Copepods in Aquaculture

Edited byCheng-Sheng Lee

Patricia J. O’BryenNancy H. Marcus

Copepods in Aquaculture

Copepods in Aquaculture

Edited byCheng-Sheng Lee

Patricia J. O’BryenNancy H. Marcus

Cheng-Sheng Lee, PhD, is the Director of theAquaculture Interchange Program, Oceanic Institutein Hawaii and the Executive Director for the Centerfor Tropical and Subtropical Aquaculture, CooperativeState Research, Education, and Extension Service,U.S. Department of Agriculture.

Patricia J. O’Bryen is the project manager for theAquaculture Interchange Program, Oceanic Institutein Hawaii.

Nancy H. Marcus, PhD, is the Robert O. LawtonDistinguished Professor and Mary Sears Professor ofOceanography, Florida State University.

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Library of Congress Cataloging-in-Publication DataCopepods in aquaculture / edited by Cheng-ShengLee, Patricia J. O’Bryen, Nancy H. Marcus.—1st ed.

p. cm.Papers presented at a workshop held in Honolulu,

Hawaii, May 5–8, 2003,Includes index.ISBN-13: 978-0-8138-0066-0ISBN-10: 0-8138-0066-81. Fishes—Feeding and feeds—Congresses.

2. Copepoda—Congresses. I. Lee, Cheng-Sheng. II. O’Bryen, P. J. (Patricia J.) III. Marcus, Nancy H.

SH156.C67 2005639.3�2—dc22

2004029918

The last digit is the print number: 9 8 7 6 5 4 3 2 1

Contents

Contributors viiPreface xiiiCheng-Sheng Lee

01 Calanoid Copepods, Resting Eggs, and Aquaculture 3Nancy H. Marcus

02 The Potential to Mass-Culture Harpacticoid Copepods for Use as Food for Larval Fish 11John W. Fleeger

03 Symbiotic Copepods as Live Feed in Marine Finfish Rearing 25Ju-shey Ho

04 Birth Control Effects of Diatoms on Copepod Reproduction: Implications for 31Aquaculture StudiesAdrianna Ianora

05 Maximizing the Nutritional Values of Copepods in Aquaculture: Managed versus 49Balanced NutritionG. S. Kleppel, Sarah E. Hazzard, and Carol A. Burkart

06 Formulated Feeds for Harpacticoid Copepods: Implications for Population Growth and 61Fatty Acid CompositionAdelaide Rhodes and Leon Boyd

07 A Brief Review of Mass Culture of Copepods Used for Fish Food in Japanese Mariculture 75and a Proposed Plan to Use High Biomass Natural Populations of Brackish-Water CopepodsShin-ichi Uye

08 Behavioral Characteristics of Copepods That Affect Their Suitability as Food for 91Larval FishesEdward J. Buskey

09 Suitability of the Copepod Gladioferens imparipes for Intensive Cultivation for 107AquacultureRobert J. Rippingale and Michael F. Payne

v

10 Development of Feeding Mechanics in Marine Fish Larvae and the Swimming 119Behavior of Zooplankton Prey: Implications for Rearing Marine FishesRalph G. Turingan, Jessica L. Beck, Justin M. Krebs, and Jason D. Licamele

11 Copepods as Live Prey: A Review of Factors That Influence the Feeding Success of 133Marine Fish LarvaeEdward J. Chesney

12 Intensive and Extensive Production Techniques to Provide Copepod Nauplii for 151Feeding Larval Red Snapper Lutjanus campechanusRonald P. Phelps, Gede S. Sumiarsa, Emily E. Lipman, Hsiang-Pin Lan,Komarey Kao Moss, and Allen D. Davis

13 Studies on the Use of Copepods in the Semi-intensive Seed Production of Grouper 169Epinephelus coioidesJoebert D. Toledo, Ma. Salvacion Golez, and Atsushi Ohno

14 Culture of Copepods and Applications to Marine Finfish Larval Rearing in Taiwan 183Huei-Meei Su, Shin-Hong Cheng, Tzyy-Ing Chen, and Mao-Sen Su

15 Copepods as a Live Feed for Striped Trumpeter Latris lineata Larvae 195David T. Morehead, Stephen C. Battaglene, Ephrime B. Metillo,Matthew P. Bransden, and Graeme A. Dunstan

16 Intensive Cultivation of a Subtropical Paracalanid Copepod, Parvocalanus sp., as 209Prey for Small Marine Fish LarvaeRobin J. Shields, Tomonari Kotani, Augustin Molnar, Kimo Marion, Jon Kobashigawa, andLarren Tang

17 Characterization of an Extensive Zooplankton Culture System Coupled with Intensive 225Larval Rearing of Red Snapper Lutjanus campechanusJohn T. Ogle, Jason T. Lemus, L. Casey Nicholson, Donald N. Barnes, and Jeffrey M. Lotz

18 Culture of Copepods and Applications to Marine Finfish Larval Rearing Workshop 245Discussion SummaryPatricia J. O’Bryen and Cheng-Sheng Lee

Index 255

vi Contents

Donald N. Barnes [17] University of SouthernMississippi-Gulf Coast Research Laboratory, P.O. Box7000, Ocean Springs, Mississippi 39564-7000, USA.

Stephen C. Battaglene [15] Marine Research Labora-tories, Tasmanian Aquaculture and Fisheries Instituteand Aquafin Cooperative Research Centre, Universityof Tasmania, Hobart, Tasmania 7001, Australia.

Jessica L. Beck [10] Department of BiologicalSciences, Florida Institute of Technology, 150 WestUniversity Blvd., Melbourne, Florida 32901, USA.

Leon Boyd [6] Department of Food Science, NorthCarolina State University, Raleigh, North Carolina27695, USA.

Matthew P. Bransden [15] Marine Research Labora-tories, Tasmanian Aquaculture and Fisheries Instituteand Aquafin Cooperative Research Centre, Universityof Tasmania, Hobart, Tasmania 7001, Australia.

Carol A. Burkart [5] Mountain Empire CommunityCollege, Big Stone Gap, Virginia 24219, USA.

Edward J. Buskey [8*]The University of Texas atAustin, Marine Science Institute, 750 Channel ViewDrive, Port Aransas, Texas 78373, USA. buskey@utmsi.utexas.edu

Edward J. Buskey is a professor of marine science inthe Department of Marine Science and a senior re-search scientist at the Marine Science Institute of theUniversity of Texas at Austin. Dr. Buskey’s research in-terests focus on the behavioral ecology of zooplanktonand include studies of planktonic predator—prey inter-actions, aggregative behavior of zooplankton, adaptivevalue of marine bioluminescence, and the role of zoo-plankton grazers in harmful algal blooms.

He received his A.B. in biology from Brown Univer-sity, his M.Sc. in zoology from the University of BritishColumbia, and his Ph.D. in biological oceanographyfrom the University of Rhode Island.

Tzyy-Ing Chen [14] Tungkang Marine Laboratory,Fisheries Research Institute, Council of AgricultureR.O.C., Tungkang, Pingtung, 92845 Taiwan.

Shin-Hong Cheng [14] Tungkang Marine Laboratory,Fisheries Research Institute, Council of AgricultureR.O.C., Tungkang, Pingtung, 92845 Taiwan.

Edward J. Chesney [11*] Louisiana UniversitiesMarine Consortium, Chauvin, Louisiana 70344, USA.echesney@lumcon.edu

Edward J. Chesney is an associate professor at theLouisiana Universities Marine Consortium. Dr. Ches-ney has been involved in research and management re-lated to fisheries, coastal oceanography, and aquacul-ture for over 30 years in such diverse locations as theGulf of Mexico, and the east and west coasts of theUnited States, and Spain.

