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      RNAi-Based Functional Genomics in Hemiptera

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          Abstract

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          RNA interference (RNAi) is a powerful strategy to understand the function of novel and critical insect genes. In this review, we highlight the pros and cons of using RNAi as a functional genomics tool, the range of applications and explore RNAi delivery approaches such as topical and carrier/nano-particle-mediated RNAi for silencing insect genes in Hemiptera. We explore factors contributing to observed variations in RNAi efficiency and possible solutions to improve RNAi based investigations. We briefly summarise and provide experimental insight on the key RNAi studies in agricultural hemipteran pests that will be applicable to lesser-studied hemipteran’s, as well as informing functional genomics studies across diverse insect pests.

          Abstract

          RNA interference (RNAi) is a powerful approach for sequence-specific gene silencing, displaying tremendous potential for functional genomics studies in hemipteran insects. Exploiting RNAi allows the biological roles of critical genes to be defined and aids the development of RNAi-based biopesticides. In this review, we provide context to the rapidly expanding field of RNAi-based functional genomics studies in hemipteran insects. We highlight the most widely used RNAi delivery strategies, including microinjection, oral ingestion and topical application. Additionally, we discuss the key variables affecting RNAi efficacy in hemipteran insects, including insect life-stage, gene selection, the presence of nucleases, and the role of core RNAi machinery. In conclusion, we summarise the application of RNAi in functional genomics studies in Hemiptera, focusing on genes involved in reproduction, behaviour, metabolism, immunity and chemical resistance across 33 species belonging to 14 families.

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          Most cited references124

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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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            A genome-wide transgenic RNAi library for conditional gene inactivation in Drosophila.

            Forward genetic screens in model organisms have provided important insights into numerous aspects of development, physiology and pathology. With the availability of complete genome sequences and the introduction of RNA-mediated gene interference (RNAi), systematic reverse genetic screens are now also possible. Until now, such genome-wide RNAi screens have mostly been restricted to cultured cells and ubiquitous gene inactivation in Caenorhabditis elegans. This powerful approach has not yet been applied in a tissue-specific manner. Here we report the generation and validation of a genome-wide library of Drosophila melanogaster RNAi transgenes, enabling the conditional inactivation of gene function in specific tissues of the intact organism. Our RNAi transgenes consist of short gene fragments cloned as inverted repeats and expressed using the binary GAL4/UAS system. We generated 22,270 transgenic lines, covering 88% of the predicted protein-coding genes in the Drosophila genome. Molecular and phenotypic assays indicate that the majority of these transgenes are functional. Our transgenic RNAi library thus opens up the prospect of systematically analysing gene functions in any tissue and at any stage of the Drosophila lifespan.
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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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                Author and article information

                Journal
                Insects
                Insects
                insects
                Insects
                MDPI
                2075-4450
                20 August 2020
                September 2020
                : 11
                : 9
                : 557
                Affiliations
                Queensland Alliance for Agriculture and Food Innovation, Centre for Horticultural Sciences, The University of Queensland, Brisbane 4072, Queensland, Australia; r.jain1@ 123456uq.edu.au (R.G.J.); s.fletcher@ 123456uq.edu.au (S.J.F.); n.mitter@ 123456uq.edu.au (N.M.)
                Author notes
                [* ]Correspondence: k.robinson2@ 123456uq.edu.au ; Tel.: +61-7-3346-2295
                Author information
                https://orcid.org/0000-0002-2315-5270
                https://orcid.org/0000-0003-1360-5669
                https://orcid.org/0000-0001-6146-6179
                Article
                insects-11-00557
                10.3390/insects11090557
                7564473
                32825516
                2ef35b92-d78d-477b-a634-d67bc7d11150
                © 2020 by the authors.

                Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ( http://creativecommons.org/licenses/by/4.0/).

                History
                : 20 July 2020
                : 17 August 2020
                Categories
                Review

                rnai,hemipteran insects,functional genomic studies,hemiptera

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