Medaka

Medaka

Biology, Management, and Experimental Protocols

Volume 2

Edited by

Chief Editors

Kenji MurataUniversity of California, Davis, CA, USA

Masato KinoshitaKyoto University, Sakyo-ku, Kyoto, Japan

Kiyoshi NaruseNational Institute for Basic Biology, Okazaki, Aichi, Japan

Editors

Minoru TanakaNagoya University, Nagoya, Aichi, Japan

Yasuhiro KameiNational Institute for Basic Biology, Okazaki, Aichi, Japan

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Library of Congress Cataloging-in-Publication DataNames: Murata, Kenji, 1961- editor. | Kinoshita, Masato, editor.Title: Medaka : biology, management, and experimental protocols / edited by

Chief editors, Kenji Murata, University of California Davis, CA, USA, Masato Kinoshita, Kyoto University, Sakyo-ku, Kyoto, Japan ; editors, Yasuhiro Kamei, National Institute for Basic Biology, Okazaki, Aichi, Japan, Minoru Tanaka, Nagoya Uiversity, Nagoya, Aichi, Japan, Kiyoshi Naruse, National Institute for Basic Biology, Okazaki, Aichi, Japan.

Description: Second edition. | Hoboken, NJ : Wiley-Blackwell, [2020] | Includes bibliographical references and index. |

Identifiers: LCCN 2019011589 (print) | LCCN 2019011759 (ebook) | ISBN 9781119575344 (Adobe PDF) | ISBN 9781119575306 (ePub) | ISBN 9781119575290 (hardcover)

Subjects: LCSH: Oryzias latipes.Classification: LCC QL638.O78 (ebook) | LCC QL638.O78 M43 2020 (print) | DDC

597.53–dc23LC record available at https://lccn.loc.gov/2019011589

Cover Design: WileyCover Images: courtesy of Kiyoshi Naruse. Medaka with rainbow color image courtesy of Lazaro Centanin and Jochen Wittbrodt

Set in 10/12pt Times LT Std by SPi Global, Chennai, India

10 9 8 7 6 5 4 3 2 1

v

Contents

List of Contributors xvPreface xxi

1 Medaka Management 11.1 Introduction 11.2 Medaka Management for Scientific Research 1

1.2.1 Outline of medaka life‐cycle in the wild 21.2.2 Preparation of normal rearing conditions of medaka in 

the laboratory and procedures for breeding 21.2.2.1 Breeding system set‐up 21.2.2.2 Obtaining medaka 31.2.2.3 Collecting eggs in a laboratory setting 31.2.2.4 Daily care and maintenance of eggs 41.2.2.5 Rearing medaka from the larval stage to adulthood 41.2.2.6 Anesthesia and euthanasia 4

1.3 Standardized Culture and Growth Curve 71.3.1 Characteristics and selection of strains 71.3.2 Management of medaka eggs and fish 8

1.3.2.1 Mating 81.3.2.2 Management of embryos 81.3.2.3 Management of embryos before hatching 131.3.2.4 Rearing from the larval stage to adulthood (to induce

earlier maturation) 141.3.3 Maintenance of breeding tanks during breeding 23

1.3.3.1 Judgment of water quality 231.3.3.2 Maintenance of breeding water 24

1.3.4 Anesthesia 251.3.4.1 Behavior under each anesthesia stage 261.3.4.2 Difference in sensitivity to anesthesia among strains 261.3.4.3 Growth stage specificity in sensitivity to MS‐222 271.3.4.4 Eugenol is recommended as an anesthetic reagent 281.3.4.5 Euthanasia 281.3.4.6 Important reminders for euthanasia 29

2 Medaka and Oryzias Species as Model Organisms and the Current Status of Medaka Biological Resources 312.1 Introduction 312.2 Common and Unique Futures of Medaka and Related Species as Model

Organisms 312.3 Phylogenetic Relationships of Medaka and Related Species 35

2.3.1 The javanicus species group 352.3.2 The latipes species group 402.3.3 The celebensis species group 42

vi Contents

2.4 BAC Resources of Species Related to Medaka 432.5 National Bio‐Resource Project Medaka (NBRP Medaka) 43

2.5.1 Support for visiting researchers 45

3 Looking at Adult Medaka 493.1 General Morphology 49

3.1.1 Secondary sexual characters 493.1.1.1 Dorsal fin 493.1.1.2 Anal fin 493.1.1.3 Papillar processes 503.1.1.4 Urogenital papillae 50

3.1.2 Body color 513.1.2.1 Pigment cells (chromatophores) 513.1.2.2 Structures of the chromatophores 513.1.2.3 Chromatophores in medaka 513.1.2.4 Chromatophore distribution in medaka 553.1.2.5 See‐through medaka 56

3.2 Anatomy and Histology 563.2.1 Observations of internal organs 56

3.2.1.1 Observations of internal organs in the live see‐through medaka 56

3.2.1.2 Dissection of adult medaka 583.2.2 Horizontal and sagittal sections of juvenile medaka 583.2.3 Nervous system 58

3.2.3.1 Adult central nervous system 583.2.3.2 Adult peripheral nervous system 67

3.2.4 Endocrine system 743.2.4.1 Hypothalamo‐pituitary system 763.2.4.2 Pineal organ (epiphysis) 783.2.4.3 Thyroid gland 793.2.4.4 Heart 813.2.4.5 Interrenal gland and chromaffin cells 813.2.4.6 Gonads 813.2.4.7 Endocrine pancreas (islets of Langerhans) 813.2.4.8 Gastrointestinal tract 813.2.4.9 Ultimobranchial gland 823.2.4.10 Corpuscle of Stannius 823.2.4.11 Urophysis 833.2.4.12 Thymus 83

