JOURNAL OF SHELLFISH RESEARCH VOLUME 18, NUMBER 1 JUNE 1999 The Journal of Shellfish Research (formerly Proceedings of the National Shellfisheries Association ) is the official publication of the National Shellfisheries Association Editor Dr. Sandra E. Shumway Natural Science Division Southampton College. Long Island University Southampton, NY 11968 Dr. Standish K. Allen, Jr. (2000) School of Marine Science Virginia Institute of Marine Science Gloucester Point. V A 23062- 1 1 346 Dr. Peter Beninger (1999) Laboratoire de Biologie Marine Faculte des Sciences Universite de Nantes BP 92208 44322 Nantes Cedex 3 France Dr. Andrew Boghen (1999) Department of Biology University of Moncton Moncton, New Brunswick Canada El A 3E9 Dr. Neil Bourne (1999) Fisheries and Oceans Pacific Biological Station Nanaimo, British Columbia Canada V9R 5K6 Dr. Andrew Brand (1999) University of Liverpool Marine Biological Station Port Erin, Isle of Man Dr. Eugene Burreson (1999) Virginia Institute of Marine Science Gloucester Point, Virginia 23062 Dr. Peter Cook (2000) Department of Zoology University of Cape Town Rondebosch 7700 Cape Town, South Africa EDITORIAL BOARD Dr. Simon Cragg (2000) Institute of Marine Sciences University of Portsmouth Ferry Road Portsmouth P04 9LY United Kingdom Dr. Leroy Creswell (1999) Harbor Branch Oceanographic Institute US Highway 1 North Fort Pierce. Florida 34946 Dr. Lou D'Abramo (2000) Mississippi State University Dept of Wildlife and Fisheries Box 9690 Mississippi State, Mississippi 39762 Dr. Ralph Elston (1999) Battelle Northwest Marine Sciences Laboratory 439 West Sequim Bay Road Sequim, Washington 98382 Dr. Susan Ford (2000) Rutgers University Haskin Laboratory for Shellfish Research P.O. Box 687 Port Norris, New Jersey 08349 Dr. Raymond Grizzle (1999) Randall Environmental Studies Center Taylor University Upland, Indiana 46989 Dr. Mark Luckenbach (1999) Virginia Institute of Marine Science Wachapreague, Virginia 23480 Dr. Bruce MacDonald (2000) Department of Biology University of New Brunswick P.O. Box 5050 Saint John, New Brunswick Canada E2L 4L5 Dr. Roger Mann (2000) Virginia Institute of Marine Science Gloucester Point. Virginia 23062 Dr. Islay D. Marsden (2000) Department of Zoology Canterbury University Christchurch, New Zealand Dr. Tom Soniat (2000) Biology Department Nicholls State University Thibodaux, Louisiana 70310 Dr. J. Evan Ward (2001) Dept. of Marine Sciences University of Connecticut Groton, CT 06340-6097 Dr. Gary Wikfors (2000) NOAA/NMFS Rogers Avenue Milford, Connecticut 06460 Journal of Shellfish Research Volume 18, Number 1 ISSN: 0077571 1 June 1999 Journal of Shellfish Research, Vol. 18. No. 1. 1-3. 1999. MBLW/HOl Library .; DEC 6 2007 WOODS HOLE Massachusetts 02543 IN MEMORIAM DR. L. EUGENE CRONIN 1917-1998 Dr. L. Eugene Cronin. NSA member and NSA President from 1959 to 1961, died at his home in Annapolis, MD on December 18, 1998 at the age of 81. He was known nationally and internationally for his efforts on behalf of estuarine research, resource management, and habitat restoration. A native of Aberdeen. MD. Gene Cronin received an AB in Chemistry from Western Maryland College ( 1938). and MS ( 1943l and PhD ( 1946) degrees in Zoology (blue crab biology) from the University of Maryland. He was a high school Biology teacher from 1938 to 1940 in Bel Air. MD. From 1943 to 1950. he was a biologist at Chesapeake Biological Laboratory (CBL) in Solomons. MD with a focus on biology and management of blue crabs, oysters, and fish. He subsequently spent five years with the University of Delaware's Department of Biological Sciences. There he established a marine laboratory in Lewes. DE. beginning in rented space in the local high school. In 1952, he and his staff of three moved into a former restaurant while making plans for a permanent research building; a converted fishing-party boat served as the University's first research vessel. Acartia. By the time the new research building was dedicated in 1955, Gene had laid the foundation for a program in research and education that has since grown into the University of Delaware Graduate College of Marine Studies. Gene returned to Maryland in 1955 as Director of the Maryland Department of Research and Education, with headquarters at CBL. In 1961. the Department became the Natural Resources Institute (NRI). a 4-laboratory entity within the University of Maryland. In 1975. NR1 merged with the newly-established Horn Point Environmental Laboratories (now Horn Point Laboratory) to form The University of Maryland Center for Environmental and Estuarine Studies (now The University of Maryland Center for Environmental Science). In 1977. Gene became Director of the Chesapeake Research Consortium that coordinates Chesapeake Bay-related research among the area's major research universities and institutions. In 1984, he "retired" to become a consultant, while remaining active in conservation organizations such as The Chesapeake Bay Foundation and The Alliance for the Chesapeake Bay. In Memoriam: L. Eugene Cronin ,J During the course of his career. Gene was involved with the governance of a number of scientific societies. In addition to being President of The National Shellfisheries Association as noted above, he was the first President ( 1949) of the Atlantic Estuarine Research Society (AERS). His continued interactions with AERS and estuarine scientists led to his involvement in the founding of the Estuarine Research Federation (ERF), of which he was Founding President from 1971 to 1973. He was a Fellow Emeritus of the American Institute of Fishery Research Biologists. Within Maryland, he was Director of the Maryland Biology Teachers Association in his early career and a Trustee of The Natural History Society of Maryland. Gene made numerous contributions towards understanding and conserving natural resources by serving on many State and Federal committees and commissions. He was an advisor to the Atlantic States Marine Fisheries Commission from 1955 to 1969, served on a variety of committees of the National Academy of Sciences and of the US Department of State, and sat on the Marine Board of the National Research Council. Through these activities, he played important roles in influencing fisheries policies in the Chesapeake Bay region. He had a key role in involving the US Environmental Protection Agency in a 5-year study of Chesapeake Bay and in the subsequent establishment of the Chesapeake Bay Program, a state-federal initiative. His efforts brought him many awards. In 1994 he became the second recipient of the Mathias Medal. This biennial award "In Recognition of Scientific Excellence" is sponsored by the Sea Grant Programs of Maryland and Virginia and the Chesapeake Research Consortium to honor those who work to enhance understanding of the Chesapeake Bay ecosystem and to encourage application of this knowledge to solving environmental problems in the Bay. In 1997, ERF created an award in his name to recognize outstanding contributions to estuarine research by a young scientist, a fitting recognition of Gene's contributions to ERF. Other awards included recognition by the Oyster Institute of North America (1967), Chesapeake Bay Seafood Industries Association (1968), and the Isaac Walton League of America ( 1990). The Governor of Maryland appointed him as "Admiral of the Chesapeake Bay" in 1987. Gene remained active professionally to within a few weeks of his death. He was one of seven distinguished scientists who participated in a workshop in November 1998 for university students interested in learning how research, policy, and management have been applied to Chesapeake Bay problems in the past. He was also involved in co-editing a volume on the biology of the blue crab. Gene is survived by his wife, Alice, herself a chemist and founding member of AERS, three sons, and four grandchildren. Victor F. Kennedy Horn Point Environmental Laboratory PO Box 775 Cambridge, MD 21613 PUBLICATIONS Cronin. L.E. 1947. Anatomy and histology of the male reproductive system of Callinectes sapidus Rathbun. J. Morphol. 81:209-239. Cronin, L.E. 1949. Comparison of methods of tagging the blue crab. Ecology 30:390-394. Cronin, L.E.. J.C Daiber & E.M. Hulbert. 1961. Quantitative seasonal aspects of zooplankton in the Delaware River estuary. Chesapeake Set. 3:63-93. Cronin. L.E. 1967a. The role of man in estuarine processes. Pages 667-689 in G.H. Lauff (ed.). Estuaries. Publication 83. American Association for the Advancement of Science. Washington DC. In Memoriam: L. Eugene Cronin 3 Cronin. L.E. 1967b. The condition of the Chesapeake Bay. Transactions North American Wildlife and Natural Resources Conference 32:137-150. Cronin. L.E. & D.A. Flemer. 1%7. Energy transfer and pollution. Pages 171-183 in T.A. Olson and F.J. Burgess (eds.). Pollution and Marine Ecology. Enter/science Publications. New York. Cronin. L.E. 1971. IV. Prevention and monitoring. Pollution prevention. Proc. Roy. Soc. Loud. B. 177:439-450. Cronin, L.E. & A. J. Mansueti. 1971. The biology of the estuary. Pages 14—39 in A Symposium on the Biological Significance of Estuaries. Sport Fishing Institute. Washington DC. Mihursky. J. A. & L.E. Cronin. 1973. Balancing needs of fisheries and energy production. Transactions North American Wildlife and Natural Resources Conference 38: 459-476. Wiley. M.L.. T.S.Y. Koo & L.E. Cronin. 1973. Finfish productivity in coastal marshes and estuaries. Pages 139-150 in R.H. Chabreck (ed.). Proceedings of the Coastal Marsh and Estuary Management Symposium. Louisiana State University. Baton Rouge LA. Cronin. L.E.. D.W. Pritchard. T.S.Y. Koo & V. Lotrich. 1976. Effects of enlargement of the Chesapeake and Delaware Canal. Pages 18-32 in M.L. Wiley (ed.). Estuarine Processes, Volume II. Academic Press, New York. Tsai, C.-F., J. Welch. K.-Y. Chang. J. Schaeffer & L.E. Cronin. 1979. Bioassay of Baltimore Harbor sediments. Estuaries 2:141-153. Cronin. L.E. 1981. The Chesapeake Bay. Transactions of the North American Wildlife and Natural Resources Research Conference 46:223-229. Cronin. L.E. 1982. Pollution in the Chesapeake Bay: A case history and assessment. Pages 17— 16 in T.W. Duke (ed.). Impact of Man on the Coastal Environment. US Environmental Protection Agency Publication EPA-600/8-82-021. Washington DC. Roberts, M.H., Jr., J.E. Warriner. C.-F. Tsai, D. Wright & L.E. Cronin. 1982. Comparison of estuarine species sensitivities to three toxicants. Arch. Environm. Contain. Toxicol. 11:681-692. Cronin. L.E. & R.B. Biggs. 1981. Special characteristics of estuaries. Pages 3-23 in B.J. Neilsen & L.E. Cronin (eds.). Estuaries and Nutrients. Humana Press, Clifton NJ. Officer. C.B., L.E. Cronin. R.B. Biggs & J.H. Ryther. 1981. A perspective on estuarine and coastal research funding. Env. Sci. Tech. 15:1282-1285. Officer. C.B.. R.B. Biggs. J.L. Taft, L.E. Cronin, M.A. Tyler & W.R. Boynton. 1984. Chesapeake Bay anoxia: Origin, development and significance. Science 223:22-27. Cronin, L.E. 1985. Chesapeake Bay: Productive? Polluted? Planned? Pages 339-358 in N.L. Chao & W. Kirby-Smith (eds.). Proceedings of the International Symposium on the Utilization of Coastal Ecosystems: Planning. Pollution and Productivity. Fundacao Universidade do Rio Grande. Brasil. Cronin. L.E. 1986. Chesapeake fisheries and resource stress in the 19th Century. J. Wash. Acad. Sci. 76:188-198. Cronin. L.E. 1987. Actions needed to reduce contamination problems impairing Chesapeake Bay fisheries. Pages 555-566 in S.K. Majundar. L.W. Hall & H.M. Austin (eds.). Contamination Problems and Management of Living Chesapeake Bay Resources. Pennsylvania Academy of Science. Easton PA. SELECTED TECHNICAL REPORTS Cronin. L.E. 1942. A histological study of the development of the ovary and accessory organs of the blue crab. Callinectes sapidus Rathbun. MS thesis. University of Maryland, College Park, MD. 37 p. Cronin, L. E. 1947. Anatomy and histology of the male reproductive system of Callinectes sapidus Rathbun. PhD dissertation. University of Maryland, College Park. MD. 71 p. Cronin, L. E. 1950. The Maryland crab industry. 1949. Chesapeake Biological Laboratory Pub. 84. 41 p. Pyle. R. & L.E. Cronin. 1950. The general anatomy of the blue crab. Chesapeake Biological Laboratory Pub. 87. 40 p. Cargo. D. G. & L. E. Cronin. 1951. The Maryland crab industry. 1950. Chesapeake Biological Laboratory Pub. 92. 23 p. Cronin. L. E., W. A. Van Engel. D. G. Cargo & F. J. Wojcik. 1957. A Partial Bibliography of the Genus Callinectes. Virginia Fisheries Laboratory Spec. Sci. Rep. 8: Maryland Dept. Research and Education Ref. 57-26. 21 p. Cronin. L.E.. M.G. Gross, M.P. Lynch & K.J. Sullivan. 1977. The condition of the Chesapeake Bay — A consensus. Pages 37-61 in the Proceedings of the Bi-State Conference on the Chesapeake Bay. Chesapeake Research Consortium Pub. 61. Shady Side MD. Cronin, L. E. 1988. Report of the Chesapeake Bay Blue Crab Management Workshop. November 9-10, 1997. Waldorf. MD. 68 p. EDITED WORKS Cronin. L.E. (ed.). 1975. Estuarine Research. Volumes I and II. Academic Press. NY. 738 and 587 p. Neilson. B.J. & L.E. Cronin (eds.). 1981. Estuaries and Nutrients. Humana Press. Clifton NJ. 643 p. Cronin, L.E. (ed.). 1982. Chlorine - Bane or Benefit? Proceedings of a Conference on the Uses of Chlorine in Estuaries. Chesapeake Research Consortium. Shady Side MD. 212 p. Cronin, L.E. (ed.). 1983. Ten Critical Questions for Chesapeake Bay in Research and Related Matters. Chesapeake Research Consortium Pub. 1 13. Shady Side, MD. 156 p. + 1 Appendix Journal oj Shellfish Research. Vol. 18. No. 1. 5-7. ITO IN MEMORIAM TERRANCE HENRY BUTLER 1923-1998 Terrance (Terry) Henry Butler, a recognized world authority in the field of crustacean biology and crustacean fisheries, passed away on March 10, 1998 in Nanaimo, British Columbia, Canada. He was 74-years-old. Terry was born in Nelson, British Columbia, where his father worked as an engineer on the Canadian Pacific ferries. He received his elementary and secondary education in the interior of British Columbia (B.C.) and, prior to World War II, entered the engineering department of Victoria College in Victoria, B.C. In 1942 he joined the Canadian army, enlisting in the engineer corps. He transferred to the Canadian Airborne Regiment, with whom he served overseas, and was stationed in the Netherlands by the end of the War. He was discharged from the army in January 1946. Upon returning to Canada, he enrolled in the University of British Columbia where he completed a BA degree in Honors Biology in 1949. He received an MA degree from the same University in 1953; the subject of his thesis was a study of Dungeness crab biology and fisheries. During the summer of 1948. Terry had been hired as a summer student at the pacific Biological Station in Nanaimo. B.C.. where he served as a groundfish technician conducting biological sampling of commercial groundfish catches. Thus began an association with the Biological Station that lasted for the remainder of his working career. In May 1949, he accepted a permanent position at the Pacific Biological Station as a research scientist in charge of the Crab and Shrimp Investigation. He continued in this capacity, and also served for a period as head of the Shellfish Investigation, until he retired from the Pacific Biological Station in March 1984. During his working career, Terry undertook research studies on a wide range of subjects that included basic biology, population dynamics and stock assessment of some British Columbia crustacean species, exploratory fishing for harvestable stocks, and improve- ments in fishing methods. He became a recognised authority on crustacean fisheries of the West Coast of North America. As a result of his work, several crustacean fisheries were started in British Columbia. Throughout his working career, Terry maintained a close working relationship with the industry and was frequently consulted by industry for his advice. Terry published widely in the field of crustacean biology and fisheries, having over 55 publications including his "Shrimps of the Pacific Coast of Canada." which has remained a classic reference work. He received the Queen's Silver Jubilee Medal in 1977 in recognition of his career. 6 In Memoriam: Terrance Henry Butler Terry also had an active interest in crustacean fisheries in developing countries. From January 1957 to November 1958, he served with the Food and Agriculture Organization of the United Nations in Indonesia, assisting in the development and management of local shrimp fisheries. In 1988. he served with the Canadian Executive Services Organization to assist in the development of a southern king crab fishery in Chile. After his retirement in 1 984, Terry continued to work several days a week at the Pacific Biological Station, even when his health was failing, to continue with his studies and publications on British Columbia crustaceans. At the time of his passing, he was writing a major work, "The Crab Fisheries of British Columbia." Terry was highly respected for his research accomplishments and was an inspiration to younger scientists at the Pacific Biological Station and elsewhere. He was a kind and patient man who always had time to talk with younger staff members, to encourage them in their work, and to give them the benefit of his long years of experience. Terry had many outside interests. He was keenly interested in plants, shrubs, and trees, and always claimed that he was a frustrated botanist. Wherever he travelled, he was very interested in the local fauna and how it compared to that in British Columbia. He had a great appreciation of classical music and he was an enthusiastic and much respected golfer and a regular member of a Saturday morning golfing group from the Pacific Biological Station. Terry married D. Joan Abel in 1947; they celebrated their golden wedding anniversary in 1997. Together, they had six children, nine grandchildren, and a great granddaughter. Terry was a warm-hearted, kind and generous companion. Those who knew him appreciated his understanding, his guidance and his example and these qualities served to inspire his associates and to assist them in their research and in their daily lives. Terry will be sorely missed by his family and by all that knew him. James Boutillier and Neil Bourne Fisheries and Oceans Pacific Biological Station Nanaimo. British Columbia CANADA V9R 5K6 PARTIAL PUBLICATION LIST FOR T.H. BUTLER Butler. T. H. 1949. The status of the pink shrimp (Prawn). Pandalus bo- realis Kroyer, in the commercial shrimp fishery of English Bay British Columbia. B.A. Thesis. Univ. of British Columbia, 37 p. Butler. T. H. 1950. The commercial shrimps of British Columbia. Fish. Res. Bd. Canada. Pacific Coast Sta., Prog. Rep. 83:30-34. Butler. T. H. 1950. Two records of shrimps from English Bay. B.C. Can. Field Nat. 64(5): 188 p. Butler. T. H. 1951. The 1949 and 1950 tagging experiments in the Graham Is. Crab fishery. Prog. Rep. Pac. Coast Stations, Fish. Res. Bd. Can. 89:88-87. Butler. T. H. 1953. The appearance of a new commercial shrimp in a newly developed shrimp fishery. Fish. Res. Board Can. Progr. Rep. Pac. Coast Stn. 94:30-31. Butler. T. H. 1953. A shrimp survey by the investigator No. 1". April. 1953. Fish. Res. Bd. Canada. Nanaimo Biol. Sta. Circular 55. 4 p. Butler. T. H. 1953. The Life of the Commercial Crab. Western Fisheries. January, 1953. Butler. T. H. 1954. Food of the commercial crab in the Queen Charlotte Islands Region. Canada. Fish. Res. Bd.. Pac. Prog. Rep. No. 99. pp 3-5. Butler. T. H. 1955. Re-discovery of the parasitic cirripede. Mycetomorpha vancouverensis Potts. In British Columbia waters. J. Parasit. 41(3): 321. Butler. T. H. 1956. The distribution and abundance of early post-larval stages of British Columbia commercial crab. Fish. Res. Bd. Canada. Pacific Prog. Rep., No. 107. pp. 22-23. Butler. T. H. 1956. A first British Columbia record of a cragonid shrimp. Can. Field Nat. 70(30): 142. Butler. T. H. 1957. The tagging of the commercial crab in the Queen Charlotte Islands region. Fish. Bd. Canada. Pacific Prog. Rep.. No. 109, pp. 16-19. Butler, T. H. 1959. Results of shrimp trawling by investigator No. 1', June 1959. Fish. Res. Bd. Canada. Nanaimo Biological Station Circular, No. 55. 7 p. Butler. T. H. 1960. Maturity and breeding of the Pacific edible crab. Can- cer magister Dana. J. Fish. Res. Bd. Canada. 1 7(51:641-646. Butler. T. H. 1961. Records of decapod Crustacea from British Columbia. Can. J. Zool. 39:59-62. Butler, T. H. 1961 . Growth and age determination of the Pacific edible crab Cancer magister Dana J. Fish. Res. Board Can. 18:873-891. Butler. T H. 1963. An improved prawn trap. Fish. Res. Bd. Canada. Nan- aimo Biol. Station Circular. No. 67. 7 p. Butler. T. H. 1963. The prawn Pandalus platyceros Brandt 1851. F. A. O. Fish. Rep.. 57(4). Butler. T. H. 1964a. Records of shrimps (Order Decapoda) from British Columbia. J. Fish. Res. Board Can. 21:419-121. Butler. T. H. 1964b. Redescription of the parasitic isopod Holophrysxus alaskenis Richardson And a note on it synonymy. J. Fish. Res. Board Can. 21:971-976. Butler. T. H. 1964c. Growth, reproduction, and distribution of pandalid shrimps in British Columbia. J. Fish. Res. Board Can. 21:1403-1452. Butler. T. H. 1967. A bibliography of the Dungeness crab. Cancer magister Dana. Fish. Res. Bd. Canada. Tech. Rep. 1. 12 p. Butler. T. H. 1967. Shrimp exploration and fishing in the Gulf of Alaska and Bering Sea. Fish. Res. Rd. Canada, Tech. Rep.. 18. 49 p. Butler. T. H. 1968. The shrimp fishery of British Columbia. FAO Fish. Rep. 57(21:521-526. Butler. T. H. 1969. Catch and effort statistics on the Canadian shrimp fishery on the Pacific Coast in 1967 Fish. Res. Bd. Canada. Manuscr. Rept. Ser. No. 1031.4 p. Butler. T. H. 1970. Synopsis of biological data on the prawn Pandalus platyceros Brandt, 1851. FAO Fish. Rep. 57(4): 1289-1315. Butler. T. H. 1971a. A review of the biology of the pink shrimp, Pandalus borealis Kroyer 1838. Can, Fish. Rep. 17:17-24. Butler. T. H. 1971b. Eualus berkeleyorum n.sp., and records of other cari- dan shrimps (Order Decapoda) from British Columbia. J. Fish. Res. Board Can. 28:1615-1620. Butler. T H. 1980. Catch and effort statistics of the Canadian shrimp fishery on the Pacific Coast in 1979. Can. Data Rep. Fish. Aquat. Sci. 224. 5 p. Butler. T. H. 1980. Shrimps of the Pacific coast of Canada. Can. Bull. Fish. Aquat. Sci. 202. 280 p. Butler, T. H. 1981. Dungeness Crab: A Primer. Western Fisheries, vol 103. no 3, pp 26-27. Butler, T. H. & G. V. Dubokovic. 1955. Shrimp prospecting in the offshore In Memoriam: Terrance Henry Butler Region of the British Columbia coast. June to August. 1953. Fish. Res. Bd. Canada. Nanaimo Biol. Sta. Circular 39. 33 p. Butler. T. H. & G. V. Dubokovic. 1955. Shrimp and prawn prospecting on the British Columbia coast. June to December. 1954. Fish. Res. Can. Nanaimo Biol. Stn. Core. (Gen. Ser.) 35: 92 p. Butler. T. H. & D. G. Hankin. 1992. Comment on mortality rates of Dunge- ness crabs Cancer Magister. Can. J. Fish. Aquat. Sci. 49: 1518-1521 p. Butler. T. H. & H. E. J. Legare. 1954. Shrimp prospecting in regions of the British Columbia coast. November 1953 to March 1954. Fish. Res. Board Can. Gen. Ser. Circ. 31. 42 p. Butler. T. H.. J. G. Lindsay & C. B. Chic. 1975. Prawn trap exploration British Columbia Central coast November 1974 to February 1975. Fish. Res. Board Can. MS Rep. 1357, 1 19 p. Butler. T. H. & M. S. Smith. 1968. Shrimp sampling and Temperature Data obtained during Exploratory fishing off British Columbia. 1966 and 1967. Fish. Res. Bd. Canada, Tech. Rep.. 61. 92 p. Butler. T. H. & M. S. Smith. 1968. Shrimp sampling and Temperature Data obtained during Exploratory fishing off British Columbia. 1966 and 1967. Fish. Res. Bd. Canada. Tech. Rep.. 61. 92 p. Butler. T. H. & M. Stocker. 1990. Surplus production model analysis of crab (Cancer magister Dana) fisheries of British Columbia. Canada. Fish. Res. 9:231-254. Butler. T. H.. A. N. Yates & C. C. Wood. 1973. G. B. Reed shrimp cruise 73-S-l. May 7-23. Fish. Res. Board Can. MS Rep. 1255. 46 p. Butler. T. H.. A. N. Yates & D. C. Miller. 1974. G. B. Reed shrimp cruise in Queen Charlotte Sound, April 1974, Fish. Res. Board Can. Nanaimo Biol. Stn. Circ. (Gen. Ser.) 98. 44 p. Allen. J. A. & T. H. Butler. 1994. The Caridea (Decapoda) collected by the Mid-Pacific Mountains expedition, 1968. Pac. Sci. 48(4):410-445. Boutillier. J. A.. T. H. Butler, el al. 1998. Assessment of the 'Area A' Crab (Cancer magister) Fishery in British Columbia. Can. Stock Assessm. Seer. Res. Doc. 98/86. 39 p. Boutillier. J. A.. J. R. Carmichael & T. H. Butler. 1978. Shrimp population survey, west coast of Vancouver Island. May 1978. Fish. Mar. Serv. Data Rep. 100. 84 p. ( 1 ) Boutillier. J. A., A. N. Yates & T. H. Butler. 1976. B. B. Reed Shrimp Cruise 76-S-l. May 3-19. 1976. Fish. Mar. Serv. Dat Rep. 13. 45p. Boutillier. J. A., A.N. Yates & T. H. Butler. 1977. B. B. Reed Shrimp Cruise 77-5-1. May 3-14. Fish. Mar. Serv. Data Rep. 37. 42 p. Hankin, D. G. and T. H. Butler. 1997. Does intense fishing on males impair success of female Dungeness crabs? Can. J. Fish. Aquat. Sci. 54(3): 655-669. Jamieson. G. S.. C. K. Robinson & T. H. Butler. 1986. King and Tanner Crabs in northern British Columbia mainland inlets. Can. Manuscr. Rep. Fish. Aquat. Sci.. no. 1880. 130 pp. Ketchen. K. S.. N. Bourne & T. H. Butler. 1983. History and present status of fisheries for marine fishes and invertebrates in the Strait of Georgia. British Columbia. Can. J. Fish. Aquat. Sci. 40:1095-1119. Margohs. L. & T. H. Butler. 1954. An unusual and heavy infection of a prawn. Pandalus borealis Kroyer. by a nematode. Contracaecum sp. J. Patasitol. 40(6):649-655. Schrivener. J. C. & T. H. Butler. 1971. A bibliography of shrimps of the family Pandalidae. Emphasizing economically important species of the genus Pandalus Fish. Res. Board Can. Tech. Rep. 241. 42 p. Wicksten. M. K. & T. H. Butler. 1983. Description oiEualus lineatus new species, with a redescription of Heptacarpus herdmani (Walker). (Car- idea: Hippolytidae). Proc. Biol. Soc. Washington. Washington D.C.. v 96. no 1, pp 1-6. Journal of Shellfish Research Vol. 18. No. I. 4-17. 1999. OBSERVATIONS ON THE BIOLOGY OF THE VEINED RAPA WHELK, RAPANA VENOSA (VALENCIENNES, 1846) IN THE CHESAPEAKE BAY JULIANA M. HARDING AND ROGER MANN Department of Fisheries Science Virginia Institue of Marine Science College of William and Mary Gloucester Point, Virginia 23062 ABSTRACT The recent discovery of the Veined Rapa whelk {Rapana venosa. Valenciennes. 1846) in the lower Chesapeake Bay provides an opportunity to observe the initial biological and ecological consequences of a novel bioinvasion. These large predatory gastropods occur in subtidal. hard bottom habitats in the lower Bay and are capable of feeding, mating, and moving while completely burrowed. Hard clams (Mercenaria mercenaria) are consumed preferentially in the laboratory when offered concurrently with oysters (Crassostrea virginica), soft clams {Mya arenaria), and mussels (Mytilus edulis). Chesapeake Bay R. venosa readily open and consume large hard clams (30 to 85 mm SH) leaving no visible signs of either drilling or boring behavior. Shell morphology and thickness may provide an inherent size-selective predation refuge for Rapa whelks in the Bay. These same shell characteristics may change the dynamics of shell selection by local hermit crabs, particularly the striped hermit crab. Clibanarius vittatus. Recent collections of striped hermit crabs from the Hampton Roads area indicate that very large striped hermit crabs are using empty Rapana shells as shelters. KEY WORDS: Rapana venosa. Veined Rapa whelk. Muncidae. Thaididae. ballast water, bioinvasion, Chesapeake Bay. Clibanarius vittatus, Mercenaria mercenaria INTRODUCTION The Veined Rapa whelk, Rapana venosa, (Valenciennes, 1846) is a large, predatory gastropod that has recently been found in the lower portion of the Chesapeake Bay. As with other representa- tives of the Thaididae family [Earlier classifications of the Neo- gastropods place Rapana sp. in the family Muricidae. Recent taxo- nomic revisions include Rapana in the Thaididae (R. Germon, Smithsonian Institution, Washington, D.C., pers. comm.)], this animal is a carnivore whose principal prey items include many commercially valuable bivalves. Rapana venosa is one of several modern Rapana species including R. bezoar and R. rapiformis. Although R. thomasiana was originally described by Crosse in 1861 as a separate species (Thomas's Rapa whelk), it is currently recognized as a synonym fori?, venosa (R. Germon. Smithsonian Institution. Washington, DC. pers. comm.). Rapana venosa is native to the Sea of Japan, the Yellow Sea, the East China Sea, and the Gulf of Bohai (Tsi et al. 1983, Chung et al. 1993. Zolotarev 1996. Chung and Kim 1998). Three species of Rapana occur sympatrically in Chinese waters: R. venosa. R. bezoar, and R. rapiformis (Tsi et al. 1983). All three species are found in coastal subtidal habitats and are commercially harvested (Hwang etal. 1991. Chung et al. 1993, Morton 1994). Rapa whelks were discovered in the Black Sea in 1947 (Drapkin 1963) and have subsequently spread throughout the Black Sea and into the Sea of Azov as well as the Aegean (Koutsoubas and Voultsiadou- Koukoura 1990, Zolotarev 1996) and Adriatic (Bombace et al. 1994) Seas. R. venosa from Korean waters described by Chung et al. (1993) ranged from 32.5 to 168.5 mm shell length (the maxi- mum distance from the tip of the spire to the bottom of the col- umella. SL). Rapana venosa is easily distinguished from native gastropods of the Chesapeake Bay. It has a short spired, heavy shell with a large inflated body whorl and a deep umbilicus (Fig. I ). The slightly concave columella is broad and smooth. Small, elongate teeth are present along the edge of the large, ovate aperture's outer lip. External shell ornamentation includes smooth spiral ribs that end in regular blunt knobs at both the shoulder and the periphery of the body whorl. In addition, fine spiral ridges are crossed by low vertical riblets. Older specimens can be eroded, but the color is variable from gray to orange-brown (one specimen is atypically blonde), with darker brown dashes on the spiral ribs. The aperture and columbella vary from deep orange-red to yellow or off-white. Spiral, vein-like coloration, ranging from black to dark blue, oc- casionally occurs internally, originating at the individual teeth at the outer lip of the aperture. The first collection of Rapana venosa in the Chesapeake Bay was made in the summer of 1998 during a routine trawl collection by the Virginia Institute of Marine Science (VIMS) trawl survey in the vicinity of the Monitor-Merrimac Tunnel (Fig. 2). This speci- men was positively identified as Rapana venosa by Drs. Jerry Harasewych (Smithsonia Institution. Washginton. DC) and Yuri Kantor (Russian Academy of Science, Moscow). A subsequent sampling trip specifically for Rapana venosa in the same vicinity on August 24, 1998 yielded two masses of R. venosa egg cases (Fig. 3; a total of 50+ egg cases) but no live animals. The egg cases were returned to VIMS and maintained at ambient temperature and salinity conditions on a 14 h light: 10 h dark regime. Within a week postcollection, individual egg cases began hatching with the last egg case hatching on September 21, 1998. Larvae were cultured and used in salinity tolerance experiments (Mann and Harding, in review). Given the size of the specimens collected to date from the lower Bay (68 to 165 mm SL) and the presence of viable egg cases, it seems reasonable to assume that the local Rapa whelk population is sexually mature and actively breeding. As in the eastern Mediterranean and Black Seas (Zolotarev 1996), ballast water from commercial and/or military ship traffic is the probable source of introduction into the Chesapeake Bay. R. venosa larvae are planktonic for 14 to 17 days (Chung et al. 1993. Mann and Harding in review). Normal transit time to the Hampton Roads/Norfolk area from the Baltic, Black, Adriatic, or Aegean Seas is approximately 10 to 24 days (G. Ruiz. Smithsonian Envi- ronmental Research Center, pers. comm.). This time interval is well within the temporal window for survival of viable planktonic R. venosa larvae. At certain times during the year (e.g.. May 10 Harding and Mann Figure 1. Picture of an adult Rapana venosa 115(1 mm SL) from the Chesapeake Bay. The arrows highlight the hroad columella, opercular teeth, and bright orange aperture. through October) temperature and salinity regimes on both ends of the trip are similar (see Mann and Harding, in review for a detailed discussion). The Hampton Roads/Norfolk area is a major foci of container, coal transport, and military ship activity. The area ranks third among U.S. ports in terms of volume of ballast discharged on an annual basis (G. Ruiz. Smithsonian Environmental Research Center, pers. comm.). Given the sheer volume of ballast water arriving in Chesapeake Bay annually from ports with active Rapa whelk populations (15 million metric tons; G. Ruiz. Smithsonian Environmental Research Center, pers. comm.). the possibility of obtaining sufficient numbers of Rapa whelk larvae needed to even- tually establish a breeding population in the Chesapeake Bay may be quite high. International traffic aside, the Hampton Roads/ Norfolk area is also a major hub for coastal shipping along the eastern seaboard of North America (G. Ruiz, Smithsonian Envi- ronmental Research Center, pers. comm.). If a local population of Rapa whelks becomes established in the Bay, it is likely that the Chesapeake would eventually become a source population for other coastal ports with similar habitat conditions. This scenario places ports throughout the Middle Atlantic Bight (e.g.. New York, Boston) as well as the South Atlantic Bight (e.g., Charleston) at higher risk for introduction of the species in that they would he receiving both international and local inoculations. Since the discovery of Rapana venosa in the Chesapeake Bay. live Rapa whelks have been under observation in wet laboratory tanks at VIMS. To date, 412 animals have been donated to VIMS (these numbers include live animals, dead animals with shells, or shells only), mostly by commercial watermen and seafood pro- cessing companies, indicating the presence of an established popu- lation of Rapana venosa in the lower portion of the Chesapeake Bay. Observations to date on the basic biology and ecology of Rapana venosa in the Chesapeake Bay are described herein and placed in the context of potential trophic interactions of this animal in the lower Chesapeake Bay. Current Distribution The current distribution of Rapana venosa in the Chesapeake Bay extends from the Chesapeake Bay Bridge Tunnel northward along the western shore line in a continuous swath across Little Creek. Ocean View, Fort Monroe, and Buckroe Beach (Figs. 2 and 4). Several unconfirmed reports from the Poquoson flats area are punctuated by two confirmed discoveries of Rapana at Tue Marshes Light in the York River (Fig. 2). The northernmost report of a Rapa whelk in the Bay is from Butler's Hole, a small oyster rock near the mouth of the Rappahannock River; this 130 mm SL Figure 2. Map showing known Rapana venosa distribution as of March 1999 in the Chesapeake Bay proper (A.) and the Ocean View/Hampton Roads/James River region (B.). The black circles (A.) indicate the Rappahannock and York River collection sites. The black zone (B.) shows the known distribution within the lower Bay/Hampton Roads/James River. The first collection location is indicated with an asterisk (B.). Biology of Rapana venosa ll Figure 3. Rapana venosa egg cases collected from Hampton Roads, VA in August 1998. The yellow egg case cluster was attached basally to a hydroid mat (A.). Note the broad phyllopodus egg case tops and egg pores shown in the top view (B.I. individual was collected by the authors during an annual oyster stock assessment dredge survey. The majority of Rapa whelks have been collected by either commercial clammers or crab dredgers working in the lower Bay. In early September 1998, VIMS established an ongoing Rapana bounty system with the help of the Virginia Saltwater Commerical Fishing Develoment Fund and, as of January 1999, the Virginia Sea Grant program. A bounty is paid for each snail turned in to VIMS personnel, provided that collection information (i.e.. loca- tion, gear, depth, and bottom type) are reported at the time of donation. The bounty program yielded an average of 8 to 10 ani- mals per week through the end of November 1998 donated pri- marily by clammers working off Ocean View and Buckroe Beach (Fig. 2). Clammers in the lower Chesapeake fish for hard clams or quahogs {Mercenaria mercenaria) with patent tongs. Quahogs > 50 mm shell height are abundant (approximately 1 to 1 1 animals m 2 ) in portions of the lower bay (Roegner and Mann 1991). and the commercial hard clam fishery in the reigon is economically important, annually landing 1.1 million pounds with a dockside value of approximately $6 million (Kirkley 1997). The lower Bay also supports a winter crab dredge fishery tar- geting blue crabs (Callinectes sapidus) that burrow into the sand/ mud bottom to overwinter. When crab dredge season opened in the lower Bay on December 1 . 1998. Bay water temperatures were still 8 to 12°C. During the first 2 weeks of December 1998. over 30 Rapa whelks/day were donated to the VIMS colleciton by crab Chesapeake Bay James Rivet >^kk Jk * 4 V 4 -*<*> dp t 1 N 10 km 10 Figure 4. Distribution map of Rapana venosa from the lower Chesapeake Bay showing collection zones: 1.) Above the SR 258 James River Bridge, 2.) Between the James River Bridge and the Monitor-Merrimac Bridge Tunnel, 3.1 Between the Monitor-Merrimac Bridge Tunnel and the Hampton Roads Bridge Tunnel, including the Hampton Bar area, 4.1 the Lafayette River, and 5.) the Buckroe Beach. Fort Monroe, Ocean View, and Little Creek areas in the bay proper. 12 Harding and Mann TABLE 1. Summary of Rapana venosa collections through March 1, 1999 from the lower Chesapeake Bay and its tributaries. Collection Location Average Shell Length (mm) Standard Error (SE. mm) Lower Bay: Little Creek/Ocean View/Buckroe Beach James River: Hampton Bar James River: between Monitor-Merrimac Bridge Tunnel and the James River Bridge James River: above the James River Bridge Lafayette River Nansemond River York River: Tue Marshes Light Rappahannock River: Butler's Hole 185 7 198 II 7 1 2 1 141.8 138.8 132.7 131.3 102.7 135.0 149.0 1 30.0 0.93 5.23 0.84 3.63 4.9 9.00 "n" refers to the number of animals collected from each location. Locations are shown in relation to the mainstem Chesapeake Bay and each other in Figures 2 and 4. dredgers and seafood processing companies. The arrival of a cold front just before Christmas 1998 caused water temperatures to fall below 5°C and coincided with a reduction in both fishing activity and R. venosa donations. Throughout January and February 1999. crab dredgers working the lower Bay have reported few R. venosa. Presumably, the sustained colder temperatures have driven them either into deeper waters as reported in their home range (Wu 1988) or deeper into the sediment below the zone of dredging activity. Although donations from crab dredgers in the lower Bay es- sentially stopped in January 1999. Rapa whelk donations from clammers working in the James Rivet continued until the closing of the area to commercial fishing in mid-March at an average rate of 6 animals/day" 1 . As of this writing, there have been no R. venosa reported by commercial oystermen working on extant oys- ter beds in the James River upstream of the Route 258/17 James River bridge (Haven and Whitcomb 1983). A majority of the ani- mals collected to date from all sources have been collected from regions with hard sand bottom in depths ranging from 10 to 60 m at salinities of 18 to 28 ppt. The collection data from commercial sources do not lend them- selves to an accurate Rapa whelk stock assessment, because it is impossible to separate the effects of fishing effort in a particular location from potential gear biases in that both crab dredges and patent tongs selectively catch larger snails (>100 mm SL) given standard ring size for both (0.06 m). However, an examination of R. venosa length-frequency distributions for sites in the lower Bay and Hampton Roads area yields an interesting pattern. The shell lengths (SL. mm) of animals from the five different regions with >5 confirmed Rapa whelk reports (Table I ) were compared with an ANOVA followed by Fisher's test for multiple comparisons (per Zar 1996). Data satisfied assumptions of both homogeneity of variance and normality without transformation. Animals collected from the Ocean View/Buckroe Beach/Little Creek area (Fig. 4) or from regions outside the Hampton Roads Bridge tunnel are sig- nificantly larger than animals collected from either the Lafayette River, above the James River Bridge, or between the Route 258/17 James River bridge (hereafter JRB) and the Monitor-Merrimac bridge tunnel (Figs. 4 and 5; ANOVA. p < .05; Fisher's test, p < .05). Animals collected from the Lafayette River are significantly smaller than Rapa whelks collected from any other site (Figs. 4 and 5: ANOVA. p < .05; Fisher's test, p < .05). It is interesting to note that the Little Creek/Ocean View area is immediately adjacent to 1 ' 1 ' 1 ' 1 ' 1 ' 1 ' 1 ' 1 ' 1 ' 1 ' 1 ' i ' i 1 ' 1 ' 1 ' • 50 Zone 2 40 2 30 20 r ■ 10 r —■■■-'-■-'-■-■ ' ' ^^ ■ ■■la I'IM'I' Zone 3 i.i.i.i nil L Zone 4 H H II i . i . i ■ i . i . i . i . i . i 50 _ Zone 5 1 i ! . | ■ | i | i | i | i , i 2 40 - 30 j 20 : 10 ■ Jail n --■-■■■-•■ ...'■■II !■■■■■_ Shell length (mm) Figure 5. Length-frequency distribution of Rapana venosa collected from the Chesapeake Bay. Zones 1 through 5 correspond to the zones shown in Figure 4; i.e., 1.) Above the SR 258/17 James River Bridge (JRB. n = ID, 2.) Between the JRB and the Monitor-Merrimac Bridge Tunnel tn = 194), 3.) Between the Monitor-Merrimac Bridge Tunnel and the Hampton Roads Bridge Tunnel, including the Hampton Bar area (n = 7), 4.) the Lafayette Ri\er (n = 7), and 5.) the Buckroe Beach, Fort Monroe, Ocean View, and Little Creek areas in the Bay proper (n= 181). Biology of Rapana venosa 13 both the anchorage for commercial and military ships awaiting pilots and clearance to enter the port and the Thimble Shoals shipping channel (Figs. 2 and 4). The area between the JRB and the Monitor-Merrimac bridge tunnel includes the Newport News coal container terminal, a major site of deballasting for interna- tional ships awaiting coal. Age Estimates In the absence of age and growth estimates for Chesapeake Bay Rapana venosa, age and growth estimates for a Knobbed whelk (Busycon carica) population from Virginia's Eastern Shore may offer a conservative estimate of potential Rapa whelk growth rates. Kraeuter et al. ( 1989) and Castagna and Kraeuter 1 1994) provide growth and length-at-age estimates for B. carica from both labo- ratory and field studies extending over a 14-year period. Growth rates for B. carcia were greatest during the first year (approxi- mately 32 mm/y -1 ) and then subsequently decreased to 14.4 mm/ yr~' for the first 10 years followed by growth rates of 6.5 to 9.5 mm/y -1 for animals older than 10 y and/or greater than 160 mm SL (Fig. 6; Kraeuter eta 1. 1989. Castagna and Kraeuter 1994). In the absence of any data on Rapana growth rates in the Chesapeake Bay. it is reasonable to consider growth rates of such sympatric species as B. carica for initial estimates of Rapa whelk age. Ninety-five percent of all R. venosa collected thus far in the Chesa- peake Bay are between 110 and 160 mm SL. If this range of Rapana shell lengths is overlaid onto the B. carica growth curve presented by Kraeuter et al. (1989) and Castagna and Kraeuter (1994). the resulting age distribution extends from approximately 7.5 to 13 years (Fig. 6). These are conservative growth estimates when considered in relation to the growth rates for Black Sea Rapana reported by Chukhchin (1984). Rapa whelk length-at-age relationships have been described by * • 12 24 36 48 60 72 84 96 108 120 132 144 156 168 Age (months) Figure 6. Plot at Busycon carica length-at-age relationship from labo- ratory and field observations of an Eastern Shore, VA B. carica popu- lation after Kraeuter et al. (1989) and Castagna and Kraeuter (1994). The shaded zone indicates the size range (SL, mm I and corresponding age estimate for 95% of the Rapana venosa collected in the Chesapeake Bay thus far. Chukhchin (1984) for animals from the Black Sea. Chukhchin ( 1984) estimates reports growth rates for individuals in Sevastopol Bay of 20 to 40 mm during year 1. with mean shell length (SL) values of 64.6 mm, 79.4 mm. 87.5 mm, and 92.1 mm in years 2 through 6. respectively. This terminal size is smaller than the maximum SL of 120.1 mm reported by Smagowicz (1989) for a specimen in a collection from Bulgaria and Georgia, whose exact collection location was not reported. Chukhchin (1984) correlates shell thickening with spawning events and notes that the first spawning occurs in the second year at sizes ranging from 35 to 78 mm SL with a mean value of 58 mm SL. Habitat Preferences Both field collections and laboratory observations confirm that Rapana venosa prefers hard sand bottom habitats. These animals are avid burrowers and remain completely burrowed for more than 95% of the time in the laboratory. A 150 mm SL R. venosa can burrow into a sand bottom so that its shell is completely covered in less than 1 h. The only visible sign of a burrowed Rapa whelk is the maroon U-shaped siphon that is usually extended 1 to 3 cm above the surface of the sand. Rapa whelk siphons are sensitive to both light and motion and are retracted immediately at the slightest disturbance. Siphonal sensitivity combined with the animal's bur- rowing speed and low visibility conditions in the Bay may make conventional benthic survey methods that relay on direct observa- tion of the animal (diver transects, video surveys) difficult as non- invasive stock assessment techniques. Bombace et al. (1994) ob- served an apparent increase in R. venosa biomass after artificial reef deployment in the Adriatic Sea. It is possible that there were burrowed Rapana at the sites at the time of reef deployment and that increases in Rapana sightings after reef construction are at- tributable to the emergence of local snails to feed on the reefs not the arrival of snails from other areas. Laboratory observations indicate that Rapa whelks are capable of both feeding and mating while burrowed. They move reason- ably quickly while burrowed (approximately 1 body length per minute). Hard sand bottom habitat is relatively common in the lower Chesapeake Bay (Fig. 7) and is not likely to be a limiting factor for potential range expansion of the animal in the bay. Prey preferences Rapana bezoar was described by Morton (1994) as "a gener- alist predator of subtidal molluscs." This description is certainly apt for R. venosa in the Chesapeake Bay. In laboratory feeding studies. Chesapeake Bay R. venosa prefer hard clams to oysters ( Crassostrea virginica), soft clams (Mya arenaria), or local mus- sels {Mxtilus edulis). although they will eat these other bivalves when hard clams are rare or unavailable (Fig. 8). A 140 mm SL Rapa whelk is capable of consuming a 75 to 80 mm hard clam in less than 1 h. Previous reports on the feeding behavior of R. thomasiana (now recognized as R. venosa) from the Black Sea place R. venosa among the gastropods that drill their prey (Gomoiu 1972. Carriker 1981) or use paralytic toxins during feeding (Chukhchin 1984). Morton ( 1994) describes feeding behavior of R. bezoar in terms of boring or crude rasping usually on the posterioventral shell margin. Similar rasping behavior has been observed for Chesapeake Bay Rapana venosa feeding on small hard clams [<30 mm shell height. SH (distance from hinge to the opposite shell margin)]. Small chips or rasp marks are visible on the posterioventral shell margin 14 50 Okm 50 100~ Harding and Mann B. A x Atlantic Ocean so Okm 50 100 A .0 Atlantic Ocean Figure 7. Maps of the lower Bay showing sand bottom habitat (A.) and hard clam populations (B.l in black per Roegner and Mann ( 1991 1 Ocean View/Hampton Roads/James River region is indicated by a square in both maps. The of some small clams attacked and eaten by large R. venosa. How- ever, Chesapeake Bay R. venosa readily open and consume large hard clams (30 to 85 mm SH) leaving no visible signs of either drilling or boring behavior. R. venosa grasps its prey along the shell margin and covers the clam with its foot until the clam gapes slightly (Fig. 8). When the clam gapes, the Rapana inserts its proboscis between the clam valves and begins feeding. The entire clam is consumed leaving clean, empty, articulated valves with no visible predation signature as the end product. Food is not likely to be a limiting factor for Rapana venosa in the Chesapeake Bay. Rapa whelks seem to share habitat preferences with their favored food item: the preferred habitat for both hard clams and Rapa whelks is sand bottom. The known Rapana venosa distribution overlaps regions of moderate to high hard clam densities in the lower bay (Fig. 7). The absence of a predation signature on large hard clams con- sumed by Rapa whelks is troubling in light of recent conversations with commercial clammers working in the Ocean View and Hamp- ton Roads area (Fig. 4). The clammers report an increase in the number of empty shell valves caught within the last 1 to 2 years and attribute the increase in empty valves to a corresponding in- crease in natural clam mortality. Given the number and size of Rapana venosa reported from these same areas during 1998 and the absence of a predation signature on large hard clams consumed A. B. Figure 8. Adult Rapana venosa consuming a hard clam (A.) and an oyster (B.t. Biology of Rapana venosa 15 in the laboratory, it is possible that the recent increase in empty, articulated shell valves observed by local watermen is attributable to Rapa whelk predation and not natural mortality. Rapa whelks have also been described as scavengers consum- ing carrion (Chukhchin 1984. Morton 1994). Laboratory observa- tions indicate that Rapa whelks prefer to capture and kill their own food; they will not feed on carrion in the presence of live prey. However. Chesapeake Bay Rapana have been caught incidentally by recreational fishermen that were using fresh squid as bait. Potential Predators: Rapa Whelks Rapana venosa are prey for native octopods in their native waters. Few of the habitats that Rapana have invaded include resident octopods as upper-level predators enabling Rapana popu- lations to grow quickly and inflict considerable damage on local shellfish resources; for example, the decimation of the Black Sea oyster population as described by Chukhchin (1984). Within the Chesapeake Bay. the only upper-level or apex predators that might be capable of using Rapa whelks as a food resource are those that currently eat the local whelk species; that is. Channeled whelks (Busycotypus canaliculatus) and Knobbed whelks (Busycon carica). Crabs and other gastropod species are potential predators for very small Rapana. Sea turtles may be capable of eating Rapa whelks Os O* Os Os Os OS Year Year Year Figure 1. Production trends in China (MAC 1985-97). A, aquaculture versus capture fishery; B, total mariculture production versus molluscan aquaculture; and C, aquaculture production of major molluscan groups from 1986 to 1996. Productions are in wet. whole body weight. lasted for 4 weeks and covered much of the central coast of China, from Qingdao in Shandong province to Wenzhou in Zhejiang province (Fig. 2). It consisted of on-site visits to aquaculture fa- cilities and discussions with local scientists, managers, and farm- ers. Major aquaculture sites we visited included those at and around Qingdao. Rizhao, Ganyu (Jiangsu). Lianyungang, Dafeng (Jiangsu), Qidong (Jiangsu). Wenzhou, and Yueqingjiang. In Oc- tober 1997. the first author visited the north and south coasts of China, which were not covered by our 1996 visit. The 1997 visit was part of a study on the Chinese molluscan aquaculture industry Figure 2. A map of China's coastal provinces show ing major cities and areas visited during this study. conducted by the first and third authors under sponsorship of the US-China Living Marine Resources Exchange Program. The 1997 visit covered major aquaculture facilities and research institutions in Liaoning and Shandong provinces in the north, and Fujian. Guangdong and Guangxi provinces in the south. Cities and areas covered by the 1997 visit included Dalian. Yantai. Rongcheng. Qingdao. Wenzhou, Xiamen. Guangzhou. Shenzhen (bordering Hong Kong), and Beihai. Production figures cited in this paper are mostly from official statistics published by the Ministry of Agriculture of China (MAC 1986-1997). Collecting accurate production data for molluscan aquaculture is always challenging, which is particularly true in China for several reasons. First, production of most molluscan species in China is estimated by multiplying the total culture area by an average yield per unit area. This method alone contributes a large degree of uncertainty. Second, the culture of a large number of species in various forms and across culturally diverse regions is a source of confusion and error. For example, the word "clam" (Ge in Chinese) may include different species in different areas. Oyster production from south China is traditionally reported in meat weights and that from the north in whole weight (with shells). Certain types of culture practices may be considered as aquacul- ture in one area, but not in another. Finally, production may be over- or underestimated by local officials for management and/or political reasons. Despite all these factors, the official figures are still the best or only available statistics. Some Chinese scientists and managers believe that the official statistics may overestimate the overall production by 20 to 30%. For some species, such as blood cockles and razor clams, the official statistics are very close to estimates from local scientists. The decline of the shrimp aqua- culture industry because of diseases is reflected in the official statistics, which corresponds well to expert estimates. (We should see a decline in scallop production, because of mortalities, in 1997 to 1998 if the official statistics are accurate.) Starting in 1996, a new reporting system, or standard, was implemented, which cor- rected some problems. For example, oyster and some clam pro- duction before 1996 were reported as meat weight, and the new statistics converted all molluscan production to whole body weights. For this report, oyster production data prior to 1996 were converted to whole body weight using a factor of 6.1 1, as recom- mended by the Ministry of Agriculture of China (MAC 1997). Scientific names are presented for all species discussed in this paper. The English common names, if available and generally accepted, are also used. For species with no common English Moi.luscan Aquaculture in China 21 names or conflicting ones, the Chinese common name is given in standard pinyin to avoid confusion with different translations. OVERVIEW OF PRODUCTION Mollusks are cultured all along China's 18,000-km coastline. The scope of molluscan aquaculture is reflected by the large num- ber of species cultured. A quick survey indicates that at least 32 species of marine mollusks are cultured commercially in China (Table 1 ), and the list is probably incomplete and growing. The list contains five gastropods and 27 bivalves. The bivalve species in- clude three oysters, four scallops, five mussels, one pearl oyster, and 14 clams. The list in Table 1 is arbitrarily divided into "major" and "minor" species. Most of the 14 major species are well-known mollusks that support large aquaculture industries in China. The minor species are also cultured at commercial scales, but with less significant production. In 1996, China produced 6.4 million metric tons of mollusks (whole body wet weight) from aquaculture, which is about eight times that of 1986 (Table 2). Oysters topped the species list, with production of 2.3 million tons, or more than one-third of the total. Clam production ranked second, with 1.6 million tons. The actual clam figure may be much higher, because some clam species from certain areas are reported under "others mollusks." Most of the 1 .2 million tons of "other" mollusks listed in Table 2 are probably TABLE 2. Aquaculture production I "hole weight in metric tons) of mollusks in China; 1986 vs. 1996/ Species Group 1986 1996 Oyster Clam Scallop Mussel Abalone Pearl Oyster Others Total 336.013 191,951 23,686 210,657 41.592 803,899 2,284,663 1,568.325 999.573 366,251 700 10.000" 1.187.083 6,416,595 'From MAC (1987, 1997) Oyster production in 1986 was converted to whole weight using a factor of 6. 