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

Background: Several mutations in voltage gated sodium channel (Na) have been identified in populations worldwide. However, only few are related to knockdown resistance to pyrethroids, most of which with variations in the 1016 and 1534 Na sites. In Brazil, at least two alleles are known: NaR1, with a substitution in the 1534 (1016 Val + 1534 Ile ) and NaR2, with substitutions in both 1016 and sites (1016Ile + 1534Cys ). There is also the duplication in the 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 Na allele and the duplication on pyrethroid resistance and (2) to screen the frequency of the alleles in 27 several natural populations from the metropolitan region of Rio de Janeiro.

Methods: Pyrethroid resistance was evaluated by a knockdown time (T) assay, an adaptation of the WHO test tubes with paper impregnated with deltamethrin. We used laboratory-selected lineages: R1R1 and R2R2 (homozygous for the NaR1 and NaR2 alleles, respectively), Dup (with duplication in the gene), Rockefeller (the susceptibility reference control), and F1 hybrids among them. Genotyping of both 1016 and 1534 Na sites was performed in 811 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 all the insects exposed to the pyrethroid, being the T R2R2 > R1R2 > R1R1, corroborating the recessive nature of the mutations. Frequency of alleles NaR1 and NaR2 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 populations from Rio de Janeiro.

Conclusions: The NaR1 and NaR2 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 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 natural populations from Rio de Janeiro.