Nuclease activity decreases the RNAi response in the sweetpotato weevil Cylas puncticollis

RNA interference (RNAi) refers to the process of suppression of gene expression in eukaryotes, which has a great potential for the control of pest and diseases. Unfortunately, the efficacy of this technology is limited or at best variable in some insects. In the African sweet potato weevil (SPW) Cylas puncticollis, a devastating pest that affects the sweet potato production in Sub-Saharan Africa (SSA), the RNAi response was highly efficient when dsRNA was delivered by injection, but it showed a reduced response by oral feeding. In the present study, the role of nucleases in the reduced RNAi efficiency in C. puncticollis is investigated. Several putative dsRNases were first identified in the transcriptome of the SPW through homology search and were subsequently further characterized. Two of these dsRNases were specifically expressed in the gut tissue of the insect and we could demonstrate through RNAi experiments that these affected dsRNA stability in the gut. Furthermore, RNAi-of-RNAi studies, using snf7 as a reporter gene, demonstrated that silencing one of these nucleases, Cp-dsRNase-3, clearly increases RNAi efficacy. After silencing this nuclease, significantly higher mortality was observed in dssnf7-treated insects 14 days post-feeding as compared to control treatments, and the gene downregulation was confirmed at the transcript level via qPCR analysis. Taken together, our results demonstrate that the RNAi efficiency is certainly impaired by nuclease activity in the gut environment of the SPW Cylas puncticollis