Nothobranchius Furzeri Research Articles

Generation-specific and interactive effects of pesticide and antidepressant exposure in a fish model call for multi-stressor and multigenerational testing

Ecological risks of a pollutant are typically assessed via short-term exposure of model organisms to that single compound. Such tests are informative, but cannot ascertain effects of long-term and multigenerational mixed-stressor exposure with which organisms are often confronted in their natural environment. Therefore, full life-cycle and multigenerational tests are needed. Yet, these are hampered due to long lifespans and generation times of many standard laboratory species, in particular for vertebrates such as fish. With a typical lifespan of 6 months and a generation time of about 3 months, the turquoise killifish (Nothobranchius furzeri) may be an ideal model for multigenerational testing. In this study, we assessed the impact of full life-cycle exposure to the emerging pollutant fluoxetine (0, 0.5 μg/L) in combination with chronic exposure during adulthood to the pesticide 3,4-dichloroaniline (0, 50, 100 μg/L) over two successive generations of N. furzeri. Overall, both life-history and behaviour were affected by exposure to fluoxetine and 3,4-DCA. Inhibitory effects of single chemical exposure on growth and fecundity were generation-dependent, while enhanced swimming acceleration and feeding in response to fluoxetine were dependent on the presence of 3,4-DCA. Together, these findings show the relevance of a multi-stressor approach across successive generations. Although full life-cycle and multigenerational tests are typically assumed to be impractical and costly for fish, we deliver an effective demonstration that such studies are possible within a timespan of less than 6 months with the killifish N. furzeri as a model organism.

Development of the African Killifish as a New Model to Study Aging and Suspended animation

We have pioneered a new model organism for aging research, the naturally short-lived African killifish Nothobranchius furzeri. The African killifish lives in ephemeral pools of water in Africa, and has evolved a short life cycle adapted to this habitat. Its embryos can also resist drought until the next wet season in a state of ‘suspended life’. In laboratory conditions, the African killifish has a maximal lifespan of about 4-6 months, and is, so far, the shortest-lived vertebrate that can be bred in captivity. We have successfully transformed this natural short-lived vertebrate into a usable model organism for aging research, including de novo assembly of the genome and CRISPR-Cas9 mediated genome-editing. Our goal is to use this model to discover new principles underlying aging, longevity, and ‘suspended animation’ in vertebrates.

Id(entifying) the inhibitor of DNA binding 3 in the brain of Nothobranchius furzeri upon aging.

Inhibitors of DNA (Id) are key transcription factors (TFs) regulating neurogenic processes. They belong to the helix-loop-helix (HLH) TF family and are dominant negative regulators of basic HLH proteins (bHLHs). Specifically, they inhibit cell differentiation and enhance cell proliferation and motility. The Id family includes four members, Id1, Id2, Id3, and Id4, which have been identified in nearly all vertebrates. The transcript catalog of the African turquoise killifish, Nothobranchius furzeri, contains all four TFs and has evolved showing positive selection for Id3. N. furzeri, a teleost, is the short-lived vertebrate and is gaining increasing scientific interest as a new model organism in aging research. It is characterized by embryonic diapause, explosive sexual maturation, and rapid aging. In this study, we investigated both the expression and the role of Id3 in the brain of this model organism. Interestingly, Id3 was upregulated age-dependently along with a distribution pattern resembling that of other vertebrates. Additionally, the gene has undergone positive selection during evolution and shows a high degree of conservation relative to that of other vertebrates. These features make N. furzeri a valid tool for aging studies and a potential model in translational research.

NETO2 Is Deregulated in Breast, Prostate, and Colorectal Cancer and Participates in Cellular Signaling.

The NETO2 gene (neuropilin and tolloid-like 2) encodes a protein that acts as an accessory subunit of kainate receptors and is predominantly expressed in the brain. Upregulation of NETO2 has been observed in several tumors; however, its role in tumorigenesis remains unclear. In this study, we investigated NETO2 expression in breast, prostate, and colorectal cancer using quantitative PCR (qPCR), as well as the effect of shRNA-mediated NETO2 silencing on transcriptome changes in colorectal cancer cells. In the investigated tumors, we observed both increased and decreased NETO2 mRNA levels, presenting no correlation with the main clinicopathological characteristics. In HCT116 cells, NETO2 knockdown resulted in the differential expression of 17 genes and 2 long non-coding RNAs (lncRNAs), associated with the upregulation of circadian rhythm and downregulation of several cancer-associated pathways, including Wnt, transforming growth factor (TGF)-β, Janus kinase (JAK)-signal transducer and activator of transcription (STAT), mitogen-activated protein kinase (MAPK), and phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) pathways. Furthermore, we demonstrated the possibility to utilize a novel model organism, short-lived fish Nothobranchius furzeri, for evaluating NETO2 functions. The ortholog neto2b in N. furzeri demonstrated a high similarity in nucleotide and amino acid sequences with human NETO2, as well as was characterized by stable expression in various fish tissues. Collectively, our findings demonstrate the deregulation of NETO2 in the breast, prostate, and colorectal cancer and its participation in the tumor development primarily through cellular signaling.

