Abstract
RNA interference (RNAi) techniques are being developed for a range of pest insect control technologies, including the sterile insect technique (SIT) and double-stranded RNA (dsRNA)-based insecticides. In SIT applications, where >99% of the released males should be sterile to meet industry standards, the efficiency of RNAi will need to be improved for many insect species if this technology is to be adopted. Endogenous dsRNases can impede dsRNA delivery in some insects, and, here, we investigated whether dsRNases in the midgut could limit RNAi efficacy in the mosquito Aedes aegypti. Ten putative dsRNases were identified in the Ae. aegypti genome, with two highly expressed in the midguts of larvae. Using an ex vivo assay, we observed that dsRNA was rapidly degraded within the mosquito larva’s gut. Double-stranded RNA targeting these two dsRNases, when fed to the larvae, effectively reduced gut dsRNase activity. When these dsRNase-specific dsRNAs were co-delivered with dsRNA targeting a cyan fluorescent protein (CFP) reporter gene, greater knockdown of CFP fluorescence was observed. These results suggest that inhibiting dsRNase activity could enable the implementation of RNAi-based mosquito control methods.
Highlights
RNA interference (RNAi) is a well-established reverse genetics method used to explore gene functions in insects
Using bacterially produced short
Phylogenetic analyses revealed that aedine mosquitoes have a diversity of dsRNases, with the greatest number found in
Summary
RNA interference (RNAi) is a well-established reverse genetics method used to explore gene functions in insects. The variability in RNAi responses in different insects has been attributed to a broad range of factors, including variations in the double-stranded RNA (dsRNA) stability within different insects, differences in cellular uptake of the dsRNA trigger molecules, differences in intracellular distribution and processing of the dsRNAs, and differences in systemic distribution of the effector molecules [6,7]. Of these factors, the most extensively studied is the stability of the dsRNAs when delivered to the insects. While many successful dsRNA feeding experiments have been reported [6,8], a growing number of research groups have observed that dsRNases within the insects can greatly reduce the efficacy of RNAi [1,7]
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