Characterization of double-stranded RNA (dsRNA) degrading nuclease genes and strategies for enhancing dsRNA stability in the European corn borer

RNA interference (RNAi) is a revolutionary technique for silencing gene expression, but the success of this technique is largely dependent upon the stability of double-stranded RNA (dsRNA). Unfortunately, degradation of dsRNA by dsRNA-degrading endonucleases (dsRNases) and RNAi efficiency-related nucleases (REases) limit RNAi efficiency in lepidopterans and other insects. To investigate the contribution of these nucleases to dsRNA instability and low RNAi efficiency in the European corn borer, Ostrinia nubilalis (Lepidoptera: Cramibidae), we identified four OndsRNase genes and one OnREase gene, sequenced their open reading frames and characterized their developmental stage- and tissue-specific expression profiles.  High expression levels of OndsRNase2 and OnREase transcripts in larval gut contents and OndsRNase1 transcript in larval hemolymph suggest that these genes contribute to low dsRNA stability in these environments. Indeed, quantification of intact dsRNA remaining after incubation in the tissue extracts showed that dsRNA was rapidly degraded in both the hemolymph and gut contents. Furthermore, OnREase and OndsRNase4 were upregulated in response to exogenous dsRNA exposure. To determine if different strategies can help stabilize dsRNA, we quantified the dsRNA that remained intact after incubation in the tissue extracts and found that chitosan-based nanoparticles, Metafectene Pro, EDTA, and Zn²⁺ enhanced dsRNA stability ex vivo, but Lipofectamine RNAiMax, Mn²⁺, or Co²⁺ did not. Our results suggest that some of these reagents could be useful for improving RNAi efficiency in lepidopteran insects.