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Weste co rootworm (WCR,Diabrotica virgifera virgifera LeConte) is highly sensitive to orally delivered double-stranded RNA (dsRNA).RNAi in WCR is systemic and spreads throughout the insect body.This raises the question whether transitive RNAi is a mechanism that functions in WCR to amplify the RNAi response via production of secondary siRNA.Secondary siRNA production is achieved through RNA-dependent RNA polymerase (RdRP) activity in other eukaryotic organisms,but RdRP has not been identified in WCR and any other insects.This study visualized the spread of the RNAimediated knockdown of Dv v-ATPase C mRNA throughout the WCR gut and other tissues using high-sensitivity branched DNA in situ hybridization.Furthermore,we did not detect either secondary siRNA production or transitive RNAi in WCR through siRNA sequence profile analysis.Nucleotide mismatched sequences introduced into either the sense or antisense strand of v-ATPase C dsRNAs were maintained in siRNAs derived from WCR fed with the mismatched dsRNAs in a strand specific manner.The distribution of all siRNAs was restricted to within the original target sequence regions,which may indicate the lack of new dsRNA synthesis leading to production of secondary siRNA.Thus,the systemic spread of RNAi in WCR may be derived from the original dsRNA molecules taken up from the gut lumen.These results indicate that the initial dsRNA dose is important for a lethal systemic RNAi response in WCR and have implications in developing effective dsRNA traits to control WCR and in resistance management to prolong the durability of RNAi trait technology.