1986). Following maturation, up to 30 gravid females were selected and individually placed in 50-ml covered Stendor dishes. Each dish contained about 20 ml of ster- ilized seawater to which algae (a 1 : 1 mixture by cell num- ber of/, galbana and T. pseudonana) was added to bring the food concentration to 2.5 X 10 5 cells ml" 1 . Females were fed three times weekly, and the seawater was changed once a week. To further minimize culture effects on egg traits, development times were determined only after the female produced a second or third clutch. The third clutch was studied in the case of females al- ready carrying egg sacs. Usually, these females were pro- ducing additional eggs visible in the oviducts. To estimate egg development times, observations were made at 4-h intervals. Total time (h) was calculated from the extrusion of eggs in sacs, to naupliar hatching. Egg volume. To determine whether females collected from the Saco River estuary produced larger eggs than those from the Sheepscott River and Patuxent River es- tuaries, females with egg clutches were preserved accord- ing to the procedures outlined by Gallager and Mann ( 1 98 1 ). To determine whether changes in egg volume are associated with embryogenesis, some females were pre- served within 4 h of clutch formation (t = h) and others after 24, 48, 72, or 96 h (for each locale and time condi- tion, n = 3-4 females except n = 2 for SHP at 96 h due to the low number of fecund females). For each clutch, dimension measurements were made on six eggs. Egg volume was calculated (after Allan, 1984) according to the formula: volume (^ni 3 ) = % TT r,^ 2 , where r, and r : are the radii of the long and short dimensions, respec- tively. Egg dimensions and lengths of females (^m) were measured with an Optical Pattern Recognition System COPEPOD GROWTH AND SURVIVAL 115 Table 11 Mean (95% confidence interval) ( 7 length (urn) and total development time (h) to CI for Scotlolana canadensis collected from three locale* (SR. SUP. ami MD) and reared at 20\ ' and two food concentrations (2.5 and 0.5 x H) ! cell\ ml ' ) Food Locale Length Development time 2.5 SR 341 (330-352) 112(106-1 18) SHP 329(318-341) 112(105-120) MD 313(302-324) 103(96-11 1) 0.5 SR 308(301-316) 164(135-201) SHP 297(268-329) 163(136-195) MD 293(278-309) 141 (1 10-180) (Biosonics, Inc.) at 400 X enlargement under a Zeiss compound microscope. Female dry mass was estimated by the equation: Y = 8.415 X 1957 , where Y = M g dry mass, and X = length in mm (Lonsdale and Levinton, 1985b). Naupliar growth and survival. Newborn nauplii, ob- tained from the egg development time studies, were placed individually in wells of a multi-depression dish that was contained within an airtight opaque plastic box. Distilled water in the bottom of the box reduced evapora- tion from the wells. Four sibs from five (SHP) or six fami- lies (SR. MD) were followed at each food concentration (0.5 and 2. 5 X 1 5 cells mr 1 ). The carbon concentration of the algal suspensions ( ~ 577 and 2887 /ug C 1 ' , respec- tively) were within the range found in many estuaries (e.g., 240-39 1 Mg C 1' ' for East Lagoon, Texas (Ambler, 1986) and 500-3388 /ug C T 1 for Narragansett Bay (Dur- bin et at.. 1983). Six additional sibs were preserved in 5% buffered formalin for measurements of body dimensions (length and width) that were determined at lOOx en- largement with a Wild inverted microscope. (Nauplii from one SHP family were inadvertently not sampled for measurement.) The algal suspensions in the wells were completely replaced daily at 1200 h, and a 50% replace- ment occurred at 2400 h. The algal suspensions were pre- pared fresh each day from sterilized, filtered seawater ( 1 5%o) and algae. The copepods were observed about ev- ery 4 h, and the time to each stage (Nil to CI) was noted. Molt lengths were measured during the 100% medium replacement. Equality of variances in the data sets was tested using the F max -test and, if necessary, the data were log R) trans- formed prior to analysis of variance (Sokal and Rohlf, 1981). Most statistical tests were conducted using the packages of Sokal and Rohlf ( 1 98 1 ) or SAS. Results Egg development time A significant difference in egg development time was found among locales (One-way ANOVA; df = 2,27, F = 1 7.03, P < 0.000 1 ). Mean egg development time (95% confidence interval) of MD females was significantly less than that of SR and SHP females [74. 1 (72.3-75.9) h ver- sus 99.2 (93.9-104.5) and 98.0 (81.4-1 14.6) h, respec- tively; 5% critical value, Tukey-Kramer method for un- planned comparisons among means]. There was no sig- nificant difference in development time among the Maine eggs. Mean egg development times were very sim- ilar to those previously obtained at 20C (97.3 and 70.4 h for SR and MD locales, respectively; see Table II, Lons- dale and Levinton, 1985a). SR = Saco Riven SHP = Sheepscott River; MD = Patuxent River. ^ S ' =e Mean egg volume of a clutch (^m 3 ) was not signifi- cantly affected by total incubation time (0 to 72 h for MD and to 96 h for SR and SHP), as the regression equations were not significant for several models tested (e.g., linear, P > 0.5, 0.1, and 0.1, respectively). Thus, the incubation time series data were pooled by collection locale. Both locale and family within locale influenced egg volume (Nested ANOVA using log,,, transformed data sets: df = 2,217, F = 44.94, P < 0.001 and df = 41,217, F= 5.99, P< 0.001, respectively; Fig. 1 ). MD females produced significantly smaller eggs than either SR or SHP females [6.18 (5.75-6.64) X 10 4 M m 3 versus 8.37 (7.91-8.86)and 8.09 (7.59-8.62) XlOVm 3 , respec- tively] and there was no difference between the latter two locales (5%. critical value, Tukey-Kramer method). The mean dry mass of females was 8.2 (7.2-9.2), 9.1 (8.3-9.9), and 7.4 (6.7-8.1) ng for MD, SR, and SHP, respectively. Differences in dry mass did not account for the locale effects on egg volume ( ANCOVA for dry mass DSR OSHP AMD m O 1.4 1-0 volume o 0.2 n n A n T n A 10 1 1 Dry mass (//g) Figure 1. Mean egg volume (+95% confidence interval) and dry mass of females ofScottolana canadensis collected from three locales [Saco River (SR), Sheepscott River (SHP), and Patuxent River (MD)] and reared at 20C and 2.5 X 1 5 cells ml ' . 116 D. J. LONSDALE AND S. H. JONASDOTTIR D DSR (2.5) O OSHP(2.5) A- -AMD (2.5) BSR(O.S) SHP(O.S) A- -AMD (0.5) 1.0 M A > \X S -E 0- - -A- _ 0.9 A\ ^S n g "6- n \\i a ~i-. ^ 0.8 \\ > 0.7 , \ \ v N. f : j C/l 0.6 \ Bj\. A- - -A. _ 0.5 -A. ^ n A "A- - - Nl Nl! Cl Figure 2. when reared ml '. Mill NIV NV NVI STAGE Survival of Scottolana canaJcnsis at each naupliar stage at 20C and at concentrations of 2.5 and 0.5 > lO 5 cells advantage of Maine nauplii was lost during the NVI stage (Fig. 2). Molt lengths of MD nauplii were in general shorter than those of Maine S 1 . canadensis (Fig. 3). The molt length of CI copepodites was affected by collection locale and by food concentration (Two-way ANOVA using logm transformed data sets; df = 2,74, F = 6.73, P < 0.01 and df = 1 ,74, F = 25.50, P < 0.0001 , respectively; Table II. The influence of family within locale was not investi- gated because some families had no CI survivors at the low food concentration). The low food concentration re- sulted in smaller copepodites, and those from MD were smaller at the high food concentration than either SR or SHP copepodites (5% critical value, Tukey-Kramer method). At the low food concentration, the molt lengths of the copepodites collected from different locales were not significantly different. The low food concentration also resulted in a longer total development time (h) to CI (Two-way ANOVA us- adjusted volumes; df = 2,257, F = 21.24, P < 0.001, Fig. 1). Naupliar newborn size, survivorship, and growth rale The length (^m) of newborn nauplii was influenced by both locale and family within locale effects (Nested ANOVA using Iog 10 transformed data sets; df = 2,78, F = 1 1 .45, P < 0.00 1 and df = 1 5,78, F = 2.46, P < 0.0 1 , respectively). MD newborns were significantly smaller than those from SHP and SR, and there was no differ- ence among the latter two groups [9 1 (90-92) /urn versus 99 (98-101) and 100 (99-101) ^m length, respectively; 5% critical value, Tukey-Kramer method]. Similarly, lo- cale and family within locale were significant factors affecting maximum head width (Nested ANOVA; df = 2,78, F = 42.96, P < 0.00 1 and df = 1 5,78, F = 4. 1 1 , P < 0.001, respectively). All locales were significantly different from one another [54 (53-55), 57 (56-59), and 61 (60-63) Mm for MD, SHP, and SR; 5% critical value, Tukey-Kramer method]. Naupliar survival was poorer at the low food concen- tration than at the high food concentration for all locales (Fig. 2). There were no significant differences among lo- cales in the proportion of nauplii surviving through the NV stage at the high food concentration (paired t-test using arcsine transformed proportions of survivors at each stage; df = 4, t s = 0.9 1 3, P > 0.4 in a comparison of SHP versus SR, and t s = 0. 1 1 2, P > 0.9 in a comparison of pooled Maine data versus MD). At the low food con- centration, no significant difference in survivorship was found among SR and SHP nauplii (t s = 0.755, P > 0.5), but the survival of MD nauplii was significantly less than that of Maine nauplii (t, = 3.733, P < 0.05). The survival 280 T 240- QSR OSHP n 2.5 . 10 5 cells mf 1 9 A 200- T a 9~4 160- O e A on A -^ 120- f 80- (J z UJ _i o 280 - O m 240- 200- D A O D i A n1 n2 n3 n4 n5 n6 5-1 T T -0.5 x 10 cells ml J i I ' L }I i-T 160- 120- T 1 I ??* 80- : B nl n2 n3 n4 n5 n6 STAGE Figure 3. Mean molt lengths (95% confidence interval) of Scotto- lana canadensis nauplii collected from three locales [Saco River (SR), Sheepscott River (SHP), and Patuxent River (MD)] and reared at 20C and either 2.5 or 0.5 x I0 5 cells ml" 1 . Missing confidence intervals are smaller than the height of the symbol. COPEPOD GROWTH AND SURVIVAL 117 ing log,,, transformed data sets; df = 1,79, F = 62.00, P < 0.0001; Table II) and although MD nauplii had a shorter mean development time at both food concentra- tions, locale variation was marginally not significant (df = 2,79, F = 3.03, P = 0.054). Regressions of molt length (log transformed) versus to- tal development time were significant for all of the growth rate studies (P < 0.001 for all regressions; Fig. 4). At the high food concentration, the regression coefficient for MD was significantly higher than either SR or SHP. and the latter two were not different from one another (Tukey-Kramer method for unplanned comparisons among a set of regression coefficients using 5% critical values). However, there were no significant locale differ- ences among regression coefficients at the low food con- centration, and all those coefficients were significantly lower than the ones obtained under the high food con- centration. Discussion Our results did not support the "nauplius develop- ment time restriction" hypothesis (Lonsdale and Levin- ton. 1985a). Total development time to CI was not less for SR nauplii than for those from either MD or SHP at either food concentration. Scottolana from the Saco River estuary were also similar to copepods from the Sheepscott River estuary in other life-history traits, such as egg development time, egg volume, and newborn size. This research, therefore, provides more evidence for ge- netically based, latitudinal differences in life-history traits ofS. canadensis (between Maine and Maryland). The adaptive significance of latitudinally related varia- tion in newborn size in S. canadensis remains unclear and may. in fact, be due to genetic drift of isolated popu- lations (see Hines, 1986, and Slatkin. 1987, for general reviews). On the other hand, the attainment of a larger newborn size appears to encumber a fitness cost in terms of prolongation of embryogenesis. In turn, this cost may be offset by the survival advantages of a larger body size. For example, the predation rate of the adult copepod Actinia tonsa on copepod nauplii was inversely related to naupliar body size (when NI-III and NIV-VI were compared), although prey swimming ability and behav- ior also may have been important (Lonsdale ct ai, 1979; Tackx and Polk, 1982). In MD. however, invertebrate predation is an important regulator of S. canadensis pop- ulation growth (Lonsdale. 1981), and thus, the labora- tory differences in naupliar size per se are not likely a reflection of habitat variation in this selective pressure. We suggest an additional fitness advantage of larger body size. A preliminary examination of eggs preserved within 4 h of mean hatching time (72 h for MD copepods and 96 h for Maine) revealed that the total lipid staining area (MITT using Oil Red O;Gallagerand Mann, 1981) of the eggs was not different among locales [62 ( 1 1 - 1 1 3), 89 (19-159), and 112 (-32-256) ^m : for MD, SR, and SHP, respectively)]. Although these area measurements are not an optimal estimate of lipid content (Gallager and Mann, 1986), they do suggest that variation in ma- ternal reserves did not contribute to the survival differ- ences of the nauplii under low food stress. Thus, there may have been size-related differences in naupliar feed- ing efficiency (energy ingested-energy expended; Hall el til.. 1976; Sebens, 1982). Larger body size may result in a greater filtering capacity (per animal), or greater differ- ence between weight-specific energy acquisition and ex- penditure, as compared to smaller forms (Hall ct til., 1976; Gliwicz, 1990). The survival patterns of 5. cana- densis may indicate that under the high food concentra- tion, differences were not found because the energetic de- mands of growth were met for both MD and Maine nauplii. But under the low food concentration, the ener- getic impact of lower feeding efficiency resulted in higher mortality of the MD copepods. The survival advantage of Maine nauplii was lost during the NVI stage, perhaps for several reasons. First, at NVI, the molt lengths of the Maine and MD nauplii were similar (Fig. 3B) and thus, there would be no size-related advantage. Second, the metamorphosis of 5. canadensis nauplii to copepodites was associated with the highest stage-specific growth rate (^m h"') compared to all other stages of nauplii and re- quired substantial morphological change. In previous studies, the mean growth rate (/ug dry mass d~') of SR and MD females was usually not different, particularly at 20C, nor were the components of growth rate (i.e., adult dry mass and total time from NI to the adult molt; Lonsdale and Levinton, 1985b. 1986). Yet growth rate differences during the naupliar stages have been shown in this study. However, in this study, the growth rates of SR and MD female copepodites (CI to adult molt) were similar [log Y = 2.35 + 0.00187 (0.00023) X and log Y = 2.32 + 0.00195 (0.00036) X where Y = ^m and X = h, respectively and at the high food concentration; unpubl.]. This result may help ex- plain the lack of concordance between mean growth rate to adult and naupliar growth rate with regard to the in- fluence of collection locale. The egg volumes determined in this study were ~25- 30% less than those reported by Lonsdale and Levinton (1985a) for both the Maine and MD locales. This dis- crepancy may be due to differences in the preservation process; in the previous study copepods were not narco- tized with MgCl : prior to preservation in formalin. Ini- tial estimates of egg volumes of Maine S. canadensis in- dividuals collected from the field, and which were not narcotized prior to formalin preservation, were more similar to those reported by Lonsdale and Levinton 118 2.6 D. J. LONSDALE AND S. H. JONASDOTTIR SHP 24- CP c en o 22- 20 /low ^ logy = 2 02 + 00424 l2 5xlO' 4 )X --logy = 2 05+0 00260(*22xlO~ 4 )x 2.6 2 4- 2 2H 20 /low log y= 2 01 + 00415 (2.7xlO' 4 )X -- logy =2 03 + 0.00270 (+2.6 x 10" 4 )x 40 80 120 160 200 240 280 Time (hrs) 40 80 120 160 200 240 280 Time (hrs) 2.6 MD high vy ox / 'low log y = I 99+ 0.00469 (+2 9x I0' 4 )x log y = 2 04 + 00260(+ 3 6x lO'^Jx 40 80 120 160 200 240 260 Time (hrs) Figure 4. Growth rate (log,,, ^m h ') of Scottolana canadensis nauplii collected from three locales [Saco River (SR), Sheepscott River (SHP), and Patuxent River (MD)] and reared at 20C and either 2.5 X 10 5 cells ml ' (solid lines and x data points) or 0.5 x 1 5 cells mr 1 (dashed lines and O data points). The 95% confidence interval of the slope is provided in the regression equation. ( 1985a). The average egg volume from field-collected fe- males ranged from 1 . 1 7 to 1 .40 X 10 5 ^m 3 when temper- atures ranged from about 1 3 to 1 8C (unpubl.). An alternate hypothesis of the "nauplius development time restriction hypothesis" to explain differences in newborn size of S. canadensis is that variation in pri- mary productivity between latitudinally separated lo- cales may be significant (Lonsdale and Levinton, 1 985a). In Chesapeake Bay during the spring bloom, the chloro- phyll concentration exceeded 40 mg m ' in the euphotic zone and 29-48 mg m~ 3 over the entire water column [Malone et al., 1988. Data were converted from mg irT 2 by using the average euphotic depth (5 m) or range of channel depths (25-42 m), respectively]. In the Damans- COPEPOD GROWTH AND SURVIVAL 119 cotta River estuary (just northeast of the Sheepscott River estuary), spring bloom chlorophyll concentrations occurred in late February and early to mid-March with amaximum valueof lO^gT 1 (Townsend, 1984). During May and June when S. canadensis have been found in high abundance in Maine (Lonsdale and Levinton. 1985a), the chlorophyll values ranged from 4 to 5 ^g 1" ' (Townsend. 1984). Although chlorophyll concentration per se is not always an adequate indicator of food avail- abiliu (Bellatoniand Peterson. 1987). we suggest that the variation in newborn size found among S. canadensis nauplii may reflect differences in food availability be- tween the Maine and MD habitats. At present, biochemical measurements of genetic vari- ation among and within .S. canadensis populations are lacking. Despite common-rearing, some of the variation found may not be related to genetic differences, but to other irreversible, non-genetic effects U'..?., maternal effects due to the handling history of individual females within a locale). Ecological studies of the energy de- mands of copepod nauplii during development and the coupling of primary production to patterns of copepod reproduction and survival in the field would further our understanding of the evolution of life-history strategies of marine invertebrates. Acknowledgments The authors thank a previous reviewer who pointed out the unique properties of the Saco River and who orig- inally put forth the hypothesis tested in this paper. Two anonymous reviewers also provided important com- ments on this paper. The laboratory assistance of P. Weissman and the Research facilities made available at the Chesapeake Biological Laboratory. University of Maryland, were greatly appreciated. This research was supported by a Faculty Grant-in-Aid. State University of New York at Stony Brook and the National Science Foundation (OCE 83-08761). This is Contribution Number 730 from the Marine Sciences Research Center. State University of New York at Stony Brook. 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Comparison of inshore zooplankton and ich- thyoplankton populations of the Gulf of Maine. Mar. Ecol. Prog. Ser. 15: 79-90. Willey, A. 1923. Notes on the distribution of free-living Copepoda in Canadian waters. Contr. Can Biol (n. 5:)1(16): 303-324. Reference: liil. Hull 179: 121-133. (August, 1990) Consequences of the Calcite Skeletons of Planktonic Echinoderm Larvae for Orientation, Swimming, and Shape J. TIMOTHY PENNINGTON 1 * AND RICHARD R. STRATHMANN 2 l Kewalo Marine Laboratory, University of Hawaii. 41 Atnii Street, Honolulu, Hawaii 96813 and 2 Friday Harbor Laboratories and Department of Zoology, University of \\ 'ashington, 620 University Road. Friday Harbor, Washington 98250 Abstract. How the echinoderm larval skeleton is used for support of larval arms, passive orientation, and swim- ming was examined by experimentally removing the skeletons of plutei and by comparing feeding larvae from four echinoderm classes. All four types of echinoderm larvae oriented with their anterior ends upward in still water, but removing the skeletons of both live and dead four-armed echinoplutei demonstrated that their skele- tons enhanced passive vertical orientation with their an- terior ends upward. In comparisons of dead four-armed echinoplutei with and without skeletons, the skeleton contributed more than half of the excess density and sinking speed. In comparisons of all four types of feeding echinoderm larvae, larvae with a greater volume of skele- ton and a smaller volume of tissues and body cavity were densest. The calculated work necessary to prevent the plutei from sinking was much less than 1% of the total aerobic energy expenditure. Thus calcite skeletons are not essential for passive vertical orientation by echino- derm larvae but enhance it, and the increased density and sinking rates impose little energetic cost in locomo- tion. The evolution of larval skeletons may have been in- fluenced by the benefits of passive orientation and by the low costs of swimming with a skeleton. Whatever the pri- mary function of skeletons, the position and form of skeletal elements is influenced by the functional require- ment for higher mass posteriorly for passive orientation. Features that enhance passive vertical orientation in- Received 31 July 1989; accepted 25 May 1990. * Present address: Hopkins Marine Station, Stanford University, Pa- cific Grove, California 93950. elude posterior ossicles and skeletal rods, posterior thick- ening of skeletal rods, and formation of juvenile parts near the posterior ends of larvae. Introduction Some echinoderm larvae (echinoplutei, ophioplutei) have a skeleton with calcite rods that support projecting arms, but others (auriculariae, bipinnariae) do not. It is peculiar that any small gelatinous planktonic animals should have an internal mineral skeleton for body sup- port, but a supporting function for some internal skeletal elements is obvious. Calcite rods support the soft parts of a pluteus against the contraction of the muscles that dilate its mouth during the rejection of large particles from its oral cavity (Strathmann, 1971). Calcite rods pro- vide the attachment and leverage for muscles that spread the arms of some plutei, presumably in defense (Mor- tensen, 1 92 1 , 1938). Other supporting functions are also likely. Gustafson and Wolpert ( 1961 ) suggested that the arm rods of plutei support an epidermis under tension. Emlet(1982, 1983) measured the stiffness of calcite skel- etal rods of plutei and analyzed the forces that swimming imposes on projecting arms. He concluded that fenes- trated skeletal rods are stiffer than needed to support the arms against forces generated in ciliary swimming and suggested that the stiffness may protect the larvae against predators. Some calcite skeletal elements, however, do not sup- port soft parts. Some, such as the posterior ossicle in the auricularia, are only solitary lumps that have no imagin- able supporting function. Also, the posterior ends of the body rods of four-armed echinoplutei are much thicker 121 122 J. T. PENNINGTON AND R. R. STRATHMANN than the arm rods hut appear to he subject to no greater forces and are resorbed at later stages. Runnstrom (1918) and Pennington and Emlet (1986) suggested that the body skeleton of the echinopluteus is a counterweight that orients the larva with its arms upward. Echinoderm larvae have no known statocyst or organ for sensing grav- ity, yet all feeding larvae tend to swim with their anterior ends upward. Mechanisms enhancing passive orienta- tion may help them maintain a favorable position in the water column. We are concerned here with the hypothesis that pas- sive vertical orientation has been an important func- tional requirement in the evolution of echinoderm larval body plans and especially of larval skeletons. That re- quirements for passive vertical orientation might con- strain larval body plans or entail ecological consequences has been little appreciated or studied. If the skeleton contributes substantially to passive ver- tical orientation, it also contributes to the excess density of the larva. Excess density is the difference between the density of an object and the density of seawater. Excess mass is the excess density of an object times its volume. The larval center of gravity is the center of its excess mass, and its center of buoyancy is the center of its vol- ume. The weight of a body acts at the center of gravity, and the upthrust from weight of fluid displaced acts at the center of buoyancy (Alexander. 1968). If the skeleton orients the larva passively with anterior end upward, then the larva's center of gravity, as controlled by the skeleton and other parts, must be posterior to the center of buoyancy. If the density of the soft parts (tissues and body cavi- ties) of a larva was equal to that of seawater, then the effect of the skeleton on passive vertical orientation might be deduced from anatomical measurements of skeletal components alone. However, some soft parts are denser than seawater; some may be less dense than sea- water; and the volume of soft parts greatly exceeds the volume of skeleton. For example, Pennington and Emlet (1986) found that four- and six-armed echinoplutei of Dendraster excentricus were denser than eight-armed plutei or even newly metamorphosed juveniles and sug- gested that accumulation of lipids in the gut (Burke, 1981) might account for the lower density at advanced stages. In addition, the density of the contents of the pri- mary body cavity may differ from the density of cells, and the primary body cavity can be small or extensive, depending on the stage and type of larva. Thus passive vertical orientation by an appropriate distribution of density along the body axis would constrain the distribu- tion of cells, body cavities, and skeleton. Demonstrating a role of the skeleton in passive orientation therefore re- quires more than anatomical measurements. In this study we examined the calcite skeletons of planktotrophic echinoderm larvae to identify those func- tional consequences of skeletal size and shape that may have influenced the evolution of these larval forms. To examine the role of calcite skeletons in passive vertical orientation, we ( 1 ) experimentally removed skeletons from echinoplutei and (2) compared types of plankto- trophic echinoderm larvae that differ in skeletal develop- ment. These studies also addressed two related topics: the role of the calcite skeletons in the maintenance of body shape, and the contribution of calcite skeletons to excess density and sinking rate. Materials and Methods Rearing The methods of obtaining gametes, fertilizing ova, and maintaining embryos and larvae are those in Strath- mann (1987). Most larvae were reared in 1.5 to 3 liter cultures in mechanically stirred jars. Some cultures of 0- to 4-day-old larvae were kept in smaller containers and not stirred. Rearing temperatures were about 1 0- 1 4C at the Friday Harbor Laboratories on San Juan Island and 23-27C at the Kewalo Marine Laboratory on Oahu, within a few degrees centigrade of the local sea tempera- tures. At the Kewalo Marine Laboratory, larvae were fed Rhodomonas lens (as described in Leahy, 1986), occa- sionally supplemented with Dunaliella tertiolecta. These larvae were the echinoplutei of Echinometra mathaei (Blainville), Tripneustesgratilla(lAtma&is), and Colobo- centrotus atratns (Linnaeus). Most larvae at the Friday Harbor Laboratories were fed natural phytoplankton supplemented with Dunali- ella tertiolecta and occasionally also Thalassiosira weiss- Jlogii or Isochrysis galbana. These larvae were the auric- ulariae of Parastichopus califomicus (Stimpson). the bi- pinnariae of Asterina ininiata (Brandt) and Evasterias troseheln (Stimpson), the ophioplutei of Ophiura sarsi Liitken and Ophiopholis aculeata (Linnaeus), and the echinoplutei of Strongylocentrotus droebachiensis (O. F. Miiller), S. pallidus (G. O. Sars), 5. purpuratus (Stimp- son), and Dendraster excentricus (Eschscholtz). Killing Dead larvae were required for several experiments. At Kewalo Marine Laboratory larvae were killed with 2% formalin buffered with calcium carbonate in seawater. At the Friday Harbor Laboratories larvae were killed with sodium cyanide (about 0.005 to 0.001 M) in sea- water. Decalcification Different methods were used to decalcify dead or live echinoplutei. The skeletons of dead, formalin-fixed lar- LARVAL SKELETONS AND ORIENTATION 123 vae were removed by placing the larvae in a 1:1 mixture of seawater and EDTA-saturated seawater for 2-10 min. Complete decalcincation was verified by viewing larvae under a dissection microscope with crossed polarizing filters: any remaining skeleton produced a bright glow. The skeletons of live echinoplutei were removed by the method of Pennington and Hadfield (1989). Batches of several hundred early four-armed plutei (3-5 days old) were pipetted into synthetic seawater (MBL recipe; Cav- anaugh. 1956) buffered at pH 5.5 or 6.0 with a final concentration of 0.03 M MES (2-[N-Morpholino]eth- anesulfonic acid). Although the rate of decalcification varied with the species, number, and stage of larvae, the plutei usually decalcified in 2 to 5 h; they were then re- moved to natural seawater. Because this treatment tem- porarily suppressed motility, the larvae were allowed to recover overnight before behavioral observations were made. Decalcified echinoplutei were maintained in groups of 1-50 larvae in 20-ml finger-bowls and fed R. lens. Orientation, density, and sinking Orientations and relative densities were compared for dead larvae sinking in density gradients. At Friday Har- bor at least three and at Kewalo at least two larvae of each type were pipetted into the top of cuvettes about 3 cm tall by 1 cm wide and deep or in some cases about 10 cm high by 1.3 cm wide and deep. The cuvettes con- tained seawater at the top and a denser fluid at the bot- tom; partial mixing of the two fluids produced a density gradient. Except where noted, the denser fluid was Per- coll in a solution of NaCl isosmotic with seawater. Per- coll is a colloidal suspension of silica particles coated with polyvinylpyrrolidine and was obtained from Phar- macia. Some observations were repeated with sucrose is- osmotic with seawater as the denser solution. Resting ori- entation at neutral buoyancy was checked by reorienting the larva with a glass needle and then waiting several minutes to see if it would return to its original position. (The needle was swept past the larva so that the return stroke did not rotate the larva back to its original posi- tion.) The densities of echinoplutei with and without skele- tons were estimated in three experiments. Dead four- armed plutei of T. gratilla (4-5 days old) were suspended in seawater layered over a series of Percoll dilutions of decreasing specific gravity but isosmotic with seawater. If larvae floated above the Percoll for 30 min, their den- sity was recorded as less than that of the Percoll solution: if they sank to the bottom, their density was greater than that of the Percoll solution. The densities of the Percoll dilutions were obtained by weighing known volumes. Is- osmotic Percoll solutions were used instead of the su- crose solutions of Pennington and Emlet (1986), who used sucrose solutions without adjusting osmolarity. When the Percoll method was compared with sucrose solutions of unadjusted osmolarity for both normal and decalcified plutei of T. gratilla, higher estimates of larval density were produced with the sucrose. To compare the orientation and sinking rates of echi- noplutei with and without skeletons, we used four-armed plutei of T. gratilla (3 and 5 days old). Observations of dead larvae with and without skeletons were made in cu- vettes, as described above. In seawater without a density gradient, currents were eliminated by minimizing air currents, covering the cuvettes, using indirect backlight- ing, and equilibrating air and water temperatures. Mo- tionless suspended particles indicated an absence of cur- rents. These observations may underestimate sinking rates because of wall effects in the small cuvettes. Larger cuvettes in water baths were impractical because plutei without skeletons were difficult to see. We measured the sinking rates of larvae that were away from the walls. The observations were adequate to demonstrate an effect of the skeleton and to provide a check on other measure- ments of the contribution of the skeleton to the density of the larval body. Stability of vertical orientation was compared for echi- noplutei with and without skeletons. Convection cur- rents in the cuvettes were enhanced by shining a lamp on the cuvette wall so that the current rose on one side and descended on the other. Dead four-armed echi- noplutei of T. gratilla (3 days old), either with or without skeletons, were pipetted into the top of the descending current. The vertical orientations of the first larvae car- ried upward past mid-depth in the ascending current were scored as ( 1 ) arms-up, (2) arms-down, or (3) hori- zontal. Orientation during swimming was compared for live echinoplutei with and without skeletons. Decalcified and normal plutei of T. gratilla and C. atratus were video- taped. Upward swimming indicated orientation to grav- ity. Swimming in other directions or tumbling indicated deficient orientation to gravity. Results Effects of experimental removal oj skeletons Shape. Decalcified dead echinoplutei retained the plu- teus form despite the complete loss of skeleton (Fig. 1 ). Decalcified live echinoplutei of E. mathaei, T. gratilla, and C. atratus were maintained for 74, 48, and 19 days, respectively. The larval shape changed during this time. Larvae without skeletons initially retained some features of the pluteus shape (Fig. 2A, B). Larval arms were shorter than arms of control plutei with skeletons (Fig. 2C) but were still present (Fig. 2B). Arms without a sup- 124 J. T. PENNINGTON AND R. R. STRATHMANN Figure 1. Echinoplutei of Tripneustes gratilla, 6 days old: (A) normal control larva with 1(10 ^m scale bar and (B) larva killed with formalin and decalcified with EDTA, same scale. Late eight-armed echinoplu- teus of Tripncuslcs Krutilla with echinus rudiment: (C) whole larvae with 300 ^m scale bar and ( D) stomach with retractile droplets that are absent in the stomach wall of larvae at earlier stages(3 times the magnifica- tion of C). porting skeleton did not bend noticeably during swim- ming. The esophagus was initially constricted in T. gra- tillu. but later opened. The mouth, stomach, and intes- tine appeared normal in all three species, and the larvae without skeletons captured and ingested phytoplankton cells. Some plutei did not resecrete a skeleton, and thev completely resorbed their arms over the course of several days. These larvae became bowl or saucer shaped but re- tained distinct oral hoods and concave circumoral fields (Fig. 2D). The ciliated band persisted as the rim of the bowl. However, most larvae resecreted abnormal skele- tons (Fig. 2E, F). Their arms developed to various LARVAL SKELETONS AND ORIENTATION 125 B Figure 2. Live four-armed echinoplutei of Colobocentrotus airattix: (A) oral and (B) lateral views of recently decalcified plutei, (C) ventral view of normal control larva of same age as larvae in A and B, (D) oral view of larva that had been decalcified several days previously and did not regrow a skeleton, (E) and (F) oral views of plutei that had been decalcified several days previously and did regrow a skeleton. lengths and in odd directions, and their body skeletons were equally abnormal. Most developed four arms, the correct number, though some had missing or extra arms. In C. atratus and T. gratilla, the postoral arm rods were usually fenestrated as in normal control larvae. No decal- cified larvae developed to six-armed or later stages, but conditions in 20-ml culture dishes were not favorable. Skeletons in echinoplutei appeared to play a small role in immediate mechanical support and a greater role in long-term maintenance and development of larval form. Passive orientation. In still seawater, almost all four- armed echinoplutei of T. gratilla with and without skele- tons oriented passively with their arms upward. Nearly 100% of dead larvae with skeletons sank with arms up- ward, and about 80% of those without skeletons sank with arms upward. However, removal of the skeleton did change the stability in the arms upward orientation. In a cuvette with 100% seawater and side illumination, there were convection currents and velocity gradients; dead echinoplutei with skeletons were oriented arms upward, but dead echinoplutei without skeletons tumbled. Of lar- vae at mid-depth in the ascending current, 100% of those with skeletons were arms-upward (n = 30) and only 28% of those without skeletons were arms-upward with the remainder arms-downward or horizontal (n = 40). We expected that with random orientation about 25% of lar- vae should have been arms upward. The plutei without skeletons did not differ significantly from this expecta- tion (Chi-square, P > 0.9), while the plutei with skele- tons were clearly non-random in their orientation (P < 0.005). Removal of the skeleton greatly decreased pas- sive stability. Orientation of living echinoplutei. The vertical orienta- tion of live four-armed echinoplutei of C. atratus, E. ma- thaei, and T. gratilla was compared with and without skeletons. Most plutei with skeletons swam with their arms upward. Forward swimming produced upwards movement. The arms-upward orientation was less pro- nounced after decalcification. Plutei of C atratus were the most vigorous after decalcification. Almost all of these plutei tumbled in an anterior-posterior direction with little or no net movement. A few decalcified plutei 126 J. T. PENNINGTON AND R. R. STRATHMANN Table 1 Ranking oj density of some Hawaiian eehinoplulei from least dense In most dense with larvae nl nearly equal density lumped at the same rank Rank Species Stage Reorientation 1 Tripneustes gran/la four-armed decalcified * 1 Triplicates granlla eight-armed with skeleton ^ Echtnomelra malhaei eight-armed with skeleton 3 Tripneusles gran/la four-armed with skeleton * 3 Colobocentrotus alratus eight-armed with skeleton * = Those that reonented as they sank toward neutral buoyancy. did swim forward without tumbling, though they swam downwards as well as upwards with no clearly discernible orientation to gravity. When disturbed, larvae with and without skeletons stopped or swam backwards. Results with E. mathaei and T. gratilla were similar, though de- calcified larvae of these species swam less vigorously. Density. When the skeletons of four-armed (4 and 5 day old) echinoplutei of T. gratilla were removed, the density of the larval body decreased. The density of lar- vae with skeletons was estimated to be about 1 .06 g/ml. because all dead larvae with skeletons sank through a Percoll solution of density 1.042 g/ml, and all floated above a solution of density greater than 1.078 g/ml. More than half (ca. 70%) sank in a solution of density 1 .054 g/ml. The density of larvae without skeletons was estimated to be about 1 .04 g/ml because all dead larvae without skeletons sank through a solution of den- sity 1.037 g/ml and all floated in a solution of density 1.042 g/ml. In a density gradient, the terminal depth of four-armed plutei of T. gratilla without skeletons was less than the depth of control plutei with skeletons (Table I). This re- sult again indicates that the skeleton increases the density of the larval body. Sinking speed. Removal of skeletons of four-armed echinoplutei of T. gratilla decreased their sinking rates in seawater. When 3 days old, echinoplutei with skele- tons sank at 0.29 mm/s (S.D. = 0.076 mm/s, n = 10), and plutei without skeletons sank at 0.052 mm/s (S.D. = 0.022 mm/s, n = 10). When 5 days old, echinoplutei with skeletons sank at 0.44 mm/s (S.D. = 0.21 mm/s, n = 10), and plutei without skeletons sank at 0.052 mm/s (S.D. =0.018 mm/s, n = 10). In both comparisons the sinking speeds were significantly less for plutei without skeletons (Mest, P < 0.00 1 ). Comparisons of larval forms and stages Passive orientation. Echinoderm larvae of four classes passively oriented with their anterior ends upward while sinking in seawater at the top of Percoll gradients. The dead larvae that sank with their anterior ends upward were auriculariae of P. californicus, bipinnariae of A. miniata and E. troschelii. ophioplutei of O. aculeata (four-armed) and O. sarsi (six-armed), and echinoplutei of/), excentricus (four- and eight-armed), S.frandscanus (four- and eight-armed), S. pallidus (four-, six-, and eight-armed), S. purpuratus (four-armed), C. atratus (eight-armed), and T. gratilla (four-armed). All were tested in gradients made with seawater as the less dense solution and with a mixture of Percoll and isosmotic NaCl as the denser solution. Many were also tested in gradients made with seawater and an isosmotic sucrose solution, with no difference in results. At the seawater end of the density gradient, sinking dead larvae reori- ented with their anterior ends upward almost immedi- ately after being turned anterior end downward. The two exceptions were eight-armed echinoplutei of T. gratilla and E. mathaei, which did not clearly orient with their arms upward while sinking into the top of a Percoll gra- dient. Dead larvae of all four types also oriented anterior end upward when sinking in 100% seawater with no density gradient, except that the larvae were sometimes turned by the convection currents that developed under uncon- trolled conditions in cuvettes lacking a density gradient. Sinking was not required for passive orientation. Ori- entation was stable for larvae suspended at neutral buoy- ancy. However, some larval forms reversed their orienta- tion as they sank into denser fluid because the distribu- tion of excess mass was different in denser media. These results are addressed below, following the observations on density. Density. Calcite skeletons affected larval density. In a density gradient, dead larvae with more skeleton in pro- portion to body volume came to rest in denser fluid (Ta- bles I, II); larvae with little or no skeleton (auricularia and bipinnaria, Fig. 3 A, B) or with the skeleton removed (pluteus of T. gratilla. Fig. 1 B) were least dense. For com- parisons among a range of larval forms, the larvae in Ta- ble II were ranked according to the proportion of body volume composed of calcite skeleton: 1 for the bipinna- riae, with no skeleton; 2 for the auriculariae, with only a small ossicle; 3 for the plutei of Strongylocentrotits from four- to early eight-armed stages, with thin skeletal rods, an incomplete basket skeleton, and large spaces between skeleton and body wall; and 4 for the remaining plutei, with a relatively large skeleton because of thick rods, fen- estrated rods, a more complete basket skeleton, or juve- nile skeletal elements of the later rudiment. These ranks for skeleton as a proportion of body volume were corre- lated with the whole body densities in Table II (Spear- man rank correlation 0.86, P < 0.01). These rank data comprise a broad range of forms of planktotrophic echi- noderm larvae. LARVAL SKELETONS AND ORIENTATION 127 Figure 3. (A| Bipinnana of Aatcnmi muuala, (B) auricularia of Parastichopus californicus, (C, D, E) view of broad side and (F) view of narrow side of ossicles at the posterior, left corner of the body of P. californicus, (G) and (H) ophiopluteus ofOphiiira tarsi, (I) echmopluteus of Dendraster excentricus. Cal- cite skeletons shown with polarizing filters partially crossed in (C) and (G) and fully crossed in (D, E, F, H, I). Scale bar is 400 ^m in (A, B. G.H.I) and 160/imin(C, D. E, F). Among echinoplutei, the stage of development affected density. In a sample of 32 larvae of S. francis- canus, the eight-armed larvae with two or fewer pedicel- lariae and no juvenile spines or plates (Fig. 4E) were less dense than both the earlier stage four- to six-armed larvae (Fig. 4C, D) and the more advanced stage eight-armed larvae with three pedicellariae (Fig. 4F. G). (Juvenile plates and spines were forming in some of the larvae with three pedicellariae.) The differences in density among stages were statistically significant (P < 0.001, Kruskal- Wallis test). Eight-armed plutei were similarly less dense than four-armed plutei within the species T. gratilla (Ta- ble I, Fig. 1 A, C) and D. excentricus (Table II). Very ad- vanced eight-armed plutei were more dense than four- armed plutei in S. pallidus (Table II, Fig. 4H). The den- sity changes during development were what one would have predicted from skeletal development. In early eight- armed stages of Strongylocentrotus species, the volume of skeleton relative to the volume of soft parts had de- creased because the heavy ends of the body rods had dis- appeared and because the soft parts had grown more than the skeletal rods (Fig. 4). Also in T. gratilla, the plu- tei at the eight-armed stage (Fig. 1C) were much more fleshy than the plutei at the four-armed stage (Fig. 1A), and the density of eight-armed T. gratilla was almost as low as the density of the decalcified four-armed plutei (Table I). In these comparisons among stages, greater de- velopment of skeleton relative to soft parts was associ- ated with greater density. However, Pennington and Em- let (1986) did not find an increase in density during rudi- ment development of larval D. excentricus. They suggested that lipids in cells of the gut (Burke, 1981) 128 J. T. PENNINGTON AND R. R. STRATHMANN Table II Ranking oj i/cnsily <>t larvae Irani ICIIM /us/ ilen\c with approximately ct/ital/v Jcn\c larvae lumped together Rank Form Species Stage Reorientation 1 auricularia Parastichopus califomicus 1 bipinnaria l^li'iiih/ ninuala 2 echinopluteus Sin 1/11:1 'A iccnlri iln.\ Iranei \cuini\ eight-armed, small echinus rudiment * 3 echinopluteus Strongylocentrotus Iruncucunit* tour-armed * 3 echinopluteus Strongylocentrotus pallidui four-armed * 4 echinopluteus Strongylocentrotus pallidus eight-armed, advanced echinus * rudiment and postlarval plates and spines 5 echinopluteus Dendraster excentricus eight-armed 6 echinopluteus Dendraster excentricus four-armed 7 ophiopluteus Ophiopholis aculeata four-armed = Those that reoriented as they sank toward neutral buoyancy. might compensate for skeletal development at this stage. Retractile droplets appeared in the stomach cells of nearly competent plutei of T. gratilla (Fig. ID), but we were not able to compare the specific gravities of all stages of T. gratillci. Differences in whole body density among plutei of different species (Table II) were related to larval shape and skeletal development. The early four-armed pluteus of S. purpuratus (Fig. 4A, B) was denser than the early four-armed pluteus of .S. pallidus. S. purpuratus develops from a smaller egg than does S. pallidus. and its pluteus begins with a larger skeleton relative to its body volume. Larvae of 5. purpuratus converge on the form of other Strongylocentrotus larvae as they grow (Sinervo and McEdward, 1988), and presumably they converge in density at later stages also. Echinoplutei of D. excentricus have thick fenestrated arm rods and an extensive body skeleton (Fig. 31) and, as expected, were denser than plu- tei of Strongylocentrotus. Four-armed ophioplutei of O. aculeata were the densest larvae examined (Table II), and ophioplutei in general have thick skeletal rods and a relatively small body cavity (Fig. 2G, H). (Plutei of S. purpuratus were not tested against plutei of D. excentri- cus or O. aculeata and therefore could not be included in Table II.) These comparisons indicate that the ratio of skeleton to whole body volume is a major determinant of density of the whole larval body. Some differences in skeletons were too small to over- ride other determinants of density of the larvae. The au- riculariae of P. californicus, with their small posterior os- sicles(Fig. 3B, C), andthebipinnariaeof,^. miniata, with no skeleton (Fig. 3A), sank to about the same depths in the density gradient. The ossicles of the auriculariae made no discernible difference in overall densities of the larval bodies. The auriculariae and bipinnariae had different lengths and body volumes, and this could have affected overall body density more than a small ossicle. Similarly, in comparisons among auriculariae of P. californicus, density of the larval body was not discern- ibly affected by the presence or absence of a posterior ossicle, but it did vary with larval size. Size, shape, and location of posterior ossicles varies; so in a sample of 30 auriculariae from 2 pairs of parents, 7 auriculariae lacked any posterior ossicle, 20 had a posterior left ossicle, and 3 had an additional posterior right ossicle. In this sample there was no relation between density of the larval body (depth of neutral buoyancy in a Percoll gradient) and the occurrence of posterior ossicles. Most posterior ossicles were broad in two dimensions (commonly 30 to 50 /^m) but thin in the third (about 10 ^m) (Fig. 3D, E, F). Vol- umes of ossicles ranged from about 5,000 to 1 3,000 ^m 3 (calculated as spheroids; sample of 4 larvae). In the sample of 30 auriculariae, longer larvae were less dense than short larvae (P < 0.02, two-tailed rank- sum test, and Spearman rank correlation 0.59, P < 0.001). Longer auriculariae were also less dense in a comparison of 12 auriculariae, all from the same pair of parents and ranging in length from 650 to 870 nm (P < 0.02, two-tailed rank-sum test, and Spearman rank correlation 0.95. P < 0.001). Larger echinoderm larvae have larger primary body cavities but about the same thickness of body wall ( McEdward, 1 984), which is a sin- gle layer of cells. As an approximation, the volume of the body cavity should increase in proportion to the cube of body length and the volume of body wall in proportion to the square of length. The lower densities of longer au- riculariae were therefore consistent with the hypothesis that their cells are denser than the contents of their pri- mary body cavity, but other aspects of larval condition could have been confounded with size. Effect of density of surrounding fluid on orientation Passive orientation depends on the density of the sur- rounding fluid. All dead larvae passively oriented ante- LARVAL SKELETONS AND ORIENTATION 129 Figure -4. Echinoplutei of Strongylocentrolus species: (A) ventral and (B) lateral views of four-armed 5. purpuratitx. (C) and (Dl four-armed S. frandxcanux, (E) eight-armed S Irandscaints without pedicel- lariae, (F) and (G) eighl-armed S. franascanus with three pedicellariae, and (H) eight-armed 5. pallidiis with juvenile plates and spines. Calcite skeleton shown with polarizing niters partly crossed in (C, E, F) fully crossed in (A, B, D, G. H). Because of their optical axis the preoral arm rods are not shown. Scale bar is 400 ^im for all photos. rior end upward when in seawater or in low-density mix- tures of seawater and isosmotic NaCl and Percoll or in seawater and isosmotic sucrose. However, echinoplutei of C. atratus, E. mathei, T. gratilla, S.franciscanus, and S. pallidus reoriented with their arms downward as they sank into the denser fluid in the density gradients and approached the depth at which they were neutrally buoy- ant (Tables I, II). The four-armed plutei of S. purpuratus and the plutei of D. exccntricitx and O. aculeuta did not reorient at neutral buoyancy, nor did auriculariae and bipinnariae. Stability of orientations at neutral buoyancy was demonstrated by changing the positions of larvae with a needle and observing their return to their undis- turbed positions. The reorientation of some plutei in denser fluids was caused by a different distribution of excess mass in dense media. The center of buoyancy was anterior to the center of gravity when the surrounding fluid was seawater, but as these plutei sank into denser fluid, the excess density of the skeleton relative to the fluid decreased, and the excess density of soft tissues and body cavities ap- proached zero and then became negative. The center of buoyancy did not move because the larvae did not change shape, but the center of gravity (defined in terms of excess mass) moved as the density of the surrounding fluid changed. In a denser fluid, therefore, the center of gravity was displaced anterior to the center of buoyancy, and the plutei reversed orientation. These plutei were those with relatively larger body cavities at their posterior ends or relatively lighter skeletons overall (for examples. Fig. 1 A, C; 4C-H). These plutei did not reverse orienta- tion simply because they sank into denser fluid than did those that remained anterior end upward; the plutei that remained arms upward at neutral buoyancy (O. acu- leata. D. excentricus, and four-armed S 1 . purpuratus) sank into denser fluid than did plutei that reversed their 130 J. T. PENNINGTON AND R. R. STRATHMANN orientation (S. franciscanus and S. pallulus) (Table II). Thus the differences among plutei in shape and skeletal development and the depths at which their stable orien- tation was anterior end upward fit our interpretation of the reorientation. The auriculariae and bipinnariae did not reverse ori- entation, but that does not imply greater passive stability than the plutei that reversed orientation because the au- riculariae and bipinnariae did not sink as far in the den- sity gradient as did these plutei. Nevertheless, the decalci- fied four-armed plutei of T. gratilla reversed their orien- tation to anterior end downward at neutral buoyancy, whereas the bipinnariae and auriculariae remained ori- ented anterior end upward. This suggests that a pluteus without a skeleton may have less vertical stability than the bipinnaria and auricularia. This could result from the distribution of epidermal cells, which form a thick co- lumnar layer at ciliated bands and are very thin else- where; the ciliated band is placed more anteriorly in plu- tei than in auriculariae and bipinnariae. Perhaps the skeleton is necessary for the pluteus form because of its contribution to vertical stability, in addition to its role in body support. Effect of drag on orientation So far we have ignored the effects of drag on orienta- tion. Drag on swimming larvae could be important but drag cannot account for the following observations on orientation. (1) Echinoplutei of 5" pallidus and S. fran- ciscanus species reoriented with their arms downward as they sank into denser fluid. If drag were responsible for an orientation with arms upward, the larvae would not have reoriented with arms pointing downward as they continued to sink. (2) All four types of echinoderm lar- vae passively reoriented when turned with a glass needle even when they were at their depth of neutral buoyancy and were no longer sinking. Drag associated with sinking was not necessary to produce stable orientations. Discussion The existence and forms of calcite skeletons in feeding echinoderm larvae pose difficult questions. Why should gelatinous planktonic animals have internal mineral skeletons? Why do these larvae have calcite skeletal ele- ments that are unsuited for supporting body parts? How did the complex skeletons of plutei evolve? Answers to these questions depend on functional consequences of larval skeletons. We have examined the importance of the skeleton in passive orientation with two approaches: ( 1 ) experimental removal of skeletons from echinoplutei and (2) comparisons among larval forms. We have ex- amined the role of larval skeletons in passive vertical ori- entation both directly and by the contributions of skele- tons to relative density and sinking rates. In this discus- sion we first review other functional roles that the larval skeleton may have and then explore ways in which a re- quirement for passive vertical orientation could influ- ence the form and distribution of the calcite skeleton. Skeletal support for arms. The primary body cavity of echinoderm larvae contains elastic gelatinous material that appears adequate to oppose muscles and to support convoluted body surfaces (Strathmann. 1989). Is a skele- ton really needed for the support of larval arms? Experi- ments indicate that the skeleton does indeed play a role in the development of arms of echinoplutei, but compar- isons show that not all arms require a skeleton and that not all calcite skeletal elements support arms. Experi- ments by Horstadius (1939), Okazaki (1956), and others showed that an interaction between skeletal rods and the epidermis is necessary for normal development of arms of echinoplutei, though short and rudimentary arms form without skeletal rods. Our removal of skeletons from plutei confirmed these observations. When living plutei with well-developed arms were decalcified, the arms initially became shorter and thicker than the arms of controls (Fig. 2A-C). Within several days the arms completely disappeared in echinoplutei that did not rese- crete a skeleton (Fig. 2D). In echinoplutei that resecreted skeletal rods, the arms formed normally, except that length and direction were usually abnormal (Fig. 2E, F). The skeleton was necessary to maintain the arms of echinoplutei, but skeletal rods are not necessary to sup- port all arms of echinoderm larvae; asteroid larvae lack a calcite larval skeleton, but many late stage asteroid larvae nevertheless develop arms (Mortensen, 1921, 1938). Also, not all skeletal elements support arms; e.g., parts of the body skeleton of plutei and the posterior ossicles of auriculariae. Thus experiments on echinoplutei indicate that the calcite skeletal rods maintain the larval arms; but in some of the other feeding echinoderm larvae skeletal rods are not necessary for the development or support of arms, and the support of arms is not the function of all skeletal elements. Skeletal defense against predators. Emlet ( 1983) deter- mined that the fenestrated skeletal rods of arms were stiffer than necessary to support them against forces gen- erated in swimming. Arms of our decalcified live plutei did not discernibly bend or flex during swimming, though admittedly these decalcified arms were very short. Because many arm rods appeared to be much heavier than necessary for support, Emlet (1983) sug- gested that the skeleton plays a role in defense against predators. The hypothesis is plausible, but direct evi- dence is lacking. In laboratory experiments, echinoid prism and pluteus stages, which have skeletons, were less vulnerable to predators than were earlier stages that lacked skeletons (Pennington el ai. 1986), but this could LARVAL SKELETONS AND ORIENTATION 131 have been from predator avoidance following develop- ment of ciliary arrest and reversal at the late prism stage (Rumrill et at., 1985) rather than from skeletal protec- tion, and the skeleton does not prevent ingestion by crab zoeae (Rumrill and Chia, 1986). A defensive role for the larval skeleton is possible but not yet tested. Vertical orientation. Runnstrom (1918) and Penning- ton and Emlet (1986) suggested that the echinopluteus skeleton weights a larva so that it passively orients with its arms upwards. In such an orientation, forward swim- ming produces upwards movement that counters sink- ing. We tested this hypothesis by comparing the vertical orientation of control and decalcified larvae. Vertical ori- entation and stability of both live and dead four-armed echinoplutei was reduced by removal of the skeleton. Al- though other effects of temporary exposure to low pH or the shape changes could have affected the vertical orien- tation of decalcified live larvae, such objections do not apply to the orientation of the dead four-armed echi- noplutei without skeletons. Does the larval skeleton contribute a sufficient part of the total excess mass that it could influence passive orien- tation? For plutei the answer is "yes," because the small skeleton of a pluteus has a large effect on whole body density and sinking rate. At a salinity of 33% and tem- perature of 25C the density of seawater is 1.02 g/ml. Therefore, for T. gralilla, the estimated excess density of the four-armed echinopluteus without a skeleton was roughly the difference between 1 .04 and 1 .02 g/ml. The additional contribution of the larval skeleton was the difference between 1 .06 and 1 .04 g/ml. By this estimate, the skeleton of the four-armed echinopluteus contrib- uted half the excess density of the larva. A similar rough estimate based on the difference in densities of prisms and four-armed plutei of). t>.YCT/ncw(Pennington and Emlet, 1986) suggests that the skeleton of some plutei may contribute as much as 77% of the excess density. If the body volume of four-armed T. gratilla did not change with the removal of the skeleton (Fig. 1A, B), then the proportion of the total body mass that was skele- ton can be estimated as ( 1 .06- 1 .04 )/ 1 .06, or only about 2% of the body mass. If the density of the skeleton is the same as the density of mineral calcite (2.71 g/ml), then the volume of the skeleton can be estimated as approxi- mately [(1.06-1.04)/2.71j (body volume), or less than 1% of the volume of the larva. These approximate esti- mates demonstrate that a skeleton of relatively small mass and volume contributed much of the excess mass of the larval body. The estimated sinking rates of four-armed plutei of T. gratilla suggest an even greater contribution of the skele- ton to excess density. For slowly sinking objects of this size and the same shape, the sinking rate should be pro- portional to the excess density ( Vogel, 1981). According to the estimated sinking speeds, the plutei with skeletons sank about 5.5 to 8.4 times as fast as the plutei without skeletons. By this estimate, the skeleton provided up to % of the excess mass of the whole larva. The discrepancy between estimates of sinking rates and estimates of den- sity could result from errors in both estimates. The mea- sured sinking rates were quite variable, and the estimate of density of the larval body could have been biased by the intervals between the densities of test solutions. Al- though the exact contribution of the skeleton to excess mass is uncertain, the estimated contribution of approxi- mately '/> to % of excess mass is substantial and indicates that the distribution of skeletal elements is important for the passive orientation of plutei. Does passive vertical orientation depend on a calcite skeleton for all types of feeding echinoderm larvae? All four types of feeding larvae, even the bipinnariae (with no skeleton), were stable with their anterior ends up- ward. Also, prior to the deposition of any skeleton, swim- ming echinoid blastulae and gastrulae swim upward in culture (Lyon, 1906), a presumably passive orientation to gravity. A calcite skeleton can enhance passive vertical orientation, but some degree of vertical orientation and gravitational stability is clearly possible without it. Does excess density depend on skeletal development for all planktotrophic echinoderm larvae? Or do other features have a greater effect on buoyancy? Comparisons of the varied types of echinoderm larvae showed that lar- vae with a larger volume of skeleton relative to the vol- ume of soft parts were denser. The plutei were denser than the auriculariae and bipinnariae, and plutei with large skeletal volumes relative to their body volumes were denser than the plutei whose skeletons were rela- tively small. Also, the passive orientation of plutei with relatively small skeletons was more affected by the distri- bution of soft parts, as was shown by resting orientation of plutei in solutions denser than their soft parts. Because whole body density increases with the amount of skele- ton, there is a functional requirement for the distribution of skeletal parts within the body: if larvae with large skel- etons are to maintain passive vertical orientation, the skeleton must have a large posterior component. Does a skeletal element as small as the posterior ossicle of an auricularia enhance passive orientation? Our ob- servations suggested little contribution of the ossicle of the auricularia of P. califomicus to excess mass, but we cannot rule out a function in passive orientation. The auriculariae with posterior ossicles had whole body den- sities similar to those of bipinnariae and of auriculariae without posterior ossicles, and measurements of the ossi- cles also suggested only a small contribution to excess mass. Most posterior ossicles of P. califomicus were very flat (Fig. 3D, E, F ) and therefore had very small volumes. Nevertheless, the small mass of the ossicle could affect 132 J. T. PENNINGTON AND R. R. STRATHMANN passive orientation because of its extreme posterior posi- tion. Costs of swimming with a skeleton. Larvae with large excess densities must swim to counter sinking. What en- ergetic cost does this impose? To estimate the work nec- essary to counter sinking, we have combined our esti- mates for T. grati/la with published data for four-armed D. e.\ccntriciis. If a four-armed pluteus has a volume of 2.6 X KT 6 ml (McEdward. 1984) and an excess density of 0.04 g/ml. then the excess mass is 10~ 7 g, and the downward force (F = mg) is 10 4 dyne. The product of this force and our estimated sinking velocity of 0.044 cm/s is 4.4 X 10"" J/s, the rate of work done against gravity. A similar calculation for decalcified plutei with 0.02 g/ml excess density, that sank at 0.0052 cm/s, gives 0.26 X 10~ L1 J/s, so that the skeleton is responsible for 94% of the work done against gravity. This may be an exaggeration because of the discrepancy between our es- timates of sinking rates and excess densities, but even if sinking rates differed by only a factor of 2, as expected from our estimate of excess density, the skeleton would be responsible for 75% of the work done. However, the total work against gravity appears to be trivially small when compared to total respiratory rate of the larva. An estimate of metabolic capacity for a four- armed pluteus can be converted to a rate of oxygen con- sumption (McEdward and Strathmann. 1987) and then converted to energy units (4.65 X 10" mol O ; /h/larva x 22.4 1 : /mol X 4800 cal/1 2 ; conversion factors from Schmidt-Nielsen, 1979) to get 5.8 X 10~ 9 J/s. The work done against gravity is thereby estimated to be less than 0.01% of the larva's total energy expenditure. The errors in the estimates used in this calculation are undoubtedly large, and the efficiency of the work of swimming is un- known, but the energetic cost of swimming with a skele- ton appears to be small. Calculations of work done by swimming copepods (Vlymen, 1970; Alcaraz and Strickler, 1988) and ciliates (Fenchel. 1987) are also a very small part of energy expenditure, as estimated from oxygen consumption. Swimming to counter an in- creased sinking rate caused by the skeleton apparently does not present an energetic problem for the larvae. Another consideration is the effect of excess density on rate of ascent. Estimated swimming speeds for feeding larval stages of echinoderms are commonly about 0.3 to 0.5 mm/s (Strathmann, 1971). Konstantinova( 1966) es- timated 0.3 mm/s for a bipinnaria. but Konstantinova's estimate of 0. 1 mm/s for an ophiopluteus appears to be an underestimate. In extensive but unpublished observa- tions, H.-t. Lee measured horizontal swimming speeds of 0.3 to 0.5 mm/s for bipinnariae. up to 0.4 mm/s for auriculariae of P. califomicus. 0.4 mm/s for two-armed ophioplutei of O. aculcata, and 0.4 to 1 .9 mm/s for four- to eight-armed echinoplutei of D. excentricus and S 1 . droebachiensis. The sinking rates of 0.3 and 0.4 mm/s measured for dead four-armed plutei of T. gratilla are comparable to these swimming speeds. Plutei would need more propulsion than bipinnariae or auriculariae to achieve the same rate of vertical ascent. Ultimately, costs of maintaining depth might be inde- pendent of excess mass. Echinoderm larvae swim when they feed because the ciliated band produces currents for both swimming and feeding (Strathmann, 1971). If lar- vae must feed much of the time and if sinking speeds are less than or equal to swimming speeds produced during feeding, then the larvae need only to orient upward to maintain or decrease their depth. Passive orientation and skeletal form. Because some types of feeding echinoderm larvae orient to gravity without any skeleton, it seems doubtful that the elabo- rate skeletons of plutei evolved primarily to produce pas- sive vertical orientation. Nevertheless, if vertical orienta- tion is important, its requirements constrain the form of the pluteus skeleton and may explain many features of pluteus morphology. A larger and more elaborate poste- rior body skeleton is correlated with a larger anterior arm skeleton, as shown by examples in Mortensen (1921, 1938). The thickened posterior body rods in early echi- noplutei of the Strongylocentrotidae and Echinidae and the posterior rods of spatangoid plutei may serve as a counterweight for the larval arms. Similarly, juvenile skeletal elements may be con- strained to develop in positions that maintain passive orientation. The asteroid, echinoid, and ophiuroid juve- nile rudiments develop in a posterior position. A role of the skeleton in passive orientation also sug- gests a functionally advantageous and simple first step in the evolution of larval skeletons. All that is required for passive orientation is a posterior position of the skeletal element; the posterior ossicles in auriculariae may be an example. In contrast, a supporting skeleton must de- velop as a system of rods favorably placed for supporting muscles or extensions of the ciliated band. Improve- ments in the skeletal form for support of muscles or pro- jecting arms may have evolved after early formation of calcite ossicles was established. Acknowledgments This study was supported by NSF grants OCE8606850 to R. R. Strathmann and DCB-8602149 to M. G. Had- field. Four classes of echinoderm larvae were compared at the Friday Harbor Laboratories of the University of Washington. Skeletons were removed from plutei at the Kewalo Marine Laboratory of the Pacific Biomedical Research Center of the University of Hawaii. R. B. 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Lite in Moving fluids. Willard Grant Press. Boston. 352 pp. Vlymen, W. J. 1970. Energy expenditure by swimming copepods. Limnol. Oceanogr. 15: 348-356. Reference: Bin/. Hull 179: I 34-1 39. (August. Pressure-Temperature Interactions on M 4 -Lactate Dehydrogenases From Hydrothermal Vent Fishes: Evidence for Adaptation to Elevated Temperatures by the Zoarcid Thermarces under soni, but not by the Bythitid, Bythites hollisi ELIZABETH DAHLHOFF, SABINE SCHNEIDEMANN 1 , AND GEORGE N. SOMERO Marine Biology Research Division, A-002. Scripps Institution of Oceanography, University oj California, San Diego. La Jo/la, California 92093-0202 Abstract. Lactate dehydrogenases (LDH; M 4 isozyme) were purified from skeletal muscle taken from two fishes endemic to hydrothermal vents, Thermarces andersoni (Zoarcidae; 13N, East Pacific Rise, depth ~ 2600 m) and Bythites hollisi (Bythitidae; Galapagos Spreading Center, depth ~ 2500 m), and from the cosmopolitan deep-sea rattail Coryphaenoid.es annul us (Macrouridae; depth of occurrence to ~5000 m). The effects of pressure and temperature on the apparent Michaelis-Menten constant (K m ) of cofactor (NADH) were measured to compare sensitivities to temperature, at in situ pressures, of enzymes from hydrothermal vent fishes and from a species adapted to cold, stable deep-sea temperatures. At 5C, the K m of NADH of the M 4 -LDHs of the three spe- cies varied only slightly between measurement pressures of 1 and 340 atmospheres (atm), in agreement with ear- lier studies of M 4 -LDHs of deep-sea fishes. At higher measurement temperatures, marked differences were found among the enzymes. For the M 4 -LDHs of C. ar- matus and B. hollisi, increases in temperature (10 to 20C), at in situ pressures, sharply increased the K m of NADH to values higher than those predicted to be physi- ologically optimal. The M 4 -LDH of T. andersoni exhib- ited only minimal perturbation by elevated temperature under in situ pressures. The different temperature-pres- sure responses of these LDHs suggest that enzymes of Received 26 February 1 990; accepted 18 May 1990. ' Present address: Department of Cell Biology, Eidgenossische Tech- nische Hochschule. CH-8093 Zurich, Switzerland. deep-sea fishes not endemic to hydrothermal vents are not adapted for function at the higher temperatures found at vent sites, and that T. andersoni is better adapted than B. hollisi for sustained exposure to warm vent waters. The importance of adaptation to warm tem- peratures in the colonization of vent habitats is dis- cussed. Introduction The hydrothermal vent sites at seafloor spreading cen- ters in the Eastern Pacific are, in several ways, unusual deep-sea environments: the food-chain is based on bacte- rial chemosynthesis rather than photosynthesis; a high degree of endemism characterizes the fauna (Newman, 1985); animal biomass is enormous; and water tempera- tures are much higher than is typical of the deep sea (~2-3C) (Hessler and Smithey, 1984; Grassle, 1985). The primary focus of physiological and biochemical re- search with vent organisms has been on the chemosyn- thetic processes supporting the food web, and on the ad- aptations of vent animals to withstand hydrogen sulfide, the primary energy source for chemosynthesis (Grassle, 1985; Somero et al., 1989). Less attention has been paid to the potential importance of temperature as a factor influencing the physiologies of the vent organisms and effecting the distribution of endemic vent species and other deep-sea animals in and near the vent fields. Temperature typically is a major influence on organis- mal distribution patterns and physiological function (Hochachka and Somero, 1984; Cossins and Bowler, 134 LDHs OF HYDROTHFRMAL VENT FISHES 135 1987). and the steep temperature gradients found at the hydrothermal vents up to ~380C over distances of several cm (Fustec ct al., 1987) could present challeng- ing thermal adaptation problems to the vent fauna. Many vent invertebrates encounter temperatures con- siderably higher than those experienced by deep-sea spe- cies living outside the vents. Sessile invertebrates, in par- ticular, live continuously in the warm vent effluents in which temperature can vary between about 2 and 1 5C at the Galapagos Spreading Center sites (Hessler and Smithey, 1984; Johnson et al.. 1988). and between 2 and at least 20C at the 1 3N site on the East Pacific Rise (EPR) (Fustec el al., 1987). The motile brachyuran crab Bythograea thermydron also forages for extended peri- ods in the warm vent waters, and this species appears well adapted for function under conditions of high pres- sure and elevated temperatures (Arp and Childress, 1981; Mickel and Childress. 1982a, b). Adaptations of hydrothermal vent fishes to high temperature and pres- sure have not previously been investigated. Although about 20 species of fishes have been described in the gen- eral area of the vents (Cohen and Haedrich, 1983), only three fishes, all endemic species, occur within the vent field, and are potentially exposed to waters with elevated temperatures. Two are zoarcids: Tlicrmarces cerberus has been identified at both the Galapagos and 21N site on the EPR; and T. andcrsoni is found at the 1 3N EPR site (Rosenblatt and Cohen, 1986). Geistdoerfer (1985), however, regards these two zoarcids as one species. The hydrothermal vent zoarcids have been observed resting on the basaltic seafloor and, at EPR sites, on the rough surfaces of "smoker" chimneys. EPR sites are characterized by these chimneys which emit hot (up to ~380C; black smokers) and warmed (~20C; white smokers) waters (Hekinian et al.. 1983). At the EPR sites, cooler water is emitted from fissures in the seafloor. Each vent type the hot black smokers, the white smokers, and the warm seeps from fissures has a distinct faunal assemblage associated with it. At all three vent types, Thermarces are found in close association with the ben- thic invertebrates (Fustec et al.. 1987). The exact water temperatures encountered by the zoarcids are not known. But, because they have been observed to rest mo- tionless on the bottom among the vestimentiferan tube worms and other invertebrates that live in the warm vent effluents, they may experience warm temperatures for periods long enough to effect thermal equilibration of their bodies with the warm vent waters (Fustec et al., 1987). The third vent fish described, Bythitex hollisi (family Bythitidae) (Cohen et al.. 1990), has been collected only at the Galapagos Spreading Center, although fishes of similar appearance have been observed from submers- ibleson the EPR. B. hollisi 'is the only endemic vertebrate common to the Galapagos site (Hessler and Smithey, 1984). Individuals have been observed hovering over warm water vent openings, sometimes with their heads protruding into the cracks from which the warm water is seeping. Given this behavior, B. hollisi probably is ex- posed to water temperatures warmer than ambient deep- sea temperatures. However, the extreme steepness of the thermal gradients above the Galapagos-type warm water vents (up to ~ 1 3C differences over a few cm; see Hessler and Smithey, 1984; Johnson el al., 1988) precludes accu- rate estimates of the temperatures encountered by B. hol- lisi. Smoker chimneys are absent at the Galapagos site, so there is no potential for B. hollisi of this vent habitat to encounter the high temperatures that might confront fishes inhabiting the EPR sites. A number of fishes typical of the cold deep sea, includ- ing rattail fishes (Macrouridae), have been observed swimming near the Galapagos and EPR vent sites (Co- hen and Haedrich, 1983). The cosmopolitan rattail Cor- yplwenoides annatits is likely to be found at the depths of the Galapagos Spreading Center and at the 13N and 2 1N EPR sites. M 4 -LDHs have been studied extensively in shallow- and deep-living fishes (Siebenaller, 1 987; Siebenaller and Somero, 1978, 1979, 1989), but only at a measurement temperature of 5C. At this low temperature, the M 4 - LDHs of adult fishes occurring at depths greater than 500-1000 m (51-101 atm pressure), differ adaptively from the M 4 -LDH homologs of shallow-living, cold- adapted fishes. For example, the effects of pressure on the apparent Michaelis-Menten constant (K m ) of cofac- tor (NADH) are small or non-existent for the M 4 -LDHs of deep-sea species, but very large in the case of the M 4 - LDHs of shallow-living fishes. These sharp differences in the effect of pressure on the K m of cofactor and substrates for LDHs and other enzymes (Siebenaller and Somero, 1989) are hypothesized to play important roles in estab- lishing the depth distribution patterns of marine fishes. Analogously, differences among deep-sea species in the effects of temperature on their enzymes under //; situ pressures might play a role in determining horizontal dis- tribution patterns related to temperature gradients near hydrothermal vent sites. To determine whether differences in temperature ad- aptation exist between the biochemistries of endemic vent fishes and deep-sea fishes from cold, thermally sta- ble waters, we studied the skeletal muscle isozymes (M 4 = A 4 )oflactatedehydrogenase(LDH: EC 1.1.1.27); the kinetic and structural properties of this enzyme strongly reflect the temperatures and pressures to which an organ- ism is adapted (Yancey and Somero, 1978; Siebenaller and Somero, 1989). M 4 -LDHs from T. andersoni, B. hoi- 136 E. DAHLHOFF ET Al. list, and C. armatus were purified and studied kinetically over a range of pressures and temperatures to determine how temperatures typical of warm water vents affect the response of M 4 -LDHs to in situ pressures. Materials and Methods Collection ami preservation of specimens The specimen of B. hollisi (initial description by Co- hen et ai. 1990) was captured by net from the DSV A/vin at the Galapagos Spreading Center during the Galapa- gos- 1988 expedition. The specimen was returned to the surface in an insulated container and immediately dis- sected. Muscle samples were frozen immediately in liq- uid nitrogen, and returned to the Scripps Institution of Oceanography (S1O) for analysis. The specimen of T. andersoni was captured in a baited trap at the 13N EPR site during the autumn 1987 French-US Hydronaut expedition. Recovery was achieved using the French submersible DSV Nautile. The specimen was frozen immediately upon return to the ship, returned to SIO. and stored at -80C until ana- lyzed. C. armatus was collected by otter trawl in Monterey Canyon at a depth of ~3000 m. White muscle was dis- sected from the fish, wrapped in aluminum foil, and fro- zen immediately on dry ice. Tissues were returned to SIO and stored at -80 until analyzed. Enzyme purification and determinations ofK m ofNADH The M 4 isozyme of LDH was purified with an oxamate affinity column, as described by Yancey and Somero ( 1 978). Native starch and polyacrylamide gels stained for LDH activity revealed a single band of activity, the M 4 - LDH. SDS-polyacrylamide gels stained with Coomossie blue showed a single protein band corresponding in M r to LDH. The K m of NADH was determined using an 80 mM imidazole/Cl buffer (pH 7.0 at 20C). This buffer was chosen, rather than the Tris/Cl buffer used in earlier studies of the effects of pressure on LDH (cf. Siebenaller and Somero, 1978, 1979), because the pK of imidazole varies with temperature in parallel with the intracellular pH (pHj) offish muscle (Reeves, 1977). The pH values of imidazole/Cl buffers, like those of Tris/Cl buffers, are virtually unaffected by pressures in the range used in these studies (Kauzmann et a!., 1962). Except for the differences in assay medium (buffer species and KC1 con- centration; cf. Siebenaller and Somero, 1978), the high pressure assays were made following the protocol of Sie- benaller and Somero (1978). Seven to nine concentra- tions ofNADH spanning the value of K m were used to determine each K m value. The K m values were computed according to the weighted linear regression method of Wilkinson (1961) (Wilman4 software; Brooks and Suelter, 1986). Standard deviations of the K m values did not exceed 12% of the K m values (Fig. 1 ). Results The effects of temperature on the pressure sensitivities of the K m ofNADH for the M 4 -LDHs of the three species are illustrated in Figure 1. At 5C, the kinetics of these enzymes resembled those of the high pressure-adapted M 4 -LDHs of other deep-sea fishes (see Siebenaller, 1987; Siebenaller and Somero, 1989). Increased pressure caused at most a slight increase in the K m ofNADH, and this increase occurred over the first 68 atm rise in mea- surement pressure. Pressures above 68 atm caused no further increase in K m . At temperatures above 5C, the M 4 -LDH of T. ander- soni differed from the homologs of the other two deep- sea species (Figs. 1, 2). At in situ pressures (~250 atm; dashed vertical line in Fig. 1). the M 4 -LDH of T. ander- soni exhibited no increase in K m of NADH between 5 and 10C, and only a slight increase between 10 and 20C. The M 4 -LDHs of C. armatus and B. hollisi exhib- ited an approximate doubling of the K m ofNADH as the temperature increased to 1 5 or 20C. Discussion The K m of substrate or cofactor for a given type of en- zyme is strongly conserved among species at their physi- ological temperatures (Yancey and Siebenaller, 1987; Yancey and Somero, 1978) and pressures (Siebenaller, 1984, 1987; Siebenaller and Somero. 1978, 1989). The K m ofNADH for M 4 -LDH varies at most by about 10 fj.M, both among species at their physiological tempera- tures and pressures, and across a single species' normal range of body temperatures and pressures. At tempera- tures or pressures above the normal physiological range, the K m ofNADH typically exhibits a large temperature- or pressure-related increase, and reaches values that no longer lie within the conserved range that is viewed as physiologically optimal. Similar trends have been seen for several enzymes, which emphasizes that enzymatic kinetic properties must be maintained within narrow ranges that are optimal for catalysis and regulation (re- viewed by Hochachka and Somero, 1984; Siebenaller and Somero, 1989). Conservation of K m and other ki- netic parameters may only be observed when compara- tive studies of enzyme homologs are all performed in the same in vitro milieu; differences in ionic strength, for ex- ample, can affect the absolute values of K m (cf. Siebe- LDHs OF HYDROTHERMAL VENT FISHES 137 o E 50 100 150 200 250 300 350 400 50 100 150 200 250 300 350 400 50 100 150 200 250 300 350 400 Pressure (atm) Figure 1. The effects of measurement temperature and pressure on the apparent Michaelis-Menten constant (K m ) of NADH for M 4 -LDHs of the cosmopolitan deep-sea rattail fish Coryphaenoides armaim, the hydrothermal vent hythitid Bytlules hollisi. and the hydrothermal vent zoarcid Tlwrmanvs ander.wni. The dashed vertical line indicates the approximate habitat pressure at the two vent sites. nailer and Somero, 1978, with Yancey and Siebenaller, 1987). For the M 4 -LDHs of C. annatus and B hollisi, tem- peratures of 10 to 20C increased the K m of NADH by ~ 1 5-20 /uM at in situ pressures (Fig. 2). In contrast, the K m of NADH for the M 4 -LDH of T. andersoni increased by only approximately 8 pM as temperature increased from 5 to 20C. Therefore, temperatures characteristic of warm water vents perturbed the K m of NADH of the M 4 - LDHs of C. annatus and B. hollisi sufficiently to increase their values beyond the physiologically conserved range noted for other species. The M 4 -LDH of T. andersoni retained its K m of NADH within the physiologically con- served range across the span of measurement tempera- tures at in situ pressure. The different responses of the M 4 -LDHs of these three species to changes in temperature at in situ pressure lead us to propose two hypotheses concerning the relation- ship between species distribution patterns and tempera- ture and pressure influences on enzymatic function. First, we propose that the M 4 -LDHs of cold-adapted deep-sea fishes are not pre-adapted for function at the elevated temperatures found at the warm water vents. Thermal perturbation of the kinetic properties of en- zymes under pressure may restrict the endemic fauna of the cold deep sea from exploiting hydrothermal vent habitats. Thus, as much as interspecific differences in the pressure sensitivities of enzymes may be important in es- tablishing species' vertical distribution patterns in the marine water column (Siebenaller and Somero, 1989), interspecific differences in the responses of enzymes to elevated temperatures, at deep-sea pressures, may be in- strumental in establishing horizontal distribution pat- terns in temperature gradients near the deep-sea hydro- thermal vents. This conjecture is not meant to imply that temperature is the only factor restricting typical deep-sea animals from the vent environment. Mechanisms for overcoming the toxic effects of hydrogen sulfide also ap- 5 10 15 20 Temperature (C) Figure 2. The effect of measurement temperature, at the approxi- mate habitat pressure of the two hydrothermal vent sites ( 250 atm), on the K m of NADH for the M 4 -LDHs of the three species shown in Figure 1. K m values at 250 atm were estimated by the intersection of the vertical dashed line (corresponding to 250 atm pressure) with the lines connecting the K m values at each temperature (see Fig. 1 ). 138 E. DAHLHOFF /: 7 Al. pear to be important components of adaptation to the vent environment (Somero el at., 1989). Second, we hypothesize that, among endemic vent species, there may be substantial differences in tolerance of high temperature and, therefore, in the microhabitats they experience. The interacting effects of elevated tem- perature and pressure on its M 4 -LDH suggest that B. hol- lisi from the Galapagos Spreading Center is not adapted for continuous existence in the warmest waters found at this site. In contrast, by our enzymatic criterion, T. an- dersoni appears well adapted to body temperatures as high as 20C. Because the exact temperatures experienced by en- demic vent fishes, and the times over which they remain in warm waters, are not known with accuracy, links be- tween enzymatic properties and environmental distribu- tions remain speculative. However, the contrasting ther- mal properties of their environments suggest that the two vent fishes used in these studies have different thermal experiences. At the Galapagos Spreading Center site, where B. hollisi is the most abundant endemic verte- brate, smoker chimneys are absent, and the highest tem- perature recorded in the warm water vents was ~ 1 5C (Johnson el al. 1988). Although B. hollisi is commonly found hovering over the vent openings, and may even enter the sites of venting (Robert R. Hessler, Scripps In- stitution of Oceanography, pers. comm.), the extremely steep thermal gradients characteristic of the vents make precise estimates of the fish's body temperature impossi- ble. B. hollisi, unlike T. andersoni, appears to spend most of its time swimming and, therefore, may select water temperatures that are lower than those encountered by the demersal zoarcid, which commonly rests among ses- sile invertebrates living directly in the warm vent efflu- ent. At the 1 3N EPR site, where T. andersoni is the most abundant endemic vertebrate, the temperatures of the warm water vents reach at least 20C (Fustec el al.. 1987). Zoarcids are also found on the walls of smoker chimneys, where waters much hotter than those at the Galapagos Spreading Center are emitted. Zoarcids are observed to swim very rapidly out of hot smoker-vent waters, so they may not experience these high tempera- tures for more than a few seconds per encounter. Recent studies of the effects of pressure and tempera- ture on the K m s of NADH of malate dehydrogenases (MDHs) of invertebrates from the hydrothermal vents and several other shallow- and deep-water marine habi- tats support the hypothesis that adaptation to elevated temperatures is important for vent species exposed to warm vent effluents for extended periods (Dahlhoff, 1989; Dahlhoff and Somero, in prep.). Although all of the MDHs from deep-sea invertebrates were found to be pressure insensitive at 5C. only the warm-adapted hy- drothermal vent species exhibited the pattern of stability of the K. m of NADH under high pressure and elevated temperature shown here for the M 4 -LDH of T. under- soni. We propose, then, that the hydrothermal vent ani- mals, which attain thermal equilibrium with the warm vent waters, are characterized by pervasive biochemical adaptations to elevated temperatures, and these adapta- tions are prerequisite to an exploitation of the warm mi- crohabitats in the vent field. Acknowledgments These studies were supported by National Science Foundation grants OCE83-00983 and DCB88- 1 2 1 80 to G. N. Somero, and by facilities support grant OCE- 8609202 to James J. Chidress. We gratefully acknowl- edge the assistance provided by the captains and crews of the research vessels R/V Thomas Thompson (Univer- sity of Washington), R/V Melville (SIO), R/V Atlantis If and DSV Alvin (Woods Hole Oceanographic Institu- tion), and R/V Nadir and DSV N ant He (IFREMER- Brest), and the help of the chief scientists of these vessels, Ms. Anne-Marie Alayse (Nadir), Dr. Horst Felbeck (Thomas Washington), and Dr. James Childress (Mel- ville). We thank Dr. Robert R. 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Jan- nasch, R. E. Marquis, and A. M. Zimmerman, eds. Academic Press, London. Siebenaller, J. F., and G. N. Somero. 1978. Pressure-adaptive differ- ences in lactate dehydrogenases of congeneric fishes living at differ- ent depths. Science 201: 255-257. Siebenaller, J. F., and G. N. Somero. 1979. Pressure-adaptive differ- ences in the binding and catalytic properties of muscle-type (M 4 ) lactate dehydrogenases of shallow- and deep-living marine fishes. / Comp. Physiol 129: 295-300. Siebenaller, J. F., and G. N. Somero. 1989. Biochemical adaptations to the deep sea. CRC Cm. Rev Aquat. Sci. 1: 1-25. Somero, G. N., J. J. Childress, and A. K. Anderson. 1989. Transport, metabolism, and detoxification of hydrogen sulfide in animals from sulfide-nch marine environments. CRC Crit. Re\\ Aqual. Sci. 1: 591-614. Wilkinson, G. N. 1961. Statistical estimation in enzyme kinetics. Bio- cliem J 80: 324-332. Yancey. P. H., and J. F. Siebenaller. 1987. Coenzyme binding ability of homologs of Mj-lactate dehydrogenase in temperature adapta- tion. Bioc/nm Biophys. Aeta 924: 483-49 1 . Yancey, P. H., and Somero, G. N. 1978. Temperature dependence of intracellular pH: its role in the conservation of pyruvate K. m values of vertebrate lactate dehydrogenases. J. Comp. Physiol. 1 25: 1 29- 134. Reference: Biol. Bull 179: 140-147. (August, 1990) Extraction of a Vanadium-Binding Substance (Vanadobin) from the Blood Cells of Several Ascidian Species HITOSHI MICHIBATA 1 , HISAYOSHI HIROSE 1 , KIYOMI SUGIYAMA 1 YUKARI OOKUBO 2 , AND KAN KANAMORI 2 [ Biological Institute and 2 Department of Chemistry, Faculty of Science, Toyama University, Gofuku 3190, Toyama 930, Japan Abstract. A combination of techniques, including chromatography on Sephadex G-15 and SE-cellulose columns and neutron activation analysis for vanadium determination, was used to extract (at low pH) a vanadi- um-binding substance (vanadobin) from the blood cells of the ascidian species: Ascidia ahodori OKA. A. gem- mat a SLUITER, A. :ara OKA, Core/la japonic a HERD- MAN, and dona intestinalis (UNNE). In general, ascid- ians can be classified into two different categories based on vanadium content: species of the family Ascidiidae contain high levels of vanadium, whereas those in the Cionidae and Corellidae do not always have such high amounts. Because Ciona intestinalis and Corel/a japon- ica do have vanadobin in their blood cells, vanadobin may well be a universal complex in ascidians, having the role of accumulating vanadium in blood cells and main- taining its concentration. The blood cells of A. gemmata contained the highest amount of vanadium. Vanadobin extracted from these cells exhibits absorption spectra, not only in the ultraviolet region, but also in the visible region; such spectra correspond to those observed in va- nadium complexes in oxidation states of +3 and +4. Introduction The unusual ability of ascidian blood cells to accumu- late vanadium in excess of one million times its level in seawater has attracted the interest of investigators from various fields of study. In particular, the chemical form of the vanadium complex present in ascidian blood cells Received 29 December 1989; accepted 24 May 1990. has long been a subject of discussion. Sometime after Henze's first discovery of vanadium in ascidian blood cells (Henze, 191 1), the element was believed to occur as part of a sulfated nitrogenous compound known as haemovanadin (Califano and Boeri. 1950; Webb. 1956; Bielig el a/.. 1966). Kustin's group claimed that haemo- vanadin is an artificial product generated by air oxida- tion (Kustin etai, 1976; Macaraet al., 1979a, b). The vanadium ion dissolved in seawater seems to exist as the vanadate(V) anion in the +5 oxidation state (McLeod et a!., 1975), but this is still experimentally un- resolved (Biggs and Swinehart, 1976). The vanadium ion contained in ascidian blood cells is, however, reduced to +4 or +3 oxidation states, i.e., vanadyl cations (cf. Mi- chibata and Sakurai. 1990). It has therefore been as- sumed that agents causing the reduction of vanadate ion to vanadyl ion must occur within ascidian blood cells and there bind with the vanadium ion. Kustin's group isolated a tunichrome from ascidian blood cells: it was proposed as being involved in the accumulation of vana- dium and in its reduction from seawater (Macara et a/., 1979a, b; Bruening et al., 1985). However, there is still no evidence that this tunichrome fulfills those functions in vanadium-containing blood cells (vanadocytes). Fur- thermore, the fluorescence due to the tunichrome is cer- tainly not detected in the signet ring cells that have been identified as the vanadocytes (Michibata et al., 1988, 1990a). While extracting tunichrome, Gilbert et al. ( 1 977) and Macara et al. (1979a) found a vanadium-containing band upon Sephadex column chromatography, but they continued to focus mainly on characterizing the tuni- 140 VANADOBIN FROM ASCIDIANS 141 chrome. More recently, in contrast, while characterizing a vanadium-binding substance extracted from the blood cells of Ascidia xydneienxix xumcti OKA under acidic- conditions, we showed that this substance, named vana- dobin, could maintain the vanadium ion in the vanadyl form (VO(IV)), had an apparent affinity for vanadium ion, and contained a reducing sugar (Michibata el ai, 1986a). The present investigation was designed to determine whether vanadobin could be extracted from the blood cells of other ascidians that contain significant amounts of vanadium and, thus, to ascertain whether vanadobin is a universal characteristic of blood cells in vanadium- containing ascidians. In fact, vanadobin was extracted from the blood cells of all ascidian species examined. Furthermore, the vanadobin extracted from A. gemmata shows an absorbance in the visible range resembling that of a vanadium compound. Materials and Methods Ascidia ahodori OKA, A. zara OKA, and dona intes- tinal is (LINNE) were collected from the Ushimado Marine Biological Station of Okayama University in Ushimado, Okayama Prefecture, Japan. A. gemmata SLUITER was obtained from the Asamushi Marine Bio- logical Station of Tohoku University in Asamushi, Ao- mori Prefecture. Corellajaponica HERDMAN was gath- ered in Yamada Bay near the Ootsuchi Marine Research Center, Ocean Research Institute of the University of Tokyo in Ootsuchi, Iwate Prefecture. Blood from each species was collected by cardiac- puncture under an anaerobic atmosphere of nitrogen gas to preclude air-oxidation; subsequent techniques were also carried out under the same conditions. The blood cells were separated from the blood plasma by centrifu- gation at 3000 X g for 20 min at 4C, and were pooled at -80C before use. A 10 mA/glycine-HCl buffer solution at pH 2.3 was added to the cell pellet, and the suspension was then ground in a glass-Teflon homogenizer at 4C. For A. gemmata and Corel/a japonica. glycine-HCl buffer was substituted with a 50 mA/ HC1 solution throughout the experimental process. The homogenate was loaded onto a column of Sepha- dexG-15 (Pharmacia Fine Chemicals). The column size and the chromatographic parameters used with each spe- cies is described in the appropriate figure legend. The col- umn was equilibrated with the glycine-HCl buffer solu- tion, and the elutant was collected in 3 ml or 5 ml frac- tions with monitoring for UV absorbance. The amounts of vanadium in each blood cell pellet and in the fractions were measured by neutron activation analysis or atomic absorption spectrometry. For neutron activation analysis, the elutant of each fraction was re- packed in a polyethylene capsule and irradiated with thermal neutrons having a flux of 5 x 10" n/cnr X s~' for 2 min in a TRIGA MARK II nuclear reactor at the Institute for Atomic Energy, Rikkyo University, Yoko- suka, Japan. The radioactivity of 52 V produced in the ir- radiated sample was measured with a 50-cm 3 Ge(Li) -y- ray spectrometer (Canberra Inc.) 2 min after the irradia- tion. Using a photon energy for 5: V of 1432KeV, the amount of vanadium in the sample was determined by comparison with that of a standard, as described pre- viously (Michibata et ai, 1986b). Flameless atomic ab- sorption spectrometry was applied to the samples that contained relatively higher amounts of vanadium. For these measurements we used a Hitachi GA-2 with a graphite furnace, and an absorption line of 3183.9 A was used for vanadium determination. The absorbance of fractions observed to contain vana- dium was measured with a spectrophotometer (Hitachi U-32 10); the fractions were then pooled and loaded onto a column of SE-52 (Serva Feinbiochimica) for ion-ex- change chromatography (Whatman W. & R.) and, thus, for further purification. Non-absorbed substances could be washed off the column with a sufficient volume of 10 mA/glycine-HCl buffer, and the purer vanadobin could then be eluted with a linear gradient of KC1, from to 0.5 M in the buffer solution. The vanadium content in each fraction obtained was also measured by the meth- ods described above, and absorbance was monitored with a spectrophotometer. We examined whether a simulated absorption spec- trum of vanadobin from A. gemmata could be repro- duced using spectra of inorganic vanadium complexes. Vanadium(III) sulfate (V : (SO 4 ),) was dissolved in 30 mA/ H : SO 4 , in a concentration of 40 mA/ at pH 1 , under an anaerobic atmosphere of argon gas, and vanadi- um(IV) oxide sulfate (VOSO 4 ) was dissolved in distilled water (10 mA/, at pH 2.0 to 3.5 adjusted with 1 A/HC1). Each absorbance was measured with a spectrophotome- ter, and each molar extinction coefficient was calculated. Based on these data, a spectrum closely resembling that of vanadobin was calculated. Results Ascidia gemmata SLUITER This species contained a higher level of vanadium in its blood cells than any other species examined (8.75 ^g/ mg wet weight) (Table I). Figure 1 shows the elution pro- file that resulted when a homogenate of the blood cell pellet ( 1 .64 g wet weight) of A. gemmata was loaded onto Sephadex G-15 and eluted. A large peak in fractions 8 through 22 was observed. As shown in Figure 2. the spec- 142 H. MICHIBATA ET AL Table I Extraction process ofvanadohmfrom the Mood cells of several ascidian species through chromatography Pellet of blood cells SephadexG-15 SE-cellulose Wet weight (mg) V content (Mg) V content (Mg) Abs. max. (nm) V content (Mg) Abs. max. (nm) Ascidia gemmata 1640.0 14,350.0 13,043.5 245.410,756 8347.8 238. 756 (100.0%) (91.0%) (64.0%) Ascidia ahodori 49.7 101.4 62.5 254 25.9 232 (100.0%) (61.6%) (25.5%) Ascidia :ara 40. 1 140.5 30.6 236 (100.0%) (21.3%) Corel la japonica 13,900.0 269.0 236.0 262 222.8 260 (100.0%) (87.7%) (82.8%) Ciona intestinalis 86.3 22.5 6.9 246 (100.0%) (30.7%) V content: vanadium content, Abs. max.: absorption maximum. The recovered vanadium through the chromatography is expressed as percent of the initial amount in the pellet of blood cells in parentheses. trum of the peak fraction ( fraction 1 3) had a clear ultravi- olet peak at 245 nm, and absorption peaks at 410 nm and 756 nm in the visible range with a shoulder at 620 nm. The elution profile of vanadium coincided with the peak of absorbance. The total vanadium content de- tected in fractions 8 through 20 was 1 3043.5 ng. The fractions from Sephadex G-15 column chroma- tography that contained vanadium were loaded onto a column of SE-52, providing the profile shown in Figure 3. By eluting with a gradient of KC1, several small peaks containing no vanadium were washed of? the column, and then a sharp peak containing vanadium was ob- g I E s 10 - 10 15 20 25 Fraction Number 30 35 Figure 1. Elution profile of the blood cell homogenate of Ascidia gemmata on Sephadex G-15 column chromatography. Pelleted blood cells ( 1 .64 g) were homogenized in about 50 mM HCI buffer solution, which was adjusted at pH 2.3. The homogenate (14 ml) was loaded onto a column (1.5 cm x 48 cm) and eluted with the same solution in 5-ml fractions. The total bed volume (Vt) and the void volume (Vo) of the column were 85 ml and 39 ml, respectively. The elution volume ( Ve) of vanadobin was 65 ml. tained in fractions 199 through 216. The amount of va- nadium recovered was 8347.8 ng. The peak fraction (fraction 205) exhibited absorption maxima at 236 nm and 756 nm, as shown in Figure 4. A. gemmata was the only species in which vanadobin exhibited absorption peaks in the visible range. Absorption spectra ofvanadium(III) sulfate and vanadiwn(IV) oxide sulfate Absorption spectra of vanadium(III) sulfate and va- nadium(lV) oxide sulfate are shown in Figures 5a and b, respectively. The former had absorption peaks at 400 nm and 610 nm, and the latter a peak at 760 nm with a shoul- der at 625 nm. When vanadium(III) sulfate (V : (SO 4 ),) 20 03 0? 300 400 500 600 WavKength(nm) 600 Figure 2. Absorption spectrum of vanadobin eluted from Sephadex G-15 column chromatography. The spectrum was recorded for the peak fraction 12 shown in Figure 1. Absorbance at 245 nm, 410 nm. and 756 nm with a shoulder 620 nm was observed. VANADOBIN FROM ASCIDIANS 143 175 185 195 Fraction Number Figure 3. Elution profile of the vanadium-containing substance (vanadob'm)ofAscidiagemmata on SE-cellulose column chromatogra- phy. Vanadium-containing fractions (45 ml) obtained after passing through Sephadex G-15 were loaded onto a column (3.7 cm $ ' 25 cm). After non-absorbed substances were washed off the column by elutmg with 50 mA/ HC1 solution at pH 2.3 in 5 ml fractions, the vana- dobin was obtained by elution with a linear gradient of KC1 at a concen- tration of 0.25 A/ and vanadium(IV) oxide sulfate (VOSO 4 ) were mixed together at concentrations of 4.8 mM and 5.9 mAl, re- spectively, an absorption spectrum closely resembling that of vanadobin after elution through Sephadex G-15 (Fig. 2) was obtained, as shown in Figure 5c. This simula- tion clearly suggested that the vanadobin of.^. gemntata, which waseluted from Sephadex G-15, contained vana- dium in the +3 and +4 oxidation states in the ratio of 45:55, whereas for the purer vanadobin eluting from SE- cellulose, the absorption peak at 410 nm disappeared, and a peak at 756 nm with a shoulder at 620 nm was seen (Fig. 4). These findings indicate that, although vana- dobin originally contains both vanadium forms in the + 3 and +4 oxidation states, vanadium in the +3 oxida- 40 30 200 250 300 500 600 Wavelength ( nm) 700 800 Figure 4. Absorption spectrum of vanadobin eluted from SE-cellu- lose column chromatography. The spectrum was recorded for the peak fraction 205 shown in Figure 3. Absorbance at 238 nm and 756 nm was observed. tion state becomes oxidized to the +4 oxidation state during the purification. Ascidia ahodori OK.4 When a homogenate containing 49.7 mg wet weight of the cell pellet was loaded on the Sephadex G-15 col- ( 6 10 02 300 400 500 600 700 Wavelength (nm) 800 900 Figure 5. Absorption spectra of vanadium complexes, a. Vanadi- um! Ill) sulfate (V,(SO 4 ) 3 ) was dissolved in 30 mA/H,SO 4 in a concen- tration of 40 mA/ at pH 1 under an anaerobic atmosphere of argon gas. Absorbance is expressed as (molar extinction coefficient), b. Vanadi- um! IV ) oxide sulfate ( VOSO 4 ) was dissolved in distilled water in a con- centration of 10 mA/ at pH 2.0 to 3.5. Absorbance was expressed as (molar extinction coefficient), c. Simulated spectrum, closely resem- bling that of the vanadobin shown in Figure 2, was obtained when va- nadium(III) sulfate (V 2 (SO 4 ) 3 ) and vanadium(IV) oxide sulfate ( VOSO 4 ) were mixed together at respective concentrations of 4.8 mA/ and 5.9 mA/. This simulation suggested clearly that the chemical forms of vanadium in the +3 and +4 oxidation states were approximately in the ratio 5:6 in the vanadobin of A. gemmata eluted from Sephadex G- 15 column (Fig. 2). 144 H. MICHIBATA ET AL 254nm 030 200 300 Wavelength ( nm ) 15 20 25 30 Fraction Number F'igure 6. Elution profile of the blood cell homogenate of Ascidia aliixlon on Sephadex G-15 column chromatography. Pelleted blood cells (49.7 mg) were homogenized in 10 mA/ glycine-HCl buffer solu- tion at pH 2.3. The homogenate ( 1.2 ml) was loaded onto a column (1.5 cm x 48 cm) and eluted with the same buffer solution in 3 ml fractions. Vt and Vo of the column were 85 ml and 39 ml, respectively. Ve of vanadobin was 63 ml. The inset shows the UV spectrum of the peak fraction (fraction 2 1 ). Absorbance at 254 nm was observed. umn, the elution profile obtained was somewhat more complicated (Fig. 6). A scan of UV wavelengths for frac- tion 21 (Fig. 6, inset) indicates that 254 nm is the peak of absorption. A total of 62.5 /^g of vanadium was eluted in fractions 17 through 26, and its peak was in frac- tion 21. As shown in Figure 7, when these vanadium-contain- ing fractions were loaded onto a column of SE-52, non- absorbed substances were first washed off the column by eluting with the glycine-HCl buffer solution, and a big peak with no vanadium was eluted with a linear gradient of KC1 dissolved in buffer solution. The vanadobin was obtained thereafter in fractions 54 through 58 with a peak in fraction 56. The vanadium content recovered was 25.9 ^g. The absorption peak, observed in fraction 56, was 232 nm (Fig. 7, inset). Vanadobin was extractable from the blood cells of three other ascidian species, A . :ara OKA, Corel/a japon- ica HERDMAN, and dona intcstimilis (LINNE); the procedures and results were similar to those described above (data not shown). The process of vanadobin ex- traction by chromatography is summarized in Table I. The highest vanadium content of 8750 ng/mg wet weight (14350 ng/1640 mg wet weight) in the blood cells was observed in A. gemmata. The next highest value was 2040 ng/mg wet weight detected in the blood cell pellet from A. ahodori. The blood cells of dona intestinal is had a much smaller content of 261 ng/mg wet weight of vanadium. Corella japonica, a vanadium-poor species. contained the smallest amount in its blood cells 19 ng/ mg wet weight). Recovery rates of vanadium ion in frac- tions eluted through Sephadex G- 1 5 were in the range of about 21-90%. The rates through SE-cellulose were in the range of about 26-83% of each initial amount. Peak wavelengths of UV absorbance in the peak fraction of Sephadex G-15 were observed between 246 nm and 262 nm, and the values observed in the peak fraction of SE- cellulose were shifted to shorter wavelengths, specifically to about 232 nm. Discussion Previous observations notwithstanding (Michibata et a/., 1986a), our present experiments show that vana- dobin extracted from ascidian blood cells does, in fact, exhibit clear absorption peaks in the visible range when its contained vanadium in vanadobin is at a significantly high concentration. In the case of A. gemmata, the con- centrations of vanadium in the peak fractions eluted from Sephadex G-15 and SE-cellulose columns were es- timated to be 12.3 mA/ and 5.3 mAf. respectively. The successful detection of this visible absorption is due to the high level of vanadium in the blood cells of A. gt'in- niata than any other ascidian, and to the extremely large volume of sample loaded onto the column (cf. Table I). Because the absorption spectrum of vanadobin from A. gemmata resembled those of vanadium compounds, an attempt was made to obtain a simulated spectrum. 232 nm 1 010 20 30 40 50 Fraction Number Figure 7. Elution profile of the vanadium-containing substance (vanadobin) of Ascidia ahodori on SE-cellulose column chromatogra- phy. Vanadium-containing fractions (15 ml) obtained after passing through Sephadex G-15 were loaded onto a column (1.2 cm x 12 cm). After non-absorbed substances were washed oft the column by eluting with 10 mA/ glycine-HCl buffer solution at pH 2.3 in 3 ml frac- tions, vanadobin was obtained by elution with a linear gradient of K.C1 (0. 