Killifish as an aging animal model
The African turquoise killifish (Nothobranchius furzeri) is the shortest lived vertebrate known to date, with a median lifespan of only 4 to 8 months. This short lifespan is closer to that of unicellular and invertebrate research organisms (such as the fruit fly) than to vertebrate research organisms such as mice or zebrafish. This places the Turquoise Killifish in the unique position of allowing high repeatability and feasibility for experiments, whilst better recapitulating human aging traits.
Habitat and life cycle of the turquoise killifish
The turquoise killifish's natural habitat is in freshwater ponds throughout central and eastern Africa, specifically the regions of Zimbabwe and Mozambique.
Due to alternating dry and rainy seasons, these regions have pronounced seasonal differences in water availability, causing the turquoise killifish to inhabit reservoirs of water that fill up during short rainy seasons and dry out entirely during the longer dry seasons.
To survive as a species, the turquoise killifish has developed a unique annual life cycle in which it can persist periods of drought through an extended period of embryonic stasis called diapause. With the onset of the wet season, the turquoise killifish then switches to a mode of explosive growth and sexual maturation, resulting in females laying up to 120 eggs per day.
The process from hatching to sexually mature fish takes no more than 14 days, making it the fastest known rate of sexual maturation for vertebrates. Subsequently, the fertilized eggs enter the diapause state to endure the following dry season, and the circle starts again. As a result of this annual life cycle, the turquoise killifish has a short adult lifespan of four to eight months and displays aging-related transformations like lose of body colour and specific patterning (see section Aging features).
While other standard model organisms such as mice and zebrafish have an average life expectancy of over 2.5 years in captivity, turquoise killifish live only four to eight months on average. Thereby, the lifespan of turquoise killifish is strongly strain-dependent. The original laboratory strain GRZ has the shortest lifespan of 11 to 18 weeks , while longer-lived strains like MZM0403 have a lifespan of 30 weeks. Besides the strain, the lifespan also depends on diet, feeding frequency, and housing conditions.
Although no sex-dependent difference in life expectancy is found in captivity, significant differences in sex ratios are observed in the wild. During the rainy season, the proportion of the male population decreases, so that after three months at least two thirds of the population are female.
Dietary restriction can increase maximum lifespan of both the short-lived GRZ laboratory strain and the longer-lived wild-derived strain MZM-04/10P. However, in the wild strain MZM-04/10P, lifespan extension is associated with increased baseline mortality. In addition to dietary restriction, lowering the water temperature can also increase median lifespan significantly. This demonstrates that lifespan is malleable in killifish when subjecting them to specific interventions, as similarly observed in other animal models.
Like most animals, killifishes show macroscopic signs of aging like a loss of color and pigmentation, emaciation, and a curved spine. The loss of color is more pronounced in males as they are more colorful than females, whereas females tend to lose their rotund appearance due to a prominent curved spine.
Killifishes show substantial strain-dependent variation in the duration of this decrepit state. Fish from wild strains can remain in this state for several weeks before they finally die, while fish of the short-lived GRZ strain usually die before developing a macroscopic phenotype.
Besides an overall decrepit appearance, killifishes also show an impaired ability to regenerate the caudal fins with age, whereas young fish can regenerate them almost completely.
Open-field exploration is a standard behavioural test used in rodents that quantifies the amount of time an individual explores a new environment. Old killifish spend significantly less time exploring new environments compared to young fish and show a decreased moving velocity. Killifishes generally show less spontaneous movement and swimming as they age. However, interventions with resveratrol or reducing the water temperature to 22 °C (instead of 25 °C) significantly reduces age-related mobility deficits.
To evaluate cognitive decline in aged killifish the active avoidance test is used. In this test, fish make an association between a red light and punishment. Both young and old fish succeed in learning the task, but young fish show significantly higher success rates than old fish.
Remarkably, the age-related decline in cognitive performance is completely prevented in old killifish treated with resveratrol. In addition, dietary restriction and reducing temperature from 25 °C to 22 °C also attenuates age-related cognitive decline.
