Knock down of Whitefly Gut Gene Expression and Mortality by Orally Delivered Gut Gene-Specific dsRNAs

Abstract

Control of the whitefly Bemisia tabaci (Genn.) agricultural pest and plant virus vector relies on the use of chemical insecticides. RNA-interference (RNAi) is a homology-dependent innate immune response in eukaryotes, including insects, which results in degradation of the corresponding transcript following its recognition by a double-stranded RNA (dsRNA) that shares 100% sequence homology. In this study, six whitefly ‘gut’ genes were selected from an in silico-annotated transcriptome library constructed from the whitefly alimentary canal or ‘gut’ of the B biotype of B. tabaci, and tested for knock down efficacy, post-ingestion of dsRNAs that share 100% sequence homology to each respective gene target. Candidate genes were: Acetylcholine receptor subunit α, Alpha glucosidase 1, Aquaporin 1, Heat shock protein 70, Trehalase1, and Trehalose transporter1. The efficacy of RNAi knock down was further tested in a gene-specific functional bioassay, and mortality was recorded in 24 hr intervals, six days, post-treatment. Based on qPCR analysis, all six genes tested showed significantly reduced gene expression. Moderate-to-high whitefly mortality was associated with the down-regulation of osmoregulation, sugar metabolism and sugar transport-associated genes, demonstrating that whitefly survivability was linked with RNAi results. Silenced Acetylcholine receptor subunit α and Heat shock protein 70 genes showed an initial low whitefly mortality, however, following insecticide or high temperature treatments, respectively, significantly increased knockdown efficacy and death was observed, indicating enhanced post-knockdown sensitivity perhaps related to systemic silencing. The oral delivery of gut-specific dsRNAs, when combined with qPCR analysis of gene expression and a corresponding gene-specific bioassay that relates knockdown and mortality, offers a viable approach for functional genomics analysis and the discovery of prospective dsRNA biopesticide targets. The approach can be applied to functional genomics analyses to facilitate, species-specific dsRNA-mediated control of other non-model hemipterans.

Prospects, challenges and current status of RNAi through insect feeding

“…Successful gene silencing through this methodology has been achieved in several species and some of them and the targeted genes are compiled in Table . Notably, many of the targeted genes, such as chitin synthase, α‐tubulin , and Vacuolar‐type H + ‐ATPase , are vital for the insects, and suppressing their translation can result in lethality . A plausible explanation for the inflated interest in this type of targets could be the enthusiasm in exploring this technique as an alternative pesticide method .…”

“…Notably, many of the targeted genes, such as chitin synthase, -tubulin, and Vacuolar-type H + -ATPase, are vital for the insects, and suppressing their translation can result in lethality. 61,62 A plausible explanation for the inflated interest in this type of targets could be the enthusiasm in exploring this technique as an alternative pesticide method. 22 Nevertheless, RNAi strategies have also been used for the control of diseases in economically important insects such as honey bees and silkworms.…”

Genome‐wide identification and analysis of genes associated with RNA interference in Bemisia tabaci

“…A better understanding of this cohort of genes in the RNAi processing in sap‐sucking hemipterans will facilitate the integration of this novel biotechnology into the management of this highly invasive insect pest, either through screening for the new molecular targets (e.g. a suite of genes in gut and involved in osmotic pressure) or developing transgenic RNAi traits (e.g. in plant system, iPS) …”

“…RNAi‐based technology can potentially be developed for pest control applications. Previous studies in B. tabaci demonstrated a general 30%–70% decrease in the expression of target genes via dsRNA feeding or the use of transgenic pants . RNAi efficiency may be the strongest limiting factor for the wide application of this method.…”

Potential of RNA interference in the study and management of the whitefly, Bemisia tabaci

“…Furthermore, osmoregulation, sugar metabolism, and sugar transport‐associated genes such as α‐glucosidase 1 , aquaporin 1 (AQP1), trehalase 1 , and trehalose transporter 1 , were found to be vital for whitefly survival (Vyas et al, ). Other genes like acetylcholine receptor subunit α and hsp70 showed lower mortality upon silencing by RNAi.…”