|Kim, Hee Shin|
Submitted to: Insect Biochemistry and Molecular Biology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/9/2011
Publication Date: 3/1/2012
Citation: Merzendorfer, H., Kim, H., Chaudhari, S.S., Kumari, M., Specht, C.A., Butcher, S., Brown, S.J., Manak, R., Beeman, R.W., Kramer, K.J., Muthukrishnan, S. 2012. Genomic and proteomic studies on the effects of the insect growth regulator diflubenzuron in the model beetle species Tribolium castaneum. Insect Biochemistry and Molecular Biology. 42(4): 264-276. http://dx.doi.org/10.1016/j.ibmb.2011.12.008. Interpretive Summary: The insect exoskeleton (cuticle) has many unique functions vital for insect survival, and is therefore an attractive target for new biopesticide design. One of the older pesticides, diflubenzuron, has already been shown to disrupt insect cuticle, but exactly how this occurs has never been demonstrated. We used modern genomic techniques to assess the effects of diflubenzuron toxicity on gene expression for 11,000 of the 16,000 total genes in the red flour beetle genome. Interestingly, genes for metabolism of chitin (a component of insect cuticle) were not affected, but cuticle proteins did show abnormal expression. Genes for diflubenzuron detoxification showed increased activity. This work demonstrates the complexity of diflubenzuron’s mechanism of toxicity, which could explain why this mechanism has remained elusive for decades. Modern genomic technology should enable this mystery to be solved, opening the door for better design of biopesticides that target the insect cuticle.
Technical Abstract: Several benzoylphenyl urea-derived insecticides such as diflubenzuron (DFB, Dimilin®) are in wide use to control various insect pests. Although compounds in this class are known to disrupt molting and to affect chitin content, their precise mode of action is still not understood. To gain a broader insight into the mechanism underlying the insecticidal effects of benzoylphenyl urea compounds, we conducted a comprehensive study with the model beetle species and stored-product pest, Tribolium castaneum (red flour beetle), utilizing genomic and proteomic approaches. DFB was added to a wheat flour-based diet at various concentrations and fed to larvae and adults. We observed abortive molting, hatching defects, and reduced chitin amounts in the larval cuticle, the peritrophic matrix, and eggs. Electron microscopic examination of the larval cuticle revealed major structural changes and a loss of lamellate structure of the procuticle. We used a genomic tiling array for determining relative expression levels of about 11,000 genes predicted by the GLEAN algorithm. About 6% of all predicted genes were more than 2-fold up- or down-regulated in response to DFB treatment. Genes encoding enzymes involved in chitin metabolism were unaffected, but genes encoding cuticle proteins were affected. In addition, several genes presumably involved in detoxification of DFB were up-regulated. Comparative 2D gel electrophoresis of proteins extracted from the midgut revealed 388 protein spots, of which 7% were significantly affected in their levels by DFB treatment as determined by laser densitometry. Mass spectrometric identification revealed that UDP-N-acetylglucosamine pyrophosphorylase and glutathione synthetase were up-regulated. In summary, the red flour beetle is a good model organism for investigating the mode of action of bioactive materials such as insect growth regulators and other insecticides. The results of this study support the hypothesis that DFB treatment of Tribolium leads to changes in the expression level of many different genes and that the mode of action of DFB at the molecular level is very complex.