Additionally, old Killifish show molecular since of neurodegeneration, as detected by Fluoro-Jade B staining. and neurodegeneration is accelerated in short-lived strains compared with longer-lived ones. Dietary restriction and resveratrol can reduce age-dependent neurodegeneration. Furthermore, old Killifish can show pathological phenotypes similar to Parkinson’s disease.
Molecular markers of ageing
Telomeres are protective caps at the ends of chromosomes that become shorter with age in various organisms. The telomere length of the short-lived killifish strain GRZ does not shorten with age, suggesting that the shorter lifespan of the strain is not a result of short telomeres. On the other hand, the telomeres of the longer-lived strain MZM0403 show significant telomere shortening within 16 weeks of life. Surprisingly, no upregulation of the telomere-preserving enzyme telomerase can be observed in old individuals of the short-lived strain GRZ, but upregulation can be observed in the longer-lived strain MZM0403.
Lipofuscin, or age pigment, is an autofluorescent pigment that accumulates progressively with age within the cells of many species. In old killifish, elevated lipofuscin levels are detected in various cell types such as heart and liver cells. Thereby, lipofuscin accumulation is faster in the short-lived strain GRZ than in the longer-lived strain MZM0403, suggesting that the short lifespan of the GRZ strain is associated with faster histological aging.
Lowering water temperature (for instance from 25°C to 22°C) as well as dietary restriction can reduce age-related lipofuscin accumulation in old killifish.
Mitochondria are the primary energy providers of most eukaryotic cells, and unlike other organelles, they are unique in that they contain their own DNA (mtDNA). Although large-scale, age-dependent mtDNA deletions are not observed in old killifish (unlike in mammals), the DNA copy number in old killifish decreases with age. Overall, old killifish show lower expression of mitochindria-associated proteins such as Pgc-1a, Tfam, and mtSsbp, and old muscle tissue has decreased levels of respiratory chain complexes. This indicates that oxidative phosphorylation (the process of energy production in mitochondria) might be impaired in old killifish (see also Mitochondrial dysfunction).
After a certain number of replications, cells enter a state of growth arrest and altered function called replicative senescence. Replicative senescence is considered a hallmark of aging, and senescent cells can be detected by markers such as the appearance of senescence-associated β-galactosidase (β-GAL). Old killifish show increased β-GAL activity, which is attenuated with lifespan-extending interventions like lowering water temperature.
Animal model for aging research
Dr Dario Valenzano, trained at Brunet lab in Stanford and currently at the Leibniz Institute on Aging, is especially known for his contribution in characterising the turquoise killifish. He is considered to have established this species as a novel research model and as an aging animal model.
One of the main advantages of using killifish as an aging animal model is its close resemblance to human aging, usually seen in much longer lived animal models such as mice and zebrafish, and its short lifespan of 4 to 8 months, which allows for greater experimental scalability.
The turquoise killifish has been recently established as a model for age-related eye disease. Considering vision decline as a conserved aging hallmark, the aging-associated decline of the killifish visual system has been proposed as a useful in vivo model to study brain aging and rejuvenation.
Genetic tools available
The group led by Anne Brunet in Stanford University has developed a genotype-to-phenotype platform using de-novo-assembled genome and CRISPR/Cas9 technology, which allows for high-throughput and high efficiency knock-out and knock-in studies in killifish.
A key tool for generating transgenic species in fish is the transposase system. Transposon elements (TE) are genes capable of changing position within the genome, which can sometimes result in de novo mutations or changes in genome size. Transposase enzymes bind to the end of TE and catalyze their movement to other parts of the genome. In killifish, the Tol2 transposase system has been adapted from other model animals by Valenzano to integrate genes of interest into the host's genome in a stable and efficient manner.
Additional progress has been made in developing other genetic tools for killifish.  Due to the fast life cycle of killifish, new stable transgenic lines can be generated as rapidly as in 2 to 3 months.
Limitations of killifish
Whilst killifish can be a great alternative compared to more conventional animal models, they also suppose some limitations:
- Teleost fish such as killifish, which includes a large and very diverse group of ray-finned fishes, all possess a duplicated genome. This whole-genome duplication (WGD) occurred in an ancient common ancestor of all teleost fishes. Duplicated genes may sometimes serve different functions or lead to the non-functionalization of one of the genes. This WGD might have occurred at least twice during evolution and may have led to highly fish-specific adaptations. This poses significant limitations on findings derived from killifishes.
