Annual Killifish Research Articles

Life in the fastlane? A comparative analysis of gene expression profiles across annual, semi-annual, and non-annual killifishes (Cyprinodontiformes: Nothobranchiidae).

The Turquoise Killifish is an important vertebrate for the study of aging and age-related diseases due to its short lifespan. Within Nothobranchiidae, species possess annual, semi-annual, or non-annual life-histories. We took a comparative approach and examined gene expression profiles (QuantSeq) from 62 individuals from eleven nothobranchid species that span three life-histories. Our results show significant differences in differentially expressed genes (DEGs) across life-histories with non-annuals and semi-annuals being most similar, and annuals being the most distinct. At finer scales, we recovered significant differences in DEGs for DNA repair genes and show that non-annual and semi-annuals share similar gene expression profiles, while annuals are distinct. Most of the GO terms enriched in annuals are related to metabolic processes. However, GO terms, including translation, protein transport, and DNA replication initiation also are enriched in annuals. Non-annuals are enriched in Notch signaling pathway genes and downregulated in the canonical Wnt signaling pathway compared to annual species, which suggests that non-annuals have stronger regulation in cellular processes. This study provides support for congruency in DEGs involved in these life-histories and provides strong evidence that a particular set of candidate genes may be worthy of study to investigate their role in the aging process.

Assessing reproductive effects and epigenetic responses in Austrolebias charrua exposed to Roundup Transorb®: Insights from miRNA profiling and molecular interaction analysis

This study examines the effects of Roundup Transorb® (RDT) exposure on reproductive functions and ovarian miRNA expression in Austrolebias charrua. Exposure to RDT (at 0.065 or 5 mg. L−1 for 96 h) significantly disrupts fertility, evidenced by changes in fertilization rates and egg diameter. Profiling of ovarian miRNAs identified a total 205 miRNAs in A. charrua. Among these, three miRNAs were upregulated (miR-10b-5p, miR-132–3p, miR-100–5p), while ten miRNAs were downregulated (miR-499–5p, miR-375, miR-205–5p, miR-206–3p, miR-203a-3p, miR-133b-3p, miR-203b-5p, miR-184, miR-133a-3p, miR-2188–5p) compared to non-exposed fish. This study reveals that differentially expressed miRNAs are linked to molecular pathways such as steroid hormone biosynthesis, lipid and carbohydrate metabolism, bioenergetics, and antioxidant defense. It also analyzes molecular interactions between miRNAs and target genes during RDT exposure in annual killifish, providing insights into biomarkers in ecotoxicology. Moreover, it provides scope for developing environmental health assessment models based on epigenomic endpoints, supporting the protection of biodiversity and ecosystem services through the quantification of stress responses in living organisms exposed to pesticides.

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 5mg/L GAE) on A. charrua exposed for 96h. 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.

Intra-sexual selection in a North American annual killifish: does the color-polymorphism matter?

Intra-sexual selection in a North American annual killifish: does the color-polymorphism matter?

Axis formation in annual killifish: Nodal and β-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 β-catenin, is truncated and inactive in Nothobranchius furzeri. Nuclear β-catenin is not selectively stabilized on one side of the blastula but accumulates in cells forming the aggregate. Blocking β-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 β-catenin and Nodal regulate morphogenesis.

Exploring the molecular basis of diapause I induction in the annual killifish Garcialebias charrua: a transcriptomic approach

Exploring the molecular basis of diapause I induction in the annual killifish Garcialebias charrua: a transcriptomic approach

The complete genome sequence of the protogynous hermaphrodite North American annual killifish Millerichthys robustus (Rivulidae, Cyprinodontiformes).

The Mexican Rivulus, Millerichthys robustus, is an enigmatic species of seasonal killifish endemic of the Southeast Mexico that has changed paradigms on the evolution of annualism in killifishes. This species survives in ephemeral environments that experience a period of seasonal drought that causes the death of all adult fish. However, populations persist due to their drought-resistant embryos capable of arresting their development in diapause until the next the rainy season. Millerichthys evolved unique characteristics within annual killifishes as a functional sequential hermaphroditism, in which females change to males (protogynous) under perceived conditions of mate competition. Also, M. robustus express different color phenotypes in both sexes: five-color phenotypes continuously distributed in various perceptual units between yellow and red in males, and different number of ocelli disposed in caudal peduncle in females. The phylogenetic relationships of M. robustus revealed that it is a sister clade to two non-annual species found exclusively in Cuba (Rivulus cylindraceus and Rivulus berovidesi), indicating that the annual life cycle, through the acquisition of embryonic diapause, has evolved independently in this species. Here, we present the complete genome sequences for the North American annual killifish Millerichthys robustus. The raw data and assembled genome are available in GeneBank.

