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      Clathrin-dependent endocytosis is associated with RNAi response in the western corn rootworm, Diabrotica virgifera virgifera LeConte

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          Abstract

          The cellular uptake of dsRNA after dietary exposure is critical for RNAi efficiency; however, the mechanism of its uptake in many insects remains to be understood. In this study, we evaluated the roles of the endocytic pathway genes Clathrin heavy chain ( Chc), Clathrin adaptor protein AP50, ADP ribosylation factor-like 1 ( Arf72A), Vacuolar H + ATPase 16 kDa subunit ( Vha16), and small GTPase Rab7 and putative sid- 1-like genes ( silA and silC) in RNAi response in western corn rootworm (WCR) using a two-stage dsRNA exposure bioassay. Silencing of Chc, Vha16, and AP50 led to a significant decrease in the effects of laccase2 dsRNA reporter, indicating that these genes are involved in RNAi response. However, the knockdown of either Arf72A or Rab7 did not suppress the response to laccase2 dsRNA. The silencing of the silC gene did not lead to a significant reduction in mortality or increase in the expression of V-ATPase A reporter. While the silencing of the silA gene significantly decreased insect mortality, significant changes in V-ATPase A expression were not detected. These results suggest that clathrin-dependent endocytosis is a biological mechanism that plays an important role during RNAi response in WCR adults. The fact that no definitive support for the roles of silA or silC in RNAi response was obtained support the idea that RNAi response varies greatly in different insect species, demanding additional studies focused on elucidating their involvement in this mechanism.

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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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            Silencing a cotton bollworm P450 monooxygenase gene by plant-mediated RNAi impairs larval tolerance of gossypol.

            We identify a cytochrome P450 gene (CYP6AE14) from cotton bollworm (Helicoverpa armigera), which permits this herbivore to tolerate otherwise inhibitory concentrations of the cotton metabolite, gossypol. CYP6AE14 is highly expressed in the midgut and its expression correlates with larval growth when gossypol is included in the diet. When larvae are fed plant material expressing double-stranded RNA (dsRNA) specific to CYP6AE14, levels of this transcript in the midgut decrease and larval growth is retarded. Both effects are more dramatic in the presence of gossypol. As a glutathione-S-transferase gene (GST1) is silenced in GST1 dsRNA-expressing plants, feeding insects plant material expressing dsRNA may be a general strategy to trigger RNA interference and could find applications in entomological research and field control of insect pests.
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              RNA interference in Lepidoptera: an overview of successful and unsuccessful studies and implications for experimental design.

              Gene silencing through RNA interference (RNAi) has revolutionized the study of gene function, particularly in non-model insects. However, in Lepidoptera (moths and butterflies) RNAi has many times proven to be difficult to achieve. Most of the negative results have been anecdotal and the positive experiments have not been collected in such a way that they are possible to analyze. In this review, we have collected detailed data from more than 150 experiments including all to date published and many unpublished experiments. Despite a large variation in the data, trends that are found are that RNAi is particularly successful in the family Saturniidae and in genes involved in immunity. On the contrary, gene expression in epidermal tissues seems to be most difficult to silence. In addition, gene silencing by feeding dsRNA requires high concentrations for success. Possible causes for the variability of success in RNAi experiments in Lepidoptera are discussed. The review also points to a need to further investigate the mechanism of RNAi in lepidopteran insects and its possible connection to the innate immune response. Our general understanding of RNAi in Lepidoptera will be further aided in the future as our public database at http://insectacentral.org/RNAi will continue to gather information on RNAi experiments. Copyright © 2010 Elsevier Ltd. All rights reserved.
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                Author and article information

