Live Feeds in Marine Aquaculture

Live Feeds in Marine AquacultureLive Feeds in Marine Aquaculture Live Feeds in Marine Aquaculture Edited by Josianne G. Støttrup, PhD Danish Institute for Fisheries Research, Charlottenlund, Denmark and Lesley A. McEvoy, PhD North Atlantic Fisheries College, Shetland Isles, UK © 2003 by Blackwell Science Ltd, a Blackwell Publishing Company Editorial Offices: 9600 Garsington Road, Oxford OX4 2DQ, UK Tel: 44 (0)1865 776868 Blackwell Publishing, Inc., 350 Main Street, Malden, MA 02148-5018, USA Tel: 1 781 388 8250 Iowa State Press, a Blackwell Publishing Company, 2121 State Avenue, Ames, Iowa 50014-8300, USA Tel: 1 515 292 0140 Blackwell Publishing Asia Pty Ltd, 550 Swanston Street, Carlton South, Victoria 3053, Australia Tel: 61 (0)3 9347 0300 Blackwell Wissenschafts Verlag, Kurfürstendamm 57, 10707 Berlin, Germany Tel: 49 (0)30 32 79 060 The right of the Author to be identified as the Author of this Work has been asserted in accordance with the Copyright, Designs and Patents Act 1988. All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, recording or otherwise, except as permitted by the UK Copyright, Designs and Patents Act 1988, without the prior permission of the publisher. First published 2003 by Blackwell Science Ltd Library of Congress Cataloging-in-Publication Data is available ISBN 0-632-05495-6 A catalogue record for this title is available from the British Library Produced and set in Times by Gray Publishing, Tunbridge Wells, Kent Printed and bound in Great Britain by MPG Books, Bodmin, Cornwall For further information on Blackwell Science, visit our website: www.blackwell-science.com 5 Problems and Prospects with Alternatives to Live Feed 1.4 North America 1.1 A Historical Perspective 1.1 General biology 2. Present and Future David A.6 South America.2 Marine Aquaculture Today and in the Future 1.4 Why is Live Feed Necessary? 1.7 References 2 Production and Nutritional Value of Rotifers Esther Lubzens and Odi Zmora 2.3 Europe 1.Contents Foreword Preface Contributors Abbreviations 1 Status of Marine Aquaculture in Relation to Live Prey: Past.3.2.3.6 Conclusions 1. including Central America and the Caribbean 1.2 Digestion xiii xv xvi xviii 1 1 5 7 7 8 9 10 10 11 11 12 13 13 17 17 19 19 21 21 23 23 25 . Bengtson 1.1 Introduction 2.3.2 Biology and Morphological Characteristics of Rotifers 2.5 Oceania 1.3.2.1 Feeding 2.3 Morphology and physiology 2.2.2.2 Asia 1.2.1 Africa 1.2 Taxonomy 2.2.3.3.3 The Status of Larviculture and Live Feed Usage 1.1 The genus Brachionus 2.3.3.2. 3.5.1 Selection of species and/or strain 2.3.3.3 Body fluids and excretion 2.6.2 Ecology and natural distribution 3.6 Diseases Nutritional Quality of Rotifers 2.2 Maintaining water quality in culture tanks 2.3.3 2.3.2 Dry weight and caloric value 2.3.1 Number of rotifers consumed by larvae 2.4.2.5 Nervous system and sensory organs 2.4.2.3.1 Protein and carbohydrate content 2.3.5.2.3 Taxonomy 65 67 67 73 75 .4.3 Ingestion rate 2.1 Organic particles 2.4 Reproduction 2.4.1 Small-scale laboratory cultures 2.7 2.3 Biochemical composition 2.2.1 Asexual and sexual reproduction 2.2.4.6.2.2.3.4 Effects of starvation Preserved Rotifers 2.3.5. Tank Production and Nutritional Value of Artemia Jean Dhont and Gilbert Van Stappen 3.5 Enzyme activity 2.5.2 Lipid composition 2.2.vi Contents 2.5.2 Biology of Artemia 3.3 Choosing the most appropriate culture techniques 2.2.3 Vitamin enrichments 2.2 Cryopreservation 2.2 Mass cultures Advanced Warning on State of Cultures 2.3.4.2.1 Introduction 3.5 2.5.1 Egg ratio 2.5.2.3.2 Bacteria and other organisms in the culture tanks 2.4.3 Resting eggs Future Directions References 26 26 26 27 27 29 31 31 31 32 33 33 34 35 36 43 43 44 44 44 44 44 45 45 46 46 46 47 48 48 49 49 50 50 52 52 65 3 Biology.6 2.6.1 Preservation at low temperatures 2.3.2.4 Movement 2.4.8 2.2 Reproductive rates 2.4 2.3.4.1 Morphology and life cycle 3.3.4 Viscosity 2.3 Sexual reproduction and resting egg formation Culturing Rotifers 2.2 Swimming velocity 2. 4 Growth rate of nauplii 3.9 Production figures of intensive Artemia cultures 3.2 Hatching quality 3.5 Enrichment with prophylactics 3.1 Lipid enrichment 3.5. Biochemical composition 3.3.5.4.Contents vii 3.5.3.7 Control of infections 3.2.4.4.2.3 Production Methods: Tank Production of Artemia Biomass 3.1.2 Phospholipid enrichment 3.2.3 Protein enrichment 3.3.4.6 Culture techniques 3.6 Life-history traits and reproductive capacity 3.1 Advantages of tank production and tank-produced biomass 3.3 Diapause 3.5.4.3.1.3.5.2.5.1 Cysts and decapsulated cysts 3.3.8 Harvest and processing of cultured Artemia 3.4.2 Nauplii 3.2.5.4.7 Nutritional value 3.1 The future use of Artemia in aquaculture 3.5.4.2.4.3 Proteins 3.2 Physicochemical conditions 3.4.4 Strain-specific characteristics 3.2.5.2 Hatching 3.2 Cyst metabolism and hatching 3.5.4 Vitamins 3.4.4.2.5.5.5 Infrastructure 3.6 Enrichment with other products 76 77 77 77 78 78 78 79 79 79 80 81 83 83 84 86 86 88 91 92 93 93 94 94 94 94 95 96 96 97 97 98 99 99 99 102 104 105 105 107 108 109 110 110 .2.4.3 Harvesting hatched nauplii 3.1 Proximate composition 3.4.5.1 Cyst morphology and physiology 3.3.1 Size and energy content 3.3.3.1 Cysts and nauplii 3.4 Decapsulation 3.4.5 Enrichment 3.2.5.5.3 Diapause characteristics 3.2 Lipids 3.5.5 Applications of Artemia 3.4.2.4 Feeding 3.5 Cyst biology and diapause 3.2 Ongrown Artemia 3.1.5.5 Temperature and salinity tolerance 3.5.2.2.4 Vitamin enrichment 3.2.5.5.4.3 Artemia strain selection and culture density 3.3 Juveniles and adults 3. 3.2 Dike construction 4.1 Climatology 4.3 Density separation in brine 110 110 110 111 111 112 122 122 123 123 124 125 126 126 126 126 127 127 128 128 128 128 129 130 130 130 131 131 131 132 132 133 133 134 134 135 137 137 137 137 139 139 .2.2.5.1 Artemia strain selection 4.2.5.2 Abiotic parameters influencing Artemia populations 4.6.2.4 Pond adaptation 4.2 Maintenance of nutritional value 3.5.2.4.6 Cold storage 3.2.3 Other advantages 3.2.2 Pond Production of Artemia Cysts and Biomass 4.6.1 Liming 4.2.5 Organic fertilisers 4.1 Harvesting techniques 4.4.1 Brine dehydration 4.2.2.3 Fertilisation 4.4 Artemia Cyst Harvesting and Processing Techniques 4.5. Harvest and Processing of Artemia from Natural Lakes Gilbert Van Stappen 4.3.2 Size separation in brine 4.5.2 Processing techniques 4.6 References 4 Production.2.2.7 Use of juvenile and adult Artemia 3.2.7.4.3 Screening 4.5 Preparation of ponds for Artemia cultivation 4.3 Artemia Harvesting and Processing Techniques 4.5.4.5.2.2.2.2.1 Harvesting techniques 4.6.5.1 Monitoring the Artemia population 4.4.2 Inoculation procedures 4.7.2.4 Inorganic fertilisers 4.2 Predator control 4.1 Survival at low temperatures 3.5.2.3 Soil conditions 4.3.2.2 Seasonal units 4.4.2.1 Introduction 4.6 Combination of organic and inorganic fertilisers 4.3.7 Monitoring and managing the culture system 4.2.7.6.2.3.1 Permanent solar salt operations 4.2.2.3 Site selection 4.2 Brine processing 4.5.6.2.4.2 Topography 4.1 Deepening the ponds 4.3 Biotic factors influencing Artemia populations 4.5.viii Contents 3.4.2.6 Artemia inoculation 4. 5 Cold storage 4.3.2.1.1 Introduction 5.2.4 Generation time 5.4.2 Harpacticoids 5. food quality and food availability 5.3 Cyclopoids 145 145 145 145 146 149 149 149 152 153 153 153 155 156 156 156 157 158 158 159 159 161 163 168 168 168 168 171 175 175 181 187 .5.4.3.2.4.3 Production Methods 5.1.2 Mortality 5.2 Harpacticoids 5.2.3.2.4 Initial (or ‘raw’) storage 4.3 Size 5.3.2 Layer drying in oven 4.1 Life cycle 5.5.2.6.4.2.2.3.2 Copepod morphology 5.4.1 Calanoids 5.Contents ix 4.2.2. size and growth 5.3 Fluidised bed drying 4.6.1 Calanoids 5.2.1 Harvest of wild zooplankton 5.4.4.2 Biology 5.2.2.2.2.2 Harpacticoida 5.4.1 General characteristics 5.4 Resting or diapause eggs 5.3.2.1.6 Feeding.2 Production in enclosed fjords or sea areas 5.2. packaging and storage 4.1.4.1 Layer drying in open air 4.2.6.2.3 Reproduction 5.2.2.1 Digestive system 5.2.2 Intensive culture of copepods 5.2 Circulatory system 5.2.3 Cyclopoids 5.4.5 Development.2.5 Prepackaging.2.3 Nervous system 5.4 Drying 4.3 Production in outdoor ponds or large tanks 5.4.1.2.1 Extensive and outdoor cultures 5.3.5. Støttrup 5.5 References 139 140 140 140 141 141 141 143 143 5 Production and Nutritional Value of Copepods Josianne G.1.5.2.3 Freshwater processing 4.1 Calanoida 5.3.2.2.3 Cyclopoida 5.4 Reproductive system 5. 8 Enzymes 5.7 References 6 The Microalgae of Aquaculture Arnaud Muller-Feuga.2.3.1 Bacillariophyceae 6.4.2.3 Substrates of photoautotrophy 6.3.5 Other factors affecting growth 6.4.2.1 Temperature 6.1 Introduction 6.5 Cryptophyceae 6.1 Light 6.3.6 Application in Marine Aquaculture 5.1 Carbon 5.2.3.5.3.3.2.2.4.3.4. Jeanne Moal and Raymond Kaas 6.1 Bacillariophyceae 6.5 Mixing 6.4 Chlorophyceae 6.x Contents 5.3.3.6 Carotenoids 5.3.5.3 Protein 5.2.3.3.2 Growth 6.2.4.2 Prymnesiophycaea 6.3 Biochemical Composition of Microalgae 6.3.5 Nutritional Value for Fish Larvae 5.4.2 Biology of Microalgae 6.3 Prasinophyceae 6.5.3 Sterols 6.2 Prymnesiophycaea 6.4 Biochemical Composition 5.4.4.4.5.7 Chitin 5.3 Metabolites 6.4 Free amino acids 5.4.4.4 Substrates of heterotrophy 6.2 Salinity 6.2 Vitamins 6.1 General characteristics of microalgae 6.3 Prasinophyceae 6.2.4 Cryptophyceae 6.4.3.2 Mineral nutrients 6.4 Fatty acids 6.3.4 pH 6.3.2 Lipids 5.1 Gross biochemical composition 6.2.5.3.4.2.2.3.6 Eustigmatophyceae 189 189 190 190 191 191 191 191 191 191 194 195 206 206 206 206 209 213 213 217 217 218 218 219 219 220 220 221 222 223 223 225 227 227 228 228 229 229 230 231 232 232 .4.3.3.5 Vitamin C 5. 1 State of the art of microalgal production techniques in hatcheries 6.3.1 Continuous cultures 6.3.4.4.4.2.1 Development of penaeid shrimp 7.2.7 Feeding microalgae to shrimp juveniles and adults 7.4 Efficiency 6.3.3 Feeding spat 7.2. J.3.2.2 The increase in production yields 6.3.1.4.5 Substitution of spray-dried algae or microparticulate compound diets for live algae 7.Contents xi 6.6 Other roles of algae in shrimp larval growth 7.2.4.1 Microalgae as a potential food source in mollusc hatcheries 7.1.3.2 Digestibility 7.1.4 Nutrient supply from algae in relation to larval shrimp requirements 7.5 References 7 Uses of Microalgae in Aquaculture A.4.2.4.4.2.1.2.3 Microalgal substitutes for bivalve feeding 7.1. Divanach 7.2. R.1 Asepsis and quality controls 6.3 Other lipid components 233 233 234 236 236 236 237 238 238 240 242 243 253 253 254 255 255 256 257 257 257 258 259 261 261 263 263 263 265 266 268 269 269 270 270 271 271 272 274 . C. Robin and P.3 Running the cultures 6.4 Production Methods for Aquacultural Microalgae 6.2.4.2.2.2.4.2 Nutritional value of algae for live prey 7.1.2 Microalgae as Food for Molluscs 7.2.4 Microalgae as Food for Live Prey 7.2.2 Fatty acids 7.4.2.4.4.4 Microalgae bulk production 7.1 Size 7.1 Feeding broodstock 7.2.4.4.3 Heterotrophic production 6. Cahu.3 Microalgae as Food for Shrimp 7.3.3 Ingestion and filtration rates for shrimp larvae fed microalgae 7.2 Methods of improvement 6. Muller-Feuga.2 Microalgal requirements in mollusc hatcheries 7.1.3 Nutritional value: biochemical composition of microalgae 7.2.2 Feeding larvae 7.1 Feeding live prey with live microalgae 7.1 Proteins and proximate composition 7.2 Culture medium and temperature 6. Robert.1 Introduction 7.2 Selection of algal species used for rearing shrimp larvae 7.4 Discussion 6.1. 1 Drinking and ingestion of dissolved organics 7.5.4.3.5 Process and efficiency of first feeding 7.7 Stimulation of digestive functions and gut flora 7.5.4 Minerals 7.4.5.6 Effect on survival and growth efficiency at first feeding 7.5.5.5.2 Effects on endotrophic larval stages 7.5 Importance of Microalgae in Marine Finfish Larviculture 7.4 Resistance to delay in first zooplanktonic feeding 7.3.5 Influence of algae on live feed and larval microbiology 7.5.5.5.6 References Appendix I Appendix II Appendix III Appendix IV Taxonomic Index Common Names Index Subject Index 275 275 275 276 279 279 279 280 280 281 282 283 283 284 285 286 286 288 288 300 304 306 307 309 312 313 .5.5.4.3 Effects on the yolk-sac drinking stage 7.3 Vitamins 7.3.xii Contents 7.10 Future developments 7.5.6 Substitutes for live microalgae 7.2 Ingestion of microalgae 7.5.4.9 Indirect effects of microalgae on larvae 7.1 Range of microalgal action 7.3 Digestion and assimilation of microalgae 7.8 Effects on early exotrophic larvae 7. not necessarily in a one-to-one ratio. it is still rather easy to find enthusiastic and dedicated authorities willing to contribute to essential reviews of the state-of-the-art of.Foreword In the preface the editors point out that marine aquaculture has shown an important evolution from a relatively modest operation to a mature bio-industry. and a part-time plumber. Trial and error ruled. Prof. Various indeed: if one feature can typify aquaculture research it is multidisciplinarity. The maturation of the commercial ventures is seriously indebted to the huge progress in research and development in various disciplines relevant to aquaculture. physiologist. It is therefore a pleasure and an honor to introduce Live Feed in Marine Aquaculture wherein all its relevant issues are covered by representatives of some of world’s finest aquaculture research groups. especially at times when upscaling from laboratory and pilot trials to large industrial units. Dr Patrick Sorgeloos . As aquaculture developed. experiments. for instance. With the drive for specialization it became harder for the individual scientist to keep track of all pertinent information as well as new developments in research fields other than his or her own. Originally. the typical aquaculture researcher was a combination of a marine biologist. biochemist. And although some degree of (mostly healthy) competition exists. engineer. But as knowledge has broadened and deepened. ecologist (at best). aquaculture scientists became more specialized and fundamental research gradually came to underpin empirical findings. Common knowledge on distinct topics was limited and progress was often achieved through sound. live feed technology. To initiate such an initiative may well turn out to be a tedious job. like this book. but fortunately strong bonds were forged between leading research groups back in the days when the aquaculture ‘who’s who’ could still be printed on a single sheet of paper. This explains and justifies the multiple initiatives to provide publications. live feed has often been a bottleneck in the larviculture of many species of fish and shellfish. both in its research and development as well as for the industry. offering comprehensive updates on a selected topic. albeit empirical. are still evident in some species and in other cases subtle differences such as decreased tolerance to low temperatures observed during the juvenile stages in marine fish are attributed to poor nutritional diets during the larval stages. It will also serve as a practical guide. their production and nutritional value. Marine larviculture without live feed. harvest and processing of Artemia from natural lakes. This book provides the reader with the compiled information on most of the live feeds used in modern marine aquaculture. Three chapters deal with the hatchery production. A further chapter deals with the production. The different species used in marine aquaculture differ in their biology and culture requirements. despite the development of formulated diets. however. It may also be relevant to experimental researchers working on physiology. In shrimp culture. . behaviour or energetics in these species. postgraduates and researchers in the field of marine aquaculture. The book is intended for advanced undergraduates. use and nutritional value of respectively Artemia. Filtering molluscs and penaeid shrimps require microalgal diets at least during some stage in their development. the industry continues the struggle to produce stable quantities of high-quality live feeds.Preface The past two decades have witnessed a dramatic expansion in the culture of marine finfish and crustaceans. are rarities in commercial aquaculture. Nutritional defects. Survival and growth in marine fish larvae can be improved by the addition of live cultures. or to hatchery biologists who may wish to diversify or improve their culture methods. or crustacean cultures without microalgae. The development of commercial formulated feeds remains today’s upcoming challenge. The development of mollusc culture is closely related to the quantity and quality of phytoplankton produced. Two chapters on microalgae deal with their use and production in aquaculture providing the reader with a broad insight on the importance of phytoplankton in marine aquaculture. rotifers and copepods. Although their role is not fully understood. it will supply the basic information needed on the biology of the species and an introduction to the relevant literature. intended to provide the reader with a good overview on culture techniques for the different species involved and with substantial reference to related literature. Although it may not be exhaustive. This book includes information on the biology and culture of copepods as well as of the better-known traditional live feeds such as rotifers and Artemia. their positive effects are well documented. With the increasing emphasis on fish welfare and the need to produce high quality fish both for the aquaculture industry and for stocking purposes. In the meantime. phytoplankton is still used in hatcheries to supplement the diet during the larval stages. Enrichment techniques for rotifers and Artemia have greatly improved their nutritional value for marine fish species and have contributed to the expansion of the industry. providing ample challenges for the novice and requiring expertise in a commercial enterprise. larval nutrition will continue to be a main focus area for research within marine aquaculture. Laboratory of Aquaculture & Artemia Reference Center. Crete. Laboratoire Algae Biotechnology. 44 311 Nantes cedex 03. 44 311 Nantes cedex 03. Laboratoire Nutrition. Centre de Brest.ac.fr . fax: (33) 2 40 37 40 71. Greece. e-mail: chantal. IFREMER. Tel. fax: (33) 2 98 22 45 45. e-mail: imbc@imbc. Ghent University. Kingston. Animal and Veterinary Science. BP 70. USA.il Jeanne Moal Senior research scientist. BP 70. 29280 Plouzané. fax: (972) 4 8511911. University of Rhode Island. Tel. Fax: (32) 9 264 4193. e-mail: raymond. Department of Fisheries.: (972) 4 8515202. e-mail: amuller@ifremer.: (1) 401 874 2668. France. BP 21 105. Laboratoire Algae Biotechnology. Bengtson Professor and Graduate Program Director.fr Esther Lubzens Department of Marine Biology and Biotechnology. France. PO Box 2214. Laboratoire Invertebrate Physiology.: (33) 2 98 22 40 40. Tel. e-mail: jeanne. Tel. PO Box 8030.: (33) 2 98 22 40 40.: (308) 81 24 15 43. 9000 Gent. Rozier 44. IFREMER.cahu@ifremer. France. Haifa 31080. France. Post of Poros.Contributors David A.fr Arnaud Muller-Feuga Head. BP 21 105. Tel.be Raymond Kaas Senior research [email protected] Pascal Divanach Head of Aquaculture Department. Centre de Nantes.edu Chantal Cahu Senior research scientist.: (33) 2 40 37 42 20. 29280 Plouzané. Tel.: (33) 2 40 37 41 09.kaas@ifremer. Israel. fax: (33) 2 98 22 45 45.moal@ifremer. Centre de Brest. Institute of Marine Biology of Crete. fax: (33) 2 40 37 40 71. e-mail: jean. National Institute of Oceanography. Centre de Nantes.: (32) 9 264 3754. Tel. IFREMER. e-mail: esther@ocean. fax: (1) 401 874 4017. RI 02881. Tel. 71003 Iraklion. IFREMER.gr Jean Dhont Researcher. Israel Oceanographic and Limnological Research. Belgium.org. e-mail: bengtson@uriacc. : (972) 7 6361442. 29280 Plouzané. Rozier 44. e-mail: [email protected] Gilbert Van Stappen Researcher. fax: (33) 2 98 22 45 45.robin@ifremer. Laboratory of Aquaculture & Artemia Reference Center.vanstappen@rug. Tel.: (45) 3396 3394.il . Danish Institute for Fisheries Research. France. France. fax: (33) 2 98 22 45 45. Tel. Laboratoire Invertebrate Physiology. e-mail: rene.ac. fax: 972 7 6375761. PO Box 1212. IFREMER.fr Josianne G.huji. e-mail: gilbert.René Robert Senior research scientist. DK-2920 Charlottenlund. Israel Oceanographic and Limnological Research Ltd.: (33) 2 98 22 40 40. Charlottenlund Castle.robert@ifremer. fax: (45) 3396 3333. Tel. Denmark. Støttrup Senior research scientist. Department for Marine Ecology and Aquaculture.be Oded Zmora National Center for Mariculture.: (33) 2 98 22 40 40. Eilat 88112.: (32) 9 264 37 54. e-mail: zmora@agri. Belgium. fax: (32) 9 264 41 93. BP 70. Israel. Centre de Brest.min. IFREMER. Ghent University. Laboratoire Nutrition. 29280 Plouzané. B-9000 Ghent.ac. email: jean. BP 70.fr Jean Robin Senior research scientist. Tel. Tel. Centre de Brest. 20:4n-6 Ascorbic acid Ascorbic acid-2-sulfate Adenosine triphosphate Air-water lift system Biochemical oxygen demand Docosahexaenoic acid.Abbreviations AA ARA AscA AscAS ATP AWL BOD DHA DPH DPF DPPC DW EEZ EFA EPA ESD FAO FCE FW GMO GSL HUFA ILL ISA LC50 LNA L-type PAR PL PLa PUFA SCP SFB SGR SL S-type TAG WW Amino acids Arachidonic acid. 20:5n-3 Equivalent spherical diameter United Nations Food and Agriculture Organisation Food conversion efficiency Weight after preservation in buffered saline formaldehyde Genetically modified organisms Great Salt Lake Highly unsaturated fatty acids with 20–22 carbon atoms and more than three double bonds Incipient limiting level International Study on Artemia Lethal concentration for 50% of the sampled population Linolenic acid. 18:3n-3 Large type Photosynthetically active radiation Phospholipid Post-larvae Polyunsaturated fatty acids with more than one double bond Single cell proteins San Francisco Bay Specific growth rate Standard length Small type Triacylglycerols Wet weight . 22:6n-3 Days post-hatching Days post-(first) feeding Dipalmitoyl phosphatidylcholine Dry weight Exclusive economic zones Essential fatty acids Eicosapentaenoic acid. especially of salmonid culture. In response to the fishery crisis at that time.1 A Historical Perspective It is difficult to determine exactly where and when marine aquaculture began. The reason for the release at such an early stage of development was simple: there was no convenient live feed with which to provide them for their postlarval survival and growth. developing embryos and larvae for distribution back into the ocean. many of these programmes were sustained for decades until the lack of evidence of any success from them became apparent.Chapter 1 Status of Marine Aquaculture in Relation to Live Prey: Past. We will never know whether earlier discovery of easily culturable live . ‘hatcheries’ were constructed in several countries for the purpose of providing fertilised eggs. Liao 1991). based on the capture of fry from the wild (Pamplona & Mateo 1985. Britain. Cod larvae were raised in concrete ponds in Flødevigen. Norway. owing to the high mortality rates of fish early life-stages in the oceans. haddock (Melanogrammus aeglefinus). Given the knowledge of freshwater fish culture in Europe and the Americas. sometimes on-board ship (some of the hatcheries were in fact ships). transportation and introduction of salmonid populations (Stickney 1996). sometimes on shore. turbot (Scophthalmus maximus = Psetta maxima). this was not an unreasonable hope for the times. The prevailing practice was to obtain gravid adults of a given species. Milkfish culture has been conducted in Asia for centuries. It is only with the benefit of hindsight that we know that these ocean stocking efforts were doomed to fail. such as cod (Gadas morhua). but apparently the results of this ‘experiment’ were interpreted to mean that the larvae should survive in nature. and maintain them no longer than the prolarva stage prior to release back to the ocean. so that modern rearing methods and live feed in the hatchery were not required. Canada and the USA all had fish hatcheries devoted to the propagation of commercially important species. France. Bengtson 1. not that juveniles could be reared for release. The hope was that these would thrive and be recruited into the commercial fisheries. in the 1880s on a diet of natural zooplankton and in the absence of predators (Rognerud 1887). winter flounder (Pleuronectes americanus) and lobster (Homarus sp. strip them of their gametes for purposes of controlled fertilisation. By the 1890s.). Present and Future David A. Nevertheless. The efforts to repopulate the seas of Europe and North America in the late 1800s may provide a more useful starting point for a brief historical review of the modern methods. which had been rapidly developing since the mid-1800s and the attendant propagation. despite the contention that ‘large-scale cultivation of microalgae … was probably first considered seriously in Germany during World War II’ (Becker 1994). Spawning and successful larval culture of mussels was not achieved until the early 1950s (Loosanoff & Davis 1963). Fertilisation of large tanks of filtered seawater to induce mixed phytoplankton blooms as food for molluscan larvae was carried out continuously beginning in 1938 (Loosanoff & Davis 1963). not only for fish. Secondly. without losing the larvae (Wells 1920). beginning with Dr M. Wales. in the 1930s. Gross 1937. Fujinaga. interrupted unfortunately by World War II. Penaeus japonicus. Rollefsen 1939). expanded in Japan in the seventeenth century with the finding that oyster larvae would settle on bamboo stakes. The use of Artemia nauplii as a convenient live feed. Just as many of the ocean stocking programmes of the late 1800s and early 1900s were being phased out. molluscan culture always relied on the settling of larvae from the natural zooplankton (and still does in many areas). Artemia. Similarly. has perhaps done as much for the explosion of marine aquaculture in the late 1900s as any other development. Meanwhile.2 Live Feeds in Marine Aquaculture feeds would have allowed hatchery culture of these species to a later stage when they might have had better chances of oceanic survival. Wells (1927) then went on to raise clam larvae as well. and expanded further in Europe. nauplii of the brine shrimp. That research. Marine algal culture lagged behind its freshwater counterpart. which has been known since Roman times. techniques were developed in the 1920s and 1930s that led to the development of molluscan hatcheries. Schreiber 1927) in the early 1900s. Fisheries Experiment Station (Walne 1974) and the Milford. japonicus was finally achieved. However. North America and Australia in the nineteenth century based on bottom culture (Bardach et al. continued through the 1960s. Investigations of algal feeds for the rearing of molluscan larvae took place in the 1930s at both the Conwy. two developments occurred half a world apart that paved the way for much of the development of modern marine aquaculture. the field of stock enhancement might have been advanced by several decades had convenient live feeds been available in the late 1800s. which subsequently led to the development of the shrimp industry that we know today (Liao & Chien 1994). began research on the culture of the kuruma prawn. A significant advance in marine algal culture was reported by Gross (1937). This allowed the culture of at least some fish species (those with mouths large enough to ingest Artemia nauplii as a first food). clam culture has been known in Japan and mussel culture has been known in France for several hundred years (Bardach et al. USA. Indeed. 1972). but also (and especially) for crustaceans. The culture of algae seems to have its origins in the late 1800s and was enabled by the methods developed by bacteriologists (Bold 1950). Bureau of Commercial Fisheries Biological Laboratory (Loosanoff & Davis 1963). when commercial culture of P. were found to be a good food for raising both freshwater and some marine larval fish (Seale 1933. Oyster culture. Although hatchery spawning of oysters had been demonstrated as early as 1879. but whose . First. Japanese researchers. no one had been able successfully to change oyster culture water. 1972). Wells (1920) used a milk clarifier to retain oyster larvae while their water was being changed. who tried to culture diatoms and dinoflagellates. and therefore replenish the algal food. which successfully used uncomplicated media (Pringsheim 1924. Decades of work at the Conwy and Milford laboratories paved the way for hatchery production of molluscs for commercial aquaculture in which natural settling of larvae was either impossible or undesirable. interest in larval fish biology from a fisheries perspective caused many laboratories to begin rearing larval fish on fieldcollected zooplankton. From early reports suspecting pollutants (Bookhout & Costlow 1970) to later. research conducted primarily in the 1970s led to the development of the French sea bass and turbot industries in the 1980s (Person-LeRuyet et al. first from a research perspective. among others. Another extraordinarily important advance was made in the 1960s. when Japanese researchers discovered that rotifers. This advance clearly allowed the culture of many more species whose larvae hatched at such a small size that their mouth gapes were insufficient for the ingestion of the larger Artemia prey.Status of Marine Aquaculture in Relation to Live Prey: Past. the debt to those initial Japanese culturists is profound. Present and Future 3 attention was drawn to ‘nannoplankton flagellates. In retrospect. In Britain. began pond culture of cod larvae using natural zooplankton in the mid-1970s. Jones 1972). most of them probably unknown systematically’ of about 2–10 m in size.g. Clearly. Many of the above efforts documented the difficulty of rearing the extremely delicate marine larvae through the first-feeding stages and on to metamorphosis and subsequent grow-out (e. efforts were made to culture larvae of red sea bream. more definitive. Houde 1972). In many countries. Improved methods for monospecific algal cultures allowed expansion of hatcheries for molluscan aquaculture and enabled culture of live invertebrates as feed for larval fish and crustaceans. He summarised his work by writing ‘All these experiments led me to the conclusion that the autotroph nannoplankton flagellates are of great importance in the food economy of the sea. The 1960s saw widespread interest in the culture of commercially important marine fish species.’ Little did he know that they would also be of great importance in aquaculture. and several papers from the International Study on Artemia: see Persoone et al. rotifers or Artemia. sometimes supplemented with rotifers and Artemia nauplii (e. whether using natural zooplankton. In France. in order to conduct fisheries research (e. could be used as a first food for larvae of both freshwater and marine fish species (Hirata 1979). As the 1970s saw the beginning of commercial production of several marine finfish and penaeid shrimp species. Brachionus plicatilis. including the USA. and followed that with a major research programme on halibut culture beginning in the 1980s. 1981). In Japan. the White Fish Authority engaged in activities particularly in the area of flatfish culture (Shelbourne 1964) that ultimately led to the first commercial production of turbot in 1976 (Person-Le Ruyet et al. this decade is also noteworthy for the discovery that live feeds vary significantly in quality. to the point that the famous Kyoto conference in 1976 declared larval rearing a major bottleneck in marine aquaculture (Pillay 1979). Coves et al. using some pertinent results from the cod-spawning and restocking efforts 100 years earlier. were fraught with difficulties. 1991. The finding that the differences were due primarily to . flounder and puffer fish. previously considered a pest in culture ponds. 1991). 1978. He was able to culture these and use them as feed for harpacticoid copepods over three copepod generations.g. Methods for marine algal culture continued to advance during the middle of the twentieth century with the development of artificial media (Provasoli et al. Norwegian scientists.g. Laurence et al. early efforts at rearing larval marine fish. it became very obvious that different geographical strains of brine shrimp differed in their ability to support good survival and growth of marine larvae. 1980). 1957) and the development of ‘f’ medium for the enrichment of seawater (Guillard & Ryther 1962). 1991). considering the large number of commercially important marine fish species that have been brought into culture and that rely on rotifers as first food in culture facilities. studies (Watanabe et al. ) and cod. As part of this effort. Investigations into the improvement of live feed. the recent trend has been toward hatchery production. 1986) and to explain much of the high quality of natural zooplankton as a food item.4 Live Feeds in Marine Aquaculture fatty acid profiles led to productive collaborations between aquaculturists and biochemists that have resulted in literally hundreds of publications on the subject. among other species. as well as commercial products that have played no small role in the development of marine aquaculture. National and prefectural hatcheries now produce millions of fish. flounder and sole species currently poised to make their debut appearances on the world’s commercial aquaculture stage. which is heavily dependent on microalgae and Artemia nauplii as larval feeds. Research on other fish species in various areas of the world has led to large-scale aquaculture production of gilthead sea bream (Sparus aurata) and sea bass (Dicentrarchus labrax) in the Mediterranean region. One of the more interesting controversies in the live feed area is the view that natural or cultured copepods are necessary for at least some species. Japanese research on red sea bream (Pagrus major) similarly led to the development of that species for commercial aquaculture in Japan as well as for stock enhancement. 1984). This event convinced the Japanese that they needed to become self-sufficient in seafood production. The aforementioned Japanese work on kuruma prawn led ultimately to the culture of numerous penaeid species around the world. The former view seems to come from the . Japanese researchers have often led the way in marine aquaculture research and the practical applications of that research can be seen around the world. Although commercial aquaculture of these species has become well established. again. 1991).) can be routinely fed formulated diets directly upon hatching. all dependent on live feed in the hatchery stage. for example. The Japanese government responded by embarking on a massive research and hatchery-building campaign (Davy 1990. turbot in western Europe and olive flounder (Paralichthys olivaceus) in east Asia. several groupers (Epinephelus spp. The last quarter of the twentieth century saw the explosion of marine aquaculture. of all the marine fish species in production or in the research and development pipeline. to be intimately connected to algal food supply (Léger et al. The lessons learned from brine shrimp were also shown to apply to rotifers (Lubzens et al. both shrimp and fish. It appears likely that live feed will be required well into the future. Although postlarval shrimp for stocking into grow-out ponds were for years collected from the wild. it seems that only the wolfish species (Anarhichas spp. because they could no longer fish at will in the coastal waters of many nations and because they saw that an interruption of supplies on an international scale was a real possibility (Sproul & Tominaga 1992). Atlantic halibut (Hippoglossus hippoglossus). The necessity for aquaculturists to understand in detail the physiology and biochemistry of the organisms that they raise has contributed much to making marine aquaculture a sophisticated industry. especially stock enhancement: the establishment of exclusive economic zones (EEZs). Asian sea bass (Lates calcarifer) in the Indo-West Pacific region. a variety of species is still undergoing commercial growing pains. prawns and crabs for release into Japanese waters each year through the efforts of the national and prefectural Sea Farming Associations. but also for the plethora of new bream. as opposed to the view that rotifers and Artemia nauplii are quite sufficient. especially rotifers and Artemia. One event of the 1970s played a major role in the development of marine aquaculture. all require live feed in the hatchery. have certainly been a major contribution of the Japanese research programme. not only for the established and nearly established species. sciaenid. Indeed. 13. 1993. Thus.1 million t from marine) (FAO 2000). In addition.9 million tonnes (19. as rotifers have been to the development of marine fish larviculture. enclosed systems primarily through the use of natural zooplankton. can introduce to the fish tanks xenobiotics from wherever in the world the Artemia cysts originated. 373). larvae can be reared in a bag enclosure and provided with additional zooplankton that has been collected from the adjacent waters by the use of a plankton wheel (see van der Meeren & Naas 1997. A company in Denmark produces turbot larvae in large concrete tanks in which zooplankton ‘blooms’ are induced. While larvae of all these species grow extremely well on the natural zooplankton. Furthermore. such as copepod nauplii. indeed critical. abundant rationale exists for research on the mass production of copepods. it is well known that the nutritional value of copepods is better than that of the convenient live feeds such as rotifers and brine shrimp. almost by definition in the temperate zone.. seasonal and not amenable to more intensive production methods in which juveniles must be produced year-round. Vadstein 1997). 1. which indicate that world aquaculture production was 32. bacteria from Artemia hatching water. The procedure requires either the filtration of incoming water or the use of rotenone to kill any predators. the production at such facilities is. Thus. Live feeds thus have both good and bad aspects. the use of live prey in hatcheries may be strongly related to one of the banes of the marine larviculturist.8 million t from freshwater. it has been known for some time that rotifer cultures fed to a tank of fish can also carry pathogenic bacteria. the United Nations Food and Agriculture Organisation (FAO) had just released its preliminary estimates for fisheries and aquaculture statistics from 1999. Van der Meeren and Naas (1997) provide an excellent review of larval fish rearing in large. The enclosures can be fertilised to increase the phytoplankton productivity within. if the Artemia cysts have not been decapsulated or otherwise disinfected. Disease has become a major consideration in hatcheries and the microbial ecology of hatchery tanks has become an area of intense research which one hopes will lead to more predictable hatchery outputs (Vadstein et al.2 Marine Aquaculture Today and in the Future At the time of writing. aquaculture makes up . Oddly. p. However. at least one of the tanks is devoted exclusively to extensive copepod culture and used to feed the larval fish tanks if live prey levels therein fall too low. Norwegian scientists have pioneered this field of larviculture in ponds and natural inlets that have been closed off from the sea. that can lead to subsequent disease problems (Gatesoupe 1982). Research efforts into rearing copepods in intensive indoor systems have shown some promise. such as Vibrio spp. As beneficial.Status of Marine Aquaculture in Relation to Live Prey: Past. and one challenge of the future is to minimize the bad aspects. but lacking in rotifers and Artemia. In a similar way. A valuable result of this controversy has been the extensive biochemical analyses of natural zooplankton to determine whether particular nutritional ingredients are present there. Alternatively. but commercial-scale production has not been achieved (see review by Støttrup 2000). so that large populations of copepods are available on which the larvae can feed. would be necessary to culture such species. some species produce larvae with mouths that are too small to ingest even rotifers at first feeding and an alternative live prey. disease. Cod have been raised successfully in Norwegian lagoons using these methods and halibut have been raised there in bags. Present and Future 5 Nordic countries. particularly Norway and Denmark. 355. mostly shrimp (1. its commercial development can be slowed or impaired (as in the case of Atlantic halibut). In 1998. etc. if live feed other than rotifers and Artemia are required. Finally. and diadromous fish. Production reliability is being improved by several strategies.g. For example. SSFA & NAFC 2000) and there is cause for optimism that improved practices will be the norm in the future. The basis of this area of research is the production of very high-quality juveniles from hatcheries (using only first-generation broodstock to maintain genetic integrity with the natural population). The fish are generally released as juveniles and therefore well adapted to formulated diets.g. The overriding one is ‘How do we make aquaculture sustainable?’ The environmental consequences of the explosion of marine aquaculture in the last quarter of the twentieth century have become a major international concern within the past decade. .000 t. thus lagging behind crustaceans. As we proceed into the future. 1999).9 million tonnes per year in recent years. Improved management of microbial ecology in hatchery tanks through better husbandry.. 1989. to allow the fish to make the transition from hatchery to natural environment with maximum likelihood of survival. Selective breeding programmes for both fast growth and disease resistance should result in improved hatchery production in future years and those for improved flesh quality should ultimately yield a better product going to market. delivered by injection to older juveniles and by immersion to younger juveniles. the search for replacements for live feed proceeds apace as the world-wide availability of Artemia remains a question (see below) and the culture of algae and rotifers continues to be a labour-intensive requirement for marine hatcheries. and maximising the survival probability in the wild for hatchery-reared fish in stock enhancement programmes. the last year for which full statistics are available. Leber et al. release site) and the use of conditioning methods. should likewise aid in the minimisation of disease problems. The development of culture methods for new species tends to demonstrate the similarity of the requirements for raising different marine fish species.6 Live Feeds in Marine Aquaculture more than 35% of the total 92. but clearly the use of high-quality live feed is necessary earlier in the hatchery to produce the high-quality juveniles needed for release. the development of a new species is immediately hindered. a few big questions dominate the landscape. The major research endeavours in marine hatchery aquaculture today can be divided into three broad categories: improving reliability of production for existing species. Yamashita et al. Otterå et al.6 million tonnes of fisheries products consumed by humans. the identification of optimal release strategies (fish size. rather than differences between them. while the growth of freshwater aquaculture has been closer to 1.909.000 t). use of probiotics. the ecological insults brought about by marine aquaculture are trumpeted to the world’s consumers by environmental groups. The research in this area generally involves the fine-tuning of widely accepted principles and procedures for application to the new species in question. but their methods have more recently been adapted by others (e. and far behind freshwater fish. 1994). if the culture of a species requires more than fine-tuning. The global aquaculture industry is responding (Boyd 1999.564.000 t). should also help production reliability. season. 1997.000 t) (FAO 2000). both in the hatchery and in the wild. From shrimp farming in mangrove areas to organic enrichment from salmon net-pen culture.1 million tonnes per year. The maximisation of survival of hatchery fish in the wild has been primarily the province of Japanese researchers (e. Development of vaccines. Marine aquaculture has been growing by about 0. development of culture methods for new species. aquaculture production of purely marine fish was 781. Tsukamoto et al. mostly salmonids (1. mostly carp (17. diagnosis and treatment of disease. 1. Egypt is the leading producer. Present and Future 7 A second major question is ‘What will we feed aquacultured organisms in the future?’ This question applies both to hatchery-reared fish and crustaceans and to those in grow-out operations. 2000). It is likely that the answers to those questions will become apparent with GM products from terrestrial agriculture before aquaculture will address them in a major way. with over 16. 1997. This is due in large part to recent poor harvests from the traditionally productive Great Salt Lake. The identification of new sources of Artemia cysts for harvest. based on production figures supplied by FAO for the calendar year 1997 (FAO 1999) and various articles as cited. While this is a question primarily for grow-out producers. the biotechnology industry is already playing a role in products for the prevention. along with increased regulation of harvests from those waters. Clearly. 1. the ramifications will certainly be felt all the way back to the hatchery phase of the industry (Will we no longer grow species that require fish meal? Should we select for individuals that have minimal fish meal requirements?). but . however. in alphabetical order. one wonders what the current crisis will yield. In a manner similar to the Artemia crises. personal communication). Recently. One final major question concerns the role that biotechnology will play in aquaculture.3. the industry is currently undergoing a kind of crisis in Artemia cyst availability. periodic shortages of fish meal world-wide (usually due to climatic conditions off western South America) bring about intensive research into fish meal replacements. Genome mapping is beginning for some of the major aquaculture species (M.000 t of mullet production. as well as environmental groups. Yúfera et al. Gomez-Chiarri.Status of Marine Aquaculture in Relation to Live Prey: Past. the aquaculture industry. An even greater question is whether consumers will accept such products.3 The Status of Larviculture and Live Feed Usage It may be useful in this introductory chapter to describe the status of marine finfish and crustacean larviculture and live prey usage in different regions of the world. Recalling that the last Artemia crisis in the mid-1970s led to the discovery of new geographical strains and focused research on Artemia cyst quality. A renaissance in research on formulated diets to replace Artemia is already underway (Kolkovsky et al. 1999) and one hopes that the results will be more commercialisable than those from the flurry of research on microdiets that arose from the last Artemia crisis. The review will be presented continent by continent. allow some hope that this crisis will soon fade. It appears that partial or complete replacement of fish meal in the formulation of diets for some species will be necessary or desirable if the industry hopes to grow to the degree necessary. Utah.1 Africa Africa’s marine finfish and crustacean production comes largely from countries bordering the Mediterranean. but the question of whether genetically modified organisms (GMO) will be allowed in the marketplace is still a question for regulators. for example in Asian countries that once belonged to the Soviet Union. USA. have questioned whether the projected growth of the industry over the next 30 years is possible in the light of fish meal availability even in the best of times (Naylor et al. At the hatchery level. so that the reader receives a broad overview on a global scale. 000 t). with significant hatchery production relying on the standard formula of rotifers and Artemia.000 t in 1997 (FAO 1999). and threadfin. Hong Kong and Taiwan are similar to each other in having their fish and crustacean mariculture activities dominated by finfish culture. It should be pointed out that both Indonesia and the Philippines are predominated by milkfish culture. japonicus. Thus. Acanthopagrus schlegeli. Nien & Lin 1996). Singapore. having predominantly shrimp culture with P. Shetty & Satyanarayana Rao 1996). 1. Morocco and Tunisia also produce hundreds of tonnes each of sea bass and sea bream. use of algae and Artemia is extremely heavy. Although some extensive culture using wild-caught shrimp still exists in India and Vietnam (Binh & Lin 1995. 800 t) of silver bream. and ca. Since this production is almost exclusively hatchery based. based primarily on P. but hatchery production is expanding. Epinephelus spp. 80.2 Asia Moving out of Africa and proceeding through Asia from west to east. Thailand also has significant production of Asian sea bass. . these countries also have significant hatchery production of both shrimp (using algae and Artemia) and fish (using rotifers and Artemia). groupers) in the case of the Philippines and Malaysia. snappers. so no live feed is used (Wassef 2000). Bangladesh ( 50. basses.g. like Egypt. as well). monodon. Thailand is the world’s largest shrimp producer (FAO 2000). Indonesia and Malaysia are somewhat similar to Thailand. so also require hatchery production using rotifers and Artemia (Romdhane 1992). among many others. including over 4000 t of black sea bream. the fry are mostly collected from the wild. all apparently from hatchery production and requiring rotifers and Artemia. therefore requiring the use of rotifers and Artemia (Wassef 2000). all of which require algae and Artemia as live feeds in the hatchery. penaeid shrimp culture. Culture of these high-value species is quite industrialised. They all culture Asian sea bass.000 t). L. calcarifer in the case of Indonesia and a variety of species (e. but that industry still depends largely on capture of fry from the wild and therefore does not require live feed for larviculture. 400 t). produce significant quantities of sea bass and sea bream. rabbitfish. 50 t). The Philippines. one finds that Israel and Turkey. with relatively little.000 t). and South Africa has a small production of Penaeus indicus and P. Egypt also produces more than 2000 t each of sea bass and seabream. 5000 t).8 Live Feeds in Marine Aquaculture these are grown from wild-captured fry.3. (ca. Iran and Saudi Arabia both report production of hundreds of tonnes of penaeid shrimp. 8 t) and Vietnam (ca. grouper. India ( 50. Sri Lanka (ca. Eleutheronema tetradactylum (ca. if any. requiring the use of algae and Artemia in hatcheries. 800 t). but Taiwan produces a wide variety of marine finfish. Penaeid shrimp culture dominates the mariculture of Pakistan (ca. L. and Hong Kong produces significant quantities (ca. groupers and snappers to greater or lesser degrees. calcarifer ( 4000 t). monodon as the major species (although with substantial culture of Penaeus merguiensis and Metapenaeus spp. A small amount of marine finfish culture is reported from Kuwait and Qatar. the majority of the above production appears to rely on hatchery production using the normal methods with algae and Artemia (Shetty & Satyanarayana Rao 1996. Pagrus major. with production of 200. but also exhibiting increasing production levels of finfish. Penaeus monodon. requiring hatchery usage of rotifers and Artemia. 400 t of red sea bream. Myanmar (ca. Madagascar and the Seychelles Islands produce significant quantities of shrimp. Rhabdosargus sarba. . reported for 1997 (FAO 1999). Seriola quinqueradiata. It is impossible to determine from FAO statistics the production of individual marine fish species in China. Tetraodontidae (nearly 6000 t) and jack mackerels. with both rotifers and Artemia required as live feed. with over 15. Their marine finfish and crustacean production is a fairly minor component of their total production. with 11 species receiving more than 10 million seed and 33 species receiving at least 1 million seed (Fushimi 1998). as in Japan. the dominant species are sea bass. with over 24. olivaceus ( 26.000 t. which requires algae and Artemia in the hatcheries. Other major finfish produced commercially include red sea bream ( 80. These impressive numbers are. is still produced commercially in Japan.Status of Marine Aquaculture in Relation to Live Prey: Past.000 t of various other species. yellowtail. Asian sea bass. still exceeds 100. however. produce prodigious amounts of both finfish and shrimp for stock enhancement and sea ranching efforts. with nearly 30. In the Mediterranean countries plus Portugal. P. prefectural and local hatcheries). tilapia. japonicus. rotifers and Artemia for marine finfish and crustacean culture in both commercial aquaculture and governmental stock enhancement efforts. but Cen and Zhang (1998) report 145.000 t. The flounder culture. Oddly.3 Europe Although minimal production of penaeid shrimp species is reported in Albania. Greece. ( 5700 t). Shrimp production. schlegeli (6 million) (Fushimi 1998). Greece. olive flounder ( 8500 t). This production included molluscs and echinoderms as well as fish and crustaceans. the production of marine finfish far outweighs that of marine crustaceans in Europe. Only a little more than 2000 t of kuruma prawn. is totally dependent on hatchery production of fingerlings. 1. Cyprus. while maintaining production of a few hundred tonnes of red sea bream and yellowtail and 12. Overall.3. primarily P. France. the species that began the industry. 305 million. major (19 million) and A. surpassed by those for kuruma prawn.000 t). Finally. Cyprus. puffer fish and olive flounder. the fish with the largest production in commercial aquaculture (nearly 140.000 t of sea bass and over 18. Trachurus spp. Cen and Zhang (1998) state that all shrimp seed for production now comes from ‘a controlled environment’. Japanese hatcheries. breams.000 t) is still dependent on wild-caught fry. P.000 t of sea bream. France. Japan produces far more marine finfish than shrimp. and sea bream. These require hatchery production using the rotifer and Artemia techniques that the Japanese largely developed. Major species with numbers of finfish fry released in 1995 are: P. but clearly Japan is a major user of live feeds such as algae.000 t). olivaceus (23 million). mostly Penaeus chinensis. responsible for a remarkable two-thirds of all aquaculture production globally. groupers.000 t per year despite problems with disease epidemics in the 1990s. Hatchery rearing with rotifers and Artemia is also necessary for the production of fry for the stock enhancement programmes. Present and Future 9 The People’s Republic of China is the world’s largest aquaculture producer. however. rather than being collected from the wild. Greece is by far the leader. Japan produced seed for stock enhancement of 80 species in a total of 284 facilities (such as national. but still dwarfs that of most other countries. including mullets. Italy and Spain (requiring use of algae and Artemia). South Korea has been rapidly expanding its marine finfish culture.000 t by 1997).000 t of production in 1995 (which had apparently increased to 250. or in place of. Since the early 1980s. which are fertilised in spring to induce blooms of phytoplankton and zooplankton before the introduction of the fish larvae (Harrell 1997). FAO includes hybrid striped bass (Morone saxatilis Morone chrysops) as a marine fish species in its statistics. In northern Europe. In the USA. All are from hatchery origin. Oddly. 1. with slightly more than 300 t of commercial production in 1997. whereas sea bream also require rotifers prior to feeding on Artemia. Pittman. In addition. saxatilis). using rotifers and Artemia as live feed for larvae. 1.3. rotifers and Artemia. Although research on culture of both species has been going on since the early 1970s. Sea bass can feed on Artemia as a first feed. for an additional total of between 4500 and 5000 t.10 Live Feeds in Marine Aquaculture Italy. the USA reported production of 1200 t of Litopenaeus vannamei in 1997. commercial production is reported for red drum (Sciaenops ocellata) and summer flounder (Paralichthys dentatus). requiring algae and Artemia. with over 17. Culture of cold-water finfish in Canada. The larvae of those bass are mostly raised in earthen ponds. ponds or blocked-off sections of fjords. ocellata) and spotted sea trout (Cynoscion nebulosus) are the most noteworthy of these. but Norwegian scientists have also been engaged for a number of years in production of cod fingerlings for stock enhancement projects.3. the major species are cod and halibut and there is much greater usage of natural zooplankton in addition to.5 Oceania The majority of production here is penaeid shrimp. red drum (S. striped bass (M. originating from intensive hatcheries with heavy use of algae and Artemia. personal communication). Cod larvae are produced in tanks.4 North America Relatively little culture of marine finfish and crustaceans is reported from North America. Production of halibut has more recently been effected in Iceland. but growing. and are fed natural zooplankton obtained from the same or similar enclosed bodies of water which have been fertilised to bring about phytoplankton blooms (Huse 1991). is still in the trial phases. Norway is the leader in cod production. Australia reported nearly 1600 t of P. despite the fact that the halibut larvae in Iceland are raised without natural zooplankton (K. . Other island nations (Fiji Islands. monodon production in 1997 and New Caledonia over 1100 t of Penaeus spp.000 t of production reported in 1997. which has now become the leading producer of halibut juveniles. Halibut are produced in a variety of enclosed systems and can eat Artemia as first food. vannamei). both of which require rotifers and Artemia as prey in the hatchery. restoration of stocks or mitigation of environmental impacts. Mexico has become a large producer of shrimp (L. even though the fish are reared in fresh water. hatchery production of a few species has been necessary for enhancement. All of this production is based on hatchery-raised fry. but Norwegian producers argue that natural zooplankton is also necessary during the larval stages for production of good-quality fry. Portugal and Spain all produce minor to significant quantities of other finfish species as well. the actual commercial production is still rather small. 1986). The use of natural zooplankton. with algae and Artemia as the live feeds of choice. but there may be other reasons besides the digestibility question. Usage of formulated diets to supplement and eventually replace Artemia is apparently increasing (see below). lacking a stomach. The hatchery techniques are by now quite standard throughout the region. the trend is for increased reliance on hatchery production of postlarvae. and much of the protein digestion takes place in hindgut epithelial cells (Govoni et al. Honduras. 1997). hatchery production of juveniles globally is normally accomplished just with Artemia. Altricial larvae therefore appear to require live feed.Status of Marine Aquaculture in Relation to Live Prey: Past. It should be pointed out that algae is routinely used in marine fish culture of the so-called ‘green-water’ method. but live feed is still dominant at this point.3. The digestive system is still rudimentary. but it is still not clear to what degree the algae may be contributing directly to the nutrition of the larvae (Reitan et al.000 t. Guam and the Solomon Islands) all report minor production ( 50 t each) of various penaeids.6 South America. Nicaragua. rotifers or Artemia. when the yolk sac is exhausted. including Central America and the Caribbean With the exception of Chile. Precocial larvae are those that. Australia also reported over 500 t of Asian sea bass production. Such fish can ingest and digest formulated diets as a first food and are best exemplified by the salmon and trout raised extensively in hatcheries around the world without the benefit of live food. Ecuador is the clear leader. exhibiting fully developed fins and a mature digestive system including a functional stomach. Altricial larvae are those that. remain in a relatively undeveloped state. with 1997 production of over 120. Chile has been rapidly increasing its finfish aquaculture industry and is poised to become the world leader in salmon production. Other countries producing between 2000 and 10.000 t include Brazil. hatchery production of penaeid shrimp postlarvae around the world depends on the use of live algae for the early stages and Artemia for the later stages. but it also is producing turbot in significant quantities for export to Europe. 1. Hatchery rearing of these turbot depends on both rotifers and Artemia in the same way that they are used by the European turbot industry. when the yolk sac is exhausted. Such a digestive system seems (at this point) to be incapable of processing formulated diets in a manner that allows survival and growth of the larvae comparable to those fed on live feed. or with rotifers and Artemia. Colombia. if a smaller initial feed is required. just as they are elsewhere in the world. For marine finfish. 1. the culture of marine finfish and crustaceans in this region is overwhelmingly dominated by penaeid shrimp. Peru and Venezuela. or the use of cultured foods other than algae. Although wild seed is still used in some places. Live feeds are able to swim in the water column and are thus constantly available to the . appear as mini-adults. is limited to a few places in the world. Guatemala.4 Why is Live Feed Necessary? Fish biologists categorise larvae of two types: precocial and altricial. Panama. To summarise this geographical review. if the mouth gape is large enough at first feeding. but it can be very important in those particular places. Present and Future 11 French Polynesia. Costa Rica. any foods must enter the mouth whole (i. inert diets or prepared diets) could not provide nearly as good survival and growth of marine larvae as could live prey. This last point is rather critical. the larva’s mouth gape must be of sufficient size for particle ingestion to occur) and they are quickly either accepted or rejected on the basis of palatability.e. They are filter feeders as early larvae and by the time they can feed on live zooplankton. Commercial microencapsulated larval feeds were developed in the 1980s and showed much greater success with shrimp larvae than . Crustacean larvae such as shrimp are qualitatively different from fish larvae. live prey. Beck & Bengtson 1979). may be more palatable to the larvae once taken into the mouth. As a result. more commonly. Finally. towards the bottom. it needed to be unimpeded by a shortage of live feed.12 Live Feeds in Marine Aquaculture larvae. 1997a).5 Problems and Prospects with Alternatives to Live Feed Pelleted diets became common in the salmonid industry during the middle of the twentieth century. In addition. so that the formulated feeds are better able to stay in the water column and the shrimp can capture the diets with their feeding appendages rather than having to ingest them in a single gulp as larval fish must do. compared with the hard. aquaculturists perceived that. since evolutionary history has probably adapted them to attack moving prey in nature. they were still not as convenient as a formulated diet would be. The results were not encouraging (Girin 1979).g. dry formulated diets. especially when considered in light of the fish larva’s absence of feeding appendages. but also a gut morphology and physiology with which to digest formulated diets more effectively (Jones et al. The natural tendency was to enlist the aid of fish nutritionists who specialised in pelleted diets for grow-out and to convince them somehow to make those pelleted diets small enough for fish or crustacean larvae to eat. Formulated feeds (often referred to as artificial diets. came the hope that this technology might be applied to the development of successful microdiets for larviculture as well. sink quickly to the bottom. if marine aquaculture were to achieve its potential rate of expansion. although some studies indicated that partial substitution of Artemia with formulated feeds did yield survival and growth of larvae equal to that of larvae fed Artemia alone (e. and are thus normally less available to the larvae than are the live feeds. Larval shrimp tanks tend to have greater water movement than do larval fish tanks. Jones et al. with a thin exoskeleton and high water content. even though encysted crustaceans such as Artemia provided a convenient live feed. whereas fish larvae do not (Holt 2000). the aforementioned Artemia crisis of the 1970s suggested that. 1997b). they possess not only feeding appendages with which to manipulate the prey organisms captured. primarily for the pharmaceutical industry. With the advent of microencapsulation technology. In addition.b) reviewed the digestive physiology and nutrition of larval crustaceans and remarked that larval penaeid shrimp and late larval Macrobrachium can successfully use artificial diets. (1997a. As marine larviculture developed in the 1960s and 1970s. 1. Formulated diets tend to aggregate on the water surface or. Formulated diets are generally capable of moving only in a downward direction. the movement of live feed in the water is likely to stimulate larval feeding responses. shrimp larvae have been shown to survive and grow well on formulated feeds (Jones et al. & Lin. J.. 1993. The fact that development of new feed(s) is not required for the development of each new species has greatly facilitated aquaculture expansion. When new species are developed or new geographical regions opened up to aquaculture.H. Vol.. 11–17. Beck. (1979) Evaluating effects of live and artificial diets on survival and growth of the marine atherinid fish Atlantic silverside. 27–33. I (Ed. A. by J. In: The Culturing of Algae (Ed. C. nutritionists.C.) (1994) Microalgae: Biotechnology and Microbiology. chemical engineers and others all working with aquaculturists. but so far the ‘magic bullet’ remains elusive. thanks to the tireless efforts of researchers around the world. rotifers and alternative prey such as copepods) be minimised so that hatcheries can produce fingerlings/postlarvae at lower cost? The Kyoto conference identified larviculture as a bottleneck in aquaculture primarily on technical grounds. E. 24. there is now a need to reduce the economic bottleneck that larviculture still exerts on the commercial culture of many species. it is a great benefit to have a standard ‘menu’ with a minimal number of well-established ‘entrees’ to use as live feed in the hatchery.Status of Marine Aquaculture in Relation to Live Prey: Past. Tiews). As argued previously (Bengtson 1993). but the usage in fish hatcheries appears to be less (although the exact numbers are not known). D. In recent years. (1950) Problems in the cultivation of algae. J. Heenemann. 20. & Bengtson. As many of the technical problems have been overcome.7 References Bardach. Bookhout. (1993) A comprehensive program for the evaluation of artificial diets. Meeresunters. & Costlow. & McLarney. Wiley-Interscience. New York. enabled in large part by the development of fairly standard hatchery protocols for live feed usage around the world. Charles F. C. (ed. World Aquacult. Ryther. New York. Person Le Ruyet et al.E. Kettering Foundation. by J. Jr (1970) Nutritional effects of Artemia from different locations on larval development of crabs.E.W. . World Aquacult.. Bold. C. 1.H. Halver & K. Soc. J.A.. In: Finfish Nutrition and Fishfeed Technology.6 Conclusions Marine finfish and crustacean aquaculture has greatly expanded since the early 1980s. Menidia menidia. Binh. 285–293. 1993).D. W. 479–489.A. 26. Cambridge University Press. pp. Brunel. pp.W. and at what cost? How can the costs of live feed culture (primarily algae.O. 1.G. Berlin. (1972) Aquaculture: The Farming and Husbandry of Freshwater and Marine Organisms.T.. D. Cambridge. Present and Future 13 with fish larvae (Jones et al. G. Bengtson. J. breakthroughs in the development of microdiets for marine fish larvae require the multidisciplinary efforts of biochemists. These multidisciplinary teams have been forming and conducting research. Becker. there has been a tendency to use formulated feeds in shrimp hatcheries to minimise usage of Artemia. The major questions that remain for the future involve the availability and costs of feed for larvae: To what degree will Artemia availability and cost limit the expansion of marine aquaculture? To what degree can formulated diets replace live feeds generally. H. Prescott & L.B.K. Helgoländ. 435–442. (1995) Shrimp culture in Vietnam. Tiffany). Davy. 6.E.. (1962) Studies on marine planktonic diatoms. H. 155. 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(1994) Effects of release size on survival and growth of Japanese flounder Paralichthys olivaceus in coastal waters off Iwate Prefecture. Bull. L. Biol. Dept. Van der Meeren.. Watanabe. Shetty. Fish. Aquacult. 29–32. Yamada. Aquaculture. 35A. Rotterdam. Farnham. (1920) Artificial propagation of oysters. C. New York. 31. (1933) Brine shrimp (Artemia) as a satisfactory food for fishes. 367–390. 44. World Aquacult. Pascual. 2. northeastern Japan. Sproul.R. (1974) Culture of Bivalve Mollusks: 50 Years Experience at Conwy. E. 50. 16. turbot (Scophthalmus maximus). Støttrup 2000. while rotifers must be cultured in hatcheries.). including the wide range of body sizes both within and between species. Brachionus rotundiformis is also known as the S-type (small-type) rotifer and B. Copepods are a major component of the natural diet of marine fish larvae.1). Rotifers are also used as food for culturing penaeid shrimp (Samocha et al. The incidental choice of rotifers (Ito 1960. 1989) and crabs (Keenan & Blackshaw 1999). Rotifers are not. gilthead sea bream (Sparus aurata) and the European sea bass (Dicentrarchus labrax) (FAO 1998).Chapter 2 Production and Nutritional Value of Rotifers Esther Lubzens and Odi Zmora 2. rotifers have been used as food organisms for cultured marine fish larvae. fish larvae are fed on two or three organisms during the initial 10–30 days of exogenous feeding. been proven a success. providing the nutrients required by the cultured marine fish larvae for proper development. However. A continuous. Artemia cysts are obtained mostly from natural sources. The specific . Hagiwara et al. pufferfish (Fugo rubripes). Epinephelus sp. mullet (Mugil cephalus). Rotifers serve as a ‘living capsule’. the natural food of marine fish larvae. plicatilis as the L-type (large-type) rotifer (Fig. Major fish species produced today using rotifers during the early developmental stages include yellowtail (Seriola quinqueradiata). as there is no other large supply of them. which have at their disposal a wide range of food organisms in their natural habitats. Copepods do have some advantages over rotifers. Attempts to culture copepods have met with some success in recent years (Støttrup & Norsker 1997. 2. therefore. Hagiwara et al.g. see reviews by Hirata 1980. it is unlikely that copepod cultures will replace rotifers as an economically viable alternative in the near future. the grouper. 2001. Asian sea bass (Lates calcarifer). Nagata & Hirata 1986. red sea bream (Pagrus major). In culture. given the relative ease and low cost of culturing rotifers in high densities.1 Introduction For almost four decades. Payne & Rippingale 2001). 2001) as food for early developmental stages of small-mouthed larvae has. stable and reliable supply of nutritionally adequate rotifers is the key to the flourishing culture of marine finfish in various parts of the world. the early stage nauplii and copepodites can be extremely useful as initial prey for species that have very small larvae with small mouth gape at first feeding (e. In particular. These include rotifers of the species Brachionus rotundiformis and/or Brachionus plicatilis and brine shrimp (Artemia) nauplii. in most cases. depending on culture facilities Can be manipulated and regulated Lorica and eggs not digested Minimal Feasible Natural zooplankton Variable but includes organisms less than 60 m in size Variable with many spiny species Variable with some benthic species and fast-moving species Some species (e. reliable.18 Live Feeds in Marine Aquaculture Fig. plicatilis) were found important in raising early developmental stages of cultured crustaceans. without spines Usually planktonic and relatively slow moving Tolerance of high densities Tolerance to a wide range of salinities Manipulated and regulated. 2.) Table 2.1. plicatilis (right).g. Characteristic Size Body shape Distribution in water column and swimming Density Salinity Supply Nutritional quality Digestibility Transmission of parasites and predators of fish larvae Transmission of therapeutic agents and probiotics Rotifers 90–350 m.1 A comparison between cultured rotifers (Brachionus plicatilis and Brachionus rotundiformis) and natural zooplankton harvested in the wild as food for early developmental stages of marine fish larvae. Rotifers (B. as opposed to zooplankton. depending on species and developmental stage Round and flat. such as the mud crab (Scylla serrata) up to zoea 5 stage (Baylon & . varies daily between locations High but unpredictable and variable Variable Realistic Doubtful or unlikely advantages of using rotifers as food for early stages of development of marine fish larvae. are summarised in Table 2.1 Rotifers used in raising marine fish larvae: Brachionus rotundiformis (left) and B. copepods) with low tolerance to high densities Species-specific and variable tolerance Unpredictable. (Photograph: Irena Pekarsky. although B. 2001). edible and ornamental fish species (De Luca et al. Wallace & Snell 1991. Lim & Wong 1997. The amount needed ranges from 20. 1989. pseudocoelomate. About 2000 species populate freshwater lakes and ponds. Nogrady et al. Lubzens et al. Ruttner-Kolisko 1974. with a specific emphasis on the genus Brachionus (Family Brachionidae. Quinitio et al. rotifers are extremely important in the freshwater environment. Nogrady et al.g. Ludwig 1994. 1999. 1999. 1997. 1987). 2. Pontin 1978. 1999. Assistance in evaluating the state of cultures is provided in Appendix I. 1990. Hagiwara et al. plicatilis rotifers were found to be helpful in culturing freshwater ornamental carp larvae (Lubzens et al.1 General biology The phylum Rotifera (previously known as the Rotatoria. Most rotifers are free-crawling or swimming.b.2 Biology and Morphological Characteristics of Rotifers 2. Raising freshwater Brachionus calyciflorus in culture systems has been examined for freshwater. Huge numbers of rotifers. Nagata & Hirata 1986. and some of them have focused on production of rotifers as live food in aquaculture (e. but sedentary and colonial forms are also known (Ruttner-Kolisko 1974. the rotifer culturist must be aware of and able to deal with occasional unpredicted events that can lead to low production. pollution of the culture water with waste products. Wallace & Snell 1991.2. 1999) or the marine shrimp (Penaeus semisulcatus) (Samocha et al.b. This basic information provides the culturists with the tools for adapting rotifers for mass culture techniques and in solving problems arising during their culture. 2001). see Ricci 1983) consists of a relatively small group of minute. Several reviews have been published on rotifer morphology. The culture of freshwater rotifer species has not yet had the same impact on aquaculture as that of the marine species. Rico-Martinez & Dodson 1992. may be required each day for raising marine fish larvae in commercial hatcheries (Lubzens et al. Awïass 1991. Hagiwara et al.Production and Nutritional Value of Rotifers 19 Failama 1999. physiology and biochemistry. Mann et al. The nutritional quality of the rotifers must be assured and controlled by the use of well-established and tested culture and enrichment methods. easily reaching several billions. Awaïss & Kestemont 1997a. 2001). 1989). 1993. 2001). 1997. Isik et al. Lubzens 1987. quality and quantity of food provided to the rotifers. Fukusho 1989a. 1993). Order Monogononta) and equip the newcomer to this field with the basic concepts and tools for using rotifers as food for cultured fish larvae. but several species are known from brackish or marine waters and from mosses and lichens in moist terrestrial habitats. Although Rotifera is a small phylum. reproductive strategies.000 to 100. genetics. Furthermore.000 rotifers per fish larva during the 20–30 days of culture (Kafuku & Ikenoue 1983. These may result from inadequate seawater supply. Li et al. Awaïss et al. By . Fulks & Main 1991. unsegmented. biology. Lubzens et al.b. disease or insufficient biological information. Keenan & Blackshaw 1999. feeding. aquatic invertebrates with bilateral symmetry. 1992a. 2001. contributing up to 30% of the total plankton biomass. The methods developed for providing an adequate supply of rotifers to a variety of small-mouthed fish larvae rely on extensive studies into their biology. The present review attempts to provide information on euryhaline rotifers. taxonomy and culture. Clément & Wurdak 1991. eye. a.2 Morphology and inner organisation of a Brachionus sp. oesophagous. as loricated and illoricated forms may be found within one genus. stomach gland. mu. egg. v. Fig. lateral antenna. g. bt. e. o.) . mastax. foot gland. (From Koste & Shiel 1987. bladder. giving the illusion of two turning wheels. Rotifers have several distinctive features (Fig. they form the link between primary producers and secondary consumers or predators such as fish and insect larvae. and the metachronal movements of the cilia in the anterior rotating apparatus. eg. p. sg. a neck of variable length. A dense intracytoplasmic lamina located within the syncytial integument forms the peripheral skeleton that serves for muscle attachment. trophy. a body and a foot. 2. m. female (left) and male (right). Species in which extensive regions of the integument are thickened are known as loricated forms (including Brachionidae) and others with a thin. fg. tr. testis. c. pe. Volume 7 with permission of CSIRO Publishing. t. central ganglion. apical. typically possessing two toes (but the number may range from none to four) that usually retract during swimming. Several regions with thinner intracytoplasmic lamina provide flexibility in loricated forms. f. s.2). ov. sensory cirri. muscle. ovary. corona. dorsal antenna. penis. is one of the distinctive characteristics of organisms belonging to this group. Reproduced from Invertebrate Taxonomy. te. They are characterised by an anterior. The presence of the corona distinguishes rotifers from all other metazoans. foot and articulations between movable spines and the body. These regions include the corona. b. 2. ciliated corona (‘crown’). foot. stomach. la. The pedal glands found in the foot secrete a sticky cement material for temporary attachment of rotifers to the substratum. The body of rotifers is covered by an extracellular cuticle that is a gelatinous secretion of the underlying integument and has no skeletal function (Clément & Wurdak 1991).20 Live Feeds in Marine Aquaculture consuming bacteria and/or algae. functional in swimming and feeding. buccal tube. st. toe. vas deferens. Their body shape ranges from saccate to cylindrical and typically four regions can be distinguished: a head bearing the corona. more flexible integument are known as illoricated. prostate. The thickness of the body wall has little taxonomic significance. bathing the internal organs. sensory bristles between them. Fig. The vitellarium carries several cells (see below) and eggs are carried at the foot opening. 1998). There are four flame cells along the protonephridial duct on each side of the body connecting with a large urinary bladder. Cell membranes in tissues disappear after embryonic development. if they occur. 2. The corona has five distinct lobes with long. The family of Brachionidae comprises six genera of common rotifers. and each egg is individually attached to the female body. Its composition is regulated by the protonephridia and it is replenished by the digestive tract. known as eutely. protonephridial and reproductive). The oesophagous is thin-walled and the cellular stomach is clearly separate from the intestine.2. One female can carry one to three amictic eggs or several male eggs. with more than 1600 species. two resting eggs and these resting eggs are easily distinguished from other eggs by their darker orange–brown colour and a space or vacuole between the two egg membranes (see below). All are assumed to be dioecious with one gonad. see Section 2.2. is equivalent to the circulatory system.2. The mastax (see below) is large with malleate trophi.2 Taxonomy Taxonomic classification of rotifers is under constant review (Garey et al. a female carries only one or. Traditionally. Rotifers are also characterised by the syncytial structure of their body parts. All individuals of a species have a consistent number of nuclei in each organ. Two of these (B. the latter consisting of two classes: the Bdelloidea with two gonads. 2. annulated.1 The genus Brachionus The distinctive morphological characteristics of the genus Brachionus are an oval body that is flattened dorsoventrally. 1998. Females possess one ovary with a vitellarium and males. Segers 1998). including Brachionus with about 25 species of littoral and planktonic rotifers. A cerebral eye is always present. 2. fully retractable into the lorica and relatively long. The Monogononta contain over 90% of all rotifer species. plicatilis and B. rarely. are structurally reduced with a vestigial gut. This situation. is also found in nematodes. rotundiformis. in about 95 genera of benthic. The foot is very mobile. and the pseudocoelom internal fluid.2. In the early days of devising methods for mass culture of rotifers. The family Brachionidae comprises a large number of species.1) are being used extensively in mass cultures and serve as food for early developmental stages of marine fish larvae. 2. ranging from 900 to 1000. forming multinucleated or syncytial tissues.2. is fixed for life during embryonic development. Usually. the cultured species was identi- . and a lorica with six spines on the dorsal anterior margin (Fig. A few species are known from the marine environment and several from euryhaline waters. indicating a limited capacity for repairing damage. and the Monogononta with one gonad (Melone et al. There are no respiratory or circulatory systems in rotifers.3). 1998. There are two gastric glands of varying shape and the anus is terminal. most of them inhabiting freshwater. Melone et al.Production and Nutritional Value of Rotifers 21 Rotifers possess an internal fluid-filled space known as a pseudocoelom that is bound externally by the integument and internally by the epithelial cells of the various organs (digestive. free-swimming and sessile forms. Males are present for brief periods (a few days or weeks). The total number of nuclei. the phylum is divided into two superclasses: the Seisona and Eurotatoria. B. 2. 1993. Fig. 1991a. 1998. However. rotundiformis SM they are retained within the body of the female (Serra et al. plicatilis is a euryhaline. in contrast to the pointed spines of B. 1991). Gomez & Carvalho 2000. the groups show different mictic responses to density.b). recent data indicate that B. 1998. and mating experiments showed that most copulations occurred within a group and no hybrids could be obtained in laboratory cross-mating experiments. B. 2. 1998). Hagiwara et al.3). rotundiformis SS. 1995b. plicatilis is significantly larger than that of B. rotundiformis (Hagiwara et al. Caution is advised when considering older publica- . (Photograph: Irena Pekarsky. 2000) suggest genetic differences between these two species. In B. 1995b. rotundiformis is composed of two genotypes.3 The lorica of Brachionus plicatilis (left) and B. rotundiformis SM and B. Boehm et al. rotundiformis and the anterior spines on the lorica of B. rotundiformis (right) showing the anterior spines. rotundiformis SM is adapted to high temperatures and low salinities and B. Rico-Martinez & Snell 1995. Chromosome number (Rumengan et al. while in B. In addition. allozyme profiles (Fu et al.4). plicatilis are obtuse. 2. plicatilis and B.22 Live Feeds in Marine Aquaculture Fig. their preservation and hatching are further discussed in later sections in this chapter. with no evidence of gene flow between them (Serra et al. plicatilis and B. There are also some differences in the way rotifers carry their resting eggs. salinity and temperature. resting eggs are carried outside the female lorica in the same way as amictic or male eggs. Accumulating data showing that these morphotypes do not differ in size and shape alone led to taxonomic identification as two species. low-temperature group. The lorica of female B. Production of resting eggs. B. These studies show that B. reproductive isolation (Fu et al. Gomez & Serra 1995) and microsatellites (Gomez et al.) fied as B. plicatilis showing two morphotypes known as L-type (large type) and S-type (small type). rotundiformis SS. rotundiformis SS is adapted to high-temperature and highsalinity conditions. rotundiformis (Segers 1995). Fig. a supple myelin-like structure whose function is to prevent rejection of food that has reached the pharynx. plicatilis and B. cirri and light-sensitive ocelli (Fig. The head also carries the ventral mouth opening and several sensory organs. (From: M. however.) tions. as there is evidence of confusion of B. A. 2. plicatilis (L). rotundiformis. Reproduced from Hydrobiologica 387/388. With kind permission of Kluwer Academic Publishers. rotundiformis SM (SM) and B.3 Morphology and physiology The anterior end. which is responsible for swimming and the inner one.Production and Nutritional Value of Rotifers 23 Fig. Carmona. or ‘head’.4 Differences between Brachionus plicatilis SS (SS). 2. is composed of cilia organised into membranelles or cirri (Clément & Wurdak 1991). The buccal tube is equipped with a variety of sensory receptors and ends in the buccal velum.3. Gomez & M. which is comprised in Brachionus females of two concentric ciliary crowns. Average body length (top number) and width (lower number) and their standard deviations (in parentheses) are shown below each rotifer. 373–384. It may be assumed that B.J. 1998. Serra.2. The pseudotrochus sweeps the food particles towards the mouth.2). B. including antennae. rotundiformis is the main cultured species in tropical areas. 2.2. the outer one is the cingulum. where the translucent hard trophi break down . carries the corona. The longitudinal and circular muscle sheaths surrounding the buccal tube can. 2. the pseudotrochus. enters the mouth opening and passes through the buccal tube to the pharynx. The pharynx houses the muscular mastax. evoke rejection of the ingested food at this level.1 Feeding Food is captured by the corona. The type of food consumed. However. and the chemical or tactile recognition at this site will determine the continuation or cessation of mastax grinding movements. with changes in efficiency with different particle size. If the food is found to be unsuitable. 1997). composed of hard parts. particularly its size. 1983. The contraction of these muscles will result in rejection of food particles if they are deemed unsuitable. The trophi. Secondly. Optimal grazing was reported on Tetraselmis suecica. calyciflorus and these are probably also operating in B. . The rotational movement of the cilia directs a water current containing food particles towards the mouth and those that are suitable are swallowed. Clément et al. physiological condition of algal cells and algal motility (summarised in Hansen et al. the anterior ciliated apparatus composed of tactile and/or chemical receptors may regulate the muscles governing the position of the pseudotrochus cilia in such a way that they would form a screen to keep certain particles from entering the mouth. Brachionus plicatilis seems to have few food preferences and can reproduce when fed with various species of algae.4 to 4838 m3.4 to 21 m of equivalent spherical diameter (ESD). the sensory receptors will evoke expulsion of the food from the buccal canal. probably with the aid of enzymes produced by ‘salivary glands’. yeast or bacteria (Clément et al. and these centres regulate the movement of circular and longitudinal muscles surrounding the buccal cavity and mastax. This type of feeding. respectively (Clément & Wurdak 1991). Large syncytial gastric glands empty into the alimentary tract at the junction between the ciliary oesophagous and the stomach and their secretion aids in the extracellular digestion occurring within the stomach lumen. 1983). The range covers sizes from bacteria to dinoflagellates (Hansen et al. Hansen et al. genera or even species. plicatilis (Clément et al. Their ingestion rates reflected the exact proportion of the volume of each species of algae (Hansen et al. also described as ‘microphagus feeding’ (Pourroit 1977. selectivity was reported in several species and depended on cell surface. Several studies indicate that food selectivity by suspension feeders such as rotifers is mainly based on prey size (Rothhaupt 1990a. Sensory receptors are located all along the alimentary canal.b. which voids fluid from the bladder or paired protonephridia.3 m. indicating a sensory mechanism regulating food selection. with a prominent sensory organ between the trophi on the mastax floor and sensory receptors on the mastax ceiling. Finally. The most common method of feeding in planktonic brachionid rotifers is by filter feeding. Hansen et al. are of extreme taxonomic importance for the characterisation of rotifer families. with an ESD of 8. The prey size spectrum for B. Rotifers were described as mechanical grazers as they were found to graze non-selectively when offered two algal species with different cell size. Three sites were suggested to regulate the intake of food in B. or 1.24 Live Feeds in Marine Aquaculture the food. and eggs from the oviduct. 1997). First. 1997) and the largest particle size caught by rotifers is dependent on its body size (Hino & Hirano 1980). The food leaving the mastax enters the cuticular region of the oesophagus and passes through the ciliary region before entering the stomach. 1983). 1997). chemoreceptors in the buccal tube may activate the longitudinal and circular muscles. is found in rotifers having a developed ciliary corona and a crushing type of mastax. plicatilis ranges from approximately 1. The stomach leads into the intestine and then into a cloaca. The sensory receptors that participate in feeding behaviour are connected either to the brain or to the mastax ganglion. is directly dependent on the size and form of the ciliary apparatus and of the mastax. 1997). the ground particles come into contact with sensory receptors in the mastax. reflecting their different body size. 4–8 106 cells ml 1 for feeding with Monochrysis (now Pavlova) lutheri and Saccharomyces cerevisae (Lebedeva & Orlenko 1995). ingestion rates remain constant. depending on the species of algae used as food and on the food density (Hirayama 1990). Finally. in the sigmoid model. the filtration rate is adjusted to the food concentration and maintains a full gut at low energy expenditure. 1997) or sigmoid (Navarro 1999) feeding models for B.2. the ILL was twice that of B. When fed with N. clearance rates increase up to a certain food concentration that is termed the incipient limiting level (ILL). rotundiformis (Navarro 1999). Interference could be indicated by mechanical interference caused by. In the rectilinear model. Additional ILL values for B. for example. oculata. Above the ILL. rotundiformis (approximately 2 106 and 1 106 cells ml 1. clearance or filtration rates increase sigmoidally with increasing food particle concentration until a maximal value is reached. 1. and is expressed by Michaelis–Menten (Michaelis & Menten 1913) or Ivlev (1960) equations.1 106 cells ml 1 (Chotiyaputta & Hirayama 1978). The filtration or clearance rate is high at low food particle concentrations and declines in a curvilinear manner with the increase in food concentration. Hirayama & Ogawa 1972). Studies have indicated curvilinear (Hansen et al.5 106 cells ml 1 with Chlamydomonas and 0. plicatilis were obtained for other food sources. Maximal ingestion rates are probably determined by the gut packing and the rate of gut evacuation and. (probably known today as Nannochloropsis sp. plicatilis. without the interference of the particles with the feeding process until the ILL is reached. 2. B. rotundiformis consumed 160% of their dry weight per day (Navarro 1999). The stomach is sometimes pigmented by recently ingested food and the colour depends on the type of consumed food. during which there is an interruption in collection of new particles. In this model there is a gradual increase in ingestion rates until a plateau is reached. while B. and then they decrease with any further increase in food particle concentration. Thus. plicatilis. plicatilis. In experiments using Nannochloropsis oculata as food for B. . There were no significant differences for values obtained for filtration and ingestion rates between live (N. The curvilinear model proposes continuously decreasing clearance rates with increasing food concentration. 