Centrality to the metapopulation is more important for population genetic diversity than habitat area or fragmentation.

Drift and gene flow affect genetic diversity. Given that the strength of genetic drift increases as population size decreases, management activities have focused on increasing population size through preserving habitats to preserve genetic diversity. Few studies have empirically evaluated the impacts of drift and gene flow on genetic diversity. Kryptolebias marmoratus, henceforth 'rivulus', is a small killifish restricted to fragmented New World mangrove forests with gene flow primarily associated with ocean currents. Rivulus form distinct populations across patches, making them a well-suited system to test the extent to which habitat area, fragmentation and connectivity are associated with genetic diversity. Using over 1000 individuals genotyped at 32 microsatellite loci, high-resolution landcover data and oceanographic simulations with graph theory, we demonstrate that centrality (connectivity) to the metapopulation is more strongly associated with genetic diversity than habitat area or fragmentation. By comparing models with and without centrality standardized by the source population's genetic diversity, our results suggest that metapopulation centrality is critical to genetic diversity regardless of the diversity of adjacent populations. While we find evidence that habitat area and fragmentation are related to genetic diversity, centrality is always a significant predictor with a larger effect than any measure of habitat configuration.

A Pilot Study of Gene Expression Modulation from Antioxidant System of Killifish Austrolebias charrua After Exposure to Roundup Transorb®.

Roundup Transorb® (RDT) is the most popular glyphosate-based herbicide (GHB) used in agriculture, and its impact extends to non-target organisms. The annual killifish Austrolebias charrua is an endangered species endemic to southern South America and inhabits temporary ponds. This study evaluates the effects of RDT concentrations (0.065 and 5 mg/L GAE) on A. charrua exposed for 96 h. Gene expression of cat, sod2, gstα, gclc, and ucp1 was evaluated on the liver and gills. Highlighting that even at low concentrations permitted by Brazilian legislation, the RDT can have adverse effects on A. charrua.

Evolution of diapause in the African turquoise killifish by remodeling the ancient gene regulatory landscape.

Suspended animation states allow organisms to survive extreme environments. The African turquoise killifish has evolved diapause as a form of suspended development to survive a complete drought. However, the mechanisms underlying the evolution of extreme survival states are unknown. To understand diapause evolution, we performed integrative multi-omics (gene expression, chromatin accessibility, and lipidomics) in the embryos of multiple killifish species. We find that diapause evolved by a recent remodeling of regulatory elements at very ancient gene duplicates (paralogs) present in all vertebrates. CRISPR-Cas9-based perturbations identify the transcription factors REST/NRSF and FOXOs as critical for the diapause gene expression program, including genes involved in lipid metabolism. Indeed, diapause shows a distinct lipid profile, with an increase in triglycerides with very-long-chain fatty acids. Our work suggests a mechanism for the evolution of complex adaptations and offers strategies to promote long-term survival by activating suspended animation programs in other species.

Validation of qPCR reference genes in the endangered annual killifish Austrolebias charrua considering different tissues, gender and environmental conditions.

The annual killifish Austrolebias charrua is an endangered species, endemic to the southern region of South America, which inhabits temporary ponds that emerges in the rainy season. The main anthropogenic threat driving the extinction of A. charrua stems from extensive agriculture, primarily due to the widrespread use of glyphosate-based herbicides near their habitats. Annual killifishes have been used as models for ecotoxicological studies but, up to now, there are no studies about reference genes in any Austrolebias species. This represents an obstacle to the use of qPCR-based technologies, the standard method for gene expression quantification. The present study aimed to select and validate potential reference genes for qPCR normalization in the annual killifish Austrolebias charrua considering different tissues, gender and environmental conditions. The candidate reference genes 18 s, actb, gapdh, ef1a, shox, eif3g, and the control gene atp1a1 were evaluated in male and female individuals in three different tissues (brain, liver, and gills) under two experimental conditions (control and acute exposition to Roundup Transorb®). The collected tissues were submitted to RNA extraction, followed by cDNA synthesis, cloning, sequencing, and qPCR. Overall, 18 s was the most stable reference gene, and 18 s and ef1a were the most stable combination. Otherwise, considering all variables, gapdh and shox were the least stable candidate genes. Foremost, suitable reference genes were validated in A. charrua, facilitating accurate mRNA quantification in this species, which might be useful for developing molecular tools of ecotoxicological assessment based on gene expression analysis for environmental monitoring of annual killifish.

