Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Laboratory breeding of the short-lived annual killifish Nothobranchius furzeri

Nature Protocols volume 11, pages 1396–1413 (2016)Cite this article

Subjects

Abstract

Turquoise killifish, Nothobranchius furzeri, have an intrinsically short life span, with a median life span of <6 months and a maximum (90%) life span of 9 months. This short life span, which is unique among vertebrates, evolved naturally and has resulted in N. furzeri becoming a widely used laboratory model species in aging research and other disciplines. Here, we describe a protocol for the maintenance and breeding of the species under laboratory conditions. We provide details for egg incubation, hatching, everyday care of juvenile and adult fish, breeding and treatment of most common diseases. Emphasis is given to the fact that the requirements of N. furzeri substantially differ from those of other fish model taxa; N. furzeri live brief lives and in nature undergo nonaquatic embryo development, with consequences for their laboratory culture.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Learn more

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Annual fish Nothobranchius furzeri.
Figure 2: Developmental trajectories of N. furzeri in aquatic medium (egg incubation at 25 °C).
Figure 3: External appearance of fish infected by common N. furzeri pathogens.
Figure 4: Male N. furzeri infected with Glugea sp.
Figure 5: Schematic overview of the sequence and duration of different steps in the husbandry regime of N. furzeri.
Figure 6: Basic equipment needed for husbandry of N. furzeri.
Figure 7: Gross staging of N. furzeri eggs incubated in peat.
Figure 8: Setup for hatching peat-incubated N. furzeri eggs.
Figure 9: Differences between a fertilized and an unfertilized N. furzeri egg.

References

  1. Gerhard, G.S. Small laboratory fish as models for aging research. Ageing Res. Rev. 6, 64–72 (2007).

    Article  PubMed  Google Scholar 

  2. Schartl, M. Beyond the zebrafish: diverse fish species for modelling human disease. Dis. Model. Mech. 7, 181–192 (2014).

    Article  CAS  PubMed  Google Scholar 

  3. Maan, M.E. & Seehausen, O. Ecology, sexual selection and speciation. Ecol. Lett. 14, 591–602 (2011).

    Article  PubMed  Google Scholar 

  4. Sloman, K.A. & McNeil, P.L. Using physiology and behaviour to understand the responses of fish early life stages to toxicants. J. Fish Biol. 81, 2175–2198 (2012).

    Article  CAS  PubMed  Google Scholar 

  5. Comfort, A. Age and reproduction in female Lebistes. Gerontologia 5, 146–149 (1961).

    Article  CAS  PubMed  Google Scholar 

  6. Egami, N. & Etoh, H. Life span data for the small fish, Oryzias latipes. Exp. Gerontol. 4, 127–129 (1969).

    Article  CAS  PubMed  Google Scholar 

  7. Di Cicco, E., Tozzini, E.T., Rossi, G. & Cellerino, A. The short-lived annual fish Nothobranchius furzeri shows a typical teleost aging process reinforced by high incidence of age-dependent neoplasias. Exper. Gerontol. 46, 249–256 (2011).

    Article  Google Scholar 

  8. Wildekamp, R.H. A World of Killies: Atlas of the Oviparous Cyprinodontiform Fishes of the World Vol. 4. (American Killifish Association, 2004).

  9. Valdesalici, S. & Cellerino, A. Extremely short lifespan in the annual fish Nothobranchius furzeri. Proc. Biol. Sci. 270 (suppl. 2), S189–S191 (2003).

    Article  PubMed  PubMed Central  Google Scholar 

  10. Wang, A.M., Promislow, D.E. & Kaeberlein, M. Fertile waters for aging research. Cell 160, 814–815 (2015).

    Article  CAS  PubMed  Google Scholar 

  11. Terzibasi, E. et al. Large differences in aging phenotype between strains of the short-lived annual fish Nothobranchius furzeri. PLoS ONE 3, e3866 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Tozzini, E.T. et al. Parallel evolution of senescence in annual fishes in response to extrinsic mortality. BMC Evol. Biol. 13, 77 (2013).

