Fenvalerate

For feeding assays, D. melanogaster wild-type or Cyp4e3 transgenic 7-day-old flies were briefly anesthetized on CO₂, starved for 4–6 h and then placed in 30-ml cellulose-capped vials containing 5 ml of 1% aqueous agarose/1% sucrose medium (control) or fed with either 1% H₂O₂ or specified concentration of permethrin or fenvalerate (all Sigma–Aldrich). The vials were placed in an incubator at 22 °C, 55% humidity with 12:12 h light:dark photoperiod and checked for dead flies every 3–6 h until no living flies were left. All experiments were run in triplicate with at least 30 flies in each run of a specified genotype. For topical application, D. melanogaster wild-type or transgenic 7-day-old male flies were briefly immobilised on CO₂, and a 69 nl volume of permethrin (0, 2, 5, 6, 8, 10, 12.5, 15, 17.5 and 20 ng/fly doses which were then converted to ng/mg by dividing the dose by the average weight, n = 25 flies with at least 3 biological replicates) was applied to the thoracic notum using a microinjector (NanojectII, Drummond Scientific Company). Treated flies were placed in an inverted food vial to prevent mortality through adherence to the food and vials were placed in the same incubator at 22 °C (55% humidity with 12:12 light:dark h photoperiod) because pyrethroids have a temperature dependent toxicity. Mortality data was recorded 24 h after application.