3.2.5 Gonads 833.2.5.1 Ovary 833.2.5.2 Testis 85

3.2.6 Kidney 853.2.6.1 Pronephros 863.2.6.2 Mesonephros 863.2.6.3 Histology of the kidney 86

Column 3.1 How to make sections of a mature ovary for  histological analysis 88

Contents vii

4 Looking at Medaka Embryos 974.1 Development of Various Tissues and Organs 97

4.1.1 Developmental stages 974.1.1.1 Stage 0: unfertilized egg (Figure 4-1) 974.1.1.2 Stage 1: activated egg stage (3 minutes) (Figure 4-1) 994.1.1.3 Stage 2: blastodisc stage (Figure 4-1) 994.1.1.4 Stage 3: two‐cell stage (1 hour 5 minutes) (Figure 4-1) 994.1.1.5 Stage 4: four‐cell stage (1 hour 45 minutes) (Figure 4-1) 1004.1.1.6 Stage 5: eight‐cell stage (2 hours 20 minutes)

(Figure 4-1) 1004.1.1.7 Stage 6: 16‐cell stage (2 hours 55 minutes) (Figure 4-2) 1004.1.1.8 Stage 7: 32‐cell stage (3 hours 30 minutes) (Figure 4-2) 1004.1.1.9 Stage 8: early morula stage (4 hours 5 minutes)

(Figure 4-2) 1004.1.1.10 Stage 9: late morula stage (5 hours 15 minutes)

(Figure 4-2) 1004.1.1.11 Stage 10: early blastula stage (6 hours 30 minutes)

(Figure 4-2) 1004.1.1.12 Stage 11: late blastula stage (8 hours 15 minutes)

(Figure 4-2) 1024.1.1.13 Stage 12: pre‐early gastrula stage (10 hours

20 minutes) (Figure 4-3) 1024.1.1.14 Stage 13: early gastrula stage (13 hours) (Figure 4-3) 1024.1.1.15 Stage 14: pre‐mid‐gastrula stage (15 hours) (Figure 4-3) 1024.1.1.16 Stage 15: mid‐gastrula stage (17 hours 30 min-

utes) (Figure 4-3) 1024.1.1.17 Stage 16: late gastrula stage (21 hours) (Figure 4-3) 1024.1.1.18 Stage 17: early neurula stage (1 day 1 hour) (Figure 4-3) 1034.1.1.19 Stage 18: late neurula stage (1 day 2 hours)  (Figure 4-4) 1044.1.1.20 Stage 19: two‐somite stage (1 day 3 hours

30 minutes) (Figure 4-4) 1044.1.1.21 Stage 20: four‐somite stage (1 day 7 hours

30 minutes) (Figure 4-4) 1044.1.1.22 Stage 21: six‐somite stage (1 day 10 hours) (Figure 4-4) 1044.1.1.23 Stage 22: nine‐somite stage (1 day 14 hours) (Figure 4-4) 1044.1.1.24 Stage 23: 12‐somite stage (1 day 17 hours) (Figure 4-4) 1044.1.1.25 Stage 24: 16‐somite stage (1 day 20 hours) (Figure 4-5) 1064.1.1.26 Stage 25: 18–19‐somite stage (2 days 2 hours)

(Figure 4-5) 1074.1.1.27 Stage 26: 22‐somite stage (2 days 6 hours) (Figure 4-5) 1074.1.1.28 Stage 27: 24‐somite stage (2 days 10 hours) (Figure 4-5) 1074.1.1.29 Stage 28: 30‐somite stage (2 days 16 hours) (Figure 4-5) 1074.1.1.30 Stage 29: 34‐somite stage (3 days 2 hours) (Figure 4-5) 1084.1.1.31 Stage 30: 35‐somite stage (3 days 10 hours) 

(Figure 4-6) 1084.1.1.32 Stage 31: gill blood vessel formation stage (3 days

23 hours) (Figure 4-6) 108

viii Contents

4.1.1.33 Stage 32: somite completion stage (4 days 5 hours) (Figure 4-6) 108

4.1.1.34 Stage 33: stage at which notochord vacuolization is completed (4 days 10 hours) (Figure 4-6) 108

4.1.1.35 Stage 34: pectoral fin blood circulation stage (5 days 1 hour) (Figure 4-6) 110

4.1.1.36 Stage 35: stage at which visceral blood vessels form (5 days 12 hours) (Figure 4-6) 110

4.1.1.37 Stage 36: heart development stage (6 days) (Figure 4-7) 1104.1.1.38 Stage 37: pericardial cavity formation stage

(7 days) (Figure 4-7) 1104.1.1.39 Stage 38: spleen development stage (8 days) (Figure 4-7) 1104.1.1.40 Stage 39: hatching stage (9 days) (Figure 4-7) 1104.1.1.41 Stage 40: first larval stage (Figure 4-8) 1124.1.1.42 Stage 41: second larval stage (Figure 4-8) 1134.1.1.43 Stage 42: third larval stage (Figure 4-8) 1134.1.1.44 Stage 43: first juvenile stage (Figure 4-8) 1134.1.1.45 Stage 44: second juvenile stage (Figure 4-8) 1134.1.1.46 Stage 45 (Figure 4-8) 113

4.1.2 Brain 1134.1.2.1 Gastrula step (stages 13–17) 1144.1.2.2 Neurula step (stages 17–18) 1144.1.2.3 Neural rod step (stages 19–22) 1164.1.2.4 Neural tube step (stages 23–27) 1164.1.2.5 Late embryonic brain step (stages 28–34) 1174.1.2.6 Larval brain step (stages 35–42) 119

4.1.3 Hatching gland 1194.1.3.1 Origin of fish hatching gland cells 1194.1.3.2 Secretion of hatching enzymes from hatching gland cells 122

4.1.4 Eye development 1244.1.4.1 Specification of the anterior neural plate 1244.1.4.2 Eye field determination and establishment of retinal

identity 1254.1.4.3 Splitting of the retinal anlage into two retinal primordia 1254.1.4.4 Morphogenesis I: evagination of the optic vesicle 1254.1.4.5 Morphogenesis II: formation of the optic cup 1274.1.4.6 Retinal differentiation I: central retina 1274.1.4.7 Retinal differentiation II: Ciliary Marginal Zone 1274.1.4.8 Retinotectal projection 128

