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Medaka: Biology, Management, and Experimental Protocols

Medaka: Biology, Management, and Experimental Protocols

Masato Kinoshita, Kenji Murata, Kiyoshi Naruse, Minoru Tanaka

ISBN: 978-0-813-81884-9

Apr 2012, Wiley-Blackwell

444 pages

Select type: Online Book


Medaka: Biology, Management, and Experimental Protocols, written by experienced researchers and reviewed by international leaders in the medaka field will provide details on how to set up and maintain medaka colonies in animal facilities, how to troubleshoot systems, how to handle the fish when applied to experimental methods, and most importantly it will introduce the researcher to cutting edge research in basic and applied biology using medaka as a model animal. The book will include well-written descriptions of experimental methods and protocols designed to educate the reader how to understand and handle medaka effectively.

Medaka: Biology, Management, and Experimental Protocols will serve as the definitive reference on the species providing essential information on medaka biology, genetics, and genomics, practical guidance to maintenance of fish stocks, and valuable experimental protocols all in a single volume. This book will be a must have addition to the library of fish researchers and those using medaka as a model organism within their laboratories.



1 History and Features of Medaka.

1.1 History.

1.2 Phylogeny.

1.2.1 Phylogeny and distribution of medaka and relatives.

1.2.2 Genetic diversity of medaka.

1.3 Advantage of Medaka as a Model Fish.

1.3.1 Advantageous features in general.

1.3.2 Color mutants. Introduction and history. Body color and chromatophores. Genes mutated in body-color mutants. Future use of body-color mutants.

1.3.3 Wild strains.

1.3.4 Inbred strains. History for establishing inbred lines. Characteristics of medaka inbred strains.

Column 1.1 For those who cannot decide which medaka to use. Polymorphic variation among inbred strains. To generate and maintain medaka inbred strains.

Column 1.2 Variation among strains.

1.3.5 Differences from zebrafish.

2 Medaka Management.

2.1 How to Obtain Medaka.

2.1.1 Obtain medaka from researchers who are culturing medaka.

2.1.2 Contact the National Bio-Resource Project (medaka) in Japan.

2.1.3 Purchase medaka from commercial vendors (aquarium shops).

2.1.4 Catching medaka from the wild.

2.2 Rearing Medaka.

2.2.1 Breeding program.

2.2.2 Recirculating system (mid-scale system). Aquarium system. Maintenance of recirculating system.

Column 2.1 Soft water is suitable for medaka breeding.

2.2.3 Large-scale breeding. Outline of large-scale water system at JST Kyoto. Water system at JST Kyoto facility. Water condition.

2.2.4 Rearing without water circulation (small-scale system). Room condition, racks, and tanks. Water. Daily care.

2.2.5 Outdoor breeding.

2.3 Feeding.

2.3.1 Feed for adult fish and larvae.

2.3.2 Feeding schedule.

2.3.3 Feed. Brine shrimp (Artemia). Dry feed. Paramecium. Other feed types.

2.4 Diseases.

2.4.1 Tail rot disease.

2.4.2 Matsukasa disease.

2.4.3 Trichodina.

2.4.4 Water mold.

2.4.5 White spot disease.

2.4.6 Water mites.

2.4.7 Gyrodactylus.

2.5 Goods for Medaka.

3 Reproduction of Medaka.

3.1 Sex Determination.

3.1.1 Sex determination in medaka.

3.1.2 Sex determination in the genus Oryzias.

3.1.3 Spontaneous sex reversals in medaka.

3.2 Hormonal Control of Gonadal Development.

3.2.1 Hypothalamic–pituitary–gonadal axis.

3.2.2 Oocyte growth and maturation.

3.2.3 Spermatocyte growth and maturation.

3.3 Oogenesis and Spermatogenesis.

3.3.1 Oogenesis.

3.3.2 Spermatogenesis.

3.4 Egg Envelope (Chorion).

3.4.1 Morphology and biochemical characters of the medaka egg envelope.

3.4.2 Origin of the egg envelope in medaka fish.

3.4.3 Gene structure of egg envelope glycoproteins in medaka .

3.4.4 Molecular mechanisms of liver-specific expression of Choriogenins’ Genes.

3.4.5 Assembly of the Choriogenins into the egg envelope in the ovary.

3.4.6 Egg envelope glycoproteins as the substrates for the hatching enzyme.

3.4.7 Conclusion.

3.5 Necessary Conditions for Spawning.

3.6 Reproductive Behavior.

3.7 Mating.

3.8 Embryo Collection.

3.8.1 Embryo collection directly from females.

3.8.2 Embryo collection from the bottom of the tank.

3.9 Embryonic Culture.

3.9.1 Cleaning.

3.9.2 Incubation.

3.10 Larval Culture.

3.11 Generation of Sex-Reversed Medaka.

3.11.1 Treatment with androgen to generate XX males.

3.11.2 Treatment with estrogen to generate XY females.

3.11.3 High-temperature treatment to generate XX males.

Column 3.1 Interstrain Variation in Reproductive Performance.

