A Targeted RNAi-of-RNAi Strategy to Overcome Nuclease-Mediated RNAi Suppression and Enhance RNAi Efficacy in Lepidoptera Pest, Cydia Pomonella

The codling moth has developed resistance to chemical insecticides and biological agents, necessitating alternative control strategies. RNA interference (RNAi) offers considerable potential. However, variable efficacy across target genes remains a major barrier in lepidopterans. In this study, we identified three REase genes in the codling moth, each containing a conserved PIN domain. CmREase1 and CmREase2 were strongly induced by exogenous dsRNA and showed high expression in midgut and hemolymph. RNAi-mediated silencing of these genes reduced dsRNA degradation in hemolymph and significantly enhanced RNAi efficiency, confirming their key roles in dsRNA degradation and suppression of RNAi. In contrast, CmREase4 was dsRNA-responsive but had no detectable effect on RNAi efficiency. Moreover, transcriptional profiling of Dcr-2 and AGO2 revealed that pretreatment with nonspecific dsEGFP followed by target-specific dsRNA markedly improved RNAi efficacy, comparable to REase knockdown. Collectively, these results demonstrate that REases are critical modulators of RNAi efficiency in codling moth and provide a framework for RNAi-based biopesticide development, offering new strategies to overcome variability in RNAi responsiveness among lepidopteran pests.