Quantitative analysis of gene amplification in insecticide-resistant Culex mosquitoes.

The amplification of carboxylesterase structural genes followed by their overexpression is the most common mechanism of resistance to organophosphorus insecticides in Culex mosquitoes. Most resistant Culex quinquefasciatus mosquitoes have co-amplified estalpha2(1) and estbeta2(1) genes. Recently, Southern, DNA dot-blot analysis and phosphorimaging technology were used to quantify the est gene copy number in aphids and mosquitoes. Although more accurate than autoradiography, this method relies on probe hybridization, which can be variable. We have directly measured gene and mRNA copy number by using real-time quantitative PCRs in mosquitoes. The acquisition of fluorescence from incorporation of the double-strand-specific dye SYBR GreenI into a PCR product once per cycle is used to provide an absolute quantification of the initial template copy number. Thus it has been possible to show that estalpha2(1) and estbeta2(1) are co-amplified approx. 80-fold in the genome of the resistant PelRR strain of C. quinquefasciatus. The two genes, although co-amplified in a 1:1 ratio, are differentially transcribed: the estbeta2(1) gene from this amplicon has greater transcription than estalpha2(1) in all individual mosquito larvae tested, with an average ratio of 10:1. Purified esterases from mosquito homogenates were found in a ratio of 3:1, which, combined with the quantitative mRNA data, suggests the operation of both transcriptional and translational control mechanisms to regulate the expression of the amplified genes in C. quinquefasciatus insecticide-resistant mosquitoes.