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Nothobranchius furzeri is an important new model organism. This Protocol aims to help laboratories establish healthy breeding colonies of this species and to standardize husbandry methods, which differ from those for other model fish because of dry incubation of the eggs. 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.

Gut bacteria occupy the interface between the organism and the external environment, contributing to homeostasis and disease. Yet, the causal role of the gut microbiota during host aging is largely unexplored. Here, using the African turquoise killifish (Nothobranchius furzeri), a naturally short-lived vertebrate, we show that the gut microbiota plays a key role in modulating vertebrate life span. Recolonizing the gut of middle-age individuals with bacteria from young donors resulted in life span extension and delayed behavioral decline. This intervention prevented the decrease in microbial diversity associated with host aging and maintained a young-like gut bacterial community, characterized by overrepresentation of the key genera Exiguobacterium, Planococcus, Propionigenium and Psychrobacter. Our findings demonstrate that the natural microbial gut community of young individuals can causally induce long-lasting beneficial systemic effects that lead to life span extension in a vertebrate model. Our bodies are home to lots of microorganisms, many of which are found throughout the gut. Gut microbes play important roles in human health, where they cooperate with our own cells to develop the immune system, synthesize essential vitamins, and help to absorb nutrients. When the cooperation between our own cells and the gut microbes fails, the microbial community within the gut can become a source of infection, sometimes leading to life-threatening diseases. Healthy individuals typically have many different types gut microbes, whereas people with poor health, or older individuals, will often have less diverse and a higher percentage of disease-causing microbes. For example, African turquoise killifish only live a few months, during which the composition of their gut microbes undergoes dramatic changes. While young fish harbor highly diverse microbial communities, older fish have less diverse communities and more microbes associated with disease. Until now, it was not known whether manipulating the gut composition could affect the aging process. By using the killifish as a model for their study, Smith et al. revealed that gut microbes affect how the fish survived and aged. When the guts of middle-aged fish were colonized with microbes transferred from younger fish, the older fish lived longer and were more active later in life. These fish also maintained a more diverse microbial community throughout their adulthood and shared key microbes with young fish – possibly associated with the improved health benefits. These results suggest that controlling the composition of the gut microbes can improve health and increase life span. The model system used in this study could provide new ways to manipulate the gut microbial community and gain key insights into how the gut microbes affect aging. Manipulating gut microbes to resemble a community found in young individuals could be a strategy to delay the onset of age-related diseases.

Intra-sexual variation in coloration is often linked to comprehensive differences in reproductive behavior and life histories. Both males and females can express intrapopulation variation in coloration, and facultative hermaphrodism adds further complexity to the social and reproductive system. In replicated experimental tanks, we studied social interactions and reproductive behavior in males, females and sex-changing (transitional) intermediate individuals of the Mexican annual killifish, Millerichthys robustus , a facultative protogynous hermaphrodite with distinct intraspecific variation in male coloration (red, orange and yellow morphs) and female ornamentation (variable number of melanin-based ocelli). Territoriality was expressed in males and females but not in intermediates. Among males, the yellow morph was competitively inferior, but males of any morph were capable of establishing territories. Red and orange males possessed territories near the substrate. The territories of yellow males were located near the water surface. Males of all morphs also engaged in sneaking behavior and attempted to mate with females outside their own territory. Females with more ocelli were more aggressive and more likely to become dominant but were not courted more often by males. Intermediates rarely engaged in any reproductive behavior. Overall, we demonstrate differences in social and reproductive behavior corresponding to male and female coloration and associate them with variation in social and reproductive niches.

The turquoise killifish, Nothobranchius furzeri, is a promising vertebrate model in ageing research and an emerging model organism in genomics, regenerative medicine, developmental biology and ecotoxicology. Its lifestyle is adapted to the ephemeral nature of shallow pools on the African savannah. Its rapid and short active life commences when rains fill the pool: fish hatch, grow rapidly and mature in as few as two weeks, and then reproduce daily until the pool dries out. Its embryos then become inactive, encased in the dry sediment and protected from the harsh environment until the rains return. This invertebrate-like life cycle (short active phase and long developmental arrest) combined with a vertebrate body plan provide the ideal attributes for a laboratory animal.

This study presents, for the first time, a comprehensive dataset that documents the range of inter- and intraspecific otolith variation in aplocheiloid killifish, based on a total of 86 individuals representing five extant species of Nothobranchius PETERS, 1868, from East Africa: the sympatric pairs N. rubripinnis SEEGERS, 1986 and N. ruudwildekampi COSTA, 2009 (Eastern Tanzania), and N. orthonotus (PETERS, 1844) and N. furzeri JUBB, 1971 (Southern Mozambique), and two isolated populations of N. korthausae MEINKEN, 1973 (Eastern Tanzania). Otolith characters were analysed based on SEM images, and otolith morphometry was conducted using uni- and multivariate statistics. Two ancient clades of probably Early to Middle Miocene age in eastern Tanzania and southern Mozambique can be recognized based on otolith morphologies, which is consistent with previous work based on molecular data. The distinctive sulcus morphologies in the otoliths of sympatric species may be linked to species-specific hearing capabilities, perhaps constituting a case of character displacement in an area of secondary sympatry. The otoliths of the studied species of Nothobranchius are diagnostic at the species level, even in the case of closely related species diagnosable otherwise only by minor differences in coloration. The two populations of N. korthausae also displayed some differences in their otolith characters. The new data may facilitate future recognition of fossil species of Nothobranchius. As no fossil remains of extant aplocheiloid killifishes have yet been described, the discovery of fossil otoliths of Nothobranchius would significantly advance understanding of the evolutionary history of this interesting group of fishes.

