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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

Author

Listed:
  • 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)

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.

Suggested Citation

  • Arnaud Segers & Joachim Carpentier & Frédéric Francis & Rudy Caparros Megido, 2023. "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," Agriculture, MDPI, vol. 13(2), pages 1-19, February.
    • Handle: RePEc:gam:jagris:v:13:y:2023:i:2:p:412-:d:1063694
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    References listed on IDEAS

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    1. Andrew Fire & SiQun Xu & Mary K. Montgomery & Steven A. Kostas & Samuel E. Driver & Craig C. Mello, 1998. "Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans," Nature, Nature, vol. 391(6669), pages 806-811, February.
    2. Scott M. Hammond & Emily Bernstein & David Beach & Gregory J. Hannon, 2000. "An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells," Nature, Nature, vol. 404(6775), pages 293-296, March.
    3. Gunter Meister & Thomas Tuschl, 2004. "Mechanisms of gene silencing by double-stranded RNA," Nature, Nature, vol. 431(7006), pages 343-349, September.
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