He received his B.S. from the University ofMichigan School of Natural Resources and his Ph.D.from the University of Rhode Island, Graduate Schoolof Oceanography in 1984. He is currently collaboratingdirectly with the aquaculture industry to improvehatchery and rearing techniques of marine finfish. Re-cent collaborative projects related to aquaculture in-clude studies of the role of gamete quality in thespawning of fishes and the subsequent effects on thequality of fish eggs and larvae, ways to improve hatch-ery production techniques, the use of recirculating sys-tems for the spawning and culture of marine finfish,studies to improve production techniques for live feeds,and a feasibility study for offshore aquaculture in theGulf of Mexico.

Allen D. Davis [12] Department of Fisheries andAllied Aquacultures, Auburn University, Auburn,Alabama 36849, USA.

Graeme A. Dunstan [15] CSIRO Marine Researchand Aquafin Cooperative Research Centre, Hobart,Tasmania, 7001 Australia.

vii

ContributorsChapter numbers in brackets follow each name. *Indicates the corresponding author.

John W. Fleeger [2*] Department of BiologicalSciences, Louisiana State University, Baton Rouge,Louisiana 70803, USA. zoflee@lsu.edu

John W. Fleeger holds the George C. Kent Chair inBiological Sciences at Louisiana State University,Baton Rouge. Dr. Fleeger has been an active researcherspecializing in the ecology, evolution, and ecotoxicol-ogy of harpacticoid copepods for more than twodecades. His research with harpacticoids and otherbenthic invertebrates includes topics in systematics(traditional and molecular), culture procedures, behav-ior, toxicology, and diet. Field-related research in-cludes examination of factors (including nutrients, pre-dation, hydrodynamics, and competition) that influencedistribution and abundance patterns.

He received his Ph.D. from the University of SouthCarolina in Columbia. He is a former chair of theInternational Association of Meiobenthologists andPresident of the Gulf Estuarine Research Association.He served as chair of the Department of Zoology andPhysiology for 6 years and has been a co-organizer ofthe Benthic Ecology Meetings (in 1999) and anInternational Conference of Meiobenthology (in 2001).

Ma. Salvacion Golez [13] Tokyo University of Fish-eries, 4-5-7 Konan, Minato-ku, Tokyo 108-8477, Japan.

Sarah E. Hazzard [5] Institute of Ecosystem Studies,Millbrook, New York 12545, USA.

Ju-shey Ho [3*] Department of Biological Sciences,California State University, 1250 Bellflower Blvd., LongBeach, California 90840-3702, USA. jsho@csulb.edu

Ju-shey Ho is a professor of biology at CaliforniaState University, Long Beach. Dr. Ho has worked onsymbiotic copepods since 1960 and published morethan 200 articles on the taxonomy, systematics, and bio-geography of symbiotic copepods. He has received sev-eral honors connected to his work, including an Awardin Science and Technology from the Taiwanese-American Foundation and a Life Time AchievementAward from The 8th International Conference onCopepoda. He is also frequently called upon for help inthe identification of copepods parasitic on various typesof marine animals ranging from sponges to mammals.

He received his B.S. at National Taiwan Universityin Taipei, Taiwan, in 1958 and obtained advanced de-grees (M.S. and Ph.D.) in biology from BostonUniversity. Dr. Ho has traveled to many parts of theworld to collect and study symbiotic copepods. Thatlong-term experience in search for the symbiotic cope-pods led him to conceive the idea of using symbioticcopepods for mass production of nauplii to obtain livefeed for marine finfish larvae rearing.

Adrianna Ianora [4*] Ecophysiology Laboratory,Stazione Zoologica A. Dohrn, Villa Comunale 80121,Naples, Italy. iannora@alpha.szn.it

Adrianna Ianora is senior scientist of theEcophysiology Laboratory at the Stazione Zoologicain Naples, Italy. Dr. Ianora received her degree at theUniversity of Naples in 1980 and has been working atthe Stazione Zoologica since 1983 on copepod ecol-ogy, reproduction, development, nutrition, and physi-ology. She is an editorial board member of the Journalof Plankton Research, Scientia Marina, and MarineEcology: PSZNI, and review editor of the journal,Marine Ecology Progress Series. She is author of over90 scientific publications and coeditor of three bookspublished by Springer-Verlag on zooplankton taxon-omy and Antarctic biology. Currently, she is studyingthe effects of phytoplankton chemical defenses oncopepod reproduction and development and screeningfor the presence of new antiproliferative compoundsin diatoms, dinoflagellates, and other microalgalgroups.

Gary S. Kleppel [5*] Department of BiologicalSciences, University at Albany, State University ofNew York, 1400 Washington Ave., Albany, New York12222, USA.

Gary S. Kleppel is a professor of biological sciencesat the University at Albany, State University of NewYork. His interests are in the nutritional ecology of zoo-plankton and sustainable development. Dr. Kleppel’sresearch in zooplankton nutrition began in the early1980s with studies on ichthytoplankton off southernCalifornia and has continued with research on the roleof nutrition in defining ecosystems and ecological in-teractions within and among copepod populations incoastal and ocean waters. Dr. Kleppel is director of theBiodiversity, Conservation and Policy Program at theUniversity at Albany and the Land Use-CoastalEcosystem Study (NOOA/COP), which seeks sustain-able solutions to the impacts of development along rap-idly urbanizing coastlines.

Dr. Kleppel received his Ph.D. from Fordham Uni-versity in 1979 and conducted postdoctoral research atthe University of Southern California from 1981 to1986. He taught at the Oceanographic Center of NovaSoutheastern University, in Fort Lauderdale, Florida,and the University of South Carolina, Columbia priorto coming to the University at Albany.

Jon Kobashigawa [16] The Oceanic Institute, 41-202Kalanianaole Hwy., Waimanalo, Hawaii 96795, USA.

Tomonari Kotani [16] Industrial Promotion Founda-tion, Ikeda 2-1303-8, Oomura 856-0026, Japan.

viii Contributors

Justin M. Krebs [10] Department of BiologicalSciences, Florida Institute of Technology, 150 WestUniversity Blvd., Melbourne, Florida 32901, USA.

Hsiang-Pin Lan [12] Department of Fisheries andAllied Aquacultures, Auburn University, Auburn,Alabama 36849, USA.

Cheng-Sheng Lee [Editor, 18] The Oceanic Institute,41-202 Kalanianaole Hwy., Waimanalo, Hawaii 96795,USA. cslee@oceanicinstitute.org

Cheng-Sheng Lee is the Director of the AquacultureInterchange Program at the Oceanic Institute in Hawaiiand the Executive Director for the Center for Tropicaland Subtropical Aquaculture, Cooperative State Re-search, Education, and Extension Service, U.S.Department of Agriculture. His research interests havefocused on control of reproduction and the early lifehistory of marine finfish and shrimp, rotifer biologyand culture, and general aquaculture management.

Jason T. Lemus [17] University of SouthernMississippi-Gulf Coast Research Laboratory, P.O. Box7000, Ocean Springs, Mississippi 395-7000, USA.

Jason D. Licamele [10] Department of BiologicalSciences, Florida Institute of Technology, 150 WestUniversity Blvd., Melbourne, Florida 32901, USA.

Emily E. Lipman [12] Department of Fisheries andAllied Aquacultures, Auburn University, Auburn, Ala-bama 36849, USA.

Jeffrey M. Lotz [17] University of SouthernMississippi-Gulf Coast Research Laboratory, P.O. Box7000, Ocean Springs, Mississippi 39564-7000, USA.

