Long dsRNAbut not siRNAinitiatesRNAi in western corn rootworm larvae and adults

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