Effectiveness of orally-delivered double-stranded RNA on gene silencing in the stinkbug Plautia stali.

Yudai Nishide,Daisuke Kageyama,Yoshiaki Tanaka,Takema Fukatsu,Kakeru Yokoi,Ryo Futahashi,Akiya Jouraku,Zach N. Adelman

doi:10.1371/journal.pone.0245081

Abstract

Development of a reliable method for RNA interference (RNAi) by orally-delivered double-stranded RNA (dsRNA) is potentially promising for crop protection. Considering that RNAi efficiency considerably varies among different insect species, it is important to seek for the practical conditions under which dsRNA-mediated RNAi effectively works against each pest insect. Here we investigated RNAi efficiency in the brown-winged green stinkbug Plautia stali, which is notorious for infesting various fruits and crop plants. Microinjection of dsRNA into P. stali revealed high RNAi efficiency-injection of only 30 ng dsRNA into last-instar nymphs was sufficient to knockdown target genes as manifested by their phenotypes, and injection of 300 ng dsRNA suppressed the gene expression levels by 80% to 99.9%. Knockdown experiments by dsRNA injection showed that multicopper oxidase 2 (MCO2), vacuolar ATPase (vATPase), inhibitor of apoptosis (IAP), and vacuolar-sorting protein Snf7 are essential for survival of P. stali, as has been demonstrated in other insects. By contrast, P. stali exhibited very low RNAi efficiency when dsRNA was orally administered. When 1000 ng/μL of dsRNA solution was orally provided to first-instar nymphs, no obvious phenotypes were observed. Consistent with this, RT-qPCR showed that the gene expression levels were not affected. A higher concentration of dsRNA (5000 ng/μL) induced mortality in some cohorts, and the gene expression levels were reduced to nearly 50%. Simultaneous oral administration of dsRNA against potential RNAi blocker genes did not improve the RNAi efficiency of the target genes. In conclusion, P. stali shows high sensitivity to RNAi with injected dsRNA but, unlike the allied pest stinkbugs Halyomorpha halys and Nezara viridula, very low sensitivity to RNAi with orally-delivered dsRNA, which highlights the varied sensitivity to RNAi across different species and limits the applicability of the molecular tool for controlling this specific insect pest.

Highlights

Since its discovery at the end of the previous century, gene silencing mediated by administration of double-stranded RNA, called RNA interference (RNAi), has been a powerful technique for unveiling gene functions
RNAi of PsMCO2, PsvATPase, PsIAP, and PsSnf7 by injection with 300 ng double-stranded RNA (dsRNA) resulted in high mortality of the injected fifth-instar nymphs, the mortality and the timing of death varied among the different genes
The nymphs injected with PsIAP dsRNA died 2 to 3 days after injection, and those injected with PsSnf7 dsRNA died 3 to 4 days after injection, with no relation to the molting events (Fig 1B)

Summary

Introduction

Since its discovery at the end of the previous century, gene silencing mediated by administration of double-stranded RNA (dsRNA), called RNA interference (RNAi), has been a powerful technique for unveiling gene functions. RNAi experiments have relied on dsRNA injection into insect bodies because of generally efficient gene silencing by this method. A critical challenge in developing insect-specific molecular biopesticides is to find effective and reliable methods for delivery of dsRNA into pest insect bodies. Arming plants with dsRNA has been suggested [10], but all such methods hinge on the idea that crop is deployed with the corresponding dsRNA of essential insect genes. This approach could reduce our dependence on chemical insecticides and could combat insect resistance to chemical insecticides [11, 12]. The efficiency of RNAi delivered by feeding varies considerably among different species including hemipterans [20]

Methods

Results

Conclusion