Nancy H. Marcus [Editor, 1*] Department ofOceanography, Florida State University, Tallahassee,Florida 32306, USA. marcus@ocean.fsu.edu

Nancy H. Marcus is the Robert O. LawtonDistinguished Professor and Mary Sears Professor ofOceanography at Florida State University. Her researchhas focused on the induction, maintenance, and termi-nation of diapause in marine copepods, specificallycoastal species that produce diapause eggs.

Kimo Marion [16] The Oceanic Institute, 41-202Kalanianaole Hwy., Waimanalo, Hawaii 96795, USA.

Ephrime B. Metillo [15] Department of BiologicalSciences, Mindanao State University-Iligan Institute ofTechnology, A. Bonifacio Ave., Iligan City, Philippines9200.

Augustin Molnar [16] The Oceanic Institute, 41-202Kalanianaole Hwy., Waimanalo, Hawaii 96795, USA.

David T. Morehead [15*] Marine Research Labora-tories, Tasmanian Aquaculture and Fisheries Instituteand Aquafin Cooperative Research Centre, Universityof Tasmania, Hobart, Tasmania 7001, Australia.

David T. Morehead is involved in striped trumpeterresearch at the Tasmanian Aquaculture and FisheriesInstitute’s Marine Research Laboratories (University ofTasmania). He received his bachelor of applied sciencedegree at Curtin University (Western Australia) in 1991and a graduate diploma in Antarctic and southern oceanstudies with honors at the University of Tasmania in1992. In 1997, Dr. Morehead completed his Ph.D. inthe management of reproduction in striped trumpeter,focusing on reproductive endocrinology. Since thattime, Dr. Morehead has concentrated on larviculture,with emphasis on nutrition, health, and system design.

Komarey Kao Moss [12] Myron B. ThompsonAcademy, PCS, 629 Pohukaina St., Suite 3, Honolulu,Hawaii 96813, USA.

L. Casey Nicholson [17] University of SouthernMississippi-Gulf Coast Research Laboratory, P.O. Box7000, Ocean Springs, Mississippi 39564-7000, USA.

Patricia J. O’Bryen [Editor, 18*] The OceanicInstitute, 41-202 Kalanianaole Hwy., Waimanalo,Hawaii 96795, USA. pobryen@oceanicinstitute.org

Patricia J. O’Bryen is the Project Manager for theAquaculture Interchange Program at the OceanicInstitute in Hawaii. She has co-edited the proceedingsof five previous workshops held by the OceanicInstitute. Ms. O’Bryen has a B.A. in Zoology and anM.A. in English as a Second Language from the Uni-versity of Hawaii.

John T. Ogle [17*] University of SouthernMississippi-Gulf Coast Research Laboratory, P.O. Box7000, Ocean Springs, Mississippi 39564-7000, USA.

John T. Ogle is a senior research scientist with TheUniversity of Southern Mississippi, Gulf Coast Re-search Laboratory at Ocean Springs, Mississippi, USA.He received his B.S. and M.S. from Texas A&M Uni-versity and has worked in aquaculture for the past 30years.

Atsushi Ohno [13] Tokyo University of Fisheries, 4-5-7 Konan, Minato-ku, Tokyo 108-8477, Japan.

Michael F. Payne [9] Seahorse Sanctuary, Red BluffRoad, Kalbarri, Western Australia 6536, Australia.

Contributors ix

Ronald P. Phelps [12*] Department of Fisheries andAllied Aquacultures, Auburn University, Auburn, Ala-bama 36849, USA.

Ronald P. Phelps is an associate professor in theDepartment of Fisheries and Allied Aquacultures atAuburn University. Dr. Phelps has worked in the areaof fish reproduction and hatchery management since1977. Much of his recent research efforts have concen-trated on the reproduction of the marine fish red snap-per Lutjanus campechanus, its larval rearing, and pro-duction of live feeds. He received his B.S. at AuburnUniversity in fisheries science in 1969 and his Ph.D. in1975.

Adelaide Rhodes [6*] Essential Live Feeds, 343Soquel Ave. #197, Santa Cruz, California 95062, USA.

Adelaide C.E. Rhodes recently received her Ph.D. inzoology from North Carolina State University. Her dis-sertation research focused on developing a simple tech-nique for rearing copepods to use in aquaculture. Dr.Rhodes started her career in aquaculture in 1993, whenshe joined the U.S. Peace Corps program in Hondurasas a freshwater fish culture extension worker. In 1996,she was selected as a Knauss Sea Grant Marine PolicyFellow in the National Sea Grant Office in the NationalOceanic and Atmospheric Administration (NOAA),where she assisted in the development of the NOAAand Department of Commerce aquaculture policies.She received her B.A. in physics from the University ofVirginia in 1990 and her M.S. in oceanography fromthe Florida Institute of Technology in 1992.

Robert J. Rippingale [9*] 15 Stuart Crescent,Lesmurdie, Western Australia 6076, Australia.

Robert J. Rippingale retired from Curtin Universityof Technology in Perth, Western Australia, where hewas a senior teaching fellow. Dr. Rippingale taughtaquatic ecology and focused research activity on estu-arine ecosystems. Under a grant from the AustralianFisheries Research and Development Corporation, hisparticular interest in estuarine copepods led to workwith copepods in cultivation and eventually linkingcopepod production to providing food for larval fish.

He received his Ph.D. from the University of West-ern Australia and has authored numerous articles inAquaculture and the Australian Journal of Marine andFreshwater Research. In 2001, he and coauthorMichael F. Payne published a guide to procedures forcultivating Gladioferens imparipes, a calanoid copepodfrom the Swan River Estuary in Australia.

Robin J. Shields [16*] School of Biological Sciences,University of Wales Swansea, Singleton Park, SwanseaSA2 8PP, UK.

Robin J. Shields is director of research for theAquaculture Wales sustainable development group, lo-cated at the University of Wales, Swansea, UK. He waspreviously finfish program manager at the OceanicInstitute in Hawaii. Dr. Shields has been actively re-searching the biology and dietary requirements of lar-val marine fish for more than 10 years, since obtaininghis Ph.D. from the University of Wales Bangor. This re-search is directed at developing reliable productiontechniques for juvenile marine fish, ranging from tem-perate to tropical species. He is a technical reviewer forseveral scientific journals and has acted as an advisor toorganizations, including the U.S. Center for Tropicaland Subtropical Aquaculture and the U.S. NationalMarine Fisheries Service.

Huei-Meei Su [14*] Fisheries Research Institute,Council of Agriculture R.O.C., Tungkang, Pingtung,92845 Taiwan.

Huei-Meei Su is a senior researcher at the TungkangMarine Laboratory, Fisheries Research Institute, Coun-cil of Agriculture, Republic of China. Dr. Su hasworked on live food culture and utilization for marinelarviculture since 1978 and since 1985 on harmful mi-croalgae occurring in aquaculture ponds. She receivedher B.S. in botany at National Taiwan University in1972 and has advanced degrees from the Institute ofOceanography (M.S. and Ph.D.). Dr. Su maintains aculture collection of microalgae and rotifers and hasprovided organisms as starting cultures to farmers andresearchers in Taiwan, and has also served to extendeducation for farmers.

Mao-Sen Su [14] Tungkang Marine Laboratory,Fisheries Research Institute, Council of AgricultureR.O.C., Tungkang, Pingtung, 92845 Taiwan.

Gede S. Sumiarsa [12] Department of Fisheries andAllied Aquacultures, Auburn University, Auburn, Ala-bama 36849, USA.

Larren Tang [16] The Oceanic Institute, 41-202 Kala-nianaole Hwy., Waimanalo, Hawaii 96795, USA.

Joebert D. Toledo [13*] Aquaculture Department,Southeast Asian Fisheries Development Center, Tig-bauan, Iloilo 5021, Philippines.