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.

An independent origin of an annual life cycle in a North American killifish species

Abstract An annual life cycle is characterized by short-lived adults that exploit seasonally productive but temporary habitats, paired with a hardy embryo stage that survives unfavourable environmental conditions, such as winter or the dry season. This life cycle is rare amongst vertebrates but has evolved independently several times in African and South American killifishes adapted to life in seasonally ephemeral aquatic pools. A single species of North American annual killifish, with a limited geographical distribution in Mexico, also exhibits this seasonal life cycle. This enigmatic species, Millerichthys robustus, is geographically separated from the nearest South American annual killifish species by >2000 km. Millerichthys robustus exhibits morphological features that have led to several competing phylogenetic hypotheses. To date, its phylogenetic relationships are unclear because no molecular data have been available. Here, we sequence the mitochondrial genome of Millerichthys robustus and generate a molecular phylogeny of killifishes that includes this species. Our results indicate that, rather than being most closely related to South American annual killifishes, this species is sister to two non-annual killifish species from Cuba (Rivulus cylindraceus and Rivulus berovidesi). Ancestral state reconstruction strongly supports an independent origin of an annual life cycle and embryonic diapause in Millerichthys robustus.

Neurotrophins and Trk Neurotrophin Receptors in the Retina of Adult Killifish (Nothobranchius guentheri).

Specific subpopulations of neurons in nerve and sensory systems must be developed and maintained, and this is accomplished in significant part by neurotrophins (NTs) and the signaling receptors on which they act, called tyrosine protein kinase receptors (Trks). The neurotrophins-tyrosine protein kinase receptors (NTs/Trks) system is involved in sensory organ regulation, including the visual system. An NTs/Trks system alteration is associated with neurodegeneration related to aging and diseases, including retinal pathologies. An emergent model in the field of translational medicine, for instance, in aging study, is the annual killifish belonging to the Nothobranchius genus, thanks to its short lifespan. Members of this genus, such as Nothobranchius guentheri, and humans share a similar retinal stratigraphy. Nevertheless, according to the authors' knowledge, the occurrence and distribution of the NTs/Trks system in the retina of N. guentheri has never been investigated before. Therefore, the present study aimed to localize neurotrophin BDNF, NGF, and NT-3 and TrkA, TrkB, and TrkC receptors in the N. guentheri retina using the immunofluorescence method. The present investigation demonstrates, for the first time, the occurrence of the NTs/Trks system in N. guentheri retina and, consequently, the potential key role of these proteins in the biology and survival of the retinal cells.

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.

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.

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

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

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.

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

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

Is African non-annual killifish Fundulopanchax gardneri (Teleostei; Cyprinodontiformes; Nothobranchiidae) true non-annual?

Annual or seasonal killifishes (Cyprinodontiformes: Nothobranchiidae) are unique among fish in their ability to enter into developmental arrests (diapauses: DI, DII, and DIII). They have a short lifespan and their embryos are exceptionally tolerant to a variety of environmental stresses. These traits make them a popular model for studying vertebrate diapause, aging, stress tolerance, genome adaptation, and evolution. In such issues, in a comparative evolutionary framework, Fundulopanchax gardneri, a popular aquarium fish from Africa, is commonly used as a representative non-annual model though its development is not studied in detail and whether it includes diapauses remains uncertain. We described in detail for the first time embryonic development of F. gardneri and revealed it to resemble that in the undoubtedly annual Austrofundulus limnaeus killifish in displaying two developmental depressions. However, if compared with A. limnaeus, these developmental states look like "less intense" versions of DII and DIII rather than true diapauses. To determine whether developmental depressions in F. gardneri represent "true" diapauses or only their functional equivalents, detailed studies of embryonic development of different killifish both annual and non-annual are needed. Before that, acceptance of F. gardneri as a representative non-annual fish seems premature.

Localization of Calretinin, Parvalbumin, and S100 Protein in Nothobranchius guentheri Retina: A Suitable Model for the Retina Aging.

