Laboratório de Fisiologia e Controle de Artrópodes Vetores, Instituto Oswaldo Cruz, FIOCRUZ, Rio de Janeiro, Brazil.
Laboratório de Entomologia, Instituto de Biologia do Exército, Rio de Janeiro, Brazil.
Biomed Res Int. 2018 Jul 11;2018:2410819. doi: 10.1155/2018/2410819. eCollection 2018.
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.
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.
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.
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.
世界各地的人群中已经发现了几种电压门控钠离子通道（Na）的突变。然而，只有少数与击倒抗性有关，其中大多数与 1016 和 1534 Na 位点的变化有关。在巴西，至少有两个 等位基因已知：NaR1，在 1534 处（1016 Val + 1534 Ile ）有取代，NaR2，在两个 1016 和位点（1016Ile + 1534Cys ）有取代。基因也存在重复，其中一个拷贝携带 1011Ile 取代，尽管其对拟除虫菊酯抗性的影响仍有待澄清。我们在这项研究中的目标是（1）确定每个 Na 等位基因和重复对拟除虫菊酯抗性的作用，以及（2）筛选来自里约热内卢大都市区的 27 个自然 种群中 等位基因的频率。
通过击倒时间（T）测定法评估拟除虫菊酯抗性，这是一种改良的世卫组织测试管，用浸渍了溴氰菊酯的纸张。我们使用了实验室选择的谱系：R1R1 和 R2R2（分别为 NaR1 和 NaR2 等位基因的纯合子）、Dup（ 基因中的重复）、Rockefeller（易感性参考对照）以及它们之间的 F1 杂种。在来自巴西里约热内卢州的 27 个地点（17 个）、尼特罗伊（6 个）和新伊瓜苏（4 个）的 811 个样本中，采用 TaqMan 实时 PCR 方法对 1016 和 1534 Na 位点进行了基因分型。
实验室谱系 R1R1、R2R2 和 R1R2 是唯一需要超过 60 分钟才能使所有接触拟除虫菊酯的昆虫全部击倒的谱系，T R2R2 > R1R2 > R1R1，证实了 突变的隐性性质。在野外捕获的蚊子中，NaR1 和 NaR2 等位基因的频率从 0 到 52%和 43 到 86%不等，这表明存在高水平的“抗性基因型”（R1R1、R1R2 和 R2R2），这些基因型在里约热内卢的 种群中总计达到 60%到 100%。
NaR1 和 NaR2 等位基因赋予了对拟除虫菊酯溴氰菊酯的抗性，在纯合子和 R1R2 杂合子中表现为 R2R2 最具抗性。在 基因中含有 1011 位点突变的重复等位基因在测试条件下并未赋予抗性。在来自里约热内卢的自然 种群中，“抗性基因型”的频率升高。