1 1 . Clam statistics include razor clam and cockle species. h Estimated from the production of 20 tons of pearls. miscellaneous clams, and the total clam production from aquacul- ture could be anywhere between 1.6 to 2.5 million tons. Scallops ranked third, with a production of I million tons. Abalone produc- tion was negligible in weight but significant in value. An estimated 20 tons of marine pearls was produced, corresponding to about 200 million pearl oysters or 10,000 tons in whole weight. TABLE 1. A list of marine mollusks cultured in China. English Name Scientific Name Major Culture Areas, Notes Major species Zhe oyster Suminoe oyster Pacific oyster Manila clam Colorful clam Meretrix clam Mud cockle Razor clam Zhikong scallop Bay scallop Wrinkled abalone Colorful abalone Pearl oyster Blue mussel Minor species Huagui scallop Japanese scallop Thick-shell mussel Green-jade mussel Senhouse mussel Penshell Xishishe surfclam Square surfclam Chinese surfclam Cyclina clam Chinese glaucomya River corbula Morella clam Hairy cockle Giant cockle Mud snail Red conch Sea hare Crassostrea plicatula Gmelin Crassostrea rivularis Gould Crassostrea gigas Thunberg Ruditapes philippinarum Adams and Reeve Ruditapes variegata Sowerby Meretrix meretrix Linnaeus Tegillarca granosa Linnaeus Sinonovacula constricta Lamarck Chlamys farreri Jones and Preston Argopecten irradians Lamarck Haliotis discus hamuli [no Haliotis diversicolor Reeve Pinclada martensii Dunker Mytilus edttlis Linnaeus Chlamys nobilis Reeve Patinopecten yessoensis Jay Mytilus comscus Gold Pema viridis Linnaeus Musculus senhousei Benson Pinna pectinatu Linnaeus Mactrn antiquata Spengler Mactra veneriformis Reeve Mactra chinensis Philippi Cyclina sinensis Gmelin Glaucomya chinensis Gray Potamocorhula rubromuscula Zhuang and Cai Moerella iridescens Benson Scapharca subcrenata Lischke Scapharca broughtonii Schrenck Bullacla exarala Philippi Rapana venosa Valenciennes Notarchus leachii cirrosus Stimpson Fujian. most important oyster, wild seeds Guangdong. Fujian. low salinity, wild seeds Liaoning. Shandong, hatchery seeds, longlines All coast, most common clam, mostly wild seeds Fujian. Guangxi, some hatchery seeds Jiangsu. Shandong, extensive culture Zhejiang to Guangdong, high-value, some hatchery Zhejiang, Fujian, wild seeds, intertidal flats Shandong, Liaoning, wild seeds, cage culture Shandong, Liaoning, Fujian, hatchery seeds, cage Liaoning, Shandong, hatchery seeds, cage, raceway Guangdong, Fujian, hatchery seeds, cage culture Guangdong. Guangxi, hatchery seeds, cage culture Shandong, Liaoning. wild seeds, longline culture Guangdong. Fujian Liaoning Zhejiang. Fujian Fujian. Guandong Fujian, Guangdong Zhejiang. Fujian Shandong. Jiangsu, Fujian Jiangsu. Liaoning Jiangsu Jiangsu. salt Guangdong. Fujian Jiangsu Zhejiang Hebei, Liaoning, Shandong Hehei. Liaoning. Shandong Zhejiang Liaoning, Shandong Fujian 22 GUO ET AL. With the exception of mussels, production in all species groups increased several fold over the 10-year period between 1986 and 1996 (Fig. 1C). Production statistics before 1986 did not separate species groups, so it is impossible to make comparisons with ear- lier years. Economic reform and government promotion are prob- ably the dominant factors behind the rapid growth. Mussel pro- duction has been declining since 1992 probably because of com- petition from high-value species such as scallops, oysters, and abalone. Molluscan aquaculture is widely distributed among the Chinese coastal provinces (Table 3). Shandong province in the north, with a production of 2. 1 million tons, or one-third of the national total, is the largest producer of cultured mollusks and leads other prov- inces in scallop, clam, and mussel culture. Yantai. Rongcheng, and Rizhao are the major mariculture areas in Shandong. Liaoning is the leader in abalone aquaculture and also contributes significantly to scallop and mussel production. Jiangsu province cultures a va- riety of clam species and little else. Zhejiang province leads the nation in cockle and razor clam culture. Fujian. Guangdong, and Guangxi are major oyster culture provinces. Fujian is also a major producer of clams, especially razor clams. Guangdong and Guangxi are the dominant producers of pearl oysters. Hebei and Hainan are relatively poor in coastal resources and contribute little to molluscan production. As a generalized pattern, the Chinese molluscan aquaculture can be characterized, with some exceptions, by the scallop, Manila clam, and abalone culture in the north, oyster and pearl oyster culture in the south, and a variety of clams in the middle. We could not find any statistics on the trading of cultured mollusks. Most of the abalone facilities we visited ship a large portion of their product to markets in Hong Kong and Japan. A significant portion of cultured scallops is also exported. Some Meretrix and Manila clams are exported to Japan, and some oys- ters are sold to Hong Kong. Most of the cultured mussel, razor clams. Ruditapes clams and blood cockles, oysters, and scallops are consumed in China. China also imports some mollusks, such as geoduck and squid from North America, green mussel from New Zealand, and blood cockle from Korea. A hotel in Wenzhou, where we stayed in 1996. was selling geoducks from Canada at US$33/ kg. We were told by sources that China has become a net seafood importer in recent years, which may be partly because of the large quantities of fish and fishmeal imported for processing. OYSTERS China produced over 2.3 million tons of cultured oysters in 1996. By weight, oysters are the largest molluscan group cultured in China, and most of the output comes from three species. The most important species is the zhe oyster, Crassostrea plicatula Gmelin. Official statistics do not distinguish individual oyster spe- cies, but experts estimate that the zhe oyster accounts for 50-60% of the total oyster production. The second largest production comes from the Suminoe oyster. C. rivularis Gould (or C. aria- kensis Fujita). which accounts for 20-30% of the total. The other major species is the Pacific oyster. Crassostrea gigas Thunberg. which may account for 10-20% of the national production. China has about 20 recorded species of oysters occurring along its coast, and the classification is sometimes problematic. There are three points of uncertainty concerning aquaculture species. First, oyster farmers in southern China recognize two forms of oysters traditionally regarded as C. rivularis. One is called the "white" oyster, and the other is called the "red" oyster (referring to the meat color). Some experts now believe that the "white" oyster is actually true C. rivularis, and the "red" oyster may be C. iredalei (Li et al. 1988). The two oysters are usually present in the same area at variable proportions. Another uncertainty is about the spe- cies status of the Dalianwan oyster. C. talienwhanensis Crosse, which is cultured in the Dalian area. Some people believe that the Dalianwan oyster is actually a variety of C. gigas. Also, people disagree on whether the monk-hat oyster. C. cucullata, is synony- mous with the zhe oyster (C. plicatula Gmelin). and both names are used in the literature. Zhe Oyster The zhe oyster is found along the entire coast of China. It is small as compared with the Suminoe and Pacific oysters, and thin-shelled. The left shell is deeply cupped, similar to that of Kumomoto oyster (C. sikamea). but even more pronounced (Fig. 3A). The right shell is flat and covered with radial plates. The zhe oyster grows rapidly during the first year, after which shell growth usually stops. Zhe oysters are cultured primarily in Fujian province and other parts of the southern coast. In Fujian alone. 23.000 hectares are used for oyster culture, 80% of which are for zhe oysters. Tradi- tionally, the zhe oyster is cultured on stone pilings, vertical stone TABLE 3. Aquaculture production of major molluscan groups (whole weight in metric tons) from nine coastal provinces of China/ Province Total Oyster Clam b Scallop Mussel Razor Cockle Other Liaoning 871.657 95.969 224,787 223,286 104.888 7.012 1.119 214.596 Hebei 51.568 — 31.591 8.882 1.900 — 9,195 8.493 Shandong 2,144.166 338.209 481,974 753.902 140,983 39,997 31.356 357.745 Jiangsu 122.012 — 105.820 — — 3,338 4.361 8,493 Zhejiang 357,861 62,459 19,121 877 11.826 189.521 56.288 17,769 Fujian 1.138.226 804.845 106.605 12.325 64.281 102.651 3.363 44,156 Guangdong 1,204,212 558.950 39,534 130 40.352 — 20.222 545.024 Guangxi 5 1 1 .885 422.555 82,765 15 2.02 1 — 4,529 Hainan 5,008 1,676 1.751 156 — — 1 .425 — Total 6,406,595 2.284,663 1,093.948 999.573 366.251 342.519 131.858 1.187,783 "From MAC (1997). ' Clam here refers to Ruditapes clams for some provinces and may include meretrix and other clams (excluding razor clams and blood cockles provinces. for other MOLLUSCAN AQUACULTURE IN CHINA 23 Figure 3. Oyster culture. A, The deeply cupped left shells of the zhe oyster cultured in Fujian and Guangdong. B. intertidal racks and stakes and suspended longlines used for the culture of zhe oyster in Xiamen. C, Suminoe oysters cultured on cement stakes in Beihai area. D, a hatchery in Wenzhou area where Pacific oyster spat are heing set on shell-strings. The large concrete tanks (20-5(1 nr'l are typical for all hatcheries and are used for larval culture of a variety of species. E, bamboo rafts used to culture Pacific oysters on shell-strings in Vueqingjiang, Wenzhou. F. Pacific oysters cultured in Dalian reach 8-1(1 cm in 6 months. strips (over 1-m tall), and bamboo or wooden stakes. These ma- terials are arranged in a variety of configurations and formations in intertidal areas (Cai and Li 1990). and oyster-growing areas filled with these structures cover miles of coastline (Fig. 3B). Culture of zhe oysters depends entirely on natural seeds. Large stone strips are permanent structures used for both seed collection and grow- out. Often, seeds are collected on stones, shells, bamboo, and cement blocks in one area and moved to another area for culture. Recently, suspended longlines (Fig. 3B) and raft culture of shell strings has gained popularity with farmers, because it results in better growth and allows for utilization of open-water areas. Stone and bamboo racks in traditional oyster fields have been abandoned in some areas because of their lower productivity. Seeds are usu- ally collected in May and September. Seeds collected in May are usually harvested starting in December. Production peaks in Feb- ruary, around the Chinese New Year, and ends in March. The total culture time is under 1 year. For the fall seeds. 14 to 17 months are needed to reach a market size of 6-7 cm. Suminoe Oyster The Suminoe oyster occurs naturally along most of the Chinese coast, where it is called the Jinjiang ("close-to-river") oyster in Chinese. It tolerates a wide range of salinity, but prefers low sa- linity estuaries and riverbeds, especially for settlement. Local oys- ter farmers recognize two forms of the oyster: the white and red oysters. The former is preferred for its flavor and valued more than the red oyster. The Suminoe oyster is cultured primarily in two southern prov- inces, Guangdong and Guangxi. Culture methods are similar to those used for the zhe oyster. At the Guangxi site we visited. concrete stakes, 50-cm long and 6 x 6 cm at the cross section, are used for both spat collection and growout (Fig. 3C). The stakes are transported to upper river sites for spat collection in the spring. After the spat set. the stakes are planted along the lower river beds for growout. A dozen oysters per stake are considered optimal. About 30.000 stakes are planted per hectare, which produce about 1 2,000 pounds of oyster meat at harvest. At low tide, the river beds are covered with a forest of oyster-carrying stakes. Suminoe oys- ters are also cultured on shell strings hanging on rafts and long- lines. Unlike the zhe oyster, which usually stops shell growth after the first year, the Suminoe oyster maintains rapid growth through out the first 3 years. Oysters are usually harvested in 2 to 3 years at a size of 10-15 cm. In some areas, oysters are moved to pro- ductive areas for fattening before being harvested. Pacific Oyster Pacific oysters occur naturally along the Chinese coast. How- ever, most of the Pacific oysters being cultured in China were originally introduced from Japan. This species is cultured in all parts of the coast, but the major producers are Liaoning and Shan- dong provinces in the north, and Guangdong in the south. Pacific oyster culture depends exclusively on hatchery- produced seeds (Fig. 3D). As with most other bivalves, larvae are cultured in large concrete tanks (10-100 m 3 ). Vitamin supplements and antibiotics are often used during larvae culture to maximize yields. Spat are set on strings of scallop or oyster shells. The recommended density is about 20 to 30 spat per shell, but. in practice, the density is often two to three times that. Farmers may break the shell in half for growout if the density is high. Spatted shells sell for about US$0.0 1-0.02 per piece, depending upon sea- 24 GUO ET AL. son. They are inserted into nylon ropes and cultured on suspended longlines (Fig. 3B) or rafts (Fig. 3E). Bottom culture is also prac- ticed in certain areas. Pacific oysters grow rapidly. Oysters at most sites we visited, including Dalian in the north, reach 8 to 10 cm after the first growing season (Fig. 3F). One factor is that the seeds are produced early in the spring so that they have a full season to grow. Another factor may be the high productivity of the culture sites. Depending upon demand, oysters may be harvested within the first year. Oys- ters cultured in the intertidal areas may need 2 to 2.5 years to reach market size. Triploid Pacific oysters, which undergo little or no gametogenesis. are used for production in Shandong and Liaoning provinces because of their superior growth and improved sur\i\al against "summer mortality." a syndrome linked to reproduction (Perdue et al. 1981). Some oysters are sold live on local markets or to restaurants. Vendors in the market would shuck the oysters if requested. Oys- ters are cooked in a variety of recipes, but rarely consumed raw in China. In southern China, most oysters are cooked and dried for storage, and the broth from processing is condensed into oyster sauce, a popular seasoning in Chinese cooking. CLAMS At least 14 species of clams are cultured in China (Table 1 ). The major species include the Manila clam (Ruditapes philippi- narum Adams and Reeve), the colorful clam (R. variegata Sow- erby). the Meretrix clam (Meretrix meretrix Linnaeus), the mud cockle (Tegillarca granosa Linnaeus), and the constricted razor clam (Sinonovacula constricta Lamarck). In addition to the five major species, at least nine others are commercially cultured in China (see Table 1 ). The official figure for clam production in 1996 is 1.6 million tons, but the actual figure may be higher, because some clam species may not be listed as clams in official figures. On the other hand, clam production listed as from aqua- culture may not be cultured in a strict sense. Culture of most clams, such as razor clams, cockles, and most of the Ruditapes clams, involves seed collection or hatchery production, nursery, and planting, and can be clearly classified as aquaculture. In some areas, however, culture of Meretrix and some other clams may simply mean protective enhancement of natural resources. The culture fields are protected from predators and poachers, but no seed collection and planting are involved. In some cases, a mixture of culture and wild harvest is practiced. Seeds of one species are planted, but multiple species are harvested from the same planting ground. All considered. China's clam production from aquaculture may be anywhere between 1.6 and 2.5 million tons. Ruditapes Clams Ruditapes clams are the most widely cultured clams in China and account for probably 60-70% of the total clam production. Two species predominate: the Manila clam. R. philippinarum Ad- ams and Reeve and the colorful clam R. variegata Sowerby (Fig. 4A). The two are often not distinguished in China, and both are referred to as "Gezi" in Chinese. The Manila clam is cultured along most of the coast and accounts for most of the production. The colorful clam is cultured in the southern provinces. Most seeds are collected from the wild, with Shandong and Fujian provinces being the major producers. Seed collection in- volves selection and building of seed-collection beds, eradication of predators, and routine maintenance. In southern China, clam Figure 4. Clam culture. A, Manila (upper) cultured in Jiangsu and the colorful Ruditapes clam (lower) cultured in Fujian. B, the cyclina (upper) and meretrix (lower) clams cultured in Daxing, Jiangsu: the dark coloration is characteristic of clams cultured in shrimp ponds. C, the square surfclam (upper) and hairy cockle (lower) cultured in Ganyu, Jiangsu. D, salt ponds (upper) in Qidong used for the production of cyclina clam seeds (lower), which usually reach a commercial size of 1 cm in a year. E, unprotected clam beds in Qidong used to culture meretrix, cyclina clams, square, and xishishe surfclams. F, net-fenced clam plots (upper) in Ganyu. Jiangsu. used to culture various clam species; large shrimp ponds (lower) in Wenzhou area, used for polyculture of shrimp, mud cockle and other clams. G, beds for the collection of the constricted razor clam seeds in Wenzhou area; the beds should be completely drainable during collecting season. MOLLUSCAN AQUACULTURE IN CHINA 25 seeds are also produced from hatchery production. Clam seeds, 5 to 10-mm in size, are planted at a density of about 35 million per hectare, but density may vary, depending upon the size of seeds and sediment type. In most cases, clam beds are not protected with nets. Clams are harvested at a size of 30 mm or larger, which is usually reached in 10 to 16 months. Manila clams are one of the most common seafoods in coastal regions of China. Most clams are sold live in local markets, for about USS0.5/kg. They are either stir-fried or used in soup with shells on. Ruditapes clams are usually not depurated before reach- ing the local market. "De-sanding" by placing clams in saltwater for an hour or two is usually the first step in the kitchen. Now there is a frozen product that uses depurated clams, vacuum-packed and frozen in a microwave oven-ready plastic bag. Some frozen clams and clam meat are sold to Japan. Meretrix and Other Clams in Jiangsu The meretrix clam (Fig. 4B) is found in most parts of China's coast. It resembles the hard clam (Mercenaria mercenaria) in bi- ology. It prefers sandy substrates and Jiangsu province, which has a long sandy coast, is the major producer of meretrix clams. Sev- eral other minor clam species are also cultured in Jiangsu using the same culture system as for meretrix clams, including the cyclina clam (Cyclina .sinensis Gmelin) (Fig. 4B). the square surfclam (Mactra venerifonnis Reeve) (Fig. 4C). and the xishishe surfclam (M. antiquum Spengler). Seeds of meretrix and most other clams are collected from the wild. Most cyclina clam seeds are artificially produced in earth ponds (Fig. 4D). Earth pond seed production is a low-tech and low-cost substitute for hatcheries and is effective for certain mol- lusks, including the Ruditapes, the cyclina clam, razor clam, and oysters. Large earth ponds, usually 1-3 hectares in area and 1-m deep, are dried and treated with bleach or herbal poisons before filling with filtered seawater (to 0.1 mm). Brood stocks are in- duced to spawn either in the pond or inside the hatchery. A density of i_4 D-stage larvae/mL is desired. Ponds are fertilized to boost algae growth. No water is discharged before larvae are completely settled. Seeds that settle in the ponds may be thinned if the density is too high. About 3,000 seed clams/nr. at a commercial size of 10 mm, can be expected from earth pond production by the end of the first year. The seed clams are mostly planted in unprotected intertidal flats (Fig. 4E). although planting areas are usually treated with herbal pesticides to remove predator species before planting. After the seed stage is passed, predation is not a major problem. Meretrix and surfclams live close to the surface and are capable of moving and relocating, particularly during storms; therefore, some clam beds are protected with net fences to prevent escape (Fig. 4F). In Jiangsu. the same bed may be used for meretrix and other clams. The clam beds are continuously harvested by manually picking out clams that have reached market size. Another major form of cul- ture for meretrix and other clams is in shrimp ponds (Fig. 4F). Because of the shrimp disease problem, most shrimp ponds are now used for other species or polyculture with other species, es- pecially clams. In the polyculture ponds, shrimp are stocked at a low density and harvested at a small size before mortality starts. One or two clam species are selected and planted in the shrimp ponds. One Jiangsu farm we visited grows both meretrix and cy- clina clams in their shrimp-clam polyculture system. Each shrimp pond is about 3.3 hectares and produces about 10 tons each of meretrix clams and shrimp. Because the harvested shrimp are small, clams constitute a significant proportion of the farm's in- come. Abandoned salt ponds are also used for clam culture. Mor- tality has been reported for meretrix clams cultured in ponds, which coincides with spawning of the clams in August when the water temperature reaches 30-3 1°C. The mortality is usually 30- 40%. but may reach 80-90%. Growers believe the problem is related to high temperature and poor water quality (e.g.. low oxy- gen and, possibly, high bacterial loads) adding to the "stress" of spawning. Mortality in other clam species is rare. Blood Cockles Three species of blood cockles are cultured in China: the mud cockle (Tegillarca granosa Linnaeus), the hairy cockle (Scapharca subcrenata Lischke), and the giant cockle (Scapharca broughtonii Schrenck). The mud cockle is the most desired and widely cultured of the three. Total cockle production from aquaculture was about 131,858 tons in 1996. mostly from the mud cockle. The mud cockle is found in muddy sand beaches of the Shandong peninsula and south. Larval settlement is found to be best on substrates that are more on the sandy side, but adult mud cockles usually grow faster on muddy sediments. The mud cockle is a small and slow growing species, usually taking 2 to 3 years to reach a market size of 2.5 cm. Both wild and hatchery-produced seeds are used in cockle cul- ture. Considerable experience in seed collection and nursery rear- ing has been accumulated throughout the history of cockle culture (1.700+ years). Cockle seeds, called "cockle-sand" (about 0.5 mm), are usually collected in September and October, using cloth bags or nytex screens. Cockle sand sells for about US$l,200/kg. but prices vary greatly from year to year. Seeds of the cockle-sand size are usually cultured in nursery beds for a year before reaching the next stage— "cockle-beans" (about 2.000 cockles/kg). Nursery beds are elevated so water can drain off completely at low tide. Predator species are eradicated before planting by applying herbal poisons before spat fall. A layer of fine sand is added to overly muddy substrates. Growout planting density varies greatly among different areas and depending upon seed size, ranges from 150 to 1 .500 seeds/nr (Wang et al. 1993). Hatchery techniques have been developed to meet the increasing demand for cockle seeds. The Zhejiang Mariculture Institute in Wenzhou we visited is a leader in cockle hatchery technology. Larval culture is the same as for other bivalves, but a unique practice in cockle hatcheries is that mud is added as a substrate to induce settlement. Mud cockle is cultured in intertidal plots and ponds. The yield from cockle ponds is about 7 to 10 tons/hectare. The mud cockle is a delicacy in the Shanghai and Zhejiang areas. It is sold for about US$1 0/kg in Zhejiang area, but imports from South Korea drove the price down by about 30% in 1997. Cockles are prepared by briefly dipping them in boiling water, after which they are consumed semiraw. Because of incomplete cooking and poor sanitation in some areas, cockle consumption, particularly with hairy cockle, has occasionally been associated with Hepatitis infection. Constricted Razor Clam in Zhejiang and Fujian The constricted razor clam (Sinonovacula constricta Lamarck) is found in most parts of the Chinese coast. It tolerates wide temperature and salinity ranges and prefers substrates with a muddy top layer and fine sand bottom. This species is primarily 26 GUO ET AL. cultured in Zhejiang and Fujian provinces and is probably the most important mollusk for Zhejiang province, where its production is greater than all other mollusks combined. Zhejiang produced 189.521 tons of razor clams in 1996. which represents 55% of the national total. Zhejiang and Fujian together account for 85% of all razor clams produced in China. Most razor clam seeds are collected from the wild. Hatchery and earth pond production are used when wild seeds are insuffi- cient. Wild seed collection has a long history and well-established protocols. Collection beds are elevated plots with drainage chan- nels so that the beds are completely exposed at low tide (Fig. 4G). Newly settled razor clam spat would move away from areas that are submerged at low tide. Larval settlement occurs in the fall, and seeds (1-2 cm) are harvested 3 month later and planted at a density of 1.000-1,500 kg/hectare for growout. The razor clams typically grow to about 5 cm in 5-8 months after planting ( 1 year of age). Large clams are harvested at 1 year of age. and small ones are cultured for a second year. Razor clams have several predators, including moon snails, crustaceans, and several fish, especially eels. Herbal pesticides are used to control predators. The razor clam is the first intermediate host of a parasitic worm ( Vesisocoe- liiini solenophagum Tong) and suffers frequent mortalities as a result (DFC 1979; Wang et al. 1993). Several finfish species are the final host of this parasite. SCALLOPS China produced 1 million metric tons of scallops from mari- culture in 1996. Most of the production was from two major spe- cies: the native zhikong scallop (Chlamys farreri Jones and Pres- ton) and the introduced bay scallop (Argopecten irradians La- marck) (Fig. 5A). The native zhikong scallop accounted for about three-quarters to four-fifths of the total, and the bay scallop ac- counted for one-fifth of the total or about 200.000 tons annually. There are two other species, Patinopecten yessoensis Jay (Fig. 5B ) and Chlamys nobilis Reeve, being cultured along the coast with little output. P. yessoensis was introduced from Japan. Its life history resembles that of the sea scallop. Placopecten magellani- cus, of the North Atlantic Ocean. It is a low-temperature species and is cultured only in the northern provinces. Liaoning and Shan- dong. It is larger in size and commands a higher market price than the zhikong and bay scallops, although its yield is low. C. nobilis occurs naturally in the South China Sea and southern Japan It is cultured in southern China on a limited scale. Zhikong Scallop The zhikong scallop is naturally found in north China. Korea, and Japan. It can survive at -1.5°C but does poorly when the temperature exceeds 25°C (DFC 1979. Wang et al. 1993). Most of the zhikong scallop culture is in Shandong and Liaoning provinces. Rizhao. in southern Shandong, probably represents the southern- most extent of its range. Shandong province is the leading pro- ducer, accounting for more than 80% of the national total. Zhikong scallop culture was first developed, between 1973 and 1983. using hatchery seed. Large-scale culture has led to the es- tablishment of breeding populations in many areas of Shandong. Now zhikong scallop culture uses almost entirely natural seed. Breeding grounds on Shandong peninsula currently provide suffi- cient seed for the scallop culture industry. One of the most pro- ductive bays in Shandong produced about 130 billion scallop seeds in 1996. The zhikong scallop has two natural spawning seasons in most areas, one in the early summer and one in the fall. Protocols for seed collection have been well established through years of Figure 5. Scallop culture. A. the introduced ba> scallop (upper) and the native zhikong scallop (lower) cultured in Shandong (photo by H. Yang). B, the Japanese scallop cultured in Dalian. C, ropes made from palm tree fibers used for the collection of scallop spat and seaweed seedlings in hatcheries. D. suspended longlines used for the culture of scallops, ahalone. Pacific oyster, and seaweeds in Rongcheng, covering most of the bay. E, lantern nets used for scallop culture. K, zhikong scallops are harvested at 1.5 years of age (photo by H. Vang). MOLLUSCAN AQUACULTURE IN CHINA 27 research and experience (DFC 1979, Wang et al. 1993). Successful collection involves site selection, preparation of collection mate- rial, and forecasting collection dates by obtaining data on gonadal development, larval stages, and density at the collection site. Seeds are usually collected using bags (30 x 40 cm) stuffed with nylon screens. Each bag may collect 100 to 1,000 spat, depending upon location, season, and year. Scallop culture primarily uses summer seeds, which set be- tween late May and early July. Commercial seeds, about 1-cm in size, are harvested and sold in October. They are then put in lantern nets and reared in a nursery area until the following March, when they reach about 3 cm and are ready for growout. Lantern nets on suspended longlines are used for scallop culture (Fig. 5D). The lantern nets are about 35-cm in diameter with 8-10 layers (Fig. 5E). In the nursery, about 200-300 seed scallops are stocked in one layer, or 2.000-3,000 per cage. The growout density is usually 30 to 50 scallops per layer, although higher densities are often used by farmers. One of the scallop farms we visited in Rizhao produces about 2.500 tons of whole zhikong scallops per year. Total annual production in the Rizhao area is about 80,000 tons. Typically, 1-cm scallop seeds are bought from northern Shandong in October. The seeds are maintained in a nursery area at five times growout density until the following March, when the scallops have reached 2.5-3.0 cm. Young scallops are thinned to 50-80 per layer or 400-500 per cage for growout. They usually reach market size. 6-7 cm, by December (Fig. 5F). The lantern nets are hung on longlines, which are usually 80 to 100-m long and supported by rubber floats. The water depth at this site is about 20 m. At the time of our visit (September), the lantern nets were heavily fouled by a variety of organisms. Scallop culture in Shandong is experiencing a mortality prob- lem. Mortality generally begins in early August as the water tem- perature reaches and exceeds 28°C. It lasts for about 20 days and ends when the temperature begins to decrease. Mortalities at the Rizhao site are in the 20 to 30% range each year, but they may reach 80% farther north, in sites nearer to where the seeds origi- nate. To the north, the timing of the deaths is similar to that in Rizhao, and the temperature is about the same or perhaps slightly (~1°C) warmer. The mortalities were first observed in 1994 at the Rizhao site and have continued since then. Both 1994 and 1995 were warmer (by 2 and 1°C. respectively) than normal. Tempera- tures in 1996 were more typical, and the death rate lessened. Mor- tality worsened in 1997 to 1998, reaching 80% or more in many areas throughout Shandong. There are several suspected causes for the scallop mortality, although they have not been studied extensively. Most scientists in China believe that the mortalities are caused by a combination ot overcrowding, high summer temperature, and deteriorating water quality. Scallop farmers often culture scallop at 2 to 3 times the density (30 to 50/layer) recommended by local scientists. The number of longlines and culture plots (not just for scallop culture) has been increasing rapidly in recent years and may have exceeded the carrying capacity of many coastal areas. Overcrowding at both cage and bay level may have added considerable stress to the culture environment. A haplosporidan parasite of the type respon- sible for extensive mortalities in oysters in the United States was identified in bay scallops in China, but there was no evidence that it was causing mortalities in that species or that it had been trans- ferred to zhikong scallops (Chu et al. 1996). Finally, there is also suspicion that the scallop stock is deteriorating. Although all seeds are collected from the wild, they are collected from a restricted area where the wild population is believed to have originated from hatchery production during the late 1970s and early 1980s. Bay Scallop The bay scallop is not native to China. It was introduced from the United States in 1982 by the third author and colleagues. Of the original shipment, a total of 26 bay scallops survived and were spawned in January 1983. producing the first generation of bay scallops in China (Zhang et al. 1986). The juveniles reached an average 6.9 mm in May and were transferred to culture sites in Shandong and Fujian provinces. The scallops grew to 50 mm by September and 59 mm by December 1983. Market size. 50 to 60 mm. can therefore be reached within a year. This is a major ad- vantage over the zhikong scallops, which usually take 1 .5 to 2.0 years to reach market size. The shorter turn-around time of bay scallops is partly attributable to its faster growth, and partly to the fact that they are spawned in the early spring (or late winter) so that they catch a full growing season. Because zhikong scallop seeds are collected in the fall, they miss most of the first growing season. Because of the short growout time, bay scallops quickly gained acceptance by scallop farmers, and aquaculture expanded rapidly after 1984. By early 1990, the annual production of bay scallops had reached about 200,000 tons. Bay scallop culture pro- duction has declined somewhat in the past few years. Because of the recent summer mortality problem in zhikong scallops, bay scallop culture may increase again. Bay scallop seeds are produced exclusively in hatcheries, where thousands of mature adults, which are hermaphroditic, are placed in lantern nets and induced to spawn in large concrete tanks, ranging from 10 to 100 cubic meters. When the desired egg density is reached (about 50/mL), the adults are moved to the next tank to continue spawning. The first water change is made as soon as the larvae reach D-stage, which is usually 24 h after spawning. Larvae reach the eyed stage in 10 to 14 days at a size of 170-190 p.m. at which time spat collectors are placed in the tanks. Two common types of spat collectors are used. One is a rope curtain made from natural palm tree fiber (Fig. 5C). The other is polyethylene or nylon nets/screens. Spat, which attach to the collectors by byssal threads, are cultured in the hatchery until they reach 500-600 p.m, after which they are transferred to shrimp ponds or nursery areas. Commercial seeds are sold at a size of 0.5 to 1 .0 cm. Lantern nets on suspended longlines is the predominant form of culture for all scallops. The culture density of bay scallops is about the same as the zhikong scallops. 30 to 50 scallops per layer, 250 to 400 per net. Hatchery production of larvae usually occurs between March and May, and scallops are harvested between November and De- cember. Most of the bay scallop production continues to be from the 26 scallops first introduced in 1982. and there have been signs of inbreeding, such as larval and juvenile mortality. Several new broodstock introductions have been made to expand the gene pool, but offspring of the recently introduced scallops have not yet en- tered the mainstream production. Most of the scallops are processed upon harvest. Adductor muscles are either individually frozen, or cooked and then dried. A small fraction of scallops is sold live to local restaurants. MUSSELS Mussel culture is relatively new in China. It started with blue mussel in the 1950s as a byproduct of seaweed {Laminaria 28 GUO ET AL. japonica) culture. Five species of mussel are cultured commer- cially. The most widely cultured is the blue mussel. Mytilus edulis Linnaeus (Fig. 6A). which is produced chiefly in Shandong and Liaoning provinces. Four other species, the thick-shell mussel {Mytilus coruscus Gould), the Senhouse mussel [Musculus sen- housei Benson), the green-jade mussel {Perna vividis Linnaeus) (Fig. 6B), and the penshell (Pinna pectinata Linnaeus) (Fig. 6C). are also cultured in Guangdong and other southern provinces on limited scales. The Senhouse mussel is primarily used for shrimp, fish, and chicken feed. All five species are native to China. For blue mussel culture, hatchery-produced seeds were used to supplement wild set during the 1970s. Now wild mussel seeds are abundant, and hatcheries are used for other molluscan species. Seeds are usually collected in May and June, occasionally also in the fall. When the juveniles reach about 10 mm, they are thinned and reattached to ropes for growout. Culture on suspended long- lines is the predominant form of growout for the blue mussel. Bottom culture is also used for the green and thick-shell mussels in the south. A portion of the juveniles that set in May and June are har- vested between October and December, at a size of 60 to 70 mm. Smaller mussels are returned and cultured for longer periods, but most mussels are harvested within a year. Mussels with full gonads are considered most desirable on the market. Some mussels are sold fresh on local markets, but most are steamed and dried into a traditional product called "Dan-cai." Mussel meat that is dried without cooking is called "butterfly meat." Some mussels are cooked to produce "oyster sauce." Mussels are also used as feed for cultured shrimp and Rapana snails. Mussel culture has been in decline in recent years probably because of competition from other high-value species, such as scallops, oysters, and abalone. ABALONE AND OTHER GASTROPODS Abalone culture is new in China. Large-scale culture started in the late 1980s, and it has been developing rapidly in the past decade. Now it is one of the largest components of the molluscan culture industry by value. It is probably also the most sophisticated industry in terms of production technology. Abalone is not indi- vidually listed in official statistics, and production estimates vary greatly. The annual production is probably between 500 to 900 tons. Two species of abalone are cultured: the wrinkled abalone, Haliotis discus hannai Ino (Fig. 7E) and the colorful abalone, Haliotis diversicolor Reeve. The colorful abalone is a southern species and is cultured mainly in Guangdong and Fujian. The wrinkled abalone is the major species and accounts for about three- quarters of the total production. It is a northern species and is cultured mainly in Shandong and Liaoning provinces. Liaoning's Dalian area is the leader in abalone aquaculture. and much of the technology has been developed there. There are three major aba- lone culture companies in Dalian: the Bilong Seafood Co.. Pacific Seafood Co., and Xinda Products Co. We visited all three facili- ties. The Bilong Seafood Co. has been the leader in abalone pro- duction for some time. The Pacific Seafood Co., which was es- tablished in 1993 with an investment of US$8.5 million, is posed to become the largest abalone culture company in China. It is designed to produce 600 tons of abalone per year, although it had not produced the first crop when we visited in 1997. Xinda has successfully used hybrid abalone to combat disease problems. Abalone culture in Shandong province is catching up fast. Over 40 large abalone facilities with investments of more than US$1 mil- lion each, have been built in Shandong's Rongcheng area. Most of the new facilities have not reached their full production capacity, and many are running at a loss because of disease problems. On the other hand, one facility in Rizhao that we visited has recovered all its investment in 3 years. The Rizhao facility is primarily designed for hatchery production of seed and has little growout production. All abalone seeds are hatchery produced with well-developed technology. Production starts in early spring with broodstock con- ditioning at elevated temperatures. After about 1.000 degree-days of conditioning (at about 18-20°C), abalone are ready to spawn. To induce spawning, they are left without water for 1 hour and then exposed to UV-treated seawater (600 u.W/h/nr 1 ). Males usu- ally begin to spawn 1 hour after being in UV-treated water, and females within 30 to 40 min after the males. Fertilized eggs are incubated at density of 15-20 mL. Eyed larvae are set on corru- gated plastic plates, which are precoated with a layer of diatoms to induce settlement (Fig. 7A). Abalone spat remain on the settlement plates until they reach 3 mm. After that, they are separated from the settlement plates and transferred to large punctured plastic plates for nursery culture (Fig. 7B). The holes allow for better water circulation and for the young abalone to move from side to side. The plates are supported in net-pans and placed in raceways, typically 0.5-m deep. 1 to 2-m wide, and 10 to 20-m long (Fig. 7C). Commercial diets (formulated) are used during the nursery phase. Juvenile abalones are cultured to a size of 1 to 2 cm in the nursery before growout. Abalone seeds sell for about $0.25 each. Three major forms of growout are used in abalone culture. The N& &JP&T V* A--*" Figure 6. Mussel culture. A, the blue mussel harvested as a byproduct from seaweed and oyster longlines in Dalian. B, green-jade mussel cultured in Guangdong and Fujian. C, the penshell cultured in Zhejiang. MOLLUSCAN AQUACULTURE IN CHINA 29 Figure 7. Abalone culture. A, settlement plates are coated with diatoms before use in a Dalian hatchery. B. juvenile abalones are cultured on plates in indoor raceways in a Dalian hatchery. C, typical indoor raceways used for abalone larval culture and nursery. D, large and specially designed cages for abalone culture on suspended longlines, Dalian. E, close to market size abalone cultured in raceways inside an abandoned air-raid bunker in Lianyungang. F. sea urchin and sea cucumber are alternative species cultured in abalone hatcheries. first, and most widely used, is culture in cages on suspended long- lines. Most abalone cages are much larger and more sophisticated than the lantern nets used for scallops (Fig. 7D). They are about 70-cm in diameter and l-m tall with 4—5 layers. One type of cage is made from large plastic tubes (35-cm in diameter and 60-cm long), with screens on each end and 1-cm holes on the side. Some farmers also use scallop lantern nets for abalone culture. The sec- ond form of culture is on plastic plates placed in indoor concrete raceways (Fig. 7E). Abandoned air-raid bunkers are now popular places for indoor abalone culture. The third form, which is not widely used, is in intertidal ponds. Intertidal net fences are also used in some areas for abalone culture. Fresh kelp, mostly Lcuni- naria japonica, is the primary food during growout, and artificial diets are used when algae are not available. Abalone is considered to be one of the best and most valuable seafoods in Chinese culture and other parts of Asia. Commercial size abalone (7 to 9 cm) is priced at S3 to $4 per animal, and a large proportion of the abalone produced in China is sold live to markets in Hong Kong and Japan. Some abalone are sold to local restau- rants. Abalone is also valuable as an ingredient in Chinese medi- cine. Abalone culture was highly profitable in the late 1980s and early 1990s, but recent disease problems have been plaguing aba- lone culture in the north. Many abalone facilities in Shandong and Liaoning have reduced or stopped abalone culture and started growing sea urchins and sea cucumbers (Fig. 7F), which have similar culture requirements. One of the diseases is known as the pustule disease and is caused by Vibrio fluvialis-U (Li et al. 1998). Another major dis- ease affecting the wrinkled abalone has a distinct syndrome. It includes a long incubation period and slow disease progression and is characterized by a shrinking of the meat within the shell. The condition is reminiscent of "Withering Syndrome", which affects wild black abalone (Haliotis cracherodii Leach) along the Cali- fornia coast. This disease is transmissible and is strongly associ- ated with a rickettsial infection (Gardner et al. 1995, Friedman et al. 1997). A similar syndrome of Nordotis discus discus in Japan is associated with a virus (Otsu and Sasaki 1997). Whether these syndromes are related is presently unknown. Several other gastropods are also cultured in China, including the red conch (Rapana venosa Valenciennes), the mud snail iBul- lacta exarata Philippi) and the sea hare (Notarchus leachii cirro- sus Stimpson). For the red conch, juveniles are collected from the wild and cultured in cages or lantern nets. They are fed with blue mussels. The mud snail is a small gastropod, belonging to Order Cephalaspidea (bubble shells), with an adult size of 2 to 3 cm. It has a large foot that covers much of its thin shell. The culture of mud snails involves the selection of muddy flats that have heavy larval settlement. The flat is treated with pesticides to remove predators and fertilized to stimulate diatom growth just before settlement occurs. No other management is required. Production at harvest usually ranges between 35-75 metric tons per hectare. The mud snail, pickled in liquor, is a delicacy in the Zhejiang and Shanghai areas and is priced at about $l/lb. Mud snail culture is a significant industry in Zhejiang and is highly profitable. The sea hare has been cultured for hundreds of years in southern China, where juveniles are collected from the wild and cultured in ponds. 30 GUO ET AL. Sea hares are not cultured for their meat, but for their egg cases, which when processed, constitute a valuable remedy in Chinese medicine, referred to as "Sea Powder." PEARL OYSTER Pearls are produced from both freshwater and marine bivalves. China has a long history of culturing freshwater pearls and cur- rently produces about 800 tons annually, mostly from the fresh- water mussel Hyriopsis cumingii Lea. Freshwater pearls are not only marketed as jewelry, but also used as ingredients in Chinese medicine and cosmetic creams. Marine pearls have a higher market value than freshwater pearls. Wild marine pearls have been har- vested for several thousand years in China, but artificial culture is less than 50 years old. China now produces about 20 tons of marine pearls annually, second to Japan's production of about 40 tons. Marine pearls are produced by species of Pteriidae. In China, almost all cultured marine pearls are from the Martensii pearl oyster, Pinctada martensii Dunker (Fig. 8A). This species is also the major species cultured in Japan and accounts for over 95% of worldwide marine pearl production by weight. Other species such as Pinctada maxima Jameson and Pinctada margaritifera Lin- naeus are also cultured experimentally in China. Pearls from the latter two species are more valuable because of their larger size, unique coloration, or both. Marine pearls are primarily cultured in provinces on the South China Sea. Guangdong and Guangxi provinces produce over 90% of China's total. We visited Beihai, the pearl city of China, where the famous Hepu pearls are produced. This area has about 360 pearl oyster hatcheries and 2,000 farms, and produces about 40- 50% of the national total. All seeds are hatchery produced using methods similar to those used for bay scallops. As in other mol- luscan hatcheries, simple technologies are often practiced. One example is the culture of age in plastic bags (Fig. 8B). Hatchery production usually starts in April. When spat reach 1 mm, they are put into fine-mesh bags and moved to nursery areas in the sea. Seeds are transferred to growout cages at a size of 5-8 mm and a density of about 5,000 per cage. Unlike the multiple-layer lantern nets used for scallops, cages used for pearl oyster culture are small (25 x 25 x 10 cm), single compartment units (Fig. 8C). They consist of metal frames with nylon net sides. Some cages are made with a metal ring, about 30-cm in diameter (Fig. 8D|. These cages are hung on suspended rafts, longlines. or intertidal longlines that are supported by wood stakes about 60-cm tall. Juveniles are thinned five to seven times during a 2 to 3-year period to a final density of 30-50 adults per cage. Pearl oysters are cultured for 2 to 3 years to a size of 50-70 mm before being used for pearl production. Marine pearls are produced by inserting a nucleus (5-8 mm) attached with a small piece of mantle (2-3 mm) from a donor oyster into the mantle of the re- cipient pearl oyster. The attached mantle tissue will grow, encap- sulate the nucleus, and produce nacre. Freshwater pearls are usu- ally produced by inserting a piece of mantle only. Nucleus pearls have recently been introduced to freshwater pearl production and have become strong competitors with marine pearls. Nuclei are usually made from molluscan shells or synthetic materials. The number of nuclei inserted per oyster depends upon the size of the nucleus and the oyster, but on average, about two nuclei per oyster are inserted. Nucleus insertion is performed in spring or fall. Sum- mer is avoided because of the additional stresses of high tempera- ture and reproduction. When nuclei are inserted between February and April, pearls are harvested in November and December of the Figure 8. Pearl oyster culture. A, the outside (upper) and inside (lower) appearance of the Martensii pearl oyster used for the production of marine pearls in southern China. B, plastic bags are used for algae culture (second stage) in many molluscan hatchery; the last stage culture is usually done in shallow concrete tanks. C and D, single compartment cages used to culture pearl oysters on intertidal longlines in Beihai. E, pearls are harvested by sacrificing oysters; one pearl per oyster is expected, on average. F, pearl necklaces on display at markets in Beihai. the pearl city of China. M( 1LLUSCAN AQUACULTURE IN CHINA 31 same year. The recovery rate is about 50% or one pearl per oyster. It takes about 10 million pearl oysters at harvest to produce one ton of pearls. At harvest, pearl oysters are sacrificed to collect the pearls (Fig. 8E). The meat is used for chicken and duck feed. Raw pearls come in various shapes and colors. They are sorted and processed in a series of chemical treatments before reaching the jewelry market (Fig. 8F). They are classified into four size categories: large (>8 mm), medium (6-8 mm), small (5-6 mm), and fine (<5 mm). Large pearls are worth considerably more than smaller ones. Nucleus-free pearls are produced by inserting only donor tissue and are intended for use in Chinese medicine. Pearl powder from pearls and shells is used in toothpaste and facial creams. One problem in pearl production is sexual maturation. When pearl oysters are full of gametes, nucleus insertion is difficult, and the survival and pearl recovery rates are low. Some farmers inhibit maturation in the fall by placing oysters at high density and in deep water. Mature oysters are sometimes induced to spawn beforehand to improve their condition for nucleus insertion. Sterile triploids are being tested for pearl production at the South China Sea In- stitute of Oceanology in Guangzhou and Guangxi Institute of Oceanology in Beihai. and preliminary results are encouraging. PERSPECTIVES Molluscan aquaculture in China is impressive in both scope and magnitude. It is practiced in almost all inhabited parts of the coast and covers all major molluscan species. A wide range of produc- tion technology is used, ranging from sophisticated intensive cul- ture of abalone, scallop, and pearl oysters, to primitive extensive culture of certain clams and snails. Some practices are distinctive. One example is the "semiartificial" collection of clam seed, which involves site selection, bed construction, substrate modification, larval forecast, and predator control. Another example is seed pro- duction from earth ponds, which seems to be an effective approach under low-tech conditions. Polyculture of mollusks, shrimp, and/or fish in ponds is widely practiced. The past decade represents the fastest growing period for mol- luscan aquaculture in China. This period coincides with rapid growth in the overall Chinese economy and is primarily influenced by China's economic reforms. The replacement of central planning with a market economy is probably the leading force responsible for the rapid growth. Marine mollusks are among the best-loved seafood in China. As the Chinese economy grows and people's income rises, demand for mollusks will continue to increase and prompt further growth of the aquaculture industry. The rapid development of molluscan aquaculture has brought with it some problems, the most pressing of which is the deterio- ration of the culture environment. The expansion of mariculture during the past decade has put considerable stress on the marine environment, and the carrying capacity of the coastal water may be exceeded in many areas. Longlines and rafts often cover much of a bay (Fig. 3B. E; Fig. 5D). Large shrimp ponds are densely situated (Fig. 4F). Scallops are often cultured at excessive densities at both cage and baywide levels. Excessive feeding from shrimp and abalone culture leads to accumulations of tremendous waste. Red tides have become more frequent along China's coast. Over- crowding and poor water quality, coupled with high water tem- perature, are believed to be the leading causes for the massive scallop mortalities in 1997 to 1998. Abalone culture has been seriously affected by diseases. There is a great need for the devel- opment of new management strategies to maintain yields while minimizing conditions that degrade the environment, cause dis- ease, or both. Future growth of the molluscan aquaculture industry may largely depend upon technological advances that make mol- luscan aquaculture more efficient and environmentally friendly, instead of crude expansion in scale. ACKNOWLEDGMENTS We thank all our hosts for their hospitality and assistance dur- ing our visits. Many Chinese scientists and aquaculturists contrib- uted personal knowledge to this paper. We particularly thank Pro- fessors Rucai Wang, Zhaoping Wang, Huiping Yang, Guofan Zhang. Zhihua Lin, Aimin Wang, and Zhinan Zeng for discussion and comments. This study and our visits were supported partly by NOAA's US-China Joint Program in Marine Living Resources. China's State Bureau of Foreign Experts, Institute of Oceanology Chinese Academy of Science, and Rutgers University. This is publication No. 99-14, IMCS/NJAES. LITERATURE CITED Cai, Y. & X. Li. 1990. Oyster culture in the People's Republic of China. World Aquacult. 