- Each killifish requires approximately 1 liter of water. This aspect becomes very troublesome for high throughput assays when taking into account space logistics. Furthermore, it may even prohibit pharmacological approaches, given that incredibly large amounts of drugs would need to be diluted in 1L of water to potentially have an effect.
- ↑ https://en.wikipedia.org/wiki/Nothobranchius_furzeri
- ↑ 2.0 2.1 Hu, C., & Brunet, A. (2018). The African turquoise killifish: A research organism to study vertebrate aging and diapause. Aging Cell, 17(3), e12757. doi: 10.1111/acel.12757
- ↑ 3.0 3.1 3.2 Valenzano, D. R., Benayoun, B. A., Singh, P. P., Zhang, E., Etter, P. D., Hu, C.-K., Clément-Ziza, M., Willemsen, D., Cui, R., Harel, I., Machado, B. E., Yee, M.-C., Sharp, S. C., Bustamante, C. D., Beyer, A., Johnson, E. A., & Brunet, A. (2015). The African Turquoise Killifish Genome Provides Insights into Evolution and Genetic Architecture of Lifespan. Cell, 163(6), 1539–1554. https://doi.org/10.1016/j.cell.2015.11.008
- ↑ Nothobranchius furzeri summary page. FishBase. Retrieved July 22, 2022, from https://www.fishbase.de/summary/Nothobranchius-furzeri.html
- ↑ Poeschla, M., & Valenzano, D. R. (2020). The turquoise killifish: A genetically tractable model for the study of aging. Journal of Experimental Biology, 223(Suppl_1), jeb209296. https://doi.org/10.1242/jeb.209296
- ↑ Vrtílek, M., & Reichard, M. (2016). Female fecundity traits in wild populations of African annual fish: the role of the aridity gradient. Ecology and Evolution, 6(16), 5921–5931. https://doi.org/10.1002/ece3.2337
- ↑ Vrtílek, M., Žák, J., Pšenička, M., & Reichard, M. (2018). Extremely rapid maturation of a wild African annual fish. Current Biology, 28(15), R822–R824. https://doi.org/10.1016/j.cub.2018.06.031
- ↑ Anon. 2017. ‘Non-Canonical Aging Model Systems and Why We Need Them’. The EMBO Journal 36(8):959–63. https://doi.org/10.15252/embj.201796837.
- ↑ 9.0 9.1 Dodzian, Joanna, Sam Kean, Jens Seidel, and Dario Riccardo Valenzano. 2018. ‘A Protocol for Laboratory Housing of Turquoise Killifish (Nothobranchius furzeri)’. JoVE (Journal of Visualized Experiments) (134):e57073. https://doi.org/10.3791/57073
- ↑ 10.0 10.1 10.2 Kirschner, Jeanette, David Weber, Christina Neuschl, Andre Franke, Marco Böttger, Lea Zielke, Eileen Powalsky, Marco Groth, Dmitry Shagin, Andreas Petzold, Nils Hartmann, Christoph Englert, Gudrun A. Brockmann, Matthias Platzer, Alessandro Cellerino, and Kathrin Reichwald. 2012. ‘Mapping of Quantitative Trait Loci Controlling Lifespan in the Short-Lived Fish Nothobranchius Furzeri– a New Vertebrate Model for Age Research’. Aging Cell 11(2):252–61. https://doi.org/10.1111/j.1474-9726.2011.00780.x
- ↑ 11.0 11.1 11.2 11.3 11.4 11.5 11.6 11.7 11.8 Valenzano, Dario R., Eva Terzibasi, Tyrone Genade, Antonino Cattaneo, Luciano Domenici, and Alessandro Cellerino. 2006. ‘Resveratrol Prolongs Lifespan and Retards the Onset of Age-Related Markers in a Short-Lived Vertebrate’. Current Biology 16(3):296–300. https://doi.org/10.1016/j.cub.2005.12.038