4.1.5 Branchial arch and jaws 1284.1.5.1 Skeletal development 1284.1.5.2 Muscle development 130

4.1.6 Vasculature 1314.1.6.1 Vascular anatomy of the developing medaka 1314.1.6.2 Origin of the medaka endothelial lineage 1424.1.6.3 Abbreviations 1424.1.6.4 Acknowledgment 143

Contents ix

4.1.7 Blood cells (hematopoiesis) 1434.1.7.1 Overview 1434.1.7.2 Observation of embryonic and adult blood cells 144

4.1.8 Heart 1464.1.8.1 Overview 1464.1.8.2 Heart architecture 1464.1.8.3 Heart morphogenesis 1474.1.8.4 Observation of the developing heart 156

4.1.9 Kidney 1594.1.9.1 Overview 1594.1.9.2 Nephrogenesis 1594.1.9.3 Pronephros 1604.1.9.4 Mesonephros 160

4.1.10 Thymus 1604.1.10.1 Overview 1604.1.10.2 Early development of the thymus 1604.1.10.3 Cortex and medulla 1614.1.10.4 Involution of the thymus 162

4.1.11 Gut and liver 1624.1.12 Bones 164

4.1.12.1 Vertebral column 164Column 4.1 Key words in bone formation 172

4.1.13 Fins 1734.1.13.1 Overview 1734.1.13.2 Fin anatomy 1734.1.13.3 Embryonic fin development (from fertilization

to stage 39 [hatching stage]) 1744.1.13.4 Fin development after hatching (after stage 39) 1754.1.13.5 Gene expression during fin development 175

4.1.14 Gonads 1764.1.14.1 Overview 1764.1.14.2 PGC specification 1774.1.14.3 Formation of gonadal primordium (Figure 4-60b) 1774.1.14.4 Sexual dimorphism in germ cell proliferation

(Figure 4-61) 1794.1.14.5 Posthatching period in XX gonads 1804.1.14.6 Posthatching period in XY gonads 180

4.2 Medaka EGG Envelope and Hatching Enzyme 1814.2.1 Overview 1814.2.2 Preparation of a hatching enzyme solution from

hatching liquid 1824.2.2.1 Procedure 182

4.2.3 Simple method for preparing hatching enzyme solution 1834.2.3.1 Procedure 183

4.2.4 Solubilization of the egg envelope using hatching enzyme 183Column 4.2 Easy method for preparation of a small amount

of hatching enzyme solution (see website for figure) 184

x Contents

4.3 Observation of Embryos (Embedding Embryos) 1854.3.1 Anesthesia of embryos using MS‐222 185

4.3.1.1 Equipment and reagents 1854.3.2 Observation of embryos (mounting) 185

4.3.2.1 Living embryos 1854.3.2.2 Processed embryos 188

4.4 Whole‐Mount In Situ Hybridization (see section 4.1.8.4 for a similar protocol) 1894.4.1 Fixation and storage 189

4.4.1.1 Procedure 1 1894.4.2 Rehydration, proteinase K treatment, and postfixation at RT 190

4.4.2.1 Procedure 2 1904.4.3 Hybridization and washing 190

4.4.3.1 Procedure 3 1904.4.4 Immunoreaction and washing antibodies 191

4.4.4.1 Procedure 4 1914.4.5 Staining 191

4.4.5.1 Procedure 5 1914.5 Embedding in a Plastic Resin (Technovit 7100) 192

4.5.1 Equipment and reagents 1924.5.2 Agarose mounting (Figure 4-68) 192

4.5.2.1 Procedure 1 1924.5.3 Dehydration and infiltration (Figure 4-68) 192

4.5.3.1 Procedure 2 1924.5.4 Polymerization (Figure 4-68) 193

4.5.4.1 Procedure 3 193Column 4.3 Pigment Cells (Figure 4-69) 194Column 4.4 Kupffer’s Vesicle 195

5 Reproductive Behavior of Wild Japanese Medaka 2055.1 Wild Japanese Medaka 2055.2 Reproductive Behavior of Wild Medaka 206

5.2.1 Aggressive behavior 2075.2.2 Spawning behavior 2075.2.3 Egg deposition behavior 2105.2.4 Egg discarding behavior 2105.2.5 School and aggregation 211

5.3 Conclusion 211

6 Cryopreservation and Transplantation of Medaka Germ Cells 2156.1 Introduction 2156.2 Cryopreservation of Medaka Testes 215

6.2.1 Solutions 2166.2.2 Materials 2176.2.3 Procedures 217

6.3 Transplantation of Thawed Testicular Cells into Recipient Larvae 218

Contents xi

6.3.1 Solutions 2186.3.2 Materials 2196.3.3 Procedures 219

Column 6.1 Production of triploid medaka 222

7 Genome Editing 2257.1 Introduction 2257.2 Outline of Targeted Genome Editing Using Nucleases 2257.3 Preparation of CRISPR/Cas9 Genome Editing Tools 226

7.3.1 Materials 2277.3.2 Production of custom‐designed sgRNA 228

7.3.2.1 Preparation of the bsai‐digested sgRNA backbone 2287.3.2.2 Design and production of customized sgRNA 228

7.3.3 Production of capped RNA encoding a Cas9 nuclease 2327.4 Preparation of Custom‐Designed TALENs 234

7.4.1 Materials 2347.4.2 Preparation of the TALEN assembly system 236

7.4.2.1 Preparation of TAL modules (HD1‐6, NG1‐6, NI1‐6, and NN1‐6) 236

7.4.2.2 Preparation of array backbone plasmids (pFUS vectors) 236

7.4.2.3 Preparation of last repeat modules (LR‐HD, NG, NI, and NN) 237

7.4.2.4 Preparation of TALEN backbone plasmids (pCS2TAL3DD and RR vectors) 238

7.4.3 Design and construction of custom‐designed TALENs 2397.4.3.1 Design of TALEN using TALE‐NT 2397.4.3.2 First assembly: construction of 6‐modules array vectors 2407.4.3.3 Second assembly: construction of TALEN

expression vectors 2427.5 Heteroduplex Mobility Assay – A Simple Method to Detect

