Evidence for selfing in a vertebrate from whole-genome sequencing.

A growing number of recent genomic studies report asexual parthenogenetic reproduction in a wide range of taxa, including vertebrate species from the reptile, bird, and fish lineages. Yet, self-fertilization (selfing) has been recorded only in a single vertebrate, the mangrove killifish Kryptolebias marmoratus In cichlid fishes, sex determination is notably diverse and can be influenced by the environment, and sequential hermaphroditism has been reported for some species. Here, we present evidence for a case of facultative selfing in the cichlid fish Benitochromis nigrodorsalis, which is otherwise known as biparentally reproducing ovophilic mouthbrooder from Western Africa. Our laboratory observations revealed that a wild-caught individual produced repeatedly viable offspring in absence of a mating partner. By analyzing genome-wide single-nucleotide polymorphism (SNP) data, we compare that individual and two of its offspring to shed light on its reproductive mode. First, our results confirm uniparental reproduction. Second, overall heterozygosity is reduced in the offspring compared with outbred individuals. Retained maternal heterozygosity in the offspring is ∼51%, which is close to the theoretically expected value of a heterozygosity reduction of 50% by selfing. Heterozygosity patterns along individual chromosomes do not point to alternative parthenogenetic reproductive mechanisms like automixis by terminal or central fusion. Facultative selfing may represent an adaptive strategy ensuring reproduction when mating partners are absent and, hence, contribute to the cichlids' enormous evolutionary success.

Salinity and prolactin regulate anoctamin 1 in the model teleost, Fundulus heteroclitus.

To maintain internal ion balance in marine environments, teleost fishes leverage seawater (SW)-type ionocytes to actively secrete Na+ and Cl- into the environment. It is well established that SW-type ionocytes use apically expressed cystic fibrosis transmembrane conductance regulator 1 (Cftr1) as a conduit for Cl- to exit the gill. Here, we investigated whether the Ca2+-activated Cl- channel, anoctamin 1 (Ano1), provides an additional path for Cl--secretion in euryhaline mummichogs (Fundulus heteroclitus). Two ano1 gene isoforms, denoted ano1.1a and -1.1b, exhibited higher expression in the gill and opercular epithelium of mummichogs long-term acclimated to SW versus fresh water (FW). Branchial ano1.1b and cftr1 expression was increased in mummichogs sampled 24 h after transfer from FW to SW; ano1.1a and -1.1b were upregulated in the gill and opercular epithelium following transfer from SW to hypersaline SW. Alternatively, the expression of ano1.1a, -1.1b, and cftr1 in the gill and opercular epithelium was markedly decreased after transfer from SW to FW. Given its role in attenuating ion secretion, we probed whether prolactin downregulates ano1 isoforms. In addition to attenuating cftr1 expression, a prolactin injection reduced branchial ano1.1a and -1.1b levels. Given how Ano1 mediates Cl- secretion by mammalian epithelial cells, the salinity- and prolactin-sensitive nature of ano1 expression reported here indicates that Ano1 may constitute a novel Cl--secretion pathway in ionocytes. This study encourages a wider evaluation of this putative Cl--secretion pathway and its regulation by hormones in teleost fishes.NEW & NOTEWORTHY In this study, we provide evidence in a teleost fish that the Ca2+-activated Cl- channel, anoctamin 1 may provide an additional path for Cl- secretion by seawater-type ionocytes. Not only is this the first report of a Cftr-independent Cl--secreting pathway conferring survival in seawater but also the first description of its regulation by the pituitary hormone prolactin.

Impacts on Atlantic Killifish from Neurotoxicants: Genes, Behavior, and Population-Relevant Outcomes.

