D3040: (2nd Place) Functional characterization of kdr mutations in the sodium channels of Aedes aegypti

Knock-down resistance (kdr) of insects to insecticides evolves due to naturally occurring mutations in target proteins. Many kdr mutations in insects' voltage-gated sodium channels are known to confer resistance to pyrethroid insecticides. Such mutations continue to emerge in mosquito populations and demonstrating their molecular mechanisms is paramount for a better understanding of the mechanisms of pyrethroid action and insect resistance.  Recent studies identified five new sodium channel mutations in Aedes aegypti mosquito populations: E478K and F174I in Florida, as well as L199F, A434T, and L982W in Vietnam and Cambodia. However, only mutation L982W has been experimentally demonstrated to confer mosquito sodium channel resistance to pyrethroids. Here we report the results of functional studies of the five mutations in Ae. aegypti sodium channel AaNav1-1. PCR-mediated site-directed mutagenesis was used to generate respective mutants. The mutant channels were expressed in Xenopus oocytes. Two-electrode voltage clamp technique was used to evaluate the gating properties and sensitivity of the mutant channels to pyrethroids deltamethrin (DMT) and transfluthrin (TF). Mutations F174I, L199F, and L982W statistically significantly reduced the channel sensitivity to DMT, but only mutation L982W statistically significantly reduced the channel sensitivity to TF. Mutations F174I, L199F, and L989W shifted the voltage dependence of channel activation but not inactivation. Meanwhile, mutations E478K and A434T had no effect on channel properties and sensitivity to DMT and TF. Our data will be valuable for providing early warning of pyrethroid resistance and developing molecular diagnostics tools to monitor frequencies of kdr mutations in field populations of insects.