2 1 :67-72. Chu. F.-L. E., E. M. Burreson, F. Zhang & K. K. Chew. 1996. An uniden- tified haplosporidian parasite of bay scallop Argopecien irradians cul- tured in the Shandong and Liaoning provinces of China. Di.s. Ai/imr. Org. 25:155-158. DFC (Dalian Fishery College). 1979. Molluscan aquaculture. Agriculture Press. China (in Chinese). FAO. 1997. Review of the state of world aquaculture. FAO Fisheries Circular No. 886. Rev. 1. Rome. Friedman. C. S.. M. Thomson, C. Chun, P. L. Haaker & R. P. Hedrick. 1997. Withering syndrome of the black abalone, Haliotis cracherodii (Leach): water temperature, food availability, and parasites as possible causes. J. Shellfish Res. 16:403^1 1 Gardner. G. R.. J. C. Harshbarger, J. L. Lake. T. K. Sawyer. K. L. Price, M. D. Stephenson, P. L. Haarker & H. A. Togstad. 1995. Association of prokaryotes with symptomatic appearance of withering syndrome in black abalone Haliotis cracherodii. J. Invertebrate Pathol. 66:111- 120. Li, G, Y. Hu & N. Qing. 1988. Population gene pools of big-size cultivated oysters (Crassoslrea) along the Guangdong and Fujian coast of China. Proceedings of Marine Biology of the South China Sea. 51-70. Li. T., M. Ding, J. Zhang. J. Xiang & R. Liu. 1998. Studies on the pustule disease of abalone (Hatliotis discus hanni Ino) on the Dalian Coast. J. Shellfish Res. 17:707-711. MAC (Ministry of Agriculture of China). Bureau of Aquatic Products. 1986-1997. China Fishery Annual Statistics. Beijing, China (in Chi- nese). Otsu, R. & K. Sasaki. 1997. Virus-like particles detected from juvenile abalone (Nordotis discus discus) reared with an epizootic fetal wasting disease. J. Invert. Pathol. 70:167-168. Perdue, J. A., J. H. Beattie & K. K. Chew. 1981. Some relationships be- tween gametogenic cycle and summer mortality phenomenon in the Pacific oyster (Crassoslrea gigas) in Washington state. J. Shellfish Res. 1:9-16. Wang. R., Z. Wang & J. Zhang. 1993. Marine molluscan culture. Qingdao Ocean University Press, Qingdao, China (in Chinese). Zhang. F.. Y. He. X. Liu. J. Ma. S. Li & L. Qi. 1986. The introduction, hatchery rearing, and culture of bay scallops. Oceanol. Limnol. Sinica 17:367-374 (in Chinese with English Abstract). Journal oj Shellfish Research, Vol. 18, No. 1. 33-39. 1999. SETTLEMENT OF THE BLUE MUSSEL MYTILUS GALLOPROVINCIALIS LAMARCK ON ARTIFICIAL SUBSTRATES IN BAHIA DE TODOS SANTOS B.C., MEXICO SERGIO CURIEL RAMIREZ AND JORGE CACERES-MARTINEZ Centra de Investigation Cientifica y de Education Superior de Ensenada Departamento de Acuicultura Apartado. Postal 2732, 2800 Ensenada, Baja California, Mexico ABSTRACT The culture of the blue mussel Mytilus galloprovincialis in Bahi'a de Todos Santos is a growing economic activity. This culture depends upon mussel seed collection from artificial collectors; however, there are no studies on time and duration of the mussel settlement season, collector material, and settlement pattern. Between December 1994 and November 1995, pieces of about 163 cm 2 of nylon ropes, polypropylene ropes, ropes made with polypropylene and cotton, pads of synthetic fibrous material, and dried Luffa, sp. were used as collectors and were deployed at 2- and 5-m depths. Mussel settlement occurred during all the period of study, and its fluctuation was similar for all collectors tested. Major settlement occurred in December and January for all collectors and depths studied. The observed settlement pattern indicates that direct settlement of competent pediveligers from the plankton onto the substrates is the main source of recruitment in the area {929c ). There was a trend of greater settlement on pads of synthetic material than on the other collectors. This material seems to be appropriate for scientific studies; whereas, for commercial activity, any filamentous rope collectors are recommended. KEY WORDS: settlement. Mytilus galloprovincialis, mussel seed collectors, dispersion and culture INTRODUCTION The culture of the blue mussel Mytilus galloprovincialis using submerged longlines in Bahi'a de Todos Santos, Baja California (Fig. 1) started in 1991 (Caceres-Martfnez 1997). At present, the annual production is around 150 metric tons and it is marketed in Mexico and in the United States. Submerged longlines. 200-m long, are suspended from 200-L plastic floating barrels and are anchored with 0.8 or 1.2 ton concrete anchors. The main line is placed at a 5-m depth, from which culture ropes, 7-m long, are suspended. Mussel seed is obtained from nature on artificial col- lectors placed from late November to December. The collectors consist of a polypropylene rope placed inside a thin plastic net and suspended from surface longlines (Caceres-Martfnez 1997). This practice and type of collectors were established on the basis of the experience of mussel growers. On the other hand, it is known that colonization on natural and artificial substrates by 116- «'\ 1 >0- 05' "31" 50' ^^Ensenada Babia de Todos \ sanlos V Pacific Ocean A Mussel cultured ^ I) area 1 J V lslas Todos ,— ^?L/ Xi " a J a Santos ^s_^ California " 31* «' \ r.lK.r.1. !^_ \Js \ Figure 1. Map showing the study area, filled oval indicates the culture area were collectors were deployed. mussels, Mytilus sp. may occur by settlement of competent pedi- veliger larvae and/or by settlement of drifting postlarvae (Davies 1974, King et al. 1989, Caceres-Martmez et al. 1993, Caceres- Martfnez et al. 1994). This settlement pattern could have practical importance for the mussel grower, because its knowledge in a given area may improve the chance of collecting mussel seed from nature (Caceres-Martfnez and Figueras 1998). However, there are no scientific studies in the area to corroborate or improve mussel seed collection practices. The aim of the present study is to determine the time and duration of the mussel settlement season and the relative percent- age of competent pediveligers to postlarvae during settlement on different artificial collectors deployed at two depths in Bahi'a de Todos Santos. MATERIALS AND METHODS The study was performed in the mussel culture area of Bahfa de Todos Santos, which is approximately 18-km long and 14-km wide and has a surface area of 252 km 2 ; it has a sandy bottom, and it is partially separated from the ocean by the two small islands. Islas de Todos Santos. The study was carried out from December 1994 to November 1995. Pieces of 25-cm long and 2-cm diameter (163 cm 2 ) of nylon ropes, material similar to that used by mussel growers (FN); polypropylene ropes (FP); ropes made with poly- propylene and cotton (FPC); pads of 25-cm long and 6.5-cm wide (163 cm 2 ) of synthetic fibrous material (Commercial Scotch Brite™) (SF) and similar pieces of dried fibrous Luffa sp. (Cucur- bitacea) (L) were used as collectors. Ropes were unraveled by passing them through a grinding machine to increase their fila- mentous nature. A comparison among surface area of the sub- strates was relative because of the difficulty for determining their exact surface area. The five collectors were attached to a PVC tube and hung at 2- and at 5-m depths, covering part of the depth at which mussel growers place their collecting ropes (1 to 7 m-depth). Collectors 33 34 Ramirez and Caceres-Martinez III.. I I, I ill. ..Ill 3 "5 to 5 2 E o ■ S3 I 1 c en o _l DJ F MAMJJ ASO N FCP I hitafc ft H nini DJ FMAMJJA SON I J, ■ II M. U DJFMAMJJASON DJFMAMJ JASON I DJFMAM JJASON Months Figure 2. Logarithm of the mean number of mussels settled (+ standard error) on different collectors placed at 2- (open bars) and 5-m (filled bars) depth, during a study period in Bahia de Todos Santos, Mexico. This graphic representation allows better visualization of results during low settlement periods. were replaced after periods of 30 ± 5 days. Each collector was taken to the laboratory in one plastic bag, then the collectors were immersed individually in a 10% solution of commercial sodium hypochlorite (Na CIO) for 5 min. (to dissolve organic material and to facilitate detachment of mussels) and were rinsed with running water directly onto a 0.09-mm sieve. After that, the seed was dried in an oven at 70°C for 24 h and passed through a series of sieves TABLE 1. Expected size of mussels during time intervals studied. Theorical Theorical Date of Time for Expected Expected Collector Temperature Colonizing Size (mm) Size (mm) Replacement <°C) (days) 2-m depth 5-m depth 15.12.94 35 1.212 1.210 08.02.95 54 1.687 1.685 27.02.95 19 0.812 0.810 29.03.95 17 30 1.087 1.085 25.04.95 18 27 1.012 1.010 18.05.95 17.6 23 0.912 0.910 16.06.95 20 29 1.062 1.060 13.07.95 20.7 27 1.012 1.010 24.08.95 21.4 42 1.387 1.385 28.09.95 18.6 35 1.212 1.210 30.10.95 18.6 32 1.137 1.135 22.11.95 17 23 0.912 0.910 of 0.09 to 0.7-mm mesh to facilitate mussel separation by size. Three subsamples of mussels were obtained from each sieve and counted. For graphic representation, data for Fig. 2 were log trans- formed. Thirty individuals per fraction were measured with an ocular micrometer under a stereoscopic microscope or with an electronic caliper if the mussel size was >5 mm to determine size distribution. Competent pediveliger larvae were separated considering mus- sels with shell lengths from 0.250 to 0.470 mm (mean 0.360 mm) according to the minimum and maximum size values recorded for this stage of Mytilus sp. (Rees 1954, Bayne 1965, Widdows 1991 ). Moreover, the growth rate of recently settled Mytilus galloprovin- cialis [ca. 25 u.md _1 at 15 to 17°C (Aguirre 1979)] was considered to estimate the probable increase in shell length of mussels during the sampling period, mussels greater than expected will confirm the attachment on collectors of drifting postlarvae. Water tempera- ture was recorded during samplings. The number of spat from different collectors and depths studied were compared using an analysis of variance (ANOVA). RESULTS Settlement fluctuations throughout the study period on different collectors are shown in Figure 2. In general, in all the collectors, a similar fluctuation was detected at the two depths studied: the settlement began to rise in December, with a peak in January (in these months, the 78% of the total spat obtained during all the Ramirez and Caceres-Martinez 35 study was recorded), and it remained low throughout the other months, except September, when a light increase was detected. This general fluctuation was best recorded on collector SF where the greater quantity of spat was detected. Particular differences are observed among the obtained spat on the studied collectors and depths: settlement seems to be more abundant at 2- rather than 5-m depth. This occurs during December and January in FP, January in FN, December in FPC, December and January in SF. and Decem- ber in L. The loss of FPC and L in January prevent a comparison in these collectors. During the months of low settlement, this dif- ference is not clear. The weakness of L favored their frequent loss during the study period. Statistical comparison of the spat among collectors showed that differences were neither significant (F = 2.18. p = .7) during the study period, nor between depths (F = 0.92. p = .34). Expected sizes of spat during permanence time on collectors and the temperatures recorded are shown in Table 1. Size dis- tributions of the spat in different collectors are shown in Figures 3 to 7. In general, a similar distribution was recorded in all collec- tors and depths. The maximum size recorded in December was 1.59 mm in all the collectors and at both depths; whereas, in January, it was 5.99 mm in FP at 2-m depth. 3.74 and 3.70 in FN and L. respectively, at 2-m depth, and <3.59 in the other collectors and depths. During December and January, about 7% of mussels in all the collectors and depths reached a size greater than ex- pected, lately this percentage accounts for \ c /c. During the low settlement period, the maximum sizes recorded were 12.49 mm during April in SF at 2-m depth, 10.32 mm during February in FP at 5-m depth, and 5.33 during August in FPC at 2-m depth. In the other collectors and depths, the maximum sizes were <3.59 mm. Sizes of <0.470 mm were recorded during all the year and in all collectors. DISCUSSION The presence of mussels <0.470 mm throughout the year and their abundance in December and January reflects the presence of spawning mussels throughout the year and the occurrence of a major spawning period during late autumn. The reproductive cycle of Mytilus galloprovincialis in Bahfa de Todos Santos has not been studied; however, in the west coast of North America, the major spawning season of M. edulis (after the works of Harger ( 1972) in Santa Barbara, California, the Mytilus edulis-like in southern Cali- fornia was identified as M. galloprovincialis (see Mc Donald and Koehn 1988. Koehn 1991)) takes place in winter (see Suchanek 1981). It is known that in populations of M. edulis and M. gallo- December n 2=2 56 n5=159 Hk March n2=43 n5=46 100 January n2=1079 n5=458 80 60 40 _^ — , — 20 TlT~L _j)M_JB_j»=b 100 April n5=9 80 60 40 . f 20 -II 100 July n2=17 n5=8 100 February n2=41 n5=18 80 60 40 • | 20 100 May n2=12 n5=11 IL. 100 80 August n2=46 D □ September n2=60 n5=69 [L 0.2S 0.471 0.9 1.6 2.6 >3.6 0.47 0.899 1.599 2.699 3.699 100 October n2=22 n5=33 100 November n2=2 n5=6 80 80 60 60 40 r| ■ 40 "I 1 . 20 3 0.26 0.47 I 1 2 °o j 1 I ■ 2.6 3.599 0.471 0.9 1.6 0.899 1.699 2.699 2.6 3.699 >3.6 0.26 0.47 0.471 0.9 1.6 0.899 1.699 2.599 >3. Size classes of mussels Figure 3. Size distribution in percentage of mussels settled on collectors FP placed at 2- (open bars) and 5-m (filled bars) depth, during a study period in Bahia de Todos Santos, Mexico. 36 Ramirez and Caceres-Martinez i 40 December January n2=151 n5=206 too so 60 40 n2=739 n5=638 L 20 JO S 60 in 3 E ■8 20 £. ° s c O 100 a. March n2=26 n5=41 [K June n2=8 nS=8 Jl □ September n2=52 n5=51 [L 0.25 0.471 0.9 1.6 2.S >3.6 0.47 0.899 1.599 2.599 3.599 April n2=3 n5=6 .■I October n2=26 n5=13 0.25 0.471 0.9 1.6 2.6 >3.6 0.47 0.899 1.599 2.599 3.599 February n2=24 n5=14 II 100 80 May n2=12 n5=2 CD August n2=33 dD □ D November n2=39 n5=13 0.25 0.471 0.9 1.6 2.6 >3.6 0.47 0.899 1.599 2.599 3.599 Size classes of mussels Figure 4. Size distribution in percentage of mussels settled on collectors FN placed at 2- (open bars) and 5-m (filled bars) depth, during a study period in Bahia de Todns Santos, Mexico. provincialis from different areas of the world, after a main spawn- ing season, minor spawning during the year may occur (Seed 1976. Ferran 1991. Vfflalba 1995. Caceres-Martinez and Figueras 1998). Differences between reproductive cycles from mussels located in different environmental conditions could be explained by tempera- ture, salinity, photoperiod. food, nutrient reserves, hormonal cycle, and genotype differences (Seed 1976. Devauchelle and Mingant 1991, Robinson 1992. Seed and Suchanek 1992. Couturier 1994). It is important to note that M. califomianus is present in the exposed rocky shores of the ocean side of the Bahia de Todos Santos, where M. galloprovincialis is found only rarely. To the contrary, M. galloprovincialis is clearly dominant in the protected areas of the bay and obviously in culture area, where M. califor- nianus is rarely seen. However, the occasional presence of M. califomianus in the culture area, suggests that some larvae of this species may reach and survive culturing conditions, then it is rea- sonable to assume that some settled larvae may belong to this species. However, morphological identification of mussels <3 mm is practically impossible (Rees 1950. Loosanof et al. 1966. Hines 1979, personal observation). To resolve this point, it is necessary to do detailed studies on identification throughout the molecular genetics of mussel larvae and postlarvae. In this study, we assume that the major recorded spat corresponds to M. galloprovincialis, taking into account the prevalence of M. galloprovincialis and that its reproductive cycle presents one major spawning season in the west coast of North America with respect to the reproductive cycle of M. califomianus without a major spawning season (Young 1946. Suchanek 1981. Hines 1979, Curiel-Ramirez and Caceres- Martinez unpublished data). The similarities found in the settlement of mussels in different collectors and depths suggest a relatively uniform presence of competent pediveligers and postlarvae in the study area. A trend similar to a major settlement in surface collectors was found by Fuentes and Molares (1994) and Molares and Fuentes (1995) in Ria de Arosa, Spain, but a contrary tendency was found by Cac- eres-Marti'nez and Figueras (19981 in Ria de Vigo. Spain. These results have been explained by the presence of the thermocline and by the behavior of larvae and postlarvae settlement (Fuentes and Molares 1994, Caceres-Martinez and Figueras 1998). The prefer- ence of mussels to settle in substrate has been widely studied (de Blok and Geelen 1958. Bohle 1971, Davies 1974. Dare et al. 1983. Eyster and Pechenik 1987. King et al. 1990. Caceres-Martinez et al. 1994). and it has been found that rugose and filamentous sub- strates are the best for settlement, because thev are related to the Ramirez and Caceres-Marti'nez 37 December n2=279 n5=63 100 80 60 40 20 January n5=531 III. February ioo n2=18 n5=171 J March n2=33 n5=22 June n2=14 n5=14 April n2=9 n5=9 .ll I July n2=5 n5=3 May n2=14 n5=4 August n2=38 □ D D □ □ September n2=77 D 0.26 0.471 0.9 1.6 2.6 0.47 0.899 1.S99 2.599 3.599 October n2=27 n5=30 100 n2=27 n5=2 so 60 I ■Qhu 0.25 0.471 0.9 1.6 2.6 >3.6 0.47 0.899 1.599 2.599 3.599 Size classes of mussels November n2=11 n5=29 .1 0.26 0.471 0.9 1.6 2.6 >3.6 0.47 0.899 1.599 2.599 3.599 Figure 5. Size distribution in percentage of mussels settled on collectors FPC placed at 2- (open bars) and 5-m (filled bars) depth, during a study period in Bahia de Todos Santos. Mexico. use of long contact mucous threads of competent pediveliger and postlarval stages that are easily jammed between filaments and rugosities (de Blok and Tan Mass 1977, Caceres-Marti'nez et al. 1994). However, comparison among efficiencies of filamentous substrates is difficult to do, not only because of the enormous surface variability of filamentous artificial substrates, but also be- cause these substrates are colonized immediately in the water by filamentous algae, hydroids, and debris that modify their surface. In laboratory studies where small pieces of substrates are used and environmental conditions are under control, image analysis has proved to be a useful tool to calculate the substrates surface area (Caceres-Martinez et al. 1994). However, this is hardly applicable to large collectors used in field studies. In these circumstances, a useful comparison among filamentous substrates is related to du- rability of collectors, handling, cost, nature of the study, and com- mercial use. Despite the fact that statistical analyses indicate that there were no differences among the spat recorded in the collectors studied, there was a clear trend of major settlement on commercial pads (SF). The observed tendency allows us to suggest the use of SF for scientific studies in the field or laboratory; however, their handling for commercial purposes is limited because of the diffi- culty of extracting the seed, in comparison with the use of fila- mentous ropes. Therefore. FP. FN. and FPC could be useful for commercial purposes. The use of L is limited because of its weak- ness for handling and its low durability in the sea. Although 92% of total spat was considered as competent pe- diveliger that arrived and grew on the collectors during the per- manence of the substrates underwater between the sample collec- tion, the presence of mussels larger than expected confirm the recruitment of at least a small proportion of postlarvae by disper- sion. However, it is important to mention than temperatures over 17°C and other environmental conditions may have a direct effect on mussel growth rates. Offshore scarcity of drifting mussels agrees with the fact that extension of postlarvae dispersion is more or less limited to the vicinity of mussel beds and high current areas (Newell et al. 1991, Caceres-Martinez et al. 1994, Caceres- Marti'nez and Figueras 1997, Caceres-Martinez and Figueras 1998). The presence of very large postlarvae ( 12.49 mm) supports the notion that the higher limit in the size of drifting postlarvae is around 10 mm (Beukema and Vlas 1989, Caceres-Martinez and Figueras 1997). ACKNOWLEDGMENTS The authors thank Oc. Sergio Guevara, from the Company Acuacultura Oceanica for allowing us to perform this study in their 38 December n2=478 n5=180 Ju 0> 60 CO » 40 E 20 **- O o 0) O) a) 100 u fe 8 ° EL March n2=41 n5=73 I. June n2=34 n5=24 XI September n2=90 n5=147 [L 0.26 0471 0.9 1.6 2.6 >3.6 0.47 899 1.699 2.699 3.699 Ramirez and Caceres-Marti'nez January n 2=1 581 nS=966 April n2=11 nS=2 nS=20 LL October n2=115 n5=91 26 0.471 0.9 1.6 2.6 >3 6 0.47 0.899 1.699 2 599 3.699 100 80 February n2=171 n6=123 May n2=1S nS=13 August n2=52 Dd November n2=74 n5=12 .i . 0.26 0.471 0.9 1.6 2.6 >3.6 0.47 899 1699 2.699 3.699 Size classes of mussels Figure 6. Size distribution in percentage of mussels settled on collectors SF placed at 2- (open barsl and 5-m (filled bars) depth, during a study period in Bahia de Todos Santos, Mexico. n2=913 n5=270 1L 0) 60 10 » 40 E 20 O o a> ra *- c 0> o c 0) 0L March n2=128 n6=43 January n6=936 III. 100 April n2=14 80 60 - I 1 40 20 100 , July n2=73 80 60 40 20 P P r- >3.6 0.26 0.471 0.9 1.6 2.6 O.t 7 0.899 1.5 99 2.699 3.599 February n2=66 n6=37 May n2=28 n5= 1, Size classes of mussels Figure 7. Size distribution in percentage of mussels settled on collectors L placed at 2- (open barsl and 5-m (filled bars) depth, during a study period in Bahia de Todos Santos, Mexico. Rami'rez and Caceres-Marti'nez 39 facilities, also Raul Silva. Hilario Cardona Sepulveda, and Victor Molina Armenta from the same company for their help and logistic support during field samplings. We also thank Rebeca Vasquez- Yeomans for her assistance in processing samples and M. C. Ig- nacio Mendez for statistical advice. This work was supported by the CICESE Project 623106. LITERATURE CITED Aguirre. M. P. 1979. Biologia del mejillon (M. eduli.s) de cultivo de la Ria de Vigo. Bol. Insr. Esp. Oceanogr. 5:107-160. Bayne. B. L. 1965. Growth and delay of metamorphosis of the larvae of Mytilus edulis (L). Ophelia 2:1-47. Beukema, J. J. & J. de Vlas. 1989. Tidal-current transport of thread-drifting postlarval juveniles of the bivalve Macoma balthica from the Wadden Sea to the North Sea. Mar. Ecol. Prog. Sen 52:193-200. B0hle. B. 1971. Settlement of mussel larvae Mytilus edulis on suspended collectors in Norwegian waters, pp. 53-69. In: D. J. Crisp led.). Pro- ceedings of the 4th European Marine Biological Symposium. Bangor. Cambridge University Press, London. Caceres-Marti'nez, J. 1997. Mussel fishery and culture in Baja California. Mexico: history, present status, and future. NOAA Tech. Rep. NMFS 128.41-55 pp. Caceres-Martines, J., J. A. F. Robledo & A. Figueras. 1993. Settlement of mussel Mytilus galloprovincialis on an exposed rocky shore in Ria de Vigo, NW Spain. Mar. Ecol. Prog. Ser. 93:195-198. Caceres-Martines, J., J. A. F. Robledo & A. Figueras. 1994. Settlement and postlarvae behavior of Mytilus galloprovincialis: field and laboratory experiments. Mar. Ecol. Prog. Ser. 112:107-1 17. Caceres-Marti'nez, J. & A. Figueras. 1997. Mussel (Mytilus galloprovin- cialis Lamarck) settlement in the Ria de Vigo (NW Spain) during a tidal cycle. J. Shellfish Res. 16:83-85. Caceres-Martines, J. & A. Figueras. 1998. Mussel (Mytilus galloprovin- cialis Lamarck) colonization on artificial substrates in the Ria de Vigo of NW Spain. J. Shellfish Res. 17:153-157. Couturier, C. 1994. Spawning in sea scallops. Placopecten magellanicus. Canadian Tech. Rep. on Fisheries and Aquatic Sciences 1994: Pro- ceedings of the 9th international pectinid workshop. Nanaimo. British Columbia. Canada. April 22-27. 1993. pp. 138-146. Curiel-Ramirez. S. & J. Caceres-Marti'nez. Reproductive cycle of coexist- ing mussel species, Mytilus galloprovincialis. Mytilus californianus. and Septifer bifurcatus in Baja California. NW Mexico (in preparation). Dare, P. J.. D. B. Edwards & G. Davies. 1983. Experimental collection and handling of spat mussels (Mytilus edulis L.) on ropes for intertidal cultivation. MAFF (Lowestoft) Fisheries Research Tech. Rept. 74:1- 23. Davies. G. 1974. A method for monitoring the spatfall of mussel (Mytilus edulis L.). ./. Cons. Int. Explor. Met: 36:27-34. de Blok, J. W. & H. J. Geelen. 1958. The substratum required for the settling of mussels (Mytilus edulis L.). Arch Neerl. Zool. Vol. Juhilaire 13:446-460. de Blok. J. W. & Tan Maas. 1977. Funtion of byssus threads in young postlarva Mytilus. Nature 267:558. Devauchelle. N. & Ch. Mingant. 1991. The conditioning of scallop spawn- ers: practical aspects. Aquaculture and the environment. Special Publ. Eur. Aquacult. Soc 14.. p. 89. Eyster. L. S. & A. J. Pechenik. 1987. Attachment of Mytilus edulis L. larvae on algal and byssal filaments is enhanced by water agitation. J. Exp. Mar. Biol. Ecol. 114:99-110. Ferran, A. E. 1991. Ciclo gonadal y del tejido de reserva del mejillon de las Rias de Galicia Mytilus galloprovincialis Lmk. Doctoral thesis, Uni- versidad de Santiago de Compostela, Spain. 229 pp. Fuentes, J. & J. Molares. 1994. Settlement of the mussel Mytilus gallo- provincialis on collectors suspended from raft in the Ria de Arousa (NW Spain); annual pattern and spatial variability. Aquaculture 122: 55-62. Harger. J. R. 1972. Variation and relative "niche" size in the sea mussel Mytilus edulis in association with Mytilus californianus. Veliger 14: 275-283. Hines. H. A. 1979. Effects of a thermal discharge on reproductive cycle in Mytilus edulis and Mytilus californianus (Mollusca. Bivalvia). Fish. Bull. 77:499-503. King, P. A., D. McGrath & E. M. Gosling. 1989. Reproduction and settle- ment of Mytilus edulis on an exposed rocky shore in Galway Bay. West Coast of Ireland. / Mar. Biol Ass. U.K. 69:355-365. King, P. A., D. McGrath & W. Britton. 1990. The use of artificial sub- strates in monitoring mussel (Mytilus edulis L.). Settlement on an ex- posed rocky shore in the west of Ireland. J. Mar. Biol. Ass. U.K. 70:371-380. Koehn. R. K. 1991. The genetics and taxonomy of species in the genus Mytilus. Aquaculture 94:125-146. Loosanof, V.L., C. H. Davies & P. E. Chanely. 1966. Dimensions and shapes of larvae of some marine bivalve mollusks. Malacologia 25. McDonald. J. H. & R. K. Koehn. 1988. The mussels Mytilus galloprovin- cialis and Mytilus trossulus on the Pacific coast of North America. Mar. Biol. 99:111-118. Molares. J. & J. Fuentes. 1995. Recruitment of the mussel Mytilus gallo- provincialis on collectors situated on the intertidal zone in the Ria de Arousa (NW Spain). Aquaculture 138:131-137. Newell. C. R., H. Hidu, B.J. McAlice. P. Podniesinski, F. Short & L. Kindblom. 1991. Recruitment and commercial seed procurement of the blue mussel Mytilus edulis in Maine. J. World Aquae. Soc. 22:134- 152. Rees, C. B. 1950. The identification and classification of lamellibrach lar- vae. Hull. Bull. 19:73-104. Rees, C. B. 1954. Continuous plankton records: the distribution of lamel- libranch larvae in the North Sea, 1950-51. Bull. Mar. Ecol. 4:21-46. Robinson, A. 1992. Gonadal cycle of Crassostrea gigas Kumamoto (Thun- berg) in Yaquina Bay. Oregon and optimum conditions for broodstock oysters and larval culture. Aquaculture 106:89-97. Seed, R. 1976. Ecology, pp. 13-65. In: B. L. Bayne (ed.). Marine Mussels: Their Ecology and Physiology. Cambridge University Press, Cam- bridge, UK. Seed, R. & T. H. Suchanek. 1992. Population and community ecology of Mytilus. pp. 87-157. In: E. Gosling (ed.). The Mussel Mytilus: Ecol- ogy, Physiology, Genetics, and Culture. Elsevier. Amsterdam. Suchanek. T. H. 1981. The role of disturbance in the evolution of life history strategies in the intertidal mussel Mytilus edulis and Mytilus californianus. Oecologia (Berl) 50:143-152. Villalba, A. 1995. Gametogenic cycle of cultured mussel, Mytilus gallo- provincialis. in the bays of Galicia (NW Spain). Aquaculture 130:269- 277. Widdows. J. 1991. Physiological ecology of mussel larvae. Aquaculture 94:147-163. Young, R. T. 1946. Spawning and setting season of the mussel, Mytilus californianus. Ecology 27:354-363. Journal of Shellfish Research, Vol. IS. No. 1, 41-46. 1999. INDUCTION OF SETTLEMENT AND METAMORPHOSIS OF THE SCALLOP ARGOPECTEN PURPURATUS LAMARCK BY EXCESS K + AND EPINEPHRINE: ENERGETIC COSTS G. MARTINEZ, 1 C. AGUILERA, 1 AND E. O. CAMPOS 2 1 Facultad de Ciencias del Mar, Universidad Catolica del Norte, Coquimbo, Chile Vnidad de Neurobiologia Molecular, Facultad de Ciencias Biologicas, Pontificia Universidad Catolica de Chile, Coquimbo, Chile ABSTRACT Settlement and metamorphosis of marine invertebrate larvae is known to be triggered by specific environmental cues. Neuroactive compounds, particularly some monoamines, have been implicated in this process, and depolarization of receptor cell membranes has been suggested to occur as a response to them. An increase of extracellular K + in seawater has been used as an effective inducer of these processes for some species. This study describes work designed to assay effects of epinephrine and excess K* as inducers of settlement and metamorphosis of larvae of the scallop Argopecten purpuratus. Epinephrine and excess K* increased the percentages of settlement, metamorphosis, and survival of these larvae. Responses were dose-dependent, with a maxima under 10" 5 M (epinephrine) and 10 mM (K + ). In the case of epinephrine, the responses did not vary significantly with the time of exposure. An analysis of size and energy content of larvae induced to metamorphosis by the different methods showed that larvae induced with epinephrine produced postlarvae that were significantly smaller in size and energetically weaker than postlarvae produced using excess K* or no added exogenous inducer. KEY WORDS: Argopecten purpuratus larvae, metamorphosis, scallops, settlement INTRODUCTION The scallop Argopecten purpuratus, as do many benthic marine invertebrates, produces pelagic larvae that spend days or weeks in the plankton before settling and metamorphosing into adult forms. Important physiological, morphological, and biochemical changes occur during the transition from pelagic to benthic existence. Settlement and metamorphosis processes are triggered by larval sensory recognition of, and responsiveness to, exogenous chemical and other environmental stimuli (Morse 1990). Various types of settlement-inducing cues have been described, including: (1) physical [e.g., illumination, physical texture (Hadfield and Pen- nington 1990), vibration (Rittschof et al. 1998)]; (2) biological [e.g., conspecific individuals, microbial films, prey species (re- viewed by Rodriguez et al. 1993)]; and (3) chemical [e.g., cues of natural or artificial origin (Yool et al. 1986)). Many of these chemical cues are compounds whose roles as neurotransmitters are broadly known (Rodriguez et al. 1993) although other chemically similar substances and some fatty acids have also been described to induce settlement and metamorphosis (Pawlik 1988, Kitamura et al. 1993). The ability of epinephrine to induce settlement and metamorphosis has been reported by Coon et al. ( 1985) (Crasso- strea gigas); Beiras and Widdows ( 1 995 ) ( C. gigas). Kingzett et al. (1990) (Patinopecten yessoensis), Tan and Wong (1995) (C. belcheri), Chevolot et al. 1991. and Nicolas et al. (1996) (Pecten maximus). It has been assumed that the inducers act on external cellular receptors somewhere on the larva (Hadfield and Pennington 1990). The rapidity and cascade of events in settlement and metamoiphic induction suggest the existence of a preformed larval nervous sys- tem capable of detecting a specific signal (Hadfield and Penning- ton 1990, Fenteany and Morse 1993). Although precompetent lar- vae may have receptors, these may be not sufficient to induce settlement behavior; attainment of competency may be determined by the accumulation of a threshold number of receptors (Barlow 1990). It is known that the response of receptor cells to an appropriate stimulus is the depolarization of specialized cells. On this basis, Baloun and Morse (1984) have suggested that the perception of inductive cues by larvae may rely on the stimulus-mediated depo- larization of cells in a sensory-inductive pathway. Given the knowledge that an increase of extracellular K + induces membrane depolarization, several authors have successfully assayed the ef- fects of an excess of this ion in seawater as an inducer of larval settlement and metamorphosis (Baloun and Morse 1984. Yool et al. 1986; Inestrosa et al. 1993a, Campos et al. 1994: Pechenick et al. 1995). The molecular events mediating the inducer-receptor interaction and the hypothesized membrane depolarization are not understood, but it has been suggested that a possible second mes- senger such as cAMP and IP, may participate (Leitz and Muller 1987, Morse 1990; Baxter and Morse 1992; Inestrosa et al. 1993b. Clare et al. 1995). These messengers might regulate ion channel activity (Wickman and Clapham 1995). Besides regulating mem- brane permeability to ions, these messengers are known to increase the activity of several catabolic enzymes through phosphorylation (Krebs 1985), a fact that must be considered when a compound is chosen for experimental induction of metamorphosis. Metamorphosis is an energetically costly process (Holland and Spencer 1973, Lucas et al. 1979). Storage reserves of energy-rich organic substrates are usually observed to increase before meta- morphosis (Lucas et al. 1986). Studies to this point have focused on variations in the composition of energy reserves and measure- ments of oxygen consumption during this process (Holland and Spencer 1973, Lucas et al. 1979, Rodriguez et al. 1990. Shilling et al. 1996). The present study evaluated the effects of excess K + and epi- nephrine as inducers of settlement and metamorphosis in the pec- tinid Argopecten purpuratus. Because epinephrine is a powerful catabolic stimulant, it was important to evaluate the comparative energy costs between individuals stimulated to metamorphosis by epinephrine and those stimulated by excess K + (plus nontreated 41 42 Martinez et al. controls). A null hypothesis was tested that there was no difference in energy cost to the larvae or postlarvae between treatments. The practical importance of this work is that there has been interest on the part of commercial scallop hatchery managers in obtaining reagents for the massive induction of metamorphosis in cultured scallop larvae destined for aquaculture growout. If a safe and reliable chemical inducer were available, it would significantly reduce production and labor costs and add reliability to this newly evolving industry. MATERIAL AND METHODS Assay of Larval Settlement, Metamorphosis, and Survival Larvae of A. purpuratus were obtained from a commercial scallop hatchery in Tongoy Bay, Chile (30°S). Larvae were mass cultured using methodology similar to that of DiSalvo et al. (1984) and transferred to our laboratory for experimentation at a stage when they were entering competence for metamorphosis (length near 200 u,m, with presence of eyespot and foot). Larvae were maintained in 0.45 p.m filtered seawater at 20°C and fed daily with mixture of the microalgae Isochrysis galbana (T-iso), Pavlova lutheri, Chaetoceros calcitrans, and Chaetoceros gracilis. Experimental configuration included the use of six 1-L plastic containers, each fitted with rippled plastic plates (ca. 150 cm 2 ) to increase the surface area for larval settlement. Each replica con- tained 800 mL of test water containing 1,500-1,600 larvae. Test solutions included 0, 5, 10, 20, and 30 mM K + ion. above the normal concentration in local seawater (9 mM); epinephrine con- centrations were 10", 10", and 10^* M, produced by dilution of a stock solution of the amine diluted in 0.0005 N HCI. Larvae were exposed to test solutions for 48 hours, after which, the free larvae were suspended into fresh, filtered seawater; settled and metamor- phosed larvae were quantified in three of the replicates. The ex- periment was then continued for 96 hours with the remaining replicates, with daily changes of filtered seawater and quantifica- tion of settled and metamorphosed larvae at required time inter- vals. Unattached, living larvae were also included in counts to account for all surviving individuals. Larvae were considered settled when swimming ceased, they had settled on container and plate surfaces and could not be detached by gentle washing with fresh seawater. Larvae were considered metamorphosed if the ve- lum had been resorbed and if they showed development of the ctenidium and deposition of dissoconch. Effect of Exposure Time A second set of experiments was carried out to determine how the time of exposure to excess K + or epinephrine could affect the response to these signals. Using the same experimental configu- ration described above, and the optimal inducer concentration then obtained, groups of competent larvae were exposed for periods of up to 96 h (excess K + ) or 48 h (epinephrine). At each time interval (see Fig. 3), larvae were rinsed and placed in fresh, filtered sea- water. After 144 h. metamorphosed, attached, and unattached or- ganisms that remained alive were counted to calculate final per- centages of survival and metamorphosis. Comparative Energetic Cost Between Inducers Energy depletion during metamorphosis induced by the two different treatments was determined by direct calorimetry of the test organisms. This measurement was carried out in triplicate for each treatment. For each replicate. 80.000 larvae were placed with filtered seawater in 10-L plastic containers, where they were ex- posed to either 10 mM K + or to 10" M epinephrine over a period of 24 h. After this, the medium was changed to filtered seawater and maintained with daily changes of water for 6 days, at which time most larvae seemed to have passed metamorphosis. Postlar- vae were then removed from the containers with a small paint- brush, and collected on 250-|xm mesh plastic screen. These organ- isms were washed with isotonic ammonium formate, dried to con- stant weight, made into pellets, and ignited in an OSK calorimeter. Sub samples were used to count the postlarvae and measure their lengths. Results were analyzed using one-way analysis of variance (ANOVA) and a Tukey test was applied to evaluate probable differences (p < .05) between treatments. In the case of percentage values, these were subjected to an arc-sin transformation for cal- culations. RESULTS Effects of A* Concentration After 48-h exposure to increased external K + , the percentage of larvae settled with 10 mM was statistically higher (Tukey, p < .001) than that of larvae incubated under normal K + (Fig. l.A); however, very few of the larvae had passed metamorphosis (Fig. LB), with no statistical difference between percentages of the different experimental groups at this time. At 144 h (96 h after removal of larvae from inductor solutions), almost half of the larvae from the 10 mM treatment had passed metamorphosis, with values for larvae from other concentrations either near or below those obtained with no treatment (Fig. 1 .B) The survival of larvae submitted to 10 mM excess K* was significantly higher than that of the other experimental groups (Fig. l.C). Effects of Epinephrine After 48 h in different concentrations of epinephrine, it was shown that the highest percentages of larval settlement were ob- tained with 10" and 10" M (Fig. 2. A). Although few larvae had metamorphosed after 48 h with epi- nephrine, significantly higher values were observed at 10" and 10" M (Fig. 2.B). On the following days, the number of larvae that metamorphosed increased, and at 144 h (48 h after removal of larvae from the inductor solutions), nearly all the settled larvae had passed metamorphosis, with the most effective concentrations hav- ing been 10" and 10" M (Fig. 2.B). Survival of larvae submitted to 10" and 10" M treatments was significantly higher than that of the other experimental groups (Fig. 2.C). Effect of Exposure Time When 10 mM excess K + was used to induce metamorphosis, the percentage of larvae that metamorphosed increased as the time of exposure increased, from 6 to 48 hours. No greater increase was detected when larvae were under this treatment for 96 hours (Fig. 3. A). In the case of larvae exposed to 10" M epinephrine, from 6 to 48 hours, the time of exposure to this amine did not affect either the percentage of mortality or that of metamorphosis of larvae (Fig. 3.B). Argopecten Purpuratus Metamorphosis by K + and Epinephrine 43 60 SO , . c p 40 d) *s CO 30 c 8 20 in a. 10 60 r"' CO SO CD a. (A 1 x: e- £ c 8 60 SO 40 30 20 10 60 50 40 -I, I- 90 c • m I > 70 y^^. e / X. S ^r ^< at t v^ ^S. c 50 — "^-^"^ x ^S. 8 """"""""^T ^T £ 40 3 CO 30 c 8 20 2L 10 10" 6 10* 10 -4 epinephrine concentration (M) ---• 48h 144 h Figure 2. Percentages of A. purpuratus larvae that settled (A), meta- morphosed (B), and survived (C) in different concentrations of epi- nephrine. Values represent the means ± SE of groups of larvae from three replicate treatments; (*) significantly different from the other values (Tukey's test, p < .05). Energetic Analysis of iMrvae Induced to Metamorphosis As it is seen in the Table 1, the energy content of A. purpuratus post larvae decreased to less than half the values they had previous to metamorphosis. In the case of larvae induced to metamorphosis by excess K + . the decrease in energy content was similar to that of the control group; when epinephrine was used as an inducer, the decrease was considerably greater. Although postlarvae showed considerably increased size over the larvae, no significant differ- ence was found between the groups of larvae induced to metamor- phosis by any of the three different treatments. DISCUSSION Raised levels of the K + ion have not previously been demon- strated as an inducer of metamorphosis in A. purpuratus larvae. The present results now add this species to a group of other mol- lusks that have been shown to be induced to settle and metamor- phose by this ion. An early study was that of Baloun and Morse (19S4). who showed this effect on larvae of Haliotis rufescens. They showed that the larval response to 7-aminobutyric acid (GABA) may be inhibited by a decrease in external K + . Yool et al. (1986) showed that an increase in the concentration of this ion induced settlement and metamorphosis in larvae of the mollusks 44 Martinez et al. 80 c 0) u 40 di Q. 20 A r" " c C ..-I b y /■-[■■ t i a _ 12 24 48 hours with excess K 96 80 « 60 c 8 V a 40 I-- I- 20 6 12 24 48 hours with epinephrine — ' — metamorphosis mortality Figure 3. Effect of exposure time to 10 mm excess K + (A) and to 10" 5 M epinephrine (B) on metamorphosis and survival of competent larvae of A. purpuratus. Values represent means ± SE of groups of larvae from three replicate treatments. Means with same letter are not sig- nificantly different (Tukey's test, p < .05). Phestilla sibogae, Haliotis rufescens, and Astraea undosa and in larvae of the marine annelid Phragmatopoma califomica. Positive results were also shown for the mollusk Concholepas concholepas by Inestrosa et al. (1993a). In each case (as now with A. Purpu- ratus), the effect of potassium has been dose dependent, although optimal inductive dose may vary among species. Present results also show that, as time of exposure to excess K + increases, up to 48 hours, the percentage of A. Purpuratus larvae that metamorphose also increases. In this respect, our results agree with those of Baloun and Morse (1984), Yool et al. (1986), and Inestrosa et al. ( 1993a). These authors showed that, not only dif- ferent doses of excess K + , but also cumulative time of exposure, produced a progressive increase in larval metamorphosis in the species cited above. The present results have shown that 10" 6 and 10" 5 M epineph- rine were the optimal concentrations for significantly increasing the percentage settlement and metamorphosis of A. purpuratus larvae; a higher dose (10~ 4 M) did not show any significant effect. TABLE 1. Size and energy content of Argopecten purpuratus larvae before and after metamorphosis induced by 10"' M epinephrine or by 10 mM excess potassium. Energy Content (mJoule/Larva) Larvae Length (urn) Before metamorphosis Competent larvae After metamorphosis Control Induced by epinephrine Induced by excess K + 4.0113 ±0.2665 1.6983 ±0.0516 0.5168 ±0.0368* 1.6477 ±0.108 216.47 ± 10.57 406.00 ± 33.68 364.17 ±26.51 399.55 ± 25.69 Values are mean ± SD (n = 3). * Significantly different from control (p < 0.001 ). These values do not agree with those described for other bivalve species, which are also induced by this monoamine. Kingzett et al. (1990) assayed three concentrations of epinephrine OCT 6 , 10 _s , and 1CT 4 M) on larvae of the scallop Patinopecten yessoensis and showed similar increases in percentage metamorphosis with each dose. In the case of the scallop Pecten maximus, epinephrine ex- hibited an optimal action between 0.5 to 2.5 x 10~ 4 M (Chevolot et al. 1991, Nicolas et al. 1996). Coon et al. 1985. Coon et al. 1986. and Beiras and Widdows (1995) have described maximum induc- tive activity in the oysters Crassostrea virginica and C. gigas with 10" 4 M epinephrine. Tan and Wong ( 1 995 ) described 10" 5 M as the best concentration of epinephrine to induce metamorphosis in the oyster. C. belcheri. Larvae of the polychaete Phragmatopoma lapi- dosa californica did not respond to epinephrine assayed at any subtoxic concentration. Thus, sensitivity to inducer compounds may vary from one species to another. Moreover, in the assays described for oysters, most of the metamorphosed individuals in- duced by epinephrine were "unattached" spat (Coon et al. 1985, Coon et al. 1986. Beiras and Widdows 1995). a fact that was not observed for pectinids. Pawlik (1990) has suggested that different routes to metamorphic activation may be involved in the responses to different cues by the species. It is apparent in the present data that metamorphosis is more rapidly triggered by the monoamine than by excess K + . When epinephrine was used as an inducer cue on A. purpuratus, a sig- nificant number of individuals metamorphosed within the first 48 hours. On the contrary, in that period, no larvae had metamor- phosed using excess K + . Pechenik and Gee ( 1993) and Pechenik et al. (1995) have shown that larvae of the gastropods Crepidula fornicata and Phestilla sibogae become responsive to excess K + at a different time than to "natural cues." suggesting possible differ- ent sites of action of the inducers. In the case of P. sibogae, larvae become responsive first to the natural cue before they do to excess K + ; the authors suggested that this ion acted internally rather than directly on surface receptors, showing increasing accessibility to those internal sites as larvae age. The assays of exposure time of A. purpuratus larvae to the different cues, agree with the preceding concept. It was shown that, as the time with excess K + increases, more larvae pass metamorphosis. Meanwhile, using epinephrine, the percentage of metamorphosis did not increase with exposure time and even was less than the maximum obtained using ex- cess K + . The use of exogenous inducers for metamorphosis has pres- Argopecten Purpuratus Metamorphosis by K + and Epinephrine 45 enlly been shown to increase the survival of A. purpuratus indi- viduals after metamorphosis. This may be of practical consequence for use in commercial production of hatchery "seed." However, in the case of individuals that had been induced to metamorphose by epinephrine, the energy content of postlarvae was significantly lower than that of individuals that had been treated with excess K + or had metamorphosed without any added inducer. Depolarization of externally accessible, excitable cells has been suggested to be a mechanism common to the induction of settle- ment and metamorphosis for a number of species (Baloun and Morse 1984. Yool et al. 1986). It is known that the mechanism of action of neurotransmitters, many of which have been reported to induce settlement and metamorphosis of larvae (Rodriguez et al. 1993), is through changing the membrane permeability, and then (Wickman and Clapham 1995) changing the degree of polarization of the cell. The mechanism by which some of these neurotrans- mitters change this permeability is unclear. These compounds may, through G proteins, increase the concentration of any second messenger (cAMP, IP,, Ca +2 ). triggering the phosphorylation of some key proteins (Krebs 1985. Ho 1994). Some of these proteins may be intrinsic to membranes playing a part or a total role as an ion channel, and therefore, its phosphorylation may be changing the corresponding ion conductance of the membrane (Wickman and Clapham 1995). In addition to membrane proteins, there are some enzymes that are phosphorylated, changing their activity ( Krebs 1985). Many of the enzymes whose activity is enhanced by phosphorylation are catabolic (e.g., glycogen phosphorylase, li- pases). Such a cascade of events, could explain how, when neu- rotransmitters are used to induce the metamorphosis of inverte- brate larvae, the normally high energy cost of the process may be further incremented, perhaps to the point of stress. Existence of a common mechanism for the inductive effects o\ potassium and neurotransmitters action is not probable. The effects of K + may be explained in terms of their ability to change mem- brane potential. It is well known that potassium gradients through cell membrane determine the degree of cellular polarization. This type of effect should not mobilize energy reserves of individuals over the degree expected in normal (noninduced) metamorphosis. In conclusion, we reject the hypothesis that there was no difference between treatments, having observed significantly greater energy depletion in organisms treated with epinephrine. In light of possible anomalous effects of membrane depolar- ization (K + ). or possible energy stress associated with neurotrans- mitters, it is not possible at this time to recommend the practical use of chemical inducers in scallop hatcheries, because the simple measurement of percentage of metamorphosis is not indicative of several other factors that may affect viability of the larvae. ACKNOWLEDGMENTS This research was supported by FONDECYT (project # 1960058) and by a Program "FONDAP de Oceanografia y Biolo- gfa Marina." We are grateful to Alejandro Abarca and to Pesquera San Jose Scallop Hatchery, from Tongoy, for their help and supply of larvae. We thank Dr. Louis DiSalvo for his help with the En- glish lansuaee. LITERATURE CITED Baloun, A.J. & D. E. Morse. 1984. Ionic control of metamorphosis in larval Haliotis rufescens (Gastropoda). Biol. Bull. 167:124-138. Barlow. L. A. 1990. Electrophysiological and behavioral responses of lar- vae of the red ahalone {Haliotis rufescens) to settlement-inducing sub- stances. Bull. Mar. Sci. 46:537-554. Baxter. G. T. & D. E. Morse. 1992. Cilia from abalone larvae contain a receptor-dependent G protein transduction system similar to that in mammals. Biol. Bull. 183:147-154. Beiras, R & J. Widdows. 1995. Induction of metamorphosis in larvae of the oyster Crassostrea gigas using neuroactive compounds. Mar. Biol. 123:327-334. Campos, E. O.. A. Pinto, A. Bustos, S. R. Rodriguez & N. C. Inestrosa. 1994. Metamorphosis of laboratory-reared larvae of Concholepas con- cholepas (Mollusca; Gastropoda). Aquaculture 126:299-303. Chevolot. L„ J. C. Cochard & J. C. Yvin. 1991. Chemical induction of larval metamorphosis of Pecten maximus with a note on the nature of naturally occurring triggering substances. Mar. Ecol. Prog. Ser. 74:83- 89. Clare, A. S.. R. F. Thomas & D. Rittschof. 1995. Evidence for the involve- ment of cyclic AMP in the pheromonal modulation of barnacle settle- ment. J. Exp. Biol. 148:655-664. Coon. S. L.. D. B. Bonar & R. M. Weiner. 1985. Induction of settlement and metamorphosis of the Pacific oyster, Crassostrea gigas (Thunberg) by L-DOPA and catecholamines. J. Exp. Mar. Biol. Ecol. 94:21 1-221. Coon, S. L.. D. B. Bonar & R. M. Weiner. 1986. Chemical production of cultchless oyster spat using epinephrine and norepinephrine. Aquacul- ture 58:255-262. DiSalvo, L. H., E. Alarcon. E. Martinez & E. Uribe. 