Embryonic developmental arrest in the annual killifish Austrolebias charrua: A proteomic approach to diapause III.

Diapause is a reversible developmental arrest faced by many organisms in harsh environments. Annual killifish present this mechanism in three possible stages of development. Killifish are freshwater teleosts from Africa and America that live in ephemeral ponds, which dry up in the dry season. The juvenile and adult populations die, and the embryos remain buried in the bottom mud until the next rainy season. Thus, species survival is entirely embryo-dependent, and they are perhaps the most remarkable extremophile organisms among vertebrates. The aim of the present study was to gather information about embryonic diapauses with the use of a "shotgun" proteomics approach in diapause III and prehatching Austrolebias charrua embryos. Our results provide insight into the molecular mechanisms of diapause III. Data are available via ProteomeXchange with identifier PXD025196. We detected a diapause-dependent change in a large group of proteins involved in different functions, such as metabolic pathways and stress tolerance, as well as proteins related to DNA repair and epigenetic modifications. Furthermore, we observed a diapause-associated switch in cytoskeletal proteins. This first glance into global protein expression differences between prehatching and diapause III could provide clues regarding the induction/maintenance of this developmental arrest in A. charrua embryos. There appears to be no single mechanism underlying diapause and the present data expand our knowledge of the molecular basis of diapause regulation. This information will be useful for future comparative approaches among different diapauses in annual killifish and/or other organisms that experience developmental arrest.

Annual killifish adaptations to ephemeral environments: Diapause i in two austrolebias species.

BACKGROUND: Many organisms are able to survive in extreme environments by entering a state of dormancy. In dormancy, vital activities are reduced until environmental conditions are compatible with active life. Annual killifishes show a special developmental pattern characterized by a phase of dispersion-reaggregation of the blastomeres that separates epiboly from organogenesis, and the capability to enter dormancy in diapause. High tolerance to environmental stress confers annual killifish embryos the condition of extremophiles. At present, the questions of our research group are focused on the understanding of the mechanisms involved in diapause regulation through an interdisciplinary approach. As a first step, it is necessary to characterize diapauses at morphological and physiological levels and to evaluate induction cues under laboratory conditions. In this context, we characterized diapause I in two Austrolebias species. RESULTS: Our experimental approach to induce diapause I was successful and revealed the co-existence of two diapause I phenotypes named A and B instead of one. These phenotypes showed a tendency for lower total extractable RNA content compared with active developmental stages (80-100% epiboly and early reaggregate). CONCLUSIONS: These phenotypes are alternative diapause I stages and may have ecological relevance because both were found in embryos in natural ponds. Developmental Dynamics 246:848-857, 2017. © 2017 Wiley Periodicals, Inc.

Sex determination in annual fishes: Searching for the master sex-determining gene in Austrolebias charrua (Cyprinodontiformes, Rivulidae).

Evolution of sex determination and differentiation in fishes involves a broad range of sex strategies (hermaphroditism, gonochorism, unisexuality, environmental and genetic sex determination). Annual fishes inhabit temporary ponds that dry out during the dry season when adults die. The embryos exhibit an atypical developmental pattern and remain buried in the bottom mud until the next rainy season. To elucidate genomic factors involved in the sex determination in annual fish, we explored the presence of a candidate sex-specific gene related to the cascade network in Austrolebias charrua. All phylogenetic analyses showed a high posterior probability of occurrence for a clade integrated by nuclear sequences (aprox. 900 bp) from both adults (male and female), with partial cDNA fragments of A. charrua from juveniles (male) and the dsx D. melanogaster gene. The expressed fragment was detected from blastula to adulthood stages showing a sexually dimorphic expression pattern. The isolated cDNA sequence is clearly related to dsx D. melanogaster gene and might be located near the top of the sex determination cascade in this species.

Characterization of partial Hox gene sequences in annual fish of the subfamily Cynolebiatinae (Cyprinodontiformes, Rivulidae)

Hox genes encode a family of transcription factors implicated in conferring regional identity along the anteroposterior axis in developing animal embryos. These genes are organized in genomic clusters, expressed collinearly and highly conserved in vertebrates. Among teleost, South American annual killifishes of the Cynolebiatinae subfamily represent an excellent model in development studies because their embryos are capable of undergoing reversible developmental arrest (diapause) at three well-defined morphological stages. They are also an excellent model for evolutionary studies due to the high rates of mutation of their mitochondrial genome, their karyotypic divergence and their morphological variability. In this study, three partial homeobox sequences were isolated from different species of the Cynolebiatinae subfamily. Phylogenetic analyses and sequence comparisons revealed that they belong to the anterior Hox complex group, specifically to paralogue groups 1 and 3. This is the first time that partial Hox genes have been described in species of the Cynolebiatinae subfamily.