Molecular, cellular, and organismal alterations are important descriptors of toxic effects, but our ability to extrapolate and predict ecological risks is limited by the availability of studies that link measurable end points to adverse population relevant outcomes such as cohort survival and growth. In this study, we used laboratory gene expression and behavior data from two populations of Atlantic killifish Fundulus heteroclitus [one reference site (SCOKF) and one PCB-contaminated site (NBHKF)] to inform individual-based models simulating cohort growth and survival from embryonic exposures to environmentally relevant concentrations of neurotoxicants. Methylmercury exposed SCOKF exhibited brain gene expression changes in the si:ch211-186j3.6, si:dkey-21c1.4, scamp1, and klhl6 genes, which coincided with changes in feeding and swimming behaviors, but our models simulated no growth or survival effects of exposures. PCB126-exposed SCOKF had lower physical activity levels coinciding with a general upregulation in nucleic and cellular brain gene sets (BGS) and downregulation in signaling, nucleic, and cellular BGS. The NBHKF, known to be tolerant to PCBs, had altered swimming behaviors that coincided with 98% fewer altered BGS. Our models simulated PCB126 decreased growth in SCOKF and survival in SCOKF and NBHKF. Overall, our study provides a unique demonstration linking molecular and behavioral data to develop quantitative, testable predictions of ecological risk.

Host ecology and biogeography drive parasite community composition in Atlantic killifishes.

Understanding the mechanisms of parasite community assembly can be confounded by phylogenetic distance among host species. Addressing this requires focusing on parasite communities within closely related taxa. Thus, we took a macroecological approach to examining parasite community structure within Killifish species in the genus Fundulus to disentangle the effects of host phylogeny and ecological variables. We constructed a database of parasite communities within Fundulus species from 15 published and unpublished surveys covering the Atlantic coast of the US and Canada. The database was expanded by sampling sites in underrepresented provinces and states, totaling 10 Fundulus species from 57 unique geographic sites. Univariate analysis of observed parasite species richness among Fundulus populations in the dataset found that latitude, climate type, and salinity were the dominant factors determining parasite species richness. Multivariate analysis found that host species and landscape type were the most important factors in determining the similarity of parasite assemblages. Unexpectedly, parasite species richness decreased in low latitudes, and host phylogenetic distance was not found to be a significant factor in the similarity of parasite communities. These results indicate that commonly reported large-scale drivers of parasite community structure, such as latitude and phylogeny, could have diminished significance at the host genus level relative to host ecology, biogeography, and local landscape factors.

Persistent interactive effects of developmental salinity and temperature in Atlantic killifish (Fundulus heteroclitus).

Climate change alters multiple abiotic environmental factors in aquatic environments but relatively little is known about their interacting impacts, particularly in developing organisms where these exposures have the potential to cause long-lasting effects. To explore these issues, we exposed developing killifish embryos (Fundulus heteroclitus) to 26 °C or 20 °C and 20 ppt or 3 ppt salinity in a fully-factorial design. After hatching, fish were transferred to common conditions of 20 °C and 20 ppt to assess the potential for persistent developmental plasticity. Warm temperature increased hatching success and decreased hatch time, whereas low salinity negatively affected hatching success, but this was only significant in fish developed at 20 °C. Temperature, salinity, or their interaction affected mRNA levels of genes typically associated with thermal and hypoxia tolerance (hif1a, hsp90b, hsp90a, hsc70, and hsp70.2) across multiple developmental timepoints. These differences were persistent into the juvenile stage, where the fish that developed at 26 °C had higher expression of hif1a, hsp90b, hsp90a, and hsp70.2 than fish developed at 20 °C, and this was particularly evident for the group developed at both high temperature and salinity. There were also long-lasting effects of developmental treatments on body size after four months of rearing under common conditions. Fish developed at low salinity or temperature were larger than fish developed at high temperature or salinity, but there was no interaction between the two factors. These data highlight the complex nature of the developmental effects of interacting stressors which has important implications for predicting the resilience of fishes in the context of climate change.

Differential DNA methylation and metabolite profiling of Atlantic killifish (Fundulus heteroclitus) from the New Bedford Harbor Superfund site.

