Site-Directed Mutagenesis of an Acetylcholinesterase Gene from the Yellow Fever MosquitoAedes aegyptiConfers Insecticide Insensitivity

Abstract

Insecticide resistance is a serious problem facing the effective control of insect vectors of disease. Insensitive acetylcholinesterase (AChE) confers resistance to organophosphorus (OP) and carbamate insecticides and is a widespread resistance mechanism in vector mosquitoes. Although the point mutations that underlie AChE insensitivity have been described fromDrosophila,the Colorado potato beetle, and house flies, no resistance associated mutations have been documented from mosquitoes to date. We are therefore using a cloned acetylcholinesterase gene from the yellow fever mosquitoAedes aegyptias a model in which to perform site directed mutagenesis in order to understand the effects of potential resistance associated mutations. The same resistance associated amino-acid replacements as found in other insects also confer OP and carbamate resistance to the mosquito enzyme. Here we describe the levels of resistance conferred by different combinations of these mutations and the effects of these mutations on the kinetics of the AChE enzyme. Over-expression of these constructs in baculovirus will facilitate purification of each of the mutant enzymes and a more detailed analysis of their associated inhibition kinetics.