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JOURNAL OF SHELLFISH RESEARc 



VOLUME 19, NUMBER 1 



JUNE ^000 




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, VA 23062-1 1346 

Dr. Peter Beninger (2001) 

Laboratoire de Biologic Marine 

Faculte des Sciences 

Universite de Nantes 

BP 92208 

44322 Nantes Cedex 3 

France 

Dr. Andrew Boghen (2001) 
Department of Biology 
University of Moncton 
Moncton, New Brunswick 
Canada El A 3E9 

Dr. Neil Bourne (2001) 
Fisheries and Oceans 
Pacific Biological Station 
Nanaimo, British Columbia 
Canada V9R 5K6 

Dr. Andrew Brand (2001) 
University of Liverpool 
Marine Biological Station 
Port Erin, Isle of Man 

Dr. Eugene Burreson (2001) 
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 Crcswell (2001) 
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 (2001) 
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 (2001) 
Randall Environmental Studies Center 
Taylor University 
Upland, Indiana 46989 



Dr. Mark Luckenbach (2001) 
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 Slate Uni\ersity 
Thibodaux, Louisiana 70310 

Dr. J. Evan Ward (2001) 
Dcpt. of Marine Sciences 
University of Connecticut 
Grolon, CT 06340-6097 

Dr. Gary Wikfors (2()()()) 

NOAA/NMFS 

Rogers Avenue 

Milford, Connecticut 06460 



Journal of Shellfish Research 

Volume 19, Number 1 

ISSN: 00775711 

June 2000 



Journal of ShcUfish Research. Vol. 19. No. 1. 1-?. 2000. 




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IN MEMORIUM 

JOHN CARL MEDCOF 

1911-1997 



J. Carl Medcof, a well-known and highly respected authority in the field of molluscan biology, oyster biology, and shellfish 
management in Atlantic Canada, died on 28 February 1997, in St. Andrews. New Brunswick. He was 86 years old. 

Carl was bom into a family with a strong academic background in Ruthven. Ontario in 1911. and the family moved soon after his 
birth to Toronto. His father, stepmother (his mother died when he was quite young), and uncles were all teachers. He received his 
elementary and secondary education mostly in Toronto and enrolled in the University of Toronto, where he received his B.A. in biology 
in 1932. He received an M.A. degree from the University of Western Ontario in 1934, undertaking a study of a snail, Campeloma, in 
a small river in southern Ontario as his thesis topic. Thus, began his lifelong interest in the field of molluscan biology. He received his 
Ph.D. degree from the University of Illinois in 1938. studying under Dr. H. J. Van Cleave, who was very influential in his early career. 
While at the University of Illinois, he was elected to the Phi Beta Kappa Society. 

During his senior year at the University of Toronto and throughout his graduate years, Carl served as a teaching assistant. He 
maintained a keen interest in young people and always welcomed opportunities to talk with students and young scientists and encourage 
them in their work. He was a lifelong teacher, and many summer students benefited from his store of knowledge. 

While an undergraduate, Carl began work as a summer student with the Biological Board of Canada, later the Fisheries Research 
Board of Canada. His first job was with investigations of Atlantic salmon. Subsequently, he worked as a summer student at Ellerslie, 
Prince Edward Island, where he came under the influence of Drs. Alfreda B. Needier for "red tides" and Alfred W. H. Needier, the 
director of the Station. Ellerslie was established to study oyster culture and foster development of the industry in the Canadian Maritimes. 
Thus, began Carl's enduring association with oyster research and the oyster industry of the Canadian East Coast. The title of his Ph.D. 
dissertation was "Studies on the larva of the Canadian oyster" and was focused on experiments with Ostrea (Cnis.wslreal virginica in 
the Bideford River. Prince Edward Island. Canada. 

On completion of his Ph.D.. Carl joined the staff of the Fisheries Research Board of Canada and was employed first at the Ellerslie 
Station, where he assumed responsibility for oyster research. He moved to the Biological Station in St. Andrews. New Brunswick in 
1940, where he continued his work with oysters and also assumed responsibility for research work on all molluscan species including 
soft-shell clams, Mya arenaria, quahaugs, Mercenaria mercenaria, and sea scallops Placopecten magellaniciis with his technician, Mrs. 
Esther Lord. Up until 1944. he moved with his family in the summers to Ellerslie and to St. Andrews in the winters. 

Carl undertook a wide range of studies on oysters, including investigations on breeding to ensure a supply of juveniles for the 
industry, work to improve culture methods, and studies to improve harvesting and marketing. He maintained a close working relationship 
with the industry during his career, and much of his work focused on developing methods to improve it. He enjoyed working with people 
in industry and had their complete trust. Much of his work with oysters culminated with the publication. Oyster Farming in The 
Maritimes. which became a standard text for oyster culture in eastern Canada. This expertise took him to Cape Breton. Nova Scotia, 
where he worked with natives from the Eskasone Indian Band to grow oysters. He was also involved in experiments in the mid-1950s 
to transplant European oysters {Ostrea edulis) from France to the Bay of Fundy. 

Carl worked extensively on the soft-shell clam with Mr. Stuart MacPhail from the Fisheries Research Board as well as with colleagues 



Bourne and Robinson 

from New England such as Mr. Dana Wallace. His clam work took him to eastern Nova Scotia, the Bay of Fundy. and the Gulf of St. 
Lawrence, where he worked with local harvesters to survey their areas and methods to increase production. He and Stuart MacPhail 
introduced the first water jet harvesters for soft-shell clams to the local industry and built prototypes for hand-held models as well as 
a vessel-equipped escalator harvester. Other clam species were studied as well. He and Ross Chandler from the St. Andrews Biological 
Station did the early biological work for the ocean quahaug clam fishery in southeast Nova Scotia. 

Another major scientific contribution Carl made was in the field of shellfish toxins, particularly paralytic shellfish poisoning (PSP). 
As a result of his work and leadership, much of the history of PSP in the Canadian Maritimes was recorded and the etiology of outbreaks 
established. Results of this work led to establishment of a monitoring system for PSP to ensure that safe shellfish were marketed. Carl 
was the guest of honor at the Third International Conference on Toxic Dinoflagellates in 1985 and was awarded with a plaque in 
recognition of his contribution in this field. Later in his life, Carl said, "The most important work I did as a scientist was on resolving 
many paralytic shellfish problems. In addition I was able to work on methods of producing reliable forecasts about the expectancy of 
one of the major poisonous agents." 

Carl also had a vision for the future. As early as the 1950s he was promoting the concept of aquaculture in marine production and 
was actively working on developing the protocols for producing soft-shell clams. He gave several media (including television) interviews 
and lectures on this topic. In addition to clams, he also predicted the development of the mussel culture industry in the Maritimes and 
an industry for sea urchin roe; something that is only just developing 25 years later. 

Because of his expertise in shellfish, Carl was seconded to the Colombo Plan for 2 years in 1953 and 1955 and worked in Sri Lanka, 
assisting with development of invertebrate and other fisheries there. In 1955. he went to Europe to tour the various shellfish industries 
for information exchange with colleagues and for technology transfer. In the late 1960s, he undertook a similar trip to Japan with a group 
of Canadian scientists. 

Carl retired from the Fisheries Research Board of Canada in 1973 and then spent a year in Australia, where he worked as a consultant 
to the shellfish industry through the University of Southern Australia in New South Wales. One project he undertook there was an 
investigation of the introduction of organisms via ballast water in ships, a subject that has become of great interest recently interna- 
tionally. 

Carl received numerous awards, both scientific and nonscientific, for the contributions he made during his lifetime. He was a 
long-standing member of the National Shellfisheries Association and served on the Editorial Board for the Proceedings and the Journal 
for many years. He was made an Honorary Member of the Association in 1973. He received a Centennial Medal from the Canadian 
Federal government in 1967 for his contributions to the Fisheries Research Board of Canada. 

During his retirement, he taught a course in Marine Ecology at the Huntsman Marine Science Centre in St. Andrews. The course 
involved a rich mixture of basic biology, natural ecology, and the geological and paleological history of the Charlotte County area of 
New Brunswick. 

Cari had a multitude of other interests and on his retirement began another career, recording the history of Charlotte County, New 
Brunswick, an offshoot of his previous hobby. He had long had an interest in the history of the area and in 1961 was a co-founder of 
the Charlotte County Historical Society, serving as its first president. He encouraged people to record what information they possessed 
of the area, and it was through his efforts as editor over a period of 21 years that an 12-volume collection of papers was published as 
Contributions From Tlic Charlotte County Historical Society. As a result of his work with the Historical Society. Carl was presented with 
an Award of Merit from the Canadian Museums Association in 1981. 

He was a devoted citizen of St. Andrews and took an active part in the affairs of the town, contributing to it in many ways over the 
years. During his lifetime, he was a Scoutmaster, Chairman of the Board of School of Trustees, a member of the local Kiwanis Club, 
and served as president. He was a member of the St. Andrews Anglican Church and published a history of that church. He served as 
vestryman, was an honorary church warden, and with his joy of singing, was a member of the church choir for many years. In 1987, 
the local Kiwanis Club selected him as their Man of the Year in recognition of his numerous contributions to the town. 

Carl was a kind, gentle, humanitarian who brought out the best in people. He enjoyed working quietly, smoking his pipe (although 
more matches were burned than tobacco!), and he always had a package of dulse in his pocket, which he chewed and offered to anyone 
he met; he was one of the few people in the world who actually enjoyed chewing dulse! Those of us who were privileged to work under 
his direction will always remember the kind, thoughtful guidance and encouragement he gave us in our careers and his kind advice not 
only to be good scientists but to be good citizens. It was a joy to work and go on field trips with him, where we could enjoy his 
companionship and take part in long philosophical discussions on a wide range of biological and other topics. 

Cari is survived by his wife of 55 years, Bessie, their three children. Susan, John, and Ranby. by three grandchildren, and by a great 
many people whose lives have been made more meaningful through their association with him. 



ACKNOWLEDGMENTS 



NEIL F. BOURNE' AND SHAWN M.C. ROBINSON' 



We thank Mrs. Lsiher Lord and Mr. Ross Chandler lor reading 
an eariier draft of the manuscript and sharing their insights on 
Cari's life. We also thank Mrs. Bessie Medcof for helping us with 
the chronology of the events and her perspective on her husband. 
Marilynn Rudi of the St. Andrews Biological Station librarv kindly 
provided some of the historical information. 



'Pacific Hiolofiical Station 

Nanaimo, British Columbia 

V9R 5K6. Canada 

'Biological Station 

St. Andrews. New Brunswick 

E5B 2L9, Canada 



John Carl Medcof 3 

publications 

Medcof. J. C. 1935. Margaree salmon investigations 1935. Scotsville: notes on the various marine species of animals. Manuscript Reports of the 

Biological Stations 1 17:89 pp. 
Smallman. B. N.. J. C. Medcof. 1935. Margaree salmon investigation. 1935. Manuscript Reports of the Biological Stations No. 116. 
Medcof J C. 1936. Report of oyster studies at the P.E.I. Biological Station, June to August. Manuscript Reports of the Biological Stations 143:23 pp. 
Medcof J. C. 1937. Report of oyster studies at the P.E.I. Biological Station June to September. 1937. Manuscript Reports of the Biological Stations 160:63 pp. 
Medcof. J. C. 1938. Excerpts from "Studies on the larvae of the Canadian oyster." Manuscript Reports of the Biological Stations 292:27 pp. 
Medcof, J. C. 1938. Oyster investigations in Bras d'Or Lakes. 1938. Manuscript Reports of the Biological Stations 159:64 pp. 
Medcof. J. C. 1938. Studies on the Larva of the Canadian oyster. Ph.D. Dissertation, University of Illinois. Champaign-Urbana. IL. 74 pp. 
Medcof. J. C. 1939. Additional records of the terrestrial amphipod, Talirrus allaudi Chevreux, in North America. Am. Midland Naturalist 22:216-217. 
Medcof J. C. 1939. Larval life of the oyster iOstrea virginica) in Bideford River. / Fisheries Res. Board Can. 4:287-301. 
Medcof J. C. 1939. Oyster investigations in the Bras d'Or lake and studies on the condition factor of oy.sters. Manuscript Reports of the Biological 

Stations 162:92 pp. 
Medcof. J. C. 1940. On the life cycle and other aspects of the snail, Campeloma. in the Speed River. Can. J. Res. 18:165-172. 
Medcof. J. C. 1940. Oyster investigations in 1940. Fisheries Research Board of Canada. Manuscript Report 184. Biological Station. St. Andrews. NB, 1940. 
Medcof. J. C. 1940. Variations in the pleopod structure of the tertestrial amphipod Talitnis allaudi Chevreux. Lloydia 3:79-80. 
Medcof J. C. 1 94 1 . Examination of oyster areas in Richibucto. Little Shemogue, and Big Shemogue Rivers, 1 95 1 . Fisheries Research Board of Canada, 

Original manuscript of the Biological Station. St. Andrews. No.686. 
Medcof J. C. 1941. Oyster investigations in 1941. Manuscript Reports of the Biological Stations 239:50 pp. 
Medcof J. C. & A. W. H. Needier. 1941. The influence of temperature and salinity on the condition of oyster (Oilrea virginica). J. Fisheries Res. Board 

Can. 5:253-257. 
Gibbard. J. A. G. Campbell, A. W. H. Needier & J. C. Medcof 1942. Effect of hibernation on content of coliform bacteria in oysters. Am. J. Puhl. Health 

32:979-986. 
Medcof, J, C. 1942. Report on 1942 investigations: principally oysters, with small note on Irish moss. Manuscript Reports of the Biological Stations 

331:102 pp. 
Medcof. J. C. 1943. La besoin d'une production d'huitres d'ensemencement("seed oysters") a Shippegan. Fisheries Research Board of Canada. Oyster 

Farming Circular 17:1 p. 
Medcof. J. C. 1943. Memorandum of oyster problems deserving biological investigations. Fisheries Research Board of Canada, original manuscript 749, 

Atlantic Biological Station, December 1943. 
Medcof J. C. 1943. Need for production of seed oyster at Shippegan. Fisheries Research Board of Canada, Oyster Farming Circular No. 17, May 1943. 
Medcof J. C. 1944. Structure, deposition, and quality of oyster shell iO.strea virginica Gmelin). / Fisheries Res. Board Can. 6:209-216. 
Medcof J. C. & E. I. Morrison. 1943. Report on 1943 shellfish investigations. Manuscript Reports of the Biological Stations 370:65 pp. 
Medcof J. C. 1944. 1944 investigations: oysters and clams. Manuscript Reports of the Biological Stations 378:90 pp. 

Medcof J. C. 1944. How relaying and transferring at different seasons affects the famess of oysters. Fisheries Res. Board Prog. Repts. 35:1 1-14. 
Medcof J. C. 1944. Report of information on shellfish gathered during the Massachusetts trip April 10-17, 1944. Original manuscript 780. St. Andrews 

Biological Station. New Brunswick. Canada. 
Medcof. J. C. 1945. Green oysters from New Brunswick. Acadian Naturalist 2:40^3. 

Medcof J. C. 1945. The mud-blister worm. Polydora. in Canadian oysters. J. Fisheries Res. Board Can. 7:498-505. 
Medcof J. C. & R. J. Gibbons. 1945. Paralytic shellfish poisoning in Nova Scotia and New Brunswick. Manuscript Reports of the Biological Stations 

376:39 pp. 
Medcof J. C. 1946. Effect of relaying and transferring on fatness of oysters. / Fisheries Res. Board Can. 6:449—155. 
Medcof J. C. 1946. More reversed winter flounder. Science 103:488. 

Stinson, R. H. & J. C. Medcof 1946. Observations on the natural history of clam drills {Polinices). Manuscript Reports of the Biological Stations 383:63 pp 
MacPhail. J. S. & J. C. Medcof 1947. 1947 Clam investigations. Fisheries Research Board of Canada, Atlantic Biological Station, original manu- 
script 855. 
MacPhail. J. S. & J. C. Medcof 1947. Report on information of the bar-clam {Mactra) gathered during a trip to New York state in August. 1946. Fisheries 

Research Board of Canada, original manuscript 698. December. 1947. 
Medcof. J. C. 1947. Clam farming in the Maritimes — preliminary information. Fisheries Research Board of Canada. General Series, 

No.9. Circular. Atlantic Biological Station. St. Andrews. N.B.. May. 1947. 
Medcof, J. C. A. H. Leim. A. B. Needier. A. W. H. Needier. J. Gibbard & J. Naubert. 1947. Paralytic shellfish poisoning on the Canadian Atlantic Coast. 

Fisheries Research Board of Canada. Bulletin No.75. Ottawa, 1947. 
Medcof J. C. & F. S. Schiffman. 1947. Recent records of the sea sunfish {Mola mola L.) in the Gulf of St. Lawrence. Acadian Naturalist 2:63-66. 
Medcof J. C. 1948. A snail commensal with the soft-shell clam. J. Fisheries Res. Board Can. 7:219-220. 

Medcof J. C. 1949. Dark-meat and the shell disease of scallops. Fisheries Research Board of Canada, Atlantic Biological Station Progress Report 45. 
Medcof. J. C. 1949. Meat yield from Digby scallops of different sizes. Fisheries Res. Board Prog. Repts. 44:6-9. 
Medcof, J, C, 1949. "Puddling" — a method of feeding by herring gulls. The Auk 66:204-205. 
Thurber. L. W. & J. C. Medcof 1949. Meat yield of clams (-) and percentage total dry solids of clam meats. Manuscript Reports of the Biological Stations 

399:.30 pp. 
Medcof J. C. 1950. Burtowing habits and movements of soft-shelled clams. Fisheries Res. Board Prog. Repts. 50:17-22. 
Medcof, J. C. & J. S. MacPhail. 1951. 1945 Investigations — clams and oysters. Manuscript Reports of the Biological Stations 414:92 pp. 
Medcof. J. C. & J. S. MacPhail. 1952. The winter flounder — a clam enemy. Fisheries Res. Board Prog. Repts. 52:3-8. 
Mullan, M. W., A. B. Williams, A, D, Tennant, 1. E. Erdman. V. C. Dohaney & J. C. Medcof 1953. 1952 clam cleansing studies (Wvo) — combined 

reports. Manuscript Reports of the Biological Stations 503:65 pp. 
Durairatnam. M. & J. C. Medcof 1954. Ceylon's red seaweed resources. Ceylon Trade J. 19:1-6. 
Medcof. J. C. 19.54. How to improve Ceylon's small-boat fisheries. Paper delivered at 1954 meeting of Ceylon Association for Advancement of Science. 



4 Bourne and Robinson 

Medcof, J. C. 1955. Day and night characteristics of spatfall and of behaviour of oyster larvae. J. Fisheries Res. Board Can. 12:270-286. 

Medcof. J. C. & L. M. Dickie. 1955. Watch for the green crab — new clam enemy. Fisheries Research Board of Canada. General Series Circular 26. 

Atlantic Biological Station. St. Andrews, N.B., July. 1955. 
Medcof. J. C. & J. S. MacPhail. 1955. Survey of bar clam resources of the Maritime Provinces. Fisheries Research Board of Canada, Bulletin 102, Atlantic 

Biological Station, St. Andrews, N.B. 
Canagaratnam, P. & J. C. Medcof. 1956. Ceylon's beach seine fishery. Fisheries Research Station. Dept. of Fisheries, Ceylon. Bulletin 4. 
Dickie, L. M. & J. C. Medcof. 1956. Environment and the scallop fishery. Can. Fisherm. 7:9, 

Bond. R. M. & J. C. Medcof. 1957. Epidemic shellfish poisoning in New Brunswick, 1957. Dept. of Fisheries, Fish Inspection Laboratory, St. Andrews, N,B. 
Medcof, J, C. 1957. Nuptial or prenuptial behaviour of the shad. Alusa sapidissinui (Wilson). Copeia 1957:252-253. 
Bond, R. M. & J. C. Medcof. 1958. Epidemic shellfish poisoning in New Brunswick, 1957. Can. Med. Assoc. J. 79:19-24. 
Medcof, J. C. 1958. Mechanized gear for shellfish harvesting and shellfish culture. Manuscript Reports of the Biological Stations 644:16 pp. 
Medcof, J. C. 1958. Stock-taking of molluscan shellfish resources and prospects for improvement. Reprinted from Progress Reports of the Atlantic Coast 

Stations of the Fisheries Research Board of Canada. Issue 71, pp. 21-26, December, 1958. 
Medcof, J. C. 1958. Studies on stored oysters {Crassosuea virginica). Proc. Nail. Shellfisli. Assoc. 47:13-28. 
Medcof, J. C. 1958. Useful publications for oyster farmers of the Maritimes. Fisheries Research Board of Canada. General Series Circular 32. Biological 

Station. St. Andrews. N.B.. October.l95S. 
Medcof. J. C. & J. E. Mortimer. 1958. Introducing European oysters to the Maritimes. Reprinted from Progress Reports of the Atlantic Coast Stations 

of the Fisheries Research Board of Canada. Issue 71. pp. 27-29. December. 1958. 
Medcof. J. C. & L. W. Thurber. 1958. Trial control of the greater clam drill (Lunatia heros) by manual collection. J. Fisheries Res. Board Can. 

15:1355-1369. 
Bell, M. C. V. M. Brawn. C. H. Clay. C. J. Kerswell. J. E. H. Legare.D. C. Maclellan. W. C. Martin, F. D. McCracken, R. A. McKenzie, 

J. C. Medcof, L. W. Scattergood, R. F. Temple, S. N. Tibbo & D. G. Wilder. 1959. Report to International Joint Comission, Ottawa, Ontario. 

Washington DC. Appendi.x II. Biology — Canada: studies in fisheries biology for the Passamaquoddy Power Project. 
MacPhail. J. S. & J. C. Medcof. 1959. Ocean quahog explorations. Trade News -6. 
Medcof, J. C. 1959. Report on visits to British Columbia and Washington state shellfish industrial and research centres. Available from: Library, 

Biological Station, St. Andrews, N.B., E5B-2L9. 
Medcof, J. C. I960. Shellfish poisoning — another North American ghost. Can. Med. Assoc. J. 82:87-90. 
Drinnan, R. E. & J. R. Medcof. 1961, Progres du Reetablissement des Stocks d'Huitres Decimes par la Maladie. Office des Recherches sur les Pecheries 

du Canada, Circulaire No 34 de la Serie Generale, Octobre 1961 (version Fran^aise), Station Biologique. St. Andrews.N.B. 
Drinnan. R. E. & J. C. Medcof. 1961. Progress in rehabilitating di.sease-affected oyster stocks. Fisheries Research Board of Canada. General Series 

Circular 34. Biological Station, St. Andrews, N.B., October, 1961. 
Medcof, J. C. 1961. Effect of hydraulic escalator harvester on undersize soft-shell clams. Proc. Natl. Sliellfish. Assoc. 50:151-161. 
Medcof, J. C. 1961. Fuller exploitation of natural spatfall. Fisheries Research Board of Canada, original manuscript of the Biological Station. St. Andrews. 

No.932. April 10. 1961. 
Medcof. J. C. 1961. Oyster farming in the Maritimes. Fisheries Research Board of Canada. Bulletin 131. Biological Station. St. Andrews, N.B., 1961. 
Medcof, J. C. 1961. Present shellfish fishery needs for oceanographic and biological research on Canadian Atlantic. Conference of A.O.G. and St. 

Andrews Staff at St. Andrews, October 19, 1961. 
Medcof, J. C. 1961. Trial introduction of European oysters (Ostrea ediilis) to Canadian East Coast. Proc. Natl. Shellfish. As.soc. 50:1 13-124. 
Medcof, J. C. 1961. Loss of the barque James W. Elwell and tragic experiences of a St. Andrews sailing captain 1872. Charlotte County Archives. St. 

Andrews. New Brunswick. BOG 2X0. 18 pp. 
MacPhail. J. S. & J. C. Medcof. 1962. Fishing Efficiency Trials with a hydraulic clam [Mya) Rake — 1961. Fisheries Research Board of Canada. 

Manuscript Report Series 724. Biological Station. St. Andrews. N.B.. July. 1962. 
Medcof. J. C. 1962. 1961 Tests of spat collection in the Intertidal /one. Fisheries Research Board of Canada, original manuscript of the Biological Station. 

St. Andrews. No.943. June, 1962. 
Medcof, J. C. 1962. Collecting spat and producing bedding oy.sters on shell strings. Fisheries Research Board of Canada. General Series Circular 36. 

Biological Station, St. Andrews, N.B., July, 1962. 
Medcof, J. C. 1962. Hydraulic escalator oyster harvesters. Address by J.C. Medcof at the 1962 July-August Convention of the Oyster Institute and the 

National Shellfisheries Association at Baltimore, Maryland. 
Medcof, J. C. & J. S. MacPhail. 1962. Fishing efficiency of clam hacks and morlalities incidental to fishing. Fisheries Research Board of Canada. 

Manuscript Report Series 784. 
Medcof. J. C. 1962. Land reconnaissance of Darnley-New London. P.E.I.. region to judge its oyster spat rearing potenlial. Fisheries Research Board of 

Canada, original manuscript of (he Biological Station. St, Andrews. N.B.. No.944. June 1962, 
Medcof. J. C. 1962. Possible elfecls of Passamaquoddy power project on clams, scallops, and shipvvorms in Canadian w;ilers. J. Fisheries Res. Board 

Can. 19:877-889. 
Prakash. A. & J. C. Medcof. 1962. Hydrographic and meteorological factors affecting shellfish toxicity at Head Harbour. New Brunswick. J. Fisheries 

Res. Board Can. 1 9: 1 1 - 1 1 2. 
Dickie, L. M, & J. C. Medcof. 1963. Causes of mass mortalities of scallops [Placopeclcn ma.t;ellaniciis) in the Southwestern Gulf of St. Lawrence. J. 

Fisheries Res. Board Can. 20:451-^82. 
MacPhail, J. S, & J. C. Medcof. 1963. A new digger for sofl-shcll clams, Reprlmcd liom March. I9(i3, issue of "Trade News" of the Dcpl. of Fisheries 

of Canada. 
Medcof, J C. 1963. Molluscan shellfish. Shellfish course, given by Dr. J. C. Medcof. 1-ebruary 12. 1963, Si. .Andrews, N.B. .'Available from: Library, 

Biological Station. St. Andrews. N.B., H5B-2L9. 
Medcof, J, C. 1963. Partial survey and critique of Ceylon's marine fisheries, 195.3-55. Bulletin of the Fisheries Research Station. Ceylon 16:29-1 18. 
Medcof, J. C. 1963. Puzzling clay tubes from the sea bottom. Can. Field-Namrulist 77:179-242. 
Medcof, J. C. 1963. Shell strings for collecting spat and rearing bedding oysters — 1962 tests. Fisheries Research Hoard of Canada, original manuscripi 

of the Biological Station, St. Andrews, No.962, April, 1963. 
Medcof, J. C. 1964. Subareas 4 and 5. Reproduced by permission Ironi ICNAI Kcdhook. I4h4. Pan II. pp. 21-34. 



John Carl Medcof 5 

Medcof. J. C. & N. Bourne. 1964. Causes of mortality of the sea scallop, Placopecten magellanicus. Proc. Natl. Shellfish. Assoc. 53:33-50. 
Bearisto. F. 1.. J. C. Medcof & E. I. Lord. 1965. Clam drill (Polinices) investigations at St. Andrews. 1948. Manuscript Reports of the Biological Stations 

845:11 pp. 
Medcof. J. C. 1965. .\ recording caliper for measuring oysters and clams. Fisheries Research Board of Canada, original manuscript of the Biological 

Station. St. Andrews. No. 1001. 
Medcof J. C. 1965. Study of Lower Oyster Pond at Pleasant Point, Halifax County, N.S. Fisheries Research Board of Canada. Manuscript Report Series 

829, Biological Station. St. Andrews. N.B., August 27, 1965. 
Medcof, J. C. A. H. Clarke & J. Erskine. 1965. Ancient Canadian East Coast oyster and quahaug shells. J. Fisheries Res. Board Can. 22:631-634. 
Medcof, J. C. & C. J. Kerswill. 1965. Effects of light on growth of oysters, mussels, and quahogs. J. Fisheries Res. Board Can. 22:281-288. 
White. H. C, J. C. Medcof & L. R. Day. 1965. Are killifish poisonous? J. Fisheries Res. Board Can. 22:635-638. 
Medcof, J. C. 1966. Incidental records on behaviour of eels in Lake Ainslie, Nova Scotia. J. Fisheries Res. Board Can. 23:1 101-1 105. 
Medcof J. C. 1966. The rough whelk fishery at Godbout, P.Q. Fisheries Research Board of Canada, original manuscript of the Biological Station, St. 

Andrews. No.l038. July 19. 1966. 
Medcof, J. C, N. Morin, A. Nadeau & A. Lachance. 1966. Survey of incidence and risks of paralytic shellfish poisoning in the province of Quebec. 

Fisheries Research Board of Canada. Manuscript Report Series 886, Biological Station. St. Andrews. N.B.. December. 1966. 
Clarke. A. H.. D. J. Stanley. J. C. Medcof. & R. E. Drinnan. 1967. Ancient oyster and bay scallop shells from Sable Island. Nature 215:1146-1148. 
Medcof, J. C. 1967. Third survey of Eel River Cove, N.B., soft-shell clam (Mvn arenaria) population. Manuscript Report Fisheries Research Board of 

Canada 941:57 pp. 
Medcof, J. C. 1968. L'ostreiculture dans les provinces Maritimes. Bulletin Office des Recherches sur les Pecheries du Canada 131:178 pp. 
Medcof J. C. 1968. Medcof s visits to European molluscan shellfish industrial and research centres 1955. Fisheries Research Board of Canada, 

Manuscript Report Series 988, Biological Station. St. Andrews, N.B.. June. 1968. 
Medcof J. C. & R. A. Chandler. 1968. Exploring for uses of ocean quahogs obstacles and opportunities. Fisheries Research Board of Canada. Technical 

Report 101. 1968. 
Medcof, J. C. & E. I. Lord. 1968. Strange catch — a walrus tusk. Notes from the Fisheries Research Board of Canada 21:19-20. 
White, H. C. & J. C. Medcof 1968. Atlantic salmon scales as records of spawning history. J. Fisheries Res. Board Can. 25:2439-2441. 
Chandler. R. A. & J. C. Medcof 1969. A "catch" 20 million years old. Notes from the Fisheries Research Board of Canada 21:15-16. 
Medcof J. C. 1969. Fishermen's reports of freshwater and saltwater migrations of Nova Scotia eels (Anguilla rostratu). Can. Field-Naturalist 83:132-138. 
Medcof J. C. D. F. Alexander & R. A. Chandler. 1969. Promising places to look for ocean Quahogs and bar clams and trial fishing with a rocker dredge 

off Richibucto. N.B.. and Clark's Harbour, N.S. Fisheries Research Board of Canada. Manuscript Report Series 1068. Biological Station, St. Andrews. 

N.B.. December. 1969. 
Medcof J. C. & M. L. H. Thomas. 1969. Canadian Atlantic oyster drills (Urosalpin.x) — distribution and industrial importance. J. Fisheries Res. Board 

Can. 26:1121-1131. 
MacPhail. J. S. & J. C. Medcof 1970. Observations on marine bait worm fisheries in the state of Maine. U.S.A.. June 1950. Fisheries Research Board 

of Canada, original manu.scnpt of the Biological Station. St. Andrews, No. 1095. December 1970. 
Medcof J. C. 1971. Winter variability in paralytic shellfish poison scores for Crow Harbour, New Brunswick. Fisheries Research Board of Canada, 

Manuscript Report Series 1163, Biological Station, St. Andrews, N.B., December, 1971. 
Medcof J. C. & J. F. Caddy. 1971. Underwater observations on performance of clam dredges of three types. International Council for the Exploration 

of the Sea. Gear and Behaviour Committee. 1971. 
Prakash, A., J. C. Medcof & A. D. Tennant. 1971. Paralytic shellfish poisoning in eastern Canada. Manuscript of Fisheries Research Board of Canada 

177:10 pp. 
Lister. D. B., J. C. Medcof & T. W. Rowell. 1972. Maritimes Region Task Force position paper on aquaculture. Canada. Fisheries Service (Maritimes 

Region), 24 pp. 
Medcof J. C. 1972. The St. Lawrence rough whelk fishery and its paralytic shellfish poison problems. Fisheries Research Board of Canada. Manuscript 

Report Series 1201:26 pp. 
Medcof J. C. 1973. Pacific oyster industries in Tasmania and South Australia and potential problems in oyster-pest and disease control. Manuscript 

Report to New South Wales State Fisheries, 12 January 1973. 
Medcof J. C. 1973. Some thoughts on the New South Wales oyster industry and New South Wales State Fisheries Research and Development 

Programmes. New South Wales Fisheries. Sydney Laboratory, October 15, 1973. 
Medcof J. C. & W. B. Malcolm. 1973. Oyster culture in New South Wales. The Fishennan 4:1-2:22-23. 
Medcof J. C. & P. H. Wolf 1973. Possibilities of oyster culture in the Northern Ten-itory. Depl. of the Chief Secretary. New South Wales State Fisheries, 

Sydney Laboratory, Sydney. 
Prakash, A., J. C. Medcof & A. D. Tennant. 1973. L'intoxication paralysante par les mollusques dans I'est du Canada. Bulletin Office des Recherches 

sur les Pecheries du Canada 177:90 pp. 
Medcof J. C. 1974. Some notes on trial fishing, processing and storage of ocean quahogs with appendix on search for ocean quahogs in Port Medway 

Harbour, N.S. Fisheries Research Board of Canada, Manuscript Report Series 1322. Biological Station. St. Andrews, N.B., September, 1974. 
Medcof J. C. & J. F. Caddy. 1974. Underwater observations on performance of clam dredges of three types. Fisheries Research Board of Canada, 

Manuscript Report Series 1313. Biological Station. St. Andrews. N.B.. June. 1974. 
Medcof J. C. & W. B. Malcolm. 1974. Making the best use of a natural resource — oyster culture in New South Wales. The Fisherman 40: 1-2:22-23. 
Medcof J. C. & M. L. H. Thomas. 1974. Surfacing on ice of frozen-in marine bottom materials. J. Fisheries Res. Board Can. 31:1195-1200. 
Medcof J. C. 1975. Living marine animals in a ship's ballast water. Proc. Natl. Shellfish. As.wc. 65:1 1-12. 
Medcof J. C. & P. H. Wolf 1975. Pacific oysters [Crassostrea gigasi in New South Wales. Australia. Available from Library, Biological Station, St, 

Andrews. N.B.. E5B-2L9. 
Medcof J. C. & P. H. Wolf 1975. Spread of Pacific oyster won-ies NSW culturists. Ansl. Fisheries 34:1-7. 

Medcof J. C. 1976. Australian oyster and oyster culture: a partial bibliography. New South Wales State Fisheries, Technical Report 1, January. 1976. 
Medcof J. C. 1979. Iron-manganese concretions from New Brunswick lakes. J. New Brunswick Museum 4:125-131. 
Medcof J. C. 1979. Lake Utopia Concretions "Still a Mystery". The St. Croix Courier. 1 August 1979. 



Joiirmil uf Shellfish Research. Vol. 19, No. 1,7-12. 2000. 




IN MEMORIAM 

RUTH DIXON TURNER 

1914-2000 



Ruth Turner was bom in Melrose, Massachusetts December 7. 1914. She attended Bridge water State College, MA and graduated with 
a B.S. in 1936. She became a school teacher, teaching in both Bondville, Vermont and North Reading. Massachusetts before accepting 
the appointment of Assistant Director of Education at the New England Museum of Natural History (now the Boston Museum of 
Science). Ruth subsequently became Assistant Curator of Birds at the Museum, before moving to Vassar College as an Instructor in the 
Biology Department. During this same period she completed a M.A. at Cornell. By this time Ruth was an accomplished ornithologist, 
an interest that she maintained throughout her life. Indeed, it was her interest in birds that first lead her to the Museum of Comparative 
Zoology at Harvard. While serving as a volunteer in the Ornithology Department she met William J. Clench, Curator of MoUusks. Clench 
introduced Ruth to Dr. William Clapp, a pioneer in the study of marine wood borers, and in 1944 she moved to the William F. Clapp 
Laboratories in Duxbury. Massachusetts. It was here that her career as a malacologist became firmly established. She returned to Harvard 
University two years later to work with Clench. 

Harvard remained her scientific home and a source of great pride to her throughout her career. One of her early field trips with Clench 
was to Cuba in 1949 to examine local terrestrial and marine mollusks. She received her Ph.D. from Radcliffe College. Harvard University 
in 1954. Her dissertation on the Teredinidae remains a standard work to this day. From 1954 through 1975 she served as Research 
Associate in the Department of Mollusks at the Museum of Comparative Zoology, Alexander Agassiz Fellow in Zoology and Ocean- 
ography, and Lecturer in Biology at Harvard. In 1976 she became Professor of Biology, Curator in Malacology at the Museum of 
Comparative Zoology, and joint editor of the scientific journal Johnsonia. Ruth received honorary D.Sc. degrees from New England 
College and Plymouth State College of the University of New Hampshire, and held honorary appointments at the Academy of Natural 
Sciences in Philadelphia, the Woods Hole Oceanographic Institution, the Gray Museum at the Marine Biological Laboratory at Woods 
Hole, Leigh University. CSIRO and the University of New South Wales in Australia, the University of Puerto Rico, and as an FAO 
Fellow in India. Ruth was honored by the Woods Hole Oceanographic Institution as a "Women Pioneer in Oceanography."" On a lighter 
but no less important note. Ruth was named "'Diver of the Year"" by the Boston Sea Rovers, an educational society of which she was 
a very proud member. Ruth served terms as President of both the American Malacological Union and the Boston Malacological Club. 
She was a Honorary Life Member of the National Shellfisheries Association. 

Ruth was a pioneer in the field of marine biodeterioration research, and enjoyed a long term relationship with the Office of Naval 
Research. This, combined with her work in invertebrate and larval ecology, took her to many corners of the globe including France, 
Belgium. Netherlands, England, Germany, Denmark. Puerto Rico, India, Pakistan, many locations in Australasia. South America, and 
the former Soviet Union. Although a leading researcher, she enjoyed teaching at all levels from special courses for public school teachers, 
to undergraduate and graduate teaching, to her gentle persuasion of fellow researchers to look at a problem or a data set in another light. 
Ruth"s work also took her on many oceanographic cruises and to the depths of the ocean. On August 13, 1971, she became the first 
woman to dive in the deep submergence research vehicle ALVIN. This was the first of many dives and a deep sea career that included 
long term biodeterioration and species diversity work in the deep ocean (it was Ruth who explained why so little wood remained on the 
Titanic when it was found deep in the North Atlantic Ocean), and participation in multi-investigator cruises to the Galapagos Rift system. 



8 In Memoriam: Ruth Dixon Turner 

Despite a career filled with discovery, innovation, and firsts, the most memorable component of Ruth"s character that remains with 
the majority of people who met her is her warmth and friendliness, and her desire to show the excitement of science to all. Her love of 
science was effusive. She had a unique ability to share her science with audiences of all ages and skill levels. She was equally a superb 
teacher, illustrator, and practical scientist from fine work with the electron microscope to dissections of material from the field. Ruth 
taught and mentored many scientists at many levels, unselfishly giving of her time and energy to advance their careers. I consider myself 
fortunate to have enjoyed such direction from Ruth. Ruth shared much of her science through her publications, over 100 in all. Ruth 
worked actively until well after her 80th birthday. She left unfinished two major works, a monograph on her studies of deep sea borers 
and a comprehensive illustrated catalog of the pholads. Her colleagues have committed to finish these. 

Throughout her career Ruth made unique contributions in malacology and deep sea biology. She was an internationally respected 
educator and researcher, an ambassador for marine and biodeterioration studies, and an outstanding role model for women in science. 
Ruth is survived by her sisters Winifred Garrity and Lina MacNeil. Ruth was predeceased by her brothers Henry and Arthur, and her 
sisters Jessie. Mary, and Frances. Ruth never married, but had a large extended family of colleagues and students. She will be sadly 
missed, 

Roger Mann 
Professor of Marine Science- 
School of Marine Science 
Virginia Institute of Marine Science 
College of William and Mary 
Gloucester Point. VA 23062 

PUBLICATIONS 

1942. Editor. Biillelin of New Bird Life. vol. 6(8-1 2):56-104. 

1943. Birding the first year of the war. Bull. Mass. Audubon Society 28(2):33-42. 

1944. Vassar birds. Vassar Alumnae Magazine 33(4):15-17 

1946. The genus Bankia in the Western Atlantic. Johnsonia 2(l9):l-28. 16 pis. (with W. J. Clench). 

1946. John Gould Anthony, with a Bibliography and Catalogue of his Species. Occ. Papers on MoUusks 1(8):81-108, 15 pi. 

1947-1948. Republication; Henry Krebs — 1864 The West Indian marine shells. Rev. Soc. Malacologica Carlos de la Torre (Habana) 5:23—10; 59-80; 
91-116 and 6;1 1-43; 45-48. (with W. J. Clench and C. G. Aguayo). 

1947. Review; Fauna of New England. List of Mollusca, by C. W. Johnson, 1915. Johnsonia 2(231:92. 

1947. Review: A List of the Mollusca of the Atlantic Coast from Labrador to Texas, by Johnson, C. W. 1934. Johnsonia 2(23):92. 

1947. Procedimientos para recolectar bromas y otros moluscos perforentes marinos. Rev. Soc. Malacologica Carlos de la Torre (Habana) 5(2):43— 14. 

1947. Collecting shipworms. Limnological Soc. America, spec. publ. no. 19:1-8, text figs. 

1948. A new Thais from Angola and notes on Thais haemastoma Linne. American Mus. Novilates, no. 1374:1-14. 1 pi. 
1948. The genus Truncatella in the western Atlantic. Johnsonia 2:149-164, pis. 65-73 (with W. J. Clench). 

1948. William Henry Fluck. 1870-1948. Nautilus 62:69-70. 

1948. A catalogue of the family Truncatellidae with notes and descriptions of new species. Occ. Papers on MoUusks 1:157-212, pis. 22-24. (with W. J. 
Clench ). 

1948. The family Tonnidae in the western Atlantic. Johnsonia 2(26):165-192. 1 1 pis. 

1949. Sea shells |determination of all shells figured). Life Magazine 27(7):72-75. (with W. J. Clench). 

1949. Review: A manual of the Recent and fossil marine pelecypod moUusks of the Hawaiian Islands, by W. H. Dall, P. Barlsch and H. A. Rehder, 1938. 

Occ. Papers on MoUusks l(14):231. 
1949. Review; Reef and Shore Fauna of Hawaii, by H. H. Edmondson 1933. Occ. Papers on MoUusks 1( 14):231-232. 

1949. Review: A collection of Japanese shells with illustrations in natural color, by S. Hirase, 1934. Occ. Papers on MoUusks 1(I4):232. 

1950. The western Atlantic marine mollusks described by C. B. Adams. Occ. Papers on MoUusks l( 151:233-403. pis. 29—49. (with W. J. Clench). 
1950. The voyage of the Tomas BaiTera. Johnsonia 2(28):220. 

1950. The genera Sihenorylis. Cirsolrenui. Acirsa. Opalia. and ,'\inaea in the western Atlantic. Johnsonia 2:221-248. pis. 96-107. (with W. J. Clench). 
1950. Review: Sullivan, M. C. 1942. Bivalve Larvae of Malpeque Bay. Prince Edward Id.. Bulletin 77. Fisheries Research Board of Canada, pp. 1-36, 
22 pis. Johnsonia 2, p. 248. 

1950. Edward Chitty, with a bibliography and a catalogue of his species of Jamaican land mollusks. Oic. Papers Mus. Inst. Jamaica, no. 1:1-12. 1 pi. 
(with W.J. Clench). 

1951. The genus Epitonium in the western Atlantic. Part I: subgenera Epiioniuni s.s.. Cycloscala. G\roscala. Johnsonia 2:249-288, 23 pis. (with W.J. 
Clench). 

1951. Review: The Shell Collectors Handbook, by A. H. Verrill. Natural History 60(5):199. 
1951. Review: The Sea Shore, by C. M. Yonge 1949. Occ. Papers on Mollu.sks 1( 16):4I0-41 1. 

1951. Review: Natural History ot Marine Animals, by G. E. and Nettie MacGinilie 1949. Occ. Papers on Mollusks 1( 16):4I 1— tl 2. 

1952. Some problems in the Pholadidae. Hidl. Am, Malacological Union .-Xnn. Kepi, for 1951:9-10. 
1952. Me.sunella. a new genus in the Camaenidae. Nautilus 66:32 (with W. J. Clench). 

1952. La Rocolte des Tarets. Catalogues VIII, Xylophages et Petricoles Quest Africains. Institut Francais d Alriquc Noirre. pp. 130-134. figs. 156-158. 
[translation of paper published in Special Publication #19 of the Limnological Society of America]. 

1952. The genera Epitcmium (Pan II (subgenera /l.syjcri.Ko/d and Boreoscala]). Dcpressiscala. Cylindriscala. Nystiella and Soluliscala in the western 
Atlantic. Johnsonia 2:289-3.56, pis. 131-177 (with W.J. Clench). 

1953. New England malacologisls. Am Malacological I'nitm Ann. Rcpt. for 1952:4-6. 

1953. The Genera Epitonium. Opalia. and Cylindriscala in the Western Atlantic. Johnsimia 2:361-363, pi. 180, 



In Memoriam: Ruth Dixon Turner 9 

I9?3. Monographs of the Marine Mollusks of the Western Atlantic. Jolinsonia 2( l9-32):357-359. 

1953. [Supplement to| The Genus Bankia in the Western Atlantic. Johiisonia 2(34):357-359. (with D. J. Brown). 

1953. Recent works on the marine inollusks of Argentina. Johnsonia 2:380. 

1954. The family Pholadidae in the western Atlantic and the eastern Pacific, Part 1: Pholadinae. Johnsonia 3:1-63. pis. 1-34. 
1954. Supplement to John Gould Anthony (Occ. Pap. no. 8). Occ. Papers on Mollusks 1(18):442. 

1954. Supplement to the Catalogue of the Family Truncatellidae (Occ. Pap. no. 13). Occ. Papers on Mollusks 1(18):445. (with W. J. Clench). 

1954. Supplement to Western Atlantic Marine Mollusks Described by C. B. Adams. (Occ. Pap. no. 15). Occ. Papers on Mollusks 1( 18):447. (with W. J. 

Clench). 
1954. Review: Ensaio de Catalogo dos Moluscos do Brasil. by Frederico Lange de Morretes 1949. Occ. Papers on Mollusks 1(18):449. 

1954. Review: Catalogo de la Malacofauna .Antarctica Argentina by A. R. Carcelles. Johnsonia 3:64. 

1955. The family Pholadidae in the western Atlantic and the eastern Pacific. Part II: Martesiinae, Juannetiinae and Xylophaginae. Johnsonia 3:65-100, 
pis. 35-93. 

1955. The North American genus Lioplax in the family Viviparidae. Occ. Papers on Mollusks 2:1-20. pis. 1^ (with W. J. Clench). 

1955. Scaphopods of the Atlantis dredgings in the western Atlantic with a catalogue of the scaphopod types in the Museum of Comparative Zoology. 

Deep Sea Research, suppl. to vol. 3. pp. 309-320. 
1955. The Genus Melongena (abstract). A.M.U. — 20th Ann. Meeting p. 10. 
1955. Collecting shipwomis. [in] How to collect shells, pp. 32-35 (American Malacological Union). 

1955. The work of Charles B. Adams in the West Indies and Panama. Am. Malacological Union Ann. Rept. for 1955 pp. 7-8 (abstract). 

1956. The family Melonginidae in the western Atlantic. Johnsonia 3:161-188. pis. 94—109 (with W.J. Clench). 
1956. Melongena corona Gmelin. an excellent marine laboratory mollusk. Turto.x News 34:106-108. pis. 1-2. 
1956. Notes on Xylophaga washingtona Bartsch and on the genus. Nautilus 70:10-12. 

1956. Additions to the Western .Atlantic Marine Mollusks described by C. B. Adams. Occ. Papers on Mollusks 2:134-136. 1 pi. 

1956. Additions to the Pholadidae— Part II. Johnsonia 3(35):188. 

1956. The eastern Pacific mollusks described by C. B. Adams. Occ. Papers on Mollusks 2:21-133. pis. 5-21. 

1956. Freshwater mollusks of Alabama. Georgia and Florida from the Escambia to the Suwannee River. Florida State Mus. Bull. 1:97-239, 9 pis. (with 
W. J. Clench). 

1957. Charles Johnson Maynard and his work in malacology. Occ. Papers on Mollusks 2:137-152. 1 pi. 

1957. Molluscan wood borers, [in] Symposium on wood for marine use and its protection from marine organisms. American Soc. Testing Materials. Spec. 
Tech. Publ. no. 200:10-13. 

1957. The family Cymatiidae in the western Atlantic. Johnsonia 3:189-244. pi. 1 10-135 (with W. J. Clench). 

1958. The genus Hemitrochus in Puerto Rico. Occ. Papers on Mollusks 2:153-180, pis. 23-30. 

1958. The family Pinnidae in the western Atlantic. Johnsonia 3:283-326. pis. 149-171 (with J. Rosewater). 
1958. The works of Georgius Everhardus Rumphius. Johnsonia 3:326-327. 

1958. Review: Voyage Aux lies de Teneriffe. La Trinite Saint-Thomas. Saint Croix et Porto Rico by Andre Pierre LeDru. Occ. Papers on Mollusks 
2(22):179-180. 

1958. Review: The Museum Boltenianum or the Bolten Catalogue. Johnsonia 3:283-284. 

1959. Notes on the genus Taheilia {Truncatellidae) in New Guinea with the description of a new species. Occ. Papers on Mollusks 2:181-188. pis. 31-32. 
1959. The genera Hemiioma and Diodora in the western Atlantic. Johnsonia 3:334-344. pis. 176-179. 

1959. Henry A. Pilsbry. Johnsonia 3: introduction ii-iv. 2 pis. 

1959. Notes on the feeding oi Melongena corona. Nautilus 73:1 1-13. 

1959. Melongena egg cases. Nautilus 73:77. 

1959. The status of systematic work in the Teredinidae. Symposium on marine boring and fouling organisms. Univ. Washington Press, pp. 124-136. 

1959. Two new genera of land mollusks (Papuininae) from the Central Highlands of New Guinea. J Malacological Soc. Australia no. 3:4-9. pi. 1. text 
figs. 1-3 (with W.J. Clench). 

1960. Some techniques for anatomical work. Ann. Rept. Am. Malacological Union for 1959:6-8. 
1960. Land shells of Navassa Island. West Indies. Mus. Comp. Zool. Bull. 122:233-244. 7 pis. 
I960. Mounting minute radulae. Nautilus 73:135-137. 

I960. A new Meliobba from Schrader Range. New Guinea. J. Malacological Soc. Australia no. 4:30-31. 1 pi. (with W. J. Clench). 

1960. The occurrence of a nematode parasite in the genus Stylodon. J. Malacological Soc. Australia no. 4:56-59, text fig. 1, pi. 7 (with M. A. Pini). 

1960. The genus Calliostoma in the western Atlantic. Johnsonia 4:1-880, pis. 1-56. I text fig. (with W.J. Clench). 

1960. Teredo s en de mens. Correspondentiblad van Nederlandse Malacologische Vereniging. no. 91:924—925. [Translated into Dutch by C.O.V. 
Regteren. Altena]. 

1961. Heli-x pomatia Linne. colonized at Plymouth. Mass. Nautilus 74:122. 

1961. Natural history museums of Europe. Am. Malacological Union Rept. for 1960:13-14. 

1961. Report on the American Malacological Union meeting at McGill University. Am. Malacological Union Rept. for 1960:28-32. 

1961. Review: Traite de Zoologie. Vol. 5 fascicule 2. Embranchement des Mollusques pp. 1625-2164. Occ. Papers on Mollusks 2:260. 

1961. Pleurotomariidae in Bermuda waters. Nautilus 74:162-163. 

1961. Remarks on Nettastomella and Jouannetia. Am. Malacological Union Rept. for 1961:17-18. 

1961. The genus Lignopholas Turner (Mollusca: Pholadidae). Mitl. Zool. Mus. Berlin 37:287-295. 

1962. Nettastomella japonica Yokoyama in North America and notes on the Pholadidae. Occ. Papers on Mollusks 2:289-308. 7 pis. 

1962. New names introduced by H. A. Pilsbry in the Mollusca and Crustacea. Acad. Nat. Sci. Philadelphia, spec. publ. no. 4:1-218 (with W. J. Clench). 
1962. Books help beachcombers play the shell game. Natural Histoiy 71(7):4-7. 
1962. The genus Lithophaga in the western Atlantic. Johnsonia 4:81-1 16. 19 pis. (with K. J. Boss). 

1962. Review: British Prosobranch Molluscs, their functional anatomy and ecology, by V. Fretter and A. Graham. Johnsonia 4:1 16. 
1962. James H. Orton. his contributions to the Held of fossil and Recent mollusks. Rev. Mus. Argentina Cienc. Nat. Bernardino Rivadavia. Buenos Aires. 
8:89-99. 



10 In Memoriam: Ruth Dixon Turner 

1963. Monographs of the genera Pupiisnla. FoicarUa. and Meliobha (Papuininae: Camaenidae). J. Matucological Soc. Auslmliu no. 6;3-33 (with W. J. 
Clenchj. 

1963. Nest building in the bivalve moilusk genera. Musculiis and Limu. The Veliger 6:55-59 (with A. S. Merrill). 

1964. The subfamilies Volutinae. Zidoninae, Odontocymbiolinae and Calliotectinae in the western Atlantic. Johiisonia 4:129-180. 30 pis. (with W. J. 
Clench). 

1964. Snail. Encyclopedia Britannica p. 848 A-848 H. 1 1 figs. 
1964. Review: Fauna und Flora der Adria. by R. Riedl. Johiisonia 4:180. 

1964. Monographs of the genera Megalacron and Rhylidoconcha (Papuininae: Camaenidae). / Mulacologlcul Soc. Australia no. 8:36-71 (with W. J. 
Clench). 

1964. Anatomical relationships in the Teredinidae. Ann. Rept. American Malacological Union for 1964:16-17. 

1965. Mussel, [in] Encyclopedia Britannica. pp. 1096-1098. 2 figs.; 1964. ibid.. Snail, pp. 848A-848H. II figs. (Other articles in the Encyclopaedia 
Britannica include: Moilusk. Periwinkle. Cockle. Quahog. Piddock, Teredo, Whelk, Scallop, and Chiton). 

1965. Introduction. Occ, Papers on MoUusks 2:l-xvi. 

1965. Joseph C. Bequaert. Occ. Papers on MoHusks 2:i-ix. 3 pis. 

1966. Monograph of the genus Rhynchotrochus (Papuininae. Camaenidae). J. Malacol. Soc. Australia, no. 9:59-95. text figs. 1-6, pis. 15-22 (with W. J. 
Clench). 

1966. A survey and illustrated catalogue of the Teredinidae. Spec. puhl. Museum of Comparative Zoology. 265 pp. 64 pis.. 25 text figs. 

1966. Some results of deep water testing. Ann. Rept. Am. Malacological Union for 1965. pp. 9-1 1. 

1966. Report to the government of India on systematic and biological research on marine wood-boring Mollusca. FAO Report TA 2155. pp. 1-30. 

1966. Implications of recent research in the Teredinidae. Beihefte zu Material und Organismen. Berlin. Heft 1. pp. 437—446. 

1966. Marine borer research in cooperation with the Office of Naval Research. Report of First Inter-American Naval Research Congress. 

1967. A new species of Lyria (Volutidae) from Hispaniola. Nautilus 80:83-84, figs. 2-3. 
1967. Teredo. Encyclopedia Britannica pp. 861-862. 

1967. A new species of fossil Chlamys from Wright Valley. McMurdo Sound. Antarctica. New Zealand J. Geology Geophysics 10:446-455. figs. 1-5. 

1968. The Xylophagainae and the Teredinidae — a study in contrasts. Ann. Rept. Am. Malacological Union for 1967. pp. 46-^8. 

1968. Monograph of the genus Letitia (Papuininae: Camaenidae). / Malacological Soc. Australia, no. 1 1:32-49. pis. 3-7. text figs. 1-2 (with W. J. 
Clench). 

1969. Biological studies in marine wood borers. Arm. Rept. Am. Malacological Union for 1968. pp. 14-16. (with A. C. Johnson). 
1969. Review: The shell, five hundred million years of inspired design, by H. & M. Stix and R. T. Abbott. Natural History 78:60-62. 

1969. Pholadacea. [in] R. Moore (ed.). Treatise on Invertebrate Paleontology. (N) Mollusca 6(2 of 3):702-742. figs. 162-214. 

1970. Some problems and techniques in rearing bivalve larvae. Ann. Rept. Am. Malacological Union for 1969. pp. 9-12. I pi. (with A. C. Johnson). 
1970. Richard Winslow Foster. Johnsonia 4:ii-v. 2 figs. 

1970. The family Volutidae in the western Atlantic. Johnsonia 4(48):369-372. pis. 172-174. (with W.J. Clench). 

1971. Some anatomical and life history studies of wood boring bivalve systematics. Ann. Rept. Am. Malacological Union for 1970. pp. 65-66 (with John 
Culliney). 

1971. Identification of marine wood boring mollusks of the world. |in) Marine Borers. Fungi and Fouling Organisms of Wood. Chapter I. pp. 18-64. 

Published by the OECD. 
1971. Biology of the marine wood boring mollusks of the world. Ibid.. Chapter 13. pp. 259-301. (with A. C. Johnson). 
1971. Review: Beneath Australian seas, by Walter Deas and Clarrie Lavvler. .Australian Newsletter N. S. no. 2. p. 9. 

1971. Australian shipworms. Australian Natural History, Sydney. I7(4):139-145. 4 pis. 

1972. Land and freshwater snails of Savo Island, Solomons, with anatomical descriptions (Mollusca, Gastropoda). Steenstrupia (Zool. Mus. Univ. 
Copenhagen). 2( l5):207-232. pis. I-I3 (with W.J. Clench). 

1972. Results of an international cooperative research program on the biodeterioration of timber submerged ni the sea. Material und Organismen 

7(2):93-l 18 (with E. B. G. Jones. H. Kuhne and P. C. Trussell). 
1972. A new genus and species of deep water wood-boring bivalve (Mollusca. Pholadidae. Xylophagainae). Basleria 36:97-104. figs. 1-12. 
1972. Teredicola typicus C. B. Wilson. 1942 (Copepoda, Cyclopoida) from shipworms in Australia. New Zealand, and Japan. .Australian ./. Marine and 

Freshwater Res. 23( l):63-72. figs. 1-16 (with A. G. Humes). 
1972. Line photo micrography: A tool in biological studies. Bulletin of the American Malacological Union, p. 30. 
1972. Cinemicrographic studies of crawling behavior in larval and juvenile bivalves, (with J. L. Culliney). 

1972. Xyloredo. new leredinid-like abyssal wood-borers (Mollusca. Pholadidae. Xylophagainae). Breviora. MCZ. no. 397: 1-19. pis. 1-6. 

1973. Wood-boring bivalves, opportunistic .species in the deep sea. Science 180:1377-1379. 2 figs.. I table. 

1973. Deep water wood-boring mollusks. Proc. Third International Congress on Marine Corrosion and Fouling. Nat. Bur. Standards. Gaithersburg. 
Maryland, pp. 8.36-841. 

1973. The biologists view of the Tereduiidae and then" control (with a documentary film on the life history of the Teredinidae). Proc. Third Inlernational 
Congress on Marine Corrosion and F<iuling. Nat. Bur. Standards. Gaithersburg. Maryland, pp. 83-87 (with J. L. Culliney). 

1974. In the path of a warm saline eftluent. Am. Malacological Union Bull, for 1973. pp. 36—44. figs. 1-3. 

1974. A new blind F'hysa from Wyoming with notes on its adaptation to the ca\e environment. Nautilus SS(3):S()-S5. 19 figs, (with W.J. Clench). 

1974. New approaches and techniques for studying bivalve larvae |in| W. I.. Smith & M. H. Chanley (eds.). Culture of Marine Invertebrate Animals, pp. 
2.57-271, 2 figs. Plenum Publishing Corp.. N.Y. (with J. L. Culliney and P.J. Boyle). 

1975. Review: The Shell Makers, Introducing Mollusks. by Alan Solem. J. Fisheries Res. Board Canada 32( 5 ):7 19-720. 

1 975. Studies of bivalve larvae using the scanning electron microscope and critical point drying. Bull. Am. Malacological t 'nion lor 1 974. pp. 59-65 (with 
P. J. Boyle). 

1976. Larval development of the wood boring piddock Martesia striata (Linnaeus) (Mollusca: Pholadidae). 7. E.xper. Marine Biol, and Ecology 22:55-68. 
text figs. 1-4 (with P. J. Boyle). 

1976. Fixation and prcser\'ation of marine /ooplankton. |inl II. F. Slcedman (ed.). SCOR/UNESCO Handbook Zooplankton Fixation and Preservation. 
Chap. 8 section on Mollusca. Part 1 1, pp. 290-304. Lhie.sco Press. Paris. 



In Memoriam: Ri'th Dixon Turner 1 1 

1976. Search for a weak link. Proc. Workshop on Biodeterioration of Tropical Woods. (D. Bultman, editor). Naval Res. Lab. Washington. D.C.. pp. 

31-tO. 
1976. Some factors involved in the settlement and metamorphosis of marine bivalve larvae, [in] Sharpley & Kaplan (eds.i. Proc. 3rd. International 

Biodegradation Symposium, pp. 409—416. 
1976. Larval development of the deep-water wood boring bivalve Xylophaga atlaniica Richards (Mollusca. Bivalvia. Pholadidae). Ophelia 15(2):149-161 

(with J. L. Culliney). 
1976. Marine biodeteriogenic organisms. I. Lignicolous fungi and bacteria and the wood boring Mollusca and Crustacea. Intern. Biodeierior. Bull. 

12(4):120-134 (with G. Jones. S. E. Funado and H. Kuhne). 
1976. Reproductive pattern in an abyssal snail. Anwr. Zool. 16(2):269 (with M. A. Rex and C. A. Van Ummensen). 

1976. Bivalve larvae, their behavior, dispersal and identification. Proc. U.S. — U.S.S.R. Workshop in Biological productivity and biochemistry of the 
worlds oceans, pp. 23-25 [in] J. Costlow (ed.). Ecology of Fouling Communities. 

1977. Control of marine borer attack on wood. U.S. Patent 4.012.529 (with J. D. Bultman & L. Jurd). 

1977. Genetic similarities of wood-boring bivalves (Pholadidae and Teredinidae) based on comparisons of allozymes. Biol. Bull. 153(2):420 (with T. J. 
Cole). 

1977. Development, metamorphosis and natural history of the nudibranch Doriclclla ohsciira Verrill (Corambidae: Opisthobranchia). J. Exp. Mar. Biol. 
Ecol. 27:171-185 (with F. E. Perron). 

1978. Contribution of field and life history studies to an understanding of some cases of opportunism. [in| U.S.S.R. — U.S.A. Symposium on the Program 
Biological Productivity and Biochemistry of the Words Oceans, pp. 241-2-14. 

1978. Wood, mollusks. and deep-sea food chains. Bull. Am. Malacological Union for 1977, pp. 13-19, figs. 1-3. 
1978. Genetic relations of deep-sea wood-borers. Bull. Am. Malacological Union for 1977, pp. 19-25 (with T. Cole). 

1978. The feeding behaviour and diet of Calliostoma occidentale. a coelenterate-associated prosobranch gastropod. J. Moll. Stud. -14:100-103 (with F. 
Perron). 

1979. Mollusks as prey of ariid catfish in the Fly River. New Guinea. Bull. Am. Malacological Union for 1978, pp. 33—10. pis, 1-6 (with T. R. Roberts). 
1979. New techniques for preparing shells of bivalve larvae for examination vvith the scanning electron microscope. Bull. ,Am. Malacological Union for 

1978. pp. 17-24. pis. 1-3 (with C. B. Calloway). 
1979. The role of phytoplankton in the diets of adult and larval shipworms. Lyrodus pedicellatus (Bivalvia: Teredinidae). Estuaries 2(1 ):58-60 (with J. A. 

Pechenik and F. A. Perron). 
1979. Bankia neztalia n. sp. (Bivalvia: Teredinidae) from Australia-New Zealand, and its relationships. J. Royal. Soc. New Zealand 9(4):465— 173 (with 

J. L. McKoy). 
1979. High Larval Dispersal Capability of a Deep-sea Hydrothermal Vent Bivalve from the Galapagos Rift. .American Society of Zoologists Meeting 

[abstracts!. Dec. 27-30, 1979. 
1979. Reproductive pattern in the abyssal snail. Benthonella tenella (Jeffreys), [in] S. Stancyk (ed. I. Reproductive Ecology of Marine Invertebrates. Belle 

W. Baruch Library in Marine Science, publ. no. 9. pp. 173-188 (with M. A. Rex and C. A. Van Ummerson). 
1979. Biology, life history and relationships of Zachsia zenkeailschi. XIV Pacific Science Congress. Khabarovsk. Abstracts. Committee F. Sec. 1 la, pp. 

139-141 (with Y. M. Yakovlev). 

1979. Galapagos 79: Initial findings of a deep-sea biological quest. Oceanus 22(2): 1-10 (with F. Grassle and members of the cruise). 

1980. Macrobiodegradation of plastics. Proc. 4th International Biodeterioration Symposium, Berlin-Dalhem, pp. 1 17-122 (with G. J. L. Griffin). 
1980. Range extension of teredinids (shipworms) and polychaetes in the vicinity of a temperate-zone nuclear generating station. Marine Biology 5S:55-(A 

(with K. E. Hoagland). 
1980. The giant white clam from the Galapagos Rift. Calyptogena magnifica n. sp. (Bivalvia; Vesicomyidae). Malacologia 20(1 ): 161-194 (with K. J. 

Boss). 
1980. Larval dispersal of a deep-sea hydrothermal vent bivalve from the Galapagos Rift. Marine Biology 57:127-133 (with R. A. Lutz. D. Jablonski. and 

D. C. Rhoads). 
1980. Evolution and adaptive radiation of shipworms. Haliotis 10(2):68 (with K. E. Hoagland). 

1980. Effects of closed-culture competitive interactions on growth of Teredo navalis. Biological Bulletin I59(2):465 (with G. A. Tracy and C. J. Bergl. 

1981. Wood Islands and Thermal Vents as centers of diverse communities in the deep-sea. Biologia Morya. no. I. pp. .VIO [in Russian, translation by 
Plenum Publishing Co.]. 

1981. Physiological aspects of wood consumption, growth, and reproduction in the shipworm, Lyrodus pedicellatus Quatrefages (Bivalvia: Teredinidae). 

/. Exp. Mar. Biol, and Ecol. 52:63-76 (with S. Gallager and C. Berg). 
1981. Preliminary observations of bacteria and shipworms (Bivalvia: Teredinidae). Biol. Bull. 161:332 (with A. Wright. C. Cavanaugh, R. Mann). 
1981. Evolution and adaptive radiation of shipworms. Malacologia 21(1-2):1 1 1-148 (with K. E. Hoagland). 

1981. Life cycle of Zachsia zenkewitschi. bivalve mollusk with dwarf males, [in] Sixth All-Union Conference on Embryology Abstracts of paper [in 
Russian]. Nauka. Moscow p. 207. 

1982. Feeding types in vent macro-organisms. Abstracts of Papers of the 148th Natl Meeting AAAS. 1982:34. 

1983. The ecology and reproduction of Zachsia zenkewitschi, a teredinid with dwarf males. Proc. XIV Pacific Science Congress, Khabarovsk. 
USSR, August 1979. Section Marine Biology 2, Genetics and Reproduction of Marine Organisms, pp. 215-219, figs. 1-5 (with Y. Yakovlev). [in 
Russian]. 

1983. Documentation and implications of rapid successive brooding in the shipworm. Lyrodus floridanus (Mollusca: Bivalvia). Proc. XIV Pacific Science 
Congress. Khabarovsk, USSR, August 1979. Section Marine Biology 2. Genetics and Reproduction of Marine Organisms, pp. 172-177, figs, 1-2 
(with C. B. Calloway). ]in Russian]. 

1983. Dwarf males in the Teredinidae (Bivalvia: Pholadacea). Science 219:1077-1078 (with Y. Yakovlev). 

1983. A Cellulolytic nitrogen-fixing bacterium cultured from the gland of Deshayes in shipworms (Bivalvia: Teredinidae). Science 221:1401-1043 (with 
J. Waterbury and C. B. Calloway). 

1983. Some aspects of the life history of a bivalve mollusc. Zachsia zenkewitschi. Biologiya Morya 9(5):27-34 (with Y. Yakov lev and E. M. Karaseva). 
[in Russian!. 

Documentation and implications of rapid successive gametogenic cycles and broods in the shipworm. Lyrodus floridanus (Bansch) (Bivalvia: Tere- 
dinidae). J. Shellfish Res. 3(l):65-69 (with C. B. Calloway). [Sept. 1984]. 



12 In Memoriam: Ruth Dixon Turner 

1984. An iiverview of research on marine borers: past progress and future directions, [in] J. D. Costlow and R. C. Tipper (eds.). Marine biodeterioration: 

an interdisciplinary study, pp. 3-16. Naval Institute Press. Annapolis. Maryland. 
1984. Some aspects of the life history of Zachsia zenkewiischi (Teredinidae. Bivalvia). The Soviet Journal of Marine Biology 9(5):257-264. Plenum 

Publishing Corp.. N.Y.. Translation from the Russian-Biologiya Morya 1983. (with Y. M. Yakovlev with E. M. Karaseua). 
1984. Growth and distribution of moUusks at deep-sea vents and seeps. Oceaims 27(3):55-62 (with R. A. Lutzl. 
1984. Larval development and dispersal at deep-sea hydrothernial vents. Science 226:1451-1454 (with R. A. Lutz and D. Jablonski). 

1984. Larval ecology of mollusks at deep-sea hydrothermal vents. Bull. Am. Malacological Union. Annual Meeting, Norfolk, Virginia, July 1984 (with 
Phillippe Bouchet and Richard A. Lutz). 

1985. Notes on mollusks of deep-sea vents and reducing sediments. American Malacological Bulletin. Special Edition No. 1: 23-34. In Perspectives in 
Malacology: A Symposium to Honor — Dr. Melbourne and R. Carriker. 

1985. Modes of larval development at deep-sea hydrothermal vents, [in] M. L. Jones (ed. ). Hydrothermal vents of the eastern Pacific: an overview. Bull. 

Biol. Soc. Washington, no. 6. pp. 167-184. figs. 1-28. (with R. A. Lutz and D. Jablonski). 
1985. Squat lobsters. Munidopsis, associated with mesh enclosed wood panels submerged in the deep-sea. American Zoologist 25(4):141A [abstract]. 

(with A. B. Williams). 
1985. Hydrothermal vents, sulfide seeps and mollusks. Am. Malacological Bulletin 3(1):96 (abstract for 1984 meeting). 
1985. Description of a hydrocarbon seep community on the Louisiana slope. Am. Zoologist 25(4): lOA [abstract], (with C. J. Denoux, M. C. Kennicutt, 

R. R. Bidigare, J. M. Brooks, R. R. Fay, M. L. Jones). 

1985. William J. Clench. October 24. 1 897-February 1984. Malacological Rev. 18:123-124. 

1986. Larval ecology of mollusks at deep-.sea hydrothermal vents. Am. Malacological Bulletin 4( 1 ):49-54 (with R. A. Lutz. P. Bouchet. D. Jablonski. 
and A. Waren). 

1986. The language of benthic marme invertebrate development patterns: problems and needs, [in) M.-F. Thompson. R. Sarojini and R. Nagabhushanam 
[eds.). Biology of benthic marine organisms: Techniques and methods as applied to the Indian Ocean. Bombay: Oxford and IBH Publishing Co. pp. 
227-235, figs. 1-10 (with J. A. Pechenik and C. B. Calloway). 

1986. Squat lob,sters (Galatheidae: Munidopsis) associated with mesh-enclosed wood panels submerged in the deep sea. J. Crustacean Biology 6(3): 
617-624 (with A. B. Williams). 

1986. The biology of molluscan hard-substrate borers. International Conference on Marine Sciences of the Arabian Sea. March 2S-Apri! 2. 1986, Karachi. 
Pakistan. Abstracts p. 35. 

1987. Seasonal recruitment of marine invertebrates to hard substrates on Georges Bank and the eastern continental shelf of the United States. Nautilus 
101(1 ):19-24 (with C.J. Berg. B. Butman and J. A. Early). 

1987. Species pairs in the Teredinidae. International research group on wood preservation. Document No: IRG/WP/4142: 1-2 (with C. B. Calloway). 
1987. Species pairs in the Teredinidae. American Malacological Union Annual Meeting July 19-23 Key West, Florida. Program p. 44 (Abstract), (with 
C. B. Calloway). 

1987. Introduction to Symposium on Deep-Sea Hydrothermal Vents and Cold- Water Seeps. 153rd National Meeting of the American Academy for the 
Advancement of Science, Chicago, 14-18 February, Abstracts of Papers p. 21. 

1988. Biodeterioration — Multidisciplinary, collaborative research, [in) M-F. Thompson, R. Sarojini and R. Nagabhushanam (eds.) Marine Biodeterio- 
ration — Advanced Techniques Applicable to the Indian Ocean. Bombay. India Oxford and IBH Publishing Co. PVT. LTD. pp. 3-12. 

1988. Biodeterioration — Multidisciplinary, collaborative research, [in) M-F. Thompson. R. Sarojini and R. Nagabhushanam (eds.) Marine Biodeterio- 
ration — Brooding in the Teredinidae (Mollusca: Bivalvial. Bombay. India Oxford and IBH Publishing Co. PVT. LTD. pp. 215-226 (with C. B. 
Calloway). 

1988. Recruitment of marine invertebrates to hard substrates at deep-sea hydrothermal vents on the East Pacific Rise and Galapagos spreading center. 
Deep-Sea Research 35( 10/1 1 ):183-V1849 (with C. L. vanDover. and C. J. Berg). 

1988. Wood, phytoplankton. dissolved organic material and nitrogen in teredinid nutrition (Mollusca: Bivalvia: Teredinidae). [in] M-F. Thompson and 
N. Tirmizi (eds.) Marine Science of the Arabian Sea. Proceedings of the International Conference — Washington, D.C.; Institute of Biological 
Sciences, pp. 585-606. 

1988. Cellulolytic nitrogen-fixing bacteria in the Teredinidae (Mollusca: Bivalvia). ]iii| Biodeterioration 7; pp. 743-748. (Dr. Houghton, R. N. Smith and 
H. O. W. Eggins. Editors). 

1989. The Pholadacea [in] Fauna of Australia. P. Beesley Ed. 

1989. The Genera Martcsia and Ligiiopliolas in the Indo-Pacific. (Mollusca: Bivalvia: Pholadidae) Ophelia 3(.)(3):I55-156. (with L. N. Santhakumaran). 

1990. Xylophile ostracoila in the deep-sea. Proc. Aberwystwyth conference on ostracods (with P. L. Steineck. R. F. Maddocks. G. Coles and R. Whatley). 
[in) Ostracoda and Global Events, pp. 307-319 (R. Whatley and C. Maybury. Editors). 

1990. Species Richness and Diversity of Algal-A.ssocialed Micromolluscan communities from Sao Miguel. Acores. Acoreana. 

1990. Supplement: pp. 39-58 (with R. C. Bullock, and R. A. Fralick). 

1990. Bivalves of hydrothermal vents and reducing sediments. Fourth International Congress of Systematic and E\ciluiionary Biology. University of 

Maryland: College Park, Maryland. July. 1990. (Abstract) (with E. A. Cobabe). 
1992. Characterization and site description of .Solemya horealis. (Bivalvia; Solemyidae). another bivalve, bacteria symbiosis. Marine Biology 1 12:601- 

613. (with N. M. Conway, B. L, Howes, J. E. Capu/zo, and C. M. Cavanaugh). 
1992. Types and Prevention of Biodeterioration in the Deep Sea. Indo-United Slates Meeting on Recent Developments in Bioloiilmg Control. Bangalore. 

(Abstracts Plenary Session; C). 
1992. Deep Sea Wood Borers and Ancient Wrecks. American Malacological Union Bulletin. Annual Meeting August 2-7. Sarasota. Florida (Abstract 

p. 38). 



Jaiimal of Shellfish Rc.winh. Vol. 19, No. 1. 13-14. 2000. 




Kenneth Kendall Chew 
Honored Life Member 



Dr. K. (Ken) K. Chew, is a recognised authority in the field of molluscan biology who has contributed extensively to invertebrate 
research and the continuing development of the shellfish industry on the west coast of North America. His contributions in the field of 
molluscan aquaculture are recognised world wide and his advice and consultation are frequently sought by industries in many countries. 

Ken was born in Red Bluff. California in 1933 and received his elementary and secondary education there. While growing up, he 
worked in the family restaurant where he learned the fine art of Chinese cuisine. Those of us who have tasted Ken's cooking can attest 
to the fact that he mastered this fine art. 

He obtained his B.A. from Chico State College in 1955 and then decided to attend the School of Fisheries of the University of 
Washington and become a fisheries biologist. Ken received a fellowship to begin graduate work on trout and salmon but Dr. Van Cleve 
sent him to the Washington State Department of Fisheries Shellfish Laboratory at Brinnon for the summer. He became involved in 
shellfish work at the lab and decided that his future lay with invertebrates, mainly molluscs, rather than fish and he entered the world 
of molluscan biology. He obtained his M.S. degree in 1958, studying the food preference of the Japanese oyster drill, and his PhD in 
1962. The title of his PhD thesis was, "The growth of a population of Pacific oysters, Crassostrea gigas, when transplanted to three 
different areas in the state of Washington." His supervisor was Al Sparks, a former president of the National Shellfisheries Association. 

After recei\ing his PhD. Ken joined the staff of the School of Fisheries at the University of Washington and has remained there since. 
He has held several positions at the School and has taught a variety of invertebrate courses. At present he is a Professor in the School 
of Fisheries. In 1989 he became the Director of the Western Regional Aquaculture Center (WRAC) which is one of five aquaculture 
centers designated by the U.S. Department of Agriculture to foster development of aquaculture in the United States. He resigned that 
position in 1996 to be the interim director of the School of Fisheries. He was then appointed Associate Dean, College of Ocean and 
Fisheries Sciences at the University of Washington in 1998, the position he holds presently. In addition to other duties. Ken is now busily 
engaged assisting with expansion of the College and seeking support for the College from industry. 

Ken's research interests cover a wide spectrum that include shellfish biology and aquaculture, paralytic shellfish poisoning, and 
problems related to baseline ecological studies involving benthic intertidal and subtidal invertebrate communities. He has published over 
100 papers on a wide range of shellfish subjects in scientific Journals. Technical Reports. Conference Proceedings, chapters of books 
and in columns of trade publications. 



13 



14 Honored Life Member: Kenneth Kendall Chew 

Teaching and maintaining a close rapport witln students has been an important part of Ken's life and he has inspired many students 
to continue with studies in molluscan biology. During his career, about 100 students obtained graduate degrees under his supervision. 
In recognition of his outstanding teaching ability he received the 1993 Distinguished Undergraduate Teaching Award from the College 
of Oceans and Fisheries Sciences at the University of Washington. 

Throughout his professional career. Ken maintained a close working relationship with the molluscan shellfish industry, particularly 
the industry in the Pacific Northwest. Much of his research and that of his graduate students focussed on finding solutions to problems 
to aid development of the industry. The present healthy state of the shellfish industry in the Pacific Northwest is due in a large measure 
to the efforts of Ken Chew and his co-workers. His talents and devotion to the shellfish industry were recognised when he was made 
Director of WRAC. In this position he devoted considerable time and energy testifying before Congressional Committees in Washington, 
DC on issues related to development of aquaculture. 

Ken's influence in the shellfish industry has not been confined to the Pacific Northwest. He has provided advice and consultation to 
many countries throughout the world including; Australia, Canada, China, Chile, Japan, Thailand, Taiwan, Hong Kong and the 
Philippines. He has lectured on molluscan biology and culture in many countries. He was an invited guest lecturer in China and in 1987 
was appointed for life as a visiting professor at Shandong College of Oceanography. 

Ken has been actively involved with the National Shellfisheries Association since he first joined in 1958. He served on the Board, 
was Vice President from 1970-71, President from 1971-72 and served as an Associate Editor for the Journal of Shellfish Research from 
1989-92. He has organized annual meetings held in Seattle. Another important function Ken has undertaken is to arrange Chinese 
Dinners at annual meetings. Many of us have enjoyed the fine cuisine and companionship that these evenings have afforded. 

In addition to his involvement with the parent National Shellfisheries Association. Ken has played a major role to preserve and foster 
the as.sociation between the Pacific Coast Oyster Growers Association and the West Coast Section of the National Shellfisheries 
Association. From 1975-1990 Ken was the main reason this association survived and he devoted considerable time and energy into 
preserving this close association between industry and the scientific community of NSA. It is now a large and dynamic association and 
serves as an excellent forum for people from industry, government, and academia to come together and discuss shellfish work and 
problems. Many students have presented their first paper at these meetings. The present healthy state of this association is a monument 
to Ken's organisational skills and abilities. 

As a result of his work and association with NSA, Ken was awarded the first David H. Wallace Award given by the Association in 
1982 for his dedicated service in promoting research, understanding and co-operation among shellfisheries scientists, culturists, man- 
agers, producers and regulators. In further recognition of his contribution to NSA, he was elected to Honored Life Member in 1989. 

Ken has been active in other organizations as well. He was a member of the Board of the World Aquaculture Society from 1973-76, 
President in 1977 and an associate editor of the Proceedings of the Society from 1985-89. He was editor for the North American Oyster 
Workshop that appeared as a special publication of the World Aquaculture Society in 1983. He received an Honorary Life Member award 
from the World Aquaculture Society in 1995. At present he is a columnist for Aquaculture Magazine. 

Ken is an avid sportsman and, when time pemiits, relishes hunting and fishing. He is an excellent taxidermist and has mounted several 
species of birds he collected. He is a keen hand ball player and more than one meeting has been delayed so he could complete a game 
of hand ball. 

Along with all his shellfish activities. Ken has found time to be an exemplary family man. He and his wife. Maegan. have raised four 
children and now have three grandchildren. They now have time to relax and enjoy their family and the view of Puget Sound from their 
wonderful house in Seattle. The shellfish world and NSA owe much to Ken Chew for his past contributions and his friends and 
co-workers know his influence will continue to be felt for many future years. 

Neil Bourne 

Department of Fisheries and Oceans 

Pacific Biological Station 

Nanaimo, British Columbia V9R 5K6 

Canada 



Journal oj Shuiljhh Research. Vol. 19, No. 1, 15-16. 2000. 



.t >^ 





Victor Samuel Stuart Kennedy 
Honored Life Member 

Dr. "Vic" Kennedy, a long time NSA member. Vice President (1988-1989). and President (1990-1991). was born in Ireland in 1942. 
Although long established in the U.S., he still maintains a Canadian, United Kingdom citizenship. His early education was at Sir George 
Williams University in Montreal. Canada, where he received his B.Sc. in zoology (1962). He continued graduate education at Memorial 
University at St. John's. Newfoundland, with a M.S. in fisheries biology ( 1964). Vic then entered the University of Rhode Island for his 
Ph.D. where Dr. Paul Saila was his major professor. 

I first remember Vic by a phone call in 1967. whereby he introduced himself and said Dr. Saila suggested he call me because of our 
Chesapeake work in thermal ecology. He expressed an interest in doing the same with a shellfish species at our Chesapeake Biological 
Laboratory's field station at Hallowing Point about 20 miles up estuary from CBL. I invited him down and vividly remember our lunch 
at old famous Shorter' s Restaurant in Benedict on the Patuxent Estuary. He impressed me with his quick mind, familiarity with the 
literature, and obvious intense interest in aquatic and shellfish ecology. I hired him on the spot and offered a pre-doc position. I called 
Dr. Saila, (an old friend that first befriended me as a graduate student at an American Fisheries Society meeting) afterward. I thought 
a graduate student accepted by Saul was surely good enough for me too. 

Vic completed his Ph.D. on the role of temperature on the soft shell clam. Mya arcnaria. in 1970 and has maintained a peripatetic 
professional career. After completing his Ph.D.. he was visiting Assistant Professor at Chapman College in California where among other 
activities he spent two semesters traveling the world and teaching on board their "World Campus Afloat" vessel. He came back to 
Maryland in 1972-73 to continue thermal research, then left in 1973 for a Post-Doc fellowship at the University of Canterbury in New 
Zealand where he taught and completed research on mussels. In 1974. a Post-Doc was accepted at Newfoundland Biological Station in 
St. John's where he investigated the role of arsenic in a marine food web and on benthic soft bottom communities. 

In 1976 he returned to the University of Maryland System's Horn Point Laboratory as an Assistant Professor and continued his 
teaching efforts as well as benthic ecology research. Again he left the Maryland area in 1983. and spent a sabbatical year as a W. F. Jones 
Fellow at St. Francis Xavier University in Nova Scotia where he continued his benthic research and taught a marine ecology course. In 
1984 he returned to the Horn Point Laboratory, was promoted to Full Professor in 1986 and named Assistant Director in 1989. Again 
he left Maryland on a sabbatical and served as visiting Professor on board the .S"5 Universe Campus. University of PittsburgI ship, for 
the Semester at Sea Program. Vic has remained at Horn Point since! Throughout all this substantial traveling (40 countries), teaching 



15 



16 Honored Life Member: Victor Samuel Stuart Kennedy 

and current administrative duties. Vic hias continued a vigorous research! program dealing witli shiellfish reproduction and larval behavior, 
as well as crustacean and fish foraging behavior as reflected in the selected publications listing. His activities have covered both the 
littoral and sublittoral benthic habitats and communities. His long term interest in thermal ecology has now evolved into the global 
climate change arena. 

Vic has over 45 journal refereed publications, over 10 chapters in books and conference proceedings and 5 written or edited books. 
He enjoys a special reputation for his publication efforts dealing with morphology, biology, ecology, and management history of the 
eastern oyster. Crassoslrea virgiiiica. A most important current activity is completing editorship of a 13 chapter book on blue crabs, 
which he has been working on with Dr. L. Eugene Cronin. (See Vic's In Memoriam to Dr. Cronin in J. Shellfish Res. 18(1): 1-3. 1999). 
Another substantial service he has provided to the research and management communities are publications of 5 extensive bibliographies 
that have covered the world's literature in their respected areas. 

Concerning professional societies. Vic has assumed numerous responsibilities over the years, including President of the Atlantic 
Estuarine Research Society, with the aforementioned National Shellfish Association and a governing board member of the Estuarine 
Research Federation. He has had editorial responsibilities for the Transactions of the American Fisheries Society, American Malaco- 
logicai Bulletin, and Estuaries, among others. Vic's service also includes numerous requests for research propo,sal reviews by NSF, Sea 
Grant. Hudson River Foundation, and the Smithsonian Institution. Beyond his usual numerous editing services, he regularly undertakes 
requests for reviews on books dealing with aquatic and coastal habitats and processes. This extensive editing experience he has translated 
to a very popular graduate course entitled "Scientific Writing and Communication" in which his last class had 38 students, an almost 
unheard of number in a graduate course. 

In addition to the W. F. Jones Fellow honor he also won as NSA Thurlow C. Nelson award in his junior investigator days (1968). 
was noted for outstanding service by AFS. and given an Honored Life Member Award by NSA in 1995. 

Vic shows no sign of slackening in his science enthusiasm and his very active and diversified professional involvements. Indeed, with 
his two children off in Canada, one in the creative arts and the other with her family working with the native Inuits on Baffin Island, 
I suspect he may even pick up the pace if his wife Debbie will allow! 

Joseph A. Mihursky 

Professor 

Chesapeake Biological Laboratory 



Journal oj Shellfish Reseuich. Vol. 19, No. 1. 1 7- IS, 2000. 




Sammy M. Ray 
Honored Life Member 

The scientific contributions of Dr. Sammy Ray to oyster disease research are widely acclaimed, due in no small part to the diagnostic 
method he developed to detect the disease agent Dennocystidiwn mariiutm. Dr. Ray was one of a handful of investigators in the early 
1950's to explore this new oyster disease found in the Gulf of Mexico. Now the disease agent is called Perkinsiis inarimis and molecular 
techniques can be used to specifically diagnose the protozoan pathogen. Nonetheless, the highly reliable diagnostic technique developed 
by Dr. Ray is still the most widely used in oyster disease studies. 

Dr. Ray was bom in Mulberry KS, attended Mississippi Delta Junior College. Louisiana State University, and received his M.A. 
(Biology. 1952) and Ph.D. (Biology. 1954) degrees at Rice University in Texas. His postgraduate career began with the U.S. Fish and 
Wildlife Service as a Fishery Research Biologist and he joined the Texas A&M staff in 1957 at the Research Foundation Laboratory in 
Grande Isle, LA. He became an Associate Professor (1963) in Oceanography and Wildlife and Fisheries Science and was named Director 
of the Marine Laboratory at Galveston. As he reached Full Professor (1972), Dr. Ray was named Head of the Department of Marine 
Sciences. Since then he has held positions as Dean of the Moody College of Marine Technology and interim President of Texas A&M 
University at Galveston. Dr. Ray officially retired in 1990, but remains active as an ad\isor and coordinator of student programs and 
several community outreach programs. 

Several academic honors have been awarded to Dr. Ray, including a Faculty Distinguished Achievement Award in Research at Texas 
A&M University at Galveston (TAMUG). the William Paul Ricker Award for Distinguished Faculty-Staff Achievement (TAMUG), a 
Distinguished Alumnus Award from the Mississippi Delta College, and a Piper Professor Award. He was awarded a lifetime honorary 
membership in the National Shellfisheries Association at the 1990 meeting in Maine. 

Dr. Ray has been a reliable source of scientific information and advice for the State of Texas for many decades. He remains actively 
engaged in the interpretation of scientific knowledge for competent management decisions related to oyster and shrimp fisheries in the 
Gulf of Mexico. He has, over the last 10 years, participated in both the Joint Interim Committee on the Texas Shrimp and Oyster Industry 
and the Gulf of Mexico Fishery Management Council. Dr. Ray is a past chair of the Scientific and Technical advisory Committee for 
the Galveston Bay National Estuary Program and is a member of the Board of Trustees of the Galveston Bay Foundation. 



17 



18 



Honored Life Member: Sammy M. Ray 




Perhaps the most rewarding achievement of this exceptional career is the initiation of Sea Camp, "a hands-on marine adventure" for 
summer students aged 10-16. currently sponsored by TAMUG and the Texas Sea Grant College Program. Students attending the 5-day 
camps are given the opportunity to explore the Galveston Island area in research vessels, visit laboratory facilities and use scientific 
equipment to study marine organisms. Dr. Ray served as Director of the Sea Camp until 1993 and. in a similar capacity, is the Director 
of the Community & Youth Program for TAMUG. Dr. Ray and his wife Charlotte, an accomplished pianist now playing organ for the 
St. Luke's Episcopal Church, have four children and reside in Galveston. 



William Fisher 

EPA Laboratory 

GB/ERL 

Sabine Island 

Gulf Breeze. FL 32561 



Journal of Slu'llfish Research. Vol. l^. No. I. l9-:2. 2000. 



HABITAT AND REPRODUCTIVE BIOLOGY OF ANGELWINGS, 
PHOLAS ORIENTAL! S (GMELIN) 



LIBERATO V. LAURETA AND EVELYN T. MARASIGAN 

Institute of Aqiiacultiire 

College of Fisheries 

University of the Phillipines in the Visayas 

Miagao, lloilo, Philippines 5023 

ABSTRACT The anaelwina. Pholas orientaUs (Gmelin) is indigenous to the coastal waters of the Provinces of Negros Occidental. 
Caniz and Iloilo in Central Philippines. Thev burrow into either muddy sand substratum in the littoral zone or compact bluish-gray 
muddv sand in the sub-littoral zone. They burrow to a depth of over 0.3 m and once extracted can never return. Specimens studied were 
invanablv dioecious without apparent sexual dimorphism. Sexual mawrity is reached at a shell length of 59 mm and 64 mm for males 
and females respectivelv. Each sexuallv mature individual possesses a gonad that is imbedded in the ventral side of the viscera. Both 
male and female gonads are arborescent in form and have the same coloration. Samples collected from Barotac Nuevo, Iloilo showed 
that the peak of spawning occurred from June through October and gametogenesis started in October. 

KEY WORDS: Pholas orientaUs. angelwing. reproductive cycle, gonad, spawning 



INTRODUCTION 

The angelwing. Pholas orientaUs Gmelin, is one of the species 
of the family Pholadidae found in the Philippines. The other spe- 
cies are: Barneci dilatata. B. manillensis. and Martesia striata. 
Pholas orientaUs is edible and is marketed either fresh or dried in 
Hongkong (haw chung). Malaysia (sipiit selat batu). Thailand (hoy 
pirn), and Philippines (diwal) (Ablan 1938; Davidson 1976; Saraya 
1982; Young and Sema 1982; Tokrisana et. al. 1985; Amomjaru- 
chit 19881. It has a sweet, juicy and tender taste, making it one of 
the most highly sought bivalves in Central Philippines. However, 
indiscriminate harvesting has resulted in the depletion of most of 
the natural beds. 

To date, the study of Ablan (1938) contains the only available 
information on the ecology and utilization of this species. To re- 
habilitate the depleted P. orientaUs beds, detailed ecological and 
biological information is required. According to Rosell ( 1979). any 
attempt to manage the resource in the absence of baseline infor- 
mation is an exercise in futility. Thus this study was conducted to 
describe habitat and reproductive biology of the species. 

MATERIALS AND METHODS 



Habitat Adaptation 

The study sites were Barotac Nuevo, Iloilo (122°47'N and 
10°55'E) along Guimaras Strait and Roxas City. Capiz ( 122°45'N 
and 1 l''37'E) adjoining Pilar Bay. both in Central Philippines (Fig. 
I). Ecological data from five random stations in each area were 
monitored during the study period. The grain size characteristics of 
the bottom sediments were determined after the procedure de- 
scribed by Buchanan (1971). Water temperature was measured 
using a calibrated laboratory thermometer and salinity was moni- 
tored using a refractometer. The pH of the water was determined 
using a pH meter. Monitoring of the environmental parameters was 
conducted from May 1994 to August 1995 at the Barotac Nuevo 
site and August 1996 to July 1997 in the Roxas City area. 

Determination of Reproductive Biology 

The specimens (n = 6-20) used for the study on reproductive 
biology were collected every month (May 1994 to August 1995) 



from the waters of the Barotac Nuevo site. Specimens were 
brought to the laboratory, where the size lengths were measured 
using a caliper, then shucked, and the gonads dissected. A portion 
of the gonad was examined with a Nikon Optiphot microscope to 
determine the sexes. The stages of maturity and gametogenic 
cycles were determined from histological preparations. Permanent 
mounts of the gonads were prepared following the modified Bell 
and Lightner (1989) method. The description of the gonadal stages 
were made following developmental stages for other clams (Jones 
1981; Nash et al. 1986; Hesselman et al. 1989; Shafee and Daoudi 
1991; Ponurovsky and Yakovlev 1992). 

RESULTS 

Habitat Adaptation 

The characteristics of the two natural beds of Pholas orientalis 
in Central Philippines are shown in Table 1. P. orientalis from 
Barotac Nuevo were found to burrow in compact muddy sand 
(particles < 0.25 mm) covered with a thin layer of silt in littoral 
areas. No specimens were found in the sandy substratum of the 
littoral zone and on the deeper water. Few mangrove trees were 
found in the area, and seagrasses and macrobenthic algae were not 
observed. At Roxas City area, the angelwings occurred in the 
sublittoral areas to a depth of 8 m during the highest high tide, the 
bed being bluish gray compact muddy sand (coarse silt). No P. 
orientalis were found in the sandy mud bottom of the littoral zone. 
The natural bed was wholly devoid of mangrove trees and any 
rooted plants. In both locations, the angelwings burrowed in the 
substrata to a depth of about 0.3 m. On some occasions, burrows 
were found almost adjoining and may have met and crossed one 
another. 

The trends of physico-chemical parameters (temperature, salin- 
ity and pH) in the two study sites during the period of observation 
are shown in Figure 2. The ambient water temperature in Barotac 
Nuevo ranged from 28 °C to 30 X. and did not fluctuate widely. 
The lowest recorded temperature readings were in the months ot 
December through February. At the Roxas City site, wider fluc- 
tuations in water temperature were observed (24 °C to 3 1 °C) with 
December to February being the coldest months. At both sites, the 
salinity readings were between the range of 30-35 ppt. A pH range 



19 



20 



Laureta and Marasigan 





Figure 1. The natural beds { — ) of P. orientalis in Central Philippines. 
(•) Barotac Nuevo study site, and (*1 Roxas City study site. 

from 7.8 to 8.2 was recorded throughout the study period at both 
study areas. 

Reproduction 

Sex 

Out of total 147 sexually matured specimens that were used in 
this study, no hermaphroditic individuals were observed. Angel- 
wings were dioecious without apparent external dimorphism. Once 
sexual maturity was attained, the single gonad was fused or im- 
bedded on the ventral side of the visceral mass, extending from the 
anterior to the posterior part. Ripe male and female gonads had a 
creamy coloration, and were arborescent in form (Fig. 3A). 
whereas spent gonads were yellowish and flaccid (Fig. 3B). The 
epithelial walls of the viscera also reflected an almost creamy 
coloration, causing difficulty in sex differentiation and deterniina- 

TABLE 1. 

Ecological information on the two natural beds of Pholas orientalis 
in Central Philippines. 



Maximum 
Water 
Habilal Depth 

Study Site Type (ml 



Substrate Type Vegetation 



Barotac Nuevo intcrtldal 



>1 



Roxas City 



sublilloriil 



muddy sand 
(panicles 
<0.25 mm) 

compaci bluish 
gray muddy 
sand (particles 
<1.00 mm) 



mansirove 



40 

35- 

30- 

2S- 

20 

15 

10 

5 





Barotac Nuevo 



Roxas City 




-Temp 

-Sal 

-pH 



A flA ft ftAflflfla ' ^ft 



MAMJ J ASONDJ FWIAMJ J AASONDJ FMAMJ J 
1994 1995 1996 1997 

Sampling Period 

Figure 2. Some physico-chemical characteristics of water in the two 
natural beds of P. orientalis in Central Philippines. 

tion of the size of the gonad and gonadal index. Of the same 147 
total gonads that were dissected. 78 (53%) were males, and 69 
(47%) females. 

Sexual Maturity 

The specimens examined in this study ranged from 50-156 mm 
shell length. Most were found to be sexually mature. The mini- 
mum shell lenath of clams containina maturing gametes was 59 




Figure .<. /'. orienlatis with (A) ripe gonad, and (B) spent gonad. 



Habitat and Reproductive Biology of Angelwings 



21 



mm and 64 mm for males and females, respectively. The ages of 
the angelv^ings, howeser were not determined. 

Gonadal Phase and Spawning 

The gonadal state in both sexes was divided uito five phases: 
early active, late active, ripe, partially spent and spent. The per- 
centage occurrence of gonadal stages of male and female P. ori- 
ermilis from Barotac Nuevo is shown in Fig. 4A and B. respec- 
tively. 

Early active stage. Females follicles were empty and lined with 
small developing oocytes and oogonia. In males, few and loosely 
an'anged spermatozoa were found in the center of the lumen of the 
follicle. These conditions occuiTed during the months of October 
to January. During this period, 14% of the male and 33% of the 
female angelwings population were in the early active phase. 

Late active phase. In females, increased numbers of enlarging 
oocytes were freed in the lumen of the follicles. Oocytes were 
irregular in shape and had a wide range of sizes. In males, sper- 
matocytes predominated the basal membrane of the follicle and 
numerous spermatids were found at the center of the follicle lu- 
men. For both male and female gonads, about 17 to 60% were in 
the late active phase during the period December to May. 

Ripe pliase. In the female gonad rounded and ripe oocytes (with 
nucleus and nucleolus) were free in the lutnen. In males, the gonad 
was predominated by mature spermatozoa in the lumen of the 
follicle; the acidophilic sperm tails formed lines radiating from the 
center of the follicle lumen. Specimens with ripe gonads were 



collected during the months of December to July. The percentage 
of ripe females ranged from 14.3 to 66.7, whereas males with ripe 
gonads ranged between 11.1 and 66.7. 

Partially spent. Male gonads had spermatozoa missing in the 
central lumen of the follicle. Female gonads contained fewer ripe 
oocytes and appeared flaccid. Both types of gonad occurred in the 
months of May to October with percentage occurrence at 14.3 to 
57.1. 

Spent. Empty shrunken follicles were characteristic of spent 
gonads. This gonadal phase was observed from the months of June 
to October. By October, most of the angelwings had spawned. 

DISCUSSION 

P. orientalis is a commercially important yet poorly understood 
bivalve species found in Central Philippines. An early survey of 
Ablan (1938) showed that angelwings are indigenous to the coastal 
waters of Hinigaran, Pontevedra. Valladolid, and San Enrique in 
Negros Occidental, Philippines. A more recent survey indicated 
the presence of this species in the coastal waters of Barotac Nuevo 
toward San Dionisio in the Province of Iloilo and in Ivisan, Sapian, 
Panay, Pilar, Pontevedra and Roxas City all in the Province of 
Capiz (Fortes, unpublished). Apart from these areas no other site 
has been identified for the collection of the angelwings in the 
country. All the locations were within 3 to 100 miles at each other. 

In this study, angelwings were found in either compact muddy 
sand or bluish gray muddy sand (with coarse silt) of the littoral or 
sublittoral zones. Ablan (1938) found the angelwings in a muddy 




MAMJJASONDJFMAMJJA 
1994 1995 

Sampling Period 



B 100 

g 80-1 

o 60 ^ 

c 

0) 

g- 40-1 

0) 

i 20- 





IJii 



MAMJJASONDJFMAMJJA 



D Spent 

D Partially Spent 

■ Ripe 

m Late Active 

a Early Active 



D Spent 

D Rartially Spent 

■ Ripe 

II Late Active 

H Early Active 



1994 1995 

Sampling Period 

Figure 4. Reproductive cycle of P. orientalis In Barotac Nuevo, Central Philippines. Relative frequency of gonadal stages of (A) male, and (B) 
female between March 1994 to August 1995. 



22 



Laureta and Marasigan 



coastal land of Negros Occidental. A related species. Cyrtopleura 
costata has been observed to inhabit the sandy mud substratum in 
shallow waters from southern Massachusetts. USA. to Brazil 
(Turner 1954: Abbott 1974; Rios 1973). No clear explanation 
could be offered for the limited distribution of angelwings in the 
Philippines, and their contrasting ecological habitats (i.e.. type of 
bottom sediments, water depth). 

The pholads are capable of burrowing to a depth over 0.3 m 
(Ablan 1938; Allan 1959; this study). They live in the burrows for 
life (Allan 1959). and once extracted from their lodge they are 
unable to return. The burrowing ability is necessary to protect 
themselves from predators and the adverse effects of the physical 
environment as their shells are fragile. For C. costata. they begin 
burrowing after larval settlement, and recorded effective burrow- 
ing size was at a mean shell length of 1 1.7 mm (Gustafson et. al. 
1991). Larger individuals (> 15 mm) of the same species were 
unable to rebury and had to be manually placed beneath the sedi- 
ments during field-planting. However, effective burrowing size for 
the P. orientalis is not known yet. 

Angelwings seem to have an extended annual breeding cycle. 
where initiation of gametogenesis begins almost after spawning. It 
was observed that sizes of specimens had no effect on the timing 
of gametogenesis. Small or large specimens, as long as they are 
sexually mature exhibited almost simultaneous gametogenesis. 
Gametogenesis was observed in the months of October to January. 
The month of October was period when most of the clams were 



partially spent or spent. The peak of spawning occurred in the 
months of June and October, at onset of the rainy season in the 
Philippines. Chanley and Andrews (1971) reported the spawning 
period from May through September for C. costata from Virginia. 
USA. whereas specimens from subtropical Florida were ripe in the 
summer months of June through August. The cyclical reproductive 
pattern observed in P. orientalis. however, cannot be definitely and 
clearly related to temporal changes in temperature and salinity. 
The lack of effect of temperature on the reproductive cycle was 
similarly observed on venerid clams like Megapitaria auranliaca, 
M. squalida. and Dosinia ponderosa from Bahia Zihuatanejo. 
Mexico (Baqueiro and Stuardo 1977 cited by Garcia-Dominguez 
et al. 1998). and the giant reef clam Periglypta multicoslata in Isia 
Espiritu Santo. Baja California Sur. Mexico (Garcia-Dominguez et 
al. 1998). 

ACKNOWLEDGMENTS 

We wish to thank the Fisheries Sector Program of the Depart- 
ment of Agriculture and the Sangguniang Panglungsod and the 
Mayor of the City Government of Roxas City. Philippines for 
funding this work. Special thanks are due also to the lA staff 
particularly to Ms. Janet O. Fernandez. Ms. Jane Apines and Ms. 
Shirley Miagao; and the fisheries staff of LGU-Roxas City, par- 
ticularly Mrs. Belinda Garido. for their technical assistance. We 
sincerely thank Dr. Amulfo Marasigan for improving the manu- 
script. 



LITERATURE CITED 



Abbott. R. T. 1974. American Seashells. 2"" ed. Van Nostrand Reinhold, 
New York. 663pp. 

Ablan, G. L. 1938. The diwal fishery of Occidental Negros. Philipp. ./. Sci. 
66(3):379-385. 

Allan. J. I9.';9. Australian Shells. The Griftln Press. Adelaide, pp. 354-356. 

Amornjaruchit. S. 19S8. Economically important molluscan shellfish of 
Thailand. In: McCoy, E. W. and T. Chongpeepien. (Eds.). Bivalve 
Mollusc Culture Research in Thailand. ICLARM Tech. Rep. 19. De- 
partment of Fisheries. Bangkok, Thailand: International Center for Liv- 
ing Aquatic Resources Management, Manila, Philippines: and Deut- 
sche Gesellschaft fur Technische Zusammenarbeil (GTZ) GmbH, Es- 
chborn. Federal Republic of Germany, pp. 1-8. 

Baquierio. E. and J. Stuardo. 1977. Observaciones sobre la biologia, eco- 
logia y Explotacion de Megiipitaria aiiruiilimu (Sow 1835), M. 
sciuulidci (Sow 1835) Dosiniu ponderosa (Gray 1838) (Bivalvia: Ven- 
eridae) de la Bahia de Zihulanejo e Isia Ixtapa, Gro. Mexico. An. 
Centro Cienc. Del Mar y Limnol. Univ. Nat. Auton. Mexio 4:161-208. 

Bell. T. A. and D. V. Lightner. 1989. A handbook of normal penaeid 
shrimp histology. World Aquaculture Society, pp. 58-63. 

Buchanan. J. B. 1971. Measurements of the physical and chemical envi- 
ronment, pp. 30-58. In: Holme. N. and A. D. Mclnlyre (eds.). Methods 
tor the Sludy of Marine Benthos. IBP Handbook 16. Blackwell Scien- 
tific Publication. 

Chanley, P. E. and J. D. Andrews. 1971. Aids for identification of bivalve 
larvae of Virginia. Malacolngia 1 1:45-1 19. 

Davidson. A. 1976. .Seafood of South-Easl Asia. Federal Publications. 
Singapore. 366 pp. 

Garcia-Doniingue/.. F., B. P. Cehallos-Vasquez. M. Villalejo-Fuerle and 
M. Arellano-Mailine/. 1998. Reproductive cycle ofthe giant reefclam 
Periftlypui iimllicnMiiUi (Sowerby 1835) (Pelecypoda: Veneridael al 
Isia Espiritu Santo. Baja California Sur. Mexico. ./. Shclllixh Res. 17(4): 
1009-1013. 

Gustafson. R. G., R. L. Crewell, T. R. Jacobsen and I). L. Vaughan. 1991. 
Larval biology and mariculiure of the angel wing clam. Cyriopleiini 
coslalii. Ai/uucullure 95:257-279. 

Hesselman, D. M.. B.J. Barber and N.J. Blake. 1989. The reproduclive 



cycle of adult hard clams, Mercenaria spp. in the Indian River lagoon. 
Florida. J. Slwllfish Res. 8( 1 ):43-I9. 

Jones, D. S. 1981. Reproductive cycles of the Atlantic surf clam. Spisiila 
solidissiiiHi. and the ocean quahog Arcliva islnndiea off New Jersey. ./. 
ShellJ'Lsh Res. l(l):23-32. 

Nash, W. J., R. G. Pearson and S. P. Westniore. 1986. A histological study 
of reproduction in the giant clam, Tridacna .^igas in the North-Central 
Great Barrier Reef In: Giant Clams in Asia and the Pacific. Coplana. 
J. W. and J. S. Lucas (eds.) pp. 86-96. 

Ponurovsky. S. K. and Y. M. Yakovlev. 1992. The reproductive biology of 
the Japanese littleneck. Tapes philippinanim (A. Adams and Reeve 
1850) (Bivalvia: Venerididae). J. Shellfish Res. 1 1(2):265-277. 

Rios. E. C. 1975. Brazilian Marine Mollusks Iconography. Fundacao Uni- 
versidade de Rio Grande Centro de Ciencias do mar Museo 
Oceonografico. Rio Grande, Brazil, 331pp. 

Rosell. N. C. 1979. A sludy on the biology and ecology of PUwwia pla- 
centa Linne. Natural and Applied Science Bulletin. 3 1 {3^1:203-25 1. 

Saraya, A. 1982. Thailand. In: Davy. F. B. and M. Graham (eds.). Bivalve 
Culture in Asia and the Pacific, Proc. Workshop held in Singapore. 
16-19 February 1982. Int. Dev. Res. Center, Ollawa, Ontario. Canada, 
pp. 73-78. 

Shafee, M. S. and M. Daoudi. 1991. Gametogenesis and spawning in the 
carpel shell clam. Riidilapes deriissalus (L.) (Mollusca: Bivalvia). from 
the Atlanlic coast of Morocco. .'Kqiutculttire and fisheries Managenieul 
22:20.3-216. 

Tokhsana, R., S. Tugsinavisuili, M. Muangkoe and S. Kao-iun. 1985. 
Marketing system of shellfish products. Asian Fish. Soc. Res. Network 
Thailand and Dept. Agric. Econ., Kasctsarl University. Bangkok. 
264pp. (in Thaii.Turner, R. D. I9.'i4. The family Pholadidae in the 
western Allanlic and ihe easlern Pacific. Part I. Pholadidae. Johnsonia 
3:1-63. 

'loung. A. and E. .Serna. 1982. Philippines. In: Davy, F. B. and M. Graham, 
(eds.). Bivalve Culture in Asia and the Pacific: Proc. Workshop held in 
Singapore. 16-19 Fcbniary 1982. Int. Dev. Res. Center, Ottawa. On- 
tario. Canada, pp. 55-68. 