1 A/). The inset shows the UV spectrum of the peak fraction (fraction 56). Absorbance at 232 nm was observed. VANADOBIN FROM ASCIDIANS 145 Indeed, the spectrum of vanadobin eluted from Sepha- dex G-15 (Fig. 2) can be reproduced when vanadium compounds in the +3 and +4 oxidation states are mixed together in a ratio of 45:55 (Fig. 5c). We therefore con- clude that vanadobin contains vanadium in the + 3 and +4 oxidation states in this ratio. On the other hand, the peak at 410 nm disappears from vanadobin after elution through SE-cellulose (Fig. 4) because vanadium(III) is oxidized to vanadium(IV) during the experimental pro- cess. The present report is the first to document an obvious absorption peak in the visible range for the native vana- dium complex, vanadobin. Previously, an absorbance peak in the visible range at 430 nm had been reported for a blood cell lysate ofAsciiiiu ohlujun (Boeri and Eh- renberg, 1954); the 430-nm peak was attributed to the hydrolyzed ion V(OH) : V 4t (or VOV 44 ). Brand et al. (1989) also found visible-range absorption of vanadium complexes from ascidian blood cells after addition of ex- ogenous ligands, 2,2'-bypyridine, 1,10-phenanthroline. and acetylacetone. Recent analysis of ascidian blood cells by NMR (nuclear magnetic resonance). EXAFS (extended X-ray absorption fine structure), and SQUID (superconducting quantum interference device) has suggested that the va- nadium is present predominantly in the +3 oxidation state, and that the +4 state accounts for less than 10% (Carlson, 1975;TulliustV/., 1980; Lee et al.. 1988). The present results confirm that data obtained from the visi- ble spectrum are also available for further determination of the valency of vanadium in cases where a high concen- tration of the metal is present in ascidian blood cells. The possibility has existed that vanadobin is an artifi- cial complex produced during the experimental process. That is to say, although the signet ring cell, among sev- eral types of ascidian blood cells, is clearly the vanadium- containing blood cell (the vanadocyte) (Michibata el al., 1987), the vanadium contained in the vanadocyte could well be mixed with a substance that was apt to bind with the metal contained in another cell type. Consequently, an artificial compound could be produced during ho- mogenizing or chromatography. However, we had pre- viously succeeded in excluding this possibility; vana- dobin could be extracted from a homogenate of a pure subpopulation of signet ring cells (the vanadocyte), but not from that of the morula cells. In these experiments we used a combination of cell fractionation for purifica- tion of a specific type of blood cell, chromatography for extraction of vanadobin, and neutron activation analysis for determination of vanadium (Michibata and Uyama, 1990). The present results clearly indicate that vanadobin is contained in the blood cells of all the ascidians exam- ined. In general, ascidians belonging to the family Ascidi- idae contain high levels of vanadium, whereas those be- longing to Cionidae (Michibata, 1984; Michibata cl al., 1986b) and Corellidae (Hawkins ct al.. 1983) do not al- ways have such high amounts of vanadium. The amount of vanadium, even in vanadium-poor ascidians, is, how- ever, thousands of times higher than that in other ani- mals, including mammals. We may suppose, therefore, that other kinds of complexes with vanadium must be present in ascidian tissues. The occurrence of vanadobin in the blood cells ofdona intestinalis and Corellajapon- ica suggests that vanadobin is a universal complex in as- cidian blood cells, and that its role is to accumulate and maintain vanadium. One of the reasons that previous attempts to obtain vanadium-binding substances failed might be that the pH of the buffer solution used in the extraction of vana- dium-binding substances was neutral. (There is utter con- fusion about the actual intracellular pH of ascidian blood cells.) Following Henze's first discovery of 1 N acidity ( Henze, 1 9 1 1 ), it was widely accepted that a homogenate of ascidian blood cells had a low pH value. However. Dingley et al. (1982) and Agudelo et al. (1983) claimed that the methods used in those early experiments gave spurious results because the cell interior containing the high levels of vanadium was most probably a highly re- ducing environment. Therefore, the possibility has not been eliminated that the intracellular redox potential, not the pH, was measured. When a new technique with an improved trans-membrane equilibrium of '^-la- beled methylamine was used, the intracellular pH was neutral. Hawkins's group also measured nearly neutral pH in ascidian blood cells by means ofa 3 'P-NMR (Haw- kins el al.. 1983; Brand et al., 1987). Conversely, Frank ct al. (1986) reported that the blood cells possess a pH value of 1 .8 based on ESR spectrometry. These previous studies were, however, carried out on whole blood cells, without cell fractionation, and were focused on the green-hued morula cells which had been considered va- nadocytes; not examined were the signet ring cells that have been newly identified as vanadocytes (Michibata et al., 1987). In fact, we have recently demonstrated that the separated subpopulations of signet ring cells in As- cidia ahodori, A. sydneiensis samea, A. genvnata, and Corella japonica show low pH values ranging from 0.8 to 3.1, and that they also contain high amounts of vana- dium, as determined by a combination of techniques in- volving cell fractionation, microelectrode measure- ments, and neutron activation analysis (Michibata et al., 1 990b). From this angle, it seems highly possible that va- nadium binds with organic substances within the signet ring cells (strictly speaking, in the vacuole), and there- fore, the previous failures in the extracting vanadium- 146 H. MICHIBATA ET AL. binding substances were due to conditions of pH. We also could not extract vanadobin under neutral condi- tions (data are not shown). Roman el al. (1988) have recently extracted metal complexes with organic substances from the blood plasma ofPyura chilensis and Ascidia dispar. These sub- stances seem to be heavier molecules than vanadobin be- cause they eluted through Sephadex G-75. The blood plasma has been proposed to contain transferrin-like metalloprotein, although the biochemical roles in ascid- ian blood are still unknown (Roman el al.. 1988). On the other hand, because vanadobin is estimated to be a low molecular weight substance (about 1300), and the con- centration of vanadium in the blood cells is 100 or more times greater than that in the blood plasma (Michibata el al.. 1986b; Roman et al.. 1988), these two types of metal binding substances probably have different roles in ascid- ian blood. The highest concentration of vanadium was contained in the blood cells of A. gemmata; this corresponds to 4,000,000 times that of seawater (<;/. Michibata, 1989). Strictly speaking, if almost all of the vanadium observed in the blood cells is contained in the vacuoles and binds with the vacuole membranes (Scippa et al.. 1988), its concentration should be much higher. Vanadobin, which can maintain the vanadium ion in the reduced form of +3 or +4 and has an affinity for exogenous vana- dium ion (Michibata et al., 1986a), must hold the key to resolving the specific accumulation of vanadium. There- fore, the next important study is to clarify the chemical structure of vanadobin. Acknowledgments We would like to express our heartfelt thanks to the staff of marine biological stations of Tohoku University (Asamushi), Okayama University (Ushimado), and the University of Tokyo (Ootsuchi) for supplying the materi- als and facilitating parts of our work. Thanks are also due to the Yamada Culture Center for Fisheries. This work was supported in part by Grants-in-Aid from the Ministry of Education, Science and Culture, Japan (#62540540, #01480026, and #01304007) and was sup- ported financially by the Japan Securities Scholarship Foundation, the Ito Science Foundation, and the Ta- mura Foundation for the Promotion of Science and Technology. Neutron activation analysis was carried out under the Cooperative Programs of the Institute for Atomic Energy of Rikkyo University. Literature Cited Agudelo, M. I., K. Kustin, and G. C. McLeod. 1983. 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McLeod, and K. Kustin. 1979b. Tunichromes and metal ion accumulation in tunicate blood cells. Comp. Bio- chem. Phvsiol. 63B: 299-302. McLeod, G. C., K. V. Ladd, K. Kustin, and D. L. Toppen. 1975. Extraction of vanadium(V) from seawater by tunicates: a revision of concepts. Limnol. Oceanogr. 20: 491-493. Michibata, II. 1984. Comparative study on amounts of trace ele- ments in the solitary ascidians, dona inieslinalis and dona m- busta. Comp. Biochem. Phvsiol. 78A: 285-288. Michibata, H. 1989. New aspects of accumulation and reduction of VANADOBIN FROM ASCIDIANS 147 vanadium ions in ascidians, based on concerted investigation from both a chemical and biological viewpoint. Zoo/. Sci. 6: 639-647. Michibata, H., T. Miyamoto, and H. Sakurai. I986a. Purification of vanadium binding substance from the blood cells of the tunicate, Ascidia sydneiensis samea. Biochem. Biophys Res Commun 141: 251-257. Michibata, H., T. Terada, N. Anada, K. Yamakawa, and I Numaku- nai. 1986b. The accumulation and distribution of vanadium, iron, and manganese in some solitary ascidians. Biol Bull 171: 672-681. Michibata, H., J. Hirata, T. Terada, and H. Sakurai. 1987. Separation of vanadocytes: determination and characterization of vanadium ion in the separated blood cells of the ascidian, Ascidia ahodori. J Exp Zooi 244: 33-38. Michibata, H., J. Hirata, T. Terada, and H. Sakurai. 1988. Autonomous fluorescence of ascidian blood cells with special refer- ence to identification of vanadocytes. Experientia 44: 906-907. Michibata, H., T. Uyama, and J. Hirata. 1990a. Vanadium-contain- ing blood cells (vanadocytes) show no fluorescence due to the tuni- chrome in the ascidian, Ascidia sydneiensis samea Zoo/. Sci 7: 55- 61. Michibata, H., Y. Iwata, and J. Hirata. 1990b. Isolation of highly acidic and vanadium-containing blood cells from among several types of blood cells from Ascidiidae species by density-gradient cen- tnfugation. J. Exp Zoo/ (in press). Michibata, H. and H. Sakurai. 1990. Vanadium in ascidians. In Va- nadium in Biological Systems. N. D. Chasteen, ed. Kluwer Acad. Pub . Dordrecht, (in press). 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. / Exp. Zool. 254: 132-137. Roman, D. A., J. Molina, and L. Rivera. 1988. Inorganic aspects of the blood chemistry of ascidians. Ionic composition, and Ti, V, and Fe in the blood plasma of Pyiira chi/ensis and Ascidia dispar. Biol Bull 175: 154-166. Scippa, S., K. Zierold, and M. de Vincentiis. 1988. X-ray microana- lytical studies on cryonxed blood cells of the ascidian Phallusia mammillata, II. Elemental composition of the various blood cell types. J. Submicrosc. Cyloi Palhol. 20: 719-730. Tullius, T. D., W. 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. Webb, D. A. 1956. The blood of tunicates and the biochemistry of vanadium. Publ. Sta:. Zool Napoli 28: 273-288. Reference: Biol. Hull 179: 148-158. (August, 1990) Diffusion Limitation and Hyperoxic Enhancement of Oxygen Consumption in Zooxanthellate Sea Anemones, Zoanthids, and Corals 1 J. MALCOLM SHICK Department of Zoology and Center for Marine Studies, University of Maine, Orono, Maine 04469-0146 Abstract. Depending on their size and morphology, anthozoan polyps and colonies may he diffusion-limited in their oxygen consumption, even under well-stirred, air-saturated conditions. This is indicated by an en- hancement of oxygen consumption under steady-state hyperoxic conditions that simulate the levels of O 2 pro- duced photosynthetically by zooxanthellae in the hosts' tissues. Such hyperoxia in the tissues of zooxanthellate species negates the effect of the diffusive boundary layer, and increases the rate of oxygen consumption; thus, in many cases, the rate of respiration measured under nor- moxia in the dark may not be representative of the rate during the day when the zooxanthellae are photosynthe- sizing and when the supply of oxygen for respiration is in the tissues themselves, not from the environment. These results have implications in respirometric methodology and in calculating the rate of gross photosynthesis in en- ergetic studies. The activity of cytochrome c oxidase is higher in aposymbiotic than in zooxanthellate speci- mens of the sea anemone Aiptasia pulehe/la, and this may indicate a compensation for the relative hypoxia in the tissues of the former, enhancing the delivery of oxy- gen to the mitochondria from the environment. "... [zooxanthellae] certainly provide abundant supplies of oxygen, without which ii is jv&l possible that such immense aggregations of living matter which constitute a coral reef. . . could not originate and flourish. " C.M. Yonge(1930) Received 30 January 1 990; accepted 16 May 1990. 1 Presented at the 5th International Conference on Coelenterate Biol- ogy, Southampton. England. July 1989. Introduction The relatively weak oxyregulatory ability apparent in most anthozoans stems in part from their scant ability to create bulk flow in the seawater surrounding them. In the laboratory, therefore, oxyregulation depends on the amount of convection provided by the experimental ap- paratus. Under well-stirred conditions, the diffusive boundary layer at the body surface will be thin, and tur- bulence especially will reduce diffusion gradients. At lower current speeds, the boundary layer thickens, and oxygen uptake becomes more diffusion-limited; this effect is pronounced at low-to-intermediate oxygen par- tial pressures, so that there is a marked effect of convec- tion on apparent oxyregulatory ability (see Dromgoole, 1978). The earliest studies of the effect of oxygen partial pres- sure (P J on the rate of oxygen consumption in antho- zoans were made with little or no stirring of the medium, and indicated little or no oxyregulation by Actinia eqitina, Anemonia viridis (=A. sulcata) (Henze, 1910), several scleractinian corals (Yonge et a/.. 1932). Calli- actis parasitica, and several pennatulids (Braneld and Chapman. 1965). Under well-stirred conditions, the rate of oxygen consumption by most anthozoans increases curvilinearly with P ,. Recently obtained curves are hy- perbolic, and most approach an asymptote at air satura- tion (cf. Mangum and Van Winkle, 1973; Sassaman and Mangum. 1972, 1973, 1974; Shumway, 1978; Ellington, 1982; Tytler and Davies, 1984). In such studies, the rate is often explicitly or implicitly assumed to reach a pla- teau at air saturation (20.95% O : , corresponding to 21.23 kPa at one atmosphere), although Mangum and Van Winkle ( 1973) emphasized that the assumption of a plateau in the commonly used hyperbolic model is not always realized in the data. The assumption has not been 148 HYPEROX1A AND RESPIRATION IN ANTHOZOANS 149 tested in anthozoans, except by Henze's (1910) experi- ments, where a hyperoxic enhancement of oxygen con- sumption is evident, albeit under apparently unstirred conditions. Similarly, investigators of coral productivity assume that respiration proceeds at the same rate in daylight and at night. But when the illuminated zooxanthellae in sym- biotic anthozoans are photosynthesizing, oxygen levels in their tissues rise well above air saturation (D'Aoust et al.. 1976; Grassland and Barnes, 1977; Dykens and Snick, 1982). Most concerns about the high oxygen lev- els in the tissues have centered on possible photorespira- tion or inhibition of photosynthesis in the zooxanthellae ( Black ctal.. 1976; Downton etui.. 1976), or on potential oxygen toxicity in the host (D'Aoust et ui. 1976; Dykens and Shick. 1982; Shick and Dykens, 1985) and zooxan- thellae (Lesser and Snick. 1989). A role for the zooxanthellae as endogenous providers of oxygen to the host during environmental hypoxia was shown by Shick and Brown ( 1977), following the demon- stration that zooxanthellae affect the spacing between clonal anemones (Fredericks, 1976). Moreover, in- creased convection in the air-saturated medium in- creases the rate of oxygen consumption in sea anemones, octocorals, and scleractinian corals (Dennison and Barnes, 1988; Patterson and Sebens, 1989), which im- plies that a diffusive boundary layer exists and that it im- pedes the delivery of oxygen to the tissues, even under well-oxygenated conditions. Thus, depending on the ex- tent of external convection which itself may vary within a coral colony, depending on its hydrodynamic porosity (Chamberlain and Graus, 1975) oxygen gen- erated photosynthetically within the tissues might well negate the effects of the boundary layer and elevate respi- ration above that measured in darkness at air saturation. The present paper reports the effects of hyperoxia on oxygen consumption in zooxanthellate anthozoans; the degree of hyperoxia used is within the range known to occur in the tissues of the animals (see Dykens and Shick, 1982). Experimental subjects were chosen to exemplify a range of the size and morphological complexity of pol- yps, and the growth form of colonies. Finally, the maxi- mum activities of cytochrome e oxidase are presented for zooxanthellate and aposymbiotic (lacking zooxanthel- lae) specimens of Aiptasia pulchella maintained under different levels of illumination and oxygenation. Materials and Methods Specimens of the sea anemone Aiptasia pallida and the zoanthid Zoanthus sociatm were collected in the vi- cinity of the Bermuda Biological Station and were main- tained in the station's seawater system prior to use in experiments. Steady-state measurements of oxygen consumption in the dark were made in a BioMetric- CYCLOBIOS twin-flow microrespirometer fitted with Orbisphere model 2120 polarographic oxygen sensors. Millipore-filtered (0.45 nm pore size) seawater (37%oS, 25C) entering the 50 cm 3 animal chamber was equili- brated sequentially with O : :N ; mixtures of 21%:79% (normoxia), 50%:50% (hyperoxia), normoxia again, and 10%:90% (hypoxia) using Tylan FC-260 mass-flow con- trollers. Perfusion of the animal chamber via an LKB MicroPerpex peristaltic pump was varied between 25 and 75 cm 3 IT 1 , to maintain an oxygen reduction ratio (see Gnaiger. 1983) of about 2-6% between the sensors measuring the oxygenation of seawater entering and leaving the chamber. Water in the chamber was well mixed with a magnetic stirrer situated beneath a perfora- ted plate to which the specimen was attached. Stirring speed was 200 rpm, the maximum that could be used without causing the anemone to collapse or contract. Measurements at each oxygen level were continued for at least 4 h. Values were corrected for blank oxygen con- sumption at each P Q ^. In a subsequent experiment, a specimen of Aiptasia pallida was placed in the 3.5 cm 3 perfusion cell of a Ther- moMetric 2277 Thermal Activity Monitor in series with the twin-flow microrespirometer, both regulated at 20C. The chamber was perfused at a flow rate of 27 cm 3 h~' for 9 h with 30%oS seawater equilibrated with 21% O 2 : 79% N : , and for an additional 4 h with seawater equili- brated with 50%. O 2 :50%i N 2 . Simultaneous fluxes of met- abolic heat and oxygen were continuously monitored and analyzed as described in Gnaiger et al. (1989). The clownfish sea anemone Heteractis crispa, colonies of the zoanthids Palythoa tuherculosa and an unidenti- fied species of Protopalythoa, and the scleractinian coral Stylophora pistillata were collected from Davies Reef on the Great Barrier Reef, Australia. The sea anemone Pliyl- lodisciis semoni was taken from the fouling community in the seawater system at the Australian Institute of Ma- rine Science. Oxygen consumption by H. crispa. by the zoanthids and coral, and by large specimens of P. semoni was measured in the dark in a closed respirometer (2.3 dm 3 ) fitted with a Radiometer E5046 oxygen sensor con- nected to a Radiometer PHM72 Mk2 acid-base analyzer. The specimen was placed on a perforated platform above a large magnetic stirrer operated continuously at 500 rpm, the highest speed that did not disturb P. semoni. Polyps of the colonial anthozoans generally remained ex- panded under this stirring regime. Each specimen was placed in the respirometry vessel, and the seawater (32%o-34%oS, 30C) bathing it was bubbled with a mix- ture of 55% O 2 :45%. N 2 delivered by a Wosthoff type SA/ 18 gas mixing pump for at least one hour before the chamber was sealed and measurement of oxygen con- sumption begun. This equilibration period was intended to eliminate transient, diffusional redistribution of oxy- gen in which uptake of oxygen by the relatively hypoxic 150 J. M. SHICK body fluids or skeletal pore water of a specimen trans- ferred acutely from the tanks of air-saturated seawater to the respirometer might be interpreted as an initially high rate of consumption of oxygen (see Dromgoole, 1978). Dur- ing the measurements, the specimen was allowed to deplete the oxygen in the respirometer to just below air saturation; the chamber was then flushed, reequilibrated with 55% O 2 for one hour, and the measurements repeated. In cases where the two measurements of oxygen consumption over a particular range of P , (either hyperoxia 46.7-45.4 kPa, or normoxia 21.2-20.0 kPa) did not agree to within 10%, the experiment was performed a third time, and the rates of oxygen consumption at both oxygen levels were calculated as the mean of the three measurements at each level. Respiration rates at each oxygen level were corrected for the blank which, in the case of Protopalythoa sp., in- cluded oxygen uptake by the substrate from which the pol- yps were removed after the experiment. The mass of the specimens was variously measured at the end of the respiration experiments, the particular measure being largely a matter of convenience. For P. tuberculosa and Protopalythoa sp., blotted wet weight ( W H') was used, whereas freeze-dried weight ( A W) was used for Z. sociatus and P. senioni. The protein content in individual A. pallida was measured by the microbiuret method with bovine serum albumin standards, and in whole colonies of S. pistillata by the Bio-Rad Coomassie dye-binding method with bovine gamma globulin stan- dards. As an index of the hydrodynamic porosity of colo- nies of 5. pistillata, the ratio of the mean distance be- tween nearest neighbor branches, to the mean branch di- ameter, was calculated (Chamberlain and Graus, 1975). Clonal cultures ofAiptasiapulchella (obtained from L. Muscatine, University of California. Los Angeles) were maintained in artificial seawater (Instant Ocean, 30%oS) at 25C. Groups of zooxanthellate anemones were ex- posed to irradiances of 85 (Dim) or 420 (Bright) m~V under the beam of a Kratos SS1000X 1 kW xe- non arc solar simulator (air mass 1 filter). One culture of aposymbiotic anemones (Apo) was maintained in con- tinuous darkness in air-saturated seawater, and another group of aposymbiotic specimens was maintained in the dark in seawater continuously bubbled with 50% O 2 ( ApoHiO : ). After two weeks of acclimation to these con- ditions, individual anemones were homogenized (10% w/v) in 100 mM potassium phosphate buffer (pH 7.0). Homogenates were centrifuged at 500 X g for 20 min to remove intact zooxanthellae and animal debris, and cytochrome c oxidase (EC 1.9.3.1) in the animal super- natant was assayed at 25C according to the modified method of Hansen and Sidell ( 1983), using reduced cyto- chrome c 1 (Sigma Type III). Results Specific rates of oxygen consumption (^mol O 2 g 'h ' on the basis of wet or dry weight, or nmol O : mg protein ' h ' ) by the several species under various oxygen regimes are given in Figures 1-4. Interspecific comparisons of specific rates are not meaningful in the present experi- ments, owing to the different measurements of mass, which are further complicated by the variable amounts of inorganic material (e.g.. sand) in the coenenchyme of the zoanthids. The rate of oxygen consumption in Aiptasia pallida increased at a partial pressure of oxygen approximately twice air saturation (Fig. 1 ). Although slight, the 1 1% en- hancement of respiration at 50% O 2 is statistically sig- nificant (paired / = 4.04, df = 4. P = 0.016). This is be- cause most of the variance seen in Figure 1 occurred be- tween specimens; all individual anemones showed higher rates of oxygen consumption at 50% than at 21% O 2 , which accounts for the significance seen in the paired /-test. The direct calorimetric experiment confirmed that the elevated rate of oxygen uptake reflected an increase in aerobic energy metabolism, as the steady rate of heat dissipation by the anemone increased from 30 ^W at 2 1 % O 2 to 33 \i W at 50%. O 2 (Fig. 1 , inset), a 1 0% rise that closely matched the independent respirometric results. When rates of both heat dissipation and oxygen con- sumption were steady, the calorimetric-respirometric (CR) ratio was 0.451 fJpmor 1 O 2 at 21% O 2 , and 0.463 jd pmol ' O 2 at 50% O 2 . Neither of these values differs significantly from the theoretical oxycaloric equivalent of 0.450 juJ pmor 1 O 2 for fully aerobic metabolism (Gnaigert'/fl/., 1989). The much greater (43%) enhancement, by hyperoxia, of respiration in Phyllodiscus semoni (Fig. 2A) is likewise highly significant (paired t = 6.25, df = 3, P = 0.008). The rate of oxygen consumption in the specimen of Heteractis crispa increased by 26% during hyperoxia (Fig. 2B). Sample sizes for the zoanthids are small (only one or two specimens of each species). Recall, however, that there are two or three measurements (continuous mea- surements over four hours, in the case ofZoanthus socia- tus) of oxygen consumption in each species at each oxy- gen level, so that although this pseudoreplication does not permit statistical analysis, any observed difference in oxygen consumption with P , is real. Morphologies of the three species are shown in Figure 3, together with the data. Hyperoxic enhancement of respiration in Palythoa tuberculosa averaged 46% (79% in one colony and 23% in a second), whereas the average of repeated measure- ments on one colony of Protopalythoa sp. indicated a slight (8.6%) decline at 50%. O 2 . Long-term measure- ments on Z. sociatus revealed a 2 1 % increase in a colony of five closely spaced individuals, but essentially no effect (3% increase) of hyperoxia on oxygen consumption in a single polyp subsequently isolated from the colony. Data are presented separately for two ecomorphs of the scleractinian coral Stylophora pistillata, shown in HYPEROXIA AND RESPIRATION IN ANTHOZOANS 151 90 - 03 Co o o en 6| 60 - 30- Aiptasia pallida 10 10.13 9 Time(h) 21 21.23 Oxygen level I 50 50-66 kPa Figure 1. Rates of oxygen consumption in Aipui-fiu pn/luhi (n = 5; mean size = 2.862 mg protein, range = 1.37-4.18 mg protein) under conditions of hypoxia ( 10% O 2 at inflow to respirometer), normoxia (21%O 2 ), and hyperoxia (50% O : ). Vertical lines indicate 1 standard error. Inset: instantaneous heat flux (,(3. /jW) in a specimen of .-(. pallida (0.788 mg protein) exposed to 21% O ; and 50%- O 2 in an open-flow calorimeter. Figure 4. Two colonies of the ecomorph having thin, widely spaced branches consistently showed a slight (9.0% and 7.5%) decrease in respiratory rate under hy- peroxia, whereas three colonies having thick, closely spaced branches showed a mean 20% hyperoxic en- hancement of respiration that was significant (paired / = 5.69, df = 2, P = 0.030). The magnitude of the effect of hyperoxia on oxygen consumption in S. pisiillata seems to be inversely related to the hydrodynamic poros- ity of the colony (Fig. 5). Rates of cytochrome c oxidase activity in the various groups ofAiptasia pulchella are shown in Figure 6. Spe- cific activity is expressed in Units per mg protein in the supernatant, each Unit corresponding to 1 ^mol cyto- chrome c oxidized per minute. Analysis of variance indi- cated a significant effect of treatment on enzymatic activ- ity (F= 7.942, df = 3, 16. P = 0.0018). Individual means were compared using the Student-Newman-Keuls test with a significance level of 0.05. Discussion The available studies of effects of hyperoxia on the rate of oxygen consumption in fishes and aquatic inverte- brates indicate no enhancement (and perhaps a slight re- duction) of the rate relative to that under normoxia. This is largely due to decreases in the ventilatory convection requirement under hyperoxia (Dejours and Beeken- kamp, 1977; Toulmond and Tchernigovtzeff, 1984; Ber- schick et ul.. 1987), and to the presence of respiratory pigments, both of which stabilize the delivery of oxygen to the tissues over a wide range of external f ,. Lacking respiratory pigments, and having only weak powers of convection of the external medium, cnidarians are more at the mercy of the Pick equations for diffusive gas ex- change. As such, their respiratory exchange must be markedly affected by the flow regime they occupy, and in the case of species harboring algae, by provision of CK from those photosynthetic symbionts. Many anthozoans, especially sea anemones, show be- havioral compensations for varying levels of water movement and environmental oxygen supply. A positive relationship exists between the degree of inflation of the hydrostatic skeleton and current velocity in Metridium senile (Robbins and Shick, 1980). Although this seems primarily related to prey capture in this suspension feeder, full extension of the column and tentacles also simultaneously maximizes the surface-to-mass ratio and minimizes diffusion distances within the tissues, and thus maximizes oxygen delivery and hence the rate of oxygen consumption (see Shick et at.. 1979). Behaviors that increase the surface area and decrease diffusion distances in the primary gas exchange surfaces are also seen as adaptive short-term responses to hypoxia 152 J. M. SHICK A. Phyllodiscus semoni 60 I ill i: 7 40 111 T3 0) H r^ iiii o 20- n liii|i 25-i 20- 15- T3 cn _ 10 o E 5 - NORMOXIA HYPEROXIA B. Heteractis crispa ; mm :::;:. NORMOXIA HYPEROXIA Figure 2. (A) Rates of oxygen consumption in Phyllodiscus semoni (n = 4; mean A W = 0.661 g, range = 0.599-0.725 g) under normoxia (20.0-21.2 kPa O : ) and hyperoxia (45.4-46.7 kPa O 2 ). Vertical lines indicate 1 standard error. (B) Rates of oxygen consumption in a 2.898 g d I) ' aposymhiotic specimen of Heteractis crispa under normoxia and hyperoxia. in several species of sea anemones and ceriantharians (SassamanandMangum, 1972, 1974;Shick etui, 1979). Conversely, the zooxanthellate sea anemones An- t hop/ f lira elegant issima and A. xanthogrammica con- tract under peak levels of solar irradiance, a response that seems related more to avoiding the damaging photody- namic effects of interacting ultraviolet radiation and hy- peroxia, than to hyperoxia per se (Shick and Dykens, 1984). In the absence of UV, A. elegantissima remains expanded under moderate levels of irradiance that yield a net production of oxygen (and hence, tissue hyperoxia) by its zooxanthellae (Shick and Brown, 1977). Earlier studies on anthozoans centered on the respira- tory response to hypoxia and did not involve oxygen lev- els above air saturation. This is understandable, because most of the species that were studied do not contain zoo- xanthellae and would not experience hyperoxia, except perhaps in tidepools where free-living algae might pro- duce transient hyperoxia (see Truchot and Duhamel- Jouve, 1980). Nevertheless, inspection of published curves (see references in Introduction) suggests that, in most cases, an increase in respiration would occur under hyperoxia, and this has relevance particularly in zooxan- thellate species that routinely experience such oxygen levels in their tissues. The hyperoxic enhancement of oxygen consumption in Aiptasia pallida reported here is admittedly slight, but has consequences in the calculations of budgets of energy and carbon in the symbiosis. Specifically, because respi- ration under 50% O 2 (a level that occurs in the tissues of symbiotic anthozoans when their zooxanthellae are photosynthesizing: D'Aoust el a/., 1976; Dykens and Shick, 1982) is 1 1% higher than at normoxia, daytime respiration is underestimated when respiration is mea- sured at air saturation. Because the compensation irradi- ance (where respiration is balanced by photosynthesis) is less than 50 /umol m~ : s~', and the saturation irradiance for photosynthesis in the zooxanthellae is 200- m~ : s~' in Aiptasia spp. (Muller-Parker, 1984; Lesser and Shick, 1 989), and considering that irradiances in the hab- itats of Aiptasia spp. exceed 100 ^mol rrT'V for most of the daylight period (Muller-Parker, 1987; Lesser and Shick, unpubl. data), there is a net production of oxygen for most of the day. Thus, 50% CK is a realistic level of tissue oxygenation for this period (also see D'Aoust et ai, 1976). Therefore, daytime respiration in Aiptasia spp. has routinely been underestimated by = 1 1%; the appar- ent value of net photosynthesis is therefore misleading, and neglect of this would underestimate gross photosyn- thesis by a corresponding amount. In the natural habitat of A. pallida at Walsingham Pond, Bermuda, the diurnal increase in respiration ow- ing to hyperoxia is slightly offset each night, caused by a brief decline in seawater oxygenation to one-half or even one-third of air saturation (K. Eakins, pers. comm.). In- spection of Figure 1 indicates that this would result in about a 33% decline in respiration, for a period of about 3 h (Eakins, loc. cit.). Among the species studied here, Aiptasia pallida would be the least likely to be diffusion-limited in its res- piratory gas exchange, owing to its small size and mor- phological simplicity. In the sea anemones Phyllodiscus HYPEROXIA AND RESPIRATION IN ANTHOZOANS 153 A 0.6^ ra 0.3 6 1 o 2cm Palythoa tuberculosa NORMOXIA HYPEROXIA B ID, en 5 o 1 cm Protopalythoa sp. NORMOXIA HYPEROXIA CO o o 6- 3_ single polyp colony (5) 2cm, Zoanthus sociatus Figure 3. Rates of oxygen consumption in zoanthids. In colonies of (A) Palythoa tuberculosa (mean W H'= 47.25 g) and (B) Prolopalvthoa sp. ( W M' = 2.89 g), normoxia corresponds to 20.0-21.2 kPa O 2 and hyperoxia to 45.4-46.7 kPa O 2 . Vertical lines in (A) indicate 1 standard error. In Zoanthus sociatus (C), the group of 5 individuals weighed 0.0 1 1 g M ' and the single polyp 0.003 g d M '. Oxygen levels at the inflow to the respirometer were normoxia (21% O 2 ) and hyperoxia (50% O 2 ). 154 J. M. SHICK A 200- 150^ 8 | 100^ C\J O "o 50 5 cm porous ecomorph NORMOXIA HYPEROXIA B 250- ,_ 200 - Lc 'c " r . : -' :, f': 1 150- I Q. E I g 100- o 1 50- n - | 1 / mordax ecomorph NORMOXIA HYPEROXIA Figure 4. Rates of oxygen consumption in coloniesofStylophorapistillata under normoxia(20.0-21.2 kPa O 2 ) and hyperoxia (45.4-46.7 kPa O 2 ). ( A) In the hydrodynamically porous ecomorph (n = 2), average total colony protein was 369 mg. (B) In the mordax ecomorph (n = 3), mean total colony protein was 586 mg. Vertical lines indicate 1 standard error. Photographs by J.-P. Gattuso. semoni and Heteractis crispa, hyperoxia increases the respiratory rate more than in A. pallida. This seems to be related to their much larger size (5 to 10 cm, vs. 0.5 cm diameter) and perhaps to their greater morphological complexity. These factors might increase the boundary layer at the body surface, in the first case, because the thickness of the boundary layer around a cylindrical anemone is related to the square root of its diameter ( Vo- gel, 1983). In addition, water flow might be impeded among the numerous pseudotentacles in P. semoni and tentacles covering the oral disc of H. crispa. Such addi- tional roughness elements may also increase eddy cur- rents and increase the residence time of water in the boundary layer. The existence of such a boundary layer under conditions of forced convection is documented by the experiments of Patterson and Sebens (1989), who found that oxygen consumption by specimens ofMetrid- ium senile of about the same size as the Phyllodiscus in the present study increased two- to threefold as current speed increased from s7 to 15 cms '. Therefore, endog- enously produced oxygen (which need not negotiate an external boundary layer) is proportionally more impor- tant to large anemones that experience a larger boundary layer that develops even under well-stirred conditions, than to small anemones. Measurements of oxygen flux in large zooxanthellate anemones under normoxic con- ditions will accordingly underestimate their daytime res- piration (and so underestimate gross photosynthesis) more than in small anemones. Unlike unitary anemones, zoanthids form colonies of HYPEROXIA AND RESPIRATION IN ANTHOZOANS 155 E 9 CD Q_ rT > Q- X 50 40 30 20- 10 - - Stylophora pistillata 1 2 Porosity Figure 5. Relationship between the hyperoxia-indueed change in oxygen consumption and hydrodynamic porosity in colonies of Sty/it- phora pistillata. Porosity was calculated as the ratio of the mean dis- tance between nearest neighbor branches to mean branch diameter. Each point represents one colony. interconnected polyps that vary in their spacing and ag- gregate morphology, and thus in their hydrodynamic properties. Such variation has been discussed primarily with respect to the provision of food to these sessile filter feeders (f.#., Koehl, 1977), hut the hydrodynamic princi- ples apply to the delivery of O 2 as well. At one morpho- logical extreme, Palythoa tuherculosa forms platelike colonies of closely conjoined polyps embedded in a mas- sive, largely inorganic, coenenchyme. Water flow across the colony decreases with increasing distance from the periphery, as kinetic energy is extracted from the flow by skin friction and by form drag of the polyps; thus the interior polyps experience relatively stagnant conditions compared to their clonemates on the edge. Delivery of oxygen to respiring tissues is also impaired by the protec- tive coenenchyme. At the other extreme, Protopalyt/ioa sp. from Australia forms loosely aggregated colonies of tall, widely separated polyps connected only at their bases and having less coenenchyme. Accordingly, P. tuberculosa might be expected to be more diffusion-limited in its gas exchange, whereas Pro- topalythoa sp. would perform more like individual small anemones. These predictions are confirmed experimen- tally, because hyperoxia generates increases in oxygen consumption in P. tuberciilosa but not in Protopalythoa sp. (cf. Fig. 3A and B). Moreover, a group of closely spaced polyps of Zoanthus sociatus shows hyperoxic en- hancement of respiration, whereas a single small polyp responds more like a solitary Aiplasia pallida and shows minimal enhancement (Fig. 3C). The difference between individual and colonial respiratory performance in the last case seems to be related to a decrease in free surface area and restriction of water flow between polyps, and hence to a larger effective diameter, with the resultant decrease in water flow toward the center of the colony. Previous studies concerning physiological effects of water flow around scleractinian coral colonies have been focused primarily on provision of food and removal of waste, although Jokiel ( 1978) suggested that respiratory exchange is also affected. Dennison and Barnes (1988) and Patterson el al. (1990) subsequently demonstrated that respiration under normoxic conditions in Acropora formosa and Montastrea annularis does increase in mov- ing water. More to the present point, enhancement by light of calcification in Stylophora pistillata was sug- gested by Rinkevich and Loya ( 1 984) to be due to stimu- lation of (aerobic) metabolism by O : produced within the symbiosis by the zooxanthellae. Together these stud- ies indicate that a boundary layer to the delivery of oxy- gen exists even under normoxic conditions, and that un- der some circumstances its effects are negated by the pro- duction of oxygen within the host's tissues. Stylophora pistillata exhibits a particularly great diver- sity of colonial morphologies, to a large extent deter- mined by the flow regime where it occurs (Veron and Pichon, 1976), as well as by photic regime (McCloskey and Muscatine, 1984; Titlyanov, 1987). Chamberlain and Graus (1975) conclude that flow within a branching colony depends entirely on its morphology and on exte- rior hydrodynamic conditions. Therefore, the preva- lence of colonies of S. pistillata having thin, widely spaced branches in low energy habitats (Veron and Pi- chon, 1976; pers. obs.) seems related to the maintenance of adequate flow among the branches in such sheltered areas (and to the avoidance of self-shading in deep water or shaded sites). Although the thick, closely spaced branches of the mordax ecomorph of this species would seemingly restrict flow to the interior of the colony, this ecomorph inhabits high energy environments (Veron and Pichon, 1976; pers. obs.). Thus, the ecomorphs of S. pistillata exemplify the principle of dynamic simili- tude under the appropriate flow conditions, morpho- logically dissimilar colonies can have similar flow char- acteristics (Chamberlain and Graus, 1975) and respira- 0030-, 0024 o _E Q) V) II 0018 0012 Aiplasia pulche/la Apo Apo HiOn Bright Dim Figure 6. Cytochrome r oxidase activity in aposymbiotic and zoo- xanthellate specimens of Aiplasia pulchella (n = 5 in each treatment). Vertical lines indicate 1 standard error. Horizontal lines underscore groups whose means are not significantly different (P > 0.05). 156 J. M. SHICK tory rates. The corallites in this species are relatively shallow, so that diffusion distances within the corallum and tissues are short, and probably similar in the differ- ent ecomorphs. In the moderate, turbulent flow in the respirometer, colonies of the hydrodynamically porous ecomorph ex- perience no diffusion limitation, as hyperoxia does not result in an increase in oxygen consumption (Fig. 4A). Similar results are obtained with single branches of colo- nies (Shick, unpub. data; J.-P. Gattuso, pers. comm.), in which water flow and delivery of oxygen to the polyps is not hindered by any nearby branches. Colonies of the high-energy mordax ecomorph, however, do appear to be diffusion limited even under these well-mixed condi- tions, showing a 20% hyperoxic increase in respiration (Fig. 4B), probably owing to elevated O 2 levels among the interior branches of the colonies. Consequently, the magnitude of the effect of hyperoxia on respiration is in- versely related to the hydrodynamic porosity of the col- ony under these conditions (Fig. 5). Whether this diffu- sion limitation is more pronounced under unidirec- tional, laminar flow, or under the turbulent conditions in the present study is unknown, but this is testable with a flow tunnel respirometer. Compensation for different levels of oxygenation may also be manifested at the cellular level. Lacking zooxan- thellae, the tissues of aposymbiotic anemones are hyp- oxic relative to those of zooxanthellate conspecifics when the latter are photosynthesizing. In Aiptasia pulchclhi. this is associated with a significantly higher activity of cytochrome c oxidase (the terminal enzyme in the mito- chondrial respiratory chain) in aposymbiotic than in zooxanthellate clonemates. Such an elevation of mito- chondrial respiratory capacity in the relatively hypoxic anemones could result from more or larger mitochon- dria, greater specific activity of cytochrome c oxidase per mitochondrion, or a combination of these. Stereological studies of the numbers, distribution, and ultrastructure of mitochondria in anemones under these conditions are in progress. Increasing the numbers of mitochondria (and hence reducing the diffusion distance for oxygen from the cell surface to a respiring mitochondrion) is a correlate of intertidal hypoxic exposure in the anemone Anthopleura elegantissima (J. A. Dykens and J. M. Shick, unpubl. data). The data on aposymbiotic A. pulchella cultured under exogenous hyperoxia are consistent with this postulate. Its cytochrome c oxidase activity is intermediate to the high activity in relatively hypoxic aposymbiotic clone- mates and to the low values in its hyperoxic zooxanthel- late clonemates; in the latter case, the major source of oxygen for much of the time is endogenous, from the intracellular zooxanthellae. The lack of effect of irradi- ance (Dim vs. Bright) on cytochrome c oxidase activity in zooxanthellate anemones suggests that both groups (maintained at irradiances exceeding the compensation point) experienced similarly high tissue oxygenation dur- ing net photosynthesis. However, anemones maintained under bright light tend to have higher cytochrome c oxi- dase activity, which is in keeping with a lower photosyn- thetic oxygen production owing to their contraction dur- ing prolonged high irradiance (Shick, unpubl. data; see also Shick and Dykens. 1984). An alternative interpretation of the data on cyto- chrome c oxidase is that the activity of this enzyme (and that of the mitochondria! respiratory chain in general) might be lower in zooxanthellate than in aposymbiotic specimens because mitochondria experiencing high oxy- gen levels have the potential for elevated production of superoxide radicals. Much of the superoxide production in cells occurs via autooxidation of the respiratory chain components NADH dehydrogenase and ubiquinone (Turrens and Boveris, 1980), and its production in- creases with P , (Freeman and Crapo, 1981; Turrens el a!.. 1982); thus, total mitochondria! production of super- oxide radicals in a tissue is a function, both of the con- centration of respiratory chain components, and of P , (see also Shick and Dykens, 1985). Therefore, the high activity of cytochrome c oxidase in aposymbiotic Aipta- sia pulchella, compared with its symbiotic clonemates, may reflect not simply a compensation for hypoxia in the former, but also an avoidance of oxidative stress in the latter. The present study demonstrates that, depending on the size and morphology of the species being examined, respiration is variably enhanced by hyperoxia at a level that occurs in the illuminated tissues of zooxanthellate anthozoans, even under well-stirred, turbulent condi- tions that minimize the thickness of the diffusive bound- ary layer. Therefore, if the rate of respiration measured in the dark is to be taken as representative of that in the light, then dark respiration must, in some cases, be mea- sured in hyperoxic water. It should be noted that the im- position of exogenous hyperoxia does not affect the thickness of the boundary layer; rather, it steepens the diffusion gradient across the boundary and thus en- hances delivery of oxygen to the tissues. Such an en- hancement of oxygen delivery simulates the effect of pro- duction of oxygen within the tissues, a condition that prevails when the symbiosis is illuminated. Whether and how much this enhances oxygen consumption depends on the size and morphology of the polyps, the hydrody- namic porosity of the colony, and the amount and per- haps the nature (laminar or turbulent) of water flow. The elevation of respiration by hyperoxia. and the ob- servation that the symbiosis produces more oxygen than it consumes, emphasize that, during daylight, the rele- vant source of oxygen for respiration is endogenous. Therefore, water movement and the thickness of the diffusive boundary layer around the polyps or colony HYPEROXIA AND RESPIRATION IN ANTHOZOANS 157 may at that time be more relevant to the removal of en- dogenously produced oxygen, which, at high concentra- tions, inhibits photosynthesis in the zooxanthellae (Black el nl.. 1976; Downton ct a/.. 1976) and necessi- tates greater defenses against oxidative stress in the host and its symbionts (Shick and Dykens, 1985; Lesser and Shick, 1989). Elevation of respiration by hyperoxia per se occurs ir- respective of its possible further enhancement by photo- synthate translocated from photosynthesizing zooxan- thellae (Edmunds and Davies, 1988). Although the pro- vision of oxygen by the zooxanthellae to the host traditionally has been viewed as only supplementary, the current results suggest that the higher oxygen levels in the tissues of zooxanthellate cnidarians reduce the amount of respiratory apparatus (e.g., cytochromes) that the host maintains. This saving may be somewhat offset by the need for higher levels of defenses against oxygen toxicity in zooxanthellate individuals. Acknowledgments This work was supported by U. S. National Science Foundation grants DCB-8509487 (Regulatory Biology) and BBS-87 1 6 1 6 1 ( Biological Instrumentation), Wilkin- son, Lehman, and Riker Fellowships from the Bermuda Biological Station, a visiting research fellowship from the Australian Institute of Marine Science, and National Geographic Society research grant 3883-88. I thank D. W. Tapley for assembling and calibrating the twin- flow respirometer prior to my arrival in Bermuda, and for performing the protein analyses on Aiptasia spp.; B. E. Chalker and D. J. Barnes for providing laboratory space and facilities at AIMS; B. Clough for the loan of a Wosthoft pump, and M. Cuthill for photographic assis- tance, at AIMS; J. S. Ryland for identifying Pwtopaly- tfwa sp.; and W. K. Fitt, J.-P. Gattuso, E. Gnaiger, M. P. Lesser, M. R. Patterson, and two anonymous reviewers for critical comments on the manuscript. This is contri- bution number 1221 from the Bermuda Biological Sta- tion for Research. Literature Cited Berschick, P., C. R. Bridges, and M. K. Grieshaber. 1987. The influ- ence of hyperoxia, hypoxia and temperature on the respiratory physiology of the intertidal rockpool fish Gohius cohitux Pallas. J. Exp. Biol. 130: 369-387. Black, C. C., Jr., J. E. Burris, and R. G. Everson. 1976. Influence of oxygen concentration on photosynthesis in marine plants. Anst. J. 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A. vulgaris exhibits an annual repro- ductive cycle, i.e., the testes increase slowly in size during fall and winter, and reach maximal size in early spring. Slow testicular growth in the winter has been attributed to low field temperatures. Previous studies indicate that the specific activity of ornithine decarboxylase and the levels of the polyamines putrescine, spermidine, and spermine decrease in mid-winter and increase in the spring, coincident with changes in field temperatures. Kinetic studies show that ornithine decarboxylase as- sayed from individuals collected in March exhibits nega- tive thermal modulation (K m of ornithine is 0.22 mM and 0.65 mAl at 15 and 0C, respectively). Q, values are highest at low substrate concentrations and at low temperatures. We hypothesize that during the cold win- ter months a decrease in the amount of ODC and an in- crease in the apparent K m causes polyamine synthesis to decline, leading to decreased growth and development of the testis. We suggest that thermal modulation of ODC (and polyamine synthesis) is a mechanism by which sea- sonal temperature fluctuations influence seasonal sper- matogenesis in A. vulgaris. We further suggest that growth of various tissues in many other ectothermal in- vertebrates may be similarly controlled. Introduction The biogenic polyamines spermidine and spermine and their diamine precursor putrescine are organic cat- Received 19 April 1990; accepted 21 May 1990. 1 Present Address: Department of Biology, The University of Ala- bama at Birmingham, UAB Station, Birmingham, Alabama 35294 ions with multiple biological functions. Polyamines are known principally for their essential role in cell prolifera- tion and for interactions with anionic molecules such as nucleic acids and membrane phospholipids in mamma- lian cell lines and tissues, both normal and neoplastic. However, the exact mechanisms by which polyamines influence cell proliferation are not fully understood. Polyamine synthesis is controlled via the rate-limiting enzyme ornithine decarboxylase (ODC) ( 1 ). The activity of mammalian ODC varies in response to a wide variety of stimuli including growth factors, hormones, and drugs (2). The inducibility and short half life of ODC [<15 min, ( 1 )] suggest that the activity of the enzyme (and thus polyamine synthesis) is highly regulated. The activity and kinetics of mammalian ODC have been character- ized in numerous studies (reviewed by ref. 1 ). ODC has not been characterized for any invertebrate with the ex- ception of a partial kinetic analysis of the enzyme in a snail (3). The role of ODC and polyamines during cell prolifera- tion in invertebrates should be similar to cell prolifera- tion in mammals. However, relatively few studies have examined polyamine metabolism in the tissues of inver- tebrates. This is unfortunate because many inverte- brates, particularly larvae and juveniles, show high sea- sonal and yearly growth rates. Polyamines are present in tissues of several invertebrates, although their distribu- tion varies greatly among tissue types (4, 5, 6, 7, 8, 9, 10). In addition, polyamine levels increase significantly during periods of mitotic and meiotic cell proliferation in the testes of the sea star Asterias vulgaris (11). Cell proliferation in the testes, demonstrated by thymidine incorporation, also increases during exposure to extrinsi- cally applied polyamines (12). 159 160 S. A. WATTS ET AL 3.0- 0.0 2 04 06 0.8 ORNITHINE (mM) 1.2 Figure 1. Activity of ornithine decarboxylase measured at various substrate (ornithine) concentrations and at assay temperatures of 0, 5, and 1 5C. All assays were performed in duplicate. Many invertebrates are exposed to daily or seasonal changes in temperature and demonstrate temperature- dependent growth characteristics. Although temperature is often cited as an important modulator of growth, the mechanisms by which temperature-dependent growth is regulated remain obscure. We hypothesize that tempera- ture regulation of ornithine decarboxylase activity and polyamine synthesis may be one mechanism by which temperature regulates growth in ectothermal organisms. In this study we report the effect of assay temperature on the kinetic characteristics of ornithine decarboxylase extracted from the testes of A vulgaris. Materials and Methods Adult specimens of Asterias vulgaris (8-10 cm arm length) were collected from a depth of 3 meters at the mouth of the Piscatequa River in Portsmouth, New Hampshire, in March 1987. Testes were removed from three individuals and pooled for ODC extraction and analysis. Fresh testes were homogenized (20% w:v, 1:4) on ice with a glass Teflon homogenizing apparatus in a buffer containing 50 mM KH 2 PO 4 , pH 7.5, 0.2 mM EDTA, 5 mM dithiothreitol and 50 fiM pyridoxal 5-phosphate. The crude extract was centrifuged for 30 min at 20,000 X g at 0C. The supernatant was used for enzyme activity determinations. The specific activity of ODC was determined by a pro- cedure modified from Landy-Otsuka and Scheffler (13) and Smith (14). The specific activity of ODC was deter- mined by measuring the release of I4 CO 2 from DL-[1- 14 C] ornithine hydrochloride (CFA.423, Amersham, 58 mCi/mmol). The enzyme reaction was performed in a 16 mm (ID) borosilicate test tube capped with a double- seal rubber stopper (Kontes, K-8823 10) penetrated by a plastic centerwell (Kontes K-882320). The centerwell contained a 2 X 3 cm square of Whatman #1 filter paper saturated with 100 /il NCS tissue solubilizer (Amer- sham). First, 150 n\ of the above supernatant was added to each tube, and the reaction was initiated by adding 30 M l of 0.5 ^Ci DL-[1- 14 C] ornithine hydrochloride and cold L-ornithine (Sigma, final concentration of total L- ornithine, approximately 1.2 mM). The reaction was stopped after 90 min at 15C by injecting 0.5 ml 5% tri- chloroacetic acid into all tubes. These tubes stood for at least 1 h to permit maximum absorption of CO 2 . Control tubes used to determine endogenous 14 CO 2 release were prepared by adding first 0.5 ml TCA followed by 1 50 ^1 of ODC supernatant and 30 jul L-ornithine. The filter pa- per was removed and placed in scintillation vials con- taining 4.0 ml Beckmann NA scintillation fluid. The vials stood in the dark overnight so that the chemilumi- nescence would be reduced before the radioactivity was measured in an LKB Excel liquid scintillation counter. The activity of ODC was measured at seven concen- trations ranging from 0.0351 to 1.144 mM ornithine at assay temperatures of 0, 5, and 1 5C. These temperatures are within the normal range of temperature to which the sea stars are exposed during the year (-2 to 1 7C). All of the assays were performed in duplicate. Activities were expressed as nmoles ornithine converted to putrescine per h per g wet weight tissue. The apparent Michaelis constant (K m ) was determined from double reciprocal plots. Although potential problems may arise using crude supernatants to determine enzyme activity, rela- tive differences in enzyme activities should reflect biolog- ical differences with respect to changes in temperature. Results The activity of ODC increased hyperbolically with an increase in substrate (ornithine) concentration (Fig. 1). The affinity of the enzyme for ornithine decreased with the assay temperature at all substrate concentrations 6 1 o LU g i LU G i o 5 - 10 20 1/ORNITHINE (mM) 30 Figure 2. Double-reciprocal plot of the activity of ornithine decar- boxylase versus substrate concentration. All assays were performed in duplicate. THERMAL MODULATION OF ODC 16! C/5 O O cn < o 08n 06- 04- 0.2- 00 - 5 5 10 15 TEMPERATURE (C) 20 Figure 3. Relation of the apparent Michaelis contant (Km, mM). as determined from the double-reciprocal plot of the activity of orni- thine decarboxylase, and assay temperature. A decrease in the assay temperature caused an increase in K m and. therefore, a decrease in the apparent affinity of the enzyme for the substrate. tested, indicating temperature-dependent enzyme-sub- strate affinity parameters. The double reciprocal plots (Fig. 2) of ODC activity show that the assay temperature did not affect the maxi- mal velocity of ornithine decarboxylase, as V mav did not change with temperature between and 15C. An in- crease in the slope of the line was apparent with a de- crease in assay temperature, indicating an increase in the apparent K m . A plot of apparent K m versus temperature, illustrating negative thermal modulation (increased K m with a decrease in temperature), is shown in Figure 3. The Q 1U values for the ODC activities were calculated between both and 5C and 5 and 15C (Table I). Q,,, values were significantly higher (sign test, P < 0.05) at the low temperatures at all substrate concentrations, ranging from 2.21 to 2.59 between and 5C. Q 10 values were lowest at the high temperatures, ranging from 1.07 to 2.04 between 5 and 15C. Q K) values of approximately unity indicated that ODC activity was essentially tem- perature-independent at high substrate concentrations (1.14 mM ornithine) between 5 and 1 5C. These values show that ODC activity is temperature-dependent at low substrate concentrations and at low temperatures (re- gardless of substrate concentration), and temperature-in- dependent at high substrate concentrations at higher temperatures only. Discussion The effects of temperature on organismal behavior, physiology, and the biochemistry of some cellular pro- cesses has been well documented for many ectothermic invertebrates. At the cellular level, the influence of tem- perature on enzyme activities is varied and dependent on the interactions of substrates and cofactors, translational and transcriptional control of enzyme production, and a host of other factors such as the metabolic pathway, type of tissue, organism, and the physical environment to which the organism is acclimatized or adapted ( 15). Metabolic pathways influencing the rate of cell and tis- sue growth (including hyperplastic and hypertrophic growth) include those that influence the rate of energy production (glycolysis, Krebs cycle) and those that in- fluence the rates of macromolecular biosynthesis (pro- teins, lipids, nucleic acids). Temperature acclimation and adaptation of many of the enzymes involved in en- ergy production have been reviewed by Hazel and Pros- ser (16). Fewer studies have focused on temperature ad- aptation of those enzymatic reactions directly involved in macromolecule synthesis, particularly those involved in regulating macromolecule synthesis and function. Polyamine biosynthesis, regulated by the activity of ODC, is directly involved in macromolecular synthesis and is considered to be one of the rate-limiting steps in the regulation of protein and nucleic acid metabolism, as well as cell growth ( 1 , 2, 1 7, 1 8, 1 9). In this study we have investigated the effects of temperature on the kinetic characteristics of an invertebrate ODC obtained from the testes of the sea star Asterias vulgaris. A. vulgaris is sea- sonally exposed to temperatures ranging from -2 to 1 7C (11). Watts et a/. (11) found that significant de- creases in testicular growth, polyamine levels (putres- cine, spermidine, and spermine), and ODC activity were coincident with decreasing or low environmental tem- peratures. It was hypothesized that low field tempera- tures resulted in a decrease in polyamine biosynthesis by directly influencing the synthesis of ODC, either at the translational or transcriptional level. In this experiment we have shown that ODC exhibits "negative thermal modulation" (15). Decreases in assay temperature resulted in increases in the apparent Km, thereby lowering the affinity of the enzyme for the sub- strate. Reduced enzyme affinity for the substrate, as well TABLE I I 'allies ofQio as determined from the activity of ornithine decarboxvlase measured at various substrate concentrations [ORNITHINE] (mM) 0-5C 5-15C .0351 2.35 2.04 .0463 2.59 .94 .0799 2.36 .86 .1359 2.34 .69 .2479 2.29 .45 .5839 2.26 .26 1.144 7^1 .07 Values of C '10 were determined over the range of to 5C and 5 to 15C. 162 S. A. WATTS ET AL. as reduced ODC synthesis reported previously by Watts el al. ( 1 1 ) suggest that polyamine biosynthesis, which is necessary for cell and tissue growth, may be decreased during exposure to low temperature, thereby inhibiting growth. The influence of temperature on the rate of ODC ac- tivity becomes more apparent when examined in terms of Q| . The combined effects of reduced substrate bind- ing and reduced kinetic energy available for enzyme acti- vation at low temperatures produced higher Q ](l values at apparent physiological substrate concentrations (ca. 0.2 mA/ ornithine; Watts, unpub.). In addition, Q m values are highest when calculated at the reduced assay temper- atures, causing temperature-dependent ODC activity and kinetics at low temperatures. We hypothesize that the exposure ofAsterias vulgaris to low temperatures decreases growth and development of the testis by negative modulation of polyamine syn- thesis. In individuals exposed to low temperatures, we suggest that polyamine synthesis may be negatively mod- ulated by 1: a decrease in the amount of enzyme (either translational or transcriptional control), 2: an increase in the amount or binding of a proposed antizyme (1,2), or 3: a decrease in the affinity of the enzyme for the sub- strate as indicated by changes in the K m . Thermal modu- lation of polyamine metabolism may be a mechanism by which seasonal temperature fluctuations induce the seasonal patterns of growth observed in many ectother- mal organisms. Further studies are needed to determine the extent to which temperature influences these pro- cesses in these organisms. Acknowledgments We thank Dr. Larry Harris for collecting the sea stars and Dr. Adam Marsh for comments on the manuscript. This research was supported by NSF (DMB-85 17284 and DCB-87 1 1425) and NATO (04 1 3/86 ). Literature Cited 1. Russell, D. H. 1985. Ornithinedecarboxylase: a key regulatory en- zyme in normal and neoplastic growth. Dru% Mclab. Rev 16: 1- 88. 2. Pegg, A. E. 1988. Polyamine metabolism and its importance in neoplastic growth and as a target for chemotherapy. Cancer Res. 48: 759-774. 3. Dogterom, A. A., H. van Loenhout, and R. de Waal. 1980. Orni- thine decarboxylase in the freshwater snail Lvinnaea stagnalis as related to growth and feeding. Proc K Ned. Akad. \\'el 83: 25-3 1 . 4. Manen, C., and D. H. Russell. 1973. Spermine is the major poly- amine in sea urchins: studies of polyammes and their synthesis in developing sea urchins. J. Embryol. Exp. Morphol. 29: 331-345. 5. Herbst, K. J., and A. S. Dion. 1970. Polyamine changes during development of Drosophila melanogaster. Fed. Proc. 20: 1 563- 1567. 6. Watts, S. A., K. Lee, G. Mines, and C. W. Walker. 1987. Deter- mination of polyammes in reproductive and digestive tissues of As- leriax vnlgarix (Echinodermata: Asteroidea). Camp. Biochem. Physiol 888:309-312. 7. Asotra, A., P. V. Mladenov, and R. D. Burke. 1988. Polyamines and cell proliferation in the sea star Pycnopodia helianthoides. Comp Biochem. Physiol. 90B: 885-890. 8. Birnbaum, M. J., T. M. Whelan, and L. I. Gilbert. 1988. Tem- poral alterations in polyamine content and ornithine decarboxyl- ase activity during the larval-pupal development of Manduca sexta. Insect Biochem. 18: 853-859. 9. Hamana, K., M. Suzuki, T. \\akabayashi, and S. Matsuzaki. 1989. Polyamine levels in the gonads. sperm and salivary gland of cricket, cockroach, fly and midge. Comp. Biochem. Phvsiol. 928:691-695. 10. Lee, K. J., S. A. Watts, and J. B. McClintock. 1989. Distribu- tions of the polyamines putrescine, spermidine and spermine in invertebrate tissues. J. Ala. Acad. Sci. 60(3): Abstract. 11. Watts, S. A., G. Hines, K. Lee, D. Jaffurs, J. Roy, F. F. Smith, and C. W. Walker, (in press). Seasonal patterns of ornithine de- carboxylase activity and levels of polyamines in relation to the cy- tology of germinal cells during spermatogenesis in the sea star, Aste- rias vulgaris. Tissue and Ceil. 1 2. Smith. F. F., and C. W. Walker. 1990. Enhanced DNA synthesis in echmoderm testes in the presence of exogenous polyamines. Comp. Biochem. Physiol. 95B: 65-69. 13. Landy-Otsuka, F., and I. E. Scheffler. 1978. Induction of orni- thine decarboxylase activity in a temperature-sensitive cell cycle mutant of Chinese hamster cells. Proc. Natl. Acad. Sci. USA 75: 5001-5005. 14. Smith, F\ K. 1985. Changes in the biochemical composition of the testes during spermatogenesis in Aslerias vulgaris. with empha- sis on the role of polyamines in regulating proliferation. Ph.D. Dis- sertation, University of New Hampshire, Durham. Pp. 1-190. 15. Hochachka, P. W., and G. N.Somero. 1984. Temperature adap- tation. Pages 355-449 in Biochemical Adaptation. Princeton Univ. Press, Princeton, New Jersey. 16. Hazel, J. R., and C. L. Prosser. 1974. Molecular mechanisms of temperature compensation in poikilotherms. Physiol. Rev 54: 620-677. 17. Heby.O. 1981. Role of polyamines in the control of cell prolifer- ation and differentiation. Differentiation 19: 1-20. 18. Tabor, C. W., and H. Tabor. 1984. Polyamines. Annu. Rev. Bio- chem. 53: 749-790. 19. Pegg, A. E. 1986. Recent advances in the biochemistry of poly- amines in eukaryotes. Biochem. J 234: 249-262. CONTENTS Annual Report of the Marine Biological Laboratory 1 INVITED REVIEW Pardue, M. L., W. G. Bendena, M. E. Fini, J. C. Garbe, N. C. Hogan, and K. L. Traverse Hsr-omega, a novel gene encoded by a Drosophila heat shock puff 77 BEHAVIOR Buck, John Unisex flash controls in dialog fireflies 87 DEVELOPMENT AND REPRODUCTION Goldberg, Walter M., Ken R. Grange, George T. Taylor, and Alicia L. Zuniga The structure of sweeper tentacles in the black coral Antipathesfiordensis 96 ECOLOGY AND EVOLUTION Curtis, Lawrence A. Parasitism and the movements of intertidal gastro- pod individuals 105 Lonsdale, Darcy J., and Sigrun H. Jonasdottir Geographic variation in naupliar growth and sur- vival in a harpacticoid copepod 113 GENERAL BIOLOGY Pennington, J. Timothy, and Richard R. Strathmann Consequences of the calcite skeletons of planktonic echinoderm larvae for orientation, swimming, and shape 121 PHYSIOLOGY Dahlhoff, Elizabeth, Sabine Schneidemann, and George N. Somero Pressure-temperature interactions on M 4 -lactatede- hydrogenases from hydrothermal vent fishes: evi- dence for adaptation to elevated temperatures by the zoarcid Thermarces anderxoin, but not by the bythitid, Bythites hollisi 134 Michibata, Hitoshi, Hisayoshi Hirose, Kiyomi Sugi- yania. Yukari Ookubo, and Kan Kanamori Extraction of a vanadium-binding substance (vana- dobin) from the blood cells of several ascidian spe- cies 140 Shick,J. Malcolm Diffusion limitation and hyperoxic enhancement of oxygen consumption in zooxanthellate sea anemo- nes, zoanthids, and corals 148 RESEARCH NOTE Watts, Stephen A., J. Roy, and C. W. Walker Ornithine decarboxylase exhibits negative thermal modulation in the sea star Asterias vulgfiris: potential regulatory role during temperature-dependent tes- ticular growth 1 59 Volume 179 THE Number 2 BIOLOGICAL BULLETIN APR 1 6 l OCTOBER, 1990 Published by the Marine Biological Laboratory THE BIOLOGICAL BULLETIN PUBLISHED BY THE MARINE BIOLOGICAL LABORATORY APR 1 6 1993 Editorial Board Woods Hole, Mass. j ological Laboratory GEORGE J. AUGUSTINE. University of Southern JOHN E. HOBBIE, Marine Biological Laboratory California GEORGE M. LANGFORD, University of RUSSELL F. DOOLITTLE. 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Authors receive their tirst 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. Reference: Biol. Bull 179: 163-177. (October. A Practical Guide to the Developmental Biology of Terrestrial-Breeding Frogs RICHARD P. ELINSON 1 , EUGENIA M. DEL PINO 2 , DANIEL S. TOWNSEND 3 , FABIAN C. CUESTA 2 , AND PETER EICHHORN J ^Department oj Zoology, University of Toronto, Toronto M5S 1A1, Canada, -Pontificia Universidad Catolica del Ecuador, Departamento de Ciencias Biological, Quito, Ecuador, - 1 'Department of Biology, University of Scranton, Scranion. Pennsylvania 185 10, and* Institute for Cell and Tumor Biology. German Cancer Research Center, Im Neiienheimer Feld 280. D-6900 Heidelberg 1. Federal Republic of Germany Abstract. Many frogs lay their eggs in water; the devel- opment of these frogs is well-known. However, many frogs reproduce on land: their eggs are large and have an altered early development. As examples, Gastrotliecu riohambae broods its embryos in a pouch on the mother's back, and Eleutherodactylus coqui exhibits direct development with no tadpole stage. We provide practical information on obtaining eggs and embryos from these terrestrial-breeding species and on analyzing their development. Our aim is to make these species more accessible to researchers who are interested in the developmental and evolutionary consequences of terrestrial development. Introduction Our view of frog development is colored by the fact that most scientists work in the temperate climates of Eu- rope, Asia, and North America. We expect frogs and other anuran amphibians to lay their eggs in water, to develop first into tadpoles, and to metamorphose later into adults. A number of anurans, particularly those found in the tropics, do not follow this life history (Lamotte and Les- cure, 1977; Duellman and Trueb, 1986). Some anurans lack a free-living tadpole and develop directly to an adult morphology. Many anurans develop entirely on land and brood their embryos in such diverse places as the oviduct, a back pouch, the stomach, or the male's vocal sac. Given these terrestrial life histories, the name "amphibian" might Received 1 June 1990: accepted 27 July 1990. not have been applied to this class had taxonomy begun as a tropical science, and our view of frog development might be totally different. Anurans with terrestrial development generally have a small number (1-1 50) of very large eggs (3-10 mm), which differ in various aspects of development from typical an- urans. Developmental biologists have noticed only a few of these animals including Gastrotheca riobambae, Flec- tonotits pygmaeus. and Eleutherodactylus coqui. Gastro- theca riobambae is an egg-brooding frog from Ecuador. The female incubates the eggs in a pouch on her back, and the young are born as advanced tadpoles. Flectonotus pygmaeus. from Venezuela, exhibits multinucleate oo- genesis. The early oocytes have up to 2000 nuclei, of which all but one disappear (del Pino and Humphries. 1978). Eleutherodactylus coqui, from Puerto Rico, exhibits direct development. The large eggs are brooded on land by the male (Townsend et al, 1984), and the froglets hatch after about three weeks. Developmental studies to date have concentrated on oogenesis and early development in G. riobambae and F. pygmaeus (del Pino and Humphries, 1978; del Pino and Escobar, 1981; Elinson and del Pino. 1985; del Pino et a!.. 1986; del Pino, 1989) and on organ formation and direct development in E. coqui (Lynn, 1942; Lynn and Peadon, 1955; Adamson et al., 1960;Chibon, 1962; Elin- son, 1990). Our discussion of these animals will reflect this bias, as we describe how to obtain and work with adults and embryos of G. riobambae, F. pygmaeus, and E. coqui. 163 164 R. P. ELINSON ET AL. Gastrotheca riobambae and Other Egg-Brooding Frogs (Hylidae) Collection and maintenance* Sites of collection. The majority of egg-brooding frogs inhabit the humid forests of northern South America, have very limited distribution ranges, and are rarely found (Table I). Species of Gastrotheca that inhabit the highlands of the Andes, however, occur in large numbers and are more easily collected. We will mostly discuss G. riob- ambae. the species with which we have extensive expe- rience, and will make reference to G. plumbea and F. pygmaeus, which we have also maintained in captivity. Gastrotheca riobambae occurs in the interandean val- leys of northern Ecuador at altitudes of 2500-3200 m (Duellman and Hillis. 1987). Frogs are found along the banks of irrigation ditches and under stones in humid areas, as well as sitting on vegetation. This frog gives birth to tadpoles that develop in temporary pools and lakes. The free-living tadpole stage lasts several months. The large tadpoles of G. riobambae (around 70 mm total length) are the only amphibian larvae that exploit the standing bodies of water in the highlands of northern Ecuador. Gastrotheca plumbea lives in cloud forests at elevations of 1300-2350 m on the Pacific slopes of the Andes in Ecuador (Duellman and Hillis, 1987). Both adults and young are found in axils of large bromeliads. Gastrotheca plumbea exhibits direct development, which means it lacks a free-living tadpole stage. Flectonotus pygmaeus lives in axils of bromeliads, in the cloud forest at Estacion Biologica de Rancho Grande, Maracay, Venezuela. This frog gives birth to advanced, 1 South American wildlife is protected by law. Investigators interested in the collection of these frogs should check with the appropriate au- thorities in the country where they wish to work. Permission for the collection and export of frogs from Ecuador is given by the Director, Direccion de Desarrollo Forestal, Ministerio de Agriculture, Quito. Ec- uador. A proposal, written in Spanish, should be sent several months in advance to the above address with a copy to the collaborating investigator or institution in Ecuador. It should be accompanied by the curriculum vitae of the investigator and a letter of support from the researcher's home institution. Duplicate collections must remain in Ecuador, usually at the Museo de Ciencias Naturales. Casa de la Cultura Ecuatoriana, Quito, Ecuador, or at the collection of one of the universities. To obtain permits, visiting scientists are required to give lectures about their work at the Museo de Ciencias Naturales or at the collaborating institution. Upon completion of the work, a report and copies of published works should be sent to Direccion de Desarrollo Forestal and to the collaborating institutions. Upon arrival in Quito, investigators are advised to visit the Direccion de Desarrollo Forestal as soon as possible to get their permits. Investigators interested in /'. /n-t,'imicn.\ are advised to check with the Estacion Biologica de Rancho Grande. Maracay, Venezuela. A permit for collection is required from the Estacion Biologica de Rancho Grande, and permits for export are obtained from the Oficina de Fauna, Ministerio de Agriculture y Cria. Caracas, Venezuela. Table I Geographic distribution of egg-brooding lrdt>.\ Approximate Genera of egg- number of brooding frogs species 3 Distribution 3 WITHOUT POUCH Andes and Sierra Nevada de Santa Marta, Colombia Panama. Pacific slopes of Colombia and northwestern Ecuador: upper Amazon Basin in Brazil. Ecuador. Peru and Bolivia" Highlands of the Guyana Shield in Venezuela and Guyana' MARSUPIAL FROGS Coastal Cordillera of northern Venezuela. Tobago and Tnnidad d 3 Mountains of Southeastern Brazil (Guanabara, Minas Gerais, Rio de Janeiro. Sao Paulo) d 4 1 Panama, northern and western South America, southward to northern Argentina, east and southeastern Brazil Cryptobatrachus Hemiphractus Slefanui Flectonotiu Frilziana Gastroiheca ' Duellman (1977). h Trueb(1974). c Duellman and Hoogmoed ( 1984). d Duellman and Gray (1983). non-feeding tadpoles that complete metamorphosis in about a week. The tadpoles are deposited in the water collected in axils of bromeliads (Duellman and Maness, 1980). Terraria for laboratory maintenance. Our terraria for maintaining Gastrotheca and Flectonotus consist of wooden frames, 60-80 cm in length by 40 cm in width and 40-50 cm in height, with side walls of plastic mesh. The floor and the roof are made of wood. The roof has a large door, that fits tightly, and measures about half the length of the terrarium. Terraria of glass and of plastic are also used. In those cases, the cover is made of plastic or metal mesh to allow gas exchange. The number of frogs per terrarium varies according to frog size. We keep about 8-10 adults of G. riobambae, 4-5 adults of the larger G. plumbea, or 12 F. pygmaeus per terrarium. Gastrotheca riobambae have been kept for up to five years, with reproduction occurring about once a year. Gastrotheca plumbea and F. pygmaeus have been kept for about two years, but their reproductive cycle and lifespan are not known. Inside terraria we place large plastic trays with earth to cover the floor completely. Stones and hollow pieces of TERRESTRIAL-BREEDING FROGS 165 brick provide frogs with hiding places, and branches serve as perches. One or two small containers with water (about 5 cm in depth) are provided with stones to give support to the frogs. Each terrarium contains one or two small pots planted with Tradescantia. This plant grows easily and soon covers the terrarium. In addition, a bromeliad is planted, when available. Bromeliads with thorny leaves produce wounds in frogs and are not recommended. Ter- raria are placed near windows where there is ample sun- light and are kept moist by watering at least twice a week. Terraria are kept at 17-23C. In native habitats of G. riobamhac, the temperature fluctuates between 5C at night to 23C at midday. Favorite hiding places for both G. riohambac and G. plumbea are the cavities under stones and bricks and in axils of bromeliads. In addition, they hide in the crevices between plastic trays and the terrarium walls as well as under the vegetation. Flectonotus pygmaeus perches in axils of bromeliads and on the vegetation. Frogs bask in the sun and may remain in the same place for several days. Frogs are in the water often, so water must be changed frequently. We have not tried terraria of larger height. However, Zimmermann ( 1983) described taller terraria with several vegetation levels and high humidity for the maintenance of tropical frogs. Those terraria have a frontal glass door and might be useful for maintaining large arboreal species of egg-brooding frogs. Feeding. Adult G. riobambae are fed two to three times a week. The easiest, most efficient food consists of sowbugs (Porcellio sp.) or meal-worm larvae (Tenebrio mol/itor), mixed with small pieces of dry dog food. Food is placed in shallow plastic containers ( 1.5-2 cm in depth) and is always given in the same place. Meal worms remain in feeding containers and in this way, frogs learn to eat dry food when capturing prey. We have maintained G. riobambae successfully on only a sowbug diet; however, sowbugs become scarce in dry weather. To avoid food scarcity, feeding alternately with sowbugs or meal-worms and dry dog food gives excellent results. Dog food provides the frogs with carbohydrates, lipids, vitamins, and minerals. Outside the feeding con- tainers, frogs ingest dirt from the terrarium floor with their prey and may obtain needed trace elements; frogs raised without dirt become weak. This diet is also well accepted by G. plwnbea. Flectonotus pygmaeus is fed once or twice a day on large Drosophila caught from the wild. Small sowbugs and meal-worm larvae were not tried, but could probably be given successfully to these frogs. Newly metamorphosed G. riobambae readily accept small meal-worm larvae, and this type of food is a key to successfully raising these frogs when used in combination with other small prey items. We mix larvae with dry dog food in very shallow plastic containers. Newly born G. plumbea have been raised successfully on the same diet given to young G. riobambae. In addition, young G. plumbea were fed large Drosophila caught from the wild. Juvenile G. riobambae and G. plumbea reach the adult stage 8-12 months after metamorphosis. Newly meta- morphosed F. pygmaeus are quite small, and we have had no success in raising them. Very small meal-worm larvae, which are readily accepted by other species, were not tried on F. pygmaeus. Amplexits anil birth. Before amplexus, G. riobumbae males call frequently. Amplexus occurs on land and it lasts for 24-48 h before egg-laying begins. Egg laying lasts about 6-8 h. During egg laying, the male introduces his feet inside the female's pouch. As each egg leaves the fe- male's cloaca, the male catches it with his heels and toes and moves it inside the pouch, the opening of which is about a centimeter anterior. In this way, eggs do not touch the ground. Eggs leave the female's cloaca, one at a time, at intervals of 30-60 s. A few eggs are lost and remain in the soil at the end of amplexus. Fertilization probably occurs during the egg's journey from the female's cloaca to the pouch. After amplexus, the female places herself tight against the cavity of a stone or other object, probably to help in pouch distension and in the arrangement of embryos in one or two even layers (del Pino el ai, 1975). Incubation of embryos lasts a mean of 100 days, during which time the wet weight of embryos increases three-fold, while the dry weight remains constant (del Pino and Escobar, 1981). At birth, the total weight of embryos equals one third to one half the weight of the female. Females become so swollen with embryos that their movements are greatly reduced. At birth, the female enters the water, introduces the long toes of her hind legs inside the pouch, and aids in the removal of tadpoles. She supports herself with her front legs against the walls of the water container or against a stone. Tadpoles at birth measure 18-20 mm in total length (del Pino el at., 1975). In captivity, amplexus ofG. riobambae occurs between September and February, the period with heavier rainfall in Quito. Sometimes frogs mate in June. These periods seem to coincide with times when most reproduction oc- curs in nature; however, females with embryos can be collected throughout the year. By administering human Chorionic Gonadotropin (hCG) to male and female frogs, described later, amplexus and reproduction can be ob- tained in captivity at any time of the year. Amplexus in G. plumbea also occurs on land, but details have not been documented. Incubation lasts about 120 days (del Pino and Escobar, 1981). At birth, the mother actively aids in the elimination of offspring by stretching the opening of the pouch and digging young out of the pouch with her hind feet, as in G. riobambae (Duellman 166 R. P. ELINSON / / II. and Maness, 1980). Brooding females have been found in June and July, and frogs in captivity gave birth in Sep- tember (Duellman and Maness, 1980). Amplexus and birth for F. pygmaean have been described by Duellman and Maness (1980). Amplexus occurs on land, and at birth the female deposits larvae in water. Embryos were incu- bated for 29 days in captivity (del Pino and Escobar, 198 1 ). The breeding season off", pygmaeus at Estacion Biologica de Rancho Grande spans from April until early Novem- ber, the time of the year with the heaviest rainfall (Duell- man and Maness. 1980). We have not tried hormonal stimulation of reproduction in 6'. plumhea or F. pyg- miu'its. but both species have mated spontaneously in the laboratory. Care of tadpoles. The extensive use of pesticides and urban growth have diminished the populations of G. riob- ambae, so frogs must be collected from remote localities. To ensure a supply of frogs, we have developed methods for raising frogs from tadpoles. Tadpoles are best maintained in large tanks with at least one cubic meter of water, a depth of 30-50 cm, and a temperature of 17-21C, without changes of water. Tanks are provided with stones as hiding places, a large plant (Cypemceae) whose roots, floating stems, and leaves provided places for tadpoles to sit near the water surface, and a few branches of Elotiea. These tanks support an abundant population of algae and protozoans that become the main constituent of the tadpole diet. About 70-100 tadpoles are raised per tank. Little or no cannibalism is observed, and tadpoles grow normally, even when newly born tadpoles are placed with older ones. Tadpoles of G. riohamhac are voracious eaters. The easiest, most effective diet has been small pieces of dry dog food or rabbit food given once or twice a week, sup- plemented by the algal growth in the tanks. Care should be taken not to contaminate the water with too much food. Tadpoles reached 70 mm total length before meta- morphosis, at about 3 months after birth. Frogs measured 18-25 mm snout-vent length, looked healthy, and sur- vived after metamorphosis. Tadpoles have also been kept in small glass and plastic aquaria of 10-20 liters capacity. Without a stable popu- lation of algae, the water becomes contaminated and re- quires weekly changes. In some instances, aquaria were aerated by means of an aquarium pump, but we found no advantage in aeration. Tadpoles of G. riobambae have well-developed lungs by the time of birth and take oxygen from air. Population densities in aquaria correspond to 1-3 tadpoles per liter of water. Higher densities result in cannibalism and early metamorphosis. Under crowded conditions small tadpoles, 50-60 mm total length, reached metamorphosis in only 40 days at 1 8C. The newly meta- morphosed frogs were small (10-15 mm snout-vent length), extremely weak, and often died at metamorphosis or soon thereafter. We have tested a variety of other diets for tadpoles. Diets included cooked and raw meat: cooked lettuce, chard and spinach; chicken feed hardened with agar. egg yolk from cooked eggs, and fish food for aquaria (Tetra- min, Tetra Werke, 4520 Melle, Federal Republic of Ger- many). Of these diets, fish food is the best, followed by chicken feed hardened with agar. Fleclonotus pygmaeus produces about six advanced tadpoles per breeding season. In the laboratory, tadpoles have been kept in shallow water containers. These tadpoles do not eat and only need 2-4 weeks of aquatic living to reach metamorphosis (Duellman and Maness. 1980). Maintenance at lower altitudes. The habitat of G. rioh- amhae is the high montane environments in northern Ecuador (2500-3200 m altitude). Atmospheric pressure, oxygen availability, length of daylight, and amount of sunlight as well as temperature of the G. riobambae habitat differ from the conditions of most laboratories where studies on development are conducted. Change in altitude and temperature often result in frog death. We had the experience of raising G. riobambae at the aquaria of the German Cancer Research Center in Heidelberg, Federal Republic of Germany. Most female frogs that were in- cubating embryos died a few days after arrival, but one frog gave birth successfully and provided us with tadpoles, from which we obtained a supply of frogs. Tadpoles were raised in small glass aquaria at a density of three tadpoles per liter. Aeration was provided with aquarium pumps. Tadpoles were fed fish food (Tetramin) and cooked lettuce and attained the normal length of about 70 mm before metamorphosis, about one month after birth. Newly metamorphosed frogs ate flies and small crickets readily. Eight months after birth, frogs reached adult size and began to sing. Adult frogs were maintained in terraria as previously described. Temperature was ad- justed to 17-18C and the light regime was a 12-h light/ dark cycle. Frogs were fed adult house-flies and medium- size crickets, which were often dusted in powdered vita- mins and minerals, Osspulvit (Zimmermann, 1983), to supplement the diet. Amplexus was observed on several occasions; however, egg laying did not occur. Accelerated development has been reported previously at lower altitude. In Holland, Hoogmoed( 1967) obtained metamorphosis of G. riobambae tadpoles in 41 days after birth at a temperature of 21-26C. In contrast, tadpoles normally require several months of aquatic living to reach metamorphosis in Quito. Water temperature as well as the change in altitude should be factors involved in the differences observed. We induced amplexus and reproduction by adminis- tering hCG intraperitoneally to both male and female frogs, as described later. Amplexus and fertilization oc- TERRESTRIAL-BREEDING FROGS 167 curred normally. Eggs began to cleave, but died at around the time of gastrulation. Lower altitude seems to affect the physiological condition of frogs and may affect the quality of eggs. For instance, the induction of oocyte mat- uration by progesterone /// vitro, took 10 h longer in San- tiago de Chile than in Quito (de Albuja cl al.. 1983). Au- ber-Thomay and Letellier (1986) obtained regular and spontaneous reproduction of G. riobambaeio France. In- cubation took 41-74 days, but half of the eggs from 10 frogs failed to develop. In contrast, 75-108 days of in- cubation are needed in Quito, but almost all of the eggs (99.4%) developed (del Pino and Escobar, 1981). Our ex- perience, and that of others, indicate that successful re- production, as it happens in Quito, is rare at lower alti- tudes. Oogenesis, fertilization, and the culture of embryos Analysis of oogenesis. Egg-brooding frogs have syn- chronous oogenesis, which means that only one batch of oocytes grows in the ovary at one time (del Pino ct al.. 1986). Full-grown oocytes are the largest documented among anurans, reaching 2.5-10 mm in diameter, de- pending on the species (Table II). Their volume is pri- marily due to yolk, and oocytes of G. riobambae actually have less rRNA than oocytes of Xcnopus laevis. which are '/, h th the volume (del Pino et al., 1986). In some species of egg-brooding frogs, oocytes contain many nuclei (4-2000 nuclei depending on species) during the previtellogenic period (Table II; del Pino and Hum- phries, 1978). At vitellogenesis, only one nucleus remains as the oocyte's germinal vesicle while the rest degenerate. This type of oogenesis is called multinucleate oogenesis (del Pino and Humphries. 1978). and unfortunately, the analysis of it is limited by the availability of frogs. Mar- supial frogs with multinucleate oocytes are mostly large frogs (Table II) that live in cloud and humid forests and are rarely found. The most accessible species encountered so far is F. pygmaeus. To study oogenesis, ovarian pieces are removed from the body cavity of the frog or tadpole and are placed in modified Barth Solution (MBS) (Gurdon. 1968), am- phibian Ringer's, or in other amphibian saline solutions. MBS contains 88 mM NaCl, 1 mAl KC1, 2.4 mA/ NaHCO 3 , 0.82 mM MgSO,, 0.33 mM Ca(NO 3 ) 2 , 0.41 mM CaCl : , 10 mg/1 each benzylpenicillin and strepto- mycin sulphate and 2 mA/ Tris-HCl (or 10 mA/ Hepes), pH 7.6. Methods for the cytological observation of germinal vesicles in mono- or multinucleate oocytes of egg-brooding frogs do not differ from those used with other amphibia (Macgregor and del Pino, 1982). The large amount of yolk stored in large oocytes, however, is bothersome for cytology as well as for the extraction of nucleic acids. Fixation of oocytes for cytological studies needs to be modified due to the yolk content of oocytes. It is important to increase the volume of fixative and the length of fixation times. Smith's fluid (Rugh, 1965) is our fixative of choice for paraffin and plastic sections. One or two yolky oocytes of 6'. riobambae are fixed in 10-20 ml of Smith's fluid for 12-24 h in the dark, washed in distilled water with several changes for 24 h, and stored in 4% neutralized formalin until processing. Yolky oocytes fixed in Smith's are particularly easy to section, when embedded in par- affin. Material embedded in plastic resins, like JB-4 (Poly- sciences), provide better resolution than paraffin sections. Small oocytes, taken from large tadpoles and newly metamorphosed frogs, can be processed for electron mi- croscopy according to standard methods. Large, yolky oocytes should be cut into smaller pieces during glutar- aldehyde fixation to allow better penetration of fixative. Thick epon sections (0.5-1 ^m thickness) of oocytes, fixed in glutaraldehyde and postfixed in osmium tetroxide, can be used for light microscopy. Sections are placed on a drop of water over a clean slide and, after drying, are mounted with immersion oil for light microscopy (del Pino ct til.. 1986). The study of nucleic acids in large oocytes of G. riob- ambae is affected by the amount of yolk. The volume of working solutions needs to be increased 10 times in com- parison to A', laevis oocytes due to the large oocyte volume. Nucleic acids cannot be totally cleaned from yolk con- taminants without losing small RNA molecules (del Pino ci til.. 1986). The large size of oocytes in egg-brooding frogs is, at first, attractive for microinjection experiments. There are, however, several limitations. The small number of eggs and oocytes per female (Table II) prevent their extensive use. Eggs are uniformly pale, and the animal pole cannot be easily distinguished, a limitation when microinjection into the germinal vesicle is desired. Large egg size is due to yolk content, and the germinal vesicle of G. riobambae oocytes is equivalent in size to the germinal vesicle of A". laevis oocytes (del Pino et al.. 1986). Microinjection into oocytes and eggs of G. riobambae is further complicated by their high internal pressure. Oocytes and eggs often burst after microinjection, although this can be prevented by injecting oocytes kept in a humid chamber or in 5% Ficoll in an amphibian saline. The easily available oocytes of A', laevis provide superior material for microinjection experiments. Hormonal induction of oocyte maturation, ovulation, and mating in G. riobambae. Large oocytes within the ovarian follicle can be induced to undergo germinal vesicle breakdown (GVBD) in vitro by exposure to hCG or pro- gesterone. In contrast, oocytes denuded from the follicular wall undergo GVBD only in response to progesterone. Treatment of ovarian follicles with hCG results not only 168 R. P. ELINSON ET AL Table II s and development <>l egg-brooding frogs Snout-vent length of Frog species a female (mm) Number of oocyte nuclei 1 = mononucleate 2 = multinudeate Egg diameter (mm) Clutch size Development 1 = tadpole 2 = direct FROGS WITHOUT POUCH ( 'r\'i>iiihulrachn.- % of the yolk. In stage 1 1 embryos, toes reach about half of their final length and the pigmented body wall finishes enclosing the yolk reserve. Stage 12 is a longer period, during which the ECD articulate at the embryo's midline and form a broad, shallow U with somewhat broadened base and spread arms. The pigmented body wall com- pletely encloses the yolk, and the pigmentation wall is heavy enough to begin obscuring the ECD at this stage. Stage 1 3 is marked by full length toes with slight swellings at their tips indicative of incipient toe discs. The first ev- idence of pigmented eyelids appears, and the initial regression of the unpigmented tail begins. Stage 14 em- bryos possess toes with full toe discs, eyes with adult col- oration (iris golden-brown above, bronze-brown below, pupil black), and clear banding patterns on the hind legs. Coqui eggs may hatch anytime during stage 15. Em- bryos at this stage possess full coloration, including any of several distinct pattern morphs exhibited by the species. The remains of the ECD are completely masked by dorsal pigmentation. A bifurcate, black egg tooth at the sym- physis of the upper jaw, first evident in stage 12 or 13. is apparently used by the froglet to rupture the egg mem- brane and hatch. Development at 25C requires approximately 17 days (Townsend and Stewart, 1986b). Hatchlings average 6 mm in length, and possess a tail remnant that requires up to two days to resorb after hatching. The large yolk reserve usually lasts for up to a week. Manipulation of embryos The normally terrestrial embryos ofEleutherodactylus can be cultured in water or in simple salt solutions as described earlier. This permits various experimental ma- nipulations such as surgery or chemical treatments which would otherwise be very difficult. For instance, Lynn and Peadon (1955) were interested in the role of thyroxine in the development of E. marlini- ccnsix, a direct developer like E. coqui. Thyroxine causes metamorphosis from tadpoles to adults in frogs with aquatic development. Embryos were freed from the jelly and fertilization membrane at Townsend/Stewart stages 6-7. and cultured in tap water with either thyroxine or phenylthiourea, a thyroid inhibitor. Thyroxine caused regression of the tail and degeneration of the pronephros. Limb development, however, was not under thyroid con- trol as it is in metamorphosing amphibians. It is clear that thyroxine and other chemicals given exogeneously can enter these embryos. Surgery is also possible. Hughes (1962) transplanted limb buds between embryos of E. martinicensis to see the interaction between the developing limb and the nervous system. Embryos as early as Townsend/Stewart stage 6 were removed from their jelly capsules and initially cul- tured in the capsular fluid. A limb bud was cut from one embryo and grafted via a small incision to a second em- bryo. After the operation, the embryo was allowed to heal for a day in Holtfreter's solution (approximately equiv- alent to 100% Steinberg's). Thereafter the embryo was cultured in water. From these limited experiences, it appears easy to ex- periment on embryos older than Townsend/Stewart stage 6. Whether earlier embryos can be manipulated depends very much on whether they can be removed from the jelly capsule without injury. A second difficulty is that the early embryo may require the structural support provided by the jelly capsule. The use of dishes with an agar base or tilled with Percoll (Pharmacia), a high density, low tonicity solution, may permit culturing of early embryos without jelly. Conclusions Terrestrial-breeding frogs provide opportunities for analysis by both developmental and evolutionary biolo- gists (Elinson, 1987a, 1990). Obvious comparative and evolutionary questions include: a. How does large egg size affect early development? b. How are reproductive adaptations, such as the fe- male's pouch or the male's brooding behaviour, controlled hormonally? c. What is the cellular and hormonal basis of direct development, an ontogeny without metamorphosis? d. How did the different reproductive and develop- mental patterns evolve? Beyond these questions, embryos from terrestrial- breeding frogs would be ideal for examining certain de- velopmental problems. For instance, the translucent blas- tocoel roof in both G. riobambae and E. coqui would allow the migration of internal cells during gastrulation to be followed in intact embryos. The early development 176 R. P ELINSON ET AL of large limb buds in E. cot/id would permit the analysis of pattern formation in limbs. Finally, the eggs and em- bryos themselves present unique questions for cell and developmental biologists. Certainly the formation of oo- cytes with hundreds of nuclei and the controlled degen- eration of them is an intriguing problem for the future. In this article, we have described how to obtain eggs and embryos for the laboratory investigation of the un- usual ontogenies found in these frogs. While these recipes permit the investigator to work in Europe or North America, we would urge that anyone interested in these and other such anurans, travel to their country of origin. This would not only allow the observation of the animals in their native habitats, but would also promote the in- teraction between scientists from temperate and tropical zones. Acknowledgments We wish to thank the following for their help. C. M. de Albuja. the late S.G. Maness, W. E. Duellman. L. Trueb, B. Zimmerman, and J. Bogart helped obtain living animals. R. F. Laurent (Fundacion M. Lillo, Argentina), P. M. Ruiz-Carranza (Museo de Historia Natural, Uni- versidad Nacional de Colombia), J. Simmons (formerly at the California Academy of Sciences), P. E. Vanzolini (Museu de Zoologia, Sao Paulo, Brazil). C. W. Myers (American Museum of Natural History, New York), and W. E. Duellman (Museum of Natural History, Kansas) provided museum specimens. R. Fischer, H. Trosterand G. Weise (Heidelberg, FRG) and P. Pasceri (Toronto, Canada) cared for animals. K. Townsend provided field assistance; M. M. Stewart, D. Cundall, and R. Wassersug provided advice: and K.. Kao helped with the figures. E. M. del Pino received a Humboldt Fellowship to work in the laboratory of M. F. Trendelenberg, Heidelberg; D. S. Townsend was supported by NSF and the Gaige Fund of the American Society of Icthyologists and Her- petologists; and R. P. Elinson received funding from NSERC, Canada. Literature Cited Adamson, I,., R. G. Harrison, and I. Baylcy. 1960. The development of the whistling frog Eleutherodactylus maninicensis of Barbados. Proi: Zoo/. Sat: Loud. 133: 453-469. Auber- 1 homay, M., and F. Letcllier. 1986. Observations sur le devel- oppement de la rainette marsupiale, Gastmtheca riobambae(J3.y]ides). Rev. Fr. Aquariol 13: 79-86. C'hibon, P. I960. Developpement au laboratoire d'E/eiilherodaclylits maninicensis Tschudi, batracien anoure a developpement direct. Bull. Sot: Zoo/. Fr. 85: 412-418. 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Embryonic stages of Gastrotheca nohamhae (Fowler) during maternal incubation and comparison of development with that of other egg-brooding hylid frogs. J Morphol 167: 277-296. del Pino, E. M.. and A. A. Humphries Jr. 1978. Multiple nuclei during early oogenesis in Heclonolus pygnitieus and other marsupial frogs. Biol Bull 154: 198-212. del Pino, E. M., M. L. (, alar/a. C. M. de Albuja, and A. A. Humphries Jr. 1975. The maternal pouch and development in the marsupial frog Gaslrolheca nohamhae (Fowler). Biol. Bull. 149: 480-491. del Pino, E. M., H. Steinbeisser, A. Hofmann, C. Dreyer, M. Campos, and M. F. Trendelenburg. 1986. Oogenesis in the egg-brooding frog Cast i at hem nohamhae produces large oocytes with fewer nucleoli and low RNA content in comparison to Xenopus laevis. Differentia- tion 32: 24-33. Drewry, G. E. and K. I,. Jones. 1976. A new ovoviviparous frog. Eleulherodaclylus /asperi( Amphibia, Anura. Leptodactylidae). from Puerto Rico. J. llerpetol 10: 161-165. Duellman, \V. E. 1977. Liste der rezenten Amphibien und Reptilien. Hylidae. Centrolenidae, Pseudidae. In Das Tierreich. M. Mertens, W. Hennig. and H. Wermuth. eds. Walter de Gruyter & Co.. Berlin. 95: 230 pp. Duellman, \V. E. 1980. A new species of marsupial frog (Hylidae: Gas- trotheca) from Venezuela, L'niv. Mich Mas Zool. Ocean Pap 690: 1-7. Duellman. \V. E. 1983. A new species of marsupial frog (Hylidae: Gas- lrolheca) from Colombia and Ecuador. Copeia 1983: 868-874. Duellman, \V. E. 1984. Taxonomy of Brazilian hylid frogs of the genus Gaslrolheca. J. Herpelol. 18: 302-312. Duellman, \V. E., and P. Gray. 1983. Developmental biology and sys- tematics of the egg-brooding hylid frogs, genera Hectonolus and fr'ntiiana. Ilerpt'tologica 39: 333-359. Duellman, \V. E., and D. M. Hillis. 1987. Marsupial frogs (Anura: Hylidae: Gaslrolheca) of the Ecuadorian Andes: resolution of taxo- nomic problems and phylogenetic relationships. Herpetologica 43: 141-173. Duellman, \V. E., and M. S. Hoogmoed. 1984. The taxonomy and phylogenetic relationships of the hylid frog genus Stefania. Univ. Kans. Misc. Piibl. 75: 1-39. Duellman, \V. E.. and S. J. Maness. 1980. The reproductive behavior of some hylid marsupial frogs. J llcrpetol. 14: 213-222. Duellman, W. E., and R. A. Pyles. 1980. A new marsupial frog (Hylidae: Gaslrotheca) from the Andes of Ecuador. Occas Pup. Mus. i\'at. Hist. L'niv. Kans. 84: 1-13. Duellman, \V. E., and L. Trueb. 1986. Biology of Amphibians. McGraw- Hill Book Co.. New York. Elinson, R. P. 1987a. Change in developmental patterns: embryos of amphibians with large eggs. Pp. 1-21 in Development as an Evolu- tionary Process. R. A. Raff and E. C. Raft", eds. Alan R. Liss. Inc.. New York. Elinson. R. P. 1987b. Fertilization and aqueous development of the Puerto Rican terrestrial-breeding frog. Eleulherodactylus coqui. J Morphol. 193: 217-224. Elinson, R. P. 1990. Direct development in frogs: wiping the recapit- ulationist slate clean. Seinin. De\: Biol. (in press). TERRESTRIAL-BREEDING FROGS 177 Elinson, R. P. and E. M. del Pino. 1985. Cleavage and gastrulation in the egg-brooding, marsupial frog, Gaslrolheca nohamhae J Emhryol Exp. Morphol. 90: 223-232. Gitlin, D. 194-4. The development of Eleutherodaclyhis porioneensis. Copeia 1944: 91-98. Coin, C. J. 1947. Studies on the life history of Eleutherodactylus ncordn planirostns (Cope) in Florida with special reference to the local dis- tribution of an allelomorphic color pattern. L'mv Fl Stml Biol. Sci. Ser. 4: 1-66. Gosner, K. L. 1960. A simplified table for staging anuran embryos and larvae with notes on identification. Herpetologia 16: 183-190. Gurdon. .). B. 1968. Changes in somatic cell nuclei inserted into growing and maturing amphibian oocytes. J. Emhryol. Exp. Morphol 20: 401-414. Hoogmoed, M.S. 1967. Mating and early development of Gasirotheea marsiipiata (Dumeril and Bihron) in captivity (Hylidae. anura, am- phibia). Br. J Herpelol 4: 1-7. Hughes, A. 1959. Studies in embryonic and larval development in Amphibia. I. The embryology of Eleutherodactylus ncordii. with special reference to the spinal cord. J. Emhryol. Exp. Morphol. 7: 22-38. Hughes, A. 1962. An experimental study on the relationships between limb and spinal cord in the embryo of Eleutherodactylus martini- censis. J. Embryo/ Exp. Morphol 10: 575-601. Jameson, D. L. 1950. The development of Eleutherodactylus lalrans. Copeia 1950: 44-46. Jones, R. E., A. M. Gerrard, and J. J. Roth. 1973. Estrogen and brood pouch formation in the marsupial frog Gastrotheca nohamhae .1. Exp tool 184: 177-1X4. Kageyama, T. 1980. Cellular basis of epiboly of the enveloping layer in the embryo of medaka, Ory-ias talipes. I. Cell architecture revealed by silver staining method. Dev Growth Differ. 22: 659-668. Lamotte, M. and J. Lescure. 1977. Tendances adaptivcs a 1'arfran- chissement du milieu aquatique chez les amphibiens anoures. Terre I'ie3l: 225-311. Laurent, R. F., E. O. Lavilla, and E. M. Teran. 1986. Contnbucion al conocimiento del genero Gastroilieca Fitzinger (Amphibia: Anura: Hylidae) en Argentina. Aclti Zool. Lilloanu 38: 171-221. Lynn, \V. G. 1942. The embryology of Eleutherodactylus nnhicoUi. an anuran which has no tadpole stage. Contnh. Emhryol. Carnegie In- stitute Hash. 190: 29-62. Lynn, \\ . G., and B. Lutz. 1946. The development of Eleutherodactylus gucnthen Stdnr. 1846 (Salientia). Bol. Mus Nac. Brasil (Nova Ser.) 71: 1-46. Lynn, \V. G., and B. Lutz. 1947. The development of Eleutherodactylus nasutus Lutz. Bol. Mus Nac. Brasil (Nova Ser.) 79: 1-30. Lynn, \V. G. and A. M. Peadon. 1955. The role of the thyroid gland in direct development in the anuran. Eleutherodactylus inarnmcensis. Growth 19: 263-286. Macgregor, H. C., and E. M. del Pino. 1982. Ribosomal gene ampli- fication in multinucleate oocytes of the egg brooding frog hylid t'lee- tonolus pygmaeiis. Chromosoma iBerl.) 85: 475-488. Noble, G. K. 1927. The value of life history data in the study of the evolution of the amphibia. Ann ,V } Aead. Sci 30: 31-128. Rivcro, J. A. 1978. Los antihios v reptiles dc Puerto Rico. Editorial Universitaria. Universidad de Puerto Rico, San Juan. Puerto Rico. Rugh, R. 1965. Experimental Embryology. Third Edition. Burgess Publ. Co., Minneapolis. Minnesota. Sampson, I.. V. 19(14. A contribution to the embryology of Hvlodes muriinicensis. Am J Anal 3: 473-504. Stewart, M. M. 1985. Arboreal habitat use and parachuting by a sub- tropical forest frog. J Herpelol. 19: 391-401. Townsend, D. S. 1984. The adaptive significance of male parental care in a neotropical frog. Ph.D. dissertation. State University of New York. Albany. Townsend, 1). S., and M. M. Stewart. 1985. Direct development in Eleiilherodaclyliis coqui (Anura: Leptodactylidae): a staging table. Copeia 1VS5. 423-436. Townsend, D. S.. and M. M. Stewart. 1986a. Courtship and mating behavior of a Puerto Rican frog. Eleutherodactylus coqui llerpeto- lo K ica42: 165-170. Townsend, D. S., and M. M. Stewart. 1986b. The effect of temperature on direct development in a terrestrial-breeding, neotropical frog. Copeia 1986: 520-523. Townsend, D. S., M. M. Stewart, F. H. Pough, and P. F. Brussard. 1981. Internal fertilization in an oviparous frog. Seience212: 469- 471. Townsend, D. S., M. M. Stewart, and F. 1 1. Pough. 1984. Male parental care and its adaptive significance in a neotropical frog. Anim. Behav. 32:421-431. Townsend, K. 1985. Ontogenetic changes in resource use by the Puerto Rican frog Eleutherodactylus coqui. M.Sc. Thesis, State University of New York. Albany. Trueb, L. 1974. Systematic relationships of neotropical horned frogs, genus Hemiphractus (Anura. Hylidae). Occas. Pap. Mus. Nat. Hist. Univ. Kans 29: 1-60. Trueb, L. and Duellman. \V. E. 1978. An extraordinary new casque- headed marsupial frog (Hylidae: Gastrotheca). Copeia 1978: 498- 503. Valett, B. B., and D. L. Jameson. 1961. The embryology of Eleuthero- daclylm augusti lalrans. Copeia 1961: 103-109. Wake, M. H. 1978. The reproductive biology of Eleutherodactylus jasperi (Amphibia. Anura, Leptodactylidae), with comments on the evolution of live-bearing systems. J. Herpelol. 12: 121-133. Wolf, D. P., and J. L. Hedrick. 1971. A molecular approach to fertil- ization. II. Viability and artificial fertilization of Xenopus laevis ga- metes. r>ev. Biol. 25: 348-359. /.immermann, E. 1983. Das Ziichlen von Terrarientieren: Pjlege. Ver- halien. Fortpjlanzung. Kosmos Bticher, Franckh'sche Verlagshand- lung, W. Keller & Co., Stuttgart. Reference: Biol Hull 179: 178-1X2. (October. The Nocturnal Emergence Activity Rhythm in the Cumacean Dimorphostylis asiatica (Crustacea) T. AKIYAMA AND M. YOSHIDA* Ushimado Marine Laboratory, Okayama University, Ushimado 701-43. Japan Abstract. The crustacean Dimorphostylis asiatica in- habits the suhlittoral zone and actively swims in the water at night. Males are positively phototaxic, and can be col- lected on the surface by using a light at night. The timing of this emergence was investigated in the field. Nearshore collections of males have demonstrated a clear rhythmic pattern, with emergence synchronized with both day-night and tidal cycles. The remarkable feature of this record was that the tidal aspect of the pattern was modified sea- sonally. While emergence was strongly synchronized with high tide during the winter and spring, tidal synchrony was scarcely detected in summer and autumn. Environ- mental factors that affect the seasonal modification of the activity pattern are still unknown. Introduction On intertidal and estuarine shores, organisms are ex- posed to periodical changes due to tides and the day-night cycle. These organisms have often evolved, as an adap- tation to these environments, timing of activities syn- chronized with both daily and tidal cycles. The activity patterns differ, reflecting the degree of synchronization with these environmental factors, i.e., daily rhythms (En- right and Hamner, 1967; Hammond and Naylor, 1977), tidal rhythms (Morgan, 1965;Klapow, 1972), and timing with both components (Barnwell, 1966; Benson and Lewis, 1976). Although seasonal modification of rhythmic behavior is well documented for terrestrial species (see reviews by Saunders, 1976, and Gwinner, 1981), few investigations have been carried out on such aspects of intertidal and estuarine organisms. The present study deals with long- Received 18 December 1 9X9; accepted 20 July 1990. * Deceased on 29 October 1988. term field observations on the nocturnal crustacean Di- morphostylis asiatica inhabiting the nearshore sublittoral zone in Japan. The nocturnal vertical migration of other cumaceans in the field was previously reported by Corey (1970). Materials and Methods Adult males of Dimorphostylis asiatica (Order: Cu- macea), 2-4 mm in body length, living in the Inland Sea of Japan, were studied. The animals hide in the mud flat in the daytime. They swim actively after sunset, and only the males are attracted to light. In spite of many plankton tows, no individuals were caught during the day, so the study focused on nighttime activity. A tungsten torchlamp ( 100 V, 200 W) was placed on the edge of a floating pier (about 10 m from shore), and the light beam was directed to the surface of the sea. The maximum depth around the collection site is about 3-4 m at the spring high tide. D. asiatica individuals swimming under the light can be easily distinguished by their bright yellow color and unique pattern of swimming. As soon as they appeared in the illuminated collection area (about 1 m X 2 m in surface area), the animals were caught with a flat hand net (10 cm in diameter, with mesh size of about 1 mm) and brought to the laboratory. Collections were made for 30 min every hour from sunset until sunrise, after which few D. asiatica individuals were seen near the surface of the water. Following each 30-min collection, the electric torch was turned off. Nightly collections spanning more than two weeks were carried out seven times (4-22 Mar. 1987; 1-16 May 1987; 28 June 28 to 6 Aug. 1987; 1 1 Sep. to 7 Oct. 1987; 5-20 Nov. 1987; 5-20 Jan. 1988; 3-18 Mar. 1988). Times of sunset (SS), sunrise (SR), high water (HW1, HW2), and low water (LW1, LW2) were based on data published by the Japan Meteorogical Agency. 178 RHYTHMIC BEHAVIOR IN THE FIELD 179 18 6 I I I I I I I I I I I I I I I I I 18 6 I I I I I I I I I I I I I I I I 1 100 animals 15 I I I I I I I I I I I I I I I I I 18 6 Time of day Figure 1. The emergence pattern in May (1-16 May 1987) as de- termined by the number of Dimorphostylis asialica individuals captured every night from 19:00 to 05:00. The actual times of the collections are indicated by the bars on the time scale. SS: sunset; SR: sunrise; HW1 and HW2: high tides; LW1 and LW2: low tides. Results and Discussion The emergence patterns of D. asialica were summarized in each season. Emergence pattern in March and May Figure 1 shows the number of animals collected during each hour of the night in May. Most animals were col- lected near the time of the night high tide. When high tides (HW1 and HW2) occurred around the time of sun- rise and sunset (6-7 days surrounding the first quarter of the moon), the activity exhibited two peaks in the night, one shortly after sunset and another before sunrise. Col- lections in March showed a similar pattern (data not shown). 30 Aug 1 I I I I I I I I 18 Time of day Figure 2. The pattern of Dimorphosl ylix axialica emergence in July (28 June-6 Aug. 1 987). Collections were made from 19:00 to 05:00. ?: indicates that no collections were made on 1 5 August due to bad weather. Other symbols as in Figure I . Emergence pattern in July The tide-correlated timing, which was clearly evident in Figure 1, became vague during the summer (Fig. 2). Until 4 July, bimodal peaks of emergence activity ap- 180 T. AKIYAMA AND M. YOSHIDA 18 I I I I I I 6 1 I I I I I I I I I I I 1 I I 1 I 1 I I I I 18 6 Time of day Figure 3. The pattern of DimurplnKlyli\ asiatica emergence in Sep- tember-October (23 Sep.