Marine and Aquaculture

Biotechnology SUB Hamburg

Dr. S. Felix, M.F.Sc., Ph.D. Professor

Fisheries Research and Extension Centre Tamil Nadu Veterinary & Animal Sciences University

Madhavaram Milk Colony Chennai - 600 051 (T.N.)

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AGROBIOS (INDIA)

Contents

PART I: GENETICS IN AQUACULTURE BIOTECHNOLOGY

1 Genetic Selection Programme ..3

A. Individual or Mass Selection 3 B. Family Selection 4 C. Within Family Selection 4 D. Combined Selection ; 4 E. Natural Selection 5 F. Artificial Selection 5

2 Chromosome Manipulation 7

A. Gynogenesis 7 (i) Natural Gynogenesis 8 (ii) Artificial (induced) Gynogenesis 8 (Hi) Induction of Meiotic Gynogenesis in Indian Major Carps 8 (iv) Meiotic versus Mitotic Gynogenesis 9 (v) Induction of Mitotic Gynogenesis in Indian Major Carps 11

B. Androgenesis 11 (i) Natural Androgenesis 13 (ii) Artificial Androgenesis ...13

C. Polyploidy 14 (i) Natural Polyploidy (Triploidy/Tetraploidy) 14 (ii) Artificial Induction of Polyploidy in Indian Major Caips 14 (iii) Detection of Polyploidy Individuals 18

3 Hormonal Manipulation of Fishes 19 .""J

A. Environmental Factors and Diet ..i :.20 (i) Temperature , 21 (ii) Light 21 (iii) Other Factors 21 (iv) Diet 22

B. Biological and Chemical Assay .....22

(i) Problems of Standard Dosage 23 (ii) Maturity of Recipient Fish 24 (iii) Variability in Pituitary Gonadotropin Related to

Phytogeny, Sex and Season 25 (iv) Use of Mammalian Hormones 25 (v) Use and Preservation of Pituitary Extracts 26 (vi) Gonadal Steroids 26

4 Hybridization in Fishes 29

I. Intra Specific Hybrids 29 II. Inter Specific Hybrids 29 III. Inter Generic Hybrids 30

Rohu — Catla and Catla — Rohu Hybrid 30 Mrigal - Common Carp.Hybrid 31 Chinese Carp Hybrid 31 Hybridization between Two Fresh Water Catfishes 31 Inter Specific Hybridization ofPenaeus monodon and

P. esculentus 31 Introgressive Hybridization 31

5 Sex Reversal in Fishes 33

I. Hybridization and Sex Reversal 33 II. Chromosome Manipulation and Sex Reversal 33 III. Temperature and Sex Reversal 34 IV. Sex Control in Fishes 35 V. 1.7a MT. in Sex Reversal : 37

Use.of Sex Steroids in Sex Reversal 37

6 Hormones and Induced Breeding in Fishes 39

Induced and Synchronized Spawning of Captive Broodstock 40 Gonadotropin-Releasing Factor (GRF) 40 The Gonadotropic Hormones 41 Hormonal Regulation of Vitellogenesis 42 Hormonal Regulation of Oocyte Maturation and Ovulation 43 Sex Steroids 44 Hormonal Regulation of Spermatogenesis and Spermiation 44 Induced Breeding in Carps with Pituitary 45 Method of Induced Breeding 45

Hypophysation (using Pituitary Gland) 45 Ovaprim to Induce Spawning in Cultured Fish ; 49 Comparision of Ovaprim and Pituitary 51 Ovaplant to Induce Maturation in Cultured Fish 53 OvaRH .: 54 Injecting OvaRH 55

Advantages of Hormones in Induced Breeding 55

References 56

PART II: AQUACULTURE NUTRITION

1 Artemia Enrichment in Aquaculture Production 69

Biotype Distribution and Zoogeography 70 Collection from Natural Habitat 71 Artemia Cyst Production and Hatching 71