Joimuil of Shellfish Reseanli. Vol. \9. No. 1. 23-28, 2000. 

INFLUENCE OF DIET ON SURVIVAL, GROWTH, AND PHYSIOLOGICAL CONDITION OF 
FINGERNAIL CLAMS MUSCULIUM TRANSVERSUM 

TERESA J. NAIMO,' W. GREGORY COPE," EMY M. MONROE,' 
JERRY L. FARRIS,^ AND CRISTIN D. MILAM' 

^U.S. Geological Siin-ey. Upper Midwest Environmental Sciences Center, 

2630 Fanta Reed Road, 

La Crosse, Wisconsin 54603 
'North Carolina State Uriiversity, 

Department of Toxicology, Box 7633, 

Raleigh, North Carolina 27695 
^Arkansas State University, 

Department of Biology, 

P.O. Box 599, 

State Universir\; Arkansas 72467 

ABSTRACT The effects of diet and lahoratory holding time on survival, growth, and physiological condition of fingernail clams 
Musculiwn transversum were evaluated in a 1 12-day study. The diets included a commercial oyster diet, a suspension of commercial 
rabbit pellets, a suspension of fine, organic-rich sediment, and a complete sediment renewal every 14 days. Sediment and clams were 
obtained from a relatively uncontaminated site in the Upper Mississippi River. The e.\perimental design consisted of 18 370-mL 
beakers per diet, each containing 5 cm of surficial sediment and 15 clams. Survival of clams was measured daily in each unit. Three 
units from each diet were randomly removed on days 7, 14, 21. 28, 56, and 112. and clams were measured for shell length. Glycogen 
and cellulase activity were measured in composite samples (5 clams per sample) at each of the six time intervals. Cellulase activity 
did not vary among diets or with time. Survival, growth, and glycogen varied significantly among diets, and glycogen concentrations 
varied with time, regardless of diet. Clams exposed to the two sediment diets were 2.4 times more likely to survive than clams exposed 
to the commercial diets. Survival of clams in all diets exceeded 80% through day 2 1 . Although clams maintained an acceptable survival 
rate for 21 days, their physiological condition was compromised much earlier, given that glycogen reserves were reduced by 14-54% 
after only 7 days. Thus, laboratory tests with fingernail clams should include physiological measures, in addition to survival, to ensure 
that clams are in suitable condition before and during testing. 

KEY WORDS: Diet. Muscidium transversum. survival, growth, biomarker 



INTRODUCTION 

Fingernail clams are an important component in the benthic 
invertebrate community of many large rivers and. in the Upper 
Mississippi River, have undergone periodic, pronounced declines 
in abundance in recent decades (Wilson et al. 1995). For example, 
densities in Pool 19 (near Keokuk, lA) averaged 32,000/nr in 
1985 and progressively declined to in 1990, and river- wide re- 
covery has been slow. Toxicity of bulk sediment or pore water has 
been suggested as a factor contributing to the decline in fingernail 
clams in the river (Wilson et al. 1995). In particular, concentrations 
of un-ionized ammonia in sediment pore water from the Upper 
Mississippi River often exceed concentrations demonstrated to in- 
hibit growth of tlngemail clams in laboratory studies (Frazieret al. 
1996). To assess these and other potential causes of the decline in 
abundance requires that clams be collected from the field, held in 
the laboratory, and tested through controlled experimentation. 
However, information on the relative condition of clams during 
long-term holding and its effect on the outcome of laboratory tests 
is lacking (Naimo et al. 2000). 

The physiological condition of an organism is dependent upon 
its nutritional status (Lanno et al. 1989, Foster etal. 1993). Yet. the 
importance of nutrition as a factor modifying physiological con- 
dition has been largely overlooked. Data on how the condition of 
an organism responds to its nutritional status are critical for un- 
derstanding the importance of diet as a variable in designing ex- 
perimental studies with benthic organisms. 



Recently, physiological indicators of condition such as glyco- 
gen concentration and cellulase activity have been used to assess 
the relative health of bivalve mollusks (Hemelraad et al. 1990. 
Haag et al. 1993, Farris et al. 1994, Naimo et al. 1998). Glycogen 
is the most readily available storage form of glucose in many 
animals, including freshwater mussels. As such, glycogen concen- 
trations have been used successfully as an indicator of physiologi- 
cal condition in unionid mussels after exposure to contaminants 
(Hemelraad et al. 1990) and after infestation by zebra mussels 
(Haag et al. 1993). Similarly, cellulase activity is an indirect mea- 
sure of feeding because it measures the rate of breakdown of 
complex sugars into simple molecules (Farris et al. 1988). Exten- 
sive use of cellulase activity in monitoring programs for molluscs 
has shown that responses at the biochemical level can be measured 
where pollutants or stress first exert their effect (Beeby 1993. 
Milam and Farris 1998). In these studies, the predictive capability 
of the enzyme assay has been compared with extensive testing of 
more traditional biological endpoints in toxicity assessments. Con- 
trolled laboratory and field exposures have provided evidence that 
reductions in enzyme activity are related to the eventual survival of 
the animal and to more subtle changes that occur in filtration, 
growth, and bioaccumulation rates (Farris et al. 1994, Milam and 
Farris 1998). 

We examined survival, growth, and physiological condition in 
clams provided different food sources in a 112-day laboratory 
study. Our specific objective was to evaluate the effect of diet on 
the survival, growth, and physiological condition of fingernail 



23 



24 



Naimo et al. 



clams MuscLilium transversum (Say 1829). Furthermore, because 
we were interested in the transferability of these data to standard- 
ized tests with benthic invertebrates, we examined differences in 
survival, growth, and physiological condition between clams fed 
two commercially available diets (easily reproducible, but a non- 
indigenous diet) and two diets containing sediment (not as repro- 
ducible, but more indigenous). 

MATERIALS AND METHODS 

Experimental Design 

We obtained about 600 fingernail clams with a Ponar dredge 
from Pool 13 of the Upper Mississippi River for use in the labo- 
ratory test. During collection, clams were placed in ice chests 
containing sediment and water from the river. The water in the ice 
chests was aerated and its dissolved oxygen content was measured 
at .30-min intervals to maintain concentrations above 60% of satu- 
ration. To obtain an estimate of the physiological condition of 
clams at this point in time, we obtained an additional 15 clams, 
placed them on dry ice in the field, and stored them at -84 °C in 
the laboratory before analysis of glycogen concentration and cel- 
lulase activity. 

The uppermost 5 cm of sediment from a single sampling site in 
Pool 7 of the Upper Mississippi River (Lake Onalaska, river mile 
704.5) that contained an abundant fingernail clam population was 
obtained with a van Veen dredge. Sediment was placed into 4-L 
glass jars, held on ice. transported to the laboratory, and stored in 
a refrigerator for no more than 5 days before the start of the test. 
Three subsamples of homogenized sediment (each 20-25 g wet 
weight) were analyzed to describe textural composition (Guy 
1969. Plumb 1981) and volatile matter content (American Public 
Health Association et al. 1992). Sediments averaged (mean ± 1 
standard error [SE]) 4 ± 0.2% sand. 54 ± 2.4% silt. 42 ± 1.8% clay. 
and 7.8 ± 0.9% volatile matter. 

The experimental unit was a .^70-mL beaker. All experimental 
units were placed into one of two 900-L water baths (3 m length 
X 0.8 m width x 0.4 m height). Each water bath was partitioned 
lengthwise with Plexiglas to provide four compartments, one for 
each diet. Eighteen experimental units were randomly allocated 
into each compartment. A temperature of 17 ± 2 °C was main- 
tained with submersible quartz healers. About 24 h before the 
addition of clams. 1 84-1 88 g of surficial sediment (about 4-5 cm) 
and 200 niL of well water from the Upper Midwest Environmental 
Sciences Center were added to each experimental unit. On day 0. 
we randomly allocated 15 clams, each measuring 4-6 mm in shell 
length, into each experimental unit. 

We measured the temperature. pH. and dissolved oxygen of the 
overlying water every Monday. Wednesday, and Friday in five 
randomly selected experimental units in each diet. Because finger- 
nail clams are particularly sensitive to un-ioniz,ed ammonia 
(Hickey and Vickers 1994). we measured concentrations of total 
and un-ionized ammonia in three randomly selected experimental 
units every 14 days (Fra/ier et al. 1996). On days 7. 14. 2 1 . 28. 56. 
and 112. clams from three randomly selected experimental units 
from each diet were sieved from test sediments, counted, recorded 
as dead or alive, measured for shell length to the nearest 0.1 mm, 
and stored at -84°C for later analysis of glycogen concentrations 
and ccllulase activity. Glycogen concentrations (Naimo et al. 
IWS) and ccllulase activity (Farris et al. 1988) were measured on 
composite samples containing five individuals from each experi- 
mental unit. Glycogen concentrations were reported as mg/g wet 



weight, and cellulase activity was expressed as a product (exocel- 
lulase activity times endocellulase activity in [units/g dry 
weight]"). One unit of the enzyme is defined as the amount of 
enzyme required to liberate 1 mg of reducing sugar equivalent to 
that of glucose per hour with carboxymethylcellulose as a sub- 
strate. 

Diet and Ration 

Clams were fed one of four diets daily; two were commercially 
available diets, and two were formulated with sediments from the 
Upper Mississippi River (sediment diets). The commercial diets 
included an oyster diet, which was a mixture of two marine dia- 
toms (50% Thatassiosira pseudoana and 50% Skeletoneina sp.) 
fed at a rate of about 7(jLL/clam/day (8-10 x 10'^ cells/mL; Pacific 
Oyster Diet B. Coast Seafood Company. Quilcene. WA). The sec; 
ond commercial diet was a suspension of Kaytee * rabbit feed, with 
pellets made largely from alfalfa, fed at a rate of 2.5 mg/clam/day. 
The two sediment diets contained organic-rich sediments from 
relatively uncontaminated areas in the river and were the same 
sediment used as the substrate in all experimental units. One was 
a suspension of fine sediment fed at a rate of 2.5 mg/clam/day. and 
the other was a complete sediment renewal every 14 days. 

The oyster diet, rabbit pellet diet, and suspended sediment diet 
were prepared about 2 days before the start of the experiment. The 
oyster diet comes in liquid form and was kept refrigerated. The 
rabbit pellet and the suspended sediment diets were prepared by 
blending 38 g of rabbit pellets or sediment with 400 mL of well 
water in a commercial blender for 5 min. The contents of the 
blender were transferred into a l.OOO-niL volumetric tlask and 
filled to the meniscus with well water. This process was repeated 
until we obtained 32 140-mL bottles of each diet. Once a week, 
one bottle of food for each diet was removed from a -20°C freezer 
and placed into a refrigerator; the quantity of food in each bottle 
was sufficient to feed all clams receiving those diets for 1 wk. 
Clams in the sediment-renewal diet were sieved from test sedi- 
ments every 14 days, and another aliquot of sediment was replaced 
into each experimental unit. Sediments for this diet were the same 
sediments that were obtained at the start of the test, stored in a 
refrigerator until needed. 

Statistical Analyses 

Survival of clams was assessed by daily counts of dead shells 
on the sediment surface. In addition, at the six time intervals in 
which clams from three beakers were removed for physiological 
measurements, we also made direct mortality estimates; these data 
allowed us to check the accuracy of the daily mortality counts. 
Because these two estimates agreed more than 90% of the time, 
analyses of survival rate were performed on daily survival counts. 
We used the Cox proportional hazards model to determine whether 
survival rates of clams varied among diets (Cox 1972). To test for 
differences in survival between the commercial and sediment diets. 
we used the Wald lest of equality (Parniar and Machin 1945). 

We analyzed growth, glycogen concentrations, and cellulase 
activity with analysis of covariance (ANCOVA). with time in the 
laboratory as the covariale. Because most clams did not survive 
after day 56. statistical analyses were only conducted until day 56. 
Orlliogonal contrasts were used to compare differences in growth 
and physiological condition between the corumerclal and sediment 
diets when the ANCOVA was significant. We did not record the 
shell leuL'th i.-\\' each clam on dav 0; instead, we ensured that all 



Influence of Diet on Musculium 



25 



clams ranged from 4 to 6 mm in length. Because shell length did 
not differ among diets at day 1 (P = 0.21). subsequent analyses 
were performed on shell length measures from day 7 through day 
56. A type I error a of 0.03 was used to reject all null hypotheses. 

RESULTS 

The quality of the overlying test water was similar among diets 
(Fig. 1 ). For example, grand means (averaged over all diets and 
time periods) ranged from 15.4°C to 15.7°C for temperature, 8.2 to 
8.3 for pH, and 9.7 to 9.8 mg/L for dissolved oxygen. Concentra- 
tions of total (range, 0.03-0.13 mg/L) and un-ionized (0.002-0.008 
mg/L) ammonia were well below concentrations that adversely 
affect fingernail clams in laboratory exposures (Sparks and 
Sandusky 1981). 

The survival rate of fingernail clams varied significantly among 
diets (P = 0.0001). Survival rates were lowest in clams fed the 
oyster diet, whereas survival was highest in clams receiving the 
sediment-renewal treatment (Fig. 2). For example, survival aver- 
aged 44% in the oyster diet, 66% in the rabbit-pellet diet, 73% in 
the suspended-sediment diet, and 84% in the sediment-renewal 
diet at day 56. By day 112, only 6% of the clams in the sediment- 
renewal treatment were alive, and none survived in the other three 
dietary treatments. 

Survival was significantly greater in clams provided the sedi- 
ment diets, relative to the commercial diets (P = 0.0001 ). After 56 
days in the laboratory, for example, survival of clams fed the 
sediment diets averaged 79%, whereas survival averaged 55% in 



100 ■ 






18 1 
17 ■ 


M 










Temperature 


16 ■ 

15 - 


% 


^ 

d 


^ 


^ 


^ 


^ 




^ 



X 

o 



Z 9 



— o— oyster diet 
— •— rabbit pellets 
— o— suspended sediment 
— • — sediment renewal 



Dissolved oxygen 




20 40 60 

Day of experiment 



80 



Figure 1. Mean temperature, pH, and dissolved oxygen in overlying 
test water from five randomly selected experimental units containing 
flngernail clams Musculium transversum fed one of four diets daily for 
112 days. 



> 



c 
u 




Day of experiment 

Figure 2. Survival of fingernail clams Musculium transversum fed one 
of four diets in a 112-day laboratory test. 



clams fed the commercial diets. However, there was little differ- 
ence in survival of clams among diets early in the test: survival of 
clams in all diets exceeded 80% through 21 days of exposure. A 
unique feature of the proportional hazards model is the ability to 
calculate a risk ratio, or the estimated hazard of surviving in one 
diet versus another. For example, clams provided the oyster diet 
were 1.9 times more likely to die than clams fed rabbit pellets 
(Table 1). Additionally, clams fed the oyster diet were almost 5 
times more likely to die than clams in the sediment-renewal treat- 
ment. Furthermore, clams fed the commercial diets were 2.4 times 
more likely to die than clams fed the two sediment diets. 

The shell length of fingernail clams also varied significantly 
among diets (P = 0.02). Clams receiving the sediment-renewal 
treatment were significantly larger than clams in the other three 
dietary treatments. For example, clams in the sediment-renewal 

TABLE 1. 

Estimated probability values, risk ratios, and upper and lower 95% 

confidence limits from the survival rate analysis in fingernail clams 

fed four different diets in a 112-day laboratory experiment. 









Lower 95% 


Upper 95% 




P 


Risk 


Confidence 


Confidence 


Contrast 


Value 


Ratio 


Limit 


Limit 


Oyster diet. 










suspended sediment 


0.0001 


2.6 


1.9 


3.5 


Rabbit pellets. 










suspended sediment 


0. 1 1 24 


1.3 


0.9 


1.9 


Sediment renewal. 










suspended sediment 


0.0120 


0.5 


0.3 


0.9 


Oyster diet, rabbit 










pellets 


0.0001 


1.9 


1.9 


2.0 


Oyster diet, sediment 










renewal 


0.0001 


4.8 


4.0 


5.8 


Rabbit pellets. 










sediment renewal 


0.0002 


2.4 


2.2 


2.8 


Commercial diets. 










sediment diets 


0.0001 


2.4 


1.9 


3.2 



The risk ratio is the estimated hazard of surviving in one diet versus 
another diet; for example, clams fed the oyster diet were 2.6 times more 
likely to die than clams fed the suspended-sediment diet. 



26 



Naimo et al. 



treatment averaged 4.8 mm in length over the 56-day duration, 
whereas clams in the other three dietary treatments ranged from 
4.3 to 4.4 mm. Furthermore, the size of clams did not differ be- 
tween clams provided the commercial and sediment diets (P = 
0.50), nor did shell length vary with time in the laboratory (P ~ 
0.23; Fig. 3a). At day 7, clams ranged in length from 4.2 to 4.8 mm 
and at day 56, they ranged in length from 4.5 to 4.8 mm. 

Glycogen concentrations in clams varied significantly among 
diets (/* = 0.049: Fig. 3b). In particular, glycogen concentrations 
differed between the commercial and sediment diets (P = 0.02). 
For example, mean glycogen concentration was 3.5 mg/g in clams 
fed the oyster diet and 4.1 mg/g in clams fed the rabbit pellets. In 
contrast, glycogen concentrations averaged 2.8 mg/g in the sus- 
pended-sediment diet and 3.0 mg/g in the sediment-renewal treat- 
ment. However, glycogen concentrations declined significantly 
with time in the laboratory, regardless of diet {P = 0.0001). For 
example, glycogen concentrations in clams in the sediment- 
renewal treatment averaged 4.6 mg/g at day 7 and had declined to 
only 2.2 mg/g by day 56. Moreover, because there was no 
diet*time interaction (P = 0.49), the response of glycogen with 
time was similar among diets. For reference, glycogen concentra- 
tions averaged 5.4 ± 0.5 (SE) mg/g in clams when they were 
removed from the Mississippi River. 



t 




•a 

00 * 

8 I 



b 


b^/^ 


— Q-- oyster diet 
— •— rabbit pellets 
— o— ■ suspended sediment 
^-^ — •— sediment renewal 


y/^ 


F^ 


r^^ 


— -~^ ^^^~~~~~~---^ 


Y 


1^^-===^==^^ 





O IT 

p u 



o a. 



16 ' 


c 




12 • 








f^,^--^^! 


8 ■ 




J 


\J 






1 \1T 




4 ' 


\ 


/^ \ir'>» 


___^ I 




f 




^.^'^ 





Day of experiment 

Figure 3. Mean (a) srowlh, (b) glycogen concenl rations, and (c) eei- 
lulase activity in Ungernail clani.s Miisciiliiim Iransvcrsiiin fed one of 
four diets in a 1 l2-da\ lahoratorv lest. (Ilycogen (mg/g «et "eight) and 
cellulase activity (junits/g dry « eight j') were measured on a composite 
of five clams from each of lliree experimental units sampled on days 7, 
14, 21, 2S, and 56. Data point al day is the mean (±1 .SE) glycogen and 
cellulase in clams at the time they were collected from the Upper 
Mississippi River. 



In contrast, cellulase activity did not vary among diets (P = 
0.12) nor with time held in the laboratory (P = 0.32; Fig. 3c). 
Cellulase activity, averaged over the 56-day exposure, ranged from 
0.8 to 5.3 (units/g dry weight") in the oyster diet, 0.8 to 4.8 in the 
rabbit pellets, 1.1 to 14.7 in the suspended sediment, and 0.6 to 
19.8 in the sediment renewal. Likewise, cellulase activity remained 
similar throughout exposure (averaged over all diets) and ranged 
from 1.6 to 10.5 at day 7 and from 1.8 to 14.3 at day 56. The lack 
of significant diet or time effects was presumably due to the large 
variance in cellulase activity among replicates. The coefficient of 
variation (CV) usually averaged well over 50%, likely obscuring 
any diet or time effects. For reference, cellulase activity averaged 
7.3 ± 1.6 (SE) in clams when collected from the Mississippi River. 

DISCUSSION 

Survival of fingernail clams was greater in treatments contain- 
ing sediment from the Upper Mississippi River than in treatments 
with commercial diets. A similar observation was made by 
Gatenby et al. (1996) with juvenile Villosa iris. In a 45-day labo- 
ratory experiment, juvenile mussels reared on sediment and algae 
had significantly higher survival {6T7r) than juveniles reared with- 
out sediment and fed only algae (227^). Although several investi- 
gators have observed higher survival rates in molluscs in experi- 
ments with sediment, relative to no sediment (Gatenby et al. 1996, 
Naimo et al. 2000, present study), the mechanism(s) contributing 
to this are largely unknown. It has been hypothesized that the 
addition of a food source, along with fine sediments and their 
associated resident bacteria, may enhance digestion in molluscs 
(Crosby et al. 1990, Naimo et al. 2000). However, the addition of 
bacteria common to riverine systems did not improve survival or 
enhance growth in laboratory studies with juvenile Villosa iris 
(Gatenby et al. 1996). Naimo et al. (2000) hypothesized that physi- 
cal contact with sediment may enhance the survival of fingernail 
clams relative to exposures without direct sediment contact. They 
observed that Miisciiliitm transversiiiii were twice as likely to sur- 
vive when provided with direct sediment contact, suggesting that 
clams received nutritional benefit from sediment contact by feed- 
ing directly on indigenous, sediment-associated food sources. 

Although survival of fingernail clams differed substantially 
among diets after 1 12 days, survival exceeded SO'/r through day 21 
in all diets. In standardized toxicity tests with bcnthic inverte- 
brates, 21-28 days is a standard test duration (American Society 
for Testing and Materials 1992), and tests are generally considered 
unacceptable if survival of control animals is less than 80%. Thus, 
in short-term standardized tests with fingernail clams, excessive 
mortality in control organisms would not invalidate test results. 

Growth of fingernail clams in the laboratory was minimal over 
the 56-day duration. Clams in the sediment renewal treatment 
seemed to maintain their size, whereas shell growth in clams in the 
other diets was variable. Differences in shell growth in the sedi- 
ment-renewal treatment, relative to the other diets, may be related 
to the volume of available food (i.e., sediment). Clams in the 
sediment renewal treatment received about 736 g of sediment over 
56 days, whereas clams in the suspended-sediment and labbit- 
pcllel treatments received only 2.1 g of food over this duration. 
Although food quality as well as quantity are important, the mag- 
nitude of the difference in quantity may have contributed to dil- 
Icrcnces in growth among diets. In addition, the magnitude of shell 
growth observed in our study (0.1-0.6 mm over 56 days) was 
sufficicntiv small such that variation in measureinent of shell 



Influence of Diet on Muscuuum 



27 



length could be a major source of variation and uncertainty in tinis 
analysis. Thus, future studies should measure individually marked 
organisms and should use techniques appropriate for detecting 
small changes in size. The lack of shell growth in this experiment 
was not unexpected. For example. Gale (1977) observed that 
Sphaeriiim tmnsversiiiii maintained in the laboratory in chambers 
containing silt from the Mississippi River grew slowly, with a 
mean length increase of 1.3 mm after 33 days. 

Glycogen concentrations have been used extensively in bi- 
valves as an indicator of physiological health (Haag et al. 1993, 
Naimo et al. 1998); however, it is unclear how much glycogen is 
required for maintenance, growth, and reproduction. In the present 
experiment, we documented significant differences in glycogen 
concentrations among diets, particularly between the commercial 
diets and the sediment diets. However, the pattern in glycogen 
concentrations was such that glycogen was elevated in clams fed 
the commercial diets, relative to the sediment diets, in contrast to 
the patterns in survival. Two alternate hypotheses for the reduction 
in glycogen in the sediment diets include ( 1 ) clams were getting 
enough nourishment from the sediment for maintenance metabo- 
lism but were unable to store glycogen and (2) clams were not 
getting enough nourishment from the sediment and were catabo- 
lizing carbohydrate stores. Whichever the case, glycogen concen- 
trations declined with time in all dietary treatments, suggesting that 
health was declining over this time period. Glycogen concentra- 
tions declined by 14-54% by day 7 and 50-70% by day 56. rela- 
tive to concentrations in clams when they were taken from the 
river. 

Some researchers have suggested that the benefit of addition of 
sediment to juvenile bivalve cultures is to provide resident bacteria 



to enhance enzymatic activity (Crosby et al. 1990). However, we 
did not observe any enhancement in cellulase activity between 
clams maintained in sediment and clams fed commercial diets. 
Cellulase activity in clams was highly variable (mean CV = 67%), 
making detection of dietary effects at an acceptable statistical level 
difficult. To our knowledge, measurement of cellulase activity has 
not been previously performed on fingernail clams; thus, further 
refinement of methods could reduce variation associated with this 
measure. 

In conclusion, we observed significant differences in survival, 
shell growth, and glycogen concentrations of fingernail clams fed 
different diets, implying that some diets were better than others. 
However, the general negative slope of most response variables 
(survival, shell growth, and glycogen) suggests that clams were 
declining in health with time in the laboratory, regardless of diet. 
Therefore, a better diet is needed to maintain clams in a healthy 
state in the laboratory. Although clams maintained an acceptable 
survival rate for 21 days in the laboratory, their physiological 
condition was compromised much earlier. Thus, valid short-term 
toxicity tests with fingernail clams can be conducted in the labo- 
ratory, but their ability to predict toxicity to field populations is 
uncertain. Therefore, laboratory tests with clams should include a 
physiological measure, such as glycogen, in addition to survival to 
ensure that clams are in suitable condition before and during test- 
ing in laboratory studies. 

ACKNOWLEDGMENTS 

Technical assistance in the field and laboratory was provided 
by Michelle Bartsch and Peter Rust. Steve Gutreuter provided 
statistical guidance. 



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Joiinuil of Shellfish Research. Vol. 19. No. 1, 29-34. 2000. 

LOSS OF GENETIC VARIATION IN A STRONGLY ISOLATED AZOREAN POPULATION OF 

THE EDIBLE CLAM, TAPES DECUSSATUS 

KURT JORDAENS,'* HANS DE WOLF,' TANIA WILLEMS,' 

STEFAN VAN DONGEN,^ CARLOS BRITO,' 

ANTONIO M. FRIAS MARTINS,' AND THIERRY BACKELJAU' ^ 

' Department of Biology 

University of Antwerp (RUCA) 

Groenenborgerlaan 171 

B-2020 Antwerp. Belgium 
'Department of Biology 

University of Antwerp (UIA) 

Universiteitsplein I 

B-2610 Wilrijk. Belgium 
' Department of Biology 

University of the Azores 

Rua da Mae de Dens 58 

Apartado 1422 

P-9502 Ponta Delgada 

Azores, Portugal 
^Royal Belgian Institute of Natural Sciences 

Vautierstraat 29 

B-1000 Brussels, Belgium 

ABSTRACT We used allozyme electrophoresis to compare the genetic variation of an introduced and strongly isolated population 
of the edible clam Tapes decussariis in the Azores (Lagoa de Santo Cristo. Sao Jorge) with populations from the main range of the 
species (Ria and Thau). Observed and expected heterozygosity values, number of polymorphic loci, and mean number of alleles per 
locus in the main-range populations fall within the limits reported for T. decussanis and other Venerid clams. In contrast to previous 
studies on Venerid clams, we observed no heterozygote deficiencies. In the introduced Lagoa population, we observed a strong 
reduction of allelic diversity and expected heterozygosities and an effective population size of only 5.30. The Lagoa population is only 
slightly differentiated from populations from the species' main range and may thus be of low "biological value." Exploitation of T. 
decussaius could therefore be allowed to continue but must follow strict collection guidelines, especially given that only 15% of the 
area is suitable for exploitation. Otherwise, a unique component of the Azorean fauna that also serves as a fishery resource may be lost. 

KEY WORDS: Azores, allozymes, founder effect. Tapes decussaius. population genetics, conservation 



INTRODUCTION structure before adequate measures can be taken. In this study, we 

estimated the effective population size and investigated whether 

Small or isolated populations can contribute substantially to genetic variation is reduced in an introduced, isolated population 

biodiversity, and the conservation of such populations must be an of the commercial edible clam Tapes decussaius (Linnaeus. 1 758). 

important part of any effective Biodiversity Action Plan (Usher The main range of T. deai.ssaws extends from Great Britain in 

1997). The genetic effects in small populations are manifold (Har- 'he north to Senegal in the south, along the Iberian peninsula, and 

ris 1984. Usher 1987). Small effective population sizes (A',) often '"'0 the Mediterranean to the east (Tebble 1966). Outside its main 

show a loss ofgenetic variability (i.e.. founder effects, bottlenecks) ^nge. the species has been introduced in the Lagoa de Santo 

caused by genetic drift (Lacy 1987). Apart from losing (rare) al- Cristo. a small and isolated lagoon situated at the north coast of the 

leles, small populations often lose common alleles by chance (Nei '^'and of Sao Jorge in the Azores, approximately 1,400 km from 

et al. 1975. Simberloff 1988) and may show elevated inbreeding, 'he African/European coasts, where it was discovered for the first 

which mav impair reproductive fitness. In addition, the loss of "me in 1967 (Morton 1967). This lagoon has a total area of 0.86 

genetic variability may limit the ability of a population to adapt to km- (length, 500 m; width, 250 m: and maximum depth, 6 m) and 

changing environments (Frankel and Soule 1981, Thorpe et al. harbors a unique fauna (Morton 1967. Santos 1985. Santos and 

1995). Over the long term, these effects may enhance the risk of Martins 1986, Morton and Tristao da Cunha 1993, Morton et al. 

extinction (Soule 1987). Effective conservation or management '^98). The lagoon was classified as a Natural Partial Reserve in 

plans require a thorough knowledge of the genetic population 1984 on the basis of its unique origin, geology, and the presence of 

the edible clam T. decussanis. In 1989. it was also declared a 

Special Ecological Area, to safeguard the unique breeding popu- 

*Corresponding author. lation of T. decussatus and to maintain the ecological equilibrium 



29 



30 



JORDAENS ET AL. 



of the area. Although there is no written record. T. deciissatus was 
probably introduced in the lagoon by humans, especially since the 
species occurs nowhere else in the Azores (Morton 1967, Morton 
and Tristao da Cunha 1993). Moreover, the planktonic stage of the 
larvae lasts approximately 10 days, during which larvae are trans- 
ported by sea currents over a distance of 10-100 km (Borsa et al. 
1991). Larval transport from the main range to the Azores by sea 
currents seems therefore unlikely. 

At this moment, T. detussauts is the main commercially ex- 
ploited species of the lagoon (Fonseca et al. 1995). Santos and 
Martins (1987), Santos et al. (1989), and Gon(;alves and Martins 
(1991) showed that the population of T. deciissatus in the Lagoa de 
Santo Cristo was declining through overexploitation, especially in 
the intertidal parts of the lagoon, where clam collection is easy. 
The intense fishery resulted in smaller individuals in the intertidal 
area. These potential detrimental impacts on the clams and other 
species of the lagoon have obliged the Azorean government to 
establish a management program for the Lagoa de Santo Cristo. 
Therefore, the clam fishery at the lagoon is nowadays closed dur- 
ing a period that largely coincides with the breeding season of the 
species (May 15 to August 15: Santos and Martins 1987. 1991). 
The present research was performed to provide genetic data that 
may be relevant for further substantial management of the clam 
population. 

MATERIALS AND METHODS 

Four samples of T. decussatus were collected from three sites: 
Lagoa de Santo Cristo (SC: July 1992 and June 1993). Etang de 
Thau (Thau: French Mediterranean coast: August 1993). and Playa 
do Testal (Ria: Ria de Muros y Noya. Galicia, Spain: December 
1993). Specimens were immediately frozen in liquid nitrogen for 
transport to the laboratory, where they were stored at -80 °C. 

Forty specimens of each sample were surveyed for allozyme 
variation with vertical polyacrylamide gel electrophoresis (PAGE). 
Individual tissue homogenates were prepared by dissecting speci- 
mens in ice-cold distilled water and removing the digestive gland, 
the gills, the foot muscle, and the adductor muscles. Each of the 
tissues was separately weighted and homogenized in a 20% (w/v) 
aqueous sucrose solution (5 |j.L sucrose solution per mg tissue). 
Crude homogenates were centrifuged for 45 min at ±27,000 g at 
5 °C to obtain clear supernatants for electrophoresis. 

PAGE was performed as described by Backeljau (1987. 1989). 
Two electrophoretic buffer systems were used: ( I ) Tris/glycine pH 
9.0 in the tray and Tris/HCl pH 9.0 in the gels and (2) Tris/citric 



acid pH 8.0 in both the tray and the gels. Enzyme staining recipes 
were adapted from Harris and Hopkinson (1976). 

Twenty-six enzyme systems were screened in the four tissues 
(see Backeljau et al. 1994). Seven of these enzymes yielded inter- 
pretable genetic polymorphisms and were retained for further 
analysis (Table 1 ). 

Alleles were designated alphabetically according to decreasing 
electrophoretic mobilities (A = most anodal = fastest-migrating 
allele). Previously typed specimens were included with each run to 
compare different gels. The BIOSYS-1 version 1.7 package 
(Swofford and Selander 1981) was used for estimating allele fre- 
quencies, mean numbers of alleles per locus (MNA). observed 
heterozygosities (W^,. direct count) and Nei's ( 1978) unbiased ex- 
pected heterozygosities {Hj. Numbers of polymorphic loci (P) 
were simply counted. Weir and Cockerham's (1984) fixation ia- 
dices (F,J were estimated with GENEPOP version 3.0 (Raymond 
and Rousset 1995). and genotype frequencies were evaluated for 
departures from Hardy-Weinberg (HW) equilibrium expectations 
with the probability test implemented by the same program. The 
significance of F,^ values was tested with FSTAT version 1.2 
(Goudet 1995). Linkage disequilibria (LD) between loci were 
tested with the exact probability test in GENEPOP version 3.0. 
Whenever needed, testing procedures were corrected for multiple 
testing with the sequential Bonferroni method (Rice 1989). Nei's 
(1978) unbiased genetic distance between populations was calcu- 
lated with BlOSYS-1 version 1.7. 

The effective population size (A'^) of the population from the 
Lagoa was estimated in two different ways. One method estimates 
A'^ from the changes in expected heterozygosity. In a population of 
size A'^.. the initial heterozygosity (//„) will decrease to W, after / 
generations. The relationship between W,, and W, is given by the 
equation «, = //„( 1 - 1/2A'^)' (Crow and Kimura 1970). A second 
method (i.e.. the temporal method) estimates A'^. from temporal 
changes of gene frequencies as described by Waples (1989) and 
Hedgecock et al. ( 1992). Although a few T. decussatus individuals 
may spawn in their first year (Vilela 1950). the vast majority of 
individuals reach their sexual maturity at the beginning of their 
second year (Gallois 1977). Therefore, we used a generation time 
of 1 y for T. decussatus. An assumption of both methods is that the 
allozyme polymorphisms studied are selectively neutral. To test 
this, we performed the Ewens-Watterson test using the algorithm 
given in Manly (1985) and implemented by the program 
POPGENE version 1.31 (updated version of POPGENE version 
1.2 of Yeh and Boyle |1997|). 

Because many bivalves show a positive correlation between 



TABLE L 

Enzymes studied, E.C. numbers, en/.vmc codes, the tissue from which the enz>me was extracted, and the buffer system (TC 
acid; T(; = Tris/glycine) used to examine senetic variation in four T. deciissalus populations. 



Tris/citric 



Enzyme 



EC Number 



Code 



Tissue 



BulTer 



M;ilatc dchydri'gcriasc 
D-Odopinc clehydrcigcnase 
IsDcitrale dehydrogenase (NADP*) 
Phosphogluconale dehydrogenase 
.l-Hydro.xyhulyralc dehydrogenase 
Leucylalanine peptidase 
PhosphogJuconiuUise 



1. 1. 1. .^7 

1.1.1,42 

1.1.1.44 

i.l.l..^() 

.V4.LVI1 

.'i.4.2.2 



MJh 
Opdh 

Iclhp 
I'ildl, 
lllolh 
I'cp 



.Adductor muscle 
Adductor muscle 
Digestive gland 
Digestive gland 
Digestive gland 
Gills 
Adductor muscle 



TC 
TC 
TC 
TC 
TG 
TG 
TG 



Genetic Variation in Azorean Tapes decussatus 



31 



shell size and individual heterozygosity (e.g.. Zouros and Foltz 
1984), we checked for such a relationship to avoid the possibility 
that discrepancies in //„ values would merely reflect size differ- 
ences between populations. Therefore. Pearson's product-moment 
correlation was calculated between shell length and numbers of 
heterozygous loci, as outlined by Diehl and Koehn (1985) and 
Fevolden (1992). 

RESULTS 

Pep revealed two independent banding zones, the cathodal of 
which was clearly polymorphic in the Thau and Ria populations. 
but monomorphic in the Lagoa population. Yet, because the bands 
in this zone were often confused, they were not used for genotypic 
analysis. The six remaining enzymes yielded information for seven 
putative loci (Table 1 ). the population genetic data of which are 
provided in Tables 2 and 3. Out of 18 HW tests, only 2 were 
significant (Pgm in Thau and /rf/;/) in Ria; Table 2), but this was no 
longer so after sequential Bonferroni correction. Not surprisingly, 
F,^ values taken over all loci in all populations were not signifi- 
cantly different from (0.193 < P < 0.27). However, compared 
with the Lagoa population, the Thau and Ria populations had 
higher heterozygosity levels and nearly twice as many polymor- 
phic loci and mean numbers of alleles per locus (Table 2). Only 
two of the 31 LD tests were significant (data not shown), but both 
cases were no longer significant after sequential Bonferroni cor- 
rection. Nei's (1978) unbiased genetic distance between the 
samples ranged from 0.036 (between two samples from the 
Azores) to 0.23 (between Thau and two samples from the Azores) 
(Table 3). 

The estimate of N^ with the temporal method was infinity. This 
result is probably an artifact caused by the small number of loci 
analyzed in = 3) (Table 2). It simply indicates that the change in 
allozyme frequencies observed between the 2 years was not large 
enough to be distinguished from sampling error. The estimate of 
N^ obtained from the reduction of heterozygosity was 5.30. The 
test for neutrality gave nonsignificant results. 

We found no significant correlation between individual het- 
erozygosity and shell length (Thau, r = 0.173, P = 0.733; Ria, /■ 
= 0.36, P = 0.556; and Lagoa (pooled samples), /• = 0.48, P = 
0.409). 

DISCUSSION 

Observed and expected heterozygosity values, number of poly- 
morphic loci, and mean number of alleles per locus in the Ria and 
Thau populations fall within the limits reported for T. decussatus 
and the palourde Rudimpes pinlippinarum (Table 4). As in many 
other bivalve species, heterozygote deficiencies have often been 
reported in T. decussatus and R. philippinarum (see references in 
Table 4), but at present the causes of this remain unclear (Zouros 
et al. 1988). Yet, in our study, we observed no heterozygote de- 
ficiencies. Nevertheless, our population genetic data of the Thau 
population are very similar to the results obtained by Jame et al. 
(1988), Borsa and Thiriot-Quievreux (1990), and Borsa et al. 
(1994) for the same population and for the nearby population of 
Etang du Prevost (Worms and Pasteur 1982). Moreover, genetic 
distances between our populations are similar to those reported by 
Jame et al. ( 1988) (compare our Table 3 with their Table 4). 



However, in the Lagoa population of T. decussatus in the 
Azores, we observed a strong reduction of allelic diversity and 
expected heterozygosities, but not heterozygote deficiencies, com- 
pared with main-range populations. Substantial losses of genetic 
diversity have also been observed in bivalves for which hatchery 
stocks have been established from only a few individuals (e.g., the 
oysters Crassostrea gigas [Gosling 1982, Hedgecock and Sly 
1990] and C. virginica [Vrijenhoek et al. 1990, Gaffney et al. 
1992]). This may have important implications when management 
and exploitation practices are developed. Many hatchery stocks or 
introduced populations have a low N^ value despite densities that 
can be very high (e.g., Saavedra 1997 and references therein). In 
the Lagoa, population densities of T. decussatus may reach 400 
individuals/m" (Gonijalves and Martins 1991). Yet we estimated 
an effective population size of only 5.30 individuals. Founder ef- 
fects, genetic drift, intentional selection, and inadvertent selection 
during culture are likely to reduce the genetic diversity of the 
Lagoa population further. The introduction of a small number of 
individuals a few decades ago probably resulted in the loss of 
genetic variation via founder effects. The strong isolation of this 
population probably does not allow transport of larvae from nearby 
populations (see Introduction), and genetic drift and inbreeding 
may further reduce genetic variability. These effects are probably 
reinforced by human activities such as selection during harvesting 
(e.g., the collection of only large adults). Indeed, the exploitation 
of T. decussatus in the Lagoa follows a classic "fishery" picture 
with old (i.e., large) shells lacking among empty shells in the 
lagoon because they were collected for consumption when alive 
(Morton and Tristao da Cunha 1993). It is unclear whether such 
selective harvesting affects the genetic structure of the population, 
because there was no association between individual heterozygos- 
ity and size. Yet this topic deserves further study, as Borsa et al. 
(1994) and Passamonti et al. (1997) found a high level of intra- 
population structuring, probably related to year-cohort heteroge- 
neities, that perhaps indicate short-term selection or genetic drift 
(Borsa et al. 1994). Thus, harvesting a single age cohort (i.e., 
oldest and largest individuals) could affect the genetic population 
structure. 

In none of the populations did we observe a significant corre- 
lation between shell size and individual heterozygosity. Some 
other studies also failed to show a relationship between individual 
heterozygosity and morphological traits such as size and growth 
(Adamkewicz et al. 1984, Volckaert and Zouros 1989, Gaffney 
1990. Slattery et al. 1991), but others report negative (Wilkins 
1978) or positive (Garton et al. 1984, Koehn and Gaffney 1984, 
Zouros and Foltz 1984, Gaffney 1990) associations, although as- 
sociations may differ among populations (Gaffney 1990). 

A positive relation between heterozygosity, body size, and sur- 
vival was found in a population of T. decussatus that survived 
natural anoxic stress (Borsa et al. 1992). However, in other popu- 
lations of the same species, Jame et al. ( 1988) observed no asso- 
ciation between asymmetry of left and right valves (as a measure 
of fitness, i.e.. the more asymmetric the less fit) and heterozygos- 
ity, and an increased variance for morphological traits in the 
classes with low heterozygosity. This also appears to be the case 
for some of the R. philippinariini populations in the Po river lagoon 
in Italy (Fava et al. 1994). In that study, individual heterozygosity 
and phenotypic variability appeared to be negatively correlated, 
but the relationship was heterogeneous between populations (Fava 
et al. 1994). 



32 



JORDAENS ET AL. 



TABLE 2. 

Allozyme variation in four populations of T. decussatus (for full 
population names we refer to the text). 

Thau (n = 40) Ria (n = 40) SC92 (« = 40) SC93 (n = 40) 



TABLE 2. 
Continued 



Mdh 




A 


0.837 


B 


0.163 


H= 


0.272 


Ho 


0.325 


F.S 


-0.182 


p 

' exact 


0.564 


Opdh 




A 


0.625 


B 


0.213 


C 


0.162 


H. 


0.538 


Ho 


0.575 


fis 


-0.057 


p 

' exacl 


0.500 


Idhp 




A 


0.113 


B 


0.887 


/^e 


0.200 


»o 


0.125 


F\. 


0.385 


"exacl 


0.057 


Pgdh 




A 


0.138 


B 


0.200 


C 


0.349 


D 


0.175 


E 


0.138 


H. 


0.769 


Ho 


0.700 


fis 


0.102 


p 

' exact 


0.384 


Hhdh-I 




A 


0.250 


B 


0.724 


C 


0.013 


D 


0.013 


H. 


0.412 


Ho 


0.400 


f,s 


0.04 1 


"cxiict 


0.832 


Hbdh-2 




A 


0.987 


B 


0.013 


//c 


0.025 


//» 


0.025 


/^is 


-0.013 


P 

• exact 


1.000 


Pgm 




A 


0.400 


B 


0.537 


C 


0.063 


D 




H. 


0.547 


//., 


0.675 


/^,. 


-0.222 


p 


0.011* 



1.000 



0.538 
0.225 
0.237 
0.604 
0.575 
0.061 
0.801 

0.038 
0.962 
0.072 
0.025 
0.661 
0.038* 



0.225 

0.613 
0.162 
0.548 
0.525 
0.054 
0.881 

0.225 
0.762 
0.013 
0.013 
0.368 
0.275 
0.264 
0.144 

1.000 



0.586 
0.363 
0.038 
0.013 
0.522 
0.500 
0.055 
0.192 



1.000 



0.488 
0.262 
0.250 
0.631 
0.675 
-0.057 
0.526 



1.000 



0.462 

0.338 
0.200 
0.632 
0.6.50 
-0.016 
0.973 



1.000 



1 .000 



0.887 
0.113 



0.200 
0.175 
0. 1 36 
0..396 



1.000 



0,600 
0.212 
0.188 
0.560 
0.575 
-0.015 
0.458 



1.000 



0.375 

0.400 
0.225 
0.649 
0.650 
0.011 
0.378 



1.000 



1.000 



0.937 
0.063 



0. 1! 7 

0.125 

-0.054 

1.000 





Thau 


Ria 


SC92 


SC93 




(n = 40) 


(n = 40) 


(H = 40) 


(n = 40) 


Overall 










H, 


0.400 


0.306 


0.212 


0.192 


(SE) 


(0.096) 


(0.104) 


(0.114) 


(0.110) 


H. 


0.404 


0.271 


0.214 


0.193 


(SB) 


(0.100) 


(0.100) 


(0.118) 


(0.110) 


MNA 


3.0 


2.4 


1.7 


1.7 


P 


in 


5/7 


3/7 


3/7 


'^(+Pep) 


8/8 


6/8 


3/8 


3/8 



//j, expected heterozygosity; W„. observed heterozygosity; f„, fixation 
index; P^^^^' exact P-values (*P < 0.05); MNA, mean number of alleles 
per locus; P. proportion of polymorphic loci; SE, standard error. 



Our allozyme data indicate that the Lagoa population from the 
Azores is genetically depauperate and only slightly differentiated 
from populations from the main range and may thus be of low 
"biological value" (i.e., in terms of biodiversity). Gathering of T. 
decussatus could therefore be allowed to continue. Nevertheless, 
given the lower genetic diversity of T. decussatus in the Lagoa, the 
low effective population size, and the depauperate intertidal region 
(Santos et al. 1985. Santos and Martins 1987), exploitation of this 
species must follow strict collection guidelines (see also Santos 
1989), especially given that only 15% of the area is suitable for 
exploitation (Morton and Tristao de Cunha I993J. Otherwise, a 
unique component of the Azorean fauna that also serves as a small 
fishery resource may be lost. In addition, there is much to compare 
between llhtiu de Vila Franca on the island of Sao Miguel in the 
Azores and the Lagoa de Santo Cristo. The faunistic and scientific 
value of Ilhiju de Vila Franca is strongly reduced because of tour- 
ism. Thus, opening up the Lagoa for tourism could be disastrous 
for the fauna too. Therefore, in view of the unique origin, geology, 
fauna, and flora, the place should be declared a "Site of Special 
Scientific Interest" (Morton and Tristao da Cunha 1993). 



ACKNOWLEDGMENTS 

We are indebted to B. Morton (University of Hong Kong) and 
R. Tristao da Cunha (University of the Azores, Portugal) for help- 
ing to collect the Azorean T. decussatus. J. Troncoso (University 
of Vigo, Spain) provided us with the Ria population. Financial 
support was received from the EC program "Biodiversidade no 
Arquipelago dos Agores" PRAXIS XXI (EUJNICT) 2/2.1/BlA/ 
169/94. S. V. D. and H. D. W. are FWO fellows. 



TABLE 3. 

Nei's (1978) unbiased genetic distance between the four populations 
of T. decussatus (for population names we refer to the text). 



Thau 



Ria 



SC92 



SC93 



Thau 
Ria 
SC92 
SC93 



0.152 
0.239 
0.2.30 



0.129 
0. 1 29 



0.036 



Genetic Variation in Azorean Tapes decussatus 



33 



TABLE 4. 
Allozyme variation reported in other studies of T. decussatus and R. pbilippinarum. 



Species 


H„ 


//, 


MNA 


P 


Reference 


T. clecii.ssaiii.s 
















0.28 


2.75 


0.83 


Worms and Pasteur (1982) 






0.23-0.28 


2.18-2.73 


0.64-0.73 


Jarne et al. (1988) 




0.22 


0.26 


2.33 


0.78 


Borsa and Thiriot-Quievreux ( 1990) 




0.18-0.24 


0.23-0.33 


1.54-1.99 


0.54-0.66 


Passamonti et al. (1997) 




0.19-0.40 


0.19-0.40 


1.71-3.00 


0.43-1.00 


This study (all populations) 


R. philipptnanim 














0.26 


0.26 


3.18 


0.73 


Moraga (1986) 




0.16-0.20 


0.18-0.22 


2.67-3.44 


0.22-0.33 


Kijimaet al. (1987) 




0.17-0.25 


0.20-0.27 


2.6-3.6 


0.43-0.57 


Oniwaetal. (1988) 




0.33 


0.34 


2,89 


0.89 


Borsa and Thiriot-Quievreux (1990) 




0.34-0.37 




2.80-3.10 


0.80-0.93 


Fava et al. (1994) 




0.19-0.22 


0.20-0.27 


1.57-1.63 


0.54-0.75 


Passamonti et al. (1997) 




0.27 


0.27 


3.15-3.35 


0.75-0.85 


Yokogawa (1998) 



H„. observed heterozygosity; H^, expected heterozygosity; MNA. mean number of alleles per locus; P, percentage of polymorphic loci. 



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Jdiiiiuil ,>/ Shellfish Research. Vol. 19. No. 1, 35-+I. :0()0. 

LIFE HISTORY AND HABITAT OBSERVATIONS OF SOFTSHELL CLAMS MYA ARENARIA IN 

NORTHEASTERN NEW JERSEY 

CLYDE L. MACKENZIE, JR.,' AND SHAWN M. MCLAUGHLIN^ 

^National Marine Fisheries Service. National Oceanic and Atmospheric 
Administration. James J. Howard Marine Sciences Lalxjratory, 
Highlands. NJ 07732 

'National Marine Fisheries Service, National Oceanic and Atmospheric 
Administration. Cooperative Oxford Laboratory. Oxford, MD 21654 

ABSTRACT Population densities, survival, factors associated with mortalities, and growth of softshell clams. Mya arenaria. in two 
northeastern New Jersey estuaries were studied from 199.^ through 1997. The study areas were near shore where low-tide water depths 
ranged from 15 to 90 cm. Juvenile densities were high only in 199.^. Light sets of juveniles from 1994 to 1997 disappeared by the end 
of their first summer. The longest living softshells were the abundant 1993 year class, which survived for 26 mo in the Shrewsbury 
River. This contrasts with life spans of 7-12 years for softshells in New England. Mortalities of softshells were correlated with: ( 1 ) 
predation by the striped killifish. Fundulus inajalis. and mummichog, F. heterocUtus: (2) mats of sea lettuce. Ulva laclusa: and (3) high 
temperatures (30-32 °Cl. Softshell sarcoma was also present and may have contributed to mortalities. The effects of the mortality 
agents varied among locations and years. The softshells of the Shrewsbury River averaged about 23 mm and 40 mm long at the end 
of their first and second growing seasons, respectively. 

KEY WORDS: Mya arenaria. settlement densities, survival, mortality factors, growth 



INTRODUCTION 

The softshell clam, Mya arenaria. ranges along the Atlantic 
coast of North America from Labrador (Abbott 1974) to Georgia 
(Rasmussen and Heard 1995). with the highest abundances located 
from the Bay of Fundy to Chesapeake Bay. The species also occurs 
in Europe and has been successfully introduced to the coasts of 
western North America (Abbott 1974). Investigators in New En- 
gland commented on the wide variations in magnitude of annual 
sets and on the subsequent survival of softshells (Belding 19.30, 
Turner 1949. Turner 1950. Brousseau 1978b). Softshells can live 
as long as 7 y (Brousseau 1978b) to 12 y (Belding 1930), Most 
postsetting mortalities of softshells are caused by predation by 
shrimp, fish, ducks, brachyuran crabs, xiphosuran crabs, and nati- 
cid snails (Belding 1930. Turner 1949. 1950, Foley and Taber 
1952. Glude 1955. Smith et al, 1955. Cronin and Hall 1968. 
Palmer 1976. Edwards and Huebner 1977. Kelso 1979, Holland et 
al, 1980. Commito 1982, Hines et al. 1990. Eggleston et al. 1992. 
Rasmussen and Heard 1995). and by breakage and displacement in 
storm-exposed areas (Kellogg 1910, Belding 1930. Turner 1950. 
MacKenzie and Stehlik 1988). Investigators in Europe also have 
reported on the wide annual variability in densities of softshell 
juveniles and on their subsequent survival and causes of mortality 
(DeVlas 1979. Beukema 1982. Pihl 1982. Moller and Rosenberg 
1983, Kube 1996). 

Epizootics of softshells reported from New England to Chesa- 
peake Bay have been associated with disseminated sarcomas 
(Barry and Yevich 1972. Farley 1976. Yevich and Barszcz 1977, 
Brown et al. 1977. 1979. Farley et al. 1986. Brousseau 1987. 
Barber 1990). The proliferative condition is transmissible, progres- 
sive, and usually fatal (Brown 1980, Cooper et al. 1982, Farley et 
al. 1986). The etiology of softshell sarcotna is uncertain; environ- 
mental factors (Barry and Yevich 1972. Yevich and Barszcz 1977) 
and a viral agent (Oprandy and Chang 1981 ) have been suspected. 

The Navesink and Shrewsbury Rivers and nearby Raritan Bay 
in northeastern New Jersey have produced softshells since prehis- 
toric times (MacKenzie 1990, MacKenzie 1992), but in recent 
years the stocks have been small, and, consequently, commercial 



production usually has been small or nonexistent. Previous studies 
of the softshells in this area have described abundances (Dean 
1975), longevity (Appeldoorn 1983, Appeldoorn 1995), abun- 
dances and effects of stomis (MacKenzie and Stehlik 1988), and 
the incidence of sarcoma (Barber 1990). Our study was undertaken 
to characterize annual recruitment, survival, factors that cause 
mortality, and growth. 

Study Areas 

The study areas were in the Navesink and Shrewsbury Rivers, 
in New Jersey (Fig. 1 ). The primary study site in the Navesink 
River was off its southeast shore. The site comprised about 3 acres 
of firm muddy-sand bottom and extended from near the shore edge 
to about 75 m offshore; water depths were froin 15-90 cm at low 
tide. The mean tidal amplitude is about 1.7 in (Jeffries 1962). Mats 
of sea letmce, Ulva lactuca, formed in the site, and their aerial dis- 
tributions varied widely among years. The study site in the Shrews- 
bury River was off its northeast shore at a similar shore position 
and water depth, and its bottom sediments were similar. It was 
about 1 acre in size. Little sea lettuce grows in that section of the 
river. A reason for selecting the two sites was convenient access to 
the shore by foot as most all the shoreline areas of the two rivers 
are private property. The softshells in the two rivers are subtidal. 

The identified predators of softshells in the two rivers were: the 
striped killifish, Fundulus majalis; the mummichog, Fundulus het- 
erocUtus: and the blue crab. Callinectes sapidus. Schools of 
striped killifish and mummichogs were nearly always present in 
the study sites, except during the lowest tides, from at least mid- 
May into October. The blue crabs were scarce in the rivers from 
1993 to 1996, but were more abundant in 1997. 

During this study, the salinity at the Navesink River site ranged 
from 15 to 25 0/00. and at the Shrewsbury River site from 20 to 25 
0/00. Water temperatures were mostly 1 1-12 °C during early May. 
18-20 °C during June, and peaked at about 25 °C in late July and 
early August, but in mid-afternoon during late July-early August, 
1995, water temperatures ranged from 30.0 °-31.8 °C. Tempera- 
tures afterward cooled. 



35 



36 



Mackenzie and McLaughlin 



Raritan 
Bay 




a 

O 
o 

CD 
03 

3 



Figure 1. Locations of study and sampling sites in nortlieastern New 
Jersey. 



The waters of the Navesink and Shrewsbury Rivers interchange 
with Raritan Bay. which is contaminated with many types of pol- 
lutants (Pearce 1983. Breteler 1984). The pollutants consist of 
suspended particulates, oil and grease, many toxic trace metals, 
polynuclear aromatic hydrocarbons, polychlorinated biphenyls, 
DDT. and dioxins (Stanford and Young 1988, Wolfe et al. 1996). 
In 1974. the copper concentration in western Raritan Bay bottom 
water was 65 parts per billion (ppb), the highest reported for any 
estuary; the copper concentration in the surface water there was 36 
ppb. and in mid-Raritan Bay it was 7.9 ppb (Waldhauer et al. 
197S). In 1992. the copper concentration in the surface water was 
considerably lower: 4.6 ppb in western Raritan Bay, and 4.3 ppb in 
mid-Raritan Bay (Anonymous 1992). The buy has extremely high 
primary productivity with the annual value in the 1970s at 817 g 
C/nr. which was considered among the highest of any estuary 
(O'Reilly et al. 1976). In the 196()s. Raritan Bay was classified as 
an advanced eutrophic system (Federal Water Pollution Control 
Administration 1967), but since the 1970s its water quality has 
improved (Brosnan and O'Shea 199.'i). Elevated nitrogenous 
wastes nevertheless continue to stimulate the growth of dense phy- 
toplankton blooms (Draxler et al. 1984, Brosnan and O'Shea 
199.'i); Draxler et al. (1984) had reported Secchi disc readings in 
the bay of < 1 .0-2.0 m during most of the spring and summer. 
Sleimlc and Caracciolo-Ward (1989) have shown that the den- 
sity and diversity of benthic macrofauna in Raritan Bay are rela- 
tively low compared with other U.S. east coast estuaries. Similar 
determinations of pollution, phytoplankton blooms, and macro- 
fauna have not been reported in the Navesink and Shrewsbury 
Rivers. 

The eutrophicalion of waters probably v\as responsible for pro- 
ducing some large mats of sea lettuce observed in the two rivers. 
As Hull ( 1987) noted, sea lettuce begins as tiny leaves attached to 
shells and other objects in the spring, grows and persists as thick 
mats during the sunnner. antl llicn ncarlv disappears in the late fall. 



METHODS 

Sampling Procedures 

Field observations lasted 5 y, 1993 to 1997. Water salinity was 
determined quarterly by titration. Surface water temperatures were 
measured with a hand-held thermometer daily at 7:30 am at the 
Navesink River study site from May into September in 1994, 1993, 
and 1996. In 1993, following heavy sets of softshells, densities of 
this year class were estimated once a month, except in the coldest 
parts of the year, in the Navesink and Shrewsbury River study 
sites, by placing a ring that encircled a 0.28 m" area on the sub- 
strate and then removing all softshells for counting and measuring. 
Three such samples were taken for each determination. From each 
monthly sample, a subset of 100 softshells, was measured and 
lengths were plotted to determine growth rates. In 1994, samples to' 
determine the densities of young-of-the-year (juvenile) softshells 
were taken similarly at each site. In 1995, 1996, and 1997, three 
0.28-m~ areas or six O.l-m" areas were sampled at each site. Only 
two to three samplings were made in each year from 1994 to 1997, 
because the low densities of juvenile softshells fell quickly to 
nearly zero per sample following the initial samplings in June or 
July. 

Potential predators of softshells were collected by pulling a 
fine-mesh, 15-m seine for about 60 m over an inshore section of 
bottom in the study sites in the Navesink and Shrewsbury Rivers. 
A single seining was made at each site at half tide during the 
outgoing tide in July 1994. Fish and shrimp were collected, but 
only the fish were examined. They were placed in a plastic bag, 
held on ice in a cooler, and frozen the same day. Later, they were 
thawed, and the invertebrates, plants, and other contents in their 
stomachs and guts were identified and counted using a dissecting 
microscope. 

Field Experiment on Fish Gut Evacuation 

During August 1996. an experiment was conducted to deter- 
mine the evacuation rate of food from the stomachs and guts of the 
mummichog, F. hcteroclinis. One hundred mummichogs (mean 
length 79.1 mm; range 63-1 10 mm) were seined and divided into 
five groups of 20 each. The first group of fish was immediately 
iced, then frozen, and later thawed and examined for the quantity 
of food in their guts. The other four groups were held in separate 
field cages suspended above the bottom for 3, 6, 9, and 24 h at 
temperatures of 23.5 °-25.0 °C and then processed similarly to the 
first group. A visual estitnate was made of gut fullness. 

Diagnosis of Sarcomas 

The prevalences of softshell sarcomas were determined using 
histological methods (Farley et al. 1986). Samples of 50 softshells, 
40-55 mm long, were collected quarterly al four sites, namely, our 
two primary study sites in the Navesink and Shrewsbury Rivers, at 
Lewis Point (5 km west of our primary study site in the Navesink 
River), and in Raritan Bay at the Old Ferry Dock on the west side 
of Sandy Hook (Fig. 1 ). The collections eventually ended ni the 
Navesink and Shrewsbury Rivers because the softshells had died 
or had became too scarce. Following collections, the sollshells 
were transported to the Cooperative Oxford Laboratory, Oxford, 
MD. Hemolymph was drawn from the adductor muscles into ster- 
ile syringes containing ambient sterile seawater. expelled into slide 
chambers, and fixed after 30 min in I i;iiJtaraldehvde-4 formalde- 



Life History and Habitats of Softshell Clams 



37 



hyde. The hemolymph preparations were stained with fuelgen pi- 
croniethyl and were examined tor sarcomas by light microscopy. 



RESULTS 



Navesink River 



The setting densities of juvenile softshells in our Navesink and 
Shrewsbury River study sites were similar to one another each 
year. The juveniles were relatively abundant in the two rivers only 
in 1993. In the Navesink River, they had set throughout the shal- 
lows over a distance of 1 0.5 km off its south and northwest shores. 
At the study site, their density at the initial sampling in August 
1993 was 1,1 10/0.28 m". Their survival after that was fairly high: 
60-69% were alive in late April to late May 1994 (Table 1 ). 

In 1993. sea lettuce was relatively sparse in the study site, but 
by mid-June to early July 1994, a solid mat of sea lettuce had 
formed. The mat was about 25 cm thick and extended from the 
shore outward to cover about half of the 3-acre bed. In addition, 
some i.solated stationary sea lettuce mats, as small as 2 m across, 
formed in areas beyond the main mat. All the observed 1993 year 
class of softshells covered by the mats initially extended their 
siphons several centimeters out of the sediment, then emerged 
from it. laid on its surface beneath the mat. and died. In contrast, 
the softshells in unvegetated areas did not extend their siphons, 
emerge, and die. 

From 1994 through 1997. the sets of juvenile softshells were 
light in the river. In 1994, the unvegetated sediments outside any 
sea lettuce mats received a set of juveniles; on June 30 of that year, 
they had a mean density of 54.7/0.28 nr (three replicates. SE 9), 
but by July 8. 1994, their density had fallen to 2.3/0.28 m" (three 
replicates, SE 0.7). The 1995 and 1996 sets were much more 
sparse than those in 1994 and 1997. On July 28, 1997, the 1997 
juveniles had a mean density of 28.8/0.10 m- (six replicates. SE 
4.7), but by August 9. 1997, their density had fallen to 3.7/0.10 nr 
(six replicates, SE 0.6). Subsequent samplings in August and Sep- 
tember each year from 1994 to 1997 found few juveniles in the 
site. 

On July 7, 1994, when the density of the 1994 year class of 
softshells was declining rapidly, a seining was made over the bed 
to examine the stomachs and guts of fish. Forty-one of 60 striped 
killifish (average length 64 mm, range 46-78 mm) contained an 

TABLE 1. 

Densities, mean, and standard error (S.E.) of 1993 year class Mya 

arenaria at stud> sites in Navesink River and Shrewsbury River. 

Densities are expressed as mean per 0.28 m'. S.E. is based on 3 

samples on each date. 



Navesink River 



Shrewsbury River 



Date 


Mean 


S.E. 


Date 


Mean 


S.E. 


1 Sep 9.^ 


1. 110 


117 


7 Oct 93 


849 


57 


8 Oct 93 


1.170 


200 


1 1 Nov 93 


650 


62 


28 Apr 94 


668 


37 


29 Apr 94 


677 


45 


24 May 94 


767 


16 


28 Jun 94 


784 


85 


29 Jun 94 







2 Aug 94 


586 


10 








2 Sep 94 


520 


81 








26 Apr 95 


573 


9 








7 Jun 95 


456 


16 








7 Aug 95 








average of 46 juvenile softshells/fish (range 1-169 softshells). and 
one of three mummichogs (average length 97.3 mm. range 84-1 15 
mm) contained two juvenile softshells. The softshells ranged from 
2-1 I mm long. The remaining striped killifish and mummichogs 
had food in their stomachs but no softshells. 

Shrewsbury River 

In 1993. softshells set densely in the shallows along most of the 
north shore of the Shrewsbury River in a band about 7 m wide, 
over a distance of about 4.2 km. The density of the 1 993 year class 
of softshells at the study site at the initial sampling in October 
1993 was 849/0.28 m". After that, their survival was fairly high, as 
54-67% were alive in late April-early June 1995 (Table 1). By 
August 7, 1 995, about 26 months after setting, this entire year class 
of softshells was dead at the site. They died during a period of 
unusually high air and water temperatures in late July-early Au- 
gust. At 3:00 PM on July 3 1 , the water temperature was 3 1 .8 °C, the 
softshells were dying and rotting, and the water over the bed was 
a yellow-brown mixture of rotting softshell meats and brown phy- 
toplankton. Their mortality apparently was caused by the high 
temperatures, because the lethal temperature of adult softshells is 
in the temperature range of 30.5 °-32.5 °C (Kennedy and Mihur- 
sky 1971). 

From 1994 through 1997, juvenile softshells were relatively 
scarce throughout the river. At the study site, the small numbers 
observed by scraping with a sieve through the surface of sediments 
in 10 places in June and July disappeared by August or September 
in the years in which they set, similarly as the light sets had 
disappeared in the Navesink River. 

On July 8, 1994, fish were seined at the study site and their guts 
were examined for softshells and other foods. Four striped killifish 
(average length 107 mm, range 92-1 13 mm) contained an average 
of 26 juvenile softshells/fish (range 21-32 softshells per fish): 123 
of 150 mummichogs (average length 69 mm, range 40-93 mm) 
had an average of 15.5 juvenile softshells per fish (range 1-53 
softshells per fish); and one spot. Leiostomus xanthwus. had 1 15 
juvenile softshells. The softshells ranged from 4 to 1 1 mm in 
length for all fish. Other items in the guts of striped killifish and 
mummichogs in the Navesink and Shrewsbury Rivers were: juve- 
nile common Atlantic slippersnails, Crepidula fornicaui: amphi- 
pods; isopods; juvenile horseshoe crabs. Limiilus polxphemus 
(about 3 mm carapace width); polychaetes; sea lettuce; and detri- 
tus. 

Food Passage Through Mummichogs 

Mummichogs passed food through their stomachs and guts rap- 
idly (Fig. 2). In the experiment to estimate the rate, a large decline 
(80%) in fullness of their guts was evident after 3 h, and little food 
remained after 24 h. The results suggest that the softshells found in 
mummichogs that were seined at the sites were eaten within 24 h, 
and they imply a high consumption rate. 

Histology 

In the Navesink River, quarterly samples showed a low sar- 
coma prevalence in 1994, but prevalence reached 18%' in Decem- 
ber 1995 and decreased slightly to 13% and 14% for the first two 
quarters in 1996. while samples from Lewis Point were negative 
for sarcomas in 1991 to 1993 (Table 2). In the Shrewsbury River, 
quarterly samples of softshells examined for sarcomas were nega- 
tive in 1994 and 1995. At the Old Ferry Dock, in collections in 



38 



Mackenzie and McLaughlin 



Fundulus Gut Evacuation Study 
(16 Aug 96) 




Figure 2. Percentage with food in guts and average fullness of guts of 
F. heteroclitus held in field cages at spaced intervals. 0-24 h. 

1995. 1996. and 1997. from 10-20% of softshells were infected 
with sarcoma on four of seven dates, and from Q^9c were infected 
in the remaining three dates. 

Growth 

The length-frequency curves for the 1993 year class of soft- 
shells in the Navesink and Shrewsbury Rivers are presented in 
Figure 3. The curves for each time period show a single mode that 
broadens somewhat as time passes. In the Navesink River, the 
softshells had a mean length of 15.4 mm in September 1993. 22.1 

TABLE 2. 

Percent prevalences of .softshell sarcomas based on histology 
(n = 50). 





Lewis 


Navesink 


Shrewsbury 


Old Ferry 


Date 


Point" 


River 


River 


Dock 


7-9-yi 











9-4-91 











12-4-91 











.^-3-92 











6-.^-92 










9-9-92 










12-9-92 










3-25-93 










6-38-93 










9-29-93 










6-15-94 












9-12-94 




4 







12-6-94 




2 







5-22-95 












6-26-95 




2 







7-25-95 




6 







9-21-95 




2 




4 


12-5-95 




18 




12 


3-27-96 




13.3" 




10 


7-18-96 




14.4"= 







10-3-96 








10 


2-26-96 








20 


4-14-97 








2 



° Location In Navesink River, 
"n = 47. 
' n = 45. 



Shrewsbury River 



Navesink River 



^^ 



November '93 



April '94 



z^.. 



May '94 



j^ 



.y%V. 



Sept '94 



.J^. 



V 






20 
10 


A 


Sept '93 


^ 






10 


/\ 








10 


J^ 


Nov '93 








10 


^yvvyv 


Apr '94 








10 
5 


/v 


May '94 
^ - 


10 13 16 19 22 25 28 31 3J 37 


« 43 46 49 



Length (mm) 



, /I Apr -9! 



12 16 20 24 28 32 36 40 44 48 52 56 60 64 



Figure. 3. Length-frequency distributions of the 1993 year class Mya 
arenaria in the Navesink River, 1993 to 1994, and the Shrewsbury 
River, 1993 to 1995. 



mm in November 1993, and 31.6 mm in May 1994. In the Shrews- 
bury River, their mean lengths were 19.3 mm in October 1993. 
22.9 mm in November 1993. 26.6 mm in April 1994. 38.9 mm in 
November 1994, 47.6 mm in April 1995, and 48.9 mm in June 
1995. 

DISCUSSION 

In attempting to find reasons for the large annual variability in 
setting densities of softshells in Europe. Beukema (1982, 1992). 
Jensen and Jensen (1985), and Moller (1986) observed that heavy 
sets of softshells and some other bivalves occurred during sum- 
mers following cold winters and that light sets followed mild win- 
ters. The bivalves were active during the mild winters when little 
food was available in the water, and they consequently had ab- 
sorbed most of their gonads by the time spawning began in the 
spring. Our study was not continued sufficiently long enough to 
document such a correlation, but it is likely that dense sets of 
softshell juveniles result from certain weather conditions. The 
spring and early summer of 1993 when the heavy sets occurred in 
the Navesink and Shrewsbury Rivers did feature weather with no 
cold easterly winds with rain. During the springs and summers of 
1994 to 1997. however, when light sets occurred, several periods 
of cold easterly winds and rain, each of 3— t days duration, were 
interspersed with periods of v\armcr westerly and southerly winds. 
Bclding ( 1930) had noted that the numbers of larvae in the water 
declined during periods of cold rains. 

Earlier investigators ha\e noted the disappearances of softshell 



Life History and Habitats of Softshell Clams 



39 



juveniles by the end of their first summer in some years (Brous- 
seau 1978a. Moller and Rosenberg 1983. Beukema 1979. Pihl 
1982). We believe that predation by striped killifish and mummi- 
chogs was the principal reason for the sharp declines and disap- 
pearances of juxeniles in our study sites during 1994 to 1997. The 
observations suggest that any relatively light sets of softshells, as 
dense as 500/m" or even higher, could be lost to such predation 
every year whenever the fish are abundant in the two rivers. The 
fish likely were present and preyed on juvenile softshells in 1993. 
but perhaps the juveniles were so abundant that a great many 
remained alive by the time they had grown too large for the fish to 
prey on them. 

Fish also prey on softshells in other regions. Kelso (1979) 
described heavy predation of juvenile softshells by mummichogs 
in Massachusetts. In our study, the sizes of softshells (2-11 mm 
long) taken by the striped killifish and mummichogs were similar 
to those that Kelso (1979) reported; probably 1 1 mm is near the 
maximum size of a softshell that the fish can devour. More soft- 
shells were present in the guts of mummichogs in the Navesink 
River (about 46 softshells per fish) than he found in Massachusetts 
(6-9 softshells per fish). Perhaps the softshells were more abun- 
dant in the Navesink River. Medcof and McPhail (1952) stated that 
adult winter flounders. Pleiironecres aineiicainis. about 28 cm 
long, consumed whole juvenile softshells and nipped off the si- 
phon tips of adult softshells in eastern Canada. In their study, the 
softshells with nipped siphons recovered without unusual mortal- 
ity. Rasmussen and Heard (1995) stated that Atlantic stingrays. 
Dasyatis sabiiui. feed on softshells in Georgia. Pihl (1982) and 
Moller and Rosenberg (1983) observed that flounders Platichthys 
ftesiis consume large numbers of juvenile softshells. 2-12 mm 
long, in Sweden, and DeVlas (1979) observed that flounders P. 
flesus and plaice. Pleiironectes platessa. consume juvenile soft- 
shells and the siphon tips of older softshells in the Netherlands. 
Summer flounders. Paralichthys dentatus. and other fish were 
present in the Navesink and Shrewsbury Rivers and might have 
preyed on softshells. but they were not observed or collected dur- 
ing our visits to the study areas. 

Relatively scarce in our study sites from 1993 to 1996, blue 
crabs appeared to be a minor predator then, but they were abundant 
and may have killed many juvenile softshells in 1997. Since our 
observations were limited to periods of low and mid tides and 
during daylight, blue crabs and other predators may have entered 
the study sites and eaten some juveniles during high tides and at 
night during all years. Green crabs, Carcinus maenas. and naticid 
snails, both predators of softshells in New England (Belding 1930. 
Glude 1955. Smith et al. 1955. Edwards and Huebner 1977. Com- 
mito 1982), were not observed in the two rivers during 1993 to 
1997 and could not have caused much mortality of the softshells. 
Horseshoe crabs, also a softshell predator in New England (Turner 
1949. 1950). were scarce and apparently killed few softshells in 
the two rivers. The shrimp. Crangon crangon. preys on softshells 
as large as 3 mm long in Europe (Moller and Rosenberg 1983). 
The seven-spine bay shrimp. Crangon septemspinosus. and the 
marsh grass shrimp. Palaemonetes vulgaris, were abundant in our 
two study areas but were not examined as predators of small post- 
set softshells. and neither were amphipods and isopods. 

In eastern North America, greater scaup. Aytliya marila. prey 
on a variety of small clams, including softshells. blue mussels. 
Mylihis edidis. and snails (Foley and Taber 1952. Cronin and Hull 
1968. Barclay pers. commun.. 1998). Black ducks. Anus nihripes. 
prey on bivalves, including Macoma balthica. blue mussels, and 



marine snails, such as eastern mud snails, llyanassa obseleta 
(Palmer 1976). Greater scaup and black ducks were present in the 
Navesink and Shrewsbury Rivers, but there were no signs that they 
ate softshells in our study areas. 

Juvenile softshells also can be killed on exposed shallow habi- 
tats during wind storms by having their thin shells ground into 
fragments or being washed onto nearby beaches (Kellogg 1910. 
Belding 1930, Turner 1950. MacKenzie and Stehlik 1988). This 
type of mortality was not observed in our Navesink and Shrews- 
bury River study sites, but it was observed in the softshells that had 
set along the south shore of Raritan Bay. 

Once past their first summer, softshells can survive fairly well 
as long as exogenous mortality factors are absent, as shown by 
Belding (1930) and Brousseau (1978b) in New England. Kube 
(1996) in Europe, and others. In the Navesink River, the 1993 year 
class of softshells survived well from September 1993 through 
May 1994 until mats of sea lettuce killed them, and in the Shrews- 
bury River it survived well from October 1993 through June 1995 
when shortly afterward high temperatures apparently killed them. 
The age of the Shrewsbury River softshells when they died. 26 mo, 
was the maximum that any lived in the two study sites and is far 
shorter than softshells lived in New England where their habitat 
was undoubtedly much better (Belding 1930, Hanks 1963, Brous- 
seau 1978b). Appeldoorn (1995) stated that softshells in the Nave- 
sink River could live at least 15 years around the time of his 
sampling (1977), but his finding was based on shell markings and 
sizes of softshells found during a single collection and might be in 
error. Nevertheless, in an earlier paper. Appeldoorn ( 1983). report- 
ing on the same 1977 samples, stated that softshells were present 
as large as 78 mm long or even larger and were obviously older 
than the largest softshells (62 mm) that we found in the Navesink 
and Shrewsbury Rivers. The environmental conditions in the two 
rivers during 1993-1997. such as extremely high temperatures in 
1995. apparently did not allow the softshells to live as long as they 
did during the 1970s. 

Some earlier workers had shown that algal mats grow over and 
kill bivalves, but our study may be the first to document that mats 
of U. lactuca kill softshells. Thiel et al. (1998) had similarly found 
that overgrowths of the filamentous alga Enteromorpha prolifera 
kill softshells in Maine; Breber (1985) found that mats of Ulva 
rigida and Cracilaria sp. kill carpet-shell clams. Tapes deciissatits, 
in Italy; and Everett (1994) showed that the bent-nose macoma, 
Macoma nasiita. was more abundant in areas devoid of Ulva ex- 
panse than in areas where it formed mats in California. The same 
condition probably develops under U. lactuca mats that Gray 
( 1992) described under U. rigida mats in Europe: Anaerobic con- 
ditions are reached and sulfide and other toxic compounds are 
produced leading to a massive mortality of benthic organisms. 

Sarcoma infections occur seasonally (Farley 1976. Farley 1989. 
Cooper et al. 1982, Brous.seau 1987, Barber 1990). Perhaps in 
collecting the softshells quarterly, we missed detecting some sar- 
coma in them. During most collections of adult softshells. a few 
recently dead specimens with whole shells were noticed among the 
100-200 that were taken. Sarcoma might have been responsible for 
some mortality that was not identified to cause, or perhaps the 
softshells died from some other cause. We were unable to deter- 
mine whether contaminants in the waters and sediments and den.se 
phytoplankton blooms affected the longevity of the softshells. Bar- 
ber ( 1990) found sarcomas in softshells in the Shrewsbury River in 
1986 and 1987 and concluded that annual mortality due to the 



40 



Mackenzie and McLaughlin 



disease was about 3.5% at that time. Our study cannot add much 
to his estimate. 

The sizes of softshells at certain ages that Appeldoorn ( 1983) 
suggested for the Navesink River correspond with our findings in 
the Shrewsbury River. For example, at 20 months of age the soft- 
shells that Appeldoorn measured were 42.5 mm long and at 28 
months they were 47.3 mm long, or similar to the mean lengths of 
softshells in the Shrewsbury River at about the same ages in No- 
vember 1994 and June 1995. However, the comparisons are too 
crude to compare actual growth rates in the 1970s and the 1990s. 



The small and sporadic commercial harvests of softshells in 
this area likely are due to their low setting densities and poor 
survival rates in recent years. The softshells probably would sur- 
vive longer if a period of cooler summers and reduced eutrophi- 
cation of waters were to follow. 

ACKNOWLEDGMENTS 

We thank D. Jeffress and F. TrioUo for assistance with the field 
work, and J. Buckle. R. Pikanowski. R. N. Reid. and two anony- 
mous reviewers for critically reviewing the manuscript. 



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Joiirmil of Shellfish Resenrch. Vol. 19. Ni). I, 4.V50. 2000. 

QUAHOG PARASITE UNKNOWN (QPX) IN THE NORTHERN QUAHOG MERCENARIA 

MERCENARIA (LINNAEUS, 1758) AND M. MERCENARIA VAR. NOTATA FROM ATLANTIC 

CANADA, SURVEY RESULTS FROM THREE MARITIME PROVINCES. 



GREGORY S. MACCALLUM AND SHARON E. MCGLADDERY 

Fisheries and Oceans Canada 
Gulf Fisheries Centre 
PO Box 5030 
Moncton, NB EIC 9B6 

ABSTRACT A histology based survey of 3047 quahogs from various sites in three Atlantic Canadian provinces between 1990-98 
revealed Quahog Parasite Unknown (QPX) in clams ranging from 18-92 mm in length (> 1 .5 years old). Prevalences ranged from 1.7% 
in wild quahogs to SO^r in hatchery broodstock. An additional two year (1996-97) seasonal survey of four sites (St. Andrews and 
Shediac Bridge, New Brunswick; Wallace. Nova Scotia; and West River. Prince Edward Island) found QPX in quahogs 43-102 mm 
in length (n = 715) at Wallace {7c P = 6J9c) and Si. Andrews iVc P = 209c). Infections were found in spring, summer and fall 
samples and no significant difference was found between male and female infected quahogs at either site (1996-97; x". P > 0.10). The 
most commonly infected tissues were the gills, mantle and gonads. 

KEY WORDS: Quahog Parasite Unknown (QPX). Af mercenaria. M. mercenuria var. nouiia. pathology 



INTRODUCTION 

Quahog Parasite Unknown (QPX) infects the hard-shell clam 
(northern quahog) Mercenaria mercenaria and the selected vari- 
ety, M. mercenaria var. notata (Chanley 1961). It has caused cu- 
mulative mortalities ranging from 80% in quahogs from New 
Brunswick (Drinnan and Henderson 1963), and Cape Cod. Mas- 
sachusetts (Smolowitz et al. 1998) to 100% in hatchery broodstock 
in Prince Edward Island (Whyte et al. 1994; Bacon et al. 1999). It 
also occurs in apparently healthy quahogs from Atlantic Canada 
and Virginia (McGladdery et al. 1993: Ragone Calvo et al. 1998). 
QPX has also been associated with quahog mortalities from Burton 
Bay, Virginia (Ragone Calvo et al. 1997). The Gulf of St. 
Lawrence is the northern-most limit of M. mercenaria. thus the 
clams may respond differently, both in terms of growth and disease 
resistance, from clams in the middle of their geographic distribu- 
tion in the U.S. The conditions which trigger pathogenic infections 
levels, however, have yet to be determined. 

Recent interest in developing the northern quahog for aquacul- 
ture in Atlantic Canada revealed a lack of base-line information on 
normal parasite and disease profiles for this species. Because cul- 
ture involves handling and holding the clams in unnatural condi- 
tions, QPX has the potential to become a significant health prob- 
lem, especially as hatchery broodstock are developed (Whyte et al, 
1994). An accurate understanding of the seasonal and geographic 
distribution of QPX in wild and cultured populations throughout 
Atlantic Canada was. therefore, required. Throughout the past 10 
years, samples of wild and cultured quahogs have been examined 
histologically for parasites and pathology, including QPX, as part 
of diagnostic services provided by Fisheries and Oceans Canada, 
Gulf Fisheries Centre, Moncton, New Brunswick. These data, in 
addition to a seasonal histological survey of wild quahogs from 
four sites in New Brunswick (NB), Nova Scotia (NS), and Prince 
Edward Island (PEI), conducted between 1996 and 1997, were 
examined to determine if there were significant population differ- 
ences in quahog health profiles. Unlike other bivalve species cul- 
tured to date, quahogs in Atlantic Canada have undergone rela- 
tively little transfer and population mixing. This was, therefore, 



seen as an opportune time to collect base-line health information 
for subsequent development of the quahog aquaculture industry. 

MATERIALS AND METHODS 

Diagnostic Survey 1990-98 

A total of 3047 quahogs was examined (Table 1). Quahogs 
(wild and notata variety) were collected or shipped live froin aqua- 
culture sites and hatcheries in NB, NS and PEI (Table I) to the 
Gulf Fisheries Centre, Moncton, within 12-24 h of collection. 
Anterior-posterior length (mm) and weight (in shell) were mea- 
sured before shucking. A 2-3 mm dorso-ventral cross-section was 
removed and fixed in 1% gluteraldehyde/4'7c formaldehyde (How- 
ard and Smith 1983) for light microscopy. The tissues for light 
microscopy were paraffin embedded, sectioned (6 \x.m ) and 
stained using Harris' hematoxylin and eosin. 

Tissue sections were examined at 25 and 250 magnification 
using a Leitz Dialux 20 compound microscope. Prevalence of QPX 
was recorded, along with a qualitative scale for intensity of infec- 
tion (light = < 25: organisms, moderate = 25-50 organisms: and 
heavy = > 50 organisms) per tissue section. The sex ratio of 
mature quahogs. infected with and without QPX, was compared to 
a 1 : 1 ratio using a standard Chi Square test (Zar 1984) to determine 
if there was any relationship between quahog sex and presence of 
QPX. 

Seasonal Survey 1996-97 

Wild quahogs were collected during the spring (May/June), 
summer (August) and fall (October/November) of 1996 and 1997 
from: (1) St. Andrews, NB: (2) Wallace, NS: (3) West River, PEI: 
and (4) Shediac Bridge, NB (Figure 1 ). Samples of 28-30, total = 
715, quahogs were collected and processed as described above, 
between May, 1996 and October. 1997 (Table 2). Water tempera- 
ture and salinity were taken at the time of collection from all four 
sites during both years. In addition, a continuous temperature re- 
corder was placed at the Wallace location from May to October 
1996 and 1997. The sex ratio of infected quahogs was compared to 
a 1:1 ratio using a standard Chi-Square test (Zar 1984). 



43 



44 



MacCallum and McGladdery 



TABLE 1. 
Collection details and QPX results for 1990-98 survey. 







Lengths 








***Sex Ratio 






examined 




Prev. 


**Inf. 


(Infected 


Date 


Collection Site 


(mm) 


No. 


(%) 


Levels 


Quahogsl 


1990 


Ellerslie, PEI* 


>25 


5 


80.0 


H 


4U 


13-5-91 


Bouctouche. NB 


55-81 


16 











22-5-91 


Shippagan. NB* 


72-90 


15 


13.3 


H 


2M 


17-6-91 


Cocagne. NB 


78-85 


30 











18-7-91 


Cocagne. NB 


75-89 


30 











29-7-91 


Pictou, NS 


83-104 


45 











6-7-91 


Halifax. NS* 


>25 


10 











15-8-91 


Shediac Bridge. NB 


73-91 


30 











17-8-91 


Cocagne. NB 


72-88 


30 











30-3-92 


Shippagan. NB* 


25-38 


5 











1-7-92 


Cocagne. NB 


71-91 


30 











26-8-92 


Cocagne. NB 


70-90 


30 











15-10-92 


Cocagne. NB 


45-63 


30 











7-6-93 


Malagash. NS 


57-76 


30 











22-6-93 


Cocagne. NB 


48-71 


30 











22-6-93 


West River. PEI 


54-73 


30 











13-7-93 


Powell cove. NS 


32-110 


26 


7.7 


H 


IM;1F 


14-7-93 


Wallace. NS 


43-67 


39 











26-7-93 


Brule Harbour. NS 


53-180 


30 


3,3 


L 


IF 


3-8-93 


West River. PEI 


52-82 


30 











4-8-93 


Wallace. NS 


49-62 


30 











8-8-93 


Cocagne, NB 


43-61 


30 











12-10-93 


Ellerslie. PEI* 


8-15 


22 











19-10-93 


Malagash. NS 


52-80 


30 











26-10-93 


West River. PEI 


51-96 


30 











7-6-94 


West River. PEI 


53-91 


30 











28-6-94 


Cocagne. NB 


65-75 


30 











12-7-94 


Malagash. NS 


52-63 


30 











22-8-94 


West River. PEI 


57-91 


30 











24-8-94 


Cocagne. NB 


63-71 


30 











13-9-94 


Malagash. NS 


54-84 


30 











2-11-94 


West River. PEI 


51-96 


30 











22-11-94 


Malagash. NS 


43-83 


30 











14-6-95 


Shippagan. NB* 


47-69 


120 











20-6-95 


Shippagan. NB 


<2 


60 











7-10-95 


Shippagan. NB 


<8 


60 











22-8-95 


Bouctouche. NB 


50-79 


6 











27-10-95 


Little Harbour, NS* (m 


>25 


8 


2.5 


H 


IM 


27-10-95 


Ellerslie. PEI* 


>25 


4 











14-5-96 


Ellerslie. PEI 


2-6 


60 











3-6-96 


Ellerslie, PEI* 


28-53 


25 


8.0 


L 


2M 


11-6-96 


Shippagan. NB* (n) 


30-50 


15 


47.0 


H 


2M:4F:U 


27-7-96 


Ellerslie. NB* 


30-40 


6 











27-7-96 


Ellerslie. NB 


<8 


20 











11-8-96 


Little Harbour. NS* 


72-105 


60 











29-8-96 


Little Harbour. NS* 


72-81 


1 











9-9-96 


Orwell. PEI 


3-7 


\5() 











11-4-97 


Corkumsls. NS*(n) 


3.3-67 


26 


31.0 


H 


5M:3F 


7-6-97 


Ellerslie. PEI 


36-71 


29 


3 1 .0 


H 


6M:3F 


7-6-97 


Vernon River. PEI 


>25 


30 











9-6-97 


Pugwash. NS 


42-86 


60 


1.7 


L 


IM 


9-6-97 


Powell Cove. NS 


45-75 


60 


(1 








9-6-97 


Tatamagouche. NS 


42-90 


60 











24-06-97 


Shippagan. NB (nl 


<I0 


48 











20-10-97 


Shemoguc, NB (n) 


7-22 


121 











20-10-97 


Bouctouche, NB (n) 


14-21 


56 











22-10-97 


Vernon, R, PEI (n) 


>25 


30 












QPX IN THE Northern Quahog 



45 



TABLE 1. 

Continued. 







Lengths 








***Sex Ratio 






examined 




Prev. 


**Inf. 


(Infected 


Date 


Collection Site 


(mm) 


No. 


(%) 


Levels 


Quahogs) 


22-10-97 


Tatamagouehe. NS (nl 


>25 


38 


(1 


(1 





23-10-97 


Bale Ste- Anne. NB (n) 


>25 


30 











11-12-97 


Ellerslie. PEl 


28-33 


30 











15-01-98 


Ellerslie. PEl* (n) 


>25 


25 











15-01-98 


Ellerslie. PEl* 


>25 


4 











4-5-98 


Little Harbour. NS* 


70-98 


60 











8-5-98 


Shemogue. NB (n) 


<10 


10 











12-5-98 


Bouctouche. NB (n) 


<10 


10 











14-5-98 


Ellerslie. PEl 


<10 


40 











14-5-98 


Ellerslie. PEl 


<5 


60 











24-6-98 


Ellerslie. PEl 


29-95 


60 


6.7 


H 


1M:3F 


26-5-98 


Shediac Bridge. NB 


89-102 


6 











5-6-98 


StCecile. NB(n) 


<10 


26 











15-7-98 


St Andrews, NB 


44-87 


40 


10.0 


H 


3M:1F 


30-7-98 


Vernon River, PEl 


18-25 


30 


6,7 


M 


2U 


30-7-98 


Vernon River. PEl (n) 


20-25 


30 











4-8-98 


Wallace. NS 


<10 


29 











4-8-98 


Wallace, NS(n) 


<I0 


31 











28-8-98 


West River. PEl 


80-100 


30 


3.3 


M 


IF 


22-9-98 


Shippagan, NB (n) 


16-22 


21 











23-9-98 


St Andrews 


40-77 


29 


6.9 


M 


2M 


6-10-98 


StCecile. NB(n) 


11-24 


40 











9-10-98 


StCecile. NB(n) 


17-27 


19 











13-10-98 


Bouctouche. NB (n) 


16-22 


9 











15-10-98 


St Mary's Bay, NS 


32-63 


60 











20-10-98 


Shippagan. NB 


13-30 


60 











20-10-98 


Shippagan. NB (n) 


15-20 


23 











26-10-98 


Vernon River. PEl 


19-25 


31 


42.0 


M 


4M;9U 


26-10-98 


Vernon River. PEUn) 


19-31 


32 











27-10-98 


Baiede Vin. NB(n) 


>25 


45 











27-10-98 


Bale de Vin, NB 


9-14 


60 











27-10-98 


Percival River, PEl 


43-63 


30 


3.3 


M 


IF 


2-11-98 


Wallace, NS (n) 


7-16 


27 











2-11-98 


Wallace. NS(n) 


19-31 


30 













Total 




3047 









*- hatchery broodstock 

(n) - Meicenaiia mercenaria variety notula 

**- H-heavy, M-moderate, L-light 

*** - M-male. F-female. U-undetermined (restinn/immature) 



RESULTS 

Diagnostic Survey 1990-98 

No gross clinical signs were observed in any of the quahogs 
examined for tfie diagnostic survey, including clams with high 
intensities of infection detected using histological examination. 
QPX was found in M. mercemma and M. m. var. notata from all 
three provinces. Prevalences ranged from 1.7% in wild quahogs 
from Pugwash, NS, in 1997, to SO'/r in moribund broodstock from 
the Ellerslie hatchery, PEl, in 1990 (Table 1). Of 3047 quahogs 
examined, 64 showed evidence of QPX infection i% P = 2,2) 
(Figure 2). Intensity of infection ranged from light to heavy. The 
size range of quahogs infected by QPX ranged from 18.3-92.5 mm 
(Table 1), The sex ratio of infected quahogs was 30 male: 1 8 fe- 
male: 16 undetermined (resting stage or immature), which was not 
significantly different from 1:1 (x". P > O.IOl. The sex ratio of 



uninfected clams, however, was significantly different from 1 : 1 
(996 male: 862 female: I 125 unidentified (resting stage or imma- 
ture): X". P < 0.005). 

Of all the infected clams, the most commonly infected tissues 
were the gills (34%). mantle (3l7f ) and gonads Ol'^r) (Table 3). 
The digestive gland and foot were less commonly infected (12 and 
5%, respectively). 

Seasonal Suney 1996-97 

No gross clinical signs were observed during necropsy of the 
quahogs collected for the seasonal survey. Clams from two of the 
four sites showed evidence of QPX infections: Wallace (1996 
only) and St. Andrews (1996 and 1997) (Table 2), The summer 
sample of quahogs from Wallace had a prevalence of 6,7'* QPX 
(light intensity). Quahogs from St. Andrews showed prevalences 
of QPX ranging from 3.3%^ (spring and fall, 1996), at light inten- 
sities, to 209^ (summer 1997) at heavy intensities (Figure 3). 



46 



MacCallum and McGladdery 




Figure I. Map of Atlantic Canada showing sampling sites positive for 
QPX from all surveys and diagnostic material examined. The circle 
denotes the 1959-63 QPX study of Drinnan and Henderson (1963), 
diamonds denote the 1990-98 diagnostic survey, stars denote the 1996- 
97 survey and triangles show sites with QPX in hatchery broodstock. 
The dashed lines represents the northern-most limit of M. mercenaria. 



The mean sample lengths of the quahogs examined (n = 715) 
ranged from 60.0 (± 10.7) to 83.5 (± 6.3) mm in 1996 and 61.9 (± 
9.9) to 83.2 (± 4,9) mm in 1997. It was difficult to tell whether the 
same cohorts were sampled over the two year seasonal survey. 
because quahogs grow slower, once mature, in cooler northern 
waters than in warmer waters to the south. The highest water 
temperatures occurred in August at all sites and temperature ranges 
(8-24'C) were relatively consistent between sites for both years 
(Table 2). All four sites had moderate to high salinities (20-329?() 
which were consistent over the survey period (Table 2). The high- 
est prevalence (20'}f ) was found in clams from St. Andrews in the 
summer of 1997 (Tabic 2). The second highest prevalence ( 13.3%) 
was found in the spring, 1997, sample. QPX was detected in one 
sample of clams from Wallace, in the summer of 1996 (6.7%). The 
temperature recorder on the Wallace bed recorded air tempera- 
tures, at low tide, as low as "C in May. 1996 and as high as 34 
"C in August. 1996 and 1997. The sex ratio of QPX-positive 
quahogs was 5 male:l female in 1996 and 8 males:4 females in 
1997, which was not significantly different from 1 : 1 (x". P > 0. 10. 
1996 and P > 0.25, 1997). The sex ratio of uninfected clams was 
173 male: 178 female: 1 unidentified (resting s(agc or immature) in 
1996. and 187 male: 159 female: 1 unidentified (resting stage or 
immature) in 1997. which was not significantly different from 1:1 
(X-, P>0.90. I996andx-. P>0.I0, 1997). Of the infected clams, 
the most commonly infected tissues were the gonads (28'7r ) and 
mantle (22%). although (he digestive gland and foot (17%) and 
gills (1 1%) also showed high levels of infection (Table 3). 



DISCUSSION 

QPX or QPX-like organisms were first found in Atlantic 
Canada in the late 1950's/ early 1960's in wild M. mercenaria 
from Neguac. NB (Miramichi River estuary) in the Gulf of St 
Lawrence (Drinnan and Henderson 1963). Prevalences ranged 
from 50% in weak and dead quahogs to 5% in apparently healthy 
quahogs (Drinnan and Henderson 1963). Accumulated mortalities 
in grow-out tests conducted between 1959 and 1960 ranged from 
60-90%' in native quahogs to 20-25% in apparently healthy qua- 
hogs transplanted from nearby Miramichi beds (Drinnan and Hen- 
derson 1963). QPX was not investigated further until the early 
1990's when it was found in moribund quahogs (15-30 mm in 
length) being conditioned for spawning at a hatchery in PEI 
(Whyte et al. 1994). The connective tissue and muscle were found 
to be infected with "an invasive eukaryote organism" identical to 
that described by Drinnan and Henderson ( 1 963 ) and was given 
the non-taxonomic acronym "QPX" for "Quahog Pararsite Un- 
known" (McGladdery et al. 1993; Whyte et al. 1994). 

QPX or QPX-like organisms have been found in quahogs from 
New Jersey in 1976 (Smolowitz et al. 1998) and more recently in 
quahogs from Virginia (Ragone Calvo et al. 1997. Ragone Calvo 
et al. 1998) and Massachusetts (Smolowitz ef a/. 1998). During the 
summer of 1995. 1.5-2 year old quahogs planted on aquaculture 
leases in Cape Cod, experienced mortalities with prevalences rang- 
ing from 10% in "non diseased" clams to 90% in di,sea.sed clams 
(Smolowitz et al. 1998). Cultured 1-2 year old clams (19-89 inm) 
from the eastern shore of Virginia ranged from 8-20% in 1996. to 
4-48%' in 1997. with associated mortalities estimated at 10-20% in 
the latter (Ragone Calvo et al. 1998). 

The Miramichi Estuary of the Gulf of St. Lawrence is the 
northern-most geographic limit of M. mercenaria. thus QPX does 
not occur in the St. Lawrence River as mentioned in Ford et al. 
(1997) and Smolowitz et al. (1998). Prevalences of QPX in M. 
mercenaria and M. m. var. notata in the 1990-98 diagnostic survey 
ranged from 1.7% in M. mercenaria in Nova Scotia, to 80% in 
broodstock being conditioned for spawning at the Ellerslie Shell- 
fish Hatchery. PEI (Table 1 ). No mortalities attributed to QPX 
have been found in wild quahogs in Atlantic Canada since the 
original cases reported by Drinnan and Henderson (1963). how- 
ever, open-water mortalities aie known lo have occurred without 
being investigated (Drinnan. pei's comm.). The highest prevalences 
of QPX recorded in the 1 990-98 diagnostic survey were in both 
cultured native and notata variety broodstock from all three Mari- 
time provinces (Table 1 ). The 1996-97 survey found 6.7% preva- 
lence of QPX in clams from Wallace. N.S. and 3.3-20% QPX in 
an isolated native population at St. Andrews. Prevalences in qua- 
hogs at both sites were comparable to those found in US wild 
clams (8-90%, Smolowitz et al. 1998, Ragone Calvo et al. 1997 
and 1998). 

The si/e range of infected quahogs in this study I'anged from 1 8 
lo 1 10 mm (Tables I and 2). Before 1998, the reported size range 
of QPX infected quahogs was > 35mm shell length. Despite their 
small size, the 18-25 mm cultured M. mercenaria from Vernon 
River. PEL had been in the field for one year and were approxi- 
malcly 1.5 years old (Burleigh pers comm.). Ford et al. (1997) 
examined tissue .sections of 2203 seed quahogs (< 1-20 mm and no 
more than a few months old) from 13 different hatcheries in six 
Stales. No evidence of QPX or QPX-like organisms was detected. 
QPX was also not detected in 756 hatchery-produced quahogs 
after a year of field grow-out (Ford el al. 1997), thus, it was 



QPX IN THE Northern Quahog 



47 



TABLE 2. 
Collection details and QPX results for 1996-97 survey 







Water 


temperature 


Lengths 














(°C) and salinity 


examined 




Prev. 


**Inf. 


***Sex Ratio 


Date 


Collection Site 


{"r,) at collection 


(mml 


No. 


(%) 


Levels 


(Infected Quahogs) 


2-5-96 


Wallace. NS 


8° 


25%<. 


63-78 


28 











21-5-96 


St Andrews, NB 


10° 


26%c 


58-83 


30 


3.3 


L 


IM 


5-6-96 


West River. PEI 


15° 


25%. 


69-91 


30 











11-6-96 


Shediac Bridge. NB 


15° 


26%. 


56-89 


30 











1-8-96 


Wallace. NS 


24° 


30%<. 


65-78 


30 


6.7 


L 


2M 


19-8-96 


West River. PEI 


22° 


26%c 


51-86 


30 











23-8-96 


St Andrews. NB 


22° 


26%o 


43-79 


30 


6.7 


M 


2M 


27-8-96 


Shediac Bridge. NB 


23° 


26%c 


57-81 


29 











1-10-96 


Wallace. NS 


10° 


20%o 


64-84 


30 











17-10-96 


West River. PEI 


10° 


23%o 


52-91 


30 











21-10-96 


St Andrews. NB 


11° 


26%o 


43-76 


30 


3.3 


H 


IF 


25-10-96 


Shediac Bridge. NB 


9° 


30%r 


73-95 


30 











27-5-97 


St Andrews. NB 


10° 


25%r 


53-87 


30 


13.3 


L 


3M:IF 


5-6-97 


Shediac Bridge, NB 


14° 


M%c 


38-101 


30 











5-6-97 


Wallace. NS 


10° 


26%c 


51-88 


30 











9-6-97 


West River. NS 


16° 


26%o 


50-95 


30 











13-8-97 


St Andrews. NB 


21° 


32%,. 


46-78 


30 


20,0 


H 


4M:2F 


18-8-97 


Wallace. NS 


24° 


31%<, 


66-93 


30 











26-8-97 


West River. PEI 


24° 


29%o 


50-93 


29 











29-8-97 


Shediac Bridge. NB 


21° 


31%<, 


54-99 


29 











9-10-97 


St Andrews. NB 


10° 


32%<, 


50-79 


30 


6.7 


H 


1M:1F 


16-10-97 


West River. PEI 


8° 


27%,, 


54-102 


30 











20-10-97 


Wallace. NS 


11° 


32%o 


71-93 


30 











24-10-97 


Shediac Bridge, NB 


10° 


32%. 


44-90 


30 













Total 








715 









** - H-heavy, M-moderate, L-lighi 

*** - M-male. F-female, U-undeterniined (resting/immature) 



concluded that hatchery-produced seed are unlikely to be infected 
by QPX. Conversely. Whyte et al. (1994) found QPX in infected 
hatchery-reared quahogs ranging from 15-30 mm in shell length. 
The report did not distinguish the exact size or age of infected 
quahogs. and no attempt was made to characterize the relationship 
between individual quahog size and presence of QPX (Whyte. 
pers, comm,). Due to colder growing conditions in the Gulf of St. 



+ 



■ N.R 

ap.E.1 

■ N.S. 



I 



m 



■nmefYcais) 

Figure 2. Historical and geographic summary of QPX in M. merce- 
naria and M. mercenaria variety notata from Atlantic Canada. 



Lawrence, compared with Massachusetts and Virginia, it is pos- 
sible that the < 20mm quahogs examined by Whyte et ai. (1994) 
could have been the same age as larger quahogs from further south. 
All QPX findings to date in the US have been from quahogs 
typically 1 to 2 years-old (Ragone Calvo et al. (1997 and 1998) and 
Smolowitz et al. (1998)), 

The taxonomic affinity of QPX is currently under investigation 
in both Canada and the U.S, (Smolowitz et al. 1998: Maas et al. 
1999). Whyte et al, (1994) suggested that the QPX was similar to 
the labyrinthulids and thraustochytrids. belonging to the Phylum 
Labyrinthomorpha (Pokorny 1985), Members of these groups are 
common saprophytes in marine environments (Porter 1990), and 
have also been reported to cause disease in a number of molluscs 

TABLE 3. 
Prevalence of QPX in different tissues of infected quahogs. 



Tissues 

Gill 

Mantle 

Gonad 

Digestive gland 

Foot 



1990-98 


1996-97 


percent of 


percent of 


nfected clams 


infected clams 


(n = 64) 


(n = 18) 


34 


11 


31 


22 


31 


28 


12 


17 


5 


17 



48 



MacCallum and McGladdery 



25 



^ 20 



«j 15 

c 

es 10 









1 


■ St. Andrews 
D Wallace 






1 






1 1 






■n 


1 


■ 


1 


r 


iT 


1 



May-96 Aug-95 OcI-96 Ma.v-97 Aug-97 Oct-97 

Time (months) 

Figure 3. Prevalence of QPX from the two year repeated survey 1996- 
97. Solid black represents St. Andrews (Sam Orr Pond), N.B.) clams; 
unfilled box represent Wallace, N.S. clams. Clams from Shediac 
Bridge, N.B. and West River, P.E.I, were negative for QPX. 



(Polglase 1980: McLean and Porter 1982; Jones and O'Dor 1983; 
Bower 1987a). One Labyrinthulid. Liibynnthidoides haliotidis, has 
been linked to mortalities of up to 100% of nursery-held juvenile 
abalone, Haliotis kamtschatkana. in British Columbia (Bower 
1987a). Further investigation found that L. haliotidis is transmitted 
directly from abalone to abalone by a flagellated zoospore stage 
(Bower 1987b). Motile zoospores have been identified in both 
Canadian (Whyte et al. 1994) and U.S. (Kleinschuster et al. 1998) 
QPX cultures, therefore, it is likely that QPX is also transmitted 
directly. The likelihood of direct transmission would also be ex- 
pected to be heightened in holding facilities or nurseries where 
clams are held in close proximity to each other in raceways, down- 
wellers or upwellers. Further research on QPX transmission to 
both M. mercenaria and M. m var. notatci is needed to ftilly un- 
derstand the epizootiological potential of this parasite. 

Smolowitz et al. (1998) noted thickened, retracted, light tan. 
swollen mantle edges in diseased clams from Cape Cod. Occa- 
sionally, yellow/tan nodules, 1—4 mm in diameter, were also ob- 
.served along the mantle edges or in the mantle areas adjacent to the 
anterior adductor muscle. Shell margins were chipped and diseased 
quahogs showed variable amounts of sand embedded between the 
mantle edge and shell (Smolowitz et al. 1998). Smolowitz el al. 
(1998) postulated that shell chipping was a result of quahogs at- 
tempting to close their shells on the sand and sediment caught in 
the quahog's mucus. Soft tissues and shells were examined for all 
clams used in this study, however, no gross pathological changes 
have been seen, to date, in infected quahogs from Atlantic Canada, 
including heavily infected individuals. 

Both sexes of quahog were infected with QPX . Prevalences in 
males were significantly higher than in females in the 1990-98 
survey, but no significant differences were found between males 
and females in the 1996-97 seasonal survey. Uninfected quahogs 
examined in both surveys had a sc\ ratio of 1:1. No diffcrejices 
between the sex o( infected clams have been reported elsewhere, to 
date. 

The most commonly infected tissues in infected clams from the 
1990-98 diagnostic survey were the gills iM'/i I. mantle (.M '/,) and 
gonad (.^K/r). Similar results were found in the 1996-97 seasonal 
survey (gonad-28'f and mantle-227f ). Smolowitz et al. (1998) 
found that the most commonly infected tissues of infected quahogs 
from Cape Cod were the mantle (917,) and gill (6.'^7f). Ragone 
Calvo c't ill. (1998) also found the mantle (ft.V;-; ) and gills (.^.S'f i to 
be ihe most tVcqucnIly inleclc(.l tissues in infected i.|ualu)gs from 



Virginia. The digestive gland (12-17%) and foot (5-17%) were 
less commonly infected in both 1990-98 and 1996-97 surveys. 
Smolowitz et al. ( 1998) also found the kidney (20-25%), adductor 
muscle (0-6%), foot (3-13%), digestive gland (0%), ganglia/ 
mantle nerves (0%) and palps (0%) to be less heavily infected. 
Ragone Calvo et al. (1998) also observed infections in the mus- 
culature of the foot, sinuses and connective tissue of the kidney 
and connective tissue of the digestive glands (4. 11, and 15%, 
respectively). Drinnan and Henderson (1963) found QPX in the 
gill, kidney, connective tissue, foot, and heart of infected quahogs 
from New Brunswick but did not differentiate between levels of 
infection and tissue site. Although not quantified for this study, we 
found no evidence of palp, nerve or adductor muscle infections. 

There are at least three environmental factors which may 
favour the proliferation of QPX in both hatchery and wild clams: 
i) stocking density; ii) water temperature; and iii) genetic suscep- 
tibility. Stocking density may have played an important part in the 
epizootic incident of QPX in wild quahogs from Neguac, N.B. 
(Drinnan 1961). The typical or natural stocking density of wild 
adult (> 20 mm) quahog populations in Atlantic Canada is ap- 
proximately 4-5 clams m"~ (T. Landry. Fisheries and Oceans 
Canada, pers. comm.). Historically some quahog farming opera- 
tions have planted seed (< 3 mm) at densities ranging from 357- 
43,01 1 m"- (Judson et al. 1977: MacPherson et al. 1978: Wither- 
spoon 1984) with no outbreaks of QPX reported. To date, only one 
report by Kraeuter et al. ( 1998) has examined the effects of plant- 
ing density on proliferation of QPX. Juvenile quahogs (< 10 mm) 
from New Jersey were planted on intertidal and subtidal sites at 
three densities; 215. 430. and 860 clams nr" per plot. The preva- 
lence of QPX increased during the four-month experiment, but no 
significant effect, due to density or location, was detected (Kraeu- 
ter et al. 1998). 

