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.

Advancing human disease research with fish evolutionary mutant models.

Model organism research is essential to understand disease mechanisms. However, laboratory-induced genetic models can lack genetic variation and often fail to mimic the spectrum of disease severity. Evolutionary mutant models (EMMs) are species with evolved phenotypes that mimic human disease. EMMs complement traditional laboratory models by providing unique avenues to study gene-by-environment interactions, modular mutations in noncoding regions, and their evolved compensations. EMMs have improved our understanding of complex diseases, including cancer, diabetes, and aging, and illuminated mechanisms in many organs. Rapid advancements of sequencing and genome-editing technologies have catapulted the utility of EMMs, particularly in fish. Fish are the most diverse group of vertebrates, exhibiting a kaleidoscope of specialized phenotypes, many that would be pathogenic in humans but are adaptive in the species' specialized habitat. Importantly, evolved compensations can suggest avenues for novel disease therapies. This review summarizes current research using fish EMMs to advance our understanding of human disease.

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.

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.

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.

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.

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.

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.

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.

Transplant experiments demonstrate that larger brains are favoured in high-competition environments in Trinidadian killifish.

The extent to which the evolution of a larger brain is adaptive remains controversial. Trinidadian killifish (Anablepsoides hartii) are found in sites that differ in predation intensity; fish that experience decreased predation and increased intraspecific competition exhibit larger brains. We evaluated the connection between brain size and fitness (survival and growth) when killifish are found in their native habitats and when fish are transplanted from sites with predators to high-competition sites that lack predators. Selection for a larger brain was absent within locally adapted populations. Conversely, there was a strong positive relationship between brain size and growth in transplanted but not resident fish in high-competition environments. We also observed significantly larger brain sizes in the transplanted fish that were recaptured at the end of the experiment versus those that were not. Our results provide experimental support that larger brains increase fitness and are favoured in high-competition environments.

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.

Genetic tools for the study of the mangrove killifish, Kryptolebias marmoratus, an emerging vertebrate model for phenotypic plasticity.

Kryptolebias marmoratus (Kmar), a teleost fish of the order Cyprinodontiformes, has a suite of unique phenotypes and behaviors not observed in other fishes. Many of these phenotypes are discrete and highly plastic-varying over time within an individual, and in some cases reversible. Kmar and its interfertile sister species, K. hermaphroditus, are the only known self-fertile vertebrates. This unusual sexual mode has the potential to provide unique insights into the regulation of vertebrate sexual development, and also lends itself to genetics. Kmar is easily adapted to the lab and requires little maintenance. However, its internal fertilization and small clutch size limits its experimental use. To support Kmar as a genetic model, we compared alternative husbandry techniques to maximize recovery of early cleavage-stage embryos. We find that frequent egg collection enhances yield, and that protease treatment promotes the greatest hatching success. We completed a forward mutagenesis screen and recovered several mutant lines that serve as important tools for genetics in this model. Several will serve as useful viable recessive markers for marking crosses. Importantly, the mutant kissylips lays embryos at twice the rate of wild-type. Combining frequent egg collection with the kissylips mutant background allows for a substantial enhancement of early embryo yield. These improvements were sufficient to allow experimental analysis of early development and the successful mono- and bi-allelic targeted knockout of an endogenous tyrosinase gene with CRISPR/Cas9 nucleases. Collectively, these tools will facilitate modern developmental genetics in this fascinating fish, leading to future insights into the regulation of plasticity.

Nephrotoxicity of iopamidol is associated with mitochondrial impairment in human cell and teleost models.

Iopamidol is a nonionic, low-osmolar iodinated contrast agent used for angiography. Its clinical use is associated with renal dysfunction. Patients suffering from preexisting kidney disease have an increased risk of renal failure upon iopamidol administration. Studies in animals confirmed renal toxicity, but the involved mechanisms remain unclear. Therefore, the aim of the present study was to use human embryonic kidney cells (HEK293T) as a general cell model of mitochondrial damage, as well as, zebrafish larvae, and isolated proximal tubules of killifish to investigate factors promoting renal tubular toxicity of iopamidol with a focus on mitochondrial damage. Results from in vitro HEK293T cell-based assays indicate that iopamidol affects mitochondrial function Treatment with iopamidol induces ATP depletion, reduces the mitochondrial membrane potential, and elevates mitochondrial superoxide and reactive oxygen species accumulation. Similar results were obtained with gentamicin sulfate and cadmium chloride, two well-known model compounds associated with renal tubular toxicity. Confocal microscopy confirms changes in mitochondrial morphology, such as mitochondrial fission. Importantly, these results were confirmed in proximal renal tubular epithelial cells using ex vivo and in vivo teleost models. In conclusion, this study provides evidence for iopamidol-induced mitochondrial damage in proximal renal epithelial cells. Teleost models allow studying proximal tubular toxicity with translational relevance for humans.