4 Strain Preservation and Related Techniques.

4.1 Shipping.

4.1.1 Scheduling.

4.1.2 Sorting of eggs or fish.

4.1.3 Procedures for packaging.

4.1.4 Transgenic medaka.

4.1.5 MTA (Material Transfer Agreement).

4.2 Quarantine and Pasteurization.

4.2.1 Materials for pasteurization of eggs/embryos.

4.2.2 Procedure (Movie M4-1).

4.3 Cryopreservation of Medaka Sperm.

4.3.1 Overview.

4.3.2 The procedure for cryopreservation (Figure 4-2 and Movies M4-2).

4.3.3 Materials.

4.3.4 Solutions.

4.3.5 Procedures.

4.4 Artificial Insemination Using Frozen Medaka Sperm.

4.4.1 Overview.

4.4.2 Solutions.

4.4.3 Materials.

4.4.4 Procedures (Movies M4-3).

Column 4.1 Infertile mating method for collecting unfertilized eggs.

5 Looking at Adult Medaka.

5.1 General Morphology.

5.1.1 Secondary sexual characters.

5.1.2 Body color. Pigment cells (chromatophores). Structures of the chromatophores. Chromatophores in medaka. Chromatophore distribution in medaka. See-through medaka.

5.2 Anatomy and Histology.

5.2.1 Observations of internal organs. Observations of internal organs in the live see-through medaka. Dissection of adult medaka.

5.2.2 Horizontal and sagittal sections of juvenile medaka.

5.2.3 Nervous system. Adult central nervous system. Adult peripheral nervous system.

5.2.4 Endocrine system. Hypothalamo–pituitary system. Pineal organ (epiphysis). Thyroid gland. Heart. Interrenal gland and chromaffin cells. Gonads. Endocrine pancreas (islet of Langerhans). Gastrointestinal tract. Ultimobranchial gland. Corpuscle of Stannius. Urophysis. Thymus.

5.2.5 Gonads. Ovary. Testis.

5.2.6 Kidney. Pronephros. Mesonephros. Histology of the kidney.

Column 5.1 How to make sections of a meture ovary for histological analysis.

6 Looking at Medaka Embryos.

6.1 Development of Various Tissues and Organs.

6.1.1 Developmental stages. Stage 0; Unfertilized Egg – Figure 6-1. Stage 1; activated egg stage (3 minutes) – Figure 6-1. Stage 2; blastodisc stage – Figure 6-1. Stage 3; two-cell stage (1 hour 5 minutes) – Figure 6-1. Stage 4; four-cell stage (1 hour 45 minutes) – Figure 6-1. Stage 5; eight-cell stage (2 hours 20 minutes) – Figure 6-1. Stage 6; 16-cell stage (2 hours 55 minutes) – Figure 6-2. Stage 7; 32-cell stage (3 hours 30 minutes) – Figure 6-2. Stage 8; early morula stage (4 hours 5 minutes) – Figure 6-2. Stage 9; late morula stage (5 hours 15 minutes) – Figure 6-2. Stage 10; early blastula stage (6 hours 30 minutes) – Figure 6-2. Stage 11; late blastula stage (8 hours 15 minutes) – Figure 6-2. Stage 12; pre-early gastrula stage (10 hours 20 minutes) – Figure 6-3. Stage 13; early gastrula stage (13 hours) – Figure 6-3. Stage 14; pre-mid-gastrula stage (15 hours) – Figure 6-3. Stage 15; mid-gastrula stage (17 hours 30 minutes) – Figure 6-3. Stage 16; late gastrula stage (21 hours) – Figure 6-3. Stage 17; early neurula stage (1 day 1 hour) – Figure 6-3. Stage 18; late neurula stage (1 day 2 hours) – Figure 6-4. Stage 19; two-somite stage (1 day 3 hours 30 minutes) – Figure 6-4. Stage 20; four-somite stage (1 day 7 hours 30 minutes) – Figure 6-4. Stage 21; six-somite stage (1 day 10 hours) – Figure 6-4. Stage 22; nine-somite stage (1 day 14 hours) – Figure 6-4. Stage 23; 12-somite stage (1 day 17 hours) – Figure 6-4. Stage 24; 16-somite stage (1 day 20 hours) – Figure 6-5. Stage 25; 18–19-somite stage (2 days 2 hours) – Figure 6-5. Stage 26; 22-somite stage (2 days 6 hours) – Figure 6-5. Stage 27; 24-somite stage (2 days 10 hours) – Figure 6-5. Stage 28; 30-somite stage (2 days 16 hours) – Figure 6-5. Stage 29; 34-somite stage (3 days 2 hours) – Figure 6-5. Stage 30; 35-somite stage (3 days 10 hours) – Figure 6-6. Stage 31; gill blood vessel formation stage (3 days 23 hours) – Figure 6-6. Stage 32; somite completion stage (4 days 5 hours) – Figure 6-6. Stage 33; stage at which notochord vacuolization is completed (4 days 10 hours) – Figure 6-6. Stage 34; pectoral fin blood circulation stage (5 days 1 hour) – Figure 6-6. Stage 35; stage at which visceral blood vessels form (5 days 12 hours) – Figure 6-6. Stage 36; heart development stage (6 days) – Figure 6-7. Stage 37; pericardial cavity formation stage (7 days) – Figure 6-7. Stage 38; spleen development stage (8 days) – Figure 6-7. Stage 39; hatching stage (9 days) – Figure 6-7. Stage 40; first larval stage – Figure 6-8. Stage 41; second larval stage – Figure 6-8. Stage 42; third larval stage – Figure 6-8. Stage 43; first juvenile stage – Figure 6-8. Stage 44; second juvenile stage – Figure 6-8. Stage 45 – Figure 6-8.