2. Gastric glands in the anterior part of the stomach may aid in extracellular digestion. the increased handling time necessary for processing large prey items. therefore. will be reached at lower food concentration with larger food items. This model indicates a typical increasing interference with the feeding process and is observed with larger than optimal food particle size.Production and Nutritional Value of Rotifers 25 Three models have been proposed to describe the effect of food particle concentration on feeding rate of filter-feeding zooplankton (Rothhaupt 1990b).3.1 106 cells ml 1 with Chlorella sp. plicatilis and a curvilinear feeding model (but not significantly different from the sigmoid model) for B. Several digestive enzymes have been reported from B.. oculata) and freeze-dried algae in these experiments (Navarro 1999). The ingestion rate remains constant after the peak value has been reached by the clearance rates. plicatilis rotifers ingested between 60 and 90% of their dry weight per day.2 Digestion The crushed food particles pass through the oesophagous into the stomach and intestine.64–10 l h 1 per individual) in filtration rates was found for B. A large range (0. This model represents a feeding mode where numerous small particles can be collected simultaneously. respectively). vitellarium and urinary bladder. Like other pseudocoelomates. into the cloaca. -amylase. probably indicating absorption of the digested food (Clément & Wurdak 1991). dorsally below the corona.3-glucanase (Kuhle & Kleinow 1985. with longitudinal muscles shortening and circular bands elongating the shape of the body. is carried out by protonephridia. They are inserted onto the integument. or forming part of the viscera. The protenephridial system comprises two lateral parallel tubules with several fan-shaped flame cells opening into them. the body fluid osmolarity was 59 mosmol l 1. or join the integument and internal organs. Kuhle & Kleinow 1985. the contraction of muscles inserted on the integument and the resistance of the body fluids facilitate the movement of the animal in the water. Mechanoreceptor bristles are located on the corona and on antennae distributed on various regions of the . ventral neurons proceed from the brain along the length of the body into the foot. 2. Osmoregulation. where they are drained into the urinary bladder. branching off to various organs. laminarinase. The flame cells are equipped with cilia that pump the body fluids and pass them into the tubules. facilitate the contraction of organs such as the digestive gland.3. Muscular contraction of the body aids in circulating the body fluid. Muscles inserted in the viscera. The bladder empties the accumulated fluids.3. Wethmar & Kleinow 1993). indicating that they are essentially osmoconformers.2. rotifers do not have a respiratory system or a circulatory system and body fluids are located in the pseudocoelom.b. at an external concentration of 32 mosmol l 1. The muscular system consists of striated and smooth muscles occurring in small longitudinal and circular bands.5 Nervous system and sensory organs The co-ordination of all of these functions is under the regulation of a nervous system that consists of a single large cerebral ganglion (‘brain’) that is located in the anterior part of the body.2. 1990. 2. Paired. In loricate species such as the Brachionidea. lysozyme and -1. at least in freshwater species.3. 1984a.4 Movement The body fluids function as a hydrostatic skeleton.26 Live Feeds in Marine Aquaculture including proteases (Hara et al. The membrane lining the stomach lumen shows invagination of vesicles that coalesce into vacuoles and large oil droplets towards the interior. Brachionus plicatilis was found to adjust the body fluid osmolarity to that of the external concentrations ranging from 32 to 957 mosmol l 1. cellulase. interacting with the muscular system. Contraction of longitudinal muscles that are inserted on the corona or foot facilitate their retraction into the lorica under unfavourable conditions or during swimming. The sensory organs can be divided into mechanoreceptors. by contraction. cellobiohydrolase. Hara et al. demonstrating that these rotifers are unable to tolerate low external concentrations (Epp & Winston 1977). chemoreceptors and photoreceptors. 1989. There are a few ganglia located on the mastax and foot and at the exit points for lateral nerves. 2. However. rotifers exchange gases and dispose of nitrogenous wastes by diffusion through their body surface. 1997).3 Body fluids and excretion As mentioned before.2. In B. meaning that asexual reproduction is prevalent.1). Monogonont species (e.4. haploid eggs via meiosis. amictic females produce parthenogenically diploid amictic eggs and mictic females produce parthenogenically haploid male eggs or sexually diploid resting eggs that hatch into diploid amictic females. plicatilis. the haploid eggs form into males. In general.5). the yolkproducing syncytial vitellarium and the follicular layer that surrounds the ovary and vitellarium and forms an oviduct leading to the cloaca. The total number of ovocytes is present at birth.4 Reproduction The reproductive organs of female Monogononts are. with embryos developing outside the maternal body. but under certain circumstances sexual reproduction may occur. These also function in copulation in males.1 Asexual and sexual reproduction Nearly all rotifers seen in nature are females. 2. Parthenogenically formed eggs (diploid or haploid eggs) will develop immediately into embryos and hatch. 2001). Females are always diploid and males. 1993. they are also attached by a thin thread to their mother until the end of their formation and later . Asplanchna.g. In the Brachionidae. B. plicatilis. where the resting eggs are formed outside the female’s body (Fig. Thus. it is easy to obtain genetic clones from cultures originating from one amictic female. Rotifers are generally oviparous. If a mictic female does not mate and is not fertilised. Several species possess one or more pigmented photoreceptive eyespots. Thus. Males occur only for short periods and in many species have never been observed. Amictic females produce parthenogenetically diploid eggs that develop mitotically into females. and the embryos of amictic eggs hatch and are released from the maternal body leaving the egg shells still attached to their mother. parthenogenetically. are haploid and very much reduced in size compared with females. when they appear. as the name implies. In some rotifer genera (e. Resting eggs will hatch under appropriate conditions into amictic females. by detecting the female’s pheromone (see Section 2. composed of a single gonad. while mictic females produce. amphoteric females that produce diploid and haploid eggs have been observed (King & Snell 1977). Production of resting eggs or subitaneous eggs via parthenogenesis and the occurrence of diapausing amictic eggs have also been observed (see discussion in Nogrady et al. Lubzens et al. A red eyespot is very distinctive in the anterior part of Brachionus species that have been fed on algae. The gonad consists of the syncytial ovary that contains the ovocytes. Conchilus and Sinantherina). including the foot region. Fig.2. Diploid females can either be amictic or mictic and morphologically they are indistinguishable.2. rotifers of this group reproduce by cyclic parthenogenesis. 2. after a dormant period (Hagiwara 1996.2.5) can reproduce either by parthenogenesis or through sexual reproduction.g. but a mated mictic female that is fertilised will form diploid resting eggs (subitaneous eggs or cysts).Production and Nutritional Value of Rotifers 27 integument. Resting eggs at the initial stages of formation cannot be distinguished from those of amictic eggs. 2. During favourable conditions. whereby diploid females produce diploid eggs known as amictic eggs. The corona also carries chemoreceptors that function in accepting or rejecting food particles. the population increases through diploid parthenogenesis.4. Gilbert & Schreiber 1995). the eggs are attached to the body of the female by a thin thread. 2. but it is generally assumed that all members of this group are capable of producing males. pond or lake sediment. 2.) released and sink to the bottom of the culture vessel. the resting eggs of B. 2001). Resting eggs survive for long periods and have been hatched from sediment samples more than 60 years after their formation (Kotani et al.5 Schematic explanation of sexual and asexual cycles of reproduction in Brachionus plicatilis. rotundiformis (SM type rotifer strains) that develop within the maternal organism are not released from it. (Photograph: Gidon Minkoff and Esther Lubzens. However. They will sink to the bottom of the culture vessel. Males are known from a limited number of monogonont species. pond or lake sediment with the death of their mother and are finally released only after the decomposition of her body. including the .28 Live Feeds in Marine Aquaculture Fig. rotundiformis and this may serve as a species-specific barrier limiting interbreeding between these species (Kotani et al. Serra and King (1999) showed that the frequency of mictic females that will maximise the population long-term fitness depends on population mortality and birth rates. Males are much smaller than females and typically very fast moving. Males attempt copulation with amictic or mictic females and mating occurs at the region of the corona or cloaca. This means that optimal culture conditions will result in higher production of resting eggs. but from laboratory studies they include nutritional. with a threshold for zero population growth and a plateau for maximal population growth values (Rothhaupt 1990c). These groups tend to inhabit time-varying environments that may become periodically unsuitable and therefore must be recolonised (Serra & King 1999). Snell & Nacionales 1990). The resting egg is the life-cycle stage having the greatest capacity to disperse in both time and space. see below) increase exponentially with increasing food concentration. 1995. Its molecular structure probably differs from that of B. It is a 29 kDa glycoprotein and is found in amictic and mictic females (Snell et al. plicatilis and suggested 50% as the highest optimal mictic ratio in nature. B. A pheromone produced by females was identified first in B.Production and Nutritional Value of Rotifers 29 brachionid species. rotundiformis. salinity and genetic factors (Pourriot & Snell 1983. The parameters of this model depend on the food . Genetic recombination occurs during sexual reproduction that leads to the formation of resting eggs. 1997).2. ameiotic parthenogenesis with the advantages of sexual recombination and is found in monogonont rotifers and cladocerans and in some other animal phyla such as aphids. Serra & King 1999. Ricci 2001). population density. Parthenogenic reproduction is predominant in the initial stages of colonisation as it produces rapid population growth and during this phase the genotype is copied without genetic recombination. The cues initiating meiosis in diploid mictic females are not well understood. Lubzens & Minkoff 1988). This means that the population growth will not increase beyond a specific concentration of food and may even decrease at relatively high food concentrations. This means that a population may adjust its relative rates of mictic and amictic production in response to environmentally induced changes. the amictic female will produce amictic and mictic daughters. Using a demographic model. The mode of cyclical parthenogenesis combines the advantages of rapid clonal propagation via diploid. 1988.2 Reproductive rates The intrinsic reproductive rates of rotifer populations (r or G values. They have a rudimentary digestive gut and a sac-like testis containing free-swimming spermatozoa.4. Successful fertilisation occurs in newly emerged females for a very limited period (Snell & Hawkins 1983). A vas deferens leads from the testis to a penis and one or two prostate glands discharge into it. 1985. and depends on food quality and salinity (Lubzens et al. plicatilis and B. and different mixis patterns are expected in different types of habitats or culture conditions. They concluded that intermediate mictic ratios are optimal in density-dependent growth conditions and that optimal mictic ratios are higher when habitat conditions are better.84% (mean SD) for B. 2. Snell (1987) reported an average mictic ratio of 21. In response to a mictic cue.2 3. The proportion of mictic daughters of B. plicatilis is variable (18–66%). and the relationship can be described by a modified Monod model. plicatilis. Lubzens 1989. Table 1). The number of eggs produced by a female (R0) is dependent on the food algal species.0 4–20 82. as the optimal temperatures for B. plicatilis (10–30°C) are lower than those for B. fed on Chlorella stigmatophora (3 106 cells ml 1) and incubated at 25 1°C under constant illumination. the production of amictic eggs is. 1979). Lubzens et al. Values are given as means SD. as discussed above) and on the nutritional quality of the food provided to the rotifers. differences have been found in the reproductive rates under the same culture conditions.35 42. The detailed experimental set-up is described in Lubzens and Minkoff (1988.2 Life tables of amictic females (AM).57 0. The optimal temperature for culturing rotifers depends strongly on the species. Rumengan & Hirayama 1990.26 43 15. of replicates 7.96 287. of eggs per female Range Rate of egg production Hours Eggs produced day 1 No.5 43. The parent females were also transferred every 8–10 h to new wells containing freshly prepared medium.61 11 0.97 17.2 35 Brachionus plicatilis rotifers were individually incubated in 1 ml seawater (at a salinity of 9 ppt). The fecundity of rotifers depends on whether asexual or sexual reproduction takes place.06 23.07 169. AM Average life span (Σlx/n) Days Hours Range (days) Preoviposition period Hours % of total lifespan Oviposition period Hours % of total lifespan Postoviposition period Hours % of total lifespan No. New offspring were separated from their mothers every 8–10 h and placed individually in new wells. .19 0.5 0. 10 times faster (about 5 eggs/day) than that of the resting eggs during a similar oviposition period (approximately 108 h) (Table 2.44 10. Lubzens et al. non-fertilised mictic females producing male eggs (M) and fertilised mictic females producing resting eggs (FM). 1989.09 34 76.05 14 0.8 4–11 41. plicatilis amictic females produced 17–24 eggs during their lifetime.2). In addition. but these did not contribute to the increase in population as they form males.08 14 21.43 17–24 4.45 23 61.21 11. compared with 1–5 resting eggs produced by fertilised mictic females (Lubzens & Minkoff 1988. 1987.30 Live Feeds in Marine Aquaculture Table 2. Within each species. Hagiwara 1994).31 1–5 0. An example given in Table 2.8 FM 11.18 9–19 3.0 25 107. rotundiformis (24–35°C) (see reviews by Hirano 1987.63 6.93 M 7.00 38 3. unpublished results of Experiment 1).8 22 108. Hirayama & Rumengan 1993).11 0.39 11.2 shows that B.45 178.8 64 24. The unfertilised mictic females produced 9–19 eggs. indicating intraspecific variability (Hino & Hirano 1977. 1989.84 16. on average.71 4–9 36.75 5. with Synechococcus elongates and Tetraselmis tetrathele supporting the highest rates of reproduction (Hirayama et al.66 37 108.8 0.7 1.56 12. particle size since this determines the relative rate of consumption (or clearance rate.59 0.70 16. Differences have also been found between B. Size. Snell & Hoff 1985. this factor has not been identified. As mentioned before. It has been shown that lower temperatures and lower salinities are required for B. plicatilis and B. Preliminary experiments were reported on testing the effect of invertebrate and vertebrate hormones for encouraging mixis in B. plicatilis rotifers. plicatilis and B. type of reproduction (asexual versus sexual) and reproductive rates are species or strain specific. Fresh or preserved algae (frozen and/or concentrated) have been used in the production of resting eggs. Snell & Boyer 1988. rotundiformis. maintaining water quality in culture tanks and choosing the most appropriate culture technique.3. To date.3 Culturing Rotifers The success of rotifer cultivation is dependent on selecting the most suitable rotifer species or strain for local culture conditions. Sexual production has more constraints than asexual reproduction. One of the dominating factors is the rotifer culture density. 2.2. and the occurrence of a ‘density-dependent factor’ associated with resting egg production has been suggested (Hino & Hirano 1976).4. rotundiformis is genetically determined (Hino & Hirano 1976.1 Selection of species and/or strain The selection of the strain is the most crucial step in initiating mass cultures. there is a large variation between cultures in the number of resting eggs they will produce under identical culture conditions. Frequent renewal of culture media has been implicated in the reduced production of resting eggs. even if the optimal environmental conditions are provided. as well as in mass production of rotifers (Hagiwara et al. Sexual reproduction is restricted by population density (Snell & Boyer 1988) and its occurrence requires more optimal conditions of food availability. densities exceeding about 150 rotifers/ml result in a lower production of resting eggs (Hagiwara et al. plicatilis (Gallardo et al. While a threshold density (approximately 10 rotifers/ml) is required to attain successful fertilisation. Lubzens 1989). the stimulus for initiating sexual reproduction is still poorly understood. salinity and temperature than those supporting asexual reproduction (Lubzens et al. 1999). plicatilis rotifers but reduced resting egg production in B. Snell 1986. . rotundiformis (Hagiwara & Lee 1991). This means that not all rotifer cultures will show sexual reproduction. rotundiformis in the environmental conditions that encourage resting egg production. 1997). 1994). Hagiwara et al. 1997. 1993. Moreover.3 Sexual reproduction and resting egg formation It is widely accepted that the occurrence of sexual reproduction in B. within each species.Production and Nutritional Value of Rotifers 31 2. while higher temperatures and relatively higher salinities will encourage higher resting egg production in B. although several studies have been conducted to elucidate the environmental requirements for amictic and mictic production. 1985. 2. 1988b. the selection of the appropriate strain or culture is imperative for successful production of resting eggs. A semicontinuous system that maintained rotifer density improved resting egg production of B. The optimal conditions for encouraging the production of resting eggs also differ between B. Serra & King 1999). 1997). Specific bacterial strains were reported to encourage resting egg production (Hagiwara et al. rotundiformis cultures. plicatilis and B. Therefore. 15.. Lubzens 1989. Monochrysis (now Pavlova) lutheri.5–8. frozen or dried algae. As reproductive rates increase in a curvi-linear manner with the food concentration. Nitzschia closterium.23 to 1. The rate of reproduction of cultures is determined as r (sometimes referred to as G): r 1 ln ( Nt T N0 ) (2. rotundiformis rotifers were 0. Hagiwara et al. The r-values for B.96 when rotifers were fed with Cyclotella cryptica. 0. 30 or 35°C. frozen or live algae support higher reproductive rates than yeast or dried algae (Lubzens et al. type of food and its quantity all modulate the type of reproduction and its rates. The routine practice is to make a daily count of the number of rotifers and the number of eggs they carry in 1 ml samples. rotifer species and rotifer density. Lubzens et al.90.69. since sexual reproduction results in males and resting eggs. calculating the increased daily increment. 1–4 g of baker’s yeast (or 30% of this weight as dry yeast) is supplied per million rotifers per day. rotundiformis values from 0. reviewed in Fulks & Main 1991) and the pH affects the percentage of un-ionised ammonia (NH3-N) in the . The amount varies according to the temperature of the culture. dry yeast. 1989.51.1) where T duration of culture in days. 30 or 35°C. 1995a.2 Maintaining water quality in culture tanks The optimal range of pH for culturing rotifers is 7. Eutreptiella sp. and for B. plicatilis (Nagasaki strain) cultured in seawater at a salinity of 17 ppt and fed with N.54 to 1. respectively (Hirayama et al.92 or 0. rotundiformis rotifers (Hirano 1987). plicatilis strains usually range from 0. comparisons between the efficacy of various food sources should be made at the Km or saturation values for each food type (Hansen et al. For example. 1997).. Male are nutritionally inferior to females owing to their fast swimming behaviour and lack of a digestive system. were 0.15 or 0. salinity (more is needed at lower salinities and at higher culture temperatures). live. A careful calculation is required on the optimal trade-off between the higher cost of algae and increased rotifer production. Hansen et al. salinities. oculata. 2001) and its amount directly affects reproductive rates. or Synechococcus elongates.20 at 25. 0. Mass production of rotifers is better achieved by encouraging rotifers to reproduce asexually. 1995b. Under the same culture conditions. The amount of food that has to be supplied daily to each culture tank depends on the reproductive rate of the rotifers. 2001). 1979). The maximal r-values were 0. and resting eggs do not hatch immediately.5 (Hirano 1987. and 1 g of yeast can produce about 80.74. 1979. The type of food (wet yeast. It has been calculated that 105–107 yeast cells need to be supplied daily for each cultured rotifer.000 B.52 at culture temperatures of 25. which means they cannot be enriched with essential nutrients required by fish larvae. Chlamydomonas sp. the r-values for B. 1995b) that do not reproduce sexually or by culturing rotifers at relatively high salinities. plicatilis or 100.77 and 1. respectively (Hagiwara et al. 0. 0. depending on salinity and temperature (Hirayama et al. 0. 1995b). the r-values observed for B.3. Lubzens et al. The number of resting eggs produced by one female is significantly lower than the number of eggs produced parthenogenically.000 B. 2. N0 initial number of rotifers and their eggs. In general. 1. Nt total number of rotifers and their eggs after T days of culture.32 Live Feeds in Marine Aquaculture Culture temperatures.55. Usually.54. Sexual reproduction can be avoided by using specific genetic strains (Hino & Hirano 1976. respectively. 1997).37 have been recorded. Hagiwara et al. two avenues have been explored: first. Adequate aeration should be provided using perforated polyvinyl chloride (PVC) tubes. plicatilis cultures. The pH of the cultures plays an important role since the toxicity of NH3-N released from ammonium (NH 4 -N) is a function of the pH. or Euplotes sp. nitrite levels by 85% and nitrate levels by 67%. to avoid the accumulation of excess organic matter in rotifer culture tanks. it encourages bacterial growth.Production and Nutritional Value of Rotifers 33 water. 2. or sink to the bottom. At lower temperatures (20–25°C). Moreover.2. The problem is particularly acute in cultures with low rotifer density (less than approximately 50 ml 1) that are fed with baker’s yeast. temperature and salinity. there is a lower rate of reproduction of bacteria. the success or failure of rotifer cultures in the presence of specific bacterial .2. air-stones or small-diameter open-ended tubes. The use of ozone is extremely useful for high-density rotifer cultures in a recycled system (Suantika et al. because they are maintained at lower temperatures than B. forming particles. Zoothanmium sp. the food is not consumed quickly enough and significant amounts will either remain suspended. In general.. There is no need to illuminate rotifer cultures and in most facilities they are exposed to natural daylight. resulting in a lower bacterial load in the culture and several contaminant protozoans (e.) do not proliferate as quickly as at higher temperatures ( 25°C or even slightly higher). This can easily be avoided by dividing the daily food ration into four to six meals a day or by continuous feeding using a peristaltic pump. thereby increasing ammonia and nitrates. The limited capacity of rotifers to filter the culture medium means that this volume should contain all the nutrients required to meet metabolic needs and support reproduction. At low rotifer density. Rotifer cultures require aeration and the dissolved oxygen level should be maintained above 4 ppm (Fulks & Main 1991). Ammonia levels in this system were reduced by 67%.3. Ozone also reduced the number of particles and the number of bacteria in the culture water of B. In addition to its effect on lowering pH.2 Bacteria and other organisms in the culture tanks When considering the role of bacteria in rotifer culture tanks. Pure oxygen gas should be provided through perforated tubes in high-density rotifer cultures (see below).g. Direct illumination or exposure to sunlight may encourage uncontrolled growth in some filamentous algal species that are not consumed by rotifers. The optimal level for ammonia is 1 mg l 1 and the acceptable range for ammonia and nitrate levels is 6–10 mg l 1. 2. Vorticella sp. as it takes longer for a culture to collapse. especially if the rotifers are only fed once daily. plicatilis cultures. it is highly advisable to consult the relevant tables in Bower and Bidwell (1978). A careful balance must be maintained between the density and number of rotifers and the allotted food ration. 2001). it is possible to take preventive measures to avoid the complete loss of B. the water quality parameters are better in B. rotundiformis. Owing to the extreme importance of these parameters. plicatilis.1 Organic particles Surplus food is one of the main contributors to the deterioration in water quality in culture tanks. more oxygen can be dissolved in seawater (a higher saturation point).3. and this may lead to increased pollution of the culture media. as the volume filtered by all the rotifers is not sufficient to clear the water of the food particles. 1989). 1999a). spores that were introduced into the culture medium of rotifers increased rotifer production and growth of turbot larvae (Gatesoupe 1991. 1991a. Several bacteria species are beneficial to rotifer culture since they produce important metabolites such as vitamin B12 (Scott 1981. Hirayama & Funamoto 1983. Two types of culture can be distinguished. 2. Hino et al. The bacterial microflora of rotifers from culture tanks can be artificially changed by incubation for 1 h in bacterial suspensions consisting of one or more probiotic strains. and act as a direct source of food (Aoki & Hino 1996. fungi or yeast that may be harmful to rotifers. non-pathogenic bacteria may not only serve to curtail the proliferation of pathogenic bacteria attacking rotifers or fish larvae. may also be present (Colorni et al. 1995a. In addition to bacteria. 1997). those species that may positively or negatively affect the culture of fish larvae when they are introduced to fish larvae tanks along with the rotifers. 1991. Maeda & Hino 1991. Hagiwara et al. 1997). or for maintaining species and genetic strains. such as ciliates. Yu et al.3. rotifers can serve as vectors for probionts that will colonise the larval gut (Fjelheim et al. but also be used to introduce beneficial microbial fauna (‘probiotics’) into the digestive system of the fish larvae via the rotifers. Probiotics are defined as ‘microbial cells that are administered in such a way as to enter the gastrointestinal tract and be kept alive. 1999) and prevent disease outbreaks (Grisez et al. encourage sexual reproduction and resting egg formation (Hagiwara et al. Comps & Menu 1997. Comps et al. Introducing selected. copepods or cladocerans. the culture water may harbour many other organisms such as viruses. Lactic acid bacteria and Bacillus sp. Alternatively. 1993).3 Choosing the most appropriate culture techniques Rotifers can easily be maintained in all scales of culture. with the aim of improving health’ (Gatesoupe 1999). and this temperature will generally be lower for B. It should also be noted that the culture temperature may have an important effect on both the type and rate of proliferation of bacteria.b. bacteria that are toxic to rotifers have also been reported (Yu et al. 1988. It is extremely difficult to obtain bacteria-free rotifer cultures. the first of which are small-scale laboratory cultures for studying rotifer biology and physiology. 1997.34 Live Feeds in Marine Aquaculture species and. The Vibrio spp. 2000) and the treated rotifers will transmit this bacterial gut flora to the fish larvae’s guts after ingestion. In these cultures. 1990). 1994). However. The probiont bacteria persist in the rotifers’ microflora for 4–24 h (Markridis et al. are among the main pathogenic bacteria infecting fish and they grow especially quickly under the low oxygen concentrations that may prevail in rotifer cultures. plicatilis rotifer cultures. Organisms that may compete with the rotifers for their food. Rombaut et al. secondly. as sterilisation methods and the use of antibiotics are not very effective (Maeda et al. non-pathogenic species. 1997). possibly increasing their stability by reducing the reproductive rate of bacteria. Jung et al. they may grow more rapidly than other bacteria and eventually dominate the bacterial assemblage. These cultures are maintained as a ‘live library’ in small volumes and serve to initiate mass cultures with . Opportunistic bacteria that are pathogenic to fish are common in seawater and may proliferate in the seawater used for live food cultures owing to the high loads of organic matter (reviewed in Skjermo & Vadstein 1999). One way of overcoming this problem is to regulate the bacterial species occurring in the rotifer cultures by introducing selected. 3. while B.Production and Nutritional Value of Rotifers 35 specific important traits. The cultures are fed ad libitum every 2 days with concentrated algae and the amounts will vary with the reproductive rate of the cultured rotifers. 2. under constant illumination. 30 or 35°C. The old cultures are kept (even without feeding) at room temperature. Caution is needed not to provide too much food as this may reduce reproductive rates and result in the collapse of the culture. Culture procedure Ehrlenmeyer flasks (100 ml in volume) or 50 ml sterile disposable tubes can be used for culture.2 m membrane filter and heat sterilised at 100°C at atmospheric pressure for 30 min to avoid the formation of insoluble precipitates. rotundiformis cultures should be kept at 25. two or more copies of each culture are maintained at all times. sterile seawater can be kept for several days at room temperature or several weeks in a refrigerator.3. Rotifers in this type of culture can also be maintained in seawater prepared from dry sea salts or in artificial seawater. This depends on the species. Algae are . temperature and salinity. depending on the strain or species of the rotifer. Usually. Each heat-sterilised flask or sterile tube is filled with 10–20 ml sterile seawater and 40–60 rotifers are added. Culture of algae Culture of Nannochloropsis sp. The following description demonstrates the culture procedure for rotifer species and strains in the authors’ laboratory using Mediterranean seawater (40 ppt). Brachionus plicatilis cultures are best kept at 20 and 25°C.1 Small-scale laboratory cultures The aim of these cultures is to maintain their specific genetic traits for long periods and facilitate their availability to mass culture facilities whenever they are needed. 25 or 35°C. a salinity of 30 ppt is suitable for most strains and also ensures that in most cultures asexual reproduction will prevail. and salinity. Seawater Natural seawater should be filtered through a 0. was found to be the most convenient source of food for rotifer cultures. until the cultures are renewed. adaptation to high or low culture temperatures. such as size. A drop of concentrated algae (see below) is added to each culture and the flasks or tubes are incubated at one or more of the following temperatures: 20. by placing each one at a different culture temperature. and empirical experience is the best way to determine the appropriate amount. if the bottles are kept sealed. Sterile distilled water is used for dilution of seawater whenever required. as a back-up. Cultures are renewed every 7–10 days. While these cultures do not reproduce at optimal rates. The second type comprises mass cultures for supplying rotifers in the quantities required for intensive larval fish production. The culture medium is prepared from natural seawater enriched with a modification of Guillard f/2 medium (Lubzens 1981). The cooled. Nannochloropsis sp. originated from Japan and is cultured in 2–3 litre batches at 25°C. To avoid loss of cultures. the rotifers remain alive and can be used for replacement in case of failure of the previous renewed cultures. strain. Four types can be described for this system (Table 2. A small number is used to inoculate newly prepared ‘green Table 2. In Type I cultures.3. rotifers are introduced at low density into ‘green water’ produced in fertilised tanks or ponds.3. 20–100 t Growth of phytoplankton (‘green water pond’) Type II Indoor. 2. and are used as food for fish larvae.000 ml 1) Feed with concentrated Chlorella Step 2 Inoculate rotifers at low density (1–5 ml 1) Culture rotifers until density reaches 20–50 ml 1 Step 3 Increase the volume Culture until rotifer Culture rotifers of the culture density reaches for 2–3 days according to the 100–200 ml 1 reproductive rate of rotifers to maintain relative high density Sieve the whole culture to harvest rotifers Reinoculate a new tank and clean old tank and pipes with chlorine solution Enrich rotifers 4–5 days Sieve the whole culture to harvest rotifers Reinoculate a new culture tank and clean old tank and pipes with chlorine solution Enrich rotifers Depends on reproductive rate Sieve rotifers Step 4 Sieve the whole culture to harvest rotifers Reinoculate a new ‘green pond’ with part of the rotifers Step 5 Reinoculate a new culture tank and clean old tank and pipes with chlorine solution Enrich rotifers 2–3 days Step 6 Duration of culture Enrich rotifers Unpredictable . The algal pellet is resuspended in a small volume of culture medium and kept in 1–2 ml aliquots (in Eppendorf tubes) at 4°C for 2–4 days or frozen at 20°C for up to 12 months. 1980.3 Types of batch culture method. Batch cultures This type of culture was initially developed in 1964 (Hirata 1964. 2001).3). Type I Place and volume of cultures Step 1 Outdoor. semicontinuous cultures and continuous cultures. 1–5 t Inoculate rotifers at high density (50–100 ml 1) in one-fifth volume of the culture tank Feed with yeast Type III Outdoor.36 Live Feeds in Marine Aquaculture cultured for 4–6 days and harvested in the log phase period of growth by centrifugation at 4200 g. for 15 min at room temperature. Rotifers are collected (‘harvested’) after all the algae have been consumed. 10–100 t Inoculate rotifers into a large tank at a low density (10–20 ml 1) Feed with yeast Type IV Indoor. 1 t Inoculate rotifers into 1000 l tank at high density ( 5000– 10. see Hirata 1979.2 Mass cultures Three methods are used today for obtaining large numbers of rotifers: batch cultures. for reviews. Lubzens 1987) and has been greatly modified in recent years by many fish hatcheries (Lubzens et al. 1997. Aquafauna. Rotifers are inoculated at a density of 250 ml 1 and harvested after 3 days at 750 rotifers ml 1 and the cultures seem very stable.. rotundiformis) are batch-cultured at 2–3 day intervals (Type IV). The culture system depends on short (1 week) production cycles with a cleaning interval of tanks and aeration tubing. owing to the short culture cycle. The volumes of the tanks range between 3 and 10 m3. Most of the concentrated rotifers are enriched (see below) with essential fatty acids and proteins before being fed to fish or crustacean larvae and a small fraction is used to inoculate the new culture tanks. One of the advantages of using the Algamac 2000 is that the rotifers are already enriched during their culture period. Rotifers are fed mainly with baker’s yeast and the daily ration is divided into three to five meals. one tank is harvested and another is inoculated. Each day. unpredictable collapse of at least one culture per week. The final volume of the tank is reached after 4–5 culture days. other dry food or concentrated algae. USA) per 106 rotifers per day. Another approach to mass culture is based on inoculating rotifers into the culture tank that contains the final volume of culture medium (Type III). Practical experience has shown that rotifers are not affected by this brief immersion period in freshwater. This may reach 300–500 rotifers ml 1 at the end of the culture period. and their number should take into account an occasional. A recent adaptation (Lubzens et al. followed by thorough rinsing with seawater before reuse. 2001). at an initial low rotifer density. 1996. The concentrated rotifers are immersed briefly (5–15 min) in dechlorinated freshwater to remove ciliates and other undesired organisms. These high-density culture systems consist of 1 m3 tank units in which rotifers (B. The rotifers are then sieved and concentrated. The introduction of refrigerated and condensed freshwater Chlorella enriched with vitamin B12 (Chlorella Industry Co. using 1 g (dry weight) of yeast and 0.000–30. and the culture tank and its accessories (sieves. This is in addition to the low cost of food for this rotifer production system. at the early stages of the culture. As the density of rotifers increases. Balompapueng et al. 1997.000 rotifers ml 1. plicatilis batch culture system in 600 litre cylindroconical fibreglass tanks. aeration tubes) are sterilised in hypochlorite solution (10 ppm of commercial grade bleach) for 24 h. The volume of culture medium is increased daily with seawater depending on the reproductive rate. Cultures are initiated at a density of 10. and harvesting is performed when high densities ( 200 rotifers ml 1) are reached. 1997a). the surplus of food remaining in the tanks will encourage bacterial growth and possible reduction in the pH. Japan) has facilitated the maintenance of extremely highdensity rotifer mass cultures and changed dramatically the future potential for providing rotifers for fish and crustacean hatcheries (Yoshimura et al. at 22–23°C and 20–25 ppt salinity. This system is less stable than the one described previously since. The rotifers are fed with yeast. depending on the number of rotifers needed in the hatchery. This safeguard can be reduced as experience is gained in the hatchery. were reported recently on a 3 day cycle of B. Preliminary experiments (Ressem et al. 1997) for batch culture (Type II) relies on a series of tanks. The large number of rotifers needed for starting the first round of these cultures can originate from scaling-up a small initial culture by . The culture in each tank is started with one-fifth of its volume with rotifers at a density of approximately 200–300 ml 1.1 g (dry weight) of Algamac 2000 (Bio-Marine. and after 2–3 days with concentrated algae the culture density reaches 20. thus maintaining a relatively high rotifer density in the culture.000 rotifers ml 1. This system is based on six tanks.Production and Nutritional Value of Rotifers 37 water’ ponds. the amount of surplus organic material from food is reduced and the system becomes more stable. Semi-continuous cultures This culture system relies on periodic (usually daily) harvesting of rotifers by removal of part of the culture medium and replacing it with new seawater (see Hirata 1980). At the same time. In general. alternatively. in turn. it is used as fertiliser for algal cultures. fungi and bacterial flocculations. viruses. which increases with increased pH. these large-volume tanks harbour many other organisms that either compete for food with the rotifers (e. In hatcheries. copepods. A special nylon filtration mat is used for removal of large amounts of suspended organic material that may also include protozoans. without enlarging the hatchery facilities. This method has been termed the ‘thinning method’ (Fukusho 1989b). ciliates. after its breakdown to nitrogen. from hatched resting eggs if they are available (see below for further discussion of this issue). as mentioned previously. Fukusho 1989b. fungi or yeast). In these ‘feedback’ cultures (Hirata 1980). 1989). The small space and reduced labour required are the main advantages of this ultra-high-density culture system. The pH of the culture also increases. The relatively high culture temperatures ( 25°C) required by small rotifers can be easily maintained in these compact systems.0 by hydrochloric acid minimises these effects. Hirano (1987) suggested that 6–7% of the biomass can be removed daily in rotifer cultures maintained on baker’s yeast. Since these . excretory products (solid wastes and nitrogenous products) that accumulate in the tank lead to their collapse. 1997). or populations of different sizes.38 Live Feeds in Marine Aquaculture traditional mass culture methods or. at these high densities.g. Regulation of the pH at 7. The volume removed depends on the reproductive rate of the rotifers and harvesting removes only the number of rotifers gained by reproduction from the previous harvesting period. and providing them with this size variation increases their survival (Lubzens et al. and these can be more easily provided by the compact. In addition. fish cultured at low temperatures can benefit from this system by using rotifers cultured at relatively low temperatures. these systems often rely on large volume tanks ranging from 3000 to 300. Fish larvae prefer smaller rotifers immediately after hatching and larger ones as they grow. eventually. Lubzens et al. However. This.g. which may be harmful to the fish larvae. The convenience and accessibility of concentrated algae encourage its use also in intensifying traditional culture systems. results in a higher proportion of toxic un-ionised ammonia of the total ammonia. this relatively compact. Continuous removal of the solid waste enhances rotifer reproduction and results in densities reaching 400 individuals ml 1 and the cultures are stable for over 30 days with daily harvesting periods. bacteria. Cultures continue for several days or even weeks but. Thus. These cultures are characterised by relatively lower rotifer density (100–300 rotifers ml 1) and baker’s yeast is used as food. it assists in providing a variety of rotifers for larvae of different fish species. carbon and phosphates. the solid waste is transferred to decomposing tanks and. closed system permits the maintenance of separate cultures of several genetic rotifer strains. salinity and the culture temperature. ammonia excreted by the rotifers becomes a significant problem. Most hatcheries aim to culture a variety of fish species that may differ in their nutritional requirements during early life stages. presumably owing to the liberation of carbon dioxide from the water. cladocerans) or harm them (e.000 litres (Hirata 1980. Oxygen gas has to be supplied to these cultures to overcome the shortage of dissolved oxygen that results from the high amounts of food and the subsequent increase in the rotifer population. by the oxygen gas supplied to the culture. high-density cultures. 1983. 1991. Log-phase produced rotifers can be harvested continuously and their nutritional quality is maintained by providing adequate food organisms (James et al. Markridis et al. At these dilution rates. plicatilis. plicatilis) culture tank. 2000. oculata powder and the effect of daily dilution rates of 0. 1990). at a salinity of 18 ppt and under 2500 lux of continuous illumination. a settlement tank for suspended particulate matter. Blanch et al. 1999). 1997). respectively. The average conversion efficiency at these conditions was 0. 2001) improved its performance significantly by supporting a higher rotifer biomass (16. the cultivation of rotifers with selected. This system consists of a 100 litre rotifer (B.000 rotifers ml 1) and prolonging by 4 days the duration of rotifer production. with the density reaching 3000 ml 1 after 8 days of culture and being maintained at about this level for over 1 month. They offer easy manipulation of rotifer physiological and nutritional quality.6 was investigated to establish the best production (mg rotifers day 1) and food conversion efficiencies (mg rotifer developed mg 1 microalgae consumed) for B. the authors recommend that rotifer production should be performed at a lower density. The amount of food provided to the rotifers in this system is adjusted to the circulation flow rate and loss of feed by the protein skimmer.b) to curtail the growth of undesired micro-organisms and contribute to the stabilisation of the rotifer cultures (for further details see Section 2.5 and 0. 1999. rotundiformis may be attributed to the relatively low culture temperature (25°C) used for this thermophilic species. semi-continuous culture system using dried Nannochloropsis powder was described recently (Navarro & Yufera 1998). Verdonck et al. 0. 1987. The application of this system on a large scale awaits additional experimental results. rotundiformis. High-density semi-continuous culture. Walz et al. Rombaut et al.30 mg rotifers mg 1 algae for B.2).12 0. using a modified commercially available formulated rotifer diet with recirculation of the culture media. respectively. Introducing ozone into this system (Suantika et al.3 for B. 0. the rotifer production reached 16. rotundiformis and B. oxygen supply and density of cultured organisms) and highly dependable (Walz 1993. A more recent adaptation of this method involves high-density cultures (excess of 10. The total rotifer production in this system is 1. plicatilis or B.2. plicatilis and 6.76 and 0. An experimental. James & Abu-Rezeq 1989a.08 mg l 1 day 1 for B. rotifers were cultured in 1 litre flasks. pH.1.75 0. Adequate water quality is maintained by a daily 500% recirculation rate. Munro et al. Continuous cultures These cultures are based on the chemostat or turbidostat models of micro-organisms and are fully controlled (temperature.Production and Nutritional Value of Rotifers 39 and other pathogens can be transferred to the fish tanks (Gatesoupe 1990. 0. rotundiformis. 0. 2000).4.3. The lower efficiency for B. a protein skimmer and a biofilter.3. They were provided daily with 25 mg of dried N. Although it is possible to obtain densities of 8000 rotifers ml 1 after 8 days of culture following inoculation.2 and 0.37 mg l 1 day 1 for B. was described recently (Suantika et al. with gentle aeration in a thermoregulated chamber at 25°C.7 109 rotifers over 32 culture days. In this system. Rotifer cultures are initiated at a density of approximately 250 rotifers ml 1. This means that under optimal conditions.b. rotundiformis. 76 and 30% of the microalgae biomass was transformed into rotifer biomass of the respective species.2. 1997. About 20% (3–6 107 rotifers) of the standing stock is harvested daily. The results showed that the optimal dilution rates were 0.000 rotifers ml 1) using the . plicatilis and B. non-pathogenic bacterial strains has been suggested (Gatesoupe 1999. 1999a. where the system is more stable. it should be noted that their biomass (depending on their size) is at least three times higher (expressed as dry weight. plicatilis. Moreover. An example of an indoor 100 m3 rotifer culture tank is shown in Fig. and mass cultures are performed in volumes ranging from 150 to 300.6 Concrete rectangular culture tank with maximum capacity of 100 m3 seawater used in culturing rotifers in Japan. The high rotifer density cultures relying on Fig. Hino et al. While more work is needed to optimise the culture system for B. Culture tanks A very large array and configuration of culture tanks is used for semi-continuous and batchculture rotifer mass production systems.6 and an example of a 500 litre cylindrical tank with a conical outlet is shown in Fig.7–3. and 500 litres provides 0. 2. (Photograph: Esther Lubzens. 2.40 Live Feeds in Marine Aquaculture concentrated freshwater Chlorella (Fu et al. rotundiformis. polycarbonate or plastic. plicatilis daily).7. square. 1997) than that of B. 1997). However. fibreglass. pp. continuous culture system described by James & Abu-Rezeq (1989a. cylindrical. their initial cost exceeds that of more conventional installations and they depend on a constant supply of concentrated algae. in terms of both volume and shape. chemostat-like systems may be attributed to bacterial growth and consumption by the cultured rotifers.000 litres (or 300 m3). rotundiformis daily. plicatilis produced per day may also be attributed to their lower metabolism and rate of reproduction at the optimal culture temperatures (20–24°C). 2. Bacterial nitrogen corresponding to approximately 20% of the algal feeding was consumed by rotifers in systems operating with unlimited food supply (Aoki & Hino 1996.27 billion B. conical or rectangular. These systems are compact (1000 litres of culture provides 1. or nylon disposable bags hanging on metal frames. and these have been summarised by Fulks and Main (1991.b) relies on 1000 litre culture tanks.) . This means that a similar biomass per culture volume will consist of a smaller number of individuals. 1997). Yufera et al. These culture tanks are made from concrete. Part of the efficiency of rotifer production in these intensive.5 billion B. 323–326). The chemostat-type.13–0. the lower number of B. They are round. the old system of rotifer culture tanks at the National Mariculture Center (Eilat. Eilat. This Fig. near the outlet of the concrete tank.) Fig. 2. (2000) consisted of 100 litre cylindroconical tanks. 2. The large 6–300 m3 concrete tanks are usually used for outdoor. For this reason.8 Round 20 m3 concrete tanks for culturing rotifers at the National Mariculture Center. used for rotifer collection (or ‘harvesting’). Israel). (Lubzens et al. These tanks are difficult to clean.Production and Nutritional Value of Rotifers 41 concentrated Chlorella cells use 100 or 1000 litre cylindrical tanks (Fu et al. In some places. 1997). 1997). and the system designed by Suantika et al. (Photograph: Esther Lubzens. where rotifer density is relatively low. (Photograph: Esther Lubzens.7 Cylindrical 500 litre fibreglass culture tanks. Eilat. Note the small tank at the lower part of the picture. semi-continuous cultures. Israel. and bacteria or other organisms may accumulate in crevices that are abundant on their rough surface. or is used for harvesting of rotifers.8) has been abandoned and replaced with an adaptation of the system devised at the Israel Salt Co.) . consisting of six. 20 m3. 2. these tanks have a conical bottom with an outlet that permits the removal of accumulated debris at least once a day. concrete. with conical bottom outlets for culturing rotifers at the National Mariculture Center. Israel. circular tanks (Fig. the policy that can be adopted by a new facility is to plan on a tank containing a volume that will suffice for 1 day’s supply of rotifers in the hatchery. For example. Most batch culture or continuous systems are performed in relatively small volumes. the accidental loss of a culture in a large-volume tank will obviously entail the loss of a larger number of rotifers. The tanks can be easily cleaned with bleach and freshwater every 5–7 days. The decision on the choice of the type and shape of culture tanks depends greatly on the available facilities and budget. One of the advantages of the smaller volume batch cultures is that they can be placed indoors (in contrast to the large-volume.000 litres. The tanks are shaped with a slanting bottom.000 litres. until the culture systems have become completely reliable and predictable. The volume of the culture tank depends on the expected production in the hatchery and the convenience of handling. Co. the use of fibreglass rectangular or circular tanks has proved to be beneficial in the authors’ facilities.42 Live Feeds in Marine Aquaculture Fig.9 Rectangular bathtub-shaped fibreglass tanks (3000 litres) for culturing rotifers (Salt. Note the cylindrical tanks with conical outlets in the background used for nutrient enrichment of rotifers or Artemia. Taking into consideration the greater reliability of the batch culture system. 2. independent of the volume. with volumes ranging from 3000 to 10.9). Israel).) alteration also involved changing from a semi-continuous culture system to batch culture production. New culture facilities should also maintain one extra tank. at the end of each batch culture. However. ranging from 150 to 3000 or even 10. leading to an external outlet that facilitates removal of debris and rotifers. This means that it is more cost-effective to maintain a small number of large-volume tanks than a large number of small-volume tanks. 1997). The smaller volumes (150–2000 litres) can be cultured in nylon disposable plastic bags that are placed on a circular or rectangular metal frame and have the great advantage of being used only once for 2–6 days. .. concrete tanks that are usually placed outdoors). thus avoiding the colonisation of the rotifer cultures with other organisms. Rotifers have to be counted and inspected daily from each culture tank. 2. The most reliable system in Israel consists of smooth-surface rectangular ‘bathtub’-shaped tanks made from fibreglass (Fig. This will mean that the hatchery’s culture will consist of six or seven tanks. (Photograph: Esther Lubzens. where every day one tank is harvested and one tank is inoculated with a new culture (see also Lubzens et al. culture temperature. reliability and the practical experience of the staff are the main factors that dictate the choice of system. pH and ammonia levels. 2001). the cost of labour is one of the main concerns in these calculations. Six parameters have been used as early warning signals for the state of the culture and these may be strain specific. The production rate can also be improved by using concentrated algae. 2001) on successful incorporation of n-3 highly unsaturated fatty acids (HUFA) by Chlorella cells will eliminate the need to enrich rotifers mass cultured with this alga. Several other companies have been involved in marketing concentrated algae with prices ranging from US $300 to 400 kg 1 of dried algae or US $50–65 for 1 litre of concentrated algae (16–20% dry matter). the egg ratio depends on food quality and quantity and is affected by abiotic parameters such as oxygen level. Usually. Therefore. depending on the species. salinity. North America and Japan. purchasing quantities and shipment destinations.Production and Nutritional Value of Rotifers 43 Choosing the best system Cost of production. and higher resting egg production. they offer several advantages: higher rotifer reproductive rates. Any deviation from optimal conditions of these parameters will be . 1997). 2. The cost of production of rotifers depends greatly on the total scale of production. Recent reports (Hayashi et al. prior to feeding them to the fish larvae. is an important indicator of the status of a culture. improved stability of rotifer cultures. whereas the cost of equipment is the main concern in developing countries. allowing them to be used in high-density culture systems.4 Advanced Warning on State of Cultures Evaluating the physiological state of the rotifer culture is extremely important in hatcheries since larval production depends on a predictable and reliable daily supply of rotifers. The cost of concentrated 18 litre canisters containing concentrated Chlorella at a density of 20 billion cells ml 1 ranges from US $140 to 150 (Hagiwara et al. with an estimated cost of US $0.15 in a 1 billion per day production system (Lubzens et al. Three different 1 ml aliquots should be counted to obtain a more reliable estimate and more aliquots should be counted if the variation exceeds 10% between the counted rotifers in three aliquots. The total number of females and the total number of eggs in 1 ml subsamples (from the 30–40 ml sample) is counted and the ratio E/N is calculated for each sample. In general.4. determined as the number of eggs (E) divided by the total number of females (N) in a sample. The reliability of each system dictates the number of replicate culture tanks that should be installed to meet the required production.04 per million rotifers in a system producing 4 billion per day. and US $0. 2. a sample of 30–40 ml or even larger is removed daily from each culture for determining the state of the culture (see Appendix I) and for evaluating the amount of food that should be provided to that specific culture. the egg ratio (E/N). In Europe. with fewer replicate cultures needed in more reliable systems.1 Egg ratio The number of eggs predicts the state of the culture for the forthcoming 24 h. Although these are more expensive than yeast. 1995). An adaptation of this test to testing water from rotifer mass cultures should be considered in the future. 2. 1998).4. 2001). of the state of a rotifer culture. plicatilis cultures indicated their instability and possible future collapse (Snell et al. size and physiological condition are provided from hatched resting eggs. ingestion rates. The tests are based on reduced enzyme activity in rotifers exposed to toxins (Burbank & Snell 1994. except that a short duration at low temperatures will not reduce the egg number. in addition to others. Rotifers in mass cultures have . resulting in reduced swimming speed. The critical ratio is strain specific.4. 2. rotifers of similar age.4. has been suggested as a monitoring tool for water quality in rotifer culture tanks (de Araujo et al. direct measurements of viscosity could be an indicator of approaching problems in maintaining culture stability.13 for B. 2000. in one study an egg ratio of less than 0. In these tests.6 Diseases Occurrence of disease will eventually lead to the collapse of the culture and. Snell & Janssen 1995.4 Viscosity It has been demonstrated that the relative viscosity of the culture medium increases with the age of the culture. This phenomenon is of great importance in the high-density continuous cultures. a low or high egg ratio should be considered as one of the indicators. Therefore. For example. early detection is important for taking appropriate measures. therefore.2 Swimming velocity Swimming velocity is a quick indicator of the current state of the culture (Snell & Hoff 1988. Hagiwara et al.3 Ingestion rate Ingestion of fluorescent labelled beads by neonates hatching from eggs was reported to be related to water quality parameters (Juchelka & Snell 1994). 2. 2.4. It is not yet known whether rotifers removed from mass cultures are suitable candidates for these tests.4. It is reduced at high un-ionised ammonia concentration and at starvation. 2. Therefore. 2001. where the culture media contain high concentrations of concentrated algae and excretory products (Hagiwara et al. Extremely low or high values of temperature and pH have the same effect. mean longevity and mean number of offspring of the rotifers.44 Live Feeds in Marine Aquaculture reflected by low egg ratios in the rotifer cultures. Korstad et al. 1998). 1987). phospholipases and glucosidases.5 Enzyme activity Changes in the activity of endogenous esterases. the incidence of specific infections. In red sea bream. 1995b). and incubated for 8–20 h with enrichment dietary components that are specifically required by the fish larvae. Zmora. rotundiformis. While there are no known remedies for rotifer diseases.Production and Nutritional Value of Rotifers 45 been observed to be infected by fungal. temperature or diet (Snell & Carillo . Comps et al. physiological processes such as satiation. Markridis & Olsen 1999).5. 1991. it has been observed (Zmora 1991) that providing Nannochloropsis to infected yeast-fed rotifer cultures will reduce the infection rate. caloric value and chemical composition (reviewed in Lubzens et al. As mentioned before. where the rotifers are collected or harvested from the culture tanks into containers where they are kept at very high densities (usually more than 100. 1991a. 2. This is usually performed by a step known as ‘enrichment’. 2000). but the accelerated reproduction of the cultured rotifers fed algae results in new generations that show a low incidence of the disease (O. 1990. Øie & Olsen 1997.1 Number of rotifers consumed by larvae The nutritional value of rotifers depends on their dry weight. the culture temperature may affect the type of bacteria or other opportunistic organisms and the rate of their proliferation. Therefore. In addition to enrichment with protein. Fig.b. egg ratio and health conditions must be inspected daily on samples removed from each culture tank. viral and yeast-like organisms (Colorni et al. Dynamic. These cultures remain susceptible and the diseases may recur if the cultures are exposed to additional stress. plicatilis and B. plicatilis and B. The number of rotifers consumed by the larvae determines the quantity of food reaching their gut. 1997. 1989). size changes occur during the life cycle of rotifers within each population or species. 1989.000 ml 1). the number of rotifers consumed daily increases with the size or age of the larva. as they are cultured at relatively low or high temperatures. Moreover. Polo et al. and this is reflected in their nutritional quality and consumption by fish larvae (Lubzens et al. 1992). Olsen et al. unpublished results). Comps & Menu 1997. 1992) or with probiotic bacteria (Markridis et al. 1989). Yufera et al. from 55–72 rotifers per 3. 1991a. 2.10). Hagiwara et al.4 mm length larva (Fukusho 1989b. 2. it is necessary to ensure that the rotifers are nutritionally adequate for the fish larvae. There is a large variation in sizes between B. Amictic rotifer eggs and the loricae of rotifers are not digested by larval fish in their early developmental stages (Lubzens et al. their type and rate of infection may vary between B. The algae do not cure the rotifers.b. respectively. with rotifers growing in size from the time of hatching from the amictic egg until they reach sexual maturity. 1993. Swimming speed. starvation and reproduction also affect the chemical composition of rotifers (Yufera & Pascual 1989. lipids or carbohydrates.5 Nutritional Quality of Rotifers After establishing mass culture techniques. 1999. Zmora 1991). the rotifers can be enriched with antibiotics (Verpraet et al. 1993. rotundiformis rotifers and between various populations within each species (Fu et al.9 mm length larva to 4700 per 11. Size also depends on culture conditions such as salinity. rotundiformis (approximately 200 ng) and this changes with their reproductive rate (Yufera et al. lipid and carbohydrate content. Markridis & Olsen 1999). 1993. Frolov & Pankov 1992.) 1984). Reitan et al. 9–18% ribose and 0. Nagata & Whyte 1992. Fernandez-Reiriz et al.10 Number of rotifers consumed daily by fish larvae: red sea bream.1 Protein and carbohydrate content Rotifer protein content ranges from 28 to 63% and lipid content from 9 to 28% of the dry weight (Lubzens et al. 1993.8–7. B. 2. At approximately 600–800 ng. Food ration affects the reproductive rate of rotifers and their protein. 1994. The protein content of individual rotifers increases by 60–80% with increasing . mannose. fucose and xylose (Nagata & Whyte 1992). 1993). 1997). Rainuzzo et al.2 Dry weight and caloric value The dry weight of rotifers depends on their size and nutritional state (Lubzens et al. Frolov et al. Nagata & Whyte 1992.0% of galactose.00 × 10 3 cal per rotifer after 6 h enrichment with a formulated enrichment diet (Fernandez-Reiriz et al. Øie & Olsen 1997. (Adapted from Fukusho 1989b. deoxyglucose. The carbohydrate content ranges from 10. Frolov et al. The caloric value was found to depend on the diet and ranged from 1. 1991.3 Biochemical composition 2. plicatilis rotifers are three to four times heavier than B. 1991. 2. Frolov & Pankov 1992.5. 2.3.46 Live Feeds in Marine Aquaculture Fig. 1993) and it is composed of 61–80% glucose (which is present mainly as glycogen). 1989. Fernandez-Reiriz et al. striped knifejaw and black sea bream.5.34 × 10 3 cal per rotifer fed on baker’s yeast to 2.5 to 27% of the dry weight (Whyte & Nagata 1990. All of these factors contribute to the difficulties in comparing results from different publications. 1989).5. 1987. unequivocally. 1983). 1997.3. 1997. since they cannot synthesise them from linolenic acid (18:3n-3). 1997). Nagata & Whyte 1992. Frolov et al. 1995. DHA is present in high concentrations in neural and visual membranes. but the amino acid profiles of rotifers are unaffected by either food ration or type of food provided to rotifers (Lubzens et al. Rainuzzo et al. Eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids (20:5n-3 and 22:6n-3. Accumulating evidence points to the importance of supplying an optimal blend of EPA. respectively) have been known for several decades to be essential fatty acids for the survival of marine fish larvae (Owen et al. phospholipids rather than triacylglycerols are the preferred vehicle for delivery of these polyunsaturated fatty acids (PUFA). or can function in response to hormonal stimulation. which has gained increased attention in recent years (Sargent et al. these acids are essential dietary constituents. In general. 1999). 1993. 1999. sterols. lipid emulsions. 1989. Tamaru et al. EPA or ARA. It has been shown to improve stress tolerance in fish larvae (Koven et al. thromboxanes and leukotrienes. 1991. While it is well known that rotifers can be easily cultured on yeast. 1975. Frolov et al. Moreover.2 Lipid composition The lipid content of rotifers varies generally between 9 and 28% of their dry weight and has been found. or lipids with protein and carbohydrates (reviewed in Lubzens et al. 1999) and that excess ARA may have a deleterious affect (Bessonart et al. including arachidonic acid (ARA. Frolov & Pankov 1992. Nagata & Whyte 1992. microparticulates or microcapsules containing lipids. Estevez et al. 2. Fujita 1979. 1997). rotifers reared in this way are nutritionally inadequate for marine fish larvae as they lack adequate amounts of DHA. These include impaired pigmentation and vision at low light intensities. The quantitative and qualitative lipid content of rotifers can be manipulated by short or long enrichment periods on various diets containing lipid emulsion.8:1:0.12 for turbot (Sargent et al. Sargent et al. DHA and ARA in the diet of marine fish larvae. 1991. About 34–43% of the lipids in rotifers are phospholipids and 20–55% are triacylglycerols. 1991. Rotifers have to be enriched with these fatty acids and enrichment methods include feeding rotifers with algae.Production and Nutritional Value of Rotifers 47 food ration. Sargent et al. Rotifer phospholipids were less influenced by these . 20:4n-6). More specifically. 1999. This probably relates to the limited ability of fish larvae to synthesise phospholipids de novo. 1997. Fernandez-Reiriz et al. 1993. Estevez et al. Watanabe et al. diacylglycerols. leading to low hunting capabilities of the developing larvae and impaired development of the neuroendocrine system (Bell et al. but these are greatly affected by the lipids provided in their diet. Similarly. to have the greatest influence on growth and survival of marine fish larvae. 1989. Fernandez-Reiriz et al. 2001).5. 1993. fish have a limited capacity to convert linoleic acid (18:2n-6) to n-6 HUFA. 1999). and insufficiency in the larval diet may result in serious consequences for a wide range of physiological and behavioural processes. sterol esters and free fatty acids (Teshima et al. It has been suggested that the optimal ratio for DHA:EPA:ARA is 1. Øie & Olsen 1997). ARA is the main precursor fatty acid of eicosanoids that are converted to biologically active compounds including prostaglandins. 1999) and is now also considered an essential fatty acid. with small amounts of monoacylglycerols. Rotifer phospholipids and triacyglycerols display similar fatty acid profiles. Rainuzzo et al. marine fish contain large amounts of EPA and DHA in the phospholipids of their cellular membranes and. Rotifers utilise more DHA in highly reproducing cultures (Øie & Olsen 1997) and lipid utilisation is temperature dependent (Olsen et al. e. Another lipid enrichment source is the freshwater Chlorella (Hayashi et al. They accumulate about three to five times more total lipids when they are kept at 10°C than at 25°C (Lubzens et al. as a live or frozen concentrated paste or after freeze-drying (Watanabe et al. Takeyama et al. 1996). 1997). These oils are usually incorporated into most artificial diets for rotifers. Its content in rotifers was found to depend on the diet. where reproductive rates and utilisation rates are slowed down.48 Live Feeds in Marine Aquaculture diets than the triacylglycerol fraction (Rainuzzo et al. 1995a. Maximal levels were achieved in rotifers enriched with ascorbyl palmitate (Merchie et al. discussed in Lubzens et al. 1983. Vitamin C (ascorbic acid) not only stimulates rotifer growth (Satuito & Hirayama 1991) but also contributes significantly to the survival of fish larvae (Dabrowski & Ciereszko 1993. However. 2001).3 mol% in whole cell extracts of the mutant strain versus 4. Commercial fish oils that consist mainly of triacylglycerols are the main source of DHA or EPA. 1997). The algae can be supplied directly from cultures. 1995b) and these results suggest that higher enrichment levels will be obtained if this procedure is performed at relatively low temperatures (depending on the rotifer strain). 1993). 2. Lubzens et al.2 mol% in the wild type) and can be used as an alternative source of this fatty acid. were suggested as a potential alternative enrichment food for rotifers (Nichols et al. Rotifers fed on algae contain sufficient amounts of these vitamins to meet the nutritional requirements of fish larvae (Lie et al. indicating that lipids are utilised by the rotifers. were quickly depleted when the rotifers were switched to a diet of Isochrysis. 1997). Navarro & Yufera 1998). both of which contain ARA. 1997). fat-soluble vitamins (A. Dabrowski & Blom 1994). A mutant strain of Nannochloropsis that was found to be deficient in EPA (Schneider et al. The most significant increase was in the content of ascorbic acid and thiamin. Their content depends on the fish species. as it is highly abundant in several species of algae. In addition to vitamin B12. 1996. D and E) were found to promote rotifer reproduction (Hirayama 1990). caused by feeding them fish oil emulsions. Nevertheless. except for tuna orbital oil that may contain about 2% of ARA. containing high levels of EPA. 2. More recently.g. The content of water-soluble vitamins in rotifers increased after changing their diet from baker’s yeast and lipid emulsion to Isochrysis.3. The lipid content of rotifers is usually lower than that of their food organism. but they usually are poor in ARA. EPA-rich Nannochloropsis and DHA-rich Isochrysis.4 Effects of starvation One of the main problems in providing rotifers to larvae is the deterioration in their nutritional quality due to starvation that results from extended periods of residence in the fish .5. their final content in rotifers exceeded the recommended levels needed for proper growth of fish larvae (Lie et al. enhanced levels of lipid-soluble vitamins in rotifers. Lipids can also be provided by feeding algae to rotifers. 1995) contains abundant ARA (23. 1991. which was mentioned previously.5. Frolov et al. bacteria isolated from Antarctica. 1989 and in Fulks & Main 1991.3 Vitamin enrichments The importance of enriching rotifers with vitamins has not been studied extensively. 2.1 Preservation at low temperatures Frozen rotifers are not usually adequate as food because of leaching of nutrients after thawing.Production and Nutritional Value of Rotifers 49 tanks. The mobilisation of sterols and wax esters follows the hydrolysis of triacylglycerols and carbohydrates.6 Preserved Rotifers Meeting the demands of fish larvae is a continuous effort from the day of first feeding up to the time that larvae are fed on other food sources (e. It can also be helpful in storing surplus rotifers produced on one day for later use. While storage at 4°C is feasible in most places. preferential degradation of lipids. free amino acids are used as the main energy source by rotifers. Lipids serve as the main source of energy and different lipid classes are mobilised at different rates during starvation. . providing a safeguard against unpredicted culture crashes.b. for at least 1 month (Lubzens et al. and while large differences may occur in the content per rotifer (especially during starvation). their lack of buoyancy and motility.6. protein content) can be presented in either mg g 1 dry weight or ng rotifer 1. About 40–50% of the rotifer body mass is lost during 4–5 days of starvation at 18–20°C and the rate of decrease is positively related to temperature (Markridis & Olsen 1999). 2001a. these may not be reflected in the ratio of mg g 1 dry weight. monoacylglycerols. see below). 1990). These results suggest that newly fed and enriched rotifers should be supplied daily to cultured larvae. and they may also cause a deterioration in the water quality if introduced into the culture tanks. but storage at this temperature requires more specific equipment. which may be performed every few days. The effect of starvation can be partially overcome by supplying algae to the fish tanks (Markridis & Olsen 1999). and this also leads to an increase in the proportion of polar lipids. various methods of storing rotifers have been explored. and the content of n-3 fatty acids is reduced more rapidly than other lipids (Olsen et al. The loss of lipids depends on temperature and can reach 19% of total lipids per day at 18°C. 1995b). An increase in the proportion of diacylglycerols. During starvation. rotundiformis strains (Assavaaree et al. sterols and free fatty acids results from mobilisation of triacylglycerols. During the first 8 h of starvation. carbohydrates and amino acid takes place. 1993). and facilitating transport of rotifers between sites of production and culture facilities. Storage at low temperature can help to reduce the daily tasks of harvesting and enriching rotifers. rotifers continue to reproduce at this temperature and require periodic feeding and exchange of culture media. The chemical composition of rotifers (e. plicatilis can be stored at 4°C at relatively high densities. with lipids and carbohydrates being utilised later. but this period is shorter for the thermophilic B.g. Live B. and coincides with increased mortality of rotifers (Frolov & Pankov 1992). While the usual practice is to depend on daily harvesting of rotifers from live cultures. Rotifers can be kept at 1°C without feeding or water exchange for about 2 weeks (Lubzens et al. leading to an increase in the proportion of proteins in the dry weight.g. 2. Artemia). The protein content of each rotifer is reduced during starvation. but the amino acid composition is rather stable (Frolov & Pankov 1992). Resting egg production is genetically determined (Hino & Hirano 1976. plicatilis strains ranged from 26. 1985. with a lower survival of SS-type strains at low temperatures. 1988a. These results indicate the difficulties in attempting to keep small-type rotifers at low temperatures for extended periods. with large variations between rotifers originating from eggs produced by one clone (Lubzens 1989). plicatilis and B. A clear variation was found between S and SS type strains. 1993. Hagiwara et al.6. Toledo et al. and varies between B. while the SS-type strain survived best at 17 ppt. and the optimal conditions have to be empirically determined for each strain. 2001a. This method ensures full preservation of genetic traits of importance to aquaculture and is especially important for those strains that do not produce resting eggs.2 Cryopreservation Long-term preservation of genetically important strains can be achieved by cryopreservation. extending this method to B. The production of these eggs can be manipulated by environmental factors. exchange of culture media and temperature. Snell & Hoff 1985. . A small collection of cryopreserved B. Since this is a relatively expensive method. 1977. rotundiformis rotifer strains are less tolerant to 4°C than B. rotundiformis strain survived after 15 days at this temperature and only four strains survived after 7 days. (Hino & Hirano 1976. rotundiformis has been examined recently (Assavaaree et al. such as salinity. Hamada et al. rotundiformis species.6. 2.3 Resting eggs Artificially produced rotifer resting eggs have been offered as an alternative route for supplying rotifers without depending on the daily production cycle used in marine hatcheries. rotundiformis rotifers (of an S-type strain and an SS-type strain) survived after 10 days. Hadani et al. Survival of an S-type strain was higher at 35 ppt. before transferring of rotifers from their usual culture temperature (28°C) to 12°C. Lubzens et al. rotundiformis strains is kept at the authors’ laboratory and serves as an alternative source for live cultures. An acclimation period of 24 h at 20°C. plicatilis and B. plicatilis rotifers. Snell 1986. more than 80% of B. it is not suitable for preservation of large numbers of rotifers for direct use as food after thawing. Amictic eggs (but not adults) are preserved in liquid nitrogen after they have been impregnated with cryoprotective agents such as dimethyl sulfoxide (DMSO) or propanediol (Toledo & Kurokura 1990.7% after 30 days at 4°C. 1991. 1988. 1997). Kogane et al. plicatilis rotifer strains. rotifer culture density.9 to 63. 1977). resulted in higher survival of SS-type rotifers but had no effect on S-type rotifers. Lubzens & Minkoff 1988. Lubzens 1987.b.b). 1984. While the mean survival of B. The results show that B. Hagiwara & Hirayama 1993. 1993. Feeding rotifers at intervals of 2 days improved their survival over those fed only at the beginning of the experiment or at intervals of 4 days. and changing the culture media every 4 days suppressed survival. 1992). food quantity and quality. 1985. 1989. 2. only one B. At 12°C. and the strains known as SS type were the most susceptible and showed the lowest survival. Hagiwara & Lee 1991.50 Live Feeds in Marine Aquaculture As storage at low temperature was found to be relatively easy and successful for B. Production and Nutritional Value of Rotifers 51 Basically, it is relatively easy to obtain rotifer resting eggs, but the main problem is the relative number produced in relation to the invested effort. The first step is choosing an appropriate strain, as their production is genetically determined. For B. plicatilis, cultures should be maintained at salinities below 30 ppt and temperatures should preferably range from 12 to 25°C. Rotifer density should be kept low, not exceeding 150 rotifers ml 1, and food (preferably Nannochloropsis sp.) should be provided in adequate quantities. After introducing a rotifer inoculum of 5–10 individuals ml 1, the rotifers will start reproducing and, depending on the culture temperature, mictic females carrying male eggs will appear after 2–3 days. This will be followed by the appearance of males and of fertilised females carrying resting eggs, after 4–6 days. These resting eggs will be released from the females at the end of their development and will sink to the bottom of the culture container. With the appearance of resting eggs, the number of females carrying male eggs will start to decline and this will be followed by a decline in the relative abundance of males and reduced production of resting eggs. Most often, cultures at this stage collapse and production of resting eggs is stopped. The production cycle lasts for 10–24 days. The resting eggs can be collected from the debris found at the bottom of the culture container, by sieving the culture water with agitation, through 200 m mesh plankton net, through which the eggs will pass. The material collected on the sieve should be resuspended in clean seawater and the sieving process repeated several times, until most of the eggs have been freed from the debris. Removal of the remaining debris can be achieved by suspending the eggs in clean, diluted seawater (e.g. 10 ppt) in a separating funnel and introducing an aeration tube just above the bottom outlet. A very fine airstream will result in flotation of the debris and sinking of the eggs to the funnel outlet, where they can be collected. The collected eggs should be stored in the dark (in aluminium foil-covered glass tubes or any other container) at a low temperature. The simplest method is to suspend the eggs in capped tubes containing clean or sterile 10 ppt seawater and store them in a refrigerator (Minkoff et al. 1983). More recent results showed that eggs may also be stored in a dried form: desiccated, lyophilised or canned under pressure (Balompapueng et al. 1997b). Hatching of resting eggs is achieved by transferring them from the stored container into fresh seawater and incubation at 10–25°C, with illumination (Minkoff et al. 1983; Hagiwara & Hino 1990). Kogane et al. (1997) showed that a low-temperature treatment could improve the efficiency of resting egg production by culturing B. plicatilis for 20 days at 12°C before transferring them to 25°C. The optimal conditions for production of B. rotundiformis differ from those of B. plicatilis, with resting egg production encouraged at higher salinities in B. rotundiformis (Hagiwara et al. 1989). Mass production of rotifer (B. plicatilis) resting eggs has been reported in 50 m3 tanks (Hagiwara et al. 1993a) and improvements in culture techniques have been tested, including the use of a semi-continuous culture method to maintain relatively low rotifer density and the use of a nylon filter system for removing excess debris (Hagiwara et al. 1993a, 1997; Hagiwara & Hirayama 1993; Hagiwara 1994; Balompapueng et al. 1997a). Methods have been devised for optimal storage of large quantities of resting eggs, including removal of attached bacteria by rinsing eggs with hypochlorite (1.0 mg l 1) or sodium nifurstyrenate (5.0 g ml 1) for improving hatchability (Balompapueng et al. 1997a,b). Hatching conditions are well established, facilitating the use of resting eggs at the required time (Minkoff et al. 1983; Pourriot & Snell 1983; Hagiwara et al. 1985, 1995c; Hagiwara & 52 Live Feeds in Marine Aquaculture Hino 1989, 1990; Hagiwara 1996; Balompapueng et al. 1997a). These eggs can be used directly for feeding fish larvae (Hagiwara & Hirayama 1993; Hagiwara et al. 1993b) or for initiating mass cultures, but asexual reproductive rates in rotifers hatched from resting eggs may show large fluctuations from those of the parent culture (Lubzens 1989). The cost of producing resting eggs exceeds several-fold that of producing mass-cultured rotifers (Lubzens 1989; Lubzens et al. 2001) and therefore has not yet been extensively adapted as a direct source of food for fish larvae. Moreover, rotifers hatched from artificially produced resting eggs may show a high occurrence of mixis, since the strain chosen for production of resting eggs has been selected for this purpose. Therefore, caution is required in using commercially available resting eggs for initiating mass cultures, as these cultures may show a high incidence of males under specific culture conditions (e.g. low salinity for B. plicatilis). 2.7 Future Directions The prospects of replacing live rotifers as food for early developmental stages of fish larvae are far from feasible, despite considerable efforts in this direction (Tandler 1984, 1985; Lubzens 1987; Kolkovsky & Tandler 1995). Generally speaking, current methodologies of producing and enriching rotifers have succeeded in meeting the demands of the industry. However, the current pressing need for very small live food is difficult to meet, although several ‘super small’ genetic strains have been found and cultured (Hagiwara et al. 2001). Improved methods for predicting the physiological state of rotifers in mass cultures could be helpful in avoiding the collapse of cultures. Using preserved rotifers may alleviate the immediate dependence of hatcheries on the daily production of rotifers. This includes keeping live rotifers at low temperatures or, alternatively, as resting eggs. Cheaper methods for resting egg production of inbred lines will be of great advantage in reaching this goal. Acknowledgements The financial support for rotifer species and strain cultures (1991–2000) by the Kunin Lunenfeld Foundation is greatly acknowledged. The advice, support and patience of the editor, Dr J. 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With the development of improved techniques for cyst and nauplii applications (Léger et al. thus realising the first breakthrough in the culture of commercially important fish species (Sorgeloos 1980). 1987a) and the exploitation of new natural resources. During the 1980s.1). In the 1950s. 3. by the end of the 1980s. The cyst shortage also simultaneously encouraged the search for alternatives for Artemia such as microencapsulated diets (Jones et al. However. California. The dramatic impact of the cyst shortage on the expanding aquaculture industry encouraged research on rationalising the use of Artemia and exploration of new cyst resources. Australia. On the initiative of the Artemia Reference Center (Ghent University.1 Introduction Artemia has probably been known and used within its natural distribution areas for centuries. Samocha et al. increased demand. possibly. Canada. USA). However. its fame elsewhere only began to rise in the 1930s when some investigators adopted it as a convenient replacement for the natural diet of fish larvae. by the mid-1970s. USA) and the Great Salt Lake (GSL. Tank Production and Nutritional Value of Artemia Jean Dhont and Gilbert Van Stappen 3. high import taxes in some developing countries and.Chapter 3 Biology. Fig. During that period. namely . improved techniques for harvesting from the open water and favourable hydrological and climatic conditions enabled a 10-fold increase in the yields from GSL ( 200 t processed product. Belgium) the International Study on Artemia (ISA) was established to co-ordinate a variety of different research initiatives (Sorgeloos 1979). cyst prices returned to normal and annual market supply reached over 50 t by the 1980s. 1991). There were only two commercial sources: the coastal saltworks in the San Francisco Bay (SFB. cyst supply was more than 90% dependent on one resource. With fish and shrimp aquaculture developing from the early 1960s. while the hatching quality was also improved. Artemia cysts were still predominantly marketed for the aquarium and pet trade at costs as low as US $10 kg 1. declining harvests from the GSL. new marketing opportunities were created for Artemia cysts. Thailand) occurred. a process that continues today with slow but steady progress. France. Kara Bogaz Gol in Turkmenistan. cysts with good hatching quality could be purchased for as low as US $20 kg 1. Although these sites did not necessarily contribute substantially to the world supply of cysts.000 6. the remainder went to marine fish larviculture in Europe. they provide interesting opportunities for local commercial development. cyst prices inflated to levels around US $100 kg 1 for GSL product and nearly US $200 kg 1 for the EPA-rich product.000 9. prices of small sized cysts with high eicosapentaenoic acid (EPA. the GSL remains a natural ecosystem subject to climatic and other influences. In 1997. some 6000 hatcheries required over 1500 t of cysts annually.000 Harvested raw product (t) 8. Whereas in the early 1990s.000 1. the consumption of cysts dropped from 10 kg per million . Bolshoye Yarovoye in Siberia. This situation urged producers in the 1990s to explore new sites such as Lake Urmia in Iran.1). 1998. In addition.000 4.1 Cyst harvest (in tonnes of raw product) from the Great Salt Lake (Utah. mainly in China and south-east Asia. For instance. With the severe cyst shortage in the mid-1990s and at the end of the twentieth century (Fig. Aibi Lake in China. cyst consumption has increased exponentially as a consequence of the booming shrimp and marine fish industries. 2001) has enabled a dramatic reduction in the required amount of cysts per unit of fish or shrimp produced. and this has been illustrated by unpredictable and fluctuating cyst harvests. 3. in shrimp hatcheries. as well as to the pet fish producers. China and Japan. a typical Mediterranean sea bass and sea bream hatchery would have been using some 150 kg cysts to produce 1 million fry. prices started to fall again.000 0 ’84 ’86 ’88 ’90 ’92 Year ’94 ’96 ’98 2000 Fig. However. formerly. Since the early 1990s. (Data from USGS 2001.000 7. Some 80–85% of the total sales of Artemia went to shrimp hatcheries.000 5.66 Live Feeds in Marine Aquaculture 10. the rationalisation of the use of Artemia in hatcheries (Sorgeloos et al. USA). 3. 20:5n-3) levels could reach over US $100 kg 1 at times of short supply owing to their critical role as starter food for marine fish larvae.000 3.000 2. as well as Ecuador and a few other Latin American countries. and several lakes in Kazakhstan (Lavens & Sorgeloos 2000).) GSL. Despite its size. numerous managed ponds and saltworks world-wide provided small quantities (1–20 t each). whereas nowadays the required amount of cysts is only 90 kg for bass and 70 kg for bream. Likewise. Following the superharvest of over 9000 t of raw cysts from the GSL in 2000–2001. New insights into hatching characteristics and nutritional essentials gave rise to the segregation of different cyst qualities. wolf fish. These cysts are metabolically inactive and do not develop further as long as they are kept dry. surrounded by the hatching membrane (Fig.3).Biology. sturgeon.4). the embryo resumes its interrupted metabolism. often referred to as ‘biomass’. Today. Juvenile and adult Artemia. Although live biomass has a higher nutritive value.5. They are also the easiest and earliest live food. different carp and catfish species and whitefish species. Artemia is essential only for those species that require live food in their early life stages. (1998. Comprehensive literature reviews on the use of Artemia as live food in fish and shellfish larviculture have been published by Léger et al.2. crawfish. it should be borne in mind that any farmer would switch to formulated feed as soon as this proves to be more cost-effective than Artemia. it is obvious that the use of nauplii will continue to be market driven for at least a few more years and that record harvests at GSL and new locations may relieve the pressure or even reverse the current trends. Artemia is used in the mass culture of different sea bream species. As a consequence. Hatching procedures can be simplified and improved through prior ‘decapsulation’ of cysts (see Section 3. 2001). In general.2). the biconcave cysts hydrate and become spherical and. Artemia produces cysts that float at the water surface and are driven ashore by wind and waves. sea bass species. but also a matter of the developmental stage and efficiency of the digestive system. thousands of tonnes are collected on an annual basis from the Bohai Bay salt ponds and are used in the local culture of Chinese white shrimp. most of the 3000 t that is harvested annually is marketed in frozen form.5. halibut. Penaeus chinensis (Tackaert & Sorgeloos 1991). most fish and shrimp larvae accept formulated feed more easily as they grow bigger. This switch will be triggered not only by the constantly improving quality of formulated feed. The same is true for commercially important crustaceans such as several shrimp and prawn species.5). Although there is no doubt that Artemia will gradually be replaced by formulated diets. several edible crab species and lobster. the most widely used forms of Artemia in aquaculture. 1996). flounder species and other flatfish. 3. However. Brine shrimp are mostly used as freshly hatched nauplii or as ‘enriched’ nauplii (see Section 3. dried or incorporated in compound diets. Upon immersion in seawater. In China.3.1) or can be harvested from salt ponds or lakes (Baert et al. (1986) and Sorgeloos et al. being obtained directly from the cysts. Tank Production and Nutritional Value of Artemia 67 postlarvae to less than 5 kg. Part of it is also flaked. undoubtedly. While the embryo hangs underneath the empty shell (the ‘umbrella’ stage) the development of the nauplius is completed and within a short period the hatching membrane is ruptured (‘hatching’). turbot. within the shell. milkfish. can be obtained through culturing (see Section 3.1 Morphology and life cycle In its natural environment under certain conditions. giving rise to the free-swimming nauplius (Fig. This is not only a matter of size of mouthparts and particle size.2 Biology of Artemia 3. Nauplii in instar I and II stages are. After about 20 h the outer membrane of the cyst bursts (‘breaking’) and the embryo appears. . 3. 3. but also by price and quality of Artemia. cod. a process that also improves the quality of poor or nonhatching cysts. 2 Cyst in breaking stage. (3) antenna. 3. 400–500 m in length) has a brownish-orange colour. Small food particles (e.68 Live Feeds in Marine Aquaculture Fig. seven postmetanaupliar and five postlarval stages have been described (Hentschel 1968. the second antennae (locomotory plus filter-feeding function) and the mandibles (food uptake function). 3.3 Embryo in ‘umbrella’ stage (left) and instar I nauplius (right). On both sides of the nauplius eye lateral complex eyes begin to develop . The instar I larva does not take up food as its digestive system is not yet functional. the animal moults into the second larval stage (instar II). Schrehardt 1987). generally one naupliar. it relies completely on its yolk reserves. a red nauplius eye in the head region and three pairs of appendages: the first antennae (sensorial function). The first larval stage (instar I. After about 8 h. The larva grows and differentiates through a number of moults. Fig. bacteria.4). (1) Nauplius eye. four metanaupliar. The ventral side is covered by a large labrum (food uptake: transfer of particles from the filtering setae into the mouth). (2) antennula. although there has been considerable disagreement about the exact number of larval stages. 3. Paired lobular appendages appear in the trunk region and differentiate into thoracopods (Fig. (4) mandible.g. algal cells. detritus) ranging in size from 1 to 50 m are filtered out by the second antennae and ingested into the now functional digestive tract. (1) Nauplius eye. 3.e. 3. 3. The thoracopods are now differentiated into three functional parts (Fig. a pair of functional thoracopods on each of the 11 thoracal segments (Figs 3.8). flexible exoskeleton of chitin to which muscles are attached internally. (2) telopodite. i. Fig. (3) exopodite. 