Water temperature and/or salinity may also be significant fac- 
tors influencing the prevalence of QPX. All four sites in the sea- 
sonal survey experienced relatively similar temperature regimes, at 
the time of collection, ranging from 8 °C (May, 1996, and October, 
1997) to 24 °C (August, 1996-97). Salinities ranged from 25-32%r 
between 1996 and 1997. The clam beds at St. Andrews, Shediac 
Bridge and West River are all sub-lidal ( 1-3 m depth depending on 
tide level), whereas the Wallace site is completely exposed during 
each low tide. As a result, seasonal temperatures at the Wallace 
site, ranged from 8-28 °C (from May to October, 1996 and 1997). 
with air temperatures reaching as high as 34 "C at low tide. QPX 
was detected at the St. Andrews location in temperatures ranging 
from 10-22 C and salinities ranging between 25-32^^1. Histori- 
cally, water temperatures at the St. Andrews site (Sam Orr Pond) 
range from -0.1-25 °C (Medcof 1961, S.M.C. Robinson. Fisheries 
and Oceans Canada, pers. comm.). The single QPX infection de- 
lected at Wallace occurred in August. 1996. when the waler tem- 
perature was 24 "C and salinity was 30'/t'(. 

Ragone Calvo et al. ( 1998) collected quahogs from 1 S different 
sites in Chesapeake Bay and coastal Virginia, where salinities 
ranged from 15 to ?i47ti. QPX was only detected in clams from 
three coastal lagoons, where salinities ranged from 30 to 34%f 
(Ragone Calvo et al. 1998). Theses authors point out that the 
absence of QPX from more moderate salinities (l5-25%f) may 
have been related to a limitation in QPX's salinity tolerance or 
have reflected sampling bias (Ragone Calvo et al. 1998). In sea- 
sonal colleclions from one Virginia coastal site, between July. 
IWfi and June. 1W7. Raizone Calvo et al. (19981 observed the 



QPX IN THE Northern Quahog 



49 



highest prevalences and most severe infections in November and 
May samples. Smolowitz et al. (1998) reported that quahog mor- 
talities in Massachusetts, associated with QPX infection, were 
highest in August and October. Temperature and salinity are 
known to be related to proliferation of other bivalve parasites such 
as Perkinstis niariniis. Haplosporidium costale and Haplospo- 
ridiiim nelsoni (Bower et al. 1994, Ford et al. 19991, thus it is 
possible that QPX proliferation and pathogenicity may also be 
influenced by temperature and/or salinity. 

Clam harvesting practices may also influence QPX prolifera- 
tion. Harvesting of quahogs in Atlantic Canada has, traditionally, 
been done by hand (forks, tongs and rakes), although hydraulic 
harvesters are also used (Bourne 1989). The population of quahogs 
in Neguac, N.B.. were harvested using an escalator harvester when 
mortalities started to increase, both in air storage and at Hay Island 
holding beds, between 1957 and 1959 (Drinnan 1960). Although 
no clear association between harvest methodology and QPX has 
been determined, its effect on physiological stress and defense 
capability seems worth investigating further. 

In conclusion, QPX seems to be ubiquitous in both wild and 
cultured quahogs from the Maritime Provinces and is reported for 
the first time in quahogs from the Bay of Fundy. In light of past 
mortalities associated with this parasite, especially in hatchery 
broodstock being conditioned for spawning, QPX may present a 
significant challenge to development of quahog aquaculture in our 
region. The dynamics of infection and pathogenicity under differ- 
ent holding and handling conditions require more investigation to 
manage pathogen proliferation. This is especially important as 
uninfected populations seem to be few. if any, in Atlantic Canada, 
making selection of QPX-free broodstock an impractical solution. 

ACKNOWLEDGMENTS 

We wish to thank Dr. S.M.C. Robinson, J. Martin, R. Chandler 
(Dept. Fisheries and Oceans, Canada), E. Semple (Wallace, N.S.), 
J. and R. Caissie (Caissie Cape, N.B.), B. Murley (New Haven, 
P.E.I. ), P. Burleigh, N. McNair (P.E.I. Dept. Fisheries and Envi- 
ronment) and D. Methe (N.B. Dept. of Fisheries and Aquaculture) 
for their assistance with collections. Dr. B.A. MacDonald (Uni- 
versity of New Brunswick. Saint John) and R.E. Drinnan (Mus- 
quodoboit Harbour, N.S.) kindly reviewed early draft manuscripts. 
Mrs. M. Stephenson, Dr. M. Maillet (DFO, Canada) and W. Morris 
(U.N. B.S.J) provided valuable technical and statistical support. 
This project was funded in part by the New Brunswick Alternate 
Shellfish Aquaculture Species Project, part of a Canada/New 
Brunswick Cooperation Agreement on Economic Diversification. 

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X ("QPX") of hard-shell clams, Mercenaria mercenaria and M. mer- 
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Bower, S.M. 1987a. Lahyrinthidoides hallolidis n.sp. (Protozoa: Laby- 
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Bower. S.M., S.E. McGladdery & I.M. Price. 1994. Synopsis of infectious 
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Chanley, P.E. 1961. Inheritance of shell markings and growth in the hard 
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Drinnan, R.E. 1960. Quahog Mortalities at Neguac. N.B. Annual Report 
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Drinnan. R.E. 1961. Mortalities in Quahogs at Neguac, N.B. Annual Re- 
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disease organisms in Neguac quahogs Annual Report No Bll. Bio- 
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Ford. S.E.. R Smolowitz. L.M. Ragone Calvo, R.D. Barber & J.N. 
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521 

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Howard, D.W. & C.S. Smith. 1983. Histological Techniques for Marine 
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setts), 96p. 

Jones, G., & R.K, O'Dor. 1983. Ultrastructure observations on a Thraus- 
tochytrid fungus parasite in the gills of squid Ulle.x dlecebrosus 
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Judson, W.I., R.C. MacPherson, P.S. Stewart & W.N. Carver. 1977. Cul- 
ture of the quahog from hatchery-spawned seed stock. Prince Edward 
Island Dept. Fish. Tech. Rept.185. lip. 

Kleinschuster. S.J., R. Smolowitz & J. Parent. 1998. //; Vitro Life Cycle 
and Propagation of Quahog Parasite Unknown. / Shell. Res. 17:75-78. 

Kraeuter, J.N., S.E. Ford & R. Barber. 1998. Effects of planting density and 
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Leibovitz, L.L. 1989. Chlamydiosis: a newly reported serious disease of 
larval and postmetamorphic bay scallops, Argopeclen irradians (Lama- 
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Maas, P.A.Y., S.J. Kleinschuster, M.J. Dykstra, R. Smolowitz & J. Parent. 
1999. Molecular characterization of QPX (Quahog Parasite Unknown), 
a pathogen of Mercenaria mercenaria. J. Shell. Res. 18:561-567. 

MacPherson, R., P.S. Stewart & W.N. Carver. 1978. Culture of the quahog 
from hatchery-spawned seed stock 1975-1978. Prince Edward Island 
Dept. Fish. Tech. Rept.189. 14p. 

Medcof, J.C. 1961. Trial introduction of European oysters iOslrea ediilis) 
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sociation 50:1 13-124. 

McGladdery, S.E., R.E. Drinnan & M.F. Stephenson. 1993. A manual of 
parasites, pests and diseases of Canadian Atlantic bivalves. Canadian 
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McLean, N. & D. Porter. 1982. The yellow spot disease of Tritona di- 
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of the Truastochytreaceous parasite by host ameobocytes. J. Parasitol. 
68:243-252. 

Pokomy, K.S. 1985. Phylum Lahyrimhomorpha. hi: J.J. Lee, S.H. Hunter 
& E.C. Bovee (eds). Illustrated Guide to the Protozoa Society of Pro- 
tozoologists, Lawrence, KS. pp. 318-321. 

Polglase, J.L. 1980. A preliminary report on the Thraustochytrid(s) and 
Labyrinthulid(s) associated with a pathological condition in the lesser 
octopus (Eledone cirrhosa). Bot. Mar. 23:699-706. 

Porter. D. 1980. Phylum Labyrinthomycota. /«: L. Margulis. J.O. Coriiss. 
M. Meilkonian & D.J. Chapman (eds). Handbook of Protoctista. (Jones 
& BartleU, Boston), pp. 388-398. 



50 



MacCallum and McGladdery 



Ragone Calvo, L.M., J.G. Walker & E.M. Burreson. 1997. Occurrence of 
QPX. Quahog Parasite Unknown in Virginia hard quahogs, Mercemiria 
mercenaria. J. Shell. Res. 16:335-336 

Ragone Calvo. L.M.. J.G. Walker & E.M. Burreson. 1998. Prevalence and 
distribution of Quahog Parasite Unknown, in hard quahogs. Merce- 
naria mercenaria. in Virginia. USA. D/.v. Ac/iiar. Org. 33: 209-219. 

Smolowitz. R. 1998. QPX. a Protozoan parasite of hard quahogs Proceed- 
ings from the 3rd International Symposium on Aquatic Health August 
30-September 3. 1998. Baltimore. Maryland, pi 46 

Smolowitz. R.. D. Leavitt & F. Perkins. 1998. Observations of a Protistan 



disease similar to QPX in Mercenaria mercenaria (hard quahogs) from 
the coast of Massachusetts. / of Invert. Path. 71:9-25 

Whyte. S.K.. R.J. Cawthorn & S.E. McGladdery. 1994. QPX (Quahog 
Parasite X). a pathogen of northern quahog Mercenaria mercenaria 
from the Gulf of St Lawrence. Can. Dis. Aq. Org. 19:129-136. 

Witherspoon. N.B. 1984. An investigation of the aquaculture potential of 
the bay quahog {Mercenaria mercenaria). the American oyster {Cras- 
soslrea virginica). and the blue mussel {Mytiliis edulis) in three estu- 
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Zar. J.H. 1984. Biostatistical Analysis (Prentice Hall, New Jersey). pp718. 



Journul of Shellfish Research. Vol. 19, No. 1. 51-56. 2000. 

AGE AND SIZE OF MERCENARIA MERCENARIA IN TWO SISTERS CREEK, 

SOUTH CAROLINA 



ARNOLD G. EVERSOLE,' NATHALIE DEVILLERS,' and 
WILLIAM D. ANDERSON- 

^ Department of Aquacidture. Fisheries and Wildlife 

Clemson University 

Clemson, South Carolina 

29634-0362 
'South Carolina Department of Natural Resources 

Charleston. South Carolina 

29422-2559 

ABSTRACT Northern quahogs. Merceiiaria mercenaria (L.). were sampled from four sites in Two Sisters Creek, South Carolina. 
Shell lengths (SL) were measured and ages estimated from increments in shell sections. Mean SL of individuals collected at the two 
sites near the mouth of the creek were significantly larger than those collected in the upper reaches of the tidal creek. The back- 
calculated mean SL. however, were similar among sites within most age classes. Mean ages of individuals near the mouth were 
significantly older than those from the upper reaches. Differences in the population age structure were also observed among sites. 
Several factors are explored to explain the upstream pattern of decreasing SL and ages of quahogs in Two Sisters Creek. 

KEY WORDS: Mercenaria. quahogs. clams, age. size, growth 



INTRODUCTION 

Commercial densities of northern quahogs. Mercenaria merce- 
naria (L.), occur in small creeks that dissect extensive tidal 
marshes of South Carolina and Georgia (Anderson et al. 1978, 
Walker 1987, 1989). Two Sisters Creek, South Carolina, which is 
representative of this type of habitat, became part of a State Shell- 
fish Ground (SSG-134) in 1986. Reported landings from SSG-134. 
which also included Ashepoo River, Rock Creek, Atlantic Intra- 
coastal Waterway, and Ashepoo-Coosaw Cut, averaged only 145 
bags/year before an exploratory survey of quahog resources in 
Two Sisters Creek (S.C. Department of Natural Resources, unpubl. 
data). Resource managers perceived that quahog exploitation was 
limited before and after Two Sisters Creek became part of SSG- 
134. The mean (± SD) shell lengths (anterior posterior axis, SL) of 
quahogs collected during an earlier exploratory survey of May 6, 
1987 indicated that individuals sampled from two sites nearer the 
mouth were larger (88.1 ± 10.37 mm and 87.1 ± 10.30 mm) than 
those collected from a mid-way site (69.0 + 14.81 mm) and a site 
farther up Two Sisters Creek (61,6 ± 19.26 mm). A similar trend 
was observed in Christmas Creek. Cumberland Island. Georgia. 
where relatively higher numbers of larger quahogs (i.e., chowders) 
were found near the creek's mouth than in the upper reaches of the 
tidal creek (Walker 1987). Differences in quahog size among sam- 
pling sites could have resulted either from variations in growth rate 
or age of the respective populations. 

The objectives of this study were to test the null hypotheses that 
growth rate and age were similar among quahog populations in- 
habiting four different upstream sites in Two Sisters Creek, South 
Carolina (Fig. I ). Age estimations, based on annual growth incre- 
ments within the shell (e,g„ Arnold et al, 1991, Jones et al. 1990, 
Peterson et al. 1985), were used to compare age and SL of differ- 
ent-aged quahogs from the four sample sites in Two Sisters Creek. 



MATERIALS AND METHODS 



Study Sites 



Quahogs were sampled from four sites within Two Sisters 
Creek, South Carolina, on February 25. 1994. The site closest to 



the mouth was designated site 1, and sites 2, 3, and 4 were located 
progressively farther up the tidal creek (Fig. 1 ). Midchannel depths 
of the four sites at flood tide were 7.60 m, 8.20 m, 4.30 m. and 3.35 
m. respectively. Tidal range was about 2 m. Bottom water tem- 
peratures and salinities at the time of collection ranged from 12 to 
14 °C and 21 to 25 gl"'. An estimate of bottom types indicated that 
sites 1, 2, and 3 were a mixture of mud, sand, and shell; whereas, 
site 4, although similar, seemed to contain more clay. 

Sampling 

Quahogs were collected at flood tide from subtidal sites with a 
hydraulic escalator harvester configured with a Maryland-type 
head. The mesh size of the escalator conveyor would retain qua- 
hogs >32 mm SL if not covered by mud or shell. In this event, 
smaller quahogs would be harvested. Subtidal bottoms at depths of 
2-8 m were sampled across the width of the creek. Sampling at 
each site continued until sample sizes were ^ 100 individuals. 
Quahogs were returned to Clemson University and frozen until 
analysis. Shell length and height (lateral axis. SH) were measured 
with calipers to the nearest 0.1 mm. After measuring, individuals 
were categorized according to the following commercial size 
groups: sublegals. < 44.4 mm SL; littlenecks, 44.4-67.9 mm SL; 
cherrystones, 68-78 mm SL; and chowders > 78 mm SL, A sub- 
sample of 50 quahogs per site, representative of the distribution at 
that site, was used for aging. 

Age Determination 

Quahogs were shucked, and the better valve was selected for 
sectioning. The valve of larger shells was cut from the ventral 
margin through the umbo, with a high-speed geological saw 
mounted with a diamond blade. Smaller shells were embedded in 
resin epoxy to avoid fracture during the sectioning (Kennish et al. 
1980). Similarly, embedded shells were cut with a slow-speed saw 
mounted with a high-density diamond blade. Valves were polished 
with various grit carborundum papers and then etched in \9c hy- 
drochloric acid. Age was obtained by counting translucent (dark) 
bands on the polished surface of a cut valve. Bands were counted 



51 



52 



EVERSOLE ET AL. 




ST. HELENA SOUND 
Figure 1. Sampling sites in Two Sisters Creeli, South Carolina. 

three times with two bhiid counts by the saine observer. Values 
difficult to read were then washed and exposed to acetone before 
pressing against an acetate sheet (Kennish et al. 1980, Ropes 
1984). The age of these clams was obtained by counting bands 
using a microfilm projector. A pattern of alternating translucent 
(dark) and opaque (light) increments on sectioned valves of known 
aged quahogs cultured in South Carolina waters was used to verify 
the formation of annual shell growth increments in the study (De- 
villers 1994). 

Back-Calculated Shell Length 

Shell heights from the umbo to the translucent increment for 
each age increment in the sectioned valves of shells were measured 
to the nearest 0.1 mm (see Jones et al. 1990). Measurements were 
limited to the first 12 increments, because of the difficulty asso- 
ciated with correctly measuring small increments thereafter. These 
measurements were then converted to SL using the equation (Ever- 
sole, unpubl. data): 



In SL 



.049.^ In SH - 0.0136; r- = 0.997. n 



1.171 



Statistical Procedures 



Analysis of variance (ANOVA) was used to determine signifi- 
cant differences in SL between the field sample and subsamples. 
Significant differences in age, SL. and back-calculated SL were 
also determined by ANOVA. Paired means were compared with 
the least significant difference (SAS I98.'i). Alpha level was set at 
0.05 for these analyses. 



RESULTS 



Shell Lengths 



Mean SL of the quahogs sampled from sites 1 and 2 were 
similar but significantly (P < 0.03) larger than those animals 
sampled at sites .3 and 4 (Table 1 ). Individuals from site 3 were 
also significantly larger than those quahogs sampled at site 4. The 
mean SL and ranges of these quahogs used for age determination 
were similar to that observed in the field sample (Table 1 ). 

The frequency distributions ot commercial qualiog sizes col- 
lected trom the four sites are presented in Figure 2. Chowders 
dominated the collections at site I (94.2%). site 2 (96. 2'*). and site 
3 (69.8'/f ). Site 4 contained 37.8% littlenecks and similar percent- 
age of cherrystones (26..3%) and chowders (27.8%). Only 1.0%. 



TABLE L 

Mean (± SD) and range (in parenthesis) of the shell lengths (mm) of 

Mercenaria mercenaria from four sites in Two Sisters Creek, South 

Carolina. Values in a column not sharing the same letter superscript 

are significantly different at P < 0.05. There was no significant 

difference between field and subsample mean SL. 



Field Sample 



Subsample 



Site 



N 



Shell Length 



104 
104 
106 
151 



93.27 ± 11.2-V' 
(41.9-110.7) 

94.27 ± 1 1 .60-' 
(36.0-117.5) 

79.05 ± 17.57" 
(32.2-101.9) 

67.84 ± 14.32' 
(33.2-97.6) 



N 


Shell Length 


50 


93.08 ±12.68" 




(41.9-110.7) 


50 


94.10+ 11.32" 




(37.7-108.8) 


50 


78.39 ± 17.81" 




(32.2-100.*) 


50 


67.92 ±14.59' 




(33.2-93.5) 



' Site 1 was closest to the mouth while sites 2, 3 and 4 were progressively 
further upstream in Two Sisters Creek. 

1.9%, 8.5%, and 7.9% of the quahogs were sublegal size (< 44.4 
mm SL) in collections from sites 1. 2. 3, and 4. respectively. 

Age 

The oldest age of the sampled quahogs was 29 years from site 
1. and the youngest was 1 year from sites 2 and 4 (Fig. 3). Sig- 
nificant differences (P s 0.05) were detected among the four sites 
in the average age of quahogs. Individuals on average (± SD) from 
site 2 ( 16.7 ± 4.70 years) were older than animals from site 1 (14.9 
± 5.37 years) and in turn, quahogs from site 3 (8.4 ± 3.96 years) 
and site 4 (4.7 ± 2.30 years) differed from each other and were 
significantly younger than those from sites 1 and 2. The range of 
ages in the sample from site I was 28 years with several (n = 10) 
unrepresented age classes in the distribution (Fig. 3). The distri- 
bution of ages from site 2 was similar (range and number of 
missing age classes) but differed from site 1 in having an obvious 
dominant age class at 15 years. The range of ages from site 3 





100 




80 




60 


? 


40 


>> 


?n 


o 




c 





0) 




1 




c 


lOOi 


0) 






Site 2 
(n=104) 



Site 3 
(n = 106) 



80 

I6C 
40 
2C 
- 



(n = 151) 



11 



■ ■■ 



y.' 









.6* 



Figure 2. Relative fre(iuency of commercial sizes of Mercenaria mer- 
cenaria from four sampling sites in Two .Sisters Creek, South Carolina. 
ComnuTcial sizes were suhlegals (< 44.4 mm SLl, littlenecks (44.-Mi7.9 
mm SI.), cherr\ stones (68-78 mm SI.) and chowders (> 78 mm SL). 



Age and Size of Quahogs 



53 



30- 








20 




Site1 




10 


.»««. . 


ILlUllL^ 


•-, 



2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 



Site 2 




2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 



Site 4 




2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 



Age (years) 

Figure 3. Age structure of 50 Mercenaria mercenaria collected from 
each of four sites in Two Sisters Creek, South Carolina. 



encompassed 18 years with six missing age classes: whereas, those 
animals aged from site 4 spanned 10 years with only one missing 
age class at 9 years. None of the quahogs sampled from site 4 were 
older than 10 years; whereas. 90%. 96% .and 22% of quahogs from 
sites 1. 2, and 3 were older than 10 years of age. 

Back-Calculated Shell Lengths 

The back-calculated mean SL of quahogs from sites 1. 2. and 3 
were similar in size from age 1 through 11 years (Table 2). One 
significant difference among the four sites occurred from ages 1 to 
4 years when quahogs from site 4 were significantly larger (P :£ 
0.05) than those quahogs from sites 1. 2. and 3. The only other 
significant size difference in back-calculated SL occurred at 12 
years (Table 2). Quahogs reached reproductive size (35 mm SL, 
Eversole in press) between 1 and 2 years and commercial size 
(44.4 mm SL) between 2 and 3 years. 

DISCUSSION 

Comparison of quahog sizes between this and other studies 
needs to be done with caution because of different sampling gear 
efficiencies and the common problem of the under representation 
of small individuals in samples (Fegley. in press). Although 
Walker (1987, 1989) used a different collection method, he did 
collect a similar range of sizes as those collected in this study and 
observed that chowders (> 78 mm SL) were the dominant com- 
mercial size class in 43% of the 40 sites sampled in Georgia 
waters. He also determined that chowders were more abundant in 
areas with little or no harvesting; whereas, littlenecks were more 
abundant in heavily fished areas. In a statewide survey of quahog 
habitat, littlenecks were found to be the most abundant commercial 
size class in South Carolina, which has a viable fishery (Anderson 
et al. 1978). In Two Sisters Creek, which has not been extensively 
harvested, the dominant commercial size was the chowder in three 
of the four sites sampled. Greene and Becker (1978). Malinowski 
(1985). Rice et al. (1989). and Walker (1989) have suggested that 
the gear used to harvest quahogs is biased towards the larger sizes 
resulting in differential removal of larger individuals and a shift in 
the population structure toward smaller commercial sizes. Con- 
versely, it is anticipated that the larger commercial sizes would 



TABLE 2. 

Mean (± SD) of the back-calculated shell length (mm) by age (years) of Mercenaria mercenaria from four sites' in Two Sisters Creek, South 
Carolina. Values in rows not sharing the same superscript are significantly different at P < 0.05. 



Site 1 



Site 2 



Site 3 



Site 4 



Age 



1 

2 

3 

4 

5 

6 

7 

8 

9 

10 

11 

12 



50 
50 
49 
48 
47 
46 
46 
46 
46 
46 
45 
40 



Mean SD 



27.4 


± 6.95-' 


47.2 


± 9.28" 


61.8 


± 8.69" 


72.2 


± 8.08" 


79.4 


± 7.60" 


83.8 


± 7.29" 


88.2 


±7.21" 


92.0 


± 7.24" 


95.8 


±7.01-' 


97.3 


± 7.49" 


99.9 


± 7.74" 


101.7 


± 8.40" 



50 
50 
49 
49 
48 
48 
48 
48 
48 
48 
48 
48 



Mean SD 



28.1 


± 6.73" 


49.6 


± 9.09" 


62.3 


± 7.86" 


72.0 


± 6.72" 


79.0 


± 6.23" 


84.4 


± 6.75" 


88.6 


± 6.85" 


92.3 


±7.22'' 


94.8 


±7.16" 


97.5 


± 7.52" 


00.2 


± 7.76" 


02.5 


± 7.90" 



50 
50 
44 
41 
41 
41 
39 
34 
26 
14 
11 
7 



Mean SD 



27.3 


± 9.35" 


47.4 


± 9.00" 


63.4 


±9.27" 


74.3 


± 8.87" 


80.4 


±9.12" 


84.8 


± 9.34" 


88.6 


± 8.83" 


91.8 


± 9.24" 


93.2 


± 8.76" 


93.0 


± 6.79" 


95,4 


±7.12" 


95.6 


± 8.45" 



50 
46 
38 
32 
28 
20 
12 

6 

1 

1 



Mean SD 



32.4 ± 6.60" 
52.1 ±8.31" 
65.9 ± 8.07" 

76.1 ±8.54" 
81.9 ±8.99" 
87.3 ±8.15" 

91.2 + 8.72" 
95.7 ± 8.23" 

101.4 
102.3 



Site 1 was closest to the mouth while sites 2, 3 and 4 were progressively farther upstream in Two Sisters Creek. 



54 



EVERSOLE ET AL. 



accumulate in areas not heavily harvested. This accumulation may 
explain why sites I through 3 in Two Sisters Creek were domi- 
nated by chowders (Fig. 2), but it does not adequately explain the 
dominance of littlenecks at site 4, which would be the least likely 
of the four sites to be commercially harvested or poached because 
of its size and location. In addition to the affect of harvesting, size 
and age structure of populations are also influenced by growth 
rates, recruitment, and mortality (Cerrato 1980). Because quahogs 
at site 4 grew at the same rate or faster than the quahogs at the 
other three sites, different growth rates can not solely be used as an 
alternative hypothesis for explaining differences in sizes among 
sampling sites. 

Annual cycles of shell growth increment formation have been 
observed in shells of quahogs sampled from Rhode Island to 
Florida (Arnold et al. 1991. Fritz and Haven 1983, Jones et al. 
1989, 1990, Kennish 1978. Peterson et al. 1985) and from South 
Carolina (Devillers 1994). Mean age determined from sectioned 
shells revealed that animals sampled from the sites nearest the 
mouth of Two Sisters Creek were significantly older than indi- 
viduals collected from site 4 in the upper reaches of the tidal creek. 
Collections from sites 1 and 2 closest to the mouth also contained 
the oldest quahogs and the widest spread of ages. Differences in 
mean age and age frequency distribution among the sampling sites 
could have resulted from sampling error (e.g.. small and under 
representative samples); however, on two separate sampling occa- 
sions and using the same gear, quahog size (age) decreased from 
sites near the mouth to upstream sites in Two Sisters Creek. If 
sampling error occurred, it was similar among sites and sampling 
occasions. 

The absence of quahogs older than 10 years in the collection 
from site 4 may have been the consequence of a catastrophic event, 
intense predation or the recent successful establishment of the 
population. Low salinity periods resulting from hurricanes are re- 
ported to cause extensive quahog mortalities (Wells 1961). Al- 
though site 4 is more likely to be influenced by a catastrophic 
event than the other three sampling sites because of its smaller 
size, we have no evidence to indicate such an event occurred in this 
section of the coast 10 years ago. Furthermore, it is unlikely that 
such a large-scale event would have a stratified effect over such a 
restricted area as from site I to site 4. 

The subject of settlement and postsettlement roles in defining 
macroinvertebrate soft-sediment communities has been exten- 
sively reviewed by Butman (1987). Olafsson et al. (1994), and 
Snelgrove and Butman (1994). Although these authors discuss 
several factors important in defining adult assemblages in soft 
sediments, it has not been clearly established whether adull spatial 
patterns result from differential larval settlement, differential post- 
larval survival, or redistribution (Armonies 1996, Bachelet et al. 
1992, Peterson 1986, Wilson 1990). 

Existing data indicate that hydrodynamic processes play a ma- 
jor role in determining the settlement of bivalves in soft marine 
sediments (e.g.. see the review by Butman 1987). Near-bottom 
hydrodynamic fortes determine the fate and tlux of bivalve larvae 
over a patch of bottom. These forces are particularly important in 
the case with M. merceiuuia larvae because of their weak swim- 
ming ability (Bachelet et al. 1992). M. nwrcciKiriii exhibited pas- 
sive sctlleiiient when exposed to different sedimenl types in still 
and tlume-tlow water tests (Butman 1987. Butman et al. 1988). 

Field studies evaluating the importance of hydrodynamics to 
recruitment are few (e.g., Carriker 1961, Mitchell 1974, Petersen 
1986, Pratt 19.53. Wilson 1990). Pratl ( 1953) provided the earliest 
suggestion that the distribution of quahogs was similar to the sedi- 



ment panicles in Narragansett Bay. Rhode Island, implying hy- 
drodynamic processes were important in quahog distribution. He 
concluded from measurements of current patterns that early stage 
larvae coincided with the dense assemblages of adults and that 
hydrographic processes mixed and transported the larvae with time 
to potential settlement sites. Carriker (1961) commented that the 
mo.st striking feature of the horizontal distribution of larvae in 
Little Egg Harbor, New Jersey, was its unevenness and as a con- 
sequence, quahogs set in areas that did not have adults. After 
studying quahog abundance and distribution in Southampton Wa- 
ters. England. Mitchell (1974) came to a similar conclusion that 
the distribution of adult quahogs is in part controlled by tidal 
transport of the larvae produced by spawning beds. Mitchell 
(1974) also hypothesizes that variation in recruitment among years 
in different sites in Southampton Waters was related to the suc- 
cessful transport and settlement of competent larvae. 

Andrews ( 1983) observed that most of the oyster larvae carried 
upriver during flood tide were transported down river during ebb 
tide with the exception of those few oyster larvae trapped upriver 
in oyster beds and small tidal creeks of James River. Virginia. 
Andrews (1983) also postulated that upriver entrainment was more 
successful in systems with low flushing rates than highly flushed 
systems. The four sites in Two Sisters Creek, because of their 
channel width and depth, have different flushing rates, with site 4 
having the highest projected rate of the sites sampled. Quahog 
larvae produced in the main body of Two Sisters Creek probably 
could have been entrained in a tidal excursion at site 4. However, 
considering the patchy distribution of larval quahogs and the short 
window competent larvae have to set at slack tide (Carriker 1961. 
Armonies 1996). the probability of setting before being flushed 
from the small tidal creek was probably low. If entering and setting 
larvae survived predation pressures, perhaps a resident population 
of quahogs would have been established and served as a source of 
larvae for future recruitment at site 4. Because quahogs have a 
tendency to spawn at ebb slack water and be transported upstream 
with the subsequent flood tide (Carriker 1961), larvae from an 
established population at site 4 would have an increased probabil- 
ity of being retained in the lidal creek and recruiting to the popu- 
lation. 

Another explanation for the different age distributions of qua- 
hogs in Two Sisters Creek involves the resuspension and distri- 
bution of postlarval individuals. Shifts from the initial distribution 
of recently settled Macoma hahhica have been observed in the 
Wadden Sea (Armonies and Hellwig-Armonies 1992). Although 
postlarval M. meirenarici possess a temporary byssus thread (Car- 
riker 1961). it can be released or broken resulting in dislodgment 
and resuspension by water flov\ (Butman et al. 1988). Resettlement 
of postlarval quahogs at site 4 in Two Sisters Creek would also 
require the appropriate hydrodynamic forces for transport and the 
subsequent survival of post larvae. 

Predation helps shape quahog population structure (Bricelj 
1993) by selecting the smaller (younger) moic \ulnerable indi- 
viduals in the population (Whetstone and Eversole 1978. 1981 ). Of 
the suite of predators consuming quahogs (Gibbons and Blogo- 
slawski 1989), crabs are the most important predators in South 
Carolina (Whetstone and Eversole 1978). Crab-related mortalities 
up lo lOO'/f were observed in juvenile quahogs planted in unpro- 
tected sites in Georgia and Florida (Men/el and Sims 1962. God- 
win 1968). Quahog survival is improved if small individuals are 
provided some protection or if predators are removed (Eldridge et 
al. 1979. Peterson 1982). Greene and Becker (1978) observed an 
increase in quahog recruitment after a severe winter reduced the 



Age and Size of Quahogs 



55 



number of blue crabs in Great South Bay, New York. Peterson 
(1982) demonstrated that the roots and rhizomes of seagrasses 
provide protection for infaunal species such as quahogs from some 
predators. Both Peterson (1982) and Wilson (1990) concluded that 
as much as 507c of the difference in quahog density between 
vegetated and unvegetated areas was attributable to enhanced post- 
larval survival. There is adequate information to indicate that post- 
settlement processes (predation) play an important role in inver- 
tebrate populations in soft marine sediments (see Olafsson et al. 
1994 review). Although the distribution and abundance of preda- 
tors within Two Sisters Creek could have played a role in the age 
distribution of quahogs among the four sites, we have no evidence 
to indicate predators either eradicated all the quahog sets 1 l-t- years 
ago or selectively preyed on the older, larger individuals in site 4. 

The maximum ages of quahogs are lower in faster-growing 
populations in southern latitudes along the United States coast than 
those observed in the slower-growing, more northerly populations 
of quahogs (Ansell 1968, Jones et al. 1990). Fewer quahogs would 
be expected to attain an older maximum age in site 4 if the faster- 
growing individuals at this site died at a younger age than at the 
other sites. Differential mortality of the faster-growing quahogs 
also helps explain why fewer chowders were observed in site 4 
than in the other sites. 

Our results illustrate that the differences in quahog sizes among 
sampling sites in Two Sisters Creek was attributable to different 



age structures at the four sites. Quahogs collected from the upper 
reaches of tidal creek (site 4) were younger than those collected 
downstream, and none of the quahogs from site 4 was older than 
10 years; whereas, the oldest quahogs from sites 1, 2, and 3 were 
29. 27, and 19, respectively. Although the first steps in establishing 
a population of quahogs involves settlement, the importance of 
hydrodynamic processes or predation (mortality) effects on post- 
larvae cannot be underestimated. Unfortunately, we have very 
little data to support a hypothesis to explain the observed age 
distribution in Two Sisters Creek. Future efforts to investigate 
quahogs recruitment should include an integrated approach that 
simultaneously considers factors such as hydrodynamic processes 
and post-settlement survival. Unraveling these causes of recruit- 
ment variation will be crucial to understanding the distribution and 
abundance of quahogs. 

ACKNOWLEDGMENTS 

The authors thank Chris Kempton for his help in cutting shells 
and preparing illustrations. Special thanks go to Dr. L. W. Grimes 
for his help with statistical analysis. Drs. Randy Walker and John 
Kraeuter generously provided comments on an earlier draft, which 
greatly improved the manuscript. This research was supported by 
the S.C. Agriculture Experiment Station, Clemson University, and, 
as such, is Technical Contribution No. 4576. 



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Jounml of Shellfish Research. Vol. 19. No. 1. 57-62, 2000. 

MODELING GEODIJCK, PANOPEA ABRUPTA (CONRAD, 1849) POPULATION 

DYNAMICS. I. GROWTH 



A. HOFFMANN,' A. BRADBURY," AND C. L. GOODWIN' 

Washington Department of Fish & Wikllife 

600 Capitol Way North 

Olympia. Washington 98501 
'Washington Department of Fish & Wildlife 

Point Whitney Shellfish Laboratoiy 

1000 Point Whitney Road 

Brinnon, Washington 98320 
' 750 Mountain View Road 

Qiiilcene, Washington 98376 

ABSTRACT In Washington State, target fishing monahty rates (f ) for the geoducl< clam, Panopea abnipta (Conrad, 1849), are based 
on relative changes in biomass and therefore depend on growth patterns. With these policies, higher growth rates lead to larger harvest 
quotas so that applying higher rates to areas with slower growth would cause overharvesting. Therefore, in estimating growth patterns, 
it is important to recognize the scale to which estimates of growth rates should be applied. In this study, we tested whether growth 
parameters differed among regions and among local sites within regions in Washington, and whether they differed enough to compel 
managers to create location-specific policies. Von Bertalanffy growth parameters were estimated for 1 1 sites dispersed among four 
regions. Among those sites, L, ranged from 13.2 to I7..1 cm. k ranged from 0.1 13 to 0.23.S. and ;„ ranged from -0.029 to 0.806. Of 
the three parameters, the growth constant k had far more influence on target fishing mortality rates (F) than either L, or /„. Statistically 
significant differences in k were found among all local sites within geographic regions. However, only some of the differences were 
of a magnitude to concern management policies. We have proposed a general method for calculating and then tesfing for managerial 
significance when a linear relationship exists between k and the fishing mortality rate (F). Our results implied that managerially 
significant differences in *: existed among local sites within Washington's geoduck management regions, posing a dilemma for 
managers who, by convention, propose a single target fishing mortality rate for each region. 

KEY WORDS: Geoduck, growth, hypothesis testing, managerial significance. Panopea abnipta. von Bertalanffy 



INTRODUCTION 

The Pacific geoduck clam Panopea abnipta is a large hiatellid 
bivalve that occurs from Alaska to Baja, CA, and west to southern 
Japan (Bernard 1983). Geoducks are one of the largest burrowing 
clams in the world, reaching a live whole weight of .3.2.'i kg (Good- 
win and Pease 1987). Adults are buried to 1 m in sand and mud 
substrates from the lower intertidal to depths of more than 110 m 
(Jamison et al. 1984). They dominate the biomass of benthic in- 
faunal communities in many parts of Puget Sound, WA, where 
they have supported a commercial dive fishery in subtidal waters 
since 1970 (Goodwin and Pease 1991). Commercial dive fisheries 
also exist in Alaska and British Columbia (Campbell et al. 1998), 
and geoducks now provide the most valuable commercial clam 
harvest on the Pacific Coast of North America. The average annual 
ex-vessel value of Washington's geoduck harvest from 1990 to 
1998 was US$14 million. From 1971 through 1997 annual land- 
ings have averaged 1 ,540 tons. 

The Washington Department of Fish and Wildlife and several 
of the Washington tribes manage commercial geoduck harvest on 
a regional basis. There are six regions statewide that are based 
largely on legally defined tribal fishing boundaries. By coinci- 
dence, these boundaries also roughly conform to major oceano- 
graphic basins within Puget Sound (Ebbesmeyer et al. 1984). Cur- 
rently, four of the regions (Fig. 1 ) are surveyed for biomass. and 
geoduck quotas are calculated annually for each of these regions as 
the product of biomass and a target fishing mortality rate. The 
target fishing mortality rate (F) is based on the output of an age- 
based equilibrium yield model (Bradbury and Tagart 20001, which 
relies in part on a three-parameter von Bertalanffy growth func- 



tion. Past studies on geoduck growth (Goodwin 1976, Breen and 
Shields 1983, Anderson 1971 ) have only provided point estimates 
for annual growth increments, making it impossible to determine 
whether growth rates differed significantly among geographic ar- 
eas. In this paper, we first estimated von Bertalanffy growth pa- 
rameters for individual geoducks at 1 1 Washington sites and then 
conducted hypothesis tests for differences in growth parameters 
within and among the management regions. 

In conducting a hypothesis test, statistical significance is not 
always biologically meaningful. In this study, statistical signifi- 
cance refers to whether or not the growth parameters change; 
biological significance refers to how much the growth parameters 
change. Statistical significance is well defined: however, biologi- 
cal significance is not. In this study, determining biological sig- 
nificance stemmed from the decision processes that were in place 
for managing the geoduck harvest and thus are more appropriately 
termed "managerial significance." Managerial significance was 
determined by how much the growth parameters must change be- 
fore management decisions would be altered, and this degree of 
change was factored into the hypothesis-testing procedure. We 
concluded that according to the management criteria given, not 
only should regional specific growth parameter estimates be used, 
but within some regions site-specific estimates should also be 
used. 



METHODS 



Data 



Geoducks were collected from 1979 to 1982 at 11 previously 
unfished sites in Washington (Fig. 1). The sites were chosen op- 



57 



58 



Hoffmann et al. 



Strait 



Dallas Bank 

Tala Point 

Port Gamble control 

Port Gamble dredged 

Thorndyke Bay 

Bangor 

Fishermans Point 

8 Agate Passage 

9 Blake Island 

10 Herron Island 

11 Hunter Point 




Figure T. Sampling sites for geoduck growth. Also shown are bound- 
aries for four of Washington's geoduck management regions; the two 
regions not shown contained no sampling sites and no surveyed geo- 
duck biomass. 

portunistically from among those scheduled at the time for pre- 
fishing surveys. However, they were spread out over the entire 
commercial fishing range of Washington geoducks. Preliminary 
dive surveys were conducted at each of the 1 1 study sites to map 
their boundaries. The shallow to deep boundaries of a commercial 
geoduck tract were set by management to be between 6 and 23 m 
mean lower low water. The along-shore boundaries of a commer- 
cial tract were subjectively defined on the basis of drops in geo- 
duck densities, suitability of substrate, proximity to sewer outfalls 
or ferry traffic lanes, etc. 

Geoducks were sampled from a series of transects. The 
transects were approximately 0.91 m wide, ran perpendicular to 
the shore from 6 to 23 m, and were spaced approximately I km 
apart (Fig. 2). In some of the larger sites, transect lines were spaced 
at systematic intervals wider than 1 km. In each site, the first 
transect was located opportunistically along the shoreline at one 
end of the mapped bed. Because the divers were unable to see 
either the substrate or the geoducks from the survey boat before 
selecting the starting point, we made the assumption that the se- 
lection represented a random starting point. 

For logistical purposes, each transect was divided into 4.i.72- 
m-long subsections. Divers used a commercial water jet to dig 
geoducks from the approximate center of selected subsections. 
They were instructed to dig the first 10 geoducks seen without 
regard to size or any other criterion. The subsections selected were 
every fourth one, ignoring transect identity, i.e.. as if the transects 
were laid end to end. For example, if the first two transects were 
each made up of 10 subsections, then the first, fifth, and ninth 





-6 m depth 


contour 


-23 m 


depth contour 




- 


loj 


.9 ml at 


1 (3) 1 m 




Hi (61 


|(7) 


1 


I 


1 km 


Transect (a) 










(8) 


to 


1 llOi 1 (II) 


|<.:. 


1 (13) 


4 d") 


i 


Transect (b) 

i 


1 IIS) 


1 (16) 


1 (17) 1 (18) 


1 (19) 


1 (20) 


1, (21) 


1 


Tran.sec( (c) 


(22) 


1 (2!) 


1 wi LOSJ- 


, !i '2'*l 


1 (27) 


1 (28) 


1 






Transect (d) 











Figure 2. A schematic of the sampling design (not to scale or number). 
The smaller rectangles represent the hypothetical 46-m-long subjec- 
tions (1-28) that make up the hypothetical transects (a-d). Ten geo- 
ducks were collected from every fourth subsection (shaded) as if the 
transects had been laid end to end. 



subsections of the first transect would have been sampled and the 
third and seventh subsection of the second transect would have 
been sampled, etc. This procedure resulted in samples ranging 
from 21 to 258 geoducks, depending on the size of the site 
(Table I ). 

A total of 1 ,2 16 geoducks were sampled from the 1 1 sites. They 
were held in saltwater and returned to the laboratory within a few 
days of collection for processing. All geoducks were numbered 
and separated by site before processing, and the greatest anterior- 
posterior length of the right valve was measured with calipers. 
Thirty geoducks per site were subsampled for this growth analysis. 
The subsample from each site was drawn randomly from the num- 
bered shell samples, ignoring the sub.section identity. Of the 330 
sampled, 234 were used in the growth analysis. The 96 geoducks 
eliminated from the subsample were discarded either because they 
were unreadable or because they were <15 years old. 

Annual growth increments were determined using the acetate- 
peel method developd by Thompson et al. (1980) and described for 
geoducks in Shaul and Goodwin (1982). Growth increments for 
ages beyond 25 years were not calculated, because geoducks reach 
their asymptotic size between the ages of 15 and 25 years (Shaul 
and Goodwin 1982). The yearly rings on individual geoducks were 
measured to provide length at age data for individuals. Thus, in 
the nh region (/ = 1 . . . 4), the data for the /th individual {/ = 
1 . . . n^) consisted of paired observations il^^^, a,^^. j = I, . . . J,^) 
where 1,^^ was the length measured for age ringy and fl^^ was the 
age assigned to age ring j. The number of individuals varied by 
region and the number of age rings varied by individual (because 
some geoducks were less than 25 y old). From the paired obser- 
vations on each individual, length was regressed on age with a 
nonlinear von Bertalanffy function: 



/;, 



L,.J\ 



'H '^.1. 



where Ki,, ~ MO.ir;;). Thus, each nonlinear regression produced a 
set of estimated parameter values {Z.^,^,^,,. f„„| for the /th indi- 
vidual (/ = I . . . n^) in the nh region (;• = I . . . 4). The variance 
(t;, represented both the measurement error and the uncertainty 
caused by the absence of old growth rings. Therefore, the vari- 



Geoduck Growth 



59 



TABLE 1. 

Sample size, mean shell length and von Bertalanffv growth parameter estimates (±SE) derived from shell length at age for P. abrupta at 11 

sites in Washington. 







No. 


No. 


Mean Shell 








Region 


Site 


Dug 


Subsampled 


Length (cm) 


L^ (cm) 


k 


'o 


South Sound 


Hunter Point 


71 


21 


15.2 


16.4 (±0.357) 


0.2283 (± 0.009) 


0.719 (±0.040) 




Herron Island 


36 


23 


12.5 


13.2 (±0.158) 


0.1 544 (±0.006) 


0.422 (±0.074) 


Central Sound 


Agate Passage 


208 


20 


13.6 


15.8 (±0.383) 


0.1964 (±0.009) 


0.183 (±0.066) 




Blake Island 


19 


18 


13.0 


14.6 (±0.283) 


0.1586 (±0.006) 


0.806 (±0.071) 


Hood Cunal 


Bangor 


98 


25 


13.5 


14.3 (±0.252) 


0.1569 (±0.007) 


0.545 (±0.055) 




Tala Point 


96 


24 


12.4 


13.6 (±0.361) 


0.1435 (±0.009) 


-0.029 (±0.071) 




Port Gamble (dredged) 


180 


21 


13.1 


15.2 (±0.283) 


0.1810 (±0.007) 


0.661 (±0.052) 




Port Gamble (control) 


80 


21 


12.7 


14.0 (±0.390) 


0.1610 (±0.007) 


0.599 (± 0.075) 




Thorndyke Bay 


258 


21 


12.2 


13.0 (±0.201) 


0.1421 (±0.005) 


0.550 (±0.097) 




Fishermans Point 


21 


19 


16.8 


17.3 (±0.251) 


0.2353 (±0.009) 


0.552 (±0.059) 


Strait 


Dallas Bank 


149 


21 


12.0 


13.3 (±0.405) 


0.1131 (±0.005) 


0.334 (± 0.096) 



ances for older geoducks with more growth rings were likely to be 
more precise than for younger geoducks. The resulting heterosce- 
dasticity for geoducks >15 years of age was thought to be minimal 
and was ignored. 

Hypothesis Testing 

The experimental design was a two-factor analysis of variance 
(ANOVA) in which the first factor was region and the second was 
sites nested within regions. We first conducted a hypothesis test on 
site effects within each region and only conducted a test among 
regions if the site effects were nonsignificant. In this case, non- 
significance meant not managerially significant. Thus, for all sta- 
tistically significant tests. Tukey multiple comparisons (Neter et al. 
1985) were used to test for managerial significance. The Tukey 
multiple comparisons yielded confidence intervals for the differ- 
ences in growth among locations. For any one comparison to be 
managerially significant, the difference in growth had to be at least 
some constant c. These comparisons were identified by confidence 
intervals that excluded the interval {-c. c). 

Calculating Managerial Significance (c = 0.027) 

Most U.S. and Canadian fisheries, including all of those under 
U.S. federal jurisdiction, are managed using biological reference 
points (BRPs). BRPs are calculable quantities that describe a popu- 
lation's state and are usually used as targets for optimal fishing 
(National Research Council 1998). A BRP is most often expressed 
as a fishing mortality rate (F); examples include f msy- /^max- ^^'^ 
fj,9i,. These are the fishing mortality rates that are expected to 
achieve, over the long term, maximum sustainable yield, maxi- 
mum yield per recruit, and a spawning stock biomass that is xx% 
of the unfished level, respectively. 

We considered two management criteria in calculating the 
threshold of managerial significance for geoduck growth param- 
eters: (1) the BRP used by managers in setting the target fishing 
mortality rate and (2) the number of significant digits to which this 
target fishing mortality rate was calculated. Geoduck managers in 
Washington currently use as a BRP the fishing mortality rate cor- 
responding to F^tfcf.. a reference point that is widely used for U.S. 
West Coast groundfish (Clark 1993). Managers have agreed to 
calculate this target fishing mortality rate to three decimal places. 
For example, there is a managerially significant difference be- 



tween annual fishing mortality rates of 0.027 and 0.028. but not 
between 0.027 and 0,0273. 

The three von Bertalanffy growth parameters were first evalu- 
ated to determine which had the most influence on yield model 
predictions. The equilibrium model described in Bradbury and 
Tagart (2000) was used to calculate F^^rt, for different values of 
{L.^_,kJf,} in the range observed in the data. L^, while it affected 
model predictions of absolute yield, did not affect relative 
spawner-per-recruit biomass or relative yield per recruit and was 
therefore eliminated from further analysis. Figure 3 shows that the 
growth parameter k is more infiuential on ^409^ than ?„. Because L-^ 
had no impact on F^^^^ and t^ had only minimal impact, we con- 
ducted univariate hypothesis tests on k. 

Changes in k would only affect management decisions if they 
caused the model-based fishing mortality rate to change by 0.001 
or more. In general, whenever a linear relationship exists between 
k and F with slope (3. 



0.000 



Figure 3. Surface plot of F^a, 
and fnS. 




080 

values as a function of the observed ks 



60 



Hoffmann ft al. 



Ak 



= 3 => AA- = — ^ 



AF, 



In this example, a linear regression of the level of change Fj,,,.^ on 
k yielded a highly significant slope {P < 0.001) of p = 0.0366. 
Using this slope and Washington's management decision to cal- 
culate annual fishing mortality rates to three significant digits, c = 
0.001/0.0366 = 0.027. Thus, absolute differences in k among sites 
(or regions) s0.027 were managerially significant. 

RESULTS 

Table 1 shows the von Bertalanffy parameter estimates and 
their variances for the 1 1 study sites. The resulting growth curves 
for the fastest-growing site (Fishermans Point) and the slowest- 
growing site (Dallas Bank) are shown in Figure 4; the growth 
curves for all other sites lie between these two. Also shown for 
comparison is Anderson's (1971) growth curve for geoducks at 
Big Beef Creek and Dosewallips beaches in Hood Canal. 

Test for Nested Site Effects 

The test for site effects within regions was statistically signifi- 
cant {Fj22^ = 24.72. P = 0*) for all regions. Further testing for 
managerial significance produced mixed results. The Tukey mul- 
tiple comparisons between sites within regions showed four com- 
parisons in which the differences were managerially significant 
(Table 2): between the sites in the South Sound region and among 
several sites in the Hood Canal region. 

Power of Tukey Multiple Comparisons 

Because the null hypothesis of the growth parameters not being 
significantly different was not rejected for sites within Hood Canal 
and for the two sites in the Central Sound region, we conducted a 
power analysis to assess whether or not the nonrejection was 
meaningful. The power analysis estimated the probability that any 
one of the Tukey multiple comparisons would have excluded the 
interval (-0.027, 0.027) if in fact the differences in k among sites 
had been at least 0.027. To estimate this probability, we used the 
two-sample /-test power analysis option of Power Analysis and 
Sample Size (PASS version 6.0, Hintze, 1996). Each of the mul- 




10 15 20 
Age (yrs) 

Figure 4. The von RcrtalanfTy growth curves for geoduck growth ul 
the fiistisl gniHth site ( FishiTmans Point! and the slowest growth site 
(Dallas Itanki In this stud). .Mso shown is Anderson's (1971) growth 
curve for Big Becf/Dosewallips. 



tiple comparisons was a /-test that needed a Tukey multiplier. To 
adapt the software into giving the appropriate power estimates 
(Table 3), we in flated the estimated standard deviation of the com- 
parison, (Vm5£ = 0.0309, calculated by the ANOVA) by the ratio 
of the Tukey multiplier (3.217) to the analogous Z multiplier 
(1.96): 



= 3.2l7/1.96VM5e 



Power in the Hood Canal Region 



0.0507. 



In the Hood Canal region, there were 6 sites and 15 compari- 
sons, 3 of which were significant. With an average sample size of 
22, the estimated probability was 0.4233 (Table 3) for detecting a 
0.027 difference in any one of the comparisons. If the actual dif- 
ferences in k among sites had been at least 0.027, then one would 
expect to detect more than three of them. In fact, the probability of 
detecting a difference of 0.027 in at most three comparisons, where 
the probability of detection was 0.4233 per comparison, is the 
probability that a binomial random variable with N = \5 and P = 
0.4233 was less than or equal to 3. This probability was 0.0645. 
Given that this probability was very low. there is evidence that 
among the sites in Hood Canal, other than Fishermans Point, the 
differences in k are not likely to be greater than 0.027 and thus 
need not be estimated separately. 

Power in the Central Sound Region 

In the Central Sound region, there were two sites, and the 
comparison was not significant. With a power of 0.4233 of detect- 
ing significance in a comparison, the chance of not rejecting the 
null hypothesis was 1 - 0.4233 = 0.5767. Because this probability 
is high, nonrejection of the null hypothesis was not meaningful: 
i.e.. the results are inconclusive. 

Because Hood Canal was the only region producing evidence 
for common growth rates among sites, we did not pursue a test of 
regional differences. Thus, we recommend that with the given 
management criteria, separate growth models should be used in the 
regions given in Table 4. For the sites within Hood Canal other 
than Fishermans Point, the average growth parameter was calcu- 
lated as the mean of the average growth parameters in each site 
(Table 4). 

DISCUSSION 

The first result to note is the difference in the growth curves 
presented here and that from Anderson ( 197 1 ). We estimated both 
a lower rate of growth (k) and a smaller asymptotic size {LJ for 
geoducks: however, differences in the target population explain 
this discrepancy. Anderson's target population consisted of sub- 
tidal and intertidal geoducks between the presumed ages of I and 
5 years. Our target population consisted of subtidal geoducks older 
than 15 years. Because mean geoduck shell length is inversely 
proportional to water depth (Goodwin and Pease 1991). it is ex- 
pected that Anderson's sample would ha\e a higher estimate of Z,.,. 
Likewise, a higher estimate of A- is expected with a vounger target 
population. 

Of the three von Bertalanffy giowih paranielers. oiilv one was 
determined to be iiinucnlial: (he parameter k. For the criteria given, 
managerial significance was calculated to be differences in k of 
0.027 or greater among locations. That is, if the growth parameter 
differed by more than 0.027 among locations, then location- 
specific growth estimates should be used for setting harvest quotas. 

Data that were collected in four different regions encompassing 



Geoduck Growth 



61 



TABLE 2. 
Confidence intervals for the multiple comparisons of Test 1. 











Lower 


Upper 


Site 


Comparison (x,/x,) 


A = v, - X, 


SD(A) 


Bound* 


Bound 


South Sound 


Herron/Huntert 


-0.0739 


0.0102 


-0. 1 1 64 


-0.0314 


Central Sound 


Agate/Blake 


0.0379 


0.0109 


-0.0076 


0.0834 


Hood Canal 


Bangor/Tala 


0.0133 


0.0088 


-0.0151 


0.0417 




Bangor/Gamdredge 


-0.0242 


0.0092 


-0.0536 


0.0053 




Bangor/Thomdyke 


0.0147 


0.0092 


-0.0147 


0.0442 




Bangor/Gamcontrol 


-0.0042 


0.0092 


-0.0336 


0.0253 




Bangor/Fishermans Pointt 


-0.0784 


0.0094 


-0.1087 


-0.0481 




Tala/Gamdredge 


-0.0375 


0.0092 


-0.0672 


-0.0077 




Tala/Thorndvke 


0.0014 


0.0092 


-0.0283 


0.03 1 1 




Tala/Gamcontrol 


-0.0175 


0.0092 


-0.0472 


0.0123 




Tala/Fishermans Pointt 


-0.0917 


0.0095 


-0.1223 


-0.0612 




Gamdredge/Thomdyke 


0.0389 


0.0095 


0.0082 


0.0696 




Gamdredge/Gamcontrol 


0.0200 


0.0095 


-0.0107 


0.0507 




Gamdredge/Fishernians 


-0.0542 


0.0098 


-0.0858 


-0.0228 




Thomdyke/Gamcontrol 


-0.0189 


0.0095 


-0.0496 


0.0118 




Thomdyke/Fishermanst 


-0.0931 


0.0098 


-0.1247 


-0.0616 




Gamcontrol/Fi shernianst 


-0.0743 


0.0098 


-0.1058 


0.0428 



* The confidence intervals were calculated using a Tukey multiplier of 3.2 1 7, i.e.. A 
distribution 95th'7r quantile with 1 1 and ^ degrees of freedom. 
t Statistically significant data in these rows. 



: 3.217* 5D{A). The multiplier corresponded to a studentized range 



1 1 different sites were tested for differences among growth pa- 
rameters. Statistically significant differences in k were detected 
among most of the sites within the three regions Central Sound, 
Hood Canal, and South Sound. Further testing showed that in the 
South Sound, the sites were also significantly different. In Hood 
Canal, only one site was significantly different from the others. In 
the Central Sound, the results were inconclusive. Therefore, to 
preserve the management sensitivity criterion of 0.001 in the es- 
timated Fjijr; levels, we recommend different growth parameter 
estimates be used for each site in Straight. Central Sound, and 
South Sound and that one common model for the sites in Hood 
Canal other than Fishermans Point be used. 

We speculate that environmental factors related to tidal flow 
may have been a primary cause of the differential growth rates. 
Goodwin and Pease ( 1991 ) found that the average shell length of 
geoducks in Puget Sound was greatest in sandy substrates and 
decreased in both muddier substrates and those composed of pea 
gravel. Because size and growth are related, it is reasonable to 



conclude that growth is greatest in sites that are subject to inter- 
mediate tidal flow (i.e.. those composed primarily of sand) and 
decreases in both low-energy (muddy) and high-energy (gravelly) 
environments. The substrate was primarily composed of sand at 
the three sites in our study with the highest k values (Fishermans 
Point. Hunter Point, and Agate Passage). The three sites with the 
lowest k values were Dallas Bank, a site composed primarily of 
pea gravel, and Tala Point and Thorndyke Bay. both of which are 
muddy. Goodwin and Pease (1991) also suggested relationships 
betwen geoduck size and environmental factors such as primary 
productivity and water temperature. Along with tidal currents, 
these factors are likely to vary from site to site, resulting in dif- 
ferential growth parameters. 

Evidence for site-specific growth differences poses a dilemma 
for managers who must recommend a single regional harvest rate. 
If growth rates were common ainong sites, a regional estimate 
based on any selection of sites would be unbiased. However, we 
found that the growth constant can be site specific, requiring ad- 



TABLE 3. 

Power estimates for a single Tukey multiple comparison for various 
sample sizes.* 



Sample Size 



Power 



TABLE 4. 
Growth Parameter k estimated by region and site. 



20 
21 
22 
23 
24 
25 
30 



Region 



0.3914 


South Sound 


0.4075 


Central Sound 


0.4233 




0.4389 


Hood Canal 


0.4542 




0.4693 




0.5409 





* Power was estimated using the two-sample 7"-test option of PASS version 
6 (Hintze 19961 with a standard deviation of 0.0507 and a difference in 
means of 0.027. 



Strait of Juan de Fuca 



Site 



Estimated k 



Hunter 


0.2283 


Herron 


0.1544 


Agate 


0.1964 


Blake 


0.1586 


Bangor 


0.1569 


Tala 


0.1569 


Gamdredge 


0.1569 


Thorndyke 


0.1569 


Gamcontrol 


0.1569 


Fishermans Point 


0.2353 


Dallas 


0.1131 



62 



Hoffmann et al. 



justments to a sampling plan for estimating an unbiased regional 
parameter. Because the sites in this study were not selected at 
random, a regional k that is an average of the estimated site As will 
be biased. Managers might consider using the lowest estimated 
A-value with the expectation that that would be a conservative 
approach. Alternatively, another study could be conducted using a 
sampling plan designed to yield unbiased regional estimators. 



ACKNOWLEDGMENTS 

Warren Shaul and Conrad Budd assisted C.L.G. in collecting, 
preparing, and analyzing the age-growth data. Michael Ulrich pre- 
pared the map. We thank Tom Jagielo for computing assistance in 
writing the growth parameter estimation program. 



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73 pp. 



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49-58. 

Goodwin. C. L. & B. C. Pease. 1987. The distribution of geoduck (Panope 
uhriipta) size, density, and quality in relation to habitat characteristics 
such as geographic area, water depth, sediment type, and associated 
flora and fauna in Puget Sound. Washington. Washington Department 
of Fisheries Technical Report No. 102. 44 pp. 

Goodwin, C. L. & B. C. Pease. 1991. Geoduck (Panope abrupla (Conrad, 
1849)) size, density, and quality as related to varous environmental 
parameters in Puget Sound. Washington. J. Shellfish Res. 10:65-78. 

Hintze, J. L. 1996. Power Analysis and Sample Size (PASS) User's Guide, 
Version 6.0. Number Cruncher Statistical Systems, Kayesville, UT. 
245 pp. 

Jamison. D., R. Heggen & J. Lukes. 1984. Underwater video in a regional 
benthos survey, pp. 15-17. In: Proceedings of the Pacific Congress on 
Marine Technology. Marine Technology Society. Honolulu. 

National Research Council. 1998. Improving Fish Stock Assessments. Na- 
tional Academy Press, Washington. D.C. 177 pp. 

Neter, J., W. Wasserman & M. H. Kutner. 1985. Applied linear statistical 
models. 2nd ed. Richad D. Irwin. Inc.. Homewood. IL. 1127 pp. 

Shaul, W. & C. L. Goodwin. 1982. Geoduck (Panope generosa: Bivalvia) 

age as determined by internal growth lines in the shell. Can. J. Fish. 

Aqual. Sci. 39:632-636. 
Thompson. I.. D. S. Jones & D. Dreibelbis. 1980. Annual internal growth 

banding and life history of the ocean quahog Arclica islandica (Mol- 

lusca: Bivalvia). Mar Biol. 57:25-.34. 



Journal of Shellfish Research. Vol. 19, No. 1. 63-70. 2000. 

MODELING GEODUCK, PANOPEA ABRUPTA (CONRAD, 1849) POPULATION DYNAMICS. II. 
NATURAL MORTALITY AND EQUILIBRIUM YIELD 

A. BRADBURY' AND J. V. TAGART^ 

' Washington Department of Fish and Wildlife 

Point Whitney Shellfish Laboratory 

1000 Point Whitney Road 

Brinnon. Washington 98320 
'Washington Department of Fish and Wildlife 

600 Capitol Way North 

Olympia. Washington 98501 

ABSTRACT The natural mortality rate of geoduck clams. Panopea ahrupia (Conrad. 1 849). was estimated from data collected at 14 
previously unfished sites in Washington State in order to predict the potential yield of the commercial fishery under various harvest 
rate strategies. The instantaneous rate of natural mortality (A/) estimated by the catch curve method for geoducks of ages 28-98 was 
0.0226 y"'. Other important life history parameters — growth, schedules of sexual maturity, weight-al-age, and fishery selectivity — 
were estimated from the literature and file data. These parameter estimates were used to drive an age-based equilibrium yield model 
that predicted yield per recruit (YPR) and spawning biomass per recruit (SPR) over a range of fishing mortality rates. The model 
produced values of the instantaneous fishing mortality rate (F) for five commonly used constant harvest rate strategies. The fishing 
mortality rate producing maximum YPR (f„a,) ranged from 0.053-0.100 depending on the site growth parameters, but reduced SPR 
to 15-21% of the unfished level, f-values for the Fq., strategy ranged from 0.28 to 0.37. reducing SPR to 35-37% of the unfished level. 
Three harvest rate strategies that reduce SPR to either 35%, 40%, or 50% of the unfished level were also evaluated, with F-values 
ranging from 0.018 to 0.036. The F„j, strategy, currently adopted by Washington managers, was achieved with F = 0.028 (averaged 
over all sites), corresponding to an annual harvest rate of 2.7% of the exploitable biomass. The model was most sensitive to estimates 
of M. whereas growth, fishery selectivity, and sexual maturity schedules had relatively little effect on yield or SPR. Apparent shifts 
in recruitment during the past 30-45 y may have biased the estimate of M. Direct estimates of M and recruitment are therefore a high 
research priority if the model outputs are to remain useful. 

KEY WORDS: Geoduck. Panopea ahrupia. natural mortality, yield, harvest rate, spawning biomass 



INTRODUCTION 

The geoduck clam Panopea abrupta (Conrad. 1849) supports 
the most economically important clam fishery on the Pacific Coast 
of North America (Campbell et al. 1998. Hoffmann et al. 2000). 
Since 1967. the Washington Department of Fish and Wildlife has 
performed annual dive surveys to estimate the exploitable biomass 
of geoducks in Washington. "'Exploitable biomass" here refers 
only to geoducks within the legally fishable water depths of 6-23 
m. in areas that are not polluted or otherwise unsuitable for com- 
mercial fishing and of sufficient size for their siphons to be visible 
to divers. Based on market and survey samples in Washington, this 
excludes most geoducks <300 g. Of 11.181 geoducks randomly 
sampled using commercial methods, only 2'7c were <300 g (Good- 
win and Pease 1987). Geoducks usually attain this size in 5-7 y 
(Hoffmann etal. 2000). 

Exploitable geoduck biomass on a commercial bed is estimated 
as the product of the total bed area, the mean weight per geoduck. 
and the mean density of geoducks. Mean density is estimated by 
counting geoduck siphon "shows'" using a systematic strip transect 
technique (Goodwin 1978). Mean weight per geoduck is estimated 
from a series of samples dug at systematic intervals along the 
transect lines. The sum of the most recent biomass estimates on all 
surveyed beds within a management region constitutes the regional 
biomass estimate. There are currently six geoduck management 
regions in Washington, based largely on legally defined tribal 
fishing boundaries. Because only a few beds can be surveyed 
intensively in this manner each year, regional biomass estimates 
consist of the most recent estimate for each bed. with known 
catches subtracted from those beds as they are fished. 

To establish annual fishing quotas, managers apply a target 



harvest rate to the exploitable biomass estimate in each manage- 
ment region. Beginning in 1981. the target harvest rate was fixed 
at 2% of the estimated virgin (unfished) biomass on surveyed, 
commercially viable beds. This target harvest rate was based on a 
Ricker (1975) yield per recruit (YPR) model, but the model out- 
puts were never explicitly documented. Furthermore, emphasis in 
fisheries management has shifted since that time; harvest strategies 
based on YPR analyses (e.g., F^^^ and Fq ,) are now often sup- 
planted by strategies that seek instead to preserve the reproductive 
potential of the population. These spawning biomass per recruit 
(SPR) strategies are increasingly being used in marine fmfisheries 
(Clark 1991) and to a lesser extent in shelltTsheries (Quinn and 
Szarzi 1993). 

In this study, we derive estimates of the natural mortality rate 
(M) from geoducks sampled at previously unfished sites in Puget 
Sound and the Strait of Juan de Fuca. We also construct schedules 
of sexual maturity, weight-at-age, and fishery selectivity from the 
literature and tile data. We use these estimates to drive an age- 
based equilibrium yield model that predicts YPR and SPR over a 
range of fishing mortality rates. We also explore the limitations of 
the model and conduct sensitivity tests to determine which param- 
eters most influence the model's predictions. Finally, we use this 
information to recommend research aimed at refining the most 
important parameter estimates. 

METHODS 

Sampling Sites and Procedures 

Geoducks were sampled between 1979 and 1981 at 14 previ- 
ously unharvested sites in Puget Sound and the Strait of Juan de 



63 



64 



Bradbury and Tagart 



Fuca to n'jiain information on age distribution (Fig. 1 ). The sites 
span fcuf of the current six management regions, with six sites in 
the Hood Canal region, two sites in the Central Sound region, one 
site in the Strait region, and two sites in the South Sound region. 
Sample.s were taken randomly within each site at depths of 10-20 
m by washing geoducks from the substrate with a commercial 
water jet. Age was determined from annual growth increments in 
the hinge plate using the acetate-peel method (Shaul and Goodwin 
1982). 

The instantaneous rate of natural mortality (A/) was estimated 
from the geoduck age-frequency distribution using two different 
catch curve models (Robson and Chapman 1961, Ricker 1975). 
Both models assume that mortality is constant for all ages used in 
the catch curve. The Robson and Chapman model is based on a 
geometric distribution and assumes that year-class survival and 
recruitment are constant and all ages are equally selected. Geo- 
ducks are extremely long-lived, so that the number of animals 
observed in each l-y age class is typically low. even for sample 
sizes in which n > 1,000. Despite this problem, we chose to pre- 
serve the data in l-y age classes rather than aggregating ages, a 
procedure that potentially ignores real variability in the original 
data and may slightly inflate estimates of M (Noakes 1992). It was 
not possible to estimate site-by-site mortality rates, because no 
individual site contained enough data to construct reliable catch 
curves. Age frequencies were therefore pooled from all 14 sites in 
order to create the catch curve. 

To avoid arbitrary choices of the upper and lower ages used in 




Figure 1. Sampling sites for geoducit natural mortality and growth. 
Also shiiwii are boundaries lor four of Washington's geoduck man- 
agement regions: the t«o not shown contained no sampling sites and 
no surveyed geoduck hiomass. 



the catch curve "right limb," we established a protocol for data 
inclusion: The initial upper age limit for the catch curve was the 
first age at which our sample contained no geoducks (i.e.. the first 
gap in frequency). We then excluded younger age frequencies if 
they were identified as outliers by Wei.sberg"s (1985) outlier test. 
Two methods were used to select the lower age limit for the catch 
curve: ( I ) The Chi-square procedure described in Robson and 
Chapman (1961) was used to differentiate partially .selected ages, 
and (2) catch curve regressions were calculated for all possible 
lower age limits, and we used an ad hoc procedure to optimize the 
coefficient of determination (r") and the linearity of positive and 
negative residuals plotted against age. Once the lower and upper 
age limits for the catch curve were identified, a Chi-square formula 
was then used to test goodness of fit of fully selected ages to a 
geometric distribution (i.e.. the Robson and Chapman model). The 
von Bertalanffy growth parameters estimated at 1 1 Washington 
sites from Hoffmann et al. (2000) were used as site-specific growth 
inputs. Sexual maturity, weight-at-length. and fishery selectivity 
parameters were derived on the basis of published literature from 
Washington and British Columbia. 

Yield Model 

Geoduck yield was modeled using a deterministic, age- 
structured equilibrium yield model. Given a set of parameter es- 
timates for mortality, maturity, growth, and selectivity, the model 
collapses the number of geoducks at age for all cohorts in the 
population to a single cohort, assumed to represent the stable age 
distribution of the population. Population size was based on an 
initial unfished spawning population, by a declining exponential 
function for survival at age, and by the Baranov catch equation 
(Ricker 1975). Baranov's catch equation says that annual catch is 
a simple linear function of instantaneous fishing mortality and 
mean population size. The derivation of Baranov"s catch equation 
is presented in Seber (1982). Seber cites Baranov (1918) as the 
origin of the catch equation, hence its common name. The model 
assumed continuous recruitment, the magnitude of which was 
based on a Beverton-Holt stock-recruitment relationship (Ricker 
1975). The Beverton-Holt stock-recruitment relationship, com- 
monly used with marine fish, is an asymptotic function that esti- 
mates annual recruitment based on parent stock size. The impli- 
cation of this relationship is that over a broad range of parent stock 
size, recruitment is stable, but as parent stocks reach critically low 
levels, recruitment drops precipitously. A maximum age (o,,,^^) in 
the model served as an "accumulator age" category that encom- 
passed all ages a > rt„,,,^. The assumption implicit in this formu- 
lation is that no significant changes in growth, weight, maturity, or 
selectivity occurred beyond «,„;,„. In the case of geoducks. this 
assumption was reasonable and is addressed below. For other ap- 
plications, the model could be simply extended to accommodate an 
unlimited nimiber of older age classes. The model was constructed 
as a QuattroPro for Windows (version 5.0) spreadsheet. 

Table I lists the user-supplied inputs required by the model. 
These include estimates of the natural mortality rate, the growth 
rate, the stock-recruit (S-R) relationship, the unfished spawning 
hiomass. fishery selectivity, sexual maturity, and the population 
sex ratio. Table 2 shows the parameters derived from the user 
supplied inputs, listed in computational order. To run the model, 
fishing mortalily (/'I was stepped from to a specified upper limit 
while computing YPR and SPR for each value of /■". 

The model is capable of returning a suite of fishing mortalily 



Modeling Geoduck P. abkupta Population Dynamics 



65 



TABLE 1. 

Geoduck life history parameter estimates iield constant for all 
study sites. 



Parameter Description 



Parameter 
Symbol 



Value, Notes 



Unfished ("virgin") 

spawning stock biomass 
(in kg); the spawning 
biomass when F = 

Instanlaneous natural 
mortality rate (assumed 
constant for all ages) 

Weighi-at-age (in g) based 
on length-at-age as 
derived from the von 
Bertalanffy growth 
function 

Maturity-at-age; the 
proportion of female 
geoducks of age a (in 
years) that are sexually 
mature 

Fishery selectivity-at-age; 
the proportion of 
geoducks of age a (in 
years) selected by the 
fishery 

Beverton-Holt 

spawner-recruit shape 
parameter (Kiinura 1988) 

Proportion of males in 
population 

Maximum (accumulator) age 



BO, 



M 



*. 



100.000 kg (only required 
to scale absolute 
biomass) 

0.0226 



»\, = "L/ 

L^ = length (cm) at age a 

X = 0.349127 

y = 2.972807 

cj)^ = 1/(1 +exp"*'') 
X = -1.9 y = 9.5 



V, = 1/(1 +exp~"'') 
X = -1.5 y = 8.0 



0.5 

2^ 



benchmarks, such as f „„^, fo.,- and F„,;. . For example, the fishing 
mortality rate that produces, over the long run. the maximum YPR 
corresponds to the F,„„^ strategy, whereas F„ , represents a rale of 
harvest less than f,„„, (Deriso 1987, Gulland 1968). 

The fraction of the unfished spawning weight per recruit re- 
maining at a given level of fishing mortality was calculated as 
SPR/SPRO and is achieved at a corresponding fishing mortality 
rate F„..,^ where .v.v represents the ratio (SPR/SPRO)IOO. Model 
predictions of this fraction formed the basis for SPR-based fishing 
strategies. For example, the fishing mortality rate that resulted in a 
value of SPR/SPRO = 0.35 corresponds to the F,v; strategy. 



RESULTS 



Natural Mortality 



Sampled geoducks from 14 previously unfished sites ranged in 
age from 2 to 131 y (Fig. 2a). The mean age of geoducks was 46 
y (standard error [SE] = 0.56. n = 2.157). The initial upper age 
limit for the catch curve was 1 10 y, because no 1 1 l-y-old geo- 
ducks were in our sample. Examination of residuals showed a 
single large negative residual at the 99-y age class (only one geo- 
duck of this age was in our sample), and this age class was elimi- 
nated from the analysis as an outlier, based on the test given in 
Weisberg (1985). Both the Robson and Chapman (1961) Chi- 
square procedure and our ad hoc optimization procedure identified 
age 28 as the lower age limit for the catch curve. A Chi-square was 
used to test goodness of fit of fully selected ages (28-98) to a 



geometric distribution. The resulting Chi-square was highly sig- 
nificant (X" = 326.56. degrees of freedom = 68). indicating that 
the age frequency was not geometric in distribution and that data 
requirements for the Rob.son and Chapman model were not met. 
Ricker (1975) pointed out that in most stocks, difference in year- 
class strength is the major source of variability, in which case the 
best estimate of survival would be obtained from a catch curve 
analysis with equal weighting. The Ricker catch curve based on 
ages 28-98 (Fig. 2b) produced an estimate of M = 0.0226 y"' 
(±0.0018 SE. /! = 71. r- = 0.70). 

Other Model Parameters 

Goodwin (1976) calculated an allometric length-weight rela- 
tionship for Washington geoducks in log-log form. We converted 
this to the more familiar power curve form h'„ = .vL„', where w.^ 
= weight (in g) at age a. L^ = shell length (in cm) at age a (Table 
1 ). The proportion of males (/),„) in the geoduck population was set 
to /J„, = 0.5 based on a 50:50 sex ratio for geoducks older than 10 
y (Goodwin and Pease 1989). 

The proportion of sexually mature geoducks at age (<l>) was 
estimated by fitting a simple logistic curve to maturity data from 
published sources. Anderson (1971 ) found that 50% of his sample 
of geoducks was mature at 75 mm and an age that he estimated to 
be 3 y. The Washington growth curves described above suggest 
that this length would be attained in roughly 5 y. depending on the 
site. Sloan and Robinson (1984) reported that geoducks mature at 
5 y and reproduce for at least a lOO-y period with no "reproductive 
senility." They stated that "unequivocally mature geoducks" were 
6-103 y old (late-active males) and 12-95 y old (late-active fe- 
males). On the basis of these two sources, we fit a logistic curve 
with the least-squares method and two data points, whereby 50% 
of the female geoducks would mature at 5 y and 100% by 12 y 
(Table 1). 

The proportion of geoducks at age a selected by the fishery (i'„) 
was based loosely on Harbo et al. (1983), who reported that re- 
cruitment to the British Columbia geoduck fishery begins at 4 y 
and is complete by 12 y. To more conservatively model fishery 
selectivity, we fit a simple logistic curve using the least-squares 
method and two data points, whereby geoducks enter the fishery at 
roughly 4 y and are fully selected by 8 y (Table I ). 

Nothing is known about the form or steepness of the S-R re- 
lationship for geoducks. We therefore set the Beverton-Holt shape 
parameter (A) equal to 1.0 for all model runs. In other words, we 
assumed that recruitment was independent of spawning stock 
abundance. This assumption is reviewed below in Discussion. 

As a practical convenience, the equilibrium yield model uses an 
"accumulator age" category («„„,) as the final age category, en- 
compassing all ages a > n,„^^. For this study, we set a^^^ = 25, 
which implicitly assumes that there are no significant changes in 
growth, selectivity, or maturity beyond age 24. This assumption is 
reasonable for geoducks, which reach asymptotic size between the 
ages of 10 and 20 y (Hoffmann et al. 2000). 

Fishing Mortality Rates for Five Harvest Strategies 

We ran the model for each site, varying only the growth pa- 
rameters based on the analysis of growth presented in Hoffmann et 
al. (2000). The only sites where growth parameter estimates (spe- 
cifically, the growth constant A) could be pooled were five of the 
six Hood Canal sites. In all other cases, site-specific growth pa- 
rameters could not be pooled, and therefore separate model outputs 



66 



Bradbury and Tagart 



TABLE 2. 
Description of derived parameters used in tiie geoducli equilibrium yield model. 



Description 



Derived Parameters 



Notes 



Number of geoducks surviving to the first age 

class (a = 1 year) 
Instantaneous rate of fishing morlahty at age u 



Instantaneous rate of total mortality at age a 

Annual rate of survival 

Number of geoducks surviving to age a for a > 1 

Average number of geoducks at age u 

Average biomass (in kg) of geoducks at age a 

Yield per recruit (in kg) at age a 

Total yield per recruit (in kg) for all ages 

Spawning weight per recruit (in kg) 



Fraction of unfished spawning stock biomass 
remaining at a given level of fishing mortality 

Spawning biomass (in kg) when F > 

Recruitment (in numbers) 

Yield (in kg) 

Harvest rate for fully selected age classes (iv, = I ) 



W, = /),„ for males 

N^ = I - /),„ for females 

F^ = Fv.^ 



Z^ = M^ + F, 

S^ = exp(-ZJ 

Wj = W„(l -SJ/Z, for o<a^,„ 
^ = ;^/Z„ for a = o„„, 
B. = N,w, _ _ 

YPR„ = v.,f B., = F,B„ 

YPR = X'',/B: = fXi'A 

SPR, = W,<t>, for age a 

SPR = ^'B^,<i>„ for all ages 

P = \ - (1M)(1 - SPR/SPRO) 



B. 



P BO. 



R = (BySPRO)/[l - A(l - P)] 

Y = YPR(R) 

|x = F/Z[\ - exp(-Z)] 



/),„ = proportion of males in the population (see 

Table 1) 
F = instantaneous rate of fishing mortality for 

fully selected age classes (v^ = 1); 

user-supplied. \\, = fishery selectivity at age a 

(see Table 1 ) 
M^ = instantaneous natural mortality rate (see 

Table 1) 



maximum (accumulator) age (see Table 1) 



<i>, = proportion of mature females at age a (see 
Table 1) 

A = Beverton-Holt shape parameter (see Table 1). 

SPRO = unfished spawning weight per recruit 

(total SPR when F = 0) 
BOs = unfished spawning stock biomass (see 

Table 1) 
Reference: Kimura ( 1988) 

Reference: Ricker (1975) 



were calculated for each site. All inputs except growth paratnelers 
were identical for each model run (Table I ). Growth parameters 
used as site-specific input are shown in Table 3. 

Values of the instantaneous fishing mortality rate (F) for five 
commonly used constant harvest rate strategies are shown in Table 
3. fn,^^ is the fishing mortality rate that produces, over the long 
run. the maximum YPR. f,, , is a common alternative to F„„^ and 
is the rate of fishing mortality at which the marginal YPR is 10% 
of the marginal YPR for a lightly exploited fishery (Deiiso 1987). 
F,5,;j, ^409^, and F^,,,. are SPR-based harvest rates that reduce SPR 
to either 35%. 40%, or 50% of the unfished level (Clark 1991 ). 

F,„,,^ ranged from 0.053 to 0.100 depending on the site (Table 
3). These rates correspond to annual harvest rates ((jl) of 5.1-9.4% 
of the exploitable geoduck biomass. The Strait of Juan de Fuca 
region, represented by the single sampling site at Dallas Bank, 
produced the lowest value, whereas Fishermans Point in Hood 
Canal produced the highest value. The F,„,^ strategy reduced SPR 
to 15-21% of the unfished level, depending on the site. Values for 
F,| I ranged from 0.028 to 0.037, corresponding to annual harvest 
rates of 2.7-3.67r. This strategy reduced SPR to 35-37% of the 
unfished level, depending on the site. 

Values for F,,,,, were, predictably, nearly identical to the F,, , 
rates, ranging from 0.30 to 0.36 ((jl = 2.9-3.5%). F values for the 
F4o-;^ strategy ranged from 0.025-0.030 ((x = 2.4-2.8%), whereas 
those for the Fs,„ strategy ranged from 0.0 1 8-0.020 ((jl = 1.8- 
2.0'/r ). 

Model Sensitivily to Parameter Estimates 

All of the parameter estimates used to drive the model arc 
subject to varying degrees of uncertainty. It is therefore reasonable 



to ask what might happen to our predictions if the true values of W 
or A:, for example, were much lower or higher than our estimates. 
We tested the sensitivily of the model by running it with a range 
of values for each parameter in turn while holding all other pa- 
rameters constant. Values ranging from one-tenth the "best" pa- 
rameter estimate (from Tables 1 and 2) to three times the estimated 
value were used in the analysis. Only the fishing mortality rates 
corresponding to the F_^^y,, strategy were calculated, but the trend 
for other strategies would be similar. 

The model was most sensitive to the estimate of M, with F^^^, 
values ranging from 0.003 to 0.068 as M was increased from 
one-tenth to three times our "best" estimate of A^ = 0.0226 (Fig. 
3). The model was far less .sensitive to the other parameter esti- 
mates, as evidenced by the relatively flat Fjii,^ trajectories for 
values of the growth coefficient k. the selectivity constant y, and 
the maturity constant y. For example, varying the value of A- from 
one-tenth to three times our best estimate resulted in Fj,,,, values 
that ranged only from 0.02 1 to 0.033. 

Use of Model Results to Set Annual Fishing Quotas 

The model results presented above, together with an estimate of 
exploitable biomass, may be used to set annual fishing quotas. The 
first step in such a process is for managers to recommend one of 
the five harvest strategies described above, or an alternate strategy; 
the model is capable of returning /-'-values for any desired level of 
equilibrium spawning biomass or yield. The decision process in- 
volved in recommending a particular harvest strategy is by no 
means clear-cut, but some guidelines on risk-averse strategies from 
the recent fisheries literature are reviewed below in Discussion. 



Modeling Geoduck P. abrupta Population Dynamics 



67 



70 



^60 
■| 50 

3 
C 

•-^40 

>- 

Z 30 
LU 

O 20 
lU 

£ 10 



1979-1981 
mean age = 46.27 yr 
SE = 0.56 
n = 2157 



I 



.ijlliiliilillillliihl:] 



LMlIk 



5 ' 10 20 30 40 5b 60 70 80 90 lOOTio 

AGE (yrs) 



mis 



130T4bT50 



4 

>- 
O 

Z 3 

o 

LU 2 
OH 









B 


' m 










^^?^ 


■ 


1979-1981 
M = 0.0226 
ages 28 - 98 
r'^2 =0.70 
n = 71 






■ 1 

■ ■ 




■■ 




■ ■ ■ 




-■ — 1 — I — h — 




-r- ^ 1 


■^^ "P ' 



10 20 30 40 50 



60 70 

AGE 



80 90 

(yrs) 



100 110 120 130 140 150 



Figure 2. (A) Age frequency of geoducks sampled at 14 sites in Wash- 
ington. (B) Catch curve used to estimate the instantaneous natural 
mortalit> rate (M) of geoduclis. 

Once managers reach a decision on the "best" harvest strategy, the 
corresponding F-value may be taken directly from the mean values 
in Table 3. This mean F-value is then converted to the harvest rate 
(jjl) for fully selected age classes (Ricker's equation from Table 2). 
To produce the recommended annual fishing quota, the harvest 
rate is then simply multiplied by the estimate of harvestable bio- 
mass. For example. Washington managers have recommended and 
adopted an F^„r; strategy for geoducks in all six management 
regions. This strategy is achieved with an instantaneous fishing 
mortality rate of F = 0.028 (mean value for all sites. Table 3): the 
corresponding annual harvest rate for fully selected age classes (|j.) 
is 0.027. or 2.79^ of the exploitable biomass. Annual dive survey 
data provide an estimate of exploitable biomass for each of six 
management regions. As an example, exploitable geoduck biomass 
in the Hood Canal Region in 1999 was estimated to be 18.185 t 
(Sizemore and Ulrich 1999). and the resulting annual quota was 
{0.027)( 18.185 t) = 491 t. 

DISCUSSION 

Our primary objective in equilibrium modeling was to simulate 
the long-term results of various geoduck fishing strategies, both in 
terms of yield and SPR. Before discussing our results, it is perhaps 
necessary to explain why we attach such importance to geoduck 
harvest rate strategies, particularly since the differences between 
many of the modeled options may appear trivial. 

In many fisheries, especially those in which biomass is small or 



estimated with great uncertainty, debating a 1% difference be- 
tween annual harvest rate options would indeed be trivial. But in 
Washington's geoduck fishery, where the exploitable biomass is 
large (73.843 t in 1999; Sizemore and Ulrich 1999) and the price 
is high, even tiny incremental differences in the recommended 
harvest rate have tremendous economic significance. Moreover, 
because geoducks have a low M (and presumably a low intrinsic 
rate of increase), small differences in annual harvest rates can have 
profound cumulative effects on stock size, especially if the harvest 
rate is set too high. This is not to discount the importance of good 
biomass estimates, but we believe there are several reasons why 
Washington managers should place the greatest emphasis on im- 
proved harvest rate strategies rather than improved biomass esti- 
mates. First, biomass estimates for individual geoduck beds in 
Washington have coefficients of variation (CVs) averaging about 
11%. Simulation tests suggest that biomass estimation errors of 
this magnitude are unlikely to result in substantial degradation of 
long-term harvest performance (Frederick and Peternian 1995). 
Second, even greatly increased sampling is not likely to improve 
biomass estimate CVs very much. Third and most importantly, 
errors in biomass estimation are assumed to be reasonably unbi- 
ased. An error in setting the annual harvest rate, on the other hand, 
will have a persistent and cumulative effect on stocks in only one 
direction, either underharvest or overharvest. We therefore believe 
that, given reasonable estimates of stock size, choosing a harvest 
strategy remains the most critical aspect of geoduck management. 

In this study, we evaluated five common harvest strategies. Our 
model predicts that fishing at F,„ ,^ will eventually reduce SPR to 
less than 20% of the unfished level, a threshold below which many 
fish stocks are assumed to collapse (Thompson 1993). Therefore, 
F^^^ should be considered a high-risk strategy for geoducks. 

Less risky are the SPR-based strategies, three of which were 
evaluated here. In this study, we assumed that recruitment was 
independent of stock size at all levels of fishing (Beverton-Holt 
parameter /4 = 1.0). Although this is the common default assump- 
tion in cases in which the S-R relationship is unknown, the risk 
inherent in this assumption is that given an existing but undetected 
S/R relationship. F^^,,, can be greater than F^,sy (the preferred 
fishing rate with a known S/R function; MSY, maximum sustain- 
able yield). As an alternative to F„^^, SPR-based strategies seek to 
preserve some minimum level of spawning biomass and at the 
same time produce yields that are close to the MSY. In an attempt 
to find fishing strategies that are robust for any likely S-R rela- 
tionship, recent modeling studies have simulated groundfish yields 
using a range of typical life history parameters and realistic S-R 
models. Clark ( 1991 ) showed that fishing at F,5^; would achieve at 
least 75% of MSY for a wide range of detemiinistic S-R relation- 
ships. On the basis of his results, F^^^,;, has been adopted as a target 
rate for a number of fish stocks in Alaska and the U.S. Pacific 
coast. Clark (1993) later revised his recommendation to Fj,,,- after 
considering variability in recruitment, but remarked that "it would 
be silly to argue very hard for or against any specific rate between 
F,5<7, and F^^.^^,." Mace (1994) also recommended Fj^^j. which she 
claimed was a modest improvement over Fjj.^^. She states that 
Fjii^j represents a risk-averse fishing strategy in the common situ- 
ation in which there is adequate information to place bounds on all 
relevant life history parameters except the S-R relationship. Quinn 
and Szarzi (1993) modeled clam fisheries in Alaska and recom- 
mended SPR-based strategies equivalent to a range of F309j,-f45*- 

As noted earlier. Washington managers have adopted an Fj,,^^ 
strategy for geoducks. which corresponds to F = 0.028 (averaged 



68 



Bradbury and Tagart 



TABLE 3. 
Benchmark instantaneous fishing mortality rates for fully selected geoducks (v^ = 1.0) from seven sites in Washington. 







n 


I.= 
















Region 


Site 


(sites) 


(cm) 


k 


'ii 


fm.v 


fo.i 


F,sr, 


''40% 


^^50% 


South Sound 


Hunter Point 


1 


16.4 


0.23 


0.72 


0.090 


0.036 


0.036 


0.029 


0.020 




Herron Island 


1 


13.2 


0.15 


0.42 


0.064 


0.031 


0.032 


0.027 


0.018 


Central Sound 


Agate Passage 


1 


15.8 


0.20 


0.18 


0.085 


0.035 


0.035 


0.029 


0.020 




Blake Island 


1 


14.6 


0.16 


0.81 


0.064 


0.031 


0.032 


0.027 


0.019 


Hood Canal 


Five sites pooled 


5 


12.8 


0.16 


0.47 


0.067 


0.032 


0.033 


0.027 


0.019 




Fishermans Point 


1 


16.8 


0.24 


0.55 


0.100 


0.037 


0.036 


0.030 


0.020 


Strait 


Dallas Bank 


1 


12.0 


0.11 


0.33 


0.053 


0.028 


0.030 


0.025 


0.018 


Mean of all sites 












0.075 


0.033 


0.033 


0.028 


0.019 



Model inputs except growth parameters are from Table 1. Growth parameter estimates are from Hoffmann et al. (2000). 



over all sites) and annua! harvest rate (fj,) of 2.7% of cuirent 
exploitable biomass. British Columbia managers calculate annual 
quotas using a fixed harvest rate of Wc (Campbell et al. 1998), but 
this rate is applied to the estimated virgin biomass rather than 
current biomass estimates, as is done in Washington. 

A secondary objective of our study was to detertnine which of 
the estimated geoduck life history parameters were most influen- 
tial in predictions of yield and SPR. The model was most sensitive 
to the estimate of natural mortality (AT), whereas growth, selectiv- 
ity, and maturity parameters had relatively little effect on SPR- 
based fishing mortality rates. This suggests that future research 
monies are best spent making more reliable estimates of M. 

Because our model is an equilibrium model and admittedly 
sensitive to the estimate of M, one could ask how it might cope 
with time varying natural mortality. If it were possible to construct 
a functional relationship between specific, measurable categorical 
variables — such as predator density, or sea temperature and natural 
mortality rates — and if these categorical variables were themselves 
predictable, one could estimate the expected changes in M. With a 
credible estimator, the equilibrium model could be conxerted to a 
dynamic pool model and revised estimates of F could be derived 
for a specific future time interval of interest. Such an application 
would be highly dependent on the accuracy and precision of the 
predictive functions, not only the functions related to M but also 
the expected annual recruitment. We are doubtful that this ap- 

0.08 



0.06 



<i> 



0.04 



o 

li. 



0.02 



0.00 




matunty y 



0.5 1 1.5 2 2.5 3 

multiple of parameter estimate 

Figure .3. The effect of difTerenl paranieter estimates on model- 
derived /•■411.. values. Numbers (m the \-a\is represent nuilliples of the 
"hesi" parameter estimates from fable 1 (inortalitv. selectivity, and 
maturity) and Tahle 3 (growth parameter k). 



proach would become profitable. Alternatively, annual or fixed 
interval updates of the equilibrium F could be computed using 
revised estimates of M. 

If natural mortality varies over time, the true F^f^,-^ would 
rise and fall proportionately with the change in M (Fig. 3). We 
would err in the application of our equilibrium F dependent on the 
trend in M. If M fluctuates around some normally distributed 
mean, then on average our equilibrium F is probably reasonable. If 
there is a significant periodicity in the trend in M (a long duration 
decline, for example) and it goes unrecognized, application of the 
equilibrium F risks overharvest of the resource. Managers could 
impose a safety valve by creating a harvest policy that reduces the 
exploitation rate below that derived from the preferred F (e.g., 0.75 
F). but it would be speculative whether this precaution was suffi- 
cient to account for real variability in M. Models of sto- 
chastic variability in recruitment have led scientists to suggest 
maintaining a larger spawning biomass and therefore adoption of 
a lower prefeired F (e.g., Fj^'; rather than ^,5,,) (Mace 1994, 
Clark 1993). 

Our estimate of M = 0.0226 is similar to estimates from British 
Columbia. Sloan and Robinson (1984) estimated M = 0.035 at a 
single site, while Breen and Shields (1983) reported M = 0.01- 
0.04 in five populations. Noakes (1992) estimated M = 0.03-0.04 
at three sites. Both our estimate and the British Columbia estimates 
relied on the catch curve method, which assumes that mortality 
rate is unifortn with age and that recruitment has been constant 
over the range of age groups analyzed. There is some suggestion 
in our age-frequency data that a shift in geoduck recruitment has 
occurred that could have biased the estimate of M. Age frequencies 
did not begin to decline until about age 25. a pattern in catch 
curves that is often due to inefficient sampling of younger age 
classes. But for geoducks, which grow quickly and are fully se- 
lected by the commercial fishery at half this age (Harbo et al. 
1983). sampling inefficiency is not a plausible explanation for the 
low numbers of geoducks in ihe l()-25-y age group. Instead, low 
numbers of IO-25-y-old geoducks may indicate poor recruitment 
during the 15-y period before sampling. This suggests that recruit- 
ment declined during the period 1955-1970 (before the advent of 
a fishery) and perhaps more recently. Sloan and Robinson ( 1984) 
suggested Ihe possibility of a similar decline in recruitment during 
the same time period in British Columbia. 

Thus, catch curve estimates of M for geoducks based on older 
age classes may not accurately represent current (rends in natural 
mortality. They likewise reveal nothing about M for younger geo- 
ducks. In either case, our results indicate that biases in the estimate 



Modeling Geoduck P. abrupta Population D>namics 



69 



of M will have a major influence on model-based predictions of 
yield and SPR. Independent estimates of M should therefore be a 
high priority for research. Given the fact that geoducks are entirely 
sedentar) . direct or "known fate"" estimates of M may be possible 
if a reliable and noninvasive tag can be developed. Such straight- 
forward measurements of annual mortality would rely on fewer 
assumptions than the catch curve method and might also provide 
age-specific and area-specific estimates M. 

A final caveat related to the use of simple yield models such as 
ours is that they do not take into account the spatial distribution of 
harvested animals. Spatial structure is frequently ignored in the 
management of finfish stocks, because it is assumed that survivors 
are being continually mixed by movement. Under this "dynamic 
pool" assumption, it does not matter whether the annual quota is 
taken in small amounts over the entire fishing area or taken en- 
tirely within a tiny comer of that area. But as Orensanz and Jamie- 
son (1998) point out. the dynamic pool assumption may be risky 
when applied to sedentary benthic species such as geoducks. More 
research should therefore be devoted to the long-terin effects of 
various spatial harvesting strategies on yield and spawning bio- 
mass of geoducks. An experiment of this sort is underway in 



Washington, where geoduck densities at 15 commercial beds are 
being monitored before and after fishing to estimate an empirical 
rate of population recovery. If it is based on a long span of time, 
an empirically determined turnover (i.e.. recruitment) rate for com- 
mercially fished geoduck beds could be used to validate, improve, 
or replace the harvest rate strategies on the basis of structural 
models. 

ACKNOWLEDGMENTS 

We thank Tom Jagielo and Dr. Annette Hoffmann for statistical 
advice and reviews of an earlier draft. Lynn Goodwin. Warren 
Shaul. and Conrad Budd collected and read the age samples. Don 
Rothaus and Bob Sizemore provided extensive reviews of earlier 
drafts. Michael Ulrich drew the site map. Don Flora, Dr. Bob 
Conrad (Northwest Indian Fisheries Commission), and Dr. J. M. 
("Lobo"") Orensanz (University of Washington) reviewed earlier 
drafts and made helpful suggestions. We thank Dr. W. G. Clark for 
providing the original FORTRAN-coded equilibrium yield model. 
Finally, we thank the anonymous reviewers who made suggestions 
on the final draft. 



LITERATURE CITED 



Anderson. A. M., Jr. 1971. Spawning, growth, and spatial distribution of 
the geoduck clam, Panope generosa (Gould) in Hood Canal. Wash- 
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Baranov. T. I. 1918. On the question of the biological basis of fisheries, pp. 
81-128. Report of the Division of Fish Management and Scientific 
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Breen. P. A. & T. L. Shields. 1983. Age and size structure in five popu- 
lations of geoduck clams (Panope generosa) in British Columbia. Can. 
Tech. Rep. Fish. Aquat. Sci. No. 1 169. 62 p. 

Campbell. A., R. M. Harbo & C. M. Hand. 1998. Harvestmg and distri- 
bution of Pacific geoduck clams. Panopea abniphi. in British Colum- 
bia, pp. 349-358. In: G. S. Jamieson and A. Campbell (eds.). Proceed- 
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and Management. Can. Spec. Publ. Fish. Aquat. Sci. 125. 

Clark, W.G. 1991. Groundfish exploitation rates based on life history pa- 
rameters. Can. J. Fish. Aquat. Sci. 48:734-750. 

Clark. W.G. 1993. The effect of recruitment variability on the choice of a 
target level of spawning biomass per recruit, pp. 233-246. In: G. Kruse. 
D. M. Eggers, R. J. Marasco, C. Pautzke, and T. J. Quinn II (eds). 
Proceedings of the International Symposium on Management Strate- 
gies for Exploited Fish Populations. Alaska Sea Grant College Program 
Report No. 93-02. University of Alaska Fairbanks. 

Deriso. R. B. 1987. Optimal f o , criteria and their relation to maximum 
sustainable yield. Can. J. Fish. Aquat. Sci. 44(Suppl. 2):339-349. 

Frederick, S. W. & R. M. Peterman. 1995. Choosing fisheries harvest 
policies: when does uncenainty matter? Can. J. Fish. Aquar. Sci. 52: 
291-306. 

Goodwin. C. L. 1976. Observations of spawning and growth of subtidal 
geoducks {Panope generosa. Gould). Proc. Nat. Shellfish. As.soc. 65: 
49-58. 

Goodwin, C. L. 1978. Puget Sound subtidal geoduck survey data. Wash. 
Dept. Fish. Prog. Rep. No. 36. 107 pp. 

Goodwin, C. L. & B. C. Pease. 1987. The distribution of geoduck (Panope 
abrupta) size, density, and quality in relation to habitat characteristics 
such as geographic area, water depth, sediment type, and associated 
flora and fauna in Puget Sound. Washington. Wash. Dept. Fish. Tech. 
Rep. 102. 44 p. 

Goodwin. C. L. & B. Pease. 1989. Species profiles: life histories and 
environmental requirements of coastal fishes and invertebrates (Pacific 
Northwest): Pacific geoduck clam. 14 pp. U.S. Fish. Wildl. Serv. Biol. 



Rep. 82( 1 1 .120). U.S. Army Corps of Engineers (TR EL-82-4), Wash- 
ington, D.C. 

Gulland. J. A. 1968. The concept of maximum sustained yield and fisheries 
management. FAO Fish. Tech. Paper 70. 30 pp. 

Harbo. R. M., B. E. Adkins, P. A. Breen & K. L. Hobbs. 1983. Age and 
size in market samples of geoduck clams (Panope generosa). Can. MS 
Rept. Fish. Aquat. Sci. No. 1714. 77 pp. 

Hoffmann. A., A. Bradbury & C. L. Goodwin. 2000. Modeling geoduck, 
Panopea abrupta (Conrad 1849) population dynamics. I. Growth. J. 
Shellfish Res. (in press). 

Jamison, D., R. Heggen & J. Lukes. 1984. Underwater video in a re- 
gional benthos survey, pp. 15-17. In: Proceedings of the Pacific Con- 
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Hawaii. 

Kimura, D. K. 1988. Stock-recruitment curves as used in the stock- 
reduction analysis model. / Cons. Int. Explor. Mer. 44:253-258. 

Mace. P. M. 1994. Relationships between common biological reference 
points used as thresholds and targets of fisheries management strate- 
gies. Can. J. Fish. Aquat. Sci. 51: 1 10-122. 

Noakes, D. J. 1992. On growth and mortality of geoduck clams (Panope 
abrupta). pp. 22-34. In: G. Thomas (ed.). Shellfish stock assessments 
for the west coast of Canada in 1991 as reviewed by the Pacific Stock 
Assessment Review Committee (PS.ARC). Can. Manuscr. Rep. Fish. 
Aquat. Sci. 2169. 

Orensanz, J. M. & G. S. Jamieson. 1998. The assessment and management 
of spatially structured stocks: an overview of the North Pacific Sym- 
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459. In: G. S. Jamieson and A. Campbell (eds.). Proceedings of the 
North Pacific Symposium on Invertebrate Stock Assessment and Man- 
agement. Can. Spec. Publ. Fish. Aquat. Sci. 125. 

Quinn. T. J. II & N. J. Szarzi. 1993. Determination of sustained yield in 
Alaska"s recreational fisheries, pp. 61-84. In: G. Kruse, D. M. Eggers. 
R. J. Marasco. C. Pautzke, and T. J. Quinn II (eds.). Proceedings of the 
International Symposium on Management Strategies for Exploited Fish 
Populations, Alaska Sea Grant College Program Report No. 93-02. 
University of Alaska Fairbanks. 

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70 Bradbury and Tagart 

Seber. G. A. F. 1982. The Estimation of Animal Abundance and Re- the geoduck clam /"anope oftrapfa (Conrad) from southern British Co- 

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Shaul. W. & C. L. Goodwin. 1982. Geoduck (Panope generosa: Bivalvia) Thompson, G. 1993. A proposal for a threshold stock size and ma.ximum 

age as determined by internal growth lines in the shell. Can. J. Fish. fishing mortality rate. pp. 303-320. In: S. J. Smith, J. J. Hunt, and D. 

Aqiiat. Sci. 39:632-636. Rivard (eds.). Risk Evaluation and Biological Reference Points for 

Sizemore, B. & M. Ulrich. 1999. 1999 Geoduck Atlas: Atlas of Major Fisheries Management. Can. Spec. Piihl. Fish. Aiiuat. Sci. 120: 303- 

Geoduck Tracts of Pugel Sound. Washington Department of Fish and 320. 

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Joiirihil ofSlwllfhli Resfiiirh. Vol. 19, No. 1, 71-75. 2U00. 

MICROSPORIDIOSIS IN QUEEN SCALLOPS (AEQUIPECTEN OPERCULARIS L.) FROM 

U.K. WATERS 

KARIN B. LOHRMANN,' ^ ' STEPHEN W. FEIST," AND 
ANDREW R. brand' 

' Universidad Catolica del Norte. Fucultad de Ciencias del Mar. Coquimbo. 

Chile 
'CEFAS Weymouth Laboratory. Barrack Road. The Nothe. Weymouth. 

Dorset DT4 SUB. UK 
^The Uuiversitx of Liverpool. Port Erin Marine Laboratoiy. Port Erin. Isle 

of Man IM9 6JA. UK 

ABSTRACT Spores of a microsporidian parasite were found in the queen scallop, Aequipecten opercularis (L.), collected from 
several coastal sites around the United Kingdom. Developing spore stages were detected in contact with the host cell cytoplasm. 
Infected host cells formed aggregates in the connective tissue of the digestive gland. Fully mature spores were found free within blood 
vessels. These spores exhibited a dome-shaped polaroplast. a diplokaryon. a posterior vacuole, and an isofilar polar tube, with seven 
to eight coils in a single row . In immature spores, the number of coils in the polar tube varied, with some having 7 to 8 coils and others 
having 10 to 12 coils. 

KEY WORDS: Microsporidian, spores, scallop, Aequipecten opercularis. parasite 



INTRODUCTION 



MATERIALS AND METHODS 



Scallop species are of commercial interest in many parts of the 
world. In Europe, the main species exploited is the great scallop 
(Pecten maximits), which is the subject of important fisheries in 
France, Ireland, the United Kingdom, and Norway, where it is 
cultivated on a small scale. There is also a significant natural 
fishery for the queen scallop. Aequipecten opercularis. This spe- 
cies is distributed from northern Norway and the Faroe Islands to 
the Mediterranean and Adriatic Seas, It lives in depths ranging 
between 18 and 46 m on fine sand, fine gravel, or sandy gravel. 
The main areas of fisheries for this species lie along the western 
coasts of the British Isles and France but also include the Shetland 
Isles and Moray Firth in the northeast and both sides of the English 
Channel (Anseil et al, 1991, Brand 1991), 

Since little is known on the natural disease status in these two 
species, a survey was undertaken to collect baseline data on para- 
sites and possible pathogens in natural, apparently healthy popu- 
lations. During this study, a microsporidian was found in the di- 
gestive gland of the queen scallop, and electron microscopy (EM) 
studies were undertaken for characterization and identification of 
this parasite. 

Microsporidians are eukaryotic, obligate intracellular parasites 
of almost all animal phyla. The most common hosts are arthropods 
and fish (Canning 1990), Only a few microsporidians have been 
reported from bivalves. Comps et al. (1975) found an unidentified 
species in Cardium edule and Jones (1981) described Microspo- 
ridium rapua from the oyster Ostrea lutaria in New Zealand. In 
mytilids, a microsporidian parasitizing the oocytes has been de- 
scribed by Figueras et al. (1991a. 1991b) who found Steinhausia 
mytilnvum in Mytilus galloprovincicdis from Spain and in Mytilus 
edulis from the United States, Villalba et al. (1997) observed the 
same parasite in ova of Mytilus galloprovincialis. and Sagrista et 
al. (1998) described the developmental cycle and ultrastructure of 
this protistan in M. galloprovincialis. 

In this study, the spores of a microsporidian parasite from the 
queen scallop are described. 



A total of 454 adult queen scallops (A. opercularis) were 
sampled during the period July 1997 to April 1998, The sampling 
localities are shown in Figure 1, The localities and dates of sam- 
pling are shown in Table 1. 

For histology, transverse tissue sections were taken that in- 
cluded the digestive gland, kidney, gills, gonad and mantle, and 
they were fi.xed in Davidson's fixative (Shaw and Battle 1957) for 
24 h. The tissues then were dehydrated, cleared, infiltrated with 
paraffin wax, and sectioned at 5-6 \i.m. The routine stain used for 
all samples was Gill's hematoxylin and aqueous eosin. Selected 
samples were stained with special stains: Farley-Feulgen (Farley 
1969) for identifying DNA. and Giemsa for staining presumptive 
parasites. For each sample, one section was cut, and the slides were 
examined using a Reichert Polyvar microscope. Photographs were 
taken with a photomicroscope (E800 Eclipse, Nikon. Tokyo. Ja- 
pan), 

For transmission EM. l-mm' pieces of digestive gland from 
each specimen sampled for histology were fixed in 3% glutaral- 
dehyde in 0.2M cacodylate buffer with 1.75% NaCI, for 2 h at 
room temperature, and were washed in the same buffer. After 
histological assessment, those samples found to harbor the mi- 
crosporidian were further processed for EM, Tissues were washed 
another three times in 0,2M cacodylate buffer with 1.75% NaCl, 
and were postfixed for I h in 1% osmium tetroxide in the same 
buffer. After washing twice with buffer, they were rinsed in dis- 
tilled water, stained for I h en bloc with 2% aqueous uranyl ac- 
etate, dehydrated in ethanol, washed in propylene oxide, and em- 
bedded in Epon 812 (premix. BDH). Semithin sections. 1 p.m 
thick, were cut on a Reichert Ultracut S microtome and were 
stained with toluidine blue, Ultrathin sections were cut with a 
diamond knife and were stained with aqueous uranyl acetate and 
lead citrate. The sections were viewed and photographed with an 
electron microscope (EM 900. Zeiss) at 50 kV. 

Measurements from stained histological or semithin sections 
were made using a Nikon E800 microscope with LUCIA screen 



71 



72 



LOHRMANN ET AL. 



SiteD 



Site A 




TABLE 2. 
Prevalence of microsporidiosis in A. opercularis. 



Site C 

Figure 1. Map of England and Wales showing the sampling sites. 

measurement system. The number of longitudinally sectioned 
spores that were measured is indicated in each case. 

RESULTS 

The prevalence of microsporidiosis in A. opercularis is given in 
Table 2. 

Spores were found in two different locations in the digestive 
gland o{ A. opercularis. Immature spores were found in the cyto- 
plasm of connective tissue cells, and mature spores were observed 
free in blood vessels. In some scallops, both kinds of spores were 
seen together in the same section; in others, only one kind of spore 
was found (Fig. 2). 

The cells with maturing spores formed aggregates measuring 
approximately .^00 (xni in diameter. They were found in two of the 

TABLE I. 
Localities and dates of sampling. 