-? Oct. 1987). Collections were carried out from 18:00 to 06:00. Symbols as in Figure 1. peared after sunset and before sunrise. Thereafter, those peaks of activity disappeared, and most of the emergence took place on the ebb tide (5-10 July). Compared with the data illustrated in Figure 1, the activity pattern in this season was not as clearly synchronized with the time of high water. For six days surrounding the last quarter moon (18 July), large peaks of emergence were recorded just after sunset. Tidally correlated timing of emergence was weak and disappeared thereafter. Emergence pattern in September and November Tidally synchronized timing was scarcely detectable in either the data from September or from November; the pattern is characterized instead by a peak coinciding with the time of dusk (Fig. 3). The pattern in January The data obtained in January (Fig. 4) show the reap- pearance of tidally correlated timing in the emergence activities. Although the tidal component is not strongly distinguished in the first half of the record, it constitutes the main activity in the latter half of the data. Figure 4 further shows that the timing was gradually modified from being dusk-correlated to tide-synchronized. The time dif- ference between sunset and the following activity peak (see the data of 5- 1 4 January) was an hour or two greater in January than at other seasons (Figs. 1, 3). Number oj animals emerging per day through a year The number of animals that were captured on each night is summarized in Figure 5. There was no clear ev- 18 6 i i i i i i i i i i i mals 20 I I I I I I I I I I I I I I I I I 18 6 Time of day Figure 4. The pattern of Dimorphostylis asiatica emergence in Jan- uary (5-20 Jan. 1 988). Collection times were 18:00-06:30. Symbols as in Figure 1. o JjlulLclil 10 15 20 March RHYTHMIC BEHAVIOR IN THE FIELD CO C B I C 181 D C 15 20 September 30 1 5 October O 10 15 November Figure 5. The number of animals captured on each night of collection. A: the data from the first series of investigations; B: the second; C: the third; D: the fourth; E: the fifth; F: the sixth. Abscissa: the dates. Ordmate: number of males. ?: signifies that no collections were made on that date. idence of lunar or semilunar rhythmic patterns. Although there is some suggestion of a monthly cycle in the record of June-August (Fig. 5C), similar trends are not clearly evident at other seasons. As indicated by the present observations, a distinct emergence rhythmicity was observed for the male pop- ulation of D. asiatica through the year in the field. The swimming activity was synchronized with environmental day-night and tidal cycles, showing a complex activity pattern. The remarkable feature of the records is the an- nual modification of the tidal timing involved in the pat- tern. To explain such a difference in the activity patterns, it is possible to postulate that the animals synchronize their activity with seasonally changing environmental factors. Intertidal organisms respond to physical factors associated with on-shore tides, e.g., changes of hydrostatic pressure (Enright, 1962; Morgan. 1965; Naylor and Williams, 1984) or cycles of water turbulence (Enright, 1965). If such environmental factors fluctuate seasonally, they might affect the timing of the animals, resulting in the different activity pattern in each season. However, the similar pattern of the fluctuation of tidal amplitude in spring and autumn (not illustrated) makes it difficult to consider that the emergence pattern of D. asiatica is di- rectly affected by such factors. Another possibility is that the internal timing of animals was somewhat different in each season, which might cause the annual modification of the emergence pattern in this population. This possibility is presently under investiga- tion in the laboratory. Acknowledgments We thank professor Y. Chiba of Yamaguchi University for his invaluable criticisms of the manuscript. Literature Cited B.ir nurll. F. H. 1966. Daily and tidal patterns of activity in individual fiddler crab (genus Vca) from the Woods Hole region. Bitil. Bull. 130: 1-17. Benson, J. A., and R. H. Lewis. 1976. An analysis of the activity rhythm of the sand beach amphipod, Talorchestia quoyana. ./. Ciiinp. Physio/. 105: 339-352. Corey, S. 1970. The diurnal vertical migration of some Cumacea (Crustacea, Pericarida) in Kames Bay, Isle of Cumbrae, Scotland. Can.J. Zoo/. 48: 1385-1388. Enright, J. T. 1962. Response of an amphipod to pressure changes. J. Omip. Bioc-hem. Phyxiol. 1: 131-145. Enright, J. T. 1965. Entrainment of a tidal rhythm. Science 147: 864- 867. Enright, J. T., and \V. M. Hamner. 1967. Vertical diurnal migration and endogenous rhythmicity. Science 157: 937-941. 182 T AKIYAMA AND M YOSHIDA Gwinner. E. 1981. Annual rhythms: perspective. Pp. 391-410 in Bio- Morgan, E. 1965. The activity rhythm of the amphipod Corophiiim logical Rhvlhm\ llumlhook o/' Behavioral Neurobiology. Vol. 4. J. voluilor (Pallasl and its possible relationship to changes in hydro- Aschoff, ed. Plenum Press. London, static pressure associated with tides. J Annual Ecol. 34: 731- II immmicl. R. D., and E. Naylor. 1977. Effects of dusk and dawn on 746. locomotor activity rhythms in the Norway lobster Ncphrop\ norvegi- Naylor, E.. and B. G. \\ illiams. I9S4. Environmental entrainment of < d\ Mar. Kiol 39: 253-260. tidally rhythmic behavior in marine animals. /<><>/ ./. Linnean Soc. Klapo, L. A. 1972. Natural and artificial rephasingof a tidal rhythm. 80: 201-208. J. Comp. Miyxinl. 79: 233-258. Saunders, D. S. 1976. Insect Clocks. Pergamon Press, London. Reference: Binl. Bull. 179: 183-185. (October, 1440) Occurrence of Partial Nuclei in Eggs of the Sand Dollar, Clypeaster japonicus MITSUKI YONEDA 1 AND SHIN-ICH1 NEMOTO 2 { Department of Zoology, Faculty of Science. Kyoto University. Kyoto 606 and -Tateyama Marine Laboratory, Ochanomizu University. Tateyama. Cluha 2im. eggs activated by treatment with 10 Mg/ml Ca-ionophore (A 23187) for 2 min (Fig. 5), although the migration started later and was slower (Fig. 6) than that observed for normally fertilized eggs (also noticed by Mar, 1980). In any event, partial nuclei in activated eggs behaved sim- ilarly to single nuclei. Microtubule inhibitors suppress the migration of nuclei towards syngamy (Zimmerman and Zimmerman, 1967; Schatten and Schatten, 1981; Hamaguchi and Hiramoto 1986). Movement of Clypeaster nuclei was stopped by treating eggs with 1 mAl colchicine in seawater 5 min after activation. The movement of partial nuclei was sim- ilarly suppressed, and they remained separated, failing to form a single nucleus (Fig. 7). Yet both nuclei eventually exhibited the breakdown and reformation of their nuclear envelopes, as was observed in mononucleate eggs treated with either Colcemid (Sluder, 1979. 1986) or colchicine (Yoneda and Schroeder, 1984). In several aspects, therefore, the partial nuclei behave very similarly to single nuclei. I 8 * " ' QJ / ^c 6 roO 5 b-i Q '0567 8 9 10 Diameter of major nuclei (//m) Figure 2. Diameters of partial nuclei measured in living unfertilized binucleate eggs. The diameters of smaller nuclei are plotted (open circles) on a cubic scale against the diameter of larger nuclei also on a cubic scale. Data are from a batch of eggs from a single female. The area between two diagonal lines ("9" and "10") indicates the domain within which the summed volumes of major and minor nuclei is equal to a sphere with a diameter between 9 ^m and 1 nm. The diameters of single nuclei from the same batch were the control (filled circles). 20 //m o> o 03 4-1 C/) b 15 20 TO Time after fertilization (min) Figure 4. Migration of female pronuclei in normal (a) and binucleate (b) eggs after fertilization at 26C. Migration indicated by the decrease in the distance (in ordinates) between pronuclei and the center of the eggs. Stars in (b) indicate fused nuclei. PARTIAL NUCLEI IN SAND DOLLAR EGGS 185 Figure 5. Migration and fusion of two partial nuclei on activation with Ca-ionophore. Times after activation are indicated by numerals. 26C. Scale bar = 30 /*m. Remarks Describing the presence of partial nuclei, Boveri's paper (1918) warns us of failure if we enucleate sea-urchin eggs hv manual bisection, but overlook the occurrence of mul- '10 20 30 Time after activation (min) Figure 6. Migration of "female pronuclei in normal (a) and binudeate (b) eggs on activation at 26C, as indicated by decrease in the distance (ordinates) between pronuclei and the centers of the eggs. Stars in (b) mark fused nuclei. Data in Figures 4 and 6 are derived from a single batch. Note that the migration starts later and is slower than that observed in fertilized eggs (cf. Fig. 4). Figure 7. A pair of partial nuclei in activated eggs treated with 1 m.l/colchicine 5 min after activation. Numerals indicate the time after activation. Both nuclei remained separated, but their nuclear envelopes still broke down (40 mm) and reformed (79 min). 26C. Scale bar = 25 tiple nuclei. Thanks to the natural transparency of Cly- pcustcr eggs, we can easily detect, with a low power mi- croscope, batches of eggs including those with partial nu- clei. Using eggs with partial nuclei may give us some insights into the process of nuclear migration and nuclear fusion in echinoderm eggs. Literature Cited Boveri, I. 1918. Zwei Fehlerquellen bei merogonischen und die Ent- wicklungsfahigkeit merogonischer und partiell-merogonischer Seei- gelbastarde. Arch. Entwicklungsmech. 44: 419-471. Chambers, R., and E. L. Chambers. 1961. Explorations into the Nan/re <>/ the Living Cell Harvard University Press, Cambridge. Hamaguchi, M., and Y. Hiramoto. 1986. Analysis of the role of astral rays in pronuclear migration in sand dollar eggs by the Colcemid- UV methods. Dt-\: Growth Differ. 28: 143-156. Mar, II. 1980. Radial cortical fibers and pronuclear migration in fer- tilized and artificially activated eggs of Lytecliinus pictus. Dev. Biol. 78: 1-13. Schatten, G., and Il.Schatten. 1981. Effects of motility inhibitors during sea urchin fertilization. E.\p. Cell Res. 135: 31 1-330. Sluder, G. 1979. Role of spindle microtubules in the control of cell c\cle timing. ./ Cell Biol 80: 674-691. Sluder, G. 1986. The role of spindle microtubules in the timing of the cell cycle in echinoderm eggs. J Exp. Zoo/. 238: 325-336. Von Ledebur-Villeger, M. 1972. Cytology and nucleic acid synthesis of parthenogenetically activated sea urchin eggs. Exp. Cell Res. 72: 285-308. Yoneda M., and I. H. Schroeder. 1984. Cell cycle timing in colchicme- treated sea urchin eggs: persistent coordination between the nuclear cycles and the rhythm of cortical stiffness. J. Exfi. Zoo/. 231: 367- 378. Zimmerman, A. M., and S. Zimmerman. 1967. Action of Colcemid in sea urchin eggs. / Cell Biol. 34: 483-488. Reference: Bw/ Bull 179: 186-190. (October, Drag Coefficients of Swimming Animals: Effects of Using Different Reference Areas DAVID E. ALEXANDER Department of Physiology and Cell Biology, 5057 Haworth Hall. University of Kansas, Lawrence, Kansas 66045-2106 Abstract. The drag coefficient (C D ) is useful for com- paring the hydrodynamic drag among different swimming animals. However, C D is calculated using an arbitrary ref- erence area for which there is no uniform convention; both total surface area ("wetted area") and maximum cross-sectional area ("frontal area") are widely used. The choice of reference area can have a profound effect on calculations of drag coefficient. To illustrate this problem, drag measurements from two isopod crustacean species were used to calculate C D based on both wetted and frontal areas. Idotea wosnesenskii had a higher mean C D based on wetted area (0.084) than Idotea resecata (0.059), but a lower mean C D based on frontal area (0.95) compared to /. resecata (1.22); both differences are statistically sig- nificant. Given that there is no powerful hydrodynamic basis for choosing either reference area, and that conver- sions between wetted area C D and frontal area C D cannot accurately be made for complex shapes, I suggest reporting both wetted area and frontal area C D 's wherever practical. Introduction Students of animal swimming often find it useful to measure the hydrodynamic drag experienced by an ani- mal. In steady swimming, thrust equals drag, and data on thrust production are requisite to study such topics as swimming biomechanics (e.g.. Webb, 1975; Wu, 1977) and energetic costs of transport (e.g.. Hargreaves, 1981; Daniel. 1983). To facilitate comparisons among individ- uals or among different species, many investigators have borrowed, from engineers, the concept of "drag coeffi- cient" (C D ) defined by: C D = 2D/pv 2 S Received 1 I April 1990; accepted 20 July 1990. (1) where p = fluid density, v = speed, S = reference area, and D = drag force (Fox and McDonald, 1978)'. The drag coefficient is dimensionless and is typically used to compare the effects of drag on objects of different config- urations or morphologies. For a given shape, the drag coefficient is a function of the Reynolds number (Re): Re = (2) where 1 = reference length (usually the length parallel to movement or fluid flow), and n = dynamic viscosity (Hoerner, 1965; Fox and McDonald, 1978). The Reynolds number may be interpreted as a dimensionless index of the relative importance of pressure (inertial or form) drag versus viscous (friction) drag (Fox and McDonald. 1978; Vogel, 1981). For simple shapes at Re < 1. viscous drag predominates, and the drag coefficient is a simple log- linear decreasing function of the Reynolds number. How- ever, at higher Reynolds numbers, pressure drag is most important, and the behavior of C D with increasing Re is very complex and may be strongly influenced by turbu- lence (although C D changes very gradually at Re > 10 6 ) (Hoerner, 1965). Most swimming animals have "inter- mediate" Reynolds numbers (10 2 -10 5 ), where neither viscous nor inertial forces dominate the flow (Hoerner, 1965), and it may be difficult to predict what shapes will give the lowest drag (Vogel, 1981). Biologists have thus found it convenient to compare drag coefficients of a va- riety of animals to determine which morphologies generate the least drag. For example, Blake ( 1985) used drag coef- ficient measurements to show that an actively swimming decapod crab species had a lower drag morphology than two other benthic species. Similarly, Gal and Blake (1987) ' Note that this equation, often given as D = '/!p\~SC D [e.g.. Hargreaves, 1981: Blake. 1985], defines the "drag coefficient." not the "drag." 186 DRAG COEFFICIENTS OF SWIMMING ANIMALS 187 compared drag coefficients of a frog species that is entirely aquatic with one that is more amphibious. Comparisons based on drag coefficients can be com- plicated by the choice of reference area. The drag coeffi- cient in the form of equation ( I ) is derived from dimen- sional analysis (Fox and McDonald, 1978), and the ref- erence area is an arbitrary scale factor with dimensions of (length) 2 . The choice of reference area can have a sig- nificant effect on the magnitude of the drag coefficients. For example, Webb (1975) reported a C D of 0.015 for a small trout, whereas Nachtigall's beetles had C D 's from about 0.3 to 0.4 (Nachtigall, 1977). Some difference might be expected between fish and beetles on morphological grounds, but the major reason is that different reference areas were used to compute the drag coefficients: the fish drag coefficients were based on total surface area or "wet- ted" area, C Dw (Webb, 1975), but the drag coefficients of the beetles were based on maximum cross-sectional or "frontal" area. C D , (Nachtigall, 1977). Both reference areas are commonly used in engineering practice (e.g.. Hoerner, 1965; Fox and McDonald, 1978; Bertin and Smith, 1979). Hoerner (1965) and Fox and McDonald ( 1 978) generally use frontal area for the drag coefficients of simple shapes (spheres, cylinders, etc.) and wetted area for streamlined objects and whole vehicles or vehicle models (but without an explicit statement of conventions). Frontal area is typ- ically much easier to measure than wetted area (see below), but wetted area is probably more appropriate in most cases, as animals rarely have simple shapes (if they did, there would be little point in measuring their drag coef- ficients!). Two other reference areas do have explicit usage conventions: vertically projected area, or "planform" area, is used for the drag coefficient of wings (or other lifting surfaces) and volume" 73 is used for airship drag coefficients (Vogel, 1981). In the present paper, data from two species of swimming isopods (Alexander, 1988; Alexander and Chen, 1990) are used to show how the choice of reference area can have a profound effect on drag comparisons. Choosing the appropriate reference area in different situations is also discussed. Materials and Methods All drag measurements and wetted area measurements are taken from Alexander and Chen ( 1990). Briefly, spec- imens of Idotea resecata and Idotea wosnesenskii (Iso- poda: Crustacea) were preserved, fixed in a life-like swim- ming posture, and mounted on a force transducer; they were placed in a flow tank at a flow speed equal to a realistic swimming speed, and the drag was measured with the force transducer. Wetted area was estimated by ap- proximating animals as oblate spheroids, with body lengths used for major axes, and the means of maximum body height and width used for minor axes (Alexander and Chen, 1990). The frontal area of these same animals was measured as follows. The preserved isopods were mounted directly head-on in the field of view of a closed-circuit, solid-state Sony video camera equipped with a Nikon 55mm macro lens. The image was displayed on a Burle high-resolution television monitor (38 cm diagonal screen). A l-cm : graph paper grid was also in the field of view of the camera. Each isopod's frontal image was traced onto a plastic transparency sheet, along with the 1-cirr grid. The isopod tracing and the area grid were cut out and weighed to the nearest 0. 1 mg on an electronic balance, and the weight of the area grid was used to calculate the area of the isopod tracing. Each isopod was traced and cut out three times, and the average weight used to calculate the frontal area; typically, tracings for one individual varied by about 3%, never more than 8%. Drag coefficients were calculated using equation ( 1 ). Statistical analyses were based on pro- cedures given in Zar ( 1984) and the microcomputer ver- sion of "Minitab" software. Results and Discussion Figure la shows the drag coefficients based on wetted area for the two species. Idotea resecata had significantly lower CD.'S than I. wosnesenskii (P < 0.001, F [U8 ] = 19.38); the mean C Da for /. resecata was 0.059, versus 0.084 for / wosnesenskii. Using the same drag data, but recalculating the drag coefficients using frontal area. Figure Ib shows that the situation is reversed; in this case, /. wosnesenskii has a significantly lower mean C Df (0.95) relative to /. resecata (mean C Df = 1.22) (P < 0.01, F luit] = 12.6). The data in Figure la and b are from the same animals in the same orientation and posture. The only difference is the choice of reference area. How can such a seemingly trivial change cause such a drastic reversal in the results? Vogel mentioned that when frontal area is used, "stubbier" (shorter, blunter) shapes should have the lowest drag coefficient, but if wetted area is used, elongate shapes will generally have lower drag coefficients (Vogel, 1981, p. 112). Consider two elongate (not bluff) objects with the same frontal area but differing substantially in length: the short object will typically have lower drag, and thus, lower C Df . In contrast, if two objects have the same wetted area, but one is shorter, the short one will necessarily be more bulbous or "bluff" and, hence, generally have a larger wake. Where Re > 1, the wider wake of the short object is likely to cause more drag and hence, a larger C Dw . As Figure 2 shows, the two Idotea species exactly fit these descriptions: Idotea resecata is more elongate and has a lower C Dw , whereas Idotea wos- nesenskii is blunter and shorter, and has a lower C Df . The startling aspect is that the differences between the species 188 D. E. ALEXANDER a 01 (a) O.IO o o 01 -t-i i o ro 0.08 o ^ o o m c 0.08 01 ^ o ^1 0.04 ** (D " 0.02 CD /. resGcoto o /. wosnesenskii a ^ a n nn i i i . i , 0.0 4.0 8.0 12.0 18.0 Speed Ccm/s) 20.0 24.0 28.0 a 0) .8 .5 o 1.2 c 0) 0.9 01 o 0.3 01 a 0.0 Cb) o o o 0.0 4.0 8.0 12.0 Speed 16.0 ( cm/s) 20.0 24.0 28.0 Figure 1. The relationship between swimming speed and drag coefficients for two species of Idotea. Each symbol represents a different individual, and shows the mean of 6 to 12 swimming speed trials and 3 drag measurements. The same individuals are represented on both graphs, (a) The drag coefficient calculated using wetted area, (b) The drag coefficient using the same data as in (a) but with drag coefficient calculated using frontal area. are statistically significant in both cases, even though re- versed. Engineers tend to use the frontal area for drag coeffi- cients where the viscous drag is important, and wetted area where pressure drag is more important (Fox and McDonald, 1978). This is reasonable: at low Reynolds numbers details of shape and orientation have little influ- ence on drag, so frontal area is an adequate scale factor; DRAG COEFFICIENTS OF SWIMMING ANIMALS 189 /. resecota I- wosnesens/iii Figure 2. Dorsal views of the bodies of male (upper) and female (lower) individuals of Idoieu rexecutu and / wnsiiesenskii (traced from video images). The mean fineness ratio (length/width) for the individuals in this study was 4.5/1 for /. rcsccala and 2.9/1 for / mm at high Reynolds numbers, streamlining and orientation are important in that the length of an object in the direc- tion of flow (or movement) affects boundary layer sepa- ration and wake size. Thus, at high Reynolds numbers, frontal area would be a poor scale factor, as it would be the same for a sphere and a well-streamlined object. How- ever, the choice of a reference area is ultimately arbitrary, and the typical types of objects to which engineers apply C Df or C Du may be as much a matter of convenience, as due to fluid mechanical principles. The problem for biologists is that many (if not most) macroscopic swimming animals operate at intermediate Reynolds numbers. In such cases, the relationship between drag coefficient and Reynolds number is strongly depen- dent on the geometry of the object and the presence or amount of turbulence in the fluid (Hoerner, 1965: p. 16.6). Furthermore, one cannot be sure a priori whether viscous or pressure drag is most important. Therefore, as Vogel (1981) noted, it is not clear what shapes will give the lowest drag. Frontal area is attractive simply because it is much eas- ier to measure accurately. Wetted area is difficult to mea- sure on any but the simplest shapes, and virtually im- possible on an object as morphologically complex as an arthropod. Thus, for all practical purposes, an estimate for wetted area must be used, as in Cowles cl at. (1986) or Alexander and Chen (1990). Such wetted area data are very likely to be underestimates for arthropods, as they do not include appendages or surface irregularities due to segmentation; an underestimate of the reference area would lead to an overestimate of the drag coefficient. Because frontal area will typically be more accurate, its use might seem to be preferable. But, as most animals do not have simple shapes, and as typical swimming animals are elongate in the direction of swimming, wetted area is the reference area of choice. However, the wetted area will necessarily be an estimate (and probably a slight un- derestimate) for animals with complex shapes, so com- parisons among animals with different shapes must be made with due caution. Frontal area is appropriate in some situations, primarily for sessile organisms, or motile organisms with no preferred directionality or which do not move with their longest dimension parallel to the di- rection of travel. If a set of drag coefficients is meant as an index for comparing groups of animals, then both C Dw and C Df should be provided. If a study is meant to investigate the relationship between C D and some other variable (c. ,(,'., Reynolds number or speed) where complete presentation of C Dw and C Df would be redundant, then future research- ers may find the data most useful if major relationships are presented using C Dw , with average or typical C Df values being included for reference. Drag coefficient data pre- sented without an explicit statement of the choice of ref- erence area are useless; biologists have no clear conven- tions for choosing reference area. Other possible reference areas ignored so far in this discussion are planform area and (volume) 2 ' 3 . Planform area is appropriately used when investigating drag on an object that is also generating a significant amount of lift. Such an object will have an additional drag component, induced drag, produced by the same process that generates lift. Indeed, to correctly determine the lift-drag ratio, the planform area must be used to calculate the drag coeffi- cient (Hoerner and Borst, 1975; Blake, 1985). Finally, Vogel (1981) suggests using vol 2/1 because, as with lighter- than-air vehicles, the internal volume of a swimming an- imal is likely to be more biologically relevant than other measures of surface area; also, vo! 2/3 can be measured as accurately as desired. However. C D 's based on vol :/1 will be lower for blunt shapes than elongate ones (for the same reasons as for frontal area) (Vogel, 1981), which de-em- phasizes streamlining. Also, drag coefficients based on vo! 2/1 are exceedingly rare in the biological literature, so for comparative purposes it may be advantageous to in- clude C Dw data in such studies as well. Acknowledgments I thank T. Chen for help with data collection, and S. Vogel and H. M. Alexander for useful comments on the manuscript. This work was supported in part by a Biomedical Sciences Support Grant from the University of Kansas. Literature Cited Alexander, D. E. 1988. Kinematics of swimming in two species of Idoteu (Isopoda: Valvifera). J. Exp. Biol. 138: 37-49. Alexander, D. E., and T. Chen. 1990. Comparison of swimming speed and hydrodynamic drag in two species of Idolea (Crustacea: Isopoda). J. Crustacean Biol. 10(3): 406-412. Berlin, J. J., and M. L. Smith. 1979. Aerodynamics for Engineers. Prentice-Hall, Englewood Cliffs, New Jersey. 410 pp. 190 D. E. ALEXANDER Blake, R. \V. 1985. Crab carapace hydrodynamics. J. /.ool. (Land.) (At 207: 407-423. Cowles, D. I,., J. J. Childress, and D. L. Gluck. 1986. New method reveals unexpected relationship between velocity and drag in the bathypelagic mysid Gnathophausia ingens Deep-Sea Res. 33: 865- 880. Daniel, T. L. 1983. Mechanics and energetics of medusan jet propulsion. Can. J. tool. 61: 1406-1420. Fo\, R. \V., and A. T. McDonald. 1978. Introduction In l-'litul Me- chanics. 2nd ed. John Wiley & Sons. N.Y. 684 pp. Gal, J. M., and R. \V. Blake. 1987. Hydrodynamic drag of two frog species: Hymenochirns hoellgeri and Ruiui pipiens Can. J Zool 65: 1085-1090. Hargreaves, B. R. 1981. Energetics of crustacean swimming. Pp. 453- 490 in Locomotion & Energetics in Arthropods. C. F. Herreid and C. R. Fortner, eds. Plenum Press, New York. Hoerner, S. F. 1965. Fluid-Dynamic Drag Hoerner Fluid Dynamics. Bricktown. New Jersey. 444 pp. Hoerner, S. F., and H. V. Borst. 1975. Fluid-Dynamic Lift Hoerner Fluid Dynamics, Bricktown. New Jersey. 494 pp. Nachtigall, \V. 1977. Swimming mechanics and energetics of loco- motion of variously sized water beetles-Dytiscidae, body length 2 to 35 mm. Pp. 269-284 in Scale Effects in Animal Locomotion. T. J. Pedley, ed. Academic Press, New York. Vogcl, S. 1981. Life in Moving Fluids Willard Grant Press. Boston 352 pp. Webb, P. \V. 1975. Hydrodynamics and energetics offish propulsion. Bull Fish Res Board. Can. 190: 1-158. \Vu, T. Y. 1977. Introduction to the scaling of aquatic animal loco- motion. Pp. 203-232 in Scale Effects in Animal Locomotion. T. J. Pedley. ed. Academic Press, New York. Zar, J. H. 1974. Biostatistical Analysis. Prentice-Hall, Englewood Cliffs, New Jersey. 620 pp. Reference: Biol. Bull 179: 14 1 -200. (October. Promotion and Inhibition of Calcium Carbonate Crystallization In Vitro by Matrix Protein From Blue Crab Exoskeleton M. E. GUNTHORPE 1 , C. S. SIKES 1 , AND A. P. WHEELER 2 1 Department of Biological Sciences, University of South Alabama, Mobile, Alabama 36688 and -Departmenl of Biological Sciences, Clemson University, Clemson, South Carolina 29634-1903 Abstract. Soluble organic matrix isolated from dorsal carapaces of the blue crab, Callinectes sapidus, inhibited CaCO, crystallization when free in solution. Immobilized matrix complexes, prepared by crosslinking soluble matrix to decalcified crab carapace, promoted CaCO 3 formation in that crystallization in the presence of the immobilized soluble matrix complexes began sooner than in solution controls. In the experimental treatments, deposition of crystals occurred only within the complexes and not in the crystallization solutions. Chitin, a polymer of N-acetyl- D-glucosamine, and chitosan, a deacetylated chitin, which are both insoluble products of the organic matrix of the crab carapace containing little to no matrix protein, did not promote CaCO, crystallization. Complexes of im- mobilized polyanionic synthetic peptides on chitosan also promoted CaCO; crystallization. Addition of a hydro- phobic tail (Ala 8 ) to the polyanionic peptide (Asp 20 ) re- duced the rate of promotion, possibly because the hydro- phobic tail formed a diffusion barrier around crystal nuclei growth sites, suppressing interactions of nascent crystal nuclei with ions in the bulk solution. Introduction The majority of biominerals are formed by an organic matrix-mediated process (Lowenstam, 1981). Organic matrix, extracted by dissolving biomineral in EDTA or dilute acid, is composed of two components that are sep- arated based on solubility. The soluble component des- ignated soluble matrix (SM) is typically composed of anionic proteins, and the insoluble component desig- nated insoluble matrix (IM) often contains more hy- Received 27 November 1989; accepted 20 July 1990. drophobic proteinaceous material that may be crosslinked (Crenshaw, 1972, 1980, 1982; Krampitzrf / CaCOj crystallization by coupons 15. 1 cur) of decalcified crab carapaces at various stages of the molt cyc/e Treatment n Wet weight (mg) Dry weight (mg) Induction period (min) Maximum growth rate (pH/min) Control Formaldehyde 28 16.2 _j_ *) .9 0.0280 0.0040 0.074% 3 21.8 + 5 .1 0.0280 0.0030 Chum 3 221.0 88.6 43 .5 14.0 22.3 7 .5 0.0280 0.0020 2% acetic acid Stage D 4 258.5 43.5 46 .8 10.0 9.3 4 2 0.0024 0.0012 Stage C 3/4 2 280.0 19.0 55 ,0 2.8 4.8 1 .4 0.0029 0.0001 New cuticle Pre-ecdysial 3 38.0 8.2 4.6 0.3 15.3 0.3 0.0005 0.0001 Post-ecdysial 5 56.8 3.0 9 .5 1.4 2.5 I .0 0.0028 0.0005 4% Formaldehyde in 2% acetic acid Stage D 5 225.7 25.9 42 ,5 3.9 4.5 2 .2 0.0035 0.0070 Stage C 3/4 3 229.1 18.4 64 .3 0.5 3.5 1 .4 0.0027 0.0030 New cuticle Pre-ecdysial (F) 3 57.6 1.5 11 .8 0.6 1.6 0.8 0.0046 0.0009 Post-ecdysial 5 55.6 10.2 12 .0 3.3 1.6 .8 0.0032 0.0006 10', IDT A Stage D 4 183.8 34.5 28 .4 + 4.5 5.8 i 1 0.0024 0.0001 4% Formaldehyde in 10%. EDTA Stage D 2 219.9 9.5 42 .0 + 6.9 2.1 1 0.0037 0.0010 Postecdysial = Stage A-B cuticle at the leathery phase before becoming brittle with CaCO, Stage C VJ = intermolt stage. Stage D = calcified cuticle covering the pre-ecdysial cuticle. (F) = 4% formaldehyde only. Means standard deviations. PROMOTION AND INHIBITION BY MATRIX 195 8.3: 8.30 Control Pre-ecdysis/A Pre-ecdysis/F Postecdysis/A PostecoVsis/FA 37 46 56 65 Time (minutes) Figure 1. Promotion of CaCOi crystallization by coupons cut from new cuticle of blue crabs at pre-ecdysis and postecdysis (Stage A-B), following decalcincation in either 2'S acetic acid (A), 4"i formaldehyde (F) to crosslink soluble proteins, or 4% formaldehyde in 2% acetic acid (FA). Crystallization was measured as a downward pH drift, reflecting the removal of CO, : from solutions ol supersaturated artificial seawater. Promotion of crystallization was indicated by a reduction of the induction period prior to the pH decrease in control solutions. Error bars represent typical standard deviations (n = 3). lutions with and without formaldehyde. However, there was no significant difference in the ability of coupons to promote crystallization. The ability of coupons to promote crystallization was reduced with prolonged storage (data not shown). Acid-washed pre-ecdysial cuticle did not pro- mote crystallization, presumably because there were few immobilized matrix proteins present. In contrast, cross- linking of soluble protein on pre-ecdysial cuticle promoted crystallization equal to that of formaldehyde-treated or acid-washed postecdysial (stage A-B) cuticle of compa- rable dry weight (Fig. 1 ). Coupons of chitin, which should not have protein, did not promote crystallization (Table II), indicating that the protein components may function as nucleating sites. That the downward pH shifts during the crystallization assays did indeed indicate growth of CaCO ? crystals was verified in three ways: by measurements of calcium on the coupons, by comparison of these measurements to calculations of the amount of carbonate removed from solution, and by the presence of birefringence on the cou- pons that was acid labile. For example, the coupon that was treated in 4% formaldehyde in 2% acetic acid (Stage D) (Table II) had 20.7 //mole Ca :i deposited on it after crystallization. This compared very well with 20.6 //mole CO 3 2 ~ calculated as removed from solution in that same experiment. Finally, birefringence that was present on the coupons after the crystallization experiments was lost fol- lowing treatment of pieces of coupons with 0.01 A' HC1, which is consistent with dissolution of CaCOi crystals. When a crystal-promoting coupon was suspended in the crystallization solution, the rate of crystal growth was reduced approximately ten fold relative to controls with- out coupons. The bulk solution was visibly turbid with crystals in controls, but when a coupon was suspended in the solution, crystals were not visible in the bulk so- lution. SM from crab carapace, when free in solution, inhibited crystallization (Table I); thus, when present in the coupons, some of it may have diffused into the so- lution, reducing crystal growth rates. In addition, the low- ering of the pH of the bulk solution, due to the growth of crystals on the coupons, would itself lead to a lack of precipitation in the bulk medium. The lack of bulk pre- cipitation was not due to formaldehyde diffusing from coupons in that high doses of formaldehyde, when free in solution, did not inhibit crystallization (Table II). Coupons of chitin or chitosan, which presumably had no protein, did not suppress crystallization in the bulk solution (Tables II. Ill, respectively). However, Gunthorpe ( 1989) demonstrated that small coupons of acid decalci- fied carapace and the membranous layer that underlies the cuticle inhibited crystallization. But when these cou- pons were extensively washed in distilled water, some TABLE III Effect of coupons (5.1 cnr) of chitosan. glutaraldehyde-treated cliitoxan. and various peptidt'-cltitosan complexes on CaCO 3 crystallization Complex n /imole protein on coupon Wet weight (mg) Dry weight (mg) Induction period (mm) Maximum growth rate (pH/min) Control 25 _ _ 15.4 2.5 0.0300 0.0040 Chitosan 5 230.4 67.6 71.8 16.6 18.8 4.7 0.0230 0.0060 PolyAsp(MW 11,500) 3 0.040 0.011 182.9 45.4 65.4 10.5 2.9 1.8 0.0036 0.0002 Asp,o 4 0.373 0.033 237.4 56.7 64.3 16.7 1.8 0.9 0.0056 0.0009 Asp 30 Ala g 3 0.320 0.080 189.0 35.9 63.4 14.6 3.1 0.3 0.0042 0.0008 Asp w 4 0.152 0.040 172.0 25.9 57.4 8.2 5.7 1.0 0.0026 + 0.0001 Asp 4 oAlaj( 3 0.126 0.012 180.8 22.0 61.5 2.0 6.2 0.9 0.0022 0.0008 Values are given as mean standard deviation for n coupons. 196 M. E. GUNTHORPE ET AL 8.33 8.30 8.26 a 30 4O Time (minutes) Figure 2. The effect of coupons (5. 1 cm 2 ) of chitosan (without cross- linked peptide or protein) and a peptide-chitosan complex (polyaspartate, MW 11,500, immobilized on chitosan by glutaraldehyde) on CaCO, crystallization. Error bars represent typical standard deviations (n = 3). promotion of crystal growth was observed with the washed decalcified carapace present compared to the nearly con- trol levels of crystal growth observed with the washed membranous layer present (Gunthorpe, 1989). Thus, loosely associated protein in the crystal-promoting cou- pons may have diffused into the solution and inhibited crystal growth. Deposition of crystals was localized on insolubilized protein complexes, giving rise to a slow rate of crystallization that began significantly sooner than ob- served in controls. Suspensions of chitosan, a partially cationic surface at pH 8.3. did not promote CaCO 3 crystallization (Table III). Various immobilized peptide-chitosan complexes promoted crystallization (Table III; Fig. 2). The presence of an Ala s tail on Asp :( ) showed suppression of promotion (P < 0.01; balanced, incomplete block design), but this tail did not affect promotion when attached to Asp 40 (Fig. 3). The quantities of the various peptides crosslinked to chitosan were statistically the same. Control solutions containing peptides but not chitosan coupons indicated a 4.2 1.6% decrease in the amount of peptides (n = 3) due to interactions with glutaraldehyde after the cross- linking procedure. When the coupon was present, there was a 28.2 to 65.1% decrease in the amount of peptide left in the solution after 1 h. The larger peptides were crosslinked to chitosan to a lesser extent than were the smaller peptides. As also seen in the studies using the crab carapace sys- tem (Table II and Fig. 1), the rate of crystal growth was reduced when coupons with immobilized peptides were suspended in the solution. Crystallization did not occur in the bulk solution in these experiments, rather crystal formation as determined by pH change occurred only on the coupons. This was confirmed by the absence of a pellet of crystals produced by centrifugation of a sample of the bulk solution at 735 X g. An equivalent analysis of the bulk solution in control treatments without coupons pro- duced a white pellet of CaCO, crystals. Discussion Inhibition hy soluble matrix and by synthetic analogs Soluble matrix isolated from dorsal crab carapaces, and synthetic peptide analogs of matrix, inhibited CaCO, crystallization when free in solution. Organic matrices isolated from other organisms are also inhibitors of CaCO, crystallization when free in solution (Termine el al.. 1980; Borman et al., 1982; Doi el al.. 1984; Sikes and Wheeler, 1983, 1986; Swift et al.. 1986; Wheeler et al.. 1988a, b; Wheeler and Sikes, 1984, 1989). Inhibition has been cor- related with the affinity of matrix molecules for crystal surfaces and adequate coverage of growth sites on crystal surfaces (Aoba et al., 1984; Wheeler and Sikes, 1989). 21 32 43 Time (minutes) 54 8.22 Time (minutes) Figure 3. The effect of addition of a polyalanine tail at the C-terminus on polyaspartates of different sizes (A, Asp 20 : B, Asp 40 ) on promotion of CaCO, crystallization by immobilized peptide-chitosan complexes. PROMOTION AND INHIBITION BY MATRIX 197 Immobilized matrix complexes Decalcified dorsal carapaces of the blue crab (immo- bilized matrix complexes) and immobilized peptide-chi- tosan complexes shortened induction periods for crystal- lization /// vitro. Shortened induction periods were also observed in collagen systems (Endo and Glimcher, 1988), and similarly, apatite crystals were formed within a shorter period in the presence of proteolipids (Boskey el at.. 1988). Shortened induction periods are often considered indic- ative of an increased rate of nucleation, which can result from a reduction of the energy that is required for nuclei formation (Garside. 1982). A likely cause of such a de- crease in nucleation free energy would be a decrease in surficial energy of the nuclei (Wheeler and Sikes, 1989) by the immobilized matrix complex. Crystal nuclei bind- ing by immobilized matrix complexes have been dem- onstrated (Termine el ul.. 1981a. b; Addadi and Weiner, 1985, 1986). The immobilized matrix complex is thought to be formed by an association of soluble matrix with insoluble matrix. The IM framework of crab carapaces is chitin, which, when suspended in a solution supersaturated with respect to calcium and carbonate ions, did not promote crystallization. Further, a derivative of chitin (chitosan) did not promote crystallization when suspended in the solution. With decalcification by acetic acid or EDTA, protein remains associated with the chitin framework (Hunt, 1970; Welinder, 1974; Muzzarelli, 1977; Brine and Austin, 1981). As demonstrated, these latter complexes promoted crystallization. An immobilized matrix com- plex (whole matrix) from Nautilus induced mineral de- position, whereas a preparation of the IM framework alone did not (Greenfield, 1987). To further clarify that SM is the functional molecule, SM was crosslinked to various solid supports. Mineral induction was demonstrated by phosphoproteins crosslinked to collagen (Boskey el al. 1988; Endo and Glimcher, 1988; Linde and Lussi, 1988) or AH-sepharose beads (Lussi et al.. 1988; Linde el al.. 1989). Soluble matrix from Mytilus californianus im- mobilized on polystyrene films also induced mineral on the film (Addadi et al.. 1987). Mineral induction was demonstrated by IM frameworks of other organisms, but the presence of SM was not clarified (Bernhardt el al.. 1985; Watabe t>/ a/., 1986). The relative proportions of matrix that was free in so- lution and that immobilized on the IM framework may correlate with the amount of crystal deposition observed in these studies. Lussi et al. (1988) demonstrated that in- duction by immobilized rat dentin phosphoproteins on AH-sepharose beads can be fully inhibited at high con- centrations (>160 Mg/ml) of the phosphoprotein when added before the experiment began. A similar phenom- enon may, in part, explain the temporal control of cal- cification in the new cuticle of blue crabs during the molt cycle. The pre-ecdysial layers are not calcified, and only the epicuticle layer is tanned (Roer and Dillaman, 1984; Freeman and Perry, 1 985 ). It may be that the pre-ecdysial cuticle contains more unbound protein that inhibits crys- tallization, preventing calcification. In contrast, cross- linking of protein onto chitin fibers after ecdysis may pro- mote calcification of the postecdysial cuticle. Accordingly, pre-ecdysial cuticle that had been acid washed thereby removing SM, did not promote crystal- lization. With immobilization of pre-ecdysial SM proteins by formaldehyde, crystallization was enhanced. In con- trast, postecdysial cuticle promoted crystallization signif- icantly, regardless of the treatment. Roer et al. (1988) demonstrated that formaldehyde-treated pre-ecdysial cu- ticle of fiddler crabs ( Uea pugi/alor) induced crystalliza- tion, but not to the extent of formaldehyde-treated post- ecdysial cuticle. Control of calcification was hypothesized to occur by an alteration of the organic matrix resulting in the removal of blocked nucleating sites at ecdysis (Roer et al.. 1988). The immobilized matrix complex can be further char- acterized by studying specific, synthetic analogs immo- bilized on a natural IM framework such as chitosan. In so doing, functional groups can be identified. Cationic groups, such as the free amine groups of chitosan. did not promote crystallization. Anionic groups, such as carboxyl groups of aspartate residues, promoted crystallization when immobilized on chitosan supports. In contrast, Ad- dadi et al. (1987) demonstrated that polyaspartate (M r = 6000) adsorbed on polystyrene films did not nucleate CaCO 3 crystals on the films, perhaps due to differences in the amounts of polyaspartate immobilized and spatial relationships between the polyaspartate and the different solid supports used in the different studies. However, Ad- dadi et al. (1987) also showed that blocking carboxyl groups of protein assemblages from mollusc shells reduced the amount of crystals induced on polystyrene films, and presented other evidence for cooperative influences of SO 4 2 ~ and carboxyl groups in promoting crystallization. The importance of anionic groups such as sulfate and phosphate have also been discussed in other studies (Greenfield et al., 1984; Endo and Glimcher, 1988). In addition to the polyanionic regions, matrix proteins frequently contain substantial hydrophobic domains (Schlesinger and Hay, 1977; Hay et a/.. 1979; Butler et al., 1983; Schlesinger et al.. 1986; Gorski and Shimizu, 1988). The hydrophobic domains are required for com- plete activity of the proteins as inhibitors of crystallization (Hay et al., 1979, Aoba et al.. 1984; Aoba and Moreno, 1990). Therefore, Sikes and Wheeler (1988b) evaluated the effect of attaching a polyalanine domain onto polyas- partate molecules of 15 residues. An enhancement of in- hibition of CaCO, crystal nucleation by polyaspartate- 198 M. E. GUNTHORPE ET AL. polyalanine molecules in solution was observed. Perhaps this enhanced inhibition is due to the disruption of the diffusion of lattice ions to the crystal nuclei by the presence of a hydrophobic layer provided by the polyalanine tails of the molecules. The hydrophobic regions of matrix molecules could also affect their behavior as promoters of crystallization. In this study, therefore, a polyalanine domain of 8 residues was attached to polyaspartate molecules of 20 or 40 res- idues. These polyanionic-hydrophobic peptides were then crosslinked to the insoluble chitosan coupons so that the effects on CaCO, crystallization could be evaluated. At- taching the hydrophobic tail to polyaspartate of 20 resi- dues significantly suppressed promotion of crystallization by the immobilized peptide. However, the polyalanine domain attached to polyaspartate of 40 residues had no measurable influence on the promotion of crystallization by the molecules. This is consistent with the observation of Sikes c/ ul. (1990), who reported that polyaspartate molecules of about 15 to 20 residues had the optimal size for interaction with CaCO 3 crystal nuclei. Although por- tions of the molecules were occupied by adsorption to crystal nuclei, polyaspartate molecules of 40 residues seemed sufficiently large so as to occupy the zone of dif- fusion around the crystal surfaces, with or without the additional polyalanine tail. In the context of the present studies, the results sug- gest that the negatively charged residues of the aspartateiualaninex molecules may have been occupied principally with the surface of the crystal nuclei, and the hydrophobic region may have been extruding from the surface, impeding access to the nucleation template. On the other hand, the aspartate4 alanine x molecules may have presented a significant number of negatively charged residues that were not associated with the surface of the crystal nuclei, and were thus available as possible sites for interaction with crystal nuclei, regardless of the presence of the polyalanine domain. In any event, the relative sizes of polyanionic and hydrophobic regions can clearly influ- ence the tendency for a peptide or protein to function as a promoter of crystallization, and one possible function of the hydrophobic zone of matrix molecules is to regulate the promotion of crystallization. Other possible effects of the hydrophobic region, such as changes in secondary structure that might favor peptide interactions with crys- tals, as described by Addadi and coworkers (1985, 1986, 1987, 1990), also need to be evaluated. Although the immobilized matrix complexes in this study promoted crystallization, crystal growth rates were suppressed relative to control experiments. That is, crystal growth did not occur in the bulk solution when immo- bilized matrix complexes were suspended in the solution. However, crystal growth (white, cloudy precipitate) did occur in the bulk solutions of controls and experiments with chitosan coupons without crosslinked peptide or protein. Similarly, Bernhardt el ul. ( 1985) observed that mineral was induced on the IM framework, and that no crystals were visible in the solution. Typically, mineral induction has been demonstrated by the presence of crys- tals on immobilized matrix complexes (Greenfield et a!.. 1984; Bernhardt et at., 1985; Lussi et at.. 1988; Roer et al.. 1988) rather than in the solutions. Roer et at. (1988) demonstrated, by polarized light microscopy, the presence of crystals grown on decalcified pre- and post-ecdysial cu- ticle after //; vitro mineralization. In the present study, crystal deposition on the immobilized matrix complex was observed by acid-labile birefringence of a sample after suspension in the bulk solution. Calcium measurements by atomic absorption spectrophotometry, before and after the experiment, also indicated crystal deposition on the coupon in amounts that were consistent with the down- ward pH shifts that accompanied the removal of carbonate from solution during crystallization. Deposition of crystal on the immobilized matrix complex and inhibition by diffusible inhibitors such as SM may account for the lack of crystal growth in bulk solutions. Various mechanisms have been proposed to explain nucleation events. For example, the epitaxial hypothesis requiring spatial matching between immobilized anionic groups and the Ca-Ca distance in the lattice suggests spe- cific amino acid sequences that might function as nucle- ation sites. Therefore, studies involving formation of pep- tide-chitosan complexes are underway to test possible mechanisms of matrix-crystal interactions. Literature Cited Addadi, L., J. Moradian, E. Shay, N. G. Maroudas, and S. VVeiner. 1987. A chemical model for the cooperation of sulfates and car- boxylates in calcite crystal nucleation: relevance to biomineralization. Pntc. Nat. Acad Sci. 84: 2732-2736. Addadi, L., J. Moradian-Oldak, and S. VVeiner. 1990. Macromolecule- crystal recognition in biomineralization: studies using synthetic polycarboxylate analogs. In Surface Reactive Peptides ami Polymers: Discovery ami Commercialization, C. S. Sikes and A. P. Wheeler, eds. ACS Books, Washington. DC. (in press). Addadi, L., and S. \\einer. 1985. 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Bull, 179: 201-206. (October. 1990) Rhizophydium littoreum on the Eggs of Cancer anthonyi: Parasite or Saprobe? JEFFREY D. SHIELDS Department of Biological Sciences, and the Marine Science Institute, University of California. Santa Barbara. California. 93106 Abstract. The relationship between host and symbiont is often difficult to assess and quantify. A novel technique that may help assess the host-symbiont relationship of organisms found in crab egg masses is described. This technique may have application in determining the re- lationship of other host-symbiont associations. Crab eggs were killed cryogenically and exposed in combinations with live eggs to a previously unrepoited symbiont of crab egg masses. The results indicated that the chytrid Rlu- :ophydium littoreum is primarily a saprobe that attacks dead eggs; yet at high zoospore densities, it attacks and kills live eggs. Furthermore, R. litton'um is the first chy- tridiomycete to be reported from a marine crustacean host. It was highly prevalent on the eggs of its host and was found throughout the year. Introduction Symbionts are, broadly speaking, two organisms living in association together (de Bary, 1879). There is a wide range of relationships that symbioses encompass e.g., mutualism, commensalism. and parasitism (Noble and Noble, 1985) and these relationships are often difficult to define. Several symbionts live in the broods of com- mercially important crabs and lobsters [e.g., Callinectes sapidus ( Rogers-Talbert, 1948), Cancer anthonyi (Shields eta/.. 1990), Cancer magister (Fisher and Wickham, 1976; Wickham, 1986), Homarus americanus (Aiken el ai. 1985; Campbell and Brattey, 1985), Paralithodes cam- tschatica (Wickham el ai. 1985; Kuris et a/.. 1991)]. In- deed, some of these symbionts are egg parasites or pred- Received 12 December 1989; accepted 20 July 1990. Present address: Department of Parasitology, University of Queensland, St. Lucia. Brisbane. 4067. Australia. ators that may cause widespread brood losses in certain commercial stocks of crustaceans (Wickham, 1986: Kuris and Wickham, 1987; Runs et ai. 1991). Species of bac- teria, zoosporic fungi, nemerteans, and amphipods have been found together on individual crab hosts, and all have been implicated as agents that cause egg mortality. The contributions of these symbionts to egg mortality in pop- ulations of some of these decapod hosts have only recently been elucidated (Shields and Kuris. 1988; Kuris et ai, 1991). This is the first report of a chytrid symbiont infesting a marine crustacean host, the yellow rock crab. Cancer anthonyi. The fungus-like chytrid, Rhizophydium litto- reum Amon, 1984, was recovered and isolated from the eggs of C. anthonyi during a field survey for the presence of egg mass symbionts (Shields and Kuris, 1988; Shields et ai. 1990). The prevalence of the chytrid in the broods of C anthonyi prompted an investigation into its role as a possible agent of egg mortality. The host-symbiont re- lationship was examined in laboratory studies that in- cluded a novel experimental protocol. Materials and Methods Ovigerous crabs were trapped in the Santa Barbara Channel, between Summerland and Gaviota, California. The crabs were collected at depths of 10- 100 m by a com- mercial fisherman, transported directly to the laboratory, and maintained at ambient seawater temperatures in 280- 1 flow-through fiberglass aquaria. The presence ofR. littoreum was established as follows. The egg samples were removed to sterile petri dishes con- taining UV-filtered seawater (2 X 35 ^m activated charcoal filters, one ultraviolet-light filter. Rainbow Plastics, Filter Division, El Monte, California) for direct examination 201 202 J. D. SHIELDS with a stereo microscope. After three days the samples were observed again and streaked with sterile pipettes onto a sterile modified Vishniac medium (M V): 1 .0 g glucose. 1 .0 g gelatin hydrolysate, 0. 1 g bacto-peptone, 0. 1 g yeast extract, 1.0 1 seawater, 15.0 g agar (modified from Fuller ct ul.. 1964), containing antibiotics (500 mg each of pen- icillin-G and streptomycin sulfate per liter). Seawater controls were also cultured. After an additional 3-5 days the plates were examined lor the presence or absence of chytrid thalli. Other substrates from the habitat of C. an- thonyi were not examined for chytrids. Pure cultures of R. littoreum were isolated from the eggs of different crabs on numerous occasions. Isolated cultures of R. littoreum were grown in sterile liquid MV medium (MV as above with 1.0 g agar instead of 15.0 g agar). Cultures were maintained both with, and without, antibiotics (500 mg/1 each of penicillin-G and strepto- mycin sulfate, Sigma Co.) at 15 and 20C. To establish the host-symbiont relationship, live and dead crab eggs (see below) were exposed to the chytrid separately and in combination. Before exposure, egg- bearing setae were removed from the pleopod and placed in UV-filtered seawater. Samples consisting of 80-300 eggs that were attached to individual and intertwined setae were counted, and the number of dead eggs and their apparent cause of death (e.g., mechanical disruption, in- fertility, etc.) were noted. After they had been counted, the samples were washed in UV-filtered seawater con- taining 1.0% bleach for 3-5 min to kill or remove micro- organisms. They were then placed in 35 X 10 mm plastic petri dishes with 3.0 ml of UV-filtered seawater containing antibiotics (500 mg/1 each of penicillin-G and strepto- mycin sulfate). Combinations of live and dead eggs (80- 300 of each per replicate) were then exposed to approxi- mately 1000 zoospores of R. littoreum. Samples of egg-bearing crab setae (80-300 eggs/sample) were plunged into liquid Freon (Pelco) in a metal dish jacketed with liquid nitrogen. The eggs thus killed were thawed in ice cold seawater, and equilibrated to 15C. The samples were then counted and the few broken eggs were recorded. The coats of the eggs killed in this manner were not grossly disrupted. The effect of zoospore density on mortality was deter- mined by exposure of eggs to 10, 100, and 1000 zoospores/ ml at 1 5C. Zoospores from cultures ofR. littoreum were counted with the aid of a hemocytometer (Levy counting chamber). Three replicates of the culture were counted and the appropriate dilutions were made to give estimated densities of 10, 100, and 1000 zoospores/ml. From eight to ten replicates were examined in each treatment. A sep- arate treatment of eggs exposed to antibiotics and diluted MV medium served as the control. Crab egg mortality (i.e., the number of living or dead eggs attacked by the chytrid) was assessed every two to three days for ten days. Eggs in control exposures experienced negligible mortality. Results Rhi:ophydium littoreum was identified by Dr. D. J. Barr (Fig. 1 ). Representative specimens (Barr #580) have been deposited at the Biosystematics Research Centre (Wm. Saunders Bldg., C.E.F. Ottawa, Ontario, Kl A OC6, Canada). The monocentric thalli of R. littoreum ranged from 30 to 90 pm wide on crab eggs and in M V medium. Smaller immature thalli were also observed. The thallus was epibiotic and typically resided externally on the crab egg with the rhizoids penetrating through the egg coat into the contents of the egg. An apophysis was occasionally observed in MV culture. In culture, the life cycle of the chytrid took approximately 3-5 days from the zoospore stage to the production of a mature sporangium (at 15C). Figure 1. (A) Rhizophydium littoreum from culture; sporangium (I) and rhizoids (arrow); bar = 50 /im. (B) Rhi:0.05). The stage of egg development did not influence the proportion of dead eggs attacked by the chytrid. In con- trast, on live eggs, the chytrid preferred eggs in later stages of embryogenesis (Fig. 2). Significantly fewer live eggs were attacked in early stages of embryogenesis (EDS I) than in later stages (ANOVA, arcsin transformation of propor- tions, Sidak's inequality, P < 0.01), but this pattern was not consistent between experiments (e.g., compare Fig. 3B, E). (A) Live eggs BO r L 60 S 40 u 20 III \ (with dead) 01 3456 9 10 (B) Dead eggs 00 80 III // ^\ l 4J i : y 1 60 1 ''/' I a tn 1 40 ^./' 20 //'^ i -=-*' '0123456789 Days post-exposure 10 Figure 2. Infection dynamics of Rhiiophydmm litloreuin on (A) live eggs and (B) dead eggs after exposure to 1000 zoospores. Live eggs in the presence of dead eggs are noted (with dead). Roman numerals refer to early (EDS I), middle (EDS II), and late (EDS III) stages in embryo- genesis (Shields and Kuris, 1988; Shields ct ai. 1990). Error bars not shown. *P < 0.05. significantly different from treatment with live eggs alone (/-test between EDS I live eggs alone and live eggs with dead eggs, at day 10). Not shown is the dead eggs in the presence of live eggs treat- ment. The data were not significantly different from the dead eggs only treatment. Discussion The results show conclusively that R. littoreurn can kill live eggs, but it prefers dead eggs. Under natural conditions (i.e., low zoospore density), R. littorewn may be a facul- tative parasite, but it is more likely a saprobe that lives on dead eggs. Indeed, in some cases, fewer live eggs were attacked by the chytrid when dead eggs were present than when dead eggs were absent. Less conclusive in the laboratory was the relationship between crab embryogenesis and chytrid-related mortal- ity. Consistent patterns were not observed (e.g.. Fig. 3B, E). Variations in fungal pathogenicity or host resistance/ susceptibility manifested by slower growth rates of the thalli or decreased zoospore production may account for EDS I Live eggs In presence of dead eggs 1 2 4 5 6 7 8 9 10 (B) Llve 100 580 3 60 40 20 0123456789 10 (C) Dead eggs 100 0123456789 10 Days post-erposure EDS II (D) Live eggs in presence of dead eggs 5 012345 7 8 9 10 Live eggs I 3 0* 0123456789 10 (F) Dead eggs 100 n I 80 o 60 40 20 0123456789 10 Days post-exposure Figure 3. Infection dynamics of Rhizophydium liiloreitm on eggs of different developmental stages (EDS I or II) after exposure to different zoospore densities. Eggs were exposed to densities of 10 zoospores (triangles and dashed lines). 100 zoospores (boxes and dotted lines), and 1000 zoospores (circles and solid lines). Error bars not shown. *P < 0.05. significantly different from treatments with live eggs alone (Mest, day 10). Not shown is the dead eggs in the presence of live eggs treatment. The data were not significantly different from the dead eggs only treatment. 204 Rfll/Ol'l/YD/L'M ON THE EGGS OF CANCER 205 the differences in attack rates of the chytrid between EDS classes. Pathogenicity and resistance have been examined in fungi-plant associations {e.g.. Miedaner, 1988; De Nooij and Van Damme, 1988; Alexander, 1989), but have re- ceived scant attention in marine associations. The exper- imental protocol can easily be manipulated to examine these factors in more detail. Chytridiomycetes develop in a variety of live and dead fungi, algae, diatoms, and higher plants (Sparrow, 1963). They damage host cells or tissues by direct penetration of rhizoids. which in the living host results in death. Chytrids can occur as saprobes, and facultative and obligate par- asites in nature (Barr, pers. comm.). Previously, Rhizophydium liitoreuiu has only been re- ported from the siphonous green algae, Bryopsis plumosa and Codium sp. (Kazama, 1972; Amon, 1984). It can be readily established in different culture media (Kazama, 1972; Amon, 1984, 1986). Chytrid parasites of other Me- tazoa have been reported from the eggs of cestodes and rotifers (Sparrow. 1963) and from aquatic copepods and ostracods (Whisler et til., 1974; Weiser. 1977). A chytrid- like organism from the branchial lamellae of a shrimp (Uzmann and Haynes, 1969) may be a thraustochytrid, Schizochytrium. Dead eggs are abundant in the broods of many decapod crustaceans (for review see Kuris. 1991). Indeed, popu- lations of several commercially harvested species have re- cently suffered catastrophic brood losses to symbiotic agents (Wickham, 1986; Kuris et ai. 1991). Cancer an- thonyi. however, experiences a relatively small degree of egg mortality (~5.0%, Shields et ai. 1990). This small degree of egg mortality translates into several thousand dead eggs per brood because a large (> 140 mm carapace width) Cancer anthonyi can oviposit up to 3 million eggs (Shields, 1991). Hence, sufficient dead eggs to provide a substrate for R. litiorenm may occur in the egg masses of the majority of the ovigerous population of C. anthonyi. In addition, female Cancer crabs bury themselves in the substrate during oviposition; their broods may become infested with the chytrid at that time. Several species of zoosporic fungi occur on the eggs of decapods (e.g.. Atkinsiella duhia. Haliphthoros milfor- densis, Lagenidium callinectes. Leptolegniella marina, Pvthiwn thalassium). The use of live and dead egg treat- ments may help to establish the role of these zoosporic fungal "pathogens." In addition, the experimental pro- tocol may help to elucidate the roles of egg parasites in other systems. Acknowledgments Drs. D. J. Barr and A. Ulken identified the chytrid. Gil Crabbe collected the crabs. Drs. A. M. Kuris, I. Ross, and R. K. Zimmer-Faust provided encouragement and criti- cisms. Special thanks to Robert Paris, and to Drs. Page Erickson and Steven Fisher for the use of cryogenic de- vices. This work was funded, in part, by a patent grant from UCSB general funds and is also a result of research sponsored in part by NOAA, National Sea Grant College Program. Department of Commerce, under grant number NA80AA-D-00120, project number R/F-75 (Drs. A. M. Kuris and D. E. Wickham), through the California Sea Grant College Program, and in part by the California State Resources Agency. The U. S. Government is authorized to reproduce and distribute this article for governmental purposes. Literature Cited Aiken, D. E., S. L. Waddy, and L. S. Uhazy. 1985. Aspects of the biology of Pseudocarcinonemertes homari and its association with the American lobster. Homurm ainericanux din. J Fish Aanat. Sci. 42:351-356. Alexander, H. M. 1989. An experimental field study of another smut disease of Silent alba caused by Ustilago violacea genotypic vari- ation and disease incidence. Evolution 43: 835-847. Amon, ,). P. 1984. Rhi:or>hydiuni litiorenm. a chytnd from siphona- ceous marine algae an ultrastructural examination. Mycologia 76: 132. Amon. J. P. 1986. Growth of marine chytrids at ambient nutrient levels. Pp. 69-80 in The Biology of Marine Fungi. Cambridge University Press. de Bary, H. A. 1879. Die Erscheinung der Symhiose. Strassburg, Karl J. Tiibner. Campbell, A., and J. Brattey. 1985. Egg loss from the American lobster. Homarus aincncanus. in relation to nemertean. Pseudocarcinone- mertes homari. infestation. Can. J. Fish. Aqual. Sci 43: 772-780. De Nooij, M. P., and J. M. M. Van Damme. 1988. Variation in host susceptibility among and within populations of PlantagO lanceolata L. infected by the fungus Phomopsis subordinaria (Desm.) Trav. Oeculogia 75: 535-538. Fisher, \V. S., and D. E. Wickham. 1976. Mortality and epibiotic fouling of eggs from wild populations of the Dungeness crab. Cancer inagisler. Fish. Bull. NOAA 74: 201-207. Fuller, M. S., B. E. Fowles, and D. J. McLaughlin. 1964. Isolation and pure culture study of marine phycomycetes. Mycologia 56: 745- 756. Kazama, F. Y. 1972. Development and morphology of a chytrid isolated from Bryopsis plumosa. Can J Hot. 50: 499-505. Kuris, A. M. 1991 . A review of patterns and causes of crustacean brood mortality. In Crustacean Issues 6, Crustacean Egg Production. A. M. Wenner and A. M. Kuris, eds. Balkema Rotterdam, (in press). Kuris, A. M.. and D. E. Wickham. 1987. Effect of nemertean egg pred- ators on crustaceans. Bull. Mar. Sci. 41: 151-164. Kuris, A. M., J. D. Shields, S. F. Blau, A. J. Paul and D. F. Wickham. 1991. Infestation by brood symbionts and their impact on egg mortality of the red king crab, Paralithodes camschatica, in Alaska: geographic and temporal variation. Can. J. Fish. Aqual. Sci. (in press). Miedaner, T. 1988. The development of a host-pathogen system for evaluating Fusarium resistance in early growth stages of wheat. J. Phytopathoi (Berl.) 121: 150-158. Noble, E. R., and G. A. Noble. 1982. Parasitology. The Biology of Animal Parasites. Lea and Febiger, Philadelphia. 206 J. D. SHIELDS Rogers-Talbert. R. 1948. The fungus Lagenidium cuHinecle\ Couch (1942) on eggs of the blue crab in Chesapeake Bay. Biol. Bull 94: 214-228. Shields, J. D. 1991. The reproductive ecology and fecundity of Omar crabs. In Crustacean Issues 6. Crustacean Egg Production, A. M. Wennerand A. M. Kuris, eds. Balkema Rotterdam, (in press). Shields, J. D., and A. M. Kuris. 1988. An in \-ilro analysis of egg mor- tality in Cancer unllinnyi the role of symbionts and temperature. Biol. Bull 174: 267-275. Shields. J. D., R. K. Okazaki, and A. M. Kuris. 1990. Brood mortality and egg predation by the nemertean. Carcinonemertes epialii. on the yellow rock crab. Cancer iiiiilnnivi. in southern California. Can. J. Fish. Aquat Sci 47: 1275-1281. Sparrow, F. K. 1963. Aquatic Phycomyivti-s, 2nd ed.. University of Michigan Press, Ann Arbor. I /numn, J. R., and E. B. Haynes. 1969. A mycosis of the pandalid shrimp, Dichelopandalus leptuceros (Smith). J Invertehr. Pulhol. 1 2: 275-277. \Veiser, J. 1977. The crustacean intermediary host of the fungus Coe- IniHiinn'ccs chirontmii Rasin. Ci'ska Mykol. 31: 81-90. V\ ickham, D. E. 1986. Epizootic infestations by nemertean brood par- asites on commercially important crustaceans. Can. ./. f-'ish Actual Sci. 43: 2295-2302. \\ ickham, D. E., S. K. Blau, and A. M. Kuris. 1985. Preliminary report on egg mortality in Alaskan king crabs caused by the egg predator Carcinonemertes. Pp. 265-270 in Proceedings oj the International King Crab Symp. Ser.. Alaska Sea Grant Rep. 85-12. \\hisler, H. C., S. L. Zebold. and J. A. Shemanchuk. 1974. Alternate host for mosquito parasite Coelomomyces. Nature 251: 715-716. Reference: Bio/ Bull. 179: 207-213. (October. 1440) Collagen in the Spicule Organic Matrix of the Gorgonian Leptogorgia virgulata RONI J. KINGSLEY 1 , MARI TSUZAKI 2 , NORIMITSU WATABE 3 , AND GERALD L. MECHANIC 24 ^Department of Biology, University of Richmond, Richmond. \'irginia 23173. 2 Denial Research Center ami ^Department of Biochemistry and Nutrition, University of North Carolina at Chapel Hill. North Carolina 27599-7455. and ^Electron Microscopy Center. The University of South Carolina. Columbia. South Carolina 29208 Abstract. Decalcification of the calcareous spicules from the gorgonian Leptogorgia virgulata reveals an organic matrix that may be divided into water insoluble and sol- uble fractions. The insoluble fraction displays character- istics typical of collagen, which is an unusual component of an invertebrate calcium carbonate structure. This ma- trix fraction exhibits a collagenous amino acid profile and behavior upon SDS-PAGE. Furthermore, the reducible crosslink, dihydroxylysinonorleucine (DHLNL), is de- tected in this fraction. The composition of the matrix var- ies seasonally; i.e.. the collagenous composition is most prevalent in the summer. These results indicate that the insoluble matrix is a dynamic structure. Potential roles of this matrix in spicule calcification are discussed. Introduction The mesoglea of the gorgonian Leptogorgia virgulata contains microscopic calcite (calcium carbonate) spicules (Kingsley and Watabe, 1982). Isolation and decalcification of the spicules yield an organic matrix, which is intimately involved in calcification (see Wilbur and Simkiss, 1968: Weiner and Traub, 1981; Watabe, 1981). Unlike vertebrate osseous tissues that consist of hy- droxyapatite (calcium phosphate) and collagen, collagen has not been associated with the formation of invertebrate calcium carbonate structures (Jope, 1967; Watabe, 1981; Benson et ai. 1983; Swift et al.. 1986). The presence of Received 20 April 1990; accepted 25 July 1990. Abbreviations: EDTA, ethylenediaminetetraacetic acid; PAGE, poly- acrylamide gel electrophoresis; SDS. sodium dodecyl sultate; PAS, pe- riodic acid-SchifTs reagent; TES, N-Tris-(hydroxymethyl) methyl-2- aminoethane sulfonic acid; DHLNL, dihvdroxylvsinonorleucme. collagen, or a collagen-like component, in the spicule proteins of gorgonians had been suggested previously (Sil- berberg et ai. 1972; Goldberg. 1988), but this could not be confirmed (Kingsley and Watabe, 1983). This report presents conclusive evidence for a predom- inant, insoluble "classic" collagen component within iso- lated and apparently homogeneous calcite spicules of L. virgulata collected in the summer months. The collagen is partially characterized, and noncollagenous components of the insoluble matrix are examined as well. Potential roles of the insoluble matrix in spicule calcification are discussed. Materials and Methods Colonies of the gorgonian Leptogorgia virgulata were collected at low tide from Sixty Bass Creek of North Inlet Estuary, Georgetown. South Carolina, in the summer of 1985 and from the subtidal waters off Morehead City, North Carolina, in March, July, and December of 1987. Colonies were immediately cleaned of adhering organisms and debris, frozen, and transported on dry ice and stored at -30C. Organic matrix preparation All preparations were conducted at 4C unless other- wise indicated. The tissues of the colonies that contain the spicules were stripped from their axes, weighed, and washed with 0.02 M NH 4 HCO 3 . The tissue was suspended in 1 volumes of 0.25 M NaCl in 0.2 AI NH 4 HCO 3 buffer adjusted to pH 8.0. The spicules remained insoluble under these conditions. They were released from their sur- rounding tissues by digestion (24 h, at 37C, with shaking) 207 208 R. J. K.INGSLEY ET AL with 1% papain (substrate/enzyme, w/w) activated with 0.005 M cysteine. The supernatant was decanted, and any undigested tissue was treated with additional papain, as described above, for another 24 h. The tissues surrounding the spicules were completely digested following these treatments. The remaining spicules were washed thor- oughly with the above buffer and retained on a 250 ^m mesh sieve. The spicules. while contained in the sieve, were washed thoroughly with the same buffer to avoid any possible contamination by enzyme or solubilized material. Examination under a microscope indicated an apparently homogeneous preparation of spicules. Spicules were suspended in an equal volume of 0.5 M potassium EDTA in 0.05 M NH 4 HCO 3 . pH 8.0, and de- mineralized by dialysis (with 3500 dalton cut off tubing) against the same solution. Following demineralization, the total content was recovered from the dialysis tubing and centrifuged. The insoluble organic matrix residue was washed 3 times with 0.2 M NH 4 HCO,, 6 times with 0.05 M NH 4 HCOi, and 3 times with distilled water. The washed insoluble matrix was lyophilized. The supernatant and the washings, which contained the soluble matrix proteins, were dialyzed exhaustively against distilled water (same cut off tubing), and then lyophilized. Aniino acid analysis The amino acid compositions of: ( 1 ) total insoluble matrices from South Carolina, and March. July and De- cember samples from North Carolina; (2) subtractions of the North Carolina July insoluble matrix (described be- low); and (3) the soluble matrices from July and December were determined. Each sample was hydrolyzed in 200 ^1 of 6 N HC1 in an N : atmosphere, for 20 h at 1 10C. Amino acid analysis was performed on a Varian 5560 Liquid Chromatograph using a stainless steel cation-ex- change column (0.4 X 25 cm, AA 911, Interaction) with post column ninhydrin detection. Color was developed at 135C in a stainless steel reaction coil (Yamauchi et al.. 1986). Collagen characterization The insoluble matrix was treated with 5% pepsin (w/ w) in 10 volumes of 0.5 M acetic acid for three days at 21C. The reaction mixture was centrifuged at 25,000 RPM in an ultracentrifuge for 1 h. The insoluble portion was washed thoroughly with 0.5 M acetic acid and ly- ophilized. The supernatant was brought to 3.0 M NaCl in the cold and allowed to stand overnight. The precipitate, after centrifugation at 10,000 RPM for 20 min, was re- dissolved in 0. 1 M acetic acid, exhaustively dialyzed against 0. 1 A/ acetic acid, and lyophilized. The supernatant of the 3.0 M NaCl solution was dialyzed exhaustively against distilled water, and lyophilized. No material was observed. The original insoluble matrix, pepsin solubilized material and pepsin-insoluble material were hydrolyzed as described above and subjected to amino acid analysis. Cyanogen bromide (CNBr) cleavage The various fractions obtained above were treated with 25% mercaptoethanol in 0.2 \/NH 4 HCO, at 55C over- night to completely reduce any methionine sulfoxide res- idues back to Met (methionine). These fractions were then lyophilized, digested with CNBr in 70% formic acid in an N 2 atmosphere for 4 h at room temperature, diluted with distilled water, and lyophilized again. Polyacrylamide gel electrophoresis (PAGE) Portions of each fraction, both treated and untreated with CNBr, were subjected to PAGE in 0. 1% SDS (Laem- mli and Favre, 1973). Soluble type I collagen from foetal bovine skin was used as a standard. Tube gels (4% acryl- amide) and gradient slab gels (3-17%. acrylamide) were employed. Some of the very insoluble samples were treated at 100C for 10 min with 0.2% SDS electrode buffer (Laemmli and Favre, 1973) to which 2 M urea was added. Gels were stained for protein with 0.05% Coomassie Bril- liant Blue, and for carbohydrates with the PAS reagent (Zacharius et al.. 1969). Determination of cross-links Samples of lyophilized untreated insoluble spicule ma- trix were suspended in 0.15 Al TES buffer, pH 7.5, and reduced with standardized NaB'H., (Fukae and Mechanic 1980; Yamauchi et al.. 1986). The reduced insoluble ma- trix was hydrolyzed in vac no with 3 N HC1 for 48 h at 115C (Yamauchi el al.. 1986). Analysis of cross-links was performed on a Varian 5560 Liquid Chomatograph as described previously (Yamauchi et al.. 1986). Hy- droxyproline (Hyp) analysis was performed by amino acid analysis and residues of cross-link per mole of collagen was calculated on the basis of 300 residues of Hyp per molecule collagen. Results Amino acid compositions The amino acid compositions of the matrix fractions of gorgonians collected from South Carolina and North Carolina are shown in Table I. Also presented are amino acid compositions of an invertebrate collagen and a mammalian mineralized collagen (Table I I. J, respec- tively). The compositions of the insoluble matrices of the two summer samples are similar (Table I A, C). Both samples display compositions typical of a collagen, since they contain significant amounts of Hyp, Hyl (hydroxy- COLLAGEN IN GORGONIAN SPICULES TABLE I Anuno uciil cimipmilnms />! matrix protein fractions from isolated calcilc xpimlcs <>j Leptogorgia virgulata 209 A TIM S.C. B TIM 3/87 c TIM 7/87 Residues per 1000 D E TIM Pepsin 12/87 sol Total Residues F Pepsin res G SM 12/87 // SM 7/87 / Sea anemone collagen" 1 J Bovine bone e Hyp 74 8 82 31 80 1 1 1 103 98 Asp 93 132 76 130 84 153 516 577 73 45 Thr a 36 57 34 48 32 63 18 16 37 17 Ser a 43 57 42 53 43 78 16 13 41 34 Glu 109 87 94 92 112 76 34 32 97 74 Pro 66 67 65 59 64 50 17 19 67 123 Gly 295 158 332 225 335 161 210 185 339 337 Ala 83 78 82 76 77 67 1 30 108 65 104 Val 27 58 26 37 >> 55 17 16 25 20 Cys b /2 2 16 1 10 17 Met c 4 1 1 7 10 5 10 5 Ik- 18 38 15 26 13 46 6 5 22 1 1 Leu 27 53 26 42 23 58 7 6 30 25 Tyr 8 24 8 20 6 32 2 3 4 Phe 9 31 9 22 7 35 4 4 8 13 His 3 17 4 1 1 3 11 2 1 1 4 Hyl 33 7 34 15 34 6 25 6 Lys 15 44 12 33 10 33 12 9 16 26 Arg 56 55 50 53 50 39 8 5 68 50 TIM Total Insoluble Matrix. S.C. Summer collection in South Carolina. 3/87. 7/87. 12/87 Dates of collection in North Carolina. Pepsin sol 7/87 collection, pepsin soluble material. Pepsin res 7/87 collection, pepsin insoluble material. SM Soluble Matrix. a Uncorrected for hydrolysis. h Half Cys. sum of cysteic acid and cystine. c Sum of methionine sulpho.xide and methionine. d Nowack and Nordwig 1974. c Herring 1972. lysine) and 33% Gly (glycine). The composition in column C is extremely close to that of the pepsin solubilized in- vertebrate collagen of the sea anemone (column I). The amino acid composition of the insoluble matrix from samples collected in March 1987 displays markedly lower values of Hyp. Hyl and Gly (Table IB). Gly, Asp (aspartic acid) and Glu (glutamic acid) are the most abundant amino acids. The insoluble matrix from samples collected in December 1987 displays an amino acid composition that is intermediate to the March and July compositions (Table ID). The July insoluble spicule matrix, which was partially solubilized by pepsin digestion in 0.5 M acetic acid and precipitated by addition of 3.0 A/NaCl, also has the com- position typical of a collagen (Table IE). This precipitated collagen is a white fibrous material that displays an amino acid composition similar to that of the whole insoluble matrix (Table 1C) and sea anemone collagen (Table II). The amino acid composition of the fraction of the July insoluble matrix that is not soluble in pepsin and acetic acid (Table IF) is similar to that of the insoluble matrix of the spicules collected in March (Table IB). The soluble matrix fractions are off-white and extremely hygroscopic. Samples from December and July display similar amino acid compositions. The most prominent feature of this fraction is its extremely high aspartic acid content (>50%). PAGE The pepsin solubilized spicule collagen was subjected to 4% acrylamide SDS-PAGE and compared to type I bovine soluble skin collagen (Fig. 1 ). Although the bands of the spicule matrix are faint, they can be seen to travel in the range of type I collagen. The spicule collagen con- tains a component similar in mobility to the 1 (I) chain. 210 R. J. K.INGSLEY ET AL. a Kn- ot 2 (I)' Figure 1. SDS-PAGE of(A) type I collagen, and (B) the total insoluble matrix of Lci>tf!<'Kia \'iri;iiltitu. Also present are molecular weight components equal to the ft and y chains of type I collagen. A CNBr digest of the solubilized collagen from the July insoluble matrix showed, on 3-17% acrylamide gradient gels, different patterns from that of a CNBr digest of type I collagen (Fig. 2). Treatment with urea did not change the pattern of the insoluble matrix. The total insoluble matrix apparently did not enter the gel and therefore re- mained uncleaved by CNBr. SDS-PAGE of the matrix not solubilized with pepsin revealed low molecular weight proteins that stained with PAS (Fig. 3), indicating the presence of glycoproteins in this matrix. Following the NaB 3 H 4 reduction of the insoluble ma- trix, cross-link analysis indicated that the only reduced cross-link present was DHLNL (Fig. 4). Each nmole of collagen contained 2.87 nmoles of DHLNL. No stable non-reducible cross-links were detected. Discussion This report is the first to demonstrate conclusively the presence of a "classic" collagen (i.e.. a protein containing Hyp, Hyl, and 33% Gly as well as a typical collagen cross- link) as part of the organic matrix of a calcite (calcium carbonate) invertebrate skeletal structure. The insoluble organic matrices from calcite spicules of Leptogorgia vir- gulata collected in the summer months have amino acid compositions characteristic of collagen (Table IA, C). Those compositions are similar to other soft tissue coel- enterate collagens (Franc, 1985). When the amino acid composition is viewed in toto. strong similarities to the pepsin soluble fraction, and sea anemone and vertebrate collagen are evident (see Table I C, E, I, J). The SDS- PAGE pattern of the July insoluble matrix (after pepsin digestion) also displayed the characteristic of type I col- lagen (100,000-300.000 daltons). However, the Lepto- gorgia matrices do not exhibit periodic bandings of typical vertebrate collagens (Watabe and Kingsley. unpub.). The intermolecular reducible cross-link, dihydroxyly- sinonorleucine (DHLNL), was clearly detected in the col- lagen of the July insoluble matrix. Little if any other re- ducible and non-reducible cross-links were present. Sim- ilar observations have been made in other invertebrate collagens (Shadwick, 1985) including coelenterates (Bailey, 1971). The latter were from non-mineralized tis- sues. DHLNL is the most prevalent reducible cross-link in the type I collagen of bovine tendon, bone, and dentin ( Mechanic etai. 1971). Mechanic et ai (1985) have dem- onstrated that DHLNL is distributed in different molec- ular locations in these three tissues and may determine the functional properties of collagen. They suggest that the presence of multifunctional cross-links (i.e., histidi- nohydroxylysinonorleucine, pyridinoline, and histidi- nohydroxymerodesmosine) in the nonmineralized colla- gen tissues holds the molecules at a shorter distance than do the bifunctional cross-links of the mineralized collagen in bone, thereby physically precluding the entrance of ions and the subsequent formation of hydroxyapatite crystals. Conversely, once bone collagen is calcified, the mineral does not allow close enough juxtaposition of the molecules to form multifunctional cross-links (Mechanic et ai. 1985). The molecular location of DHLNL is not known in the spicule matrix of gorgonians, however, consistent with this theory, multifunctional cross-links are not present. We have used enzymic digestion by papain at pH 8.0 at 37C in order to isolate a homogeneous population of calcite spicules. The mineral contained in the collagen of a mineralized tissue protects collagen from denaturation. as well as from enzymatic degradation at neutral pH and B Figure 2. Cyanogen bromide cleavage patterns of (A) type I collagen; (B) pepsin solubilized insoluble matrix; and (C) pepsin solubilized in- soluble matrix treated with urea. COLLAGEN IN GORGONIAN SPICULES B Figure 3. SDS-PAGE of the traction of the insoluble matrix not soluhilized in acetic acid and pepsin, stained for (A) protein with Com- massie blue, and (B) carbohydrate with PAS. above (Bonar and Glimcher, 1970). The physiological, relatively gentle digestion procedure used in this study degraded and solubilized all the collagenous and non-col- lagenous protein that was not protected by mineral. Pre- vious methods used to isolate "calcined" structures used NaOCl or a strong base to destroy organic material. How- ever, NaOCl will produce anorganic bone from normal mammalian bone and is often used to examine bone ar- chitecture by scanning electron microscopy. This also de- grades as well as destroys a significant portion of the or- ganic matrix of mineralized tissue. The above analysis is substantiated by a comparison of the current results with a previous failure to detect collagen in the insoluble matrix fraction (Kingsley and Watabe, 1983). Kingsley and Wa- tabe (1983) isolated spicules in 5.25% NaOCl and demin- eralized in 0. 1 N HC1 at room temperature. The isolated spicules that were analyzed were, in both cases, from an- imals collected during the summer, so the previous meth- ods were harsh and yielded erroneous results. The spicule insoluble matrix of the gorgonian Briareitm asbestinum consisted of very small peptides (1600-5000 daltons) and contained 22 Hyp residues/ 1000, approxi- mately 20% Gly and no Hyl (Silberberg el ai. 1972). These features only suggest the presence of collagen-like com- ponents. The amino acid composition of the whole spicule matrix of the gorgonian Pseudoplexaura flagellosa re- vealed 48, 237, and 9 residues/1000 total residues of Hyp, Gly, and Hyl, respectively (Goldberg, 1988). Silberberg el ai (1972) air dried, ground the whole animals, digested the organic material with 30% KOH, and demineralized in concentrated HC1 at 4C. These procedures are ex- tremely severe in the processing of biological material and may have resulted in the loss of collagenous components. The isolation of spicules in 1 N NaOH by Goldberg (1988) may have caused similar problems. However, such dif- ferences in the matrix compositions of the gorgonian spe- cies may also be the results of other factors including spe- cies variations, and the season and the environment from which specimens were collected (see below). The results of the present study indicate that the amount of collagen in the insoluble matrix is directly related to the season of collection and, therefore, to temperature or other environmental conditions. The matrices isolated from animals collected in the summer from both South Carolina and North Carolina display Hyp and Gly levels similar to those of vertebrate collagen, as well as large amounts of Hyl (Table I A, C). Matrices from animals collected in March and December are different from the above: i.e.. they are less collagen-like in composition. In December, both the Hyp and Hyl contents were less than half of those of summer samples, and the Gly was reduced to 225 residues/ 1000. By March, the Hyp, Hyl, and Gly contents continued their reduction to 8, 7, and 158 resi- dues/1000, respectively. These results clearly indicate a seasonal variation in the insoluble matrix. Had the anal- yses of the organic matrix compositions only been con- ducted on specimens collected in March, the predomi- nance of the collagenous component of the matrix might 15 30 45 60 Elution time (mm) Figure 4. Elution patterns of reduced collagen from (A) bovine bone and (B) Leptogorgia virgiilata spicule insoluble matrix. The positions of the intermolecular cross-links dihydroxylysmonorleucine (DHLNL) and hvdroxvlvsinonorleucine (HLNL) are shown. 212 R. J. KINGSLEY ET .11. have been overlooked. The seasonal changes in the in- soluble matrix are not simply shifts of amino acid residues from insoluble to soluble fractions, because the soluble fractions in summer and winter have relatively similar amino acid compositions (Table I G. H). The apparent seasonal variation suggests a remodeling or turnover of the collagen component, and thus a degree of demineralization and remineralization (turnover) in the spicules. A comparison of the amino acid composition of the non-solubilized portion of the insoluble matrix and the total insoluble matrix collected in March reveals strong similarities. If indeed there is partial demineralization of spicules in winter months, and the portion of the collagen that is pepsin soluble is lost, then the amino acid com- position of the winter matrix and that of the non-solu- bilized fraction of the summer matrix should be similar. Certainly, in the remodeling of vertebrate bone, demin- eralization must occur prior to degradation of collagen. Spicules in L. virgulata are initially formed intracellularly, but later, they become exposed to the extracellular en- vironment (Kingsley and Watabe, 1982). It is not clear whether spicule growth and maturation continues once these structures are in the extracellular environment, al- though this apparently occurs in other gorgonian species (Goldberg and Benayahu, 1987). The present results in- dicate that the spicules are dynamic structures even after they emerge from the cell. The pepsin solubilized insoluble matrix shows a col- lagenous composition similar to total insoluble matrix. However, CNBr digestion of this solubilized collagen pro- duced peptide patterns distinct from bovine skin type I collagen. Samples of the total insoluble matrix treated with CNBr remained insoluble even after reduction of any methionine sulphoxide to Met with 25% mercapto- ethanol. No material was found to enter the gel. It is ob- vious that another insoluble protein is present that protects the collagen from digestion by CNBr. The fraction of the insoluble matrix that was not sol- ubilized by pepsin did not display a typical collagenous amino acid composition. Because it did contain some Hyp and Hyl, however, a portion of the collagen in the matrix is probably blocked from enzymic digestion. This pre- dominantly non-collagenous matrix fraction on SDS- PAGE revealed low molecular weight glycoproteins, as is seen by Coomassie Blue and PAS staining. The presence of carbohydrates in invertebrate calcifying matrices is common and has been described previously in gorgonians (Kingsley and Watabe, 1983; Goldberg, 1988). The pos- sibility that glycoproteins play a role in calcification has been proposed for several organisms, (see Crenshaw, 1972; deJong ct a/.. 1976; Fichtinger-Schepman ct ai, 1979; Marsh and Sass, 1984). The roles of collagen in biomineralization have been examined extensively in bones and teeth and may now be extended to the invertebrates. Some of the major the- ories of how collagen may be involved in the regulation of calcification involve its association with other non-col- lagenous components such as phosphate and osteonectin (see Prockop and Williams, 1982). Similarly, in L. vir- gulata the structure-functional relationship between the non-collagenous matrix and the insoluble matrix, may determine how the total organic matrix regulates spicule formation. Both Kingsley and Watabe (1983) and the present report indicate that the soluble spicule matrix of L. virgulata has a soluble acidic protein containing more than 50% Asp. Much of the calcium binding capacity of invertebrate mineralizing matrices has been attributed to the soluble fraction (see Watabe and Kingsley, 1989). Although this is the first conclusive report of the pres- ence of collagen within the organic matrix of a calcium carbonate skeletal structure, collagen has been found to be associated, indirectly, with a number of calcium car- bonate structures. In the echinoderms, collagen is not found within the sea urchin larval spicule matrix (Blank- enship and Benson, 1984; Wilt el ai, 1985); however, collagen metabolism is critical for normal spicule for- mation. Collagen is apparently necessary for providing a permissive substratum in which spicule formation may occur (Blankenship and Benson, 1984). Similarly in the pennatulids, another coelenterate, collagen is closely as- sociated with the calcitic crystals but is not responsible for the nucleation of the mineral (Ledger and Franc. 1978). Other such indirect roles of collagen in invertebrate calcification are discussed by Watabe and Dunkelberger (1979) and Kingsley (1984). Acknowledgments This work was supported in part by grants from NSF #DCB 8502698 and DCB 8801809, NIH #AR19969 and AR30857, and NASA NAG 2-181. We thank Betty M. Bynum and Robert T. Whitaker for their assistance in preparing the manuscript. Literature Cited Bailey, A. ,). 1971. Comparative studies on the nature of the cross- links stabilizing the collagen fibres of invertebrates, cyclostomes and elasmobranchs. FEBS Lett. 18: 154-158. Benson, S., E. M. E. Jones, N. C rise-Benson, and F. Wilt. 1983. Morphology of the organic matrix of the spicule of the sea urchin lar\a. /I'v/v (.'ell Re-, 148: 249-253. Blankenship, J., and S. Benson. 1984. Collagen metabolism and spicule formation in sea urchin micromeres. E.\p. Cell Rex. 152: 98-104. Bonar, 1.. C., and M. J. Glimcher. 1970. Thermal denaturation of mineralized and demineralized bone collagens. J. i'liruslriu: Res. 32: 545-551. Crenshaw, M. A. 1972. The soluble matrix from Mercenaria mercen- ui'iii shell. Biomineralization Rex 6: 6-1 1. 1'ichlinner-Schepman, A. J., J. P. kamerling, J. F. G. Vliegenthart, COLLAGEN IN GORGONIAN SP1CULES 213 E. W. deJong, L. Bosch, and P. YVeslbroek. 1979. Composition of a methylated, acidic polysaccharide associated with coccoliths of Emiliitniti hitxleyi (Lohmann) Kamptner. Carbohydrate Res 69: 181- 189. Franc, S. 1985. Collagen of coelenterates. Pp. 147-210 in Biology <>/ Invertebrate and Lower Vertebrate Collagens, A. Bairatiand R. Gar- rone, eds. Plenum Press. New York. Fukae, M., and G. 1.. Mechanic. 1980. Maturation of collagenous tissue. Temporal sequence of formation of peptidyl lysine-derived cross- linking aldehydes and cross-links in collagen. ,/ iiioi Clicni. 255: 651 1-6518. Goldberg, W. M. 1988. Chemistry, histochemistry and microscopy of the organic matrix of spicules from a gorgonian coral. Relationship to Alcian blue staining and calcium binding. Histochemistry S9: 163- 170. Goldberg, W. M., and Y. Bcnayahu. 1987. Spicule formation in the gorgonian coral Pseudoplexaura llagellosa. I: Demonstration of in- tracellular and extracellular growlh and the effect of ruthenium red during decalcitication. Bull. Mar. Set. 40: 287-303. Herring, G. M. 1972. The organic matrix of bone. Pp. 127- 184 in The Biochemistry and Physiology of Bone-Structure, Vol. l.G. H. Bourne, ed. Academic Press, New York. deJong, E. W., L. Bosch, and P. \\eslbroek. 1976. Isolation and char- acterization of a Ca :+ -hmding polysaccharide associated with coc- coliths of Emiliania huxlcyi (Lohmann) Kamptner. Eur. J Biochem 7(1:611-621. Jope, M. 1967. The protein ot brachiopod shell-I. Ammo acid com- position and implied protein taxonomy. Comp. Biochem l'liY\n>l 20: 543-600. kingsley, R. .1. 198-4. Spicule formation in the invertebrates with special reference to the gorgonian Leptogorgia virgulata Am. '/Mil. 24: 883- 841. Kingsley, R. .1., and N. \Vatabe. 1982. Ultraslructural investigation of spicule formation in the gorgonian Leptogorgia virgulata (Lamarck) (Coelenterata: Gorgonaceae). Cell 7V.vv. Rex. 223: 325-334. Kingsley, R. J., and N. \Vatabe. 1983. Analysis of proteinaceous com- ponents of the organic matrices of spicules from the gorgonian Lep- togorgia virgnlala Comp. Biochem. Physiol. 76B: 443-447. I .1. iiiiiili. I'. K., and M. Havre. 1973. Maturation of the head of bac- tenophage T4 I. DNA packaging events. J Mol Biol 80: 575-599. Ledger, P. \V., and S. Franc. 1978. Calcification of the collagenous axial skeleton of \'cretiUum cynomorium Pall (Cnidaria: Pennatu- lacea). Cell Tiss. Res. 192: 244-266. Marsh, M. K., and R. L. Sass. 1984. Phosphoprotein panicles; Calcium and inorganic phosphate binding. Biochemistry 23: 1448-1456. Mechanic, G., P. M. Gallop, and M. L. Tanzer. 1971. The nature of crosslinking in collagens from mineralized tissues. Biochem. Biophys Rex. Comin 45: 644-653. Mechanic, G. L., A. J. Banes, M. Henmi, and M. Yamauchi. 1985. Possible collagen structural control of mineralization. Pp. 98-108 in The Chemistry and Kiologv ol Mineralized Tissues. W. T. Butler, ed. EBSCO Media. Birmingham. Norwack, H., and A. Nordwig. 1974. Sea-anemone collagen: isolation and characterization of the cyanogen-bromide peptides. Eur. J. Biochem 45: 333-342. Prockop, D. J., and C. J. Williams. 1982. Structure of the organic matrix: collagen structure (chemical). Pp. 161-177 in Biological Mineralization and Demineralization. G. H. Nancollas, ed. Spnnger- Verlag. Berlin. Shadvtick, R. E. 1985. Crosslinking and chemical characterisation of cephalopod collagens. Pp. 337-343 in Biology of Invertebrate and Lower 1 'erlelirule t 'ollugens, A. Bairati and R. Garrone, eds. Plenum Press, New York. Silberberg, M. S., L. S. Ciereszko, R. A. Jacobson, and E. C. Smith. 1972. Evidence lor a collagen-like protein within spicules of coel- enterates. Comp Biochem I'hvsiol 43B: 323-332. Swift, D. M., C. S. Sikes, and A. P. Wheeler. 1986. Analysis and func- tion of organic matrix from sea urchin tests. / Exp. Zool 240: 65- 73. \\atabe, N. 1981. Crystal growth of calcium carbonate in the inver- tebrates. Prog. On/i// lirowih Charact 4: 99-147. Watabe, N., and D. G. Dunkelberger. 1979. Ultrastructural studies on calcification in various organisms. S E.M. II: 403-416. Watabe, N., and R. J. Kingsley. 1989. Extra-, inter-, and intracellular calcification in invertebrates and algae. Pp. 209-223 in Origin. Evo- lution, and Modern Aspects of Biomineralization in Plants and An- imals. R. E. Crick, ed. Plenum Press, New York. \\einer, S., and W. Traub. 1981. Orgamc-matrix-mineral relationships in mollusk-shell nacreous layers. Pp. 367-482 in Structural Aspects oj Recognition and Assembly m Biological Macromolecules, M. Bal- aban, J. L. Sussman, W. Traub, and A. Yonath. eds. Balaban I SS, Rehvot. Philadelphia. Wilbur, K. M., and K. Simkiss. 1968. Calcified shells. Pp. 229-295 in Comprehensive Biochenu.stry. Vol. 26A, M. Florkin and E. H. Stotz. eds. Elsevier, New York. Wilt, F. H., S. Benson, and J. A. Uzman. 1985. The origin of the micromeres and formation of the skeletal spicules in developing sea urchin embryos. Pp. 297-310 in The Cellular and Molecular Biology nl Invertebrate Development. R. H. Sawyer and R. M. Showman, eds. LIniversily of South Carolina Press, South Carolina. Yamauchi, M., E. P. Katz, G. L. Mechanic. 1986. Intermolecular cross- linking and stereospecific molecular packing in type I collagen fibrils of the periodontal ligament. Biochemistry 25: 4907-4913. /.acharius, R. M., T. E. /ell, J. II. Morrison, and J. J. Woodlock. 1969. Glycoprotein staining following electrophoresis on acrylamide gels. Anal. Biochem. 30: 148-152. Reference: Bml Hull 179: 214-218. (October. 1990) Functional Autonomy of Land and Sea Orientation Systems in Sea Turtle Hatchlings KENNETH J. LOHMANN 1 *, MICHAEL SALMON 2 , AND JEANETTE WYNEKEN 2 'Neural and Behavioral Biology Program, University of Illinois. Urbana, Illinois 61801, and Department of Biological Sciences, Florida Atlantic University, Boca Raton, Florida 33431-0991 Sea turtle hatchlings emerge from underground nests on oceanic beaches and immediately confront two sepa- rate problems in orientation. First they must locate the ocean and crawl to it; then they must orient offshore while they swim out to sea in a migration lasting several days. Visual cues guide hatchlings from the nest to the sea (1, 2), but little is known about the cues used by turtles in the ocean. Nevertheless, the crawl across the beach has long been considered essential to swimming orientation because hatchlings released offshore without a crawl re- portedly fail to orient seaward (3, 4). Here we report that hatchling leatherback (Dermochelys coriacea) and green (Chelonia mydas) sea turtles released offshore consistently swam toward approaching waves and oceanic swells. Wave tank experiments confirmed that swimming hatch- lings oriented into waves. A crawl across the beach was not a prerequisite for wave orientation in either the field or lab, indicating that hatchling sea turtles possess two separate orientation systems, each based on different sen- sory cues and capable of functioning autonomously. The first guides hatchlings on land to the sea; the second, based on wave detection, functions during the ocean migration. In five field experiments with green turtles and five oth- ers with leatherbacks, we monitored the swimming ori- entation of hatchlings released at various distances off- shore near Fort Pierce, Florida. A total of 45 green turtle and 48 leatherback hatchlings were tested. All experiments were conducted between July and September in 1988 and 1989. Hatchlings were obtained from nests deposited on beaches in the Fort Pierce area. Nests were checked daily Received 18 April 1990; accepted 2(1 July 1990. * Present address: Friday Harbor Laboratories, 620 University Road, Friday Harbor, Washington 98250. until a depression formed above the eggs (indicating the eggs had hatched and an emergence would probably occur that evening). We then carefully dug in the sand and re- moved hatchlings, placed them into styrofoam boxes, and transported them by motorboat to testing sites 2.0-30.0 km offshore. All turtles were tested and released within 48 h of capture. Each turtle was placed into a nylon-lycra harness (5) tied by a short line to the side of a spherical, half-sub- merged floating buoy (Fig. 1A). The buoy was attached by another line to the submerged center of a floating cage (Fig. 1A). Swimming hatchlings exerted sufficient force to easily rotate the buoy. Markings on the buoy were clearly visible from the boat, enabling observers to deter- mine the orientation of the buoy (and thus of the turtle) as the hatchling swam in place. Previous reports have in- dicated that migrating hatchlings do not change course or alter their behavior in response to nearby boats (3, 5, 6). In the present experiments, driving the boat around the cage at distances of 10-20 m also had no detectable effect on hatchling orientation. When released into the cage, harnessed hatchlings often dove or circled for their first few minutes in the water. After 2-5 min, nearly every hatchling established an es- sentially constant swimming course from which it only occasionally deviated. Once a turtle settled on its course (or 6 min elapsed), its orientation was determined once each minute for five minutes with a sighting compass. These five readings were used to calculate the mean angle and vector length for each hatchling, following standard procedures for circular statistics (7). Hatchlings consistently oriented into approaching waves in all experiments, regardless of distance from shore. Data from two different green turtle experiments, in which waves approached from nearly opposite directions, are 214 SEA TURTLE WAVE ORIENTATION 215 FRAME GLOBE W 270- S 180- C mydos (n = 45) D coriaceo (n = 48) E 90- .&*> "* *i . t *' 6 ' ' N W on f *'"** -90 90 180 270 W N E S W Direction of Wave Approach (in degrees) figure I. Results ol ticld experiments HW/I the floating cage (At The floating orientation cage, globe, and tethered turtle. Thecage was constructed of eight canal lengths ofPVCpipe(5 cm diameter) fused end to end to form an Maximal frame 112 m in diameter The frame /loafed in lite water so that only about 1 cm of its upper surface was exposed Stainless sieel supports converged on a baseplate 2? cm below the water. The hati tiling was tethered 10 a buoyant globe anchored to the baseplate by a linn, nylon line The turtle could thus swim in nn\' direction, while markings on the globe enabled observers in a nearby boat to monitor its swimming orientation- The bottom ol the cage was encased in a mesh net to exclude' predators A detailed description of the floating cage is provided elsewhere (6). (Bl Results from an experiment with green turtle hatchlings in which waves ap- proached from approximately north The range of wave directions during the ex- periment is indicated by marks outside the edge of the circle The mean angle of orientation /or each hatehling is indicated by a line originating at the circle center (7). Line length is proportional to mean vector length, with a line reaching the edge of the circle corresponding to r = 1 Long lines thus indicate consistent orientation toward a single direction throughout the test period: shorter lines indicate more directional variability F and r values were calculated with the lloielling lest (7). The mean angle of the group (a) is indicated by the large arrow outside of the circle. The coastline was approximately parallel to a line defined by the 343-161 axis (westward, or to the left) but was not visible because we u rrc J,V-^0 km from shore CO A second experiment with green turtles in which waves approached In mi approximately southeast Conventions as in (B> (D) Summary of the five green turtle and five leatherback orientation experiments conducted 2 0-jO.O km from shore Orientation of each haichling is plotted as a junction ol direction of wave approach during the lest Direction of wave approach was defined a.s the mean direction of propagation for all wave n/'c\ present One or more of three wave types (swells, waves, and wind ripples) were present in all trials Oceanic swells are generated by prevailing winds over large expanses of water in the open ocean: near Fort Pierce, swells consistently approach from 70 -140 shown in Figures 1 B and C. These results are represen- tative of responses shown by both species. The orientation angles of all hatchlings are plotted as a function of wave approach direction in Figure 1 D. Jupp-Mardia circle-circle correlation analysis (7) indicated that wave direction and hatehling orientation were significantly (P < 0.001 for each species) related. None of the hatchlings used in these experiments crawled across the beach; all were taken directly from the nest to the testing site. Thus, turtles can clearly orient in the ocean without crawl experience. But hatchlings might still acquire directional information during the crawl that could alter their orientation while swimming. Turtles with crawl experience, for example, might use different ori- entation cues than those released offshore without a crawl, or the two groups might use the same guideposts in dif- ferent ways. To examine these possibilities, hatchlings with and without beach crawl experience were tested offshore in the floating cage on the night of their expected emergence. Hatchlings were removed from nests before sunset and placed into one of two styrofoam boxes. After sunset both boxes were transported to a nesting beach. The hatchlings in one box were released on dry sand at distances of 10- 20 m from the edge of the water. They were permitted to crawl to the wet sand at the edge of the wave wash zone, then retrieved and placed into styrofoam coolers. The lid of the second box was removed during the time the first group was crawling, so that the turtles inside could crawl in the box with a view of the sky. Thus, turtles in both groups crawled, but only one group crawled across the beach. The orientation of hatchlings with and without a beach crawl was statistically indistinguishable for both green turtles (Fig. 2A-B; Watson test. U 2 = 0.097, P > 0.20) and for leatherbacks (Fig. 2C-D; U 2 = 0.045, P > 0.20). Nearly all of the turtles swam in the general direction of approaching waves, suggesting that hatchlings released (E-SE) during the summer and arc largely unaffected by weather conditions near land. Waves generated by local wind patterns were often present and usually moved in directions similar (within 80) to those of swells A few minutes after abrupt changes in wind direction, wind ripples 1-5 cm in height tracked the new wind direction, while larger "old" waves continued to track the previous wind direction- No trials were conducted undci sii nmy conditions when the various wave types could not be clearly resolved or when waves (or swells) exceeded 1 5 in in height The direction of each wave type was measured easily by sighting down the axis of wave propagation with a digital hand-bearing A utohe/m" compass. Because we had no a priori reason to consider one iu;ir type more important than another, we calculated a mean direct ion of wave approach for this study However, hatchlings may actually i iricni i 'illy to the largest waves present (regardless of type) while ignoring the others. For a detailed discussion of wave tvpes. see (10). Circle-circle correlation analysis (7) indicated that direction of wave approach and hatehling orientation were significantly related (see text) There was no evidence that responses varied with distance from shore 216 K. J. LOHMANN ET AL C. mydas NO CRAWL 270 D. coriacea CRAWL NO CRAWL Figure 2. Result <>/ ollshore orientation e\/'erinienf, null liatclil/ngs that hail ami had not crawled across it heach prior to testing I A) Results of green turtle hatehlmgs thai crawled across the hem It Inner circle indicates r value of 0.5 (outer circle still corresponds to r = ID). All oilier conventions as in Figure IB Mean angle Jor the group is 99. IB) Results ol green turtle halchhngs deprived of a heacli eran'l Convention* <;\ in Figure _?. I .Mean angle lor the group is 100. CO Results ol Icaiherhack liulchlings that crawled across the heach. Conventions as in Figure 2A Mean angle lor the group = 124 (D) Results of Icatherback hatehlings deprived ol a heach eran-l. Conventions as in Figure 2A. Mean angle for the group = 124 offshore orient toward waves regardless of whether they first experienced a beach crawl. To study the relationship between turtle orientation and waves more rigorously, we monitored the orientation of hatchlings swimming in a wave tank. Under dim light, each turtle was tethered to a central post (made of nylon fishing line strung vertically from the bottom of the tank to a rod across the top). Thus, hatchlings could swim in any direction in the wave tank but could not contact the sides. All lights were then turned off except for a single in- frared source [a Kodak darkroom light with a 40 W bulb covered by an Edmunds infrared transmitting filter (#8247-29-1)]. After a 5-min acclimation period, an ob- server using a night vision scope recorded the orientation of each hatchling at 30-s intervals for 5 min; these mea- surements were used to calculate the mean angle and vec- tor for each turtle (7). One group of hatchlings (for each species) was tested with the wave tank motor off so that no waves were generated and the room was silent. A sec- ond group was tested with the motor running but the drive disconnected so that hatchlings were exposed to motor sounds and vibrations, but not to waves. The third group was tested with the motor on and the drive engaged so that waves were generated. Results for green turtles and leatherbacks were quali- tatively identical. Neither species was significantly oriented in the absence of waves (Fig. 3). When waves were present, however, the hatchlings oriented toward the direction of wave approach (Fig. 3). These experiments confirmed that hatchling sea turtles can use waves as an orientation cue, even in the absence of visible light. Our results suggest that sea turtle hatchlings sequentially employ two separate orientation systems, each based on different cues. While on the beach, hatchlings find the sea by seeking out bright, open horizons (1,2). Our field and wave tank experiments provide evidence that hatchlings released in the ocean orient by swimming toward waves. Because sea-finding orientation is not a prerequisite for wave orientation, the land and sea orientation systems can function independently. Our results do not demonstrate, however, that the two systems never interact under natural conditions. Imme- diately after entering the sea, for example, hatchlings might use visual cues to establish an offshore course. Later, when visual contact with land is lost, hatchlings could maintain their orientation by swimming at a fixed angle relative to waves. Visual cues experienced during either the beach crawl or near land might also be critical for hatchlings that emerge on nesting beaches surrounded by exceedingly calm seas. Additional experiments are re- quired to determine whether the land and sea orientation systems are completely independent under all conditions, or if they interact in some way as hatchlings migrate away from land. During daylight hours, especially near shore, local winds can generate waves which induce hatchlings to swim in directions other than directly offshore (Fig. ID) However, Florida sea turtle hatchlings nearly always emerge from nests and enter the sea shortly after dark (8, 9). Oceanic swells, produced by prevailing easterly winds, are the prevalent waves during this time (10). The swells move toward the Florida coast, where the propagation direction becomes oriented perpendicular to the shore as waves en- ter shallow water and approach a beach (11). Wave prop- agation direction thus provides a consistent, reliable cue for offshore orientation during the time hatchlings usually enter the ocean. Several marine molluscs (12, 13) and crustaceans (14, 15) can orient using waves or wave surge in shallow water near shore. Sea turtle hatchlings, however, continued to SEA TURTLE WAVE ORIENTATION 217 270 MOTOR WAVES D. coriacea SILENCE MOTOR WAVES Figure 3. Results ol the wave lank experiments (. Results ol green turtle hatch/ings fill Results nl Iculhcrhack halchltngs All conventions follow //imc in l-igure _M l-'nr each \/)iv;i'\. "Silence" indicate* refill': ol llalch/ings tested with the wave tank motor of/ "Mod// ' indicates the motor was on hut the drive was disconnected so no waves were produced "Waves" indicates result* i>l hatchlings tested when waves were generated: waves approached /mm (tit/* of the diagram) Each specie.* mis significantly oriented only when mire* were picsenl The mean angles of the Imi mire groups {jar right, upper and lower) were directed almost .straight toward the direction of ware approach II II 1. 7'.-IA'A. The ware lank, located a! the l-'loi ula Institute ol Technologr in Melbourne, Florida, was v [ m in length (I <> m high, and 0.6 m in width It was tilled with water to a depth ot l> 5 m A paddle al one end mi.\ driven hv a lit ' motor I slopmv plrwood plallorm 4 V m in length al the op/>oMie end absorbed wave energy and ininimi-ed ware re/leelion llatchlmgs were tethered (see fe\lf so liter could swim in anr diicclton while an ohsenei watched with a night vision scope i/ir/iuglt a i ircnlar opening in a siiro/oam sheet wedged across the lop o/ the tank The sirroloam was maiked in 5 increments, providing a reference lor determining orientation Throughout the espenments iu- alternated helween trials under the three treatments described in the le.