Atlantic killifish (Fundulus heteroclitus) is a valuable model in evolutionary toxicology to study how the interactions between genetic and environmental factors serve the adaptive ability of organisms to resist chemical pollution. Killifish populations inhabiting environmental toxicant-contaminated New Bedford Harbor (NBH) show phenotypes tolerant to polychlorinated biphenyls (PCBs) and differences at the transcriptional and genomic levels. However, limited research has explored epigenetic alterations and metabolic effects in NBH killifish. To identify the involvement of epigenetic and metabolic regulation in the adaptive response of killifish, we investigated tissue- and sex-specific differences in global DNA methylation and metabolomic profiles of NBH killifish populations, compared to sensitive populations from a non-polluted site, Scorton Creek (SC). The results revealed that liver-specific global DNA hypomethylation and differential metabolites were evident in fish from NBH compared with those from SC. The sex-specific differences were not greater than the tissue-specific differences. We demonstrated liver-specific enriched metabolic pathways (e.g., amino acid metabolic pathways converged into the urea cycle and glutathione metabolism), suggesting possible crosstalk between differential metabolites and DNA hypomethylation in the livers of NBH killifish. Additional investigation of methylated gene regions is necessary to understand the functional role of DNA hypomethylation in the regulation of enzyme-encoding genes associated with metabolic processes and physiological changes in NBH populations.

Scaling of fast-start performance and its thermal dependence in mummichog Fundulus heteroclitus.

Fast-start predator-escape performance and its sensitivity to temperature (24, 30, and 36°C) were evaluated in mummichog Fundulus heteroclitus across a range of body sizes spanning YOY to adult (35-68 mm standard length). Mummichogs exhibit isometry of body dimensions and areas of the dorsal and anal fins but negative allometry of the caudal fin area. These scaling relationships are consistent with observed decreases in fast-start angular velocities with increasing body size. Linear velocity, on the contrary, does not vary with size, and both large and small mummichogs are capable of traversing similar distances in a given amount of time. In addition, temperature influences fast-start performance in similar ways over the size range, though the magnitude of the effect varies with size for some performance measures. In general, fast-start performance increases with test temperature, but mummichogs acclimated to warmer temperatures exhibit lower performance at each test temperature. Altogether, our results suggest that mummichogs across the adult size range may suffer decreases in their predator-escape performance as increasing sea temperatures combine with short-term temperature fluctuations in the estuaries these fish occupy.

Conserved satellite DNA motif and lack of interstitial telomeric sites in highly rearranged African Nothobranchius killifish karyotypes.

Using African annual killifishes of the genus Nothobranchius from temporary savannah pools with rapid karyotype and sex chromosome evolution, we analysed the chromosomal distribution of telomeric (TTAGGG)n repeat and Nfu-SatC satellite DNA (satDNA; isolated from Nothobranchius furzeri) in 15 species across the Nothobranchius killifish phylogeny, and with Fundulosoma thierryi as an out-group. Our fluorescence in situ hybridization experiments revealed that all analysed taxa share the presence of Nfu-SatC repeat but with diverse organization and distribution on chromosomes. Nfu-SatC landscape was similar in conspecific populations of Nothobranchius guentheri and Nothobranchius melanospilus but slightly-to-moderately differed between populations of Nothobranchius pienaari, and between closely related Nothobranchius kuhntae and Nothobranchius orthonotus. Inter-individual variability in Nfu-SatC patterns was found in N. orthonotus and Nothobranchius krysanovi. We revealed mostly no sex-linked patterns of studied repetitive DNA distribution. Only in Nothobranchius brieni, possessing multiple sex chromosomes, Nfu-SatC repeat occupied a substantial portion of the neo-Y chromosome, similarly as formerly found in the XY sex chromosome system of turquoise killifish N. furzeri and its sister species Nothobranchius kadleci-representatives not closely related to N. brieni. All studied species further shared patterns of expected telomeric repeats at the ends of all chromosomes and no additional interstitial telomeric sites. In summary, we revealed (i) the presence of conserved satDNA class in Nothobranchius clades (a rare pattern among ray-finned fishes); (ii) independent trajectories of Nothobranchius sex chromosome differentiation, with recurrent and convergent accumulation of Nfu-SatC on the Y chromosome in some species; and (iii) genus-wide shared tendency to loss of telomeric repeats during interchromosomal rearrangements. Collectively, our findings advance our understanding of genome structure, mechanisms of karyotype reshuffling, and sex chromosome differentiation in Nothobranchius killifishes from the genus-wide perspective.

