Evidence for gene duplication in the voltage-gated sodium channel gene of Aedes aegypti

Background and objectives: Mutations in the voltage-gated sodium channel gene (Na(V)), known as kdr mutations, are associated with pyrethroid and DDT insecticide resistance in a number of species. In the mosquito dengue vector Aedes aegypti, besides kdr, other polymorphisms allowed grouping AaNa(V) sequences as type ‘A’ or ‘B’. Here, we point a series of evidences that these polymorphisms are actually involved in a gene duplication event. Methodology: Four series of methods were employed: (i) genotypying, with allele-specific PCR (AS-PCR), of two AaNa(V) sites that can harbor kdr mutations (Ile1011Met and Val1016Ile), (ii) cloning and sequencing of part of the AaNa(V) gene, (iii) crosses with specific lineages and analysis of the offspring genotypes and (iv) copy number variation assays, with TaqMan quantitative real-time PCR. Results: kdr mutations in 1011 and 1016 sites were present only in type ‘A’ sequences, but never in the same haplotype. In addition, although the 1011Met-mutant allele is widely disseminated, no homozygous (1011Met/Met) was detected. Sequencing revealed three distinct haplotypes in some individuals, raising the hypothesis of gene duplication, which was supported by the genotype frequencies in the offspring of specific crosses. Furthermore, it was estimated that a laboratory strain selected for insecticide resistance had 5-fold more copies of the sodium channel gene compared with a susceptible reference strain. Conclusions and implications: The AaNa(V) duplication here found might be a recent adaptive response to the intense use of insecticides, maintaining together wild-type and mutant alleles in the same organism, conferring resistance and reducing some of its deleterious effects.