Molecular evidence of sequential evolution of DDT- and pyrethroid-resistant sodium channel in Aedes aegypti

Background Multiple mutations in the voltage-gated sodium channel have been associated with knockdown resistance (kdr) to DDT and pyrethroid insecticides in a major human disease vector Aedes aegypti. One mutation, V1016G, confers sodium channel resistance to pyrethroids, but a different substitution in the same position V1016I alone had no effect. In pyrethroid-resistant Ae. aegypti populations, V1016I is often linked to another mutation, F1534C, which confers sodium channel resistance only to Type I pyrethroids including permethrin (PMT), but not to Type II pyrethroids including deltamethrin (DMT). Mosquitoes carrying both V1016G and F1534C exhibited a greater level of pyrethroid resistance than those carrying F1534C alone. More recently, a new mutation T1520I co-existing with F1534C was detected in India. However, whether V1016I or T1520I enhances pyrethroid resistance of sodium channels carrying F1534C remains unknown. Methodology/Principal findings V1016I, V1016G, T1520I and F1534C substitutions were introduced alone and in various combinations into AaNa(v)1-1, a sodium channel from Aedes aegypti. The mutant channels were then expressed in Xenopus oocytes and examined for channel properties and sensitivity to pyrethroids using the two-electrode voltage clamping technique. The results showed that V1016I or T1520I alone did not alter the AaNa(v)1-1 sensitivity to PMT or DMT. However, the double mutant (TI)-I-1520+(FC)-C-1534 was more resistant to PMT than (FC)-C-1534, but remained sensitive to DMT. In contrast, the double mutant (VI)-I-1016+(FC)-C-1534 was resistant to DMT and more resistant to PMT than (FC)-C-1534. Furthermore, (VI)-I-1016/G and (FC)-C-1534 channels, but not (TI)-I-1520, were resistant to dichlorodiphenyltrichloroethane (DDT). Cryo-EM structures of sodium channels suggest that T1520I allosterically deforms geometry of the pyrethroid receptor site PyR1 in AaNa(v)1-1. The small deformation does not affect binding of DDT, PMT or DMT, but in combination with F1534C it increases the channel resistance to PMT and DDT. Conclusions/Significance Our data corroborated the previously proposed sequential selection of kdr mutations in Ae. aegypti. We proposed that mutation F1534C first emerged in response to DDT/pyrethroids providing a platform for subsequent selection of mutations V1016I and T1520I that confer greater and broader spectrum of pyrethroid resistance. Author summary Intensive use of pyrethroids has led to the selection of resistance in mosquitoes, and knockdown resistance (kdr) is one of the major mechanisms of pyrethroid resistance. So far, eleven kdr mutations were identified to be associated with pyrethroid resistance in Aedes aegypti. Among the mutations, the V1016I and T1520I substitutions were found to be associated with F1534C but rarely found alone. F1534C confers sodium channel resistance to Type I pyrethroids including permethrin (PMT). However, whether V1016I or T1520I enhances the F1534C-mediated sodium channel resistance remain unknown. In this study, our electrophysiological results confirmed their involvement in kdr and corroborate the previously proposed sequential selection of kdr mutations in Ae. aegypti: F1534C likely emerged first in response to DDT and/or pyrethroids, whereas V1016I and T1520I appeared later under more intensive selection from pyrethroid use.