Joebert D. Toledo is a scientist at the SoutheastAsian Fisheries Development Center (SEAFDEC)Aquaculture Department (AQD) in Iloilo, Philippines.He now heads the grouper research program at ADQ,where he documented the natural spawning of grouperin concrete tanks and floating net cages. His technicalskills significantly contributed in the generation of

x Contributors

technologies for the breeding and seed production ofmilkfish Chanos chanos, sea bass Lates calcarifer, andmangrove red snapper Lutjanus argentimaculatus. Heoften serves as a resource person in local and interna-tional training courses and in-situ seminars and is aconsultant for several fish hatcheries.

Dr. Toledo received his B.Sc. in marine fisheries atthe University of the Philippines in 1980 and his M.Sc.from Hiroshima University in 1990. As a RonpakuFellow, he earned his Ph.D. from Hiroshima Universityin 2002.

Ralph G. Turingan [10*] Department of BiologicalSciences, Florida Institute of Technology, 150 WestUniversity Blvd., Melbourne, Florida 32901, USA.

Ralph G. Turingan is an associate professor of bio-logical sciences at Florida Institute of Technology inMelbourne, Florida. He has taught fish biology and fin-fish aquaculture. His research centers on the ecologyand evolution of functional design in fishes. He re-cently applied his field of research into aquaculture. Heis now conducting research on the development offeeding functional morphology and prey-capture per-formance in hatchery-reared fish larvae. The centralhypothesis of his research is that the development of

the larva’s prey-capture mechanism influences its abil-ity to capture prey. Understanding how the feedingmechanism constrains the type of prey a fish larva isable to capture and ingest underlies the ability of hatch-ery managers to select the optimal prey for a given on-togenetic stage of fish larva.

Shin-ichi Uye [7*] Graduate School of BiosphereSciences, Hiroshima University, 4-4 Kagamiyama 1Chome, Higashi-Hiroshima 739-8528, Japan. suye@hiroshima-u.ac.jp

Shin-ichi Uye is a Professor of Biological Ocean-ography at Hiroshima University. Dr. Uye has workedon zooplankton production ecology in coastal waters ofJapan, mainly in the Inland Sea of Japan (Seto InlandSea). His research field has expanded from the copepodautoecology to trophodynamics of the plankton com-munity in relation to anthropogenic environmental im-pacts. One of his current interests is jellyfish bloomsand their impact on fisheries.

He received his D.Agr. from Tohoku University in1981. He is currently the president of the PlanktonSociety of Japan, and vice-president of the World Asso-ciation of Copepodologists.

Contributors xi

Rotifers and brine shrimp nauplii are the twocommon live-food organisms for early life stagesof marine finfish in hatcheries. Copepods provideadditional desirable characteristics such as sizeand nutritional value to finfish larvae and, untilnow, have played a supplemental role in larvalrearing. Nauplii of some copepod species havebeen used to successfully raise fish species thatcannot use rotifers as a first feed. Copepodids ofsome species provide greater amounts of thehighly unsaturated fatty acids eicosapentaenoicacid and docosahexaenoic acid than rotifers orbrine shrimp. Unless, however, mass-productiontechnology for copepods is established, copepodswill not be a major live-feed organism for finfishlarvae in hatcheries. Limited information aboutcopepod culture is available, but it is generallyfound from among diverse sources.

A workshop, Culture of Copepods andApplications to Marine Finfish Larval Rearing,was held in Honolulu, Hawaii, from May 5 to 8,2003, to gather information about the culture ofcopepods in hatcheries and basic biological stud-ies on various types of copepods with a potentialapplication to larval rearing in hatcheries.Funding for the workshop was provided under agrant from the National Oceanic and AtmosphericAdministration (NOAA; grant no. NA07RG0579,Amendment 2) to The Oceanic Institute (OI),

Hawaii. With the assistance of Dr. Nancy H.Marcus, copepod biologists and scientists work-ing on copepod culture were invited to presenttheir work and discuss key issues at the work-shop. Copepod biologists and ecologists coveredthe production of resting eggs, reproductive per-formance, behavior, and natural productivity ofvarious types of copepods, and identified poten-tial species for aquaculture purposes. Scientistsspecializing in live feed and early life stages ofmarine finfish presented their studies on feedingbehavior, culturing copepods, and applying cope-pods to larviculture. Ample time was allowed forinformation exchange and discussion among theparticipants from a diversity of disciplines repre-sented at the meeting.

This book is a peer-reviewed collection of pa-pers that were presented at the workshop, with afinal chapter summarizing the discussions thattook place. It is hoped that these studies and theknowledge gained thereby will help advance thetechniques for culturing copepods and applyingthem to larviculture. The opinions expressed inthese papers reflect those of the authors and notnecessarily those of NOAA or OI. Warmestthanks are extended to the contributors and re-viewers for the quality of the papers and the valu-able information presented.

xiii

PrefaceCheng-Sheng Lee

Copepods in Aquaculture

.

ABSTRACTA major bottleneck in the cultivation of many ma-rine fish species for commercial purposes is thelack of a suitable food for the first-first-feedinglarval stages. The standard feeds, Artemia and ro-tifers (Brachionus spp.), are not always effectivefoods. This could be due to several factors, in-cluding size, biochemical composition, andswimming behavior. Studies of wild-caught fishlarvae report that calanoid copepod nauplii are animportant item in the diet of various fish species.Hence, establishing a protocol for the large-scalecultivation of calanoid copepods would appear tobe a fruitful direction for further research. Indeed,a few studies have already been conducted, de-monstrating the suitability of species of Acartiaand Gladioferens for raising some fish species. Inview of the fact that people are trying to cultivatea fairly wide variety of fish species, e.g., small or-namentals to large consumables, from estuarineto open ocean habitats, a single calanoid species,however, may not solve the needs of the aquacul-ture industry. This paper discusses several of thecalanoid copepods, especially in terms of the phe-nomenon of dormancy, and makes suggestions asto species that hold promise for meeting the needsof aquaculture. Promising species are found ingenera including Centropages, Labidocera, Acar-tia, and Eurytemora.

INTRODUCTIONDuring the last decade a number of excellent re-views and manuals addressing the use of copepodsas a live feed by aquaculturists have been pro-duced. These include Manual on the Productionand Use of Live Food for Aquaculture (Lavens and

Sorgeloos 1996), Intensive Cultivation of a Cala-noid Copepod for Live Food in Fish Culture (Rip-pingale and Payne 2001), and Live Feeds inMarine Aquaculture (Støttrup and McEvoy 2003).Nevertheless copepods are still not being usedroutinely by the aquaculture industry.

Of the ten orders of copepods, the Calanoida,Harpacticoida, Cyclopoida, and Mormonilloidahave pelagic representatives in marine systems. Ofthese four taxa, the calanoid copepods are particu-larly abundant members of the pelagic realm in es-tuaries and other coastal habitats and generallyrepresent an important link between the phyto-plankton and fish in these inshore nursery sys-tems. Largely for these reasons, the calanoid cope-pods have received considerable attention byresearchers (see review by Mauchline 1998). Mostspecies are approximately 1.0 mm in total lengthwith some being as small as 0.4 mm and some aslarge as 10.0 mm (see Fig. 3 in Mauchline 1998).Based on citations in the scientific literature,Acartia clausi and Calanus finmarchicus are themost widely studied species (Mauchline 1998),followed by Temora longicornis, Paracalanusparvus, Calanus helgolandicus, Pseudocalanuselongatus, Acartia tonsa, Centropages hamatus,Centropages typicus, and Temora stylifera.