3.10). 3.7. (Figs 3.7) has a paired penis on the first of the eight abdominal segments (Fig. Female Artemia can easily be recognised by the brood pouch or uterus situated in the same segment. In males (Figs 3. The ovaries are paired tubular structures extending into the abdomen . From the 10th instar stage onwards.5 Head and thoracic region of young male. sensorial antennulae. Adult Artemia are typical primitive arthropods (8–12 mm in length) having an elongated segmented body with two stalked complex eyes. a linear digestive tract. (3) antenna. (5) budding of thoracopods.8. (2) lateral complex eye.10). and the membranous exopodites (gills). The female reproductive system consists of ovaries and oviducts leading into the single. just behind the 11th pair of thoracopods (Figs 3. The male (Fig. 3.9): the telopodites and endopodites (locomotory and filter-feeding).5). (4) labrum. The entire body is covered with a thin.4 Instar V larva. 3.Biology. 3. Tank Production and Nutritional Value of Artemia 69 Fig. 3.8) and a furca on the last abdominal segment.6. (1) Nauplius eye. the antennae lose their locomotory function and undergo sexual differentiation. wherein several clusters of shell glands open.4. (6) digestive tract. while the female antennae degenerate into sensorial appendages (Fig. (1) Antenna. 3.7) they develop into hooked graspers. important morphological and functional changes begin to take place. median uterus. 70 Live Feeds in Marine Aquaculture Fig. (3) lateral complex eye.8 Adult female. Fig. (2) antennula. (4) mandible.6 Head of an adult male. (1) Antenna. . 3. Fig. 3. 3.7 Adult male. . (1) Ovary with eggs. (1) Exopodite.9 Detail of anterior thoracopods in adult Artemia. Fig.10 Artemia couple in riding position.11 Uterus of ovoviviparous Artemia filled with nauplii (first larvae are being released). 3. (2) penis. 3. 3. Tank Production and Nutritional Value of Artemia 71 Fig.Biology. Fig. (1) Uterus. (2) telopodite. (3) endopodite. low oxygen levels) the embryos only develop up to the gastrula stage.g. Morris & Afzelius 1967. The lateral pouches function as seminal receptacles during the time between copulation and fertilisation (within 1 h) (Benesch 1969. 3. The cysts usually float in the high-salinity waters and are blown ashore where they accumulate and dry. Criel 1980a. Lochhead & Lochhead 1940. both oviparity and ovoviviparity are found in all Artemia strains. Dutrieu 1960a. (1) Brown shell glands. The shell glands consist of several cell clusters. 3. Fertilised eggs normally develop into free-swimming nauplii (ovoviviparous reproduction) (Fig. spawning is followed by a moult. . 3. Adult females ovulate approximately every 140 h. Each oviduct empties into the anterolateral border of the uterus. Once ripe. The oviducts emerge from the ovaries near the anterior part of the third abdominal segment (Cassel 1937). not all encysted embryos produced by oviparous animals enter diapause. depending on rearing conditions and whether development of embryos occurs oviparously or ovoviviparously. At this point they are surrounded by a thick shell (secreted by the brown shell glands located in the uterus). 3. enter a state of metabolic dormancy (diapause) and are then released by the female (oviparous reproduction) (Fig. the eggs developing in the ovaries become spherical and migrate via two oviducts into the unpaired uterus. and nauplii emerge from some cysts without dehydration or other treatment (Jensen 1918.11). (Fig.12 Uterus of oviparous Artemia filled with cysts. However.72 Live Feeds in Marine Aquaculture Fig.b. depending on reproductive strategy.b). and can vary from dark brown to white or even colourless. Mathias 1937. grow from nauplius to adult in only 8 days and reproduce at a rate of up to 300 nauplii or cysts every 4 days. and females can switch reproductive modes from one ovulation to the next. cysts are now in a state of quiescence and can resume their further embryonic development when hydrated in optimal hatching conditions. In extreme conditions (e. In females. In principle. Although females may differ in their genetic tendency to reproduce either ovoviviparously or oviparously. after which ovulation takes place. high salinity. As a result of this dehydration process the diapause mechanism is generally inactivated.12). which are released by the mother. Under optimal conditions brine shrimp can live for several months.11). no Artemia are known to lack completely the ability to produce ovoviviparous nauplii. 2. (Modified from Morris & Afzelius 1967. especially in continental Asia. and the hypochlorite-resistant embryonic cuticle (Morris & Afzelius 1967) (see also Section 3.13 Schematic diagram of the ultrastructure of an Artemia cyst. Anderson et al. separating chorion from embryonic cuticle.5.Biology. 3. As a result. The cryptobiotic cyst shell has two important layers in addition to the hypochlorite-soluble. USA.) Benesch 1969. especially in inland lakes that are sufficiently large and productive to justify commercial exploitation. and these cysts are being used world-wide in aquaculture. mainly reflecting sampling and exploration activities (Fig.13). The decline of Artemia cyst harvests from the GSL in Utah. several sites. since 1977 (Lavens & Sorgeloos 2000) has intensified the search for alternative resources. These are the outer cuticular membrane. along coastlines as well as inland.14). which delineates the embryo from the fibrous layer of the embryonic cuticle (Fig. 1970).2 Ecology and natural distribution Artemia populations are found in about 500 natural salt lakes and artificial salterns scattered throughout the tropical. The distribution of these sites over the continents is very uneven. are exploited occasionally or on a regular basis (with some local investment). The identity or location of these sites has still not reached scientific literature. and the inner cuticular membrane.1). 3. Tank Production and Nutritional Value of Artemia 73 alveolar layer outer cuticular membrane embryonic cuticle inner cuticular membrane embryo Fig. These cysts are surrounded by a much thinner shell than those that enter diapause (Lochhead & Lochhead 1940). subtropical and temperate climatic zones. 3.2. double-layered outer chorion secreted by the shell glands. As such. changes in cysts post release suggest that diapause is established gradually after release (Jardel 1986). it does not give a precise picture of the actual global occurrence of Artemia. Moreover. 3. and attempts are seldom made to perform a systematic . light intensity. whether or not the seasonality of the environment is predictable (Lenz 1987. No Artemia are found in cold tundra or frost climates.14 World distribution of Artemia. characterisation of the respective strains. as Artemia cysts will only start to develop when the salinity of the medium drops below a certain threshold value. 3. Two critical factors determine the population dynamics of Artemia and its biogeographical distribution: first. secondly. For physiological reasons the salinity optimum is situated towards the lower end of the salinity range. Other variables (temperature. as brine shrimp possess: • • • a very efficient osmoregulatory system the capacity to synthesise very efficient respiratory pigments to cope with the low oxygen levels at high salinities the ability to produce dormant cysts when environmental conditions endanger the survival of the species. The common feature of all Artemia biotopes is their high salinity. or may cause only a temporary absence of brine shrimp. 1995). Salinity is without doubt the predominant abiotic factor determining the presence of Artemia and consequently limiting its geographical distribution.74 Live Feeds in Marine Aquaculture Fig. as the year-round extremely low temperatures preclude Artemia development. Ambient salinity also plays a role in cyst metabolism. Amat et al. primary food production) may have an influence on the quantitative aspects of the Artemia population. Its physiological adaptations to high salinity provide a very efficient ecological defence against predation. The maximum temperature tolerated by . whether water body conditions allow the animals to survive throughout the year and. as higher ambient salinity requires higher energy costs for osmoregulation. Most strains do not seem to survive prolonged temperatures below 5°C unless in the form of cysts. A continued survey will undoubtedly lead to the discovery of many more Artemia biotopes in different parts of the world. physiological adaptation of SFB Artemia to high temperatures (40°C) after a number of generations in Vietnamese salt ponds has also been reported (Clegg et al. 1997b). they remain intact for at least 2 days in the digestive tract of birds. parthenogenetica (with different levels of ploidy. The ametabolic dehydrated cysts are resistant to a wider temperature range than would ever occur in nature. Endemic to the Old World are the parthenogenetic types designated by Barigozzi (1974) as A. and many parthenogenetic strains is currently acknowledged. temperature optima are difficult to define and are strain dependent. however. 3. and nuclear and mitochondrial DNA sequencing. however. multidimensional process involving a variety of environmental and genomic factors. when ingested.3 Taxonomy The brine shrimp Artemia comprises a group of zygogenetic and parthenogenetic. Artemia salina is now only recognised as a valid name for the zygogenetic species found in the Mediterranean area (Mura 1990.2. and Leach in 1818 renamed the brine shrimp as Artemia salina (Artom 1931). including cross-breeding tests. morphological differentiation. Speciation in the genus should be regarded as a complex. Triantaphyllidis et al. salinas along the north-east coast of Brazil) are not naturally inhabited by brine shrimp. in general. origin and amount of clonal diversity. Tank Production and Nutritional Value of Artemia 75 Artemia populations has repeatedly been reported to be close to 35°C. parthenogenetica by Barigozzi (1974). as well as to gaining insight on population structure. In 1755 Schlosser described the brine shrimp based on material collected from the solar saltworks near Lymington. With the exception of cross-mating. The identification of zygogenetic Artemia species has been established by a multidisciplinary approach. a temperature often attained in the shallow tropical salterns that constitute a large part of the Artemia habitats. all of these techniques have also contributed to identifying the parthenogenetic types described as A. Consequently. Very often authors have named all brine shrimps A. As Artemia is incapable of active dispersion. the optimum for Artemia is in the range 25–30°C. Badaracco et al. The floating cysts adhere to feet and feathers of birds and. Linnaeus in 1758 classified it as Cancer salinus. Africa.g. strain dependent. England (no longer in existence) (Kuenen & Baas-Becking 1938).Biology. Asia and Australia). tunisiana was used. the absence of migrating birds is probably the reason why certain areas that are suitable for Artemia (e. The differentiation of seven zygogenetic species. While for some time the name A. Artemia is a non-selective filter feeder of organic detritus. salina. salina. defined primarily by the criterion of laboratory reproductive isolation. Moreover. This tolerance threshold is. As for salinity.5 million years ago (Abreu-Grobois & Beardmore 1982. the zygogenetic A. Leach 1819 (Mediterranean area) (Triantaphyllidis . found in Europe. wind and waterfowl (especially flamingos) are the most important natural dispersion vectors. 1987). allozyme studies. Abreu-Grobois 1987. morphologically similar species very likely to have diverged from an ancestral form living in the Mediterranean area some 5. microscopic algae and bacteria. The Artemia biotopes typically show a very simple trophic structure and low species diversity: the absence of predators and food competitors allows brine shrimp to develop into monocultures. cytogenetics. 2001). conditions of salinity. pentaploid). stressful. 1991).76 Live Feeds in Marine Aquaculture et al. Browne & Halanych 1989). 1997b). with A. sinica (Cai 1989) (continental China). belonging to different species. fed Dunaliella) resulted in the dominance of A. but Artemia cysts are commercially available from various production sources in America. triploid. but not completely. While the nutritional value can be manipulated through enrichment. Triantaphyllidis et al. 80 g l 1. A.2. and cytological and allozyme studies (Abreu-Grobois & Beardmore 1982. Parthenogenetic types tend to predominate in more disturbed. tetraploid. or genetically different populations within the same sibling species. persimilis (Piccinelli & Prosdocimi 1968) (Argentina) and A. Artemia sp. over 90% of all marketed cysts originate from the GSL. franciscana over parthenogenetic populations on the one hand. Central and South America). In spite of the fluctuations in the harvest. urmiana (Günther. i. 1986). . they have become genotypical as a result of long-term adaptations of the strain to the local conditions. A. other qualities favourable for aquaculture can be obtained by selection of strains and/or their crossbreeds. 1993) and different ploidy levels (diploid. tibetiana (Abatzopoulos et al. Endemic to the New World are A. and parthenogenetic populations over A. Lenz & Browne 1991). Coexistence of two species in the same saline habitat is possible: mixtures of parthenogenetic and zygogenetic populations have been reported in Spain. Abreu-Grobois 1987. 1890) (Iran). salina on the other (Browne 1980. Other characteristics such as cyst diameter and resistance to high temperature are considered strain-specific and remain relatively constant (Vanhaecke & Sorgeloos 1980a). Australia and Europe. franciscana (Kellogg 1906) (North. The genus Artemia is thus a complex of sibling species and superspecies. The parthenogenetic Artemia with their great clonal diversity. (franciscana) monica being a special case of a population described for an ecologically unique habitat (Mono Lake. (Pilla & Beardmore 1994) (Kazakhstan) and A. Abatzopoulos et al. Among these strains a high degree of genetic variability as well as a unique diversity in various quantitative characteristics have been observed (Browne et al. Some of this variability is phenotypical.e. Knowledge of the characteristics (both genotypic and phenotypic) of a particular batch of cysts can greatly increase the effectiveness of its use in a fish or shrimp hatchery. as evident from morphology (Hontoria & Amat 1992. such as the nutritional composition of the cysts (Léger et al. and central and northern China. were raised together and reproduced for a maximum of 3 months (25°C. 3. temperature and food availability (Browne & Bowen 1991. and changes from batch to batch. Very rarely. 1998) (Tibet). USA). by the criterion of reproductive isolation. Italy. Asia.4 Strain-specific characteristics The world-wide distribution of the brine shrimp Artemia in isolated habitats (about 500 natural salt lakes and artificial salterns) with specific ecological conditions has resulted in numerous geographical strains. defined largely. display a wide genotypic variation. it has been shown that genetically extremely distinct and allopatric species can produce laboratory hybrids (Pilla & Beardmore 1994). Laboratory competition experiments where Artemia adults. 1997a). instar I naupliar length.55 2. A. rate and efficiency (Vanhaecke & Sorgeloos 1982.2.Biology. show a high correlation with the cyst diameter (Vanhaecke et al.4. and to help define the origin of unknown or even mixed cyst samples (Vanhaecke & Sorgeloos 1980a). as well as production conditions affecting the parental generation.1 Size and energy content The nutritional effectiveness of a food organism is primarily determined by its ingestibility and. Canada Tanggu. USA Shark Bay.4. In spite of small variations between batches of the same strain. and to synchronise population developments to the variations of their specific biotype. salina. possibly caused by environmental and/or processing factors.63 1. However.2 Hatching quality Comparative studies of hatching behaviour of cysts of different origin show a considerable variation in hatching percentage. none of these parameters is strain-specific as they are influenced by a wide array of factors such as harvesting. For optimal use of Artemia in aquaculture the hatching characteristics of each batch of cysts being used should be known.03 — Energy content (10 3 J) 366 392 541 576 448 681 — — — Cyst source San Francisco Bay. by its size and form. 25°C). In response to simple dehydration by storage in a highly saline medium or by air-drying. PR China Yuncheng. Other biometrical characteristics. Tank Production and Nutritional Value of Artemia 77 Table 3. such as cyst volume. as a consequence. Australia Chaplin Lake. 1983). Some general correlations can also be made between sibling species and size: parthenogenetic Artemia produce large cysts. 3. As a consequence.2. individual naupliar weight and naupliar volume. Data on biometrics of nauplii from various Artemia strains are given in Table 3. USA Macau. cyst dry weight. are good tools to characterise Artemia strains.1.4. Length (mm) 428 447 486 458 475 515 515 460 497 Dry weight ( g) 1.47 2.04 3.09 4. franciscana and A.42 2. large cysts with a thick chorion. the process of diapause and its deactivation is likely to be adapted to the population’s habitat (Lavens & Sorgeloos 1987). persimilis. SFB-type cysts are gradually released . in particular cyst diameter. biometrical parameters. Bohai Bay.74 2.3 Diapause characteristics As diapause can be considered as a life-cycle strategy to overcome temporarily adverse conditions.1 Size. A. processing.2. and energy content. PR China Aibi Lake. 3. Iran 3. Adaptations to local conditions may have contributed to strain-specific differences in diapause sensitivity. small or intermediate cysts with a thin chorion. Brazil Great Salt Lake. generally the cyst diameter of different production batches of the same strain remains rather constant. storage and hatching techniques. PR China Lake Urmiah. individual dry weight and energy content of Artemia instar I nauplii from different cyst sources hatched in standard conditions (35 g l 1. 1983). and the rate of colonisation of new environments with limited nutrient resources. These are all favourable characteristics compared with those of Old World zygogenetic and parthenogenetic Artemia (Browne et al. if environmental preferences and nutritional factors do not interfere.4. but not among batches of the same strain (Vanhaecke & Sorgeloos 1980b). after inoculation) is determined by a variety of factors. New World (zygogenetic) populations have a very large number of offspring per brood. In addition. 3. the latter in their turn predominating over Old World zygogenetic species. southern Siberia) need a period of cold storage or hibernation of several weeks to break diapause.5 Temperature and salinity tolerance Both temperature and salinity significantly affect survival and growth. 1984.3) may be partially genetic and thus strain-specific. especially when competition with a local strain is to be expected. New World zygogenetic species generally outcompete parthenogenetic strains. 3.2.6 Life-history traits and reproductive capacity Life history and reproductive characteristics of Artemia strains are important factors when an introduction of brine shrimp to a new habitat is considered. 3. total lifespan.g. . Inoculation experiments in natural habitats therefore require prior screening of candidate strains and of indigenous local populations.2. At elevated temperatures the survival of the GSL strain is significantly higher than for other strains. etc.4.4 Growth rate of nauplii Standard culture tests with brine shrimp from different geographical origins show important differences in growth rate even within the same sibling species. the effect of temperature being more pronounced. prereproductive and postreproductive periods. 1991). where mortalities are 10%. Vu Do Quynh & Nguyen Ngoc Lam 1987). A broad range of temperatures and salinities meets the requirements for 90% survival (Vanhaecke et al. Consequently. 1998). Strains from thalassohaline biotopes share a common preferred temperature range around 20–25°C.2. 1984). interval between broods.5. These competitive abilities are related to factors such as the length of reproductive. while cysts from inland salt lakes (GSL. In general. a large number of offspring per female per day and a fast development time to sexual maturity. broods per female. a limited screening of different combinations is therefore needed when the hatching of new batches is being optimised (Van Stappen et al. as well as the study of prevailing environmental conditions. Although general recommendations can be formulated with regard to H2O2 concentration and exposure time. number of offspring per brood. Age at first reproduction is a key factor determining the population growth rate. substantial differences in tolerance have been recorded at low salinities (around 5 g l 1) and high temperatures (30–34°C). Although the population growth of Artemia in the field (e.2.4. differences in tolerance and responsiveness of different strains to hydrogen peroxide (H2O2) treatment during diapause deactivation (see Section 3.78 Live Feeds in Marine Aquaculture from diapause (Versichele & Sorgeloos 1980. selection of a strain with a high potential growth rate will have a positive impact on maximal production output. Interaction between temperature and salinity is limited. 5 Cyst biology and diapause 3. 3. but correspond to different production conditions.5. In particular. 1978.Biology.2. seemed to differ widely from strain to strain. when many fish and shrimp hatcheries started to commercialise. Bruggeman et al.2. The haematin concentration determines the colour of the shell. pigments (canthaxanthin).4.2. Tank Production and Nutritional Value of Artemia 79 3. vitamin C. see Section 3. which destroy specific enzymatic systems in the ametabolic Artemia cysts. Their effects on larviculture success are far less significant than nauplii fatty acid composition. The embryonic cuticle is apparently impermeable to non-volatile solutes (De Chaffoy et al. the lipid and fatty acid compositions. The embryo is an undifferentiated gastrula. as well as the metabolisation of fatty acids in the Artemia. Cyst products from inland resources are more constant in fatty acid composition.e. Clegg & Conte 1980). In most cases these variations are not strain-specific. Applying simple methods.1 Cyst morphology and physiology A schematic diagram of the ultrastructure of an Artemia cyst is given in Fig.13. cosmic radiation results in the formation of free radicals. Appropriate enrichment techniques have thus been developed to improve the lipid profile of deficient Artemia strains. The viability is affected when water levels are higher than 10% (start of metabolic activity) and when cysts are exposed to oxygen. in the presence of oxygen. acts as a permeability barrier). A number of other compounds also varies from strain to strain: nutritional components such as total amount of free amino acids. as well as contamination with chemicals such as pesticides and heavy metals.5. This layer can be completely removed (dissolved) by oxidation treatment with hypochlorite (cyst decapsulation. taking advantage of the indiscriminate filter-feeding behaviour of Artemia (Léger et al. i. . The cyst shell consists of three layers: • • • Alveolar layer: This hard layer consists of lipoproteins impregnated with chitin and haematin. i.4.7 Nutritional value In the late 1970s. Its main function is to provide protection for the embryo against mechanical disruption and ultraviolet (UV) radiation.e. 3. albeit at suboptimal low levels. minerals and trace elements. which is ametabolic when water content is below 10% and can be stored for long periods without losing its viability. 1987b). Outer cuticular membrane: This protects the embryo from penetration by molecules larger than the carbon dioxide molecule (multilayer membrane with a very special filter function.5). 1987a). and even from batch to batch. 1980). as a consequence of the fluctuations in biochemical composition of the primary producers (mainly unicellular algae) available to the adult population (Léger et al. Aquaculturists even noticed highly significant differences when using different batches from the same geographical origin (Léger & Sorgeloos 1984). switching from one source of Artemia to another provoked unexpected problems. lipophilic compounds can easily be incorporated into Artemia before being offered as live feed (see Section 3. Embryonic cuticle: This transparent and highly elastic layer is separated from the embryo by the inner cuticular membrane (develops into the hatching membrane during hatching incubation). from pale to dark brown.5. 15 Cellular metabolism in Artemia cysts (as a function of hydration level).80 Live Feeds in Marine Aquaculture 3. Fig.15) are very resistent to extreme temperatures. the cyst shell (including the outer cuticular membrane) bursts (breaking stage) and the embryo surrounded by the hatching membrane becomes visible. . hatching viability is not affected in the temperature range 273°C (Skoultchi & Morowitz 1964) to 60°C.5.e. in the order of 8–24 h depending on temperature and salinity. The embryo then leaves the shell completely and hangs underneath the empty shell (the hatching membrane may still be attached to the shell). RNA and protein synthesis begin within minutes (Clegg & Conte 1980). a reversible interruption of the metabolism (viability not affected) occurs between 18 and 4°C and between 33 and 40°C. After a period of postdiapause development. Within this range. Active cyst metabolism occurs between 4 and 33°C. Respiration. 3. i.2 Cyst metabolism and hatching Given favourable environmental conditions the metabolism and development of encysted embryos are rapidly reinitiated. the hatching membrane breaks open (hatching) and the free-swimming larva emerges head-first. Shortly thereafter. When incubated in seawater the biconcave cyst swells and becomes spherical within 1–2 h. supporting the conclusion that encysted embryos contain all the components needed for these activities.2. above 60°C and up to 90°C only short exposures can be tolerated. Through the transparent hatching membrane one can follow the differentiation of the pre-nauplius into the instar I nauplius which starts to move its appendages. Dry cysts (water content from 2 to 5%. Fig. although the upper and lower temperature limits vary slightly from strain to strain. 3. Hydrated cysts have far more specific tolerances: mortalities occur below 18°C and above 40°C. the hatching percentage remains constant but the nauplii hatch earlier as the temperature increases. Finally.3 Diapause As Artemia is an inhabitant of biotopes characterised by unstable environmental conditions. its survival during periods of extreme conditions (e. The exact optimal water level within the cyst is not known. The basic mechanisms involved in this switch are . Tank Production and Nutritional Value of Artemia 81 As for other environmental conditions. 3. Even correctly packaged cysts are preferentially stored at low temperatures. cleaning.g. in the worst case.15). Subsequent dehydration of these cysts will. Sufficiently dehydrated cysts only keep their viability when stored under vacuum or in nitrogen. Above a threshold salinity (varying from strain to strain.5. the addition of NaHCO3 (up to 2 g l 1). As a consequence. hatching in a higher salinity medium will consume more of the energy reserves of the embryo. When frozen. need a minimal light triggering for the onset of the hatching process. This may be related to the optimal pH activity range for the hatching enzyme. The presence of oxygen results in a substantial reduction in hatching success as a result of the formation of highly detrimental free radicals. Cysts exposed for too long a period to water levels exceeding 65% will have completed their pre-emergence embryonic development. at least during the first hours after complete hydration. when hydrated and in aerobic conditions. eventually reducing the energy content to levels insufficient to reach the state of emergence under optimal hatching conditions (Fig.5. Increased hatching has been reported with increasing oxygen level in the range 0.4). desiccation. Furthermore. and maximal hatching above this concentration. result in the killing of the now differentiated embryos. high salinities) is ensured by the production of dormant embryos. optimal hatching outputs are reached in the pH range 8–8.15). but generally situated in the range 15–70 g l 1.6–2 ppm. however. to artificial or diluted seawater or to dense suspensions of cysts. hatchability of cysts is largely determined by the conditions and techniques applied for harvesting. brine shrimp cysts. be retarded when the cysts are stored at freezing temperatures. results in improved hatching. This process may. Water levels in the range 30–65% initiate metabolic activity. drying and storing of the cyst material (see Section 4. but 90 g l 1 for most strains). the cysts should be acclimated for 1 week at room temperature before hatching. Artemia females can indeed easily switch from the production of live nauplii (ovoviviparity) to cyst formation (oviparity) in response to fluctuating circumstances. 3. related to light intensity and/or exposure time. Water content of about 5% is a reasonable value. Hatching quality in stored cysts slowly decreases when cysts contain 10–35% water (Fig. but generally strong illumination (about 2000 lux at the water surface) is recommended. Little is known about the exact light requirements. extreme temperatures. the amount of water that can be taken up is insufficient to support the embryo’s metabolism. To avoid oxygen gradients during hatching a good homogeneous mixing of the cysts in the incubation medium is required. Long-term storage of such material may result in a substantial decrease in hatching success. Although the physiological role of light during the hatching process is poorly understood. 3.2. Optimal salinity for hatching is equally strain specific. a depletion of the energy reserves occurs when the cysts undergo subsequent dehydration/hydration cycles. As stated above.Biology. although there are indications that too severe dehydration (down to 1–2%) results in a drop in viability. the usual cyst processing techniques do not yield a sufficiently high hatching quality. Lavens & Sorgeloos 1987. other diapause termination techniques (cyclic dehydration/hydration. As some form of dehydration is part of most processing and/or storage procedures. synchronous hatching occurs. In some cases no effect at all is observed. one should keep in mind that the increase in hatching percentage after any procedure may (even partially) be the result of a shift in hatching rate (earlier hatching). diapause or a similar state can be reinduced by exposure to anoxia (Drinkwater & Clegg 1991. with some strains of Artemia cysts. eventually resulting in hatching. The termination of diapause is a complex process. This allows effective colonisation in temporal biotopes. but sudden fluctuations in. influenced by multiple and mutually interfering genotypical and environmental factors. diapause termination does not require any particular extra manipulation. It is important to note here that the sensitivity of Artemia cysts to these techniques shows strain. Drinkwater & Clegg 1991). In principle. In many cases.2. and preliminary tests are needed to provide information about the optimal dose and period to be applied. For the user of Artemia cysts. Upon the deactivation of diapause by environmental factors (Drinkwater & Crowe 1987). The study of the mechanisms of diapause induction and deactivation can help to maximise the hatching yield of the commercially important Artemia cysts. USA. the removal of cyst water is an efficient way to terminate the state of diapause. cysts enter the stage of quiescence and metabolic activity can be resumed when they are exposed to favourable conditions in terms of temperature. hence the difficulty in predicting the effect on hatching outcome. other chemicals) give rather erratic results and/or are not user friendly. When working with new or relatively unknown strains. resulting in a fast start and consequent development of the population shortly after the re-establishment of favourable environmental conditions. 2000) or by a well-dosed heat shock (Abatzopoulos et al. As a result.4).or even batch-specificity (see Section 3. Nevertheless. The phenomenon of diapause in Artemia cysts has been the subject of numerous studies and reviews (Clegg & Conte 1980. Moreover. Overdosing results in reduced hatching or even complete mortality as a result of toxicity of the chemical. In this phase the metabolic arrest is uniquely dependent on external factors. Utah. continental Asia). Artemia embryos released as cysts in the medium are. Clegg et al. diapausing (Clegg et al. or soon become. This can be achieved by drying the cysts at temperatures not exceeding 35–40°C or by suspending the cysts in a saturated NaCl brine solution (300 g l 1). • • Freezing: This ‘imitates’ the natural hibernation period of cysts originating from continental biotopes with low winter temperatures (GSL. Drinkwater & Crowe 1987. for example. decapsulation. 1994). The triggering mechanism for the induction of the diapause state is not yet known. and about the maximal effect that can be obtained. indicating that a more specific diapause deactivation method is necessary. The following procedures have proven to be successful when applied with specific sources of Artemia cysts. 1996). the relative success or failure of certain methods has to be found out empirically. In general. Clegg 1993. Incubation in H2O2 solution: In most cases. several techniques have proven successful in terminating diapause. oxygen and light.82 Live Feeds in Marine Aquaculture not yet fully understood. oxygen levels and salinity seem to trigger oviparity. . the sensitivity of the strain (or batch) to this product is difficult to predict. However. France. e. Depending on the selected culture technology and implantation facilities. Tank Production and Nutritional Value of Artemia 83 3. First. In practice. As a consequence very high production yields per volume of culture medium can be obtained with tank rearing systems. particles. the UK and Australia. to supply local demand. justify its application: • • • year-round availability of ongrown Artemia. free from diseases). The abiotic and biotic conditions relevant for Artemia culture are: • • • • physicochemical conditions: – ionic composition of the culture media – temperature – salinity – pH – oxygen concentration – water quality (nitrogen metabolites. depending on the local condition. in the USA.g. specific stages (juveniles. since the culture procedure does not require highly saline waters or specific climatological conditions. preadult. quality of the Artemia can be better controlled (e.1 Advantages of tank production and tank-produced biomass Although tank-produced Artemia biomass is far more expensive than pond-produced brine shrimp its advantages may. etc. controlled production can be performed with very high densities of brine shrimp. independent of climate or season. with wholesale prices varying from US $25 to 100 kg 1. Since the early 1990s. e. several thousand animals per litre versus a maximum of a few hundred animals per litre in outdoor culture ponds.) Artemia: – strain selection – culture density feeding: – feeding strategy – selection of suitable diets infrastructure: – tank and aeration design – filter design – recirculation unit . High-density intensive culture techniques offer two main advantages compared with pond production techniques.3. Secondly. production costs are estimated at US $2.5–12 kg 1 live weight Artemia. nutritional content.3 Production Methods: Tank Production of Artemia Biomass 3. adults) or prey with uniform size can be harvested as a function of size preferences of the predator. when setting up an Artemia culture one should start by listing the prevailing culture conditions and available infrastructure.g.g. there is no restriction with regard to production site or time.Biology. several superintensive Artemia farms have been established. However. it is generally accepted that the pH tolerance of Artemia ranges from 6. in terms of growth rate and food conversion efficiency.5 mg O2 l 1 in seawater of 32 g l 1 salinity). The pH tends to decrease during culture as a result of denitrification processes. Since ionic composition is so important. is at salinities from 32 to 65 g l 1. concentrated brine (150 g l 1) from salinas can also be transported to the culture facilities and diluted with freshwater before use. It is important to remember that for a given air flow. several artificial media with different ionic compositions are used with success in indoor installations for brine shrimp production.e. depending on the cultured strain. biomass production will decrease at concentrations below 2 mg l 1. continuously maintaining oxygen levels higher than 5 mg l 1 will result in the production of pale animals (low in the respiratory pigment haemoglobin). Small adjustments in salinity can be made by adding brine or diluting with tap water free from high levels of chlorine. .2 Physicochemical conditions Salinity and ionic composition of the culture media Although. For most strains a common range of preference is 19–25°C (Table 3. For optimal production. A dark red coloration (high haemoglobin content) is easily obtained by applying regular oxygen stresses (by switching off the aeration for a few minutes several times a day) a few days before harvesting. When the pH drops below 7. Temperature. oxygen concentrations higher than 2. When oxygen occasionally drops below 30% saturation (i. aeration intensity should be increased temporarily or air stones added. possibly with a lower individual dry weight. Oxygen levels should be checked regularly as they may drop significantly. The best physiological performance. If oxygen levels remain low.84 Live Feeds in Marine Aquaculture • – heating – feeding apparatus culture techniques: – stagnant culture – open flow-through system – recirculation type. With regard to oxygen. the use of natural seawater of 35 g l 1 is the most practical. For Artemia culture. In the latter case.3. However. which may therefore be less perceptible to and attractive for the predators. the method must be suitable for seawater. the oxygen level is increased more effectively by small air bubbles than by large ones.5 mg l 1 are suggested.5 small amounts of NaHCO3 (technical grade) should be added to increase the buffer capacity of the culture water. especially after feeding.5 to 8. the aeration capacity should be increased. brine shrimp do thrive in natural seawater. The pH is commonly measured using a calibrated electrode or with simple analytical test kits. causing the animals to float and congregate at the surface. Beside natural seawater or diluted brine. in the wild. very small air bubbles can become trapped between the thoracopods. 3. pH and oxygen concentration Temperature must be maintained between the specific optimal levels of the selected Artemia strain.2). Oxygen is conveniently measured in the culture tank with a portable oxygen electrode. 2. In the literature. Artemia only occurs in highly saline waters (mostly above 100 g l 1). Biology, Tank Production and Nutritional Value of Artemia 85 Table 3.2 Effect of temperature on different production parameters for various geographical strains of Artemia. Temperature (°C) Geographical strain 20.0 22.5 25.0 27.5 30.0 32.5 San Francisco Bay, California, USA Survival (%) 97 Biomass production (%)a 75 Specific growth rateb 0.431g Food conversionc 3.89e Great Salt Lake, Utah, USA Survival (%) Biomass production (%)a Specific growth rateb Food conversionc 77 69 0.392g 3.79h 97 101 0.464d 3.35d 85 104 0.437f 2.90e 75 102 0.452d,e 3.00d 94 61 0.343e 5.42e 94 100 0.463d,e 3.64e 89 122 0.454e 2.65d,e 77 108 0.459d 3.03d 91 80 0.371d 4.46d,e 91 94 0.456e 3.87e 89 128 0.460d,e 2.62d 65 106 0.456d 3.11d 93 92 0.387d 3.84d 66 88 0.448f 4.15f 87 135 0.465d 2.40d 50 90 0.437e,f 3.72d 84 85 0.378d 4.22d,e na na na na 88 78 0.406g 4.14g na na na na 54 16 0.208g 22.04g Chaplin Lake, Saskatchewan, Canada Survival (%) 72 Biomass production (%)a 78 0.422f Specific growth rateb c Food conversion 3.42e Tanggu, PR China Survival (%) Biomass production (%)a Specific growth rateb Food conversionc 95 41 0.299f 7.22f Data compiled from Vanhaecke and Sorgeloos (1989). a Expressed as % recorded for the Artemia reference strain (San Francisco Bay, batch 288–2596) at 25°C after 9 days’ culturing on a diet of Dunalliella cells; bSpecific growth rate k ln(Wt W0) T 1, where T duration of experiment in days ( 9); cFood conversion F(Wt W0), where F g dry weight Dunalliella offered as g dry weight Artemia biomass after 9 days’ culturing, and W0 g dry weight Artemia biomass at food, Wt start of experiment; d–gMeans with the same superscript letter are not significantly different at the p 0.05 level. na, not analysed. Water quality The quality of the culture medium is primarily affected by excess particles as well as by soluble waste products such as nitrogenous compounds. High levels of suspended solids will affect production characteristics, either by their interference with uptake of food particles and propulsion by the Artemia, or by enhancing bacterial growth that will compete for oxygen and eventually infest the culture tank. Soluble waste products give rise to toxic nitrogenous compounds. The tolerance levels in Artemia for ammonia, nitrite and nitrate in acute and chronic toxicity tests with, for instance, GSL brine shrimp larvae showed no significant effect on survival [median lethal concentration (LC50)] or growth for concentrations up to 1000 mg l 1 NH 4 , 320 mg l 1 NO 2 (Chen et al. 1989). For nitrate, no effects were observed at 1000 mg l 1 and it is therefore considered non-toxic. It is therefore unlikely that N-components will interfere directly with Artemia cultures. Nevertheless, the presence of soluble substances should be restricted as much as possible since they are an ideal substrate for bacteria. Excess soluble waste products can only be eliminated by diluting the culture water with clean water, be it new or recycled. 86 Live Feeds in Marine Aquaculture 3.3.3 Artemia strain selection and culture density Strain selection Based on laboratory results (Table 3.2), guidelines are provided for strain selection as a function of optimal temperature and culture performance. The optimal strain should be selected according to specific culture conditions. Culture density of Artemia Unlike other crustaceans, Artemia can be cultured at high to very high densities without affecting survival. Depending on the applied culture technique, inoculation densities up to 5000 larvae per litre for batch culture, 10,000 for closed flow-through culture and 18,000 for open flow-through culture can be maintained without influencing survival and growth. Above these densities, culture conditions become suboptimal (water quality deterioration, lower individual food availability), and growth and survival decrease (Table 3.3). Crowding seems to affect ingestion rate and thus growth. In stagnant systems, a clear decrease in growth rate with increasing animal density has been observed (Dhont et al. 1993). The costeffectiveness of a culture increases with increasing Artemia density. In an open flow-through system, maximal densities will be limited by feeding rate, while in recirculating and stagnant system the preservation of water quality will determine a safe feeding level, which in turn determines the animal density at which the individual feed amount still allows a satisfactory growth rate. After some culture trials with increasing animal densities, the maximal density can be identified as the highest possible density where no growth inhibition occurs. 3.3.4 Feeding Artemia is a continuous, non-selective, particle-filtering organism. Various factors may influence the feeding behaviour of Artemia by affecting the filtration rate, ingestion rate and/or assimilation: the quality and quantity of the food offered, the developmental stage of the larvae and the culture conditions. More detailed information about these processes is given in Coutteau and Sorgeloos (1989). Selection of a suitable diet Artemia can take up and digest exogenous microflora as part of the diet. Bacteria and protozoans, which develop easily in the Artemia cultures, are able to biosynthesise essential nutrients as they use the supplied brine shrimp food as a substrate. In this way they compensate for possible deficiencies in the diet composition. The interactions with bacteria make it a hard task to identify nutritionally adequate diets per se, and growth tests are difTable 3.3 Directive animal densities under different culture conditions. Culture system Open flow-through Closed flow-through Stagnant Animals/litre 18,000 10,000 5000–10,000 5000 20,000 Culture period To adult To adult To adult 7 days 7 days Growth High Moderate High High Low Reference Tobias et al. (1979) Lavens et al. (1986) Dhont et al. (1991) Biology, Tank Production and Nutritional Value of Artemia 87 ficult to run under axenic conditions. As a consequence, nutritional composition of the diet does not play the most critical role in the selection of diets suitable for high-density culture of brine shrimp. The criteria used generally include: • • • • • • • • availability and cost particle size composition (preferentially 50 m) digestibility consistency in composition among different batches and storage capacity solubility (minimal) food conversion efficiency (FCE) buoyancy. Commonly used food sources are listed below. Microalgae: These undoubtedly yield best culture results but it is rare that sufficient algae are available at a reasonable cost. Mass culturing of suitable algae for Artemia is most often economically unrealistic, so their use can only be considered in locations where algal production is an additional feature of the main activity. Furthermore, not all species of unicellular algae are considered capable of sustaining Artemia growth (D’Agostino 1980); For example, Chlorella and Stichococcus have a thick cell wall that cannot be digested by Artemia, Cocochloris produces gelatinous substances that interfere with food uptake and some dinoflagellates produce toxic substances. Dried algae: In most cases algal meals give satisfactory growth performance, especially when water quality conditions are kept optimal. Drawbacks in the use of these feeds are their high cost ( US $12 kg 1), as well as their high fraction of water-soluble components, which cannot be ingested by the brine shrimp, but will interfere with the water quality of the culture medium. Bacteria and yeasts: Single-cell proteins (SCP) have several characteristics that make them an interesting alternative to microalgae: – the cell diameter is mostly smaller than 20 m – the nutritional composition is fairly complete – the rigid cell walls prevent the leakage of water-soluble nutrients in the culture medium – products are commercially available at acceptable cost (e.g. commonly used in cattle feeds). The highly variable production yields that often occur when feeding a yeast mono-diet are assigned to nutritional deficiencies of the yeast diet and should therefore be met by supplementation with other diets. For some SCP, digestibility by the Artemia can be a problem. Complete removal of the complex and thick yeast cell wall by enzymatic treatment and/or supplementation of the diet with live algae significantly improve the assimilation rate and growth rate of the brine shrimp (Coutteau et al. 1992). By-products from the food industry: Non-soluble by-products from agricultural crops or from the food-processing industry, such as rice bran, corn bran, soyabean pellets and lactoserum, appear to be a very suitable feed source for high-density culturing of Artemia (Dobbeleir et al. 1980). Their main advantages are their low cost and world-wide availability. Equally important in the evaluation of dry food is the consistency of the food quality and supply, and the possibility for storage without loss of quality. Bulk products must be stored in a dry and, preferentially, cool place. • • • 88 Live Feeds in Marine Aquaculture 1 3 2 T Fig. 3.16 Feeding strategy with cultured Artemia. (1) Look through looking glass to turbidistick (or submerge stick in cylinder with appropriate mesh). (2) Submerge turbidistick until contrast between black and white disappears. (3) Read depth of submergence in cm T. During first week: T 15 cm: stop feeding and/or increase water renewal; 15 cm T 20 cm: maintain actual feeding ratio; T 20 cm: increase feeding ratio and/or add food manually. During next week: T 20 cm: stop feeding and/or increase water renewal; 20 cm T 25 cm: maintain actual feeding ratio; T 25 cm: increase feeding ratio and/or add food manually. Feeding strategy Since Artemia is a continuous filter-feeding organism, highest growth and minimal deposition of unconsumed food is achieved when food is distributed as frequently as possible. When feeding SCP, algae or yeast, concentrations should be maintained above the critical minimum uptake concentration, which is specific for the algal (or other) species and the developmental stage of Artemia (Abreu-Grobois et al. 1991). Levels of dry feeds, consisting of fragments and irregular particles, cannot be counted in the culture tank. Therefore, a correlation between optimal feed level and turbidity of the culture water has been developed, whereby the feed concentration in a culture tank is determined by measuring the turbidity of the water with a simplified Secchi-disc (Fig. 3.16). 3.3.5 Infrastructure Tank and aeration design Artemia can be reared in containers of any possible shape as long as the installed aeration ensures proper oxygenation and adequate mixing of feed and animals throughout the total culture volume. However, aeration should not be too strong. Thus, aeration and tank design must be considered together as the circulation pattern is determined by the combination of both. A wide variety of culture tanks has proven to be suitable (Dhont & Lavens 1996). For cultures up to 1 m3, rectangular tanks are most convenient. They can be aerated either with an air–water lift (AWL) system (Fig. 3.17), by an aeration collar mounted around a central standpipe or by perforated polyvinyl chloride (PVC) tubes fixed to the bottom of the tank. Biology, Tank Production and Nutritional Value of Artemia 89 Fig. 3.17 Air–water lift. For large volumes ( 1 m3), it is advantageous to switch to cement tanks covered on the inside with impermeable plastic sheets or coated with special paint. These large tanks are traditionally operated as raceway systems. They are oblong, approximately 1.5 m wide and with a height:width ratio kept close to 1:2. The length is then chosen according to the desired volume. The corners of the tank may be curved to prevent dead zones where sedimentation can take place. A partition, to which AWLs are fixed, is installed in the middle of the tank and ensures a combined horizontal and vertical movement of the water, which results in a screw-like flow pattern (Bossuyt & Sorgeloos 1980). If axial blowers are used for aeration, the water depth should not exceed 1.2 m to ensure optimal water circulation. Filter design The most important and critical equipment in flow-through culturing is the filter used for efficient evacuation of excess culture water and metabolites without losing the brine shrimp from the culture tank. These filter units should be able to operate without clogging for at least 24 h, to reduce risks of overflowing. Traditionally, filters are constructed as a PVC frame around which an interchangeable nylon screen is fixed. The aeration is positioned at the bottom of the filter, ensuring a continuous friction of air bubbles against the sides of the filter screen and thus reducing filter-mesh clogging (Fig. 3.18). A more sophisticated type of cylindrical filter system consists of a welded-wedge screen cylinder, made of stainless steel. This welded-wedge system has several advantages with respect to the filter-screen types: • • Larger particles with an elongated shape can still be evacuated through the slit openings. The specially designed V-shape of the slit openings creates specific hydrodynamic suction effects, as a result of which filter particles that are only slightly smaller than the slit opening are sucked through. 90 Live Feeds in Marine Aquaculture 65 air 20 70 PVC frame 55 60 filter bag 15 20 65 aeration collar 25 4 Fig. 3.18 Construction of filters used in Artemia culture (dimensions in cm). 55 • Possible contact points between particles and filter are reduced to two instead of four mesh borders, which consequently reduces the chances of clogging. This filter can be operated autonomously for much longer periods than traditional mesh filters. Therefore, proportionally smaller welded-wedge filters can be used, leaving more volume for the animals in the culture tank. As brine shrimp grow, the filter is regularly switched for one with a larger mesh or slit opening to achieve maximal evacuation of moults, faeces and other waste particles from the culture tanks. A set of filters covering a 14 day culture period should consist of approximately six different slit or mesh openings ranging from 120 to 350 m. Heating When ambient temperature is below the culture optimum range (25–28°C), heating is imperative. Small volumes ( 1 m3) are most conveniently heated using electric thermoregulated resistors. Depending on the ambient temperature, a capacity up to 1000 W m 3 must be provided. For larger volumes, a heat exchanger consisting of a thermostatically controlled boiler with copper tubing under the bottom of the culture tank is recommended. Heat losses can be avoided by insulating the tanks with Styrofoam and covering the surface with plastic sheets. Feed distribution apparatus Dry feed cannot be distributed directly to the culture tank, but should be suspended by mixing it in tap water or seawater beforehand. The feed suspension is distributed to the culture tanks via a timer-controlled pump. The volume of the food tank should be big enough to hold the highest daily food ration at a concentration maximum of 200 g food l 1. Even at those concentrations, the food suspension is so thick that there is a high risk of blocking of the food lines. 60 6 Culture techniques Depending on the objectives and the opportunities. The pilot system consists of six oval raceway tanks of 1 m3 and six reservoir tanks of the same capacity placed above each culture tank. The culture system should be designed in such a way that the water quality can be maintained as close to optimal as possible. This means that the concentration of particles and soluble metabolites should remain minimal to prevent toxicity problems. The water retention time is chosen so as to reach an optimal compromise between efficient evacuation of wastewater and minimal food losses. and flow rate is easily adjusted by means of a siphon of a selected diameter. or where large quantities of algal food are available. a small part of the culture water must be regularly renewed. different culture procedures for highdensity intensive Artemia production may be applied. Retention time is at least 12 h. which are between those obtained in batch and flow-through systems (see Section 3. In reality. ranging from open flow-through with 0% recirculation to closed flow-through with 100% recirculation. Decisions need to be made as to: (a) whether or not the water should be renewed (open flow-through). tertiary treatment systems or intensive grow-out ponds of shrimp. whether a particular water treatment should be applied (closed flow-through or stagnant or batch system). and need manual refilling only once or twice a day.3. (1992).Biology.9 for production figures). The culture is started in an aerated tank and biomass is harvested after a reduced culture period. is limited to those situations where large volumes of sufficiently warm seawater (or brine) are available at relatively low cost. proliferation of micro-organisms and interferences with the filter-feeding apparatus of the brine shrimp. however.3. production needs and investment possibilities. A very simple semi flow-through system has been developed by Dhert et al. will result in the best possible culture conditions and highest production capacities. Application of an open flow-through culture technique. 1993). Tank Production and Nutritional Value of Artemia 91 3. such as from effluents from artificial upwelling projects. These reservoir tanks hold seawater and food (squeezed rice bran suspension). with consequent dilution of particulate and dissolved metabolites. This technique involves minimal sophistication and appears to be very predictable in production yields. The system does not require the use of feeding pumps and involves minimal care. even at complete recirculation. Stagnant systems Stagnant systems are the simplest concept for intensive Artemia culture: no wastewater evacuation. They are slowly drained to the culture tanks. and (b) in the latter case. The main disadvantage is that high animal densities do not allow for extended culture periods because of the degradation of the water quality. The final selection of the type of culture installation will be subject to local conditions. There are many kinds of transition types. The culture effluent is drained using weldedwedge filters as described above. Open flow-through A discontinuous or continuous renewal of culture water by clean seawater. Successful trials with 10 animals l 1 on micronised soya pellets yielded Artemia juveniles of 3 mm in length and over 75% survival in 7 days (Dhont et al. . filter systems or water treatment are involved. 4. Treatment Mechanical treatment Type Plate separator Cross flow sieve Foam fractioner or protein skimmer Rotating disc contactor Reference Lavens et al. open flow-through systems cannot be considered. debris. In general. particles. at least one option will be most appropriate to a given situation when local conditions are taken into account. and overfeeding of the tanks occurs. however. by preference on the thoracopods. Black spots appear primarily on the extremities. a recirculation unit consists of a mechanical treatment that removes flocculations.. In high-density cultures of Artemia using agricultural by-products as a food source. A unit that removes the soluble fraction is also included. such as on the thoracopods and antennae. the black disease is observed when water quality deteriorates (probably interfering with the composition of the bacterial population and consequently the diet composition) and/or when feeding rates are not optimal. The brine shrimp suffer physically. proving that more than one solution exists to treat effluents from intensive Artemia cultures. The Leucothrix colonies fix on to the exoskeleton. as the movements of their thoracopods become affected and. An overview of the most frequently used treatment components is presented in Table 3. consequently. This disease consists of the detachment of the epidermis from the cuticula. However. eventually resulting in a collapse of the Artemia culture. One cure may be the application of tetramycin. A second observed disease in Artemia cultures is the ‘black disease’. which particularly occur in nutrient-rich media (Solangi et al. This unit should be designed to remove particles and decrease levels of harmful nitrogen components. and a biological treatment that breaks down ammonia to nitrite and nitrate. (2001) Bossuyt & Sorgeloos (1980) Biological treatment . Raising salinity from 35 to 50–60 g l 1 appears to be the most practical solution. and is caused by a dietary deficiency. coupled with a higher water renewal rate of 25% instead of 10% on a weekly basis (Lavens et al.3. Table 3. 1979).4 Water treatment systems used in intensive Artemia culture. High-density flow-through culturing of Artemia can only be sustained by recirculating the culture water via a water treatment unit. antibiotics cannot be used in recirculation systems as they will affect the biological treatment unit. Several suitable recirculation systems have been designed. (1982) Lim et al. (1986) Brisset et al. etc. and become visible only from instar V/VI stage onwards.7 Control of infections Heavy losses of pre-adults may be due to infections with the filamentous bacterium Leucothrix. Improving these conditions does not save the affected animals. and lowers the biological oxygen demand (BOD). 3. which interferes with lipid metabolism (Hernandorena 1987). 1986).92 Live Feeds in Marine Aquaculture Closed flow-through (recirculation) systems When only limited quantities of warm seawater are available. growth and moulting are arrested. Ultimately. but appears to avoid further losses. filtration rates are reduced. . which should be partially submerged. ( ) 300 litre closed flow-through culture on a mixture of soyabean waste and corn bran (modified from Lavens 1989). The biomass should be spread out in thin layers (1 cm) in plastic bags or on ice trays.19 provides a summary of average production data expressed as Artemia survival and length. ( ) 3 m3 closed flow-through raceway culture on rice bran diet (modified from Lavens 1989). When the aeration in the culture tank. ( ) 1000 litre open flow-through culture on rice bran diet (Dhert et al. unpublished data).Biology. 3. and its body fluids have a constant and low salt content of about 9 g l 1. and transferred to a quick freezer (at least 15°C). In seawater. Live brine shrimp can be transported in plastic bags containing cooled seawater under oxygen. The harvested Artemia can then be offered as live food for freshwater as well as marine predators. as Artemia is a hypo-osmoregulator. which do not pollute the water by leaching of body fluids. can easily be scooped out with a net of an appropriate mesh size. after about 30 min the Artemia respond to the oxygen depletion by concentrating at the water surface. When the culture water is not loaded with particles. After 100 80 growth (mm) 2 6 survival (%) 60 40 20 0 0 4 8 10 12 14 time (days) 8 7 6 5 4 3 2 1 0 0 2 4 6 8 10 12 14 time (days) Fig. together with the flow-through and the automatic feeding are interrupted. Artemia will continue to swim for another 5 h. ( ) 200 litre open flow-through culture on Chaetoceros diet (modified from Lavens 1989).3. Tank Production and Nutritional Value of Artemia 93 3. obtained in the different culture systems described in this chapter.8 Harvest and processing of cultured Artemia Harvesting of high-density cultures of Artemia can be facilitated by taking advantage of the surface respiration behaviour of the animals. The adult exoskeleton is not damaged when the biomass is frozen properly. 3. The Artemia should be washed thoroughly in freshwater or seawater. brine shrimp can be harvested by draining the complete culture over a sieve.19 Production figures of various intensive Artemia cultures. brine shrimp biomass must be frozen immediately after thorough washing with freshwater when still alive. they remain alive without feeding for several days.3. Upon thawing. When transferred into freshwater. To ensure optimal product quality. The salinity of the predator culture water is of no concern. oxygen levels in the water drop very quickly and all waste particles sink to the bottom. after which time they eventually die as a result of osmoregulatory stress. ( ) 500 litre batch culture on a mixture of pea and corn bran (Dhont. The concentrated population. free from suspended solids.9 Production figures of intensive Artemia cultures Figure 3. 1992). Harvested Artemia that are not for immediate consumption can be frozen or dried in flakes. the Artemia cubes yield intact animals. where they perform surface respiration. its composition is highly dependent on its diet. there is a significantly higher mortality during the end of the first week of culture. 3. distinctions between species. owing to its specific nature and the range of its applications. to adult biomass. which is nonfeeding.4. 3. which have a functional digestive . However. In stagnant cultures. probably because the early naupliar stages are more sensitive than the juvenile or pre-adult stages. different life stages of Artemia are used as larval food: from the embryonic form (as decapsulated cysts).4) the remaining embryo is digestible.5. milkfish and some marine shrimp. which can be explained by the deterioration of the water quality. Since Artemia is a non-selective filter-feeder with a relatively high ratio of gut content to body volume. These life stages may show important biochemical differences. pre-adult or adult Artemia with an average length of 5 mm or more can be harvested. No significant differences in survival are observed between open flow-through and recirculating cultures (Fig. Average production yields harvested after 2 weeks (live wet weight Artemia biomass relative to tank volume) amount to 5. 3. Presented figures inevitably consist of average values that may conceal subtle changes or intriguing differences.19). the most important differences in composition can be observed with the exogenous feeding stages (instar II and later).1. Unlike algae or rotifers. and flow-through systems using micronised feeds and live algae.1.1 Proximate composition Dealing with the biochemical composition of any living organisms usually involves a great deal of generalisation. and has been used successfully in the larviculture of carp. catfish. This section aims to give a comprehensive picture of the composition of various Artemia forms (see Table 3. However.5). These differences in production figures are mainly the result of differences in maximum stocking density at the start and survival at the end of the culture trial. sources and life-stage must be made. 15 and 25 kg m 3 for batch production. since they cannot be used as a food source. and subsequent stages (instar II and further). the biochemical composition of intact cysts could be considered irrelevant.4. Different Artemia strains or even Artemia from the same strain but from another batch or a different seasonal harvest may show variation in their composition.1 Cysts and decapsulated cysts Since the chorion of Artemia cysts is completely indigestible by all known cultured species. once this chorion has been removed by chemical decapsulation (see Section 3. 3.4 Biochemical Composition 3.94 Live Feeds in Marine Aquaculture 2 weeks of culturing.2 Nauplii There is a crucial distinction between the first larval stage or instar I.4. respectively. In a flow-through culture there is a slight but continuous mortality during the whole of the culture period. In the case of Artemia. through non-feeding nauplii and enriched nauplii. 1 1. dry weight. USA (c) PR China (b) France (b) SFB.8–67. However. USA (d) Nauplii GSL. USA (d) Italy (g) Protein (%) Lipid (%) Carbohydrate Ash (%) (%) Fibre (%) DW ( g) 55.4–19.4 56. d.0 41.0 12. they often remain a significant extra investment. Several publications offer data on naupliar composition.5 10.4 4.7 15.c.2 5. Great Salt Lake. USA (a) SFB.4 15.9 5.6 9. at least with respect to its protein quality and individual energetic content. (1987). USA (b) Italy (g) Adults: cultured GSL. García-Ortega et al. the nutritional value of ongrown Artemia compared with freshly hatched nauplii is superior.0 20.2 61.4 11.8–30. —. Correa Sandoval et al. 3.9 — — — — — — 4.6 — 7. but few authors make a clear distinction between instar I and instar II stages. Correa Sandoval et al. (2001).4 8. (1994). Lim et al. f.0 5.31 1. not mentioned.8–12. culturing Artemia requires a considerable amount of labour and infrastructure.45–2. Tank Production and Nutritional Value of Artemia 95 Table 3. San Francisco Bay. Their initial composition reflects the parental characteristics.17 20. for the reason that. (1986).3 Juveniles and adults Ongrown Artemia are used much less frequently in aquaculture than nauplii.6 — 20. USA (b.6 — 3.9 0.4–50.6 67.1. e. Even though various culture techniques have been developed to suit all kinds of local conditions (Section 3.9–59. Dhont & Lavens 1996). USA (b.4 12. This is understandable given the fact that asynchronous hatching produces batches of nauplii consisting of different stages.7 41.5 Proximate composition of different developmental stages of Artemia (% on a dry weight basis).2 64.4. b.0 14.1 21.d) Adults: wild population San Diego. Dendrinos and Thorpe (1987).3 — — 20.3 36.4 53.0 2.7–3.3.0 6.5 10. GSL.4 6. Instar I nauplii survive by depleting their yolk reserves.6 10.2 3.2 — 4.2 41. while from instar II nauplii onwards.9 47. Trotta et al.3 3. USA (a) SFB.1 4. system.0 55.6 10.5 7.6 21.Biology.2 — 5.6 9. (1993).9–27.1 14.4 15.2–13. USA (b) GSL. both genetic and phenotypic.6–47.0 50.9–23.4–64. c. g.6 — — — — — — — 5. (1998).42 — 2.2 — — — 4.2 50.6 29. USA (a) GSL.70 — 3.7 17. Léger et al. Artemia stage Source (refs) Cysts GSL.2 — 17. SFB.7 39.83 — — 3.09 2. and gradually decrease in nutritional value and in energetic content.6 — — 11.8 45.2 12.f) France (b) SFB.9 36.3–1.2 41. . while nauplii can easily be obtained through simple hatching of widely available and storable cysts.5–12. the composition will also be influenced by the diet.2–58.0–12. USA (e) Mexico (e) Decapsulated cysts GSL. USA (b) SFB. DW.87 — — — — — — — References: a.65–2.9 50.3 55. (1989).7 1.h) Urmia.b) France (b) Nauplii GSL.6 20.6 1. 2000b) can reflect either genetic characteristics or the lipid profile of the food of the parental population.7 1. docosahexaenoic acid.2.0 3. Han et al. 1984.5 2. l.5 27.5 13.9 9.4. persimilis.8 4.0–17. Triantaphyllidis et al.4 7. Published values seem to exhibit important differences (Table 3. d.0–8.4 References: a. Han et al.7 tr 1. SFB.7 4.5–3. these differences may also arise from different analytical methodologies or inaccurate definitions of what exactly was analysed (e.2–49. (2000b). (1998).2 17. Artemia stage Source (refs) Cysts GSL.7 2.2 0.4 15.6 Fatty acid composition of different developmental stages of Artemia (mg g Palmitic acid (16:0) Palmitoleic acid (16:1n-7) Oleic acid (18:1n-9) Linoleic acid (18:2n-6) Linolenic acid (18:3n-3) 1 dry weight).3 9. USA (a.9 1.96 Live Feeds in Marine Aquaculture 3.2 11.2 3.2 20. (1996).1 6.1 4. as several authors have demonstrated that the fatty acid profile of Artemia adults and their offspring clearly reflects the composition of the parental diet. k. (1993).1–7.2–1. 18:3n-3 and 20:5n-3) actually make up Table 3.1 28.7 24.3 0.1–25.7–30.9 5.1 17. 18:2n-6. GSL.4–7. Dendrinos and Thorpe (1987) (adults fed on yeast).4 0. Han et al. tibetiana.0 3.4 23.5 2.5 0.0 0.1 0. Many publications include values for 15 or more different fatty acids in Artemia nauplii. eicosapentaenoic acid.6 12. tr.c.4.8 2.7 3.8 8. Lavens (1989) (adults fed on soya pellets). Dhert et al.6–40. j.2 3. 3. The differences in lipid profile that are observed between strains (Triantaphyllidis et al. the lipid profile is considered to be environmentally rather than genetically determined. c.4 0. Lavens et al. USA (l) SFB.5–14. yielding a wealth of published lipid analysis on all kind of Artemia strains and live stages. However.0–0.4 7.d) SFB.2 7. García-Ortega et al.0–0. g.5–8.8 6.2 15. f.7 21.3 22.8 6.9 40. San Francisco Bay.g.7–10. (1995). exact strain or larval stage).4 0.3 0. 1989. USA (a) Decapsulated cysts GSL.8 0.7 5.0 0.1 34.8 23.4 3.6 3.7 18. . Navarro & Amat 1992).0 1. (1986) only six fatty acids (16:0.7 6.2–19.h) Madagascar (i) A. but according to Léger et al. 1995.1 9.9 0.4 18. Lim et al. e. Iran (g) A. Millamena et al.6).1–0. 16:1n-7. Lavens et al.4 8. USA (e) PR China (f. EPA.7 16.5 3.9 20. (1999). Besides natural fluctuations. regardless of the strain (Vos et al. b. 1988. USA (a. trace.7 0.4 0.5 6.1 24. h.1 6.3 12. Great Salt Lake.1 5.3–34.25 16. USA (k) GSL.5–14. (2001) (adults fed on rice bran).7 16.6 15.0 19.9 0.2 3.0 44. i. parthenogenetica (g.g.9–11. 18:1n-9. USA (c) EPA (20:5n-3) DHA (22:6n-3) 12.2 9. Estevez et al.2 Lipids The lipid fraction has undoubtedly received most attention in marine larviculture.6 14. DHA.4 1. (1998). Triantaphyllidis et al.6–39. Arg (h) A.9–4.1 Cysts and nauplii The lipid content and profile of cysts and instar I nauplii are not affected by diet or environmental conditions.5 tr 0. China (j) Adults GSL. Han et al.5–13. (2000a).0 6. 3.6. the lipid fraction (Table 3. (1986) compiled data on almost 150 fatty acid analyses of nauplii from about 20 different Artemia sources and came to the following conclusions. Nevertheless. This is the basis of the enrichment technique that is discussed in detail in Section 3. which was not reported earlier. generally. their lipid profile will quickly reflect the profile of their diet. some authors also detected significant levels of 18:1n-7. while 43% of the samples contain less than 10% linolenic acid. The level of 16:0 is fairly constant over different strains. it should be borne in mind that data on lipid composition of Artemia biomass reflects a variety of influences such as diet. (1986) concluded that Artemia nauplii contain between 0. 1998. Oleic acid (18:1n-9) is often the most abundant fatty acid. Just as for nauplii. Han et al. but that the distribution of these levels is actually bimodal: 36% of the samples contain more than 20% of their total fatty acids as linolenic acid. 1998). which is composed of lipoprotein impregnated with chitin and haematin (García-Ortega et al.Biology. Even so. life stage and physiological stage. Thus. Only data expressed in mg g 1 dry weight have been listed here.2 Ongrown Artemia As soon as brine shrimp start feeding (at instar II). for example. It also has the most stable occurrence (lowest coefficient of variation).4 and 33.5. it seems correct to state that protein levels and amino acid profiles show much less fluctuation between strains or life stages than. geography. methionine seems to be the first . strain. and as such the figures presented only offer an indication of possible levels. Léger et al. but levels of 16:1n-7 are more variable. probably reflecting increased chromatographic resolution (Estevez et al. 2000b).3 Proteins Some confusion appears when reviewing amino acid profiles of Artemia because of different methods of analysis or reporting the data.4. Tank Production and Nutritional Value of Artemia 97 about 80% of the total fatty acid pool in an Artemia sample.2.6% linolenic acid (18:3n-3). 3.7). season. Indications that Artemia converts docosahexaenoic acid (DHA) into EPA (McEvoy et al. Léger et al. Together with palmitic (16:0) and palmitoleic acids (16:1n-7). 1995) were proven to be correct by Navarro et al. Most of these patterns can be recognised in the more recent compilation presented in Table 3. In later publications. The levels of EPA (20:5n-3) seem to be inversely related to linolenic acid levels. Nauplii contain markedly lower levels of free amino acids compared with wild copepods (Tonheim et al. Adult Artemia have a slightly higher protein content than nauplii and contain slightly more essential amino acids than nauplii. The conversion rate seems to vary according to the strain (Evjemo et al. 2000) but. as these not only reflect the relative proportions of the various fatty acids. The metabolic pathways and abilities of Artemia to convert one fatty acid to another are not yet entirely elucidated. the lipid composition of natural Artemia biomass will reflect the composition of its diet. (1999). Artemia nauplii as well as adults contain sufficient levels of the 10 amino acids that are considered essential for fish larvae. but also indicate their quantities with reference to Artemia body weight. The higher protein content of cysts compared with decapsulated cysts or nauplii is due to the presence of the chorion.5. it accounts for 40–60% of the total fatty acids in Artemia.4. 1997). 0–9. Trotta et al.4 and 49.7 Amino acid composition of different developmental stages of Artemia (g 100 g 1 protein).0 3.5–13.2 7. With the exception of AscA and thiamine.6–6.2 85.6 4.1 1. b.2 2.3 3.6 4.4 4.4.3 85.3–5.9 2.2–12.8 5.3 4. 1998) Artemia seems to cover the minimal dietary requirements of .0 5. (1987).98 Live Feeds in Marine Aquaculture Table 3.1 4.5–111.9–4.2–9.. The presence of these low molecular weight peptides and free amino acids in nauplii.2 4.7 7.8 1.0–6.1 References: a.6–9.2–5.0 5.9–4.7–7.9 2.7–5.2 — 52. An account of differences between strains could only be found for ascorbic acid (AscA) forms in cysts in Merchie et al.1 1.2 kDa (García-Ortega 1999). limiting amino acid when feeding nauplii to fish larvae (Fyhn et al.2–5.5–3.4–5.9 4.3–10.5 2.e) 5.5–8. Kaushik et al.3–4.2 2.0 4. c.1–1. Decaps.5–7. Stage (refs) Cysts(b) Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Cystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Histidine Lysine Arginine Tryptophan Total amino acid 8.4 0. García-Ortega et al.4 0.6 3.7 11.2 7.8–16. 2000) and according to the NRC standards (NRC 1993.c) 7.4 Vitamins Most data on vitamins relate to Artemia franciscana (Table 3. e.3–6. decapsulated.1 6.4–6. 3.8–9.7 7.8–10.3 4. Bond et al.1–125.6 3.4 2.2 2.3 4.6 4.6 4.4 1.1 6.9–5. Whether AscAS serves as a storage form of AscA to satisfy the brine shrimp’s larval requirements after hatching (Mead & Finamore 1969). 1993. Most protein in Artemia nauplii consists of small size proteins with a molecular weight between 7. (1998).2 5. Artemia nauplii contain higher vitamin levels than natural marine zooplankton (Mæland et al.7 Juveniles and adults (b. together with their autolytic capacity and high solubility. (2001) (adults fed on rice bran).c.3–1.7 0.5 11.7 3.4 3.1 1.4 3.6 Nauplii (a. (1995).7–3.3–5. 1972) is unclear.0 2.3 10.3 4. Lim et al.8). They observed considerable differences in ascorbic acid 2 sulfate (AscAS) concentrations (296–517 g g 1 dry weight expressed as AscA) when comparing 10 different strains.8 Decaps.4–3.7–8.9 4. Dendrinos and Thorpe (1987) (adults fed on yeast).5 7.2 3.3–8.1–2.1–4.9–6.2–7. cysts (a.9 2.9 3. 1999).9 87. accounts for the easy digestion of the proteins by fish larvae.9 2. Furthermore.3 1.1–9.3 5.5–3.3 2.1 4.9 3.7–11. 1997.5–105.1–1.0–13.7–2.0–4.4 2.4 6. or acts as a sulfating agent during embryonic development (Mead & Finamore 1969. they provided further evidence for the complete conversion of the ascorbic acid 2 sulfate form to free ascorbic acid.3–3.7–11. Conceição et al.9–5.6–6.9 6.3 3.3 6.c) 7.1 6. Helland et al.0 4. several factors are critical for the successful hatching of the large quantities needed in larval fish production.5 Applications of Artemia 3. (1986).2d 108. (1995).5 47. (1983).4 3.0a 0. but it seems far from redundant to improve and promote the existing applications of Artemia in live food production. Tank Production and Nutritional Value of Artemia 99 Table 3.5 18.0a 8a 19a 5. cMerchie et al.6a 3. exact dietary vitamin requirements have not been established for many marine fish larvae. fish larvae.1a 6.5.6d 10.2 Hatching Although hatching Artemia cysts appears to be simple. the use of nauplii will continue to be market driven for at least a few more years (see also Section 3. (2000).0 3. Cysts Ascorbic acid Ascorbic acid 2 sulfate Thiamin Riboflavin Niacin Pantothenic acid Vitamin B6 Biotin Folate Vitamin B12 Choline chloride Inositol Piridoxine HCl a Nauplii 692 89a 1.5 d Mæland et al.0 minimum oxygen levels of 2 mg l 1. and hence the production cost of the harvested Artemia nauplii. Best hatching results are achieved in containers . Optimal hatching conditions are (Lavens & Sorgeloos 1996): • • • • • • constant temperature of 25–28°C 15–35 g l 1 salinity pH around 8. 3. However. All of these factors will affect the hatching rate and maximum output.1).5d 7. bSimpson et al.8a Adults 49b 27b 17b 130b 68b 1b 1b 3b 6100b 1200b 8b 861 16c 7.4a 0.1 The future use of Artemia in aquaculture Although there is no doubt that Artemia will gradually be replaced by formulated diets. Léger et al.13d 23.7d 72.Biology. preferably 5 mg l 1(see below) maximum cyst densities of 2 g l 1 strong illumination of 2000 lux. 3.5.3 187 86 9. Increased harvests at GSL and new locations may relieve the pressure or even reverse the current trends.8 Vitamin levels in different developmental stages of Artemia ( g g 1 dry weight). and the nauplii have a higher energy content (Table 3. Above threshold salinity.20). For some sources of cysts. The optimal aeration rate is a function of the tank size and the density of cysts incubated. preferably 5 mg l 1.20 Large-scale hatching set-up. For practical convenience. natural seawater is most often used to hatch cysts. the incubation salinity will interfere with the amount of glycerol that needs to be built up to reach the critical osmotic pressure within the outer cuticular membrane of the cysts. for instance when low-salinity water is used. The underlying physiological processes of hatching are described in detail in Section 3. especially when harvesting. Quantitative effects of the incubation salinity on cyst hatching are related primarily to the hydration level that can be reached in the cysts. aerated from the bottom with air-lines (Fig. Cylindrical or squarebottomed tanks will have ‘dead spots’ in which Artemia cysts and nauplii accumulate and suffer from oxygen depletion. The effect of more extreme temperatures on the hatching output is largely strain specific. silicone antifoam). the cysts do not absorb sufficient quantities of water.100 Live Feeds in Marine Aquaculture Fig. Transparent or translucent containers will facilitate inspection of the hatching suspension. The aeration intensity must be sufficient to maintain oxygen levels above 2 mg l 1. since it will take less time to reach breaking.2. This threshold value varies from strain to strain. 3. with a conical bottom. Secondly. 3. the buffer . Excessive foaming can be reduced by disinfection of the cysts before incubation and/or by the addition of a few drops of a non-toxic antifoam agent (e. hatching at low salinity results in higher hatching efficiencies.g.9). The temperature of the seawater should be kept in the range of 25–28°C. The pH must remain above 8 during the hatching process for optimal functioning of the hatching enzyme.5. Optimal hatching can be obtained in the range 15–70 g l 1. If necessary. but practical implications are reiterated here. but is approximately 85–90 g l 1 for most Artemia strains.2. The fastest hatching rates will thus be noted at the lowest salinity levels. below 25°C cysts hatch more slowly and above 33°C cyst metabolism is irreversibly stopped. Strong illumination (about 2000 lux at the water surface) is essential.8 0.5 73. Cyst density interferes with other abiotic factors that are essential for hatching.2 4.5 1.4 406.5 537. USA Macau. PR China Hatching output (mg nauplii g San Francisco Bay. Australia Chaplin Lake.28 3. an impressive bacterial load rapidly develops (Dehasque et al.35 2. (1984). Although this level of illumination can generally be attained in daytime by using transparent tanks set up outdoors in the shade. Salinity Cyst source Hatching percentage San Francisco Bay. Australia Chaplin Lake. Australia Chaplin Lake. so as to ensure good standardisation of the hatching process.5 68.1 2.4 45.5 19.Biology. Brazil Great Salt Lake.0 256.1 1.7 257. Tank Production and Nutritional Value of Artemia 101 Table 3. individual nauplius weight and hatching output for Artemia cysts from different geographical origins. This is a potential source of pathogens.0 43.1 1. 1993).64 2.8 199. a competitor for oxygen and a general threat to hatchery hygiene.63 1.0 563.5 6.76 2.0 1.1 6. oxygen and illumination.42 2.3 85.74 2. it is advisable to keep the hatching tanks indoors and to provide artificial illumination.47 2. USA Macau. through decapsulation (see Section 3.0 86.9 Effect of incubation at low salinity on hatching percentage. Brazil Great Salt Lake.9 87.5 133.9 11.0 6. PR China 1 35 g l 1 5gl 1 % diff. Brazil Great Salt Lake. It can be achieved through simple disinfection of the cysts using liquid bleach solution. The density may be as high as 5 g l 1 for small volumes ( 20 litres) but should be decreased to maximum 2 g l 1 for larger volumes. at least during the first few hours after complete hydration.3 3.2 75.07 440. 71. Reducing bacterial development during hatching will improve the hygienic status of nauplii and may result in better hatching yields. capacity of the water should be increased by adding up to 1 g NaHCO3 l 1.0 4.73 1.3 1.8 400. Canada Bohai Bay. Increased buffer capacity is also essential when high densities of cysts are hatched (because of high carbon dioxide production). to minimise mechanical injury to the nauplii and to avoid suboptimal water conditions.2 563.5 529.6 2. Canada Bohai Bay.4 82.9 167. to trigger the start of embryonic development. USA Shark Bay. USA Macau.4 Modified from Vanhaecke et al. such as pH. USA Shark Bay. When hatching large quantities or high densities of cysts.09 cysts) 435.8 52.5.6 0. Canada Bohai Bay.3 400.8 5. PR China Naupliar dry weight ( g) San Francisco Bay.4) or through the use of recently .6 1.04 3. USA Shark Bay. i. as soon as Fig.5. 3. unhatched cysts.e.102 Live Feeds in Marine Aquaculture developed cyst and enrichment products that achieve disinfection during the course of the hatching process (Sorgeloos et al. 3. while nauplii and unhatched cysts will concentrate at the bottom (Fig. cyst shells will float and can be removed from the surface. Attention should be paid to the selection of Artemia cyst batches with good hatching synchrony (less than 7 h between hatching of first and last nauplii) and high hatching efficiency (more than 200. debris. Since instar I nauplii rely solely on their endogenous yolk reserves they should be harvested and fed to the fish or crustacean larvae in their most energy-rich form. and even among batches from the same strain (Vanhaecke & Sorgeloos 1982).21 Hatching tank after switching off aeration. 3.e. when using cysts of a lower hatching quality). Since nauplii are positively phototactic. . unhatched cysts and other debris that have accumulated underneath the nauplii are siphoned or drained when necessary (i. which should be submerged at all times to prevent physical damage to the nauplii. 3. their concentration can be improved by shading the upper part of the hatching tank (use of cover) and by focusing light on the bottom part of the conical tank. as they will quickly suffer from oxygen depletion. First.22) allows fast harvesting of large volumes of Artemia nauplii and complete removal of debris from the hatching medium. In commercial operations the use of a concentrator/rinser (Fig.3 Harvesting hatched nauplii After hatching and before feeding to fish/crustacean larvae. the nauplii should be separated from the hatching wastes (empty cyst shells. They are rinsed thoroughly with water to remove possible contaminants and hatching metabolites such as glycerol. as considerable variation has been demonstrated for cysts from different sources.21). Nauplii should not be allowed to settle for too long in the bottom of the conical container. 2001). micro-organisms and hatching metabolites). Five to ten minutes after switching off the aeration. Then the nauplii are collected on a filter with a fine mesh screen ( 150 m).000 nauplii per gram product). 3. (Modified from Léger et al. cold stored nauplii and decapsulated cysts. 1986). Fig. At the high temperatures that occur during cyst incubation.23). instar II Artemia are less visible as they are transparent.23 Energy content and dry weight of instar I.22 Concentrator/rinser used for an efficient harvest of large amounts of hatched Artemia.) . and their lower individual organic dry weight and energy content will reduce the energy uptake by the predator per hunting effort.Biology. and increased Artemia cyst usage and cost. instar II. 3. Moreover. It is important to feed first instar nauplii to the predator rather than starved second instar meta-nauplii which will already have consumed 25–30% of their energy reserves within 24 h after hatching (Fig. Fig. 1987a. 3. Farmers often overlook the fact that an Artemia nauplius in its first stage of development cannot take up food and thus consumes its own energy reserves. the freshly hatched Artemia nauplii develop into the second larval stage within a matter of hours. They are also larger and swim more rapidly than first instar larvae. Furthermore. As a result they are less accessible as prey. as about 20–30% more cysts need to be hatched to feed the same weight of starved meta-nauplii to the predator (Léger et al. they contain lower amounts of free amino acids. Tank Production and Nutritional Value of Artemia 103 possible after hatching. All this may be reflected in a reduced growth of the larvae. 10). Older penaeid larvae. The use of decapsulated cysts as a food source is much more limited than the use of Artemia nauplii. thus reducing their availability for fish larvae feeding in the water column unless adequate mixing of the culture water is applied. where cysts have a relatively low energy content. the complete separation of Artemia nauplii from their shells is not always possible.104 Live Feeds in Marine Aquaculture 3. .5. Nevertheless. and marine shrimp and milkfish larvae (Verreth et al. Australia Chaplin Lake. (1980). 1995. the common carp (Cyprinus carpio). dried decapsulated Artemia cysts have proven to be an appropriate feed for larval rearing of various species such as the freshwater catfish (Clarias gariepinus). PR China Hatchability 15 12 24 4 132 4 Modified from Bruggeman et al. Cysts with poor hatching quality or even non-hatching cysts can still be used as a food source. Decapsulated cysts. however.7). Stael et al. have the disadvantage that they are non-motile and thus less visually attractive to the predator. Decapsulation results in complete disinfection of the cyst material. Table 3. is avoided. Moreover. a labour-intensive job that requires additional facilities. Using decapsulated cysts in larval production offers a number of advantages over nauplii and non-decapsulated cysts: • • • • • The daily production of nauplii. USA Shark Bay. 1987. decapsulated cysts dehydrated in brine sink rapidly to the bottom. Cyst shells are not introduced into the culture tanks. the gross biochemical composition of decapsulated cysts is comparable to that of freshly hatched nauplii (García-Ortega et al. however. From the nutritional point of view.10 Improved hatching characteristics (%) of Artemia from different geographical origins as a result of decapsulation.5–3. Naupliar dry weight 7 2 2 6 5 6 Hatching output 23 14 21 10 144 10 Cyst source San Francisco Bay. Vanhaecke et al. are mainly bottom feeders and do not find this a problem. because of the lower energy requirement to break out of a decapsulated cyst (Table 3. Nauplii that are hatched out of decapsulated cysts have a higher energy content and individual weight (30–55% depending on strain) than ‘regular’ instar I nauplii from nondecapsulated cysts. Sui 2000). In some cases. Brazil Great Salt Lake. When hatching normal cysts. 1990. because they do not expend energy breaking out of the shell.4 Decapsulation Decapsulation is the process whereby the chorion that encysts the Artemia embryo is completely removed by a short exposure to a hypochlorite solution (Bruggeman et al. USA Macau. Canada Bohai Bay. Ribeiro & Jones 1998. Unhatched cysts and empty shells cannot be digested by fish or shrimp larvae and may obstruct the gut when ingested. 1998) (Tables 3. the hatchability may be improved by decapsulation. 1980). the decapsulated cysts can be transferred into a saturated brine solution. and start filtering particles smaller than 25 m irrespective of their nature (Makridis & Vadstein 1999. 3. instar II. brine shrimp moult to the second naupliar stage. crab. After harvesting of these cysts on a mesh screen they should be stored cooled in fresh brine.23).5. 1991). These decapsulated cysts can be directly hatched into nauplii.5 Enrichment The nutritional value of Artemia nauplii is easy to manipulate thanks to their primitive feeding characteristics. Fig. their individual dry weight and energy content is on average 30–40% higher than for instar I nauplii (Fig. Gelabert Hernandez 2001). This ‘bioencapsulation’ or ‘enrichment’ is a now very common practice in fish and crustacean hatcheries for enhancing the nutritional value of this live feed or for delivering specific ingredients to cultured larvae. the now coffee-bean-shaped decapsulated cysts settle out. 3. and upon interruption of the aeration. 3. 3. or dehydrated in saturated brine and stored for later hatching or for direct feeding.24 Principle of bioencapsulation or enrichment. Tank Production and Nutritional Value of Artemia 105 In addition. removal of the brown shell in a hypochlorite solution and deactivation of the remaining hypochlorite by washing.5.1 Lipid enrichment In the early 1970s. The decapsulation procedure (Appendix II) involves the hydration of the cysts (as complete removal of the envelope can only be performed when the cysts are spherical).5. If storage for prolonged periods is needed (weeks or few months).Biology. 3. Since they lose their hatchability when exposed to UV light it is advisable to store them protected from direct sunlight. After considerable efforts by diverse research groups. simple methods were developed to incorporate various kinds of products into nauplii before feeding them to predatory larvae (Fig. lobster and marine fish larvae when using Artemia sources other than SFB Artemia. After about 8 h posthatch.24). . During overnight dehydration (with aeration to maintain a homogeneous suspension) cysts have released over 80% of their cellular water. prawn. these striking differences in culture results could be related to the origin of the Artemia strain used (Bengtson et al. Taking advantage of this non-selective filter feeding. They can be stored for a few days in the refrigerator at 0–4°C without a reduction in hatchability. several authors reported problems with the larviculture of shrimp. 