Analysis of the promoters for the β-esterase genes associated with insecticide resistance in the mosquito Culex quinquefasciatus

Abstract

Resistance to organophosphorus (OP) insecticides in the mosquito Culex quinquefasciatus is primarily due to the amplification and over-expression of non-specific esterases. Co-amplification of two esterase genes, estα2 1 and estβ2 1 , is the most common resistance genotype. In both resistant and susceptible mosquitoes the α- and β-esterase genes are oriented in a head-to-head arrangement, the intergenic sequences containing promoter elements for the divergent transcription of both esterases. Transient transfection of luciferase reporter gene constructs into a C. quinquefasciatus cell line was used to study these promoters. A control vector was constructed with the strong Drosophila actin 5c promoter driving expression of β-galactosidase. The β-esterase promoters from both insecticide resistant and -susceptible insects were highly active in directing luciferase expression. Transfections with panels of deletions revealed several regions where binding sites for positive and negative regulatory elements are located, and candidate transcription factor sites have been identified. Gel shift assays have identified one DNA–protein interaction that is stronger with the resistant than with the equivalent but slightly altered susceptible sequence. The arthropod initiator site TCAGT 135bp upstream of the ATG in both β-esterase promoters is essential for transcription initiation, but a putative TATA box is not involved.