Niche conservatism and spread explain introgression between native and invasive fish.

Hybridisation can be an important driver of evolutionary change, but hybridisation with invasive species can have adverse effects on native biodiversity. While hybridisation has been documented across taxa, there is limited understanding of ecological factors promoting patterns of hybridisation and the spatial distribution of hybrid individuals. We combined the results of ecological niche modelling (ENM) and restriction site-associated DNA sequencing to test theories of niche conservatism and biotic resistance on the success of invasion, admixture, and extent of introgression between native and non-native fishes. We related Maxent predictions of habitat suitability based on the native ranges of invasive Eastern Banded Killifish (Fundulus diaphanus diaphanus Lesueur 1817) and native Western Banded Killifish (Fundulus diaphanus menona Jordan and Copeland 1877) to admixture indices of individual Banded Killifish. We found that Eastern Banded Killifish predominated at sites predicted as suitable from their ENM, consistent with niche conservatism. Admixed individuals were more common as Eastern Banded Killifish habitat suitability declined. We also found that Eastern Banded Killifish were most common at sites closest to the presumed source of this invasion, whereas the proportion of admixed individuals increased with distance from the source of invasion. Lastly, we found little evidence that habitat suitability for Western Banded Killifish provides biotic resistance from either displacement by, or admixture with, invasive Eastern Banded Killifish. Our study demonstrates that ENMs can inform conservation-relevant outcomes between native and invasive taxa while emphasising the importance of protecting isolated Western Banded Killifish populations from invasive conspecifics.

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.

Hypoxia-inducible factor 1 alpha protein increases without changes in mRNA during acute hypoxic exposure of the Gulf killifish, Fundulus grandis.

The hypoxia inducible factor 1 (HIF1) is a central regulator of the molecular responses of animals to low oxygen. While the hypoxia-responsiveness of HIF1 is generally attributed to the stabilization of the alpha protein subunit (HIF1α) at low oxygen, several studies on fish report increased tissue levels of HIF1A mRNA during hypoxia, suggesting transcriptional regulation. In the current study, HIF1α protein and HIF1A mRNA were determined in parallel in tissues of Gulf killifish, Fundulus grandis, exposed to short-term hypoxia (24 h at 1 mg O2 l-1). HIF1α protein was higher in brain, ovary, and skeletal muscle from fish exposed to hypoxia compared with normoxic controls by 6 h, and it remained elevated in brain and ovary at 24 h. In contrast, HIF1A mRNA levels were unaffected by hypoxia in any tissue. Moreover, HIF1α protein and HIF1A mRNA levels in the same tissues were not correlated with one another during either normoxia or hypoxia. Hence, an increase in HIF1α protein does not depend upon an increase in HIF1A mRNA during acute exposure to low oxygen in this species. The results support the widely accepted mechanism of post-translational protein stabilization, rather than new transcription, during the initial response of fish to hypoxia.

Fast satellite DNA evolution in Nothobranchius annual killifishes.

Satellite DNA (satDNA) is a rapidly evolving class of tandem repeats, with some monomers being involved in centromere organization and function. To identify repeats associated with (peri)centromeric regions, we investigated satDNA across Southern and Coastal clades of African annual killifishes of the genus Nothobranchius. Molecular cytogenetic and bioinformatic analyses revealed that two previously identified satellites, designated here as NkadSat01-77 and NfurSat01-348, are associated with (peri)centromeres only in one lineage of the Southern clade. NfurSat01-348 was, however, additionally detected outside centromeres in three members of the Coastal clade. We also identified a novel satDNA, NrubSat01-48, associated with (peri)centromeres in N. foerschi, N. guentheri, and N. rubripinnis. Our findings revealed fast turnover of satDNA associated with (peri)centromeres and different trends in their evolution in two clades of the genus Nothobranchius.