Enrichment of Artemia 72 Enrichment Procedure 74 Artemia and Fatty Acid Enrichment 75 Enriching Artemia Nauplii 76 Preparation of Enrichment Diets 78 Enrichment with Nutrients 78 Direct use of Decapsulated Cysts 80 Evaluation of Artemia Cysts. . 82

Farming Practices in India 82 Economic Viability of Artemia Production ; 83

Establishment of Artemia Processing Plant...: 83 Research Needs on Artemia 84 Conclusion 84 References 85

2 Aquaculture Feed Technology 87 r

Formulated Larval Feeds 87 Artificial Feed - Rotifer (AFR) 89 Microencapsulated Diet for Larval Rearing (Micro Mac) 89 Power Omega 89 Preparation and Enrichment 90 ENCAP Shrimp Hatchery Microencapsulated Feed. 91 Microparticulate Microbound Diet 92 GAP- Genchem Artificial Plankton 92

HIFACS : 92-. Spirulina Micro Algae 92 BP 1000E ; ...93 GSP 500... : .-.93

Microencapsulated Egg Diets for Fish Larvae 93 Composition of Egg Components 94 Composition of (chicken) Egg. 94

Microencapsulated Diets Made from Whole Egg 94 (i) Wholesomeness 95 (ii) Consistency 95 (iii) Water Stability 95 (iv) Low Biological Oxygen Demand (BOD) 95

(v) Good Shelf Life 95 (vi) Ease for Preparation 95 Processing Techniques : 96 Advantages of the Novel Diets 96 Formulating Encapsulated Egg Diets 97

Feeding Methods - Complete Diet Feeding 97 Manufacture and Storage of Complete Pelleted Diets 97 Grinding 98 Pelleting 99 Storage 101

Feeding Practices . 103 Dietary Feeding Regimes and Allowances 106 • Choice of Complete Diet Feeding Strategy 108

Selection Criteria for Choice of Hatchery Feeding Strategy 109 In-House Production of a Dry or Moist Artificial Diet 109

Feed Performance 110 References ; 111

3 Nutrition and Health Management 113

Feed Additives 113 Enzymes 113 Probiotics 115 Prebiotics 116 Immunostimulants 117

Dietary Aminoacids I 1 8 Toxin Binders 118 Minerals and Vitamins.. 118

Vitamin C (Ascorbic Acid) 119 Vitamin E *. ...; 119 Astaxanthin : 119 Feed Additives used in Aquaculture Feeds 121 Antibacterial Agents 122 4-Quinolones 123 Therapeutants other than Antibacterials 124 Copper Compounds (Aquatrine®) 125

References -...126

PART III: BIOREMEDIATION IN AQUACULTURE SYSTEMS

1 Biosecured Aquaculture System 131

Concept of Raceways 132 Zero Water Exchange System 132 Bioremediation in Shrimp Raceways 133

Principle of Bioremediation 133

Application Aspects of Bioremediation 134 Constraints of Bioremediation 135

Sustainability : 135 Issues in Genetic Diversity 136 The Aqueous Medium 137 SPFShrimp Population 137

The Raceway Tank and Associated Equipment/Structure 137 Environmental Impact 138 Advantages of Raceway Culture System 138

2 Harmful Algal Blooms and Control 141

Harmful Algal Blooms •. 142 Specific Impacts 143 Adverse Effects on Human Health 143 Commercial Fisheries 143 Subsistence Fisheries ! 143 Recreational Fisheries 144 Ecosystem and Environment 144 Organisms Accumulate Toxins 144 Impact of HABs Problems 144 Mariculture and the Incidence or Severity of Harmful Algal

Blooms 145 Control of Algal Blooms 147

Control of HABs 148 Mitigation Measures to Reduce Impacts on Fish Farms 150 Effective Measures to Mitigate the Impacts of Toxic HABs on