- ↑ Reichard, M., M. Polačik, and O. Sedláček. 2009. ‘Distribution, Colour Polymorphism and Habitat Use of the African Killifish Nothobranchius Furzeri, the Vertebrate with the Shortest Life Span’. Journal of Fish Biology 74(1):198–212. https://doi.org/10.1111/j.1095-8649.2008.02129.x
- ↑ Vrtílek, Milan, Jakub Žák, Matej Polačik, Radim Blažek, and Martin Reichard. 2018. ‘Longitudinal Demographic Study of Wild Populations of African Annual Killifish’. Scientific Reports 8(1):4774. https://doi.org/10.1038/s41598-018-22878-6
- ↑ 14.0 14.1 14.2 14.3 14.4 Terzibasi, Eva, Christel Lefrançois, Paolo Domenici, Nils Hartmann, Michael Graf, and Alessandro Cellerino. 2009. ‘Effects of Dietary Restriction on Mortality and Age-Related Phenotypes in the Short-Lived Fish Nothobranchius Furzeri’. Aging Cell 8(2):88–99. https://doi.org/10.1111/j.1474-9726.2009.00455.x
- ↑ 15.0 15.1 15.2 15.3 15.4 15.5 15.6 Valenzano, Dario R., Eva Terzibasi, Antonino Cattaneo, Luciano Domenici, and Alessandro Cellerino. 2006. ‘Temperature Affects Longevity and Age-Related Locomotor and Cognitive Decay in the Short-Lived Fish Nothobranchius Furzeri’. Aging Cell 5(3):275–78. https://doi.org/10.1111/j.1474-9726.2006.00212.x
- ↑ 16.0 16.1 16.2 Kim, Yumi, Hong Gil Nam, and Dario Riccardo Valenzano. 2016. ‘The Short-Lived African Turquoise Killifish: An Emerging Experimental Model for Ageing’. Disease Models & Mechanisms 9(2):115–29. https://doi.org/10.1242/dmm.023226
- ↑ 17.0 17.1 17.2 17.3 17.4 17.5 17.6 17.7 Terzibasi, Eva, Dario Riccardo Valenzano, Mauro Benedetti, Paola Roncaglia, Antonino Cattaneo, Luciano Domenici, and Alessandro Cellerino. 2008. ‘Large Differences in Aging Phenotype between Strains of the Short-Lived Annual Fish Nothobranchius Furzeri’. PLOS ONE 3(12):e3866. https://doi.org/10.1371/journal.pone.0003866
- ↑ 18.0 18.1 18.2 18.3 18.4 Genade, Tyrone, Mauro Benedetti, Eva Terzibasi, Paola Roncaglia, Dario Riccardo Valenzano, Antonino Cattaneo, and Alessandro Cellerino. 2005. ‘Annual Fishes of the Genus Nothobranchius as a Model System for Aging Research’. Aging Cell 4(5):223–33. https://doi.org/10.1111/j.1474-9726.2005.00165.x
- ↑ Wendler, Sebastian, Nils Hartmann, Beate Hoppe, and Christoph Englert. 2015. ‘Age-Dependent Decline in Fin Regenerative Capacity in the Short-Lived Fish Nothobranchius Furzeri’. Aging Cell 14(5):857–66. https://doi.org/10.1111/acel.12367
- ↑ Matsui, Hideaki, Naoya Kenmochi, and Kazuhiko Namikawa. 2019. ‘Age- and α-Synuclein-Dependent Degeneration of Dopamine and Noradrenaline Neurons in the Annual Killifish Nothobranchius Furzeri’. Cell Reports 26(7):1727-1733.e6. https://doi.org/10.1016/j.celrep.2019.01.015
- ↑ 21.0 21.1 21.2 Hartmann, N., Reichwald, K., Lechel, A., Graf, M., Kirschner, J., Dorn, A., Terzibasi, E., Wellner, J., Platzer, M., Rudolph, K. L., Cellerino, A., & Englert, C. (2009). Telomeres shorten while Tert expression increases during ageing of the short-lived fish Nothobranchius furzeri. Mechanisms of Ageing and Development, 130(5), 290–296. https://doi.org/10.1016/j.mad.2009.01.003
- ↑ Brunk, U. T., Jones, C. B., & Sohal, R. S. (1992). A novel hypothesis of lipofuscinogenesis and cellular aging based on interactions between oxidative stress and autophagocytosis. Mutation Research, 275(3–6), 395–403. https://doi.org/10.1016/0921-8734(92)90042-n
