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

Arnaud Segers (), Joachim Carpentier, Frédéric Francis and Rudy Caparros Megido
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Arnaud Segers: Functionnal and Evolutionnary Entomology, University of Liège–Gembloux Agro-Bio Tech, Passage des Déportés, 2, 5030 Gembloux, Belgium
Joachim Carpentier: Functionnal and Evolutionnary Entomology, University of Liège–Gembloux Agro-Bio Tech, Passage des Déportés, 2, 5030 Gembloux, Belgium
Frédéric Francis: Functionnal and Evolutionnary Entomology, University of Liège–Gembloux Agro-Bio Tech, Passage des Déportés, 2, 5030 Gembloux, Belgium
Rudy Caparros Megido: Functionnal and Evolutionnary Entomology, University of Liège–Gembloux Agro-Bio Tech, Passage des Déportés, 2, 5030 Gembloux, Belgium

Agriculture, 2023, vol. 13, issue 2, 1-19

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.

Keywords: RNAi; Bruchidae; Bruchinae; reference gene; RT-qPCR; RNA interference; dicer; argonaute; RISC (search for similar items in EconPapers)
JEL-codes: Q1 Q10 Q11 Q12 Q13 Q14 Q15 Q16 Q17 Q18 (search for similar items in EconPapers)
Date: 2023
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