6.1.2 Brain. Gastrula step (stages 13–17). Neurula step (stages 17–18). Neural rod step (stages 19–22). Neural tube step (stages 23–27). Late embryonic brain step (stages 28–34). Larval brain step (stages 35–42).

6.1.3 Hatching gland. Origin of fish hatching gland cells. Secretion of hatching enzymes from hatching gland cells.

6.1.4 Eye development. Specification of the anterior neural plate. Eye field determination and establishment of retinal identity. Splitting of the retinal anlage into two retinal primordia. Morphogenesis I: evagination of the optic vesicle. Morphogenesis II: formation of the optic cup. Retinal differentiation I: central retina. Retinal differentiation II: CMZ. Retinotectal projection.

6.1.5 Branchial arch and jaws. Skeletal development. Muscle development.

6.1.6 Vasculature. Vascular anatomy of the developing medaka. Origin of the medaka endothelial lineage. Abbreviations. Acknowledgment.

6.1.7 Blood cells (hematopoiesis). Overview. Observation of Embryonic and Adult Blood Cells.

6.1.8 Heart. Overview. Heart architecture. Heart morphogenesis. Observation of the developing heart.

6.1.9 Kidney. Introduction. Nephrogenesis. Pronephros. Mesonephros.

6.1.10 Thymus. Overview. Early development of the thymus. Cortex and medulla. Involution of the thymus.

6.1.11 Gut and liver.

6.1.12 Bones. Vertebral column.

Column 6.1 Key words in bone formation.

6.1.13 Fins. Introduction. Fin anatomy. Embryonic fin development (from fertilization to stage 39 [hatching stage]). Fin development after hatching (after stage 39). Gene expression during fin development.

6.1.14 Gonads. Introduction. PGC specification. Formation of gonadal primordium (Figure 6-60B). Sexual dimorphism in germ cell proliferation (Figure 6-61). Post-hatching period in XX gonads. Post-hatching period in XY gonads.

6.2 Medaka EGG Envelope and Hatching Enzyme.

6.2.1 Overview.

6.2.2 Preparation of a hatching enzyme solution from hatching liquid.

6.2.3 Simple method for preparing hatching enzyme solution.

6.2.4 Solubilization of the egg envelope using hatching enzyme.

Column 6.2 Easy method for preparation of a small amount of hatching enzyme solution (see DVD for figure).

6.3 Observation of Embryos (Embedding Embryos).

6.3.1 Anesthesia of Embryos using MS-222.

6.3.2 Observation of embryos (mounting). Living embryos. Processed Embryos.

6.4 Whole Mount in situ Hybridization (see Section 6.1.8. for a similar protocol).

6.4.1 Fixation and storage.

6.4.2 Rehydration, proteinase K Treatment and post-fixation at RT.

6.4.3 Hybridization and washing.

6.4.4 Immunoreaction and washing antibodies.

6.4.5 Staining.

6.5 Embedding in a Plastic Resin (Technovit 7100).

6.5.1 Agarose mounting (Figure 6-68).

6.5.2 Dehydration and infiltration (Figure 6-68).

6.5.3 Polymerization (Figure 6-68).

Column 6.3 Pigment cells (Figure 6-69).

Column 6.4 Kupffer’s vesicle.

7 Transgenesis.

7.1 Microinjection Technique for Medaka Eggs.

7.1.1 Equipment required. Egg holder. Glass needles for microinjection. Injector and manipulator with needle holder. Microscope and light. Other tools and fertilized eggs.

7.1.2 Microinjection procedure.

Column 7.1 Microinjection into nuclei.

