Spatio-temporal patterns of ovarian development andVgRgene silencing reduced fecundity of parthenogeneticArtemia

ABSTRACT Halophilic zooplankton brine shrimp Artemia has been used as an experimental animal in multidisciplinary research. However, the reproductive patterns and regulation mechanisms of Artemia remain a mystery. In this study, the ovarian development process of parthenogenetic Artemia (P. Artemia ) was distinguished into five stages, and oogenesis or egg formation process was identified with six phases. The mode of oogenesis was assumed polytrophic. Moreover, we traced the dynamic translocation of germline stem cells (GSCs) by EdU labeling, elucidated several key cytological events in oogenesis through HE staining and florescent image. Notably, distinguished from the ovary structure of insects and crustaceans, P. Artemia germarium originated from ovariole buds and located in the base of the ovarioles. RNA-seq based on five stages of ovarian development revealed that 2657 up-regulated genes related to reproduction were obtained by pair-to-pair comparison of RNA-seq. Furthermore, Gbb, Dpp, piwi, vasa, nanos, VgA and VgR associated with GSCs recognition and reproductive development genes were screened and also verified by qPCR. Silencing of the VgR gene in P. Artemia ( PA-VgR ) at stage L of ovarian development led to a low level of gene expression (<10%) within five days, which resulted variations of oogenesis-related gene expression and significantly inhibited vitellogenesis, impeded oocyte maturation, and eventually decreased the offspring number. In conclusion, we illustrated the patterns of ovarian development, outlined the key spatio-temporal features of oogenesis and established a platform for gene function research using P. Artemia as an experimental animal. These results lay the foundation for studying the reproductive biology of invertebrates, may also provide a novel insight in the reproductive regulation mechanism of aquatic animals in extreme environments. SUMMARY STATEMENT Artemia is a novel experimental platform and potential “model” crustacean for studying specific process and mechanism of reproduction.