Transcriptomes of aging brain, heart, muscle, and spleen from female and male African turquoise killifish.

The African turquoise killifish is an emerging vertebrate model organism with great potential for aging research due to its naturally short lifespan. Thus far, turquoise killifish aging 'omic' studies have examined a single organ, single sex and/or evaluated samples from non-reference strains. Here, we describe a resource dataset of ribosomal RNA-depleted RNA-seq libraries generated from the brain, heart, muscle, and spleen from both sexes, as well as young and old animals, in the reference GRZ turquoise killifish strain. We provide basic quality control steps and demonstrate the utility of our dataset by performing differential gene expression and gene ontology analyses by age and sex. Importantly, we show that age has a greater impact than sex on transcriptional landscapes across probed tissues. Finally, we confirm transcription of transposable elements (TEs), which are highly abundant and increase in expression with age in brain tissue. This dataset will be a useful resource for exploring gene and TE expression as a function of both age and sex in a powerful naturally short-lived vertebrate model.

Taxonomic Profiling of Fecal and Intestinal Microbiota in the African Turquoise Killifish Nothobranchius furzeri.

Killifish are emerging as a new laboratory system in which to study a range of questions, from the genetic basis of embryo dormancy to life history trait evolution, age-dependent neurodegeneration, and the connection between microbial community structure and biology of aging. Over the past decade, advances in high-throughput sequencing have helped uncover the vast diversity of microbial communities present in environmental samples and on host epithelia. Here, we describe an optimized protocol to study the taxonomic composition of intestinal and fecal microbiota in laboratory-raised as well as natural killifish populations and provide comprehensive step-by-step instructions for tissue sampling, high-throughput genomic DNA extraction, and the generation of 16S V3V4 rRNA and 16S V4 rRNA gene libraries.

From fish to cells: Establishment of continuous cell lines from embryos of annual killifish Nothobranchius furzeri and N. kadleci.

There is a growing need of alternative experimental models that avoid or minimize the use of animals due to ethical, economical, and scientific reasons. Surprisingly, the stable embryonic cell lines representing Nothobranchius spp., emerging vertebrate models in aging research, regenerative medicine, ecotoxicology, or genomics, have been not derived so far. This paper reports establishment and deep characterization of ten continuous cell lines from annual killifish embryos of N. furzeri and N. kadleci. The established cell lines exhibited mostly fibroblast- and epithelial-like morphology and steady growth rates with cell doubling time ranging from 27 to 40 h. All cell lines retained very similar characteristics even after continuous subcultivation (more than 100 passages) and extended storage in liquid nitrogen (∼3 years). The cytogenetic analysis of the cell lines revealed a diploid chromosome number mostly equal to 38 elements (i.e., the native chromosome count for both killifish species), with minor but diverse line/passage-specific karyotype changes compared to the patterns observed in non-cultured N. furzeri and N. kadleci somatic cells. Based on transcriptional analysis of marker genes, the cell lines displayed features of an undifferentiated state without signs of senescence even in advanced passages. We confirmed that the cell lines are transfectable and can form viable 3-D spheroids. The applicability of the cell lines for (eco)toxicological surveys was confirmed by assessing the effect of cytotoxic and growth inhibitory agents. Properties of established Nothobranchius embryonic cell lines open new possibilities for the application of this model in various fields of life sciences including molecular mechanisms of aging, karyotype (in)stability or differences in lifespan.

Molecular and Biochemical Analyses of Aging Hallmarks in the Central Nervous System of the Fast-Aging African Turquoise Killifish.