Tissue stretching is a confounding factor for the evaluation of neurodegeneration in the fast-ageing killifish.

The fast-ageing killifish has gained increasing attention as a promising gerontology model to study age-related processes and neurodegeneration. Interestingly, it is the first vertebrate model organism that shows physiological neuron loss at old age in its central nervous system (CNS), including its brain and retina. However, the fact that the killifish brain and retina are ever-growing tissues complicates studying neurodegenerative events in aged fish. Indeed, recent studies showed that the method of tissue sampling, either using sections or whole-organs, has a large effect on the observed cell densities in the fast-expanding CNS. Here, we elaborated on how these two sampling methods affect neuronal counts in the senescent retina and how this tissue grows upon ageing. Analysis of the different retinal layers in cryosections revealed age-dependent reduction in cellular density but evaluation of whole-mount retinas did not detect any neuron loss, as a result of an extremely fast retinal expansion with age. Using BrdU pulse-chase experiments, we showed that the young adult killifish retina mainly grows by cell addition. However, with increasing age, the neurogenic potency of the retina declines while the tissue keeps on growing. Further histological analyses revealed tissue stretching, including cell size increase, as the main driver of retinal growth at old age. Indeed, both cell size and inter-neuronal distance augment with ageing, thereby decreasing neuronal density. All in all, our findings urge the 'ageing science' community to consider cell quantification bias and employ tissue-wide counting methods to reliably quantify neuronal numbers in this unique gerontology model.

Optimizing preclinical models of ageing for translation to clinical trials.

Preclinical models have been the backbone of translational research for more than a century. Rats and mice are critical models in the preliminary stages of drug testing, both for determining efficacy and ruling out potential human-relevant toxicities. Historically, most preclinical pharmacological studies have used young, relatively healthy, inbred male models in highly controlled environments. In the field of geriatric pharmacology, there is a growing focus on the importance of using more appropriate preclinical models both in the testing of therapeutics commonly used in older populations, and in the evaluation of potential geroprotective drug candidates. Here we provide a commentary on optimizing preclinical models of ageing for translation to clinical trials. We will discuss approaches to modelling clinically relevant contexts such as age, sex, genetic diversity, exposures and environment, as well as measures of clinically relevant outcomes such as frailty and healthspan. We will identify the strengths and limitations of these approaches and areas for improvement. We will also briefly cover new preclinical models that move beyond rodents. We hope this commentary will be a springboard for larger discussions on optimizing preclinical ageing models for testing therapeutics.

Fish Models for Exploring Mitochondrial Dysfunction Affecting Neurodegenerative Disorders.

Neurodegenerative disorders are characterized by the progressive loss of neuronal structure or function, resulting in memory loss and movement disorders. Although the detailed pathogenic mechanism has not been elucidated, it is thought to be related to the loss of mitochondrial function in the process of aging. Animal models that mimic the pathology of a disease are essential for understanding human diseases. In recent years, small fish have become ideal vertebrate models for human disease due to their high genetic and histological homology to humans, ease of in vivo imaging, and ease of genetic manipulation. In this review, we first outline the impact of mitochondrial dysfunction on the progression of neurodegenerative diseases. Then, we highlight the advantages of small fish as model organisms, and present examples of previous studies regarding mitochondria-related neuronal disorders. Lastly, we discuss the applicability of the turquoise killifish, a unique model for aging research, as a model for neurodegenerative diseases. Small fish models are expected to advance our understanding of the mitochondrial function in vivo, the pathogenesis of neurodegenerative diseases, and be important tools for developing therapies to treat diseases.