Site 


Number 


Date of 


Locality Name 


Reference 


Sampled 


.Sampling 


Isle of Man. Bradda Offshcire 


.Sile A 


.5 


05/08/97 


Isle of Man. Douglas 


Site A 


150 


18/11/97 


Red Wharl Bay 


Site B 


59 


1 WO 1/98 


West ol Portland Bill 


Site C 


33 


24/09/97 


West of Portland Bill 


SiteC 


117 


14/10/97 


West of Portland Bill 


Site C 


50 


24/04/9,8 


Hunibcr Rough 


Site D 


40 


08/01/98 



Isle of 
Man (A) 



Red Wharf 

Bay (B) 



Portland 
Bill (C) 



Humber 
Rough (D) 



4.5% 



10.2% 



I2.57f 



20% 



80 resin blocks examined. The whole aggregate (Fig. 2) as well 
as each infected host cell was surrounded by layers of fibroblast- 
like cells, as shown at the EM level (Fig. 3). In infected cells, the 
nucleus could be observed in some sections. The cytoplasm gen- 
erally was degraded, although mitochondria could still be recog- 
nized. The presence of a sporophorous vesicle was not confirmed, 
the spores being in direct contact with the host cytoplasm. The 
spores measured 2.3 (range 1.8-2.8) x 1.3 (range 1.1-1.9) (i.m (h 
= 20). had an elongate-ovoid shape, and showed different degrees 




Figure 2. Histological section shoeing nialure spores (msl, free in a 
blood vessel (l)\ », and Iho aggregates of host cells containing immature 
spores (isl. The upper aggregate is less mature, and both aggregates 
are surrounded h\ I'lhrohlast-like cells of host origin (arrows): dl = 
digestive gland tuhule ((ilenisa stain: bar = 50 pm). 
Figure 3. Transmission electron micrograph of one infected cell v»ith 
immature spores (isl. surrounded h\ nbroblast-like cells of host origin 
( Fl. fhe cell membranes of these cells form layers around each infected 
cell (arrows) (bar = 1 ^ml. 



MiCROSPORIDIOSIS IN QliEEN SCALLOPS 



73 




Figure 4. Mature spore viewed in longitudinal section. The exospore (Ex) and the endospore (En) can be observed. Internally, the polaroplast 
(P) and oblique sections of the polar tube can be seen (*) (bar = 0.1 fini). 

Figure 5. Electron micrograph of a slightly oblique section of a mature spore. The outer covering consists of an exospore (Ex), endospore (En), 
and the cell membrane (arrow). The polaroplast can be observed at the anterior end of the spore, with the two nuclei (N) forming a diplokaryon 
in the central region of the spore. The coils of the polar tube in transverse section (*), the posterior vacuole (Pv), and in close proximity Golgi-like 
membranes (G) can also be seen (bar = 0.1 jim). 



of maturation. It was not possible to determine whether the im- 
mature spore contained a single nucleus or a diplokaryon, since 
areas considered to be nuclear did not appear to be delimited by an 
envelope. The polar tube was isofilar, with 10 to 12 coils in a 
single row. Some spores showed a shorter polar tube, with seven 
to eight coils. The spores were limited by an inner electron-lucent 
endospore and an outer electron-dense exospore. 

Mature spores were found in only one of the resin blocks ex- 
amined. They were located in blood vessels and were elongate- 
ovoid in shape, measuring 2.3 (range 1.9-3.2) x 1.2 (range 0.8- 
1.7) |jLm (/! = 9). They were limited by an outer exospore and an 
electron-lucent endospore covering the plasma membrane (Figs. 4, 
5). The polar tube was inserted into the anterior anchoring disc 
(Fig. 6). passing through the center of the spore, and then, in the 
posterior two thirds of the spore, were wound in most cases into 7 
to 8 coils (Fig. 7), and exceptionally into 9 coils (Fig. 5), and were 



aligned in a single row. The polar tube was isofilar. measuring 83.5 
nm in diameter. A conspicuous, dome-shaped polaroplast occupied 
the anterior third of the spore, enclosing the straight region of the 
polar tube and terminating close to the coiled polar tube (Fig. 4). 
Two spherical nuclei were closely apposed, forming a 
diplokaryon. Each measured up to 0.88 |jim in its longest axis and 
was flattened in the zone of contact with the other nucleus (Fig. 7). 
The diplokaryon was located in the central third of the spore, 
between the polaroplast and the posterior vacuole. The latter was 
limited by a single membrane, with Golgi-like membranes often 
present in close association (Fig. 5). 

DISCUSSION 

This is the first time that a microsporidian infection has been 
reported in any scallop species. 



74 



LOHRMANN ET AL. 




Figure 6. Two electron micrographs showing the anterior pole of the spore. In these sections the anchoring disc (Ad) with the polar tube (*) 
attached can be seen. The polaroplast (P) is also apparent (bar = 50 nm). 

Figure 7. Electron micrograph from a spore sectioned through the nuclei (Nl of the diplokaryon. The nuclear envelope can be clearly observed 
(arrows). The polar tube (*) has seven coils, and the posterior vacuole (Pv) is also present in this section (bar = (1.5 nm). 



Spores were found in two dift'erent locations in the digestive 
gland. Immature spores were located intracellularly. and mature 
spores were located within blood vessels. 

We were unable to find any of the earlier stages of this parasite 
in the scallops examined. Unlike in other microsporidian species, 
where developmental stages and spores are present concurrently 
(Comps et al. 1979, Amigo et al. 1996, Johnson et al. 1997. Larsson 
et al. 1997), in this species only one developmental stage, the spore, 
could be observed. It is possible that the early stages were present 
in a tissue other than that of the digestive gland. .Since the cells 
infected by the microsporidian appear to be hemocytes, the poten- 
tial for infection in tissues apart from the digestive gland, such as 
the intestine, stomach, and gills should be recognized. Additional 
EM studies are needed to investigate this possibility. That the 
microsporidian has an intermediate host within which the earl\ 
developmental stages could be present should also be considered. 
Although most microsporidians have only one host, there are sev- 
eral examples of the requirement for an intermediate host (i.e., 
Amhlyiyspora) (Andreadis 19S.'S. Beciiel 1992). 



The immature spores differed slightly in the length of the polar 
tube, some having 7 to 8 coils, and others showing 10 to 12. The 
presence of two types of spores differing mainly in the length of 
the polar tube has been described for other microsporidian species, 
i.e., Noseinii spp. (Iwano and Ishihara 1991 ) and Noseiua miiscidi- 
fiiracis (Becnel and Geden 1994). These authors suggest that a 
shorter polar tube is characteristic of spores involved in infection 
of other cells in the same host, the longer polar tube belonging to 
spores that are involved in transmission from host to host. All the 
mature spores examined in the present study had a short polar tube, 
but they were all from one specimen and from the same resin 
block. We presiMiie that mature spores with a long polar tube also 
exist, because ue found them in the immature spores. Despite the 
large number of resin blocks examined, we did not succeed in 
finding the mature spores with 10 to 12 coils of the polar lube. 

Fully mature spores showed clear evidence of a diplokaryon. 
This feature, together with a polar tube consisting of eight coils in 
a single row, the overall dimensions, and the fact that it is infecting 
an invertebrate host, places this microsporidian near to the genus 



MlCROSPORIDlOSlS IN QUEEN SCALLOPS 



75 



Pseiidopleistophora (Sprague et al. 1992). a microsporidian first 
described as Pleistophora sp. parasitizing eggs of the annelid Ar- 
mandia brevis by Szollosi (1971). 

One important point that needs to be explained is the mecha- 
nism by v\ hich immature spores contained in individual cells later 
appear as free, mature spores in blood vessels. No transitional 
forms were seen in the current study, but in some reports, as the 
maturation of the spores progresses, the host cells start to loose 
their plasma membranes and become a syncytium (Weiser 1976). 
In this way. the spores would be released to reinfect adjacent cells 
or to become phagocytosed and perhaps migrate to other tissues. 

No host reaction against this microsporidian was seen, other 
than a thin capsule made by fibroblast-like cells. This protistan 
does not seem to be a threat to queen scallops, as those sampled 
showed no evidence of poor condition. However, if these scallops 
become stressed due to changes in temperature, salinity, or crowd- 
ing, as occurs in culture situations, the parasite could potentially 
become harmful to the host (Sindermann 1990). Despite the high 



prevalence of microsporidiosis in animals from a variety of loca- 
tions around the United Kingdom, the impact of this parasite on 
wild populations oi A. opercidaris remains unknown. Further stud- 
ies are needed to investigate the pathogenicity of the microsporid- 
ian in A. openidwis held in laboratory conditions under different 
temperatures and stocking densities. In addition, the identification 
of potential interinediate hosts and early developmental stages of 
the parasite are required for a specific identification of this mi- 
crosporidian. 

ACKNOWLEDGMENTS 

K.B.L. thanks The British Council for a fellowship that allowed 
her to work for one year in the United Kingdom, and also MAIT 
funding, which contributed to this work. The authors also want to 
thank Dr. Eduardo Couve for access to the electron microscope at 
the Universidad de Valparaiso. Valparaiso, Chile, and Mr. Fidel 
Vargas for his skillful technical assistance. 



LITERATURE CITED 



Andreadis. T.G. 1985. Experimental transmission of a microsporidian 
pathogen from mosquitoes to an alternate copepod host. Proc. Natl. 
Acad. Sci. U.S.A. 82:5574-5577. 

Amigo. J.M.. H. Salvado. M.P. Gracia & C.P.Vivares. 1996. Ultrastructure 
and development of Microsporidium ovoidewn (Thelohan. 1895) Spra- 
gue. 1977, a microsporidium parasite of the red band fish iCepola 
macroplnhalma L.). Europ. J. Protistol. 32:532-538. 

Ansell, A., J.- C. Dao & J. Mason. 1991. Three European scallops: Pecten 
maxiinits. Chlamys (Aeqiiipecten) opercularis and C. (Chlamys) vaiia. 
In: Sandra E. Shumway (ed.). Developments in Aquaculture and Fish- 
eries Science, vol. 21. Scallops: Biology. Ecology and Aquaculture. 36 
pp. 

Brand, A.R. 1991. Scallop ecology: distributions and behaviour. In: Sandra 
E. Shumway (ed.). Developments in Aquaculture and Fisheries Sci- 
ence, vol. 21. Scallops: Biology. Ecology and Aquaculture. 48 pp. 

Becnel. J.J. 1992. Horizontal transmission and subsequent development of 
Amblyospora califomica ( Microsporida: Amblyosporidae) in the in- 
termediate and definitive hosts. Dis. Aquat. Org. 13:17-28. 

Becnel. J.J. & C.J. Geden. 1994. Description of a new species of microspo- 
ridia from Muscidifura.x raptor (Hymenoptera: Pteromalidae). a pupal 
parasitoid of muscoid flies. / Eiik. Microbiol 41:236-243. 

Canning. E. 1990. Phylum Microspora. In: Margulis. Corliss, Melkonian 
and Chapman (eds). Handbook of Protoctista. Jones and Bartlett Pub- 
lishers. Boston. 18 pp. 

Comps. M.. H. Grizel. G. Tige & J.-L. Duthoit. 1975. Pathologie des 
Invertebres. Parasites noveaux de la glande digestive des mollusques 
marins Mytihis edulis L. et Cardium edule L. Note. C.R. Acad. Sc. 
Paris 281:179-181. 

Comps. M.. Y. Pichot & J. -P. Deltreil. 1979. Mise en evidence d'une 
microsporidie parasite de Marteilia refringens agent de la maladie de la 
glande digestive de Ostrea edulis L. Rev. Trav. Inst. Peches Marit. 
43:409-412. 

Farley, C.A. 1969. Probable neoplastic disease of the hematopoietic system 
in oysters, Crassostrea virginica and Crassostrea gigas. In: C.J. Dawe 
and J.C. Harshbarger (eds). Neoplasms and Related Disorders of In- 
vertebrate and Lower Vertebrate Animals, vol. 31. National Cancer 
Institute, Bethesda, MD. pp 541-555. 

Figueras, A.J., C.F. Jardon & J.R. Caldas. 1991a. Diseases and parasites of 



rafted mussels (Mytilus galloprovincialis Lmk): preliminary results. 
Aquaculture. 99:17-33. 

Figueras. A.J.. C.F. Jardon & J.R. Caldas. 1991b. Diseases and parasites of 
mussels (Mytilus edulis. Linneaus. 1758) from two sites on the east 
coast of the United States. ./. Shellfi.di Res. 10:89-94. 

Iwano. H. & R. Ishihara. 1991. Dimorphism of spores of Nosema spp in 
cultured cell. / Invertebr. Pathol. 57:211-219. 

Johnson, M.A.. J.J. Becnel & A.H. Undeen. 1997. A new sporulation se- 
quence in Edhazardia aedis (Microsporidia: Culicosporidia), a parasite 
of the mosquito Aedes aegypti (Diptera: Culicidae). J. Invertebr. 
Pathol. 70:69-75. 

Jones. J.B. 1981. A new microsporidian from the oyster Ostrea lutaria in 
New Zealand. J. Invert. Pathol. 38:67-70. 

Larsson, J.I.R., D. Ebert & J. Vavra. 1997. Ultrastnictural study and de- 
scription of Ordospora colligata gen et sp. Nov. (Microspora, Or- 
dosporidae fam. Nov.), a new microsporidian parasite of Daphnia ma- 
gna (Crustacea, Cladocera). Europ. J. Protistol. 33:432^443. 

Shaw, B.L. & H.I. BaUle. 1957. The gross and microscopic anatomy of the 
digestive tract of the oyster Crassostrea virginica (Gmelin). Can. J. 
Zool. 35:325-347. 

Sindermann C. J. 1990. Principal Diseases of Manne Fish and Shellfish, 
vol. 2. Academic Press, San Diego, CA. 516 pp. 

Sagrista, E., M.G. Bozzo, M. Bigas, M. Poquet & M. Durfort. 1998. De- 
velopmental cycle and ultrastructure of Steinhausia mytilovum. a mi- 
crosporidian parasite of oocytes of the mussel, Mytilus galloprovincia- 
lis (Mollusca. Bivalvia). Europ. J. Protistol. 34:58-68. 

Sprague. V.. J.J. Becnel & E.I. Hazard. 1992. Taxonomy of Phylum Mi- 
crospora. Cril. Rev. Microbiol. 18:285-395. 

Szollosi, D. 1971. Development of Pleistophora sp. (Microsporidian) in 
eggs of the polychaete Armandia brevis. J. Invertebr. Pathol. 18:1-15. 

Villalba. A.. S.G. Mourelle. M.J. Carballal & C. Lopez. 1997. Symbionts 
and diseases of farmed mussels Mytilus galloprovincialis throughout 
the culture process in the Ri'as of Galicia (NW Spain). Dis. Aquatic 
Org. 31:127-139. 

Weiser, J. 1976. Microsporidia in invertebrates: host-parasite relations at 
the organismal level. In: Bulla & Cheng (eds.) Comparative Pathobi- 
ology, vol. 1 . Biology of the Microsporidia. 38 pp. 



Jourmil of Shellfish Reseurch. Vol. 19, No. 1. 77-83. 2000. 

EVALUATION OF THREE METHODS OF BOTTOM CULTURE OF THE TROPICAL SCALLOP 

EUVOLA (PECTEN) ZICZAC (L. 1758) 



LUIS FREITES V,' ANIBAL VELEZ AND CESAR LODEIROS 

Depuruimeiuo de Biologia Pesqitera 

Instituto Oceanogrdfico de Venezuela 

Universidad de Oriente 

P.O. Box 245 

Ciimaiui 6101 

Venezuela 

ABSTRACT Three methods were used to study the growth and survival of juvenile Euvola ziczac (initial shell height of 40.4 mm 
SD = 4.21. and initial dry mass tissues of 0.35 g (SD = 0.01). which were set out at a density of 15 individuals m"- on a sandy bottom 
at Turpialito in the Golfo de Cariaco. Venezuela. The first method was applied on an area of 3 x 5 m (15 m*) with minimum 
demarkation (0.20-m low walls) on the bottom, the second method was applied on 1 x 1 m corrals with 1-m high walls, and the third 
method on 1 x 1 x 1 m cages with bottom and top covers. Both treatments with high walls were conducted with 15 replicates. We could 
not quantify growth and survival in the first treatment, because the rate of escape was >80'7f month"' (12 scallop m~- month"' ). In the 
corrals, the escape rate increased progressively from 4% ( 1 scallop m"- month"') to 36% (5 scallop m"- month"'), suggesting that the 
swimming ability of Euvola ziczac increased with size from an initial 40.4 mm to final 69.7 mm in shell height obtained in this study. 
No scallops escaped from the cages, but survival was less than in the corrals. Our observations suggest that the most appropriate bottom 
culture method would be corrals with walls higher than 1 m. 

KEY WORDS: Euvola ziczac. bottom culture, scallop, enclosure, grow-out 



INTRODUCTION 

Euvola ziczac is a functional hermaphrodite scallop present 
from Cape Hatteras, North Carolina, throughout the Gulf of 
Mexico and the Caribbean Sea to southern Brazil off Santa Cata- 
rina (Abbott 1974). Although Euvola ziczac does not form dense 
natural banks able to support commercial fisheries activity, the 
species is considered to have great potential for commercial aqua- 
culture activity off the Bermudas, Columbia, Venezuela, and Bra- 
zil (Hernandez 1990. Velez and Lodeiros 1990, Waller 1991, Cas- 
tellanos et al. 1997). In Venezuela, several studies have determined 
aspects of biological feasibility for culture in the marine environ- 
ment under hanging culture conditions (Freites et al. 1993, Freites 
et al, 1995, Freites et al. 1996, Lodeiros and Himmelman 1994). In 
this manner, rapid growth (up to 30-35 mm) and high survival rate 
have been attained. However, in larger sizes, diverse factors in- 
trinsic to suspended culture, such as fouling (Lodeiros and Him- 
melman 1996), wave action (Freites et al. 1999). and food quality 
(Hunaulth et al. unpublished data), linked with unfavorable periods 
of high temperature, low available food, and reproduction effort in 
this species, generating stressful conditions, which lead to a de- 
crease in growth and survival, have been noted (Lodeiros and 
Himmelman 1994, Lodeiros and Himmelman 2000). However, 
when Euvola ziczac is cultured in contact with the sandy substra- 
tum on the seabed (its natural habitat), high growth and survival 
rates have been noted, considering bottom culture as the most 
appropriate for the grow-out stage of the species (Velez et al. 1995, 
Hunaulth et al. unpublished data). 

Studies of the feasibility of various bottom culture techniques 
have been made for numerous pectinid species including Chlamys 
farreri (Wang et al. 1992), Placopecten magellanicus (Kleiman et 
al. 1996). Pecten maximus (Cliche et al. 1994, Dao et al. 1995). 



'Address correspondence to: E-mail: lfreites@cumana.sucre. udo.edu. ve or 
lfreites@iim.csic.es 



Patynopecten yessoensis (Aoyama 1989, Ito 1991), Argopecten 
circularis (Caceres-Marti'nez et al. 1986: Maeda-Marti'nez et al. in 
press), and Pecten novaezelandicie (Bull 1991 ). So. bottom culture 
is an alternative that has shown important levels of profitability in 
other scallop species. This is because of a lower investment in 
equipment, consumables, and maintenance than with the hanging 
method (Frishmand et al. 1980, Felix-Pico et al. 1991, Gilbert and 
Leblanc 1991. Wang et al. 1992, Kleinman et al. 1996). 

In this manner, the aim of this study was to evaluate the growth 
and survival of scallop Euvola ziczac applying two bottom culture 
grow-out methods: with barriers in the cage and corral enclosures 
and with no barriers, to obtain market size. 

MATERIALS AND METHODS 

This study was conducted over a 6 month period (February 
27-September 7, 1994) off the south coast of the Golfo de Cariaco, 
eastern area of Venezuela (Fig. 1 ). The individuals used in the 
experiment were obtained from a hatchery under controlled con- 
ditions at the end of August 1993, following the methodology 
described by Velez and Freites (1993). Scallops were held in sus- 
pension for intermediate culture following the methodology de- 
scribed by Freites et al. (1993, Freites et al. 1995) until the initial 
mean shell height for the study of 40.4 mm (SD = 4.20) and initial 
dry mass tissues of 0.35 g (SD = 0.01) was obtained. A total of 
720 individuals oi Euvola ziczac were divided into three batches of 
240 individuals each and thereafter, we took 15 individuals for 
each batch to the initial sample. Later, the remaining 225 individu- 
als of each batch were allotted to the cages, corrals, and the barrier- 
free method. In the case of the enclosures. 15 replicates were 
introduced. 12 of which were experimental and three replace- 
ments. The latter were introduced to maintain density of the indi- 
viduals reduced by the effects of mortality and escape. In the case 
of the barrier- free method, a total area of 15 m" was evaluated. 

The cages measured 1 x 1 x 1 m, built with galvanized iron bars 
8 mm in diameter, lined on the six sides by a galvanized wire mesh 



77 



78 



Freites et al. 



100° 90° 



40° 



30° 



20° 



10° 



0° 




Figure 1. Geographical location of the study area. 

with a 30-mm diameter opening (Fig. 2a). The corrals were built of 
the same size and with the same materials as the cages, except that 
the galvanized mesh was not fitted on the top and bottom parts 
(Fig. 2b). Both types of enclosures were buried 7-8 cm into the 
sand to allow the scallops in cages also to bury, and in case of the 
corrals, to avoid the escape of individuals under the enclosure and 
at the same time, to avoid entry of such predators as gastropods 
and crabs. The individuals in the bamer-free method were distrib- 
uted ill the area marked out beforehand by galvanized mesh, but 
with an edge of 20 cm. This was used to mark out the original area 
and thus enabling control of the density but not to act as a barrier 
(Fig. 2c). Both cage and corral methods were randomly placed at 
a depth of 7-8 m by a SCUBA diver. Density was a common 
parameter ( 15 individuals m"^) both for the enclosures and for the 
barrier-free method. 

Growth of individuals in the enclosures was followed by sam- 
pling the three replicates of five individuals ( 1 5 individuals) taken 
randomly, by previously allotting them random numbers. These 
samples were obtained over appro.ximately 60 days. Also, the 
number of dead and live individuals was quantified monthly in all 
the experimental replicates in terms of determining moilality. es- 
cape, and monitoring the density of individuals. 

The paramclers for evaluating growth were shell height (dis- 
tance between the anterior-posterior margins taken with a Vernier 
calliper with 0.01 accuracy) and the dry mass of ihe shell, gonad, 
muscle, digestive gland, and remaining somatic tissues (dried at 
80 ' C for 72 h). 

Because there was an initial escape rate on the order of 84% of 
the individuals placed in the original area with the barrier-free 
method and because these could not be recovered, the evaluation 
of Ihis method could no! continue. Moreover, because of scallop in 



corrals escaping, we could not continue the evaluation of methods 
for longer than the 6 months of the study. To evaluate the results 
on the enclosures, cages, and corrals during the experimental pe- 
riod, the paired student's t test was applied to all growth param- 
eters. Also, in terms of evaluating the masses and heights attained 
at the end of the study, the nonpaired student's ; test was applied. 
To evaluate the survival rate, because the data were incompatible 
with assumed normal levels, analysis was conducted by nonpara- 
metric tests not correlative to those previously noted (Wilcoxon 
and Mann-Whitney range tests, respectively, following the re- 
comendations in Zar ( 1984). For all test a a = 0.05 was applied. 



RESULTS 



Escape 



At the start of the experiment, an 84'7f escape rate was found 
(12 scallops m"~ month"') from the original area using the barrier- 
free method (Table 1). Furthermore, despite having searched an 
approximate area of 2500 m" taking the original area as the center, 
none of the individuals (C/r recovery) was recovered, so that we 
were unable to continue with the evaluation. In the corrals, a 
progressive increase in monthly escapes was noted, from 4% (1 
scallop m"- month"') rising to TibVc (5 scallops m"" month"'), 
observed at the end of the experiment (Table 1 ). In this way, the 
ratio of growth in shell height with the percentage increase in 
escape of individuals reared in cage was directly proportional (P < 
0.05, r" = 0.89: b = 2.15). No scallops escaped from the cages. 

Survival Rate 

Monthly survival rates in the two enclosures showed similar 
trends (Fig. 3a) (Wilcoxon test, P = 0.679). At the end of the 
study, however, accumulated mortalities result in a significantly 
lower survival rate of the individuals in cages (51%); whereas, in 
the corrals, it was in the order of 78% (Fig. 3b) (Mann-Whitney 
test, P < 0.05). 

Shell Size and Mass 

Growth curve trends in shell height, both for indisiduals in 
cages and in corrals, were similar throughout the study period 
(paired student's /-test, P = 0.912). with the exception of the last 
sampling, where a reduction in the growth rate of cage-reared 
individuals was observed (Fig. 4a). At the end of the experimental 
period, the individuals reared in conals had an average of 73.1 ± 
2.34 mm; whereas, the average for the cages was 69.7 ± 3.93 mm. 
These differences, however, were not significant (nonpaired stu- 
dent's /-test, P = 0.082). The dry mass of the shell showed a 
growth pattern similar to that of shell length during almost the 
entire study period (Fig. 4a. b). but in this case, significant differ- 
ences were noted (paired student's /-test. P < 0.05). So, there were 
significant differences (nonpaired student's /-test, P < 0.05) noted 
in the shell growth rates at the end of the study between scallops 
in corral (26.5 ± 2.61 g) compared to the indi\ iduals maintained in 
cages (23.9 ± 3.72 g). 

Somatic Tissue Mass 

The groulh trend for somatic tissues muscle, digestive gland, 
and the remaining somatic tissues observed during the study period 
in cages and corrals, (Fig. 4c, d, e), showed no significant differ- 
ences (paired student's /-test, P = 0.719, 0.679, and 0.369, re- 
spectively), despite the fact that these showed divergences in the 



Bottom Culture of Euvola zkzac 

b 



79 



Galvanized net 



1 m 




1 m 



Cage 



1 m 



close ' f 

bottom 



Galvanized iron rod 




open top 



open 
bottom 



Corral 




yyyyy^yyyyyy^vvyyywyyyyyvf^yvyyyv^ | 0.20 m in height 

»- 

5.0 m 

Barrier-free 

Figure 2. Design of enclosure cages (a) and corrals (b) and tlie barrier-free method (c). 



latter period of the sampling, particularly in the remaining somatic 
tissues of both groups of individuals. Also, the decrease in growth 
of the mass of remaining tissues and gonads of individuals main- 
tained in cages, observed at the end of the experimental period, 
contrasted with the increase in mass of these tissues in the corral- 
reared individuals (Fig. 4d, f). in such a manner that these were 
significantly greater at the end of the experimental period (non- 
paired student's ?-test, P < 0.05). In the case of the muscle mass in 
individuals reared in cages and corrals, no significant differences 
were shown at the end of the study period (nonpaired student's 
r-test. P = 0.947). 

DISCUSSION 

Our results showed that by applying the bottom culture method, 
in the course of approximately I year (from fertilization of the 
oocytes), sizes and biomasses considered as commercially feasible 



TABLE 1. 

Monthly escape ( % ) of the scallop Euvola ziczac observed during the 
study in the methods evaluated: barrier-free and cage. 



Month 


Methods 


Shell Height 


% Escape 


April 


Barrier-free* 




84 




Corrals 


54 mm 


4 


May 


Corrals 


59 mm 


10 


June 


Corrals 


65 mm 


18 


July 


Corrals 


67 mm 


33 


September 


Corrals 


70 mm 


36 



In the cages, the percentage of escape was always of 0%. 
* We were unable to continue with the evaluation. 



80 



Freites et al. 



100 




80 9*^ 



-©- Corrals 
-O- Cages 




J J 

Month 



Cages Corrals 



Figure 3. Monthly (a) and cumulative survival (b) of Euvola ziczac in bottom culture (vertical bars represent the standard deviations of the 
values). 



for the scallop Euvola ziczac may be obtained. Thus, the size range 
in adults located on natural banks is between 65 and 95 cm (Him- 
melman and Lodeiros, unpublished data), and sizes for individuals 
reared both in corrals (73 mm) and in cages (70 mm) were within 
this size interval. These sizes of scallop had wet muscle weights of 
7-8 g. which are considered excellent for scallop commercializa- 
tion (Dore 1991). 

The rate of growth observed in this study (approx. 0.16 mm 
d"') was similar to that observed in Euvola ziczac (approx. 0.15 
mm d~') for Velez et al. ( 1995) in the same locality, for a similar 
period of year (70% of the same period) and in the same period of 
190 days, but with a higher initial high density (64 individuals 
m"~) than in this study ( 1 5 individuals m""). These similar growth 
rates, despite the different densities in both .studies, suggest that the 
growth observed in this study was not more influenced by the 
density used. This also suggests that the bottom culture produc- 
tions of this species can be increased with the use of higher den- 
sities in the methods studied. 

The high escape rate of individuals with the barrier-free method 
led to the discontinuation of this method. This suggests that Euvola 
ziczac has a high dispersion capacity, which would lead to a low 
recovery rate of the stock originally used for cultivation with no 
barriers. Therefore, we considered that it is necessary to develop a 
new experiment with a more adequate scale before suggesting the 
use of this barrier-free method. In any ca.se, in other countries, such 
as Canada, Japan, and France, the use of the barrier-free bottom 
culture method in more adequate scales had a low recovery rate of 
the initial stock because of the high escape rates of scallops (Wild- 
ish et al. 1988. Aoyama 1989, Cliche et al. 1994, Dao et al. 1995). 
Nevertheless. Wang et al. (1992) showed that even when the re- 
covery rate of the initial bottom stock was on the order of 54'/r, the 
low production costs exerted an influence on the high profitability 
of scallop cultivation of Chlaniys farrcri. In our case, the recovery 
rate of individuals with the barrier-tree method was 16%. This may 
be considered as very low it ue take into account thai it was 
obtained after only .^0 study days. This escape capacity was also 
evident in the corrals where, despite l-m high barriers, escape 
gradually increascil unlil the end of the study (.^6% escape). Fur- 



thermore, it was observed that some specimens cultured with this 
method, when unintentionally disturbed for the purpose of taking 
samples, showed a clearly evident capacity to escape beyond the 
1 -m high barriers. In these observations, we noted the increase in 
the vertical displacement capacity of Euvola ziczac as size in- 
creases. However, we do not exclude the possibility of the increase 
in the rate of depredation by some fish, octopus, and crab decapods 
during the time of the experinient. Nevertheless, this phenomenon 
was not noted in the course of our frequent observations. 

At the end of the study, the cage-reared individuals presented a 
significantly lower survival rate than those reared in corrals. In the 
cages, greater protection from predators was expected because of 
the presence of netting on all sides that, theoretically, would im- 
pede their entrance of the same. In the cages, however, several 
fragmented shells were collected, a fact that indicated the action of 
predators. For this reason, a detailed search was conducted, and the 
presence of decapod crab juveniles Calappa cinerea was discov- 
ered. These had gone unnoticed until that point because of their 
strategy of burying themselves in the substrata. This decapod has 
strong chela that allow it to fragment Euvola ziczac shell. Judging 
by the condition of the shells. This ability has also been noted in 
the species Calappa ocelluia. as a result of its preying action on the 
bivalve Brcuhidonlcs doiuiitf^cnsis (Hughes and Finer 1989). This 
suggests that the decapod C. cinerea apparently entered the cages 
at its juvenile stage, when the opening in the mesh still made this 
possible, so that it was also able to take advantage of the protection 
afforded by the cage. This situation helped avoid competition for 
food and being preved upon. One observation that supports this 
hypothesis is that in the corrals, where there was no upper netting, 
the dead individuals of Euvola ziczac showed no shell fragmenta- 
tion and nor were any detected C. cinerea. These observations 
differ from those for the cultivation of temperate water scallop 
species where the use of nets substantially decreased predation 
(Morgan et al. 1980, Quayle and Newkirk. 1990). 

Because this study was conducted in a certain season of the 
year, possible biocontrol of decapod C. cinerea juveniles may not 
be present throughout the year. Furthermore, one of the predators 
that nui\ possibly exert a dramatic effect on scallop survival under 



Bottom Culture of Euvola ziczac 



81 



8-1 


a 


Shell height 




7- 




Cages,^ 


V^ 


^ 


^ 6- 

B 

u 

"-^ 5 J 




/ 


Ct>rrals 


4- 




/ 


3- 




-1 1 1 r— I I 1 1 



30 



20 



bo 



F M A M J J A S 



10- 



Dry mass shell 




f'm' a'm' J ' j' a' s' 



1.6-] 


C 


Dry 


mass muscle 




1.4 




^^^^ 




1.2- 




y^yr^^^^^^*^ 


•• 


^ 1.0- 

130 
^ 0.8- 




/ 


K 




0.6- 




/ 




0.4- 


/ 


f 




0.2- 




1 1 1 1 T I 


1 



bo 



l.On 


d 


Dry mass remaining tissues 


0.8- 




y. 


fi 


0.6- 




.^'^^ 




0.4- 




A 


0.2- 


/ 


/^ 


0.0- 


— 1 


1 1 1 1 1 1 1 



F M A M J J A S 



F M A M J J A S 



(30 



0.5 

0.4-1 

0.3 

0.2 
0.1 



0.0^ 



Q Dry mass digestive gland 




F ' M ' A 'm 



J 
month 



— I 1 — 

J A S 



1.0' 


f 


Dry mass gonad 




0.8- 




^^ 


z 


^0.6- 
bo 




,^ 




0.4- 




A 


y 




0.2- 


A 


/ 




0.0- 


-X- 


— 1 1 1 r— 1 r 


1 



F M A M J J A S 
month 



Figure 4. Growth in siieli iieiglit (a) and dry mass of the shell (b) muscle (c), remaining soft tissues (d), digestive gland (e), and gonad (f) of the 
cultivated specimens oi Euvola ziczac from bottom culture (vertical bars represent the standard deviations of the values). 

bottom culture conditions, as noted on natural scallop banks, are in future studies aimed at determining the effect of predators on the 

the cephalopods Octopus spp. (Freites, personal observations), survival of bivalves in corrals, it is advisable to cover different 

Nevertheless, despite the fact that the period of greatest influence periods or seasons of the year. 

by these predators fell within the experimental period of this study Also, in the Golfo de Cariaco. hanging culture of these bivalves 

(from June to September), they were not observed. For this reason. does not guarantee a lesser impact of predation compared to some 



82 



Freites et al. 



temperate water species (Quayle and Newkirk 1990, Hickman 
1992). This is because of the recruitment of some predatory de- 
capod and gastropod species during their planktonic larval stage, 
which allows them to gain access to the hanging baskets. Once 
inside these baskets, if uncontrolled, their growth is so fast that, in 
some cases, they have caused substantial mortality rates (>60%) in 
the cultivation of several bivalve species with culture potential, 
including Euvola ziczac (Freites et al. 1995, Freites et al. 2000). 
Pinna carnea (Narvaez 1999) and pearl oyster Piiutcula inihricala 
(Pico D., unpublished data). 

The growth pattern for the corral and cage-reared individuals 
was similar, except in the latter sampling period, when the cage- 
reared individuals showed lower growth rates. These differences 
may not be attributable to a differential colonization by fouling 
organisms in nets of the enclosures or on shell that may, in the long 
term, affect food availability for the scallops, because the nets in 
both enclosures were cleaned throughout the experimental period 
because of the action of "grazing" of some fish and benthonic 
invertebrates on the net of the corrals and cage (personal obser- 
vation), and because virtually no organisms colonized the shells of 
scallops in both enclosure, probably because the scallops were 
usually recessed in the sand. This together with maintaining the 
same density of individuals in the enclosures suggests that food 
availability was not a factor in the decreased growth observed in 
the cages. 

One possible explanation is based on the fact that, as the Euvola 
ziczac individuals reared in cages increased in size and even when 
new C. cinerea decapod recruits were observed, they were physi- 
cally unable to prey on the scallop because of the larger, more 
resilient shell, as evidenced by the subsequent lack of fractured 
shells. We do not rule out the fact, however, that the decapod 
juveniles may cause some disturbance leading to a defensive be- 
havior, so that the bivalves close their valves, thus restricting fil- 
tration time and, con.sequently, affecting growth. 

As we have seen earlier, the growth of juvenile scallops reared 



in the two types of enclosures may not be used as a selection 
criterion for recommending the use of dismissal of one of these 
two types of enclosures studied, particularly if we take into ac- 
count that at the end of the experimental period, no significant 
differences were noted in muscle weight. Survival, however, may 
be used as a selection criteria, because, in the case of corrals, the 
rate was 27% higher. This difference would significantly affect the 
production level of the culture in favor of corrals. Furthermore, 
corrals involve a lower investment cost, and it is likely that op- 
erational costs would also be lower, because of a need for less 
material to construct the enclosure, and while seeding, supervision 
and harvesting tasks are easier. Taking the above into account, the 
use of corral-type enclosures is advisable, with a height of over I 
meter, to minimize escape. 

Finally, during this study, an average growth rate of 6 mm 
month"' was found. This is high if we compare it to growth rates 
of other scallop species of commercial importance, such as Pecten 
maxinnis (2 mm month"'), P. siilsicostatus (2 mm month"'), P. 
albicans (3 mm month"' ), and P. novaezelandiae (4 mm month"') 
(Mottet 1 979). Only Amusimn halloti (Williams and Dredge 198 1 ), 
Clilaiiiys piiipiiratiis (DiSalvo et al. 1984), and Arf>opecren ciicu- 
laris (Felix-Pico 1991 ) scallops attained similar rates of growth. In 
this manner, the growth rate of bottom-reared Euvola ziczac. its 
survival and relatively low cost with this culture method (Ventilla 
1982) offer clear possibilities for further investigations in the de- 
velopment of commercial culture of this species. 

ACKNOWLEDGMENTS 

We thank the valuable cooperation of the personnel at the Tur- 
pialito Hydrobiology Station of the Instituto Oceanografico de 
Venezuela, Universidad de Oriente: Maximiano Nfmez, Antonio 
Sotillet, Aquiles Rojas and Eduardo Gonzales. This research work 
was funded by grants from the Consejo de Investigacion de la 
Universidad de Oriente. Finally, we thank Ian Emmett for trans- 
lation of this article. 



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Journal of Shellfish Reu'iiich. Vol, I*). Nd. I. 85-8S. 2000. 

ALLOZYME AND BIOCHEMICAL VARIATION AT THE OCTOPINE DEHYDROGENASE 

LOCUS IN THE SCALLOP EUVOLA ZICZAC 



J. E. PEREZ,' O. NUSETTI,- N. RAMIREZ," AND C. ALFONSi' 

Instinito Ocecmogrdfwo de Venezuela, and 
'Departamento de Biologia 
Esciiela de Ciencias 
Universidad de Oriente 
Nucleo de Sucre, Venezuela 

ABSTRACT High activities of octopine dehydrogenase (Odh) in the adductor muscle of bivalve molluscs are associated with a 
dependence on anaerobic glycolysis during swimming. The Odh locus is polymorphic in the scallop EiivoUi ziczcic. Estimated apparent 
Odh A',„s for arginine were not different among nine Odh genotypes; however. A'„,s for pyruvate differed significantly (P < 0.001) 
between heterozygous and homozygous scallops. The estimated apparent A'„, values of Odh for arginine and pyruvate are dependent 
on their respective cosubstrate concentrations. Possible mechanisms for this overdominance include null alleles, aneuploidy. and higher 
fitness of the heterozygous. Our data suggest that heterozygous superiority in fitness is the most likely explanation for the apparent 
o\erdominance at the Odh locus. 

A'£}' WORDS: Arginine. bivalves, molluscs, octopine dehydrogenase, overdominance. pyruvate, scallops 



INTRODUCTION 

Numerous studies demonstrate how allozymes influence vari- 
ous components of fitness. For example, allozymes differ in their 
catalytic properties, including kinetic parameters (K„^ and V,„^^), 
and heterozygous genotypes may show overdominance (exceeding 
the two corresponding homozygous genotypes) (Sarver et al. 
1992); be intermediate in catalytic efficiency between homozy- 
gous genotypes (Hoffmann 1981, 1983): or exhibit dominance, 
having catalytic efficiencies similar to the most efficient genotype 
(Hillbish and Koehn 1985; Nirchio et al. 1991). 

Allozyme heterozygosity and growth rate are positively corre- 
lated in many bivalve species (Beaumont and Zouros 1991 ; Zouros 
et al. 1992; Hedgecock et al. 1996). Higher levels of heterozygos- 
ity are associated with a lower basal metabolic rate that allows 
heterozygous individuals to devote more of their aerobic scope to 
growth and reproduction (after meeting basal requirements) 
(Koehn and Shumway, 1982). Recently, Hedgecock et al. (1996) 
found in the Pacific oyster Crassostrea gigas (Thunberg) that not 
only are oxygen consumption rates lower for hybrid compared 
with the inbred larvae, but also the net efficiency of protein syn- 
thesis is much higher for the hybrids. Several authors (Carton et al. 
1984; Rodhouse et al. 1986: Volckaert and Zouros 1989) have 
suggested that organisms use this energy surplus for functions that 
increa.se fitness. In sedentary molluscs such as mussels and oys- 
ters, metabolic energy would be better invested in growth during 
the juvenile stages and in reproduction in adults. However, scal- 
lops are active bivalves that avoid predation by vigorous swim- 
ming; thus, selection of an allozyme could result in an increased 
locomotion capacity. 

Scallops display sudden bursts of muscle activity, initially sus- 
tained by arginine phosphate breakdown, followed by the activa- 
tion of glycolytic pathways that result in rising levels of octopine 
(Chih and Ellington 1983: Bricelj and Shumway 1991). Octopine 
is produced by the reductive condensation of arginine and pyruvate 
catalysed by octopine dehydrogenase (Odh, EC 1.5.1.11). in the 
presence of NADH. High activities of Odh occur in the adductor 
muscles of scallops (Chih and Ellington 1983: Alfonsi et al. 1995). 
The advantage of octopine formation in adductor muscles may be 



that oxidation of NADH removes arginine and thereby facilitates 
the formation of ATP from arginine phosphate. 

The energy needs among molluscs vary, as scallops, which 
swim, require higher instantaneous rates of ATP production than 
sedentary bivalves such as mussels and oysters. Phosphoarginine is 
the principal fuel during valve snapping, and octopine accumulates 
during the subsequent recovery phase under functional anaerobio- 
sis (Bricelj and Shumway 1991 ). Genetic effects on glycolitic ATP 
production are correlated with increased ability for burst activity in 
pectinids. Volckaert and Zouros (1989) found in the scallop Pla- 
copecten magellanicus (Gmelin) that heterozygosity and octopine 
accumulation after burst activity are correlated. The degree of 
heterozygosity and the maximal activity of pyruvate kinase and 
Odh are positively correlated in the adductor muscle of the scallop 
Eitvoki ziczac (Alfonsi et al. 1995). 

In scallops, the primary function of Odh is to maintain the 
redox balance of the muscle during exhaustive exercise. The 
present study was designed to determine whether allozymes of 
Odh. which is polymorphic (Coronado et al. 1991), differ in cata- 
lytic properties. We determined this by measuring the apparent A"^ 
of Odh in the scallop. E. ziczac, under varying concentrations of 
pyruvate and arginine. 

MATERIALS AND METHODS 

Adult scallops. E. ziczac {n = 103), were collected in 1998 
during their sexual resting period from the waters of the Gulf of 
Cariaco (Chacopatica) on the northeastern coast of Venezuela 
(10°30'I0"N. 64°13'06"W). They were maintained in running aer- 
ated seawater. 

To determine the genotype at the Odh locus, the adductor 
muscle from each individual was excised, minced, and centrifuged, 
and the supernatant was analyzed by horizontal 12% starch gel 
electrophoresis. The activity of Odh was identified using the stain- 
ing procedures described by Morizot and Schmidt ( 1990). Allelic 
variants were designated by letters, with "a" being always the most 
anodic. To prepare the enzyme extracts, the frozen adductor 
muscle of each specimen was chopped and homogenized in 20% 
w/v cold 50 niM imidazole-HCI buffer in ice, pH 7.5, with 2 mM 
ethylenediaminetetra-acetic acid. The homogenized tissue was 



85 



86 



Perez et al. 



centrifuged at 27,000 ,1; for 20 min at 4 °C. Solid ammonium 
sulphate was added to the supernatant to reach 70% saturation. The 
resulting suspension was stirred at 4 'C for 30 min and then cen- 
trifuged at 20,000 g for 20 min. The pellet was dissolved in a small 
volume of the homogenizing medium, applied to a Sephadex 
G-lOO column equilibrated with 50 mM Tris-HCl (pH 7.6) at 
24 °C, and eluted with the same buffer. The eluted fraction with 
highest Odh activity was used for kinetic analyses. 

Odh activity was measured by recording changes in optical 
density (OD; 365 nm) that were caused by the oxidation of NADH. 
Reactions were run using 25 |j,L of the enzyme preparations in 
1.25 mL of incubation mixture. The routine enzyme assay for 
maximal activity was 0.2 mM NADH, 2.5 mM pyruvate, and 5.0 
mM arginine in a 50 mM imidazole buffer at pH 7.5. All of the 
assays were run at 24 °C. The enzyme activity was expressed as 
spectrophotometric units (OD). Maximal activity was recorded 
between pH 6.0 and 7.5 in pilot enzymatic assays. 

Odh followed Michaelis-Menten kinetics for both arginine and 
pyruvate, at saturation concentrations of the other substrate and of 
NADH. Substrate inhibition by pyruvate was observed at concen- 
trations over 2.5 mM. Accordingly, the apparent Michaelis con- 
stants (apparent AT,,,) for the substrates arginine and pyruvate were 
estimated from the Michaelis-Menten equation, according to Chur- 
chill and Livingstone (1989): 



Estimated apparent K„ 



(V„,^,yV) - 1/S 



where V represents the initial reaction velocity at either a pyruvate 
or arginine subsaturating concentration, when the respective co- 
substrate was at a saturating level. Before applying this formula, 
the maximal velocity was calculated from the Lineweaver-Burke 
plots (Segel 1975), in which the concentration of one substrate (A) 
was varied and the concentration of the other substrate (B) kept 
constant. The data were fitted to the following equation: 



lA' 



0/V„_(l +^„;VA) I/B+ 1/V„ 



The initial velocity was recorded against the pyruvate concentra- 
tion (0.10, 0.20, 0.83, and 2.50 mM) at fixed arginine concentra- 
tions (0.5, 1.5, 3.0, and 5.0 niM). The inverse of the initial velocity 
(lA') was plotted against the inverse of the pyruvate concentration 
(l/S) for each arginine concentration. The Y intercepts of the 
Lineweaver-Burke lines estimated by linear regression analysis 
were plotted against the inverse of arginine concentration. The 
maximal velocity was determined from the value of the resulting Y 
intercept, which was essentially similar to that estimated by the 
routine enzyme assay. Likewise, the data were plotted as a func- 
tion of the arginine (0.5, 1.5, 3.0, and 5.0 niM) concentration. The 



V,„^^ value estimated agrees with that obtained using varying con- 
centrations of pyruvate at fixed concentrations of arginine. Sub- 
strate inhibition of V,,,^^ was observed for pyruvate concentrations 
over 2.5 mM at each arginine concentration. 

Deviations from expected values of allele frequency for Hardy- 
Weinberg equilibrium were tested by using a Chi-square analysis. 
The deficiency or excess of heterozygotes (analyzed by the F 
statistic) and the effective number of alleles at this locus (N^, the 
reciprocal of the sum of squares of the allele frequencies) were 
calculated by using the statistical program Genes in Populations, 
version 2 (Perkins and Paul 1995). 

RESULTS AND DISCUSSION 

The sample of 1 03 individuals from the population of Chaco- 
patica contained nine Odh genotypes: c/c, d/d, e/e, b/c, c/d, c/e, c/f, 
d/e, and d/f, determined by five alleles Odh'', Odh'. Odh'', Odh^ 
and Odh'. Because genotypes that include alleles a and b are very 
rare (Table 1), it was not possible to obtain sufficient samples to 
study their catalytic properties of these rare alleles. Allele frequen- 
cies have been stable since the first sample was examined in 1984. 
All three samples were in Hardy-Weinberg equilibrium. Hetero- 
zygote superiority probably provides the best explanation for the 
maintenance of the polymorphism. 

Apparent A',„ for arginine and pyruvate were related to varia- 
tions in the concentrations of the respective cosubstrates, because 
K,„ decreased as the concentration of cosubstrate increased (Table 
2). This suggests a mechanism that favors the formation of oc- 
topine when the concentration of the two substrates increases si- 
multaneously, as is seen in active individuals. The availability of 
arginine and pyruvate could be the two limiting factors in the 
regulation of Odh activity for maximal glycolytic capacity during 
the escape response and recuperation of E. ziczac. In addition, 
specific genetic influences affect the regulatory properties of the 
enzyme by acting on their relative substrate affinities. 

Results for the K^ of pyruvate and arginine at different cosub- 
strate concentrations were separated into two groups: homozygotes 
and heterozygotes. Table 3 indicates no significant differences for 
the A",,, of arginine (pyruvate as cosubstrate) (F = 0.017; P>Q.05), 
whereas highly significant differences were detected between ho- 
mozygotes and heterozygotes for the K^ of pyruvate (arginine as 
cosubstrate) (F = 29.33; P < 0.00 1 ). These results indicate that the 
affinity of the Odh enzyme for pyruvate was predominantly greater 
in heterozygous than in homozygous individuals. Similar results 
were observed by Walsh ( 1981 ) for three phenotypes of Odh in the 
anemone Metridium senile (L.), in which (he heterozygotes 
showed a higher affinity for pyruvate. Sarver ct al. (1992) mea- 



TABLE 1. 



Allele frequencies, effeclive number of alleles (A'^), observed (W,,) and expected (//,.) values for hetero/.vsi'sitv, and values of 7' are tests of 
goodness of fit to Hardy-VVeinberg proportions for the Odh locus in samples of C'hacopatica collected in 1984, 1994, and 1998. 









Allele Frequency 






N 


N, 


"„ 


H. 


F;. 


y' 






a 


b 


c 


d 


e 


f 


P 


1984* 
1994t 
1998 


0.0 II 
0.000 

o.ooo 


0.033 
0.000 
0.005 


0.456 
0.500 
0.495 


0.244 
0.310 
0.3 1 1 


0.244 
0.1.50 
0.165 


0.01 1 
0.0.50 
0.024 


45 
113 
103 


3.05 
2.75 
2.66 


0.533 
0.655 
0.62 1 


0.609 
0.636 
0.63 1 


0.12 
0.03 

0.014 


8.82 
4.76 
3.72 


>0.l 
>0.1 
>0.3 



N = sample size. F,^ indicaies deficiency or excess of heterozygotes. 
*Coronadoet al. 1991. 
t Fernande/ 1 995. 



Polymorphism at the Odh Locus in Euvola ziczac 



87 



TABLE 2. 

Means and standard deviations for the estimated apparent A„, for both substrate arginine and p.vru>ate for the different genotypes, at the 

different concentrations. 





N 




Cosubstrate Arginine (mM) 






Cosubstrate Pyruvate (mM) 




Genotypes 


1.5 


3.0 


5.0 


0.025 


0.83 


2.5 


be 


1 


0.25 


0.18 


0.11 


0.91 


0.83 


0.42 


cc 


7 


1.70± 1.15 


0.58 + 0.14 


0.35 ± 0.09 


2.80 ± 1.38 


1.61 ±0.60 


0.82 ±0.29 


cd 


7 


1.20 ±0.33 


0.57 ±0.1 7 


0.19 ±0.07 


1 .50 ± 0.59 


1.18 ±0.47 


0.61 ±0.21 


ce 


7 


1.52 ±0.62 


0.48 ±0.1 5 


0.25 ± 0.08 


4.60 ± 1 .60 


1.99 ±0.61 


0.99 ± 0.35 


cf 


1 


0.19 


0.13 


0.06 


2.39 


1.44 


0.76 


dd 


7 


2.33 ± 1.44 


1.04 ±0.27 


0.42 ±0.11 


2.48 ±1.65 


1.71 ±0.93 


0.83 ±0.33 


de 


7 


0.67 ± 0.34 


0.35 ± 0.09 


0.17 + 0.04 


2.41 ±1.15 


1.31 ±0.46 


0.67 ±0.19 


df 


4 


0.49 ±0.21 


0.26 ±0.11 


0.16 ±0.06 


1.86 ±0.63 


1.40 ±0.43 


0.59 ±0.16 


ee 


3 


0,69 ± 0.33 


0.22 ± 0.05 


0.14 ±0.02 


3.56+ 1.24 


1.71 ±0.52 


0.53 ±0.11 



N is the number of animals examined. 



sured specific Odh activities in a large number of individuals of the 
mussel Mytilus tiossulus Gould for Odh and found that the mean 
Odh activity was greater in heterozygotes than homozygotes. 

Multiple range analysis (least-significant difference) indicate 
three groups in increasing order of A",,,: (1) d/f, e/e, d/e. c/d, and 
c/e: (2) c/d, c/e, and c/c; and (3) d/d. Genotypes c/d and c/e be- 
longed to the groups with higher and medium affinities [ 1 1 and [2], 
Table 3). By increasing Odh affinity for pyruvate, heterozygous 
individuals could enhance the ability of the muscle to maintain the 
NADH/NAD* redox balance during the glycolytic flux which can 
occur during high-intensity muscle work. This affinity would be 
particularly useful during the initial phase of glycolysis when py- 
ruvate concentration is low and arginine levels begin rising (argi- 
nine phosphate pool is depleted). Moreover, the shunting of pyru- 
vate to mitochondrial metabolism or cytoplasmic synthesis of ala- 
nine could be inhibited to rapidly meet the energy demands under 
functional anaerobiosis of the contracting fibers. On the other 
hand, it appears that arginine does not represent a control or lim- 
iting factor for the anaerobic glycolitic capacity for the fast muscle 
contraction of E. ziczcic. This assertion, however, does not exclude 
the possibility of other genetic influences on the enzymatic con- 
version of arginine into arginine phosphate, which would assure a 
faster recuperation after a burst exercise. 

Heterozygotes show an apparent overdominance in A',,, for py- 
ruvate thai may be a fitness component if the concentration ot 

TABLE 3. 

Means and standard deviations from the estimated apparent A„, for 
pyruvate for heterozygous and homozygous individuals. 





A' 


Cosubstrate Arginine 


(mM) 


Genotype 


L5 


3.0 


5.0 


Heterozygous 
Homozygous 


27 
17 


0.72 ±0.49 0.32 ±0.1 6 0.16 ±0.06 
1.57±0.68 0.61 ±0.33 0.30±0.12 

Cosubstrate Pyruvate (mMl 




0.205 


0.83 


2.5 


Heterozygous 

Homozygous 


27 
17 


2.84 ± 1 .30 
2.64 ± 1.16 


1.49 ±0.36 
1.66 ±0.33 


0.75 ±0.1 6 
0.83 ± 0.2 



N is the number of animals examined. Means are different (P < 0.001). 



pyruvate is low. Apparent overdominance (heterozygous geno- 
types are phenotypically superior to homozygous genotypes), in 
fitness components such as growth, viability, and fecundity, has 
been observed in many species of marine bivalves (Sarver et al. 
1992). Possible explanations for overdominance, as well as the 
commonly reported deficiencies of heterozygotes. include null al- 
leles (Foltz 1986) and aneuploidy (Thiriot-Quievreux et al. 1988). 
Several studies of allozyme inheritance have found substantially 
higher frequencies of null alleles in bivalves than found in other 
organisms, suggesting that null alleles or segmental aneuploidy 
may play a role in the apparently lower fitness of allozyme het- 
erozygotes (Gaffney 1994). Null alleles or missing chromosomes 
(and therefore missing alleles) could contribute to fitness advan- 
tages of heterozygotes. In Mylihis ediilis (L.), Hoare and Beaumont 
(1995) found not only heterozygotes, but also homozygotes for a 
null Odh allele. We believe this situation unlikely to occur an 
active species, such as E. ziczac. 

Considering an alternate explanation of heterozygote superior- 
ity in fitness or other phenotypic attributes to explain our results, 
there are two possible scenarios, as follows. 

( 1 ) Individuals that appear single banded (homozygous), which 
were numerous in our sample, may really be heterozygous (active/ 
null alleles); if so. we would expect a bimodal distribution, with a 
resulting higher variance of activity in homozygous compared with 
heterozygous. However, this was not the case for our samples of £. 
ziczac (see Table 3). 

(2) Results obtained in the analysis of other enzymes (pyruvate 
kinase, glucose 6-phosphate dehydrogenase, isocitrate dehydroge- 
nase, and malate dehydrogenase) in E. ziczac indicate that their 
specific activity is correlated positively with heterozygosity. Be- 
cause the Odh activity in homozygotes and heterozygotes scallops 
increases with heterozygosity at multiple loci, it seems highly 
unlikely that null alleles (or missing chromosomes) would occur at 
all of these loci (Alfonsi et al. 1995). 

Therefore, our results can be best explained by assuming an 
overdominance at the Odh locus, which could enable the heterozy- 
gotes to increase their ability to escape from predators. 

In conclusion, the pyruvate affinity of the adductor muscle Odh 
allozyme in £. ziczac appears to be a catalytic target upon which 
genetic influences act to determine the tissue's capability for main- 
taining a steady NADH/NAD ratio, that would support the rate of 
anaerobic glycolysis at the burst working muscle, such as the 



88 



Perez et al. 



sudden escape behavior commonly observed among scallops when 
they tlee from predators. During routine work, we have observed 
that E. ziczac scallops are easily induced to vigorously snap their 
valves (swimming) when approached by gastropods, crabs, star- 
fish, and human divers. This predator-avoidance behavior may be 
repeated for several minutes before entering in a variable resting 
period. However, the nature of the relationship that exists between 
the capability to sustain muscle contraction in response to preda- 
tory stimulus and the Odh genotypic variants in E. ziczac is not 
clear. 

Finally, further research is required on some biochemical and 
physiological events associated with activity in different genotypic 
variants of E. ziczac, both under laboratory bioassays and field 
work conditions, because predation is a significant component in 
the life history of adult scallops, we are currently searching for a 



possible relationship between the escape reaction and the different 
genotypes, and because polymorphism at the Odh locus in E. zic- 
zac seems to be important for understanding genetic variation in 
molluscs, we are also searching for the presence of polymorphism 
in other, sedentary and motile, species of molluscs. Additionally, 
we are examining stronibine and alanopine dehydrogenase (which 
also serve as hydrogen and carbon sinks in maintaining redox 
balance) in several species of molluscs during anaerobic metabo- 
lism, for possible polymorphisms and their maintenance mecha- 
nisms. 

ACKNOWLEDGMENTS 

We thank Dr. Kent Rylander, Texas Tech University, as well as 
two anonymous reviewers, for critically reading the first version of 
the manuscript. 



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Beaumont. A. R. & E. Zouros. 1991. Genetics of scallops, pp. 58.'i-623. In: 
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Gaft'ney, P.M. 1994. Heterosis and heterozygote deficiencies in marine 
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Hoffman, R. J. 1981. Evolutionary genetics of MetriJiuni senile. I. Kinetic 
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Joiinwl of Shellfish Rfsearch. Vol. 19. No. 1. 89-93, 2000. 

ENVIRONMENT AND POPULATION ORIGIN EFFECTS ON FIRST SEXUAL MATURITY OF 
CATARINA SCALLOP, ARGOPECTEN VENTRICOSUS (SOWERBY II, 1842). 



PEDRO CRUZ. CARMEN RODRIGUEZ-JARAMILLO, AND 
ANA M. IBARRA 

Centre) de Investigaciones Bioldgicas del Noroeste, S.C.. 

Km. I Can: a San Juan de la Costa, 

El Comitdn, A. P. 128. 

La Pa: B.C.S. 23000. Mexico 

ABSTRACT Two populations of catarlna scallop, and their cross ( F 1 ). were evaluated for the age at first se.\ual maturity, and for their 
gonadal development in both populations' native environments. All experimental groups were hatchery produced. Differences in mean 
gonad index (MGI) were seen between the different environments. In Bahia Magdalena. a site characterized by high productivity and 
lower average water temperatures, the MGI was higher than for scallops grown in Bahia Concepcion, a bay with lower productivity 
and higher average water temperatures. Differences in age and size at first sexual maturity, defined as tho,se when 50% of the scallops 
in any group were sexually mature, were seen between the populations when grown at Bahia Magdalena but not when grown at Bahia 
Concepcion. At Bahia Concepcion. none of the groups had reached their first sexual maturation after the experimental period of 7 mo. 
At Bahia Magdalena. the Magdalena population and the Fl reached sexual maturity at an eariy age of 4 mo, whereas the Concepcion 
population reached maturity when 5 ino old. Also for the Bahia Magdalena environment, the gonad index (GI) estimated at first sexual 
maturity for the Magdalena population and the Fl was significantly higher than that estimated for the Concepcion population at that 
same age. There were no significant differences in GI values when the groups were grown at Bahia Concepcion. The differences 
between populations in age at first sexual maturity suggest that a triggering mechanism exists in catarina scallop for the initiation of 
sexual maturation, whereas the differences between environments suggest that regardless of that mechanism, environmental conditions 
have a significant role in further maturation processes. 

KEY WORDS: Argopecten venlricosus, environment, gonad index, populations, maturation 



INTRODUCTION 

The catarina scallop, Argopecten ventricosus (Sowerby II, 
1842), which is a functional hertnaphrodite species, is an important 
fishery and aqiiaciilture resource on both coasts of the Baja Cali- 
fornia Peninsula, Mexico. The geography of the peninsula results 
in this species distributing and growing in different environmental 
conditions: semitropical to temperate on the Pacific Ocean side 
and tropical on the Gulf of California side. Because of this, as well 
as the presumed isolation caused by the Peninsula barrier itself, 
natural populations existing on both sides are expected to be ge- 
netically different, that is, to have evolved differently in response 
to environmental conditions on each side. In fact, we have dem- 
onstrated that there are differences between these two populations 
in growth and survival (Cruz and Ibarra 1997, Cruz et al. 1998). 

An additional important trait to compare in populations on both 
sides of the peninsula is the age and size at which each population 
reaches its first sexual maturity. It has been stated that the repro- 
ductive cycle of scallops is a genetically controlled response to 
environmental conditions (Sastry 1970, Sastry 1979, cited by Bar- 
ber and Blake 1991). which depends on the optimum interactions 
between exogenous and endogenous factors. When the appropriate 
combination of exogenous and endogenous factors occurs, a mini- 
mum age (or size) has to be reached before the beginning of 
gametogenesis (Barber and Blake 1991). Differences in the onset 
of sexual maturity and reproductive cycle have already been re- 
ported for other bivalve species (Dalton and Menzel 198.3. Knaub 
and Eversole 1988. Barber et al. 1991. Mackie and Ansell 1993). 
Among different bivalves studied simultaneously at different sites. 
or through transplantation, there are differences in the onset of 
gametogenesis (Newell et al. 1982. Barber and Blake 1983. 
Walker and Heffernan 1994). spawning time (Brousseau 1987. 



Emmett et al. 1987. Paulet et al. 1988). fecundity (Bricelj et al. 
1987). and gatnetogenic cycle (Wilson 1987, Thorarinsdottir 1993. 
Sbrenna and Campioni 1994). Some of the previously reported 
differences are not necessarily caused by genetic factors but by 
different environmental conditions at each site studied. 

Different studies with catarina scallop have been peiformed 
regarding sexual maturation and gametogenic cycles (Baqueiro et 
al. 1981. Tripp-Quezada 1985. Villalejo-Fuerte 1992. Felix-Pico 
1993. Villalejo-Fuerte and Ochoa-Baez 1993). However, differ- 
ences between populations in age at first sexual maturity or in the 
effects of different environments on gametogenic cycles and matu- 
ration have not been investigated. 

In this study, we evaluated the onset of first sexual maturity and 
the gametogenic cycles for two populations of catarina scallop and 
their cross (Fl). The two populations were Concepcion in Bahia 
Concepcion on the Gulf of California side of the Baja California, 
and Magdalena in Bahia Magdalena on the Pacific Ocean side of 
the Baja California peninsula. All groups, Magdalena, Concep- 
cion. and their Fls. were simultaneously evaluated in both envi- 
ronments. 

MATERIALS AND METHODS 

Populations and Fl 

Spawners used, conformation of the experimental groups, and 
larvae rearing have been described by Cruz and Ibarra (1997). In 
summary, four groups were produced by mass spawning; 
Magdalena. Concepcion, and both reciprocal Fls. At a spat size of 
1.5 cm length and 2.5 mo old. 9 (pseudo) replicates, each with 100 
scallop spats, were formed within each group by randomly sam- 
pling 900 spats from the total group pool. 



89 



90 



Cruz et al. 



Grow-Out 



Statistical Analyses 



The spat contained within each of the nine replicates per group 
were simultaneously transported to each of the experimental field 
areas. Bahia Magdalena and Bahia Concepcion, where they were 
maintained for 5 mo. At each site, the scallops contained in each 
replicate were kept in a Nestier tray suspended from a long-line for 
45 days and then were transferred to bottom culture to avoid po- 
sition effects on growth caused by water temperature stratification. 
Nestier trays were attached to a metal structure anchored to the 
bottom. Maintenance was performed monthly. Densities were the 
same for all replicates within groups and in both environments 
(Cruz et al. 1998). 

Gonad Sampling and Histology Analysis 

Sampling for gonad tissue began after 1 .5 mo of grow-out. at 4 
mo age. Three individuals were sampled per replicate (27 per 
group) at ages 4. 5. 6, and 7 mo. Samples were fixed in Davidson's 
fixative and were preserved in 70% alcohol. The hematoxylin- 
eosin staining technique was used. Sexual maturity was evaluated 
with a modified Villalejo-Fuerte (1992) scale for this hermaphro- 
dite scallop, where seven stages are included for the female portion 
of the gonad (Stage = undifferentiated or virginal; Stage I = 
resting: Stage II = start of gametogenesis; Stage III = advanced 
gametogenesis; Stage IV = maturity; Stage V = spawned; Stage 
VI = postspawned). 

Age-Size at First Sexual Maturity 

Age and size at first maturity were established by a different 
criterion than that commonly used when populations are evaluated 
following a field-born cohort (Nikolsky 1969). Under that meth- 
odology, age-size at first maturity is estimated when the cumula- 
tive frequency of mature individuals reaches 50% in the cohort. 
Field-born individuals of a population are of different ages because 
spawning of the whole population usually last from days to weeks. 
In the present study, all individuals were the same age. Therefore, 
in this study, age and size at first sexual maturity were defined as 
the age when 50% of the organisms within any group were in the 
"maturity"" gonadal stage, or Stage IV as defined above, or when 
the sum of individuals in Stage IV (maturity). Stage V (spawn). 
and Stage VI (post-spawn) was 2 50%. Only the female gonad 
portion was used for the establishment of the age and size at sexual 
maturity. 

Gonad Indices 

Gonad indices (GIs) were calculated for each replicate within 
each group based on a calculation by Seed (1976) by using the 
number of individuals and the stage at each age (4, 5, 6. and 7 mo) 
to find u GI at age for each group as follows: 

GI„K = [(0*N„) + ( 1*N,) + (2*N„) -h (.V'^N,,,) -I- (4*N,v) 
-K5*Nv)-H(6*Nv,)]/N,„„„„ 

where 01,^,^ is the GI for replicate / (/ = 1,2,. ^,...9), of the group / 
(/ = 1,2,3), in the environment k {k = 1,2): N^uhsiMpi 's 'he number 
of individuals in that gonadal stage for replicate /: and N,,,„,||^, is 
the total number of individuals in that replicate of that group in that 
environment. 



The GIs estimated for each replicate within the groups were 
analyzed by a complete two-factor. Model I, analysis of variance, 
where age was taken into consideration as a covariable. After 
establishing the lack of differences between the reciprocal Pis {P 
> 0.05). for all further analysis the Fls were pooled into what is 
defined as the Fl between these two populations. The effects of 
group (Magdalena, Concepcion. and Fl), environment (Bahia 
Magdalena and Bahia Concepcion), and their interaction on GI 
were analyzed. Effects means were compared with a Tukey test, 
setting a = 0.05. Additionally, at the age when first sexual matu- 
ration was observed, as defined above, a second partial Model I 
analysis of variance was made. This was performed to establish the 
effect of groups and environments on GIs at the age of first sexual 
maturity and to find out whether there was a group by environment 
interaction for GI. All statistical analyses were performed using a 
computer software (Statistica, version 5; StatSoft, Inc.; Tulsa, 
OK), and significance for all analyses was set to P < 0.05. 

RESULTS 



First Sexual Maturity 

At Bahia Magdalena. the age at first sexual maturity (Stage IV) 
for the Magdalena population was 4 mo. However, at this age, 
which corresponds to the first sampling time during grow-out (1.5 
mo of grow-out), 56% of the individuals were scored as matured, 
but 9% were spawned, and 13% postspawned. This indicated that 
first sexual maturity occurred slightly before this time. Shell height 
at 4 mo of age was 20.0 mm (SD ± 0.88 mm). At this same age, 
the Fl also reached sexual maturity, as defined in this study, since 
it had 41% individuals in the maturity stage, 19% spawned, and 
5%^ postspawned (Table 1). Shell height was 21.2 mm (SD ± 0.85 
mm). At the age of 4 mo, the Concepcion population had no 
mature or spawned individuals, but 4% were postspawned (Table 
1 ). At 5 mo of age, the Concepcion population had 75% mature 
individuals, reaching sexual maturity (Fig. 1) at a shell height of 
32.9 mm (SD ± 1.34 mm). 

At Bahia Concepcion, sexual maturity was not reached by any 
group during the experimental period (Fig. 1 ). Although sexual 
maturity was not detected in this environment, a differential pat- 
tern between groups was evident from 5 mo to the end of the study: 
a larger percentage of individuals from the Magdalena population 
and the Fl were postspawned than the percentages seen for the 
Concepcion population. Also, at 7 mo of age, corresponding to the 
last sampling date, 16%- of the individuals within the Magdalena 
group and 4% of the Fl were already matured, v\hercas within the 
Concepcion group there were no mature, spaw ned, or postspawned 
individuals (Fig. 1 ). 



GIs 



Bolh main effects (group antl en\ ironmentl were significant for 
both analyses, the whole grow-out period and the age (4 mo) at 
sexual maturity in the Magdalena population and the Fl. There 
was no interaction between groups and environments (Table 2). 
For the whole grow-out period, mean GIs (MGIs) for all groups at 
Bahia Concepcion (MGI 1 .63) were significantly less than those at 
Bahia Magdalena (MGI 3.72) (Table 3). These MGI values indi- 



Environment and Population Effects on First Sexual Maturity of Catarina Scallop 



91 



TABLE 1. 

Frequencies (in percentages), of A. ventricosus at 4 mo of age. in each gametogenic stage within each experimental group when grown at 

Bahia Magdalena and Bahia Concepcion. 







Bahia Magdalena 






Bahia Concepcion 






Magdalena 




Concepcion 


Magdalena 




Concepcion 


Stage 


Population 


Fl 


Population 


Population 


Fl 


Population 


Undifferentiated 





5 


22 


75 


89 


100 


I Resting 





5 





25 


6 





II Initial gametogenesis 





5 


48 





5 





III Advanced gametogenesis 


22 


17 


26 











IV Maturity 


56" 


4r 














V Spawned 


9 


19 














VI Postspawned 


13 


5 


4 












' Indicates whether sexual maturity of female gonad portion was reached for that group at this age. 



cated that, over the grow-out period, scallops at Bahia Magdalena 
were between the advanced gametogenesis (Stage III) and 
spawned (Stage V) stages, whereas those at Bahia Concepcion 
were between resting (Stage I) and initial (Stage II) gametogenic 
stages. Within environments and for the whole grow-out period, 
there were significant differences between groups only when 
grown at Bahia Magdalena. where the two populations were dif- 
ferent, and the Fl was in an intermediate maturity stage, which is 
not different from either population. The largest GI was that of the 



Magdalena population (GI 4.02), followed by the Fl (GI 3.81 ) and 
the Concepcion population (GI 3.33) (Table 3). 

At 4 mo of age. when sexual maturity had occurred, the MGl 
at Bahia Concepcion was lower (MGI 0.5) than that at Bahia 
Magdalena (MGI 3.17). There were significant differences be- 
tween groups only in Bahia Magdalena. with no significant dif- 
ferences in GI between the Magdalena population (GI 4.08) and 
the Fl (GI 3.57). whereas the Concepcion population had the 
lowest GI ( GI 1.85) (Table 3 ). At Bahia Concepcion. GIs were not 



Magdalena 



BAHIA MAGDALENA 

Fl 



Concepcion 





Age (months) 



Age (months) 



CD 1 



Age (months) 



Magdalena 



BAHIA CONCEPCION 

Fl 



Concepcion 




100 
90 






^ V. 



■^S5' 




£13 » w 



ES3 V 



EST 



r'.-'i-'A 



ES3 s 



5 6 

Age (months) 



Figure. 1. Frequencies of gonadal developmental stages in A. ventricosus at the ages of 4, 5, 6, and 7 mo, for each experimental group at each 
environment. Stage = undifferentiated: Stage I = resting; Stage II = start of gametogenesis; Stage III = advanced gametogenesis; Stage IV = 
maturity; Stage V = spanned; and Stage VI = postspawned. 



92 



Cruz et al. 



TABLE 2. 

Results of the analyses of variance testing significant effect on 

female GIs of A. venlricosiis for the complete model during the 

grow-out period, and the partial model only at 4 mo of age (see 

Materials and Methods section). 



Source of Variation 



Full Model 



Partial Model 



Environment 

Group 

Interaction 



0.0000-' 
0.0015" 
0.8373 



O.OOOO" 
0.0069-' 
0.3340 



' Indicates significance at the pre-established level of P < 0.05. 

different between groups ( 1 .0. 0.5, and 0.0, respectively, for the 
Magdalena population, Fl, and Concepcion population). 

DISCUSSION 

Differences between the two populations in age and size at 
sexual maturity were clearly evident when grown at Bahia 
Magdalena but not at Bahia Concepcion. At Bahia Concepcion. 
sexual maturity was not reached by any of the groups. However, at 
7 mo, some mature individuals were already present for the 
Magdalena population, but not for the Concepcion population. 
Previous work by Villalejo-Fuerte and Ochoa-Baez (1993) indi- 
cates that the native population at Bahia Concepcion reaches sex- 
ual maturity at the age of 1 y and a 58-mm shell height. For other 
Argopecten species, as for example Argopecten irradians, the 
maximum gonad weight was reported to be at 57 mm shell height 
(Bricelj et al. 1987), whereas 'tax Argopecten gihhits. ripe individu- 
als as small as 20 mm shell height have been reported (Miller et al. 
1979). In fact, precocious individuals such as those seen in the 
population of A. ventricosus from Bahia Magdalena have only 
been reported for A. gihhus. which reaches sexual maturation when 
only 71 days old (see review by Barber and Blake 1991 ). 

The failure of all groups to reach sexual maturity during our 
experimental period when grown at Bahia Concepcion can be ex- 

TABLE 3. 

GIs (SD) in A. ventricosus, for the whole grow-out period and for 

the age at sexual maturity (reached when grown at Bahia 

Magdalena at 4 mo of age I for each envinmment and for each 

experimental group." 







Whole 


Age 4 mo 






grow-out 


(Sexual 


Environment 


(■roup 


period 


Maturity) 


Bahia Magdalena 


Magdalena GI 


4.02(0.4.5)" 


4.08 (0.75)" 




Fl ni 


3.S1 (O.0)2)-''' 


3.57 (0.94)" 




Concepcion Gl 


3.33(1.14)" 


1.85(1.28)" 




BM MGI 


3.72 (O.Xl)'^ 


3.17(1. .32)^ 


Bahia Concepcion 


Magdalena Gl 


1.81 (1.26)'- 


1.0(2.24)'- 




Fl GI 


1.75(1.-30)' 


0.5(1.58)' 




Concepcion Gl 


!..34(0.96)' 


0.0 (0.00)' 




BC MGI 


l.fi3(1.22)" 


0.5(1. .54)" 



MGI (SD) is the average female Gl of all groups within that environment. 
GIs for gonad by group are given. Means with the same letter wilhin gonad 
part (female or male) are not significantly dilTerent. Group means dilTer- 
ences within environment and sc\ in lower case. Capital case lellers tor 
dilTcrences between environments. 



plained by the environmental conditions which characterize this 
bay; low productivity (chlorophyll-o 0.38-1.63 mg/m') and high 
average annual temperature (24.9 °C) with a wider range (17.7- 
32.1 °C) (Martinez-Lopez and Garate-Lizarraga 1994, Reyes- 
Salinas 1994). Bahia Magdalena is characterized as a more benign 
environment. Average temperature is 22 "^C, with a small range 
(20-26.6 °C) (Hemandez-Rivas et al. 1993), and a high chloro- 
phyll-a concentration (1.5-5.1 mg/m') (Acosta-Ruiz and Lara- 
Lara 1978). Poor environmental conditions are known to affect 
gonad development (i.e.. decreases in reproductive output seen in 
Placopecten inagellaiucus) (Macdonald and Thompson 1985). 
Barber and Blake (1991) proposed that the oocyte reabsorption 
seen in different species of Pectinids could be caused by unfavor- 
able temperatures that inhibit full gonad development. This was 
probably the case in the Bahia Concepcion population, where de- 
spite the fact that mature individuals 4-6 mo old were not detected, 
there were some classified as postspawned. Furthermore, rather 
than in the undifferentiated stage, most scallops in Bahia Concep- 
cion were in a resting stage during most of the experimental pe- 
riod, which could have been caused by attempted maturation fol- 
lowed by follicular atresia because of high temperatures and low 
productivity. 

The mechanism that detains the maturation process under in- 
adequate environmental conditions is not known. However, it is 
known that in A. irradians, the regulation of the gametogenic cycle 
is controlled by a neurosecretory cycle with a checkpoint that 
seems to act as a switching mechanism, allowing or delaying oo- 
cyte growth depending on food and temperature (Barber and Blake 
1991). When scallops are subjected to prolonged threshold tem- 
peratures after the neurosecretory cycle enters the neurosecretory 
stage (NS) corresponding to cytoplasmic growth phase (NS III) or 
vitellogenesis (NS IV), scallops do not regress in NS, and vacu- 
olization of cytoplasm and lysis of oocytes can occur (Sastry, 
1966a, 1968, cited by Barber and Blake 1991). Whether a similar 
mechanism exists in the catarina scallop is not known, but it could 
explain the presence of atresias in scallops when they are grown in 
Bahia Concepcion. 

The differences between populations in age at sexual maturity 
suggest that a genetic triggering mechanism might exist for the 
onset of sexual maturity in the catarina scallop. When grown in 
Bahia Magdalena. an environment characterized by high produc- 
tivity and lower temperatures, the mechanism of early maturation 
in the Magdalena population and the Fl is triggered, and because 
the prevailing environmental conditions at this site (low tempera- 
ture and abundant food), full development is reached at an early 
age. A suggestion that the mechanism is genetically controlled 
comes from the age and size at which the two populations reached 
their first sexual maturity. The Concepcion population reached 
sexual maturity in this environment at least 1 mo later than the 
Magdalena population. Inheritance (from the Magdalena popula- 
tion to the Fl) in the dominant fashion of an early triggering 
mechanism is suggested by the Fl reaching sexual maturity at the 
same age as the Magdalena population and by the fact that the GIs 
of the Fl showed no significant differences with the Magdalena 
population at first sexual iT)aturity. However, the GI of the Fl 
group was intermediate to the GIs of the two populations for the 
whole grow-out period, indicating more of an additive form of 
inheritance for this trait. Furthermore, whereas at Bahia Concep- 
cion sexual maturity was not reached for any group in this study. 
at 7 mo of ane there were 16'/f mature indi\'iduals wilhin the 



Environment and Population Effects on First Sexual Maturity of Catarina Scallop 



93 



Magdalena population, but only 4"^^ mature and 2'7r post spawn for 
the Fl (6';*-). Further research, with segregation studies included, is 
required to provide a definitive answer to the inheritance of this 
trait. Whereas the inheritance of reproductive traits has been sug- 
gested in other mollusk species, no study has attempted to dem- 
onstrate it at the genetic level. For example. Knaub and Eversole 
(1988) found that the Fl between two populations o{ Mercenaria 
mercenaria resembled the paternal population in some reproduc- 
tive traits and the maternal population in others. Also, each of two 
lines of Crassosirea virginica. derived from two populations 5-6 



generations before, still followed the reproductive pattern of their 
original populations (Barber and Blake 1991). 

ACKNOWLEDGMENTS 

We thank MAZAVI enterprise for help during field mainte- 
nance of the experimental groups, and M. Romero from SEMAR- 
NAP, Guy. A. Garcia and Jose L. Ramirez from CIBNOR for 
technical support during this research. This research was partially 
supported by CONACyT grants 720-N9204 and 1473PB to A.M. 
Ibarra. Dr. Ellis Glazier edited the English-language text. 



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Cruz. P., J. L. Ramirez. G. A. Garcia. & A. M. Ibarra. 1998. Genetic 
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venlricosiis) for adaptations for growth and survival in a stressful en- 
vironment. Aquaculture. 166:321-335. 

Dalton. R. & W. Menzel. 1983. Seasonal gonadal development of young 
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Emmett. B.. K. Thompson & J. D. Popham. 1987. The reproductive and 
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B.C.S., Mexico. 

Knaub, R. S. & A. G. Eversole. 1988. Reproduction of different stocks of 
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Journal of Shellfish Research. Vol. 19. No. 1. 95-99, 2000. 

REPRODUCTIVE CYCLE OF THE RUGOSE PEN SHELL, PINNA RUGOSA SOWERBY, 1835 

(MOLLUSCA: BIVALVIA) FROM BAHIA CONCEPCION, GULF OF CALIFORNIA AND ITS 

RELATION TO TEMPERATURE AND PHOTOPERIOD 



BERTHA PATRICIA CEBALLOS-VAZQUEZ, 
MARCIAL ARELLANO-MARTINEZ, 
FEDERICO GARCIA-DOMINGUEZ, AND 
MARCIAL VILLALEJO-FUERTE 

Centra Interdisciplinario de Ciencias Marinas 

Institiito Politecnico Nacional 

Apartado Postal 592 

La Paz. B.C.S. 23000. Mexico 

ABSTRACT This study describes, through monthly histological examinations of gonadal tissue samples, the reproductive cycle of 
Pinna nigosa and relates gametogenesis to temperature and photoperiod. Monthly gonadal samples were obtained from February 1993 
to February 1994. in Bahi'a Concepcion. Gulf of California. Mexico. Five stages of gonadal development were characterized: indif- 
ferent, developing, ripe, partially spawned, and spent. Histological evidence revealed hermaphroditism in 20.9% of animals sampled. 
Gametogenesis commenced in March, with ripe and spawning stages occurring from April to November, and no gametogenic activity 
occurring from December to February. From March to November, water temperature ranged from 20 °C to 31 °C. with an average 
range of light of 650-820 min/day. P. nigosa had a seasonal gametogenic cycle directly related to water temperature and photoperiod. 

KEY WORDS: Reproduction, bivalve. Pinna, histology. Gulf of California 



INTRODUCTION 

The rugose pen shell. Pinna nigosa Sowerby, 1835, is com- 
monly known in Mexico as "hacha" (hatchet). This bivalve is of 
commercial importance and supports a fishery in the northwestern 
area Gulf of California. Mexico. P. nigosa is greatly appreciated 
by consumers because of its tasty, large adductor muscle, com- 
monly refeired to as "callo." The pen shell fishery has been an 
important economic activity in Mexico for many years. Production 
trends, however, have drastically declined over the past years, and 
some populations have been depleted (Reynoso-Granados et al. 
1996). Few biological studies of P. nigosa have been conducted 
(Arizpe and Felix 1986, Arizpe and Covairubias 1987, Mazon- 
Suastegui and Aviles-Quevedo 1988. Rui'z-Verdugo and Caceres- 
Martinez 1990, Arizpe 1995). 

Documentation of the reproductive biology of P. nigosa in the 
Gulf of California is extremely scarce. Noguera and Gomez- 
Aguirre (1972) described the reproductive cycle of P. nigosa in 
Laz Paz Bay, B.C.S.. Mexico, and they showed that gametogenesis 
commenced in mid-spring and that the animals spawned in late 
summer. 

Because of the economic importance and high price obtained 
by the callo. efforts have recently been under way to cultivate this 
species. Therefore, studies of its reproductive biology are essential 
to achieve reproduction in a laboratory setting. This study docu- 
ments the reproductive cycle of P. nigtKsa from Bahi'a Concepcion. 
Gulf of California, Mexico, and examines the relationship of ga- 
metogenesis to temperature/photoperiod. 

MATERIALS AND METHODS 

Bahi'a Concepcion. Mexico, is located on the western coast of 
the Peninsula of Baja California, between 26'55' and 26°30'N and 
1 12° and 1 1 l°40'E. The bay is approximately 40 km long and 10 



km in its widest part and oriented in a NW-SE direction (McFall 
1968). 

Monthly, between 13 and 35 specimens of rugose pen shell 
were collected by a scuba diver at a 2- to 8-ni depth from February 
1993 to February 1994. Animals were collected from a wild popu- 
lation located off Santispac Beach in Bahia Concepcion. Gulf of 
California. The individuals were collected and fixed in 10'7f for- 
malin solution. When the biological samples were collected, water 
temperature at the collection site was recorded. 

The visceral mass (gonad included) was dissected from each 
pen shell and stored in 70% alcohol. Later, a slice of tissue of the 
dorsal area of the visceral mass was cut. This tissue samples were 
dehydrated in an ethanol series of progressive concentrations, 
cleared in toluene, and embedded in paraffin. Serial sections 7-9 
|jLm thick were obtained with a rotary microtome. Preparations 
were stained with hematoxilyn and eosin. The gonad structure was 
examined under a microscope, and the sex was determined for 
each animal by the presence of egg or sperm in the tissue section. 

Each tissue section of P. nigosa was categorized on the basis of 
the qualitative characteristics of five stages of maturation (indif- 
ferent, developing, ripe, partially spawned, and spent) as described 
by Villalejo-Fuerte and Garcia-Domi'nguez (1998). The monthly 
relative frequencies of the stages of gonadal development through- 
out the annual cycle were obtained. This enabled the description of 
the reproductive cycle. The spawning season is defined as the time 
period containing ripe and partially spawned individuals. 

To obtain a quantitative value that represents the reproductive 
activity, a monthly gonad index (GI) was computed (Heffernan et 
al. 1989) utilizing a numerical grading system. Three categories 
were established according to the degree of development of the 
gonad, with 1 = indifferent and spent. 2 = developing, and 3 = 
ripe and partially spawned. The monthly GI was determined by 
multiplying the number of specimens ascribed to each category by 
the category score, summing all such values, and dividing the 
resulting value by the total number of pen shells analyzed. The 



95 



96 



Ceballos- Vazquez et al. 




Figure I. Photimiicrographs of gonadnl stsiRcs of P. nif-osa. (a) (ionad classified as developing female; small oocytes groHing attached to the 
follicle wall, male spent, lb) Developing male; thick layer of spermatocytes developing, (c) Mature female; large oocytes free In the lumen of 
follicles, (dl Mature male; large (juantity of spermalo/oa tilling the follicles, (el Partially spawned female; empty follicle with some residual 
oocytes. (f| Partially spawned male; a marked decrease in the nunihcr of spermatozoa lllling the lumen, igl Indifferent goniid; follicles with total 
absence of gametes, (h) Gonad spent; follicles collapsed, aniebocytes phagocytizing residual gametes. Scale bar = ?l) pm. 



Reproductive Cycle of Pinna rugosa 



97 



values obtained permit us to realize the correlation analysis of 
reproductive activity with temperature and photoperiod. 

Data for photoperiod for this study were not determined di- 
rectly by the authors. Data from nautical almanacs of the Secretaria 
de Marina of Mexico were used to define the photoperiod. The 
data correspond to the daily period of illumination, and an average 
in minutes of illumination was calculated for each month, between 
February 1993 and February 1994, for the latitude corresponding 
to Bahi'a Concepcion. 

A Spearman rank order correlation analysis was used to inves- 
tigate the relationship between GI, temperature and photoperiod. 
Correlation analysis were carried out with the monthly values {n 
= 13). 

RESULTS 

A total of 3 1 1 specimens was collected, 33 females (10.6%), 55 
males (17.7%). 65 hermaphrodites (20.9%). and 158 indifferenti- 
ated (50.8%). The range in shell length of pen shells was from 134 
to 366 mm (258 mm average, 29 mm standard deviation). 

In the hermaphrodite gonads, the development of both sexes 
was not synchronous. On the contrary, one sex was always in a 
more advanced stage of development {i.e.. the female phase was 
developing, whereas the male phase was spent) (Fig. la). 

To describe the reproductive cycle, all of the organisms were 
considered, including the hermaphrodites. In the case of hermaph- 
rodites, they were each considered as one individual accordingly 
with the more advanced developing stage. The similar range of 
gonadal development for small to large individuals indicated that 
all pen shell sampled were reproductively active. All five stages of 
gonadal development were observed (Fig. 1). 

The reproductive cycle of P. rugosa from Bahi'a Concepcion, 
Gulf of California, is summarized in Figure 2. Indifferent indi- 
viduals were observed all year, except in June. In February 1993 
and from December 1993 to February of 1994, most pen shell were 
indifferent staged (94.1%, 100%, 93%, and 100%. respectively). 
Gametogenesis commenced in March. Maturation was continuous 
through November. Ripe stage was present from April to Novem- 
ber, except in September. The partially spawned stage was present 
in May and from July through November. Spent specimens oc- 
curred from May to September, except in June. 

Monthly quantitative assessments of histological reproductive 
condition are illustrated in Figure 3a. From these data, it is appar- 
ent that the GI has a seasonal tendency along the year, with high 
values coinciding with ripe individuals and the fall of values co- 
inciding with spawning activity. The values of GI were higher in 
April, June, and October and were lower from December to Feb- 
ruary. The GI values indicated that the gametogenesis started in 
March and continued until November, with pen shell quiescent 
from December to February. 

Water temperature showed considerable seasonal variation 
(Fig. 3b) with extreme values of 31 °C in August and 19 °C in 
February. 

The photoperiod (minutes of daily illumination) is illustrated in 
Figure 3c. The longest monthly average daily illumination in the 
study area occurred during May to July, with the highest in June 
(820 min). The minutes of daily illumination presented a decreased 
tendency during July through November. The shortest time of 
illumination occurred in November/December and January (640 
min). 



100 



80 



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cc 

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20 



17 26 13 25 30 35 18 27 25 20 19 29 27 



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M J J 
1993 





J F 
1994 



H INDIFFERENT \ 
■ PART SPAWN [ 



1 DEVELOPING ^RIPE 
! SPENT 



Figure 2. Reproductive cycle of P. rugosa from Bahia Concepcion, 
Gulf of California, Mexico. Relative frequency of gonadal stages be- 
tween February 1993 and February 1994. Observations of males and 
females are combined. Numbers at top indicate the sample sizes for 
each month. 



In all cases significant correlation (P < 0.05) was found. The GI 
presented a positive correlation with temperature (/; = 13: r = 
0.85: P = 0.000192) and photoperiod (n = 13: r = 0.69: P = 
0.008980). Temperature and photoperiod were positively corre- 
lated (n = 13: '• = 0.59: P = 0.031929). 

DISCUSSION 

In Bahfa Concepcion, the rugose pen shell exhibits an annual 
gametogenic cycle, which commences in March with rapid prolif- 



o 
o 



o E 



D. .5- 
O ro 

o -s 




Figure 3. Monthly variation of GI (a), water temperature (b). and 
photoperiod (c) in Bahia Concepcion, B.C.S., Mexico. 



98 



Ceballos-Vazquez et al. 



eration of gametes and ends by December. According to the his- 
tological analysis, the spawning occurs from May to November, 
except in June. Our results are in agreement with the reproductive 
cycle of P. rugose occurring in La Paz Bay. B.C.S.. Mexico, as 
described by Noguera and Gomez- Aguirre (1972). who docu- 
mented that sexual maturation begins in mid-spring, with spawn- 
ing occurring by late summer. 

The characteristics of gametogenesis in P. rugosci from Bahfa 
Concepcion were similar to those described for Spondyliis leuca- 
canthus from Isla Danzante (Villalejo-Fuerte and Garci'a- 
Dominguez 1998). The gonad off. nigosa has oocytes with the 
same degree of development, common for bivalves with a syn- 
chronic development. The histological examination additionally 
showed that P. nigosa is a hermaphrodite species; in this study 
20.9% of pen shell presented this condition. Hermaphroditism is 
common in bivalves (Tranter 1958, Garcfa-Domi'nguez et al. 1996, 
Villalejo-Fuerte and Garcia-Dominguez 1998). 

There are two basic types of reproductive pattern exhibited by 
marine bivalves in the Gulf of California waters. Many bivalve 
species have no seasonal reproductive cycle, and their spawning 
activity is continuous, for example. Megapitaria aiinintiaca (Gar- 
cfa-Donii'nguez et al. 1994) and Pinctada mazatlanica (Garcia- 
Domi'nguez et al. 1996). Other bivalve species exhibit distinct 
seasonal reproductive cycles, such as Dosinia ponderosa (Arreola- 
Hernandez 1997). Chione undatella (Baqueiro and Masso 1988), 
and M. sqiialida (Villalejo-Fuerte et al. 1996). which usually are 
related to temporal variations of environmental factors such as 
food availability, water temperature, and/or photoperiod. 

The reproductive activity of P. nigosa was significantly corre- 
lated to the water temperature and photoperiod. The protracted 
period of reproductive activity (March to November 1993) of P. 
nigosa from Bahi'a Concepcion coincides with the gradual increase 
of sea-surface temperature (from 20 °C until a maximum of 31 



°C), and with increased values of photoperiod (720 min/day). The 
period of reproductive inactivity was clearly distinguished in win- 
ter (November 1993 to February 1994), and coincides with an 
abrupt decrease of 3.5 °C in the sea-surface temperature (26 °C) 
and with the photoperiod minimum values (640-650 min/day). 

The Spearman correlation analyses indicated that the major 
environmental factor that directly influences the gonadal growth is 
the water temperature, suggesting that the production of gametes is 
stimulated by increases in temperature. The same has been ob- 
served for other bivalve species, such as Spondyliis leiicacanthus 
(Villalejo-Fuerte and Garcia-Domi'nguez 1998) and Argopecten 
circiilaris (Villalejo-Fuerte and Ochoa-Baez 1993). However, in 
other bivalves from the Gulf of California, no clear relationship 
exists between gonadic development and water temperature (e.g.. 
M. aiirantiaca [Garci'a-Domi'nguez et al. 1994] and P. mazatlanica 
[Garcia-Dominguez et al. 1996]). Although water temperature af- 
fects reproduction, other environmental factors may well play an 
integral role in determining the pattern of annual gonad activity for 
species in a given geographical area (Sastry 1970). 

Giese and Pearse ( 1974) have reported photoperiod as a factor 
that influences spawning of invertebrates. However, it has not been 
widely studied in bivalves (Villalejo-Fuerte and Ochoa-Baez 
1993). The temperature and photoperiod are positively coiTelated. 
But it may not be possible to separate the effects of these two 
factors with the data presented in this paper. 

ACKNOWLEDGMENTS 

We are grateful to Direccion de Estudios de Postgrado e In- 
vestigacion del Instituto Politecnico Nacional for funding this 
work and to Comision de Operacion y Fomento de Actividades 
Academicas for the fellowships to F. Garci'a-Dominguez and M. 
Villalejo-Fuerte. Thanks to Ma. Consuelo Gonzalez Ordonez for 
her editorial help on English manuscript. 



LITERATURE CITED 



Arizpe, C. O. 1995. Mortality, growth and somatic secondary production of 
the bivalve. Pinna nijio.sa (Sowerby), in suspended and boltoni culture 
in Bahia de La Paz. Mexico. Aiiiiaciilt. Res. 26:843-833. 

Arizpe, CO. & O. Covarrubias. 1987. Reclutamiento y mortalidad de 
Pinna nigosa (Sowerby. 1835) en condiciones semicontroladas en Ba- 
hi'a de La Paz, Mexico. Anales del Inslitiito de Ciencias del Mar 
y Limnologiu. Universidad Nacional Aut6noma de Mexico. 14:249- 
254. 

Arizpe. C. O. & U. R. Felix. 1986. Crecimientode Pinna itigosa (Sowerby. 
1835) en la Bahi'a de La Paz. Mexico. Anales del Instituto de Ciencias 
del Mar y Limniilitgia. Universidad Nacional Autonoma de Mexico. 
13:167-172. 

Arreola-Hernandez, F. 1997. Aspectos reproductivos de Dosinia ponder- 
osa. Gray, 1838 (Bivalvia: Veneridae) en Punta Arena, Bahia Concep- 
cion. B.C.S., Mexico. M.S. Thesis. Instituto Polilccnico Nacional 
CICIMAR, La Paz, Mexico, 85 pp. 

Baqueiro, L. & J. A. Masso. 1988. Variaciones poblacionales y reproduc- 
ciiin de dos poblaciones de Chione undatella (Sowerby. 1835) bajo 
difcrenlcs rcgi'menes de pesca en la Bahia de La Paz, B.C.S.. Mexico. 
Ciene. Fescj. Insl. Nal. de la Pesca. Mexico 6:51-67. 

Garci'a-Doniinguez. P., S. A. Garci'a-Gasca & J. L. Caslio-Ortiz. I9')4. 
Spawning cycle of the red clam Megapitaria aiirantiaca (Sowerby. 
1831) (Veneridae) al Ma Lspirilu Santo. Mexico. J. Shellfish Res. 
13:417^23. 

Garci'a-Domi'nguez. F., B. P. Ceballos-Va/que/ & A. Tripp-Que/ada. 
1996. Spawning cycle <il' the pearl oy^ler I'inctada ina:atlanica (Han- 



ley. 1836) (Pteriidae) at Isla Espi'ritu Sanio, Mexico. / Shellfish Res. 

15:297-303. 
Giese. A. C. & J. S. Pearse. 1974. Introduclion: general principles, pp. 

1-49. In: A. C. Giese & J. S. Pearse (eds.). Reproduction of Marine 

Invertebrates, vol. I. Academic Press, New York. 
Hcffeman. P. B., R. L. Walker & J. L. Carr. 1989. Gametogenic cycles of 

three bivalves in Wassaw Sound, Georgia. 1. Mercenaria mercenuria 

(Linnaeus, 1758). / Shellfi.sh Res. 8:51-60. 
Maziin-Suastegui. J. M. & M. A. Aviles-Quevedo. 1988. Ensayo prelimi- 

nar sobre la alimentacion de bivalvos juveniles con dietas artificiales. 

Re\isla Latinoamericana de Aciiiciilnira. 36:56-62. 
McFall, C. C. 1968. Reconnaissance Geology of the Concepcion Bay Area, 

Baja California Sur, Mexico, vol. 5. Stanford University. Publications, 

Geological Sciences. 25 pp. 
Noguera. O. M. & S. Gomez-Aguiire. 1972. Cicio sexual de Pinna nigosa 

Sowerby, 1835 (Lanicllibranchia, Pinnidae) de La Paz, B.C., Mexico. 

pp. 273-283. In: J. Carran/a (ed.). Meinorias IV Congreso Nacional de 

Occanograli'a. Mexico City. Mexico. 
Reynoso-Granados, T.. A. Maeda-Marti'nez. F. Caidoza-Velasco, & P. 

MonsaUo-Spencer. 1996. Cullivo de hacha. pp. 545-550. In: Casas- 

Valde/. M. & G. Poncc-Dia/ (eds.). Estudio del Potencial Pesquero y 

Acuiola en Baja California Sur, La Pa/, B.C.S.. Mexico. 
RuiV-Verdugo. C. A. & C. Caceres-Martinez. 1990. Estudio preliminar de 

captaci6n de juveniles de moluscos bivalvos en la Bahi'a de la Paz, Baja 

California Sur. Mexico. Invest. Mar. CICIMAR. 5:29-38. 
Saslry. A. N. 1970. Reproductive physiological variation in lalitudinally 



Reproductive Cycle of Pinna rugosa 99 

separated populations of the bay scallops, Aequipeclcn irnuliuns La- Villalejo-Fuerte. M., G. Garci'a-Melgar. R. L Ochoa & A. Garci'a-Gasca. 

marck. Biol. Bull. 138:?6-65. 19%. Ciclo reproductive de MegapiUuia squalida (Sowerby, 1835) 

Tranter, D. J. 1958. Reproduction in Australian pearl oyster (Lamellibran- (Bivalvia: Veneridae) en Bahi'a Concepcion, Baja California Sur, 

chia). II. Pincmda cilhiiui (Lamarck) gametogenesis. Aii.it. J. Mar. Mexico. Bol Cienlifico (Santa Fe de Bogota) 4:29-39. 

Freshwater Res. 9:44-158. Villalejo-Fuerte. M. & R. I. Oclioa-Baez. 1993. The reproductive cycle of 

Villalejo-Fuerte. M. & F. Garci'a-Domi'nguez. 1998. Reproductive cycle of the scallop Argopecten circularis (Sowerby. 1835) in relation to tem- 

SpondyUis leiicacantlnts Broderip. 1833 (Bivalvia: Spondylidae) at Isla perature and photoperiod, in Bahfa Concepcion, B.C.S., Mexico. Cien- 

Danzante, Gulf of California. / Shellfish Res. 17:1037-1042. cias Ma. 19:181-202. 



Joiirnai of Shi-ilfish Resfiirch, Vol. 19. No. 1. 101-105. 2000. 

CHROMOSOME SEGREGATION IN FERTILIZED EGGS FROM ZHIKONG SCALLOP 
CHLAMYS FARRERI (JONES & PRESTON) FOLLOWING POLAR BODY 1 INHIBITION 

WITH CYTOCHALASIN B 



HUIPING YANG, HUAYONG QUE, YICHAO HE, AND 
FUSUI ZHANG 

Experimental Marine Biology Laboraton- 

Institute of Oceanology, Chinese Academy of Sciences, 

Qingdao, Shandong 266071. China 

ABSTRACT Chromosome segregation in fertilized eggs of the zhikong scallop. Chlamys farreri. following polar body 1 (PBl) 
inhibition with cytochaUisin B (CB) was studied. The fertilized eggs were treated with CB (0.75 mg/L) at 7-10 min postfertilization 
until polar body 2 (PB2) was released in control groups. The embryos were sampled every 5-10 min after fertilization and fixed in 
Carney fixative. Chromosome segregation in both control groups and treated groups were analyzed using a hematoxylin stain method. 
In fertilized eggs of control groups, the 19 tetrad chromosomes went through meiosis I and II, and released PBl and PB2, finally 
reaching 19 chromatids. In CB treated groups, meiosis I proceeded normally and produced two groups of dyads, 19 in each group. With 
the CB treatment, both of the two dyad groups were retained in the eggs and entered meiosis II. The segregation in meiosis II had four 
patterns: bipolar, tripolar, tetrapolar, and unsynchronized segregation. When the two groups of dyads from meiosis 1 united, the treated 
eggs entered meiosis II through tripolar (40.9'7f) and bipolar (11.4%) segregation patterns. Otherwise the two groups of dyads 
segregated separately and formed tetrapolar segregation ( 15.7%). Also a small proportion of treated eggs (4.0%) underwent meiosis 
II in an "unsynchronized segregation" pattern, which means that the two groups of dyads from meiosis I did not segregate synchro- 
nously. There were 28.0% of treated eggs that could not be classified. The four segregation patterns produced different ploidies of 
embryos in CB treated groups, such as triploids, tetraploids. pentaploids, and aneuploids. 

KEY WORDS: Zhikong scallop, Cliluiiiys farreri. chromosome segregation, triploid, tetraploid, polar body 



INTRODUCTION 

Triploids can be induced by blocking the first polar body (PB I ) 
in some mollusk species, such as American oyster, Crassostrea 
virginica (Stanley et al. 1981), Pacific oyster. Crassostrea gigas 
iThunberg) (Quillet and Panelay 1986). Pacific abalone. Haliotis 
discus hamuli (Aral et al. 1986). pearl oyster, Pinctada martensii 
(Jiang et al. 1987). and blue mussel. Mytiliis ediilis (Yamamoto 
and Sugawara 1988). Tetraploids were also reported among trip- 
loids in American oyster (Stanley et al. 1981 ) and in other mollusk 
species (Arai et al. 1986. Yamamoto and Sugawara 1988). 
Stephens and Downing (1988) reported that 917f tetraploid at 24-h 
postfertilization (PF) was produced by inhibiting PBl in fertilized 
eggs from the Pacific oyster. In similar work, Guo et al. (1992a) 
reported that many aneuploids embryos (57.6%) were also pro- 
duced. All of these results indicate that PB 1 inhibition results in 
complicated chromosome segregation. Observation of chromo- 
some segregation in the Pacific oyster explained the mechanism 
for formation of different ploidies when PB 1 was blocked (Guo et 
al. 1992b). Three different types of segregation, including "tripolar 
segregation," "united bipolar .segregation." and "separated bipolar 
segregation" were evident. Later, the chromosome segregation in 
triploid Pacific oysters was also studied when eggs from triploids 
were fertilized with diploid sperm and PBl was blocked with CB 
(Que et al. 1997). The observation showed that there were also 
three types of segregation patterns, confirming the mechanism by 
which viable tetraploid Pacific oysters can be successfully induced 
through blocking PBl in fertilized eggs from triploids (Guo and 
Allen 1994). 

In the zhikong scallop. Clilamys farreri. blocking PBl in fer- 
tilized eggs from normal diploids can result in triploid, tetraploid, 
pentaploid, and aneuploid embryos. We have also found that both 
triploids and tetraploids can survive to 2-3 mm juvenile stage 
(unpublished). In this paper, the behavior of chromosome segre- 



gation was observed in fertilized eggs from normal diploids when 
PBl was blocked with CB, offering an explanation for the forma- 
tion of embryos with different ploidies. 



MATERIALS AND METHODS 



Gametes 



Parent scallops were from Rizhao and Qingdao, Shandong, 
China. The scallops were conditioned indoors to accelerate gonad 
maturity. Gametes were obtained through natural spawning. Eggs 
were collected with a 25-[j, screen and resuspended into 2 ~ 3 L 
seawater at 20 °C, ready for fertilization. Sperm were prepared by 
screening sperm suspension through a 25-p. nylon screen. For 
fertilization, sperm were added to the egg suspension at a final 
density of 5-7 sperm per egg. Fertilization, treatment, and embryo 
culture were all conducted at 20 °C. 

Treatment and Sampling 

PBl in fertilized eggs was blocked with 0.75 |jLg/mL CB dis- 
solved in dimethyl sulfoxide (DMSO-final concentration 0.1%). 
CB treatment began at 7-10 min PF and ended when the second 
polar body (PB2) in control groups was observed under micro- 
scope. Fertilized eggs in both control groups and CB-treated 
groups were .sampled every 5-10 min during development until 75 
min PF. Samples were directly fixed in Carnoy fixative (methanol: 
acetic acid = 3:1), which was changed twice, and the samples 
were then stored at 4 °C before analysis. The experiment was 
repeated four times using different parent scallops. 

Chromosome Observation 

Slides for observing chromosomes were made by a modified 
squashing method. The staining solution was made by dissolving 
0.5%' hematoxylin in 45% acetic acid, with ammonium iron sulfate 



101 



102 



Yang et al. 



dodecahydtate as a mordant (about 0.5%). Embryo samples were 
dropped and spread on clean slides. Excessive fixative was al- 
lowed to run off the slides, and then drops of staining solution were 
added onto the samples just before the fixative evaporated. A clean 
cover glass was placed gently on the samples. Before squashing on 
filter paper, slides were warmed slightly by passing them across an 
alcohol burner. Then, the cover glass was sealed on all four sides. 
Alternatively, the whole cover glass was sealed with neutral bal- 
sam after removal by icing the slides. 

Slides were examined with a Nikon compound microscope. 
Photographs were taken using LUCKY black and white film (ASA 
100 and 400). 

RESULTS 

Initially, normal diploid eggs were observed in prophase of 
meiosis I (Fig. la). Zhikong scallop has a diploid number of 38 
chromosomes (Wang et al. 1990). Nineteen tetrads were observed 
in the unfertilized eggs. 

In control groups, the 19 tetrads began to segregate at about 
9-10 min PF, then the tetrads in the majority of fertilized eggs 
segregated into 38 dyads, and then divided into two groups, 19 in 
each group (Fig. lb). Later, one group of dyads condensed and 
released as PBI (Fig. Ic). The remaining 19 dyads continued 
meiosis II and segregated into two groups of chromatids (Fig. Id). 
One of the groups of chromatids was released as PB2 in most 
fertilized eggs at 40^3 min PF. Normally, the two polar bodies 
were positioned next to each other (Fig. le). As for the chromatids 
from sperm, at first, they could only be observed as dark-stained 
material. Only during mitosis I, did the chromatids from egg and 
sperm unite, yielding 38 chromosomes. 

In CB-treated groups, chromosome segregation was compli- 
cated. After fertilization, the 19 tetrads segregated into 38 dyads 
(Fig. Ig). Under the microscope, no PBI was released in the ma- 
jority of treated, fertilized eggs during CB treatment. Thus, 38 
dyads in fertilized eggs entered meiosis II, and chromosome seg- 
regation differed greatly from that in control groups. Four patterns 
of segregation were observed: bipolar, tripolar. tetrapolar, and un- 
synchronized. Some segregations could not be classified. When 
the 19 dyads from PBI united with the other 19 dyads, the chro- 
mosome segregation in meiosis II proceeded with bipolar or tri- 
polar segregation. 

Bipolar Segregation 

The 19 dyads from PB 1 united with the remained 19 dyads, and 
went through meiosis II together (Fig. Ih). All 38 dyads segregated 
in a bipolar pattern just like normal meiosis II, and divided into 
two groups of sister chromatids, 38 in each group (Fig. 1 i). One of 
the two groups of chromatids was released as PB2 after CB treat- 
ment. This pattern of chromosome segregation could result in Irip- 
loids. 

Tripolar Scungalioii 

The 38 united dyads divided into three groups, apparently al 
random (Fig. Ij), and the dyads in each group .segregated in two 
directions. Finally, the chromatids migrating in one direction 
united with the chromatids from its neighboring group at a pole, 
forming three groups of chromatids (Fig. Ik). The number of chro- 
matids in the three groups varied considerably, apparently depend- 
ing upon random distribution of dyads before meiosis II. Rarely, 
one of the three groups had exactly 19 chromatids. In this pattern 



of chromosome segregation, the three groups of chromatids had 
probability of being released as PB2 after CB treatment. 

Sometimes the 19 dyads from the unreleased PBI fail to unite 
with the remaining 19 dyads in the fertilized eggs (Fig. 11). They 
entered meiosis II independently, resulting in two patterns of chro- 
mosome segregation: tetrapolar and unsynchronized segregations. 

Tetrapolar 

In both dyads groups, the chromosomes segregated in a normal 
bipolar pattern. The final result was that four groups of chromatids 
formed, 19 chromotids in each group (Fig. Im). 

Unsynchronized Segregation 

The unreleased dyads and the remaining dyads in the fertilized 
egg went through meiosis II asynchronously. Sometimes one 
group of dyads did not go through meiosis II. but remained un- 
changed, and another group of dyads went through meiosis II and 
underwent bipolar segregation to anaphase (Fig. In). 

