Background: Austrofundulus limnaeus is an annual killifish from the Maracaibo basin of Venezuela. Annual killifishes are unique among vertebrates in their ability to enter into a state of dormancy at up to three distinct developmental stages termed diapause I, II, and III. These embryos are tolerant of a wide variety of environmental stresses and develop relatively slowly compared with nonannual fishes. Results: These traits make them an excellent model for research on interactions between the genome and the environment during development, and an excellent choice for developmental biology laboratories. Furthermore, A. limnaeus is relatively easy to maintain in a laboratory setting and has a high fecundity, making it an excellent candidate as an emerging model for studies of development, and for defining the limits of developmental buffering in vertebrates. Conclusions: This study reports for the first time on the detailed development of A. limnaeus and provides a photographic and illustrated atlas of embryos on the two developmental trajectories possible in this species. Developmental Dynamics 246:779-801, 2017. V C 2017 Wiley Periodicals, Inc.

BackgroundThe annual killifish Austrofundulus limnaeus inhabits ephemeral ponds in northern Venezuela, South America, and is an emerging extremophile model for vertebrate diapause, stress tolerance, and evolution. Embryos of A. limnaeus regularly experience extended periods of desiccation and anoxia as a part of their natural history and have unique metabolic and developmental adaptations. Currently, there are limited genomic resources available for gene expression and evolutionary studies that can take advantage of A. limnaeus as a unique model system.ResultsWe describe the first draft genome sequence of A. limnaeus. The genome was assembled de novo using a merged assembly strategy and was annotated using the NCBI Eukaryotic Annotation Pipeline. We show that the assembled genome has a high degree of completeness in genic regions that is on par with several other teleost genomes. Using RNA-seq and phylogenetic-based approaches, we identify several candidate genes that may be important for embryonic stress tolerance and post-diapause development in A. limnaeus. Several of these genes include heat shock proteins that have unique expression patterns in A. limnaeus embryos and at least one of these may be under positive selection.ConclusionThe A. limnaeus genome is the first South American annual killifish genome made publicly available. This genome will be a valuable resource for comparative genomics to determine the genetic and evolutionary mechanisms that support the unique biology of annual killifishes. In a broader context, this genome will be a valuable tool for exploring genome-environment interactions and their impacts on vertebrate physiology and evolution.Electronic supplementary materialThe online version of this article (10.1186/s12864-018-4539-7) contains supplementary material, which is available to authorized users.

Annual killifishes exhibit a number of unique life history characters including the occurrence of embryonic diapause, unique cell movements associated with dispersion and subsequent reaggregation of the embryonic blastomeres, and a short postembryonic life span. Insulin-like growth factor (IGF) signaling is known to play a role in the regulation of metabolic dormancy in a number of animals but has not been explored in annual killifishes. The abundance of IGF proteins during development and the developmental effects of blocking IGF signaling by pharmacological inhibition of the insulin-like growth factor I receptor (IGF1R) were explored in embryos of the annual killifish Austrofundulus limnaeus. Blocking of IGF signaling in embryos that would normally escape entrance into diapause resulted in a phenotype that was remarkably similar to that of embryos entering diapause. IGF-I protein abundance spikes during early development in embryos that will not enter diapause. In contrast, IGF-I levels remain low during early development in embryos that will enter diapause II. IGF-II protein is packaged at higher levels in escape-bound embryos compared with diapause-bound embryos. However, IGF-II levels quickly decrease and remain low during early development and only increase substantially during late development in both developmental trajectories. Developmental patterns of IGF-I and IGF-II protein abundance under conditions that would either induce or bypass entrance into diapause are consistent with a role for IGF signaling in the regulation of developmental trajectory and entrance into diapause in this species. We propose that IGF signaling may be a unifying regulatory pathway that explains the larger suite of characters that are associated with the complex life history of annual killifishes.

Our results suggest that female bias can be generated during the gametophytic generation, before plants reach sexual maturity. These results, combined with those of previous work, suggest that both the gametophytic and the sporophytic stages drive population sex ratios in C. purpureus, thus indicating that multiple mechanisms operate to create biased population sex ratios.

Embryos of the annual killifish Austrofundulus limnaeus are the most anoxia-tolerant vertebrate. Annual killifish inhabit ephemeral ponds, producing drought and anoxia-tolerant embryos, which allows the species to persist generation after generation. Anoxia tolerance and physiology vary by developmental stage, creating a unique opportunity for comparative study within the species. A recent study of small ncRNA expression in A. limnaeus embryos in response to anoxia and aerobic recovery revealed small ncRNAs with expression patterns that suggest a role in supporting anoxia tolerance. MitosRNAs, small ncRNAs derived from the mitochondrial genome, emerged as an interesting group of these sequences. MitosRNAs derived from mitochondrial tRNAs were differentially expressed in developing embryos and isolated cells exhibiting extreme anoxia tolerance. In this study we focus on expression of mitosRNAs derived from tRNA-cysteine, and their subcellular and organismal localization in order to consider possible function. These tRNA-cys mitosRNAs appear enriched in the mitochondria, particularly near the nucleus, and also appear to be present in the cytoplasm. We provide evidence that mitosRNAs are generated in the mitochondria in response to anoxia, though the precise mechanism of biosynthesis remains unclear. MitosRNAs derived from tRNA-cys localize to numerous tissues, and increase in the anterior brain during anoxia. We hypothesize that these RNAs may play a role in regulating gene expression that supports extreme anoxia tolerance.