Since most of this basic research on thecalanoid copepods has been limited to only a fewspecies, it is difficult to make informed choicesabout the best candidates for aquaculture because,as noted by Støttrup (2000), “A basic knowledgeof physiological processes and population dy-namics of a species is a prerequisite for the devel-opment of rearing techniques.” Calanoid cope-pods are consumed by many larval fish in the wild(e.g., Pepin and Penney 1997) and some species

3

1Calanoid Copepods,

Resting Eggs, and Aquaculture

Nancy H. Marcus

(e.g., A. tonsa, C. hamatus, Eurytemora affinis,and Gladioferens imparipes), have already beenused for the cultivation of fish (see review byStøttrup and McEvoy 2003). A feature that thisauthor believes makes most of these species par-ticularly good candidates for finfish aquacultureis that they undergo a dormant phase during theirlife cycle.

DORMANCYDormancy or suppressed development is a char-acteristic feature of the life cycle of many cope-pod species (Grice and Marcus 1981; Dahms1995; Williams-Howze 1997). Depending on thespecies, the suppression of development mayoccur at the embryonic, naupliar, copepodid, oradult phase. Dormancy has been described as rep-resenting a spectrum of suppressed development,ranging from quiescence (retarded) to diapause(arrested) (see Grice and Marcus 1981). Quie-scence occurs as an immediate response to ad-verse conditions in the environment; diapausetypically occurs in response to a cue (e.g., pho-toperiod) that occurs prior to the onset of deterio-rating environmental conditions (Grice andMarcus 1981). Generally, quiescent individualsresume development as soon as the immediate en-vironmental conditions improve. For example,the development rate of many copepods, espe-cially warm-water species, is slowed when theyare exposed to cold temperatures, but speeds upwhen temperatures increase again. In the case ofdiapause, an organism generally undergoes bio-chemical, physiological, and/or endocrinalchanges and enters diapause itself or produces di-apause eggs (see Grice and Marcus 1981). In dia-pausing individuals development resumes onlyafter completion of a refractory phase that mightlast days to months, depending on the species(Marcus 1979; Grice and Marcus 1981). Duringthe refractory phase individuals do not resume de-velopment even if conditions are favorable(Watson and Smallman 1971). The distinctionbetween quiescence and diapause is important be-cause for at least one copepod species, C. hama-tus, diapause eggs are capable of long-term sur-vival (several months) even when exposed totoxic chemicals such as hydrogen sulfide (Marcusand Lutz 1998).

The existence of arrested development in cope-

pods has been recognized for decades. The dor-mant CV copepodid stage of Calanus spp. at-tracted the attention of researchers early on in the20th century, but the factors controlling this de-velopmental delay still remain elusive (seeMarcus and Boero 1998). While dormancy duringthe CV stage has been noted for several open-ocean copepod genera, dormancy during the egg(embryonic) phase is characteristic of manycoastal and estuarine copepod species (Grice andMarcus 1981). In fact, most of the copepodspecies mentioned previously in the context ofrearing larval fish are also known to exist as dor-mant/resting eggs in the environment. Indeed, thelist of copepod species that exist as resting eggs inthe environment has gradually grown over the lastthree decades as more geographic regionsthroughout the world’s oceans have been studied.In the first review of the topic, Grice and Marcus(1981) noted that since the initial observation ofcopepod resting eggs in A. tonsa (Zillioux andGonzalez 1972), 15 more marine calanoid cope-pod species had been shown to have a dormantegg phase as part of the life cycle. More recently,Mauchline (1998) noted the existence of a restingegg phase in 44 calanoid copepod species. Inmany cases this categorization was based on ob-servations of nauplii hatching from samples ofsediments that were collected from the seabedand incubated in the laboratory. While the naupliipresumably came from resting eggs existing inthe sediments, the time at which the eggs wereoriginally spawned and settled to the seabed inthe field was not known. Thus it cannot be con-cluded with certainty that the nauplii developedfrom diapause eggs, quiescent nondiapause eggs,or some other intermediate egg type (Chen andMarcus 1997). A few studies (see review Marcus1996), however, have either raised copepods inthe laboratory and revealed conditions that led tothe production of diapause and nondiapause eggs,collected eggs from wild females and revealeddifferent hatching responses under existing fieldconditions and after incubation in the laboratory,or documented morphological features that canbe used to distinguish diapause and nondiapauseeggs that are found in the field. Labidocera aes-tiva, C. hamatus, A. clausi, A. tonsa, and E. affi-nis are among the calanoid copepod species thathave been studied in this regard (e.g., Marcus1980; Uye 1985; Ban 1992; Chen and Marcus

4 Chapter 1

1997; Castro-Longoria 2001), that is, species forwhich diapause eggs have been clearly shown toexist.

RESTING EGGS ANDAQUACULTUREThe remainder of this chapter will address the rel-evance of copepod resting eggs to aquaculture.Naess (1991a) believed that resting eggs of cope-pods were important for the maintenance of zoo-plankton populations in outdoor “extensive” pondsystems that were used to rear fish. As a result, heexamined the tolerance of copepod eggs to freez-ing, desiccation, and the insecticide rotenone,since the procedures for rearing fish involved an-nual draining of the ponds and exposure torotenone. Rotenone was applied to kill parasitesand predators. Based on the results, Naess con-cluded that subjecting ponds to these treatmentsled to reduced hatching of copepod resting eggsfrom the bottom sediments. At the time of thisstudy the eggs were not identified as to species,but previous work (Naess 1991b) had shown thatA. clausi, E. affinis, and C. hamatus were presentin the ponds. Despite the fact that these treat-ments resulted in reduced egg hatching, Naesssuggested that differences in egg type (quiescentnondiapause versus diapause) and differences inthe tolerance of the various species to environ-mental stressors would eventually lead to domi-nance in the ponds of resistant copepod speciesby directional selection. If the surviving specieswere still suitable for the fish larvae, this evolu-tionary response would be beneficial, because itwould allow treatment of the pond systems withcompounds that reduce harmful parasites andpredators.

In a subsequent study Naess (1996) quantifiedthe abundance of copepod resting eggs in the sed-iments of outdoor marine ponds used for raisingfish. The ponds were located along the coast ofNorway. Five of the ponds were used exclusivelyfor raising zooplankton. The bottoms of thesesystems were described as muddy, anoxic, andrich in sulfide. The other two ponds had moresandy, oxygenated seabeds and were used forraising cod (Gadus morhua). Copepod eggs werefound in the seabed of all ponds, but the densitiesvaried both within and between ponds. Densitiesranging from 104 to 106 eggs/m2 were found.

Eggs of E. affinis, Acartia teclae, A. clausi, and T.longicornis were all found at densities exceeding104 eggs/m2. Centropages hamatus was repre-sented in the form of resting eggs, but the densi-ties were generally less than 103 eggs/m2. Thehatching success of the eggs varied. Naess sug-gested that one reason for reduced egg abundancewas the presence of predators in the sediments.Hence, in ponds that were subjected to rotenone,densities were higher because presumably thebenthic predators had been reduced. This sugges-tion is supported by more recent studies that indi-cate benthic amphipods can have a negative im-pact on the survival of benthic resting eggs ofplanktonic copepods (Albertsson and Leondard-sson 2001). More recently, Engell-Sørensen et al.(2004) reported on the rearing of flounder(Platichthys flesus) juveniles in semiextensivesystems associated with a Danish fjord. Some ofthe copepods used for food were derived fromresting eggs obtained from the bottom of rearingponds.

In the book, Manual on the Production andUse of Live Feeds for Aquaculture, Lavens andSorgeloos (1996) suggested that copepod restingeggs could be used as an inoculum to initiate cul-tures of copepods. The suggestion is reasonable,but this author believes that the value of copepodresting eggs to aquaculture extends beyond theiruse as an inoculum. Since it is possible to storecopepod resting eggs for days to years and obtainnewly hatched nauplii, the risk of having no nau-plii available to feed to fish larvae can be reduced.A second advantage is that since the restingstages of some taxa are resistant to surface disin-fection agents commonly used in aquaculture, itmay be possible to develop protocols that reducethe risk of parasitic contamination of larval fishcultures when nauplii derived from copepod rest-ing eggs are used as a source of food.