Unclassified 

In addition to the above segregation patterns, there were also 
other patterns that could not be classified. In fertilized eggs with 38 
united dyads, only some began meiosis II segregation; whereas, 
some were left as dyads. In some treated fertilized eggs, the 38 
dyads went through meiosis II. but the 76 chromatids distributed 
themselves randomly. No segregation poles were observed (Fig. 
lo). 

The frequencies of the four segregation patterns were calcu- 
lated from embryos where PBI had been blocked (Table I). On 
average, the majority of treated eggs went through meiosis II as 
tripolar segregations (40.9%), 11.4% were bipolar, and 15.7% 
were tetrapolar. Only a small proportion of treated eggs (4.0%) 
went through unsynchronized segregation. Finally, 28.0% of seg- 
regations in treated eggs could not be classified. 

DISCUSSION 

We made slides for observing chromosome segregation by a 
modified squashing method using a hematoxylin stain. For fertil- 
ized eggs and embryos from the zhikong scallop, this procedure 
was quite useful. In fertilized eggs of the Pacific oyster, orcein 
dissolved in 60% acetic acid employed chromosome observations 
(Guo et al. 1992a, Que et al. 1997). In the zhikong scallop, we have 
tried orcein staining, but it produced poor contrast. Hematoxylin is 
a typical chromosome stain (Sharma and Sharnia 1980). Normally 
hematoxylin solution must be made in advance to ripen for a few 
weeks. In this experiment, the stain solution was modified as 0.5% 
dissolved in 457r acetic acid with ammonium iron sulfate as mor- 
dant and could be used instantly. In addition, this method produced 
satisfactory results for observing chromosomes in the fertilized 
eggs and embryos from the jinjiang oyster, Cni.\si'strc'ii ariakensis 
(unpublished). We suggest this new staining method for chromo- 
some observations in bivalve mollusk. 

Chromosome Segregation 

Unfertilized eggs of normal diploid zhikong scallops are ar- 
rested al late prophase of meiosis I. Only after fertilization, did the 
eggs continue meiosis I and II. releasing PBI and PB2. In the end, 
19 maternal chomatids remained in the lertili/ed eggs. This pattern 
of chromosome segregation is common among bivahe mollusks 
(Longo and Anderson 1969). 



Chromosome Segregation in PB 1 Blocked Eggs 



103 



> V 




. V 







J h 



m 






^2 



ri*i 



j^ 



1 



** 



,*■'. 1' tM', 






r-'f 



> >• 



•** 4 



\e 




( 



^ 






r 






^\ * 



il 



n 



•^ if-' 



^^' 






Figure 1. Segregation patterns observed in fertilized eggs from diploid zhikong scallop, CMamys farreri. following normal fertilization (a-e) and 
PBl blocking with Cvtochalasin B (f-ol. a-e: meiosis in normal fertilized eggs; f: two polar bodies positioned side-by-side in fertilized eggs 
following PBl blocking with CB: g: the united 38 dyads, h-i: bipolar segregation pattern: j-k: tripolar segregation pattern; 1-m: tetrapolar 
segregation pattern. 1 and 2 indicated two separate poles; n: unsynchronized segregation pattern. 3 indicated dyad groups; o: undassifled 
segregation pattern. 



104 



Yang et al. 



TABLE 1. 
Chromosome segregation patterns (%) in fertilized eggs wlien PBl was blocl^ed with CB in zhikong scallop Chlamys farreri. 



Replicate 






Chromosome Segregation 


Patterns 




















(Number) 


n 


Bipolar 


Tri polar 


Tetrapolar 




Unsynchronized 


Unclassiried 


1 


109 


11.0 


4.'i.9 


17.4 




1.8 


23.9 


2 


162 


7.4 


46.9 


17.3 




4.3 


24.1 


3 


106 


13.2 


46.2 


11.4 




2.8 


26.4 


4 


85 


14.1 


24.7 


16.5 




7.1 


37.6 


Average 




11.4 


40.9 


15.7 




4.0 


28.0 



In CB-treated groups, chromosomes in fertilized eggs, follow- 
ing PBl blocking, segregated in four patterns: bipolar (11.4%), 
tripolar (40.9%), tetrapolar (15.7%), and unsynchronized (4.0%). 
Bipolar, tripolar, and tetrapolar segregation patterns were similar 
to those reported in diploid (Guo et al. 1992b) and triploid Pacific 
oysters (Que et al. 1997) when PBl was blocked. 

In addition, in the zhikong scallop a small proportion of treated 
eggs (4.0%) went through meiosis II asynchronously. Blocked 
dyads from PBl failed to unite and segregated asynchronously 
from the remaining dyads. Sometimes the remaining dyads went to 
anaphase of meiosis II and divided into two groups of chromatids, 
while the dyads from blocked PBl remained paired and skipped 
meiosis II. leaving three chromatin groups. In eggs of triploid 
Pacific oysters, asynchronous segregation was also observed when 
crossed with a normal sperm of diploid followed by PBl inhibition 
by CB (Que et al. 1997). In the Japanese pearl oyster, Pinctada 
fiicala inanensii. Komaru et al. (1990) reported that three groups 
( 20.6% ) and four groups ( 1 7.6% ) of maternal chromatin were pro- 
duced by blocking PB 1 . The observation of three groups of chro- 
matin might be explained in two ways: asynchronous segregation 
or tripolar chromosome segregation, both resulting in three chro- 
matin groups. The percentage of fertilized eggs with three groups 
of maternal chromatin (20.6%) as observed by Komaru was much 
lower than our observations of tripolar segregation (40.9%) and 
asynchronous segregation (4.0%) in the zhikong scallop. This is 
possibly caused by differences in chromosome segregation be- 
tween the different species or because the conditions of CB treat- 
ment were different. In diploid Pacific oysters, chromosome seg- 
regation following PBl inhibition was observed to pass through 
meiosis II synchronously (Guo et al. 1992b). 

In addition to the described tour segregation patterns, there was 
a large proportion of chromosome segregations (28.0%) that could 
not be classified, such as 76 chromatids scattered randomly. Un- 
classified .segregation patterns have also been observed in both 
diploid and triploid Pacific oysters (Guo et al. 1992b. Que et al. 
1997). Considering the results of this experiment and those in 
diploid and triploid Pacific oysters and pearl oysters, we suggest 
that tripolar, tetrapolar, bipolar, and unsynchronized segregation 
patterns are the normal ways for fertilized eggs to go through 
meiosis II alter PBl blocking. 

Observations of chromosome segregation using the st|uashing 
method provide an incomplete picture of cytological events, be- 
cause compression of the eggs transforms the three dimensionality 
of the meiotic plates into a plane, thus rearranging the position of 
chromosomes. This method also fails to display centrosomes and 
spindles that play an important role in meiosis. Observing cen- 
trosomes and spindles might provide a clearer picture of how 
chromosomes segregated. Especially for the centrosome. its num- 
ber and replication are critical factors in the chroiiiosome segre- 



gation. Normally the centrosome from sperm does not participate 
in meiosis (Sluder et al. 1993), and the centrosome from maternal 
replicates two times with each meiotic stage, resulting in the nor- 
mal bipolar segregation. In this experiment, we hypothesize that 
centrosome number is the primary factor controlling patterns of 
chromosome segregation. With PBl blocked in eggs, centrosome 
number could change profoundly, affecting chromosome segrega- 
tion in meiosis II. Centrosome numbers could range from 2—4, 
depending upon whether centrosomes replicated, and could result 
in bipolar, tripolar, or tetrapolar chromosome segregation patterns. 
This supposition must be tested by visualization of the cen- 
trosomes, spindles, or both. 

Ploidy Consequences 

In the zhikong scallop, diploid, triploid, tetraploid, pentaploid. 
and aneuploid 2—4 cell stage embryos were all produced when PBl 
was blocked in fertilized eggs. Both triploid and tetraploid zhikong 
scallops survived to juvenile stage (21.3% triploid and 1.9% tet- 
raploid in one group, unpublished). The various ploidy conse- 
quences of PB I blocking relate to the different chromosome pat- 
terns, as observed in this study. 

First, bipolar segregation patterns formed two groups of 38 
chromatids. Either of the two chromatids group could be released 
as PB2, leaving 38 chromatids. No matter which group was re- 
leased, triploids would be produced by bipolar segregation v\ith 19 
chromosomes contributed by the sperm. 

For tetrapolar segregation patterns, four separated chromatids 
groups were formed after meiosis II, 19 chromatids in each group. 
The ploidy consequences would depend upon how many chroma- 
tid groups would be released with PB2. Release of one group 
would produce tetraploids; whereas, release of two groups would 
produce triploids and release of three would produce diploids. 
After CB was washed off, embryo development showed that .some 
fertilized eggs in the treated groups released one PB, and some 
fertilized eggs released two PBs positioned side-by-side (Fig. If) 
or separated from each other on the egg. Rarely were these two 
polar bodies positioned next to each other, as in Figure le. It was 
impossible by our methods to observe total number of chromatids 
in released PBs. This problem might be resolved by using special 
staining methods to label chromatids individually, such as //; siiu 
fluorescent hybridization. 

The ploidy consequences of embryos after tripolar segregation 
were the most complicated because of random allocation of chro- 
matids at three poles and the random release of PB2. The meta- 
pliase and anaphase period In meiosis II were very short, so it was 
not practical to count numbers of chromatids at the three poles in 
most fertilized eggs. By counting the chromosome of 2-4 cell 
embryos, we could infer that chromosome number varied highly. 



Chromosomh Segregation in PB 1 Blocked Eggs 



105 



In tripolar segregations, tetraploids would be produced only when 
one pole had exactly 19 chromatids, and the chromatids at this pole 
were released as PB2. If the 19 chromatids at one pole remained in 
the eggs, and the chromatids at the other two poles were released 
as PB2. diploids would be produced. Otherwise, aneuploids re- 
sulted. The majority of fertilized eggs proceeded by tripolar seg- 
regation (40.9'/(-), explaining why about 23.3% of 2-4 cell stage 
embryos were aneuploid (unpublished). In most aneuploids. chro- 
mosome numbers were distributed mainly into three groups: 42- 
48, 62-69. or 83-89. most likely the result of random allocation of 
chromatin from the three poles. 

Unsynchronized segregation resulted in three groups of chro- 
matin, two with 19 chromatids in each and the other with 19 dyads 
from blocked PBl. Diploids, triploids. and tetraploids could pos- 
sibly be produced, depending upon which group was relea.sed as 
PB2. Supposing one group of 19 chromatids was released as PB2. 
tetraploids would be produced. If 19 dyads were released as PB2, 
triploids would be produced. If two groups of chromatin were 
released as PB2. triploids or diploids would be produced. 

Pentaploids were also observed at the 2^ cell embryo stage 
(unpublished). The formation of pentaploidy was probably caused 
by the failure of PB2 to be released in fertilized eggs after PB 1 was 
blocked with CB. Thus, the 76 chromatids from maternal chro- 
motids plus the 19 chromatids from sperm formed pentaploids. 

Clearly, differences in chromosome segregation resulted in dif- 
ferent ploidy consequences and agree with the proposed mecha- 



nism to form different ploidies in diploid Pacific oyster when PBl 
was blocked (Guo et al. 1992b), PBl blocking is also an effective 
way to induce triploids and tetraploids. Both triploid and tetraploid 
embryos have been produced through blocking PBl in fertilized 
eggs from normal diploid, such as in Pacific oyster (Guo et al. 
1992a), American oyster (Stanley et al. 1981). Pacific abalone 
(Aral et al. 1986) and blue mussel (Yamamoto and Sugawara 
1988). 

In summary, the inhibition of PBl in fertilized eggs of zhikong 
scallop with CB resulted in complicated chromosome segregation 
patterns, including bipolar, tripolar, tetrapolar. unsynchronized. 
and unclassified segregations, producing diploid, triploid, tetra- 
ploid, pentaploidy, and aneuploid embryos. This study provided 
cytological evidence about possible formation of different ploidies 
and valuable information on polyploid induction. 

ACKNOWLEDGMENT 

The authors thank Drs. Ximing Guo and Standish K. Allen Jr. 
for their constructive comments on the manuscript. This study is 
supported by Chinese postdoc fund (No. 6975), Grant 819-01-07 
from China's National High-Tech Development Program (863), 
the "100 Scholar" program of the Chinese Academy of Science 
and China's Natural Science Foundation (No. 39825121). This is 
publication No. 3682 of the Institute of Oceanology, Chinese 
Academy of Sciences. 



LITERATURE CITED 



Aral. K. F.. F. Naito & K. Fujino. 1986. Triploidizalion ot the Pacific 
abalone with temperature and pressure treatments. Bull. Japan Soc. Sci. 
Fish. 52:417-422. 

Guo. X.. K. Cooper, W. K. Hershberger & K. K. Chew. 1992a. Genetic 
consequence of blocking polar body I with cytochalasin B in fertilized 
eggs of the Pacific oyster. Crassostrea gigas: I. Ploidy of resultant 
embryos. Biol. Bull. 183:381-386. 

Guo. X.. W. K. Hershberger. K. Cooper & K. K. Chew. 1992b. Genetic 
consequence of blocking polar body I with cytochalasin B in fertilized 
eggs of the Pacific oyster. Crassostrea gigas: II. segregation of chro- 
mosomes. Biol. Bull. 183:387-393. 

Guo, X. & S. K. Allen Jr. 1994. Viable tetraploids in the Pacific oyster. 
Crassostrea gigas (Thunberg). produced by inhibiting polar body I in 
eggs from triploids. Mol. Mar. Biol. Biotechnol. 3:42-50. 

Jiang. W.. G. Li & Y. Lin. 1987. The polyploid induction in Pearl oyster. 
Pinctada martensii. Tropic Oceanog. 6:37^5 (in Chinese). 

Komaru. A.. H. Matsuda. T. Yamakawa & K. T. Wada. 1990. Chromo- 
some-behavior of meiosis-inhibited eggs with cytochalasin B in Japa- 
nese pearl oyster. Nippon Suisan Gakkaishi 569:1419-1422. 

Longo. F. J. & E. Anderson. 1969. Cytological aspects of fertilization in 
the Lamellibranch. Mytilus edulis. I. polar body formation and devel- 
opment of the female pronucleaus. J. Exp. Zool. 172:69-96. 



Que. H.. X. Guo. F. Zhang & S. K. Allen Jr. 1997. Chromosome segre- 
gation in fertilized eggs from triploid Pacific oyster. Crassostrea gigas 
(Thunberg). following inhibition of polar body I. B/o/. Bull. 193:14-19. 

Quillet. E. & P. J. Panelay. 1986. Triploidy induction by thermal shocks in 

the Pacific oyster. Crassostrea gigas. Ac/uaculture 57:271-279. 
Sharma. A. K. & A. Sharma. 1980. Chromosome techniques: theory and 

practice. 3rd ed. Butterworth. London, pp. 1 1 1-1 13. 
Sluder. G.. F.J. Miller & K. Lewis. 1993. Centrosome inheritance in 

starfish zygotes 11: selective suppression of the maternal in centrosome 

during meiosis. Dev. Biol. 155:58-67. 
Stanley. J. G., S. K. Allen Jr. & H. Hidu. 1981. Polyploidy induced in the 

American oyster, Crassostrea virginica, with cytochalasin B. Aqiiacul- 

ture 12:1-10. 
Stephens. L. B. & S. L. Downing. 1988. Inhibiting first polar body forma- 
tion in Crassostrea gigas produces tetraploids. not meiosis triploid. / 

Shellfish Res. 7:550-551. 
Wang. M.. J. Zheng & H. Wang. 1990. The karyotype of Zhikong scallop, 

Chlamys farreri. J. Ocean Univ. Qingdao 20:81-85 (in Chinese). 
Yamamoto. S. & Y. Sugawara. 1988. Induced triploidy in the mussel, 

Mytilus edulis. by temperature shock. Aquaculture 72:21-29. 



Journal of Shellfish Research. Vol. 19. No. 1. 107-112. 2U()(). 

REPRODUCTIVE CYCLE OF ARGOPECTEN VENTRICOSUS (SOWERBY 1842) (BIVALVIA: 

PECTINIDAE) IN THE RADA DEL PUERTO DE PICHILINGUE, B.C.S., MEXICO AND ITS 

RELATION TO TEMPERATURE, SALINITY, AND FOOD 

ANTONIO LUNA-GONZALEZ,* CARLOS CACERES-MARTINEZ,' 
CLAUDIA ZUNIGA-PACHECO,' SILVERIO LOPEZ-LOPEZ," AND 
BERTHA PATRICIA CEBALLOS-VAZQUEZ" 

' Departamento de Ingenieiia en Pesquen'as 
Universidad Autonoma de Baja California Sur 
Lahnratorio Experimental de Maricultura. Apartado Postal I9-B. 
La Paz. B.C.S.. 23081 Mexico. 

'Centra Interdisciplinario de Ciencias Marinas 
Apartado Postal 592. 
La Paz. B.C.S.. 23000 Mexico 

ABSTRACT The reproductive cycle of the catarina scallop Argopecten ventricosiis and its relation to temperature, salinity, and 
quantity of food was studied in the Rada del Puerto de Pichilingue. B.C.S. Mexico, from April 1995 to March 1996. Organisms were 
obtained from a hatchery and grown on the bottom. Ripe organisms occurred throughout the year showing the lack of seasonality in 
its reproduction. No consistent relation between reproductive cycle and environmental factors or food was evident. The muscle yield 
index showed a significant positive correlation with temperature, but it had no correlation with gonadosomatic index. The relation 
between the muscle yield index and seston with the reproductive cycle suggested the transference of energy from the muscle to the 
gonad and directly from the seston ingested. This relation suggested that A. ventricosus is a conservative and opportunistic species 
depending on the available food. Histochemical analysis revealed the transference of carbohydrates from the intestinal loop to the 
gonad and therefore to the oocytes. 

KEY WORDS: Argopecten. reproductive cycle, bivalves, histochemistry, seston, food index 



INTRODUCTION 

The scallop Argopecten ventricosus (Sowerby 1842) is distrib- 
uted from Isla Cedros and the Gulf of California to Peru (Keen 
1971). A. ventricosus supports an important fishery in northwest 
Mexico, especially in Baja California Sur (Chavez- Villalba and 
Caceres-Martinez 1992). It is an important resource because of the 
high commercial value of its adductor muscle (Villalejo-Fuerte 
and Ochoa-Baez 1993). 

The necessity of measures for the regulation of the fishery has 
prompted several studies about reproduction of the catarina scallop 
in Baja California Sur (Baqueiro et al. 1981, Caceres-Martinez et 
al. 1990, Villalejo-Fuerte and Ochoa-Baez 1993. Felix-Pico et al. 
1995). 

The reproductive cycles of scallops are influenced by changes 
in environmental variables, such as temperature and food (Mac- 
Donald and Thompson 1985. Barber and Blake 1991). and by 
genetic characteristics (Barber and Blake 1991). Gametogenesis 
needs a lot of energy (Sastry 1979). This energy is obtained di- 
rectly from the seston or from storage organs or tissues, like the 
digestive gland, mantle, and adductor muscle (Ansell 1974, Gab- 
bott 1975, Barber and Blake 1983). 

The seston includes live plankton, organic detritus, and inor- 
ganic particles (Navarro and Thompson 1995). The quantity and 
quality of seston varies in response to physical and biological 
factors such as tides, storms, wind, bacteria, fungi, and primary 
consumers (Berg and Newell 1986, Mann 1988). 

The objective of the study is to examine the reproductive cycle 
of A. ventricosus in relation to its condition, histochemical com- 
position of somatic and reproductive tissues, temperature, salinity, 
and quantity of available food. 



MATERIALS AND METHODS 

Between April 1995 and March 1996, 30 specimens of A. ven- 
tricosus (shell height mean ± SD = 5.25 ± 0.02 cm) were col- 
lected randomly per month by diving between 3- and 4-m depth 
from a population grown in the Rada del Puerto de Pichilingue, 
B.C.S. , Mexico (24°16'N; 1 10°19'W). These organisms were ini- 
tially produced in September 1994 at our hatchery at Universidad 
Autonoma de Baja California Sur and seeded on the bottom in 
February 1995. The surface water temperature and salinity were 
recorded at the time of sampling. Total soft body, adductor muscle, 
and gonad wet weights were recorded for each specimen. 

Reproductive Cycle 

The scallops were fixed in 10% formalin. Tissue sections were 
taken through the middle of the gonad, dehydrated in alcohol, and 
embedded in paraffin wax. Sections (5 |j.m) were placed on slides 
and stained with hematoxylin-eosin (Humason 1979). Gametoge- 
nesis (either spermatogenesis or oogenesis) of A. ventricosus was 
divided into five stages (undifferentiated, developing, ripe, spawn- 
ing, and spent) on the basis of the developmental stages defined by 
Villalejo-Fuerte and Ochoa-Baez (1993) for the same species and 
our own observations. 

Undifferentiated Stage 

Abundant connective tissue, without germ cells or residual ga- 
metes. It was not possible to distinguish the sex. 

Developing Stage 

In the female, this stage is characterized by the presence of 
variable quantities of developing oocytes attached to the follicle 



107 



108 



LUNA-GONZALEZ ET AL. 



wall. Some detached ripe oocytes occurred in the lumen of the 
follicle. In the male, this stage had variable quantities of germinal 
cells, spermatocytes, spennatids, and ripe spermatozoa. Interfolli- 
cular connective tissue decreases and follicles increase in area as 
the result of the accumulation of ripe gametes. 

Ripe Stage 

In the female, there were abundant, ripe polygonal-shaped oo- 
cytes free within the follicles. Yolk droplets were observed in the 
oocyte cytoplasm. Some developing oocytes remained attached to 
the follicle wall by a slender stalk. In the male, this stage was 
characterized by follicles filled with ripe spermatozoa arranged in 
characteristic radial bands with tails pointing toward the center of 
the lumen. Almost all the connective tissue has been completely 
replaced by follicles forming the gonadic tissue, which is occupied 
by gametes. 

Spawning Stage 

The walls of follicles become broken. Variable quantities of 
unspawned oocytes and spermatozoa were observed into the fol- 
licles. Free spaces inside the follicles were abundant. Some fol- 
licles are completely devoid of gametes. 

Spent Stage 

The follicles were empty, with the exception of some residual 
oocytes and spermatozoa. Connective tissue begins increasing. 
The broken follicles are invaded by phagocytes. The relative fre- 
quencies of the stages of gonadal developinent throughout the year 
were obtained. This enabled the description of the reproductive 
cycle. 

Gonadosomatic Index (GSI) 

This index in wet weight was calculated according to Sastry 
and Blake (1971). 

GW 

Where GSI is the gonadosomatic index, GW is the gonad weight 
in grams, and TSBW is the total soft body weight in grams. 

Muscle Yield Index (MYI) 

The muscle yield index was calculated as an indicator of the 
condition of the scallops (Caceres-Martinez et al. 1990). 

MW 

Where MYI is the muscle yield index. MW is the weight of muscle 
in grams, and TSBW is the total weight of the soft body in grams. 

Histochemical Analysis 

Four .scallops corresponding to each stage of gonadal develop- 
ment (twenty in total) were collected in September 1995 for his- 
tochemical analysis (qualitative analysis) of gonad, mantle, and 
muscle to delcrminc carbohydrate and lipid content. Unfortu- 
nately, we did not take samples since April 1995 to get an annual 
cycle. Tissue sections were embedded in paraffin wax and O.C.T. 
compound (an embedding medium for frozen tissue specimens). 
Sections 5-(j.m thick from paraffin wax and sections 16-^m thick 
from O.C.T. were placed on slides. The oil red technique (Spann- 



hof 1966, Martoja and Person 1970) was used on frozen cuts to 
determine unsaturated lipids. Periodic acid of the Schiff-Malt tech- 
nique was used to determine glycogen (Martoja and Person 1970, 
Sheehan and Hrapchak 1973, Humason 1979), and the blue alzian 
technique was used to detect acid mucopolysaccharides (Spanhoff 
1966, Martoja and Person 1970). 

Seston Analysis 

During the study period, every 15 days, 12-L of unfiltered 
seawater samples of the scallop-sampling area were collected in 
clean plastic containers and transported to the laboratory. The 
seawater samples were collected at 3.5-m depth, close (about 15 
cm) to the sandy bottom on which the scallops grew. The water 
was screened through a 180-|jLm Nitex mesh to eliminate large 
zooplankton and debris before analysis. 

For dry weight and chemical analysis, 2-L of seawater for each 
filter (six filters in total every 15 days) were immediately filtered 
under gentle vacuum through washed, precombusted, preweighed 
Whatman GF/C filters, 4.7-cm diameter. Three filters for chemical 
analysis were stored at -40 °C until the analysis was done. Three 
filters for dry weight were dried in an oven at 80 °C for 24 h. Then 
they were weighed and combusted at 475 °C for 4 h. Finally, filters 
were reweighed after cooling in a desiccator. The particulate or- 
ganic matter (organic seston) was obtained by difference of both 
weights. 

For chemical analysis, two filters per month (one filter per 
sampling) with 2-mL of distilled water were ground at 5 '^C in an 
ice bath. A 400-|jlL aliquot was used for lipid determination using 
the Bligh and Dyer (1959) method. Carbohydrates were analyzed 
in a 300-p.L sample by the method of Dubois et al. (1956), modi- 
fied by Malara and Charra (1972a). Proteins were analyzed in a 
300-(i,L aliquot by the method of Lowry et al. (1 95 1 ), modified by 
Malara and Charra (1972b). Results of chemical analysis were 
standardized for volume of seawater filtered. 

Total Seston ( TS) 

The TS was obtained as the sum of inorganic seston and or- 
ganic seston (dry weight). 

Inorganic Seston/Organic Seston Ratio (IS/US ratio) 

This ratio was obtained to relate (monthl) ) inorganic seston to 
organic seston. 

Food Index (FI) 

An evaluation of the nutritional value of the seston throughout 
the annual cycle in the Rada del Puerto de Pichilingue was done 
using the 3 major biochemical components of the seston (lipid, 
carbohydrate, and protein). Thus food quantity was defined as the 
sum of these components and a food index was calculated accord- 
ing to Widdows et al. (1979) as the percentage of food nialerial 
contained in the total seston. 



Fl= — * 100 

Where 1-1 is the food index, F is the lood material (mg/L), and TS 
is the total seston (ma/L). 



Reproductive Cycle of A. ventricosus 



109 



RESULTS 



Reproductive Cycle 



The scallop A. ventncosiis is a functional hermaphrodite. In the 
female and male follicles, the gametes were in the same develop- 
mental stage. The gonad showed well-differentiated male and fe- 
male areas. Figure 1 summarizes the reproductive cycle of A. 
ventricosus. The presence of ripe gonads throughout the year in- 
dicated a prolonged reproductive period with a lack of a clear 
seasonal pattern. Despite this, there was a major resting period in 
June and September 1995 where the undifferentiated stage reached 
a maximum (91.3 and 46.15%. respectively). The spawning stage 
was observed in 9 of the twelve months sampled but reached the 
maximum \alue in August 1995 (50%). 

Environmental Parameters 

Temperature and salinity fluctuated relatively little (Fig. 2a). 
The maximum water temperature was in September 1995 
(29.5 °C). and the minimum (20.5 °C) in January 1996. The maxi- 
mum salinity was in January. February, and March (37 7cc). and the 
minimum in August and September 1995 (34 %o). 

Gonadosomatic Index 

The GSI supported the results obtained in the histological 
analysis (Fig. 2b). The values were at a minimum in April, May. 
June. September. January, and increased drastically from January 
(4.08%) to February (7.79%) and March (8.75%). 

Muscle Yield Index 

The MYI was at a maximum in June and September 1995 (45.8 
and 46.9%, respectively) and was at a minimum in April 1995 
(33.7%) and from December 1995 to March 1996 (Fig. 2c). The 
MYI showed a significant positive correlation with temperature (r 
= 0.797; P = 0.001; n = 12). and a significant negative corre- 
lation with salinity (r = -0.788; P = 0.002; n = 12). With the 
GSI, there was no significant correlation (r = -0.405; P = 0.190; 
n = 12). 

Histochemical Analysis 

The results of the histochemical analysis of gonad, adductor 
muscle, and mantle are in Table 1. Positive results were found for 
glycogen in the female area of the gonad (developing and ripe 




I I Undifferentiatec |~p Developing 
^■Spawning ^H Spent 



I Ripe 



35 " 


r 




—-—Temperature -*— Salinity 


9 30- 


-^----^^ 






<u 


""\_ / "*^ ^V^ 






3 25 - 


/C / \ 










QJ 


■ ^ ^v 


^ 20. 


X---' • 


H 




15 - 





38 




36 


2 


34 


>. 
c 



32 



12 



SI 


10- 


T) 




_C 


8 - 


o 




ra 


fi - 


l- 




o 
(/I 


4 - 


o 




■n 




ro 


? - 


r 




o 


- 


O 


^ 


55 


V 


50 


0) 




-o 

c 


45 


-D 


40 


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>- 


3b 



() 


30 


m 




D 


25 





A M J 



J A 
1995 



S O N D 



J F 
1996 



M 



Figure 1. Reproductive stages of Argopecten ventricosus in the Rada 
del Puerto de Pichilingue, B.C.S., Mexico (n = 30). 



Figure 2. Water temperature and salinity in the Rada del Puerto de 
Pichilingue, B.C.S., Mexico (a) and gonadosomatic (b) and muscle 
yield index (c) of Argopecten ventricosus. (Error bars = SD). 

oocytes) and in muscle fibers. Unsaturated lipids were found in the 
female area of the gonad (developing and ripe oocytes). Acid 
mucopolysaccharides were found in the interfollicular connective 
tissue of developing stage gonads (male and female areas), epi- 
thelium and food content of intestinal loop, and in the epithelium 
and connective tissue of mantle. 

Total Seston, Inorganic Seston, and Organic Seston 

Sediment resuspension was caused mainly as a consequence of 
tidal currents and wind. In this area, maximum values of TS and IS 
were found in April. July. December, and January. Higher values 
of OS were found in April. December, and January (Fig. 3a). 

Inorganic Seston/Organic Seston Ratio 

The IS/OS ratio had no clear relation with the reproductive 
cycle (Fig. 3a). However, it showed a significant positive correla- 
tion with salinity (r = 0.676; P = 0.015; n = 12). 

Food index 

The FI expresses the quality of the diet available to a filter- 
feeding organism. This FI showed maximum values in July, Sep- 
tember, and November 1995 (4.99, 5.03, and 4.97%, respectively) 
and minimum values in April-May 1995 (2.79 and 2.92%, respec- 
tively) and February 1996 (2.78%) (Fig. 3b). It did not show a 
clear relation with the reproductive cycle. 



110 



Luna-Gonzalez et al. 



TABLE 1. 
Histochemical tests performed on gonad, muscle, and mantle of Argopecten ventricosus. 



Substance Tested 



Technique 



Control 



Gonad 



Muscle 



Mantle 



Glycogen 



Unsaturated lipids 



Acid mucopolysaccharides 



Pas-Mall 



Oil Red 



Al/ian Blue 



Rat liver 


- 00 




++ ro 




++ do 


None 


- oo 




++ ro 




++do 


None 


++ ifd 




+ ifr 



Abbreviations: -, not detected; +. positive reaction; ++, strong positive reaction; do. developing oocytes; ifd, interfollicular connective tissue of 
developing stage gonads (male and female area), epithelium and food content of intestinal loop; ifr. interfollicular connective tissue of ripe stage gonads; 
mec, mantle epithelium and connective tissue; oo. oogonias; ro, ripe oocytes. 



DISCUSSION 

The cytological characteristics of the gonad of A. ventricosus in 
the Rada del Puerto de Pichilingue, B.C.S. were similar to those 
described by Villalejo-Fuerte and Ochoa-Baez ( 1 993) for the same 
species in Bahi'a Concepcion. B.C.S.. and for other pectinids. like 
Patinopeclen yessoensis (Motavkine and Varaksine 1983) and Pla- 
copecten magellanicus (Beninger 1987). Male and female follicles 
developed simultaneously and the gametes were spawned at about 
the same time. 

The gonads of A. ventricosus contained gametes in different 
stages of development in all the months during the annual cycle, 
although in lesser amount in June when the majority of the speci- 
mens were in the undifferentiated stage. Ripe organisms were 
present throughout the year, which suggests that this species re- 
produces throughout the year. Similarly, the presence of ripe or- 
ganisms of A. ventricosus all year has been reported in other lo- 
cations of Baja California Sur (Baqueiro et al. 1981, Felix-Pico et 
al. 1995). 

Although the temperature is an important environmental factor 
in the regulation of bivalve reproduction (Sastry 1979). in this 
work, neither temperature nor salinity showed a clear relation with 
the reproductive cycle of A. ventricosus in the Rada del Puerto de 
Pichilingue because partly spawning scallops appear throughout 
the annual cycle. Maximum and minimum water temperatures co- 
incided with the spawning (histologicaly detected) of August- 
September and December, as did the minimum and maximum 
values of salinity. The above suggests that the changes in tempera- 
ture and salinity may be responsible for triggering spawning, but 
did not affect directly the gonadal maturation process. 

In this work, the MYI did not show a significant negative 
correlation with GSI, but reproductive activity was present year 
around. An explanation of this unclear relation of the MYI with Ihc 
reproductive activity is that in the Rada del Puerto de Pichilingue 
this species uses the available food in the environment more than 
inuscle reserves for the gonadal maturation when the food is abun- 
dant, and they use the muscle reserves when the food abundance is 
poor. A transference of energy from the muscle to the gonad in A. 
ventricosus had been suggested by Caceres-Martine/ et al. ( 1990) 
and Villalejo-Fuerte and Ceballos-Va/c|ue/ (1996). 

Bayne (1976) divided the bivalves into two groups based on 
their gametogenic pattern; 1 ) "conservative" species where game- 
togenesis occurs from energy stored in the tissue, and 2) "oppor- 
timistic" species where gametogenesis occurs when theic \v;is 



abundant phytoplankton. In this case. A. ventricosus would be 
named both opportunistic and conservative depending on the avail- 
able food. 

The MYI had a positive correlation with temperature. This may 
be the environmental variable that influences the transference of 
stored reserves from the adductor muscle to the gonad of A. ven- 
tricosus. as happens in A. irradians (Sastry and Blake 197 1 . Barber 
and Blake 1981. MacDonald and Bourne 1987). For salinity, a 
negative correlation with MYI was observed but the influence of 
salinity in the transference of nutrients remained unclear. 

Le Pennec and Beninger (1991) observed that through most of 
the energy supplied to the developing gametes comes from protein 
and glycogen reserves in the adductor muscle, there is also energy 
transference from the reabsorption of residual oocytes and from 
the transference of nutrients from the intestinal loop to the gonad. 
The intestinal loop penetrates into the gonad and has a digestive 
function (epithelium with intracellular and extracellular digestion) 
and there is a direct transference of the metabolites from the in- 



18 

16 

3 14 

o> 12 

£ 10 ^ 



6 1 
4 
2 





4 

■ 3.5 .o 
ra 

cr 

3 to 

O 

w 

2.5 - 



— •— Inorganic Seston —*— Organic Seston 
-•- Total Seston -.- IS/OS Ratio 



5,5 

5 
4.5. 

4 

35 

3 

2,5 




M 



M 



JJASOND JF 
1995 1996 

Figure ,'. Changes in the seston and IS/OS ratio (a), and food index (b) 
throughout the annual cycle from the Kada del Puerto del Pichilingue, 
B.C.S., Mexico. (Krror bars = SD). 



Reproductive Cycle of A. ventr/cosus 



111 



testinal epithelium to the gonad and therefore to the developing 
oocytes. In this work, we found a lot of acid mucopolysaccharides 
in the intestinal loop, the mantle, and the perigonadal connective 
tissue of the developing gonads (male and female) of A. ventrico- 
siis. In the gonads, these carbohydrates can be the result of the 
transference from the intestinal loop |Le Pennec and Beninger 
1991) or from the mantle (Barber and Blake 1983). In contrast, in 
the ripe gonad the acid mucopolysaccharides were few. so we 
believe that they were used in the maturation of the gametes. 

In the developing and ripe oocytes, we observed a lot of gly- 
cogen that probably was the result of the transformation of the acid 
mucopolysaccharides. This carbohydrate is converted into triglyc- 
erides and is stored in the oocytes to be used as a future energy 
source for the larvae (Gabbott 1975). To support this, we found 
much unsaturated lipids (oil droplets) in the cytoplasm of devel- 
oping and ripe oocytes. 

The gross analysis of the seston or the measurement of a single 
chemical variable cannot describe fully the nutritive value of 
seston. To understand seston as food, it is necessary to determine 
its major biochemical constituents (lipid, protein, and carbohy- 
drate) (Navarro et al. 1993). These components form the food 
material available for scallops and their larvae. TS. IS/OS ratio, 
and FI showed no clear relation to the reproductive cycle. It seems 
the reproductive cycle was influenced by a combination of the 
quantity of food and the muscle reserves. 



A. ventricosiis exists in large stocks in the bays of Baja Cali- 
fornia Sur (Tripp 1985, Aurioles-Gamboa 1992) but some of these 
stocks have been overfished (Chavez-Villalba and Caceres- 
Martinez 1992, Caceres-Martinez et al. 1993). This is true in Bahi'a 
de La Paz. in which the study area of this work is included. There 
is no fishery in this bay now because of the depletion of the A. 
venrricosiis population. A management option is the culture of the 
species and this idea directed this study. From our results, we can 
say that the Rada del Puerto de Pichilingue is not an appropriate 
zone for the culture of catarina scallops. This is because the quality 
and quantity of food is poor and cannot support commercial pro- 
duction. Though reproductive activity was observed throughout 
the year as in other locations of Baja California Sur (Baqueiro et 
al. 1981, Villalejo-Fuerte and Ochoa-Baez 1993. Felix-Pico et al. 
1995), the GSI values in the Rada del Puerto de Pichilingue were 
lower (2% less) than those obtained for A. ventricosiis from Bahi'a 
Concepcion. B.C.S.. Mexico (Villalejo-Fuerte and Ochoa-Baez 
1993). 

ACKNOWLEDGMENTS 

This study was supported by the UABCS research for the Mas- 
ter of Sciences in Aquaculture Program. Special thanks are due to 
Javier Cortes Salazar for his technical support during the field 
studies. Thanks to Dr. Ellis Glazier (CIBNOR) for editing this 
English-language text. 



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Journal of Shellfish Research. Vol. 19, No. I. 113-124. 20UU. 

BIOECONOMIC ANALYSIS OF A SEA SCALLOP, PLACOPECTEN MAGELLANICUS, 
AQUACULTURE PRODUCTION SYSTEM IN NEWFOUNDLAND, CANADA 

R. W. PENNEY' AND T. J. MILLS" 

' Department of Fisheries and Oceans 

P. O. Box 5667 

St. John's. Nfld. Canada 

AIC 5 XI 
'Thimble Bay Farms Ltd. 

P. O. Box 381 

Botwood, Nfld. Canada 

AOH lEO 

ABSTRACT We report the results of 2-year pilot scale scallop, Phuopecten nuigelhmicus. culture trials at Charles Arm. in Notre 
Dame Bay on the northeast coast of Newfoundland during 1989-1991. We used extrapolations of the growth and survival data from 
these trials, as well as records of all capital, labor, and operational costs, to conduct simulation modeling of cash flows associated with 
start-up and operation of scallop farms scaled up to commercial size. Our aim was to determine the economic viability of sea scallop 
farming in Atlantic Canada using the standard economic evaluation methods of Net Present Value (NPV) and Internal Rate of Return 
(IRR), based on production of 55-65 mm (shell height) live, whole, scallops using a suspended pearl net culture system. Two separate 
pilot trials were carried out concurrently. In the first, the effect of stocking density and culling on growth and survival were determined 
by stocking scallop seed in peari nets at five different starting densities; 50, 75, 100, 150, and 200/net with subsequent culling (thinning) 
at two different times during grow-out. In the second trial, the effect of seed grading and net mesh size on growth and survival were 
determined by grading seed into two nominal sizes based on shell height, small (< 18 mm) and large (> 18 mm), followed by stocking 
in pearl nets of varying mesh size: 4.5, 6.0, and 9,0 mm, at a starting density of 50/net for grow-out. After 2 years of grow-out, size 
at age (growth) was significantly related to initial seed stocking density, culling, net mesh size, and seed grading. Survival was 
significantly related to initial stocking density, culling, and seed grading but not to net mesh size. The model simulations predict scallop 
farming enterprises with vertically integrated culture farms and processing plant operations, and with annual stocking rates of about 
1 million or more seed per year, are economically viable in current market and financial conditions. Sen.sitivity analyses indicate farm 
business viability is relatively sensitive to changes in sale price received for harvested product, but relatively insensitive to changes 
in capital costs, labor, other operational costs, or to mortality. The simulations also predict farm ownership of a processing plant 
enhances the economic viability of farming enterprises. These favorable economic projections support the contention that continued 
private and government-assisted investment in expansion of a whole scallop culture industry in Atlantic Canada is warranted. 

KEY WORDS: Scallop. Placopeclen magellaiiicus. aquaculture, bioeconomics 

INTRODUCTION duction of Atlantic sea scallop: ( 1 ) industry dependence on annual 

wild seed collection that has proved to be unreliable with wide 

The sea scallop. Placopeclen mogellanicus. has been the main- interannual fluctuations in seed settlement (Couturier et al. 1995); 

stay of the traditional commercial scallop capture fishery in At- and (2) high production costs for meats that marginalized eco- 

lantic Canada for many years. Beginning in the 1960s, consider- nomic viability (Frishman et al. 1980, Gilbert 1987, Gilbert and 

able effort has been expended to explore the comtnercial aquacul- LeBlanc 1991, Wildish et al. 1988). 

ture potential of the sea scallop (Couturier et al. 1995, Naidu et al. These economic analyses, which focused on "'meat" production 
1987) using technology, equipment, and reaiing practices widely as the sole source of farm revenue, cited high meat production 
used in the extensive Japanese scallop culture industry (Taguchi costs as the principal obstacle to viability but did not consider the 
1977 Aoyama 1989) for the indigenous Japanese scallop. Pali- harvesting and sale of alternative products. However, markets are 
nopecten yessoensis. North American markets for scallop products reported outside North America for "meats with roe." whole, live 
have traditionally been almost exclusively restricted to the white scallops and various "value-added" products. In Japan, a large- 
adductor muscle, or "meats." as they are known in the industry. scale culture industry producing Japanese scallop. Patinopecten 
Early efforts to commercialize culture of the giant sea scallop, yessoensis. for sale in a variety of product forms, including whole, 
Placopeclen magellaniciis. in Canada's Atlantic provinces were in-shell product has thrived for many years (Ikenoue and Kafuku 
based on production of meats intended to compete in these existing 1992). Whole, in-shell queen. Chhiniys operciilaris. and king, 
markets. Peclen ma.ximus, scallops are marketed in several European coun- 

Development of the culture industry has been slow. Total an- tries (de Franssu 1990, Hardy 1991). but availability typically is 

nual Canadian production of cultured scallop has averaged < 100 restricted to markets near fishing ports, because scallops have a 

mt since 1993 (Dept. of Fisheries and Oceans Statistics Rept. relatively short shelf life in air (De Franssu 1990). In British Co- 

1998). This figure includes both sea scallop. Placopeclen magel- lumbia, live, in-shell, pink scallops, Chlamys nibida. and spiny 

lanicus, production from Atlantic Canada as well as production of scallops. C. haslala. < 80 mm in shell height have been supplied 

the introduced Japanese scallop, Patinopecten yessoensis. in Brit- in small quantities (< 100 mt per annum) to both domestic and U.S. 

ish Columbia on Canada's West Coast. Two factors are considered markets for several years (W. Heath. BC Min. of Fisheries, pers. 

to be primarily responsible for the slow increase in cultured pro- comm.). Since 1996, this has been augmented by cultured scallop, 

113 



114 



Penny and Mills 



P. xessoensis, production, which has been sold throughout Canada. 
the U.S., and Asia. Cultured, in-shell sea scallops are also pro- 
duced in small quantities (< 30 mt/annum) from farms in Nova 
Scotia and Newfoundland for sale into domestic Canadian mar- 
kets. 

Beginning in 1992. promotional and market development ini- 
tiatives carried out by Thimble Bay Farms Ltd.. have identified a 
niche market in Canada and the northeastern United States for a 
55-65 mm SH (shell height) sea scallop product, sometimes 
termed "princess"" or "•cocktail"" scallops by the seafood industry, 
depending upon its intended retail presentation. These are intended 
for retail and seafood service industry presentations similar to 
those in existing large volume North American markets for soft- 
shelled clam (A/ra arenaria). steamers, littleneck. and cherrystone 
clams {Mercenaria mercenaria), and oysters (Crassostrea vir- 
ginica) (De Franssu 1990). Sale of live, in-shell sea scallops now 
account for most of the annual farmed scallop production from the 
private company. Thimble Bay Farms Ltd in Newfoundland. 

However, efforts to increase market volume have been limited 
by unavailability of product attributable in large part to production 
bottlenecks caused by unstable seedstock supply. This has limited 
total cultured scallop production in Newfoundland to 10-19 mt 
annually since 1994 (Dept. of Fisheries and Oceans Statistics Re- 
port, 1998). Recent construction of a new scallop hatchery at Bel- 
leoram in Newfoundland with an estimated annual production ca- 
pacity of 20 million seed (G. Deveau, Ntld. Dept. Fisheries and 
Aquaculture. pers. comm.) may resolve the immediate seedstock 
supply problem and allow significant expansion in the industry. 
This has rekindled industry attention toward production and mar- 
keting issues, including whether it is economically advantageous 
to invest in market development for whole scallop products. 

Harvest and sale of small, in-shell scallops, in particular, may 
have a dramatic effect on scallop farm viability. In the United 
Kingdom, harvest and sale of small (5-6 cm), in-shell queen scal- 
lop, Chlamys opercularis. is considered to be financially advanta- 
geous for scallop farmers, because it reduces labor costs associated 
with shucking meats and greatly shortens production time (Hardy 
1991). This may also be true for sea scallop culture in Atlantic 
Canada. Recent consulting studies commissioned by the Provincial 
Government of Newfoundland and Labrador seem to support this 
contention (Atlaniecon 1992. ARA Consulting Group 199.^). Both 
studies developed financial projections suggesting the economic 
viability of commercial sea scallop culture might be enhanced, as 
compared to meat production, by developing markets for alterna- 
tive products, particularly whole scallops < 70 mm in shell height. 
The purpose of this paper, is to determine whether continued 
private industry and government investment in commercial expan- 
sion of this sector is warranted. In this paper, we report the results 
of pilot-scale sea scallop culture trials conducted at Thimble Bay 
Farms" s leased acreage at Charles Arm in Newfoundland, Canada 
during 19X9-1991. These trials sought to determine the biological, 
technological, and economic factors associated with producing for 
market a whole, in-shell scallop product using a Japanese sus- 
pended pearl net culture system. During the pilot trials, scallop 
growth and mortality, as well as labor, capital, and operating costs 
were recorded during a 2-year production cycle. Data collected 
during the pilot trials were used as input into a financial model to 
forecast the economic viability of commercial scale farms and, 
thus, provide both biological and economic bases for capital in- 
vestment decision making for the Atlantic Canadian shellfish cul- 
ture induslrv. 



This paper has three goals: ( 1 ) to quantify the effects of stock- 
ing density, culling during grow-out. initial spat grading, and net 
mesh size on scallop growth and survival observed in pilot-scale 
culture trials and use these values to define the optimum husbandry 
techniques appropriate for future scaled-up commercial opera- 
tions: (2) to conduct model simulations forecasting the economic 
viability of commercial-scale sea scallop culture farms utilizing 
production methodology similar to that used in the pilot trials to 
produce a 55-65 mm whole scallop: and (3) to assess the impact 
of vertical integration (e.g., farm ownership of a processing plant 
along with the culture farm) on projections of economic viability 
for scallop farming enterprises. 



METHODS 



Culture Trials 



Pilot-scale culture trials were conducted at Charles Arm. in 
Notre Dame Bay on the northeast coast of Newfoundland. This site 
is one of two shellfish production areas leased by Thimble Bay 
Farms Limited, a private shellfish aquaculture company specializ- 
ing in sea scallop and blue mussels. In October, 1989, 50,000 
approximately 1 -year-old sea scallop seed, originating from stocks 
in Port au Port Bay in western Newfoundland were purchased and 
transferred to the Charles Ann site. Scallop seed were stocked into 
standard 34-cm square Japanese pearl nets. The pearl nets were 
hung using a longline, suspended culture system in vertical arrays 
of 10 nets (Fig. 1). Each vertical array was repeated at 0.7-m 
intervals along a horizontal subsurface headline suspended at 3-m 
water depth and supported by surface floats. 

Two separate trials were carried out concurrently. In the first, 
scallop seed were stocked at five different starting densities: 50, 
75. 100. 150. and 200/pearl net. All nets were 6-mm mesh size. 
During the first year of culture in May, 1990, and again, in Sep- 
tember, 1990, some of these nets were selected for culling (thin- 
ning), while others were left unculled. The nets originally stocked 
at 50/net were culled to 25/net, while all others were culled to 
50/net. In the second trial, seed were graded into two sizes based 
on shell height, nominally referred to as small (< 18 mm) and large 
(> 18 mm). The graded seed were stocked in pearl nets of varying 
mesh size: 4.5, 6.0. and 9.0 mm. at a starting density of 50/net, 
with the exception of the small size grade which, because of their 
small size, could not be stocked into the 9-mm nets, because they 
readily fell through the mesh. All experimental trials were repli- 

Floats 




Bottom 
Contour 



Rock Anchors Pearl Nets 

Fisure I. Diajjranimatic cross-sectional representation of the longline 
seallop eiillure system used in the pilot trials at Charles Arm, New- 
I'oundiand. 



BioECONOMic Analysis of P. magellanicus in Newfoundland 



115 



cated such that each trial had a minimum of six pearl nets (some- 
times as many as 10) in each category. In May and September of 
both 1990 and 1991 (September. 1990 and 1991 only for the size 
grade-mesh size trial), all pearl nets were retrieved and cleaned by 
a pressure washer, all scallops were measured for shell height, 
counted, and all mortalities were removed. 

SAS statistical software (SAS Institute. Inc. 1985) was used for 
all statistical analyses of the biological data from the pilot trials. 
We used a nested, main effects analysis of covariance model 
(ANCOVA. SAS Institute Inc.. 1985) to determine the relation- 
ships among stocking density, culling, size at age (shell height). 
and survival on each sampling date. Initial starting size of seed- 
stock was the covariate to control for initial variation in shell 
height among pearl net groups. 

Economic Model Parameter Selection 

During the culture trials and including the post-trial harvest in 
September. 1991, records were kept of all capital and operational 
costs, as well as all labor incurred. These records, as well as the 
pilot trial growth and survival results, were used as input data to a 
spreadsheet-based Lotus'"''^' financial model (Table I ) and extrap- 
olated to commercial scale to simulate the startup and operational 
costs of commercial-scale farms and to forecast their economic 
viability using the standard financial evaluation methods of net 
present value (NPV) and internal rate of return (IRR) (Lusztig and 
Schwab 1977). All equipment, supply, and labor costs were 
sourced from commercial equipment suppliers as of March. 1999 
and are quoted in Canadian dollars. Estimates of useful life span of 
various equipment were based on practical experience of Thimble 
Bay Farms. The purchase price of scallop seed and the sale price 
of harvested product are the most recent values quoted for Thimble 
Bay Farms. Ltd. 

Selection of specific husbandry practices used in the models 
can have a major impact on the outcome of the model simulations. 
To ensure parameter values selected were as realistic as possible, 
we used the results from the stocking density-culling and net 
mesh-seed grade trials to select appropriate model input values for 
several key parameters. These included net mesh size, time to 
harvest for each seedstock cohort, the annual production cycle, 
stocking density, survival rate to harvest, and frequency of culling 
and handling for net cleaning. 

Although a larger mesh size is expected to yield a faster growth 
rate, particulariy in the second year of the production cycle, the 
6-mm mesh is the largest mesh size capable of accommodating the 
smallest of the purchased seedstock (10-15 mm) in year 1. Any 
economic advantage attributable to slightly faster growth in 9-mm 
mesh nets, as compared to the 6-mm mesh, is outweighed by cost 
considerations because of the need to stock nets of two or more 
mesh sizes, the utility of which will vary annually, depending on 
interannual variations in shell height of the seedstock supply. 
Therefore, use of the 6-mm mesh size was assumed in the simu- 
lation modeling exercise. 

The minimum time to harvest for each seedstock cohort was set 
at 15 months. Because the harvesting schedule must be year round, 
the annual production cycle from each annual seedstock cohort 
was set at January (year 2) to January (year 3) or, in other words, 
a 15-27 month production cycle. This production schedule was 
determined by analysis of the variability in the size at age data 
from the pilot trials. 

Assuming no seasonality in the harvesting schedule, we se- 



TABLE 1. 

Selection of key model parameter values used in the economic 
model simulations. 



Key Model Parameters 



Pearl net (square) specifications: 
Cage/mesh size 
Stocking density (% of stock 



' # per net) 



Net clean (# of times per year) 

Culling/thinning of stock 

Cost/life span (years) of capital equipment; 

Pearl nets (bulk order) 

Mainline. 365-m coil. 16-mm polypropylene 

Anchor and float lines. 365-m coil, 19-mm 
polypropylene 

Pearl net droplines. 365-m coil. 7-mm polypropylene 

Floats. 34 cm 

Floats, 200 L 

Work boat, 6.8-m aluminum 

Outboard motor, 40 hp 

Boat eqmt.. star wheel and hydraulics 

Vehicle, '/: ton pickup with cap 

Processing plant/work shed, 9.3 x 6.2 m 

Plant water pumps 
Hourly labor rate 
Owner/manager's annual salary 
Per unit fuel cost (liter, gasoline) 
Unit cost of autumn-delivered Spat ( 10-25 mm) 
Survival rate to harvest 
Time to reach harvest size 
Harvest schedule 
Product specifications; 

Market required product size (shell height) 

Ex-plant, per unit scallop sale price 
Business and startup fees (Year 1 ) 
Crop insurance (per million stock) 
NPV discount rate (prime + 2%) 



34 cm/6 mm 
50% @ 25 
25% @ 50 
25% @ 75 
1 
None 

Sl.80/10 
$98.60/8 

$233.10/8 
$29.00/8 
$10.50 
$35.00 
$9,400/15 
$3,695/5 
$4,000/5 
$23,800/5 
$19,300/20 
$2,000/5 
$8.50 
$18,000 
$0.60 
$0.04 
85% 
15-27 months 
Year Round 

55-65 mm 

$0.25 

$9,205-11.505" 

$4,000 

8.75% 



■' Varies with farm size. 

Individual equipment costs were obtained from commercial supply 
sources. All other values were obtained from analysis of the pilot trial data 
or from Thimble Bay Farms Ltd. records. 



lected the following stocking scenario for use in the model simu- 
lations: 50% of seedstock would be set at 25 scallops/net; 25% at 
50/net: 25% at 75/net. Based on the growth data from the pilot 
trials, this stocking scenario should ensure year-round availability 
of a 55-65 mm product for harvesting and minimize the likelihood 
of scallops exceeding the maximum product size specification be- 
fore being harvested. Although the pilot trials had no peari nets 
initially stocked at 25/net, we consider the data from the 50/25 
stock culled in May 1990 to represent a reasonable estimate of the 
probable growth performance of scallops initially stocked at 25/net 
for use in the model simulations. However, because scallops 
stocked at 25/net would exceed the maximum acceptable market 
size in less than 27 months, to ensure year-round availability of 
55-65 mm product some seed scallops must be stocked at higher 
densities. Analysis of the variability in the size at age data from the 
pilot trials indicated unculled peari nets initially stocked at 50 or 
75 scallops/net best matched the required market size during the 
15-27 month production cycle. 



116 



Penny and Mills 



In the pilot trials, all experimental groups with stocking of 
50/net or less achieved survival rates > 85%. Most were > 90%. 
Therefore, we considered a survival rate of 85% to be a reasonably 
conservative estimate of survival for the model simulations. 

Ideally, operational costs are minimized by selection of hus- 
bandry practices that allow individual scallops to be handled as 
little as possible during the production cycle. Because there are 
indications from the pattern of survival data, as well as from other 
concurrent farm operations, that excessive handling has a negative 
impact on survival, we chose no culling as the preferred production 
method for the simulations. This also lowered labor costs. For the 
model simulations, it also necessitated optimizing production 
solely by varying initial seed stocking density rather than by a 
combination of stocking density and culling. 

The discount rate for NPV calculations was the small business 
cost of borrowing, as of March. 1999, used by the Canadian bank- 
ing industry and is calculated as bank prime rate -i- 2%. A 50:50 
split between bank loans and owner equity for capital infrastruc- 
ture and equipment as well as an operating line of bank credit with 
a monthly repayment schedule of 3% of the outstanding balance is 
assumed. 

Economic Model Simulations 

We selected three hypothetical commercial farm sizes, based 
on annual seed stocking rates, for the model simulations: 500,000 
('/: M), I million (1 M), and 3 million (3 M) seedstock per year. 
The half million size model represents a farm size consistent with 
a part-time or family operation worked as an income supplement; 
whereas, the other two represent possible full-time commercial- 
scale farms consistent with the amount of leased acreage currently 
utilized by shellfish farms in Newfoundland. Model simulations 
assume a year-round market requirement for 55-65 mm SH. live, 
whole product that is fully processed in accordance with all ap- 
plicable Canadian seafood processing regulations in a farm-owned 
federally registered processing plant. 

The spreadsheet-based financial model (Lotus"") forecasted 
the potential economic viability of each of these three model farm 
sizes using the NPV and IRR values. We used a sensitivity analysis 
procedure to simulate the effect of variability in specific model 
input parameters on the model output. For the sensitivity analyses, 
we used an iterative procedure, changing the value of the most im- 
portant input variables (as a proportion of cash outflow) individu- 
ally by a pre-scl percentage until the NPV at year 10, NPV 10 =0. 

To assess the effect of vertical integration (e.g., culture farm 
plus a farm-owned processing plant) on over-all economic viabil- 
ity of scallop fanning enterprises, we recalculated the model simu- 
lations with the capital and operational costs of the processing 
plant deleted. This farm model requires assumption of sale of 
unprocessed scallops to an ex-farm seafood processor. We used an 
iterative process, adjusting the ex-farm price for harvested scallops 
in $0,005 intervals to determine the ex-farm price for unprocessed 
scallops needed to: ( 1 ) achieve minimal standards of economic 
viability (e.g., < NPVIO < $1000: 8.75% < IRR 10 < 9%); and (2) 
achieve economic viability projections for farms without the pro- 
cessing plant comparable to those for the same size farm with the 
processing plant included. 

RKSULTS 

Peiisily and Culling Trials 

At the outset of the culture trial, the mean shell height of all 
groups ranged from 19-21 mm (Fig. 2). Beginning with the first 



sampling in May. 1990, shell height was significantly related to 
stocking density (P < 0.0001). This relationship was maintained 
through all sampling periods. The pattern in least-square means 
(LSM) among the five initial stocking densities was also signifi- 
cant (f < 0.01 or greater) and consistent across all stocking den- 
sities (LSM^,, > LSM75 > LSM|„o > LSM,.,,, > LSM.oo)- 

Shell height was also significantly related to culling {P < 
0.0001 ). The LSMs of culled (thinned) scallop groups were con- 
sistently larger in shell height than their unculled counterparts {P 
< 0.0001) at the same initial stocking density (LSM^.„„,„,.,,,y,i > 
LSM^.^,i..,epc,„ > LSM„„,.„|,^.j). The interaction term of stocking den- 
sity X culling date was also significant (P < 0.0001 ) throughout the 
sampling period. By the end of the second year of the trial (Sep- 
tember 1991) an increase in shell height attributable to culling 
(Table 2) was noted at most initial stocking densities. In Fig. 2, the 
slope of the lines between adjacent sampling times indicate the 
mean growth rate during that interval. Growth rates were highest 
during the May to September period, 1990 (first summer season) 
and declined considerably thereafter. The highest mean growth 
rates, observed in the May 1990 cull group, ranged from 0.142 to 
0.176 mm day"', depending upon stocking density, during this 
time. 

The effects of initial stocking density and culling on survival 
were less consistent (Fig. 3). Survival was significantly related to 
both initial stocking density and culling date (P < 0.0001 ). How- 
ever, the pattern in LSMs was inconsistent among initial stocking 



densities (LSM^,, = LSM, 



= LSM,,u > LSM75 > LSM,„„). 



LSM patterns with respect to culling date was also inconsistent 
(LSM,„,|.,,,,c„ > LSM,,.„.,,p«„ = LSM„„,„„,,,) although the May 
1990 cull group were consistently larger than the other two groups. 
All except the unculled 200/net group had mean survival rates > 
80% at the end of the pilot trials. Most exceeded 85%. Overall, the 
change in survival attributable to culling was much less pro- 
nounced than that for shell height among the experimental groups 
(Table 2). 

Maximum growth was achieved in the 50/25 cull groups. In 
these groups, more than 90% of all scallops were greater than the 
minimum acceptable market size by May of Year 2 in the produc- 
tion cycle. Back-calculation of the size at age data from May in 
Year 2 ( 1991 ) based on the mean monthly growth rate during the 
September, 1990 to May, 1991 period projected that 90% of the 
50/25 stock culled in May 1990 were probably in excess of the 55 
mm minimum market size in January of Year 2 (1991). Thus, the 
minimum time to first harvest is approximately 15 months. 

Seed Gradin/i and Mesh Size Trials 

We used a similar anahtical approach (o determine the rela- 
tionships among seed grading and net mesh size with size at age 
(shell height) and survival. When graded, the mean shell height of 
scallop seedstock in the two nominal size grade categories were 
15.3 mm (small grade) and 22.5 mm (large grade). At the end of 
Year 2 of the pilot trial in September, 1991, size at age was 
significantly related to both mesh size {P < 0.0001 ) and initial size 
grade (/-" < 0.03l. The interaction term was not significant {P > 
0.05 ). All groups exceeded 45 mm shell height by the end of Year 
I and exceeded 60 mm shell height by the end of Year 2 (Fig. 4a). 
Increasing mesh size had a positive effect on mean size at age for 
both size grades. Howe\er, the mean shell heights of small size 
grade groups were sometimes larger at the end of 'tear 2 compared 
to large size grade seed in nets of the same mesh size (LSM,,, = 
LSM„s > LSM,,, > LSMj ,s = LSM^ „ ). 



BioECONOMic Analysis of P. magellanicvs in Newfoundland 



117 





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Figure 2. Mean size at age (shell height) over time of scallops in the stocking density-culling trials. The figure legends follow the convention 
"original stocking density / culled density, and date of culling" (e.g., 50/25 May90 means original stocking density = 50, culled density = 25, and 
May 1990 was the date of culling). Standard error bars (± 2 SE) are plotted but are obscured by the datapoints. 



118 



Penny and Mills 



TABLE 2. 

Percentage i%) change in mean shell height and mean survival at 

Year 2 attributable to culling in Spring (May) and Autumn 

(September) of Year 1, compared to the unculled stock of the same 

initial stocking density. Stocking density values are number of 

scallops net^'. 





Shell 


Height 


Surv 


ival 


Stocking Density 


May Cull 


Sept. Cull 


May Cull 


Sept. Cull 


50 


16.2 


14.2 


0.5 


-2.7 


75 


2.5 


2.6 


12.0 


5.3 


100 


20.2 


L<i.7 


-3.7 


-6.7 


150 


29.4 


22.7 


0.1 


4.4 


200 


42.5 


33.9 


24.4 


5.7 



Survival through Year 2 was not significantly related to net 
mesh size (P > 0.05) but was weakly related to initial size grade 
(P < 0.04). The small size grade experienced a relatively lower 
survival during the trials (Figure 4b) than did the large grade 



(LSM^L = LSM4 , 



LSMft, > LSM, 



LSMj ,s). Survival 



through Year 2 in all groups exceeded 83%, while mean survival 
of the large grade exceeded 91%. In September, 1991, the ob- 
served mean size at age and mean survival of scallops in the mesh 
size-seed grade trials were comparable to those observed in the 
stocking density-culling trials. 

Effect of Farm Size 

The key parameter values used as input to the model simula- 
tions are given in Table 1. Net cashflow projections for all three 
farm sizes followed similar patterns of an initial cash investment in 
business startup (year 0), a further negative net cash outflow in the 
first year of operation, followed by a series of positive net cash 
inflows in subsequent years, the magnitude of which increased 
with increasing farm size (Fig. 5). Net present value (NPV) and 
internal rate of return (IRR) values derived from the model simu- 
lations indicate both the 1 M and 3 M farms are projected to be 
economically viable using a 10-year forecast horizon at present 
commercial bank interest rates and market prices (Figure 6). The 
smaller ('/: M) farm size is not considered economically viable. 
The model simulations predict a trend of increasing NPV and IRR 
values with increasing farm size indicating the influence of 
"economy of scale" in farm operations. 

Annual labor and .seedstock acquisition costs represent > 50% 
of the total cash outflow for all farm sizes (Fig. 6). Acquisition of 
capital equipment and infrastructure is a relatively smaller propor- 
tion of cash outflow when annualized over the 10-year model 
simulation cycle. However, much of the cost for capital equipment 
and farm infrastructure are concentrated in Year I (processing 
plant, work boat, culture equipment, etc.). Labor and debt servic- 
ing costs as proportions of total cash outflow over a 10-year cycle 
do not vary with farm size. However, acquisition of capital equip- 
ment and operational costs both decline proportionally with in- 
creasing farm size; whereas, purchase of annual seedstock propor- 
tionally increases over a 10- year period. The payback period, 
defined as the time to recoup the initial investment assuming op- 
erating profits arc retained within the business, is estimated al 4.2 
and 3.4 years for the 1 M and 3 M farms, respectively. 

Sensitivity Analyses 

To determine how robust our viability projections from the 
model simulations were, we recalculated the sinuilalions for the 



two model farm sizes deemed economically viable with the base 
input assumptions (1 M and 3 M farms). We used an iterative 
procedure, changing the value of each of the most important input 
variables (as a proportion of cash outflow) individually by a preset 
percentage until the NPV 10 = 0. Projections of economic viability 
for both the 1 M and 3 M farm models are relatively insensitive to 
changing value assumptions for most major input variables, in- 
cluding capital, operational costs, and mortality (Fig 7). However, 
both models are relatively sensitive to changes in sale price. Re- 
duction in sale price obtained for harvested product in the order of 
20% and 28%, for the 1 and 3 M farms, respectively, reduced the 
NPV to zero. This is equivalent to a minimum sale price of $0.20 
and $0.18/scallop, respectively. 

Effect of Farm-Owned Processing Capacity 

Without the processing plant, the '/: M farm is still not con- 
sidered to be viable economically (NPV 10 < 0; IRR 10 < 8.75%) 
under assumptions of current scallop sale prices. Farms of this size 
only become marginally economically viable (e.g., < NPVIO < 
$1000; 8.75% < IRRIO < 9%) if the ex-farm sale price for un- 
processed scallops exceeds $0.26/scallop. a price that exceeds the 
current sale price for processed scallops. For the 1 M and 3 M 
farms without processing plants, economic viability becomes mar- 
ginal as the ex-farm sale price for unprocessed scallops approach 
$0,185 and $0.165/scallop, respectively. To achieve economic vi- 
ability projections comparable to those for farms with processing 
plants (equivalent NPV or IRR), the sale price for ex-farm unproc- 
essed scallops must exceed $0.235/scallop for both the I M and 3 
M farms, a difference of only S0.015/scallop for unprocessed ver- 
sus processed scallops at current prices. Obtaining such a small 
price differential (approximately 6%) for sale of unprocessed scal- 
lops to an ex-farm processor may not be realistic, because it would 
seem to allow a rather small profit margin for the processor. With 
this considered, scallop fanning enterprises with owner-operated 
processing capacity are likely more economically attractive than 
farms without owner-operated plants. 

DISCUSSION 

In commercial production systems, growth and survival are the 
two major biological rates of importance to cultured seafood grow- 
ers. For bivalve mollusks, many factors influence these two vari- 
ables. Some are environmental, such as food availability and water 
temperature, and others are physiological related to age, size, and 
reproductive maturity of the animals themselves (see Shumway 
1991 for review). For suspended culture systems, additional stock 
husbandry factors must also be included, such as gear depth, type 
of gear and mesh size, current velocity, stocking density, and 
extent of biofouling (Claereboudt et al. 1994a, Claerboudt et al. 
1994 b. Cote et al. 1993, Parsons and Dadswell 1992, Parsons and 
Dadswell 1994, Shellfresh Farms Ltd. 1993). 

In this paper, we have examined the effect on scallop growth 
and survival of the major variables that can be readily manipulated 
by scallop farmers, assuming use of a basic pearl net culture sys- 
tem styled after the equivalent Japanese industry for the Japanese 
scallop. Palinopccten yessoensis. These are selection of stocking 
density, gear mesh size, culling (thinning) practices, and seed grad- 
ing. In their review of sea scallop culture in Atlantic Canada, 
Couturier et al. (1995) considered stocking density the single most 
important factor affecting cultured sea scallop growth rates. Al- 
Ihough it is tlifficult lo compare growth rates, size at age. or sur- 



BioECONOMic Analysis of P. magellanicus in Newfoundland 



119 



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90 

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Figure 3. Mean survival over time of scallops in the stocking density-culling trials. The figure legends follow the convention "original stocking 
density / culled density and date of culling" (e.g.. 50/25 May90 means original stocking density = 50, culled density = 25, and May 1990 was the 
date of culling). Standard error bars are ± 2 SE. 



120 



Penny and Mills 



(a) --i'^ 
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Figure 4. Mean size at age (shell height) and mean survival of scallops 
in the spat grading-net mesh size trials. The figure legends follow the 
convention "net mesh size in millimeters / nominal spat size grade at 
time of stocking" (e.g.. 4.5 / small means mesh size 4.5 mm and stocked 
spat were small grade). Nominal spat size grades were < 18 mm (small) 
and > 18 mm (large). Standard error bars are ± 2 SE but are obscured 
by the datapoints in (a). 

vival data among different studies, gear, and locations, the growth 
and survival rates observed in this study seem consistent with 
those reported for sea scallops in suspension culture grown else- 
where in Atlantic Canada (Cote et al. 1993, Dadswell and Parsons 
1991. Parsons and Dadswell 1992. Parsons and Dadswell 1994. 
Wildishelal. 1988). 

In our study, size at age and survisal varied with stocking 
density, gear mesh si/e. culling practices, and seed grading. Both 
size at age and survival tended to decrease with increasing stock- 
ing density, a result consistent with previous studies on several 
scallop species including the .lapanese scallop. PuUno\H'cU'n yes- 
soensis (Yamamoto 1978. Ventilla 1982). the bay scallop. Ar- 
gopi'cten inadiaits (Duggaii 1973, Rhodes and Widman 1984), 
and the sea scallop. I'Uuopeili'u imifU'lUmkus (Cole et al. 1993). 
However. Penney ( 1 99.S ). in a study ol large scallops > 75mm shell 
height, did not find a significant relationship between slocking 
density and survival. Parsons and Dadswell (1992) also found 
survival in sea scallops from New Brunswick to be unrelated lo 
stocking density. In the present work, nonrandom handling moi- 





Figure 5. Annual net forecasted future cashflows over a 10-year pe- 
riod from initial startup for three sizes of sea scallop farms with farm- 
owned processing plant included: ": million seed yr"', 1 million seed 
yr"', and i million seed yr '. Year I) is the initial capital investment 
before startup. 

talily, an artifact of our "hatch processing" style sampling proce- 
dure, was likely implicated in the inconsistent survival patterns 
among the different density groups observed in the pilot trials. This 
might explain the anomalous high mortality among the 75/net 
groups. Similar instances of nonrandom apparent handling mor- 
tality among adjacent groups were noted periodically during the 
farm's other commercial operations. We, therefore, consider ex- 
cessive or improper handling of gear lo be a more imporlanl factor 
influencing survival than stocking density, a finding that would be 
consistent with that of other studies (Parsons and Dadswell 1992, 
Ventilla 1982, Wildish et al. 1988). 

In this work, size at age in pearl net culture was improved by 
early culling and by increasing the initial net mesh size. Survival 
was also improved by early culling but was unrelated to net mesh 
size. Larger sized seed did not maintain their size at age advantage 
over small grade seed after 2 years of grow-out. This suggests shell 
height variation in I -year-old seed scallops from wild sources is 



BioEcoNOMic Analysis of P. magellanicus in Newfoundland 
500,000 Seed / Year ^ 



121 



IRR5 = -9.9% 
NPV5 = -$78,000 



IRR10 = 8.5% 
NPV10 = -$1,800 



Operations 
19% 




Capital Equipment 
18% 



1M Farm 



—Sale Price 

- - Labor 

■ ■ Seedstock 

- Other O & M 

- — Mortality 
—Cost of Peari Nets 

-•Total Capital Cost 




100 ISO 

NPV ( % OOO's) 



1 Million Seed / Year 



IRR5= 10.2% 


IRR10 = 25.8% 


NPVS = $9,500 


NPV10 = $237,000 


Operations 




18% ^„g«^^S 


rr*?**,,^ Seed 


/^fl 


::-:-x:x^xN. 2®* 


D btS / **iilll^H 


"M^^^M^y^ 



11% 



Capital Equipment 
16% 



3 Million Seed / Year 



IRRS = 26.2% 
NPV5 = $308,800 



IRR10 = 39.4% 
NPV10 = $1.12 Million 



Operations 

15% 




Capital Equipment 
15% 



Figure 6. Mean annual cash outflows by category over a 10-year cycle, 
with NPV and IRR values for model simulations of three sizes of farm 
operations. (NPVS = NPV calculated over 5 years, etc.). NPV and IRR 
calculations were based on cashflows from Figure 5. 




200 «0 600 800 1000 1200 
NPV ( S OOO's) 

Figure 7. Sensitivity analysis of the effect of changing value assump- 
tions of the major model input variables (reduction in sale price; in- 
crease for all cost variables and mortality) on projected NPVIO values 
(NPV calculated over 10 years) for the I M and 3 M farm models. 
Percentage change in input variables at NPV = indicate the propor- 
tional change from the base values for each variable required to re- 
duce NPVIO to zero. 

likely the result of variation in environmental factors or seed col- 
lection husbandry practices rather than within population genetic 
variation. However, seed grading before initial stocking may have 
commercial value as a means to reduce size at age variation within 
individual pearl nets at harvest time. 

Selection of production practices for the model simulations was 
guided by two general considerations; ( 1) the intended product was 
a 55 to 5 mm, whole, live scallop; and (b) harvesting and sale of 
product was required year-round with no seasonality in production 
volume. Ensuring year-round availability of a 55 to 65 mm product 
requires adjustment of farm production practices, including selec- 
tion of stocking densities, deciding whether or not to cull and 
when, and selecting a harvesting schedule to minimize labor costs, 
while still meeting market demands. Variable stocking densities 
are required to ensure correctly sized stock are available for har- 
vest throughout the year. Economic viability is optimized by early 
generation of sales revenue, achieved through timely harvesting. 



122 



Penny and Mills 



However, harvesting should not be so early that within-net shell 
height variation is such that a significant proportion of scallops in 
harvested nets are under the minimum acceptable market size. This 
situation would necessitate the return of large numbers of under- 
sized scallops to pearl nets for further on-growing, resulting in 
added labor cost. 

It would seem unlikely that further minor changes to the basic 
pearl net culture system or husbandry practices are capable of 
affecting significant improvement in labor costs and, hence, the 
outcome of the model simulations. The sensitivity analyses indi- 
cate the viability of model farms is relatively insensitive to 
changes in capital costs, labor, or other operational costs. Further 
significant reductions in labor costs are likely to be achieved only 
by substitution to another culture gear entirely or by use of in- 
creased mechanization during farm operations. Parsons and Dad- 
swell (1994) suggested that, although the initial capital cost of 
pearl nets was much lower compared to lantern nets, when han- 
dling times and their associated labor costs were factored in, lan- 
tern nets and Shibetsu nets gave the lowest over-all cost of pro- 
duction. However, this suggestion must be viewed with some cau- 
tion. In a previously reported scallop rearing trial in Newfoundland 
using scallops > 75 mm shell height, scallops raised in pearl nets 
were larger than those raised in lantern nets at comparable stocking 
densities (Penney 1995). In the same study, survival was unaf- 
fected by gear type. 

The reason for the better growth in pearl nets is unknown, but 
it may be attributable to differences in water flow (and, hence, 
food availability) around and within the two net types. All sus- 
pended net culture systems impede water flow, which, in turn, 
negatively affects production, a condition that is exacerbated by 
increased stocking density, reduced net mesh size, and biofouling 
(Claereboudt et al. 1994b. Devaraj and Parsons. 1997. Parsons and 
Dadswell 1994). Pearl nets are estimated to reduce water flow by 
46-61'^ (Claereboudt et al. 1994b), but no comparative measure- 
ments are available for lantern nets. The better growth of scallops 
in pearl nets compared to lantern nets (Penney 1995) support se- 
lection of the basic pearl net system as the more appropriate net 
type compared to lantern nets. In addition, some growers find large 
lantern nets clumsy to handle from small boats similar to those 
used in our simulations. 

Projections of economic viability derived from the model simu- 
lations indicate commercial sea scallop farms marketing a whole, 
55-65 mm product can be profitable enterprises in Atlantic 
Canada. Our favorable projections are in sharp contrast to earlier 
economic analyses for culture systems based on production of 
adductor meats alone for sale into traditional North American scal- 
lop markets (Frishman et al. 1980. Gilbert 1987, Wildish el al. 
1988. Gilbert and LeBlanc 1991 ). 

These conflicting economic projections for in-shell versus meat 
production are likely because of a combination of factors. Rev- 
enues from in-shell product sales begin about 15 months after 
stocking in our model simulations. In contrast, production of meats 
in the .■^0-40 count range (North American scallop markets quote 
in number of pieces to make one pound weight) would require an 
extra 15 to 20 months of culture (Penney 1995; Penney and Mc- 
Kcn/ie. 1996) and would likely generate less ex-farm revenue per 
scallop at current North American market prices (L'rner Barry 
1999). This protracted culture time woukl also increase capital 
costs, because extra pearl nets and other gear are required for each 
annual seed cohort, as well as increase labor costs for stock thin- 
ning, gear deployment, and in-plant moat shucking. 



In Newfoundland, only two companies are currently in com- 
mercial production although this will likely increase quickly, be- 
cause a total of nine companies and 1 1 culture sites in various 
stages of development totaling nearly 400 leased hectares are now 
in operation (G. Deveau. Nfld. Department of Fisheries and Aqua- 
culture, pers. comm.). Recent annual scallop production by the two 
farms currently selling cultured scallops has varied since 1994 
from 10-19 mt, the majority of it marketed in whole form. Esti- 
mated annual production from a single 1 M farm would be about 
17 mt. This is approximately 10-20'^ of all in-shell scallop prod- 
ucts currently being sold in Canada from Canadian sources. 
Clearly, considerable developmental marketing initiatives would 
be required by industry to expand significantly North American 
market share for in-shell scallop products sufficient to absorb the 
production of a new Atlantic industry composed of several such 
farms. Alternatively, the potential for increased development of 
other international export markets into such countries as France, 
with an existing tradition of acceptance of alternative scallop prod- 
ucts (de Franssu 1990) should be determined. 

A trend of increasing NPV and IRR values with increasing 
farm size indicates significant economies of scale accrue to larger 
farms. Despite farm size, annual labor and seedstock acquisition 
costs are the largest factors in over-all annual cash outflows. The 
cost of labor has been previously recognized as an important com- 
ponent of over-all production costs for scallop culture (Atlantecon 
1992. Parsons and Dadswell 1994). In our model simulations, 
labor is reduced through elimination of the need for culling during 
grow-out by selecting appropriate initial stocking densities. This 
tends to improve the over-all survival rate as well. Larger sized 
farms ( 1 M and 3 M models) are projected to be more profitable 
than smaller operations ('/2 M model). 

In Atlantic Canada, many shellfish culture farms, particularly 
in mussel and oyster culture, have been started as "cottage-style" 
ventures operated on a part-time basis as a source of supplemen- 
tary family income by persons employed in other industries. If 
started by families already employed in the fishing industry, eco- 
nomic viability forecasts using NPV or IRR calculations typically 
remove from consideration certain capital costs (e.g., cost of boat, 
motor, truck, ropes, etc.) that are shared with the fishing enterprise 
(e.g.. Ridler 1995). These capital costs are considered to have been 
already compensated by the fishing enterprise. The ', : M model is 
sufficiently small in scale to be considered this way. If calculated 
using these assumptions, the V2 M model is forecasted to be eco- 
nomically viable (NPV = $34,000: IRR = 16.59^). 

Of particular interest for industry development purposes, is the 
effect the owner-operated processing plant has on projected eco- 
nomic viability. Vertical integration and increased farm size are 
known to have a positive effect of the viability of other shellfish 
aquaculture operations (Adams and Pomeroy 1992, Lambregts et 
al. 1993). For scallop farming enterprises, irrespectixe of farm 
size. NPV- and IRR-bused projections of economic viability 
changed only slightly with elimination of the owner-operated pro- 
cessing plant as part of the over-all enterprise. This is attributable 
to two factors. First, the capital investment in processing capacity 
is quite small for processing whole scallops (see Table 1 1. Because 
product processing of whole scallops consists of a fairly simple 
process of washing and cleaning shells, sorting empty shells, and 
packaging, a relatively small building with minimal equipment is 
needed. Second, the labor costs for such a simple processing op- 
eration are also relatively minor. Processing in-shell scallops 



BiOECONOMic Analysis of P. magellanicus in Newfoundland 



123 



eliminates the need for shucking, which is the most labor-intensive 
component of in-plant scallop "meat" processing. 

For either the 1 M or 3 M farms, comparable NPV and IRR 
values were projected for enterprises with and without processing 
capacity at a sale price difference of only $0,015 per scallop for 
ex-farm processed scallops versus ex-farm unprocessed scallops. It 
seems unlikely that ex-farm processing companies would pay to 
the farmer such a small price differential for unprocessed scallops, 
because this leaves them a very tight margin for their own capital 
and operational processing costs and potential profit. It is far more 
likely that processing companies would pay farmers a lower price 
for unprocessed scallops, which would have a negative impact on 
the economic viability projections for farming enterprises. Thus, 
incorporation of an owner-operated processing plant as part of the 
business venture would be likely to enhance business viability. 
Ex-farm prices for unprocessed scallops in the range of $0.16- 
$0.18/scallop would make scallop farming not economically viable 
under current conditions regardless of farm size. Nevertheless, 
product processing with subsequent direct sale of product to sea- 
food buyers and brokers represents a level of business manage- 
ment and marketing activity that some prospective farmers may 
not choose to pursue. This may be especially true for the Vz M farm 
size model that may be a part-time or family operation. 

Favorable NPV and IRR projections are not the only criteria 
upon which to evaluate the potential for success of any new busi- 
ness venture. It should be recognized that many other factors can 
and do influence individual business investment decisions that are 
not considered in NPV or IRR calculations. Other factors, such as 
timing of large cash outflows versus revenues, debt repayment 
schedules, other financing arrangements, personal, biological, le- 
gal, and regulatory considerations, all may vary on an individual 
business and location basis and may also affect the success of any 
business venture (Lusztig and Schwab 1977). 

Both the NPV and IRR financial forecasting methods used in 
our analyses are based on the estimation of future cash flows 
generated by an initial capital investment and are commonly used 
as decision-making tools by financial analysts to guide investment 
in new businesses. Both give explicit consideration of the time 
value of money, incorporated through the discounting of cash 
flows, which is often related back to the cost of credit (borrowing) 
from banks. Accurate forecasting of future cash flows, the basis of 
successful NPV and IRR applications, is often a challenge (Lusztig 
and Schwab 1977) and cannot anticipate aperiodic potentially cata- 
strophic events, such as disease outbreak, major loss of gear be- 
cause of ice damage, etc. Despite these indi\ idual situational con- 
siderations, favorable general NPV and IRR values such as we 
have forecaste from our model simulations indicate the underlying 
potential profitability of sea scallop farming in Atlantic Canada. 
Our positive NPV and IRR projections for in-shell scallop farms 
indicate continued industry and/or government investment to en- 
courage commercial expansion in this sector is warranted. 

Sensitivity analyses indicate the forecasted profitability is fairiy 
robust with respect to anticipated variability in capital and oper- 
ating costs, and stock mortality, but is relatively sensitive to fluc- 
tuations in sale price for harvested product. This last point must be 
closely considered in the start-up of any commercial business ven- 
ture of the scale outlined by the model simulations. Greatly in- 
creased product availability in the marketplace may exert down- 
ward pressure on prices, particularly in the presence of inadequate 
marketing efforts. Given the in-shell nature of the product, prices 
may not be affected by trends in market prices for traditional 



scallop meats, a critical point considering the continuing increase 
in Chilean and Chinese cultured meat production as well as past 
fluctuations in both price and supply of meats from the North 
American fishery (de Franssu 1990). In existing North American 
markets, an in-shell sea scallop product would be more likely to 
compete (and, hence, to be affected by price fluctuations) with 
soft-shelled clam {Mya arenaria). steamers, littleneck. and cher- 
rystone clams {Meirenaria mercenaria). and oysters (Cnissostrea 
virginica). 

We consider further development of a sea scallop farming in- 
dustry in Atlantic Canada to be constrained by four factors; (1) 
availability of a reliable large-volume annual seedstock supply at 
commercially acceptable prices; (2) market development neces- 
sary to substantially increase the current, small volume niche- 
market status of North American markets for whole, 55-65 mm 
products; (3) the reported short shelf life of live scallops (de 
Franssu 1990) and; (4) the long-term frequency and severity of 
shellfish site closures because of outbreaks of biological toxins. 
The first two are inextricably linked. Resolution of the seed supply 
problem that has plagued industry expansion for years (Couturier 
et al. 1995), possibly by increased hatchery production of seed- 
stock, will greatly increase the volume and interannual stability of 
available harvested product and, thus, encourage greater market 
penetration of the 55-65 mm whole product. Large-scale markets 
for whole scallop products will only be developed when produc- 
tion volume is sufficient to warrant the required financial invest- 
ment for promotional market development. However, live scallops 
are reported to have a relatively short ex-farm shelf life as com- 
pared to other molluscan shellfish such as clams, oysters, or mus- 
sels (de Franssu 1990). Increasing market volume for whole scal- 
lop products may require a shift from sale of live product to a 
frozen in-shell or other secondarily processed form. This would 
have a negative impact on our projections of farm enterprise eco- 
nomic viability unless accompanied by commensurate farm-gate 
price increases. 

Ultimately, the limitation to increased production of whole 
scallop products in Atlantic Canada most difficult to mitigate may 
be that caused by the distribution, frequency, and prevalence of 
biological toxin outbreaks. Scallop species are well known for 
their propensity to sequester biological toxins in their mantle, roe, 
and hepatopancreas tissue at relatively high levels as compared to 
other bivalve species (Shumway and Cembella 1993; Douglas et 
al. 1997). Detoxification of affected scallops may be quite slow, 
exceeding several months in duration and be quite variable among 
individuals (Shumway and Cembella 1993). Frequent and severe 
toxin outbreaks may limit expansion of scallop farms to areas 
where toxin outbreaks are relatively infrequent and of short dura- 
tion. Although this has been the case in Newfoundland, thus far, 
continued industry expansion, particularly elsev, here in the Atlan- 
tic Canadian provinces may be seriously impeded by toxic event 
considerations. 

ACKNOWLEDGMENTS 

We thank members of the Mills and Jewer families of Bot- 
wood, Newfoundland who willingly helped pick, sort, and other- 
wise handle scallops during the 2- year pilot trials as well as staff 
of Thimble Bay Farms, Ltd. for their patience and help with many 
of the labor costing activities. We especially thank Frank Corbett, 
DFO Economics Branch, St. John's for much advice and assis- 



124 



Penny and Mills 



tance to set up and i^n the economic analyses. K. S. Naidu and J. 
Davis. DFO St. John's provided many useful comments on an 
earlier draft. Funding assistance for the pilot trials was provided by 



the Department of Fisheries and Oceans. St. John's. Newfoundland 
and by the Canada / Newfoundland Inshore Fisheries Development 
Aareement. 



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Journal of Shellfish Resaiirh. Vol. 19, No. 1, 125-128. 2000. 

THE EFFECT OF CYTOCHALASIN B DOSAGE ON THE SURVIVAL AND PLOIDY OF 
CRASSOSTREA VIRGINICA (GMELIN) LARVAE 



JOHN E. SUPAN,' CHARLES E. WILSON^ AND 
STANDISH K. ALLEN, JR' 

^Office of Sea Grant Development 
Louisiana State University 
Baton Rouge, Louisiana 70803 
'Coastal Fisheries Institute 
Louisiana State University 
Baton Rouge, LA 70803 
Virginia Institute of Marine Science 
College of William & Man- 
Gloucester Point, VA 23062 

ABSTRACT Survival and ploidy of D-stage oyster larvae (Crassosrrea virginica) were determined following the rearing of embryos 
exposed to CB dosages of 0.5 mg/L. 0.25 mg/L. and 0.125 mg/L for 10 minutes, with O.OS'/r DMSO and ambient seawater as controls. 
The experiment was replicated three times on the same day with the same procedures and partially stripping the same male oysters; 
different females were used for each replicate. CB dosage treatments began when 509c of the eggs reached PBI (24-3 1 min). Embryos 
were reared for 48 h at ambient temperature and salinity. Resulting triploid percentages were 13% ± 6.7% (0.125 mgCB/L), 61.8% ± 
6.2% (0.25 mgCB/L). and 68.2% ± 14.1% (0.5 mgCB/L). No significant difference {P s 0.05) in mean survival was found between 
the three CB treatments. Significant differences in mean survival between the three replicates implies variability because of different 
sources of eggs. 

KEY WORDS: Crassoslrea virginica. oyster, triploid. cytochalasin B. dosage 



INTRODUCTION 

Cytochalasin B (CB), a cytokinetic inhibitor, was first used to 
produce triploid Crassostrea virginica and Crassoslrea gigas 
(Thunberg) over a decade ago (Allen 1986. Stanley et al. 1981). 
Optimal treatments; that is. those yielding high proportions of 
triploids, have been reported for C. gigas. based on temperature, 
dosage, time of application, and duration; namely, 0.5 mgCB/lmL 
dimethyl sulfoxide (DMSO)/L of seawater for 20 min at 25 °C, 
when 507f of the eggs were at meiosis I (Allen et al. 1989, Down- 
ing and Allen 1987). Because C. virginica is less fecund than C. 
gigas (Galtsoff 1964), there is more concern for egg survival. 
Lower dosages and treatment times of 0.5 mgCB/L for 15 min at 
25 "C (Shatkin and Allen 1990) and 0.25 mgCB/L for 10 to 15 
min, at 27 to 29 °C (Barber et al. 1992) were suggested to increase 
the survival of embryos while maintaining high yields of triploids. 

We tested the feasibility of triploid C. virginica production in 
Louisiana, based on the premise that higher summertime meat 
yields resulting from triploidy could be profitable for the oyster 
industry. Triploid induction, using 0.5 mg/L CB. was variable with 
commercial size broods (a 4 million eyed larvae). During the first 
summer of commercial-scale production, survival of CB-treated 
embryos was < 5% compared to s 21% for diploid controls using 
stripped gametes. Differences between the salinity at our hatchery 
and salinities at sites where broodstock were obtained affected 
development time, in particular meiotic synchrony, and have been 
identified as major causes of this variation (Supan 1995). 

The objective of this study was to investigate the effect of CB 

dosage (H,|:p.|,5„„CB ~ M-0.25mgCB ~ M-0.12SmgCB ~ M-OmgCB- 

H^:=^) on survival and triploidy induction in C. virginica, and to 
determine what component of the variability was attributable to 
females, held in identical environments. 



METHODS AND MATERIALS 

Survival and ploidy of oyster larvae were estimated after ex- 
posing embryos to CB dosages of 0.5 mg/L. 0.25 mg/L, and 0.125 
mg/L for 10 min. with 0.05% DMSO and ambient seawater as 
controls. The experiment was replicated three times on the same 
day with the same procedures by partially stripping the same male 
oysters; different females were used. 

Preparation of Gametes 

Gametes were obtained for each replicate in a fashion similar to 
the method described by Allen and Bushek (1992). Oysters were 
collected from nearshore containers, opened, and their gender was 
determined microscopically using gonadal smears. Ripeness was 
visually recognized by the presence of prominent genital canals. 
Female and male oysters were placed in separate areas to avoid 
cross contamination. 

Eggs were obtained from three ripe females, randomly chosen 
for each replicate. Females were individually dry-stripped (i.e.. 
without using seawater) to ensure equivalent periods of hydration 
(defined as the length of time eggs are exposed to seawater after 
stripping) and simultaneous fertilization. The resulting eggs were 
pooled and washed of gonadal debris with filtered ( 1 (jim) ambient 
(24%c) seawater (FAS) by passing them through a 75 |xm Nytex 
screen onto a 15 |xm screen. They were then resuspended for 
enumeration and brought to a volume of approximately 8 million 
in 1 L FAS. The eggs were allowed to hydrate for 60 min at 28 °C 
before fertilization and treatment. 

Three male oysters were partially stripped for each replicate by 
scraping away only a portion of the gonad into a beaker and then 
covering the oyster with plastic wrap to prevent desiccation. Sperm 
from the three inales was pooled in a beaker after being washed of 
gonadal debris by passage through a 15 |j.m screen. 



126 



SUPAN ET AL. 



100 



80 



i60 



40 



20 








I 



Replicate 1 



Replicate 2 
Replicate 3 



>?J-'-'-'?'-'::H 



o.'s 



0.1'25 025 

CBTreatment (mg/L) 



mn 



control DMSO 



Figure 1. Percentage triploidy in C. virginica D-stage oyster larvae after treatment witii cytochalasin B, by replicate. 



Fertilization and Treatment 

Pooled eggs were fertilized with approximately 10 sperm/egg 
and stirred regularly. After fertilization of the 8 million eggs, they 
were divided into five treatment beakers each containing 800 niL 
of FAS, bringing the eggs per treatment to about 1 .5 M eggs/L. 
Eggs from individual beakers were examined microscopically for 
polar body formation at appropriate intervals. Treatments began 
when approximately SO'/r of the eggs reached PBI (24 to 31 min 
among replicates). 

Treatments consisted of adding the appropriate aliquot of 1 mg 
CB/1 ml DMSO to the beakers of developing eggs to obtain dos- 
ages of 0.5 mg/L. 0.25 mg/L. and 0.125 mg/L. Our control con- 
sisted of 0.05'/f DMSO (v/v) dissolved in FAS and FAS alone 
served as a normal. Treatments lasted for 10 min. Afterward, each 
CB-treatment group of embryos was rinsed of CB with FAS over 
a 15 \xm screen then placed in separate beakers containing 0.05% 

TABLE 1. 

Results of analysis of variance (.\N()VAl: Kffect of cytochalasin B 

treatment and experimental replication on the percentage Iriploidy 

of C. virginica oyster larvae. 



Sources of 
Variation 



DF 



F-ratio 



I'rob > F 



Treatment 
Replicate (error) 



4y.7() 



O.OOOI 

()..s2yi 



DMSO-FAS solution for 15 min. The embryos from each beaker 
were then rinsed of the DMSO solution and put into separate, 
labeled culture vessels containing 15 L of FAS for a final culture 
density of 15 embryos/L. Culture vessels were aerated and equal 
volumes Isochrysis aff. galbana clone CISO added. Embryos were 
incubated for 48 h at ambient temperature and salinity until they 
reached D-stage. All counts were obtained using triplicate 1 mL 
subsamples from each culture vessel. At 48 h, each vessel was 
individually drained onto a 40 jjim screen, and subsamples were 
placed into 1.5 mL centrifuge tubes and shipped overnight to Rut- 
gers University's Haskin Shellfish Research Laboratory for ploidy 
determination using fiow cytometry. 



TABLE 1. 

Results of ANOVA: Post lioc comparisons of mean percentage 
triploidy of C. virginica lar>ae b) treatment. 



Triploidy* 



Treatment 


Mean 


SD 


Comparisons** 


0.1 2.S mgCB 


0.3594 


0.110 


A 


0,25 mg CB 


0.90.52 


0.063 


B 


0.5 mg CB 


0.97S.^ 


0.1.56 


B 


Ccnirol w/i) DMSO 


0.1862 


0.009 


A 


Control w/DMSO 


0.0949 


0.0S4 


A 



R- = 0.9616. 



'Triploidy = arcsin (\(';'rTripk)iiil (0.01)). 
■ * Tukoy's honestly signitlcanl difference (^ 
SD = Standard deviation. 



0.05). 



CB Dosage Effects on Larval Ploidy and Survival 



127 



Data Analyses 

Differences among treatment means for survival and percent- 
age triploidy were detennined using analysis of variance (SAS 
1991). Percentage triploidy was determined as a proportion of 
triploid cells among the total number analyzed by the curve-Fitting 
program ModFit (Verity Software House. Topsham. ME) (Allen 
and Bushek 1992). Survival and percentage triploidy met the as- 
sumptions of normality and variance homogeneity after angular 
transformation (Dowdy and Wearden 1991). The models used sur- 
vival and percentage triploidy as separate dependent variables and 
treatments and experimental replicates as independent variables. 
Tukey"s Honestly Significant Difference Procedure was u,sed to 
test the difference among the treatments and replicates (a = 0.05). 

RESULTS 

Percentage triploidy and survival were not different between 
0.5 mg/L and 0.25 mg/L CB treatments. 

Percentage Triploidy 

In treatments, mean percent triploidy was 13% + 6.7% for 
0.125 mgCB/L. 61.8% ± 6.2% for 0.25 mgCB/L. and 68.2% ± 
14.1% for 0.5 mgCB/L. In controls, 1.4% ± 1.3% of the 0.05% 
DMSO treatment and 3.4% ± 0.3% of the FAS normal larvae were 
triploid. Figure 1 depicts percentage triploidy by treatment and 
replicate. Variation seems high among the three replicates; how- 
ever, transformed data revealed no significant difference (P < 
0.05). 

The model (% triploidy = treatments, replicates) defined the 
relationship between the treatment effects and percentage triploidy 



and explained mo.st of the variability (R~ = 0.9616). Treatment 
was highly significant (f < 0.0001), and the replicate effect was 
not significant (P = 0.5291) (Table 1). Post hoc comparisons of 
mean percentage triploidy found neither significant differences 
between the 0.125 mgCB/L and the two controls, nor between the 
0.25 mgCB/L and 0.5 nigCB/L dosages (Table 2). 

Survival 

Figure 2 shows survival by treatment and replicate. On average, 
the results demonstrate an inverse relationship between survival 
and CB dosage and a lack of effect (slight enhancement) with 
DMSO e.xposure. Although there was moderate variability among 
the replicates, overall, they all demonstrated the same trends across 
treatments. 

The model explained a reasonable amount of variation in sur- 
vival (R" = 0.7172). Both replicate and treatment were highly 
significant {P < 0.0001, Table 3). For treatments, both control and 
norinal were the same, and all CB groups were the same (Table 4). 
Overall, CB groups had about 20% lower survival than did the 
controls. 

DISCUSSION 

These results support previously reported findings that 0.25 
mgCB/L (Barber et al. 1992) and 0.5 mgCB/L (Shatkin and Allen 
1990) are appropriate dosages for inducing triploidy in C. vir- 
i^inica. However, results are variable depending upon egg or sperm 
quality or some other factor (Allen and Bushek 1992). 

Treatment Recommendations 

We found no statistical difference in percentage triploidy or 
survival between the two higher CB dosages. Considering the cost 





0.7 




0.6 




0.5 


03 

> 


0.4 



CO 



0.1 



Replicate 1 



Replicate 2 



;W;W>W;W;WJ 



£ 0.3 - 



0.2 



-■ 



Replicate 3 






I 






0.125 0.25 



0.5 



control DMSO 



CB Treatment (mg/L) 

Figure. 2. Survival of C. virginica embryos to D-stage larvae after cytochalasin B treatment, by replicate. 



128 



SUPAN ET AL. 



TABLE 3. 

Results of ANOVA: Effect of cytochalasin B treatment and 
experimental replication on survival of C. virginica larvae. 



Sources of 
Variation 



DF 



F-ratio 



Prob > F 



Treatment 
Replicate (error) 



11.54 
2?. 1 1 



0.0001 
0.0001 



R- = 0.7172 



of CB {$10/mg in the U.S.). economics suggest that the lower 
effective dosage is preferable, at 28 °C for 10 min. However, with 
a range of 54 to 82% triploidy (0.5 mgCB/L) versus 55 to 67% 
(0.25 mgCB/L), one is inclined to use the higher dosage. Greater 
triploidy might have resulted from a longer (15 min) treatment 
time, at the sacrifice of lower survival. For maximum triploid 
production, embryos should be exposed to CB for a period of time 
long enough to have a high proportion captured at PBI extrusion 
but short enough to minimize mortality (Barber et al. 1992). Allen 
and Bushek ( 1992) attributed low variance in triploid production to 
using meiosis I as a benchmark to begin treatment, claiming to 
have effectively removed meiotic rate as a factor. Although the 
time of initiation is determined by an appropriate developmental 
milestone (i.e., 50% PBI). the duration is fixed and does not ac- 
commodate varying meiotic rates. We suggest that appropriate 
duration of treatment be addressed by using a developmental 
benchmark to cease treatment as it is used for beginning it. Ob- 
servation of a subsample of eggs, held at the same temperature but 
without treatment, could provide such a cue. Although this cue 
must be determined empirically, we suggest 2-5% cleavage might 



TABLE 4. 

Results of ANOVA: Means and standard deviations (SD) of survival 
of C. virginica by treatment with comparisons. 

Survival* 



Treatment 


Mean 


SD 


Comparisons** 


0.125 mg cb 


0.5961 


0.1204 


A 


0.25 mg cb 


0.5795 


0.0860 


A 


0.5 mb cb 


0.4748 


0.(»I3 


A 


Control w/o dmso 


0.7.\M 


0.2035 


B 


Control w/dni.so 


0.7585 


0.2126 


B 











* Survival = arcsin (V(Normal larvae/embryos)). 

** Tukey's honestly significant difference (.'-^ = 0.05). 

be appropriate. This benchmark could be used for C. virtfinica or 
any bivalve species. 

The real solution to improving efficiency of triploid production 
is the development and use of tetraploid broodstock. Tetraploid 
male oysters produce diploid sperm; when used to fertilize eggs 
from diploid females. 100% triploid offspring result (Guo and 
Allen 1994). 

ACKNOWLEDGMENTS 

We are grateful to Jordan Bradford. Lee Hanson, the late Tony 
Venterella and his wife. Gayle. and the late Carlo Venterella for 
their logistical support during the project, and Mr. Ron Becker for 
his guidance and insight. We are also grateful to Wilbert Collins, 
Jules Melancon. and Al Sunseri for their donation of broodstock. 
Financial support was provided by grants from the Louisiana 
Board of Regents [LEQSF (93-96)-RD-B-08| and the Louisiana 
Sea Grant College Program [NA89AA-D-SG226]. 



LITERATURE CITED 



Allen. S. K. Jr. 1986. Genetic manipulations: critical review of methods 
and performances in shellfish. //;.■ K. Tiews (ed.). Selection. Hybrid- 
ization, and Genetic Engineering in Aquaculture.. Proceedings of a 
World Symposium. Schriften der Bundesforschungsanstalt fiir Fis- 
cherei Hamburg Band 18/19. Berlin. 

Allen. S. K. Jr. 1988. Triploid oysters ensure year-round supply. Occainis 
31:58-63. 

Allen, S. K. Jr. & D. Bu.shek. 1992. Large-scale production of triploid 
oysters Crassoxrrea virginica (Gmelin). using "stripped" gametes. 
Aquaculmrc 103:241-251. 

Allen. S. K. Jr.. S. L. Downing & K. K. Chew. 1989. Hatchery manual for 
producing triploid oy.sters. Washington Sea Gram Publ. WSG-89-3. 
University of Washington, Seattle, WA. 27 pp. 

Barber, B. J., R. Mann & S. K. Allen Jr. 1992. Optimization ol triploidy 
induction for the oyster. Crassoslreii virginica (Omelin). ./. Shellfish 
Res. 11:189. 

Chailon. J. A. & S. K. Allen Jr. 1985. Eariy detection of triploidy in the 
larvae of Pacific oysters. Crassoslrea gigas. by fiow cytometry. Aiiiiii- 
ciilliire 48:35-43. 

Downing. S. L. & S. K. Allen Jr. 1987. Induced triploidy in the Pacific 



oyster. Crassostrea gigas: optimal treatments with cytochalasin B de- 
pend on temperature. Aqiiaciiluire 61:1-15. 

Dowdy. S. & S. Wearden. 1991. Statistics for research. John Wiley & Sons 
Publishing Co.. New York. 629 pp. 

Galtsoff P. A. 1964. The American oyster. Crassoslrea virginica [Gme- 
lin|. Fisheries Bulletin. U.S. Fish and Wildlife Service. 480 pp. 

Guo, X. & S. K. Allen Jr. 1994. Viable letraploids in the Pacific oyster 
[Crassoslrea gigas Thunberg) produced by inhibiting polar body 1 in 
eggs from triploids. Mol. Mar. Biol. Bioteclmol. 3:42-50. 

SAS Institute. 1991. SAS system for linear models. 3rd ed. SAS Institute. 
Cary. NC. 327 pp. 

Shatkin. G. M. & S. K. .Mien. 1990. Recommendations for commercial 
production of triploid oysters. / Shellfish Res. 8:449. 

Stanley. J. G., S. K. Allen Jr. & H. Hidu. 1981. Polyploidy induced in the 
American oyster, Crassoslrea virginica. with cytochalasin B. Aquacul- 
lure 23:1-10. 

Supan. J. E. 1995. The effects of salinity on the production of triploid 
oyster larvae [Crassoslrea virginica Gmelin) in Louisiana. Ph.D. Dis- 
sertation, LSU. Baton Rouge, LA. 



Jniinuil of Shellfish Research. Vol. 19. No. 1. 129-1.^2. 2000. 

DELIVERY OF RIBOFLAVIN TO LARVAL AND ADULT PACIFIC OYSTERS, CRASSOSTREA 

GIGAS THUNBERG BY LIPID SPRAY BEADS 

C. J. LANGDON.' C. SEGUINEAU," B. PONCE,^ J. MOAL,^ 
J. F. SAMAIN- 

^ Coastal Oregon Marine Experiment Station 

Hatfield Marine Science Center and Department of Fisheries and Wildlife 

Oregon State University 

Newport. Oregon 97365 
-IFREMER 

Lahoratoire de Physiologie des Invertebres 

BP70. 29280 Ploiizane, France 

ABSTRACT Lipid spray beads (SB) were prepared containing 13% w/w particulate riboflavin. Beads suspended in seawater lost 73% 
riboflavin after 24 h. Release of riboflavin from SB ingested by Pacific oyster (Cmssostrea gigcis) larvae was observed under 
epifluorescent light. Riboflavin concentrations in tissues of adult oysters fed on riboflavin-SB were significantly (SNK; P < 0.05) 
greater than those of oysters fed on seawater-filled SB. Concentrations of riboflavin in oysters exposed to dissolved riboflavin were 
not significantly greater than those of oysters fed on seawater-filled SB, indicating that elevated riboflavin concentrations in oysters 
fed on riboflavin-SB were attributable to breakdown of ingested beads rather than uptake of dissolved riboflavin leaked from SB into 
the culture medium. SB seem to be a promising means of delivering water-soluble nutrients to bivalve suspension feeders 

KEY WORDS: Spray beads, lipid, riboflavin, oyster, larvae. Crassostrea gigas 



INTRODUCTION 

Little is known of the nutritional requirements of bivalve mol- 
lusks despite their obvious importance in aquaculture and natural 
ecosystems. The main reason for this lack of knowledge is that 
nutritionally satisfactory, defined artificial diets are not available. 
The development of microparticulate diets that both retain dietary 
ingredients when suspended in seawater and are digestible by bi- 
valve mollusks has been a difficult goal to achieve. High surface 
area to volume ratios of microparticulate diets together with low 
molecular weights of essential nutrients, such as water-soluble 
vitamins, results in their rapid loss. For example. Lopez-Alvarado 
et al. (1994) reported that > 80% amino acids were lost from 
microgel particles after only 2 minutes in aqueous suspension. 

To address the problem of rapid loss of water-soluble nutrients 
from microparticulate feeds, Langdon and Siegfried (1984) devel- 
oped lipid-walled microcapsules for the delivery of water-soluble 
vitamins to juvenile oysters [Crassostrea virginica). Later Buchal 
and Langdon (1998) and Langdon and Buchal (1998) developed 
lipid spray beads (SB) for the delivery of water-soluble nutrients 
and therapeutic substances to bivalves. Buchal and Langdon 
(1998) found that it was important to soften the walls of lipid- 
walled capsules and SB by adding 40% w/w fish oil to the tripal- 
mitin walls of the particles in order to make them digestible by 
clams {Tapes philippinarwn): however, softening the walls of SB 
in this way lowered 24-h retention efficiencies for encapsulated 
riboflavin from 97.9 to 85.1% (Buchal and Langdon 1998). 

Seguineau et al. (1996) reported that the microalgal species 
Isochiysis galbana. Pavlova lutheri. and Skelelonema costatiim 
contained high concentrations of riboflavin and thiamine; how- 
ever, the concentrations of these two vitamins in scallop (Pecten 
ina.ximiis) larvae fed on a mixture of these algal species declined 
during growth and development. Seguineau et al. (1996) suggested 
that microencapsulated supplements of riboflavin and thiamine 
could be used to study the requirements of scallop larvae for these 
vitamins. 



In this paper, we describe the results of feeding experiments in 
which larval and adult oysters ( Crassostrea gigas Thunberg) were 
fed on SB containing particulate riboflavin to evaluate the poten- 
tial usefulness of SB in delivering low-molecular weight, water- 
soluble nutrients to bivalve mollusks. 



METHODOLOGY 



Spray Beads 



Preparation of Spray Beads Containing Riboflavin 

Spray beads were prepared containing micronized, particulate 
riboflavin (Sigma) based on the method described by Buchal and 
Langdon (1998). Briefly, riboflavin crystals were ground to a fine 
powder (< 5-(xm particles; McCrone micronizing mill, McCrone 
Scientific Ltd). Two grams of ground powder were mixed by soni- 
cation at 90 °C with 8 g of a lipid mixture made up with 4.8 g 
tripalmitin (Fluka Chemical Co.) and 3.2 g of menhaden oil (light 
cold pressed; Zapata Haynie Ltd.). The heated mixture was then 
forced under pressure (90 psi) through at atomizing nozzel (SUE- 
25B; Spraying Systems Ltd.) supplied with pressurized nitrogen at 
10 psi. The beads were collected in a stainless steel cylinder cooled 
with liquid nitrogen then stored in the dark at -20 "C until use. 

