Project Number: 3020-43000-033-09-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Sep 15, 2015
End Date: Sep 14, 2020
To design new control strategies based on the genetic manipulation of stored product pests, including oral RNAi.
Our goal is to develop economical and eco-friendly control strategies for coleopteran storage pests based on oral RNA interference (RNAi). The cooperator will work with ARS scientists to evaluate the potential of oral RNA interference (RNAi) to target specific genes in T. castaneum using bacterial and yeast systems. ARS scientists have identified a number of potential targets in the gut of the feeding stages of T. castaneum, larvae and adults. We anticipate that some candidate genes will be easier to knockdown than others. For example, genes that play an important role in the insect gut will likely be easier to knockdown than those that play roles in distant tissues (e.g. brain); genes expressed at lower levels should also be easier to knockdown. For this reason our list of candidate genes has been selected from the T. castaneum gut transcriptome. Candidate genes include those previously demonstrated as successful in other RNAi systems (vATPase), and those identified as lethal in injection studies (specific cathepsin L genes), as well as those identified in injection studies with increased mortality (enzymes involved in cuticle metabolism). ARS will incorporate dsRNA into biological organisms, such as Escherichia coli and Saccharomyces cerevisiae, and the cooperator will work with ARS scientists to evaluate the efficacy of different delivery vehicles and targets in T. castaneum through bioassays. Unlike plasmid-based systems, the yeast-based system uses homologous recombination to incorporate the transgene directly into the S. cerevisiae yeast chromosome. This is an important consideration because use of plasmid DNA, like those used in E. coli for triggering oral RNAi in C. elegans, requires constant antibiotic selection, while genomic integration does not. This reduces costs, is safer to the environment, and adds stability. Also, since S. cerevisiae lacks RNAi machinery, it may prove to be superior in bioassays. Specifically, S. cerevisiae lacks Dicer, an enzyme required for dsRNAs to be processed in to siRNAs. Therefore, this yeast species will produce dsRNAs, but will leave them uncut. RNAi is present in other budding yeast species, including S. castellii, which will be tested if S. cerevisiae fails to trigger a robust effect. Each dsRNA construct will be tested in small-scale bioassays to determine how well it triggers an RNAi affect. These bioassays will include; direct feeding, mixing bacteria or yeast in diet, and assays (applying yeast to bioassay diets and scoring percent kill). If necessary, dsRNA can be purified from the recombinant strains for more precise dosing. Strains showing a strong effect will be tested for off-target effects in other closely related species, such as T. confusum and Tenebrio molitor (we expect these assays to be negative), and then tested for percent kill on a larger-scale. The project will include a Ph.D. student from NCSU and will constitute work towards a degree program. These data will be used to develop new control products for the control of damaging coleopteran storage pests.