Single housing of juveniles accelerates early-stage growth but extends adult lifespan in African turquoise killifish.

Within the same species, individuals exhibiting faster growth tend to have shorter lifespans, even if their fast growth arises from early-life pharmacological interventions. However, in vertebrates, the impact of the early-life environment on the growth rate and lifespan has not been fully elucidated. In this study, by utilizing the short-lived African turquoise killifish, which is suitable for a comprehensive life-stage analysis in a brief timeframe, we explored the effects of housing density during the juvenile stage on holistic life traits. As a result, we found that lower housing densities resulted in faster growth, but led to longer adult lifespan, which was contrary to the common notion. Furthermore, the single-housed adult fish displayed a longer egg-laying period than did their group-housed counterparts. Our transcriptome analysis also demonstrated that, in terms of internal transcriptional programs, the life stage progression and aging process of single-housed fish were slower than those of group-housed fish. Collectively, our results suggest that sharing housing with others in early life might influence whole-life attributes, potentially leading to specific life history traits beyond the typical relationship between the growth rate and lifespan.

Effective toxicity of pH to embryonic development of the banded lampeye killifish Aplocheilichthys spilauchen (Duméril, 1861).

The banded lampeye killifish, Aplocheilichthys spilauchen, feeds on larvae of the anopheles mosquito, which makes the fish a good candidate for biological control of malaria. Aquatic organisms require optimal hydrogen ion concentration (pH) for healthy aquatic life, but the impact of pH alteration on A. spilauchen reproductive potential is not known. This study investigated the toxic effect of pH on the embryonic development of A. spilauchen using a static renewal exposure method. Newly fertilised embryos were exposed to a range of pH treatments (5-6 acidic; 7 neutral; 8-9 alkaline) until hatching. All pH media involved triplicates and pH 7 was used as a control. The pH media were checked every 24 h for change and adjusted if necessary. Dissolved oxygen (DO), electrical conductivity (EC), and temperature were monitored throughout the experiment. The endpoints investigated were hatching success, hatchling length, and hatching period. Results indicated a mean hatching success of 95.83 % for pH 7 (neutral) group, while embryos exposed to acidic and basic media recorded lower hatching successes of 60.42 % and 83.34 %, respectively. Hatchling length and hatching period were longer in the control group than in groups of acidic and basic media. Effective toxic pH values of 4.5, 6 and 6.5 were estimated for the hatching period, hatching success, and hatchling length, respectively. These results suggest that sharp changes in the pH of aquatic environments from neutral may adversely impact the reproductive potential of A. spilauchen via embryonic development. This is the first time effective toxic pH values have been estimated for A. spilauchen embryonic development.

Single-cell sequencing unveils the impact of aging on the progenitor cell diversity in the telencephalon of the female killifish N. furzeri.

The African turquoise killifish (Nothobranchius furzeri) combines a short lifespan with spontaneous age-associated loss of neuro-regenerative capacity, an intriguing trait atypical for a teleost. The impact of aging on the cellular composition of the adult stem cell niches, leading to this dramatic decline in the postnatal neuro- and gliogenesis, remains elusive. Single-cell RNA sequencing of the telencephalon of young adult female killifish of the short-lived GRZ-AD strain unveiled progenitors of glial and non-glial nature, different excitatory and inhibitory neuron subtypes, as well as non-neural cell types. Sub-clustering of the progenitors identified four radial glia (RG) cell types, two non-glial progenitor (NGP) and four intermediate (intercell) cell states. Two astroglia-like, one ependymal, and one neuroepithelial-like (NE) RG subtype were found at different locations in the forebrain in line with their role, while proliferative, active NGPs were spread throughout. Lineage inference pointed to NE-RG and NGPs as start and intercessor populations for glio- and neurogenesis. Upon aging, single-cell RNA sequencing revealed major perturbations in the proportions of the astroglia and intercell states, and in the molecular signatures of specific subtypes, including altered MAPK, mTOR, Notch, and Wnt pathways. This cell catalog of the young regeneration-competent killifish telencephalon, combined with the evidence for aging-related transcriptomic changes, presents a useful resource to understand the molecular basis of age-dependent neuroplasticity. This data is also available through an online database (killifishbrain_scseq).