Shellfish 150 References ; 151

PART IV: BIOTECHNOLOGY BASED HEALTH MANAGEMENT IN AQUACULTURE

1 Vaccination for Sustainable Aquaculture 157

Vaccines 157. Concept of Vaccination 157 Reports on Fish Vaccination : 158 Role of Vaccines 159

Types of Vaccines..... 159 A. Active Immunization 159 B. Passive Immunization 160 C. Dead and Live Vaccines 160 D. Modern Vaccines 161

Recombinant DNA to Produce Fish Viral Vaccines 163

Characterization of Vaccines 163 1. Safety 163 ii. Immunogenicity 164 iii. Stimulation of Protective Immunity 164

Efficacy of Vaccines 164 Specificity 164 Safety and Efficacy of Live Agent Recombinant Vaccines 164

Vaccine Development 164 Steps involved in the Development of Fish Vaccines 165

Methods of Vaccination \ : 166 /. Injection Vaccination 166 2. Oral Vaccination 166 3. Immersion Vaccination 166

Vaccines for Sustainable Aquaculture 167 Potential to Develop Vaccines and Immunostimulants for

Crustaceans 169 Vaccines and Antibiotics 169 Vaccination for Fish Disease Control 171 Advantages of Vaccination 171

References i 171

2 Immunostimulants in Aquaculture 173

Essential Features of Immunostimulants 174 Immunostimulants Act through following Mechanisms 174 Immunostimulants - Scientific Background 174 Immunostimulants in Aquaculture 175 Immunostimulants in Fish Diets 177 Immunostimulant for Higher Survival Rate 177 Characteristics ofLamal Fish Immune System 177 Immunostimulation of Larvae - Possibilities and Constraints... 178 Immunostimulation of Larval Fish: Nonspecific Defences 179 Fish Defense System Stimulation by Immunostimulants 180 Immunostimulants used in Fish and Shrimp 181 Immunostimulation of Shrimps ! 181 Natural Immunostimulants ...182 Immunostimulant Property of Botanical Additives 183 . Lipo Poly Saccharides ..183 Beta Glucans as Immunostimulants in Fish 183

Peptide and Other Compounds 184 Levamisole 184 FK-565 . 185 ISK ; 185 Chitin and Chitosan 185 EF203 : 185

Ete 185 PKS 186 Bacterial Derivatives 186

Animals and Plant Components 187 Animal Extracts 187

. Glycyrrhizin 187 Other Immunostimulants 187 Diet Components ' 187 Vitamin C 188 Vitamin E 188 Vitamin A 188 Hormones, Cytokines and Others 188

The use of Immunostimulants in Fish Larval Aquaculture...! 190 Prospects 190 Advantages 191

References.... 191

3 Probiotics 193

Probiotics in Aquaculture Health Management 193 What are Probiotics? 194 Mechanism of Action of the Probiotic Bacteria 194 Types of Probiotics 195 Probiotics Administration 195 Feed Probiotics 195 Water Probiotics 195

Probiotics in Aquaculture 196 Microbial Supplements - The Broader Perspective 197 Probiotic Role of Lactic Acid Bacteria in Aquatic Systems 198 Intestinal Environment of Aquatic Animals 199 Action of Probiotics in the Gut 200 Preconditioning to Establish Probiotics in the Gut 200

Probiotics and their Role in Shrimp Hatcheries 201 Influence of Probiotics on Growth of Indian White Shrimp

Fenneropenaeus indicus 202 Use of Probiotics in Penaeid Shrimp Grow out Systems 203 Probiotics to Assure Nutritional Security 203

Conclusion 204 References 204

4 Fermentation in Aquaculture Systems 207

Factors Influencing Fermentation 207 Influence of Mode of Fermentations on Microbial Enzyme

Production 207 Fermented Products to Sustain Microbial Population 208

Anaerobic Fermentation 209 Ethanol Fermentation 210

Fermentation Biotechnology in Aquaculture 211 Fermentors 211 Components and Functions of fermentor 211 Fermentation Technology 212 Microbial Supplements :.. 212

