1a. Objectives (from AD-416):
Develop an effective method for oral RNAi in coleopteran storage pests.
1b. Approach (from AD-416):
The cooperator and ARS have recently reviewed the field of RNAi (Aronstein et al., 2011). We have research experience in injection-based RNAi (Brown et al., 1999; Lorenzen et al., 2002; Oppert et al., 2009), but efforts to establish oral delivery of dsRNA have been unsuccessful. Development of oral RNAi is necessary to develop RNAi as a new control method for storage pests. To develop an effective oral RNAi method, we will identify the most likely candidate genes from the genome of Tribolium castaneum. The cDNA for each gene will be isolated using standard molecular protocol. We will manufacture dsRNA either with commercial kits in vitro or by a bacterial system in vivo. dsRNA will be introduced to insects via different methods (application to diet or encapsulated and fed via liquid dose). Phenotype and genotype will be compared in treated and control insects. Successful methods will be evaluated in Tenebrio molitor, Rhyzopertha dominica, and Sitophilus spp., and optimized for large-scale screening of potential biopesticides.
3. Progress Report:
We continue to work on effective delivery methods for oral RNAi. Several gene candidates have been identified from the red flour beetle genome as the most likely candidate genes for RNAi, and constructs for dsRNA have been made. dsRNA was administered to flour beetle larvae by droplet and microinjection. With one of the constructs, 85% mortality for oral-dosed and 100% mortality for injected larvae were recorded in the first week post treatment. We are continuing to screen to identify dsRNA constructs that may be used for large-scale screening of potential biopesticides.