Abstract
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.
Highlights
RNA interference (RNAi) is an evolutionarily conserved mechanism, in which double-stranded RNA molecules trigger gene silencing in a sequence specific manner [1,2,3]
RNAi or loss-of-function experiments for yellow genes have been performed in other insects, some of which have resulted in reduction of melanin-based pigmentation [31,32,33]. laccase genes code for phenol oxidases (POs). laccase 2 has been identified as a critical PO for body wall pigmentation and sclerotization in the red flour beetle, Tribolium castaneum, as well as in some other insects including WCR [34,35]. ebony codes for NBAD (N-beta-alanyl dopamine) synthetase, which is critical for the formation of NBAD sclerotin [26,28]
We have established an in vivo assay system in WCR that will allow a quick survey of the genes that may be involved in environmental RNAi in WCR
Summary
RNA interference (RNAi) is an evolutionarily conserved mechanism, in which double-stranded RNA (dsRNA) molecules trigger gene silencing in a sequence specific manner [1,2,3]. RNAi has provided a promising new trend to the pest management field [5], as RNAi-based pest control strategies have the potential to target pest species with great specificity. Application of RNAi to pest control is still a challenge in part due to the difficulty of effectively delivering dsRNA molecules into organisms [5]. In some organisms including several pest insects, RNAi can be triggered via feeding with dsRNA molecules (feeding RNAi or environmental RNAi), and RNAi in these organisms often works systemically as well The ease of inducing a systemic RNAi response via dsRNA feeding opens the door to the practical application of RNAi-based techniques in crop pest management
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