The core circadian component, Bmal1, is maintained in the pineal gland of old killifish brain.

SummaryCircadian rhythm is altered during aging, although the underlying molecular mechanisms remain largely unknown. Here, we used the turquoise killifish as a short-lived vertebrate model to examine the effects of aging on the major circadian network comprising the four mammalian clock protein homologs, Bmal1, Clockb, Cry1b, and Per3, which are highly conserved in the killifish with 50%–85% amino acid sequence identity to their human counterparts. The amplitude of circadian rhythm was smaller in old fish (14 weeks) than in young fish (6 weeks). In old fish brain, the Bmal1 protein level was significantly downregulated. However, the Bmal1 interaction with Clockb and chromatin binding of Bmal1 to its downstream target promoters were retained. Furthermore, Bmal1 was relatively well maintained in the pineal gland compared with other regions of the old fish brain. The results suggest that the circadian clock system in the killifish becomes spatially confined to the pineal gland upon aging.

Nothobranchius furzeri as an emerging model for mate choice: female choice revealed by animations

Abstract The African turquoise killifish (Nothobranchius furzeri) is the shortest-lived vertebrate research model. It is also sexually dimorphic, making it suitable for studying sexual selection. We take advantage of a natural tail colour polymorphism in males and investigate female responses to computer animations of males that differ in this phenotype. Our findings indicate that GRZ (Gonarezhou) females prefer animated males with traits specific to their strain (a yellow tail with a black band) compared to males exhibiting traits from another strain of the same species (a red tail). When females were simultaneously shown animations of both males, they spent significantly more time on the side of the tank where the yellow-tailed animation was visible, and significantly more time interacting with the yellow-tailed animation. Given these repeatable responses and the availability of genomic resources, N. furzeri represents an excellent, untapped model for studying the genetic basis of preferences and reproductive behaviours.

Reproductive senescence in a short-lived fish.

Reproductive senescence is an age-associated decline in reproductive performance, which often arises as a trade-off between current and future reproduction. Given that mortality is inevitable, increased allocation into current reproduction is favoured despite costs paid later in life. This assumption is violated in organisms with post-maturity growth whose reproductive output increases long after maturity. While reproductive senescence is frequently studied in animals with determinate growth at maturity, such as insects or mammals, we have very limited understanding of reproductive senescence in organisms with an extensive post-maturity growth period. The fact that many post-maturity growers experience strong adult mortality leads to conflicting expectations for reproductive senescence. The aim of this study was to investigate how co-occurrence of rapid life history and post-maturity growth mould reproductive senescence in a short-lived killifish, Nothobranchius furzeri, using longitudinal data on laboratory and wild-type populations. We followed the individual fecundity, fertility and fertilization of 132 singly housed fish from the perspectives of chronological and biological age. At the onset of senescence, the sex-specific contribution to decrease in fertilization capacity was investigated. Allocation trade-offs were estimated through the association between reproductive parameters and life span, and between early-life and late-life fecundity. We demonstrate that female fecundity increased steadily after maturity and reproductive senescence occurred long after the growth asymptote. The prime age for fecundity coincided with 50% female survival and consequent decline in fecundity implies an association with somatic deterioration. Reproductive senescence in fertilization rate was stronger in females than in males. Females with high early fecundity experienced a long life span and high late-life fecundity, discounting the role of allocation trade-offs in reproductive senescence. The present study reports a clear case of reproductive senescence in a fish with a long post-maturation growth period, unusually rapid development and short life span. The onset of reproductive senescence was postponed compared to animals that cease growing at sexual maturity. Fish and other animals with post-maturity growth have long been considered insusceptible to ageing but this conclusion may be related to the previous lack of longitudinal data rather than to the absence of reproductive senescence in such organisms.

Conspecific density and environmental complexity impact behaviour of turquoise killifish (Nothobranchius furzeri).