Preparation of High-Viability Single-Cell Suspensions from African Turquoise Killifish Brain Tissue.

Studying the brain at the single-cell level has become increasingly popular in recent years. This, however, remains challenging, especially in emerging model organisms. To carry out single-cell sequencing, the preparation of a high-viability single-cell suspension is critical. In this protocol, we describe how to prepare a high-viability single-cell suspension starting from brain tissue of the African turquoise killifish (Nothobranchius furzeri). The protocol consists of dissection, enzymatic and mechanical dissociation of the brain tissue, and debris removal. The protocol described here has been successfully used for 10× Genomics single-cell sequencing of the telencephalon of adult killifish, which requires a cell viability of at least 70%. In addition to single-cell sequencing experiments, the single-cell suspension generated can be used for other applications, including cell culture and flow cytometry.

Comprehensive Colony Health Management and Emerging Pathogens of the Annual Killifish Species Nothobranchius furzeri.

The Leibniz Institute on Aging has maintained killifish colonies for over 15 y. Our veterinarians, scientists, and animal technicians developed a fish health scoring system and routine colony health surveillance program for our colonies. Over a 4-y period, health data from the African turquoise killifish Nothobranchius furzeri colony were systematically collected and analyzed. The fish health assessment system facilitated categorization of clinical signs and differentiation of fish with mild clinical signs from fish that required euthanasia. This report provides new information on clinical signs and conditions that may occur in young and aged N. furzeri. To be comprehensive, a colony health surveillance program incorporates animal health at both the individual and the population levels. The quarterly routine health monitoring program identified Mycobacterium spp. as the most common agent in our facility and identified the killifish pathogen (Loma acerinae) for the first time. Taken together, these findings demonstrate the importance of a comprehensive colony health management system in a fish research facility. By improving the health and welfare of fish used for research, the scientific community will benefit from less variable and more reliably reproducible research results.

Ecological character displacement among Nothobranchius annual killifishes in Tanzania.

Divergent ecological character displacement (ECD) is the competition-driven divergence in resource use-related phenotypic traits between coexisting species. It is considered one of the primary drivers of ecological diversification and adaptive radiation. We analyzed phenotypic and ecological variation in 2 African annual killifish species of the genus Nothobranchius: N. eggersi and N. melanospilus in sympatry and N. melanospilus in allopatry. Our aim was to test whether allopatric and sympatric populations of N. melanospilus differ morphologically from each other and from N. eggersi and examine whether these differences are consistent with the predictions of ECD. We find that sympatric N. melanospilus differ from allopatric N. melanospilus and differ from N. eggersi more strongly than the latter. Our data satisfy four criteria for demonstrating ECD: Differences in phenotypes between allopatric and sympatric N. melanospilus are greater than expected by chance; the divergence pattern between allopatric and sympatric N. melanospilus results from an evolutionary shift rather than from ecological sorting; morphological differences observed reflect differences in resource use; and, lastly, sites of allopatry and sympatry do not differ in food resource availability or other ecological conditions. Our results suggest that competition is the main driver of the observed divergence between two N. melanospilus populations.

The African Turquoise Killifish: A Scalable Vertebrate Model for Aging and Other Complex Phenotypes.

The African turquoise killifish Nothobranchius furzeri is currently the shortest-lived vertebrate that can be bred in captivity. Because of its short life span of only 4-6 months, rapid generation time, high fecundity, and low cost of maintenance, the African turquoise killifish has emerged as an attractive model organism that combines the scalability of invertebrate models with the unique features of vertebrate organisms. A growing community of researchers is using the African turquoise killifish for studies in diverse fields, including aging, organ regeneration, development, "suspended animation," evolution, neuroscience, and disease. A wide range of techniques is now available for killifish research, from genetic manipulations and genomic tools to specialized assays for studying life span, organ biology, response to injury, etc. This protocol collection provides detailed descriptions of the methods that are generally applicable to all killifish laboratories and those that are limited to specific disciplines. Here, we give an overview of the features that render the African turquoise killifish a unique fast-track vertebrate model organism.

