Identification and characterization of ace1-type acetylcholinesterase likely associated with organophosphate resistance in Plutella xylostella

Insensitive acetylcholinesterase (AChE) was determined to be primarily involved in a prothiofos-resistant (PR) strain of diamondback moth (DBM, Plutella xylostella L.), as judged by the AChE inhibition assay using paraoxon, where the PR strain exhibited ca. 26-fold increased I50 value. Extensive sequence analysis of the previously reported ace2-type DBM AChE gene revealed no difference between the susceptible and PR strains. To elucidate the molecular basis of the prothiofos resistance mechanism mediated by insensitive AChE, we cloned and characterized a second AChE gene from DBM. The deduced amino acid sequence of the novel AChE showed the highest homology to ace1, the second copy of insect ace, and was determined in fact as the predominant AChE in DBM compared to the ace2-type with 13- to 250-fold higher transcription levels depending on different tissues. Sequence comparison of the ace1-type cDNA between the susceptible and PR strains of DBM revealed that a total of three amino acid substitutions are closely associated with the PR strain. Among these, the Gly227Ala1 mutation, exclusively present in the PR strain, was located at the same position of the organophosphate resistance-conferring Gly-to-Ala mutation on the ace2 of the fruit fly and house fly. This finding suggests that the Gly227Ala mutation along with two other ones on the ace1 are likely responsible for the AChE insensitivity in DBM.