Euschistus heros RNase silencing: dsRNA stability and effects on RNAi susceptibility

Nuclease (RNase) activity is a major limiting factor that reduces the effectiveness of RNAi-induced gene silencing across many insect species. In Euschistus heros, dsRNA degradation occurs predominantly upon contact with saliva, while midgut fluids exhibit moderate degradative activity and the hemolymph shows minimal or no RNase activity. High expression levels of RNase genes have been confirmed in the salivary glands, and to a lesser extent, in the midgut. To assess the impact of this barrier on RNAi efficacy, a RNAi-of-RNAi-based strategy was designed, involving the microinjection of dsRNA targeting the RNase gene (dsRNase), followed by the oral delivery of dsRNA targeting the V-ATPase gene (dsVATP-ase). This approach successfully reduced RNase gene expression and significantly decreased dsRNA degradation in saliva and midgut extracts. However, RNase suppression did not result in a significant enhancement on the E. heros sensitivity to dsVATP-ase, suggesting the presence of additional barriers that limit RNAi efficacy in this species. These results highlight the importance of mitigating enzymatic degradation while also emphasizing the need for complementary approaches to overcome other physiological barriers and enhance the viability of RNAi as a control tool in E. heros.