Two different amino acid substitutions in the ali-esterase, E3, confer alternative types of organophosphorus insecticide resistance in the sheep blowfly, Lucilia cuprina

Abstract

Two types of organophosphorus (OP) insecticide resistance are associated with reduced `ali-esterase' (E3 isozyme) activity in Lucilia cuprina. The `diazinon' resistance type shows generally greater resistance for diethyl than dimethyl OPs but no resistance to malathion. The `malathion' resistance type shows generally greater resistance for dimethyl than diethyl OPs, low level diazinon resistance, but exceptionally high malathion resistance (600 × susceptible), the last being attributed to hydrolysis of the carboxylester groups which are peculiar to malathion (malathion carboxylesterase, MCE). E3 variants from diazinon resistant strains have previously been shown to have a Gly 137 → Asp substitution that structural modelling predicts is only about 4.6 Å from the γ oxygen of the catalytic serine residue. Here we show that E3 variants from malathion resistant strains have a Trp 251 → Leu substitution predicted to be about 4.3 Å from that serine. We have expressed alleles of the gene encoding both resistance variants of E3 and an OP susceptible variant in a baculovirus system and compared the kinetics of their products. We find that both resistance substitutions reduce ali-esterase activity and enhance OP hydrolase activity. Furthermore the Gly 137 → Asp substitution enhances OP hydrolase activity for a diethyl OP substrate (chlorfenvinphos) more than does the Trp 251 → Leu substitution, which is consistent with the OP cross-resistance patterns. Trp 251 → Leu also reduces the K m for carboxylester hydrolysis of malathion about 10-fold to 21 μM, which is consistent with increased MCE activity in malathion resistant strains. We then present a model in which the malathion carboxylesterase activity of the E3-Leu 251 enzyme is enhanced in vivo by its OP hydrolase activity. The latter activity enables it to reactivate after phosphorylation by malaoxon, the activated form of malathion, accounting for the exceptionally high level of resistance to malathion. We conclude that the two types of resistance can be explained by kinetic changes caused by the two allelic substitutions in the E3 enzyme.