1984. Progress in mass culture of Chlamys (Argopecten) purpurata Lamarck (1819) with notes on its natural history. Rev. Chil. Hist. Nat. 57:35-45. Fenteany. G. & D. E. Morse. 1993. Specific inhibitors of protein synthesis do not block RNA synthesis or settlement in larvae of a marine gas- tropod mollusk (Haliotis rufescens). Biol. Bull 184:6-14. Hadfield. M. G & J. T. Pennington. 1990. Nature of the metamorphic signal and its internal transduction in larvae of the nudibranch Pheslilla sibogae. Bull. Mar. Sci. 46:455—164. Ho Ren-Jye. 1994. Transmembrane signaling and animal evolution. Zoolog. Studies 33:1-28. Holland, D. L. & B. E. Spencer. 1973. Biochemical changes in ted and starved oysters, Ostrea edulis (L.) during larval development, meta- morphosis and early spat growth. J. Mar. Biol. Assoc. U.K. 53:287- 298. Inestrosa, N. C. M. Gonzalez & E. O. Campos. 1993a. Metamorphosis of Concholepas concholepas (Bruguiere 1789) induced by excess potas- sium. J. Shell. Res. 12:337-341. Inestrosa, N. C, M. Gonzalez & E. O. Campos. 1993b. Molecular changes induced by metamorphosis in larvae of the prosobranch Concholepas concholepas Bruguiere (Mollusca: Gastropoda; Muricidae). J. Exp. Mar. Biol. Ecol. 168:205-215. Kingzett. B. C. N. Bourne & K. Leask. 1990. Induction of metamorphosis of the Japanese scallop Patinopecten yessoensis Jay. J. Shellfish Res. 9:119-124. Kitamura. H.. S. Kitahara & H. B. Koh. 1993. The induction of larval settlement and metamorphosis of two sea urchins. Pseudocentrolus depresus and Anlhocidaris crassispina, by free fatty acids extracted from the coralline red alga Corallina pilulifera. Mar. Biol. 115:387- 392. Krebs, G. E. 1985. The phosphorylation of proteins: a major mechanism for biological regulation. Biochem. Soc. Trans. 13:813-820. Leitz. T. & W. A. Midler. 1987. Evidence of the involvement of Pl- signaling and diacylglicerol second messengers in the initiation of 46 Martinez et al. metamorphosis in the hydroid Hydractima echinata Fleming. Dew Biol. 121:82-89. Lucas, M. I.. G. Walker, D. L. Holland & D. J. Crisp. 1979. An energy budget for the free-swimming and metamorphosing larvae of Balanus balanoides (Crustacea:Cirripedia). Mar. Biol. 55:221-229. Lucas, A.. L. Chebab-Chalabi & P. Beninger. 1986. Variation of relative organic matter in Mytilus edulis L. larvae and postlarvae. J. Exp. Mar. Biol. Ecol. 95:99-103. Morse, D. E. 1990. Recent progress in larval settlement and metamorpho- sis: closing the gaps between molecular biology and ecology. Bull. Mar. Sci. 46:465^83. Nicolas. L., R. Robert & L. Chevolot. 1996. Effect of epinephrine and seawater turbulence on the metamorphosis of the great scallop. Aqua- cull. Int. 4:293-297. Pawlik, J. R. 1988. Larval settlement and metamorphosis of sabellanid polychaetes, with special reference to Phragmatopoma lapidosa, a reef-building species, and Sabellaria floridensis. a nongregarious spe- cies. Bull. Mar. Sci. 43:41-60. Pawlik. J. R. 1990. Natural and artificial induction of metamorphosis of Phragmatopoma lapidosa californica (Polychaeta: Sabellariidae). with a critical look at the effects of bioactive compounds on marine inver- tebrate larvae. Bull. Mar. Sci. 46:512-536. Pechemk. J. A. & C. C. Gee. 1993. Onset of metamorphic competence in larvae of the gastropod Crepidula fomicata (L), judged by a natural and an artificial cue. J. Exp. Mar. Biol. Ecol. 167:59-72. Pechemk. J. A.. M. G. Hadfield & L. S. Eyster. 1995. Assessing whether larvae of the opisthobranch gastropod Phestilla sibogae Bergh become responsive to three chemical cues at the same age. /. Exp. Mar. Biol. Ecol. 191:1-17. Rittschof, D„ R. B. Forward, Jr. G. Cannon. J. M. Welch. M. McClary. Jr. E. R. Holm. A. S. Clare. S. Conova. L. M. McKelvey, P. Bryan & C. L. Van Dover. 1998. Cues and content: larval responses to physical and chemical cues. Biofoulirtg 12:31-44. Rodriguez, J. L., F. J. Sedano, L. O. Garcia-Martin. A. Perez-Camacho & J. L. Sanchez. 1990. Energy metabolism of newly settled Ostrea edulis spat during metamorphosis. Mar. Biol. 106:109-1 1 1. Rodriguez, S. R.. F. P. Ojeda & N. C. Inestrosa. 1993. Settlement of benthic marine invertebrates. Mar. Ecol. Prog. Ser. 97:193-207. Shilling, F.M., O.Hoegh-Guldberg & D. T. Manahan. 1996. Sources of energy for increased metabolic demand during metamorphosis of the abalone Haliotis rufescens (Mollusca). Biol. Bull. 191:402-412. Tan. S. H. & Wong, T. M. 1995. Induction of settlement and metamor- phosis in the tropical oyster. Crassostrea belcheri (Sowerby). by neu- roactive compounds. J. Shellfish Res. 14:435^138. Wickman. K & D. E. Clapham. 1995. Ion channel regulation by G proteins. Physiolog. Rev. 75:865-885. Yool, A. J., S. M. Grau, M. G. Hadfield, R. A. Jensen. D. A. Markell & D. E. Morse. 1986. Excess potassium induces larval metamorphosis in four marine invertebrate species. Biol. Bull. 170:255-266. Journal of Shellfish Research. Vol. 18, No. 1, 47-58, 1999. EVIDENCE FOR FALL SPAWNING OF NORTHERN BAY SCALLOPS ARGOPECTEN IRRADIANS IRRADIANS (LAMARCK 1819) IN NEW YORK STEPHEN T. TETTELBACH, 1 CHRISTOPHER F. SMITH, 2 ROXANNA SMOLOWITZ/ KIM TETRAULT, 2 AND SANDRA DUMAIS 2 ^Natural Science Division Southampton College Long Island University Southampton, New York 11968 'Marine Program Cornell Cooperative Extension Riverhead, New York 11901 Marine Biological Laboratory Woods Hole, Massachusetts 02543 ABSTRACT Spawning of Argopecten irradians irradians is generally believed to occur between late May to August; however, some literature reports and anecdotal observations have suggested that npe individuals may be present well into the fall. This paper reports on evidence for fall spawning of bay scallops that we sampled from different populations in Long Island. New York, waters, in different years. At two sites, a spawning peak in September followed a discrete spawning peak in early summer (late June/early July). Scallops at one or more of four different sites were conclusively shown to spawn well into the fall (late September, October, or early November) during 3 different years: one in which a brown tide (Aureococcus anophagefferens) algal bloom occurred (1995) and during nonbrown tide years (1993, 1994). Our work, coupled with reports of other researchers, suggests that fall spawning of A. i. irradians in NW Atlantic waters does not seem to be uncommon and may be important in some populations during particular years. KEY WORDS: Argopecten, spawning. New York, reproduction, scallop, seasonality, brown tide INTRODUCTION Much of the basic biological knowledge of the northern bay scallop, Argopecten irradians irradians (Lamarck 1819), is based on the early work of Risser (1901) and Belding (1910). These authors determined that the typical lifespan of this subspecies is 18-22 months; however, some individuals may reach the age of 3 years (Marshall 1960. S. Tettelbach, pers. obs.). Bay scallops are hermaphroditic and generally are regarded as semelparous, al- though Belding ( 1910) estimated that 10-20% of a given year class may survive to spawn in 2 successive years if not removed by the fishery. Bricelj et al. (1987a) reported that perhaps 30% of caged scallops at one site in Long Island, NY survived to spawn during their second year. Spawning of Argopecten irradians irradians over its natural geographic range is generally believed to occur between late May to midAugust (see Table 1 ), as water temperatures are rising (Sas- try 1963). Spawning of northern bay scallops has been described by some authors as a single, catastrophic event; whereas, others report spawning over much of the summer (see Table 1), some- times with a distinct secondary or tertiary peak later in the period (MacFarlane 1991 ). A few authors have reported early September spawning in Massachusetts (Kelley and Sisson 1981, Taylor and Capuzzo 1983, Hampson and Capuzzo 1984). Kelley and Sisson (1981) surmised, on the basis of scallop seed sizes, that spawning occurred after September in Nantucket Island. MA. MacFarlane (1991) concluded, on the basis of gross observation, that scallops in Orleans, MA spawned into October during 1980. The belief is still widely held, however, that spawning does not occur past early September; hence, sampling in many studies has been terminated prior to the fall. This paper reports on evidence for fall spawning of Argopecten irradians irradians that we obtained through the analysis of tem- poral patterns of bay scallop reproduction in different populations in Long Island. NY waters, during different years. This work was prompted by anecdotal observations made by baymen and our personal observations of visually ripe scallops in the Peconic Bay system during fall months between 1992 to 1996. Histological analyses on archived bay scallop gonadal samples were performed to determine if there was any concrete evidence of spawning be- tween September to November during 1993 to 1995. The 1995 data also provided the opportunity to determine whether bay scal- lops were able to spawn during a bloom of brown tide, Aureococ- cus anophagefferens (Sieburth et al. 1988). MATERIALS AND METHODS Analyses of reproductive activity primarily focused on three groups of scallops transplanted from natural populations in eastern Long Island, NY during 1994 and 1995. During 1994. scallops were dredged from Northwest Harbor (NWH) on 25 April and 1 May and free-planted directly on the bottom (density = 9.6/m") to the south of Red Cedar Point (RCP) in Flanders Bay (Fig. 1 ). In 1995, scallops were dredged from Sag Harbor and either deployed in rafts (density = 80.7/m 2 ). on 2 May. in East Creek (EC). South Jamesport. NY or free-planted (density = 5/nr) on 4 May to the south of RCP (Fig. 1 ). These three groups of scallops were moni- tored as part of broader studies (Smith and Tettelbach 1996. Smith 47 4S Tettelbach et al. TABLE 1. Summary of previous studies assessing temporal patterns of reproduction in populations of northern bay scallops {Argopccten irradians irradians) in waters of the northeastern United States, in order of decreasing latitude. Peak Water Spawning Spawning Depth Temp. Means of Location Period Period! s) 16.5 <1910 GO. MO Belding 1910 Woods Hole, MA Mid-July to late Aug August 1965 Gl. H Sastry 1970 Narragansett Bay. Rl June to July Mid-June <1901 GO Risser 1901 Nantucket Hbr, MA Mid-June to early Sept Late June to early July 1980 GO, S. L. H Kelley & Sisson 1981 Niantic R.. CT Mid-June to late July Mid-June to July 19-28 1955 GO. S Marshall I960 Pocjuonock R.. CT Mid-June to August Mid-June to early July 0.3-0.8 17-24 1983,84 GO. S Tettelbach 1991 Lake Montauk. NY Early June to August Mid-June to early July 0.6-2.6 -17-23 1974 Gl. L Hickey 1977 Accabonac Hbr. NY 'late May to August Early June 0.6-2.3 -17-23 1974 GI. L Hickey 1977 Three Mile Hbr. NY Early June to July Mid-June 0.6-3.3 -17-23 1974 Gl, L Hickey 1977 Three Mile Hbr. NY Early June to Aug Early June 2 > 16-26 1984 Gl, GW Bncelj et al. 1987b Northwest Hbr. NY Early June to Aug Early to mid-June 2 £16-25 1984 GI. GW Bricelj et al. 1987b Northwest Hbr. NY Early June to Aug Early to late June 3.5 a 1 5-24 1984 GI. GW Bncelj et al. 1987b Sag Hbr. NY Late May to Aug Late May to mid-June 3.5 > 14.5-27 1984 GI. GW Bricelj et al. 1987b Flax Pond, NY Late June to mid-July Late June to mid-July 2 19-23 1985 GI, H Eppetal. 1988; Epp 1989 All analyses are based on samples taken from natural populations, except for Flax Pond. At the latter site, scallops were held in cages on the bottom, after being dredged from Northwest Harbor, New York. Means of Assessment: GO = gross observation; MO = microscopic observation; GW = gonad weight; GI = gonad index; H = histology, with oocyte measurement (Oo); L = larval abundance; S = spat abundance (partially adapted from Barber and Blake 1991). and Tettelbach 1997) intended to evaluate different reseeding tech- niques (see Tettelbach and Wenczel). Temporal changes in reproductive condition were evaluated by means of analyses of gonad dry weights (GDW) and gonad in- dexes (Gil (Barber and Blake 1991). as well as by histological examination (see below). For each individual, shell height was measured to the nearest mm, and then the gonad was dissected proximal to the foot (so the foot remained attached to the excised gonad). For the GDW and GI analyses, the gonad and remaining tissues were weighed separately for each individual after they were dried to a constant weight (>48 h) at ~82°C. A GI value was calculated for each individual, as follows: GI = Gonad Wt (g) x 100/[Gonad Wt + Remaining Tissue Wt (g)] (Barber and Blake 1991 1. Temporal changes in mean GDW and GI values were ex- amined to determine if spawning had occurred, as inferred from a significant drop in GDW or GI. At the times when scallops were LONG ISLAND SOUND Figure 1. Map of the Peconic Bay system in eastern Long Island, New York, USA, showing the location of bay scallop and Aureococcus sampling sites. Fall Spawning of Argopecten in New York 49 East Creek -1995 B 100°/c 57.0 57.3 53.8 (3.6) (3.2) (3.5) 57.9 51.4 55.2 (2.9) (3.3) (33) 53.2 67.3 70.8 (29) (4.6) (2.7) June 9 June 21 July 7 July 20 Aug 4 Aug 18 Sept 5 Oct 3 Nov 6 es Recovering |H Early Maturation m Mid-maturation ■ Ripe ■ Early-spawn g Mid-spawn 3 Late-spawn Very late-spawn Spent Figure 2. Temporal change in reproductive condition of bay scallops (male. A, and female. B, gonadal portions) deployed in rafts in East Creek, South Jamesport, New York during early May 1995. Percentage of individuals in a given gonadal stage were determined by histological analysis (n = 1 1-12 scallops per date) and scored using a modification of Naidu's ( 1970) method: stage 4 = recovering from spawning: stage 5 = early maturation of eggs and sperm; stage 6 = midmaturation of eggs and sperm: stage 7 = mature eggs and sperm (ready to spawn); stage 8 = spawning, where: 8e = early-spawn, (<25% of eggs or sperm were spawned); 8m = midspawn. (25-85% of eggs or sperm were spawned); 81 = late spawn, (85-95% of eggs or sperm were spawned); 8vl = very late spawn, (between 96-100% of eggs or sperm were spawned); stage 9 = spent (immediately postspawn). Bay scallop shell height data [mean, (SD)] are given at the top of the figure. transplanted from Northwest Harbor to RCP (29 April 1994) and from Sag Harbor to EC and RCP (2 May 1995). GI values (13.59 and 15.25. respectively) were marginally different (t = 2.14. p = .04, n = 20 and 25. respectively, for 1994 and 1995). During 1995, GDW and GI data (n = 23-25 scallops per sample) were collected biweekly between late May to midAugust (RCP) or early September (EC); histological sampling (n = 10-15 scallops per sample) started in early June, then followed the same schedule. Additional histological samples were collected monthly from early September through early October (at RCP) or Novem- ber (at EC). (On 21 July 1995, only, it was also necessary to include in the histological analysis scallops that were transplanted to RCP on 25 May.) During 1994. initial sampling of GDW and GI was done in late April; then GDW, GI, and histology samples were taken biweekly from around late May through late September. GDW/GI sample size was 13-20 scallops, except on 25 May 1994 (n = 8). For the 1994 histological analyses, sample size ranged from two (25 May only) to nine scallops, but was usually six. Eight scallop gonads sampled in 1993 from natural populations in NWH were also examined histologically to provide additional information on the possible occurrence of fall spawning. These samples were taken opportunistically (i.e.. tissues were only ar- chived from scallops that visually appeared to be very ripe (ovar- ian portion of gonad was bright orange with evident veins)) during October and November off Barcelona Neck and south of Alewife Creek, respectively (Fig. 1 ) and preserved in 70% ethanol. The methods employed for the fixation and preservation of scallop gonadal tissues for histological analyses (total n = 276) followed procedures described by Humason ( 1979). After fixation, gonadal tissues from each scallop were processed in paraffin. Six micron (6 p,m) sections were cut and stained with hematoxylin and eosin using standard histologic methods (Humason 1979). These cross sections (along the dorsoventral axis) were taken -1/3 of the way from the proximal and distal end of each gonad. The proximal and distal end sections contained predominantly male and female tubules, respectively. Gonadal developmental stages (see Figs. 2—1) were scored via 50 Tettelbach et al. Red Cedar Point - 1995 B 55.8 (4.1) 100% 52.9 (3.8) 51.3 53.9 455 47 6 47.8 60.3 (6.1) (6.1) (6.0) (5.1) (6.7) (3.6) JunelO June23 July5 July21 Aug3 Aug"! 7 Sept5 Oct3 Recovering Ripe Late-spawn Early Maturation | Early-spawn Very late-spawn Mid-maturation Mid-spawn Spent Figure 3. Temporal change in reproductive condition of bay scallops (male, A. and female, B. gonadal portions) free-planted to the south of Red Cedar Point in Flanders Bay, New York during early May 1995. Gonadal stages were determined by histological analysis In = 10-15 scallops per date) and scored using a modification of Naidu's (197(1) method: gonadal stages, scallop shell size data as given in Figure 2. traditional, subjective tissue evaluation methods performed hy a trained pathologist (RS), using a modification of Naidu's (1970) method. In the present study, stage 8 of Naidu (1970) was divided into four substages: 8e (early spawn) in which fewer than 25% of the eggs or sperm were spawned, 8m (midspawn) in which 25 to 85% of the eggs or sperm were spawned, 81 (late spawn) in which 85 to 95% of the eggs or sperm were spawned, and 8vl (very late spawn) in which between 96 to 100% of the eggs or sperm were spawned (see Fig. 5. A to D). The percentage of eggs or sperm spawned was subjectively determined by visual examination of the stained tissue section and was based on the amount of empty space within the mature tubules resulting from the loss of spawned ga- metes [in stage 7 ( = ripe), eggs and sperm are tightly packed within the gonadal tubules]. In cases where more than one stage was clearly evident in the male or female portion of the gonad from a given individual, partial designations were made (e.g.. 81/4). The degree of reproductive synchrony was assessed histologi- cally at two levels in this study: among scallops from the same sample group ( = interanimal) and within the same individuals ( = intra-animal). Synchronous interanimal maturation was identified when the male and female gonads of the animals examined varied little from the most common stage within that sample group (Fig. 2: 9 June female and male gonadal portions). Asynchronous inter- animal maturation was identified when numerous developmental stages were present in animals sampled at one given time (Fig. 2: 6 November female and male gonadal portions). Synchronous in- tra-animal maturation was most obvious in tubules of female go- nads that were in stages 6, 7, and 8. For example, normal synchro- nous maturation in stage 7 (Fig. 6A) was characterized by centrally located mature or submature eggs occupying >85% of the tubule lumen surrounded by lesser numbers of mid- to small-sized eggs lining the tubules (Naidu 1970). Asynchronous intra-animal matu- ration in stages 6. 7. and 8e female gonads was characterized by variable numbers of mature (70-85%) and submature eggs cen- trally located in tubules associated with increased numbers of im- mature eggs ranging from mid- to small sizes in outer layers of the tubules (Fig. 6B). Asynchronous intra-animal developmental maturation in male gonads was defined by alternating areas, within the same tubule or different tubules, of tufts at primarily later stages of sperm development (e.g., stage 8) interspersed with tufts at an earlier stage of development (e.g.. stage 6). Water temperature was monitored through the course of the field sampling during 1993 to 1995. A continuous temperature recorder (Ryan RTM2000). deployed at the bottom [-3 m at mean low water (MLW)|. was employed at the RCP site from May to Fall Spawning of Argopecten in New York Red Cedar Point - 1994 51 56.0 52.8 53.7 (1.4) (3.3) (1.2) 58.3 56.6 57.3 59.5 58.4 65.8 (3.2) (3.0) (2.1) (4.5) (2.8) (1.3) B R»™ + ^ri + jmmiL, ™ | I 1 I l H May 25 June 9 June 23 July 8 July 21 Aug 3 Aug 26 Sept 16 Sept 30 Recovering I Ripe Late-spawn | Early Maturation | | Early-spawn Very late-spawn Mid-maturation | Mid-spawn Spent Figure 4. Temporal change in reproductive condition of bay scallops (male, A. and female, B. gonadal portions) free-planted to the south of Red Cedar Point in Flanders Bay, New York during late April 1994. Gonadal stages were determined by histological analysis (n = 2 scallops on 25 May and 4-9 individuals on other dates) and scored using a modification of Naidu's ( 1970) method: gonadal stages, scallop shell size data as given in Figure 2. September 1994. In 1995, continuous temperature recorders (On- set Stowaway® Model #1405) were attached to midwater nets (depth = 1-2 m at MLW) at EC and RCP (May-September) and to one of the rafts (depth = 0.15 m at MLW) at EC (May- October). Readings were taken every 0.5 h. Continuous tempera- ture recorders were calibrated against a NITS standardized ther- mometer to within 0.1°C. Water temperature readings at other times and sites were taken in situ with a hand-held thermometer. Salinity samples were taken periodically during 1994 and 1995 and analyzed in the laboratory with a Beckman induction salinom- eter. Between 12 April and 6 October 1994. surface salinity ranged from 24.06-28.79 ppt at a site in central Flanders Bay [Sta. #170 of the Suffolk County Dept. of Health Services (SCDHS)] (see Fig. 1). -2200 m from our RCP site. During 1995. surface salinity at our EC site ranged from 28.21-29.99 ppt between 9 April and 5 September; surface salinity at our RCP site ranged from 27.20- 29.26 ppt between 10 June and 5 September. Cell concentrations of Aureococcus anophagefferens (Siehurth et al. 1988) were monitored throughout 1993 to 1995 by SCDHS. Sampling was done on a biweekly basis at East Creek (Sta. #101 ), s 100 m from our EC site, and weekly at Sta. #170 (see Fig. 1 ). For Flanders Bay, parameters of the Tetra Tech ( 1997) hydrodynamie model of the Peconic Bay system were used to calculate the av- erage transit time for tidal movement of surface waters from SCDHS Sta. #170 to our RCP site. At an averaged residual veloc- ity of 1.14 cm s _I , this transit time was calculated to be 69.1 h. (Tettelbach, unpubl. data). Using a maximum doubling time of 0.8 day -1 ( = every 30 h) for Aureococcus anophagefferens (based on laboratory studies by Cosper et al. 1989. Gobler 1995), cell con- centrations in Sta. #170 and our RCP site would be expected to vary by a factor of no more than 2.3. For 1993, biweekly Aureo- coccus cell counts were available for SCDHS Sta. #1 18. -1.5 km from our sampling sites in Northwest Harbor. RESULTS Histological analyses revealed that initial spawning of the RCP scallops in 1994 and of the RCP and EC scallops in 1995 occurred between late June and early July (Figs. 2^4); most scallops were spent (immediate postspawn) by the time of the late (20 and 21) July sampling dates. In 1994, the RCP free-plant group had just begun to spawn by 8 July, at which time 17 and 50% of the male and female gonads, respectively, were in early spawning condition (stage 8e). In 1995, by contrast, 100% of the RCP free-plants were 52 Tettelbach et al. wmM^ Figure 5. Photomicrographs of four spawning substages of female gonads from the bay scallop {Argopecten irradians irradians ) ( 25x ); A. early spawning (8e). only a few eggs are missing from the central lumens of the tubules: B. midspawning (8m), large numbers of eggs are missing from the tubules; C. late spawning (81), most eggs are missing from the tubular lumens; D. very late spawning (8vl), only rare eggs remain within the tubular lumens. past early spawning condition (stages 8m-9) on 5 July. Fifty-nine and 839}- of the male and female gonads, respectively, from EC raft scallops were in mid to late spawning condition (stages 8m-81) by 7 July 1995 (Figs. 2-4). Spawning continued after July in all three groups of scallops, but additional peaks of spawning activity occurred at different times. In both the 1994 RCP free-plants and the 1995 EC raft scallops, histological analyses revealed that gonadal maturation proceeded steadily from late July until mid to late August, and then a second spawning peak occurred between late August through late September to early October (Figs. 2. 4). In the 1995 RCP free- planted scallops, however, a second period of spawning was al- ready in progress by early August (Fig. 3). Spawning of these animals continued through early October, although a dramatic- spawning peak was not obvious in this group. The times of spawning suggested by the GI/GDW analyses, where they overlapped with histological analyses, agreed with the above results, except for the respective periods of spawning ini- tiation in the 1994 and 1995 RCP free-planted scallops. In the 1994 RCP group. GI/GDW trends (see Fig. 14) suggested that spawning commenced between late May and early June, rather than between late June to early July. In the 1995 RCP group, GI/GDW trends (see Fig. 13) showed a more gradual decline from peak values in late May. An analysis of variance (ANOVA) of differences in GI of RCP scallops between late May to early July 1995 (F = 104.65. 96 df, p < .0001 ) and subsequent Bonferroni multiple comparisons showed that there was no significant difference between GI values on 27 May and 10 June, but these GI values were both significantly greater than those on 23 June (p < .001). which were, in turn, significantly greater than those on 5 July (p < .001). These GI analyses suggest that spawning of the RCP free-planted scallops in 1995 commenced between 10 and 23 June rather than between 23 June and 5 July, as suggested by the histological analyses. Possible reasons for these apparent disagreements are discussed below. For the most part, inter- and intra-animal gonadal development stages were fairly comparable among the male and female portions of scallops sampled from a given site at a given time. Develop- mental stages of male and female gonadal portions from the same animal were almost always within one developmental stage of one another. The interanimal range of designated histological stages never exceeded two to three adjacent developmental stages for a given sample group at a given sampling period. Interanimal asynchrony was evident in the female and male gonads of scallops at EC and RCP following the end of the first major spawning in late July, as compared to the period between late May to early June. Interanimal asynchrony was more pro- nounced in 1995 than in 1994, particularly at RCP, where it was first noted in free-planted scallops sampled on 3 August 1995. In East Creek rafts, interanimal asynchrony (first noted on 7 July 1995) was not as severe. Interanimal asynchrony became common in both groups by August and September of 1995 (Figs. 2, 3), then began to decline. Interanimal asynchrony also was observed in the 1994 RCP samples, but was mild as compared to that seen in 1995 samples. Intra-animal asynchrony was also evident in female gonads sampled from the three groups described above. In 1995, it tended to become more apparent during midsummer to early fall (7 July-5 September) at both sites, but was most pronounced in the East Creek raft scallops. Intra-animal developmental asynchrony was noted in the 1994 samples during the second spawn, but was mild in comparison to that seen in both groups sampled in 1995. Spawn- ing of animals with marked intra-animal developmental asyn- chrony showed evidence of retention of several undeveloped eggs in the tubules at late (stage 81) spawning (Fig. 7). Interestingly, in Fall Spawning of Argopecten in New York 53 Figure 6. Photomicrograph of a bay scallop"s female gonadal tubules (50x). A. In this stage (7|. gonad tubules are filled with mature eggs. Rarely, eggs of smaller sizes are noted at the edges of the tubules. B. These female gonadal tubules are in stage 7/8e. but. in addition to the mature eggs, the tubules also contain many immature eggs (arrows). samples taken at both sites in September to October 1995. female gonads in stage 81 (late spawning condition) or 8vl (very late spawning condition) also often showed early proliferation of the germinal epithelium with small developing eggs (stage 4) (Fig. 8). Male gonadal tubules did not exhibit detectable intra-animal asynchrony. Male gonadal tubules from animals sampled in late spring rarely were observed to empty completely, as described by Naidu (1970). However, male gonads of scallops sampled in late summer, which were staged as 81 and 8vl. often showed active, mature, sperm-producing germinal epithelium that was sometimes only a few cells thick. Rarely, intra-animal developmental asyn- chrony was identified in these animals by the appearance of tufts of less differentiated spermatogenic cells interspersed with sper- matocytes and spermatids within the more mature (although greatly thinned) epithelium (Fig. 9). Postspawn or very late spawn intratubular invasion by hemocytes of the female and male gonadal tubules was rarely noted and was minimal when seen. Intratubular inflammation usu- ally consisted of a few hemocytes (usually between two and 20 cells) associated with degenerating retained eggs (Fig. 10). In cross sections of a single male gonad, approximately five closely asso- ciated tubules contained numerous hemocytes. resulting in signifi- cant inflammation that filled and slightly extended the walls of the tubules. The cause of inflammation in this male gonad was not apparent. Interestinglv. in one male and one female sonad from two different animals, rare foci consisting of tumorous proliferations formed cell mounds that projected into the tubular lumen from the germinal epithelium (Fig. 11). No tumorous cells were noted in- vading through the basement membranes. In both cases, the cells were undifferentiated. -10 pjn in diameter with a high nuclear/ cytoplasmic ratio and mitoses of 1/higher power field. Water temperatures recorded at the EC rafts were slightly higher than those recorded at the lantern nets at RCP; however, temporal trends were very similar in 1995 (see Figs. 12, 13). At RCP. peak temperatures for the 2 years of study were 29.4°C (on 3 August 1995) and 28.6°C (on 9 July 1994). More significantly, perhaps, there was a sharp drop in water temperature just before spawning seemed to commence during both years (from -26 to 20.5°C between 18-21 June 1995 and from -27 to 23°C between 19-22 June 1994) and then a more or less steady rise in tempera- ture after that (to ~27.4°C by 14 July 1995 and to ~28.6°C on 9 July 1994). Concentrations of Aureococcus anophagefferens were 2-10 5 cells mL" 1 from 20 June through at least 18 July 1995 at East Creek and near Red Cedar Point (Figs. 12. 13). with respective recorded peaks at these two sites of 9 x 10 5 and 1.2 x 10 6 cells mL" 1 on 3 July 1995 (SCDHS 1995). Thus, commencement of spawning at both sites coincided with the time of rising Aureo- coccus concentrations, before peak bloom conditions. The second spaw ning peak exhibited by EC raft scallops, which started in early September 1995. also coincided with rising Aureococcus concen- trations before the second brown tide peak (3.8 x 10 5 cells mL -1 ) on 12 September 1995. In 1994. Aureococcus concentrations at these sites did not exceed 1.5 x I0 3 cells mL -1 at any time (SCDHS 1994). At the time the last histological samples were taken in late September to early October or early November, all three of the above groups of scallops included individuals that were still in spawning condition (Figs. 2—1). This was not a rare phenom- enon, because about one-third of the EC raft scallops sampled on 6 November 1995 were still in spawning condition (either wholly or in part), while other individuals were ripe or in midmaturation (stage 6). Direct evidence of spawning was obtained on the after- noon of 3 October 1995, when East Creek raft scallops were seen to be spawning in situ. Water temperature on this day ranged from 17.0°C (at -0530 h) to I9.9°C (at -1300 h). Gametes collected from a few individuals yielded viable trocophore larvae by the next day. Additional evidence of fall spawning was provided by means of histological analyses of scallops that had been sampled from two different natural populations in NWH during 1993; GL/GDW data were not collected at that time. Of the six individuals sampled off Barcelona Neck on 14 October, all of the female gonadal portions and five of six male portions were in early to late spawning con- dition (stages 8e-81). The sixth male gonad was ripe (stage 7). One of two individuals sampled from south of Alewife Creek on 7 November was in midspawning condition (stage 8m). with the female gonadal portion also showing some sections that were re- covering from spawning (stage 4). The second individual was ripe (stage 7). but had not begun spawning. Only these eight individu- als had been selectively archived out of larger samples of scallops, because they visually appeared to be very ripe (ovarian portion of gonad was bright orange with evident veins). Thus, we can con- clude that the minimum proportions of scallops in the process of spawning at NWH and Barcelona Neck on 14 October and 7 November 1993 were 6/49 (12.2%) and 1/88 (1.1%). respectively. 54 Tettelbach et al. 9W W$Mit mm Figure 7. Photomicrographs of a bay scallop's female gonad in stage 8m/l sampled in the late summer. Most mature eggs have been spawned; however, numerous immature eggs of various sizes remain within the tubules (arrows) (25x) (5 September 1995. East Creek rafts). Figure 8. Photomicrographs of a bay scallop's female gonad in stage Svl. Although only few mature eggs remain in the tubules, early regeneration of the tubule epithelium has already begun (arrows) (25x) (3 October 1995, East Creek rafts). Figure 9. Photomicrograph of a bay scallop's male gonad in stage 8vl. (1) Foci of maturing spermatids alternate with (2) some foci of spermatocyte proliferation (25x) (7 July 1995, East Creek rafts). Figure 10. Photomicrograph of a bay scallop's female gonad in stage 81. Hemocytes surround and engulf portions of eggs remaining within the tubular lumens (1: hemocytes; 2: eggs) (lOOx) (7 July 1995, East Creek rafts). Figure 11. Photomicrographs of a bay scallop's female gonad. A. Nodules of tumor cells project into the tubular lumens ( lOOx). B. Mitotic figures are identified within the mass (arrow) (250x) (14 October 1993, Barcelona Neck). Water temperature on the latter date was 10.0°C. During 1993, Aureococcus coneentrations never exceeded 1.1 x 10 3 cells mL _1 in Northwest Harbor (SCDHS 1993). DISCUSSION Temporal patterns of bay scallop reproductive condition re- vealed by our histological analyses and GI/GDW monitoring agreed in many, but not all, instances in this study. Histological analyses may simply have missed the late May to early June 1994 spawning that was suggested by the Gl/GDW analyses of the RCP free-plants because of the small sample size (n = 2) on 25 May. However, we might then have expected that some evidence of postspawning recovery would show up in the next group of samples (n = 6) on 9 June 1994. It did not. Further work is needed to elucidate the apparent disagreement between the two methods: however, because histology is considered the most definitive way to assess reproductive condition in scallops (Barber and Blake 1991). we base the ensuing discussion on these results. Two clear spawning peaks (in late June to July and late August to September) were evident in the 1994 RCP free-plants and the 1995 EC raft scallops; whereas, spawning of the 1995 RCP free- plants showed one distinct peak (late June to early July) followed by a prolonged and less dramatic period of spawning. The latter group also showed the least synchronous pattern of reproductive development following the end of the first spawn in late July. The mean shell size of the 1995 RCP free-planted scallops was con- siderably smaller, several weeks after deployment, than the 1994 Fall Spawning of Arcopecten in New York 55 EAST CREEK -1995 RED CEDAR POINT -1995 J 1200- '■= 1000- u> 800 - O 600- • • g 400- 8 200- 5 i: 0- — ■«- • I ■ • (*- • • — I P-* — I — -*-* — 70 60 .s> X 150 V) 40 ' ( |fHH n . Gonad Index Gonad Wt Total Body Wt Figure 12. Temporal changes in water quality parameters iAureococ- cus cell concentrations, water temperature), and size (shell height) and reproductive condition (gonad index, gonad dry weight, total body weight) of bay scallops deployed in floating rafts at East Creek, South Jamesport, New York in early May 1995. Initial values were obtained from a sample of scallops collected from Sag Harbor at the time when transplants were initiated. Datapoints for scallops are mean values ± 1 SD; n = 24-25 scallops per sampling date. RCP free-plants and the 1995 EC raft scallops (see Figs. 2-4, 12-14) (this may have been a sampling artifact, but the reason for the apparent decline in size of the surviving 1995 RCP scallops is unclear). Dry tissue weights are highly correlated with shell size in A. i. irradians (Epp et al. 1988). however, the exhibited magnitude of size differences for this group is not expected to have affected temporal spawning patterns: bay scallops of three groups ("large" natural, "small" natural, and hatchery-reared animals with respec- tive mean sizes of 53.4, 37.6, and 32.6 mm at the time of deploy- ment in pearl nets at the same site in Hallock Bay. NY during 60 50 40 Hi — i — i — >— ( — >- -•- — i — ^ 1 — i — i — i — i — i — - m - Gonad Index Gonad Wt Total Body Wt Figure li. Temporal changes in water quality parameters {Aureococ- cus cell concentrations, water temperature), and size (shell height) and reproductive condition (gonad index, gonad dry weight, total body weight) of bay scallops free-planted south of Red Cedar Point in Flanders Bay, New York in early May 1995. Initial values were ob- tained from a sample of scallops collected from Sag Harbor at the time when transplants were initiated. Datapoints for scallops are mean val- ues ± 1 SD; n = 23—25 scallops per sampling date. spring 1994) showed nearly identical temporal patterns of repro- duction as shown by changes in GI and GDW (Smith and Tettel- bach 1996). Several authors have suggested that when environmental con- ditions are less than favorable for synchronous spawning of scal- lops, "dribble" spawning may help to ensure that some larvae are able to survive (Langton et al. 1987, Paulet et al. 1988). The different patterns of inter- and intra-animal developmental asyn- chrony within scallop groups we observed in this study may sim- ply fall within the range of normal variability between different populations (see Bricelj et al. 1987b) and different years. However. 56 Tettelbach et al. RED CEDAR POINT -1994 -m- Gonad Index Gonad Wl Total Body Wt Figure 14. Temporal changes in water temperature, and size (shell height) and reproductive condition (gonad index, gonad dry weight, total body weight) of bay scallops free-planted south of Red Cedar Point in Flanders Bay, New York in late April 1994. Initial values were obtained from a sample of scallops collected from Northwest Harbor at the time when transplants were initiated. Datapoints are mean val- ues ± 1 SD; n = 8 scallops on 25 May and 13-2(1 individuals on other sampling dates. the differences in reproductive patterns exhibited by the 1995 RCP free-planted scallops warrant further examination of the possible effects of the brown tide (vs. nonbrown tide years) and depth in the water column (i.e.. surface rafts vs. on-bottom). Our finding that scallops at EC and RCP first spawned during the period of rising Aureococcus concentrations, before the peak of the brown tide bloom in 1995, is of particular interest because: ( 1 ) it demonstrates, for the first time, that bay scallops definitely spawned during a brown tide algal bloom (this seems to be coin- cidental rather than to have been a causative factor in spawning); and (2) it helps to answer the question posed by Bricelj et al. (1987b) and Bricelj and Kuenstner (1989) as to whether a temporal decline in scallop gonad weights or gonad indexes during a brown tide bloom represents actual spawning or gamete resorption. If gamete resorption had occurred, it is expected that extensive in- filtration of the gonadal tubules by hemocytes and the presence of numerous degenerative eggs within the tubules would have been apparent. However, only mild inflammation was noted in tubules and then only in association with a few degenerative eggs. This low level of inflammation was noted in scallops sampled during both 1994 and 1995 and thus probably represents a low level of atresia of unripened, but normal, eggs, as occurs in most other types of animals (Coe and Turner 1938, Van der Kraak et al. 1998). The occurrence of tumorous proliferations of cells of gonadal epithelial origin in gonads from animals that have actively pro- duced gametes for 3 to 4 months may parallel the phenomena of hormonally stimulated tumors as seen in other animals (Jubb et al. 1985) and at least partially result from repetitive gonadal cycling. Laboratory studies have shown that 3- to 10-day old bay scal- lop larvae experienced reduced growth and elevated mortality when exposed to Aureococcus concentrations of > 1.8 x 10 5 cells mL" 1 (Gallager et al. 1989); thus, it seems unlikely that spawning of scallops during brown tide bloom conditions in 1995 resulted in successful recruitment of scallop larvae. This was borne out by the virtual absence of scallop seed in any part of the Peconic Bays during the fall of that year: the commercial harvest of adult (1+ v) bay scallops in 1996 was among the poorest in New York over the last 50 years. Histological analyses conducted in the present study conclu- sively demonstrated that spawning occurred at least through late September to early October in the 1994 and 1995 RCP free-plants and into November during 1993 and 1995 for the NWH and EC scallops, respectively. These are the latest spawning dates yet re- ported for Argopecten irradians irradians at this latitude. Based on the data we have presented here and prior reports from other locations (see Kelley and Sisson 1981, MacFarlane 1991) we believe that fall spawning of northern bay scallops is not an unusual phenomenon. Fall spawning probably has been missed in other studies, because reproductive sampling is usually termi- nated before the end of the summer, although Bricelj et al. (1987b) did conduct GI analyses through October 1984 in Northwest Har- bor, NY and found no evidence of a fall spawn. Research on other pectinid species has demonstrated the occurrence of "late" spawn- ing; for example, Placopecten magellanicus (Mac Donald and Thompson 1988) and Argopecten irradians concentricus (Bologna 1998). In the latter species, reproduction may occur throughout the year in St. Joseph Bay, FL (Bologna 1998). We do not yet know the full significance of fall spawning in Argopecten irradians irradians, especially given the reduced fer- tilization success and recruitment that may result when broodstock densities are low and/or when spawning individuals are separated by some critical distance (Levitan et al. 1992, Peterson and Sum- merson 1992). Virtually nothing is known about the latter phe- nomena in Argopecten irradians irradians. The problem of ensur- ing successful fertilization later in the fall is also likely to be exacerbated because of a reduced proportion of spawning indi- viduals in the population. Even if bay scallop spawning occurred throughout the Peconic Bay system after the brown tide subsided in 1995, the very low adult population size relative to 1994 and other years (based on reported commercial bay scallop landings by the NY State Dept. of Environmental Conservation) suggests that potential recruitment resulting from the 1995 fall spawn was prob- ably unimportant. Nevertheless, the occurrence of unusually small Argopecten irradians irradians seed at the end of some fall growing seasons or of adults with growth rings very close to the hinge (Kelley and Sisson 1981, MacFarlane 1991. Tettelbach et al. 1994) suggests that "late" spawning may be important to some bay scallop popu- lations in certain years. Tettelbach et al. (1994) found that during the winter of 1990 to 1991. following a nonbrown tide year, the Fall Spawning of Argopecten in New York 57 percentage of "small" (S20 mm) seed ranged from 0-9% in eight different bay scallop populations in eastern Long Island. In 1992, 1 year after a brown tide bloom, 100% (n = 268) of the adult scallops sampled at one of the same sites (south of Alewife Creek in NWH) had growth rings that were 2-7 mm from the hinge, indicating that the adults had only reached this size, as seed, at the end of their first growing season. These small seed may have resulted from late spawning, an extended larval period and/or slow growth following larval settlement, but our present findings lend further credence to the possibility that these small seed resulted from a fall spawn. In that case, fall spawning clearly may be essential to the persistence of certain populations during some years. Larvae of A. i. irradians have been shown to exhibit >90% survival, 8 days after fertilization in the laboratory, at a tempera- ture of 10°C and salinity of 25-30 ppt (Tettelbach and Rhodes 1981 ); thus, larval survival is probable well into November in local waters. However, although larval growth also occurred in these laboratory conditions, it was an order of magnitude slower than at 25°C (Tettelbach and Rhodes 1981 ). Our histological results suggest that eggs were not routinely resorbed during the spring to fall months when samples were taken, but continued to mature until they were spawned. The en- vironmental stimuli that induce spawning of Argopecten irradians irradians in the fall, however, are presently unknown. Barber and Blake (1991) have suggested that there does not seem to be any critical temperature, per se, at which scallop reproduction occurs and. furthermore, that a rapid temperature change (AT) is probably a more important spawning trigger than an absolute temperature or the direction of chan«e. Direct disturbance of the raft during the process of sampling on 3 October 1995 may possibly have trig- gered spawning, but the fact that water temperature at the EC rafts did spike to over 22°C at the end of September 1 995 and rose from 17 to 19.9'C on 3 October, just before the time when spawning was observed in situ in East Creek rafts suggests that these con- ditions may have provided the appropriate stimuli for spawning. Further work is necessary to elucidate the mechanisms that trigger fall spawning of northern bay scallops and the importance of this phenomenon in bay scallop populations. ACKNOWLEDGMENTS Many thanks to Ed Decort of Southampton College and Chris Pickerell, Gregg Rivara, and Mark Cappellino of Cornell Coop- erative Extension for assistance with field and lab work, and to Southampton College for a Faculty Research Release Time award (to STT). We thank the personnel of the Bureau of Shellfisheries, New York State Department of Environmental Conservation for their cooperation and assistance, and Dr. Bob Nuzzi, Vito Minei. and Mac Waters of the Suffolk County Department of Health Services. Office of Ecology, for Aureococcus and salinity data. We also thank the Town of Riverhead Shellfish Program for use of rafts at East Creek. We gratefully acknowledge funding for this work by the National Marine Fisheries Service, the Environmental Protection Agency's Near Coastal Waters Program, and the New York Sea Grant Institute. In particular, we thank Cornelia Schlenk of NYSGI, Jon Gorin, Rick Balla, and Felix LoCicero of USEPA, and Vito Minei and Walt Dywidiak of the Peconic Estuary Pro- gram Office. A special thanks to bayman Peter Wenczel for the many ways in which he inspired and assisted in this study. LITERATURE CITED Barber. B. J. & N. J. Blake. 1991. Reproductive physiology, pp. 377-428. In: S. E. Shumway (ed.). Scallops: Biology. Ecology, and Aquaculture. Elsevier. New York. Belding. D. L. 1910. The scallop fishery of Massachusetts. Marine Fish- eries Service — No. 3, Division of Fisheries and Game. Department of Conservation, Commonwealth of Massachusetts, Boston. MA. 51 pp. Bologna, P. A. X. 1998. Growth, production, and reproduction in bay scal- lops Argopecten irradians concentricus (Sayl from the northern Gulf of Mexico. J. Shellfish Res. 17:91 1-917. Bricelj, V. M., J. Epp & R. E. Malouf. 1987a. Comparative physiology of young and old cohorts of bay scallop Argopecten irradians irradians (Lamarck): mortality, growth, and oxygen consumption. J. Exp. Mar. Biol. Ecol. 112:73-91. Bricelj. V. M.. J. Epp & R. E. Malouf. 1987b. Intraspecific variation m reproductive and somatic growth cycles of bay scallops Argopecten irradians. Mar. Ecol. Prog. Ser. 36:123-137. Bricelj, V. M. & S. H. Kuenstner. 1989. Effects of the "brown tide" on the feeding physiology and growth of bay scallops and mussels, pp. 491- 509. In: E. M. Cosper. V. M. Bricelj, and E. J. Carpenter (eds.). Novel Phytoplankton Blooms: Causes and Impacts of Recurrent Brown Tides and Other Unusual Blooms. Coastal and Estuanne Studies 35. Springer. New York. Coe. W. R. & H. J. Turner. Jr. 1938. Development of the gonads and gametes in the softshell clam (Mya arenaria). J. Morphol. 62:91-1 I I. Cosper. E. M., W. Dennison. A. Milligan. E. J. Carpenter. C. Lee. J. Holza- pfel & L. Milanese. 1989. An examination of the environmental factors important to initiating and sustaining "brown tide" blooms, pp. 317- 340. In: E. M. Cosper. V. M. Bricelj. and E. J. Carpenter (eds.). Novel Phytoplankton Blooms: Causes and Impacts of Recurrent Brown Tides and Other Unusual Blooms. Coastal and Estuarine Studies 35. Springer. New York. Epp. J. 1989. Energy storage and utilization in the bay scallop, Argopecten irradians irradians (Lamarck). M.S. thesis. SUNY-Stony Brook. Stony Brook. New York. 83 pp. Epp, J., V. M. Bricelj & R. E. Malouf. 1988. Seasonal partitioning and utilization of energy reserves in two age classes of the bay scallop Argopecten irradians irradians (Lamarck). J. Exp. Mar. Biol. Ecol. 121:113-136. Gallager, S. M.. D. K. Stoecker & V. M. Bricelj. 1989. Effects of the brown tide alga on growth, feeding physiology, and locomotory be- havior of scallop larvae (Argopecten irradians). pp. 51 1-541. In: E. M. Cosper. V. M. Bricelj. and E. J. Carpenter (eds.). Novel Phytoplankton Blooms: Causes and Impacts of Recurrent Brown Tides and Other Unusual Blooms. Coastal and Estuanne Studies 35. Springer, New York. Gobler. C. J. 1995. The role of iron in the occurrence of Aureococcus anophagefferens blooms. M.S. thesis, SUNY-Stony Brook. New York. 127 pp. Hampson, G & J. M. Capuzzo. 1984. Growth and reproduction of bay scallops in shallow and deepwater embayments. WHOI Tech. Rept. WHOI-84-38:4-5. Hickey. M. T. 1977. Age. growth, reproduction, and distribution of the bay scallop. Aequipecten irradians irradians (Lamarck), in three embay- ments in eastern Long Island. New York, as related to the fishery. M.S. thesis, C. W. Post College, Long Island University, Brookville. New York. 101 pp. Humason. G. L. 1979. Animal Tissue Techniques. W. H. Freeman & Co.. San Francisco. Jubb. K. V. F.. P. C. Kennedy & N. Palmer. 1985. Pathology of Domestic Animals, vol. 3: 3rd ed.. Academic Press. Orlando. FL. Kelley, K. M. & J. D. Sisson. 1981. Seed sizes and their use in determining spawning and setting times of bay scallops on Nantucket, pp. 43^49. 58 Tettelbach et al. In: K. M. Kelley (ed.). The Nantucket Bay Scallop Fishery: The Re- source and Its Management. Shellfish and Marine Department. Nan- tucket. MA. Langton. R. W., W. E. Robinson & D. Schick. 1987. Fecundity and repro- ductive effort of sea scallops Placopecten magellanicus from the Gulf of Maine. Mar. Ecol. Prog. Ser. 37:19-25. Levitan, D. R.. M. A. Sewell & F. S. Chia. 1992. How distribution and abundance influence fertilization success in the sea urchin Strongylo- centrotus franciscanus. Ecology 73:248-254. MacDonald. B. A. & R. J. Thompson. 1988. Intraspecific variation in growth and reproduction in latitudinally differentiated populations of the giant scallop Placopecten magellanicus (Gmelin). Biol. Bull. 175: 361-371. MacFarlane, S. L. 1991. Managing scallops Argopecten irradians irradi- ans (Lamarck. 1819) in Pleasant Bay. Massachusetts: large is not al- ways legal, pp. 264-272. In: S. E. Shumway and P. A. Sandifer (eds, I An International Compendium of Scallop Biology and Culture. World Aquaculture Society. Baton Rouge. LA. Marshall, N. 1960. Studies of the Niantic River. Connecticut with special reference to the bay scallop. Aequipeclen irradians. Limnol. Oceanogr. 5:86-105. Naidu. K. S. 1970. Reproduction and breeding cycle of the giant scallop Placopecten magellanicus (Gmelin) in Port au Port Bay. Newfound- land. Can. J. Zool. 48:1003-1012. Paulet, Y. M., A. Lucas & A. Gerard. 1988. Reproduction and larval de- velopment in two Pecten maximus (L.) populations from Brittany. J. Exp. Mar. Biol. Ecol. 119:145-156. Peterson. C. H. & H. C. Summerson. 1992. Basin-scale coherence of popu- lation dynamics of an exploited marine invertebrate, the bay scallop: implications of recruitment limitation. Mar. Ecol. Prog. Ser. 90:257- 272. Risser, J. 1901. Habits and life-history of the scallop (Pecten irradians). pp. 47-55. In: 3 1st Annual Report to the Rhode Island Commissioners of Inland Fisheries. (Cited in: Barber. B. J. & N. J. Blake. 1991. Re- productive physiology, pp. 377-428. In: S. E. Shumway (ed.). Scal- lops: Biology, Ecology, and Aquaculture. Elsevier, New York). Sastry, A. N. 1963. Reproduction of the bay scallop Aequipecten irradians Lamarck, influence of temperature on maturation and spawning. Biol. Bull. 125:146-153. Sastry, A. N. 1970. Reproductive physiological variation in latitudinally separated populations of the bay scallop. Aequipecten irradians Lama- rck. Biol. Bull. 138:56-65. SCDHS. 1993-1995. Brown Tide Cell Counts— 1993-1995. Monitoring Reports. Suffolk County Department of Health Services, Office of Ecology, Riverhead, NY. Sieburth, J. M.. P. W. Johnson & P. E. Hargraves. 1988. Ultrastructure and ecology of Aureococcus anophagefferens gen. et sp. nov. (Chrysophyceae): the dominant picoplankter during a bloom in Nar- ragansett Bay, Rhode Island. Summer 1985. J. Phycol. 24:416-425. Smith, C. F. & S. T. Tettelbach. 1996. Bay Scallop Restoration: Western Peconic Bay. Final report submitted to the Environmental Protection Agency. 44 pp. Smith. C. F. & S. T. Tettelbach. 1997. Restocking Bay Scallops. Final Report to the National Marine Fisheries Service. 76 pp. Taylor. R. E. & J. M. Capuzzo. 