VIABILITY OF RESTING EGGS

Research to date has shown that survival of cope-pod eggs with storage varies with egg type (non-diapause or diapause), species, and conditions ofstorage. When held under conditions that preventhatching, nondiapause eggs of copepods gener-ally do not remain viable for as long as diapauseeggs. For example, Marcus et al. (1997) collectedeggs from wild-caught females of C. hamatus, A.tonsa, and L. aestiva. Control eggs were incu-

Calanoid Copepods, Resting Eggs, and Aquaculture 5

bated under conditions suitable for development,and within a few days, hatching was generally84% or greater, indicating that the eggs were non-diapause. Experimental eggs were stored undersulfidic and/or anoxic (283–352 μM) conditionsto prevent hatching. When the experimental eggswere returned to normoxic and nonsulfidic condi-tions within 7 days, viability (as evidenced byhatching) ranged between 50% and 80% (depend-ing on the species). Longer exposures to sulfideand/or anoxia (e.g., 4 weeks) resulted in furtherreductions in hatching for all species. In a subse-quent study, Marcus and Lutz (1998) collectedeggs from wild-caught females of C. hamatus at atime of the year when the species was producingmostly diapause eggs (Chen and Marcus 1997).The eggs were incubated in the laboratory for 5days to allow nondiapause eggs to hatch. The re-maining unhatched presumptive diapause eggswere exposed to sulfide and/or anoxia as great as1,363 μM. These eggs survived 20 weeks of suchexposure with little or no reduction in viability, asshown by the hatch of more than 80% of the eggswhen they were returned to normoxic and nonsul-fidic conditions. In fact, some eggs survived ex-posure to anoxia for 437 days (Marcus and Lutz1998). Studies of E. affinis also indicate that dia-pause eggs survive storage longer than nondia-pause eggs (Ban and Minoda 1992). Generally,studies such as these are designed to gain insightinto how long copepod resting eggs survive in thefield. Hence, they do not typically consider stor-age conditions that may not have any relevance tosurvival of eggs in the field but that might extendshelf life in the laboratory and thus be of great im-portance to aquaculturists. There is a need forstudies of the combined effects of anoxia and coldon shelf life. For example, nondiapause eggs ofAcartia steuri and A. clausi that were covered bymuddy sediments survived 100 and 165 days, re-spectively, when stored at 5°C, but only 75 and 70days at 20°C (Uye 1980). Similar results were re-ported by Ban and Minoda (1992) for eggs of E.affinis stored in sediments at 15°C and 4°C. It islikely that dissolved oxygen concentrations werereduced in the sediments.

While the focus of this chapter is on restingeggs, it is important to note a study by Payne andRippingale (2001). They stored nauplii of G. im-paripes in static containers of seawater at 8°C.The nauplii were not fed. When these nauplii

were held under these conditions for up to 12days and then transferred to 20°C and fed, sur-vival was excellent (> 99%), but for nauplii heldlonger, survival declined. Payne and Rippingale(2001) emphasized the minimal maintenance ef-fort required for storing the nauplii in this wayand hence the advantage this afforded in holdingnauplii to feed fish larvae when needed. The au-thors did not indicate whether or not the naupliideveloped while held at 8°C, but it is unlikely,since they were not fed.

NAUPLII FROM RESTING EGGS

If copepod eggs (and other stages as well) are tobe stored for later use as a food source, not onlymust they survive storage, but also the nutritionalquality of the nauplii that hatch from the eggsmust be maintained. Highly unsaturated fattyacids (HUFAs), especially docosahexaenoic acid(DHA) and eicosapentaenoic acid (EPA), are es-sential components of the diet of fish (Sargent etal. 1997). Støttrup et al. (1999) and Støttrup andMcEvoy (2003) reported that copepod nauplii areexcellent sources of these essential fatty acids, un-like Artemia and rotifers (Brachionus spp.), whichmust be treated with fatty acid supplements toachieve a good nutritional status for feeding fishlarvae. Moreover, Støttrup et al. (1999) showedthat there was no change in the EPA content of A.tonsa eggs that were stored for 12 weeks at 4°C,but the amount of DHA decreased.

RESISTANCE OF RESTING STAGES TO

SURFACE DISINFECTION AGENTS

A problem associated with using live feeds inaquaculture is the introduction of pathogens andother contaminants into larval fish cultures in as-sociation with the food. This risk could be mini-mized with copepod resting eggs if surface disin-fection protocols could be developed for theiruse. There is evidence that resting stages of sev-eral taxa, including the eggs of copepods, surviveexposure to surface disinfection agents. Naessand Bergh (1994) subjected resting eggs of A.clausi and E. affinis to three disinfection agents(glutaraldehyde, FAM-30, or Buffodine). Eggswere washed with 0.2 μm filtered sterile seawaterand monitored for hatching and survival.Bacterial growth was reduced on the eggs follow-ing treatment, but hatching and survival differedamong the treatments. Lavens and Sorgeloos

6 Chapter 1

(1996) suggested that nondiapause eggs may ex-perience higher mortality than diapause eggs fol-lowing such treatment because the outer coveringof nondiapause eggs is thinner than the coveringof diapause eggs (Ianora and Santella 1991;Fanelli et al. 1992). Other disinfection agentshave been tested with resting stages of other taxa.Douillet (1998) reported that sodium hypochlo-rite was an effective disinfection agent for rotifercysts. When cysts were exposed for 3 minutes to0.5% sodium hypochlorite, hatching remainedhigh, and in 97% of the trials the resulting popu-lations were bacteria free. Higher concentrationsreduced viability. Similarly Pati and Belmonte(2003) exposed cysts of Artemia, a rotifer, and aciliate to five disinfectant agents commonly usedin aquaculture. They reported that some cystswere able to survive commonly used dosage lev-els of these chemicals. While the goal of the studyhad been to determine levels of the disinfectantsthat would successfully kill cysts because theycould be the source of contamination in aquacul-ture tanks, the fact that some survived suggeststhat certain concentrations might be effective forsurface disinfection of live feeds that are derivedfrom resting cysts or eggs.

One of the limitations of using the diapauseeggs of some species (e.g., L. aestiva and C.hamatus) for aquaculture is that based on re-search to date, they must be stored for a lengthyperiod (weeks to months) before they can be in-duced to hatch, and eggs stored for a very longterm (years) require more time to hatch whenplaced under conditions suitable for hatching(personal observation). For example diapauseeggs of L. aestiva must be stored at 5°C for ap-proximately 10–30 days before they can be in-duced to hatch at 19°C (Marcus 1987). Con-versely, diapause eggs of C. hamatus must bestored for approximately 4 months at 25°C beforethey can be induced to hatch at temperatures lessthan 20°C (personal observation). In addition di-apause eggs of C. hamatus that have been storedfor approximately 4 months at 25°C normallyhatch within 5–7 days when placed at 15°C.Diapause eggs that were stored for 22 months at25°C, however, required 10 days of subsequentincubation at 15°C to hatch (personal observa-tion). Research that elaborates the hatching re-sponse of dormant eggs under various conditionsis needed so that better control of the process is

achieved. For example Jo and Marcus (2004) sub-jected diapause eggs of C. hamatus to differenttemperature regimens following storage underanoxic conditions at 25°C for approximately 8months. They found that for eggs stored for thisperiod of time, the time to hatching could beshortened by exposing the eggs to 15°C for only36 hours followed by re-exposure to 25°C. It mayalso be possible to reduce the time to hatching byexposing eggs to certain chemical conditions. Forexample, normally dehydration breaks the dia-pause of Artemia cysts, and cysts must be rehy-drated for development to resume, followed byhatching. Clegg and Jackson (1998), however,showed that diapause could be broken in Artemiacysts, which were prevented from dehydrating, byexposing them to ammonium chloride.

