Quantitative analysis of gene amplification in insecticide-resistant Culex mosquitoes

Abstract

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.

Quantitative real-time PCR with SYBR Green detection to assess gene duplication in insects: study of gene dosage in Drosophila melanogaster (Diptera) and in Ostrinia nubilalis (Lepidoptera)

“…This had been previously observed with DNA from cancer samples [30] and with virus variants [15] and can be a source of additional information about the number and frequency of alleles in the studied population. In fact, differences in the dissociation temperature of PCR products have been used to distinguish between alleles of the estβ gene in Culex [16]. We have observed that when the polymorphism affected the nucleotide sequence complementary to the primers, Ct values were higher than expected.…”

“…SYBR Green I may even detect DNA variants present in the target sequence that TaqMan probes would miss [15]. SYBR Green I has been applied to the detection of chorion genes in Drosophila (about 30-fold amplification) [4] and to determine the gene copy number of esterase genes (about 30-fold amplification) in insecticide-resistant Culex mosquitoes [16]. However, to our knowledge, it has never been applied for determining gene dose in low copy number scenarios, probably because of the possibility to generate false positive results.…”

Transcriptome Profiling and Genetic Study Reveal Amplified Carboxylesterase Genes Implicated in Temephos Resistance, in the Asian Tiger Mosquito Aedes albopictus

“…albopictus also indicated that gene regulation might have an important role in the OP resistance for some individual mosquitoes. The operation of both gene amplification, transcriptional and translational control mechanisms to regulate the expression of CCE genes involved in insecticide resistance has been previously shown [ 38 ], and it is not clear whether gene regulation or amplification (or both) is the determining factor in resistance. Furthermore, it has been shown in population studies that amplification levels vary between individuals over time through variation in organophosphate selection pressure, and that the loss or gain of gene copies, possibly through unequal sister-chromatid exchange is also a common phenomenon in mosquitoes and aphids [ 35 , 39 ].…”

Analysis of ovary-specific genes in relation to egg maturation and female nutritional condition in the mosquitoes Georgecraigius atropalpus and Aedes aegypti (Diptera: Culicidae)

“…qPCR master mixes were prepared as above with serially diluted PCR product (1 ng, 100 pg, 10 pg, 1 pg, 100 fg or 10 fg/PCR) to which primers for the gene of interest were added. The standard line was then used to calculate the transcript copy number in each of the qPCR reactions as described by Paton et al (2000). This method has been successfully used to analyze genes expressed in ovaries of Ae.…”