Fish models are essential for research in many biological and medical disciplines. With a typical lifespan of only 6 months, the Turquoise killifish (Nothobranchius furzeri) was recently established as a time- and cost-efficient model to facilitate whole-life and multigenerational studies in several research fields, including behavioural ecotoxicology. Essential information on the behavioural norm and on how laboratory conditions affect behaviour, however, is deficient. In the current study, we examined the impact of the social and structural environment on a broad spectrum of behavioural endpoints in N. furzeri. While structural enrichment affected only fish boldness and exploratory behaviour, fish rearing density affected the total body length, locomotor activity, boldness, aggressiveness and feeding behaviour of N. furzeri individuals. Overall, these results contribute to compiling a behavioural baseline for N. furzeri that increases the applicability of this new model species. Furthermore, our findings will fuel the development of improved husbandry protocols to maximize the welfare of N. furzeri in a laboratory setting.

Intra-species differences in population size shape life history and genome evolution.

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.

Fluctuating temperatures extend median lifespan, improve reproduction and reduce growth in turquoise killifish.

In natural populations, individuals experience daily fluctuations in environmental conditions that synchronise endogenous biorhythms. Artificial alterations of environmental fluctuations can have negative consequences for life history traits, including lifespan. In laboratory studies of aging, the role of fluctuating temperature is usually overlooked and we know little of how thermal fluctuation modulates senescence in vertebrates. In this longitudinal study we followed individually-housed turquoise killifish, Nothobranchius furzeri, from two thermal regimes; ecologically relevant diel fluctuations (20 °C - 35 °C) and stable temperature (27.5 °C), and compared their survival, growth and reproduction. Fish experiencing fluctuating temperatures had a longer median lifespan but reached smaller asymptotic body size. Within-treatment variation indicated that extended lifespan in fluctuating temperatures was not causally linked to decreased growth rate or smaller body size, but occurred solely due to the effect of thermal fluctuations. Male body size was positively associated with lifespan in stable temperatures but this relationship was disrupted in fluctuating thermal regimes. Females exposed to fluctuating temperatures effectively compensated egg production for their smaller size. Thus, there was no difference in absolute fecundity between thermal regimes and body-size corrected fecundity was higher in females in fluctuating temperatures. Overall, despite a brief exposure to sub-optimal thermal conditions during fluctuations, fluctuating temperature had a positive effect on survival and reproduction. These results suggest that the expression of life history traits and their associations under stable temperatures are a poor representation of the relationships obtained from ecologically relevant thermal fluctuations.

Comparative analysis of metabolic and transcriptomic features of Nothobranchius furzeri.

Some species have a longer lifespan than others, but usually lifespan is correlated with typical body weight. Here, we study the lifetime evolution of the metabolic behaviour of Nothobranchius furzeri, a killifish with an extremely short lifespan with respect to other fishes, even when taking into account rescaling by body weight. Comparison of the gene expression patterns of N. furzeri with those of zebrafish Danio rerio and mouse (Mus musculus) shows that a broad set of metabolic genes and pathways are affected in N. furzeri during ageing in a way that is consistent with a global deregulation of chromatin. Computational analysis of the glycolysis pathway for the three species highlights a rapid increase in the metabolic activity during the lifetime of N. furzeri with respect to the other species. Our results highlight that the unusually short lifespan of N. furzeri is associated with peculiar patterns in the metabolic activities and in chromatin dynamics.

Antidepressant exposure reduces body size, increases fecundity and alters social behavior in the short-lived killifish Nothobranchius furzeri

Social and mating behavior are fundamental fitness determinants in fish. Although fish are increasingly exposed to pharmaceutical compounds that may alter expression of such behavior, potential effects are understudied. Here, we examine the impact of lifelong exposure to two concentrations (0.7 and 5.3 μg/L) of the antidepressant fluoxetine on fecundity and social behavior (i.e. sociability and male-male aggression) in the turquoise killifish, Nothobranchius furzeri. When exposed to the highest concentration of fluoxetine (5.3 μg/L), fish were smaller at maturation but they more frequently engaged in mating. In addition, in both fluoxetine treatments females roughly doubled their overall fecundity while egg fertilization rates were the same for exposed and unexposed fish. Although aggression of male fish was not impacted by fluoxetine exposure, exposed male fish (5.3 μg/L) spent more time in the proximity of a group of conspecifics, which implies an increased sociability in these individuals. Overall, the results of this study indicate that exposure to fluoxetine may result in disrupted male sociability, increased mating frequency and an increased reproductive output in fish populations.

Cholinergic System and NGF Receptors: Insights from the Brain of the Short-Lived Fish Nothobranchius furzeri.