CONCLUSIONClearly, the production of large numbers of cope-pod resting eggs, especially diapause eggs, couldfacilitate the availability of copepod nauplii foraquaculture. Sufficient information on the culti-vation requirements of several copepod species(e.g., L. aestiva, C. hamatus, Acartia spp., and E.affinis) that produce diapause eggs or other eggtypes capable of surviving lengthy periods of sup-pressed development exists to support continuedexploration of this approach. Labidocera aestivais a temperate warm-water free-spawning species.Diapause and nondiapause egg production areprimarily controlled by photoperiod and tempera-ture (Marcus 1980). Both egg types are smooth.Generally the diapause eggs must be stored for atleast 1 month at 5°C before they can be inducedto hatch by placing them at 25°C (Marcus 1987).Diapause eggs survive storage at 5°C for severalmonths. Centropages hamatus is a temperatecold-water free-spawning species. Diapause andnondiapause egg production are controlled byphotoperiod and temperature (Marcus and Mur-ray 2001). Both egg types are spiny, which can besomewhat of a problem, because the spines pro-vide a surface to which debris readily clings (per-sonal observation). Diapause eggs must be storedat 25°C for approximately 4 months before plac-ing the eggs at temperatures less than 20°C in-duces hatching (Chen and Marcus 1997). Dia-pause eggs of C. hamatus can survive as long as22 months when held at 25°C (Marcus and

Calanoid Copepods, Resting Eggs, and Aquaculture 7

Murray 2001). Eurytemora affinis is a widespreadspecies that occurs in brackish environments.Photoperiod plays a role in the type of eggs pro-duced, as does population density (Ban 1992).Females carry eggs in sacs, but sacs with diapauseeggs are released in the water column and sink tothe seabed. Diapause eggs of E. affinis survivestorage for as long as 20 months (Ban unpub-lished, as cited in Ban and Minoda 1992). SinceE. affinis tolerates reduced salinity, and somepopulations occur in freshwater lakes (Ban andMinoda 1991), it may be possible to maintain cul-tures free of marine parasites by rearing animalsin freshwater. Acartia spp. spawn eggs freely intothe water column, and several species produce di-apause eggs (see Marcus 1996; Castro-Longoriaand Williams 1999). A. clausi and A. bifilosa aretemperate cold-water species; A. tonsa, Acartiacaliforniensis, and Acartia latisetosa are temper-ate warm-water species. Diapause egg productionby A. clausi is controlled by photoperiod (Uye1985); the factors controlling diapause egg pro-duction by A. tonsa are not well understood (seeWilliam-Howze 1997). Nevertheless A. clausiand A. tonsa have been cultivated extensively andin some cases for feeding larval fish. Nondia-pause eggs of species of Acartia are amenable tosome manipulation aimed at suppressing develop-ment. For example, eggs stored in the cold remainviable for several weeks (Støttrup et al. 1999).

In conclusion, dormancy during the egg (em-bryonic) phase is a feature that potentially makescertain copepod species particularly suitable foraquaculture. If eggs can be stored for later use,then it is no longer necessary to maintain simulta-neous cultures of copepods and fish larvae.Moreover, having a stock of eggs facilitates re-covery from population crashes that may occurduring cultivation phases. Hence, research thatdetermines the most effective storage conditionsto prolong survival and maintain nutritional qual-ity is needed. In addition, dormant eggs may lendthemselves more to disinfection than later stages,thus reducing the chances for contamination offish cultures.

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Zillioux, E. and Gonzalez, J. 1972. Egg dormancy inthe neretic calanoid copepod and its implications tooverwintering in boreal waters. Archives ofLimnology. European Marine Biology Symposium5:217–230.

Calanoid Copepods, Resting Eggs, and Aquaculture 9

ABSTRACTHarpacticoid copepods are a promising alterna-tive food resource for larval and juvenile fishesthat require live feeds in mariculture. Harpac-ticoids, fed alone or as a supplement, have beenshown to promote faster growth than rotifers,brine shrimp, or both together. Research suggeststhat this is most likely related to small body sizefrom first nauplius to adult, which favors inges-tion by gape-limited fishes, and the observationthat harpacticoids are naturally rich in essentialfatty acids needed for marine fish growth. In ad-dition, cultures of harpacticoids have the poten-tial to be productive enough to be of value in mar-iculture because harpacticoids (a) have highreproductive output and short generation times;(b) are able to withstand variable conditions oftemperature, salinity, and waste accumulation;and (c) have flexible food requirements regardingnutritional content and particle shape and sizethat facilitate the use of formulated feeds, mono-specific algae, or both. As a result harpacticoiddensities may exceed 100,000 individuals perliter in mass cultures. Finally, harpacticoid biodi-versity is high both within and among habitats,and there are many species available for massculture. Characteristics associated with a prima-rily benthic lifestyle, however, render manyharpacticoid species inappropriate for large-scalemariculture applications. Most have been cul-tured only in the presence of substrates, compli-cating the harvest of early developmental stagesbecause few harpacticoid nauplii swim. Further-more very few species encyst or undergo dia-

pause, requiring that high yields from mass cul-tures be synchronized with the birth or arrival oflarval fish. Escape responses and behavioral at-tributes have been little studied. Many adult andjuvenile harpacticoids are relatively poor swim-mers, suggesting a reduced escape response. Al-ternatively, harpacticoids are commonly pro-tected by long, heavy caudal spines that may aidresistance to ingestion. Currently, the search con-tinues for harpacticoids with favorable featuresfor mass culture that are also high-quality foodfor mariculture applications. Additional specieswith a small body size, good swimming abilitiesin all developmental stages, and ability to be cul-tured in the absence of substrate are needed.Species in the genera Tisbe and Nitokra displaymany of these features and should be the focus ofadditional research to determine their utility infish mariculture.

INTRODUCTIONMany marine fishes produce small pelagic eggs.Larvae hatched from small eggs require a sourceof live food very soon after the onset of exoge-nous feeding. Larvae of these species raised inmariculture are usually fed the rotifer Brachionusplicatilis followed by the brine shrimp Artemia.The early feeding regime in the rearing processoften limits survival and growth, and thus, thesuccess and/or cost of fish production. Severallines of reasoning suggest that harpacticoid cope-pods have potential as an alternative food re-source in larval fish mariculture that might re-

11

2The Potential to Mass-Culture

Harpacticoid Copepods for Use as Food for Larval Fish

John W. Fleeger

place or supplement rotifers, brine shrimp, orboth. The purpose of this review is to summarizecurrent understanding of this potential. Mass-culture techniques and population growth charac-teristics of harpacticoid species known to havepotential for mariculture applications are consid-ered below. To complement the recent review ofCutts (2002), features of the biology of harpacti-coids that may be construed as positive and nega-tive regarding their use in larviculture are dis-cussed, and suggestions for future avenues ofresearch to foster harpacticoid use in larvicultureare highlighted.

There are over 3,000 described species in theOrder Harpacticoida, one of 10 orders in the sub-class Copepoda of the crustacean class Maxillo-poda (Huys and Boxshall 1991). The number ofrecently confirmed cryptic species (e.g., Rocha-Olivares et al. 2001) and the high number of rela-tively poorly explored habitats (e.g., tropicalmangrove forests, the deep sea) suggest that thetrue number of harpacticoids worldwide is muchhigher.

