Mode of action of the insect growth regulator diflubenzuron: A comprehensive study in Tribolium castaneum utilizing genomic tiling array and immunolocalization approaches

Several benzoyl-phenyl-urea-derived insecticides such as diflubenzuron (DFB, Dimilin®) are widely used to control various insect pests. Although this class of compounds disrupts molting and affects chitin content, their precise mode of action is not known. To evaluate the mechanism underlying the insect growth regulating effects, we are conducting a comprehensive study utilizing genomics, and immunolocalization tools in the model insect species and stored product pest, Tribolium castaneum (red flour beetle). The results showed that DFB affects larval-larval and larval-pupal molting. In addition, the ensuing adults had walking defects and died prematurely after adult eclosion. The chitin content of whole larvae or isolated larval midguts was reduced by ~50% after DFB treatment as revealed by the Morgan-Elson method. This result was consistent with confocal analysis of elytra of control and DFB-treated pharate adults using a chitin-binding probe. In contrast, chitosan content was elevated significantly after DFB treatment. Results of RT-PCR and immunolcalization studies of several genes involved in chitin metabolism as well as a gene encoding a putative receptor for DFB will be presented. In summary, DFB treatment results in an increase chitosan, a reduction in chitin and transcripts for specific genes of chitin metabolism, cuticle formation and insecticide metabolism. We also observed defects in the mobility (leg movement) of adults that eclosed following DFB-treatment of larvae. Hence, our results support the hypothesis that DFB affects the expression of multiple genes involved in cuticle chitin metabolism, which are critical for normal insect growth and development.