\t (silence, motor, wavesi in semi-random order (blocks ol 1-3 trials under one condition were to/lowed />v .similar bloek.s nf trials tinder the other two conditions! Each hatchling was tested unly once under one ol the three conditions Waves generated in green turtle experiments were s cm ipeak ion ought m height (frequency about 4t> waves/mm t \\ares in lealherback experiments were }-J cm m height, also at 40 wares/mm II e have observed wares of these approximate heights and frequencies at sea on calm days during the summer at Fort Pierce. Natural waves during the summer are only occasionally smaller than this and are often considerably (2-50 times! larger. swim toward waves even when out of sight of land (beyond about 1 8 km from shore), indicating that wave propaga- tion direction can be used by animals as a cue for ori- entation in the open ocean. This cue might well be used by other long-distance ocean migrants such as fish and cetaceans. The responses of the hatchlings reported here must be viewed as the earliest manifestations of a sophisticated orientation system, one that allows these animals as adults to complete migrations between nesting sites and feeding grounds located hundreds or thousands of kilometers apart (16, 17). Further ontogenetic analysis may provide insight into how an orientation system that initially guides hatchlings offshore develops into one that provides adults with the ability to complete complex navigational tasks. Acknowledgments We thank M. Flaherty, J. Norton, and T. H. Frazzetta for technical assistance, B. Dally and the Florida Institute of Technology for the use of the wave tank, E. Martin and R. Ernest for locating turtle nests, C. M. F. Lohmann for critically reading the manuscript, and the Harbor Branch Oceanographic Institution for providing lab and 218 K J. LOHMANN KT AL tank space. 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Orientation cues used by hatchling loggerhead sea turtles (Carelta earetla L.) dunng their off- shore migration. Ethology 83: 2 1 5-228. 7. Batschelet, E. 1981. Circular Statistics in Biology Academic Press, London. 8. Bustard, R. 1973. Sea Turtles: Natural History and Conservation. Taplinger Publ.. New York. 10. 12. 13. 14. 15. 16 17 Demmer, R..1. 1981. The Hatching and Emergence of Loggerhead Turtle /Caretta carettai Halchlings. M.S. thesis. Univ. Central Flor- ida. Orlando. Florida. Bascom, \V. 198(1. H 'u\r\ and Beaches Anchor Press Doubleday. New York. Denny, M. VV. 1988. Biology ami the Mechanic* of the Wave-Swept l-jmronment. Princeton University Press, Princeton, New Jersey. Gcndron, R. P. 1977. Habitat selection and migratory behaviour of the intertidal gastropod Littorina litlorea (L.). J. Am in Ecol. 46: 79-42. Hamilton, P. V., and B. J. Russell. 1982. Field experiments on the sense organs and directional cues involved in offshore-oriented swimming by Aplysia brasiliana (Rang) (Mollusca: Gastropoda). J Exp. Mar. Biol. Ecol. 56: 123-143. Walton, A., and \V. Herrnkind. 1977. Hydrodynamic orientation of spiny lobster. Panulims argits (Crustacea: Palinuridae): wave surge and unidirectional currents. Proceedings of the Annual Northeastern Mtg-of the Animal Behavior Soc. 1977 Plenary Papers. Memorial University of Newfoundland. Marine Sci. Res. Lab, Tech. Report No. 20: 184-211. Nishimoto, R. T., and W. F. Herrnkind. 1978. Directional ori- entation in blue crabs, Callinecles sapiittix Rathburn: escape re- sponses and influence of wave direction. J. Exp. Mar. Biol Ecol. 33:93-112. Carr, A. 1965. The navigation of the green turtle. Sci. Am. 212(5): 78-86. Carr, A. 1984. The Sea Turtle: So Excellent a Fishe. University of Texas Press. Austin. Reference: Bn>l Bull. 179: 219-235. (October, WO) Abstracts of Papers Presented at the General Scientific Meetings of The Marine Biological Laboratory August 20-22, 1990 Abstracts arc arranged alphabetically hy first author within i he following categories: cell moii/ity, developmental biology, ecology and population biology. fertilization and early development, mariculture and the marine environ- ment, neurobiology and biophysics, physiology and be- havior, and sensory biology. A uthor and subject references will he found in the regular volume index in the Decem- ber issue. Cell Motility Detailed observations on the migration oj Limulus amoe- bocytes hy video microscopy. E. L. BEARER. M. STROUT, AND DAVID DEMERS (Dept. of Biochem.. University of California, San Francisco). The amoebocyte the blood cell of the horseshoe crab is a composite neutrophyl-thrombocyle capable of either directed migration and phagocytosis or activation, degranulation, and clot formation. The mi- gratory behavior has been difficult to study because the amoebocyte is extremely sensitive to endotoxin, rapidly degranulating in its presence. By putting fresh cells upon a lawn of spread amoebocytes in serum on baked glass coverslips, one of us (E.L.B.) has observed in detail the mi- gratory behavior of these cells. The amoebocyte crawls at a maximum rate of 20-30 /jm/min. It extends long filopodia rich in actm filaments in the general direction of movement. Initially, these extend above the surface of the substrate, then come to rest upon it. An attenuated sheet of membrane advances between forward-most filopods, and is subse- quently inflated by cytoplasm. By phalloidin staining, this attenuated membrane does not contain actin ruffles, and no retrograde movement can be observed. An apparent contractile event squeezes the nucleus and posterior cytoplasm containing the large granules forwards. During this process, tilopods that have extended, but no submerged in membrane, are collected to the rear, such that the cell resembles a pin cushion. By phalloidin staining, actin filament bundles extend from the leading fil- opods along the ventral surface of the cell to the posterior pin cushion. Filopods initially extend at the rate of 3-5 ^m/s, but sufficient G-actin for such a rate of polymerization could not be detected. Amoebocytes will continue to crawl for 15-30 min after being permeabilized briefly with 0.1 mg/ml saponin in a magnesium-EGTA buffer. If 2 mAI ATP is added, some cells can continue to crawl for up to 5 h. These results suggest that the filopods are extended by an ATP-dependent sliding pro- cess rather than hy polymerization of new filaments, supporting a role for actin-based motors in the leading edge. To identify such motors, we have adopted a biochemical approach and have isolated a fraction en- riched in actin-dependent ATPase activity that retains actin filaments in an in vitro motility assay. By Coomasie staining of protein gels, this fraction contains no proteins in the molecular weight range of myosin II (220 kDa), but has several bands. Supported by the Frederick Bang Fellowship and the American Society for Cell Biology. Effects oj cAMP-dependent protein kinase inhibitor on or- ganelle movement in Y-I adrcnocortical tumor cells. GEORGE M. LANGFORD (Department of Physiology, University of North Carolina, Chapel Hill, NC 27599), EDWARD E. LEONARD, DIETER G. WEISS, AND SAN- DRA A. MURRAY. The saltatory motion of lysosomes in Y-I adrenal cells was quanti- tatively analyzed in control populations and in cells into which protein kinase inhibitor (PK.I) was introduced by electroporation. Organelle mo- tion was studied to determine whether a change in movement pattern occurred in the presence of a factor that suppressed steroidogenic activity. Organelle motion was analyzed from the positional data obtained at 100 ms time resolution. The movement of lysosomes (0.5-0.8 ^m diameter) was the Interrupted Motion Type II as defined by Weiss el at. (1986, (.'ell Mulil. Cyltixkel. 6: 128-135). In control populations of cells, these organelles exhibited periods of rapid directed movement interrupted by pauses that were most often followed by a reversal of direction of move- ment along the same or adjacent parallel tracks. The average velocity of lysosome movement in both the anterograde (toward the cell periphery) and the retrograde (toward the nucleus) directions was 1.2 0.8 pm/s with a V ma> of 2.1 0.7 jirn/s. The average duration of pauses was 17 5.3 s, while the average duration of movement was 8 4.9 s. In PK.I- treated cells, the average and maximum velocities (1.11 0.5 and 2.73 0.8 ^m/s, respectively) were not significantly different from those mea- sured in control cells. However, the ratio of the average duration of pauses (4.7 3.3 s) and the average duration of movement (3.5 2.7 s) in the PKI-treated cells was significantly different. We conclude that PKI had no apparent effect on the motors of Organelle movement because the maximum and average velocities were not affected. The reduction in the duration of pauses may be due to changes in the degree to which the cytomatrix is crosslinked. We interpret these results to mean that phosphorylation increases the degree of cytomatrix crosslinkage and that such linkages influence the pattern ol'organelle motion. 219 220 (III MOTILITV Supported by NSF grants BNS-9004526 (G.M.L.) and DCB-8910545 (S.A.M.): DFG grant We 790/12 (D.G.W.); ASCB/MBL research fel- lowship (E.E.L.). Reorganization of cytoske/eton during cell fusion induced hy electric field. Q. ZHENG AND D. C. CHANG (Dept. of Mol. Physiology and Biophysics. Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030). Cultured mammalian cells can he fused with high efficiency by applying a pulse of radio-frequency electric field. We have used the electrofused CV- 1 cells to study the reorganization of cytoskeletal proteins during the various stages of cell fusion. The structures of microtubules (MTs) and the intermediate filament vimentin were studied by means of immu- notluorescence microscopy. The structure of F-actin was examined using rhodamme-labeled phalloidin. We observed that stress libers disappeared quickly after the initiation of fusion. Later. F-actin bundles were formed at the cell peripheries or at the edges of cyloplasmic bridges; these bundles may help to extend the cytoplasmic bridges. The stress fibers reappeared about 2 h after the electrical treatment. MTs began to extend into the cyloplasmic bridges within a few minutes after the initiation of cell fusion. Later, a network of parallel MT bundles appeared between the adjacent nuclei of the fusing cells. After the nuclei of the fusing cells had aggregated, the MT networks reorganized into a network similar to that of the control cells with a single MT organizing center. A high concentration of col- chicine (0.1-1 m.U) could disrupt MT bundles and block nuclear ag- gregation in some of the fusing cells. Colchicine treatment could also impede the merging of cytoplasms. The vimentin proteins extended into the cyloplasmic bridges during cell fusion, but with a slight time delay. The distribution of vimentin proteins generally followed that of MTs. Thus, the formation of parallel MT bundles in fusing cells may provide the mechanical links for nuclear aggregation. Elasmobranch eye lens: act in and Ul' radiation. SEY- MOUR ZIGMAN (University of Rochester School of Medicine), NANCY S. RAFFERTY, AND KRIS C. LOWE. The contribution of actin integrity to the stability of dogfish (Mustelus ci/(\l and skate (Rii/a crcnacia) eye lens epithelial cells was investigated. The role of near-UV radiation as a cytoskeletal actin-damaging agent was further investigated. Three procedures were used to analyze fresh elasmobranch eye lenses that had been incubated for up to 22 h in \-iirn. with elasmobranch Ringer's medium, and with and without exposure to a near-UV lamp (emission at 365 nm 30 nm; irradiance of 4 mW/ cm'). The lenses were observed histologically with phalloidin-rhodamme specific staining: by transmission electron microscopy, with and without gold-labeled antibodies; and by polyacrylamide gel electrophoresis with immunoblotting. In addition, solutions of purified polymerized rabbit muscle actin (supplied by Dr. Thomas Pollard. MBL) were exposed to the same UV conditions, and depolymerization was assayed by ultra- centrifugation and high pressure liquid chromatography. Actin monomer increased due to UV exposure from 3 to 18 h. We found that skate and dogfish lenses developed superficial opacities due to II V exposure. In whole mounts of lens epithelium, actin filaments in the basal region of the cells were broken down within 18 h of UV exposure. TEM confirmed the breakdown of actin filaments due to UV exposure. PAGE and immunoblotting positively identified actin in these cells. Direct exposure of purified polymerized actin in polymerizing buffer, led to a 10 to 20% increase in actin monomer in the test solutions within 3 to 18 h. whether assayed by ultracentrifugation or HPLC. We conclude that elasmobranch lens epithelial cells contain UV labile actin filaments, and that near-UV radiation, as is present in the sunlit environment, can break the filaments down in these cells. Furthermore, breakdown of purified polymerized actin does occur due to near-UV light exposure. While the damage appears to be a direct effect of the UV radiation in the actin. the damage could be at the sites at which actin bind to the supporting cell architecture. Support: N. I. H. (N.E.I. #EY 00459) and RPB. Inc. (S.Z.). N.I.H. (N.E.I. #EY 00698) (N.S.R.). Developmental Biology S.E.M. observations oj early cleavage in Hoploplana in- quilina. JOHN M. ARNOLD (University of Hawaii), HYLA C. SWEET, AND BARBARA C. BOYER. Many "lower" invertebrates display "mosaic development" in which the fates of the early blastomeres are determined very early in ontogeny. By studying very primitive animals, which can be thought of as being "frozen in evolution," we can gain insight into the origin of mosaic development. The current observations were made over the last seven summers on the primitive flatworm Hoploplana inquilina using standard fixation techniques for scanning electron microscopy. Our results indicate that some embryos, when viewed from the side or vegetral pole, show fur- rowing beginning unipolarly at the animal pole and proceeding to the vegetal pole. This results in the two blastomeres being temporarily con- nected by a large cyloplasmic bridge that is eventually reduced to a mid- body. This mid-body is later lost, but the cells remain with close mem- brane contact. The bridge is covered with cytoplasmic prolusions and folds, which appear to indicate subsurface tensions. It has been dem- onstrated in echinoderm embryos that the position of the cleavage furrow is determined by the position of the mitotic apparatus. Further, in asym- metrical cleavage, the yolk concentration in the vegetal region of the egg may displace the influences of the M.A. so that the microfilaments causing cytokinesis form at the animal pole and progress vegetally around the zygote to eventually meet at the vegetal pole. Cleavage and mosaic development are highly variable in these embryos: both bipolar and unipolar cleavage occur in the same batch of eggs. This may indicate that early development in this flatworm is a constant, on- going evolutionary experiment. Supported by a NSF grant DCB 8817760 to Barbara Boyer. Morphogenesis ofascidian ampullae and polarized move- ments of tunic extracellular matrix components along ampullae. WILLIAM R. BATES (Dept. of Biology, Carle- ton University, Ottawa, Canada). The ampullae of solitary ascidians are epidermal structures that attach the juvenile to specific kinds ot substrates and are thought to secrete the extracellular matrix (ECM) components of the tunic. Each ampulla grows out from the body epidermis as a fluid-filled tube, surrounded by a single layer of cells, and encased within the tunic ECM. In the present study, the movements of contractile rings along ampullae 200 to 800 jim in length were investigated using Molgula manhattensis (a urodele species) and M. provisional^ (an anural species). Similar results were obtained for both species. Within the ampullar lumen, near the base of each am- pulla and in close proximity to the developing zooid (at days 2, 3. and 4), an oscillating bubble-like region formed before each contraction wave. Each of these regions functioned autonomously for several days before their pulsations stopped. These regions may be localized centers of varying hydrostatic pressure that may trigger the polarized movements of the contractile rings in a proximal to distal direction. Contractile rings were ABSTRACTS FROM MBL GENERAL MEETINGS 221 produced at regular time intervals over several days and could he blocked with cytochalasin D (at 10 fjg/ml). Previous TEM studies of an anural species, Molgulu paci/ica, demonstrated a circumferential pattern of mi- crofilaments in the basal cytoplasm of ampulla cells (Bales and Mullctt. Can J /<>/ in press). Together these results suggest that ampullar con- traction waves are mediated by micronlaments. When clusters of chalk particles were positioned on the tunic ECM near the tips of day 2 am- pullae, the particles were translocated in a distal to proximal direction and. by day 3. decorated the surface of the tunic that encased the zooid. These experiments suggest that tunic ECM components are synthesized by ampullar epidermal cells, and that there is a polarized movement of these components along ampullar surfaces. W.R.B. is supported by NSERC of Canada. Macromere control of early development in the polyclad flatworm Hoploplana. BARBARA C. BOYER (Union College, Schenectady, NY 12308). Either one, two, three, or four macromeres were deleted from eight- cell stage embryos of the polyclad flatworm Hoploplana nii/iti/nni. with multiple deletions involving various combinations of cross-furrow (IB and I D) and non-cross-furrow ( I A and 1C) cells. Normal Muller's larvae developed in 28% of embryos from which one blastomere was removed, regardless of whether it was a cross-furrow or a non-cross-furrow ma- cromere. Deletion of more than one macromere never produced normal larvae. Although first quartet micromere deletions produced loss of e\es (Boyer, 1989, liio/ Bull. 177: 338-343), macromere ablations usually resulted in supernumerary eyes. As more macromeres were removed, the number of larvae with extra eyes also increased, with cross-furrow macromere deletions producing additional eyes more often than non- cross-furrow deletions (e.g., 52% with loss of 1 B and 1 D versus 11% with deletion of 1A and 1C). Similarly to first quartet deletions, as increasing numbers of macro- meres were removed, the frequency of bilaterally symmetrical larvae with Muller's morphology declined. Most of the abnormal larvae were "swollen," consisting of lobeless spheres with undilferentiated masses of internal cells surrounded by a cavity and usually possessing supernu- merary eyes. However, bilaterally symmetrical larvae developed more frequently when non-cross-furrow hlastomeres were deleted. For example, deletion of one cross-furrow cell produced approximately the same per- centage of Muller's-type larvae (64%) as deletion of both non-cross-furrow macromeres (62%). Similarly, deletion of IB and ID, and deletion of three macromeres including 1 A and 1C, led to 19% and 18% bilaterally symmetrical larvae, respectively. Loss of all four macromeres produced 100% radial or asymmetric larvae. These results suggest that determi- nation of symmetry in Hoploplana involves a mechanism similar to that of the equally cleaving mollusks in which micromere-macromere inter- actions specifically involving a cross-furrow macromere establish bilateral symmetry and determine the dorsal quadrant. This work was supported by NSF grant DCB-88 17760. Effects of tumor promoters (okadaic acid, TPA) and an- ticarcinogens (nicotinamide, sarcophytol A) on Arbacia development. KRYSTYNA FRENICEL', HIROTA FujiKi 2 , ALBERT GROSSMAN', AND WALTER TROLL' ('New York University Medical Center and : National Cancer Center Research Institute, Japan). The sequence of cell divisions that follows fertilization ol sea urchin eggs by sperm may serve as a model for tumor promotion and progression in mammalian systems. One observation that supports such a notion is that H ; O : is produced immediately after fertilization and also is one of the earliest effects elicited by tumor promoters in human neutrophils. Two types of tumor promoters were used: okadaic acid, a potent inhibitor of protein phosphatases I and 2A, and TPA ( 1 2-O-tetradecanoylphorbol- 13-acetate). an activator of protein kinase C. Okadaic acid (in the range of 10"* M) and TPA (in the range of 10 " A/) inhibited development of sea urchin embryos when given 15 min prior to fertilization. Although fertilization and the subsequent 3 to 4 divisions appeared to be normal, development was significantly retarded thereafter. Lower concentrations of these tumor promoters seemed to accelerate development, whereas higher concentrations were more toxic. The anticarcinogens nicotinamide and sarcophytol A inhibited oxygen radical release by tumor promoter-activated human neutrophils in a time- and dose-dependent manner. Preliminary evidence suggests that such anticarcinogenic agents may also be effective in overcoming the inhibitory action of tumor promoters on sea urchin development. That ability may become the basis for screening the agents for their potential anticarcinogenic activity. Supported by N1EHS Center Grant ES 00260 and Superfund Grant 1 P42 ES 04895, and NIH Grants CA 37858 and CA 53003. Morphology and reproductive tract development in winter and summer populations oj the male spider crab Lihinia emarginata: a proposed life history and regulatory mechanism. HANS LAUFER, ELLEN HOMOLA, AR- MAND M. KURIS, AND AMIR SAGI (Molecular and Cell Biology, University of Connecticut, Storrs, CT). We investigated a possible role for the mandibular organ (MO) and its secretion, methyl farnesoate (MF). in the regulation of reproduction and morphogenesis. Two different subpopulations were observed in win- ter (1989) and summer (1990) populations, with respect to relative claw size (propod us/carapace). The shell texture of small-clawed males was always pubescent, whereas large-clawed males were either pubescent, with velvety carapaces indicating a recent molt, or abraded, with a worn carapace, indicating a prolonged intermolt. All males had sperm and were in their intermolt phase. Abraded winter males were the largest, with heavily calcified carapaces. In the summer, no differences in cal- cification were noted, and abraded crabs of different sizes were present. Variation in carapace, from smooth to velvety pubescent, was noted. The reproductive system was significantly more developed in abraded males, and reproductive system size correlated well with body size. Large- clawed pubescent crabs had small reproductive systems that were not correlated to their body size. Small-clawed males possessed a small re- productive system that was correlated with their body size. MF in the hemolymph and its synthesis by the MO was two to five times higher in animals with the larger reproductive systems. Mature L emarginula males molt in the fall. We suggested that the abraded crabs were in their terminal molt, and that they transformed in the fall of 1988 (or before). Full reproductive tract development followed. The pubescent large-clawed males, having similar body sizes to the abraded males, seem to have had their terminal molt in the fall of 1989 and still have a growing reproductive system. Small-clawed males con- tinue to molt. The evidence strongly supports a role for MF as a gonad- otropin, which directs the growth of the reproductive system; it may also play a role in the development of male polymorphism. Supported by the Sea Grant College Program, a Fulbnght Fellowship, and BARD. Metalloproteinases of the developing sea urchin embryo. J. QuiGLEY (Pathology, SUNY, Stony Brook, NY 1 1794), R. S. BRAITHWAITE, AND P. ARMSTRONG. Proteolytic enzymes play an active role in inflammation and in tumor cell metastasis. Of particular importance are the enzymes that degrade 222 DEVELOPMENTAL BIOLOGY the basal lamina, because il is a major barrier to cell motility. Gelatinases pla> a key role in basal lamina degradation, because they degrade collagen type IV. a major constituent of the basal lamina. We have investigated the activity of gelatinases in Arhaciu embryos to observe their role in the extracellular matrix remodelling that occurs in normal embryonic development. Gelatinase activity is detected by SDS-polyacrylamide gel electrophoresis, with gelatin incorporated into the resolving gel. Washing with Triton removes the SDS. and an incubation period immediately thereafter allows the enzymes to degrade the gelatin. This activity is revealed subsequently by bands of clearance in Coomassie blue stain. We find four prominent zones of lysis (gelatinases) at approximate mo- lecular weights of 51). 45, 37, and 33 kDa. As the embryos advance through the blastula. gastrula, and prism stages, the 37 and 33 kDa bands increase in activity, and the 44 kDa hand decreases in activity. The activity of these enzymes is increased after an incubation with I mm APMA, an organomercurial that activates mamallian gelatinases. These enzymes are completely inhibited by 1 mm 1,10-phenanthroline and EDTA, and are unaffected by the senne protease inhibitor PMSF, demonstrating that they are metalloproteinases. The involvement of these enzymes in embryonic extracellular matrix remodelling is under study. The repetitive calcium mm-s in the fertilized ascidian egg arc initiated in the vegetal hemisphere by a cortical pacemaker. J. E. SPEK.SNUDER (Dept. Exp. Zoology, University of Utrecht. The Netherlands). Ascidian eggs respond to fertilization with a series of calcium pulses that continues until meiosis is completed (Speksmjder el al.. 1989, Dev. Hinl. 135: 182-190). In the egg of Phalluvu inainnullala. the first large (activating) pulse is initiated by the sperm at its entry point, usually in the animal hemisphere, and spreads across the egg as a wave (Speksnijder clal.. 1990,7. Cell Biol. 110: 1589-1598). The remaining calcium pulses also travel as waves, but their initiation point is located mainly near the vegetal pole, where the developmentally important, mitochondria-rich myoplasm is concentrated (Speksnijder el al.. 1990 Dev. Biol. 142, in press). The object of this study is to determine whether the pacemaker of these calcium waves is associated with the myoplasm. or located in the vegetal cortex underlying the myoplasm. Therefore unfertilized eggs were centrifuged al 2000 x g for 5 min to displace their myoplasm. In D1C optics, three cytoplasmic layers are visible in such centrifuged eggs: a centrifugal yolk zone, a myoplasmic zone rich in mitochondria, and a clear zone. Vital staining of mitochondria with diOC2 (0.5 Mg/ml, 10 min). and of DNA with H33342 ( 10 Mg/ml. 10 min), reveals that the myoplasm is displaced randomly with respect to the animal pole, which is marked by the female chromosomes. The three cytoplasmic zones remain clearly distinguishable for at least 1 h after centrifugation, and no visible rearrangements of the zones take place at fertilization. Imaging of the cytosolic calcium in 14 stratified, aequorin-injected eggs reveals a series of calcium waves following fertilization. About 60-70% of these waves start near the vegetal pole, which corresponds to the number ot waves previously found to start near the vegetal pole in non-centrifuged eggs. In contrast, only 20-30% of the waves start in the vicinity of the displaced myoplasm. 1 conclude that the main wave initiation site is not displaced by the centrifugal forces used, which suggests that a cortical component located in the vegetal hemisphere is involved in initiating these repetitive calcium waves in the fertilized ascidian egg. Supported by a MBL Summer Fellowship to J.E.S. and NIH grants HD 1 88 1 8 and RR 1 395 to L. F. Jaffe. Single and multiple micromere deletions in first quartet emhryos ot Ilyanassa obsoleta. HYLA C. SWEET AND BARBARA C. BOYER (Union College, Schenectady, NY 12308). Following the deletion of the la or Ic micromeres of eight-cell llyunuxxa embryos. Clement (1967, J. Exp. /-ooi 166: 77-88) found that, at seven days, the veliger larvae were missing the left or right eye, respectively. To determine whether the experimental larvae can replace the missing eye, as is the case in Bilhynia (van Dam and Verdonk. 1982 RHILX'X Arch. Dev. Biol. 191: 1 12-1 18), Ilyanassa embryos were raised to an average of 14 days after the deletion of la or Ic. None of the vehgers formed a second eye. indicating that eye development in Ilyanassa is not as regulative as it is in Bithynia. Combinations of first quartet micromeres were deleted to further test the mosaic verxus regulative nature of the Ilyanassa embryo. The removal of the whole first quartet resulted in several larvae that had stump-like structures instead of a velum, suggesting that the first quartet is determined at the eight-cell stage to form head structures, with little contribution from other blastomeres. Clement found considerable variation in Ib-deleted veligers. but little difference between Id-deleted and control larvae. The results of exper- iments in this study, in which both Ib and Id were removed, support Clement's suggestion of a general unstabilizing effect created in Ib-deleted larvae. However, the degree of variation was higher than in Clement's single Ib deletions and occurred mainly in the size and shape of the velum and in the number of eyes. This suggests that the deletion of Ib and Id together causes a loss of regulation among the first quartet mi- cromeres. Because little variation was found among multiple micromere deletions involving Id, and either la or Ic, a destabilizing effect may be created by the loss of Id only when it is deleted with Ib. Thus Ib and Id may work together during the early development of Ilyanassa to establish some sort of stability, or regulation, in forming the head struc- tures. This work was supported by NSF grant DCB-88 17760 to B. Boyer and by the Llnion College Internal Education Foundation. Ecology and Population Biology Feeding and predation rates of winter flounder, Pseudo- pleuronectes americanus, and blue crabs. Callinectes sapidus, in an eelgrass bed in \Vaa.uoit Bay. ROBERT BILLARD (Marine Biological Laboratory), LAURA LYNCH, LINDA A. DEEGAN, JOHN T. FINN, AND SUZ- ANNE G. AYVAZIAN. Development around Waquoit Bay, Massachusetts, is adding nutrients that may adversely effect young of the year (YOY) nnfish and shellfish populations. This nutrient deposition causes dense macroalgal growth, which may inhibit these animals from finding adequate protection from predation and food supply. A field study was conducted to investigate the impact of macroalgae in an eelgrass habitat on the feeding habits of. and the predation rates on, YOY winter flounder and blue crabs. Four (25 25 m) plots of varying macroalgal density were established. First, algae were removed from one plot (removal) and added to a second plot (addition). A control plot with normal amounts of algae and a dis- turbance control plot from which the algae were removed and then re- turned, were also sampled. Predation rates were determined by tethering YOY of each species in the four plots for 6 (flounder) or 24 h (blue crabs). At the end of the period, the presence or absence of these animals was noted. Absence was assumed to be due to predation. Feeding habits of the two species were determined by placing starved animals in the plots and allowing them to feed for 2 h. We conclude that macroalgal density may influence the predation rates on, and the feeding habits of, YOY winter flounder and blue crabs. The predation rate on winter flounder was significantly higher in plots with high algae concentrations. There was no observable difference in predation rates on the blue crab. Initial results indicate that winter floun- ABSTRACTS FROM MBL GENERAL MEETINGS 223 der fed when dissolved oxygen and temperature were within tolerance limits. The blue crab feeding experiment was done in late summer, and very few crabs fed. We believe feeding stopped in the late summer due to anoxic conditions and high temperatures. Stochastic and deterministic models of niche displacement. JOHN F. BOYER (Union College, Schenectady, NY 12308). I used computer simulation to study the interaction of two species, each of which had two phenotypes determined by one diallelic locus. One phenotype was a generalist predator on five different prey types; the other was a specialist on only one kind of the prey. The fitness (repro- ductive rate) of each phenotype depended on its predation success. In the deterministic experiments, a second-order difference equation was used to calculate predation rates, and genotypic frequencies at birth were always in Hardy-Weinberg proportions. In the stochastic experiments, predation was a consequence of random encounters, and genotypic fre- quencies were subject to genetic drift. When the two species were allopatnc (non-competing), the specialist individuals persisted at a (usually) low equilibrium frequency in the deterministic experiments, and were only rarely persistent in the stochastic experiments, in which fixation of the generalist allele was the predominant outcome. When the two species were sympatric and the generalists of both species competed for one or more prey types, the specialist individuals attained a higher average fre- quency than did their counterparts in the non-competing populations, thus showing niche displacement. The predation level (or efficiency), the magnitude of the satiation (or handling-time) parameters, and the resource coarseness (few large prey versus many small prey) all had a significant effect on the equilibrium frequencies or fixation probabilities. Almost all combinations of values for the five major experimental variables re- sulted in niche displacement, but the frequency-dependent selection forces were not strong enough to prevent allelic fixation in the majority of the stochastic populations. These results confirm the theoretical ubiquity of niche displacement, but also suggest that the magnitude of this displace- ment is likely to be sufficiently small that clearly discernable instances of this phenomenon will be quite rare in nature. This research was supported in part by a grant from Union College. The effects of macroalgae on the abundance oj eelgrass fZostera marinaj in the Waquoit Bay Estuary. HEIDI E. GEYER (The Marine Biological Laboratory), LINDA A. DEEGAN, JACK T. FINN, AND SUZANNE G. AY- VAZIAN. Within the last 20 years, estuarine eelgrass (Zostera marina) bed com- munities within the Waquoit Bay area have diminished in size and abun- dance. Concurrently, macroalgal abundance has increased. The impact of macroalgal development on Zoxiera manna in the Waquoit Bay Es- tuary was examined. Four plots (25 x 25 m) of varying macroalgal density were estab- lished. Macroalgae were removed by SCUBA from one plot (removal plot) and placed in another (addition plot). A disturbance control plot was established by removing the algae and replacing them in the same plot. A control plot was established in which no algal mani- pulation was done. Macroalgal density varied significantly in the removal and addition plots. The abundance of eelgrass was determined by counting tufts at the stem base along 0.5 m > 35 m transect lines within each plot both before (May 1990) and after (August 1990) the manipulation. Removal of macroalgae from a pre-existing eelgrass bed appears to cause increased tuft abundance in Zoslera marina. From May to August, the average number of tufts increased significantly in the removal plot and decreased significantly in the disturbance control plot. No significant difference in tuft abundance was seen in either the addition plot or in the control plot. The influence of macroalgae in decreasing eelgrass beds could be the result of direct and indirect effects. Macroalgae can directly over-grow eelgrass. It also causes a strongly anoxic benlluc layer, which is difficult for eelgrass to sprout in. Additionally, on sunny days we observed large mats of algae, filled with oxygen bubbles, lift off the bottom and float away, taking rooted eelgrass with them. Ex- perimental results and observations lead us to believe that dense macroalgae mats may contribute to the loss of healthy eelgrass beds in Waquoit Bay. The effects of macroalgae on the abundance and diversity of free-swimming invertebrates in eelgrass beds of Wa- quoit Bay, MA. KRISTIN M. O'BRIEN (Marine Biolog- ical Laboratory) LINDA A. DEEGAN, JOHN T. FINN, AND SUZANNE G. AYVAZIAN. Macroalgae in eelgrass beds of Waquoit Bay may decrease the suitable area of nursery habitats for juvenile fishes by affecting food abundance and availability. The effects of macroalgae on the abundance and diversity of free-swimming invertebrates were studied in a large scale field exper- iment. Four 25 by 25 meter plots with varying densities of macroalgae were constructed. A removal plot, containing minimal amounts of algae, an addition plot with algae taken from the removal plot, a control, and a disturbance control were all maintained from June through August. Mo- bile invertebrates were sampled with activity traps. Six traps were deployed in each plot for 24 h during July and August. An increase in macroalgal density apparently increases free-swimming invertebrate abundance, but invertebrate diversity is decreased. Typical species found were Gammams Oceanians. Erichsonclla atlcmtala. l-'.ilnlcu inliihii. Calanoid copepoda, and Palaemonetes pugio DuringJuly. there were significantly fewer invertebrates in the removal plot compared to both the control and addition plots, but during August there was no difference in abundance among the plots. The decreased invertebrate abundance was correlated with decreased algal density. When we com- bined all of the data we found that invertebrate abundance was inversely correlated with invertebrate diversity. Therefore, free-swimming inver- tebrate populations, which are important food sources for juvenile fishes, are increased in abundance and decreased in diversity in high density macroalgae areas. Inlluence of macroalgae in eelgrass beds on finfish abun- dance and dissolved oxygen in Waquoit Bay. REBEKA J. RAND (Marine Biological Laboratory), LINDA A. DEEGAN, JOHN T. FINN, AND SUZANNE G. AYVAZIAN. Waquoit Bay is an important estuarine area for resident, catadromous, and seasonally migrating finftsh. as well as the nursery habitat for some commercial fish species. An increase of macroalgal abundance has de- creased dense eelgrass beds. Therefore, we hypothesize that this macroalgal infestation of Zoxiera manna beds has reduced species diversity, total number, and total biomass of fishes. Four experimental plots (25 , 25 m) were established: removal, ad- dition, disturbance, and control. The macroalgae were removed from one plot (removal) and placed into the addition plot. Fish were collected using I - 1 m fish traps with '/,,, inch mesh netting. Four traps per plot were taken monthly from May to July. Samples were identified, counted, weighed, and measured. 224 ECOLOGY AND POPULATION BIOLOGY Our results indicate a greater number and biomass of fishes in the removal plot. Cumulative total fish biomass was highest in the removal plot (n = 33.6 g), followed by the control (n = 25.2 g), disturbance (n = 21.1 g). and addition (n = 17.6 g) plots. The total number of fish was highest in the removal plot (n = 70), compared to the control (n = 24). addition (n = 30). or disturbance (n = 36) plots. There was no trend in the species number among the plots. Juvenile winter flounder were only observed in the removal plot. Fourspine sticklebacks were the dominant species in the samples and were primarily found in the removal plot. Other species showed a mixed distribution among the plots. The observed distribution in fishes may be due to an increase in bare substrate for benthic fishes (i.e., juvenile winter flounder and benthic egglayers) and an increase in habitat for eelgrass dependent species (i.e.. fourspine stick- leback). Fertilization and Early Development Where does the calcium lost by fertilizing Arbacia eggs go? I. GILLOT, J. CHRYSTAL, L. F. JAFFE, AND W. M. KUHTREIBER (Marine Biological Laboratory, Woods Hole. MA 02543). After eggs of the sea urchin Arbacia are fertilized, a calcium wave traverses the eggs at about 14 ^m/s. This wave is immediately followed by an exocytotic wave that releases the contents of the cortical granules, resulting in the lifting off of the fertilization membrane. In this study, we have used our recently developed vibrating ion selective probe system (KuhtreiberandJaffe. 1990,/ Cell Biol. 110: 1565-1573) to investigate which mechanisms are responsible for returning the calcium concentra- tion to its resting level. Arbacia punctulata eggs were fertilized in artificial seawater (ASW) containing I mM Ca ++ . We could measure a calcium efflux during the 215 10 s (S.D.: n = 5) before the signal was lost in the noise, with a peak efflux of about 1 1 pmol/cnr/s. The total calcium lost during this period was calculated to be about 1 1 .7%, which is in excellent agree- ment with the results of Azarnia and Chambers (1976. J. E\p. Zoo/. 198: 65-78). We also performed measurements on eggs of Phallusia mammilata. Although the eggs of this ascidian do not contain cortical granules, we nevertheless measured a post-fertilization calcium efflux with a duration and magnitude similar to that of Arbacia (265 18s; S.D. n = 4). Moreover, post-fertilization measurements with a proton-selective vi- brating probe showed that proton release by exocytosis lasts less than 20 s. Presumably, calcium release by exocytosis would also last for this short period. In summary, fertilized sea urchin and ascidian eggs use a calcium pump that actively transports intracellular free calcium out of the cell. Only a small part, if any. of the calcium lost from Arbacia eggs to the medium can be due to cortical granule release. This work was supported by NIH grant #RR01395 to L. F. J. The path of calcium in fertilization and other endogenous oscillations: a unifying view. LIONEL F. JAFFE (Marine Biological Laboratory, Woods Hole, MA 02543). Throughout the vertebrate line, eggs are activated by a calcium wave that crosses them at about 10 /im/s. In tunicates, as well as mammals, these fertilization waves are followed by a long, periodic series of after- waves (Speksnijder el a/.. 1990, Dev Biol. 142). A growing number of the periodic calcium pulses found in various cultured cells also consist of calcium waves. This abstract makes five postulates. ( 1 ) Every calcium pulse takes the form of a calcium wave. (2) Every medium speed (i.e.. 3-300 fim/s) calcium wave is initiated and then propagated by two distinct modes of calcium-induced calcium release: first, in the lumenal mode, a slow rise of calcium within the lumen of the endoplasmic reticulum (E.R.) reaches a trigger level that initiates fast, localized release into the cytosol; then the well-known cytosolic mode drives a reaction-diffusion wave across the cell. (3) The velocities (v) of such calcium waves are governed by the Luther equation: where D is the diffusion constant of an autocatalytic substance (here Ca ++ ), t is the time taken to rise e-fold during the autocatalytic rise, and K is about one (Arnold el ai. 1987. / Chem. Ed. 64: 740-744). (4) During the latent periods that precede fertilization, as well as some agonist-induced oscillations, calcium is pumped into a wave initiation or pacemaker region of the E.R. (5) In fertilization, this calcium comes from the medium via the inner acrosomal membrane of the fused sperm. Supported by NIH Grant No. RR01395. The gigantic germinal vesicles of elasmobranchs. C. D. LEIDIGH, N. H. KIM, R. D. GOLDMAN, A. GOLDMAN, M. V. L. BENNETT (Albert Einstein College of Medi- cine), AND G. D. PAPPAS. Germinal vesicles (GVs) of the skate. Raja erinacea. are readily visible as small clear regions just beneath the surface of immature and mature oocytes. The GVs are exceptionally large, and their diameters range from 280 to 500 fim in oocytes from 0.7 to 20 mm in diameter. Slightly smaller GVs were seen in the larger (40 mm diameter) oocytes of one specimen of the dogfish shark. Sunaliix acanthias. GVs are readily isolated by dissection and are nearly spherical, very transparent, and quite robust. Light microscopy of isolated GVs and light and electron microscopy of GVs fixed in siln revealed structural details. The nuclear envelope is somewhat convoluted and consists of two membranes with abundant nuclear pores about 60 nm in diameter. The inner aspect of the envelope is covered by a thick (50 nm) layer, probably of lamin. The nucleoplasm contains some granular material, but no apparent structure such as nu- cleoli. DNA staining with Hoechst 33342 or RNA staining with thyazol orange was negative, presumably because the chromalin is dispersed. SDS-PAGE of isolated vesicles reveals several bands between 60 and 70 kDa, which are likely to be lamin. as well as a band at 45 kDa ascnbable to actin. The lamin may account for the strength of the GV envelope. Isolated GVs were bathed in fluorescent tracers in elasmobranch phys- iological saline and penetration and washout observed. The nuclear en- velope is permeable to fluoresceinated dextrans of both 10 kDa and 70 kDa molecular size and is less permeable to the larger tracer. Consistent with the permeability to large molecules, we could detect no membrane potential or resistance. These GVs should be a valuable preparation for characterization of properties of the nuclear envelope. Calcium waves spread beneath the furrows of cleaving Oryzias latipes and Xenopus laevis eggs. ANDREW L. MILLER (Marine Biological Laboratory), RICHARD A. FLUCK, JANE A. MCLAUGHLIN, AND LIONEL F. JAFFE. To monitor cytosolic [Ca 2+ ] during cytokinesis, recombinant aequorin was microinjected into eggs of Oryzias and Xenopus. Photon emission was monitored with an ultra-sensitive imaging photon detector, and the mechanical events accompanying cytokinesis recorded using time-lapse video microscopy. Most of our observations were made on the two fur- ABSTRACTS FROM MBL GENERAL MEETINGS 225 rows that form during the second cleavage of the blastodisc in Oryziax. where cytokinesis consists of two distinct processes. In the first, a furrow cuts through the blastodisc from the external surface, cleaving the cy- toplasm; in the second, the membranes of the daughter cells "zip up." becoming closely apposed. Both processes begin near the center of the blastodisc. at right angles to the first furrow, and end at the edges of the blastodisc. At 19C. the second furrow forms in 7 mm. begins to zip 3 min later, and finishes zipping 14 min later: the entire process takes 24 min. We observed two distinct calcium waves along the furrow during cytokinesis: the first approximately coincided with the formation of the furrow, and the second approximately coincided with the zipping up of the furrow. That is to say, a calcium hot spot appears near the center of the blastodisc when the furrow begins to form and travels to the edge of the blastodisc, arriving there when the furrow does. A second hot spot appears at the center of the blastodisc when zipping up begins, and it also travels to the edge of the blastodisc. Both waves spread at 0.3-1 urn s ' and are thus in the class of slow calcium waves, which may be mechanically propagated via stretch activated channels. Simi- lar waves were also detected during first cleavage of Xcnapux eggs. This is the first report of the visualization of cytosolic calcium changes during cleavage. Supported by NSF DCB-881II98 (Amendment 01) and NIH RR01395. Sea urchin embryos: suitability jor e.\ogenoiis expression of gap /unction channels. ALONSO P. MORENO (Albert Einstein College of Medicine) AND DAVID C. SPRAY. Gap junction channels in \ertebrates are formed of a group of ho- mologous proteins termed connexins. The tissue-specific expression of various connexins gives rise to gap junction channels with different phys- iological properties [e.g.. voltage and pH dependence, unitary conduc- tance |")j) of the channels]. Because certain tissues may express multiple connexins, it has become desirable to study the properties of connexins expressed individually in exogenous expression systems, including \en- o/w.s oocytes. This preparation has certain limitations, such as endogenous connexins, that complicate the interpretation ol data from mRNA-in- jected cell pairs. In addition, single channel studies (where channel cur- rents are detected between two voltage clamped cells) are compromised by the large size and low input resistance of the oocyte. We have attempted to use sea urchin embryos for these studies, because they lack gap junc- tions at early developmental stages (Spiegel and Howard. 1483. / Cell Sci. 62: 27-48). and have small blastomeres. Using Lucifer Yellow in- jection to test for dye coupling, we have found that blastomeres at early cleavage stages (<16-cell) are not coupled following cell division. At later stages (16-, 32-cell). dye transfer is limited to sister blastomeres. The input resistance of the blastomeres (> 800 MB) is high enough to calculate the -), of the endogenous channels, which averaged about 220 pS (>16- cell stages). To test for the expression of exogenous mRNAs. mRNA encoding rat connexin32 and transcribed in \'itn>. or. poly A + RNA extracted from the ctenophore .\fneiniop.iis, or deionized water were injected into sea urchin oocytes immediately after fertilization. Subse- quent cleavages were judged normal in about 50% of the embryos. Em- bryos (<16-cell stage) injected with ctenophore mRNA or water showed no dye coupling, whereas in the connexin32-injected blastomeres. dye spread rapidly to sister cells and was detectable throughout the embryo within 5 min (n = %). We conclude that connexin32 is expressible in sea urchin embryos, where the lack of endogenous coupling and small size should permit the characterization of gap junction channel properties inaccessible in Menopus oocytes. Supported in part by a Grass Foundation Fellowship to A. P.M. An aerial vibrating probe. RICHARD H. SANGER (Marine Biological Laboratory), ERIC KARPLUS, AND LIONEL F. JAFFE. We are improving a technique for observing the electrical behavior of a biological system developing in air using the AC Kelvin method. An electrical current travelling through a resistive element generates an electric field outside the element. One can construct a capacitor by placing a conductive plate in the electric field. The charge on the capacitor will be proportional to the local voltage on the resistive element (produced by a current flowing through the element) divided by the distance from the element to the plate. With the aerial vibrating probe, a biological system behaves as a resistive element with a current flowing in it, and a vibrating plate is used as a probe to construct a variable capacitor. Sub- surface currents in the biological system generate the electric field to be measured. Vibrating the plate changes the distance between the plate and the source of the electric field, hence changing the charge on the effective capacitor. This changing charge is amplified and processed with a lock-in amplifier. The present device uses a common Field Effect Tran- sistor (FET) as a head stage amplifier. The probe that serves as the de- tecting plate is the factory-supplied lead on the FET. which is approxi- mately 430 n in diameter. We have been vibrating the entire FET with a piezoelectric element at about 130 Hz. With a 30-ji vibration at 60 >* from the source, we have already been able to resolve signals of several millivolts, similar to those found on corn coleoptiles in response to light or gravity (Grahm and Hertz. 1962. Pliysiol. Plant. 15: 96-113). We hope to reduce the size of the probe by a factor of 10 and increase the sensitivity of the system by a factor of 100. This will involve improving the electronics and physical geometry of the system. Supported by NIH grant RR01395. Gap junction eluinnels in marine embryos: comparison of properties in late b/aslit/ae of squid CLoMgo paeleij and skate (Raja erinaceaA D. C. SPRAY (Albert Einstein College of Medicine), A. C. CAMPOS DE CARVALHO, A. P. MORENO. E. SCEMES, C. LEIDIGH. N. H. KIM, G. D. PAPPAS, AND M. V. L. BENNETT. Gap junctions are abundant in most early embryos; their role, pre- sumably, is to facilitate the exchange of developmentally relevant mol- ecules between the differentiating cells. In previous studies, the physio- logical properties of gap junctions in ascidian. amphibian, and teleost blastulae have been characterized. In all these groups, gap junctions exhibit voltage sensitivity; i.e.. a voltage imposed across the junctional membrane closes the channels. The sensitivity in amphibian and ascidians, but not in teleosts. is great enough to suggest that resting potential differences could act to establish developmental compartments. We therefore ex- amined other embryos. We have now recorded currents through gap junction channels from pairs of cells dissociated from late blastulae of skate (Raja erinaceu) and squid (Loligo pealei) using the dual whole cell recording method with patch pipettes. In skate, we recorded from 18 cell pairs; in the 8 pairs that were coupled [i.e.. junctional conductance (gj) was greater than 10 pS] g ; ranged from 2 to 20 nS, with a mean of 10 nS. In squid. 18 of 23 cell pairs were coupled, with a mean g ; of 13.5 nS. In neither cell type was there a marked degree of voltage dependence. In both types, g, was reversibly reduced by exposure to 2 mAt halothane in the bathing solution. At low values of g r single channel currents were observable from which the unitary junctional conductance (7,) was cal- culated. For skate. 7, values peaked near 100 pS; for squid, y, was much larger, with most values above 250 pS. The skate 7, is similar to that recorded from cells expressing connexins of adult mammals, whereas the squid y, is substantially larger. The larger 7, for the squid junctions 226 FERTILIZATION AND EARLY DEVELOPMENT is consistent with their larger channel diameter inferred from previous permeability measurements. Cyclic AMP-dcpciiilcnl p/iosphorylu/ion of dynein licavy chain,*, in Mytilus edulis sperm ftagella. R. E. STEPHENS ANDG. PRIOR (Marine Biological Laboratory, Woods Hole. MA 02543). Lateral ciliary activity in Mytilitii citulis gill is mediated by serotonin through the cAMP-dependent phosphorylation of presumptive dynein light chains. Because MvliliLf sperm are also reported to be serotonin- activated, we investigated cAMP-dependent phosphorylation of sperm dynein. Sperm were decapitated by homogenization and the flagella were recovered by differential centrifugation. The flagella were permeabilized with 0.012% NP-40 and then incubated with -y- 3: -P-labeled ATP. either with or without added cAMP. The reaction was stopped, and the mem- branes were removed by extraction with 0.25% NP-40. Outer arm dynein was produced by extracting the resulting 9 + 2 axonemes with 0.6 M NaCl for 15 min on ice, while inner arm dynein was obtained by a second extraction with 0.6 M NaCl containing 0.25% NP-40. The dynein fractions were analyzed by sucrose gradient centrifugation, SDS-PAGE. and autoradiography. Each fraction represented >45% of the total ATPase. The dynein sedimented at 18-2 IS and consisted of equimolar a and li heavy chains, intermediate chains of 95 kDa and 80 kDa, and three light chains. No light chain phosphorylation was observed. However, the a heavy chains of both the outer and the inner dynein arm fractions were singularly and equally labeled. Phosphorylation occurred maximally at cAMP levels above 1 micromolar; cGMP was also effective, but at > 10-fold higher levels. The phosphorylation was extremely rapid, highly stable, and calcium-independent. Photocleavage at 360 nm. in the pres- ence of vanadate and Mg-ATP, yielded products with molecular weights of 225 and 215 kDa (HUV fragments) and 185 and 175 kDa (LUV fragments). Phosphorylation occurred exclusively on the 185 kDa LL1V fragment, establishing its derivation from the heavy chain and arguing against multiple phosphorylation sites. Dynein heavy chain phos- phorylation was similarly obtained with Spisula solidixxima sperm, also known to be serotonin-activated. These are the first definitive examples of dynein heavy chain phosphorylation in response to cAMP. Supported by USPHS GM 20.644. Gossypol-binding proteins from marine species. HIROSHI UENO (Rockefeller University), SHELDON J. SEGAL, AND S. S. KOIDE. During the past few years, various biological activities of gossypol have been explored. Besides male antifertility or antispermacidal activity, we have reported antiparasite activity (Eid et al, 1988, Exp. Parasilol. 66: 140) and antiHIV activity (Polsky el al.. 1989, Contraception 39: 579). This wide range of biological actions suggests that there might be a parameter common to the cell types affected. In this study, we aim to establish the methodology to identify this common parameter, a protein that is responsible for gossypol binding. We used sperms from marine organisms as a model system because of their various advantages over other cells. Sperm proteins from marine species (Spisula and Arbacia) were collected and washed with MBL ASW. The sperm pellet was treated with methanol and chloroform to remove lipophilic materials. A protein material was extracted from this lipid-free sperm material by incubation with PBSTDS buffer which contains Triton X-100, deoxycholate. and SDS in phosphate buffered saline. This was incubated with ' 4 C-gossypol (500 p.M) for 30 min at 25C. then a reducing agent. NaBH 4 , was added. Incorporation of ' 4 C-gossypol was both time and dose dependent. Free gossypol was removed by the chromatography on a Pharmacia Fast De- salting column. Gossypol-binding protein was fractionated by gel filtration on Pharmacia Superose 6; it was eluted with bisTris-EDTA-Tween 20 buffer, at pH 7.5. and at a flow rate of 0.3 ml/min. The protein extract of I rhacia sperm contained two fractions with equal amounts of radio- activity: one at the void volume, and the other at 45 min (estimated molecular weight at around 100 kDa). Similar results were obtained from the protein extract ofSpisiilu sperm; but in this case, the radioactivity was found predominantly in the second fraction. Analysis on SDS-PAGE showed that fraction I gave one major band at 16 kDa with two minor bands at the top, and fraction 2 gave one band at 2 1 kDa. These results suggest that there are specific proteins modified by gossypol, and these proteins could be a target for gossypol action. The method described here could be used for the isolation of gossypol-binding protein from other cells. This research was supported by funds from the Rockefeller Foundation. Mariculture and the Marine Environment Submersible vehicle observations of deep sea red crabs, Chaceon quinquedens, off of the U.S. continental shelf ROBERT A. BULLIS (University of Pennsylvania. Lab- oratory for Marine Animal Health. Marine Biological Laboratory). The purpose of this study was to assess the ability of submersible technology to provide on-site inspection of Crustacea for signs of gross pathology. Concurrently, we sought to discover whether a gradient of shell disease exists as a result of oceanic dispersal of sewage sludge from the Deep Water Municipal Sewage Sludge Dump Site 106. The Harbor Branch Oceanographic Institution submersible Johnson Sea Link II was used for in suit observations. Rated to a depth of 3000' (9 1 m ), the Sea Link provides unexcelled visibility and maneuverability for observational and experimental studies of midwater benthic communities. Red crabs were monitored for shell disease, and corresponding sediment samples were analyzed for indicator bacteria. Bacterial isolates were successfully obtained from nephloid (surface) sediments at all sampling stations. Greater numbers of bacteria were isolated from shallow (<800 ft.) stations than from deep stations (>2000 ft.). Significant numbers of bacteria, typically associated with shell disease, were isolated from affected, in contrast to non-affected, areas of red crab exoskeletons. Shell disease appeared to occur randomly throughout the population. Of particular note was the observation that, while standing or walking, all red crabs support themselves on the tips of their walking legs. The ventral surfaces of the appendages and the sternum do not scrape the sediment except when the animal is entering or leaving its burrow. Indeed, after the red crabs leave their burrows, nephloid sediments adhere to their carapaces. After a time, sediment remains only in "suture depressions" of the car- apace. This prolonged contact between the sediments and certain areas of the exoskeleton may help to explain why hyperpigmentation of red crab carapaces is often bilaterally symmetrical. This work was sponsored through the generosity of the National Un- dersea Research Program (NOAA-UCAP) and a grant from the Division of Research Resources, NIH (P40-RR1333-10). Calibration of the effect of the air-sea interface on mea- surement ofee/grass and macroalgal density in Waquoh Bav. JOHN T. FINN (University of Massachusetts), LINDA A. DEEGAN, AND DAVID PATON. Waquoit Bay is an important nursery ground for fish and shellfish. Nutrients have caused an increase in macroalgae at the expense of eelgrass ABSTRACTS FROM MBL GENERAL MEETINGS 227 beds over the last two years. To examine its effect on eelgrass, we removed macroalgae from one 25 25 m plot in an eelgrass bed in Hamhlin Pond, a part of Waquoit Bay. The algae removed were added to a second plot, and there were two control plots: one untouched, and one with algae removed and replaced. Can the amount of algae and eelgrass in the experimental plots 90- 120 cm deep be determined using aerial photography? If aerial pho- tography is to be successful, we must identity the plots from the air and distinguish between the bottom, the macroalgae, and the eelgrass. To test this question several problems had to be addressed. Surface glare was minimized by using a polarizing filter and by taking pictures at times of low sun angle. Ektachrome 100 film was used. Distortion was addressed by placing targets at the plot corners and correcting the photographs to match the true size and shape of the plots. Targets of known color were used to correct for color distortion of dissolved and suspended matter. To test the feasibility of aerial photography, white locational and color strip targets were photographed in water from 35 to 100 cm deep. Targets disappeared at 55 cm, and colors became indistinct at 45 cm depth. To do the aenal photography, Secchi depth would have to be at least twice what it was during the calibration test. In May or June, when the water is clear, this procedure should allow determination of macroalgal density, although distinguishing between eelgrass and macroalgae would still be a problem. Intentional catch (Busyconj and unintentional catch (Ho ploplanaj hy fishermen and the question of. seafood in- spection. ILENE M. KAPLAN, BARBARA C. BOYER, AND DANIELA E. HOFFMANN (Union College, Schenectady, New York 12308). Data from a longitudinal study on socio-economic and ecological trends in the New England conch (Biisycon) fishery are reported together with related work on the commensal relationship of the tlatworm Ho- p/i'pliinu Special attention was given to marketing conditions and the policies (both formal and informal) that protect these species as well as their consumers. Interviews with fishermen, seafood buyers, processors, and fish market and restaurant owners were conducted, and participant observations were made. New England conch is different from, but may be confused with. South Atlantic conch (Strumhus) because they have the same commercial name. Fishermen receive 30 to 50 cents a pound for conch in the shell, which are then sold in New England markets for two to three times their initial worth. Conch meat removed from the shell sells for $3.99 to $4.25 per pound, and in a salad from $3.49 to $5.99 a pound. Customers are typically from Italian and Oriental ethnic groups; foreign export sales include Asian and European markets. The volume of conch sales vary markedly; wholesalers' figures range from 300 pounds to over 8000 pounds per week. Inconsistencies in the reports of sales were found; government reports of sales appear to be more conservative. Variability in the conditions of processing and marketing of conch were also observed. Markets follow inspection guidelines on a volunteer basis, but there are no formal rules for seafood inspection. Although conch is categorized as a shellfish, enforcement of shellfish tagging rules may be variable. Overfishing of New England conch is a potential problem as com- mercial interest increases. The number of conch pots has increased dra- matically in the last few years, threatening not only the conch, but the flatworm Hupbplana that lives commensally in its mantle cavity. A total of 579 specimens ofBusycon canaliculatum were examined (4 1 2 females and 167 males). Twenty-two percent yielded 190 worms. The occurrence of multiple worms per specimen has decreased from previous years, and more worms were found in males than females, also a departure from previous observations. Formal and unified seafood inspection programs and education and clarification of policies regarding the commercial fishing of conch are suggested. The authors gratefully acknowledge the support of the Woods Hole Oceanographic Institution, the Marine Biological Laboratory, the Na- tional Marine Fisheries Service and Dana Fellowships/Union College. Ozonation of natural seawater affects the embryology of Hermissenda crassicornis. ALAN M. KLJZIRI AN (Marine Biological Laboratory). CATHERINE T. TAMSE, AND MARK HEATH. The new MBL Marine Resources Center (MRC) will have three dif- ferent uses for ozonated seawater: in the recirculating system, in the treated, flow-through system, and in the effluent system. Major aquaria and commercial hatcheries operate at ozone dosages ranging from O.I to 2 mg/l. However, the exact dosage appropriate for the new MRC is unknown. We undertook a study to ascertain a preliminary working ozone dosage, using the embryonic development of Hermissenda m/.v- Wivnm as a bioassay. The following treatment solutions were tested: (1) ozone levels (12.8 min contact time) at 2.2 mg/l. and 0.24-0.28 mg/l; (2) ozone plus 0.1 mAf Na thiosulfate to remove hypobromic acid; (3) ozone plus NH, (0.6- 1 .2 mg/l). NO, (0.05 mg/l) (N-cmpds); and (4) ozone plus thiosulfate and N-cmpds. Controls consisted of 0.2 ^-filtered natural seawater (NSW). Replicates of the solutions, with and without 0.25 mg/l EDTA were used, and each solution was run in duplicate. A single egg mass from Hermissenda was cut and distributed equally for each run, which lasted 6 days. Ozone dosage of 2.2 mg/l resulted in a very unstable ozone residual of <0. 1 mg/l; this level should require only minimal aeration for post- treatment. Ammonia concentration did not decrease with ozonation, but all nitrites decreased below detectable limits. Total residual oxidants (TRO). as mg/l C1 2 , registered 0.9 mg/l and slowly decayed to nonde- tectable limits (70-80 min). TRO for 0.28 mg/l ozone was undetectable. Bioassay results for 2.2 mg/l ozonation indicated that EDTA or thio- sulfate was required for normal development. Ozone plus N-cmpds also produced normal survival rates, but the number of fouling organisms on the shells increased. Ozone with N-cmpds and thiosulfate were del- eterious to larval hatch and survival. Dosage of 0.28 mg/l ozone was insufficient to inhibit fouling organisms (diatoms, ciliates); mortality was high for all conditions. Normal development was correlated with the presence of EDTA. Ozone treatment of MBL seawater is effective at 2.2 mg/l dose levels. Residual ozone and TRO must be removed to insure the survival of sensitive organisms. Biofiltration is supplemented by residual ozone, which oxidizes toxic nitrites to nitrates. The lower ozone dose tested was insufficient as a primary treatment to inhibit the growth of fouling organisms. Hermissenda proved to be a sensitive animal for use in this bioassay. This research was supported in part by a grant to A.M.K. (NIH, RR03820). Greenough Pond project planning: Yarmouthport, Mas- sachusetts: acid water and macro flora is the base line ofH. Sverdop and P. Wurfmgers ' chain of response de- cision tree. DAVID PATON (BUMP affiliate, Marine Biological Laboratory). The Greenough kettle pond is spring fed from the unused cranberry bogs at its south west corner. Ground water flows from this direction and includes the Little Greenough Pond. It is one of the more elevated ponds on the Cape and is located near the center of the single source 228 MARICUI JURE AND THE MARINE ENVIRONMENT aquifer. This fresh water lens lies beneath a glacial till of sandy soils, sweetened by gravels and dotted with granite boulders, that form the substrata that form the basin of Greenough Pond. The bottom of the pond is silt overlaid with litter from trash that is flown in by seagulls. Milfoil plants dominate the below-surface water weeds, and their growth appears to be depth dependent as they emerge in the shallow coves; they seldom occur below depths of 12'. White lily pads and water shield float on the surface in the quiet water of the west and north coves of the pond. The pond is acidic. Samples taken in January. Apnl, July, and October, as part of an ongoing monitoring of acid rain deposition taken throughout the Cape, have a typical pH of 5.18. This acidity does not vary more than two tenths of a pH unit over a data set that was compiled from as far back as April 1985. EPA alkalinity is zero or slightly higher. Halichoerus grypus at Monomoy Island, winter of 1988- 1989: photogrammetry \howing shift east and north of traditional birthing sites. DAVID PATON (Marine Bio- logical Laboratory). On 13 February 1989, high oblique aerial photographs were taken of the seal habitats in Martha's Vineyard and Nantucket Sounds. This was the second of two flights made that winter which were the only surveys attempted during 1989 to test a behavior prediction model. High density black and white 70-mm film was used. Positive enlarge- ment transparencies were then projected on a silver screen for viewing. Prints were offered to four biologists for comment. ITEK. international Corp. of Bedford. Massachusetts, was consulted for photointerpretive advice. Two of the seal pups appear to have completed the molt from white coat. This represents a shift from the place where these animals have been seen at Nomans Island during the 1930s, and off Nantucket during 1987-1988, and in 1963. These results are dedicated to the memory of my father. His meteo- rological career and dedication to education represented his unfailing belief in our stewardship of the land and sea. The Dikcnson family sup- ported the winter observations for definition of critical marine habitat. Neurobiology and Biophysics Antimalarial drugs block calcium currents in Paramecium. SUSAN R. BARRY (University of Michigan), JUAN BERNAL, AND BARBARA E. EHRLICH. We studied the effects of antimalarial drugs (quinacnne, chloroquine, and quinine) on calcium currents in Paramecium calkinxi. These com- pounds are structurally similar to W7, a drug that blocks calcium channels in paramecia. As an initial test of the effects of the antimalarial drugs on calcium currents, we observed the actions of these drugs on calcium-dependent swimming behavior in paramecia. When the paramecium is placed in a high potassium medium (31 mUl KCI, 94 mA/NaCI, 1 mA/CaCl 2 , and 10 mA/ MOPS, pH 7.3). the cell swims backward for about 50 s. The application of calcium channel blockers, such as W7, reduces the duration of backward swimming. Quinacrine, chloroquine, and quinine all reduced the duration of backward swimming in a concentration-dependent man- ner. At a concentration of 10 >iA/. quinacrine inhibited backward swim- ming by 88%, chloroquine by 37%, and quinine by 29%-. The effects of quinacrine were tested directly on calcium currents using a two-microelectrode voltage clamp. The paramecia were bathed in a sodium-free recording medium containing potassium channel blockers (125 mA/TEA-Cl. 10 mA/ CsCl. 5 mA/4-AP. 5 mA/ 2,4-DAP, 15 mA/ Cad,, 10 mA/ MOPS), impaled with microelectrodes filled with 300 mA/ cesium citrate, and held at a resting potential of 40 mV. Depo- larizing voltage steps evoked an inward calcium current, the peak am- plitude of which was reduced by 15% in 10 fiM quinacnne, by 51% in 100 ^A/ quinacnne. and by 91% in 1 mA/ quinacrine. In summary, the effects of quinacrine, chloroquine. and quinine on backward swimming behavior suggest that these drugs inhibit calcium currents. Subsequent voltage clamp studies demonstrated that quinacnne indeed blocks calcium currents. Antimalanal drugs may also reduce cal- cium currents in other protozoans, including plasmodia, the protozoan parasites that cause malaria. Calcium channel blockade may explain in part the therapeutic effects of these drugs. Supported by the Grass Foundation and NIH grant GM39029. B.E.E. is a Pew Scholar in the Biomedical Sciences. Calcium-dependence of inositol 1,4,5-trisphosphate-gated calcium-channels from endoplasmatic renculum of cer- ebellum is hell-shaped. II.YA B. BEZPROZVANNY, JAMES WATRAS. AND BARBARA E. EHRLICH (Univer- sity of Connecticut Health Center). Regulation of inositol 1,4,5-trisphosphate (IP,)-induced calcium (Ca) release from intracellular stores has been hypothesized to occur by a direct effect of cytoplasmic Ca on the IP 3 -receptor. To study the char- acteristics of this regulation, we incorporated native membrane vesicles from cerehellar endoplasmic retieulum into planar lipid bilayers. Channels were studied with 53 mA/ Ca(OH) 2 in 250 mA/ HEPES. pH 7.35 on the tram side of the membrane and 250 mA/HEPES-Tns. 1 mA/ HEEDTA, and 1 mA/ EGTA, pH 7.35, on the m side of the membrane (cix cor- responds to the cytoplasmic side). Addition of 2 fiAt IP 3 to the cis chamber activated Ca-permeable channels with four conductance states, each a multiple of the lowest conductance (20 pS). Experiments on the Ca-dependence of these chan- nels were performed in the presence 330 M AMP-PCP, because we found that adenine nucleotides activated the channels about 10 fold. As the free Ca concentration was increased, the probability of observing open channels changed biphasically. with the maximum at about 0.25 nM Ca. This value is in a good agreement with data obtained with IP,- induced Ca-release from permeabilized smooth muscle (lino, 1990. J. Gen. Physiol. 95: 1 103-1 122). The Ca-dependence of the channels was very sharp, with half-maximum activation and inhibition at 0.1 ^A/Ca and 0.5 \iM Ca, respectively. To fit the data with a theoretical curve, we assumed that each subunit of the receptor had two sites and that binding of Ca to one of these sites leads to activation of the channel, and that binding to the other leads to inhibition. We had to assume the existence of a tetrameric structure for the IP-rreceptor complex to make the Ca-dependence sharp enough to be in agreement with the data. The existence of the tetrameric structure is supported by four subconductance states observed in our experiments and by electron-microscopic analysis of the purified IP,-receptor. Supported by NIH grants HL-33026 and GM-39029. B.E.E. is a Pew Scholar in the Biomedical Sciences. Calcium dynamics in the presynaptic terminals oj barnacle photoreceptors. JOSEPH C. CALLAWAY (University of North Carolina, Chapel Hill), NECHAMA LASSER-ROSS, ANN E. STUART, AND WILLIAM N. Ross. We have measured the time course and magnitude of the [Ca], in the presynaptic arbors of individual photoreceptor neurons of the barnacle (Balanux nubiliis) injected with FURA-2. The ocellus, containing the light-sensitive dendntes. was cut off, and the FURA-filled electrode was used to stimulate the terminal and record membrane potential. The (Ca], ABSTRACTS FROM MBL GENERAL MEETINGS 229 was recorded al even point along the arbor (about 3 fim resolution), from the primary branches to the spray of smaller processes housing the release sites. Depolarizations with injected current caused a rapid rise in [Ca] : initially localized to the tips of the spray. The [Ca], in proximal parts of the arbor rose more slowly and with a delay as the Ca diffused backwards from the tips to these locations. The threshold voltage for Ca entry was between -55 and -62 mV. During maintained depolarization, Ca entry was continuous and the [Ca], at any point in the arbor reached a steady state; as the Ca diffused longitudinally from the release sites into the primary branches, a gradient of [Ca], was established. The [Ca]j at the release sites increased less than 100 nAI in response to depolarizations that were just above threshold and sufficient to cause transmitter release. The [Ca], increased to 400-500 nAl when the terminals were depolarized to potentials normally achieved by steady light of moderate intensity (-45 mV). When the PR terminal was repolarized from a depolarized level, the [Ca], at the tips decreased quickly, as might be expected from this synapse, where decn'ii.w* in transmitter release constitute the im- portant signal. Computer simulations indicate that longitudinal diffusion alone can account for this decrease. Supported by grants NS16295 to W.N.R. and EY03347 to A.E.S. Cross-correlations in the spike activity of neurons in the Aplysia abdominal ganglion during the gill-withdrawal reflex. LARRY COHEN, CHUN XIAO FALK, DAVID SCHIMINOVICH, JlAN-YOUNG WU, AND RAY FALK (Dept. of Physiology, Yale University School of Med- icine). In previous attempts, we were unable to find significant correlations between the spike times of the 100-200 neurons in the Aplnia abdominal ganglion that are activated by a light touch to the siphon skin. In these experiments, we weighted the correlation scores inversely to the overall spike density. In this study, we have not made such a correction. We used two methods (raw scores and normalized scores) to evaluate correlations. For the normalized score, the raw score was divided by the number of spikes in the cell of the pair having the smaller number of spikes. We scored approximately 5000 pairs of cells for each data set. Because the number of pairs and the number of spikes is large, it is not surprising that we detect correlations. To test the possibility that these correlations are due to chance, we generated a random data set from the real data b\ replacing each spike time with a randomly chosen time point within a 64-ms interval surrounding the real time. We made 10 random sets and compared the correlation scores from the real data with the scores from the 10 random sets. In the trials thus far examined, the correlation scores from the real data were bracketed by the largest and smallest correlation scores from the random data. Thus we did not find correlations larger than might be expected by chance. If the Aplysiu nervous system used many large, excitatory synaptic potentials to generate the gill-withdrawal reflex, then we would expect to find larger than random correlations between the activity of cell pairs. Our results suggest that large excitatory synaptic connections are not common. Further, correlation analysis may contribute relatively little to working out the map of synaptic connections among the neurons of the abdominal ganglion. Supported by PHS grant #NS08437. Effects of synapsin I on synaptic facilitation at crayfish neiiromusadar junction. K. R. DELANEY, Y. YAMAGATA. D. W. TANK, P. GREENGARD, AND R. LLINAS (Marine Biological Laboratory, Woods Hole, MA 02543). The effects of presynaptically injected phospho- and dephospho-syn- apsin I on transmitter release were studied in excitatory claw opener junctions in crayfish. We examined release evoked by presynaptic action potentials delivered at 0.5 Hz, which does not produce facilitation, and at higher frequencies (5-50 Hz) where facilitation is prominent. Consistent with previous work on squid giant synapse (Llinas el al.. 1985, PNAS 82: 3035) and Mauthner cell synapses (Hacket cl al., 1990. ./ Ncimi- phyxiol. 63: 701), reduction of excitatory junction potentials (EJPs) ob- tained with 0.5 Hz stimulation was seen 5-30 min after injection. This reduction was coincident with the appearance of Texas red labeled syn- apsin I in the preterminals contacting the postsynaptic muscle liber (8 of 9 expts.). The EJP amplitude continued to decline linearly over the course of 60-120 min to near zero. In addition, the rate of facilitation of the EJP during short stimulus trains at 5. 20, and 50 Hz was reduced following this injection. Moreover, high rates of release, which are nor- mally produced and maintained during several minutes of stimulation at frequencies around 30 Hz, were not maintained following dephospho- synapsin 1 injection. These effects were not seen following the injection of phosphorylated synapsin I (n = 1). We conclude that synapsin 1 can modulate facilitated and unfacilitated transmitter release at this tonic junction. Application of a magnetic current probe to map axial in- homogeneities in a siniid giant axon. J. M. VAN EGE- RAAT AND J. P. WIKSWO JR. (Vanderbilt University. Nashville, TN). The magnetic current probe developed by Wikswo (Gielen el al., 1986, IEEE Trans Bnmml Eng BMK-33: 910-921) enables us to measure accurately intracellular action currents in nerve fibers and bundles without the need for physical contact between the probe and the preparation. We exploited this feature to map axial inhomogeneities in the squid giant axon by scanning the probe along the fiber and measuring the action current at many different positions. Similar measurements with mtracellular microelectrodes would be hard to perform without damage to the preparation, particularly because a single experiment may involve 250 measurements at 50 different positions. With our probe, we studied the sequence ofevents that follows a crush injury to a squid giant axon over a period of 3 h under normal physio- logical conditions. We observed that the crush blocked propagation and caused a depression of the action currents in the region proximal to the crush. The proximal depression spread retrograde with time. This led to the conclusion that the crushed axon did not seal. Other preparations have been reported to seal and. in addition, to form a partition-like structure in the intracellular space, blocking propagation (Yawo and Kuno. 1985, J. Neiirnsc. 5: 1626-1632). We simulated this by injecting the axoplasm with a small amount of paraffin oil and verified that our crush measurements never showed any correspondence to the sealed situation. Yawo and Kuno also reported that an increased extracellular calcium concentration contributes to sealing. In the squid giant axon, we observed that a five-fold increase of the extracellular calcium con- centration slowed the spreading of the depression but did not lead to complete sealing. A second case of an axial inhomogeneity was studied in preliminary measurements on axons temporarily exposed to a local cold block. These measurements indicated that the effects of the cold block could be detected in a region that is 3 times larger than the cooled axon segment, even 20 min after removal of the cold block. We are currently investigating whether such effects can be attributed to the block of axonal transport. This work was supported by the Grass Foundation and NIH grant I- R01 NS 19794. Preliminary optical measurements on the Melibe leonina buccal ganglion. CHUN X. FALK, WIN WATSON III, 230 NEUROBIOLOGY AND BIOPHYSICS JIM TRIMARCHI, JIAN-YOUNG Wu, AND LARRY B. COHEN (Dept. of Physiology; Yale University School of Medicine). Molluscan buccal ganglia have been used extensively to study the neural basis of feeding behavior. However, detailed information about the underlying neural circuits is lacking for most buccal preparations because of their complexity. A previous study, done by Watson et ai. (1989. Soc. Neurosd. Ahslr. 15: 1139) introduced a relatively simple system, the buccal ganglia in the nudibranch, Mclihc lamina, which has only about 50 neurons. Their work indicated that the role of the Mclihc buccal ganglia is to control swallowing. We monitored the activity from the four nerves leaving the ganglion. When the circumesophageal ganglia were removed, the buccal ganglion alone generated rhythmic activity. We attempted to use optical recording on the Mclihc buccal ganglion to see if we could measure every action potential in every cell in the ganglion. We did optical recording on preparations stained with a mero- cyanine dye (JPW1 124) or an oxonol dye (NK3041). We did complete- ness tests on several ganglia. In one ganglion we detected the activity of more than 90'!; of the cells. In an optical recording done during a rhythm, activity was detected in more than half of the neurons. We achieved a reasonably good signal-to-noise ratio. By comparing optical and nerve recording, we determined the locations of some cells that have their outputs in the nerves. At this writing, two cells have appeared consistently in all the experiments we have analyzed. Photo-activation during the optical recording was a serious problem. Photo-activation only occurred in dye-stained preparations. It could be that the dye, alone or together with light, lowers the threshold. We plan to try additional dyes. Supported by PHS Grant #NS08437 and a UNH Summer Faculty Scholarship. Segments! location of cranial nerve roots and motor nuclei in Squalus acanthias. E. GILLAND AND R. BARER (De- partment of Physiology and Biophysics, NYU Medical Center, New York, NY 10016). Cranial nerve roots III-X were exposed by dissection in paraformal- dehyde-tixed embryos of S acanlhius between Scammon stages 25 and 30. Crystals of fluorescent lipophilic dyes (Dil and DiA) were applied to the nerve roots in phosphate buffer, and the embryos were incubated at 40C for 4-12 h. Neuroepithelial wholemounts were cleared in glycerol and viewed with combined epilluorescent-bnght field illumination that allowed clear visualization of neuromeres, cranial nerve roots, and motor nuclei. Six interneuromenc borders were observed to delineate rhom- bomeres (r) 1-7. Sensory and motor roots of nerves V, VII, and IX entered and exited the neuroepithelium ot r3, r4, and r7, respectively. The majority of motor neurons contributing to each of these branchio- motor nerves were found at the segmental level of the roots, but in all cases a small number of motor neurons were also located in adjacent rhombomeres (r2 for V, r5 for VII, r6 for IX). At these stages, central sensory tracts of V and VII traversed all hindbrain segments. The motor neurons of nerve X were located posterior to those of IX in a region that lacked any clear segmental demarcation but which may represent an eighth rhomhomere. Small clusters of nerve III motor neurons appeared in the mesencephalon. each giving rise to a separate root traceable to a distinct eye-muscle mesenchymal condensation. Motor neurons of nerve IV arose only from rl. and those of nerve VI from four distinct clusters in r6. These data largely confirm Neal's 1918 segmental scheme for the cranial nerve roots and motor neurons of S acanthias. The overall neu- romenc pattern of the embryonic shark rhombencephalon is clearly ho- mologous to that of the chick; however, differences in the segmental relations of the roots and motor nuclei of nerves V. VI, VII. and IX suggest that a general vertebrate plan for neuronal organization within a given neuromere cannot be denned at the present time. The Limulus-eye view oj the world. ERIK HERZOG AND ROBERT BARLOW JR. (Syracuse University, NY). The horseshoe crab, Limitlii\ polyphemus, is an excellent model for studying the neural basis of visual behavior. Extensive information exists about the anatomy and physiology of its visual system, and we are be- ginning to understand the role of vision in the animal's behavior. To relate physiology and behavior, we need to know what the animal sees. Here we examine the eye-world interface by measuring the orientation of the lateral compound eyes in the animal and the lens facets in the eyes. The orientation of the eye in sini is defined by a plane tangential to a marked, central ommatidium. This "eye plane" was determined in two steps with a large, two-dimensional goniometer. First, the animal was rotated about an axis parallel to its horizon and its longitudinal axis, and passing through the ommatidium. Next, a laser was rotated along the horizon about an axis that also passes through the ommatidium until the laser beam reflected back on the face of the laser. The two angles denned the eye plane at the marked ommatidium with respect to the horizon and the animal's longitudinal axis. The eye was then fixed, excised, mounted in seawater on a second two-dimensional goniometer, and the eye plane was re-established. Axial illumination of the eye reveals a pseudopupil that moves as the eye is rotated. The pseudopupil is composed of a ring of ommatidia that appear dark (because they absorb axial light) surrounding a central ommatidium that glows brightly (because it maximally reflects axial light). The deep aperture of the glowing ommatidium can be centered by rotation of the eye. and the angular settings of the goniometer then define the optic axis of that ommatidium. This procedure was repeated for hundreds of om- matidia in the eyes of five adult males. The optics of the lateral eye are not homogeneous. The array of lens facets is not regular, and the angles between adjacent ommatidia are not constant. The optical axes of horizontal neighbors diverge most greatly in the ventro-posterior region of the eye (approx. 7.0). The axes of dorso-anterior ommatidia diverge the least (approx. 3.3). Ventro-anterior and dorso-ventral ommatidia have intermediate angles of divergence. The optic axes of vertical neighbors diverge 10.4 on average in the dorso-posterior part of the eye. whereas those in the ventro-anterior region diverge the least, at 3.2. Intermediate divergence (approx. 6) is found in the other two parts of the eye. Combining all measures of optic axes yields an overall held of view of the eye of about 187 along the azimuth and 87 along the vertical. In sum, the Ltmiiliis-eye view of the world is large, especially in the horizontal direction, and has the highest spatial resolution in the forward direction which is the direction of movement. Research supported by NIH and NSF grants. Inaclivation oj squid rhodopsin in the absence of phos- phorylation. ALON KAHANA, PHYLLIS R. ROBINSON, AND JOHN E. LISMAN (Brandeis University). Visual transduction involves a cascade of biochemical reactions that are transiently activated by a flash of light. In the first stage of the cascade, light-activated rhodopsin (R*) activates G-protem molecules by catalyzing the exchange of GTP for GDP. Termination of transduction requires inactivation of R*. We monitored R* in suspensions of squid outer segments by measuring light-activated GTP>S binding by G-protein. ABSTRACTS FROM MBI GENERAL MEETINGS 231 Inactivation of R* is defined as the time-dependent reduction in the rate of light-activated nucleotide binding. Up to 50-fold inactivation occurred in the absence of added ATP or in the presence of kinase inhibitors (AMP-PNP or sangivamycin), indicating that inactivation does not re- quire phosphorylation. Furthermore, added ATP did not significantly enhance inactivation. Further experiments were conducted to test whether inactivation is due to an intramolecular thermal transition of R* into an inactive state, or to interaction of R* with another protein. When the retinal suspension was diluted or hypotonically washed, R* inacti- vation became minimal. Addition of supernatant back to the washed membranes restored inactivation. These results suggest that inactivation of squid rhodopsin occurs through interaction with a soluble factor. SDS- gel electrophoresis of light and dark suspensions shows a single protein band around 55 kDa that undergoes light-dependent binding to the membranes, making this protein a good candidate for the factor partic- ipating in inactivation. Squid rhodopsin belongs to a family of structurally related trans- membrane receptors that includes vertebrate rhodopsin, 0-adrenergic and muscarinic receptors. These receptors require phosphorylation for inactivation to occur. Our finding of an ATP-independent inactivation process in squid rhodopsin indicates that phosphorylation subsequent to activation is not a general requirement for inactivation in this receptor family. Supported by EY01496 to J.E.L. and the Dons Brewer Cohen and Richter awards to A.K.. Slices of mouse suprachiasmatic nucleus with attached optic nerve: recording ofglutaminergic and GABA-ergic postsynaptic potentials using a voltage-sensitive dye. H. KOMURO, A. L. OBAID, S. S. KUMAR, AND B. M. SALZ- BERG (University of Pennsylvania School of Medicine). The suprachiasmatic nucleus of the hypothalamus plays a critical role in the generation and entrainment of circadian rhythms in mammals. Visual input, through the retinohypothalamic tract terminating in the suprachiasmatic nucleus (SCN), participates in the light cycle regulation of circadian rhythms, and ablation of the SCN produces severe disruption or loss of periodicity. However, because of its size, location, and inherent complexity, the properties of the SCN are difficult to study with con- ventional electrophysiological techniques. Here, we report the use of multiple-site optical recording with voltage-sensitive dyes to facilitate the identification of the important neurotransmitters of the retinohy- pothalamic pathway to the neurons of the SCN. We used an acute, 350- fjtn thick, slice preparation from the CD-I mouse, cut in an oblique plane that included the optic chiasm, one SCN, and the intact optic nerve. After cutting, the slice was rested for at least 1 h in oxygenated mouse Ringer's solution containing 0.003' r hydrogen peroxide. The slice was then stained for 30 min in a I00^g/ml solution of the pyrazo-oxonol dye, RH 155, and changes in transmitted light intensity were measured with a photodiode array, providing millisecond, time-resolved readout of electrical activity from 124 regions of the slice simultaneously. A suc- tion electrode was lilted to the intact optic nerve, and a single SO-^s shock was delivered to the axons of the retinal ganglion cells. The optical signals recorded from the SCN reflect the superposition of numerous postsynaptic membrane potentials. When Ca :+ was lowered from 2.2 mA/toO.l mA/, with Mg- + substitution, the amplitude of the signal was dramatically reduced, while raising Ca : * to 7 mA/ increased it. In the high Ca 2+ Ringer's solution, a prominent hyperpolarizing phase lasted hundreds of milliseconds. CNQX (6-cyano-7-nitroquinoxaline-2,3-dione), a potent inhibitor of non-NMDA type glutamate receptors, at 2 nM concentration, completely eliminated the compound postsynaptic potential in 30 mm. Kynurenic acid, another blocker of these glutamate receptors, also reduced the am- plitude of the signal. These experiments and others suggest that non- NMDA type glutamate receptors play a central role in mediating synaptic transmission in the retinohypothalamic pathway to the SCN. The long-lasting hyperpolanzation, that is particularly evident in high Ca 2+ Ringer's, might represent the activation of inhibitory pathways within the SCN. Block of GABA A receptors with 10 nM bicuculline had no effect on the amplitude of the signal, nor on the rising phase, but the width of the signal was greatly increased, as though an inhibitory com- ponent of the signal had been reduced or eliminated. Following a 5-min exposure to 1 fiM baclofen, a GABA B agonist, the compound postsynaptic potential was markedly reduced in size. In this case, the activation of GABA B receptors had its effect very early in the time course of the optical signal, compared with the effect of bicuculline. This suggests that the GABA A and GABA B pathways are anatomically distinct, with a longer latency assigned to the hicucullme-sensitive pathway. Baclofen block, unlike the effects of CNQX and bicuculline. was fully reversible. In summary, we have demonstrated the application of multiple site optical recording techniques to a SCN brain slice preparation having the visual input pathway intact. Also, we have provided physiological evi- dence for the involvement of a non-NMDA type glutamate receptor in an excitatory pathway, and of both GABA A and GABA B -mediated in- hibitory synapses. Supported by NS 16824 and a grant from the Government of Japan to H.K GABA-induced currents oj internal horizontal cells of the skate retina. ROBERT PAUL MALCHOW (Dept. Oph- thalmology, University of Illinois College of Medicine, Chicago, IL 60612), RICHARD L. CHAPPELL, PAUL GLYNN, AND HARRIS RIPPS. Two classes of horizontal cells are morphologically and electrophys- iologically distinguishable in the all-rod retina of the skate: the large, distally located external horizontal cells, and the slender, more proximal internal horizontal cells. Previous studies have shown that the external horizontal cells possess an electrogenic transport mechanism for GABA (Malchow, 1989, Bid. Hull. Ill: 324). The present experiments were designed to investigate whether internal horizontal cells display a similar uptake system. Isolated internal horizontal cells from the retinas of Raja mnacca and R mvlliila were obtained by enzymatic dissociation, and the whole-cell version of the patch clamp technique was used to monitor currents induced by GABA and various agonists applied by pressure ejection from nearby pipettes or by bath supertusion. Pressure ejection of 500 ^M GABA elicited an inward current of about 500-750 pA in internal horizontal cells. This current was not mimicked by I mA/ muscimol, a GABA A agonist, or by 1 mA/ of the GABA B agonist ( - Ibaclofen. The current was abolished when cells were bathed in a physiological saline in which all of the sodium had been replaced with lithium, and the response was markedly reduced when the chloride concentration in the saline was lowered from 266 to 16 mA/ by the substitution of sodium isethionate for sodium chloride. The current induced by GABA persisted when cells were bathed in a physiological saline containing 10 mA/ 4-ammopyndine, 10 mA/ cesium chloride, 4 mA/ cobalt chloride, 25 mA/ tetraethylammonium, and 1 nM tetrodotoxin, a solution that blocks the majority of voltage-activated conductances. Fi- nally, the GABA-induced current did not reverse into an outward current over the range of - 1 10 to +70 mV. These findings suggest that internal horizontal cells of the skate retina possess an electrogenic transport mechanism for GABA similar to that present in external horizontal cells. This study was supported by grants (EY-06516 and EY-00777) from the National Eye Institute and a Steps Towards Independence Fellowship to R.P.M. from the Marine Biological Laboratory. 232 NEUROBIOLOGY AND BIOPHYSICS Desperately seeking se.x neurons: detection <>/ 'projections in the male sex nerve of Hirudo medicinalis using nickel and horseradish peroxidase backfills. MICHAEL NITA- BACH AND EDUARDO MACAGNO (Columbia Univer- sity). Backfills of the male sex nerve of the medicinal leech, Hirudo medici- nalis. were used to find those neurons in the sixth midbody ganglion that send processes out this nerve. The male sex nerve is a branch of the anterior root of ganglion six that innervates the male sexual organs. For this reason, neurons backfilled from this nerve are good candidates for playing specific roles in the sexual behavior of the leech. Three dyes were used: 5% biocytin. 30% horseradish peroxidase (HRP). and 5% nickel chloride. Since we already knew that the rostral and lateral penile evertor motor neurons and the Retzius cell send processes out this nerve, the presence of dye in these cells was used as the sine qua nun for the success of the backfill. With biocytin we often failed to backfill one or more of these cells. With HRP we usually labeled the penile evertor motor neurons, but often failed to label the Retzius cell. This may be because the Retzius cell has a smaller diameter axon in the male sex nerve than the penile evertors. In contrast, in the eight nickel chloride backfills we have tried thus far, both penile evertors and the Retzius cell were invariably labeled. We conclude from this that nickel chloride is the most useful of these dyes lor backfilling the male sex nerve of Hirudo. Of course, the purpose of these experiments was to identify new cells that send processes out the male sex nerve. We were quite successful in this respect; we consistently backfilled several neurons that were readily identifiable from experiment to experiment, but that were hitherto un- known. We must now use further anatomical, physiological, and neu- roethological methods to elucidate the roles, if any, that these newly identified cells play in the sexual behavior of Hirudo medicinalis. FTX, an HPLC-pwified fraction of funnel web spider venom, blocks calcium channels required for normal re- lease in peptidergic nerve terminals of mammals: optical measurements with and without voltage-sensitive dyes. A. L. OBAID, H. KOMURO, S. S. KUMAR, M. SUGI- MORI, J.-W. LIN, B. D. CHERKSEY, R. LLINAS, AND B. M. SALZBERG (University of Pennsylvania School of Medicine). We have been using intrinsic (light scattering) and extrinsic (poten- tiometnc dye absorption) optical signals to study the ionic basis of the action potential in the nerve terminals of the frog neurohypophysis, and the detailed role that Ca 2+ channels play in the coupling of excitation to secretion. We have already shown (Salzberg el at., 1990. Biophys. J. 57: 305a) that specific channel blocking agents, FTX (Llinas el al.. 1989, PNAS 86: 1689) and w-conotoxin GVIA (Cruz and Olivera. 1986, J. Bio/. CliL'iii 261: 6230), can be used to characterize the Ca 2+ channels of intact nerve terminals in Xenopiix. FTX, an HPLC-purified polyamine fraction of funnel web spider venom, and a 385-Da synthetic analog, eliminated the undershoot of the normal action potential and reduced the magnitude of the calcium spike in TTX-TEA pretreated preparations. Both of these effects are indicative of Ca 2+ channel block. In preparations maximally blocked by 10 pAI m-conotoxin GVIA, the synthetic analogue of FTX exhibited further effect but did not entirely eliminate the active calcium response. Further addition of 200 pM Cd 2+ left only the passive electrotonus. These results suggest that at least two, and probably three, populations of Ca 2+ channels are present in the intact nerve terminals of the frog neurohypophysis. In the mouse neurohypophysis, the S-wave, a component of the light scattering signal that is strongly correlated with peptide secretion, was reduced significantly by dilutions as high as 1 : 10,000 of the HPLC-punfied funnel web spider venom fraction. Neither FTX. nor u>-conotoxin. were able to eliminate completely the S-wave, which could be blocked entirely by 200 n.\I Cd 2+ . Our results suggest that, in the mouse neurohypophysis. there are at least two, and possibly three, different populations of Ca 2 * channels, all involved in secretion. Supported by NS 16824. NS 13742, EY 08002, AFOSR85-0368 and a grant from the Government of Japan to H.K. Lyoluminescence. G. T. REYNOLDS (Dept. of Physics, Princeton University). Lyoluminescence (LL) is the term applied to the emission of light when certain materials go into solution. Almost all recent studies have been of LL from irradiated materials. It is natural to think of the phe- nomenon as a potential technique for dosimetry. Certain organic and inorganic materials, after irradiation, exhibit LL upon dissolution. Certain organics (sacchandes. ammo acids) closely resemble soft tissue and are therefore of particular interest. In general, the literature to date reports results from very high doses (up to 10 6 rad). Certain irradiated inorganic- phosphors (alkali halides) exhibit LL due to the formation of F- and V- centers, which release hydrated electrons upon dissolution. Our work has concerned observations of LL from some of the organics used in liquid scintillators. and from NaCl, with particular interest in detection at doses less than 1 rad. An interesting comparison of reagent grade NaCl and a sample of non-iodized table salt has been made. None of the ingredients ("impurities") of the table salt explains by itself the order of magnitude greater light output over that of the reagent grade. LL can be enhanced by the addition of selected molecules or ions to the solvent. In the case of NaCl. for example, the addition of 3 X 10~ 4 M Cu ++ increases the light output by an order of magnitude (Kalkar and Ramani, 1981, Radiochem Radioanul Leu 47: 203-210). Using such techniques, we have detected doses less than 1 rad with very simple equipment. It is possible to design detection systems permitting mea- surements of 100 samples at a time, involving multianode photomulti- pliers. coupled to irradiated samples by tapered light pipes. Also, based on experience with liquid scintillator components, an efficient a detector (for radon) may be possible. This work was supported by Princeton University DOE Grant #DE- FG02-87ER 60522. Calcium channels in identified neurons of Hermissenda crassicornis. EBENEZER YAMOAH, NORMAN STOCK- BRIDGE, AND ALAN KUZIRIAN (Marine Biological Laboratory). We have studied calcium currents in neurons of the pedal ganglion that innervate visceral organs of the nudibranch mollusk. Hermissenda crassicornis. The whole-cell patch-clamp technique was used. The bathing medium consisted of (m.U concentrations) 300 choline chloride, 100 tetraethylammonium chloride, 5 4-aminopyridine, 50 magnesium chloride, 10 potassium chloride. 10 HEPES buffer. 10 glucose, and 10 calcium chloride. The pipette solution contained 400 cesium chloride. 50 HEPES, 20 sodium chloride. 10 reduced glutathione. 5 EGTA, 5 MgATP, and 1 GTP. Depolarization from -80 mV produces an inward current that activates at about -30 mV and peaks al about +20 mV. The current inactivates with two time constants. Depolarization from 40 mV also elicits current with a biphasic inactivation. The total current is therefore composed of components with similar activation kinetics, but different inactivation time constants. ABSTRACTS FROM MBL GENERAL MEETINGS 233 Both components of inactivation are voltage dependent. With barium ( 10 m/l/) as charge earner, the fast component of inactivation is unaffected and the slow component is further slowed. Both current components are insensitive to cobalt (600 p.M) and blocked by cadmium (300 /j,U). Lanthamum. however, blocks the slow component fully, but leaves about 50''.' of the fast component unaffected. The dihydropyndines, nitrendipine (300 M). and mfedipme (400 M .U| selectively inhibit the slowly inac- tivating component. These findings suggest the presence of two types of calcium channels with similar activation kinetics, but different inactivation kinetics and pharmacology, in an identified class of neurons of the pedal ganglion of Hfrmisxcmla cra\\ia>rnis. E. Yamoah thanks members of the Laboratory for Cellular* Molecular Neurobiology, NINDS-NIH. for their support. Physiology and Behavior Structure of alpha-2 macroglobulin from the horseshoe crah. Limulus polyphemus. PETER B. ARMSTRONG (Laboratory for Cell Biology, Department of Zoology, University of California. Davis. CA 95616-8755), WALTER F. MANGEL, ATSUSHI IK.AI, K.ENSAL E. VAN HOLDE, AND JAMES P. QUIGLEY. A structural and functional homologue of vertebrate alpha-2-macro- globulin has been identified in the hemolymph and blood cells of the arthropod Limulus polyphemus (Quinsy and Armstrong, 1985. / Biol Chcm 260: 12,715-12,719). The subunit molecular mass, determined by SDS-polyacrylamide gel electrophoresis (reducing conditions), is 185 kDa. The native molecular mass, determined by scanning transmission electron microscopy (STEM) under conditions in which the linear re- lationship between the STEM large angle detector signal and specimen mass thickness allows the determination of the total molecular mass, was 354 35 kDa. Sedimentation equilibrium measurements gave a value of 366 kDa. independent of solute concentration. Sedimentation velocity experiments indicated a homogeneous component with a fric- tional ratio of 1.41. Thus, the native structure appears to be a dimer. with a somewhat extended conformation. The behavior during gel per- meation chromatography was anomolous. yielding an apparent molecular mass approximately half-way between that expected for the dimeric (370 kDa) and tetrameric (740 kDa) configurations. N-terminal peptide se- quence analysis yielded a single sequence, indicating a homo-multimenc structure. Transmission electron microscopy of negatively stained prep- arations revealed a dimeric butterfly-like structure that collapsed following reaction with chymotrypsin. Gel permeation chromatography also showed a more compact structure for the chymotrypsin-reacted molecule. Reaction with trypsin and plasmin results in multiple products which, when analyzed by reducing SDS-polyacrylamide gel electrophoresis, in- cluded two prominant bands of higher molecular mass than the unreacted alpha-2-macroglobulin. Surprisingly these high molecular mass products do not contain covalently bound trypsin. Supported by NIH Grant No. GM-35185. Lever-press conditioning in the crab. Green crabs perform well on fixed ratio schedules, but can they count? RICH- ARD D. FEINMAN, HESTER KORTHALS ALTES, SAM KINGSTON, CHARLES I. ABRAMSON, AND ROBIN R. FORMAN (State University of New York Health Science Center at Brooklyn). We previously showed that the green crab, Carciimx nuicnaa. could be taught to press a lever if every response was reinforced with food [fixed ratio of 1 schedule (FR II] or if every other response was reinforced (FR 2) (Abramson and Feinman, 1990. Pliy.tiol. & Be/uiv. 48: 267-272). We have now extended this work to include more protracted schedules of reinforcement. We found that crabs showed high rates of responding on progressively increased fixed ratio schedules, one animal reaching FR 9. Blank controls with an inactive lever (but with food present in the food dispenser) showed much poorer rates of responding but performed well when switched to FR schedules. Cumulative records show that an- imals frequently paused immediately after reinforcement as seen with vertebrates (on much higher FR schedules). To test whether responses were actually controlled by the number of responses, we subjected a group of four animals to four days of FR 6 training. We followed this with four days of a regimen in which they were rewarded on an FR 6 schedule until three reinforcements had been obtained; they were then switched to extinction (no reward). All animals performed well during acquisition, but only one showed consistent high rates of responding during extinction. Several individual records showed that responses were frequently grouped before reward, but an analysis of intervals revealed this was not a predictable effect. Thus, although crabs are capable of perseverance in fixed ratio schedules, it is unlikely that the numerical value of the required responses enters into the behavior. This work was supported by grant BNS 8819830 from the National Science Foundation and funds from the Research Foundation of the State University of New York. Search for the biological stimulus oj the dishing response in hluefish (Tomatomus saltatrixj. STEPHEN H. Fox, CHRISTOPHER S. OGILVY, AND ARTHUR B. DuBois (John B. Pierce Laboratory, New Haven, CT 06519). We have found that increasing intracranial pressure (ICP) almost in- variably produces an increase of heart rate and blood pressure (BP) in the ventral aorta of bluefish (n = 30). Indeed. BP changes are prompt and almost in direct proportion to the increase of ICP. Cooling the water delays the onset, but does not decrease the magnitude of the BP response. However, oxygenation of the cooled water not only delays the onset, but also reduces the amplitude of the BP response (n = 6). Therefore, cerebral hypoxia seems to be the trigger for the Cushing response in these fish. We then looked for the environmental stimulus that might cause this response in nature. Bluefish can accelerate 3 G, exerting great force be- tween the tail and head. We speculated that the spine might be compressed during swimming, raising ICP. To test this, we produced head to tail compression of the vertebral column in anesthetized bluefish, in dead fish, and in vertebral and cranial preparations, and coupled this with side-to-side flexure, hyperextension, or torsion of the body and spine to simulate fast swimming. These motions, which distorted the shape of the spinal canal, displaced fluid into the head and raised ICP by 20 mm Hg. Next, we measured ICP using a 5-m tube connected to a cranial implant in a bluefish swimming (after recovery from anesthesia) in a 3.7 m diameter round tank at 1.3 m/s. ICP fluctuations of 20 mm Hg (unrelated to depth) were recorded. Tubing flexion artifact accounted for only 4 mm Hg of this fluctuation. We conclude that the natural role of this BP response may he to maintain cerebral blood flow during ICP changes produced by swimming at speeds sufficient to cause lon- gitudinal compression which together with lateral or vertical flexion, or torsion of the body, displaces fluid contained in the spinal canal into the head. 234 SENSORY BIOLOGY Sensory Biology Boundary layers and microsculc fluid dynamics around I lie lobster's (Homarus americanusj chemosensory ap- pendages. JELLE ATEMA AND PAUL A. MOORE (Boston University Marine Program, Marine Biological Labo- ratory'). The three major chemosensory appendages of the lobster, H'-iiii'n\ a Drosophila lieat shock puff BEHAVIOR Buck, John Unisex flash controls in dialog fireflies DEVELOPMENT AND REPRODUCTION Goldberg, Walter M., Ken R. Grange, George T. Taylor, and Alicia L. Zuiiiga The structure of sweeper tentacles in the black coral Antipathes fiordensis ECOLOGY AND EVOLUTION Curtis, Lawrence A. Parasitism and the movements of intertidal gastro- pod individuals Lonsdale, DarcyJ., and Sigrun H. Jonasdottir Geographic variation in naupliar growth and sur- vival in a harpacticoid copepod 77 87 96 105 GENERAL BIOLOGY Pennington, J. Timothy, and Richard R. Strathmann Consequences of the i ale it e skeletons of planktonic echmoderm larvae for orientation, swimming, and shape 12 1 PHYSIOLOGY Dahlhoff, Elizabeth, Sabine Schneidemann, and George N. Somero Pressure-temperature interactions on M 4 -lactatede- hydrogenases from hydrothermal vent fishes: evi- dence tor adaptation to elevated temperatures by the /oaicid T/ii'i'in\ matrix protein from blue crab exoskeleton 191 Shields, Jeffrey D. Rhizophydium li/iun'ia// on the eggs of Cancer anthonyi: parasite or saptobe? 