Life stage-dependent RNA interference in the tropical rock lobster Panulirus ornatus: mechanistic insights from RNA-seq and functional studies

RNA interference (RNAi) has emerged as a key molecular tool in various commercially important decapod crustaceans, offering potential biotechnological applications in aquaculture. However, in the tropical rock lobster Panulirus ornatus, gene silencing through RNAi has proven difficult to achieve despite the availability of extensive omics data. This study investigates the RNAi response across life stages in P. ornatus, focusing on larvae and juveniles to determine when the species is most receptive to RNAi. Late-stage phyllosoma larvae and early juveniles were microinjected with dsRNA for the insulin-like growth factor binding protein encoding gene to determine silencing efficiency. Our results show that while juveniles exhibit an efficient systemic RNAi response with robust silencing across tissues, larvae display limited silencing capacity. A key finding is the differential expression of RNAi pathway components, including SID1, which facilitates dsRNA uptake in juveniles but is less active in larvae. Fluorescent microscopy revealed that dsRNA is rapidly sequestered and expelled by the antennal gland in larvae, potentially limiting RNAi efficacy. To further explore the mechanisms underlying RNAi in P. ornatus, RNA-seq analysis was conducted on pleopods collected across time points after dsRNA exposure in juvenile lobsters. Transcriptomic analysis identified significant upregulation of RNAi machinery, including Dicer-2, Argonaute-2, and SID1, which are critical for silencing. Additionally, several genes associated with antiviral responses were differentially expressed, suggesting broader involvement of RNAi in immune regulation. These findings highlight the potential to enhance RNAi strategies in P. ornatus juveniles, advancing the development of RNAi-based tools for disease resistance and productivity in aquaculture.