Abstract
The sequence specificity of the endogenous RNA interference pathway allows targeted suppression of genes essential for insect survival and enables the development of durable and efficacious insecticidal products having a low likelihood to adversely impact non-target organisms. The spectrum of insecticidal activity of a 240 nucleotide (nt) dsRNA targeting the Snf7 ortholog in Western Corn Rootworm (WCR; Diabrotica virgifera virgifera) was characterized by selecting and testing insects based upon their phylogenetic relatedness to WCR. Insect species, representing 10 families and 4 Orders, were evaluated in subchronic or chronic diet bioassays that measured potential lethal and sublethal effects. When a specific species could not be tested in diet bioassays, the ortholog to the WCR Snf7 gene (DvSnf7) was cloned and corresponding dsRNAs were tested against WCR and Colorado potato beetle (Leptinotarsa decemlineata); model systems known to be sensitive to ingested dsRNA. Bioassay results demonstrate that the spectrum of activity for DvSnf7 is narrow and activity is only evident in a subset of beetles within the Galerucinae subfamily of Chrysomelidae (>90 % identity with WCR Snf7 240 nt). This approach allowed for evaluating the relationship between minimum shared nt sequence length and activity. A shared sequence length of ≥21 nt was required for efficacy against WCR (containing 221 potential 21-nt matches) and all active orthologs contained at least three 21 nt matches. These results also suggest that WCR resistance to DvSnf7 dsRNA due to single nucleotide polymorphisms in the target sequence of 240 nt is highly unlikely.Electronic supplementary materialThe online version of this article (doi:10.1007/s11248-013-9716-5) contains supplementary material, which is available to authorized users.
Highlights
RNA interference (RNAi) technology can achieve sequence-specific gene silencing in some insects by feeding double-stranded RNAs (Baum et al 2007; Whyard et al 2009; Swevers and Smagghe 2012)
RNAi insect control technology has the potential for high taxonomic specificity and to target only one or a group of closely related species (Whyard et al 2009)
Selecting test species based upon their phylogenetic relationship to the target organism is a powerful approach to characterize the spectrum of activity for genetically engineered crops, as this focuses testing to those species most likely to be susceptible due to sequence similarity (Romeis et al 2013)
Summary
RNA interference (RNAi) technology can achieve sequence-specific gene silencing in some insects by feeding double-stranded RNAs (dsRNAs) (Baum et al 2007; Whyard et al 2009; Swevers and Smagghe 2012). In addition to sequence specificity, additional barriers exist to oral toxicity in insects These include potential degradation of the dsRNA prior to ingestion, barriers to cellular uptake, instability of the dsRNA within the insect following ingestion, and the inherent sensitivity of the organism to ingested dsRNA (Huvenne and Smagghe 2010; Allen and Walker 2012; Garbutt et al 2013). It has been established with Caenorhabditis elegans that cellular uptake of a dsRNA and subsequent spreading involves the transmembrane receptor molecules SID-1 and SID-2 (Feinberg and Hunter 2003; Winston et al 2007). Rapid degradation of dsRNA in the hemolymph of Manduca sexta has been reported and attributed to nuclease activity, indicating that sensitivity to RNAi may be influenced by the instability of dsRNA within the insect (Garbutt et al 2013)
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