Find in Library

Bruchids are the most important pests of leguminous seeds in the world. In this study, the focus was done on <i>Callosobruchus maculatus</i>, a serious pest of <i>Vigna unguiculata</i> 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 <i>C. maculatus</i> genome (<i>c.f.</i>, <i>dicer-2, argonaute-2, R2D2,</i> and <i>laccase 1</i>), (ii) the identification of suitable reference gene for RT-qPCR analyses, and (iii) the micro-injection of dsRNA coding for <i>laccase 1</i> to adults of <i>C. maculatus</i> 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 <i>laccase 1</i> in <i>C. maculatus</i>. A new reference gene was identified (i.e., <i>alpha-tubuline 1</i>) and coupled with <i>glutiathone S transferase</i> for RT-qPCR analyses. Double-stranded RNAs coding for <i>laccase 1</i> and <i>green fluorescent protein</i> (control) were produced and 400 ng of each dsRNA were micro-injected into <i>C. maculatus</i> adults. RT-qPCR analyses revealed a stable significant decrease in <i>laccase 1</i> expression in about 80% of adults treated with <i>laccase 1</i> 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.