The elytron as a model system for the study of the mode of action of the chitin inhibitor, diflubenzuron

We have focused on the elytron of the red flour beetle, Tribolium castaneum, as the model tissue for studying the mode of action of the “chitin inhibitor”, diflubenzuron (DFB). In addition to the ease of isolation and manipulation, this specialized wing tissue is essentially made-up of epidermal cells and is amenable to immunolocalization and transmission electron microscopic (TEM) studies.  We have standardized the protocol for topical administration of DFB on precisely timed 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. However, Global analysis of transcripts by RNA Seq indicates differential expression of several critical genes of chitin metabolism in these insects compared to mock-treated controls.  While immunolocalization of CHS-A also did not detect any significant change in the level or localization of this enzyme inside the cell following DFB-treatment. there were changes in the distribution of other  chitin metabolism and cuticular proteins in DFB-treated insects.  Adults fed  a diet containing 100 ppm chitin laid eggs with significantly reduced chitin and even though the embryos appeared to have completed their larval development, they failed to hatch. These results will be presented.