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The discovery of environmental RNA interference (RNAi), in which gene expression is suppressed via feeding with double-stranded RNA (dsRNA) molecules, opened the door to the practical application of RNAi-based techniques in crop pest management. The western corn rootworm (WCR,  Diabrotica virgifera virgifera ) is one of the most devastating corn pests in North America. Interestingly, WCR displays a robust environmental RNAi response, raising the possibility of applying an RNAi-based pest management strategy to this pest. Understanding the molecular mechanisms involved in the WCR environmental RNAi process will allow for determining the rate limiting steps involved with dsRNA toxicity and potential dsRNA resistance mechanisms in WCR. In this study, we have established a two-step  in vivo  assay system, which allows us to evaluate the involvement of genes in environmental RNAi in WCR. We show that  laccase  2 and  ebony , critical cuticle pigmentation/tanning genes, can be used as marker genes in our assay system, with  ebony  being a more stable marker to monitor RNAi activity. In addition, we optimized the dsRNA dose and length for the assay, and confirmed that this assay system is sensitive to detect well-known RNAi components such as  Dicer-2  and  Argonaute-2 . We also evaluated two WCR  sid1- like  ( sil ) genes with this assay system. This system will be useful to quickly survey candidate systemic RNAi genes in WCR, and also will be adaptable for a genome-wide RNAi screening to give us an unbiased view of the environmental/systemic RNAi pathway in WCR.

Establishing an In Vivo Assay System to Identify Components Involved in Environmental RNA Interference in the Western Corn Rootworm