RNA interference in the Asian Longhorned Beetle:Identification of Key RNAi Genes and Reference Genes for RT-qPCR

Thais B Rodrigues,Jian J Duan,Ramesh Kumar Dhandapani,Subba Reddy Palli

doi:10.1038/s41598-017-08813-1

Thais B Rodrigues, Jian J Duan + Show 2 more

Open Access

https://doi.org/10.1038/s41598-017-08813-1

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Abstract

Asian Longhorned Beetle (ALB) Anoplophora glabripennis is a serious invasive forest pest in several countries including the United States, Canada, and Europe. RNA interference (RNAi) technology is being developed as a novel method for pest management. Here, we identified the ALB core RNAi genes including those coding for Dicer, Argonaute, and double-stranded RNA-binding proteins (dsRBP) as well as for proteins involved in dsRNA transport and the systemic RNAi. We also compared expression of six potential reference genes that could be used to normalize gene expression and selected gapdh and rpl32 as the most reliable genes among different tissues and stages of ALB. Injection of double-stranded RNA (dsRNA) targeting gene coding for inhibitor of apoptosis (IAP) into larvae and adults resulted in a significant knockdown of this gene and caused the death of 90% of the larvae and 100% of adults. No mortality of both larvae and adults injected with dsRNA targeting gene coding for green fluorescence protein (GFP, as a negative control) was observed. These data suggest that functional RNAi machinery exists in ALB and a potential RNAi-based method could be developed for controlling this insect.

Highlights

Five days after injection of dsRNA targeting iap gene (dsIAP), the IAP mRNA levels were reduced by 81% when compared to their levels in control larvae injected with dsGFP (Fig. 4A)
When dsRNA targeting an essential insect gene was injected into larvae and adults of A. glabripennis, nearly complete mortality of the treated insects occurred after target gene silencing in different insect tissues
The present study identified and validated the RNAi machinery in A. glabripennis, further studies to address the function of the AgladsRNase enzymes and their effects on RNAi response after ingestion of dsRNA need to be conducted

Summary

Introduction

The gapdh gene identified as the most stable gene across different tissues of A. glabripennis larvae in the current study has been identified as one of the most stable reference genes in other insects including Apis mellifera[33], Spodoptera exigua and B. We prepared dsRNA targeting iap gene (dsIAP) and tested its efficiency in knocking down this gene in larvae. Five days after injection of dsIAP, the IAP mRNA levels were reduced by 81% when compared to their levels in control larvae injected with dsGFP (Fig. 4A).

Results

Conclusion