1983. The reproductive cycle of the bay scallop, Argopecten irradians irradians (Lamarck), in a small coastal embayment on Cape Cod, Massachusetts. Estuaries 6:431^t35. Tetra Tech, Inc. 1997. Surface Water Quality Modeling of the Peconic Estuary — Calibration of EFDC Hydrodynamic Model. Interim Rept. 2. submitted to Peconic Estuary Program, Suffolk County Department of Health Services. Suffolk County, New York. Tettelbach. S. T. 1991. Seasonal changes in a population of northern bay scallops. Argopecten irradians irradians (Lamarck, 1819). pp. 164- 175. In: S. E. Shumway and P. A. Sandifer (eds). An International Compendium of Scallop Biology and Culture. World Aquaculture So- ciety. Baton Rouge, LA. Tettelbach, S. T. & E. W. Rhodes. 1981. Combined effects of temperature and salinity on embryos and larvae of the northern bay scallop, Ar- gopecten irradians irradians. Mar. Biol. 63:249-256. Tettelbach. S. T. & P. Wenczel. 1993. Reseeding efforts and the status of bay scallop Argopecten irradians (Lamarck, 1819) populations in New- York following the occurrence of "brown tide" algal blooms. /. Shell- fish Res. 12:423-431. Tettelbach, S. T.. P. Wenczel & S. W. T. Hughes. 1994. Size variability of juvenile (0+ yr) bay scallops in Long Island, New York populations. /. Shellfish Res. 13:284. Van der Kraak, G.. J. P. Chang & D. M. Janz. 1998. Reproduction, pp. 480-483. In: D. H. Evans (ed.). The Physiology of Fishes, 2nd ed. CRC Press. Boca Raton. FL. Journal of Shellfish Research, Vol. 18, No. 1. 59-66, 1999. SOME METHODS FOR QUANTIFYING QUALITY IN THE SCALLOP PECTEN MAXIMUS (L.) JULIE A. MAGUIRE, PIERRE G. FLEURY, 1 AND GAVIN M. BURNELL Aquaculture Development Centre Department of Zoology and Animal Ecology Lee Makings, Prospect Row University College Cork Cork. Ireland ABSTRACT Because biological systems do not work in isolation, behavioral, biochemical, and physiological tests can give an overview of an individual's vital processes and reaction to stress. Two stress gradients were applied in this study, a short acute desiccation stress and a long-term density stress. These stress gradients were used to assess the usefulness of various techniques for quality assessment; namely, a standard salinity stress test, condition index, recessing speed of the scallop, adenylic energetic charge (AEC). and percentage carbohydrate content of the striated muscle. The results showed that AEC could be used effectively to measure the effect of a short-term stress. In the striated muscle. AEC levels were useful in discriminating between good and poor quality scallops. The total carbohydrate content in the striated adductor muscle and condition index were useful in assessing the effect of long-term stress on scallop quality. The most promising results arose from the recessing trials, because this nondestructive test successfully discriminated the different qualities of scallops arising from both long- and short-term stress. KEY WORDS: Pecten maximus, stress, quality, desiccation, density INTRODUCTION Juvenile scallops are either collected by natural settlement onto artificial collectors or produced in a hatchery. Intermediate culture of spat then takes place in suspended culture or cages on the sea bottom until the scallops reach a size (35-50 mm) that offers some protection from predation. Final outgrowth can take place in sus- pended cage culture or by ranching them on the seabed. Large variations in the survival and performance of spat and juveniles during transport, nursery, and outgrowth have demonstrated the need for research into the effect of stress on the quality of the scallop Pecten maximus (Maguire 1998). Stress has been defined as "the effect of any environmental alteration or force that extends homeostatic or stabilizing processes beyond their normal limits at any level of organization." (Esch and Hazen 1978). Chronic sublethal stress, such as pollution from heavy metals or stocking at high densities, can cause an even or negative scope for growth (Thompson and MacDonald, 1991) and can occur over months or even years. Short acute stresses can occur over hours or days for example, desiccation, thermal shock, and salinity, but both types of stress can eventually result in mortality. The stress effect of various husbandry practices on the physiology of bivalve mollusks is virtually unknown but is believed to be significant. Dhert et al. (1992a). Dhert et al. ( 1992b) considered stress tests to be invaluable in testing the nutritional requirements of aquacul- ture species at various stages of their development and established a standard stress test to determine the quality of shrimp and fish fry. in which they used elevated salinity as a stressor. Duran- Gomez et al. ( 1991 ) developed a test to be performed on postlarval prawns Penaeus japonicus (Bate) using salinity and pH shocks as stressors. Likewise, Ashraf et al. (1992) employed a standard sa- linity stress test to detect differences in nutritional studies when no 'Direction des Resources Vivantes. IFREMER. Centre de Brest. BP 70, 29280 Plouzane, France. Corresponding Author: Gavin M. Burnell. Tel-(353)21 904192. Fax-(353) 21 270562. email: g.burnell@ucc.ie. differences existed in survival and growth using larval striped bass Morone saxatilis (Walbaum) and the silverside Menidia beryllina (Cope) as the experimental organisms. Because biological systems do not work in isolation, a combi- nation of physiological, biochemical, and behavioral tests can give a more complete picture of an individual organism's reaction to stress. Examples of some techniques used for assessing quality in bivalve molluscs are listed in Table 1. Scallops have some unique behavioral traits among bivalves in that they have the ability to swim relatively long distances in an oriented way. They can also recess into the sediment, first de- scribed by Baird and Gibson (1956). Therefore, potential behav- ioral tests could include recessing and righting behavior (turnover after being placed flat side down), which would affect their ability to withstand predation. Recessing requires a large energetic cost, and scallops that are already weakened by the stress of handling or exposure to air during transport would be less able to escape from predators by recessing or swimming when returned to the sea. Fleury et al. (1997) completed a study of the recessing behavioral of three sizes of ranched scallops during three seasons and three sizes and used adenylic energetic charge as an index. They dis- covered that the best seeding time was in the spring and summer and that within this period, medium sized scallops (30 mm) re- cessed more effectively than the small (15 mm) or larger (42 mm) sized scallops. In our study, recessing speed was used as a method for stress assessment. The effect of a short-term stress on the biochemistry of the animal can be measured by its level of adenylic energetic charge (AEC). AEC is defined by the ratio: AEC = (ATP + 0.5 ADP) -^ (ATP + ADP + AMP) where (ATP = adenosine triphosphate, ADP = adenosine diphosphate, AMP = adenosine monophos- phate). The triphosphate bond of the ATP molecule has maximum energy, the diphosphate bond of ADP is half as rich, and the monophosphate bond (AMP) lacks energy. The AEC ratio ranges from to 1; that is, (when 0, all nucleotides are AMP, and when 1. all nucleotides are ATP). Therefore, the relative level of these bonds can be used as a measure of the energy directly available to 59 60 Maguire et al. TABLE 1. A review of techniques used for quality assessment. General Category Technique Used Species Stress Reference Standard stress test "Aerial exposure Mytilus edulis (L.) Chronic Veldhuizentsoerkan et al. (1991) Acute Viarengo et al. ( 1995) Biometrics ""Condition index Flesh condition Oslrea edulis (L. ) M. edulis Pinctada fucata martensii (Dunker) Crassostrea virginica (Gmelin) Ruditapes philippinarum (Adams and Reeve) Argopecten ir radians i /radians (Lamark) M. edulis Chronic Rogan et al. ( 1991 ) Lundebye et al. (1997) Numaguchi (19951 Fisher et al. (1996) Isonoet al. (1998) Rheault and Rice (1996) Agirregoikoa et al. ( 1991 ) Behavior "Recessing Pectin maximus Fleury et al. (1997) Biochemical "Adenylic energetic charge "Carbohydrate content Lipid content Total oxyradical scavenging capacity RNA:DNA Bivalve mollusks C. gigas (Thunberg) Dreissena polymorpha (Pall.) M. edulis Eiinila ziczac (L.) P. maximus Placopecten magellanicus (Gmelin) Acute Moal et al. (1989a) Chronic Kaufmann et al. ( 1994) Short Sprung and Borcherding (1991) Chronic Regoli et al. (1998) Lodeiros et al. (1996) Robbins et al. (1990) Kenchington (1994) Cytochemical Lysosomal membrane fragility M. edulis Mya arenaria (L.) Digestive tubule and vesicular C. virginica connective tissue condition Chrome Pelletier et al. (1991) Tremblay et al. (1997) Fisher et al. (1996) Physiological Scope for growth Oxygen consumption: ammonia excretion Lipofusin accumulation O. edulis Pcrua viridis < L. ) M. edulis Amhlema plicata (Say) P. viridis Siinena scripla (L) Chronic Hutchinson and Hawkins ( 1992) and acute Chronic Cheung and Cheung ( 1995) Hatcher et al. (1997) Acute Barker and Horbach (1997) Mathew and Damodaran (1997) ' Techniques used to measure stress in this study. the cells at that particular time. For example, empirical studies have shown that a very weak, stressed scallop would have an AEC level (measured from the striated muscle) of 0.3 to 0.5 (Fleury. pers. comm.). Such a scallop would have a negative scope for growth and would have a poor chance of recovery. A scallop recording a level of 0.5 to 0.7 would have reduced growth, would not reproduce but could recover to its original quality. A healthy scallop would have an AEC level of 0.8 to 1. Adenylic energetic charge was first proposed as a stress index by Atkinson (1968). who suggested that modulations in the levels of adenylphosphate reflected variations of enzyme activity at key points in metablic pathways that yield energy in the form of high energy adenine- phosphate bonds. These variations are a result of external stress. In other words, the more stressed an animal becomes, the more en- ergy it uses to counteract the stress, thus lowering its AEC level. Many studies have been carried out using AEC as a stress index or in nutritional studies on different marine animals; for example, the marine isopod Cirolana boreulis (Lijborg) (Skjoldal and Bakke 1978). the European sea bass Dicentrarchus labrax (L.) (Reali et al. 1987), the oyster Crassostrea gigas (Moal et al. 1989b). the spider crab Hyas araneus (L.) (Harms 1992). the sturgeon Aci- penser beari (Brandt) (Salin 1992). the oyster C. angulata (Lamark) (Madureira et al. 1993) and the scallop P. maximus (Fleury et al. 1997). In juvenile scallops, the level of AEC varies between tissues. Le Coz (1989), in a comparative study of different tissues in the juvenile scallop P. maximus. found the highest AEC ratios in the adductor muscle. Within the muscle, the highest level was found in the striated part (mean = 0.93). which is concerned with the fast repetitive opening and closing of the valves of the scallop, thus enabling the animal to swim, to escape from predators, and to recess. In the smooth part of the muscle, the AEC results were Quantifying Scallop Quality 61 more variable. The smooth muscle has slower contractions and is capable of keeping the scallop shell closed for long periods, with little energy expenditure (Chantler 1991). Energy is transported from the muscle to the various organs via the haemolymph. The haemolymph of bivalves is also concerned with a variety of physiological functions; that is, transport of nu- trients and wastes, gas exchange, osmoregulation, and defence (Benniger and Le Pennec 1991). Therefore, in this study, we looked at the effect of a desiccation stress on AEC levels in the smooth and striated part of the adductor muscle and in the hae- molymph of P. maximus juveniles. The effect of a long-term stress on the biochemistry of an animal can be measured by the carbohydrate content of the smooth and striated adductor muscle, respectively. The adductor muscle is the main storage area for energy reserves. Many studies have concentrated on the seasonal partitioning of energy reserves in bivalves: for example. Epp et al. ( 1 988) studied energy partitioning of the bay scallop A. irradians. Walne ( 1970) assessed the seasonal variation of the glycogen content of seven populations of the oys- ter O. edulis. De Zwaan and Zandee (1972) studied the utilization of glycogen and accumulation of some intermediates during anaerobiosis in M. edulis. In this study, the effect of high stocking density on the carbohydrate content of cultured scallop spat was assessed. The criteria for a useful "stress detector" are that it should be reliable and significant; that is with little individual variation within the populations and significant differences between popu- lations. Quality in this study was defined by the degree of acute (emmersion) or chronic (density) stress endured by the scallops during these trials. Therefore, the objectives of this study were divided into two parts. First, to create different juvenile scallop qualities using a desiccation stress gradient and to use these ref- erence animals to compare different laboratory techniques, (stan- dard salinity stress test, recessing behavior and level of adeny lie- energy charge) for quality assessment. Second, to use the same laboratory techniques, (including total carbohydrate content in- stead of level of AEC) to measure quality in a case study where scallops were cultured at three different densities. MATERIALS AND METHODS The scallop spat (30 mm) used in this experiment were col- lected from the Rade de Brest, France. Shell length, height, depth, and total wet weight measurements were taken from a subsample of 100 spat used in each experiment, and a condition index was compiled: condition index = [Weight/! Height x Length x Depth)] x 10.000 (Fleury, pers. comm.). The scallops were acclimated in tanks and were maintained at a temperature of 15°C and a salinity of 35%e and fed an equal mixture (1 x 10 7 mL~') of batch cultured algae Pavlova lutheri (Droop). Isochrysis galbana, and Chaetoceros calcitrans (Paulsen) in volumes sufficient to give a tank concentration of 30-50 cells p-L. -1 . The scallop were scrubbed clean of epibiota and used in experiments within 2 weeks. Creation of a Gradient of Scallop Qualities Using a Desiccation Stress (Short-Term Stress) Four batches (A-D) containing three replicates each of healthy scallop spat (n = 30) were used for each of three experiments (Expt. 1-3). The spat were individually weighed, labeled with a permanent marker, and placed out of water in a constant tempera- ture room for 0, 3, 6. and 12 h ( = A. B, C and D, respectively). The air temperature used to stress the scallops was 19°C for the first experiment, 15°C for the second, and 1 7°C for the third. The stress detector tests (standard stress test, recessing ability, and level of adenylic energetic charge) were carried out on each batch (A-D) to determine whether the tests could discriminate among the batches. Determination of Various Scallop Qualities L'sing Scallop Spat Cultured at Different Densities (Long-Term Stress) The scallop postlarvae (2 mm), were taken from Tinduff Hatch- ery/Nursery in April 1995. They were transferred to the Bay of St. Brieuc. Three months later (July 5). the scallops were removed from the cages and graded by shell size (mean ± SD height 12 ± 2 mm). They were placed in new cages (0.75 m 2 ) with a larger mesh size (5x5 mm). A range of stocking densities from 700 to 900 to 1,250 scallops per tier was set up and was referred to as density 1, 2. and 3. Nine replicates of each experimental density were used. After a 3-month period (October 5). the scallops were retrieved by SCUBA diving from the cages at each density. During transport (4 h), the spat were wrapped in towels soaked with seawater. The juveniles were then stored in aerated seawater tanks at 16°C over- night. Over the next 2 weeks, various stress tests were carried out. These were a standard salinity stress test (2-wk duration), recessing ability (2-wk duration), and total carbohydrate content fixed im- mediately. A description of these tests follows. Standard Stress Test A useful stress test will pick up differences induced by a stress gradient. The ultimate reaction to stress is mortality, so this was used as a standard assessment. Shell height, length, depth, and various wet weight measurements were taken before and after the standard stress was completed to enable condition indices to be computed. The standard stress tests were performed in a cubic recirculat- ing tank (1.5 x 1.5 m). The experimental salinity was 25%c tem- perature 15 ± 1°C for experiments 1 and 2. this was made up using seawater and distilled water. This acted as a semi-severe stress to the already stressed spat to hasten mortality. The experiments took 2 weeks to complete. In experiment 3, the salinity stress test was carried out using freshwater (temperature 14 ± 1°C) to achieve a quicker result. The spat were given food daily at the same rate with the same species of algae used during their acclimation period. However, even in experiments 1 and 2 (257(c) the scallops were so stressed that they did not seem to feed. Survival was monitored twice per day over a 2-wk period in experiments 1 and 2 and every 15 min over a 2 h period for the freshwater test (experiment 3). The criterion for death was open valves with a lack of valve contraction when touched by a glass rod. All scallops were then reweighed and the shell length, height, and depth were recorded. Recessing Behavior Twenty scallops each from the different groups of spat were quickly measured for shell length, height, depth, and total wet weight. The spat were color labeled and placed in a tank (length 2m. width 0.5 m) with recirculating seawater (salinity 35% 5 mm particle size. 58% 2 to 5-mm. 35% 1 to 2-mm, and 3% < I -mm particle size. The juveniles were fed a mixture of batch-cultured algae, at the same volume used during their acclimation period. Recessing time was monitored every 4 h. and scallops were recorded as recessed (completely covered by substrate), semi-recessed (half covered by sediment), or not recessed. Extraction and Analysis of Nucleotides Scallop parameters (shell length, height, depth, and total wet weight) were quickly measured for each batch of spat. The scallop was rapidly dissected and the striated and smooth muscle sepa- rately removed and frozen in liquid nitrogen. There it was stored until analysis (within a few days). Moal et al. (1989a) found that a better nucleotide extraction was obtained when the required tis- sue, rather than the whole animal, was frozen. At the time of the analysis, the striated and the smooth part of the muscle were withdrawn from the liquid nitrogen. One mL of 0.5M ice-cold TCA was then added immediately to each sample, as better recovery of ATP was observed using TCA as compared to other acids; for example perchloric acid (PCA) (Moal et al. 1989a). Preliminary crushing of the extracts increases the stability of the neutralized extracts. The tissue (still frozen) was instanta- neously homogenized at 25.000 rpm for 10 s. The homogenate was centrifuged for 10 min at 4.500 rpm. and the supernatant was neutralized with 0.5 m fresh amine freon solution. The neutralized sample was either stored at -18°C or immediately analyzed by high-performance liquid chromatography (HPLC). Analysis The HPLC apparatus was composed of a pump (Waters model 510). an automatic injector (Kontron 460), and a spectrophotom- eter (Merck L4250). The separation took place in a C18 column of length 150-mm. diameter 4.6-mm (model SFCC/Shandon Spher- isorb 3u-OD52). and ultraviolet light (254 nm) was used for the detection of the nucleotides. An isocratic NaH 2 P0 4 (0.15 m) buffer (pH 6) containing an ion-pairing agent (0.005 M tetrabuty- lammonium) and 5% methanol was used to elute the nucleotides. All chemicals were of analytical grade and supplied by Sigma. Separation took approximately 30 min at a flow rate of 1 mL/min. Carbohydrate Content Biometric measurements were taken for each scallop from the different spat groups. The animals were rapidly dissected, and the striated muscle was removed and immediately placed in liquid nitrogen. At the time of analysis, the samples were withdrawn and freeze dried using a HETOSICC CD 53-1 freeze dryer. The car- bohydrate content was analyzed using a miniaturization of the Dubois et al. (1956) method. Twenty (xg of the muscle sample were crushed and resuspended in 1 mL of distilled water. Fifty p.L of the mixture was placed in an epindorff tube. 50 yiL of 5% phenol was added, and the resultant solution was allowed to stand for 20 min at 15°C. Five hundred u.L of 98% H 2 S0 4 was added, and the tube was placed on ice. After centrifugation. the absor- bance of the supernatant was read at 492 and 620 mu, using a spectrophotometer model SLT Spectra. A glucose standard was used at concentrations of 0. 50. 100. 150. and 200 p,g of glucose per mL of distilled water, and blanks were made using distilled water. Statistical Analyses Nonparametric data were normalized by log transformation or arcsine square root transformation for percentage data. One-way analyses of variance (ANOVAs) were used to test significant dif- ferences among treatments, and a posteriori Tukey test was used to contrast treatments. The level of significance was set at 0.05. RESULTS Standard Stress Test Figure I shows the mean survival times (over 2-week test pe- riod) of each population for each test (desiccation temperature 19° and 15°C). It showed that the degree of desiccation endured (0-12 h) by each group was directly proportional to the mortality rate of each group. However, the desiccation temperature of 1 9°C was too high, because all the spat from group D (12-h emmersion) died either during the last hour of desiccation or immediately after reimmersion. Despite this, a significant difference was found be- tween the spat groups created by using the higher desiccation temperature. The data for test 2 (desiccation temperature 15°C) showed a significant difference in the mean survival times between groups A/B, C, and D (0, 3, 6. and 12-h desiccation) with similar mortalities occurring between groups A and B (0- and 3-h desic- cation). Figure 2 shows the survival of the four populations (A-D) in test 3. using a freshwater standard stress (temperature 14°C). The data showed no significant difference between the populations (/> > 0.05). The stress used in this test was too severe to pick up the subtle differences in quality between the populations. The standard salinity stress test (water temperature 15°C, sa- linity 25%) was carried out on the groups 1-3 of the spat density experiment, and no significant difference was found between the survival of the different density treatments. Only 10% mortality was recorded in the test. Recessing Behavior Table 2 shows the recessing time of the four scallop groups in the desiccation experiment and the three groups in the density experiment. Recessing speed was directly proportional to the des- iccation endured (0-12 h) by the spat and the density (700-1,250 spat per tray). A significant difference was found among the treat- I mmersion Time (hours) Figure 1. The mean survival times over a 2-week period of four dif- ferent qualities (desiccation times: A = 0h, B = 3h, C = 6h, and D = 12 h) of juvenile scallops to a standard salinity stress of reduced sa- linity (S = 25% r , T = 15 C and 19°C). Quantifying Scallop Quality 63 45 60 75 Time (minutes) Figure 2. The = h, B = 3 h. salinity stress survival of four different qualities (desiccation times: A C = 6 h, and D = 12 h( of juvenile scallops to a standard of freshwater ( 14°C). merits in the desiccation experiment (F 76 3 = 74.2. p < 0.01) and the density experiment (F 1172 = 13.67. p < 0.01 ). Adenylic Energetic Charge Table 3 shows the relationship between the striated and smooth adductor muscle of the four populations of spat. The highest levels of AEC for all groups was found in the striated adductor muscle. The AEC level in the striated muscle was significantly higher than the AEC level in the smooth muscle for each population (group A t 34 = 19.56. p< 0.01; group B t 36 = 12.12, p< 0.01; group C t 3S = 3.34, p < 0.01; and group D t 34 = 7.32, p < 0.01 ). The AEC levels in the striated adductor muscle clearly showed two significant groups (F 72 3 = 24.15. p < 0.01). Scallops from group A/B had higher AEC levels (> 0.85) than the scallops from group C/D (< 0.75). In the smooth muscle, the highest levels of AEC were found in population B, and again levels significantly decreased from this in group C and D (F 65 3 = 4.53, p < 0.01). Hemolymph was also extracted, but the AEC results were deemed to be unreliable because of the difficulty of extracting the hemolymph. Carbohydrate Content Table 4 shows percentage carbohydrate in dry weight of the striated adductor muscle and the condition index of scallops cul- TABLE 2. Mean ± SD recessing time of different spat qualities in the short- (desiccation) and long-term (density) experiments. Spat Group Experiment 1 Average Recessing Time (Days) A (0-h desiccation) B (3-h desiccation) C (6-h desiccation) D ( 12-h desiccation) Spat Group Experiment 2 1.6±0.4 J 2.4 ± 0.6 b 3.5±0.T 5.7 ± 1.6 d Average Recessing Time (Days) Group 1 (density 700 per tray) Group 2 (density 900 per tray) Group 3 (density 1250 per tray) 1.73 ±0.93" 2.28 ± 1.24" 3.13 ± 1.38 h Any two means sharing a common letter in each column are not signifi- cantly different at p < 0.05 (Tukey test). TABLE 3. Levels of AEC (mean ± SD) in the adductor muscle of four different groups of scallop spat. Group Striated Muscle Smooth Muscle A (0-h desiccation) B (3-h desiccation) C (6-h desiccation) D (12-h desiccation) 0.89 ±0.04'' 0.87 + 0.1 I" 0.71 ±0.1 b 0.72 + 0.08 b 0.55 ± 0.07 ab 0.65 + 0.11- 0.61 ±0.08" b 0.53 + 0.08 b Any two means sharing a common letter in each column are not signifi- cantly different at p < 0.05 (Tukey test). tured at three different densities. The carbohydrate content in- creased significantly from spat cultured at a density of 700 and 900 per tray to those cultured at 1,250 per tray (F 56 2 = 5.25, p < 0.01). Biometrics Shell length, height, depth, and total wet weight measurements were taken from all spat held at each stocking density, and a condition index was calculated: condition index = [Weight/ (Height x Length x Depth)] x 10,000. Table 4 represents the average value calculated per scallop at each density. Spat cultured at a density of 700 and 900 per tray had a similar condition index. The condition index decreased signifi- cantly for those cultured at a density of 1,250 per tray (F n72 = 4.188, p< 0.05). DISCUSSION Standard Stress Test In our study, salinity was reduced to 25 and 0%o, respectively, with the aim of inducing stress and, hence, mortality, which could be used to quantify the quality of the spat. Quality in this study was defined by the degree of acute (emmersion) or chronic (high- density) stress endured by the scallops during these trials. Simi- larly Viarengo et al. (1995) reported that a simple secondary stress response in mussels showed a sensitivity in the same range as other commonly used general stress indices at the cellular level. The results showed that short-term exposure of mussels to sublethal concentrations of pollutants significantly reduced mussel survival in air. Dredge (1997) suggested that saucer-shaped scallops Amu- sium japonicum balloti (Bernardi) can withstand exposure to air for up to 2 h before suffering significant mortality. TABLE 4. Mean ± SD percentage carbohydrate content of dry weight in the striated adductor muscle and the mean condition index of scallops cultured at three different densities. Scallop Density per Tray % Carbohydrate Content Condition Index Value 700 900 1,250 8.9 ± 2.4° 8.8 ± 2.5 a 11.5±3.8 b 5.81+0.42" 5.81 ±0.46 a 5.55 ± 0.48" Any two means sharing a common letter in each column are not signifi- cantly different at p < 0.05 (Tukey test). 64 Maguire et al. Although different spat qualities were obtained when the spat were removed from water for 0. 3. 6, and 12 h (groups A-D) at a desiccation temperature of 19°C. this temperature was considered too high, particularly for the group D scallops, because some of the spat from this group died during the 12-h desiccation period. Therefore, an air temperature of 15°C is recommended to give a wider range of spat quality. Similarly. Hutchinson and Hawkins (1992) measured stress in the oyster O. edulis using scope for growth as an index. A severe reduction in scope for growth was observed when oysters were placed in conditions where high tem- peratures were combined with low salinity. The third test using freshwater as the stress test was found to be too severe, and no difference was found among the treatments because of the rapid mortality (within 1 20 min) of all groups. This is contrary to a study by Dhert et al. (1992b), who worked on the use of stress evaluation as a tool for the quality control of hatchery-produced shrimp and fish fry. In their experiments, the best results were achieved with stress tests performed within a 60 to 90 min period containing 15 to 30 evaluation points. A standard salinity stress test (water temperature 15°C, salinity 25%o) was carried out on the groups 1 to 3 of the density experi- ment, and no difference was found among the populations. Very few mortalities were recorded in the test. Dhert et al. (1992b) emphasized the importance of using the appropriate salinity level for each species and for each larval stage. Apparently, in our test, the salinity level was not severe enough to differentiate the differ- ent densities, or there was no difference in the quality of spat. Recessing Behavior It is not surprising that the best quality scallop recessed into the sediment quickest (Table 2|. Dao et al. (1985) found that when seeding scallop spat on the seabed, success seemed to depend upon three factors; namely, the quality, the size of the scallop, and the time of year that seeding takes place. By removing seasonal and size variables, we were able to demonstrate a relationship between quality and behavior in juvenile scallops. This is beleived to be the first time this has been demonstrated experimentally. Tyurin (1991) worked on the behavioral reactions of the scallop Mizu- hopecten yessoensis (Jay) to reduced salinity and oxygen exposure to synthetic detergents. Under unfavorable conditions, the test scallops were stressed, could not recess, and elicited an escape response instead. In this study, the recessing speed of scallops deteriorated significantly as the stocking density increased. The recessing test is, therefore, not only sensitive to subtle changes in the spat quality, but is also a very quick and simple test to perform. Adenylic Energetic Charge In general, the results indicated that as the stress level in- creased, the AEC level decreased in the striated muscle, to a cer- tain point where the AEC level did not decrease any more. This seems to be the threshold level for this test. Similar results have been shown by Madureira et al. (1993). who looked at the effect of polychorinated biphenyl (PCB) on adenylic energetic charge in the oyster C. angulata, which was fed a PCB-contaminated algal cock- tail. They found that the level of PCB increased with time within the animal and that this sublethal stress resulted in the reduction of AEC levels as PCB concentration increased. In our study, the striated muscle was found to be the best tissue to use when measuring AEC levels, because there was little indi- vidual variation within the groups and a large difference between stressed (group C and D) and unstressed (group A and B) treat- ments. Similarly, Le Coz (1989) reported that highest levels of AEC were found in the striated adductor muscle of P. maximus and that AEC results were more variable in the smooth muscle. In prior studies, (Fleury et al. 1997) the level of AEC in the striated muscle seemed to be a better measure of stress and quality, than the smooth muscle, because the decrease of AEC in this part of the muscle attributed to stress was more pronounced. In our study, as well, a similar AEC decrease was found in the striated muscle. The hemolymph of bivalves is concerned with a variety of physiologi- cal functions, but also the transport of ATP from the striated muscle to various organs. We expected similar results as those found in the muscle. Our results, however, were unreliable because of the difficulty of the hemolymph extraction procedure. The results of our study were consistent with those found by Moal (1989a. 1989b. 1991 ). who showed that the effect of short- term desiccation on the oyster C. gigas was dependent upon sea- son. AEC levels remained high after 3 h of desiccation in January, but decreased after 3 h of desiccation in May and July. Therefore, there is a negative correlation between AEC and season. Our study was only carried out in May, and the results showed a decrease in AEC levels after a 3-h desiccation period. Further experiments would have to be carried out to determine whether AEC levels could be used to quantify stress in P. maximus at other times of the year. Overall, the recessing test was just as reliable as the level of AEC in measuring the effect of stress on scallops. The recessing test is nondestructive: therefore it can be used for continuous monitoring of the same scallop and is more cost effective than the biochemical test. However, to monitor stress, the testing of the shellfish must take place immediately after sampling so that the condition of the scallop will not be altered by handling. Therefore, because a sample can easily be frozen for biochemical analysis later, it may be more convenient to use AEC rather than have to set up a recessing trial immediately after sampling. Carbohydrate Content The main energy reserve in scallops is glycogen, which is stored in the adductor muscle. It is mobilized and converted into usuable energy (ATP) when needed. In general, scallops contain relatively low levels of glycogen in the adductor muscle attaining maximum levels of up to 24% in P. maximus (Ansell 1978). 23- 25% of dry muscle weight in A. irradians (Epp et al. 1988) and 18% in Chlamys islandica (Muller) (Vahl 1981): whereas, the mussle A/, edulis attains glycogen levels of 42-53% in the mantle (Gabbott 1983). The percentage carbohydrate content in this study measured in October was quite low. ranging from 8.8-11% dry weight of the adductor muscle. This is the period when maximum levels of carbohydrate should be found in the adductor muscle after the summer period. Ansell (1978) suggested that the carbo- hydrate content varies among sites and among years in the maxi- mum levels found but generally varies from a minimum in March (2.5%) to a maximum in September (24%). Table 4 showed a surprising result, with the highest level of carbohydrate found in scallops cultured at the highest density ( 1 ,250 juveniles per tray). However, the microanalytical technique used for carbohydrate analysis was precise, and the coefficient of variation between subsamples of a single sample was 2.0. This result was contrary to a study by Kaufmann et al. (1994). who reported that the glycogen content of the Pacific oyster C. gigas Quantifying Scallop Quality 65 decreased by 90% after 5 weeks during a growth trial in Maderia Island. This decrease was attributed to a combination of stress factors. Biometrics The use of condition indices are the traditional method for measuring quality. In this study, the condition index used was sensitive enough to pick up a significant difference in quality between the scallops held at the lower density treatments (700 and 900 scallops per tray) and the high-density treatment (1,250 scal- lops per tray). Similarly, Rheault and Rice ( 1996) found that dou- bling the stocking density of the eastern oyster C. virginica re- sulted in a 20% reduction in the condition of the bivalve. CONCLUSIONS Scallop spat of significantly different quality were obtained and were used as a reference to test techniques for quality assess- ment. It was possible to detect a significant decrease in the qual- ity of scallops with increasing stress conditions in both experi- ments. Both AEC and recessing speed detected acute differences in spat quality in the desiccation experiment. Recessing speed, carbohydrate content, and condition index detected chronic dif- ferences in spat quality brought about by varied stocking densi- ties in the density experiment. These tests can now be reliably used to measure quality or the effect of a chronic or acute stress on scallops. LITERATURE CITED Agirregoikoa. M. G.. M. A. Perez, J. A. Marigomez & E. Angulo. 1991. Relationships between quantitative indexes of individual and digestive cell conditions in the common mussel, Mytilus edulis L. from the Biscay coast. Acta Hydrochim. Hydrobiol. 19:1:29-37. Ansell, A. D. 1978. Storage and utilization of reserves in Pectinid bivalves with particular reference to the adductor muscle. Proceedings of the Scallop Workshop. Brest. France 8-13 May 1978. 17 pp. Ashraf. M. K. L. Simpson, & D. A. Bengston. 1992. Development of sa- linity stress tests for larval striped bass. Morone saxatilis, and inland silversides, Menidia beryllina, used in nutritonal studies. Proceedings of Aquaculture '92, Growing Toward the 21st Century. 31 pp. Atkinson. D. E. 1968. The energy charge of the adenylate pool as a regu- latory parameter. Interaction with feedback modifiers. Biochemistry 7:4030-4034. Baird, T. H. & F. A. Gibson. 1956. Underwater observations on escallop {Pecten maximus L.) beds. J. Mar. Biol. Assoc. U.K. 35:555-562. Baker, S. M. & D. J. Hornbach. 1997. Acute physiological effects of zebra mussel (Dreissena polymorpha) infestation on two unionid mussels. Actinonaias ligamenta and Amblema plicata. Can. J. Fisheries Aquat. Sci. 54:3:512-519. Benninger, P. G. & M. Le Pennec. 1991. Functional anatomy of scallops, scallops: biology, ecology, and aquaculture. pp. 133-223. In: Shum- way S. (ed.). Developments in Aquaculture and Fisheries Science. Elsevier, New York. Chantler, P. D. 1991. The structure and function of scallop adductor muscles, scallops: biology, ecology, and aquaculture. In: Shuinway S. (ed.). Developments in Aquaculture and Fisheries Science. Elsevier, New York. Cheung, S. G. & R. Y. H. Cheung. 1995. Effects of heavy metals on oxy- gen consumption and ammonia excretion in green-lipped mussels (Perna viridis). Mar. Poll Bull. 31:4-12:381-386. Dao, J.C., D. Buestel. A. Gerard. C. Halary & J. C. Cochard. 1985. Le programme de replement de coquilles Saint-Jacques {Pecten maximus L.) en France: finable, resultats et prespectives. Colloque Franco- Japonais D'oceanographie: Marseille France, September 1985. De Zwaan, A. & D. I. Zandee. 1972. The utilization of glycogen and the accumulation of some intermediates during anaerobiosis in Mytilus edulis L. Comp. Biochem. Physiol. 43B:47-54. Dhert, P., P. Lavens & P. Sorgoloos. 1992a. A simple test for quality evaluation of cultured fry of marine fish. Me d. Fac. Landbouww. Univ. Gent 57/4b. 8 pp. Dhert. P., P. Lavens & P. Sorgoloos. 1992b. Stress evaluation: a tool for quality control of hatchery-produced shrimp and fish fry. Aquacul. Eur. 17:2:6-10. Dredge, M. C. L. 1997. Survival of saucer scallops, Amusium japonicum balloti. as a function of exposure time. J. Shellfish Res. 16:1:63-66. Dubois. M., K. A. Gilles, J. K. Hamilton, P. A. Rebers & F. Smith. 1956. Colorimetric method for determination of sugars and related sub- stances. Anal. Chem. 28:350-356. Duran-Gomez, R.. J. M. Rodriguez & J. Morales. 1991. Stress tests: a practical tool to control larval shrimp quality. In: P. Lavens, P. Sorgeloos. E. Jaspers and F. Ollevier (eds.l. Larvi '91, No. 15. Epp. J.. V. M. Bricelj & R. E. Malouf. 1988. Seasonal partitioning and utilization of energy reserves in two age classes of the bay scallop Argopecten irradians irradians (Lamark). ./. Exp. Mar. Biol. Ecol. 121:113-136. Esch, G. & T. C. Hazen. 1978. Thermal ecology and stress: a case history for red-sore disease in largemouth bass. pp. 331-363. In: J. D. Thorpe and J. W. Gibbons (eds.). Energy and Environment Stress in Aquatic Ecosystems. Technical Information Center. U.S. Department of En- ergy. CONF-771114. Fisher, W. S., J. T. Winstead. L. M. Oliver. H. L. Edmiston & G. O Bailey. 1996. Physiologic variability of eastern oysters from Apalachi- cola bay. Florida. J. Shellfish Res. 15:3:543-553. Fleury. P. G.. C. Mingant & A. Castillo. 1997. A preliminary study of the behavior of reseeded juvenile great scallops of three sizes in three seasons. Aquacul. Int. 4:325-337. Gabbott, P. A. 1983. Developmental and seasonal metabolic activities in marine molluscs, pp. 165-219. In: Hochachka P. W. (ed.). The Mol- lusca, 2, Environmental Biochemistry and Physiology. Academic Press, New York. Harms, J. 1992. Effects of nutrition (herbivore vs. carnivore) on energy charge and nucleotide composition in Hyas araneus larvae. Helgol Meeresunters. 46:1:29^14. Hatcher. A., J. Grant & B. Schofield. 1997. Seasonal changes in the me- tabolism of cultured mussels (Mytilus edulis L.) from a Nova Scotian inlet: the effects of winter ice cover and nutritive stress. J. Exp. Mar. Biol. Ecol. 217:1:63-78. Hutchinson. S. & L. E. Hawkins. 1992. Quantification of the physiological responses of the European flat oyster Ostrea edulis L. to temperature and salinity. J. Moll. Stud. 58:215-216. Isono, R. S.. J. Kita & C. Kishida. 1998. Upper temperature effect on rates of growth and oxygen consumption of the Japanese littleneck clam. Ruditapes philippinarum. Nippon Suisan Gakkaishi 64:3:373-376. Kaufmann, M. J., M. N. L. Seaman, C. Andrade & F. Buchholz. 1994. Survival, growth, and glycogen content of pacific oyster. Crassostrea gigas (Thunherg 1793), at Madeira Island (subtropical Atlantic). J. Shellfish Res. 13:2:503-505. Kenchington, E. L. R. 1994. Spatial and temporal variation in adductor muscle RNA:DNA ratio in sea scallops (Placopecten magellanicus) in the Bay of Fundy. Canada. J. Shellfish Res. 13:1:19-24. Le Coz. J. R. 1989. La charge energetique adenylique: mise au point, application a trois mollusques bivalves, synthese et perspectives. Mem- oire IFREMER, Centre de Brest, Direction des Ressources Vivantes. Lodeiros, C. J. M.. R. I. Fernandez. A. Boumati. J. H. Himmelman & K. S. Chung. 1996. Relation of RNA/DNA ratios to growth for the scallop Euvola [Pecten) ziczac in suspended culture. Mar. Biol. 126:2:245- 251. Lundbye, A. K.. W. J. Langston & M. H. Depledge. 1997. Stress proteins 66 Maguire et al. and condition index as biomarkers of tributyltm exposure and effect in mussels. Ecotoxicology 6:3:127-136. Madureira. M. J.. A. M. Picado. A. M. Ferreira. F. Mendonca & Y. Le-Gal. 1993. PCB contamination in the oyster Crassostrea angulata: effects on lipids and adenylic energetic charge. In: W. Sloof and H. de-Kruijf (eds.). Proceedings of the Second European Conference on Ecotoxi- cology 1993, vol. suppl. pts. 1-2. Maguire, J. A. 1998. Aspects of the biology of cultured scallops (Pecten maximus L.) with particular reference to stress. Ph.D. thesis. National University of Ireland. 152 pp. Mathew, S. & R. Damodaran R. 1997. Effect of ambient oxygen concen- tration on lipofuscin accumulation in a clam Sunelta scripla and a mussel Perna viridis. Indian. Mar. Sci. 26:1:57-63. Moal. J.. J. R. Le Coz, J. F. Samain. & J, Y. Daniel. 1989a. Nucleotides in bivalves: extraction and analysis by high-performance liquid chroma- tography (HPLC). Comp. Biochem. Physiol. 93B:307-316. Moal. J.. J. R. Le Coz, J. F. Samain. & J. Y. Daniel. 1989b. Responses and adaptations of adenylate energy charge and digestive enzyme activities to tidal emmersion of Crassostrea gigas population in Marennes. Ole- ron Bay. Sci. Mar. Bare. 53:2-3. 699-704. Moal. J.. J. R. Le Coz. J. F. Samain, & J. Y. Daniel. 1991. Oyster adenylate energy charge (AEC) and its natural variability: implications for envi- ronmental monitoring. Oceanis. Doc. Ocenogr. 17:3:279-280. Numaguchi. K. 1995. Effects of water temperature on catabolic losses of meat and condition index of unfed pearl oyster Pinciada fucata mar- tensii. Fisher. Sci. 61:5:735-738. Pelletier. E.. S. Ouellet & M. Paquet. 1991. Long-term chemical and cy- tochemical assessment of oil contamination in estuarine intertidal sedi- ments. Mar. Poll. Bull. 22:6:273-281. Reali, N.. A. Casti. G. Orlandini & R. Viviani. 1987. Effects of temperature on muscle adenylic nucleotides of European sea bass {Dicenlrarchus labrax L.). Ital. J. Biochem. 36:2: 136A-138A. Regoli. F., G. W. Winston, V. Mastrangelo. G. Principato & S. Bompadre. 1998. Total oxyradical scavenging capacity in mussel Mytilus sp. as a new index of biological resistance to oxidative stress. Chemosphere 37:14-15.2773-2783. Rheault, R. B. & M. A. Rice. 1996. Food limited growth and condition index in the eastern oyster, Crassotrea virginica (Gmelin 1791), and the bay scallop. Argopeclen irradians irradians (Lamark 1819). J Shellfish Res. 15:2:271-283. Robbins. I., P. Lubet & J. Y. Besnar. 1990. Seasonal variation in the nucleic acid content and RNA:DNA ratio of the gonad of the scallop Pecten maximus. Mar. Biol. 105:191-195. Rogan, E.. S. C. Culloty, T. Cross & M. F. Mulcahy. 1991. The detection of Bonamia ostreae (Pichot et al. 1980) in frozen oysters (Ostrea edulis L.) and the effect of the parasite on condition. Aquaculture 97:311-315. Salin, D. 1992. The ammonia toxicity for sturgeon Acipenser haeri: mor- phological, physiological, and metabolic effects of an exposure to sub- lethal and lethal doses. Thesis. Bordeaux University, France. 176 pp. Skjoldal. H. R. & T. Bakke. 1978. Relationship between ATP and energy charge during lethal metabolic stress of the marine isopod Cirolana borealis. J. Biol. Chem. 253:3355-3356. Sprung, M. & J. Borcherding. 1991. Physiological and morphometric changes in Dreissena polymorphs (Mollusca, Bivalvia) during a star- vation period. Malacologia 33:1-2,179-191. Sunila. I. 1991. Respiration of sarcoma cells from the soft shell clam Mya arenaria L under various conditions. J. Exp. Mar. Biol. Ecol. 150:19-29. Thompson. R. J. & B. A. MacDonald. 1991. Physiological interactions and energy partitioning, scallops: biology, ecology, and aquaculture. In: Shumway S. (ed.l. Developments in Aquaculture and Fisheries Science 21:347-376. Tremblay, R. & J. Pellerin-Massicotte. 1997. Effect of tidal cycle in lyso- somal membrane stability in the digestive gland of Mya arenaria and Mytilus edulis L. Comp. Biochem. Physiol. 117:199-104. Tyurin, A. N. 1991. Behavioral reactions of the scallop. Mizuhopecten yessoensis and the mussel. Crenomytilus gray anas, to reduced salinity and oxygen and exposure to synthetic detergents. /. Hydro, biol. 24: 13-19. Vahl. O. 1981. Energy transformations by the Iceland scallop. Chlamys islandica (O. F. Muller) from 70°N.I. the age-specific energy budget and net growth efficiency. J. Exp. Mar. Biol. Ecol. 53:281-296. Veldhuizentsoerkan, M. B., D. A. Holwerda. A. M. T. Debon. A. C. Smaal & D. I. Zandee. 1991. A field study on stress indexes in the sea mussel. Mytilus edulis — application of the stress approach in biomonitoring. Arch. Environ. Contamination Toxicol. 21:497-504. Viarengo. A., L. Canesi. M. Pertica. G. Mancmelli, R. Accomando. A. C. Smaal & M. Orunesu. 1995. Stress on stress response — a simple moni- toring tool in assessment of a general stress syndrome in mussels. Mar. Environ. Res. 39:1-4.245-248. Walne. P. R. 1970. The seasonal variation of meat and glycogen content of seven populations of the oyster Ostrea edulis L. and a review of the literature. Series Invest. Series II XXVLT-33. Journal of Shellfish Research, Vol. IS. No. I. 67-70. 1999. CLONING AND CHARACTERIZATION OF TROPOMYOSIN cDNAs FROM THE SEA SCALLOP PLACOPECTEN MAGELLANICUS (GMELIN, 1791) MOHSIN U. PATWARY, 1 MICHAEL REITH, 2 AND ELLEN L. KENCHINGTON 3 Department of Biology Medgar Evers College of The City University of New York Brooklyn, New York 11225 'Institute for Marine Biosciences, National Research Council of Canada Halifax, Nova Scotia, Canada B3H 3Z1 ^Science Branch. Bedford Institute of Oceanography Department of Fisheries and Oceans Dartmouth. Nova Scotia. Canada B2Y 4A2 ABSTRACT Two different complimentary DNAs (cDNAs) encoding tropomyosin have been characterized from adductor muscle of the sea scallop Placopecten magellanicus. These cDNAs fall into two size classes of approximately 2,540 and 2.030 base pairs with the larger clones containing a longer 3' untranslated region. This difference apparently arises from the utilization of two different polyadenylation signals. All clones are identical in both coding and noncoding regions, indicating that they represent the same gene. Northern analysis indicates that this gene is expressed highly in adductor muscle and at a much lower level in several other tissues. Southern blots indicate a small (1—3) number of tropomyosin genes in the sea scallop, and population studies detect a high degree of individual polymorphism at this locus. KEY WORDS: Placopecten magellanicus, sea scallop. cDNA, tropomyosin INTRODUCTION Tropomyosins are highly conserved, actin-binding proteins present in virtually all eukaryotic cells (see Lees-Miller and Helf- man 1991 for review). Different tropomyosin isoforms are ex- pressed in developmental and tissue-specific patterns and are broadly catagorized into three major classes: nonmuscle (cytoplas- mic), smooth muscle, and striated muscle specific. Tropomyosin mediates Ca 2+ -dependent actomyosin contraction through its in- teraction with troponins in striated muscle or caldesmon in smooth muscle and nonmuscle cells. In addition to this importance as an essential structural and functional component of the actin mi- crofilament system of the cell, tropomyosins have also been iden- tified as the major protein causing allergic reactions to shrimp (Shanti et al. 1993. Daul et al. 1994, Leung et al. 1994. Witteman ct al. 1994). Like many cytoskeletal proteins, the diversity of tropomyosin isoforms is generated from a few genes through alternative RNA processing or expression from alternate promoters rather than through individual genes for each isoform (Pittenger et al. 1994). In the rat, at least 16 different tropomyosin isoforms have been identified that are encoded by only four genes (Balvay and Fisz- man 1994). The four genes are the a-gene, p-gene, TM-4 gene, and hTMnm gene, each named after a protein they encode (striated muscle a- and B-TM, fibroblast TM-4, and human nonmuscular TM-30. respectively). The a-gene encodes at least nine different isoforms that are generated from two promoters (which results in the use of two different initial exons) and alternative splicing of exons 2. 6, and 9 (two alternate exons are encoded for exons 2 and 6, and four different exons are available for exon 9). The alterna- tive exons have been shown to encode tropomyosin sequences essential for critical interactions with other proteins. For example, the 9a exon of the a-gene, which is only expressed in striated muscle, is required for troponin to mediate high-affinity actin bind- ing (Hammell and Hitchcock-DeGregori 1996). The use of alter- nate promoters and splicing to generate multiple tropomyosin iso- forms has been found in all vertebrates investigated as well as Drosophila (Hanke and Storti 1988). In this communication, we describe the isolation and charac- terization of cDNA clones encoding tropomyosin from sea scallop adductor muscle. This paper contributes to a better understanding of the stucture-function relationship of the tropomyosin gene in bivalves, because there have been no detailed studies previously. We demonstrate that the region surrounding the tropomyosin gene is highly polymorphic in individual scallops and may prove to be an excellent marker for genetic studies of sea scallop. The mo- lecular characterization of tropomyosin cDNA will also be useful to future studies defining the physiological and molecular basis of allergic sensitivity. MATERIALS AND METHODS Sea scallops were obtained from commercial beds near Yarmouth and Sable Island. Nova Scotia and from St. Pierre Bank near Newfoundland, Canada. DNA extraction. cDNA library con- struction and screening, probe preparation for southern blot hy- bridization, and the preparation of genomic blots were as described previously (Patwary et al. 1996). Using a commercial RNA isolation kit (Stratagene). total RNA was extracted from pooled sea scallop adductor muscle, gill, go- nad, heart, liver, and mantle tissues from several individuals that had been snap-frozen in liquid nitrogen immediately after collec- tion and stored at -70°C. All RNAs were further extracted twice with phenolchloroform and once with chloroformisoamyl alcohol (24:1). precipitated with 7.5 m NH 4 C1 (DEPC-treated) and 2.5 volume ethanol and dissolved in DEPC-treated water. For northern blots, 15 jig of each RNA were electrophoresed on a 0.8% aga- rose-formaldehyde gel according to standard methods (Sambrook 67 68 Patwary et al. 1 ccctttcgagtctctgggagcccggtgtgtgttaggaataaggcagaagtcaggaggctctcgtctgcagttgttcagca 80 81 tttcccttctgcttcacacttcttcttcatctttctatttagataccgttaattctcaaacaacaaa AGT GAT GCT 156 1 M D A 3 15 7 ATC AAG AAG AAG ATG CAG GCC ATG AAG GTC GAC AGG GAG AAT GCC CAG GAC ATG GCC GAA 216 4 IKKKMQAMKVDRENAQDMAE 23 217 CAG ATG GAG CAG AAA TTG AAG GAC ACC GAG ACA GCC AAG GCA AAG TTG GAG GAA GAT TTC 276 24 QMEQKLKDTETAKAKLEEDF 43 2 77 AAC GAA CTC CAG AAG AAG CTC GGC ACC ACC GAA AAC AAC TTT GAT ATA GCC AAC GAA CAA 336 44 NELQKKLGTTENNFDIANEQ 63 33 7 TTG CAG GAA GCT AAT ACC AAG CTC GAA AAC TCA GAC AAA CAG ATC ACC CAG CTA GAA AGT 396 64 LQEANTKLENSDKQITQLES 83 397 GAT GTT GCT GGA CTC CAG AGG AGG CTC CAA CTG CTG GAA GAC GAT TAT GAG CGA TCT GAA 45 6 84 DVAGLQRRLQLLEDDYERSE 103 457 GAG AAG CTT AAC ACA ACA GCA GAG AAA TTG GAA GAG GCA TCC AAA GCT GCA GAT GAG AGT 516 104 EKLNTTAEKLEEASKAADES 123 517 GAG AGA AAT CGC AAG GTG TAT GAA GGC AGG AGT AAC ACT TGT GAG GAG AGG ATT GAT GAG 576 124 ERNRKVYEGRSNTCEERIDE 143 577 CTA GAA AAA CAG TTG GAT ACT GCT AAA ACC ATT GCA ACA GAT GCT GAC TCT AAG TTT GAT 636 144 LEKQLDTAKTIATDADSKFD 163 637 GAG GCC GCC CGT AAG CTT GCT ATT ACA GAA GTG GAC CTT GAG CGC GCC GAG ACT AGG CTG 696 164 EAARKLAITEVDLERAETRL 183 697 GAG GCC GCT GAC GCC AAA GTA CAC GAA CTC GAA GAA GAG CTC ACT GTT GTT GGT TCA AAT 756 184 EAADAKVHELEEELTVVGSN 203 75 7 ATC AAA ACC CTT CAG GTG CAA AAC GAT CAG GCA TCA CAG AGA GAG GAT AGC TAC GAG GAA 816 204 IKTLQVQNDQASQREDSYEE 223 817 ACC ATT AGA GAT CTC ACC AAA AGC CTG AAG GAT GCT GAG AAC AGG GCC ACA GAA GCT GAT 876 224 TIRDLTKSLKDAENRATEAE 243 87 7 AGA CAA GTA GTC AAA CTC CAG AAA GAG GTG GAC AGA CTC GAA GAT GAG CTG CTT GCG GAG 936 244 RQVVKLQKEVDRLEDELLAE 263 937 AAG GAA AGA TAC AAG GCA ATC AGT GAC GAA CTG GAC CAG ACC TTT GCC GAG ATT GCT GGT 996 264 KERYKAISDELDQTFAEIAG 283 997 TAC TAA tgtgtccagcaaggacatattcccctcaccaaatttatatcataaattacgaggaatgacagacaaagaaaa 1074 284 Y * 285 1075 gactgtcaattggaaaaataatattacatcagctttgtacagctatacaaatcgtcgtgcaagaattcgaaacagagaac 1154 1155 ctgccataaaggatccaacatttctttctcaatgtgtgtaatggtacagacgatgaaattccaaatttataaattattat 12 34 1235 taaqaaatqctaatctcta tqtqaccttqccqcqatattqcqacccaqcqcqqqaqtqaqaqactaqaqqqcaaqqacqq 1314 1315 qtqqqqtcqaccqqqtccqctttttaacctctctactcttcqtqtatttqtatqtatqttacttttqtqaacqqtttctt 1394 1395 ttcqaacacctqtcaqacttctqcaaaqctactacttctqqqqqqtaaaataatqttcatttctatatttatatacaatq 1474 1475 tatatataactqatacatacatqqattcatatttttcqttattttattatatcatgttatqacatcqqaacacqacacaq 1554 1555 aaqattqtqttatccatqcctqqcqcattctqtacttqaqqctcqgqaaqqcaatqctaqcqqcacatqqttaccattta 1634 1635 aq-taqtaqctacqcaqaqq-tttqqaccaqqcqtacaaaaaccaatcqqqqqtaatattqcqaqatacqacaqtqq-tqqaa 1714 1715 atttqacaqctttaqacatqtaqaqtcqtttataqatacaaqttaqttqtaqqaqtqtaqaqaqactqtaaqtaqtacat 1794 1795 cctqtaqtcctaqaqaqqcqqtcacatctqcccttacattcaaaaqcqacqaccaaqaaattccaqcattcat-tttaacc 1874 1875 accacatqactatattttttttctatttttgttttatataaaaaqacttaatqqcaatatccaqacaacqccattqtqat 1954 1955 qattttttttcaaaqaaaaaactaaaaqctttaaatt-tccacqqcttctqq-tcctgcccctatttaaataaaqaaqqtql: 2034 * 20 35 atqtactatgtctttggaatgatttattttgcatgttgtttgtgtatagaagaatgtgttgtatggactacaaacaaaac 2114 2115 gtagctggctatattattaattgaaaaacaaatttatacattttccttcacagattatttaccttatatattatactttt 2194 2195 gattgagaattgatttttctcatctataaaatatccttattatggtacgaaatttttatcactatacatatatgtgtaga 22 74 2275 cacagataaagagaacttttgtaggtgtactaatttcgtggacattgttcatgttacatttgccatgtgaccaagactag 2354 2 355 ttagctgtacaggagggaaaagcgagaactattttgtcatgtgacaactgtagacggaagactctagtgtttgtcatgtg 24 34 24 35 actgtcatgtgacaactgtagacggaagactcctagtattcctcacaattcagcttccattgcatttccctggatattgc 2514 * 2515 caatttgttttaaccaaca a t a aacttgtattgcttacaaaaaaaaaaaaaaaaaaaaaaaaaaaaa 25 80 Figure 1. Nucleotide sequence and the derived amino acid sequence for sea scallop tropomyosin (nucleotides and amino acids follow standard abbreviations). The nucleotide residues are numbered from the 5' end of clone PmC 128. The amino acid residues are numbered from first in-frame methionine (M). The polyadenylation signals are in bold. The stars indicate the end of transcripts attributable to polvadenylation at different sites. Sequence for the 3' UTR probe is underlined. Tropomyosin cdna in Sea Scallops 69 Kb 4.40 2.37 . 