Targeted Genome Modification 2447.5.1 Materials 2467.5.2 Procedure 246

7.5.2.1 Identification of the wild type, heterozygotes, and homozygotes 246

7.5.2.2 Evaluation of the efficiency of targeted genome modifications 246

7.6 How to Establish Gene Knock‐out Strains 2477.6.1 Design and synthesis of genome‐editing tools 2477.6.2 Evaluation of genome‐editing activity with fertilized

medaka eggs 2477.6.3 Microinjection of the selected genome‐editing tool(s) 2487.6.4 Selection of founder fish by genotyping F1 embryos 2497.6.5 Selection of F1 fish carrying the same mutation and the

establishment of mutant strain 2497.6.6 Selection of homozygous mutant fish in the F2 family 251

xii Contents

7.7 How to Establish Gene Knock‐in Strains 2527.7.1 Design and synthesis of CRISPR/Cas9 components 2537.7.2 Evaluation of genome‐editing activity with fertilized medaka eggs 2537.7.3 Construction of donor plasmid with homology arms (Ca. 0.5 kbp)

and bait sequences 2537.7.4 Microinjection for establishing knock‐in strains 2547.7.5 Selecting G0 founders harboring the insert gene in the

genomic target site 254Column 7.1 Utilization of crRNA, tracrRNA, Cas9 Protein 2557.A Simple Genomic DNA Preparation by an Alkaline Lysis Method 2567.A.1 Materials 2567.A.2 Procedure 256

8 Photo‐Inducible Gene Expression in Medaka 2618.1 Outline of IR‐LEGO 2618.2 Practical Strategies of IR‐LEGO in Medaka Study 262

8.2.1 Selection of heat shock promoters and application studies 2628.3 Laser Irradiation Conditions and Sample Preparation 2658.4 Caution in Maintaining Strains 2678.5 Other Uses of IR‐LEGO 2678.6 Summary and Future Prospects 268

9 Screening and Testing Methods of Endocrine‐Disrupting Chemicals Using Medaka 2719.1 Applied Toxicity Tests for Endocrine Disruptors 2719.2 Detection of Androgenic and Antiandrogenic Chemicals Using Medaka 275

9.2.1 The formation of papillary processes on anal fin rays as an  indicative phenotype for exposure of androgenic and/or antiandrogenic chemicals 275

9.2.2 Candidate biomarkers for assessing the action of androgenic and  antiandrogenic chemicals 276

9.2.3 Visualization of androgenic and antiandrogenic activity as green fluorescence with spiggin‐GFP medaka 276

10 Application of the Seawater Medaka Oryzias melastigma (McClelland) for Marine Ecotoxicology 281

10.1 Background and Development of Oryzias melastigma for Marine Ecotoxicology 281

10.2 Marine Medaka Developmental Staging 28310.3 Standard Breeding and Rearing Conditions 284

10.3.1 Seawater 28510.3.2 Temperature 28510.3.3 Photoperiod 28510.3.4 Feeding 28610.3.5 Embryo collection and rearing 28610.3.6 Hatching and larvae collection 287

Contents xiii

10.3.7 Larvae rearing 28810.3.8 Larvae feeding 288

10.4 Raising Marine Medaka for Experimental Use 28910.4.1 Experiments using adult fish 28910.4.2 Experiments using larvae 289

10.5 Troubleshooting 28910.5.1 Mass mortality 28910.5.2 Low egg production 28910.5.3 Extensive algal growth 290

10.6 How to Obtain Marine Medaka O. melastigma 29010.7 Experimental Protocols Using Marine Medaka 29010.8 Immunotoxicity Assessment: Bacteria Challenge Assays 290

10.8.1 SOP for adult bacterial challenge assay 29110.8.2 SOP for larval bacterial challenge assay 29210.8.3 Age selection for larval bacterial challenge 293

10.9 Fish Dissection and the Whole Adult Histoarray 29410.9.1 SOP for fish dissection 29510.9.2 SOP for adult medaka histoarray 295

10.10 Embryo Chip 29710.10.1 SOP for embryo and larvae histoarray 297

10.A Materials for SOP for Adult Medaka Histoarray (see section 10.9.2) 299

11 Telomerase and Telomere Biology in Medaka 30311.1 Introduction 30311.2 SOP for Quantification of Telomerase Activity Using

the Real‐Time Quantitative Telomeric Repeat Amplification  Protocol (RTQ‐TRAP) 30811.2.1 Procedures for sample extraction 30811.2.2 Procedures for determination of protein

concentration 30811.2.3 Procedures for RTQ‐TRAP linearity test 30811.2.4 Calculation of telomerase activity 309

11.3 SOP for Quantification of Telomere Length Using Southern Blotting Analysis 30911.3.1 Procedures for genomic DNA extraction and digestion

with restriction enzymes 30911.3.2 Procedures for probe preparation 31111.3.3 Procedures for electrophoresis and southern blotting 31111.3.4 Procedures for hybridization and detection 31211.3.5 Procedures for computerized telomere analysis 312

11.4 SOP for Quantification of Telomere Length Using Fluorescence In Situ Hybridization 31311.4.1 Procedures for fluorescence in situ hybridization 31311.4.2 Procedures for confocal microscopy detection 31311.4.3 Procedures for ImageJ analysis 314

xiv Contents

12 Assessments of Medaka Skeletal Toxicity 31712.1 Introduction 31712.2 Methods 318

12.2.1 Embryonic exposures: dioxin 31912.2.2 Embryonic exposure: dithiocarbamates 31912.2.3 Whole‐mount alcian blue staining of hatchlings/larvae a 32012.2.4 Whole‐mount Alizarin red S staining of hatchlings/larvaec 32012.2.5 In vivo Alizarin complexone fluorescent staining

for  mineralized bone matrix 32112.2.6 In vivo calcein fluorescent staining for mineralized bone matrix 32112.2.7 Confocal imaging of embryo/hatchling medaka 32112.2.8 Morphological assessments 323