    Article  PubMed  Google Scholar 

  13. Wit, J., Loeschcke, V. & Kellermann, V. Life span variation in 13 Drosophila species: a comparative study on life span, environmental variables and stress resistance. J. Evol. Biol. 28, 1892–1900 (2015).

    Article  CAS  PubMed  Google Scholar 

  14. Wendler, S., Hartmann, N., Hoppe, B. & Englert, C. Age-dependent decline in fin regenerative capacity in the short-lived fish Nothobranchius furzeri. Aging Cell 14, 857–866 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Reichard, M., Polačik, M. & Sedláček, O. Distribution, colour polymorphism and habitat use of the African killifish, Nothobranchius furzeri, the vertebrate with the shortest lifespan. J. Fish Biol. 74, 198–212 (2009).

    Article  CAS  PubMed  Google Scholar 

  16. Wourms, J.P. The developmental biology of annual fishes. III. Pre-embryonic and embryonic diapause of variable duration in the eggs of annual fishes. J. Exp. Zool. 182, 389–414 (1972).

    Article  CAS  PubMed  Google Scholar 

  17. Furness, A.I., Lee, K. & Reznick, D.N. Adaptation in a variable environment: phenotypic plasticity and bet-hedging during egg diapause and hatching in an annual killifish. Evolution 69, 1461–1475 (2015).

    Article  PubMed  Google Scholar 

  18. Blažek, R., Polačik, M. & Reichard, M. Rapid growth, early maturation and short generation time in African annual fishes. EvoDevo 4, 24 (2013).

    Article  PubMed  PubMed Central  Google Scholar 

  19. Cellerino, A., Valenzano, D.R. & Reichard, M. From the bush to the bench: the annual Nothobranchius fishes as a new model system in biology. Biol. Rev. 91, 511–533 (2016).

    Article  PubMed  Google Scholar 

  20. D'Angelo, L. Brain atlas of an emerging teleostean model: Nothobranchius furzeri. Anat. Rec. 296, 681–691 (2013).

    Article  Google Scholar 

  21. Valenzano, D.R., Sharp, S. & Brunet, A. Transposon-mediated transgenesis in the short-lived African killifish Nothobranchius furzeri, a vertebrate model for aging. G3 (Bethseda) 1, 531–538 (2011).

    Article  CAS  Google Scholar 

  22. Hartmann, N. & Englert, C. A microinjection protocol for the generation of transgenic killifish (species: Nothobranchius furzeri). Dev. Dyn. 241, 1133–1141 (2012).

    Article  CAS  PubMed  Google Scholar 

  23. Allard, J.B., Kamei, H. & Duan, C. Inducible transgenic expression in the short-lived fish Nothobranchius furzeri. J. Fish Biol. 82, 1733–1738 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Petzold, A. et al. The transcript catalogue of the short-lived fish Nothobranchius furzeri provides insights into age-dependent changes of mRNA levels. BMC Genomics 14, 185 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Baumgart, M. et al. Age-dependent regulation of tumor-related microRNAs in the brain of the annual fish Nothobranchius furzeri. Mech. Age. Dev. 133, 226–233 (2012).

    Article  CAS  Google Scholar 

  26. Valenzano, D.R. et al. The African turquoise killifish genome provides insights into evolution and genetic architecture of lifespan. Cell 163, 1539–1554 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Reichwald, K. et al. Insights into sex chromosome evolution and aging from the genome of a short-lived fish. Cell 163, 1527–1538 (2015).

    Article  CAS  PubMed  Google Scholar 

  28. Harel, I. et al. A platform for rapid exploration of aging and diseases in a naturally short-lived vertebrate. Cell 160, 1013–1026 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Polačik, M. & Reichard, M. Diet overlap among three sympatric African annual killifish species (Nothobranchius spp.) from Mozambique. J. Fish Biol. 77, 754–768 (2010).

