DEVELOPMENT OF A GENE SILENCING METHOD FOR BIVALVE BASED ON BACTERIA-EXPRESSED DSRNA POOL STRATEGY

RNA interference (RNAi) represents a promising technique for gene functional studies, yet its implementation in bivalve mollusks has been hindered by two persistent obstacles: inefficient in vivo delivery and substantial off-target effects. To overcome these challenges, we developed an integrated RNAi strategy combining bacterially expressed double-stranded RNA (dsRNA) with a pooled dsRNA approach and applied it to silence the nuclear receptor nr5a2 gene in the zhikong scallop (Chlamys farreri). Five non-overlapping dsRNA fragments targeting the nr5a2 open reading frame were expressed in RNase III-deficient Escherichia coli HT115 and delivered orally via an established Spirulina-based feeding system. Comparative assessment revealed that feeding achieved higher and more prolonged nr5a2 knockdown than intramuscular injection, with 60.5% silencing on day 1 and remaining 33.4% on day 7. Temporal profiling indicated effective silencing for up to 12hours post-feeding, supported by bacterial viability assays that justified a 12h feeding interval. Crucially, the dsRNA pool strategy minimized off-target effects: in silico analysis identified only 110 potential off-target transcripts, and RNA-seq validation showed merely 4 (females) and 8 (males) with modest expression changes, none of which exhibited typical RNAi suppression patterns. In summary, we present a scalable, cost-effective RNAi platform that combines bacterial dsRNA production, algal delivery, and a pooled dsRNA design. This system achieves efficient (>50%) and specific gene silencing in a bivalve species, offering a robust tool for functional genomics and potential applications in aquaculture breeding.