12.3 Results and Discussion 32412.3.1 Dithiocarbamates 32412.3.2 Dioxin 325

Appendix A Solutions 329Attributions 331Index 335

xv

List of Contributors

Satoshi AnsaiLaboratory of Bioresources,National Institute for Basic Biology,Japan

Doris W.T. AuState Key Laboratory of Marine Pollution, Department of Chemistry,City University of Hong Kong,Hong Kong

Napo K.M. CheungState Key Laboratory of Marine Pollution, Department of Chemistry,City University of Hong Kong,Hong Kong

Michael W.L. ChiangState Key Laboratory of Marine Pollution, Department of Chemistry, City University of Hong Kong,Hong Kong

Shin-ich ChisadaDepartment of Preventive Medicine and Public Health,Kyorin University, School of Medicine,Japan

Tomonori DeguchiBiomedical Research Institute, National Institute of Advanced Industrial Science and Technology,Japan

Misato FujitaDepartment of Biological Sciences,Faculty of Science, Kanagawa University,Japan

Shoji FukamachiLaboratory of Evolutionary Genetics,Department of Chemical and Biological Sciences, Japan Women’s University,Japan

Hisashi HashimotoBioscience and Biotechnology Center, Nagoya University,Japan

Narisato HiraiNational Research Institute of Fisheries and Environment of Inland Sea, Japan Fisheries Research and Education Agency,Japan

Taisen IguchiNanobioscience, Yokohama City University,Japan

Masayuki IigoDepartment of Applied Biological Chemistry, Faculty of Agriculture,Utsunomiya University,Japan

Keiji InohayaSchool of Life Science and Technology, Tokyo Institute of Technology,Japan

xvi List of Contributors

Yuji IshikawaNeurobiology Lab,Japan

Yasuko IsoeGraduate School of Natural Science and Technology,Okayama University,Japan

Ichiro IuchiDepartment of Materials and Life Sciences, Faculty of Science and Technology,Sophia University,Japan

Norimasa IwanamiDepartment of Regenerative Medicine, National Center for Geriatrics and Gerontology,Japan

Eri IwataLaboratory of Veterinary Ethology,Faculty of Veterinary Medicine, Okayama University of Science,Japan

Yasuhiro KameiSpectrography and Bioimaging Facility, NIBB Core Facilities,National Institute for Basic Biology,Japan

Sakurako KamideLaboratory of Aquatic Biology,Department of Natural Sciences,International Christian University,Japan

Takashi KawasakiMedical and Biological Engineering Research Group,Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST),Japan

Fumi KezukaDepartment of Aquatic Biosciences,Tokyo University of Marine Science and Technology,Japan

Masato KinoshitaDepartment of Applied Biosciences, Graduate School of Agriculture,Kyoto University,Japan

Shin-ichi KitamuraCentre for Marine Environmental Studies,Ehime University,Japan

Daisuke KobayashiDepartment of Anatomy and Developmental Biology,Kyoto Prefecture University of Medicine,Japan

Makito KobayashiLaboratory of Aquatic Biology,Department of Natural Sciences,International Christian University,Japan

Akira KudoInternational Frontier, Tokyo Institute of Technology,Japan

Seth W. KullmanDepartment of Biological Sciences,North Carolina State University,USA

Rie KusakabeLaboratory for Evolutionary Morphology,RIKEN Center for Biosystems Dynamics Research,Japan

List of Contributors xvii

Sungki LeeBiological and Genetic Resources Assessment Division,National Institute of Biological Resources,South Korea

Kouichi MaruyamaNational Institute of Radiological Sciences, National Institutes for Quantum and Radiological Science and Technology,Japan

Shinichi MiyagawaDepartment of Biological Science and Technology,Faculty of Industrial Science and Technology, Tokyo University of Science,Japan

Helen O.L. MokState Key Laboratory of Marine Pollution, Department of Biomedical Science,City University of Hong Kong,Hong Kong

Yu MurakamiDepartment of Applied Biosciences, Graduate School of Agriculture,Kyoto University,Japan

Kenji MurataCenter for Health and the Environment,University of California Davis,USA

Yuki NakataniDepartment of Biological Sciences,Tokyo Institute of Technology,Japan

Kiyoshi NaruseLaboratory of Bioresources,National Institute for Basic Biology,Japan

Yukiko OginoCentre for Promotion of International Education and Research, Faculty of Agriculture,Kyushu University,Japan

Kataaki OkuboDepartment of Aquatic Bioscience,Graduate School of Agricultural and Life Sciences, University of Tokyo,Japan

Drew R. RetersonState Key Laboratory of Marine Pollution,Department of Chemistry,City University of Hong Kong,Hong Kong

Daisuke SaitoResearch Center for Systems Immunology,Kyushu University,Japan

Anthony SèbillotDépartement Adaptations du vivant,"Évolution des régulations endocrini-ennes", Muséum National d’Histoire Naturelle,France

Shinsuke SekiBioscience Education and Research Support Center,Akita University,Japan

Eriko ShimadaLaboratory of Cell Biology,Cellevolt,Japan

Ai ShinomiyaDivision of Seasonal Biology,National Institute for Basic Biology,Japan

xviii List of Contributors

Ian T. StancilDepartment of Biological Sciences,North Carolina State University,USA

Yoshiro TakanoDepartment of Cell Biology and Neuroscience,Juntendo University School of Medicine,Japan

Yusuke TakehanaDepartment of Animal Bioscience,Nagahama Institute of Bio‐Science and Technology,Japan

Minoru TanakaDivision of Biological Science,Nagoya University,Japan

Yoshihito TaniguchiDepartment of Preventive Medicine and Public Health,School of Medicine, Kyorin University,Japan

Norihisa TatarazakoGraduate School of Agriculture, Department of Science and Technology for Biological Resources and Environment,Ehime University,Japan

Yuko WakamatsuPhysiological Chemistry I, Biocenter,University of Würzburg,Germany

AtLee T.D. WatsonDepartment of Biological Sciences,North Carolina State University,USA

Joachim WittbrodtCOS Heidelberg, Heidelberg University,Germany

Rudolf S.S. WuDepartment of Science and Environmental Studies,Education University of Hong Kong,Hong Kong

