Gene Silencing of laccase 1 Induced by Double-Stranded RNA in Callosobruchus maculatus (Fabricius 1775) (Coleoptera: Chrysomelidae) Suggests RNAi as a Potential New Biotechnological Tool for Bruchid’s Control

Abstract

Bruchids are the most important pests of leguminous seeds in the world. In this study, the focus was done on Callosobruchus maculatus, a serious pest of Vigna unguiculata seeds. As no efficient control methods preventing collateral effects on beneficials currently exist, this study investigated whether RNA interference (RNAi) could provide a new biotechnological and selective tool for bruchids control. Three principal objectives were followed including (i) the identification of all RNAi machinery core components and a key protein to silence in C. maculatus genome (c.f., dicer-2, argonaute-2, R2D2, and laccase 1), (ii) the identification of suitable reference gene for RT-qPCR analyses, and (iii) the micro-injection of dsRNA coding for laccase 1 to adults of C. maculatus to assess gene expression levels by RT-qPCR and potentially related mortalities. Phylogenetical analyses performed from transcriptomic information successfully identified all necessary proteins in the RNAi mechanism and also the open reading frame of laccase 1 in C. maculatus. A new reference gene was identified (i.e., alpha-tubuline 1) and coupled with glutiathone S transferase for RT-qPCR analyses. Double-stranded RNAs coding for laccase 1 and green fluorescent protein (control) were produced and 400 ng of each dsRNA were micro-injected into C. maculatus adults. RT-qPCR analyses revealed a stable significant decrease in laccase 1 expression in about 80% of adults treated with laccase 1 dsRNA after three days post-injection. No significant mortalities were observed which is probably related to the non-exposure of adults to anti-nutritional factors that are usually regulated by laccase. Further research should focus either on the feeding larval stage which is directly exposed to anti-nutritional factors, or on other target genes to induce dead phenotypes. This study is the first gene silencing report on a bruchid species and supports RNAi as a potential future method of control.