201 PHYSIOLOGY Kingsley, Roni J., Mari Tsuzaki, Norimitsu Watabe, and Gerald L. Mechanic Collagen in llir spiuile otgaitii matrix ol the got - goman Leptogorgia virgulata 207 RESEARCH NOTE Lohmann, Kenneth J., Michael Salmon, and Jeanette Wyneken KutKtion.il autonomy ol land and sea orientation systems in sea tut tie hatchlings 214 ABSTRACTS Abstrac Is ol papers presented at the General Scientific Meetings of the Marine Biological L.iboralorv 219 No. 3. DK KMIUR 1990 CELL STRUCTURE Weidner, Earl, Robin M. Overstreet, Bruce Tedeschi, and John Fuseler Cytokeratin and desmoplakin analogues within an intracellular parasite 237 DEVELOPMENT AND REPRODUCTION Carroll, David J., and Stephen C. Kempf Laboratory culture ol the aeolid nudibranch Bf>v/ini vfiiuin<>i->ii.<, (Mollusia. Opisthobranchia): some as- pects of its development and life history 243 Komatsu, Mieko, Yasuo T. Kano, and Chitaru Oguro Development ol a true ovoviviparous sea star, ,4\- li'inni pseudoexigua /itni/nn lla\ashi 254 Maruyama, Yoshihiko K. Roles of the polar cytoplasmic region in meiotic di- visions in oocytes of the sea cucumber, Hnlntliitriii leucospilota l!li i ECOLOGY AND EVOLUTION Amano, Shigetoyo Self and non-self recognition in a calcareous sponge, Leucandra abratsbo 272 ll.ui. Mil h.i. and Yossi Loya Ontogenetic variation in sponge histocompatibility responses 279 Berges, John A., John C. Roff, and James S. Ballan- tyne Relationship between body size, growth rate, and maximal enzyme activities in the brine shrimp, A>- teinia fraiifi.Mdiiu 287 Coon, S. L., M. Walch, W. K. Fin, R. M. Weiner, and D. B. Bonar Ammonia induces settlement behavior in oyster larvae 297 Pearce, Christopher M., and Robert E. Scheibling Induction of metamorphosis of larvae of the green sea urchin, Strongylocentrotus ilrot'l>(icltiensi.\. by cor- alline red algae 304 Rawlings, Timothy A. Associations between egg capsule morphology and predation among populations of the marine gastro- pod, Xiiit'llti I'litiii-gintitit 312 GENERAL BIOLOGY MacColl, Robert, John Galivan, Donald S. Berns, Zenia Nimec, Deborah Guard-Friar, and David Wagoner The chromophore and polypeptide composition of Aplyuii ink 326 McFall-Ngai, Margaret, and Mary K. Montgomery The anatomy and morphology of the adult bacterial light organ of Euprymna scolopes Berry (Cephalo- poda:Sepiolidae) 332 PHYSIOLOGY Bowlby, Mark R., Edith A. Widder, and James F. Case Patterns of stimulated bioluminescence in two py- rosomes (Tnnicata: Pyrosomatidae) 340 Kallen, Janine L., S. L. Abrahamse, and F. Van Herp Circadian rhythmicity of the crustacean hypergly- cemic hormone ((II 1H) in the hemolymph of the crayfish 351 Kuhns, William J., Gradimir Misevic, and Max M. Burger Biochemical and functional effects of sulfate restric- tion in the marine sponge, Murticitma prolif'eru . . . 358 Toulmond, Andre, Fouzia el Idrissi Slitine, Jacques de Frescheville, and Claude Jouin Extracellular hemoglobins of hydrothermal vent annelids: structural and functional characteristics in three alvinellid species 366 Trapido-Rosenthal, Henry G., Richard A. Gleeson, and William E. S. Carr The efflux of amino acids from the olfactory organ of the spiny lobster: biochemical measurements and physiological effects 374 Index to Volume 179 . 383 Erratum The Biological Bulletin, Volume 179, Number I, page 91 The following correction should be made in the article by John Buck, titled "Unisex flash controls in dialog fireflies" (Biol. Bull. 179: 87-95). The words "found to be" were deleted from the sentence beginning on line 1 7 in the right hand column of page 9 1 . The sentence should now read: "By programmed stimulation, the triggering was found to be confined to the latter half of the flashing cycle . . ." Reference: ;<>/ Hull. 179: 237-242. (December. 1490) Cytokeratin and Desmoplakin Analogues within an Intracellular Parasite EARL WEIDNER', ROBIN M. OVERSTREET : , BRUCE TEDESCHI 1 , AND JOHN FUSELER 4 ^Department of Zoology and Physiology, Louisiana State University, Baton Rouge. Louisiana 70803: 2 Gulf Coast Research l.ahoralory. Ocean Springs, Mississippi 39564; 3 'Department of Anatomy anil Cell Biology, Eastern I'irgmia Medical School. Norfork. Mrginia 23501 and 4 Department oj Cellular Biology and Anatomy, Louisiana State University Medical Center. Shreveport, Louisiana 71130 Abstract. A significant amount of the total protein in the spore sacs of the microsporidian Thelohania sp. con- sisted of the cytoskeletal elements, cytokeratin interme- diate filaments, and the desmosomal analogues. The cy- tokeratin and desmosomal analogues were organized as cage envelopes surrounding the spores within the spore sac stage. Thelohania sp. parasitizes the skeletal muscle of Callinectes sapidus, a crustacean that does not appear to have cytokeratins or desmosomes. Immunoprobe data indicate Thelohania sp. has a 240 kDa desmoplakin pro- tein and 48, 5 1, 54 and 56 kDa cytokeratin polypeptides responsive to antibodies developed against bovine cyto- skeletal counterparts. The cytoskeletal envelopes within the Thelohania sp. spore sac stage appear to enhance the stability and viability of the spores. Introduction Cytokeratin intermediate filaments and desmosomal proteins are specific markers for epithelial cells in verte- brates (Cowin ct ai. 1985; Romano el ai. 1986). Using high resolution two-dimensional gel electrophoresis, over 1 5 distinct cytokeratin polypeptides have been character- ized from vertebrates (Cooper et ai, 1984). Monoclonal antibody studies indicate that some of these cytokeratins have analogues present in lower vertebrates (Rungger- Brandle et a/.. 1989). Indeed, antibody cross-reactivity studies indicate that intermediate filament (IF) epitopes are shared among the different IF families; this cross-reac- tivity extends to IFs present in many invertebrate groups (Bartnik and Weber, 1989). However, there is no evidence Received 29 May 1990; accepted 25 September 1990. that a monoclonal antibody, directed to mammalian cy- tokeratin, cross-reacts with presumptive cytokeratin counterparts present in epithelial cells of invertebrates (Fuchs and Marchuk. 1983; Weidner, unpub. data). The desmosomal plaque elements consist of desmo- plakin 1 (240-250 kDa), a protein that localizes to the region of the desmosomal plaque where cytokeratin binds (Jones and Goldman, 1985). Desmoplakin antibody shows cross-reactivity to this protein in epithelia from various vertebrate groups (Rungger-Brandle et ai, 1989). While desmosome assemblages and cytokeratin-like IFs are apparent in invertebrate epithelial cells, these proteins are reported to be absent in arthropods (Bartnik and Weber, 1989). The absence of these proteins in arthropods is noteworthy because we report here that cytokeratin and desmoplakin analogues are present in an intracellular parasite found within arthropods. The cytokeratin and desmoplakin analogues present in the microsporidian parasites cross-react with monoclonal antibodies directed to mammalian cytokeratins and desmoplakin. Materials and Methods Animal and cell preparations Thelohania sp. was taken from blue crabs (Callinectes sapidus) collected from Mississippi Sound and the west coast of Florida near St. Petersburg. After removing the Thelohania sp.-infected muscle, the dissociated infected muscle fibers were applied to glass slides and fixed, per- meabilized in 100% methanol, and further processed for immunofluorescence microscopy. Other infected muscle was washed in 0.5 mM CaCl : , and the Thelohania sp. spore sac stage was liberated and purified into populations 237 238 E. WEIDNER ET AL. of spore sacs following a wash cycle described elsewhere (Weidner. 1976). l-'/cciroii microscopy Infected skeletal muscle with Thelohania sp. spore sac stages from the blue crab, C'tillinccli's sapidus. were pre- pared so that the stages of spore sac development could be examined. Also, isolated spore sacs were fixed, washed, embedded, and processed for electron microscopy as de- scribed elsewhere (Overstreet and Weidner, 1974). Antibodies The following primary antibodies were used: mouse cytokeratin antibody clones Lu5, AE1 and 3 (Boehringer Mannheim, Indianapolis. Indiana), K.8.12, K8.13. DK802 (Sigma Chemical Co., St. Louis, Missouri), and mouse desmoplakin antibody clones PD2. 1 5 and PD2. 1 7 (ICN, Costa Mesa. California). Whereas, clones K8.12, K8.13, Lu5 and AE1 and 3 react to a number of epitopes com- mon to cytokeratins, DK802 has affinity for desmosomal- binding cytokeratin 8. Second antibodies were rabbit im- munoglobulins against mouse immunoglobulins coupled to alkaline phosphatase, FITC, or peroxidase (Sigma Chemical). Immunofluorescence and immuno-electron microscopy Infected blue crab muscle fibers or isolated Thelohania sp. spore sacs were fixed and permeabilized in 100% methanol and processed for indirect immunofluorescence as described elsewhere (Pasdar and Nelson, 1988). Cells were washed in PBS and incubated with anti-desmoplakin or anti-cytokeratin diluted 1:100 with PBS for 30 min at 37C. After five washes in PBS. cells labeled with anti- mouse immunoglobulin coupled to FITC (Sigma Chem- ical). After five washes with PBS, cells were mounted in 20% glycerol and viewed with a 60X objective on a Nikon Microphot FXA equipped with epifluorescence illumi- nation: images were recorded on Tri-X film (Eastman Kodak, Rochester, New York). For immuno-electron microscopy, cells were permeabilized with methanol and further fixed in 1% glutaraldehyde for 20 min. Cells were then washed in cacodylate buffer, transferred to PBS, and later immersed into primary antibody in PBS for 1 h. After 30 min of repeated washings in PBS, cells were ex- posed to anti-mouse conjugated to peroxidase (Sigma Chemical) for 30 min, washed in PBS. and exposed to diaminobenzidine working medium and processed for electron microscopy as described earlier (Pleshinger and Weidner, 1985). Gel electrophoresis of Thelohania sp. spore sac proteins and immunoblotting Spore sacs were liberated from Thelohania sp.-infected blue crab muscle with a glass homogenizer. The cell sus- pension was placed in 0.5 mM CaCl 2 washed and pro- cessed to purification with the wash cycle described earlier (Weidner, 1976). The spore sac desmosomal analogues were disrupted by immersing the sacs in 0.5 mM EGTA for 30 min. Protein samples were prepared by homoge- nization in boiling SDS sample buffer. The discontinuous buffer system of Laemmli (1970) was used in polyacryl- amide gradient gels (7.5-15%.). Samples with 15-20 ^g protein per lane were heated for 5 min with 2% SDS and 3% 2-mercaptoethanol before loading for electrophoresis. Proteins were stained with Coomassie blue. Proteins for duplicate unstained gels were transferred electrophoreti- cally to nitrocellulose membrane using a Bio-Rad trans- blot apparatus (Bio-Rad Laboratories, Palo Alto, Califor- nia) overnight at 4C 50V in Tris-glycine buffer. pH 7.5 with 20% methanol. The nitrocellulose was treated with blocking buffer (1% milk powder, 0.02% Tween 20, 0.02% sodium azide in PBS) for 3 h before incubating in primary antibody (1:100 dilution in blocking buffer overnight). The nitrocellulose membrane was washed five times (10 min/wash) in PBS and transferred to anti-mouse coupled to alkaline phosphatase (1:100 dilution in blocking buffer) for 6 h. The nitrocellulose membrane was then washed five times (10 min/wash) in PBS and the antibody was visualized in incubating nitrocellulose in alkaline phos- phatase substrate ( 100 mA/Tris, pH 8.8, 0.01% nitroblue tetrazolium and 0.005% 5 bromo-4-chloro-3-indolyl phosphate) (Sigma Chemical). Results A large percentage of the microsporidian parasites de- velop a sporophorous vesicle (spore sac) stage that sur- rounds the spores. Within Thelohania sp.. the extraspor- ular space within the spore sac stage is filled with inter- mediate filaments (IFs) that appear attached to desmosomal or half-desmosomal plaques as illustrated in Figure 1 A. Within Thelohania sp., eight spores are tightly packed within a spore sac (Fig. IB). The spore sacs were extremely stable and resisted dissociation in dithiothreitol, 2-mercaptoethanol, SDS. 10 M urea, methanol, or organic solvents such as choloroform. However, Thelohania sp. spore sacs were partially permeabilized with 0.5 EGTA and subsequent shearing with a glass homogenizer caused up to 20%- of the spores to liberate from the spore sacs as shown in Figure 1C. Fluorescent antibody labeling for cytokeratins and des- mosomal proteins showed a strong fluorescence primarily on the spore envelopes of Thelohania sp. Figure 2A depicts phase optical imaging of spore sacs recovered from in- fected muscle of Callinectes sapidus. On the basis of an- tibody labeling, only 15-20% of the spore sacs were suc- cessfully permeabilized for antibody labeling. All spores liberated from the sacs were reactive to specific antibodies PROTISTAN CVTORERATIN AND DESMOPLAKIN ANALOGUES 239 Figure 1. Low magnification images of Tlwlohunia sp. spore sacs. Figure A is a diagram of a spore sac with spores. Note the arrow indicating a plaque envelope bearing cytokeratin analogues surrounding spores; plaque surrounding spores appear as half-desmosomes while plaque connecting spores appear as desmosomes. Figure B is an electron micrograph of a spore sac with spores. Figure C is an electron micrograph of a spore sac with plaque envelopes but without spores. Note the IF-bearing half-desmosome plaques that originally enveloped spores in Figure C appear like those illustrated in diagram in Figure A. Bar = 1 ^m Figure 2. Light microscopy images of Tliclohaniu sp. spore sacs. Figures A and B are phase and im- munofluorescence imaging of the same field of spore sacs. About 30% of the spore sacs of Thelohania in Figure B were permeabilized adequately to enable anti-cytokeratin binding; all liberated spores were positive for cytokeratins. Figure C is an enlargement of Figure B; note that the fluorescence of individual cytokeratin bundles produces a fuzzy impression at the spore surface. The single spores in Figure C are 4 pm in length. Figure 3. Electron micrographs of desmosomes within a Thelohania spore sac. Figure A is a lead-stained image of desmosomes. Figure B shows desmosome plaques immunoperoxidase-labeled for desmoplakin; there is no staining of the cytokeratins. Bar = 50 nm. 240 I \\ FIDNER ET AL Figure -I. Electron micrographs of Thelohania sp. spore sacs. Figure A shows a portion of spore sac without spores. Note the hall-desmosome plaques with cytokeratin analogues attached. Figure B shows the half-desmosome plaques with immunoperoxidase labeling for desmoplakin I; note that the cytokeratin an- alogues within the spore sac appear unstained. Figure C shows a contrasting image of half-desmosomal plaques immunoperoxidase stained for cytokeratins: note that the filaments stand out much more clearly than observed in Figure B. Bar = 0.5 urn. for cytokeratins or for the desmosomal constituent, des- moplakin. Figure 2B shows fluorescent antibody activity for cytokeratins in field of spore sacs viewed in Figure 2A with phase optics. Figure 2C shows an enlargement of a few fluorescent antibody-labeled Thelohania sp. spores. The hairy surface of fluorescence at the spore surface is attributed to the spore-bound IFs. Both desmoplakin and cytokeratin antibody activities were confined to the The- lohania sp. spore sacs throughout the different stagings in microsporidian development: thus, host muscle tissue domains were negative for cytokeratin and desmoplakin analogues. Ultrastructure of Thelohania sp. spore sacs showed an abundance ofcvtokeratin IFs attached to half-desmosome- PROTISTAN CYTOKERATIN AND DESMOPLAKIN ANALOGUES 241 like plaques enveloping the spores (Fig. 4A). The half- desmosomal plaques that envelope each spore join to form desmosome-like attachments between the spores (Fig. 1 A, Fig. 3A). Immunostaining with peroxidase conjugate di- rected to desmosomal indicator, desmoplakin 1, yielded peroxidase staining on both the desmosome-like structures (Fig. 3B) and the half-desmosomal analogues surrounding the spores within the Thclohania sp. spore sacs (Fig. 4B). Immuno-localization ofantibody-peroxidase forcytoker- atin analogues revealed obvious staining for bundles of IFs bound to the plaques enveloping the microsporidian spores (Fig. 4C). For further analyses of the number of cytokeratin an- alogues reactive to keratin antibodies, proteins recovered from purified Thclohania sp. spore sacs were subjected to gel electrophoresis and immunoblotting. Monoclonal an- tibodies AE 1 and AE3, K.8. 1 3, and Lu5 reacted to epitopes common to cytokeratin analogues from Thelohania sp. spore sacs. Immunoblots show response bands near po- sitions 50. 54, and 56 kDa (Fig. 5A). Monoclonal antibody K.8.12 reacted to two bands, indicating the presence of cytokeratin analogues 13 and 16 (51 and 48 kDa). For identifying desmosomal proteins, monoclonal antibodies DP2.15 and DP2.17 were used. These antibodies were responsive to a single 240 kDa band corresponding to desmoplakin 1 in Thclohania sp. spore sacs (Fig. 5B). In the controls. DP2. 1 5 antibody responded only to the 240 kDa desmoplakin band from bird (turkey) wing tegument (Fig. 5C). Discussion The results of this study indicate that cytokeratin IFs and desmosomal proteins appear to be expressed within the microsporidian spore sac stages found within the skel- etal muscle of the crustacean, Callinectes sapidus. Mono- clonal antibody labeling, applied to immunofluorescence and immuno-electron microscopy, indicate an abundance of cytokeratin IFs and desmoplakin analogues within Thclohania sp. spore sacs. Curiously, these proteins are reported to be absent in arthropods (Bartnik and Weber, 1989). Immunolabeling data, supported by gel electro- phoretic and immunoblot analyses, indicate that the cy- tokeratin and desmoplakin analogues recovered from the microsporidian spore sacs were immunologically respon- sive to antibodies prepared against bovine cytoskeletal counterparts. This is surprising because cytokeratins have diversified rather significantly among vertebrates. Anti- bodies directed to bovine cytokeratins would not be ex- pected to respond to cytokeratin analogues from a lower eukaryote (Fuchs and Marchuk. 1983). Additionally, it was unexpected to find cytokeratin IFs in blue crab skeletal muscle because neither skeletal muscle nor arthropods appear to express cytokeratins. However, the cytokeratins 65 - 55 50 240 200** 240 r Figure 5. Immunohlot analysis of Thelohania sp. spore sac proteins. (A) Lane one shows standard cytokeratins resolved (from human epi- dermis) into bands in the 65. 58, 56 and 50 kDa range. Lane 2 shows distinct bands of 56. 54. 52. 50 and 48 kDa for cytokeratins from Thc- liiluiniti spore sacs. (B) Lane 2 shows a 240 kDa response band for des- moplakin I; lane 1 shows the molecular weight marker myosin (200 kDa). (C) Control showing desmoplakin I. Lane 1 shows stained proteins from turkey epidermis. Lane 2 shows an immunoblot of lane 1 with a response band to desmoplakin I. and desmoplakin analogues were confined to the micro- sporidian spore sac domains within the skeletal muscle. Two major lines of evidence indicate that the spore sac structure represents a stage in microsporidian life cycles. First, the majority of microsporidian species have spore sacs as a stage in their life cycle (Canning el ill.. 1982; Becnel et a!.. 1986). Second, during microsporidian spore sac development, the spores differentiate internally within a progenitor cell in which the extrasporular cytoplasmic domain becomes the spore sac (Overstreet and Weidner, 1974). The binding of cytokeratin analogues to desmoplakin- bearing plaques in Thelohania sp. spore sacs resembles the cytokeratin IF attachments in vertebrate tegumental epitheilium (Kelly, 1966). However, the binding patterns differ because cytokeratin in epithelial cells binds only to membrane after it is stabilized by attachments to adjoining membrane from another cell. In Thclohania sp., however, the cytokeratin and desmoplakin plaque binding is pri- marily to that membrane which is attached to spore sur- faces. Thus, cytokeratin and plaque analogues in Thelo- hania spore sacs bind to membrane that has firm attach- ments to spore surfaces. The identification of cytokeratin polypeptides in The- lohania sp. spore sacs is very preliminary, nevertheless, cytokeratin 13 and 16 appear to be present because 51 and 48 kDa proteins respond to monoclonal antibody K.8. 12. Also, there is some evidence of cytokeratin 8 (des- 242 E WEIDNER ET AL mosomal cytokeratin), a protein which has a 52 kDa mo- lecular weight. The origin* <>/ desmoplakin-cytokeratin IF expression in Thelohania sp. It is unlikely that IF and desmosomal cytoskeletal genes are native to the microsporidian species in general because only a small percentage of the microsporidians express these proteins. Furthermore, it is unlikely that the micro- sporidians acquired the capabilities for expressing these proteins from arthropod hosts because these animals do not appear to express the cytokeratins or desmosomes (Bartnik and Weber. 1989). However, it would seem more likely that Thelolwnui sp. may have acquired the cyto- keratin and desmosomal genes from a vertebrate source. This is within the realm of possibility because nearly all microsporidians begin growth in epithelial cell lines; and, nearly 100 species of microsporidians have been reported parasitizing aquatic vertebrate animals (Canning and Lorn, 1986). Acknowledgments We thank Phil Steele. Department of Natural Re- sources, Bureau of Marine Research, St. Petersburg, Flor- ida, for providing infected crabs from Florida. The study was conducted in cooperation with the U.S. Department of Agriculture, CSRS, Grant No. 88-38808-3319 and the U.S. Department of Commerce, NOAA, National Marine Fisheries Service, Grant No. NA90AA-D-1J217. Literature Cited Bartnik, K., and K. Weber. 1989. Widespread occurrence of inter- mediate filaments in invertebrates; common principles and aspects of diversion. Eur J. Cell Bin/ 50: 17-33. Becnel, J. J., E. I. Hazard, and T. Fukuda. 1986. Fine structure and development of Pilos/wrellu chapmani (Microspora: Thelohaniidae) in the moquito. Aedes inxeruilii\ (Say). J Protozool. 33: 60-66. Canning, E. I'., and J. l.om. 1986. The Microsporidae <>/ I 'enehruies. Academic Press, New York. NY. Canning, E. LI., J. l.om, and J. P. Nicholas. 1982. Genus (iliinai The- lohan 1891 (phylum Microspora): redescnption of the type species (iliiKca cinoniiiUi (Moniez 1887) and recognition of its sporogonic development within sporophorous vesicles (pansporoblastic mem- branes). ProtisloloKiai 18: 192-210. C'owin, P., H. P. Kapprt'll, and W. W. Eranke. 1985. The complement of desmosomal plaque proteins in different cell types. / Cell Biol. 101: 1442-1454. Cooper, D., A. Schermer, R. Pruss, and I . Sun. 1984. The use of alF. AE1. and AE3 monoclonal antibodies for the identification and clas- sification of mammalian epithelial keratins. Differentiation 28: 30- 35. Euchs. E.. and D. Marchuk. 1983. Type I and type II keratins have evolved from lower eukaryotes to form the epidermal intermediate filaments in mammalian skin. Pnte. Nail Aaiil. Sci- 80: 5857-5861. Jones, J. C. R.. and R. I). Goldman. 1985. Intermediate filaments and the initiation of desmosome assembly. J. Cell Biol. 101: 506-517. Kelly, D. E. 1966. Fine structure of desmosomes. hemidesmosomes and an adepidermal globular layer in developing newt epidermis. J. Cell Biol. 28: 51-72. l.aemmli, U. K. 1970. Cleavage of structural proteins during the as- sembly ot the head of bactenophage T4. Mature 221: 680-685. Overstreet, R. M., and E. Weidner. 1974. Differentiation of micro- sporidian spore-tails in lnodosporu\ \-/>r<"t'i gen. et sp. n.. '/.. Par- axilenkiintte. 44: 164-186. Pasdar, M., and W. J. Nelson. 1988. Kinetics of desmosome assembly in Madin-Darby canine kidney epithelial cells: temporal and spatial regulation of desmoplakin organization and stabilization upon cell- cell contact. II. Morphological analysis. / Cell Biol. 106: 678-695. Pleshinger, J., and E. Weidner. 1985. The microsporidian spore in- vasion tube. IV. Discharge activation begins with pH-triggered Ca 2+ influx./ Cell Biol. 100: 1834-1838. Romano, V., M. Hatzfeld, T. M. Magin, R. Zimbelmann, W. W. Eranke, G. Maier, and II. Ponstingl. 1986. Cylokeratin expression in simple epithelia. I. Identification of mRNA coding for human cytokeratin no. 18 by cDNA clone. Differennalion 30: 244-253. Rungger-Brandle. E., T. Achtstatter, and W. W. Franke. 1989. An epithelium-type cytoskeleton in a glial cell: astrocytes of amphibian optic nerves contain cytokeratin filaments and are connected by des- mosomes. / Cell Biol. 109: 705-715. Weidner, E. 1976. The microsporidian spore invasion tube. The ul- trastructure, isolation, and characterization of the protein comprising the tube. / Cell Biol. 71: 23-34. Reference: Biol Bull 179: 243-253. (December. 1990) Laboratory Culture of the Aeolid Nudibranch Berghia verrucicornis (Mollusca, Opisthobranchia): Some Aspects of Its Development and Life History DAVID J. CARROLL AND STEPHEN C. KEMPF Department of '/.oology and \\'ildlile Science and . l/ahama Agricultural Experiment Station. 101 Can- Hall. Auburn l'ni\-er\itv. Alabama 36849-5508 Abstract. Adult Berghia verrucicurnis individuals lay white, spiral egg masses containing zygotes. Egg masses are easily cultured in aerated, Millipore-filtered, seasoned aquarium water. Development proceeds quickly, with the hilobed velum apparent by the end of the second day. and the larval shell appearing at the beginning of the third day after oviposition. Hatching occurs 1 1 to 12 days after oviposition (23.9 1.3C). If egg masses are incubated without aeration, poecilogonous development is observed; both larvae and juveniles hatch from the same undisturbed egg mass. The larvae metamorphose soon after hatching, losing the velum and larval shell. A habitat-specific in- ducer is not required for metamorphosis; but a factor as- sociated with the sea anemone Aipiasia pa/1 it/a appears to enhance a larva's tendency to metamorphose. Juveniles begin feeding on A. pal/itla three to four days after meta- morphosis. Reproductive maturity is achieved as early as 47 days after oviposition. Because B. verrucicornis can be cultured, along with its prey A. pallida, at inland facilities, this nudibranch species may be a useful model for labo- ratory-oriented life history and neurobiological investi- gations. Introduction Successful opisthobranch culture has been limited to species with life histories that tie them to fresh seawater. Among the limiting factors have been: ( 1 ) a planktotrophic Received 13 April 1990; accepted 25 September 1990. Abbreviations: MFSA = Milliporc-tiltered. seasoned, aquarium water; MFSA + A = MFSA plus the sea anemone Aiptasia pallida; MFSA-A = MFSA from aquaria containing A. pallida; AFSA = Aiptasia-ftee, seasoned, aquarium water; MFSA from aquaria that had never contained A. pallida; BF = bacterial film culture. larval stage that requires long-term culture best accom- plished with fresh, natural seawater. or (2) a prey species for both juveniles and adults that cannot be easily cultured in sufficient quantity in the laboratory (Kriegstein et a/.. 1974; Harris, 1975; Kempf and Willows, 1977; Switzer- Dunlap and Hadfield, 1977; Bickell and Kempf, 1983; Paige. 1988). Thus, an opisthobranch species that could be reliably cultured through successive generations in a laboratory environment lacking ready access to fresh sea- water would be a valuable source of developmental stages, juveniles, and adults for research in such diverse areas as behavior, development, and ecology (e.g.. Bonar and Hadfield, 1974; Kandel, 1979;Todd, 1981; Marcus et a!.. 1988; Kempf, 1989a; Kempf and Todd, 1989). In partic- ular, opisthobranch mollusks have become premier mod- els for neurobiological investigations, because neurons in their central nervous system are large, repeatably identi- fiable, and easily manipulated (Willows, 1971, 1973; Kriegstein, 1977a, b; Hening et ai. 1979; Jacob, 1984; Kempf <>//., 1987; Cash and Carew, 1989; Chia and Koss, 1989; Marois and Carew, 1989). We selected the aeolid nudibranch Berghia verrucicor- nis as a likely candidate for successful laboratory culture. This mollusk occurs on coral rubble in the shallow waters of southern Florida and feeds on the common anemone Aiptasia pallida. The adult attains lengths of 2-3 cm, and its dorsum is covered with cerata that appear brown when the nudibranch has been feeding. Among the characteristics that anticipate the successful culture of this species in laboratories lacking a ready supply of fresh sea water are: (1) a short generation time (adult- hood is reached five to six weeks after oviposition), (2) lecithotrophic larvae that undergo metamorphosis within days of hatching from the egg mass, and (3) a juve- 243 244 D. J. CARROLL AND S. C. KEMPF nile and adult prey that can also be reared in the lab- oratory. We suggest that Berghia vernuicornis could be a useful opisthobranch species for investigators at inland, as well as marine facilities. Herein we describe the culture tech- niques used to rear and maintain this nudibranch in the laboratory and give an overview of embryonic and larval development. Certain life history traits, such as: (1) in- duction of metamorphosis; and (2) the potential for de- velopmental variability in this species (poecilogony), are also discussed. Materials and Methods Collection of adult animals Adult individuals of Berghia verruciamiis were col- lected in southern Florida, in abandoned coral quarries on Grassy Key and near Bahia Honda Key, at depths of less than 6 feet during late December 1987, 1988, and March 1988. 1989. They were transported to Auburn, Alabama in aerated buckets of seawater with fresh sea- water changes every 4-5 h. The sea anemone Aiptasia pal/iiici was also collected in the Florida Keys and trans- ported to Auburn University as food for the juvenile and adult B. verniciconiis. Culture of the sea anemone Aiptasia pallida Aiptasia pallida may be cultivated in typical salt-water aquaria with undergravel filtration; wet/dry trickle filters should also be adequate. Our own system consists of a number of individual aquaria, as well as a large-scale cul- ture system consisting of one 1 10-gallon, four 30-gallon, and two 20-gallon aquaria connected together with flow- through water circulation. One week after set-up, new aquaria with undergravel filtration are conditioned by the addition to each of a few small salt-water fish (e.g.. clown- fish or damsel fish) or invertebrates (e.g.. hermit crabs, anemones). These animals are fed and maintained for at least one month; their metabolic and digestive wastes provide for the growth of essential, gravel associated, bac- terial populations that detoxify ammonia and nitrites. At the end of the conditioning period, a number of Aiptasia pallida (20-30) are added to each aquarium. Anemones may be obtained from the field or from various suppliers such as Carolina Biological Supply Co. The ane- mones are maintained under a combination of "Grow- Lux" and Actinic Blue fluorescent lighting and are fed newly hatched brine shrimp every two days. With appro- priate care, the number of A. pallida will gradually increase as clones develop from pedal laceration. Regular replen- ishment of aquarium water with freshly prepared, artificial seawater is important. Individual aquaria are most easily replenished each day, when "seasoned aquarium water" is removed for use in the culture of egg masses, larvae, juveniles, and adult Berghia verrucicornis (Fig. 1 A, B, C). Healthy anemone colonies should be established before an attempt is made to establish a colony of B. verruci- cornis. Other, more detailed, methods for culture of Aip- tasia have been described by Hessinger and Hessinger (1981). Culture and feeding methods Jor Berghia verrucicornis Figure 1 summarizes the culture methods used. Pairs of adult Berghia verrucicornis used for egg mass produc- tion were kept in glass bowls containing 300-350 ml of 0.45 yum Millipore-filtered artificial seawater (Instant Ocean or Tropic Marine Systems, Inc.) that was obtained from established saltwater aquaria supplied with CaCOi gravel and undergravel filtration. This water is designated as "Millipore-filtered, seasoned, aquarium water" (MFSA). The water and bowl were changed daily for each culture. As opisthobranch egg masses were laved, they were transferred either to aerated 500-ml beakers con- taining 350 ml of MFSA or, in the case of experiments concerned with direct development, to unaerated 300-ml glass crystallizing dishes containing 100 ml of MFSA. Water and containers were changed daily. Harvested Aip- tusia pallida. used for food, were also kept in bowls of MFSA; both bowls and water were changed every few days. Two days before the expected date of hatching, two or three small Aiptasia pallida were placed in fresh dishes containing MFSA. The MFSA in these cultures was changed each day. When the larvae hatched, we concen- trated them by pouring the culture water through a Nitex strainer (see Switzer-Dunlap and Hadfield, 1981 ); we then pipetted them into the dishes containing anemones, taking care to release them underwater so that they would not be trapped at the air- water interface. The number of larvae in each metamorphosis dish varied depending upon the experiment. Metamorphosis usually occurred in one to three days, and crawling juveniles were present by the third day. After a successful metamorphosis, and until the nudibranchs began feeding, the culture water was changed on alternate days. Thereafter, sea anemones were added as needed, and the culture water and container were changed daily. See Figure 1 for additional details about the frequency with which the water and culture containers may be changed for early juveniles. Antibiotics were not used in any stage of culture. Reg- ular water changes were sufficient to prevent problems with protist and bacterial contaminants. Embryonic and lamil development Zygotes could only be obtained from egg masses that had been collected directly after oviposition. At this time. INLAND CULTURE OF A NUDIBRANCH 245 B vacuum MFSA l I D i *aftU$] juvenile larval experiments /K egg experiments mas ses T adult air V f \ J larvae experments juveniles /N 1 1 adults \\E , / > , \\ F \- L G L larvae "> : r juveniles 1 \\ > Cj* [j^^t ^ adults Figure 1. Algorithm for the culture of Hcrxhui trn m /iwm.v. (A) Aquaria with undergravel filtration and a 3-4 inch thick bed of CaCO, gravel are filled with freshly prepared artificial seawater. A few inverte- brates or fish are added to each aquarium after 3-7 days of operation. After one month of operation, additional anemones or other animals are added to the aquaria. Salinity is adjusted each week. Water from these aquaria is designated as "seasoned aquanum water." (B) Seasoned aquarium water is regularly removed from established aquana and used lor egg mass, larval, juvenile and adult cultures. (C) Seasoned aquarium water is filtered through a 0.45-^m Millipore filter for culture use. (MFSA: Millipore-filtered seasoned aquarium water.) (D) Adults for stock cultures are maintained in small bowls (about 1 3 cm diameter, 5 cm deep). Bowl and water are changed daily. Appropriately sized anemone prey are added as needed. (E) Egg masses are collected from adult cultures and placed in 500-ml beakers containing 350 ml of MFSA. Aeration is provided through Pasteur pipettes. Beaker and water are changed daily. As egg masses hatch, larvae are concentrated with a Nitex filter (see Switzer- Dunlap and Hadfield. 1981, p. 208) and used in larval experiments or metamorphosis cultures. (F) For metamorphosis cultures, larvae are placed in crystallizing dishes containing MFSA and a few small anemones. Water is left unchanged for 5-10 days and then the water is changed daily, and the bowl every few days as needed, until juveniles are large enough to transfer to finger bowls (2-3 weeks after metamorphosis). Juveniles are harvested for experiments or stock juvenile cultures. (G) Juvenile stocks are cultured as are the adults (see D above). Appropriate sized juveniles or adults are used for experiments or stock adult cultures. Transfer of larger juveniles and adults between cultures is easily accom- plished with a "reversed" Pasteur pipette, the rubber bulb being placed over the end from which the tapered tip has been broken off. the egg could be observed with a dissecting microscope, and its diameter measured with an ocular micrometer. Ten egg masses were cultured as described above and were observed at hourly intervals with a compound microscope until the cleavage divisions were finally obscured by the growing number of cells. Subsequently, until hatching, observations were made several times a day so that the appearance of characteristic larval structures, such as the velum, foot, left and right digestive diverticula, larval re- tractor muscles, eyespots, and propodium could be re- corded. The number of egg masses laid per week was re- corded for more than a month, for 22 adult nudibranchs. Induction of metamorphosis We assessed the effect of the sea anemone Aiptasia pal- lida on metamorphosis, by observing the following four metamorphosis cultures each containing 50 larvae: ( 1 ) MFSA in dishes containing the anemone A. pallida (MFSA 4- A); (2) MFSA in dishes without A. pallida (MFSA - A); (3) a bacterial film culture (BF) (see below); and (4) MFSA from an aquarium that had never con- tained A. pallida (Aiptasia-free, seasoned, aquarium water, AFSA). MFSA for treatments 1, 2, and 3 was taken from aquaria that contained A. pallida. Twenty replicates were performed for treatments 1 and 2, and ten replicates for treatments 3 and 4. New glassware was used to filter water and hold AFSA cultures. The bacterial film culture was prepared by removing a few pieces of gravel from an aquarium containing A. pallida and placing them in MFSA overnight. The next day the gravel was removed, the MFSA in the dish was changed and, finally, the larvae added. The number of larvae undergoing metamorphosis in each of the cultures was counted daily over three days. Care was taken to count all juveniles on the bottom, sides, and water surfaces of the culture. The mean standard deviation was calculated for each treatment and day. A Student /-test was used to determine whether the difference between the means was significant (Steel and Torrie. 1980). Culture of egg masses for direct development Single intact egg masses were cultured in dishes of MFSA without aeration. MFSA was changed daily in these cultures; the medium was decanted and fresh MFSA was gently added. Results Collection oj adult animals Berghia vemicicornis never inhabits the mangrove roots where its juvenile and adult food, the sea anemone Aip- tasia pallida, occurs in abundance. Rather, the nudi- branch is found in another habitat of A. pallida, among coral rubble in shallow, sub-tidal waters. The nudibranchs are relatively difficult to spot on the darkly colored coral rocks because the dorsally positioned cerata appear brown when the animal has been feeding. A typical adult of B. 246 D. J. CARROLL AND S. C. KEMPF vcmicicornis is shown in Figure 2A. The white appearance of its egg mass (Fig. 2B) facilitates the collection of B. vernicicornis by providing evidence of the adults' presence. The mass The gelatinous egg masses are laid as untwisted strings in a dextral or sinistral spiral (Fig. 2B). In the laboratory, each pair of adults (n = 1 1 pairs) laid an average of 4 1 egg masses weekly. The egg masses were found attached to the sides and bottom of the culture dish, and some were floating at the air-water interface; the site of ovipo- sition seemed unaffected by the presence or absence of Aiplasia palliila. In the field, these egg masses are depos- ited on the underside of coral "rocks." The embryos, whether in the field or in culture, are contained within two membranes: one, a primary mem- brane or capsule, surrounds each individual embryo; the secondary membrane encases all of the capsules (Fig. 2B, C). Empty primary egg capsules are located at both ends of the egg string. Embryogenesis Egg masses were cultured at 23.9 1.3C; the range was 21-26C (Table I). Cleavage proceeded quickly at this temperature, and because the divisions within a given egg mass were asynchronous, both two-celled embryos and zygotes could be seen. This asynchrony was evident throughout cleavage, until the later blastula stage, when the opacity of the blastomeres reduced the accuracy of the cell count. All of the larvae hatched from the egg mass at the same morphological stage of development. At 2.2 0.3 days after oviposition, the velar rudiment was evident at the future anterior end of the embryo, and the embryo began to move. At first, the cilia were difficult to detect, and we could not be sure that they were beating. Nevertheless, the cilia are probably the cause of the move- ment of the embryos. Larval structures The velum is the first larval structure evident. It de- velops as a ridge on the anterior end of the embryo and assumes its characteristic bilobed appearance on the third day. The velar lobes are located anterolateral to the mouth and possess pre-oral and post-oral ciliary bands. By 4.7 0.9 days, the embryo can partially retract the velum into the shell, indicating the presence of a retractor muscle. Large retractile cells, located around the periphery of the velum, are visible from day 8 until metamorphosis. The velum is lost during metamorphosis. The larval foot (metapodium) appears soon after the velum, during the second day of development (2.8 0.2 days), as a flat, blade-like metapodium. As development continues, the foot thickens and lengthens. The operculum is present by the fourth day. Cilia appear along the ventral length of the foot on the fifth day. The metapodium thickens considerably by the seventh day, and the posterior aspect develops into a definitive propodium soon there- after (7.4 0.5 days). During metamorphosis the foot becomes longer and wider; eventually it occupies the ven- tral surface of the juvenile. The larval shell appears concurrently with the foot. The shell is clear, allowing an unobstructed view of the larval viscera. Shell length increases from 143.7 21.4 ^m on day 3 to a plateau of 251.4 7.0 /urn on day 8 (Fig. 3). When shell deposition has been completed, the mantle fold begins to withdraw from the shell edge. This occurs 7.0 0.3 days after oviposition. The shell stopped growing once the mantle fold retracted, and was shed by the larva during metamorphosis. The larval gut of Berghia vernicicornis occupies most of the space within the shell in the early days of devel- opment. It appears milky white and presumably contains yolk reserves that maintain the nudibranch throughout its embryonic and larval periods, and probably during early juvenile development. The various components of the viscera were not discernible early in development, but became evident 4.1 0.2 days after oviposition. The stomach and the left and right digestive diverticula con- stituted most of the viscera. Initially, those three visceral organs are of similar size. As development proceeds, the right digestive diverticulum decreases in size (from 93.7 9.6 /im on day 4, to 31.6 5.0 /um on day 10), pre- sumably reflecting the use of yolk reserves in this structure. The size of the left digestive diverticulum remains un- changed throughout development; Figure 4 shows the re- lationship between the diverticula from day 4 to day 10. The intestine could be seen looping anteriorly, from the posterior portion of the stomach to the anus on the right side of the mantle cavity, at the same time that the stom- ach and digestive diverticula were evident. Sense organs present in the embryo and larva are the eyespots and statocysts. The statocysts are situated in the larval foot near its attachment to the body; they become apparent four days after oviposition. The two eyespots are located dorsally. directly posterior to the velar lobes: they appeared 6.3 0.4 days after oviposition. Hatching Hatching occurred 1 1.6 0.5 days after oviposition; the range was 9-14 days (T = 23.9 1.3C). In aerated laboratory cultures, the secondary egg mass membrane begins to break down first, releasing intact primary egg capsules. These primary egg capsules appear more pliable than earlier in development and change shape as the em- bryo moved within them. The primary egg capsules soon INLAND CULTURE OF A NUDIBRANCH 247 V' Figure 2. Photographs of several stages in the life history ofBerghia n'micicornix. (A) Adult specimen. Scale bar = 2.0 mm. (B) Egg mass. Scale bar = 2 mm. (C) Example of direct development. A newly metamorphosed juvenile is still present in an egg capsule. Scale bar = 140 nm. (D) Juveniles of Berghia vernicii'urnix. Scale bar = 0.3 mm. Legend: a = anemone; c = ceratal tuft; e = eyespot; j = juvenile; 1 = larva; ot = oral tentacle; pc = primary capsule membrane; sm = secondary membrane; s = larval shell. 248 D. J. CARROLL AND S. C. KEMPF Table I Developmental m'>\ during the luhorainrv culture 0.05). A comparison of the number of metamorphoses in the control cultures (BF. AFSA, and MFSA - A), revealed that the BF and AFSA cultures were significantly different only on Day 1 after hatching (P < 0.05). No difference was seen between the BF cultures and the MFSA - A cultures on any day tested (P > 0.05 ). Similarly, the num- ber of larvae metamorphosing in the AFSA cultures was, statistically, the same as in cultures containing MFSA - A (P>0.05). Direct development The hatching times for veliger larvae in aerated and unaerated cultures were not different (P > 0.5). The av- rupture, releasing veliger larvae. Neither the mouth nor the foot appear to participate actively in the hatching pro- cess. Metamorphosis The lecithotrophic larval stage o(Bert;hitt verrucicornis is released from the egg mass and undergoes metamor- phosis as early as 1 day thereafter. Within metamorphosis cultures, the larvae swim vertically upward immediately after release from the primary egg capsules, and occa- sionally become trapped at the air-water interface. The larvae trapped at the water surface can move around be- cause the velar cilia are submerged. They are apparently able to undergo metamorphosis without the benefit of attachment of the foot to the substratum. In metamor- phosis cultures containing the anemone A. pallida, the larvae show no preference to settle near the anemones. Marked changes in morphology take place during metamorphosis. After a short planktonic larval phase, generally 1-3 days, the larvae settle on the bottom and sides of the metamorphosis dish. Once settled, they appear to crawl randomly along the bottom, slowly beating their velar cilia. The velum is lost first, and the larvae continue 300- 250- iE 200 H T C/} 150- 100- 50- Mantle Retracts Hatching 3 4 ~i i i i i r~ 8 9 10 11 12 13 DAY AFTER OVIPOSITION Figure 3. Changes in shell length during embryonic and larval de- velopment, n = the number of shell lengths measured. Day = 3 n = 26 5 95 6 100 7 91 8 96 9 81 10 50 I I 30 12 10 INLAND CULTURE OF A NUDIBRANCH 249 100- 90- _ 80- E b E 70- 60- 50- 40- 30- n = LDD O = ROD 10 -100 -90 -80 -70 -60 -50 -40 r DAY AFTER OVIPOSITION Figure -I. Changes in the diameter of the left and right digestive di\crticuladunngembryogenesis. LDD, left digestive diverticulum; ROD, right digestive diverticulum. n = the number of diverticula measured. Day = 4 5 6 7 8 9 10 n, DD = 10 40 60 61 60 60 40 n RDD = 10 20 30 31 30 18 5 erage time from oviposition to hatching in unaerated cul- tures was 1 1.7 1.1 days (n = 30); in aerated cultures it was 1 1.6 0.5 days(n = 8). However, both lecithotrophic larval and direct development were seen in egg masses from unaerated cultures (Fig. 2C). This type of variable development is known as poecilogony, which has been denned as "intraspecinc variation in the mode of larval development" (Bouchet, 1989). In some instances, meta- morphosis occurred within the primary egg capsule and the individuals hatched as juveniles, leaving the shell in the egg capsule; other capsules released lecithotrophic lar- vae that could metamorphose soon thereafter. Intracap- sular metamorphosis occurred after some of the larvae had already hatched from the same egg mass. Veliger lar- vae hatching from the same egg masses underwent meta- morphosis after a short planktonic period. The total number of hatchlings of each type varied considerably from one egg mass to the next. This may be due to dif- ferences between the embryos themselves, as the culture conditions were identical for each egg mass. The external appearance of juveniles was identical, whether develop- ment was direct or via a lecithotrophic larva. Juvenile and adult Newly metamorphosed juveniles were oval and white, with eyespots indicating the anterior end, and the rem- nants of the site of larval attachment to the shell at the posterior end. The juveniles initially crawled randomly over the bottom of the culture dish without feeding. One day after metamorphosis, a slender tail-like extension of the foot projected posterior to the elongated body of the juvenile. Rhinophore rudiments were present as small dorsal projections anterior to the eyes at this time. Stiff, cirrus-like projections extended from the anterior end of the juvenile and in opposed pairs along the dorsum. The foot was tightly associated with the body along the entire ventral length of the juvenile. In culture dishes, the ju- veniles gathered at the base of the anemones one day after metamorphosis. They then dispersed before returning one to two days later to begin feeding ( 1 5.3 0.8 days after 30- 20- T z O \. cr D 1 Z CO T LU O T < X 3o 10- \ N t / fel - s CD A f _L ~ , -L, \ ~/ s t t / / 5 Z) R ~\ \ -z. LU ^ EJ N s t / X \ / / \ -/ \ DAY AFTER HATCHING FigureS. The mean cumulative number of larvae undergoing meta- morphosis in four different culture media. Each culture contained 50 larvae initially, a = MFSA from aquaria containing Aiplasia pallida. but no anemones present (MFSA - A); b = MFSA plus the sea anemone A. pallida (MFSA + A); c = bacterial film culture (BF); d = Aiptasia- free, seasoned, aquarium water (AFSA). Twenty replicates were performed for treatments a and b. and 10 for treatments c and d. Error bars represent one S.D. 250 D. J. CARROLL AND S. C. KEMPF oviposition). The paired, dorsal cerata appeared 8-1 1 days after metamorphosis (Fig. 2D). Reproductive maturity was reached 50 3 days after oviposition, but had been observed in the laboratory as early as 47 days. The first egg masses were small and contained less than 100 em- bryos. Discussion Bcrghia vcmtcicnnii.'i is the first opisthobranch mollusk to be cultured through successive generations and main- tained as a viable population of experimental animals at an inland facility. This species has several characteristics that suggest it will be a useful model for laboratory ori- ented research. These include ease of maintenance, regular oviposition throughout the year (3 + egg masses/pair of animals/week), a prey organism (Aipiasia pallula) that can be cultured in the lab, a short embryonic (9-14 days) and lecithotrophic larval period ( 1 -3 days), and a gen- eration time (egg to egg) as short as 47 days. These traits should make B. verrucicornis a convenient organism for research in larval ecology, energetics, neurodevelopment, and, probably, neurophysiology. In addition, this species appears to maintain a symbiosis with a zooxanthclla that it obtains from its prey (Kempf. 1989b), so the association can be used to investigate the establishment, energetics, and evolution of algal-invertebrate endosymbioses. In general, the development of Berghia veirucicornix follows that reported for other opisthobranch mollusks with lecithotrophic larvae (Thompson, 1958, 1962: Bonar and Hadfield, 1974; Harris, 1975; and others). It is char- acterized by the major life cycle stages that Kriegstein ( 1977b) noted for Aplysia californica. i.e.. ( 1 ) embryonic, (2) planktonic, (3) metamorphic, (4) juvenile, and (5) adult. The morphological descriptions given for post- hatching stages of A. calijornica cannot be applied directly to B. verrueicornix because the larvae of B. verrueieornix are lecithotrophic and undergo what might be considered homologous developmental stages as an embryo, rather than as a feeding larva. In lecithotrophic larvae, such as these, the major increase in size of the viscera, develop- ment of the eyespots and propodium, and maximum shell length are all attained prior to hatching. In opisthobranch species with obligate planktotrophic larvae, such as A. californica, these events occur after hatching. Thus, B. verrucicornis might be included in the non-feeding, non- growing group of veliger larvae proposed by Hadfield (1963). But more recent investigations into the feeding potential of lecithotrophic larvae suggest that such dis- tinctions may be hazy at best, because the lecithotrophic larvae of at least some species are capable of feeding if the opportunity presents itself (Kempf and Hadfield, 1985; Emlet, 1986; Kempf and Todd, 1989). A safer develop- mental designation would be that of type 2 larval devel- opment proposed by Thompson ( 1967); these larvae hatch from their egg capsules as late veligers, and undergo meta- morphosis shortly thereafter. The early cleavages of the aeolid nudibranch Phestilla melanobranchia are synchronized (Harris, 1975). Con- versely, early cleavages in egg masses of B verrucicornis are asynchronous. For instance, in a given egg mass, both zygotes and two-celled embryos can be observed simul- taneously, although embryos more than one cleavage stage apart are never observed. Since this asynchrony in de- velopmental stage is seen in embryos situated side by side, it is probably not due to differences in oxygen diffusion through the egg mass (Chaffee and Strathmann, 1984; Strathmann and Chaffee, 1984). Mediation of early de- velopment by a cue intrinsic to the egg mass (Harris, 1975) is a possibility, though the early development is certainly not as well synchronized as that reported for P. niekuio- hninclua. As development proceeds, the discrepancy in cleavage rates becomes less noticeable, with later stages of B. verrucicornis developing at the same apparent rate. As a result, all sibling embryos incubated in an aerated culture hatch together at essentially the same morpho- logical stage of development. Lecithotrophic development, such as that characterized by the embryos and larvae of Berghiu verrucicornis, is generally thought to use maternally derived yolk reserves to fulfill energetic needs. Recent investigations (Jaeckle and Manahan, 1989; Manahan. 1989; Manahan el ai. 1989; Shilling and Manahan, 1990) indicate that cellular endocytic systems in tissues other than those of the diges- tive tract may allow dissolved organic material from sur- rounding seawater to make a significant contribution to the energetic requirements of larval and possibly embry- onic development. Although no conclusions with respect to the developmental importance of DOM can be drawn from this study, the decrease in size of the right digestive diverticulum during embryonic development in B. ver- rucicornis suggests that yolk reserves stored in this organ are an important nutrient source used to support embry- onic energetic needs. The mechanism of hatching has been described for sev- eral opisthobranchs. The aeolid, Phestilla melano- branchia. hatches through a hole made in the capsule wall by repeated contact with the mouth (Harris, 1975). In Adalaria proximo, a dorid, hatching appears to result from mechanical buffeting of the primary capsule wall with the velar cilia and, perhaps, the secretion of an enzyme, be- cause the capsule wall becomes more pliable as the hatch- ing date approaches (Thompson, 1958). Larvae ofBerghia verrucicornis appear to effect their release from the pri- mary egg capsule in a manner similar to that described for A. proximo. As hatching approaches the capsule membrane becomes more flexible. Continuous buffeting and distortion of this barrier bv the velar cilia mav be INLAND CULTURE OF A NUDIBRANCH 251 sufficient to eventually tear an opening that allows release of the larva. Bacteria, protozoans, and other microfauna may also contribute to the lysis of egg mass membranes during hatching. In opisthobranchs, as in many other invertebrates, ex- ternal cues are often responsible for the onset of meta- morphosis (Thorson, 1946; Thompson, 1958, 1962;Bonar and Hadtield, 1974; Harris, 1975). Metamorphosis of the larvae of such species is triggered by chemicals or factors associated with a specific aspect of the mature animals habitat, often the presence of the food of adults (Hadneld, 1977, 1984, 1986; Burke, 1983, 1986; Hadneld and Scheuer. 1985; Morse, 1985; Fitt ct til., 1987; and many others). Our experiments demonstrate that a habitat-spe- cific inducing factor is not an absolute requirement for Berghia vem/cicornis. A mean of 26% of the veligers re- leased from the egg masses of this species metamorphosed in the absence of a habitat-specific inducing molecule (AFSA cultures). But the treatments used to examine in- duction do not preclude the presence of some ubiquitous inducing molecule common to seawater or to habitats in general (e.g., from bacterial or algal films). Further analysis of our experiments on metamorphic induction requires consideration of three components of the results. ( 1 ) The presence of the anemone Aiptasia pallicla always resulted in the greatest number of larvae completing metamor- phosis. (2) The number of larvae metamorphosing in ane- mone-containing treatments was always significantly dif- ferent from that in controls containing only (a) Millipore- filtered, seasoned aquarium water from aquaria containing A. pallida (MFSA A), or (b) Aiptasia-free, seasoned aquarium water (AFSA). (3) Bacterial film controls were only different from the AFSA controls on the first day after hatching. These results suggest that a factor (pre- sumably chemical) enhancing a larva's tendency to me- tamorphose is associated with the anemone A juilliiki. The enhancement of metamorphosis by a substance as- sociated with the prey of juveniles and adults, rather than a habitat-specific metamorphic induction, would be con- sistent with proposals recently made by Kempf and Todd ( 1989) concerning the functional aspects of evolutionary selection for direct development (see below). The juveniles that gather at the base of the anemones one day after metamorphosis may be responding to the same anemone-associated factor that enhances meta- morphosis. The presence of such a response would help to ensure that they gathered in areas containing their prey organism. After the initial "discovery" of the anemone, juveniles in culture bowls disperse, presumably searching out other areas where prey are present. The re-aggregation of juveniles at the bases of culture bowl anemones 1-2 days after dispersal may well be an artifact produced by confinement in the culture container. The nudibranchs simply "rediscovered" the same anemones in the small container and began feeding as their maternally derived yolk reserves were depleted. Classically, most invertebrate species have been con- sidered to undergo a single type of development; but re- ports of poecilogony occurring in a number of invertebrate species, including opisthobranchs, suggest that intraspe- cific developmental flexibility is greater than was previ- ously thought possible (Clark ct a/.. 1978; Eyster. 1979; Gibson and Chia, 1989; Hoagland and Robertson, 1988 review). Our observations indicate that, when egg masses of Bcrgliia vemicicornix are agitated by aeration, only ve- liger larvae hatch from the primary egg capsules. An in- advertently forgotten culture in our lab led to the seren- dipitous observation that, if egg masses of this species are left undisturbed by aeration, both veligers and juveniles will be released from primary capsules of the same egg mass. Repeated observations of additional unaerated egg masses suggest that the factors affecting the type of de- velopment expressed by egg masses of Berghia vcrmci- cornix may be extrinsic rather than intrinsic. One expla- nation for this phenomenon is that aeration of egg mass cultures causes a mechanical break down of egg mass membranes, resulting in the "premature" release of only veliger larvae from the primary egg capsules. Lack of aer- ation (i.e.. agitation) would allow for greater integrity of the egg mass membranes, thus allowing time for some embryos to undergo metamorphosis within the primary capsule. This scenario seems unlikely, because aerated and unaerated cultures show no significant difference in time to hatching at similar temperatures. Perhaps a more acceptable explanation is that agitation due to aeration somehow interferes with either (a) the physical act of metamorphosis itself or, (b) the intrinsic larval systems responsible for attaining competence or initiating meta- morphosis. Whether the poecilogony described above ac- tually occurs in the field has not been determined; but, two facts suggest that it does: (1) Berghia vermcicornis lays its egg masses closely attached, along the egg string's entire length, to the bottoms of coral rocks, thus reducing the effects of agitation; and (2) egg masses are laid in sub- tidal habitats of low energy flux in some instances. Thus, Berghia verrucicornis may enjoy the best of two worlds, being able to benefit from both the advantages of direct development and dispersal via a larval stage. Kempf and Todd ( 1989) have proposed that, as a spe- cies evolves toward a reproductive strategy characterized by lecithotrophic development, a loss in habitat-specific induction of metamorphosis would usually be necessary prior to the establishment of a direct mode of develop- ment. Berghia verrucicornis appears to exhibit develop- mental characteristics that are consistent with this hy- pothesis. It has lost the need for a habitat-specific inducer and, given the right environmental conditions, can re- produce via both a larval stage and direct metamorphic 252 D. J. CARROLL AND S. C. KEMPF development as described by Bonar (1976). As such, B. nrnnitarn/.s appears to be poised on the cusp between indirect (larval) and direct development, suggesting that it will be useful in future studies concerning the evolution of invertebrate reproductive strategies. Acknowledgments We wish to thank Drs. Christine Sundermann, Michael L. Williams, and Gary Miller for providing photographic equipment and lab facilities for parts of this research. Dr. John Miller of Baldwin-Wallace College pointed out the interesting nudibranch that started multiplying in one of his laboratory aquaria. Dr. Tern, Gosliner (California Academy of Science) kindly provided a taxonomic iden- tification of Bcrghia vcrnicicornix. Comments on the manuscript made by Drs. George Folkerts. Ray Henry, James Bradley, and anonymous reviewers were extremely helpful. This research was supported by funds allocated to SCK by the Alabama Agricultural Experiment Station. Project #ALA00780 (AAES Journal No. 15-902548P). Literature Cited Bickell, L. R., and S. C. Kempf. 1983. Larval and metamorphic mor- phogenesis in the nudibranch Melihe Icomna (MolluscarOpisthob- ranchia). Biol. Bull. 165: 119-138. Bonar, I). B. 1976. Molluscan metamorphosis: a study in tissue trans- formation. .-(HI. /.out. 16:573-591. Bonar. I). B., andM.G. Hadfield. 197-4. 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Reference: fl/'o/ Hull 179: 254-263. (December. 1990) Development of a True Ovoviviparous Sea Star, Asterina pseudoexigua pacifica Hayashi MIEKO KOMATSU, YASUO T. KANO 1 , AND CHITARU OGURO Department of Biology, Faculty of Science, Toyania University, Toyama 930, and { Uo:u Aquarium. Uo:u, Toyama 937. Japan Abstract. Asterina pseudoexigua pacifica is a true ovo- viviparous asteroid in that its development and meta- morphosis occur within the spatial hermaphroditic gonad. From the middle of June to the middle of July, the gonad contains numerous embryos and juveniles in various stages through metamorphosis. The opaque, greenish yel- low mature ovum is 450 /jm in diameter. Development is direct. Embryos develop through wrinkled blastula and gastrula stages into a pear-shaped brachiolaria with three arms. The general process is similar to that of asteroids having direct development. Newly metamorphosed ju- veniles are released from the gonopores. Peak release oc- curs in the middle of July. The maximum number of juveniles released from an adult is about 1 300. The ju- venile is 900 nm in diameter and has two pairs of tube- feet in each arm; the skeletal plates are well developed. The present results are compared with those of other true viviparous echinoderms. Introduction The specific name vivipara has often been given to spe- cies that are believed to be viviparous, and a number of echinoderm species bear this name. Hendler ( 1 979) noted about 60 viviparous ophiuroids, and many holothuroids also have this mode of development. The term "vivipar- ity," however, should be restricted to the case in which embryos develop within the gonad or the genital tract, a portion of which may be specialized for incubating em- bryos. Coelomic or bursal incubation is a specialized type of brooding. If we accept this definition, only five species of echinoderms are known to be viviparous: Ophiuroidea, Ophionotus hexactis (by Mortensen, 1921), Holothuro- idea, Leptosynapta c/arki (by Everingham, 1961; cited Received 15 March 1990; accepted 25 September 1990. from McEuen, 1987) and Taeniogyruscontortus(byBoo- lootian, 1966), Asteroidea, Paliriella vivipara (by Dartnall. 1 969; Chia, 1976), and Concentricycloidea, Xyloplax me- dusijormis (by Rowe et a/., 1988). Descriptions of repro- duction and development in these species, although suf- ficient to establish viviparity, are fragmentary. Development through metamorphosis is known in about 40 of the 2500 extant asteroid species. Among these, reproduction and development are best known in several species of Asterina. The entire development through metamorphosis has been reported in A. gibbosa (Ludwig, 1882; MacBride, 1896), A. eoronata japonica (Komatsu, 1975), A. batheri (K.ano and Komatsu. 1978), and A mi- nor (Komatsu et al., 1979). Larval development has been reported in A. exigua. A. pectimfera. and A. regularis (Mortensen, 1921; Komatsu, 1972). Sexuality and repro- duction have been reported in some asterinid species (Cuenot, 1898; Ohshima, 1929; Bacci, 1949; Cognetti, 1954; Delavault, 1966; Dartnall, 1970; Emson and Crump, 1979; Komatsu et al.. 1979). Some species have direct development (yolky eggs and development only through the brachiolaria stage), while others have indirect development (non-yolky eggs and development through both the bipinnaria and brachiolaria stages). As to the sexuality of asterinids, some are gonochoric and some are hermaphroditic. Among the latter are A. bat fieri. A. gib- bosa, A. minor, A. pancerii, A. phylactica, and A. scobi- nata. Asterina minor shows a breeding assemblage (Ko- matsu et al., 1979). Thus, the diversity of development and reproduction occurring in various species belonging to the genus Asterina is well documented. Asterina pseudoexigua pacifica was described by Haya- shi (1977) as a new subspecies of A. pseudoexigua Dart- nall. This subspecies differs from A. pseudoexigua in that its gonopores are on the abactinal side, and it is ovovi- viparous. Development through metamorphosis takes 254 DEVELOPMENT OF A VIVIPAROUS SEA STAR 255 Table I \iinihci ami w;c distribution of juveniles* of Asterina pseudoxigua pacifica cnlU'clcil in ilic lickl Individual Number Number of April May June tube-feet in the longest July 30, 31 Sept. 11, 12 Dec. 7, 8 Feb. 16 23 25 28 1. 2 14, 15 13 21-25 27-30 arm (in pairs) 1974 1473 1972 1976 1986 1975 1976 1985 1973 1976 1987 1986 2 37 3 4 1 1 3 1 2 4 9 5 6 6 3 T 13 1 5 9 20 8 7 1 1 14 4 2 29 7 6 4 34 16 8 26 45 1 6 58 22 7 1 2 16 15 28 56 2 11 32 34 8 1 8 4 5 1 1 1 30 1 19 36 21 9 12 2 1 1 10 32 9 * Juveniles bear fewer than nine pairs of tube-feet on the longest arm. place in the gonad. and juveniles are released from the adult. The present report describes ovoviviparity and the entire process of development of A. pseitdocxigua pacifica. Materials and Methods From time to time, between 1974 and 1987, specimens of Asterina pseiidoexigua pacifica Hayashi were collected from the undersurface of stones in the intertidal zone of Kushimoto, Wakayama Prefecture (Fig. 1 A). The number and size distribution of the juveniles collected are given in Table I. The specimens were kept alive in the labora- tory. Adults were cultured individually in small glass jars so that the release of embryos could be observed. During culture, the temperature was maintained similar to that of the natural habitat (20-25C). Embryos of various stages of development were ob- tained by dissecting the gonad at appropriate intervals between the middle of June to the middle of July. General observations were made using dissecting and light microscopes. Living embryos were measured with an ocular micrometer. For microscopic examination of the skeletal system, juveniles were fixed in 70% alcohol, then macerated in a 10% aqueous solution of potassium hydroxide. For histological observations of the gonad, some specimens were fixed with Bouin's solution im- mediately after collection. The fixed material was embed- ded in paraffin, serially sectioned at 6 pm. and stained with Delafield's hematoxylin and eosin. Gonads and embryos obtained from the gonad by dis- section were fixed for scanning electron microscopy in 2% OsO 4 in 50 mM Na-cacodylate buffer (pH 7.4); the osmolarity of the fixative was adjusted by the addition of sucrose (final concentration, 0.6 AI). The fixed materials were dehydrated in ethanol, dried with a critical-point dryer (Hitachi, HCP-2), and observed with a scanning electron microscope (Hitachi, S-510) after being coated with gold-palladium (Hitachi. E101 Ion Sputter). Results Ovoviviparity The gonad is composed of clusters of lobules arranged in pairs in each interradius. Each gonad opens on the aboral side of the disk through a gonoduct (Fig. IB). In early June, each gonad consists of ovarian and testicular portions (Fig. 1C, D). The majority of the eggs in the ovarian part are fully grown, nearly spherical (about 400 /jm in diameter), and pale green. The head of the sperm contained in the testicular part is spherical (2-3 /urn in diameter); the total length, including the tail, is 50 nm. This species is a spatial hermaphrodite, containing full- grown ova and active sperms simultaneously in each gonad. The breeding season of Asterina pseiidoexigua pacifica is from the middle of June to the middle of July, and most adults have many developing embryos or juveniles in their gonads. Developing embryos of particular stages can be obtained if the gonad is dissected at the appropriate time during the season: early cleavage and early gastrula stages in the middle of June (Fig. 2A. B); late gastrula stage from the middle to the end of June (Fig. 2C); bra- chiolariae from the end of June to the beginning of July (Figs. 1G. 2D and 2E); and metamorphosing larvae and juveniles from the beginning to the middle of July (Figs. 1H; 2F, G). There are some individual and yearly varia- tions. 256 M. KOMATSU ET AL Figure I. A. Specimens (arrows) of adult Asierina pseudoexigua pucilti'a, attached to the undersurface of a stone on the shore of Kushimoto. Bar scale = 50 mm. B. Sagittal section of the gonad (g) of A. p. pacifica. Note gonoduct (gd) passing through the dorsal wall (dw). Bar scale = 200 /im. he. hepatic caecum; is. interradial septum: vw, ventral wall. C. Section of the hermaphroditic gonad ot a specimen of .-1. p. pacijica. Note full grown ova and mature sperms. Bar scale = 200 ^m. o, ovum; t, testicular portion. D. Magnified picture of the hermaphroditic gonad. Bar scale = 100 ^m. gv, germinal vesicle; o, ovum; s, sperm. DEVELOPMENT OF A VIVIPAROUS SEA STAR '-*.- 257 1 "S3* " ., I "' ! ftfw&fJ'^y ] JES ' .?; *- M >.v* n Figure 2. Micrographs of sections of the gonad of a speeimen ofAsterina pseudoexigua pucitica. Bar scale = 100 pm. A. Arrows point to three embryos in early cleavage. B. Wrinkled hlastula with grooves (arrows). C. Sagittal section of a gastrula with a differentiated archenteron. Arrow indicates blastopore. D. Many embryos developing simultaneously in the gonad. E. Sagittal section of a brachiolaria with brachiolar arms (arrows). F. Sagittal section of a metamorphosing brachiolaria. Arrows show brachiolar arms. G. Hor- izontal (long arrow) and cross (short arrows) sections of juveniles in the gonad. E. Dorsal view of a birthing specimen of.-l. /> pacil'wa. Short and long arrows indicate juveniles after birth and just appearing from gonopores. respectively. Bar scale = 1 mm. m, madreporite. F. Living specimen of a juvenile of.-l. />. pacijiat just after birth, dorsal view. Bar scale = 200 ^m. tf. tube-foot. G. Scanning electron micrograph of the inside of the gonad of a specimen of A /> pucilica. Note brachiolaria (arrow) and metamorphosing larvae (asterisks). Bar scale = 100 //m. H. Same as Figure 1G, showing juveniles (j) with tube-feet (arrows) just prior to birth. 258 M. K.OMATSU ET AL Development Developing embryos of various stages are easily ob- tained by dissecting the gonads during mid-June to mid- July. The embryos usually fill the coelom of the adult. The developmental stages shown in Figures 3 and 4 rep- resent embryos removed from dissected gonads: organ- ogenesis (Fig. 2) was studied in sections of the gonad. Cleavage is total, equal, and radial (Fig. 4A). The early blastula is 450 /urn in diameter and composed of equal- sized blastomeres (Fig. 4B). Figures 3C and 4C show blas- tulae in the most wrinkled stage. The surface of the blas- tula is divided by furrows into several portions, each con- sisting of clusters of blastomeres (Fig. 2B). Gastrulation takes place by invagination; the blastopore is circular and small (30 ^m in diameter). Early gastrulae are 440 jum long and 380 ^m wide. Hatching must follow the wrinkled blastula stage, because no fertilization membrane is ob- Figure 3. Scanning electron micrographs of specimens ofAsterina pseudoexigua piii'ifica. The specimens shown in A-H were dissected out of the gonad. The juvenile shown in I was born from the gonopore. Bar scale = 100 fim. A. Embryo of an early cleavage with a fragment of the removed fertilization membrane (arrow). B. Early blastula. C. Wrinkled blastula in its most conspicuous stage. Arrow shows a fragment of the removed fertilization membrane. D. Early gastrula with blastopore (arrow) after hatching. E. Ventral side of a brachiolaria hearing three brachiolar arms (short arrows) and a central sucker (long arrow) among them. F. Magnified view of the anterior part of the specimen shown in Figure 3E, illustrating ciliation. G. Anterior view of the metamorphosing larva. Long and short arrows indicate stalk of larva and hydrolobes, respectively. Each hydrolobe has rudiments of a terminal tentacle and two pairs of tube-feet. H. More advanced metamorphosing larva with terminal tentacle (long arrow) and tube-feet (short arrows), st, stalk of larva. I. Juvenile alter birth. Long and short arrows show a terminal tentacle and tube-feet, respectively. DEVELOPMENT OF A VIVIPAROUS SEA STAR 259 Figure -4. Development of Aslcrinu pseudoexigua pacifica Even, 1 drawing was made from a living spec- imen. Specimens in A-N and in O-S were either dissected out of the gonad, or horn from the gonopores, respectively. A. Early hlastula. earlier stage than that shown in Figure 3B. hm, hlastomere; fm. fertilization membrane. B. Wrinkled hlastula in earlier stage than that shown in Figure 3C. em, cell mass. C. More advanced wrinkled hlastula than that shown in Figure 3C. cm, cell mass. D. Early gastrula. same stage as shown in Figure 3D. E. Early brachiolana. ventral view. F. Same as Figure 4E, left lateral view. G. Brachiolaria in earlier stage than that shown in Figures 3E and 41. H. Same as Figure 4G, ventro-lateral (left) view, bra, brachiolar arm. I. More advanced brachiolana, same stage as shown in Figure 3E, right lateral view. J. Metamorphosing larva in earlier stage than that shown in Figure 3G, anterior (future oral) view, st, stalk of larva. K. Same as Figure 4J, left lateral view. ra. rudiment of adult; st, stalk of larva. L. More advanced metamorphosing larva than that shown in Figure 3G, future oral view. M. Same as Figure 4L, future aboral view. N. Metamorphosing larva just before completion of metamorphosis, future oral view. es. eye-spot; tf. tube-foot; tt. terminal tentacle. O. Juvenile after birth, same stage as shown in Figure 31, oral view. mo. mouth. P. Same as Figure 40. aboral view. tf. tube-foot. Q. Schematic drawing of aboral skeletal system, same stage as shown Figure 4O. cp, central plate; irp, interradial plate; rp, radial plate; tp, terminal plate. R. Skeletal plates and spines of a ray of a specimen shown in Figure 4Q. aboral view, cp, central plate; irp. interradial plate; rp. radial plate; tp. terminal plate; ts. terminal spine. S. Same as Figure 4R, oral view, ap, ambulacral plate; op, oral plate; tp, terminal plate; ts, terminal spine. served around the gastrula. Coelomic pouches emerge from the tip of the archenteron during the gastrula stage (Fig. 2C). Many mesenchyme cells are present in the blas- tocoel. The larva of this sea star is a pear-shaped brachiolaria. Early brachiolariae with rudiments of brachiolar arms are shown in Figure 4E and F. Brachiolariae. which grow to become 600 /urn long and 350 ^m wide, bear three ap- parent brachiolar arms (Fig. 4G, H). Brachiolar arms are short; the lengths of the ventro-anterior arm and of the 260 M. KOMATSU ET AL. ventro-lateral arms are about 150 jum and 75 ^m. re- spectively. At this stage, the blastopore is closed. Bra- chiolariae taken from the gonad can swim in seawater. The body surface of the larva is covered by cilia (Fig. 3E); they are about 10 ^m long and are uniformly distributed at about 15/100 ^m 2 . Figures 2E, 3F. and 41 show more developed brachiolariac than those shown in Figure 4G and H. The posterior part of the larval body of this stage, which corresponds to the larval disk, becomes transformed into a subpentagonal form. A small hydropore is present near the center of the right side of the body. Three bra- chiolar arms become longer and project beyond the "cen- tral sucker," the triangular region denned by the bases of the three arms. The ventro-anterior arm is 175 ^m long and ventro-lateral arms are 100 ^m. The anterior part of the body, designated as the "stalk" of the larva, becomes translucent except for the tip of the brachiolar arms. At metamorphosis, the stalk is absorbed (Fig. 2G). The posterior portion of the metamorphosing larva is hemi- spherical with a subpentagonal margin, being 450 nm in diameter. The metamorphosing larva, shown in Figure 4J and K, bears the shrunken stalk. The bulges of the hydrolobe become recognizable on the future oral side of the disk. Three brachiolar arms are still distinguishable at this stage. Tube-feet appear on the future oral side of the body in more advanced larvae (Fig. 4L, M). At this stage, the stalk is further reduced and situated in one in- terradius (Fig. 3H). The larva shown in Figure 4N has almost completed metamorphosis, and its diameter is 650 /um. The stalk has been completely absorbed. Two pairs of tube-feet and one terminal tentacle, which has a red eye-spot at the basal portion, are developed in each ray (Fig. 2G). When removed from the gonad, the larvae use their tube-feet to move on the substratum. Re/ease Three adults, collected 4-7 July 1974 and kept indi- vidually in small jars, began to release juveniles from their gonopores on 10 July (individuals A. C. and I in Table II; Fig. IF). Soon after release, the juveniles leave their mother and move around on the substratum with their tube-feet. The juveniles, about 900 nm in diameter, are white with a yellow tint (Figs. IE; 4P, Q). They have two pairs of tube-feet in each arm, and their mouths are open. Skeletal plates ( 1 central, 5 interradial, and 5 radial plates on the aboral side; 5 pairs of oral and ambulacra! plates on the oral side; and 5 terminal plates) are well developed (Fig. 4R, S, T). The release of juveniles by adults in the laboratory after mid-July has been observed since 1975. The number of juveniles released from adults is shown in Table II. The peak season of release is from 11 to 20 July. The maximum number of juveniles born from one adult was 1288. Table II Number of juveniles released from un adult j Astenna pseudoxigua pacifica July August 1-1(1 Total Individual 10 11-20 21-31 A 20 1,179 82 7 1.288 B 996 4 15 1.015 C 3 957 1 961 D 263 3 32 298 E 257 1 2 260 F 158 158 G 113 113 H 63 17 80 1 3 50 16 69 J 46 46 K 28 5 9 42 L 17 22 39 M 35 35 N 6 9 15 Most juveniles collected at the end of July 1974 (Table I) had two pairs of tube-feet on each arm. so they had been born less than one month previously. In September and February, the juveniles collected from the field are on average larger than those collected in July. We conclude from these data that juveniles have more than five pairs of tube-feet (some with 6 or 7 pairs) in each arm one year after birth. Discussion Many echinoderms have been described as viviparous. These include 2 species of crinoids, 7 of holothuroids. and 70 of ophiuroids. Isometra vivipara, a crinoid, broods its eggs in a chamber, formed by pinnules, called a mar- supium (Andersson, 1904; Mortensen, 1920). Chiridota rolifcra. a holothuroid, broods its eggs in the coelom (Clark, 1910; Boolootian, 1966). Stegophiura sculpta, an ophiuroid. broods its larvae in the bursa (Murakami, 1941). But none of these species is truly viviparous, be- cause development proceeds outside of the ovary or the genital tract. Mortensen (1921) reported that development in Opluonotus hexactis begins in the ovary and. indeed, that the embryos develop entirely within the ovary. Ever- ingham (1961; cited from McEuen, 1987) observed that Leptosynapta c/arki is an intraovarian brooder. Taenio- gyrux contonus was listed by Boolootian (1966) as a pre- sumptive ovarian incubator. Dartnall ( 1969) reported that in Patiriella vivipara the embryonic development occurs in a sac derived from the gonad. However, he later noted that this species is a coelomic incubator ( Dartnall, 1971). Although there is some discrepancy about the portion of the incubation (Dartnall, 1969, 1971), Chia (1976) re- DEVELOPMENT OF A VIVIPAROUS SEA STAR 261 ported that P. vivipara is an intraovarian brooder. Thus, P. vivipara seems to be a viviparous species. In Xyloplax medusiformis, development occurs in the ovary (Rowe el a/.. 1988). Although this species is dioecious, eggs, sperms, embryos at various stages, and juveniles are present in the ovary. Thus, this species is truly ovoviviparous. Re- cently, Concentricycloidea, to which X medusiformis be- longs, has been regarded as a member of the subphylum Asterozoa. rather than of the Crinozoa or Echinozoa (Barker a til.. 1986). Therefore, further studies on the developmental process in A meditsiformis may provide important informations about true ovoviviparity in As- terozoa. Thus, previous descriptions suggest that the last mentioned five species are truly viviparous. The present study has shown that development of Asterina pseudoex- /Xiia pacifica commences and proceeds throughout meta- morphosis within the gonad; the resulting juveniles are released from the gonopore. Thus, A. pseudoexigua pae- ifica is the sixth species of truly viviparous echinoderms. Embryos of A. pseudoexigua paeifica have no tissue connection with the adult. Rather, the nutritional re- quirements of the embryos in these viviparous echino- derms seem to be supplied by nutrient reserves in the egg. Embryos of the bursal brooding ophiuroids. Ampliip/iolis laponica and Amphipholis sqmunata. do have an organic connection with the bursal wall, and these ophiuroids were thought to be viviparous, the larva being brooded in the bursa and nourished by the adult (Murakami. 1940: Fell, 1 946). But the tissue connection now appears to be a sup- porting structure, as had been suggested by Fell ( 1946). Both male and female elements mature simultaneously in the gonads of A. pseudoexigua paeifica. Therefore, this species is spatially hermaphroditic. Spatial hermaphro- ditism has been reported in a few asterinid species: As- terina gibbosa (Cuenot, 1898; Bacci, 1949, 1951: Dela- vault, 1966), Asterina minor (Komatsu el til.. 1979) and Asterina phylactica (Emson and Crump, 1979). suggesting that spatial hermaphroditism is not rare in this genus. Chia (1976) mentioned that P. vivipara is probably self- fertilizing. Asterina minor is self-fertilizing (Komatsu el ai. 1979). Fertilization in Echinodermata generally occurs in seawater. but Mortensen ( 192 1 ) reported that ripe eggs of a true viviparous ophiuroid. O. liexactix. are fertilized in the ovary. Internal fertilization has not been described previously in any asteroid species. Although we have not observed natural self-fertilization in A. pseudoexigua pae- ifica, the development was initiated in the hermaphroditic gonads by injecting l-methyladenine into the coelomic cavity of the adult in June. Therefore, we assume that self-fertilization takes place by the sperm of the same in- dividual (Komatsu, unpubl.). The gonads of A. pseu- doe.\igua paeifica have mature sperms and full-grown ova, simultaneously, in early June. The gonads contain many embryos or juveniles from mid-June to mid-July. These facts suggest that self-fertilization takes place internally. Brooding occurs in many asteroid species, but the brooding habits differ with species (Feder and Christen, 1966; Hayashi, 1972). The protective location of the em- bryos varies: underneath the disk in Leptaxterias ocho- tensis similispinis (Hayashi, 1943; Kubo, 1951); in a brooding chamber beneath the disk in llenricia xanxni- nulcnta (Sars, 1844; Masterman. 1902), llenricia tumida (Hayashi, 1940). and Leptasterias hexactis (Osterud, 1918; Chia, 1966); in the nidamental chambers that are formed by interlocking spines at the arm bases in Odinella nutrix (Fisher, 1940): among the bases of the outspread spinelets of the dorsal paxillae in Ctenodiscus aitstralis (Lieberkind, 1926); in the nidamental cavity between the aboral body wall and the supradorsal membrane in Pieraster militaris (Koren and Danielssen. 1857) and Pteraster obseurus (D'yakonov, 1968); and in the stomach in Leptasteriax gr0enlandeca (Lieberkind, 1920; Fisher, 1930). Ovoviviparity may be considered to be a type of brood- ing. However, a definite difference exists between the vi- viparity and brooding outside the gonad. In asteroids, maturation of ova takes place during their release from the gonads. On the other hand, ova of the viviparous as- teroid should mature within the gonads. Furthermore, many physiological changes must have occurred in the embryos during the change in adaptation, from the sea- water environment, to the intraovarian circumstances. Viviparity is thus a very unique and specialized way of protecting the embryos in asteroids. As adaptations for protecting offspring, specific methods and sites have been developed in each species during evo- lution. The ovoviviparity of .-1. pseudoexigua paeifica should reflect a period of evolution unsuitable for free larval life, during which the present species evolved. Observations on the development of truly viviparous echinoderms are limited. Even in O. hexactis, the devel- opment of which has been thoroughly reported, the pro- cess of metamorphosis is unknown (Mortensen, 1921). Thus, the entire process of development of a true vivip- arous species is reported for the first time in the present study. Eggs of A. pseudoexigua paeifica are 450 ^m in diameter, more than twice the size of those of P. vivipara (Chia, 1976; 150Mm)orofa hexactis (Mortensen, 1921; 200 MID). PatirielUi vivipara has no larval stage. Devel- opment of A', medusiformis is direct, and embryos are present in the gonad (Rowe el a/.. 1988). In L. tiarki, egg diameters range from 240 to 404 ^m, and development proceeds, within the ovary, to tentacled juveniles through pentactula larvae (Everingham, 1961; cited from McEuen, 1987). Ophionotus hexactis has an ophiopluteus larva that is rudimentary, because the arms develop poorly and the anus is not open. The larva of A. pseudoexigua paeifica is a pear-shaped brachiolaria, similar to that of asteroids 262 M. KOMATSU ET AL. with direct development. Differences in the egg and the mode of development in these viviparous echinoderm species may indicate that ovoviviparity in echinoderms is a result of convergence. Development of . 1. pseudoexigua pacifica is direct, with only a brachiolaria stage and without a bipinnaria. Direct development has been reported in other Asterina species: . 1. halhcri, A. hurloni, A. cornnata japonica, A. e.\igua. A. gihhosa. and A. minor (MacBride, 1896; Mortensen. 1921; James, 1972; Komatsu, 1975; Kano and Komatsu, 1978; Komatsu el a/., 1979). The eggs of these asterinids are yolky and large in diameter. The morphology of the larva of A. pseudoexigua pacifica resembles that of other aster- inid species having direct development, especially A. co- ronata japonica. Brachiolariae of . ) pseudoexigua pacifica and A. coronata japonica have short brachiolar arms and a poorly developed central sucker. All asterinids under- going direct development have a pelagic larval phase, ex- cept for A, exigua, A. gihhosa. and .1 minor. Although the larvae of A. pseudoexigua pacijtcu remain in the gonad throughout their development, they have cilia on the body surface and can swim in seawater when removed from the ovary. Furthermore, the larvae continue developing in seawater (Komatsu, unpubl.). Hence, the larva of the ancestor of A. pseudoexigua pacifica may have been free- swimming in seawater like the larvae of some other as- terinids. Although A. pseudoexigua pacifica is ovovivi- parous, it has the same developmental type, egg, and bra- chiolaria as other non-viviparous asteroids. This suggests that ovoviviparity in A. pseudoexigua pacifica evolved re- cently from the direct type with free-swimming brachio- laria. Acknowledgments The authors are indebted to Dr. Hiro'omi Uchida and members of the Sabiura Marine Laboratory' of the Marine Parks Center of Japan for providing facilities and collec- tion of specimens. They thank Mr. Makoto Murase for his cooperation in preparation of scanning electron mi- crographs. They are also obliged to Professor John Law- rence, University of South Florida, for his kind suggestions during the preparation of the manuscript. The present study was supported in part by Itoh Science Foundation toMK. Literature Cited Andersson, K. A. 1904. Brutflege bei Anledon Inrsina Carpenter. Mm Ergeb. Schwedische Sudpolar-Exped. (1901-1903)5: 1-7. Bacci, G. 1949. Ricerche su Asterina gihhoxa (Penn.) II. L'ermafrod- itismo in una popolazione di Plymouth. li\ h /ool. Ital. 34: 49-74. Bacci, G. 1951. On two sexual races of Aslerina gihhoxa (Penn.). AA- perientia 7: 31-33. Barker, N. N., F. \V. E. Rowc, and H. E. S. Clark. 1986. 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DEVELOPMENT OF A VIVIPAROUS SEA STAR 263 Kubo. K. 1951. Some observations on the development ot'the sea-star. Leptasterias ochotensis similispinis (Clark). J. l-'ac. Set . Hokkaulo Imp Univ. Scr. 6 Zooi 10: 97-105. I iflKTkiml. 1. 1920. On a starfish (Asterias graenlandeca) which hatches its young in its stomach. I 'idcnsk. Medd. Dansk Nalurh. I-'oren. Scr A' 72: 121-126. Lieberkind, I. 1926. Ctenodiscus auxtralis Liitken. A brood-protecting asteroid. I u/cnsk Medd. Dansk Naturh. h'oren. Scr. 882: 183-196. I.udwig, II. 1882. Entwicklungsgeschichte der Astcrina gihhma Forbes. Z U7.v.v. /,>/. 37: 1-98. MacBride, F.. \V. 1896. The development of Astenna gihhusa. Q J. Hficrosc. 5(7. 38: 339-41 I. Masterman, A. T. 1902. The early development of Crihrclla oculala (Forbes), with remarks on echinoderm development. Tranx. R Sue Edinburgh 40: 373-418. McEuen, F. S. 1987. Phvlurn Echmodermata, Class Holothuroidea. Pp. 574-596 in Reproduction and Development <>t Marine Invene- brali"* i 'I I he \nrilieiii Pacific Coast, M. F. Stratmann ed.. University of Washington Press, Seattle and London. Mnrtensen, Th. 1920. Studies in the development of crinoids. Papers Depl Marine Biol., Carncxic IIIM H<;v/; 16: 1-94. Mortenscn, Th. 1921. Studies <>l I lie DcvclnpniCHl anil Larval l-'nrins / Kchinoiicrms G. E. C. Gad. Copenhagen. 21ft pp. Murakami, S. 1940. On the development of the calcareous plates of an ophiuran, Amphipliiilix lapomea Matsumoto. Jpn. J /.not 9: 19- 33. Murakami, S. 1941. On the development ol the hard part of a vivip- arous ophiuran. Stegophiura .//>// nii'ioiic Antilles and polar liuilif, in nocivf.s under compression* Meiotic spindles 3 Initiation ot Polar body 4 compression No. of oocytes Distance 1 + formation Before migration 31 (9) 98 [70-148] T (I) 29(8) 0(0) During migration 34(13) 35(12-110] 31 (12) 3(1) 17(6) After migration 26(9) 5 [0-28] 26 (9) 0(0) 22(8) 1 Oocytes were induced to mature by treatment with 1 mM DTT or radial nerve extracts, compressed, and observed successively (24-27C). Results from the latter were given in parentheses as fractions of cases. 2 The "Distance" is the mean value with its range (brackets) in ^m between the pole and the nucleus, measured for each oocyte at a time just before or after breakdown ot the germinal vesicle. 3 The meiotic spindle formation was examined through successive observations from about 30 min up to about 70 min. +, spindle formed; -, did not. 4 Polar body formation was examined through further observations ol each oocyte up to 96-190 min. chromosomes appearing in the nucleoplasmic area re- mained there without forming karyokinetic figures (Fig. 2E-J). Thus, most (29/31) of the oocytes failed to form meiotic spindles (Table I). Nevertheless, a small fraction (3/29) of these oocytes did form a metaphase-like chro- mosome configuration at a later time (at 120-140 min). In the remaining 2 out of 31 oocytes, metaphase to ana- phase figures were detected at 50-70 or 55-80 min (Ta- ble I). Maturing oocytes at 11-16 min (i.e.. during GV mi- gration) were then compressed (Table I). The GV broke down near the pole (cf. Fig. 3N, O). A pair of asters were first detected, either at the margin of the nucleoplasmic area facing the pole, or in the cytoplasm adjacent to the pole. The asters then moved into the nucleoplasmic area, and chromosomes in the nucleoplasmic area were aligned to form a metaphase plate at 30-50 min. Most (31/34) of the oocytes formed meiotic spindles (Table I). When oocytes were compressed as late as 19-20 min (i.e.. after GV migration), the nucleoplasmic area formed at the pole (Fig. 3A), and in these cases, meiotic spindles formed (Fig. 3B-L, Table I). In summary, meiotic spindle formation in compressed oocytes is apparently dependent upon the time of compression. Polar bodies formed in oocytes under compression (Table I). This occurred in cases where the metaphase Figure 4. Transection of prophase-arrested oocytes. The oocytes were transected at one of the planes indicated (A-D) with reference to the protrusion marking the presumptive animal pole. On transection, the GV ends up in one of the daughter fragments. spindle moved, and "attached" to the pole (Fig. 3B-E). The first polar body formed by a pinching-off of the pro- trusion at its base (Figs. 3F-I), and the second polar body formed just beneath the first (Figs. 3J-L), as in uncom- pressed oocytes. In contrast, when the metaphase spindle failed to move and attach to the pole, polar bodies failed to form, but anaphase movement of chromosomes still occurred (Fig. 3M-O). As shown in Table I, spindle and polar body formation failed, even in those compressed oocytes that had been obtained from ovaries stimulated with radial nerve ex- tracts. Thus, such failures are not due to the effects of DTT used for maturation. The distance between the pole and the nucleus at the time of GV breakdown may be important for the successful formation of the meiotic spindle and polar bodies in oocytes under compression (Table I). The results show that a pair of asters develop in the cytoplasmic region at the pole after GV breakdown, and that the asters are required for organizing the meiotic spindle. The pair of asters required for meiotic spindle formation are derived from the pole of the prophase-arrested oocyte Prophase-arrested oocytes were microsurgically tran- sected (Fig. 4), and the GV-enucleate fragments, whether containing the pole or not, were treated to induce mat- uration and observed (Table II). Fragments containing the pole developed a pair of asters in the cytoplasmic region at the pole (Fig. 5 A-D), whereas fragments lacking the pole did not. Two asters were in- variably observed at 20-40 min (Table II); later (at 80 min or more), however, about half of the fragments had four asters. This shows that in fragments lacking the con- tents of the GV, each of the asters had split into two. Autonomous replication of centrosomes has been shown in starfish oocytes deprived of GV materials (Picard el POLARITY AND ME1OSIS IN OOCYTES 269 ill., 1988) and in enucleated sea urchin embryos (Lorch, 1952; Sluder <>/oc\'ii'.i Fragments' No. of asters 2 Transection Pole No. 2 1 Undetected A + 23 22(12) 1 B + 21 21 (21) C + 14 14(8) Q D 15 15 ' Prophase-arrested oocytes were transected in various directions (Fig. 4). and GV-enucleate fragments either containing the pole ( + ) or lacking the pole (-) were treated with 1 m.l/ DTT and observed from about 20 mm to about 40 min at room temperature (24-27C). The fragments lacking the pole were examined further up to about 100 min. : The number of asters detected is indicated. The number in paren- theses indicates the fraction of cases in which the site of the pole was recognizable. In these cases, the asters appeared in a cytoplasmic region close to the pole. Discussion Poles as associated sites ofceiilrosomes The present study has revealed a pair of asters at the pole of living sea cucumber oocytes during maturation. The occurrence of fibrillar structures (Ohshima, 1921, 1925; Inaba. 1930) and microtubules (Smiley and Cloney, 1985) at the pole in prophase-arrested oocytes suggests that the asters may pre-exist in some form in immature oocytes. And Oka ( 1 940) reported a fibrillar structure even in growing oocytes. The present study demonstrates that the organizing centers of the meiotic spindle are derived from the pole in prophase-arrested oocytes. These findings imply that the fibrillar structure at the pole is a structure Figure 5. Aster formation in a GV-enucleate fragment with the pole (A-D), and chromosome arrange- ments in GV-nueleate fragments without (E), and with (F), the pole. The fragment in A-D was obtained from transection A (Fig. 4). and those in E and F were obtained from transections C and D, respectively. A: 9 min. The pole (arrow-head) looks clear. B: 15 min. C: 21 min. A pair of asters (arrows; also in D) form at the pole. D: 30 min. E: 93 min. Chromosomes (arrow) show no karyokinetic figures. F: 65 min. Anaphase chromosomes (arrow). Temperature: 24-27C. Bar in A: 50 ^m (for A-F). 270 Y. K. MARUYAMA similar to the pre-meiotic aster seen in starfish oocytes (Schroeder, 1985a). I conclude that the pole (i.e.. the pre- sumptive animal pole) of the prophase-arrested oocyte of the sea cucumber is the site of the centrosome(s) or mi- crotubule-organizing center(s). With maturation (perhaps, at a time prior to GV breakdown), the centrosome may change to form a pair of asters, and these asters (centro- somes) then function as the organizing centers of the meiotic spindle. In starfish, the pre-meiotic asters are associated with the cortex at the presumptive animal pole (Schroeder and Otto, 1984, Schroeder, 1985a, b; Picard ct a!.. 1988). A similar microtubule-array containing a microtubule-or- ganizing center occurs in association with the cell surface inGV stage oocytes of sea urchins (Boyle and Ernst, 1989). Hence, the association of the centrosome with the cell surface of the presumptive animal pole where polar bodies later form may be a characteristic common to all echi- noderm oocvtes. Poles and meiotic divisions As can be seen in Figure 1, features specific to oocytes of the sea cucumber, Holotlntna leucospilota, are the mi- gration of the GV to the pole and the subsequent for- mation of a meiotic spindle in the polar region. Because the centrosome participating in meiotic spindle formation is initially dissociated from the GV and resides at the pole, a direct outcome of the GV migration is 'restoration' of the spatial association of the centrosome(s) with the nu- cleus to form the meiotic spindle. The pole, embracing the centrosome (microtubule-organizing center), micro- tubules, and the cortex, may participate in the migration of the GV to the pole. Nuclear migration, or spindle migration, and subse- quent attachment of the spindle pole to the cortex results in unequal divisions: e.g.. polar body formation in surf clam oocytes (Dan and Ito. 1984; Dan and Inoue, 1987), Chaetopterus oocytes (Hamaguchi el a/.. 1983) and Cre- pidula oocytes (Conklin, 1917); micromere formation in sea urchin embryos (Dan. 1979, 1984; Dan el at.. 1983; Schroeder, 1987); and ganglion cell formation in neuro- blasts of grasshoppers (Kawamura, 1977). The present study of the meiotic divisions of sea cucumber oocytes has revealed that a meiotic spindle eventually "attaches" to a definite site the pole to form the polar bodies. Thus, the cortex of the pole is a site specialized for inter- acting with the spindle-pole aster, and for anchoring the microtubule-organizing center or the asters originating from it. The cortex or cell surface of the pole may contain local factors responsible for binding with asters or the organizing center. Polar protrusion and clear spot as markers oj the animal-vegetal axis in sea cucumber oocytes The presence of the "clear spot," a special cytoplasmic islet, in the cytoplasm near the cell surface opposite the presumptive animal pole (Maruyama, 1981) could be traced back to prophase-arrested oocytes. With matura- tion, the clear spot exhibits a rather strong birefringence. Further studies are needed to define its significance in development. In any event, there are now two visible structures, the polar protrusion and clear spot, both serv- ing as markers for the main axis (animal-vegetal axis) of the oocyte. They could be useful for analyzing localized morphogenetic determinants, as has been done in eggs and embryos of sea urchins and starfish (Maruyama et nl.. 1985; Maruyama and Shinoda, 1990). Acknowledgments I thank the Director, Professor E. Harada, and the staff of the Seto Marine Biological Laboratory of Kyoto Uni- versity for providing the facilities for this work. I am grateful to Professor M. Yoneda of Kyoto University for critically reading the manuscript. Literature Cited Boyle, J. A., and S. G. Ernst. 1989. Sea urchin oocytes possess elaborate cortical arrays of microfilaments, microtubules. and intermediate fil- aments. Dc\: Biul. 134: 72-84. Conklin, K. G. 1917. Effects of centrifugal force on the structure and development of the eggs of Crepidula. J. E.\p. Zoo/. 22: 311-419. Dan, K. 1979. Studies on unequal cleavage in sea urchins I. Migration of the nuclei to the vegetal pole. Dev. Growth Differ. 21: 527-535. Dan, K. 1984. The cause and consequence of unequal cleavage in sea urchins. Zoo/. Sci. 1: 151-160. Dan, K.. S. Endo, and I. I'emura. 1983. Studies on unequal cleavage in sea urchins II. Surface differentiation and the direction of nuclear migration. Dcv. Growth Differ. 25: 227-237. Dan, K., and S. Ito. 1984. Studies of unequal cleavage in molluscs: I. Nuclear behavior and anchorage of a spindle pole to cortex as revealed by isolation technique. De\: Growth Differ. 26: 249-262. Dan, K., and S. Inoue. 1987. Studies of unequal cleavage in molluscs II. Asymmetric nature of the two asters. //;/ J. Invert. Rep Dcv 11: 335-354. Gerould, J. II. 1896. The anatomy and histology ofCaudina arcnata Gould. Bull Mus Camp, tool 29: 123-190. I l:ui i. iuii. In, Y., D. A. Lutz, and S. Inoue. 1983. Cortical differentiation, asymmetric positioning and attachment of the meiotic spindle in Chaetopterus pergamt'iilact'tnts oocytes. J. Cell Bio/. 97: 245a. Holland, N. D., J. C. Grimmer, and II. Kubota. 1975. Gonadal de- velopment during the annual reproductive cycle of Comanthus ja- />omo;(Echinodermata: Crinoidea). Biul. Bull 148: 219-242. Inaba, D. 1930. Notes on the development of a holothurian. Caudina clulcnsis (J. Muller). Sci Rep Tohoku Imp. I'mv. Ser IV. 5: 215- 248. .IriikmscMi. J. W. 191 1. On the origin of the polar and bilateral structure of the egg of the sea-urchin. Arch. Entwicklungsmech. 32: 699-716. Kanamura, K. 1977. Microdissection studies on the dividing neuroblast of the grasshopper, with special reference to the mechanism of unequal cytokinesis. Exp. Cell Re* 106: 127-137. POLARITY AND MEIOSIS IN OOCYTES 271 I.induhl, P. K. 19.12. Zur Kenntnis des Ovarialeics bei dem Seeigel. Arch I-:nl\\'iMiin.i>\nieih 126:373-390. l.orch, I. J. 1952. Enuclealion of sea-urchin blastomeres with or without removal of asters. Q .1 Micro* Sci 93:475-486. Maru>ama. Y. K. I9SO. Artificial induction of oocyte maturation and development in the sea cucumbers Holothuria leucospilota and Ifol- othurui rtmliili* Biol Bull 158:339-348. Marinama, V. K. 1981. Precocious breakdown ol the germinal vesicle induces parthenogenetic development in sea cucumbers. Biol. Bull 161: 382-391. Maruyama. Y. K. 1985. Holothunan oocyle maturation induced by radial nerve, liiol Bull 168: 249-262. .Maruyama, Y. K., Y. Nakaseko, and S. Yagi. 1985. Localization of cytoplasmic determinants responsible lor primary mesenchyme for- mation and gastrulation in the unfertili/ed egg of the sea urchin Hemicentrotus pulcherrimus .1 E\p /.ool 236: 155-163. Maruyama, Y. K., K. Yamamuto, I. Mila-Miya/awa, I. Kotninami. and S.-l. Nemoto. 1986. Manipulative methods for analyzing embryo- genesis. Methods Cell Biol 27: 325-344. Maruyama, Y. K., and M. Shinuda. 1990. Archenteron-forming ca- pacity in blastomeres isolated from eight-cell stage embryos of the starfish. Antenna pecliin/mi Dcv Growlh Differ. 32: 73-84. Monnc, 1.. 1946. Some observations on the polar and dorsoventral organization of the sea urchin egg. Ark. /.ool. 38A No. 15: 1-13. Ohshima, H. 1921. On the development of Cnciiinuiui ccln/uiui v. Marenzeller. Q. J Microsc. Sci. 65: 173-246. Ohshima, II. 1925. Pn la matungo kaj fekundigo ce la ovo de I'mar- kukumoj. Sci. Bull Fac Agric Ai'r.v/m i'niv I: 70-102. Oka, T. B. 1940. Chromatoid fibnllar structure of the micropyle-canal ofgrowingoocytes of Holothuria munacarui. /.ool. Sci 52: 138-140. Picard, A., M.-C. Harrieane, J.-C. Labbe, and M. Durev. 1988. Germinal vesicle components are not required for the cell- cycle oscillator of the early starfish embryo. >cv Biol 128: 121-128. Schroedur, I . K. 1985a. Physical interactions between asters and the cortex in echinoderm eggs. Pp. 69-89 in Molecular Biologv ol In- vcnchnilc Development. R. H. Sawyer and R. M. Showman, eds. Belle W. Baruch Lib. Mar. Sci. 15. Schroeder. T. K. 1985b. Cortical expression of polarity in the starfish oocyte. /Vr. Growth Differ. 27: 31 1-321. Schroeder, T. K. 1987. Fourth cleavage of sea urchin blastomeres: mi- crotubule patterns and myosin localization in equal and unequal cell divisions. Dev. Biol 124: 9-22. Schroeder, T. K., and J. J. Otto. 1984. Cyclic assembly-disassembly of cortical microtubules during maturation and early development of starfish oocytes. Dcv. Biol 103: 493-503. Sluder, G., F. J. Miller, and C. L. Rieder. 1986. The reproduction of centrosomes: nuclear versus cytoplasmic controls. / Cell Biol 103: 1X73-1881. Smiley, S., and R. A. Cloney. 1985. Ovulation and the fine structure of the Stichopus califomicus (Echinodermata: Holothuroidea) fecund ovarian tubules. Biol Bull 169: 342-364. \\ iNon, E. B., and A. P. Mathews. 1895. Maturation, fertilization, and polarity in the echinoderm egg. New light on the "Quadnlle of the centers."/ Morphol. 10: 319-342. Reference: Biol. Bull 179: 272-278. (December. Self and Non-Self Recognition in a Calcareous Sponge, Leucandra abratsbo SHIGETOYO AMANO Cancer Research Institute, Kanazawa University. Kanaiawa. Isliikawa 920, Japan Abstract. Discrimination between self and non-self has been shown in many demosponges, but calcareous sponges have not been studied. Allorecognition in a cal- careous sponge, Leucandra abratsbo, was analyzed in al- logeneic combination assays. Most allogeneic combina- tions were incompatible, and the low rate (4.8%) of al- logeneic acceptances suggests an extensive polymorphism in those genes that may control allorecognition. However, histological studies of the rejection process revealed that the first reaction consisted of strong adhesion of allogeneic pieces. Thereafter, the rejection reaction that followed was accompanied by the accumulation of archeocytes in the contact region. Vigorous cytotoxic reactions occurred within this region, and the degenerated cells were probably phagocytosed by archeocytes, which suggests that they are the primary effector cells for cytotoxicity and phagocytosis. Because L. abratsbo is a solitary sponge, armed with pro- truding spicules that prevent contact of the pinacoderm with that of conspecific individuals, allorecognition may not prevent the formation of allogeneic chimeras in the natural habitat. Introduction The immune systems of invertebrates have interested investigators who believe that such systems might be pre- cursors of the vertebrate immune system (Coombe et a!., 1984; Stoddart el ai. 1985). In the last decade, compre- hensive studies have provided much information on sponge allorecognition (Hildemann el ill., 1979, 1981; Kaye and Ortiz, 1981: Curtis el a/.. 1982: Jokiel et al., 1982; Van de Vyver and Barbieux, 1983; Buscema and Van de Vyver, 1984a-c; Neigel and Schmahl, 1984; Neigel and Avise, 1985; Mukai and Shimoda, 1986; Smith and Hildemann, 1984, 1986a, b). The resulting indisputable Received 3 December 1989; accepted 29 August 1990. evidence suggests that allorecognition is the rule in de- mosponges: alloincompatibility can be induced in most orders of the class Demospongiae. The allogeneic reactions of demosponges, however, are remarkably variable, so a thorough understanding of sponge allorecognition has been difficult. First, allografts are rejected in some sponges, but accepted in others (Jokiel et al.. 1982: Buscema and Van de Vyver. 1984c). Second, the rejection reaction varies considerably from species to species. According to present information, allografts are rejected by cytotoxic reactions (Hildemann et al., 1979, 1981; Buscema and Van de Vyver, 1984b; Mukai and Shimoda, 1986; Smith and Hildemann, 1986a), by the formation of a collagenous barrier ( Buscema and Van de Vyver, 1984a, c), or by nonfusion (Buscema and Van de Vyver, 1984c; Mukai and Shimoda, 1986). Moreover, two or three types of rejection reactions have been observed in some species (Van de Vyver and Barbieux, 1983; Bus- cema and Van de Vyver. 1984c; Mukai and Shimoda, 1986) and the type of allogeneic rejection is independent of sponge phytogeny. Third, various effector cells partic- ipate in the rejection reaction. Although several effector cells including archeocytes. collencytes. lophocytes, phagocytes, and amoebocytes have been identified thus far(Vande Vyver and Buscema, 1977; Van de Vyver and Barbieux, 1983; Buscema and Van de Vyver, 1984b, c; Smith and Hildemann, 1986a), we cannot predict which of these cells actually effects rejection reactions (Smith. 1988; Van de Vyver, 1988). Furthermore, we know very little about their origins and transitions as these cells de- velop normally. Calcareous sponges diverged from the ancestral sponge before the Devonian period (Hyman, 1940). The shapes and composition of their spicules are distinctly different from those of demosponges, and their allorecognition systems may also be different. In this paper allorecognition in a calcareous sponge (Leucandra abratsbo) is presented. 