Nerve growth factor (NGF) receptors are evolutionary conserved molecules, and in mammals are considered necessary for ensuring the survival of cholinergic neurons. The age-dependent regulation of NTRK1/NTRKA and p75/NGFR in mammalian brain results in a reduced response of the cholinergic neurons to neurotrophic factors and is thought to play a role in the pathogenesis of neurodegenerative diseases. Here, we study the age-dependent expression of NGF receptors (NTRK1/NTRKA and p75/NGFR) in the brain of the short-lived teleost fish Nothobranchius furzeri. We observed that NTRK1/NTRKA is more expressed than p75/NGFR in young and old animals, although both receptors do not show a significant age-dependent change. We then study the neuroanatomical organization of the cholinergic system, observing that cholinergic fibers project over the entire neuroaxis while cholinergic neurons appear restricted to few nuclei situated in the equivalent of mammalian subpallium, preoptic area and rostral reticular formation. Finally, our experiments do not confirm that NTRK1/NTRKA and p75/NGFR are expressed in cholinergic neuronal populations in the adult brain of N. furzeri. To our knowledge, this is the first study where NGF receptors have been analyzed in relation to the cholinergic system in a fish species along with their age-dependent modulation. We observed differences between mammals and fish, which make the African turquoise killifish an attractive model to further investigate the fish specific NGF receptors regulation.

Good performance of turquoise killifish (Nothobranchius furzeri) on pelleted diet as a step towards husbandry standardization

Dietary alteration is one of the most universally effective aging interventions, making its standardization a fundamental need for model organisms in aging. In this dietetic study we address the current lack of standardized formulated diet for turquoise killifish Nothobranchius furzeri – a promising model organism. We first demonstrated that N. furzeri can be fully weaned at the onset of puberty onto a commercially available pelleted diet as the sole nutrition when kept in social tanks. We then compared nine somatic and six reproductive parameters between fish fed a typical laboratory diet - frozen chironomid larvae (bloodworms) and fish weaned from bloodworms to BioMar pellets. Both dietary groups had comparable somatic and reproductive performance. There was no difference between diet groups in adult body size, specific growth rate, condition or extent of hepatocellular vacuolation. Fish fed a pelleted diet had higher juvenile body mass and more visceral fat. Pellet-fed males had lower liver mass and possessed a lipid type of hepatocellular vacuolation instead of the prevailing glycogen-like vacuolation in the bloodworm-fed group. No considerable effect was found on reproductive parameters. The negligible differences between dietary groups and good acceptance of pellets indicate their suitability as a useful starting point for the development of standardized diet for Nothobranchius furzeri.

Reduced proteasome activity in the aging brain results in ribosome stoichiometry loss and aggregation

A progressive loss of protein homeostasis is characteristic of aging and a driver of neurodegeneration. To investigate this process quantitatively, we characterized proteome dynamics during brain aging in the short‐lived vertebrate Nothobranchius furzeri combining transcriptomics and proteomics. We detected a progressive reduction in the correlation between protein and mRNA, mainly due to post‐transcriptional mechanisms that account for over 40% of the age‐regulated proteins. These changes cause a progressive loss of stoichiometry in several protein complexes, including ribosomes, which show impaired assembly/disassembly and are enriched in protein aggregates in old brains. Mechanistically, we show that reduction of proteasome activity is an early event during brain aging and is sufficient to induce proteomic signatures of aging and loss of stoichiometry in vivo. Using longitudinal transcriptomic data, we show that the magnitude of early life decline in proteasome levels is a major risk factor for mortality. Our work defines causative events in the aging process that can be targeted to prevent loss of protein homeostasis and delay the onset of age‐related neurodegeneration.

The Core Circadian Component, BMAL1, is Maintained Preferentially in the Pineal Gland of Aged Killifish Brain

Circadian rhythm controls various physiological aspects for an organism to adapt to the daily repeated changes of environments. As an organism ages, a circadian rhythm is altered, yet the underlying molecular mechanism remain largely elusive. Here, we revealed, using the short-living vertebrate model, the turquoise killifish, how organismal aging alters the major circadian clock components and their spatial distribution in the brain. Old fish showed faster dampening of the free-running rhythm in swimming behavior than young fish. The regulatory modes of circadian components were distinctive in the turquoise killifish; the protein level of BMAL but not CLOCK was highly reduced. Both BMAL1 and CLOCK proteins were detected in the whole brain of young fish but they are partially co-localized. Upon aging, BMAL1 was preferentially maintained in the pineal gland than. Thus, aging is associated with not just the reduced level but also the spatial confinement of a core circadian component in the pineal gland of the turquoise killifish brain.

Extreme genomic volatility characterizes the evolution of the immunoglobulin heavy chain locus in cyprinodontiform fishes.