5. most attention was focused on the presence of EPA (20:5n-3) in Artemia and its importance in the successful production of marine fish and crustacean larvae (Watanabe et al. Initially. Altering the dietary dose of one of them will influence the ARA:EPA:DHA balance owing to competitive interactions and metabolic conversions (Sargent et al. 1983a) revealed differences in levels of specific polyunsaturated fatty acids. and EPA is important in modulating eicosanoid production by competing for the same enzyme systems that convert ARA to eicosanoids (Sargent 1995). The requirements for these essential fatty acids cannot be considered separately. 1985). 1999b). 1994. as in mammals (Castell et al.5. 1983b. Dietary HUFA requirements seem to be. the reader is referred to the reviews by Sargent et al. 1998b). 1999. Reitan et al. Kraul 1993. Copeman et al. EPA or arachidonic acid (ARA. (1999a. 1983b. at least to some extent. species specific. DHA is a major constituent of neural and visual cell membranes and. Besides absolute HUFA requirements. A myriad of studies has been carried out on the essentiality of long-chain. 1993). especially in marine flatfish (Watanabe et al. Kraul 1993. marine zooplankton generally have ratios substantionally higher than 1:1. 1997). more particularly the requirement for high DHA:EPA ratios (Lavens et al. 1999).b). . While the DHA:EPA ratio in enriched Artemia rarely exceeds 2:1. EPA is present in large amounts in the cellular membranes of marine fish larvae and is also a precursor of eicosanoids (Sargent et al. 1999b). 1981. Mourente et al.2). the importance of polar lipids and the distribution of HUFA between dietary phospholipids and triacylglycerols (TAG) should not be overlooked (see Section 3. Consequently. • • • • • • • • Most marine fish larvae cannot synthesise DHA. Léger et al. 1979) and with other zooplankton (Nellen et al. To provide fish larvae with adequately enriched Artemia. 1993). thus. Eicosanoids formed from EPA are less biologically active than ARA-derived eicosanoids. 1995). 20:4n-6) from shorter chain precursors and they must be provided preformed in the larval diet (see review by Sargent et al. attention shifted to DHA when several authors documented the importance of DHA. zooplankton-fed halibut larvae have a much higher DHA level and DHA:EPA ratio than enriched Artemia-fed larvae (McEvoy et al. ARA (20:4n-6) is the major precursor for eicosanoids in fish. 1995. 1993. In the late 1980s and early 1990s. with the ultimate goal of optimising larval feed and/or enrichment products. often 4:1 and higher (Shields et al. see also Chapter 5). Bell et al. is essential for a range of physiological processes that are crucial to fast-growing marine fish larvae (Sargent et al. 1994. Watanabe et al. the following points should be considered. For the latest developments and insights into larval fish lipid nutrition. 1994. stress resistance and proper pigmentation. Reitan et al. It has been proven that optimised DHA levels and high DHA:EPA ratios improve growth. These facts have various consequences on the usefulness of enriched Artemia as larval food and the modalities of enrichment procedures. highly unsaturated fatty acids (HUFAs) in several fish and shrimp species to gain a better understanding of their true requirements. Mourente et al.106 Live Feeds in Marine Aquaculture Comparative studies with different strains of Artemia (Kanazawa et al. However.5. Estevez et al. as excessive ARA:EPA ratios tend to exert negative effects on pigmentation (McEvoy et al. Velazquez 1996) and to maintain them during subsequent starvation (Evjemo et al. 3. Moreover. physiological functions and dietary requirements. It is difficult to maintain high DHA levels in enriched A. 1996). franciscana because of the rapid retroconversion of DHA to EPA. (1987b). the highest enrichment levels are obtained using emulsified concentrates (Table 3. involves the incubation of freshly hatched nauplii in an enrichment emulsion for a period up to 24 h (for detailed procedure. developed by Léger et al. we are still far from understanding every species’ requirements and it therefore seems a logical approach to tune the composition of the larval food and associated enrichment products to reflect the natural diets of the larvae. 1999b). they are likely to be species specific. Copeman et al. 18:3n-3) in the phospholipids of enriched Artemia will hinder fish larvae in assimilating sufficient EPA and DHA (mainly . resulting in decreased DHA:EPA ratios as soon as enrichment is interrupted (Navarro et al. yolk and zooplankton (Sargent et al. 2000a). In parallel to this relentless unravelling of the biochemical pathways. 1993. Nevertheless. they are increasingly supported by sound insight into the true dietary requirements of marine fish larvae. yeast and/or emulsified preparations. Although initially the composition of enrichment products was often based on empirical trials of variable components. Although fish larvae appear to have a certain ability to convert fatty acids between phospholipids and TAG. and the constant improvement of enrichment products ensure its continued use in marine fish larviculture. see Appendix III). enriching Artemia with traditional products seems to increase the fraction of TAG at the expense of the phospholipid fraction (McEvoy et al.Biology. This procedure. the relatively high proportion of linolenic acid (LNA. numerous enrichment products and procedures were developed.11). Although retroconversion of DHA to EPA may also occur in rotifers. namely. Although the exact requirements and effects of ARA in relation to EPA and DHA are not fully understood. Tank Production and Nutritional Value of Artemia 107 • • Although ARA is an essential precursor of eicosanoids. (1997) demonstrated that the observed positive effect of phospholipid supplementation was not connected to the role of phospholipids as an additional HUFA source. which suggested that dietary phospholipids were necessary to compensate for a limited ability for de novo biosynthesis by the fish larvae.2 Phospholipid enrichment Several marine fish and shrimp larvae seem to have a requirement for phospholipids (see review by Coutteau et al. it seems to occur at a lower rate than in Artemia and it is easier to maintain high DHA levels in rotifers. Although Artemia is often an inferior food source for fish larvae compared with wild zooplankton. in contrast to zooplankton. 1998a. Han et al. An interesting solution to this problem may stem from the capacity of some Artemia strains to reach high DHA levels during enrichment (Dhert et al. Currently. 1999. 1999). the ability to produce any amount of biomass within 24 h. 1996). the dietary requirements for ARA are relatively low. self-emulsifying concentrates and/or microparticulate products (reviewed by McEvoy & Sargent 1998). using selected microalgae and/or microencapsulated products. 1997.5. dietary levels must be carefully chosen. Geurden et al. 1999). (1999) Narciso et al.8 32.9 0.3 0.1–39.4 LNA. especially in methionine. In further studies.11 Overview of lipid levels obtained through enrichment of Artemia franciscana by various authors (mg g 1 dry weight).5 0.1–14.5. indigestible dipalmitoyl phosphatidylcholine (DPPC.4 2.7–40.7 1. using liposomes.5–45.9 4. DHA.5 25.1–20.9 4. (1998) Estevez et al.1 36.2–4. docosahexaenoic acid.6 9.0 1. incorporated to stabilise the liposomes) remaining in the guts of the Artemia and.0–7.3–1.9 10.5.4 9. (1995) Evjemo et al. Moreover.6 1.2 23.9 1.1 5.108 Live Feeds in Marine Aquaculture Table 3.4 — 3.2–28. DHAethyl esters were compared with DHA-containing phospholipids. this increase in polar lipid may have represented solidified. EPA. (1996) obtained significantly higher levels.2 — — 2. 3.2 29.4–0.7 — — 25.1 5.3 Protein enrichment Compared with lipids.7 0.5 1.7 0.6–21.0 14. despite the fact that amino acid catabolism is a major source for energy in fish larvae (Dabrowski 1983) and amino acids are essential for the synthesis of proteins and enzymes (Conceição 1997). (1997) Harel et al.8 4.5 27.) LNA (18:3n-3) ARA (20:4n-6) EPA (20:5n-3) DHA (22:6n-3) DHA:EPA 39. In an effort to determine the most effective molecular carrier of DHA for Artemia.0–25. Increasing the polar lipid level in Artemia with emulsions is difficult but. (1993) Triantaphyllidis et al.3 0. much less research has been carried out on the role and requirements for protein in larval nutrition. ARA.6 3.4–2.9 — 2. 1999) and may be used to increase the polar lipid content in larval live food. Stage Reference Nauplii Dhert et al.6–53. 2000).2 4. Although it is believed that Artemia contain adequate levels of most amino acids.0 1.0–0.7–17. arachidonic acid. it was observed that mixtures of phospholipids with DHA sodium salts resulted in maximal absorption of DHA phospholipids in Artemia (Harel et al.8 10.5 17. (1997) observed growth retardation in turbot larvae fed on Artemia and suspected it to be related to methionine deficiency.6 21.8 1. 2001). Conceição et al.4–19.0 2. (2001) Dhont (unpubl.9 13.7–3.6–45. (1993) Dhert et al. the fraction of free amino acids is low compared with levels in wild copepods (Tonheim et al. not determined.3 1.5–11.7 2.3 0. if so.1 5. (1998) found significantly higher absorption of DHA using 10% dietary phospholipids compared with 5%. eicosapentaenoic acid. as these authors point out. (1999) Estevez et al. McEvoy et al. . However.1–1.0 1.4–11.3 — — — 20.2 0. it is questionable whether this DPPC could be digested by fish larvae.5–33. while no further improvement in absorption was obtained at higher phospholipid percentages.4–4. —. linolenic acid. (2000b) Adults Han (2001) Lim et al. (1999) Han et al. larvae seem to have higher amino acid requirements than juvenile or adult fish (Dabrowski 1986. Fiogbé & Kestemont 1995).1 1.8–4. present in the TAG of the ingested Artemia) to replace the LNA in the ingested phospholipids (Bell et al.1 11.9–26.8–9.7 17. Harel et al.2–16.0–10.3–0. Tank Production and Nutritional Value of Artemia 109 Table 3.12). the larvae given the high AscA treatment showed a better pigmentation rate compared with the control group. (2000). tyrosine. isoleucine.5. (1999). This increase could be doubled (to 60-fold of the unenriched control) when incorporating methionine in liposome droplets.2 3. or vitamin B6 or B12 (Mæland et al. riboflavin. However.) (a) (c) (b) (c) (b) (b) (b) (b) (b) (b) (b) References: a.4 Vitamin enrichment Enrichment of Artemia for 48 h with DHA-Selco (Inve. When these vitamin C-enriched Artemia were fed to turbot larvae. The pattern for levels of essential amino acids was not very clear: some levels clearly increased (leucine.Biology. 1996). Olsen et al. Evaluation of the physiological condition through a salinity stress test also revealed an improvement. Mæland et al. (1995).8 20–40 38. High levels of -tocopherol can be bioaccumulated and maintained in Artemia nauplii. In a 24 h enrichment with self-emulsifying concentrates containing 10–20% ascorbyl palmitate (AscP). lysine) but others remained unchanged or decreased slightly (phenylalanine. Belgium) led to increased levels of thiamin. no differences in growth or overall survival could be detected compared with those fish fed the non-enriched live food containing 500 g AscA g 1 dry weight. c. Cumulative mortalities after challenge with Vibrio anguillarum amounted to 50% for the control versus 40% for the AscA-supplemented fish. (2000) demonstrated that methionine levels in nauplii could be boosted 20–30-fold in 16 h by simply adding dissolved methionine in the water.7 4.000 8.0 12. Merchie et al.5. b. with a slower onset of mortality for the AscA-fed fish (Merchie et al.3 187 86 9. histidine. 3. making this live food delivery system useful for studying dietary requirements as well as antioxidative effects of vitamin E (Huo et al.5 47. Better results for selected vitamins are obtained using specific enrichment preparations. 1995). arginine).0 3. Tests have been conducted to incorporate extra AscA into Artemia nauplii in a stable and bioavailable form (see Table 3.4 3.5 18. niacin and pantothenic acid. 2000).12 Vitamin levels obtained by enrichment ( g g Unenriched nauplii 692 7. levels up to 2. Dendrinos and Thorpe (1987) cultured Artemia on different types of protein and observed an increase in total protein content when feeding Candida utilis and Saccharomyces cerevisiae. but no changes in the content of AscA. 1995).5 mg free AscA g 1 dry weight were achieved in brine shrimp nauplii (Merchie et al.9 Vitamin Ascorbic acid Thiamin Riboflavin Niacin Pantothenic acid Vitamin B6 Biotin Folate Vitamin B12 (Ref.0 202 81 6. . 24 h enriched nauplii 3100 1000–12. Tonheim et al.5 1 dry weight). Evjemo et al. 2001).110 Live Feeds in Marine Aquaculture Vitamin A levels in Artemia nauplii could be raised from 1. (2001) kept nauplii after enrichment at moderate densities ( 100. 3. Rønnestad et al.23).000 l 1) and recorded survival above 70% for temperatures between 8 and 19°C.5. but fatty acid levels. while . More detailed analysis revealed that protein is fairly well conserved even after 96 h (Evjemo et al. especially the lipid content (Léger et al. Although the pigmentation status was not systematically recorded. it was generally accepted that nauplii stored at low temperatures maintained their biochemical composition (Fig. Nauplii stored at 20 million/l showed good survival ( 70%) even after 72 h when kept at 12°C with a slight injection of pure oxygen (Anbaya Almalul 2000).5. watersoluble compounds via liposomes (Hontoria et al.6. e. 3. decrease significantly. Only slight aeration is needed to prevent the nauplii from accumulating at the bottom of the tank where they might suffocate. the authors observed a higher incidence of malpigmentation with Artemia-fed larvae. it may be of interest to mention that techniques have been developed for oral biomedication. However. disapproved of or even banned.6.1 Survival at low temperatures Moulting of the Artemia nauplii to the second instar stage can be delayed and their energy metabolism greatly reduced by storage of the freshly hatched nauplii at a temperature below 10°C at densities of up to 8 million/l (Léger et al. (1998) demonstrated that up to 70% of the DHA obtained through enrichment was catabolised. Nauplii can be stored for more than 24 h without significant mortalities and a reduction in energy of less than 5%. for each nutrient. 3. mainly DHA. 1982). 1996). respectively.5.5 Enrichment with prophylactics Although the use of antibiotics in larviculture is rightfully questioned. 1996.2 Maintenance of nutritional value Initially.5.6 Enrichment with other products The effectiveness of Artemia nauplii as a dietary carrier system could be tested for various other nutritional components. 3. 3. the usefulness of the Artemia bioencapsulation method remains to be verified by chemical analysis.g. rather than administration via the culture water (‘bath treatments’). Doses ranging from 20 to 100 ppm sulfadrugs can be incorporated in sea bass and turbot larvae tissue. Estevez et al.5. 1983).5. 1983). liposoluble products administered via an emulsion. Gapasin et al.6 Cold storage 3. At 5°C and above 19°C survival decreased rapidly after 48 h. 1994) and microcapsule delivery (Sakamoto et al. However. 1995).5. within less then 4 h by feeding them with specifically enriched Artemia (Chair et al.3 to 1283 IU g 1 dry weight over an 18 h period through the addition of vitamin A palmitate to an egg-yolk-based emulsion (Dedi et al. (1998) found striking differences in vitamin A and carotenoid composition between halibut larvae fed SuperSelco-enriched Artemia and a species of copepod (Temora). franciscana catabolised DHA at a rate that increased with temperature but demonstrated that. Similarly. but the quality of the halibut larvae (proper pigmentation. The growth performance of shrimp reared from PLa-4 to PLa-25 on juvenile Artemia live prey is identical to the growth obtained when feeding newly hatched Artemia.3 Other advantages Cold storage enables the farmer to reduce hatching efforts (less frequent hatching and harvesting. 3. Dhert et al.6. 1991. Naessens et al. less labour-intensive and nutritionally suitable alternative to the traditional Moina culture. the stress sensitivity index dropped from 138 with freshly hatched nauplii to 36 when feeding juvenile Artemia. after enrichment. Artemia biomass is apparently a good food for the maturation of several species of penaeid shrimp. (2001) developed a pilot-scale culture unit for ongrown Artemia for use in ornamental fish in Singapore and proved it to be cost-effective. For example. With poor hunters such as the larvae of turbot. using cold-stored. Lim et al. sinica remained at almost constant high levels after enrichment and at temperatures ranging from 6 to 22°C. 1999) may also account for the reduced losses of EPA compared to DHA.5.e. farmers often experienced juvenile Artemia in their larviculture tanks. An interesting observation was made by Evjemo et al. It offers local fish breeders a cheaper. with a payback period of less than 18 months. but the PLa-25 reared with juvenile brine shrimp display significantly better resistance in salinity stress tests. less active Artemia as live prey resulted in a much more efficient food uptake (Léger et al. (1997): they confirmed that. (1993) developed a simple culture system for juvenile and adult Artemia as food for postlarval (PLa) Penaeus monodon. freeze-dried or acid-preserved for later use (Abelin et al. The fact that Artemia retroconvert DHA to EPA (Navarro et al. Although the fresh.7 Use of juvenile and adult Artemia Besides nutritional and energetic advantages. DHA levels in A. A. eye migration and lack of deformities) was significantly higher. 1997). 1995). applying one or two feedings per day. . i. 1986). (1999) proved that halibut larvae fed with gradually increasing sizes of nauplii showed the same satisfactory growth and survival as larvae fed short-term enriched nauplii. Tank Production and Nutritional Value of Artemia 111 losses of EPA were more moderate and depended on the type of enrichment received. which have been identified as a critical fresh-food component in the maturation diet of Litopenaeus vannamei (Bray & Lawrence 1991).Biology.5. Using cold storage also allows for more frequent and automated distribution of nauplii to larvae. Recent culture tests in Ecuador and the USA have shown that polychaetes. 3. harvested Artemia can also be frozen. the use of Artemia biomass for feeding postlarval shrimp also results in improved economics. This appears to be beneficial for fish and shrimp larvae as food retention times in the larviculture tanks can be reduced and hence growth of the Artemia in the culture tank can be minimised. fewer tanks. or made into flakes or other forms of formulated feed (Sui 2000). under similar circumstances. live form has the highest nutritive value. larger volumes). 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Aquacult. 6. 155.R. & Sargent. 159–164. B. Skoultchi.. Quantz.. Matsumoto. Aquaculture. (1993) Docosahexaenoic acid and the development of brain and retina in marine fish. (1998) The potential of dried. Bengtson. & Torano. 421–440. Burkhäuser Verlag. J. (1999a) Recent developments in the essential fatty acid nutrition of fish. Samocha.. J. Drevon. Aquaculture. W.. Jensen. & Prosdocimi. Sci. 3. & Jones. 51. 179. D. A. J. & Olsen. Decleir & E. low-hatch. Basel. Sargent. & Olsen.I.. 335. Hydrobiologia. P. pp. 147–159.120 Live Feeds in Marine Aquaculture Solangi. Wetteren.. et al.. & Sorgeloos. R. G. Tackaert. Tianjin. P. 129. Roels & E..J.A. O. G. Baton Rouge. 3 (Ed. Artemia spp.. Artemia spp. pp... Jaspers & I. In: Larvi ’95.A. by P. D. Tonheim. Sci. (1979) A filamentous bacterium on the brine shrimp and its control. & Ronnestad.V. 155–163. P. G. 459–463. Rep. Special Publication No.J.. & Sorgeloos.V. Ghent. Forneris. (1997a) International study on Artemia.usgs. P.A... Villani. S. 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In: Larvi ’95. (1980) Life history of the brine shrimp Artemia. Triantaphyllidis. In: Artemia Research and its Applications.J. Rev.L.M. LVII.. by T. 190. O.. 223–235. Sanggontanagit. W. (1996) International study on Artemia. Tunsutapanich. Fishing News Books. Dhert. 342–345. Vols 1–3 (Ed. T. 17–21 September 1990 (Ed. P. & Sorgeloos. XIX–XXIII. European Aquaculture Society. Morphological and molecular characters suggest conspecificity of all bisexual European and North African Artemia populations. T. Merchie. & Lavens. in marine fish larviculture.. & Candreva. P. PR China. P. Jaspers). L. 24. Palmegiano.V..J. Ghent. 275–281. European Aquaculture Society. Ghent. Persoone. (2000) Use of Artemia biomass in practical diets and decapsulated cysts as food source for common carp (Cyprinus carpio L. by P. p. Farnham. In: Proceedings of the 10th Annual Meeting of the World Mariculture Society (Ed. pp. LA. in larval crustacean nutrition: a review. P. (1987) Laboratory-grown Artemia as reference food for weaning fish fry and shrimp postlarvae. Miasa. Sorgeloos.. In: Proceedings of the International Symposium Biotechnology of Solar Saltfields. P. G. P. Coutteau. Jaspers).W.. Tang Gu.R. M. G. Tanggu.gov/greatsaltlake/ . Sorgeloos. Criel. Jaspers & I. Vol. Triantaphyllidis. 55–68. Bengtson.R. Wetteren. pp. P. Dhert.. (1979) The technical feasibility of massculturing Artemia salina in the St. E. (1997b) International Study on Artemia. Tobias. & Gannam. Sorgeloos. Artemia salina: a bottleneck in mariculture? In: FAO Technical Conference on Aquaculture (Ed. Van Ballaer. Trotta. T. M. Cheng). & Lavens.. W. Gulf Res. Universa Press. E. G. P. Parthenogenetic populations. Puwapanich. LIV. W. Koven. by G. 200. P. 24.. Salt Research Institute. by L... Croix ‘artificial upwelling’ mariculture system. P. LVI. N. by P. Abatzopoulos. (1995) The stability of (n-3) highly unsaturated fatty acids in various Artemia populations following enrichment and subsequent starvation. Stael. Avault). G.V..A. 477–487. Abatzopoulos. 203–214.K. II. Universa Press.J. MSc Thesis. Sorgeloos. C. Sorgeloos. Morphological study of Artemia with emphasis to Old World strains. W.R. 357. Decleir & E. A. Overstreet. Roelants).B. I. & Sorgeloos. Biol.. 97–106. (2001) Use of brine shrimp. G. Vol. P. Issues Adv. & Sorgeloos. 7–23. XVIII. P. MSc Thesis. H. A. Soc. 119. The biometrics of Artemia strains from different geographical origin. P. Vanhaecke. 155–164. De Vrieze. by G. P. 113. Exp. The hatching rate of Artemia cysts – a comparative study. Jaspers). 259–275... & Fujita. XIV. pp. S. (1982) International study on Artemia. 3 (Ed. Decleir & E.. & Sorgeloos. & Sorgeloos. micro-encapsulated egg diets and enriched dry feeds for Clarias gariepinus (Burchell) larvae. P. larvae.. Belg. P. 34. Combined effects of temperature and salinity on the survival of Artemia of various geographical origin. P. Vos. P. Vanhaecke. 30. 52. & Sorgeloos. Aquacult. & Sorgeloos. M. Bengtson. (1983) International Study on Artemia. 63. J. Tank Production and Nutritional Value of Artemia 121 Vanhaecke. P. XLVII. 393–405. Aquaculture. Vanhaecke. Wetteren. 231–246. W. XXXII.. J. C.. (1983) International Study on Artemia. J. (1980b) International Study on Artemia. D. P. (1996) Characterization of Artemia urmiana Gunther (1900) with emphasis on the lipid and fatty acid composition during and following enrichment with highly unsaturated fatty acids. & Storch. 253–269. & Sorgeloos. XIX. Vanhaecke. Velazquez. & Fujita. Hatching data for ten commercial sources of brine shrimp cysts and re-evaluation of the ‘hatching efficiency’ concept. S. 21. Vanhaecke. D. P. Soc.. & Sorgeloos. L.. (1987) A comparative study on the nutritional quality of decapsulated Artemia cysts. P. (1983b) Improvements of dietary value of live foods for fish larvae by feeding them on (n-3) highly unsaturated fatty acids and fat-soluble vitamins.P. C. Mar. Persoone. Aquaculture. (1989) International study on Artemia. pp.. Ecol. (1980a) International Study on Artemia. 49.A. 281–296. Spec. Ann. M. Segner.. In: Artemia Research and its Applications. W. P. Scient. P. T. Universa Press.. 3 (Ed. S. Hydrobiol. Mar. Limnol. Tackaert. P. Universa Press. Versichele.. Biol. R. 303–307. Jaspers). P. Sorgeloos. Belg. 1.. Tamiya. P. 68 pp. 80. T. Jpn. & Sorgeloos. XVII. Prog.. IV. T. Roels & E. P. Roels & E. Lavens. P.Biology. Zool. by P.. Ghent. Eng. Growth and survival of Artemia larvae of different geographical origin in a standard culture test. 3 (Ed. & Sorgeloos. P. 471–479. Persoone. Siddall. Energy consumption in cysts and early larval stages of various geographical strains of Artemia. Vol. Verreth. Vol. 263–273. 269–282. Hirata. RUG. O. J. (1998) Effects of hydrogen peroxide treatment in Artemia cysts of different geographical origin. 3. Soc. Ecol. Kitajima. (1984) International study on Artemia. Vanhaecke. Aquaculture. Vu Do Quynh & Nguyen Ngoc Lam (1987) Inoculation of Artemia in experimental ponds in central Vietnam: an ecological approach and a comparison of three geographical strains. Kitajima. R. Lavens. pp. (1983a) Nutritional values of live organisms used in Japan for mass propagation of fish: a review. 43–52. Van Stappen. & Sorgeloos.. In: The Brine Shrimp Artemia. In: The brine Shimp Artemia. Zool. Fish. World Aquacult. 257–262. (1980) Controlled production of Artemia cysts in batch cultures. Vanhaecke. Leger. Oka. Jaspers). V. (1984) Quality evaluation of brine shrimp Artemia cysts produced in Asian salt ponds.E. 17–23. Arch. Watanabe. Vanhaecke.. Soc. (1990) The use of decapsulated cysts of the brine shrimp Artemia as direct food for carp Cyprinus carpio L. Sorgeloos. O. Ann. & Sorgeloos. P. Ser.. 115–143. Sorgeloos. Watanabe. 108. P. . G.. The effect of temperature on cyst hatching larval survival and biomass production for different geographical strains of brine shrimp Artemia spp. Universa Press. P. Hydrobiologia. P. P. Wetteren. by G. Wetteren.. Bull.. Chapter 4 Production. 4.1 Introduction Highly saline lakes with natural Artemia populations can vary in size from a few hectares to large inland lakes like Great Salt Lake (GSL. Fig. Utah. both 4000–6000 km2 in size. at GSL and other sites. . this harvesting was performed at most sites without too much concern for sustainable exploitation or the carrying capacity of the available Artemia population. Until recently. population densities are usually low and fluctuate mainly as a function of food availability. USA. several population monitoring and modelling studies have been realised. The size and lack of suitable infrastructure make management of such lakes very difficult. temperature and salinity. Since the low harvests at GSL in the late 1990s (see Chapter 3). In these inland lakes. 4. Only the sharp decrease in the harvests in GSL at the end of the 1990s has urged both the Artemia industry and wildlife authorities.1) and Lake Urmiah (Iran).1 Harvesting of Artemia cysts from Great Salt Lake. and research-based management of the Fig. USA. to perform population assessment studies. restricting the main activity to extensive harvesting of Artemia biomass and/or cysts. Utah. Harvest and Processing of Artemia from Natural Lakes Gilbert Van Stappen 4. The population density depends on food availability. In these salt operations.1 Permanent solar salt operations Mechanised salt production operations consist of several interconnected evaporation ponds and crystallisers. and links are made with the population’s age structure and reproduction characteristics (Stephens 2000. Moreover. Belovsky et al. the stable conditions prevailing in the ponds of these saltworks often result in stable populations in which the ovoviviparous reproduction mode dominates. the Utah Division of Wildlife Resources. A co-operative research programme was launched by the Utah State Department of Natural Resources. On an industrial scale (e. the need for increased osmoregulatory activity. animal density decreases. 4. where NaCl precipitates. MgSO4 and KCl. 4.g. each with depths of 0. this ‘mother brine’ is pumped into the crystallisers. negatively influences growth and reproduction. 80–140 g l 1). when boats are not available. must be drained off. Seawater is pumped into the first pond and flows by gravity through consecutive evaporation ponds. then concentrated (Fig.2 Pond Production of Artemia Cysts and Biomass 4. the Utah State University and the United States Geological Survey. .2. which start precipitating at this elevated salinity. the NaCl deposits will be contaminated with MgCl2. It includes the study of the abiotic parameters and the phytoplankton population. as this product contains fewer impurities and has a higher viability. the lowest salinity at which animals are found is also the upper salinity tolerance level of possible predators (i. at GSL).e. eventually leading to starvation and death. Once the seawater has evaporated to about one-tenth of its original volume (about 260 g l 1). best practice is to collect the cyst accumulations from the water surface. pond size can vary from a few to several hundred hectares.Production. requiring higher energy inputs. Although live animals can be found at higher salinity.2). floating cyst accumulations are spotted by aircraft. To ensure that the different salts precipitate in the correct pond. by studying its population dynamics and formulating quantitative population predictions.1) and pumped into vessels. now called bittern. Before all NaCl has crystallised. However. As Artemia have no defence mechanisms against predators.5–1. Harvest and Processing of Artemia from Natural Lakes 123 lake started in 1996. and to open and close the harvesting season. Cysts are produced in ponds having intermediate and high salinity (80–250 g l 1). While passing through the pond system. salinity gradually increases as a result of evaporation. Harvesting cysts is sometimes undertaken by collecting cysts from the shore. Through the identification of a minimal viable population size. 4. 2000). Otherwise. Above 250 g l 1. Within such salt production facilities. The technique of salt production thus involves fractional crystallisation of the salts in different ponds. but generally numbers of animals and thus cyst yields are low.5 m (Fig. salinity in each pond is strictly controlled and during most of the year kept at a constant level. and salts with low solubility precipitate as carbonates and sulfates. This research programme focuses on the study of the lake’s ecosystem and the sustainable exploitation of its brine shrimp resources. the concentration of cysts found is used as a criterion to determine the harvestable amounts. the mother liquor. temperature and salinity. brine shrimp are mainly found in ponds at intermediate salinity levels. The ponds are just a few hundred square metres in size and are 0. For the remainder of the season water is kept in each pond until the salinity reaches a predetermined level and is then allowed to flow into the next pond. Salinity : 35ppt – 80ppt Second evaporator = 100ha. At the beginning of the production season. Once the salinity reaches 260 g l 1. the shallow ponds. they are fairly easy to manipulate. production methods differ (Vu Do Quynh & Nguyen Ngoc Lam 1987). Salt production is abandoned during the rainy season. all ponds are filled with seawater. Artemia thrive in ponds where salinity is high enough to exclude predators (between 80 and 140 g l 1).1–0. In addition.2. Sometimes. Egypt) : First evaporator = 600ha. 4. Although salt production in these saltstreets is based on the same chemical and biological principles as in the large saltfarms.2 Schematic outline of a typical permanent saltwork. Egypt. where NaCl precipitates. Salinity : 80ppt – 140ppt Third evaporator = 75ha. Salinity : 180ppt – 260ppt Crystallisers = 125ha. During that time the bottom heats up. The salinity in the different ponds is not kept constant as in continually operated saltworks. oxygen and salinity to fluctuate. water is pumped to the crystallisers. creating an unstable environ- .3). As seasonal systems are often small. small-scale artisanal saltworks (saltstreets) operate during the dry season.6 m deep (Fig. Port Saïd.2 Seasonal units In the tropics and subtropics. 4. Salinity > 260ppt Fig. Hence. when evaporation of water from the ponds exceeds precipitation.124 Live Feeds in Marine Aquaculture Crystallisers Fourth evaporator Third evaporator Second evaporator First evaporator Pump1 Dike Pump2 Large salt operation (Port Saïd. high algal density. higher food levels and thus higher animal densities can be maintained. use of organic manure and discontinuous pumping cause abiotic factors such as temperature. to increase evaporation further. which further enhances evaporation. ponds are not refilled immediately but left dry for 1 or 2 days. Salinity : 140ppt – 180ppt Fourth evaporator = 70ha. when evaporation ponds are often turned into fish/ shrimp ponds. 4. holding water of a higher salinity. as ponds are managed intensively (i. As the Artemia population is maintained throughout the culture season. Ponds can be constructed close to evaporation ponds with . in contrast. Fig. Salinity: 80ppt – 160ppt Crystallisers = 0. 1997).5ha.5ha. Salinity: 40ppt – 120ppt Evaporation pond (second series) = 0. 120ppt – all water evaporated. together with the fact that population cycles are seasonally interrupted. this system can be described as ‘one-cycle’ (Baert et al. Vietnam) Storage and evaporation area = 0. Harvest and Processing of Artemia from Natural Lakes 125 Storage and evaporation area Evaporation pond (first) Evaporation pond (second) Crystalliser Evaporation pond (first) Evaporation pond (second) Crystalliser Canal Storage and evaporation area Evaporation pond (first) Evaporation pond (second) Crystalliser Evaporation pond (first) Evaporation pond (second) Crystalliser Small salt operation (Vinh Tien.2.4ha – 1ha. This.e. 1996). More stable cyst yields can be obtained by the multicycle system. Salinity: 10ppt – 50ppt Evaporation pond (first series) = 0.3 Layout of a typical artisanal saltfarm.2ha – 0. allow higher numbers of reproducing adults and larger brood sizes. as the pumping rates (limited by salinity constraints) and the use of organic manure are already maximised. ponds are managed as separate units.2ha – 0. ment. and animals are introduced only once per season. seems to favour oviparous reproduction. Once stocked. 1997). Food levels in this system. manipulation of primary and secondary production. This culture system is referred to as semi-intensive (Tackaert & Sorgeloos 1991) and static (Vu Do Quynh & Nguyen Ngoc Lam 1987).Production. Vietnam. 4. inoculation of selected strains. and green water is pumped from a common fertilisation pond and. 4. In the one-cycle system. the food levels can only be increased to a limited extent.3 Site selection Integrating Artemia production in an operational solar saltwork or shrimp/fish farm improves the cost-effectiveness. where only the first generation is allowed to develop and release cysts. mixed with brine to maintain high salinity levels in the culture ponds (Baert et al. predator control) and are not interconnected. where ponds are drained and restocked several times per season (Baert et al. if necessary.2ha – 0.5ha. Artemia culture is mostly found in areas where evaporation rates are higher than precipitation rates during extended periods of the year (e.2. a dry season of more than 4 months in the tropical–subtropical belt). Heavy clay soils with minimal contents of sand are the ideal substrate. which may have a negative effect on the phytoplankton population. An additional problem may be the presence of acid sulfate soils.2 Topography Flat land allows easy construction of ponds with regular shapes. 4. where organic material is decomposing. Evaporation rates depend on temperature. this climatological factor should receive special attention.3. When exposed to air such soils form sulfuric acid. adaptation of the existing ponds is normally not possible and generally not needed. Making use of gravity or tidal currents to fill the ponds.2. but these problems will gradually disappear when the same pond is used for several years.4 Pond adaptation In large salt operations. resulting in a pH reduction in the water. even if only partially.1 Climatology The presence of sufficient amounts of highly saline water is imperative. or low-salinity ponds already existing in the salt operation can be modified (Tackaert & Sorgeloos 1993). The choice between dug-out (entirely excavated) and level ponds (bottom at practically the same depth as the surrounding land and water retained by dikes or levees) will depend on the type of ponds already in use. Temperatures that are too low will result in slow growth and reproduction. Therefore. problems can arise with oxygen depletion at the pond bottom. Moreover. wind velocity and relative humidity.3. such earth tends to shrink. often found in mangrove or swamp areas. 4. 4. the most important food source for Artemia. will reduce pumping costs.2. as the water flow into the ponds is much higher than the outflow (usually ponds are only drained at the end of the culture season).g. Optimal culture temperatures are strain-dependent. 4. thus lowering the dike height considerably. The presence of high levels of organic material in the pond bottom may also cause problems.3 Soil conditions Because long evaporation times are needed to produce high-salinity water. The presence of solar saltfarms in the neighbourhood is a clear indication that Artemia pond culture is possible during at least part of the year. Locating the Artemia ponds lower than all other ponds is good practice.3. As temperature also influences population dynamics directly. especially when used for dike construction. Several criteria have to be taken into account when selecting a site for Artemia production.126 Live Feeds in Marine Aquaculture the required salinity. leakage and/or infiltration rates should be minimal. whereas high temperatures can be lethal. The only adaptation needed is the installation of screens to reduce . A gradual slope can eventually facilitate gravity flow in the pond complex.2. 4. sometimes resulting in water temperatures that are too high for Artemia ( 40°C) and promoting phytobenthos rather than the required phytoplankton. Fig. 4.4.2 Dike construction To prevent leakage. at the same time.4 Floating bamboo poles used as wave breakers for the harvesting of Artemia cysts. High water levels are needed not only to prevent lethal water temperatures but. ponds are often very shallow.4). Under windy conditions. This is especially important in regions where predators are found at high salinity (e. However.g. To prevent such leaks from occurring. high wave action will enhance evaporation. 4.2. Dikes are often inhabited by burrowing crabs that cause leakage and destabilisation. to reduce growth of benthic algae.2. When heightening old dikes. Ponds are usually deepened by digging a peripheral ditch and using the excavated earth to heighten the dikes. dikes heightened and screens installed to prevent predators from entering the culture ponds. newly constructed dikes need to be well compacted (Kungvankij & Chua 1986). 4. the old dike should first be wetted and ripped before new soil is added. . In artisanal saltworks. 4. the Cyprinodont fish Aphanius). Depths of 40–50 cm are to be recommended.1 Deepening the ponds In regions with high air temperatures. Filling nests with CaO and clay will reduce leaks caused by these crabs. wave breakers should be installed (Fig. Harvest and Processing of Artemia from Natural Lakes 127 the number of predators entering the evaporators (see below). Integration of Artemia production with such systems requires that ponds should be deepened. to reduce foam formation (in which cysts get trapped) at the downwind side of the pond. leaks will occur most frequently at the interface of old and new soil. Dike slopes should have a 1:1 ratio (height:width) to prevent excessive erosion.Production. deepening the ponds is crucial. which will also act as cyst barriers and facilitate harvesting. 4.007–0.5 and stimulating an algal bloom proves difficult. Thus.4) and restricting the culture of Artemia to high-salinity ponds are of the utmost importance. this is not true for acid sulfate soils. When exposed to the air.2. For small ponds. The liming substances most often used in aquaculture are agricultural lime. welded-wedge filter is installed under an adjustable angle.2 Predator control Removal of predators in large salt operations is very difficult. even small competitors such as copepods can be removed (up to 90%).01 kg 75% bleaching powder m 3) will be necessary.015 kg urea m 3 and 0. However. Careful screening of intake water (see Section 4. often found in mangrove areas.1 Liming Normally. which will kill possible pathogens and predators. After 2–3 days. CaO and Ca(OH)2 are therefore often used to disinfect the pond bottom. As intake water is often heavily loaded with particles.5. The lime requirement is highest for clay bottoms and acid bottoms. the pH drops to 7. A simpler method to reduce acidity is flushing the ponds repeatedly after oxidation (exposing the soil to the air) but. by trawl nets) and killing fish/shrimp accumulating at the gates using a mixture of urea and bleach (0. If large numbers of predators are found in the culture ponds.2. Whereas drying can be beneficial for most soils.2. 4. Results are especially good when Artemia culture periods are relatively short (6–8 weeks). is used. ponds used to culture Artemia do not need liming. In small artisanal systems. CaO. culturing brine shrimp in regions with acid sulfate soils should be avoided. Liming of these soils requires large amounts of lime.2. where an array of different screens. manual removal (i. to ensure maximum evaporation. each with a smaller mesh size than the previous one.5. ponds should initially be filled only to a level of 10–15 cm.01–0. Water is lifted by a pump into an overhead compartment from where the water is drained over the filter screen.e. Both stainless-steel screens and filter bags should be cleaned regularly. quicklime and Ca(OH)2 or hydrated lime (Boyd 1990). 4. sequential screening is recommended. in general. after which normal mineralisation takes place. and when the pond water has low concentrations of Ca2 and Mg2 . When using such filters. polyurethane or nylon) or stainless-steel screens. . Liming should be considered when culture water has a pH of less than 7. the pyrite in these soils oxidises to form sulfuric acid. the use of filter boxes can be considered.5 Preparation of ponds for Artemia cultivation 4. Liming ponds with such hardness will not further improve yields. The highly saline water often has a hardness of more than 50 mg CaCO3 l 1 (owing to the presence of carbonates). Two types of filter can be used: filter bags (in plastic mosquito-screen.3 Screening Intake waters should be screened to prevent predators from entering the culture ponds. the high cost of these units restricts their use to regions where highly saline water is not abundant and/or where the presence of (small) predators seriously hampers Artemia culture. Using CaO and Ca(OH)2 will result in a quick pH rise to about 10.2.5.128 Live Feeds in Marine Aquaculture 4. a salinity lethal to predators will rapidly be obtained. In such boxes a stainlesssteel. . sunlight) and species composition [nitrogen:phosphorus (N:P) ratio. nitrogen and phosphorus. CaO or derris root (1 kg 150 m 3). 40 cm). as phosphorus has poor solubility in saltwater and is quickly absorbed at the pond bottom. Secchi depth. Besides the N:P ratio. pH and pond bottom. Oscillatoria).e. whereas if tea-seed cake or dipterex is used. used by photosynthesising algae.0 mg l 1). An N:P ratio of 10 is desirable for the growth of green algae (Tetraselmis.3 Fertilisation Fertilisers are added to the culture ponds to increase primary production. Ideally.Production. However. The inorganic nutrients carbon. Nitschzia). The most dominant algae in the intake water will often also be the dominant ones after fertilisation. Some green algae (Nannochloropsis. Lyngbya. a combination of urea and hypochlorite (5 mg l 1 urea and 24 h later 5 mg l 1 hypochlorite).05–2. especially at high temperatures ( 28°C) and in the case of low turbidity (bottom visible). i. fish. high N:P ratios stimulate green algae more than diatoms. Regular pumping is often more effective in controlling the Artemia standing crop. at high pumping rates algae will not have time to take up nutrients). As some algae are better suited as food for Artemia than others. The degradation rate of rotenone. a high N:P ratio is usually recommended. temperature. When pumping. If too much phosphorus is added. In areas where intake water is nutrient rich (turbidity readings. salinity.5. fertilisation should be combined with lower pumping rates in systems with short retention times. salinity. 4. ponds should be flushed prior to the stocking of animals. Navicula.e. Harvest and Processing of Artemia from Natural Lakes 129 Screening of the intake water will further reduce the number of predators in the pond. Numerous factors influence the chemistry of the fertilisers. and algal growth (temperature. especially when temperatures are high. light intensity and pumping rates (input of new nutrients and carbon dioxide) also play an important role. selective grazing pressure] (Tackaert & Sorgeloos 1991). Dipterex (2 mg l 1) will kill smaller predators such as copepods and is also very toxic to shrimp. such as the ion composition of seawater. Both algae are often too large in size for ingestion by Artemia. As pumping influences the retention time of the nutrients in the ponds (i. growth of benthic algae is promoted.2. no additional fertilisation should be used. tea-seed cake (15 mg 1 1). crabs and shrimp left in puddles may be killed using rotenone (0. N:P ratios of 3–5 may be more appropriate. algal turbidity readings should be kept between 20 and 40 cm in the Artemia culture ponds by regular water intake from the fertilisation ponds. enter the system via the photo-autotrophic pathway. or are consumed directly by the Artemia.g. costs may limit the use of fertilisers. At lower salinity and higher light intensities. chlorine and CaO to non-toxic forms is fairly rapid (24–48 h). high phosphorus concentrations combined with low salinity seem to induce the growth of filamentous blue–green algae (e. whereas organic nutrients are processed through the heterotrophic pathways of heterotrophic bacteria. new nutrients and carbon dioxide enter the culture ponds that stimulate algal growth. In large salt operations. Turbidity readings of less than 20 cm may result in oxygen stress at night. manipulation of algal populations also depends on the composition of the local algal community. Dunaliella) and diatoms (Chaetoceros. Likewise. As ponds often cannot be drained completely. Finally. Chlamydomonas) are poorly digested by Artemia. fertilisers with a small grain size. However. The algae and organic matter created in the low-salinity ponds are drained to the high-salinity ponds. further stimulate algal .5. by reacting with Ca2 . Finally. i. Inorganic fertilisers stimulate algal growth and mineralisation of the organic fertiliser by lowering the carbon:nitrogen (C:N) ratio. Phosphorus fertilisation As with nitrogen. pig and goat dung have also been used. Cow. The organic fertilisers most often used in aquaculture are chicken. 4. nitrogen often limits algal growth. nitrates [e.130 Live Feeds in Marine Aquaculture 4. especially phosphorus. via slow release of nutrients.6 Combination of organic and inorganic fertilisers A common practice is to use a combination of inorganic and organic fertilisers. In cases where the use of phosphorus fertilisers is desirable. which dissolve easily in water.5 Organic fertilisers Organic fertilisers present some distinctive advantages as they contain. Usually.2. Phosphorus is also quickly absorbed at the pond bottom. Among the most common inorganic nitrogen fertilisers are ammonium salts [e. should be selected. The need for nitrogen fertilisation varies considerably and should be determined experimentally at each site. as algal densities here are limited not by the nutrient concentrations but rather by the grazing pressure imposed by the brine shrimp. The use of inorganic fertilisers in Artemia culture ponds is not recommended (except before introducing the nauplii). Their use results in a higher biological oxygen demand (BOD) in the pond and they may carry pathogens. adding between 1 mg l 1 (eutrophic intake water) and 10 mg l 1 (oligotrophic water) nitrogen will induce an algal bloom.2. but seem to stimulate phytobenthos (Tackaert & Sorgeloos 1991). the use of organic fertilisers implies the recycling of a waste product.4 Inorganic fertilisers Nitrogen fertilisation As the nitrogen influx to the system depends solely on biochemical processes (degradation of organic matter by bacteria) and the nutrient level in the intake water.g.5. (NH4)2SO4]. especially in saltwater ponds. It is best to fertilise only the low-salinity ponds in a flow-through system. organic fertilisers are used directly as food for the Artemia and. where they are available as food. However.e. Most phosphorus fertilisers precipitate. where it is bound in the form of AlPO4 2H2O or FePO4 2H2O. Initiating an algal bloom in high-salinity ponds is difficult and can take more than 1 month. Predissolving the fertiliser in freshwater will improve its availability. the use of organic fertilisers is difficult to standardise in view of their variable composition. besides nitrogen and phosphorus. phosphorus enters the culture ponds with the intake water in the form of organic material that only becomes available through bacterial decomposition. Ca(NO3)2]. other minerals beneficial for phytoplankton growth. The use of nitrogen fertilisers is therefore widespread. quail and duck manure.g. Fertiliser particles coated with bacteria enhance the microflora and can be used directly as food by Artemia. amides and urea.2. 4.5. Phosphorus is also found in the soil. followed by dispersion by wind and local waterbirds over a distance of more than 1000 km.6.1 Artemia strain selection While Artemia introduction frequently ensures social and economic benefits. inorganic fertilisers are added to the fertilisation ponds or canals.2. a stocking density of 5–10 nauplii l 1 should be considered. of one or both competitors. Strain selection can be based on the literature data for growth.2 Inoculation procedures Standard hatching procedures (described in Section 3. 4. For large salt operations. Animals should be stocked as early as possible in the brine circuit where no predators are found.2. at worst. which may lead to the extinction of some genotypes or. may further limit the stocking density. such measures include the establishment of gene banks (cysts). process and store a sufficient quantity of good quality cysts of the autochthonous gene pool that have a proven high hatch rate. Normally. 4. large cysts. Stocking density is determined by the nutrient level and temperature found in the culture ponds. such as facilities to hatch out the required amount of cysts. while manure can be added directly to the Artemia culture ponds or to the fertilisation ponds. However. The effect of one introduction will not remain local but may have consequences over large areas: many saltworks in north-east Brazil are now populated with Artemia since the human intervention in Macau in 1977. Competition experiments suggest that Artemia franciscana may outcompete others (Browne 1980. is still valid: ‘… the 2nd International Symposium resolves that all possible measures be taken to ensure that the genetic resources of natural Artemia populations are conserved.2. In summary.6. depending on the size of the ponds. particularly in developing countries.6 Artemia inoculation 4. reproductive characteristics and especially temperature/salinity tolerance. or its unsuitable characteristics for use in aquaculture (e. in large operations practical considerations. The resolution put forward at the 2nd International Symposium on Artemia.5. It is essential to harvest the nauplii in the first instar stage. Browne & Halanych 1989).g. and where possible the use of indigenous Artemia for inoculating Artemia-free waters’ (Beardmore 1987). in September 1985. all possible efforts should be made to collect. Harvest and Processing of Artemia from Natural Lakes 131 growth. particular diapause or hatching characteristics). Competition with local (or nearby) strains or species of Artemia may occur (Geddes & Williams 1987). Older instar stages will not survive the salinity shock when transferred from the hatching vessel (20–35 g l 1) to the culture ponds (80 g l 1 upwards). in terms of its limited effect on algae removal in the salt production process. the initial stocking density can be as high as 100 nauplii l 1 in ponds with turbidity between 15 and 25 cm. it also bears certain risks (Beardmore 1987). close monitoring of inoculation policies. However. at such high stocking densities oxygen . Belgium. in Antwerp.2) should be followed wherever possible.Production. In small pond systems. a strain exhibiting maximal growth and having a high reproductive output at the prevailing temperature/salinity regime in the ponds should be selected. When the idea is thus to replace a poorly performing strain. However. at least (Eliot 1977. since more food is available per individual. or by dragging a conical net over a certain distance through the water. the mesh size and diameter of the sampling net depend on the volume of water sampled.1 Monitoring the Artemia population Samples should be collected at fixed sampling stations located in as many different strata as possible. The type of sampling programme depends largely on the goals. number of females and brood size). Samples are fixed with formalin and carefully examined. for instance. Wet weight should not be used. Krebs 1989).2.132 Live Feeds in Marine Aquaculture may become limiting.7 Monitoring and managing the culture system Very regular monitoring of the ponds is necessary to allow correct management. However. turbidity. the use of fixed well-maintained and operational equipment. where it is allowed to settle for 10 min. . 5–10 litres of water over a 100 m sieve. especially when water temperatures are high. More extensive sampling programmes are required when research programmes are carried out in the culture ponds. 4. whereas stocking at lower density may increase the proportion of ovoviviparity. A quick way to estimate standing crop is to use sample volume as an estimate. stocking density should be decreased to 50–70 nauplii l 1. Although such estimates do not give the exact number of animals per litre. As a result. after which the volume is read. only those variables necessary to provide essential decision-making information should be monitored (temperature. only the distal part of the net has a small mesh size (100 m). appropriate techniques when measuring parameters or when analysing samples. then biomass is transferred to a measuring cylinder. Stocking at high density is thought to stimulate oviparous reproduction. and keeping accurate data records. The reproductive status of the females can also be used as an indicator for the health status of the Artemia population. The most important rule when collecting data is standardisation. such as nauplii (no thoracopods). by filtering. Large broods and short interbrood intervals show that pond conditions are good. allowing for relative estimates of population numbers.7.2. When food for the nauplii is less abundant (turbidity readings of more than 25 cm). If production is the main objective. Using dry weight as an estimator is only possible if samples can be cleaned properly. which in turn depends on the population density in the pond. juveniles (developing thoracopods clearly visible) and adults (sexual differentiation apparent). dividing animals into age or size classes. final cyst yields do not necessarily decrease when lower stocking densities are applied. This includes selection and marking of fixed sampling stations at every site. as it is very imprecise and inaccurate. To prevent clogging. The scores for each life stage of all samples taken in one pond are summed and plotted in time. Drags can be horizontal or vertical. animals grow more rapidly and females have larger broods. 4. while the remainder of the net can have a mesh size of 300–500 m. The sample is fixed with lugol or formalin. salinity. they correctly reflect the variations in abundance and allow for adaptation of the management procedures. indicated by large clouds of deep red Artemia at the water surface.e. the water may be stratified and temperatures at the surface and bottom may differ considerably. Turbidity readings between 25 and 35 cm are optimal.7. swim slowly and start surfacing.2. precipitation and leakage.7. effects of pH on growth and reproduction have not been studied so far. lowering the algal concentration or circulating the water in the pond will increase oxygen levels. At lower turbidity levels. Regular pumping or raking of the pond bottom will prevent stratification.2.2 Abiotic parameters influencing Artemia populations Temperature In deeper ponds.g.Production. extra pumping of nutrientrich water is needed. especially in situations of salinity stratification (i. the risk of oxygen depletion at dawn increases. Furthermore. oxygen) and therefore should also be recorded. However. . evaporation. Extra pumping. problems with pH are rare. temperature.g. 1995). Artemia turn red. Concentrations are lowest at dawn (algal respiration) and highest in the afternoon (algal photosynthesis). Turbidity fluctuates during the day. Water at greater salinity than 250 g l 1 is toxic for Artemia. Oxygen Often oxygen levels will be higher at the surface than at the bottom. but as seawater is usually well buffered. In extreme situations this can lead to lethally high temperatures and low oxygen concentrations at the pond bottom. fluctuations in pond depth give information on pumping rates. fish. Algal blooms can affect the pH (consumption of carbon dioxide). Such conditions. Harvest and Processing of Artemia from Natural Lakes 133 4. the upper salinity tolerance level of predators (e. except in areas with acid sulfate soils.8 and 8. a greenhouse effect resulting from the low-salinity top layer). and wind (concentration of algae in the downwind corners) as well as suspended solids (e. and growth is retarded. oviparous reproduction is often found at high salinity. Salinity Salinity is an important factor in determining the success of Artemia populations (Triantaphyllidis et al. As mentioned previously. especially when ponds are stratified. Oxygen levels also exhibit daily cycles. probably as a result of oxygen and food stress. have a negative influence on growth and survival. Under field conditions. which is often given as the optimal range. The water depth influences several of the other measurements (e. 4. When exposed to oxygen stress. At higher turbidity. Corixidae) determines the threshold beyond which reasonable numbers of Artemia can be found.3 Biotic factors influencing Artemia populations Algae The easiest way to estimate algal abundance is by the measurement of turbidity with a Secchi disk. clay) can affect turbidity readings. Water depth Depth is best measured using calibrated sticks placed in the pond. pH In their natural habitat Artemia are mostly found in a pH range between 7.2.g. 5 Raking the Artemia pond to remove the benthic algae. Algal numbers can be increased through fertilisation.5).7). Careful screening of the intake waters and increasing salinity will keep their numbers within acceptable limits. . various species of insect (Corixidae) and some copepods. and herons. pond bottoms are raked daily to remove benthic algae (Fig. 4. 4. but also has an effect on the nutritional value of the biomass and the cysts (e. fatty acid composition). 4. Tilapia). avocets.6. shallow ponds with conical nets mounted in front of a motorboat or pulled by people (Figs 4.1 Harvesting techniques Adult Artemia biomass can be collected from large. As wading waterbirds (e. In Vietnam. The colour of the water can give useful indications concerning the type of organisms present in the culture ponds.134 Live Feeds in Marine Aquaculture Fig. Rotifers and ciliates (Fabrea) are possible food competitors.g.3 Artemia Harvesting and Processing Techniques 4. the algal density can be estimated by analysis of the chlorophyll concentration. In small ponds. A problem often encountered in Artemia ponds is the presence of benthic and/or filamentous algae. raking brings detritus (extra food for Artemia) as well as inorganic nutrients back into suspension. Ardea) also take adult Artemia. Development of these algae can be prevented by keeping pond water turbid and deepening the ponds. 4. If time and equipment are available. Moreover. more thorough analysis of the algal samples is recommended. Both types of alga are unsuitable as food for the Artemia. If problems are encountered.g. bird scarers and wires stretched above the water near shallow places may be used to control these predators. Recurvirostra. Predators and competitors Possible predators of Artemia include fish (Aphanius. if combined with a proper sampling programme. Algal composition not only influences growth and reproduction of the Artemia.3. 8). 4. biomass should be prepared for transport and further use or treatment. nets can be installed (temporarily) at the pond outlet and biomass is then collected automatically when water flows (by pumping or gravity) to the next pond (Fig. 4. Alternatively.2 Processing techniques Artemia biomass should be used immediately after harvesting as live food. Harvest and Processing of Artemia from Natural Lakes 135 Fig. Live transportation for marketing as a live product ( 90% survival after 24 h) is undertaken using similar techniques to those for the transportation of live fish and shrimp . or should be frozen or dried. Harvested biomass can be stored temporarily in nets installed in the pond (Fig. 4.7 Small net used for Artemia biomass harvesting. 4.6 Raft with conical net used for Artemia biomass harvesting. After collection. Fig. dip-nets can be used.Production.9). 4.3. 8 Installation of filter nets at the sluice gate for Artemia biomass harvesting in solar saltworks.136 Live Feeds in Marine Aquaculture Fig. 4. . Fig.9 Storage net for Artemia biomass harvested from seasonal salt ponds integrated for Artemia production. 4. biomass can be dried and used as an ingredient for larval feeds (flakes or particulate diets). or they can be applied simultaneously. strain/batch specific characteristics and local conditions (e. Basically. If these cysts are produced in low-salinity ponds ( 100 ppt) or when salinity stratification takes place after rainfall.2. as slow freezing will result in proteolytic activity and leaching of essential nutrients when used subsequently. 4. site location. Freezing should be done as quickly as possible (thin layers.4. the processor will choose a combination of processing stages and diapause deactivation techniques depending on a number of factors such as trade-off between required final quality and economic viability. marketable product with acceptable hatching properties and shelf-life. low temperature). these cysts may also become airborne. cysts float on the water surface and are washed ashore by winds and waves. Since Artemia is rich in proteolytic enzymes it is essential to process the biomass alive. Artemia biomass can be frozen for subsequent use as a food source in fish/shrimp hatcheries or for the pet market. prepackaging. They are transported in plastic bags.1 Brine dehydration To improve storage conditions and/or to deactivate diapause. packaging and dry storage. ultraviolet (UV) irradiation and repeated hydration/dehydration cycles. a number of processing steps should be carried out to obtain a clean. When dry.4.10): harvesting. Alternatively. In places with changing wind direction. The rest of the bag is inflated with oxygen and closed off with a rubber band. drying. Harvest and Processing of Artemia from Natural Lakes 137 larvae. and stocked with Artemia at a density of 100 g live wet weight biomass per litre.1 Harvesting techniques After being released. locally available equipment and scale of operation). When size . Cysts washed ashore may be exposed to high temperatures.4 Artemia Cyst Harvesting and Processing Techniques Once the cysts have been harvested. filled to one-third of their capacity with seawater. The best quality biomass meal is obtained with freeze-drying or spray-drying. storage facilities. 4. When water is very agitated and foam develops. cysts may be carried around for a long period before they are thrown ashore. quiescent cysts may hatch. which in many cases decrease the viability of the final product. The production of good quality cysts with reduced contamination by impurities is maximised when they are harvested from the water surface on a regular basis. cysts can become trapped in airborne foam. 4. freshwater processing. The processing can be classified into a number of successive processing stages (Fig. some processing activities may be omitted.Production. According to specific requirements.g.4. The bags are packed in polystyrene boxes filled with ice. 4. The processing activities within a processing stage can vary. brine processing. cysts are usually dehydrated (to a water content of 20–25%) in saturated brine immediately after harvesting.2 Brine processing 4. stones. sand. organic matter) temporary storage before or in-between brine processing steps storage for diapause deactivation storage before use as wet dry product (within 2 to 3 months) dehydration dehydration dehydration aging in brine/hibernation hydration/dehydration cycles peroxide treatment FRESH WATER PROCESSING removal excess brine density separation disinfection rinsing removal excess water to avoid salinity increase of freshwater and sub-optimal separation to separate high sinking fraction (mainly cysts) from low density floating fraction (mainly empty/cracked shells. light organic matter) reduce bacteria load remove salt just before drying to improve drying efficiency storage before use as a clean wet-dry product (within 2 to 3 months) dehydration aging in brine/hibernation dehydration BRINE PROCESSING brine dehydration raw storage DRYING PRE-PACKAGING size separation air classification temporary packaging mixing PACKAGING DRY STORAGE for long time storage remove cyst aggregates remove remaining empty shells and non-cyst material optimal storage prior to final packaging obtain constant hatching quality oxygen free conditions to permit long time storage (>1 year) special storage conditions to increase shelf life Fig.10 Overview of cyst processing.DIAPAUSE DEACTIVATION ACTIVITIES DESCRIPTION OF CYST PROCESSING STEPS HARVESTING BRINE PROCESSING dehydration size separation density separation raw storage AIM OF PROCESSING STEPS AND/OR PROCESSING apply proper harvesting procedures to ensure better quality of the cysts to prevent quality decrease during storage and/or to deactivate diapause removal of light and heavy debris in different size range of cysts removal of heavy debris (e.g. (Modified from Lavens & Sorgeloos 1987. high dens.) . 4. 4.g. Raw storage in low-saline brine (e. brine dehydration is either combined with or performed immediately after density separation and size separation (see below). it is more efficient to perform a density separation in brine (see below) before size separation.g. pond brine) Many strains can be stored in pond brine with salinity as low as 100 g l 1 for several days at ambient temperature without a decrease in the viability. . alternatively. It is essential that the cysts remain under hypoxic conditions to prevent initiation of the hatching metabolism. For cyst material containing a lot of heavy debris (e.4. The cysts can be stored in containers submerged in brine or. hand squeezing) and the wet–dry product can be stored in bags made of cotton or jute.15 mm).Production. When stored as a wet–dry product over longer periods ( 1 week).g.2.2. 4.g. the storage (ageing) in brine may further deactivate the diapause in certain strains and batches. excess brine can be removed (e. Besides diapause deactivation as a result of the dehydration process itself. 0. However. sand. Cysts immersed in brine float. It can be combined with brine dehydration or cysts can be transferred to a special brine dehydration tank or pond following density separation. 4. Density separation is often performed soon after harvesting near production sites because of the availability of saturated brine. Raw storage in saturated brine After brine dehydration cysts can be stored safely for up to 1 month at ambient temperature.2 Size separation in brine This involves the separation of debris from the cysts (e. small stones. is done through density separation in brine.4 Initial (or ‘raw’) storage Cysts are usually stored in a raw condition as follows: • • • • temporary storage (days or weeks) before the next brine processing stage temporary storage before the freshwater processing stage combination of raw storage and specific diapause deactivation methods raw storage for use as a wet–dry product (within 2–3 months). sand. it is advisable to perform brine dehydration before size and density separation so as to avoid any loss of cyst quality. Harvest and Processing of Artemia from Natural Lakes 139 and density separation equipment is located near the collection sites.3 Density separation in brine Removal of heavy debris in the same size range as the cysts. in areas of high relative humidity. crude salt should be mixed with the cysts to prevent rehydration of the highly hygroscopic cysts.g. heavy organic matter) sinks. when collected from the shore).g. wood. 1 mm.5 mm. when performed subsequent to size separation.4. 0. while heavy debris (e. 4. feathers.2. by storing them at a relatively high ratio of cysts to brine (20–80% volume:volume) without aeration. stones) by screening the harvested product over different mesh sizes (e. when there is a long period (up to several weeks) between collection and further processing. 4. as a clean wet–dry product. rinsing and collection in bags. In freshwater.g. the cysts can be dried immediately for long-term storage. Usually. they must always be rinsed thoroughly with freshwater to avoid crystallisation of any remaining salts during drying and consequent damage to the cyst shells. the cysts are further cleaned by density separation and prepared for subsequent drying.140 Live Feeds in Marine Aquaculture 4. After the freshwater treatment. within 1–3 months. 4. e.2. Alternatively. Few data are available on possible quality improvements when the drying time is very . To reduce the bacterial load of the final cyst product the cysts can be disinfected during the freshwater treatment. the bulk of the freshwater can be removed by firm squeezing of the cysts. to prevent further metabolism and decreased hatchability. consequently. The best results are obtained when a water content of 10% is reached within 8 h or less. if they remain hydrated for too long a period under aerated conditions. The cysts can be dehydrated in saturated brine for raw storage and used. cysts are usually dehydrated in saturated brine and packed as a wet–dry product before cold storage. freshwater processing should be limited to a maximum of 30 min. the water content of the cysts should be reduced as soon as possible below the critical level of 10% to arrest metabolic activity and. If cysts are to be dried. Although many strains and batches have been stored safely without proper dehydration. Following separation. little is known about the actual relation between water content and subsequent quality and shelf-life (Clegg & Cavagnaro 1976). Cyst material immersed in freshwater will separate into a high-density (sinking) fraction and a low-density (floating) fraction. the cysts will partially hydrate and. the embryos will eventually reach a stage of initiation of the hatching process that is irreversible and they cannot then be dehydrated without affecting the viability of the embryos (Clegg & Cavagnaro 1976). a final water content between 3 and 8% is the objective. Hence. excess brine must be removed to prevent a salinity increase in the water and. Below a water content of 10%. The sinking fraction contains mainly full cysts and some non-cyst material of similar density and similar size as the full cysts. Before density separation in freshwater.3 Freshwater processing During the freshwater processing stage. by adding hypochlorite (liquid bleach) to the freshwater separation tanks before adding the cyst material. The concentration of active chlorine in the freshwater of the separation tanks should be less than 200 g l 1. consequently. to ensure a long shelflife.4 Drying The type of drying procedure used can affect the quality of the cysts in terms of hatching percentage and rate. their energy reserves may have been depleted to levels that result in a decrease in hatchability. Further removal of excess water can be achieved by centrifugation. Drying procedures differ in a number of factors. For certain species and strains.4.4. cold storage for several months is an adequate diapause deactivation method. 4. The floating fraction contains mainly empty and cracked cyst shells and light non-cyst material of a similar size range.5 Cold storage The cysts of many strains and batches may be stored for several months to a year at temperatures between 20 and 4°C. suboptimal separation. Moreover. 4.Production. Moreover. A final equilibrium will be reached.4. they may withstand higher temperatures (for some strains/batches. thus improving the drying. eventually not reaching a water content of 10%. Harvest and Processing of Artemia from Natural Lakes 141 short ( 3 h). without exposing the cysts to critical temperatures. 1996). if dried for long enough. Consequently. Depending on the available equipment and financial considerations. If the freshwater processing cycle is limited in time and if excess water is properly removed. at a relative humidity of 70–75% cysts may reach a water content of about 10–15% after a maximum of 48 h.11. heterogeneous drying will result in some cysts drying very slowly.2 Layer drying in oven Drying racks are placed in a temperature-controlled room or oven with good air exchange. Exposure to direct sunlight may result in critical temperature increases within the cysts (through heat absorption by the dark shell) or in UV damage to the embryos. and a reduced shelf-life. cysts are only partially hydrated (water content between 40 and 45%). The maximal drying temperature is both strain specific and dependent on the degree of dehydration of the cysts. temperatures below 35°C are usually safe.4. 24 h) results in a decreased hatching percentage. The relationship between temperature tolerance and water content of the cysts should be checked to determine the most efficient temperature cycle and so avoid overheating.12). Prolonged drying (e. In areas with variable air humidity. This may cause both a decrease in hatching percentage and hatching rate. between the water content in the cysts and the relative humidity of the air. poor standardisation and slow drying. 4. Heating incoming air significantly decreases the relative humidity. Finally. may result in fluctuating cyst quality.4. possibly caused by a decrease in energy reserves. As the drying proceeds. 4. The blower forces air over the heating unit into the drying chamber. for example. a blower and a heating unit with temperature control device (Bosteels et al. optimal results are obtained when ensuring a fast ( 8 h) and homogeneous drying stage to a water content below 10%.4. The conical shape of the drying .4. For fully hydrated cysts. drying may still be quite slow and the problem of cyst aggregation remains. In summary. 4. owing to poor mixing.3 Fluidised bed drying The most efficient and most versatile drying is obtained by means of a fluidised bed dryer (Figs 4. sheltered from direct sun irradiation and provided with good air exchange. which consists of a conical drying chamber. up to 60°C). small aggregations of cysts may form which may affect the overall quality of the final product.g. water content decreases and cysts tend to be resistant to higher temperatures. Although a better standardization is possible. 4. different drying techniques can be applied. Sieves at the inlet and outlet of the drying chamber allow free airflow without loss of air-suspended (fluidised) cysts.1 Layer drying in open air Cysts are spread in thin layers of uniform thickness (a few millimetres only) on a drying rack (trays made with 120 m screen). inherent to this method. 12 Fluidised bed dryer for Artemia cysts. . Fig. 4.142 Live Feeds in Marine Aquaculture 100 µm screen Removable part of cone for addition or removal of cysts Temperature sensor (control of temperature in cone) Temperature control device Temperature sensor (control of inflowing air) Blower unit 100 µm screen Heating unit Fig.11 Schematic drawing of a fluidised bed dryer for Artemia cysts. 4. Anh. provided cysts are not exposed to high humidity (to avoid rehydration). Genetics. T.T. Strain Characterization. Vu Do Quynh & Hoa. W. This can be achieved by vacuum or nitrogen packing.. storage temperatures below 10°C are usually recommended. Vol. 196–251.A. P. D. 809–814.. small aggregations of cysts are formed. Toxicology (Ed. Wetteren. which results in homogeneous drying without excessive formation of cyst aggregations. and give lower hatching values postdrying. N. Some species and strains of cysts seem unsuited to the intense turbulence inherent to this type of drying. 4. Morphology. 4. P. the shelflife of dry cysts is strain.4. N. the fluidised bed dryer may include cylindrical drying chambers. Lavens & P. Baert. 28. pp. Rome. Bengtson. & Sorgeloos. Air separation of cysts is often applied to separate any remaining empty and cracked shells that were not removed during freshwater separation. In: Artemia Research and its Applications. ecotoxicology. To cope with these cysts.5 Prepackaging. genetics.A. Harvest and Processing of Artemia from Natural Lakes 143 chamber ensures optimal mixing of the cysts throughout the drying process. the air inflow can be regulated to allow empty cysts to be blown through the mesh. 1. P. Aquacult.. If the inlet and cone temperature are limited to 80 and 40°C. radiobiology.V. which can be removed by dry sieving to improve the visual appearance of the final product. 345–346. . Although some strains may be stored at room temperature. Any type of mixing equipment may be used. into which hot air is introduced from below at a moderate rate. Dry cysts should be packed in oxygen-free conditions to prevent formation of free radicals resulting in the irreversible interruption of the hatching metabolism. respectively.Production.and batch-specific. The air inflow is insufficient to blow the cysts high into the air. by P. a unit as described above will dry approximately 35 kg of wet cysts in less than 3 h to a water content below 10%. Properly packed cysts may be stored for months or even years without any significant decrease in hatching success. Furthermore. pp. Sorgeloos.. packaging and storage During drying (especially layer drying).N. Bosteels. Variations in hatching quality of the dry cysts may require mixing of different cyst batches to ensure a marketable product of constant quality. Beardmore. Sorgeloos). (1996) Pond production of Artemia. Res. Decleir & E. J. Jaspers). and so homogeneous mixing of the cysts is achieved by a series of blades rotating about a central vertical axis within the drying chamber. Universa Press. FAO Fisheries Technical Paper 361. (1987) Concluding remarks for Symposium Session I: morphology. (1997) Increasing cyst yields in Artemia culture ponds in Vietnam: the multi-cycle system. Food and Agriculture Organisation of the United Nations. This can be effected in a horizontal air stream in which heavy particles tend to fall down more quickly than light particles.5 References Baert. by P. but it is often combined with the fluidised bed drying process itself: if a mesh is installed at the top outlet of the air chamber. In: Manual on the Production and Use of Live Food for Aquaculture (Ed. Improved drying efficiency is further obtained by the heating unit. reproductive and lifespan characteristics of a bisexual and a parthenogenetic population of Artemia. 302. pp. Sirikarnpimp. (1995) International study on Artemia. & Sorgeloos. Jaspers). Sorgeloos & C. (1987) Comments on Artemia introductions and the need for conservation. Artemia salina. Amsterdam. & Halanych. Decleir & E. Clegg. & Sorgeloos. Vol. by R. Krebs. K. USA.J. Use in Aquaculture (Ed.A.A. Biophys.. Salt Lake City. Freshwater Biology Association. Ecology.S. Jr. Boyd. R.. Boca Raton..C. Anostraca). pp. & Cavagnaro. P. Progress report. 617–622. Triantaphyllidis. C.A. Bosteels. & Sorgeloos. NACA Training Manual Series No. Culturing. W. In: Proceedings of the 7th International Symposium on Salt. Browne. H. 19–26. growth. .A. (1986) Shrimp culture: pond design. G. 61. D. Larson. biometrics.C. J. T.. CRC Press. Kakihana. its diapause deactivation and hatching: a review. 25. Vol. Hoshi & K. 68 pp. 2. & Chua.A.D. Sorgeloos. Bengtson. 253–269. In: Artemia Biology (Ed. Bangkok.A. Progress Report to the Utah Division of Wildlife Resources. (1991) Semi-intensive culturing in fertilized ponds. P. New York. pp. Poulopoulou. & Mellison. Kungvankij. D. (1993) The use of brine shrimp Artemia in biological management of solar saltworks. Geddes. Universa Press. Browne. J. P. Hardy. (1989) Competition between sexual and parthenogenetic Artemia: a re-evaluation (Branchiopoda. Abatzopoulos. J. & Sorgeloos. Biochem. 57–71. W. 3. Bengtson.M. Birmingham Publishing. Wetteren. Jaspers). Jaspers). Tokyokura). 215–227. by P. Decleir & E.. D. 287–315. Cytol. Universa Press. Wetteren. Use in Aquaculture (Ed. T. (1987) The cryptobiotic state of Artemia cysts. Eliot.E. Wetteren. 466–470. Tackaert.N. IV. P. Great Salt Lake Ecosystem Project. maturity. Hydrobiologia. 169–179. In: Artemia Research and its Applications. & Sorgeloos. by H. Tackaert. July (1998) through June (1999). C. 3 (Ed. In: Artemia Research and its Applications. (2000) Brine shrimp population dynamics and sustainable harvesting in the Great Salt Lake. Vu Do Quynh & Nguyen Ngoc Lam (1987) Inoculation of Artemia in experimental ponds in Central Vietnam: an ecological approach and a comparison of three geographical strains. AL. 159–166. Crustaceana. P. C. D. Aquacult. Decleir & E.R. Browne. 27–63.A.. Sorgeloos. W. T. T. FL. Pinto Perez. J. Lavens. ATP and cyst hydration. 1 (Ed. by P. Tackaert. Utah. W. W. Ecology.A. pp.. Universa Press. P.E. Scientific Publication No.. by P. Trotman). J. (2000) Brine shrimp ecology in the Great Salt Lake. pp. R. S. Salt Lake City. Eng. W. G. (1977) Statistical Analysis of Samples of Benthic Invertebrates.. (1989) Ecological Methodology. (1990) Water Quality in Ponds for Aquaculture. Culturing. C. M. Van Stappen. G. P. (1980) Competition experiments between parthenogenetic and sexual strains of the brine shrimp. Utah. operation and management. Bengtson. K. prepared in cooperation with Utah Division of Wildlife Resources.E. Sorgeloos. Stephens. Salinity effects on survival. Vol. Birmingham. & Williams.W. Utah.144 Live Feeds in Marine Aquaculture Belovsky. USA.V. (1976) Interrelationships between water and cellular metabolism in Artemia cysts. (1996) Improved use of the fluidized bed dryer for Artemia cysts. Ecology. Utah. Harper & Row. W. P. In: Artemia Research and its Applications. 57. Elsevier. XLIX. 15. 3. 88. Vol.M. In the open water marine environment. The name copepod is derived from the Greek kope meaning ‘oar’ and podos meaning ‘foot’. . harpacticoids are an important constituent in the diet of larval and juvenile fish. calanoids dominate the herbivorous zooplankton and provide the food-chain base for practically all marine fish larvae and planktivorous fish (Pauly & Christensen 1995). free-living copepod species. Aquaculturists may involuntarily be better acquainted with fish parasites than with the more numerous. Almost one-third of marine copepod species are parasites or live in symbiotic relationship with other organisms. mostly marine.2. copepods constitute a first vital link in the marine food chain leading from primary producers to fish. culture and use of free-living copepod species as live feed in aquaculture.1 General characteristics Copepods are aquatic animals.2 Biology 5. Around 200 families with some 1650 genera and 11. paddle-like swimming legs. less harmful. The most commonly used species in aquaculture are free-living copepods belonging to three of the ten orders of copepod (reviewed by Huys & Boxshall 1991): Calanoida. to subterranean species living in groundwater or in deep-sea hydrothermal vents. through benthic species that live on the surface of macroalgae or inhabit microscopic spaces in marine sediments. This chapter deals with the biology. copepods play a central role in the global production of fish. Freeliving copepods inhabit a variety of habitats.1 Introduction In nature. flatfish and salmonids (Hicks & Coull 1983.500 species were classified by 1993 (Humes 1994). Støttrup 5. In estuaries and coastal areas. Thus.Chapter 5 Production and Nutritional Value of Copepods Josianne G. Huys & Boxhall 1991). which by 1999 supported an estimated total capture fishery production of around 92 million tonnes (FAO 2000). ranging from the planktonic copepods that inhabit the world’s oceans. although many species occupy freshwater or estuarine habitats. 5. and refers to the flat. For a comprehensive review on the biology of calanoid species consult Mauchline (1998).146 Live Feeds in Marine Aquaculture Harpacticoida and Cyclopoida.1. 5.1 Calanoida The calanoids are predominantly pelagic. gracilis. (d) N1 T. with up to 27 segments and biramous antennae used as accessory locomotory appendages (Huys & Boxshall 1991. Dussart & Defaye 2001). 5. Dahms & Bergmans 1988. occurring at all depths. as long as the body itself or even longer. Calanoida: (a) stage CV Calanus finmarchicus.] .1. (From Lebour 1916–1918.2. Cyclopoida: (e) C5 Cyclops strenuous. Leiden. Dussart & Defaye 2001). The position of the prosome–urosome articulation is between the fifth and sixth postcephalosome somite (Mauchline 1998. or predators feeding on a variety of animal prey including copepod eggs. (b) stage I nauplius C. Dussart & Defaye 2001. finmarchicus. Calanoid species used in aquaculture as live prey for marine fish larvae are listed in Table 5. Figure 5.1 depicts an adult and a nauplius representative from each order. They are selective feeders feeding on small phytoplankton cells by filtration. (f) N1 Eucyclops serrulatus. with some near-bottom and benthic species.) [Parts (e) and (f) reproduced with kind permission of Backhuys Publishers. They are distinguished by their long antennules. Harpacticoida: (c) male CV Tisbe gracilis.1 Schematic diagrams of an adult and nauplius representative from each of the three orders: Calanoida. Harpacticoida and Cyclopoida. Fig. Eurytemora affinis Eurytemora affinis Eurytemora affinis. plumose Acartia sinjiensis Acartia tonsa Acartia tonsa Acartia spp. Loligo pealie Golden snapper. Anarhichas lupus Halibut. Morone saxatilis Turbot. Calanoid species Acartia longiremis Acartia pacifica. (1982) Schipp et al. some harpacticoids Outdoor and indoor systems Collected from wild Co-fed with rotifers Co-fed with rotifers Collected from wild Outdoor tanks Laboratory culture Origin or application Collected from wild Collected from wild Marine fish species Wolf fish. Gadus morhua Fundulus spp. (1997b) Jung & Clemmesen (1997)a Turk et al. Anarhichas lupus Asian sea bass.Table 5. (1995) Doi et al. (1981) Rippingale & MacShane (1991) Rippingale & MacShane (1991) Payne et al. (1987) Sunyoto et al. A. mangrove jack. Coryphaena hippurus Sea horse. Glaucosoma hebraicum Pink snapper. (1999)a (continued) ... Lutjanus argentimaculatus Striped bass. Lutjanus johnii. Pseudodiaptomus spp. Gadus morhua Grouper. (2001)a Ringø et al. Lates calcarifer. Pagrus aurata Wolf fish. (2001)a Payne et al.. Hippocampus angustus West Australian dhufish. Hippoglossus hippoglossus Cod. Epinephelus fuscoguttatus Red snapper. Oithona sp. grouper. Lutjanus argentimaculatus Cod. (1998) Gamble & Houde (1984) Toledo et al. Elops saurus and squid. Epinephelus coioides References Ringø et al. (1999) Chesney (1989) Tsai (1991) Kuhlmann et al.1 Species of calanoids used in aquaculture as live prey for marine fish species. (1987) Rønnestad et al. Scophthalmus maximus Dolphin fish. Morone saxatilis Striped bass. Acartia tonsa and others Gladioferens imparipes Gladioferens imparipes Gladioferens imparipes Gladioferens imparipes Metridia longa Temora longicornis Mixed zooplankton/ Pseudocalanus elongates Mixed copepods: Acartia tsuensis. Hippoglossus hippoglossus References Kvenseth & Øiestad (1984) Battaglene & Fielder (1997) Mixed species: Eurytemora affinis. or higher rates of normal pigmentation in fish compared with diets comprising rotifers or Artemia nauplii. Sillago ciliata Halibut. Argyrosomus hololepidotus. Macquaria novemaculeata. wild zooplankton Wild zooplankton Marine fish species Cod. C. . Eurytemora hirundoides Frozen copepods a Collected from wild/culture ponds McEvoy et al. (1984) Showed improved growth or survival. hamatus.Table 5. (1998)a Collected from wild or outdoor pond cultures. Hippoglossus hippoglossus Næss & Lie (1998)a Flounder. Acartia teclae. Gilthead sea bream Sparus aurata Bedier et al. sand whiting. Acartia spp.. Gadus morhua Barramundi.1 (continued) Calanoid species Mixed zooplankton Mixed zooplankton Origin or application Outdoor enclosures. Centropages hamatus Mixed species dominated by Eurytemora affinis or Centropages hamatus Mixed copepods: Temora longicornis. mulloway. Lates calcarifer. Platichthys flesus Engell-Sørensen (1997) Added on days 35–55. Centropages typicus. Supplement over a short period Collected from wild and cultured for three generations Halibut. Australian bass. 3.1. The urosome ends in a furca formed of two symmetrical rami ornamented with setae. 5. The genital opening is usually located in the first abdominal segment. less robust fish larvae (Cooper 1996). the position of the prosome–urosome articulation is between the forth and fifth postcephalosome segment (Dussart & Defaye 2001). being shortest in harpacticoids and longest in calanoids. with no appendages except for the caudal rami.Production and Nutritional Value of Copepods 149 5. This appendage bears different setae with chemosensory or mechanosensory functions. This is rarely the case in harpacticoids and . or living on sediment or plant surfaces (epibenthic). (Dussart & Defaye 2001). 5. The antennules in cyclopoids are shorter than in calanoids. 5. epibenthic. The third family contributing to the free-living cyclopoids. and have six to 17 segments (Huys & Boxshall 1991. although they are far more abundant in freshwater. which include over 50% of copepod species. freeliving. which inhabit saline waters.3 Cyclopoida The cyclopoids include pelagic. whereas the last segment bears the anal opening (anal somite). This anterior part has been designated as the prosome. etc. The antennules vary in size and may be used to distinguish among the three genera.2. and inhabit both freshwater and marine environments. The position of the prosome–urosome articulation is between the fourth and fifth postcephalosome segment (Dussart & Defaye 2001). They inhabit sediments occupying spaces between sand particles (interstitial).2. The abdomen is generally narrower than the thorax.2 lists the harpacticoid species used in aquaculture as live food for marine fish larvae. fewer than 10 segments. benthic.2. In the marine environment. They are distinguished by their short antennules. broader bodies and/or dorsoventrally compressed forms.2). The trunk is composed of a thorax (metasome) and an abdomen (urosome) (Fig. and biramous antennae. benthic organisms. In contrast to calanoids and harpacticoids. burrowing into sediment (burrowers). The head (cephalosome) is fused with the thorax and bears anteriorly a typical median naupliar eye. Dussart & Defaye 2001). littoral. the antennules help to slow down the sinking rate in calanoids (Mauchline 1998). Table 5.1. cyclopoids belonging to the family Cyclopinidae are predominantly benthic and those of the Oithonidae planktonic (Huys & Boxshall 1991). As in harpacticoids.2 Copepod morphology Free-living copepods have generally cylindrical bodies with a narrow abdomen (planktonic forms) or. cyclopoids have uniramous antennae used to help catching food. as evident in Table 5. are primarily marine. rarely reaching beyond the cephalothorax. and the form of these antennules is closely related to the lifestyle of the copepod: pelagic. attacking predominantly smaller. benthic and parasitic species. Several of the freeliving forms are predatory. When held out laterally from the body. is the Cyclopidae. although the majority of species in this family are freshwater species.2 Harpacticoida The harpacticoids. Cyclopoid species are not frequently used in the larviculture of marine species. a conspicuous set of antennae and the various appendages used for feeding and swimming. in the case of benthic or surface-living forms. Tisbe sp. Acartia clausi. Epinephelus striatus Northern anchovy.. (1991) Mud dab. Engraulis mordax Turbot. or higher rates of normal pigmentation in fish compared with diets comprising rotifers or Artemia nauplii. Acanthopagrus latus Fukusho (1980) Kahan et al. Pacific white shrimp. Coryphaena hippurus Black sea bream. (1981) Tucker & Woodward (1996) Hunter (1976) Støttrup & Norsker (1997) Heath & Moore (1997)a Jinadasa et al. (1991. (1981)a Lee & Hirano (1979) in Lee et al. Gobionellus boleosoma Grey mullet. Schizopera elatensis Tigriopus japonicus a Nanton & Castell (1999) Kraul (1983)a Kraul et al. Mylio macrocephalus Sand borer. Solea solea References Sun & Fleeger (1995) Amonardia sp. Harpacticoid species Amphiascoides atopus Origin or application Marine fish species/crustaceans Grass shrimp. . Solea solea Dover sole. Tigriopus japonicus Tisbe holothuriae.(dominant) Temora longicornis. Limanda yokohamae Sea bream. Mugil cephalus Mahimahi. Centropages hamatus. Scophthalmus maximus Dover sole.Table 5. Sparus aurata Yellow-fin sea bream. (1982) Tseng & Hsu (1984) Showed improved growth or survival. 