272 AI.LORECOGNITION IN SPONGE 273 Most allogeneic combinations were incompatible, sug- gesting the existence of an extensive polymorphism of histocompatibility genes in natural populations. Vigorous cytotoxic reactions by archeocytes were observed in the contact region. The ecological significance of self and non- self recognition in these sponges is discussed. Materials and Methods All specimens of Leucandra uhratsho. a calcareous sponge with a leuconoid canal system, were collected from a raft at the Breeding Center of Aomori Prefecture in northern Japan. They were abundant in scallop-breeding baskets that were hung a few meters below the water's surface. The size of the raft is about 1 5 X 20 m, so the maximum distance between any two specimens is about 20 m. The sponge was easily freed from the substratum because it is upright and has a stout body. Once collected, the sponges were put in water-tight containers, brought to the Asamushi Marine Biological Laboratory, and placed immediately in running seawater where they could be maintained for more than ten days. The largest specimen was about 8 cm in length; only those larger than 4 cm were used. Assessment of incompatibility Because parabiosis experiments were not feasible with this sponge, "the allogeneic combination test" was per- formed as an alternative method. Sponges that had been selected for the allogeneic combination assay, were cut into slices 2 to 3mm thick, and two sponge pieces derived from different individuals were bound together with a piece of cotton thread. The flatness of their opposed cut surfaces allowed the sponge pieces to be closely appressed, and caused the deeper sponge tissues to be in direct con- tact. To ensure reliability, the test was first performed with ten replicates of each sponge pair. Because all rep- licates of a pair showed similar allogeneic reactions, two replicates of each combination were usually performed in this study, unless otherwise mentioned. The polarity of the sponge pieces exerted no influence on their reactions in either allogeneic or autogeneic combinations. Bound sponge pieces were supplied with clean running seawater during the experiments and were as healthy as intact sponges under laboratory conditions. They regenerated the dermal layer and pinacoderm on the free cut surface during the allogeneic combination test. The bound sponge pieces were examined daily, and most of them were distinctly rejected in four days. In pre- liminary experiments, five allogeneic combinations that were not rejected in four days did not reject in an addi- tional four days. Thus, all allogeneic combinations that showed no external signs of rejection were fixed with the Bouin's solution five days after binding. To provide a time- series analysis of the rejection process, ten replicates of the same allogeneic combination were constructed from an allogeneic sponge pair. Two of these replicates were fixed daily, embedded in Palaplast, sectioned, and stained with haematoxylin and eosin. Results A uiogi'iick' m/c7/< w.v The fusion process was analyzed morphologically using sponge pieces in autogeneic combinations derived from one sponge specimen. One day after binding, these au- togeneic sponge pieces were firmly adherent (Fig. 1 ). One striking feature in the contact region of such autogeneic combinations is the development of a dermal layer-like tissue between the sponge pieces. Development of this Figure I. Fusion of a one-day autogeneic combination of Leucandra ahrufibo The mid-horizontal line of this photomicrograph is in the in- terface of the sponge pieces. The dermal layer-like tissue (asterisks) has developed in the contact region. Scale bar = 100 ^m. Figure 2. Fusion of a two-day autogeneic combination of L ahralsbo. Mid-horizontal line is in the interface, however, choanocyte chambers are arranged almost regularly. No dermal layer-like tissue is observable. Scale bar = 100 /jm. 274 S. AMANO -- ! i- 4***^ *<. Jjr *"*^ "3,** r '>^ .; .;* ^ e ..' -' .;^Ttr /. I? -. ' -vH5_-.i.a ''SiSc ^ Figure 3. Rejection reaction in a one-day allogeneic combination of Lciicandra abratsbo. Although these sponge pieces look like fusion from external observation, many archeocytes have already gathered in the contact region. The dermal layer-like tissue (asterisks) is observable on the both sides of the archeocyte accumulation. Scale bar = 100 ^m. Figure 4. Archeocytes accumulated in the contact region of a one- day allogeneic combination. These archeocytes are in contact with each other, and most of them already show nuclear condensation. Scale bar = 50 urn. tissue seems to be necessary for the fusion process, facil- itating the adhesion of the sponge pieces during the early stages. Only a few archeocytes were found within the con- tact region. Two days after binding, the autogeneic sponge pieces were more intimately fused than on day one, so that their external boundaries became obscure. Figure 2 shows the contact region of such sponge pieces. The dermal layer- like tissue has disappeared, and the choanocyte chambers are arranged almost regularly, with no evidence of cyto- toxic or phagocytic reactions. The autogeneic sponge pieces fused rapidly, and after four days, the interface be- tween the sponge pieces was almost undetectable micro- scopically, this signaled that the fusion process was com- plete. Allogeneic reaction* The rejection process occurring in allogeneic combi- nations was studied histologically using daily samples from sets of coupled sponge pieces, each set derived from two physiologically discrete individuals of the same species. Four incompatible sponge pairs were thus observed, and they all showed a similar rejection process. The rejection process of only one allogeneic sponge pair is represented. One day after binding, allogeneic sponge pieces had ad- hered firmly and their pinacoderms were already fused. They are therefore difficult to distinguish from autogeneic combinations by external observation. Nevertheless, the rejection process has already begun microscopically. Fig- ure 3 shows the contact region of a one-day allogeneic combination. As in autogeneic fusions, dermal layer-like tissue has developed in the contact region, and the sponge pieces are firmly adhered. In contrast to autogeneic fusion, archeocyte accumulations are already visible in the contact region (Fig. 4, an enlargement). The archeocytes are con- gregated and in close contact with each other. Within this Figure 5. Rejection reaction in a two-day allogeneic combination of Leitcamlru uhrafiho. Extensive degeneration of the archeocyte accu- mulation is shown. These sponge pieces are splitting off (arrows). Scale bar = 100 pm. Figure 6. Phagocytes and archeocytes with conspicuous nuclear condensation (arrows) in a two-day allogeneic combination. Scale bar = 50 urn. AL I ORECOGNIT10N IN SPONGE 275 t , t w* on Figure 7. Rejection reaction in a four-day allogeneic combinatio.. of Lcuauuira uhriil\h<>. The aggregates of the degenerated archeocyles have split off. Scale bar = 100 ^m. Figure 8. A large aggregate of degenerated cells in a four-day allo- geneic combination. Scale bar = 50 ^m. cell accumulation, cytotoxic reactions are evident, because numerous cells show degenerative nuclear condensation. The cytotoxic reaction mediated by the archeocytes ap- parently begins soon after they accumulate and make cell contact. Two days after the allogeneic sponge pieces had been bound, external signs of rejection are already evident. The fused pinacoderm begins to break along the boundary between the sponge pieces. Figure 5 shows massive ac- cumulations of archeocytes in the contact region. Within this cell accumulation, tissue degeneration is obvious, and the sponge pieces begin to split oft" (Fig. 6, enlargement). Nuclear condensation is clearly visible in the degenerated cells. Phagocytosis is already discernible, and phagocytes that had engulfed several degenerated cells can be seen. Four days after binding, extensive necrotic tissue (about 0.5 mm thick) is visible between allogeneic sponge pieces. Because it has become frail, the sponge pieces fall apart if the binding thread is removed (Fig. 7). The necrosis is, however, limited to the contact region, and the sponge tissues external to it show no degenerative signs. Thus, archeocytes, at least at four days, had apparently not in- vaded far into the allogeneic tissues. In the necrotic cell Figure 9. Many phagocytes and degenerated cells in a four-day al- logeneic combination. Scale bar = 50 ^m. Figure 10. A large phagocyte that has engulfed more than ten de- generated cells. Scale bar = 10 ^m. masses, nuclear condensation is evident in most of the cells, and cell lysis prevails (Fig. 8). The numbers of phag- ocytotic figures have increased considerably, and Figure 9 shows such phagocytosis in the contact region. Figure 10 reveals a phagocyte that has engulfed more than ten 1234 Figure 1 1 . Reactions of allogeneic and autogeneic combinations be- tween the seven individuals of Leuiwiitni ahratsbo. H, allogeneic rejec- tion; U, weak rejection; autogeneic fusion or allogeneic acceptance. 276 S. AMANO Table I imx in Leucandra abratsbo Number of combinations scored Ot Acceptance 6 4.8 Weak rejection 9 7.1 Reiection 112 88.2 Total 127 100.1 degenerated cells. Later, these sponge pieces were com- pletely disjoined, and regeneration of pinacoderm on the re-exposed surface was observed. Patterns and frequencies ofallorecognition Figure 1 1 represents an example of autogeneic and al- logeneic combinations among seven individuals. All the combinations were tested twice and gave similar results. One acceptance and one weak rejection was found among the allogeneic combinations, but all of the other allogeneic combinations rejected vigorously. Seven autogeneic com- binations fused completely. Table I shows the cumulated results of 127 allogeneic combinations. About 5% of the combinations were ac- cepted, while the others were incompatible. Histological examination of the allogeneic acceptances revealed no re- jection reactions; neither archeocyte accumulation nor cytotoxic reactions were observed at the interface of the accepted sponge pieces. Accordingly, they were indistin- guishable from autogeneic fusions. In about 7% of the allogeneic combinations, the sponge pieces rejected, but weakly. These weak rejections were hardly distinguishable from the allogeneic acceptances by external observation, because the fused pinacoderm was not broken. Neverthe- less, histological examination revealed distinct rejection reactions in the contact region. Parabiosis experiments Parabiosis experiments, in which two sponge pieces are touching surface-to-surface, were tried. Figure 12 shows the result of an autogeneic parabiosis, and Figure 1 3 shows an allogeneic combination, both five days after binding. Both figures show a wide gap between the sponge surfaces because the densely protruding spicules, which have been dissolved in the Bouin's solution, prevented contact be- tween the opposing pinacoderms. Obviously, no fusion or rejection reaction has occurred in these sponge pieces. Discussion Alloincompatibility in a calcareous sponge, Leucandra uhraisho, is shown for the first time in this report. These results suggest an extensive polymorphism of histocom- patibility genes, because most individuals are alloincom- patible. Obviously, further studies on other calcareous sponges are necessary to determine whether allorecogni- tion specificity is a general phenomenon in the class Cal- carea. In this calcareous sponge, allogeneic pieces were rejected by cytotoxic reactions. Neither collagen deposition (chronic rejection) nor nonfusion was observed, although they have been shown in demosponges (Buscema and Van de Vyver, 1984a-c; Mukai and Shimoda. 1986). Many archeocytes accumulated in the contact region of allo- geneic combinations of this calcareous sponge; mesohyl cell accumulations have been observed in many demos- ponges (reviewed by Smith, 1988). Direct contact between 71 ]. , iv. '" ' .,"." \ ~\ . ^ i 12 13 Figure 1 2. A parabiosis experiment of autogeneic sponge pieces five days after binding. They have not fused, and there is a wide space between their opposing pinacoderms. Calcareous spicules that prevented contact of the sponge surfaces have been dissolved in the Bouin's solution. Scale bar = 25 pm. Figure 13. A parabiosis experiment of allogeneic sponge pieces five days after binding. There is no sign of a rejection reaction. Scale bar = 25 m. ALLORECOGNITION IN SPONGE 277 archeocytes is most probably necessary for the cytotoxic reaction to be triggered, because it occurred only within cell accumulations in which archeocytes were in close contact with each other. In demosponges, the necessity for contact between mesohyl cells has been suggested (Bigger ct a/-. 1981), but the involvement of diffusible substances is also plausible (Smith and Hildemann, 1986a, b). Until now, there has been no evidence that archeocytes selectively come into contact with allogeneic cells. To an- swer this question, ;/; vitro studies may be helpful. In a solitary ascidian. Halocynthia mretzi. Fuke ( 1980) showed that the cytotoxic reaction between allogeneic coelomo- cytes in vitro, termed the "contact reaction," occurs after close contact between allogeneic cells. Bound sponge pieces of L. ahratsho adhered firmly within 24 h in allogeneic combinations, as well as in au- togeneic ones. By this time, the dermal layer-like tissue has developed in the contact region, and this intervening tissue may play an important role in the adhesion of sponge pieces. Because this dermal layer-like tissue formed similarly in allogeneic and autogeneic combinations, its formation is not an allogeneic reaction but more likely a regenerative event induced by the exposure of inner tissues to the exterior. Indeed, the composition of the dermal layer-like tissue was similar to that of the dermal layer that regenerated on the reverse side of the sponge pieces. Pinacoderm was formed on the surface of the dermal layer-like tissue after the sponge pieces were disunited. In autogeneic fusions, however, it disappeared from the con- tact region within a few days. Therefore, the dermal layer- like tissue is conceivably a regenerated dermal layer in the contact region. About 95% of the allogeneic combinations of L. abratsbo were incompatible in natural populations col- lected from a raft (15 X 20 m). This high rate of alloin- compatibility reflects extensive dispersion of sponge lar- vae. This calcareous sponge released amphiblastula larvae in the morning, and they swam actively, settled, and metamorphosed on the substratum (Amano, in prep.). Before settlement, they crawled about on the substratum for several hours. In demosponges, also, larval release is controlled by light (Amano, 1986. 1988); phototaxis and geotaxis enable the swimming larvae to settle in a suitable site, often at a considerable distance (Bergquist ct a/.. 1970). Twenty-four hours of swimming and transport by water currents are probably sufficient for the released am- phiblastula larvae to be dispersed beyond the limits of the raft ( 15 X 20 m). Therefore, some specimens used in this study may be kin, and allogeneic combinations of the sponges with kinship may result in fusion. Not knowing the genealogies of the tested specimens, however, we can- not know whether allogeneic acceptances necessarily im- ply genetic identity of the combined individuals of L. abratsbo (Grosberg, 1988). Because L. ahratsho is densely covered with protruding stout spicules, they prevented sponge pieces from touching each other when parabiosis experiments were tried. With- out immediate contact between their opposing pinaco- derms, the sponge pieces did not fuse, nor were there re- jection reactions even in autogeneic or allogeneic indi- viduals. Therefore, allorecognition in this sponge is not required to avoid fusion and the formation of allogeneic chimeras in nature. But if it is so, why has this sponge developed a recognition system that can be revealed only in the laboratory? Grosberg (1988) has discussed the evo- lution and ecological significance of allorecognition sys- tems in clonal invertebrate-organisms that have numerous opportunities for tissue contacts between isogeneic and allogeneic individuals. In solitary invertebrates, however, conspecific interactions rarely occur during the life cycle. Accordingly, we cannot assume that allorecognition spec- ificity is the only phenotypic effect of genes controlling allorecognition, particularly in solitary invertebrates (Grosberg, 1988, 1989; Grosberg and Quinn, 1988). This study indicates that L. ahratsho, a solitary sponge, has few opportunities for tissue contacts in nature. Thus, al- lorecognition specificity may be an epiphenomenon re- sulting from pleiotropic genes. Although the ecological significance of allorecognition specificity is as yet un- known in invertebrates, pleiotropic models have been proposed and supported experimentally; e.g., the control of gametic incompatibility (Oka, 1970; Scofield cl a/., 1982; Fuke, 1983), and the discrimination of food bacteria (Wilkinson. 1984; Wilkinson ct a/.. 1984). In conclusion, this study supports the idea that self and non-self recog- nition is a general phenomenon in the lowest metazoan phylum, the sponges. Acknowledgments I am grateful to Dr. T. Numakunai and to the staff of the Asamushi Marine Biological Laboratory for their hos- pitality and help during my stay. Mrs. T. Mayama, S. Tamura, and M. Washio helped collect the sponges. I also thank the lunch-time conference group of biologists at Kanazawa University for their enlightening discussions, and Professor Edwin L. Cooper (UCLA), who read the manuscript. Literature Cited Amano, S. 1986. Larval release in response to a light signal by the intertidal sponge Halichondria panicea. Bio/. Bull. 171: 371-378. Amano, S. 1988. Morning release of larvae controlled by the light in an intertidal sponge, Callyspongia ranwxa. Bin/. Bull. 175: 181-184. Bergquist, P. R., M. E. Sinclair, and J. J. Hogg. 1970. Adaptation to intertidal existence: reproductive cycles and larval behaviour in de- mospongiae. Symp. Zool. Soc. Loud. 25: 247-27 1 . Bigger, C. H., W. H. Hildemann, P. L. Jokiel, and I. S. Johnston. 1981. Afferent sensiti/.ation and efferent cytotoxity in allogeneic 278 S. AMANO tissue responses of the marine sponge Callyspongia dit/uxu. Trans- plantation 31: 461-464. Buscema, M., and G. Van de Vyver. I984a. Allogeneic recognition in sponges: development, structure, and nature of the nonmerging front in Ephydatia fluvialilis ./. Morphol. 181: 279-303. Buscema, M., and G. Van de Vjver. I984b. Cellular aspects of alloim- mune reactions in sponges of the genus Axinclla I. Axinclla polypoides J .v/>. Zoo/ 229: 7-17. Buscema, M., and G. V an de Vyver. 1984c. Cellular aspects of alloim- mune reactions in sponges of the genus Axinclla II. Axinella vemicma and Axinella damicomis .1 I \i> /not 229: 19-32. Coombe, D. R., P. L. Ey, and C. R. Jenkin. 1984. Self/nonself recog- nition in invertebrates. Q Rev liitii 59: 231-255. Curtis, A. S. G., J. Kerr, and N. knovtlton. 1982. Graft rejection in sponges. Genetic structure of accepting and rejecting populations. Transplantation 33: 127-133. Fuke, M. T. 1980. "Contact reactions" between xenogeneic or allo- geneic coelomic cells of solitary ascidians. Biol. Bull 158: 304-315 Fuke, M. T. 1983. Self and non-self recognition between gametes of the ascidian. Halm i wlim rorcin Roux's Arch. Dev Hint 192:347- 352. Grosberg, R. K. 1988. The evolution of allorecognition specificity in clonal invertebrates. Q Rev Biol. 63: 377-412. Grosberg, R. K. 1989. The evolution of selective aggression conditioned on allorecognition specificity. Evolution 43: 504-515. Grosberg, R. k., and J. K. Quinn. 1988. The evolution of allorecognition specificity. Pp. 157-167 in Invertebrate IliMorecoxnition. R. K. Grosherg. D. Hedgecock. and K.. Nelson, eds.. Plenum Press. New York. Ilildemann, \\ . H., I. S. Johnston, -and P. L. Jokiel. 1979. Immunocompetence in the lowest metazoan phylum: transplantation immunity in sponges. Science 204: 420-422. Ilildemann, \\ . H., and D. S. Linthicum. 1981. Transplantation im- munity in the Palaun sponge. Xc^io^pon^ia c\ii;iont>ia dif/usa (Ponfera; Demospongia). Proc. R Soc. Lond. B226: 445- 464. Smith, L. C., and \V. H. Ilildemann. 1986b. Allogeneic cell interactions during graft rejection in ( 'allyspongia dilhixa ( Porifera: Demospongia); a study with monoclonal antibodies. Proc. R Soc. Lund. B226: 465- 477. Stoddart, J. A., and D. J. Ayre. 1985. Self-recognition in sponges and corals? Evolution 39: 461-463. Van de Vyver, G. 1988. Histocompatibility responses in freshwater sponges: a model for studies of cell-cell interactions in natural pop- ulations and experimental systems. Pp. 1-14 in I nvcnchrate Hislo- iC(.oxnilinn, R. K. Grosberg. D. Hedgecock. and K. Nelson, eds.. Plenum Press. New York. Van de Vyver, G., and B. Barbieuv 1983. Cellular aspects of allograft rejection in marine sponges of the genus Polymaslia. J Exp. /<><>l 227: 1-7. Van de Vyver, G., and M. Buscema. 1977. Phagocytic phenomena in different types of fresh-water sponge aggregates. Pp. 3-8 in Devel- opmental Immunobiology. J. B. Solomon and J. D. Horton, eds., Elsevier/North-Holland Biochemical Press. Amsterdam. \\ ilkinson. C. R. 1984. Immunological evidence for the Precambrian origin of bacterial symbiosis in marine sponges. Proc. R Soc. l.ond B220: 509-517. \\ilkinson. C. R., R. Garrone, and J. Vacelet. 1984. Marine sponges discriminate between food bacteria and bacterial symbionts: electron microscope radioautography and in situ evidence. Proc. R. Soc. Lond B220: 519-528. Reference: Bn>l Bull 179: 279-286. (December, Ontogenetic Variation in Sponge Histocompatibility Responses MICHA ILAN 1 AND YOSSI LOYA Department of Zoology, Tel Aviv University, Ranun Aviv. Tel Aviv 69978, Israel Abstract. Grafting of adult sponge fragments (Clitil inn/a sp.) led to isograft fusion and allograft nonfusion in both parahiotic and implant grafts. We conclude that adult CtiiilinnUi sp. individuals discriminate between self and nonself, and fuse only isogeneic fragments. In the labo- ratory, however, larvae and early juveniles fuse. Larvae used in the experiments were probably genetically differ- ent, even if they were asexually reproduced. These results indicate that the capacity for fusion between allogeneic individuals disappears during ontogenesis in this sponge. In some cases, multichimeras were formed when up to five larvae fused to yield a single sponge. All 37 chimeras metamorphosed and survived during 17 days of obser- vation. Possible mechanisms for the formation of sponge chimeras during early development are discussed, as are the costs and benefits of chimera formation at juvenile versus adult stages. We propose that, if fusion exists in the field, it occurs between kin larvae. Introduction Sessile marine organisms frequently contact each other. In many instances this contact induces a recognition pro- cess during which self/nonself histocompatibility is estab- lished, resulting in acceptance or nonacceptance of the tissues involved. Intraspecific (allogeneic) encounters are frequently characterized by visible recognition events in which various responses may occur. Allogeneic histoin- compatibility (nonacceptance of tissues from different conspecific individuals) has been observed in various groups of invertebrates during the past two decades: as- cidians, bryozoans, stony corals, sea anemones, gorgon- ians, hydrozoans, and sponges (reviewed in Grosberg, Received 1 I June 1990; accepted 25 September 1990. 1 Present address: Marine Science Institute. University of California. Santa Barbara, CA 93106. 1988). The pioneering work of Wilson ( 1907) led to ex- tensive research on cellular events during the reaggregation of sponge cells (reviewed in Smith. 1988; and Gramzow ct al.. 1989), and on the consequences of sponge grafting (e.g.. Hildemann et al.. 1979; Curtis el al.. 1982; Smith and Hildemann, 1986). The general view is that, even if all sponges cannot be claimed to manifest allorecognition, increasing evidence suggests that many of them are characterized by a high degree of polymorphism, and usually will not accept al- lografts( Smith, 1988; Van de Vyver. 1988). These results demonstrated highly diverse reactions among sponges, to both allografts (contact between individuals of the same species) and xenografts (contact between individual of dif- ferent species). The reactions vary from xenograft (Paris, 1961) and allograft acceptance (formation of stable chi- meras) in various sponges (Evans and Curtis, 1979; Kaye and Ortiz, 1981; Zea and Humphreys, 1985), to allograft rejection (Smith, 1988). The genetics of sponge allorecognition, currently, is poorly known, but can be explored through allograft ex- periments on transitivity relationships. Transitive com- patibility is defined as a situation in which individual A is compatible with B, B is compatible with C, and A is compatible with C. A non-transitive situation occurs when A is compatible with B and C, but B and C are not com- patible with each other. Allograft studies of marine sponges have demonstrated transitivity (Neigel and Avise 1983, 1985; Wulff, 1986), and may imply that complete allotypic matching is required for compatibility. A diffi- culty in such studies among sponge populations in the field is the possible existence of clonemates derived through asexual propagation (e.g.. Neigel and Avise 1983; Wulff, 1986). Therefore, the tested sponges must be widely separated from each other in the field (more than the dis- persal distance for an asexual propagule). to reduce the possibility of their being clonemates. 279 280 M. II AN AND V. LOYA The phenomenon of fusion between sponge larvae has been sporadically reported (Wilson, 1907; Burton, 1949; Warburton, 1958: Borojevic, 1967; Van de Vyver, 1970; Fry, 1971; Van de Vyver and Willenz, 1975). but has received inadequate attention. Fusion between larvae de- rived from the same parent might be considered as an autograft if the larvae were produced parthenogenetically. as is known in corals (Stoddart, 1983, 1984) and has been suggested for some sponges (reviewed by Fell. 1974; Bergquist, 1978; Simpson, 1984). On the other hand, if the larvae are not clonemates, then their fusion may be regarded as allograft fusion, resulting in the creation of a chimera. If adult sponges do not fuse, but their allogeneic larvae fuse, then questions of variable self/nonself histo- recognition responses during a sponge's lifetime, and the capacity of larvae to distinguish between self and nonself, are raised. In the present study, we address the following questions. How do Chalimda sp. adults react toward isografts and allografts? How do Chalimda sp. larvae derived from the same parent react to each other? And what are the con- sequences of encounters between larvae that originated from different parents? Materials and Methods We studied the brooding sponge Chalimda sp. from the coral reefs of Eilat, Israel, on the Red Sea (2930'N: 3455'E), after establishing its reproduction and settlement (Ilan and Loya, 1990). Larvae were obtained by slicing adult sponges and collecting the well-developed free- swimming larvae. Two sets of experiments on larvae were conducted. In the first set, larvae were derived from the same parent, and in the second, from different parents. In the first set of experiments. 224 larvae were obtained from 25 indi- vidual sponges. Two to ten larvae were placed in each petri dish (all derived from the same parent) to assess the possibility of fusion between two larvae (bichimera) or more (multichimera). In the second set, 3 experiments were conducted with 104 larvae taken from 14 different individuals. Every petri dish contained only two larvae, each derived from a different adult sponge. The petri dishes (bottom surface area 9.6 cm 2 ) were filled with 9 ml unfiltered seawater. The adult sponges used in the second set of experiments grew in the sea, 1 to 300 m apart from each other. Such distances have been considered beyond fragment dispersal in cases of frequently fragmenting sponges growing in areas affected by storms (Jokiel el a/., 1982; Kaye and Ortiz, 1981; Wulff, 1985). Because frag- mentation and budding are not common phenomena in Chalimda sp., and no frequent storms occur in the study area, such a distance between the parental sponges, di- minishes the possibility that these sponges could be ge- netically identical clonemates. The experiments in this second set were designed to introduce larvae of every sponge to larvae from each of the other sponges. The lar- vae for the 3 experiments in this set were obtained from 6. 4. and 4 parental sponges and had 1 5, 6, and 6 possible combinations of parents, respectively (25 out of the 27 possible combination were performed in duplicate). All experiments were conducted at ambient seawater tem- perature (25 1C). The tendency of larvae to aggregate was tested in the second set. using the statistical analysis of the goodness offit(Sokal and Rohlf, 1969). According to our definition, aggregation occurs when two larvae establish and remain in contact. To calculate this occurrence, a hypothetical Chalimda sp. larva was considered to be a rectangle of 1 X 0.5 mm (0.5 mm 2 ). These two figures are larger than those of any Chalimda sp. larva measured (Ilan and Loya, 1990) and are therefore considered to be conservative. In a petri dish of 960 mm 2 bottom area, there are 1920 rect- angles of 0.5 mm 2 . The second larva will contact the first one only if it settles on top of the first larva, or in one of its four neighboring rectangles. Given a random larval settlement in a dish, the probability of two larvae con- tacting is: 5 X (1/1920). Because 52 pairs of larvae were used in these experiments, there would be random contact between the larvae in 52 X 5 X (1/1920) = 0.135 of the pairs. Any significantly higher value than this prediction, implies larval aggregation. Two grafting experiments between fragments of adult sponges, involving two different protocols, were conducted /// situ in front of the Marine Biological Laboratory, Eilat. at least 1 m below lowest tide. Chalimda sp. fragments of about 3 X 4 cm were attached to each other and to a fiberglass net anchored to the bottom. Fragments were taken from sponges situated 10 to 300 m apart from each other on the coral reef. We used five sponges in each of the two experiments, with all cross combinations (with duplicates) of allogeneic interaction made. To determine whether this species is capable of fragment fusion, all the experiment sponges were also isogeneically grafted. In the first experiment, intact external surfaces of sponges (pin- acoderms) were placed in contact (parabiotic grafts). Nei- gel and Avise( 1985) considered this technique to be more reliable than implant grafts in which a block of donor's tissue is implanted into a recipient. However, following the suggestion of Johnston and Hildemann (1982) that a reaction may be very slow, and a review by Smith ( 1988) on the involvement of mesohyle (inner nonfiltering) cells in the process of acceptance or rejection of grafts, we set up a second experiment in which contacts were made between fragments of mesohyle to speed the reaction pro- cess. We used equal sized fragments, and not the implant technique, to avoid a possible effect of recipient size on SPONGE HISTOCOMPATIBILITY RESPONSES 281 the donor's block (Hildemann ct a/.. 1980). The grafting experiments were observed for three months. Samples of grafted zones on the sponges were fixed in 2.5% glutaraldehyde buffered in seawater for scanning electron microscopy, then washed, dehydrated in graded ethanol series, critical-point-dried, coated with gold-pal- ladium and viewed in a JEOL JSM-840A SEM. Results When free-swimming Chalinula sp. larvae (n = 224), derived from the same parent sponges, were put together in a dish, 44% fused (Table I). In dishes with more than two larvae, there were cases of fusion between two larvae (bichimera as in Fig. la); 3 to 5 larvae (multichimera) also fused, metamorphosed successfully, and gave rise to single sponges (Fig. Ib). In all, 37 chimeras were observed (Table I). Chalinula sp. larvae fused during different stages: as free swimming larvae (Fig. la. Ib), or as post larvae shortly after attachment and metamorphosis, when they grew toward each other and fused at the contact zone (Fig. Ic). In some cases, free-swimming larvae settled on, and fused with, recently metamorphosed sponges. In the second set of experiments, each of the pair of larvae in a dish was taken from a different sponge. In 19 pairs out of 52 (36.5%), the larvae fused. The observed number of fused pairs is significantly higher than the ex- pected (0.135) from random settlement (P< 0.001, good- ness of fit test). The larvae settled in all areas of the dishes and were not confined to certain microhabitats (e.g.. cor- ners, center): therefore the aggregation was not due to external pressures. In two cases, chimeras redivided into two distinct individuals, after one to three days, although the duplicate of one of these pairs, which also produced a chimera, did not separate. Larvae and chimeras re- mained alive during the 17 days of observation in the first larval experiment, and in the second, they remained alive for 39 days of observation. Isografts conducted between fragments of adult Chal- inula sp., fused within three to ten days, whether the con- tact zone was between exopinacoderms (parabiotic grafts) or between mesohyles (Fig. 2). Fusion was characterized by a continuum of the choanosome, with no apparent boundary at the grafted zone. These fragments remained fused over 3 months of observation. When allografting was performed between fragments taken from the same sponges that had been used for the isografting, no fusion was observed between the 20 allografts (Fig. 3). Scanning electron micrographs of allografts attached at the internal (choanosomal) zone of the fragments, revealed that within 3 d, a gap of about 100 ^m was formed between the frag- ments, with spicules erected toward this zone (Fig. 3b, 3c). Each fragment developed a pinacoderm at the grafted area, with a separation between them (Fig. 3d). No ag- gressive interactions were observed between the nonfusing fragments in the allografts. When parabiotic grafts were employed (20 pairs), both fragments remained intact, but fusion did not occur, nor was any rejection phenomenon observed over the three months of the experiment. Discussion The existence of self/nonself recognition among adult ( 'halinula sp. is strongly indicated in this study. Fusion between all fragments involved in isografts occurred, re- gardless of the grafting method used (parabiotic versus implant grafts), establishing that members of this species can fuse isogeneically. However, when grafts were made between allogeneic fragments of the same individuals used in isografting, fusion did not occur in any of the paired fragments. Chalinula sp. larvae have a statistically significant ten- dency to aggregate. Molecules termed aggregating factors occur in some sponges (Moscona, 1968), and such mol- ecules are known to facilitate species-specific and non- Table I Occurrence of fusion between larvae taken from the same Chalinula s/> individual # Larvae in dish # Dishes % Dishes with fusion % Fused larvae # Chimeras of 2 3 4 5 Larvae 7 8 37.5 37.5 3 3 4 50.0 41.7 1 1 4 27 55.6 30.6 12 3 5 4 75.0 65.0 2 1 6 3 33.3 11.1 1 7 3 100.0 71.4 2 1 > 9 1 100.0 66.7 2 10 2 100.0 95.0 d 5 1 Total 52 57.8 44.2 19 12 282 M. II.AN AND Y. LOYA Figure I. Cliulinulu sp. larval and post-larval fusion, (a) A pair of fused larvae, 2 h after initial contact, (h) A pair of fused larvae. 24 h after fusion, start to fuse with a third larva, (c) Fusion of two post-larvae. Fusion followed their settlement in proximity, (d) A new larva starts to settle on and fuse with a two-day old post-larva. In all the light micrographs, arrows indicate the contact zone (scale bar = 200 ^m). specific reaggregation of dissociated sponge cells (reviewed by Muller, 1982; Coombe and Parish, 1988). The fusion of larvae derived from the same parent may have occurred for several reasons: ( 1 ) the larvae may lack a capacity for self/nonself discrimination; (2) they may possess such discrimination, but may also express an in- hibition of the rejection mechanism; and (3) the larvae may have been genetically identical (products of parthe- nogenetic reproduction), thus resulting in the fusion of grafts that were actually isografts and not allografts. The last cause of larval fusion is less likely, because the larvae were taken from sponges 10 to 300 m apart from each other in their natural habitat; therefore they were probably genetically different. Thus, the larvae that fused in the experiments were probably genetically different, even if asexual development of larvae occurs in Chalinula sp., which is unlikely (Ilan and Loya, 1990). These results differ from the situation reported by Van de Vyver and Willenz (1975), who studied the freshwater sponge Ephy- datia fluviatilis and described larval fusion as occurring only between larvae belonging to the same strain. If indeed larvae were incapable of self/nonself discrim- ination, the results with adult grafting indicate acquisition of this capability during ontogenesis. Juvenile immuno- logical incompetence is well known among vertebrates (Cooper, 1 976) and has been suggested also for corals (e.g., Duerden, 1902; Lang, 1971; Hidaka, 1985) and hydroids (Teissier, 1929; Schijfsma. 1939). The tendency ofChal- SPONGE: HISTOCOMPATIBILITY RESPONSES 283 Figure 2. Chalinula sp. adult isograft viewed through scanning electron microscope, (a) Two complete grafted fragments, with arrows indicating toward the fusion at the contact zone (scale bar = I mm), (h) Higher magnification of the contact zone shows a continuum of cells (scale bar = 100 nm). hntla sp. larvae to form intraspecific aggregates demon- strates, however, some recognition capacity (though only on the species level). Lack of reaction against nonself might have been due to a lag period after which a rejection, separation, or resorption of one partner in the chimera by the other could have occurred, as is known for tunicates (Scofield ct ai, 1982; Rinkevich and Weissman, 1987, 1989). The present study indicates that if any lag period exists, it must be at least 17-39 days long. However, a process that cannot be excluded is cell lineage competition (Buss, 1982), in which cells with different genotypes within one body may compete for position in the germ line. Fi- nally, another option is that, although capable of differ- entiating self from nonself, larvae were unable to inhibit fusion due to lack or inactivation of a rejection mecha- nism, a situation analogous to self-tolerance in vertebrate T or B-cells (e.g., Basten. 1989; Nossal, 1989; Schwartz, 1989). Conspecific larval aggregation by Chalinula sp., fol- lowed by fusion with no rejection raises the question; what are the benefits of creating chimeras during larval or early post-larval stages, in contrast to the possible disadvantages (reflected by allograft incompatibility) of having chimeras at the adult stage? In the juvenile stage, the most important advantage may be the chimera size, which is larger than any of the individuals that created it. Small body size in marine invertebrates is often accompanied by high mor- tality, whereas larger size results in higher survivorship (t',?.. Loya. 1976; Ayling, 1980; Hughes and Connell, 1987). Hence, individuals that fuse, forming a chimera, immediately increase their total size and probably also their survivorship. Another possible benefit suggested for chimeras is early reproduction, because sexual maturity is also often size-related (reviewed in Harvell and Gros- berg, 1988). Thus, reducing generation length may yield an increasing number of offspring per unit time, compared with a similar genotype having a longer generation time. Buss ( 1982) argued that chimeras might be advantageous if there is mixing of cells from all partner genotypes. Chi- meras, being larger are more likely to suffer partial- rather than whole-colony mortality, with surviving cells bearing all genotypes. Finally, having a compound genotype, a chimera may gain more physiological resistance to dif- ferent environmental conditions than any of its members separately (Buss, 1982; Grosberg and Quinn, 1986). Most of the proposed benefits (except for physiological resistance) are consequences of larger body size of a chi- mera versus its members. Therefore, adults, which have already reached a substantial size, do not need to fuse with others to raise their survivorship or to reduce the 284 M. ILAN AND V. LOYA Figure 3. Chalinula sp. adull allografts, made by placing together the mesohyl of two fragments from different individuals and viewed through a scanning electron microscope, (a) Two allogeneic fragments. Arrow indicates the grafted area (scale bar = 1 mm), (b) Fragments A and B with a gap at the contact zone (scale bar = 100 ^m). (c) Higher magnification, reveals spicules erected toward the contact zone, presumably due lo cell disappearance from this area (scale bar = 100 ^m). (d) Formation of a pinacoderm (P) layer by each fragment at the contact zone (scale bar = 30 ^m). time to onset of reproduction, which they have already started. We suggest, therefore, that the nonfusion of adult Chalinula sp. evolved because the disadvantages and risks involved are not outweighed by the chimeric benefits of larger total size. Considering the costs of participating in a chimera, we first assume that an organism acts to maintain its integrity, in order to pass on its genotype to the next generation. Several potential deleterious consequences of creating chimeras were proposed in the literature. Buss (1982, 1983) suggested a possible parasitism by one member of a chimera on the other: by differentiating its germ cells to gametes, it would take advantage of the other member's investment in somatic tissues for maintenance. Other workers have demonstrated oriented translocation of ma- terial in coral chimeras (Rinkevich and Loya, 1983), pos- sible transmission of pathogens (Buss, 1982), or in an as- cidian, total resorption of one member's soma by its part- ner, under laboratory mariculture (Rinkevich and Weissman, 1987. 1989). In this study, although all the chimeras survived at least 17 days, the fate of the cells of each partner was not determined. Chalinula sp. larval fusion has been observed exper- imentally in this study in the laboratory. However, its frequency in nature is unknown. Theoretically, the chances of contact between Chalinula sp. larvae from different sources in the field are small. Its year-round reproductive pattern (Ilan and Loya, 1990) leads to a small number of free-swimming larvae in the popula- tion at any given time. This fact, together with the large distance between adult colonies, relative to larval size, plus rapid larval settlement ( 1 to 8 h after release) (Ilan and Loya, 1990), contributes to the low probability of larval contact. Nonetheless, larvae brooded in the same sponge, even if they are genetically different (produced sexually), may overcome most of the barriers to larval fusion in the field. Such larvae are in close proximity and. if spawned synchronously, may settle together and SPONGE HISTOCOMPATiBILITY RESPONSES 285 fuse. Fusion between kin larvae may provide an addi- tional selective advantage. Kin larvae partially share genotypes, therefore the survival of each is a partial success for the other genotype. Thus, if larval and newly post-larval chimeras of Chalinnla sp. do occur in the field, we assume they will be primarily among kin. Acknowledgments We are grateful to B. Rinkevich for many valuable dis- cussions. We thank R. Seggev Ben-Hillel who helped in maintaining the larvae, and Z. Goldberg for assistance in the field work. A. Colorni and A. Shoob took the light microscope photographs. Y. Delarea and F. Skandrani helped with the electron microscope. We appreciate the time and effort spent by T. P. Hughes and B. Rinkevich and two anonymous reviewers in critically reading earlier versions of the article. 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Cabrie et 0.07 in all cases). In the following discussion, the term "global" refers to the scaling exponent for all data combined, while "spe- Table I ParaiiH'lcn iwith e s ' A) Protein mass Culture Mo (asymptotic 95% CI) g (asymptotic 95% CI) LI 0.962(0.195-1.72) 0.279 (0.077-0.482) L2 0.560(0.431-0.689) 0.342 (0.303-0.380) Ml 1.06 (0.52-1.60) 0.479 (0.363-0.595) M2 0.626 (0.256-0.995) 0.560 (0.466-0.652) HI 0.881 (0.782-0.979) 0.826 (0.802-0.850) H2 1.050(0.54-1.56) 0.495 (0.387-0.605) B) Dry mass Culture M,, (asymptotic 95% CI) g (asymptotic 95% CI) LI 12.5 (7.94-14.3) 0.110(0.036-0.256) L2 5.81 (3.15-9.09) 0.305(0.180-0.429) Ml 7.84 (5.92-9.82) 0.399(0.298-0.501) M2 3.38 (2.74-4.04) 0.524 (0.469-0.587) HI 5.83 (3.40-9.84) 0.783 (0.708-0.859) H2 5.80 (3.66-7.94) 0.509 (0.363-0.656) * M,, is initial mass (fig). M, is mass at time t. and t is time in days. Culture treatments are concentrations of the alga Dunaliella salina (L = 2000 cells- mr 1 , M = 10, 000 cells- ml"' and H = 50.000 cells- ml' 1 ). 292 J. A. BERGES ET AL o 'E 10.00 CO CJ too 100.0 10000 moss (fj.g) L 100.00 o E _ 010 10.0 100.0 10000 OL1 L2 DM2 < AH1 A H2 mass (fj.g) c Z! 1 00- i o 100 mass D o 10000 E OL1 DM2 .< AMI A H2 E 100 1000 10000 nass (/xg) 100 100.0 mass (/tig) F 0- !> O OL1 L2 DM2 mu2 A HI AH2 G 100 1000 10000 mass Figure 5. Log-transformed enzyme activity versus log-transformed dry mass and protein mass for A, citrate synthase (CSV, B. lactate dehydrogenase (LDH); C. pyruvate kinase (PK): D. alanme aminotransferase (ala AT): E. aspartate aminotransferase (asp AT); F, glutamate dehydrogenase (GDH): and G. glucose-6- GROWTH. SIZE. AND ENZYMES IN ARTEMIA 293 cific" refers to scaling relationships in individual culture treatments (see Table II). CS activity scaled to a global exponent of 1.13 based on dry mass. Specific exponents were generally near 1 .0, except culture L 1 . which was significantly higher than 1 .0 (P < 0.02). Tests revealed significantly different slopes between cultures (P < 0.03). LSD tests identified LI as the sole distinct culture. In terms of protein mass, the global exponent was 0.864, significantly different from 1.0 and 0.75. As observed for dry mass data, scaling ex- ponents of LI and H2 cultures were significantly different from those of other cultures. LDH scaled to a global exponent of 1 .07 for dry mass data and 0.812 for protein data. Significant differences in specific scaling exponents were found for dry mass data (P < 0.01), and for protein data (P < 0.01). However, only L 1 and H2 cultures were significantly different when examined with LSD tests. PK. data yielded global scaling exponents of 1.56 for dry mass, and 1.18 for protein mass, both significantly higher than 1.0. Significant differences were detected in both the dry mass (P < 0.01) and protein mass data (P < 0.01). LSD tests showed culture LI to be distinct. Asp AT activity scaled to a global exponent generally higher than 1 .0 ( 1 .2 1 ) for dry mass, yet lower than unity (0.94) for protein mass. Heterogeneity of scaling exponents was found in both cases: P < 0.01 for dry mass, P < 0.01 for protein, with LSD tests identifying H2 and L2 cultures as significantly different from others. For ala AT activity, the global scaling exponent was 0.984 for dry mass (not significantly different from 1.0, P < 0.67). but 0.747 (significantly lower than 1 .0, but not different from 0.75 (P < 0.01, P < 0.91. respectively) for protein data. In both cases, specific scaling exponents were not significantly different from one another (P < 0.39 for dry mass. P < 0.39 for protein). GDH activity scaled to a global exponent of 1.28 for dry mass, significantly higher than 1.0 (P < 0.01); and 0.978 for protein data, not significantly different from 1 .0 (P < 0.45). As was the case for CS activity, specific slope differences were detected for dry mass (P < 0.02) and protein mass (P < 0.03). LSD tests showed that only cul- ture H2 was significantly different. NDPK data were more variable with respect to specific scaling exponents for both protein and dry mass than for any other enzyme. The global exponent for dry mass was 1.24, significantly greater than 1.0 (P < 0.01). Specific exponents varied from 0.327 to 2.06 and significant dif- . D_ O - : Q_ Q OL1 DU1 AMI 300 500 mass (fj,g) Figure 6. A. Log-transformed nucleoside diphosphate kinase (NDPK) activity versus log-transformed dry mass and protein mass in Anemia Iranciscana SFB under different culture treatments (L = 2000, M = 10,000 and H = 50.000 cells -ml" 1 of Dunaliella salina) for trial 1 data. Solid line represents protein mass regression, dashed line represents dry mass regression. Data points are presented only for protein mass data. Linear regression parameters are given in Table II. B. Linear plot of the same data, dry mass only. ferences were found (P < 0.01 ). A similar picture emerged from protein mass analysis; the global exponent of 0.942 (not significantly different from 1.0, P < 0.35) had a spe- cific range of 0.189 to 1.57 (significant differences found, P < 0.01). LSD tests identified four groupings for both protein and dry mass data; LI and L2 were distinct, while HI and Ml fell together, and H2 and M2 formed another grouping. For G6Pdh. activity scaled to a global exponent of 1 .26 for dry mass (significantly greater than 1.0, P < 0.01) and 0.961 for protein mass (not significantly different from 1.0, P > 0.21). The specific scaling exponents were not phosphate dehydrogenase (G6Pdh) in Anemia frantiscana SFB under different culture treatments (L = 2000, M = 10.000 and H = 50.000 cells- ml"' of Dunaliella salina). Solid line represents protein mass regression, dashed line represents dry mass regression. Data points are presented only for protein mass data. Linear regression parameters are given in Table II. 294 J. A. BERGES ET AL. I'tirameters (S,E.M.) for renown for Artemia franciscana Sh'B under different culture treatments Table II / of cniymc activity I Units- animat~') versus mass ln.t;i Protein mass Dry mass Enzyme Cullure log a b Ho test log a b Ho lest r 2 "90% of larvae exposed to 310 ^M NH, responding within less than 5 min. After 15 to 30 min, larvae become habituated to NH 3 and resume swimming so that the percent exhib- iting settlement behavior after 30 min is <10%. Other weak bases, such as methylamine and trimethylamine, induce similar behavior suggesting that NH 3 acts by in- creasing intracellular pH. Evidence that NH, and L-3,4- dihyrodxyphenylalanine (L-DOPA) induce settlement behavior through different mechanisms is presented. Ammonia may be a natural environmental cue that promotes oyster settlement behavior and, ultimately, recruitment. Introduction Many marine invertebrates, including oysters, have planktonic larvae that are recruited preferentially to hab- itats suitable for subsequent survival (Thorson, 1950). Recruitment of invertebrate larvae often involves a ste- reotyped series of search and crawl behaviors that is called settlement, followed by a morphogenetic phase called Received 4 June 1990: accepted 25 September 1990. Contribution #141 from the Center of Biotechnology. Manne Bio- technology Institute, University of Maryland. metamorphosis (Burke, 1983). The settlement behavior of oyster larvae has been well characterized and includes swimming with the foot extended forward followed by a series of increasingly localized crawling maneuvers (Pry- therch, 1932; Cranfield. 1973; Coon et a!., 1985). If the habitat in which the larva has settled is suitable, the larva will cement permanently to the substratum and meta- morphose. Settlement is reversible and does not neces- sarily culminate in metamorphosis once initiated; if the habitat is unsuitable, the larva may resume swimming and repeat the process elsewhere. Invertebrate larvae are often induced to settle and me- tamorphose by environmental cues, typically chemical, associated with the adult habitat (Crisp, 1974; Chia and Rice, 1978). Microbial films play an important role in the development of many invertebrate assemblages (Zobell and Allen. 1935: Meadows and Campbell, 1972; Schel- tema, 1974; Bonar et ai. 1986). Both soluble and surface- associated bacterial products are important in recruiting invertebrate larvae to surfaces containing bacterial films (Wilson, 1955; Scheltema, 1961; Gray, 1967; Muller, 1973; Neumann, 1979; Kirchman et ai, 1982), although some larvae prefer unfilmed surfaces (Crisp and Ryland, 1960). A bacterium, Altenmionas colwelliana (originally called LST), was found to enhance the recruitment of oyster larvae to colonized substrates (Weiner et ai. 1985; 1989). Supernatants from cultures of A. colwelliana, as well as other bacteria, contain one or more soluble factors that induce settlement behavior in oyster larvae of the genus Crassostrea. Preliminary studies showed that the soluble inducer has a low molecular weight (<300 dal- tons), and that supernatants have increased inductive po- tency commensurate with the age of the bacterial culture (Fitt etui. 1990). 297 298 S. L. COON ET I/ Experiments reported in this paper demonstrate that solutions of NH 4 C1 induce settlement behavior, and that NH,, not NH 4 + , is the active chemical species. Additional experiments further explore the relationship between the mechanism of NH 3 induction and induction of settlement behavior by L-3,4-dihydroxyphenylalanine (L-DOPA). another known soluble inducer of oyster settlement be- havior (Coon el nl.. 1985, 1990). Preliminary results of this work have been presented (Coon ct til., 1988; Bonar el al, 1990). Materials and Methods Obtaining ami maintaining larvae Larvae of the Pacific oyster, Crassostrea gigas, were obtained from the Coast Oyster Company of Quilcene, Washington, and maintained in the laboratory (Coon el al., 1990). Larvae were used within one week of arrival. Bioassay procedure Experiments were conducted as previously described (Coon ft al.. 1990). Aliquots of 20-50 larvae were assayed in 24-well tissue culture plates (Falcon #3047) in a final volume of 1.0 ml. Antibiotics were not used, but all ex- periments were conducted in 0.2 ^m filtered seawater. Each treatment was duplicated or triplicated, and results are expressed as the mean standard error. All chemicals were obtained from Sigma Chemical Company (St. Louis, Missouri). Larval settlement behavior was defined as in Coon et al. (1990), the basic criterion being active foot extension beyond the ventral margin of the shell. Behavior in each well was monitored with a dissecting microscope for 30 s at the times noted. The length of each experiment was between 30 and 40 min, as noted. Statistical tests were performed on arcsine-transformed data using a one-way analysis of variance (ANOVA) within time points. The ANOVA was followed by a Stu- dent-Newman-Keuls pair-wise comparisons test when significant differences were detected (Zar, 1974). Differ- ences were considered significant if P < 0.05. Effects of ammonia, ammonium, ami ptf Larvae were exposed to a range of concentrations of NH 4 C1, in the first series of experiments. Stock solutions of NH 4 C1 were made in seawater at twice the final con- centration and adjusted to pH = 8.0 with NaOH. At the beginning of each bioassay, 0.5 ml of stock solution was added to an equal volume of seawater (pH = 8.0) con- taining swimming larvae. This experiment was repeated using (NH 4 ) 2 SO 4 and other chloride salts (NaCl, KC1) at concentrations up to 10 mM. Approximately 96% of the total (NH, + NH 4 + ) in sea- water at pH = 8.0 is present as the ammonium ion. NH 4 + (Bower and Bidwell. 1978). To determine whether NH, or NHj" 1 was the active chemical species, larval settlement responses were observed while the concentrations of NH 3 and NH 4 + were varied under two different regimes. In the first, pH was held constant at 8.0 and the total (NH 3 + NH 4 + ), as NH 4 C1, was varied as described for the initial experiments above. In the second regime, total (NH 3 + NH 4 4 ) was held constant at 5.0 mM and the proportion of NH 3 to NH 4 + was varied by altering the pH. The ab- solute concentrations of NH 3 and NH 4 + were calculated by means of a hydrolysis constant for ammonium ion in seawater of pK a s = 9.39. at 30% salinity, 23C and 1 atm pressure (Bower and Bidwell, 1978). Because ammonia is a weak base (pK a = 9.25), its effects might result from an increase in intracellular pH (pH,). Therefore, two other weak bases, methylamine (pK a = 10.7) and trimethylamine (pK a = 9.8 1 ), were tested for their ability to induce settlement behavior. The inductive activities of these two compounds, along with those of NH 4 C1, were investigated according to the original pro- tocol described above; concentration was varied while the pH remained constant at 8.0. Relationship ofNH 3 -inditciion to L-DOPA-induction of settlement behavior To determine whether NH 3 and L-DOPA induce set- tlement behavior through the same mechanisms, we tested sulpiride, a dopaminergic receptor antagonist (Stoof and Kebabian, 1984) and potent inhibitor of L-DOPA-in- duced settlement behavior (Coon and Bonar, 1987), for its ability to block NHi-induced settlement behavior. Ammonium chloride stock solutions were made 10 times the final concentration in filtered seawater and adjusted to pH = 8.0. Solutions of L-DOPA and sulpiride were made 10 times their final concentrations in 0.002 N HC1. All larvae were pre-incubated in either 100 nM sulpiride or seawater for 12 min. then exposed to either 10 mM NH 4 C1 or 100 ftM L-DOPA. The effects of seawater and 0.002 A' HC1 were appropriately controlled. The pH of the final solutions was 7.7, yielding a calculated NH 3 con- centration of 270 nAf. In other experiments, larvae that were "habituated" to NH 3 (see Results) were tested to see whether they would still respond to L-DOPA. Larvae were exposed to 5.0 mM NH 4 C1 for 18 min until they began to habituate. They were then removed, rinsed, and exposed to either: ( 1 ) 100 nM L-DOPA; (2) fresh 5.0 mM NH 4 C1; (3) the NH 4 C1 solution from which they had just been removed; or (4) filtered seawater. Control groups were pre-exposed to fil- tered seawater instead of NH 4 C1, then rinsed and put in AMMONIA INDUCES OYSTER SETTLEMENT 299 o z K m x o LJ > UJ < > 100 ^- 80-- fi Li_ O UJ O < z LU O LU Q_ CD 60-- 40-- 20-- Concentrotion of NH 4 CI [NH-j] 7.9 [0.31] mM A A 6.3 [0.25] mM A A 5.0 [0.20] mM D D 4.0 [0.16] mM V V 2.5 [0.10] mM T V Contra 10 15 20 TIME (min) 25 30 Figure 1. Percentages of O90% within 5 min of exposure to an NH 4 C1 solution of 7.9 mA/at pH = 8.0. Responses to higher concentrations of NH 4 C1 are not shown because larvae in these solutions exhibited re- duced activity levels after short exposures. Between 2.5 and 7.9 mM (pH = 8.0), the larval response to NH 4 C1 was concentration dependent. As the NH 4 C1 concentra- tion increased, the percentage of larvae exhibiting settle- ment behavior increased, and the length of time required for the maximum percentage of larvae to respond de- creased. Following the maximum larval response, the percentage of larvae continuing to exhibit settlement be- havior rapidly declined so that 30 min after the initial exposure, almost all the larvae had "habituated" to the NH 4 C1 solutions and had resumed normal swimming. No subsequent metamorphosis was observed after 24 to 48 h. Larvae also exhibited high levels of settlement behavior in response to (NH 4 )iSO 4 , indicating that either NH, or NH 4 + was the active chemical species. This was corrob- orated by the observation that larval settlement behavior was not induced by Cl as NaCl or KC1 at concentra- tions comparable to inductive NH 4 C1 solutions (data not shown). Methylamine and trimethylamine, which are weak bases like NH 3 , induced high levels of oyster settle- ment behavior (Table I). The active species is AT/,- rather than AT// Ammonia has a pK a s of 9.39 in seawater and its cal- culated speciation as a function of pH is shown in Figure 2 A. As the pH of the NH 4 C1 solution decreases from 8.0 to 7.0, which is within the physiological tolerance range for oyster larvae, the NH, concentration changes much more dramatically (89.6% decrease) than the NH 4 + con- centration (3.6% increase) (Fig. 2B). Theoretically, the chemical species, NH^ or NH 4 + , to which the larvae are responding, would have the same dose-response curve, whether the concentrations are adjusted by varying the NH 4 C1 concentration under constant pH, or by keeping the NH 4 C1 concentration constant and varying the pH. This experiment shows that the maximal percentage of larvae exhibiting settlement behavior in response to NH 3 was independent of the regime used to vary the NH 3 con- centration (Fig. 3 A). The difference between these two curves represents less than 0. 1 pH unit, which was within experimental error. In contrast, the larval response to NH/ was highly dependent on the regime used to vary the NH 4 + concentration: the larval response increased with increasing NH 4 + concentration when the pH was held constant while the NH 4 C1 concentration was varied, but the larval response decreased with increasing NH/ con- centration when the NH 4 C1 was held constant while the pH was varied (Fig. 3B). These results indicate that, in these solutions, NH,. not NH/, was the active chemical species inducing settlement behavior in oyster larvae. and L-DOPA induce settlement behavior through different mechanisms The dopaminergic antagonist, sulpiride, blocked the ability of L-DOPA to induce settlement behavior (Fig. Table I Maximal percentage of oyster larvae exhibiting settlement behavior in response to exposure to weak bases at pH = 8.0 pK a 1 .0 mM 3.3 mM 10 mM NH 4 C1 9.25 3.2 0.4 51.2 4.0 90.3 2.3 Methylamine 10.7 4.4 1.4 47.6 14.2 94.6 2.4 Trimethylamine 9.81 19.7 7.6 91.6 5.4 Data are means of duplicates standard error. 300 S. L. COON ET AL. < o bJ O LU or LJ Q_ 100 80- 11.0 pH o od X Q. O a: u. UJ o X o 2U- pH D! -20- 0-0 NH 3 / NH 4 + / To P -40- / 7.8 -60- / 7.6 X 7.4 -80 7.2 ( 7.0 1 00 7 7.5 8 PH L-DOPA was added (data not shown). The small, tran- sient, increase in settlement behavior following transfer to a new NH 4 C1 solution was an artifact of the procedure. Discussion This study demonstrates that NH, in the surrounding medium induces oyster larvae to exhibit settlement be- havior. The onset of settlement behavior is rapid, high percentages of larvae are induced to behave, and the larvae quickly resume swimming without cementing to the plas- tic culture plates (a suboptimal settlement surface) in which the experiments were conducted. The results also indicate that, although NH, and L-DOPA induce similar settlement behaviors, the biochemical mechanisms by which they do so are different. pH DROP FROM pH 8.0: Figure 2. Calculated effect of pH on NH 3 and NH 4 * speciation. (A) Percentage contribution to the total (NH, + NH 4 + ) by each species as a function of pH. pK a 5 = 9.39. (B) Percentage change in NH 3 and NH 4 + as the pH of the solution drops from pH = 8.0 to the specified value. Actual calculated changes in speciation are tabulated for clarity. 4A) but did not block the ability of NH, to induce settle- ment behavior (Fig. 4B). However, two small effects of sulpiride on NH,-induced settlement behavior were noted: ( 1 ) settlement behavior was more rapidly expressed; and (2) the maximum percentage of larvae induced to exhibit settlement behavior was slightly lower (/-test; P < 0.1). The differential effects of sulpiride on the abilities of NH, and L-DOPA to induce settlement behavior indicate that NH, functions through a mechanism that does not require the dopaminergic receptor involved in the induction of settlement behavior by L-DOPA (Coon and Bonar, 1987). The effects of NH, and L-DOPA are not completely independent. Although larvae that had habituated to NH, were almost completely refractory to fresh NH,, they could still respond to L-DOPA (Fig. 5). However, fewer of these larvae exhibited settlement behavior, and they responded more slowly to L-DOPA than larvae that had not been habituated to NH,. The larvae also showed an attenuated response to L-DOPA in additional treatments during which they were left in the presence of NH, when % CHANGE 100- O s*\ -^ ~\~j~-~~- * AvJ^ ^--^ A A Vnru PNM Pll / A V varyLiNri^^ij / ^ NH, NH/ > 80- O O Vary pH / / -36.0 + 1.5 11 / / 1 -59.2 + 2.4 LJ CO 60- / -74.1 + 3.0 h- / -83.6 + 3.4 z i^ 40- / -89.6 + 3.6 J> / LU 1 / / 1 % |= 20- o-y LJ / ' cn / H/ sneci ition. (A) r *! n i , . . i . . t 99 IE x LJ LJ < ai LJ O LJ O a: LJ o_ 0.010 0.100 1-000 NH3 CONCENTRATION (mM) 100- 80- R - D Vary [NH 4 C1] 1 O O Vary pH 60- ) 40- [ / 20- ( } n ( D i 1 0.100 1.000 10.000 NH 4 + CONCENTRATION (mM) Figure 3. Maximal percentages of Crassostrea gigas larvae exhibiting settlement behavior as a function of the concentration of either NH, or NH 4 *. In one regime. pH was held constant while the NH 4 C1 concen- tration was varied (data calculated from Fig. 1); in the other regime. NH 4 C1 concentration was held constant while the pH was varied. The concentrations of NH, and NH 4 + were calculated from pH values and NH 4 C1 concentration. (A) Larval response as a function of the calculated concentrated of NH 3 under the two regimes. (B) Larval response as a function of NH 4 + under the two regimes. Data are means of duplicates. AMMONIA INDUCES OYSTER SETTLEMENT 301 100- o: O 80-- - Sulpiride + L-DOPA Sulpiride -A L-DOPA D_ TIME (min) Figure 4. Percentages of Ciuwnlmi xixax larvae exhibiting settle- ment behavior as a function of length of the duration of their exposure to NH, or L-DOPA in the presence of Sulpiride. Sulpiride is a dopa- minergic receptor antagonist. (A) Effect of sulpiride on the ability of L- DOPA to induce settlement behavior. (B) Effect of sulpiride on the ability of NH, to induce settlement behavior. Larvae were pre-incubated in sulpiride (100 it.M) or seawater for 12 min. then L-DOPA (100 n\I). NH 4 C1 (10 m.M). sulpinde ( 100 nAI) or seawater were added as indicated by the arrows. Data are means standard error of triplicates followed through time. For each time point, treatments with the same letter, or no letter are not significantly different from each other. Only statistics for relevant time points are shown for clarity. Ammonia, as a by-product of protein catabolism, is excreted by most marine bacteria and animals (Campbell. 1970; Billen. 1984). Therefore, in areas of high biological activity and reduced mixing (such as in boundary layers near surfaces). NH, might reach levels high enough to induce settlement behavior in oyster larvae. Total (NH, + NH/) concentrations of 10 mA/ have been reported in interstitial waters from marine sediments (Bruland, 1983). Stevens (1983) found that total (NH, + NH 4 + ) concentrations in association with oyster reefs may reach greater than 200 ^M in sediment waters and 3 nM in overlying waters 10 cm above the sediment interface. The presence of high levels of NH 3 in the environment, the rapid induction of settlement behavior bv NH,, and the quick reversibility of inductive effects of NH,, strongly suggest that NH, is a natural environmental cue for re- cruitment of oyster larvae. Its actual involvement in larval recruitment, however, has not yet been demonstrated. If NH, is a natural inducer of settlement behavior, then it must be a relatively non-specific indicator of biologically rich environments. Ammonia alone could not account for the specificity observed in natural oyster settlement and metamorphosis. We hypothesize that NH, acts as a chemokinetic agent that induces settlement behavior once a threshold concentration is encountered, bringing oyster larvae into contact with substrates and other potential contact-dependent and soluble cues (<./.' Crisp, 1974). Once settlement behavior has been initiated, oyster larvae rely on other cues from the environment to indicate that the habitat is suitable for cementation and metamorpho- sis. If these secondary cues are not present, the larvae habituate to NH, and swim away. This scenario is con- sistent with models and observations of oyster settlement (Prytherch, 1934; Cranfield, 1973; Coon et ai, 1985; Werner etui.. 1989; Coon cl ai. 1990). Although the mechanism by which NH, induces set- tlement behavior in oysters is unknown, NH, acts by in- creasing pH, in other invertebrate systems (Boron and DeWeer, 1976; Roos and Boron, 1981; Dube and Guer- rier, 1982; Ward eta/.. 1983; Bibring *>//., 1984; Williams et til.. 1984; Busa, 1986). Weak bases, such as NH,, raise pH, by penetrating the cell membrane as the uncharged O O NH 3 -- L-DOPA A ASW L-DOPA NH, NH-, A ASW-^NH-, Q_ 12 18 24 30 36 42 TIME (min) Figure 5. Percentages of Crassoslrea gigas larvae exhibiting settle- ment behavior as a function of length of time exposed to various regimes of NH 4 CI and L-DOPA. Larvae were exposed to either NH 4 Cl (5 mA/) or seawater for 18 min, then removed (downward pointing arrow) and put into either NH 4 C1 (5 mM) or L-DOPA ( 100 pM). Data are means standard error of triplicates followed through time. For each time point, treatments with the same letter, or no letter, are not significantly different from each other. Only statistics for relevant time points are shown for clarity. 302 S. L. COON ET \L species. then reprotonating in the cytoplasm (Roos and Boron. 1981). The induction of settlement behavior by other weak bases, such as methylamine and trimethyl- amine. is consistent with NH 3 acting by increased intra- cellular alkalization. An increase in pH, would not be expected to be cell-type specific and so may affect a diverse range of cell types in the larvae. Larvae of the hydroid, Hviiractinia, are induced to metamorphose by NH/, not NH 3 , through a mechanism that may involve regulation of intracellular transmethylation rather than pH, (Berking, 1988). Whatever its mode of action, induction of settlement behavior by NH 3 clearly involves a mechanism different from that of L-DOPA induction, though they are probably related. Ammonia and L-DOPA have different time courses. Larvae respond quickly to NH,, then soon ha- bituate to it; in contrast, larvae respond more slowly to L-DOPA, and the effects are longer lasting. Induction of settlement behavior by NH, is not mediated through the same dopaminergic receptors required for induction by L-DOPA, but is effected slightly by blocking these recep- tors with sulpiride. Conversely, larvae habituated to NH, can still respond to L-DOPA but to a lesser degree. There may be some interaction between pH, and signal trans- duction through the dopaminergic receptors. Further ex- periments are underway to resolve the mechanism of NH 3 - induction. Acknowledgments The authors gratefully acknowledge the Coast Oyster Company of Quilcene, Washington, for providing the oyster larvae for this study. This research was supported by the National Science Foundation (PCM 831678), Maryland Department of Natural Resources (S- 124-88- 008), Maryland Sea Grant (NOAA-NA-86AAD-SG006) and the Maryland Industrial Partnership Program (#119.23). Literature Cited Berking, S. 1988. Ammonia, tetramelhylammonium. barium and amiloride induce metamorphosis in the marine hydroid Hydractinia. Koux'x Arch. Dcv. Bid 197: 1-9. Bibring, I., J. Baxandall, and C. C. Harter. 1984. Sodium-dependent pH regulation in active sea urchin sperm. Dev. Biol. 101: 425-435. Billen, G. 1984. Heterotrophic utilization and regeneration of nitrogen. Pp 313-355 in Heterotrophic Activity in the Sea. J. E. Hobbie, and P. J. leB. Williams, eds.. Plenum Press, New York. Bonar, D. B., S. L. Coon, M. Walch, R. M. Weiner, and \V. Fitt. 1990. Control of oyster settlement and metamorphosis by endog- enous and exogenous chemical cues. Bull Mar. Set 46: 484-498. Bonar, D. B., R. M. Weiner, and R. R. Colwell. 1986. Microbial-in- vertebrate interactions and potential for biotechnology. Microb. Ecol 12: 101-110. Boron, XV. F., and P. DeXX'eer. 1976. Intracellular pH transients in squid giant axons caused by CO 2 . NH 3 , and metabolic inhibitors. J. (jen I'hysiol 67: 91-1 12. Bower, C. F., and J. P. Bidwell. 1978. lonization of ammonia in sea- water: effects of temperature, pH, and salinity. J Fish. Res Board Can 35: 1(112-1016. Bruland. K. XX'. 1983. Trace elements in seawater. Pp. 157-220 in C 'hcnucal Oceanography. J. P. Riley, and G. Skirrow. eds.. Academic Press, New York. Burke, R. D. 1983. The induction ol marine invertebrate larvae: stim- ulus and response. Can. J /.ool. 61: 1701-1719. Busa, XX'. B. 1986. Mechanisms and consequences of pH-mediated cell regulation. Aim Rev Plivsioi 48: 389-402. Campbell, .) . XX'., ed. 1 970. Comparative Biochemistry ol Nitrogen \le- tabo/iMii I I he Inveriehrates. Academic Press. New York. 493 pp. Chia, F.-S., and M. F. Rice, eds. 1978. Settlement and Metamorphosis of Marine Invertebrate Larvae. Elsevier. New York. 290 pp. Coon, S. L., and D. B. Bonar. 1987. The role of DOPA and dopamme in oyster settlement behavior. Am Zool 27: 128A. Coon, S. L., D. B. Bonar, and R. M. Weiner. 1985. Induction of set- tlement and metamorphosis of the Pacific oyster, Cruxsoxtreu gigus (Thunberg). by L-DOPA and catecholamines. J E.\p Mar Biol h'cul 94:211-221. Coon, S. L., XV. K. Fitt, and D. B. Bonar. 1990. Competence and delay of metamorphosis in the Pacific oyster. Crasxoslrea gigax. Mar. Biol. 106: 379-387. Coon, S. L., M. XValch, W. K. Fitt. D. B. Bonar, and R. M. Weiner. 1988. Induction of settlement behavior in oyster larvae by ammonia. Am. Zool. 28: 70A. Cranfield, II. J. 1973. Observations on the behavior of the pediveliger of Oxlrea edulis during attachment and cementing. Mar. Biol 22: 203-209. Crisp, D. J. 1974. Factors influencing the settlement of marine inver- tebrate larvae. Pp. 177-265 in Chemoreeeption in Marine Organisms, P. T. Grant, and A. M. Mackie, eds.. Academic Press, London. Crisp, D. J., and J.S. Ryland. I960. Influence of filming and of surface texture on the settlement of marine organisms. Nature 185: 1 19. Dube, F., and P. Guerrier. 1982. Activation of Barnea Candida (Mol- lusca. Pelecypoda) oocytes by sperm or KC1, but not by NH 4 CI, re- quires a calcium influx. Dev. Biol. 92: 408-417. Fitt, XX'. K., S. L. Coon, M. XX alch, R. M. XX einer, R. R. Colwell, and D. B. Bonar. 1990. Settlement behavior and metamorphosis of oyster larvae ofCrassoslrea gigax in response to bacterial supernatants. Mar Biol 106: 389-394. Gray, J. S. 1967. Substrate selection by the archianellid. Prolodnlus nihropharyngeiix Jagersten. Helgol. HV.v.s Mecresuniers. 15:253-269. Kirchman, D., S. Graham, D. Reish, and R. Mitchell. 1982. Bacteria induce settlement and metamorphosis of Janua (Dexiospira) braxi- IICHXIX Grube (Polychaeta: Spirorbidae). J. E\p. Mar. Biol. Ecol. 56: 153-163. Meadows, P. S., and J. I. Campbell. 1972. Habitat selection by aquatic invertebrates. Adv. Mar. Biol. 10: 271-382. Muller, XV. A. 197.3. Induction of metamorphosis by bacteria and ions in the planulae of Hydractinia echinata: an approach to the mode of action. Puhls. Selo Mar Biol Lab. 20: 195-208. Neumann, R. 1979. Bacterial induction of settlement and metamor- phosis in the planulae larvae of Cassiopea andromeda (Cnidana: Scyphozoa, Rhizostomeae). Mar. Eeol. Prog. Ser. 1: 21-28. Prytherch, II. F. 1934. The role of copper in the setting, metamorphosis and distribution of the American oyster, Ostrea virginica. Ecol. Mon- OKr 4: 45-107. Roos, A., and XV. F. Boron. 1981. Intracellular pH. Phyxiol. Rev 61: 296-434. AMMONIA INDUCES OYSTER SETTLEMENT 303 Scheltema, R.S. 1961. Metamorphosis of the veliger larvae of A'uw ni\ obsolctiix (Gastropoda) in response to bottom sediment. Biol. Bid! 120:92-109. Scheltema, R. S. 1974. Biological interactions determining larval settlement of marine invertebrates. Thalassia Jugoslav. 10: 263- 296. Stevens, S. A. 1983. Ecology of mtertidal oyster reefs: food, distribution and carbon/nutrient flow. Ph.D. dissertation, Unnersity of Georgia. 195pp. Sloof, J. C., and J. \V. Kebabian. 1984. Two dopaminc receptors: biochemistry, physiology and pharmacology. Lite Set- 35: 2281- 2296. Thorson, G. 1950. Reproductive and larval ecology of marine bottom invertebrates. Bin! Rev 25: 1-45. \\ard, S., E. Hogan, and G. A. Nelson. 1983. The initiation of sper- miogenesis in the nematode Caenorhabditis cUyans. Dcv. Bml 98: 70-79. \\ einer, R. M., A. M. Segall, and R. R. Colwcll. 1985. Characterization of a nianne bactenum associated with Ous.vni//vrni\ Savigny. / Mar. Biol. .-l.v.vof. V. A' 34: 531-543. /,ar, J. H. 1974. Biiuliili.'iiietil . l/;u/r.v/\ Prentice-Hall. New Jersey. 620 pp. /85% of laboratory-reared lar- vae of Strongylocentrotus droehacliiensis to metamor- phose. Larvae must contact live L. glaciate or its spores for metamorphosis to occur; the inducer is not sensed in the water column. However, aqueous extracts of L. gla- ciale can induce metamorphosis, suggesting that the in- ducing factor is chemical. Neither ashed nor boiled L. glaciate induces metamorphosis, indicating that the factor is heat-labile and that thigmotaxis, per se, is not important in the response. The amino-acid, -y-aminobutyric acid (GABA), which induces settlement of other marine in- vertebrate larvae, also induces significant rates of meta- morphosis of S. droebachiensis at concentrations > 1CT 4 M. A reduction (with antibiotics) in the number of live bacteria on the surface of L. glaciate does not affect the rate of metamorphosis of larvae. Introduction The larvae of a variety of benthic marine invertebrates are known to settle and metamorphose in response to coralline red algae, including: corals. Agaricia agaricites danai. A. agaricites humilis, and A. tenuifolia (Morse et ul ., 1988); chitons, Tonicella lineata (Barnes and Gonor, 1973), Mopalia nntscosa (Morse et at., 1979a), and Ka- thanna tunicata (Rumrill and Cameron, 1983); limpets. Acmaea testitdinalis (Steneck, 1982); trochid gastropods, Trochus niloticus (Heslinga, 1 98 1 ); abalone, Haliotis spp. (Shepherd. 1973; Morse et at.. 1979a, 1980a; Morse and Morse, 1984; Shepherd and Turner, 1985); tubeworms. Received 27 March 1990; accepted 25 September 1990. * Present address for correspondence and reprint requests: GIROQ. Departement de Biologic. Universite Laval. Ste. Foy. Quebec, Canada. G1K 7P4. Spirorhis corallimu'(de Silva, 1 962) and S. rupestris (Gee. 1965); sea urchins, Strongylocentrotus purpwatiis (Row- ley, 1989); and seastars, Acanthaster planci (Henderson and Lucas, 1971; Yamaguchi, 1973; Lucas and Jones, 1976) and Sticliaster cmstralis (Barker, 1977). The relationship between some grazers and coralline algae may be mutually beneficial, and the species may have co-evolved. For example, by preferentially settling and metamorphosing on crustose coralline algae, the aba- lone Haliotis rufescens gains obligate chemical cues for the induction of metamorphosis and further development, micro-refuges from predation, adequate food to support early growth (e.g., mucous exudates of the coralline alga, diatoms, bacteria, and other epiphytes), and camouflage (the red pigment of the coralline alga is incorporated into the shell of the developing abalone). In turn, the coralline alga is cleaned of epiphytic algae (which reduce photo- synthesis and can potentially kill the coralline) by the abalone's grazing activity ( Morse et at. , 1 980a; Morse and Morse, 1984). A similar, mutualistic relationship has been shown with the limpet, Acmaea testiidinalis, and the cor- alline alga. Clat/iromorpfium circumscription (Steneck. 1982). In the shallow rocky subtidal zone of north temperate oceans, strongylocentrotid sea urchins are generally as- sociated with coralline algal-dominated communities, de- scribed by various workers as "barren grounds" (Pearse et at., 1970; Lawrence, 1975), "Isoyake areas" (Hagen, 1983), or "coralline flats" (Ayling, 1981). In many cases, the destructive grazing of kelps and other fleshy macroal- gae by expanding populations of sea urchins has led to the establishment of these coralline communities, which are maintained by continued intensive grazing (see reviews by Lawrence, 1975; Lawrence and Sammarco, 1982; and Chapman, 1986). This has been well documented for Stwngvlocentrotus droebachiensis in eastern Canada 304 INDUCTION OF URCHIN METAMORPHOSIS 305 (Mann and Breen, 1972; Breen and Mann, 1976a, b; Lang and Mann. 1976; Mann, 1977; Breen, 1980; Chapman, 1981; Wharton and Mann, 1981), where the common shallow-water species of coralline algae are Clathromor- phuiu circuinscriptum. Corallina officinalis. l.ithotham- nion glaciate. Phymatolithon laevigutiim. and P. nigu- losiun. Recruitment ofS. droebachiensis (Langand Mann, 1 976; Wharton and Mann, 1981; Miller, 1 985; Scheibling, 1986) and other strongylocentrotid species (Pearse et at.. 1970; Tegner and Dayton. 1981) is lower in kelp beds than in coralline barren grounds, and selective settlement of sea urchin larvae on coralline substrata may account, at least in part, for these differences (Raymond and Schei- bling. 1987). In this study, we show that larvae of S droebachiensis are induced to settle and metamorphose in the presence of coralline algae. In a series of laboratory experiments with L. glaciate, we investigate the potential mechanism of settlement induction. We discuss the implications of this result to settlement patterns in the field. Scotia in June and July when larvae of S. droebachiensis are settling (Raymond and Scheibling, 1987). Fluorescent lighting provided a light intensity (at culture jar level) of Materials and Methods Larval rearing Adults of Strongylocentrotus droebachiensis were col- lected at 5-10 m depth at Sandy Cove (Digby County), Nova Scotia, Canada (44 29' N. 66 05' W). They were maintained in the laboratory in running seawater and fed kelp (Lanunaria tligitata and L. longicruris) at regular intervals. Gametes from adults of S. droebachiensis (50-85 mm test diameter) were obtained by peristomial injection of 2.5-4.0 ml of 0.53 M K.C1. Females shed their eggs into glass bowls of chilled 0.45 jim Millipore*-filtered seawater (hereafter referred to as filtered seawater); males shed sperm into dry, chilled bowls. After ~20 min of spawning, the eggs were rinsed three to four times with filtered sea- water. Several drops of sperm (checked under a micro- scope for motility) from one male were mixed with the eggs from one female for ~ 10 min. The eggs were then rinsed another three to four times with filtered seawater. Mean (SD) percentage of fertilized eggs, as judged by the presence of a fertilization membrane, was 99.4 0.6% (n = 15). Early-stage embryos were reared in standing cultures in small glass bowls for ~ 72- 120 h post-fertilization. When blastulae were seen swimming at the surface of the water, they were transferred to 4-1 glass jars containing ~3 1 of filtered seawater which was stirred constantly by T-paddles attached to 10-rpm motors. Larval densities, after the first week in stirred cultures, were maintained at <2 individuals ml" 1 . All culturing was carried out in fil- tered seawater at 10.8 1.4C (mean SD, n = 535), approximating ambient seawater temperatures off Nova 99.6 1 1.6 ME m V (mean SD, n = 3) on a 12 L:12 D photoperiod. Every second day (occasionally every third day) 50-75% of the culture water was removed by reverse filtration and replaced with fresh filtered seawater and microalgal food. The larvae were fed Dunaliella tertiolecta (a unicellular green alga) at a concentration of 1 x 10 4 cells ml ' of culture water. Algae were cultured at 23C under constant fluorescent illumination in f/2 nutrient medium (Guillard and Ryther, 1962). Only larvae that were deemed competent were used in experiments. Com- petence was indicated by the presence of large juvenile rudiments and a high rate of metamorphosis (>60%) in trial assays with coralline algae. The time from fertilization to competency ranged from 33 to 5 1 days. Experimental proti >c< >/.v For any experiment, only larvae from the same batch were used. If more than one culture jar of larvae was re- quired for an experiment, larvae from different jars were thoroughly mixed before allocation to treatments. Ex- periments were run in 250-ml glass jars with ~ 150 ml of filtered seawater and a test substratum or ~ 150 ml of a test solution. Five replicate jars (each with 25 larvae) were used per treatment (except where noted). Larvae were transferred into experimental jars with a syringe. Exper- iments were run for ~24 h (range: 24-28 h) in the same environmental chamber and at the same temperature and photoperiod as larval cultures. The light intensity at ex- perimental jar level (shaded during the light period) was 2.8 0.1 ^E rrrV (mean SD, n = 3). After 24 h, larvae and recently metamorphosed juve- niles were located in jars using a dissecting microscope and classified as: ( 1 ) free-swimming (larvae only), (2) on test alga (when an algal substratum was present), or (3) on bottom or sides of experimental jar. To facilitate the location of recently metamorphosed individuals (221-392 ^m test diameter) on coralline algal substrata, the follow- ing technique was used. After counting and removing any free-swimming larvae from ajar, the coralline algal sub- stratum was removed and immersed in an isotonic so- lution of MgCl in water (72 g mT 1 ) to narcotize any ju- veniles or larvae on the alga. These would then easily be displaced by gentle agitation or washing of the substratum. In some cases, 3-5 ml of buffered 10% formalin in sea- water were added to the MgCl samples so that counting could be postponed. Because counting was time consum- ing (requiring 2-14 h), replicates were set up in a com- pletely randomized block design, and blocks of treatments were counted in succession. However, only one experi- ment (with coralline algal extract) had a significant block 306 C. M. PEARCE AND R. E. SCHEIBLING effect, indicating that the majority of larvae that meta- morphosed did so during the experimental period and not during subsequent counting of individuals. Two controls were used for each experiment: ( 1 ) filtered seawater without any test substratum, to ensure that larvae were not metamorphosing in response to handling pro- cedures or other unknown factors, and (2) a cobble en- crusted with Lithoifiainninn glaciate (occasionally Phy- maioliilion iaevigatitiii or P. rugulosum), to assess the proportion of larvae capable of metamorphosing, because the rate of metamorphosis is generally maximal in re- sponse to coralline algae (see Results). The rate of metamorphosis was expressed as the num- ber of individuals metamorphosed divided by the total number of individuals recovered (n) (usually >90% of individuals were recovered). An individual was scored as metamorphosed if the larval arms had been resorbed and the globular test, tube feet, and spines of the juvenile were apparent. To compare the rate of metamorphosis in response to morphologically different types of coralline red algae. Corallina officinalis (finely branched, arborescent form). Lithot/uiinnion glaciate (rugose, crustose form), and Phy- inalolhhon laevigaium or P. rugulosum (smooth, crustose forms) (the latter two species were not distinguished and hereafter are referred to collectively as Phymatolithori) were collected subtidally at Eagle Head (44 04' N, 64 36' W) and Mill Cove (44 36' N, 64 04' W). Nova Scotia. Lithothamnion glaciate and Phynuitolitlion were collected as monocultures totally encrusting cobbles. Cobble sizes were: length, 34.6-56.4 mm; width, 24.0-44. 1 mm; height, 12.9-38.5 mm. Tufts of C. ofjicinalis, of similar dimen- sions, were presented upright in experiments. Algae were immediately transported to the laboratory in coolers where they were maintained in separate 91 X 61 X 45 cm fi- berglass aquaria with running seawater. All algae were carefully cleaned of epibionts and debris and thoroughly rinsed with filtered seawater prior to use in experiments. Lithothamnion glaciate and Phymatolithon also were scrubbed with a stiff plastic brush. To examine the effect of surface contour, in the absence of living tissue, on metamorphosis of 5. droehachiensis, L. glaciate was killed either by ashing at 500C for 4 h in a muffle furnace or by vigorous boiling in deionized water for two 15-min periods. Killed L. glaciate was washed in running seawater prior to experimental use (ashed for 7 days, boiled for 30 min). To test whether L. glaciate released a chemical into the water that could induce metamorphosis of free-swimming larvae, five cobbles encrusted with L. glaciate were placed in 2 1 of filtered seawater in the environmental chamber for 24 h. The L#/z0r/zam/0-conditioned filtered seawater was then decanted and used in an experiment with filtered seawater and L. glaciate controls. To test whether urchin larvae metamorphosed in re- sponse to a diffusion gradient of inducer molecules sur- rounding L. glaciate, treatments with Lithothamnion-en- crusted cobbles conducted under static and agitated (on a shaker table at 126 rpm) conditions were compared (this was the lowest possible speed of rotation capable of totally dispersing 1 ml of concentrated methylene blue dye in 150 ml of fresh water in under 10 min in test trials). To test whether a water-soluble extract of L. glaciate would induce metamorphosis of urchin larvae, fragments of the alga were chiselled off of cobbles, scrubbed with a brush, and washed with seawater. Four hundred grams of cleaned L. glaciate were finely ground up in 800 ml of filtered seawater (at 1 1-1 5C) with a mortar and pestle. The supernatant was decanted and refrigerated overnight at ~4C, then centrifuged at 27138 X gfor 10 min at 2- 3C to remove particulates. To test whether larvae re- sponded in a concentration-dependent manner, this su- pernatant was then serially diluted to 1:5, 1:10, 1:100, 1: 1000. 1:10,000. and 1:100,000 with filtered seawater. These dilutions were left overnight in the environmental chamber, and the following day 150 ml of each dilution were added to experimental jar replicates along with lar- vae. Protein concentration of the undiluted crude extract, as measured at the onset of the experiment using a Sigma Diagnostics micro-protein determination kit. was 305 Mg ml' 1 . The amino-acid neurotransmitter. -y-aminobutyric acid (GABA) is known to induce settlement of several benthic marine invertebrates including the chitons, Mopalia muxcosa (Morse et at., 1979a) and Katharina lunicala (Rumrill and Cameron, 1983), and several species of aba- lone of the genus Haliotis (c.f. Morse, 1984); GABA-mi- metic molecules, present in coralline red algae, have been shown to be the inducers of metamorphosis in H ntfescens (Morse el at., 1979a. b, 1980b: Morse and Morse, 1984; Morse, 1985). To test whether induction of metamor- phosis of urchin larvae by coralline algae could also be mimicked by GABA, solutions of GABA (obtained from the Sigma Chemical Company) were prepared in filtered seawater and tested for their ability to induce metamor- phosis of larvae of S. droebachiensis over the concentra- tion range of 10~ 7 -10~' A/, 10 larvae per replicate were tested. During an experiment, Lithothamnion glaciate occa- sionally released minute spores (mean diameter SD: 109 16 urn, n = 250) that were found on the bottom of jars. To test whether these spores could induce meta- morphosis of larvae, a Lithothamnion-encrusled cobble was placed in each of 20 experimental jars with filtered seawater for 78 h and the spores collected. The five jars with the most spores (>50) were rinsed three to four times with filtered seawater (spores stayed attached to glass) and INDUCTION OF URCHIN METAMORPHOSIS 307 XI). Figure 1. Mean percentage of Strongylocentrotus droebachiensis lar- vae that metamorphosed in response to filtered seawater (FSW) and the coralline red algae, Corallina ntlicmalis (CORA). I.iihi>lluiiiiiini xlactalc (LITH). and I'liviiiulnlithon (PHYM). Each treatment consists of 5 rep- licates with 25 larvae per replicate. Error bars indicate standard error. denotes that no metamorphosed individuals were found. used as a treatment in an experiment with filtered seawater and L. glaciale controls. To test whether a reduction in the number of live bac- teria on the surface of L. glaciale would reduce the rate of metamorphosis of larvae, five Lithothamnion-encrusted cobbles were scrubbed with a brush, rinsed with filtered seawater. and put in 1 1 of unfiltered seawater containing a mixture of penicillin and streptomycin (1000 units ml" 1 each). After 42 h, the cobbles were removed (with sterile gloves) and rinsed with filtered seawater to remove the antibiotics and dead bacteria. Bacterial samples were col- lected from three antibiotic-treated cobbles and three un- treated cobbles (which had been similarly scrubbed and rinsed) by swabbing a 1-cnr area twice (for 1 min each) with a cotton swab. The adherent material was suspended in 5 ml of artificial seawater and serially diluted in artificial seawater before being plated on marine agar plates. Bac- terial colonies were counted after 5 days of development at room temperature. After swabbing, the cobbles were placed in filtered seawater (antibiotic-treated and un- treated pieces in separate containers) and left overnight in the environmental chamber before use in the experi- ment. All statistical tests were carried out on arcsine-trans- formed data. This transformation helped to normalize the data and reduce heteroscedasticity. Replicates that had 0/n (no) or n/n (all) larvae metamorphosed were replaced with values of l/4n and l-l/4n, respectively, to improve the transformation (Bartlett. 1937). Normality wasjudged by examination of cumulative probability plots, and het- erogeneity of variances was assessed with Cochran's test ( = 0.01). All statistical analyses were carried out with the SYSTAT (Wilkinson, 1986) statistical computer package. Untransformed values are presented in graphs. Results Larvae of Strongylocentrotus droehacluensis showed similar, high rates of metamorphosis in response to three morphologically different coralline algae: Cora/Una offi- cinalis, Lithothamnion glaciale, and Phymatolithon (Fig. 1 ). Differences in mean rates among coralline treatments (range: 85-91%) were not statistically significant (F : , 2 = 0.45, P > 0.05), indicating that morphology does not affect metamorphic rate under static laboratory condi- tions. No larvae metamorphosed in a concurrent filtered seawater control, indicating the requirement for an ex- ternal cue. The mean rate of metamorphosis of S. droebachiensis in response to live L. glaciale did not differ significantly among different batches of larvae from different parentage (range: 62-98%, grand mean SE: 86.9 2.6%, n = 18) (Kruskal-Wallis test, P > 0.05). There also was no signif- icant difference among these batches of larvae in their response to concurrent filtered seawater controls (range: 0-10%, grand mean SE: 2.3 0.6%, n = 18) (Kruskal- Wallis test, P > 0.05). In experiments with /,. glaciale, killing the coralline alga markedly reduced the numbers of metamorphosing larvae (Fig. 2). The rate of metamorphosis with ashed L. glaciale was less than a tenth of that with live L. glaciale. although it was significantly greater than that in a filtered seawater control (Mann-Whitney U-test. P < 0.05). There was no significant difference in the rate of metamorphosis between the filtered seawater control and boiled L. glaciale (Mann-Whitney U-test, P > 0.05). Neither ashing nor boiling appeared to alter the macroscopic structure of L. glaciale. Metamorphosis in Lithothamnion-condiiioned filtered seawater was not significantly different from a filtered sea- water control (Mann-Whitney U-test, P> 0.05). indicat- ing that inducers are not leaking into surrounding seawater (Fig. 3). Thus, metamorphosis of urchin larvae in response to L. glaciale appears to require contact with the alga. The larvae are probably not responding to a diffusion gra- dient of inducer about live L. glaciale, since mild agitation 100 60. 40. 20- FSW BOIL ASH LIVE LITH LITH LITH Figure 2. Mean percentage of Strongylocentrotus droebachiensis lar- vae that metamorphosed in response to filtered seawater (FSW). boiled Lithothamnion glaciale (BOIL LITH). ashed L glaciale (ASH LITH). and live I. glaciate (LIVE LITH). Each treatment consists of 5 replicates with 25 larvae per replicate. Error bars indicate standard error. 308 C. M. PEARCE AND R. E. SCHE1BLING IUU - 80. 1 60- I 40. g 5 20. Q _ FSW LITH LITH COND FSW Figure 3. Mean percentage of Strongylocentrotus droebachiensis lar- vae that metamorphosed in response to filtered seawater (FSW). filtered seawater conditioned with live Lilholhanininn xlaaalc (LITH COND FSW), and L glaciate (LITH). Each treatment consists of 5 replicates with 25 larvae per replicate. Error bars indicate standard error. denotes that no metamorphosed individuals were found. ii ill EXTRACT DILUTION Figure 5. Mean percentage of Strongylocentrotus druchaclucnsis lar- vae that metamorphosed in response to serial dilutions of an extract of Lithnthamnion glaciate and to intact L. glacialeCLYTK). Each treatment consists of 5 replicates with 25 larvae per replicate. Error bars indicate standard error. (which would disrupt any such gradient) did not reduce the rate of metamorphosis with L. glaciate ( Mann-Whit- ney U-test, P> 0.05) (Fig. 4). This result provides further evidence of contact dependence. Induction of larval metamorphosis in S. droebachiensis by a crude extract of L. glaciate in filtered seawater was concentration-dependent (Fig. 5); high rates of metamor- phosis occurred at 1:5 (92%) and 1:10 (78%) dilutions, and these rates did not differ significantly from that with intact L. glaciate (88%) (Mann- Whitney U-test, P > 0.05 for both comparisons). Metamorphosis was minimal (<9%) at higher dilutions. The protein concentration of the algal extract within the range of effectiveness was be- tween ~30 ^g ml ' (1:10 dilution) and 60 ^g ml' ( 1:5 dilution). Induction by GABA also was concentration-dependent (Fig. 6). GABA induced larval metamorphosis at concen- trations > 10~ 3 M; the weakest concentration of GABA that induced metamorphosis in a proportion of larvae T T 80. n Slalic d Ag![aled(126rpm) t>0- 40 _ 20- r^-, - Figure 4. Mean percentage of Strongylocentrotus droebachiensis lar- vae that metamorphosed in response to filtered seawater (FSW) and Litliulhamniim glaciate (LITH) under static (light bars) and agitated (dark bars) conditions. Each treatment consists of 5 replicates with 25 larvae per replicate. Error bars indicate standard error. similar to that of a coralline algal control (Phymatolithon) was 10 3 M(Dunnett'stest. />> 0.05). About 20 juveniles that metamorphosed in response to GABA were placed in running seawater and observed for a period of about 2 weeks. They appeared normal and active during this time. Larvae metamorphosed in response to spores of L. gla- ciale adhering to the glass bottom of jars. The rate of metamorphosis in a treatment with spores was signifi- cantly higher than that in a filtered seawater control (Mann-Whitney U-test, P < 0.05), but significantly lower than with live L. glaciale (Mann-Whitney U-test, P <0.01) (Fig. 7). This latter result may be explained by the surface area covered by spores which was only a small fraction of that covered by the alga (spores: <1 mm 2 ; L. glaciale: >900 mirr). Settlement and metamorphosis in response to spores may have accounted for some of the |GABA](M) Figure 6. Mean percentage of Strongylocentrotus droebachiensis lar- vae that metamorphosed in response to various concentrations of 7- ammobutyric acid (GABA) and a Phymatolithon control (PHYM). Each treatment consists of 5 replicates with 10 larvae per replicate. Error bars indicate standard error. INDUCTION OF URCHIN METAMORPHOSIS 309 recently metamorphosed individuals found on the bottom and sides of experimental jars after treatment with live L. glaciate. In treatments with live L. glaciate (pooled from 21 experiments). 24.5 3.2% (mean SE) of all individ- uals were juveniles located on the bottom or sides of jars, whereas 60.9 3.6% (mean SE) were juveniles on the alga. Treating L. glaciate with antibiotics did not affect the rate of metamorphosis of 5. droebachiensis (Fig. 8), al- though live bacterial numbers were significantly reduced with antibiotics (mean SD, treated: 5.60 X 10 : 3.68 X 10' bacteria cm : , untreated: 9.86 X 10 4 3.65 X 10 4 bacteria cirT : ) (one-tailed /-test. P < 0.005). Discussion Under static laboratory conditions. Strongylocentrotus droebachiensis showed a high rate of metamorphosis in response to three different morphological types of coralline red algae: a finely branched erect form (Coral/inn ol/icin- alis), a rugose crust with short nubby branches (Lithoth- aninion glaciate), and a smooth crust (Phymatolithon). In the field, however, passive entrapment of larvae may result in higher settlement on the more structurally com- plex branched and rugose corallines than on relatively smooth crusts. Dense aggregations of juveniles of S. droe- bachiensis (Scheibling, pers. obs.) and other small inver- tebrates (Keats el at.. 1984) have been observed on C. qfficinalis in the field. In eastern Newfoundland, Keats et at. (1984) found that juveniles of S. droebachiensis (2-6 mm test diameter) were most abundant on L. glaciate and rare on Phymatolithon laengaliini. P rugulosum, and Clathromorphum circuniscripium (another smooth crust). However, the extent to which these observed distributions of juveniles in the field are determined by settlement pro- cesses or by differential mortality or migration is un- known. Flume experiments, examining settlement on al- gae with various morphologies, would be helpful in es- o < 60- 40. 20. FSW SPORE LITH Figure 7. Mean percentage of Strongylocentrotus droebachiensis lar- vae that metamorphosed in response to filtered seawater (FSW), spores of Lilholhanmion glacial? (SPORE), and L glaciate (LITH). Each treat- ment consists of 5 replicates with 25 larvae per replicate. Error bars indicate standard error. o o LITH ABT- LITH Figure 8. Mean percentage of Strongylocentrotus droebachiensis lar- vae that metamorphosed in response to filtered seawater (FSW). Lilli- othammon glaaale(U1W, and L glaciate treated with antibiotics (ABT- LITH). Each treatment consists of 5 replicates with 25 larvae per replicate. Error bars indicate standard error. tablishing the role of passive settlement in determining juvenile distribution patterns. Metamorphosis of the larvae of S. droebachiensis ap- pears to involve contact chemoreception. There is no ev- idence (from experiments with Lithothamnion-condi- tioned filtered seawater) that a chemical inducer is released into the water column (at least at concentrations that lar- vae can detect) or that larvae are responding to a diffusion gradient surrounding the alga. Boiling or ashing /.. glaciate inactivates the inducing factor, suggesting that the inducer of metamorphosis of S. droebachiensis is a heat-labile molecule. Because these treatments kill algal tissues but do not visibly alter the surface contour of L. glaciale [the term contour is used to indicate that the scale of roughness is larger than the larva itself (Crisp, 1976)], thigmotaxis per se is probably not important in initiating metamor- phosis. Although induction of metamorphosis of S. droeba- chiensis may require contact with L. glaciale. recently metamorphosed individuals were not always located on the alga. Because larvae can be induced to metamorphose by isolated algal spores, some of these juveniles may have metamorphosed directly upon contact with spores released from L. glaciale onto the glass bottom of the jars. Alter- natively, some larvae may land on the alga and receive a cue for metamorphosis, but then swim or crawl to adjacent areas before, or shortly after, metamorphosis. The latter phenomenon has been observed with the coral, Agaricia tenuifolia: the larvae require contact with the surface of crustose coralline algae to metamorphose, but subsequent attachment does not always occur directly on the algae (Morse et at.. 1988). In contrast, larvae of the abalone, Haliotis rufescens, settle and metamorphose exclusively on crustose coralline algae and not on adjacent non-algal surfaces (Morse et ai. 1980a). Aqueous extracts of L. glaciale can induce metamor- phosis of larvae of S. droebachiensis. indicating that 310 C. M PEARCE AND R. E. SCHEIBLING grinding releases a water-soluble chemical cue. Larvae of the sea urchin, Strongylocentrotus pitrpnratus (Rowley, 1 989), are induced to settle and metamorphose in response to the same small peptide inducer. purified from extracts of crustose coralline red algae (Lithothamnium cali/nr- nicum), that induces the larvae of // nitc\ccns to meta- morphose (Morse ct ai. 1984). These surface protein- linked oligopeptides have been demonstrated to be GABA-mimetic in their interaction with the larval recep- tors controlling metamorphosis of//, rufcsccns (Trapido- Rosenthal and Morse, 1986). GABA also triggers the metamorphosis of S. droebachiensis, but at higher con- centrations (10~ 4 -1CT 3 M range) than those recorded for //. rufescens (1(T 6 M) (Morse ct ai. 1980b). A metamorphosis-inducing factor may be produced by coralline algae per se or by some component of the mi- crobial film associated with these algae. Treating L. gla- ciate with antibiotics did not reduce the rate of meta- morphosis of S. droebachiensis, even though the number of live bacteria on the surface of the alga was reduced by two orders of magnitude. However, some residual bacteria or other microbes (such as diatoms and protozoa) unaf- fected by antibiotics may be responsible for the production of an inducing factor. Other laboratory studies of Strongylocentrotus spp. have shown that the larvae metamorphose in response to var- ious substrata besides coralline red algae. Larvae of 5. purpuratiis showed similar rates of metamorphosis on rocks covered with coralline red algae and those with a marine microbial film and no coralline algae (Cameron and Schroeter, 1980). Rowley (1989) found that coralline red algae and red algal turf induced similar numbers of larvae of S. purpuratiis to metamorphose, but that meta- morphosis was significantly lower with filmed rocks. We have observed a high rate of metamorphosis of larvae of .S. clroehachiensis in response to a variety of macroalgae, including non-coralline brown, green, and red algae, as well as microbial and algal films (Pearce and Scheibling. in prep.). Thus, although adults of S. droebachiensis are frequently associated with coralline substrata, the factors triggering metamorphosis are apparently not specific to coralline red algae. This suggests that selective settlement of S. droebachiensis in coralline algal barren grounds rather than kelp beds may be less important than factors that limit larval supply to kelp beds [e.g.. deflection of water currents by kelp plants (Jackson and Winant, 1983)], larval predation by planktivorous fish (Tegner and Dayton, 1981; Gaines and Roughgarden, 1987) and sus- pension feeders (Pearse el ai. 1970; Bernstein and Jung, 1979)], or early post-settlement survival (Cameron and Schroeter. 1980; Harris el ai. 1984; Rowley, 1989). Acknowledgments We thank T. 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Bull. 179: 312-325. (December. 1990) Associations Between Egg Capsule Morphology and Predation Among Populations of the Marine Gastropod, Nucella emarginata TIMOTHY A. RAWLINGS* Department of Zoology. University of British Columbia, I'ancouver, British Columbia, Canada, l'6T 2A9 Abstract. Intraspecific variation in the morphology of egg capsules is ideal for assessing the costs and benefits of encapsulation, yet little is known about the extent of such variation among populations of a single species. In the present study, I compared capsule morphology among three populations of the intertidal gastropod, Nucella emarginata. Significant differences were found both in capsule wall thickness and capsule strength. Mean capsule wall thickness varied as much as 25% among populations, with the dry weight of capsular cases differing accordingly. Capsule strength, measured as resistance to puncturing and squeezing forces, also varied among populations, but did not directly reflect differences in capsule wall thickness. Despite extensive variation in capsule morphology within this species, the number and size of eggs contained within capsules of equal volume did not differ significantly among populations. I also compared the type of capsule-eating predators that were present at each site. Shore crabs, Hemigrapsus spp., were abundant at all three sites; however, the pred- atory isopods Idotea wosnesenskii were only present at sites containing relatively thick-walled capsules. Although Hemigrapsus and Idotea were able to chew through both thick- and thin-walled capsules, laboratory experiments revealed that Idotea preferentially opened thin-walled capsules. These results suggest that variation in capsule morphology among populations of N. emarginata may, at least in part, reflect selection for the protection of em- bryos against predation. Received 9 May 1990: accepted 25 September 1990. * Present Address: Department of Zoology. University of Alberta. Ed- monton. Alberta. Canada. T6G 2E9. Introduction The confinement of developing embryos within elab- orate egg capsules is a common phenomenon among ma- rine invertebrates. Although this trait is widespread, few studies have addressed the benefits and costs associated with the production of encapsulating structures. Egg cap- sules may protect embryos from such environmental stresses as: predation (Pechenik, 1979; Perron, 1981), bacterial attack (Lord, 1986), osmotic changes (Pechenik, 1982, 1983; Hawkins and Hutchinson, 1988), desiccation (Spight, 1977; Pechenik, 1978), temperature shock (Spight, 1977; Pechenik, 1986), and wave action (Perron, 1981). Yet the ability of capsule walls to resist such stresses is known for only a few species (Emlen, 1966; Spight, 1977; Pechenik, 1978; 1982; 1983; Brenchley, 1982; Lord. 1986; Hawkins and Hutchinson, 1988), and only limited data are available on the survivorship of encapsulated embryos in the field (Spight, 1977; Pechenik, 1978; Bren- chley. 1982). The production of egg capsules must also have associated costs. Capsule walls can divert a substan- tial amount of energy away from the production of eggs (Perron, 1981) and may also limit the availability of ox- ygen and nutrients to encapsulated embryos (Strathmann and Chaffee, 1984). If the adaptive significance of encap- sulation is to be understood, these benefits and costs must be assessed. Encapsulation of developing embryos is widespread among the more advanced gastropods (Pechenik. 1986). Neogastropod mollusks enclose embryos within structur- ally complex proteinaceous capsules and attach these structures to firm substrata in the marine environment. Within this group, egg capsule morphology varies tre- mendously (e.g., Ostergaard, 1950; D'Asaro, 1970, 1988: Perron. 1981). Subtle differences in the properties of these 312 ,Y EMARGINATA EGG CAPSULES 313 capsules may reflect tradeoffs between benefits and costs of encapsulation (Perron, 1981; Perron and Corpuz. 1982). For instance. Perron ( 198 1 ) found that capsule wall strength, and the proportion of reproductive energy in- vested in capsular cases, varied among closely related spe- cies of Conns. These differences were directly related to the development time of encapsulated embryos, such that embryos with long-term development were enclosed in thicker, stronger, and energetically more expensive cap- sules than those with short-term development (Perron, 1981; Perron and Corpuz, 1982). Thus, energetic costs associated with the production of strong capsular cases may be compensated for by the benefits of increased em- bryonic protection in species with protracted intracapsular development. If variation in the morphology of egg capsules does re- flect tradeoffs associated with specific benefits and costs of encapsulation, then intraspecific variation in capsular structure offers an ideal opportunity to assess such benefits and costs. Unlike interspecific comparisons, which may be subject to potentially confounding phylogenetic effects, studies of variation within a species can determine the importance of ( 1 ) physical constraints in female body size, (2) phenotypic responses to variation in environmental conditions and, (3) genetic divergence resulting from se- lection, in accounting for differences in capsule mor- phology. In no studies, however, have the structures of egg capsules in different populations of a single species been compared, and little is known about the extent of intraspecific variation in capsule morphology. The widespread geographic distribution and direct de- velopment of the marine intertidal snail Niict'lla cmar- g/Hflta (Deshayes, 1839)(Prosobranchia: Muncidae) make this species an ideal candidate for studies of intraspecific variation in capsule morphology. These snails are com- mon inhabitants of rocky shores from California to Alaska and range across wide extremes in wave exposure. N. emargmata deposit eggs year-round within 6-10 mm-long vase-shaped capsules and attach these structures directly to the substratum. After approximately 80 days of encap- sulation (Emlen, 1966), embryos emerge as juvenile snails. Due to the absence of a planktonic larval stage in this species, gene flow among geographically separated habitats may be low (Palmer, 1 984). As a consequence, snail pop- ulations may have become adapted to localized environ- mental conditions. In this study, I examined variation in capsule mor- phology among three populations of A', emarginata sep- arated along a gradient of wave-exposure. I compared capsule size, protective quality (as determined by wall thickness and capsule strength measurements), and cap- sule contents, among these study populations. Intraspecific differences in capsule morphology were examined with respect to site differences in the presence and abundance of capsule-eating predators. Previous studies, such as those by Emlen (1966) and Spight ( 1977), have indicated that predation on encapsulated Nucella embryos can be severe. Materials and Methods Si ml v sites This study was conducted at the Bamfield Marine Sta- tion on the west coast of Vancouver Island, British Co- lumbia. Three study sites were established in Barkley Sound along a gradient of wave-exposure from sheltered to exposed: Grappler Inlet (4849'N, 12507'W), Ross Is- lets (4852'N, 12509'W), and Seppings Island (4850'N, 12512'W). Although no empirical studies have ranked these areas with respect to wave-exposure, my rating sys- tem, based on visual observations, corresponded with ex- posure scales used by others in the same geographic region (Austin eta/.. 1971; Kitching, 1976;Craik, 1980; Crothers, 1984). Intraspecific variation in capsule morphology Snail size and capsule size. To compare the size of capsules produced by snails from each site, I collected 1 00 snails from each study area in March 1988. Collections were made by removing all living Nucella emarginata in- dividuals from a given area, except for snails smaller than 10 mm in length, which were considered to be reproduc- tively immature. Snails were brought into the laboratory, measured for shell length (apex to tip of siphonal canal) with Vernier calipers, tagged for identification, and then placed in mesh-panelled plastic containers (32 X 26 X 12 cm). Approximately 40-50 snails were held in each con- tainer and provided with barnacles (Balaniis glandula) for food. Containers were kept submerged in seawater tanks and supplied with a continuous flow of fresh sea- water. A snail was recognized to be laying an egg capsule only if it was found molding a new capsule with its ventral pedal gland. Other capsules were considered to be part of the same clutch if they were laid within a few millimeters of the freshly spawned capsule and were similar in shape and orientation (see Gallardo, 1979). Five capsules were collected from each female, and then preserved in 5% formalin (in seawater) for subsequent measurement. Egg capsule proteins are known to be stable in this fixative (Hunt. 1971). I recorded the total capsule length, chamber length, and chamber width for each egg capsule. Total capsule length was the length of the capsule including the plug, but excluding the stalk, as the stalk length was known to be highly variable (Spight and Emlen, 1976). Chamber length and chamber width were measures of the maximum dimensions of the region housing developing embryos and 314 T. A. RAWLINGS nutritive nurse eggs. The volume of the capsule chamber was estimated from these measures using the formula for a prolate ellipsoid, V = 4/37r(a/2)(b/2) 2 , where a = cham- ber length and b = chamber width (Pechenik. 