The evolution of the adaptive immune system has provided vertebrates with a uniquely sophisticated immune toolkit, enabling them to mount precise immune responses against a staggeringly diverse range of antigens. Like other vertebrates, teleost fishes possess a complex and functional adaptive immune system; however, our knowledge of the complex antigen-receptor genes underlying its functionality has been restricted to a small number of experimental and agricultural species, preventing systematic investigation into how these crucial gene loci evolve. Here, we analyse the genomic structure of the immunoglobulin heavy chain (IGH) gene loci in the cyprinodontiforms, a diverse and important group of teleosts present in many different habitats across the world. We reconstruct the complete IGH loci of the turquoise killifish (Nothobranchius furzeri) and the southern platyfish (Xiphophorus maculatus) and analyse their in vivo gene expression, revealing the presence of species-specific splice isoforms of transmembrane IGHM. We further characterize the IGH constant regions of 10 additional cyprinodontiform species, including guppy, Amazon molly, mummichog and mangrove killifish. Phylogenetic analysis of these constant regions suggests multiple independent rounds of duplication and deletion of the teleost-specific antibody class IGHZ in the cyprinodontiform lineage, demonstrating the extreme volatility of IGH evolution. Focusing on the cyprinodontiforms as a model taxon for comparative evolutionary immunology, this work provides novel genomic resources for studying adaptive immunity and sheds light on the evolutionary history of the adaptive immune system.

Risk-taking behaviour in African killifish – a case of parasitic manipulation?

Parasites commonly manipulate host behaviour to increase transmission success between hosts. While most behavioural changes comprise slight alterations to host activity patterns and habitat use, some represent impressive alterations to routine behaviour which, while having direct positive effects on parasite transmission, compromise host survival. Here, we report conspicuous risky behaviour in an African annual killifish, Nothobranchius furzeri, infected by metacercariae of a strigeid trematode, Apatemon sp., residing in their cranial cavity. We demonstrate a striking contrast in the spatial and temporal behavioural responses of fish from populations naturally infected with Apatemon sp. and fish from two control populations with either a similar baseline parasite fauna but lacking Apatemon, or an overall low-level of infection. During routine activity, fish from Apatemon-infected populations positioned themselves just below the water surface, while other fish spent most of their time near the bottom. During a simulated avian attack, killifish from Apatemon-infected populations jumped above the water surface, moved in an uncoordinated manner, and rotated in the upper water layer, while fish from the control populations rapidly escaped into deeper water and ceased moving. The same self-exposing behaviour (jumping out of the water and lying on floating lily pads for extended periods) was also observed under natural conditions. Such behaviour greatly facilitates location of Apatemon-infected host fish by avian definitive hosts, especially in turbid pools. Moreover, the nothobranchiid killifish host's own life history, i.e. an extremely short lifespan limited to several months, may represent an important driver in the evolution of behavioural manipulation.

Optimization of mass spectrometry settings for steroidomic analysis in young and old killifish

Steroids are essential structural components of cell membranes that organize lipid rafts and modulate membrane fluidity. They can also act as signalling molecules that work through nuclear and G protein–coupled receptors to impact health and disease. Notably, changes in steroid levels have been implicated in metabolic, cardiovascular and neurodegenerative diseases, but how alterations in the steroid pool affect ageing is less well understood. One of the major challenges in steroidomic analysis is the ability to simultaneously detect and distinguish various steroids due to low in vivo concentrations and naturally occurring stereoisomers. Here, we established such a method to study the mass spectrometry behaviour of nine sterols/steroids and related molecules (cholesterol precursors: squalene, lanosterol; sterol metabolites; 7 Dehydrocholesterol, 24, 25 and 27 Hydroxycholesterol; and steroids: progesterone, testosterone, and corticosterone) during ageing in the African turquoise killifish, a new model for studying vertebrate longevity. We find that levels of all tested steroids change significantly with age in multiple tissues, suggesting that specific steroids could be used as biomarkers of ageing. These findings pave the way for use of Nothobranchius furzeri as a novel model organism to unravel the role of sterols/steroids in ageing and age-related diseases.Graphical abstract

Nothobranchius furzeri (African Turquoise Killifish)

The African turquoise killifish (Nothobranchius furzeri) is the shortest-lived vertebrate known to science and is characterized by embryonic diapause, explosive sexual maturation and rapid aging. Thanks to the ease of laboratory husbandry and amenability to genetic manipulation, this species is emerging as a new model organism for aging and biomedical research. Sequence variation is associated with natural variations of lifespan in different populations of turquoise killifish and other closely related killifish species.