Fig. 1: kdr mutations para^L1014F and para^I1011M confer insecticide resistance in Drosophila melanogaster.

A Generation of kdr mutations. First, a GFP transgene was inserted at the L1014 site using Cas9-mediated editing. Next, the GFP insertion was deleted with a pair of gRNAs cleaving adjacent sequences and replaced with a donor template containing one of the desired mutations (L1014F, I1011M or I1011V). Successful transformants were screened as GFP negative and later confirmed by genotyping. B DDT resistance: Flies (total number of flies n = 200; number of independent replicates N = 8) were exposed to different concentrations of DDT. Survival, a proxy for DDT resistance, was determined 24 h post exposure. At the reference dose of 50 ppm DTT, the mean resistance values ± s.e.m were: WT = 8.4 ± 4.3; L1014F = 100 ± 0; I1011M = 65.6 ± 6.15; I1011V = 8.75 ± 3.6. C Survival plot for para^WT (Median lifespan = 52), para^L1014F (Median lifespan = 33), para^I1011M (Median lifespan = 33), para^I1011V (Median lifespan = 32) males in absence of any insecticide. All three mutants displayed shortened lifespans compared to the control. Data were analyzed using the Log-rank test for trend and the Mantel-Cox test. D Comparison of embryonic survival. No significant differences in hatching rate were observed among WT, para^L1014F and para^I1011M genotypes. N = 4; n = 400. E para^L1014F are resistant to oxidative stress. para^L1014F flies had significantly higher survival rates at 48 h compared to WT or to para^I1011M mutants. N = 5; n = 125. F para^L1014F are paralytic at high temperature. Male flies were exposed to 37 °C for 4 h and their ability to climb above 5 cm was recorded. N = 4; n = 100. G, H para mutants manifested abnormal sleep patterns in both females and males. Significant day-time sleep latency for para^I1011M mutants is indicated by the arrowhead. Control = Black; para^1014F = Orange; para^1011M = Blue; para^1011V = purple. N: independent biological replicates, n: number of animals tested; Means ± s.e.m are plotted; B two-way ANOVA with Sidak’s multiple comparison, D, E, F one-way ANOVA with Sidak’s multiple comparison. *p < 0.033, **p < 0.0021, ***p < 0.0002, ****p < 0.0001, and ns not significant.