1.35 abed e f g Kb M A B « Figure 2. Northern analysis of sea scallop tropomyosin cDNA. Fifteen ug of total RNA in each lane was elect rophoresed on a formaldehyde agarose gel, blotted to a nylon membrane, and probed with a ,2 P- labeled 3'UTR probe. Source of RNAs are from adductor muscle (lanes a & b), gonad (lane c), heart (lane d). liver (lane e), mantle (lane f), and gill (lane g). The membrane was exposed for one hour at -70 C with an intensifying screen. 2.02- I Figure 4. Polymorphisms in the region surrounding the sea scallop tropomyosin gene. Each lane contains Hae Ill-digested DNA (10 ug) from a different animal hybridized with a tropomyosin cDNA coding region probe. DNAs in panel A lanes are from Yarmouth, Nova Scotia, panel B lanes are from Sable Island, Nova Scotia, and panel C lanes are from Newfoundland, Canada. M is digoxigenin-laheled DNA mo- lecular-weight marker (Boehringer Mannheim). el ul. 1989) and transferred to a nylon membrane (Boehringer Mannheim) with a Pharmacia Vacu-Gene XL unit following the manufacturer's protocol No 4. Tropomyosin cDNA probes were amplified from a plasmid clone by the polymerase chain reaction (PCR) using two nested primers. Amplification products were visualized on agarose gels, and the PCR products were excised and purified using a QIAquick Spin PCR Purification Kit (Qiagen Inc). PCR products (25-50 ng) were labeled by random priming with 32 P-dCTP(3000 Ci/mmol) using a Ready-to-go labeling kit (Pharmacia Biotech), and the kb abode 2.69- 1.4 0.42- Figure 3. Southern hybridization of the sea scallop tropomyosin cod- ing region to genomic DNA. The blot contains DNA from a single animal digested with EcoR I (lane a), EcoR V (lane b). Hind III (lane c), Sal I (lane d), and Xba I (lane e). unincorporated nucleotides were removed using Nick columns (Pharmacia Biotech). Prehybridization and hybridization of northern blots were car- ried out in a hybridization oven at either 55 or 65°C in 15 mL of hybridization buffer (0.25 m Na,HP0 4 , pH 7.2. 7% SDS, 50 mg/ mL sheared, denatured salmon sperm DNA). Hybridization was for 24-36 h with 1-2 x 10 6 cpm denatured probe. The membranes were then washed twice for 40-50 min each in 20 mm Na,HP0 4 . pH 7.2, 5% SDS and twice for 35-45 min each in 20 mm Na 2 HP0 4 . pH 7.2, 1% SDS at the same temperature used for hybridization. To characterize the major transcripts of scallop adductor muscle, 130 plaques from an adductor muscle cDNA library were randomly selected and sequenced on each end. About 4% of these clones were identified as encoding tropomyosin. Inserts from five clones (PmC 60, PmC 92. PmC 104, PmC 118. PmC 128) were subcloned and sequenced completely in both directions. RESULTS AND DISCUSSION The five DNAs characterized by sequencing (PmC 60, PmC 92, PmC 104, PmC 1 18, PmC 128) can be divided into two groups on the basis of size. PmC 60. PmC 104, and PmC 1 18 are approxi- mately 2.030 base pairs (bp) in length (excluding the polyA tail), and PmC 90 and PmC 128 are 2,531 and 2,546 bp. respectively. The slight differences in length within each group are caused by incomplete first strand cDNA synthesis; whereas, the difference between the two groups is caused by different lengths of the 3' untranslated region (3'-UTR). The two larger clones have a 1,550 nucleotide 3'-UTR. and that of the shorter clones is 1.036 nucle- otides. The difference in the 3'-UTR region seems to arise from polyadenylation at two different sites, with the shorter clones re- sulting from the recognition of a polyadenylation signal (AATAAA) at position 2020, whereas, the longer clones result from utilization of the polyadenylation signal at 2533 (Fig. 1 ). The use of alternative polyadenylation sites has been found previously in tropomyosins from a wide range of organisms (Balvay and Fiszman 1994). All five cDNA clones represent transcripts from the same tro- 70 Patwary et al. pomyosin gene, because they are identical in both their coding and noncoding nucleotide sequences. The cDNAs encode an open reading frame of 284 amino acid residues, with a predicted mo- lecular mass of 30.280 d. Sea scallop tropomyosin is approxi- mately 70% identical to other molluscan tropomyosins. 60% iden- tical to those of flukes. 55% identical to those of insects and worms, and 52% identical to vertebrate tropomyosins: the tro- pomyosin cDNA described herein may be useful as a heterologous probe. The observation that all five cDNA clones encode the same protein suggests that in adductor tissue, which contains both stri- ated and smooth muscle (Chantler 1991), both muscle types ex- press this tropomyosin isoform. Northern hybridization with a 3'-noncoding region probe to total RNA from several sea scallop tissues revealed intense signals only in the adductor muscle lanes (Fig. 2). Two bands consistent with the sizes of the two cDNAs that were isolated were seen, with the smaller, approximately 2.1 kilobase (kb) band present in greater abundance. Upon longer exposure, faint signals were also detected in lanes from other tissues (results not shown), indicating that the gene represented by this cDNA is also expressed in many scallop tissues. The cDNA we report here seems to represent the principal tropomyosin gene expressed in sea scallop adductor muscle. The number of genes encoding tropomyosin in sea scallop was estimated by southern hybridization with a probe covering most of the coding region (Fig. 3). The results indicate that there are only a few (1-3) tropomyosin genes in sea scallop, and similar results were obtained with a shorter probe from the 5' end of the coding region (not shown). As a part of an effort to develop DNA-based genetic markers to conduct population genetic studies on sea scallop (Patwary et al. 1994a. Patwary et al. 1994b. Patwary et al. 1996). we examined the utility of tropomyosin cDNA as a probe to reveal polymorphisms. A genomic blot containing Hae Ill-digested DNAs from 12 sea scallops from three distant locations was probed with a tropomyo- sin coding region probe. The probe revealed a highly polymorphic locus with a total of six alleles (Fig. 4). Although heterozygote deficiency has been reported to be common in sea scallop popu- lation (Foltz and Zouros 1984. Beaumont and Zouros 1991). this locus is highly heterozygous. Although the polymorphisms re- vealed here are limited by a small sample size, the tropomyosin probe seems to be a useful marker for various genetic studies in sea scallop. ACKNOWLEDGMENTS This project was supported in part by funds from the Depart- ment of Fisheries and Oceans. Canada through a contract to the NRC Institute for Marine Biosciences and in part by an NSERC Strategic Grant to E.K. and Prof. E. Zouros. Dalhousie University, Canada. LITERATURE CITED Balvay. L & M. Y. Fiszman. 1994. Analyse de la diversite des isoformes de tropomyosine. C. R. Soc. Biol. 18:527-540. Beaumont. A. R. & E. Zouros. 1991. Genetics of sea scallops. In: S. E. Shumway (ed.). Scallops: Biology. Ecology, and Aquaculture. Elsevier, Amsterdam. Chantler, P. D. 1991. The structure and function of scallop adductor muscles. In: S. E. Shumway (ed.). Scallops: Biology. Ecology, and Aquaculture. Elsevier. Amsterdam. Daul, C. B.. M. Slattery. G. Reese & S. B. Lehrer. 1994. Identification of the major brown shrimp (Penaeus aztecus) allergen as the muscle pro- tein tropomyosin. Int. Arch Allergy Immunol. 105:49-55. Foltz. D. W. & E. Zouros. 1984. Enzyme heterozygosity in the scallop Placopecten magellanicus (Gmelin) in relation to age and size. Mar. Biol. Lett. 5:255-263. Hammell. R. L. & S. E. Hitchcock-DeGregori. 1996. Mapping the func- tional domains within the carboxyl terminus of a-tropomyosin encoded by the alternatively spliced ninth exon. J. Biol. Chem. 271:4236—1242. Hanke, P. D. & R. V. Storti. 1988. The Drosphila melanogaster tropomyo- sin II gene produces multiple proteins by use of alternative tissue- specific promoters and alternative splicing. Mol. Cell. Biol. 8:3591- 3602. Lees-Miller, J. P. & D. M. Helfman. 1991. The molecular basis for tro- pomyosin isoform diversity. BioEssays 13:429—437. Leung. P. S. C. K. H. Chu, W. K. Chow. A. Ansari. C. 1. Bandea. H. S. Kwan. S. M. Nagy & M. E. Gershwin. 1994. Cloning, expression, and primary structure of Metapenaeus ensis tropomyosin, the major heat- stable shrimp allergen. J. Allergy Clin. Immunol. 94:882-890. Patwary. M. U.. E. L. Kenchington, C. J. Bird & E. Zouros. 1994a. The use of random amplified polymorphic DNA markers in genetic studies of the sea scallop Placopecten magellanicus (Gmelin, 1791). J. Shellfish Res. 13:547-553. Patwary. M. U.. R. M. Ball. C. J. Bird. B. Gjetvaj. S. Sperker, E. L. Kench- ington & E. Zouros. 1994b. Genetic markers in sea scallop and their application in aquaculture. Bull. Aquacult. Assoc. Can. 2:18-20. Patwary, M. U.. M. Reith & E. L. Kenchington. 1996. Isolation and char- acterization of a cDNA encoding an actin gene from sea scallop (Pla- copecten magellanicus). J. Shellfish Res. 15:265-270. Pittenger. M. F.. J. A. Kazzaz & D. M. Helfman. 1994. Functional prop- erties of nonmuscle tropomyosin isoforms. Curr. Opinion Cell Biol. 6:96-104. Sambrook. J.. E. F. Fritsch & T. Maniatis. 1989. Molecular cloning — a laboratory manual. Cold Spring Harbor Laboratory Press. New York. Shanti, K. N., B. M. Marin. S. Nagpal. D. D. Metcalfe & P. V. S. Rao. 1993. Identification of tropomyosin as the major shrimp allergen and characterization of its IgE-binding epitopes. J. Immunol. 151:5354- 5363. Witteman. A. M., J. H. Akkerdaas. J. V. Leeuwen. J. S. van der Zee & R. C. Aalberse. 1994. Identification of a cross-reactive allergen (pre- sumably tropomyosin) in shrimp, mite, and insects. Int. Arch Allergy Immunol. 105:56-61. Journal of Shellfish Research, Vol. IS. No. 1. 71-76, 1999. GROWTH CHARACTERISTICS OF CHLAMYS FARRER1 AND ITS RELATION WITH ENVIRONMENTAL FACTORS IN INTENSIVE RAFT-CULTURE AREAS OF SISHILIWAN BAY, YANTAI HONGSHENG YANG, TAO ZHANG, JIAN WANG, PING WANG, YICHAO HE, AND FUSUI ZHANG Institute of Oceanology Chinese Academy of Sciences Qingdao 266071 Peoples Republic of China ABSTRACT The growth characteristics of the scallop Chlamys farreri under intensive raft-culture and its relationships with major environmental factors were studied. The experiment was conducted from May 1997 to April 1998 in four farming areas of Sishiliwan Bay, Yantai, China. The instantaneous growth rates of shell height, wet weight, fresh and dried soft tissue of scallops were measured and calculated during the course of this study. The results showed obvious seasonal variation in the growth in Sishiliwan Bay. The main factors affecting the growth rate of the scallops were water temperature and food supply in the farming areas. The growth rates of the scallops cultured in Jinggouwan and Yueliangwan areas were faster than that in Kongtongdao and Qingqiangzhai areas. The rela- tionships between the instantaneous growth rates in dried tissue weight of C. farreri with water temperature, and particulate organic matter (POM) in Jinggouwan area and Yueliangwan area were simulated. Growth rate declined when water temperature was below 5°C. Between 5 and 23°C, growth rate increased with the increasing of water temperature. Growth rate sharply declined when water temperature was above 23°C. The scallops stopped growing when POM was less than 0.90 mg/L and grew rapidly with increasing POM. When POM was above 3.67 mg/L. the growth rate of the scallops decreased again. KEY WORDS: Sishiliwan Bay, C. farreri. instantaneous growth rate, intensive raft-culture, temperature, particulate organic matter, scallop, manculture INTRODUCTION The scallop C. farreri is the main cultured species over the coastal farming areas in the northern China Sea. In the recent decade, the growth rate of C. farreri sharply declined because of high culture density and exhaustion of food supply. Furthermore, spats used for farming were mostly collected from the recruitment reproduced by the cultured and possibly inbred stock in recent years. Mass mortality of this species occurred in most of the farm- ing areas in the northern China Sea, especially during the summer and the autumn 1997, and the mortality rate was above 60.0%. The exact cause for the mortality remains unknown. Therefore, it is necessary to study the growth characteristics of cultured scallops systematically among the different farming areas, and its relation- ship with the major environmental factors, such as water tempera- ture and availability of natural food supply. The main factors influencing growth are water temperature and the amount of food ingested. From feeding experiments carried out by Winter and Langton (1976) with Mytilus edulis, it is ob- vious that with increasing amounts of food available, there is an increase in growth rate. Since 1970s, the growth and its relation- ship with the main environmental factors have been studied in detail, in many pectinids, including Amusium japonicum balloti (Williams and Dredge 1981), Argopecten irradians (Briceli et al. 1987, Cahalan et al. 1989, Duggan 1972. Kirby-Smith and Barber 1974. Rhodes and Widman 1984, Zhang et al. 1987, Zhang et al. 1991a, Zhang et al. 1991b). Chlamys islandica (Vahl 1980), Chlamys opercularis (Broom and Mason 1978. Taylor and Venn 1978), Chlamys varia (Conan and Shafee 1978, Shafee 1980), Pecten alba (Gwyther and Mcshane 1988), Patinopecten caurinus (Haynes and Hitz 1971), Placopecten magellanicus (Macdonald 1986, Shumway et al. 1987). and Pecten maximus (Mason 1970). MATERIALS AND METHODS Sishiliwan Bay, Yantai, and its near sea areas, located on near Yantai city ( 121°20'-40'E, 37°25'-40'N) including Zhifuwan Bay, Jinggouwan Bay, and Sishiliwan Bay, is 26 km in width, and 13 km farthest from the shore. It is ear-shaped and half enclosed. The mouth of the bay faces eastward and is divided into two parts by Kongtondao Island. The smaller one is between Zhifudao Island and Kongtondao Island, and the larger one between Kongtongdao Island and Yangmadao Island (See Fig. 1). The bay has a muddy and sandy bottom. The total area is about 13,000 ha. and the depth is about 9-15 meters. Sishiliwan Bay is one of the earliest farming areas, where the kelp Laminaria japonica raft-culture was developed in 1949. At the end of 1960s, researchers from the Institute of Oceanology, Chinese Academy of Sciences, and other institutions had studied the artificial collection of mussel M. edulis spats and tested raft- culture techniques. The mariculture farms of Yantai had also col- lected C. farreri spats in Jinggouwan area in the middle of 1970s. Between the end of 1970s and the early 1980s, hatchery production of C. farreri seed was successfully developed and used, and the scallop was cultured in large scale. The northern bay scallop Ar- gopecten irradians has been one of the main species cultured in the bay since 1986. Now, M. edulis. C. farreri. A. irradians. and the kelp Laminaria japonica are the primary species under raft-culture in the bay. The culture areas include 830 ha ( 1 ha = 6.000 cages, the same as for mussels) for scallops, 460 ha for mussels, 250 ha ( 1 ha = 6000 strings) for kelps. Mussel and scallop seeds are mainly collected from the wild, and the spats of the bay scallop and the seedling of the kelp are hatchery-produced. Set-Up of Research Stations Four farming areas, Jinggouwan farming area, Yueliangwan farming area. Kongtongdao farming area, and Quingquanzhai 71 72 Yang et al. 1.4 Figure 1. Main farming areas in the Sishiiwan Bay. Yantai. A: Jing- gouwan, B: Yueliangwan, C: Kongtongdao, and I): Qingquanzhai; 1: Zhifudao Island, 2: Kongtongdao Island, 3: Yangmadao Island. farming area in Sishiliwan Bay were chosen in this study. In each farming area, three stations were set, totaling 12 stations. Sampling The specimens sampled in May to September 1997. were 1-year scallops, which were collected in the spring of 1996, and those sampled in October 1997 to April, 1998 were 1-year scallops collected in the spring of 1997. Fifty scallops were collected monthly at each station. After being taken back to the laboratory, the specimens were cleaned (the epibionts were cut off) and boiled, the adductor muscle and the viscera were divided, and the shell height, wet weight, shell weight, wet and dried weight of soft tissue (65°C. for 48 h) were measured. Water temperature, salinity, the biomass of seston and particulate organic matter (POM) were determined at the same time. The seston was filtered by GF/C and dried at 65°C for 48 h and weighed, and then washed at 450°C for 6 h and weighed again. The biomass of POM equals the difference between the weight of dried sestons minus that of ash. Calculation The instantaneous growth rates of the shell height, wet weight, the fresh and dried weight of the soft tissues were calculated using the following equation IGR = ((Ins, - Ins, )/t) x 100. IGR stands for instantaneous growth rate, s, stands for initial shell height, wet weight, the fresh or dried weight of the soft tissues measured first 1.2 1 ia> u 0.8 u sz 06 o a. o 0.4 -D — Jinggouwan area -• — Yueliangwan area :v*: > • S-0 N-D F-M A-M Month J-J A-S Figure 3. Annual variations of instantaneous growth rate in shell height of ('. farreri in Jinggouwan area and Yueliangwan area. time, s 2 for ending shell height, wet weight, the fresh or dried weight of the soft tissues, and t for the interval days between initial and ending measurement. RESULTS AND ANALYSIS Annual Variation of the Growth of C. farreri in Relation to Water Temperature The surface water temperature and salinity variations of Sishi- liwan Bay. Yantai are illustrated in Figure 2. The lowest and highest water temperatures are observed in February and August, respectively. The annual fluctuation of salinity is small, around 29.95 ± 0.51. The annual variations of instantaneous growth rates of shell height, wet weight with shell, fresh and dried weight of soft tissue in Jinggouwan and Yueliangwan farming area are shown in Figures 3. 4, 5. and 6. It is obvious that the growth of C. farreri in two areas varies with the season. The growth rate of the scallops is fastest from May to July. The relationships between the instantaneous growth rate of soft tissue and water temperature are similar at these two areas (Fig. 7). The regression equations are as follows. Jinggouwan farming area IGR = - 0.003 IT 1 + 0.091 IT 2 - 0.4568T + 0.6218, r = 0.9788 Yueliangwan fanning area IGR = - 0.0027T 3 + 0.0780T 2 - 0.3801T + 0.4635. r : 0.9916 J F M A M J J Month A S O N D Z 3.5 3 2.5 2 1.5 I 0.5 S-0 Jinggouwan area Yueliangwan area N-D F-M A- Month ■M J-J A-S Figure 2. Annual variation of water temperature and salinity in the Figure 4. Annual variations of instantaneous growth rate in wet Sishiiwan Bay, Yantai. weight of C. farreri in Jinggouwan area and Yueliangwan area. Growth Characteristics of C. farreri 73 s-o N-D F^Vl A~M Month J-J A-S Figure 5. Annual variations of instantaneous growth rate in fresh soft weight of C. farreri in Jinggouwan area and Yueliangwan area. a y // ' ^^x / ^\ / ° \ D Jinggouwan area /a Yitliangwanarea o\ 8^ a/ ) 5 10 15 20 25 Temperature(°C) Figure 7. The relationship between water temperature and the instan- taneous growth rate of dried soft tissue of C. farreri in Jinggouwan and Yueliangwan areas. IGR stands for the instantaneous growth rate of soft tissue and T for water temperature (°C) These equations clearly show that there is a strong correlation between the growth of C. farreri and water temperature. The growth rate is slow when water temperature is below 5°C, and then increases sharply with increasing temperature. The fastest growth rate is at 16-18°C. The growth rate sharply declined when water temperature was over 23°C. Variation Among Different Culture Areas Results from the main growth period. May to September, show that there are differences in instantaneous growth rate of shell height, wet weight, fresh and dried weight of soft tissue in the four culture areas (Figs. 8, 9, 10, 1 1). especially from May to August. The dried weight of soft tissue in Yueliangwan and Jinggouwan areas increases fastest from May to July, and the growth rate in these two areas is faster than those in Kontongdao and Qingquan- zhai areas. The growth rate of C. farreri in Kontongdao and Qingquanzhai areas is slow, and their instantaneous growth rate varies little. That is quite different from the scallops cultured in Yueliangwan and Jinggouwan areas, where the growth rate is rela- tively faster in May to July, and gradually declines after that pe- riod. It is necessary to note that scallops in these four farming areas mostly died by September 18. 1997, up to 80.0%. The instanta- neous growth rate of the survivors decreased considerably, espe- cially the instantaneous growth rate of dried soft tissue. Relationship Between the Growth and POM Biomass The measurements of the biomass of seston and POM are listed in Table 1. The differences in the biomass of seston and POM are obvious among different culture areas. The relationship between the instantaneous growth rate of dried soft tissue and the biomass of POM can be formulated as IGR = -0.5695[POM] : + 4.1780[POM] - 3.4031. r = 0.9338 (Fig. 12). It shows that the instantaneous growth rate of dried soft tissue of C. farreri tends to be zero when the biomass of POM is less than 0.90 mg/L and increases with the increasing POM biomass. The scallop growth rate declines when the POM is more than 3.67mg/ L. It is clear that the growth of C. farreri is limited in some degree by the abundance of natural foods in the farming area. DISCUSSION Pectinids can be partitioned into four broad groups according to their patterns of life history (Orensanz et al. 1991 ): ( 1 ) long-lived, iteroparous species; (2) short-lived, iteroparous species: (3) short- lived, semelparous or quasi-semelparous species; and (4) small- sized, presumably short-lived, brooding species. The first group o o oi O 3.5 3 2.5 2 Jinggouwan area Yueliangwan area _J N-D F-M A- Month M J-J A-S OB J3 O 1.6 1.2 0.8 0.4 M-J ---A -B a -D „ ■ "~~"~~4 J-J Month J-A A-S Figure 8. The instantaneous growth rate in shell height of C. farreri in Figure 6. Annual variations of instantaneous growth rate in dried soft the culture areas. A: Jinggouwan, B: Yueliangwan, C: Kongtongdao, weight of C. farreri in Jinggouwan area and Yueliangwan area. and D: Qingquanzhai. 74 Yang et al. m-j J-J J-A A-S Month Figure 9. The instantaneous growth rate in wet weight of C. farreri in the culture areas. A: Jinggouwan, B: Yueliangwan. C: Kongtongdao, and I): Qingquanzhai. can be further divided into two types: large-sized (above 100 mm), relatively long-lived (maximum longevity usually above 12 years) species, and medium-sized (60-100 mm) species with maximum longevity usually less than 10 years. The scallop C. farreri belongs to the second type of the first groups. There are a lot of environ- mental factors influencing the growth of scallops, mainly being water temperature, water current, the biomass of natural food, culture density, and the amount of other filter-feeding animals within the farming area (Zhang et al. 1987. Zhang et al. 1991a, Zhang et al. 1991b). Water temperature and the biomass of natural food in culture area might be the most important factors affecting the growth of C. farreri. In most previous studies (Lou 1991. Wang et al. 1993. Zhang et al. 1956). the shell height of C. farreri was used to measure the growth of the scallops. Their results reflected that. C. farreri grow fast in the months in which water temperature is high, and vice versa in normal culture conditions. In the winter. C. farreri totally stop growing. From March in each year, the growth of C. farreri increases gradually with the increasing water temperature and reaches its peak in July. When water temperature is above 25°C, the growth obviously decreases. From January to March, the water o et Z Month Figure 11. The instantaneous growth rate in dried soft weight of C. farreri in the culture areas. A: Jinggouwan, B: Vueliangwan, C: Kong- tongdao, and D: Qingquanzhai. temperature is lower than 5°C, the growth of shell is nearly zero. In this paper, the instantaneous growth rate was used for the first time to describe the growth of this species, and the resulting model with the instantaneous growth rate and water temperature support the viewpoints above. A combination of many environmental fac- tors might have strongly affected the growth of C. farreri, leading to the death of most C. farreri in this area in 1997. The results of this study differ from previous studies on the influence of high temperature on the growth of C. farreri: when temperature is over 23°C, the instantaneous growth rate of dried weight of soft tissue of C. farreri sharply declines. Our findings indicate that the growth of C. farreri varies dif- ferently with different areas in the same season. The environmental differences of farming areas include the difference in water cur- rent, concentration of nutrients, primary production, and stocking density. The variation in water quality and food supply, especially the food supply, has an obvious influence on the growth of C. farreri. The biomass of POM is higher in the Yueliangwan and Jinggouwan areas than that in the Kongtondao and Qingquanzhai fanning areas. The Yueliangwan area lies upstream to the Sishi- liwan Bay (following the direction of water current), and it is the at o Month Figure 10. The instantaneous growth rate in fresh soft weight of C. farreri in the culture areas. A: Jinggouwan. B: Vueliangwan, C: Kong- tongdao, and D: Qingquanzhai. 4.5 * 3.5 3 2.5 Pi O 2 - 1.5 - 1 < 0.5 J 0<5> O 0.5 1 1.5 4.5 5 2 2.5 3 3.5 POMbioinass(mg/l) Figure 12. The relationship between the instantaneous growth rate in dried soft weight of C. farreri and the biomass of POM. Growth Characteristics of C. farreri 75 TABLE 1. Biomass of seston (S, mg/L) and particulate organic matter (POM, mg/L) in the farming areas of Sishiliwan Bay. Jinggouwan Yueliangw an Kongtongdao Qingqianzhai Sampling Date S POM S POM S POM S POM May 15 11.28 2.70 8.55 4.72 4.47 0.99 13.80 2.28 June 18 3.66 2.17 6.26 4.69 3.11 1.52 3.03 1.70 July 15 2.07 1.15 3.46 2.19 2.54 1.29 2.30 1 .03 Aug. 20 2.12 1.24 2.84 1.57 1.97 1.11 2.18 1.02 Sept. 18 3.48 1 .05 3.64 1 .53 4.37 2.03 3.93 1.07 front part of the whole culture area (Yantai Port is above it). The Jinggouwan farming area is downstream of it. and the Qingquan- zhai farming area is at the bottom of the bay. and water current in the Kongtongdao farming area is slow, and the culture density is highest. The modeling results of this study suggest that, when the bio- mass of POM is lower than 0.90 mg/L, the instantaneous growth rate of dried soft tissue tends to be zero; the growth rate increases with the increasing biomass of POM, and then the growth rate declines as the biomass of POM is above 3.67 mg/L. Roland and Brown (1990) established the relationships between POM and the growth of the Crassostrea gigas. which is similar to ours, except that the upper limit of POM that restricts the growth of C. farreri is 5.00 mg/L. The metabolism of C. farreri increases with the increasing temperature from 10°C to 23°C (Yang et al. 1998). The depiction of natural foods and the food selection of C. farreri (Wang et al. 1989) make the energy consumed by the scallops for metabolism, rather than for growth. The overabundance of natural foods limits the growth of filtering-food bivalves, because of the increased mucus excretion and the pseudofeces production of scal- lops, which consume large amounts of energy and lead to the discharge of carbon and nitrogen of the scallops. ACKNOWLEDGMENTS This work was supported by the National Commission of Sci- ence and Technology of China, Grant No. 96-922-02-04. and by Chinese Academy of sciences. Grant No. KZ951-A 1-102-02. Con- tribution No. 3501 from the Institute of Oceanology, Chinese Academy of Sciences. LITERATURE CITED Broom. M. J. & J. Mason. 1978. Growth and spawning in the pectinid Chlamys opercularis in relation to temperature and phytoplankton con- centration. Mar. Biol. 47:277-285. Bricelj, V. M. & S. E. Shumway. 1991. Physiology: energy acquisition and utilization, pp. 305-346. In: S. E. Shumway (ed.). Scallops: Biology. Ecology, and Aquaculture. Elsevier. Amsterdam. Bricelj, V. M. J. Epp & R. E. Malouf. 1987. Intraspecific variation in reproductive and somatic growth cycles of bay scallops Argopecten irradians irradians (Lamarck): mortality, growth, and oxygen con- sumption. J. Exp. Mar. Biol. Ecol. 112:373-397. Cahalan J. A., S. E. Siddall & M. W. Luchenbach. 1989. Effects of flow velocity, food concentration, and particle flux on growth rates of ju- venile bay scallops Argopecten irradians. J. Exp. Mar. Biol. Ecol. 129:45-60. Conan, G. & M. S. Shafee. 1978. Growth and biannual recruitment of the black scallop Chlamys varia (L.) in Lanveoc area. Bay of Brest. J. Exp. Mar. Biol. Ecol. 35:59-71. Duggan. W. P. 1972. Growth and survival of the bay scallop. Argopecten irradians, at various locations in the water column and at various densities. Proc. Natl. Shellfish Assoc. 63:68-71. Gwyther. G & P. E. Mcshane. 1988. Growth rate and natural mortality of the scallops Pecten alba Tate in Port Phillip Bay, Australia, and evi- dence for changes in growth rate after 20-year period. Fish. Res. 6: 113-131. Haynes, E. B. & C. R. Hitz. 1971. Age and growth of the giant Pacific sea scallop. Patinopecten caurinus. from the Strait of Georgia and outer Washington coast. J. Fish. Res. Bd. Can. 28:1335-1341. Kirby-Smith. W. W. & R. T. Barber. 1974. Suspension feeding aquaculture systems: effects of phytoplankton concentration and temperature on growth of the bay scallop. Aquaculture 3:135-145. Lou, Y. 1991. China, pp. 809-824. In: S. E. Shumway (ed.). Scallops: Biology. Ecology, and Aquaculture. Elsevier. Amsterdam. MacDonald. B. A. 1986. Production and resource partitioning in the giant scallop Placopecten magellanicus grown on the bottom and in sus- pended culture. Mar. Ecol. Prog. Ser. 34:79-86. Mason, J. 1970. The age and growth of the scallop. Pecten maximus L„ in Manx waters. J. Mar. Biol. Assoc. U.K. 37:653-671. Orensanz, J. M., A. M. Parma & O. O. Iribarne. 1991. Population dynamics and management of natural stocks, pp. 625-713. In: S. E. Shunway (ed.). Scallops: Biology. Ecology, and Aquaculture. Elsevier. Amster- dam. Rhodes. E. W. & J. C. Widman. 1984. Density-dependent growth of the bay scallop, Argopecten irradians Indians, in suspension culture. ICES CM 1984/k:18. 12 pp. Roland. W. G & J. R. Brown. 1990. Production model for suspended culture of the Pacific oyster, Crassostrea gigas. Aquaculture 87:35-52. Shafee. M. S. 1980. Application of some growth models to the black scal- lop. Chlamys varia (L.) from Lanveoc, Bay of Brest. J. Exp. Mar. Biol. Ecol. 43:237-250. Shumway S. E.. R. Selvin & D. E. Schick. 1987. Food resources related lo habitat in the scallop. Placopecten magellanicus (Gmelin. 1791): a qualitative study. J. Shellfish Res. 6:89-95. Taylor, A. C. & T. J. Venn. 1978. Growth of the queen scallop. Chlamys opercularis, from de Clyde Sea area. J. Mar. Biol. Soc. 54:9-28. Vahl, O. 1980. Seasonal variation in seston and in the growth rate of the Iceland scallop. Chlamys islandica (O. F. Muller) from Balsfjord. 70°N. J. Exp. Mar. Bio. Ecol. 48:195-204. Wang. R.. X. Lan & L. Liu. 1989. Analysis of the scallop Chlamys farreri food. J. Ocean Univ. Qingduo 19:12-18. Wang, R., Z. Wang & J. Zhang. 1993. Molluscan mariculture. Ocean University of Qingdao Press. Qingdao. China, pp. 155-204. 76 Yang et al. Winter. J. E. & R. W. Langton. 1976. Feeding experiments with Mytitm editlis L. at small laboratory scale. I. The influence of the total amount of food ingested and food concentration on growth, pp. 565-581. In: G. Persoone and E. Jaspers (eds. ). Proceedings of the 10 lh European Sym- posium on Marine Biology Ostend. Belgium. Sept. 17-23, 1975. Uni- versa Press. Wetteren. Williams, M. J. & M. C. L. Dredge. 1981. Growth of the saucer scallop Amusium japonicum balloti Bernardi in central eastern Queensland. Aust. J. Mar. Freshwater Res. 32:657-666. Zhang. X., Z. Qi & J. Li. 1956. Observation on the reproduction and growth of scallop. Chlamys farreri. J. Zool. China 8:235-253. Zhang. F.. Y. He & J. Ma. 1987. A repon on the experimental cultivation of bay scallop and kelp by turns. Marine Sci. 6:1-6. Zhang. F.. Y. He & X. Liu. 1991a. Growth and mortality of bay scallop Argopecten irradians cultured at various water layers in Jiaozhou Bay. J. Fisheries China 15:42^17. Zhang. F., J. Ma & Y. He 1991b. Growth and mortality of bay scallop Argopecten irradians cultured in different vessels and different density in Jiaozhou Bay. Mar. Sci. 2:1-2. Yang, H.. T. Zhang, Wang P. et al. 1998. Effects of temperature on oxygen consumption and ammonia-N excretion of Chlamys farreri. Chinese J. Oceanol. Limnol. 16:167-172. Journal of Shellfish Research. Vol. IS, No. 1. 77-83. 1999. CULTURE OF MERCENAR1A MERCENARIA (LINNAEUS): EFFECTS OF DENSITY, PREDATOR EXCLUSION DEVICE, AND BAG INVERSION EVA M. FERNANDEZ, 1 JUNDA UN, 1 AND JOHN SCARPA 2 'Florida Institute of Technology Melbourne, Florida 32901 'Harbor Branch Oceanographic Institution, Inc. Fort Pierce, Florida 34946 ABSTRACT Growth, survival, and condition index (CI) of the northern quahog, Mercenaria mercemiria (Linnaeus, 1758), cultured in nylon mesh bags ( 1.2 x 1.2 m) were assessed against density and predator exclusion device (PED: Vexar net with 2.5-cm openings) in the northern Indian River Lagoon at Oak Hill. Florida. Nursery seed [mean ± SD: 6.0 ± 0.8 mm shell length (SL)] were stocked in February 1997 at densities of 7.500 (5.210). 10.000 (6.944), and 12,500 (8,680) clams/bag (clams/nr) (n = 4) and monitored until the end of May 1997. Two replicates of each treatment were inverted 5 weeks before harvesting to smother fouling organisms and examine their influence on growth. Growout seed (mean ± SD: 21.1 ± 1.7 mm SL) were stocked in October 1996 at densities of 750 (521 ), 1.000 (694). and 1.250 (868) clams/bag (clams/irr) (n = 4) and monitored until early June 1997. At the end of the nursery seed experiment, the average final SL of clams was significantly different among the density treatments (p = .03) and not significantly different between the PED (p = .31) treatments. Nursery seed in the inverted bags were significantly larger (p = .03). and a higher percentage of them reached growout seed size ( 12 mm in SL). Density (p = .60) did not have a significant effect on survival; whereas, the bags with PED had significantly (p = .005) lower survivorship than that of the bags without PED. Density (p = .15) and PED (p = .79) did not significantly affect mean CI at the end of the study, but inversion significantly (p = .002) increased CI. At the end of the growout seed experiment, SL was not significantly different among the treatments (density, p = .40; PED. p = .17). There was a significant (p = .04) effect of density on percentage of the seed that reached legal harvest size (16 mm in shell thickness). In general, percentage of seed that reached harvest size decreased with increasing density. The effects of density (p = .04) and PED (p = .0009) on survival were significant, but there was no apparent pattern. Density (p = .29) and PED (p = .88) did not affect mean final CI. Chlorophyll a concentration and water current speed measured in April and May. 1997 indicated that food was not a limiting factor on growth of the northern quahog at the study site. Our recommendations for northern quahog culture in the Oak Hill area are: ( 1 1 use a planting density of 7.500 clams/bag for nursery seed and 750 clams/bag for growout seed; (2) could use PED to reduce fouling on the culture bags, although PED may not improve clam survivorship; and (3) invert culture bags periodically. KEY WORDS: Mercenaria mercenaria, density, Florida, fouling INTRODUCTION The northern quahog. Mercenaria mercenaria (Linnaeus, 1758). is a commercially important shallow-water species that has been harvested by subsistence fishermen since pre-Columbian times and today supports an important commercial and recre- ational fishery (Crawford 1992). Since the early 1950s, the feasi- bility of artificially enhancing the commercial crop of cultured northern quahog has been studied intensively (MacKenzie 1979). Some of the factors affecting the growth and survival of commer- cial northern quahog are planting density and predation (Flagg and Malouf 1983). Most studies dealing with densities found that high planting density resulted in slower growth of clams (Eldridge et al. 1976, Eldridge et al. 1979. Hadley and Manzi 1984, Walker 1984). although others indicated no significant difference in final size (Godwin 1968. Summerson et al. 1995). A major inhibition to commercially viable northern quahog culture has been predation (Carriker 1959. Menzel and Sims 1964. Haven and Loesch 1973. Menzel et al. 1976. MacKenzie 1977. Whetstone and Eversole 1978, Steinberg 1980. Castagna and Kraeuter 1981. McHugh 1981. Peterson et al. 1995). with crabs representing the most serious problem from Massachusetts to Texas (MacKenzie 1977. Virnstein 1977, Boulding and Hay 1984. Jory et al. 1984, Hines et al. 1990, Eggleston et al. 1992, Sum- merson et al. 1995. Kraeuter et al. 1998). Several techniques have been developed to minimize predation, including rafts, trays, cages, nets (Manzi et al. 1980). addition of gravel to inhibit for- aging effectiveness (Castagna and Kraeuter 1977). introduction of organisms that consume clam predators (Castagna and Kraeuter 1981, Castagna 1984, Jory et al. 1984, Bisker and Castagna 1989) and combinations of the various methods (Kraeuter and Castagna 1985). Despite the partial effectiveness of these control measures, predation remains a critical factor in northern quahog aquaculture. The field culture of bivalve mollusks is also dependent on the production and supply of phytoplankton and other food sources. Seston depletion is a major influence on cultured suspension feed- ers whose growth can be limited by both food quality and quantity (Fegley et al. 1992). It has proved most economical to grow quahogs in the natural environment at controlled densities, because space and food re- quirements increase exponentially as clams grow (Castagna and Kraeuter 1977). Although field nursery and growout offer a low- cost production system for the shellfish, one of the disadvantages is fouling. Fouling can affect production in various ways. The most obvious is a reduction of water flow through the enclosure, which, in turn, decreases food availability (Paul and Davies 1986, Wildish and Kristmanson 1984). In addition, fouling organisms are often themselves filter feeders, so they compete with the cultured species for food resources. Finally, fouling may reduce oxygen supply (Wallace and Reinsnes 1985). Several solutions have been used to remove fouling: addition of animals that prey upon the biofoulers (Flimlin and Mathis 1993). cleaning and changing structures often (Claereboudt et al. 1994), and inversion of bags (Mojica and Nel- son 1993). In Florida, growth of northern quahogs is more rapid than that observed for northern populations (Jones et al. 1990, Arnold et al. 1 99 1 ) because of the wanner temperature and longer growing 77 78 Fernandez et al. season. The best growth occurs in fall and spring (Eldridge et al. 1976. Eldridge et al. 1979. Menzel 1961). Fouling of culture bags and heavy predation force modifications in culture techniques. such as different mesh size of bags and predator excluding devices (Vaughan et al. 1988), as well as maintenance and cleaning of the system. In Florida, cultured clams reach legal harvest size ( 16 mm in ST) in approximately 9 to 10 months, and the commercial "littleneck" size (25.4 mm in ST, legal harvest size of wild clams) in approximately 12 to 18 months (Vaughan et al. 1988). Clam farming is an important industry in the Indian River Lagoon (IRL) of east central Florida. During the early to middle 1980s, a successful fishery for naturally occurring northern qua- hogs developed in the IRL. Landings from this fishery peaked in 1985 at more than 1.5 million pounds with an estimated value of more than US $8 million (Florida Department of Natural Re- sources 1986). Landings decreased substantially since then. The magnitude and success of this fishery influenced the development of a culture-based fishery in the IRL. The IRL possesses the nec- essary components of a suitable growout site for northern quahog aquaculture (Arnold et al. 1990): area of the lagoon is consider- able, water quality is generally good, and extensive shoreline is available from which to monitor and maintain aquaculture opera- tions. The present study was designed to determine how clam growth, survival, and CI was affected by planting density, predator exclusion device (in addition to the culture bag), and bag inversion at Oak Hill, Florida. MATERIALS AND METHODS Experimental Design This study took place in the IRL at Oak Hill. Florida (Fig. I ). In the IRL. water depth generally does not exceed 1-1.5 m, except near the Atlantic Intracoastal Waterway, and tidal range does not exceed 0.5 m (Sheng et al. 1990). Hatchery-reared northern quahog seed used in this study were produced by Harbor Branch Oceanographic Institution, Inc. in Fort Pierce. Florida. The seed were stocked in bags made of a flexible nylon mesh material. The experiment consisted of two different growing periods. Nursery seed clams were planted on February 27. 1997 and monitored until May 29, 1997 (13 weeks), and growout seed clams were planted on October 10, 1996 and monitored until June 6. 1997 (34 weeks). The experimental densities utilized for the nursery seed were 7.500/bag (5.210/nr), 10,000/bag (6.944/ nr ). and 12.500/bag (8.680/nr). with four replicates for each den- sity. Growout seed densities were 750/bag (521/nr), 1,000/bag (694/nr). and 1,250/bag (868/nr). with four replicates for each density. For both nursery and growout seed experiments, an addi- tional four replicates were planted and covered with PED for each density treatment. The PED was a 1 .6 x 1 .5 m Vexar cover net of 2.5-cm mesh size laid over the bags to exclude predators. Water temperature (to the nearest 0.LC with a thermometer), salinity (to the nearest 1 ppt with a hand-held temperature-compensated re- fractometer (Atago S/Mill), dissolved oxygen (to the nearest 0.01 ppm with a temperature-compensated dissolved oxygen meter (YSI Model 57), current speed (to the nearest 0.01 cm/second) and direction (with a mechanical flowmeter (Model 2030R, General Oceanics Inc.), and Secchi disc depth (to the nearest 1 cm with a 15-cm diameter Secchi disc) were measured weekly. Nursery Seed Nursery seed [mean ± SD shell length (SL): 6.0 ± 0.8 mm; n = 100] were stocked in 24 3-mm mesh size bags (1.2 x 1.2 m) on :a*3Q OAK HILL TrniSVIU-E.:: :3°oo m - atlant;c qcsan SS3AS71AN CHEH!Cp\o, VEHO 3EACW.V 27*-Q'- FT. PIEHCc-rA -E.NSEN 3EAC STUAffS^ ll u 10"30' 10" Figure 1. Map of the Indian River Lagoon, including the study area (Oak Hill). February 27. 1997. The number of seed placed in each bag was determined volumetrically. The bags and cover nets were kept in place on the bottom with metal stakes. PVC pipes were placed underneath the PED at the corners to maintain tension and prevent predators from entering the bags. Growth was assessed by measuring 100 clams per bag for SL, shell height (SH). and ST to the nearest 0.01 mm with Vernier calipers every 4 weeks. Only SL was used in further analysis because of the high correlations between the measurements (r = 0.97 for SL and SH: r = 0.80 for SL and ST). The measured clams were then returned to the bags. At the beginning of the study. 100 clams were sacrificed and dried in an oven at 65°C for 48 hours to determine shell and soft tissue dry weight (Walne and Mann 1975). At the end of the 13-week trial, another sample of 100 animals per bag was similarly sacrificed and measured. During weekly monitoring, any dead clams found were removed and re- corded, but not replaced. Bags and cover nets were inspected weekly and cleaned biweekly to assure proper water flow. Clean- ing consisted of manually removing fouling organisms that grew on the bags. Two replicates from each density and PED treatment combination were inverted on April 27. 1997. 5 weeks before harvesting to smother fouling organisms further and to examine their influence on growth. The 24 bags containing nursery seed clams were harvested on May 29. 1997. Surviving clams were counted, and the percentage of seed reaching the growout size was determined by sieving (10 to 11-mm mesh screen) clams from each bag. Clams that were retained on the screen were large enough for the growout phase, whereas, seed that passed through Culture of Northern Quahog 79 the sieve were not. The percentage of growout seed from each bag was calculated based on the total number of seed harvested. Growout Seed Growout seed (mean ± SD SL: 21.1 ± 1.7 mm: n = 100) were stocked in 10.5-mm mesh size bags (1.2 x 1.2 m, n = 24) and planted on October 10. 1996 following the same method described for nursery seed. One hundred clams per bag were sampled every 4 to 5 weeks. Growth was assessed by measuring SL, SH, and ST. but only analysis on SL was conducted, because that shell mea- surement was highly correlated (r = 0.94 for SL and SH; r 2 = 0.83 for SL and ST). One hundred clams were sacrificed to mea- sure shell and tissue dry weight, as described earlier, at the begin- ning and again at the end of the study. Inversion of bags to smother fouling organisms was not performed on the growout seed. The 24 bags containing growout seed clams were harvested on June 5, 1997. Surviving clams were counted and the percentage of seed reaching legal harvest size was determined by grading (16 mm width on bar grader) clams from each bag. Clams that were re- tained on the bar grader had reached legal harvest size for cultured clams in Florida. The percentage of harvestable clams from each bag was calculated based on the total number of clams harvested. Food A vailability Chlorophyll a concentration and current speed were estimated at five locations in the study site to assess food availability along the prevailing flow gradient. Over a period of several days, float- ing objects were placed in the water during incoming and outgoing tides and followed to establish the prevailing current pattern in the area. Once the prevailing current pattern was established, five locations (two before, one inside, and two after the clam bed) were chosen to estimate food availability. Three 1-Liter water samples were taken at each location, on 3 days during incoming tide (April 10, May 8, and May 22, 1997) and 3 days during outgoing tide (April 18, May 15, and May 29, 1997). Water samples were taken to the laboratory and kept cool and dark until analyzed within a few hours. An appropriate volume (500 to 1000 mL) of seawater was vacuum filtered onto a synthetic filter (Millipore AA 47-cm diameter). Chlorophyll a was measured by spectrophotometries analysis (Strickland and Parsons 1976). Pigments were extracted from the filter with 90% acetone, and pigment absorbance was estimated spectrophotometrically at 750. 660, 647. and 630 nm wavelengths. A standard concentration curve was produced using a commercial chlorophyll a extract (SIGMA Chemical Company, St. Louis, MO). Dalit Analysis All data were examined for variance heteroscedascity using F max test (Sokal and Rohlf 1995), and no data transformation was necessary. Final SL of the clams was analyzed by a two-way (density and PED) ANOVA for growout seed and a three-way ANOVA (density. PED. and inversion) for nursery seed. Bonfer- roni's multiple comparison test was used to compare the means if there was a significant difference in the ANOVA (Sokal and Rohlf 1995). Survival of nursery seed was analyzed by a three-way ANOVA (density. PED. and inversion), and survival of growout seed was analyzed by a two-way (density and PED) ANOVA. Condition index was calculated using the formula: dry soft tissue wt. (g)* 1000/ dry shell wt. (g) (Walne and Mann 1975) and was analyzed by a three-way ANOVA (density, PED, and inversion) for the nursery seed and a two-way (density and PED) ANOVA for the growout seed. Percentage of clams that reached growout size was analyzed by a three-way (density. PED, and inversion) ANOVA. and percentage of clams that reached legal harvest size was analyzed by a two-way (density and PED) ANOVA. Pearson product moment correlation was used to correlate growth with temperature, salinity, and Secchi disc depth (Sokal and Rohlf 1995). The significance level (a) for all statistical tests was 0.05. RESULTS Environmental Parameters Water temperature ranged from 8.8 to 28.8°C during the study period, with a mean (± SD) of 21.7 (±4.1 )'C (n = 32). Mean (± SD) salinity was 32 (±1.7) ppt (n = 32) with a range of 29 to 34 ppt. Mean (± SD) dissolved oxygen concentration (D.O.) was 1 1.6 (±4.7) ppm (n = 32) at the surface (range: 4.8-20.0 ppm) and 1 1.3 (±4.8) ppm (n = 32) at the bottom (range: 5.0-19.0 ppm). Water depth was between 1 and 1.5 m. Mean (± SD) Secchi disc depth was 0.86 (±0.14) m (range: 0.57-1.00 m. n = 32). Mean (± SD) current speed was 6.8 (±4.2, n = 32) cm/s at the surface (range: 2.0-15.0 cm/s. n = 32) and 6.6 (±3.3. n = 32) cm/s at the bottom (range: 3.0-13.8 cm/s). Water temperature, salinity, D.O.. Secchi disc depth, and current speed did not show significant (p > .05) correlation with SL. Nursery Seed Nursery seed clams of all treatments grew at almost perfect linear rates over time, from an average initial SL of 6.0 mm to a mean final SL of 14.6 mm in the 13-week study. Density (p = .03) and inversion (p = .03) had a significant effect on the final SL; whereas, PED did not (p = .31 ). Low density clams tended to be larger than those of medium and high density treatments; and clams from inverted bags tended to be larger than those from the noninverted bags (Table 1 ). Mean percentage of nursery seed that reached growout size ranged from 25.5 to 91.8% (Table 1 ). There was a significant effect of density (p = .02) and inversion (p < .001) on percentage of clams attaining growout size. A higher percentage of clams in the low-density treatment reached growout size than that of clams in the medium- and high-density treatments (Table 1 ); inversion resulted in a higher percentage of nursery seed that reached the growout size (Table 1 ). Survival at the end of the study ranged from 59.0 to 94.5% (Table 1 ). PED (p = .005), and inversion (p = .002) effects were significant, but density effect was not (p = .60). Surprisingly, survival of clams in bags with PED was lower than that of clams in bags without PED (p < ,05, Bonferroni's test) (Table 1); inver- sion resulted in higher survivorship (p < .05, Bonferroni's test) (Table 1). Mean (± SD) initial condition index (CI) of nursery seed was 49.6(±28.3)(n = 100), and it changed little after the 13-week trial period (Table 1). The density (p = .15) or PED (p = .79) had no significant effect: whereas, the inversion (p = .002) did. The CI of the clams in the inverted bags was larger than that of the clams in the noninverted bags (p < .05. Bonferroni's test) (Table 1). Growout Seed Growout seed clams grew from an initial SL of 21.1 mm to a mean final SL of 33.3 mm. There was no significant difference (density: p = .40, PED: p = .17) in the final SL among the 80 Fernandez et al. TABLE 1. Mean )± SD) of shell length, percentage reached growout size, survival, and CI of nursery seed grown for 13 weeks. % Reached Inversion Densitv PED Shell Length Growout Survival Condition (I. NI) (Clams/Bag) (N, C) (mm) Size (%) Index I I 1 I 1 I NI NI NI NI NI NI 7.500 N 17.1 ±0.0 a 91.8 + 9.8" 94.5 ± 6.4 a 55.2±0.1 abc 10,000 N 14.1 ±0.5 ab 62.5 ± 13.4 abc 76.0 ± 11.3 abc 65.3 ± 3.2 a 1 2,500 N 14.2±0.8 ab 45.5 ± 6.4 cd 81.0±5.7 ab 52.9±6.1 abc 7.500 C 15.1 ±0.2 ab 67.5 ± 7.8 ab 82.0±5.7 ab 56.5 ± 2.6 ah 10.000 C 14.4±0.3 ab 56.0 + 7. 1 KJ 83.5 ± 7.8 ab 52.3±4.1 abc 12.500 c 13.7±0.7 b 57.5 + 0.7 abcd 75.5 ± 3.5 abc 42.9 ± 2.0 bc 7,500 N 14.2±2.0 ab 40.5 ± 3.5 bcd 78.0 ± 2.8 abc 45.9 + 9.0 K 10.000 N 13.1 ± 1.0" 29.5 ± 6.4 td 77.0 ± l.4 abc 36.5 ± 8.0 C 12.500 N 14.3+ l.l ah 37.0 ± 1.4 ud 81.0 + 1.4 ab 43.2 ± 4.5* 7,500 C 14.1 ± 1.4 ab 35.5 ± 28.9 bcd 59.0±8.5 C 52.8 + ICO 60 10,000 c 13.3 ±0.8" 29.5 ± 10.6" 1 69.5 ± 9.2 C 43.5 ± 5.7 bc 12.500 c I3.9±0.6 b 25.5 ± 4.9 d 62.5 ± 6.4 C 47. 