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.

Genomes of the Orestias pupfish from the Andean Altiplano shed light on their evolutionary history and phylogenetic relationships within Cyprinodontiformes.

BACKGROUND: To unravel the evolutionary history of a complex group, a comprehensive reconstruction of its phylogenetic relationships is crucial. This requires meticulous taxon sampling and careful consideration of multiple characters to ensure a complete and accurate reconstruction. The phylogenetic position of the Orestias genus has been estimated partly on unavailable or incomplete information. As a consequence, it was assigned to the family Cyprindontidae, relating this Andean fish to other geographically distant genera distributed in the Mediterranean, Middle East and North and Central America. In this study, using complete genome sequencing, we aim to clarify the phylogenetic position of Orestias within the Cyprinodontiformes order. RESULTS: We sequenced the genome of three Orestias species from the Andean Altiplano. Our analysis revealed that the small genome size in this genus (~ 0.7 Gb) was caused by a contraction in transposable element (TE) content, particularly in DNA elements and short interspersed nuclear elements (SINEs). Using predicted gene sequences, we generated a phylogenetic tree of Cyprinodontiformes using 902 orthologs extracted from all 32 available genomes as well as three outgroup species. We complemented this analysis with a phylogenetic reconstruction and time calibration considering 12 molecular markers (eight nuclear and four mitochondrial genes) and a stratified taxon sampling to consider 198 species of nearly all families and genera of this order. Overall, our results show that phylogenetic closeness is directly related to geographical distance. Importantly, we found that Orestias is not part of the Cyprinodontidae family, and that it is more closely related to the South American fish fauna, being the Fluviphylacidae the closest sister group. CONCLUSIONS: The evolutionary history of the Orestias genus is linked to the South American ichthyofauna and it should no longer be considered a member of the Cyprinodontidae family. Instead, we submit that Orestias belongs to the Orestiidae family, as suggested by Freyhof et al. (2017), and that it is the sister group of the Fluviphylacidae family, distributed in the Amazonian and Orinoco basins. These two groups likely diverged during the Late Eocene concomitant with hydrogeological changes in the South American landscape.

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.

Increased eye size is favoured in Trinidadian killifish experimentally transplanted into low light, high competition environments.

Intraspecific variation in vertebrate eye size is well known. Ecological factors such as light availability are often correlated with shifts in relative eye size. However, experimental tests of selection on eye size are lacking. Trinidadian killifish (Anablepsoides hartii) are found in sites that differ in predation intensity. Sites that lack predators are characterized by lower light, high killifish densities, low resource availability, and intense competition for food. We previously found that killifish in sites that lack predators have evolved a larger "relative" eye size (eye size corrected for body size) than fish from sites with predators. Here, we used transplant experiments to test how selection operates on eye size when fish that are adapted to sites with predators are translocated into sites where predators are absent. We observed a significant "population × relative eye size" interaction; the relationship between relative eye size and a proxy for fitness (rates of individual growth) was positive in the transplanted fish. The trend was the opposite for resident fish. Such results provide experimental support that larger eyes enhance fitness and are favoured in environments characterized by low light and high competition.

Identification of protein aggregates in the aging vertebrate brain with prion-like and phase-separation properties.

