Multiplex qPCR assay detects genomic copy numbers of two esterase genes associated with insecticide resistance in Culex pipiens complex mosquitoes in the US

Abstract

Insecticide resistance in vector mosquitoes can be the result of a variety of changes to genes and metabolic functions. In Culex pipiens complex mosquitoes, 2 esterase genes (CPIJ013917 and CPIJ013918) confer resistance to organophosphate (OP) insecticides either through upregulation or gene amplification. Previous work to quantify gene copy numbers has examined the 2 genes separately, or considered only 1 of the members of the Cx. pipiens complex. We sought to develop an assay that examines copy numbers of both target genes simultaneously and works in both Cx. pipiens and Cx. quinquefasciatus in the US. The resulting real-time PCR assay uses primer pairs with fluorescent probes to calculate genomic copies of the target genes relative to the known single-copy reference gene Ace2 (CPIJ000662). Starting with DNA from pools of 10 Sebring colony individuals, we created standard curves for each of the 3 genes separately, and then as a multiplex. After verifying that the levels of efficiency and reproducibility were acceptable, each of the 3 amplicons was cloned using TOPO-TA cloning kits to create single-copy plasmid DNA, which was mixed and run on every plate in triplicate as a positive control. The assay was tested on DNA from adults raised from field-collected egg rafts whose resistance levels to malathion and permethrin had been assessed with a bottle bioassay. The number of gene copies was variable within the populations tested. In addition, there was a positive correlation between copy numbers of each target gene and levels of resistance.