Silencing of Double-Stranded Ribonuclease Improves Oral RNAi Efficacy in Southern Green Stinkbug Nezara viridula

SIMPLE SUMMARY: RNAi interference (RNAi) is a conserved mechanism found in all eukaryotes. This mechanism is initiated by the presence of double-stranded RNA in the cells and leads to the blockage of protein synthesis of a target gene. This technique is being explored to develop species-selective biopesticides, where insect-specific double-stranded RNA would be delivered to an insect via the oral route. However, orally delivered double-stranded RNA leads to a variable RNAi-interference efficacy in different insect orders. Previous studies have shown rapid degradation of double-stranded RNA in the saliva of the southern green stinkbug. In this study, we identified and characterized the protein associated with double-stranded RNA degradation and provided evidence of the involvement of this protein in limiting RNAi efficacy in this pest. Our results revealed that one protein, a double-stranded RNA nuclease, is associated with double-stranded RNA degradation. Further, the blockage of double-stranded RNA nuclease synthesis by RNAi-interference significantly enhances the death-rate in the southern green stinkbug. These findings will be useful in the development of RNAi-interference-based pest control strategies. ABSTRACT: Variability in RNA-interference (RNAi) efficacy among different insect orders poses a big hurdle in the development of RNAi-based pest control strategies. The activity of double-stranded ribonucleases (dsRNases) in the digestive canal of insects can be one of the critical factors affecting oral RNAi efficacy. Here, the involvement of these dsRNases in the southern green stinkbug Nezara viridula was investigated. First, the full sequence of the only dsRNase (NvdsRNase) in the transcriptome of N. viridula was obtained, followed by an oral feeding bioassay to evaluate the effect of NvdsRNase-silencing on oral RNAi efficacy. The NvdsRNase was first silenced in nymphs by NvdsRNase-dsRNA injections, followed by exposure to an artificial diet containing a lethal αCop-specific dsRNA. A significantly higher mortality was observed in the NvdsRNase-silenced nymphs when placed on the dsαCop-containing diet (65%) than in the dsGFP injected and dsαCop fed control (46.67%). Additionally, an ex vivo dsRNA degradation assay showed a higher stability of dsRNA in the saliva and midgut juice of NvdsRNase-silenced adults. These results provide evidence for the involvement of NvdsRNase in the reduction of oral RNAi efficacy in N. viridula. This information will be useful in further improving potential RNAi-based strategies to control this pest.