Determination of Encapsulation Efflciency 

To determine encapsulation efficiencies. 10 g of SB were first 
dissolved in 3 mL chloroform and the vitamin core material ex- 
tracted by addition of 3 mL distilled water with shaking. The 
aqueous supernatant was removed and the extracfion repeated 
three times. Aqueous extractions were combined and the concen- 
tration of dissolved riboflavin determined spectrophotometrically 
(absorbance at 267 nm). 

A subsample of 0.5 mL of chloroform was removed from the 
capsule extraction and transferred to a dry. tarred weighing boat. 
The chloroform was removed by heating for 24 h at 50 °C. and the 
boat was reweighed to determine the weight of extracted lipid. 



129 



130 



Langdon et al. 



Encapsulation efficiencies were expressed as the weight of encap- 
sulated vitamin (mg) per 100 mg of lipid. 

Retention of Riboflavin by SB Suspended in Seawater 

Retention of riboflavin by SB was determined by measuring the 
proportion of initially encapsulated riboflavin remaining after 24 h 
suspension in seawater. To prepare SB for a leakage experiment, 
beads were first suspended in 2% polyvinyl alcohol with sonica- 
tion. SB were then sieved using a 40-|jim sieve, and beads under 40 
p,m were collected on a GF/C filter and rinsed with cold (5 °C) 
distilled water. The beads were then washed from the filter with 
cold distilled water and collected in a sealed vial and stored at 5 °C 
in the dark. 

At the start of a leakage experiment. 75 to 1 00 mg of the sieved 
(< 40 p,m) SB were suspended in 15 mL seawater (20 °C) by 
vigorous shaking. Immediately after suspension (t = 0), 1 mL of 
the bead suspension was taken and filtered onto a GF/C filter. The 
filtered SB were then washed with 1 mL of chilled (5 °C) seawater. 
The filtrate and washings were pooled and stored in the dark at 5 
°C for analysis. Riboflavin concentrations were determined as de- 
scribed above. The remainder of the SB suspension was placed on 
a continuous agitator at 20 to 22 °C. Samples of suspended SB 
were taken over a period of 24 h to determine changes in the 
retention of riboflavin over time. Retention efficiency (RE) was 
defined as 

riboflavin retained on the filter 

RE= ., „ . T—rr, X 100 

riboflavm on inter and m riltrate 

Breakdown of SB and Release of Riboflavin by Larvae 

Feeding experiments were conducted to determine if oyster 
larvae could inge.st and breakdown SB, thereby releasing ribofla- 
vin into the digestive system. Broodstock oysters were spawned, 
and the resulting larvae were raised on a mixed diet of Isochiysis 
gulhaiui (T-ISO) and Chaetocems cakitnins (Breese and Malouf 
1975). After 8 days of culture, larvae were sieved onto a 45-p.m 
screen, divided into two groups and each resuspended in two liters 
of seawater at a density of 1 larvae per mL. 

Riboflavin-SB at a concentration of 50 SB/p,L were fed to one 
group of larvae with gentle aeration to maintain SB in suspension. 
After 1 hour, larvae were sieved from suspension using a 45-|jLm 
sieve, rinsed with seawater, then resuspended in two liters of fil- 
tered seawater and fed on T-ISO alone for 2 hours. The larvae were 
then sieved from the culture medium and preserved with 0.59f- 
formaldehyde (final concentration made up in seawater, buffered 
at pH 8.0 with borax) for microscopic analysis. The second group 
of 8-day old larvae were fed on T-ISO alone for 2 hours, then 
sampled and preserved for microscopic analysis (as described 
above). 

Sampled larvae were examined using an epifluorescent micro- 
scope (Leica DMRBE: excitation 355-^25 nm. emission 525 nm) 
at x400 magnification. Green and yellow fluorescence indicated 
the presence of dissolved and particulate riboflax in. respectively, 
while red fluorescence indicated the presence of chlorophyll de- 
rived from ingested algae. 

Breakdown of SB and Uptake of Released Riboflavin by Adults 

An experiment was carried out to determine if adult oysters 
could breakdown ingested SB and absorb released vitamin into the 
hemolymph and tissues, lliree groups of six adult oysters were 



separately held in 20 L of seawater and fed for 6 hours on T-ISO 
at a concentration of 50 cells/(j.L in combination with one of the 
following additions: 

1. 20 riboflavin-SB/|jiL (equivalent to a concentration of 1.15 
mg riboflavin/L or a vitamin dose of 3.8 mg riboflavin/ 
oyster): 

2. 20 seawater-filled SB/p.L; or 

3. dissolved riboflavin at the same concentration provided in 1. 
Two grams of riboflavin-SB were suspended in distilled water, 

and the suspension was then poured through a 20fjLm mesh sieve. 
SB smaller than 20 ixm were collected and filtered onto a GF/C 
filter, rinsed, and resuspended in 10 mL of distilled water. Aliquots 
of 100 |j,L SB suspension were taken to determine riboflavin and 
bead concentrations. Riboflavin concentrations were determined as 
described above. SB concentrations were determined using a 
Coulter Counter (Model TA2). Seawater and food additions were 
replaced every 2 hours over a period of 6 hours. The cultures were 
gently aerated to maintain beads in suspension. 

After 6 hours, oysters were removed and dissected. 
Hemolymph samples were taken with a syringe from both the heart 
and the sinus of the adductor muscle. Samples of stomach contents 
were removed with a Pasteur pipette inserted through the mouth. 
Tissue samples of mantle and adductor muscle were also removed. 
The samples were frozen at -20 °C for protein and riboflavin 
analysis. 

Riboflavin and Protein Analysis 

Hemolymph samples were centrifuged, and riboflavin concen- 
trations of the supernatant fraction were determined by high- 
pressure liquid chromatography (HPLC) (Seguineau et al. 1996). 
Mantle and adductor muscle samples were ground in 0.2M HCl 
and 0.6N perchloric acid (PCA) and centrifuged. Supematants 
were then removed, their volumes adjusted to 2 mL with distilled 
water and riboflavin concentrations determined by HPLC 
(Seguineau et al. 1996). 

Protein concentrations of hemolymph samples were determined 
by the method of Bradford (1976), using bovine serum albumen as 
a standard. Treatment of adductor muscle and mantle samples with 
HCl and PCA for the extraction of riboflavin probably resulted in 
the precipitation of most proteins in these samples; therefore, 
Bradford assays indicated concentrations of PCA-soluble protein 
and peptides in muscle and mantle samples. Riboflavin concentra- 
tions were expressed in terms of ng ribofla\ in per mg protein in 
tissue samples or per mL of stomach extract. 

Statistics 

The rate of loss of riboflavin from SB suspended in seawater 
was analy/.ed by regression analysis. Riboflavin concentrations in 
oyster samples from the three experimental irealments were com- 
pared by analysis of variance (ANOVA) (Model 111: Super 
ANOVA, Abacus Concepts). Concentrations were log-trans- 
formed to ensure homogeneity of variances, as determined by plots 
t)f residual values against fitted values. If ANOVA indicated a 
significant treatment effect on riboflavin concentration, individual 
treatments were compared by Student-Newman-Keuls (SNK) 
nuiltiple range test (significance level P < 0.05). 

RKSULTS 

Encapsulation and Retention Efficiencies 

SB were lound to ha\e an encapsulation efficiency of 13'7r w/w 
(mg riboflavin per 100 mg lipid I. Leakage experiments indicated 



Delivery of Riboflavin to Oysters by Lipid Spray Beads 



131 



that almost half the encapsulated ribonavin was lost from SB 
during the first 3 hours of suspension in seawater, followed by a 
more gradual loss over the subsequent 21 hours (Fig. I). Approxi- 
mately 27% of the initially encapsulated riboflavin was retained 
after 24 hours of suspension, equivalent to 3.3 mg of riboflavin 
retained per 100 mg of lipid. 

Regression analysis indicated that there was a significant {P = 
0.0012) relationship between the log of the fraction of riboflavin 
retained and the duration (log time (h)] that SB were suspended in 
seawater (Fig. 1 ). The rate of loss of riboflavin could be expressed 
in terms of the equation; 



Log fraction retained 
0.994 



0.116 - [0.333 X log time (h)] r" = 



Breakdown of SB and Release of Riboflavin by Larvae 

Larvae were able to ingest and breakdown SB. liberating en- 
capsulated riboflavin into the digestive system. Free riboflavin was 



100 - 


1 1 1 1 1 1 1 1 1 1 




[ 


-;? 80 - 


\ 


£ 


\ 




V 


>. 


^v 


o 


^ 


C 60 - 


^. 





#(.^^ 


o 


^^-^^_^ 


it 


^■^-^^ 


0) 




_ 40 - 


^""^■^^,,___^ 


C 


^""^~— ^__^ 


o 


^^^~^^^_^ 


^H* 




c 


^^ ^ 


B 20- 




(D 




tr. 

n - 





10 12 14 16 18 20 22 24 

Time (h) 




.2 .4 .6 .8 1 

Log Time (h) 

Figure 1. Retention of riboflavin by lipid spray beads suspended in 
seawater. Top: Change in percentage riboflavin retained by beads over 
a 24-h period Bottom: Relationship between log of fraction retained 
and log time duration of beads suspended in seawater. Log fraction 
retained = -0.116 -[0.333 x log time (h)l; r" = 0.994 




Figure. 2. Eight-day-old larvae of the Pacific oyster {Crassostrea gigas) 
viewed under epifluorescent light (excitation 355—125 nm, emission 525 
nm) at x400 magnification. Average larval shell length = 122 pm. Top: 
Larvae fed on ribofiavin-containing lipid spray beads (50 beads/pL) 
for 1 hour, followed by a 2- hour period of feeding on T-ISO alone. 
Bottom: Larvae fed on Isochrysis sp. (T-ISO) alone for 2 hours. 

observed as a diffuse greenish fluorescence in the guts of larvae 
fed on SB. and riboflavin crystals present in intact or partially 
digested SB were evident as bright yellow points (Fig. 2). The 
digestive systems of larvae fed on algae alone fluoresced red be- 
cause of the presence of chlorophyll from ingested algae but no 
yellow or green fluorescence was evident (Fig. 2). 

Breakdown of SB and Uptake of Released Riboflavin by Adult Oysters 

ANOVA of log-transformed riboflavin concentrations in oys- 
ters fed on riboflavin-SB were significantly greater (SNK; P < 
0.05) than those of oysters either fed on seawater-filled SB or 
exposed to riboflavin dissolved in seawater (Table 1). The pres- 
ence of significantly higher concentrations of riboflavin in the 
hemolymph. adductor muscle, and mantle of oysters fed on ribo- 
flavin-SB indicated that oysters were able to digest the lipid walls 
of SB and absorb released riboflavin. There were no significant 
differences in riboflavin concentrations in hemolymph sampled 
from either the heart or adductor muscle (ANOVA: P > 0.05). 

Concentrations of riboflavin in the tissues of oysters exposed to 
dissolved riboflavin were not significantly (SNK; P > 0.05) dif- 
ferent from those of oysters fed on seawater-filled SB, indicating 
a limited ability of adult oysters to take up dissolved riboflavin 
from seawater. 

CONCLUSIONS 

Feeding experiments indicated that both oyster larvae and 
adults were able to breakdown SB and release riboflavin. Free 



132 



Langdon et al. 



TABLE 1. 

Concentration of riboflavin in tissues of adult Pacific oysters exposed to either lipid sprav beads (SB) containing 13% riboflavin at a 
concentration of 20 SB/pm, seawater-filled SB at a concentration of 20 SB/pL or dissolved riboflavin at the same concentration as that 

delivered by riboflavin-SB (1.15 mg/L). 





Stomach 


Hemolymph 


Mantle 


Adductor 
muscle 










Contents 


Heart 


Muscle 


(ng/mg PCA-soluble 


(ng/mg PCA-soluble 


Treatment 


(ng/mL) 


(ng/mg protein! 


(ng/mg protein) 


protein) 


protein) 


Seawater-filled SB 


20 ±4 


47 ± 19 


21 ±10 


198 ±94 


177± 115 


Dissolved riboflavin 


24 ±4 


84 ± 53 


51 ±32 


112 ±22 


67 ± 20 


Riboflavin-SB 


7274 ± 1619 


1844±66(J 


1 1 65 ± 247 


1633 ±573 


10636 ±3808 



Values are given as means (±1 SE. /; 



6). 



riboflavin was evident in the stomachs of larvae and elevated 
riboflavin concentrations were evident in the tissues of adult oys- 
ters after being fed on riboflavin-SB. Adult oysters exposed to 
concentrations of dissolved riboflavin, equivalent to those supplied 
by encapsulated riboflavin, did no show elevated tissue concen- 
trations of riboflavin, indicating that uptake of dissolved riboflavin 
lost from SB was not a significant source for adult oysters. 

About half of the riboflavin was lost during the preparation of 
SB, based on a comparison between the measured encapsulation 
efficiency of 13% and a maximum theoretical encapsulation effi- 
ciency of 25%. Further losses of riboflavin from SB occurred after 
suspending SB in seawater; for example, it can be estimated (based 
on Eq. 1) that 39% of encapsulated riboflavin would have been 
lost at the end of each 2-hour period of the adult feeding experi- 
ment. 

In this study, retention of riboflavin by SB suspended in 
seawater for 24 h was only 27% compared with 85% reported 
by Buchal and Langdon (1998). This difference may have been 
attributable to higher encapsulation efficiencies of SB used in 
this study, because riboflavin-SB prepared by Buchal and Lang- 
don (1998) had an encapsulation efficiency of 4.89^ compared 
with an encapsulation efficiency of 13% for SB used in this 
study. 

The effects of additions of riboflavin-SB on the growth and 
survival of oysters needs to be determined in future experiments. 
Because of the need to prepare SB with a high proportion (> 60%) 
of lipid wall material to ensure encapsulation of the core material, 
it is unlikely that SB will be useful in delivering bulk dietary 
ingredients, such as protein and carbohydrate, to oysters. However. 
SB may be useful in supplementing algal or artificial diets with 



water-soluble micronutrients. such as essential ainino acids or wa- 
ter-soluble vitamins (Seguineau et al. 1996). 

ACKNOWLEDGMENTS 

This research was supported by a fellowship from the Organi- 
zation for Economic Cooperation and Development (OECD). co- 
operative research program: Biological Resource Management for 
Sustainable Agricultural Systems. We also thank Jacques Panfili 
for his help in taking photographs of larvae fed on SB. 

LITERATURE CITED 

Bracit'ord. M. M. 1976. A rapid and sensitive method for the quantitation of 
microgram quantities of protein utilizing the principle of prolein-dye 
binding. Anal. Biochein. 72:248-254. 

Breese. W. P. & R. E. Malouf 1975. Hatchery Manual for the Pacific Oys- 
ter. Oregon State University Sea Grant Program. Pub. No. ORESU-H- 
75-002. 22 pp. 

Buchal. M. A. & C. J. Langdon. 1998. Evaluation of lipid-spray beads for 
the delivery of water-soluble materials to a marine suspension-feeder, 
the Manila clam Tupcs philippinanim (Deshayes 1853). Acjuacull. Nii- 
lii. 4:263-274. 

Langdon. C. ,1. & M. A. Buchal. 1998. Comparison of lipid-walled micro- 
capsules and lipid spray beads for the delivery of water-soluble, low- 
molecular weight materials lo aquatic animals. Aiiiiciciili. Nutri. 4: 275- 
284. 

Langdon. C. J. & C. A. Siegfried. 1984. Progress in the development of 
artificial diets for bivalve filter-feeders. Aqiuiciilture 39:135-153. 

Liipez-Alvarado. J., C. J. Langdon. S -I. Teshima & A. Kanazawa. 1994. 
Effects of coating and encapsulation of crystalline amino acids on 
leaching in larval feeds. Aiiiiaciiliure 122:335-346. 

Seguineau. C. A. Laschi-Loquerie. J. Moal & J. F. Samain. 1996. Vitamin 
requirements in great scallop larvae. Aqiuiciill. In!. 4:315-324. 



Jvuimil of Shellfish Raeciich. Vol. 19. No. I. I3.V138, 2000. 

MODELING SEASONAL PROLIFERATION OF THE PARASITE, PERKINSUS MARINUS 
(DERMO) IN FIELD POPULATIONS OF THE OYSTER, CRASSOSTREA VIRGINICA 



D. J. BROUSSEAU' AND J. A. BAGLIVO^ 

Department of Biology 
Fail fie Id University 
Fairfield. Connecticut 06430 
'Department of Mathematics 
Boston College 
Chestnut Hill. Massachusetts 02467 

.ABSTRACT A temperature-disease course model was developed to predict the effect of seasonal water temperature changes on 
disease progression of Dermo in field populations of Crassostrea virgiiiica. A linear model was used to describe the relationship 
between weighted prevalence (disease intensity) and lagged cumulative temperature, where cumulative temperature was used as an 
estimate of cumulative harm. The model developed for Long Island Sound showed the strongest relationship between cumulative 
temperature and disease intensity when a lag time of 53 days was used. Point and interval estimates for the day(s) of the year when 
a mean weighted prevalence of 2 (Mackin Index) is expected at four sites in Long Island Sound are given. This model allows the 
grower/manager to predict Dermo intensity in shellfish beds if field water temperature patterns are known. Such information can be 
used to select oyster growout beds and determine optimal time to harvest. 

KEY WORDS: Perkinsii.'s mariiiiis. Dermo. temperature-disease course model. Long Island Sound 



INTRODUCTION 

Perkinsus marimis (commonly known as "Dermo"). a proto- 
zoan pathogen of uncertain phylogenetic affinities (Siddall el al. 
1995) is now well established in Long Island Sound (Brousseau et 
al. 1994, Brousseau 1996, Ford 1996, Brousseau et al. 1998) and 
has been reported as far north as the Damariscotta River in Maine. 
This pathogen is a major cause of oyster inortality in the Gulf of 
Mexico and Chesapeake Bay. Its introduction to Long Island 
Sound, the third largest producer of commercial oysters in the 
U.S.. has prompted efforts to develop management strategies and 
husbandry protocols to help control the spread of this disease. 

The influence of temperature on the activity of Perkin.sus mari- 
mis is well documented. Temperature affects multiplication rates. 
virulence (Andrews 1988) and zoosporulation of the parasite (Chu 
and Greene 1989), and disease intensity in the host increases with 
increasing temperature (Chu and LaPeyre 1993). Temperature is 
also believed to be the most important factor affecting the geo- 
graphic distribution and seasonal cycle of this pathogen (Andrews 
1988, Andrews and Ray 1988. Crosby and Roberts 1990. Soniat 
and Gauthier 1989). 

Modeling studies also point to the importance of temperature in 
the development and maintenance of Perkinsus marinus infections. 
Simulations have shown that the timing and duration of long-term 
climatic changes are important in determining levels of infection in 
diseased (coinfeclion by MSX and Dermo) oyster populations 
(Powell et al. 1992); whereas, the results of Hofmann et al. ( 199.5) 
suggest that temperature is the primary factor regulating the para- 
site in the Gulf of Mexico. 

Soniat and Kortright (1998) recently developed a model to 
estimate the time to a critical level of Perkinsus marinus in eastern 
oysters using a long-term dataset of temperature, salinity, and 
parasite infection level. Their model indicates that both tempera- 
ture and salinity are important variables in predicting Dermo pro- 
gression in areas such as the Terrebonne estuary of Louisiana, 
where fluctuations in salinity are high and salinity levels often fall 
below 10 ppt. In high-salinity, oyster-producing regions such as 



Long Island Sound; however, it is likely that water temperature is 
the more important factor in controlling parasite proliferation. 

This paper reports the results of a modeling study aimed at 
predicting the effect of short-term (seasonal) temperature changes 
on disease progression of "Dermo" in oyster populations from 
New York and southern New England. The annual cycle of Per- 
kinsus marinus infections in oysters from six locations in Con- 
necticut, Massachusetts, and New York is presented, and a pre- 
dictive temperature -disease course model developed for wild and 
commercial oyster beds in Long Island Sound is described. A 
discussion of the usefulness of this model to oyster aquaculturists 
is also presented. 



MATERIALS AND METHODS 



Data Collection 



Oysters (Crassostrea virginica) were collected twice a month 
from six locations in Connecticut. New York, and Massachusetts 
from January to December 1997 (Figure 1). Most samples con- 
tained 25 oysters; a total of 3,786 animals were studied. Sampling 
locations, site descriptions, sample sizes, and ages (juvenile vs. 
adult) of oysters sampled are provided in Table 1 . Tissue diagnosis 
of Perkinsus iiuuinus was done by culture of rectal and mantle 
tissue in fluid thioglycollate medium, as described by Ray ( 1954), 
Infections were scored for intensity of disease by use of the mea- 
sure originally described by Mackin (1962) as the weighted inci- 
dence and later renamed weighted prevalence (Ragone and Bur- 
reson 1994). On the Mackin Index, scores of 0.5-1.0 indicate light 
infections, scores of 2.0-3.0 indicate moderate infections and 
scores of 4.0-5.0 are considered heavy. 

Temperatures (°C) were monitored at each site using an Optic 
Stowaway"^' Temperature Logger (Onset Computer Corp.). At in- 
tertidal sites, the recorder was attached to a stake driven into the 
flat, and at subtidal locations, it was suspended over the shellfish 
bed along a buoy system anchored to the bottom. As a backup 
against loss or failure, teinperatures were also taken by hand sev- 
eral times a month. Periodic salinity measurements were taken to 



133 



134 



Brousseau and Baglivo 



74'-'W 



4h^N 



420 N _ 



NEW YORK 



40" N -i 



NEW 
.JERSEY 




Figure 1. Map showing tiie locations of the six study sites used in this study: (A) Oyster Bay, NY, (B) Saugatuck River, Westport, CT, (C) Blacli 
Rock Harbor, Bridgeport, CT, (D) Thames River, Waterford, CT, (E) Mystic River, Stonington, CT, and (F) Cotuit, MA. 



substantiate earlier reports thiat salinities at tine study sites routinely 
run in the 20 to 30 ppt. range (Brousseau 1996, S. Ford pers. 
comni). They are shown in Table 2. 

Mean Temperature Model 

The mean temperature. T(x), for sampling day x can be mod- 
eled as a cyclic function 



T(x) 



A cos[c (X - X|^„,,)| 



where p. is mean temperature for the year. A is one-half the range 
of mean temperatures (the amplitude). X|„„ is the day with the 
lowest mean temperature, and c is the constant needed to make the 
period equal to one year (c = 2tt/365.25). 

For X between the time of lowest mean temperature and highest 



TABLE 1. 

Age (adult vs. juvenile) and sampling location, number of samples collected, size of oysters sampled, site description (intertidal vs. subtidal). 

and type of oysters sampled (wild vs. cultivated). 









Mean 








Shell 


Shell 




Age and 


Number of 


Length 


Length 




Sampling Location 


.Samples 


(mm) 


(mm) 


Comments 


Adult populations: 










Black Rock Harbor. Bridgepon, CT 


25 


38..'i- 133.7 


70.4 


1. 3 


Mystic River, Stonington, CT 


23 


36.6-130.7 


76.2 


2.4 


Thames River. Waterford. CT 


2.^ 


30.1-122..') 


62.1 


2,4 


Saugatuck River, Wcslport. CT 


2.5 


.34..3- 112.7 


69.4 


1.3 


Juvenile popukilions ( IW.S/yfi YOY): 










Black Rock Harbor. Bridgeport. CT 


10 


14.8-69.1 


34.8 


1. 3 


Cotuit. MA 


24 


42.2-104.1 


69.6 


2.4 


Oyster Bay. NY 


23 


42.S-96.y 


65.6 


2.4 



1 = Interlidal sampling site. 

2 = Suhiidal sampling site. 

3 = Wild population. 

4 = Cultivated population. 



Modeling Dermo Progression in Field Oysters 



135 



TABLE 2. 

Salinity measurements taken at tlie six study sites durin!> 1997. In 
sample size). 







Mean ± SE 


Range 


Study Site 


n 


(PPt) 


(ppti 


Black Rock Harbor. Bridgeport, CT 


128 


23.1 ±0.2 


15,5-31.0 


Mystic Ri\er. Stonington, CT 


-11 


25.1 ±0.5 


18.1-28.0 


Thames River, Waterford. CT 


3 


14.9 ±4.9 


5.4-21.7 


Saugatuck River. Westport. CT 


55 


22.1 ±0.4 


11.5-27.0 


Oyster Bay. NY 


36 


24.8 ±0.2 


22.0-26.0 


Cotuit. MA 


7 


26.5 ± 0.3 


25.0-27,0 



mean temperature, the cumulative temperature. CT(x). is the area 
under the temperature curve from time X|^,„ to time x: 



CT(x) = JJL (X 



J - A sin[c (X 



Jl/c. 



Separate temperature models were developed for each site 
(Black Rock Harbor, n = 309; Cotuit. n = 93: Mystic River, n = 
365; Oyster Bay. n = 146; Thames River, n = 365; Westport, n 
= 365), Weighted nonlinear least squares analysis was used to fit 
the parameters. 

Temperature — Disease Course Prediction Model 

A linear model was used to describe the relationship between 
weighted prevalence y and lagged cumulative temperature for 
sampling day x and site s: 

y = a + b CT, (X - lag). 

In this fomiula. CT, (x - lag) is the area under the mean 
temperature curve for site s from the time of lowest mean tem- 
perature to time x minus the lag. Cumulative temperature is used 
as an estimate of cumulative harm; parasite proliferation is as- 
sumed to be a function of ambient water temperature patterns at 
each site. 

Samples with sampling day on or after the mean low tempera- 
ture day for the site formed the working set for the analyses. A 
total of 87 samples were used (Bridgeport, n = 23; Mystic River, 
n = 20; Thames River, n = 21; Westport, n = 23). The lag was 
chosen to maximize the correlation between weighted prevalence 
and lagged cumulative temperature. The slope and intercept were 
then estimated using linear least squares. The model with best 
overall fit was chosen. Bootstrap analysis using 1.000 resamples 
was used to estimate the sensitivity of the choice of lag time in the 
model (Efron and Tibshirani 1993). 

Model Predictions 

To make predictions, a weighted prevalence of 2.0 was selected 
as the parameter of interest since we considered it a critical stage 
in the progression of the disease. Andrews (1988) reported that 
some mortalities are likely to occur when the mean intensity for a 
population exceeds 1.0; however, severe mortalities (5()-757f) are 
not expected until the wp reaches 3.0 (Ray and Chandler 1955, 
Mackin 1961. Mackin and Hopkins 1961). Site-specific tempera- 
ture models were then used to obtain point estimates for the day 
with mean weighted prevalence of 2.0. A bootstrap analysis with 
1000 resamples was used to construct 95% confidence intervals for 
the day with mean weighted prevalence of 2.0 at each site. 



1 

0.8 
0.6 


' — 1 — ' 

• 
• 

• 

• 1 


• 

• 
1 • ♦ 












U.4 

0.2 






I 











Bpt-A Mys Thm Wpt Bpt-J Cot OB 

Figure 2. Side-by-side box plots of disease prevalence in juvenile and 
adult oy.sters. Adult populations in Bridgeport (n = 25 samples, median 
= 100% infected). Mystic River (n = 23 samples, median = 96% in- 
fected), Thames River (n = 23 samples, median = 100% infected), and 
Westport (n = 23 samples, median = 100% infected) and juvenile 
populations in Bridgeport (n = 10 samples, median = 42% infected), 
Cotuit (n = 24 samples, median = 30% infected), and Oyster Bay In = 
23 samples, median = 68% infected) are represented. 

RESULTS 

Descriptive Statistics 

Disease prevalence in adult and juvenile oysters from all sites 
during 1997 is shown in Figure 2. Median values were between 96 
and 100% for adult samples and between 30% and 68%- for juve- 
nile samples. 

Distributions of weighted prevalences among sites are shown in 
Figure 3. The highest median weighted prevalence was found at 
the Saugatuck River site, followed by the Thames River and Black 
Rock Harbor sites. The median weighted prevalence was highest at 
sites where adult oysters were sampled ( 1 .4-2.2); median weighted 
prevalence in juvenile oyster samples did not exceed 0.5. Distri- 
butions of proportions of oysters in all samples with intensity score 
of 2.0 or more on the Mackin scale is given in Figure 4. Median 
values for adult samples were between 32 and 60%; median values 



4 

3 




































































1 












A 














1 


































1 
















|J 


'— 1 




i 


1 




1 




1 1 






















' 1 

























■ 






























1 




1 





OB 



Bpt-A lyiys Thm Wpt Bpt-J Cot 

Figure 3. Side-by-side box plots of weighted prevalence in juvenile and 
adult oysters. Adult populations In Bridgeport (n = 25 samples, median 
= 1.76 wp). Mystic River In = 23 samples, median = 1.40 wp). Thames 
River In = 23 samples, median = 1.90 wpl, and Westport (n = 25 
samples, median = 2.20 wp) and juvenile populations in Bridgeport (n 
= 10 samples, median = 0.49 wp), Cotuit (n = 24 samples, median = 0.44 
wp), and Oyster Bay In = 23 samples, median = 0.40 wp) are repre- 
sented. 



136 



Brousseau and Baglivo 



0.8 • 



0.6 




Bpt-A Mys Thm Wpt Bpt-J 

Figure 4. Side-by-side box plots of proportion with intensity score of 
2.0 or more. Adult populations in Bridgeport (n = 25 samples, median 
= 40%), Mystic River (n = 23 samples, median = 32% ), Thames River 
(n = 23 samples, median = 48%), and Westport (n = 25 samples, 
median = 60%) and juvenile populations in Bridgeport (n = 10 
samples, median = 13% ): Cotuit (n = 24 samples, median = 12%); and 
Oyster Bay (n = 23 samples, median = 4%) are represented. 

for juvenile samples were between 4 and W/c The largest pro- 
portions were observed at the Mystic River, Saugatuck River, and 
Thames River sites. 

In adult oysters from Bridgeport, Thames River. Mystic River, 
Cotuit and Westport, weighted prevalence values increased dra- 
matically during a 50-day period from the beginning of June to the 
middle of July. The proportion of individuals with infection inten- 
sities of 3.0 or higher also climbed during that time interval. In 
oysters from Oyster Bay, the shift from lower to higher weighted 
prevalences, and from a low to high proportion of moderate to 
severely diseased individuals also occurred over a 50-day period, 
but it happened later in the year (Figure 5). This result suggests a 
pattern of seasonal parasite proliferation for a population that be- 
gins in late spring or early summer and continues over a 7-week 
period, before reaching a plateau in mid- to lale summer. 

Temperature Models 

Temperature model parameter estimates for each site are given 
in Table 3. Temperature patterns during 1997 were most similar at 
the Black Rock Harbor, Oyster Bay, Thames River and Westport 
sites, where mean maximum temperatures were between 21 and 23 
°C. At Cotuit. on the other hand, mean temperatures peaked at 26 
°C: whereas, in the Mystic River, mean temperatures reached a 
maximum of only 19 °C (Figure 6). The percentage of variation 
explained by the models ranged from 95 lo 98%. The on.set of 
seasonal proliferation of Pcrkiiisiis iiitiriiiiis at the study sites (Fig- 
ure 5) coincides with approximate ambient water temperatures of 
13 °C at the Mystic River, 16 "C at Bridgeport. Westport and the 
Thames River, and 20 "C at Cotuit and Oyster Bay (Figure 6). 

Temperature — Disease Course Prediction Model 

The prediction inodel with a lag time of 53 days gave the best 
over-all fit. explaining 45.1% of the variation in the data. The 
niiidel equation 

y = 0.9461 1 -t- 0.000899753 CT, (x - 53) 

is ba.sed on 50 samples satisfying the condition that the sampling 
day minus 53 is between the mean low and mean high temperature 
days. Point and interval estimates for the day of the year with mean 
weighted prevalence of 2.0 are shown in Table 4. 



FMAMJJASOND 



Month 




Month 



Figure 5. Graphs of weighted prevalence over time. Top plot: Bridge- 
port adults (n = 25 samples, solid black). Mystic River (n = 23 samples, 
solid gray), Thames River (n = 23 samples, dashed black), Westport (n 
= 25 samples, dashed gray). Bottom plot: Bridgeport juveniles (n = 10 
samples, solid black), Cotuit (n = 24 samples, solid gray). Oyster Bay 
(n = 23 samples, dashed black). 

For comparison, separate site-based models were developed 
and gave similar predictions. 

DISCUSSION 

Many of the characteristics of the Dermo epizootic in the north- 
east are similar to those described for epizootics in other areas. 
Disease prevalence is higher in adult oysters than in juveniles. 
Infection levels differ among size clas.ses (ages): higher parasite 
burdens are found in adult oysters throughout the year. The lower 
infection intensities generally reported for juvenile oysters (Ray 
1954) are believed to be the result of the relative growth rates of 
host and parasite (Mackin 1951. Hofmann et al. 1995). The plateau 
of high infection intensity seen in Ihe northeast during the summer 

TABLE 3. 

Temperature model parameter estimates i\i: mean temperature; X|„„ 

= time of minimum average temperature: A = amplitude) for each 

studv site. 



Sampling Site 




M 


\nv. 


A 


Black Rock Harbor. Bridgeport. 


CT 


12.19 


i5.\b 


10.. sy 


Coluil. MA 




I.V7() 


2,V47 


12.49 


Myslic River. .Stciniiigloii. (T 




1 1 ..^ 1 


%).{)! 


s.oo 


Oyster Bay. NY 




11.41) 


4(1.14 


11.67 


Thames River. Waterford. CT 




\}.l} 


.W..^4 


9.36 


Saugaluck River. Westport. CT 




11..^.^ 


3S.68 


10.49 



Modeling Dermo Progression in Field Oysters 



137 



has been reported for other infected populations as well (Crosby 
and Roberts 1990. Soniat 1985). The simulation study by Hofmann 
et al. (1995) has suggested that this buffering of infection intensity 
at levels of 3 to 4 on the Mackin scale may be attributable to two 
factors: (1) a decrease in parasite division rate at high parasite 
density; and (2) replacement of oysters that reach a lethal level of 
infection with less heavily infected oysters. 

The northward spread of Perkinsiis mariiiiis into New England 
was not widely anticipated, because it had been viewed as a 
"warm-water" pathogen, which required minimal temperatures of 
20 °C and extended periods of temperatures above 25 °C to es- 
tablish an epizootic (Andrews 1988). Failure to predict the range 
expansion that has occurred may be attributable in part to lack of 
reliable water temperature data for oyster-growing areas. The most 
northerly site in this study. Cotuit, a shallow embayment on Cape 
Cod, experienced the highest mean water temperatures with tem- 
peratures consistently above 25 °C for over a month (Table 3; 
Figure 6), conditions similar to those reported for Delaware Bay 
(Ford 1996). The lowest mean water temperatures were recorded at 
Mystic, a deep-water site strongly affected by tidal exchange in 
and out of Long Island Sound. Site characteristics such as tidal 
exposure, water depth, tidal currents, and proximity to rivers or 
substantial freshwater inflow can be more important factors in 
determining the temperature characteristics of an area than its geo- 
graphic location. 

Infection levels in oyster populations began climbing when 
water temperatures reached 13-16 °C at the Bridgeport. Mystic 
River, Thames River, and Westport sites. This finding supports 

Temp 



TABLE 4. 

Point and interval estimates for day of year with mean weighted 
prevalence of 2.(1 determined for each study site. 




Month 



Month 



Figure 6. Mean temperature curves. Top plot: Bridgeport (maximum 
= 23 °C, solid black). Oyster Bay (maximum = 23 C, solid gray), 
Thames River (maximum = 23 'C, dashed black), Westport (maximum 
= 22 °C, dashed gray). Bottom plot: Cotuit (maximum = 26 °C, solid 
black), Mystic River (maximum = 19 C, solid gray). 







Day of Y 


ear 




Study Site 


Point Estimate 




Interval Estimate 


Bridgeport 
Mystic River 
Thames River 
Westport 


237 
215 
226 






208, 237 
221, 254 
200, 231 
211. 241 



earlier observations made for the Bridgeport population (Brous- 
seau 1996). A later onset of parasite proliferation occurred among 
the juvenile oyster populations at Oyster Bay and Cotuit. when 
temperatures of 20 °C were reached, but the reason for the differ- 
ence in timing is not known. Nonetheless, these results show a 
significantly different pattern of infection development from those 
reported in oysters from locations further south, where tempera- 
tures >20 °C are required for parasite proliferation (Andrews 
1988). The reason for these observed differences in the tempera- 
ture-time course of the disease are unknown, but possible hypoth- 
eses include: ( 1 ) the existence of a low temperature-adapted strain 
of the parasite (Bushek and Allen 1996. Dungan and Hamilton 
1995) and/or (2) physiological differences in the immune systems 
of oysters from different geographic areas. 

Soniat (1985) failed to find a correlation between water tem- 
perature and prevalence or intensity of Peikiiisiis inaiiniis. but 
Crosby and Roberts (1990) found a statistically significant but 
weak correlation between water temperature and Dermo intensity. 
In a .study that introduced lags into the relationship. Burreson and 
Calvo ( 1996) found significant correlation between water tempera- 
ture and both prevalence and intensity of Perkinsiis inarinus in the 
Chesapeake Bay when lags of 2 to 4 months were used. The 
strongest relationship was with a 3-month lag; 46% of the vari- 
ability in weighted prevalence and 39% of the variability in preva- 
lence was explained. 

The model developed for Long Island Sound showed the 
strongest relationship between cumulative temperature and Perk- 
insus marimts intensity when a lag time of 53 days was used. This 
result is similar to previous reports in the literature of significant 
correlations between temperature and parasite intensity when tem- 
perature was lagged by 60 days or more (Burreson and Ragone- 
Calvo 1996). It predicted that the oyster population from the 
Thames River would reach critical disease intensity levels (wp = 
2) by late July/early August; whereas, similar intensity levels 
would not appear in the Mystic River until a month later. The 
eventual impact of the disease may depend on the time of the year 
when critical disease levels are attained. Very high oyster mortali- 
ties were experienced in the Thames River after mid-August 1997 
(Janke pers. comm.) but no unusual mortalities were reported in 
the Mystic River during the year. The oyster mortality at the 
Thames River site may be attributable to high infection levels early 
in the season (Fig. 5) and higher mean temperatures during the 
year (Table 3). Any mortalities that may have occurred at the 
uncultivated sites (Bridgeport and Westport) went largely undocu- 
mented. 

Water temperature is likely the most important single factor 
responsible for the establishment of Perkinsiis marinus in the re- 
gion of study, and although not controllable, knowledge of how the 
disease responds to differing environmental temperature patterns 



138 



Brousseau and Baglivo 



can be helpful in managing oyster stocks in the face of disease 
pressure. Unlike most previous attempts to model the effects of 
environmental factors on the development and activity of P. mari- 
iiiis epizootics (Powell et al. 1992, Hofmann et al. 1995), this 
model has the advantage of being simple to use and having modest 
data requirements. It allows the grower to predict disease intensity 
in shellfish beds if field temperature patterns are known. The 
grower can then use this information in selecting oyster growout 
beds and determining optimal harvesting times for his product. 

Admittedly, one drawback of using such a simple model for 
predicting parasite proliferation in the field is its failure to take into 
account additional factors that may affect local patterns of disease 
progression such as changing size frequency distributions within 
the population, yearly variations in food supply and annual 
changes in disease prevalence (Soniat et al. 1998). Also, this model 
was developed using only one year of data; it would benefit from 
additional tests over a longer time period to substantiate its general 



applicability. In spite of these shortcomings; however, the model- 
ing approach presented here shows promise, and with further test- 
ing could prove to be a useful tool in industry efforts to minimize 
the impact of Dermo disease. 

ACKNOWLEDGMENTS 

We thank S. Ford and R. Smolowitz for providing unpublished 
data on Oyster Bay and Cotuit oysters. Our thanks also go to D. 
Relyea, F. M. Flower & Sons, and T. Janke, Ram Island Oyster 
Co. for providing oysters and valuable conversations during the 
course of this study. The following students; K. Cuniff, J. Guedes, 
C. Infantolino, C. Lakatos, G. LeCleir, R. Pinsonneault, and A. 
Takesy are also appreciated for their help with data collection. The 
final version of the manuscript benefited greatly from the com- 
ments of reviewers, E. Powell and T. Soniat. This research was 
supported by NRAC Grant No. 94-38500-0044. 



LITERATURE CITED 



Andrews, J. D. 1988. Epizootiology of the disease caused by the oyster 
pathogen Perkinsus mariims and its effects on the oyster industry, pp. 
47-63. In: W. S. Fisher (ed.). Disease Processes in Marine Bivalve 
Molluscs. American Fisheries Society Special Publication, American 
Fisheries Society. Bethesda. MD. 

Andrews. J. D. & S. M. Ray. 1988. Management strategies to control the 
disease caused by Perkinsus marinus. pp. 257-264. In: W. S. Fisher 
(ed.). Disease Processes in Marine Bivalve Molluscs. American Fish- 
eries Society Special Publication. American Fisheries Society. Be- 
thesda, MD. 

Brousseau, 0. J., C. Orsine, M. Rios & W. Zavadoski. 1994. Preliminary 
results on Perkinsus prevalence in oyster populations from western 
Long Island Sound (Abstract). J. SItellfish Res. 13:312-313. 

Brousseau, D.J. 1996. Epizootiology of the parasite, Perkinsus nuiriniis 
(Dermo) in intertidal oyster populations from Long Island Sound. / 
Shellfish Res. 15;583-.'i87. 

Brousseau, D. J.. J. C. Guedes, C. Lakatos, G. LeCleir & R. Pinsonneault. 
1998. A comprehensive survey of Long Island Sound oysters for the 
presence of the parasite, Perkinsus marinus. J. Slu-llfish Res. I7;255- 
258. 

Burreson, E. M. & L. M. Ragone-Calvo. 1996. Epizootiology of Perkinsus 
marinus disease of oysters in Chesapeake Bay, with emphasis on data 
since 1985. / Shellfish Res. 15:17-34. 

Bushek, D. & S. K. Allen. 1996. Races of Perkinsus marinus. J. Shellfish 
Res. 15:103-107. 

Chu, F-L. E. & K. H. Greene. 1989. Effect of temperature and salinity on 
the in vitro culture of the oyster pathogen Perkinsus marinus ( Apicom- 
plexa: Perkinsea). / Invertebr. Palhol. 53:260-268. 

Chu, F-L. E. & J. F. LaPeyre. 1993. Perkinsus marinus susceptibility and 
defense-related activities in eastern oysters, Crassostrea virginica tem- 
perature effects. Dis. Aquai. Org. 16:223-234. 

Crosby, M. P. & C. F. Roberts. 1990, Seasonal infection intensity cycle of 
the parasite Perkinsus marinus (and absence of Haplosporidium spp.) 
in oysters from a South Carolina sail marsh. /)/.v. .Aquai. Org. 9:149- 
155. 

Craig, A., E. N. Powell. R. R. Fay & J. M. Brooks. 1989. Distribution of 
Perkinsus marinus in Gulf coast oysler populations. Estuaries 12:82- 
91. 

Dungan, C. F. & R. M. Hamilton. 1995. Use of a tctrazolium-ba.sed cell 
proliferation assay to measure effects of in vitro conditions on Perk- 
insus marinus (Apicomplexa) proliferation. / Pukaryotir Microbiol. 
42:379-388. 

Efron, B. & R. J. Tibshirani. 1993. An inlrodiiclion lo the boolslnip. Chap- 
man & Hall, Inc., New York. 



Ford. S. 1996. Range extension by the oyster parasite Perkinsus marinus 
into northeastern United States: response to climate change? J. Shellfish 
Res. 15:45-56. 

Hofmann, E. E., E. N. Powell. J. M. Klinck & G. Saunders. 1995. Model- 
ing diseased oyster populations. I. modeling Perkinsus marinus infec- 
tions in oy.sters. J. Shellfish Res. 14:121-151. 

Mackin. J. G. 1961. Mortality of oysters. Proc. Natl. Shellfish Assoc. 50: 
21-40. 

Mackin. J. G. 1962. Oyster disease caused by Denytocystidium marinum 
and other microorganisms in Louisiana. Tex. Inst. Mar. Sci. Puhl. 7: 
132-229. 

Mackin. J. G. & S. H. Hopkins. 1961. Studies on oyster mortality in rela- 
tion to natural environments and oil fields in Louisiana. Publ. Inst. 
Mar Sci. Univ. Te.x. 7:1-131. 

Powell, E. N., J. D. Gauthier, E. A. Wilson, A. Nelson, R. R. Fay & J. M. 
Brooks. 1992. Oyster disease and climate change. Are yearly changes 
in Perkinsus marinus parasitism in oysters [Crassostrea virginica) con- 
trolled by climate cycles in the Gulf of Mexico? P.S.Z.N.I.: Mar. Ecol. 
13:243-270. 

Ragone, L. M. & E. M. Burreson. 1994. Characterization of overwintering 
infections of Perkinsus marinus (Apicomplexa) in Chesapeake Bay 
oysters. J. Shellfish Res. 13:123-130. 

Ray. S. M. 19.54. Biological studies of Dermocystidiu/n marinum. a tungus 
parasite of oysters. Rice Institute, Houston, TX. 1 14 pp. 

Ray. S. M. & A. C. Chandler. 1955. Dermocystidium marinum a parasite of 
oysters. Exp. Parasitol. 4:172-200. 

Siddall. M. E., N. A. Stokes & E. M. Burreson. 1995. Molecular phyloge- 
nelic evidence that the phylum Haplosporidia has an alveolate ancestry. 
Mol. Biochem. Evol. 12:573-581. 

Soniat, T. M. 1985. Changes in levels of infection of oysters by Perkinsus 
marinus with special reference to the interaction of temperature and 
salinity on parasitism. N. E. Gulf Sci. 7:171-174. 

Soniat, T. M. & J. D. Gauthier. 1989. The prevalence and intensity of 
Perkinsus marinus from the midnorthern Gulf of Mexico, with com- 
inenls on the relationship of the oyster parasite lo temperature and 
salinity. Tulane Stud. Zool. Sot. 27:21-27. 

Soniat, T. M. & E. V. Kortrighl. 1998. Estimating lime to critical levels of 
Perkinsus marinus in eastern oysters, Cras.wstrea virginica. J. Shell- 
fish Re.s. 17:1071-1080. 

Soniat, T M., E. N. Powell, E. E. Hofmann & J. M. Klinck. 1998. Under- 
standing the success and failure of oyster populations: the importance 
of sampled variables and sample liming. ./. Shellfish Res. 17:1149- 
1165. 



Joiirmil of Shellfish Research. Vol. 19. No. 1. 139-145. 2000. 

OSMOTIC TOLERANCE AND VOLUME REGULATION IN IN VITRO CULTURES OF THE 

OYSTER PATHOGEN PERKINSUS MARINUS 



CAROLINE L. O'FARRELL,'* JEROME F. LA FEYRE,'t 
KENNEDY T. PAYNTERr AND EUGENE M. BURRESON'^ 

^ Departmem of Fisheries Science 

Virginia Institute of Marine Science 

College of William and Mary 

Gloucester Point. Virginia 23062 
'Department of Zoology 

University of Maiyland 

College Park. Maryland 20742 



ABSTRACT Growth rate, cell size, osmotic tolerance, and volume regulation were examined in cells of Perkinsus mariiuis cultured 
in media of osmolalities ranging from 168 to 737 mOsm (6.5-27.0 ppt). Cells cultured at the low osmolalities of 168 and 256 mOsm 
(6.5 and 9.7 ppt) began log phase growth 4 days postsubculture. whereas cells cultured at the higher osmolalities 341. 433. and 737 
mOsm (12.7. 16.0, and 27.0 ppt) began log phase growth 2 days postsubculture. During log phase growth, cells from the higher 
osmolalities 341, 433. and 737 mOsm had shoner doubling times than cells from the lower osmolalities 168 and 256 mOsm. During 
both log and stationary phase growth, the mean cell diameter of cells cultured at 168 mOsm was significantly greater than cells cultured 
at 341 and 737 mOsm: the mean diameters of cells cultured at 341 and 737 mOsm did not differ significantly from each other. P. 
mariniis cells cultured in various osmolalities were exposed to artificial seawater treatments of 56-672 mOsm (2.5-24.7 ppt). After the 
hypoosmotic treatment of 56 mOsm, cells that had been cultured in medium of low osmolality. 168 mOsm. showed only 41% mortality 
whereas the cells from the 34 1-. 433-. and 737-mOsm culture groups experienced 100<7f mortality. During the hyperosmotic shock, all 
of the groups exhibited mortalities of less than 107r. In P. mariiuis cells cultured in medium of 737 mOsm and then placed in a 50% 
dilution, cell diameter increased 13%, which was a volume increase of 44.5%. but cells returned to baseline size (size before osmotic 
shock) within 5 minutes. P. marinits cells cultured at low osmolalities can withstand both hypo- and hyperosmotic stress and use 
volume-regulatory mechanisms during hypoosmotic stress. Results suggest that transferring infected oysters to low salinity will result 
in strains of P. marinus acclimated to low salinity that will be able to withstand periodic events of extremely low salinity. 

KEY WORDS: Osmotic tolerance, volume regulation. Perkinsus mariiuis 



INTRODUCTION 

Perkinsus mariiuis. a parasite of the eastern oyster, Crassostrea 
virginica (Gmelin), was first reported in the Gulf of Mexico 
(Maekin el al. 1950) but is now observed in C. virginica along the 
Atlantic west coast from Maine to Florida and in the Gulf of 
Mexico from Florida to Mexico (Andrews and Hewatt 1957, 
Maekin 1962, Burreson et al. 1994a). Since the 1950s and espe- 
cially since 1986, P. marinus has been a major cause of mortality 
in the eastern oyster in the Chesapeake Bay (Burreson and Ragone 
Calvo 1996). 

The eastern oyster, C. virginica, is an osmoconformer. but the 
osmotic tolerances of the parasites Haplosporidium nelsuni (MSX) 
and P. marinus living within the oyster are not clearly defined 
(Ford and Haskin 1988). Salinity is believed to be an important 
environmental factor that regulates the prevalence and intensity of 
H. nelsoni and P. mariiuis. These two common oyster parasites, 
however, appear to have differing tolerances to hypoosmotic con- 
ditions. Ford (1985) reported a reduced prevalence of H. nelsoni in 
oysters in salinities lower than 15 ppt. Ford and Haskin (1988) 
showed that some killing of H. nelsoni occurred at 15 ppt with 
maximum elimination at 9 ppt. suggesting that the pathogen is 



*Present address: School of Fisheries, University of Washington, Seattle. 
WA 98195. 

tPresent address: Department of Veterinary Science. Louisiana State Uni- 
versity. Baton Rouge. LA 70803. 

ICorresponding author: Eugene M. Burreson. Virginia Institute of Marine 
Science, Box 1346, Gloucester Point, VA 23062-1346. 



physiologically unable to tolerate low salinities. P. marinus toler- 
ates salinities lower than 12 ppt, but the mechanisms that allow 
survival in low-salinity environments have not been clearly de- 
fined (Ragone and Burreson 1993, Burreson and Ragone Calvo 
1996). Studies have shown that low salinity has a retarding effect 
on P. marinus developinent (Ray 1954, Maekin 1962, Soniat 1985, 
Burreson and Ragone Calvo 1996). In addition, it has been re- 
ported that infection intensity of P. marinus is positively correlated 
with temperature and salinity (Soniat 1985, Soniat and Gauthier 
1989. Crosby and Roberts 1990, Burreson and Ragone Calvo 
1996). An in vivo study of oysters infected with P. marinus de- 
termined the critical salinity range for pathogenicity to be between 
9 and 12 ppt. and that P. marinus was less virulent below 9 ppt 
(Ragone and Burreson 1993). Also, the study reported that lower 
salinities (6 and 9 ppt) delayed disease development, whereas in- 
fections at higher salinities (12 and 20 ppt) increased in intensity 
and resulted in higher levels of oyster mortality. 

Despite these findings, little is known about the osmotic toler- 
ance of P. marinus when faced with hypo- and hyperosmotic 
stress. Studies with both free-living and parasitic protozoa have 
shown that many protozoa have the ability to adjust their cell 
volumes when faced with external osmotic changes (Kaneshiro et 
al. 1969, Da Silva and Roitman 1982, Geoffrion and Larochelle 
1984, Ahmad and Hellebust 1986, Andre et al. 1988, Cronkite and 
Pierce 1989, Hellebust et al. 1989, Darling and Blum 1990, Dar- 
ling et al. 1990). Similarly, P. marinus may also utilize physiologi- 
cal mechanisms to adjust to its changing osmotic environment. 
Only one previous study has been conducted on the osmotic tol- 
erance of P. marinus in the absence of host influences (Burreson 



139 



140 



O'Farrell et al. 



et al. I994b|. This study reported that cells cultured at 22 ppt and 
placed in extreme low-salinity treatments ofO and 3 ppt had higher 
than 90% mortality. As a continuation of this work, we investi- 
gated the osmotic tolerances and volume-regulatory abilities of P. 
maiinus cells, which have been cultured in a range of osmotic 
conditions ( 168-737 mOsm or 6.5-27.0 ppt) and exposed to vari- 
ous osmotic treatments (56-672 mOsm or 2.5-24.7 ppt). 

MATERIALS AND METHODS 

In Vitro Cultures of P. marinus 

Cultures of P. marinus were maintained in Jeronie La Peyre- 
Oyster Disease Research Program- 1 (JL-ODRD-1) medium (La 
Peyre et al. 1993) (approximately 737 mOsm or 27.0 ppt) without 
bovine serum albumin (BSA) in a humid atmosphere at 28 °C in a 
5.0% CO, incubator. Cells from the BSA-free acclimated cultures 
were transfened from 737-mOsm culture medium into 168. 256, 
341, and 433 mOsm (approximately 6.5, 9.7, 12.7, and 16.0 ppt) 
media in a gradual procedure in which cells from 737 mOsm were 
placed into 433, 433 into 341 mOsm, etc., with the stepwise trans- 
fer occurring every 3 days. For culture maintenance, subculturing 
occurred every 2^ wk. Cultures were seeded at a density of 5 x 
10* cells per 5 mL of medium for all experiments, and during these 
experiments, subculturing occurred every 2 wk. Growth curves for 
the groups cultured at 168, 256, 341, 433, and 737 mOsm were 
determined by obtaining cell counts with a hemacytometer (Fisher 
Scientific) every day for 12 days starting the day after subculture 
to determine the time period of log phase growth. The growth rate 
study used cells approximately 20 generations (about 1 y ) descend- 
ing from the original cultures that were first acclimated to the 
different osmolalities. A generation is defined as one subculture. 
Cell size experiments used cells that were approximately 25-30 
generations descended from the acclimated cultures. The osmotic 
tolerance experiments used cultures that were 7-10 generations 
descended from the original groups acclimated to the different 
osmolalities. 

Culture Media 

The cell culture medium used for the P. iiniriinis cultures was 
the JL-ODRP-1 (La Peyre et al. 1993) without BSA. Media ( 100 
mL) equivalent to 168. 256, 341, 433. and 737 mOsm were pre- 
pared before each subculture for the different culture groups fol- 
lowing methods described by La Peyre et al. ( 1993). In addition to 
the reported constituents, the culture medium, depending on the 
desired osmolality (168, 256. 341, 433, and 737 mOsm), also 
included basal synthetic sea salts (0.3, 0.6, 0.9, 1.2, or 2.2 g), 0.2 
g NaHCO,. and KCI (0.0061, 0.0079. 0.0097,0.01 15, or 0.0 177 g) 
dissolved in 91.5 mL tissue culture-grade water. 

Cell Sizes of Cultured Cells 

Cell diameters of the various P. marinus groups in both l<ig and 
stationary growth phase were measured by using the NIH Image 
Analysis (Version 1.56) Macintosh computer program for particle 
size analysis and the MediaGrabber Macintosh program with Ras- 
terOps video digitizer board to capture live microscopic images 
from an inverted Zeiss light microscope (4()x objective used in all 
of the cell size experiments). Cell measurement teclini(.|ues with 
image analysis were based on methods described by Weeks and 
Richards (1993). 

Baseline measurements were initially couducletl to dclermine 
whether the groups cullmcd in media with osmolalities ol 168. 



341, and 737 mOsm varied in cell size. For log phase growth size 
distributions, cells cultured in 168-, 34 1-, and 737-mOsm media 
were harvested 6 days after subcultured and transferred to 15-mL 
microcentrifuge tubes. Each group of cells was declumped by 
repeatedly withdrawing the cells and passing them through a 3-mL 
syringe (25G 7/8-inch hypodermic needle). Cells were centrifuged 
at 800 g for 15 minutes, the medium decanted, and the cells re- 
suspended in 10 mL of isotonic seawater. Seawater solutions (173, 
365, and 740 mOsm or 6.7, 13.6, and 27.1 ppt) that were isotonic 
to the culture medium of each group consisted of 97.5 mL tissue 
culture-grade water, basal synthetic sea salts (0.45, 1.05, or 2.35 
g), 0.2 g NaHCO,, KCI (0.0061, 0.0097, or 0.0777 g), and 2.5 mL 
HEPES buffer (original concentration = 239.02 mg/mL). After 
resuspending the cell pellets in the isotonic artificial seawater so- 
lutions, cell solutions were stirred with a vortex mixer (Fisher 
Scientific), and a 10-(xL sample was withdrawn from each group 
for cell counts using a hemacycometer. Volumes containing 1 x 
10°^ cells from each group (168, 341. and 737 mOsm) were cal- 
culated, and these cell solutions were added to three different cell 
wells (three wells per group) in a cell well plate. From each well 
of each of the three groups, three to four images were captured. 
The number of cells per image ranged from approximately 40 to 70 
cells. Clumped cells that could not be easily distinguished were 
excluded. This cell sizing protocol was also followed to measure 
cells cultured at 168, 341. and 737 mOsm in stationary phase 
growth (2 wk after subculture). Mean cell diameters were calcu- 
lated for the culture groups from both log and stationary growth 
phase, and the relationship between culture medium osmolality 
and cell diameter was examined by a one-way analysis of variance. 
Significant differences between the groups cultured at the three 
different osmolalities were determined by using the Scheffe post 
hoc multiple comparison test. 

Osmotic Tolerance 

Buffered artificial seawater (ASW) treatment solutions of 56, 
135. 222. 305, 386, and 672 mOsm (approximately 2.5, 5.3. 8.5, 
1 1.4. 14.4. and 24.7 ppt. respectively) were prepared by dissolving 
synthetic basal salts (Sigma Chemical Co.) (0.0. 0.3. 0.6. 0.9, 1.2, 
or 2.2 g), 0. 1 1 76 g NaHCO,, KCI (0.0014, 0.0044, 0.006 1 . 0.0078, 
0.0097. or 0.0156 g). and 2.5 mL HEPES buffer (original concen- 
tration = 239.02 mg/niL) in 97.5 niL of tissue culture-grade water. 
After adding these constituents, the mixtures were adjusted to a pH 
of 7.5 and then filter sterilized. All of the treatment .solutions, the 
BSA-free media for the culture groups, and the isotonic seawater 
solutions (used for cell size experiments) were analyzed on a vapor 
pressure osmometer (Wescor) to determine osmolalities. Cell den- 
sity by hemacytometer and cell viability of the P. marinus cultures 
were assessed in each culture group ( 168. 256. 341. 433. and 737 
mOsm). To determine cell viability, a lOO-jxL subsample was 
placed in a microcentrifuge tube and 10 p-l of 0.05% neutral red 
stain added. After 10 min. two 1()-|jl1 aliquots were placed on the 
hemacytometer. Both live (stained) and dead (unstained) cells 
were counted for at least 200 cells. From each group. 2.0 x 10'' 
cells were added to sterile 15-mL centrifuge tubes and the volumes 
raised to 7 ml, with the treatment ASW at the osmolality equiva- 
lent to the medium osmolality. Then, 1 niL of each of these cell 
suspensions was centrifuged at 470 i; for 5 min. The supernatant 
was decanted and the pellet resuspended in I niL of each of the 
ireatment osmolalities (ASW) in a 24-wcll tissue culture plate. 
Thus. P. marimis cells cultured in media ol 168, 256. 341, 433. and 



Pf.rkinsi!s Marinus Volume Regulation 



141 



737 mOsm were placed in ASW treatment osmolalities of 56, 135, 
222. 305, 386, and 672 mOsm for 24 hours in 24-well microliter 
plates at 28 °C in an incubator without CO,. After the 24-hour 
incubation. 100 (j.L of neutral red was added and gently mixed 
with a pipette tip. Mortality was assessed by counting live and 
dead cells in two to three random grid fields with an inverted light 
microscope (Zeiss) and a 10 x 10-mm ocular micrometer grid. The 
experiment was repeated three times. Logistic regression analyses 
with SAS procedure Catmod were utilized to examine the response 
of the population (culture group) to the treatment osmolality and to 
calculate predicted mortalities (which describe the response of 
each population) with 95%- confidence intervals for each of the 
culture groups at each treatment osmolality. A logistic regression 
model was chosen to represent the binary response of mortality 
(live versus dead). In addition, the actual live and dead cell counts 
were used for calculating percent mortalities and for an analysis 
that compares proportions from independent samples (Fleiss 
1981). 

Cell Size after Hypoosmotic Shock 

Cell diameter changes following a hypoosmotic shock were 
measured with the MediaGrabber and NIH Image Analysis sys- 
tems. Cells cultured in medium of 737 mOsni were harvested 2-3 
wk after subculture, declumped with a 3-inL syringe (25G 7/8-inch 
hypodermic needle), and centrifuged at 800 g for 15 min. The 
medium was decanted, and isotonic seawater was added to obtain 
a volume of 10 mL. Cell density was determined with a hemacy- 
tometer, and a volume containing 1x10"^ cells was added to a cell 
well. A volume of 173 mOsm ASW was added to the well to result 
in a 50'7f dilution of the original seawater solution. Before adding 
this calculated volume of the hypoosmotic shock solution, an im- 
age was captured to represent time 0. Ten to twenty seconds after 
the 50% dilution, a second image was captured as time 1 . Images 
were then captured at 1, 3, 5, 7, 10, 12, 15, 20. 30. 45, and 60 min 
after dilution. The same cells from the same plane were captured 
as images, and thus, the same population experiencing the shock 
was represented. These images were analyzed with the NIH Image 
Analysis system to determine cell diameters at each time interval. 
The experiment was repeated five times. The first experiment used 
cells 19 days postsubculture. The second experiment used cells 
from a different culture 18 days postsubculture and included time 
points of and 10-20 sec, and 1, 3, 5, 10, 15, 20, and 30 min. The 
third, fourth, and fifth experiments used cells 20 days postsubcul- 
ture and were performed consecutively on the same day with cells 
from the same tlask. Experiments 3. 4. and 5 included images 
captured at 10-20 sec and 1. 3. 5. 10, 15, 20, and 30 min. Cell 
viability was assessed with the vital stain neutral red before the 
shock and 30 min after adding the shock solution. The control 
experiment used cells 21 days postsubculture and followed the 
protocol described above without adding the shock solution; im- 
ages were obtained at 0, 1, 3, 5, 10, 15, 20, and 30 min. Cell sizes 
after hypoosmotic shock were analyzed with the nonparametric 
Kruskall-Wallis test to first examine the effect of each experiment. 
To separate out the significant effect of each experiment but still 
look at the results of all trials together to examine the overall effect 
of the treatment osmolality on cell size, a mean center standard- 
ization was used by subtracting the mean cell diameter (total mean 
diameter for all time points within each experiment) from each 
data point. A second Kruskall-Wallis test was run on the standard- 
ized data to examine whether each experiment continued to have a 



significant effect on cell diameter. The effect of the experiment 
was no longer significant, and the experiments were pooled. A 
third Kruskall-Wallis test was used to determine whether time had 
a significant effect on cell diameter. Lastly, the Tukey-Kramer 
multiple comparison iiosf hoc analysis was implemented to deter- 
mine at which time points the mean cell diameters were signifi- 
cantly different from each other. An unpaired r-test was used to 
determine whether there was a significant difference between the 
standardized control diameters and the standardized replicate di- 
ameters (experiments pooled) both before the shock and 1 min 
after the shock. 



RESULTS 



Growth Rate 



The results of the growth rate study indicated that log phase 
growth began approximately 2 days postsubculture for P. marinus 
cells cultured in 341, 433, and 737 niOsm (12.7, 16.0, and 27.0 
ppt) media. Cultures from the 168 and 256 mOsm (6.5 and 9.7 ppt) 
media began log phase growth approximately 4 days postsubcul- 
ture (Fig. 1 ). The groups cultured at the higher osmolalities of 341, 
433, and 737 mOsm had shorter doubling times compared with the 
groups cultured at the low osmolalities of 168 and 256 mOsm. For 
the 168-mOsm cells, 35.2 h were required for one doubling and 
35.7 h for the 256-mOsm cells. For the higher osmolality cells 
from 341, 433, and 737 mOsm, one doubling required 22.8, 25.9, 
and 24.4 h. respectively. 

Cell Sizes of Cultured Cells 

During log phase growth, the mean diameters (± standard error) 
of P. marinus cells cultured in media of 168, 341, and 737 mOsm 
were 11.8 (±0.191), 9.6 (±0.108), and 9.2 (±0.106) p.m, respec- 
tively. The effect of culture medium osmolality on cell diameter 



CD 
O 

1 — 

X 

a5 
O 



E 

2 



uu- 


Culture Group (mOsm of media) P 




•••■■••■ 


168 / 


50- 




/ 




— G- 


256 / 




— .^- 


341 >° 


40- 


- X- 


433 y^ 




-D- 


737 / /.■*^-~-^ 


30- 




.-/^'■■' 






/ J X 






//,»-^' 


20- 




/rtl «' 

if 


10- 




IM / m^T^-'-e ■ 

/ '^. ■■■-■'' ' 


0- 


_D=Q=d 


^^4-' 



10 11 12 



Days Post-Subculture 



Figure L Growth curve of P. marinus cells cultured in media of 168, 
256, 341, 433, and 737 mOsm (6.5, 9,7, 12.7, 16.0, and 27.0 ppt). 



142 



O'Farrell et al. 



was statistically significant (P = 0.0001). Cells cultured at 168 
niOsm were significantly larger than cells at either 341 (P < 
0.0001) or 737 mOsm (P < 0.0001). The differences in diameter 
between cells at 341 and 737 mOsni were not significant {P = 
0.1565). The mean diameters of stationary phase P. marinus cells 
cultured at 168. 341. and 737 mOsm were 8.4 (±0.165), 4.7 
(±0.070). and 5.1 (±0.093) |jim, respectively. As observed with 
cells from log phase growth, the effect of culture medium osmo- 
lality on cell size was statistically significant (P < 0.0001). Cells 
cultured at 168 mOsm were significantly larger than cells at either 
341 (P < 0.0001 ) or 737 mOsm (P < 0.0001 1, whereas the differ- 
ence in cell diameter between the 341- and 737-mOsm groups was 
only significant at the 5% level of significance (P = 0.021). 

Osmotic Tolerance 

Before osmotic shock, the mean viabilities of the P. marinus 
cells cultured at 168, 256, 341, 433. and 737 mOsm were 88.2%. 
96.2%. 99. 1 %, 99.3%. and 98.8%, respectively. After hypoosmotic 
treatments, the percent mortality was lower in groups that were 
cultured in low-osmolality media than in groups from higher os- 
molalities (Fig. 2). For example, in the extreme hypoosmotic shock 
of 56 mOsm (2.5 ppt). mortality was 41% in cells cultured at an 
osmolality of 168 mOsm but was 100% in cells that were cultured 
at 737 mOsm. Conversely, in the hyperosmotic shock of 672 
mOsm (24.7 ppt), groups that had been cultured at low osmolali- 
ties as well as high osmolalities all experienced mortalities of less 
than 10% (Fig. 2). A logistic regression analysis showed that a 
significant relationship existed between treatment osmolality as a 
function of mortality [P < 0.001). A comparison of proportions 
from independent samples test showed that the mortality response 
of the 168-mOsm group was significantly different {P < 0.001) 
from the mortality observed for the 737-mOsm culture group at the 
56-mOsm treatment. Predicted mortalities determined from a lo- 
gistic regression analysis indicated that in low-osmolality treat- 



100 



90 

80- 

^ 70- 

1 60- 
o 

^ 50- 

I 40- 

°- 30- 

20- 

10- 




Culture Group (mOsm of media) 
-K- 168 

-0-- 256 

-h- 341 
- • - 433 
-B- 737 



100 200 300 400 500 
Treatment Osmolality (mOsm) 



' I ' 

600 



700 



Figure 2. PiTccnl miir(:ilily of /'. marinus ci'lis ciilliired in nu-diii of 
16S. 256. .141. 4.VV unci 7.17 iiiOsin (6.5, 9.7. 12.7. I6.(). and 27.(1 ppll 
and placed in trratinent osnioiaiilii's of 56. 1.15. 222. .1(15, .186, and 672 
mOsni (2.5. 5..1. 8.5. 1 1.4, 14.4. and 24.7 ppl) for 24 h. Krror bars = 
standard error. 



ments. groups cultured at 168 and 256 mOsm have lower mortali- 
ties than the groups that had been cultured at 341. 433. and 737 
mOsm. 

Cell Size after Hypoosmotic Shock 

Cell viability was not affected by the 50% dilution; the results 
of a viability test indicated a 99% viability before the dilution 
(time 0) and 97% viability 30 min after the dilution. From the first 
nonparametric analysis, it was difficult to examine the effect of 
osmolality on cell size because of variability between experiments 
and variability between experimental conditions. After implement- 
ing a mean center standardization, however, nonparametric analy- 
sis indicated that each experiment did not have a significant effect 
on cell diameter (P = 0.8976). Therefore, the results from each 
experiment could then be pooled. The overall response to the 50% 
hypoosmotic shock was an initial swelling followed by a return to 
baseline size (Fig. 3). When placed in the 50% dilution treatment, 
P. marinus cells that were cultured at 737 mOsm experienced an 
initial swelling between and 30 sec after hypoosmotic shock. 
Cells swelled and returned to baseline size within about 5 min. The 
mean diameter change during swelling was 0.7 (jim. The initial 
mean cell diameter was 5.7 (xm. and thus, the percent diameter 
increase during initial swelling was approximately 13%. which 
was a 44.5% change in cell volume. The nonparametric analysis on 
the pooled, standardized data showed that time had a significant 
effect on cell diameter, with a tied P- value of <0.0001. The post 
hoc multiple comparison analysis with a P < 0.05 level of signifi- 
cance indicated that significant differences existed between the 
following time points: and <30 sec. <30 sec and 5 min, <30 sec 
and 15 min, <30 sec and 20 min, and <30 sec and 30 min. The 
unpaired r-test showed no significant difference between the mean 
diameters of the control group and the experimental groups (all 
experiments pooled) at time (P = 0.2931), but there was a 
significant difference at a significance level of P < 0.05 between 
the control group and the experimental groups 1 min after the 
shock (P = 0.0022). 

DISCUSSION 

Continuous cultures of P. marinus can be maintained in low- 
osmolality environments (as low as 168 mOsm or 6.5 ppt). Fur- 




5 10 15 20 25 30 

Time (min) 

I'lgure .1. Standardised mean cell diameter (pm) of P. marinus cells 
cultured al 7.17 niOsm (27.(1 ppl) and placed in 5(l'^f hvpoosmolic 
shock (arrow) wilh evperimenis 1-5 pooled and Ihe control experi- 
ment. Arrow indicates aclual lime of shock: time represents lime after 
shock. Krror bars = standard error. 



Perkinsus Mar/nus Volume Regulation 



143 



therniore. cells maintained in osmolalities ranging from 168 to 737 
mOsm (6.5-27.0 ppt) are tolerant of hypo- and hyperosmotic con- 
ditions in the treatment range of 222-672 mOsm (8.5-24.7 ppt). 
Cells cultured at low osmolalities can also withstand extreme low 
osmolalities such as 56 mOsm (2.5 ppt) for at least 24 hours. Thus, 
these experiments have shown that cultured cells of P. marinus can 
survive both hypo- and hyperosmotic stress. During hypoosmotic 
stress, cells increased in diameter, followed quickly by a return to 
baseline size (size before osmotic shock), which indicates a vol- 
ume-regulatory response. This response helps explain why P. 
marinus continues to persist in the Chesapeake Bay despite periods 
of low salinity that occur during times of high rainfall and runoff 
into the tributaries. 

The growth rate study showed that P. inaiiniis cells that were 
cultured at osmolalities of 341. 433, and 737 mOsni (12.7, 16.0, 
and 27.0 ppt) reached log phase growth before cells cultured at 
lower osmolalities of 168 and 256 mOsm (6.5 and 9.7 ppt). In 
addition, cells from higher osmolalities had greater rates of mul- 
tiplication (shorter doubling time) during log phase than cells cul- 
tured in low osmolalities. These results correspond to a study with 
trypanosome cultures that showed that media of high osmolality 
supported greater multiplication rates than low-osmolality media 
(Da Silva and Roitman 1982). 

The osmotic tolerance study indicated that P. marinus cells 
cultured at low osmolalities experienced reduced mortality when 
placed in extreme hypoosmotic conditions when compared with 
the groups cultured at higher osmolalities. Because the cells were 
already acclimated to the stress of a low-osmolality environment, 
they were able to withstand an extreme low osmolality of 56 
mOsm better than cells cultured at much higher osmolalities. Ap- 
proximately 60% of the 168-mOsm cultured cells survived the 
extreme low osmolality of 56 mOsm for at least 24 hours. In this 
study, all of the culture groups had low mortalities (<10'7r) after 
hyperosmotic stress. Consequently. P. marinus was more tolerant 
of hyper- than hypoosmotic shock. 

This research showed that the stressor did not seem to be the 
magnitude of the shock, but instead the type of shock (hypo- or 
hyperosmotic) and the actual osmolality of the challenge treat- 
ment. For example, the 737-mOsm cells placed into 222-mOsm 
treatment (a difference of 515 mOsm) had much higher mortality 
than cells from 168-mOsm placed into 672-mOsm treatment (a 
difference of 504 mOsm). Although the magnitude of the shock 
was about the same, the hypoosmotic rather than the hyperosmotic 
environment was more stressful, as indicated by higher mortality 
levels. In addition to the type of stress, the actual osmolality of the 
stress affected the level of mortality. For instance, cells acclimated 
to 737 mOsm and placed into 386-mOsm treatment (a difference 
of 351 mOsm) had much lower mortality «10'7r) than cells from 
433 mOsm placed into 56-mOsm treatment (a difference of 377 
mOsm), which resulted in 100'7f mortality. Although the magni- 
tude of both of the hypoosmotic shocks was similar, mortality was 
higher in the treatment with the lowest absolute osmolality, indi- 
cating the cells may have a threshold osmolality level needed for 
survival. 

The results from the osmotic tolerance experiment differ from 
the study by Burreson et al. (1994b), which reported much higher 
mortality levels in cells acclimated to 737 mOsm and placed in the 
same hypoosmotic treatments. The study by Burreson et al. 
( 1994b) showed greater than 607^ mortality for cells acclimated to 
737 mOsm and placed in treatments of 1 36 mOsm and 2 1 3 mOsm, 
whereas this study reports 15^0% mortality in the same low- 



osmotic treatments. One difference is that Burreson et al. (1994b) 
used P. marinus cells cultured in medium with BSA (known as 
JL-ODRP-1 media), whereas this study used cells cultured in 
BSA-free medium. However, comparative experiments with cells 
acclimated to either medium with BSA or BSA-free medium 
showed no significant difference between the effects of the two 
types of media on osmotic tolerance. Other factors that may have 
contributed to the differences in mortality between this experiment 
and the previous one include reported differences in experimental 
design such as the age of the cells (numbers of subcultures since 
isolation and initiation), growth phase of the cells, and type of 
incubator used (CO, or without CO,). For example, the cells in the 
previous study were transferred to an incubator without CO, for 1 
week before use, whereas cells in our experiment were in an en- 
vironment without CO, for only 1 day. The prolonged exposure to 
an environment without CO, may have stressed the cells in the 
previous study, making them more susceptible to mortality after 
osmotic shock. Growth rates are reduced in cultures that have been 
transferred to an incubator without CO, when compared with cul- 
tures maintained in a 5.09<- CO2 incubator (La Peyre, personal 
observation). 

Cells cultured at the low osmolality of 168 mOsm were sig- 
nificantly larger than cells cultured at the high osmolalities of 341 
and 737 mOsm during both log and stationary growth phases. The 
cells cultured at the high osmolalities of 341 and 737 mOsin. 
however, were not significantly different from each other in size. 
The difference in cell size may be due to increased water content 
required to match the low osmolality of the dilute external me- 
dium. A study with red coelomocytes of the euryhaline polychaete 
Gtycera dibranclnata showed cells acclimated to a lower osmo- 
lality had a higher "body-wall-tissue water content" and greater 
cellular volume than cells acclimated to a higher osmolality (Costa 
et al. 1980). An experiment with the amoeba Acanthamoeba cas- 
tellanii indicated that the amount of intracellular water increased 
when cells were placed in a severe hypoosmotic shock (Geoffrion 
and Larochelle 1984). Similarly, the gradual acclimation of the P. 
marinus cells from high- to low-osmolality media when develop- 
ing low-osmolality cultures may have caused an increase in size as 
water initially diffused into the cells, and the cells cultured in the 
low osmolality may not have been able to completely return to 
baseline size during volume regulation because of the stress of the 
prolonged hypoosmotic environment. Cells must maintain certain 
levels of metabolites to survive the stress of a low-osmolality 
environment. These levels of solutes attract water molecules be- 
cause of simple diffusion, and therefore, an increased intracellular 
water content results. Studies on the erythrocytes of the bivalve 
Noetia ponderosa (Amende and Pierce 1980. Smith and Pierce 
1987) and a report on the euryhaline ciliate Paramecium callcinsi 
(Cronkite and Pierce 1989) indicated that cells may not always 
completely return to baseline (size before osmotic shock) after 
volume regulation. 

Alternatively, the difference in cell size of the groups cultured 
at low versus high osmolalities may be due to a difference in life 
stages of the groups that were measured. Cells of P. marinus 
divide by schizogony with a cell increasing in size, acquiring a 
vacuole, and then releasing several daughter cells (La Peyre and 
Faisal 1997). This process could have been occurring with some of 
the cells from the low-osmolality cultures during the cell-size ex- 
periment, as both small cells and large cells with smaller cells 
inside them were observed, whereas the groups measured at higher 
osmolalities mainly consisted of small cells. Thus, because the 



144 



O'Farrell et al. 



low- and high-osmolality groups had different growth rates, they 
may not have been at the same growth stage when their cell di- 
ameters were measured, which could account for the differences in 
size between the groups. The cells from higher osmolalities were 
not observed as a large parent cell dividing into several smaller 
cells, but instead, one cell often appeared to divide into two (data 
not shown). P. marinus cells with high growth rates appear to 
divide as one small cell dividing into two cells (La Peyre 1996). 
The cells at the low osmolality, however, may be larger in size 
even before schizogony because of an increased internal water 
content. Further studies examining the relationship between me- 
dium osmolality. P. marinus growth stage, and cell size would be 
useful in understanding the role of osmolality in P. marinus growth 
and survival. 

During the short-term hypoosmotic stress experiment in this 
study, P. nmriniis cells followed a typical cell volume response 
that is observed in other organisms by experiencing an initial 
swelling and then shrinkage back toward baseline (Costa et al. 
1980, Smith and Pierce 1987. Cronkite and Pierce 1989, Darling et 
al. 1990). The results indicate that P. marinus cells do not resist 
swelling during sudden or extreme external osmolality changes. 
The size at the maximum swell was significantly different from the 
initial baseline and the acclimated sizes. The erythrocytes of the 
clam N. ponderosa exhibited a similar pattern when cells accli- 
mated to 935 mOsm were placed in a hypoosmotic shock of 560 
mOsm; the cells swelled, thereby increasing their volume by 50% 
within 5 min followed by a gradual return toward ba.seline (Smith 
and Pierce 1987). Because the cells in this study did swell and 
return to baseline size, the results suggest that P. marinus regulates 
the intracellular osmotic concentration to regulate cell volume dur- 
ing changing external osmolalities. The results reported here along 
with other studies by our laboratory (data not shown) and by 



Paynter et al. (1997) on intracellular osmolytes (i.e., free amino 
acids) used by P. marinus indicate that P. marinus cells utilize 
volume-regulatory mechanisms to compensate for osmotic 
changes in the external medium. These mechanisms enabled cells 
in this study to survive a 50% dilution of the external medium. But 
to better describe the specific volume-regulatory mechanisms used 
by P. marinus. current studies are focusing on measuring the levels 
of intracellular inorganic ions and organic molecules before, dur- 
ing, and after osmotic shock to determine their role in volume 
regulation. 

The results of these experiments help explain why P. marinus 
continues to persist in the upper portions of the Chesapeake Bay 
tributaries despite periods of low salinities. Periodic increases in 
stream flow causing lowered salinities have not greatly affected 
the abundance of P. marinus in Chesapeake Bay tributaries (Bur- 
reson and Ragone Calvo 1994, Ragone Calvo and Burreson 1995). 
The fact that low salinities have not eradicated the pathogen from 
these areas may be explained by the results in this osmotic toler- 
ance study that indicate P. marinus can use volume-regulatory 
mechanisms to adapt to changing external osmolality and become 
acclimated to extreme low osmotic conditions. Transferring in- 
fected oysters to low salinities may exacerbate the P. marinus 
problem by allowing acclimation of the parasites to lower salini- 
ties, thereby making them more tolerant of extremely low salini- 
ties. As a result, strains of P. marinus that are tolerant of a wide 
range of fluctuating salinities, including extremely low-salinity 
environments, may develop. 

ACKNOWLEDGMENTS 

We appreciate the assistance of Lisa Ragone Calvo in experi- 
mental design and Robert Diaz in statistics. VIMS contribution 
number 2295. 



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Journal uf Shellfish Research. Vol. 19. No. 1. 147-151, 2000. 

PRODUCTION OF TETRAPLOID PEARL OYSTER (PINCTADA MARTENSU DUNKER) BY 
INHIBITING THE FIRST POLAR BODY IN EGGS FROM TRIPLOIDS 



MAOXIAN HE, YUEGUANG LIN, QI SHEN, JIANXIN HU, AND 
WEIGUO JIANG 

South China Sea Institute of Oceanology 
The Chinese Academy of Sciences 
164 West Xingang Road 
Guangzhou. China 510301 

ABSTRACT All previou,s attempts to produce viable tetraploid pearl oyster (Pinctudu martcnsii Dunker) by inhibiting the first polar 
body and the first mitosis have failed. This study aims to test the possibility of producing viable tetraploids by the intentional process 
of crossing triploid females and diploid males following the inhibition of the first polar body. When 0.5mg/L CB was applied to inhibit 
the release of the first polar body, about 16.69% of embryos developed as tetraploids, the majority of embryos were aneuploids 
(65.48%); about 82% embryos developed as aneuploids in the control group (TDl ). but no tetraploid embryos were found. Ploidy of 
embryos in the TDl group mainly fell between 2n and 3n. but ranged from 2n to 5n in the TDCB group. During rearing period, larvae 
died heavily. At Day 51 post-fertilization. 2125 spat were harvested, averaging 0.033% of D-larvae cultured. Chromosome analysis 
revealed that 1 15 one-year-old pearl oysters consisted of 28.70% diploids (n = 33), 40.87% triploids (n = 47), 1.74% tetraploids (n 
= 2) and 28.70% aneuploids (n = 33) with 29, 30, 40, 41. and 43 chromosomes. Comparison of growth showed that aneuploids was 
not significantly different from diploids in both shell length and body weight (f > 0.1 ). but significantly smaller than triploids (P < 
0.05). This study demonstrated that the production of viable tetraploid pearl oysters with eggs from triploids is possible, and certain 
levels of aneuploidy can be tolerated in this species. 

KEY WORDS: Tetraploid. aneuploid. triploid. Pinctada marlensii (D.) 



INTRODUCTION 

Artificial triploid pearl oysters, Pinctada inartensii (D.), have 
been successfully obtained (Jiang et al, 1987), Because of their 
reduced gonadal development, triploid pearl oysters grow faster 
than diploids (Jiang et al. 1993), and pearls cultured in triploids are 
significantly bigger than pearls from diploids in pearl size, weight, 
and pearl layer (Lin & Jiang 1993). On the other hand, the mor- 
tality of triploids isn't different from that of diploids during the 
adult stage (Lin et al. 1996). All of which suggest a promising 
future for pearl culture by using triploid pearl oysters. Now pilot- 
scale testing of pearl culturing in triploids is being conducted in 
China. However, the method of inducing triploids by inhibiting 
polar bodies rarely produces 100% triploids, and treatment of in- 
duction may have deleterious effects on the survival and growth of 
induced triploids. If crossing tetraploids and diploids could pro- 
duce all-triploids as expected, the use of tetraploids may eliminat 
these problems. Tetraploid is commonly induced by inhibiting the 
first polar body, the first mitosis division or cell fusion. However, 
most of previous attempts to produce viable tetraploids in several 
species have failed (Stephens & Downing 1988; Diter & Dufy 
1990: Guo et al. 1994; Jiang et al. 1998), which has eluded re- 
searchers leading to doubt that tetraploids were inviable in shell- 
fish. Tetraploid embryos of pearl oyster were produced with sev- 
eral methods, including inhibition of the first polar body, the first 
cleavage division with cytochalasin B (CB) or pressure, and cell 
fusion with PEG, but none lived to adult age (Jiang et al. 1998). 

Although many attempts to induce viable tetraploids in mol- 
lusks have failed, there are a few reports of success. For example. 
Scarpa et al ( 1993) produced tetraploid mussel {Mytilus gallopro- 
vincialis) as an incidental product by inhibiting both the first and 
second polar bodies with CB treatment. Out of 29 mussels sampled 
at 82 days after fertilization, 5 were tetraploids (17%). Tetraploid 
Manila clams. Tapes pliilippinarum (Adams and Reeve), were 
found in offspring produced by blocking the first polar body to 



induce triploids (Allen et al. 1994). Guo and Allen (1994a) re- 
ported that 67% of tetraploid juveniles produced by the inhibition 
of the first polar body of eggs from triploid Pacific oysters (Cras- 
sostrea gigas Thunberg), and all-triploid Pacific oysters have been 
produced by mating tetraploids and diploids (Guo et al. 1996). 
These reports renew interests in tetraploid induction in shellfish. 
This study aims to induce tetraploidy with pearl oysters by 
crossing triploid females and normal diploid males following the 
inhibition of the first polar body, and look into the possibility of 
this intentional process to induce tetraploid peari oysters. 

MATERIALS AND METHODS 

Triploid pearl oysters, Pinctada marlensii (D.), used in this 
study were produced from 2n x 2n crosses by inhibiting the first 
polar body with CB treatment in 1996. Ploidy was confirmed by 
chromosome count prior to spawning. Gametes were obtained by 
dissecting gonads, and were passed through a 100 \xm screen to 
remove the large tissue debris. Fertilization was conducted at 24- 
25 °C. Eggs from triploid females (about 7 cm in shell length and 
2.5 cm in shell width) were fertilized with sperm from normal 
diploid males in 0.6%c ammonia-seawater and treated with 0.5 
mg/L CB to block the release of polar body 1 (as TDCB groups). 
CB treatment started at 6 min after fertilization and lasted 15 min 
After the treatment, eggs were rinsed with 0.1 7f DMSO in seawa- 
ter and cultured at a density of about 1/mL in filtered seawater. The 
remains of feed and dead larvae were removed at regular intervals 
to maintain water quality. The resulting spat were cultured in the 
sea. The first treated group (TDCB 1 ) had one female parent; 
the other three groups had two triploid females respectively. The 
group receiving no CB treatment is as the control (TD), only the 
first group had a control (TDl). All groups shared one diploid 
male. The experiments were conducted on April. 23, 1998. 

To examine the ploidy of embryos, samples of developing zy- 
gotes of 2-cell stage were taken, and treated with 0,05% colchicine 



147 



148 



He et al. 







TABLE 1. 






The ploidy 


of embryonic 


cells in the treated groups 


and the control. 




Diploid 


Triploid 


Tetraploid 


Aneuploid 


Group 


(%) 


(%) 


(%) 


(%) 


TDCBl 


15.89 


11.21 


20.56 


52.34 


TDl 


12.00 


6.00 


0.00 


82.00 


TDCB2 


13.73 


8.82 


22.55 


54.90 


TDCB3 


5.83 


5.85 


11.65 


76.69 


TDCB4 


5.00 


5.00 


1 2.00 


78.00 



for 15 min, then fixed with Camoy's solution (1;3 glacial acetic 
acid and absolute methanol). Fixatives were changed twice. Chro- 
mosomes were observed by acetic orcein stain. Briefly, drops of 
fixed samples were spread on a slide, stained with 1-2 drops of 
orcein stain (2% orcein in 50% acetic acid), and after 15-30 sec, 
covered with a cover glass and pressed gently. Slides were exam- 
ined with a LEICA DMLS microscope; photographs were taken 
with black-and-white film with speed set at 100 ASA. Ploidy of 
embryos were determined according to 2n = 28 ± 2, 3n = 42 ± 
2, 4n = 56 ± 2, others as aneuploids (normal diploid pearl oyster 
has 28 chromosomes). About 100 embryonic cells with good 
metaphases were counted for chromosome analysis in each group. 
When pearl oysters reached 4 to 6 cm in shell length (on June 
6, 1999), 230 pearl oysters were sampled. Each was numbered and 
measured for shell length (SL) and whole body weight (BW); a 
piece of gill was removed for chromosomal analysis. Gill tissues 
were treated with 0.05% colchicine in 50% seawater for Ih. then 
treated with 25% seawater ( 1 part seawater/ 3 parts distilled water) 
for 30 min, and fixed in a freshly prepared Carney's solution with 
three changes of 20 min duration. The tissue was stored overnight 
in fixatives in 4 °C. The next day the fixative was replaced by 50% 
acetic acid, gill tissue was treated for 10-30 min. then 2-3 drops 
of the resulting cell suspension were dropped onto a warmed slide 
(40-50 °C) and dried. Slides were stained with 10% Giemsa 
(pH6.8) for 40-60 min. Ploidy was determined by examining no 
less than five chromosome metaphases with the same chromosome 
numbers from gill cells. Individuals with 28. 42. and 56 chromo- 
somes were classified as diploid, triploid and tetraploid respec- 
tively; any derivation from the euploid chromosome numbers was 
classified as aneuploid. Growth comparison between aneuploid 
and ployploid was conducted by Student's r-test. 

RESULTS 

The female parent used in TDCBl had approximately 61.92 
million eggs, almost equal to normal diploids of the same size. The 
number of eggs obtained from seven triploid females in this study 
varied between 2.07 and 61.92 million. The average diameter of 



2b 




20 


I 








\ 


— •-TUBi 

— o— TOI 




=1 
£ 10 


\M\f\ 


5 
n 


mAmJD^^ 


-oSSASi 



(N n fO 



s 



Chromosome numljer 
Figure 1. The distribution of chromosome number of embryonic cells 
in TDCBl and TDl. 

eggs from triploids was 57 jjim, 14% larger than eggs from diploids 
(50 (Jim), which corresponded to about 50% increase in cell vol- 
ume. 

Chromosome examination showed that the ploidy level differed 
greatly among groups (Table I ). On average, there were about 
16.69 ± 5.68% tetraploid embryos in the treated groups, while 
most of embryos were aneuploids, averaging 65.48 ± 13.75%. The 
percentage of aneuploids (82.00%) in TDl was higher than that of 
TDCBl (52.34%). but the percentage of polyploid (18.00%) was 
significantly lower than that of TDCBl (47.667f ). no tetraploids 
were found in the control. The distribution of chromosome num- 
bers among embryos in TDl and TDCBl is showed in Figure I. 
Ploidy of embryos in TDl mainly fell between 2n and 3n. but 
chromosome numbers in TDCB I ranged considerably, from 2n to 
5n. and there was a peak in 4n. 

The survival of the developing eggs in several stages is pre- 
sented in Table 2. Due to a heavy mortality in TDCB2 and TDl. 
larvae were too few to be sampled for collecting data. The survival 
of treated groups varied greatly. At 6 days after fertilization, the 
mean survival rate was 24.01% of the total number of D-stage 
larvae cultured. At Day 22, only 2.05% of larvae survived. At Day 
51,2125 spat of 0.5cm in size were harvested. In TDCB 1,355 spat 
were harvested (0.0295% of D-larvae cultured). 737 spat in 
TDCB3 (0.0164% of D-larvae cultured). 1033 spat in TDCB4 
(0.0531% of D-larvae cultured), the mean harvest rate was 
0.033%. 

After I -year culture in the sea. shell length had reached 4 to 6 
cm. 230 pearl oysters were sampled for size and weight, and ploidy 
determination. One hundred and fifteen pearl oysters had good 
chrtimosome metaphases and their ploidy was determined, the 
ploidy of others could not be determined due to too few 
metaphases. Among these 115 samples, two (1.74%) were tetra- 
ploids with 56 chromosomes. 33 (28.70%) were diploids, 47 
(40.87%) were triploids. and 33 (28.70%) were aneuploids with 
29. 30, 40. 41. and 43 chromosomes (Table 3). Reprcsentati\'e 



TABLE 2. 
The number of D-larvae cultured and the survival at several stages in TDCB groups. 





Number of D-larvae 
cultured (xlO") 






% Surv 


val 






Group 


Day 6 (D-stage) 


Day 12 


Day L«! 




Day 22 (eyed stage) 


Day 51 (spat) 


TDCBl 
TDCB3 
TDCB4 


1.2(1 
4.50 
1.95 


30.4 1 
23.17 
18.45 


24.78 
8.50 
9.99 


3.31 
5.90 
6.31 




0.96 
1.63 

3.57 


0.0295 
0.0164 
0.053 1 



Production of Tetraploid Pearl Oyster 



149 



TABLE 3. 
Individuals observed and the ploidy of adult pearl oysters from induced groups of triploid females x diploid males 





Total 


Tetraploid 


Diploid 


Triploid 






Aneuploid 








Total 


29 


3(1 40 


41 


43 


Number 
Percentage 


115 


1.74 


33 
28.70 


47 
40.87 


33 
28.70 


10 


5 2 


13 


3 



metaphases of ployploids and aneuploids are presented in Figure 2. 
Analysis of ;-test showed tliat triploids were significantly big- 
ger than diploids (P < 0.03). suppoiling our previous findings 
(Jiang et al. 1991). Aneuploids were significantly smaller than 
triploids in mean shell length and body weight (P < 0.05). but were 
not significantly different from diploids (P > 0. 1) (Table 4) One 
tetraploid had the size of 5.38 cm in SL and 22. 2g in BW, one was 
5.35 em in SL and 21g in BW. The body weight distribution of 1 15 
samples is presented in Figure 3. 

DISCUSSION AND CONCLUSIONS 

Triploid shellfish are commonly assumed to be sterile due to 
their retarded gonadal development. Retarded gonadal develop- 
ment and abnormal gametogenesis have been confirmed in 
Pinctada martensii (Komaru and Wada 1990; Jiang et al. 1990) 
and several other species. It is interesting that some female and 
male triploids in mollusks can produce numeral gametes and fer- 
tilize with normal diploids, even produce offspring (Allen 1987; 
Allen and Downing 1990; Guo 1991; Komaru and Wada 1994; He 
et al.l996). In this study, 2-year-old triploid females had between 
2.07 and 61.92 million eggs. Reasonable fecundity in triploid fe- 
males makes it possible to produce tetraploids through this tech- 
nique, but this maybe puts breeders in the unusual position of 







*fl 











U 



5t. 



f\- 



*v 



rAi 







i. 




HSJ^^J- 



Figure 2. Representative metaphases of adult Pinctada martensii (D.I. 
A: 2n = 28, B: 2n + 1 = 29, C: 2 n + 2 = 30. D: 3n - 2 = 40, E: 3n - I 
= 41, F: 3n = 42, G: 3n + 1 = 43, H: 4n = 56. 



needing non-reproductive triploids for commercial culture. How- 
ever, the previous research has demonstrated that the daily growth 
rate of triploids is obviously greater than that of diploids during the 
reproductive period, and there is no significant difference in most 
months of the non-reproductive period (Jiang et al. 1991). This 
result implies that a small proportion of matured triploids has no 
obvious effect on the advantage of faster growth. On the other 
hand, retarded gonadal development is not the only reason why 
triploids grow faster than diploids. 

The TD cross primarily produced aneuploid embryos with 
chromosome number between 28 and 42, with an average ploidy of 
2.5n (35 chromosomes), which agrees with previous observations 
(He et al. 1996). However, no larvae survived through metamor- 
phosis in our experiments. An exception is that juvenile Japanese 
pearl oysters. Pinctada fticata martensii (a subspecies, Jiang et al. 
1993). survived in TD with ploidy of 2n and 3n (Komaru and 
Wada 1994). This result differs from the ploidy composition (2n, 
3n, and 4n) of Pacific oyster offspring in TD (Guo and Allen 
1994b). In TDCB, tetraploid embryos were 16.69%. yet at adult 
age, only 1.74% of tetraploids survived. But, 67% tetraploid Pa- 
cific oysters were produced by this method (Guo and Allen 1994a). 
The percentage of adult aneuploid was 28.70%', smaller than that 
of early embryo stage. The ratio of diploid and triploid in adult age 
increased to 69.57% from 16.37% in early embryo stage. Spat 
harvested were about 0.033% of D-stage larvae cultured. These 
data suggested that most of tetraploids and aneuploids died during 
rearing or culturing. Guo and Allen ( 1994a) reported that spat of 
Pacific oysters were harvested from only one of three replicates, 
which were about 0.0738% of the developing eggs. These showed 
there was a heavy mortality of larvae produced by crossing triploid 
females and diploid males. It is concluded that lower fecundity of 
triploids and lower survivorship of larvae may restrict the potential 
of this technique for producing viable tetraploids. 

Guo ( 1991 ) suggested that the inviability of induced tetraploid 
oysters might be due to a cell-number deficiency caused by the 
cleavage of eggs of normal volume with a large, tetraploid nucleus. 
In oysters and most other mollusks, development is mosaic. Unlike 
shellfish, tetraploid fish and amphibians have been obtained; their 

TABLE 4. 

Comparison of body size and weight between aneuploids and 
euploids in Pinctada martensii (D.). 











Shell 




Chromosome 


Individuals 


Body 


length 


Ploidy 


numbers 


observed 


weight (SE)/g 


(SE)/cm 


Diploid 


28 


33 


23.38(4.75) 


5.07 (0.40) 


Triploid 


42 


47 


28.01 (7.80) 


5.37 (0.65) 


Aneuploid 




33 


20.62(7.83) 


4.91 (0.78) 


Tetraploid 


56 


2 


21.60(0.851 


5.37 (0.02) 



150 



He et al. 



en 

D) 

O 
CO 



4 






tl^ 



»- 



t 



t 



26 28 30 32 34363840 42 44464850 52 545658 

Chromosome numbers 

Figure 3. The body weight distribution of Piiictada martensii (D.) with 
different chromosome numbers. 

development is not affected by the cell number deficiency prob- 
ably because the development is regulative. The problem of cell 
number deficiency in tetraploid embryos might be eliminated by 
an increase in the egg volume. Eggs from triploids are larger than 
eggs of diploids, probably led to a significant reduction in cell 
number deficiency and therefor the survival of tetraploids. Pro- 
duction of viable tetraploid Pacific oysters (Guo and Allen 1994a) 
and pearl oysters in this study supported the cell number defi- 
ciency hypothesis. But, why are tetraploids of Mytilus gallopro- 
vincialis and Tapes philippiiuintm produced from eggs of diploids 
viable, whereas tetraploid Pacific oysters and pearl oysters pro- 
duced from diploid eggs are not? Eggs of Mytilus galloprovincialis 
have a diameter of about 66-70 [jtm and eggs of Tapes philippi- 
nanon are 55-60p.m, larger than the diameter of eggs from Pacific 
oysters (47.8 |j.m) and pearl oysters (50 (xm). The remarkable 
increase in egg volume may account for it. It may reflect species- 
species difference in tolerance to tetraploidy. Although only two 
tetraploids were produced in this study, this finding indicated that 
tetraploidy can be tolerated in Piiictada martensii (D.), and dem- 
onstrated that this method of producing tetraploids is viable. 

It is seen that inhibition of the first body release increased the 
ratio of polyploid embryos, which maybe result from a variety of 
segregation patterns in meiosis (Guo et al. 1992b). Que et al. 
(1997) reported that the pattern of chromosome segregation in 
meiosis was changed when CB was applied to inhibit the polar 
body in eggs from Iriploids. Four types of segregation such as 



tripolar segregation, united bipolar segregation, separated segre- 
gation and incomplete united bipolar segregation were observed. 
Similar patterns of chromosome segregation were found in pearl 
oysters (unpublished data). Guo and Allen (1994a) and Que et al 
(1997) suggested the production of tetraploids was as a result of 
united bipolar segregation. According to this type of segregation, 
the united chromosome will undergo equational division, 42 chro- 
mosomes are rejected as the first polar body, and 42 chromosomes 
remain in the eggs, combining with haploid sperm (14 chromo- 
somes) producing tetraploid. In the TD cross, the majority of fer- 
tilized eggs went through two meiotic divisions and released two 
polar bodies, the extra set of chromosomes segregated randomly. 

In addition to ployploid pearl oysters, this process also pro- 
duced many aneuploids. This study provided another evidence that 
certain aneuploids are viable in shellfish. The viability of aneu- 
ploid has been reported in Pacific oysters (Guo and Allen 1994a: 
Wang et al. 1999) and Pacific abalone (Haliotis discus liaimi) 
(Fujino et al. 1990). The data showed that aneuploid pearl oysters, 
as a group, are not significantly different from diploids in shell 
length and weight. In contrast, aneuploid Pacific abalone shows no 
growth retardation, actually they are bigger than normal diploids 
(Fujino et al. 1990); aneuploid Pacific oysters (3n ± n) are larger 
than diploids (Wang et al. 1999), but probably because of their 
triploidy, not aneuploidy. 

It is interesting to note one pearl oyster with 43 (3n -i- I) 
chromosomes is the largest in body size and the second largest in 
body weight in this study. Guo and Allen (1994a) reported that one 
of the aneuploid oysters with 38 (4n - 2) chromosomes is the 
largest by whole body weight. These findings suggested that some 
aneuploids have the growth advantage and the potential applica- 
tion to aquaculture through breeding and selection. Certain aneu- 
ploids may also be useful in genetic manipulation. For example, 
trisomies and monosomies are of use of the gene transfer or gene 
identification. The use of aneuploid has successfully lead to the 
transfer of leaf rust resistance from a w ild grass {Aegilops umbel- 
lidata) to wheat (Sears 1956). 

Further research will focus on the growth and use of aneuploid 
pearl oysters, and on how to raise the survival rate of tetraploids. 

ACKNOWLEDGMENTS 

This study was supported by "863" Project in China (#863- 
819-01-03) and Natural Science Foundation of Guangdong. China 
(#990315). 



LITERATURE CITED 



Allen. S. K. .Ir. 1987. Reproductive sterility ol IriploiJ shelltlsh and lish, 
Ph.D. dissertation. University of Washington, Seattle, Washinglon, 

Allen, S. K. Jr. & S. L. Downing. I'M). Performance of triploid Pacific 
oyster, Cnissoslreu gigas: Gametogenesis. Cun. J. Fi.sli. Aijiiat. Sci. 
47:1213-1222. 

Allen. .S. K. Jr.. M. .Shpigel. S. Uning & B. Spencer. 1994. Incidental 
production of tetraploid Manila clams. Tiipcs pliilippiihiniin. Ai/iiariil' 
lure 128 (1-21:13-19. 

Diter. A. & C. Duty. 1990. Polyploidy in the manila clams Riuliiapes 
philippinarum. Chemical induction of tetraploid embryos. Aqual. Liv- 
ing Resntir. 3:107-112. 

Fujino. K., K. Aral, K. Iwadave. T. Yoshida & S. Nakajima. 1990. Induc- 
tion of gynogenelic diploid by inhibiting 2nd meiosis in the Pacific 
abalone. Hull. Jap. Soc. Sci. Fisheries 5&.\lf<5-\^f>^. 



Guo. X. 1991. Studies on tetraploid induclion in tlie Pacific oyster, Cra.v- 
scisirea gigas (Thunberg). Ph.D. dissertation, Unixersity of Washing- 
ton. Seattle. Washington. 

Guo, X. & S. K. Allen, Jr. 1994a. Viable tetraploids in Ihe Pacific oyster 
[Crcis.soslreci gigus Thunberg) produced by inhihiling polar body I in 
eggs from Iriploids. Mol. Mar. Biol. Bimcchnol. 3( 1 1:42-50. 

Guo, X. & S. K. Allen, Jr. 1994b. Reproductive potential and genetics of 
Iriploid Pacific oyster, Crassoslrea giga.'i (Thunhergj. Biol. Bull. IS7: 
.109-318. 

Guo, X., G. Debrosse & S. K. Allen, Jr. 1996. All-triploid Pacific oyster 
(Crii.s.^oslreu gigas Thunberg) produced by maling tetraploids and di- 
ploids. Aquaculture 142: 1 49- Id I . 

Guo, X.. K. Cooper, W. K. Hershberger & K. K. Chew. 1994. Telraplold 
induclion with mitosis inhibition and cell lusion in the Pacific oyster, 
Cru-mMrea gigas (Thunberg). J. Sliclll'ish Res. 13(11:193-198. 



Production of Tetraploid Pearl Oyster 



151 



Guo, X., W. K. Hershberger. K. Cooper & K. K. Chew. 1992b. Genetic 
consequences of blocking polar body 1 with cytochalasin B in fertilized 
eggs of the Pacific oyster. Crassostrea gigas: Segregation of chromo- 
somes. Biol. Bull. 183:387-393. 

He. M.. Y. Lin & W. Jiang. 1996. Studies on the sterility of iriploid pearl 
oyster. Pimuida muriensii (D.). Tropic Oceunology 15(2):17-21 (in 
Chinese). 

Jiang. W., G. Li. G. Xu, Y. Lin & N. Qing. 1993. Growth of the induced 
triploid pearl oyster Pinctada mariensii (D.). .\qiiaculture lll:24.'i- 
253. 

Jiang, W.. G. Li. Y. Lin &. N.Qing. 1987. Induced polyploidization in pearl 
oyster, Pinctada martensii (D.). Tropic Oceanology 6(l):37-45 (in 
Chinese). 

Jiang. W.. G. Li. Y. Lin. G. Xu & N. Qing. 1990. Obser\ation on the gonad 
of triploidy in Pinctada martensii (D.). Tropic Oceanology 9(1):24— 30 
(in Chinese). 

Jiang. W., Y. Lin & M. He. 1998. A study on induction of tetraploid in 
pearl oyster. Pinctada martensii (D.). Tropic Oceanology 17(2):45-51 
(in Chinese). 

Jiang.W., G. Xu. Y. Lin & G. Li. 1991. Comparison of growth between 
triploid and diploid Pinctada martensii Dunker. Tropic Oceanology 
10(3): 1-7 (in Chinese). 

Komaru. A. & K. T. Wada. 1990. Gametogenesis of triploid Japanese peari 
oyster, Pinctada fiicata martensii. In: M. Hoshi and O. Yamashita 
(Editors). Advances in Invertebrate Reproduction 5. Elsevier, Amster- 
dam, pp. 469-474. 



Komaru, A. & K. T. Wada. 1994. Meiotic maturation and progeny 
of oocytes from triploid Japanese pearl oysters {Pinctada fiicata mar- 
tensii) fertilized with spermatozoa from diploids. Aquaculture 120:61- 
70. 

Lin. Y. & W. Jiang. 1993. A preliminary study on comparison between 
triploid and diploid in the cultured pearls of pearl oyster. Tropic Ocean- 
ology 12(3):90-94 (in Chinese). 

Lin, Y.. M. He & W. Jiang. 1996. An observation on the mortality of 
triploidy in Pinctada martensii (D.). Tropic Oceanology 15(2):80-84 
(in Chinese). 

Que. H.. X. Guo, F. Zhang & S. K. Allen, Jr. 1997. Chromosome segre- 
gation in fertilized eggs from triploid oyster, Crassostrea gigas (Thun- 
berg), following inhibition of polar body 1. Biol. Bull. 193:14-19. 

Scarpa, J., K. T. Wada & A. Komaru. 1993. Induction of tetraploidy in 
mussels by suppression of polar body formation. Nippon Suisan Gak- 
Aoii/ii 59(1 2):201 7-2023. 

Sears. E. R. 1956. The transfer of leaf rust resistance from Aegilops um- 
bellulata to wheat. Brookhaven Symp. Biol. 9:1-22. 

Stephens. L. B. & S. L. Downing. 1988. Inhibiting first polar body forma- 
tion in Crassostrea gigas produces tetraploids. not meiotic triploids. J. 
Shellfish Res. 7(3):550-551 (Abstract only). 

Wang. Z.. X. Guo, S. K. Allen & R. Wang. 1999. Aneuploid the Pacific 
oyster. Crassostrea gigas (Thunberg) as incidental from triploid pro- 
duction. .Aquaculture 173:347-357. 



Jourmil of Shellfish Reseiiirh. Vol. 19. No. 1. 153-L>7. 2000. 

EVALUATION OF FIVE MICROALGAL SPECIES FOR THE GROWTH OF EARLY SPAT OF 
THE JAPANESE PEARL OYSTER PINCTADA FUCATA MARTENSII 



KATSUYUKI NUMAGUCHI 

National Research Institute of Fisheries Science 
6-31 Nagai. Yokosuka 
Kanagawa 238-0316. Japan 

ABSTRACT To estimate the food value of five microalgal species, early spat of the Japanese pearl oyster, Pincuula fucata martensii, 
were fed five algal species separately; Pcniovu lutheri. Chaeloceros calchrans. Tetruselmis tetnnhele. Nitzschiii closreriiim. and 
Nannochloropsis oculata. The food value of each microalgal species was estimated from the growth rate of hinge length, dry whole 
spat, dry shell weight, and dry flesh weight of spat fed each microalgal diet over 19 days in the laboratory. C. calcitrans produced the 
best growth of the pearl oyster spat. P. hillwn and T. wtrathele supported moderate growth of the spat. However, growth rate of the 
spat fed Nilzschia closlerium was very low and spat fed Nannochloropsis oculata showed negligible growth. These results indicate that 
C. calcitrans is an appropriate microalgal diet for rearing pearl oyster spat. P. lutheri and T. tetrathele are also suitable diets for rearing 
early spat of pearl oysters. 