Kazunori YamahiraTropical Biosphere Research Center,University of the Ryukyus,Japan

Shigeki YasumasuDepartment of Materials and Life Sciences, Faculity of Science and Technology,Sophia University,Japan

Ryohei YatsuDepartment of Integrative Biology,University of Texas,USA

Roy R. YeState Key Laboratory of Marine Pollution, Department of Chemistry,City University of Hong Kong,Hong Kong

Bill W.P. YipState Key Laboratory of Marine Pollution, Department of Chemistry,City University of Hong Kong,Hong Kong

Hiroki YodaDevelopmental Biology Unit,European Molecular Biology Laboratory,Germany

List of Contributors xix

Hirofumi YokotaDepartment of Biosphere Sciences,School of Human Sciences, Kobe College,Japan

Goro YoshizakiDepartment of Aquatic Biosciences,Tokyo University of Marine Science and Technology,Japan

xxi

Preface

Ten years after publishing our first book (Volume 1) in 2009, this second book (Volume 2) of Medaka: Biology, Management, and Experimental Protocols presents significant progress in technological innovation and development in the fields of biological and med-ical science. The purpose of Volume 2 is to familiarize scientists worldwide with the advan-tages of using medaka in experimental designs, to facilitate research using medaka, and address the value to science of adopting medaka as a model animal.

In Volume 2, the authors provide additional information and current protocols that have been recently developed, or modified, to successfully raise medaka under stable laboratory culture conditions and how to use medaka as a model animal. This Volume 2 describes new technologies developed after 2009, using the fish as a molecular tool in the fields of life science, evolution, ecology, and toxicology. It provides an informational bridge that spans the varied research disciplines and abilities ranging from undergraduate education through senior researchers.

Contributing authors were chosen because of their expertise and demonstrated ability to conduct experiments involving medaka, and because they are recognized pioneers in the use of medaka as a model animal in their scientific fields. The authors were also asked to describe their experimental protocols in detail, and explain their rationale for the chosen protocols in achieving their conceptual goals. The editors also recommend that users read the procedures described in the first edition that describe the maintenance of medaka, and use that information to create or modify the current fish maintenance systems.

Chapter 1: Dr. Chisada and colleagues describe contemporary procedures used to main-tain medaka in culture facilities.

Chapter  2: This chapter covers the current phylogenetic relationship of medaka and other Oryzias species, and the geographical distributions of each species. In this chapter, Dr. Naruse and colleagues also describe and update the present status of medaka resources available through the National Bio‐Resource Project Medaka (NBRP Medaka) that has been supported by the Japanese government since 2002.

Chapters 3 and 4 introduce the reader to the medaka fish: Chapter  3 covers general information about adult medaka, including secondary sexual characters, body color, and internal organs. Section  3.2, “Anatomy and Histology,” provides details of the nervous system, endocrine system, kidneys, and gonads. Chapter 4 covers the characteristics of the developing embryos. It includes brief outlines of the development of the fish’s organs and tissues, with an emphasis on histology rather than developmental mechanisms. Chapter 4 also discusses the basic procedures for preparing and mounting embryos, and performing in situ hybridization. These similar chapters appear as Chapter 5 and 6 in Volume1; how-ever, the authors retained the material in Chapter 3 and 4 of volume2, because of the impor-tance of the information.

To address medaka reproduction, two groups of researchers were chosen to describe procedures and applications for the preservation of genetic resources of the fish, and for the fish’s reproductive ecology.

xxii Preface

Chapter 5: Dr. Kobayashi and colleagues, experts on wild medaka fish, address sexual behavior and reproduction of wild populations of medaka. The chapter’s focus is on wild populations as opposed to laboratory‐maintained fish.

Chapter 6: Dr. Kezuka and colleagues introduce methods of cryopreservation of whole fish testes, the preparation of testicular cell suspension, and testicular cell transplantation into recipient fish. This method will potentially contribute not only to saving and recov-ering populations of endangered species, but also to increasing the numbers of commer-cially important, high‐quality food fishes.

Since publication of the first Volume, new technologies using medaka have been devel-oped and these advances contribute to the identification and function of genes and their products in the body. Two useful technologies are addressed as follows:

Chapter 7: In this chapter, Dr. Ansai and colleagues describe recent developments and progress in the amazing application of molecular biology used in gene editing using medaka. They address the basic theory and applications used to create and apply the estab-lishment of transgenic, gene knock‐out medaka, and conditional knock‐out medaka.

Chapter 8: In this chapter, Drs. Isoe and Kamei describe newly developed technology using medaka and the infrared laser‐evoked gene operator (IR‐LEGO) system. They explain how to use the system not only for research using medaka, but also with cultured cells and plants. The authors also address how to analyze the function of genes in vivo, in developmental biology and related fields, and the development of a new microscopic tech-nology used to control the expression of genes using the combination of the heat shock promoter system and an infrared laser, by focusing on the targeted cells.

Chapters 9–12 introduce the use of medaka in the fields of toxicology and medicine; and address recently developed methodologies and new technologies.

Chapter  9: Dr. Iguchi and colleagues describe several test guidelines used by the Organization of Economic Cooperation and Development (OECD) employing medaka in standardized testing methods to screen and/or access potential endocrine‐disrupting chemi-cals, and how the fish is used to identify adverse effects of toxic chemicals. They also describe how toxicologists are using medaka to assess hazardous chemicals in both drinking water and sewage effluent, and to identify endocrine disruptors in natural waters. The authors address endocrine disruptors, not only restricted to chemicals with estrogenic activities but also those with androgenic activities. Dr. Ogino and colleagues also describe the use of medaka to detect androgenic and antiandrogenic chemicals.

Chapter 10: This chapter introduces the basic biology of the marine medaka, Oryzias melastigma (McClelland), and how its use has been applied to research in marine ecotoxicology. Dr. Peterson and colleagues describe the fish’s biological characterization, how to maintain the species in the laboratory, and how to use it in research on marine ecotoxicology.

