The search for the target of the chitin inhibitor, dilfubenzuron (DFB) in the red flour beetle

We have used the elytron of the red flour beetle, Tribolium castaneum, as the model tissue for studying the mode of action of the “chitin inhibitor”, DFB and its molecular target(s). Elytron is a specialized wing tissue essentially made-up of epidermal cells associated with the multilayered laminar cuticle, which is amenable to immunolocalization and transmission electron microscopic (TEM) studies. We have standardized the protocol for topical administration of DFB on precisely-aged prepupae to achieve the desired level of mortality (50-90%) on day 5 of the pharate adult stage when the elytra (and other cuticle forming tissues) can be analyzed for expression of genes of by RNASeq and by immunolocalization of proteins involved in chitin metabolism and cuticle assembly. Exposure of prepupae to DFB at 1000 ppm results in near complete loss of chitin in the newly forming procuticle of the elytron and body wall. Global analysis of transcripts by RNA Seq indicates differential expression of several critical genes of cuticle assembly in these insects compared to mock-treated controls.  Interestingly  genes directly involved in the biosynthetic pathway of chitin were not among those affected by DFB. On the other hand, several other proteins involved in chitin-cuticle assembly were mislocalized following DFB treatment. RNA interference studies are ongoing to identify the precise molecular targets of DFB and these results will be presented.