As modern society is graying, aging research and biogerontology models, in which the aging process can be studied, are becoming increasingly important. A proper aging model can be defined as one that displays many of the aging hallmarks. Here, we provide two different practical approaches-namely, real-time quantitative polymerase chain reaction (RT-qPCR) and western blotting-that can be used to investigate cellular senescence (RT-qPCR for p21 and p27), altered intercellular communication/inflammaging (RT-qPCR for il-10, sirt-1, il-6, il-1b, il-8, and tnf), and oxidative stress (western blotting for 4-HNE) in the killifish central nervous system, and, more specifically, in the retina, optic tectum, and telencephalon. These molecular and biochemical analyses are a first step in confirming the aging characteristics but should preferably be combined with morphological analyses.

Evidence of prezygotic isolation, but not assortative mating, between locally adapted populations of Fundulus heteroclitus across a salinity gradient.

Selection along environmental gradients can drive reproductive isolation and speciation. Among fishes, salinity is a major factor limiting species distributions, and despite its importance in generating species diversity, speciation events between marine and freshwater are rare. Here, we tested for mechanisms of reproductive isolation between locally adapted freshwater and brackish water-native populations of killifish, Fundulus heteroclitus, from either side of a hybrid zone along a salinity gradient. There was evidence for pre-zygotic endogenous reproductive isolation with reduced fertilization success between crosses of freshwater-native males and brackish water-native females. Exogenous pre-zygotic isolation was also present where females had highest fertilization in their native salinity. We used a replicated mass spawning design to test for mate choice in both brackish and fresh water. After genotyping 187 parents and 2523 offspring at 2347 SNPs across the genome, 85% of offspring were successfully assign to their parents. However, no reinforcing mate choice was observed. These results therefore demonstrate emerging, yet limited, reproductive isolation and incipient speciation across a marine to freshwater salinity gradient and suggest that both endogenous and exogenous mechanisms, but not assortative mating, contribute to divergence.

Reproductive isolating mechanisms contributing to asymmetric hybridization in Killifishes (Fundulus spp.).

When species hybridize, one F1 hybrid cross type often predominates. Such asymmetry can arise from differences in a variety of reproductive barriers, but the relative roles and concordance of pre-mating, post-mating prezygotic, and post-zygotic barriers in producing these biases in natural animal populations have not been widely investigated. Here, we study a population of predominantly F1 hybrids between two killifish species (Fundulus heteroclitus and F. diaphanus) in which >95% of F1 hybrids have F. diaphanus mothers and F. heteroclitus fathers (D♀ × H♂). To determine why F. heteroclitus × F. diaphanus F1 hybrids (H♀ × D♂) are so rare, we tested for asymmetry in pre-mating reproductive barriers (female preference and male aggression) at a common salinity (10 ppt) and post-mating, pre-zygotic (fertilization success) and post-zygotic (embryonic development time and hatching success) reproductive barriers at a range of ecologically relevant salinities (0, 5, 10, and 15 ppt). We found that F. heteroclitus females preferred conspecific males, whereas F. diaphanus females did not, matching the observed cross bias in the wild. Naturally rare H♀ × D♂ crosses also had lower fertilization success than all other cross types, and a lower hatching success than the prevalent D♀ × H♂ crosses at the salinity found in the hybrid zone centre (10 ppt). Furthermore, the naturally predominant D♀ × H♂ crosses had a higher hatching success than F. diaphanus crosses at 10 ppt, which may further increase their relative abundance. The present study suggests that a combination of incomplete mating, post-mating pre-zygotic and post-zygotic reproductive isolating mechanisms act in concert to produce hybrid asymmetry in this system.

Dynamic regulation of gonadal transposon control across the lifespan of the naturally short-lived African turquoise killifish.

