RNA interference (RNAi) has become a widely used reverse genetic tool to study gene function in eukaryotic organisms and is being developed as a technology for insect pest management. The efficiency of RNAi varies among organisms. Insects from different orders also display differential efficiency of RNAi, ranging from highly efficient (coleopterans) to very low efficient (lepidopterans). We investigated the reasons for varying RNAi efficiency between lepidopteran and coleopteran cell lines and also between the Colorado potato beetle, Leptinotarsa decemlineata and tobacco budworm, Heliothis virescens. The dsRNA either injected or fed was degraded faster in H. virescens than in L. decemlineata. Both lepidopteran and coleopteran cell lines and tissues efficiently took up the dsRNA. Interestingly, the dsRNA administered to coleopteran cell lines and tissues was taken up and processed to siRNA whereas the dsRNA was taken up by lepidopteran cell lines and tissues but no siRNA was detected in the total RNA isolated from these cell lines and tissues. The data included in this paper showed that the degradation and intracellular transport of dsRNA are the major factors responsible for reduced RNAi efficiency in lepidopteran insects.

Evolution of resistance threatens sustainability of transgenic crops producing insecticidal proteins from the bacterium Bacillus thuringiensis (Bt). The fall armyworm (Spodoptera frugiperda) is a devastating pest of corn in the Western Hemisphere initially controlled by transgenic Bt corn producing the Cry1Fa insecticidal protein (event TC1507). However field-evolved resistance to TC1507 was observed in Puerto Rico in 2007 and has subsequently been reported in a number of locations in North and South America. Early studies on Puerto Rico fall armyworm populations found that the resistance phenotype was associated with reduced expression of alkaline phosphatase. However, in this work we show that field-evolved resistance to Cry1Fa Bt corn in Puerto Rico is closely linked to a mutation in an ATP Binding Cassette subfamily C2 (ABCC2) gene that functions as a Cry1Fa receptor in susceptible insects. Furthermore, we report a DNA-based genotyping test used to demonstrate the presence of the resistant (SfABCC2mut) allele in Puerto Rico populations in 2007, coincident with the first reports of damage to TC1507 corn. These DNA-based field screening data provide strong evidence that resistance to TC1507 in fall armyworm maps to the SfABCC2 gene and provides a useful molecular marker for detecting the SfABCC2mut allele in resistant fall armyworm.

Field tests of corn co-expressing two new delta-endotoxins from Bacillus thuringiensis (Bt) have demonstrated protection from root damage by western corn rootworm (Diabrotica virgifera virgifera LeConte). The level of protection exceeds that provided by chemical insecticides. In the bacterium, these proteins form crystals during the sporulation phase of the growth cycle, are encoded by a single operon, and have molecular masses of 14 kDa and 44 kDa. Corn rootworm larvae fed on corn roots expressing the proteins showed histopathological symptoms in the midgut epithelium.

RNAi shows potential as an agricultural technology for insect control, yet, a relatively low number of robust lethal RNAi targets have been demonstrated to control insects of agricultural interest. In the current study, a selection of lethal RNAi target genes from the iBeetle (Tribolium castaneum) screen were used to demonstrate efficacy of orthologous targets in the economically important coleopteran pests Diabrotica virgifera virgifera and Meligethes aeneus. Transcript orthologs of 50 selected genes were analyzed in D. v. virgifera diet-based RNAi bioassays; 21 of these RNAi targets showed mortality and 36 showed growth inhibition. Low dose injection- and diet-based dsRNA assays in T. castaneum and D. v. virgifera, respectively, enabled the identification of the four highly potent RNAi target genes: Rop, dre4, ncm, and RpII140. Maize was genetically engineered to express dsRNA directed against these prioritized candidate target genes. T0 plants expressing Rop, dre4, or RpII140 RNA hairpins showed protection from D. v. virgifera larval feeding damage. dsRNA targeting Rop, dre4, ncm, and RpII140 in M. aeneus also caused high levels of mortality both by injection and feeding. In summary, high throughput systems for model organisms can be successfully used to identify potent RNA targets for difficult-to-work with agricultural insect pests.

Transgenic maize plants expressing dsRNA targeting western corn rootworm (WCR, Diabrotica virgifera virgifera LeConte) v‐ATPase subunit C mRNA for RNAi provided significant root protection from WCR larval feeding damage in greenhouse assays compared to negative controls. Transcribed hairpin dsRNA in WCR‐resistant maize plants was present as both intact hairpin‐derived dsRNA and plant‐processed siRNA. Therefore, the ability of dsRNA and siRNA targeting Dv v‐ATPase CmRNA to cause an RNAi response was studied in both WCR larvae and adults. In 9‐day diet‐based feeding assays, dsRNA of at least 60 bp in length resulted in high levels of larval mortality. In contrast, 15‐, 25‐ or 27‐bp dsRNAs or pooled 21‐bp siRNAs did not cause mortality of exposed larvae. When larvae were fed with diet overlaid with siRNAs, Dv v‐ATPase C transcript levels did not change. Conversely, when WCR larvae were fed with diet overlaid with 184‐bp dsRNA, the mRNA level was reduced by >20‐fold relative to yfp dsRNA negative control. Similarly, 184‐bp dsRNA caused 100% mortality of WCR adults, whereas the mortality of adults fed on diet treated with siRNAs was similar to the negative control. Feeding adults with siRNAs on diet did not affect the level of Dv v‐ATPase CmRNA transcripts, whereas adults fed with the 184‐bp dsRNA showed approximately 35‐fold reduction in the target mRNA level. Similar results were obtained with the WCR adults injected with 184‐bp dsRNA or 21‐bp siRNA. These results suggest that only long dsRNA or hairpin‐derived dsRNA is effective in causing lethal knock‐down of Dv v‐ATPase CmRNA. These results have implications for efficacious plant‐delivered dsRNA for the protection of transgenic maize from WCR feeding damage and for the risk assessment of transgenic maize expressing insecticidal dsRNA.