Preparation of Fermented Product for Raceway System : 214 Aspergillus oryzae Fermentation Product 215 Bacillus subtilis Fermentation Product : 215 Biological Control Agents 215 Application of Microbial Inoculants 216 Application of Fermentation Technology 216

Fermentation Biotechnology: Future Prospects... 217 References 218

PART V: MOLECULAR DIAGNOSTIC METHODS IN AQUACULTURE MANAGEMENT 1 Polymerase Chain Reaction Techniques 221

Production of Multiple Copies of DNA Fragments.. ......221 Components of PCR 222

1. Primers ' .' 222 2. PCR Buffers : 223 3. DNA Polymerase 224 4. Deoxyribonucleotide Triphosphates 225 5. Template DNA 225

The Cycling Reactions.... 225 1. Denaturation at 94°C 225 2. Annealing at 54°C 226 3. Extension at 72°C 226

Selection of Primers 226 DNA Polymerases for DNA Amplifications 227 DNA Polymerases for DNA Amplifications 227

Modifications of PCR 230 1. Competitive PCR 230 2. Hot-start PCR ...: 230 3. RT-PCR (Reverse Transcriptase Polymerase Chain

Reaction) 230 4. Nested PCR 231 5. Inverse PCR 232 6. Vectorette PCR 232 7. Panhandle PCR 232 8. Anchored PCR 233

9. Multiplex PCR .... 233 10. Coupled Amplification and Sequencing (CAS) 234 11. Self Sustained Sequence Replication (SSSR) 234 12. Expand Long Template PCR 234 13. In-situ PCR 234 14. Competitive RT-PCR 234 15. Non-competitive RT-PCR 235 16. Kinetic RT-PCR £ 235 17. Quantitative PCR 235 18. Real Time PCR 235 19. Relative Quantitative RT-PCR 236 20. Asymmetric PCR 236

Mutagenesis by PCR 236 Linear DNA Mutagenesis 236 Circular DNA Mutagenesis 238 A. Recombinant Circle PCR (RCPCR) 238 B. Recombination PCR (RPCR) : 238 C. Marker Coupled PCR 239 PCR in Viral Disease Diagnosis 242 Applications of PCR 244

2 Nucleic Acid Hybridization 247

Principles and Strategies 247 What is Nucleic Acid Hybridization? 247 Various Approaches and Kinetics of Nuecleic Acid

Hybridization 248 I. Solution Hybridization 248 II. Hybridizations on Solid Support 248

Various types of Solid support Hybridizations 248 1. Dot Blot or Slot Blot Hybridization 249

Rapid Detection Disease Diagnosis in Shrimp 250 1. Dot-Blot Hybridization Technique 250 2. Colony or Plaque Hybridization 251 3. Southern Blot Hybridization .' 252 4. Northern Blot Hybridization 254 5. Immunoblotting 254

References..... 256

3 Prophenoloxidase (Pro-PO) Based Shrimp Disease Diagnosis 257

Immune Mechanism of Shrimp 257 Prophenoloxidase (proPO) System •. 258 Pro-PO based Screening of Seaweeds as Immunostimulants 260

ProPO System using Immunostimulants for Immune Enhancement 260

References 261

PART VI: MARINE BIOACTIVE COMPOUNDS

1 Marine Bioactive Compounds 265

Biotechnological Improvements of Aquatic Products 265 Marine Natural Products , 266 Bioactive Molecules of Therapeutic Importance 271

Jellyfish Green Fluorescent Proteins 272 Improved GFP 273

Biomaterials and Nanotechnology . 274 Biofuel and Biofouling 275

Enzymes and Biochemicals 276 Biopolymer/Biomaterials 278 Seaweed 279 Novel Compounds from Seaweeds ..279

Pigments 280 Corals 281 Green-lipped Mussels 282 Sponges 282

Biotoxins 285 Insulin from Fish ; 286 Mineral Gold 286 Fish Toxins 286 Insecticides ! : 286 Heparin 287 Horse Shoe Crab 288

. Metabolites from Marine Bacteria 289 Marine Microalgae Metabolites 290 Molecular Probes and Cnidarians 292

Drugs from Bryozoans .293 Biochemical Probes from Molluscs 294 Bioactive Molecules of Ascidians 294

Biotechnology and Bioactive Compounds 295 The Major Goals of Marine Biotechnology 295 Genomic-based Approaches 296 Antagonistic Microbes in Aquaculture 297 Sustainable and Safe Aquaculture ! 298 .