1992)a Lee et al.2 Species of harpacticoids used in aquaculture as live prey for marine fish species. darter goby. Penaeus vannamie. Sillago sihama As a supplement Co-fed with rotifers Sole and co-fed with rotifers Sole diet and co-fed with Artemia Outdoor tanks with multiple copepod species Nassau grouper. Paracalanus sp. Euterpina acutifrons Euterpina acutifrons Tigriopus japonicus Tigriopus japonicus Tigriopus japonicus Tisbe furcata Tisbe holothuriae Tisbe holothuriae Tisbe spp. In benthic species the antennules help in anchoring the copepod to the substratum (Björnberg 1986). and maxillae. Co-fed with rotifers and Artemia Origin or application Sole diet or co-fed (1:1) with Artemia nauplii Marine fish species Acanthopagrus cuvieri Grouper. which help to macerate the food. (1996) a Showed improved growth or survival. Epinephelus spp. Eugerres brasilianus References James & Al-Kars (1986)a Su et al. Striped patao. or higher rates of normal pigmentation in fish compared with diets comprising rotifers or Artemia nauplii.2 Diagrammatic illustration of the external morphology and relevant terminology of a female calanoid. Rudiments appear during the naupliar stages but are first fully functional during the copepodite stages. 5. In filter-feeders the maxillae bear setae where . (1997) Alvarez-Lajonchère et al. In some species of all three genera one or both antennules are used to grasp the female during copulation. The structure of the oral aperture and surrounding appendages differs with species according to their feeding mode. maxillules adapted to grasp and break up the food. Calanoid species Apocyclops borneoensis Apocyclops royi Oithona sp.Production and Nutritional Value of Copepods 151 Table 5. Fig. but exceptions do exist such as the planktonic species Oithona. The oral aperture is situated ventrally and is surrounded by mandibles. whereas the uniramous form in cyclopoids is used to help catch and handle the food (Dussart & Defaye 2001).3 Species of cyclopoids used in aquaculture as live prey for marine fish species. which adopt different forms according to the feeding mode of the species and help in capturing the food. The antennae are used as accessory locomotory appendages in calanoids and harpacticioids. cyclopoids. during the first three naupliar stages (Hicks & Coull 1983. resulting in simple.2. The external skeleton or integument consists of several layers arranged in three distinct structures: an outer cuticle composed of a thin epicuticle and a thicker procuticle. has no oral aperture and lives on its vitelline reserves. is involved in digestion. In many species the first naupliar stage. this appendage may also be modified. Dahms 1993. but they contain glucids. essential for locomotion. glycoproteins and mucopolysaccharides. The cuticle is not entirely impermeable but is perforated by many pores.2. In several species of calanoids and harpacticoids an extension from the midgut leading dorsally and anteriorly. Price et al. collecting particles on the setae. In cyclopoids the labrum. This appendage is very developed in Oithona species where it is used for catching food (Björnberg 1986). called the midgut diverticulum. Cyclopoid nauplii have a pear-shaped body (Dussart & Defaye 2001). together with the middle section. accumulated and transferred to the mouth (Mauchline 1998). is dorsoventrally compressed and unsegmented. In calanoids and some harpacticoids. (1983) describe the maxillae periodically combing the feeding appendages. which produce secretions that may bind the food together and initiate digestion (Dussart & Defaye 2001). 5. antennae and mandibles. The musculature is most primitive in cyclopoids. From the buccal cavity a dorsal oesophagus leads to the midgut. which protects the oral aperture anteriorly. being often reduced in females and enlarged and asymmetrical in males and used to grasp the female during mating. Attached to the thorax are four or five pairs of swimming legs. which produce egg sacs. In male calanoids this appendage may be modified so as to enable it to grasp the female with one leg. During the remaining three naupliar stages there is a progressive development of setae and of appendages in the posterior end of the body. In free spawners the fifth pair of swimming legs is reduced or absent in order to facilitate swimming movements (Björnberg 1986). functions as a hepatopancreas. In cyclopoids this pair of legs is similar in both species. Dussart & Defaye 2001). abbreviated to NI. The oesophagus is lined with chitin and its wall has longitudinal folds and strong muscular tissue. erratic jerks forwards or sideways.1 Digestive system The oral aperture is formed by the anterior labrum and the posterior labium. barely moving. but considerably reduced or even absent (Dussart & Defaye 2001). At the articulation points the cuticle is not chitinised to allow flexibility. The body of newly hatched nauplii has an oval shape. Its body is unsegmented and has three pairs of appendages: antennules. The labrum contains the labral glands. The maxillipeds generally appear during the sixth naupliar stage and are situated between the maxillules and aid in scraping the substratum or in cleaning the other appendages. the last pair is often modified. somersaulting or helical movements (Björnberg 1986). In females. while the other is used to transfer the spermatophore to the female. Absorption and the . whereas calanoids exhibit circular. The precise composition of the secretions is not known. They are also useful in taxonomy because the distribution of spines and setae on the legs is species specific. The procuticle consists of chitin in a protein/lipid matrix arranged in two layers in different orientation to each other.152 Live Feeds in Marine Aquaculture particles are retained. with relatively few appendages and a single naupliar eye. is armed with teeth whose number and shape are used in taxonomy. the epidermis and the basal lamina (Dussart & Defaye 2001). This diverticulum forms part of the first section of the midgut which. Phosphate. A sympathetic nervous system innervates the digestive tract.Production and Nutritional Value of Copepods 153 formation of faecal pellets take place in the posterior section of the midgut. branching off to segmental and intersegmental nerves. which extends dorsally along the digestive tract. also lined with chitin and with longitudinal folds. innervating longitudinal body muscles and flexor muscles of the swimming legs.2. and consists of a brain situated dorsally and anterior to the oesophagus. 5. which also functions as a primitive kidney. 5. The posterior end.2. In most copepod females the genital system consists of a single ovary.3 Nervous system The central nervous system is simple and relatively uniform in copepods. The naupliar eye is characteristic in all stages of copepods and is generally red and located anterodorsally (Fig. ducts and a ventrally placed genital aperture. where they grow in size and undergo . urea and dissolved primary amines are also excreted (Dussart & Defaye 2001). ammonia production was shown to reflect feeding activity and suggested as an ideal feedback parameter for feeding control in a continuous production system (Støttrup & Norsker 1997).3). From the brain extends a ventral nerve chord that extends to the posterior end and comprises both sensory and motor nerves. to be expelled via the anus placed dorsally on the anal somite (Mauchline 1998. Dussart & Defaye 2001).2.2.2. Anteriorly. it consists of paired glands.4 Reproductive system The genital system is located dorsally in the prosome and lies along the digestive tract. The new oocytes are located anteriorly. It is located dorsally between the second and third thoracic segments and consists of one ventricle. A giant fibre system formed by two pairs of interneurons and numerous giant motor fibres is involved in the rapid escape movements in copepods. The faecal pellets are generally ovoid. is where the oogonia multiply. In this region of the midgut. 5. 5. these fibres leave the brain to innervate the antennules.4) and some harpacticoids. In general. 5. The main nitrogenous compound excreted by copepods is ammonia (Le Borgne 1986). It arises posteriorly from the brain and extends ventrally to the base of the labrum at the oral aperture (Dussart & Defaye 2001). coloured droplets or concentrated in an oil sac. lipid reserves are accumulated and distributed throughout the body in small. the germinal site of the ovary. The colourless blood is circulated through an anterior aorta and a system of sinuses. The pigments are carotenoids associated with proteins or pro-melanins (Dussart & Defaye 2001). From here the pellets move through the hindgut. The giant fibres run along the nerve chord.2 Circulatory system A heart is only found in calanoids. The male genital system is unpaired in calanoids and most harpacticoids and paired in cyclopoids (Fig. peristaltic movements of the muscles of the alimentary tract ensure blood circulation (Dussart & Defaye 2001).2. In harpacticoids and cyclopoids with no distinct heart. In the harpacticoid Tisbe holothuriae. 154 Live Feeds in Marine Aquaculture Eye Fig.) Fig. Støttrup. (Photograph: J.3 Tisbe holothuriae nauplius with a characteristic nauplius eye. (Photograph: E. 5.4 Picture of the cyclopoid Apocyclops panamensis with paired egg sacs.) . 5.G. Lipman.E. some species of Acartia and several Calanus species. Tisbe battagliai produces up to nine egg sacs. shedding eggs singly into the water. 1999). Copepods can produce eggs that are non-viable or unfertilised. including cyclopoids and harpacticoids. Females from different Tisbe species were capable of fertilising up to 12 broods from a single fertilisation and up to around 21 in other harpacticoid species (Hicks & Coull 1983). which remain attached to the female genital segment until they hatch. The male deposits a sac containing viable sperm called a spermatophore near the genital aperture of the female. Free-spawning species such as various Acartia species may produce between 11 and 50 eggs female 1 day 1. Egg production is measured as number of eggs female 1 day 1. the harpacticoid T. and an average of 10 offspring O 1 day 1 (Williams & Jones. and that of free spawners between 30 and 700 eggs (Mauchline 1998). 5. In cyclopoids. which run laterally along the thorax and enter the genital somite. holothuraie fed artificial diets produced 258–416 nauplii per female (Gaudy & Guerin 1977). . 5. producing a total of up 1200 from one single spawning. fecundity in calanoids with egg sacs is about 7. The number of eggs spawned in a single event may vary from a few eggs to 50 or more eggs. In most cases a new mating is necessary for the female to produce eggs again. A review covering 27 different harpacticoid species showed a range of 3–229 eggs sac 1 (Hicks & Coull 1983). In cyclopoids the eggs are contained within paired egg sacs (Fig. Dussart & Defaye 2001). Each egg sac or egg mass may contain a few to 50 or more eggs and may be produced at frequent intervals of a few days. such as Centropages furcatus. Since a new egg sac cannot be produced before + the nauplii have hatched from the previous one and because of their larger size.Production and Nutritional Value of Copepods 155 differentiation. Several calanoids. independently of the spawning method.4) (Huys & Boxshall 1991). the male transfers paired spermatophores from its genital aperture to the ventral surface of the female genital somite. The ripe oocytes occupy the oviducts. Fecundity in species with egg masses is within the range of 10–200 eggs. Most calanoids are broadcasters. are known to spawn at night.5 times lower than in free spawners (Kiørboe & Sabatini 1995). There are very few reported cases of repeated mating in harpacticoids and it is assumed that the female dies after having produced one or a number of batches of eggs from a single spermatophore. In laboratory experiments. Average brood sizes for Tisbe holothuriae reared on three different artificial diets ranged from 58 to 86 eggs sac 1 (Gaudy & Guerin 1977). Both sexes of cyclopoids and harpacticoids have paired genital apertures located ventrally (Huys & Boxshall 1991). and each spawning event may occur about once every 24 h for extended periods. In calanoids the eggs are not contained in a membrane but adhere to each other as an egg mass and remain attached to the female (Mauchline 1998).3 Reproduction Most copepods reproduce sexually. where they merge into a genital cavity (Mauchline 1998. have their eggs contained within one or two egg sacs (ovisac). 1994). and Calanus species between 15 and 230 eggs female 1 day 1 to a total of up to 3800 eggs female 1 (Mauchline 1998). produced at intervals from 1 day to 4 days (Hopcroft & Roff 1996). Other copepods. Higher fecundity has been shown for some harpacticoid species. Clutch size in marine cyclopoid species varies from a few eggs up to around 100. depending on food conditions (Poulet et al.2. 5.5.4 Resting or diapause eggs Resting eggs are produced by several species of copepod and are the primary mode of dormant state in calanoids. a brackish-water harpacticoid copepod. 1975) and up to 5. In culture. has been demonstrated for a few harpacticoid species under laboratory conditions. Egg viability was not registered. diapause egg production can be stimulated by the culture conditions such as short daylength and enhanced by low temperature (Ban 1992) or by subjecting the culture to high densities (Ban & Minoda 1994). (1999) stored normal eggs under anoxic conditions at 4°C for up to 12 weeks to examine the effect of cold storage on lipid composition. Harpacticoida and Cyclopoida.5 million eggs m 2 in a Norwegian enclosed pond system (Næss 1996). although summer encystment of adult harpacticoids and in cyclopoids has been reported.2. although in the latter no dormancy is reported in marine species (Dahms 1995).2. More recently. Resting eggs are able to withstand long periods of desiccation. asexual reproduction through generations of females. In the laboratory. Close to 100% survival was achieved when nauplii and adults of Gladioferens imparipes were stored at 8°C for up to 12 days (Payne & Rippingale 2000d).2 million eggs m 2 in the Inland Sea of Japan (Kasahara et al. They may occur in high quantities in the sediments up to 3. they were observed to withstand disinfection of the egg surface (Næss & Bergh 1994: Acartia clausi and Eurytemora affinis). Dormancy occurs more frequently in winter in species from temperate regions and higher latititudes. 5. In the latter case. and possess an additional external envelope of variable thickness. Most of the resting eggs in the top 8 cm of the sediment remained viable for several years (Katajisto 1996). Mauchline 1998). Further. the induction was chemically mediated. Dormancy in fertilised females was reported to occur exclusively in the freshwater cyclopoid copepod Cyclops strenuous (Næss & Nilssen 1991). Resting eggs have only been reported for calanoids (Grice & Marcus 1981. The egg is spherical and . Scottolana canadensis).e. but dormant nauplii or copepodites are known from free-living representatives of all three copepod taxa discussed in this chapter. possibly through metabolic products in the culture medium. Once fertilised. reproductive-resting females were reported for Coullana canadensis (i. Daylength and temperature were the principal environmental cues that induce females to switch from active reproductive to a resting reproductive state. Støttrup et al. size and growth 5. namely the Calanoida. the eggs pass into the water or into an egg sac. and may also occur in other species (Lonsdale et al.1 Life cycle Copepod species belonging to these three orders considered for aquaculture have similar life stages. although hatching rates were lower than when hatching eggs directly.156 Live Feeds in Marine Aquaculture Parthenogenesis. Normal eggs have been stored under cool conditions in aquaculture to ensure sufficient quantities for rearing fish. rearing conditions during the naupliar stages may determine whether the resultant adults produce diapause eggs (Ban 1992). whereby development is arrested. heat or cold (Dussart & Defaye 2001).5 Development. 1993).2. These copepod eggs are laid during development. but there is little information on this phenomenon or its occurrence in nature (Dussart & Defaye 2001). develops through five (a few calanoid species) or six moults before passing onto the copepodite stage where they display the general adult features. 1988) and even fewer during the first naupliar stage (Mauchline 1998). 14%. whereas the larger Calanus pacificus nauplii measure around 220 m (Greene & Landry 1985). Calanus and Pseudocalanus and the pelagic harpacticoid Euterpina acutifrons are reported to exhibit isochronal development where the different stages have virtually the same duration (Neunes & Pongolini 1965. whereas in Apocyclops royi the digestive tract is incomplete during the first stage. In cyclopoids. 5.2 Mortality Mortality rates. Nauplii of the epibenthic harpacticoids S. but within the optimal range other factors such as food quantity and quality may influence development (Williams & Jones 1994. Temora longicornis and Centropages hamatus. the nauplius (NI). Guérin et al. 1982). which are rarely larger than 200 m (Mauchline 1998). Klein Breteler et al. holothuriae.2. Newly hatched nauplii such as A. T. Volkmann-Rocco & Battaglia 1972. (Jinadasa et al. The rate of development is dependent primarily on temperature. canadensis and Tisbe sp. 1982.g. Berggreen et al. The nauplii of Gladioferens imparipes measuring 126 67 m was an ideal size for first-feeding seahorse Hippocampus subelongatus (Payne & Rippingale 2000b). Schipp et al.Production and Nutritional Value of Copepods 157 protected by a chitinised envelope. the principal (46%) copepod species to be found in the water column were young stages of Tisbe spp. The larva that hatches from copepod eggs. The harpacticoid Euterpina acutifrons is pelagic and some harpacticoid species have planktonic immature stages (Neunes & Pongolini 1965). clodiensis. which is of a very short duration. . Klein Breteler (1980) estimated 46–75% survivial during one generation for three calanoid species: Acartia clausi. for Tisbe species measured in the laboratory range from 2 to 23% (Bergmans 1981). Harpacticoid nauplii are believed to be able to feed from the first stage and undergo six moults.5. measuring 65 m in width an ideal size for first-feeing Lutjanus johnii. Acartia tonsa. Corkett et al. although a few commence feeding during the second (e. 5. Several harpacticoid species tend towards progressively longer stage durations (Bergmans 1981). 2001. Size ranges of newly hatched nauplii also vary. In other species such as Calanus finmarchicus the stage durations differ (Tande 1988). lasting for a few minutes (Chang & Lei 1993). Various species of calanoids of the genera Acartia.5. Mauchline 1998). In an extensive cultivation system for sole. (1999) found Acartia spp.g. 16%). 1991). and more recent reports on mortality rates for different species of Tisbe lie within this range (e. are pelagic (Hicks & Coull 1983). T. 1986). tonsa measure less than 100 m in body length (Klein Breteler et al. the final one resulting in the first copepodite stage (CI) (Hicks & Coull 1983. Williams & Jones 1994). the first nauplii of the cyclopoid Oithona ovalis have been shown to have a functioning gut (Fanta 1976).2. Most species commence feeding during the third or fourth naupliar stages. in cultures with Tisbe species and under appropriate conditions a survival rate of 75% to adults could be expected. Thus.3 Size Calanoid eggs produced in egg sacs range in diameter from 70 to around 800 m and are generally larger than freely spawned eggs. in terms of naupliar survival to adulthood. Although shorter in length. The first naupliar stage of the cyclopoid Apocyclops royi is the larger specimen. gracilis Apocyclops royi Fig.7–1. Chang & Lei 1993.2. Canada. the width of the first naupliar stage of Tisbe cucumariae is 68 m (Dahms et al. Once they reach maturity. For example.4 0. furcata T. measuring around 1 mm in length (Fig. The small nauplii of three different harpacticoid species commonly used for cultures are similar in size. defined as the time interval between hatching of an individual and the hatching of its progeny.5 Sizes of naupliar (N) of three Tisbe species and copepodite (C) stages of T. Adult sizes are generally around 0. The adult females are generally larger than the males. The larger forms are epibenthic and have the ability to swim or are among the few truly planktonic harpacticoids. Dahms et al.6 0. Cyclopoids are generally smaller than calanoids. gracilis and a cyclopoid species.4 mm (Böttger-Schnack 1988). 1991). 1991.5).5 mm in body length (Ikeda 1973). somatic growth ceases in adult females and egg production is thus often used as an expression of growth (Kiørboe et al. Dahms & Bergmans 1988. 1985). differs from species to species and is positively correlated with .2 0 NI NII NIII NIV NV NVI CI Copepod stages CII CIII CIV F CV F CVI F Tisbe cucumariae T.4 to 1. their width is similar to that of small calanoids. 5. 5. The carnivorous adult Euchaeta elongata measures 6.3–7.5 mm (Hicks & Coull 1983). 5.8 0. growth can be expressed in dry weight. The harpacticoids are smaller and range in size from 0. Newly hatched nauplii of a Tisbe sp. which increases almost exponentially with development (Mauchline 1998) in most species.4 Generation time The generation time.5).4 mm in total length (Greene & Landry 1985). 5. Apart from growth in length (total length or prosome length). measured 90 m in length and females grew to around 2 mm in total length (Nanton & Castell 1998). whereas the adults are relatively small.) Marine free-living copepods may measure up to 10 mm in length. collected near Halifax. Apocyclops royi.2 1 Length in mm 0.2 to 2.158 Live Feeds in Marine Aquaculture 1. A study of the female size of 11 species of the family Oithonidae from the central Red Sea showed a range in body length from 0. the adults measuring less than 1 mm (Fig.5. (Drawn from Johnson & Olsen 1948. such as demonstrated for A. tonsa 7 days at 25°C) to months (Mauchline 1998. Kiørboe et al. Huntley et al. Artemia nauplii were used to investigate predation rates in adult Aetideus divergens. food quality and food availability 5. Artemia nauplii were suggested to be easier prey than copepod nauplii (Mullin & Brooks 1967).6.1 Calanoids Feeding Calanoids have a non-visual. Males are often smaller than females. (A.11 to 11 prey adult 1 day 1. In laboratory experiments. Unpalatable feed. able to distinguish between particles and selecting between different food particles based on size or taste (Donaghay & Small 1979. was regurgitated by Eurytemora affinis (Powell & Berry 1990) and the toxic dinoflagellate Gonyaulax grindleyi by Calanus finmarchicus (Sykes & Huntley 1987). generation times varied from around 1 week in Acartia spp. The duration of development from the first naupliar stage to adult in Tigriopus japonicus was shorter with increasing salinity (Hagiwara et al. 1995). In calanoids reared at different temperatures. Calanoids are generally herbivorous filter-feeders. The functional response (rate of consumption of algal particles) is influenced by the size. quantity and quality of the food (Paffenhöfer 1976. reared at 17°C. 1986). including yolk-sac cod larvae as was observed for the carnivorous Euchaeta norvegica (Yen 1987). such as inert beads. sinjiensis 5–6 days at 28–30°C. Støttrup & Jensen 1990. However. may develop more rapidly (shorter generation time) and have a shorter longevity. (1988) and vary from 0.2. a copepod adapted to living below the euphotic zone and to feeding on larger particles such as faecal pellets or large phytoplankton cells (Roberston & Frost 1977). 1996). Hicks & Coull 1983.Production and Nutritional Value of Copepods 159 increasing temperature.6). One outlier. tonsa was estimated at 2–4 m (Berggreen et al. 5. Dahms 1987). a phytoplankton species widely used in aquaculture (see Chapters 2 and 7) as a diet for rotifers. Centropages and Temora feeding on nauplii of their own or other species are reviewed by Daan et al. Similar population characteristics are found in harpacticoids (Bergmans 1981. Kiørboe et al. However. Huntemannia jadensis. Acartia tonsa was shown to be able to switch between particle feeding and a predatory mode (Kiørboe et al. 1985.2. capturing and ingesting a variety of animal prey (Tiselius & Jonsson 1990). active raptorial mode of feeding. requiring higher concentrations relative to other algal species to reach maximum ingestion rates (Støttrup & Jensen 1990) (Fig. Food supply and salinity may also influence development rates. Small size was suggested to be one of the probable causes for poor survival. 1996). The lower size limit for particle capture in A. had a generation time of 167 days. tonsa feeding on Isochrysis galbana. . development and fecundity in Gladioferens imparipes fed Nannochloropsis oculata (Payne & Rippingdale 2000a).6 Feeding. 1988). Table 47). A. predation rates on nauplii decreased in the presence of sufficient alternative (phytoplankton) food. Predation rates of different surface-dwelling calanoid species of the genera Acartia. but the culture temperature was only 8°C (Hicks & Coull 1983). Size is important relative to the structure of the oral appendages. Copepod species may also create feeding currents that entrap non-evasive prey such as copepod eggs and nauplii (Yen & Fields 1992). 5. The generation time in 34 different harpacticoid species ranged from 7 days in Tisbe holothuriae reared at 22°C to 42 days in Robertgurnenya sp. and copepods may be less efficient in retaining small cells. tonsa. Acartia tonsa produced more eggs when feeding on I. Miralto et al.160 Live Feeds in Marine Aquaculture 140 Cell numbers per ml 120 100 80 60 40 20 0 0 5 10 15 20 Cell size (µm) 25 30 Fig. 1985. In A.6. Ban 1999) or be deleterious to copepod embryogenesis. resulting in variable hatching success due to the presence of toxic aldehydes (Ianora et al. 1980). Jónasdóttir (1994) found that the age of the diatom culture influenced the ability of Acartia sp. 1994) (Fig.7). despite similar ingestion rates in terms of carbon and nitrogen (Støttrup & Jensen 1990). Ban 1999. 5. ◆: Støttrup & Jensen 1990. eggs to hatch. Ingestion rates in terms of carbon. Ban (1999) reviewed the literature on diatom effects and found 31 combinations involving 12 estuarine or coastal ocean copepod species and 13 diatom species where either reduced fecundity or reduced hatching. was reported. or both. volume or weight copepod 1 day 1 and percentage copepod body weight ingested day 1. values from 6 to 360% are reported. galbana than on Thalassiosira weissflogii. helgolandicus (Gatten et al. 1999). Egg production The relationship between the rate of egg production and phytoplankton concentration in copepod species for which experimental data are available indicates that the food concentrations at which egg production commences and at which it attains a maximum level differ between species. 5. (Drawn from : Kiørboe et al. ■: Cowles et al. The mean daily ration of adult calanoids feeding on phytoplankton and expressed as a percentage of copepod body weight ingested day 1 ranged from 2 to 360% (Mauchline 1998. Earlier findings have long established the relationship between copepod egg production and lipid levels in the diet in C. 187). Food quality also influences growth and reproduction. 1995).6 Relationship between maximum ingestion rate and algal cell size in the adult female calanoid Acartia tonsa. 1988. The different algal species used for different copepods in culture are given in Tables 5. (1995) also showed low hatching success in Calanus helgolandicus feeding on dense cultures of Phaeodactylum tricornutum. Kleppel & Burkart 1995. Some species of diatom used as food for copepods have been reported to reduce fecundity (Kleppel et al. 1985.4–5. 1995. Egg production per female per day increases with increasing food concentrations to an asymptotic level (Kiørboe et al. A diet of Skeletonema costatum resulted in sterility or death in Temora stylifera (Ianora et al. 1991. and it has been . Kleppel & Burkart 1995. p. Dam et al. nitrogen or protein are also available in the literature.) Ingestion rates Ingestion rates are calculated in laboratory experiments from the number of cells removed from a volume of water per unit time and expressed in different units such as numbers. Poulet et al. tonsa in laboratory experiments (Jónasdóttir 1994).2 Harpacticoids Feeding Harpacticoids are primarily detritivorous. 22:6n-3) and eicosapentaenoic acid (EPA. tonsa (Jónasdóttir 1994). Koski et al. When fed Dunaliella tertiolecta. tonsa (Støttrup & Jensen 1990. Components other than lipids may also be important for copepod fecundity. (1998) showed the dietary content of amino acids to be correlated to egg production in A. Amino acids are important sources of organic carbon and total nitrogen in copepods (Cowie & Hedges 1996). Thus. Protein and nitrogen content in food was shown to be positively correlated with fecundity in two species of Acartia. to ensure high fecundity in calanoids. algal biofilm. helgolandicus (Lacoste et al. (Drawn from Kiørboe et al. eggs per female per day 45 40 35 30 25 20 15 10 5 0 0 5 10 15 20 25 30 35 40 45 50 Food concentration. 1999).) suggested that low levels of docosahexaenoic acid (DHA. marsh grass. although lipids were shown to exert a superior influence on fecundity in A. ample food of the right size and nutritional quality must be supplied. Jónasdóttir & Kiørboe 1996) and G.2. but they are reported to be important in maintaining membrane fluidity in cold environments (Benson & Lee 1975) and facilitating catabolism of long-chain monoenoic fatty acids (Sargent & Henderson 1986). 1985.6. 1998. The role of these n-3 polyunsaturated fatty acids in copepods is not clear. in Pseudocalanus elongatus (Koski et al. tonsa. a chlorophyte deficient in n-3 polyunsaturates. Kleppel et al. diatoms. 1998) and in C. macroalgae. imparipes (Payne & Rippingdale 2000a).7 Rate of egg production by Acartia tonsa as a function of food concentration of Rhodomonas baltica.Production and Nutritional Value of Copepods 161 Egg production. Although no clear relationship could be established between copepod fecundity and EPA or DHA concentration in diets (Støttrup & Jensen 1990. Jónasdóttir 1994. cells per ml Fig. 20:5n-3) in the diet may reduce fecundity (Lacoste et al. 5. the highest rate of egg production coincided with the highest DHA:EPA ratio in both A. 5. efficiently utilising various food sources such as bacteria (Rieper 1978). fecundity was irreversibly blocked in A. benthic grazers. 2001). 2001). . The importance of n-3 fatty acids compared with n-6 fatty acids was demonstrated for A. Lee et al. tonsa (Støttrup & Jensen 1990). Egg production ceased in the harpacticoid Scottolana canadensis at concentrations below 2. development and reproduction in harpacticoids. 1997).5 104 cells ml 1 but increased exponentially with increasing concentrations to a symptotic level (Harris 1977). Egg production Food supply in terms of quality and quantity affects feeding. In a study on a harpacticoid. survival and population densities obtained were highest for Tisbe furcata fed the diatom Skeletonema costatum at densities of 80 cells l 1. Indeed. Fecundity. this does not imply that they are nonselective feeders and that their offspring production is independent of the type of diet offered (i. Ingested food is thus assumed to be invested directly into energy resources of the yolk (Hicks & Coull 1983).2%. resulted in higher percentages of fecund females and higher total egg production of the culture. and Guérin et al. Cannibalistic behaviour towards non-related nauplii has also been shown in a harpacticoid (Lazzaretto & Salvato 1992). and Fryfood® and lowest on Spirulina. longevity. Further. despite the low PUFA content in the yeast.4 nauplii daily when fed a diet of Rhodomonas baltica alone. Whereas calanoids obtain copious amounts of n-3 polyunsaturated fatty acids (PUFA) from their phytoplankton diet. whereas a diet of either Rhinomonas reticulata or Pavlova lutheri gave poorer results at maximum ingestion densities (Abu-Rezq et al. The number of offspring per female in T.6 nauplii female 1 day 1) compared with a single algal diet (3.2 on I. Tetra Min. as in many calanoids. a phytoplankton very high in protein (68%) (Miliou & Moraïtou-Apostolopoulou 1991). holothuriae was highest when fed a mixture of Ulva sp. PUFA content in both copepod species was high.e.2 on T.162 Live Feeds in Marine Aquaculture polychaete meat. galbana alone and 4. Nanton (1997) fed two harpacticoid species yeast with a dietary lipid content of 2. harpacticoids feed on other sources lacking or containing low levels of these fatty acids. Although harpacticoids eat practically anything. shrimp solids (Norsker & Støttrup 1994) and artificial diets such as Tetramin (Guérin & Gaudy 1976). compared with 2. or lie within the lower range of that in calanoids. whereas its sibling species T. Tisbe furcata exhibited a preference for dead bacteria to dead Dunaliella tertiolecta. Carbon derived heterotrophically was assimilated at a rate eight to 10 times higher than autotrophically derived carbon in certain harpacticoids (Brown & Sibert 1977). holothuriae and T. pseudonana alone). Detritus and associated micro-organisms have been suggested as important nutritional elements in the harpacticoid diet.5 on a diet of Dunaliella tertiolecta (Norsker & Støttrup 1994). the rate of daily naupliar production was higher when fed a mixture of Isochrysis galbana and Thalassiosira pseudonana (5. Harpacticoids are not known to store lipids as an energy source during or prior to reproduction. Williams and Jones (1999) showed that low algal concentrations decrease fecundity. Tisbe holothuriae females produced approximately 8. mixed cereals (Guidi 1984). yet. Because of the difficulty in assessing the nutritional contribution from bacteria. . battagliai were indiscriminate in their feeding (Vanden Berghe & Bergmans 1981). (2001) showed that the addition of small amounts of bacteria and vitamin D2 to a commercial pet food. it may be hard to establish harpacticoid production on the merit of various feeds unless the experiments are carried out axenically. Tisbe battagliai. lipid levels in harpacticoids are generally low. food quality). . Although HUFA content may be less important for production in harpacticoids. Tisbe sp. the DHA:EPA ratio remained high ( 2) in both copepod species despite their dietary fatty acid content (Nanton 1997). but although bacteria containing EPA have been found in deep-sea or low-temperature environments and to a lesser extent in warm-water environments. a chlorophyte with trace amounts of n-3 PUFAs. 1978). with egg sacs being laid down at dawn and hatching at dusk on the following day (Hopcroft & Roff 1996). turbulence is important for encounter rates with prey (Kiørboe & Saiz 1995). Similarly. this has not been tested directly. These workers observed that this species attacks and ingests Artemia nauplii ( 1 mm). both species ingested bacterial matter but only H. Tigriopus japonicus. Nanton 1997). Although there is evidence of the ability of harpacticoids to synthesise PUFA from HUFA precursors such as 18:3n-3. Gyllenberg and Lundqvist (1978) showed that two cyclopoid species. an alga with a relatively high content of long-chain polyunsaturates (Norsker & Støttrup 1994). these results suggested that this harpacticoid was able to synthesise significant amounts of EPA and DHA to maintain a high production level and to incorporate quantities of these fatty acids in its offspring. and Amonardia sp. compared with Rhodomonas baltica. When fed an EPA-rich diet. switching to predominantly carnivorous feeding during the later copepodite stages. Another explanation for the high PUFA levels in harpacticoids fed low PUFA diets may be their ability to utilise marine bacteria in the culture. Harpacticoids utilise bacteria as a food source (Rieper 1978).3 Cyclopoids Feeding Cyclopoids are considered omnivorous. 1997).2. The highest number of ovigerous females was registered on diets containing 49–52% protein (% dry weight).Production and Nutritional Value of Copepods 163 Egg production in Tisbe holothuriae was not reduced significantly when fed Dunaliella tertiolecta. In tropical waters. were shown to synthesise significant amounts of EPA and DHA when fed a PUFA-poor diet (Nanton 1997). ingest and assimilate dissolved glucose. holothuriae and in a Canadian Tisbe sp. In the absence of dietary n-3 PUFA. dominance of single species among planktonic cyclopoids is not as pronounced as in calanoids (Böttger-Schnack 1988).6. Thus. In nature. diel periodicity was demonstrated for Oithona plumifera. curticorne assimilated living bacteria. feeding on planktonic and benthic prey and even on their own progeny. For many cyclopoids. Like harpacticoids. Chang and Lei (1993) found that Apocyclops royi is herbivorous in the early stages. Because of the ability of the copepod to manipulate its relative contents of various fatty acids. EPA was converted to DHA. Levels of arachidonic acid (20:4 n-6) in T. the content of protein seems to play an important role. a light regimen would be important in the culture of these species. making bacteria an unlikely source of DHA. In another harpacticoid. DHA in bacteria has so far only been found in deep-sea samples (Yano et al. were high ( 1%) even when only trace amounts were present in the diet (Norsker & Støttrup 1994. high levels of DHA (12%) and EPA (7%) were observed when fed exclusively on baker’s yeast (Watanabe et al. including EPA and DHA. 5. Guidi (1984) showed that even though food composition did not influence ingestion rate. attacking nauplii or copepodites. Cyclops oithonoides and Halectinosoma curticorne. the content of nitrogen (protein) in the diet played an important role in egg production in Tisbe cucumariae. Oxyrrhis marina Dinoflagellates.000 cells ml 1) Isochrysis galbana (up to 50. (1977) 1 Acartia clausi Acartia clausi 22 litres. Isochrysis galbana. checked 1–2 times daily Tetraselmis suecica Twice weekly: Rhodomonas baltica (up to 50. Isochrysis galbana. Isochrysis galbana (up to 50. seawater filtered through 5 m 15°C 20–24°C — — Iwasaki et al.5 106 cells ml 1) Rhodomonas sp.000 cells ml 1) Thalassiosira pseudonana.4 Cultivation techniques for different calanoid species. Monochrysis (now Pavlova) lutheri (1 106 cells ml 1) T/S 15°C Harvest — References Zillioux (1969) Acartia clausi Acartia clausi 1 year Multiple generations Multiple generations 55 generations 20 generations 20°C 15°C 20 litres — 300–350 adults l 1 — 16 eggs adult day 1 25 eggs adult 1 day 1 Person-Le Ruyet (1975) Iwasaki et al.Table 5. 88 litres 10 day batch cycle 40 litres 100 litres. water exchange 3 times a week 11–95 l 1 19 eggs adult day 1 1 Ogle (1979) . equal volume 3 times weekly Natural phytoplankton blooms..000 cells ml 1) Tetraselmis suecica Equal density of Isochrysis galbana.000 cells ml 1). (1977) Acartia clausi Equal density of Isochrysis 20°C galbana. Duration of culture 14 months Culture volume/ system 100 litres recirculation Densities obtained 40 l 1 Species cultured Acartia clausi Food organisms Twice weekly: Rhodomonas baltica (up to 50. recirculation 1000 ind l 1 mixed species 40 l 1 Klein Breteler & Gonzalez (1982) 70 eggs F 1 day Khanaichenko (1998) 1 Acartia clausi Tisbe furcata Acartia tonsa Multiple generations 10 months 15°C 15°C Person-Le Ruyet (1975) — Zillioux (1969) Acartia tonsa Several generations 6 months 18°C 1500 ml Parrish & Wilson (1978) Acartia tonsa Ambient 6–28°C/ 1–26 psu 1890 litres circular outdoor tanks. concentrations at 3–10 g ml 1. Monochrysis (now Pavlova) lutheri (1. Chroomonas salina. 88 litres Klein Breteler & Gonzalez (1982) 87 adults l 1 Centropages typicus Centropages typicus Eurytemora affinis 1 year 1 year Multiple generations 15°C 20°C 15–20°C 40 litres 20 litres 23 litres water exchange every 2–3 weeks 92 adults l 1 435 ind l 1 Person-Le Ruyet (1975) Person-Le Ruyet (1975) Katona (1970) (continued) . Isochrysis galbana Dinoflagellates. aeration 40 l 1 — Person-Le Ruyet (1975) Person-Le Ruyet (1975) Klein Breteler (1980) Centropages hamatus 55 generations 15°C 22 litres. Tetraselmis sp.. Skeletonema costatum every 2–3 weeks 15°C/32 psu 100 litres. Rhodomonas baltica (3–11 104 cells ml 1) Isochrysis galbana. (total 20. and a diatom Defatted rice bran. aeration 40 l 1 — Klein Breteler (1980) 17°C 20–25°C/ 15–25 psu 16–18°C/ 35 psu 20–24°C 1500 ml 170 litres 870–1680 N l 1 170–1520 adults l 1 100 adults l 1 Zillioux & Wilson (1966) Turk et al. Isochrysis sp. Cyclotella nana. checked 1–2 times daily Rhodomonas sp. 6 months 28–32°C/ 30–34 psu 15°C 20°C 15°C/28 psu Schipp et al. Rhodomonas sp.. Oxyrrhis marina Tetraselmis suecica Tetraselmis suecica Isochrysis galbana..000 cells ml 1 ratio of 2:1:1) Tetraselmis suecica Tetraselmis suecica Daily: Isochrysis galbana (3–8 104 cells ml 1) Rhodomonas baltica (2–5 104 cells ml 1) Rhodomonas sp. (1982) Acartia tonsa Acartia tonsa 70 generations 20 generations 200 litres 10 day batch cycle 100 litres or 1000 litres batch aeration 23–27 eggs F day 1 70 eggs F 1 day 1 2000 N l 1 harvested after 8 days 1 Støttrup et al.. 1–3 g l 1 culture fed twice daily Rhodomonas baltica. Platymonas sp. Isochrysis galbana. concentrations at 3–10 g ml 1.Acartia tonsa Multiple generations Acartia tonsa Acartia tonsa 12 generations 4 months Daily: Isochrysis galbana (3–18 104 cells ml 1). (1986) Khanaichenko (1998) Acartia spp. (1999) Centropages hamatus Centropages hamatus Centropages hamatus 1 year 1 year Multiple generations 40 litres 80 adults l 1 20 litres 110 adults l 1 100 litres. 88 litres 2 litres 20.000 N day 1 Rippingale & MacShane (1991) Klein Breteler & Gonzalez (1982) Davis (1983) Rhincalanus nasutus 7 generations 12°C 19 litres. Barthel (1983) Chesney (1989) Tsai (1991) Gladioferens imparipes Pseudocalanus sp. 7. Thalassiosira pseudonana clone 3H. stirred aeration 45 eggs F 1 Mullin & Brooks (1967) Temora longicornis Temora longicornis 2 months Multiple generations 55 generations 20°C 15°C/ 28 psu 15°C 40 litres 100 litres. Isochrysis galbana. Pseudocalanus sp.. 5 104 to 5 105) 28 generations 55 generations 70 days Isochrysis sp. Ditylum brightwellii.4 (continued) Duration of culture 45 days Culture volume/ system 27 m3 in outdoor tanks.. Thalassiosira weissflogii clone Actin Diatoms Cyclotella nana. Coscinodiscus wailesii naupliar Artemia salina Tetraselmis suecica Daily: Isochrysis galbana (3–8 104 cells ml 1) Rhodomonas baltica (2–5 104 cells ml 1) Rhodomonas sp. 12–28°C/ 2–38 psu 15°C 5°C 15 litres 22 litres. Isochrysis galbana.Table 5. Oxyrrhis marina 19°C 50 litres 3000 litres 1 Species cultured Eurytemora affinis co-cultured with other species Eurytemora affinis Eurytemora affinis Eurytemora affinis Food organisms Nannochloris T/S 15–20°C/ 16 psu 15°C/ 12 psu Densities obtained Harvest 1–2000 N l day 1 1 Reference Nellen et al. Thalassiosira pseudonana (ratio 1:1. aeration 22 litres.5% harvested daily 150 litres 2000 litres Isochrysis galbana. Thalassiorsira fluviatilis. Pavlova sp. Skeletonema costatum clone Skel. 88 litres 100 adults l 40 l 1 1 Person-Le Ruyet (1975) — Klein Breteler (1980) Temora lonicornis Klein Breteler & Gonzalez (1982) .. Oxyrrhis marina Isochrysis galbana clone Iso. (1980) Nannochloris sp. Phaeodactylum tricornutum Rhodomonas sp.. Centropages hamatus.. temperature.. F. outdoor 21–238 N l day 1 1 Engell-Sørensen (1999b) Gaudy (1978) 15 weeks 10–20°C/ 28–33 psu 85 m3. flowthrough. outdoor aeration 25 ind l 1 Jinadasa et al. Mixed copepods: Temora longicornis. mixed with seawater Eutrophic ponds 20 m3 10. Selected copepod species (starter cultures) Selected copepod species (nursery ponds) Selected copepod species (grow-out ponds) min. Female. Paracalanus sp. . water exchange every 20–30 days 5–200 ha 1000–6000 ind l 1 Quin (1993) 100 ind l 1 Quin (1993) T. Tisbe sp. salinity. Acartia clausi. S.Mixed copepods Mixed copepods: Acartia sp. individuals. once weekly second month. mixed with seawater Algae from aerobic waste stabilisation ponds.000 l 1 Harvested to inoculate nursery ponds Batch harvest used to inoculate grow-out ponds Up to 8 107 ( 200 m) 6 107 (200– 500 m) h 1 harvest Quin (1993) 100 m3. 1/6 volume added daily with enriched seawater phytoplankton bloom 1/3 water exchange first month. (1991) Algae from aerobic waste stabilisation ponds. Oithona sp. twice weekly thereafter Ambient 1000 m3 30 m3. N. Tisbe sp. 3 generations 6 weeks Natural phytoplankton Approx. ind. nauplii.. In some systems. In others. and subsequently sorting these into size fractions and concentrating these in separate containers. It consists of a flow-through system equipped with rotating discs mounted with plankton gauze of different sizes (Fig. often from fjords or inlets where natural densities are high.3. 1998) (Fig.2 Production in enclosed fjords or sea areas Copepods occurring naturally in enclosed or semi-enclosed ‘polls’ with volumes of 37. with a filtering capacity of 35 m3 min 1 (Fig.3. enough food could be collected theoretically to feed 40. The UNIK filter system described by van der Meeren and Naas (1997) represents a more sophisticated plankton-collecting device.3 Production Methods 5. and 250 m was sufficient to retain adult copepods. or harvested and frozen.000 turbot fry. Potential predators in the enclosed system were initially killed off with rotenone (Naas 1990). whereas numerous copepod nauplii were retained. The Baleen filter described by Quin (1993) is mounted on a craft and is capable of filtering 200 l s 1 on a 63 m screen without damaging the zooplankton. By increasing the mesh size of the larger gauze. whereas sizes between 70 and 100 m retained rotifers almost exclusively. The phytoplankton production is enhanced by adding agricultural fertilisers and where possible water-flow to maintain a high and stable production of zooplankton. wild zooplankton are collected from the sea and transferred to the . the copepod starting culture is derived from resting eggs in the sediment. Huse (1994) used this system to collect wild copepods.3. dried or freeze-dried for later use as an inert diet. They are collected directly from nature.1.8). 5. 5.9). Even larger collection devices have since been developed which enable the filtration of larger volumes of water per hour. Above 120 m few rotifers were retained. inoculated into outdoor tanks on land to produce live zooplankton for fish larval rearing. 5.000 m3 have been utilised directly for rearing marine fish in Norway (Svåsand et al.1 Extensive and outdoor cultures 5. the size fraction can be broadened to include copepodites (350 m) and adult copepods (600 m) (van der Meeren & Naas 1997). using mesh sizes 80 and 250 m to obtain equal quantities of Acartia teclae and Centropages hamatus. and used directly as live prey.1. This size range collects almost exclusively nauplii.1 Harvest of wild zooplankton Copepods have been used for culturing marine species for several decades.000–100. A further development of the UNIK filter is used in the lagoon Parisvatnet in Norway. Mesh sizes below 70 m quickly became clogged.000–510. Barnabé (1980) described a floating propeller-induced device that directed the water current through an elongated plankton net at around 146 m3 h 1. This calculation was based on a bioenergetic food consumption model developed by van der Meeren (1991). 5. Several types of filtering device have been developed for this purpose. and consists of a floating device with a large rotor at the bottom driving a water flow upwards and through the rotating filters.168 Live Feeds in Marine Aquaculture 5. capable of concentrating a defined size fraction of copepods at a rate of 1–5 m3 min 1. When operated at 1 m3 min 1 in a eutrophic (artificially fertilised) saltwater lake with copepod densities of 20–50 l 1.10). see text for more detail. which are then flushed by water jets (visible in the bottom picture) onto a collection pipe. The two wheels have different mesh sizes and the copepods entrapped on each of these wheels are collected in two separate containers (top). (Photographs: J. Top: a UNIK filter viewed from the collection side. 5.8 UNIK filter. Støttrup.G.Production and Nutritional Value of Copepods 169 Fig. The gauze on the rotating wheels (bottom) entraps copepods. Bottom: inside the UNIK filter.) . 5.) . (Photograph: J.10 Parisvatnet (top): a natural semi-enclosed ‘poll’ used for rearing cod juveniles in Norway. (Photographs: J.G. Støttrup. Juveniles are maintained within the fjord (bottom). Støttrup.9 Free-floating zooplankton filtering unit used in the extensive rearing of cod in Norway to concentrate zooplankton from a fjord at a capacity of 35 m3 min 1.G.170 Live Feeds in Marine Aquaculture Fig.) Fig 5. copepodite stages (80–350 m) or primarily adult stages (250–600 m) to inoculate the rearing tanks. 1998).3. and to examine nitrogen and phosphorus levels before the time of these occurrences and add fertilisers to achieve similar levels. the restrictions and unpredictability of such systems are well recognised.3 Production in outdoor ponds or large tanks Outdoor production in 350–5000 m3 ponds and tanks is carried out in Europe and Asia for the culture of round fish species. 1991). natural phytoplankton can be transferred to the ponds without the accompanying zooplankton or potential predators. In this system. (1991). In a Norwegian manipulated seawater enclosure. 1982). Blooms of toxic algae can result in total mortalities for a year class. In conditions of non-limiting nitrate levels ( 5 M) and high oxygen concentrations. Collecting preweaning juveniles has been tried without much success (Svåsand et al. cod production from such systems varies from 0. For this reason the nutrients in the water intake and the type of phytoplankton found in the rearing tanks are closely monitored in the Danish extensive system for rearing marine fish larvae (Engell-Sørensen 1999a. From 1986 to 1994 a total of around 2 million juvenile cod was produced (Svåsand et al.b). such as turbot. and flatfish. Another disadvantage is that it is difficult to assess the number of surviving juveniles and thus difficult to ration food at the time of weaning to dry diets. In the experiment carried out by Naas et al. 1978.32 l 1. which are preferable to the development of the smaller flagellates. Despite improvements and experience in managing these systems.6 cod juveniles m 3. In Norway.g. and adding nutrients. The larvae are then transferred to these enclosures at densities of 0. Low nitrate concentrations favour algae with low Ks (concentration at which the uptake rate of nitrate is at half the maximum rate) values of nitrate. a good rule of thumb is to identify local fjords or inshore waters where there are regular algal blooms. and mixing of the water layers by generating flow and adding filtered seawater. 1998). Takahashi et al.5 M. In Asia a mesh size of 400–600 m was used to inoculate outdoor tanks for grouper rearing with copepodite and adult stages 3 days before stocking the newly hatched fish larvae at densities of 5 m 3 (Toledo et al. NPK complex) in small quantities can induce a bloom. larger diatoms were favoured. 1999). Silicate is sometimes added and this encourages the development of diatoms.01–0. 5. Filtering devices that allow for selective sieving are used to collect primarily nauplii (80–250 m). using wild-harvested copepods (chiefly Acartia tsuensis . but experience has enabled the Norwegian culturists to prevent such blooms by nutrient manipulation to encourage diatom blooms rather than flagellate blooms. Regarding the amounts to add.Production and Nutritional Value of Copepods 171 enclosures. Lack of food results in differential growth in fish larvae. giving rise to a prolific copepod production. Additional prey may be added during the larval rearing when necessary to maintain prey densities in the range of 200–500 l 1.1. small flagellates dominated during the initial period before fertilisers were added and where the nitrate levels were registered at 0. such as cod and grouper. the increase in diatom production and improved oxygen conditions resulted in an increased production of calanoid nauplii (Naas et al.02 to 1. The phytoplankton can be monitored. By using filters of around 20–40 m. which in cod leads to higher rates of cannibalism. Filtered seawater is generally used in these systems. generally commercial fertilisers (e. such as small flagellates (Parsons et al. Disadvantages with this type of system include the inability to control production and thus food levels and predators. Centropages typicus. In a similar system in France. Obtaining reliable density estimates of the . Typically.000 cod juveniles were produced in such systems. C. During the initial period a bloom of rotifers reaching well over 10. Acartia spp. 10–50 l 1. Tisbe sp. Artemia nauplii are sometimes added as a supplement when copepod densities are low (Naas 1990.000 flounder juveniles each year (one season) (EngellSørensen 1999b). Eurytemora hirundoides and occasionally Oithona similis (Engell-Sørensen 1997.172 Live Feeds in Marine Aquaculture with smaller amounts of Pseudodiaptomus spp.. resulting in the production of 3. Oithona sp. 1999a). hamatus. Calanus finmarchicus. van der Meeren & Naas 1997). longicornis and the concurrent dominant algal species was Thalassiosira nordenskjoldii.000 individuals m 3 developed. Newly hatched flounder larvae were stocked at around 0. whereupon they were replaced by copepods and especially nauplii. Paracalanus parvus and Pseudocalanus elongatus.4% at harvest corresponded to an average production of 0. Copepods identified in this system were Temora longicornis. within a couple of years of its establishment. The rearing system for flounder consists of three outdoor ponds (1200 m3). Temora longicornis. The Danish turbot-producing hatchery described by van der Meeren and Naas (1997) and Støttrup (2000) has. At least one of the concrete tanks is used entirely for the zooplankton cultures.. an average survival of 3. extensive systems use either outdoor concrete tanks or tarpaulin-lined earth-ponds (Fig. two used for fish larval rearing and one for copepod culture. and a very few harpacticoids). the dominant copepod species developing in the system were Acartia sp. In Denmark. around 140. In the Norwegian systems.17 grouper Epinephelus coioides juveniles m 3. but other copepods such as Oithona similis and Tisbe sp.4–2. rotifers dominated at the outset. A similar sequence has been observed in some of the productions in a tarpaulin-lined pond system used to culture flounder. lasting for around 8 days. The plankton found in these systems consists of primarily calanoid species: Eurytemora affinis. produced around half a million turbot juveniles each year. 5. in two out of five successful productions over a 3 year period. have been reported (van der Meeren & Naas 1997). the dominant species in two productions was T. Because of the relatively high energetic demands of the fast-growing fish larvae. (Gaudy 1978).05 yolk-sac turbot larvae l 1 are added to each pond and during successful runs a production of around 20 juvenile turbot m 3 can easily be achieved. Regular monitoring of densities of the live prey in these outdoor systems is important for the successful rearing of marine fish larvae.8–40 cod juveniles m 3 (Svåsand et al. Around 0. Good techniques for filtering zooplankton are essential as this system relies heavily on the addition of zooplankton during the fish larval rearing. The zooplankton most commonly reported in these systems is calanoids of the genera Acartia.08 l 1 and 11–47 flounder juveniles m 3 were produced. In this system. lasting for around 20 days. During 1997. one species dominated and between two and five other calanoid species were identified.11).. Platicththys flesus (Engell-Sørensen 1999b). Centropages hamatus. In Norway between 1989 and 1993.8 fish larvae l 1 are added. The naupliar concentrations around first feeding were generally in the lower range compared with those reported in the Norwegian systems. Centropages and Temora. prey densities range from 10 to 300 l 1 and around 1. and Oithona sp. 1998. Acartia spp. 1998). and has produced up to 82. The duration of rotifer dominance was shorter in this system. A bloom of nauplii and other zooplankton then replaced the rotifers.. from which water can be filtered and zooplankton added to the fish rearing tanks. peaking at approximately 350–450 rotifers l 1. Pseudocalanus elongates. 1991. (Photograph: J. Toledo et al.Production and Nutritional Value of Copepods 173 Fig. In general. Støttrup. a large volume (10–25 litres) from various positions in the tanks is sampled and filtered.11 Harvesting juvenile cod reared in earth-ponds lined with black tarpaulin in Denmark.) copepod populations within the larger outdoor systems is not an easy task owing to their patchy distribution and changes in their distribution throughout the day. Van der Meeren . 1999). 5. and the copepods are identified and counted (Jinadasa et al.G. personal communication). 1998) (Fig. It is easier in these systems to estimate the numbers of surviving juveniles and thus to ensure an adequate food supply. The juveniles are usually metamorphosed when they are collected from the ponds or tanks and transferred to indoor tanks for weaning to a dry diet and ongrowing. The formula for such a preservative fluid is given in Mauchline (1998. Since many of these parasites use copepods as intermediate hosts between compulsory hosts. The bags used in Norway are cylindrical. Marcogliese 1995). An advantage of outdoor ponds over the extensive systems that rely on the local production of zooplankton is the possibility of ‘culturing’ the zooplankton over one generation before using them as food. 1998). less harmful to health. care should be taken when working with this preservative. This production system.12). which infect marine fish. the use of the first generation nauplii in the system is sufficient to reduce the risk of parasite transfer for those parasites that use copepodites or adult copepods as intermediate hosts. In some cases around half the water is left in the outdoor tanks. the tanks are emptied and left dry during the winter.174 Live Feeds in Marine Aquaculture (1991) used a long tube. 2–8 m in diameter and 4–6 m deep. With the falling temperatures. At the end of the season. Thus. The method described by Mauchline (1998) is very practical and convenient. Trematodes and cestodes. 5. needs to be developed further to ensure predictable production cycles. After 10 days. the copepods produce resting eggs. This consists of adding 30 g of borax to 1 litre of analytical reagent-grade formalin (40%). the sample can be filtered onto a sieve. Both the transfer and weaning may be very stressful to the fish and heavy losses may occur during this period. The volume of the settled copepod sample should not exceed 20–25%. Since formalin is detrimental to health. Regardless of the sampling technique. p. like many of the other extensive or semiextensive systems.5 litres from a 3 m water column. . enough to ensure that the whole water column does not freeze. glass vials with screw-on caps with easily identifiable millilitre marks are ideal for formalin storage. and the total sample (copepods seawater) not exceed 90% of the volume of the bottle. live prey and phytoplankton. K. It is important to ensure that the material used is non-toxic to the fish larvae. which sampled 11. Dussart and Defaye (2001) recommend the plankton trap of Schindler-Patalas (Schindler 1969. Robert & Gabrion 1991. Vaccination against Vibrio shortly after the transfer has been shown to mitigate these losses. which survive in the sediment until the following year and are used as starter zooplankton cultures in the following year when the larval rearing season recommences (Næss 1996. in Dussart & Defaye 2001) for quantitative sampling in lakes. 10). feeding wild zooplankton directly to the fish increases the risk of infection. The final 10% is filled with the buffered formalin and should be stored in the dark for at least 10 days. The advantage here is that the number of bags used to rear fish can be regulated according to the zooplankton supply. Tarpaulin bags were placed in the polls or fjords in an attempt to increase control and allow manipulation of the biotic and abiotic parameters (Svåsand et al. have been identified in copepods (Bristow 1990. washed in seawater and transferred to another preservative fluid. it is advisable to take the samples at the same time each day. The bags are filled with filtered (80 m) seawater and copepod nauplii added to densities of 100–500 l 1. The zooplankton samples can be preserved in 4% formalin buffered with borax (sodium tetraborate). The plankton sample should be concentrated and added to a bottle of known volume. with a flow-through system with a bottom outflow (van der Meeren 1991. van der Meeren & Naas 1997). Engell-Sørensen. In Norway almost half a million cod juveniles were produced in such systems between 1988 and 1994 (Svåsand et al. Several attempts to mass-culture copepods in intensive systems have been undertaken with varying success and have resulted in the development of different systems for particular species of copepods (Tables 5.G. or with a small mouth size. Species inhabiting coastal environments are normally more tolerant to variations in salinity and temperature and have a wider thermal and salinity tolerance. 5. such as marine ornamental fish species (Payne & Rippingale 2000b) or species difficult to rear on the traditional live prey. 1997a). Eurytemora and Temora (Table 5. such as those of the genera Acartia.2. the species possibly taking advantage of some environmental parameter to outcompete other species.Production and Nutritional Value of Copepods 175 Fig. production of toxic metabolites and genetic adaptation to particular environmental conditions (niches) have been proposed (Bergmans & Janssens 1988).) 5. Rearing in larger volumes ( 10 litres for calanoids and 2 litres for harpacticoids) may be more representative of the conditions required for mass rearing.3. 1999). such as grouper (Epinephelus sp. consult Ikeda (1973) and Paffenhöfer and Harris (1979). Although the mechanisms for competition have not been fully explored. dhufish (Glaucosoma sp. Støttrup.2 Intensive culture of copepods Since the early 1990s there has been a revival of attempts at intensive cultures of specific species of copepods.6).4). partly in response to a global shortage of Artemia eggs (see Chapters 3 and 4) and partly to diversification to new culture species with very small larvae. For further references including small-scale (millilitre to litre) cultures. 5.) (Toledo et al. with relatively short generation times and a wide thermal and .3. Species with relatively short generation times at ambient temperatures are best suited for aquaculture purposes. These copepods are small. Centropages. One calanoid or harpacticoid species often dominates. These dominant species are ideal candidates for intensive rearing.4–5.12 Experimental floating tarpaulin bag (foremost) placed in an enclosed fjord for rearing cod juveniles in Norway.1 Calanoids The most frequently cultured calanoid species belong to the genera found in coastal waters.) (Payne et al. rotifers or Artemia nauplii. (Photograph: J. 2001) and red snapper (Lutjanus argentimaculatus) (Doi et al. A smaller sized gauze (80–120 m) would also have been sufficient. 2001). Failing to do this would result in a culture crash.6). although Turk et al. high in DHA (DHA:EPA 29. 5.6 for A. waste products and superfluous feed may accumulate and generate problems with ciliates and other contaminants.176 Live Feeds in Marine Aquaculture salinity tolerance. 3:1 by cell numbers). faecal matter and associated ciliates (Fig. Food is provided at regular intervals (daily to two or three times weekly). During the siphoning. In culture systems where the culture medium is not exchanged daily. which may not comply with all the requirements for maximum egg production. and of a size that can be utilised by both the feeding naupliar stages and the copepodite and adult stages. As a general rule. (1982) demonstrated that it was possible to culture Acartia tonsa on rice bran. tonsa. and are easily adaptable to laboratory conditions. this would also ensure a suitable fatty acid distribution in the copepods used as live feed. which can be filtered from the water. tonsa (Støttrup et al. Food and feeding Most calanoids require the provision of phytoplankton. galbana. The removed debris was checked daily. (1986). A combination of at least two algal species with high n-3 polyunsaturated lipid content. as discussed earlier.4 105 cells ml 1 day 1. high in EPA (DHA:EPA 0. imparipes (Payne & Rippingale 2000b.3). quantity and quality of the algae provided. A combination of I. allowing most of the debris and ciliates to pass through and be removed from the culture. the eggs sedimented to the bottom from where they were siphoned daily. This is also illustrated in Fig. simultaneously siphoning out debris. around 104 cells ml 1 using smaller cells such as Rhodomonas baltica (around 5–12 m) and 105 cells ml 1 using yet smaller cells such as Isochrysis galbana or Pavlova lutherii (Støttrup & Jensen 1990. The adults were then used to inoculate . heralded the deterioration of water quality and a thorough water exchange was necessary. was used for the culture of A. they tend to dominate with time. The food ration was adjusted according to water turbidity. high ingestion rates and high egg production rates. In many cases.2–1. the eggs were concentrated on a 45 m sieve. The culture was filtered through a 180 m sieve submerged in seawater to retain the adult population and wash out the ciliates. The daily removal of eggs eliminates the potential loss of nauplii through cannibalism by the adult population. baltica. 5. the rate of egg production in copepods is dependent on the size. Somatic growth ceases in adult copepods and growth rate is more or less equivalent to the rate of egg production. Since the fatty acid distribution in adults and their non-feeding offspring reflects that of the adult diet. When reared in outdoor multispecies cultures.13). Lacoste et al. which may cause the culture to collapse. to reach food saturation. Larger calanoid species may be less efficient in feeding on the smaller algal sizes. and food density is either counted to ensure specific densities or regulated according to the water turbidity in the culture tanks. the copepods are reared on monoalgal diets. cell concentrations of around 103 cells ml 1 would be sufficient using larger cells such as Thalassiosira weissflogii and Ditylum brightwellii ( 12 m in mean diameter). probably comprises an adequate diet for culture. Payne & Rippingale 2000c. The algal concentration at which egg production commences or is at its maximum rate differs in copepod species depending on the diet.. In the culture system described by Støttrup et al. Similar to ingestion rates. and the presence of a particular protozoan. the ciliate Euplotes sp. 1986) and G. ranging from 6–8 104 to 1. and R. together with the egg count. In general. Gadus morhua (Grønkjær et al. may have alleviated this problem. The frequency of this water exchange varied. A reliable batch culture system for rearing Acartia sp. A further advantage of this system and that of Støttrup et al. or in this case a smaller surface area. and Elops saurus (Turk et al. The eggs were siphoned daily off the bottom of the 180 litre black tanks (left). By growing the nauplii for a set number of days. tonsa can be obtained by separating out eggs or nauplii and maintaining each day’s production in separate tanks. but was generally done around every 2–4 months. but because many copepods would be lost with each siphoning. 1986). 5. cod. The greatest advantage of this culture method is that it does not rely on algal cultures. In many cases no particular light is provided and ambient light levels are not recorded. 1982). These authors used defatted rice bran sieved through a 73 m mesh and mixed in deionised water before adding twice daily to the copepod culture at concentrations of 1–3 g l 1 culture. the feeding level was reduced. is described in Schipp et al. Fundulus spp. The culture of Acartia tonsa without cultured algae has been achieved using rice bran over a 4 month experimental period (Turk et al. 1995). Hirtshals. 1985).Production and Nutritional Value of Copepods 177 Fig. Clupea harengus (Kiørboe et al. showing consistent production results over an 8 day cycle in three 1000 litre tanks run concurrently over a period of 7 weeks. If the culture water did not clear between feedings. (1999). the culture contained after 7 days around 2000 nauplii. As discussed later (culture tank size and shape). (1986) is that known age (and size) cohorts of A. a new tank filled with filtered (1 m) seawater. Light Light levels and periodicity are rarely reported in the literature. Starting with an inoculum of around 50–100 adults and 150–250 copepodites per litre. this was done as infrequently as possible. Two or three times weekly the bottom was siphoned. one could ensure the availability of nauplii of the required size as live feed for larvae of herring. 750 copepodites and 300 adults per litre. Davis (1983) used a light/dark (L/D) cycle of 10/14 h at an intensity of . concentrated on a 45 m filter and transferred to 100 litre containers (right). plaice. Pleuronectes platessa (Støttrup et al. Denmark. low light levels are applied.13 System for culturing the calanoid Acartia tonsa at the North Sea Centre. a deeper tank with a smaller diameter. 1982). Eurytemora longicornis and E. High density fluctuations were observed in the cultures independent of light regime. (1986) for culturing Acartia tonsa through several hundred generations are cylindrical with a capacity of 200 litres and a centrally placed air-stone with very gentle aeration (Fig. as was shown for Eurytemora affinis exposed to 10 h light (60 lux)/14 dark (Ban 1992). affinis (Katona 1970. the most favourable regimen seems to be a photoperiod of at least 12 h of light. 5. In outdoor ponds used for rearing marine fish larvae. Aeration also helps to prevent anoxic conditions. in that it increases the encounter rate (Kiørboe & Saiz 1995). which helps to distribute the copepods within the culture tanks and prevent ambient low algal concentrations due to patchy distribution of predator and prey. Photoperiod (8L/16D) and constant illumination at an intensity of 1000–1200 lux at the water surface was used for rearing four calanoid species (Person-Le Ruyet 1975). adults show negative phototaxis during the day and positive phototaxis during the night (Dussart & Defaye 2001). Several calanoids are known to spawn at night (Mauchline 1998). Hence. Higher densities have been achieved in Acartia clausi.178 Live Feeds in Marine Aquaculture 18 E m 2 s 1 for rearing Pseudocalanus sp. Tanks used by Støttrup et al. in culture. air-stones or air bubbles may be used to support a gentle upward and circulating flow. In nature. and air bubbles that are too small may become entrapped in the copepod appendages and should be avoided. Aeration and oxygen Aeration is required to help to maintain phytoplankton in suspension and to create small turbulence. but it has no negative effect on these species. (1986) the light intensity was 25 E m 2 s 1 and lids were placed over the production tanks during the night. Too vigorous aeration should be avoided and is unnecessary. In a continuous recirculation system provided with constant illumination at 1500 lux. It is also important to bear in mind that in some species a short photoperiod is the primary cue for the production of diapause eggs. The bottom is flat to enable siphoning of the eggs from the bottom. which establish their own feeding currents. A high tank height to . turbulence is important in ambush-feeding copepods such as Acartidae. maximum egg release occurred during the dark phase and a rhythmic pattern was evident under constant dark conditions but not under constant light conditions. copepods live in well-oxygenated environments. Chesney 1989). oxygen supersaturation of up to around 160% has been registered (Engel-Sørensen 1998). In the system of Støttrup et al. Gentle air-lifts.13). Coastal species produce eggs that sink to the bottom sediment and are able to survive anoxic conditions. In nature. Culture tank size and shape Most calanoids require large volumes and the adult density rarely exceeds 100 per litre. Zillioux (1969) reported that the copepods gathered in the corners. high solar radiation is harmful to copepods. hence. Egg production in the calanoid Labidocera aestiva was examined under cycling light/dark conditions and compared with constant light or darkness (Marcus 1985). In contrast. In this study. with no evidence of adverse effects on the copepod population. Positive phototaxis in Gladioferens imparipes nauplii was used advantageously to concentrate nauplii before harvest from 500 litre culture units (Payne & Rippingale 2000c). thus facilitating harvest. Person-Le Ruyet 1975. Turbulence is less important for suspension feeders. Eurytemora. 1999). In cultures where the bottom is siphoned regularly. 1. sampling devices) that may be used in each tank. In laboratory . use of the same siphon for all copepod tanks should be avoided at all costs. It is therefore important to keep these cultures strictly apart. Turk et al. These species are probably the most suitable candidates for culture. a central air-stone may be sufficient to ensure proper circulation within the tank column without too vigorous aeration. The presence of other copepods may pose a problem. Mauchline 1998). However. The bottom was flat to enable debris to be siphoned off the bottom. Estuarine species are more tolerant to lower salinities and temperate species to temperature changes. other copepods or rotifers may pose a problem. such as species belonging to the genera Acartia. The low tank height to bottom surface area may result in high numbers of adult copepods being siphoned together with the bottom debris. tonsa (Støttrup 2000). The successful batch culture of the calanoid Acartia sp. Centropages or Temora.g. Furthermore. The same rule stands for other devices (e. it is clear that certain species of calanoid often become dominant. Temperature and salinity Temperature plays a fundamental role in the life of the copepods. Mauchline 1998). although the presence of the harpacticoid Tisbe furcata in a culture of Acartia clausi helped to increase the calanoid production. few studies have dealt with the influence of different pH levels on copepod development or production. outdoor rearing tanks or bag enclosures used in extensive rearing of marine fish larvae. Geographical populations are genetically adapted to the conditions of their natural habitat. From experience gained from extensive copepod production. it is preferable to choose species with similar thermal–salinity optima to those present in the rearing facility. (1982) used square tanks 100 50 50 cm with air-lifts in two opposite corners. since the rotifers with their higher reproductive rate would quickly outcompete the copepods.3 m in diameter with a conical base. but they suggested doing this as rarely as possible to prevent loss of copepods. contamination by rotifers is the most likely cause of the collapse of a copepod culture. These tanks were emptied after the 8 day batch cycle and cleaned.Production and Nutritional Value of Copepods 179 tank diameter (tank bottom area) is considered advantageous in reducing the surface area to be siphoned and thus the loss of copepods. pH Extreme pH values are often observed in eutrophic ponds. as in the case of A. Contamination Contamination of copepod cultures by bacterial blooms. but their ability to adapt to temperatures even beyond their natural range is remarkable. was achieved in 1000 litre polyethylene tanks. In culture. ciliate infections. and separate siphons used for each tank to avoid contamination. ensuring a natural cleaning of the tank (Person-Le Ruyet 1975). although it is possible to obtain species from one temperature–salinity regimen and adapt them to another. Coastal species have wider thermal and salinity tolerances than oceanic species (Bradley 1986. Ciliates are utilised by copepods and may in periods of low phytoplankton concentrations constitute the major dietary source (Poulet 1983. In commercial facilities. and a new batch culture was started (Schipp et al. Copepods can survive for short periods on the gauze as they are transferred from one tank to another or when refilling a tank. it is advisable to empty the culture using a 60 or 80 m mesh size gauze. Should ciliates be observed. Freely spawned calanoid eggs sink to the . the presence of certain ciliates. This hypotrichid ciliate is a common contaminant of live prey cultures and is easily identified under a light microcope. and in bioaccumulation studies (Toudal & Riisgård 1987. cod) in extensive systems (Næss 1991). Hook & Fisher 2001). 1982). or continuous water recirculation (Sun & Fleeger 1995) also helps to reduce accumulation of contaminants. Submerged air-lift foam filters maintained good water quality in 500 and 1000 litre cultures of G. Even when assorted sizes are used simultaneously. Takano (1971) also speculated that the bacteria on the cereal fed to G. such as Vibrio sp. storage and transport The zooplankton are hardy enough to withstand repeated filtering. Some bacteria. The culture can then be started afresh. imparipes.g. They can also survive for an extended time (a few hours) at very high densities. Cultures may succumb to uncontrolled proliferation of bacteria. clausi (Buttino 1994). Payne and Rippingale (2000c) found higher concentrations of ammonia and nitrate in cultures of G. Kusk & Wollenberger 1999. are known to infect copepods in eutrophic coastal waters. although submerged filters should be used at all times. In intensive cultures. such as Euplotes species. although the clearance rates on ciliates decreased with increasing phytoplankton concentrations (Stoecker & Egloff 1987). natural or anthropogenic. copepods are used in toxicity tests to determine acute and sublethal effects of water-soluble chemicals and effluents.180 Live Feeds in Marine Aquaculture studies. Because of their high sensitivity to a great variety of chemicals. Water renewal in batch systems or water flow-through in continuous systems (Støttrup & Norsker 1997). Mauchline 1998). Copepods are sensitive to pesticides. but the extent to which this effected fecundity was not examined. imparipes might have been the major nutritional source. imparipes through rapid removal of faecal pellets (Payne & Rippingale 2000c). It was suggested that bacteria colonising the rice bran might also have served as food in the culture of A. resulting in lower survival rates (Nagasawa & Nemoto 1986). but allows the ciliates to be washed out. providing there is sufficient oxygen. tonsa on this inert diet (Turk et al. Calanoids are sensitive to high ammonia concentrations. which retains the adult copepods. Ammonia concentrations of 0. heavy metals and chlorinated compounds. is often an indication of overfeeding and should be avoided.. even though bacteria often constitute a part of the diet of copepods. Heavy metals such as cadmium or copper and chlorinated compounds are toxic to copepods (Toudal & Riisgård 1987. ciliates were demonstrated to be nutritionally adequate. Harvest.12 ppm resulted in an increase in egg production but negatively affected egg viability in the calanoid A. Fungicide-free silicone produced for aquarium purposes should be used and corrosive materials avoided in a saltwater laboratory. Copepod populations are drastically reduced when exposed to rotenone. a pesticide commonly used to kill parasites or predators before rearing marine fish larvae (e. Bacteria-colonised faecal pellets or detritus have been found in the guts of calanoids and some of the ingested bacteria were absorbed and nutritionally utilised (Mauchline 1998). the filters should be constructed such that all are submerged during the filtering process. Care should be taken concerning materials used to rear copepods in the laboratory. which is still within the range found in wild zooplankton. since only the nauplii can be separated. Fukusho (1991) found that the harpacticoid Tigriopus japonicus was the only species to be successfully mass cultured from a list of 11 different species examined. unpublished data). There are several advantages in using harpacticoids in culture: • • • • • • • • high tolerance to a wide range of environmental conditions (Lee & Hu 1981.3. 1999).2. Fukusho 1980. Newly hatched nauplii that had been cold-stored as eggs for a period of 12 weeks had lower DHA levels than and similar EPA levels to nauplii from newly spawned eggs (Støttrup et al. Kept cool. Khanaichenko (1998) registered hatching rates down to 55% in batches stored at 4°C. 12–21 days. Tigriopus spp. The day’s production can be transferred to individual hatching tanks and grown to the required size to feed as live feed for fish larvae. Eurytemora and Pseudodiaptomus (Calanoida) and species of the genera Oithona (Cyclopoida). . including species of the genera Acartia.7. 5. other copepod cultures or larval tanks (Person-Le Ruyet 1975. Uhlig 1984) ability to feed on a wide range of live or inert diets (Uhlig 1984) high reproductive capacity (Uhlig 1984) relatively short life cycles (Tisbe spp. The DHA:EPA ratio had decreased from 2. The method for harvest is different for copepods with egg-bearing females. Methods for harvesting nauplii are described by Payne and Rippingale (2000c).Production and Nutritional Value of Copepods 181 bottom and can be harvested by siphoning the bottom once daily. Støttrup & Norsker 1997) planktonic naupliar stages (Hicks & Coull 1983. it is usually of so short a duration that phytoplankton can be added to the hatching tank once hatching has commenced. and include manual collection of nauplii using buckets with meshed sides or automatic collection of nauplii using light to concentrate the nauplii. Although the first naupliar stage does not feed in most calanoids.G. 1981. these eggs can be transported to another site or stored for later use. although no attempts were made to quantify this (J.3 to 1. Uhlig 1984) ability to be cultured in high densities (Uhlig 1984) requirement for surface area rather than volume (Uhlig 1981. Støttrup. transferred to small vials. compared with 96% for newly spawned eggs of Acartia sp. Uhlig 1984). Eggs from the daily collections can be concentrated.2 Harpacticoids Harpacticoids have been cultured in batch and continuous systems to provide food for marine fish larvae. The aim was to produce high-density cultures and highest densities were obtained in extensive co-cultures of T. deoxygenated and sealed before being stored in a refrigerator for weeks to months. Jinadasa et al. Støttrup & Norsker 1997). The eggs can be transferred directly from storage at 4°C to the hatching tanks at 16–18°C. to be ideal candidates for cultivating in large cultures (Rothbard 1976. japonicus and rotifers. The viability of the eggs decreases with storage time. 7–29 days. and several studies have demonstrated improvements in growth and survival when using harpacticoids either as the only food source or as a supplement to traditional feeds (Lee et al. Eggs were also transferred directly from the storage temperature (4°C) to 20–24°C. Heath & Moore 1997). Schizopera elatensis 8 days) (Hicks & Coull 1983. 1991) can be used as tank cleaners in rotifer cultures. Several workers have declared harpacticoids. in particular species of the genera Tisbe or Tigriopus. are very appropriate for benthic copepods. Dahms et al. 1991). and the mixture of algae and bacteria may be a superior dietary combination for harpacticoids. This simplifies the culture method and eliminates the need for cultures of phytoplankton. Algae such as Skeletonema costatum. resulting in around 30% larval mortality (Zhang & Uhlig 1993). which quickly sediment. Crowding affects both female productivity and egg viability in T. Although HUFA do not seem to affect fecundity or the content of HUFA in the offspring. and a whole range of inert food is acceptable to many harpacticoid species (see Table 5. Criteria for culture conditions for harpacticoids are less demanding than those for calanoids. 2001). This may not be valid for all harpacticoids. gracialis: NI 73 m. This was demonstrated in experiments comprising three successive generations of T. The use of inert feed may cause hygiene problems in the culture tank. A similar reliance on photoperiod for the synchronisation of egg production was found in Euterpina acutifrons in tropical waters (Hopcroft & Roff 1996). are adequate as a starter feed for small marine fish larvae. with equal success. Sun and Fleeger (1995) fed Amphiascoides atopus either the phytoplankton Chaetoceros muelleri. The protein content of the diet affected both the development time and fecundity in Tisbe cucumariae (Guidi 1984). Light Photoperiod influences both offspring production and their sex ratio. a diet containing HUFA is recommended until conclusive evidence on the role of HUFA in harpacticoid diets has been provided. Algae. NVI 207 m. as discussed earlier. Filtered. and should be considered carefully. Harpacticoids can be reared on a variety of inert feed. producing the least number of offspring and lowest percentage of females (20–23%). Food and feeding If algae are readily available. artificial or non-treated seawater may be used. The best density for naupliar production was 180 individuals cm 2. possibly because bacteria colonise these cells.5). Rhodomonas baltica and Tetraselmis suecica quickly sediment. cucumariae: NI 72 m. A mixed diet may provide the best nutritional value (Zhang & Uhlig 1993). holothuriae (MoraitouApostolopoulou et al.182 Live Feeds in Marine Aquaculture Many of the techniques described for calanoids can be used to culture harpacticoids and cyclopoids. Continuous light was the least favourable. T. a mixture of two algal species would be the preferred choice. 1982). NVI 186 m. The addition of vitamins (vitamin D2) to inert food was shown to improve fecundity (Guérin et al. Dahms & Bergmans 1988. Daily naupliar yield was highest when females were kept at a density of 40 cm 2. Only the points where they may differ. or peculiarities to these genera are dealt with in the following sections. Tisbe species. A photoperiod of 12 L/12 D was shown to be most favourable for offspring production. However. holothuriae (Zhang & Uhlig 1993). and both photoperiod and continuous dark conditions resulted in 40–48% females among the offspring. food quality affects development and fecundity. whereas species such as Isochrysis galbana remain in suspension and may be less available to the harpacticoids. with their small nauplii (T. commercial fish flakes or a mixture of algae and commercial fish flakes. . but it may be appropriate to ensure a diet containing around 50% protein for most harpacticoids. Table 5. Duration of culture 17 weeks Culture volume/ system 1440 litres recirculation Densities obtained Species cultured Amphiascoides atopus Food organisms Chaetoceros muelleri 1 106 cells ml 1 2 day 1. lettuce leaves 1 18°C 21–24°C 3 litres 600 litre batch 15 day cycle inoculated with 50 litres 500 adults l 8900 ind l 1 1 Neunes & Pongolini (1965) Alessio (1974) Euterpina acutifrons Euterpina acutifrons Euterpina acutifrons Nitocra spinipes Schizopera elatensis Schizopera elatensis 18°C/37– 38 psu 22°C 22–23°C/ 34 psu or 28–32°C/ 25 psu 25°C/ 35 psu 21–22°C/ 40 psu 50 litres 10 litres aeration 0. (1987) Szyper (1989) Gopalan (1977) 1 Kahan (1981) Kahan et al. (1978) Ben-Amotz et al. diatoms.5 Cultivation techniques for different harpacticoid species. Chlorella ovalis. Flagellates. Platymonas suecica. static 1.5 106 ind day 1 for 10 weeks. Peridineae Several generations Algae or yeast Chaetoceros gracilis 56 days 55 days 13–21 days Chlorella 1–3 106 cells ml shrimp head meal Lettuce leaves Mytilus powder 5–150 mg. Phaeodactilum tricornutum. or mixture of algae 20 g commercial fish flakes 2 day 1 T/S 23–26°C/ 30–34 psu Harvest 0.5 litre baskets in 200 litres 11 ind ml 412 ind ml 29 ind ml 1 1 356 eggs F 1 Zurlini et al. Chroomonas fragarioides. (1982) (continued) . Glenodinium sp.5–6 litres. then 2–4 106 day 1 Reference Sun & Fleeger (1995) Euterpina acutifrons (pelagic) Euterpina acutifrons 1 year 90 days Dunaliella salina. Thalassiosira pseudonana weekly Chlorella and soya cake Artificial fish feed 3 g crushed seaweed. Platymonas suecica. faecal pellets of Nereis Dunaliella salina. baker’s yeast.Table 5. Glenodinium sp. then 1 Gaudy & Guerin (1978) . -yeast Seaweed. once weekly Green algae or artificial foods Chlorella minutissima.9 kg day 1 Fukusho (1980) 1–3 generations 90 days 513 eggs F 1 Johnson & Olson (1948) Alessio (1974) Tisbe holothuriae for 30 days. dried mussels and scallops. 25°C 22–25°C Densities obtained Harvest 10 eggs F day 1 1 Reference Harris (1977) Kitajima (1973) Kitajima (1973) 26.000 N l 1 Rothbard (1976) Tigriopus japonicus Tigriopus japonicus co-cultured with rotifers Tisbe furcata Tisbe furcata Several generations 89 days 15. Tetramin 1 g day 2 g day 1 Tetramin 1 g day 2 g day 1 1 T/S 20°C. recirculation 12 l h 1 6700 ind l 1 Takano (1968) in Lee & Hu (1981) — 1.5 (continued) Duration of culture Multiple generations 1 month 2 months 26 days Culture volume/ system 2 litres. batch 150 F with eggs l 1 aeration 22°C 2 litres semicontinuous aeration 3500 ind l 1000 F l 1 1 Species cultured Scottolana canadensis Tigriopus japonicus Tigriopus japonicus Tigriopus japonicus Food organisms Isochrysis galbana. Chlorella ovalis.6°C/ 36 psu Ambient. Chroomonas fragarioides. 5–25°C 17–21°C 21–24°C 200 m3 in outdoor ponds 50 ml 600 litre batch 15 day cycle inoculated with 50 litres 100 litres recirculation 12 l h 1 100 litres 10 m2 surface area. Phaeodactilum tricornutum. then 20 ind l day 1 70 ind l day 1 1 Gaudy & Guerin (1978) Tisbe holothuriae 1 for 30 days. Ulva petrusa. lettuce leaves Rhodomonas baltica. (1982) 10 N F day 1 1 Uhlig (1984) Nanton & Castell (1997) Coli (2000) 16 days 0. S. 400 ml added daily with water renewal Rhodomonas baltica 1 106 cells ml 1. continuous system. Tisbe sp. batch 150 litres filled with balls. lettuce leaves Granulated Mytilus edulis 21–22°C/ 40 psu 28 psu 20°C 1. (1982) Støttrup & Norsker (1997) Tisbe holothuriae Støttrup & Norsker (1997) Tisbe reticulata Battaglia (1970) Tisbe sp.5 g Microfeast L-10® twice per week Tetraselmis suecica. Rhodomonas baltica and two algal pastes 1 adult 31 N C ml 1 Bioballs© T.000 N l day 1 1533 N and 1800 C l 1 day 1 1 Kahan et al. airlift pump 70 litres 115 ind l 25 F cm 2 1 Kahan et al. Tisbe sp. nauplii. 13–21 days Mytilus powder 5–150 mg. copepodites. 20 l day 1 Ulva fragments Dunaliella. Dunaliella tertiolecta. C. Isochrysis galbana tahiti.Tisbe holothuriae Tisbe holothuriae 13–21 days Multiple generations Multiple generations Mytilus powder 5–150 mg. . ind. Tisbe sp. temperature.5 litre baskets in 200 litres 3 litres in flat trays 40 60 cm. salinity. continuous 20 cm3 100. F. females. N. individuals.5 litre baskets in 200 litres Floating sieves 32 litres. Pheodactylum or Nitzschia and boiled wheat grain fragments 21–22°C/ 40 psu 18°C/ 34 psu 18°C/ 34 psu 1. During a 5 month harvest period this system averaged a production of 0. Both copepodites and nauplii were harvested daily and sufficient nauplii would remain in the culture unit to supplement continuously the breeding population. Very successful mass culture of the harpacticoid Amphiascoides atopus was achieved in a continuous system with a relatively small basal surface area (4 m2) and a water volume of 1440 litres (Sun & Fleeger 1995). but may be less important in recirculation systems where the water volume is replaced more than once daily. the units are filled with small balls. whereas the nauplii did not seem to be affected (or were poorer swimmers) and remained in the water column close to the outlet where the harvest took place. This form of harvest was not totally effective. some aeration may be applied to maintain an even distribution of food and zooplankton. Temperature and salinity Most harpacticoids have wide thermal and salinity tolerances.8 ppm after a feeding event (Støttrup & Norsker 1997). reflective of the variable environment they inhabit. or other material. Amphiascoides atopus also possesses a wide thermal and salinity tolerance range and survived salinities from 10 to 60 psu (Sun & Fleeger 1995). Thus. the effect of ammonia levels on fecundity was not examined. which was allowed to function for approximately 1 year (Støttrup & Norsker 1997). which provide surface area for the copepods (Gaudy & Guerin 1978.000 nauplii per litre. Ciliates may also compete for food and be detrimental . A strong light source was thus switched on for a few minutes before the daily harvest.2 to 1. However. Harpacticoid cultures contaminated with rotifers tend to be outcompeted by the rotifers. is dependent on the available surface area rather than culture volume (Uhlig 1981).000 nauplii day 1 was produced from four trays measuring 40 60 cm and filled with around 3 litres of filtered (0.5 million individuals day 1. Støttrup & Norsker 1997.8 psu. The copepodite and adult harpacticoids were negatively phototactic and were observed to swim energetically away from a sudden light source.186 Live Feeds in Marine Aquaculture Light was used to harvest selectively harpacticoid nauplii from an automated continuous culture system. a very effective yet gentle mode of aeration and circulation within the culture system. Contaminants The ammonia concentration in high-density cultures of T. holothuriae varied from 1. In continuous or recirculation systems. Nanton (1997) used an air-lift pump. Coli 2000). Culture tank size and shape The mass culture of the benthic harpacticoid Tisbe spp. The harpacticoid Tigriopus japonicus is very tolerant to salinity changes and can survive a change from 36 to 1. or in batch systems where the whole water volume is filtered and replaced daily or every 2 days. an average of 300.22 m) seawater. In the batch system described by Støttrup and Norsker (1997). Aeration Batch or continuous systems used for cultures of harpacticoids generally lack mechanical aeration. although it is doubtful that this species could be reared at this low salinity (Lee & Hu 1981). This corresponds to a daily volume output of 100. ciliates and rotifers would not survive. and possibly depending on when during development the egg sac is lost. or Apocyclops spp.3. holothuriae (Støttrup & Norsker 1997). Chang and Lei (1993) reported great ease in culturing A. Although the Tisbe sp.Production and Nutritional Value of Copepods 187 to the culture performance. Norsker. but the adults were retained. 