- ↑ 23.0 23.1 Ahuja et al. (2019). Loss of genomic integrity induced by lysosphingolipid imbalance drives ageing in the heart. EMBO Reports, 20(4), e47407. https://doi.org/10.15252/embr.201847407
- ↑ 24.0 24.1 24.2 Hartmann, N., Reichwald, K., Wittig, I., Dröse, S., Schmeisser, S., Lück, C., Hahn, C., Graf, M., Gausmann, U., Terzibasi, E., Cellerino, A., Ristow, M., Brandt, U., Platzer, M., & Englert, C. (2011). Mitochondrial DNA copy number and function decrease with age in the short-lived fish Nothobranchius furzeri. Aging Cell, 10(5), 824–831. https://doi.org/10.1111/j.1474-9726.2011.00723.x
- ↑ Dimri, G. P., Lee, X., Basile, G., Acosta, M., Scott, G., Roskelley, C., Medrano, E. E., Linskens, M., Rubelj, I., & Pereira-Smith, O. (1995). A biomarker that identifies senescent human cells in culture and in aging skin in vivo. Proceedings of the National Academy of Sciences of the United States of America, 92(20), 9363–9367. https://doi.org/10.1073%2Fpnas.92.20.9363
- ↑ Hu, C., & Brunet, A. (2018). The African turquoise killifish: A research organism to study vertebrate aging and diapause. Aging Cell, 17(3), e12757. doi: 10.1111/acel.12757
- ↑ 27.0 27.1 Vanhunsel, S., Bergmans, S., Beckers, A., Etienne, I., Van houcke, J., & Seuntjens, E. et al. (2021). The killifish visual system as an in vivo model to study brain aging and rejuvenation. Npj Aging And Mechanisms Of Disease, 7(1). doi: 10.1038/s41514-021-00077-4
- ↑ 28.0 28.1 Harel, I., Benayoun, B., Machado, B., Singh, P., Hu, C., & Pech, M. et al. (2015). A Platform for Rapid Exploration of Aging and Diseases in a Naturally Short-Lived Vertebrate. Cell, 160(5), 1013-1026. doi: 10.1016/j.cell.2015.01.038
- ↑ Bourque, G., Burns, K., Gehring, M., Gorbunova, V., Seluanov, A., & Hammell, M. et al. (2018). Ten things you should know about transposable elements. Genome Biology, 19(1). doi: 10.1186/s13059-018-1577-z
- ↑ Valenzano, D., Sharp, S., & Brunet, A. (2011). Transposon-Mediated Transgenesis in the Short-Lived African KillifishNothobranchius furzeri, a Vertebrate Model for Aging. G3 Genes|Genomes|Genetics, 1(7), 531-538. doi: 10.1534/g3.111.001271
- ↑ Harel, I., Valenzano, D., & Brunet, A. (2016). Efficient genome engineering approaches for the short-lived African turquoise killifish. Nature Protocols, 11(10), 2010-2028. doi: 10.1038/nprot.2016.103
- ↑ Platzer, M., & Englert, C. (2016). Nothobranchius furzeri: A Model for Aging Research and More. Trends In Genetics, 32(9), 543-552. doi: 10.1016/j.tig.2016.06.006
- ↑ Glasauer, S., & Neuhauss, S. (2014). Whole-genome duplication in teleost fishes and its evolutionary consequences. Molecular Genetics And Genomics, 289(6), 1045-1060. doi: 10.1007/s00438-014-0889-2
- ↑ Dehal, P., & Boore, J. (2005). Two Rounds of Whole Genome Duplication in the Ancestral Vertebrate. Plos Biology, 3(10), e314. doi: 10.1371/journal.pbio.0030314
- ↑ Dodzian, J., Kean, S., Seidel, J., & Valenzano, D. (2018). A Protocol for Laboratory Housing of Turquoise Killifish (<em>Nothobranchius furzeri</em>). Journal Of Visualized Experiments, (134). doi: 10.3791/57073