1982). Micromorphology o/'N. emarginata egg capsules. To examine the microstructure of N. enuirginala capsules from each site, representative capsules were collected from field populations and then sectioned (unfixed) along sag- ittal and transverse planes using a freeze microtome. The wall microstructure of these sections was viewed under a compound light microscope and interpreted with refer- ence to previous histological studies of the egg capsules of A', lapillus and other muricids (Bayne, 1968; Tamarin andCarriker, 1967; Sullivan and Maugel, 1984; D'Asaro, 1988). The thickness of capsule walls was examined by taking serial cross-sections down the length of the chamber. Capsules were always selected from separate clutches to ensure that at least some were laid by different snails. Representative capsules were then measured, emptied of all contents by removing the capsular plug, and individ- ually frozen on a freeze microtome. One section (10-12 nm thick) was taken at 10 percentile intervals along the length of the capsule chamber, starting at the opening of the plug region into the capsule chamber (0%) and ending at the base of the capsule chamber ( 100%). Sections were mounted in a seawater-soluble medium and then mea- sured using a compound microscope with a calibrated ocular micrometer. Eight measurements of wall thickness were taken at approximately equal intervals around the circumference of each capsule section. The average wall thickness within each section was used in all subsequent data analyses. To examine wall thickness differences among a large number of egg capsules, I established a laboratory pop- ulation of 60 snails (30 males; 30 females) from each site. For each population, five male and five female snails were allocated to one of six replicate mesh-panelled plastic containers (26 X 16.5 X 13 cm), and were maintained in the laboratory as described above. Every two weeks, freshly laid capsules were collected from each container and placed in small mesh-panelled vials. Vials were then la- belled, dated, and immersed in flowing seawater. Capsules required for experiments were selected by removing an equal number (whenever possible) from each replicate vial. Only relatively fresh capsules (within 4-weeks after deposition) were used in the following experiments, since capsule wall properties may change with age (see Roller and Stickle, 1988). To examine variation in capsule wall thickness within and among populations of N. emarginata. 30 capsules were selected from each laboratory population. Each cap- sule was marked at a point 70% along the length of the capsule chamber. Preliminary data on wall thickness variation within a capsule indicated that capsule walls were thinnest and least variable in this region. For sub- sequent measurement, capsules were frozen on a freeze microtome and then sectioned at the marked region. In addition, I examined variation in capsule wall thick- ness within and among clutches to determine whether females from the same population produced capsules of a similar wall thickness. For each laboratory population, egg capsules were collected from five different females by removing one clutch of capsules from each replicate con- tainer. Depending on clutch size, four to six capsules were selected from each clutch and then sectioned as described above. To determine whether intraspecific variation in capsule wall thickness resulted in differences in the total amount of material allocated to capsular cases, I compared the dry weights of capsular cases from each site. Represen- tative capsules were collected from each laboratory pop- ulation, measured, and emptied of all contents. Stalks were removed from capsules to minimize variability in weight among capsules. Capsules were rinsed twice in dis- tilled water, dried for 48 h at 75C. and then weighed to 0.01 mg. Capsule wall strength. I used two indices to measure the strength of egg capsule walls. The first index deter- mined the resistance of capsule walls to puncturing forces and was based on Perron's (1981) procedure for Conns egg capsules. Freshly laid capsules were collected from laboratory populations and marked at a point 70%. along the length of the capsule chamber. Capsules were then bisected by cutting along the two seams of the capsule chamber. Each capsule half was mounted individually between two pieces of Plexiglas (8.5 X 5 cm) and orien- tated such that a 1 mm diameter hole in each piece of Plexiglas was positioned directly over the marked region of the capsule chamber. A blunt-ended needle (0.36 mm 2 area), mounted beneath a flat weighing pan, was posi- tioned over the Plexiglas such that the needle was per- pendicular to the exposed capsule wall. Five-gram weights were sequentially loaded onto the weighing pan until the needle punctured the capsule wall. Each capsule half was punctured once. The mean puncturing force per capsule was used in all subsequent data analyses. The second index of capsule strength measured the force needed to squeeze the plug out of intact capsules. Shore crabs (Hemigrapsus spp.) often ruptured N. emar- ginata egg capsules in this way by squeezing them in their chelae. Individual capsules were glued to a metal plate, which was then bolted to a vertical piece of Plexiglas mounted with strain gauges. A second metal plate was attached to a spindle so that this plate could be hand- cranked towards the mounted capsule. A chart recorder provided a record of the force required to rupture each A'. EMARGINATA EGG CAPSULES 315 egg capsule. This system was calibrated with known weights. Capsule com cms. Egg capsule contents were examined to determine whether the number or size of eggs per cap- sule differed among populations with respect to differences in capsule structure. Eighteen freshly laid capsules were collected from each laboratory population. Each capsule was measured and then emptied of all contents. As it proved difficult to distinguish between early developing embryos and nurse eggs, no attempt was made to separate nurse eggs from embryos. Before egg counts were made, however, a few embryos from each capsule were examined to ensure that they had not advanced past the second veliger stage. At this stage, embryos are able to feed on nurse eggs (LeBoeuf. 1971; Lyons and Spight, 1973). Egg size was also compared among sites. As egg size was relatively constant within a capsule, only five eggs were sampled from each capsule. Length and width were measured for each egg using a compound microscope equipped with an ocular micrometer. As eggs were off- round in shape, volume was estimated by using the above formula for a prolate ellipsoid. Predation on N. emarginata egg cupsu/es Laboratory experiments. A variety of abundant inter- tidal organisms was collected from each field site to de- termine which species might prey on A', emarginata cap- sules (nemertineans: Emplectonema gracile: annelids: Nereis vexillosa: mollusks: Mopalia spp., Littorina scittnlata. Onchiclella horealis, Searlesia dim. Tegula fu- nebralis. Nueella emarginata: arthropods: Pagurus gra- nosimamis, P. hirsutiusculus, Hemigrapsus nitdits, Hemigrapsus oregonensis, hlotea wosnesenskii, Gnori- mosphaeroma oregonense, Cirolana harfordi: echino- derms: Leptasterias hexaetis, Pisasier ochraceus; chor- dates: Oligocotlits nwculosiis, Anoplarehus purpurescens). Groups of individuals from each species were placed in appropriately sized mesh-panelled vials (3X3X6 cm) or containers (8 X 8 X 10 cm or 20 X 20 X 10 cm), and were provided with intertidal shells or bare rocks for shel- ter. Containers were partially immersed in seawater tanks and provided with a continuous flow of fresh seawater. Test animals were starved for 24 h before being presented with 8 intact A', emarginata egg capsules. Capsules were mounted on small Hat rocks using a cyano-acrylate glue and arranged in a circular configuration. A predator was defined to have opened an egg capsule only if it ruptured or ate through the chamber containing developing em- bryos. Capsules were checked every 1 -2 days for evidence of predation, and experiments were continued for at least two weeks or until all capsules had been opened. Five to ten replicates, including controls consisting of cages with no predators, were conducted for each species. b'leld censuses oj predation. In May 1988, two transects ( 10 m in length) were established parallel to the shoreline at Grappler Inlet (4849'55"N; 12507'03"W) at tidal heights of 1 .2 and 2.2 m above extreme low water, spring [ELWS] (Canadian datum). Quadrats (0.25 m 2 ) were sampled at 0.5-1.0 m intervals along these transects to determine the abundance of A', enuirginata and their egg capsules. Egg capsules were categorized on the basis of whether the capsule chambers were intact or ruptured. The age of intact capsules was estimated by noting the developmental stage of the embryos. New capsules were identified by the presence of nurse eggs, while older cap- sules contained well-developed shelled embryos. Ruptured capsules were also examined to determine whether they had been attacked by predators or whether developing embryos had hatched naturally. If capsules were empty, but had been chewed into the capsule chamber, they were considered to have been opened by predators. Such cap- sules were described by distinctive bite marks left on the capsule walls (see Fig. 9 below). The abundance of po- tential predators (identified from laboratory studies) was also censused along each transect. In June 1988. three transects were established parallel to the rocky shoreline at the Ross Islets site (4852'12"N, 12509'36"W) at tidal heights of 1 .9, 2.3 and, 2.6 m above ELWS. The two highest transects were positioned along a steeply sloping granite wall sparsely covered with Fucus distic/ius. Balanus glandula, and Semibalanus cariosns. The lowest transect was set along a boulder-covered beach directly below the higher transects. Data were collected as described above for the Grappler Inlet site. A large rocky outcropping (2.8-3.1 m above ELWS) adjacent to the study site was also censused in August 1988. This 16 m 2 area was divided into six equal-sized grids and a 0.25 m : quadrat was thrown haphazardly into each region. Snail density, egg capsule density, and predator abundance were recorded. Censuses of snail density or predator abundance were not made at the Seppings Island site due to its extreme exposure to wave action. Capsule remains were regularly collected, however, to compare the type of predation among sites. Susceptibility of capsules to predators I also conducted a series of laboratory experiments to determine whether the intertidal isopods Idotea wosne- senskii could differentiate between thick- and thin-walled capsules. My field observations, and also those by Emlen ( 1966), indicated that these were important predators of Nueella egg capsules. As adult isopods were able to chew through all capsules regardless of wall thickness, I chose to compare the overall preferences of these predators for thick- and thin-walled capsules. 316 T. A. RAWLINGS O O SEPPINGS ROSS ISLETS krl 3RAPPLER INLET 10 1.5 2.0 2.5 30 3.5 40 SHELL ^ Figure 1 . Size-frequency histograms of the first 50 \uivlla emarximitu individuals collected from each study site in March 1988. Snails smaller than 1.0 cm in shell-length are not included. Wave-exposure levels were predicted to be lowest at Grappler Inlet, highest at Seppings Island, and intermediate at Ross Islets. Mean shell lengths of snails are 1.9, 2.1. and 2.7 cm for Seppings, Ross Islets, and Grappler Inlet populations, re- spectively. Adults ofldotea (mean length = 2. 1 cm) were collected from Grappler Inlet in November 1988. Groups of three hlotea were placed in mesh-panelled cages (8 X 8 X 10 cm), and were then partially immersed in trays of fresh seawater. Predators were starved for an initial period of 24 h and then given five capsules from each of two snail populations (10 capsules in total). Predator preferences were tested for (1) thick versus thin-walled capsules (Grappler vs. Ross, and Seppings vs. Ross) and, (2) thick- versus thick-walled capsules (Grappler vs. Seppings). Capsules were arranged in a circular configuration, such that capsules from each population were interspersed. The number of capsules opened was recorded daily. Experi- ments were terminated when 4-6 out of 10 capsules had been opened. Five to ten replicate cages were used for each experimental combination. Results Intnispeci/ic variation in capsule morphology Snail size and capsule size. Snail size varied considerably among sites, with mean shell length increasing from wave- exposed to wave-sheltered shores (Fig. 1). Snail size at re- productive maturity also varied among populations. The smallest snails to spawn were 1.7, 2.1, and 2.7 cm in shell length from Seppings, Ross, and Grappler sites respectively, even though laboratory populations greatly overlapped in size (Seppings 1.4-2.2 cm; Ross: 1.4-3.0 cm; Grappler: 1.8-3.5 cm). Differences in the size of mature females within and among populations were reflected in the length of capsules produced (Fig. 2). Within each population, larger snails laid significantly longer capsules than smaller snails. Among populations, this trend was also apparent, although Seppings snails produced disproportionately large capsules per unit shell length (ANCOVA for slopes; F = 1.20, P > 0.25; ANCOVA for elevations: F = 14.84; P < 0.001). Hence, capsule size differed markedly among sites. Micromorphology of N. emarginata egg capsules. Cap- sule walls of N. emarginata were composed of three lam- inae (L, , L 2 , and L 3 ; Fig. 3A,B) and were similar in struc- ture to the capsule walls of other muricids (Sullivan and Maugel, 1984; D'Asaro, 1988). All measurements of cap- sule wall thickness were taken from the thick middle lam- ina (L;), which consisted of a dense, fibrous middle layer (L 2b ), sandwiched between two transparent, homogeneous layers (L 2a and L 2c ). The outermost lamina (L,) was ex- tremely thin and often formed elaborate projections from the capsule wall. Consequently, this lamina was too dif- ficult to measure reliably. The innermost capsule lamina (L,) lined the capsule chamber and formed a transparent bag that enclosed developing embryos, nurse eggs, and intracapsular fluid. Sections in the apical region of the capsule indicated that this lamina was actually connected 10 ? 9 E I a 13 < O : O 1.5 1.9 2.3 2.7 3.1 SHELL LENGTH (cm) 3.5 Figure 2. Relationship between total capsule length (excluding stalk) and shell length for laboratory-laid capsules from three populations of .\itic/lu emarginata. Snails smaller than 1.7. 2.1. and 2.7 cm from Sep- pings. Ross Islets, and Grappler populations did not spawn. Least-squares linear regression equations for each site are: Seppings: Y = 3.185X - 0. 1 1 1. r = 0.548. n = 23; Ross Islets: Y = 3. 1 39X - 1 .232, r = 0.752. n = 23; Grappler Inlet: Y = 2.206X + 1.481. r = 0.423, n = 20. \ i:\llKiil\ll\ EGG CAPSULES 317 B . - D / - St Figure 3. Microstructure of Nucella cman>inahi egg capsules: (A), (B) transverse sections taken 70% along the chamber of a capsule from Ross Islets (mean thickness = 60 ^m) and Grappler Inlet (mean thickness = 90 ^m), respectively; (C) longitudinal section through the capsule stalk; (D) longitudinal section through the capsule plug. Outer (L,), middle (Li), and inner (L 3 ) capsule wall laminae are indicated, as are the three component layers (L ;abc ) of the middle lamina, although note the disappearance of L 2b and L 2c in the vicinity of the stalk. The capsule plug (P) is also shown. to the capsule plug and appeared to he composed of a similar material (Fig. 3D). The structure of the capsule wall was not homogeneous throughout the capsule, as is shown by longitudinal sections through the stalk and plug regions (Fig. 3C, D). Serial sections along the chamber revealed considerable variation in capsule wall thickness (Fig. 4). Walls tended to be thickest in the plug and stalk regions and thinnest at a position 75% along the capsule chamber. Although capsule width also varied along length of the capsule 318 T. A. RAWL1NGS 180-1 ' GRAPPLER - 160< o o SEPPINGS A 140- \\ \\T / \ O / 120- \t\ / V \- / 1 1 i X T LJ ~' ! - I '----,- o ~~~m- 20 40 60 80 100 POSITION ALONG CAPSULE CHAMBER (%) Figure 4. Variation in wall thickness along the capsule chamber of field-collected Kitccllti cnnir^innUi capsules from Grappler Inlet, Ross Islets, and Scppings Island. Serial sections were taken at 10% intervals along the capsule chamber, starting at the plug (0%) and ending at the stalk ( 100%). Values are expressed as mean 1 S.E. for 8 capsules sec- tioned from each population. 110- VUKAHH - - RC "g" 100- oSEPPlNGS a. n * ' ' V 90- O V v 'v 17 - 8 o ^ ^ ^ *: o 80- QOo ^ %o ^ ^ v O V V V i 8 00 'V i 70- o _ i ^* UJ , 60- ! " Q_ 5 ' - O 50- 4.0 5.0 6.0 7.0 8.0 TOTAL CAPSULE LENGTH (mrr Figure 5. Variation in capsule wall thickness with total capsule length (excluding stalk) for 30 \itci'lla I'lnarximilu capsules from each laboratory population. Each value represents a mean of eight measurements taken from one section at a point 70'" along the capsule chamber chamber, there was no correlation between capsule width and wall thickness (see Rawlings, 1989). Capsule wall thickness also varied among populations (Fig. 4). At the 70th percentile division along the capsule chamber, Ross Islets capsules were significantly thinner than capsules from Seppings and Grappler (means of 61, 80 and 8 1 ^m, respectively; ANOVA: F = 29.9, P < 0.00 1 ; Fig. 3A,B). This trend in wall thickness was apparent throughout the length of the capsule chamber. Differences in capsule wall thickness among populations resulted from variation in the thickness of all three component layers of the middle lamina (i.e., L 2a . h . c ), rather than in one com- ponent alone (data not shown). Significant differences in capsule wall structure were also evident among laboratory-laid capsules (mean = 60, 78, and 83 ^m, for Ross, Seppings, and Grappler capsules, respectively: ANOVA; F = 81.32, P< 0.001; Fig. 5). The wall thickness of these capsules did not differ significantly from capsules previously collected in the field (ANOVA; Grappler: F = 0.54, P = 0.47; Ross: F = 0.09, P = 0.77; Seppings F = 0.29, P = 0.60). Long-term exposure to the laboratory environment did not affect the morphology of capsules laid by these snails. Even after five months, snails still continued to produce their respective thick- or thin- walled capsules (data not shown). Although the size of capsules varied extensively within and among snail populations, no relationship was evident between wall thickness and total capsule length (Fig. 5). Because capsule length was related to female shell length (Fig. 2), differences in capsule wall thickness within each population were probably not related to female size. Also, differences in capsule wall thickness among sites did not appear to reflect differences in snail size, as small Seppings snails (mean shell length = 1.9 cm) and large Grappler snails (mean shell length = 2.7 cm) both produced rela- tively thick-walled capsules. Capsule wall thickness varied significantly among clutches within each population (ANOVA; Grappler, F = 3.44, P = 0.02; Ross, F = 32.05. P < 0.001; Seppings, F = 47.52, P < 0.001; Fig. 6). Variation in capsule wall thickness among clutches, however, did not obscure dif- 105-, 95- 85- 75- o I z> uo D. 65- 55- 45 CLUTCH #: 12345 12345 12345 SITE: SEPPINGS ROSS GRAPPLER Figure 6. Variation in capsule wall thickness within and among clutches of Nucella cmarginala capsules. Five clutches, each from a dif- ferent female snail, were sampled from all three laboratory populations, with n = 6. n = 4, and n = 6 capsules/clutch for Seppings, Ross Islets, and Grappler populations, respectively. Each data point represents the mean of eight measurements taken from one section at a point 70% along the capsule chamber. Each vertical group of points represents one clutch of capsules. N. EMARG1NATA EGG CAPSULES 319 o - If 4.0 -I 3.5- 3.0- 2.5- 2.0- 1.5- 1.0 4.0 5.0 6.0 7.0 80 90 I DIAL CAPSULE LENG~- nrr Figure 7. Dry weight of empty capsular eases as a function of total capsule length (excluding stalk) for each laboratory population. Each data point represents one capsule. Least-squares linear regression equa- tions for each population are: Seppings: Y = 56.859X - 142.901, r = 0.701, n = 27; Ross Islets, V = 41.359X - 79.242. r = 0.523, n = 33; Grappler Inlet, Y = 53.873X - 100.746. r = 0.740. n = 28. ferences in capsule wall thickness among sites. Ross Islets capsules ranged in wall thickness from 50 to 76 ^m, while Seppings and Grappler capsules ranged from 71 to 92 ^m and 72 to 102 /urn, respectively. Longer capsules had significantly heavier dry weights for each laboratory population of snails (Fig. 7). Although the slopes of these site-specific relationships were not sig- nificantly different (ANCOVA for slopes; F = 1.20, P > 0.25), the elevations did vary significantly (ANCOVA for elevations; F = 50.52; P < 0.001). These differences corresponded well with those in capsule wall thickness among populations, as thin-walled Ross Islets capsules weighed significantly less for a given length than thicker- walled capsules from the other two sites. Grappler capsules were also significantly heavier than Seppings capsules, again reflecting the differences reported above in wall thickness. Capsule wall strength. The force required to puncture capsule walls differed among the three laboratory popu- lations (Table I). Grappler capsules were significantly more resistant to puncturing than capsules from the other two sites (ANOVA, F = 1 1.77, P < 0.001; Tukey Multiple Comparison Test. P < 0.05). Ross Islets and Seppings capsules did not differ significantly in puncturing resis- tance (Tukey M.C.T., P > 0.05), despite the fact that Sep- pings capsules had substantially thicker walls (Fig. 5). The force needed to rupture capsules by squeezing also varied among laboratory populations (Table I). Grappler capsules required significantly larger forces to rupture the capsular plug than did either Ross Islets or Seppings cap- sules (ANOVA. F = 18.67, P < 0.001; Tukey M.C.T., P < 0.05). Thick-walled Seppings capsules were also slightly more resistant to squeezing than thin-walled Ross Islet capsules, however, this difference was not significant (Tu- key M.C.T., P>0.05). Capsule contents. No significant differences were ob- served in the total number of eggs allocated to Seppings, Ross Islets, and Grappler capsules (Fig. 8). Neither the slopes (ANCOVA for slopes: F = 0.65, P > 0.50) nor elevations (ANCOVA for elevations: F = 0.36; P > 0.50) of these relationships differed significantly among popu- lations. The size of N. emarginata eggs was also relatively con- stant. Although Ross Islets capsules contained slightly larger eggs than Grappler or Seppings capsules (mean egg volume = 40.5 X 10~ 4 mm 3 . 39.6 X 10 4 mm 3 , and 38.1 X 10~ 4 mm 3 , respectively), these differences were not sig- nificant (ANOVA: F = 1.55, P> 0.22). Predation on N. emarginata egg capsules Laboratory-identified predators. Only three types of in- vertebrates opened Nucella emarginata egg capsules in the laboratory: isopods (Idoteu wosnesenskii), shore crabs (Ilemigrupsus mulits and H. oregonensis), and chitons (Mopalia spp.). Idotea \msnesenskii regularly preyed upon egg capsules in laboratory experiments. These predators usually opened capsules by chewing through the side of the capsule chamber and left bite-marks as shown in Figure 9 (A, E). Table I Intraspecific variation in the wall thickness and strength of Nucella emarginata capsules Mean S.E. a Grappler Inlet (n) Seppings island (n) Ross Islets (n) h Capsule wall thickness (^m) Puncturing force (MN/m : ) Popping force (N) 83.3 1.6 (30) (10) (19) 78.4 1.4 (30) (10) 59.9 0.9 (30) (15) (21) 6.18 0.20 4.92 0.14 5.17 + 0.17 14.5 1.0 9.7 1.0 (15) 7.5 0.6 1 For each index, populations not connected by a horizontal line are significantly different from one another (Tukey M.C.T. at a = 0.05). '(n) refers to the number of capsules sampled from each population. 320 T. A. RAWLINGS Caged Idotca (1.6-3.2 cm in body length: mean = 2.2 cm) opened a mean ( S.E.) of 4.6 0.8 capsules over a five-day period (n = 11). Predation rates varied among these individuals, but not in relation to size or sex, al- though newly hatched Idotea (5 mm in length) did not open egg capsules in the laboratory. Two other species of intertidal isopods. Gnorimosphaeroma oregonense (mean length = 0.9 cm) and Cirolana harfardi (mean length = 1 .4 cm), nibbled capsules extensively, but never chewed through capsule walls. The shore crabs Hemigrapsus nudus and H. orcgonensis also readily opened N. enuirginata capsules in the labo- ratory. Predation rate was dependent on crab size. Small and medium-sized H. nudus (carapace widths of < 1.5 cm and 1.5-2.5 cm, respectively) opened a mean ( S.E.) of 3.3 0.8 (n = 10) and 6.0 0.7 (n = 18) capsules re- spectively over a 5-day period. Larger crabs (carapace width > 2.5 cm) opened all eight capsules after only 3 days (n = 9). Hemigrapsus spp. exhibited two methods of opening Nucella capsules. Larger crabs typically rup- tured the capsular plug by squeezing the capsule chamber in their chelae. More often, however, crabs tended to chew through the plug region directly into the capsule chamber, as shown in Figure 9(B, F). Few N. emarginala capsules were opened by Mopalia spp. in laboratory tests. Over a 2-week period, 2 1 chitons only opened 6 of 56 capsules. These predators usually rasped capsules open near the base of the chamber (Fig. 9C, G), and sometimes completely severed the capsule chamber from the stalk. Field censuses ofpredation. Snails and egg capsules were most abundant in the lower regions of the intertidal chan- nel at Grappler Inlet (Table II), with egg capsules being deposited deep within a dense meshwork of mussels and barnacles. Egg capsule predators Hemigrapsus oregonensis ( 1 .0-2.2 cm in carapace width). Idotea wosnesenskii (2. \ - 2.9 cm in body length) and Mopalia spp. (2-6 cm in body length), were present in this region, with Mopalia spp. being the most numerous. Eighteen percent of capsules collected along this lower transect had been opened by predators (Table II). Predators of many of these capsules could be identified by distinctive bite marks left on capsule walls (Fig. 9). The majority of capsules showed evidence of predation by Idotea, even though these isopods were scarce at the time of censusing. Capsules collected from three intertidal boulders showed similar types ofpredation, with the percentage of capsules opened by predators rang- ing from 1 1 to 26% (mean = 18%). Although there was no direct evidence of predation by Hemigrapsus spp. at this site, these crabs may have been responsible for open- ing many torn and chewed capsules whose bite marks could not be readily identified. Some capsules were also emptied by means of bevelled holes (0.4 X 0.2 mm; Fig. 9D, H). Predators of these capsules may have been inter- 1300 LJ _1^ 1000- 5 900 ] a 800 700- UJ 600 Ld m 1 500 z 400 GRAPPLER v S SEPPINGS o 10 20 30 40 50 6070 CAPSULE VOLUME (fj.\) Figure 8. Relationship between the number of eggs per capsule and the volume of the capsule chamber for Nucella emarginala individuals from Seppings. Ross Islets, and Grappler Inlet. Counts of eggs include both developing embryos and non-developing nurse eggs. Least-squares linear regression equations for each population are: Seppings: Log Y = 0.676 Log X + 1 .954. r = 0.55 1 , n = 18; Ross Islets: Log Y = 0.669 Log X + 1.962. r = 0.340. n = 18; Grappler: Log Y = 0.492 Log X + 2.192, r = 0.410, n = 18. tidal gastropods, because they have been reported to make similar holes in other gastropod egg capsules (Abe, 1983). The density of snails and egg capsules was lower along the high transect at Grappler Inlet (Table II). In contrast, Hemigrapsus and Idotea. were notably more abundant, and the percentage of capsules opened was also higher, with 32% of capsules showing evidence ofpredation. Ido- tea bite-marks were found on all capsular remains. Densities of snails and egg capsules varied markedly among transects at the Ross Islets site (Table II). All cap- sules at this site were attached to vertical surfaces or ov- erhangs. Encapsulated embryos were also generally further developed than those at Grappler Inlet, reflecting the fact that censuses were made approximately a month later. Hemigrapsus nudus (0.6-2.4 cm in carapace width) were the only known predators of Nucella egg capsules at this site, with densities ranging up to 360/m 2 . The majority of opened egg capsules also appeared to have been preyed upon by Hemigrapsus (Table II). Egg capsules from Seppings Island showed evidence of bite-marks by both Idotea and Hemigrapsus. Despite the extreme levels of wave action at this site, these predators were abundant, especially within the thick beds ofAIytilus californianus. Capsules were also found with bevelled holes identical to those collected from Grappler Inlet (Fig. 9D, H). Susceptihility of capsules to predators Idotea opened thin-walled capsules from Ross Islets more frequently than thick-walled capsules from either N. EMARG1NAT.I EGG CAPSULES 321 Figure 9. Charactenslics of species-specific predation on N. cmari>inata egg capsules by: Idolea iro.v- nc\cii\kii (A. El. llcmigrapxus spp. (B, F), Mupalia spp. (C, G). and an unknown predator (D. H), possibly an intcrtidal gastropod. For each type of predator, a whole mount of the opened capsule is shown (Mag: 8X), with a close-up below illustrating the characteristic bite-marks (Mag: 25-50X). Grappler Inlet or Seppings Island (Table IIIA, B). In 10 trials, 35 Ross Islet (thin-walled) capsules were opened compared with 15 Grappler (thick-walled) capsules (Fish- er's Exact Test, P = 0.000 1 ). Ross Islets (thin-walled) cap- sules were also opened more frequently than Seppings (thick-walled) capsules, although this difference was not quite significant (16 Ross Islets capsules versus 10 Seppings capsules; Fisher's Test, P = 0.08). In contrast, isopods did not exhibit any preferences for Seppings versus Grappler capsules ( 1 Grappler versus 9 Seppings capsules; Fisher's Test, P = 0.50; Table IIIC), and in two of six trials no capsules were eaten during a ten-day period. Hence, thick- walled capsules from Grappler Inlet and Seppings Island were more resistant to predation than thin-walled capsules from Ross Islets. Discussion Intraspecific variation in capsule morphology The morphology of N. enuirginata capsules varies ex- tensively among populations. In the present study, both capsule wall thickness and strength differed significantly among the three intertidal locations examined. Such vari- ation in capsular structure may reflect ( 1 ) physical con- straints associated with female body size, (2) phenotypic differences in response to variable environmental condi- tions, or (3) genetic divergence caused by selection. Intraspecific differences in the wall thickness and strength of TV. emarginata capsules may reflect constraints associated with female size. Although the morphology of neogastropod egg capsules is governed by the size of the capsule gland, which, in turn, is restricted by female shell- length (Spight etal., 1974; Spight and Emlen, 1976; Perron and Corpuz, 1982; present study), little is known about the direct effect of female size on capsule wall structure. Perron and Corpuz (1982) reported that wall thickness and strength of Conus pennaceus capsules increased with capsule size and snail shell-length. Their results suggested that the structure of capsule walls may be limited by the size of the capsule gland. In the present study, capsule size and snail shell-length varied markedly within and 322 T. A. RAWLINGS Summary of Nucella emurginata (',%' capsules, and potential at (i nippier Inlet and Ross Islets study sites Table II capsule predators censused along transects Census of egg capsules* 1 Chamber ruptured Density J (Mean/nr S.E.) Chamber intact Predators of opei led fi'ucella eniurginulii Egg Heintgrapsus capsules spp. Opened capsules 6 hlolea .Mopalia # of Early Late Hatched by spp. spp. capsules embryos embryos naturally predators H I M U.G. UNID Grappler Inlet Tidal height 1.2 m (n = 8) 29.6+ 8.8 126.0 47.6 4.9 0.5 0.4 0.4 9.3 2.1 255 35 22 22 18 4 43 2 17 34 494" 24 50 18 3 82 1 14 2 m (n = 8) 14.0 6.0 9.6+ 5.2 10.2 3.1 8.0 3.7 19 68 32 100 Ross Islets Tidal height 1 9 m (n = 6) 26.0 10.4 24.8 19.2 360.8 38.6 37 46 5 24 24 100 23 m (n = 8) 6.4 4.4 70.0 30.4 140 11 13 45 22 77 23 2.6 m (n = 8| 289.6 97.6 5.2 4.8 10 10 0000 2.8-3.1 m (n = 6) 203.3 + 30.3 744.6 17.2 24.6 6.4 1117 7 70 22 73 00 26 "Censuses were made in May 1988 and June 1988 for Grappler Inlet and Ross Islet study sites, respectively. The number (n) of 0.25 irr quadrats used to estimate these densities is shown lor each transect. h Intact capsules were aged by examining the developmental stages of enclosed embryos. Empty capsules were categorized according to whether embryos had hatched naturally or had been opened by predators. Data are expressed as a percentage of the total number of capsules along each transect that were found in each category. Percentages may not always add up to 100%, because some capsules were found intact but their contents were dead. 1 Predators responsible for opening capsules were identified by means ot bite-marks left on the capsule chamber. Abbreviations: H = H&nigrapsus spp.. I = Idolea spp., M = Mnpalia spp.. U.G. = unknown gastropod, and UNID = unidentified predators. Capsules in the "UNID" category 1 had been opened by predators, but bite-marks could not be accurately identified. Data in each category represent a percentage of the total number of capsules opened by predators along each transect. " These capsules were collected in Aug 1988 from three inlertidal boulders in Grappler Inlet. among the Grappler, Ross Islets, and Seppings popula- tions. Capsule wall thickness, however, did not differ as predicted with either capsule length or snail shell-length. Hence, variation in the thickness of capsule walls among N. i'lnurghuila populations was not the result of allometric contraints associated with female size. Differences in capsule structure among populations of N. emarginata also did not appear to be the result of phe- notypic plasticity. Variation in capsule wall thickness within a clutch was low compared to variation among clutches produced by different individuals. Hence, within a spawning period, individual females deposited capsules of relatively consistent wall thickness. Also, snails contin- ued to produce their respective thick- or thin-walled cap- sules even after five months in the laboratory, a period during which snails from all three populations were kept under similar environmental conditions. Thus, differences in the structure of egg capsules were not likely to be short- term phenotypic responses to site differences in diet, food abundance, or levels of environmental stress. Such results suggest that the production of thick or thin capsule walls may be an adaptive response to environmental conditions. Costs associated with producing thick-walled capsules There are likely to be both costs and benefits associated with the production of thick-walled capsules. Thick-walled capsules may incur a greater energetic cost than thin- walled capsules based on their greater dry weight per unit length. For instance, thin-walled capsular cases from the Ross Islets (6.5 mm in length) weighed 24% less than thick- walled capsules from Grappler Inlet, and 16% less than thick-walled capsules from Seppings Island. As capsular cases can account for more than 50% of the dry weight of intact capsules (i.e.. including the eggs; Roller and Stic- kle, 1988; Rawlings, unpub. data), and as N. emarginata capsular material has almost the same energy content per unit weight as the eggs (22.6 KJ per ash-free gram com- pared to 25. 1 KJ per ash-free gram of embryos; J. Davis, unpub. class project, Friday Harbor Laboratories, 1984), N. EMARGINATA EGG CAPSULES 323 Table III Preferences <>/ Idotea wosnesenskii fur thick- or thin-walled e^K capsules <>/ Nucella emargmata Mean (S.E.) number of capsules opened a Site Compansons Thin-walled Thick-walled A| Ross vs. Grappler (n = 10) 3.5 0.3 1.5 0.3 B) Ross i'.v. Seppings (n = 5) 3.2 0.4 2.0 0.5 C) Grappler v.v. Seppings (n = 6) h 2.5 2.3 0.3 0.3 a Average number of egg capsules opened by / mnnesenskii when given a choice of capsules from two different study sites. Predators were placed in cages with 10 capsules (5 from each site), and the first 5 capsules to be opened were recorded. Data are expressed as the mean number (1 S.E.) of capsules selected from each site, where (n) refers to the number of replicates performed for each comparison. h I n 2 out of 6 replicates, no capsules were eaten over a 1 0-day period. the energy spent in producing thicker capsule walls must represent either a substantial decrease in the energy avail- able for egg production or an increase in the reproductive effort of an individual. In fact. Perron (1982) found that the production of thick, puncture-resistant, capsule walls among Conns spp. was associated with a higher annual reproductive effort than the production of weak, thin- walled capsules. In the present study, I did not compare reproductive effort among populations. The production of thick-walled capsules, however, was not associated with a reduction in egg size or number of eggs contained per unit capsule volume. Hence, on a per capsule basis, there was no evidence of a tradeoff between the amount of en- ergy invested in capsular cases versus eggs. Other potential costs still remain to be tested. For in- stance, Strathmann and Chaffee (1984) have suggested that thick encapsulating structures may reduce the avail- ability of nutrients and oxygen to developing embryos. Hence, (1) the density of embryos per capsule, (2) the developmental rate of embryos, or (3) the proportion of embryos surviving, may differ between thick- and thin- walled capsules. Although preliminary results have indi- cated that there are no significant differences between the number of embryos contained within thick- and thin- walled capsules (Rawlings, 1989), further comparisons still need to be made. Benefits of enclosing eggs within I hick-walled capsules Numerous studies have examined interspecific differ- ences in the properties of gastropod egg capsules (Perron, 1981; Perron and Corpuz. 1982; Pechenik, 1983, D'Asaro, 1988). The degree to which thick-walled capsules protect developing embryos better than thin-walled capsules, however, is still unclear. Pechenik (1983), for example, found that the rate of salt movement across the walls of Nucella lamcllosa. N. lapillus, and N. lima capsules did not vary systematically with capsule wall thickness. Hence, the resistance of capsule walls to osmotic shock or des- iccation stress might not differ between thick- or thin- walled structures. Such interspecific comparisons may be confounded by differences in the structural components of capsule walls, however, which vary considerably among Nucella species (pers. obs.). The only previous evidence to support the hypothesis that strong, thick-walled capsules are more protective than weak, thin-walled capsules has come from positive cor- relations between capsule strength, the proportion of re- productive energy invested in capsule walls, and devel- opmental time of encapsulated embryos among Conns species (Perron, 1981; Perron and Corpuz, 1982). Al- though capsule wall thickness was not compared among all species. Conns pcnnaccus. with encapsulated devel- opment times of 26 days, was found to have significantly thicker capsule walls than Conns minis, with encapsulated development times of 1 1 days (Perron and Corpuz, 1982). These results indicate that strong, thick-walled capsules may reflect selection for increased protection of embryos when exposure to environmental stresses is long. As yet, however, no selective mechanism has been identified to explain this pattern. Perron ( 198 1 ) has suggested that egg capsule predators may be the agent of selection for strong, energetically ex- pensive capsule walls. Indeed, predation appears to be an important source of mortality among encapsulated em- bryos. For instance, Brenchley ( 1982) found that 52% of the capsules of the mud snail, flyanassa ohsoleta, were opened by crabs or snails during 10 days of a development period lasting up to 3 weeks. Spight (1972) noted that predators had opened 77% of Nucella lamcllosa capsules in some spawning aggregations. Other studies, such as those by MacKenzie (1961). Haydock (1964), Emlen (1966), and Abe (1983), have also documented high levels of predation on gastropod egg capsules. In the present study, one-time field censuses of predation on N. emar- ginata egg capsules indicated that up to 32% of capsules had been opened by crabs, isopods, and other predators. Therefore, predators are responsible for considerable mortality among encapsulated embryos. Thick-walled capsules may be more difficult to open or require longer handling times by predators than thin- walled capsules. Hence, the former might be selected for in areas where predators are abundant. The production of thick-walled N. emarginata egg capsules was not related to the relative abundance of Hemigrapsus spp. among 324 T. A. RAWLINGS Grappler Inlet, Seppings, and Ross Islet study sites. In fact, thin-walled capsules were found at Ross Islets, where crabs densities reached up to 360/rrr. In contrast, the predatory isopod Idoica wosenesenskii was found only at the two sites where thick-walled capsules were present. Embryos contained within thick-walled capsules were also less likely to be eaten by hlotca than those contained within thin-walled capsules. Hence, these results indicate not only that thick capsule walls protect developing em- bryos better against hlotca than thin capsule walls, but also that these predators may have resulted in selection for thick-walled capsules at Grappler Inlet and Seppings Island study sites. Although capsule wall thickness varied in accordance with the presence of hlotea. capsule strength did not. The fidelity with which puncture-resistance and squeezing forces my measures of capsule strength simulate methods used by Idotea to open capsules is not known. Possibly, however, these measures of capsule strength could reflect the action of other environmental stresses affecting encapsulated embryos. Desiccation (Feare, 1970; Spight, 1977: Pechenik. 1978), osmotic stress (Pechenik, 1982; 1983; Hawkins and Hutchinson, 1988), wave-action (Perron, 1981), bacterial attack (Lord, 1986), and thermal stress (Spight, 1977; Pechenik. 1986) are all potentially important sources of mortality for encapsulated embryos. These stresses may have independently resulted in the selection of different properties of capsule walls. Confounding influences in intraspecific comparisons Although intraspecific variation in capsule morphology may provide the best opportunity to address costs and benefits of encapsulation, interpretations of differences among populations may be confounded by the effects of environmental stresses on adult snails. Environmental stresses, such as wave-exposure, affect the reproductive effort of gastropods profoundly. For instance, wave-ex- posed snails typically mature at smaller sizes and exhibit higher reproductive efforts over shorter lifespans than longer-lived, wave-sheltered snails (Roberts and Hughes, 1980; Calow, 1981; Etter, 1989). Similarly, TV. emarginata from Seppings matured at smaller sizes and produced proportionally larger capsules than those from Grappler Inlet (Fig. 2). How such differences in reproductive effort might be reflected in the partitioning of energy between eggs and extraembryonic products is unclear. Neverthe- less, the type of capsule produced should still depend on the relation between energetic cost and the defensive ef- fectiveness of capsular material. Acknowledgments I would like to thank Dr. T. H. Carefoot, Dr. D. Padilla, L. Taylor, D. Gamier, A. Martel, G. Jensen, and G. Gib- son for their input into this study, and K. Durante and Dr. A. R. Palmer for reviewing earlier draft? of this manu- script. I am also grateful to J. Ferris for providing me with a never-ending supply of Nucella egg capsules for labo- ratory predation experiments, and to the director and staff of the Bamfield Marine Station for making this research possible. This project was supported by an NSERC op- erating grant to Dr. T. H. Carefoot and a McLean-Fraser Memorial Fellowship to T.A.R. Literature Cited Abe, N. 1983. Breeding of Thais davigera (Kuster) and predation of its eggs by Cronia margariticola (Brodenp). Pp. 381-392 in Pro- en-iJings of i he Second International Workshop on the Malacofauna nl Hung Kong and Southern China. Morton, B. and D. Dudgeon. eds. Hong Kong University Press. Hong Kong. Austin, \V. C., I,. C. Druehl, and S. B. Haven. 1971. Bamfield survey: marine habitats and biota. Bamfield Survey Report 2: 1-30. Bayne, C. J. 1968. Histochemical studies on the egg capsules of eight gastropod molluscs. Proc. Malawi. Sac. Loiul. 38: 149-212. Brenchley, G. A. 1982. Predation on encapsulated larvae by adults: effects of introduced species on the gastropod Ilyanassa ohsoleta Mar Eeol. Prog. Ser. 9: 255-262. Calow, P. 1981. Adaptational aspects of growth and reproduction in Lvmiuiea peregra (Gastropoda: Pulmonata) from exposed and shel- tered aquatic habitats. Malacologia 21: 5-13. Craik, G. J. 1980. Simple method for measuring the relative scouring of intertidal areas. Mar. Biol. 59: 257-260. Crothers, J. II. 1984. Some observations on shell shape variation in Pacific Kueella. Biol J. Linn. Sue. 21: 259-281. D'Asaro, C. N. 1970. Egg capsules of prosobranch mollusks from south Florida and the Bahamas and notes on spawning in the laboratory. Bull Mar. Sei. 20:414-440. D'Asaro, C. N. 1988. Micromorphology of neogastropod egg capsules. Nautilus 102: 134-148. Kmlen, J. M. 1966. Time, energy and risk in two species of carnivorous gastropods. Ph.D. Thesis. University of Washington. Seattle. Etter, R. J. 1989. Life history variation in the intertidal snail Nucella lapillus across a wave-exposure gradient. Ecology 70: 1857-1876. Keare, C. J. 1970. Aspects of the ecology of an exposed shore population of dogwhelks Nueella lapilhis (L.). Oecologia 5: 1-18. Gallardo, C. S. 1979. Development pattern and adaptations for repro- duction in Kiicella erassilahrum and other muricacean gastropods. Biol. Bull 157: 453-463. llaydock, C. I. 1964. An experimental study to control oyster drills in Tomales Bay, California. Calif. Fish and Game 50: 1 1-28. I la" kins, L. E., and S. Hutchinson. 1988. Egg capsule structure and hatching mechanism of Oeenebra erinaeea (L.) (Prosobranchia: Muncidae). / E\p. Mar. Biol. Eeol. 119: 269-283. Hunt, S. 1971. Comparison of three extracellular structural proteins in the gastropod mollusc Buccinum undaluin L.. the periostracum. egg capsule, and operculum. Comp. Bioehem. Physiol. 40B: 37-46. kitching, J. A. 1976. Distribution and changes in shell form of Thais spp. (Gastropoda) near Bamfield. B. C. J. E.\p. Mar Biol Eeol. 23: 109-126. I.eBoeuf, R. 1971. Thais emarginata (Deshayes). Description of the veliger and egg capsule. I 'eliger 14: 205-2 10. Lord. A. 1986. Are the contents of egg capsules of the marine gastropod Nucella lapillus (L.) memc"? Am. Malaeol. Bull 4: 201-203. Lyons. A., and T. M. Spight. 1973. Diversity of feeding mechanisms among embryos of Pacific Northwest Thais. Veliger\6: 189-194. A i:\t.\RHINATA EGG CAPSULES 325 Mackenzie, C. I.., Jr. 1961. Growth and reproduction of the oyster drill Euplciira cainlma in the York River. Virginia. Ecology 42: 3 1 7- 338. Oslergaard, J. M. 1950. Spawning and development of some Hawaiian marine gastropods. Pac Sci 4: 75-1 15. Palmer, A. R. 1984. Species cohesiveness and genetic control of shell color and form in 77/n cmargmala (Prosobranchia. Muricacea): preliminary results. Ululucologia 25: 477-491. Pechenik, J. A. 1978. Adaptations to mtertidal development: studies on A'imw/in / a/., 1965). Unsuccessful attempts have been made to learn how the sea hare uses ink in its natural environment. Carew and Kupferman (1974) and Kupferman and Carew (1974) reported on lengthy observations of Aplysia calif ornica in a variety of habitats; inking was never observed. Further- more, they did not observe an Aplysia being attacked by a predator. Inking occurred routinely, however, if the mollusks were roughly handled by the investigators. These results suggest, but do not prove, that the ink is a defense mechanism used only in rather extreme cases. The func- tion of the ink would then be produced by its chemical composition rather than its optical properties. Inking has been studied by the techniques of neuro- physiology (e.g., Byrne, 1981 ), and it was determined that an electric-shock stimulus must cross a high threshold before inking occurs (Carew and Kandel, 1977). Chapman and Fox (1969) studied the correspondence between diet and the presence of ink in Aplysia. After inducing complete discharge of the ink by tactile stimu- lation, they fed the spent organisms either brown or red algae and found that only after feeding with red algae was the ink replenished. Rudiger (1967) had shown that the major pigment in the ink has the structure of a mono- methyl ester of phycoerythrobilin. a chromophore of the 326 APLYSIA INK 327 m oc O 0.2 - 300 400 500 600 WAVELENGTH (nm) 700 Figure 1. Absorption spectra of .!;>/rw6 h) in 0.1 M sodium caco- dylate buffer with 0.45 or 0.50 M NaCl. Some components of the light organ showed enhanced fixation with 0.45 M NaCl while others showed similar improvements with 0.50 M NaCl (see figure legends). Results The fully developed light organ of Euprymna scolopes is a bilobed structure that occupies a significant portion of the mantle cavity (Fig. 1 A, B). Ten specimens, ranging in size from 4.0 mm to 25 mm in dorsal mantle length (ML), were used for microscopy. The light organ ranged in anterior-posterior length from 1.5 mm in the smallest animal to 7.5 mm in the largest individual. Although on- going studies in our laboratory have shown that the de- velopment of the E. scolopes light organ involves a set of complex stages, all components of the light organ are present and appear mature in juveniles as small as 4 mm ML. Histological analysis of the light organ of this species (Fig. 2) revealed that the tissues and their anatomical re- lationships were similar to those described in the light organ system of another sepiolid, Sepiola atlantica (Her- Figure 2. Light micrograph and opposing diagram of a typical 1 -^m histological cross-section through the light organ of Euprymna scolopes showing the various associated tissues. The ink is lost from the ink sac during dissection, and shrinkage that occurs during fixation and embed- ding procedures causes the tissue containing the bacteria to pull away from the reflector. 20X. (bet, tissue containing bacteria). 334 M. McFALL-NGAI AND M. K. MONTGOMERY Figure 3. Composite transmission electron micrograph of the bacteria-containing central core of the light organ of Euprymna scolopes. Numerous electron-dense vesicles (arrows) are concentrated in portions of the host cell adjacent to the tubules that contain bacteria. Primary fixative used was 2.5% glutaraldehyde in 0.2 M sodium cacodylate buffer with 0.45 A/ NaCI. Bar scale = 10 ^m. (bet, bacteria; bv. blood vessel) ringer ai, 1981). The bacteria occur in animal tissue that is surrounded by a thick reflector, which is, in turn, sur- rounded by diverticula of the ink sac. In some preserved specimens, the medioventral portion of the reflector was pulled back, and ink in the ventral portions of the ink sac shunted medially (see Fig. 2). Histological analysis of a large number of specimens showed considerable vari- ability in the positions of the reflector and ink sac in re- THE EVPRYMNA SCOLOPES LIGHT ORGAN 335 Figure 4. High magnification transmission electron micrographs of the light organ tissue that contains the bacteria. A. Abundant mitochondria occur in the host cell adjacent to the microvillous border lining the tubule that houses the bacteria. Primary fixative used was 2.5% glutaraldehyde in 0.2 M sodium cacodylate buffer with 0.50 M NaCl. Bar scale = 1 ^m. B. Vibrio fischeri cell as it appears in symbiosis with squid tissue. This single bacterium, although extracellular, appears almost completely surrounded by host cell membrane. Primary fixative is the same as above. Bar scale = 0.25 fim. C. The lower osmolarity of this buffer resulted in poor fixation of the mitochondria, but better fixation of the electron-dense vesicles, both of which appear in abundance in the portion of the animal cell adjacent to the microvillous border that lines the tubules containing bacteria. Primary fixative used was 2.5% glutaraldehyde in 0.2 M sodium cacodylate buffer with 0.45 M NaCl. Bar scale = 1 nm. (bet, bacteria: m, mitochondria; n. nucleus) 336 M. MrFALL-NGAI AND M K. MONTGOMERY Figure 5. Composite electron micrograph of the ciliated duct of the light organ of Enprymna scolopes. A. The composite resulted from a transverse section of the light organ and shows the ciliated duct that leads to the lateral pore on each lobe. Branches of the duct shown in this micrograph are contiguous with the tubules that contain bacteria. Primary fixative used was 2.5% glutaraldehyde in 0.2 M sodium cacodylate buffer with 0.45 M NaCl. Bar scale = 5 ^m. B. High magnification showing the cilia in cross-section. Primary fixative as above. Bar scale = 0.25 ^m. lation to the tissue that contains the bacterial symbionts. These data, coupled with observations of the behavior of light organ tissues of anesthetized, dissected animals, in- dicate that the expression of light is controlled by move- ments of both the reflector and ink sac. The entire ventral surface of the light organ is covered by a thick, transparent lens, the outer edge of which is continuous with the ink sac lining (Fig. 2). Microscopic examination of the intact light organ revealed a pore on the lateral face of each light organ lobe. Histological sections through this area revealed that the pore is continuous with the light organ tissue that contains bacteria (data not shown). Low magnification transmission electron micrographs of light organ tissue that contains bacteria (Fig. 3) reveal that the bacteria occur in narrow channels, usually only a few bacteria in width, in most portions of the light organ. The narrow channels are surrounded by a single layer of animal cells, which are surrounded by a layer of blood vessels and other connective tissue elements. This pattern is repeated through the bacterial core tissue. Microvilli from the epithelial cells of the animal invest the bacterial culture. Electron-dense vesicles occur in the portions of the animal cells adjacent to the bacterial culture, sug- gesting the exchange of materials. Observations at higher magnifications of the animal cell/bacterium interface (Fig. 4) revealed that, in addition to electron-dense vesicles. Figure 6. Accessory structures of the light organ of Euprymnu MVI/O/V.V. A. Transmission electron mi- crograph of the light organ with some of the structures associated with light modulation. This section is through a particularly narrow portion of the reflector (r) so that several layers of the system are viewed. The reflector is closely associated with the lining of the ink sac (see also 6D) and surrounds much of the central core tissue, which contains the bacterial symbionts. Primary fixative used was 2.5% glutaraldehyde in 0.2 M sodium cacodylate buffer with 0.45 M NaCl. Bar scale = 2 ^m. B. High magnification micrograph of the reflector showing the membrane bound platelets. Primary fixative same as above. Bar scale = 0.25 ^m. C. Transmission electron micrograph of the lens exposing its lack of detailed cell structure but indicating its THE ELI PRY MN A SCOLOPES LIGHT ORGAN 337 . * -. ,'. , S _ ' 8 development from muscle-derived tissue. Note the numerous, thin, aligned filaments filling most of the cells. Primary fixative used was 2.5% glutaraldehyde in 0.2 M sodium cacodylate buffer with 0.5 M NaCI. Bar scale = 2 pm. D. Transmission electron micrograph of the lining of the ink sac showing its ciliated ink- producing cells and its close association with the reflector. Primary fixative used was same as for 6A. Bar scale = I tim. (b, bacteria; i, ink sac; r, reflector) 338 M. McFALL-NGAI AND M. K. MONTGOMERY high densities of mitochondria exist in portions of the animal cell adjacent to the bacteria. Tubules containing bacteria empty into a common, ciliated duct that is con- nected with the pore on the lateral face of each light organ lobe (Fig. 5). This duct provides a direct connection be- tween the bacterial culture and the mantle cavity of the animal. The tissue containing bacteria is surrounded by reflec- tive tissue, which is itself enclosed by the ink sac (Fig. 6A). The reflector is made up of cells containing platelets oriented perpendicularly to the bacterial culture, and has a similar structure to that reported for the reflective tissues of other cephalopods (Arnold el at.. 1974; Brocco and Cloney, 1980; Cloney and Brocco, 1983). The width of the reflector is several dozen to hundreds of platelets thick, depending on the location within the light organ. These electron-dense reflector platelets, which appear to be membrane bound (Fig. 6B), averaged 100 nm wide with cytoplasmic spacing that varies from 50 nm to 200 nm. Because of shrinkage during preparation, however, this may not represent the actual spacing. The ventral portion of the reflector abuts the lens of the light organ (see Fig. 2), the ultrastructural characteristics of which suggested that it was derived from muscle tissue (Fig. 6C). The cells of the lens had little structural detail except for the pres- ence of numerous, thin, aligned elements. The ink sac lining appeared as a mitochondrion-rich tissue with elec- tron-dense vesicles, presumably packaged ink (Fig. 6D). Discussion Progress toward an understanding of developmental processes in higher animal/bacterial mutualisms has been slow because of the lack of tractable experimental systems. Unlike plant mutalisms, such as the leguminous plant/ Rhizobium symbiosis (Long, 1989), animal mutualisms usually involve a variety of different species of microor- ganisms in a single host, or are characterized by a host that cannot live axenically or by symbionts that cannot be cultured. One of the few higher animal/bacterial mu- tualisms the study of which does not suffer from these drawbacks, is the symbiotic relationship between the se- piolid squid, Euprymna scolopes. and its luminous bac- terial symbiont, Vibrio fischeri. The squid host, which can be raised in the laboratory (Arnold el ai, 1972; pers. obs.), hatches without its luminous symbiont (Wei and Young, 1989). V. fischeri. which occurs freeliving in the water and is readily culturable (Ruby and McFall-Ngai. 1989; Boettcher and Ruby, 1990), is picked up by the newly hatched squid within hours after hatching (Wei and Young, 1989). A prerequisite to the studies of the development of the light organ is a description of the morphology and anat- omy of the adult association. Although the adult light organ of E. sco/opes. described here, is similar to that of other sepiolids (Kishitani, 1932; Herring el ai, 1981), some ultrastructural differences have emerged. Herring el a/. (1981) reported that the bacteria of another sepiolid squid, Sepio/ci atlantica. are loosely associated with the animal cells, which lack microvilli. In contrast, the bac- terial culture in the light organ ofE. sco/opes is in intimate contact with the microvillous border of the host cells. Caution must be exercised in interpreting these differ- ences, which could be due to differences in the quality of fixation. The high concentrations of mitochondria in the por- tions of the animal cells adjacent to the bacteria may be of particular significance in the physiology and metabolic dynamics of the light organ association. Monocentrid fishes also have light organs containing an abundance of mitochondria in the cells next to the bacterial symbionts (Tebo et ai. 1979). Under laboratory culture conditions of low oxygen, \ 'ibrio fischeri grows poorly, but luminesces brightly and excretes pyruvate (Ruby and Nealson, 1976; Nealson and Hastings, 1977). The ultrastructure of the monocentrid light organ and the physiology of the bacteria in culture has led to a model for this symbiosis (Nealson, 1979). The model holds that pyruvate excreted by the bacteria fuels the mitochondria, the respiratory activity of which keeps the oxygen tension low around the bacterial culture, thus promoting the slow growth but high lumi- nescence of the bacteria in the association. However, while the light organ tubule cells of E. scolopes also have high densities of mitochondria, the symbiotic strain of I', fis- cheri they surround does not show enhanced luminescence under low oxygen tensions (Boettcher and Ruby, 1990). Thus, physiological behavior of the E. scolopes bacteria in culture and the ultrastructural characteristics of the light organ are inconsistent with the model developed for the monocentrid fish symbiotic association. During the development and ontogeny of a complex light organ, such as that of Euprymna scolopes. tissues must be recruited and modified to form the various com- ponents of the organ. Not only must tissue be adapted so that the squid can efficiently use the light produced by the bacteria, but the light organ and the bacterial culture must be supported by recruited vascular and nervous tis- sue. Further, the squid host must produce a site that pro- motes the growth and luminescence of the native sym- biotic bacterium, while excluding other bacterial species. How these processes are orchestrated to create the complex adult structure, and the part played by the bacteria in the morphogenesis of the light organ, should be revealed through experimental manipulations of the developing system. Acknowledgments We thank R. E. Young (University of Hawaii) for as- sistance in collecting the animals and for helpful discus- THE EVPRYMNA SCOLOPES LIGHT ORGAN 339 sions, and N. Holland (Scripps Institution of Oceanog- raphy) for help and advice with TEM procedures. We also thank E. G. Ruby for help with collecting animals, and E. G. Ruby and O. Hoegh-Guldberg for critical com- ments on the manuscript. We are grateful for technical assistance from A. Thompson and W. Ormerod of the Center for Electron Microscopy at USC, and for field as- sistance from the staff of the Hawaiian Institute for Marine Biology (HIMB). This paper is publication number 821 from the HIMB. This work was supported by NSF Grant No. DCB-89 17293 and by the Faculty Research Inno- vation Fund of USC. Literature Cited Arnold, J., C. Singley, and L. Williams-Arnold. 1972. Embryonic de- velopment and post-hatching survival of the sepiolid squid Euprymna xeolnpes under laboratory conditions. Veliger 14: 361-364. Arnold, J. M., R. E. Young, and M. V. King. 197-4. Ultrastructure of a cephalopod photophore. II. Iridiophores as reflectors and trans- mitters. Biol. Bull 147: 522-534. Berry, S. 1912. The Cephalopoda of the Hawaiian Islands. Bull U S Bur. Fish 32: 255-362. Bocttcher, K. and E. Ruby. 1990. Depressed light emission by symbiotic I 'ihnu tixeheri of the sepiolid squid Euprymna scolopes ./. Bacterial. 172: 3701-3706. Boletzky, S. 1970. On the presence of light organs in Senurin.\iu Steenstrup. 1887 (Mollusca:Cephalopoda). Bull. Mar Sci 20: 374- 388. Brocco, S. L. and R. A. Cloney. 1980. Reflector cells in the skin of Octopus dotleini. Cell Tissue Rex. 205: 167-186. Cloney, R. A. and S. L. Brocco. 1983. Chromatophore organs, reflector cells, iridocytes and leucophores in cephalopods. Am. /.ool. 23: 581- 592. Herring, P., M. Clarke, S. Boletzky, and K. Ryan. 1981. The light organs of Sepiola allanlica and Spirutu spirilla (MolIusca:Cephalo- poda): bacterial and intrinsic systems in the order Sepioidea. / Mar. Biol. Assoc. U. A 61: 901-916. Kishilani, T. 1932. Studiend uber Leuchtsymbiose von Japanischen Sepien. Folia Anal Jupn. 10:317-416. Long, S. 1989. Rhizobium-legume nodulation: life together in the un- derground. Cell 56: 203-214. McFall-Ngai, M., and K. Ruby. 1989. The changing host-tissue/sym- biont relationship in the developing light organ of Euprymna sec/opes (Cephalopoda:Sepiolidae). Pp. 319-322 in Endocylobiology 11 4lli International Colloquium on Emlocytobiology and Symbiosis. P. Nardon, V. Gianinazzi-Pearson. A. Grenier, L. Margulis and D. Smith, eds. INRA Service des Publications, Versailles, France. Moynihan, M. 198.3. Notes on the behavior of Euprymna xcolopes (Cephalopoda:Sepiolidae). Behavior 85: 25-41. Nealson, K. 1979. Alternative strategies of symbiosis of marine lumi- nous fishes harboring light-emitting bacteria. Trends Biochem. Sci. 4: 105-110. Nealson, K., and J. Hastings. 1977. Low oxygen is optimal for luciferase synthesis in some bacteria: ecological implications. Arch. Microbiol. 112: 9-16. Ruby, E., and M. McFall-Ngai. 1989. Morphological and physiological differentiation in the luminous bacterial symbionts of Euprymna scolopes Pp. 323-326 in Endocylohiology II': 4th International Col- loquium on Endocytobiology and Symbiosis, P. Nardon, V. Gian- inazzi-Pearson, A. Grenier. L. Margulis and D. Smith, eds. INRA Service des Publications, Versailles. France. Ruby, E., and K. Nealson. 1976. Symbiotic association of Photobac- lernim fischen with the marine luminous fish Monocentris japonica: a model of symbiosis based on bacterial studies. Biol. Bull. 151: 574- 586. Singley, C. 1983. Euprymna scolnpes. Pp. 69-74 in Cephalopod Lije Cycles. I V)/ 1 Academic Press, London. Spurr, A. 1969. A low-viscosity epoxy resin embedding medium for electron microscopy./ Ullraxlruel. Res 26: 31-43. Tebo, B., D. Linthicum, and K. Nealson. 1979. Luminous bacteria and light emitting fish: ultrastructure of the symbiosis. BioSystems 11: 269-280. Wei, S., and R. Young. 1989. Development of a symbiotic bacterial bioluminescence in a nearshore cephalopod, Euprymna scolopes. Mar. Biol. 103: 541-546. Reference: Biol. Bull, 179: 340-350. (December. 1990) Patterns of Stimulated Bioluminescence in Two Pyrosomes (Tunicata: Pyrosomatidae) MARK R. BOWLBY 1 . EDITH A. WIDDER : , AND JAMES F. CASE Marine Science Institute and Department of Biological Sciences, University of California, Santa Barbara. California 93106 Abstract. Pyrosomes are colonial tunicates that, in contrast with typical luminescent plankton, generate bril- liant, sustained bioluminescence. They are unusual in numbering among the few marine organisms reported to luminesce in response to light. Each zooid within a colony detects light and emits bioluminescence in response. To investigate the luminescence responsivity of Pyrosoma at/anticum and Pyrosomella verticillata. photic, electrical, and mechanical stimuli were used. Photic stimulation of 1.5 X 10 9 photons -s" 1 -cm" 2 , at wavelengths between 350 and 600 nm, induced bioluminescence. with the maxi- mum response induced at 475 nm. The photic-excitation half-response constant was 1 . 1 X 10 7 photons -s~' -cm' 2 at 475 nm for P. atlanticwn; P. verticillata had a signifi- cantly higher half-response constant of 9.3 X 10 7 pho- tons- s" 1 cirT 2 . Individual zooids within a colony, how- ever, appeared to have different half-response constants. Stimulus strength influenced recruitment of zooids and, in turn, luminescent duration and quantum emission. Image intensification revealed saltatory propagation of luminescence across the colony, owing to photic triggering among zooids. Repetitive, regular mechanical or electrical stimulation elicited rhythmic flashing characterized by alternating periods of high and low light intensities. Introduction Pyrosomes are holoplanktonic colonial tunicates found at depths to 1000 m (Soest, 1981). Their remarkable ca- pacity to luminesce and their occasional presence in very Received 18 May 1990; accepted 25 September 1990. ' Present address: Department of Neurobiology, Harvard Medical School, 220 Longwood Ave.. Boston. MA 021 15. 2 Harbor Branch Oceanographic Institution. Fort Pierce, FL 34946. large numbers at the ocean surface occasioned T. H. Huxley to write in his diary in 1849: "I have just watched the moon set in all her glory, and looked at those lesser moons, the beautiful Pyrosoma, shining like white-hot cylinders in the water" (Huxley, 1936). Colonies may be of highly variable size, reaching lengths of 30 m in some species (Griffin and Yaldwyn, 1970), owing to their growth habit of budding successive rings of zooids around the periphery of an elongating cylinder. The zooids are arranged so that their exhalent currents are conducted into the hollow core of the cylinder to gen- erate a communal locomotor current. Each zooid contains a pair of luminescent organs, bilaterally flanking the in- current siphon, and lying at the periphery of the colonial cylinder (Panceri, 1873). The organs are external to the pharyngeal epithelium, but protrude into the pharyngeal cavity (Mackie and Bone, 1978). Closely packed cells in the luminescent organs are filled with luminous organelles, which may be intracellular luminescent bacteria (Pier- antoni, 1921; Neumann. 1934; Buchner, 1965; Mackie and Bone, 1978). Bacterial luciferase activity similar to that of the luminescent bacteria Photobacterium has been found in Pvrosoma sp. (Leisman el ai, 1980). The mechanism of luminescence propagation within the colony is not neural. Neither innervation nor an ep- ithelial conduction pathway is in evidence (Mackie and Bone, 1978). Photic stimuli are presumably received by a photoreceptor lying just above the brain, triggering brain-induced arrests of gill basket cilia (Buchner. 1965; Mackie and Bone, 1978). This ciliary arrest, and the con- comitant gill collapse, might reduce blood flow and de- crease the supply of oxygen or metabolites to the light organ, thereby indirectly controlling light emission (Mackie and Bone, 1978; Mackie, 1986). 340 PYROSOME BIOLUMINESCENCE 341 Pyrosomes have the remarkable ability to detect exter- nal light flashes and to respond by luminescing (Polimanti. 1911). Colonies respond to conspecifics( Burghause, 1914) and simulated bioluminescence (Mackieand Bone, 1978). Localized stimulation of one area of a colony produces a wave of light that travels across the colony from the point of stimulation (Panceri, 1873). Intact tissue connections are not necessary among zooids, indicating that wave propagation within the colony may occur photically (Burghause. 1914; Mackie and Bone, 1978). Luminescence may also be photically induced in other organisms, including ctenophores (Labas, 1980), ostracods (Tsuji ci a/.. 1970). copepods (Lapota ci a/.. 1986), eu- phausiids (Kay, 1965: Ten, 1969, 1972), and a decapod shrimp (Herring and Barnes, 1976). Pulsed, colored light at 700 m in situ enhanced bioluminescence activity from unidentified organisms (Neshyba, 1967). Some of these organisms undergo inhibition of their luminescence when exposed to constant, "bright" illumination (Burghause, 1914;Nicol, 1960). The organisms used in this study were Pyroxoma at- lanticwn Peron, a cosmopolitan form, and Pyrosomella verticillata (Neumann), which occurs in tropical and sub- tropical waters (Soest, 1981). Although we investigated bioluminescence produced by photic, electrical, and me- chanical stimulation, photic stimulation was our major concern because it is the least well understood among the bioluminescence excitatory modes, has never been inves- tigated quantitatively, and has significance in the inter- pretation of the roles of bioluminescence in the behavior of marine animals. In our work, photic stimuli varying in wavelength and irradiance were related to their bio- luminescent responses and compared with the effects of other stimulus modes. The pattern of the luminescent wave and the mode of its transmission through the colony are discussed. A preliminary report of this work has ap- peared (Bowlby and Case, 1988). Materials and Methods Spec 11 ut'n collect ion Mature specimens of Pyrosoma atlanticwn and Pyro- somella verticillata were studied during July 1986 and 1987, aboard the R.V. New Horizon off the southwest coast of Oahu, Hawaii. Collections were made at approx- imately 21 N 158 W, with an opening-closing Tucker Trawl (length, 30 m; mouth, 10 m : ). The trawl was equipped with an insulating cod end (Childress el al., 1977), towed at depths ranging from 400 to 800 m, and brought to the surface every 4 to 6 hours. Specimens were sorted under ambient light, and maintained in darkness in 12C seawater for 8 to 24 hours. The colonies studied ranged in length from 1.4 to 7.7 cm; surface area was calculated from measurements of colonial length and di- ameter. Colonies up to 30 cm in length were captured, but were not included in the investigation due to space limitations in the experimental apparatus. Experimental procedure Individual colonies were placed in covered Plexiglas chambers holding 25 to 125 ml of filtered seawater. The dimensions of the chamber substantially exceeded those of the colony to minimize luminescence induced by con- tact with the container. A 25-cm diameter integrating sphere, coated internally with white Polane polyethylene paint (97% reflectance at 500 nm), surrounded the animal to insure maximal reflectance and detection of biolumi- nescence irrespective of orientation (Latz et al., 1987). Bioluminescence was detected by a photon counting pho- tomultiplier tube (RCA Model 8850), which viewed the interior of the sphere through a 4.5-cm diameter port. A baffle between the source and detector only allowed light that had undergone multiple reflections within the sphere to be measured. This apparatus provides a directionally unbiased, quantifiable measure of bioluminescence from non-isotropic sources. Radiometric calibrations were made with an Optronics Laboratory Model 310 multifilter calibration source referenced to an NBS standard. At sea, the system calibration was maintained with a C' 4 phos- phor referenced to the Optronics source. The calibration corrected for the spectral responsivity of the sphere and photomultiplier tube, as well as for the bioluminescence spectrum of both pyrosome species, as measured by an optical multichannel analyzer (Widder et al., 1983). The photomultiplier signal was monitored for 40 to 200 s with a Norland Model 5400 multichannel analyzer (MCA) and stored on a diskette in a microcomputer for subsequent analysis. Temporal resolution ranged from 10 to 50 ms per channel. Bioluminescence was stimulated by light, electrical, and mechanical excitation. Electrical stimuli (0.5-50 Hz, 5 ms duration, 50 V) from a Grass S48 stimulator were delivered by tungsten electrodes projecting into the chamber. A 1-cm diameter fiberglass rod driven by a so- lenoid to produce a displacement of 1 cm in 0.5 s applied mechanical stimulation. In some trials the colony was stimulated with the rod manually until bioluminescence was no longer produced. Photic stimuli, produced by a Bausch and Lomb monochromator with a tungsten light source, were delivered through a 5-mm diameter fiber optic into the sphere. Stimuli entering the sphere were deflected by a stimulus baffle placed at 45 to the fiber optic, providing a uniform stimulus illumination over the entire colony. Stimulus wavelength (FWHM = 21 nm) was either varied between 350 nm and 800 nm (in 25 nm 342 M. R BOWLBY ET AL. Table I kinetics and intensities ofpyrosome llasht"< stimulated at effective wavelengths (350-550 nm) and irradiances (6.8 X W W 4.3 X 10'" photon^. s Species Latency (s) Rise time (s) 98% Duration (s) Maximum tlux (photons -s"') Mean emission (photons -s^') Quantum emission (photons -flash"') Pyrosoma atlanticum (n = 6) 1.4 0.2 4.9 1.6 16.0 3.8 1.2 x 10" 1.0 X 10" 4.8 x 10' 3.8 x 10' 1.4 10' 2 1.1 x I0' ; P\ '/v iMimella renicillaia (n = 9) 1.4 0.1 4.3 1.1 11.6 2.9 1.1 X 10' 4.3 x 10 9 4.5 x 10 9 2.1 x 10' 1.0 x 10" 5.0 X I0' Values represent the mean standard error of the mean. Means are not significantly different between species (Mest, P > 0.05). increments) at constant quantal irradiance, or irradiance was varied with neutral density niters, between 6.8 X 10 6 and 4.3 X 10' photons -s" 1 -cm 2 , at constant wave- length. The stimulus duration was controlled by a Uniblitz electronic shutter at 0.5 s for all trials. Individual colonies were allowed to dark adapt for a minimum of one hour before testing. In preliminary trials to determine the op- timum interstimulus period, colonies produced less con- sistent responses (flash strength and number) to inter- stimulus periods of less than 3 min. Increasing the inter- stimulus interval beyond 5 min made no further improvement in response uniformity. Consequently, stimuli were delivered every 4 to 5 min, with the colony remaining undisturbed in the light-tight sphere during the interstimulus periods. The temperature of the seawater gradually increased from 12 to approximately 18C during an experimental session, but no change in excitability was observed. Colonies produced few flashes in the absence of applied stimuli. Stimulus irradiance was calibrated with a radiometer (United Detector Technology, Model S370) equipped with o 0.3- *O A x "*" a * 02- 1 1 - 0- D l'6 20 30 4C Time (s) 10 20 30 Time (s) Figure 1. Varying luminescent responses ofpyrosome colonies to photic stimuli at 475 nm, measured in an integrating sphere. The first flash is the stimulus artifact: it is followed by the bioluminescent response after a brief latency. The height of the stimulus flash does not represent the true intensity ot the stimulus, due to detector saturation. (A) A simple response from Pyrosomella vcmcillata. due to simultaneous zooid light production. (B) A complex response from Pyrosoma atlanticum with two distinct peaks of luminescence. a silicon photodiode detector and a 180 cosine diffuser in the test specimen position. The presence of the stimulus baffle created a uniform diffuse stimulus: therefore, the radiant energy arriving at the surface of the sphere, as measured with the silicon photodiode cosine collector, is a measure of spherical irradiance. Because the pyrosome tissue is very clear, the zooid light receptor receives input from all directions, so the measured irradiance was mul- tiplied by four to convert to scalar irradiance which is the energy per area arriving at a point from all directions about the point (Tyler and Preisendorfer, 1962). Stimulus irradiance was finally converted into quantal units, as ( 1 ) the number of photons may be more important than total energy in stimulating pyrosomes to produce light, and (2) to aid in comparisons with bioluminescence measure- ments. Measured flash characteristics were: (a) Latency time from stimulus onset to flash onset; (b) Rise time time from flash onset to maximum photon flux of the flash: (c) 98% response duration time from flash onset to when photon flux has declined to 2% of maximum; (d) Maximum flux maximum flash intensity; (e) Quantum emission total integrated photons emitted over 98% response duration: and (f) Mean emission average integrated photons per second emitted during 98%- response duration. Images were intensified with an ISIT (Dage) low light level video camera with a 105 mm Nikon f/4 lens. Spec- imens were placed in Plexiglas chambers and enclosed in a light-tight container with white reflective internal sur- faces. A photon counting photomultiplier system viewed the interior of the box, permitting simultaneous recording of relative flash kinetics and intensified video images. In some cases, specimens were examined with a dissecting microscope, with the ISIT camera recording the image through the photographic tube. Stimuli identical to those described above were used to elicit bioluminescence. PVROSOME BIOLUMINESCENCE 343 P atlanticum P verticillata -A- 300 400 500 600 700 Stimulus wavelength (nm) Figure 2. Normalized spectral responsivity ofPyrosoma atlanticum and PyrofiimiclUi vcrticillaia. Mean relative response magnitude is plotted as a function of stimulus wavelength; error bars represent standard errors of the mean. The stimulus scalar irradiance (500-ms pulse) for I' allan- //<.; was 1.5 x I O g photons -s~' cm" 2 , and 7.1 . I O 8 photons -s~'- cm 2 for P \-crlicilliiia Peak response for both was at approximately 475 nm. Data fit a quadratic regression for both species, according to the equation y = -19.07 + 0.0887x - (9.82 x l(T 5 )x 2 . r = 0.98 for P. allanliaim. and y = -8.34 + 0.039x - (4.32 X Ifr-V- r = 0.98 for P. vcrnalltiui. n = 4 colonies of each species. The resulting MCA recorded waveform was stored and analyzed as previously described, while the video images were viewed at slow speed to analyze the propagation of signals. Video images were enhanced with a Megavision 1024XM image-analysis system for final presentation. The relative light emission of individual zooids was also ex- amined with the image analysis system. In this analysis, the gray scale of the luminescent signal indicated the rel- ative flash intensity of the region measured. This analysis was performed only on: ( 1) data collected with the ISIT video camera set to the manual gain setting, and (2) data not saturating the gray scale levels. Results Photic stimulation In response to light stimuli, colonies often produced 25 to 30 flashes over approximately a 2-h period. Char- acteristic flashes had long latencies and durations and large quantum emissions (Table I). Flash latency and rise time were much less variable than quantum emission. Kinetic values for the two species (Table I) were not significantly different (ANOVA, P > 0.05). Light emission was inde- pendent of the colony surface area. Colonies responded to spatially diffuse photic stimu- lation with varying flash displays, ranging from the most commonly observed simple flash (Fig. 1A), in which the responding zooids react approximately simultaneously, to more complex emission patterns (Fig. IB). Such pat- terns may result from a variable latency in response to the initial stimulus, or to zooid reexcitation after a re- fractory period. The spectral responsivity curves for both P. atlanticum and P. verticillata lay between 400 and 550 nm, and are described by a quadratic regression (Fig. 2). The spectral responsivity maxima were approximately 475 nm. The half-response constant of photic excitation was de- termined by exposing specimens to between three and eight stimuli of identical scalar irradiance. The percentage of stimuli eliciting a response to 475 nm, regardless of magnitude, was plotted as a function of stimulus scalar irradiance (Fig. 3). Most stimuli elicited a response in either or 100% of trials, except in a narrow range of irradiances. Three wavelengths were examined (graphs not shown for 400 nm and 600 nm). with similar response patterns observed. Investigations of single visual receptor cells in insects produce similar results (Laughlin and Hardie, 1978; Har- die, 1979). In insects, the intensity response function fol- lows the form V/V (1) where I is the stimulus intensity. V is the response am- plitude, V mav is the maximum response amplitude, and 100 c o Q. 60 40 20 P. atlanticum P, verticillata __j 10 100 1000 Scalar irradiance (photons s' 1 cm" 2 x 10 7 ) Figure 3. Bioluminescent response to 475 nm photic stimulation. The percentage of stimuli that elicited a response is shown as a function of the log of stimulus scalar irradiance. Data are grouped for all specimens. Calculated half-response constants are shown in Table 11. Responses between and 100% fit the linear regression y = 10.13 + (1.9 X 10~ 6 )x, r = 0.73 for Pywaoma ulluinuwii: and y = 4. 12 + (3.2 x 10~ 7 )x, r = 0.91 for Pyroaimiella verticillata n = 3 colonies of each species. 344 M. R. BOWLBV El I/ H is the sensitivity parameter and equals the reciprocal of the intensity required to produce a response 50% of max- imum (Laughlin, 1975). The half-response constant is the level at which the slope of the V/log I curve is maximal, and is denned in this study as the threshold level of photic stimulation that produces a bioluminescent response. Us- ing formula (1), the half-response constant for P. atlan- ticinn to 475 nm was significantly lower than for P. ver- licilluui (Table II; Mest of slopes and points of linear regressions. P < 0.05). Half-response constants to 400 and 600 nm stimuli were not significantly different (Table II). The half-response constant to photic stimulation was in- dependent of the overall colony length. Colony quantum emission and flash duration were proportional to stimulus irradiance. Relative quantum emission (Fig. 4A) and 98% flash duration (Fig. 4B) varied logarithmically with stimulus scalar irradiance. The max- imum and mean emissions, though, did not vary consis- tently with the stimulus scalar irradiance. Thus the scalar irradiance effect on flash duration may account for the change in quantum emission. To clarify these relation- ships, the relative light emission of individual zooids was examined with the image analysis system. Individual zooids in unvarying orientation during flash events elicited by photic, mechanical, and electrical stimuli had remark- ably constant flash intensities. The quantum emission per zooid varied by less than 10% among flashes, independent of the colony quantum emission. Differences between flashes were often less than 2%. Light emission of zooids began to decrease only after about 10-15 flashes spaced about 30 s apart. Image analysis also revealed that more intense or repeated stimuli caused increasing numbers of zooids to respond asynchronously, thus increasing the to- tal flash duration of the colony. This relationship between stimulus scalar irradiance and the fraction of zooids re- sponding is evidence for a variation in the half-response Table II Half-response constants (photons s~' cm'-) lo photic stimulation C' m 0.05). (B) Relative 98% flash durations. The slope of the logarithmic regression for P. allanticiim is 0.10 (r = 0.86); that for P verticillata is 0. 16 (r = 0.91 ). These slopes are significantly different (Mest, P < 0.05). constant among zooids. Thus, the dependence between quantum emission and stimulus scalar irradiance is due to the asynchronous triggering of greater numbers of zooids, leading to longer colonial flash durations. Image intensification revealed strikingly different co- lonial patterns of luminescence in the two species inves- tigated. In P. atlanticum, small and large zooids are in- termixed throughout the colony. This pattern is evident as an irregular pattern of small and large luminescent sources distributed over the colony surface (Fig. 5 A). The zooid light organs lie close together, often producing ap- PYROSOME B1OLUMINESCENCE 345 parent single points of light, which arc resolved under higher magnification into pairs of luminous sources. P. verticillala. in contrast, possesses zooids of uniform size in distinct rows, with a wider spacing between the pair of light organs in each zooid. This results in uniform rows of luminescent sources over the colony surface (Fig. 5B). These obvious differences in light patterns, rooted in the colony morphology, make the two species easily distin- guishable by their luminescent patterns. Bioluminescence propagation ISIT video records of events caused by a single me- chanical stimulus revealed that luminescence begins from the point of stimulation and slowly spreads across the colony in all directions, at an overall rate of 2.1-4.1 mm s~', at temperatures of 12 to 16C (Fig. 6). The light usually travels across the colony by saltatory conduction, the nodes being either single zooids or groups of zooids 0.5 to 1.5 cm apart, with a latency between nodes of about 3 s. Zooids between the responsive regions begin to lu- minesce after being bypassed, while the wave continues to the next responsive site. This disjointed wave of bio- luminescence is characteristic of both species examined. Electrical stinu//ali< i Single electrical stimuli produced flashes of simple shape, with few irregularities in the waveform (Fig. 7 A; Table III). Temporal summation of luminescence was in- duced by electrical pulses at 0.5 and 1 Hz (Fig. 7B). Video analysis revealed that this summation was due to increas- ing recruitment of zooids with successive stimuli and, to a lesser extent, an increase in zooid light emission. In trials with constant stimulus rates of 5 to 50 Hz, lumi- nescence was produced initially, and was often followed by a series of shorter, repeating flashes (Fig. 7C) with an average intertlash period of 18 s. In a few trials, however, light was elicited at a similar initial rate, but no repetitive flashing pattern was observed (Fig. 7D). Multiple stimu- lation elicited a significantly larger response duration, maximum flux, and mean emission than single pulses (/- test, P < 0.05; Table III). Rise time, quantum emission, and flash duration in response to electrical stimuli were significantly different from these parameters for photic excitation (Tukey test, P < 0.05). The maximum flux and quantum emission were again independent of colony sur- face area. Mechanical stimulation Repetitive mechanical stimulation induced a signifi- cantly greater light emission than any other stimulus method employed in this study (Tukey test, P < 0.05; Table IV). The total duration of light emission in all cases exceeded the 200-s collection period, with a mean flash duration of 59 s. Within this period a repeating flash pat- tern, with similar kinetics to that for electrical stimuli, was observed (Fig. 8A). Many colonies also produced bioluminescent flashes with simple kinetics (Fig. 8B) in the absence of any obvious external stimuli except ship movement (Table IV). Bio- luminescent events of this type occurred randomly during the interstimulus resting periods and were thus easily sep- arated from flashes elicited by photic or other stimuli. Unlike photically or electrically stimulated organisms, the light emission for mechanically induced bioluminescence was directly related to the colony surface area (Fig. 9). Figure 5. Single ISIT video frames of patterns of luminescence in response to photic stimuli. Arrangement of luminous sources is based upon colony morphology. (A) Pyrosoma atlanlicum. showing an irregular pattern of luminescent sources. Bar = 1 cm. (B) Pymsomella venicillala, exhibiting uniform distribution of luminescent sources. Bar = 0.5 cm. 346 M. R BOWLBY ET AL Figure 6. A bioluminescent wave traveling across Pyr.\tiui allanlicum. The colony is oriented in the same position in (A) through (F). (A) Image of the colony with red illumination. Arrow indicates the point of mechanical stimulation. Bar = 1 cm. (B) The flash begins at the point of stimulation (time = s). (C) After 3 s the luminescent response has spread to other nearby zooids. Progressive bidirectional conduction of the light wave across the colony at (D) 6 s and (E) 9 s after the beginning of the response. (F) Decay in intensity of the response (time = 15s). Therefore, most zooids in the colony probably responded to mechanical stimuli, in contrast to responses to the other stimulation methods. Discussion Bioluminescence of pyrosome colonies begins at the location of the stimulation, and slowly propagates by a photic, saltatory conduction process in all directions. The photic propagation of light along the colony is supported by several pieces of evidence. First, the similarity between our action spectrum and the luminescent emission spec- trum (Swift ct ui. 1977; Widder et <;/.. 1983) indicates that the colony responds to, and emits, the same wave- lengths of light. Second, light from one zooid is also easily able to surpass the half-response constant of other nearby zooids. According to Allard's law E x = Ie- c Yx 2 , (2) light of intensity I will be attenuated in the sea, at a dis- tance x, to an irradiance E, where c is the light attenuation coefficient (Jerlov, 1968). Using formula (2) and an at- tenuation coefficient of 0.05 for Type I Hawaiian waters (Jerlov, 1968), the approximate luminescent output of an individual P. vertidllata zooid, calculated from the colony maximum flux and total number of zooids, decays to the colonial photic half-response constant at 2.6 m, assuming no absorption or scattering due to pigments in the colony. Finally, the variation among zooids in their half-response constants reflects their ability to respond independently to light. Progression of the colonial luminescent wave is not de- pendent upon intact connections between the zooids, clearly indicating that the wave propagates by a photic process (Burghause, 1914). Mackie and Bone (1978) photically stimulated the tetrazooid of P. at/anticum and calculated that, if the luminescent wave were propagated PYROSOME BIOLUMINESCENCE 347 4- A 3- 2- X *- 1- 'co n. 3- 2- 40 80 120 160 200 20 40 60 80 9H 6J O CL 3- 6- 3- 40 80 120 160 200 Time (s) o- 20 40 60 80 100 120 Time (s) Figure 7. Examples of electrically induced bioluminescence of Py- rimtmella verticillata. Fifty-volt, 5-ms duration pulses were applied to the medium. (A) Single stimulus delivered at 80s, 120s, and 165 s; each produced a simple response. (B) Stimuli delivered at 1 Hz for 60 s. starting at time = 0, showing temporal summation. (C) Constant 5 Hz stimu- lation, yielding a regular (lashing pattern, perhaps due to an 1 X-s refractory period characteristic of the zooids. (D) Constant 10 Hz stimulation, elic- iting light at a similar initial rate to (C). but without a regular flashing pattern. by the serial excitation of zooids, the colony propagation velocity, taking into account the response delay and the distance between zooids, would be 2.0-4.0 mm s~'. This value is remarkably similar to the observed saltatory propagation rate of 2. 1-4. 1 mm s ' found in this study of mature colonies. Mackie and Bone also found no nerves or gap junctions associated with the light organ, and deemed it unlikely that conducting epithelia could prop- agate this activity, because the normal rate of epithelial conduction in tunicates is about 20 cm s* 1 . In addition, no specific cellular depolarizations were associated with Hashing, and the mantle epithelium, to which the light organ is attached, is not a conducting type in the taxon- omically similar ascidians. Luminescent waves typically propagate in the colonial coelenterate Renilla at 6-10 cm-s ' (Nicol, 1955: Morin and Cooke, 1971), and 20- 50 cm s 'in hydrozoa (Widder et al.. 1989). These high propagation rates are enabled by the underlying nervous tissue. These data indicate that the luminous wave is propagated by a photic chain reaction. The mechanism underlying the saltatory conduction of luminescence may derive in part from the different photic half-response constants of the zooids. This is a rea- sonable finding, as each zooid contains its own light de- tection and production organs (Bone and Mackie, 1982; Mackie, 1986). The zooids thus seem to act independently of one another, rather than as a single, integrated colonial receptor. The variation in colony half-response constant may be due in part to colony size. Larger colonies necessarily ab- sorbed a larger number of photons entering the sphere; less light is therefore incident per zooid for a given number of photons. Thus, half-response constants may have been artificially elevated for larger colonies. Continuous excitation of a colony often produced a rhythmic colonial flashing pattern, characterized by al- ternating periods of high and low light emission. Although some light is produced between flashes under these con- ditions, the majority of zooids are quiescent. Zooids thus appear to possess a refractory period of about 1 8-s dura- tion, during which their half-response constant is greater than the stimulation that they receive. The existence of a refractory period is further supported by the quenching of a single wave of luminescence elicited by a local mechanical stimulus. In long colonies, light Table III Kinetics and intensities oj Pyrosomella verticillata flashes stimulated with single or repetitive electrical pulses. 50- 1 ', 5-m.v pulses were applied in the medium \~alnes represent the mean standard error o) the mean Stimulus type Latency (s) 98% Rise time Duration (s) (s) Maximum flux ( photons -s~') Mean emission (photons -s' 1 ) Quantum emission (photons- flash"') Single (n = 4) 5.6 2.1 0.3 8.8 1.6" 2.3 x 10' 6.6 x 10 9 6.1 x I0 9a 1.3 x 10 9 5.9 > 10' 2.1 X 10'" Multiple (n = ID 3.4 1.2 10.1 2.7 78.3 21.8 a - b 6.6 x 10' 1.4 x 10' 2.7 X 10' 0a 5.7 X 10 9 3.7 x 10' : 1.5 X 10 12 * Means are significantly different ((-test, P < 0.05). h Mean underestimates the actual value, because some responses persisted beyond the data collection period. 348 M. R. BOWLBY /;7' .11. Table IV A'mtY/o and inli'H( mechanically Mimulnlcd /'irosomc tla\hc\. I 'allies represent llic mam standard error Species Stimulus type Rise time (si 98% Duration (s) Maximum flux (photons -s~') Mean emission ( photons -s~') Quantum emission (photons- Hash" ') l'vm\i>ma atlanticum Constant prodding (n = 9) 20.0 1.6 59.2 14.6 3.3 x 10' 2a 3.1 x 10" 6.6 x 10" 6.0 x 10" 2.3 X 10" 1.9 X 10" Pyrosome sp. Ship movement (n = 6) 8.3 3.0 25.2 6.7 7.5 x 10" a 6.8 x 10" 1.7 X 10" 1.3 X 10" 3.8 > 1() |: 2.7 x 10' 2 1 Means are significantly different between stimulus methods (Mest, P < 0.05). from the stimulus area was extinguished when the wave of light was at the far end of the colony. As a zooid's maximum flux decays to the colonial half-response con- stant at about 2.6 m. the wave of light should be able to reexcite the previously responsive parts of the colony. Reexcitation, however, is rarely observed, with a colonial response to a single stimulus usually subsiding after one pass across the colony. The bioluminescent response to light flashes implies several uses of light for the colony. Using Allard's law for Hawaiian waters, the maximum flux for P. atlanticum (mechanical stimuli) would decline to the photic half-re- sponse constant at 78 m. Few zooids in a colony would respond, however, to this dim level of luminescence. The maximum quantum emission observed to photic stimuli in this study could be induced at a distance of 17 m, indicating that flash entrainment among colonies may occur at large distances. Roe ct al. ( 1987) report a max- imum of 85 colonies per 10,000 m' at 800 m in the At- lantic near the Canary Islands. The model of closest pack- ing of equal spheres allows for one colony every 5.5 m, indicating that, in some areas of the ocean, flash entrain- o o .c CL O o Q. 40- 30- \ A 20- I | 10- n \ ill I II 3- 40 80 120 160 200 Time (s) 10 20 30 Time (s) 40 Figure 8. Examples of luminescence by Pyrosoma atlanticum in- duced by mechanical stimulation. (A) Periodic stimuli (1-2 Hz) produced a regular flashing pattern similar to that caused by repetitive electrical stimulation. Individual flashes within the pattern averaged 59-s duration. (B) A simple flash presumably induced by ship motion. ment among colonies may produce widespread displays (Mackieand Mills, 1983). Flash entrainment may also occur interspecifkally. Most other planktonic organisms produce biolumines- cence of the same wavelengths as that of pyrosomes (Young, 1981; Herring, 1983; Widder et al.. 1983; Latz el u/.. 1988); this light could also stimulate pyrosome lu- minescence if it were of sufficient intensity. For example, a flash from the common copepod Pleuromamma xipluas in Hawaiian waters would be sufficient to elicit lumines- cence in P. atlanticum at 14 m (Latz et al., 1987). Luminescence is often produced in response to a dis- turbance by a predator, and is conventionally thought to confer protection by startling or blinding the predator, or by attracting a secondary predator (David and Conover, 1961; Monn, 1983; Young, 1983; Buskey and Swift, 1983, 1000 r o Q. E 'x 03 100 co co c o 'o Q. 10 10 20 30 A Colony surface area (cm 50 Figure 9. Maximum bioluminescence production as a function of colony surface area, including both species. Luminescence was induced by mechanical stimulation, as this method produced the greatest light emission and thus most closely approximated a colonial response of all zooids. The slope of the exponential regression is 0.15 (r = 0.55). PYROSOME BIOLUMINESCENCE 349 1985). Pyrosomes also display an additional set of behav- iors in response to photic stimuli; zooids close their oral openings, arrest their cilia (resulting in the suspension of locomotion), and produce luminescence (Mackie and Bone, 1978: Mackie. 1986). Being negatively buoyant, the colony would sink into deeper layers until the recom- mencement of ciliary action, thus perhaps evading pre- dation by leaving a depth of high predator density (Mackie and Bone, 1978). Flashing may serve as a means of com- munication between distant zooids or colonies, enabling them to close protectively and sink before oncoming harmful stimuli can arrive. Photically stimulated biolu- minescence may also discourage predators by making the colony, or a group of adjacent colonies, loom up out of darkness, perhaps giving the impression of a very large source that should not be trifled with. Simultaneous lu- minescence from many spatially separated sources might also distract a predator from a single target, analogous to the simultaneous displays of fish schools in the photic zone(Radakov, 1973; Morin, 1983). Acknowledgments The authors are grateful to the captain and crew of the RV New Horizon and to J. Favuzzi, T. Frank, M. Latz, A. Mensinger, and S. 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K 57:817-823. van Soest, R. W. M. 1981. A monograph of the order Pyrosomatida (Tunicata, Thaliacea). ./ Plunk. Rc\ 3(4): 603-631. Tett, P. B. 1969. The effects of temperature upon the flash stimulated luminescence of the euphausiid Thysanoessa raschn J Mar. Biol. Assoc. U. K. 49: 245-258. Tett, P. B. 1972. An annual cycle of flash induced luminescence in the euphausiid Thysanoessa rasehn. Mar. Biol. 12: 207- 218. Tsuji, F. I., R. W. Lynch, and Y. Haneda. 1970. Studies on the bio- luminescence of the marine ostracod Btol Hull. 139: 386-401. Tyler, J. E., and R. W. Preisendorfer. 1962. Transmission of energy within the sea. Pp. 397-451 in The Sea, M. N. Hill. ed. Interscience. New York. \\ idder, E. A., S. A. Bernstein, D. F. Bracher, J. F. Case, K. R. Reisen- bichler, J. J. Torres, and B. H. Robison. 1989. Bioluminescence in the Monterey Submarine Canyon: image analysis of video recordings from a midwater submersible. Mar. Biol. 100: 541- 551. \\ idder, K. A., M. I. Latz, and J. F. Case. 1983. Marine hiolumines- cence spectra measured with an optical multichannel detection sys- tem. Hiol Hull 165: 791-810. Young, R. K. 1981. Color of bioluminescence in pelagic organisms. Pp. 72-81 in Bioluminescence: Current Perspectives. K. H. Nealson, ed. Burgess Publishing, New York. Young, R. E. 1983. Oceanic bioluminescence: an overview of general functions. Bull. Mar. Set. 33(4): 829-845. Reference: Biol Bull 179: 35 1-357. (December. 1940) Orcadian Rhythmicity of the Crustacean Hyperglycemic Hormone (CHH) in the Hemolymph of the Crayfish JAN1NE L. KALLEN, S. L. ABRAHAMSE, AND F. VAN HERP Zoiilogiscli Lahonitorniiu. Faculleil Natuurwetenschappen, Katholieke Univcrsiteit. Toemooiveld, 6525 ED Nijmegen. The Netherlands Abstract. The crustacean hyperglycemic hormone (CHH) is involved in the regulation of endogenous blood glucose metabolism. In this paper we describe the daily rhythmicity in the blood glucose and the blood CHH con- tent of the crayfish Orconectex linwxns. Both blood CHH and blood glucose levels increase during the first hours after the beginning ot darkness. The bioactivity of released CHH is far higher than that of CHH stored in the sinus gland. Moreover, the released hyperglycemic material shows an affinity for high molecular weight proteins in the hemolymph. Preliminary results suggest that subunits of hemocyanin may act as potential carrier-proteins for bioactive CHH. Introduction The neuroendocrine system producing the crustacean hyperglycemic hormone (CHH) of decapod crustaceans forms part of the medulla terminalis ganglionic X-organ (MTGX). The MTGX lies at the outer edge of the medulla terminalis the most proximal optic ganglion in the eyestalk and contains several hundred neuroendocrine cells. In crayfish, such as Astacnx leptodactylus and Or- conectes limosiis, about 35 to 40 CHH-producing cells form a distinct group located latero-ventrally on the MTGX. Neurosecretory granules containing CHH are transported via a tract that leads across the neuropil of the medulla terminalis to a neurohemal region, the sinus gland. Immunocytochemical and morphometric research indicates that about 40% of the sinus gland axon terminals Received 15 March 1990; accepted 25 September 1990. are filled with neurosecretory granules containing CHH, which is released into the hemolymph by exocytosis. The hyperglycemic hormone in the blood regulates blood sugar levels to meet physiologically required metabolic energy needs (Strolenberg and Van Herp, 1977; Strolenberg el at., 1977; Van Herp and Van Buggenum, 1979; Gorgels- Kallen and Van Herp, 1981; Gorgels-Kallen et at.. 1982; for a review see Kleinholz, 1985). The glucose level in the hemolymph of decapod crus- taceans reveals a day/night rhythmicity, characterized by a low basal level during the light period, and a peak in glucose content appearing several hours after the onset of darkness (Hamann, 1974; Strolenberg, 1979; Reddy et at.. 1981). The basal level during the day, as well as the height and duration of the nocturnal peak, are species- dependent and are affected by seasonal influences (unpub. obs.) as well as physiological events, such as molting (Kal- len, 1985). Physiological research in crayfish strongly in- dicates an endogenous circadian blood glucose rhythm entrained by the light/dark schedule (Kallen et at.. 1988). In previous studies, we investigated the secretory dy- namics of the CHH-system of Aslacus leptodactylus. Im- munocytochemical staining combined with morphomet- ric analyses at the light and electron microscopic level revealed a daily rhythmicity in the synthetic activity of the perikarya, the transport of CHH-material to the sinus gland, and the release of CHH into the hemolymph (Gor- gels-Kallen and Voorter, 1984, 1985). In this study, we report and discuss the immunochemical detection of cir- culating bioactive CHH in the hemolymph during a 24- h period. We present a preliminary molecular character- ization of the bioactive CHH present in the blood and 35! 352 J L. K.ALLEN ET AL compare it to the molecular form of the hormone stored in the sinus gland 1 . Materials and Methods \niinuls Crayfish (Orconectes liminrU"~ //wnvin). T = total hemolymph; 31, 32, 33 = immuno- and bioactive hemolymph fractions: 25-27 and 35-40 = immuno- and bioactively negative hemolymph fractions. (A) Coomassie Brilliant Blue staining. (B) Immunoblotting with &nti-Orconectes- CHH mouse serum. levels during daytime and an increase of blood CHH con- tent during the first hours of darkness. In previous studies on the diurnal cycle of the CHH cells in Astacus, we gath- ered information on the secretory dynamics of the peri- karya and the rate of exocytoses of CHH granules, both events preceding nocturnal hyperglycemia (Gorgels-Kal- len and Voorter, 1985). The increased blood CHH content described in this paper for Orconectes occurs in the period of expected high exocytosis of CHH into the hemolymph. Our results show further that the application of a DAS- ELISA is a suitable method for determining hormone lev- els in crustacean hemolymph. Previously, the ELISA- technique was successfully applied by Quackenbush and Fingerman (1985) to determine the level of black pigment dispersing hormone (BPDH) in the blood of the fiddler crab. Our ELISA results are presented as optical densities. If we compare those with a standard curve of purified CHH, blood CHH levels during the day are estimated at about 1 ng per 100 n\ hemolymph. The nocturnal peak in blood CHH content is comparable to about 10 ng pu- rified hormone per 100 jul hemolymph. However, super- fusion experiments have shown that the released bioactive CHH-peptide undergoes molecular changes that increase its potency relative to the storage pool in the sinus gland (unpub. obs.). Furthermore, our results point to the affin- ity of the released CHH to high molecular weight proteins in the hemolymph. We can only speculate about the pos- sible effect of these molecular changes on the immunodetectability of the hormone in the blood. Therefore we cannot presently draw any conclusions about the actual quantity of bioactive hormone in the hemolymph. Previous research on the chemical nature of CHH has focussed on the isolation, characterization, and physio- 356 J. L. KALLEN ET AL. logical effects of CHH material in the sinus gland. In this neurohemal organ, the agent causing hyperglycemia in various species of decapod crustaceans has been described primarily as a neuropeptide with a molecular weight of around 7000 Da (for a review see Kleinholz, 1985). Kegel ct al. (1989) described the amino acid sequence of the CHH (8524 Da) from the crab Cardnus maenus. Recent work in our laboratory on the sinus gland of the lobster Homams amcncanus has shown that several CHH and CHH-like molecular forms occur in this organ (Jensen et al., 1989). Furthermore, limited research on the chem- ical nature of newly synthesized CHH points to the pres- ence of a prohormone or precursor in the perikarya (Stuenkel, 1983: Van Wormhoudt et al.. 1984a, b; Kallen et al., 1986). Weidemann el al. (1989) sequenced the cDNA encoding a precursor for the CHH from the crab Cardnus maenas. Although we know much about CHH in the sinus gland, our knowledge of the chemical nature of the CHH material released into the hemolymph is extremely lim- ited. Our results have frequently pointed to substances of high molecular weight in the hemolymph that show strong affinity for hyperglycemic factors from the sinus gland. For instance, purification of sinus gland extract by gel nitration has always resulted not only in the purification of a hyperglycemic factor with a molecular weight of around 6500 Da. but also in immunological and biological activity in the void volume. Moreover, mixing the purified 6500-Da material with hemolymph always caused the low molecular weight form to disappear, leaving the immuno- and bioactivity exclusively in the high molecular weight void volume fraction (unpub. obs.). These observations, together with the strong immunopositive reaction in the hemolymph, encouraged us to search for more informa- tion about the molecular characteristics of the circulating hyperglycemia-producing material in the blood. Our preliminary results presented in this paper show that, after gel filtration of hemolymph on Sephadex G- 200 sf. high molecular weight proteins are detectable with both a CHH-immunopositive reaction and a strong hy- perglycemic activity. Moreover, the bioactivity of released CHH is far higher than that of the CHH stored in the sinus gland. This might be caused by molecular changes in the hyperglycemic hormone just before or after release. Stuenkel and Cooke (1988) suggested that only small amounts of neurohormones must be released into the blood to meet the physiological needs. These authors sug- gest the presence of a "readily releasable pool" that is distinguishable from the bulk of stored material. Our re- sults are consistent with this idea. They also point to the existence of a large non-active storage pool, as opposed to a small amount of bioactive neurohormone in the neu- rohemal organ. The high bioactivity of the CHH in the blood could also be caused by binding of the released factor to a carrier-protein. The results of SDS-PAGE and immunoblotting show immunoreactivity associated with several proteins of high molecular weight: 1 50, 80. 74, 72, and 56 kDa. The pattern of this electrophoresis cor- responds to the electrophoretic behavior of crustacean hemocyanins as described by Markl et al. ( 1979). Our standard analytical methods did not reveal any low molecular weight CHH-active proteins, but the pos- sibility of their presence should not be excluded. We in- tend to continue the search for the role of subunits of hemocyanin as potential carrier-proteins for bioac- tive CHH. Acknowledgments The authors thank Prof. Dr. J. M. Denuce and Dr. R. A. C. Lock for reading the manuscript. Secretarial as- sistance by Mrs. E. A. J. Derksen is gratefully acknowl- edged. Literature Cited Davis, B. J. 196-4. Disc electrophoresis. II. Method and application to human serum proteins. Ann. ,\ Y. Acad. Sci 121: 404-427. Gorgels-Kallen, J. L., and F. Van Herp. 1981. Localization of crus- tacean hyperglycemic hormone (CHH) in the X-organ sinus gland complex in the eyestalk of the crayfish Asiacits leptodactylus (Nord- mann. 1842). J. Morphol. 170: 347-355. Gorgels-Kallen, J. L., and C. E. M. Voorter. 1984. Secretory stages of individual CHH-producing cells in the eyestalk of the crayfish Aslantx leptodactylus, determined by means of immunocytochemistry. Cell Tissue Rex. 237: 291-298. Gorgels-Kallen, J. L., and C. E. M. Voorter. 1985. The secretory dy- namics of the CHH-producing cell group in the eyestalk of the crayfish. Asuicus li'pttidaelylus. in the course of the day/night cycle. Cell 7'miif Rex. 241: 361-366. Gorgels-Kallen, J. L., F. Van Herp, and R. S. E. \V. Leuven. 1982. A comparative immunocytochemical investigation of the crustacean hyperglycemic hormone (CHH) in the eyestalks of some decapod Crustacea. J Morphol 174: 161-168. Hamann. A. 1974. Die neuroendokrine Steuerung tagesrhythmischer Blutzuckerschwankungen durch die Sinusdriise heim Flusskrehs. / Comp. Physwi 89: 197-214. Kallen. J. L. 1985. The hyperglycemic hormone producing system in the eyestalk of the crayfish Asiaais leptodactylus. Thesis. Catholic University Nijmegen, The Netherlands. Kallen, J. I-., and S. I.. Abrahamse. 1989. Functional aspects of the hyperglycemic hormone producing system of the crayfish Orcneele\ lintuHix in relation to its day/night rhythm. Gen. Comp. Endocrinol. 