Chapter 11: Dr. Au and colleagues, experts in telomere biology using medaka, introduce their SOPs and procedures, which have great potential for research in the fields of toxi-cology, senescence, and aging. They address medaka as a unique, alternative vertebrate model for studying telomere and telomerase function in a cross‐disciplinary range from environmental toxicology to biomedical research on aging, as well as for cancer and tissue regeneration research.

Chapter 12: Dr. Watson and colleagues describe the application of medaka in research on human skeletal biology and toxicity and as a human disease model.

Preface xxiii

The format of Volume 2 is designed to capture the thoughts and methods of researchers that use medaka as a model animal and to make this expertise accessible to students, beginning researchers, and senior researchers who might become intrigued with using medaka as the model animal in their own work. To accomplish this, and following a reading of Volume 1, the reader is given step‐by step specifics for each protocol that allows appli-cation of the fish in their own work. The information includes specific information to facil-itate ease of adoption, including details such as reagents used, instrumentation, and other essential requirements. It is anticipated that this highly practical format will encourage the reader to use medaka as a model animal and permit the reader to bring new concepts into personal practice in a more efficient manner.

The use of medaka fish as a model animal requires experimental insight and practical troubleshooting of experimental designs. Of equal importance is an overall appreciation of both the power and limits of using medaka as a model animal. To assist in visualizing and understanding medaka and the research protocols used, the editors strongly suggest that readers refer to Volume 1 of Medaka: Biology, Management, and Experimental Protocols (2009) (ebook: http://onlinelibrary.wiley.com/book/10.1002/9780813818849) as a refer-ence. All figures and videos in both Volumes are shown at https://medaka-book.org/.

As a final note, the preparation of this book would not have been possible without the dedication of the excellent array of contributing authors. We also thank the staff of Wiley‐Blackwell Publishers, specifically Justin Jeffryes, Shelby Hayes, Rebecca Ralf, Antony Sami, Holly Regan-Jones and Vivek Jagadeesan as they have demonstrated great patience with our efforts and provided excellent guidance and assistance. Finally, we also express our thanks to Ms. Robin Lee Kingsley (Deceased) and Dr. Fred S. Conte (University of California Davis) for their assistance in editing each chapter.

Kenji Murata, Masato Kinoshita (Chief Editors)Yasuhiro Kamei, Minoru Tanaka, Kiyoshi Naruse (Editors)

Medaka: Biology, Management, and Experimental Protocols, Volume 2, First Edition. Edited by Kenji Murata, Masato Kinoshita, Kiyoshi Naruse, Minoru Tanaka, and Yasuhiro Kamei. © 2020 John Wiley & Sons Ltd. Published 2020 by John Wiley & Sons Ltd.

1

1.1 Introduction

This chapter provides supplemental information about medaka breeding described in Chapter 2 Medaka Management of the first edition of Medaka: Biology, Management, and Experimental Protocols (2009). In this decade, the biological usefulness and convenience of medaka as a model animal have been recognized and the number of research fields and researchers using medaka has been expanding. In these situations, strict and sophisticated rearing methods are required to evaluate the effects of the gene of interest, chemicals, environmental conditions, and so on. Well‐designed and regulated feeding is especially critical to evaluate growth and appetite. Additionally, ethical issues such as animal welfare have been emphasized including the “3Rs” concept: Replacement, Reduction, and Refinement. Therefore, it has become important to understand anesthesia and euthanasia methods.

Understanding the life‐cycle of medaka in the wild is important to construct a research plan for successful breeding in the laboratory. First, in this chapter, the life‐cycle of medaka in the wild is described. Then, the outline of the breeding process is mentioned before the detailed breeding procedure is explained. Finally, anesthesia methods are discussed.

1.2 Medaka Management for Scientific Research

In breeding model animal strains, the most critical point is to maintain the phenotypical characteristics of the various strains in order to obtain experimental reproducibility. Although the same model animal strain is subjected to a certain experiment, the results may differ due to different breeding and maintenance conditions. Therefore, it is very important to describe the proper breeding and maintenance conditions as well as the significant results in the research paper so that other researchers can reproduce these procedures as accurately as possible. In this section, the standard breeding procedures based on the life‐cycle of medaka in the wild are described; they are helpful in setting up a medaka breeding system as a first step to begin a research project.

Chapter 1Medaka Management

2 Medaka

1.2.1 Outline of medaka life‐cycle in the wild

Medaka (Japanese medaka, Oryzias latipes) has a widespread distribution in Japan, pri-marily in small ponds and rice paddies (See 1.2 Phylogeny in Chapter 1 in the first edition of Medaka: Biology, Management, and Experimental Protocols (2009) and Chapter 2 in this book). Medaka can live throughout the year in Japan (Shima and Mitani 2004), but has a limited spawning period in the wild. More than 12 hours of daylight and water tem-perature higher than 13 °C are required for oogenesis and spermatogenesis to take place. Then, the fish start to mate and the females spawn eggs. These climate conditions match those during spring and summer in Japan. Actually, the combination of a 14 hour light/10 hour dark (14L–10D) cycle and temperatures of 25–28 °C (which are consistent with those of early summer in Japan) and sufficient food provides the best conditions for spawning eggs in the wild. In spring, in the wild, when the water temperature is lower than 25 °C, larvae hatch in 10 or more days. The larvae reach maturity in the early summer. They mate and spawn eggs until the hours of daylight become shorter than the hours of darkness (late summer to autumn). A small number of the next generation survives the winter and a smaller number may survive two winters. The average lifespan in the wild is considered to be around one year because predation and/or seasonal environment change are the leading causes of death of medaka in the wild. Egami reported that the average lifespan is less than three years and the maximum lifespan is approximately five years under experimental conditions (Egami 1971).

1.2.2 Preparation of normal rearing conditions of medaka in the laboratory and procedures for breeding

The outline of the rearing schedule is shown in Figure 1-1. To obtain eggs, creating the conditions that are required for successful mating and spawning is important. Details of these conditions are described in 3.5 Necessary Conditions for Spawning in Chapter 3 in the first version of this book. Briefly, these conditions consist of feeding three times a day, a 14L–10D light cycle at 25–28 °C, and avoiding keeping the fish at a high density.

