Isolation and Characterization of an Insecticide-Resistance-Associated Esterase in the Tobacco Budworm Heliothis virescens (F.)

Abstract

1-Naphthyl acetate (1-NA) esterase activity was elevated in whole body homogenates of first, third, and fifth stadium tobacco budworms resistant to thiodicarb and cypermethrin compared to susceptible budworms at the same developmental stage. Increased esterase activity was attributed mostly to three esterases designated A1, B1, and C1 with isoelectric points of 4.8, 5.1, and 5.3, respectively, as determined by isoelectric focusing-polyacrylamide gel electrophoresis (IEF-PAGE). These three esterases were purified by preparative liquid column isoelectric focusing followed by slab IEF-PAGE and sodium dodecyl sulfate-PAGE. Specific antibody to the resistance-associated esterase of the house fly (anti-E1, N. Motoyama) and the green peach aphid (anti-E4, A. Devonshire) was immunoreactive on Western blots with purified esterase A1, while minimal or no reactivity was noted for esterases B1 and C1. Anti-E4 detected A1 in 10,000g supernatant from homogenates of resistant and susceptible tobacco budworms. Kinetic studies demonstrated that methyl paraoxon, cypermethrin, DDT, and thiodicarb were competitive inhibitors of the 1-NA esterase activity of A1, whereas eserine hemisulfate had no effect on activity. An amino-terminal sequence analysis revealed that residues Gly-12, Arg-15, and Gly-16 were conserved with Culex esterase B1 (CuB1), Drosophila melanogaster esterase-6 (DmE6) and acetylcholinesterase, H. virescens JH esterase, human butyrylcholinesterase, and rabbit liver esterase form 2, while Arg-15 was missing in E4 from Myzus persicae. A1 demonstrated the greatest sequence homology with that of the DmE6 of Drosophila and the resistance-associated esterase, CuB1, of Culex.