Ecdysone receptor in the mud crab Scylla paramamosain: a possible role in promoting ovarian development

In arthropods, it is known that ecdysteroids regulate molting, limb regeneration, and reproduction through activation of the ecdysone receptor (EcR). However, the ecdysteroid signaling pathway for promotion of ovarian development in crustaceans is still unclear. In this study, three cDNA isoforms of EcR were cloned from the mud crab Scylla paramamosain. qRT-PCR revealed that the SpEcR mRNA was abundant in the eyestalk, ovary and epidermis. During ovarian development, the SpEcR transcripts increased from stage I (undeveloped stage) and reached a peak at stage IV (late vitellogenic stage) before dropping to a lower level at stage V (mature stage). Meanwhile, levels of 20-hydroxyecdysone (20E) in the hemolymph, detected by HPLC-MS, displayed a similar pattern of increase with ovarian development. Results from in situ hybridization indicated that SpEcR mRNA was present in the follicular cells during vitellogenesis. Results from in vivo experiments revealed that 20E at 0.2 μg/g body weight significantly stimulated the expression of SpEcR and vitellogenin (SpVg) in female crabs during the early vitellogenic stage but not during the previtellogenic stage. This was confirmed by results from in vitro experiments which indicated that SpEcR and SpVg expression levels were significantly upregulated in early vitellogenic ovarian explants incubated with 5.0 μM 20E at 3 and 6 h but not in previtellogenic ovarian explants. Finally, results from in vitro gene silencing experiments indicated that the expression of SpEcR and SpVg in the ovary was significantly inhibited by SpEcR dsRNA. All these results together indicated that in S. paramamosain, 20E, and SpEcR, located in the follicular cells, play important roles in the promotion of ovarian development via regulating the expression of SpVg