Transport of dsRNA in lepidopteran and hemipteran insects

The primary goal of this project is to increase our understanding of RNAi mechanisms in insects to facilitate application of this technology against a greater diversity of arthropod pests. Research supported by CAMTech showed that RNAi works well and is systemic in two coleopteran insects tested. However, RNAi works inefficiently in two lepidopteran insects tested. Enhanced dsRNase activity and entrapment of dsRNA in acidic bodies in the cells are among the main reasons for inefficient RNAi in lepidopteran insects (7). Research supported by CAMTech showed that naked dsRNAs taken up by lepidopteran cells are trapped in the endosomes resulting in inefficient knockdown of target genes (8). We developed constructs for labeling cell organelles (early and late endosomes, P-bodies, stress granules and D2-like bodies), stable Sf-9 cell lines expressing the luciferase reporter gene and the Caenorhabditis elegans SID1 transporter gene, methods for radioactive and fluorescent labeling of dsRNA and their detection by microscopy and biochemical methods. We propose to utilize these resources to determine differences and similarities in the transport of naked, lipoplex and polyplex conjugated dsRNA. There is very little information on the differences and similarities of pathways used by siRNA, dsRNA (different lengths) and shRNA for transport into and within lepidopteran cells and tissues. We will conduct experiments to compare the transport of various types of dsRNAs into and within lepidopteran cells.