Silencing of Double-Stranded Ribonuclease Improves Oral RNAi Efficacy in Southern Green Stinkbug Nezaraviridula.

Rohit Sharma,Guy Smagghe,Olivier Christiaens,Clauvis Nji Tizi Taning

doi:10.3390/insects12020115

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 Nezaraviridula 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.

Highlights

This article is an open access articleRNA-interference (RNAi) has been exploited in molecular biology for various purposes since the discovery of its mechanism at the end of the 20th century [1]
One hit was identified in this N. viridula and to obtain the full length of its open reading frame (ORF), a forward primer was designed based on the homologous sequence of H. hayls, as the Sequence Read Archive (SRA) did not cover the full-length mRNA of the DNA/RNA non-specific endonuclease of E. heros and H. hayls, and a reverse primer was designed from the sequence read of
To obtain the full ORF, a PCR was performed using a reversed primer designed based on the in-silico analysis of the SRA database of N. viridula and a forward primer coming from the homologous sequence in the closely related stinkbug

Summary

Introduction

This article is an open access articleRNA-interference (RNAi) has been exploited in molecular biology for various purposes since the discovery of its mechanism at the end of the 20th century [1]. Coleopterans are generally considered to be highly sensitive; on the other hand, other insect orders such as dipterans, hemipterans and lepidopterans show a more variable or low response to the orally delivered dsRNA [2,13,14,15,16,17]. This variation can be attributed to different factors such as dsRNA cellular uptake, dsRNA-nuclease activity, dsRNA length and other factors [18]. RNAi-mediated silencing of dsRNases, followed by an RNAi-treatment targeting an essential gene

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Discussion

Conclusion