Age-Related Alterations in the Level and Metabolism of Serotonin in the Brain of Males and Females of Annual Turquoise Killifish (Nothobranchius furzeri).

The annual turquoise killifish (Nothobranchius furzeri) is a laboratory model organism for neuroscience of aging. In the present study, we investigated for the first time the levels of serotonin and its main metabolite, 5-hydroxyindoleacetic acid, as well as the activities of the key enzymes of its synthesis, tryptophan hydroxylases, and degradation, monoamine oxidase, in the brains of 2-, 4- and 7-month-old male and female N. furzeri. The marked effect of age on the body mass and the level of serotonin, as well as the activities of tryptophan hydroxylases and monoamine oxidase in the brain of killifish were revealed. The level of serotonin decreased in the brain of 7-month-old males and females compared with 2-month-old ones. A significant decrease in the tryptophan hydroxylase activity and an increase in the monoamine oxidase activity in the brain of 7-month-old females compared to 2-month-old females was shown. These findings agree with the age-related alterations in expression of the genes encoding tryptophan hydroxylases and monoamine oxidase. N. furzeri is a suitable model with which to study the fundamental problems of age-related changes of the serotonin system in the brain.

Physiological and behavioural strategies of aquatic animals living in fluctuating environments.

Shallow or near-shore environments, such as ponds, estuaries and intertidal zones, are among the most physiologically challenging of all aquatic settings. Animals inhabiting these environments experience conditions that fluctuate markedly over relatively short temporal and spatial scales. Living in these habitats requires the ability to tolerate the physiological disturbances incurred by these environmental fluctuations. This tolerance is achieved through a suite of physiological and behavioural responses that allow animals to maintain homeostasis, including the ability to dynamically modulate their physiology through reversible phenotypic plasticity. However, maintaining the plasticity to adjust to some stresses in a dynamic environment may trade off with the capacity to deal with other stressors. This paper will explore studies on select fishes and invertebrates exposed to fluctuations in dissolved oxygen, salinity and pH. We assess the physiological mechanisms these species employ to achieve homeostasis, with a focus on the plasticity of their responses, and consider the resulting physiological trade-offs in function. Finally, we discuss additional factors that may influence organismal responses to fluctuating environments, such as the presence of multiple stressors, including parasites. We echo recent calls from experimental biologists to consider physiological responses to life in naturally fluctuating environments, not only because they are interesting in their own right but also because they can reveal mechanisms that may be crucial for living with increasing environmental instability as a consequence of climate change.

Molecular targets of prolactin in mummichogs (Fundulus heteroclitus): Ion transporters/channels, aquaporins, and claudins.

Prolactin (Prl) was identified over 60 years ago in mummichogs (Fundulus heteroclitus) as a "freshwater (FW)-adapting hormone", yet the cellular and molecular targets of Prl in this model teleost have remained unknown. Here, we conducted a phylogenetic analysis of two mummichog Prl receptors (Prlrs), designated Prlra and Prlrb, prior to describing the tissue- and salinity-dependent expression of their associated mRNAs. We then administered ovine Prl (oPrl) to mummichogs held in brackish water and characterized the expression of genes associated with FW- and seawater (SW)-type ionocytes. Within FW-type ionocytes, oPrl stimulated the expression of Na+/Cl- cotransporter 2 (ncc2) and aquaporin 3 (aqp3). Alternatively, branchial Na+/H+ exchanger 2 and -3 (nhe2 and -3) expression did not respond to oPrl. Gene transcripts associated with SW-type ionocytes, including Na+/K+/2Cl- cotransporter 1 (nkcc1), cystic fibrosis transmembrane regulator 1 (cftr1), and claudin 10f (cldn10f) were reduced by oPrl. Isolated gill filaments incubated with oPrl in vitro exhibited elevated ncc2 and prlra expression. Given the role of Aqps in supporting gastrointestinal fluid absorption, we assessed whether several intestinal aqp transcripts were responsive to oPrl and found that aqp1a and -8 levels were reduced by oPrl. Our collective data indicate that Prl promotes FW-acclimation in mummichogs by orchestrating the expression of solute transporters/channels, water channels, and tight-junction proteins across multiple osmoregulatory organs.