Although germline cells are considered to be functionally "immortal," both the germline and supporting somatic cells in the gonad within an organism experience aging. With increased age at parenthood, the age-related decline in reproductive success has become an important biological issue for an aging population. However, molecular mechanisms underlying reproductive aging across sexes in vertebrates remain poorly understood. To decipher molecular drivers of vertebrate gonadal aging across sexes, we perform longitudinal characterization of the gonadal transcriptome throughout the lifespan in the naturally short-lived African turquoise killifish (Nothobranchius furzeri). By combining mRNA-seq and small RNA-seq from 26 individuals, we characterize the aging gonads of young-adult, middle-aged, and old female and male fish. We analyze changes in transcriptional patterns of genes, transposable elements (TEs), and piRNAs. We find that testes seem to undergo only marginal changes during aging. In contrast, in middle-aged ovaries, the time point associated with peak female fertility in this strain, PIWI pathway components are transiently down-regulated, TE transcription is elevated, and piRNA levels generally decrease, suggesting that egg quality may already be declining at middle-age. Furthermore, we show that piRNA ping-pong biogenesis declines steadily with age in ovaries, whereas it is maintained in aging testes. To our knowledge, this data set represents the most comprehensive transcriptomic data set for vertebrate gonadal aging. This resource also highlights important pathways that are regulated during reproductive aging in either ovaries or testes, which could ultimately be leveraged to help restore aspects of youthful reproductive function.

RNAseq Analysis of Brain Aging in Wild Specimens of Short-Lived Turquoise Killifish: Commonalities and Differences With Aging Under Laboratory Conditions.

A vast body of studies is available that describe age-dependent gene expression in relation to aging in a number of different model species. These data were obtained from animals kept in conditions with reduced environmental challenges, abundant food, and deprivation of natural sensory stimulation. Here, we compared wild- and captive aging in the short-lived turquoise killifish (Nothobranchius furzeri). These fish inhabit temporary ponds in the African savannah. When the ponds are flooded, eggs hatch synchronously, enabling a precise timing of their individual and population age. We collected the brains of wild fish of different ages and quantified the global age-dependent regulation of transcripts using RNAseq. A major difference between captive and wild populations is that wild populations had unlimited access to food and hence grew to larger sizes and reached asymptotic size more rapidly, enabling the analysis of age-dependent gene expression without the confounding effect of adult brain growth. We found that the majority of differentially expressed genes show the same direction of regulation in wild and captive populations. However, a number of genes were regulated in opposite direction. Genes downregulated in the wild and upregulated in captivity were enriched for terms related to neuronal communication. Genes upregulated in the wild and downregulated in captive conditions were enriched in terms related to DNA replication. Finally, the rate of age-dependent gene regulation was higher in wild animals, suggesting a phenomenon of accelerated aging.

Sex chromosome differentiation via changes in the Y chromosome repeat landscape in African annual killifishes Nothobranchius furzeri and N. kadleci.

Homomorphic sex chromosomes and their turnover are common in teleosts. We investigated the evolution of nascent sex chromosomes in several populations of two sister species of African annual killifishes, Nothobranchius furzeri and N. kadleci, focusing on their under-studied repetitive landscape. We combined bioinformatic analyses of the repeatome with molecular cytogenetic techniques, including comparative genomic hybridization, fluorescence in situ hybridization with satellite sequences, ribosomal RNA genes (rDNA) and bacterial artificial chromosomes (BACs), and immunostaining of SYCP3 and MLH1 proteins to mark lateral elements of synaptonemal complexes and recombination sites, respectively. Both species share the same heteromorphic XY sex chromosome system, which thus evolved prior to their divergence. This was corroborated by sequence analysis of a putative master sex determining (MSD) gene gdf6Y in both species. Based on their divergence, differentiation of the XY sex chromosome pair started approximately 2 million years ago. In all populations, the gdf6Y gene mapped within a region rich in satellite DNA on the Y chromosome long arms. Despite their heteromorphism, X and Y chromosomes mostly pair regularly in meiosis, implying synaptic adjustment. In N. kadleci, Y-linked paracentric inversions like those previously reported in N. furzeri were detected. An inversion involving the MSD gene may suppress occasional recombination in the region, which we otherwise evidenced in the N. furzeri population MZCS-121 of the Limpopo clade lacking this inversion. Y chromosome centromeric repeats were reduced compared with the X chromosome and autosomes, which points to a role of relaxed meiotic drive in shaping the Y chromosome repeat landscape. We speculate that the recombination rate between sex chromosomes was reduced due to heterochiasmy. The observed differences between the repeat accumulations on the X and Y chromosomes probably result from high repeat turnover and may not relate closely to the divergence inferred from earlier SNP analyses.

