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      RNAi-mediated knockdown of the voltage gated sodium ion channel TcNa v causes mortality in Tribolium castaneum

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          Abstract

          The voltage-gated sodium ion channel (VGSC) belongs to the largest superfamily of ion channels. Since VGSCs play key roles in physiological processes they are major targets for effective insecticides. RNA interference (RNAi) is widely used to analyse gene function, but recently, it has shown potential to contribute to novel strategies for selectively controlling agricultural insect pests. The current study evaluates the delivery of dsRNA targeted to the sodium ion channel paralytic A (TcNa v) gene in Tribolium castaneum as a viable means of controlling this insect pest. Delivery of TcNa v dsRNA caused severe developmental arrest with larval mortalities up to 73% post injection of dsRNA. Injected larvae showed significant (p < 0.05) knockdown in gene expression between 30–60%. Expression was also significantly (p < 0.05) reduced in pupae following injection causing 30% and 42% knockdown for early and late pupal stages, respectively. Oral delivery of dsRNA caused dose-dependant mortalities of between 19 and 51.34%; this was accompanied by significant (p < 0.05) knockdown in gene expression following 3 days of continuous feeding. The majority of larvae injected with, or fed, dsRNA died during the final larval stage prior to pupation. This work provides evidence of a viable RNAi-based strategy for insect control.

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          Control of coleopteran insect pests through RNA interference.

          Commercial biotechnology solutions for controlling lepidopteran and coleopteran insect pests on crops depend on the expression of Bacillus thuringiensis insecticidal proteins, most of which permeabilize the membranes of gut epithelial cells of susceptible insects. However, insect control strategies involving a different mode of action would be valuable for managing the emergence of insect resistance. Toward this end, we demonstrate that ingestion of double-stranded (ds)RNAs supplied in an artificial diet triggers RNA interference in several coleopteran species, most notably the western corn rootworm (WCR) Diabrotica virgifera virgifera LeConte. This may result in larval stunting and mortality. Transgenic corn plants engineered to express WCR dsRNAs show a significant reduction in WCR feeding damage in a growth chamber assay, suggesting that the RNAi pathway can be exploited to control insect pests via in planta expression of a dsRNA.
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            Ingested double-stranded RNAs can act as species-specific insecticides.

            A serious shortcoming of many insecticides is that they can kill non-target species. To address this issue, we harnessed the sequence specificity of RNA interference (RNAi) to design orally-delivered double-stranded (ds) RNAs that selectively killed target species. Fruit flies (Drosophila melanogaster), flour beetles (Tribolium castaneum), pea aphids (Acyrthosiphon pisum), and tobacco hornworms (Manduca sexta) were selectively killed when fed species-specific dsRNA targeting vATPase transcripts. We also demonstrate that even closely related species can be selectively killed by feeding on dsRNAs that target the more variable regions of genes, such as the 3' untranslated regions (UTRs): four species of the genus Drosophila were selectively killed by feeding on short (<40 nt) dsRNAs that targeted the 3' UTR of the gamma-tubulin gene. For the aphid nymphs and beetle and moth larvae, dsRNA could simply be dissolved into their diets, but to induce RNAi in the drosophilid species, the dsRNAs needed to be encapsulated in liposomes to help facilitate uptake of the dsRNA. This is the first demonstration of RNAi following ingestion of dsRNA in all of the species tested, and the method offers promise of both higher throughput RNAi screens and the development of a new generation of species-specific insecticides.
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              RNAi-mediated crop protection against insects.

              Downregulation of the expression of specific genes through RNA interference (RNAi), has been widely used for genetic research in insects. The method has relied on the injection of double-stranded RNA (dsRNA), which is not possible for practical applications in crop protection. By contrast, specific suppression of gene expression in nematodes is possible through feeding with dsRNA. This approach was thought to be unfeasible in insects, but recent results have shown that dsRNA fed as a diet component can be effective in downregulating targeted genes. More significantly, expression of dsRNA directed against suitable insect target genes in transgenic plants has been shown to give protection against pests, opening the way for a new generation of insect-resistant crops.
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                Author and article information

                Journal
                Sci Rep
                Sci Rep
                Scientific Reports
                Nature Publishing Group
                2045-2322
                14 July 2016
                2016
                : 6
                : 29301
                Affiliations
                [1 ]Newcastle Institute for Research on Environment and Sustainability, School of Biology, Newcastle University , Newcastle Upon Tyne NE1 7RU, UK
                [2 ]Entomology Department, Faculty of Science, Benha University , Benha, Egypt
                [3 ]Department of Zoology, University of, Swabi , KPK, Pakistan
                [4 ]Zoology Department, Faculty of Science, Benha University , Benha, Egypt
                Author notes
                Article
                srep29301
                10.1038/srep29301
                4944135
                27411529
                d1496d24-d884-41d4-a442-1fc9e8ef28c2
                Copyright © 2016, Macmillan Publishers Limited

                This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/

                History
                : 16 January 2016
                : 24 May 2016
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