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.

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.

Fish (eggs) out of water: evolutionary divergence in terrestrial embryonic plasticity in Trinidadian killifish.

Externally laid eggs are often responsive to environmental cues; however, it is unclear how such plasticity evolves. In Trinidad, the killifish (Anablepsoides hartii) is found in communities with and without predators. Here, killifish inhabit shallower, ephemeral habitats in sites with predators. Such shifts may increase the exposure of eggs to air and lead to possible desiccation. We compared egg-hatching plasticity between communities by rearing eggs terrestrially on peat moss or in water. The timing of hatching did not differ between communities when eggs were reared in water. Eggs from sites with predators responded to terrestrial incubation by hatching significantly earlier compared with water-reared eggs. These responses were weaker in sites with no predators. Such divergent trends show that the presence of predators is associated with evolutionary shifts in hatching plasticity. Our results provide evidence for local adaptation in embryonic plasticity at the population scale.

Axis formation in annual killifish: Nodal and ß-catenin regulate morphogenesis without Huluwa prepatterning.

Axis formation in fish and amphibians typically begins with a prepattern of maternal gene products. Annual killifish embryogenesis, however, challenges prepatterning models as blastomeres disperse and then aggregate to form the germ layers and body axes. We show that huluwa, a prepatterning factor thought to break symmetry by stabilizing ß-catenin, is truncated and inactive in Nothobranchius furzeri. Nuclear ß-catenin is not selectively stabilized on one side of the blastula but accumulates in cells forming the aggregate. Blocking ß-catenin activity or Nodal signaling disrupts aggregate formation and germ layer specification. Nodal signaling coordinates cell migration, establishing an early role for this signaling pathway. These results reveal a surprising departure from established mechanisms of axis formation: Huluwa-mediated prepatterning is dispensable, and ß-catenin and Nodal regulate morphogenesis.

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.

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.

Age-related dysregulation of the retinal transcriptome in African turquoise killifish.

Age-related vision loss caused by retinal neurodegenerative pathologies is becoming more prevalent in our ageing society. To understand the physiological and molecular impact of ageing on retinal homeostasis, we used the short-lived African turquoise killifish, a model known to naturally develop central nervous system (CNS) ageing hallmarks and vision loss. Bulk and single-cell RNA-sequencing (scRNAseq) of three age groups (6-, 12-, and 18-week-old) identified transcriptional ageing fingerprints in the killifish retina, unveiling pathways also identified in the aged brain, including oxidative stress, gliosis, and inflammageing. These findings were comparable to observations in the ageing mouse retina. Additionally, transcriptional changes in genes related to retinal diseases, such as glaucoma and age-related macular degeneration, were observed. The cellular heterogeneity in the killifish retina was characterized, confirming the presence of all typical vertebrate retinal cell types. Data integration from age-matched samples between the bulk and scRNAseq experiments revealed a loss of cellular specificity in gene expression upon ageing, suggesting potential disruption in transcriptional homeostasis. Differential expression analysis within the identified cell types highlighted the role of glial/immune cells as important stress regulators during ageing. Our work emphasizes the value of the fast-ageing killifish in elucidating molecular signatures in age-associated retinal disease and vision decline. This study contributes to the understanding of how age-related changes in molecular pathways may impact CNS health, providing insights that may inform future therapeutic strategies for age-related pathologies.

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.

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.

Morphofunctional analysis of the gastrointestinal tract of an annual killifish: In search of structural traits related to the annual life history.

Annual killifishes have active and voracious rates in acquisition of food resources with display of behaviors that allow them to maintain high metabolic rates to deal with the harsh and unpredictable conditions prevalent in temporary pools where they inhabit. The objective of this research was to describe histologically and ultrastructurally, the digestive system of the annual killifish Millerichthys robustus to identify morphological traits related to its annual life history and digestive physiology. Also, we quantify food items along the intestine as a proxy for rates of digestion. Millerichthys has a short digestive system, associated with a carnivorous diet, with no evidence of a stomach. Instead, the presence of pharyngeal jaws with caniform teeth was documented, related to the breakdown of invertebrate exoskeletons, allowing prey fluids to be tasted by taste corpuscles related to selection of food items, and that digestive enzymes penetrate once in the intestine. The histological morphology of the intestine showed four different regions, associated with its digestive rates: (i) reception of food from the esophagus with intact pray; (ii) digestion with enzymes from the pancreas and liver/gallbladder of simple exoskeleton prey (Entognatha), and beginning of absorption; (iii) absorption of nutrients, and digestion of large-complex exoskeleton prey (Hexanauplia, Brachiopoda, and Ostracoda); and (iv) probable absorption of intact macromolecules. The second region of the intestine presented two anatomical loops and the highest thickness that may be related to reducing the speed of food transit, allowing for more efficient digestion given the large amount of food ingested by this species.

Method dependency of maximum oxygen uptake rate and its repeatability in the Gulf killifish, Fundulus grandis.

The maximum rate at which fish can take up oxygen from their environment to fuel aerobic metabolism is an important feature of their physiology and ecology. Methods to quantify maximum oxygen uptake rate (MO2), therefore, should reliably and reproducibly estimate the highest possible MO2 by an individual or species under a given set of conditions (peak MO2). This study determined peak MO2 and its repeatability in Gulf killifish, Fundulus grandis, subjected to three methods to elevate metabolism: swimming at increasing water speeds, during recovery after an exhaustive chase, and after ingestion of a large meal. Estimates of peak MO2 during swimming and after an exhaustive chase were repeatable across two trials, whereas peak MO2 after feeding was not. Peak MO2 determined by the three methods was significantly different from one another, being highest during swimming, lowest after an exhaustive chase, and intermediate after feeding. In addition, peak MO2 during recovery from an exhaustive chase depended on the length of time of recovery: in nearly 60% of the trials, values within the first hour of the chase were lower than those measured later. A novel and important finding was that an individual's peak MO2 was not repeatable when compared across methods. Therefore, the peak MO2 estimated for a group of fish, as well as the ranking of individual MO2 within that group, depends on the method used to elevate aerobic metabolism.

