Characterization of the spectrum of insecticidal activity of a double-stranded RNA with targeted activity against Western Corn Rootworm (Diabrotica virgifera virgifera LeConte)

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.

Fig. 1

WCR and CPB bioassays demonstrate specificity between two closely related species in the family Chrysomelidae. Conspecific and heterospecific Snf7 dsRNAs from WCR and CPB were tested at in 12-day diet incorporation bioassays. LdSnf7 and DvSnf7 dsRNAs were tested against WCR at concentrations of 15,000 and 50 ng/mL diet, respectively. DvSnf7 and LdSnf7 dsRNAs were tested against CPB at concentrations of 5,000 and 50 ng/mL diet, respectively

Fig. 2

ClustalWS multispecies alignment of Snf7 240 nt orthologs in selected Chrysomelid species. The coloring is based upon the percent of shared sequence identity. A representative of the family Tenebrionidae (T. castaneum) was used to anchor the tree. Created using Jalview Version 2.8. The alignment order parallels that shown for the phylogenetic tree in Fig. 3

Fig. 3

Average distance phylogenetic tree of Snf7 orthologs based upon percent identity and ClustalWS alignment. The tree illustrates the rapid divergence of Snf7 sequences within the coleopteran family Chrysomelidae. A representative of the family Tenebrionidae (T. castaneum) was used to anchor the tree. The tree was calculated on distance matrices using the percent identity between sequences (represented numerically below) and created using Jalview Version 2.8