The evolutionary forces shaping life history divergence within species are largely unknown. Turquoise killifish display differences in lifespan among wild populations, representing an ideal natural experiment in evolution and diversification of life history. By combining genome sequencing and population genetics, we investigate the evolutionary forces shaping lifespan among wild turquoise killifish populations. We generate an improved reference genome assembly and identify genes under positive and purifying selection, as well as those evolving neutrally. Short-lived populations from the outer margin of the species range have small population size and accumulate deleterious mutations in genes significantly enriched in the WNT signaling pathway, neurodegeneration, cancer and the mTOR pathway. We propose that limited population size due to habitat fragmentation and repeated population bottlenecks, by increasing the genome-wide mutation load, exacerbates the effects of mutation accumulation and cumulatively contribute to the short adult lifespan.

The ability to attract mates, acquire resources for reproduction, and successfully outcompete rivals for fertilizations may make demands on cognitive traits—the mechanisms by which an animal acquires, processes, stores and acts upon information from its environment. Consequently, cognitive traits potentially undergo sexual selection in some mating systems. We investigated the role of cognitive traits on the reproductive performance of male rose bitterling (Rhodeus ocellatus), a freshwater fish with a complex mating system and alternative mating tactics. We quantified the learning accuracy of males and females in a spatial learning task and scored them for learning accuracy. Males were subsequently allowed to play the roles of a guarder and a sneaker in competitive mating trials, with reproductive success measured using paternity analysis. We detected a significant interaction between male mating role and learning accuracy on reproductive success, with the best-performing males in maze trials showing greater reproductive success in a sneaker role than as a guarder. Using a cross-classified breeding design, learning accuracy was demonstrated to be heritable, with significant additive maternal and paternal effects. Our results imply that male cognitive traits may undergo intra-sexual selection.

Brood parasites are involved in coevolutionary arms races with their hosts, whereby adaptations of one partner elicit the rapid evolution of counter-adaptations in the other partner. Hosts can also mitigate fitness costs of brood parasitism by learning from individual or social experience. In brood parasites, however, the role of learning can be obscured by their stealthy behaviour. Cuckoo catfish ( Synodontis multipunctatus ) parasitise clutches of mouthbrooding cichlids in Lake Tanganyika and are the only non-avian obligate brood parasites among vertebrates. We experimentally demonstrate that cuckoo catfish greatly enhance their efficiency in parasitising their hosts as they learn to overcome host defences. With increasing experience, cuckoo catfish increased their parasitism success by greater efficiency through improved timing and coordination of intrusions of host spawnings. Hence, within the coevolutionary arms races, brood parasites learn to overcome host defences during their lifetime. The importance of learning for brood parasites is explored using cuckoo catfish. The catfish increase their parasitism success as they gain experience, mainly by improving their social coordination and timing of intrusions to cichlid host spawnings.

Facilitative and competitive interactions among coexisting parasite species, as well as among alternative host species, produce considerable ecological and evolutionary responses to host-parasite relationships. Such effects can be illuminated by impacts of non-native species on relationships in local communities. We used the association between parasitic European bitterling fish ( Rhodeus amarus ) and unionid mussels (which host bitterling eggs in their gills) to test the effects of the invasive Chinese pond mussel ( Sinanodonta woodiana ) and the presence of non-bitterling mussel macroparasites on the pattern of host mussel use by the bitterling across 12 unionid mussel communities with the absence or presence of S. woodiana (and variation in duration of coexistence with local species). While all six European mussel species were used by the bitterling (with the prevalence of > 30% in Unio spp.), no S. woodiana individual was infected by the bitterling. The presence of S. woodiana did not affect bitterling eggs distribution in native mussels. Large native mussels hosted more bitterling. Infection by non-bitterling parasites, mostly water mites (prevalence 47%) and trematodes (25%), did not affect rates of bitterling parasitism. We discuss our results in the context of the rapid evolution of non-native species in their new range and its implication on mussel conservation.

The natural history of model organisms is often overlooked despite its importance to correctly interpret the outcome of laboratory studies. Ageing is particularly understudied in natural populations. To address this gap, we present lifetime demographic data from wild populations of an annual species, the turquoise killifish, Nothobranchius furzeri , a model species in ageing research, and two other species of coexisting annual killifishes. Annual killifish hatch synchronously, have non-overlapping generations, and reproduce daily after reaching sexual maturity. Data from 13 isolated savanna pools in southern Mozambique demonstrate that the pools supporting killifish populations desiccated 1–4 months after their filling, though some pools persisted longer. Declines in population size over the season were stronger than predicted, because they exceeded the effect of steady habitat shrinking on population density that, contrary to the prediction, decreased. Populations of N. furzeri also became more female-biased with progressing season suggesting that males had lower survival. Nothobranchius community composition did not significantly vary across the season. Our data clearly demonstrate that natural populations of N. furzeri and its congeners suffer strong mortality throughout their lives, with apparent selective disappearance (condition-dependent mortality) at the individual level. This represents selective force that can shape the evolution of lifespan, and its variation across populations, beyond the effects of the gradient in habitat persistence.