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.

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.

Widespread sex dimorphism across single-cell transcriptomes of adult African turquoise killifish tissues.

The African turquoise killifish (Nothobranchius furzeri), the shortest-lived vertebrate that can be bred in captivity, is an emerging model organism for aging research. Here, we describe a multitissue, single-cell gene expression atlas of female and male blood, kidney, liver, and spleen. We annotate 22 cell types, define marker genes, and infer differentiation trajectories. We find pervasive sex-dimorphic gene expression across cell types. Sex-dimorphic genes tend to be linked to lipid metabolism, consistent with clear differences in lipid storage in female vs. male turquoise killifish livers. We use machine learning to predict sex using single-cell gene expression and identify potential markers for molecular sex identity. As a proof of principle, we show that our atlas can be used to deconvolute existing bulk RNA sequencing (RNA-seq) data to obtain accurate estimates of cell type proportions. This atlas can be a resource to the community that could be leveraged to develop cell-type-specific expression in transgenic animals.

A scalable and tunable platform for functional interrogation of peptide hormones in fish.

Pituitary hormones play a central role in shaping vertebrate life history events, including growth, reproduction, metabolism, and aging. The regulation of these traits often requires precise control of hormone levels across diverse timescales. However, fine tuning circulating hormones in-vivo has traditionally been experimentally challenging. Here, using the naturally short-lived turquoise killifish (N. furzeri), we describe a high-throughput platform that combines loss- and gain-of-function of peptide hormones. Mutation of three primary pituitary hormones, growth hormone (gh1), follicle stimulating hormone (fshb), and thyroid stimulating hormone (tshb), alters somatic growth and reproduction. Thus, suggesting that while the killifish undergoes extremely rapid growth and maturity, it still relies on vertebrate-conserved genetic networks. As the next stage, we developed a gain-of-function vector system in which a hormone is tagged using a self-cleavable fluorescent reporter, and ectopically expressed in-vivo through intramuscular electroporation. Following a single electroporation, phenotypes, such as reproduction, are stably rescued for several months. Notably, we demonstrate the versatility of this approach by using multiplexing, dose-dependent, and doxycycline-inducible systems to achieve tunable and reversible expression. In summary, this method is relatively high-throughput, and facilitates large-scale interrogation of life-history strategies in fish. Ultimately, this approach could be adapted for modifying aquaculture species and exploring pro-longevity interventions.

In humans and other vertebrates, a pea-size gland at the base of the brain called the pituitary gland, produces many hormones that regulate how individuals grow, reproduce, and age. Three of the most prominent hormones are known as the growth hormone, the follicle-stimulating hormone, and the thyroid-stimulating hormone. It is important that the body precisely controls the levels of these hormones throughout an individual's life. One way researchers can investigate how hormones and other molecules work is to artificially alter the levels of the molecules in living animals. However, this has proved to be technically challenging and time-consuming for pituitary gland hormones. Moses et al. studied the growth hormone, follicle-stimulating hormone, and thyroid-stimulating hormone in the turquoise killifish, a small fish that grows and matures more rapidly than any other vertebrate research model. The experiments revealed that mutant fish lacking one of the three primary pituitary hormones were smaller, took longer to reach maturity, or were completely sterile. This suggests these three hormones play a similar role in killifish as they do in other vertebrates. The team then developed a new experimental platform to precisely control the levels of the three hormones in killifish. Genes encoding individual hormones were expressed in the muscles of the mutant fish, effectively making the muscles a 'factory' for producing that hormone. Treating mutant fish this way once was enough to restore growth and to fully return reproduction to normal levels for several months. Moses et al. also demonstrated that it is possible to use this platform to express more than one hormone gene at a time and to use drugs to switch hormone production on and off in a reversible manner. For example, this reversible approach made it possible to effectively adjust fertility levels. The new platform developed in this work could be adapted for modifying a variety of traits in animals to explore how they impact health and longevity. In the future, it may also have other applications, such as optimizing how farmed fish grow and reproduce and regulating hormone levels in human patients with hormone imbalances.

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.

Bioenergetic Effects of Polycyclic Aromatic Hydrocarbon Resistance Manifest Later in Life in Offspring of Fundulus heteroclitus from the Elizabeth River.

Shifts in key physiological processes can confer resistance to chemical pollutants. However, these adaptations may come with certain trade-offs, such as altered energy metabolic processes, as evident in Atlantic killifish (Fundulus heteroclitus) in Virginia's Elizabeth River (ER) that have evolved resistance to polycyclic aromatic hydrocarbons (PAHs). We seek to understand the bioenergetic costs of PAH resistance among subpopulations of Atlantic killifish with differing contamination levels in order to examine how these changes manifest across multiple life stages and how these costs might be exacerbated by additional stressors. Bioenergetics data revealed differences in metabolic rates between offspring of PAH-resistant fish and reference fish were absent or minimal in both the embryo and larval stages but pronounced at the juvenile life stage, suggesting that bioenergetic changes in pollution-adapted killifish manifest later in life. We also provide evidence that killifish from remediated sites are more sensitive to PAH exposure than killifish from nonremediated sites, suggesting loss of PAH tolerance following relaxed selection. Collectively, our data suggest that the fitness consequences associated with evolved resistance to anthropogenic stressors may manifest differently over time and depend on the magnitude of the selection pressure. This information can be valuable in effective risk and remediation assessments as well as in broadening our understanding of species responses to environmental change.