title: 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.
authors.affiliation: School of Plant Sciences, University of Arizona, Tucson, Arizona, United States of America.
authors.forename: Meenal
authors.initials: M
authors.lastname: Vyas
authors.affiliation: School of Plant Sciences, University of Arizona, Tucson, Arizona, United States of America.|National Institute for Biotechnology & Genetic Engineering (NIBGE), Faisalabad, Pakistan.
authors.forename: Amir
authors.initials: A
authors.lastname: Raza
authors.affiliation: School of Plant Sciences, University of Arizona, Tucson, Arizona, United States of America.|National Institute for Biotechnology & Genetic Engineering (NIBGE), Faisalabad, Pakistan.
authors.forename: Muhammad Yousaf
authors.initials: MY
authors.lastname: Ali
authors.affiliation: Centre of Excellence in Molecular Biology (CEMB), University of Punjab, Lahore, Pakistan.
authors.forename: Muhammad Aleem
authors.initials: MA
authors.lastname: Ashraf
authors.affiliation: National Institute for Biotechnology & Genetic Engineering (NIBGE), Faisalabad, Pakistan.
authors.forename: Shahid
authors.initials: S
authors.lastname: Mansoor
authors.affiliation: Centre of Excellence in Molecular Biology (CEMB), University of Punjab, Lahore, Pakistan.
authors.forename: Ahmad Ali
authors.initials: AA
authors.lastname: Shahid
authors.affiliation: School of Plant Sciences, University of Arizona, Tucson, Arizona, United States of America.
authors.forename: Judith K
authors.initials: JK
authors.lastname: Brown
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D001681:Biological Assay; D017931:DNA Primers; D005786:Gene Expression Regulation; D015723:Gene Library; D020868:Gene Silencing; D006430:Hemiptera / Q000235:genetics*; D007306:Insecticides; D007413:Intestinal Mucosa / Q000378:metabolism*; D010641:Phenotype; D034622:RNA Interference; D012330:RNA, Double-Stranded / Q000378:metabolism*; D060888:Real-Time Polymerase Chain Reaction; D009435:Synaptic Transmission; D013696:Temperature; D059467:Transcriptome
chemical_list: D017931:DNA Primers; D007306:Insecticides; D012330:RNA, Double-Stranded