1.2.2.1 Breeding system set‐up

Select the water system (flowing, recycled, or static water) according to the laboratory situation or the requirements of the experiment, and prepare the water to fill the fish tanks. Soft water (this means the water contains a concentration of calcium and magnesium ions lower than 120 mg/L) without chlorine is recommended. The light/dark cycle (14L–10D) and the room temperature (25–28 °C) of the breeding room should be controlled respectively using a timer and an air conditioner, before bringing medaka into the room. In case it is difficult to control the lighting conditions of the room where they have been set for the experiments and the regulation of the water tem-peratures in each breeding tank in the room as a single unit, the light/dark cycle and the temperatures of the water in the tanks can be independently regulated at each breeding tank or aquarium system. (See Chapter 2 Medaka Management in the first version of this book.)

Medaka Management 3

1.2.2.2 Obtaining medaka

It is strongly recommended that medaka be obtained from researchers who are culturing medaka or from the National Bio‐Resource Project (NBRP) Medaka (https://shigen.nig.ac.jp/medaka) to prevent the contamination and/or introduction of pathogens or parasites into the aquarium(s). The NBRP Medaka at the National Institute for Basic Biology in Japan distributes medaka strains, including wild, inbred, transgenic, and mutant strains, domestically and internationally.

In the wild, medaka habitats are located in areas that encompass tropical, temperate, and cold climate zones within various types of aquatic habitats, with water conditions ranging from still, freshwater lakes, flowing freshwater streams, and rice paddies to brackish and sea water; medaka have adapted to all these conditions. However, to avoid problems such as the contamination of pathogens and/or parasites from the environment as described above, the medaka used for research should be kept away from all wild aquatic animals, and water taken directly from the natural environment should never be used, since untreated water from natural habitats may harbor pathogens and/or parasites.

If it should be necessary to use medaka obtained from the wild or pet shops, these fish should be reared in an independent tank to collect more than 20 eggs from them. After pasteur-izing (see section 1.3.2.2) these eggs, the larvae that hatch from these eggs can be introduced into the aquarium. Also see Chapter 2 Medaka Management in the first version of this book.

1.2.2.3 Collecting eggs in a laboratory setting

In order to obtain eggs from medaka to use for your experiment, introduce a pair of adult medaka into a half‐full breeding tank. The volume of the tank should be at least 1 L

Tools Fish Tanks and Dishes Water Foods Light and Timer

Adult culture(a pair)

Stock center(e.g. NBRP Medaka)

Daily care of medaka culture

Embryo culture(9-day incubation)

9-dayincubation

Larvae are cultured in a small tank in0.1 x sea water under 25–27°C.Frequent feeding and water change.Nursery will be last for a month.

Larvae

Adults

Daily cyclefor medaka culture

A monthnursery

Feeding: Egg collection:24

12

189

Embryos

Larva culture(A month nursery)

Nextgeneration

hatching

hatching

Continue daily care

New strain

Water change etc.

Young fish

Eggs

Eggs

3 pairs of adult

Embryos are cultured in a small dish in0.1 x sea water under 27°C incubator.Embryos will hatch at day 9.

Figure 1-1. Outline of the rearing schedule.

4 Medaka

(10 × 10 × 10 cm). The fish will spawn eggs (10–30 eggs a day per a female) within two weeks with enough food and proper water temperature (25–28 °C).

1.2.2.4 Daily care and maintenance of eggs

The collected eggs are cultured with embryo culture medium* (ECM) or 0.3% (W/V) artificial sea water in a plastic dish (a, b, and c in Table 1-1) at 25–28 °C. Since a lack of oxygen causes delay of ontogeny and/or abnormal development of the embryos, an exces-sive number of embryos in a dish should be avoided. The proper number of embryos in a dish is shown in rows a, b, and c in Table 1-1. The dish should be examined every day for abnormal and/or dead embryos which should be promptly removed; otherwise fungi and bacteria will multiply sufficiently to kill the embryos. The details of sorting and cleaning methods of embryos are described in sections 1.3.2.2 and 1.3.2.3.

* 0.1% (W/V) NaCl, 0.003% (W/V) KCl, 0.004% (W/V) CaCl2‐2H

2O, 0.016%(W/V)

MgSO4‐7H

2O.

1.2.2.5 Rearing medaka from the larval stage to adulthood

After bringing the newly acquired larvae to the breeding facility, transfer them to a culture tank (see d or e in Table 1-1) as soon as possible. Larvae can start eating in one or two days after hatching, therefore it is necessary to start feeding them just after the transfer of hatched larvae into the tanks. The amount of food and frequency of feeding depend on the type of food. The feeding methods using paramecia and commercial powdered food are described in section 1.3.2.4, and Table 1-2.

Leftover food on the surface and bottom causes deterioration of water quality. In breeding with static water, leftover food and debris should be removed every day and half of the breeding water should be changed every day. When breeding fish in a tank that has a water recycling system with filtration, it is recommended that leftover food and debris are removed every day. One method for removing them from the bottom of the tank is described in section 1.3.3.2. The amount of food should be increased as the larvae grow. The size of the food particles is another important matter and should be changed as the fish grow. Information about the particle size and the amount of commercial powdered food needed is described in Tables 1-2 and 1-4. Additionally, since the density of larvae in the tank/aquarium affects the growth rate, the size of the tank and the number of fish should be adjusted to the optimal conditions as shown in Table 1-3.

The fish grow to sexual maturity within three months in proper breeding condi-tions  and they can continue spawning for at least for three months. Rearingmulti-generation will allow to obtain eggs throughout the year (Figure 1-2). It is helpful to keep in mind that a lower density of fish in a breeding tank results in a shorter time between generations.

1.2.2.6 Anesthesia and euthanasia

The ethics of animal welfare have become an important issue in daily life and even affect conditions in scientific fields. Therefore, the fish used in experiments should be treated properly after the experiments have finished. Principally, these fish should be treated according to the guidelines of experimental animals established by each institute or