1 ± 4.9 abc Values within a column with different superscripts were significantly different. Under inversion. "I" means inverted. "NI" means noninverted: under PED. "N" means not cover, and "C" means cover. treatments (Table 2, Fig. 2). Mean percentage of growout seed that reached 16 mm in ST (legal harvest size for cultured northern quahog) at the end of the study ranged from 30.1 to 66.8% (Table 2). The density effect was significant (p = .04); whereas, the PED effect was not (p = .25). In general, the percentage decreased with increasing density (Table 2). Survival at the end of the study ranged from 75.0 to 87.0% (Table 2). The effects of density (p = .04) and PED (p = .0009) were significant, but there was no apparent pattern (Table 2). Mean (± SD) initial CI of growout northern quahog seed was 65.4 (+28.3). The CI decreased after the 34-week trial period to an average of 36.3, with no significant difference among the treat- ments (Table 2). Food Availability Generally, chlorophyll a concentration was similar among the stations and between incoming and outgoing tides at a given date. Average chlorophyll a concentration in the April 1997 samples was 0.0642 u.g/L. Mean surface current during the April sample days was 1 1.3 cra/s, and mean bottom current was 9.5 cm/s. May 8 and May 15 samples showed an order of magnitude increase in chlorophyll a concentration to 0.96 p-g/L. Average late-May mea- surements of chlorophyll a was 0.81 fxg/L. In May. mean surface current speed was 6.7 cm/s and mean bottom current speed was 6.1 cm/s. DISCUSSION Ansell (1968) reviewed the growth of northern quahog in vari- ous locations along the eastern coast of the United States and concluded that the optimum temperature for growth was approxi- mately 20°C and that shell growth ceased below 9°C or above 31°C. In the present study, the mean water temperature over the 34-week period ranged from 15.1 to 26.3°C. Small changes in salinity do not have a major influence on growth rates, unless the salinity goes below 20 ppt (Castagna and Kraeuter 1981). The optimal salinity for the growth of northern quahog is reported to be about 26 and 27 ppt (Rice and Pechenik 1992). In the present study, salinity ranged from 29 to 36 ppt. In the Oak Hill area, D.O. was high (mean = 13 ppm) during the 34-week period. High D.O. has been found to be common in the southern IRL as well (Arnold et al. 1990, Dierberg et al. 1986). Manzi et al. ( 1981) recommended that intensive field culture is best initiated with seed size larger than 10 mm in SL. However, larger seed are more expensive, and their cost may be >60% of the total cost of producing the final product in northern quahog aqua- culture (Adams et al. 1991). In a study conducted in New Jersey TABLE 2. Mean (+ SD) of shell length, percentage reached legal harvest size, survival and CI of growout seed grown for 34 weeks. Shell "fc Reached Densitv PED Length Legal Harvest Survival Condition (Clams/Bag) (N, C) (mm) Size (%) Index 750 1,000 1,250 750 1.000 1.250 N N N C C C 33.5 ±2.1 32.9 ±2.1 32.8 ± 2.5 34.0 ± 2.2 33.0 ± 2.2 33.8 ± 2.5 53.7 ± 4.3" 49.1 ± U.2" b 30.1 ±4.8 b 66.8 ± 8.2 J 38.9 ± 10.0" 49.5 ± 3.8 ab 75.0 ± 3.4" 87.0 ± 1 .3" 75.0 ± 3.1 b 81.0±3.4 al 81.0 + 2.3 al 82.0±1.8 al 37.2 ±6.0 34.8 + 4.9 35.5 ± 5.7 35.3 + 5.8 34.1 ±5.5 38.6 ± 6.4 Values within a column with different superscripts were significantly different. Under PED. "N" means not cover and "C" means cover. Culture of Northern Quahog Time (week) Figure 2. Mean (n = 4) shell length of growout seed over time at the different density and PEI) treatment combinations. (I,n: low-density bags without PEI); m.n: medium density without PKI); h,n: high density without PED; l.c: low with PED; m,c: medium with PED; h,c: high with PED). (Kraeuter et al. 1998) and the present study, successful culture was achieved with nursery seed of 5 and 6 mm SL. respectively. Our nursery seed grew on average 2.76 mm/month in SL. similar to that found by Kraeuter et al. ( 1998) in New Jersey in the summer and by Sturmer et al. (1995) on the west coast of Florida, and higher than that found by Summerson et al. (1995) in North Caro- lina. The growth rates of nursery seed were similar among the months. The winter months had much smaller effects in reducing grow rates on the nursery seed, as it did on the growout seed (Fig. 2). Growout clams in high density bags showed retarded growth during the winter months (Fig. 2). when environmental conditions were not optimal for clam growth. As soon as environmental con- ditions became optimal, clams in the high-density bags grew rap- idly to reach similar SL as the clams of the other treatments (Fig. 2). However, a lower percentage of clams in high-density bags reached the legal harvest size (Table 2). Menzel et al. (1976) and Kraeuter et al. ( 1998) suggested that survival of planted clams should be more than 40 to 50% for the commercial culture to be profitable. In the present study, average survival of nursery clams ranged from 59.0 to 94.5%. This survival is similar to the 87% found by Summerson et al. ( 1995) for the nursery seed grown in raceways in North Carolina and to the 58 to 88% found by Sturmer et al. ( 1995) in a 3-month field study on the west coast of Florida. Densely packed seed clams without PED in southeastern states have demonstrated massive losses to predation in very short peri- ods of time (e.g., Menzel et al. 1976. Gibbons and Castagna 1985. Peterson 1990, Kraeuter et al. 1998). Predators did not seem to be a significant problem in the present study. Although crabs and sheepshead were observed in the area, and sometimes small crabs were found inside the bags. Also, some of the dead clams found showed signs of crab predation: chipped margins or crushed shells (Vaughan et al. 1988). The PED used in the present study did not improve clam survival, and the maintenance of the PED was time consuming. However, biofouling on the culture bags w ithout the PED was heavier than those with the PED. Some PEDs were found float- ing or folded over the bags during the study and were re-installed. The use of PED may affect such biological processes as growth (Virnstein 1977, Dayton and Oliver 1980, Riese 1985). In the present study, survival was slightly higher in bags with PED in the growout period, and clams in bags with PED were found to grow slightly, but not significantly, faster than those in the bags without PED. Water flow in bags without PED seemed to be retarded because of fouling (by drift algae and sea squirts) of the bags. Fouling was found to be higher in bags without PED than in bags with PED where fouling was mainly observed on the PED itself. Fouling organisms diminish the water flow that passes through a bag. preventing clams from getting food necessary for optimal growth (Flimlin and Mathis 1993. Mojica and Nelson 1993). Drift algae (Gracilaria sp.) and tunicates were the most abundant foul- ing organisms in the present study. They grew rapidly if left un- checked. Inversion of the bags to smother fouling organisms was found to increase the growth of clams in the area. Inversion in- creased the percentage of nursery clams that reached the growout size and resulted in higher CI. Inversion also increased survival of the clams. A large increase in chlorophyll a concentration was observed from late April to late May. This increase in food supply in con- junction with increasing temperature may explain the rapid growth of the clams during this period. Since the pioneering work of Kellog (1903), it has been recognized that current speed has a major effect on the growth of northern quahog (Kerswill 1949, Haskins 1952. Hadley and Manzi 1984, Manzi et al. 1986). Grizzle and Morin ( 1989) and Grizzle and Lutz (1989) suggest that north- em quahog growth is primarily determined by horizontal seston flux past the animals and that intermediate seston flux rates pro- duce the highest growth rates in Mercenaria mercenaria in sandy sediments. In the present study, mean current velocity was 6.7 cm/s for the 34-week period, and mean chlorophyll a concentration was 0.61 u,g/L in April and May. Cahalan et al. (1989) indicated that growth rate of scallops peaked at 6.5 cm/s at 6.000 algal cells/mL. In conclusion, a planting density of 7,500 and 750 clams/bag for nursery seed and growout seed, respectively, should be used in the Oak Hill. Florida area, because the highest percentage of seed that reached growout seed or legal harvest size, respectively, was 82 Fernandez et al. found at these low densities. The PED used in this study did not improve survival, and its maintenance is time consuming. How- ever, it could be used to reduce biofouling on the culture bags. It is much easier to clean the PED (with larger mesh size and without clams and sediment inside) than to clean the culture bags. We rec- ommend periodic inversion of bags to smother biofouling organisms. ACKNOWLEDGMENTS Harbor Branch Oceanographic Institution. Inc. supplied the clam seed. We thank Sean Reif for his help in the field. An anony- mous reviewer provided valuable comments to an earlier version of the manuscript. LITERATURE CITED Adams, C. J. Cato. J. E. Easley. Jr.. S. Kemp. W. Mahan, J. J. Manzi, M. Oesterling, R. Pomeroy. E. Thunberg, D. Vaughan & R. Walker. 1991. Investing in commercial hard clam culture: a comprehensive guide to the south Atlantic states. Florida Sea Grant College. Rep. 104. Gaines- ville. Florida. 128 pp. Ansell, A. D. 1968. The rate of growth of the hard clam Mercenaria mercenaria throughout the geographical range. /. Cons. Int. Expl. Mar. 31:364-409. Arnold, W. S„ D. C. Marelli & C. Lund. 1990. Suitabdity of the Southern Indian River Lagoon for hard clam {Mercenaria spp.) culture. Aqua- culture report series. Final reports for the Aquaculture Market Devel- opment Aid Program 1989-1990 and 1990-1991. vol. III. Florida De- partment of Agriculture and Consumer Services, Tallahassee. Florida. Arnold. W. S„ D. C. Marelli. T. M. Bert. D. S. Jones & I. R. Quitmyer. 1991. Habitat-specific growth of hard clams Mercenaria mercenaria from the Indian River. Florida. J. Exp. Mar. Biol. Ecol. 147:245-265. Bisker. R. & M. Castagna. 1989. Biological control of crab predation on hard clams Mercenaria mercenaria by the toadfish Opsanus tau in tray cultures. J. Shellfish Res. 8:33-36. Boulding, E. G. & T. K. Hay. 1984. Crab response to prey density can result in density-dependent mortality of clams. Can. J. Fish. Aquat. Sci. 41:521-525. Cahalan. J. A., S. E. Siddall & M. W. Luckenbach. 1989. Effects of flow velocity, food concentration, and panicle flux on growth rates of ju- venile scallops Argopeclen irradians. J. Exp. Mar. Biol. Ecol. 129:45- 60. Camker. M. R. 1959. The role of physical and biological factors in the culture of Crassostrea virginica and Mercenaria mercenaria in a salt water pond. Ecol. Monogr. 29:219-266. Castagna, M. 1984. Methods for growing Mercenaria mercenaria from postlarval to preferred size seed for field planting. Aquaculture 39:355- 359. Castagna, M. A. & J. N. Kraeuter. 1977. Mercenaria culture using stone aggregate for predator protection. Proc. Nat. Shellfish. Assoc. 67:1-6. Castagna, M. & J. N. Kraeuter. 1981. Manual for growing the hard clam Mercenaria. VIMS Special Rept. in Applied Marine Science and Ocean Engineering, No. 249. 110 pp. Claereboudt, M. R., D. Bureau. J. Cote & J. H. Himmelman. 1994. Fouling development and its effect on the growth of juvenile giant scallops in suspended culture. Aquaculture 121:327-342. Crawford. B. 1992. Oysters and clams. Aquaculture Species Resource Series. Final Repts. for the Aquaculture Market Development Aid Pro- gram 1989-1990 and 1990-1991, vol. III. Florida Department of Ag- riculture and Consumer Services, Tallahassee, Florida. Dayton, P. K. & J. S. Oliver. 1980. An evaluation of experimental analysis of population and community patterns in benthic marine environments, pp. 93-120. In; K. R. Tenore and B. C. Coull (eds.). Marine Benthic Dynamics. University of South Carolina. Columbia. South Carolina. Dierberg. F., J. Ryther. C. Traintafyllidis. R. Creswell. T. Debusk & M. Schilling. 1986. An inventory of bivalves and their food supply in the Indian River. Brevard County, Florida. Brevard County Water Re- sources Department. 61 pp. Eggleston, D. B., R. M. Lipcius & A. H. Hines. 1992. Density-dependent predation by blue crabs on infaunal clam species with contrasting dis- tribution and abundance patterns. Mar. Ecol. Prog. Ser. 85:55-68. Eldridge, P. J.. W. Waltz. R. C. Gracy & H. H. Hunt. 1976. Growth and mortality rates of hatchery seed clams Mercenaria mercenaria in pro- tected trays in waters of South Carolina. Proc. Natl. Shellfish. Assoc. 66: 1 3-20. Eldridge, P. J.. A. G. Eversole & J. M. Whetstone. 1979. Comparative survival and growth rates of hard clams Mercenaria mercenaria planted in trays subtidally and intertidally at varying densities in a South Carolina estuary. Proc. Natl. Shellfish Assoc. 69:30-39. Fegley, S. R . B. A. MacDonald & T. R. Jacobsen. 1992. Short-term varia- tion in the quantity and quality of seston available to benthic suspen- sion feeders. Est Coast. Shelf. Sci. 34:393— J 12. Flagg. P. J. & R. E. Malouf. 1983. Experimental plantings of juveniles of the hard clams Mercenaria mercenaria in the waters of Long Island, New York. J. Shellfish Res. 3:19-27. Flimlin. G. E. & G. W. Mathis. 1993. Biological biofouling control in a field-based nursery for the hard clam. World Aquaculture 24:47—48. Florida Department of Natural Resources (FDNR). 1986. Summary of Florida commercial landings, 1985. FDNR. Tallahassee, Florida. Gibbons. M. C. & M. A. Castagna. 1985. Biological control of predation by crabs in bottom cultures of hard clams using a combination of crushed stone aggregate, toadfish. and cages. Aquaculture 47: 101-104. Godwin. W. F. 1968. The growth and survival of planted clams. Merce- naria mercenaria. on the Georgia coast. Georgia Game Fish Coram. Contr. Series. No. 9. Brunswick, Georgia, pp. 1-16. Grizzle. R. E. & P. J. Morin. 1989. Effect of tidal currents, seston. and bottom sediments on growth of Mercenaria mercenaria: results of a field experiment. Mar. Biol. 102:85-93. Grizzle. R. E. & R. A. Lutz. 1989. A statistical model relating horizontal seston fluxes and bottom sediment characteristics to growth of Merce- naria mercenaria. Mar. Biol. 102:95-105. Hadley. N. H. & J.J. Manzi. 1984. Growth of seed clams Mercenaria mercenaria at various densities in a commercial scale nursery system. Aquaculture 36:369-378. Haskins. H. H. 1952. Further growth studies on the quahog Mercenaria mercenaria. Proc. Natl. Shellfish. Assoc. 42:181-187. Haven. D. S. & J. G. Loesch. 1973. An investigation into commercial aspects of the hard-clam fishery and development of commercial gear for the harvest of mollusks. Virginia Institute of Marine Science, An- nual Contract Rept. 3-124R. pp. 1-92. Hines, A. H.. A. M. Haddon & L. A. Weichert. 1990. Guild structure and foraging impact of blue crabs and epibenthic fish in a subestuary of the Chesapeake Bay. Mar. Ecol. Prog. Ser. 67:105-126. Jones. D. S.. I. R. Quitmyer. W. S. Arnold & D. C. Marelli. 1990. Annual shell banding, age. and growth rate of hard clams {Mercenaria spp.) from Florida. J. Shellfish Res. 9:215-225. Jory. D. E.. M. R. Carriker & E. S. Iverson. 1984. Preventing predation in molluscan mariculture: an overview. /. World Maricult. Soc. 15:421- 432. Kellog. J. I. 1903. Feeding habits and growth of Venus mercenaria. N.Y. State Mus. Bull. 71. Zool. 10:1-28. Kerswill. C. J. 1949. Effects of water circulation on the growth of quahogs and oysters. J. Fish. Res. Bd. Can. 7:545-551. Kraeuter. J. N. & M. Castagna. 1985. The effects of seed size, shell bags, crab traps, and netting on the survival of the northern hard clam Mer- cenaria mercenaria. J. Shellfish Res. 5:69-72. Kraeuter. J. N.. S. Fegley, G. E. Flimlin, Jr., & G. Mathis. 1998. The use of mesh bags for rearing northern quahog (hard clam). Mercenaria mercenaria. seed. J. Shellfish Res. 17:205-209. Culture of Northern Quahog 83 MacKenzie. C. L. 1977. Predation on hard clam Mercenaria mercenaria populations. Trans. Amer. Fish. Soc. 106:530-537. MacKenzie. C. L. 1979. Management for increasing clam abundance. Mar. Fish. Rev. 41:10-22. Manzi, J. J.. V. G. Burrell. Jr. & W. Z. Carson. 1980. A mariculture dem- onstration project for and alternative hard clam fishery in South Caro- lina: preliminary results. Proc. World. Maricult. Soc. 11:79-89. Manzi. J. J.. V. G. Burrell & H. Q. M. Clawson. 1981. Commercialization of hard clam. Mercenaria mercenaria, mariculture in South Carolina: preliminary report. J. World. Maricult. Soc. 12:181-195. Manzi. J. J.. N. H. Hadley & M. B. Maddox. 1986. Seed clam Mercenaria mercenaria culture in an experimental scale upflow nursery system. Aquaculture 54:301-31 I. McHugh, J. L. 1981. Recent advances in hard clam mariculture. J. Shellfish Res. 1:51-55. Menzel. R. W. 1961. Seasonal growth of the northern quahog Mercenaria mercenaria and the southern quahog M. campechinensis in Alligator Harbor. Florida. Proc. Nat. Shellfish. Assoc. 52:37-46. Menzel. R. W. & H. W. Sims. 1964. Experimental farming of hard clams. Mercenaria mercenaria in Florida. Proc. Nat. Shellfish. Assoc. 53: 103- 109. Menzel. R. W.. E. W. Cake. M. L. Haines. R. E. Martin & L. A. Olson. 1976. Clam mariculture in northwest Florida: a field study of predation. Proc. Nat. Shellfish. Assoc. 65:59-62. Mojica. R. Jr. & W. G. Nelson. 1993. Environmental factors of a hard clam aquaculture site in the Indian River Lagoon. Florida. Aquaculture 1 13: 313-329. Paul. J. D. & I. M. Davies. 1986. Effects of copper- and tin-based anti- fouling compounds on the growth of scallops and oysters. Aquaculture 54:191-203. Peterson. C. H. 1990. On the role of ecological experimentation in resource management: managing fisheries through mechanistic understanding of predator feeding behavior. In: R. N. Hughes (ed.). Behavioral Mecha- nisms of Food Selection. Spnnger-Verlag. Berlin, pp. 821-846. Peterson. C. H.. H. C. Summerson & J. Huber. 1995. Replenishment of hard clam stocks using hatchery seed: combined importance of bottom type, seed size, planting season, and density. J. Shellfish Res. 14:293- 300. Rice, M. A. & J. A. Pechenik. 1992. A review of the factors influencing the growth of the northern quahog Mercenaria mercenaria. J. Shellfish Res. 11:279-287. Riese. K. 1985. Predator control in marine tidal sediments. In: P. E. Gibbs (ed.). Proceedings of the 19th European Marine Biology Symposium. Plymouth. Cambridge University Press. Cambridge, pp. 311-321. Sheng. Y. P.. S. Peene & Y. M. Liu. 1990. Numerical modeling of tidal hydrodynamics and salinity transport in the Indian River Lagoon. Florida Scientist 53:147-168. Sokal, R. R. & F. J. Rohlf. 1995. Biometry. 3rd ed. W. H. Freeman and Co.. New York. 850 pp. Steinberg. M. N. 1980. Preliminary system dynamic model of the effec- tiveness of shellfish hatcheries on increasing harvestable yields. Pro- ceedings of the International. Conference on Cybernetics and Society. Institute of Electrical and Electronic Engineering New York. pp. 895- 900. Strickland, J. D. H. & T. R. Parsons. 1976. A practical handbook of sea- water analysis. Fish. Res. Bd. Can. Bull. 167:31 1. Sturmer. L. N„ E. Quesenberry. J. Scarpa & D. E. Vaughan. 1995. Devel- opment of a shellfish aquaculture industry on the west coast of Florida: seed production, growout monitoring, and species diversification. Ab- stracts from Aquaculture '95, San Diego. California, p. 279. Summerson. H. C. C. H. Peterson & M. Hooper. 1995. Aquacultural pro- duction of northern quahog, Mercenaria mercenaria (Linnaeus. 1758): high water temperatures in the nursery and growth penalties of predator control by gravel. J. Shellfish Res. 14:25-31. Vaughan. D.. L. Creswell & M. Pardee. 1988. A manual for farming the hard-shell clam in Florida. Aquaculture Report Series. Final Repts. Aquaculture Market Development Program 1989-1990 and 1990- 1991. vol. II. Florida Department of Agriculture and Consumer Ser- vices. Tallahassee, Florida. Virnstein, R. W. 1977. The importance of predation by crabs and fishes on benthic infauna in Chesapeake Bay. Ecology 58:1199-1217. Walker, R. L. 1984. Effects of density and sampling time on the growth of the hard clam Mercenaria mercenaria planted in predator-free cages in coastal Georgia. Nautilus 98: 1 14-1 19. Wallace. J. C. & T. G. Reinsnes. 1985. The significance of various envi- ronmental parameters for growth of the iceland scallop [Chlamys is- landica) m hanging culture. Aquaculture 44:229-242. Walne, P. R. & R. Mann. 1975. Growth and chemical composition in Ostrea edulis and Crassostrea gigas. pp. 587-607. In: Proceedings of the 9th European Marine Biology Symposium. Aberdeen University Press. Aberdeen. Scotland. Whetstone, J. M. & A. G. Eversole. 1978. Predation on hard clam Merce- naria mercenaria by mud crabs. Proc. Nat. Shellfish Assoc. 58:42-48. Wildish. D. J. & D. D. Kristmanson. 1984. Importance to mussels of the benthic boundary layer. Can. J. Fish. Aquat. Sci. 41:1618-1627. Journal of Shellfish Research. Vol. 18. No. 1. 85-89, 1999. RELATIONSHIP BETWEEN THE BURROWING WORM POLYDORA SP. AND THE BLACK CLAM CHIONE FLUCT1FRAGA SHOWERBY JORGE CACERES-MARTINEZ, 1 GISSEL DALILA TINOCO, 1 MARCO LINNE UNZUETA BUSTAMANTE, 2 AND IGNACIO MENDEZ GOMEZ-HUMARAN 3 Laboratorio de Patologia de Mohtscos del Departamento de Acuicultura. Centro de Investigation Cientifica y de Education Superior de Ensenada Apdo. Postal 2732, 2800 Ensenada Baja California, Mexico ~Centro de Investigaciones Biologicas del Noroeste, S.C. Unidad Guaymas Apdo. Postal 349, 85469 Guaymas, Sonora, Mexico ' Departamento de Estadistica del Colegio de la Frontera Norte. Zona Rio, Tijuana, B. C. 22320, Mexico ABSTRACT The black clam Chione fluctifraga is collected for human consumption in both coasts of the peninsula of Baja California. Mexico. An epibionts survey on the black clam from Bahfa Falsa, B.C. and Bahi'a de Guasimas, Sonora revealed an association between the clam and the burrowing worm Polydora sp. Approximately 94% of the worms were located around the siphon area in both valves of the clams taken from Bahia Falsa and 54.5% in clams from Bahi'a de Guasimas. The number of worms per host varied from 1 to 48 in Bahia Falsa and 2 to 15 in Bahia de Guasimas. There was a trend of increased intensity of worm infestation with increased clam size. After a period of 10 months under aquarium conditions, mean percentage of occupation of the siphon area by the worm varied from 94.2% at the beginning of the observation period to 88.3% at the end in clams from Bahia Falsa, and from 54.5% at the beginning of the observation period to 43.4% at the end in clams from Bahia de Guasimas. There was an increase in the mean number of worms on the clams after the observation period, from 9 to 15.7 worms in organisms from Bahi'a Falsa and from 5.2 to 9.5 worms in clams from Bahia de Guasimas. Worms may survive on the shell after the host is dead. Temperature during observation period varied from 20 to 25.5°C. The U-shape channels of the worm result in a very porous and brittle host shell. In heavily infested clams the shell is broken and this impinges on the clams ability to close its valves. This is the first record of burrowing worms associated with the siphon aperture area of the shell of C. fluctifraga. KEY WORDS: Polydora. Chione fluctifraga, burrowing worm, infestation INTRODUCTION worm can also render oyster shells brittle and easily broken during shucking, packaging, and transport (Korringa 1951). The named "polydorid" complex of the family Spionidae com- The black clam. Chione fluctifraga is a highly regarded food prises a number of highly diverse but closely related species, all and supports an extensive sport and commercial fishery in south- characterized by a modified fourth or fifth setiger (Light 1978). ern California (Haderlie and Abbott 1980). This species is also Among them, the genera Polydora and Boccardia contain a large gathered for human consumption on the Pacific coast of Baja number of species able to bore into calcareous substrates including California and the Gulf of California, Mexico (Martinez-Cordova shells of such commercially important bivalves as mussels, oys- 1988, Martinez-Cordova 1996). A survey of clams from Bahia ters. cockles, and scallops (Read 1975, Sato-Okoshi et al. 1990. Falsa on the Pacific coast of Baja California, and Bahia de Guasi- Blake 1996, Handley and Bergquist 1997). These species live in a mas, Sonora. Mexico revealed the presence of polychaetes on tube inside the hole bored in the shell of the host with two exterior shells. The aims of this study were to determine the identity of the apertures. The anterior end of the worm emerges from the tube and polychaete and to document some aspects of the relationship be- feeds from particles taken from the sediment surface or from the tween this worm and the black clam, overlying water column with the aid of its palps (Daro and Polk 1973, Blake 1996). Its boring activity may reach the inner surface MATERIALS AND METHODS of the mollusk's shell and induce the host to secrete calcite and conchiolin layers, forming a blister to isolate the worm (Kent In April 1997. a sample of 48 live Chione fluctifraga from 1979, Lauckner 1983). As a consequence, these worm species are Bahia Falsa. Baja California was collected and subsequently, in also named "mudworms" or "blisterworms" (Lauckner 1983). A June, a sample of 71 dead clams (empty paired shells) were ob- deviation of host energy for growth and reproduction to build a tained. Finally, in July 1997. a sample of 125 live black clams from blister has been suggested by some authors (Williams 1968. Kent Bahia de Guasimas, Sonora was also collected (Fig. 1). Bahia 1979). In edible oysters, the blisters affect the half-shell market. Falsa has a muddy bottom, and Bahia de Guasimas has a sandy and because the blisters can be punctured and release anaerobic me- muddy bottom. After washing the clams in running seawater, each tabolites. including hydrogen sulphide (Handley 1995). A blister- live clam was measured (length from the umbo to the posterior 85 86 Caceres-Martinez et al. 1 1 \ 116 t, 00W \ _-/•? Bahia \\ ^} 30°25'N- __ J _^ MEXICO r 1 ^ 110'35-W >l Bahia X , de Guasimas^--£n] -27°50'N fl-, f\r^ Figure 1. Map showing Bahia Falsa in Baja California and Bahia de Guasimas, Sonora. Mexico. Black dots indicate sampling areas. margin of the shell I. The surface of the shell was delimited for examination in three zones (approximately 30% of the total surface area each one): zone 1 (Zl), around the siphon area; zone 2 (Z2) in the middle area, and zone 3 (Z3) opposite to the siphon area (Fig. 2). Clams were placed individually in Petri dishes filled with seawater. and the number of worms emerging from the shell were counted by zone under the dissecting microsope. Two holes from the same "U"-shaped channel were considered as one worm. Sub- sequently, clams were opened with a knife, and the meat was discarded. Then, the inner sides of the right and left valves were checked for worms by zone, and Polydora infestations visible in the inner shell were enumerated by zone. The total infestation intensity was determined by comparing the number of worms ob- served on both sides of the shell. Dead clams were cleaned under running tap water, and worm blisters and holes related to burrow- ing worms were counted by zone. Prevalence was considered as the percentage of infested clams in the sample. To determine the degree of damage of the burrowing worm on the shell of the clam. X-ray radiographs were taken from clams with different infestation intensities. The number of worms was related to the size of the clams. Fifteen worms were extracted from the shell of six clams col- lected in Bahia Falsa, and seven were extracted from six clams from Bahia de Guasimas by crushing them with nippers around the edge of the shell, where the worms were located. The worms were removed from the shell fragments with dissection tweezers and fixed in 70% ethanol for identification. To assess worm behavior. 33 infested live clams from Bahia Falsa and nine infested live clams from Bahia de Guasimas were labeled and placed separately in aquaria without sand for 10 months. The water was changed every 3 or 4 days, and the tem- perature was recorded with a manual thermometer. The clams were fed once daily with hochrisis galvana and Chaetoceros sp. The water was checked for release of burrowing worm larvae and the Bahia Falsa Bahia de Guasimas MeanZl =94.2% N = 33 11 = 262 Mean = 9 SE = 1.3 Range = 1-25 N=23 n = 303 Mean = 15.7 SE = 1.8 Range = 6-37 MeanZl =54.5% N = 9 n = 47 Mean = 5.2 SE= 1 6 Range = 2-15 MeanZl =43 4 Figure 2. Zones delimited on the valves of Chione fluclifraga to determine distribution of Polydora sp. Percentage of Polydora sp. infesting different zones (Zl, Z2, Z}) of the right (R) and left (L) valves of the black clam from Bahia Falsa, B.C. and Bahia de Guasimas, Sonora, at the beginning of the observation period (Al and the end (Bl. N = number of clams studied; n = number of Polydora sp.; Mean Zl = Mean of the percentages of occupation of worms in both valves; Mean = mean number of worms in both valves; SE = standard error of the mean number of worms in both valves; Range = minimum and maximum number of Polydora sp. found in both valves. POLYDORA SP. AND ChIONE FLUCTIFRAGA 87 C 50 o 'O 40 Mean +4.472 * SE ^_^ Mean + SE Mean - SE Mean - 4.472 * SE □ Mean 34-36 37-39 40-42 43-45 46-48 49-51 52-54 55-57 Size (mm) Figure 3. Relationship between the size of the clams and the number of worms infesting their shells. number of living worms, and worm holes per zone in the shell of the clams were counted and compared to the initial numbers at the end of the period. Statistics Mean shell length and infestation between live and dead clams were compared using t and the Kruskal-Wallis tests. A nested effect model was carried out to determine infestation differences between: ( 1 ) clams from Bahia Falsa and Bahia de Guasimas; and (2) valve side: and (3) among valve zone, assuming that the num- ber of worms per zone was nested to the corresponding valve, which was also nested to the corresponding bay. Finally, another nested effect model was applied to the study in aquaria conditions. The initial number of worms was incorporated as a covariate to analyze whether the number of worms changed at the end of the study. RESULTS The mean size of live clams from Bahia Falsa was 43.3 mm (SE 0.48) and of dead clams was 46.4 mm (SE 0.56), the difference was significant (r = -39. p < .01 ). The mean size of clams from Bahia de Guasimas was 32.9 mm (SE 0.2). Holes in the shells were occupied by a spionid polichaete from the genus Polydora, and the mean size of worms was 37.7 mm (SE 5.68). Their morphological characteristics agree with the descrip- tion of Polydora limicola (Anenekova): prostomium weakly in- cised along anterior margin, caruncle extending to setiger 3. 4 eyes present: palps and dorsum of anterior setigers with black pig- mented bands or without bands; major spines of setiger 5 with a small, triangular lateral tooth; posterior notopodial spines absent and pygidium disclike with dorsal notch. However, its borrowing behavior suggests the species P. ciliata. (Johnson) (See Blake 1996; 173). The worm produced U-shaped channels, which were filled with compacted mud. The burrows were extended by "chim- neys" composed of detritus (algae remnant), which protruded from the surface of the clams' valves. Worms were very active, their palps were continuously extended from the burrows. Polydora prevalence in live and dead clams from Bahia Falsa was 48.6 and 66%, respectively. The number of worms per host ranged from 1 to 25 in live clams and from 1 to 48 in dead clams, and infestation differences were not significant, /-test (/ = 1.345, p = .180), Kruskal-Wallis test ( X 2 = 0.110. p = .740). In gen- eral, there was a trend of more Polydora sp. in larger clams relative to small clams (Fig. 3), however, this trend was not significant. f-test (F = 1.750. p = .112). Kruskal-Wallis test (\ 2 = 10.600. p = .157). Figure 4. X-ray photograph of several black clams showing different degrees of damage in the shell; note the channels are touching each other in heavily infested clams and the borders of the shells are de- stroyed. Worm prevalence in clams from Bahia de Guasimas was 15%, and the number of worms per host ranged from 2 to 15. Between 95 and 97% of the observed worms were placed in the Zl of live and dead clams from Bahia Falsa, respectively. The corresponding intensity of infestations were 5 and 3% in Z2. there were no worms in Z3. The distribution of Polydora sp. in clams from Bahia de Guasimas was 54.5% in the Zl, 18.2% in Z2, and 27.3% in Z3. The right valve was slightly more infested than the left valve. The results of the nested effect model confirmed that the infestation in clams from Bahia Falsa was greater than in clams from Bahia de Guasimas (F = 229.370. p < .0001): the model also showed that there was a greater infestation in the right than in the left valve (F = 6.390, p = .0017): finally, it was confirmed that the infestation per valve zone was greater in Zl than Z2 and Z3 (F = 72.660. p < .0001). The damage on the shell depends upon the number of worms and size of the channels. Figure 4 shows different degrees of infestation and associated damage. The channels often extended toward the middle of the valve (Z2) were in close proximity with one another, resulting in a very brittle shell. The shell of heavily infested clams was often broken in the siphon area, hindering valve closure (Fig. 5). In aquaria, where clams could not burrow into substrate. Poly- dora sp. showed a slight tendency to spread on all the surface of 88 Caceres-Martinez et al. Figure 5. Shell of Chione fluctifraga infested by Polydora sp. around the siphon area; note the holes in the area (Al, heavy infestation results in a very brittle shell that is easily broken (B). both valves of the host. Mean percentage of occupation of Zl varied from 94.2% at the beginning of the observation period to 88.3% at the end of the observation period in clams from Bahia Falsa, and from 54.5% at the beginning of the observation period to 43.4% at the end of the observation period in clams from Bahia de Guasimas. The distribution of worms was similar in both valves (Fig. 2). There was an increase in the mean number of worms on the clams after the observation period from 9 ( 1-25) to 15.7 (6-37) worms in organisms from Bahia Falsa, and from 5.2 (2-15) to 9.5 (2-38) worms in clams from Bahia de Guasimas (Fig. 2). The results of the nested effect model showed that infestation in clams from Bahfa Falsa and Bahia de Guasimas was slightly different (F = 3.070. p = .081); the same model revealed that infestation differences between both valves were not significant (F = 0.731, p = .482). However, infestation differences among valve zone showed that it was greater in Zl than Z2 and Z3 (F = 9.250. p < .0001 ): finally, it was corroborated that there was an increase in the number of worms at the end of the study ( F = 1 38.53, p < .000 1 ). Approximately 30% of the clams from Bahia Falsa and 1 1 % of the hosts from Bahia de Guasimas died during this observation period. Worm larval stages were observed during water exchange. The number of living worms at the end of the study from clams be- longing to Bahia Falsa was 58 and in clams from Bahia de Guasi- mas was 35. Temperature ranged from 24 to 25.5°C in summer and from 19 to 21°C in winter. DISCUSSION The polydorid species Polydora limicola and P. ciliata have been found in the northwest Pacific (Radashevsky 1993). Polydora limicola has been found in southern California (Hartman 1961, Blake 1996). However, the authors recognize that there are diffi- culties in their systematics. In accordance with Blake ( 1996), Poly- dora limicola is virtually identical to that of the well-known and widely distributed shell and limestone borer. P. ciliata. Manchenko and Radashevsky ( 1994) evaluated genetic differences between P. limicola and the superficially indistinguishable shell borer (P. cf. ciliata) in the Sea of Japan and found that, although there were no morphological differences, there were clear differ- ences in 10 of 27 gene loci surveyed. These genetic differences support the separation of P. ciliata and P. limicola. which formerly had been based strictly on habitat (Blake 1996). In this sense, the species that we found in this study could be P. ciliata: however, detailed molecular genetic studies are needed to clarify its identity . This is the first record of Polydora sp. associated with the siphon area of the black clam Chione fluctifraga; however, a simi- lar observation has been recorded in C. stutchburyi infested by the polychaete Boccardia acits in Wellington Harbour, New Zealand. This worm is a common and conspicuous epibiont of the cockle C. stutchburyi. The U-shaped burrow usually follows the curve of the shell growth lines. The boring is not lined with sand apart from a sand-grain partition at the U-bend and a short external sand-grain chimney. The external chimney extends out around the siphons of the cockle, which lies buried just beneath the sediment surface (Read 1975). The polychaete Boccardia chilensis has also been recorded boring in Chione stuchburyi shells in association with Broccardia syrtis [Read (1975)]. The high percentages of worms sited around the siphon area may suggest a specialized relationship between worm and host. The specialized relationship between spionids and their hosts has been described in Polydora commen- salis: it lives in a shallow burrow excavated along the columnella of the gastropod shell occupied by a hermit crab. This specialized species has short palps with an unusually narrow food groove that seems to be adapted to capturing food particles stirred up or sus- pended by the activities of the hermit crab (Blake 1996). Our results suggest that the relationship between the worm and the clam seems to be less specialized, because the worm may be sited out of the siphon area if the surface of the clam is available, as in aquaria conditions. Moreover. Polydora sp. remain alive on the shell after host death. The trend of more worms around the siphon area between clams from Bahia Falsa and those from Bahia de Guasimas suggest that, in the latter bay, clams are more exposed to colonization by Polydora sp. than in the former. Worm prevalence could be related to the type of substrate and with particular envi- ronmental factors of the two embayments we examined. Its place- ment, exclusively around the siphon aperture, allows the worm to feed on the particles inhaled or expelled by the clam, resulting in an advantageous position relative to other surface areas of the shell, if available. We observed great motility of worm palps while the clam protruded its siphons. The preference of worms for the right valve recorded in the field study remains unknown. The prevalence and number of worms per host in studied clams could be related to age and size of the clams. Possibilities of infestation by Polydora sp. in older clams are higher than in younger clams, because the former have had more encounters with POLYDORA SP. AND CHIONE FlUCTIFRAGA 89 the worms. In addition, larger surfaces provide more area for bur- rowing worm colonization. This observation could explain differ- ences in prevalence and number of worms per host between larger and older clams from Bahfa Falsa relative to smaller and younger clams from Bahi'a de Guasimas. The different environmental con- ditions of both bays may also play an important roll in prevalence and abundance of the worm. In aquaria conditions, the increase in number of worms through time and the presence of larval stages indicated reproduction and settling of the worm species. Temperature is one of the primary factors for determining the abundance of Polydora sp. (Lauckner 1983); in other words, generation time, reproduction and, hence, transmission. In this study, temperature was maintained near the values recorded in Bahi'a Falsa (see Caceres-Marti'nez et al. 1998) and Bahi'a de Guasimas (Arreola 1998), this supported the repro- duction, setting, and the increase in the number of worms recorded in this study. However, specific studies on temperature in relation to reproduction, growth, and transmission are needed. The mean number of worms per host (initial and final) was slightly higher in larger and older clams from Bahi'a Falsa, than in those from Bahi'a de Guasimas. This observation also supports the observed rela- tionship of surface area and intensity of worm infestation (Fig. 2). Mortality of both host and worms was detected at the end of the observation period. This could be related to deterioration of aquarium conditions ( frequency of water renovation and nutrition). However, specific studies on clam mortality in relation to the presence of this worm are needed. Heavy infestation may result in severe damage to the clam shell, a brittle shell border may increase the potential for mortality because of enhanced predation as a result of holes in the valves, and problems handling the clam for packing. ACKNOWLEDGMENTS M. C. Veronica Rodriguez identified the worms and Dr. M. A. del Rio Portilla took the photographs. Vicente Guerrero provided us with the clams and logistic support during sampling in Bahfa Falsa. This work was supported by CICESE # 623106. LITERATURE CITED Arreola. L. A. 1998. Grupo de Zonas Costeras del CIBNOR Unidad Guay- mas. Serie de datos ambientales de las Bahi'as del Estado de Sonora. Cento de Investigaciones Biologicas del Noroeste. Guaymas. Sonora. Mexico. Blake, J. A. 1996. Family Spionidae. Grube. 1850. pp. 81-223. In: Taxo- nomic Atlas of the Santa Maria Basin and Western Santa Barbara Channel, vol. 6. The Annelida. Part 3. Polichaeta: Orbiniidae to Cos- suridae. Santa Barbara Museum of Natural History. Santa Barbara. California. Caceres-Martfnez. J.. P. Macias-Montes de Oca & R. Vasquez-Yeomans. 1998. Polydora sp. infestation and health of the pacific oyster Cras- sostrea gigas cultured in Baja California, NW Mexico. J. Shellfish Res. 17:259-264. Daro. M. N. & P. Polk. 1973. The autoecology of Polydora ciliata along the Belgian coast. Neth. J. Sea Res. 6:130-140. Haderlie E. C. & D. P. Abbott. 1980. Bivalvia: the clams and allies, pp. 355-411. In: R. H. Moms. DP. Abbott, and EC. Haderlie. (eds.). Intertidal Invertebrates of California. Stanford University Press. Stand- ford. California. Handley, S.J. 1995. Spionid polychaetes in Pacific Oysters. Crassostrea gigas (Thunberg) from Admiralty Bay. Marlborough Sounds, New Zealand. A'ZV. Mar Freshwater Res. 29:305-309. Handley, S. J. & P. R. Bergquist. 1997. Spionid polychaete infestations of intertidal pacific oyster Crassostrea gigas (Thunberg). Mahurangi Har- bour, northern New Zealand. Aquaculture 153:191-205. Hartman, O 1961. Polychaetous annelids from California. Part III. Sys- tematica: Polychaetes. Allan Hancock Pacific Expeditions 27:1-93. Kent. R. M. L. 1979. The influence of heavy infestations of Polydora ciliata on the flesh content of Mytilus edulis. J. Mar. Biol. Ass. U.K. 59:289-297. Korringa. P. 1951. The shell of Ostrea edulis as a habitat. Arch, neerlan- daises zoologie 10:32-136. Lauckner. G 1983. Diseases of mollusca: bivalvia. pp. 477-1038. In: O. D. Kinne (ed.). Diseases of Marine Animals. Biologische Anstalt Hego- land. Hamburg. Federal Republic of Germany. Light. W.J. 1978. Spionidae polychaeta annelida. pp. 1-211. In: W. L. Lee (ed.). Invertebrates of the San Francisco Bay Estuary system. Cali- fornia Academy of Sciences. Manchenko. G. P. & V. 1. Radashevsky. 1994. Genetic differences between two allopatric sibiling species of the genus Polydora (Polychaeta: Spionidae) from the western Pacific. Bioch. Syst. Ecol. 22:767-773. Martinez-Cordova. L. R. 1988. Bioecologia de la almeja negra Chione fluctifraga (Sowerby, 1853). Rev. Biol. Trop. 36:213-219. Martinez-Cordova. L. R. 1996. Contribution to the knowledge of the mal- acological fauna of four costal lagoons in the state of Sonora. Mexico. Ciencias Marinas 22:191-203. Radashevsky, V. I. 1993. Revision of the genus Polydora and related gen- era from the Northwestern Pacific (Polychaeta: Spionidae). Publ. Sew Mar. Biol. Lab. 36:1-60. Read. G. B. 1975. Systematics and biology of polydorid species (Polycha- eta: Spionidae) from Wellington Harbour. J. Roy. Soc. of NZ 5:395- 419. Sato-Okoshi. W. Y. Sugawara & T. Nomura. 1990. Reproduction of the boring polychaete Polydora variegrata inhabiting scallops in Abashin Bay, North Japan. Mar. Biol. 104:61-66. Williams. C. S. 1968. The influence of Polydora ciliata (Johnston) on the degree of parasitism of Mytilus edulis L. by Mytilicola intestinalis Steur. /. Animal Ecol. 37:709-712. Journal of Shellfish Research, Vol. 18. No. 1, 91-97, 1999. ADHESION OF VIBRIO TAPETIS TO CLAM CELLS LOURDES LOPEZ-CORTES, ANTONIO LUQUE, EDUARDO MARTINEZ-MANZANARES, DOLORES CASTRO, AND JUAN J. BORREGO Department of Microbiology, Faculty of Sciences, University of Malaga, 29071 -Malaga, Spain ABSTRACT The adhesive properties of Vibrio rapetis, the causative agent of the brown ring disease affecting cultured clams, were determined considering both the contribution of bacterial surface hydrophobicity and the attachment capability to different animal cells. Hydrophobicity of V. tapetis strains was evaluated by means of three different methods, most of the strains being highly hydrophobic for any of the methods used. V. tapetis showed higher adhesion capability toward the clam cells used (hemocytes and mantle cells), as compared to several fish cell lines. No significant relationship was obtained between hydrophobicity and cell adhesion, which suggests the existence of adhesion-specific mechanisms. In addition, different bacterial structures were investigated as potential adhesins of V. tapetis, including hemagglutinins, pili, flagella, and outer membrane proteins. KEY WORDS: Vibrio tapetis, brown ring disease, adhesive capabilities, hydrophobic interaction, clam cells INTRODUCTION Vibrio tapetis is the causative agent of brown ring disease IBRD), an epizootic disease that affects cultured clams (Tapes philippinarum and T. decussatus). Although experimental repro- duction of BRD symptoms in healthy clams has been achieved by means of V. tapetis inoculation (Paillard and Maes 1990, Castro 1994. Novoa et al. 1998). the precise mechanisms involved in the in vivo infection have not yet been well established. Bacterial attachment and ulterior colonization of the clam periostracal lamina seem to be the first steps in the pathogenesis of V. tapetis (Paillard and Maes 1995a, Allam et al. 1996). The colo- nization and disruption of the periostracal lamina provoke the bac- terial accumulation on the inner surface of the clam shell, thereby producing the conchiolin deposit (Paillard and Maes 1995b), which constitutes the main gross symptom of this disease. In BRD. as in other fish and shellfish diseases, bacterial adhe- sion to appropriate host surfaces is a key factor for infection es- tablishment (Daly and Stevenson 1987, Santos et al. 1991). How- ever, little is known about the factors contributing to V. tapetis adhesion to host surfaces. Several potential adherence factors have been described for Vibrio species, including surface proteins, hemagglutinins, and several types of pili (Jonson et al. 1991, Spe- randio et al. 1995). These bacterial surface structures, named ad- hesins, interact with a broad variety of molecular host-cell recep- tors (Iijima et al. 1981. Christensen et al. 1985, Nakasone and Iwanaga 1990). On the other hand, it has been reported that hy- drophobic interactions in addition to hemagglutinating capabilities could be responsible for bacterial adhesion to animate and inani- mate surfaces (Bruno 1988. Clark et al. 1989. Savage 1992, Vazquez-Juarez et al. 1994). However, several Vibrio species showed an ability to adhere to host cells and cell lines, regardless of their degree of hydrophobicity (Santos et al. 1991 ). The aim of this work is to study the adhesion properties of V. tapetis, consid- ering both the contribution of bacterial surface hydrophobicity and the specific attachment to different cells. MATERIAL AND METHODS Microorganisms and Culture Conditions Twenty-seven strains of V. tapetis were used for the hydropho- bicity studies. Bacterial strains were grown in tryptone soya broth or agar (Oxoid Ltd.. Basingstoke, Hampshire. UK) supplemented with 1.5% NaCl (TSBS and TSAS. respectively), and incubated at 22°C for 18 h. Eight V. tapetis strains, representative of each V. tapetis group established previously by Borrego et al. (1996b) and Castro et al. (1997) were used for the adhesion experiments to cells. Bacteria were grown in TSBS for 18 h at 22°C. resuspended in sterile buffered saline (BS). and washed twice by centrifugation (at 24.000 x g for 5 min at 4°C). Bacterial pellets were resus- pended in the same BS and adjusted at a bacterial concentration of 10 8 bacteria/mL. Hydrophobicity Assays To test the hydrophobic capabilities of V. tapetis strains, three different assays were performed: microbial adhesion to hexa- decane (MATH); salt aggregation test (SAT); and nitrocellulose adhesion test (NCF). MATH was performed as described by Rosenberg et al. (1980). Bacteria were centrifuged at 4,000 x g for 10 min at 4°C, washed, and resuspended in phosphate urea magnesium sulphate (PUM) buffer (22.2 g/L K 2 HP0 4 • 3H 2 0. 7.26 g/L KH,P0 4 . 1.8 g/L urea. 0.02 g/L MgS0 4 -7H 2 0. pH 7.1). or phosphate buffered saline (PBS) (0.02 M. pH 7.2) to an absorbance at 400 nm of 0.9-1.1. Bacterial suspension aliquots (2 mL) were then transferred to clean round-bottom test tubes, and 0.3 mL n-hexadecane (Sigma Chemi- cal Co., St. Louis. MO, USA) were added and incubated for 10 min. After the mix was homogenized for 2 min, the hydrocarbon phase was allowed to rinse completely, and the aqueous phase was removed to determine the absorbance at 400 nm. The percentage of adhesion to hydrocarbons was calculated using the following ex- pression: Adhesion (%) = [A 4(MI (initial bacterial suspension) - A 40n (aqueous phase)]/[A 4m (initial bacterial suspension)] x 100. The ability of the bacteria to bind to nitrocellulose filters (NCF) was determined according to the technique described by Lachica and Zink ( 1984). Bacterial cultures were centrifuged at 4,000 x g (10 min at 4°C), washed as above, and resuspended in saline solution (0.85% NaCl. pH 7.2) at an absorbance of 1 at 600 nm. Suspensions were filtered through a 13-mm NCF (type CS, 8.0- u,m pore size) (Millipore Corp.. Bedford. MA, USA). Optical den- sity of the filtrates was measured at 600 nm. and the percentage of adhesion was expressed as: Adhesion (%) = [A 60l) (initial bacte- 91 92 Lopez-Cortes et al. rial suspension! - A 600 ( filtrate )]/[A 600 (initial bacterial suspen- sion!] x 100. The SAT, described by Lindhal et al. ( 1981 ), is based on bac- terial precipitation in presence of salts. Bacterial cultures were centrifuged at 4.000 x g, washed, and resuspended in PBS (0.002 M. pH 8.6) to achieve a concentration of 5 x 10'' bacteria/mL. Then, 30-p.L aliquots of bacterial suspensions were mixed with equal volumes of decreasing molarities of buffered ammonium sulfate solutions ranging between 0.05 and 4 M. Hydrophobicity was expressed as the lowest molarity of ammonium sulfate that produced visual clumping. Kendall rank coefficients were calcu- lated to determine the correlation between the different hydropho- bicity tests assayed. Hemocytes and Mantle Cells Collection Hemolymph was taken from the posterior adductor muscle of two clam species. Tapes decussatus and T. philippinarum, using a 20-gauge needle attached to a 3-mL syringe, through a hole per- formed in the shell margin of the clams. Then, the collected hemolymph was diluted 1 :3 in a modified anti-aggregant Alsever solution (MAS) (20.8 g/L glucose. 8.0 g/L sodium citrate. 3.36 g/L EDTA. 22.5 g/L NaCl, and 100 u-L/L distilled water). Hemolymph of 5 adult specimens of each clam species was pooled and the number of hemocytes was estimated using a Coulter-counter. Be- fore the adhesion assays, hemocyte suspension in MAS was cen- trifuged at 400 x g for 10 min. supernatant removed, and hemocytes were resuspended in BS (0.58 m NaCl. 13 mm KG, 13 mm CaCL, 26 mm MgCL. 0.54 mm Na 2 P0 4 . 50 mm Tris-HCl. pH 7.4). Then, the cells were fixed with 3.7% formaldehyde for 20 min at 4°C. Formaldehyde was removed by centrifugation (400 x g for 10 min). and the pellet resuspended in BS at a concentration of 10"cells/mL. Mantle cells were collected from healthy specimens of both clam species. Briefly, the clams were opened, and the mantle was extracted in aseptic conditions and washed for 15 min in BS. for 20 min in an antibiotic solution (Sigma. 10.000 IU/mL penicillin. 10 mg/mL streptomycin and 25 u.g/mL amphotericin) 10-fold diluted in PBS, and finally washed for 15 min in BS supplemented with 2.5% trypsin. Mantle tissue was disrupted using Pasteur pipettes, centrifuged at 300 x g for 5 min at 4°C. and the pellet was resus- pended in saline solution (0.55 m NaCl in distilled water). Mantle cells were isolated using a continuous gradient of Percoll (Amer- sham Pharmacia Biotech GmbH. Barcelona. Spain) previously prepared by centrifugation (at 25.000 x g for 20 min at 4°C) of a 60% percoll in saline solution. Cells were separated by centrifu- gation at 10.000 x g for 10 min at 4°C. The band in the percoll gradient that contained the cells was collected, washed twice in BS (at 300 x g for 5 min at 4°C), and fixed with 3.7% formaldehyde for 20 min at 4°C. Then, the formaldehyde was removed by cen- trifugation (400 x g, for 10 min), and the pellet was resuspended in BS at a concentration of 10 6 cells/mL. Cell Adhesion Assays The adhesion of V. tapetis to hemocytes or mantle cells of both clam species was evaluated by two different methods: the adhesion method described by Kumazawa et al. (1991), and by an ELISA test developed in the present study. Clam cells and V. tapetis were incubated at a concentration of 1 x 10" cells/mL and 1 x 10 x bacteria/mL, respectively, in BS at room temperature (about 20°C) for 2 h with gentle aeitation. After the incubation, the nonadhered bacteria were removed by three cycles of centrifugation (at 300 x g for 5 min. 4°C) in BS. and the final pellet was resuspended in 500 p.L of BS and fixed with 0.7% formaldehyde overnight. Af- terwards, volumes of 100 u.L were deposited in microplate wells to perform the ELISA test, and volumes of 200 uL were disposed in slides, stained with Giemsa and observed under light microscopy. In the indirect ELISA test, an anti-V. tapetis serum raised in rabbit (Castro et al. 1995) was used as the first antibody, and antirabbit IgG labeled with peroxidase (Sigma) as the second an- tibody. Mixtures of bacteria and clam cells without the first anti- body, and clam cells with the first and second antibodies, were used as controls. Adhesion to Fish Cell Lines Three fish cell lines were used for adhesion assays, Chinook salmon embryo (CHSE), epithelioma papullosum of carp (EPC) and SAF-1 derived from fibroblast of gilt-head seabream fins. Cells were maintained in Eagle's minimal essential medium (MEM) (Gibco Life Technologies. Paisley, UK) or, in the case of SAF-1 cell line, in L-15 Leibovitz medium (Gibco) supplemented with 2% glutamine. both containing 10% fetal calf serum and antibiotics (1% penicillin/streptomycin). Semieonfluent monolay- ers were grown on 24-multiwell plastic dishes with glycerol- treated coverslips (12-mm diameter). Cell monolayers were fixed with 3.7% formaldehyde for 20 min at 4°C, and washed thor- oughly with PBS. To perform the adhesion assays, bacterial suspensions contain- ing 10 s bacteria/mL were placed in the multiwell dishes containing the cell-coated coverslips and incubated at 20°C with gentle shak- ing for 2 h. After being washed thoroughly with PBS. coverslips were air dried and fixed with formaldehyde for 20 min. Then, coverslips were stained with crystal violet, mounted onto micro- scope slides, and examined under light microscopy. The adherence to fish cell lines was also evaluated using the ELISA test described above. Fish cell monolayers were trypsinized and resuspended in fresh medium without antibiotics. Then, the cells were fixed and washed as described, and resuspended in PBS at a concentration of 10 h cells/mL. Adhesion assays and the ELISA test were conducted as mentioned for clam cells. Hemagglutination Tests Hemagglutination was determined using rat, horse, rabbit, and human erythrocytes according to the technique described by Larsen and Mellergaard ( 1984). Equal volumes ( 100 u.L) of bac- terial suspension (10 9 bacteria/mL) and erythrocyte suspension (3%, v/v) in PBS (0.01 m. pH 6.8) were mixed on a 96-well plate, and incubated at room temperature for 1 h. As negative controls, erythrocyte suspensions in PBS and bacterial suspensions in PBS were used. The test was considered negative if visible agglutina- tion did not occur within 10 min. Inhibition of hemagglutination was performed by mixing the bacterial suspensions with 10. 