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


METHODS
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
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.
Laboratório de Fisiologia e Controle de Artrópodes Vetores, Instituto Oswaldo Cruz-FIOCRUZ and Laboratório de Entomologia, Instituto de Biologia do Exército, Rio de Janeiro, RJ, 21040-360, Brazil, Instituto Nacional de Ciência e Tecnologia em Entomologia Molecular, Brazil, Laboratório de Biologia Molecular de Insetos, Instituto Oswaldo Cruz-FIOCRUZ, Rio de Janeiro, RJ, 21040-360, Brazil and Laboratório de Biologia Molecular de Flavivirus, Instituto Oswaldo Cruz-FIOCRUZ, Rio de Janeiro, RJ, 21040-360, Brazil.
Ademir Jesus
AJ
Martins
Luiz Paulo
LP
Brito
Jutta Gerlinde Birggitt
JG
Linss
Gustavo Bueno da Silva
GB
Rivas
Ricardo
R
Machado
Rafaela Vieira
RV
Bruno
José Bento Pereira
JB
Lima
Denise
D
Valle
Alexandre Afranio
AA
Peixoto
Aedes aegypti; gene duplication; kdr mutation; pyrethroid resistance; sodium channel