1997). Eugerres brasilianus (Alvarez-Lajonchère et al. A description of the manual harvest of harpacticoid nauplii is given by Støttrup and Norsker (1997).3 Cyclopoids Very few cyclopoid species have been reared in the laboratory (Table 5. and is similar to that used by Payne and Rippingale (2000c) for the calanoid G. it was a relatively reliable system.H. harpacticoids are sensitive to a variety of chemicals and are used in bioassays. A private hatchery in Taiwan uses the copepod Apocyclops royi as live feed for grouper larvae (Su et al. Photoperiod As in the two other genera. An automated system. are an ideal supplement to the traditional live feed for striped patao. was used for a continuous culture system for T. using light to concentrate the nauplii. Kahan et al. Excess nauplii can also be stored at 4°C for up to 1 week and used on days when the production output is below the required amount. royi in the laboratory using 600 lux and a 16 L/8 D photoperiod. Although not a very efficient collecting system. 1996). West Indies. embryonic development may be arrested.6). Harpacticoids are relatively tolerant to high stocking densities and can be transported for a period of up to 2–3 days. borneoensis was a suitable replacement for Artemia in rearing Acanthopagrus cuvieri (James & Al-Kars 1986). photoperiod affects egg production in cyclopoids. Ciliate and rotifer free cultures were obtained by exposing fecund females to weak doses ( 1%) of chlorine for a few minutes (N. imparipes. Norsker. (1982) solved the problem of harvesting nauplii for feeding fish by growing the harpacticoids in floating baskets within the fish larval tanks. 5. The egg sacs would separate from the females but remain viable. and would hatch upon being washed and transferred to normal medium. As in the case of calanoids. Harvest. storage and transport Since harpacticoids are not free spawners. Adult copepods. personal communication). and A.H. The egg sacs were laid down at dawn and the nauplii hatched at dusk on the following day. used in the laboratory was robust. Oithona spp. .000 individuals per litre (N. harvest methods for collecting nauplii need to be developed. From the available information. appear to be the best candidates and they are relatively easy to culture over several generations in the laboratory (Chang & Lei 1993). The basket had bottom sieves through which the nauplii could pass. several species may lose their egg sacs during collection.2. since many nauplii would mature within the system and several copepodites were harvested daily. This was demonstrated by Hopcroft and Roff (1996) in four species of cyclopoid copepods sampled from tropical waters around Jamaica. needing little labour. for example to examine toxic substances in sediments (Chandler & Green 1996). Oithona spp. kept cool in blood-transfusion bags (2 litres) at densities of up to 200. personal communication). S. 4400 ind l 1 Harvest 2. ind. temperature.75 106 day 1 Reference James & Al-Khars (1986) Apocyclops dengizicus Apocyclops panamensis Apocyclops royi 120 days 77 days Isochrysis galbana 5 105 cells ml 1 1 20–25°C/ 0. nauplii. C. individuals. Candida sp.6 Cultivation techniques for different cyclopoid species. F. Culture volume/ system 15 m3 Species cultured Apocyclops borneoensis Duration of culture 60 days Food organisms Marine yeast.7 N C F 4–9 days 1 1 Dexter (1993) Lipman (2001) Chang & Lei (1993) Several generations Tetraselmis chuii 105 cells ml T. female. copepodites. . salinity.Table 5. T/S 28°C/20 psu Densities obtained 2300 adults l 1.5–68 psu 30 psu 25°C/30 psu 200 ml 100 litres 400 ml 10. N. 1 Carbon Carbon content in calanoids varies from around 28 to 68%. 5. nitrogen and hydrogen in the adults and offspring (Zhang & Uhlig 1993). being generally lowest in species from low and medium latitude. ranging from 3 to 10% of body dry weight. whereas around 30% of the cyclopoid survived. with modal values of 40–46% dry weight (Båmstedt 1986). carbon values for females are around 40% of body dry weight. In the harpacticoid T. but as for most copepods vigorous aeration should be avoided.Production and Nutritional Value of Copepods 189 Aeration and oxygen Gentle turbulence is important for most cyclopoids and increases food encounter rates (Kiørboe & Saiz 1995). The content of phosphorus rarely exceeds 1% (Båmstedt 1986). mostly associated with lipid content. However. holothuriae. Hydrogen content is low. exposing the culture to low pH (3. Hydrogen content is also less than 10% (Zhang & Uhlig 1993) and C:N ratios vary between 5 and 12. Species from colder regions generally contain higher carbon levels than temperate. range 67–92%) of the copepod wet weight. and higher in those from high latitudes. The energy content ranges from 9 to 31 J mg 1 dry weight. Contamination Cultures of C.6 (Bulkowski et al. 1985). as shown in Oithona sp. When fed an algal diet.4. the variation is due primarily to changes in carbon content (Guérin & Gaudy 1977). Zhang & Uhlig 1993). Water constitutes about 82–84% (modal value. 5. depending on the diet provided. Carbon:Nitrogen (C:N) ratios are generally between 3 and 4. . with lower values in males and higher content in egg sacs (Guérin & Gaudy 1977. Nannochloris sp.4 Biochemical Composition Båmstedt (1986) provides a comprehensive review on the chemical and energy content in pelagic copepods. Harvest Some cyclopoids are sensitive to handling and fecund females may lose their egg sac.. and total organic matter 70–98% or more of the dry weight (Båmstedt 1986). pH The cyclopoid Cyclops vernalis is very tolerant to low pH and can survive for several hours at pH 3.8) for up to 7 h selectively killed off the Daphnia. vernalis could only be maintained in a Daphnia-free environment. this ratio was around 5. (Hopcroft & Roff 1996). 1985). vernalis (Bulkowski et al. subtropical and tropical species (Ikeda 1973). since Daphnia easily outcompetes C. Crowding affected dry weight and contents of carbon. especially in low and medium latitudes. and medium-latitude species have a lipid content of 8–12%. shorter chain fatty alcohols such as 16:0 and 14:0 dominate (Sargent & Falk-Petersen 1988). A similar pattern of lipid content was observed in calanoids. 1990). Lipids are stored during the autumn in the later copepodite stages and adults of many species. 5. Wax esters may make up to 90% of the total lipid in calanoids and are contained in an oil sac. (1992) was 71% and 10% of the dry weight. 1992). and a review of lipid biochemistry in copepods is provided by Sargent and Henderson (1986). the classes of lipid differ in eggs from different species. The major neutral lipid in Calanus sp.3 Protein Protein contents in marine pelagic copepods range from 24 to 82% dry weight and are highest in species from medium latitudes (Båmstedt 1986). whereas that in Euchaeta norvegica eggs is wax esters (Lee et al. 1992). 20:1 and 22:1 fatty acids. These n-3 polyunsaturates may constitute up to 40% of the total fatty acids in the wax esters. In the harpacticoid T. The classes of stored lipid can also change seasonally within a species. Hagen 1988). The protein and lipid content in female T. . Calanoid wax esters are characterised by 20:1n-9 and 22:1n-11 fatty alcohols and high levels of 18:4n-3. utilisation and transformation in marine food chains have received particular attention over several decades. with little difference relating to whether or not they were carrying egg sacs. The lipid stores also serve as a buoyancy aid (Sargent & Henderson 1986. protein content was 71% of the dry weight (Miliou et al. eggs is triacylglycerols (Gatten et al.4. but are rich in n-3 polyunsaturates. The predominant lipids during these stages are structural phospholipids (Sargent & Henderson 1986. Lipid levels are often high in newly hatched nauplii owing to residual lipid stores. but these are soon used and lipid levels fall in the later nauplii and early copepodite stages.4. In the late copepodite stages lipids are accumulated in the form of wax esters or triacylglycerol (Kattner & Krause 1987. respectively. 1974). holothuriae.2 Lipids Lipid content in marine pelagic copepods varies with latitude.and medium-latitude species. which runs parallel to the gut. Herbivorous calanoids were observed to have higher lipid content and higher content of wax esters (Sargent & Henderson 1986). with a decrease towards early copepodite stages and an increase in late copepodite and adult stages (Miliou et al. They are stored in the form of wax esters in Pseudocalanus acuspes or in the form of triacylglycerols in Acartia longiremis (Norrbin et al. Lipid storage. Similarly. Sargent & Falk-Petersen 1988). Hagen 1988). especially in situations of food limitation (Sargent & Henderson 1986). season and food availability. Most of the low. In non-calanoid zooplankton such as euphasiids. These phospholipids have negligible levels of 14:0. which fuel reproduction. 1980). and 8–73% in high-latitude species (Båmstedt 1986). Sargent & Falk-Petersen 1988). with a range of 2–61% in low.190 Live Feeds in Marine Aquaculture 5. Lipid content is therefore within the range normally found in pelagic species occurring at low and medium latitudes (Båmstedt 1986). This author attributes the need to store lipids to maintain fecundity in times of food shortage in high-latitude species as an explanation why these copepods are generally more energy rich than those in low latitudes. holothuriae (Mediterranean) examined by Miliou et al. EPA (20:5n-3) and DHA (22:6n-3) fatty acids (Sargent & Henderson 1986. longicornis. in keeping with the increasing industrial utilisation of chitin and its derivative. 1990). survival and/or rates of normal pigmentation have been documented for several marine fish species fed copepods alone or as a supplement to the traditional diets of rotifers or Artemia nauplii compared with traditional diets alone (Kraul 1983. primarily used in osmotic regulation. There is an increasing interest in chitin production. In many hatcheries. Næss & Lie 1998. chitosan. Thus. McEvoy et al. (1964) found the predominant and possibly the only carotenoid in most species to be astaxanthin or its esters.Production and Nutritional Value of Copepods 191 5. In a more recent study on T. 5.5 Nutritional Value for Fish Larvae Improved growth. 5.4.4. with lutein present at almost four times the level of astaxanthin (Rønnestad et al. 1998). with concentrations ranging from trace levels to 1133 g g 1 wet weight. copepods may be an important source of vitamin C in fish. Fisher et al. Larval nutrition is suggested to be the major factor determining pigmentation patterns. and levels ranging from 201 to 235 g g 1 were reported in nauplii of Acartia clausi and Temora longicornis (Hapette & Poulet 1990). arginine. exoproteases. 5. Nanton & Castell 1999). esterase and phosphodiesterase were high in copepods (adult Eurytemora hirundoides) (Munilla-Moran et al. proline and taurine are quantitatively the most important. Heath & Moore 1997. These carotenoids were not detected in Artemia.7 Chitin Chitin content in marine copepods ranges from 2.4 Free amino acids Free amino acids. 5. generally increase in level with increasing environmental salinity (Båmstedt 1986).6 Carotenoids In a study involving over 80 species of calanoid. amylase. Glycine.4.8 Enzymes Levels of endoproteases. .4.1 to 9. 1998. in particular omnivorous and herbivorous species. lysine. which contained primarily cryptoxanthin/canthaxanthin and an unknown retinoid component.3% dry weight (Båmstedt 1986). malpigmentation of the reared juveniles constitutes a major problem.4. alanine. two carotenoids were evident in high quantities. Vitamin C is known to stimulate reproduction in crustaceans and is suggested to induce reproduction in copepods (Hapette & Poulet 1990). 5.5 Vitamin C Copepods. contain high levels of vitamin C. 1997. contain high levels of DHA and other PUFA. 1998).5–1.2).g. Heterocapsa triquetra or Isochrysis galbana). It is suggested to play an important role in the development of normal pigmentation when provided in . 1997). although the results for the latter species were not statistically significant (Payne et al. Dietary deficiency of DHA was shown to impair vision at low light intensities in juvenile herring. Hippocampus subelongatus. DHA can be synthesised from shorter chain precursors in some marine fish larvae. Nanton & Castell 1998) and EPA:ARA ratio (Bell et al. but by the time these prey are consumed by the fish larvae n-3 PUFA levels may be reduced. Estevez et al. 1995b). 1994). A. Zheng et al. 1997). A minimum of 0. Reitan et al. wild calanoid zooplankton. 1989. Flatfish larvae fed natural or laboratory-reared zooplankton generally exhibit higher rates of normal pigmentation than do larvae fed Artemia nauplii (e. and approximately 2 in the yolks of wild halibut eggs (Parrish et al. compared with rotifers only. but at rates insufficient to meet requirements for their normal growth and survival (Sargent et al. Psetta maxima (Støttrup & Norsker 1997). Sargent et al. Reitan et al. Bell & Sargent 1996). 1994) and retroconversion of DHA to EPA takes place in Artemia nauplii (Navarro et al. either obtained through their phytoplankton diet or accumulated despite low PUFA levels in the diet (see Section 5. Marine copepods. Pagrus auratus.5% (Støttrup & Attramadal 1992) or 2.6. DHA levels in wild copepods can be more than 10 times higher than in enriched Artemia (McEvoy et al. (1983) estimated a minimum n-3 HUFA requirement of 1–3% dry weight for turbot larvae. 1996. in neural development and function. 1. Glaucosoma hebraicum. 1995b. and pink snapper. Growth and survival was higher in seahorses.2..192 Live Feeds in Marine Aquaculture possibly enhanced by suboptimal or stressful rearing conditions (McEvoy et al. McEvoy et al. tonsa nauplii from adults fed Rhodomonas baltica. the proportion of different lipids may be an important factor.2% (Gatesoupe & Le Milinaire 1984). Sargent et al. and in particular to the DHA:EPA ratio in the diet (Bell et al. Sargent et al.0% of the dry weight as n-3 HUFA is required for juvenile marine fish larvae and higher amounts are probably required for the rapidly growing fish larvae (Sargent et al. 1998. 1999. it is possible to boost DHA levels in both Artemia nauplii and rotifers to levels equivalent to zooplankton (Reitan et al. 1994).and 22-day-old turbot larvae and the rate of normal pigmentation at 27 days. 1998. Clupea harengus (Bell et al. McEvoy et al. Rotifer residence time in larval fish tanks is crucial since n-3 HUFA levels decrease with time (Reitan et al. 1995. the principal diet for most marine fish larvae in nature. Using special emulsions. fed copepods compared with Artemia nauplii (Payne & Rippingale 2000b). 1994. Japanese flounder. 1999) and probably in rotifers (Barclay & Zeller 1996). Bell et al. Atlantic halibut). Støttrup et al. A combination of rotifers and copepod nauplii increased growth and survival in larvae of turbot. 1999). dhufish. Næss et al. 1995b). Seikai et al. However. 1998). Le Milinaire et al.2% (Støttrup 1992) did not improve growth in turbot larvae. The ratio of DHA to EPA in calanoids and harpacticoids is generally 2 (Nanton & Castell 1998. Atlantic halibut. EPA and/or arachidonic acid (ARA) in the diet (Castell et al. since n-3 HUFA levels of 1. Tisbe sp. and especially in retinal development and vision (Bell & Tocher 1989. 1995a. The documented improvements in larval growth. DHA is important in maintaining structural and functional integrity in fish cell membranes. 1997. 1994. (1994) found a positive correlation between DHA:EPA ratios in 12. 1987. survival and rates of normal pigmentation are generally attributed to levels of DHA. 2001). and EPAderived eicosanoids are involved in the physiological reaction to stress and it may be that the optimal EPA:ARA ratios found in copepods allow the larval fish to cope better with stressful conditions (McEvoy & Sargent 1998). This metabolic interaction necessitates an optimal EPA:ARA ratio in the diet (Bell et al. where these essential fatty acids are present mainly as neutral lipids (McEvoy et al. 1998). PUFA. This was confirmed by Rønnestad et al. When improvements in husbandry remove these stressful conditions. Nutritional benefits of feeding copepods (in terms of improved larval fish growth. Levels of total polar lipids in wild calanoids were almost twice those in enriched Artemia (McEvoy et al. The assimilation of wax esters is a highly efficient process in fish. Apart from the superior fatty acid composition in copepods compared with Artemia nauplii or rotifers. 1994). and in the latter are independent of the dietary content. (1998). Like DHA. These authors cautioned against adding vitamin A to the enrichment emulsion of Artemia. Næss and Lie (1998) showed that malpigmentation could be avoided by providing copepods for a short period to halibut larvae before reaching 2. halibut larvae contained 50–80% lower vitamin A (retinol and retinal) than those fed zooplankton. 1998). Eicosanoids of n-6 origin are important for the normal function of vital organs such as the kidney. Since it competes metabolically for the same enzyme systems required for ARA-derived eicosanoid production. total polar lipids averaged 46% and 47% of total lipids. gill. . 1989). Levels of ARA in copepods are high ( 1%) in both calanoids and harpacticoids. suggesting that these fish larvae were unable to convert the available carotenoids (canthaxanthin) present in Artemia. the benefits of the optimal EPA:ARA ratio may become less apparent. Thus. survival and frequency of normal pigmentation) seem to lessen with improved knowledge on husbandry and optimal rearing conditions for a particular species. since canthaxanthin is not frequently encountered in the natural environment and possibly may not be easily assimilated by marine fish larvae (McEvoy et al. 1995a). 1993). EPA gives rise to less biologically active eicosanoids than those produced from ARA. intestine and ovaries of marine fish. Long-chain monoenoic fatty acids can be exploited as metabolic fuel (catabolised) in fish. EPA is very important in modulating the production of these highly biologically active eicosanoids.5 mm mytome height. especially during early development – and it is therefore essential in the diet of the fish. In A. 1999). 1998). facilitated by the presence of n-3 PUFA. The difference in carotenoid pigments between copepods and Artemia nauplii was suggested to be a possible explanation for the poor development of normal pigmentation in marine fish larvae.Production and Nutritional Value of Copepods 193 sufficient quantities at particular times during the larval stage (Reitan et al. Varying concentrations of the carotenoid astaxanthin were found in various copepods and it was suggested that its possible value for fish is as a precursor to vitamin A (Fisher et al. After 14 days of feeding on Artemia. larval fish may more easily assimilate DHA and other essential fatty acids in copepods than in Artemia. even after enrichment. even though not all of the dietary wax esters are utilised (Sargent & Henderson 1986). respectively (Støttrup et al. EPA cannot be synthesised by most marine fish – or not at a rate to satisfy demands. 1964). tonsa adults fed different monoalgal diets and their newly hatched nauplii. Polar lipids are more readily digested by larvae and may also facilitate digestion of other lipids in the undeveloped gut of marine fish larvae (Koven et al. Both ARA. copepods contain high amounts of polar lipids (Fraser et al. since excess amounts are toxic to fish larvae. minerals and enzymes. although there is an indication that copepodite stages of Acartia spp. easily assimilated carotenoids. amino acids. 1990). The species composition was not given. Harpacticoid nauplii are suitable prey for fish larvae. Copepods are also an important source of exogenous digestive enzymes and are thought to play an important role in fish larval digestion (Munilla-Moran et al. suggesting that this prey may be more visible. respectively. research and development has been focused on enhancing culture techniques or enrichment methods to improve the availability and nutritional value of rotifers and Artemia nauplii. The high levels of natural antioxidants in copepods protect these PUFA. Marine zooplankton are a valuable source of lipids. methods for the production and harvest of zooplankton in large ponds utilising sewage waste have been developed and the zooplankton used as food for marine fish species (Quin 1993). antioxidants have to be added to enrichment emulsions for both rotifers and Artemia to prevent auto-oxidation. In general. and are nutritious prey for. Nanton & Castell 1999). care should be taken using products derived solely from freshwater zooplankton. attractive or better suited in size than traditional live prey.194 Live Feeds in Marine Aquaculture particularly DHA. are highly prone to auto-oxidation. through the purchase of cysts and subsequent hatching of their nauplii (see Chapter 3). despite their proven superior nutritional value. interest in copepods has been regenerated and the use of . Heath & Moore 1997. With the rapid expansion of the sector and increasing interest in new species and the culture of ornamental species to replace wild fisheries.6 Application in Marine Aquaculture The ready availability of Artemia nauplii. since some freshwater zooplankton species may lack essential fatty acids. Little effort has been directed towards developing an adequate. and an alternative or supplement to Artemia nauplii or rotifers. large-scale simple and reliable culture technique for copepods. Watanabe et al. They could be an inexpensive ingredient to replace expensive fishmeal. and the short generation time and relatively uncomplicated culture of rotifers (see Chapter 2) make these live feeds more accessible for marine hatcheries. van der Meeren 1991. and Moina sp. 1997). was deficient in both DHA and EPA. may be less suitable due to their high escape ability (van der Meeren 1991). Because of the rapid expansion and the increasing demand for reliable large-scale production of live feeds. supplying fish with both PUFA and high levels of natural antioxidants (Sargent et al. 1991) are readily consumed by. They have been reported to predominate within the nauplii fraction of zooplankton samples (van der Meeren 1991. Indeed. However. protein. contained high levels of EPA it was deficient in DHA. in this case rotifers (van der Meeren 1991). cultures of these organisms have been responsible for the rapid progress over recent decades in the marine aquaculture sector. In freshwater systems. requirements arise that cannot be met by conventional species. Thus. The temperature within the systems where the zooplankton is harvested is also important. all stages of copepods are suitable as food. Jinadasa et al. marine fish larvae (Kraul 1983. 5. A strong selection for copepod nauplii was observed in turbot. (1978) showed that although Daphnia sp. essential fatty acids. In contrast. since PUFA levels in the phospholipids in freshwater copepods increase with decreasing temperature (Farkas 1979). For example.M. temperature. A. Soc. Biol. S. 29. the production of resting or diapause eggs for sale on a commercial scale. Ban. Oxford. G. . Soc.B. J. Payne et al. O’Hara). 1991) have further encouraged the search for alternative live prey and the renewed interest in using copepod species. & Torres Gómes. U.) (Osteichthyes. World Aquacult. Bull. Jpn. O. Tisbe furcata.S. Barclay. T. 46. Pérez Sánchez. 215–220. Idrobiol. Institut National de la Recherche Agronomique. Sparidae).K. the future expansion of marine aquaculture may further encourage work on copepods towards the development of reliable production systems or.S. pp. (1986) Chemical composition and energy content. fecundity. the Danish hatchery Maximus A/S produces around half a million turbot juveniles each year in extensive systems based on copepods with supplement of Artemia to cover the energetic demands of the rapidly growing late larval stages. Båmstedt. Paris. G. Ban. (1996) Mass production of striped patao Eugerres brasilianus juvenile in Cuba. 74. In: The Biological Chemistry of Marine Copepods (Ed. 109–118. 314–322. Hydrobiologia. T. (1974) Riproduzione artificiale de orata. (1992) Effects of photoperiod. growth rates and culture of the harpacticoid copepod. 27. but which may be expanded to year-round production in regions with more favourable climatic conditions and for rearing ornamental fish or fish with a very small mouth size. 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Further developments in classification are expected in the near future related to the implementation of molecular techniques to distinguish between species.1).1 Introduction Ocean phytoplankton. This chapter considers the biological features of microalgae most commonly used in hatcheries as well as their biochemical composition. . with a production of several hundred billion tonnes of dry weight per year (Pauly & Christensen 1995). contributing to the production of some 100 million tonnes of renewable resources per year from fishing.2 Biology of Microalgae 6. paying special attention to those compounds beneficial to marine animal nutrition. microalgae do not require a rigid skeleton for countering the force of gravity in the manner of higher plants. it is hardly surprising that the microalgae composing phytoplankton play a crucial nutritional role in marine animal aquaculture.2. some classes. shrimp and fish.Chapter 6 The Microalgae of Aquaculture Arnaud Muller-Feuga. Nevertheless. microalgal production systems are described and compared with those implemented in hatcheries. such as peridinians and diatoms. Finally. forms the base of the aquatic food chain. a provisional structure based essentially on cytomorphological analysis is presented for this polyphyletic part of the plant kingdom (Fig. Although class membership and interrelationships are still heavily debated. in the case of marine fish larvae. Several tens of thousands of species are ranked in 15 major classes.1 General characteristics of microalgae Because of their small size and aquatic existence. each exactly half the size of the mother cell. metabolism is directed towards the synthesis of carbohydrate compounds not requiring nitrogen (polymers such as starch and polysaccharides) and of valuable lipids such as carotenoids . when unavailability of nitrogen prevents algae from synthesising the structural proteins required for growth and division. They probably exhibit a greater variety of these products than terrestrial plants that have developed from only one of the two lines of the algal evolutionary ‘tree’. although gamete-like forms have been described for certain species. Thus. and some species show great resistance to dryness. etc. low light. under certain conditions.The Microalgae of Aquaculture 207 Higher plants Green algal line Chlorophyceae Charophyceae Prasinophyceae Euglenophyceae Cyanobacteria Prokaryotes Rhodophyceae Cryptophyceae Eukaryotes Dinophyceae Eustigmatophyceae Brown algal line Xanthophyceae Chloromonadophyceae Thraustochytriidae Prymnesiophyceae Phaeophyceae Chrysophyceae Bacillariophyceae Fig. Microalgal metabolism involves numerous biochemical compounds of interest in nutrition. some algae can utilise organic substrates. high salinity. Microalgae have colonised a great variety of environments. 6. from hot-water springs to polar ices. As for most micro-organisms. The daughter cells sometimes remain within the cell wall of the mother cell until two or three divisions have occurred. Their metabolic plasticity allows species adaptation to a wide range of environmental conditions. reproduction is primarily asexual. For example. However.1 Provisional phyletic relationships between algal classes. the metabolism of microalgae is directed towards cell division and increase in size. with an indication of those of importance to aquaculture (shaded boxes). Vegetative reproduction consists of the equal division of the mother cell into two daughter cells. cosmetics and the pharmaceutical industry. in a manner similar to bacteria and fungi. using light as the sole energy source. Excretion of high molecular weight carbohydrates and their inhibitory action upon copepod growth has been reported by Malej and Harris (1993). PS LPF BM. food for bivalve mollusc larvae. especially their ability to synthesise polyunsaturated fatty acids (PUFA) beneficial for animal nutrition. chuii virginica salina. PS BM. Main utilisation LPF BM. PS.1 Classes. Although toxic metabolites of dinoflagellates are frequently harmful for marine life. As indicated below. PS BM LPF. salina atomus suecica. However. only a handful is widely used today. food for live preys of fish larvae. virginica. Microalgae have also developed anti-radical mechanisms against activated oxygen species. The first microalgae species used in aquaculture have been selected from those that developed naturally in the marine environment of pioneering aquaculture farms and were probably the easiest ones to cultivate. and their main utilisation (synonymous names are in parentheses). maxima costatum. Thraustochytriidae are mentioned here because they are considered as heterokont algae by some authors Table 6. The metabolic plasticity of microalgae has been extensively studied. PS BM. Some species develop allelopathic activities and should be more suitable for high-density production. grossii tertiolecta. PS BM BM. PS BM LPF LPF. salina cohnii sp. food for penaeid shrimp larvae. all of these features impact to some extent on the nutrition of marine animals.1 summarises the 16 genera of microalgae most commonly grown for aquacultural purposes. with plastidial membranes separating their organelles. aff. a representative of this class has recently been proposed as potentially useful in aquaculture. Like other micro-organisms. . baltica. gracilis. galbana ‘Tahiti’ (T-iso) lutheri. genera and species of major currently named microalgae grown for food in aquaculture. Class Cyanophyceae (blue–green algae) Bacillariophyceae (diatoms) Genus Arthrospira (Spirulina) Skeletonema Phaeodactylum Chaetoceros Thalassiosira Chlorella Dunaliella Nannochloris Tetraselmis (Platymonas) Pyramimonas Rhodomonas Nannochloropsis Isochrysis Pavlova (Monochrysis) Crypthecodinium Schizochytrium Species platensis. PS BM. Subsequently. Among the numerous species tested. BM. but only chlorophyll a instead of a and b (with the exception of eustigmatophytes). calcitrans. Those prokaryotes have photochemical systems I and II as in eukaryotes. other collected species were investigated and the most nutritionally efficient were retained. striata.208 Live Feeds in Marine Aquaculture and fatty acids (Sukenik & Wahnon 1991). PS LPF LPF Chlorophyceae (green algae) Prasinophyceae (scaled green algae) Cryptophyceae Eustigmatophyceae Prymnesiophyceae (haptophyceae) Dinophyceae (dinoflagellates) Thraustochytriidae LPF. they show a capacity to resist competition and predation by synthesising bioactive substances. pseudocostatum tricornutum. Dunaliella tertiolecta and Pyramimonas grossii were clearly found to exhibit allelopathy (Smith 1994). BM. Table 6. reticulata oculata galbana. Most of the major microalgae classes are represented in this list. pumilum pseudonana minutissima. The majority are eukaryotic. cyanobacteria of the genus Arthrospira are also included because of the numerous examples of their utilisation as food in aquaculture for both larviculture and ongrowing. 24 day 1 (2. 1991). 6. Lewis et al. an average value of 1 day 1 can be maintained for most species. vessel shape and volume.g. so that counting is difficult and cell weight is generally not determinable. corresponding to a doubling time of about 17 h.7 h doubling time). Most of the sites providing information on microalgae are listed by the Phycological Society of America (www.org). concentration. Depending on salinity. which results from both the increase in size and the division of the cells composing it. Spirulina versus Arthrospira (Guglielmi et al. there are some discussions about the classification of some genera. while day 1 is more suitable for photoautotrophs. Smith. where C0 is the initial concentration at time t0. This section considers the practical aspects of the biology of microalgal species. where X is the algal biomass concentration and t the time. new classes have been proposed for almost all genera in Table 6. 1986). For example. In rare cases. the growth rates of S. All are pelagic and in the nannoplankton range (2–20 m). 2000). and C the final concentration at time t. Chrétiennot-Dinet et al.2.1. Thus. which is a common feature of microalgae in photoautotrophy. As shown later. Porter 1990). Nitzschia closterium (Ehrenberg) Wm. Results are very heterogeneous.g. mixing) and on the moment of measurement. However. Chlorella versus Nannochloropsis (Gladu et al. forma minutissima versus Phaeodactylum tricornutum (Wilson 1946) and Skeletonema costatum versus S. and of some species.g. Those with benthic behaviour have been excluded because they tend to settle on vessel walls and make cleaning difficult. they depend on various culture conditions (e.g. defined as dX/(Xdt). costatum vary between 0. which makes it difficult to characterise any particular species.2 Growth The growth of a population of micro-organisms. with the relation (day 1) 24 (h 1).84 and 2. . e. As no standard is available to define the conditions of measurement of the growth.88 day 1 at 135 Einstein m 2 s 1 (Curl & McLeod 1961). The growth rate is generally expressed in h 1 for heterotrophs. Gaertner 1977. for example the heterotrophs Crypthecodinium and Schizochytrium and some species of Chlorella. 1993).g. They have also been classified as fungi (e. 1994. 1995). Some web sites provide extensive information on algae and can supply strains (see Appendix IV). The microalgae of aquaculture have been well described and documented (e. Barclay et al. Microalgae species selected for aquaculture are generally free-living (Table 6. A more practical expression of over a period of time (t t0) results from integration: [log(C) log(C0)]/(t t0). especially those relevant to aquaculture.psaalgae.g. the specific growth rate can reach values as high as 6. 2000). with the progress in molecular taxonomy. is currently indicated by the specific growth rate .The Microalgae of Aquaculture 209 (e. forming chains of cells. light and nutrient concentrations. pseudocostatum (Medlin et al. More recently. e. the size and appearance of microalgae can vary markedly (Hiramatsu et al. Both diatoms and cyanobacteria cells often remain associated after division. even for a given species and a given irradiance. 1999). especially within Chlorophyceae (Marin & Melkonian 1999) and Prymnesiophyceae (Edvardsen et al. the data should be considered with due caution. The doubling time of the biomass of a population is also used to characterise growth in microorganisms and can be expressed as log(2)/ .2). Table 6.3 summarises some specific growth rate values obtained for the microalgae species most often used in aquaculture. no cell wall I. calcitrans forma pumilum Chlorella Smaller than C.6 3–5 2–4 3–6 7–9 3–6 23–47 (owb) 31 41–83 15–19 30–44 10 5–7 3–4 30–40 (owb) 102 57 (log phase) Pavlova lutheri . yellow to golden brown. Brown et al. four long diagonal setae Length width ( m) Cell weight (pg) References Vonshak et al.4 4. brown.5–10 3–7 (vulgaris) 1–11 10–30 30 5–6 2–4 2.5–3 11 31–61 8–22 (owb) C. no cell wall 6. (1993b) Gladue & Maxey (1994) Chrétiennot-Dinet et al. ovoid. (1986) Brown et al. incomplete cingulum. short haptonema with two unequal flagella.2 Features. forming cyst Free-livinga. size and weight of the microalgae currently used in aquaculture. mobile. mobile. affinis galbana (T-iso) Nannochloropsis oculata Identical to I. Genera and species Arthrospira Chaetoceros calcitrans Features Cylindrical cells forming helicoidal trichomes Free-livinga. (1986) Fernandez-Reiriz et al. (1982) Borowitzka & Borowitzka (1988) Chrétiennot-Dinet et al. galbana Free-livinga or aggregated. 2–16 autospores a Crypthecodinium Isochrysis galbana Free-livinga. cell wall. (1993b) 50–300 10 (trichome) 1–12 (cell) 5–16 – 3–8 3–7 1. no chlorophyll b Free-livinga. mobile. (1989) Brown (1991) Fidalgo et al. short subapical haptonema with two flagella. (1998) Wikfors et al. (1986) Fernandez-Reiriz et al. cell wall. (1986) Fernandez-Reiriz et al. (1997) Sournia (1986) Gladue & Maxey (1994) Chrétiennot-Dinet et al. green. (1998) Chrétiennot-Dinet et al. proportionally shorter setae Free-living .Table 6. spherical. gold–brown. (1989) Chrétiennot-Dinet (1990) Gladue & Maxey (1994) Hirata et al. ovoid. spherical or elliptic. calcitrans. (1986) Brown (1991) Lopez-Elias & Voltolina (1993) Chrétiennot-Dinet et al. (1996) Brown (1991). (1989) Brown (1991) Brown et al. mobile. oval.3 3–10 10 5 10 4 9–11 7–8 100 (reticulata) 79 Schizochytrium sp. Total weight when not specified. . (1986) Brown (1991) Cognie & Barille (1999) Renaud et al. (1989) Gladue & Maxey (1994) Wikfors et al. (1986) Fernandez-Reiriz et al.5 5. Heterokont biflagellate spherical to ovoid zoospores Chained. mobile. single valve 3–4 8 to 25–35 5–10 (owb) 77 81–140 (owb) Rhodomonas Free-livinga. spindle-shaped cells. (1986) Fernandez-Reiriz et al. (1989) Brown (1991) Fernandez-Reiriz et al. four polar flagella. (1999) Azevedo & Coral (1997) Azevedo & Coral (1997) Chrétiennot-Dinet et al. (1997) Laing & Psimopoulous (1998) Renaud et al. two subapical flagella 10 5–12 (salina) 13 8 4. (1999) Chrétiennot-Dinet et al. (1996) Laing & Psimopoulous (1998) a b Free-living: not colonial and not benthic cells. organic weight.Phaeodactylum tricornutum Chained. spherical to cylindrical cells attached together by a ring of external gutter-shaped processes 52 18 4–6 160–227 (owb) Tetraselmis Free-livinga. ow. (1996) Wikfors et al. Y or spindle-shaped. (1989) Chrétiennot-Dinet (1990) Brown et al. cyst form 7–22 10–12 8–10 (chui) 9–11 (striata) 104–135 (chuii) 64–158 (striata) 200 (suecica) Chrétiennot-Dinet et al. Skeletonema costatum Thallum attached to substrates by rhizoidal network. cell wall. 165. energy (in Einstein m 2 s 1. (1998) Droop (1974) Goldman (1979) Hiramatsu et al. 25°. (1999) Chlorella Crypthecodinium Isochrysis Nannochloropsis Pavlova Phaeodactylum Rhodomonas Schizochytrium Skeletonema Tetraselmis a Principally refers to species. 18°. depending on whether or not the illuminated reaction medium is renewed after inoculation. 18°. the reaction medium is renewed and an equivalent flow Q of culture is harvested. Figure 6. B. 20°. (2000) Ben-Amotz & Gilboa (1980) Sigaud & Aidar (1993) Brown et al. 25 g l−1 sp. light regimen (light/dark hours day 1). (1998) Renaud et al.74–1.88 2. by far the more frequent. 12/12. 100. A culture can also be run continuously.3 0. 12/12. (1994) Lau et al. B T-iso. B suecica.. various nitrogen sources. 60. (1999) Barclay et al. B galbana. 20°. heterotrophy. continuous or semi-continuous culture). 18°. B T-iso.0 1.84–2. batch. The first situation. (1991) Gonzalez-Chabarri et al. B lutheri.. C lutheri. 12/12. 80. B tricornutum.5–80. 24/0.. C. 130. The production P of biomass in this case is P (C C0)V/(t t0). 165.56 0. Specific growth rate (day 1) 2. 25 g l−1 References Zarrouk (1966) Aiba & Ogawa (1977) Fernandez-Reiriz et al. 15°. C. 332.9–1.47 2. (1999) Ryu & Tokuda (1984) Ryu & Tokuda (1984) Toro (1989) Toro (1989) Brown et al. B pyrenoidosa.2 presents a typical kinetic of growth for batchcultured microalgae. heterotrophy.98 1. axenic.03 0. 70–80. 24/0. temperature (°C). 20°.47 1. heterotrophy.36–0. B sp. 20°. N/P 2. 20°.4 0. (1988) Blanchemain (1993) Molina-Grima et al.. if unit is not specified). B. (1994a) Brown et al. 24/0. B galbana.41 Genus Arthrospira Chaetoceros Culture conditionsa maxima. 12/12.30 6.6 0. 24/0. B salina.37 1.75 1. B sp. 22°. 18°. C calcitrans. 550. (1994) Curl & McLeod (1961) Hitchcock (1980) Mortain-Bertrand et al. 273. (1998) Brown et al. open condition.5 1. 332. 20°. B lutheri. 23°. 100.3 Growth rates of the microalgae species commonly used in aquaculture. Two situations are considered.. The equation that simulates this situation . B vulgaris. 25°.25 1. 60. In this case.28 0.92 0. 18°. 12/12. 20°. Biomass production is the major criterion for assessing cultures of microalgae.16 4.4 0. B sp. Its calculation depends on the manner in which the culture is operated.2 2. (1992) Molina-Grima et al. 360. it is called productivity. 75. B T-iso.2 0. 35 g l−1 sp. B tricornutum. 36. B. 35 g l−1 gracilis. (1989) Toro (1989) Toro (1989) Mayo & Noike (1994) Lau et al. 40 W fluorescent. C.11 0. 35°.14 1. 25 g l−1 sp. B platensis. 33°. When related to the surface of illumination (g m 2 day 1) or to the volume of culture (g l 1 day 1).75 1. through strain selection. B costatum.. 15–23°.37 0. (1994) De Swaaf et al.48 0. 80. 12/12. B sp. 20°. salinity.35 1. 24/0. heterotrophy. The production P expresses the increase of biomass per unit of time. B vulgaris.68 0. B costatum. where V is the volume of culture produced.68 0. B cohnii.212 Live Feeds in Marine Aquaculture Table 6. is called batch culture and consists of no media renewal other than supplying air enriched with carbon dioxide and illumination during cultivation. where five phases can be identified. 24/0. 12/12.49 1. B vulgaris. 25°. (1994) Running et al. 135. B gracilis.87 1. B costatum. 24/0.. 24/0. (1998) Renaud et al. 24/0. 16°. 12/12. 24/0. B costatum. renewal status (B.24 0. 120. 25°. 1 Light Light. which are examined below. In addition to the production of biomass. plays a fundamental role in the development of cultivated microalgae. expresses that the variation of concentration in the reactor is the difference between the growth of the biomass and the population reduction following dilution/harvesting: dC/dt ( D) C. 6. deposits on the light wall and contaminations may result in a drop in production. It is highly variable in intensity and distribution. The low concentration and abundant nutriments result in no limitation ( max). like that of all plants. 3: limitation phase: exponential growth could not be sustained because of light and substrate restrictions. Three conditions may develop: • • • D : Growth cannot compensate for dilution and the concentration C diminishes. Growth of algae. D : The concentration stabilises around a mean value C. D : The culture has not reached its steady state and concentration still increases.2. is governed by environmental factors. 1: Lag or acclimation phase.The Microalgae of Aquaculture 213 dB/dt = k2 Biomass of the population 4 5 3 dB/dt = k1 B 2 1 Time Fig. The longest runs reported in the literature have lasted for several months. mixing and toxins. through photosynthesis. where D is the dilution rate Q/V. and other medium factors such as salinity.2 Typical growth kinetic of microalgae cultivated in a batch.2. the production of biomass per unit of time p is the product of the harvesting flow rate DV by the concentration C ( p DVC). nutrients (involved in the organic growth). the culture washes out. and both aspects have to be considered for photosynthesis. temperature (affecting biochemical reaction velocity). 6. 5: senescence phase: death and lysis of cells. its biochemical composition is affected by these factors. In a stabilised continuous culture ( D).3. . 4: plateau phase: cessation of growth. 2: exponential phase: cells divide actively. and phases. among which the most influential are the source of energy (light or organic substrates). pH. Although a continuous culture could theoretically last indefinitely.3 Substrates of photoautotrophy 6. In the first phase. for cool white fluorescent tubes. In the second case. Hence. Some units based on light perception by the human eye are also used to quantify light. which propagates in a straight line in a homogeneous medium. The protons accumulate in the thylakoid lumen of the chloroplast. where h is the Plank constant. The electrons accumulate as reduced nucleotides (NADPH) in the chloroplasts. Light of different wavelengths is utilised differently by the photosynthetic apparatus of a plant. it is characterised by its frequency and its energy ε which are related by ε h . and glycogenesis is reported to be favoured by red light (SanchezSaavedra & Voltolina 1996). In the first case. but human eye sensibility is centred on yellow light (550 nm). The reactions of biosynthesis occur according to two successive phases. which is one of the highest values encountered in the entire plant kingdom. and expressed in Einstein m 2 s 1 (or mol-quanta m 2 s 1. As a consequence. the maximum practical energy conversion efficiency for outdoor continuously mixed cultures is near 5% of total solar energy. They condition the biomass production and the overall biochemical content of the cells. whereas plants utilise blue light (440 nm) and the yellow to red range of wavelengths (620–680 nm). or light irradiance. the photosynthetic response in terms of accumulation of organic matter by a given plant will vary according to both the light spectrum and intensity of the source. The ATP and NADPH generated are then used to produce reduced organic molecules supplying chemical energy for further biosynthesis. Light is then quantified through its photon-flux density. giving readings in either lux or lumen m 2. resulting in pH gradients of up to 3 units. only about 45% (Kirk 1986) of the energy is useful for plants (found in the PAR spectrum). Light irradiance can be measured by means of several types of quantum meter. each of them under the control of photosystems I and II located in the membrane of the chloroplast. For example. the ‘light reaction’. especially of glucids. . the photons are absorbed by the chlorophyll. Protein level is thought to increase more with blue than with white light. or mol-photons m 2 s 1). The PAR corresponds roughly to the visible range of the light. This makes it necessary to consider the nature of the light source in order to convert lux to Einstein m 2 s 1. the physical energy content of ‘blue’ photons (440 nm) is higher than that of ‘red’ ones (650 nm). The behaviour of light can be simulated either as an electromagnetic wave or as particles. Taking sunlight as an example. so that only a fraction of the light is transformed into chemical energy. Light sources specialised for plant production generally produce reinforced reddish radiation. which is activated and then returns to its previous state while producing energy.214 Live Feeds in Marine Aquaculture Light is an energy flux. The wavelength c/ (where c is the light celerity) is more commonly used to characterise the distribution of the radiations (spectrum) of a light. According to Oswald (1978). the remainder being either ultraviolet (shorter than 400 nm) or infrared (longer than 700 nm). O2 and electrons. The PAR energy is dissipated in the culture. The photosynthetically active radiation (PAR) is found between 400 and 700 nm. and the nature of its photochemical apparatus. while green radiation is minimised. which is used to split water molecules into H . The proton gradient is used to produce directly usable energy under the form of adenosine triphosphate (ATP). in the cell and in the chloroplasts as fluorescence and heat. the particles composing light are called quanta or photons. 1 Einstein m 2 s 1 corresponds to 74 lux (Thimijan & Royal 1982). some of which present the PAR directly in Einstein m 2 s 1. 6. the curves of variation of the oxygen evolution rate with respect to chlororophyll a content.0 0. an example of which is given in Fig. especially the initial slope. 311–321.) . Other works were devoted to the influence of irradiance on the specific growth rate . The simplifying assumption that growth is nil at dark was frequently made.. Several fitting parameters were determined. and may even diminish at higher irradiance.5 and 1.5 0.g. with permission from Elsevier Science. Isochrysis galbana and Thalassiosira weisflogii. chlorophyll a is observed to increase almost linearly with increasing irradiance until a maximum plateau is reached. though a concept familiar to oceanographers. Specific growth rate (day 1) 2. and the use of polarographic Clark electrodes for the study of the rate of photosynthetic oxygen production. As photosynthesis/irradiance and growth/irradiance curves showed similar shapes. is not available for most aquaculture species. spectrum and regimen of delivery. and in several microalgae species by plant physiologists.3.0 0 P. Limnol. During photosynthesis in microalgae. galbana T. (b) maximum growth rate . The results showed variations in relation to time. saturation light energy Is is generally in the 300–600 Einstein m 2 s 1 range for most species. This became possible thanks to two recent techniques: the use of radioactive isotopes of carbon for measuring the rate of organic build-up of the cells (primary production).4) do not simultaneously take into consideration the three key criteria of consistency of this energy transformation (Muller-Feuga 1999): (a) loss of weight with respiration in the dark.. also called photoinhibition. K. and (c) maximum yield /I.3 Growth versus irradiance of three species of microalgae at 18°C: Prorocentrum micans. were fitted to mathematical equations. and revealed a highly unstable and adaptive capacity (e.The Microalgae of Aquaculture 215 To determine the influence of intensity.G. & Wyman. Macedo et al. P. However. the relation between photosynthesis and irradiance has been studied in wild phytoplankton by oceanographers. In an attempt to describe the influence of light on photosynthesis. 30. also called P/I curves. 1998). the maximum production and the optimal irradiance corresponding to it. The parameter values necessary to adjust these models are not yet available for all the microalgae of aquaculture. Oceanogr. micans 200 400 600 Irradiance (µEinstein m 2 s 1) I. weisflogii Fig. they were often fitted using the same equations.3 for three microalgae. Dubinsky. (1985) Growth irradiance relationship in phytoplankton. Copyright 1985.5 day 1. Most of those proposed in the literature (Table 6. As indicated in Table 6. 1993). (Reprinted from Falkowski. species and the physiological state of the cells. This loss of efficiency with high energies. 6. Compensation energy Ic.5 1. Z. has been attributed to an overdestruction rate of the high-turnover D1 protein of photosystem II (Aro et al. the highest values of the specific growth rate ( s) are between 0.0 1. the number of parameters required for their fitting. Exp. 1–13. Continuous illumination of indoor cultures was preferred for practical reasons. The gradual extinction of light penetrating into the depth of the culture. I energy. J. (1987) Muller-Feuga (1999) si n ik n in 3 4 2 3 Same model with translatory motion along y-axis i Ki K 2 i 2 2 s (1 ic )(i ic ) (1 ic )2 (i ic )2 The notations are homogenised to facilitate the comparison: s growth rate at saturation. with permission from Elsevier Science. for Chaetoceros gracilis and Isochrysis affinis galbana ‘Tahiti’ (T-iso) cultured in batch and after 14 days (Table 6. Toro (1989) observed no significant differences in growth rate and final concentrations between 165 Einstein m 2 s 1 under continuous illumination and 332 Einstein m 2 s 1 under 12/12 h L/D cycles. The question then arises of the necessity of simulating such cycles in controlled cultures. Growth as a function of rationing: a model applicable to fish and microalgae. ic normalised compensation energy Ic/Is.4 Major models of energy transformation available in literature. and their consistency. (1994b) Lee et al. (1953) Droop (1983) Kiefer & Mitchel (1983) Haldane (1930) Aiba (1982) Model 1 I 2 3 I2 K1 2 2 ) si Same model with translatory motion along y-axis sI s I Tessier (1942) van Oorschot (1955) Steele (1977) Peeters & Eilers (1978) Bannister (1979) 2 s(1 e i) i) si e(1 s (1 i2 i 2 i 1 0 3 4 n ( K1 1 in ) n Moser (1985) Molina Grima et al. i normalised energy I/Is. 236. Ecol. Biol.. Mar. Consistency Number of Weight loss Maximum Maximum parameters at dark growth yield sI s References Michaelis & Menten (1913) Monod (1942) Tamiya et al.) In nature. Copyright 1999.3). (Reprinted from Muller-Fenga (1999). sunlight is delivered according to nycthemeral light/dark (L/D) cycles varying with location and season. and this has proved suitable for most species cultured. creates a light gradient which makes the relationship between incident energy and growth much more complicated than for a suspension with low cell concentra- .216 Live Feeds in Marine Aquaculture Table 6. Is saturation energy. also called the self-shading effect. Heterotrophic cultivation was assessed with Chlorella in Asia in the early 1980s and later on with Tetraselmis (Day & Stavalos 1996). as hatcheries generally cultivate more than one species. Diatoms also require silicon.3. Cid et al. Microalgal organic matter contains about 50% carbon and 10% nitrogen. some amino acids and some carboxylic acids in different concentrations. However. Other nutrients are supplied in the liquid culture medium according to different recipes. this law expresses that the relative variation in intensity dI/I along the path length h of an absorbing medium is inversely proportional to the distance dh and the concentration C. whereas lipid content changed little. For example. In intensive cultures. This simplifies their culture to some extent. 6. comparing five media. Conway (Walne 1966).4. AM (Fabregas et al. such as glucose. Some authors (Gladue & Maxey 1994) consider that this is the main disadvantage of photoautotrophic microalgae production. the major nutrients for microalgae are carbon. These aspects will be considered in more detail in Section 6. very large amounts of water must be handled. (2000) showed that they greatly influence the fatty acid composition and the protein content of Isochrysis galbana. f/2 and Conway media provide satisfactory growth for most of the species of microalgae used in aquaculture. Because of the low concentrations attained. the increasing cell concentration diminishes the amount of energy I available for photosynthesis according to the Beer–Lambert law. concluding that Ukeles medium modified by Fabregas and Herrero (1985) provides the best compromise between optimising the biochemical profiles and the growth kinetics for this species. As indicated in Table 6. can accelerate algal growth. the main ones being Watanabe (Watanabe 1960). 1984) and Knop (Bajguz & Czerpak 1996). f series (Guillard & Ryther 1962). After 7 days. resulting in increasing requirements for space. Among the 10 genera investigated.03%. gracilis and Pavlova lutheri cultured in light increased perceptibly after the addition of 30 g l 1 of citric acid (Ohgai et al. Zarrouk (Zarrouk 1966). labour and energy. carbon dioxide is generally mixed with the air injected at the bottom of vessels. (1992) showed that the supply of both light and organic substrates could affect the biochemical composition in comparison to a control without any organic compound.4 Substrates of heterotrophy Some microalgae have the ability to grow in the dark.The Microalgae of Aquaculture 217 tion. This law is strictly valid for monochromatic lights as the coefficient of proportionality (sometimes called the coefficient of extinction) depends on the wavelength. However. the cell concentrations of C. carbon is available as carbon dioxide present in the atmosphere at a concentration of 0. in intensive cultures of microalgae.2. Written I I0 e C h. 1993).5. the composition of the culture media determines to a large extent the biochemical composition of the biomass. using organic substrates in place of light energy. depending on the species cultured. nitrogen and phosphorus in mineral form. Sánchez et al. The addition of a small range of reduced organic compounds. all other factors remaining constant. Working on Tetraselmis suecica. In nature. Kuhl (Wong 1977). Self-shading is generally the limiting growth factor in intensive culture. and highly variable quality. with much higher carbohydrates and protein content.2 Mineral nutrients As for all plants.2. . 6. (1999) Si 20 0.5 Other factors affecting growth 6. volume of gas per volume of culture and per hour. affinis galbana Tahiti (T-iso) Nannochloropsis oculata Pavlova lutheri f/2 f/2 ES Conway f/2. Gladue and Maxey (1994) showed that Cyclotella. (2000) Turner (1979) Fernandez-Reiriz et al. with respect to the . (1984) Mandalam & Palsson (1998) Sung et al. the data should be considered with due caution. Air supply CO2 (v/v/ha) (% in air) Species Arthrospira Chaetoceros Liquid medium Zarrouk Conway f/2 Si f/8 Si Knop Vitamin Autotrophic Autotrophic References Vonshak (1997) Walne (1966) Brown et al.8 h (instead of 16–24 h in photoautotrophy) and maximum cell densities can reach 40–100 g l 1. (1998) Albentosa et al. doubling times are within 3.6 summarises the growth temperatures of species used in aquaculture. (1998) Renaud et al.5 Chlorella Thiamine AM M4N Watanabe.5 0. Thus. (1998) Bonin et al. (1999) Fábregas et al. (1999) Turner (1979) Brown et al.5–1. (1993b) Burkhardt et al.1 Temperature Raising the temperature can increase microalgal growth up to an optimum point. thiamine Conway f/2 f/2 Si f/2 f/2 Si AM f/2 Conway B12 B12 B12 17 0. h/2 B12. However. As no standard is available to define minimum. (1993b) Soudant et al. (1998) Wong (1977) Turner (1979) Fábregas et al. (1986) Fernandez-Reiriz et al. (1989) Brown et al.5 Phaeodactylum tricornutum Rhodomonas Skeletonema costatum Tetraselmis a v/v/h. (1998) Bonin et al. (1989) Sánchez et al. (2000) Brown et al. maximum and optimum temperatures. (1984) Brown et al. Tetraselmis and Nitzschia have moderate to rapid growth in heterotrophy and are of potential interest for aquaculture. (1999) Brown et al.5 Autotrophic 17 0. Table 6. Ochromonas. (1993a) Price et al.218 Live Feeds in Marine Aquaculture Table 6. When the latter condition is well accepted. f/2 Bristol Isochrysis galbana I. 6. (1986) McCausland et al.5.5 Thiamine 17 0. whereas Nannochloropsis exhibit slow growth.5 Thiamine B12 Autotrophic 150 60 17 10 0. the choice of species and genera will probably differ depending on whether growth is by photoautotrophy or heterotrophy.5–4. (1999) Illman et al.5 Liquid and gaseous substrates supply conditions for the photoautotrophic cultivation of the main species of microalgae used in aquaculture.2.2. after which it is reduced. 25°C on average is suitable for nearly all the species except for Arthrospira. However. Chaetoceros sp. For example. Temperature (°C) Species Arthrospira sp. (1999) vws Fanuko (1981) Sigaud & Aidar (1993) vws Blanchemain (1993) Lopez-Munoz et al. Table 6.5.2. salinity should generally be reduced to 25–27 g l 1 for improved growth. Adaptative mechanisms producing variations in the inner concentration of small molecules such as glycerol and sorbitol compensate for ambient osmotic pressure. Although the use of seawater can be advantageous for medium preparation in intensive culture. Skeletonema costatum Tetraselmis sp. S. who studied . the cell densities in photoautotrophic cultures remain small compared with those of fermentation. vacuolata Isochrysis galbana I. However. Richmond and Zou (1999) removed the inhibitory activity of high-density suspensions by replacing the culture medium after separation.3 Metabolites As for most micro-organisms. sorokiniana C. In an attempt to determine the optimum cell density of outdoor Nannochloropsis cultures.The Microalgae of Aquaculture 219 Table 6. 6.2 Salinity Most of the microalgae used in aquaculture are euryhaline. affinis galbana ‘Tahiti’ (T-iso) Nannochloropsis oculata Pavlova lutheri Phaeodactylum tricornutum Rhodomonas sp.2. where it develops the characteristic reddish colour of -carotene.6 Temperature tolerance of the main species of microalgae used in aquaculture. C. costatum and Tetraselmis. microalgae development is generally accompanied by the excretion of metabolites that accumulate in the reaction medium and could become inhibitory for growth. and such inhibitions seldom occur. fusca var.7 shows that preferred salinity for most species is below that of seawater. vws. 6. The concentration threshold at which inhibitory activity began to reduce the growth was in the range of 6–7 g dry weight l 1. Chlorella sp. According to Javanmardian and Palsson (1991). because of light limitation. (1992) 3 15 2 11 11 2 15 32 30 32 11–16 22–25 30 20–25 24–25 22–26 20 16–26 20–25 20 20 optimum temperature for culture conditions. various web sites.5. Minimum 15 10 22 Maximum 45 40 35 26–30 38–42 34 28 36 29 Optimum 35–38 16 20–30 26 References vws Flassch (1978) Wei et al. (1986) vws Kessler (1980) Kessler (1980) Kessler (1980) vws Ryu & Tokuda (1984) vws Srisudha & Nair (1996) vws Abu-Rezq et al. saccharophila C. Dunaliella salina is known to grow in supersaturated waters. The accumulation of dissolved oxygen produced during cultivation may affect growth at high concentrations.5 by addition of acid prior to culture inoculation. the inhibitory compounds could be analogous to pheromonal mating factors and could exhibit bacteriocidal activity.g.7 Salinity tolerance of the main species of microalgae used in aquaculture.220 Live Feeds in Marine Aquaculture Table 6.e. (1999) Bonin et al. (1986) Brand (1984) Droop (1958) Fanuko (1981) Sigaud & Aidar (1993) Hill (1992) Blanchemain (1993) Laing & Utting (1980) Fabregas et al. with an optimum close to neutrality. The literature mentions the adverse effect of high oxygen contents on microalgae growth (e. mixing is an important factor affecting growth as it allows alternate exposure of the entire microalgal population to light.2. The carbon dioxide injected to supply photosynthetic carbon requirements also reduces the pH and helps to maintain this within the growth range for the algae. Isochrysis galbana I.5 20–30 15 15–25 25–35 Optimum References Borowitzka & Borowitzka (1988) Nana-Kebou-Hako (1999) Baticados & Gacutan (1977) Flassch (1978) Alias (1988) Laing & Utting (1980) Renaud & Parry (1994) Abu-Rezq et al.5.1 60 2–10 5–35 Phaeodactylum tricornutum Rhodomonas sp. Growth usually increases pH because carbon dioxide in the medium is consumed by the microalgae. and especially in closed systems (Richmond et al. initial batch pH should be as low as growth compatibility allows. Ogawa et al. 1991).0–7. Skeletonema costatum Tetraselmis sp. affinis galbana ‘Tahiti’ (T-iso) Nannochloropsis oculata Minimum 2. which tolerates pH as high as 11.3) is currently adjusted to 7.6 20 15 10 Maximum 80 88 35 36 36 35 35 15–23 20–30 Pavlova lutheri 0. This situation could occur when degassing is insufficient. platensis Chaetoceros sp. 6.2. (1984) 25 25–30 Chlorella. Using L/D cycles.5–30.5 1. Salinity (g l 1) Species Arthrospira sp. For this reason. . A.5. (1999) Renaud & Parry (1994) Wood et al. 1993). 6.5 Mixing In intensive photoautotrophic cultures. Seawater pH (around 8. (1999) Chini Zitelli et al. 2 0 10 10 63 40 33 45 36 13–16 28. inhibiting photosynthesis and even causing photo-oxidative cell destruction. from air saturation to a concentration of 29 g l 1. (1980) found that Chlorella vulgaris growth is reduced by some 20% when the partial pressure of oxygen varies from 21 to 65 kPa O2. i. and because precipitates could appear when adding the enrichment medium.4 pH With the exception of Arthrospira. Tredici et al. Chlorella sp. most species used in aquaculture require a pH between 6 and 9. 3 Biochemical Composition of Microalgae The main zoological groups produced by aquaculture. Microalgae have a limited and probably species-specific ability to ‘average’ exposure to strong light and darkness given that adequate mixing is provided (‘light integration’). 1985. but in less controlled conditions (non-axenic cultures. 1996). that are more representative of the quality of algae distributed to bivalves in commercial hatcheries. especially shear forces. For example. 5 and 6 desaturases. The microalgae species used in aquaculture have not been studied for their tolerance to mechanical stress. 1986. The data on biochemical composition presented here refer mainly to vitamins. Watanabe et al. . although some mixotrophic and hetrotrophic marine algae have also been described (Wood et al. Mixing is also necessary to provide gas exchange and possibly to break up nutrient and inhibiting metabolite gradients. Brown et al. but highly variable. 1989. 1996. dark zones may develop in the culture vessel. 1992) or culture media (Wikfors et al. Volkman et al. but the growth will eventually decrease with increasing extension of the dark zones.The Microalgae of Aquaculture 221 it has been shown that microalgae continue to grow for a while in darkness. The data presented refer mainly to photoautotrophically produced algae. 1993a). 1999). provided that previous light exposure has been long enough (e. 1998). with 20–22 carbon atoms and more than three double bonds) by desaturation and chain elongation. These results were supplemented with production data from IFREMER obtained over several years and using larger volumes (300 litre tanks). sterols and PUFA of phytoplankton species commonly used for bivalve aquaculture. 1999) and Volkman et al. Enright et al. 1999). are more tolerant. Soudant et al. marine fish. Algae constitute the basic diet of these species that cannot easily be shifted to artificial diets. 1993). 1989). Further details can be found in the well-documented reviews of Brown et al.g. shrimps and molluscs. 1989). suecica loses its flagella and stops growing with strong mixing. as can fish and crustaceans. variable greenhouse light and temperature). period of harvesting). Merchuk et al. However. Trider & Castell 1980. exhibit reduced ability to synthesise highly unsaturated fatty acids (HUFA. With increasing cell density. HUFA and cholesterol are thus essential substances that must be supplied by food sources. (1997. Microalgae can also provide a large variety of vitamins to satisfy marine animal requirements (Seguineau et al. As most of them have flagella or appendices. 6. Those microalgae with a thick cell wall. These substances are abundant in microalgae (Lin et al. (1981. conditions (batch/continuous. such as Chlorella and Nannochloropsis. The composition of microalgae is clearly related to their growth phase (exponential or stationary) and to culture conditions such as light frequency (Brown et al. light intensity (Thompson et al. low sterol synthesis and poor bioconversion ability (Teshima & Kanazawa 1974. Kanazawa et al. which possess 4. including cholesterol. because of the possible detrimental effects of mechanical forces on the integrity of the algal cells (Gudin & Chaumont 1990). 2000). temperature (Thompson et al. 1982. mixing must not be too vigorous. T. Results in the literature have been obtained mainly with small experimental volumes under various controlled. they are usually fragile. L/D cycle. allowing synthesis of essential PUFA (with more than one double bond) as well as a large variety of phytosterols. (From Brown et al. carbohydrate and protein in per cent of dry weight (aeration with 1% carbon dioxide). Gross composition does not always correlate directly with nutritional value owing to possible deficiency in some essential nutrients. Proteins range from 26 to 40%. aeration with 1% carbon dioxide. 6. The data are in the range of those reported in the literature. (1997) reported the overall biochemical composition of 40 algal species grown under standard conditions (Fig.3. However. in accordance with previous reports. the gross composition may be important. except for diatoms whose storage product is lipid. 1997. 6. 6.) Fig. Protein was the major organic component (15–52% of dry weight). Microalgae are cultured in 300 litre tanks under the following conditions: artificial sunlight. Fig. and carbohydrates and lipids are stabilised around 15–17% for most species.1 Gross biochemical composition Brown et al. when specific essential nutrients are in adequate proportion.4 Microalgal lipid. The number of replications is shown in parentheses.5 Lipid. carbohydrate and protein in per cent of dry weight in microalgae grown in the IFREMER-Brest hatchery.4). followed by lipid (5–20%) and carbohydrates (5–12%).5 shows results obtained in the IFREMER experimental hatchery with carbon dioxide-enriched aeration. being 18% on average. Lipid content was higher for diatoms.222 Live Feeds in Marine Aquaculture 6. Figure 6. acid-treated and enriched with Conway medium. showing a very consistent pattern. . filtered (1 m) seawater. 1979. (1993a) also showed that arachidonic acid (ARA) and sugar composition in T-iso did not differ for the range of irradiances tested (50–1000 Einstein m 2 s 1). Brown et al. (1986). depending on the algal species. with other amino acids ranging from 3. The greatest differences between the species concern retinol and pyridoxine in relation to the growth phase. Phaeodactylum tricornutum is unique in its rich concentration of mannose (46%). glucose appears as the predominant sugar in all species. (1999) are summarised in Table 6.2 to 13. Phytoplankton sterols are found in free form in neutral lipids (Ballantine et al. Brown & Farmer 1994. Few studies are devoted to microalgal amino acid composition. (1997) considered this feature in relation to the low nutritional value reported by Enright et al. methionine. 1979.1–12. thiamine and vitamin E increase. The data from Brown et al. Major vitamin contents are similar for all species. showing high levels of vitamin C.3. Gordillo et al. riboflavin. temperature. 1996). 1999). vitamin E. Variations in sterol levels can depend on the growth phase. 1999). 1981).9%).5%. whatever the conditions leading to this phase. tryptophan and histidine in the lowest (0. lipids and/or carbohydrates tend to accumulate in late senescence phase (Wikfors 1986. which consists exclusively of cholesterol. sampling conservation (Brown et al.3 Sterols Unlike human sterol. Polar sterols have also been reported. Despite the variability in quantitative data. Moal et al. regardless of algal class. Whyte 1987. There are few significant differences between algal classes. and vitamin A decreases (Seguineau et al. 1996. .3. 1987). Quantitative discrepancies found in the literature are probably related to the analytical and extraction methods. 1998). Brown et al.2%). 1993). vitamin C may decrease or increase depending on species (Brown & Miller 1992. With the onset of the stationary phase.2 Vitamins Most vitamins are found in algae used in aquaculture (Bayanova & Trubachev 1981. Volkman et al.The Microalgae of Aquaculture 223 The phase of harvesting and the culture conditions (nutrients. Brown et al. 1998). Brown et al. In terms of carbohydrate composition. Aspartate and glutamate occurred in the highest concentrations (7. Brown & Farmer 1994. which may in fact be glycosylated forms of sterols (Véron et al.8. Brown & Miller 1992. The 14 sterols listed below can be easily recognised in gas chromatography.4–3. 1999) and the harvesting phase. algal sterols are very complex and show species specificity. Seguineau et al. light intensity) influence the biochemical composition. These authors considered the nutritional value of proteins to be due to their composition of essential amino acids being similar to that of the animals to which the algae are offered as feed. The data of Brown et al. 1991) and recommended levels in a fish diet (Seguineau et al. the concentrations found in algae are high compared with both those in human food (De Roeck-Holtzhauer et al. 1991. and cysteine. It is generally accepted that. 6. 6. -tocopherol and niacin (vitamin PP = pellagra preventing factor. De Roeck-Holtzhauer et al. nutrients and light conditions of the culture (Ballantine et al. (1997) for 40 species show a consistent pattern. but prymnesiophytes contain more arabinose (2–12%) than other classes (0–2%). values for retinol were calculated using 3100 IU 1 mg. centrifuged and frozen at 20°C.1 60 25 75 10 50 5 0.8–7.8) na 0.4) 1.7) na 12 (7–15) 8.0) na na na Seguineau et al.5–1. centrifuged (?) and analysed immediately.24 (0.58) na 26 (7–48) 43 (15–66) na 26 (2–67) na na na na na na na Marine fishf 0.47) 0.33 (0. harvest stage not specified.4–1.5 (0.3–3.06–0.2 20 20 50 5 20 10 0.4) 0.4–0.47) 0.2 0. b 24/0 h L/D.81 (0.23 (0. (1991)d 0.3 (6.84) 1.4 (1.9) 0.81 (0.6 (0.4 (0.17 (0. sea bream and grouper.4 (1. 1 mg.025 Prawng 0.16–0.3) 0.3 (1.8 (1.6 (0.02 1 1.7) 1. (1993)b 1.3 (0. .1) na 0.6 0.1 Vitamin Ascorbic acid (C) -Carotene Niacin -Tocopherol (E) Thiamine (B1) Riboflavin (B2) Pantothenic acid Folates Pyridoxine (B6) Phylloquinone (K1) Cobalamin (B12) Biotin Retinol Ergocalciferol (D2) Cholecalciferol Units mg g mg g mg g mg g gg gg gg gg gg gg gg gg gg gg gg 2. c 24/0 h L/D.11–0.45 0. centrifuged and/or filtered.9 0. f New (1986). na.07–0.7 (0.3–2.35) 65 (40–110) 32 (28–38) 26 (14–38) 12 (7–15) 8.6 (0.11–0.6–17) na 1.12–6.8–7. d 24/0 h L/D.15 0.35 Data are average values for three to seven strains in each study (range in parentheses).7 ( 0.9) 0.14–4.2 1 1.3) 1. and those for ergocalciferol and cholecalciferol using 40. for yellowtail.24 (0.29) 61 (29–109) 32 (25–50) na 21 (17–24) 8.33) 81 (66–86) 0. e 24/0 h L/D.27 (0.7–10) na 4. Content in microalgae Brown et al.000 IU a 12/12 h L/D. log phase.7–1.2) 0.06–0.3 (6.4 (0.25–2.03–2.8 Reported vitamin content of microalgae. (1999)a 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 Animal requirements Seguineau et al.4 (1.32) 550 (290–710) 22 (6–42) na na 96 (4–180) 10 (0–28) 277 (8–1200) na na 14 (0–39) na Bayanova & Trubachev (1981)e 1.95) 1. g From Conklin (1997). Culture conditions and the method for harvesting and/or processing are indicated (from Brown et al. 1999).0) 0. late log phase.18–0.0) na na na De Roeck Holtzhauer et al.7 (3.2 0.1–1. late log phase.1 (0. (1996)c 1.2 (0.Table 6. centrifuged and frozen at 20°C. freeze-dried and stored at 70°C.4) 0.6–1.7–1. late and log phase.7–1. sea bass. cited by Tacon (1991). not analysed.7–1.04 0.2) 0.5–2.7 (0.7–10) na 4. centrifuged (?) and frozen at 30°C. 24 -dimethylcholestan-3 . 6.3.24(28)-dien-3 -ol campesterol 24 -methylcholesta-5-en-3 -ol stigmasterol 24 -ethylcholesta-5.22-dien-3 -ol 4 -methylporiferasterol 4. calcitrans. with no differences between the species mulleri. D es .6 Sterol profile of the diatom Chaetoceros sp. n 8) with results from cultures in the IFREMER-Brest hatchery (mean and standard deviation. 24 methylenecholesterol and fucosterol. The mean values for C.-methyl-24. Studies show considerable variability in composition owing to confusion between fucosterol and -sitosterol in the earliest works.22-dien-3 -ol 24-methylenecholesterol 24 -methylenecholesta-5. 6.22-dien-3 -ol trans-dehydrocholesterol cholesta-5.22-dien-3 -ol dihydrocholesterol cholesta-3 -ol cholesterol cholesta-5-en-3 -ol brassicasterol 24 -methylcholesta-5. 6.22-dien-3 -ol cis-dehydrocholesterol cholesta-5.4 -diol.The Microalgae of Aquaculture 225 Sterol nomenclature (trivial and systematic names): • • • • • • • • • • • • • • norcholesterol 22-trans-24-5.6).4 -diol ethylpavlovol 4 -methyl-24 -ethylcholestan-3 . calcitrans and gracilis (Gladu et al. calcitrans cultures grown in the IFREMER-Brest laboratory or in commercial hatcheries show the same distribution as those described in the literature (Fig. Comparison of data reported in the literature (mean and standard deviation. 100 90 weight % of total sterols 80 70 60 50 40 30 20 10 0 m os l ho tero l le D ste ih yd rol Br ro c a 24 ssic hol -m as t et hy ero l le C ne am c ho p St este l ig m rol a β.ste Si r to ol st Fu ero co l M s et hy tero l l M et por hy ife l r Et pav a hy lo l p vo av l lo vo l C yd r oc ho Chaetoceros sp.3. Chaetoceros exhibits the least diversified sterol spectrum: essentially cholesterol. 1991). n 10) for the strain C.-ethylcholesta-22-en-3 -ol -sitosterol 24 -ethylcholesta-5-en-3 -ol fucosterol 24 -ethylcholesta-5.1 Bacillariophyceae Among diatoms. (literature) Chaetoceros calcitrans (hatchery) eh D Fig.24(28)-dien-3 -ol methylpavlovol 4 . 6.1 29. 1995). campesterol.3 4. Cholesterol Brassicasterol Tetraselmis sp.7).5 52.1 7.226 Live Feeds in Marine Aquaculture The diatom Skeletonema costatum has a larger sterol spectrum.4 0. Species Thalassiosira sp.9) usually has the same sterols as Skeletonema. including cholesterol. Bergé et al. 3 17. n 8).9 17.7 3. 6.9 43 SD. costatum strain cultured in the authors’ laboratory was lower than that reported in the literature (Fig.6 14. Cholesterol Campesterol 24-Methylene Data are mean values Data from literature IFREMER-Brest hatchery Commercial hatcheries 28.3 15.5 5. 1995).8 0.8 45.5 92. 24-methylenecholesterol. Comparison of data reported in the literature (mean and standard deviation. The variability of quantitative data between studies was greater for Skeletonema than for Chaetoceros and related to culture conditions (Barrett et al.9 0. but 100 90 weight % of total sterol 80 70 60 50 40 30 20 10 0 D eh yd ro D es ch ol m o C ste ho ro l le s D ih tero y Br dro l ch as 24 ol s -m ica st et er hy ol le C ne am ch pe ol st St er ig o m as l te βr Si to ol st Fu ero co l M s et hy tero l l M et por hy ife lp r av a Et lo hy l p vol av lo vo l S.1 1. Cholesterol Desmosterol Campesterol 24-Methylene -Sitosterol Fucosterol Rhodomonas sp.6 4.1 4.12 23 4.9 78.8 0.2 97.3 3. Table 6.6 79. costatum (literature) S. -sitosterol and fucosterol (Tsitsa-Tzardis et al.8 7.4 2.3 2. Thalassiosira (Table 6.8 6.3 3 2.5 5.7 Sterol profile of the diatom Skeletonema costatum.9 Main sterol composition (weight in % of total sterols) of some microalgae: comparison of data in the literature with results from routine hatchery cultures. respectively. showing 70 and 54 fg cell 1 for Chaetoceros and Skeletonema.7 17. The weight of sterols per cell also differed for those two species.5 10. The cholesterol content of the S.3 13.7 .9 3. 1993.7 82.5 4. n 5) with results from cultures in the IFREMER-Brest hatchery (mean and standard deviation.9 0.9 5 2. costatum (hatchery) Fig. Diatoms are the only algal group containing a high level of cholesterol. Comparison of data reported in the literature (mean and standard deviation. 1979. Data obtained in routine hatchery cultures D . methylporiferasterol.e.8 mg g 1 dry weight. 1993b).3 Prasinophyceae Two main sterols are evident in Tetraselmis suecica. Chaetoceros shows the highest level (50%). stigmasterol.The Microalgae of Aquaculture 227 100 90 80 weight % of total sterol 70 60 50 40 30 20 10 0 eh yd r D es och m ol o C ste ho ro l le D ste ih yd rol Br ro 24 ass ch -m ica ol s et hy ter ol le C ne am ch pe ol st St e ig m rol as βSi tero to s l Fu tero l c M et ost er hy o M l et po l hy rif l p er Et av a hy lov lp o av l lo vo l Isochrysis sp. A peculiarity of Isochrysis is the presence of alkenones.9 and 2. The prymnesiophyte Pavlova lutheri (Fig. 6. 6.3. with only one study (Véron et al. The last two sterols are diols. the sterol essential for mollusc growth (Trider & Castell 1980). C37 and C39 ketones (Patterson et al. found exclusively in Pavlova sp. Patterson et al.9%) and 24methylenecholesterol (43%). 1991). Lin et al. namely campesterol (52. 6. n 7) with results from cultures in the IFREMER-Brest hatchery (mean and standard deviation. 6. 6.8 Sterol profile of the prymnesiophyte Isochrysis sp.3. respectively). Results in the literature are generally similar. i. 1998) showing a different pattern (equilibrated levels of brassicasterol and cholesterol). -sitosterol. with an additional 4 -hydroxyl group in the structure. This species contains much higher levels of total sterols than Isochrysis sp.3. respectively (Knauer et al. n 16) for the strain Isochrysis galbana (clone T-iso).2 Prymnesiophyceae The sterol composition of the Isochrysideae Isochrysis galbana and Isochrysis affinis galbana (clone T-iso) is simpler (Fig. (literature) T-isochrysis (hatchery) Fig. together with a low amount of cholesterol (Ballantine et al. methyl and ethyl pavlovol (Patterson et al.8) and characterised by brassicasterol ( 90%). with higher and lower percentages of 24-methylenecholesterol and cholesterol.3. Alkenones may be more concentrated than sterols. 1982. i. respectively (Gladu et al. 1999). with 31. 1994) that elute after sterols on the chromatogram. Data obtained routinely in hatcheries are very similar to those described in the literature. (290 and 30 fg cell 1.e. 1993a).9) shows a very complex composition with campesterol. which are distinctive of plants and located in the thylakoids. The mean content of total sterols in hatchery production is 250 fg cell 1. lutheri (hatchery) Fig. 1995).3. glycolipids and phospholipid. showing higher levels of campesterol in the authors’ hatchery (82%) and commercial hatcheries (78%). neutral. X the number of double bonds. which constitute the membranes. show the highest level of unsaturation.4 Fatty acids Fatty acids are named here following the C:Xn-Y formula. 6.9) show a very similar profile to that of Isochrysis. Temperature mainly affects the galactolipid composition (Henderson & McKinlay 1989).9 Sterol profile of the prymnesiophyte Pavlova lutheri. and Y the position of the first double bond counted from the CH3-terminal. 6. Total lipid content and distribution vary considerably with the status of the culture or the strain used. Glycolipids. Polar lipids (glycolipid and phospholipid). Fatty acids are distributed among three lipid classes. are different (Table 6. The fatty acid composition of microalgae generally shows a very consistent pattern in each group (Napolitano et al. Taxonomic groups exhibit a specific fatty acid composition. in routine hatchery production (Table 6. Those obtained for Rhodomonas sp. Comparison of data reported in the literature (mean and standard deviation. whereas nutrients mainly modify triglyceride and .3. the main constituent (Bergé et al. as well as with the environmental conditions of the culture. Decreasing growth rate and temperature increase the lipid content.3.228 Live Feeds in Marine Aquaculture 100 90 weight % of total sterol 80 70 60 50 40 30 20 10 0 D eh yd ro D es ch ol m o C ste ho ro le l D ste ih ro y Br dro l ch as ol 24 si ca -m st et hy ero l l C ene am c ho p St est l e ig m rol as βt Si ero to l st Fu er ol c M et oste hy ro l l M et por ife hy r l Et pav a hy lo l p vo av l lo vo l P. lutheri (literature) P. n 4) with results from cultures in the IFREMER-Brest hatchery (mean and standard deviation. 6.9). are represented by monogalactosyl and digalactosyl glycerides. in which C is the number of carbon atoms. n 2). 1990).4 Cryptophyceae No data were found in the literature concerning Cryptophyceae. lutheri.. 6.10. Sukenik et al. with increased levels of docosahexaenoic acid (DHA. although the response is moderate (Thompson et al. fatty acid content is about 1. 1989.The Microalgae of Aquaculture 229 35 30 weight % of the total FA Chaetoceros sp. except for 16:4n-1 (Ackman et al.. but to a lesser extent. costatum only) and EPA (Ackman et al. and Skeletonema sp.3. n 7) with results obtained in the IFREMER-Brest hatchery (mean and standard deviation. with a higher content for Chaetoceros (2 pg cell 1) than for S. 22:6n-3 only). Light also exerts some changes. Thompson et al. calcitrans. 6.1 pg cell 1 for T-iso and P. Volkman et al. which occurs mainly in galactolipid. monounsaturated fatty acids mainly by 16:1n-7. than in Skeletonema sp.8 pg cell 1 and 1. Comparison of data in the literature (mean and standard deviation. 6. costatum (1. respectively. 1968. Saturated fatty acids are represented predominantly by 14:0 and 16:0. eicosapentaenoic acid (EPA.2 Prymnesiophycaea According to data obtained at the IFREMER laboratory. the data are similar to those described in the literature (Figs 6.4. 20:5n-3).4 pg cell 1). which is in good agreement with results in the literature (Volkman et al. 6. 1968. n 6) for the species C. Although the latter fatty acid was not found in the present cultures. 1989).3. (1993a).1 Bacillariophyceae The mean fatty acid content for diatoms is 1.4. Saoudi-Helis et al. in Nannochloropsis sp. and PUFA mainly by 16:3n-4. 1994). Decreasing temperature increases the PUFA levels in marine phytoplankton. showing that 20:4n-6 and the EPA:DHA ratio are higher in Chaetoceros sp. The spectra are very similar for the genera Chaetoceros sp. (1996) obtained the same result for Pavlova lutheri and Thalassiosira pseudonana. According to Brown et al. 1991. However. irradiance caused very little change in the proportions of lipid classes and fatty acid composition of T-iso. phospholipid composition (Fernandez-Reiriz et al.7 pg cell 1. Bergé et al.10 Fatty acid (FA) profile of the diatom Chaetoceros sp. literature C. 16:4n-1 (in S. Wikfors & Patterson 1994). The . The fatty acid composition of diatoms is generally homogeneous within each genus.11). increases in low light conditions when galactolipid concentrations increase. 1992) and is species specific. 1995). calcitrans hatchery 25 20 15 10 5 0 14 :0 15 : 0 6: 0 8: 0 -9 -7 -9 -7 -7 -6 -4 -4 -3 -1 -3 -6 -4 -3 -6 -3 -3 -6 -6 -3 -6 -3 -3 1 1 : 1n : 1n : 1n : 1n : 2n : 2 n : 2 n : 3 n : 3n : 4 n : 4n : 2 n : 2 n : 3n : 3n : 4 n : 5n : 2 n : 4n : 5n : 5 n : 5 n : 6n 16 16 18 18 16 16 16 16 16 16 16 18 18 18 18 18 18 20 20 20 22 22 22 Fig. (1989) found that the longest fatty acid. and the main .11 Fatty acid (FA) profile of the diatom Skeletonema costatum. major fatty acids of Isochrysis sp. the monounsaturated fatty acids are 16:1n-9 and 18:1n-9.costatum (hatchery) weight % of the total FA 30 25 20 15 10 5 0 14 :0 15 : 0 6: 0 8: 0 -9 -7 -9 -7 -7 -6 -4 -4 -3 -1 -3 -6 -4 -3 -6 -3 -3 -6 -6 -3 -6 -3 -3 1 1 : 1n : 1n : 1n : 1n : 2n : 2 n : 2 n : 3 n : 3n : 4 n : 4n : 2 n : 2 n : 3n : 3n : 4 n : 5n : 2 n : 4n : 5n : 5 n : 5 n : 6n 16 16 18 18 16 16 16 16 16 16 16 18 18 18 18 18 18 20 20 20 22 22 22 Fig.8 pg cell 1. n 7) for the strain Isochrysis affinis galbana (clone T-iso).12 Fatty acid (FA) profile of the prymnesiophyte Isochrysis galbana (clone T-iso). lutheri provides high levels of both essential n-3 HUFA. n 9) with results obtained in the IFREMER-Brest hatchery (mean and standard deviation. 18:4n-3 and DHA. are 14:0. P. with an EPA:DHA ratio 1. Pavlova lutheri exhibits some differences. but these culture conditions seem to favour the PUFA content of P. 16:0. and Isochrysis sp. in that the monounsaturated fatty acid is 16:1n-7 and EPA replaces 18:4n-3. 6.230 Live Feeds in Marine Aquaculture 40 35 S. has lower levels of EPA and an EPA:DHA ratio 1. lutheri.4.12. Comparison of data in the literature (mean and standard deviation. 25 weight % of the total FA 20 T-iso (literature) T-iso (hatchery) 15 10 5 0 14 :0 15 : 0 6: 0 8: 0 -9 -7 -9 -7 -7 -6 -4 -4 -3 -1 -3 -6 -4 -3 -6 -3 -3 -6 -6 -3 -6 -3 -3 1 1 : 1n : 1n : 1n : 1n : 2n : 2 n : 2 n : 3 n : 3n : 4 n : 4n : 2 n : 2 n : 3n : 3n : 4 n : 5n : 2 n : 4n : 5n : 5 n : 5 n : 6n 16 16 18 18 16 16 16 16 16 16 16 18 18 18 18 18 18 20 20 20 22 22 22 Fig. Comparison of data in the literature (mean and standard deviation. The main saturated fatty acid is 16:0. Therefore. 18:1n-9. 6. The IFREMER hatchery data are quite similar to those in the literature (Figs 6. n 3). 6.3 Prasinophyceae The genus Tetraselmis has a mean fatty acid content of 5. 6.costatum (literature) S. Low levels of ARA (20:4n-6) are also found in both species.3.13). n 3) with results obtained in the IFREMER-Brest hatchery (mean and standard deviation. The Microalgae of Aquaculture 231 30 25 weight % of the total FA P. n 4) with results obtained in the IFREMER-Brest hatchery (mean and standard deviation. suecica (hatchery) weight % of the total FA 35 30 25 20 15 10 5 0 14 :0 15 : 0 6: 0 8: 0 -9 -7 -9 -7 -7 -6 -4 -4 -3 -1 -3 -6 -4 -3 -6 -3 -3 -6 -6 -3 -6 -3 -3 1 1 : 1n : 1n : 1n : 1n : 2n : 2 n : 2 n : 3 n : 3n : 4 n : 4n : 2 n : 2 n : 3n : 3n : 4 n : 5n : 2 n : 4n : 5n : 5 n : 5 n : 6n 16 16 18 18 16 16 16 16 16 16 16 18 18 18 18 18 18 20 20 20 22 22 22 Fig.lutheri (literature) P.4 Chlorophyceae This class is not suitable for feeding to molluscs. 45 40 T. Comparison of data in the literature (mean and standard deviation.3. together with lower amounts of 18:4n-3 and EPA.4. polyunsaturated ones are 16:4n-3 and 18:3n-3. 6. Fatty acid content is similar for Pyramimonas virginica (Chu & Dupuy 1980). However. Comparison of data in the literature (mean and standard deviation. suecica (literature) T. On average. The most unsaturated fatty acids are 16:4n-3 and 18:3n-3 (Ackman et al. 1968. 6. probably because of its PUFA deficiency. n 7) with results obtained in the IFREMER-Brest hatchery (mean and standard deviation.14). 1996). Low levels of ARA have also been found (Wikfors et al. Prasinophyceae provide fewer unsaturated fatty acids than Prymnesiophyceae and exhibit total DHA deficiency. Chu & . n 4).lutheri (hatchery) 20 15 10 5 0 14 :0 15 : 0 6: 0 8: 0 -9 -7 -9 -7 -7 -6 -4 -4 -3 -1 -3 -6 -4 -3 -6 -3 -3 -6 -6 -3 -6 -3 -3 1 : 1n : 1n : 1n : 1n : 2n : 2 n : 2 n : 3 n : 3n : 4 n : 4n : 2 n : 2 n : 3n : 3n : 4 n : 5n : 2 n : 4n : 5n : 5 n : 5 n : 6n 1 16 16 18 18 16 16 16 16 16 16 16 18 18 18 18 18 18 20 20 20 22 22 22 Fig.14 Fatty acid (FA) profile of the prasinophyte Tetraselmis suecica. 6. as indicated by the low reported standard deviation (Fig.13 Fatty acid (FA) profile of the prymnesiophyte Pavlova lutheri. cells appeared to be enriched in saturated and monounsaturated fatty acids in a reproducible manner. n 2). 6. The data obtained under IFREMER experimental conditions are in accordance with the literature. 5 1.0 1.5 23.1 6.2 6.10). Dupuy 1980).5 10. Dunaliella tertiolecta Compilationa 1.8%) than Prasinophyceae.2 Chlorella sp.6 Eustigmatophyceae This class provides EPA at a high level (19.7 1.1 13.6 7. (1968) reported data referring to an unknown cryptophycea (Table 6. 6.6 32.3.3.1 2. (1999).0 27.7 15.4 3.7 SD from Ackman et al. (1968) 1.9 0. (1968).10 Mean fatty acid composition (weight in % of total fatty acids) for two chlorophyceae.5 Crytophycea Ackman et al.4 2.1 0.4.4 1.8 16. Sukenik et al.2 4.5 Cryptophyceae Ackman et al.1 3. .10.5 1.6 7. 18:4n-3 and EPA as PUFA.2 6.3 0.5 5. and 18:2n-6.4. 6.232 Live Feeds in Marine Aquaculture Table 6. a eustigmatophycea and an unknown cryptophycea.8 0.1 1.5%) and some ARA (6. 18:3n-3.6 3.3 3.4 0.0 16.0 0. it has a high level of monounsaturated fatty acid 16:1n-7 (Table 6. This genus provides more EPA (13.2%).5 1.9 26. The fatty acid composition of Dunaliella tertiolecta and a species of Chlorella is given in Table 6.10).6 Nannochloropsis sp.7 1. However. (1989) 9. (1992) and McCausland et al.5 3.4 0.8 0. Chu & Dupuy (1980) 2.5 11.0 7.8 0.2 19. This species contains 16:0 as saturated fatty acid. 1989). 18:1n-9 and 18:1n-7 as predominant monounsaturates.2 1.2 Fatty acid 14:0 15:0 16:0 18:0 16:1n-9 16:1n-7 18:1n-9 18:1n-7 16:2n-7 16:2n-6 16:2n-4 16:3n-4 16:3n-3 16:4n-1 16:4n-3 18:2n-6 18:2n-4 18:3n-3 18:3n-6 18:4n-3 18:5n-3 20:2n-6 20:4n-6 20:5n-3 22:5n-6 22:5n-3 22:6n-3 a 0.4 6.2 14.0 0.4 18.0 28. Thompson Data for Dunalliela tertiolecta are a compilation of mean values et al. but lacks intermediary PUFA (Sukenik et al. microalgae were cultured in specialised facilities and then transported to larval or live prey rearing tanks to be used as food and/or for medium stabilisation. Later. but the high latitude of most sites requires supplementary use of artificial light. since light attenuates while penetrating the reaction medium.4. the production of energetic hydrocarbons. .03% in natural conditions. These studies were concerned successively with the production of new food resources. and for aquaculture with a dozen species currently cultivated. This lag could be due to the special nature of the energy source. This progressive attenuation of light accounts for most of the difficulties encountered during the development of photoautotrophic biotechnology. culture units consist of vertical cylindrical tanks (100–500 litres) made of transparent plastic (acrylic.0 volume per culture volume and per hour (v/v/h).15). The first research studies on microalgal production in the 1960s led to the development of most of the culture media and growth models still in use today. ensuring mixing as well as gas–liquid exchanges by direct contact. The culture techniques developed then have remained relatively unchanged.1 State of the art of microalgal production techniques in hatcheries Typically (Fig. Since then. with an emphasis on possible improvements. fibreglass polyester. in the 1970s. With some 2000–4500 t per year depending on sources. 6. it is often added to the compressed air at a concentration of 1–3% (volume/volume). This section considers the state of the art of microalgal cultivation techniques. These rudimentary rearing methods. Although the carbon dioxide content in air is 0. the preservation of the environment and the production of commercially valuable molecules. which are flat or conical at the bottom and open at the top or closed by an unsealed cap. industrial production has developed for human nutrition with Chlorella and Spirulina. The value of microalgae for aquaculture has been constant and of a strategic nature since the 1970s. aquacultural microalgae were produced by inducing phytoplankton development in ponds with enriched natural water in which the animals to be fed were then directly hatched. The airflow rate is between 0. but more slowly than that of bacteria and fungi.The Microalgae of Aquaculture 233 6. World production for aquaculture is difficult to assess. polyethylene). Injection of compressed air at the bottom of each tank produces bubbles that rise to the surface. during photosynthesis.1 and 3. 