7.2 DNA Microinjection.

7.2.1 DNA microinjection for transgenesis and transient expression.

7.2.2 DNA construction for transgenesis.

Column 7.2 Toxicity of DNA.

Column 7.3 The form of DNA for transgenesis.

7.3 RNA Microinjection.

Column 7.4 Importance of 3_-UTR.

7.4 Gene Knockdown Technology.

7.4.1 Morpholinos.

7.4.2 gripNAs.

8 Toxicology.

8.1 Status of Medaka in Toxicology.

8.2 Fish Culture for Toxicology.

8.2.1 Preparation and acclimation of fish.

8.2.2 How to expose to chemicals.

8.3 Standardized Toxicity Testings.

8.3.1 International standardization for toxicity tests.

8.3.2 Acute Toxicity Test (OECD TG203).

8.3.3 Early-life stage toxicity test (OECD TG210).

8.4 Applied Toxicity Tests for Endocrine Disrupters.

8.4.1 Screening assays using medaka.

8.4.2 Fish full lifecycle testing (FFLC) using medaka.

8.4.3 Sensitive period to estrogen substances in early life stages.

8.5 Vitellogenin as an Environmental Endocrine Disrupting Chemical Exposure Index.

8.5.1 Features of VTG.

8.5.2 Vitellogenin measurement.

8.5.3 Summary and comments.

8.6 New Techniques and Other Studies.

Column 8.1 Application of medaka and olyzias Sp. in seawater. Can medaka survive in seawater?

9 Bioinformatics.

9.1 Medaka Genome Project and Genome Sequence Database.

9.1.1 Genome database.

9.1.2 Polymorphism between the Southern and Northern Japanese populations.

Column 9.1 How to get BAC/Fosmid clones harboring the target gene.

9.2 Database for Transcribed Sequences.

9.2.1 EST database.

9.2.2 Developmental Expression database.

9.3 Positional Cloning of the Causal Gene in Mutants.

9.3.1 Mapping mutants using SSLP and RFLP markers. Creating mapping panel. Identification of the linkage group linked to a mutation using bulk segregation analysis with M markers. Low-resolution mapping. Intermediate-Resolution Mapping. High-Resolution Mapping. In silico chromosome walking. Identification of target gene.

Column 9.2 Construction of fosmid library.

10 Advanced Techniques.

10.1 Cell Culture from Medaka Embryo.

10.1.1 Flow chart of primary cell culture from medaka embryo.

10.1.2 Equipment and materials.

10.1.3 Protocol.

10.1.4 Notes.

10.2 In Vitro Spermatogenesis from Primary Spermatocytes.

10.2.1 Flow chart of in vitro spermatogenesis from primary spermatocytes.

10.2.2 Required equipment and materials for primary culture of primary spermatocytes.

10.2.3 Protocol.

10.3 Single Cell Labeling.

10.3.1 Flow chart of single cell labeling.

10.3.2 Required equipment and materials.

10.3.3 Protocol of single cell labeling.

10.3.4 Example of cell labeling and tracing.

10.4 Imaging of Living Embryos.

10.4.1 Flow chart of imaging of living embryos.

10.4.2 Fluorescent labeling.

10.4.3 Sample preparation.

10.4.4 Recording setup.

10.4.5 Data analysis.

10.4.6 Time-lapse imaging of primordial germ cell migration.

10.4.7 Conclusion.

10.5 Transplantation.

10.5.1 Cell transplantation in embryo. Figure 10-11 shows the procedure of cell transplantation in embryo briefly.

10.5.2 Scale.

10.6 Nuclear Transplantation.

10.6.1 Equipment and materials.

10.6.2 Flow chart of the method.

10.6.3 Perspectives.

10.7 Mutagenesis.

10.7.1 Benefits of using medaka.

10.7.2 Mutagens that have been used for medaka.

10.7.3 Mutagenesis screen using ENU.

10.8 Tilling (Gene Knockout).

10.8.1 Outline of the TILLING method.

10.8.2 An example of screening and quality of our library.

10.8.3 About SNPs.

10.8.4 How to obtain a Medaka TILLING library.

10.9 Cell Trace Experiment with a Caged Fluorescent Dye During Medaka Gastrulation.

10.9.1 Flow chart of cell trace experiment with a caged fluorescent dye.

10.9.2 Equipment and materials.

10.9.3 Protocol (Figure 10-2).

10.9.4 Notes.

Appendix 1 Guidelines on Using Medaka in Experiments.

Appendix 2 Internet Websites Related to Medaka Research.

Appendix 3 Solutions.

Appendix 4 Inbred Strains, Closed Colonies, and Mutant Strains.

Appendix 5 Index of Abbreviation.



  • Valuable reference on a model fish species of growing importance

  • First volume that brings together medaka research protocols and fish maintenance and care into a single volume

  • Written and reviewed by leading medaka researchers

  • CD with additional illustrations and video demonstrations of research protocols