Purification and Characterization of a Resistance-Associated Esterase from the Colorado Potato Beetle, Leptinotarsa decemlineata (Say)

Abstract

Two strains of the Colorado potato beetle (CPB), Leptinotarsa decemlineata (Say) (Coleoptera: Chrysomelidae), were found to be resistant to carbofuran and azinphosmethyl when compared to a susceptible strain in bioassays using a discriminating insecticide dose. The percentage of mortalities for carbofuran were 115.1- and 70.8-fold lower in the resistant New York (NY) and Hot Spot (HS) strains, respectively, than in susceptible beetles. When treated with azinphosmethyl, the percentages of mortalities for the NY and HS populations were 65.1- and 4.4-fold lower, respectively, than the susceptible strain. The synergist S,S,S-tributyl phosphorotrithioate (DEF) increased azinphosmethyl toxicity, suggesting esterase involvement in azinphosmethyl resistance. Although 1-naphthyl acetate esterase activities in individual fourth instars of resistant strains were not significantly different, a novel resistance-associated esterase (RAE) (p I = 6.23) was discovered in the first and fourth instars and adults of NY and HS CPBs, but not in the susceptible strain. The RAE was purified from fourth stadium NY CPBs by Rotofor followed by polyacrylamide gel isoelectric focusing. Methyl paraoxon was the most potent inhibitor of the purified RAE with an I 50 of 0.1 μ M. The esterase inhibitors octylihio-1,1,1-trifluoropropan-2-one (OTFP), DEF, and azinphosmethyl had I 50s of 1.1, 5.5, and 9.8 μ M, respectively. Carbofuran and eserine hemisulfate were poor inhibitors, with I 50s greater than 100 μ M. In substrate competition assays with the RAE, methyl paraoxon and OTFP were competitive inhibitors.