Efficient Transgenesis in African Turquoise Killifish Using a Gibson Assembly-Based Tol2 Transposon System.

The short-lived African killifish Nothobranchius furzeri is an attractive genetic model for vertebrate aging and regeneration studies. The utilization of genetically modified animals is a common strategy for unveiling molecular mechanisms responsible for a biological phenomenon. Here, we report a highly efficient protocol for generating transgenic African killifish using the Tol2 transposon system, which creates random insertions in the genome. Transgenic vectors carrying gene-expression cassettes of interest and an eye-specific marker for transgene identification can be quickly assembled through Gibson assembly. The development of this new pipeline will facilitate transgenic reporter assays and gene-expression-related manipulations in African killifish.

The African killifish: A short-lived vertebrate model to study the biology of sarcopenia and longevity.

Sarcopenia, the age-related decline in muscle function, places a considerable burden on health-care systems. While the stereotypic hallmarks of sarcopenia are well characterized, their contribution to muscle wasting remains elusive, which is partly due to the limited availability of animal models. Here, we have performed cellular and molecular characterization of skeletal muscle from the African killifish-an extremely short-lived vertebrate-revealing that while many characteristics deteriorate with increasing age, supporting the use of killifish as a model for sarcopenia research, some features surprisingly reverse to an "early-life" state in the extremely old stages. This suggests that in extremely old animals, there may be mechanisms that prevent further deterioration of skeletal muscle, contributing to an extension of life span. In line with this, we report a reduction in mortality rates in extremely old killifish. To identify mechanisms for this phenomenon, we used a systems metabolomics approach, which revealed that during aging there is a striking depletion of triglycerides, mimicking a state of calorie restriction. This results in the activation of mitohormesis, increasing Sirt1 levels, which improves lipid metabolism and maintains nutrient homeostasis in extremely old animals. Pharmacological induction of Sirt1 in aged animals was sufficient to induce a late life-like metabolic profile, supporting its role in life span extension in vertebrate populations that are naturally long-lived. Collectively, our results demonstrate that killifish are not only a novel model to study the biological processes that govern sarcopenia, but they also provide a unique vertebrate system to dissect the regulation of longevity.

Senescence-associated ß-galactosidase staining over the lifespan differs in a short- and a long-lived fish species.

During the aging process, cells can enter cellular senescence, a state in which cells leave the cell cycle but remain viable. This mechanism is thought to protect tissues from propagation of damaged cells and the number of senescent cells has been shown to increase with age. The speed of aging determines the lifespan of a species and it varies significantly in different species. To assess the progress of cellular senescence during lifetime, we performed a comparative longitudinal study using histochemical detection of the senescence-associated beta-galactosidase as senescence marker to map the staining patterns in organs of the long-lived zebrafish and the short-lived turquoise killifish using light- and electron microscopy. We compared age stages corresponding to human stages of newborn, childhood, adolescence, adult and old age. We found tissue-specific but conserved signal patterns with respect to organ distribution. However, we found dramatic differences in the onset of tissue staining. The stained zebrafish organs show little to no signal at newborn age followed by a gradual increase in signal intensity, whereas the organs of the short-lived killifish show an early onset of staining already at newborn stage, which remains conspicuous at all age stages. The most prominent signal was found in liver, intestine, kidney and heart, with the latter showing the most prominent interspecies divergence in onset of staining and in staining intensity. In addition, we found staining predominantly in epithelial cells, some of which are post-mitotic, such as the intestinal epithelial lining. We hypothesize that the association of the strong and early-onset signal pattern in the short-lived killifish is consistent with a protective mechanism in a fast growing species. Furthermore, we believe that staining in post-mitotic cells may play a role in maintaining tissue integrity, suggesting different roles for cellular senescence during life.

Intraspecific variation in thermal performance curves for early development in Fundulus heteroclitus.