Gentle Isolation of Nuclei from the Brain Tissue of Adult African Turquoise Killifish, a Naturally Short-Lived Model for Aging Research.

Studying brain aging at single-cell resolution in vertebrate systems remains challenging due to cost, time, and technical constraints. Here, we demonstrate a protocol to generate single-nucleus RNA sequencing (snRNA-seq) libraries from the brains of the naturally short-lived vertebrate African turquoise killifish Nothobranchius furzeri. The African turquoise killifish has a lifespan of 4-6 months and can be housed in a cost-effective manner, thus reducing cost and time barriers to study vertebrate brain aging. However, tailored protocols are needed to isolate nuclei of sufficient quality for downstream single-cell experiments from the brain of young and aged fish. Here, we demonstrate an empirically optimized protocol for the isolation of high-quality nuclei from the brain of adult African turquoise killifish, a critical step in the generation of high-quality single nuclei omic libraries. Furthermore, we show that the steps to reduce contaminating background RNA are important to clearly distinguish cell types. In summary, this protocol demonstrates the feasibility of studying brain aging in non-traditional vertebrate model organisms.

Phylogenomics reveals extensive introgression and a case of mito-nuclear discordance in the killifish genus Kryptolebias.

Introgression is a widespread evolutionary process leading to phylogenetic inconsistencies among distinct parts of the genomes, particularly between mitochondrial and nuclear-based phylogenetic reconstructions (e.g., mito-nuclear discordances). Here, we used mtDNA and genome-wide nuclear sites to provide the first phylogenomic-based hypothesis on the evolutionary relationships within the killifish genus Kryptolebias. In addition, we tested for evidence of past introgression in the genus given the multiple reports of undergoing hybridization between its members. Our mtDNA phylogeny generally agreed with the relationships previously proposed for the genus. However, our reconstruction based on nuclear DNA revealed an unknown lineage - Kryptolebias sp. 'ESP' - as the sister group of the self-fertilizing mangrove killifishes, K. marmoratus and K. hermaphroditus. All individuals sequenced of Kryptolebias sp. 'ESP' had the same mtDNA haplotype commonly observed in K. hermaphroditus, demonstrating a clear case of mito-nuclear discordance. Our analysis further confirmed extensive history of introgression between Kryptolebias sp. 'ESP' and K. hermaphroditus. Population genomics analyses indicate no current gene flow between the two lineages, despite their current sympatry and history of introgression. We also confirmed introgression between other species pairs in the genus that have been recently reported to form hybrid zones. Overall, our study provides a phylogenomic reconstruction covering most of the Kryptolebias species, reveals a new lineage hidden in a case of mito-nuclear discordance, and provides evidence of multiple events of ancestral introgression in the genus. These findings underscore the importance of investigating different genomic information in a phylogenetic framework, particularly in taxa where introgression is common as in the sexually diverse mangrove killifishes.

Polyphenisms and polymorphisms: Genetic variation in plasticity and color variation within and among bluefin killifish populations.

The presence of stable color polymorphisms within populations begs the question of how genetic variation is maintained. Consistent variation among populations in coloration, especially when correlated with environmental variation, raises questions about whether environmental conditions affect either the fulcrum of those balanced polymorphisms, the plastic expression of coloration, or both. Color patterns in male bluefin killifish provoke both types of questions. Red and yellow morphs are common in all populations. Blue males are more common in tannin-stained swamps relative to clear springs. Here, we combined crosses with a manipulation of light to explore how genetic variation and phenotypic plasticity shape these patterns. We found that the variation in coloration is attributable mainly to two axes of variation: (1) a red-yellow axis with yellow being dominant to red, and (2) a blue axis that can override red-yellow and is controlled by genetics, phenotypic plasticity, and genetic variation for phenotypic plasticity. The variation among populations in plasticity suggests it is adaptive in some populations but not others. The variation among sires in plasticity within the swamp population suggests balancing selection may be acting not only on the red-yellow polymorphism but also on plasticity for blue coloration.