Levels of Resistance to Pyrethroid among Distinct kdr Alleles in Aedes aegypti Laboratory Lines and Frequency of kdr Alleles in 27 Natural Populations from Rio de Janeiro, Brazil

BACKGROUND: Several mutations in voltage gated sodium channel (Na(V)) have been identified in Aedes aegypti populations worldwide. However, only few are related to knockdown resistance to pyrethroids, most of which with variations in the 1016 and 1534 Na(V) sites. In Brazil, at least two Na(V) alleles are known: Na(V)R1, with a substitution in the 1534 (1016 Val(+) + 1534 Ile(kdr)) and Na(V)R2, with substitutions in both 1016 and sites (1016Ile(kdr) + 1534Cys(kdr)). There is also the duplication in the Na(V) gene, with one copy carrying the substitution Ile1011Met, although its effects on pyrethroid resistance remain to be clarified. Our goals in this study were (1) to determine the role of each kdr Na(V) allele and the duplication on pyrethroid resistance and (2) to screen the frequency of the kdr alleles in 27 several natural Ae. aegypti populations from the metropolitan region of Rio de Janeiro. METHODS: Pyrethroid resistance was evaluated by a knockdown time (KdT) assay, an adaptation of the WHO test tubes with paper impregnated with deltamethrin. We used laboratory-selected Ae. aegypti lineages: R1R1 and R2R2 (homozygous for the kdr Na(V)R1 and Na(V)R2 alleles, respectively), Dup (with duplication in the Na(V) gene), Rockefeller (the susceptibility reference control), and F1 hybrids among them. Genotyping of both 1016 and 1534 Na(V) sites was performed in 811 Ae. aegypti sampled from 27 localities from Rio de Janeiro (17), Niterói (6) and Nova Iguaçu (4) cities, Rio de Janeiro State, Brazil, with a TaqMan real time PCR approach. RESULTS: The laboratory lineages R1R1, R2R2, and R1R2 were the only ones that needed more than 60 minutes to knock down all the insects exposed to the pyrethroid, being the KdT R2R2 > R1R2 > R1R1, corroborating the recessive nature of the kdr mutations. Frequency of kdr alleles Na(V)R1 and Na(V)R2 in field-caught mosquitoes varied from 0 to 52% and 43 to 86%, respectively, evidencing high levels of “resistant genotypes” (R1R1, R1R2, and R2R2), which together summed 60 to 100% in Ae. aegypti populations from Rio de Janeiro. CONCLUSIONS: The Na(V)R1 and Na(V)R2 kdr alleles confer resistance to the pyrethroid deltamethrin in homozygotes and R1R2 heterozygotes, being the R2R2 most resistant genotype. The allele containing duplication in the Na(V) gene, with a mutation in the 1011 site, did not confer resistance under the tested conditions. The frequencies of the “resistant genotypes” are elevated in Ae. aegypti natural populations from Rio de Janeiro.