An investigation into the RNA interference pathways in lobsters reveals key differences in immune factors across life stages.

RNA interference (RNAi) is a valuable tool for functional genomics, yet its effectiveness varies across species and developmental stages. While RNAi-mediated gene silencing is highly effective in spiny lobster (Panulirus ornatus) juveniles, silencing is inconsistent during the larval phyllosoma stages, which limits the scope of effective aquaculture biotechnologies in this species. To identify the mechanisms underlying this stage-dependent variability, we examined double-stranded RNA (dsRNA) stability, candidate uptake associated receptors,and associated immune responses in different life stages of P. ornatus. Ex vivo assays showed that 1 μg of dsRNA remained stable for up to 6 h in plasma from both phyllosoma and juvenile stages, indicating that double-stranded RNase (dsRNase) activity is unlikely to limit RNAi efficacy in either stage. Additionally, comparative transcriptomic profiling of water- and dsRNA-injected individuals revealed striking differences in differentially expressed genes across developmental stages, particularly in immune activation. In phyllosoma, dsRNA exposure primarily induced generalised antimicrobial and humoral immune pathways, including serine protease cascades, melanisation, antimicrobial peptides, and other pattern-recognition mechanisms. In contrast, juveniles exhibited enrichment of antiviral and RNAi-associated pathways almost exclusively, suggestive of a robust RNAi mechanism. Together, these findings demonstrate that the processing and utilisation of exogenous dsRNA in P. ornatus is regulated differently throughout development. This ontogenetic shift provides a mechanistic basis for stage-dependent RNAi efficiency across arthropods and sheds lights on developmental changes of the antiviral immune system and informs the development of RNAi-based applications in crustaceans.