                Contributors
                Role: ConceptualizationRole: Data curationRole: Formal analysisRole: InvestigationRole: MethodologyRole: ValidationRole: VisualizationRole: Writing – original draftRole: Writing – review & editing
                Role: ConceptualizationRole: Data curationRole: Formal analysisRole: InvestigationRole: MethodologyRole: Project administrationRole: ResourcesRole: SupervisionRole: ValidationRole: VisualizationRole: Writing – original draftRole: Writing – review & editing
                Role: ConceptualizationRole: Data curationRole: VisualizationRole: Writing – original draftRole: Writing – review & editing
                Role: ConceptualizationRole: MethodologyRole: Writing – review & editing
                Role: ConceptualizationRole: InvestigationRole: Methodology
                Role: InvestigationRole: Methodology
                Role: ResourcesRole: Writing – review & editing
                Role: Writing – review & editing
                Role: ConceptualizationRole: Data curationRole: Funding acquisitionRole: Project administrationRole: ResourcesRole: Writing – review & editing
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                9 August 2018
                2018
                : 13
                : 8
                : e0201849
                Affiliations
                [1 ] Entomology and Nematology Department, Charles Steinmetz Hall, University of Florida, Gainesville, Florida, United States of America
                [2 ] Department of Chemistry, Federal University of Lavras, Lavras, Minas Gerais, Brazil
                [3 ] Department of Entomology, Entomology Hall, University of Lincoln, Lincoln, Nebraska, United States of America
                [4 ] Corteva Agriscience™, Agriculture Division of DowDuPont™, Indianapolis, Indiana, United States of America
                [5 ] Nucleus of Applied Biology, Embrapa Maize and Sorghum, Sete Lagoas, Minas Gerais, Brazil
                [6 ] Departamento de Producción Agropecuaria, Universidad de Caldas, Manizales, Caldas, Colombia
                University of Tennessee, UNITED STATES
                Author notes

                Competing Interests: KEN is an employee of Corteva Agriscience™, Agriculture Division of DowDuPont™. EF is a former employee of Corteva Agriscience™, Agriculture Division of DowDuPont™. This does not alter the authors' adherence to PLOS ONE policies on sharing data and materials. There are no disputes pertaining to the ownership of the data presented in the paper and all contributions are attributed below. All authors state that they adhere to the PLoS One Conflict of Interest Guidelines.

                Author information
                http://orcid.org/0000-0002-8329-6243
                http://orcid.org/0000-0003-3328-2932
                Article
                PONE-D-18-10655
                10.1371/journal.pone.0201849
                6084943
                30092086
                cbf2edf8-66d9-4301-90dc-60f40382babf
                © 2018 Pinheiro et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 9 April 2018
                : 22 July 2018
                Page count
                Figures: 7, Tables: 0, Pages: 19
                Funding
                Funded by: funder-id http://dx.doi.org/10.13039/100008114, University of Nebraska-Lincoln;
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/501100003593, Conselho Nacional de Desenvolvimento Científico e Tecnológico;
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/100008114, University of Nebraska-Lincoln;
                Award Recipient :
                This work was partially supported by CNPq-Conselho Nacional de Desenvolvimento Científico e Tecnológico for DHP’s scholarship (DHP). AMV, HW, AVJ and BDS were funded by the University of Nebraska-Lincoln. Corteva Agriscience™, Agriculture Division of DowDuPont™ provided support in the form of salaries for authors KEN and EF but did not have a role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section.
                Categories
                Research Article
                Biology and life sciences
                Genetics
                Epigenetics
                RNA interference
                Biology and life sciences
                Genetics
                Gene expression
                RNA interference
                Biology and life sciences
                Genetics
                Genetic interference
                RNA interference
                Biology and life sciences
                Biochemistry
                Nucleic acids
                RNA
                RNA interference
                Biology and Life Sciences
                Cell Biology
                Cell Processes
                Endocytosis
                Biology and Life Sciences
                Cell Biology
                Cell Processes
                Secretory Pathway
                Endocytosis
                Biology and Life Sciences
                Organisms
                Eukaryota
                Animals
                Invertebrates
                Arthropoda
                Insects
                Biology and Life Sciences
                Nutrition
                Diet
                Medicine and Health Sciences
                Nutrition
                Diet
                Biology and Life Sciences
                Organisms
                Eukaryota
                Animals
                Invertebrates
                Arthropoda
                Insects
                Beetles
                Biology and Life Sciences
                Agriculture
                Pests
                Insect Pests
                Research and Analysis Methods
                Experimental Organism Systems
                Model Organisms
                Caenorhabditis Elegans
                Research and Analysis Methods
                Model Organisms
                Caenorhabditis Elegans
                Research and Analysis Methods
                Experimental Organism Systems
                Animal Models
                Caenorhabditis Elegans
                Biology and Life Sciences
                Organisms
                Eukaryota
                Animals
                Invertebrates
                Nematoda
                Caenorhabditis
                Caenorhabditis Elegans
                Biology and Life Sciences
                Genetics
                Gene Types
                Reporter Genes
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                All relevant data are within the paper and its Supporting Information files.

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