Variabilidade do domínio IIS6 do gene do canal de sódio, associada à resistência aos inseticidas piretróides, em populações de Anopheles darlingi e Anopheles marajoara da Amazônia brasileira

Abstract

Non synonymous mutations in the IIS6 domain of the voltage-gated sodium channel gene (NaV) have been frequently associated with resistance to pyrethroids in several species of anophelines. In this work, the first investigation of the nucleotide diversity of this region was carried out in Anopheles darlingi and Anopheles marajoara, two main human malaria vectors in Brazil. A partial region of the IIS6 domain was sequenced from adults females collectedindoor and outdoor from Manaus and Coari (AM), and most and least susceptible larvae to deltamethrin from Manaus, São Gabriel da Cachoeira, and Iranduba, in the Amazonas State, Rio Branco, Acre, and Macapá, Amapá. In addition, a simplified knockdown bioassay using larvae was adapted to assess the status of susceptibility or tolerance of Neotropical anophelines. The results of the bioassays revealed low susceptibility in the populations from Amazonas State (A. darlingi: Manaus; A. marajoara: Iranduba). The analysis of the nucleotide diversity in the NaV of A. darlingi showed five polymorphic sites that assembled the samples into five haplotypes (A, B, C, D and I). In A. marajoara four polymorphic sites were detected that assembled the samples into four haplotypes (E, F, G and H). All these substitutions were synonymous and the kdr (knockdown resistance) mutations at 1014 site were not detected, suggesting the occurrence of other resistance mechanisms related to the loss of susceptibility to deltamethrin, such as metabolic resistance. Although kdr mutations have not been found, it is important that its occurrence is permissive, once the codon 1014 of these two species is formed by TTA and a single mutation (TTT or TCA) can change the amino acid to phenylalanine or serine as observed in other insects. The amino acid translation of the region analyzed showed complete homology with other anophelines placed in public databases and the nucleotide differences observed were in agreement with the phylogeny of xi the species. It was not possible to correlate the frequency of some haplotypes with the loss of susceptibility to the insecticide or females captured indoor or outdoor. Our analysis of the IIS6 domain of the A. darlingi and A. marajoara’s NaV represents a unique opportunity for understanding the evolutionary diversification of a gene directly associated with pyrethroid resistance. Furthermore, we describe for the first time the padronization of a simple, quick and cost effective susceptibility test in anophelines larvae. All this information can be used by the competent institutions to direct the strategic measures to be adopted to combat malaria vectors.