PURIFICATION AND CHARACTERIZATION OF A RESISTANCE-ASSOCIATED ESTERASEFROM THE COLORADO POTATO BEETLE, LEPTINOTARSA-DECEMLINEATA (SAY)

Two strains of the Colorado potato beetle (CPB), Leptinotarsa decemlin eata (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 resi stant New York (NY) and Hot Spot (HS) strains, respectively, than in s usceptible beetles. When treated with azinphosmethyl, the percentages of mortalities for the NY and HS populations were 65.1- and 4.4-fold l ower, respectively, than the susceptible strain. The synergist S,S,S-t ributyl phosphorotrithioate (DEF) increased azinphosmethyl toxicity, s uggesting esterase involvement in azinphosmethyl resistance. Although 1-naphthyl acetate esterase activities in individual fourth instars of resistant strains were not significantly different, a novel resistanc e-associated esterase (RAE) (pl = 6.23) was discovered in the first an d fourth instars and adults of NY and HS CPBs, but not in the suscepti ble strain. The RAE was purified from fourth stadium NY CPBs by Rotofo r followed by polyacrylamide gel isoelectric focusing. Methyl paraoxon was the most potent inhibitor of the purified RAE with an I-50 of 0.1 mu M. The esterase inhibitors octylihio-1,1,1-trifluoropropan-2-one ( OTFP), DEF, and azinphosmethyl had I(50)s of 1.1, 5.5, and 9.8 mu M, r espectively. Carbofuran and eserine hemisulfate were poor inhibitors, with I(50)s greater than 100 mu M. In substrate competition assays wit h the RAE, methyl paraoxon and OTFP were competitive inhibitors. (C) 1 995 Academic Press, Inc.