6. for pharmaceuticals with -carotene from Dunaliella salina. Natural light is used whenever possible.4 Production Methods for Aquacultural Microalgae The biotechnology of microalgae and cyanobacteria has developed constantly since the 1960s. which are still used in family farms raising penaeid shrimp in south-east Asia. were the first forms of ‘green water’ and mesocosm techniques. and diets for the live prey species essential for small-mouthed fish larvae and shrimps. respectively. providing food for molluscs. Fluorescent tubes placed around the tank in a power:volume ratio of 1 W l 1 of culture provide artificial lighting. mainly because the microalgae are not harvested. but fed live to animals as raw culture. These exchanges consist of oxygen stripping and carbon dioxide enrichment as those gases are produced and consumed. Spirulina is the most widely produced genus. Initially. 6. two species of small larvae fish.0 m depth) are also used. thereby . Paralichthys dentatus. All fluids and surfaces that come into contact with the culture must be sterilised. the site needs to be carefully chosen in an area with water that is free of pollution and stable in quality. Natural water pumped near the facilities is the basis for the culture medium and larval rearing.15 Typical microalgal production facility for aquaculture at Great Bay Aquafarms.16). However. 6.4. Once again. Portsmouth. production per unit of time and volume is slightly lower. predators or toxic agents for microalgae. This hatchery produces the flounder.1 Asepsis and quality controls Certain precautions are needed to maintain culture asepsis and prevent contamination by organisms naturally present in the environment that could act as competitors. New Hampshire. Thus. If those precautions are not taken. USA. harbours and estuaries. the pumped water is prepared by passage through successive filtering devices. 6. air is injected at the bottom of the water column for mixing and gas exchange. and the black sea bass. with surface lighting provided by artificial sources similar to those used in greenhouses (Fig.5–1. Once these precautions are taken.234 Live Feeds in Marine Aquaculture Fig. inhibit growth or degrade the nutritional quality of microalgae. A sand filter reduces the size of suspended particles to 10–20 m. Rectangular tanks (0. contamination can occur and destroy the cultures. Centropristis striata. in particular at some distance from towns. These tanks are easier to clean than the transparent ones as they are largely open and do not require tilting for access to the inside.1. (Photograph: IFREMER/Muller-Feuga). headdresses. The staff can contribute to cleanliness by wearing disposable or regularly cleaned gloves. In practice. overalls. the systematic use of low melting temperature plastics such as polyethylene and polyvinyl chloride (PVC) for culture confinement and piping impedes the use of heating as a sterilising agent. as bacteria from microalgal cultures are seldom . In some cases. thereby eliminating most of the protists. Barfleur. Moreover. Additional filtration by means of cartridges or sieves reduces particle size to 1 m. these precautions are often resisted by a workforce better accustomed to handling a scoop than Pasteur pipettes. Water preparation by filtration often remains at this stage. A footbath filled with sterilising product should be placed at the entry to the culture room to avoid ground contamination. Additional filtration by active carbon is sometimes necessary to eliminate organic substances such as phytoplankton toxins. even though a culture room may be equipped with dust-control devices to prevent contamination by air. this measure is seldom applied. 6. The other procedures for keeping cultures aseptic are filtration of the gas injected at the bottom of the tanks and cleaning of intermediate surfaces and containers. These filters are easily cleaned by regular reverse flushing. they represent an important expenditure item. This consists of the addition of a product whose sterilising toxicity can be eliminated by adding a neutralising agent after a suitable contact delay. oxidant– reductant or acid–base pairs. For instance. Hypochlorite is generally reduced by thiosulfate after overnight contact. and overshoes.g. still allowing prokaryotes to pass through. Although filters are generally recyclable after washing. (Photograph: IFREMER/Barbaroux). especially at the surface of tanks.16 Microalgae production in opaque-walled tanks with an artificial light source above the surface at SATMAR.The Microalgae of Aquaculture 235 Fig. Microalgal cultures for aquaculture are not monoseptic because the means available in hatcheries and the capabilities of the staff are generally inadequate to achieve this quality level. e. eliminating most of the zooplankton. chemical sterilisation is performed within the culture tank once it is filled with filtered water. France. However. as all of these cultures are grown in the same room. The densities reached in these intermediate cultures vary from 2–3 106 cells ml 1 for T. whereas this important growth factor should be fine-tuned to each species. thereby reducing the advantages of biodiversity. For example.5 per 1000 litres of final solution. The same enrichment medium is generally used for all species of microalgae at the same facility. the most commonly used being f/2 medium (Guillard & Ryther 1962) and the medium of Conway (Walne 1966).1.12 g l 1 (the dry weight .4. They differ according to the light source and the species. temperature is often controlled between 18 and 25°C for reasons of comfort. some of these media are also marketed in the form of several solutions to be mixed just before use. phosphorus. and trace elements including iron and silicon for diatoms. 400.000 cells ml 1 are necessary for Tetraselmis suecica and 106 cells ml 1 for Pavlova lutheri with 150 Einstein m 2 s 1 from fluorescent sources. 2 litre bottles and. Vitamins such as thiamine or biotin.236 Live Feeds in Marine Aquaculture harmful for algae and larval rearing. this common environment does not make it possible to take the physiological features of each species into consideration. metabolisable nitrogen.e. i. The algal strains are provided from large culture collections or specialised public organisations in the form of a few millilitres of culture in a test-tube (generally not bacteria free). a dry weight concentration of 0. aquaculture operators have chosen to consider the current quality of their algae as acceptable. enrichment medium is added and compressed air supply plugged. for which some algae are auxotrophic. the salts required for f/2 medium can be purchased at a cost of approximately US $0. These initial concentrations should be just sufficient for providing photoprotection by self-shading and for outrunning possible competitors.4.1. For example. Once illuminated tanks have been cleaned and filled with filter-sterilised water. successive volumes of increasing size are inoculated in order to prepare the biomass required to reach inoculation concentrations in the production tanks. Starting from this sample.e. suecica to 5–15 106 cells ml 1 for P. 6. These salts constitute the enrichment media. is often added to prevent precipitation of ferric hydroxide. 10–20 litre bottles or carboys (Fig. should be added with due caution because of their rapid degradation with heat. 6. Qualitative levelling is likely to occur in these common conditions. and an inoculum is introduced into the tank in such an amount that the resulting concentration prevents the culture from failure and ensures a quick start. finally.17).1. The intermediate vessels generally used are 250 ml conical flasks. The operators can prepare enrichment media from the constitutive salts.4. 6. 6. generally ethylenediaminetetra-acetate (EDTA). For example.2 Culture medium and temperature Natural filtered water is enriched by the addition of the mineral salts required for photosynthesis.4 Efficiency The cultures obtained in current hatchery facilities seldom exceed a density of 6 106 cells ml 1 at the end of 5 days.3 Running the cultures Batch cultures are generally run according to production cycles of 3–7 days. Moreover. A chelating agent. lutheri. i. However. 6.2 Methods of improvement Productivity is often used to compare various production systems or to define the optimal conditions for operating a given system. An increase in production yields can also contribute to a reduction in cost price. can market their product at prices lower than US $200 kg 1 of dry weight.000 kWh and salts to manufacture 200 m3 of culture medium. other industrial facilities specialised in the production of microalgae and exploiting production systems in highly controlled conditions. Thus. These figures are in agreement with those of Bennemann (1992). a facility with a capacity of 5000 litres operating on 200 days per year and producing 24 kg of dry microalgae requires a full-time technician.000 worth of equipment. amortisation (6%). (Photograph: IFREMER/Barbaroux). energy (3%) and miscellaneous expenses (1%). This criterion consists of the dry biomass produced per unit of culture space and of time. France. . This suggests the possibility of a considerable reduction in costs. a decrease in production costs depends especially on a reduction of staff time. of a cell is on average 20 pg). 6. Barfleur. Thus. with no very significant increase in other expenses. uses 23. The cost price of producing hatchery microalgae includes labour (90%).17 Intermediate cultures for the inoculation of production tanks at Satmar. such as photobioreactors.4. and needs to amortise about US $10. The total production cost is approximately $1400 kg 1 of dry matter of microalgae in the conditions existing in western Europe.The Microalgae of Aquaculture 237 Fig. which should be of interest to aquaculture operators. Meanwhile. for the culture of the red microalga Porphyridium cruentum. For example. 1991. human intervention is limited to preparation of the nutrient medium upstream. This high discrepancy can be explained by the difference in light path length of the cultures in those production systems. Typically.2. To illustrate this observation. culture concentrations seldom exceed 0. This balance is characterised by the concentration of the culture C and the dilution rate D (D Q/V expressed in % per day).18 shows a comparison between 500 mm diameter hatchery tanks. which measures the output efficiency of a system. use of the harvest downstream. The dilution rate D is a major parameter since it determines the average age of the cells. and the inoculation process. 1993b). The residual volume of a culture then constitutes the inoculum of a new production cycle. and photobioreactors. is equal to the dilution rate multiplied by the concentration (P CD C/T. whose light path length ranges between 8 and 30 mm. Intermediate modes of operation between batch and continuous (called semi-batch or semi-continuous) consist of collecting only part of the culture volume and replenishing with fresh medium. Thus. Tredici et al. Pulz et al. and amortisation in the cost price of the algae. Hartig et al. Another factor is the residence time T.g. Volume productivity. Fig. such renewals may comprise two-thirds of the volume every 5 days. a continuous culture involves supplying a culture tank of volume V with fresh medium according to a renewal flow rate Q. 6. with a mean light path length of 392 mm.1 Continuous cultures The main labour-consuming operations are the distribution of cultures when mature. Grobbelaar 1981. and a general. with a reduction in the share of nutrients and electricity.2.238 Live Feeds in Marine Aquaculture 6. According to the type of facility and species. However. 6. A balance is established at the end of a few days between the renewal of the medium and the growth capacity of the algal population. whereas concentrations of photoautotrophic cultures produced in photobioreactors are reported to be some g l 1. as it has been stated that this factor has a strong inverse influence on the final concentration (e. This example shows that the final concentration C of a culture varies with . A substantial reduction in labour requirements may follow (seven-fold according to some estimates).4. the dilution rate D is between 20 and 50% per day. the cleaning of vessels and their filling with culture medium. corresponding to a residence time T between 5 and 2 days. Once a culture has been started in the continuous mode of operation.1 g l 1 (or approximately 6 million cells ml 1 for T-iso).4. 1988. this would be accompanied by an increase in investments. It is possible to increase concentrations10-fold by reducing the light path length. which has a considerable influence on their biochemical characteristics (Brown et al. 1995). in g l 1 day 1). which is the time required for the complete renewal of the volume of culture (T 1/D in days). Continuous cultures are the chief means of obtaining such results.2 The increase in production yields An increase in concentration of the cultures would result in a more efficient utilisation of the production factors. particularly for the fresh medium pumps and automatic systems such as carbon dioxide injection under the control of pH. a reduction in the time devoted to these operations presumes an increase in efficiency or removal of parts or all of these processes. In most hatcheries. rapid monitoring of parameters for the culture and the facility. P. Phycol. remains constant (S CE. This situation allows a considerable margin of progress for hatcheries. According to the Beer–Lambert law. The concept of closed tubular photobioreactors was initially explored in the early 1980s. representing a 10-fold increase. or g m 2). Volume productivity seldom exceeds 20 mg l 1 day 1 in most hatcheries. the mean value of over the spectrum was equal to 0. Meanwhile. whether cultures are operated in batch or continuously.. where I0 is the initial energy hitting the culture. 1995. in g l 1 mm. cruentum (Chaumont et al. cruentum gave 0. It is interesting to examine the practical implications of the stability of the areal density S. 6. and Gudin and Chaumont (1990) proposed detrimental effects of mixing on cells as a possible explanation. 1988). The curve corresponds to 42 g m 2 areal density. (Reprinted from Muller-Feuga. as the technologies developed for the photobioreactors could usefully improve the quality and reduce the cost price of algae.18 Culture concentration versus mean optical thickness obtained for different systems used for the production of Porphyridium cruentum (precision bars show SD). 6.) its light path length E according to a simple relation stating that the product of these two factors.The Microalgae of Aquaculture 239 2 ALP 2 ALP 1 NLP Concentration (gl 1) 1 Tanks 0 0 100 200 300 Mean culture thickness (mm) 400 Fig. In the example of Fig. (Livansky et al. Hervé. at least for a given species. platensis (Torzillo et al. This suggests that the microalgae cultures in production are unable to utilise all of the light provided and their concentration stabilises before complete extinction. However. A. R. and 66 g m 2 for Arthrospira platensis (calculated from Torzillo et al.. some results do not fit with this simple assertion. 1988). Appl. It is the energy transmitted throughout the culture and is the coefficient of extinction of the culture. which was confirmed by direct measurements. & Durand. 10.115 m2 g 1. J. and provided that their productivity remains at a maximum. Reported areal densities are in the vicinity of 40 g m 2 for Dunaliella salina (Grobbelaar 1994) and for Scenedesmus sp. Hartig et al.18. The calculation of the transmission through the continuous culture of P. where S was 42 g m 2.8% regardless of the light path length of the production system used. 1986).. 83–90: with kind permission of Kluwer Academic Publishers. also known as areal density S (Soeder 1980). (1998) Comparison of artificial light photobioreactors and other production systems using Porphyridium cruentum. production values for photobioreactors usually exceed 250 mg l 1 day 1. Le Guédes. when their efficiency was assessed for different designs and for species such as A. 1986) and P. A. Although hesitant and . the transmission It/I0 remains constant and equal to e S. representing a 100-fold increase compared . can reach 100 g l 1. Fermentation technologies. the technology developed by Muller-Feuga et al.0 cm exposed to natural light. the diameter of the photobioreactor tubes has tended to decrease from 100–300 mm in the first vessels (Torzillo et al.19).7 g l 1 day 1. The closed photobioreactors are surely the most efficient. which are widespread in the food and pharmaceutical industries. high irradiance and thermal stability. 6.b) uses artificial light. There is a risk of damage to the photosynthetic system when exposed to high light intensities. requiring the least unfavourable compromises. such as carotenoid synthesis.4. This diversity suggests that microalgal photoautotrophic biotechnology has not yet reached its maturity. provide high-quality products. Appropriate technical solutions for cooling outdoors facilitites are required. As observed by Borowitzka (1996).3 Heterotrophic production Some microalgae are able to use organic substrates efficiently in darkness and are thus potentially suitable for production by fermentation. 1994). whereas Molina-Grima et al. Tredici et al. such as Nannochloropsis sp.240 Live Feeds in Marine Aquaculture burdened by some failures. decay processes and dark respiration may result in a considerable loss of biomass at night. (1998a. but are more often close to 40 g l 1 (Running et al. As an example among others. have been developed by microalgae to survive what appears to be a recurrent crisis.8 h instead of 16–24 h). but designs and operation modes under investigation are still quite diverse. This study investigated the productivity of flat plate reactors of light path length ranging between 1. Maximum volume productivity reached 1. This approach offers many technical advantages compared with the photoautotrophic method. even though some commercial products and services have already been marketed. mixing and cell fragility. (1995) measured night losses smaller than 8% for Phaeodactylum tricornutum. 1986) to 40 mm and less in current types.5–4.3 and 17. using plates or tubes.. In addition. Doubling times are then drastically reduced compared with those of photoautotrophy (3. (1991) mentioned differences between day and night concentrations as high as 42% for the cyanobacterium Anabaena azollae. 6. As a result. glass or plastic. the most efficient protection against high irradiance remains self-shading. volume productivity greater than 200 g l 1 day 1 can be reached. industrial and commercial developments implementing photobioreactor technology have considered microalgae of interest for the production of natural substances. sun or artificial light. which reduces costs to levels of a few dollars per kilogram of dry weight. is of stainless-steel and glass modular conception. Fermentation volumes are huge (up to 500 m3). Sunlight can reach more than 2000 Einstein m 2 s 1 for PAR at midday. Such cultures are necessarily monoseptic. More recently. The production of microalgae in photoautotrophy is influenced by a series of biological antagonistic effects such as access to light and self-shading. The final concentrations of heterotrophic cultures of Chlorella sp. especially since concentrations of the organic substrate and oxygen can be as high as their solubility allows and also homogeneous in the whole reaction volume. The reduction in the light path length of a culture is generally associated with an increase in the concentration and productivity. for the feeding of aquaculture larvae (Zou & Richmond 1999). attention has been drawn to the production of algae of interest. Although several mechanisms. and allows axenic and mixotrophic cultures (Fig. an intermediate solution consisting of heterotrophy in light. 6. although some biocompounds such as pigments are not synthesised in heterotrophy (Grobbelaar 2000).5 m2. is still under investigation. illuminated surface: 4. three of them in parallel being represented here. temperature. However. pH. The synthesis of PUFA does not seem to be affected. Liquid and gaseous substrates are filter-sterilised at the inlet and at the outlet.19 Example of an artificial light photobioreactor. their application is restricted to those species that are able to grow and to produce the key metabolites in these conditions. These biochemical modifications may downgrade the nutritional value of the algae. Steam can be admitted into the reactor at several points for initial sterilisation. pressure and absorption are measured in line and regulated. Continuous cultures can be run according to both turbidostat and chemostat modes. . Culture volume: 300 litres. The culture is impelled by a propeller and an air-lift along a loop passing through eight series-connected light chambers per production set. although heterotrophic cultures are of considerable interest because of their high productivity. Mixotrophy. dissolved oxygen.The Microalgae of Aquaculture 241 Fig. with photoautotrophy. 1 Skeletonema sp. entrepreneurs are reluctant to take a technological leap that would require significant investment and the recruitment of more qualified staff.11 Comparison of the concentration.7 100–200 100 10 Chini Zittelli et al. the techniques of microalgal production used in aquaculture hatcheries are far less efficient than those of other sectors of application. (1991) Gladue & Maxey (1994) Barclay et al. the latter species is in high demand in Asia in the field of dietetics. Why do aquaculture operators fail to use more efficient technologies allowing both cost reduction and improved quality? First. it should be recalled that microalgae are intermediate products requiring further processing and not a marketable end-product on which the profitability and image of the company are based. Table 6. (1993b) Type of production system Tanks Species T-iso 0.4. productivity and cost price of some aquaculture microalgae for various types of production system. (Lee 1997). the technological advances in the production of artificial diets could make this leap unnecessary. The example of shrimp aquaculture shows that microalgae can be replaced by microencapsulated food. such as Tetraselmis suecica (Day et al. (Barclay et al. Crypthecodinium cohnii Schizochytrium sp.02 Order of magnitude of cost price Calculation (US $ kg 1) data from 1000 Bennemann (1992) Brown et al. other species have been proposed because of their high DHA content. (1999) Zou & Richmond (1999) Day et al. and Korea has an ambitious production programme of 1000 t per year (Apt & Behrens 1999). (1994) De Swaaf et al. Consequently.242 Live Feeds in Marine Aquaculture The first heterotrophic microalgae that appeared on the market as food for rotifers were chosen from those already in use. Pavlova lutheri Nannochloropsis sp.4 Discussion The productivity of the microalgal systems used in aquaculture hatcheries is10-fold lower than that of photobioreactors. 6. Photobioreactors Nannochloropsis sp. Moreover. probably because of biomass processing that decreased its nutritional value. The cost prices of the biomass produced with these systems are directly related to this evident disparity. 1994) and the dinoflagellate Crypthecodinium cohnii (De Swaaf et al. as the PUFA composition of this alga is not satisfactory for aquaculture. such as the diatom Nitzschia. The former species had no commercial success.11). The Japanese market represents 500 t per year. However.5–1. which is in turn 10-fold lower than that of fermentation techniques (Table 6. the thraustrochytrid Schizochytrium sp. Thus. 40–60 0. 1999 . a practice that could one day be extended to larval rearing of small-larva fish and most molluscs (Muller-Feuga 2000). 1991) and Chlorella sp. However. 1999). Volume productivity of dry Concentration biomass (g l 1) (g l 1 day 1) 0. 1–5 Fermentors Tetraselmis suecica Cyclotella cryptica Chlorella sp. . Aquat. 179–182. Perez-Camacho. & Coral.. J. Biophys. protein damage and turnover. (1997) Some ultrastructural observations of a thraustochytrid (Protoctista. This accounts for the success of live. Fernandez-Reiriz.. (1999) Optimum production conditions for different high-quality marine algae. Tocher. J... Aiba. 6. Inactivation. R. Microbiol. & MacLachlan J. Al-Musallam. offering constant. (1996) Metabolic activity of estradiol in Chlorella vulgaris Beijerinck (Chlorophyceae). . 85–156. P. In fact. Hydrobiologia. (1999) Commercial developments in microalgal biotechnology.J. & Dias. high culture concentrations are essential since they reduce the volume of water handled and transported. 23. 73–78. Ackman. The recent marketing of DHA-rich heterotrophic species is also a response to the demand for suitable food for live prey of fish larvae. 18. S. 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Watanabe, T., Izquierdo, M.S., Takeuchi, T., Satoh, S. & Kitajima, C. (1989) Comparison between eicosapentaenoic and docosahexaenoic acids in terms of essential efficacy in larval red seabream. Nippon Suisan Gakkaishi, 55, 1635–1640. Wei, X.M., Xu, Z.C. & He, J.J. (1986) Effects of temperature and specific gravity of seawater on the multiplication of calcareous Chaetoceros. Taiwan Strait, 5, 97–100. Whyte, J.N.C. (1987) Biochemical composition and energy content of six species of phytoplankton used in mariculture of bivalves. Aquaculture, 60, 231–241. Wikfors, G.H. (1986) Altering growth and gross chemical composition of two microalgal molluscan food species by varying nitrate and phosphate. Aquaculture, 59, 1–14. Wikfors, G.H. & Patterson, G.W. (1994) Differences in strains of Isochrysis of importance to mariculture. Aquaculture, 123, 127–135. Wikfors, G.H., Patterson, G.W., Ghosh, P., Lewin, R.A., Smith, B.C. & Alix, J.H. (1996) Growth of post-set oysters, Crassostrea virginica, on high-lipid strains of algal flagellates Tetraselmis spp. Aquaculture, 143, 411–419. 252 Live Feeds in Marine Aquaculture Wilson, D.P. (1946) The triradiate and other forms of Nitzschia closterium (Ehrenberg) Wm. Smith, forma minutissima of Allen and Nelson. J. Mar. Biol. Assoc. UK, 26, 235–270. Wong, M.H. (1977) The comparison of activated and digested sludge extracts in cultivating Chlorella pyrenoidosa and C. salina. Environ. Pollut., 14, 207–211. Wood, B.J.B., Grimson, P.H.K., German, J.B. & Turner, M. (1999) Photoheterotrophy in the production of phytoplankton organisms. J. Biotechnol., 70, 175–183. Zarrouk, C. (1966) Contribution à l’étude d’une cyanophycée. Influence de divers facteurs physiques et chimiques sur la croissance et la photosynthèse de Spirulina maxima (Setch et Gardner) Geitler. Thesis, 109 pp. University of Paris. Zou, N. & Richmond, A. (1999) Effect of light-path length in outdoor flat plate reactors on output rate of cell mass and of EPA in Nannochloropsis sp. J. Biotechnol., 70, 351–356. Chapter 7 Uses of Microalgae in Aquaculture A. Muller-Feuga, R. Robert, C. Cahu, J. Robin and P. Divanach 7.1 Introduction Unlike their terrestrial equivalents, aquatic animals used as food by humans are rarely herbivorous at the adult stage. Only filtering molluscs and a few other animals are true plankton feeders throughout their lifetime. Most farmed aquatic animals are carnivorous from their postlarval stage and sometimes omnivorous. However, microalgae are required for larval nutrition during a brief early period, either for direct consumption (molluscs and penaeid shrimp) or indirectly as food for live prey fed to small marine fish larvae. Even when necessary for a short period only, microalgae are crucial as they determine (to various extents) the supply of juveniles available for production. Freshwater species such as salmonids do not depend on microalgal production for their culture. Their eggs have sufficient reserves to hatch large larvae capable of feeding directly on dry particles. Certain marine species such as the European sea bass have larvae that are large enough to feed directly on Artemia nauplii. The main microalga-consuming aquaculture groups include filtering molluscs, penaeid shrimps and small larva fish. Overall world production of these microalga-consuming species reached 12 106 t in 1999, i.e. 28% of world aquacultural production (FAO: Shatz 2000). Filtering molluscs constitute in weight the most significant contribution to aquaculture production, with a total of 10 106 t in 1999, and a 23% increase over 5 years. Nutritionally, these harvests are dependent on wild phytoplankton in natural water masses circulating around the livestock, and juvenile supply comes mainly from natural spat collection. However, hatcheries in which larval and juvenile production depends on cultured microalgae are assuming an increasingly important role (Muller-Feuga 2000). The algal requirements of mollusc larvae are considered in Section 7.2. Farmed shrimp reached 1.2 106 t in 1999, and exhibited a 19% increase over 5 years. Production is carried out mainly in subtropical regions of America and south-east Asia. Microalgae are necessary from the second stage of larval development (zoea), and in combination with zooplankton from the third stage (mysis). Although of short duration, these larval stages require microalgal culture facilities, which vary with the size of the hatchery and the extent to which medium parameters are controlled. Although the trend is to substitute cultured microalgae with dry formulated feeds, microalgae are still necessary (Rosenberry 1998). Section 7.3 considers algal requirements for shrimp larvae. Marine finfish aquaculture reached 0.8 106 t in 1999, and is sharply increasing worldwide at a rate of 55% over 5 years. Small larva species, such as sea bream (146 103 t in 5 75 0 62.5 —a 12. This eliminates almost all of the natural phytoplankton. see Chrétiennot-Dinet et al. usually by fine filtration (0.5 considers other roles of phytoplankton in aquaculture and the possible mechanisms involved. Many attempts have been made to determine which of the microalgal species provide the best food value in terms of mollusc optimal growth and survival (for a review.5 37. mollusc development is closely related to the quantity and quality of phytoplankton produced. Consequently. Fifty species have been tested on larvae and juveniles of commercially raised bivalves.1 shows that the relative importance of some species has changed significantly in recent Table 7.2–1. Molluscs. Section 7. and a variety of intensive and semi-extensive technologies has developed along with this practice. but this is a much less efficient alternative to phytoplankton.0 m) and/or ultraviolet (UV) treatment (Robert & Gérard 1999). 1986).5 12. Class Bacillariophyceae Bacillariophyceae Bacillariophyceae Bacillariophyceae Bacillariophyceae Prymnesiophyceae Prymnesiophyceae Prymnesiophyceae Prasinophyceae Prasinophyceae Chlorophyceae Eustigmatophyceae . are fed microalgae directly. which must then be replaced by dense artificial cultures. unlike fish and crustaceans. clone 3H Isochrysis galbana Isochrysis affinis galbana (clone T-iso) Pavlova lutheri Pyramimonas virginica Tetraselmis suecica Dunaliella sp. Nannochloropsis occulata From Robert and Trintignac (1997a). to avoid bacterial diseases. 7. The survival and growth of various marine fish larvae are improved by the addition of microalgae. Moreover. Section 7.2 Microalgae as Food for Molluscs The culture of microalgae is of fundamental importance to commercial hatcheries rearing marine molluscs.254 Live Feeds in Marine Aquaculture 1999) and flatfish (31 103 t in 1999).5 62. The best results are obtained with live cultures added to. since they are currently the only suitable food source. They can be fed yeast-based artificial feeds.5 25 25 25 Coutteau & Sorgeloos (1992) 37 53 14 5 33 19 72 26 —a 35 9 —a Microalgal species Chaetoceros calcitrans Chaetoceros gracilis Skeletonema costatum Phaeodactylum tricornutum Thalassiosira pseudonana. require small live prey that feed on phytoplankton. including 12 commonly used in mollusc hatcheries. the seawater used for rearing is purified. Utilisation frequency (%) Walne (1970) 40 —a 20 50 40 80 20 70 0 60 0 0 Lucas (1980) 37. a No data. Microalgal cultures are necessary because the concentration of natural phytoplankton in the seawater used in the hatchery is generally insufficient for optimum growth of the high densities of larvae and juveniles reared. Table 7.4 considers the conditions for using microalgae as food for live prey. or grown directly in tanks with live prey and/or larvae.1 Utilisation frequency of microalgal species in a mollusc hatchery. 2. depending on population blooms (Baldwin 1995.1b).8 m). 7.2.1 Microalgae as a potential food source in mollusc hatcheries Four criteria are required for a microalga to qualify as a potential food source for bivalves in a hatchery: size. They need only yeast extract and vitamins for their larval development and adopt a particular feeding strategy after metamorphosis that consists of symbiosis with the dinoflagellate Symbiodinium microadriaticum (Fitt et al. C. . they also selected small food particles (0. However. I. 1997). lutheri and T. small larvae fed mixtures of four cultured microalgae ranging in size from 1 to 11 m preferred those of 1 m. which is widespread in France (Sauriau & Baud 1994). aff. 100–125 m. When fed on natural assemblages of phytoplankton. good nutritional value and ease of bulk production. Crassostrea virginica larvae over 300 m were able to graze prey as large as 30 m. 1984). P. whereas the low larval growth rates obtained with Phaeodactylum tricornutum. 1986) has increased its use. They also found that eight algal species (Chaetoceros calcitrans. and that most hatcheries cultured between two and five different algal species. but only 3% had cells of 15–25 m (Raby et al.Uses of Microalgae in Aquaculture 255 years. I.e. Moreover. Conversely.5 m3. more than half (55%) of all veligers of scallops (Placopecten magellanicus). (Gallager 1988). 2-day-old clam larvae (mean length 100 m) ingested on average 48 Synechococcus spp. 7. to large. such as dinoflagellates (Baldwin & Newell 1991.5 m). i. while large larvae preferred those of 11 m. When offered a 50:50 mixture of T-iso and Synechococcus spp. such as the cyanobacterium Synechococcus spp. 200–290 m) showed a strong preference for 2–4 m particles (Fig.7 m) for every T-iso cell (mean diameter 4. C. 1995). digestibility.1..1 Size Under controlled conditions. The recognition of Chaetoceros gracilis as a valuable species (Enright et al. galbana. representing 90% of the volume of algal culture produced. costatum. Thus.2–0. which is more tolerant to high temperatures (Ewart & Pruder 1981). This mean ratio dropped to 3:1 in 10-day-old larvae (mean length 234 m).5 m in diameter. reflecting a better knowledge of their food quality and/or feasibility for bulk production. this study underestimated the production of S. indicating a clear relation between prey and larval size (Gallager 1988). Skeletonema costatum. Coutteau and Sorgeloos (1992) found that 50% of hatcheries produce less than 5 m3 of microalgal culture per day. when fed natural suspensions ranging in size from 1 to 10 m. Pavlova lutheri and Tetraselmis suecica explain their decreasing use. Thalassiosira pseudonana clone 3H. Isochrysis galbana has been replaced by Isochrysis affinis galbana ‘Tahiti’ (clone T-iso). galbana. clam larvae (Mercenaria mercenaria) can actively ingest food particles as small as 0. However. mussels (Mytilus edulis) and clams (Mya arenaria) collected in the field contained cells of 5–15 m in their stomach. Baldwin & Newell 1995). 25% 10–50 m3 and 25% up to 110 m3. confirming the relation between prey and larval size (Fig. in natural surroundings.1a). However. 7. C. suecica) were most widely used. virginica larvae of all sizes (small. virginica larvae are able to graze over a wide phytoplankton size range. cells (mean diameter 0. a daily mean production per hatchery of 13. 7. Among the hatchery molluscs that do not require microalgae are the giant clams (Tridacna gigas and Hippopus hippopus. gracilis. and Chaetoceros calcitrans forma pumilum by 3–4-day-old C. (1995) Selective particle ingestion by oyster larvae (Crassostrea virginica) feeding on natural seston and cultured algae. Conversely.2 0. edulis and Pecten maximus (Babinchak & Ukeles 1979. A lack of appropriate digestive enzymes . (1984). virginica larvae above 160 m. Pro: Prorocentrum mariae-lebouriae. With permission of Springer-Verlag.7 0. but not well digested. T-iso: Isochrysis affinis galbana. 7.4 0.6 Selectivity (Wi) 0. Nannochloris atomus and Stichococcus bacillaris (Robert 1998). Tetraselmis suecica (Le Pennec & Rangel-Davalos 1985).7 Selectivity (Wi) 0. which become larger as molluscs grow. Lucas & Rangel 1981. and measure 10–20 m for C. Dunaliella primolecta. according to Ukeles and Sweeney (1969).5 0.. (Reproduced from Baldwin. these studies showed that those species mentioned above are normally ingested. some microalgae have shapes that make their ingestion more difficult. virginica. B.) 106 µm-larvae 290 µm-larvae As noted by Fritz et al. Biol. 95–107.1 Food selection (Wi) patterns of Crassostrea virginica larvae (a) on different sizes of natural phytoplankton assemblages and (b) on mixtures of cultured microalgae.0 (Syn) 4.2 0. virginica.2 Digestibility Some authors have considered that the low larval growth rates obtained with some microalgae can be explained by the thickness of their ‘cell wall’ that makes their digestion difficult. M.S. gigas larvae (Robert et al.1 0 1.4 0. Mar.2.1 0 1–2 2– 4 4– 6 125 µm-larvae 260 µm-larvae 6–8 8–10 Phytoplankton size fraction (µm) (b) 0.1. Pavlova lutheri is highly ingested and well digested by the larvae of C. This is the case with Chlorella autotrophica (Babinchak & Ukeles 1979). Dun: Dunaliella tertiolecta. Le Pennec & Rangel-Davalos 1985). 123. resulting in poor larval growth for Crassostrea gigas or C. the critical size of selected cells smaller than 10 m probably depends on mouth and oesophagus diameters. for example diatoms with long spines (Robert et al. Syn: Synechococcus bacillaris. 1989). On the basis of epifluorescence microscopy observations. Moreover. The horizontal line across each graph represents neutral selection.3 0.0 (Pro) Phytoplankton size (µm) and composition Fig.2 (Dun) 11.5 (T-iso) 6.256 Live Feeds in Marine Aquaculture (a) 0.6 0. 1989). 7.5 0.3 0. but their role in mollusc feeding requirements remains speculative.1. which may explain why they are inadequate for young mollusc larval stages. Although the importance of highly unsaturated fatty acids (HUFA) 20:5n-3 and 22:6n-3 is well documented in vertebrate nutrition. Most algae are rich in one or both of these fatty acids. Indeed. reaching 40–100 m3 of .5 fg m 3 (Brown et al. when adequate amounts of protein and lipid are supplied.2. C. 7. carbohydrates may play an important role in balancing the utilisation of protein and lipid for biosynthesis against catabolism for energy production (Whyte et al. Specific sterols are considered essential for bivalves. From a size of 1. For example. As microalgae may lack one or more key nutrients. reports concerning the importance of lipids as an energy source for the early life stages of bivalves are contradictory (for review see Knauer & Southgate 1999). Because of the variability in carbohydrate composition. Microalgae are rich sources of two key vitamins. are not used by commercial hatcheries because of difficulties in developing large-scale production for these species.4 Microalgae bulk production Some species.2.Uses of Microalgae in Aquaculture 257 in young larvae is the current hypothesis to explain why some algal species resist breakdown. However. but also lack these fatty acids. 1989). passing intact through the gut. whereas poor development is often reported when the concentration is below 0. suecica are ‘sticky’ and foul culture tank walls. there is no clear relationship between the whole protein content of microalgae and their nutritional value for bivalves. but some species lack specific vitamins (De Roeck-Holtzhauer et al. but is generally not the major factor determining their food value. 7. For example. larvae and postlarvae. 1989). microalgae containing 1–20 fg m 3 of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) produce satisfactory growth in bivalves. The phytoplankton requirements of the Japanese oyster C. and amino acid composition cannot explain differences in food quality (Webb & Chu 1983. calcitrans forma pumilum is seldom grown in volumes over 20 litres (Helm 1990). 1986).2 Microalgal requirements in mollusc hatcheries Microalgae are used in mollusc hatcheries to feed broodstock. containing high levels of carbohydrates. enhanced the growth of Ostrea edulis juveniles (Enright et al. 1991) and their composition could account for differences in their nutritional value. Some other species such as T. the use of Chaetoceros muelleri. In particular. 7. chlorophytes are not only difficult for bivalves to digest. ascorbic acid and riboflavin. gigas and the king scallop Pecten maximus at different life stages are shown in Table 7.5–2 mm. a mixed algal diet increases the chances of achieving a balanced diet. The latter authors suggested that larval molluscs in general require 30–60% protein for good growth. In general.3 Nutritional value: biochemical composition of microalgae Gross composition differs among species. there are few consistent compositional differences between algal classes. Daily consumption in these tanks is even greater than in indoor tanks.2. 1989).2. in some instances. oyster spat are usually grown in an outdoor nursery. Brown et al.1. despite having good food value. (2001) showed that a 6% (dry weight of algae/dry meat weight) addition per day of equal quantitities of Chaetoceros calcitrans and T-iso had a positive effect on C.0 mm) 10–20 litres 1–3 months 0. . Additional food for the broodstock appears to be of less importance than the initial content of glycogen reserves before conditioning. edulis. In some cases.74 million 12. suecica. the mean fertility (number of eggs per female) of fed broodstocks originating from six different French oyster areas was 5.2. . edulis must sustain egg development. More recently. edulis produced earlier broods. 7. showed more rapid larval growth and gave greater spat yields than if no additional food was provided (Helm et al. it is not surprising that food is particularly important to the flat oyster during the considerable biological effort required by the conditioning process. large-scale algal culture (106 cells ml (Bacher & Baud 1992).2.05 .2 Phytoplankton requirements of Crassostrea gigas and Pecten maximus in a hatchery. When seawater was enriched with T.2–3. embryogenesis and larval growth in the gill cavity for at least 1 week.91 versus 0.23 million 2. 1973).5–1.0 litres 106 larvae 2–4 litres 106 postlarvae (0.1 Feeding broodstock 1 mean density) for one million 6–12 mm juveniles The effect of food on broodstock conditioning is species specific for molluscs. The parent O.21 for unfed groups. As the genus Ostrea is larviparous. Similar observations have been reported in IFREMER-Brest hatchery with flow-through systems of sand-filtered water (about 50 m mesh). (1996) showed that spat produced by starved females of the Chilean oyster Ostrea chilensis exhibited low rates of growth and survival. but it is still difficult to determine the precise role of adult reserves and of additional food.5–1 month 2–3 months From Robert and Gérard (1999). However. Alga Daily consumption Multispecific mixture Microbiological quality Rearing period per batch One breeding individual 0. food availability favours growth and maintenance rather than reproduction (Donalson 1991). Chàvez Villalba et al. Similar observations were reported in summer: 30. gigas broodstock was 60% greater when fed an algal food supplement rather than starved. which reproduces externally. high requirements. Low requirements. The same is not true for the cupped oyster Crassostrea gigas. It is generally recognised that feeding is necessary during conditioning. During spring conditioning. Volume of algae refers to Isochrysis galbana equivalents at 8 106 cells ml 1. the rate of gonad development and gamete viability did not differ significantly.258 Live Feeds in Marine Aquaculture Table 7. . gigas fertility.77 million 1. medium requirements. Millican and Helm (1994) showed that an algal diet representing 3–6% of the initial meat weight of oysters (dry weight/dry weight) per day increased larval production in O. the European oyster O. whereas Muranaka and Lannan (1984) found that the fecundity of C. Wilson et al. The number of larvae released by flat oysters was also closely related to diet quality (the poorest result being obtained with a monospecific Dunaliella tertiolecta diet). Patinopecten yessoensis. gigas eggs contain sufficient reserves (particularly essential PUFA) to ensure survival throughout embryogenesis. (1996) reported similar results for Pecten fumatus. lutheri and I.9 m) was obtained at the highest temperature (25°C) and food concentration (100 cells l 1) (Fig. virginica. The effect of food on broodstock conditioning is closely related to the reproduction strategy of each mollusc species. Sprung 1984. although not for subsequent larval growth (Utting & Millican 1997). Gonad activity suffered first at lower feeding levels. specialised storage cells appear to compensate for a lack of food.2 Feeding larvae Quantitative requirements The number of algal cells eaten by a larva per day is related to both species and size.g. Similar combined effects of food and temperature on larval growth have been reported for other mollusc species receiving monospecific diets (e. for fed and unfed oysters.11 million 7. However. Endogenous reserves deposited in eggs during vitellogenesis are an important source of energy during embryogenesis. The effect on broodstock conditioning of the addition of a mixed algal diet to the circulating water seems to be more marked for P. whereas subsequent larval growth and survival are independent of egg lipid reserves (Le Pennec et al. These results clearly indicate that food demand is closely related to rearing temperature. Heasman et al. gigas. Broodstock diet has an impact on the fecundity of the broodstock. showing that gonad condition and egg production improved as feeding rates increased from 12. As in C. respectively. but only in the female part of the gonad. gigas larvae fed a mixed diet (P. Ostrea edulis shows a higher feeding requirement than C. His et al. Rhodes & Landers 1973.2. 7.). However.5 to 100% satiation (equivalent to 0. No eggs were obtained after 28 and 45 days of conditioning when scallops were kept unfed.2. . Although a suitable algal ration for bivalve broodstock is 6% (dry weight of algae/dry meat weight) per day for most species reared at 20–22°C. maximus is positively related to lipid levels in eggs. gigas (Saout 2000). in normal conditions. 1990. With C. while muscle and the digestive gland play this role in P. with the exception of R. gigas. 7. MacDonald 1988). gigas. 7. but not on subsequent larval growth (except for Ostrea sp.3). Moreover. the hatching success rate for P. The combined effects of temperature and food concentration on the growth of C. Mytilus edulis. edulis. 1989).75 to 6 109 cells scallop 1 day 1 respectively) at all test temperatures in the 12–21°C range.6 0. 1993). while the Manila clam Ruditapes philippinarum is far less demanding (Fig. maximus than for C. Beiras & Pérez-Camacho 1994.Uses of Microalgae in Aquaculture 259 versus 8. 1988. C. O. philippinarum (size 190 m).20. Devauchelle and Mingant (1991) reported that the fecundity of the hermaphroditic scallop P. maximus at similar gametogenic stages decreased with feeding levels. the effects of food on larval development are also related to environmental conditions.2). 3% may be sufficient for species reared at lower temperatures (Utting & Millican 1997). maximus. C. Delaunay et al. whereas fecundity increased by 8–25% with a food ration of 3 109 cells animal 1 day 1 and by 30–60% with 14 109 cells animal 1 day 1. especially rearing temperature (Robert et al. the feeding requirement increases as the larva grows. galbana) showed that the maximum daily growth rate (8. –.260 Live Feeds in Marine Aquaculture 140 Thousands of cells of algae eaten per larva per day 120 100 80 60 40 20 0 100 150 200 250 300 Mean shell length ( m) Fig. gigas are also greater when larvae are fed a mixed algal diet (50 cells l 1 I. galbana plus 50 cells l 1 C. galbana).–. 7. Knauer & Southgate 1999).) Ostrea edulis.–. Brown et al. Most authors consider that the higher growth and survival rates observed when larvae are fed a combination of microalgae are due to a more balanced diet. with permission. Gerdes (1983) showed that filtration activity and food uptake for C. (From Abdel-Hamid et al. Robert & Trintignac 1997a. 7. 1992. Higher growth rates Te mp era tur e( °C ) . Webb & Chu 1983.4) that a mixture of algal species provides a better food supply than any individual species (for reviews.) Qualitative requirements It has been clearly established (Fig. (-------) Ruditapes philippinarum.3 Effect of different concentrations (cells l 1) of a mixed diet (Pavlova lutheri and Isochrysis galbana) on young Crassostrea gigas larvae at different temperatures. (From Utting & Spencer 1991.) 10 1) Daily growth rate (µm day 8 6 4 2 25 0 25 Food co ncen 50 100 tration (c ells µl 1) 13 20 Fig. calcitrans) compared with an equivalent monospecific ration (100 cells l 1 I. (–. see Ukeles 1975.2 Quantitative feeding requirements of some mollusc larvae expressed as the number of algal cells (equivalent in size to Isochrysis galbana) eaten per day at 24°C. 1989. (_______) Crassostrea gigas. This was clearly apparent for larvae with a shell length of more than 120 m. 7. However. g. 1994.2. Skeleto: Skeletonema costatum. they can accept a larger phytoplankton size range. Brown et al. PT: P. growth is largely influenced by the amount of food available. Lu & Blake 1996). Flow-through systems (up. spat growth and survival rates are affected by factors such as species-specific requirements (e. whereas higher flow rates result in an increase in energy expenditure and a waste of algal culture. lutheri T-iso. 7. less than optimal flow rates result in a serious reduction in the rate of spat development.69 l larva 1 h 1. and potentially more efficient digestive tract.66 0. Laing 2000. As noted above for larvae.3 Feeding spat Nursery cultures of spat from settlement to a size suitable for growth in the sea are crucial to the success of hatchery-based mariculture operations.g. 1998. the rearing systems used cause flow rates to be critical (Rodhouse & O’Kelly 1981).81 7. spat are more tolerant than larvae to monospecific diets. Walne 1970.g. However. As spat densities are very high. 1986. the large-scale production of selected microalgae by conventional photoautotrophic means is expensive. It represents nearly 50% of the operating costs . 1992.4 Effects of monospecific and plurispecific diets on the growth of Pecten maximus larvae.18 g algal dry weight larva 1 day 1). PTS: P. At this stage. pumping) is often found at this stage (Robert & Nicolas 2000). A compromise between biological efficiency and operating costs (heating. T-iso: Isochrysis affinis galbana. Enright et al.) were achieved as a result of a nearly two-fold increase in filtering activity with a plurispecific compared with a monospecific diet (18.06 and 9. Pavlo: Pavlova lutheri.Uses of Microalgae in Aquaculture 261 250 Larval length (µm) 200 Unfed Skeleto Pavlo T-iso PT PTS 150 100 0 5 10 15 20 Time since fertilisation (days) 25 Fig. 7. Higher food uptake may also account for the faster development generally observed when larvae are fed mixed diets. respectively) and a large increase in filtered algae (0. Moreover. Nicolas & Robert 2001). Concerning this last aspect. O’Connor et al.2.3 Microalgal substitutes for bivalve feeding As indicated in Chapter 6. (From Robert & Trintignac 1997a with permission. Laing & Millican 1986. Robert & Nicolas 2000) and effects of mixed diets (e. Albentosa et al. 7.or downwelling) are generally used for spat development. physical parameters such as temperature (e. Beiras et al.85 0. even though food quality is still important. lutheri T-iso S. costatum.83 0.2. because of their greater size. 1994.45 versus 0. have the potential to replace the fresh microalgal cultures used to rear larvae (a) 250 200 150 100 50 0 Spat length (mm) (b) 6. for C. sp.. Robert & Trintignac 1997b.5 Effects of live algae and mixed (20% fresh and 80% concentrate) diet using different algal species on the growth (mean SE) of (a) Crassostrea gigas larvae 2 weeks after fertilisation and (b) spat 16 and 28 days after the beginning of the postlarval feeding trial. 1999. ten C. Concentrates produced by centrifugation and stored at 2–4°C for 1–8 weeks have been used successfully as part of mixed or complete diets for larval or juvenile bivalves (Nell & O’Connor 1991. 207. calcitrans. C. liposomes. conc: concentrate. although some have proved useful in a mixed diet with live microalgae. A 28 day feeding experiment with oyster spat showed no difference between C. calcitrans forma pumilum) live microalgae (Fig. 7. Recent studies have shown that microalgal concentrates.0 . when appropriately harvested and stored.262 Live Feeds in Marine Aquaculture in mollusc hatcheries.0 3. whereas I. gigas larvae and spat. yeasts.0 Day 16 Day 28 Larval length (µm) 5. together with 20% live microalgae. ten conc C. In general. (2002) Preparation and assessment of microalgal concentrates as feeds for larval and juvenile Pacific oyster (Crassostrea gigas). concentrates gave poorer results than live microalgae (S. aff. (tenuissimus like). The alternatives tested include bacteria.5a). S. I. calcitrans forma pumilum and C.0 4. costatum and Tetraselmis spp. microcapsules. lipid emulsions and dried microalgae (for reviews. calcitrans) or equivalent to (C. costatum. (Reprinted from Brown. Heasman et al. ten conc C. T-iso: Isochrysis affinis galbana. aff. Knauer & Southgate 1999). sp. galbana exhibited lower values (31%). C. pum 2. pum: Chaetoceros calcitrans forma pumilum. On three occasions. ‘tenuissimus-like’. Copyright 2001. pum conc T-iso (control) C. 2001). concentrates showed an efficiency better than (C. pum C. ‘tenuissimus-like’.) C. ten: Chaetoceros sp.0 C. whereas the flagellates P. The efficiency of these concentrates on larvae varied with the species tested and the experimental conditions. ten T-iso (control) C. pum conc Diet Diet Fig. & Robert. Aquaculture. galbana). 7. On other occasions. Robert et al. see Coutteau & Sorgeloos 1992. with permission from Elsevier Science.R. lutheri and Isochrysis sp. the lower nutritional value of these products makes them unsuitable as complete feeds. Five of these microalgae were stored at 4°C for 10–20 days and tested as a diet. 7. R. McCausland et al. 2000. A novel method for preparing microalgal concentrates based on chemical flocculation (Knuckey 1998) has recently been applied to seven marine microalgae grown in 300 litre tanks (Brown & Robert 2001). (T-iso) are damaged by centrifugation and deteriorate rapidly. whether live or concentrated (Fig. 289–309. Species with good nutritional value and the best shelf-life for oyster larvae and spat include C. Six species were successfully harvested with flocculation rates of 58–81%. An effort has been made to find nutritionally adequate alternatives that are more cost-effective than algae produced on-site. C. M.5b). Microalgal concentrates represent the most promising off-the-shelf alternatives. do not appear to select algae according to size or ‘taste’. After a second metamorphosis. Although the total world capture of shrimp remains at a constant level. and involves endogenous feeding. but ingest a variety in similar ratios to those in plankton. Preston et al.g. Despite the development of different inert microparticulate diets.1 Development of penaeid shrimp Two days are required for embryonic development of penaeid shrimp in the egg. concluded that penaeid zoea do not select particular algal species. Therefore.3 Microalgae as Food for Shrimp Penaeid shrimp are omnivorous during juvenile and adult life and show filter-feeding behaviour during larval stages.or large-scale hatcheries. feeding mainly on small animal prey. 7. 7. flocculation. Microalgae are the most important dietary source during larval stages in the wild and contribute to the nutrient supply for postlarvae and juveniles in estuaries. and the presence of microalgae in the rearing water improves survival and growth during these stages (Fig. Larval life then consists of a succession of moults and metamorphoses. microalgae are still cultured in hatcheries to feed shrimp larval stages and as an additional dietary source for shrimp in extensive and intensive growth ponds. Diatoms and prasinophytes are good candidates for microalgal concentrates. After a first metamorphosis. The postlarval supply for aquaculture comes partly from the wild. bulk filtration) and/or preservation techniques (e.g. algal species used to feed shrimp in hatcheries are generally chosen according to size and their ability to grow in culture conditions. However. mysis and postlarvae still ingest microalgae. The total duration of the zoea phase is around 5 days at 28°C. there is considerable scope for the development of improved microalgal harvesting (e. depending on the species.Uses of Microalgae in Aquaculture 263 and juvenile bivalves. penaeid shrimp farming has steadily increased since the early 1980s and is now an important part of world aquaculture production. such as Brachionus. naked flagellates are difficult to preserve. Brown & Robert 2002). whereas further research is required for the more fragile prymnesiophyte species. the use of antioxidants and other additives) or specific storage protocols with regard to light. Thus. The first phase includes five or six naupliar stages. 7. The highest survival rates in that study were associated with high levels of diatom flora. Regardless of the harvesting methods applied today (centrifugation. during which filter-feeding behaviour is predominant and hatchery production is based on a supply of microalgae.2 Selection of algal species used for rearing shrimp larvae Shrimp larvae.3.6). The postlarval stages begin after a final metamorphosis. Heasman et al. and the lowest with cyanobacterial flora. who studied gut content in Penaeus esculentus zoea reared in grow-out ponds. (1992).3. Mysis and postlarvae exhibit predatory behaviour. showing satisfactory shelf-life. Artemia or copepods. larvae go through three zoea stages. 2000. temperature and the mixing atmosphere. but increasingly from small. 7. unlike some other crustacean filter-feeders. the larva is in mysis phase for 3 days. The algae used are generally 5 m . With permission of SpringerVerlag. Few species have been tested in the laboratory or used in intensive shrimp hatcheries. such as Prorocentrum micans (32 25 m).) (e.000 algae cells ml 1 120–200 mg microparticles 10.5 mm No feeding Mysis 5–8 mm Fig.000 algae cells ml 1 1.000–20. but it is unclear whether these poor results were attributable to the biochemical composition or the size of the algae (Sanchez 1986). Diatoms such as Chaetoceros and flagellates such as Tetraselmis have provided good results for the growth and survival of penaeid larvae.) to 10–20 m (e.000 Artemia nauplii 15.264 Live Feeds in Marine Aquaculture Days Development stages Daily feeding ration (for 1000 larvae) 1 Nauplius 2 3 4 5 6 7 8 9 10 11 12 13 14 8 –15 mm Post-larvae 300–500 mg microparticlate diet 10. algal biochemical composition has been studied to determine which algae satisfy the nutritional requirements of shrimp larvae.000 algae cells ml 1 0. (From Cahu.g. respectively (Galgani & Aquacop 1988). In: Nutrition and Feeding of Fish and Crustaceans. Low survival was recorded in zoea stages. Isochrysis sp.6 Development and feeding sequences for penaeid shrimp larvae.5 –1. Nutrition and feeding of penaeid shrimp larae. 7. An experiment based on formulated microparticulate diets showed that zoea I and II stages of penaeid species can ingest spherical particles up to 35 and 50 m in diameter.000 Artemia nauplii 15.5–5 mm Zoea 50–100 mg microparticles 15. C. regardless of the species used. 2001. In addition to selection based on size and culture ability.g. Larger cells have been tested. D’Souza & Loneragan (1999) found that survival was two-fold higher when Penaeus . Tetraselmis chuii) in diameter. Uses of Microalgae in Aquaculture 265 Table 7. clone T-iso D’Souza & Loneragan (1999) japonicus larvae were fed Chaetoceros muelleri rather than Dunaliella tertiolecta.2) where t is the experimental time (day). but the results are not conclusive (D’Souza & Loneragan 1999). suecica References Cahu (1979) Penaeus monodon Penaeus vannamei Better growth and survival with C. shrimp larvae can ingest a sufficient amount of food per time unit to sustain their development.000 cells ml 1) may stress the larvae and induce mortality. Very poor survival with D.1) (7. 7. which have swimming ability. muelleri and Dunaliella tertiolecta T. n is the number of larvae. suecica Isochrysis galbana. the mobility of nauplii is poor. Shrimp species Penaeus japonicus Microalgal species Monochrysis lutheri Pheodactylum tricornutum Pseudo Isochrysis paradoxa Tetraselmis suecica Chaetoceros calcitrans Tetraselmis chuii Isochrysis sp. Above this concentration.3 Main algal species tested in penaeid larval rearing. The threshold concentration is estimated at 20. At low concentrations. Several experiments have been conducted to determine whether a combination of algae was better than a single species. and food availability is directly related to its concentration. As for other filter-feeders.3 Ingestion and filtration rates for shrimp larvae fed microalgae Unlike zoea. Kurmaly et al. C0 and Ct are the initial and final concentrations respectively. calcitrans Best growth and survival with Isochrysis sp.3. below which growth is reduced. an algal threshold concentration can be determined. Both rates are dependent on the developmental stages of the larvae and on cell concentration. and V is the volume of water. tertiolecta Tobias-Quinitio & Villegas (1982) Sanchez (1986) Penaeus monodon Kurmaly et al. (1989) obtained good growth and survival for Penaeus monodon larvae when Skeletonema costatum and Rhodomonas baltica were used (Table 7. Conversely. very high concentrations (more than 75.000 cells ml 1 for the alga Tetraselmis. penaeid larvae appear to be less efficient filter-feeders than copepods (Cahu 1979). The filtration rate F and the ingestion rate I can be calculated according to the formulae of Paffenhöffer (1971): F V [log(C0 ) log(Ct )] tn I V (C0 Ct ) tn (7.3). The filtration rate is . Bacteriastrum hyalinum Prorocentrotrum micans Tetraselmis chuii Dunaliella tertiolecta Rhodomonas baltica Skeletonema costatum Observations Best growth and survival with T. (1989) Penaeus monondon Penaeus japonicus Penaeus semisulcatus Chaetoceros muelleri Best growth and survival Tetrasemis suecica with C. Nevertheless. the main parts of the filterfeeding apparatus. Optimal protein content in diet dry matter is between 30% (Khannapa 1979) and 50% (Kanazawa 1990). japonicus larvae decreases from 55 to 45% when carbohydrate concentration in the diet increases from 5 to 25%. become reduced in size. but it is difficult to understand which of the algal nutritional constituents are essential for larvae. 7.000 for mysis II (Cahu 1979). algae (e. indicus fed T. need to be added to the diet.266 Live Feeds in Marine Aquaculture inversely related to algal concentration. After the mysis stages. Postlarval ingestion rate reaches only 150. The optimal protein level in a shrimp diet depends on the carbohydrate. with carbohydrate supplying up to one-quarter of this energy.g.000 and 20. Ash content is very high (nearly 40% of dry matter) and does not contribute to the energy supply. aff. suecica is 70.000 for zoea III and 400. such as Brachionus and Artemia. Only the recent use of purified microparticulate diets has allowed a better relationship to be established between larval growth and some biochemical components of algae. The energy value in algae is around 16 kJ g 1 dry weight. Experiments conducted with live food (algae.000 cells algae individual 1 day 1.4 Nutrient supply from algae in relation to larval shrimp requirements Despite extensive studies on the nutrition of juvenile shrimp. Brachionus.000 cells of Tetraselmis ml 1. The following microalgae densities are generally used for shrimp larvae reared in the laboratory as well as in the hatchery: 30. especially phospholipids or HUFA. 250. 200. The maximum ingestion rate calculated for P. Shrimp larvae have higher .000 for zoea II.3. The global effect of an algal species can be assessed. the concentration has no effect on the ingestion rate. Beyond this point. Thus. as it is indigestible. juveniles and even adult shrimp. the requirements specific to larvae are poorly known. there is some evidence that around 30% protein and 20% lipid in the dry matter of algae selected for aquaculture satisfy larval requirements. muelleri or I. Penaeus indicus can filter 1–4 ml of water day 1 at the zoea I stage and 3–10 ml day 1 at the zoea III stage when the concentration is between 75. galbana (T-iso). and a mysis III between one-and-a-half and three times. reaching a plateau. diatoms) inducing good larval shrimp development have a disconcerting biochemical composition in nutritional terms. suecica and 100. A high ingestion rate is necessary to sustain such a growth rate. Maxillae and maxillipeds.000 cells ml 1 of T. Teshima and Kanazawa (1984) showed that the protein requirement in P. Animal live prey.000 cells ml 1 of C. This accounts in part for the very high ingestion rate during filter-feeding stages. Similar values were found for P. Most of the studies conducted to determine whether algae satisfy larval shrimp requirements relate to lipids. It appears that a zoea I ingests between three and six times its own dry weight of algae. and filtration efficiency declines from mysis to postlarva stages. larvae shift towards raptorial feeding.000 cells larva 1 per day for zoea I. The zoea stages constitute a period of intense growth during shrimp development: larval dry weight is 4 g at the zoea I stage and reaches 27 g after 5 days of development to the mysis I stage. However. indicus fed Thalassiosira weissflogii (Emmerson 1980). microalgae are still ingested in postlarvae. Larval lipid requirements are also unclear. The ingestion rate increases with cell concentration. Artemia) or compound diets have given different results for protein requirement. 1 16. Although shrimp larvae have a greater ability than juveniles to desaturate and elongate 18:3n-3 to 20:5n-3 and 22:6n-3 (Teshima et al. b . was adequate to sustain growth and survival of penaeid larvae.2% and phosphatidylinostol only 0. muellerib Isochrysis sp. Isochrysis sp. From D’Souza and Loneragan (1999).2 18.6 32.5 17.. total lipids and carbohydrate) does not account for the differences observed in larval growth and survival. but low ARA and EPA content. nd. suecica. D’Souza and Loneragan (1999) attributed the good results obtained in Penaeus larvae fed C.3 1.4). 1985). such as C. EPA (20:5n-3) and DHA (22:6n-3). EPA and DHA). The minimum phospholipid requirement in larvae is considered to be 3.0 7.8 9. and some. consisting essentially of phosphatidylcholine and phosphatidylinositol.5 23. not determined.1 2. These fatty acids.0 22.2 12.9 17. fed lutheri P. tertiolecta were attributed to a low content of the three most important HUFA (ARA.3 7. but not at a sufficient rate to sustain high development during larval stages. are essential for larval development.Uses of Microalgae in Aquaculture 267 phospholipid dietary requirements than juveniles.7 37.6 45. the conversion rate is too low to meet larval requirements.9 2.7 16. 1992). which has high DHA content.7 3.5 4.6 33.5% of diet dry matter (Kanazawa et al. Shrimp have some ability to synthesise phospholipids. Thus.2 21. muelleri and T.9 0. spp.6 From Cahu et al.b Total saturated fatty acids Total monounsaturated fatty acids 20:4n-6 20:5n-3 22:6n-3 Total n-6 Total n-3 a 27. in which phosphatidylcholine represented 1. This was confirmed by intermediate results obtained with Isochrysis sp.2% phospholipid.4 Fatty acid contents in algae and in zoea fed these algae. Several authors have shown that fatty acid content at different larval stages reflects that of the food. Mourente et al.0 23. Penaeus Penaeus Pavlova indicus fed Chaetoceros Penaeus spp. (1995) found that a mixture of Tetraselmis plus Isochrysis. 1997). Different studies have led to the conclusion that the gross composition of an alga (protein.1 1. a mixture of algae with different fatty acid compositions.0 7. the composition of fatty acids could be a predominant factor for differences in larval growth and survival.0 21. Larvae fed with such an algal mixture have the average fatty acid content of larvae fed each alga separately.0 25.1 5. muelleri and T. provides an adequate fatty acid composition. providing 8. The optimal phospholipid level in a diet also depends on the dietary supply of HUFA (Cahu et al. Table 7. suecica to their high content of ARA and EPA.3 6.5 19. The poor results obtained for larvae fed with D. (1988).6 32. can be increased up to 20-fold in larvae (Table 7.2 nd 0.3 13.2 34. such as arachidonic acid (ARA. Kontara et al.2 40. at low concentrations in algae. expressed as a percentage of total fatty acids.2 20. These fatty acids can be concentrated in larvae.8 2. 1994. However. Algae constitute a substantial source of HUFA.5 8. 20:4n-6). lutheria muelleri fed C.1%.7 28.3 20. Hirata et al. studies conducted with semi-purified . 1997).3.5 in the other algae. japonicus larvae with soya-cake particles containing 28% protein and 10% fat. different ingredients were incorporated into particles.5 Substitution of spray-dried algae or microparticulate compound diets for live algae Attempts have been made to replace live microalgae with spray-dried algae in order to lower hatchery production costs. The best survival during zoea stages was obtained with 0. the lower growth observed in larvae fed Tetralsemis grown with a low-nitrogen medium can be attributed in part to the lower protein: energy ratio and mainly to the lower n-3 HUFA content. crab protein (Koshio et al. Merchie et al. which supports the notion that these micronutrients are of considerable importance during early larval development. Dried algae are often used in the hatchery as a supplement to live algae or microparticulate diets. but the essential fatty acid 18:3n-3 was 1. Nevertheless. Although levels as high as 3800 g g 1 dry weight were detected in laboratory-cultured Isochrysis or Chlorella. algae supply larvae with vitamins. (1990) showed that total replacement of live algae with spray-dried algae leads to a decrease in growth. 1979). it appears that 60% of live algae can be replaced by spray-dried T. The gross composition of larvae fed the two diets was not different. but are more likely to be due to culture conditions than to the species used.0 in the algae reared in high-nitrogen medium.6 times as high in larvae fed the second diet.268 Live Feeds in Marine Aquaculture Algal biochemical composition can be altered by that of the culture medium. (1994) recorded high survival and development of Penaeus chinensis juveniles fed artificial diet containing linolenic and linoleic acids in a 3:1 ratio. 1975). 1993). Thus. Large differences in ascorbic acid levels have been detected in algae. the ratio of n-3 fatty acids to n-6 fatty acids was 3. High concentrations of some vitamins (500 g g 1 of diet for ascorbic acid and 300 g g 1 for -tocopherol) have been found in developing embryos (Cahu et al. 1995). Despite the wide range of ascorbic acid concentrations reported in algae cultured in various conditions. mollusc meal (Jones et al. In addition to HUFA. Moreover. Although a concentration of 20 g g 1 is sufficient to sustain penaeid juvenile growth. lipid and n-3 HUFA content than Tetraselmis grown in a high-nitrogen medium. D’Souza and Kelly (2000) found that Tetraselmis reared in a low-nitrogen medium had a three-fold higher carbohydrate content and lower protein. The high concentrations found in algae (up to 2 g g 1) enhance the resistance of postlarva shrimp to stress conditions and bacterial infections. 1989). whereas it reached only 1. suecica. squid meal. (1975) reported the first results obtained by feeding P. (1997) estimated that 130 g of ascorbic acid g 1 of diet is needed to sustain requirements related to fast collagen formation during frequent moulting and metamorphosis. Concurrently. 1987) and fish meal (Teshima et al. This disappointing result was attributed to an alteration in the physical integrity of algal cells and the rapid loss of soluble nutrients upon placement in water. Biedenbach et al.16 mg zoea 1 day 1. 7. yeast (Jones et al. Xu et al. requirements are much higher during larval stages. metamorphosis and survival. This ratio may be important for prawn development. coinciding with faster development of zoea II. Subsequently. Studies have been conducted since the early 1970s to substitute a compound diet for algae. only one-third of this concentration was found in algae grown in a commercial hatchery (Merchie et al. such as chicken eggs (Jones et al. it is obvious that microalgae are a rich source of this vitamin for shrimp larvae. improves penaeid larval growth. Stable carbon. These algae contributed mainly to the particulate organic carbon in intensive ponds.3. Benthic microalgae also play an important role in shrimp feeding. compared with live prey (including algae). In extensive ponds. The main drawback of compound diets.000 cells ml 1). is their low stability in water. 1998). so that a nutritional effect of algae could not be implicated. and microalgae constitute a part of the diet of growing shrimp under natural conditions. but micro-encapsulation using a cross-linked protein has produced more stable particles (Kurmaly et al. and a highly significant linear relationship was observed between shrimp growth and the particulate organic matter concentration. Kumlu and Jones (1995) showed that a small quantity of algae. galbana (20. or may affect the bacterial population in the rearing water and thus contribute to establishing an early gut microbial flora in larvae (Skjermo & Vadstein 1993). Studies have investigated the physical characteristics of microparticles in an attempt to prevent nutrient leaching. Nevertheless. microalgae can supply up to 50% of the feeding ration of shrimp and also provide a means of recycling nitrogen and phosphorus leached from faeces and uneaten feed (Duerr et al. the contribution of algae to shrimp nutrition is variable. More efficient digestion of a microparticulate diet could account for growth enhancement in larvae reared in co-feeding.Uses of Microalgae in Aquaculture 269 casein-based diets have provided a better understanding of the dietary requirements of larvae (Teshima & Kanazawa 1984). Similar results have been obtained in fish: the addition of a very low concentration of I. Moss and Pruder (1995) showed that the presence of pennate and centric diatoms induced improved growth in Penaeus vannamei reared in intensive ponds.3.7 Feeding microalgae to shrimp juveniles and adults Juvenile and adult shrimp are omnivorous.6 Other roles of algae in shrimp larval growth The beneficial effect of the presence of algae on larval growth and survival has long been known. but algae will still be used quite often in co-feeding. Further ecological research could improve algal productivity and shrimp growth. 1998). gelatine or zein. alkaline phosphatase and maltase) and improvement of digestive tract maturation are responsible for the improved sea bass larval rearing (Cahu et al. as the temporal variability in algal cell density follows a bloom and crash cycle. Microparticulate feed may replace algae in the hatchery (Autrand & Vidal 1995). in addition to compound diet feeding. The concentration of algae was very low in this experiment (15. Other hypotheses have also been postulated to explain the beneficial effect of algal addition in fish and shrimp rearing: algae may stimulate the appetite of larvae by releasing components that act as attractants (Støttrup et al. 1989). 7.000 cells ml 1) induced 40% improvement in the growth of sea bass larvae fed compound diets as well as 26% survival enhancement. Microbound particles were manufactured using binders such as carrageenan. 7. 1995). The authors suggested that this positive effect was caused by alga-induced stimulation of larval digestive enzymes (mainly trypsin). sulfur and nitrogen isotope ratio techniques were used to evaluate the relative importance of algae in the . It has been suggested that enhancement of different enzymes (trypsin. Pleurosigma and Nitzschia. as nauplii are not actually grown. shrimp production is carried out in extensive ponds. 7.4 Microalgae as Food for Live Prey The first attempts at culturing live prey for aquaculture mimicked the marine food chain.4. Some algal species can also be detrimental to shrimp production. Animals showed necrosis of the epithelial lining of the midgut. each of which has its own biochemical characteristics.1 Feeding live prey with live microalgae Various authors have studied the optimal feeding rate of live prey in test-tubes containing low animal densities. and the dominant species are blue–green algae of the genera Oscillatoria. For the rotifer. RNA:DNA ratios were significantly greater in muscle tissue of diatom-fed shrimp. 7.270 Live Feeds in Marine Aquaculture nutrition of two penaeid prawns (Penaeus merguiensis and Parapenaeopsis sculptilis) in Malaysia. Differences are apparent in the fatty acid profile. Thus. rotondiformis. as the two were formerly regarded as a single species. Microalgae and animals potentially useful as live prey were identified by such experiments. As the hatchery artificial food chain has been simplified to reduce production costs. A large variety of microalgae is found in ‘lab-lab’. i. resulting in haemocytic enteritis. fed to improve their nutritional quality just before being used as live prey. In a great part of the world. whereas protein:lipid:carbohydrate ratios depend on culture conditions as well as the algal species. sterols and vitamins.e. For example. the nutritional composition of a given alga offered to a live prey has an influence on growth efficiency as well as on the quality of the prey produced. Hirayama et al. dorsal caecum and hindgut gland. the rotifer Brachionus plicatilis is the organism most commonly cultivated and studied today. Among them. toxic effects were suspected in the blue shrimp Penaeus stilirostris exposed to the blue–green alga Spirulina subsala (Lighner 1978). plicatilis may in fact concern B. but only enriched. which in turn was fed to fish or prawn larvae. Stable isotope analyses suggested that benthic microalgae are the major dietary component for prawns living in tidal creeks (Newell et al. A basic method in early research was to enclose and enrich natural water masses. In addition. thus encouraging the growth of prey organisms to support cultured fish and shellfish species. 1995). Phormidium and Spirulina. using phytoplankton to feed zooplankton. which suggests that diatoms make a substantial contribution to shortterm shrimp growth. as well as diatoms of the genera Navicula. Artemia is used as live prey because of its convenient ‘off-the-shelf’ availability in cyst form. Moss (1994) showed that shrimp fed a diatom culture composed primarily of Chaetoceros or a culture of Nannochloropsis oculata grew significantly better than those fed the macroalga Ulva or Enteromorpha. Its use today in larval rearing does not require algal production. where ‘lab-lab’ (a biological plant–animal complex) grows concurrently (Bai & Bensam 1993). (1973) found an optimal feeding . Some data for B. Microalgae support shrimp growth better than macroalgae. live prey are now often fed with a single microalgal species. lipid and carbohydrates. since the addition of concentrated food could result in detrimental effects on medium quality (oxygen depletion and metabolite toxicity). and the addition of microflagellates may provide better growth (Kraul 1989). and an ingestion rate of about 1. However. and underfeeding will decrease the growth rate and nutritional status of the animals. 7.2 Nutritional value of algae for live prey Various algal species have been compared for their efficiency as food for live prey. regardless of species (Sick 1976). Artemia of a given length are able to ingest a wide range of particle sizes. Microscopic observation of rotifers feeding on Tetraselmis shows faecal pellets containing undigested and even viable algal cells (Fig. 7. the total filtration capacity of 200 rotifers ml 1 is equal to the entire rearing volume within less than 1 h. optimal growth can only be transient. According to Frolov et al. with a preference for those between 2 and 12 m. Conversely. The results obtained for these comparisons are only of relative value as they depend on culture conditions.1 Proteins and proximate composition By varying the composition of the alga Brachiomonas submarina grown in a chemostat. which suggests that feeding activity may exceed digestion capacity.Uses of Microalgae in Aquaculture 271 concentration of around 10 g ml 1 for 150 104 cells ml 1 of ‘marine chlorella’ (Nannochloropsis). In optimal conditions. A common means of production is a semi-continuous culture in which a part of the population is withdrawn daily and replaced by algal culture. waste products have to be maintained at low levels. rapidly exhausting the algal population. Scott and Baynes (1978) found that the mean dry weight per rotifer reached around 600 ng during the first days of culture and then decreased to less than 300 ng after the algal population was consumed. there is a good correlation between protein and lipid content in rotifer and that in the algal diet. 7. Although the essential amino acid content of algae . Copepod species can feed on diatoms. for fish larvae. small diatoms with long setae (Chaetoceros) cannot be ingested. The amino acid composition of rotifers is constant and not correlated with the algal amino acid profile. consequently. However. The efficiency of an algal species may also be related to the capacity of live prey to digest cell walls.4 10 3 g individual 1min 1. Thus. raw algal cultures (around 100 g ml 1 dry weight) are generally not concentrated enough to meet the feeding demand. With a filtration rate of up to 10 4 ml individual 1 min 1.2.4. live prey production rate and density depend on the quantity of food delivered and on algal nutritive quality. as rotifers have been observed feeding on Arthrospira trichomes. This section will consider the nutritional value of algal species for live prey and. The rotifer B. plicatilis ingests small cells less than 20 m in diameter. the terms of which are examined later in this chapter. However. In that case. a rotifer population may double or triple daily. as mentioned previously for molluscs.4. A food supply compromise has then to be found for rotifer rearing. (1991). at maximum ingestion rate the same total volume of cells is ingested. another important food selection criterion could be the particle width rather than the mean volume.7). Scott (1980) obtained optimal growth/ingestion efficiency of B. plicatilis with algae containing approximately equal amounts of protein. ) has never been considered deficient for live prey. or from comparisons of copepods with rotifers or Artemia fed on the same algae (Witt et al. The ability to use and incorporate essential fatty acids from algal sources differs widely between prey types. 1997). some essential amino acids could exist in lower amounts in algae than in live prey. provided an experimental means for estimating the HUFA requirements of several fish species. Most marine animals have little or no capability to transform polyunsaturated fatty acids (PUFA) such as linoleic acid or linolenic acid into longer and more unsaturated fatty acids.5).7 Rotifer Brachionus rotondiformis fed on Tetraselmis suecica. The first evidence for the importance of dietary HUFA for marine larval growth and survival came from comparisons of live prey grown on algae or baker’s yeast (Watanabe et al. . and the DHA:EPA ratio is a key criterion for the efficiency of live prey as food for marine larvae (Table 7.2. Kleppel et al. 1979) or on algal species showing different HUFA levels (Scott & Middleton 1979).2 Fatty acids Most of the literature on the nutrition of live prey concerns fatty acids. even though their tissues contain high proportions of n-3 HUFA. These studies indicated that DHA is more efficient than EPA (these have been introduced earlier) as an essential fatty acid for marine fish. The ability of rotifers or Artemia to grow on yeast (low lipid content). 1984). which are considered of major importance in larval nutrition.272 Live Feeds in Marine Aquaculture Fig. and may reduce nutritive efficiency. (Photograph: IFREMER/Robin. 7. A DHA:EPA ratio above 1 and the addition of ARA were considered as optimal for feeding fish larvae (Sargent et al.e. i. with an addition of various oil emulsions (during feeding or final enrichment). 7. HUFA.4. (1998) found that egg production was poor in copepods fed a strain of Isochrysis unusually deficient in histidine. 7 5.8 8. (1997) Thinh et al. DHA: EPA References 0. Tisbe holothuriae Tisbe holothuriae Brachionus plicatilis Brachionus plicatilis Brachionus plicatilis Artemia (fed 24 h) Artemia (fed 24 h) Artemia (unfed 24 h) EPA 16.5 6. growth improved .2 6.3 11. The animals will be richer in HUFA if fed on HUFA-rich algae.0 2.4 15.6 0. (1997) Reitan et al.4 28. Støttrup and Jensen (1990) found that the copepod Acartia tonsa fed with Dunaliella tertiolecta (containing 18:3n-3 but no HUFAs) ceased egg production.5 7.4 22.7 9. Norsker and Støttrup (1994) found no difference in brood size for Tisbe holothuriae females fed either D. (1984) Witt et al.4 0. Moreno et al.9 — Copepods Marine planktonic copepods contain high amounts of n-3 HUFA.8 0. while HUFA-containing algae such as Rhodomonas baltica were suitable for egg production.8 0.1 1. such as baker’s yeast.5 0. baltica.0 3. (1999) Thinh et al. but also modified by elongation–desaturation capabilities and/or selective incorporation. Isochrysis galbana Pavlova lutheri Cryptomonas Isochrysis (T-iso) Control (starved) EPA DHA Prey 24.1 0. copepod production is positively related to the DHA:EPA ratio in the diet. Tisbe sp. tertiolecta or R.3 6.1 2. (1984) Witt et al.4 12.9 — 24.3 2.Uses of Microalgae in Aquaculture 273 Table 7.7 0.4 — — Copepod nauplii Copepodites Brachionus Artemia (fed 12 h) Tisbe sp.2 1.7 13. Nanton & Castell 1998).4 6.8 0. Harpacticoid copepods are easier to grow than calanoid copepods.9 — 24. (1999) Alga Nannochloris Nannochloris Nannochloris Nannochloris Chaetoceros calcitrans Dunaliella tertiolecta Isochrysis galbana Dunaliella tertiolecta Rhodomonas baltica Tetraselmis sp. the fatty acid content will show very low HUFA levels.9 — 33.9 2.6 1.4 24. Various food sources can be used.1 0. The fatty acid content of both copepod groups is influenced by dietary fatty acid.5 11. Rotifers: Brachionus plicatilis Rotifers can also be fed with various food sources.0 Witt et al.3 — 21. (1999) Thinh et al.3 0.5 0.9 — 24. It is questionable whether all species require n-3 HUFA or only C18 PUFA as essential fatty acids. (1984) Witt et al. (1984) Nanton & Castell (1998) Nanton & Costell (1998) Nanton & Costell (1998) Norsker & Støttrup (1994) Norsker & Støttrup (1994) Reitan et al. Harpacticoid copepods contain relatively suitable amounts of 22:6n-3 and ARA. even when raised on a diet deficient in essential fatty acids (Norsker & Støttrup 1994.7 0. (1997) Reitan et al.8 8.3 15.8 DHA 10.4 3. According to Payne and Rippingale (2000).9 32.2 0.2 25. even those nearly devoid of essential fatty acids. which are obtained from their phytoplankton diet in the natural environment.6 2.9 17. (1979) showed that Paracalanus parvus has the enzymatic capability to desaturate and elongate 18:3n-3 to n-3 HUFA.9 19. Tisbe sp.1 0.9 4.5 7.5 21.5 Effect of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) content (% total fatty acid) of algae on the same contents in prey fed these algae during culture or enrichment. In an axenic culture of rotifers fed with baker’s yeast.5 0.8 1.4 3. In this case.8 0.6 17.7 1. which suggests that they do not require dietary HUFA for growth and reproduction. but will have a higher DHA:EPA ratio than that of their food and are thus eminently suitable diets for marine fish larvae.17 3.2 7.9 10. 1998). but almost no DHA. but phospholipid levels remain constant for a given size of prey. Since these features all lower the postenrichment DHA:EPA ratio in Artemia. This aspect should also be considered in evaluating HUFA requirements. regardless of the diet and/or enrichment products used.274 Live Feeds in Marine Aquaculture slightly when marine oil was added to the medium (Hirayama & Funamoto 1983). but can convert some other sterols to cholesterol (Teshima & Kanazawa 1971). population growth appeared to be independent of algal content of n-3 HUFA.e. Some elongation of dietary fatty acids was observed in rotifers. algal enrichment is sometimes performed (mainly with Isochrysis). even though it corresponds to microalgal fatty acid composition (Thinh et al. Isochrysis affinis galbana has a good DHA:EPA ratio. freshwater Chlorella does not contain longer chain fatty acids than C18. 1999). as oil emulsion enrichment causes a disproportion between neutral and polar lipid contents in live prey. nauplii initially contain EPA (high amounts in ‘marine’ strains). algal species providing the best rotifer productivity are not those with the highest HUFA content. a common practice consists of using a microalgal species suitable for mass production of rotifers. newly hatched or shortterm enriched nauplii. galbana. Furthermore. Artemia show a clear tendency to incorporate less DHA than EPA from dietary fatty acids and demonstrate rapid retroconversion of DHA to EPA (Navarro et al. but oil emulsion enrichment is generally preferred. Unfortunately. 1999). In these circumstances. while rotifer fatty acid content reflected that of the algae used as food (Frolov et al.3 Other lipid components Crustaceans are incapable of synthesising cholesterol from lower units and require a dietary source of cholesterol or some sterol precursors. Compared with oil emulsions. before being distributed as food to larvae.4. so that the DHA:EPA ratio is lower than in their food (Rainuzzo et al. As nauplii already show a high fat content at hatching. they have a high EPA content. even though rotifers grown on yeast have been successfully used for prawn production. any noticeable modification in their fatty acid profile within a short period requires a high-lipid diet. aff. Artemia lacks sterol synthesising activity. For instance.2. but freshwater Chlorella or yeast is not. Even though Nannochloropsis and Tetraselmis are good food sources for rotifer production. Phospholipids are considered to be essential for the nutrition of prawn and fish larvae (Kanazawa 1993). (1981) found that cholesterol is mainly of dietary origin in the rotifer. 7. but no substantial amount of desaturation products appeared. Thus. 1991). Moreover. but provides rather low or unstable rotifer production. and the DHA:EPA ratio remains low. 1994). In various experiments comparing the effect of different algae on rotifer culture. Like other crustaceans. which contain very low amounts. Artemia In hatcheries. Teshima et al. Artemia are mainly used in their early life-stages. The effect of phospholipids on fish seems to be related to their role in intestinal absorption of neutral lipid fatty acids (Geurden et al. rotifers seem to catabolise more DHA than EPA. but DHA is absent except in a few strains. Nannochloropsis is a good cholesterol source. live prey can store additional neutral lipids. optimal enrichment diets must have very high DHA and low EPA levels. . which are then fed a DHA-rich microalga such as I. 24 h feeding on algae results in a relatively slight HUFA increase. Unfortunately. i. The mineral medium used to grow algae should be reappraised to optimise nutritive value through the food chain.Uses of Microalgae in Aquaculture 275 7. whose complex status in culture tanks depends on the bacterial population (which produces this vitamin at low oxygen levels) and algal content (Hirayama 1987). E) have a supplementary effect on rotifers. (1997). natural microalgal antioxidants are likely to minimise PUFA peroxidation during the enrichment procedure.4. (1983). galbana increases ascorbic acid. but none of these species was able to achieve complete development of Artemia or indefinite cultivation of Tigriopus when used alone.3 Vitamins The nutritional deficiencies that occur when a single algal species is used as a sole food source are often attributed to vitamins. some vitamins (e.4 Minerals Seawater is generally considered to be a sufficient source of minerals for most marine organisms. aff. However. Maruyama and Hirayama (1993) obtained optimal cultures of rotifer by enriching freshwater Chlorella vulgaris with B12 at 2 g g 1 of dry matter. in intensive production systems. in an analysis of various components of larval feed. considered that minerals were not a determining factor for dietary value. As bacteria are known to produce vitamins. D. high biomass levels may lead to the depletion of essential minerals. As suggested by Sargent et al. Satuito and Hirayama (1986) have shown that fat-soluble vitamins (A. thiamine and B12 content in rotifers previously grown on yeast (the last two vitamins were incorporated with biotin in ‘f/2’ algal culture medium. The rotifer Brachionus plicatilis requires vitamin B12.5 Influence of algae on live feed and larval microbiology Many substances released by marine algae influence the relationship between algae and zooplankton.g. 7. vitamin status depends on specific synthesis by the algal species.4. incorporation of the vitamins into algal culture medium and vitamin production by bacterial populations. (1997) showed that I. and even less about the effect of algal mineral content on live prey (Lie et al. studies for the determination of vitamin requirements have used bacteriafree cultures. As well as their nutrient function. C and E) have been shown to have an antioxidant function. used in this study).4. Van Alstyne (1986) found that exudates of some algal species enhance and . However. There is little information in the literature about mineral and trace element requirements. mainly of the B group. Robin (1989) obtained a significant increase in turbot growth using rotifers enriched with a mineral premix. 