Abstract
RNA interference (RNAi) has emerged as a powerful tool for knocking-down gene function in diverse taxa including arthropods for both basic biological research and application in pest control. The conservation of the RNAi mechanism in eukaryotes suggested that it should—in principle—be applicable to most arthropods. However, practical hurdles have been limiting the application in many taxa. For instance, species differ considerably with respect to efficiency of dsRNA uptake from the hemolymph or the gut. Here, we review some of the most frequently encountered technical obstacles when establishing RNAi and suggest a robust procedure for establishing this technique in insect species with special reference to pests. Finally, we present an approach to identify the most effective target genes for the potential control of agricultural and public health pests by RNAi.
Highlights
Variability and experimental difficulties encountered with RNA interference (RNAi)Delivery of double-stranded RNA (dsRNA)—environmental RNAi and putative dsRNA receptors RNAi emerged early in evolution of eukaryotes as immune response against viruses and transposons and the core components of the RNAi machinery are widely conserved [1, 11–14, 57–60]
RNA interference (RNAi) has emerged as a powerful tool for knocking-down gene function in diverse taxa including arthropods for both basic biological research and application in pest control
RNA interference in research and pest control For a long time, studying gene function was restricted to highly developed model organisms such as Drosophila melanogaster, where genetic screens and the maintenance of mutants were eased by an elaborate genetic toolkit
Summary
Delivery of dsRNA—environmental RNAi and putative dsRNA receptors RNAi emerged early in evolution of eukaryotes as immune response against viruses and transposons and the core components of the RNAi machinery are widely conserved [1, 11–14, 57–60]. Depending on the injected life stage, the application of dsRNA is often called “larval” or “pupal” or “adult RNAi” [71, 72] Another option opened by environmental RNAi is “in vitro RNAi” knocking down gene function by soaking cells in culture [73]. Once environmental RNAi has been shown to work by the above-mentioned experiment, this aspect can be tested by injection of female pupae or adults with dsRNA targeting developmental regulatory genes and scoring the hatchlings for morphological phenotypes. We noted complex and unexpected patterns of down- and up-regulation with respect to several other genes after RNAi and these highly depended on the time between injection and qPCR Given these uncertainties regarding the interpretation of qPCR results, quantification of the phenotypic readout may still represent the most meaningful readout to answer the question of whether RNAi is an adequate tool for a specific gene in a given process. Coinciding phenotypes in both treatments indicate the absence of (at least strong) off target effects, while some degree of quantitative differences between fragments can occur
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