Adult harpacticoid copepods are typicallysmall in body length (~1 mm) and width (~350μm) as well as mass (~3 μg dry mass). Mostharpacticoids are free living, although ectopara-sitic and commensal harpacticoids are commonon a variety of animals. For example, manyspecies inhabit the protected gill chambers of de-capod crustaceans. About 1,000 harpacticoidspecies live exclusively in freshwater habitats.

The diversity of habitats and lifestyles ex-ploited by marine harpacticoids is great. Althougha few harpacticoids are planktonic, the majorityare benthic. Harpacticoids are especially abun-dant in muddy sediments and on foliose macroal-gae (Hicks and Coull 1983). Harpacticoids aretypically the second most abundant taxon, afternematodes, in the sedimentary meiobenthos.Many species are associated with the epiphytes ofseagrasses and macrophytes (Hall and Bell 1988;Rutledge and Fleeger 1993). They live intersti-tially in sand, in mud as burrowers and tubedwellers (Chandler and Fleeger 1984), and onsurfaces such as at the sediment–water interfaceor on microalgal-covered hard substrates (Dano-varo and Fraschetti 2002; Atilla et al. 2003).Although many harpacticoids are passivelyeroded into the water column when currents ex-ceed some critical erosion velocity (Palmer

1988), some species actively emerge from thesediment by swimming (Armonies 1988; Waltersand Bell 1994; Suderman and Thistle 1998;Thistle 2003), potentially increasing encounterrates with predators (Gregg and Fleeger 1997).

Harpacticoids are natural prey to many larvaland juvenile fishes (Gee 1989; Coull 1990). Com-parisons between the faunal compositions foundin fish gut contents with samples from the envi-ronment suggest that juvenile fishes may activelyselect harpacticoids (Feller and Coull 1995).Strong selection may be related to the shallowdepth profile of many harpacticoids in sediments(Fleeger et al. 1995a) and the higher frequency ofemergence by harpacticoids compared with othermeiofaunal groups (McCall and Fleeger 1995).Mean abundances of harpacticoids in fish gutcontents often exceed 1,000 harpacticoids per ju-venile or adult fish (McCall and Fleeger 1993).

Harpacticoid copepods are an important foodresource for many species of marine fish (Coull1990). Many fishes undergo an ontogenetic shiftfrom a diet comprised largely of harpacticoids tolarger-bodied prey when they reach a standardlength of about 35 mm (McCall and Fleeger1995). Harpacticoid copepods, however, mayserve as prey for the entire life of some marinefishes (Tipton and Bell 1988; Toepfer and Fleeger1995) or until a larger size is reached (McCall andFleeger 1993; Feller and Coull 1995). Species offlatfish, gobies, salmonids, sciaenids, and blen-nies are sometimes considered to be obligatoryharpacticoid feeders, at least for a portion of theirlives (Coull 1990; McCall and Fleeger 1995).

HARPACTICOID COPEPODS ANDTHE GROWTH OF FISHAs has been shown for other copepods, harpacti-coids may promote rapid growth, a high repro-ductive rate, or both, in fishes and invertebrates(Cutts 2002). Volk et al. (1984) showed that foodconversion efficiency for juvenile Oncorhynchusketa fed the harpacticoid Tigriopus californicuswas higher than when fed calanoid copepods oramphipods. They attributed differences to thehigher caloric content of T. californicus comparedwith amphipods and a poor escape response com-pared with calanoid copepods. Kreeger et al.(1991) examined the nutritional value of newlyhatched Artemia, Artemia supplemented with

12 Chapter 2

lipid microspheres, and the harpacticoid copepodT. californicus. They found that mysid shrimpsurvival, growth, and the proportion of femalesbrooding offspring were not significantly differ-ent among dietary treatment groups, but that thenumber of viable offspring produced by mysidswas improved by adding lipid microspheres andT. californicus to a diet of Artemia. They attrib-uted the difference to the observation that bothlipid microspheres and T. californicus are richsources of essential fatty acids. Støttrup andNorsker (1997) found that larval turbot (Scoph-thalmus maximus) growth and survival when fedrotifers were enhanced when the harpacticoidTisbe holothuriae was provided with rotifers.Including T. holothuriae in a daily dietary regimeimproved appetite, growth rate, and pigmentationof Dover sole (Microstomus pacificus) comparedwith a diet of Artemia alone (Heath and Moore1997).

FAVORABLE LIVE FEEDCHARACTERISTICS OFHARPACTICOIDSAn increased growth rate when harpacticoids areincluded in the diet of larval fishes may be attrib-uted to many factors, including nutritional qual-ity, favorable body size, and effects on larval ap-petite. Harpacticoids have superior nutritionalqualities compared with brine shrimp and rotifersthat may stimulate fish growth (Cutts 2002).Highly unsaturated fatty acids (HUFA), espe-cially n-3 fatty acids, are typically represented inharpacticoids in high concentrations, comparedwith other live feeds used in mariculture. Table2.1 shows values of selected phospholipid fattyacids (PLFAs) from Amphiascoides atopus grownin a laboratory mass culture (the system describedby Sun and Fleeger 1995) compared with 1-dayold Artemia nauplii (analysis conducted by

Microbial Insights, Inc.). Note that A. atopus isrich in the fatty acids, 22:6n3 and 22:5n3, essen-tial to marine fish growth, while these fatty acidsare absent in Artemia. Sun and Fleeger (1995) re-port that 28% of all phospholipids in A. atopusare essential n-3 fatty acids. Other authors havefound similarly high amounts of n-3 fatty acids inharpacticoids (Watanabe et al. 1983; Norsker andStøttrup 1994; Nanton and Castell 1999, Rhodesand Boyd, this volume). Compared with brineshrimp and rotifers, harpacticoids appear to haveuniformly favorable ratios and amounts of essen-tial fish oils.

At least some harpacticoids are able to synthe-size longer-chained HUFA (Norsker and Støttrup1994), thereby increasing levels of n-3 fatty acidsthat are essential to fishes. Nanton and Castell(1998) suggest that harpacticoids have enzymes(i.e., �-5, �-6 desaturase and elongase) that arenecessary for the conversion of shorter chain n-3polyunsaturated fatty acids to the longer-chainedessential fatty acids docosahexaenoic acid (DHA)and eicosapentaenoic acid (EPA). In the absenceof this enzymatic capability, animals may requirea diet that matches their DHA and EPA require-ments. Because harpacticoids can synthesize n-3HUFA, feeds without enriched levels of marinefish oil can be used to produce a favorable DHA/EPA composition. Thus, harpacticoids may havefew specific nutritional needs in culture and maybe able to synthesize long-chained HUFAs re-gardless of their diet (e.g., harpacticoids show fa-vorable nutritional qualities when fed algal mono-cultures and formulated feeds). Fukusho et al.(1980), however, reported that harpacticoids fedn-yeast resulted in increased survival and growthof the larvae of mud dab (Limanda yokohamae)compared with harpacticoids fed baker’s yeast. Inaddition, Cutts (2002) reports that synthesis ofHUFA may be a rate-limiting step for harpacti-coid growth, and dietary supplement of fatty

Potential to Mass-Culture Harpacticoid Copepods as Food for Larval Fish 13

Table 2.1. Selected fatty acids (expressed in pmol) from a phospholipid fatty acid analysisconducted on Amphiascoides atopus and day-old Artemia

Dry wt/gSpecies tissue 18:4n3 20:5n3 20:4n3 20:2n3 22:6n3 22:5n3

A. atopus 1.41 0.25 6.3 0.05 0.08 10.8 0.95Artemia 1.92 7.9 4.0 0.07 0.02 0 0

Note: Values represent means of two replicate analyses.