1997). 7. Algae synthesise ascorbic acid (vitamin C). which stimulates rotifer growth (Satuito & Hirayama 1991) and has a beneficial effect on larval culture. However. a mixture of algal species or the addition of a mixture of vitamins can compensate for the nutritional inadequacy of algal food. Tigriopus and Artemia display diverse ability to use different species of algae as food. Lie et al. in practical mass cultivation of living prey. which suggests that no algal species is able to cover fully the various vitamin requirements (Shirashi 1966). Thus. Watanabe et al. Makridis et al. when algae are the only food source. this antibacterial effect amounting to nutritional improvement. 1994). but abnormal larval deaths and/or failures in live prey production can still occur. In general. In a clearwater system. they not only contribute to maintaining the nutritional quality of live food (Reitan et al. A balanced microflora is required in the gut of fish larvae to prevent intestinal opportunistic bacteria from causing disease (Ringo & Birkbeck 1999). None of the bacteria from the algal culture could be isolated from the corresponding rotifer culture.276 Live Feeds in Marine Aquaculture others inhibit the feeding activity of copepods in cultures. Microalgae have inhibiting properties on bacteria. their replacement by other food sources is of considerable economic importance for hatcheries. The bacteria harboured in the digestive tract of healthy and especially unhealthy larvae belong mainly to Vibrionaceae. In a later investigation. (1992) showed that T. it is possible to introduce favourable bacteria as a probiotic. including pathogenic bacteria. (2000) showed that short-term enrichment with microalgae reduces the total bacteria population harboured by live food. 1993). As live prey actively ingest bacteria. Nicolas et al. stable cultures to be established. Most of the bacteria from the rotifers were not found again in the larvae. particularly on species of the Vibrio group (Salvesen et al. (1989) compared two fish rearing systems using rotifers fed with baker’s yeast and Tetraselmis suecica or Monochrysis lutheri. These substances are also involved in the settlement of bacterial microflora within the algal population. but also have a positive influence on the settlement of a healthy intestinal microflora in fish larvae by preventing the development of opportunistic bacteria (Skjermo & Vadstein 1993). Pathogenic or undesirable bacteria should then be suspected. most of the area in larval production facilities is used for live feed production. turbot larvae ceased to ingest the rotifers and died. Douillet (2000) showed that the introduction of bacterial strains into an axenic culture of algae and rotifers could affect rotifer production positively or negatively. especially in cases where antibiotic treatment (Gatesoupe 1982) or a sanitary cleaning strategy restored production. except in the two batches of larvae. while large numbers of bacteria (particularly Vibrionaceae) were found in the rotifer population. lutheri was used. but not in algal cultures (Verdonck et al. unavoidable contamination by various micro-organisms leads to complex and unstable bacterial associations. Analysis of bacterial communities showed no evidence of a direct relationship between the bacteria in the algal culture and those in the fish larvae. The direct influence of microalgae on marine fish larvae is discussed further in Section 7.5. except for some Vibrio. Such bacteria are classically found in rotifer and Artemia samples. As noted above. In hatcheries. No common bacteria were observed in the two rearing systems.6 Substitutes for live microalgae As the major drawback in using living microalgae is production costs. 7. When M. can be transmitted to larval fish during feeding (Benavente & Gatesoupe 1988). 2000). Bacteria associated with live feed. modern husbandry methods allow good. bacterial communities developing in live prey cultures may provide micronutrients. . Austin et al.4. suecica inhibits bacterial fish pathogens. When algae are added directly to larval tanks (‘green-water’ technique). As the algae culture volume requirement is two to three times that of the rotifer rearing volume. products should be suspended in the water and homogenised. Several artificial diets for rotifer production are available as commercial products. and could be disastrous for larval production because of inefficient or collapsed live prey cultures. 1987). although frozen algae exhibit three times as much exudation of organic nitrogen (which becomes a pollutant). In contrast. requiring buoyant feed. delivered continuously. Papandroulakis et al. which may have a lower dietary value and/or show potentially detrimental physical properties (buoyancy. but results in total loss of viability. and daily rations fractionated or. Another type of food consists of marine microalgae produced on a large scale and then concentrated and preserved frozen. (1995). cultured in fermenters on . Caked live yeast. In Japan. 1994) help to avoid degradation of the medium. Yùfera and Navarro (1995) obtained good rotifer production with 25–100 mg l 1 day 1 of freeze-dried T. either alone or supplemented with living algae or oil emulsions. planktonic copepods. (1989). These problems may be reduced through improvements in feeding methodology. can be utilised by benthic harpacticoid copepods. even better. 1980). suecica. These properties are conducive to increased waste and thus higher bacterial levels. for which a recommended batch culture and feeding methodology (Lavens et al. with accumulation of packed detritus. The nutritional values of freeze-dried and live algae appear to be similar for a number of species. More recently. Substitute diets should be carefully prepared. whereas 20 years earlier Person-Le Ruyet (1976) used 200–250 mg l 1 day 1 of the same food. The general purpose of their use is to save labour and space in hatcheries by using products processed in specialised facilities. Heterotrophically grown microalgae and microalgae-like organisms have recently become available on the market. has become the most common substitute food for rotifers. Most dry products tested as food for rotifer and Artemia are commercially available as single-cell proteins. (1996a) obtained higher rotifer production with these concentrated algae than with frozen algae. freezing allows long-term preservation of Tetraselmis cell quality. Microalgae are commonly used by these copepods but can be replaced by yeasts (Fukusho et al. even with dried algae (Austin et al. The inhibition of harmful bacteria by microalgae seems effective. Spray-dried freshwater algae were tried first. According to Yamasaki et al. freshwater Chlorella. Micronised bran has also been used for Artemia rearing. Dry yeast was also used alone or in formulated diets for rotifers (Gatesoupe & Robin 1981) and Artemia (Robin et al. such as baker’s yeast. similar population growth can be obtained with rotifer fed on either fresh or preserved Nannochloropsis. including off-the-shelf products and detritus. and soluble fish protein concentrate for rotifers (although for a short period only). These animals may consume the product directly and/or microorganisms growing on it. Mesoplanktonic harpacticoids such as Euterpina or Tigriopus show a more planktonic behaviour. However. In general. the use of chilled algae as paste or concentrate slurry has been developed. This may increase the labour time required for live prey culture and harvesting. leaching from broken cells and packed cells). have a greater requirement for live microalgae. resulting in an unstable culture and/or contamination by protozoa. 1992). such as calanoids. As observed by Montaini et al. food efficiency is better with live microalgae than with substitutes. whereas concentrated cultures kept in darkness at 4°C show a high capacity for survival. any dead material can be a source of bacterial proliferation. spray-dried or chilled.Uses of Microalgae in Aquaculture 277 Many food sources. especially Scenedesmus and Spirulina (Person-Le Ruyet 1976) and Chlorella (Hirayama & Nakamura 1976). This has led to the extensive use of oil enrichment. The ideal algal species. data for Artemia oil enrichment from Curé et al. Rotifer grown on Ts Rotifer grown on yeast + fish oil Rotifer fed 8 h on Sc Rotifer fed 6 h on oil DHA/EPA = 1 Artemia fed 24 h on oil DHA/EPA = 1 Artemia fed 24 h on oil DHA/EPA = 4 Artemia fed 24 h on 0.000–25. However. regardless of the food used for mass production and enrichment of live prey. Microalgae provide other factors than HUFA in live prey production. live algae are still the best option for strain conservation and production start-up.278 Live Feeds in Marine Aquaculture organic medium and fortified with vitamin B12. resulting in high rotifer concentrations (20. Microalgae are still required as food for calanoid copepods. 1996).) . This technique consists of a 2 day batch culture with high food density (3–5 g dry weight l 1 day 1). In particular. other data from IFREMER-Brest. Sc: spray-dried Schizochytrium. Increasing the HUFA content of live prey has drastically enhanced the survival and growth of larvae. their importance has decreased since the 1980s. It requires continuous control of oxygen level. HUFA: highly unsaturated fatty acids. such short-duration cultivation does not require cell viability. (Data for Schizochytrium from Barclay & Zeller 1996.4 g l Artemia fed 24 h on 0. their effect on the bacterial environment is now recognised. still remains to be discovered.000 individuals ml 1). Microalgae were the first food commonly used for live prey production. pH (to lower un-ionised ammonia) and accumulation of particulate organic matter in the culture. so it is hardly surprising that current developments in live prey production procedures are mainly focused on providing the n-3 HUFA requirements of larvae. as more cost-effective products can now be substituted as food for rotifer production. 1995. It is likely that new techniques based on ‘off-the-shelf’ products will prevail.2 g l 1 1 DHA EPA Sc Sc 0 1 2 3 4 5 Live prey HUFA content (% dry matter) 6 Fig. but these cultures have economic disadvantages that limit their use. For enrichment of rotifers and Artemia. High DHA content can be found in the heterotrophically grown and spray-dried thraustochytrid Schizochytrium (Barclay & Zeller 1996) or the dinoflagellate Crypthecodinium cohnii. However. Such products can be an alternative to oil emulsions for live feed enrichment protocols (Fig. which is more efficient than microalgae for attaining high HUFA levels. A high HUFA value and a suitable DHA:EPA ratio are prevalent criteria for live microalgae substitutes. This highly concentrated algal biomass (135 g l 1) can be used for ‘ultra-high-density’ mass culture of rotifers (Yoshimura et al. 7.8). 7. is available commercially in refrigerated form for rotifer culture.8 Comparison of docosahexaenoic acid (DHA) and eicosapentaenoic (EPA) contents of Artemia and rotifer using various substitutes to living algae: Ts: chilled Tetraselmis. which would be inexpensive to grow and efficient for both live prey production and nutritional quality for larvae. Navarro & Sarasquete 1998). Cahu et al. analysis is difficult because phytoplankton cultures are complex mixtures of suspended (live or inert) and soluble organic and mineral substances. 1998).e. 1996a. 1998. The green-water technique (larviculture in an endogenous bloom of phytoplankton and rotifers) and the ‘pseudo-green-water technique’ (larviculture in a tank supplemented daily with exogenous phytoplankton and rotifers).5 Importance of Microalgae in Marine Finfish Larviculture Unlike bivalve and crustacean larvae.2 Effects on endotrophic larval stages Accounts of positive effects of microalgae on endotrophic larval stages have sometimes been reported. 1990. Microalgae also reduce the third day . The most spectacular effects are observed during transition from the endotrophic to the exotrophic phase (Table 7. behaviour and availability (Reitan et al. as well as with frozen or lyophilised extracts of microalgae (Scott & Baynes 1978.Uses of Microalgae in Aquaculture 279 7. Øie et al. as well as all mesocosm technologies. first large zooplanktonic prey) feeding processes. The reasons for the apparently positive effects of microalgae on fish larvae are not yet fully understood. the micronutrient stimulus for feeding behaviour or physiological processes (Hjelmeland et al. 1997. 1994. 7. the results obtained are often more positive than expected (Tamaru et al.e. most marine fish larvae do not feed directly on microalgae and cannot survive in pure microalgal cultures or on exclusive phytoplankton diets. 1997. 1994). The following hypotheses have been proposed to explain this phenomenon: stabilisation of or improvement in water quality and light contrast (Naas et al. However. 1993). Papandroulakis et al.5. endo–exogenous) and first (i.1 Range of microalgal action Positive results have been obtained with a large specific variety of live mature (but not old) microalgal cultures used at low or medium concentration (Divanach & Kentouri 2000). However. van der Meeren 1991. the regulation of bacterial opportunistic populations by antibacterial or probiotic action (in Skjermo & Vadstein 1993). the survival. A light microalgal background improves the buoyancy of eggs after handling and/or transfer.b. Scott & Baynes 1979. but these are generally unproven. Jones et al. when phytoplankton was included in larval rearing tanks. and improvements in rotifer pelagic quality. 1993. which are regular or transient microalgal feeders. 1997). 7. 1988. endotrophic stages (eggs and prelarvae) and early exotrophic stages are also affected. 1992). Reitan et al. 1970. Reitan et al. Eda et al. 2000. growth and food conversion index of more than 40 species were better than in clear-water conditions (Anon. Tamaru et al. Dhert et al. the role of direct (via drinking and gill retention) or indirect (via absorption of endogenously enriched prey) nutrition (Moffatt 1981.6).5. particularly for the two ‘mixed’ (i. Howell 1979. In particular. 1981. constitute industrial application of this phenomenon (Divanach & Kentouri 2000). Quinonez Velasquez 1989. 2002a. as well as the deployment and subsequent survival of newly hatched sea bream prelarvae. Scott & Middleton 1979. 1993. Papandroulakis et al. b). Moreover. hunting. as well as through primitive sensorial links provided by auditive capsules. 1994). After mouth opening. A. they interact with the environment through transchorionic (eggs) and integumental (prelarvae) exchanges (water. mouth opening.e.280 Live Feeds in Marine Aquaculture Table 7. to 0. Reitan et al. a counterbalance for losses due to hyperosmotic seawater pressure and/or stress conditions. photoreceptors of the pineal gland and future eyes.6 Possible early feeding phases in marine fish (shading indicates the predominant feeding mechanism). Oocyte Maternal 1 Egg Endotrophic 2 3 Prelarva Larva Endo–exotrophic 4 Exotrophic post-hatching (DPH) sinking syndrome for sea bream and the lethal consequences of mirror effects of light reflecting walls at the end of the prelarval stage for sea bass and sea bream. 1990).3.10–0. transchorionic. 1993) and environmental changes.3 Effects on the yolk-sac drinking stage At mouth opening. fish prelarvae begin a new nutritional relationship with the environment that is more efficient than the previous integumental interaction. hatching. There is thus a range of possible modes through which microalgae could interact with endotrophically feeding stages. E. and 7 DPH herring in an iso-osmotic environment (Tytler & Bell.59% larval wet weight h 1 in cod (Mangor-Jensen & Adoff 1987). drinking. Drinking rates range from 0. hepatic glycogen reserves vary considerably according to nutritional status (Guyot et al. 4. C.1 Drinking and ingestion of dissolved organics Water uptake. for 4 DPH rainbow trout (Tytler et al. with a slight increase in the last part of the yolk-sac stage (Tytler & Blaxter 1988. The mode of action of microalgae during this phase is still debated. 1994). Differences in drinking rates for stress compensation have not yet been documented in clear . fertilisation. in Tytler et al. dissolved minerals and organic substances). oral absorption of water) not only contributes to osmoregulation. B. 3. integumental. Drinking activity (i.15–0.27% larval wet weight h 1 (7–160 nl larva 1 h 1) in 20 DPH halibut. The major environmental effects on eggs and larvae have been documented in Hoar and Randall (1988). 3–7 DPH in halibut (Tytler & Blaxter 1988. is developed early in marine fish larvae: from 1 DPH in cod (Mangor-Jensen & Adoff 1987).5.5. 3 DPH in sea bream and 4 DPH in sea bass (Diaz et al. but also allows intestinal absorption of dissolved organics and ingestion of particulate matter. but may be related to a reduction in water loss experienced under stress conditions. This activity has also been reported in cod eggs (Mangor-Jensen 1987). 7. neuromasts. filter-feeding. 1994). 7. Although embryos and prelarvae are dependent on internal nutrient reserves. 2. Life stage Nutritional mode Important steps A Dominant feeding mechanisms B C D E 1. resorption of last reserves. 1990) in clear water. Reitan et al. D. At correct doses. Their role in larval nutrition during this period is probably important. 1986). are very sensitive to any change in water quality during this period and show much better behaviour in green than in clear water. 1993). 1983). Absorption of soluble organics through the gut by drinking larvae increases markedly after mouth opening: glucose in sea bream and sea bass (Diaz et al. all larvae had Chlorella cells present in the gut when examined under the microscope (Kentouri 1985). mormyrus (i. tintinids and the rotifer Synchaeta) in the gut of 1 and 2 DPF D. before intake of copepod nauplii and the rotifer Synchaeta) and as the second most dominant prey (after nude ciliates. 2000). all of these organics can induce accumulation of large amounts of glycogen in hepatocytes via neoglucogenesis and restore the deficit that occurs in clear-water controls at the end of the prelarval stage. such as the sea bream (personal observations) and halibut (Holmefjord et al. cod. the possibilities for interaction with drinking larvae are multiple. such as the anchovy. Admiral et al. none can actually sustain long life.e. Lithognathus mormyrus and Puntazzo puntazzo (Kentouri 1985). Kentouri & Divanach 1986.Uses of Microalgae in Aquaculture 281 and green water. glycerol and various neoglucogenic substrates in sea bream (Maurizi et al. Lein & Holmefjord 1992). puntazzo and L. these larvae would seem to have a considerable advantage over their clear-water counterparts. and free amino acids in cod (Fyhn & Serigstad 1987. Studies of the influence of microalgae on larval absorption of soluble organics via drinking are still preliminary.3. 1994). Fyhn 1989). Brevoortia patronus (Stoecker & Govoni 1984). menhaden. Diplodus sargus. 1998) and die a few days after the clear-water controls. Algal concentrates have systematically been found to be the most dominant prey in the gut 1–10 days post-first feeding (DPF) in P. some larvae of certain fish. Gadus morhua (van der Meeren 1991). if these few extra days’ survival represent additional time for initiation of successful exogenous feeding. ingestion of microalgae by marine fish larvae has been reported in several species. In the laboratory or in aquaculture with an oligospecific food chain. 1983. As microalgae can absorb nutrients heterotrophically or myxotrophically. Sparus aurata. halibut Hippoglossus hippoglossus (Reitan et al. and transform and excrete various soluble organic substances (Brockmann et al. Starved green-water larvae quickly present symptoms of nutritional deficiency (Diaz et al. In intensive sea bream larviculture using the pseudo-green-water method. microalgae and the greenish remains of semi-digested phytoplankton have been found in the gut of several families of marine fish larvae. However. 1991. such as clupeids (Lebour 1919). sargus and S. Although impossible to count precisely because of their small size (1–2 m in diameter) and large number. A positive effect of dissolved organic substances on fish larvae has been documented in clear and green water. 7.2 Ingestion of microalgae First ingestion of microalgae begins passively via water intake at mouth opening and becomes progressively passive (filter-feeding) or active (hunting) intake of larger microalgae and microzooplankton. In the wild or in mesocosms with a natural food chain. Engraulis mordax (Moffatt 1981). these cells never . aurata (Kentouri 1985.5. pleuronectids (Last 1978a). It always precedes the classical first (zooplanktonic) feeding. even in the gut. gadoids (Last 1978b) and scophthalmids (Last 1979). produce them photosynthetically. However. Kentouri et al. However. engraulids (Scura & Jerde 1977). Green water is added empirically to tanks to minimise larval stress and correlated sedimentation during and after transfer and handling. was found in the mouth cavity of cod. reaches a peak of 1. modes of ingestion of microalgae include specific selectivity and quantitative regulation modified by age (a proof of adaptive behaviour). 7.5% larval biomass day 1 between 43 and 48 DPH. 1994). cod larvae were still able to ingest algae. In mesocosms with a wider variety of . Similar findings were obtained with D. but not in the larval gut (van der Meeren 1991). Ellertsen et al. salina) enter the mouth cavity by accident and clog the visceral arches before being swallowed. When offered various prey. In yolk-sac halibut. salina cell density in the medium from 1 to 10 million cells l 1 resulted in an increased feeding incidence of only about 20% in 2 DPH cod larvae. northern anchovy benefited directly and indirectly from the algal supply (Moffatt 1981). (1981) considered that microalgal cells (D. (1993) concluded that both cod and halibut are active filter-feeders at this stage owing to specific adaptation of gill-arch spacing and prospective behaviour. salina. Mechanisms of microalgal ingestion are specific. which were actively concentrated from the wild. and 93–100% ingested Nannochloris atomus at 2 and 3 DPH. and then ranged from 1 to 5% (Reitan et al. In 2–6 DPH cod. up to 7% and 40% of larvae ingested Isochrysis galbana and Dunaliella salina respectively. In a mesocosm with a variety of phytoplankton and zooplankton organisms. In yolk-sac halibut. the assimilation of ingested Tetraselmis was low through the yolk-sac stage. Reitan et al. the uptake of Tetraselmis is low before 30 DPH ( 0. fish are not biologically equipped to digest microalgae (Juario & Storch 1984).2% at 7 DPH and decreased to 12. Thalassiosira sp. In halibut. B. 6–10 m) represented 492–7251 times the drinking rate (van der Meeren 1991). 1994). In complete darkness. 1–4 m) were found to enter the larval gut as a function of the drinking rate. van der Meeren (1991) and Reitan et al. but about 40% in 6 DPH larvae (van der Meeren 1991). at 1 DPH. small algae (N.6% at 12 DPH.282 Live Feeds in Marine Aquaculture represented more than a few per cent of the total volume of ingested material. 3. As for larger prey. An increase in D. patronus. Siganus sp. with no detectable (14C-labelled) respiratory products from the algae and assimilation efficiency below 1% until 55 DPH. The most characteristic gut contents in the youngest cod larvae were green spheres (10 m). including passive and active responses conditioned by biological adaptations.1% larval biomass day 1). dinoflagellates (Prorocentrum micans) and microzooplankton (tintinids). Chanos chanos. salina after microscopic studies of cod larval gut content (van der Meeren 1991). ranged from 100 times (before 30 DPH) to more than 1000 times (between 30 and 43 DPH) and 60–300 times (after 43 DPH) the drinking rate (Lein & Holmefjord 1992. atomus. Conversely. However.9–4. In cod.3 Digestion and assimilation of microalgae Except for some families (clupeids. which were selected later (Stoecker & Govoni 1984).2 mm larvae of the menhaden. Most microalgae in laboratory tests are not (or only partly) digested.2–4. Positive correlations were noted between algal size and intergill-arch distance.3. naked dinoflagellates (20 m) and short chains of Skeletonema costatum. engraulids) and to a lesser extent a few other species (Boops salpa. the clearance rate of Tetraselmis sp. and then drops again to low values (Lein & Holmefjord 1992). began feeding on phytoplankton. showing a high feeding incidence similar to (or even higher than) larvae on a diurnal photoperiod (van der Meeren 1991). but not on copepod nauplii.5. while concentrations of larger alga (D. the fraction of algal material larger than 8 m in diameter was 39.). Kentouri and Divanach (1986) noted the importance of microzooplankton [mainly small pelagic ciliates (Amphorella. . naked dinoflagellates and various (greenish. Conversely. and could initiate successful intestinal lipid absorption within 40 h after mouth opening and then remain alive. 50% consumed rotifers in green water compared with 0% in clear water. Naas et al. In rearing trials. 1992) showed an increased rate of consumption of the rotifer B.4 Resistance to delay in first zooplanktonic feeding Microalgae compensated for delays in successful initiation of first feeding in the sea bream. their ingestion is of major importance for subsequent larval adaptation to the heterotrophic stage. and better matched the moment corresponding to mouth opening (Kentouri 1985). more to begin successful feeding and induction of normal intestinal lipid absorption. defects became so great that larvae could not begin to feed at all (the ‘point of no return’) and died at 7–8 DPF. even after a 32 h delay in first feeding in green water. In clear water. Halibut larvae showed similar behaviour. brownish) remains were observed in the gut. yellowish. respectively. Feeding intensity is specific. As a result. With sea bream. This trend was maintained in 2 and 6 DPF turbot. the filter-feeder adaptations of fish larvae seem to be more efficient.5 Process and efficiency of first feeding Microalgal background has an important. Larvae in clear water showed high cholestase. 7. 50% of larvae in green water versus 0% in clear water consumed the rotifer B. up to 10 days was necessary after mouth opening for all larvae to be engaged in consumption. Similar observations applied to B. Øie et al. effect on the timing and intensity of first zooplanktonic feeding.Uses of Microalgae in Aquaculture 283 microplankton species (algae or other organisms). cholestase) and a poor (often irreversible) state of health among the population (Maurizi 2000). 1997). hepatocytic degeneration. Although microalgae probably contribute little energy to larval metabolism at this stage. 1992). 7. without further feeding for up to 9–10 days (Maurizi 2000). When first feeding was delayed for 32 h. Microalgal cells. Sparus aurata (Maurizi 2000). They ingested rotifers almost immediately after distribution (8 h maximum delay). and 100% versus 50% at 7 DPH. there was a high frequency of fasting symptoms (hepatocyte degeneration. 1993. 1997. 1997) and halibut (Reitan et al. At 3 DPF. plicatilis in tanks containing microalgae rather than clear water. A similar slight increase in survival without feeding was observed with cod in green water (Lein & Holmefjord 1992). pancreas pathology and depressed glycogenic reserves after a delay in feeding of 8 or 16 h after mouth-opening and needed 16 or 40 h. plicatilis at 5 DPH. First-feeding larvae of turbot (Reitan et al. and 14 DPF was necessary for all batches to show a high feeding incidence on this prey (Naas et al.5. larvae did not develop such symptoms of metabolic stress. first (zooplankton) feeding time in sparids was earlier in green than in clear water. plicatilis in green water. However. regardless of the rotifer strain and the type of enrichment (303–400 versus 130–318 rotifers larva 1 day 1) (Øie et al. Stenosemella. but specifically different. the same time as starved controls.5. Strobilidium) and tintinids (Favella)] as concentrators and predigestors of microalgae in the early larval nutrition of sparids. in terms of the percentage of body weight.7 30 71 3 Without algae 4–18 0–2 5–6 6–24 41–54 18–18 8 4 16 6 1. noting that the ‘gut was distended. a 82% increase in wet weight of 20 DPF seabream (Papandroulakis et al. days post-hatching. (2002a) Papandroulakis et al. such as sea bass or mullet. which is sufficient to improve the cost-effectiveness of production. 1998).8). (1997) found a greater number of rotifers in guts of turbot larvae in clear rather than green water and suggested that a longer digestion time was responsible for the better assimilation rate in clear water. Microalgae also play a role in intestinal transit and gut repletion. Øie et al. 1997) Scott & Baynes (1978) Reitan et al. (1992) Cahu et al. Reitan et al.7 Increased survival (%) with and without microalgae in larval tanks. 2002a). DPF. the gain is generally greater than 100–500% (Naas et al. (1997) Tamaru et al.7). This trend was also observed in 15 DPF sea bream larvae (343–350 versus 266–414 ng C larva 1 in green water versus clear water. 1998). Papandroulakis et al. However.6 Effect on survival and growth efficiency at first feeding Improvement in survival at first feeding is the main result of larviculture with microalgae (Table 7. 1992.9 1.2 23 DPH 13 DPH 23 DPF 23 DPH 15 DPH 15 DPH 30 DPH 20 DPH 15 DPH 21 DPF 32 DPH Reitan et al. larvae in clear water (which were smaller) consumed slightly more than those in green water. (1998) Mugil cephalus Sparus aurata Hippoglossus hippoglossus Dicentrarchus labrax DPH. (1994) Papandroulakis et al. giving the false impression of good feeding’. such as halibut. (2002b) found only a slight difference in consumption (37–42 ng C larva 1 in green water versus 33–43 in clear water. Papandroulakis et al. In 10 DPF sea bass. 2002a) and may exceed one order of magnitude (Scott & Baynes 1979). Species With algae 28–55 5–25 30–36 29–54 76–82 34–43 56 16 44 17 21. For species considered easy to rear in clear water.284 Live Feeds in Marine Aquaculture Table 7.2 60 4 % Increase Age References Scophthalmus maximus 277 1400 500 177 66 113 600 175 1042 2400 18. Improvement in growth efficiency during the rotifer period was another result of microalgal background in larval tanks (Table 7. microalgae enhances survival by 18–113% (Tamaru et al. (1993) Øie et al. in 5 DPF sea bream.5. between 22 and 75% relative difference in . depending on the photoperiod). turbot or sea bream. (2002b) Maurizi (2000) Naas et al. days post-(first) feeding. Kentouri (1985) observed similar results for 2–5 DPF sea bream. Differences in death rates were evident at less than 10 DPF and often at 2–5 DPF for difficult species. Cahu et al. this improved growth was apparent as a 40% increase in weight (as measured in formol-preserved larvae) compared with clear-water controls (Cahu et al. 1993. (1994) Tamaru et al. Part of the distension was due to accumulation of empty rotifer lorica at the end or the rectum and a very low rate of excretion. respectively). 7. (1993. Nonetheless. 1994. For species considered difficult to rear in clear water. Reitan et al.3–8.7 Species Turbot Turbot Halibut Sea bass Sea bream Mullet Criteria SGR (% day 1) SGR (% day 1) SGR (% day 1) FW (mg) WW (mg) SL (mm) Rate na na na 40% 82% na Age 8 DPF 7 DPF 11 DPF 10 DPF 20 DPF 15 DPH References Reitan et al. and improves the quality of gut flora. facilitating the onset of hydrolytic functions of cell membranes and early development of brush-border membranes lining the gut.5. At 26 DPH. (1988) for .8 Differences in initial growth with and without microalgae in the larval tanks. although not homogeneously. Cahu et al. the specific growth rate in turbot at 7 and 8 DPF (Øie et al. However. (1997) found that the ingestion rate for turbot was lower in clear than in green water and that more protein (P) and carbon (C) from rotifers were used by larvae in clear (18–28% P. 7.2 versus 12. 12–19% C) than in green water (6–9% P. wet weight. DPF. weight after preservation in buffered saline formaldehyde. na.8–20.Uses of Microalgae in Aquaculture 285 Table 7. Similar triggering of enzymatic synthesis was reported by Hjelmeland et al. alkaline phosphatase and maltase assayed in purified brush-border membranes of the intestine are significantly higher for larvae reared in green water than in clear water. days post-hatching. (1997) Naas et al.5 na 1. The use of microalgae in tanks increases the production of pancreatic and intestinal digestive enzymes. 1997. 1993). FW. DPH. galbana clone T-iso triggers digestive enzyme production at both the pancreatic and intestinal levels. With algae 28 28 9. (1992) Cahu et al. microalgae present in the sea bass larval culture water result in a marked increase in trypsin activity. (2001b) found that daily feeding rates were similar for sea bream in green and clear water for a long photoperiod.7 Stimulation of digestive functions and gut flora Early enhancement of digestive and assimilative functions improves the survival and growth of fish larvae and favours the transition to exotrophy. and (b) lesser growth of larvae in clear water compared with those in green water during the rotifer stage. These differences in growth were due to two causes: (a) negative or only slight growth during the first 2–5 days (depending on species) for larvae in clear water compared with fast initial growth for larvae in green water. The efficiency of prey assimilation was also affected by microalgae. specific growth rate.1. and 530% relative difference in the specific growth rate in 11 DPF halibut (Naas et al. (2002a) Tamaru et al.0 4. Papandroulakis et al. 1992). and that the overall food conversion index (weight of ingested food/gain of larval biomass) for rotifer carbon was better in larvae reared with microalgae than in clear water (6.1 3.0–4. not available. days post-(first) feeding. whereas amylase and chymotrypsin are not affected. (1998) Papandroulakis et al.5 na 2. Øie et al. (1998) found that I. 4–7% C). Both stimulations are correlated with better survival and growth efficiency. In sea bass larvae. (1994) SGR. possibly because of differences in feeding rates. standard length. From 8 to 16 DPH. WW. (1993) Øie et al.4 Without algae 16 23 1. SL. respectively). Similar inverse correlations between the feeding rate and the conversion index have often been found in aquaculture.5–3. Surprisingly. 15 DPH mullet larvae reared with Nannochloris atomus background showed better growth efficiency than those reared in clear water. indicating that microalgae produced substances (e.8 Effects on early exotrophic larvae Even after the endo–exotrophic phase. despite the higher concentration of un-ionised ammonia (0. 1994). Sea bass larvae reared with I. lectins. the situation is more complex than first believed. 1998).5. 1993). and the quality and accessibility of rotifers. no statistically significant difference was found beween treatments following a salinity stress test (Øie et al.9 Indirect effects of microalgae on larvae The indirect effects of microalgae on larvae are mainly related to three causes: water quality and luminosity. It consisted mainly of slow-growing bacteria. galbana) showed a lower death rate during a stress test (30 s exposure to air) than those reared in clear water (5–6% versus 29%. 1997). fluctuations in oxygen and pH are generally higher than in clear water because of difficulties in stabilising the microalgal bloom. the bacteriology of water and rotifers. ammonia and phosphate) with photosynthesis (uptake of carbondioxide. 7.13 versus 0. 7. the intestinal microflora of larvae kept in green water differed considerably from that of larvae kept in clear water.286 Live Feeds in Marine Aquaculture herring larvae. 1986). However. and I. (1994) found that the input of algal culture fertilisers increased ammonia levels in tanks with green water. taxins) that enhanced the ability of certain bacteria to grow in the gut.03 mg l 1) in phytoplankton culture (Tamaru et al. galbana clone T-iso showed a 26% improvement in survival over those in clear water when they were weaned early (15 DPF) with compound diets (Cahu et al. However. Unlike skin microflora. microalgae have a positive effect on larviculture and may increase the resistance of larvae to further stressing or adaptive conditions: • • • 23 DPF turbot larvae reared in pseudo-green water (Tetraselmis sp. With the green-water technique (based on strong lighting). . Improved larval rearing efficiency in tanks with microalgae was initially considered an effect of improved water quality due to a counterbalance of larval respiration (oxygen uptake and production of carbondioxide. which is clearly related to the flora of the water and less affected by algal addition than gut flora. Selection of bacteria in the gut was more active in green than in clear water. Stimulation of trypsin synthesis was also triggered when larval diets were supplemented with free amino acids (Zambonino Infante & Cahu 1994). suggesting that the large amounts of free amino acids in microalgae may be the cause of enhanced trypsin activity in green-water larvae (Admiral et al. nitrogen and phosphorus.5. together with oxygen production) and the stabilisation of fluctuations in pH and carbonate–bicarbonate equilibrium. Tamaru et al. The addition of microalgae to rearing water modified the bacteriology of larval skin and gut (Skjermo & Vadstein 1993). together with a smaller fraction of opportunistic bacteria (potential pathogens). respectively) (Reitan et al.g. In green water containing Pavlova lutheri. plicatilis. Except for C. were observed with all algae and mainly with Tetraselmis (Salvesen et al. This problem is currently being solved through the use of both automatic computerised feeding and a long photoperiod (Papandroulakis et al.e. 1989). accessible). Therefore. . but lost quite rapidly in tanks without microalgae (Olsen et al. are likely to have an effect on behaviour and thus on efficiency. showed a bacterial gut flora close to that of the rearing medium. low levels of Vibrio sp. with a larger fraction of slow growers and fewer opportunistic (potentially pathogens) fast growers than in clear water. which was associated with relatively high densities and a high proportion of opportunistic and haemolytic species. Estevez et al. In addition to a direct nutritional action. Nannochloropsis oculata. bacterial density was increased by 45%. meulleri. Addition of microalgae to larval rearing water modifies the bacteriology of the water and rotifers both quantitatively and qualitatively (Nicolas et al. Tamaru et al. Pavlova lutheri and Tetraselmis sp.e. Therefore. 2000). the decline in nutritive value of rotifers in clear water before consumption by larvae could be more marked than improvements in composition made by live prey enrichment before distribution (Øie et al.4). Their role in larval tanks is even more apparent. Reitan et al. For this reason. Through these behaviour modifications and alterations in prey visibility. 1997). green-water culture may also have the indirect effect of reducing larval stress levels. 1989. 1994). Chaetoceros meulleri. With other microalgae (Skeletonema costatum. Their lipid and protein content was maintained or enhanced in tanks with microalgae. The matured green water also had a more stable microflora. microalgae in larval tanks can cause behavioural effects in live prey and fish larvae. Salvesen et al. which appear to be the main environmental factors involved. 1995). The correlations between the bacteriology of water.). 2000). Skjermo & Vadstein 1993. The rotifer B. rotifers. In cultivation trials. when introduced into tanks together with microalgae. enrichment and nutritional value of rotifers before distribution has been extensively documented (see Section 7. it is generally believed that green-water culture helps to improve the success of these two parameters (Støttrup et al. 2000).e. the growth rate of rotifers was positive in all tanks with algae and negative in all tanks without algae (Øie et al. 1999). With sea bream. Reduced light intensity and changes in spectrum. and the structure of bacterial organisation was more diversified. which was less affected by modifications of the organic load or antibiotics than the microflora in clear water (Skjermo & Vadstein 1993). whereas they sink to the bottom or agglutinate on walls within 3–6 h in clear water. In green-water systems. The importance of microalgae for the production. this reduced accessibility becomes critical (i.Uses of Microalgae in Aquaculture 287 This can result in water near the tank bottom being deficient in oxygen even though surface water is oxygen saturated. detrimental) during the night and the health condition of the larvae is markedly affected in the morning. 1997). Isochrysis galbana. various authors (Scott & Baynes 1979. higher levels of bacteria were also associated with slowgrowing bacteria. 1998). Since the biochemical composition of live prey is important in pigmentation and metamorphosis of flatfish (McEvoy et al. a factor thought to be influential in flatfish pigmentation quality (McEvoy et al. any delay in feeding at this time can have severe consequences. 1998. 1994) have considered the possible effects of a modified lighting environment in green water. i. rotifers remain pelagic (i. larvae and larval efficiency indicate the importance of this factor. 1993. with fewer opportunistic bacteria than in clear water (Nicolas et al. contrast and turbidity. growth and quality in fish larviculture. the pretrophic (probably oligotrophic or non-trophic) importance of microalgae as a trigger for both physiological and behavioural processes and bacterial probiotic conditioning of water. rotifers and larval gut. 5. M. & Khalil. Assoc... 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Notice the colour. or Thalasiossira sp. but additional microscopic observations (see below) are needed to verify the presence of food in the gut and the presence of eggs.Appendix I Procedures for Assessment of Rotifer Cultures Routine Procedures Daily routine procedures for examining the state of the rotifer cultures are essential for assuring an appropriate supply for raising marine fish larvae in hatcheries. A clear appearance of the culture indicates an active culture.). flagellates (e. foam and coloured rings at the rim of the culture tank walls. The daily examination of the culture should be done at several levels: 1. identify problems before the cultures collapse and improve culture practices under the specific culture conditions prevailing in a hatchery. rotifer cultures require high concentrations of dissolved oxygen. While there are many options for culture methods. The observations should be recorded in a log-book to help in tracking problems and in formulating more reliable routine culture practices. In some cases the milky appearance of the culture medium may be caused by other reasons. Take a general look against the light of 30–40 ml samples in glass beakers. high relative levels of un-ionised ammonia and non-suitable salinity.). It may either indicate excess feeding of the culture or some other problems in the culture that prevented the anticipated filterfeeding process that may have consequently reduced reproductive rates of the rotifers. blooms of certain algae that cannot be ingested by rotifers (Chaetoceros sp. such as the proliferation of ciliates (usually Euplotes sp.g. milky. clean or dirty. Take a general look at the culture tanks. 2. low oxygen levels. The observations should reveal whether the samples are clear. usually indicates the presence of excess food in the culture. A milky appearance of the culture. Record the temperature. especially in high-density cultures. feeding. from each culture tank. daily routine monitoring of general and specific parameters will help to predict the production scale. These include: culture temperatures that are not in the optimal range. Oxyrrhis marina) or. In contrast to published data. several hours after the last feeding (this is relevant in cultures using non-continuous feeding methods). This is more typical of semi-continuous extensive cultures that are run for several weeks or months. smell. Routine practice is based on looking at each culture in the morning before initiating any treatment such as harvesting. pH and oxygen level. . until the difference between replicate counts does not exceed 10%) and calculate the average number of eggs per female (egg ratio). A ratio of less than 25% may indicate insufficient feeding of the culture or other problems that will lead to its eventual collapse. but their reproductive rates at 10°C are significantly lower than those at 20–22°C.. nematodes or copepods? (b) Are the rotifers swimming or sinking to the bottom of the examination dish? Are the rotifers clean? Do they stick to each other.)? Are there any filamentous bacteria or other undesired organisms such as amoebas. Although live rotifers may also be removed this way. 5.. These cultures show a fast growth in density. However. two or more? Are there any males? What is the relative abundance of young. Closer observations using a binocular microscope (at 20–50 magnification) should be made on the 30–40 ml sample. For example. How do the rotifers swim: Quickly? Slowly? Nervously? While fast swimming rates are a sign of good fitness. they do not necessarily indicate optimal reproductive rates. low oxygen levels. the swimming behaviour of Brachionus plicatilis rotifers at 10°C does not differ greatly from their swimming behaviour at 20–22°C. The number of rotifers and the number of eggs that they carry are used for calculating the food ration for the next 24 h. Fabrea sp. The amount will range from 1 to 4 g of wet baker’s yeast for every million rotifers (adult females plus amictic eggs). flagellates or filamentous bacteria may stick to rotifers’ lorica or may be present in the culture medium and hamper their swimming speed. or Zoothanmium sp. plicatilis rotifers.. there is an exception to this general concept in the case of young cultures that exhibit log-phase population growth rates. high un-ionised ammonia levels and too high temperatures for B. The exact amount has to be determined empirically for each culture facility. a certain rate of daily growth in the rotifer population is achieved by using a specific routine method. including: (a) How dirty is the culture? Are there many particles? Do they float or sink? Are there any protozoa in the culture? Ciliates such as Euplotes sp. immature rotifers? Count the number of rotifers and the number of eggs in 1 ml samples (three to five replicate counts per tank. forming clumps? Are there dead rotifers and/or empty loricas? Are the guts empty or full? (c) Swimming behaviour. Vorticella sp. Uronema sp. Remove the debris that has accumulated in the culture tank by stopping the aeration for a few minutes and allowing the suspended material to sink to the bottom. The egg ratio is a powerful tool for predicting problems in rotifer cultures. This value will serve as an indicator for the stability of the cultures kept in the facility and for calculating the food ratio to be supplied to the culture tanks.. Slow swimming is a good and quick indication of problems in the culture such as starvation. one. After some experience and feeding trials.. . Sessile ciliate species such as Vorticella sp.Appendix I 301 3. Paramecium sp. (d) How many eggs are the females carrying? None. The debris is removed from the bottom outlet. an abundance of young rotifers and relatively few females bearing eggs. or flagellates (Oxyrrhis sp. 4. Nervous swimming may indicate the presence of contaminants or external parasites on the rotifers. it usually has little effect on the total production. flagellates and bacteria. The removal of suspended dirt particles is more problematic. sand filtration is used to remove most live organisms in seawater. Settlement of dirt particles can also be encouraged by placing the aeration tubes a short . and has little effect on survival of the rotifers. followed by suspension in seawater at the culture salinity.302 Appendix I 6. creating an optimal habitat for harmful bacteria. A simple way to avoid the growth of algae is to cover the rotifer culture tanks. The correct appearance is gained by experience. short-term batch cultures (of about 7–10 days at the most) are easier to maintain free of contaminants than long-term semicontinuous cultures. the rotifer collection hose should be placed below the upper layer. either in the form of gravitation or by a pumping system. it may not be appropriate as food for rotifers. or Tetraselmis sp. and the best way is to filter the water through 0. this step requires an energy source. Some particles sink after cessation of aeration in tanks and can be removed easily in tanks with a bottom outlet. such as ciliates. Contaminant organisms may compete for the food with rotifers. In most places. or adhere to the walls in the fish larvae tank. Trouble Shooting Contaminant particles and cleaning of rotifer cultures The main problems in rotifer cultures are the occurrence of contaminant organisms and dirt particles. by starting rotifer cultures with a clean inoculum and culturing rotifers in clean seawater. The colour and shape of the algal cells is also a good indicator for cultures of Nannochloropsis sp. These cleaned rotifers can then be used either as an inoculum for new cultures or for nutrient enrichment before being transferred to fish larvae as food. Using old yeast may lead to a reduction in the pH of the culture. The collected rotifers should be immersed in chlorine-free freshwater. The best way of dealing with these problems is to avoid them. for 5–10 min. but this method of filtration does not eliminate eggs or cysts of various sizes. increase ammonia levels through their excretion. In general. While the yeast may still look fresh. but its more difficult to identify quickly problematic batches. The seawater used for mass cultures should be filtered and sterilised by ultraviolet light or ozone. (b) Preserved algae (concentrated chilled pastes or frozen concentrated paste) should be inspected for their colour (spoiled algae can appear brownish) and smell. (d) Freeze-dried yeast should be inspected after suspension in fresh water. One of the simplest practices is to stop aeration in the culture tank before harvesting of rotifers and allow the settlement of the dirt particles to the bottom of the tank.. reduce the dissolved oxygen levels and be harmful to the fish larvae after they are passed with the cultured rotifers to the fish larvae tanks. If there are also floating particles.2 m water filters. while motility can serve as an additional good indicator for flagellates such as Isochrysis sp. Undertake routine inspection of food quality provided to rotifers: (a) Live cultures of algae should be inspected for occurrence of contaminating organisms such as bacteria. This treatment removes several of the parasites. Dirt particles may either harbour undesired organisms that will be passed on to the fish larvae tanks. (c) Fresh yeast: the expiry date is an important indicator. However. the rotifers are not affected by the strong aeration. While the strong aeration will disconnect the eggs from the adult rotifers. The tank water content passes this filter-bed. The filter can either be submerged in the culture tank or stand as a separate unit outside the culture tank. However. Using open-ended pipes will produce large air bubbles with a small surface to volume ratio. While some of the rotifers are caught in the filter and lost from the culture. this is not important as the rotifers after this treatment serve only as food for the fish larvae. where rotifers are suspended in very high densities in nutrient-rich media. with one in use and another one in the process of being washed and dried. most particles are suspended in the water column and cannot be removed easily by sieving. which may reach tens of litres per minute per tube outlet. which reaches about 1% of the culture tank volume. One solution is to pass the water of the culture tank continuously through a filter. in many cases the total amount of air passing these diffusers is very small. Adequate oxygen supply is specifically of importance during nutrient enrichment of rotifers. being of the same size as the rotifers or causing the rotifers to cling to them. The filter material should be washed once a day and it is advisable to construct two filter beds per tank. However. This filter is constructed within a container that encloses synthetic nylon fibres or material similar to that used in scouring pads the culture water is forced through a pipe into the container by an air-lift and exits the filter directly to the culture tank. From experience.Appendix I 303 distance off the tank bottom. Oxygen supply Aeration of rotifer cultures is usually done through air-stones that provide fine air bubbles to the water. . Floating particles can be removed by siphoning the top layer of the culture. Another way of eliminating non-motile dirt particles is by using a settling device. The settled dirt can be removed either by siphoning or by a bottom outlet. the rest of the rotifers are relatively clean. which can be attached on the side of each culture tank and the culture water passed through it once or twice a day. several times a day. but much more oxygen will be dissolved in the water. Prepare the decapsulation solution with: (a) hypochlorite.4 g CaO for bleaching powder. (b) An alkaline product is necessary to keep the pH 10. This will enable the process to start with spherical cysts. Add the hydrated cysts and keep them in suspension (e. which improves the physical contact with the decapsulation solution. and (c) seawater. The solution will turn brown–red and release foam. Cool the solution to 15–20°C (e.g. Collect the now hydrated cysts on a 125 m mesh sieve. . Check the evolution of the decapsulation process regularly under binoculars. Check the temperature regularly. Decapsulation 2. Use. since the reaction is exothermic. otherwise it must be determined by titration). Hydrate cysts by placing them for 1 h in water (less than 100 g l 1).15 g technical grade NaOH when using liquid bleach. by placing the decapsulation container in a bath filled with ice water). (c) Determine the amount of seawater required to end up with a final solution of 14 ml decapsulation solution per gram of cysts. 4. Ca(OCl)2 (activity normally 70%). NaOCl (fresh product.g. Either 0. • • 3. or bleaching powder. use only the supernatants of this solution. add ice to decapsulation solution). dissolve the bleaching powder before adding the alkaline product. with an aeration tube) for 5–15 min. Use an amount equivalent to 0.Appendix II Decapsulation Procedure for Artemia Cysts Hydration 1.67 NaCO3 or 0. per gram of cysts: 0. never exceed 40°C (if necessary. (a) The hypochlorite solution can be made up with either liquid bleach.5 g active hypochlorite product per gram of cysts (the activity is normally labelled on the package. rinse and transfer to the hypochlorite solution. activity normally 11–13% w/w). When it turns whitish-yellow it is time to stop the reaction. with aeration. at 25°C. (b) an alkaline product. since this will affect their viability.Appendix II 305 Deactivation 5. . For long-term storage cysts need to be dehydrated in saturated brine solution (1 g of dry cysts/10 ml 1 of brine of 300 g NaCl l 1). washing and deactivation have to be continued. It is crucial not to leave the embryos in the decapsulation solution for longer than strictly necessary. Use of the Decapsulated Cysts 7. The decapsulated cysts can be incubated immediately for hatching or used directly as food. they can stored in the refrigerator (0–4°C) for a few days before hatching incubation. Hypochlorite residues can be detected by putting some decapsulated cysts in a small amount of starch-iodine indicator (starch. deactivate the hypochlorite with an equivalent amount of 0. H2SO4 and water). KI. Cysts should be removed from the decapsulation suspension and rinsed with water on a 125 m screen until no chlorine smell is detected anymore. The brine must be renewed once or twice after 24 h until the salinity of the brine does not drop significantly. When the reagent turns blue. When cysts turn grey (with powder bleach) or orange (with liquid bleach).1% Na2S2O3 solution. Alternatively. or when microscopic examination shows almost complete dissolution of the cyst shell (after 3–15 min). Washing 6. 7. Hatching 9. Add 0. .5. Transfer nauplii to the enrichment tank. 15. Choose a cylindroconical tank with a volume of clean seawater as to obtain a maximum density of 300. Hatch and harvest nauplii as described in Sections 3.Appendix III Enrichment Procedure Seawater Disinfection (Optional) 1. 4.000 nauplii l 1. Harvest over a 100–150 m sieve and rinse well with tap water or seawater.5 g l 1 NaHCO3 (dissolved in deionised water and filtered).6 g emulsion l 1 at the start and another 0. 6.6 g l 1 after 12 h. e. 13. 12. Harvesting 14.g.5. 3. 16. Use the nauplii as such or proceed to cold storage. Keep the temperature around 28°C and oxygen above 4 mg l 1. 2. Fill a cylindroconical tube. 8.3.2 and 3. Leave for 20 min with 200 mg l 1 NaOCl ( 2. Incubate for 1h without aeration. Add 1 mg l 1 NaOCl (100 l bleach solution per 10 litres of filtered seawater). Enrichment 10. 0. Add the enrichment product according to specific guidelines. Remove all aeration. 11. Cyst Disinfection 5. Collect and rinse nauplii over a submerged 120 m sieve as to keep the nauplii submerged at all times.0 ml bleach solution l 1) under strong aeration. Incubate cysts at a maximum density of 100 g l 1. Aerate strongly overnight. uk/ccap .Appendix IV Web Sites for Culture Collections Provasoli–Guillard National Center for Culture of Marine Phytoplankton in the United States: ccmp.org Centro de Investigaciones Biológicas del Noroeste in Mexico: www.bigelow.org Culture Collection of Algae and Protozoa in the United Kingdom: www.ac.cibnor.ife. . 265. 256 Dunaliella salina. 183. 21. 271. 17. 154. 188 Apocyclops royi. 4 Anarhichas lupus. 146. 112 Artemia tibetiana. 76 Arthrospira. 184. 183. 166 Cynoscion nebulosus. 178–180. 129. 219 Chlorella stigmatophora. 75 Candida. 148. 192. 208. 184. 233. 76. 45. 275 Chroomonas fragarioides. 212. 159 Acartia teclae. 161–163. 265–267. 175. 208. 4. 254. 300 Chaetoceros calcitrans. 272 Brevoortia patronus. 24 Brahcionus plicatilis. 256. 150. 49–52. 208 Chlorella vulgaris. 187 Acanthopagrus latus. 273 Brachiomonas submarina. 2. 150. 165. 189 Cyclotella. 283. 219 Chlorella grossii. 281 Chlorella autotrophica. 25. 172 Centropages typicus. 188 Apocyclops panamensis. 98. 187. 287 Chaetoceros calcitrans forma pumilum. 185. 166 Artemia tunisiana. 147. 18. 159. 43. 216. 176 Dunaliella. 150. 9. 165. 159. 271 Brachionus calyciflorus. 281 Ditylum brightwellii. 148. 208. 187 Apocyclops borneoensis. 110 Centropages. 104 Clupea harengus. 242. 210. 270–275. 277. 277. 184 Chlorella pyrenoidosa. 273. 210. 93. 129. 192 Cocochloris. 34 Bacteriastrum hyalinum. 271 Arthrospira platensis. 208 Chlorella minutissima. 301 Brachionus rotundiformis. 76. 159. 212 Chlorella saccharophila. 172. 165. 229. 210. 212 Asplanchna. 257. 49. 262 Crassostrea virginica. 40. 32. 9. 217. 168 Acartia tonsa. 147 Aphanius. 220. 9–13. 282 Calanus. 163. 259 Crypthecodinium. 270. 242 Cyclotella nana. 262. 147 Acartia sinjiensis. 160. 212. 167. 234 Chaetoceros. 122–143. 77. 257. 190 Acartia pacifica. 37. 240. 275. 183. 188 Apocyclops dengizicus. 76. 262. 181. 159–161. 40. 256 Chlorella fusca var vacuolata. 168. 171 Aetideus divergens. 96 Artemia urmiana. 257. 208. 258. 65–111. 155. 147. 157. 32. 19. 287 Chaetoceros pumilum. 159 Amphiascoides atopus. 27 Bacillus. 147. 258. 273 . 239 Arthrospira maxima. 187. 268. 222. 46. 266. 176–180. 167. 48. 271. 77. 190 Calanus finmarchicus. 278 Artemia franciscana. 156. 157. 232. 222. 148. 175. 243. 151. 287. 179. 30 Chlorella virginica. 25. 208–210. 282 Dunaliella tertiolecta. 17. 165. 157. 164 Clarias gariepinus. 111. 21–23. 109. 163. 232. 191 Acartia longiremis. 157. 212. 273. 157. 255 Chaetoceros muelleri. 147. 147 Acartia plumose. 10 Daphnia. 281. 208. 172 Calanus helgolandicus. 208 Chlamydomonas. 255. 284 Diplodus sargus. 147. 164. 270. 166 Coullana canadensis (ie. 217–221. 107. 96 Artemia salina. 179 Centropages furcatus. 209. 131 Artemia (franciscana) monica 76 Artemia parthenogenetica. 75. 17–19. 39. 218–220. 254. 183. 87. 177. 164. 233. 282 Chlorella. 212. 194 Acartia clausi. 265 Boops salpa. 264. 186 Amphorella. 260. 177. 270. 85. 23. 193. 157. 157 Cancer salinus. 134 Argyrosomus hololepidotus. 155. 254 Dunaliella primolecta. 273 Cyclops oithonoides. 185. 225. 225. 188 Candida utilis. 218 Cyclotella cryptica. 184 Chroomonas salina. 278 Cryptomonas. 184. 108. 187 Acartia. 134 Apocyclops. 163 Cyclops strenuous. 155 Centropages hamatus. 76. 150. 212. 75 Artemia sinica. 147. 148 Artemia. 49–51. 175. 37. 156. 129 Chanos chanos. 166. 182. 242. 148. 267. 28–33. 189. 240 Anarhichas. 150. 165. 191–195. 183. 283 Amonardia. 40. 21–35. 262 Chaetoceros gracilis. 274. 87 Conchilus. 151. 263. 208 Chlorella ovalis. 255. 182. 264. 148. 146. 225. 127. 172. 220. 210. 8. 44–46. 229. 159–161. 265–267. 112. 270. 172. 263. 111. 210 Crypthecodinium cohnii. 37. 172 Centropristis striata. 151.Taxonomic Index Acanthopagrus cuvieri. 151. 183. 282 Brachionus. 157. 255. 258. 76. 273 Acartia tsuensis. 39. 19. 219. 187. 209. 75–77. 219 Chlorella sorokiniana. 27. 150 Coscinodiscus wailesii. 35. 208. 225–227. 161 Calanus pacificus. 17. 158. 162 Crassostrea gigas. 150 Acanthopagrus schlegeli. 25. 3. 157. 218–220. 163 Anabaena azollae. 96 Artemia persimilis. 239. 188 Ardea. 183. 256. 194 Dicentrarchus labrax. Scottolana Canadensis). 266. 41. 256–260. 167. 27 Coryphaena hippurus. 156 Cyclops vernalis. 208. 176. 175. 208. 172. 281 Enteromorpha. 232. 150 Euchaeta elongata. 192 Pagrus major. 216. 256. 157. 276. 178 Eurytemora hirundoides. 287 Navicula. 187 Euplotes. 147. 1. 151. 183 Nitzschia. 176. 270 Epinephelus. 39. 148. 259–262. 254. 32. 300. 165 Platymonas suecica. 12 Macquaria novemaculeata. 261. 210. 208. 254. 172. 147. 180. 255. 147 Moina. 17. 163 Oithona similis. 302 Nannochloropsis oculata. 212. 270. 164–166. 172. 181. 175 Glaucosoma hebraicum. 287 Pavlova salina. 210. 147. 262. 172. 257–260 Oxyrrhis marina. 277 Euterpina acutifrons. 208. 218–220. 184 Nitocra spinipes. 218. 164–166. 157. 161–166. 156. 177. 147 Lutjanus argentimaculatus. 147. 281 Litopenaeus vannamei. 159. 25. 175 Epinephelus coioides. 10. 175 Lutjanus johnii. 188. 301 Parapenaeopsis sculptilis. 265 Penaeus semisulcatus. 17 Fundulus. 210. 301 Favella. 150. 48. 222. 159. 166 Rhinomonas reticulata. 258. 231. 147. 176. 259 Ostrea chilensis. 208. 39. 172 Pseudodiaptomus. 256–259. 256. 218 Oithona. 25. 172. 223 Phaeodactylum tricornutum. 192 Glenodinium. 286. 151. 187 Glaucosoma. 270 Patinopecten yessoensis. 273 Rhodomonas reticulata. 51. 222. 261 Penaeus. 10. 286 Nannochloropsis. 259 Pavlova. 150. 147. 236. 8. 211. 269 Phaeodactylum. 164. 259 Pecten maximus. 129 Macrobrachium. 229. 190 Eugerres brasilianus. 255 Metapenaeus. 147 Epinephelus striatus. 48. 189. 147. 4. 161. 8. 227. 10. 177 Gadus morhua. 183. 150 Gonyaulax grindleyi. 255. 219. 4. 255. 177 Pleurosigma. 1 Huntemannia jadensis. 208 Pyramimonas virginica. 176. 8 Meteridia longa. 222. 157 Lyngbya. 33. 32 Fabrea. 242. 273 Paralichthys dentatus. 301 Pagrus auratus. 191 Eurytemora longicornis. 19. 229. 281. 281 Gladioferens imparipes. 147. 239 Prorocentrum mariae-lebouriae. 10. 242. 217–220. 270 Penaeus monodon. 134 Rhabdosargus sarba. 183. 32. 267 Penaeus japonicus. 277. 265. 159 Halectinosoma curticorne. 10. 267. 166. 167 Paracalanus parvus. 266. 227. 8 Elops saurus. 230. 172 Epinephelus fuscoguttatus. 147 Hippocampus subelongatus. 265 Phormidium. 208. 208. 287 Labidocera aestiva. 269. 208. 270 Porphyridium cruentum. 17 Paracalanus. 148 Leucothrix. 282. 178 Euterpina. 8. 242. 282 Pseudocalanus. 227 Pavlova (Monochrysis) lutheri. 265. 181 Pseudo Isochrysis paradoxa. 266. 185. 158 Euchaeta norvegica. 166. 166. 172 Paralichthys olivaceus. 284 Hippopus hippopus. 166. 173. 285 Isochrysis galbana. 147. 223. 17. 185 Mytilus edulis. 148. 35. 273. 212. 183. 10 Morone saxatilis. 166. 259 Nannochloris. 176. 282. 178. 148. 148 Melanogrammus aeglefinus. 177 Engraulis mordax. 159. 160. 255 Homarus. 67. 165. 265. 234 Paralichthys flesus. 272. 215. 147. 192 Hippocampus angustus. 264. 265 Puntazzo puntazzo. 147. 129. 300. 187. 8. 218. 271. 111. 273–275. 194 Morone chrysops. 10. 218–220. 111 Loligo pealie. 256 Prorocentrum micans. 211. 268 Penaeus esculentus. 212. 8 Rhincalanus nasutus. 192 Hippoglossus hippoglossus. 166. 208 Pecten fumatus. 147. 166. 185. 218–220. 217–220. 150. 147. 273 Nannochloris atomus. 227–231. 182. 266. 209 Ochromonas. 238. 212. 270 Nitzschia closterium. 283 Fugo rubripes. 167. 302 Isochrysis affinis galbana ‘Tahiti’ (T-iso). 209. 150 Lithognathus mormyrus. 265. 112. 8. 218–220. 267 Penaeus chinensis. 1 Mercenaria mercenaria. 184. 192. 301 Eurytemora. 45. 263 Penaeus indicus. 9. 208. 258 Ostrea edulis. 162 Rhodomonas. 254–256. 9.310 Taxonomic Index Eleutheronema tetradactylum. 265. 8. 148. 159. 210. 147. 254–257. 182. 163 Heterocapsa triquetra. 185. 242. 175. 184 Pleuronectes americanus. 180. 284 Mya arenaria. 17. 129. 183 Eutreptiella. 8. 159 Isochrysis. 228 Rhodomonas baltica. 161. 4. 240. 270 Nereis. 258–260. 274. 240. 226. 129. 151. 92 Limanda yokohamae. 260 . 1 Pleuronectes platessa. 230. 208. 183. 262. 147. 270 Penaeus vannamei. 178 Lates calcarifer. 166. 274. 209. 152. 9 Paramecium. 25. 162. 111. 264. 181 Eurytemora affinis. 157. 9. 258. 265 Penaeus stilirostris. 172 Oscillatoria. 208. 159. 32. 157 Oithona plumifera. 228. 254. 265. 192. 212 Robertgurnenya. 134. 4. 190 Pseudocalanus elongatus. 255 Mylio macrocephalus. 184 Gobionellus boleosoma. 211 Rhodomonas salina. 150 Mytilus. 265. 148. 215. 182. 219. 157. 162. 281 Pyramimonas grossii. 164–166. 172 Platymonas. 159 Ruditapes philippinarum. 227. 268 Penaeus merguiensis. 270 Ostrea. 212. 259. 147 Mugil cephalus. 273. 211. 160. 156. 179. 185. 2. 212. 4. 254 Recurvirostra. 268. 178 Pseudocalanus acuspes. 221. 150. 150. 255 Platicththys flesus. 189 Oithona ovalis. 4. 267. 270 Placopecten magellanicus . 185. 184. 157. 227. 239. 212. 167. 181. 276. 271. 282. 150. 172. 185. 165. 276. 212. 229. 146. 208. 24. 283. 272. 147.Taxonomic Index 311 Saccharomyces cerevisae. 264. 208. 150 Simantherina. 222. 150. 158. 282 Sillago ciliata. 180. 184 Uronema. 256 Strobilidium. 211. 226. 211 Tetraselmis suecica. 242 Skeletonema costatum. 148 Sillago sihama. 164. 273 Tisbe reticulata. 262. 30 Tigriopus. 226. 275. 221. 34. 172. 157. 134 Tisbe. 182. 219. 270. 273. 270 Stenosemella. 211. 166 Thalassiosira nordenskjoldii. 208. 255 Synchaeta. 220. 160 Tetraselmis. 162. 158. 155. 184 Tisbe gracialis. 209. 208. 181. 265 Tetraselmis striata. 270 Ulva petrusa. 192. 302 Tetraselmis chuii. 9 Tridacna gigas. 163. 160. 110 Scenedesmus. 215. 1. 10 Schizochytrium. 166. 155. 176. 282 Thalassiosira fluviatilis. 217–220. 172 Thalassiosira pseudonana. 212. 265. 153–155. 162. 157. 163. 182. 261. 4. 254. 277 Tigriopus japonicus. 284 Scottolana canadensis. 256 Synechococcus elongates. 212. 287 Skeletonema pseudocostatum. 159. 278. 159. 264–268. 109. 218. 255. 208. 188. 262. 184. 162. 30. 182 Tisbe holothuriae. 87 Stichococcus bacillaris. 25. 211. 211. 233. 148. 209 Solea solea. 229. 179. 156. 192. 226. 182. 284 Spirulina. 281 Synechococcus. 172. 110 Vorticella. 129. 217. 209. 17. 165. 110. 166. 164. 182 Tisbe furcata. 162. 254. 283 Stichococcus. 185 Thalassiosira. 9. 162. 109. 184 Scylla serrata. 191 Temora stylifera. 27 Skeletonema. 32 Temora. 182. 231. 277. 150. 277 Tetraselmis tetrathele. 5. 226. 17 Siganus. 281. 242 Schizopera elatensis. 189. 150. 209. 255 Synechococcus bacillaris. 190. 174. 301 . 150. 277 Spirulina subsala. 163. 255 Ulva. 184. 208. 175. 17. 287. 4. 265–268. 181. 254–258. 184–187. 148. 164–166. 185–187. 283 Symbiodinium microadriaticum. 301 Zoothanmium. 162. 266 Trachurus. 208. 185. 301 Vibrio. 229. 230. 158. 236. 159. 277 Sciaenops ocellata. 166. 18 Seriola quinqueradiata. 242. 150. 286. 179 Temora longicornis. 160. 167. 211. 162 Tisbe cucumariae. 208. 150. 183 Scophthalmus maximus (ie Psetta maxima). 282. 230. 33. 255 Thalassiosira weissflogii. 9. 150 Sparus aurata. 163. 186 Tilapia. 33. 147. 287 Vibrio anguillarum. 162. 166. 181. 11. 274. 273 Tisbe battagliai. 157. 280–285 Australian bass. 148 Mussels. 147. 147. 150. 187 Summer flounder. 258 cupped. 268 Striped bass. 10. 281 Asian sea bass. 4. 284. 17. 255 Gilthead sea bream. 17 . 283. 67. 280–285 Herring. 187 Halibut. 283–285. 8–10. 4. 192 Plaice. 147 Striped patao. 4. 192. 8. 175 Dolphin fish. 177 Pufferfish. 107. 9. 8. 111. 265. 7. 18 Mud dab. 148. 150. 177. 111. 286 Jack mackerels. 253. 1. 10 Red snapper. 67. 151. 2. 8. 177. 148. 10. 150 Sand whiting. 280 Red sea bream. 104 Chinese white shrimp. 284. 3–5. 1 Wolfish. 9. 150 Dover sole. 280. 3–5. 192. 285 Flounder. 11. 45. 147. 4. 284 Silver bream. 150 Mullet. 147. 285 Golden snapper. 94. 147. 147. 284 Grouper. 171–175. 150. 286 Mulloway. 158. 172. 172. 9. 47. 194. 171. 9. 4. 256–259. 281. 257–260 Pacific white shrimp. 259. 287 Seahorse. 187 Black sea bass. 159. 10. 283–286 Winter flounder. 3. 10. 10 Threadfin. 3–6. 275. 9 Japanese flounder. 279–281. 255 Sea bass. 17. 112. 180. 9. 281. 192 King scallop. 148. 265. 3. 17. 256. 9. 147. 2 Chilean. 10 Squid. 148 Atlantic halibut. 268 Clams. 147. 276. 17 Rainbow trout. 234 Catfish. 4. 8 Turbot. 46 Red drum. 280. 9. 284. 148. 150 Yellowtail. 175 Sand borer. 168. 67. 17. 4. 281. 150. 269 Pink snapper. 17. 67. 1. 3. 46. 193. 8. 17. 150 Olive flounder. 195. 9. 8 Spotted sea trout. 148 Scallop. 107. 261 Kuruma prawn. 9 Oyster. 150 Dhufish. 262 European. 284–286 Sea bream. 255 Cod. 67. 34. 192. 253. 8. 67. 147. 260 Menhaden. 2. 268 Manila clam. 171. 109–112. 10. 172 Giant clams. 9. 112. 193. 151. 9. 111. 147. 147 Yellowfin sea bream. 150 European sea bass. 150. 255 Nassau grouper.-260. 281. 4. 269. 17. 4. 282 Mud crab. 157 Grey mullet. 9. 192. 280–283 Darter goby. 4. 266. 3–6. 17. 175.Common Names Index Anchovy. 148. 67. 148 Black sea bream. 10. 150. 111. 147. 301. 225–227. 270 copepods. 254. 88. 47. 272. 221. 104. 161. 280 Axenic. 269. 270. 223 Chymotrypsin. 286. 253 Carotene. 84. 103. 283. 287 Antifoam. 180. 163. 286. 28. 23. 68 70. 194. 75. 171. 231. 276. 179. 208. 42. 301–304. 221 Bio-encapsulation. 281. 269. 149. 87. 257. 164–167. 25. 233 Carbonate. 266 Aspartate. 236. 208. 92. 302 Algal blooms. 102. 86. 139–141. 300–302 Amphoteric females. 301 Amictic female. 129. 302 Batch culture. 277 grazers. 39. 276 β-carotene. 82. 30. 129. 32. 151. 42. 20. 106. 38. 147. 215. 227 Allelopathic. 286 Amictic egg. 273. 19. 206–232. 152 Antennule. 161 Bicarbonate. 279 Calanoid. 137–140. 133–134. 88. 153. 266–268. 227. 273. 171. 152. 269. 224. 240 Ascorbic acid. 233. 149. 217. 111. 35. 207. 79. 233 Carotenoid. 255. 267. 224. 105. 33. 240. 111 Biosynthesis. 307 Algae. 240 Biotin. 240 Carrageenan. 257 Chloroplast. 159. 271 Centric diatoms. 233. 141. 92. 31. 277. 133. 207. 125. 153. 240. 11. 233. 272. 93. 302 Batches. 146. 172. 97. 35. 223. 6. 275 Asepsis. 31. 301. 217 Carnivorous. 215 Chlorophytes. 79. 5. 236. 39. 217. 5. 279–281. 156 Ash. 219. 163. 158. 227 Brine. 98. 175–180. 273. 149. 254 Chlorophyll. 7. 234 Aseptic. 214. 38. 285 Alkenones. 256. 97. 97. 286 Carboxylic acids. 109. 278 Campesterol. 236. 217. 181. 275–279. 206. 45. 134. 276. 27–30 Ammonia. 277. freeze-dried/preserved. 304 Bloom. 219. 149. 147. 2. 191. 132. 272. 236 Chemostat. 235. 86. 220. 43. 34. 206. 233. 107–109. 221. 269. 87. 223 Arachidonic acid. 233–242. 228 Cannibalism. 227 Chromatography. 222. 225. 4.153 Antenna. 263. 106. 127. 239 Artemia Reference Center. 275 Bivalve. 255. 101 Antioxidant. 189. 285 Anal somite. 182. 99. 179. 300 Blue. 33. 161. 46. 41. 171. 26. 206. 178. 32. 99–109. 92. 214. 253–276. 127. 176. 191. 268. 43. 306 Airflow. 189–193. 73. 273 Broodstock. 233 Bacillariophyceae. 221. 270. 194. 111. 208 Amino acid. 286–288. 85. 302 Algae. 153. 35. 67. 27. 48. 269 Centrifugation. 279. 151–153 Antibiotic. 223. 180. 254. 214 Cholestase. AA. 207–209. 147. 24. 68. 225. 220. 209. 108. 21. 162. 45. 38. 91. 37. 34. 268. 191 Chlorophyceae. 26. 49. 140. 94. 261–263. 92 Bleach. 98. 207. 257–259 Bubbles. 48. 161–163. 68–70. 48. 157. 35. 283 Cholesterol. 229–230. 224257. 182. 20. 276–278. 279 Black disease. 194. 31. 33. 108. 163. 40. 209. 125. 268 Chitin. 286 Binders. 129. 130. 102. 33. 241. 278. 45–47. 213. 223. 151 Chelating agent. as food. 280 Chromatogram. 8. 77–79. 37.Subject Index Adenosine triphosphate. 208. 208 Allelopathy. 105. 230. 105. 128. 37. 149. 27. 133. 302 Beer-Lambert. 217. 39. 233 Air-water lift. 80. 24. 281–287. 182. 235 Asexual reproduction. 130. 110. 239 Benthic. 257 Cell wall. 269 Casein. 155. 47. 305 Bleaching. 305 Brood size. 6. 214 Aeration. 186. 79. 269. 275 Arabinose. 259. 221. 128. 131. 128. 277. 109. 77. 262. 51. 128. 129.302 Baker’s yeast. 187. 232. 146–153. 207. 269 Catabolism. 300 Algae. 228. 89 Algae. 50. 176 Carbohydrate. 155–167. 228 Brassicasterol. 51. 182. 262. 303 Buoyancy. 130. 158. 257 Biotechnology. 37–41. 193. 25. 271 Carbon. 157. 48. 183–185. 305 Bond. 285 Carbon dioxide. 89. 277. 65 Artificial light. 222. 254 Bacteria. 45. 21 algae. 129. 172. 75. 189.123. 273 Areal density. 257. 276. 161. 274 Chorion. 109. 177. 269 Auditive capsules. 207. 10. 186. 143. 36–38. 32. 235. 140. 101–103. culture of. 83. 263 Cephalosome. 212. 49. 160–162. 190. 47–49. 191. 269 Bioconversion. 88. 44. 225–227. 171. 271 Chicken eggs. 49. 32. 87. 151. 84. 36. 87. 27 Amylase. 163. 304. 134. 89. 177. 278. 232. 85–87. 106. 95. 217. 110. 38. 171 Alkaline phosphatase. 214. 94. 140. 2. 178. 223. 186. 84. 285 . 223 Attractants. 27. 87. 123. 92. 23. 176–178. 74. 257–259. 209. 43. 132. 242. 79. 77–79. 79. 103. 97. 24. 97. 140. 47. 104. 257. 35. 282 CO2. 33. 236. 191. 301 Egg sac. 222. 257. 97. 47–50. 139. 107–112. 263 Cyclopoid. 301 Corn bran. 261. 277 Continuous culture. 161. 149. 76. 189. 176. 178. 220. 163. 215 Dark-light cycles. 68. 282. 91. 238. 216 Day post-hatching. 3. 301 Feeding rate. 37. 278 Electrodes. 102. 270. 176. 79. 271. 223 D1 protein. 106. 280. 288 microphagus feeding. 269. 207. 2. 159. 195 Diatoms. 263. 215 Elongate. 274 Embryogenesis. 156. 215 Competitor. 256. 160. 271. 151–153. 305 Decapsulated cyst. 180. 49. 156. 236. 3. 191. 141. 129. 161. 181. 157. 128. 272–274 Fecundity. 43. 190. 66. 128. 17. 84. 67. 32. 268 Compensation energy. 283–287 Clupeids. 155. 214 Engraulids. 109. 7. 153. 107. 304. 68. 96. 273 Eukaryotes. 106. 131. 207. 190. enrichment. 82. 259 Feeding. 266. 23 Clam. 213. 253 Dry matter. 234. 212. 93. 30. 21. 305 Endogenous reserves. 162. 43. 172. 160. 112. 243. 282–286 Decapsulation. 302 Energy conversion. 134. 237. 267. 259 Endopodite. 37. 174. 128. 38. 172. 273. 161. 233. 273 Desaturation. 306 Collagen. 306 Enteritis. 190 Eicosapentaenoic acid (EPA). 17. 270 Environmental factors. 137. 123. 276. 222 Exopodite. 30. 106–112. 5. 269 Fasting. 125. 268. 302. 186. 100–106. 194. 221. 109 Dipterex. 302. 178. 24. 209. 159. 218. 134. 208 Euryhaline. 280. 257. 304–306 Cyst. 47. 193. 108. 242. 283. 276 Dilution rate. 96. 208. 190. 220. 109. 1. 107. 69. 103. 176. 38–42. 23. 97. 270. 112. 300. 186. 102. 222. 122. 285 Epifluorescence. 223. 270. 111. 45–49. 242. 239 Cold storage. 266 rotifers. 49 Diapause Artemia. 227. 72. 94. 70 Exotrophic. 263. 256. 267–287 Artemia. 50–52. 70. 278 DNA. 284. 32. 232. 129. 228–232. 279–281. 272–274. 192–194. 156. 93 Corona. 269. 187. 65–68. 83. 23–25. 111. 236 Copepod. 228. 232 Cyanobacteria. 79. 86. 283. 233. 151. 262. 103–106. 194. 147. 253 EDTA. 24. 221. 94–98. 271. 263. 3. 69. 237. 100. 231. 32. 98. 5. 89. 193. 305 Degassing. 257. 236 Egg production. 157. 239. 25. 193. 237. 213. 239. 286 Coefficient of extinction. 189. 98. 12. 168. 34. 79. 236. 137. 288. 152. 268. 266. 96. 17. 180. 34. 279. 111–112. 17. 253. 25. 189. 208. 138. 222. 137. 176. 278 DHA/EPA. 112. 111. 281. 161–163. 287. 13. 156. 82. 35. 73. 194. 221. 48. 220 Desaturases. 69. 263. 240. 280. 153. 25. 79. 208. 208. 266. 52. 206. 192–194. 143. 280 Energy. 106. 271–273. 44. 158. 181. 193. 31. 213. 282. 190. 176. 105. 271 Faeces. 274. 69. 261. 102. 240. 187. 275 Dry particles. 86. 24. 270. 264. 280. 258. 87. 3. 86–88. 287 f/2. 180. 269. 140. 306 Docosahexaenoic acid (DHA). 129 Disinfection. 26. 129. 2. 37. 18. 276 filter-feeding. 25. 12. 19. 194. 155. 27. 20. 225. 141. 258. 51. 283. 273. 110. 105. 213. 300–302 Cingulum. 243. 156. 194. 103. 188 Cyst. 283. 82. 11. 285 Conway. 214. 107. 288 Dry formulated feed. 141. 272–274. 146–195. 43. 101. 282 Enrich. 217. 193. 187. 240 Drinking. 238. 171. 283 Fatty acid. 283 Diols. 217. 137. 31. 107. 154. 256 Essential amino-acid. 208. 21. 87. 134. 106. 105. 159–163.163. 81. 97. 13. 270 Doubling time. 223. 218. 73. 182. 21. 89. 134. 267. 267. 283 Concentrated Chlorella. 182. 235. 133. 282. 29 Cost. 267. 74. 276–278. 227 Dipalmitoyl phosphatidylcholine (DPPC). 163.94–98. 300 first feeding. 243 Crustaceans. 149. 155. 67. 108. 52. 123. 24–26. 131. 179. 47–49. 263. 152. 190. 36. 6. 154. 11. 37. 213–217. 302 Conversion index. 40. 276. 269. 217. 106. 27. 105. 255. 178. 78. 103. 259. 272–274. 50. 272. 81. 207. 21.314 Subject Index Ciliates. 237. 279. 67. 266. 151. 103. 179. 40. 272–278. 221 Desaturate. 101. 112 copepods. 273 Egg ratio. 109. 179. 261. 259 Clear water. 75. 254 Eustigmatophyte. 140. 284 Cost price. 37. 65. 88. 126. 47. 238. 229. 159. 68. 234. rotifer 27 Cysteine. 194. 19. 160–163. 140 copepods. 91. 279. 303. 229–231. 284 Digestive tract. 38. 80–82. 42. 140. 218. 279 Cryptophyceae. 259 Embryo. 279–282. 207–209.229–232. 286 Eye. 268. 287 Enzymes. 265. 281. 94. 181. 87. 90. 276. 2 255. 24 raptorial feeding. 41 Contamination. 102. 157. 47. 195. 266–268. 70 Endotrophic. 43–45. 282–283. 157. 97. 83. 259. 30. 10. 106. 158. 268. 131. 187. 219 Eustigmatophyceae. 279. 139. 242. 47. 255. 49. 271 Digalactosyl glycerides. 267. 77. 72. 43. 42. 3–5. 2. 39. 217. 75. 256. 6–13. 103. 186. 125. 236 Concentrator/rinser. 275 Faecal pellets. 35. 152–154. 160–162. 22. 37. 254. 112. 134. 281. 91. 111. 161. 238 Dinoflagellates. 107. 187. 272 Essential fatty acid (EFA). 273 Elongation. 278. 92 . 72–82. 97. 20. 52. 186. 182. 239. 129. 48. 161–264. 272. 110. 75. 127. 180. 208 Eukaryotic. 274 Diacylglycerol. 69. 228 Digestion. 281. 217. 19. 278. 240. 42. 125. 50. 40. 112. 132. 243. 219. 209. 32. 236. 153. 106. 227. 259 Heterokont. 34. 288 algae. 4. 38. 36 Artemia. 228. 76. 149. 225. 125. 101. 37. 302 Growth rate. 277 Harvest. 234. 300–302 Flatfish. 183–189. 29. 102. 133. 48. 172. 253 Hermaphroditic. 177–182. 281 Herbivorous. 12. 280 Filamentous bacteria. 78. 222. 2. 5. 192. 283 Glycolipid. 111. 233. 172. 279. 160. 134. 233. 127. 207. 147. 279. 238. 109. 163. 302 Instar. 191. 17. 123. 181. 160. 277. 67. 103. 82. 21. 65–67. 110. 39. 270 Labrum. 112. 180. 7. 281. 168 Fucosterol. 11. 254. 79. 235. 189. 73. 7. 104. 152. 194. 25. 100. 86. 271 copepods. 221. 228 Artemia. 134–139. 177. 288. 23. 281. 168. 280. 178. 171. 302 zooplankton. 127. 273 rotifers. 85. 215–221. 263. 45. 159. 262. 222. 1. 7. 172. 76. 66. 105. 258. 254–272. 1. 281–283 algae. 91. 132. 162. 213. 66. 94.5. 189. 93. 3. 78. 66. 236. 150. 104. 228. 227 Isochronal development. 159. 153. 98. 156. 187. 266–269 Ketones. 157 Iso-osmotic. 208. 155. 159. 131. 210. 99. 193. 131 Integumental. 146. 11. 171. 154. 287 Flocculation. 171. 78. 91. 214. 285–287 Fluorescence. 230. 27. 109. 171. 288 . 192. 277 Free amino acid. 103–106. 1. 107. 168. 17. 95. 78. 108. 96. 84–87 copepods. 271. 228. 86. 158. 212. 76. 22. 274. 47–49. 133. 271. 19. 227 Kinetic. 226 Fungi. 253. 238 Artemia. 79. 258 Infra-red. 206. 306 copepods. 11–13. 180. 13. 80. 181. 21. 271. 51. 174. 274. 2. 214 Ingestion. 220. 106. 281 Husbandry. 263. 175. 3. 267. 179. 152. 28. 258 Gametogenic. 92. 194 Genetically modified organisms (GMO). 234. 10. 233. 280 Intergill-arch. 44. 49. 31. 208. 106.97–99. 73. 20 crustacean. 38. 132. 280–283. 264. 273. 176. 1. 259. 221. 52. 43. 214 Fluorescent. 3. 34. 206. 276 Hyperosmotic. 129. 240–243.3. 134. 6. 72. 277 Fertilization. 12. 86. 163. 254. 12. 46. 17–19. 222. 47. 235 Filtering. 278 copepods. 27. 79. 163. 81. 228 Gonad. 104. 225. 253. 1. 129–131. 43. 229 Gamete. 4–11. 281 Glutamate.Subject Index 315 Female. 177. 86. 278. 77. 190. 7–11. 122. 195. 266. 286 Freeze-dried algae. 108. 149. 273–275. 52. 110. 219. 81. 83. 281. 285 Artemia. 243 Fermentor. 261. 208. 3. 216. 302 Filtration rate. 107. 271 Finfish. 12. 268. 258. 5. 159. 211 Hetrotrophic. 225. 36. 230. 171. 67. 253. 221. 24. 258. 172. 234 radiation. 170. 112. 281 Glycogenic reserves. 217. 227. 284–286. 236 Isochrysideae. 182. 69 Larvae. 160. 171. 175. 152. 208. 147. 24. 287. 73. 103. 234. 272–274. 129. 163. 181. 283–288 Gross composition. 181. 215. 69. 301 Fermentation. 126. 41. 84–88. 98. 263 Flora. 44. 70. 256 rotifers. 122. 131. 93. 235. 2. 129. 191–194. 214 water. 24. 7–11. 13. 147. 36. 280 Juveniles. 209. 268 Growth. 258. 300 Hepatocytes. 286. 137. 111. 6. 181. 108. 160. 305. 243. 21. 284. 159. 95. 27. 242. 209. 171. 37–39. 260. 159. 7. 47. 253–255. 92. 282 Glucose. 279. 257. 284. 130. 37. 140. 257. 34. 285 Inhibitory. 278. 80. 212. 6. 25. 259 Great Salt Lake. 253. 176. 10. 301.65. 86. 172. 265. 51. 140. 170. 25. 94–97. 123. 105–111. 279 Fingerlings. 137. 36–38. 180. 223. 1. 209 Histidine. 235–239. 112. 270 house. 32. 191. 65–67. 163. 306 Hatchery. 171. IFREMER. 44. 67. 238 Inoculum. 187. 262–264. 72. 86. 65–67. 301 Harpacticoid. 263. 217 Lab-lab. 92. 253. 269. 208. 67. 146. 45. 84. 300–303 mollusc. 266. 97. 285 algae. 3. 191. 112. 279. 155. 276. 37. 266. 92. 187. 161–164. 155–158. 143. 107. 129. 176. 192. 276. 83. 221 Flagellates. 131. 233. 126. 223. 207. 3. 219. 262. 29. 271 shrimp. 270. 34. 68. 129. 191. 254. 171. 100. 214. 25 Generation time. 265. 190. 68. 79. 280–287. 256. 266–268. 46. 162. 111. 253. 146. 302 Hatch. 191. 276. 3. 103–108. 212. 39. 131. 217. 72. 282 Iron. 109. 276. 51. 45. 77–82. 176 rotifers. 221. 9. 233. 155–159. 278 Hunting. 43. 182. 47. 281. 131. 255. 257. 112 yeast. 5. 126. 168 Fry. 101. 266. 11. 280 Hypochlorite. 270. 89–91. 51. 156. 279 fish. 34. 139. 301 Filter. 42. 1. 281 Glycogen. 69. 211. 259 Gastric glands. 258. 149. 7 Gill-arch. 163. 7. 125. 13 Flagella. 168. 6. 263. 233. 10–13. 223. 223 Glycerol. 305. 266 Ingestion rate. 243. 106. 8. 94. 168. 67. 271. 25. 29. 182–187. 128. 36. 38.217. 141. 220 Inoculation. 193. 100–106. 229–231. 4. 123 Green. 269. 265. 260. 271–274. 187. 49. 164–167. 158. 100. 273. 224 Formulated diets. 279. 192–194. 194 rotifers. 287. 43. 283 Heterotrophs. 28. 236 Folate. 242. 304. 265.66. 38. 146. 13. 281 Galactolipid. 190. 136. 30. 106. 207. 77. 44. 52. 32. 272 Highly unsaturated fatty acids (HUFA/HUFAs). 173 algae. 163. 92. 253–263 Larviculture. 33. 195. 25. 2. 277. 236. 240. 261 Filtration. 261–264. 162. 172. 233 Gadoids. 172. 158. 277 Artemia. 86. 174. 234. 85. 258 Lectins. 111 Live prey. 181. 208. 266 Media. 264. 44. 83. 219–221. 17. 270 Males. 279. 276 Pavlovol. 65. 17. 108. 195 Osmoregulation. 268. 255. 284. 178. 274. 272. 5. 74. 137. 279. 286. 12 Penaeid. 88. 280–283 Mouthparts. 5. 209 Naupliar. 229–232 Mouth. 275. 155–159. 163. 85–93. 282 Mesoplanktonic. 77. 263. 280 Mixing. 175. 23. 163. 129. 29 . 190 Omnivorous. 181. 178. 130–132. 17. 100. 89. 253. 29. 99. 280 Osmotic. 207–209. 4. 151. 301. 281. 223. 261 Optimum temperature. 44. 107. 153 Oviparous. 160–162. 218. 268. 240 level. 269 Operating costs. 241. 275 Mineral salts. 12. 269. 257. 238–240 Pathogenic bacteria. 96. 231. 7. 174–178. 190. 179. 94–98. 269. 192–194. 3. 72. 208. 174. 281 Neoglucogenic. 214. 287 Micronutrients. 5. 3. 82. 2. 129. 266. 215. 271. 125. 215 PAR. 194. 17. 227 Microalga-consuming. 130. 181. 17. 288 Nycthemeral. 265 Artemia. 207. 214 Nutrients. 124. 189. 97. 285 Mesocosm. 227. 190. 194. 153. 271. 253. 137. 216 Off-the-shelf. 70. 182. 274. 192. 128. 235. 253 Lorica/loricated. 2–4. 21. 4. 141. 131. 105. 5. 285 Mandible. 11. 217. 219 Ornamental fish. 300 Membranes. 209. 186. 152. 195. 221. 221. 34. 238. 103. 130. 76. 219. 223 Methylenecholesterol. 236. 192. 262 Micro-encapsulated. 190. 274. 263–265. 3. 47. 21. 281. 266 Maxillipeds. 255. 263. 146. 268 Metasome. 176. 68. 130. 111. 176. 92. 217 Monogononta. 71. 128 Linoleic. 92. 227. 300–303. 149. 18. 190. 240 Monospecific. 266. 24. 281. 207. 43. 83. 180. 277–279. 81. 152. 235. 50. 206. 206. 241. 81. 152. 19. 34. 2. 26. 86. 270. 301 Parthenogenesis. 45–49. 27–30. 301 Lumen. 81. 270. 146. 283 Mictic female. 266–268. 175. 65. 67 Mouth opening. 286. 112. 287. 23. 156–159. 233. 47. 109. 24. 2. 282. 160. 276. 220. 85. 257. 47. 182. 68–70. 271. 240. 110–112. 71. 126. 258–261 Monounsaturated. 79. 238. 3. 268. 223. 263. 221. 149. 17. 123 Oxygen. 220. 88. 47. 214. 38. 187. 90. 47. 225–227 Methylporiferasterol. 2. 112. 20. 49. 191. 133 Ovisac. 87. 156 Partial pressure. 33. 143. 21. 129. 269 Microsatellite. 39. 301–303 Path length. 12. 26 Maxillae. 51 Mineral premix. 274. 50. 19. 8. 277. 274 Niacin. 106. 242. 130. 47. 21 Monoseptic. 33–38. 222. 140. 206 Peroxidation. 46. 177. 287 Lipisomes. 32. 18. 233. 156. 288 Neuromasts. 187. 278 Oil sac. 149. 161. 134. 49 Monochromatic. 3. 68. 39. 79. 214 Nannoplankton. 97. 69. 189. 281 Nutritional value. 206–243. 270–272. 134. 100–103. 8–12. 102. 109. 91. 240 Models. 2. 214. 189. 195. 95. 287. 81. 110. 257. 269. 147. 27. 213. 189. 133. 81. 171. 34. 27. 67–69. 224 Nitrogen. 271 Monoacylglycerol. 223. 286 Light. 172. 223. 126. 103. 253–288 Microalgal feeders. 233 Mollusc. 253. 151–153. 220. 240 Parasite. 102. 27. 228. 214 Lux. 209. 21. 233. 171. 3. 168. 212. 95. 132. 33. 280. 263 Mixis. 286 Nitrogenous. 28. 229. 108. 83. 217. 97. 51. 262. 84. 159. 176. 143. 65. 39. 175. 180. 72. 220 Particles. 279. 175. 23. 162. 168. 3. 74. 239–241. 94. 286. 235. 81–85. 264. 108. 209. 26. 274–278. 143. 152. 287 Livestock. 239. 25. 129. 221. 32. 262. 105. 52. 38. 187. 189. 3. 83. 263. 93. 77. 162. 263 Nauplius. 34. 37–39. 306 P/I. 22 Microzooplankton. 26. 146. 241. 219. 158. 33. 253 Microalgae. 300. 277. 272 Linolenic. 154. 174. 172. 190–194. 241 Metamorphosis. 275. 77–81. 159. 152 Mannose. 228. 81. 152 Nucleotides. 236. 106. 303 Medium. 52 Mixotrophic. 27. 217. 234. 152. 243.316 Subject Index Larviparous. 261 Mastax. 180. 79. 111. 240. 283. 128. 223 Mariculture. 23. 68. 2. 223. 143. 217. 268. 187. 143. 255. 72. 26. 74. 233. 233. 91. 269–272. 69. 23. 109. 172. 287 Pelleted. 101. 44. 108. 20–22. 278. 253–259. 248. 152. 106. 217. 81–83. 151. 72. 96. 179. 280 Neutral lipids. 86. 228. 236. 21. 212–214. 79. 151 Methionine. 283 NADPH. 155 Ovoviviparous. 178. 130. 149. 233. 228 Pelagic. 43. 278 Oil emulsions. 268. 87. 301 Maltase. 242 Microflagellates. 182. 157. 281. 222. 287. 191. 45. 271 Microflora. 12. 229. 111. 222. 306 copepods. 24. 163. 171. 280. 268. 213. 189. 354. 40. 279 Microbial flora. 38. 129. 110. 262. 84. 220. 193. 23. 48. 256. 219. 207. 275 pH. 189. 281. 27–30. 49. 269 Microcapsules. 267–270 Pennate diatoms Peridinians. 33. 9. 302 Pheromone. 177 Liming. 215. 96. 83. 112. 39. 186. 206. 287. 253. 271. 276 Microparticulate. 29. 277. 268. 33. 39. 31. 171. 156. 103. 153. 92. 277. 275. 30. 105. 262. 214 Lyophilised. 19. 67. 279 Macroalgae. 233–235. 32. 133. 268. 276 Microorganisms. 232–234. 41. 301 Neoglucogenesis. 233. 147. 163. 223. 233. 31. 286. 5. 208. 236 Minerals. 225. 217. 30. 191. 47. 213–217. 272 Lipid. 39. 219 Ovary. 152. 243. 277 Metabolites. 259. 283. 161. 300 intensity. 282 Nematodes. 23–27. 194. 278. 39. 100–106. 100–102. 45. 106–109. 195. 215. 37. 38. 253. 176. 268. 135. 174. 12. 176. 178. 33. 2. 233. 78. 47. 19. 191. 236. 79. 66. 209. 261. 50–52 Retinal. 190. 37. 281. 139. 229. 25. 215 Phototactic. 208. 285 Phosphatidylcholine. 193. 277. 159. 302. 122 Salinity. 19. 22. 123.Subject Index 317 Phosphatase. 220. 281 Seawater. 3–6. 99. 26. 274 Stomach. 270 Rotenone. 93. 98. 195. 75. 233. 186. 220 Photoperiod. 67. 74–76. 123. 105–108. 152. 168. 109. 84. 123. 159. 30 Red. 179. 237. 241. 207. 172. 110. 242. 35. 87. 131 Saltwork. 83. 215. 87. 149. 279. 229. 24–26. 6. 286 Photo-autotrophic. 45. 49. 79. 149. 133. 153. 253 Small-mouthed. 217. 220. 123–125. 191–194. 156. 93. 281. 39. 253. 215. 36. 219. 187. 283–288. 65–67. 147. 263 Pseudocoelom. 27–35. 285 Spermatophore. 130. 206. 104. 75. 8–11. 194. 273. 17. 40 Polysaccharides. 302. 26 Pseudo-green water. 240–243 Photon-flux density. 24. 81. 100. 218–221. 83. 279. 280. 126. 263 Planktonic. 253. 221 Pigment. 11. 266. 258. 90–93. 140. 174. 86. 263. 190. 208. 109. 223. 99. 22. 137. 261 Polar lipids. 37. 146. 304. 108. 153. 222. 281. 168. 93. 109. 207 rotifers. 101. 207–209. 223. 91. 273. 223. 49. 174. 37–39. 182. 88. 177. 26 Protozoa. 112. 207 Ponds. 280. 217. 257. 168. 133. 243. 268 Spat. 228. 97. 93 Soy pellets. 233. 223 Steady state. 18. 193. 149. 84. 171. 109–111. 285. 221. 29. 141. 180. 156. 259 Artemia. 181. 224 Riboflavin. 222. 123–135. 129. 2. 44. 175. 81. 80. 182. 174. 109. 286. 261. 253 Salt production. 274 Predator. 104–106. 107. 237–239. 18. 277 Starch. 27. 106. 125. 21. 275 Productivity. 34. 177. 155 Spray-dried. 87 Sinking syndrome. 38. 163. 275 Pyridoxine. 10. 51. 110–112. 214. 140. 74. 306 Self-shading. 5. 215. 307 Phytosterols. 133. 267. 287 Plankton. 143. 1. 259. 157. 95. 130. 108. 219. 180 RNA. 137. 124. 236 Photoreceptor. 280 Sitosterol. 152. 254 Prawn. 269. 48 Reserves. 281 Prey size. 276. 67. 80–84. 178–182. 228. 191–193. 45–47. 43. 240 Setae. 285. 103. 51. 280 Prelarval stage. 168. 236. 151. 156. 105. 66. 176. 212. 272. 13. 258. 137. 162. 180 Rotifer. 132–134 copepods. 126 Post-larva. 238. 193 Retinol. 171. 129. 141. 271 Sexual reproduction. 182. 107. 13. 233. 283 Prelarvae. 241. 209 Photobioreactors. 179. 219 Soybean. 1. 192. 190. 236 Single-cell-proteins (SCP). 253. 34 Probiotic. 220. 238 Resting egg copepods. 5. 178. 162. 225–227 Small larva fish. 123. 101. 32. 180. 237–239. 131 Sand filter. 52. 213 Sterilisation. 257. 229. 96. 213. 5. 163. 178. 27–32. 286. 257. 21. 176. 238. 8–13. 5. 267 Phosphatidylinositol. 162. 301. 125. 214 Photoinhibition. 45. 37. 50. 307 Prymnesiophyceae. 208. 223. 206. 206. 122–134. 242. 110 Protonephridia. 160–163. 103. 112. 213. 28. 214. 31–33. 223. 107–109. 282. 134. 215. 24. 179. 288 Production systems. 275. 263–270 Silicon. 279. 233 Sorbitol. 262 Specific growth rate. 223. 214. 194. 18. 34. 96 Soy-cake. 134. 253–255. 1. 109. 305 Salmonids. 281. 221 Shelf life. 94. 147. 171. 209. 236. 223. 171. 78. 158. 187. 21. 18. 254. 168. 269. 131. 207. 243. 72. 286 Polyunsaturated fatty acids (PUFA). 287 Photoprotection. 258. 87. 80. 274 Polycarbonate. 221. 243. 19. 160. 68. 263. 240. 189. 2. 280 Photosynthesis. 270 Population dynamics. 305 Senescence phase. 3. 11. 229–232. 217. 17. 43. Prokaryotes. 242. 168. 269. 263 Predigestors. 133. 153. 235 Protein. 101. 234 Saturation. 129. 263 Prasinophyceae. 172. 175. 213. 127–129. 27–32. 2–4. 155. 21. 300–303 Saline lakes. 128. 5. 49. 192–194. 47. 304 Reproduction. 191. 159. 280. 277 Pleuronectids. 28. 103 Phototaxis. 257 Rice bran. 282. 84. 45. 194. 153. 123. 277. 67. 137. 221. 129. 21. 235 Sterol. 123. 215 Photons. 27. 24 Probionts. 174 rotifers. 40. 274 Phosphorus. 261 Photoautotrophs. 227. 181. 175. 270. 178 Phytoplankton. 257. 47. 268. 207. 137. 186. 34. 227. 10. 152. 68. 19. 146. 257. 35. 159. 216. 266–271. 127. 38. 186. 74. 129. 281 Plurispecific. 217. 223. 124. 270. 84. 225–228. 176. 151. 273 microalgae. 153. 209. 235 Sterilising. 241 Pigmentation. 72. 221. 214 Photochemical. 224. 287 Protein enrichment. 75. 91. 98. 19. 270. 300. 4. 40. 110. 139. 91. 6. 162. 160. 33. 214 Photooxidative. 172. 220 Scophthalmids. 9. 262. 67. 124. 259. 231. 230–232. 20. 174. 192. 263 Shrimp. 213. 279. 255 . 127. 258. 80. 267 Phospholipid. 192. 152. 235. 68. 34 Shear forces. 273. 283 Residence time. 186. 286 Photosystem. 224 Quanta R0. 92. 95. 47. 270–279. 126. 94. 3–5. 217. 254 Prymnesiophyte. 17–52. 124. 234. 140. 43. 11. 207. 301 Swimming velocity. 214. 108. 49. 38. 88. 285. 48. 237. 81. 98. 180. 217. 207. 21. 139. 258–261. 271 Triglyceride. 34. 37. 32–34. 280. 93. 156. 214. 76–79. 96–98. 37. 224. 193. 23. 78. 48. 98. 278. 158. 91. 255 Viability. 112. 133. 258 Yolk-sac. 187 Trichomes. 156. 24 Trypsin. 7. 240. 128 Yeast. 263–265. 302 Swim. 81. 238. 223. 108. 236 Transchorionic. 233. 262. 276 Vitamin. 106. 214. 155 Thraustochytrid Thraustochytriidae. 70 Thiamine. 85. 228 Urosome. 110. 35. 23. 269. 181. 257. 108. 110. 301 Yellow. 172. 159. 140. 94. 182. 38. 281–283 Tocopherol. 47. 162. 280 Transmission. 32. 272–277. 254. 213. 171. 49–51 Strains. 129. 79. 270. 268. 267–269. 110–112. 149. 45. 104. 93. 98. 107. 283. 228 Telopodite. 181. 268. 44. 180. 236. 74. 254. 274. 259 Wavelength. 125. 184. 209. 107. 133. 159. 22. 39. 283–286. 272. 218. 286 Taxonomic. 45–48. 72. 87. 228 Triacylglycerol. 275. 268 Toxins. 20. 109–111. 48. 278 Vitamin enrichment. 269 Zoea. 11. 143. 304 Yields. 52. 190. 192–194. 136. 263–268 Zooplankton. 235 Thorax. 305. 82. 259. 135. 279. 181. 84. 172. 186. 217 Wax esters. 286–288 Substrates. 83. 10–12. 190 Tintinids. 109 Vitellarium. 236. 223. 148. 190. 6. 193 Weaned. 276 Stress. 263 Artemia. 87. 281. 281 Survival. 283. 239 Transport. 78. 168 Unsaturation. 139–141. 24. 193. 74. 99. 43. 82. 1. 131. 3. 86. 306 copepods. 85. 214 UNIK filter. 28. 228 Trophi. 128. 156. 47–49. 137. 277–279. 213. 91. 132. 3. 50. 147. 223 UV light. 255. 281–283 . 76. 163. 288. 79–81. 137. 258. 236. 1–5. 172. 93. 162. 18. 157. 275. 191–193. 162. 98. 111. 87. 26. 47. 235 Trace elements. 27 Vitellogenesis. 50. 79. 106 Ultraviolet. 86. 144. 277. 101. 81. 281. 180–182. 102. 286 Welded-wedge filter. 265. 275 Thiosulphate. 125. 282 Zein. 220. 140.318 Subject Index Storage. 253. 94. 107. 18. 84. 49. 1. 11. 191. 123. 38. 135. 190 rotifers. 107. 126. 24. 174. 268. 143. 91. 21. 286 Tryptophan. 77. 12. 221. 181. 174. 149 Veligers. 152. 208 Thylakoid. 90. 65. 93. 112. 49. 133. 94. 31. 69. 280. 44 Taxins. 270. 21. 139. 305 Vibrionaceae. 180. 223. 84. 221. 255. 123. 180.