Protein aggregation, which can sometimes spread in a prion-like manner, is a hallmark of neurodegenerative diseases. However, whether prion-like aggregates form during normal brain aging remains unknown. Here, we use quantitative proteomics in the African turquoise killifish to identify protein aggregates that accumulate in old vertebrate brains. These aggregates are enriched for prion-like RNA-binding proteins, notably the ATP-dependent RNA helicase DDX5. We validate that DDX5 forms aggregate-like puncta in the brains of old killifish and mice. Interestingly, DDX5's prion-like domain allows these aggregates to propagate across many generations in yeast. In vitro, DDX5 phase separates into condensates. Mutations that abolish DDX5 prion propagation also impair the protein's ability to phase separate. DDX5 condensates exhibit enhanced enzymatic activity, but they can mature into inactive, solid aggregates. Our findings suggest that protein aggregates with prion-like properties form during normal brain aging, which could have implications for the age-dependency of cognitive decline.

Evidence for Selection on Mitochondrial OXPHOS Genes in the Mediterranean Killifish Aphanius fasciatus Valenciennes, 1821.

Mitochondrial oxidative phosphorylation (OXPHOS) genes are a system subject to selection under determined environmental constraints despite a neutral evolution model that has long been hypothesized for the mitochondrial genome. In this study, the sequences of ND1, Cytb, and COI OXPHOS genes were analyzed in six populations of the eurythermal and euryhaline killifish A. fasciatus, to detect non-synonymous mutations leading to amino acid changes and to check whether selection acted on them using tests of recombination and selection. The results indicate a high COI and Cytb gene diversity and a high percentage of private haplotypes in all populations. In the Greek population, non-synonymous nucleotide substitutions were observed in the N-terminal region of COI and Cytb. Positively selected sites were also found. The information we obtained from the mitochondrial DNA sequences of A. fasciatus adds to the growing data on selective pressure acting on mitochondrial DNA in non-model species. These results should be explored from the perspective of the local adaptation of eurythermal and euryhaline species and supported using experimental evidence to better understand the interplay between historical climatic events and local adaptation and how each of them contributes to shaping the genetic structure of this species.

Aluminum and ABC transporter activity.

Aluminum is the third most common element on Earth´s crust and despite its wide use in our workaday life it has been associated with several health risks after overexposure. In the present study the impact of aluminum salts upon ABC transporter activity was studied in the P-GP-expressing human blood-brain barrier cell line hCMEC/D3, in MDCKII cells overexpressing BCRP and MRP2, respectively, and in freshly isolated, functionally intact kidney tubules from Atlantic killifish (Fundulus heteroclitus), which express the analog ABC transporters, P-gp, Bcrp and Mrp2. In contrast to previous findings with heavy metals salts (cadmium(II) chloride or mercury(II) chloride), which have a strong inhibitory effect on ABC transporter activity, or zinc(II) chloride and sodium arsenite, which have a stimulatory effect upon ABC transport function, the results indicate no modulatory effect of aluminum salts on the efflux activity of the human ABC transporters P-GP, BCRP and MRP2 nor on the analog transporters P-gp, Bcrp and Mrp2.

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.

Immune aging in annual killifish.

Turquoise killifish (Nothobranchius furzeri) evolved a naturally short lifespan of about six months and exhibit aging hallmarks that affect multiple organs. These hallmarks include protein aggregation, telomere shortening, cellular senescence, and systemic inflammation. Turquoise killifish possess the full spectrum of vertebrate-specific innate and adaptive immune system. However, during their recent evolutionary history, they lost subsets of mucosal-specific antibody isoforms that are present in other teleosts. As they age, the immune system of turquoise killifish undergoes dramatic cellular and systemic changes. These changes involve increased inflammation, reduced antibody diversity, an increased prevalence of pathogenic microbes in the intestine, and extensive DNA damage in immune progenitor cell clusters. Collectively, the wide array of age-related changes occurring in turquoise killifish suggest that, despite an evolutionary separation spanning hundreds of millions of years, teleosts and mammals share common features of immune system aging. Hence, the spontaneous aging observed in the killifish immune system offers an excellent opportunity for discovering fundamental and conserved aspects associated with immune system aging across vertebrates. Additionally, the species' naturally short lifespan of only a few months, along with its experimental accessibility, offers a robust platform for testing interventions to improve age-related dysfunctions in the whole organism and potentially inform the development of immune-based therapies for human aging-related diseases.

