Delivery, Persistence, and Functional Response of dsRNA in Acyrthosiphon pisum: Progress and Remaining Barriers.

Aphids are major agricultural pests due to their rapid reproduction, insecticide resistance, and role as vectors of plant pathogens. RNA interference (RNAi) has emerged as a promising strategy for species-specific pest control. However, RNAi in aphids remains inconsistent, limited by dsRNA instability, inefficient uptake, and variable gene silencing. Here, we investigate key biological barriers limiting RNAi efficiency by evaluating the delivery, stability, and efficacy of double-stranded (ds)RNA in the pea aphid Acyrthosiphon pisum. Feeding bioassays using food dyes and Cy3-labeled dsRNA demonstrated ingestion and persistence of fluorescent signals within the aphid midgut for up to 120 h, confirming that the feeding system effectively delivers dsRNA to the insects. Ex vivo degradation assays revealed rapid dsRNA breakdown by aphid nucleases, with complete degradation within 2 h. Formulations improved stability: chitosan-protected dsRNA resisted aphid nuclease activity, and layered double hydroxide (LDH) nanosheets induced elevated expression of RNAi-associated genes. Functional assays targeting the vacuolar H+-ATPase 26 kDa-like gene (apvhal26) produced variable knockdown but consistently induced the expression of RNAi-associated genes (ago2, dicer1), suggesting activation of the RNAi pathway or recognition of exogenous dsRNA despite inconsistent gene silencing. Our results demonstrate that several key barriers to RNAi in aphids can be partially mitigated through improved delivery and nanoparticle formulations. However, rapid nuclease-mediated degradation and inconsistent target gene silencing remain major obstacles to reliable RNAi efficacy. This study highlights both the potential and limitations of RNAi for aphid control and emphasizes the need for optimized formulations, target gene selection, and delivery methods to advance RNAi as a sustainable biopesticide strategy.