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

Abstract

Background and objectives: Mutations in the voltage-gated sodium channel gene (NaV), 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 AaNaV 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 AaNaV sites that can harbor kdr mutations (Ile1011Met and Val1016Ile), (ii) cloning and sequencing of part of the AaNaV 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 AaNaV 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.

Keywords: Aedes aegypti; gene duplication; kdr mutation; pyrethroid resistance; sodium channel.

Figure 1.

Diversity of a voltage-gated sodium channel gene region observed in Ae. aegypti Brazilian populations. Part of the region corresponding to the AaNa_V exons 20 and 21, and the intron between them, are represented. A and B indicate the type of intron, as previously stated [14]. In red, the presumed amino acids for the sites 1011 and 1016. Genomic sequences representative for each haplotype were submitted to GenBank: 1011Ile + B + 1016Val (GenBank accession number: FJ479613), 1011Ile + A + 1016Val (FJ479611), 1011Met + A + 1016Val (FJ479612) and 1011Ile + A + 1016Ile (JX275501). TIGR = sequence from Ae. aegypti genome project (Vectorbase)

Figure 2.

Schematic representation of AaNa_V haplotypes. Blue boxes indicate exons 20 and 21 with the intron between them, the latter used to classify the haplotypes as A (orange) or B (green). Sites 1011 and 1016 are represented by the variant wild-type (blue box) or mutant (red box). According to our hypothesis, there is a duplication in some populations, comprised of haplotypes 1011Ile + B + 1016Val and 1011Met + A + 1016Val. Dashed line suggests linkage of the haplotypes, but which one is upstream was not determined

Figure 3.

Three hypotheses with the expected genotypes and molecular phenotypes in the AaNa_V 1011 site for the parental and their respective expected frequency in the F1 offspring. The 1011Met mutation is shown in red. See text for further details