Bioproducts Developed/Under Development from Marine Organisms 299

Drugs 300 Marine Biotechnologies with Commercial Potential 302

9. Multiplex PCR .. .: ' ....: : 233 10. Coupled Amplification and Sequencing (CAS). 234 11. Self Sustained Sequence Replication (SSSR) 234 12. Expand Long Template PCR 234 13. In-situ PCR 234 14. Competitive RT-PCR 234 15. Non-competitive RT-PCR 235 16. Kinetic RT-PCR ...... 235 17. Quantitative PCR 235 18. Real Time PCR 235 19. Relative Quantitative RT-PCR , 236 20. Asymmetric PCR 236

Mutagenesis by PCR 236 Linear DNA Mutagenesis 236 Circular DNA Mutagenesis 238 A. Recombinant Circle PCR (RCPCR) 238 B. Recombination PCR (RPCR) 238 C. Marker Coupled PCR.... , 239 PCR in Viral Disease Diagnosis 242 Applications of PCR 244

2 Nucleic Acid Hybridization 247

Principles and Strategies '. 247 What is Nucleic Acid Hybridization? ' 247 Various Approaches and Kinetics of Nuecleic Acid

Hybridization 248 I. Solution Hybridization..:. : 248 II. Hybridizations on Solid Support ....: 248

Various types of Solid support Hybridizations..... L248 1. Dot Blot or Slot Blot Hybridization 249

Rapid Detection Disease Diagnosis in Shrimp .250 1. Dot-Blot Hybridization Technique 250 2. Colony or Plaque Hybridization 251 3. Southern Blot Hybridization 252 4. Northern Blot Hybridization 254 5. Immunoblotting.... 254

References 256

3 Prophenoloxidase (Pro-PO) Based Shrimp Disease Diagnosis 257

Immune Mechanism of Shrimp 257 Prophenoloxidase (proPO) System 258 Pro-PO based Screening of Seaweeds as Immunostimulants 260

ProPO System using Immunostimulants for Immune Enhancement •. 260

References 261

PART VI: MARINE BIOACTIVE COMPOUNDS

1 Marine Bioactive Compounds, 265

Biotechnological Improvements of Aquatic Products . 265 Marine Natural Products ! 266 Bioactive Molecules of Therapeutic Importance 271

Jellyfish Green Fluorescent Proteins 272 Improved GFP 273

Biomaterials and Nanotechnology 274 Biofuel and Biofouling 275

Enzymes and Biochemicals 276 Biopolymer/Biomaterials .' 278 Seaweed 279 Novel Compounds from Seaweeds 279

Pigments 280 Corals 281

• Green-lipped Mussels 282 Sponges 282

Biotoxins * , 285 Insulin from Fish.. 286 Mineral Gold 286 Fish Toxins 286 Insecticides 286 Heparin ; 287 Horse Shoe Crab 288 Metabolites from Marine Bacteria 289 Marine Microalgae Metabolites 290 Molecular Probes and Cnidarians 292

Drugs from Bryozoans 293 Biochemical Probes from Molluscs 294 Bioactive Molecules of Ascidians...:. 294

Biotechnology and Bioactive Compounds 295 The Major Goals of Marine Biotechnology 295 Genomic-based Approaches 296

. .. Antagonistic Microbes in Aquaculture :.... 297 Sustainable and Safe Aquaculture 298