KEY WORDS: Pearl oyster spat, microalgal diet, growth 



INTRODUCTION 



Microalgae 



Microalgal plankton is the principal food source for bivalves. 
There have been many studies on the nutritional value of cultured 
microalgae and their promotion of growth in marine bivalves lar- 
vae, spat, and juvenile (Ostrea edulis. Enright et al. 1986a. Walne 
1963; Cnissostrea virginica, Davis and Gullard 1958; Sciccostrea 
commercialis. Nell and O'Connor 1991, O'Connor et al. 1992; 
Pinctada fiicata martensii. Wada 1973. Okauchi 1990: Crawo- 
doma gigantea. Whyte et al. 1990; Riiditapes philippinanim. Sakai 
and Toba 1994), 

Pavlova lutlieri (Droop) Green is the inost popular microalgal 
species in Japanese bivalve culture and seed production studies 
(Scapharca bnniglnonii. Ohhashi and Kawamoto 1980; Pinctada 
fucata martensii. Hayashi and Seko 1986; Riiditapes philippi- 
nanim. Miyama and Toba 1990. Taba and Miyama 1993; Meretrix 
lamarckii, Shitomi and Kodama 1987a, Yanagida and Kodama 
1988; Pseudocardiitm sachalinense, Shitomi and Kodama 1987b. 
Yanagida et al. 1988). 

In a previous study. I reconfirmed that Pavlova lutheri is a 
suitable microalga for the growth of early spat of pearl oyster. 
Pinctada fucata martensii (Numaguchi 1999). However, there is 
little information that evaluates other microalgal species for the 
grow th of pearl oyster spat. The aim of this study is to evaluate five 
microalgal species as diets for pearl oyster spat. 

MATERIALS AND METHODS 



Pearl Oyster Spat 

Pearl oyster spat used were produced in the Pearl Oyster Seed 
Production Center of the Nagasaki Pearl Oyster Fisheries Coop- 
erative Association. Spat were obtained approximately 3 months 
after fertilization in the hatchery. Average hinge length of the spat 
was about 3.5 mm. These spat were reared for 2 weeks in a 30-L 
aquarium with water temperature 26-27 °C, salinity 30-32 ppl and 
fed an algal diet of Pavlova lutheri. 



The algal species used are shown in Table 1 . Algal cultures 
were produced axenically in 5-L glass flasks using modified Erd- 
Schreiber medium: 100 mg NaNO,, 20 mg Na,HP04 • 12H,0. 50 
mgNaSiO,. lOOmg Nitrilotriacetic (NTA). 100 mg Tris (hydroxy- 
methyl) aminomethane. 0.4 jjLg Vitamin B,,. 100 p-g Thiamin. I 
pig Biotin. 5 mg Clewat 32 (Teikoku Kagaku Ltd. Japan; I g 
Clewat 32 contains 3.8 mg Fe. 7.7 mg Mn, 1 .6 mg Zn, 0,07 mg Cu, 
6,3 mg Mo. 24.7 mg B. 0.23 mg Co. and some EDTA) in 1-L of 
4/5 diluted seawater. The medium was adjusted to pH 7.8 and 
sterilized by autoclaving (121 °C, 15 min). All species were batch 
cultured at 20 °C with 24-h illumination at an intensity of 5.000 
lux. 

Because cell size and volume differed for each of these mi- 
croalgae, cell size and weight were measured for each species. 
Their sizes were measured using a Coulter Counter (Model ZB) 
and a Coulter Channelyzer-(Model C-100; Coulter Electronics 
Inc. USA). The range and mode of cell diameter for each algal 
species was estimated from the histogram of algal cell vol- 
ume from Coulter Channelyzer. and the dry weight of each algal 
species was determined as follows. Initially, the algal cell con- 
centration was determined using a Coulter Counter. A known vol- 
ume (20-50 mL) was then filtered though a GF/C glass fiber filter 
(Whatman Ltd. England), which was preheated for 2 h at 500 °C 
to remove organic substances, to collect the algal cells. The filter 
was washed with 0.9% ammonium formate solution to remove 
salt and dried at 110 °C for 24 h. The dry cell weight was 
then calculated using the algal cell concentration and total 
weight of filtered cells. The dry weight of suspended solids in 
the seawater used for the experiment was also determined this 
way. 



Experimental Design 

Twenty spat were allocated to each 2-L beaker aquarium with 
seawater filtered with 1 ixm cartridge filters. During the experi- 



153 



154 



NUMAGUCHI 



TABLE 1. 

List of microalgal diets used for the experiment and their cellular characteristics. 



Phytoplankton 





Cell 


Mode 






Diameter- 


of Cell- 


Dry Weight' 


'olume' 


Range 


Diameter 


of Cell 


(Hm^) 


((im) 


(Mm) 


(pg/Cell) 



Haptophyceae 

Pavlova liitheri (Droop) Green 
Bacillariophyceae 

Chaetoceros calcilnms (Paulsen) Takano 

Nitzschia closleriiim (HER.) W.Smith 
Prasinophyceae 

Telraselmis lelralhele (West) Butcher 
Eustigmatophyceae 

Naimochtoropsis oculala (Droop) Hibberd 



57 

56 

64 

335 
9 



4.5-5.6 

4.5-5.6 
4.8-7.3 

8.0-10.7 

2.3-3.5 



4.8 

4.8 
5.0 

8.6 

2.6 



32.5 ± 2.6 

70.3 ±4.1 
30.9 ± 1.6 

251 ± 10 

4.9 ±0.1 



' Cell volume was measured by Coulter Counter and Coulter Channelyzer. 

- Cell diameter range and mode were calculated by the equation of a spherical body from the cell volume histogram measured by the Coulter counter 

and Coulter Channelyzer. 



' Values are means ± SD (n = 5). 



merit, the dry weight of suspended solids in the filtered seawater 
was 1.54 ± 0.56 mg/L (n = 4). water temperature was 26-27 °C 
and salinity was 30-32 ppt. 

Feeding trials, including an unfed control, were carried out over 
19 days. Insufficient feeding will give false evaluations of food 
value of the microalgae for the growth of pearl oyster spat, so each 
feeding diet was supplied in excess in this experiment. Numaguchi 
(1999) showed that the optimal feeding concentration of Pavlova 
luteri was 2x10^ cells/mL for maximum growth of pearl oyster 
spat at 2.6-3.0 mm hinge length. In this experiment, three times 
the concentration of P. Iiitlwri (6 x lO'^ cells/mL) was fed to pearl 
oyster spat of 3.5 mm hinge length. Other algal concentrations 
were calculated from same packed cell volume as one of P. liitheri. 
the packed cell volume calculated to product of cell concentrations 
and cell volume. The feeding concentration of each algal species 
was set as follows; P. liitheri 6 x 10"* cells/mL, Chaetoceros cal- 
citransbA x lO'* cells/mL. Tetraselmis tetrathele I x lO"" cells/niL, 
Nitzschia closterimn 5.4 x 10'* cells/mL, and Naiiiunhlornpsis 
oculata 37.5 x lO'^ cells/mL. 

Each algal diet was added to the relevant beaker each morning 
at the above concentrations. Seawater in each beaker was changed 
every day just before feeding to remove the remaining algae that 
might have negatively affected feeding. Over the rearing period. 



spat were observed to determine whether they were alive or dead. 
Spat attached to the aquarium wall were regarded as alive, and 
unattached spat, those with no viscera, or only a shell were re- 
garded as dead. Dead spat were counted and removed from the 
aquarium. 

Spat Growth Measurement 

Hinge length of each spat was measured at the beginning and 
end of the feeding experiment using a stereoscopic microscope 
with a micrometer. Growth rate of spat hinge length per day was 
calculated as follows: 

Growth rate of hinge length (jj,m/day) = (final average 
hinge length - initial average hinge length) / rearing duration 

To measure the dry weight of whole spat, the spat shell, and 
spat flesh, ten spat were collected randomly from each aquarium at 
the beginning and end of the feeding experiment. Each spat was 
washed in 0.9'7r ammonium formate solution to remove salts and 
was wiped with paper towel. Dry whole spat weight was measured 
after spat were dried at 1 10 °C for 24 h on a platinum board. Dry 
shell weight was measured after drying the spat on a platinum 



TABLK 2. 
Growth of hinge length and mortality of pearl oyster spat. 



Diet 



Hinge Length (pni) 



Initial (I) Day)' 



Final (19 Day) 



Growth 




Rate 


Mortality 


(Uni/Dayl 


(%) 


281 





146 


10 


141 


10 


66 


5 


2 


10 


2 


25 



Chaetoceros calcilrans 
Pavlova liitheri 
Telraselmis lelralhele 
Nitzschia closteriiim 
Nannochloropsis ociiUila 
Unfed control 



3.405 ± 296-' (n = 20) 

3.443 ± 337'' (n = 20) 

3.510 ±5l8-'(n = 20) 

3.338 ± .37.3" (n = 20) 

3.653 ±3.W(n = 20) 

3.525 ±.36()-'(n = 20) 



8.745 ± 1,285'' (n = 20) 
6.217 ± 881" (n = 18) 
6.183 ±92r(n = 18) 
4.587 ± 962" (n = 19) 
3,683 ± 351" (n = 18) 
3,.563 ± .346" (n = 15) 



Values are means ± SD, values within a column with different superscripts were significantly different (Duncan multiple range test, P < 0.05). 



MiCROALGAE AS FOOD FOR PEARL OYSTER SPAT 



155 



TABLE 3. 
Dry weight gain of whole spat, shell, and flesh of pearl oyster spat. 



Diet 



Whole Spat' (pg) 



Shell' (Mgl 



Flesh' (pg) 



Initial (0 day) 

Final (19 days) 

Chaeloceros calcitrans 
Pavlova liitheri 
Tetraselmis tel?'arhele 
Nitzschia closterium 
Nannochloropsis oculata 

Unfed control 



1,470 ±533 

16.709 ± 6,250'- 
9,785 ±4,180'' 
7,979 ± 3,994'' 
3,907 ± 1,447" 
2,308 ± 674" 
1,679 ±410" 



1.255 ±452 

14.015 ±4.990" 
8.280 ± 3,484'' 
6.852 ±3.185" 
3.297 ± 1.202" 
2.188 ±548" 
1.556 ±389" 



215 ±83 

2,694 ± 1,302'= 

1.505 ±7 IS' 

1,127 ±819"' 

610 ±275" 

120 ± 140" 

123 ±53" 



' Values are means ± SD (n = 10). values within a column with different superscripts were significantly different (Duncan multiple range test. P < 0.05). 



board at 500 °C for 6 h in a muffle furnace to burn away the flesh. 
Dry whole and shell weight of each spat was weighed to the 
nearest 1 jig using a Micro Balance (Mettler Type M-3; Metiler 
Toledo, Switzerland). Dry flesh weight was calculated by subtract- 
ing dry shell weight from dry whole weight. Growth rates for the 
whole spat, shell, and flesh, in dry weight per day, was calculated 
as follows: 

Growth rate of weight (jjig/day) = (final average 

dry weight - initial average dry weight) / rearing duration 

RESULTS 

Table 2 shows hinge length of the spat at the beginning and 
end of the experiment and growth rate and mortality of the spat 
during the experiment. Chaetoceros calcitrans produced the best 
growth of the pearl oyster spat in this feeding experiment. Al- 
though growth rates of the spat fed Pavlova lutheri and Telra- 
selmis tetrathele were about half those fed C. calcitrans. P. lutheri. 
and T. tetrathele. both supported good growth rates of pearl oyster 
spat. Spat growth rate with Nitzschia closterium was poor. 
Moreover, there was almost no growth of pearl oyster spat fed 
Nannochloropsis oculata. There was no mortality of the spat 
fed C. calcitrans and 5-10% mortality of the spat fed P. lutheri. 
T. tetrathele. Nitzschia closterium, and Nannochloropsis oculata. 
In contrast, mortality of the unfed control was rather high 
(25%). 

Weight gain of dry whole spat, dry shell, and dry flesh was 
greatest for the spat fed Chaetoceros calcitrans. Weight gain of the 
spat fed Pavlova lutheri and Tetraselmis tetrathele was moderate. 
Whereas, weight gain of the spat fed Nitzschia closterium was 
poor. However, spat fed Nannochloropsis oculata and the unfed 
control had very low weight gain (Table 3). Figure 1 shows the 
growth rate of dry spat weight, dry shell, and dry flesh of the spat 
fed various microalgal diets along with the unfed control. The spat 
fed C. calcitrans had the highest growth rate compared to the other 
microalgal species. In decreasing order, diets of P. lutheri, T. 
tetrathele. and Nitzschia closterium promoted the growth of pearl 
oyster spat. The spat fed Nannochloropsis oculata had a negative 
growth rate as did the unfed control. 

DISCUSSION 

Good growth rates of the bivalve are obtained with various 
algal cell because of their appropriate cell size for ingestion, their 



susceptibility to mechanical or enzymatic digestion by bivalves, 
their nutritive and biochemical composition, and their lack of toxic 
cell metabolite (Babinchak and Ukeles 1979). 

In this experiment, Chaetoceros calcitrans was the superior 
microalgal species for maximum growth rate of pearl oyster 
spat. Although Pavlova lutheri and Tetraselmis tetrathele were 
inferior diets to C. calcitrans, these species supported a moderate 
growth rate of pearl oyster spat. These results indicate that C. 
calcitrans is an appropriate microalgal diet for rearing pearl oyster 
spat; whereas, P. lutheri and T. tetrathele are also suitable diets 
for this species. However, Nitzschia closterium was an unfavor- 
able diet for the growth of pearl oyster spat. Nannochloropsis 
oculata did not promote the growth of pearl oyster spat, sug- 
gesting it is an inappropriate diet for rearing pearl oyster spat. 
Wada (1973) also showed that Chlorella sp. (now classified 
as Nanochloropsis) was a poor diet for pearl oyster larvae. 
Walne (1963) indicated that Chlorella stigmattophora. which 
has cell wall, is of little value as food for oyster, Ostrea edulis. 
larvae. Babinchak and Ukeles (1979) also described that the 
cell wall of Chlorella autotropphica was resistant to enzymatic 
breakdown by the digestive system of larvae of the oyster, Cras- 
sostrea virginica. Nannochloropsis oculata may be similarly 
resistant to mechanical or enzymatic digestion by pearl oyster 
spat. 

The biochemical composition and nutritional components of 
microalgae differ between species (Parsons et al. 1961, Epifanio et 
al. 1981, Enright et al. 1986b. Whyte 1987). O'Connor et al. 
(1992) found that suitable dietary algal species were different for 
different growth stages of the same bivalve species. For the pearl 
oyster, Wada (1973) indicated that P. lutheri was a more suitable 
algal diet than C. calcitrans for larvae; however, for the spat in this 
experiment, C. calcitrans was a more suitable diet than P. lutheri. 
Fuilhermore, Okauchi (1990) found that Isochrysis gracilis was 
suitable algal diet for pearl oyster juveniles. These results suggest 
that the nutritional demands of the pearl oyster may change with its 
growth stage. 

ACKNOWLEDGMENTS 

The author expresses gratitude to Dr. T. Horii. National Re- 
search Institute of Fisheries Science, for statistical analysis of the 
data. This study was supported in part by grants-in-aid from the 
Ministry of Agriculture, Forestry, and Fisheries, Japan. 



156 



NUMAGUCHI 



> 

a 

-o 

=1 



a 
o 



(0 

bo 

51 



t 

o 



>. 

(0 
■D 

0) 
+J 
(0 

o 
o 



1000 



800 



600 



400 



200 




800 



600 



<S 400 



200 




ItU 

120 [ ^[ 
100 jH 

40 ^H 

: 1 


1 

Dry flesh weight 

li. 


C-cal 
—on 


P-lut T-tet Ni-cio Na-ocu Unfed 



Micro-algal species 



Figure I. Variation in growth rate of pearl oyster spat fed various niicroalgal diets. [C-cal] Chaeloceros calcilrans. |P-lutl Pavlova liitheri. |T-tetl 
Tctraselmis Ulralltclc, |Ni-iio| \ilzcliia closUrium, |Na-ocu| ^tmnochloropiis oculata. 



MiCROALGAE AS FOOD FOR PEARL OYSTER SPAT 



157 



LITERATURE CITED 



Babinchak. J. & R. Ukeles. 1979. Epifluorescence microscopy, a technique 
for the study of feeding in Crassoslrea viii>iiiic{i veliger larvae. Mar. 
Biol. 5 1 :69-76. 

Davis. H. C. & R. R. Guillard. 1958. Relative value often genera of micro- 
organisms as foods for oyster and clam larvae. Fish. Bull. 58:203-304. 

Enright. C. T.. G. F. Nev\kirk. J. S. Craigie & J. D. Castell. 1986a. Evalu- 
ation of phytoplankton as diets for juvenile Oslreci echilis L. J. Exp. 
Mar. Biol. Ecol. 96:1-13. 

Ennghl. C. T., G. F. Newkirk, J. S. Craigie & J. D. Castell. 1986b. Growth 
of juvenile Ostrea ediilis L. fed ChaeWceros gracilis Schutt of varied 
chemical composition. / Exp. Mar. Biol. Ecol. 96:15-26. 

Epifanio. C. E.. C. C. Valenti & C. L. Turk. 1981. A comparison of Phaeo- 
ilacryliim iriconmmm and Thalassiosira pseudonana as foods for the 
oyster. Crassoslrea virginica. Acjuacullurc 23:347-353. 

Haya.shi. M. & K. Seko. 1986. Practical technique for artificial propagation 
of Japanese pearl oyster (Pinclada fucata). Bull. Fish. Res. Inst. Mie. 
1:36-68 (in Japanese with English abstract). 

Miyama. Y. & M. Toba. 1990. Studies on the seedling production of 
short-necked clam Ruditapes philippinarum Adams & Reeve-III food 
value of 8 microalga for the lar\a of Manila clam Ruditapes philippi- 
narum Adams & Reeve. Bull. Chiba Pref. Fish. E.xp. .Sin. 48:93-96 (in 
Japanese with English abstract). 

Nell. J. A. & W. A. O'Connor. 1991. The evaluation of fresh algae and 
stored algal concentrates as a food source for Sydney rock oyster. 
Saccostrea commercialis (Iredale & Roughley), larvae. .Aquacullure 
99:277-284. 

Numaguchi, K. 1999. Effective feeding concentration of the microalga 
Pavlova lutheri for growth of early spat of the pearl oyster Pinctada 
fiicata marlensii. J. World Aqua. Soc. 30:290-292. 

O'Connor. W. A.. J. A. Nell & J. A. Diemar. 1992. The evaluation of 
twelve algal species as food for juvenile Sydney rock oysters Saccos- 
trea commercialis llredale & Roughley). Aquacuhure 108:277-283. 

Ohhashi. H. & Y. Kawamoto. 1980. Technical development of mass cul- 
ture of ark shell. Scapharca broughtonii. Rep. Tech. Develop. Sea 
Farming Yamaguchi Naikai Sea Farming Center 6:80-135 (English 
translation: in Japanese). 



Okauchi. M. 1990. Food value of Isochrysis aff. galbana for the growth of 

pearl oyster spat. Nippon Suisan Gakkaishi 56:1343. 
Parsons. T. R.. K. Stephens & J. D. H. Strickland. 1961. On the chemical 

composition of eleven species of marine phytoplanktons. J. Fish. Res. 

Bd. Canada 18:1001-1016; 25:77-87. 
Sakai. M. & M. Toba. 1994. Mass culture of Isochrysis aff. galbana V. 

food value of mixture of two algal species for the spat of Manila clam 

Ruditapes philippinarum. Saibai Ciken 23:1-5 (in Japanese). 
Shitomi, S. & M. Kodama. 1987a. Seed production of Asiatic hard clam. 

Meretrix lamarckii. Bull. Ibaragi Pref. Fish. Exp. Stn. 61:285-291 

(English translation; in Japanese). 
Shitomi. S. & M. Kodama. 1987b. Seed production of Sakhalin surf clam. 

Pseudocardium sachalinense. Bull. Ibaragi Pref. Fish. Exp. Stn. 61: 

292-300 (English translation: in Japanese). 
Taba, M. & Y. Miyama. 1993. Gross growth efficiency in juvenile Manila 

clam Ruditapes philippinarum fed different levels of Pavlova lutheri. 

Bull. Chiba Pref, Fish. E.xp. Stn. 51:29-36 (in Japanese with English 

abstract). 
Wada, K. T. 1973. Growth of Japanese pearl oyster larvae fed with three 

species of microalgae. Bull. Natl. Pearl Res. 1Mb. 17:2075-2083 (Japa- 
nese with English summary). 
Walne. P. R. 1963. Observations on the food value of seven species of 

algae to the larvae of Ostrea edulis. 1 . feeding experiments. J. Mar. 

Biol. Ass.. U.K. 43:767-784. 
Whyte, J. N. C. 1987. Biochemical composition and energy content of six 

species of phytoplankton used in mariculture of bivalves. Aquaculture 

60:231-241. 
Whyte, J. N. C, N. Bourne & C. A. Hodgson. 1990. Nutritional condition 

of rock scallop, Crassadoma gigantea (Gray), larvae fed mixed algal 

diets. Aquaculture 86:25—40. 
Yanagida. Y. & M. Kodama. 1988. Seed production of Asiatic hard clam. 

Meretrix lamarckii. Bull. Ibaragi Pref. Fish. Exp. Stn. 62:338-346 

(English translation; in Japanese). 
Yanagida. Y.. S. Shitomi & M. Kodama. 1988. Seed production of Sakha- 
lin surf clam. Pseudocardium sachalinense. Bull. Ibaragi Pref. Fish. 

Exp. Stn. 62:347-357 (English translation; in Japanese). 



JoKimil of Shellfish Rfsfurch. Vol. 19. No. 1. 159-166, 2000. 

COMBINED EFFECTS OF TEMPERATURE AND ALGAL CONCENTRATION ON SURVIVAL, 
GROWTH AND FEEDING PHYSIOLOGY OF PINCTADA MAXIMA (JAMESON) SPAT 



DAVID MILLS 

Acjiiciciilturt' Co-operative Research Centre 

Northern Territory University 

Darwin Aqiiacultiire Centre 

Department of Primary Industry and Fisheries 

Darwin, Northern Territory, Australia 

ABSTRACT To determine a suitable culture environment to maximize growth and survival, Pinctmla maxima spat were held at 36 
combinations of temperature and algal concentration for 14 days within a flowthrough system. Survival was greatest between 23 °C 
and 32 °C. with 35 °C resulting in high mortalities. The optimum temperature range for P. maxima spat found in this study agrees well 
with the observed temperatures which limit the natural distribution of P. maxima in Australian waters. Survival of spat was highest 
at low algal concentrations. Growth was optimal between 26 °C and 29 °C and at 54 algal cells |j.L"'; however, growth was still 
acceptable at algal concentrations as low as 12 cells (jlL"'. The organic content increased with feeding rate and was positively correlated 
with specific growth rate. Spat filtration rate declined at high feeding rates, whereas grazing rate increased, with a commensurate 
decline in conversion efficiency. It is recommended that P. maxinui spat be maintained within the temperature range of 26 "C to 29 
°C and at algal cell densities between 12 and 54 cells |xL"' to maximize spat performance and minimize algal wastage. 

KEY WORDS: Pinaada maxima 



INTRODUCTION 

Following high mortalities of adult silver-lip pearl oysters. 
Pinctada maxima (Pass et al. 1987), during the 1970s and early 
1980s, there has been a focus on hatchery production for ongrow- 
ing and pearl production (Rose et al. 1990). Although there has 
been considerable work published on P. maxima spat production 
and husbandry, there has been no published investigation into the 
role of either temperature or food concentration on spat culture 
success. 

Temperature is regarded as one of the most potent factors af- 
fecting growth and metabolism of marine poikilotherms (Griffiths 
and Griffiths 1987) and has been shown to effect many physiologi- 
cal processes of bivalves, such as filtration, feeding, respiration, 
reproduction, and growth (Bayne et al. 1976). 

There is evidence that the Australian distribution of both P. 
margaritifera and P. maxima is limited to areas where seawater 
temperatures range from 18 °C to 32 °C (Hynd 1955, Pass et al. 
1987). High mortalities of up to 80% of wild fished P. maxima in 
Western Australia (WA) in the late 1970s and early 1980s were 
attributed to reduced disease resistance during periods of low tem- 
perature (Pass et al, 1987), This effect may have been enhanced by 
the change in temperature (from 19 °C to 26 °C) between the 
collection grounds and the farms during transportation. 

Rose et al, (1990) investigated the seawater temperatures of the 
main Western Australian fishing beds for P. maxima and recorded 
bottom temperatures of between 20 °C and 26,8 °C, Surface tem- 
peratures showed a larger range ( 19.8-32,3 °C). 

There have been several feeding rates used for P. maxima spat, 
without any real evidence as to their suitability. Rose ( 1990) rec- 
ommended twice-daily feedings of 55-65 cells jjlL"'. whereas 
Rose and Baker ( 1994) fed spat a mixed algal diet at 40-285 cells 
(xL"' depending on spat size. The algal concentration dynamics in 



Current address: Paspaley Pearling Co. P/L. P.O. Box 338, Darwin, NT 
0801, Australia, 



a batch-fed system will vary with tank size, stocking density, and 
feeding frequency. 

The aim of this experiment was to quantify the effects of tem- 
perature and food availability on the growth, survival, and feeding 
of P. maxima and to determine suitable regimes for spat culture. 

Materials and Methods 

Experimental animals 

P. maxima spat were obtained from the Darwin Hatchery Proj- 
ect on December 17, 1996. These spat averaged 11 ±0.7 mg with 
an initial organic content of 10.9%. Mean initial shell height (dor- 
soventral measurement) was 4.3 mm and ranged from 3.3 to 
5.2 mm. 

System 

The system used in this experiment was a modified and scaled- 
up version of that described in Mills (1997). There were three 
experimental blocks, each consisting of six 100-L temperature- 
controlled waterbaths and six elevated 100-L reservoirs. Each res- 
ervoir contained an algal suspension at one of the experimental 
concentrations and supplied one replicate in each waterbath 
through a submersible pump and 4-mm tubing manifold. The flow 
rate into each replicate was controlled with 2-L h"' irrigation 
drippers. Thus, each waterbath in each block contained one repli- 
cate tray at each algal concentration, giving one replicate of each 
combination of temperature and food concentration per block and 
three replicates of each combination. Different-colored pegs were 
used to identify replicates of each algal concentration within a 
waterbath. Both the incoming air and algal suspension were pre- 
heated to the correct temperature before entering the replicates by 
first passing through approximately 4 m of the 4-mm supply lines 
coiled within the waterbath. Spat were held individually within 
histological cassettes, with 10 spat in each replicate tray. 

Trays were supported by the rim in rectangular holes cut into a 
32-mm-thick sheet of extruded polystyrene foam, which was 
floated within each waterbath and acted as both tray support and 



159 



160 



Mills 



insulator. Irrigation drippers and trays were replaced at weekly 
intervals to prevent fouling. 

Outflow from each replicate was collected from a 4-mm tube 
connected to the tray outlet. A 60-|j.m mesh feces retainer pre- 
vented contamination of the outflow sample with feces and/or 
pseudofeces. 

Temperatures and algal concentrations 

There were six temperatures used in the experiment: 20, 23. 26. 
29. 32. and 35 °C. This temperature range was chosen as it en- 
compasses the annual range experienced in Darwin Harbour (23- 
32 "O and is similar to that recorded at Broome (Rose et al.l990). 
The ambient room temperature was maintained at 20 °C (the mini- 
mum temperature attainable in the isothermal room), and all of the 
waterbaths at higher temperatures were heated with 300-W glass 
immersion heaters. Temperatures of the replicates were checked 
twice daily and maintained within ±0.5 °C of the desired experi- 
mental temperature. Standard errors of experimental temperatures 
were generally ±0.15-0.2 °C. 

The initial algal concentrations delivered from the reservoirs 
were 10. 20. 40. 80. and 160 cells \}.L'\ with unfed controls 
exposed only to filtered seawater. All seawater was filtered to 1 
|xm and then passed through a carbon filter to remove possible 
contaminants from the intake seawater. which was drawn from a 
commercial shipping wharf. 

The algal concentration range was chosen to encompass the 
optimums found for P. fiicata (Numaguchi 1994a. Krishnan and 
Alagarswami 1993) and for P. maxima by Bellanger (1995). and 
also the commonly used feeding rates in commercial hatcheries 
(80-100 cells |jiL"'). However, the effective algal concentration 
surrounding the oyster may be better represented by the concen- 
tration in the outflow (Hildreth & Crisp 1976). That the outflow 
algal concentration was the same as that within the replicate was 
confirmed by comparing the algal concentration in samples taken 
from several replicates at 4-h intervals, with the subsequent con- 
centration in the outflow. Thus, the results presented are given 
relative to the effective (outflow) rather than initial algal concen- 
tration. The mean effective outflow concentrations were 0. 6. 12. 
23. 54. and 1 10 cells (j.L"'. Algal feeding reservoirs were cleaned 
and refilled daily with the appropriate algal suspension. 

.Spat were fed an algal diet of equal cell numbers of Tahitian 
Isdclirysis sp. (T. Iso) and Chaetoceros miwUeri. These species 
have been shown to support good growth and survival of pearl 
oyster spat (Taylor et al. 1997. Southgate et al. 1998). Mean algal 
cell dry weights were 19 and 20 pg. respectively, and were deter- 
mined by the method of Epifanio (1979). Algae was cultured in 
20-L carboys using f/2 medium with a 12:12 photoperiod and 
harvested at the late exponential stage. 

Preliminary trial 

A preliminary trial was conducted to determine whether there 
was any change in the delivered algal concentration due to cell 
damage, growth or sedimentation, or differences in delivery vol- 
umes due to differing friction head loss within the system. One 
block was run over 24 hours without animals in the system at an 
initial algal concentration of 100 cells jjiL"'. Outflow volumes and 
initial and final algal concentrations were compared by two-way 
ANOVA using a significance level (c») of 0.2. 

There were no significant differences in either the xolumes 
delivered {P = 0.56) or the oulllow concentration (/' = 0.69). 



Initial and final algal concentrations were not significantly differ- 
ent (P = 0.78). Subsequent trials showed that the volume of 
suspension delivered by a dripper was independent of the number 
of drippers on the manifold line at least up to n = 8. This occurs 
as the pumps used were not positive displacement, but rather main- 
tained a set delivery pressure and possessed a delivery capacity 
exceeding that of the combined number of drippers. Thus, the 
number or status of drippers on a manifold line had no effect on the 
delivery pressure (and hence output) of individual drippers. 

Sampling 

As it was not logistically possible to weigh and measure all of 
the 1080 spat and sample all of the 108 outflows during a single 
day. both the startup and sampling procedures were sequenced 
over 3 days. A full block could not be sampled on 1 day, as there 
was not enough floorspace for all of the outflow collection vessels; 
hence, a part of each block was sampled on each day. The se- 
quence used was designed to sample one replicate of each treat- 
ment combination on each day. 

At days 7 and 14, each spat was removed from the histological 
cassettes, washed in seawater of the appropriate temperature to 
remove adherent feces, and then weighed to the nearest 0.1 mg and 
measured to the nearest 0.1 mm (DVH). Outflow volume and 
collection duration were recorded, and outflow samples were pre- 
served with Lugols iodine for later counting and calculation of 
filtration and grazing rates. 

Growth was expressed as the daily tissue weight specific 
growth rate (SGR) and was calculated according to the following 
equation: 

SGR = (Ln final tissue weight) - (Ln initial tissue weight)/time 
[days|)x 100 

The organic content was calculated as: 

Organic content (%) = loss on ignition/dry weight x 100 

Filtration rate (FR) was calculated by the formula of Bayne et 
al. ( 1976) for flowthrough systems: 

FRa h"') = a- CO/CO x F 

where CI = the initial algal concentration. CO = the final algal 
concentration, and F = flow rate (1 h"'). 

This was converted to a weight-specific filtration rate by the 
following equation: 

FR (1 h''g-') = FR (1 h"')/tissue weight (g) 
Algal grazing rate for each replicate was calculated as: 

Grazing rate (Vr ) = C (g)/dry tissue wt (g) 

Conversion efficiency was calculated b\ the equation: 

Conversion efficiency (Vr) = SGR/giazing rate x organic 

content of algae x 100 
(modified trom De Sil\a and Anderson 1995) 

Statistical analysis 

All responses to temperature and algal concentration were ana- 
lyzed using a two-way factorial ANOVA model. Although 
samples for growth, filtration, and grazing rates were taken at 
weekly intervals, because of the plasticity of the spat organic con- 
lent the analysis was conducted only on the final values, as these 
responses were all calculated relative to spat tissue weight. 



Temperature and Algae Effects on P. Maxima 



There was very low survival at 35 °C (1.1%). and this tem- 
perature was excluded from subsequent analysis because of the 
low number of surviving individuals and hence very high selection 
pressure on the population. 

Any survival percentage data that were not normally distributed 
was arcsine transformed before being analyzed (Underwood 
1981). 

Homogeneity of variances were tested with Cochran's test with 
the critical value (CV) calculated as: 

CV = largest variance/ S variance 

and was compared with a tabulated value with (replicates/ 
treatment) - 1 and (treatment levels - 1) degrees of freedom. 

Normality of response distributions were tested on residuals 
(yij-Yi) using the Shapiro-Wilk W test (Zar 1984). If variances 
v\ere found to be unequal, or the data had a non-normal distribu- 
tion, appropriate transformations were done. Comparison of means 
w as only undertaken if the overall ANOVA model was significant, 
using the Fisher's protected least significant difference test. Rela- 
tionships between measured responses and culture conditions were 
examined using regression analysis. A P value <0.05 was consid- 
ered significant for all statistical analysis. 

Results 

Survive/ 

Both temperature {P < 0.0001) and algal concentration {P = 
0.03) affected spat survival, with temperature being a much stron- 
ger influence than algal concentration. There was no significant 
interaction (P = 0.16). Within the naturally occurring temperature 
range for Darwin Harbour (23-32 °C), there was no effect of 
temperature on mortality rates, and survival was greater than 90% 
(Fig. 1). At 35 °C mortality was almost complete (98.97f ). and at 
20 °C survival was significantly lower than at 23 °C. 26 °C. 29 °C, 
and 32 °C. 



Spat at the lowest algal concentration of 6 cells |xL"' showed 
the highest survival, which was significantly higher than those at 
23 and 110 cells (aL"' and unfed spat (Fig. 2). It is notable that the 
only survival at 35 °C was at the lower algal concentrations (6 and 
23 cells |jiL~'). 

Growth 

Tissue SGR responded significantly to both temperature and 
algal concentration (P = 0.0008 and P < 0.0001. respectively), but 
there was no significant interaction (P = 0.73). Growth increased 
with increasing temperature up to 29 °C and then declined from 
29 °C-32 °C (Fig. 3). The decrease in growth at 32 °C indicates 
that this is approaching the upper temperature limit for the species, 
as confirmed by the very low survival at 35 °C. Growth at 29 °C 
was more than twice that at 20 °C. and growth at 32 °C was similar 
to that at 23 °C. Tissue weights of unfed spat declined, indicating 
that there was no significant nutritional value in the filtered sea- 
water. In fed spat, growth increased progressively with increasing 
algal concentration up to 54 cells |jlL''. after which there was a 
slight but nonsignificant decline (Fig. 4). Growth at 54 cells jjiL"' 
was approximately 50% greater than that at 6 cells [i.L'\ This 
illustrates that P. ma.xima spat are capable of moderate growth 
even at very low algal concentrations. 

Organic content 

Increases in algal concentration were reflected in significant 
increases in spat organic content {P = 0.0002), from 9.4% in 
unfed spat to >13% at the highest concentrations (Fig. 5). The 
organic content of spat cultured at 6 and 12 cells |jiL~' was not 
significantly different from the initial value of 10.9%. Temperature 
had no significant effect on spat organic content (f = 0.8). nor 
was there any significant interaction between temperature and al- 
gal concentration (P = 0.8). There was a positive correlation 
between the SGR of spat and their organic content (;' = 0.51, P 



100 



100- 



> 




120 



Temperature (°C) 



Figure 1. Survival off. maxima spat after 14 days" culture at various 
temperatures. Figures show means ± standard error. Means with simi- 
lar subscripts are not significantly different (/* > 0.(15). 



Algal concentration (cells ijI' ') 



Figure 2. Survival of P. maxima spat after 14 days culture at various 
algal concentrations. Figures show means ± standard error. Means 
with similar subscripts are not significantly different iP > O.OS). 



162 



Mills 



T3 



O 
CO 




Temperature (°C) 

Figure 3. SGR of P. maxima spat at various temperatures. Figures 
show means ± standard error. Means with similar subscripts are not 
significantly different (P > 0.05). 



< 0.0001), with faster-growing spat having a higher organic con- 
tent. 

Feeding 

The two different algal species comprising the diet were 
counted separately in outflow samples obtained during the first 
week. There was no preferential selection by the spat for either of 
the species at any concentration or temperature, and the ratio of T. 
Iso to C. miielleri in the outflow was not significantly different 



c 
B 

o 
o 

o 

'c 

CO 

en 




100 120 



Algal concentration (cells |J I' ') 



Figure 5. Organic content [% of dry weight) of P. maxima spat after 
14 days at various algal concentrations. Figures show means ± stan- 
dard error. Means with similar subscripts are not significantly differ- 
ent (P > 0.05). 



from 1 , Henceforth, for the calculation of feeding rates it was 
assumed that there was no selection for either species by the spat. 
The weight-specific filtration rate increased with moderate in- 
creases in algal concentration up to 23 cells (xL"', before declining 
significantly at 54 and 1 10 cells |j.L"' (Fig. 6). Filtration rate was 
highest at 20 °C (54 L h"'g"') and declined significantly with 
increasing temperature to 17.3 L h"'g"' at 32 °C (Fig. 7). This is 
an inverse response to that shown in most bivalve studies, in which 



(0 






5- 



2.5 



-2.5- 




150 



Algal concentration (cells |jL ' ) 



Figure 4. ,S(;R of P. maxima spat after 14 days at various algal con- 
centrations. Figures show means ± standard error. Means with similar 
subscripts are not signincantly different (/' > (1.05). 




125 



Algal concentration (cells pi" ) 



Figure A. Filtration rates of /'. maxima spat at various algal concen- 
trations. Figures show means ± standard error. Means with similar 
subscripts are not .significantly different (/' > 0.05). 



Temperature and Algae Effects on P. Maxima 



163 




Temperature (°C) 

Figure 7. Filtration rate of P. maxima spat at different temperatures. 
Figures show means ± standard error. Means with similar subscripts 
are not significantly different (P > 0.05). 



filtration rate generally increases with increasing temperature up to 
a maximum, with a subsequent decline. 

The grazing rate (G) increased linearly with increasing algal 
concentration from approximately 15% at 6 cells |jlL"' to 136% at 
1 10 cells tJ-L"' following the equation: 

C% = 0.74 X algal concentration + 18.2 (;-- = 0.96) (Fig. 8) 

With the increase in grazing rate, there was a corresponding de- 
cline in the gross conversion efficiency from approximately 38% 
at 6 cells jxL"' to 5% at 1 10 cells |j,L'' (Fig. 8). As the production 
of pseudofeces was not quantified, the gross conversion efficiency 
refers to growth from algae grazed, rather than ingested. The loga- 



o 

c 

o 

0) 
it: 
(D 

c 
g 
(/) 

> 

c 
o 
O 

O 



-| 1 1 I 1 r 

20 40 60 80 100 120 

Algal concentration (cells \i ''^) 

Figure 8. Grazing rate and conversion efficiency of P. maxima spat at 
different algal concentrations. Figures show means ± standard error. 
Means with similar subscripts are not significantly different iP > 0.05). 




rithmic decline in conversion efficiency iCE) can be described by 
the equation: 

CE = [128.4 X algal concentration (cells [jlL'' )1-"^-" 

As the grazing rate increased greatly in response to increasing 
algal concentration without a commensurate increase in growth 
rate, it seems likely that the majority of the algae grazed were 
rejected as pseudofeces. Neither the grazing rate nor conversion 
efficiency was affected by temperature, nor was there any signifi- 
cant interaction of the two factors. 

Discussion 

Survival 

The pattern of survival exhibited in this experiment is consis- 
tent with the observations of Pass et al. ( 1 987 ), who concluded that 
the natural distribution of P. maxima was limited to areas with a 
seawater temperature range of 18-32 °C. Although it was not 
possible to examine the effects of temperatures below 20 °C. it is 
apparent from the significantly lower survival at 20 °C that the spat 
were approaching their lower tolerance limit. The very low sur- 
vival at 35 °C indicates that this is above the upper tolerance limit. 
as foreshadowed by the reduced growth at 32 °C. The reduction in 
survival at 20 °C. and the very low survival at 35 °C. reflects the 
results of Doroudi et al. (1999) for P. margaritifera larvae, in 
which there was no development at either 20 °C or 35 °C. The 
optimum range for P. margaritifera larvae was found to be be- 
tween 26 °C and 29 °C, which is slightly narrower than that found 
for P. maxima spat in the present study (23-29 °C). Numaguchi 
and Tanaka (1986) investigated the effects of temperatures from 
7.5-35 °C on P. fucata and concluded that the lower and upper 
tolerance limits were 15 °C and 32 °C, respectively, with the 
optimum range being from 17.5 °C to 28 °C. 

Temperatures on both the natural pearl oyster beds and pearl 
farms in WA would occasionally be high or low enough to be 
deleterious to P. maxima spat, as they generally range from 20 °C 
to 32 °C (Rose et al. 1990). In the Northern Territory (NT), sum- 
mer inshore water temperatures are generally 31-32 °C from De- 
cember to April (Padovan 1997) and may be approaching stressful 
temperatures. Wada (1953) observed that the temperature of the 
main deepwater commercial oyster grounds in the N.T. was the 
same throughout the water column and averaged 29 °C during the 
summer. The lower temperatures offshore may be more conducive 
to growth and reproduction than warmer inshore waters. 

As surface seawater temperatures reach higher levels than bot- 
tom waters, pearl oysters hung from longlines (typically 1-3 m 
deep) may experience temperatures greater than 32 °C. especially 
in sites farther to the north such as the Kimberly region in northern 
WA. and the NT, and in calm sheltered bays. These temperatures 
may be at or near the tolerance limit of the species, and although 
there does not seem to be any direct mortality associated with 
them, there could be significant sublethal effects such as reduced 
growth, reproductive output, pearl quality, and resistance to stress- 
ors such as cleaning and handling. This may account for the lower- 
quality spawnings and gametes produced by oysters from farm 
longlines compared with those from the offshore fishing grounds 
(Rose et al. 1990), although this may be partly attributable to the 
frequent cleaning and handling of farm oysters. 

High water temperatures may be more critical in NT hatcheries, 
as during the summer air temperatures commonly reach 34 T. and 
this may be reflected in the temperature of the rearing tanks. Cur- 



164 



Mills 



rently. the industry addresses this by shading of the seawater sup- 
ply and rearing tanks; however, temperatures may still reach 
stressful levels. Stressed larvae and spat may be more susceptible 
to disease and suboptimal water quality. Algal cultures used to 
feed larvae and spat are grown at temperate water temperatures 
(20-24 °C). and problems may occur as a result of the abrupt 
increase in temperature experienced by the algae when it is trans- 
ferred from the algal culture system to the spat culture tanks. 
Minaur (1969) noted that P. lutheri became moribund at tempera- 
tures above 30 °C and attributed this as a major problem in at- 
tempts to rear P. maxima larvae and spat. 

Numaguchi (1994b) considered that an increase in the mortality 
rates of farm-held P. fucata was due to su.stained elevated tem- 
peratures of greater than 28 °C. This may have been related to 
temperature stress combined with reduced food intake and higher 
metabolic costs, as the same author demonstrated that filtration 
rate declines dramatically at temperatures above 28 °C (Numagu- 
chi 1994c). whereas catabolic losses increase at higher tempera- 
tures (Numaguchi 1995). There appears to be a similar process in 
P. maxima. Assuming that the organic content of the spat at day 7 
was the same as that at the beginning of the experiment, then at 
35 °C the mean filtration rate over all algal concentrations at 35 °C 
was 9.6 L h~'g"' compared with >30 L h"'g"' at temperatures 
from 20-32 °C. Unfed .spat showed greater tissue weight loss with 
increasing temperature. This strongly suggests that if the experi- 
ment had been extended, there would have been large mortalities 
in unfed spat, particularly at the higher temperatures. The combi- 
nation of these two factors indicate that at 35 °C there is reduced 
feeding and increased metabolic costs, leading to negative growth 
and increased mortality. 

Algal concentration had a small but significant effect on spat 
mortality, with the survival rates at 6 cells jxL"' significantly 
greater than those of unfed spat and those at 23 and 1 1 cells p,L" ' . 
The only surviving spat at 35 "C were unfed, and at the lower fed 
algal concentration. This is probably a result of the stimulatory 
response in filtration rate at higher algal concentrations (Fig. 6). 
resulting in an increase in metabolic rate and energetic costs, and 
con.sequently a more rapid loss of body tissue and death. 

Grawlh 

As previously found by Mills ( 1997), there was no significant 
relationship between the initial weight of spat and the subsequent 
SGR within the spat si/e range used (/' = -0.11. P = 0.08). 

Increasing growth with increasing temperature up to an asymp- 
totic point, followed by a rapid decline, is a common pattern for 
bivalves (Bayne et al. 1976). A similar pattern was shown by a 
temperate strain. P. fucata (Numaguchi and Tunaka 1986), ex- 
po.sed to temperatures ranging from 7.5 °C to 35 °C. 

The relationship between preasymptote temperalure and 
growth of P. maxima spat can be described by the second-order 
polynomial equation: 

Growth (SGR 'A day ' ) = -19.85 -I- 1.6 temperature - 0.027 
tempeialure' (/~ = 0.98) 

From this equation, the calculated temperature of zero growth 
is 17.7 °C. which agrees well with the esliniale of the lower tem- 
perature limiting the distribution of /'. iiia\uua by Pass et al. 
(1987) of 18 "C. 

Growth relative to algal concentration showed a pattern similar 
to that obtained by Numaguchi ( 1994a) for P. fucata spat, in which 
growth increased rapidly up to a concentration of 20 cells p.L ', 



with no advantage of further increases in concentration. In this 
study, growth increased rapidly up to 12 cells (j.L"', with further 
increases in concentration producing slightly higher growth. This 
lower threshold value for P. maxima may reflect the very high 
filtration rates attainable in this species. Yukihira et al. (1998b) 
calculated that the algal concentration for maximum scope for 
growth (SFG) of adult P. maxima was 20-30 cells |jiL"'. Above 
this concentration the calculated SFG declined and was negative 
above 90 cells \xL'. The decline was primarily due to a large 
reduction in the absorbed energy as a result of a decrease in ab- 
sorption efficiency. Similarly, the SFG of P. maxima spat calcu- 
lated by Bellanger (1995) predicted that growth would decline at 
T. Iso concentrations greater than 17 cells |xL~'. Although the 
results of the present study indicate that low algal concentrations 
may still promote good growth, there is no evidence that higher 
algal concentrations are detrimental. Bellanger (1995) could not 
separate pseudofeces from true feces, and consequently the ab- 
sorption efficiency was underestimated. It is possible that spat may 
have different energetic characteristics than adults. Alternatively, 
there may be an acclimation to higher algal concentrations over 
time, which cannot be compensated for in short-term studies. 

Preasymptotic growth at various algal concentrations can be 
described by the equation: 

Growth (SGR^f day"') = 2.921 +0.05 algal concentration 

(cells p.L"') - 3.795"'* algal concentration (cells (xL~')~ 

(r- = 0.93) 

This equation predicts a maintenance ration (where SGR = 0) 
of 1.45 cells |j.L~'. This value is substantially lower than that of 
Bellanger ( 1995), where the SFG was calculated to be at 7.6 cells 
|i.L~'. Given that growth was still quite high at 6 cells (xL~' in this 
experiment, the estimate obtained in this study would appear to be 
a more accurate estimate of the maintenance concentration. Yuki- 
hira el al. (1998a) calculated that the SFGs for P. maxima and P. 
margaritifera were very high even when exposed to very low algal 
concentrations (5 cells |jiL"'). Hayashi and Seko (1986) monitored 
chlorophyll a levels and growth of P. fucata on pearl farms in 
Japan and concluded that maintenance requirements were met by 
algal concentrations that result in chlorophyll a levels of 3 (ig L~', 
whereas levels of 4-5 p.g L"' were required for good growth and 
reproductive development. This was equivalent to 6 and 10 cells 
(xL"' of P. lutheri. respectively. P. fucata appears to be adapted to 
more eutrophic conditions than P. nuixima. as chlorophyll a levels 
in Darwin Harbour are generally from 0.5 to 3 ixg L~' (Radovan 
1997) and similarly low levels occur in the main fishing grounds 
off Broome (Rose et al. 1990). Mean chlorophyll a le\els recorded 
at the Broome fishing grounds were from 0.3 to 0.9 |j.g L ' (Rose 
et al. 1990), equivalent to approximately 0.(j-1.8 cells jxL"'. Thus, 
the calculated maintenance ration in this experiment agrees well 
with observed food levels in the field. The ability to thrive in 
conditions of very low food concentrations is due to the ability to 
process very large volumes of water (\'ukihira et al. 1998a). As 
growth rates increased by 50% from 6 to 54 cells jiL"', it may be 
that growth of oysters in the field is commonly food limited. 

Organic coiiltnt 

Organic content is rarely determined in bi\al\e studies, and 
there are few references to pearl oyster spat. Given that spat or- 
ganic content increased with both algal concentration and SGR, 
and that SGR increased comnicnsuralcly with algal concentration, 
it is possible that the increase in organic content is related to the 
SGR rather than the algal concentration per se. This would agree 



Temperature and Algae Effects on P. Maxima 



165 



with the results of Taylor et al. (1997), who found that the organic 
content of P. wu.xiimi spat increased with higher SGR despite a 
reduced weight-specific ration fed to the fastest-growing spat. 

Feeding 

A limitation of the experimental method utilizing histological 
cassettes is that it is impossible to collect biodeposits: thus, the 
estimation of ingestion, absorption, and conversion efficiencies 
cannot be carried out. The filtration rates obtained in this experi- 
ment are very high compared with those of the previous experi- 
ment and other published filtration rates. This is probably due to 
the small size of the spat used, as the weight-specific filtration rate 
generally declines with increasing size according to the equation: 

FR (L h"') = aW'^ (Bayne et al. 1976). 

Thompson and Bay ne ( 1 972 ) found that the weight exponent for 
mussels less than 1 g dry weight was higher than that of larger 
mussels. Thus, the very high filtration rates found in this study 
may reflect the very small spat used. Yukihira et al. (1998a) dem- 
onstrated that smaller P. inaxiiiia and P. maigantifera spat had a 
considerably higher filtration rate than larger animals. Using the 
equation developed by Yukihira et al. (1998a). CR = 10.73 
W^^'^, the predicted filtration rate for the mean final spat ash-free 
dry weight used in this trial (0.0023 g) would be 0.115 L h^' 
compared with a measured value of 0.09 L h"' . 

Yukihira et al. (1998a) found that the filtration rates obtained 
for P. maxima and P. margaritifera were among the highest re- 
corded for any bivalve species. A similar result was found for P. 
margaririfera by Pouvreau et al. (1999). 

Reduction in filtration rate is a common response to increasing 
algal concentration (Bayne et al. 1976). The trends found in this 
study are similar to the findings of Bellanger ( 1 995 ), in which algal 
concentrations above 17 cells |jiL"' resulted in a decrease in the 
weight-specific filtration rate. In both cases, filtration rates initially 
increased with moderate increases in algal concentration and then 
declined at higher algal concentrations. 

Reduction in filtration rate with increasing temperature is con- 
trary to results from other studies on pearl oy.sters. Numaguchi 
(1994c) found that the filtration rate of 2-year-old P. fucata (5.7- 
6.1 cm shell height) increased with increasing temperature up to 
the tolerance limit before sharply declining. A similar pattern was 
shown for P. fucata spat (Numaguchi 1994a). The unusual results 
in this study are probably an artifact of the differences in size of 
the spat at the various temperatures, and a high rate exponent. 
Mean final dry tissue weight at 20 °C was 7 ± 1 .5 mg and increased 



commensurately with temperature up to 21 ± 1.7 mg at 32 °C. 
Filtration rates of the largest spat (17 L h^'g"' at 32 °C) ap- 
proached those obtained by Mills (1997) of 7.3 L h 'g"' and those 
of Bellanger (1995) (11.9 L h"'g-'). To eliminate any potential 
effects of different-sized spat, a short-term experiment would have 
to be conducted with similar-sized spat at all temperatures. 

The lack of a temperature effect on grazing rate may also be an 
artifact of the variations in spat size at the different experimental 
temperatures, as the increase in filtration rate by smaller spat 
would have masked the increase in grazing rate with higher tem- 
peratures. 

The increase in grazing rate with increasing algal concentration 
reflects the relatively low corresponding decrease in filtration rate. 
As growth did not increase proportionally, the extra algae grazed 
at higher algal concentrations was probably rejected as pseudofe- 
ces. This was consistent with observations that pseudofeces were 
produced at initial algal concentrations above 20 cells |a.L~'. A 
similar observation was made by Bellanger (1995) at algal con- 
centrations greater than 22 cells |xL"'. This increased rejection as 
algal concentration increases is reflected in the decrease in con- 
version efficiency from approximately 37% at 6 and 12 cells (xL~' 
to approximately 5% at 54 and 1 10 cells |jlL"'. 

Both the grazing and growth rates in this study are substantially 
higher than those recorded by Mills ( 1997), suggesting that growth 
is heavily dependent on food intake. This is consistent with the 
higher growth at higher algal concentrations. 

In the present study the growth rate at 6 cells |xL"' was still 
quite high, although Bellanger (1995) predicted it to be negative. 
It is likely that the metabolic costs in that study were overesti- 
mated, leading to erroneous conclusions as to the predicted growth 
at various algal concentrations. 

On the basis of the results of this study. P. maxima spat should 
be maintained at temperatures between 26 °C and 29 °C, and algal 
concentrations between 12 and 54 cells |jlL^'. Within these culture 
parameters, spat growth and survival will be optimal, and the 
efficient utilization of algal cultures will be maximized. 

ACKNOWLEDGMENTS 

This research was funded by the Co-operative Research Centre 
for Aquaculture and supported by the Darwin Aquaculture Centre 
of the Department of Primary Industry and Fisheries. Northern 
Territory. The author is grateful to the staff of the Pearl Oyster 
Propagators and the Darwin Hatchery Project, who supplied the 
spat and microalgae. 



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NJ. 



Journal of Shellfish Rcscanh. Vol. \9. No. 1, 167-174. 2000. 

INFLUENCE ON UPTAKE, DISTRIBUTION AND ELIMINATION OF SALMONELLA 
TYPHIMURIUM IN THE BLUE MUSSEL, MYTILUS EDULIS, BY THE CELL SURFACE 

PROPERTIES OF THE BACTERIA 

BODIL HERNROTH,' ANNHILD LARSSON,^ AND LARS EDEBO^ 

' The Royal Swedish Academy of Sciences 

Kristineherg Marine Research Station 

SE-450 34 Fiskebdckskil, Sweden 
'Department of Radiation Physics 

Sahlgrenska University Hospital 

SE-413 45 Goteborg. Sweden 

Department of Clinical Bacteriology 

Sahlgrenska UniversityHospital 

SE-413 46 Goteborg. Sweden 

ABSTRACT This study was carried out to investigate whether the cell surface charge of Salmonella typhinutrium could influence the 
kinetics of uptake, distribution, and elimination in the blue mussel. Mytiliis edulis. The bacteria (1 |j.m) were labeled with '"'Tc"' in the 
presence of stannous fluoride. Two different concentrations of stannous fluoride were used to produce differences in the cell surface 
charges of the bacteria. A set of mussels in the investigation were also given "'Sn-labeled microspheres (15 p,m) together with bacteria 
to compare the impact between particle size and cell surface properties on the distribution kinetics. The distribution of radiolabeled 
particles in the mussel was followed and analyzed with a computer-aided gamma camera that can detect two isotopes simultaneously. 
Finally, the mussels were dissected and the radioactivity in the fractions was measured with a well-shielded Nal(Tl) detector. The 
reduced cell surface charge of S. typhimitrium enhanced the preingeslive selection on the gills or labial palps as well as the postingestive 
selection in the digestive glands in such a way that it became similar to the microspheres, despite the size differences. The uptake of 
the bacteria labeled in the presence of less stannous fluoride was significantly lower. However, the subsequent absorption of these 
bacteria in the digestive gland was greater, because the recovery of radioactiv ity outside the digestive tract was higher than for the more 
manipulated bacteria and the inicrospheres. Likewise, the elimination of the more manipulated bacteria was similar to that of the 
microspheres and significantly higher than that of the less affected bacteria. It is concluded that the cell surface properties of bacteria, 
possibly the charge, influence the uptake, distribution, and elimination in M. edulis and that this factor could have the same influence 
as size on the uptake capacity. 

KEY WORDS: Mytilus edulis. bivalves, molluscs. Salmonella typhimurium. gamma camera, ""'Tc'^-labeled bacteria, surface, uptake, 
preingestive selection, postingestive selection 

INTRODUCTION microalgal metabolites have been proved to influence mussel feed- 

Because of their efficient filter-feeding mechanism, bivalves '"§ ^^^avior (Ward & Targett 1989). This indicates that the lamel- 

are capable of accumulating large numbers of microorganisms '''^''^"'-"'^ t"^^'^« have some ability of premgestive selection, pre- 

from the surrounding water. ^""^^'''>' °" '^e gills or the labial palps, which is not only related 

Problems related to microbes in terms of pathogenic bacteria '° ^'^^ t."' ^'^^ '° o*er particle characteristics. In addition, the 

and viruses in bivalves can be a major deterrent when developing possibility of postingestive selection in the digestive tract has been 

a sustainable plan for utilization of coastal resources. The plank- suggested (Shumway et al. 1985, Smith & MacDonald 1997). 

tonic bacterium Vibrio parahaemolyticus, as well as bacteria as- Digestion in invertebrates includes extracellular and intracellular 

sociated with fecal pollution, such as Salmonella. Shigella, and digestion processes. The extracellular digestion is a fast process 

Closlndiwn spp.. have caused numerous outbreaks of gastroen- "lat dominates during intestinal digestion. It results in low absorp- 

teritis in connection with consumption of seafood (Matches and tion efficiency and pooriy digested "intestinar" feces. The intra- 

Abeyta 1983, Rodrick and Schneider 1991. Wilson and Moore cellular digestion is a slow process in the diverticular folds of the 

1996). In addition, viruses, such as the small, naked viruses (e.g. stomach. The epithelial cells of the folds phagocytose and digest 

Calici and Norwalk viruses), hepatitis A. and enterovirus, are con- small particles with high efficiency resulting in good absorption 

sidered as health hazards in utilizing bivalves for food (Sinder- and well-digested "glandular feces" (Morton 1973. Decho & 

mann 1990. Enriques et al. 1992. Cliver 1997). To improve risk Luoma 1991). The hemocytes of the bivalves are also functioning 

assessment and develop satisfactory control methods in respect to in nutrient digestion and transport as well as in internal defense 

public health, the basic knowledge about the regulating mecha- (Cheng 1984). They contain numerous lysosomes capable of re- 

nisms for uptake, distribution, and elimination of microbes in bi- leasing hydrolytic enzymes and reactive oxygen radicals (Winston 

valves has to be improved. et al. 1991). Birkbeck and McHenery ( 1982) showed that bacteria 

A study on filtration capacity of particles in Mytilus edulis such as Micrococcus roseus and Staphylococcus aureus, resistant 

(Mohlenberg and Riisgard 1978) showed a marked decline in the to the lysozyme of M. edulis, were rejected intact, whereas Es- 

uptake of particles smaller than 7 jxm. which fell to 20% at 1 [xm. cherichia coli. M. luteus and Bacillus cereiis. which were sensitive 

Also, it has been shown by Allison et al. (1998) that particles to lysozyme, were killed after ingestion. Rogener & Uhlenbruck 

(3^0 jj-m) enriched in metals were rejected in M. edulis, and (1984) found that invertebrates recognized and bound so-called 

167 



168 



Hernroth et al. 



heterophilic antigens or ubiquitous chemical structures sucli as 
lipopolysaccharides and zymosan, wiiicli are often present on the 
surface of microorganisms. This indicates that recognition of cell 
surface characteristics of the prey might be a regulating mecha- 
nism for selection in the digestive gland. 

We hypothesize that not only size but also cell surface prop- 
erties of particles might influence the uptake and the subsequent 
distribution and elimination of microorganisms in ^4. ediilis. To 
test this, a gamma camera technique was used for in vivo recording 
of blue mussels with respect to uptake and elimination of radio- 
labeled Salmonella lyphinnirium (ca. 1 p,m) and microspheres ( 15 
|xm). After the gamma camera experiment, the radioactivity in 
dissected fractions of the mussel tissue was measured with well- 
shielded Nal(TI) detector to follow the distribution into different 
organs. The labeling procedure for S. nphimiiriiim has been de- 
scribed in the previous study by Hernroth et al. (2000). Bacteria 
with different cell surface charges were obtained by using different 
concentrations of stannous fluoride. 

MATERIALS AND METHODS 

Bacterial Strain and Growth Conditions 

S. typhinnuium .^95 MR 10. a nonvirulent. chemotype-Rd mu- 
tant (Edebo et al. 1980) was grown for 16 h in glucose broth 
(Lindberg et al. 1970) (pH 7.0-7.2) at 37 °C on a rotary shaker 
(200 rpm). The bacterial suspension was washed three times by 
centrifugation (2000 rpm. 10 min. 4 °C) in 3 mL 0.9% NaCI to 
remove the culture medium. The pellet was resuspended in 2 mL 
0.9% NaCl (2.5 x 10'^' mL"'). 

Radiolabeling and Chemical Modification of Bacteria 

Stannous fluoride (SnF-,) was used to reduce "'"'Tc"' to facilitate 
the labeling of the isotope (Perin et al. 1997). It also binds to 
protein structures intracellularly as well as at the cell surface 
(Rhodes 1991 ). It has been shown in a previous study by Hernroth 
et al. (2000) that stannous fluoride can chemically modify the cell 
surface charge of 5. typhimiiiiiim. The bacteria showed differences 
in electrophoretic mobility when 80 jjLg SnF, and 800 |j.g SnF,. 
respectively, were u.sed in the labeling procedure. The mobility for 
bacteria treated with 80 [x.% SnF, (5.4 x 10~" m" x V"'s"') was not 
significantly different from untreated 5. typliimurium (4.7 x 10 " 
m- X V"'s~'), whereas the mobility for bacteria treated with 800 
SnF, was significantly reduced (2.3 x 10"'^ nr x V^'s"'). The 
same treatment to label bacteria with different cell surface prop- 
erties was used for this study. 

One milliliter of bacteria suspension (2.5 x 10" mL"') was 
mixed with 2 mL of 37 "C 0.9% NaCl containing 80 or 800 fjLg 
SnF, and then incubated with approximately 50 MBq ""Tc"'- 
pertechnetate for 20 min at 37 "C on a rotary shaker (200 rpm). 
After incubation, the bacteria were cenlrifuged and washed three 
times. Ascorbic acid (0.25 mg niL ') was added to the washing 
solution to prevent reoxidation of the reduced ""Tc"'. The bacteria 
were resuspended in 1 mL 0.9% NaCl. 

To control the cell size and shape of S. lypliinuoiiini and ob- 
serve possible formation of aggregates, the batches of labeled bac- 
teria were inspected in a microscope (12.5 x lOOx magnificalion) 
before feeding took place. As references, unlabeled .V. lypliimiirinni 
bacteria were used. 

V(a/)i/i/v of '"'•"Tc-lxiheled Bacteria 

The viability of the bacteria was chccketl using a lluorescence 
assay (LIVE/DEAD «((( Light 1^1 Bacterial Viabililv Kit. Molecular 



Probes. The Netherlands). Triplicates of ''''Tc'":80 and '"*Tc"':800 
were compared with unlabeled bacteria from the same culture, 
using epifluorescence microscopy (Zeiss Axioscop, Exciterfilter 
BP450-490. Dichroic reflector 510, and Barrier filter LPS 159, 
Zeiss, Germany). The suspension of bacteria was diluted to 10^ x 
mL"' in sterile filtered (Schleicher & Schuell, Keene, NH, FP 
030/3) seawater (32 PSU) and incubated with the fluorescence 
probe for 15 tnin at 12 °C. The intact plasma membranes of live 
bacteria give green fluorescence, whereas compromised mem- 
branes of dead ones give red fluorescence (Haugland 1996). The 
viable bacteria were calculated as part of 100 cells. 

Mussel Experiment 

The experiment was carried out during April and May 1998. 
Blue mussels, M. edulis, were collected at 1 m depth in the Aby 
Fjord, on the west coast of Sweden (tidal amplitude 20 cm). The 
salinity, when sampling, was 28 PSU, and the temperature was 
6 °C. Mean shell length of the 32 mussels was 7.6 ± 0.8 cm, shell 
width was 3.4 ± 0.7 cm, and flesh wet weight was 1 1 .6 ± 3.5 g. The 
mus.sels were cleaned from epiphytes and stored in running sea- 
water (32 PSU and 8 °C). Two days before the experiment started, 
mussels were placed individually in hanging baskets in filtered 
(Millipore, 0.3 jjim) seawater (32 PSU, 1 2 °C) for adaptation to the 
experimental .setup, in which it was necessary to mix the water by 
magnetic stirring. The water (eight mussels in 15 L) was ex- 
changed daily. It was continuously oxygenated, and the mussels 
were fed the nanoflagellate Isochrysis ffulhana. One hour before 
the start of the experiment, each basket with one mussel was 
transferred to a beaker with 700 mL filtered (Millipore, 0.3 m) and 
oxygenated seawater (32 PSU, 12 °C). The beaker was placed on 
the magnetic stirrer in front of the gamma camera. Ten minutes 
before addition of bacteria, the mussel was given 1 niL of a sus- 
pension of 10" niL"' of/. gLilbana as a trigger for filtration, and the 
activity was visually confirmed. Thereafter, the radiolabeled mi- 
crospheres and/or bacteria were added to the beaker. The final 
concentration of bacteria was approximately 3 10" niL"', and ap- 
proximately 32.4 MBq of '"Tc"' activity was added to the water. 
The concentration of the microspheres was approximately 1.5 10* 
niL '. and the activity of "''Sn was approximately 1.1 MBq. All 
values used in this study have been corrected for the half-life of the 
isotopes. 

Sixteen mussels were given 5. typhinnuium labeled in the pres- 
ence of 80 |jLg SnF, (designated '"Tc'":80). and 16 mussels were 
given S. typhimurimn labeled in the presence of 800 (xg SnF, 
(designated ''''Tc"':8()0). Within each group of mussels. 50% were 
given "\Sn-labeled microspheres (NEN TRAC microspheres, Du 
Pont) together with the bacteria. The nondegradable microspheres 
were made from styrene-divinyl ben/ene resins and were uniform 
in size (15 (xm) (designated "'Sn:ms). 

Each mussel was exposed to radiolabeled microbes/ 
microspheres for 5 hours. During this time the radioactivity was 
continuously recorded by the gamma camera. Then the mussel was 
carefully rinsed and repositioned in front o{ the camera, but now in 
clean seawater for recording of elimination of radioacti\ ity. Fi- 
nally, the mussel was dissected and the radioactivity of the organs 
and tissue tractions was measured using a well-shielded Nal(Tl) 
detector ( 15 cm in diameter; Nuclear Enterprises, UK) in a low- 
activity laboratory. The dissected fractions were the fimbriae part 
of the mantle, one pair of gills, one pair of palps, pericardial gland 
including the pericardium, gonail. one pair of kidneys, digestive 



Influences of 5. Typhimurum in M. Eduus 



169 



gland, terminal part of intestine, crystalline style, mantle, posterior 
adductor muscle, foot, anterior adductor muscle, and retractor 
muscle. The digestive gland was transected to distinguish the an- 
terior part, including the esophagus, stomach, and diverticular 
folds (designated the stomach) from the posterior part, including 
the direct and recurrent intestine and the blind sac (designated the 
liver). The terminal intestine (designated the gut) was dissected 
separately. The dissected part of the kidneys was one of the lon- 
gitudinal canals that lie on either side of the body at the root of the 
gills and the closest connected tissue (designated the renal). As the 
transit time of the radioactive particles in the mussel tissue was 
unknown, the dissection was done with different time lags, and 
the.se were randomized among the mussels. Meanwhile, the mus- 
sels were stored as under the pre-experimental conditions, with a 
daily exchange of water but without any food supply. The time 
lags were 5. 20, 28, and 54 hours for the mussels fed on ''''Tc"':80 
(n = 4 in each group). The mussels fed on ''''Tc'":800 were dis- 
sected after 5 (/i = 4). 20 {n = 4). 28 (/i = 2). 54 (n = 2). and 
68 (II = 4) hours. It should be pointed out that these differences in 
numbers of mussel dissected at different times were not planned 
but were a result of the time-consuming dissection. 

Gamma Camera Technique 

The gamma camera technique (MAXI II General Electric. 
Hermes Sy.stem NuD, Nuclear Diagnostic. Hilgersten. Sweden) 
was used. The camera continuously visualizes the distribution of 
radioactivity in the mussel. Furthermore, by outlining the regions 
of interest (ROIs) on the screen, the radioactivity in the region was 
quantified and listed and displayed as curves of activity versus 
time. The ROIs chosen in this study were the images of the stom- 
ach and gut regions. The parameters were calculated from the 
stomach region as follows: 

Uptake = the fraction (% ) of the initial amount of the given 
radioactivity that was accumulated when the maximum value in 
the ROI was reached. The initial radioactivity in the beaker rep- 
resented the given activity. The maximum value was normalized 
to the initial radioactivity to avoid differences due to variations in 
the given activity, differences due to the distance between the 
mussel and the camera, and differences in the geometry of the 
mussels. 

Elimination = the reduction (%) from the maximum value 
measured in the ROI. The reduction was determined after 20 hours 
of measurement. These values were normalized to the maximum 
value in the ROI to avoid differences in the uptake capacity. 

Statistical Analysis 

The influence on the uptake and the elimination of the bacteria 
due to the chemical treatments (80 or 800 p.g SnF,) and to the 
presence or absence of "'Sn:ms was analyzed using two-way 
analysis of variance (ANOVA) and a Tukey test to allow multiple 
comparisons (Zar 1995). The variance of the uptake and elimina- 
tion of '"'Tc"':80. ''^Tc'^:800. and "''Sn:ms was analyzed using 
one-way ANOVA and a Tukey test (Zar 1995). To obtain inde- 
pendent measurements for the ANOVA analysis, the '™Tc'":80 and 
'*"Tc'":800 groups included mussels fed exclusively on bacteria, 
and the ' '^Sn:ms group included the same numbers of individuals 
randomized from the mussels fed simultaneously on bacteria and 
microspheres. 

Because of the differences between ''"Tc"':80 and ''"Tc"':800 in 
time lags before dissection, some of the mussels were excluded to 



equalize the groups when analyzing the distribution of the mi- 
crobes in the mussel tissue. The excluded mussels were the four 
fed on ''^^Tc"':800 dissected after 68 h and two mussels randomly 
chosen among those fed on "''Tc"':80 dissected after 28 and 54 h. 
respectively. The Mann-Whitney rank sum test (Sokal & Rohlf 
1995) was used to compare variances in content of radioactivity in 
the different fractions. Pearson product moment correlation 
(Snedecor & Cochran 1989) was used to investigate whether an 
increased amount of radioactivity in mussel tissue outside the di- 
gestive tract was related to a decrease in the digestive gland. In all 
of the statistical analyses, Sigma Stat version 2.0 (Jandel Scientific 
Software. San Rafael. CA) was used. 

RESULTS 

Viability of """"Tc-Labeled Bacteria 

The viability of the unlabeled bacteria was 96.4 ± 1.4%. The 
viability of '"'Tc'":80 was 95.9 ± 1.7%. and for ''''Tc'";800 it was 
89.6 ± 4.2%. The microscopic inspections showed the same size 
and shape of the bacteria compared with the unlabeled bacteria, 
and no aggregates were observed. 

Uptake and Elimination of Radiolabeled Bacteria and Microspheres in 
M. edulis 

When mussels were given S. typliiumriuin labeled with a small 
amount of stannous fluoride ('^''Tc"':80; Fig. I A), as well as ra- 
dioactive microspheres ("''Sn:ms; Fig. IB), the uptake of bacteria 
in the stomach was slow and small and in the gut it was neariy 
inconspicuous, whereas the microspheres rapidly accumulated in 
the stomach and later in the gut. The uptake when '''^Tc"':80 was 
tested alone (Fig. IC) was similar to that of the bacteria in the 
mixture (Fig. I A). Mussels given bacteria labeled with more 
stannous fluoride ('"'Tc'":800: Fig. 2A) as well as "'Sn:ms (Fig. 
2B) showed similar uptake kinetics for the two kinds of particles. 
A similar pattern appeared for '^"Tc"':800 alone (Fig. 2C). 

Two-way ANOVA confirmed that the presence of the ' '""Snims 
affected neither the uptake nor the elimination of ^''Tc"':80 and 
''''Tc'":800, but the difference due to the amount of stannous fluo- 
ride used for the labeling of the bacteria was significant (Table 1 ). 
The uptake (Fig. 3) and elimination (Fig. 4) varied on an individual 
basis. Still, the statistical analysis showed that the variances in the 
processing of ''"Tc'":80 compared with '"Tc"':800 and "'Sn:ms 
were significant. The uptake of "'Snims and of ''''Tc'";800 was 
significantly higher than that of '^''Tc"':80 [one-way ANOVA. F = 
32.4; degrees of freedom (df) between subjects = 1; P < 0.001, 
post hoc Tukey test]. Similarly, the elimination of ' '-'Snrms and of 
'''^Tc'":800 was significantly higher compared with that of ''''Tc'": 
80 (one-way ANOVA. F = 20.8, df between subjects = 5; P < 
0.001. post hoc Tukey test). 

The Distribution of Radioactivity in M. edulis 

The radioactivity in the different organs and tissues showed 
great differences between individuals. Despite this, the differences 
between the three different groups (""Tc'":80. ""Tc'":800. and 
"■^Sn:ms) were pronounced. Most of the activity of """Snims in 
the digestive tract (Fig. 5) was recovered in the stomach, in the 
liver, and in the gut. The activities of ''''Tc"':80 and '"'Tc'^rSOO 
were even higher in the stomach and liver. "'^'Tc'":80 could hardly 
be detected in the gut. The gills contained significantly more 



170 



Hernroth et al. 



Fig. 1A: ^'Tc'":80 




Fig. IB 



1200 




0,6 1 
0,4 


Fig. 1C; 


^'Tc'iSO 










0,2 






_^ 









mmmA^mJii 



600 
Time (min) 



1200 



Figure 1. Chart lines showing the radioactivity measured in the ROIs (stomach and gut) in a mussel which was simultaneously given 5. 
typhimuriiim labeled with ""Tc"' in the presence of 80 ng SnF, ((A) '"'Tc"':801 and microspheres labeled with '"Sn ((B) "'Sn:ms). (C) Shows a 
mussel which was given ""^Tc^iSO only. The amount of radioactivity is normalized to the initial amount given to the mussel. 



"''Tc'^iSO than ■''Tc"':800 (ANOVA, df = 15. P = 0.028). There 
were low values of ''*Tc"\ and ' ''^Snims were almost undetectable 
in the mussel tissue outside the digestive tract. When analyzing 
fractions with detectable activity, the gonad, posterior adductor 
muscle, mantle, and renal showed significantly (Mann-Whitney 
rank sum test) higher activities of ""Tc'";80 than of ''"Tc"';800 
(Table 2). The correlation analysis (Table 3) showed that when the 
amount of '^''Tc'":80 in the digestive gland decreased, the amount 



in gonad, adductor muscle, mantle, and renal increased. This was 
not the case when comparing the corresponding values for '^''Tc'": 
800. 

The Transit Time of "^Tc" in M. edulis 

The number of mussels dissected at 5. 20, 28, and 54 h were too 
few for any statistical ANOVA of transit time, but the general 



0,6 



> > 0.4 



0.2 



Fig. 2A: ^'Tc'^:800 



1^ 



600 
Time (mm) 



1200 



Fig. 2B: "^Sn:ms 




99T„m., 



Fig 2C: '^Tc"':800 




600 
Time (mm) 



1200 



Figure 2. C'harl liius shoHlng (he nididaclivlty nuiisurcd In (he KOIs (stomach and gut) in a mussel which \\as simultaneously given S. 
lyphimurium laheled with ''''iv'" In the presence of 8()() jig SnF, ((A) ''''rc"':8(l(() and microspheres labeled with '".Sn ((H) "'Sn:ms). (C) Shows 
a mussel which was given ''''Tc"':8U0 only. The amount of radioactivity is normalized to the initial amount given to the mussel. 



Influences of 5. Typhimurum in M. Edulis 



171 



TABLE 1. 

Two-way ANOVA table, comparing the influence on the uptake and 
the elimination of S. typhimuriuin in M. edulis due to the chemical 

treatments of the bacteria (""Tc^iSO or ""Tc'-'iSOOl and to the 

absence or presence of the microspheres in the medium (mono- or 

multiple medium). 



df 



SS 



Source of variation for uptake 

Chemical treatment ('"'Tc"':80 
or ''''Tc"':800) 

Mono- or multiple medium 

Residual 

Total 
Source of variation for elimination 

Chemical treatment ('"'Tc"':80 
or ''^Tc'":800) 

Mono- or multiple medium 

Residual 

Total 



6080 



^^9 ~> j9*** 



1 
28 
31 



0.0166 0.0001 (NS) 
4341 
1053 



6698 

1021 
(20)21 8348 
(23) 24 16.384 



1 



16.848*** 
2.568 (NS) 



*** P < 0.001. 

NS, not significant: P > 0.5. 

df, degrees of freedom; SS, sum of squares. 

patterns based on the mean values in Figure 6 gave some indica- 
tions. In the posterior adductor muscle and the mantle, there was a 
reduction of radioactivity from '''Tc":80 and '''^Tc"\800 after 28 h. 
The most rapid accumulation and the highest values were mea- 
sured in the gonads of the mussels fed '*'^Tc"^80. The amount of 
'^Tc^iSO did not decrease in the gonad and the renal during 54 h. 
The mussels fed on '''Tc'":800 did not show any reduction in the 
renal after 68 h (Fig. 6). In the digestive tract, there was also 
detectable activity after 54 h. In the digestive gland, there was 0.45 
± 0.26 MBq x g'' for '"'Tc"':80 and 0.57 ± 0.03 MBq x g"' for 
''^Tc'":800. In the gills, there was 0.16 ± 0.06 MBq x g"' from 
'^^Tc"\80 but not detectable values from '"'Tc'":800. 



80 



60 



> 40 ■ 
0) 



3 









o 

t 




f 












o 










+4- 










t 




^ 


o 






o 






^ 













99tc" 



99-, 



113c 



':80 ^"Tc'^iSOO ' '-^Stiims 

Figure 3. The uptake of radioactivity in the stomach ( % of the given 
amount) when its maximum activity was measured, in 16 mussels fed 
on S. typhimurium labeled with "Tc"' in the presence of 80 and 800 ng 
SnF,, respectively ('"""Tc:80 and '"'"'Tc:800) and the microspheres la- 
beled with "'Sn ("'Snims). Box plots display the median of the data, 
the lower and upper quartiles. and the lowest and highest values ob- 
served. 




^^Tc'^:80 ^^Tc'^:800 ''"'^Snims 



Figure 4. The elimination of radioactivity ( % of the maximum value in 
stomach) determined 20 h after the experiment started, in 12 mussels 
fed on S. typhimurium labeled with ''''Tc"' in the presence of 80 and 800 
Mg SnF,, respectively ("""'TcrSO and """'Tc:800) and the microspheres 
labeled with "'Sn (""'Sn:ms). Box plots display the median of the data, 
the lower and upper quartiles, and the lowest and highest values ob- 
served. 



DISCUSSION 

S. typhimuriuin 395 M RIO is an Rd-tnutant deficient of the 
0-antigenic polysaccharide side chain and with a pronounced nega- 
tive surface charge (Edebo et al. 1980). Hernroth et al. (2000) 
described the chemical manipulation of S. typhimurium, using 
stannous fluoride. The electrophoretic mobility toward a cathode 
was significantly reduced for ''''Tc"':800 when compared with 
99jj,m.gQ gjjj untreated bacteria. In this study we have found dif- 
ferences in the mussel processing of the differently manipulated 
bacteria. 

The preingestive selection of particles is expected to take place 
on the gills or on the labial palps. The structure of the gills is 
known to divert particles due to size (Riisgard et al. 1996), and it 



0,45 

0,4 
0,35 

0,3 
0,25 

0,2 
0,15 

0,1 

0,05 





■ 99mTc80 

[]99mTc800 

B113Sn:ms 



i 






it 



I 



Gills 



Palps 



Stomach 



Liver 



Gut 



Figure 5. Distribution of radioactivity of S. typhimurium [+ standard 
deviation) labeled with '"''Tc'" in the presence of 80 and 800 (jg SnFj, 
respectively (''"""Tc:80 and ''''"'Tc:800) and microspheres labeled with 
"-'Sn ("'Snims), in the digestive tract Igills, labial palps, posterior 
part of the digestive gland (stomach) and the anterior part (liver) and 
terminal intestine (gut)]. The columns are based on the mean values 
(per g tissue) from 12 mussels dissected within 54 h. 



172 



Hernroth et al. 



TABLE 2. 

Mann-Whitney rank sum test: comparison of median values 

(''■'Tc"' X mg"') in posterior adductor muscle, mantle, gonad, and 

renal from mussels fed on S. typhimurium. labeled in the presence of 

80 and 800 pg of SnF,, respectively. 



Group 



Median 



25% 



75% 



P < 0.05 



Adductor: 80 
Adductor: 800 
Mantle:80 
Mantle:800 
Gonad:80 
Gonad:800 
Renal:80 
Renal: 800 



12 
12 
12 
12 
12 
12 
12 



0.99 
0.29 
4.67 
1.58 
1.5 
0.63 
42.5 
17.4 



0.47 
0.15 
1.53 
0.37 
0.85 
0.12 
27.5 
5.. 36 



2.35 
0.55 
6.23 
2.61 
3.22 
1.00 

59.5 

30. 1 



Yes 
Yes 

Yes 
Yes 



has been suggested that potentially nutritive particles will be se- 
lected relative to inert particles on the labial palps (Hylleberg & 
Gallucci 1975. Newell & Jordan 1983. Bayne et al. 1993). The 
uptake of '''■^Tc"':800 and """Snims was similar and much faster 
than that of '''*Tc"':80. showing that the size alone did not deter- 
mine the uptake, because the "^''Tc"'-labeled bacteria were much 
smaller (approximately 1 \Lm) than the microspheres ( 15 (j.m). The 
higher uptake capacity of ''''Tc"':800 was correlated to a decrease 
of the net negative cell surface charge, indicating that negative 
charge might antagonize uptake. This study showed discrimination 
in ingestion of the less manipulated bacteria, and significantly 
more bacteria of this kind were "trapped"" on the gills. A proper 
explanation to this requires further studies, and we suggest that it 
should include electrostatic repulsion and also hemocytic attach- 
ment or engulfment of bacteria on the gills. 

The elimination of the bacteria was also affected by the chemi- 
cal modification of the cell surface. The elimination was signifi- 
cantly lower for ''''Tc'":80 than that for '^Tc'":800 which again was 
similar to that of "'Sn:ms. The less-modified bacteria were to a 
high degree accumulated in the stomach part of the digestive 
gland, but were hardly present in the gut. According to Birkbeck 
and MacHenery (1982). this indicates a postingestive selection 
based on phagocytic activity. These authors showed, in their study 
of M. cihilis. that the processing of bacteria after phagocytic uptake 
in the hepatopancreas digested the bacteria into polymers that were 



TABLE 3. 

Pearson Product Moment Correlati4)n tahle from dissected fractions 

of the mussel tissue. The relationship hetween the contents of 
radioactivity (over time, as described in Materials and Methods) in 

the digestive gland and the gonad, posterior adductor muscle, 

mantle, and renal in mussels in = 12) fed on S. lyphiiniiritim labeled 

in the presence of 80 and 800 pg SnF, (marked as :80 and 

:800), respectively. 





(:onad:80 


Adductor:80 


Mantle:80 


Renal:80 


Digestive 


-0.696** 


-0.898*** 


-0.846*** 


-0.825*** 


gland:8() 












(;onad:800 


Adductor:800 


Mantle:800 


Kenal:80U 


Digeslive 


-0.3.56 iNS) 


-0.395 (NS) 


-0.095 (NS) 


-0.244 (NS) 


gland:8()() 











** P<0.0\. 

*** p < ().()() I. 

NS, not significant; P > 0.05. 




>, 0,8 

I 0,6 

o 

<? 0,4 

^^ 0,2 



I Gon 80 




5 20 28 54 68 

Time (h) 



0,15 






o 

CD 



0,05 




20 



28 
Time (h) 



54 



68 



Figure. 6. Distribution of radioactivity of .V. typliiiiiiiriiiiii labeled >\ith 
''"Tc"' in the presence of 80 and 800 pg .SnF,, respectivelv (""Tc"':80 
and '"'Tc"':800) in posterior adductor muscle (Adduc), mantle (Mant), 
gonad (Gon), and renal (Ren) (NB: different scales). The columns are 
based on the mean values (per g tissue) from mussels dissected after 5, 
20, 28, 54, and 68 h. 

transferred to other sites in the mussel, whereas most of the 
lysozyme-resistant bacteria were rejected as fecal production. It 
has prc\ iouslv been shown that M. cdiilis can lyse bacteria extra- 
cellularly (Pricur 1981 ). but the slow processing and the preferen- 
tial absorption of ""Tc"':80 compared with ""Tc'":800 that evi- 
dently occurred in our study indicated a predominance of phago- 
cytosis and intracellular digestion of the less-manipulated bacteria. 
Absorption of radioactivity from ""Tc"':80 was supported by the 
relationship between the decrease of radioactivity in the digestive 



Influences of S. Typhimurum in M. Epulis 



173 



gland and the appearance in organs and tissue outside the digestive 
tract. This was not found for '*'*Tc"':800. These bacteria were less 
absorbed and were more directly transferred into the intestine. The 
faster elimination with lower absorption efficiency shown for 
'"Tc'^'iSOO and ' ''Sn:ms indicated extracellular digestion. 

Radioactivity was still detectable in the digestive tract of the 
mussels dissected after 54 hours. This observation was made in a 
closed system in which the water was exchanged only on a daily 
basis and should not be compared with the depuration study of 
Martinez-Manzanares et al. (1992). They showed a rather rapid 
elimination of Salmonella spp. after purification in running sea- 
water. However. Plusquellec et al. (1994) managed to detect Sal- 
monella spp. in air-stored mussels. 20 days after contamination. 
Minet et al. (1995) found culturable cells of 5. typhinniriiim in the 
gut 1 week after contamination. The possibility for extracellular 
survival of 5. typhimurium in the digestive tract, including the 
gills, as indicated by the presence of radioactivity after 54 hours, 
needs further investigation. Extracellular survival can thus be a 
cause for shellfish-borne gastrointestinal infections and should be 
included in risk assessment. Likewise, we found '^"''"Tc distributed 
in mussel tissue outside the digestive tract, such as gonads, kidney, 
mantle, and adductor muscle, 1-2 days after exposure to the bac- 
teria. This might be caused by degraded bacteria but might also be 
an effect of resistance to phagocytosis and killing. 

In the previous study by Hemroth et al. (2000), the stability of 
the isotope bindings to S. typhimurium in seawater was not sig- 
nificantly different for '"Tc'^iSO and ''"^^''^SOO. Fragile binding 
could increase the amount of hydrolysed, reduced technetium or 
free pertechnetate, but these radiochemical impurities did not in- 
fluence the uptake capacity of ''''Tc™ in the mussel. The possibility 



of diffusion of released '^''Tc'" into the mussel tissue could, as 
pointed out. interact with the measurement of the distribution of 
radioactivity in the mussel tissue. However, as the binding stability 
of '"Tc"' was comparable for ""Tc'":80 and '"Tc'":800, this could 
not explain the differences in the distribution of these microbes 
shown in this study. 

The viability estimated with the fluorescent probe of the la- 
beled 5. typhimurium was initially good (96% for '^''Tc'^:80 and 
90% for '''^Tc"':800), and the microscopic inspections confirmed 
intact cell size and shape and no aggregates. Thus, it was consid- 
ered that the same numbers of viable "''Tc'^'iSO and ^''Tc'":800 
were given to the mussels. The differences in the uptake between 
''"Tc"':80 and '"'Tc"':800 occurred directly from start, indicating 
that viability was not the discriminating factor for uptake. 

This investigation has shown that the uptake, distribution, and 
elimination of microbes by the blue mussel are strongly influenced 
by the cell surface characteristics of the microbe. This factor might 
be at least as important as particle size. We suggest that recogni- 
tion for phagocytic uptake might play an important role in the 
processing of microbes. 

ACKNOWLEDGMENTS 

We thank Prof. Magne Alpsten for providing us with the 
gamma camera facilities; Britta Ahlstrom, MD, for valuable advice 
concerning the culturing of bacteria: and Ann-Sofi Rehnstam- 
Holm, PhD, for discussing the manuscript. This research was sup- 
ported in part by the Adlerberth Foundation and by the Sustainable 
Coastal Zone Management within the Foundation for Strategic 
Environmental Research. 



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Joimuil of Shellfish Research. Vol. 19. No. I. I7.'i-181, :()0(). 

ON THE POSSIBILITY OF USING RADIOACTIVE LABELING AND GAMMA CAMERA 
TECHNIQUE TO STUDY SALMONELLA TYPHIMURIUM IN THE BLUE MUSSEL, 

MYTILUS EDULIS 

BODIL HERNROTH,' ANNHILD LARSSON,^ MAGNE ALPSTEN,^ 
AND LARS EDEBO^ 

' The Royal Swedish Academy of Sciences 

Kristineberg Marine Research Station 

SE-450 34 Fiskebdckskil. Sweden 
'Department of Radiation Physics 

Sahlgrenska University Hospital 

SE-413 45 Goteborg, Sweden 
^Department of Clinical Bacteriology 

Sahlgrenska UniversityHospital 

SE-413 46 Goteborg. Sweden 

ABSTRACT This paper presents a radiolabeling method for Salmonella typhimiiriiim to be used for in vivo studies on the kinetics 
of uptake in blue mussels with a gamma camera technique. S. lyphimurium bacteria were labeled with technetium-99m (''^Tc"') at 
conditions preserving the viability in seawater and the cell surface properties of the bacteria. Stannous fluoride (SnF,) was used to 
facilitate the binding of '^''Tc'" to the bacteria. The toxicity of SnF, could inhibit the growth of bacteria, and it can also bind 
extracellularly and reduce the negative cell surface charge of the bacteria. Additionally. SnF, can cause radiochemical by-products such 
as Tc-stannous colloids, which might interact with the image analysis. To optimize the labeling, two different concentrations of SnF, 
used in the labeling process were evaluated. Neither the efficiency nor the stability of the binding of '*''Tc'" to the bacteria increased 
significantly, when the amount of SnF, was increased 10-fold. Both treatments of bacteria reduced the viable counts, whereas the 
viability assessed microscopically with fluorescent probe was affected only little. However, after incubation in seawater, the viability 
was reduced for cells treated with the highest concentration of SnF,. Still, approximately 60% remained viable. Presence of radioac- 
tivity, not bound to bacteria, was investigated by centrifugation in Percoll. Less than 4% of probable Tc-stannous colloids were found, 
and they were therefore not considered to be disturbing the imaging analysis. The net negative surface charge of the bacteria, examined 
by measuring the electrophoretic mobility, was significantly reduced when the concentration of SnF, increased, but was still negative. 
Radioactive particles, formed by mixing '^''Tc"' and SnF, in the absence of bacteria, were rapidly taken up by mussels in a way similar 
to that of the more heavily labeled bacteria. When less SnF, was used for labeling of the bacteria, different uptake and processing 
kinetics were seen. Thus, to keep the natural conditions, the concentration of the labeling compounds have to be minimized. The study 
showed that it is possible to balance the labeling method and get a valuable tool for following the uptake and fate of 5. lypliinmrium 
in blue mussels. 

KEY WORDS: Gamma camera, radiolabeling, 99m-technetium, "'''"'Tc, Salmonella typhimurinm. bacteria, Mylilus ediilis, bivalves, 
molluscs, uptake, elimination, enteric infections 

INTRODUCTION fluoride (SnF,) has been used to reduce "^Tc"' from +VII to 

-i-IV, which facilitates its binding (Rhodes 1991, Perin et al, 1997). 

The blue mussel, Mytihis ediilis. is in nature exposed to a g^p^ j^ ^^^^ knovjn for its bactericidal effect (Tsao et al. 1982. 

mixture of particles and is able to accumulate high numbers of Caufield et al. 1987. Oosterwaal et al. 1989, Oosterwaal et al. 

microbes from the suiTOunding water. The potential for the mussel 1991 ), and therefore the amount used in the labeling process 

to become a carrier of food-borne diseases is therefore significant, ^i-if, to be selected with care. SnF, acts as an inhibitor of glyco- 

and several repons have pointed out the molluscs as commonly lytj^- enzymes, as it binds to SH groups. The metal ions interact 

incriminated in outbreaks of enteric infections (West et al. 1985, ^jth both Gram-positive and Gram-negative bacteria, and the 

Martinez-Manzanares et al. 1992, Wittman and Flick 1995, Ripa- antimicrobial effect depends on the concentration of free ions 

belli et al. 1999). Depuration studies on bivalves have shown great as well as the chemistry of the ions in the specific system (Scheie 

individual variations between mussels (Heffernan and Cabelli 1994). In addition, the concentration of SnF, must be used 

1971, Plusquellec et al. 1994) and vaiiations due to bacteria spe- with care, as it might influence the cell surface properties of 

cies (Plusquellec et al. 1998). This stresses the necessity of studies the bacteria. Olsson and Oldham (1978) have proved that the 

on the uptake and elimination of microorganisms in individual binding of metal ions to bacteria alters their cell surface charge 

bivalves to establish satisfying monitoring programs and to im- and adherence ability. With the amount of stannous fluoride 

prove risk assessment with respect to public health. used by Plotkowski et al. (1987) in the '-'"'Tc'" labeling of the 

Gamma-emitting radionuclides in bacteria can be used for in Pseudomonas aeruginosa, the electrophoretic mobility was not 

vivo imaging distribution. Technetiuni-99m ("'^Tc"') isotope has changed, but Perin et al. (1997) showed that the '^'Tc"' labeling of 

been used as a radiotracer when studying phagocytosis of viable S. ahortnsovis demands a higher concentration of SnF,. As it has 

bacteria in vertebrates (Plotkowski et al. 1987. Bernardo-Fiiha been shown that particle processing in M. edidis can be effected 

et al. 1991, Perin et al. 1997). In the coupling process, stannous when the particles are enriched in metals (Wang et al. 1995, Al- 

175 



176 



Hernroth et al. 



lison et al. 1998), the concentration of stannous in the labeling 
process is critical. 

An indirect labeling method has been used to follow the dis- 
tribution of leukocytes in humans (Mock and English 1987, 
Puncher and Blower 1995). In these assays, leukocytes were la- 
beled intracellularly by phagocytosis of colloidal compounds of 
'''Tc"' and SnF, (Tc-stannous colloid). The.se studies also demon- 
strated that active compounds might have occurred as nonspecific, 
cell surface-bound labeling with low affinity and soluble radiocol- 
loids. Radiochemical by-products might also occur in the direct 
labeling of bacteria. As a side reaction, the reduced technetium can 
bind to more low-affinity binding sites or together with SnF-, form 
Tc-stannous colloids. There is a possibility that released pertech- 
netate or Tc-stannous colloids might be ingested by the mussel and 
confuse the image analyses of the bacteria. Because of the com- 
plications described, the labeling method has to be optimized to 
avoid decreased viability of the bacteria and radiochemical impu- 
rities. Furthermore, alteration of the surface properties by the la- 
beling process has to be taken into consideration, as this could 
affect the processing of the bacteria in M. ediilis. 

Salmonella can appear in the marine environment because of 
fecal contamination (Prazeres Rodrigues et al. 1989, Papa- 
petropoulou & Moschopoulos 1996, Wilson & Moore 1996) and is 
of great interest in terms of shellfi.sh safety. The aim of this study 
was to investigate and evaluate the ""Tc"'-labeled Salmonella n- 
phimithum as a tool to study its uptake and fate in M. edulis. S. 
typhimurium 395 MR 10 was chosen because it is nonvirulent and 
known to be well accessible to phygocytosis, killing, and degra- 
dation in mammalian systems (Edebo et al. 1980). Considering the 
evaluation of the method used in the direct labeling of 5. abor- 
Uisovis (Perin et al. 1997), we compared the effect of two different 
concentrations of SnFj on (I) the viability of labeled 5. typhimii- 
riiim in seawater. (2) the labeling efficiency of the bacteria and the 
stability of the label in seawater, (3) the formation of Tc-stannous 
colloids during the labeling process, (4) the cell surface charge of 
the labeled bacteria, and (5) interaction of the labeled bacteria with 
M. edulis. 

MATERIALS AND METHODS 

Bacterial strain and growth conditions 

S. lyphimiirium 395 MR 10 (chemotype Rd. deficient of O- 
antigenic poly.saccharide side chain and most core sugars of the 
lipopolysaccharide) has been described by Edebo et al. (1980). A 
single bacterial colony was harvested from a nutrient agar plate 
(beef extract, Oxoid 10 g; peptone, Oxoid 10 g; NaCl 8 g; glucose 
7.5 g: and agar 1.47r) and cultured in glucose broth, pH 7.0-7.2 
(Lindberg et al. 1970) at 37 C tin a rotary shaker (200 rpm) for 16 
h. The bacteria were washed three times by centrifugation (2000 
rpm, 10 min, 4 °C) in ().9'/f NaCl and resuspended in 2 niL ().9"r 
NaCI (2.5 X 10" mL '). With these culturing conditions, the bac- 
teria are considered to reach the prestationary phase. 

Kadiolnheling of bacteria 

One millililei- of the bacterial suspension (2.5 x 10" mL"') was 
incubated with approximately 50 MBt| ""Tc"'-perlechnetate and 2 
mL of 37 "C ().9'/r NaCl containing 80 and SOO (xg .SnF,, respec- 
tively, to cause reduction of '"'Tc'". After incubation for 20 min at 
37 °C on a rotary shaker (200 rpm I. the bacteria were centrifugcd 
and washed three limes. Ascorbic aciil (0.25 mu x niL ') was 



added to the NaCl to prevent reoxidation of the isotope (Rhodes 
1991). The bacteria were resuspended in 1 mL 0.9% NaCl. 

Bacterial viability 

For estimation of the effect of the labeling procedure on the 
viability, bacteria treated with ""Tc'^-pertechnetate as well as with 
80 (n = 6) or 800 (n = 6) p,g SnF,, respectively, were compared 
with control bacteria incubated in 2 mL 0.97c NaCl (n = 6) only. 
The suspensions of bacteria were serially diluted in phosphate- 
buffered saline (NaCl 0.15 M, sodium phosphate 0.01 M, pH 7.2 
7.4), spread onto nutrient agar plates using a spiral plating system 
(Spiral System Inc.. Cincinnati, OH), incubated at 37 °C for 24-48 
h. and the colonies counted and the colony-forming units per mL 
(CFUs X mL"') calculated. 

The viability of the bacteria was also investigated using the 
LIVE/DEAD® SflcLight™ Bacterial Viability Kit (Molecular 
Probes, The Netherlands). Live bacteria appear with green fluo- 
rescence (SYTO 9), whereas the red fluorescence of membrane- 
impermeant propidium iodide dominates membrane-compromised 
bacteria (Haugland 1996). The bacterial suspensions were diluted 
(5 X 10'^' X mL"') in sterile filtered (Schleicher & Schuell. Keene, 
NH; FP 030/3) seawater (33.69 PSU, 6 °C), incubated on a rotary 
shaker (200 rpm) with the fluorescent probe for 15 and 180 min, 
observed in an epifluorescence microscope (Zeiss Axioscop, ex- 
citation filter BP450-490, dichroic reflector 510. barrier filter 
LP5I59, Zeiss, Germany), and the fraction of viable cells calcu- 
lated. After all labeling processes, the bacterial suspensions were 
observed in a light microscope ( 12.5 x 100 times magnification) to 
check possible effects on shape and size and aggregate formation. 

Efficiency and .stability of the ''''Tc'" labeling 

After the labeling process, the radioactivity of ''''Tc'"-labeled 
bacteria was measured using a well-shielded Nal(Tl) detector (15 
cm[diameter|; Nuclear Enterprises, UK) in a low-activity labora- 
tory. The labeling efficiency was expressed as percentage of the 
initial activity bound. 

The stability of the binding was tested by incubation of three 
batches of bacteria (final concentration 5 x 10'' x mL"' ), labeled in 
the presence of 80 and 800 (xg SnF, respectively, in filtered (Mil- 
lipore 0.3 fjim) seawater (33.69 PSU, 6 °C). Triplicate samples 
were taken within 3 min and then after 15. 30, 60, and 180 min. 
Particles >0.2 (jitTi were separated from the water using sterile 
filters (Schleicher & Schuell: FP 030/3), and the filtered volume 
was collected in vials and the fractions were measured in the 
well-shielded Nal(TI) detector. The bounded part was expressed as 
the particulate fractitin of the total radioactivity. 

Radiochemical by-products 

Possible formation of Tc-stannous colloids in the labeling so- 
lution was investigated by separation in Percoll (n = 3), with a 
density of 1.12 g x mL"'. One milliliter of the labeled bacteria was 
layered on the Percoll, and the tubes were centrifuged for 20 min 
at 2000 rpm. Free '"'Tc"'-pertechnetate and two colloidal suspen- 
sions were used as references. The colloidal suspensions were 
formed when ""Tc"' was incubated in the presence of 80 and 800 
(j-g SnF,, respectively, without bacteria (in this paper called Tc-h80 
and Tc-(-800). After centrifugation, the tubes were placed in front 
ol the gamma camera and the separated parts were measured and 
calculated as a percentage of the total radioactivity. 



Stl'dyinc. S. Typhimurivm in M. Edlius 



177 



Cell microelectrophoresis 

The cell surface charge of the bacteria, labeled in the presence 
of 80 and 800 \x.g SnF,, respectively, was investigated using mi- 
croelectrophoresis (Mark II. Rank Brothers Ltd., Cambridge, En- 
gland). The electrophoretic mobility (m~~ x V"'s~') of the chemi- 
cally treated bacteria was compared with that of untreated bacteria. 
The bacteria were diluted in 5 niM KCl. and the time needed to 
pass a 180-|xm grid in the electric field (90 V; distance between 
electrodes. 64.6 cm) was measured 10 times. The variances be- 
tween the treatments were analyzed using one-way analysis of 
\ariance on ranks (Student-Newman-Keuls method) (Sokal & 
Rolph 1969). 

Uptake by M. ediilis of radiolabeled bacteria and possible by-products 

Mussels were kept in circulating seawater of approximately 33 
PSU at 6 °C and fed the nanoflagellate Isochrysis galbana before 
the e.xperiment started. The mean length of the mussels was 7.1 ± 
0.5 cm. and the mean wet tlesh weight was 9.1 ± 3.0 g. Two sets 
of two mussels each were used to study the uptake of 5. ryplwmi- 
riiim labeled in the presence of 80 jjig SnF^ (S:80) and in the 
presence of 800 (xg SnF, (S:800). As control mussels, two were 
given the colloidal suspension (without bacteria) incubated with 
'"Tc"' and 80 p.g SnF. (Tc-l-80), two mussels were given the sus- 
pension incubated with '"'Tc'" and 800 |jLg SnF, (Tc-f800), and two 
mussels were given a suspension with free ""Tc"'-pertechnetate 
(free Tc). 

Single mussels were positioned in front of the gamma camera, 
and hung above the bottom in beakers containing approximately 
700 mL of filtered (Millipore 0.3 jjLm) seawater (33.7 PSU). The 
water was kept at 6 °C, well mixed with a stirrer, and oxygenated 
during the experiment. Labeled bacteria or reference solutions 
were added to the beaker. The final concentration when gi\en the 
bacteria was approximately 5 x lO*" mL"'. The distribution of 
radioactivity was visualized for 5 h using a conventional, com- 
puter-aided gamma camera technique (MAXI II, General Electric: 
Hermes-system NuD. Nuclear Diagnostic. Hiigersten Sweden) as 
shown in Figure 1 . By outlining the region of interest (ROI) of the 
image, the amount of radioactivity in the chosen region was mea- 
sured. The ROI chosen for this study was the area where the 
radioactivity was accumulated after passing the gills, identified as 
the stomach. The uptake was estimated as the accumulated fraction 
of the given amount of radioactivity (%), measured when the maxi- 
mum value in the ROI was reached and the uptake rate was cal- 
culated (7f min"'). The values were normalized to the initial 
amount of radioactivity to eliminate differences in the given 
amount of activity and geometric properties, such as mussel size 
and shape and the distance between the mussel and the camera. 
The radioactivity was corrected for the half-life of the isotope 
(6h). 

RESULTS 

Viability of the '"""Tc-labeled bacteria 

Compared with the unlabeled bacteria, the viable counts on 
agar plates were significantly reduced for both S:80 and S:8()0. 
When analyzing the unlabeled bacteria. 187 ± 29 x 10^ CFU were 
found. The CPUs for S:80 and S:800 were 33 ± 16 x 10^ and 10 
± 2 X 10'. respectively, corresponding to a reduction of 82 and 
9.'i9K compared with the control. The fluorescence assay showed 
(Fig. 2) that after incubation for 15 min in seawater, the viable 



B 


D 

• 


D 

• 


D 

% 





Figure 1. Gamma camera imaj^e of a mussel after beinji given bacteria 
labeled with ''''Tc"' in the presence of 8(M) (ig SnF,. (A) Concentrated 
along the gills of the mussel. (B) Accumulated in the stomach region. 
(C) Directed to the gut. (D) Transported as fecal content through the 
gut. 

fraction of S:80 (88 ± 6%) was similar to that of the unlabeled 
bacteria (94 ± 2%). The corresponding value for S:800 was 81 ± 
%%. After incubation for 180 min in seawater. the viable fractions 
of the unlabeled bacteria and S:80 remained unchanged, being 97.3 
± 0.6% for the unlabeled bacteria and 96.9 ± 2.2% for S:80, 
whereas for S:800 it was significantly reduced (59.2 ± 4.5%). The 
microscopic inspections showed that the cell size (approximately 1 
p.m) and shape were not altered for S:80 and S:800. and no ag- 
gregates were observed. 

Efficiency and stability of the ''"'Tc'" labeling 

The efficiency of the labeling of S:80 (77 ± 7%) and of S:800 
(70 ± 14%) was not significantly different. The amount of '^'^Tc'" 
released during the first hour in seawater was approximately 37% 
for S:80 and 30% for S:800. During the following hour, the bound 
i«j^m jfgygj more stable (Fig. 3). 

Radiochemical by-products 

After centrifugation in Percoll (Fig. 4). free Tc stayed on the 
top layer (96 ± 17r), as did the bacteria. S:80 (95 ± 2%). and S;800 



120 




D15 min 
■ 180 min 



Control S:80 S:800 

Figure 2. Viable cells (% of the total number of cells) (+SD. n=6), 
estimated by fluorescence assay, of S. typhimurium labeled in the pres- 
ence of 80 (S:8(l) and 800 (S:800) \i% SnF,, respectively, and unlabeled 
S. typhimurium (Control). The viability was estimated after 15 and 180 
min of incubation in seawater. 



178 



Hernroth et al. 



^ 100 



> 

ni 

e' 
o 

H 




50 



150 



200 



100 
Time (min) 

Figure 3. The particulate fraction ( % ) of the total amount of ^'^Tc"* 
(+SD, n=9l from S. typhimurium labeled in the presence of 80 (S:80) 
and 800 (S:800) fig SnF,. respectively, measured during 180 min of 
incubation in seawater. 

(92 ± 1 %), and less than 4% were found at the bottom. In the tubes 
with colloids formed during the incubation of '^''Tc'" with ShF, 
(without bacteria), more were found at the bottom. The bottom 
fraction increased with the amount of SnF,. showing 48 ± 17% of 
the radioactivity in Tc+80 and 71 ± 22'7o in Tc+800. 

Cell microelectrophoresis 

One-way analysis of variance on ranks (Kruskal-Wallis) 
showed that the electrophoretic mobility for S. lyphimuriuin was 
affected by the treatments of the bacteria (Table 1 ). There was a 
statistically significant reduction in electrophoretic mobility for 
S:800 compared with S:80 and untreated bacteria. The median 
value for S:800 was 2.3 x 10"" m" x V's"'. For S;80 it was 5.4 
X 10"'' m" X V~'s"' and for untreated bacteria 4.7 x 10"'' m" x 



Uptake by M. edulis of radiolabeled bacteria and possible by-products 

Preliminary studies on the uptake of labeled bacteria by mus- 
.sels showed that initially the radioactivity accumulated in the gill 
area and subsequently in the stomach and gut region (Fig. I). 
Figure 5 displays the curves from the measurements of radioac- 
tivity in the stomach from the two mussels given bacteria (S;80 
and S:800, respectively). The accumulation of S:80 in the stomach 
region was 1 1 ± 1.4% of the given amount of radioactivity and the 
process was slow (0.04 ± 0.01% min"') and nondynamic. Of the 
given activity of S:800. 32.7 ± 0.28% was measured in the stom- 




■ top 
n bottom 



Tc+80 Tc+800 S:80 S:800 free Tc 



Figure 4. The mean percentage of the total amount of "Tc"" activity 
(n=3) accumulated in the top and the bottom layers of the test tube 
after centrifugation in Percoll. The columns show '"Tc"' incubated 
only with 80 (Tc+80) and 800 (Tc+800) m8 SnF,, S. typhimurium la- 
beled with ''■'Tc™ in the presence of 80 (S:80) and 800 (S:800) fig SnF,, 
and free ''''Tc'"-pertechnetate (free Tc). 

ach. The accumulation was faster (0.36 ± 0.20% min"'). and the 
reduction came in pulses. 

Figure 6 displays the curves from the measurements of radio- 
activity in the stomach from the two mussels fed on the colloidal 
suspensions (Tc-i-80 and Tc-f800. respectively). The radioactivity 
from Tc4-80 was 21.2 ± 3.6%. and the uptake rate was 0. 14 ± 
0.01% min"'. For Tc-l-800. the uptake was 31.1 ± 2.2% and the 
process was faster (0.31 ± 0.02% min"'). The dynamic movements 
of the radioactivity in the stomach were similar between these 
mussels. The radioactivity from the two mussels given free Tc was 
below the limit of detection. 

