Evolution of kdr haplotypes in worldwide populations of Aedes aegypti: Independent origins of the F1534C kdr mutation

Aedes aegypti is the primary vector of dengue, chikungunya, Zika, and urban yellow fever. Insecticides are often the most effective tools to rapidly decrease the density of vector populations, especially during arbovirus disease outbreaks. However, the intense use of insecticides, particularly pyrethroids, has selected for resistant mosquito populations worldwide. Mutations in the voltage gated sodium channel (NaV) are among the principal mechanisms of resistance to pyrethroids and DDT, also known as “knockdown resistance,” kdr. Here we report studies on the origin and dispersion of kdr haplotypes in samples of Ae. aegypti from its worldwide distribution. We amplified the IIS6 and IIIS6 NaV segments from pools of Ae. aegypti populations from 15 countries, in South and North America, Africa, Asia, Pacific, and Australia. The amplicons were barcoded and sequenced using NGS Ion Torrent. Output data were filtered and analyzed using the bioinformatic pipeline Seekdeep to determine frequencies of the IIS6 and IIIS6 haplotypes per population. Phylogenetic relationships among the haplotypes were used to infer whether the kdr mutations have a single or multiple origin. We found 26 and 18 haplotypes, respectively for the IIS6 and IIIS6 segments, among which were the known kdr mutations 989P, 1011M, 1016I and 1016G (IIS6), 1520I, and 1534C (IIIS6). The highest diversity of haplotypes was found in African samples. Kdr mutations 1011M and 1016I were found only in American and African populations, 989P + 1016G and 1520I + 1534C in Asia, while 1534C was present in samples from all continents, except Australia. Based primarily on the intron sequence, IIS6 haplotypes were subdivided into two well-defined clades (A and B). Subsequent phasing of the IIS6 + IIIS6 haplotypes indicates two distinct origins for the 1534C kdr mutation. These results provide evidence of kdr mutations arising de novo at specific locations within the Ae. aegypti geographic distribution. In addition, our results suggest that the 1534C kdr mutation had at least two independent origins. We can thus conclude that insecticide selection pressure with DDT and more recently with pyrethroids is selecting for independent convergent mutations in NaV.
Author summary Insecticide resistance is a global threat for the control of Aedes aegypti, the mosquito vector of aboviruses such as dengue, chikungunya and Zika. Mutations in the voltage gated sodium channel (NaV), known as kdr, are one of the principal mechanisms related to resistance to pyrethroids, the class of insecticide most employed worldwide inside and around residences. We investigate whether the same kdr mutations found in Ae. aegypti populations from distinct regions of the world have a common origin and subsequently dispersed or if they emerged in unrelated populations at distinct moments. By evaluating the sequences of two fragments of the NaV gene, obtained from DNA collections of Ae. aegypti from several countries, we found at least two independent origins for the F1534C kdr mutation in American, African and Asian populations. There was no evidence for multiple origins of the common kdr mutations V1016I and P989S + V1016G, which were exclusive to American and Asian populations. Our results increase our knowledge of insecticide resistance evolution in one of the main arboviral mosquito vectors of major global diseases.