Bioproducts Developed/Under Development from Marine Organisms 299

Drugs .....300 Marine Biotechnologies with Commercial Potential 302

Marine Biotechnology- A Promising Sector of Future Scope 302 References . 303

PART VII: ADVANCED BIOTECHNOLOGICAL TECHNIQUES IN AQUACULTURE MANAGEMENT

1 Cell Culture Techniques and its Scope in Aquaculture Development 309

Historical Landmarks : 309 Reasons for Developing Cell Culture 310

Fish Cell Culture 310 Aquatic Invertebrate Cell Culture 311 Fish Cell Culture Techniques 311 Permanent Fish Cell Cultures as Novel Tools in Environmental

Toxicology ; 311 Fish Embryos for Cell Culture 312

Embryo Extract 312 Growth Medium 312 Cell Culture Quality Control : .J. 314 Steps for the Isolution of Viruses in Cell Culture 314 Identification of Betanodavirus Isolated in Cell Culture 316 Direct detection in fish tissues 317 Types of Cell Culture. 317 Basic Methods of Cell Culture:. •. 317 Procedure 318 Cell Culture Techniques 319 Short-Term Cultures 319 Long-Term Cultures 320 (i) Explant Cell Culture 321 (ii) Isolated Cell Culture.:. .....: '. 321 Characterization of Cells 322 Choice of Cell Culture System 323 Main Characteristics of Primary Cell Culture and Cell Lines 323

Physicochemical Characteristics 323 Nutrients and Metabolites 324

Amino Acids 324. Proteins.: 324 Routine Maintenance 325 Cell Culture from Crustaceans 325 Uses of Cell Culture 326 Recent Achievements 326 Advantages of Cell Culture 327 Limitations of Cell Culture ! 327

• Commonly used Fish Cell Cultures : 327

. Conclusion...'. ...328. References...; 328

2 Cryopreseryation and Biodiversity Conservation 331

Cryopreservation in Aquaculture Research 331 Cryopreservation of Spermatozoa of Oysters 332. Meeting'the' Challenge of Preserving Endangered Species '. 332

Cryopreservation of Marine Invertebrate Gametes : 333 State-of-the Art Research in Cryopreservation Technology ....335

Extenders 338 Cryoprotectant 338 Motility..... 339 Methodology ' •• 340 Cryopreservation of Gametes 340 Potential Application of Cryopreservationof Spermatozoa 341 Cryopreservation of Eggs and Embiyos 341

Biodiversity Conservation 343 Benefits of Cryopresen>ation in Aquaculture Species 345

Constraints to Management and Conservation of Aquatic Genetic Resources.... 347

Inadequate Knowledge Base.: : 347 Insufficient Attention 347 Magnitude of the Task : : 347

Human Interventions and Genetic Risks to Fish and Shellfish 348 Technical Problems in Fish Sperm Ciyopreservation 348 Future Directions 349 Recommendations 349

References.: : : .' 349

3 Transgenic Production of Fishes - Prospects and Problems 351

' Transgenic Fish Production 351 Growth Hormone Gene Cloning in Transgenics 352 Basic Studies on Gene Expression 352

Selection of Model Fish Species i 353 Preparation of the Gene Constructs : 354

Production of Transgenic Fish ! 355 Methodology of Gene Transfer 356 Microinjection 357 Electroporation..!.. 357

Fate of the Transgenes 358 ~' . Pattern of Transgene Integration 358

• . Examples of Transgenic Fish.. 359

. Other Transgenic Fish '. 359 Growth-enhanced Common Carp 360

Biotechnology and Transgenic Animals 364 Genes and the Genome : 364 Making a Transgenic Animal 365 Traits being Introduced into Animals : 365 Current and Potential Applications of Transgenic Fish 365 Objections to Transgenic Fish •. 369 Risks and Regulation of Transgenic Animals : 370 Main Findings and Outcome 370 Applications and Prospective 371