Calcium-binding proteins (CaBPs) are members of a heterogeneous family of proteins able to buffer intracellular Ca2+ ion concentration. CaBPs are expressed in the central and peripheral nervous system, including a subpopulation of retinal neurons. Since neurons expressing different CaBPs show different susceptibility to degeneration, it could be hypothesized that they are not just markers of different neuronal subpopulations, but that they might be crucial in survival. CaBPs' ability to buffer Ca2+ cytoplasmatic concentration makes them able to defend against a toxic increase in intracellular calcium that can lead to neurodegenerative processes, including those related to aging. An emergent model for aging studies is the annual killifish belonging to the Nothobranchius genus, thanks to its short lifespan. Members of this genus, such as Nothobranchius guentheri, show a retinal stratigraphy similar to that of other actinopterygian fishes and humans. However, according to our knowledge, CaBPs' occurrence and distribution in the retina of N. guentheri have never been investigated before. Therefore, the present study aimed to localize Calretinin N-18, Parvalbumin, and S100 protein (S100p) in the N. guentheri retina with immunohistochemistry methods. The results of the present investigation demonstrate for the first time the occurrence of Calretinin N-18, Parvalbumin, and S100p in N. guentheri retina and, consequently, the potential key role of these CaBPs in the biology of the retinal cells. Hence, the suitability of N. guentheri as a model to study the changes in CaBPs' expression patterns during neurodegenerative processes affecting the retina related both to disease and aging can be assumed.

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

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

Glyphosate-induced biochemical changes in female Cynopoecilus sp. inhabiting temporary wetlands

Glyphosate is Brazil’s most widely used pesticide, often applied to different crops that may be close to temporary wetlands. This pesticide poses a threat to populations of annual killifish that live in these locations. Few studies have explored the toxicodynamics of pesticides in annual killifish. Here females of Cynopoecilus sp. at different development stages (young-adult and senile) are exposed to Roundup Original DI® herbicide (65, 130 and 260 µg/L of acid equivalent of N-phosphomethyl glycine (glyphosate)) to analyze its impact on oxidative balance and lipoperoxidation, including superoxide dismutase (SOD), catalase (CAT), glutathione S-transferase (GST), and SOD/CAT ratio, as well as markers of intermediary metabolism such as total proteins (TPs), glycogen, and uric acid (UA). Senile females showed increased CAT and glutathione S-transferase activities and a decreased SOD/CAT ratio. Total protein and UA values were increased. Two-way ANOVA demonstrated correlations between age and different exposure groups for lipoperoxidation, SOD, CAT, GST, TPs, and UA, especially in senile females. Although it is not considered a target species, Cynopoecilus sp. suffers harmful effects from the interaction with this herbicide even at environmentally relevant concentrations. This impact was observed in senile females, which may result in reduced life cycle and reproduction quality.

Embryonic development of natural annual killifish populations of the genus Austrolebias: Evolutionary parallelism and the role of environment

Abstract Repeated, independent emergence of the same trait within different phylogenetic lineages is termed parallel evolution. It typically occurs as a result of similar selective pressures. Annual killifish have adapted to survive in the extreme habitat of temporary pools on three continents and present an especially amenable system for studying fundamental principles of evolutionary parallelism. When the pools dry, annual killifish embryos survive through the dry phase in the bottom substrate in a stage of dormancy—a diapause. The diapause is a complex set of three different developmental stages, none of which is obligate, thus leading to a multitude of potential developmental trajectories. While the intricacy of the killifishes' embryonic development has been thoroughly studied in the laboratory, information on their natural development is virtually absent. We hypothesised that the natural development of annual killifishes is largely synchronised and governed by ambient conditions as shown in the lineage of the African genus Nothobranchius. We sampled wild embryo banks of the South American genus Austrolebias, which evolved its diapause independently of the African lineage. We sampled during two consecutive dry seasons, using both longitudinal and snapshot monitoring, and conducted transplant experiments to determine the extent of the evolutionary parallelism and role of the environment in Austrolebias spp. embryo development. Main habitat phases were characterised by largely synchronised embryo banks. Different inter‐seasonal or local environmental conditions were reflected in a different developmental profile of the embryo banks, suggesting a high degree of environmental control. We found striking similarity in the habitat phase–embryo stage associations between the two lineages. The diapause in the two annual killifish lineages represents a unique example of evolutionary parallelism, with the analogy manifested in very close detail. We highlight the similarity of the selective forces in the two genera despite the different geographic origins, climate zones and reversed seasonality. The repeatedly occurring strict association of the same developmental stages with the same habitat conditions suggests a limited array of developmental settings that can be applied to cope with the given environmental challenges.