74: 74 (abstract). kallen, J. 1,., F. M. J. Reijntjens, D. J. M. Peters, and F. Van Herp. 1986. Biochemical analyses of the crustacean hyperglycemic hor- mone of the crayfish Astacus lepiodactylus. Gen. Comp Endocrinol. 61: 248-259. Kallen, J. L., N. R. Rigiani, and H. J. A. J. Trompenaars. 1988. Aspects of entrainment of CHH cell activity and hemolymph glucose levels in crayfish. Biol. Bull 175: 137-143. Kegel, G., B. Reichwein, S. VVeese, G. Gaus, J. Peter-Katalinic, and R. Keller. 1989. Amino acid sequence of the crustacean hyperglycemic hormone (CHH) from the shore crab, Carcimm maenax. FEES Lett 255: 10-14. RELEASED CHH IN CRAYFISH 357 Keller, R. 1977. Comparative electrophorctic studies of crustacean neurosecretory hyperglycemic and melanophore-stimulating hor- mones from isolated sinus glands. J. Comp. Physiol. 122: 359-373. klriiiluil/. I.. II. 1985. Biochemistry of crustacean hormones. Pp. 463- 522 in The Biolo K v olCntsiaeea. Vol. 9, D. E. Bliss and L. H. Mantel, eds. Academic Press, New York. Laemmli, V. K. 1970. Cleavage of structural proteins during the as- sembly of the head of bactenophage T4. failure 227: 6X0-685. I.emen, R. S. E. \V., P. P. Jaros, K. Van Herp, and R. Keller. 1982. Species or group specificity in biological and immunological studies of crustacean hyperglycemic hormone. (.ien. Comp. Emii>- ermol. 46: 288-296. Lowry, O. H., N. J. Rosebrough, and A. L. Farr. 1951. Protein mea- surement with the Folin phenol reagent. J Hiol (.'hem 193: 265- 275. Markl, J., A. Hofer, G. Bauer, A. Markl, M. Keupter, M. Brezinger. and B. Linzen. 1979. Subunit heterogeneity in arthropod hemo- cyanins. II. Crustacea. J. Comp Phyiiol. 133: 167-175. Quackenbush, L. S., and M. Fingerman. 1985. Enzyme-linked im- munosorbent assay of black pigment dispersing hormone from the fiddler crab. L'ea piigtlalnr. Gen. Comp. Endocnnol 57:438-444. Reddy, C. S. D., M. Raghupathi, \ . R. Pursushotham, and B. P. Naidu. 1981 . Daily rhythms in levels of blood glucose and hepatopancreatic glycogen in the freshwater field crab Oiioielphu\a \ene\ \ene\ (Fa- bncius). Indian J E\p Biol. 19: 403-4(14. Slrolenberg, G. E. C. M. 1979. Functional aspects of the sinus gland in the neurosecretory system of the crayfish Axiaaia leptodactylus an ultrastructural approach. Thesis. Catholic University Nijmegen, The Netherlands. Slrolenberg, G. E. C. M., and F. Van Herp. 1977. Mise en evidence du phenomene d'exocytose dans la glande du sinus d'A.itacux lep- todaclvlus (Nordmann) sous ('influence d'injections de serotonine. C R Acad. Set. Pan* 284: 57-59. Strolenberg, G. E. C. M., II. P. M. Van Helden, and F. Van Herp. 1977. The ultrastructure of the sinus gland of the crayfish Axiaenx leplodaetylus (Nordmann). Cell Tisane Res 180: 203-210. Stuenkel, E. L. 1983. Biosynthesis and axonal transport of proteins and identified peptide hormones in the X-organ sinus gland neu- rosecretory system. J Comp Physiol. 153: 191-205. Stuenkel, E. L.. and I. M. Cooke. 1988. Electrophysiological charac- teristics of peptidergic nerve terminals correlated with secretion, t 'nrr Topics Neuroendocrinol 9: 123-150. Tensen, C. P., K. P. C. Janssen, and F. Van Herp. 1989. Isolation, characterization and physiological specificity of the crustacean hy- perglycemic factors from the sinus gland of the lobster. Hoiiiurnx amerieunna (Milne-Edwards). Im: Reprod. Dev 16: 155-164. Van Herp, F., and H. J. M. Van Buggenum. 1979. Imrnuno- cytochemical localization of hyperglycemic hormone (HGH) in the neurosecretory' system of the eyestalk of the crayfish Aslacus leplo- daelylns. Expcnenlia 35: 1527-1528. Van \\ormhuudt. A., F. Van Herp, C. Bellon-Humbert, and R. Keller. 1984a. Polymorphisme de 1'hormone hyperglycemique chez Pa- laeninn aerrulna (Crustacea, Decapoda, Natantia). Vllle Reunion des Carcinologistes de Langue Francaise, Liege 1983. Ann. Sue R. Zool. Belg. 114: 179-180. Van \\ormhoudl. A., F. Van Herp, C. Bellon-Humbert. and R. Keller. 1984b. Changes and characteristics of the crustacean hyperglycemic hormone (CHH material) in Palaemon terrains Pennant (Crustacea, Decapoda. Natantia) during the different steps of the purification. Comp. Binelicm I'/IVMO/ 79B: 353-360. Voller, A., D. E. Bidwell, and A. Burllett. 1979. The enzyme linked immunosorbent assay (ELISA). A guide with abstracts of microplate applications. Nuffield Laboratories of Comparative Medicine, The Zoological Society of London, p. 125. \\ eidemann, W., J. Gromoll, and R. Keller. 1989. Cloning and sequence analysis of cDNA for precursor of a crustacean hyperglycemic hor- mone. t'KBS Lett 257: 31-34. Reference: Biol Bull 179: 358-365. (December, 1990) Biochemical and Functional Effects of Sulfate Restriction in the Marine Sponge, Microciona prolifera WILLIAM J. KUHNS,* GRADIMIR MISEVIC, AND MAX M. BURGER Hospital tor Sick Children. Toronto, Ontario, Canada, Marine Biological Laboratory, Woods Hole, Massachusetts, ami the Frieilrich Miescher Institute. L'nivcrsity Hospital of Basel. Basel, Switzerland Abstract. The functional and biochemical consequences of sulfate restriction were studied in chemically dissociated Microciona sponge cells maintained in artificial seawater with or without SO 4 2 ~. In cells pre-treated to reduce pre- formed secretions, SO 4 2 deprivation reduced cell motility judged by the lack of aggregates in rotating or stationary cultures in comparison with controls. Microscopic ex- amination showed that cells that customarily demonstrate cytoplasmic processes, such as filopodia and pseudopodia, exhibited marked decreases in these cellular processes when maintained in SO 4 2 -deprived artificial seawater. Uptake and incorporation of 35 SO 4 2 ~ by disaggregated and pre-treated cells was higher under SO 4 2 ~-free conditions relative to controls; this effect was time dependent, rising to a maximum at 12 h, when a three- to seven-fold dif- ference could be demonstrated. 3 H-leucine incorporation indicated that protein synthesis was similar in test and control populations. Comparative high voltage electro- phoresis of supernatants containing 35 SO 4 macromole- cules from chemically dissociated cells indicated deficien- cies of such 35 SO 4 macromolecules if the rotated cells that released these secretions had been pre-treated in SO 4 2 ~ free artificial seawater. The results of SO 4 2 restriction suggest that secretion of macromolecules or Microciona aggregation factor (MAP), and aggregation and locomotion of Microciona cells depend upon an adequate extracellular source of Introduction Both vertebrate and in vertebrate cells require sulfated macromolecules on cell surface receptors and in intra- Received 25 April 1990; accepted 25 September 1990. * On leave, University of North Carolina School of Medicine. De- partment of Pathology. Chapel Hill, NC. SO 4 2 . sulfate transport, and sulfation of macromolecules such as polysaccharides. cellular fluid (Cassaro and Dietrich, 1977; Hogsett and Quantrano, 1978; Mulder. 1981; Klebe et a/.. 1986; Mulder et ai. 1987). For example, mesenchymal migra- tion of sea urchin embryos is blocked in situ in sulfate- deprived medium (Katow and Solursh, 1981), and cell motility and morphology in cell cultures have been influ- enced by sulfated glycosaminoglycans (Venkatasubra- manian and Solursh, 1984). Blebbing has been observed on cell surfaces of sea urchin embryos maintained in sul- fate-free seawater. but not the prolonged processes that accompany mesenchymal cell migration. In this instance it appeared that sulfate deprivation was capable of causing an inhibition of the formation of stable cell attachments to the basal lamina (Venkatasubramanian and Solursh. 1984; Akasaka et ai. 1980). The defect could be reversed by a 6-h pre-treatment in normal seawater. Sulfate availability appears to be particularly important during early embryogenesis and differentiation in several species (Cassaro and Dietrich, 1977; Katow and Solursh, 1981; Lindahl. 1942: Immers and Runnstrom. 1965; Wenzl and Sumper, 198 1 ). Particularly vital in this regard are the sulfated mucopolysaccharides: their presence cor- relates well with tissue-level organization and normal de- velopment (Wenzl and Sumper. 1981; Kinoshita and Saiga, 1979; Yamaguchi and Kinoshita, 1985). This interesting background prompted us to address sulfation, using as a model Microciona, a relatively well studied marine sponge (Humphreys. 1963, 1967; Henkart et ai. 1973; Burger et ai. 1975; Jumblatt et ai, 1980; Misevic and Burger, 1986: Misevic et ai, 1987). These sponges are multicellular. but the relatively loose orga- nization of embryonic and differentiated cells is easily disaggregated. If divalent cations are deleted from the 358 SULFATE RESTRICTION IN MARINE SPONGE 359 supporting medium (seawater), a specific aggregation fac- tor (AF) a sulfated proteoglycan-like molecule is re- leased; this factor can then promote specific cell aggre- gation (Humphreys, 1963). AF contains two functional domains, one a cell binding portion, the other an AF in- teraction domain (Misevic and Burger, 1986). Cell aggre- gation by Microciona AP (MAF) appears to be based on multiple low affinity carbohydrate-carbohydrate interac- tions (Misevic et a/., 1987). The role of sulfate in such reactions is unknown, but other work shows that the mi- gration and release from cells of proteoglycan-containing vesicles may be related to a high sulfate content (Albedi ct al.. 1989; Takagi ct al.. 1989). Monoclonal antibodies raised against sulfated proteoglycan from rat chondrocytes were used with immunoperoxidase electron microscopy to demonstrate relatively high concentrations of mem- brane-associated sulfated proteoglycans in cell processes and filaments from which matrix vesicles are presumably released into the surrounding medium (Takagi el al.. 1989). In the studies to be described, the effects of seawater, with and without sulfate. upon subsequent aggregation of disaggregated Microciona sponge cells was examined, both in rotating and stationary cultures. The isolation of cells in a relatively simple culture medium, such as seawater, possesses advantages over such alternatives as perfusion. or //; vm> methods, or the use of complex culture media. The level of sulfation can be controlled, all the relative enzyme systems are present in the cells, and the cellular uptake of labeled sulfur can be studied. Our primary pur- pose in the initial study was to observe the effects of sulfate deficiency, or restriction, upon cell process formation and locomotion and upon cellular aggregation. Using the conditions indicated by these observations, the incorpo- ration of 35 SO 4 was carried out, and correlations with in- tracellular-free sulfate and active sulfate (PAPS, i.e., phosphoadenosine-5'-phosphosulfate) and sulfated mac- romolecules determined. Materials and Methods Sponges Live specimens of Microciona prolifera were collected by members of the Supply Department of the Marine Biological Laboratory (Woods Hole, Massachusetts) dur- ing the months of July and August. Sponges were used on the day of collection or on the following day, but could be maintained in satisfactory condition for several days in the laboratory at ambient temperature in tanks of run- ning seawater. Buffers and artificial seawater preparations Bicarbonate buffered artificial seawater (MBLSW) was made up according to the Marine Biological Laboratory formula (Humphreys, 1963: Cavanaugh, 1964). Calcium- and magnesium-free seawater (CMFSW) was prepared as described by Humphreys (1963). In aggregation assays, CMFSW was supplemented with 10 mM CaCl : . Sulfate- free seawater was prepared as follows: in the case of MBLSW, magnesium chloride was substituted for MgSO 4 - 7H : O: in CMFSW, sodium chloride was substituted for Na 2 SO 4 . The preparations were termed MBL - SO 4 and CMF SO 4 , respectively. Dissociation of sponge cells The chemical dissociation of Microciona cells (Hum- phreys, 1963) began with small lumps of tissue that were first rinsed to remove foreign material, and then blotted. Fragments ( 1-3 mm) were cut and placed in cold CMFSW in the ratio of 1 g/100 ml CMFSW. We dissociated the fragments by pressing them gently through no. 25 bolting cloth into a second volume of CMFSW. The resulting suspension contained about 2 X 10 7 cells/ml as estimated by hemocytometer counts. The suspension was spun in the centrifuge for 5 min at 2000 RPM and resuspended to make a concentration of 10 7 cells/ml. Small clumps were flushed gently with a Pasteur pipet and thereby readily broken up. The suspension was then rotated in CMFSW at 16C for 6 h. The supernatant containing aggregation factor (AF) was removed, and the cells were washed and resuspended. The rotation was then repeated, first for 6 h in CMF - SO 4 and then for 24 h in MBL SO 4 . The cells were then divided into two aliquots: one was maintained in MBL - SO 4 for an additional 24 h, and the other was placed simultaneously in MBLSW for 24 h. The preconditioned cells from both aliquots were then pelleted and each aliquot resuspended in MBL - SO 4 and used in isotope labeling experiments. Aggregation factor AF was extracted and purified according to Humphreys ( 1 963), as modified by Jumblatt et al.,(\ 980). Protein was estimated using the Bio-Rad colorimetric assay (Bradford, 1976). Sponge cell aggregation assays (Humphreys, 1963; Jumblatt et al., 1980) Serial two-fold dilutions of AF were incubated for 20 min at 22C with cells ( 10 7 /ml) in the presence of CaCK. The cells were then visually inspected for evidence of ag- gregation. Sponge cells in rotation-and petri dish cultures used to study cell motility and aggregation Cells were prepared as described above, and batches were then adjusted to a concentration of 10 7 /ml in the following buffered solutions: MBLSW, MBL - SO 4 , and 360 W. J. KUHNS ET AL CMFSW, CMF - SO 4 . From each suspension, one aliquot was rotated in covered beakers for 24 h at 16C. A second aliquot from each suspension was placed in glass petri dishes and maintained motionless at 22C for 24 h. The presence of aggregates was then determined. Microscopic studies Following the incubation period, small aliquots of cul- tured preparations were mounted on glass slides, and overlaid with cover slips, which were sealed with resin to prevent evaporation. Some cell preparations were vitally stained with a 0. 1% aqueous solution of Nile blue sulfate (Leith and Steinberg, 1972). The cells were examined by phase contrast and interference contrast microscopy with a Zeiss Axiophot microscope at magnifications of 100X and 400X. Sulfate incorporation studies using isotope-labeled, carrier-free sulfuric acid (H : 3 \SOj) Radiolabeled H : X " 1 SO 4 (2 mCi/ml) was purchased from New England Nuclear. Aliquots of each preconditioned Microciona cell preparation in MBLSW and MBL SO 4 were washed in MBL - SO 4 , then calibrated to 10 7 cells/ ml in sulfate-deficient artificial seawater, and incubated in rotating culture in medium containing 2 /uCi/ml Hi 35 SO 4 . Replicate 1-ml aliquots of cells were placed on 25-mm diameter cellulose acetate filter discs (0.45 yum), beginning at 15 min, and at intervals thereafter up to 12 h. The dried discs were treated as follows: (a) for uptake studies, duplicate dried discs were each placed in a scin- tillation vial and 15 ml Aquosol-2 liquid scintillation fluid added; (b) for incorporation studies, filter discs were treated with 100% ethanol to precipitate proteins, washed twice in ethanol, dried, and treated as in (a). Counts were carried out in a Beckman LS6000 1C scintillation counter. The results are expressed as dpm/10 7 cells. High voltage electrophoresis (Hl'E) Channels (2" wide) were pencilled on Whatman 3M filter paper (18 X 22"), which was then moistened with 1% sodium tetraborate pH 9.1. One aliquot could then be spotted on one channel for a total of nine assays on each sheet of moistened paper. Electrophoresis was carried out at 1 kV and 180 mAmp for 60 min; the current was then discontinued and the paper dried in a warm air oven. Each channel, containing one separated extract, was cut into one-inch strips and placed in vials to which was added Beckman Redi-Solv EP scintillation fluid for scintillation counting as described. 3} SO^ Incorporation into secreted extracellular macromolcculcs Microciona cell suspensions were pre-treated and chemically dissociated, as described. The suspension me- dium was either CMFSW or CMF- SO 4 . To suspensions adjusted to a concentration of 10 7 cells/ml in a volume of 50 ml, was added 100 /uCi of carrier-free 35 SO 4 ; the suspensions were rotated for 12 h at 16C. The super- natants were harvested, and concentrates were prepared and assayed for MAF (16). The MAP pellets were washed exhaustively, redissolved in a minimal volume of artificial seawater, adjusted to equal protein concentrations, and dialyzed overnight in electrophoresis buffer. Such prep- arations were assayed by HVE. Free 15 SO 4 and PAP ( 35 S) were included in the assays as reference standards. Ma- terial that remained at the origin following electrophoresis was regarded as containing macromolecules that had in- corporated 35 SO 4 . Results are expressed as dpm/mg pro- tein. Ammo-acid incorporation using 3 H-leucine Aliquots of cell preparations maintained in the presence or absence of sulfate were calibrated to 10 7 cells/ml and incubated in MBL - SO 4 in the presence of 100 /ul of a 50 juCi/ml solution of 3 H-leucine (>300 mCi/mmol New England Nuclear); aliquots were taken for counts at spaced times beginning at 2 min. The cells were treated with 100% ethanol as described above. Results Sponge cell aggregation In contrast to cells suspended in sulfate-containing me- dium, aggregation was either partially or greatly retarded in samples suspended in sulfate-free seawater. This could be demonstrated as follows: ( 1 ) test cells that were prepared in the routine manner were rotated at 1 6C in the presence of CaCli along with supernatants derived from an equal number of chemically dissociated cells rotated in either CMFSW or CMF - SO 4 . Supernatants prepared in sulfate free seawater (CMF - SO 4 ) proved relatively ineffective in aggregation assays when compared with the action of AF or supernatant derived from sponge cells that had been rotated in CMFSW (Fig. 1 ). (2) AF prepared from fresh cells under normal con- ditions and concentrated, was tested in routine assays with chemically dissociated cells rotated in either CMFSW or CMF SO 4 . When cells which had been pre-treated in CMF SO 4 were used in assay, the aggregation of sponge cells was reduced in comparison with cells that had been pre-treated in sulfate containing seawater (Fig. 2). A small rim of adherent cells that ordinarily collected at the liquid- air interface of the container was not observed in assays that included CMF - SO 4 treated cells. Petri dish cultures Chemically dissociated sponge cells were rotated in changes of CMFSW and washed with CMF - SO 4 . Ali- SULFATE RESTRICTION IN MARINE SPONGE 1/16 Figure 1. Results of aggregation assays using supernatants derived from chemically disaggregated cells rotated in sulfate-free artificial sea- water. Fresh normally processed Microciona cells and CaCli added. Pho- tograph depicts results at 1 h. Wells from left to right in first two rows ( 1 and 2) depict serial dilutions of supernatant preparations. Microciona cells and CaCl : in CMFSW are in bottom-most (31 well (2nd from left). Reading from the top: Row 1, assay contains supernatant (MAF) from CMFSW cells; Row 2, contains supernatant from CMF - SO 4 cells. Aggregation is impaired in presence of CMF - SO., supernatant and dilutions. Figure 2. Aggregation assays using Micrvcitma cells prepared from suspensions rotated in CMFSW or CMF - SO 4 . Assays were earned out in calcified dilutions of MAF prepared as described! 12. 16). Photograph depicts results at 1 h. Wells from left to right in rows 1 and 2 depict serial dilutions of MAF in CMFSW with CaCK . Wells at bottom contain samples of each cell preparation in calcified CMFSW. Reading from the top from left to right: first row, assay contains cells pre-treated in CMFSW; second row. cells pre-treated in CMF - SO.,. Aggregation is impaired in cells which had been pre-treated in CMF - SO 4 . quots of cells were then placed in even suspension in the following media: CMFSW, CMF - SO,, MBLSW, MBL - SO 4 . Each suspension was gently pipetted with a Pasteur pipet and counted; the count in each suspension was ad- justed to 10 7 /rnl. Thirty ml of suspension were then poured into large glass petri dishes and gently pipetted to assure an even distribution of cells. The dishes were then covered and were permitted to remain undisturbed for 24 h at 22C. Inspection at the end of this period revealed aggregates of varying sizes in samples that had been sus- pended in MBLSW and CMFSW. Aggregation was not observed in cell suspensions lacking sulfate (Fig. 3). ing in single cells and aggregates when sulfate was absent, whether Ca :+ and Mg 2 " were present (Fig. 4E-F). Our general impression was that processes were most often seen in relatively agranular cells of intermediate to large size. Incorpt >ral it >n of if SO 4 The results of 35 SO 4 uptake into Microciona cells are shown in Figure 5. Each sampling time point depicts the average of duplicate tests, and the values are expressed as Microscopic studies The most striking observation was a relative lack of cellular blebbing or of nlopodia or pseudopodia in cell preparations suspended in sulfate-free seawater. In con- trast, we commonly observed, in suspensions containing sulfate, slender nlopodia that sometimes extended a long distance from single cells or from aggregates (Fig. 4A-D). Other, more substantial processes were suggestive of a cell elongating in the direction of a second cell using a single pseudopod as a means of locomotion. We observed oc- casional single cells, or cell clumps, from which multiple nlopodia emerged, creating a stellate or radiating pattern (Fig. 4B). All of these appeared to be natural events in the presence of sulfate. They were greatly reduced or lack- Figure 3. Aggregation of Microciona cells maintained in stationary culture in petn dishes. Readings are at 24 h under conditions as described in the text. Aggregation is observed when cells are maintained in MBLSW (right) but not when cells are in MBL - SO 4 (left). 362 w. j. KUHNS /:/ i/ ... Figure 4. (A-B) Micrciciona cells observed by interference contrast microscopy 400 -. magnification. Cells shown here had been in rotation in MBLSW as described in text. Filopodia or pseudopodia. some of considerable length, are noted in cells in aggregates as well as single cells. Multiple filopodia produce a stellate appearance in cell or cells seen in Figure 4B. (C-D) Interference contrast study ofMicrociona cells 400X magnification. Cells shown here had been maintained stationary in petri dish cultures in MBLSW as noted in text. Filopodia or pseudopodia are frequent in single cells and in small aggregates. (E) Interference contrast study of Mk-rwiona cells which had been in rotation in MBL - SO 4 as described in text. Typically, small aggregates were produced in the relative absence of tilopodia or pseudopodia. ( F ) Interference contrast microscopy of Micrnciona cells which had been maintained stationary in MBL - SO 4 in petri dishes as described in text. The majority of cells were unattached, lacking processes, or adherent in very small clusters. dpm per 10 7 cells. The highest uptake of 15 SO 4 occurred in cell samples pre-treated in sulfate-free medium; in comparison, cells pre-treated in sulfate-containing me- dium demonstrated considerably lower levels of 15 SO 4 uptake beginning minutes after the addition of 35 SO 4 . In replicate experiments with sponge derived from two col- lecting stations, cells in MBL - SO 4 showed progressive increases in "SO 4 uptake and incorporation up to 12 h. SULFATE RESTRICTION IN MARINE SPONGE 363 o "*'- 3 4 5 6 7 8 9 10 II 13 Hours Figure 5. Uptake of 35 SO 4 by Mumcin/ia cells. Pre-lreated Mu n KYI >m/ cells were distnbuted into flasks containing (a) MBLSW, (b) MBL - SO 4 at a concentration of 10 7 cells/ml and placed in rotation for 24 h. Cen- tnfuged pellets from each tlask were washed with MBL - SO 4 and sus- pended in MBL - SO 4 : 5 M' Hv' 5 SO 4 (carrier free) was added to each tlask and rotated at 16C. Radioactivity of duplicate I -ml aliquots was monitored at intervals up to 12 h. The solid lines depict uptake of 35 SO 4 by cells pre-treated in MBL - SO 4 . The broken lines represent cells pre- treated in MBLSW. Circles and triangles signify experiments carried out on cells from sponge obtained at two different collecting stations. dpm 800 700 600 500 400 300 INCHES Figure 6. High voltage electrophoresis of 35 SO 4 macromolecules in supernatant preparations derived from rotated chemically dissociated Microciona cells with incorporated carrier free 35 SO 4 . 75 n\ placed at origin. Conditions: 1% sodium tetraborate pH 4.1. 1 KV. 180 mA, I h. CMFSW D---D; CMF - SO 4 --- in contrast to controls. At 12 h, values for 35 SO 4 were three- to eight-fold higher in sulfate deprived cells than in controls (6925 and 6603 dpm in test samples versus 882 and 2075 dpm in controls). Incorporation of ^SO 4 into macromolecules was calculated to be 70-85% of "SO 4 uptake at the 12-h sampling time. In recent separate ex- periments with extended sampling times, the differential in counts between cells pre-treated with MBLSW and with MBL - SO 4 remained for up to 5 days, at which time sampling was discontinued. In samples of cells pre-treated with MBL - SO 4 , elevated counts exhibited some fluc- tuations, but remained at high levels during the period of sampling. Cells were collected for extracts that will be analyzed by HVE for the distribution of 35 SO 4 macro- molecules. This study will be reported in a separate pub- lication. Jf SO 4 Incorporation into secreted extracellular macromolecules The protein yields in the extracellular secretion from 5 X 10 8 Microciona cells were as follows: from CMFSW cells, 1.12 mg; and from CMF - SO 4 cells, 0.37 mg. The yield of 15 SO 4 macromolecules revealed by HVE after subtraction of background values was: forCMFSWMAF, 190 dpm/50 jug protein; for CMF - SO 4 supernatant, 35 dpm/50 /jg protein (Fig. 6). Incorporation of 3 H-leucine Incorporation of 3 H-leucine into protein by Microciona cells in sulfate-free seawater was comparable to that of cells rotated in sulfate-containing seawater; aliquots from specimens obtained at two collecting stations were ex- amined. In all instances, incorporation was prompt, with mild to moderate increases over the testing period (Ta- ble I). Discussion A role for sulfated polysaccharide recognition in sponge cell aggregation was suggested by Coombe el a/. (1987) based upon an analysis of endogenous polysaccharide Table I 3 H-leucinc incorporation into Microciona aV/.v maintained under different conditions ot sulfale availability Conditions of culture Time of sampling 3 H-Leucine incorporated (dpm) la. MBL + SO 4 30 mm 2 h 17111* 20586 Ib. MBL - SO 4 30 min 2 h 19149 25644 2a. MBL + SO 4 30 mm 2 h 13688 21646 2b. MBL - SO 4 30 min 2h 12140 19781 * Average of duplicate ethanol precipitated samples after subtraction of background values. I0 7 cells per sample. Specimens 1 and 2 were obtained at two different collecting stations. 364 W. J. KLIHNS /;/ I/ from sponge cell cholate lysates. The extract possessed a high content of sulfate and inhibited the aggregation of intact sponge cells, as did the sulfated compounds poly- vinyl sulfate and dextran sulfate. The latter compounds, coupled to erythrocytes, rendered the erythrocytes agglu- tinahle in the presence of sponge cell lysates. In the present studies, pre-treated chemically dissociated cells maintained in a sulfate-free environment exhibited greatly reduced motility and marked changes in functional behavior. Aggregation of Microciona cells became im- paired under these conditions, particularly in stationary cultures, regardless of the presence of Ca :+ and Mg 2+ . The effect of a sulfate-free environment could be observed in Microciona cells, as well as in supernatants derived from cells under sulfate-free conditions. This was demonstrated in controlled aggregation assays, as follows. ( 1 ) The ca- pability of AF derived from cells in CMFSW was com- pared with that of supernatants derived from cells chem- ically dissociated in CMF - SO 4 with healthy Microciona cells and CaCl 2 being employed in the assays; and (2) cells prepared in sulfate-free artificial seawater were tested in standard assays with AF prepared in the usual manner from chemically dissociated cells. Of special note was the finding that aggregates were absent or greatly reduced in size when cells under these conditions were maintained in stationary cultures. Random collision in rotation cul- tures probably accounted for the small aggregations noted, but even in this circumstance, aggregation in the absence of sulfate was modest when compared with Microciona cells maintained in sulfate. The most obvious morphologic change in individual cells maintained in sulfate-free medium was a reduction in cell processes such as filopodia and pseudopodia. The relative lack of such processes most likely contributed to the inability of these cells to form normal contacts, es- pecially when in stationary cultures. In the presence of sulfate. the primary activity seemed to reside in medium- to large-sized cells, cells which were relatively agranular, some of which were reminiscent of choanocytes (Kuhns et al., 1980). It remains to be established whether certain cell types in Microciona, such as larval cells, possess special components that are unusually sensitive to sulfate depri- vation, and that are necessary for secretion or cellular migration, as is the case in the sea urchin embryo. The secretion of sulfated polysaccharide appears to be necessary to maintain these functions, as established ear- lier by Immers and Runnstrom (1965), and this form of secretion is diminished or sulfate-poor in parallel with sulfate restriction. However, there is no direct evidence from our work that sulfated polysaccharides were specif- ically affected by changes in seawater sulfate content. The evidence presented relates to the flow of sulfate into cells, and the biosynthesis of sulfated macromolecules as im- portant factors in cell locomotion and cell adhesion for reasons that are yet unclear. Nevertheless, we presume that MAP molecules become sulfated as part of the bio- synthetic process (Misevic el al, 1987). Although the de- sign of this study does not enable conclusions about their specific nature, 3S SO 4 macromolecules appeared to be de- ficient in Microciona supernatants derived from chemi- cally disaggregated cells, in contrast to supernatants de- rived from cells in sulfated seawater. When supernatants derived from sulfate-free cells were concentrated and pu- rified as described, the protein content, as well as the con- tent of macromolecules (denned on HVE), were reduced in relation to AF prepared from an equal number of Microcionu cells prepared in artificial seawater. Such a deficit may be caused by a defective transport of secretory vesicles to the cell surface in the absence of sulfate. We suspect, from our results, that sulfation of macro- molecules such as polysaccharides may be crucial in the trans-golgi transport of vesicles and their secretion into the extracellular matrix. These results, coupled with find- ings that sulfate depleted cells can greatly augment 3 "SO 4 incorporation relative to controls, suggest a mechanism whereby extracellular sulfate deficiency can alter the sul- fation process, perhaps by influencing membrane com- position and function. Note in this context that amino acid uptake and incorporation was similar in test and control cells as judged by experiments using 3 H-leucine. Our studies of sulfate depleted cells suggest that the sulfate assimilatory pathway is altered when sulfate be- comes rate limiting. Regulation and transport in such a system has been explained by the existence of a specific membrane permease in two bacterial species. Salmonella typhimurium and Anacystis nidulans (Green et al.. 1 989); the permease genes have been cloned and, from these, a polypeptide structure of a putative membrane component determined. Homologies with message derived from sul- fate-restricted Microciona sponge might be sought using probes derived from these bacteria. Studies in sulfate-de- ficient wheat and barley roots have also defined a sulfate transporter that was sensitive to DIDS, an inhibitor of anion transport (Clarkson and Saker, 1989). A sulfate permease. if defined in our system, would encourage fur- ther studies to define ways in which cells recognize sulfate levels and transduce this signal into altered sulfate trans- port, increased biosynthesis of sulfated glycoconjugates and altered cell locomotion. Fine structure differences in MAF derived from sulfate-deprived versus normal cells may prove important in defining extracellular prompting mechanisms which initiate or modulate such changes (Brunner. 1977). Literature Cited Akasaka, K., S. Amcniya, and H. Terayama. 1980. 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Cavanaugh, G. 196-4. I-'ormulae and Methods ol the Marine Biological I.ahoraiory. 6th ed. Manne Biological Laboratory. Woods Hole. MA. 67 pp. Clarkson, D., and L. Saker. 1989. Sulphate influx in wheat and barley roots becomes more sensitive to specific protein binding reagents when plants are sulphate deficient. Planla 178: 249-257. Coombe, D., K. Jakobsen, and C. Parish. 1987. A role for sulfated polysaccharide recognition in sponge cell aggregation. Exp. Cell Re-* 170: 381-401. Green. I... D. I.audenbach. and A. Grossman. 1989. A region of a cy- anobacterial genome required for sulfate transport. Proc \ : al. K ail Sci. L'.S.A. 86: 1949-1453. I lenkart. P.. S. Humphreys, and I. Humphreys. 197.3. Characterization of sponge aggregation factor: a unique protcoglycan complex. Bio- chemistry 12: 3045-3055. Hogsett, \V., and B. Quatrano. 1978. Sulfation of Fucoidins in l-'ncns embryos. III. Required for localization in the rhizoid wall. J. Cell Biol. 78: 866-873. Humphreys. 1 . 1963. 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Adhesive and migra- tor, behavior of normal and sulfate deficient sea urchin cells in vitro Exp. Cell Res 154:421-431. \Venzl, S. and M. Sumper. 1981. Sulfation of a cell surface glycoprotein correlates w-ith the developmental program during embryogenesis of I'olvox carteri Proc Nat. Acad. Sci. U.S.A. 78: 3716-3720. Vamaguchi. M., and S. Kinoshita. 1985. Polysacchandes sulfated at the time of gastrulation in embryos of the sea urchin Clypeaster ja- /'onicus. Exp. Cell Res. 159: 353-365. Reference: Biol. Bull. 179: 366-373. (December, 1990) Extracellular Hemoglobins of Hydrothermal Vent Annelids: Structural and Functional Characteristics in Three Alvinellid Species ANDRE TOULMOND, FOUZIA EL IDRISSI SLITINE, JACQUES DE FRESCHEVILLE, AND CLAUDE JOUIN Laboraioirc de Biologic ct Physiologic Marines, Universite Pierre-et-Marie-Curie, 75252 Paris Cct/c\ II? and C.N.R.S.. Station BiologUjue. 296X2 Roscoff. France Abstract. The polychaete annelids Alvinclla pompejana. A/vine/la candata. and Paralvinella grasslei are strictly associated with deep sea hydrothermal vents. Each species possesses an extracellular hemoglobin, Hb, which has been studied and compared to that of a common intertidal polychaete, the lugworm Arenico/a marina. The four Hbs exhibit very similar quaternary structures and spectral properties, and only small differences appeared in the gross polypeptide compositions after reduction and sodium dodecyl sulfate denaturation of the native molecules. Conversely, by a comparison of the effects of pH (6.6- 7.6) and temperature (10-40C) on their intrinsic O af- finities, Bohr factors, cooperativities, and apparent heats of oxygenation, lugworm Hb can be differentiated from that of the alvinellids, and the Hb of .1. pompejana from that of A. candata. The known biology of the lugworm and a further analysis of the data suggest several hy- potheses concerning the //; vivo O : transport function of the alvinellid Hbs, the //; vivo blood pH value in the two alvinellid species, their respective range of optimal tem- perature, and their ability to create a differentiated and stable external microenvironment. Introduction The known members of the tubicolous polychaete family Alvinellidae are associated only with deep sea hy- drothermal vents. In the East Pacific Rise region, the tubes of the closely related species Alvinclla pompejana and Al- vinclla candata form honeycomb-like structures covering the external surface of the active vents, where they are Received 15 May 1990: accepted 21 August 1990. frequently associated with the smaller species Paralvinella grasslei (Desbruyeres and Laubier, 1986). The mixing of the very hot, anoxic vent water (up to 320C) with the cold, oxygenated deep seawater (2C) occurs at random. All three alvinellid species are supposed to live on the colder edge of a very sharp thermal gradient, at temper- atures as high as 50C (Desbruyeres et a/.. 1982; Arp and Childress in Terwilliger and Terwilliger, 1984). This en- vironment is characterized by high-frequency, unpre- dictable changes in temperature, pH, oxygen partial pres- sure, and sulfide concentration (Johnson ct al. 1986, 1988). The alvinellids have well-developed gills (Jouin and Gaill, 1990) and a closed vascular system containing a high molecular weight, extracellular hemoglobin (Hb) dissolved in the blood. These Hbs have rarely been studied, and most of the available data have been obtained by Terwilliger and Terwilliger (1984) on A. pompejana Hb. Recently, one of us (A.T.) collected fresh blood directly from living specimens of A. pompejana. A. candata. and P. grasslei. We describe here the structure and some of the functional properties of the Hbs from these samples. The effects of pH and temperature on the oxygen binding properties of the Hbs were examined at constant inorganic ion concentration and at one atmosphere hydrostatic pressure. For comparison, the same studies were carried out on solutions of the extracellular Hb of a mainly in- tertidal species, the common lugworm Arenicola marina. prepared and stored in the same conditions. Materials and Methods Animals The alvinellids were collected at 2600 m depth in No- vember 1987 during the French-American "Hydronaut" 366 ALVINELLID EXTRACELLULAR HEMOGLOBINS 367 expedition on the "13N" hydrothermal vent site (East Pacific Rise region, Fustec cl ai. 1987). Large pieces of black or white smokers were plucked off by the external arm of the DSRV Nautili' and placed in an insulated, non-pressurized container, closed at depth to keep tem- perature constant as the yellow submarine surfaced. The lugworms were collected on the Penpoull beach near Ros- coff, Brittany, France. Immediately after the alvinellids were recovered on board ship, they were opened dorsally, and the blood, uncontaminated with coelomic fluid, was withdrawn from the main vessels into glass micropipettes and pooled on melting ice. In Roscoff, the same procedure was applied to lugworms kept unfed for 12 to 24 h in local running seawater (temperature 14-16C). The total blood volumes collected from the alvinellids were around 0.8 ml for A. pompejana (10 specimens), 0.7 ml for .-1. caudata (12), and 0.05 ml for P. gnissli'i (3). The blood was centrifuged at low speed for a few min- utes, and the supernatant was divided into two parts, (i) For examination of the Hb molecules by transmission electron microscopy (TEM), a few droplets of the super- natant were diluted 1 :200 in a buffer comprising 50 mAl Bis-tris-propane (BTP: Sigma) and HC1 at pH 7.4. The grids were prepared by standard techniques (Valentine el n/.. 1968), on board ship or in the Roscoff laboratory, (ii) The remaining supernatant was equilibrated against 50 mAf BTP-seawater/HCl buffer (pH 7.6) by gel nitration on Sephadex G-25, saturated with carbon monoxide, and frozen in liquid nitrogen. In Paris, these samples were thawed, and a metHb-free, HbCO-free, pure HbO 2 so- lution was prepared using standard techniques (Riggs, 1981). Spectrophotometric studies U.V./vis. absorption spectra of the Hbs were obtained at 20C with a Bausch and Lomb Spectronic 2000 spec- trophotometer. The heme concentration of the solutions was determined using a millimolar extinction coefficient t= 1 1 .0 at 540 nm for the cyanmet heme (Van Assendelft, 1970). Electrophoretic studies The Hbs were denaturated by sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) in the presence or absence of mercaptoethanol (ME). The Hbs and markers (Pharmacia) of low relative molecular mass (A/ r ) were first heated at 100C for 5 min in a 2.5% SDS solution, with or without 5% ME. The electrophoresis was then carried out on 10% polyacrylamide slab gels, in 12.5 mAl Tris/glycine buffer (pH 8.5), with 0.1% SDS. Functional properties For studies of the O : -binding characteristics of the Hbs, aliquots of the pure HbO : solutions were equilibrated against 50 mM BTP/HC1 buffer by gel filtration on Seph- adex G-25 (final heme concentration: 40-70 ^M). The buffers were adjusted in order to obtain constant pH val- ues: 6.6, 6.9, 7.25, 7.6, whatever the experimental tem- perature: 10, 20, 30, 40C. Except for Na + , which varied between 265 and 305 mAl depending mostly on the pH value, the inorganic ion concentrations, in mAl, were also kept constant: Cl = 470; SO 4 : = 30; Mg 2+ = 50; Ca :i = 10. These values are similar to those observed in the coelomic fluid of the lugworm (Robertson, 1949). The total osmolarity of the solutions was about 1.06 OsM. Oxygen-equilibrium curves (OEC) were obtained, with no carbon dioxide in the gas phase and at one atmosphere hydrostatic pressure, by a continuous spectrophotometric method; we used a Hemox Analyser spectrophotometer (TCS, Southampton, Pennsylvania) interfaced with a Hewlett-Packard 85B microcomputer and a Hewlett- Packard ColorPro Graphics plotter. The purified HbO : solution was first equilibrated against pure oxygen and then slowly deoxygenated with pure nitrogen or argon. The deoxygenation procedure lasted 60 to 90 min. and the microcomputer was programmed to store up to 300 points of the OEC on tape, each point corresponding to the coupled mean values of 30 and 60 successive mea- surements of, respectively, oxygen partial pressure (P ,) and O: saturation of the Hb. Negligible quantities of metHb were produced during these experiments, a con- sequence of the particularly high resistance of lugworm and alvinellid Hbs to oxidation (Toulmond ct ai, 1988). The P , at half saturation of the Hb (P 50 ) was calculated from the experimental values between 40 and 60% O 2 saturation by linear regression analysis, and approximate values of the dissociation constants for the R and T states [respectively, A' R and A' T (Edelstein, 1975)] were estimated graphically from the Hill plot of the OEC. The value of the Hill coefficient [ max , corresponding to the maximum slope of the Hill plot (Imai, 1982)], as well as its position on the saturation axis, were estimated graphically from the calculated first derivative of the Hill plot, the so-called cooperativity curve (Girard et ai, 1987). The Hemox technique gave highly reproducible results, especially in conditions where the Hb affinity is high. The statistical analysis of a preliminary set of 10 OECs, ob- tained at pH = 7.6 and 20C on lugworm blood, gave the following mean results (value SD): P 50 (mm Hg) = 1.82 0.04; 50 = 2.36 0.06; A T (mm Hg) =16.1 1.1; A R (mm Hg) = 1.21 0.32. Results Absorption spectra Absorption spectra were typical of hemoglobins and quite similar in all the species studied; the position of 368 A. TOULMOND ET AL. Table I Spectral position in nm o/ IIbO : / negatively sunned Hb molecules as measured on electron micrograph s Alvinella Alvinella Paralvinella Arcnicola poinpc/una caudala grassic/ manna Maximum diameter 30.4 30.2 29.6 30.0 0.8* 0.9 1.4 1.2 Side to side 27.0 27.2 26.5 27.5 width 0.8 0.7 1.1 0.7 Height 19.7 19.4 18.9 19.7 0.9 1.2 1.9 1.0 * Standard deviation; n = 30. In the three alvinellid species, denaturation and elec- trophoresis of the Hbs by SDS-PAGE yielded three major bands corresponding to proteins of A/ r about 45,000, 30,000, and 15,000. Two fainter bands were also present corresponding to proteins of M t ca. 28,000 and 22,000 in the genus Alvinella, and about 28,000 and 25,000 in the genus Paralvinella. By comparison, denaturation of Ar- enicola marina Hb gave four major bands corresponding to Al r s of about 45,000, 32,000. 28.000. and 15,000 (Fig. 3A). In the four species, reduction by ME and simultaneous denaturation by SDS produced a major band correspond- ing to polypeptides of M r between 14,000 and 16,000. Fainter bands corresponded to polypeptides of A/ r about 35,000 and 25,000 in the genus Alvinella, 30,000 and 28,000 in Arcnicola, and 28,000 in Paralvinella (Fig. 3B). Oxygen equilibrium studies Because so little P. grasslei blood was available, these studies were carried out only on A. pompejana. A. caudala. 0.6 Am * Figure I. Electron micrographs of native molecules of the four ex- tracellular Hbs, negatively stained with 2% uranyl acetate. Scale bar: 25 nm. (Ap) Alvinella pompeiana. (Ac) .1. caudala. (P) Paralvinella grasslei: (Am) Areiucola marina. 0.4 O c n) -O -?0.2- - A 6912 Elution volume, ml Figure 2. Elution profiles of Alvinella pnmpeiana Hb on a Superose 6 column, in Bis-tris-propane/HCl buffer. The arrow indicates the peak position for Areiucola marina Hb. Absorbance was measured at 280 nm (solid line) and 4IO nm (dashed line). ALVINELLID EXTRACELLULAR HEMOGLOBINS 369 A SDS-PAGE 10' 3 M, 94 60 43 30 20 14.4 Ac Ap Par M Am : SDS-PAGE * ME 10' 3 M, 94 - 67 43 30 20 14.4 Ac Ap Par M Am Figure .V SDS slab gel electrophoresis. lO'i polyacrylamide. of the four extracellular Hhs. (A) Before reduction by mercaptoethanol (ME); (B) after reduction by ME. (Ac) Alrincllu auulala. (Ap) .1 />o;/v/(/m/, (Par) Parahiiiclla wapiti: (Am) Arcnicola manna: (M) low molecular mass markers (Pharmacia), (a) Phosphorylase; (b) serum albumin; (c) catalase; (d) ovalbumin; (e) carbonic anhydrase; (f) trypsin inhibitor; (g) lactalbumin. and Arcnicola marina Hbs. Figure 4 shows the Hill plot of a typical OEC obtained on A. pompcjana Hb. In vitro. the alvinellid Hbs were characterized by a very high in- trinsic O : affinity, with P 50 values very dependent on pH and temperature (Table III). The normal Bohr effect was large, with Bohr factors that may have been lower than - 1 , and was greatest at low temperature and at low to medium O 2 saturation of the pigment (Table IV). The cooperativity was also high. The Hill coefficient. H mav , was in some cases higher than 4 (Fig. 5) and was strongly dependent on pH and temperature, being maximum for pH around 6.6-6.9 (Fig. 6). The apparent heat of oxy- genation, \H, was also very high, peaking at more than -100 kJ/mol O 2 , and strongly pH dependent (Table V). The two alvinellid Hbs differed significantly with respect to these characteristics: the Bohr effect, cooperativity, and apparent heat of oxygenation were systematically higher in A. pompejana than in A. caudata. However their Hbs shared particular properties quite different from those of the lugworm. In the same experimental conditions, the lugworm Hb exhibited a lower O 2 affinity, a lesser Bohr effect with maximum values of the Bohr factor at medium to high O 2 saturation, a lower cooperativity with maxi- mum values at rather alkaline pH (about 7.25-7.6), and lower pH-independent values of A//. Discussion Molecular structure The alvinellid and lugworm Hbs exhibit the same qua- ternary structure, and it is typical of annelid extracellular Hbs. For the four molecules, and in the same experimental conditions: (i) FPLC, as well as low-pressure column chromatography, give almost identical elution profiles (Fig. 2) indicating very similar A/ r s of about 3.6 X 10 6 ; and (ii) the native molecules measured on electron mi- crographs show only small, nonsignificant differences in dimensions (Table II). These M$ and dimensions are very close to those recorded in the literature for the Hbs of intertidal polychaetes and terrestrial or aquatic oligo- chaetes (Vinogradov ct ai. 1982), and they are very similar to those obtained in a recent small angle X-ray scattering study of lugworm Hb (El Idrissi Slitine ct ai. 1990). From these observations, we consider that the decrease in hydrostatic pressure experienced by the alvinellid blood during the submarine's rise to the surface (about 260 at- mospheres) had little or no effect on the shape, size, struc- ture, and, consequently, on the functional properties of alvinellid Hbs. Three observations support this opinion: (i) the electron micrographs show that both alvinellid and lugworm Hbs dissociate into more or less spheroidal par- ticles, probably corresponding to twelfths of the native molecules: but the proportion of these particles is nearly -2 -3 looK, \ -2 log Hg) Figure 4. Hill plot of a typical oxygen equilibrium curve of A pom- pejana extracellular Hb. Log A R and log A T were graphically estimated at the intersection of the log P 0z axis by straight lines (slope = 1 ) drawn asymptotic to the Hill plot at extreme high and low O 2 saturation values, respectively. pH 6.90; 20C; heme concentration: 370 A. TOULMOND ET AL Table III /><,, in nun lit; a.\ n function ol pi I. 6 6 in ~ ft. and temperature. ll> in -till. ' l-Mc'll i'ii/iii' \\a\ obtained Irani une, lurch' /uv). 6.6 6.9 7.25 7.6 Alvinella 10C 0.5 0.2 0.1 ND* pompeiana 20C 1.9 1.0 0.3 0.2 30C 3.9 1.8 0.9 0.5 40C 8.1 4.4 2.9 2.2 Alvinella 10C 0.5 0.4 0.2 0.1 caudata 20C 1.8 1.0 0.4 0.3 30C 3.8 2.0 1.1 0.8 40C 6.6 4.4 2.5 2.4 Arenicola IOC 5.7 4.2 2.8 1.4 manna 20C 9.5 6.5 3.7 2.1 30C 13.9 9.1 5.4 3.3 40C 15.6 10.5 5.8 4.3 Table IV Effect i>l temperature. 10 In 40 C. on the mean Bohr factor calculated between pH 6.6 and 7.6 for almost completely deoxygenated (T = \ log K T /pH). hall-o\\Keated (P SU = \ log P }u /pH). and almmt completely oxygenated (T ?, R Alvinella pompeiana 10C -1.60 -1.17 -0.05 20C -1.21 -1.18 -0.24 30C -1.20 -0.89 +0.02 40C -0.86 -0.56 -0.08 Alvinella caudata 10C -0.35 -0.76 -0.15 20C -0.93 -0.90 -0.30 30C -0.92 -0.68 -0.03 40C -0.78 -0.47 -0.03 Arenicola manna 10C -0.18 -0.62 -0.44 20C -0.34 -0.66 -0.43 30C -0.34 -0.64 -0.38 40C -0.44 -0.58 -0.38 the same for all species, indicating either their normal presence in the blood in vivo, or, most probably, their unavoidable formation during the preparation of the grids for the TEM study, (ii) A recent study has shown that hydrostatic pressure dissociates annelid extracellular Hbs significantly only when it is increased to more than 1000 atmospheres (Silva el ai, 1989); a decrease in hydrostatic pressure, from about 260 to 1 atmosphere, would be un- likely to substantially affect the quaternary structure of these Hbs. (iii) Preliminary experiments have shown that, during a progressive increase of the hydrostatic pressure up to 1500 atmospheres, followed by a progressive de- crease back to one atmosphere, the absorbance spectrum of half-oxygenated lugworm Hb is not appreciably mod- ified, indicating that no change occurs in either the O 2 saturation or the O : affinity of the Hb (Hui Bon Hoa and Toulmond, unpub.). Nevertheless, we must keep in mind that, on the basis of data obtained //; vitro at 1 atmosphere hydrostatic pressure, we compare below the properties of Hbs that function in vivo at two different values of hy- drostatic pressure ( 1 atmosphere for the lugworm Hb, 260 atmospheres for the alvinellid Hbs). We obtained some information about the detailed structure of the native Hb molecules. SDS denaturation confirms that these molecules belong to the annelid ex- tracellular Hb family, with only small variations around the general type (Vinogradov, 1980). However, small dif- ferences exist between the electrophoretic patterns of A. pompejana and A. caudata Hbs. These differences, to- gether with those concerning the functional properties discussed below, confirm the distinct taxonomic status of these two recently separated species (Autem et ul.. 1985; Desbruyeres and Laubier, 1986). Physicochemical and functional properties Alvinellid Hbs can be easily distinguished from lug- worm Hb in that there are notable differences of intrinsic O; affinity, Bohr effect, cooperativity, and apparent heat of oxygenation. In a detailed examination of these prop- erties, the Hb of A. pompejana can be distinguished from that of A. caudata. Can these differences be correlated with what is known of the specific characteristics of the animals and their environment? The high intrinsic O : affinity of A. pompejana Hb has already been reported by Terwilliger and Terwilliger (1984). We confirm here that the O 2 affinity of both A. pompejana and A. caudata Hbs is very high whatever the -2 -1 1 log (Y/[1-Y]j Figure 5. Three different calculations of the first derivative of the Hill plot of Figure 4. showing the variations of the Hill plot slope, n. as a function of log (Y/(l - Y)). n m ^: the graphically estimated value of the Hill coefficient. ALVINELLID EXTRACELLULAR HEMOGLOBINS 371 experimental conditions (Table III), 2 to 10 times higher than that of the lugworm which has a Hb affinity for O 2 that is already quite high (for a comparison with other annelid Hbs. see Weber, 1 980). In the extreme conditions of low temperature (10C) and high pH (7.6), the O : af- finity of .-1. potnpejana Hb was so high (P 5I , lower than 0. 1 mm Hg, with 1 mm Hg = 1 33.3 Pa) that it could not be measured with the Hemox technique. Hbs with high O 2 affinities are generally considered very adaptive in spe- cies lacking an efficient, specialized respiratory organ (see Weber, 1978). But alvinellid gills are characterized by the highest specific surface areas yet measured in polychaetes. low diffusion distances between the external seawater and the blood, and a branchial circulatory system with a com- plexity comparable to that of the fish gill ( Jouin and Gaill. 1990). Hbs with high O: affinity can also be advantageous to species living in a poorly oxygenated environment (Weber, 1980). But what do the alvinellids actually breathe? Ac- cording to Desbruyeres el al. ( 1 982) and Arp and Childress (in Terwilliger and Terwilliger, 1984), a mild to warm (up to 50C), hypoxic water: i.e., a mixture of the very hot, anoxic vent water and the cold, oxygenated local bottom seawater. But the oxygen concentration of this water mix has never been directly measured in aim. and the only direct evidence for low O 2 concentrations inside and out- side hydrothermal vent community come from the Rose Garden vent field in the Galapagos Rift (Johnson et al., 1986), where the O 2 content is always below 'A of the saturation at one atmosphere hydrostatic pressure. How- ever, alvinellids have never been seen at the Rose Garden site, and the conditions there are quite different from those at the 13N site. The hypothesis that alvinellids breathe hypoxic water must be considered, but is as yet not really supported. The high O 2 affinity of alvinellid Hbs is modulated by the very large Bohr effect we found. The magnitude of the Bohr effect is extremely dependent on the oxygenation Table V Heal i>/'o.\yi;cmili lii 7.6 '). U/mol O : 6.6 6.9 7.25 7.6 Alvinella -66 -76 -89 -102 poinpciuna -0.976* -0.990 -0.998 -0.999 Alvinclla -57 -63 -69 -76 caudata -0.990 -0.998 -0.999 -0.995 Arciucula -23 -23 -24 -24 marina -0.972 -0.977 -0.987 -0.993 * Correlation coefficent. of the Hb molecule (Table IV): it is maximum when the molecule, almost fully deoxygenated, is in the so-called T-state (S , ca 0%); minimum or null when the molecule, almost completely oxygenated, is in the R-state (S , fa 100%); and intermediate when the molecule is half-oxy- genated at P 50 . These S ,-dependent Bohr-effect variations must greatly facilitate the O : unloading of the pigment at the tissue level, an advantage in view of the very high intrinsic O : affinity of alvinellid Hbs. The Bohr effect of the lugworm Hb is not as strong, and the maximum values of the Bohr factor occur when the Hb is half or nearly completely oxygenated, a property that Weber ( 198 1 ) sees as favoring the O 2 loading of the pigment at the gill. The oxygen transport efficiency of a respiratory pigment also depends on its cooperativity because, in vivo, a max- imal cooperativity allows a maximal O 2 loading or un- loading of the molecule for a corresponding minimal change of blood P<>. In alvinellid as well as in Arenico/u Hbs, the O 2 -binding process is highly cooperative, with max values that can be above 4 in A. pompejana. The value of m a\ varies much more with temperature and pH in alvinellid than in lugworm Hbs (Fig. 6). In Arenicola, 4- 3- 2- Ac Am 6.6 6.9 7.2 7.5 6.6 6.9 7.2 7.5 6.6 6.9 7.2 7.5 pH pH pH Figure 6. Variations of the Hill coefficient, max , as a function of pH at 10, 20, 30 and 40C. Other experimental conditions: see text. (Ap) Alvinclla pompejana. (Ac) A caudata; (Am) Arenicola marina. 372 A. TOULMOND /,/ I/. which normally lives in cold to temperate waters (Wells, 1963), the physiological blood pH is 7.25 and 7.58 in animals acclimated at 26 and 5C, respectively (Toul- mond, 1977). The cooperativity of lugworm Hb is max- imum between pH 7.25 and 7.6, and for temperatures between 10 and 30C (Fig. 6). If the maximum cooper- ativity of the respiratory pigment is correlated with the physiological pH value in Alvinella. as it is in Arenicola, then the physiological range of blood pH in Alvinella is probably 6.6-6.9 and, in this pH range, the maximum cooperativity is obtained at 10-20C in A. caudata. and at 20-30C in A. pompejana. This blood pH range is un- usually low for annelids and only direct measurements of blood pH could validate our interpretation of the data. But it is noteworthy that in the vestimentiferan worm Riftia pachypti/a. another hydrothermal vent dweller liv- ing in sulfide-rich water, similar slightly acidotic pH values have been measured //; vivo (Childress et al., 1984). Our observations suggest two sets of hypotheses: (i) the two alvinellid species form sympatric mixed populations on the same white or black smokers, but the external mi- croenvironment is probably slightly colder for A. caudata, at 10 to 20C, than for A. pompejana. at 20 to 30C. These temperatures are well below the maximum tem- perature, 50C, that the animals are supposed to withstand in situ. Our findings would corroborate Terwilliger and Terwilliger' s ( 1984) observation that A. pompejana Hb is unstable at such a high, and probably nonphysiological. temperature, (ii) At those pH and temperature values, the //; vitro intrinsic O 2 affinities of the Hbs are finally not so high, with P 50 values ranging from 1.0 to 3.9 mm Hg in A. pompejana and from 0.4 to 1.8 mm Hg in A. caudata (Table III). The characteristics of the Bohr effect in these animals make such values quite compatible with an in vivo O 2 transport function for the Hbs. The additional hypothesis of Terwilliger and Terwilliger (1984), that the greater the depth, the greater the drop in the Hb O ; affinity caused by hydrostatic pressure, then becomes unnecessary. The lugworm and alvinellid Hbs differ by another characteristic. In the lugworm, the apparent heat of oxy- genation. A//, is quite low, about -25 kJ/mol, and pH- independent. By contrast, in alvinellid Hbs, A// is strongly pH-dependent and is about three times higher, at pH 6.6- 6.9, than in the lugworm Hb (Table V). These high A// values explain the important effects of a temperature change on the intrinsic O 2 affinity, the Bohr effect, and the cooperativity. A general inverse relationship can be established between the value of A// and the range of temperatures at which a given respiratory pigment has to function /'/; vivo: the larger the temperature range, the lower the value of A/7 (see Toulmond, 1985. for exam- ples). Since A// is higher for alvinellid than for lugworm Hbs, the alvinellids probably live in an environment better temperature-regulated than that of the intertidal lugworm. This conclusion might seem to be inconsistent with the supposed extreme environmental variability around the hydrothermal vents, but annelids, and especially those living in elaborate tubes or galleries, are capable of creating their own regulated microenvironment (Toulmond. 1990). But as for O 2 concentrations, in situ direct mea- surements of the temperature microdistributions inside and outside the alvinellid tubes are needed. In conclusion, although the alvinellid Hbs are struc- turally very similar to those of annelids living in more ordinary habitats, these Hbs clearly exhibit some distinct functional properties that are most probably directly re- lated to the characteristics of the hydrothermal vent en- vironment. Their properties suggest that the alvinellid Hbs function as O 2 carriers at slightly acidic blood pH values and at fairly constant temperatures, not exceeding 20C for A. caudata and 30C for A. pompejana. This could indicate that both species can create a differentiated and stable external microenvironment. Acknowledgments This work has been partly supported by the Centre Na- tional de la Recherche Scientifique (Paris and LP 4601. Roscoff), the Institut Francais de Recherche pour ['Ex- ploitation de la Mer (Paris and Brest), and the National Science Foundation. We thank A. M. Alayse, H. Felbeck, D. Desbruyeres, and J. J. Childress, the leaders of the French-American project Hydronaut, the captains and crews of the RV Thomas G. Thompson and RV Nadir. and the pilots, copilots, and team of the DSR V Nautile. The TEM study was made with the participation of the Service d'Accueil de Microscopic Electronique, CNRS- Paris VI. We are most grateful to C. Poyart (Institut Na- tional de la Sante et de la Recherche Medicale, U299, Paris) who introduced A.T. to the Hemox Analyser tech- nique, and to Sarah Dejours who edited the English of this article. Literature Cited Autem, M., S. Salvidio, N. Pasteur. D. Desbruyeres, and 1>. Laubier. 1985. Mise en evidence de 1'isolement genetique des deux formes sympatriques A' Alvinella pompejana (Polychaeta: Ampharetidae). annelides infeodees aux sites hydrothermaux actifs de la dorsale du Pacifique oriental. C'. R Acud. Sci. Pans. St>r. 7/7301: 131-135. Childress, J. J., A. J. Arp, and C. R. Fischer Jr. 1984. Metabolic and blood characteristics of the hydrothermal vent tube-worm Riliia pa- chrplilii. Mar Hint. 83: 109-124. Desbruyeres, D., P. Crassous, J. Grassle, A. Khripounorf, D. Reyss, M. Rio, and M. Van Pract. 1982. Donnees ecologiques sur un nouveau site d'hydrolhermalisme actif de la nde du Pacifique oriental. C R Acad. Sci. Paris. Ser. Ill 295: 489-494. Desbruyeres, D., and I.. 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Functions of invertebrate hemoglobins with special reference to adaptations to environmental hypoxia. Am. /.ool. 20: 79-101. Weber, R. K. 1981. Cationic control of O ; affinity in lugworm ery- throcruonn. Nature 292: 386-387. W ells, G. P. 1963. Barriers and speciation in lugworms. Speciation in the sea. Systematics Association, London. Publ. No. 5, pp. 79-98. Reference: Bil. Hull 179: 374-382. (December, 1990) The Efflux of Amino Acids from the Olfactory Organ of the Spiny Lobster: Biochemical Measurements and Physiological Effects HENRY G. TRAPIDO-ROSENTHAL, RICHARD A. GLEESON, AND WILLIAM E. S. CARR The \Mutncv Lahoralon: L'niversiiy of Florida. 9505 Ocean Shore Blvd.. St. Augustine. FL 32086-8623 Abstract. The amino acids taurine and glycine are odorants that activate specific chemosensory cells in the olfactory sensilla (aesthetascs) of the spiny lobster, Pan- ulinis argus. We show that the aesthetascs themselves contain large intracellular concentrations of taurine ( == 2 mA/) and glycine ( = 85 mA/); these concentrations are more than 10,000-fold greater than the response thresh- olds of the chemosensory cells. A net efflux of at least five amino acids occurs when the olfactory organ is immersed in amino acid-free seawater. With taurine and glycine. efflux continues until an apparent equilibrium is reached between the sensilla and the external medium; for taurine the equilibrium with seawater occurs at = 12 to 28 nA/. and for glycine at =100 to 500 nA/. Aesthetascs may achieve these equilibria within 300 ms. Hence, even during the brief interval between consecutive flicks of the anten- nule. olfactory receptors are exposed to a background of odorants escaping from intracellular stores. Electrophys- iological studies show that both the spontaneous and evoked activities of taurine-sensitive chemosensory cells are markedly affected by a taurine background simulating that measured in the efflux studies. Uptake systems may participate in establishing the equilibria between sensilla and seawater since ( 1 ) the net efflux of amino acids in- creases in sodium-free seawater: and (2) guanidinoethane sulfonate, a competitor for taurine uptake, selectively in- creases net taurine efflux. Effluxes from an olfactory organ may contribute noise to the chemosensory process; alter- Reeeived 19 June 1990; accepted 25 September 1990. Abbreviations: ASW: artificial seawater: GES: guanidinoethane sul- fonate; HPLC; high performance liquid chromatography; OPA: orlho- pthaldialdehyde. natively, background substances could contribute func- tionally by affecting membrane proteins. Introduction Olfactory sensilla on the antennules of the Florida spiny lobster, Panulirw argus. contain populations of chemo- receptor cells with differential specificities for taurine, gly- cine, and other amino acids (see review by Carr et al. 1987). Physiological studies have revealed that taurine- sensitive cells have response thresholds of about 10'' M (Fuzessery et al.. 1978; Ache et al.. 1988), with some cells being activated by taurine at concentrations as low as 10 ' Al (Thompson and Ache, 1980). The response thresholds of the glycine-sensitive cells ( 10~ fi A/. Ache et al.. 1988) are generally higher than those of the taurine-sensitive cells. Chemoreceptors sensitive to exogenous amino acids are not unique to the spiny lobster. Indeed, chemoreceptor cells with selective sensitivities to specific amino acids also occur in several other crustaceans including the American lobster, Homarus americanus (Derby and Atema, 1982: Johnson and Atema, 1983), a crayfish, Austropotamobius torrent htm (Halt. 1984), a prawn, Macrobrachium rosen- hergii (Derby and Harpaz. 1988). and a crab, Carcinu.\ nicienas (Schmidt and Gnatzy, 1989). In addition to serving as exogenous chemoexcitants of crustaceans, amino acids such as taurine and glycine also occur intracellularly in very high concentrations (10"' to 10~ 3 A/), and contribute to osmotic regulatory processes (e.g.. Yancey et al.. 1982; Pierce, 1982). The antennular nerve of the spiny lobster, for example, contains taurine at a concentration of about 4 mA/ (see Results). Thus, this animal maintains an intracellular concentration of taurine that is more than a million-fold higher than the 374 EFFLUX OF AMINO ACIDS FROM AN OLFACTORY ORGAN 375 response thresholds reported for its taurine-sensitive che- moreceptors. If the sensory cells themselves contain mil- limolar concentrations of taurine, then a small leakage from these cells into the receptor environment could gen- erate a high background level of taurine that might negate the apparent utility of receptors with nanomolar sensitiv- ities. The leakage of internal chemicals into the receptor environment has indeed been implicated in mammals, where chemostimulants injected into the blood stream activated olfactory (Maruniak el a/.. 1983) and gustatory receptors (Bradley and Mistretta, 1971 ). In the present study, we show that the olfactory sensilla of the spiny lobster contain high intracellular concentra- tions of free amino acids. Using an attached intact anten- nular preparation (Trapido-Rosenthal a al.. 1990), we show that there is a measurable efflux of at least five amino acids from the olfactory organ. Although the efflux of each amino acid appears to be regulated, the efflux of glycine is most pronounced and may produce glycine back- grounds of up to 5 X 10 7 Al in the receptor environment. Finally, we demonstrate that the physiological responses of taurine-sensitive cells can be affected by a taurine back- ground of about 10~ 8 M which occurs because of the efflux from intracellular pools. Materials and Methods Collection and maintenance oj animals Specimens of the Florida spiny lobster, Paimlinis argus, were collected in the Florida Keys and maintained at the Whitney Laboratory in flowing seawater on a diet offish, squid, and shrimp. Only adult intermolt animals were used. Biochemical procedures Amino acid analysis. Amino acids present in tissue samples or in aliquots of incubation media were deriva- tized with <>/v/;o-pthaldialdehyde (OPA) (Lindroth and Mopper, 1979). The fluorescent derivatives were separated by HPLC; an octadecylsilane column packed with 4 pm beads (Waters Nova-Pak C u ) and eluted with Buffers A and B was used as described by Manahan (1989). Buffer A consisted of 50 mA/ sodium acetate (pH 6.8), methanol, and tetrahydrofuran (80:19:1). Buffer B consisted of 50 mAf sodium acetate and methanol (20:80). Derivatized amino acids were eluted from the column according to a step gradient procedure modified from Manahan (1989), so that at 1.6. 11. and 16 min after sample injection, the percentage of Buffer B was increased from to 25, 50, 75 and 100, respectively. The flow rate was 1.2 ml per min. Fluorescent derivatives were detected with a Bio-Rad Model 1700 fluorometer fitted with a 360 nm excitation filter and a 440 nm emission filter; peaks were integrated by means of a Waters Model 730 Data Module. Identi- fication and quantitation of amino acid derivatives were performed by comparisons with standards. Extraction oj amino acids from tissues. Olfactory sen- silla ( = aesthetascs) were collected from lateral antennular filaments after blotting, rapid freezing in liquid nitrogen, and lyophilization. The sensilla were removed with fine- tipped forceps, and their numbers estimated by counting the antennular segments harvested. Antennular nerve sections, approximately 2 cm in length, were dissected from lateral antennular filaments at a position just prox- imal to the aesthetasc tuft. Dissections were performed in a bath of P. argus saline, and the tissue blotted, weighed, and frozen. Hemolymph samples ( 1 ml) were withdrawn at the base of a walking leg; a chilled syringe was used to minimize clotting. Samples were immediately centrifuged (12.000 X g) to remove cellular material, and the super- natant frozen. Free amino acids in tissue samples were extracted by homogenization in a solution of 80% methanol/20% so- dium acetate (50 mAl, pH 6.8) followed by centrifugation (16,000 X g). The supernatants were transferred to clean tubes, evaporated to dryness. then redissolved in 50 mA/ sodium acetate for reaction with OPA as described above. Net efflux oj amino acids from the aesthetasc sensilla of attached intact antenmdes. Lobsters were removed from the water and immobilized on racks as described previ- ously (Trapido-Rosenthal el al., 1990). The distal portions of the intact lateral antennular filaments were placed in vials containing 3.5 to 4.5 ml of artificial seawater ( ASW; see Gleeson et al., 1989) which was vigorously agitated by using magnetic stirring bars. At selected times, samples of the ASW (= incubation medium) were removed from the vials for amino acid analysis. The above procedure ensured that the only part of the lobster contacting the incubation medium was the antennular filament; all ref- erences to the use of intact antennules are references to this procedure. In one experiment, analyses were performed on amino acids released into ASW by both aesthetasc-bearing and aesthetasc-free sections of antennular cuticle. These iso- lated sections were prepared as described in Trapido-Ro- senthal et al. (1987). In experiments to investigate the effect of low concen- trations of sodium on amino acid efflux from the aesthetascs, intact antennules were subjected to three se- quential. 10-min incubations. In the first incubation, the antennule was immersed in ASW; in the second incu- bation the antennule was immersed in artificial seawater in which the sodium chloride had been replaced with equimolar choline chloride. The third incubation was again performed in ASW. The effect of the taurine-uptake competitor, guanidinoethane sulfonate (GES), on amino acid efflux was examined according to a similar protocol 376 H. G. TRAPIDO-ROSENTHAL ET AL. except that the second of the three incubations contained GES in ASW. /:'/(.( 7 n >pln -sit >/< it;ii 'til pn tcedures The responses of single cells stimulated by taurine were recorded extracellularly from the isolated perfused lateral filament of the antennule. The olfactometer and recording procedures have been described in detail previously (Gleeson and Ache. 1985). Action potentials (impulses) from single cells were discerned via an amplitude/time window discriminator, and the time intervals between impulses analyzed with a microprocessor. In this report. cell responses are quantified in two ways: (1) the total number of impulses occurring within a 5- or 10-s period following onset of the response; and (2) maximum fre- quency. defined as the mean instantaneous frequency de- termined for the four shortest intervals between successive impulses. Effects oj background taurine. Cells stimulated by tau- rine were identified by introducing a 10-^A/ search stim- ulus into the carrier stream of ASW that continuously flowed past the olfactory sensilla at a rate of 3 ml/min. Once a taurine-sensitive cell had been identified, the dose- response function was determined in the presence and absence of an imposed background of 10 nAl taurine. This background simulated a representative concentration present in the receptor environment as calculated from our measurements of the efflux of endogenous taurine (vide infra). The effect of the background on the dose- response function was determined by applying an as- cending series of taurine concentrations; each concentra- tion was tested with and without background before the next higher concentration in the series was applied. For tests in the presence of background, the carrier stream of ASW contained 10 nA/ taurine which flowed through the olfactometer for 2 min before and during the introduction of a test stimulus. The response to each concentration was monitored following the injection of 1 90 ^1 into the carrier flow of ASW. With this volume, the concentration of taurine in the olfactometer reached the injected level within 1 s and began to decline after a 2-s plateau period (Zimmer-Faust el . c Hi ' -tree ASW 0) in 400 - C.'.'.'.l ASW V) '. 300 - 1 X J 200 - 1 i LJ *1> 100 - n nil A _i_ Gly Asp Glu Ala Tau Figure 4. Efflux of amino acids from intact antennules incubated sequentially in ASW (open bars). Na + -free ASW (solid bars), and ASW (hatched bars). In Na + -free ASW. NaCl was replaced with equimolar choline chloride. Incubations were for 10 min. Values are the means + SEM of three experiments. Amino acid abbreviations as in Figure 2. ANOVA coupled with a Planned Comparison Test (CSS, StatSoft): P < 0.05, n = 3] and reversible. GES did not significantly affect the efflux of the other amino acids ex- amined. The time course for the appearance ofglycine and tau- rine in the incubation medium was determined for the antennules of three different animals (Fig. 6). Although considerable inter-animal variability existed, in each case and for both amino acids, the extracellular concentration increased until an apparent equilibrium concentration was attained. The equilibrium concentrations ranged from 100 to 500 nM for glycine (Fig. 6A) and from 12 to 28 nM for taurine (Fig. 6B). The occurrence of a rapid efflux to an equilibrium concentration was typically observed for other amino acids as well. tion. This shift is highly significant as revealed by the in- tercept differences for the linear regions (i.e.. between 0.01 and 3.3 fiM taurine) of the maximum frequency curves [Random Coefficient Regression Analysis with intercepts compared using a Wilcoxon Signed Rank Test: P = 0.004, n = 9]. A paired comparison of the maximum responses (/.c., responses to 10 and 33 pAI taurine) for cells in the presence and absence of background taurine also yielded a significant difference [Wilcoxon Signed Rank Test: P = 0.03 (maximum frequency data), P < 0.001 (impulses per 5 s data), n = 15]. The responses of taurine-sensitive cells to exogenous taurine stimuli were unaffected by the presence of taurine in the perfusion saline (Fig. 8). For the six cells examined, the mean responses to test stimuli in the presence and absence of taurine in the perfusion medium were virtually identical (Wilcoxon Signed Rank Test: P = 0.438, n = 6). Discussion Cells within the aesthetasc sensilla of the spiny lobster contain the amino acids glycine and taurine at concen- trations of about 85 and 2 mM. respectively. Following immersion of the intact lateral antennular filament in amino acid-free seawater, a net efflux ofglycine and tau- rine from the sensilla occurs until an apparent equilibrium is reached with the external medium; for glycine the equi- librium concentration in the seawater is about 100 to 500 nM. and for taurine it is approximately 12 to 28 nA/(Fig. 6). The establishment of these equilibria indicates that aesthetascs can maintain intracellular glycine and taurine at concentrations that are more than 100,000-fold greater than those in the external medium. However, the existence of apparent limits on the ratio of intracellular to extra- cellular concentrations suggests that, even in seawater free Physiological c/lccts of a taurine background In the presence of a 10 nM background concentration of taurine in the carrier stream of ASW, the spontaneous activity in all of the taurine-sensitive cells examined ob- viously increased. This increase was significant; following a 2-min exposure to the taurine background, the mean number of impulses per second was 4.26 0.28 (SEM) versus 0.88 0.08 in the absence of taurine (Wilcoxon Signed Rank Test: P < 0.01, n = 9). The dose-response function for injected (= exogenous) taurine in the presence and absence of the 10 nM taurine background is shown in Figure 7. Expressing the response in terms of either maximum frequency or impulses per 5 s reveals a dose-dependent increase in activity which ap- pears to attain a maximum level between 3.3 and 10 nM taurine. In the presence of the taurine background, there is an apparent downward shift in the dose-response func- 200 - 150 4- 100 -- r so -- I I ASW B ASW plus GES Gly Asp Glu Ala Tau Figure 5. Efflux of amino acids from intact antennules incubated sequentially in ASW (open bars), ASW containing 1 fiM GES (solid bars), and ASW (hatched bars). Incubations were for 10 min. Values are the means + SEM of three experiments. Amino acid abbreviations as in Figure 2. EFFLUX OF AMINO ACIDS FROM AN OLFACTORY ORGAN 379 10 20 30 40 50 Incubation Time (min) Figure 6. The time course of efflux for glycine (A) and taunne (B) from intact antennules of three animals. At each time point, 100-fjl al- iquots were removed from the incubation vials and the amino acid con- centrations in the incubation media were determined. Apparent equi- librium concentrations were attained in the 4.5-ml incubation volumes within 30 to 40 min. of exogenous glycine or taurine, sensillar receptors will be exposed to background ("noise") levels of these amino acids because of their efflux from intracellular pools. The effluxes of glycine and taurine from the olfactory organ create, at equilibrium, background levels that cor- relate quite well with those occurring in natural seawater. Glycine is frequently present in seawater at levels ap- proaching 100 nA/ or greater (e.g., Garrasi el al.. 1979; Braven el ai, 1984; Siebers and Winkler, 1984), whereas taurine is often not detected and seldom exceeds 10 nM (MopperandLindroth, 1982; Wright and Secomb, 1986). The existence of a low background (i.e.. low noise) level of taurine in seawater, plus the occurrence of only a slight efflux from the olfactory organ, suggests that taurine leak- ing from a prey organism would be more readily detected than glycine. For taurine, an effective signal-to-noise ratio could exist at exogenous concentrations above approxi- mately 10 nM; whereas for glycine, good signal-to-noise ratios would require concentrations greater than about 100 nA/. Indeed, the contrasts between the low (nano- molar) thresholds of taurine-sensitive cells (Fuzessery et ul-, 1978; Ache et ai. 1988). and the apparently higher (micromolar) thresholds of glycine-sensitive cells (Ache ct ul., 1988), may be expressions of receptor adaptations to the exigencies of different background concentrations. The lobster obtains discontinuous samples of its chem- ical environment by periodically flicking its antennules in a manner that rapidly exchanges water trapped between the densely arranged aesthetasc sensilla (Price and Ache, 1977; Schmitt and Ache, 1979; Moore and Atema, 1988; R. A. Gleeson, pers. comm.). The time interval between successive flicks (interflick interval) can vary from about 500 ms to over 30 s (R. A. Gleeson, pers. comm.). During this interflick period, seawater trapped between the aes- thetascs forms a large boundary layer within which odor- ant movement is essentially restricted to molecular dif- fusion (Schmitt and Ache, 1979; Moore and Atema, 1988). As a consequence, chemoreceptors within the aesthetascs are primarily exposed to whatever odorants are captured during the preceding flick, with the actual concentrations at the receptors being dependent upon the rates of odorant diffusion between the aesthetascs and the zoo A N I Control t 150 ^ u c o> D D" Plus Taurine Background 1 1 1 i v 100 9 b_ I I I I D E 50 o 1 X 2 -T 1 1 ~i I 0.01 0.1 1 10 2SU - B T [ 200 Control o Plus Taurine Background 1 1 150 I 1 T I 1 100 i I 1 ' ' 50 4 , ? 9 n 0.01 0.1 1 10 Taurine Concentration (yiiM) Figure 7. Dose-response functions for taurine-sensitive cells in the presence and absence of a 10 nM taurine background. Response mag- nitude is expressed in terms of maximum frequency (A) and total number of impulses during the first 5 s of the response (B). Points are the means SEM for nine cells. 380 H. G. TRAPIDO-ROSENTHAL ET AL \ : o CD 300 % 200 3 CL E 100 (0.1) /,\ [ ] Control Saline HI Saline plus Ta urine (100) 0) (1000) Mil 1 23456 Cell Number Figure 8. Mean responses (+SEM) of taurine-sensitive cells in the presence and absence of taurine in the perfusion saline. The micromolar concentrations of taunne in the perfusion medium are indicated in pa- rentheses. For each cell, a test-stimulus concentration close to the EC 50 was presented via the carrier stream of ASW. boundary layer of seawater. This boundary layer can also limit the rate of amino acid efflux from aesthetascs by acting as a buffer between the sensilla and seawater outside the aesthetasc tuft. If it is assumed that a major fraction of the glycine and taurine leaking from the cells of an aesthetasc during the interflick interval remains within the sensillar lymph (volume = 200 pi), then only about 300 ms would be required for the sensillum to attain the equilibrium concentrations measured in the current study (Fig. 9). Hence, during a considerable portion of each interflick interval, the sensillar receptors are probably ex- posed to backgrounds ranging from low nanomolar in the case of taurine, to as high as 0.5 micromolar in the case of glycine. When sensilla are immersed in seawater, the efflux of glycine and taurine from intracellular stores does not continue unabated until the concentration in the sensilla and medium are equal. Rather, at equilibrium the intra- cellular concentration is about 100,000-fold greater than the medium. Uptake systems in the sensilla are the most plausible mechanisms for regulating the amino acid efflux. The hypothesis that uptake systems might control the net efflux or loss of intracellular amino acids was proposed by Wright and Secomb (1986) based on studies with the gills of marine mussels. These workers noted that mussel gills contain intracellular taurine at a concentration of about 60 mM. and that a net efflux occurred into seawater. They demonstrated the existence of a taurine uptake sys- tem and showed that it was able to recapture up to 30% of the taurine escaping from the gills. They then proposed that this re-uptake conserves energy and contributes to maintaining the high intracellular concentrations of tau- rine (Wright and Secomb. 1986; Wright, 1987). Our study on amino acid efflux from the lobster olfactory organ re- veals the following parallels with these findings from mol- luscan gills: ( 1 ) immersion in amino acid-free seawater results in a net efflux of amino acids that continues until an apparent equilibrium is established with the external medium (Fig. 6); (2) the net efflux of amino acids increases in Na + -free seawater (Fig. 4); and (3) a selective competitor of taurine uptake increases the net taurine efflux (Fig. 5) (Wright and Secomb. 1984, 1986; Wright el al.. 1989). Regarding the olfactory organ of the lobster, we already know that uptake systems for taurine and other amino acids are present in the olfactory sensilla (Gleeson el al., 1987; Trapido-Rosenthal el al.. 1988). However, the ki- netics of uptake exhibited by the excised sensilla used in these earlier studies are not compatible with maintaining the equilibrium concentrations measured in the current study. The intact antennular preparation should now be employed to re-examine the kinetics of uptake. In the American lobster, Homarus americamis, the ad- aptation of NH 4 -sensitive chemosensory cells to increased background levels of NH 4 was studied in detail by Borroni and Atema (1988). For each imposed background, ad- aptation of these receptor cells included: ( 1 ) a re-setting of the response threshold to an NH 4 concentration greater than background; and (2) a concomitant, parallel right- ward shift in the stimulus-response function (ihid.). In the present study, the exposure of taurine-sensitive cells of the spiny lobster, P. argus, to a background level of taurine yielded results having both similarities and differences to those described for the NH 4 -cells of//, americamis. Unlike 500 400 S 300 \ c o o -v 200 1 o 100 100 200 300 400 500 Time (msec) 600 Figure 9. Calculated concentrations for glycine and taunne in the sensillar lymph of a single sensillum from the animal represented by open circles in Figure 6. Assumptions used were as follows. (1) At to. the antennule is flicked and the 200-pl extracellular volume within the sensillum immediately equilibrates with the amino acid-free seawater. (2) During the subsequent interflick interval, all amino acids released from the cells of the sensillum are retained in the 200-pl volume of sensillar lymph. Under these conditions, both glycine and taurine attain their equilibrium concentrations in the lymph within 300 ms. EFFLUX OF AM1NO ACIDS FROM AN OLFACTORY ORGAN 381 the responses of NH 4 -cells to NH 4 backgrounds, taurine- sensitive cells did not exhibit complete adaptation to the taurine background examined. Instead, after an initial phasic response when the background was first introduced, these cells reached a new tonic level of activity that was significantly greater than their spontaneous activity in ASW. As in NH 4 -cells. adaptation (albeit partial) was in- dicated by a rightward, or seemingly downward, shift in the dose-response function. In the taurine-sensitive cells, this shift included an apparent reduction in the maximum response; this effect might be considered functionally equivalent to either a generalized reduction in the efficacy of taurine, or to an inactivation of some proportion of the receptor population. In the current study, the responses of taurine-sensitive chemosensory cells in the antennule were not affected when taurine concentrations as high as 1 mM were pre- sented internally via the perfusion saline (Fig. 8). These results imply that a functional barrier, at least for taurine, separates the hemolymph and the sensillar lymph. These results contrast with findings in mammals where certain chemostimulants injected into the blood stream were found to stimulate olfactory (Maruniak el ai. 1983) and gustatory receptors (Bradley and Mistretta, 1971). A high intracellular concentration of low molecular weight organic substances is a characteristic feature of or- ganisms subjected to water stresses, including high or fluctuating salinity (Yancey el ai. 1982). These organic substances together with inorganic ions represent the ma- jor osmotically active solutes (osmolytes) present within all cells. Prominent among these compounds are certain amino acids including taurine, glutamate. alanine, glycine, proline, and aspartate (Clark. 1985). The similarities be- tween species in the chemical properties of organic os- molytes are remarkable; these properties parallel those of cations and anions in the Hofmeister series that favor compatibility with protein structure and function. Indeed, these intracellular substances may be important in off- setting the destabilizing or perturbing effects that high neutral salt concentrations have on macromolecules (Yancey el a/., 1982). By analogy with the intracellular effects described above, the efflux of certain amino acids (e.g., glycine) and other osmolytes into the receptor en- vironment within olfactory sensilla may play a general role in stabilizing the extracellular domains of various membrane proteins associated with the sensory dendrites. The efflux of such compounds could be important in maintaining the structure and function of receptors, channel proteins, transporters and ecto-enzymes that would otherwise be directly exposed to seawater. Indeed, certain of these substances may specifically modulate the activity of some membrane proteins. For example, extra- cellular glycine occurring in synaptic clefts binds to specific sites on the NMDA-glutamate receptor-channel complex and contributes significantly to receptor activation and channel function (C.H.. Kessler el ai. 1989; Thomson, 1989). Organic osmolytes in the extracellular lymph of olfactory sensilla may play similar regulatory roles in che- mosensory processes. Acknowledgments This research was supported by NSF Grant BNS- 8908340. We thank Ms. Marsha Lynn Milstead for pre- paring the illustrations and Dr. Charles Derby for helpful discussions during the preparation of the manuscript. Literature Cited Ache, B. W., R. A. Gleeson, and H. A. Thompson. 1988. Mechanisms for mixture suppression in olfactory receptors of the spiny lobster. Chem. Senxex 13: 425-434. Borroni, P. K., and J. Alema. 1988. Adaptation in chemoreceptor cells. 1. Sell-adapting backgrounds determine threshold and cause parallel shift of response function. J. Comp. Phytiol A 164: 67-74. Bradley, R. 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Diel and depth variations in dis- solved free amino acids in the Baltic Sea determined by shipboard HPLC analysis. Limnol. Oceano K r 21: 336-347. Pierce, S. K. 1982. Invertebrate cell volume control mechanisms: a coordinated use of intracellular amino acids and inorganic ions as osmotic solute. Bui i hucliicii\i.'i. by coralline red algae, 304 Influence of macroalgae in eelgrass beds on finfish abundance and dis- solved oxygen in Waquoit Bay, 223 Inhibition, 191 Inland culture of a nudibranch, 243 Inositol 1,4,5-trisphosphate, 228 Insect neurohiology, 87 Intentional catch (Busycon) and unintentional catch (Hoploplana) by fishermen and the question of seafood inspection, 227 Interommatidial angle. 230 Intertidal ecology, 105 Intracranial pressure, 233 Intraspecific variation in egg capsule morphology, 312 Invertebrate. 223 Isomate. 234 Isopod. 186 JAFFE, L. F., see I. Gillot, 224 JAFFE, LIONEL F., see Andrew L. Miller. 224; and Richard M. Sanger, 225 JAFFE. LIONEL F.. The path of calcium in fertilization and other endog- enous oscillations: a unifying view, 224 JONASDOTTIR, SiGRUN H., see Darcy J. Lonsdale, 1 13 JOUIN, CLAUDE, see Andre Toulmond. 366 K KAHANA, ALON, PHYLLIS R. ROBINSON. AND JOHN E. LISMAN, Inac- tivation of squid rhodopsin in the absence of phosphorylation, 230 KALLEN. JANINE L., S. L. ABRAHAMSE, AND F. VAN HERP, Orcadian rhythmicity of the crustacean hyperglycemic hormone (CHH) in the hemolymph of the crayfish, 351 KANAMORI. KAN, see Hitoshi Michibata. 140 KANO, YASUO T., see Mieko Komatsu, 254 KAPLAN, ILENE M.. BARBARA C. BOYER, AND DANIELA E. HOFFMAN, Intentional catch (Bitsycon) and unintentional catch (Hoploplana) by fisherman and the question of seafood inspection, 227 KARPLLIS, ERIC, see Richard M. Sanger, 225 KEMPF, STEPHEN C., see David J. Carroll. 243 KIM. N. H., see C. D. Leidigh. 224; and D. C. Spray, 225 KlNASE, 219 KJNGSLEY, RONI J., MARI TSUZAKI, NORIMITSU WATABE. AND GERALD L. MECHANIC. Collagen in the spicule organic matrix of the gor- gonian Leptogorgia virgukiia. 207 KINGSTON, SAM, see Richard D. Feinman. 233 KOIDE, S. S.. see Hiroshi Ueno. 226 KOMATSU, MIEKO, YASUO T. KANO, AND CHITARU OGLIRO, Devel- opment of a true ovoviviparous sea star, Asterina pseudoexigua paciftca Hayashi. 254 KOMURO, H.. A. L. OBAID, S. S. KUMAR. AND B. M. SALZBERG, Slices of mouse suprachiasmatic nucleus with attached optic nerve: re- cording of glutammergic and GABA-ergic postsynaptic potentials using a voltage-sensitive dye, 231 KOMURO, H.. see A. L. Obaid. 232 KORTHALS ALTES, HESTER, see Richard D. Feinman, 233 KUHNS, WILLIAM J., GRADIMIR MISEVIC, AND MAX M. BURGER. Bio- chemical and functional effects of sulfate restriction in the marine sponge, Microciiina prolijera, 358 KDHTREIBER, W. M., see I. Gillot, 224 KUMAR, S. S.. see A. L. Obaid. 232; and H. Komuro, 231 KURIS, ARMAND, see Hans Laut'er. 221 KUZIRIAN, ALAN M., CATHERINE T. TAMSE, AND MARK HEATH, Ozonation of natural seawater affects the embryology of Hermis- scnila crassicornis, 221 KUZIRIAN, ALAN, see Ebenezer Yamoah. 232 Laboratory culture of the aeohd nudibranch Bergliia verrucicornis (Mol- lusca, Opisthobranchia): some aspects of its development and life history. 243 LANGFORD, GEORGE M.. EDWARD E. LEONARD, DIETER G. WEISS, AND SANDRA A. MURRAY, Effects of cAMP-dependent protein ki- nase inhibitor on organelle movement in Y- 1 adrenocortical tumor cells, 219 Larvae. 297. 304 Larval aggregation, 279 development, 243 fusion, 279 skeletons and orientation, 12 1 LASSER-ROSS, NECHAMA, see Joseph C. Callaway, 228 LAUFER, HANS, ELLEN HOMOLA, ARMAND M. KURIS, AND AMIR SAGI, Morphology and reproductive tract development in winter and summer populations of the male spider crab Lihiniu emarginata: a proposed life history and regulatory mechanism. 221 LDHs of hydrothermal vent fishes, 134 Learning, 233 LEIDHIGH, C.. see D. C. Spray, 225 LEIDIGH. C. D., N. H. KIM. R. D. GOLDMAN, A. GOLDMAN, M. V. L. BENNETT, AND G. D. PAPPAS, The gigantic germinal vesciles of elasmobranchs. 224 Lens, 220 LEONARD. EDWARD E., see George M. Langford, 219 Leptogorgia virgulata, 207 Leucandra. 272 Lever-press conditioning in the crab. Green crabs perform well on fixed ration schedules, but can they count? 233 Libinia emarginata, 22 1 Light organ. Euprymna, 332 Limiiliis. 219, 233 Limu/us-eye view of the world. The. 230 LINN, J.-W.. see A. L. Obaid. 232 LISMAN, JOHN E., see Alon Kahana. 230 Litlwiliamnion glaciate. 304 INDEX TO VOLUME 179 387 LLINAS, R., see A. L. Obaid, 232; and K. R. Delaney. 229 Lobster. 234, 374 LOHMANN. KENNETH J., MICHAEL SALMON, ANDJEANETTE WYNEKEN, Functional autonomy of land and sea orientation systems in sea turtle hatchlings, 214 LONSDALE, DARCY J., AND SIGRUN H. JONASDOTTIR, Geographic vari- ation in naupliar growth and survival in a harpacticoid copepod, 113 LOWE, K.RIS C., see Seymour Zigman, 220 LOYA, Yossi. see Micha llan, 279 LYNCH, LAURA, see Robert Billard, 222 Lyoluminescence. 232 Lyosomes, 219 M MACAGNO, EDLIARDO, see Michael Nitabach, 232 MACCOLL, ROBERT, JOHN GALIVAN, DONALD S. BERNS, ZENIA NIMHC, DEBORAH GUARD-FRIAR, AND DAVID WAGONER, The chromo- phore and polypeptide composition of Aply.iia ink, 326 Macroalgae. 223 Macromere control of early development in the polyclad flatworm, //<>- ploplana, 22 1 Macromere deletions. 22 I MALCHOW, ROBERT PAUL , RICHARD L. CHAPPELL, PAUL GLYNN, AND HARRIS RIPPS, GABA-induced currents of internal horizontal cells of the skate retina. 231 MANGEL, WALTER F., see Peter B. Armstrong. 233 Marine embryo coupling, 225 Marine policy, 227 MARUYAMA, YOSHIHIKO, K., Roles of the polar cytoplasmic region in meiotic divisions in oocytes of the sea cucumber llolnilniriu Icu- cospiloia. 264 Maximal enzyme activities, 287 McFALL-NGAi, MARGARET, AND MARY K. MONTGOMLRY, The anat- omy and morphology of the adult bacterial light organ of Eiiprynimi scolopes Berry (Cephalopoda: Sepiolidae), 332 MCLAUGHLIN, JANE A., see Andrew L. Miller, 224 MECHANIC, GERALD L.. see Roni J. Kingsley, 207 Meiosis, 264 Meiotic spindle organizing centers, 264 Melibe. 229 Metalloproteinases of the developing sea urchin embryo, 22 I Metamorphosis. 243, 304 Methyl farnesoate. 221 MlCHIBATA, HlTOSHI, HlSAYOSH! HlROSE, KlYOMI SUG1YAMA, YlIKARI OOK.UBO, AND KAN KANAMORI, Extraction of a vanadium-binding substance (vanadobin) from the blood cells of several ascidian spe- cies. 140 Microciona proli/era. 358 Micromere deletions, 222 Microscale fluid dynamics, 234 Microspondian cytoskeletal elements. 237 Migration, 214 MILLER, ANDREW L.. RICHARD A. FLUCK, JANE A. MCLAUGHLIN, AND LIONEL F. JAFFE, Calcium waves spread beneath the furrows of cleaving Oryzias talipes and Xenopus lacvis eggs, 224 Mineral induction. 191 MISEVIC. GRADIMIR, see William J. Kuhns, 358 MONTGOMERY, MARY K., see Margaret McFall-Ngai, 332 MOORE, PAUL A., see Adele Pile. 234; Jelle Atems, 234; and Nat Scholz, 235 MORENO, A. P.. see D. C. Spray, 225 MORENO, ALONSO P.. AND DAVID C. SPRAY, Sea urchin embryos: suit- ability for exogenous expression of gap junction channels, 225 Morphogenesis of ascidian ampullae and polari/ed movements of tunic extracellular matrix components along ampullae, 220 Morphology and reproductive tract development in winter and surnmar populations of the male spider crab Libinia I'marginaia: a proposed life history and regulatory mechanism. 221 Mosaic development, 220 Motihty, 219 Motor nuclei, 230 mRNA expression, 225 MURRAY, SANDRA A., see George M. Langford, 219 N Naupliar growth and survival, I 13 Nematocyst, 96 NEMOTO, SHIN-ICHI, see Mitsuki Yoneda, 183 Nerve backfills, 232 sealing, 229 terminals, 232 Neuromeres, 230 Neurons. 232 Niche displacement, models of. 223 Nickel chloride, 232 NIMEC. ZENIA. see Robert MacColl, 326 NITABACH, MICHAEL, AND EDLIARDO MACAGNO, Desperately seeking sex neurons: detection of projections in the male sex nerve ot 'Hmulu mi'iiicinalix using nickel and horseradish peroxidase backfills, 232 Nocturnal emergence activity rhythm in the cumacean Dimorphostylis axiaiica (Crustacea), 1 7S Nucella imargmala, 312 Nuclear envelope, 224 fusion. 183 RNA, 77 Nudibranch. 243 o O'BRIEN, KRISTIN M., LINDA A. DEEGAN, JOHN T. FINN, AND SUZANNE G. AYVAZIAN, The effects of macroalgae on the abundance and diversity of free-swimming invertebrates in eelgrass beds of Waquoit Bay, MA, 223 OBAID, A. L., H. KOMURO, S. S. KUMAR. M. SUGIMORI, J.-W. LIN. B. D. CHERKSEY, R. LLINAS, AND B. M. SALZBERG, FTX, an HPLC- punfied fraction of funnel web spider venom, blocks calcium chan- nels required for normal release in peptidergic nerve terminals of mammals: optical measurements with and without voltage-sensitive dyes, 232 OBAID, A. L., see H. Komuro, 231 Occurrence of partial nuclei in eggs of the sand dollar, Clypcaxtcr ;<;- punicux. 183 Odor flow within normal cavity, 234 Odor plumes, 235 Odorant behavior. 374 OGILVY, CHRISTOPHER S.. see Stephen H. Fox, 233 OGURO. CHITARLI, see Mieko Komatsu, 254 Olfaction, 374 Olfactoi-y sensilla. 374 Ontogenetic variation in sponge histocompatibility responses, 279 Oocytes. sea cucumber. 264 OOKUBO, YUKARI, see Hitoshi Michibata, 140 Operant conditioning. 233 Optical. 229 Optical recording, 232 Optics, 230 Organic matrix. 191 Orientation, 214. 235 Ornithine decarboxylase exhibits negative thermal modulation in the sea star Asteriax vulgaris: potential regulatory role during temperature- dependent testicular growth, 159 Osmolytes. 374 OVERSTREET, ROBIN M., see Earl Weidner, 237 Oyster, 297 Ozonation of natural seawater affects the embryology of Hermissenda crassicornix, 227 388 INDEX TO VOLUME 179 Palylhtia liihcrci/liaa. 148 Panulinn ar\;iis. 374 PAPPAS, G. D.. see C. D. Leidigh, 224; and D. C. Spray, 225 Paralvinella, 366 Paramecium. 228 Parasitism and the movements of intcrtidal gastropod individuals, 105 PARDUE, M. L., W. G. BENDENA, M. E. FINI, J. C. GARBE, N. C. HOGAN, AND K. L. TRAVERSE, Hsr-omcga, a novel gene encoded by a Dro- sophila heat shock puff. 77 Partial nuclei in sand dollar eggs, 183 Passive orientation. 121 Path ol calcium in fertilization and other endogenous oscillations: a uni- fying view. The. 224 PATON, DAVID, Greenough Pond project planning: Yarmouthport, Massachusetts; acid water and macro flora is the base line of H. Sverdop and P. Warfingers' chain of response decision tree. 227 PATON, DAVID, Halichoerus grypus at Monomoy Island, winter of 1 988- 1989: photogrammetry showing shift east and north of traditional birthing sites, 228 PATON, DAVID, see John T. Finn. 226 Patterns of stimulated bioluminescence in two pyrosomes (Tunicata; Pyrosomatidae). 340 PEARCE. CHRISTOPHER M., AND ROBERT E. SCHEIBLING. Induction of metamorphosis of larvae of the green sea urchin, Strmg\-locentrotus droebachiensis, by coralline red algae, 304 PENNINGTON, J. TIMOTHY, AND RICHARD R. STRATHMANN, Conse- quences of the calcite skeletons of planktonic echinoderm larvae for orientation, swimming, and shape, 121 Phosphorylation, 226, 230 Photic stimulation, 340 Phyllodiscus senwni, 148 PILE, ADELE, PAUL MOORE, AND JELLE ATEMA. Three-dimensional odor flow within the nasal cavity of the bullhead catfish, 214 Pluteus, 121 Poeciloogony, 243 Polar body formation, 264 Polarity. 222 Polarity and meiosis in oocytes, 264 Polyamines. 159 Polymorphic males, 221 Polytene chromosomes, 77 Ponuitumus saltutrix. 233 Pond, acid, management, 227 Practical guide to the developmental biology of terrestrial-breeding frogs 163 Predation on egg capsules, 3 1 2 Predation rates, 222 Preliminary optical measurements on the Melihc Iconina buccal ganglion 229-230 Pressure-temperature interactions on M 4 -lactate dehydrogenases from hydrothermal vent fishes: evidence for adaptation to elevated tem- peratures by the zoarcid Thermarces amlcrsoni. but not by the by- thitid, Bythitex hollixi. \ 34 Presynaptic terminal, 228 PRIOR, G., see R. E. Stephens. 226 Promotion and inhibition of calcium carbonate crystallization in vitro by matrix protein from blue crab exoskeleton, 191 Protein content. 287 synthesis, 77 Proteolytic enzymes, 22 1 Protistan cytokeratin and desmoplakin analogues. 237 Protopalythoa sp., 148 Pyrosome bioluminescence, 340 QUIGLEY, J., R. S. BRAITHWAITE, AND P. ARMSTRONG, Metalloprotei- nases of the developing sea urchin embryo. 221 QUIGLEY, JAMES P., see Peter B. Armstrong. 233 R RAFFERTY, NANCY S., see Seymour Zigman, 220 Raja. 231 RAND, REBEKA J., LINDA A. DEEGAN, JOHN T. FINN, AND SUZANNE G. AYVAZIAN, Influence of macroalgae in eelgrass beds on finhsh abundance and dissolved oxygen in Waquoit Bay, 223 RAWLINGS, TIMOTHY A., Associations between egg capsule morphology and predation among populations of the marine gastropod, Nucclla emargmata. 3 1 2 Recruitment, 297 Red algae. 326 Red crab, 226 Relationship between body size, growth rate, and maximal enzyme ac- tivities in the brine shrimp. Anemia franciscana. 287 Relationship between flow and chemical signals in providing directional cues for chemically orientating hermit crabs. The, 235 Released CHH in crayfish. 351 Reorganization of cytoskeleton during cell fusion induced bv electric field, 220 Repetitive calcium waves in the fertilized ascidian egg are intitiated in the vegetal hemisphere by a cortical pacemaker. The. 222 Reproductive ecology, 312 Reproductive system regulation, 221 Reptile, 214 REYNOLDS. G. T.. Lyoluminescence. 232 Rhizophydium littomim on the eggs of Cancer anthonvi: parasite or saprobe 1 ? 20 1 Rhodopsin. squid. 230 Rhythmic behavior in the field, 178 RIPPS, HARRIS, see Robert Paul Malchow, 231 ROBINSON, PHYLLIS R., see Alon Kahana, 230 ROFF, JOHN C., see John A. Serges. 287 Roles of the polar cytoplasmic region in meiotic divisions in oocytes of the sea cucumber, Hololhuna leucospilola. 264 Ross, WILLIAM N., see Joseph C. Callaway. 228 ROY, J., see Stephen A. Watts, 159 S.E.M. observations of early cleavage in Hoploplana inquilina. 220 SAGI, AMIR, see Hans Laufer, 221 SALMON. MICHAEL, see Kenneth J. Lohmann. 214 SALZBERG, B. M.. see A. L. Obaid, 232; and H. Komuro. 231 Sand dollar egg, 183 SANGER, RICHARD A., ERIC KARPLUS, AND LIONEL F. JAFFE, An aerial vibrating probe, 225 SCEMES, E.. see D. C. Spray. 225 SCHEIBLING, ROBERT E., see Christopher M. Pearce. 304 SCHIMINOVICH. DAVID, see Larry Cohen, 229 SCHNEIDEMANN, SABINE, see Elizabeth Dahlhoff, 134 SCHOLZ, NAT, PAUL A. MOORE, AND JAFFE ATEMA, The relationship between flow and chemical signals in providing directional cues for chemically orientating hermit crabs, 235 Sea anemones, 148 Sea cucumber, 264 Sea turtle wave orientation, 214 Sea urchin. 224. 225. 304 Sea urchin embryos: suitability for exogenous expression of gap junction channels. 225 Seafood inspection. 227 Search for the biological stimulus of the Cushing response in bluefish 233 Seasonal variation, 178 SEGAL, SHELDON J.. see Hiroshi Ueno. 226 Segmental location of cranial nerve roots and motor nuclei in Squalus acanthias, 230 Self and non-self recognition in a calcereous sponge, Leucandra ahratsbo 272 Settlement, 304 Settlement behavior, 297 INDEX TO VOLUME 174 389 Shell disease, 226 SHICK, J. MALCOLM, Diffusion limitation and hyperoxic enhancement ot'oxygen consumption in zooxanthellate sea anemones, zoanthids, and corals. 14S SHIELDS, JEFFREY D., Rhiiophydium liiinrcnni on the eggs of Cancer anthonyi: parasite or saprohe? 20 1 SlKES. C. S., see M. E. Gunthorpe. 191 Single and multiple micromere deletions in first quartet embryos of II- yiimixsa nhso/cui. 222 Single channels, 225 Sinking, 121 Size-scaling. 287 Skate retina, 231 Skeleton, 121 Slices of mouse suprachiasmatic nucleus with attached optic nerve: re- cording of glutaminergic and GABA-enc postsynaptic potentials using a voltage-sensitive dye, 23 1 SLITINE, FOUZIA EL IDRISSI, see Andre Toulmond, 366 SOMERO, GEORGE N., see Elizabeth Dahlhoff. 134 SPEKSNIJDER. J. E., The repetitive calcium waves in the fertilized ascidian egg are intitiated in the vegetal hemisphere by a cortical pacemaker, 222 Spermatogenesis. 159 Spicule matrices, 207 Spirocyst, 96 Sponge, 272, 358 Sponge histocompatibility responses. 279 SPRAY, D. C., A. C. CAMPOS DE CARVALHO, A. P. MORENO, E. SCEMES. C. LEIDHIGH, N. H. KIM. G. D. PAPPAS. AND M. V. L. BENNETT, Gapjunction channels in marine embryos: comparison of properties in late blastulae of squid and skate, 225 SPRAY, DAVID C, see Alonso P. Moreno. 225 Squid, 332 giant axon, 229 rhodopsin, 230 STEPHENS. R. E.. ANDG. PRIOR, Cyclic AMP-dependent phosphor. lation of dynein heavy chains in Myiilux cdulix sperm flagella. 226 Stochastic and deterministic models of niche displacement. 223 STOCKBRIDGE, NORMAN, see Ebenezer Yamoah, 232 STRATHMANN, RICHARD R., see J. Timothy Pennington, 1 2 1 Strongylocenirotus droebachiensis, 304 STROUT, M., see E. L. Bearer, 219 Structure of alpha-2 macroglobulin from the horseshoe crab, 233 Structure of sweeper tentacles in the black coral Antipathes fuirilmsis. The. 96 STUART, ANN E.. see Joseph C. Callaway, 228 Slylophora pixiillala, 148 Submersible vehicle observations of deep sea red crabs. Chaccon (/IIIH- ijuedens. off of the LI.S. continental shelf. 226 SUGIMORI. M., see A. L. Obaid, 232 SUGIYAMA, KlYOMI, see Hitoshi Michibata. 140 Sullate restriction in marine sponge, 358 Suprachiasmalic nucleus. 231 Sweeper tentacles. 96 SWEET, HYLA C., AND BARBARA C. BOYER, Single and multiple micro- mere deletions in first quartet embryos of llyanassa nhxnlela. 222 SWEET. HYLA C., see John M. Arnold, 220 Swimming, 121, 186 Symbiosis, 201 Synapsin I, 229 Synaptic transmission, 229 TAMSE, CATHERINE T, see Alan M. Kuzirian, 227 TANK, D. W., see K. R. Delaney. 229 Taunne, 374 TAYLOR, GEORGE T., see Walter M. Goldberg, 96 TEDESCHI, BRUCE, see Earl Weidner. 237 Temperature adaptation. 159 Terrestrial-breeding frogs, 163 Thermal modulation of ODC, 159 Thermarcei anilcixoiu. 134 Ihrec-dimensional odor tlow within the nasal cavity of the bullhead catfish. 234 Tidal cycle, 17X TOULMOND, ANDRE, FOUZIA EL IDRISSI SLITINE, JACQUES DE FRESCHEVILLE, AND CLAUDE JOUIN, Extracellular hemoglobins of hydrothermal vent annelids: structural and luctional characteristics in three alvinellid species, 366 TOWNSEND. DANIEL S., see Richard P. Elmson, 163 TRAPIDO-ROSENTHAL, HENRY G.. RICHARD A. GLEESON, AND WILLIAM E. S. CARR. The efflux of amino acids from the olfactory organ of the spiny lobster: biochemical measurements and physiological ef- fects. 374 TRAVERSE, K. L., see M. L. Pardue, 77 Trematod parasitism, 105 TRIMARCHI, JIM, see Chun X. Falk, 229-230 TROLL, WALTER, see Krystyna Frenkel, 221 TSUZAKI. MARI. see Roni J. Kinsley, 207 Tumor promoters, 22 1 Tunicate, 140 Tuning, 234 u UENO, HIROSHI, SHELDON J. SEGAL, AND S. S. KOIDE, Gossypol-bmdmg proteins from marine species. 226 Ultrastructure, 96 Unisex Hash controls in dialog fireflies. 87 UV radiation. 220 VAN EGERAAT, J. M., AND J. P. WIKSWO JR., Application of a magnetic current probe to map axial inhomogeneities in a squid giant axon, 229 VAN HERP, F., see Janine L. Kallen, 351 VAN HOLDE, KENSAL E., see Peter B. Armstrong. 233 Vanadobin from ascidians, 140 Vibrating probe. 225 Mbrni fischeri, 332 Viviparity. 254 VoiGT, RAINER, see George Gomez, 234; and H. F. Gerardo, 234 Voltage-sensitive dyes, 231 W WAGONER, DAVID, see Robert MacColl. 326 WALCH, M.. see S. L. Coon. 297 WALKER, C. W., see Stephen A. Watts. 159 Waquoit Bay, 223 WATABE, NORIMITSU, see Roni J. Kinsley, 207 Water How, 148 WATRAS. JAMES, see Ilya B, Bezprozvanny, 228 WATSON, WIN, III, see Chun X. Falk, 229 WATTS, STEPHEN A., J. ROY, AND C. W. WALKER, Ornithine decar- boxylase exhibits negative thermal modulation in the sea star As- leruix vit/garix: potential regulatory role during temperature-depen- dent testicular growth. 159 Wave, 214 WEIDNER. EARL, ROBIN M. OVERSTREET, BRUCE TEDESCHI. AND JOHN FUSELER, Cytokeratin and desmoplakin analogues within an intra- cellular parasite, 237 WEINER, R. M., see S. L. Coon, 297 WEISS. DIETER G., see George M. Langford. 219 WHEELER, A. P., see M. E. Gunthorpe, 191 Where does the calcium lost by fertilizing Arbacia eggs go? 224 WIDDER, EDITH A., see Mark R. Bowlby. 340 WIKSWO, J. P., JR., see J. M. van Egeraat, 229 Winter flounder. 222 390 INDEX TO VOLUME 174 Wu, JlAN-YOUNG, see Chun X. Falk, 229-230; and Larry Cohen. 229 Z WYNEKEN, JEANETTE, see Kenneth J. Lohmann. 214 ZHENG, Q.. AND D. C. CHANG. Reorganization of cytoskeleton during Y cell fusion induced by electric field. 220 ZIGMAN, SEYMOUR, NANCY S. RAFFERTY, AND KRIS C. LOWE, Elas- YAMAGATA, Y., see K. R. Delaney, 229 mobranch eye lens: actin and UV radiation. 220 YAMOAH. EBENEZER, NORMAN STOCKBRIDGE, AND ALAN KUZIRIAN, Zoanthids. 148 Calcium channels in identified neurons. 232 Zoanlhus socialus, 148 YONEDA, MITSUKI, ANDSHIN-ICHI NEMOTO, Occurrence of partial nu- Zooxanthellae, 148 clei in eggs of the sand dollar, Clypeaster japonicus, 183 Zosicru manna. 223 YOSHIDA, M., see T. Akiyama, 178 ZUNIGA, ALICIA L.. see Walter M. Goldberg. 96 CONTENTS CELL STRUCTURE Weidner, Earl, Robin M. Overstreet, Bruce Tedeschi, and John Fuseler Cytokeratin and desmoplakin analogues within an intracellular parasite 237 DEVELOPMENT AND REPRODUCTION Carroll, David J., and Stephen C. Kempf Laboratory culture of the aeolid nudibranch Berghia verrucicofnis (Mollusca, Opisthobranchia): some as- pects of its development and life history 243 Komatsu, Mieko, Yasuo T. Kano, and Chitaru Oguro Development of a true ovoviviparous sea star, As- terina pseudoexigua pacifica Hayashi .- 254 Maruyama, Yoshihiko K. Roles of the polar cytoplasmic region in meiotic di- visions in oocytes of the sea cucumber, Holothuria leucospilota 264 ECOLOGY AND EVOLUTION Amano, Shigetoyo Self and non-self recognition in a calcareous sponge, Leucandra abratsbo 272 Ilan, Micha, and Yossi Loya Ontogenetic variation in sponge histocompatibility responses 279 Berges, John A., John C. Roff, and James S. Ballan- tyne Relationship between body size, growth rate, and maximal enzyme activities in the brine shrimp, Ar- temia franciscana 287 Coon, S. L., M. Walch, W. K. Fitt, R. M. Weiner, and D. B. Bonar Ammonia induces settlement behavior in oyster larvae 297 Pearce, Christopher M., and Robert E. Scheibling Induction of metamorphosis of larvae of the green sea urchin, Strongylocentrotus droebachiensis, by cor- alline red algae 394 Ran lings. Timothy A. Associations between egg capsule morphology and predation among populations of the marine gastro- pod, Nucella emarginata 312 GENERAL BIOLOGY MacColl, Robert, John Galivan, Donald S. Kerns. Zenia Nimec, Deborah Guard-Friar, and David Wagoner The chromophore and polypeptide composition of Aplysia ink 325 McFall-Ngai, Margaret, and Mary K. Montgomery The anatomy and morphology of the adult bacterial light organ of Euprymna scolopes Berry (Cephalo- poda:Sepiolidae) 332 PHYSIOLOGY Bowlby, Mark R., Edith A. Widder, and James F. Case Patterns of stimulated bioluminescence in two py- rosomes (Tunicata: Pyrosomatidae) 340 Kallen, Janine L., S. L. Abrahamse, and F. Van Herp Circadian rhythmicity of the crustacean hypergly- cemic hormone (CHH) in the hemolymph of the crayfish 351 Kuhns, William J., Gradimir Misevic, and Max M. Burger Biochemical and functional effects of sulfate restric- tion in the marine sponge, Microciona pro! if era . . . 358 Toulmond, Andre, Fouzia el Idrissi Slitine, Jacques de Frescheville, and Claude Jouin Extracellular hemoglobins of hydrothermal vent annelids: structural and functional characteristics in three alvinellid species 366 Trapido-Rosenthal, Henry G., Richard A. Gleeson, and William E. S. Carr The efflux of amino acids from the olfactory organ of the spiny lobster: biochemical measurements and physiological effects 374 Index to Volume 179 383 WH iff!" 1