Fish predation affects invertebrate community structure of tropical temporary ponds, with downstream effects on phytoplankton that are obscured by pesticide pollution.

Aquatic biota of tropical temporary ponds typically experience a wide range of stressors that can drive the structure and dynamics of natural communities. Particularly in regions with intense agricultural activity, aquatic biota may not only experience predation pressure but also stress from pesticides that inadvertently enter the ponds. We increasingly understand how these different sources of stress affect classic model taxa under controlled laboratory conditions, but how predators and pesticides may jointly affect pond invertebrate communities is still unclear, particularly for tropical systems. Here, we conducted an outdoor mesocosm experiment to study how fish predation combined with exposure to an environmentally relevant concentration of the commonly used insecticide cypermethrin (0.8 ng/L) affects the structure of invertebrate communities, and its potential effects on leaf litter decomposition and invertebrate grazing efficiency as measures of ecosystem functioning. A total of seven invertebrate taxa were recorded in the mesocosm communities. Fish predation effectively lowered the number of invertebrate taxa, with fish mesocosms being dominated by high densities of rotifers, associated with lower phytoplankton levels, but only when communities were not simultaneously exposed to cypermethrin. In contrast, cypermethrin exposure did not affect invertebrate community structure, and neither fish predation nor cypermethrin exposure affected our measures of ecosystem functioning. These findings suggest that predation by killifish can strongly affect invertebrate community structure of tropical temporary ponds, and that downstream effects on phytoplankton biomass can be mediated by exposure to cypermethrin. More broadly, we contend that a deeper understanding of (tropical) temporary pond ecology is necessary to effectively manage these increasingly polluted systems.

Impaired biogenesis of basic proteins impacts multiple hallmarks of the aging brain.

Aging and neurodegeneration entail diverse cellular and molecular hallmarks. Here, we studied the effects of aging on the transcriptome, translatome, and multiple layers of the proteome in the brain of a short-lived killifish. We reveal that aging causes widespread reduction of proteins enriched in basic amino acids that is independent of mRNA regulation, and it is not due to impaired proteasome activity. Instead, we identify a cascade of events where aberrant translation pausing leads to reduced ribosome availability resulting in proteome remodeling independently of transcriptional regulation. Our research uncovers a vulnerable point in the aging brain's biology - the biogenesis of basic DNA/RNA binding proteins. This vulnerability may represent a unifying principle that connects various aging hallmarks, encompassing genome integrity and the biosynthesis of macromolecules.

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.

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.

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.

Immunohistochemical characterisation of the adult Nothobranchius furzeri intestine.

Nothobranchius furzeri is emerging as an exciting vertebrate organism in the field of biomedicine, developmental biology and ecotoxicology research. Its short generation time, compressed lifespan and accelerated ageing make it a versatile model for longitudinal studies with high traceability. Although in recent years the use of this model has increased enormously, there is still little information on the anatomy, morphology and histology of its main organs. In this paper, we present a description of the digestive system of N. furzeri, with emphasis on the intestine. We note that the general architecture of the intestinal tissue is shared with other vertebrates, and includes a folding mucosa, an outer muscle layer and a myenteric plexus. By immunohistochemical analysis, we reveal that the mucosa harbours the same type of epithelial cells observed in mammals, including enterocytes, goblet cells and enteroendocrine cells, and that the myenteric neurons express neurotransmitters common to other species, such as serotonin, substance P and tyrosine hydroxylase. In addition, we detect the presence of a proliferative compartment at the base of the intestinal folds. The description of the normal intestinal morphology provided here constitutes a baseline information to contrast with tissue alterations in future lines of research assessing pathologies, ageing-related diseases or damage caused by toxic agents.

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.