Adult female bioassays

We did insecticide bioassays for three purposes: to determine the levels of resistance to insecticides, to determine if enzyme-mediated detoxification is a major mechanism of resistance by using synergists, and to calculate the degree of dominance of pyrethroid resistance. To determine the levels of resistance to insecticides, solutions were dissolved in acetone and serial dilutions were prepared to find the range of doses that provided mortality values between 0% and 100%. Acetone was applied as control. Dosing of mosquitoes was conducted as outlined for cyhalothrin selections except, 20 adult females were treated per dose. Bioassays for each insecticide were repeated 5 times per strain. To determine if enzyme-mediated detoxification is a major mechanism of resistance in the LM-R strain, we conducted bioassays with cyhalothrin and the synergists PBO, DEM and DEF which inhibit CYPs, GSTs and hydrolases, respectively [30]. Bioassays were done as described above, except that 2.5 μg PBO, 5 μg DEM or 0.31 μg DEF (the maximal sublethal dose of each) were applied to the thorax of each adult female 1 hour prior to cyhalothrin application. For each replicate, controls included double acetone and an acetone plus synergist application.

We determined the degree of dominance of pyrethroid resistance in LKR relative to ROCK and KDR:ROCK to understand how pyrethroid resistance is inherited when the 410L + 1016I + 1534C allele is paired with a susceptible allele or the S989P + V1016G allele. To compare the dominance of resistance between LKR and ROCK, 400 unmated females of LKR were crossed with 200 ROCK males. Bioassays were done with adult females from the F₁ using cyhalothrin (we chose cyhalothrin to calculate the degree of dominance because this is the pyrethroid used to select LM-R strains based on a previous study [31]. Reciprocal crosses were not conducted because Vssc is not sex linked [32]. The degree of dominance (D) was calculated according to Stone [33]. To compare the mode of inheritance between LKR and KDR:ROCK, 549 unmated females of KDR:ROCK strain were crossed with 220 males of LKR strain. Bioassays using permethrin and transfluthrin were done with F₁ adult females and D was calculated as described above. We tested permethrin and transfluthrin because these two pyrethroids had some of the highest fold difference in the resistance ratios between KDR:ROCK and LKR (9.2- and 9.5-fold difference between KDR:ROCK and LKR, respectively).

For each insecticide, data from all replicates were pooled and assessed by probit analysis using R software version 3.6.3 [34] and a script that is publicly available (https://github.com/JuanSilva89/Probit-analysis/commit/2eaaff05da0f89294788bd0bed564e1bf257acf2) to determine the LD₅₀ and 95% confidence intervals (CI 95%). Control mortality was corrected according to Abbott’s formula [35]. For each insecticide, the resistance ratios were calculated by dividing the LD₅₀ of LKR by the LD₅₀ of the ROCK strain. Statistical analysis between different resistance ratios was done as previously described [36].