25. 50. 75. and 100 mm solutions in PBS of D-mannose. D-fucose. L-fucose. D-glucose. D-galactose. D-fructose. D-lactose, and raffinose (Sigma). A negative control of erythrocytes plus sugar in PBS was used. Transmission Electron Microscopy ITEM) The arrangement of flagella and fimbriae was examined under TEM in 24-h V. tapetis grown in TSAS. Briefly, the samples were fixed with 2.5% glutaraldehyde in 0.01 m cacodylate buffer (pH Adhesion of Vibrio tapetis 93 7.2) for 2 h at 4°C. Then, they were stained with 1% uranyl acetate (pH 4.5) for 45 s on copper grids (400 mesh) covered with form- var. dried, and examined under TEM. Outer Membrane Protein Analyses Outer membrane protein (OMP) analyses were carried out by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). following the method described previously by Cas- tro et al. ( 1996). Briefly, bacterial cells were disrupted by sonica- tion (in an ice bath with 4 pulses of 30 s at 50 W). and the cellular envelopes were sedimented by centrifugation (100.000 x g for 1 h at 4°C). Cytoplasmic membranes were selectively solubilized with sodium lauryl sarcosinate (Sarkosyl, Sigma), and outer membranes (OM) were sedimented by centrifugation as above. OM samples were electrophoresed in discontinuous polyacryl- amide-SDS gels (4.5-12.5%). using the Laemmli"s technique (1970). Proteins were detected in the gels by staining with Coo- massie brilliant blue (Sigma). TABLE 1. Determination of cell surface hydrophobicity of Vibrio tapetis strains using different methods RESULTS Hydrophobicity Different results have been obtained depending on the method used to estimate cell surface hydrophobicity of V. tapetis strains (Table 1 ). Most of the isolates (77.8%) aggregated in the presence of less than 1 M ammonium sulfate. Adhesion to nitrocellulose filters yielded the highest values of hydrophobicity for most of the bacterial isolates, percentages of adhesion ranging between 94.9 and 99.9%. A variable degree of adhesion to n-hexadecane has been observed, percentages of adhesion ranging between 20. 1 and 64.6% when PUM buffer was used an aqueous phase and between 21.4 and 63.8% when PBS was used. The criteria of hydrophobicity proposed by Santos et al. ( 1990) and Lee and Yii (1996) were used to evaluate the hydrophobicity of the V. tapetis strains tested (Table 2). According to the criteria applied, 11.1 and 40.8% of the isolates were highly hydrophobic with MATH assay, using PUM and PBS buffers, respectively. However, 77.8% of the strains showed strong hydrophobicity with SAT and 100% with NCF assay (Table 2). Most of the isolates were included in the group of moderate hydrophobicity with MATH (88.9% for PUM buffer, and 59.2% for PBS buffer), and only 22.2% of the strains were included using the SAT test. Kendall rank coefficients have shown the presence of signifi- cant correlation (p < .05) between MATH assays in the presence of PUM and PBS buffers (p = .026). These findings suggest that adhesion to n-hexadecane is not significantly influenced by the use of PUM or PBS buffers as aqueous phase. On the contrary, no significant correlation was obtained between the rest of tests as- sayed (p > .05), with significance degree of p = .393 for MATH (PUM) versus SAT, p = .884 for MATH (PUM) versus NCF. p = .440 for MATH (PBS) versus SAT. p = .491 for MATH (PBS) versus NCF, and p = .348 for SAT versus NCF. Cell Adhesion Assays V. tapetis adhesion to different cell systems, such as clam hemocytes, mantle cells of clams, and fish cell lines, has been evaluated by two different approaches, the microscopic determi- nation of cellular adhesioin percentages and by an EL1SA (Tables 3 and 4). The results obtained varied depending both on the cel- lular system used and the V. tapetis strains tested. Thus, all the V. MATH" NCF h Strains PUM PBS SAT C V. tapetis 2.1 47.5 61.0 99.1 0.50 V. tapetis 2.3 44.9 60.0 99.2 1.00 V. tapetis 8.1 47.3 58.9 99.9 0.46 V. tapetis 8.3 42.9 63.8 99.5 0.93 V. tapetis 8.4 32.2 55.7 99.5 0.70 V. tapetis 8.5 35.4 62.6 99.1 0.46 V. tapetis 8.6 22.0 22.3 99.5 0.80 V. tapetis 8.7 29.6 37.6 99.4 0.70 V. tapetis 8.17 24.8 35.4 99.9 1.20 V. tapetis 8.19 51.3 48.5 99.4 0.80 V. tapetis 9.3 54.0 53.3 99.4 0.70 V. tapetis 9.4 30.4 44.3 99.9 0.93 V. tapetis 9.5 46.2 53.7 99.7 0.66 V. tapetis 9.7 46.0 58.0 99.4 0.93 V. tapetis 11.1 64.7 50.0 99.1 1.00 V. tapetis 11.2 26.0 21.4 99.9 0.80 V. tapetis 11.4 29.1 56.3 97.9 0.93 V. tapetis IS- 1 20.1 37.4 94.9 0.86 V. tapetis IS-5 46.9 33.5 99.4 0.80 V. tapetis IS-7 9.7 44.8 98.1 0.83 V. tapetis IS-8 27.0 35.6 99.8 0.80 V. tapetis IS-9 46.2 50.7 99.9 0.80 V. tapetis CECT 4600 T 20.7 46.5 99.6 1.00 V. tapetis 1703 23.3 31.4 98.9 0.80 V. tapetis 6301 46.0 42.0 99.5 0.66 V. tapetis 0202 40.9 44.5 99.1 1.00 V. tapetis 0705 26.2 44.4 99.0 1.00 J Percentage of adherence to n-hexadecane. b Percentage of adherence to nitrocellulose filters. c Lowest molarity of ammonium sulphate producing visible aggregation. CECT: Spanish Type Culture Collection. 1 Type strain. tapetis strains did not adhere to the fish cell lines CHSE and EPC. but the adhesion percentage to SAF- 1 cells ranged from 2 to 96% (Table 3). These values contrast with the values obtained for clam cells (hemocytes and mantle cells), varying between 68 and 100% (Table 3). Only two V. tapetis strains (8.6 and 0202) showed similar adhesion rates, regardless of the type of cells used. No significant differences (p > .05) were obtained in the adhesion capability of V. tapetis strains depending on the origin of the clam cells used, except in the case of the strain 1 1 .2 for hemocytes of T. decussatus and T. philippinamm (89% vs. 68%), and the strains 2.1 (76% vs. 100%) and CECT 4600 T (77% vs. 97%) for mantle cells of both clam species. Mean numbers of adhered bacteria per cell are also given in Table 3. SAF-1 cell line proved to be a poor system for V. tapetis adhesion, with mean values ranging between 1 .5 and 6.8 adhered bacteria per cell. In contrast, mantle cells and clam hemocytes were better matrix systems for V. tapetis adhesion, ranging be- tween 5.8 and higher than 25 adhered bacteria per hemocyte. and between 10.4 and 22.8 adhered bacteria per mantle cell. In this case, no significant differences were detected between the origin of the cells (source and species), although significant differences were obtained between the V. tapetis strains tested, strain 11.2 being the least adherent to hemocytes (Table 3). 94 LOPEZ-CORTES ET AL. TABLE 2. Hydrophobicity degree of Vibrio lapetis strains using SAT, NCF, and MATH assays according to the criteria proposed by Santos et al. (1990) and Lee and Yii (1996) Hydrophobicity Percentage of Test Values degree V. lapetis Isolates SAT <1.0M Strong 77.8 1.1-2.0 M Moderate 22.2 2.1-1.0 M Weak >4.0M Negative NCF >75% Strong 100 50-75% Moderate <50% Negative MATH >50% Strong 40.8 (PBS) J 20-50% Moderate 59.2 <20% Negative MATH >50% Strong 11.1 (PUM) b 20-50% Moderate 88.9 <20% Negative 1 PBS used as aqueous phase. h PUM buffer used as aqueous phase. Table 4 expresses the results of V. tapetis adhesion using an ELISA technique. In agreement with the results given in Table 3. V. tapetis strains possessed a low adhesion rate for SAF-1 cells, and even strain IS-7 was nonadherent. Adhesion to clam cells varied depending on the strains considered: thus, significant dif- ferences were found between the adhesion to clam hemocytes of both clam species for V. tapetis CECT 4600 T . No correlation was obtained between the hydrophobicity of the strains and their adhesion to several cells, except in the case of MATH (PUM) hydrophobicity and adhesion to hemocytes of T. philippinarutn determined by ELISA (data not shown). Hemagglutination and V. tapetis Appendages None of the V. tapetis strains presented hemagglutination of horse, rabbit, and human erythrocytes; on the contrary, all of them agglutinated rat erythrocytes, and the hemagglutination was not inhibited by any of the sugars tested at the different concentrations assayed. The presence of appendages on the V. tapetis surface was de- termined by TEM. In all the V. tapetis strains tested, the presence of a sheathed polar flagellum was recorded (Fig. 1 1, and sometimes several lateral flagella were also observed. Piliation of the strains or fimbria-like structures were not observed in the bacterial cul- tures tested. However, visualization of isolated and purified pili has not been performed yet. Analysis of the Cellular Components of the Outer Membrane The electrophoretic analyses of OMP showed that all V. tapetis strains tested, except the strain 0202. present the same band pat- tern, expressing proteins of molecular weight ranging between 78 and 15 kDa. The profile of these strains is dominated by a major outer membrane protein (MOMP) of an estimated molecular weight (MW) of 35 kDa. In the case of the strain 0202 the MOMP was of 37 kDa. and presented a high MW protein of 94 kDa (Fig. 2). DISCUSSION In BRD. an epizootic disease affecting cultured clam species, mainly T. philippinarum, V. tapetis is predominantly detected on the clam periostracal lamina (Allam et al. 1996). The capacity of this pathogen to adhere to periostracum is obviously an essential step for the bacterial colonization. However, the mechanisms by which bacterial cells adhere to this clam surface has not been elucidated completely. One hypothesis for the mechanisms by which V. tapetis adheres to clam tissues involves the concept that filamentous appendages characterized as pili (Paillard and Maes 1995a) bind the cells to periostracal lamina. However, the presence of these bacterial appendages were visualized only in some colo- nizing V. lapetis in diseased clams (Borrego et al. 1996a). On the other hand, Arp ( 1988) pointed out that before the bacterial adhe- sion, it is necessary for the bacteria to maintain their position along a mucosal surface by establishing small numbers of noncovalent bonds between the bacterial and mucosal surfaces. These bonds depend on several physicochemical mechanisms, the hydrophobic interactions being the most important (Rosenberg and Kjelleberg 1986). For these reasons, we suggest that the adhesion of V. tapetis to clam tissues may be governed by two different but complemen- tary mechanisms: ( 1 ) physicochemical forces of adsorption; and (2) specific adhesion depending on adhesive bacterial structures TABLE 3. Adhesive capabilities of Vibrio tapetis strains to different cell systems Adhesion Percentages Mean of Adhered Bacteria per Cell SAF-1 Hemocytes Mantle Cells SAF-1 Hemocytes Mantle Cells Strains T.d" T.p" T.d T.p T.d T.p T.d T.p 2.1 ~> 88 91 76 100 1.9 6.6 11.2 21.0 21.3 8.6 96 100 94 88 98 6.8 11.7 10.9 19.0 17.6 9.4 25 98 94 90 99 5.1 >25 12.1 15.1 22.8 11.1 18 88 100 96 96 1.6 10.4 15.2 21.7 10.5 11.2 46 89 68 90 91 2.2 6.6 8.1 21.1 13.7 IS-7 58 89 99 94 98 1.5 >25 16.1 17.9 16.5 CECT 4600 T 59 100 95 77 97 3.7 11.3 5.8 10.4 17.4 0202 96 100 100 90 97 6.6 >25 15.2 14.6 20.1 1 Tapes decussatus. ' Tapes philippinarum. Adhesion of Vibrio tapetis 95 TABLE 4. Adhesive capabilities of Vibrio tapetis strains to different cell systems using an ELISA technique SAF-1 Hemocytes Mantle Cells Strains T. decussatus r. philippinarum T. decussatus T. philippinarum 2.1 0.21 ' 0.23 0.24 0.28 0.34 8.6 0.19 0.22 0.25 0.28 0.33 9.4 0.18 0.23 0.24 0.28 0.31 111 0.17 0.24 0.24 0.24 0.30 11.2 0.15 0.20 0.26 0.26 0.30 IS-7 0.09 0.20 0.25 0.29 0.29 CECT 4600 T 0.22 0.17 0.38 0.24 0.30 0202 NT h NT NT NT NT ' Absorbance units at 450 nm. ' NT: Not tested because of the lack of specificity of the antiserum against V. tapetis to strain 0202. that interlock in a stereospecific manner with complementary structures on the opposing surface. Cellular hydrophobicity is known to be associated with the capacity of microbial cells of many taxonomic groups to adhere to surfaces of numerous types, including those of animal tissues (Doyle and Rosenberg 1990). The results obtained in the present study demonstrate that even strains of V. tapetis closely related at biochemical, serological, and molecular levels (Borrego et al. 1996b, Castro et al. 1996, Castro et al. 1997) may vary signifi- cantly in surface hydrophobicity as measured by any of the three assays used (Table 1 ). As with other bacteria, consistency of re- sults among the three hydrophobicity assays was only observed in strains with very hydrophilic surfaces (Mozes and Rouxhet 1987, Sorogon et al. 1991). Differences in the distribution of hydropho- bic components on the bacterial surface may account for these disparities. Although the SAT may provide a measure of overall surface hydrophobicity. the MATH and NCF assays may indicate the presence of hydrophobic domains on an otherwise hydrophilic cell surface (van der Mei et al. 1987). The lower values of hydro- phobicity obtained for V. tapetis strains using the MATH assay as compared to SAT and NCF techniques has been reported previ- ously (Sorogon et al. 1991). These authors pointed out the possi- '7\ -n*A^ '» 4 . ■L *i ^^^ '• 1 fim bility that cell surfaces of the bacteria tested were modified by the hydrophobicity assay procedures. Thus, hexadecane used in the MATH assay may extract constituents from the bacterial envelope (Dillon et al. 1986. Rosenberg and Kjelleberg 1986). Methods for measuring bacterial hydrophobicity differ some- what in the precise properties they measure, and different types of interactions are considered when different methods to estimate hydrophobicity are used. Thus, Dickson and Koohmaraie (1989) observed that the relative hydrophobicity estimated by hydropho- bic interaction chromatography. MATH and contact angle mea- surements for different bacterial species was dependent on the specific method tested. The results obtained in the present study for V. tapetis strains isolated from diseased clams show that the experimental conditions imposed by the different methods used influence the observed hydrophobicity interactions to some degree. Specific hydrophobicity assays may be useful predictors of adhesion for closely related strains of certain bacterial species (Martin et al. 1997). In the case of V. tapetis strains isolated from diseased clams, the results obtained in the hydrophobicity tests indicate that adhesion of these bacteria cannot be. explained by hydrophobic interactions alone. Rather, adhesion is likely to be mediated by an interplay of hydrophobic and hydrophilic surface components (Dickson and Siragusa 1994), or specific interactions 205 Figure 1. Electron micrograph of negative stained Vibrio tapetis CECT 4600 cells, showing the presence of a single sheathed polar flagellum. 94 67 43 30 Figure 2. Electrophoresis in SDS-polvacrvlamide gels of the outer membrane proteins from several Vibrio tapetis strains; lanes 1 to 5, strains CECT 4600 T , 2.1, 11.2, IS-7, and 0202. respectively. In lanes M, the molecular weight (in kDa) of standard proteins is indicated. 96 LOPEZ-CORTES ET AL. between bacterial adhesins and cellular receptors (Christensen et al. 1985). For many pathogenic bacterial strains, mucosal attachment is mediated by specific bacterial surface appendages. Pili are consid- ered as the most relevant adhesins and colonization factors of host tissue surface. Many types of bacterial pili have been recognized in Vibrio species, such as toxin-coregulated pili (TCP), mannose- sensitive hemagglutinin (MSHA) pili, core-encoded pili (Cep), ac- cessory colonization factor (Acf), NAGV 14, Na2, and Ha7 pili (Yamashiro and Iwanaga 1996). In the present study, we have not detected the presence of these appendages on V. lapetis surface, under TEM examination (Fig. 1). However, all V. tapetis cells examined showed the presence of a sheath flagellar structure, which may be important for attachment to host tissues (Attridge and Rowley 1983). In contrast. Paillard and Maes ( 1995a) reported that fimbria-like appendages were present in the V. tapetis colo- nizing the periostracal lamina of diseased clams. These contradic- tory results lead us to suggest the role of an unknown factor that promotes the fimbria synthesis in V. tapetis infection. Iijima et al. (1981) suggested that V. parahaemolyticus synthetize a cytotoxic factor that degenerates epithelial cells of the host and promotes its adherence. On the other hand, nutrient-limiting conditions may enhance the bacterial adhesion (Dai et al. 1992) or induce the formation of adherent structures (McCarter and Silverman 1989, Nakasone and Iwanaga 1990). The importance of the flagellum as a potential virulence factor has been demonstrated for several bac- terial species. This structure has been involved in pathogenicity as either a motility organelle or an organelle that carries an adhesive component, both roles providing an advantage to the bacterium for its invasive capabilities (Norqvist and Wolf-Watz 1993, Milton et al. 1996). The agglutination of bacteria to erythrocytes has been proposed as an efficient in vitro system to demonstrate the bacterial adhesive activity (Santos et al. 1990) and as a system to characterize the type of adhesins (Evans et al. 1980. Zunino et al. 1994). None of the V. tapetis strains tested showed hemagglutination of horse, rabbit, and human erythrocytes, but all of them were positive for rat erythrocytes. This apparent contradiction in the hemagglutina- tion results obtained may be explained by the fact that the hem- agglutination depends on the presence of specific receptors on the erythrocyte surface, and such receptors contain oligosaccharides that varied depending on the animal species (Jones and Freter 1976). Previously, adhesion to clam cell systems by V. tapetis has not been demonstrated. Therefore, this study constitutes the first report of adhesion to hemocytes and mantle cells of two clam species. All the V. tapetis strains tested showed a higher degree of adhesion to clam cells, both hemocytes and mantle cells, than to fish cells (Table 3). These findings suggest the existence of a host or tissue specificity. According to Christensen et al. ( 1984), the adhesion of a particular bacterial species may vary considerably depending on the host species, physiology, phenotype. and tissue. Miller and Mekalanos (1988) described the role of two outer membrane proteins. OmpU and OmpT. as colonization factors of V. cholerae. Later. Sperandio et al. ( 1995) verified that OmpU acts as an adherence factor involved in the colonization of epithelial cells by V. cholerae. The amino-terminal amino acid sequence of OmpU was similar to the sequences of Haemophilus influenzae HMW1 and HMW2 adhesins. and shared also similarities with the Bordetella pertussis FHA. As it can be seen in Fig. 2. all the V. tapetis strains tested presented the same OMP profile, except for 0202 strain. The MW of OmpU (32-38 kDa) is similar to the major OMP (MOMP) detected in V. tapetis (35-37 kDa), which induces to speculate about a similar function of this protein in V. tapetis strains. To verify this hypothesis, further studies of adhesion to cells using isolated MOMP and inhibition with anti-MOMP anti- serum are necessary. In short, the adhesion mechanisms of V. tapetis are complex and depend on different processes that act in several steps. In a hypothetical model, V. tapetis is directed to specific substrate of the clam tissue by means of their motility organelles, which also help the bacterium adhere to clam cells. Then, the hydrophobic forces maintain the bacterial position along a mucosal surface by establishing small numbers of non-covalent bonds, and, finally, bacterial adhesins or surface proteins of V. tapetis interlock spe- cific attachment with complementary structures on the opposing surface. ACKNOWLEDGMENTS This study was supported by a grant from the Direction Gen- eral de Ciencia y Tecnologi'a (DGICYT) (No. PB-95-0467). We thank Miss M. J. Navarrete for her help in the English revision of the manuscript. LITERATURE CITED Allam. B., C. Paillard & P. Maes. 1996. Localization of the pathogen Vibrio PI in clams affected by brown ring disease. Dis. Aquat. Org. 27:144-155. Arp, L. H. 1988. Bacterial infections of mucosal surfaces: an overview of cellular and molecular mechanisms, pp. 3-27. In: J. A. Roth (ed.). Virulence Mechanisms of Bacterial Pathogens. American Society for Microbiology, Washington, DC. Attridge, S. R.. & D. Rowley. 1983. The role of flagellum in the adherence of Vibrio cholerae. J. Infect. Dis. 147:864-872. Borrego. J. J.. A. Luque. D. Castro. J. A. Santamaria & E. Martinez- Manzanares. 1996a. Virulence factors of Vibrio PI. the causative agent of the brown ring disease in the Manila clam. Ruditapes philippinarum. Aquat. Living Resour. 9:125-136. Borrego. J. J., D. Castro. A. Luque, C. Paillard. P. Maes. M. T. Garcia & A. Ventosa. 1996b. Vibrio tapetis sp. nov.. the causative agent of the brown ring disease affecting cultured clams. Int. J. Syst. Bacterial. 46:480-484. Bradford, M. M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 72:248-254. Bruno. D. W. 1988. The relationship between autoagglutination. cell sur- face hydrophobicity. and virulence of the fish pathogen Renibacterium salmoninarum. F EMS Microbiol. Lett. 51:135-140. Castro. D. 1994. Patologi'as de origen bacteriano en almejas cultivadas Tapes decussatus y T. phillipinarum. Ph.D. thesis. University of Malaga. Spain. Castro, D.. A. Luque, J. A. Santamaria, P. Maes. E. Maninez-Manzanares & J. J. Borrego. 1995. Development of immunological techniques for the detection of the potential causative agent of the brown ring disease. Aquaculture 132:97-104. Adhesion of Vibrio tapetis 97 Castro, D., J. A. Saniamaria. A. Luque, E. Martinez-Manzanares & J. J. Borrego. 1996. Antigenic characterization of the etiological agent of the brown ring disease affecting manila clams. Syst. Appl. Microbiol. 19:231-239. Castro, D.. J. L. Romalde, J. Vila. B. Magarinos. A. Luque & J. J. Borrego. 1997. Intraspecific characterization of Vibrio tapetis strains by use of pulsed-field gel electrophoresis, ribotyping, and plasmid profiles. Appl. Environ. Micribiol. 63:1449-1452. Christensen. G. D., W. A. Simpson & E. H. Beachy. 1984. Bacterial ad- herence in infection, pp. 6-23. In: G. L. Mandell, R. G. Douglas & J. E. Bennett (eds.). Principles and Practice of Infectious Diseases, 2nd ed. John Wiley & Sons. New York. Christensen. G. D.. W. A. Simpson & E. H. Beachy. 1985. Adhesion of bacteria to animal tissues: complex mechanisms, pp. 297-305. In: D. C. Savage and M. Fletcher (eds.). Bacteria Adhesion. Plenum Press, New York. Clark, R. B„ F. C. Knoop. P. J. Padgitt. D. H. Hu, J. D. Wong & M. J. Janda. 1989. Attachment of mesophilic aeromonads to cultured mam- malian cells. Cnrr. Microbiol. 19:97-102. Dai. J. Ft., Y. S. Lee & H. C. Wong. 1992. Effects of iron limitation on production of a siderophore, outer membrane proteins, and hemolysin and on hydrophobicity, cell adherence, and lethality for mice of Vibrio parahaemolyticus. Infect. Imniun. 60:2952-2956. Daly, J. G. & R. M. W. Stevenson. 1987. Hydrophobic and hemaggluti- naling properties of Renibacterium salmoninarum. J. Gen. Microbiol. 133:3575-3580. Dickson. J. S. & M. Koohmaraie. 1989. Cell surface charge characteristics and their relationship to bacterial attachment to meat surfaces. Appl. Environ. Microbiol. 55:832-836. Dickson. J. S. & G. R. Siragusa. 1994. Cell surface charge and initial attachment characteristics of rough strains of Listeria monocytogenes. Lett. Appl. Microbiol. 19:192-196. Dillon, J. K„ J. A. Fuerst, A. C. Hayward & G. H. G. Davis. 1986. A comparison of five methods for assaying bacterial hydrophobicity. J. Microbiol. Meth. 6:13-19. Doyle. R.J. & M. Rosenberg (eds.). 1990. Microbial cell surface hydro- phobicity. American Society for Microbiology. Washington. DC. Evans. D. J. Jr.. D. G. Evans. L. S. Young & J. Pitt. 1980. Hemagglutina- tion typing of Escherichia colt: definition of seven hemagglutination types. J. Clin. Microbiol. 12:225-242. Iijima. Y., H. Yamada & S. Shinoda. 1981. Adherence of Vibrio para- haemolyticus and its relation to pathogenicity. Can. J. Microbiol. 27: 1251-1259. Jones. G. W. & R. Freter. 1976. Adhesive properties of Vibrio cholerae: nature of the interaction with isolated rabbit brush-border membranes on human erythrocytes. Infect. Iininnn. 14:240-245. Jonson, G.. J. Holmgren & A. M. Svennerholm. 1991. Identification of a mannose-binding pilus on Vibrio cholerae El Tr. Microb. Pathog. 11: 433-141. Kumazawa. N. H.. T. Tanigawa, Y. Tanaka. H. Osatake & K. Tanaka. 1991. In vitro attachment of Vibrio parahaemolyticus to hemocytes of two gastropod mollusks. J. Vet. Med. Sci. 53:297-300. Lachica. R. V. & D. L. Zink. 1984. Plasmid-associated cell surface charge and hydrophobicity of Yersinia enterocolitica. Infect. Iininitn. 44:540-543. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680-685. Larsen. J. L. & S. Mellergaard. 1984. Agglutination typing of Vibrio an- guillarum isolates from diseased fish and from the environment. Appl. Environ. Microbiol. 47:1261-1265. Lee. K. K. & K. C. Yii. 1996. A comparison of three methods for assaying hydrophobicity of pathogenic vibrios. Lett. Appl. Microbiol. 23:343-346. Lindhal. M.. A. Fans. T. Wadstrom & S. Hjerten. 1981. A new test based on "salting out" to measure relative surface hydrophobicity of bacterial cells. Biochim. Biophys. Acta 677:471-476. Martin, M. L., Y. Benito. C. Pin, M. F. Fernandez, M. L. Garcia. M. D. Selgas & C. Casas. 1997. Lactic acid bacteria: hydrophobicity and strength of attachment to meat surfaces. Lett. Appl. Microbiol. 24:14-18. McCarter. L.. M. Silverman. 1989. Iron regulation of swamer cell differ- entiation of Vibrio parahaemolyticus. J. Bacterial. 171:731-736. Miller. V. L. & J. J. Mekalanos. 1988. A novel suicide vector and its use in construction of insertion mutations: osmoregulation of outer mem- brane proteins and virulence determinants in Vibrio cholerae requires toxR. .1. Bacterial. 170:2575-2583. Milton. D. L.. R. O'Toole, P. Horstedt, & H. Wolf-Watz. 1996. Flagellin A is essential for the virulence of Vibrio anguillarunu J. Bacterial. 178: 1310-1319. Mozes, N. & P. G. Rouxhet. 1987. Methods for measuring hydrophobicity of microorganisms. J. Microbiol. Melli. 6:99-112. Nakasone. N. & M. Iwanaga. 1990. Pili of Vibrio cholerae non-Ol. Infect. biiniun. 58:1640-1646. Norqvist, A. & H. Wolf-Watz. 1993. Characterization of a novel chromo- somal virulence locus involved in expression of a major surface flagel- lar sheath antigen of the fish pathogen Vibrio anguillarum. Infect. In, mini 61:2434-2444. Novoa, B.. A. Luque, D. Castro, J. J. Borrego & A. Figueras. 1998. Char- acterization and infectivity of four bacterial strains isolated from brown ring disease-affected clams. J. Invertebr. Pathol. 71:34 — i\. Paillard. C. & P. Maes. 1990. Etiologie de la maladie de I'anneau brun chez Tapes philllipinaruni: pathogenicite d'un Vibrio sp. C.R. Acad. Sci. Paris 310:15-20. Paillard. C. & P. Maes. 1995a. The brown ring disease in the Manila clam. Ruditapes philippinarum. I. ultrastructural alterations of the periostra- cal lamina. J. Invertebr. Pathol. 65:91-100. Paillard, C. & P. Maes. 1995b. The brown ring disease in the Manila clam. Ruditapes philippinarum. 11. microscopic study of the brown ring syn- drome. J. Invertebr. Pathol. 65:101-1 10. Rosenberg. M.. D. Gutmck & E. Rosenberg. 1980. Adherence of bacteria to hydrocarbons: a simple method for measuring cell-surface hydro- phobicity. FEMS Microbiol. Lett. 9:29-33. Rosenberg. M. & S. Kjelleberg. 1986. Hydrophobic interactions: role in bacterial adhesion. Adv. Microb. Ecol. 9:353-393. Santos, Y.. I. Bandin. T. P. Nieto, D. W. Bruno. A. E. Ellis & A. E. Toranzo. 1990. Comparison of the cell surface hydrophobicity of bac- terial fish pathogens by different procedures, pp. 101-115. In: F. O. Perkins and T. C. Chen (eds.). Pathology in Marine Science. Academic Press. New York. Santos, Y., 1. Bandin. T. P. Nieto, J. L. Barja, A. E. Toranzo & A. E. Ellis. 1991. Cell-surface-associated properties of fish pathogenic bacteria. J. Aquat. Amm. Health 3:297-301. Savage. D. C. 1992. Growth phase, cellular hydrophobicity. and adhesion in vitro of lactobacilli colonizing the keratinizing gastric epithelium in the mouse. Appl. Environ. Microbiol. 58:1992-1995. Sorongon, M. L„ R. A. Bloodgood & R. P. Burchard. 1991. Hydrophobic- ity. adhesion, and surface-exposed proteins of gliding bacteria. Appl. Environ. Microbiol. 57:3193-3199. Sperandio. V., J. A. Giron. W. D. Silveira & J. B. Kaper. 1995. The OmpU outer membrane protein, a potential adherence factor of Vibrio cho- lerae. Infect. Iiiiiiiiin. 63:4433-4438. van der Mei. H. C, A. H. Weerkamp & H. J. Busscher. 1987. A compari- son of various methods to determine hydrophobic properties of strep- tococcal cell surfaces. J. Microbiol. Meth. 6:277-287. Vazquez-Juarez, R., T. Andlid & L. Gustafasson. 1994. Cell surface hy- drophobicity and its relation to adhesion of yeasts isolated from fish gut. Colloids Surf. 2:199-208. Yamashiro. T. & M. Iwanaga. 1996. Purification and characterization of a pilus of a Vibrio cholerae strain: a possible colonization factor. Infect. biimiin. 64:5233-5238. Zunino. P.. C. Piccini & C. Legnani-Fajardo. 1994. Flagellate and non- flagellate Proteus mirubilis in the development of experimental urinary tract infection. Microb. Pathog. 16:379-385. Journal of Shellfish Research, Vol. IX. No. 1. 99-105, 1999. ASSESSING MANIPULATIONS OF LARVAL DENSITY AND CULLING IN HATCHERY PRODUCTION OF THE HARD CLAM, MERCENARIA MERCENARIA CARRIE J. DEMING AND MICHAEL P. RUSSELL* Biology Department Villanova University Villanova, Pennsylvania 19085-1699 ABSTRACT Studies of the hard clam. Mercenaria mercenaria, indicate that the hatchery practice of larval culling may be coun- terproductive because of an inverse relationship in growth between larvae and postsettlement juveniles. The effects of larval culture manipulations were explored with two parallel studies: one set up in a commercial hatchery and the other in the laboratory. Both laboratory and hatchery studies started with the same cohort of larvae produced from spawning hatchery broodstock. Except for culling, these larvae were raised using standard practices in the hatchery for the first 10 days. On the tenth day of development, the cohort was sieved through 105-p.m mesh and separated into two treatments, large larvae and small larvae. In the hatchery, these samples were followed through settlement, and growth was monitored for 1 74 days postfertilization. In the laboratory, the two samples of larvae were raised under high-density (20 larvae/mL) or low-density (4 larvae/mL) conditions and thus assigned to one of four larval treatments: large/high-density, large/low-density, small/high-density. and small/low-density. These treatments were replicated (10 times each) to yield a 2 x 2 factorial, randomized block, repeated-measures analysis of variance (ANOVA) experiment. Growth in the laboratory was monitored for 276 days. Both the hatchery and laboratory studies indicate that larval culling does not increase productivity and may be counterproductive. Furthermore, larval density has an effect on subsequent juvenile growth. Larvae raised at low density produced larger clams in both the small and large larval treatments. KEY WORDS: Mercenaria mercenaria. hard clam, larval culling, aquaculture. growth INTRODUCTION ... it is reasonable to question the merits of the larval culling practices carried out worldwide in bivalve hatcher- ies . . . a substantial proportion of the lar\>▼ Larval Corral Downweller Upweller Figure 1. Experimental units. Each replicate was a 15-cm segment of PVC pipe with three 4-cm sections cut out of the bottom to form a tripod support. As the cohort developed, the size of the mesh on the bottom was increased (Table 1) to enhance flow within the approximately 8(10 ml. volume held by each unit. The units are drawn to scale; however, the sea table and upwelling table are not — all 40 experimental units were in the sea table and then moved to the upwelling table (which was held in the sea table). The thick arrows represent air and water flow. Flow rates were monitored regularly and adjusted to no less than 1 L/min in the downwellers and upwellers. A. Larval Corral. Filtered air entered the bottom of the experimental units through a 2-mm plastic tube positioned approximately 2 cm from the bottom and was regulated to I bubble/s. Seawater circulated within the unit and between the unit and the sea table through the bottom mesh ( 105 fan). B. Downweller. Seawater entered through a 1-cm tygon tube positioned directly above each experimental unit. C. Upweller. All 40 experimental units were placed in the same blocked arrangement, in an upwelling table nested within the sea table. Each unit was plumbed near the top with a sealed tygon connector attached to the upwelling table. Seawater tlowed into the upwelling table, up through the bottom of each unit, and out to the sea table via the tygon connector. 102 Deming and Russell ization) and used as a covariate in the repeated-measures ANOVA to account for the effects of juvenile density on growth. In addi- tion, an ANOVA was performed on the means of SL from each replicate on the last set of measurements (day 276 postfertilization) with average postsettlement density as the covariate. Differences in survivorship were tested using a randomized block ANOVA on the number of individuals in each replicate on the last sampling date (start density / final density). All analyses were performed using SAS (1989). RESULTS There was a significant difference (p < .0001. / = 18.06) in SL between the large and small larval treatments on day 10 postfer- tilization (Fig. 2A). On this date, approximately 5% of the culture was in the pediveliger stage of development, and all pediveligers were part of the large larval treatment. Settlement occurred earlier in the hatchery-reared clams (days 17-20 postfertilization) than in the laboratory experiment (days 23-25 postfertilization. Table 1 ). Although we tracked the growth of clams in the hatchery phase of the experiment (Fig. 3). we did not compare the two groups sta- tistically, because this part of the experiment was pseudoreplicated (Hurlbert 1984). At the end of the hatchery study ( 174 days post- fertilization), there did not seem to be a difference in SL between the two larval size groups; the mean size of clams from the large larval treatment was 4.21 mm. and the mean size of clams from the small larval treatment was 4.31 mm (Fig. 2B). The repeated-measures ANOVA revealed that there were no significant differences in SL among the four treatments attribut- able to larval size or larval density (0.10 >p> .05. Table 2) over the course of the 276-day experiment. However, the ANOVA from the last sample revealed that although larval size was not signifi- cant (p = .22, F = 1.69) larval density was significant (p = .02. F = 7.65). In both the large and small larval treatments, larvae raised at lower densities produced bigger juveniles at the end of the experiment on day 276 postfertilization (Fig. 2C). This difference in growth cannot be attributed to differences in postsettlement density (p = .39. Table 2). For most of the experiment, postsettle- ment juvenile density was highest in the large/low-density larval treatment (Fig. 4). and this treatment produced the biggest juve- niles (Fig. 2C). Figure 4 shows the average number of individuals per replicate in the four treatments over the course of the experiment. Mortality levels were highest among all treatments between days 21 and 42 postfertilization. For most of the experiment (days 42-270). sur- vivorship remained constant among all four treatments. Mortality was highest in the high larval density treatments and lowest in the large/low-density larval treatment (p < .0001, F = 4.12, Fig. 4). Larval Separation (June 15. 1996) 0.185 0.180 - bb 0.175 c "S 0.170 C/3 0.165 - 0.160 * 4.5 4.4 4 3 42 - 4.1 4.0 B C Hatchery Laboratory (November 26. 1996) (March 15. 1997) 6.00 -i 5.75 - 5.50 5.00 - 4.75 A 6 Small Large Small Large Small Large Larval Size Figure 2. Means (± 2 standard errors) of shell lengths at the beginning and end of the hatchery and laboratory phases of the study. The circles represent the small larval treatment; larvae that passed through the 105-u.m mesh and were caught on the 70-pm mesh on June 15, 1996. The triangles represent the large larval treatment: larvae caught on the 105 um mesh. A. Initial separation. These samples (n = 200 each for large and small) are significantly different (p < .0001, r-test). B. Final measurements of hatchery-reared samples on day 174 postfertilization (n = 200 each for large and small). No statistical analysis was performed on the hatchery-reared samples, because the design of this experiment was pseudoreplicated (Hurlbert 1984. see text). C. Final measurements of laboratory samples. These values are means of replicates (n = 10 for small larvae at low density, and n = 9 for the other three treatments, see text). There were 25 clams measured from each replicate. The open symbols (Lo) represent larvae raised at low density (4/mL) and the shaded symbols (Hi) represent larvae raised at high density (20/mL). Within larval size treatment, the low -density larval treatments produced larger postsettlement juveniles. There was no difference in SL associated with larval size treatment (p < .05, see text) on this date. Larval Hard Clam Density and Culling 103 4.0 - E 3 -° I 00 a 1.0 T J T T ■ ( < < T j - - t' • S k ■ 3 **** * 25 45 65 85 105 125 Age (days post-fertilization) Figure 3. Means and standard deviations (±1) of shell lengths from the two experimental groups raised in the hatchery between the initial separation and the final measurements at the end of the experiment. The circles represent the small larval treatment and the triangles represent the large larval treatment (n: 2(10-1,600). DISCUSSION Our results are consistent with previous work showing either no correlation or a negative correlation between larval and juvenile growth rates in mollusks (e.g.. Newkirk and Haley 1982. Newkirk and Haley 1983. Stromgren and Nielsen 1989. Pechenik et al. 1996). Hilbish et al. (1993) found no evidence for "positive genetic- covariation between larval and juvenile growth" in M. mercenaria and recommended against larval culling as a method for enhancing juvenile growth and improving hatchery productivity (Hilbish et al. 1993. p. 102). In contrast, Heffernan et al. ( 1991 ) found a strong negative relationship between larval and juvenile growth, but they also questioned "the merits of hatchery culling practices for smaller larvae" (Heffernan et al. 1991. p. 199). We did not address the issue of heritability of growth characteristics in this study. Instead, we focused on the practical issues and consequences of 15 o o 3 x o "el c S S 9 - left y-axis right y-axis ► 10 14 21 30 42 -//- 1000 Slid ■600 400 200 ns u c 0> a. .05). Missing data for group II are attributable to oysters lost (see Materials and Methods). Comparative Growth of Oysters Reared at Low and High Salinity hi TABLE 2. Probabilities obtained with two-way ANOVA tests on data for condition indices and glycogen concentrations values of eastern oysters in relation to site and culture method, for each of four groups (I: 2-year-old oysters in 1993, II: 4-year-old oysters in 1993, III: 2-year-old oysters in 1994, and IV: 4-year-old oysters in 19941. Probabilities Before Spawning After Spawning Before Winter Group Group C roup Factors I II III IV I II III IV I II III IV Condition Indices Site .040* .006* .006* .019* .571 .012* .102 .002* .004* .000* .000* Culture method .002* .000* .797 .003* 1.000 .017* .043* .017* .017* .801 1.000 Site* method .100 .114 .512 .517 .909 .352 Glycogen Concentrations .602 .263 .077 .339 .844 Site .188 .000* .092 .737 .027* .001* .001* .000* .000* .006* .000* Culture method .001* .197 522 .015* .187 .031* .032* .248 .000* .110 .660 Site* method .087 .125 .934 .002* .203 .010* .889 .624 .009* .045* .511 ■ Significant difference (p < .05). data not available. collected before winter revealed that condition indices were higher for oysters sampled from stations HSS than for those from stations HSB. LSS. and LSB, all of which were similar to each other. The condition indices were higher for oysters sampled from high- salinity stations (HSS and HSB) than for the low-salinity stations (LSS and LSB) for groups III and IV oysters. DISCUSSION We focused on similarities and differences in growth and body condition between 2- and 4-year-old oysters cultured in suspension and on the bottom in a high- and a low-salinity environment during two consecutive growing seasons. Indices employed in our work. 250 250 r Figure 5. Glycogen concentrations in oysters sampled from stations HSS I high-salinity surface I, HSB (high-salinity bottom), LSS (low -salinity surface), and LSB (low-salinity bottom) for groups I (2-year-old oysters in 1993). II (4-year-old oysters in 1993), III (2-year-old oysters in 1994), and IV (4-year-old oysters in 1994), before (BS) and after spawning (AS) and before winter (B\V). Sampling dates for each group are shoyyn in Table 1. Bars with similar letters are not significantly different from each other (p > .05). Missing data for group II are attributable to oysters lost (see Materials and Methods). 112 Bataller et al. Figure 6. Condition indices for oysters sampled from stations HSS thigh-salinity surface), HSB {high-salinity bottom). LSS (low-salinity surface), and LSB (low-salinity bottom) for groups I (2-year-old oysters in 1993), II (4-year-old oysters in 1993), III (2-year-old oysters in 1994). and IV (4-year-old oysters in 1994), before (BS) and after spawning (AS) and before winter (B\V). Sampling dates for each group are shown in Table 1. Bars with similar letters are not significantly different from each other (p > .05). Missing data for group II are attributable to oysters lost (see Materials and Methods). such as growth rate, glycogen concentration, and condition index have been used in the past to gauge the relative growth and health of oysters (Muniz et al. 1986. Brown and Hartwick 1988. Little- wood and Gordon 1988, Austin et al. 1993. Boghen et al. 1993). Our work is consistent with those of others who demonstrated that oysters cultured in suspension display superior growth as com- pared to other growout methods. Although this proved to be the case at each site, when the sites were grouped together, we noted that oysters cultured on the bottom at high-salinity (HSB) dis- played growth comparable to those reared near the surface at low- salinity (LSS). Salinity and temperature, acting either independently or in syn- ergy, are known to have greater effects on growth of the eastern oyster than do other factors (Butler 1949. Wells 1961. Alderdice 1972, Vernberg and Vernberg 1972). This, however, does not pre- clude the fact that such environmental variables as seston and primary production, may, either in combination with or separate from salinity and temperature, have an important effect on the physiology and growth of the eastern oyster ( Bayne and Newell 1983. Brown and Hartwick 1988. Dekshenieks et al. 1993. Shum- way 1996). The significant difference that has been observed between years for the PIM/POM. may partially explain the differences be- tween 1993 and 1994 for certain recorded values, such as growth, condition index, or glycogen content ratio (Galtsoff 1964, Bayne and Newell 1983, Wallace and Reinsnes 1985). Although there were no significant differences in primary pro- duction among stations, this does not preclude the fact that the nutritional value of individual species of phytoplancton occurring at a given site or station at any particular time, may. in the long run. represent a more critical factor in determining the suitability of a particular culture location (Haven 1960. Dunathan et al. 1969. Castell andTrider 1974). Establishment of an halocline. persisting for up to 4 weeks, in the high-salinity water column during May and June for HSS and HSB (Fig. 3 and Table 1 ). is attributable to the effects of succes- sive periods of melting snow and heavy rain (Environment Canada 1993. 1994). Although better growth is associated with higher salinity (Butler 1949. Chanley 1958, Galtsoff 1964) as observed at HSB. water temperature was lower (Fig. 3. points 1 to 5). and may. therefore, have had an opposite effect on oyster growth. The dif- ference in temperature between bottom and surface at HSB and HSS persisted during the early growing period, which may help explain the reason for better growth at HSS over the growing season. Salinity fluctuations (5-1 59^) were evident at LSB at the end of May and beginning of June (Fig. 3). and this may be attributable to heavy precipitation and tidal action. From early August to mid-October, a phase representing opti- mal growing conditions for oysters (Shumway 1996). all four sta- tions (HSS. HSB. LSS. and LSB) displayed comparable tempera- ture and salinity profiles. The 1994 data demonstrated tendencies similar to those reported for 1993, although differences in weather patterns resulted in a slight shift in temperature and salinity varia- tions. In all instances. 2-year-old oysters displayed superior growth to 4-year-olds, similar to findings reported by Carriker ( 1996). Interpretations of findings related to glycogen concentrations and condition index (dry meat weight/dry shell weight), were con- sidered for the three critical periods during the oysters' growing season: before spawning, after spawning, and before winter, as previously mentioned. For this study, animals were sampled at Comparative Growth of Oysters Reared at Low and High Salinity 113 specific dates on a biweekly basis. Therefore, it is possible that the data reported for glycogen and condition index do not necessarily represent the absolute maximum and minimum values. This may help to explain differences in recorded values between 1993 and 1994. The less favorable environmental conditions (Fig. 3) for oysters at stations LSS and LSB for groups I and II after spawning may be responsible for their reduced capacity to rebuild glycogen reserves and their increased vulnerability at a critical time during their growing cycle. In general, however, oysters grown in suspension at HSS and LSS, during the same period, displayed higher glycogen concentrations than those cultivated on the bottoms at HSB and LSB, respectively. This is consistent with our growth data. Results of the condition indices measured for oysters sampled from all stations for groups I to IV generally support the findings recorded for glycogen and growth (Fig. 6). One notable exception was that the condition index proved to be inconsistent with the glycogen data for oysters cultivated in the low-salinity environ- ment. This index may prove to be an effective tool that could correlate positively with reported glycogen concentrations, de- pending upon environmental conditions (Ingle 1949. Walne 1970, Gabbott and Stephenson 1974). The absence of a detectable rela- tionship between the condition index and glycogen concentration or even growth in certain instances may be attributable to several factors, ranging from low calcium levels in less saline waters to poor substrates and/or inferior quality and availability of food. These factors may, in tum, contribute to improper shell formation and may alter shell form and thickness, thus biasing the relevancy of condition index as based on conventional methods (Riley and Chester 1971, Wilbur and Saleuddin 1983). Moreover, a pro- nounced asynchronism in the growth rate of shell versus soft tissue may likewise influence the accuracy of interpretation of values for condition index, as has previously been demonstrated for mussels (Hilbish 1986, Rainer and Mann 1992). Various authors (Lucas and Beninger 1985, Brown and Hart wick 1988, Rainer and Mann 1992) demonstrated that static indices based on the ratio of dry meat weight/shell cavity volume or dry meat weight/shell weight are less efficient than such dy- namic indices as biochemical indicators. The value of such bio- chemical indicators as glycogen concentration, carbohydratemitro- gen, and carbon:nitrogen have been discussed by Mann (1978). Our results demonstrate that oysters grown in suspension under high-salinity conditions display growth and development superior to those cultured in a low-salinity environment. Despite the supe- riority of oyster growth recorded at station HSS. the effectiveness of oyster culture in less saline waters as determined from our study should not be overlooked. Depending upon the specific environ- mental and hydrographic conditions characterizing a given site, bottom culture may be comparable to surface culture, if not more appropriate. This possibility is supported by Newell et al. (1998). who demonstrated that the contribution of detritus as a source of nutrient for mollusks may be more important then the phytoplank- ton available in the upper portions of the water column. Such an outcome may explain the reason for superior growth of bottom- cultured oysters versus oysters grown in suspension. A comparable situation may explain the similarity in growth of oysters from stations LSS and HSB. The implications of our findings lend credence to the potential advantages of identifying new aquaculture sites, which may have been rejected for commercial oyster culture up to now. The im- portance of such studies is reinforced by the decreasing availability of traditional culture sites in Atlantic Canada because of excessive coastal development and enhanced organic pollution. Finally, the appropriateness of certain sites may be more ap- plicable for the culture of one age group versus another, as re- ported by Ortega and Sutherland 1992. Alternatively, less tradi- tional sites may also prove to be useful as secondary or provisional storage areas for established operations, particularly at certain times of the year. ACKNOWLEDGMENTS We are grateful to Drs. N. Bourne. R. Lavoie, and G. Miron for reviewing this paper and for their helpful suggestions. Cooperation provided by Aquaculture Acadienne Inc. and the Big Cove Band Council is much appreciated. We also thank C. Mallet for his assistance with some of the technical drawings. This work was partially financed by the Faculties of Graduate Studies of the Uni- versite de Moncton (Moncton, N.B.) and University of New Brunswick (Fredericton, N.B.) through grants awarded to the se- nior author. Financial assistance was also provided by the New Brunswick Department of Fisheries and Aquaculture. This study is part of the Richibucto Environment and Resource Enhancement Program. LITERATURE CITED Alderdice. D. F. 1972. Factor combinations, responses of marine poikilo- therms to environmental factors acting in concert, pp. 1659-1772. In: O. Kinne (ed.). Marine Ecology. Wiley-Interscience, London. Austin. H. D. S. Haven & M. S. Moustafa. 1993. The relationship between trends in a condition index of the American oyster, Crassostrea vir- ginica, and environmental parameters in three Virginia estuaries. Es- tuaries 16:362-374. Bayne. B. L. & R. C. Newell. 1983. Physiological energetics of marine mollusks. pp. 407-515. In: A. S. M. Saleuddin and K. M. Wilbur (eds.). The Mollusca. vol. 4. Physiology, Part 1. Academic Press, Lon- don. Boghen, A. D.. J. Allard & E. Bataller. 1993. Rapport final et recomman- dations sur le programme de monitoring pour la cote est du Nouveau- Brunswick. Centre de Recherche en Sciences de 1' Environment. Uni- versite de Moncton. Moncton. N.-B. mars 1993. Brown. J. R. & E. B. Hartwick. 1988. Influences of temperature, salinity, and available food upon suspended culture of the Pacific oyster, Cras- sostrea gigas. II. condition index and survival. Aquaculture 70:253- 267. Butler. P. A. 1949. Gametogenesis in the oyster under conditions of de- pressed salinity. Biol. Bull. 96:263-269. Carr. R. S. & J. M. Neff. 1984. Quantitative semi-automated enzymatic assay for tissue glycogen. Comp. Biochem. Physiol. 77B:447^449. Carriker. M. R. 1996. The shell and ligament, pp. 75-168. In: V. S. Kennedy, R. I. E. Newell and A. F. Eble (eds.). The Eastern Oyster Crassostrea virginica. Maryland Sea Grant College, College Park. MD. Castell, J. C. & D. J. Trider. 1974. Preliminary feeding trials using artificial diets to study the nutritional requirements of oysters Crassostrea vir- ginica. J. Fish. Res. Bet. Can. 31:95-99. Chanley. P. E. 1958. Survival of some juvenile bivalves in water of low salinity. Proc. Natl. Shellfish Assoc. 48:52-65. Dekshenieks. M. M.. E. E. Hofmann & E. N. Powell. 1993. Environmental effects on the growth and development of eastern oyster. Crassostrea virginica (Gmelin, 1791), larvae: a modeling study. J. Shellfish Res. 12:241-254. 114 Bataller et al. Dunathun. J. P., R. M. Ingle & W. K. Havens. Jr. 1969. Effects of artificial foods upon oyster fattening. Florida Department of Natural Resources. Marine Research Laboratory Report 58:1-39. Environment Canada.