Thermal performance curves (TPCs) provide a framework for understanding the effects of temperature on ectotherm performance and fitness. TPCs are often used to test hypotheses regarding local adaptation to temperature or to develop predictions for how organisms will respond to climate warming. However, for aquatic organisms such as fishes, most TPCs have been estimated for adult life stages, and little is known about the shape of TPCs or the potential for thermal adaptation at sensitive embryonic life stages. To examine how latitudinal gradients shape TPCs at early life stages in fishes, we used two populations of Fundulus heteroclitus that have been shown to exhibit latitudinal variation along the thermal cline as adults. We exposed embryos from both northern and southern populations and their reciprocal crosses to eight different temperatures (15°C, 18°C, 21°C, 24°C, 27°C, 30°C, 33°C, and 36°C) until hatch and examined the effects of developmental temperature on embryonic and larval traits (shape of TPCs, heart rate, and body size). We found that the pure southern embryos had a right-shifted TPC (higher thermal optimum (Topt) for developmental rate, survival, and embryonic growth rate) whereas pure northern embryos had a vertically shifted TPC (higher maximum performance (Pmax) for developmental rate). Differences across larval traits and cross-type were also found, such that northern crosses hatched faster and hatched at a smaller size compared to the pure southern population. Overall, these observed differences in embryonic and larval traits are consistent with patterns of both local adaptation and countergradient variation.

Assessment of oxidative stress biomarkers in the threatened annual killifish Austrolebias charrua exposed to Roundup.

This study aimed to analyze the toxic effects of Roundup Transorb® on the endangered Neotropical annual killifish Austrolebias charrua through the assessment of molecular and biochemical biomarkers. The fish were collected in temporary ponds and exposed to environmentally realistic concentrations of the herbicide (5 mg.L-1 for 96 h). The production of ROS, lipid peroxidation, DNA damage, and membrane fluidity were evaluated in the blood cells by flow cytometry. The mRNA expression of the antioxidant-related genes sod2, cat, gstα, atp1a1, gclc, and ucp1 across the brain, liver, and gills was quantified. The acute exposure of annual killifish to Roundup significantly increased ROS production, lipid peroxidation, and DNA damage in their erythrocytes. Likewise, Roundup Transorb® decreased membrane fluidity in the blood cells of the exposed fish. Gene expression analysis revealed that Roundup exposure alters the relative expression of genes associated with oxidative stress and antioxidant defense. Our results give rise to new insights into adaptive mechanisms of A. charrua in response to Roundup. Since Brazilian annual killifishes strongly risk extinction, this study paves the way for developing novel biotechnologies applied to environmental monitoring and aquatic toxicology assessment.

Validation of Aphyosemion lorai and Caeruleamsemion; a new species and subgenus of killifish (Cyprinodontiformes: Nothobranchiidae), from the continental region of Equatorial Guinea.

N/A.

A new annual killifish, Moema beltramonorum (Cyprinodontiformes: Rivulidae) from the Amazon basin, Bolivia.

A new annual killifish species, Moema beltramonorum, is described from a seasonal swamp next to arroyo Crespo, belonging to Ro San Martin, a tributary of Ro Itnez, Amazon River basin, Bolivia. The new taxon presents an unique combination of male colour pattern characters not present in any congener, and an unique caudal fin coloration.

Molecular delimitation of the seasonal killifishes of the Hypsolebias antenori species group (Cyprinodontiformes, Rivulidae), with description of two new species from the Caatinga coastal basins, northeastern Brazil.

Hypsolebia antenori is a seasonal killifish considered to be broadly distributed along the Caatingas basins of northeastern Brazil, with records in the coastal drainages of the rio Pacoti, rio Jaguaribe, crrego Virglio and rio Apodi-Mossor basins. Based on morphological characters and molecular phylogenetic reconstructions, it was possible to diagnose two new species and restrict H. antenori to the rio Jaguaribe basin. Hypsolebias gongobira new species is described from a temporary pool in the rio Pacoti basin in Cear State, in syntopy with the species threatened H. longignatus. Hypsolebias bonita new species occurs in the floodplains of the rio Apodi-Mossor basin and in the crrego Virglio microbasin in the Furna Feia National Park, in Rio Grande do Norte State. We also discuss the conservation status of these new species and how the So Francisco Interbasin Water transfer, and the agricultural and urban expansion in the semiarid may be adversely affecting these seasonal killifishes.