    PubMed  Google Scholar 

  30. Polačik, M., Donner, M.T. & Reichard, M. Age structure of annual Nothobranchius fishes in Mozambique: is there a hatching synchrony? J. Fish Biol. 78, 796–809 (2011).

    Article  PubMed  Google Scholar 

  31. Reichard, M., Polačik, M., Blažek, R. & Vrtílek, M. Female bias in the adult sex ratio of African annual fishes: interspecific differences, seasonal trends and environmental predictors. Evol. Ecol. 28, 1105–1120 (2014).

    Article  Google Scholar 

  32. Bartáková, V. et al. Strong population genetic structuring in an annual fish, Nothobranchius furzeri, suggests multiple savannah refugia in southern Mozambique. BMC Evol. Biol. 13, 196 (2013).

    Article  PubMed  PubMed Central  Google Scholar 

  33. Bartáková, V., Reichard, M., Blažek, R., Polačik, M. & Bryja, J. Terrestrial fishes: rivers are barriers to gene flow in annual fishes from the African savanna. J. Biogeogr. 42, 1832–1844 (2015).

    Article  Google Scholar 

  34. Polačik, M. et al. Alternative intrapopulation life-history strategies and their trade-offs in an African annual fish. J. Evol. Biol. 27, 854–865 (2014).

    Article  PubMed  Google Scholar 

  35. Podrabsky, J.E. & Culpepper, K.M. Cell cycle regulation during development and dormancy in embryos of the annual killifish Austrofundulus limnaeus. Cell Cycle 11, 1697–1704 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Graf, M., Cellerino, A. & Englert, C. Gender separation increases somatic growth in females but does not affect lifespan in Nothobranchius furzeri. PLoS ONE 5, e11958 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Noga, E.J. Fish Disease: Diagnosis and Treatment 367 (Blackwell Publishing, 2000).

  38. Lom, J., Noga, E.J. & Dyková, I. Occurrence of a microsporean with characteristics of Glugea anomala in ornamental fish of the family Cyprinodontidae. Dis. Aquat. Org. 21, 239–242 (1995).

    Article  Google Scholar 

  39. Lesseps, R.J., van Kessel, A.H. & Denuce, J.M. Cell patterns and cell movements during early development of an annual fish, Nothobranchius neumanni. J. Exp. Zool. 193, 137–146 (1975).

    Article  CAS  PubMed  Google Scholar 

  40. Haarlem, R.V. Contact inhibition of overlapping: one of the factors involved in deep cell epiboly of Nothobranchius korthausae. Dev. Biol. 70, 171–179 (1979).

    Article  CAS  PubMed  Google Scholar 

  41. Van Haarlem, R., Van Wijk, R. & Fikkert, A.H. Analysis of the variability in cleavage times and demonstration of a mitotic gradient during the cleavage stages of Nothobranchius guentheri. Cell Tissue Kinet. 14, 285–300 (1981).

    CAS  PubMed  Google Scholar 

  42. Markofsky, J. & Matias, J.R. The effects of temperature and season of collection on the onset and duration of diapause in embryos of the annual fish Nothobranchius guentheri. J. Exp. Zool. 202, 49–56 (1977).

    Article  CAS  PubMed  Google Scholar 

  43. Inglima, K., Perlmutter, A. & Markofsky, J. Reversible stage-specific embryonic inhibition mediated by the presence of adults in the annual fish Nothobranchius guentheri. J. Exp. Zool. 215, 23–33 (1981).

    Article  CAS  PubMed  Google Scholar 

  44. Levels, P.J., Gubbels, R.E. & Denuce, J.M. Oxygen consumption during embryonic development of the annual fish Nothobranchius korthausae with special reference to diapause. Comp. Biochem. Physiol. A 84, 767–770 (1986).