DISCUSSION 

S. rypliiinuriiini 395 MR 10, used in this study, is a deep rough 
(chemotype Rd) mutant (Edebo et al. 1980). its surface is more 
hydrophobic and negati\ely charged than in most other Salmo- 
nella, and it forms a homogenous single-cell suspension in water. 
Labeling with '""Tc"^ using the high amount of SnF^ (S:800) re- 
duced the net negative charge of the bacteria as studied by use of 
electrophoresis. When less SnF, (S:80) was used, no effect on 
charge was discerned. Wang et al. ( 1995) and Allison et al. (1998) 
have suggested that the cell surface properties of particles will 
intluence the preingestive selection on the labial palps. Our results 
are in accordance with these suggestions. The reduced net negative 
charge of .S:8()() was probably a consequence of accumulation of 



TABLE 1, 

One-way ANOVA on ranks (Kruskal-Wallis) comparing the electrophoretic mobility (nr x V"'s"'l for S. typhimurium, treated with 80 and 
800 ng SnFj, respectively (S:80, S:800), and untreated .S. typhimurium (control I (post hoc Student-New man-Keuls). 



Group 


n 


Median 


25% 


75% 


Control 


1(1 


4.7 X 10"" 


4.1 X I0-' 


5.1 X 10-' 


S:80 


10 


.'i.4x 10"' 


4.7 X 10-" 


t.f, X 10"" 


S:8()0 


10 


2..1X 10-" 


2.1 X nr" 


2.5 X lO"" 


Comparison 


DitTerence of Ranks 


P 


q 


P < 0.05 


S:800 vs. S:80 


l.'i6 


.1 


.■S.6 


Yes 


S:80 vs. control 


24 


2 


1.2S 


No 


Control vs. .S:8()() 


1,12 


2 


7.06 


Yes 



Studying 5. Typhimvrivm in M. Edulis 



179 



0,5 



o ~ 

c « 

>= 0,25 
5 



S:80 



60 



120 180 

Time (min) 



240 



300 



0,5 -1 



o -> 

£■= 0,25 - 
> 5 



o 

'■5 



Tc+80 




60 



120 180 

Time (min) 



240 



300 



Stomach 
Gut 



Stomach 
Gut 



0,5 y 



S:800 



Tc+800 




180 



240 



300 



Time (min) 



Figure 5. Curves showing the "Tc"' activity in the stomach and gut 
(observation time 5 h) of mussels fed S. typhimurium, labeled in the 
presence of 80 (S:80» and 800 (S:800) (ig SnF,. 

positively charged metal complexes at its surface. These com- 
plexes might work as ligands for binding to mussel receptors or 
mainly operate by reducing the electrostatic repulsion between the 
bacterial particles and the recipient mussel surface. The differences 
in the cell surface properties between S:80 and S:800 might be a 
possible explanation for the differences shown in uptake and ki- 
netic handling of the bacteria in the mussels. 

Previous studies by Mayhew and Brown (1981) and Tseng and 
Wolff (1991) showed that SnF, inhibits the growth of the bacteria. 
This was also the case in our study. The viability in terms of viable 
counts was significantly reduced for S:80 and still more so for 
S:800. Bacteria in the prestationary phase w,ere used for the label- 
ing experiment, but log phase might have been a better condition 
for preserving the viability. However, the suppressed growth on 
agar did not correspond to the viability estimated by use of a 
fluorescent compound, probing the integrity of the barrier of the 
cell membrane, indicating that the labeling process may impair 
growth and division without conspicuously disturbing the cell 
membrane barrier. S:80 was better maintained during the incuba- 
tion in seawater. but initially the viability of S:80 and S:800 was 
similar. Our evaluation is that the differences shown for the uptake 
should not be explained by differences in viability, since this phe- 
nomenon appeared directly from start when the viability of S:80 
and S;800 was still comparable. The similarity between S:80 and 
S:800 in cell membrane integrity, size, and shape made us judge 
them as equal prey when given to the mussels. 



> 
•a 



0,25 




60 120 180 240 300 



Time (min) 

Figure 6. Curves showing the '^Tc" activity in the stomach and gut of 
mussels (observation time 5 h) given a suspension of by-products 
(probable "Tc^-stannous colloids) formed during incubation with the 
isotope and 80 ^g SnF^ (Tc+80) and the isotope and 800 (Tc+800) ^g 
SnF,. 



According to Ross et al. (1984). the size of Tc-stannous col- 
loids is approximately 1.5 |j.m. which is close to that of 5. typh- 
imurium. When giving the mussels the suspension with complexes 
formed between '''^'Tc'" and SnF,. without involvement of biologi- 
cal material, radioactivity was also accumulated in the mussels, in 
a way very similar to that of the more heavily labeled bacteria. 
These results indicate that the metal complexes on the surface of 
bacteria play a mediating role in the uptake process and that by- 
products formed when labeling the bacteria can influence the im- 
aging analysis. The fraction of activity not bound to the bacteria 
was not greater than the fraction of by-products found when ana- 
lyzing free pertechnetate, which indicates that it might include free 
or hydrolyzed pertechnetate and not only colloids. However, these 
fractions of "probable colloids" produced less than 4% of the total 
amount of radioactivity, and the influence on image analysis was 
considered to be of minor importance for the purpose of this study. 

The amount of free or hydrolyzed pertechnetate in the bacterial 
suspension could not be established. The labeling efficiency was 
not significantly different comparing S:80 and S:800. The mean 
efficiency was 73.3%, and there is no evidence that the excess of 
'*'*Tc" was separated from the bacteria through the washing steps. 
In addition, a released fraction of radioactivity from the bacteria 
suspension appeared during the incubation in .seawater. However, 
this study showed that even though the mussels were offered only 



180 



Hernroth et al. 



free pertechnetate (free Tc), the uptake was not detectable and did 
not affect the measurements. The possibility of passive diffusion of 
free pertechnetate cannot be excluded and needs further investi- 
gation. 

The appearance of soluble '^''Tc"' was not significantly greater 
for S;800 than for S:SO after 180 min of incubation, even though 
the viability was more reduced. Thus, only a limited proportion of 
the bacteria were lysed. or lysed bacteria did not release the ra- 
diotracer. The feeding activity of the mussels can be stimulated 
both for paniculate or nonparticulate food (Thompson and Bayne 
1972). Cell leakage due to lysed bacteria could elicit a chemosen- 
sory response, which might explain the preferential uptake of S; 
800. However, the intact state of cell membranes and similar up- 
take of Tc-stannous colloids argue against such an effect. Aggre- 
gation of the bacteria would also affect the ingestion, but as no 
aggregates were found by the microscopic inspection, this expla- 
nation is rejected. 

In summary, this study showed that there seems to be a higher 
uptake capacity and a more dynamic processing of the bacteria in 



the digestive gland due to the amount of SnF, used in the labeling 
process. The disturbance of the processing stresses the importance 
to keep the bacteria in a state as natural as possible. Although 
stannous fluoride is a toxic component to bacteria, it can be used 
as a reducing agent in the labeling process to produce a stable 
gamma-emitting bacterial tracer. However, the concentration used 
for this purpose has to be taken in consideration when studying 
uptake of viable bacteria in mussels. Bacteria labeled with gamma- 
emitting radionuclides, such as '^''Tc'"-pertechnetate. have the po- 
tential to be used in numerous applications of bivalve research. 

ACKNOWLEDGMENTS 

We thank Assoc. Prof. Staffan Wall, Department of Physical 
Chemistry, Goteborg University, for helping us with the determi- 
nation of the electrophoretic mobility of the bacteria. This study 
was funded through grants provided by Adlerberth Foundation 
and the Sustainable Coastal Zone Management (SUCOZOMA) 
project of the Foundation for Strategic Environmental Research 
(MISTRA). 



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Journal of Shellfish Research. Vol. 19. No. I. I8.V186. 2000. 

ISOLATION AND CHARACTERISATION OF A cDNA ENCODING AN ACTIN PROTEIN FROM 

THE MUSSEL, MYTILUS GALLOPROVINCIALIS 



GUILLAUME MIXTA, PHILIPPE ROCH, AND 
JEAN-PAUL CADORET* 

Defense et Resistance chez les Invertebres Marins 
(DRIM) IFREMER-CNRS- Universite 
de Montpellier 11 - Case courrier 80-2 
Place Eugene Bataillon F-34095 
Montpellier Cedex 05. France 

ABSTRACT A full-length complementary DNA encoding an actin was isolated from a Mytilus galloprovincialis hemocyte library. 
This actin displays a typical 376 amino acid open reading frame. Northern blotting indicated that the expression of the actin gene is 
particularly abundant in muscular tissues. This actin cDNA will be useful as a potential genetic marker as a standard for expression 
level in genetic regulation studies and will allow screening for the whole gene as well as its upstream regulation sequences. 

KEY WORDS: Actin. Mytilus galloprovinciaUs. mollusk 



INTRODUCTION 

Actins are highly conserved contractile proteins ubiquitous in 
all eukaryotic cells. In muscle cells it is important in myofibrillar 
contraction, and in non-muscular cells these proteins play a role in 
diverse functions such as motility, phagocytosis, chromosome 
movements, and transport of niacromolecules within the cells 
(Kom 1978). Muscle-specific actins can be distinguished from 
cytoplasmic actin in vertebrates by their primary sequences 
(Vandekerckhove and Weber 1978). For example, the amino acid 
Val 10 is characteristic of cytoplasmic actin. while Val 17 is typi- 
cal of muscular actin. Wesseling proposed 3 boxes in the N- 
terminal region as diagnostic for the family to which an actin 
belongs (Wesseling et al. 1988). In invertebrates, actins also have 
both muscular and non-muscular functions, but these two classes 
are not readily distinguished on the basis of amino acid sequence. 
Indeed, invertebrate muscular forms of actins are closer to P-cy- 
toplasmic pattern of vertebrate. In practice, rigorous analysis of 
tissue expression is necessary in order to distinguish between the 
different forms. Actin genes are very abundant and constitutively 
expressed. As such they have been subject to numerous studies 
also among invertebrates (Gomez-Chiarri et al. 1994; Horard et al. 
1994; Lardans et al. 1997; Cadoret et al. 1999). 

The bivalves rely on an innate immune defence based on both 
cellular and humoral components which interplay to eliminate po- 
tentially infectious microorganisms. One such innate immune 
mechanism is the production of antimicrobial peptides which have 
been recently identified in mussels of the genus Mytilus sp. (Hu- 
bert et al. 1996; Charlet et al. 1996; Mitta et al. 1999a.b). A deeper 
knowledge of this defence systein would allow the establishment 
of health controls to detect bivalve immunodeficiency, the selec- 
tion for disease resistance with a coupling of immunology and 
genetics or by referring to classical genetics, the characterisation of 
immune genes could be exploited in genetic quantitative selection. 
Finally, genetic transformation constitutes another promising strat- 
egy to obtain resistant strains by various modifications systems. As 
part of this strategy, the identification of constitutive genes like the 
actin. that provide tools in the study of regulation mechanism of 



*Cortespondence to; jean.paul.cadoret@ifremer.t'r 



the identified peptides was undertaken. We isolated a full-length 
actin cDNA and carried out initial inapping of its expression by 
Northern blot experiments. This is a first step toward the identi- 
fication of promoter regions as well as the sequencing of the whole 
gene. 

MATERIAL AND METHODS 

Animals and Hemolymph Collection 

Adult mussels (Mytilus galloprovincialis) were obtained from a 
commercial shellfish farm (Palavas, France, Gulf of Lion) during 
winter. The hemolymph of 20 mussels (approximately 0.5 mL/ 
animal) was extracted via a 23G needle plus syringe, directly into 
an equal volume of the anti-aggregant buffer. Modified Alsever 
Solution (MAS. Bachere et al. 1988). and immediately centrifuged 
at 800 g for 15 min at 4 °C. The cell pellet was air-dried and stored 
at -80 °C until required. 

Actin-Specific and Ribosomal ISS-Specific DNA Probes and Screening 
of cDNA Library 

Poly (A)* RNA from adult mussel hemocytes were used to con- 
struct a cDNA library in the ZAP Express Vector (Stratagene. La 
Jolla). Reverse transcription and polymerase chain reaction (PCR) 
were used to prepare a DNA probe corresponding to hemocytic 
actin. Three jxg of total RNA (see below for RNA isolation) were 
submitted to reverse transcription using the Ready-to-Go You- 
prime first strand beads kit (Pharmacia). One-fifth of the reaction 
was directly used as a template for PCR with two primers designed 
from a consensus actin sequence by M. E. Unger and G. Roesijadi 
(1993) for the oyster Crassostrea virginica. and renamed Avil 
(5'TAA TCC ACA TCT OCT GGA AGG TGG 3') and Avi2 
(5'TCA CCA ACT GGG ATG ACA TGG 3'). PCR was per- 
formed in 50 (iL with 40 cycles consisting of 1 min at 94 °C, 1 min 
at 60 °C and 1 min at 72 °C with 1 .5 mM MgCL and 1 (JiM 
primers. 

The resulting 846 base pair fragment corresponding to an actin 
cDNA fragment was cloned using the pCR-Script Amp SK (-I-) 
Cloning Kit (Stratagene, La Jolla). The plasmid containing the 
actin cDNA fragment was called pBSAct.846. The pBSAct.846 
insert was labeled with |'-P] by random priming using the Ready- 



183 



184 



MiTTA ET AL. 



ARNm 



First strand cDNA 



846 bp actin cDNA 
fragment 



■ AAAAA(A)n 
TTTTTT(T),, 



Reverse transcription 

Avi2 

< 



Avil 



■ mTT(T)i 



Polymerase Cham Reaction 

(specific amplification of the actin cDNA fragment) 



Cloning, [ "P] labelling 
and cDNA library screening 



Actin cDNA 



Complete 
aclin cDNA 



• AAAAA(A), 
TTTTTT{T), 



5 ' RACE-PCR 



AAAAA(A), 



Figure I. Complete characterization of Mytilus galloprovincialis actin cDNA {IVIya2). 



to-go DNA labeling kit (Pharmacia Biotech.) and used to screen 
400,000 plaques from the cDNA library that has been transferred 
to Hybond-N filter membranes (Amersham Corp.). High strin- 
gency hybridization was carried out overnight at 65 °C in 5X 
Denhardt"s solution, 5X SSPE (Sambrook et al, 1989). 0.1% SDS, 
100 (j.g/niL salmon sperm DNA. The filters were washed in a 
solution of 0.5X SSC containing 0.1% SDS at 65 °C, followed by 
autoradiography. A secondary screening was performed to purify 
the positive clones. Phagemids were obtained by in vivo excision 
according to the manufacturer's instructions and sequenced on 
both strands. 

To compare the relative expression of actin messenger in vari- 
ous tissues of the mussel (see Northern-blot analysis), a probe 
detecting a 18s rRNA, present at the same level in all tissues was 
designed. As such, a sense oligonucleotide primer (5'TGAC- 
CTCGCGGAAAGAGCGC 3') and an antisense oligonucleotide 
primer (5'AGGGGACGTAATCAACGCGAGC 3') were de- 
signed from the sequence of the ribosomal RNA small subunit 
(Kenchington ci al. 1995) and used in PCR experiments. Five 
hundred ng of mussel genomic DNA were submitted to amplifi- 
cation in 50 \^.L using 35 cycles consisting of 1 min at 94 C, 1 min 
at 60 °C and 1 min at 72 °C with 1.5 mM MgCK and 1 |j.M 
primers (Fig. 1 ). 

Northern lilol Analysis 

The hcmocytes from 4 mussels collected together during winter 
(8 X 10'' cells per animal) were centrifuged and resuspcndcd in 1 
mL of Tri/ol (Life Technologies). Immedialcly after hemolymph 
collection, the mantle, foot, labial palps, gills, hepatopancreas, and 
adductor muscle were excised from the same animals and washed 
extensively in sterile-riltcred seawater. The tissues (100 mg of 
each) were honiogeni/ed in 1 mL of Tri/ol with 30 strokes of a 
Potter homogeni/cr to break the cells in 1 ml. of Tri/ol and total 
RNA was extracted according to the manufacturer's protocol (Life 



Technologies). Five \x.g of total RNA was isolated from each tis- 
sue, pooled from each animal (20 jj.g total per tissue) and subse- 
quently analyzed. 

Total RNA and size markers were electrophoretically separated 
on a 1.2% agarose gel containing 17% formaldehyde, transferred 
and cross-linked to a Hybond-N filter membrane (Amersham) 
which was then stained with methylene blue. The membrane was 
hybridized with the |'"Pl-labeled actin cDNA probe in a solution 
containing formamide (50%), 5X SSC, 8X Denhardt's solution, 
sodium phosphate (0.05 M pH 6.5). SDS (0.1%) and salmon sperm 
(100 iJLg/mL) at 55 'C for 12 h. The membrane was washed in 0.2 
X SSC. 0.1% SDS at 65 "C and autoradiography was carried out. 
After autoradiography, the membrane was stripped by incubating 
the blot with a boiling solution of 0.1% SDS for 1 hour and 
submitted to a subsequent hybridization with the ['•'P|-labeled 
DNA probe revealing I8S rRNA. 

Rapid Amplification of 5' cPNA end (RACE-PCR) and PCR 

To obtain the complete cDNA sequence corresponding to the 
actin mRNA. a 5' RACE-PCR was undertaken. This was per- 
formed using the 5' RACE Kit (Boehringer Mannheim) following 
the manufacturer's instruction. Briefly. 2 (xg of total RNA from the 
pooled hcmocytes were submitted to reverse transcription using 
antisense 24 nucleotides primer (5'ATGATGTC TGTTT- 
TATAAAGTTAT 3'). deduced from the actin cDNA sequence. 
After first-strand cDNA synthesis and addition of a poly(A) tail at 
its 5' end. PCR was performed with an oligo d(T)-anchor primer 
and a nested antisense primer of 24 nucleotides (5'AGAGGAG- 
TATCTCACCCTGACTTC 3) deduced from the actin cDNA se- 
quence. Amplification was performed according to the following 
program; melting at 94 "C for 1 min. annealing at 50 °C for I min. 
elongation at 72 ' C for 1 min (35 cycles). The PCR products were 
cloned using the pCR-Script Amp SK (-I-) Cloning Kit (Stratagene) 
and several dillerent cDNA clones were sequenced. 



Mytilus galloprovincialis 185 

1 tcttttacca gtctgttgta gaagtcaggg tgagatactc ctctttagcg 

MCDDKVA 7 

51 tttagtataa ctttataaaa cagacatcAT GTGTGACGAC AAAGTAGCCG 

ALVV DNG SGMC KAG FAG 24 

101 CTTTGGTAGT AGACAATGGA TCAGGAATGT GCAAAGCTGG TTTCGCCGGA 

NDAP RAV FPS IVGR PRH 41 

151 AATGATGCTC CAAGAGCCGT GTTTCCCTCC ATCGTTGGAA GACCAAGACA 

QGVMVGMGQKDSYVGD 57 

201 TCAGGGAGTC ATGGTTGGTA TGGGTCAGAA AGACTCCTAC GTAGGAGATG 

EAQS KRG ILTL KYP lEH 74 

251 AAGCCCAGAG CAAGAGAGGT ATCCTCACCC TGAAATACCC AATTGAGCAC 

GIVT NWD DME KIWH HTF 91 

3 01 GGTATCGTCA CAAACTGGGA CGATATGGAA AAAATCTGGC ATCACACCTT 

YNE LRVA PEE HPV LLT 107 

351 CTACAACGAA CTCCGTGTTG CCCCAGAAGA GCACCCAGTC CTTCTGACTG 

EAPL NPK ANRE KMT QIM 124 

4 01 AGGCTCCACT CAATCCCAAA GCCAACAGGG AAAAGATGAC CCAGATCATG 

FETF NAP AMY VAIQ AVL 141 

451 TTCGAGACCT TCAATGCACC AGCCATGTAC GTCGCTATCC AGGCCGTACT 

SLY ASGR TTG IVL DSG 157 

501 CTCACTGTAT GCTTCCGGTC GTACCACTGG TATCGTACTC GACTCTGGAG 

DGVT HTV PIYE GYA LPH 174 

551 ATGGTGTCAC ACACACCGTA CCAATCTACG AAGGTTACGC TCTTCCCCAC 

AILC LDL AGR DLSD NWM 191 

601 GCCATCCTCT GTCTAGACTT GGCCGGTAGA GATCTTAGTG ATAACTGGAT 

KIL TERG YSF TTT AER 207 

6 51 GAAAATCCTC ACCGAGAGAG GTTACTCATT CACAACCACC GCGGAGAGAG 

EIVR DIK EKLC YVA LDF 224 

701 AAATCGTTAG AGACATTAAG GAAAAATTGT GCTATGTTGC TCTTGATTTC 

EQEM STA ASS SSLE KSY 241 

751 GAGCAGGAAA TGTCAACCGC CGCTTCTTCA TCTTCCCTAG AAAAGAGCTA 

ELP DGQV ITI GNE RFR 257 

801 CGAATTGCCC GATGGACAGG TTATCACCAT TGGTAACGAA AGATTCAGGT 

CPES LFQ PSFL GME SAG 274 

8 51 GTCCAGAATC ATTATTCCAA CCATCCTTCT TGGGTATGGA ATCTGCTGGT 

IHET TYN SIM KCDV DIR 291 

901 ATCCATGAAA CCACATACAA CAGTATCATG AAGTGTGATG TCGATATCCG 

KDL YANT VLS GGT TMF 307 

951 TAAGGACTTG TACGCCAACA CCGTCTTGTC TGGTGGTACC ACCATGTTCC 

PGIA DRM QKEI TAL APS 324 j 

1001 CAGGTATTGC CGACAGAATG CAGAAGGAAA TCACAGCACT TGCTCCAAGC 

TMKI KII APP ERKY SVW 341 

10 51 ACAATGAAGA TCAAAATCAT TGCCCCACCA GAGAGGAAAT ACTCCGTCTG 

IGG SILA SLS TFQ QMW 357 

1101 GATCGGTGGT TCCATCTTGG CTTCATTGTC CACCTTCCAA CAGATGTGGA 

ISKQ EYD ESGP SIV HRK 374 

1151 TCAGCAAACA GGAATATGAC GAATCTGGCC CATCCATTGT CCACAGGAAA 

(^ p * 3 76 

1201 TGCTTCTAAa ctaaattgtt ttctaggact tatattaatt tattttcaaa 
1251 tctcgttaaa acaaaaagtt tcgtgcttgg taacatggac tttaatttat 
1301 acaaactgtc tttaaccctt tcaaacttca gatctgtact agcattgagc 
1351 Caacggtact tgtacaaata taggacagta aattattatt tgttttatgt 
1401 gaaaaagtct ggtggttcaa atgcaagaat gtggagagtt gaatgtgaaa 
1451 aagacttgta aaaatactaa acaatccgga aacatatttc aggtttccag 
1501 gggagataac tttttactaa atttgatgta catgtgg aat aaa tcatctq 
1551 cattattgtg ataaaatgac ctttatacat ccaattatat taaatcttat 
1601 aaaaaaaaaa aaaaaaaa 
Figure 2. Nucleotide sequence and deduced amino acid sequence of the Mytilus galloprovincialis actin cDNA (lVIya2). Untranslated regions in 
lower-case letters. Start codon in boldface letters. Polyadenylation signal is underlined. 

RESULTS AND DISCUSSION 1618 bp and codes for a typical 376 amino acids actin. The 5' 

RACE-PCR experiment allowed an additional 19 base pairs to be , 

After colony blot of the cDNA library, 5 positive clones among added and helped to suggest the Transcription Start Point ( + 1). j 

352 were chosen and submitted to secondary screening for isola- Best homologies in amino acid sequence were found with the i 

tion. The corresponding phagemids were obtained by in vivo ex- bivalve Placopeclen magellanicus: 97.8%, the nematode Cae- 

cision and the longest was sequenced on both strands (Fig. 2). This norhabditis elegans: 96.27f , the brine shrimp Anemia sp; 96.5% 

complete actin cDNA (named Mya2. Genbank accession number and the silk worm Boniby.x inori: 96.2%. For nucleic acid se- 

AF157491) shows a potential coding sequence stretching over quence. best homologies are found with the scallop Placopeclen 



186 



MiTTA ET AL. 



12 3 4 5 6 7 



Actin 
(1750b) 

ISsrRNA 




Figure 3. Northern blot analysis of RNAs from various tissues of the 
mussel. Twenty (ig of total RNAs from various tissues: 1, hemocytes; 2, 
mantle; 3, foot: 4, labial palps: 5, gills: 6, hepatopancreas: 7. adductor 
muscle. All were separated by 1% agarose-formaldehyde gel electro- 
phoresis, blotted and hybridized with '"P-labeled cDNA probe corre- 
sponding to actin cDNA. The RNA relative amounts of the various 
tissues are evaluated by hybridizing the same membrane with a probe 
corresponding to the 18S rRNAs because the actin mRNA probed is 
differentially expressed in the different tissues tested. 

magellanicus (85%). the zebra mussel Dreissena polymorpha 
(84%) and the oyster Crassostrea gigas (83%). According to 
Vandekerckhove and Weber, (1978) who described 20 residues 
discriminating muscular and cytoplasmic actins, Myal displays 



feature of cytoplasmic actin for 10 codons, while 3 of them show 
the mark of muscular actins. The cystein in position #2 is a com- 
mon feature among invertebrate actins. although some exceptions 
are documented. Actin mRNAs were detected in various tissues as 
demonstrated by Northern Blot experiments using the M\a2 cDNA 
as probe (Fig. 3). The signal is particularly strong in the mantle, the 
labial palps, and the adductor muscle. This strong signals, how- 
ever, are mainly due to the recognition by the probe of all forms of 
actin niRNA. Indeed, conservation is so high (particularly within 
the used probe) that both muscular and cytoplasmic forms are 
highlighted giving a cumulative signal. 

Several isoforms have been reported in higher vertebrates, di- 
vided into muscular and non-muscular actins (Rubenstein 1990). 
Due to the high level of similarity with the other actin genes, this 
sequence may not be suitable for intra- and inter-species phyloge- 
netic studies. Nevertheless, the potential availability of intronic 
non-expressed sequences within this actin gene would be of inter- 
est in developing a selectively neutral marker as has already been 
done in other bivalves (Corte-Real et ai. 1994; Ohresser et al. 
1997). Furthermore, this complete cDNA sequence can now be 
used in regulation studies as an expression level standard as well 
as an anchor in the search for the complete gene including the 
proximal promoter involved in its expression pattern. 



ACKNOWLEDGMENT 

We are indebted to Andy Beaumont for critical reading of the 
manuscript and correction of English. 



LITERATURE CITED 



Cadoret. J. -P., R. Debon. L. Cornudella. V. Lardans, A. Morvan. P. Roch 
& V. Boulo. 1999. Transient expression assays with the proximal pro- 
moter of a newly characterized actin gene from the oyster Crassostreci 
gii(as. FEES Letters. 460:81-85. 

Bachere. E.. D. Chagot & H. Grizel. 1988. Separation of Crassostrea gigas 
hemocytes by density gradient centrifugation and counterflow centrifu- 
gal elutrialion. Dev. Comp. Immunol. 12:549-559. 

Charlet. M.. S. Chernysh, H. Philippe. C. Hetrut. J. Hoffmann & P. Bulet 
1996. Isolation of several cysteine-rich antimicrobial peptides from the 
blood of a mollusc. Myllliis edulis. J. Biol. Chem. 271(.^6):2I808- 
2181-^. 

Cone-Real, H. B. S. M.. D. R. Dixon & P. W. H. Holland. 1994. Intron- 
largeted PCR: A new approach to survey neutral DNA polymorphism 
in bivalve populations. / Mar. Biol. 120:407—11.1. 

Coustau. C. 1991. Analyse genetique et physiologiquc des interactions 
hote-parasile: le systeme Prosiirhynclnis sijiuimaliis-Mylihis. In These 
Monlpellier II. pp. 1 33. 

Gomez-Chiarri. M., L. Hereford & D. Powers. 1994. Cloning of an actin 
promoter from the red Abalone Haliolis rufescens. 3rd International 
Marine Biotechnology Conference. Tromso, Norway. August 7-12. 

Gosling, E. M. 1992. Systemalics and geographic disiribulion o( Myiiltis. 
In: The Mussel Mytilus: Ecology. Physiology. Genetics and Culture. 
Ed. E. M. Gosling. 1-20. Elsevier. Amsterdam. 

Horard, B.. A. Mang^. B. Pilissier & P. Couble. 1994. Bomhy.x gene 
promoter analysis in transplanted silk gland transformed by particle 
delivery system. Insect Mol. Biol. 3(4):261-265. 

Hubert, F., T. Noel & P. Roch 1996. A member of the arthropod defensin 
family from Kcnchington. E. L. R.. D. Landry & C.J. Bird. 1995. 
CompariMin of taxa of the mussel Mytihis (Bivalvia) by analysis of the 
nuclear small-suhunit rRNA gene sequence. Can. J. Fish. Aquat. Sci. 
52:2613-2620 

Kenchinglon. E. 1.. R.. D. Landry & C. J. Bird. 1995. Comparison of laxa 



of the mussel Mytilus (Bivalvia) by analysis of the nuclear small- 
subunit rRNA gene sequence. Can. J. Fish. .Aquat. Sci. 52:261. V2620. 

Kom. E. D. 1978. Biochemistry of actomyosin-dependent cell motility (a 
review). Proc Nail Acad Sci U S A. 75:588-599. 

Lardans. V.. V. Ringaut. J. P. Cadorel & C. Dissous. 1997. Nucleotide and 
deduced amino acid sequences of Biomphalaria glabrata actin cDNA. 
DNA seq. 7:353-356. 

Mitta. G.. F. Hubert. T. Noel. & P. Roch. 1999a. Myticin. a novel cystein- 
rich antimicrobial peptide isolated from hemocytes and plasma of the 
mussel Mytilus gidloprovincialis. Eur. ,/. Biochem. 265:71-78. 

Mitta, G., F. Vandenhulcke. F. Huben. F. & P. Roch. 1999b. Mussel defen- 
sins are synthesised and processed in granulocytes and then, released in 
the plasma after bacterial challenge. J. Cell. Sci. 1 12:4233-4242. 

Ohresser, M., P. Borsa & C. Delsert. 1997. Intron-length polymorphism at 
the actin gene locus mac-l: a genetic marker for population studies in 
the marine mussels Mytihis galloprovincialis Lmj. and M. edulis L. 
Mol. Mar. Biol. Biolechiwl. 6:123-130. 

Rubenstein, P. A. 1990. The functional importance of multiple actin iso- 
forms. Bioes.wys 12:309-315. 

.Sambrcxik. J., E. F. Fritsch. & T. Maniatis. 1989. Molecular cloning, a labo- 
ratory manual. Second edition. Cold Spring Harbour Laboralors Press. 

Unger, M. E. & G. Roesijadi. 1993. Sensitive assay for molluscan metal- 
lothionein induction based on ribonuclease protection and molecular 
titration of metallolhionein and actin mRNAs. Mol. Mar Biol. Bio- 
technol. 2:319-324. 

Vandekerckhove. J. & K. Weber 1978. Mammalian cytoplasmic actins are 
the products of at leasl two genes and differ in primary structure in at 
least 25 identified positions t'roni skeletal musLic actins. Proc. Natl. 
Acad. Sci. 75:1106-1110. 

Wesseling. J. G.. J. M. de Ree, T. Ponnudurai, M. A. Smils & J. G. G. 
Schoenmakers. 1988. Nucleotide sequence and deduced amino acid 
sequence of a Plasmodium falciparutn actin gene. Mol. Biochem. Para- 
sitol. 27:31.V320. 



Joiirmil of Shellfish Research. Vol. 19. No. I. 187-19?. 2000. 

GROWTH OF SEED MUSSEL (MYTILUS GALLOPROVINCIALIS LMK): EFFECTS OF 
ENVIRONMENTAL PARAMETERS AND SEED ORIGIN 



J. M. F. BABARRO, M. J. FERNANDEZ-REIRIZ,* AND 
U. LABARTA 

CSIC In.stiiitto de Investigaciones Marinas, c/Eduardo Cabello, 6, 
E-36208 Vigo. Spain 

ABSTRACT Mussel seeds {MyliUis galh)proviiicialis Lmk) of similar weight and length from two different origins (rocky shore and 
collector ropes) were cultivated on a raft in the Ria de Arousa (northwest Spain), from seeding to thinning out, for a total period of 
208 days (November 1995 through July 1996). Weight increase rates for the seed from collector ropes were higher than those for the 
seed from rocky shore, and the growth rate variations during the cultivation period were associated with the environmental parameters 
measured (chlorophyll a and temperature). The origin of the seed was also found to be a significant factor. The condition index (CI) 
of the seed froin collector ropes was significantly greater than that of the rocky shore seed at the beginning of the cultivation time. Both 
mussel seeds showed a similar CI after 70 days and during the rest of the cultivation time. Although allometric coefficient values for 
the relation total dry weight-length showed a similar range for both types of seed, no significant differences were observed for this 
coefficient in collector rope mussels throughout the cultivation period. Rocky shore mussels showed, on the contrary, a significant 
increase for this allometric coefficient value throughout the cultivation period. These preliminary results from the total dry weight- 
length relationship obtained here and the change of CI differences serve to strengthen the hypothesis of a physiological basis for the 
differences in growth between both types of seed mussel. This finding could be related to the different features of the original habitats 
of the two types of seed, in terms of the cycles of availability of food and exposure to the air. 

KEY WORDS: Mussel, growth, environmental parameters, condition index, allometric functions 



INTRODUCTION 

Mussel (Mytilus galloprovincialis Lmk) cultivation in Galicia 
and other cultivation zones (Perez Camacho et al. 1995) is depen- 
dent on the availability of large quantities of seed, which can be 
obtained from two very different origins: coastal stocks from the 
rocky shoreline, and collector ropes suspended from cultivation 
rafts. 

Previous studies about the growth of these two types of seed in 
the Ria de Arousa disagree as to their growth potential from seed- 
ing to thinning out. On the one hand. Perez Camacho et al. ( 1995) 
found differences in growth rates and condition indices of the 
mussels that they attributed to the origin of the seed, with collector 
rope seed having the highest values. On the other hand, Fuentes et 
al. ( 1998) concluded that neither of the two types of mus.sel .seed 
(rocky shore and collector rope) has a higher growth potential, 
although the authors do recommend that mussel farmers "use seed 
from collector ropes due to their significantly larger size at harvest 
time." 

Dickie et al. ( 1984). Page and Hubbard (1987). and Fuentes et 
al. (1992) have all established that the origin of the seed has a 
significant effect on mortality rates, although not on growth. How- 
ever. Peterson and Beal (1989) and Rawson and Hilbish (1991) 
have observed a significant effect of origin on growth, which they 
explain as being due to genetic differences. 

Bayne and Newell (1983) point to the effect of endogenous 
factors (physiological condition, size, and genotype) and the spe- 
cific environmental conditions of the area in question as being two 
of the factors that most affect growth in bivalve molluscs. In the 
case of environmental factors, it has been shown that in areas 
where temperature, for example, is not a limiting factor, the avail- 
ability of food affects growth to a very large extent (Mallet et al. 
1987. Stirling and Okuinus 1994. Sukhotin and Maximovich 1994. 
Widdows et al. 1997). 



*Corresponding author. E-mail: mjreiriz@iim.csic.es 



The aim of this study was to investigate the effects of seed 
origin and environmental parameters on different growth indica- 
tors (growth rate, condition index [CI|, and the allometric relation 
weight-length). 

MATERIALS AND METHODS 
Experimental Design 

Seed of Mytilus galloprovincialis Link, approximately 20 mm 
long, was gathered from the rocky coastline and from raft collector 
ropes in the mid-to-outer area of the Ria de Arousa (Galicia, north- 
west Spain) in November 1995. Both types of seed, from the same 
year class, came from the spawning period in the previous spring- 
summer, and the sampling locations were 2 km away from each 
other. Experimental cultivation, which was carried out in a raft 
usually used for the culture in the Ria de Arousa (500 m"). com- 
menced in winter in order to minimize any possible advantages 
that collector rope seed may have as a result of its being better 
adapted to cultivation on the raft. The experiment ran until June 
1996 (208 days), thus covering the first stage in mussel cultivation, 
from seeding to thinning out (50-60 mm). Sixteen cultivation 
ropes (12 m) were used, eight for each type of seed, alternately 
placed and having a density of 19 kg of seed per rope ( 1 .6 kg/m of 
rope or 2,600 individuals per meter of rope). Sampling was per- 
formed by removing mussels from adjacent ropes at an average 
depth of 2-4 m for both types of seed. 

Initial average lengths (+ standard deviation) were 22.5 ± 1.5 
mm for the seed from collector ropes and 19.0 ± 1.9 mm for that 
from the rocky shore. Average total dry weights were 0.36 ± 0.06 
and 0.27 ± 0.06 g/individual. respectively. No significant differ- 
ences were observed for length and dry weight between both types 
of seed at the outset of the experiment (analysis of variance 
[ANOVA]: P > 0.05), 

Environmental Parameters 

Natural seston was described as total particulate matter (TPM. 
mg/L). particulate organic matter (POM. mg/L). particulate inor- 



187 



Babarro et al. 



ganic matter (PIM, mg/L). total particulate volume (Vol. mm'/L), 
and chlorophyll a (ch\-a. p,g/L). The quality of the seston was 
expressed as Q, (POM/TPM) and by the chl-a/TPM index. 

The values of chl-fl, as well as temperature (°C) and salinity 
(%t) of the water column, were supplied by the Marine Environ- 
ment Quality Control Centre of the Conselleria de Pesca. Maris- 
queo e Acuicultura (Ministry of Fisheries, Shellfisheries and 
Aquaculture) of the Xunta de Galicia (Galician Regional Govern- 
ment), chl-fl was calculated from the fluorescence data. 

Seawater samples were filtered onto pre-ashed (450°C for 4 h) 
and weighed GFC filters and rinsed with isotonic ammonium for- 
mate (0.5 M). Total dry matter was established and the weight 
increment determined after drying the filters to constant weight at 
110 °C for 12 h with an accuracy of 0.001 mg. Organic matter 
corresponded to the weight loss after ignition at 450 °C for 4 h in 
a muffle furnace. Particulate volume per liter of seawater was 
determined by counting in the range of 2-56 p.m using a Coulter 
Counter Multisizer II fitted with a 100 ixm-aperture tube. 

Mussel Sampling 

Duplicate samples of 200-350 individuals were taken from 
adjacent ropes, which corresponded to both types of seed mus.sel 
after 70, 148, and 208 days. 

Individual mussel length (L) was measured to the nearest I mm 
using calipers, and each sample was divided into 1-mm length 
classes. Adjusted length was given by the formula: L = (C, F)/N 
(Box et al. 1989), where C, is the individual length class, F is the 
frequency, and N is the total number of individuals. Subsamples of 
5-15 mussels were each taken from five to six length classes 
covering the entire size range and used to determine total dry 
weight (DW„„,,|) and organic weight of tissues (OW,|.,.,„^.). After 
cutting adductor muscles and allowing intervalvar water to drain 
by placing the mussels with their ventral edge on filter paper, 
tissues were dissected and both shell valves (DW^heii) ^nd soft 
tissues (DW,,.^.,^^.) were weighed after drying at 100°C until con- 
stant weight was obtained. We ashed the .soft tissues at 450°C for 
48 h to determine OW||.,^„^., with an accuracy of 0.01 g in all cases. 

CI was calculated from the ratio of tissue dry weight (DW,,.,,,^^.) 
and the dry weight of the valves (DW..,,^.,,) according to the equa- 
tion CI = (DW,,.,.,,,^. /DW.,,,^.,,) 100 (Freeman 1974). 

Data Analysis 

Regression models were calculated for the logarithm ot tiital 
dry weight (log DW,^,,,,), tissue dry weight (log DW,,,^.,,,^.). and 
tissue organic weight (log OW„.„.„^.) versus logarithm of length 
(log L) relationships from data obtained for five or six length 
classes covering the entire length class range from 10-15 mm 
above and below the mean length: log W = log a -h b log L. 
Analysis of covariance (ANCOVA; Snedecor and Cochran 1980) 
was used to make a comparison of these functions between both 
types of seed mussel and the change of allometric cocfficicnl (b) in 
the experiment. 

The confidence inlcr\al lor the dillerence in length and weight 
between the months of the cultivation period studied that gives the 
growth rate for each stage was g iven by the formula: X, ^ , - X, ± 
it (I - .,/2.k, Sp V(l/n,^ , + l/n,)| (Canavos 1988), where X, , , and 
X, are the mean values for length and weight al each end of the 
intervals, Sp- is the variance at each end ol' the interval, n, _, , and 
n, are the nimiher iil samples al each end >i\' (he inler\al. and 1 



, I /, ki is the Student /-distribution value with 95'7f confidence and 
k degrees of freedom (k = n, ^ , + n, - 2). 

Comparison of mean values of growth rate was carried out with 
an ANOVA. Homogeneity of variances was tested by the Bartlett 
test (Snedecor and Cochran 1980), and correction for heterogene- 
ity (when required) was performed by reciprocal or logarithmic 
transformation data. In cases in which homogeneity was not ob- 
tained after these transformations had been carried out, the 
Kruskall-Wallis nonparametric te.st was used. 

The effects of environmental parameters and origin of seed 
mussel on the growth rate were tested by stepwise multiple regres- 
sion. Seed origin was introduced with values and 1 for collector 
rope and rocky shore mussels, respectively. Length and dry weight 
values of growth rate were transformed by log|||(x -i- I ) to stabilize 
variances. 



RESULTS 



Environmental Parameters 



Variation in temperature (°C) took place within a narrow range, 
there being a difference of only 2.7 °C between the maximum and 
minimum temperatures during the whole of the experimental pe- 
riod (Fig. I A). Temperature was high at the beginning of the 
cultivation period ( 15.5 °C) and then decreased in zigzag until the 
minimum temperature was reached in February (12.9 °C). From 
then on, throughout the spring months, there was a steady increase 
in temperature. 

Salinity (%o) was dependent on rainfall. Average values for the 
area (3l.3-.35.2'^() were obtained at the outset, and they gradually 
decreased until January, when the minimum value (28.0'^r) was 
recorded. Salinity then increased during the spring months and 
finally reached its maximum value at the end of the cultivation 
period in July (35.6%p; Fig. I A). 

High values for TPM were registered in February through April 
(0.9-1.4 mg/L; Fig. IB), in contrast with the low values obtained 
throughout the winter months. However, the maximum of TPM 
occurred at the beginning of January (2.6 mg/L: Fig. IB), which 
can be related to maximums in POM (I mg/L) and especially in 
PIM ( 1.6 mg/L). With the single exception of this maximum value. 




30 60 90 120 150 180 210 240 
NDEF MAMJJ 

IWS 11996 



30 60 90 120 160 ISO 210 240 
ND EF M AMJJ 



Figure I. Viiriution of averaut values (nu'an SI)) of tcnipiTalure (°C) 
and salinity (',,) (Al: I PM tmii/],). I'OM (mj;/!.), and IMM (mtt/L) (B); 
chl-fl Ik/I.) (C): and (|ualit> iif Ihf si'sldn (Q, ti\i I'OMAII'Ml and 
chl-rt/Tl'M index (I)), during Ihc experimental period November 1995 
through July 1996. 



Growth of Mussels from Two Origins in NW Spain 



189 



POM was higher during the spring O0.5 mg/L) than during the 
winter (0.3 nig/L). Fluctuations in chl-i/ produced two peaks in 
February and April (1.4 and 2.0 |J.g/L, respectively; Fig. IC) after 
the low values recorded during the initial stages of the experiment 
(0.3-0.8 M-g/L). 

Qi varied between 0.3 and 0.6. showing a greater oscillation in 
winter and a narrower range of fluctuation around 0.5 during 
spring, which corresponds to the value that is generally obtained 
for the Ri'a de Arousa (Fig. ID). The chl-o/TPM index varies to a 
much greater extent, with low values being recorded in winter 
(0.1-1.1: Fig. ID) and then increasing from February on to reach 
a peak in April and June (2.1). 

Growth 

The growth rate in terms of length (mm/mo) shows minimum 
values in winter ( 1.5 and 2.0 mm/mo for collector rope and rocky 
shore mussels, respectively; P > 0.05) and maximum levels from 
April through June (9.1 and 6.8 mm/mo for the same two mussel 
populations, respectively; P > 0.05; Table 1). The average growth 
rates for the whole period November through June were thus simi- 
lar for both types of mussel seed, at 4.8 and 4.5 mm/nio. respec- 
tively {P > 0.05). 

Weight growth shows a trend similar to that for length over the 
cultivation period, with the minimum in winter (0.07 g DW,„,^|/mo 
for both seed types) and the maximum in the April through June 
period, when the collector rope mussels showed significantly 
higher values (1.60 g DW,^^,,^|/mo) than the rocky shore mussels 
(0.86 g DW,„,^,/mo) {P < 0.05; Table I). The overall November 
through June values for DW,p,jj| growth rates are 50% higher for 
the former (0.61 g DW„„^|/mo) than for the latter (0.41 g DW„„,,,/ 
mo) (P < 0.05; Table 1 ). The differences between these two groups 
of mussels in the final stages of cultivation (April through June) 
and in the overall average values (November through June) also 
apply to organic and dry weight of tissues (OW,,.,.,,,^. and DW„„^,,^„ 
respectively), with collector rope mussels once again showing 
higher values (see Table 1). 

The variation of growth rate in terms of both length and total 
dry weight in this study bore a significant relationship to fluctua- 
tions in the environmental parameters chl-fl and temperature of the 
water column, in this order of importance (Table 2). Both of these 
environmental variables show positive and significant coefficients 
(P < 0.001 for chl-a and P < 0.05 for temperature vs. growth rate 
for length), with chl-a being the major component of the variance 



(40.1 and 56.6% for growth rates for dry weight and length, re- 
spectively). A significant but residual effect (P = 0.040) was also 
noted for seed origin vs. growth rate for total dry weight (Table 2). 

Condition Index 

CI for collector rope mussels was 33% higher than that for 
rocky shore mussels (P < 0.001 ) at the beginning of the cultivation 
period (Table 3). After 70 days, similar values of CI were obtained 
for both groups of mussels {P > 0.05), and this remained the case 
until the end of the cultivation period without differences between 
them. The significant increase in CI (P < 0.001 ) for both groups of 
mussels between 70 and 148 cultivation days, which corresponds 
with the period February through April, is remarkable. 

Allometric Functions 

Values a and b of the allometric function weight-length (W = 
a L"^) for each mussel seed during the cultivation period are shown 
in Table 4. No significant differences among the slopes (b) of both 
groups of mussels at any time during the cultivation period were 
detected when an ANCOVA was performed on the linear trans- 
formations of these functions (P > 0.05). However, the intercepts 
for collector rope mussels were significantly higher at the end of 
the cultivation period (June) in all cases (P < 0.001 for DW,„,^, and 
OW,,^,^^, vs. L and P < 0.01 for DW„^,„^, vs. L; Table 4). Signifi- 
cant differences were also obtained for the intercepts in February 
(P < 0.05) and November (P < 0.01 ) for the relations DW„^,^^, and 
OWj.^^jj^ versus L, respectively, in which higher values were once 
more recorded for the collector rope mussels (Table 4). 

Concerning shell weight, we found no differences at the onset 
of the experiment (0.32 ± 0.05 and 0.25 ± 0.06 g for collector rope 
and rocky shore mussels, respectively; P > 0.05). The same ten- 
dency was maintained during the cultivation period except at the 
end (June), when mussels from collector ropes presented heavier 
shells (3.63 ± 0.17 g) than rocky shore ones (2.41 ± 0.23 g) (P < 
0.001). 

A second ANCOVA was performed on the fluctuation of the 
values a and b in the relation DW,^„.,,-L over the cultivation period, 
for each seed type independently. The results are shown in Table 
5. The power b gives similar values throughout the cultivation 
period for collector rope mussels (P > 0.05), yet when intercept a 
is recalculated for a common power (Rec.a), it gradually increases 
over time, with significant differences between November and 
April (P < 0.05) and maximums occurring in June (P < 0.001 ). On 



TABLE 1. 
Grovrth rates of mussels from collector ropes and rocky shore in different periods of culture. 





L (mm/mo) 


DW„„„ 


(g/mo) 


DW,„,„. (g/mo) 


ow„. 


sue (g/mo) 


Period of 


Collector 


Roclvv 


Collector 


Rocky 


Collector 


Rocky 


Collector 


Rocky 


Cultivation 


Ropes 


Shore 


Ropes 


Shore 


Ropes 


Shore 


Ropes 


Shore 


Nov-Feh 


1 .5 ± 1 .4 


2.0 ±1.3 


0.07 ±0.(17 


0.07 ± 0.05 


4.10-'±7.10" 


6.10"' ±5.10"' 


2. 10-' ±5.10"^ 


4.10-'±4.I0-' 


Feb-Apr 


4.4 ± 1.5 


4.9 ±1.3 


0.35 ± 0. 1 1 


0.37 ± 0.09 


0.12 ±0.02 


0. 11 ± 0.02 


0.10 ±0.02 


0.10±0.02 


Apr-Jun 


9.1 ±2.0 


6.8 ±2.1 


1.60 ±0.26* 


0.86 ± 0,23 


0.37 ± 0.06* 


0.19 ±0.06 


0.32 ± 0.06* 


0.17 ±0.05 


Nov-Jun 


4.8 ± 0.45 


4.5 ±0.5 


0.61 ±0.07* 


0.41 ±0.06 


0.15 ±0.02* 


0.10 + 0.02 


0.13 + 0.02* 


0.09 ±0.01 


Percentages 


6% 




50% 




55% 




52% 



Data are means (n = 5 samples) ± standard deviation. L, length: DW,„,-,|, total dry weight: DW,,^^^, dry weight of soft tissues; OW,,.,,^^, organic weight 
of soft tissues. 

* Significant differences between both sources of mussels (f < 0.05: ANOVA). Percentage values indicate how much higher is the increment of growth 
parameters in collector ropes mussels over total time of culture (November through June, 208 days). 



190 



Babarro et al. 



TABLE 2. 

Multiple regression analysis of shell length (L) and total dry weight (DW„„^,) increment on water temperature (in °C) and 

chlorophyll-a (in ^g/L). 



Parameter 






Constant 




Chlorophyll-fl 


Temperature 


Origin 


A. L. mm/mo 






-2.855 ± 1.131 




0.527 ±0.0911 (56.6%) 


0.2 10 ±0.078* (70.8%) 


-0.01 1 ± 0.008 


B. DW,„,,,. g/mo 






-2.900 ± 0.392 




0.247 ±0.035t (40.1%) 


0.197 ±0.030t (84.1%) 


-0.053 ±0.026* (87.8%) 


A. N = 18; r = 


0.708; 


F, 


1, = 18.155; P < 


0.001 








B. N = 18; r = 


0.878; 


F., 


,4 = 33.620; P < 


0.001 









Mean intercept and coefficients ± SD. Origin is defined with values and 1 for collector ropes and rocky shore mussels respectively. Percentage values 
mean proportion of accumulated variance with inclusion of different factors (NS not significant). 
* P < 0.05, significant difference from 0. 
f P < 0.001, significant difference from 0. 



the other hand, rocky shore mussels showed a steady and signifi- 
cant increase of slope (b) over time (P < 0.05; Table 5), reaching 
maximum values in June (2.507), although significant differences 
were already evident between the allometric coefficients for No- 
vember (2.276) and April (2.491) (P < 0.01). 

DISCUSSION 

The variations in factors such as temperature, salinity, and 
chl-« in the area studied are consistent with previous descriptions 
of the Galician Ri'as (Fraga 1996). Abundant rainfall and low 
levels of sunlight until February are the reason for low salinity and 
the concentration of chl-t; in the winter months. The maximum 
values of TPM and POM that occurred in January constitute an 
exception to the winter-spring pattern that characterizes the natural 
seston variability and reveal the effect that frequent storms have on 
a shallow area such as this at this time of year, leading to a 
resuspension of previously sedimented particles. The mainly sedi- 
mentary origin of this sudden increase in POM in January is sup- 
ported by the low winter values of the chl-(//TPM index. The peak 
levels of phytoplankton that occur in the Galician Ri'as can be 
related either to an increase in sunlight (the first chl-a peak occurs 
in mid-February) or to the upwelling of nitrates/silicates of the 
water caused by the appearance of North Atlantic Central Water 
(NACW). NACW is the triain reason for the spring upwelling in 
the Galician Ri'as, which is represented by a second and higher 
chl-(/ peak in mid-April. 

Among the environmental parainetcrs studied, the availability 
of plankton in the water column in the form of chl-a and water 
temperature had a significant effect on the variations in growth 

TABLE 3. 

Condition index (CI) values for hoth types of seed mussel during 
their cultivation on a raft. 





Days of 
Cultivation 




CI 




Month 


Collector 
Ropes 




Rocky 
Shore 


November 
February 
April 
June 




70 

148 

208 


15.84 ±2.44* 
1 3.27 ± 0.87 NS 
33.11 +4.10NS 
30.08 ± 2.87 NS 




11.87 + 0.97 
12.08± 1.86 
30..19 ± 2.62 

28.88 ± 3.26 



NS, not significant (N = 12 in all ca.ses). 
* Differences highly significant. 



rate. Both of these factors have previously been signalled as being 
responsible for most of the variation in the growth rate of bivalve 
molluscs (Bayne and Newell 1983). and the fact that in the present 
study chl-(/ has the greater effect of the two supports earlier results 
(Perez Camacho et al. 1995). In temperate waters, such as Ri'a de 
Arousa, temperature fluctuations are not as marked as they are in 
extreme environments where fluctuations in this factor play a more 
important role (Kautsky 1982, Sukhotin and Kulakowski 1992). 
Therefore, variations in growth rate in temperate waters have been 
associated with the availability of food (Page and Hubbard 1987, 
Thompson and Nichols 1988, Femandez-Reiriz et al. 1996). 

Growth rate variation, estimated here with low values during 
winter and maxitnums in spring, follow a pattern similar to that 
found in other studies (Freeman and Dickie 1979. Pieters et al. 
1980, Kautsky 1982. Loo and Rosenberg 1983, Skidmore and 
Chew 1985, Page and Hubbard 1987, Mallet et al. 1987). The 
maximum growth rates for length, which were recorded in spring 
(9.1 and 6.8 mm/mo for collector rope and rocky shore mussels, 
respectively), agree with the findings described by Perez Camacho 
et al. ( 1 995 ) for the same time of year and both types of seed in the 
Ri'a de Arousa. The increase in length after the experiinental period 
(31-33 mm for 208 days; 4.4-4.7 mm/mo, with both types of seed 
included) is comparable to that of a previous paper on the Ria de 
Arousa for a similar period of the year, 5.6-5.8 mni/mo (Fuentes 
et al. 1998). Perez Camacho et al. ( 1995) found higher growth rates 
of 7-9 mm/mo. However, it is necessary take into account that this 
experiment began in April and ended 90 days later, which means 
favorable conditions froin the beginning with regard to tempera- 
ture and seston availability and quality. The lower growth rates 
obtained by Fuentes et al. (1992) also with M. gaUoprovincialis in 
the Ria de Arou.sa (2.4 mm after 3 mo) can be attributed to the 
cultivation technique used (plastic cages). 

These differences also appear in the cultivation period needed 
before thinning out. which is greater in experiments that com- 
mence in winter (5 and 7 mo, respectively, for Fuentes et al. 1998 
artd the present study) than in those that start in spring (3 ino; Perez 
Camacho et al. 1995). 

Although Fuentes et al. ( I99S) recommend that seed from col- 
lector mpcs sliould he used for cultivation, since it reaches greater 
length and/or weight than rocky shore seed, they differ from Perez 
CaiTiacho et al. ( 1995) as to the existence of a difference in growth 
rates from seeding to thinning out. Their reasoning is based on the 
fact that if more than one cohort were included in the process of 
gathering the rocky shore seed, this may well explain the different 
growth rates reported by the latter authors. The results of this study 



Growth of Mussels from Two Origins in NW Spain 



191 



TABLE 4. 

Results of regression and covariance analysis on data relating weight (W mg) of A/, galloprovincialis from two sources of seed to 

length (L mm). 



Collector Ropes 



Month 



Rec. a 



a 
Common 



b 
Common 



Rockv Shore 



Rec. a 



DW,„,.,| versus 


L: 


A 








November 






0.328 NS 




"> 


Fehruarv 






0.152 NS 




2. 


April 
June 






0.232 NS 
0.644* 


0.321 




DW,„,„, versus 


L 


B 








November 






0.019 NS 




2 


February 

April 

June 






0.026t 
0.047 NS 
0.043i 


0.036 
0.039 


2 
2. 
2. 


0W|,^.,„^. versus 


L 


C 








November 






0.012+ 


0.027 


-) 


Februarv 






0.018 NS 




1 


April 
June 






0.046 NS 
0.037* 


0.035 


1 



2.247 ± 0.092 NS 0.986 10 

505 ± 0.068 NS 0.990 1 5 

397 ±0.1 96 NS 0.943 11 

212±0.118NS 0.978 10 

521 ± 0.158 NS 0.970 10 

361±0.104NS 0.976 15 

464 + 0.161 NS 0.962 11 

523 ± 0.260 NS 0.922 10 

580 ± 0.208 NS 0.951 10 

385 ±0.1 17 NS 0.980 15 

429 ±0.1 70 NS 0.958 11 

526 ± 0.275 NS 0.914 10 



0.325 


2.267 


0.326 


0.171 


2.464 


0.194 


0.207 


2.442 


0.179 




2.383 


0.167 


0.026 


2.398 


0.03 1 




2.279 


0.041 


0.040 


2.508 


0.033 




2.546 


0.030 




2.355 


0.028 


0.017 


2.380 


0.016 


0.039 


2.473 


0.032 




2.540 


0.028 



0.273 



0.03 1 



0.031 



0.018 



0.027 



2.274 ±0.056 0.996 10 

2.430 + 0.062 0.992 15 

2.491 ±0.054 0.996 11 

2.507 ±0.098 0.988 10 



2.318 
2.200 
2.557 
2.569 



: 0.087 0.988 10 

: 0.102 0.972 15 

: 0.1 23 0.980 11 

: 0.350 0.964 7 



2.223 ±0.085 0.980 10 

2.374 + 0.251 0.872 15 

2.522 ±0.126 0.978 11 

2.554 ± 0.232 0.960 7 



a and b values are parameters in the equation W = aL*": ANCOVA ANOVA was made after logarithm transformation: log W = log a + b log L. When 

there were no differences in slopes (b) of the relationship, a common exponent was therefore calculated and used to recalculate values for the parameter 

a (Rec. a). NS, not significant. 

*/'< 0.001. 

t P < 0.05. 

±/'<0.01. 



do nevenheless point out a difference in growth rates, especially 
for weight, that are also related to vaiiability of environmental 
parameters during the cultivation period. This study is also in 
concordance with Perez Camacho et al. ( 1995). who showed that 
initial size (weight/length) has no effect on the results, either be- 
cause the experiment was designed with this condition in mind (as 
in this study) or because the statistical analysis (multivariate 
ANOVA) performed on the results showed this to be the case 
(Perez Camacho et al. 1995). With reference to the study by Fu- 
entes et al. (1998), the differences observed in the initial siie of 
both types of seed (0.6 cm for rocky shore mussels and 2. 1 cm for 
collector rope mussels), as well as the high density of mussels on 

TABLE 5. 

Results of ANCOVA on data relating DW,„,3| (mg) to shell 
length (mm). 







Collector Ropes 






Rocky Shore 




Month 


b 


a 


Rec. a 


n 


b 


a 


Rec. a 


n 


November 
February 
April 
June 


2.247 
2.505 
2.397 
2.212 


0.328 
0.152 
0.232 
0.644 


0.209 
0.237 
0.249 
0.319 


10 
15 
11 
10 


2.276 
2.430 
2.491 

2.507 


0.326 
0.194 
0.179 
0.167 


— 


10 
15 
11 
10 



Collector ropes: 

Comparison among slopes. F = 1.091 (DF = 3.38) P > 0.05. 

b.„n™o„ = 2.384. 
Comparison among intercepts. F = 12.944 (DF = 3.41 ) P < 0.001. 
Rocky shore: 
Comparison among slopes, F = 3.134 (DF = 3.38) P < 0.05 

a and b are parameters in the equation DW,^,,, = aL'' (.see Table 4. top). 
Rec. a represents recalculated intercept for common slope. 



the ropes (5.000 individuals per meter), which contrasts with the 
2.600 individuals per meter of the present study and the 2,000 
individuals per meter of Perez Camacho et al. (1995), may have 
affected their results, given the effect that both of the above- 
mentioned factors (initial size and density) may have on growth 
(Sukhotin and Ma.ximovich 1994. Eldridge et al. 1979, Femandez- 
Reiriz et al. 1996). 

Although our results show a difference in growth rates between 
collector rope and rocky shore tiiussels, as was previously ob- 
served by Perez Camacho et al. (1995). it should be pointed out 
that these differences become more apparent in those months that 
most favor growth (April through June), which is precisely the 
period in which the experiment by Perez Camacho et al. (1995) 
took place, and just as was the case in their experiment, the most 
marked differences in our results are those for growth in wet 
weight and tissue weight. A higher growth efficiency for collector 
mussels when environmental conditions (temperature and overall 
quality of food) are more favorable, resulting in a more positive 
scope for growth, and the persistence of different metabolic pat- 
terns due to immersion-emersion periods that are indicative of 
anaerobic pathways for rocky shore mussels, could help us to 
understand such different growth responses. Genetic factors could 
also explain a significant proportion of the variances in production/ 
growth of mussels (Widdowset al. 1984. Mallet et al. 1987). since 
it has been described that mussels exhibit high levels of genetic 
variability measured as enzyme polymorphisms both on a micro- 
and a macrogeographic scale (see Hawkins and Bayne 1992). Ad- 
ditionally, energy-saving mechanisms related to respiration me- 
tabolism have been described for those animals, which live in the 
intertidal locations. Metabolic depression and anaerobiosis are 
clearly implicated as key factors of energy conservation to with- 
stand emersion conditions in order to compensate for reduced 



192 



Babarro et al. 



feeding time with respect to sublittoral animals (de Zwaan and 
Mathieu 1992). 

Both terms (physiological rates and metabolic patterns) are 
being tested for both types of seed mussel cultivated on suspended 
conditions in Arousa. 

The use of the allometric function weight-length in growth 
studies is firmly established (Hickman 1979. Rodhouse et al. 1984. 
Sprung 1995, Sara et al. 1998, among others). A correspondence 
has occasionally been established between the variation in the 
allometric coefficient and local food conditions (Sara et al. 1998). 
In this study, we did not measure the original weight-length rela- 
tionship of intertidal mussels before putting them on the raft. How- 
ever, the fact that the experiment began a few hours after the seed 
mussels were gathered from their environments suggests that this 
relationship is similar to that which these mussels might show in 
their original habitat. An ANCOVA that was performed for this 
weight-length relationship in rocky shore mussels with regard to 
cultivation time period showed changes in the b parameter value 
(more evident between slopes of November and April), whereas 
collector rope mussels presented no differences in this value 
throughout the cultivation period. This probably means that envi- 
ronmental changes for rocky shore mussels, when they are put 
under immersed conditions on the raft, might be responsible for 
such a response of the allometric functions. 

The initial differences in the CI of the two types of mussel seed 
can be attributed to their original habitats, which differ greatly with 
regard to the availability of food and their respective situations of 
emersion-immersion. The disappearance of these differences after 
70 days may be related to the changes in physiological responses 
resulting from a new environmental situation (Bayne et al. 1984, 
1987), although these differences may well persist for some time 
(Widdows et al. 1984. Iglesias et al. 1996). 

Given the experimental design in this study, any effects on our 
results of a genetic nature that have occasionally been used to 
explain differences in growth (Peterson and Beal 1989, Rawson 
and Hilbish 1991) would be possible when genetic factors play a 



part in the choice of substrate (rocky shore or collector rope) by 
larvae in the Ria de Arousa or when different cohorts are involved. 
As has previously been mentioned by Perez Camacho et al. ( 1995), 
an alternative hypothesis would be to consider a physiological 
adaptation response of each seed to its habitat of origin, which 
would imply that cultivation starts from different physiological 
states, which is described by Mallet et al. (1987) as ecological 
memory. This ecological memory would condition the physiologi- 
cal response of the seed to its new environmental situation, as 
shown by an increase in the CI for the rocky shore seed. 

We can consider that the aim of slowing down the initial 
growth rates in order to minimize any possible advantages for the 
collector rope mussels has been achieved. This would explain why 
the differences observed in growth rates between the two types of 
seed are less marked than those recorded by Perez Camacho et al. 
( 1995) during the first stage of the cultivation period. Although the 
allometric coefficients for both types of seed need to be tested with 
regard to their original habitat for establishing more properly habi- 
tat-dependent changes, the CI differences maintained in both seed 
types supports the hypothesis that there is an underlying physi- 
ological basis for the difference in their respective growth rates. 
Moreover, given the experimental conditions under which the 
present study was performed and taking into account the CI 
changes, the physiological parameters of the two types of seed 
could be expected to converge. 

ACKNOWLEDGMENTS 

We are grateful to Lourdes Nieto. Beatriz Gonzalez, and Sonia 
Villar for technical assistance. We also thank Juan Maneiro from 
the Marine Environment Quality Control Center of the Consellen'a 
de Pesca. Marisqueo e Acuicultura of the Xunta de Galicia for the 
determination of environmental parameters. We are also indebted 
to the crew of the Jose Maria Navaz from Instituto Espafiol de 
Oceanografia. This work was supported by Project CICYT 
MAR97-0592. J. M. F. Babarro was funded by a grant from Ex- 
cma. Diputacion de Pontevedra. 



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Peterson. C. H. & B. F. Beal. 1989. Bivalve growth and higher order 
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1404. 

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I 



Joiinwl at Shclljhh Research. Vol. 1^, No. I. 195-201. 2000. 

FEEDING BEHAVIOR OF SEED MUSSEL MYTILUS GALLOPROVINCIALIS: ENVIRONMENTAL 

PARAMETERS AND SEED ORIGIN 



J. M. F. BABARRO, M. J. FERNANDEZ-REIRIZ,* AND 

U. LABARTA 

CSIC Institiito de Investigaciones Marinas 

c/Editardo Cabello. 6. E-36208 Vigo. Spain 

ABSTRACT Mussel seed (Mylilus galloprovinciali.'i) from two original habitats (rocky shore and collector ropes) was cultivated on 
a raft in the Ria de Arousa (northwest Spain), for a period of 226 days (November 1995 through June 1996), from seeding to thinning 
out. during which time the behavior of clearance rates (CR) and ingestion rates (IR) was studied. The study of these two physiological 
parameters of energy acquisition (CR and IR) demonstrates that the two types of seed showed significant differences in these 
parameters at the start of the experiment and after the first 8 days on the raft. After 15 days, large increases in these physiological rates 
were observed for both types of seed, with the increase for the rocky shore mussels doubling that of the collector rope specimens. These 
increases led to the disappearance of the significant differences in CR and IR between both seed origins, with this situation being 
maintained for the remainder of the experimental period. The variation in CR follows a seasonal pattern, with low values being recorded 
in winter and increasing in spring and summer. Minor seasonal variations of total seston concentration are counterbalanced by an 
inverse variation in organic content, and so organic IR followed a pattern similar to that of CR. This seasonal variation can be attributed 
to fluctuations in the factors food quality (Q, ) and temperature, in this order, as the use of multiple regression analysis has proved. Seed 
origin had a significant effect as a factor of interaction with food quality Q,. probably because of differences between the original 
habitats of the seed (rocky shore and collector ropes) in the latter factor. Although in this study food quality has been expressed in terms 
of organic content (Q, = organic/total particulate matter), the content of phytoplankton as chlorophyll a may have had an important 
effect on the variation of both of these physiological rates. A significant exponential relationship has been established between the IR 
and the content in total particulate matter, which suggests regulation processes according to the amount of natural food available based 
on a decrease of CR. 

KEY WORDS: Mytilus gaUoprovinciaUs. mussel seed, clearance rate, and ingestion rate. Ria de Arousa 



INTRODUCTION 

Clearance rates (CRs) and ingestion rates (IRs) determine the 
amount of food that enters the digestive system of bivalve mol- 
luscs. The variability observed in these physiological parameters 
has been interpreted in terms of the ability of these animals to 
adapt to the specific environmental and nutritional conditions of 
their habitat (Widdows et al. 1984; Navarro et ai. 1991; Okumus 
and Stirling 1994; Iglesias et al. 1996). The relationship between 
IR and food concentration depends on CR, which in turn is af- 
fected by environmental factors. Hawkins and Bayne (1992) pro- 
posed the use of multifactorial analyses to ascertain the relevance 
and ecological complexity of the set of environmental variables, as 
well as their interaction with physiological parameters. 

When attempting to determine the extent of the influence of 
habitat, transplant experiments are considered to be the ideal way 
of analyzing the effect of the variability attached to the environ- 
ment in which the individuals lived previously, in connection with 
what Mallet et al. (1987) termed ecological memory. Previous 
comparative studies of mussel seed gathered from a rocky shore 
and from collector ropes and then cultivated on a raft established 
the existence of a significant effect of the seed origin on growth 
rate (Perez Camacho et al. 1995; Babarro et al. 2000). with this 
effect being associated with physiological parameters. 

The extent of time needed for CR and IR to acclimate to new 
environmental conditions has been reported in various studies (4.5 
mo [Okumus and Stirling 1994] and more than 2 mo [Widdows et 
al. 1984], although Hawkins and Bayne [1992] have suggested a 
period of less than 2 mo). The aim of the present study was to 
determine the extent to which differences in the feeding regime 



*Corresponding author. E-mail: mjreiriz@iim.csic.es 



and the regime of immersion-emersion in their original habitats 
(rocky shore and collector ropes) affects the behavior of CR and IR 
during the cultivation period in the raft (20-60-mm shell length). 
The study also deals with a set of factors, such as an endogenous 
factor (i.e., shell length) and the environmental and nutritional 
conditions in the cultivation area, and the effect they have on these 
physiological rates for raft-cultivated mussels. 

MATERIALS AND METHODS 

Harvesting and Maintenance of Mussels 

In November 1995, seed of Mytilus galloproviticialis of ap- 
proximately 20 mm in length was gathered from the rocky shore 
and from collector ropes on a raft, both in the mid to outer area of 
the Ria de Arousa (Galicia, northwest Spain). Both types of seed, 
from the same year class, came from a spawning period in the 
previous spring/summer. Experimental cultivation, which was car- 
ried out under production conditions on the raft (500 m"), began in 
winter — the season of minimal growth rate — with the aim of mini- 
mizing any possible advantages for the collector rope seed as a 
result of its better adaptation to raft cultivation conditions. The 
experiment continued until July 1996 (226 days) and covered the 
first stage of mussel cultivation from seeding to thinning out (50- 
60 mm). Sixteen cultivation ropes (12 m) were used, 8 for each 
type of seed, disposed alternately and with a density of 19 kg of 
seed per rope (1.6 kg/m of rope or 2.600 individuals/m of rope). 
Specimens were sampled each time from adjacent ropes from the 
stretch of 2-5 m. 

The initial length of the seed was 22.55 ± 1 .55 mm for collector 
rope seed and 1 9.02 ± 1 .93 mm for the rocky shore seed. Mean 
total dry weight was 0.36 ± 0.06 and 0.27 ± 0.06 g/individual for 
each type of seed, respectively. These differences in length and dry 



195 



196 



Babarro et al. 



weight between the mussels from the two different original habi- 
tats were found to be not significant at the beginning of the ex- 
periment (analysis of variance [ANOVA]; P > 0.05 in both cases; 
n = 96). 

Experimental Design 

Seawater was pumped from the depth where seed was sampled 
(2-5 m) into an open circuit consisting of three rectangular cages 
(45 X 40 X 14 cm = length x width x height and 19 L of capacity). 
each provided with 16 compartments set in parallel. Seed speci- 
mens from the two origins were placed in the side cages while the 
middle cage, containing no specimens, acted as the control. The 
water flowed independently into each cage from an inlet pipe, 
which went all the way around the top of the cage. The water outlet 
for each cage consisted of a single pipe leading off from the top of 
the cage. The flow in each cage was maintained at a steady rate ot 
approximately 3 L/min. so that the concentration of particles at the 
outlet would never fall below 50% of that at the inlet. The number 
of specimens used in each replica for physiological measurements 
varied according to their size (i.e., with the length of cultivation 
period). At the outset, six specimens of the 20-mm shell length 
class were placed in each compartment, and this number was also 
used for 30-mm shell length. From the 40-mm shell length onward, 
the number of animals used progressively decreased and at the end 
of the experiment there was only one specimen of the 60-mm 
length class in each compartment. Physiological measurements 
were taken weekly from November to January, fortnightly from 
February to May, and monthly in June and July. 

Measurements 

Natural seston was characterized as total particulate matter 
(TPM, mg/L), particulate organic matter (POM. mg/L), particulate 
inorganic matter (PIM, mg/L), particulate volume (Vol. mm'/L), 
and chlorophyll a (chl-a, |jig/L). The values for ch\-a. as well as for 
the temperature ( °C) and salinity (%<) of the water column were 
provided by the Centro de Control de Calidade do Medio Marino 
da Conselleria de Pesca, Marisqueo e Acuicultura (Xunta de Gali- 
cia). chl-rt was calculated from the fluorescence data. Seston qual- 
ity was expressed as Q, (POM/TPM), Q, (POM/Vol), and the 
chl-(i/TPM index. The same methodology as that used for gravi- 
metric analysis of seston was applied to characterize the feces 
produced by the mussels in the experimental system: seawater 
samples and aliquots of known volumes from each fecal sample 
were filtered onto pre-ashed (450 °C for 4 h) and weighed GFC 
Alters and rinsed with isotonic ammonium formate (0.5 M). Total 
dry matter was established as the weight increment detennined 
after drying the filters to constant weight at 110 °C for 12 h. 
Organic matter corresponded to the weight loss after ignition at 
450 ■ C for 4 h in a muffle furnace. Vol/L of seawater was deter- 
mined by counting in the range of 2-56 fji.m using a Coulter 
Counter Multisizer II fitted with a lOO-fxm aperture tube. The 
variation in these environmental and/or nutritional parameters over 
the cultivation period is shown in Table I. 

The egestion rates of inorganic matter (mg/h) were determined 
for each group of mussels and assumed to represent inorganic IR 
(i.e., no absorption of ash in the digestive tract was considered). 
CRs were then estimated indirectly, with PIM concentration (mg/L 
of seawater) as the reference for available inorganic matter: then 
CRIh ' = mgPlM 



iy9S). where PIM, 



^.„ h'/mg PlM, , L ' (Iglesias et al. 1996: 

is the amount o\' inorizanic content \(iided 



with the feces in a given unit of time (h) and PIMp„„j is the 
inorganic content of the food in a given unit of volume (L). A lag 
time of 2 h was allowed between the sampling of seawater and the 
gathering of feces, to account for the estimated time for intestinal 
transit for mussels from the Ri'a de Arousa (Navan-o et al. 1991). 
Before the start of the experiments, mussels were kept in the cages 
for 1 h with flowing seawater at the natural particle concentration 
to allow for valve opening and acclimation to cage conditions. The 
feces obtained on the bottom of cages after this time were refused. 

The organic ingestion rate (OIR mg org/h) was calculated as a 
product of CR and the organic food concentration (mg POM/L). 

For purposes of comparison with this indirect CR estimation. 
CR was also calculated by the direct estimation (flow method) 
using the Hildreth and Crisp (1976) equation: CR = f([C, - C.,]/ 
C„), where f is the flow rate, C, and C,, are food concentrations at 
the inflow and outflow of the experimental cage, and C, represents 
the particle concentration surrounding the mussel. The experimen- 
tal design used in this study enabled us to consider C„ concentra- 
tion as being close to C,, so C, was subsequently used as the 
reference concentration for calculation purposes. C, and C„ were 
determined by recording the concentration of particles 2-56 p.m in 
water sainples with a Coulter Counter Multisizer. 

The degree of correlation obtained between the two calcula- 
tions for CR was highly significant for both groups of mussels 
together, similar to those observed by Urrutia et al. (1996) and 
Iglesias et al. (1998) (Y [biodeposition] = 1.1 18 ± 0.074 x flow 
+ 0.043; r- = 0.86; P < 0.001; /; = 300). Once significant rela- 
tionships were established for both methodologies, CR here (text, 
tables, and figures) refers to indirect measurements by biodeposi- 
tion method. 

Size Standardization 

To preclude variability in physiological rates caused by size 
differences, these rates were corrected to a standard-sized indi- 
vidual. To this end, once physiological measurements were com- 
pleted, shell length of each individual was recorded to the nearest 
0.1 mm with Vernier calipers and the soft tissues excised from the 
shell, dried at 1 10 °C for 12 h, and weighed. The most commonly 
used reference for size is soft body mass; however, the weight 
standardization of CR may be somewhat arbitrary, because this 
rate is considered to be dependent on filtration (gill) area, which is 
closely related to shell length (Hughes 1969; Jones et al. 1992). As 
discussed before by Iglesias et al. (1996) and Labartaet al. (1997), 
in this study we used shell length (L) to standardize CR following 
the equation: Y, = Y^, x (LyL^.)'' where Y, and Y^, are the stan- 
dardized and the nonstandardized CRs, respectively; L, is the stan- 
dard length of the animal according to shell increment during the 
experiment (20-60 mm); L,, is the observed length of the animal; 
and b is the power that scales CR with shell length (b = 1.85. 
Perez Camacho and Gonzalez 1984). Furthermore, with the aim of 
establishing the fluctuation of clearance and IRs over the cultiva- 
tion period, 40-mm shell length was chosen as an average size for 
the experiment. 

Data Analysis 

Comparison of means for CR and OIR was carried out by 
means of standard ANOVA after data translorniation when nec- 
essary. Kruskall-Wallis and Friedman nonparametric tests were 
used when homogeneity was lacking (Bartlett's test). Multiple 
analysis (stepwise regression) was used to determine the effect ot 



Feeding Behavior of Seed Mussel M. galloprovincialis 



197 



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various factors, both endogenous (shell length) and environmental 
(TPM, POM. Vol. Q|. Q., T. and chl-</), and their interactions on 
the variation in CR. The factor seed origin was added to this 
analysis with values of and 1 for collector rope and rocky shore 
mussels, respectively. All of these analyses were performed ac- 
cording to the methods described by Snedecor and Cochran (1980) 
and Zar( 1984). 

RESULTS 

The values of CR and OIR during the experiment are shown in 
Table 2. CR ranges between 0.20 and 0.26 L/h in the initial stages 
of cultivation (20-mm mussel), rising up to 4.37-4.51 L/h for a 
60-mm mussel. 

CR values for collector rope mussels were significantly higher 
than those of rocky shore mussels in days and 8 of the experi- 
mental period (P < 0.01 in both cases; ANOVA; Table 2). How- 
ever, CR for the rocky shore mussels had increased by 35% by the 
end of the 2nd week, compared with only 16% for the collector 
rope specimens. From this point on, no further significant differ- 
ence in this physiological rate was recorded between the two types 
of mussels {P > 0.05; Table 2). In the case of OIR, both groups 
of mussels followed the same pattern as that described for CR 
(Table 2). 

VARIATION OF CR AND OIR IN CULTIVATION TIME 

Figure 1 shows the variation of standardized CR and OIR for 
the 40-mm length class in cultivation time. CR showed a clear 
seasonal pattern with low values during the winter months, in- 
creasing in spring and summer. High values recorded in January 
constituted the sole exception. These trends were also recorded for 
OIR (Fig. 1 ), amplified in this case by the coincidence of high CR 
and high POM. 

CR versus Shell Length and Environmental Parameters 

The multiple regression analysis carried out on the variation 
observed in CR during the experiment showed a significant and 
positive relation to size (L mm), food quality (Q,), and tempera- 
ture (see F-ratio, Table 3). The regression model accounted for 
76.7% of the variance for CR, which in turn is mainly accounted 
for by size (L mm 67.6%). with a coefficient of 1.762. It is im- 
portant to point out the significant negative effect of the interac- 
tions of food quality (Q,) with both origin (Q, x origin) and 
temperature (Q, x T) (Table 3). 

Organic IR versus Natural Seslon (TPM mg/L) 

A significant relationship was established between the IR 
(OIR) and the variation in seston (TPM. mg/L), that could not be 
established in the case of the CR. This response of ingestion to 
seston concentration is shown in Figure 2 and fits exponential 
functions according to the Ivlev curves IR = a ( 1 - e ): 



OIR= 1.29 ± 0.39 [1 -e 
n = 14; r- = 0.517; P< 0.0 



-a7-'i±0.J6.TPM-i 



Rocky shore 



OIR = 1.18 : 
n= 14; r- 



0.36 [1 -e 
= 0.481; P< 0.01 



0.87±0.4fi-TPM-i 



H O 



The covariance analysis performed for the linear transforma- 
tions of these exponential curves showed no significant differences 



198 



Babarro et al. 



TABLE 2. 

Values of physiological parameters (mean ± SD, n = 32) of two sources of seed muscles standardized to shell length (L) according to growth 

of Af. galloprovincialis during the experiment. 





Cultivation 


Source of 






OIR 


Date 


Days 


Seed Mussel 


L (mm) 


CR (L/h) 


(mgPOM/h) 


11/27/95 





Collector ropes 


20 


0.43 ±0.12* 


0.16 ±0.04* 






Rocky shore 




0.34 ± 0.09 


0.13 ±0.03 


1 2/5/95 


8 


Collector ropes 


20 


0.26 ± 0.09* 


0.08 ± 0.03* 






Rocky shore 




0.20 ± 0.09 


0.06 ± 0.02 


12/13/95 


15 


Collector ropes 


20 


0.50 ±0.10 


0.16 ±0.03 






Rocky shore 




0.46 ±0.1 5 


0.14 ±0.05 


12/20/95 


22 


Collector ropes 


20 


0.37 ± 0.06 


0.14 ±0.02 






Rocky shore 




0.35 ± 0.08 


0.13 ±0.03 


1/3/96 


36 


Collector ropes 


20 


0.40 ± 0.09 


0.40 ± 0.09 






Rocky shore 




0.36 ±0.10 


0.36 ±0.10 


1/17/96 


50 


Collector ropes 


20 


0.57 ±0.14 


0.26 ± 0.06 






Rocky shore 




0.60 ±0.1 5 


0.28 ± 0.07 


1/31/96 


64 


Collector ropes 


30 


0.72 ± 0.23 


0.20 ± 0.06 






Rocky shore 




0.71 ±0.25 


0.20 ± 0.07 


2/15/96 


80 


Collector ropes 


30 


0.49 ± 0.09 


0.19 ±0.04 






Rocky shore 




0.52 ±0.15 


0.20 ± 0.06 


2/28/96 


95 


Collector ropes 


30 


0.69 ±0.1 3 


0.39 ± 0.07 






Rocky shore 




0.69 ±0.14 


0.39 ± 0.08 


3/13/96 


110 


Collector ropes 


40 


1.23 ±0.27 


0.56 ±0.1 3 






Rocky shore 




1.25 ±0.38 


0.57 ±0.18 


3/27/96 


125 


Collector ropes 


40 


1.13±0.27t 


0.59 ± 0.1 4t 






Rocky shore 




1.27 ±0.29 


0.66 ±0.15 


4/10/96 


140 


Collector ropes 


40 


1.16 ±0.47 


0.74 ± 0.30 






Rocky shore 




1.17±0.23 


0.75 ±0.1 5 


4/24/96 


155 


Collector ropes 


50 


2.47 ± 0.74 


0.97 ±0.29 






Rocky shore 




2.06 ± 0.64 


0.81 ±0.25 


6/5/96 


197 


Collector ropes 


60 


4.51 ± 1.21 


1 .30 ± 0.35 






Rocky shore 




4.37 ± 1.16 


1.26 ±0.33 


7/3/96 


226 


Collector ropes 


60 


3.92 ±0.89 


1.69 ±0.38 






Rocky shore 




4.09 ± 0.83 


1 .77 ± 0.36 



CR. clearance rate length-specific (L/h) by biodeposition method: OIR. organic ingestion rale length-specific (mg POM/h). 
* P < 0.01. fP < 0.05. ANOVA and Kruskall-Wallis nonparametric test in case of heterogeneity of variances). 



between both groups of mussels for OIR (t = 0.037, df = 24. P 
> 0.05. and t = 0.358, df = 25. P > 0.05 for analysis of slopes and 
intercepts, respectively). Therefore, one exponential curve for both 
groups of mussels together is shown in Figure 2: 

OIR = 1.23 ± 0.26 [1-e^'^*"-"^™] 
n = 28; r- = 0.500; P< 0.01 

DISCUSSION 

The variation in CR and OIR during the experimental period, 
for mussels standardized to 60 mm to compare with the literature 
values, covers a wide range ( 1 .46—4.5 1 and 1 . 1 3—4.37 L/h for CR. 
0.43-2.23 and 0.33-2.04 mg POM/h for OIR. values for collector 
rope and rocky shore mussels, respectively). These data coincide 
with those obtained by Navarro et al. (1991) and Iglesias et al. 
( 1996) for M. !ialloprovincialis in the Ri'a de Arousa. In the case of 
CR. however, these values are higher than those recorded for mus- 
sels elsewhere reported by Okumus and Stirling (1994) in their 
wide-ranging review. Despite the above-mentioned differences in 
CR due to low seston loads, characteristics from Galician Rias in 
particular, the values for organic IRs reported in the present study 
are similar to those obtained hv Widdows ct al. ( 1979). also under 



environmental conditions, and by Bayne et al. ( 1989) in the labo- 
ratory, with a higher range of values for seston and organic content 
(0.79-7.43 mg TPM/L. 0.43-1.79 mg POM/L. and 0.18-0.71 for 

Qi). 

The few studies that include CR data recorded over a period of 
seasons show that CR follows a clear seasonal pattern, with maxi- 
mum values occurring in the spring and summer months and mini- 
mum values in winter. This cycle can be observed both under 
laboratory conditions, with a constant food supply a\ailable (Wor- 
rall et al. 1983). and under natural conditions (Newell and Bayne 
1980). Larretxea (1995), taking into account a previous study of 
Hawkins et al. (1985). suggests that the seasonal sequence of CR 
is persistent to a large extent, although the effect of temperature 
could be an important determinant of this seasonal response. 

In this study, rates of energy acquisition exhibit a seasonal 
pattern, with lower values occurring during the winter months and 
slightly higher during spring and summer. The range results 
greater in terms of OIR because of the simultaneous decrease in 
CR and in organic content of the seston. An exception to this 
overall behavior are those values found to deviate largely from the 
mean of the season during which they were obtained, namely 
unexpectedly high values for CR. and especially OIR. in the 
samples taken in January. These may be accounted for by the high 



Feeding Behavior of Seed Mussel M. galloprovincialis 



199 



collector ropes -o- rocky shore 



£■21 



A 




30 60 90 120 150 180 210 240 



^^■^1 




1995 I 1996 

Time (days of cultivation/month) 

Figure 1. Seasonal changes in CR and OIR standardized to 40 mm of 
shell length for both sources of seed mussel M. galloprovincialis. 

POM values ( 1 .003 mg/L) and high Q, value (0.386), which can be 
considered as a result of a process of resuspension of material from 
the bottom in the Ria de Arousa (Babarro et al. 2000). Similar 
feeding behavior has been observed by Ki0rboe et al. (1981) and 
Larretxea (1995) concerning CR increments associated with an 
increase in detritus and sediment resuspended. 

With regard to seston composition, the results of this study 
show a 33% increase in total organic content in spring/summer 
when compared with winter, which can be related to an increase in 
mean CR of 30% for collector rope mussels and 40% in the case 
of rocky shore specimens. 

However, other factors that seem to exert indirect influence on 
the energy gain should be taken into account. This is the case for 
chl-a values for the period February through July that doubles that 
of the period November through February (Babarro et al. 2000). 
During the winter months, the proportion of phytoplankton (chl-(() 

TABLE 3. 

Stepwise multiple regressions of clearance rate of mussels with log 

shell length (L), quality of seston (Q, = POM/TPM), temperature 

(T, °C), and interactions terms. 



Parameter 


CoefTicient 


SE 


F-Ratio 


P 


r- 


Constant 


-20.329 










LogL 


1.762 


0.042 


1764.516 


<0.001 


0.676 


Q, XT 


-1.297 


0.085 


230.838 


<0.00l 


0.683 


Qi X origin 


-0.070 


0.023 


9.491 


<0.01 


0.686 


T 


1.778 


0.245 


52.512 


<0.00l 


0.688 


Q, 


18.948 


1.228 


238.067 


<0.001 


0.761 


T- 


-0.039 


0.009 


20.776 


<0.001 


0.767 


r- = 0.767: n 


= 812; F,.,o5 


= 440.729; 


/'< 0.001 











"1 

n 


fl 


^/^ 


^^^^^ 


/On 






/a 






/ □ 






i- L 







Origin factor has been estimated with values and 1 for collector ropes and 
rocky shore mussels, respectively. 



TPM (mg/L) 

Figure 2. OIR versus TPM relationship for both sources of seed mus- 
sel M. galloprovincialis. Both groups of mussels (collector ropes, 
squares; rocky shore, circles) were fitted by nonlinear regression ac- 
cording to Ivlev curve: Y = atl - e*" ^) (see text for details of fitting 
equation). 



in the organic content of the diet is 4-23%, rising to 21^1% 
during spring and summer, with peaks of 37.4% and 40.9% in 
April and February, respectively, which bears a close relationship 
to the seasonal variation in CR and OIR (carbon content = chl-« 
X 54, Widdows et al. 1979: organic matter = carbon x 1.87, 
Fernandez Ri'os 1992). This incidence of phytoplankton (chl-a) can 
also be observed in the fact that when the value for POM is not 
associated with chl-« (Babarro et al. 2000), no effect on CR is 
observed. However, the effect of this factor (chl-«) has not been 
tested in the multiple model because of the use of Q, as a factor of 
food quality and in order to avoid overlapping of information. 

The multiple regression model for CR shows the importance of 
shell length, food quality (Q,), and the temperature either as an 
independent variable or as a term in interaction (Q, x T). Very 
likely the presence of a term Q, x origin could be related to 
differences in seston quality between both original locations. Pre- 
vious studies carried out with both groups of mussels showed 
higher Q, values for subtidal location than that for rocky shore 
(unpublished data). Shallow water and stronger tidal waves in the 
rocky shore spot seem to affect the relation organic:inorganic frac- 
tion, with resuspension processes of the sediment playing an im- 
portant role. Mussels seem to adjust their feeding rates in a rela- 
tively short time under environmental changes (first 8 days under 
culture conditions), and probably when animals are "adapted," 
fluctuations of Q, after this initial period of time do not cause 
different CR responses between the two populations. 

The effects of the food ration or particle concentration on fil- 
tration rates in bivalve molluscs have been widely studied over the 
years. A reduction in CR when seston concentration increase has 
been reported in several experiments (Foster-Smith 1975: Wid- 
dows et al. 1979: Riisgard and Randlov 1981). As was previously 
established by Winter (1978). it seems that the ability of bivalves 
to adjust CR in response to an increase in particle concentration 
allows the regulation of IR. 

In fact, the relationship between IR and seston concentration 
has been appropriately described by an exponential asymptotic 



200 



Babarro et al. 



function (Ivlev curve) in this study. This behavior suggests to us a 
mechanism of regulation of ingestion based, in this case, on ad- 
justing CR and taking into account that this saturating increase in 
OIR cannot be assigned to the negative organic content versus 
seston availabihty relationship, which was not observed in our 
study as significant. Although higher CRs are related with lower 
seston availability values, significant effects of either TPM or 
POM and chl-a on CR were not observed, possibly because of the 
reduced range of variation in seston concentration. 

As already been mentioned, the results of this study establish 
that temperature has a significant effect on CR. The thermodepen- 
dence of CR coincides with the observations made by Widdows 
(1976), namely that mussels living in a stable thermal environment 
(which is the case with a range of temperature variation of 2.73 °C) 
have not developed compensation mechanisms, being thermode- 
pendent. 

Although the effects associated with origin have been consid- 
ered by many authors to be indicative of genetic differences be- 
tween mussels from different original habitats. Mallet et al. (1987) 
offered an alternative explanation. In their study, the authors sug- 
gested that these effects would reflect the differential influences 
undergone by mussels during their pre-experimental stage, so con- 
forming to an "ecological memory" of the individuals with respect 
to the conditions experienced in the primary habitat (food avail- 
ability and quality, tidal regime, air exposure, etc.). Okumus and 
Stirling (1994), Navarro et al. (1996), Iglesias et al. (1996), and 
Labarta et al. (1997) all recorded differences in CR for mussels 
from different original habitats, which they attribute to this eco- 
logical memory. 



The present study shows significant differences for CR and 
OIR between different original habitats of the mussels (collector 
rope and rocky shore) at the outset of the experiment that persisted 
after 8 days' cultivation on the raft. These significant differences 
concerning the two physiological rates between both types of mus- 
sel disappeared 15 days after raft cultivation commenced. The 
initial differences in CR and OIR may be the consequence of a 
response by the rocky shore mussels to the new conditions found 
on raft cultivation (i.e., a lower concentration and higher-quality 
Qi of seston in continuous immersion: previous data unpublished) 
over a short period of time. This hypothesis is supported by results 
of the multiple regression analysis, which indicates that both Q, 
and origin, the latter being expressed as a term of interaction with 
Qi. account for apart of the variance experienced by CR according 
to the model. 

ACKNOWLEDGMENTS 

We are grateful to Lourdes Nieto. Beatriz Gonzalez, and Sonia 
Villar for technical assistance. We also thank Juan Maneiro from 
Centre de Control da Calidade do Medio Marifio da Conselleria de 
Pesca. Marisqiieo e Acuicultura da Xunta de Galicia. for the de- 
termination of environmental parameters. We are also indebted to 
the crew of the Jose Maria Navaz from Instituto Espanol de 
Oceanografia. This work was supported by Project MAR97-0592 
by the Comision Interministerial de Ciencia y Tecnologi'a CICYT. 
J. M. F. Babarro was funded by a grant from Excma. Diputacion 
de Pontevedra. 



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Fernandez Rios. A. 1992. El fitoplanclon en la Ri'a de Vigo y sus condi- 
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Ilildreth. 1). I. & D. J. Crisp. 1976. A corrected formula for calculation of 
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Okumus. I. & H. P. Stirling. 1994. Physiological energetics of cultivated 
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Journal of Shellfish Research. Vol. 19, No. 1. 203-212. 20()(). 

SALINITY TOLERANCE OF BROWN MUSSEL PERNA PERNA (L.) FROM THE GULF OF 

MEXICO: AN EXTENSION OF LIFE TABLE ANALYSIS TO ESTIMATE MEDIAN SURVIVAL 

TIME IN THE PRESENCE OF REGRESSOR VARIABLES 



DAVID W. HICKS,** DOYLE L. HAWKINS," AND 
ROBERT F. MCMAHON' 

^Department of Biology 
Box 19498 

The University of Texas at Arlington 
Arlington, Texas 76019 
^Department of Mathematics 
Box 19408 

The University of Texas at Arlington 
Arlington. Texas 76019 

ABSTRACT The nonindigenous brown mussel Periia perna was first recorded in the Gulf of Me.xico at Port Aransas, Texas in 1990. 
The association between survival time and chronic exposure to hypo- and hypersaline conditions was examined to estimate the potential 
range of habitats that P. pema could invade in coastal North American Gulf of Mexico habitats. A novel application of the discrete 
logistic failure time model (DLFTM) was used to estimate covariate-adjusted median survival times from the interval-level survival 
data collected. This method allowed factoiial-type comparisons of the covariate-adjusted medians across treatments. This analysis 
indicated that salinities ranging from 15-50 ppt are nonlethal to P. perna. under which at least 80% of individuals survived 30 days 
(720 h). Chronic exposure to salinities outside 15-50 ppt were lethal to P. perna. Lethality was size-dependent, with both smaller and 
larger individuals having reduced survival times. For an average-sized individual (shell length = 40 mm), median survival times were 
191, 268. 335, 1 19, and 1 16 h at 0, 5. 10, 55, and 60 ppt, respectively. The 15-50 ppt incipient salinity limits of Texas P. pema suggest 
that this species could potentially colonize the majority of marine and estuarine coastal habitats in the Gulf of Mexico. 

KEY WORDS: interval-level survival data, mytilacea. nonindigenous species, Perna perna. salinity tolerance 



INTRODUCTION 

Since its initial discovery in Port Aransas. Texas, in 1990, the 
marine brown mussel, Pema perna (L,), has colonized hard shores 
at intermittent locations throughout the southwestern Gulf of 
Mexico in Texas and Mexico (Hicks and Tunnel! 1993, Hicks and 
Tunnell 1995). The majority of the Gulf of Mexico's margins are 
sandy beaches, which has historically impeded development of 
natural hard-shore communities. Thus, it was not until the con- 
struction of jetties, breakwaters, and other coastal structures during 
the last century that habitat existed for true intertidal bed-forining 
mytilid genera such as Pema. These man-made structures, which 
are continuously being constructed to control coastal erosion, 
present a relatively open niche for such invasive mytilid species as 
P. pema. Generally, considered an open-water species (Berry 
1978). Texas P. perna populations have been reported from littoral 
and shallow sublittoral habitats of widely varying physiochemical 
parameters including the low salinity (20-25 ppt) Lavaca-Tres 
Palacios estuary (Davenport 1995) and the hypersaline (35—10 
ppt and occasionally higher) Laguna Madre (McGrath et al, 1998). 

The endemic range of P. perna (synonymous with Pema pitta 
(Bom) and Pema indica Kuriakose and Nair. [Siddall 1980. Va- 
kily 1989]) includes India. Sri Lanka. Madagascar, the east coast 
of Africa from central Mozambique to False Bay. South Africa, 
and the African west coast from Luderiz Bay. Nambia. north into 



'Current address: Center for Coastal and Marine Studies, Department of 
Biology, Lamar University, P.O. Box 10037, Beaumont, TX 77710. 
E-mail: hicksdw@hal.lamar.edu 



the Mediterranean from Gibraltar to the Gulf of Tunis as well as on 
the Atlantic coasts of Brazil, Uruguay. Venezuela, and in the West 
Indies (Berry 1978). 

Salinity influences many physiological functions, making it an 
important limiting factor in the distributions of estuarine and ma- 
rine bivalves (Bayne et al, 1976, Widdows 1985. Dame 1996). As 
with most marine mytilaceans. P. perna is incapable of extracel- 
lular osmotic control; thus, its extracellular fluids are nearly isos- 
motic to ambient seawater over its tolerated salinity range (Salo- 
mao and Lunetta 1989). The typical short-term response of osmo- 
conforming bivalves to salinity reductions is to close the shell 
valves temporarily isolating tissues and body fluids from poten- 
tially lethal hyposaline waters, while allowing time for intracellu- 
lar volume regulation by adjusting the concentrations of intracel- 
lular amino acids and other small organic molecules (Hawkins and 
Bayne 1992). 

We examined the effects of chronic exposure to hypo- and 
hypersaline media on survival times in P. perna. Salinity tolerance 
data have proved effective in predicting local distributions in ma- 
rine bivalves (Castagna and Chanley 1973). Thus, the incipient 
salinity limits determined in this study were used to predict the 
potential for P. pema to colonize coastal Gulf of Mexico marine 
and estuarine habitats in North America. 

We also developed a specialized methodology for analyzing 
our survivorship data that allows estimating and comparing co- 
variate-adjusted median survival times for grouped lifetime data. 
Current methods for analyzing data of this type compare treatment 
survival distributions based upon odds-ratios. Our method of using 
median survival times, as opposed to odds, provides biologically 
more meaningful interpretations of survival data. 



203 



204 



Hicks et al. 



MATERIALS AND METHODS 



Experimental Protocol 

Specimens of Penia perna were collected from intertidal rocks 
on the north jetty of Mansfield Pass (26 °34 'N) on the Texas coast 
and were transported overnight in cooled insulated containers to 
Arlington, Texas. Upon arrival, mussels were maintained in a 
284-L aerated holding tank containing artificial seawater (35 ppt) 
at a constant temperature of 20 °C on a 1 2; 1 2 hour light-dark cycle 
without feeding before experimentation. Experiments were initi- 
ated within 30 days of collection. 

Individual mussels were excised from mussel clumps by cut- 
ting byssal attachment threads with scissors before salinity toler- 
ance testing. For each salinity tested, four subsamples of 10 mus- 
sels each, visually selected to be of similar size range, were held 
for 2 weeks in a constant temperature laboratory at 20 °C (±1 °C) 
in 3.5-L plastic aquaria containing 3 L of continuously-aerated. 35 
ppt artificial sea water (Fritz Supersalt). Tank medium was re- 
placed daily. During the 2 week acclimation period, mussels bys- 
sally reattached to tank walls or other individuals. The size range 
of the subsamples utilized in each salinity test reflected the size 
range in the original sample (shell lengths - 15-70 mm). 

After acclimation to experimental conditions, subsamples were 
randomly assigned to test salinities of 0. 5. 10. 15. 20, 30, 40, 50. 
55, and 60 ppt (created with Fritz Supersalt and City of Arlington, 
Texas, dechlorinated tap water), chosen to include the range of 
salinities occurring in Texas coastal aquatic habitats. Testing was 
initiated by replacing the 35 ppt seawater medium in each tank 
with 3 L of test salinity medium. During testing, media were held 
at 20 °C (±1 °C), continuously aerated and changed daily. Byssal 
attachment, valve opening or closure, and viability of all individu- 
als was examined at 24-h intervals. Viability was detennined by 
touching the exposed mantle edges of emersed gaping mussels 
with the tip of a fine-haired brush. Individuals not closing their 
valves in response to this stimulus were considered dead, and were 
removed from the aquaria. The time of the observation was re- 
corded, and the shell lengths (SL) of dead individuals were mea- 
sured as the linear distance from the anterior to posterior margins 
of the shell to the nearest 0.1 mm using digital calipers. All non- 
gaping individuals were considered alive. Exposure to salinity 
treatments and viability testing was continued until either lOOVr 
sample mortality was achieved or individuals survived for 30 days 
(720 h). 

Statistical Methods 

The salinity resistance of specimens of P. perna was examined 
using a survival analysis strategy designed to determine the effects 
of seawater concentration on survival duration: whereas, control- 
ling for individual-specific covariates (e.g.. size). Our viability 
monitoring at 24-h intervals prevented knowledge of an individu- 
afs exact time of death. Thus, survival time was known only to fall 
within the interval Ij = |«,_|, ci,]. while «, was the current obser- 
vation time, and o^_i was the last observation time. Available 
parametric (e.g., Weibull) and nonparametric (e.g.. Kaplan-Meier, 
Cox regression) survival estimation nieihods assume that the time 
of death is known exactly. However, in the vast majority of such 
studies, as in this one. time of death is not recorded exactly, but is 
known only to have occurred within a particular interval (Hosmer 
and Lemeshow 1989). Applying continuous-data methodology to 
such interval-level data can result in serious bias, especially if the 



interval length is large relative to the average lifetimes one is 
observing. 

When survival data are recorded at the interval level, the life 
table (i.e., actuarial method) is often used to estimate survival 
probabilities. Life tables are essentially frequency tables modified 
to deal with censored observations (i.e.. those data for individuals 
that survive treatments) (Lawless 1982). The main outcome of life 
table analysis is estimation of the survival function Sicij). which is 
the probability of surviving to time a,, for all observation times 
a^ «j. However, standard life table analysis cannot incorpo- 
rate continuous regressors, such as size, which are likely to influ- 
ence individual survival times. When such regressor variables are 
present, a discrete logistic failure time model (DLFTM), which 
generalizes the life table method, can be used to estimate survival 
probabilities and allow their adjustment for regressor effects (Cox 
1972, Thompson 1977). The ability to include regressor variables 
in the DLFTM greatly broadens the scope of life table analysis by 
revealing both treatment and individual-specific regressor influ- 
ences, allowing more biologically appropriate interpretations of 
survival data (Lawless 1982. Hosmer and Lemeshow 1989). 

Although survival probabilities, when graphed into the usual 
form of survival functions, provide a summary of the survival 
experience of a population, these functions are cumbersome when 
there are many such populations to be compared (e.g.. levels of 
treatments, different values of important regressors). In such cases, 
it is useful to have a one-number summary (e.g.. the median sur- 
vival time) of each survival function to compare across many 
populations. The DLFTM can. as illustrated here, be used to pro- 
vide such median estimates for interval level data under some 
reasonable assumptions discussed below. 

We implemented the DLFTM for our data analysis in a com- 
puter program written in SAS's interactive matrix language (IML, 
SAS, Gary, NC) available from the authors. The routine was 
checked for programming errors using simulations of data from 
lifetime distributions with known parameters. 

DLFTM and Its Estimation 

For our analysis, the 720-h observation period was divided into 

24-h time intervals, /, = [o^.,, a,), y = 1 k = 30. where 

(( = 24 ■) are times of observation, a„ = and «j+, = ^. The 
data for the /th individual, 1 < / s n. consists of the vector G,- = 

(G,| G, i^^i ), where G„ = 1 if individual i dies in interval /, 

and C„ = otherwise, and a vector x, of covariates describing 
treatments and individual-specific characteristics. 

Let 7, denote the actual, but unobserved, lifetime of individu- 
als. Let S{t I X) = PrtT, > H X) denote the survival function of 
individuals with regressor x, which contains indicator variables 
describing salinity level and shell length (SL). The goal of 
the analysis was to estimate the sur\i\al probabilities Pj(\) = 

5((i, I X) at each of the observation limes (/, fl^. By a standard 

argument using conditional probabilities (see Lawless 1982. p. 55), 



^,(x) = /),(x)- ■ ■ p,i\). 1 ^j ^k. 



(1 



where /),(x) = Pr {T, > a, I T, >«, ,, x| is the probability of 
surviving the time interval /,. given alive at its outset. The /),(x)s 
are called "interval-specific" survival probabilities. It is clear from 
( I ) thai to estimate P,(x) it is enough to estimate the p^Ws. 

The method of maximum likelihood estimation was applied to 
estimate the /j,(xIs, because it is known to produce estimates sta- 
tislically optimal for large sample si/cs n. In this regard, again by 



Salinity Tolerance in Perna perna 



205 



a standard argument (see Lawless 1982. p.372). the likelihood 
function for the above data on n independent indi\iduals. assuming 
censoring only at «j. is 



^[/'lO /'*c>]=n{n[i-/',<''->]- n /v\> 



y=l 



(2) 



where: /?, is the set of individuals / who are alive just before <;,_,. 
and D, is the set of individuals / who die in interval 1^. 

The DLFTM is a model for the functional form of ^^(x). Spe- 
cifically. 



pM) 



(1 



1 



(3) 



where p = ((3, P,„)^ is an in x 1 vector of unknown regres- 



sion coefficients relating the covariate vector x = (.v. 



..V,,,) to 



/),. and the a,s are interval-specific parameters (i.e.. the interval 
effects). Because (3) implies 



I -pj(x + A) 



1 



■pM) 



pM) 



. 1 <y<A- 



(4) 



for any A. it follows that P relates the odds of death in any interval 
/, for covariate value x -i- A [i.e., the left side of (4)] to the odds of 
death in /, for covariate value x. Thus, if p^ > (<0) for some 1 s 
*• < III. the odds of dying in interval /, increases (decreases) as .v,. 
increases. The parameter a^ is seen from (3) to equal In {[1 - 
Pj{0)]/pjiO)}, the log-odds of death in interval /, when x = 0. 

For a particular choice of the covariate vector x (i.e., including 
terms to represent treatments, dependence on SL, etc.). the un- 
known parameters P and a = (a, a^V in the DLFTM (3) are 

estimated by substituting (3) into the likelihood (2). and then maxi- 
mizing the resulting "constrained likelihood" (5) with respect to p 
and a (Lawless 1982). 



L(a,P) = n 






n {1 



+ e 



a,+x,p,-l 



The logarithm of this constrained likelihood is 



logL(a,P) = 2 2<«y + ".P' - 2l"( I + ''"'"'''*' 
7=1 



(5) 



(6) 



The maximum likelihood estimators (mles) d and P of a and P are 
obtained by maximizing (6). The maximization is done by the 
Newton-Raphson algorithm, which iteratively solves the so-called 
likelihood equations (writing x, = (.v,,, . . . ,.v,„,)) 



fdogL 



.v,^ 



,3 iXv,,-!:,^, 



.j+x,P 



a;+x,p 



: 0. /•= 1 in: and 



aiogZ. 

r'*a,, 



E<i)-E 



^oiv+x,p 



isD„ 



isRm 



+ e 



+x,P 



: 0. 1) = 1 k. 



(7) 



(8) 



The interval-specific survival probability estimates, /5,(x) are 
obtained by substituting the m/es. a. and P into (3). The survival 
probability estimates P,(x) are then obtained by plugging the /5^(x) 
into ( 1 ). The issue of which variables to include in the regressor 
vector x (e.g.. linear or quadratic functions of SL. treatment by SL 
interactions, etc.) was addressed by beginning with a model a 



priori deemed sufficiently flexible and then testing a sequence of 
nested models until reaching the most parsimonious model that 
adequately explained the data. Goodness-of-fit was assessed by 
comparing the most parsimonious fitted model to the a priori 
model (i.e.. the model containing at most quadratic functions of SL 
and treatment by SL interactions) by a Wald statistic. 

To conduct inferences using the estimated P,(x)s. their covari- 
ance matrix was needed. This matrix was derived by a sequence of 
three steps, the first of which was to construct the covariance 
matrix of the estimators d and p. An estimate of the covariance 
matrix of 6 = (d,P) was obtained as the negative of the inverse of 
the matrix of the second-order partial derivatives of log L (6) (i.e.. 
-H~\ where H is the Hessian matrix). The covariance matrices of 
the derived estimates /',(x) and P,(x) were propagated, in turn, 
from the covariance matrix of 6 by the Delta method (Sertling 
1980). See Appendix A for details. 

Estimating Median Siiniial Time 

A unique aspect of our application of the DLFTM to analyze 
salinity as a lethal factor in P. perna was our incorporation of a 
method of estimating, and computing variances for, the covariate- 
adjusted median survival times. This method allows factorial-type 
comparisons of the covariate-adjusted medians across treatments. 
Median survival time. A/(x), which satisfies 5(M(x)lx) = 0.5. was 
estimated by assuming that the survival function, S{t\x). was linear 
in t (for fixed x) over each time interval [«,_;, a,). Specifically, 
given that P,(x) < 0.5 ^Aj-\W for some 1 s j < k. then the 
median estimate is (using linear interpolation) 



M/x) = aj + 



"j-\ 



1 



■ PjW 



(9) 



P/x)-/^_,(x)_ 

for the interval index 7 in which P^Cx) < 0.5 < P,_,(x). However, 
this interval is itself a random variable, so it is necessary to express 
the median estimate as 



M(x) = 2'W/x)/[P/x) < 0.5 < P,_i(x)] 



(10) 



7=1 



where M,(x) is given by (10). and the indicator variable [second 
factor in the summand in (10)] equals 1 if the pa