^References 372

4 DNA Fingerprinting in Fishes 373

DNA Typing Techniques 373 DNA Finger Printing in Fishes 374

Working Concept 375 DNA-Fingerprinting in Rainbow Trout (Oncorhynchus mykiss) 375

Wildlife Management 376

5 Genetically Mofidied Fish Production - Prospects and Problems 379

Genetically Engineered Fish 380 The First Genetically Engineered Fish /. ' 380 Marine Genetically Engineered Organisms (GEOs) 381 Marine GEOs Gain Ground 381 Advantages and Disadvantages of Marine GEOs !.... 381 Ecological and Evolutionary Issues Posed by Genetically

Modified Fishes 383 Commercial Race in GM Fish Production 385

Fish Species that have been Genetically Engineered 386 Global Concern on GMO Fish ! .:... 387 Ecological and Biodiversity Threats : ..... 387 Health Concerns 388 Transgenics verses Ecological Implications ; 388 Risk Assessment Study and Safety Aspects 389 Safety in Research and Evaluation f. ....389 Global Initiatives on Aquatic GMOs and Indigenous Efforts 390 Conclusion and Suggestions 391 Epilogue •.... 392

References '. 393

PART VIII: ENZYMES IN GENETIC ENGINEERING

1 Enzymes in Genetic Engineering '. 397

Storage of DNA and Handling of Enzymes 400 Enzyme Technology : . 400 Application of Enzyme Technology in Developing Countries ... 400 Restriction Endonucleases 401 Restriction Enzymes 401 Restriction-Modification Systems 402 Types of Restriction Enzymes 402 Restriction Enzyme Recognition Sequences 403 Restriction Recognitions Sites can be Unambiguous or

Ambiguous 403 Patterns of DNA Cutting by Restriction Enzymes 404 Methylation of DNA 405

Ligation 406 DNA Ligases 406 Activity of Ligases 407 Blunt ended Ligation 407 Sources of DNA Ligases 408 Applications of DNA Ligases 408 Polynucleotide Kinase 409 Forward Reaction 409 Exchange Reaction 409 Applications of Polynucleotide Kinase 409 Taq DNA Polymerase 409 Applications of Taq Polymerase 410 Ribonucleaase (RNase H) 410 Applications of RNase H 410 Gene Therapy 410 Gene Delivery Methods 410

References 412

PART IX: HYBRIDOMA TECHNOLOGY

1 Hybridoma Technology 415

History 416 Monoclonal Antibodies 417 Production of Monoclonal Antibodies and Vaccines 420 Monoclonal Antibody Production Strategy 422 Monoclonal Antibody in Salmon 427

Monoclonal Antibodies in Fish and Shellfish Health Management in India 428

Antibpdies in Health Management 429

Advantages of Monoclonal Antibodies 430 Disadvantages of Monoclonal Antibodies 431 Polyclonal Antiserum vs. Monoclonal Antibody 431

Uses of Monoclonal Antibodies : 432 i. Immuno Diagnostic Reagents 432 ii. Experimental uses of Monoclonal Antibodies 434 iii. Therapeutic Monoclonal Antibodies : 435

Application 436 References 436

PART X: FISH GENOMICS: TRENDS AND PROSPECTS

1 Fish Genomics: Trends and Prospects 441

I. Structural Genomics 441 II. Functional Genomics 442 Others 443

Proteomics 443 Marine Genomics 449

i. Preserve Genetic Diversity 449 ii. Protect Vulnerable Populations and Species 449 iii. Reduce the Impacts of Disease and Environmental Stress 450 iv. Improve Aquaculture Practices 450 v. Monitor Aquatic Environmental Health 450

Gene Mapping 450 a. Physical Maps 450 b. Genetic Maps 451

Genetic Distance 451 Potential Applications in Fisheries Sector 452

Applications 452 DNA Markers 453

Molecular Genetic Markers 453 Advantages of DNA Markers in Fish Biotechnology 457

References , 458