    Article  CAS  PubMed  Google Scholar 

  45. Markofsky, J. & Milstoc, M. Histopathological observations of the kidney during aging of the male annual fish Nothobranchius guentheri. Exp. Gerontol. 14, 149–155 (1979).

    Article  CAS  PubMed  Google Scholar 

  46. Cooper, E.L., Zapata, A., Garcia Barrutia, M. & Ramirez, J.A. Aging changes in lymphopoietic and myelopoietic organs of the annual cyprinodont fish, Nothobranchius guentheri. Exp. Gerontol. 18, 29–38 (1983).

    Article  CAS  PubMed  Google Scholar 

  47. van der Hoeven, J.C., Bruggeman, I.M., Alink, G.M. & Koeman, J.H. The killifish Nothobranchius rachowi, a new animal in genetic toxicology. Mutat. Res. 97, 35–42 (1982).

    Article  CAS  PubMed  Google Scholar 

  48. Haas, R. Behavioral biology of the annual killifish Nothobranchius guentheri. Copeia 1976, 80–91 (1976).

    Article  Google Scholar 

  49. Genade, T. et al. Annual fishes of the genus Nothobranchius as a model system for aging research. Aging Cell 4, 223–233 (2005).

    Article  CAS  PubMed  Google Scholar 

  50. Valenzano, D.R. et al. Resveratrol prolongs lifespan and retards the onset of age-related markers in a short-lived vertebrate. Curr. Biol. 16, 296–300 (2006).

    Article  CAS  PubMed  Google Scholar 

  51. Valenzano, D.R., Terzibasi, E., Cattaneo, A., Domenici, L. & Cellerino, A. Temperature affects longevity and age-related locomotor and cognitive decay in the short-lived fish Nothobranchius furzeri. Aging Cell 5, 275–278 (2006).

    Article  CAS  PubMed  Google Scholar 

  52. Terzibasi, E. et al. Effects of dietary restriction on mortality and age-related phenotypes in the short-lived fish Nothobranchius furzeri. Aging Cell 8, 88–99 (2009).

    Article  CAS  PubMed  Google Scholar 

  53. Hartmann, N. et al. Telomeres shorten while Tert expression increases during ageing of the short-lived fish Nothobranchius furzeri. Mech. Ageing. Dev. 130, 290–296 (2009).

    Article  CAS  PubMed  Google Scholar 

  54. Hartmann, N. et al. Mitochondrial DNA copy number and function decrease with age in the short-lived fish Nothobranchius furzeri. Aging Cell 10, 824–831 (2011).

    Article  CAS  PubMed  Google Scholar 

  55. Tozzini, E.T., Baumgart, M., Battistoni, G. & Cellerino, A. Adult neurogenesis in the short-lived teleost Nothobranchius furzeri: localization of neurogenic niches, molecular characterization and effects of aging. Aging Cell 11, 241–251 (2012).

    Article  CAS  PubMed  Google Scholar 

  56. Kirschner, J. et al. Mapping of quantitative trait loci controlling lifespan in the short-lived fish Nothobranchius furzeri - a new vertebrate model for age research. Aging Cell 11, 252–261 (2012).

    Article  CAS  PubMed  Google Scholar 

  57. Hoppe, B. et al. MiR-21 is required for efficient kidney regeneration in fish. BMC Dev. Biol. 15, 43 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Baumgart, M., Di Cicco, E., Rossi, G., Cellerino, A. & Tozzini, E.T. Comparison of captive lifespan, age-associated liver neoplasias and age-dependent gene expression between two annual fish species: Nothobranchius furzeri and Nothobranchius korthause. Biogerontology 16, 63–69 (2015).

    Article  CAS  PubMed  Google Scholar 

  59. Vrtílek, M. & Reichard, M. Highly plastic resource allocation to growth and reproduction in females of an African annual fish. Ecol. Freshw. Fish 24, 616–628 (2014).

