Submitted to: Journal of Insect Biochemistry and Molecular Biology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/15/2003
Publication Date: 2/1/2004
Citation: ZHOU, X., SCHARF, M.E., SARATH, G., MEINKE, L.J., CHANDLER, L.D., SIEGFRIED, B.D. PARTIAL PURIFICATION AND CHARACTERIZATION OF A GENERAL ESTERASE ASSOCIATED WITH METHYL-PARATHION RESISTANCE IN DIABROTICA VIRGIFERA VERGIFER (COLEOPTERA:CHRYSOMELIDAE). JOURNAL OF INSECT BIOCHEMISTRY AND MOLECULAR BIOLOGY. 2004. Interpretive Summary: This initial study was undertaken to discover pesticide resistance mechanisms in the western corn root worm. This agronomically important pest can be devastating to corn and has developed a remarkable array of insecticide resistance. If we can uncover biochemical mechanisms that impart resistance, we may be able to develop appropriate strategies to control this pest. One of the common means by which insects develop resistance is through making enzymes that can detoxify a pesticide, frequently these enzymes belong to a class called esterases. Esterases can either bind irreversibly to a toxin or catalyze their hydrolysis into chemical forms that are not toxic. In this work we have documented the presence of esterase-resistance mechanisms in the corn root worm and have partially purified one of these enzymes. These data will assist us in cloning the gene for the esterase in the future.
Technical Abstract: Insecticide resistance among Nebraska western corn rootworm populations has been shown to involve increased hydrolytic metabolism, and esterases with intermediate electrophoretic mobility (group II) have been used as a biochemical marker to monitor resistance frequencies in the field. In this study, resistance-associated group II esterases were partially purified by the combined techniques of differential centrifugation, ion exchange, and hydroxyapatite column chromatography, with a final purification factor and % recovery of approximately100-fold and 10%, respectively. Esterases from the resistant and susceptible populations displayed similar chromatographic behavior, however, the activity peak of group II esterases was consistently larger (at least 4-fold) in resistant populations. Kinetic and inhibition studies suggested that the group II esterases are a B-type esterase with similar Km but higher Vmaxin resistant relative to susceptible populations. A putative group II esterase, DvvII, was further purified to homogeneity by preparative polyacrylamide gel electrophoresis. DvvII exists as a monomer with a molecular weight around 66 kDa based on size exclusion chromatography of the native enzyme, although three distinct isoforms with similar pIs were evident based on isoelectric focusing gel electrophoresis. The combined results of this investigation suggest the underlying biochemical basis of esterase-mediated resistance is the overproduction of resistance-associated group II esterases in the resistant D. v. virgifera population.