BACKGROUND Targeting insect‐specific genes through post‐transcriptional gene silencing with RNA interference (RNAi) is a new strategy for insect pest management. However, lepidopterans are recalcitrant to RNAi, which prevents application of novel RNAi technology to many notorious pests, including Ostrinia nubilalis (ECB). Strategies for enhancing RNAi efficiency, including large doses of double‐stranded RNA (dsRNA), nuclease inhibitors, transfection reagents, and nanoparticles, have proved useful in other insects exhibiting substantial dsRNA degradation, a major mechanism limiting RNAi efficacy. To determine if similar strategies can enhance RNAi efficiency in ECB, various reagents were tested for their ability to enhance dsRNA stability in ECB tissues, then compared for their effectiveness in whole ECB. RESULTS Ex vivo incubation experiments revealed that Meta dsRNA lipoplexes, EDTA, chitosan‐based dsRNA nanoparticles, and Zn2+ enhanced dsRNA stability in ECB hemolymph and gut content extracts, compared with uncoated dsRNA. Despite these positive results, the reagents used in this study were ineffective at enhancing RNAi efficiency in ECB in vivo. To reduce assay time and required dsRNA, midguts were dissected and incubated in tissue culture medium containing dsRNA with and without reagents. These experiments showed that RNAi efficiency varied between target genes, and nuclease inhibitors improved RNAi efficiency for only a portion of the refractory target genes investigated ex vivo. CONCLUSION These results indicate that enhancing dsRNA stability is insufficient to improve RNAi efficiency in ECB and suggests the existence of additional, complex mechanisms contributing to low RNAi efficiency in ECB.

Variable RNA interference (RNAi) efficiencies limit RNAi-based pest management strategies for many pests. Previous efforts to understand mechanisms contributing to low RNAi efficiency indicate that double-stranded RNA (dsRNA) is degraded in the European corn borer (ECB), Ostrinia nubilalis, due to nuclease activity. To investigate the contribution of dsRNA-degrading endonucleases (dsRNases) and lepidopteran-specific RNAi efficiency-related nucleases (REases) to dsRNA instability and low RNAi efficiency in ECB, five complementary DNAs putatively encoding four dsRNases (OndsRNase1, 2, 3, and 4) and one REase (OnREase) were sequenced. Characterization of these transcripts revealed that substrate specificity might vary among the four dsRNases due to different amino acid combinations in the substrate-binding sites. Gene expression analysis indicated that OndsRNase2 and OnREase were highly expressed in the larval gut, and OndsRNase1 showed the highest expression in hemolymph, especially in older developmental stages. Transcript level analysis after dsRNA exposure revealed that expression of OnREase rapidly increased upon dsRNA ingestion or injection, whereas OndsRNase4 expression only increased after long-term ingestion of dsRNA. While the biological function of these nucleases remains to be verified, our results suggest that OnREase and OndsRNase2, and OndsRNase1 and OndsRNase4 may be responsible for degradation of dsRNAs in the ECB gut and hemolymph, respectively, thereby contributing to low RNAi efficiency.