    Article  Google Scholar 

  60. Polačik, M. & Reichard, M. Asymmetric reproductive isolation between two sympatric annual killifish with extremely short lifespans. PLoS ONE 6, e22684 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. Podrabsky, J.E., Garrett, I.D. & Kohl, Z.F. Alternative developmental pathways associated with diapause regulated by temperature and maternal influences in embryos of the annual killifish Austrofundulus limnaeus. J. Exp. Biol. 213, 3280–3288 (2010).

    Article  PubMed  PubMed Central  Google Scholar 

  62. Calviño, P.A., Alonso, F. & Sanjuán de Torres, J. Llenado de gas de la vejiga natatoria de post-larvas de peces anuales sudamericanos (Cyprinodontiformes; Rivulidae). Boletín del Killi Club Argentino 13, 19–38 (2007).

    Google Scholar 

  63. Podrabsky, J.E. Husbandry of the annual killifish Austrofundulus limnaeus with special emphasis on the collection and rearing of embryos. Envir. Biol. Fishes 54, 421–431 (1999).

    Article  Google Scholar 

  64. Gerhard, G.S. et al. Life spans and senescent phenotypes of zebrafish (Danio rerio). Exp. Gerontol. 37, 1055–1068 (2002).

    Article  PubMed  Google Scholar 

  65. Reed, B. & Jennings, M. Guidance on the housing and care of zebrafish Danio rerio.: Research Animals Department, Science Group, RSPCA, last updated 2011) https://www.scilifelab.se/wp-content/uploads/2013/10/Guidance-zebrafish.pdf.

  66. Davis, C.R., Okihiro, M.S. & Hinton, D.E. Effects of husbandry practices, gender, and normal physiological variation on growth and reproduction of Japanese medaka, Oryzias latipes. Aquat. Toxicol. 60, 185–201 (2002).

    Article  CAS  PubMed  Google Scholar 

  67. Denny, J.S., Spehar, R.L., Mead, K.E. & Youssuf, S.C. Guidelines for culturing the Japanese medaka Oryzias latipes. United States Environmental Protection Agency, Office of Research and Development http://nepis.epa.gov/Exe/ZyPDF.cgi/30000OXS.PDF?Dockey=30000OXS.PDF1991.

  68. Ding, L., Kuhne, W.W., Hinton, D.E., Song, J. & Dynan, W.S. Quantifiable biomarkers of normal aging in the Japanese medaka fish (Oryzias latipes). PLoS ONE 5, e13287 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  69. Hirshfield, M.F. An experimental analysis of reproductive effort and cost in the Japanese medaka, Oryzias latipes. Ecology 61, 282–292 (1980).

    Article  Google Scholar 

Download references

Acknowledgements

Financial support for our current research on Nothobranchius comes from the Czech Science Foundation (P506/11/0112) to M.R. We thank M. Vrtílek and three anonymous referees for comments, E. Řehulková for help with illustrations, A. Dorn for providing Figure 4 and helpful comments, and R. Spence and S. White for correcting the English. All procedures described herein are in accordance with Czech legal regulations and have been approved by the ethical committee of the Institute of Vertebrate Biology, Czech Academy of Sciences.

Author information

Authors and Affiliations

  1. Institute of Vertebrate Biology, Academy of Sciences of the Czech Republic, Brno, Czech Republic

    Matej Polačik, Radim Blažek & Martin Reichard

Authors
  1. Matej Polačik
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Radim Blažek
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Martin Reichard
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

M.P., R.B. and M.R. developed the protocol over the last 10 years. M.R. initiated the paper. M.P. drafted the protocol. R.B. prepared photographic documentation. All authors contributed to the final text.

Corresponding author

Correspondence to Matej Polačik.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Rights and permissions

Reprints and Permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Polačik, M., Blažek, R. & Reichard, M. Laboratory breeding of the short-lived annual killifish Nothobranchius furzeri. Nat Protoc 11, 1396–1413 (2016). https://doi.org/10.1038/nprot.2016.080

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2016.080

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing