Co-amplification explains linkage disequilibrium of two mosquito esterase genes in insecticide-resistant Culex quinquefasciatus

The mosquito Culex quinquefasciatus (Say) is a vector of human disease and a world-wide biting nuisance. Organophosphorus insecticides (OPs) have been widely used to control C, quinquefasciatus populations and this has led to the emergence of OP-resistance, Predominantly, resistance is caused by increased production of two non-specific carboxylesterases, Est alpha 2(1) and Est beta 2(1). Increased abundance of these esterases is associated with the amplification of their respective genes. The est alpha 2(1) and est beta 2(1) es were cloned and sequenced from OF-resistant Sri Lankan C, quinquefasciatus: the two adjacent genes are in a head to head configuration, within a single amplification unit (amplicon), The homology between the two genes suggests that they arose from an ancient duplication event. The two genes have different numbers of exons (est alpha 2(1) has seven and est beta 2(1) has four); however, the intron/exon boundaries in est beta 2(1) are all conserved in est alpha 2(1). The two genes are co-amplified in three other mosquito strains with the elevated Est alpha 2(1)/Est alpha 2(1) phenotype. Their complete linkage disequilibrium is explained by the location of the two genes involved in resistance within a single amplicon. In insecticide-susceptible C, quinquefasciatus, the non-amplified est alpha and est beta gene loci are also found in a similar head to head configuration, but the size of the intergenic non-coding region is approx. 1 kb less than in the amplicon. The smaller intergenic spacer is also found in a strain with amplified est beta(1), which suggests that extensive laboratory selection for this amplified esterase:has not eliminated the non-amplified genes, The intergenic spacer regions have been subcloned and sequenced. They ae contain numerous possible TATA boxes, promoters and a number of possible regulatory elements with high homology to the consensus sequence of the Barbie box, These latter putative regulatory elements are more numerous in the larger intergenic spacer, which differs from the non-amplified spacer by two large (> 420 bp) and one small (5 bp) insertions.