The larval toxicities of CINEX, the isolated sesquiterpenes, and cypermethrin were evaluated using an established assay [46 (link), 47 (link)]. In brief, five 1st-instar larvae were added to the wells of a 24-well Falcon Multiwell plate (Becton Dickinson Labware, Franklin Lakes, NJ) containing 985 μl of dH2O and 5 μl of a food solution (13 mg/ml of finely ground fish food flakes in dH2O; Tetramin, Blacksburg, VA). To each well, 10 μl of CINEX, a sesquiterpene, or cypermethrin (all dissolved in 100% acetone) at various concentrations was added. Control wells received 10 μl of 100% acetone. The plates were held under normal rearing conditions (28°C, 80% relative humidity, 12:12 light:dark) for 24 h before assessment of larvae. The efficacy of a concentration was determined as the percentage of larvae in a well that died within 24 h. Larvae were counted as dead if they did not move after gentle touching with a fine needle or pipette tip.
The adult toxicities of CINEX and the isolated sesquiterpenes were evaluated using an established assay [46 (link), 48 (link)]. In brief, groups of 10 adult female mosquitoes (3–10 days post-emergence) were immobilized on ice and treated with 500 nl (Ae. aegypti, Cx. pipiens) or 200 nl (An. gambiae) of a compound (dissolved in 100% acetone) at various concentrations. The compounds were delivered to the thorax of mosquitoes with a repeating dispenser (PB600-1, Hamilton, Reno, NV). As a control, 100% acetone was used. Immediately after treatment, the mosquitoes were transferred to small cages (32 oz. containers) with access to 10% sucrose and held under normal rearing conditions for 24 h before assessment. The efficacy of a dose was defined as the percentage of incapacitated mosquitoes (i.e., dead or unable to fly) in a cage within 24 h [46 (link), 48 (link)–51 (link)].
The concentration/dose-response curves for CINEX, the sesquiterpenes, and cypermethrin were evaluated with GraphPad Prism (version 6.07) software. In brief, percent efficacies were plotted against the log transformations of the concentrations/doses. The EC50, ED50, and Hill slope values were determined with non-linear regressions using the log(agonist) vs. normalized response function. Statistical comparisons of the best fit values were made with F-tests using GraphPad Prism software.
The adult toxicities of CINEX and the isolated sesquiterpenes were evaluated using an established assay [46 (link), 48 (link)]. In brief, groups of 10 adult female mosquitoes (3–10 days post-emergence) were immobilized on ice and treated with 500 nl (Ae. aegypti, Cx. pipiens) or 200 nl (An. gambiae) of a compound (dissolved in 100% acetone) at various concentrations. The compounds were delivered to the thorax of mosquitoes with a repeating dispenser (PB600-1, Hamilton, Reno, NV). As a control, 100% acetone was used. Immediately after treatment, the mosquitoes were transferred to small cages (32 oz. containers) with access to 10% sucrose and held under normal rearing conditions for 24 h before assessment. The efficacy of a dose was defined as the percentage of incapacitated mosquitoes (i.e., dead or unable to fly) in a cage within 24 h [46 (link), 48 (link)–51 (link)].
The concentration/dose-response curves for CINEX, the sesquiterpenes, and cypermethrin were evaluated with GraphPad Prism (version 6.07) software. In brief, percent efficacies were plotted against the log transformations of the concentrations/doses. The EC50, ED50, and Hill slope values were determined with non-linear regressions using the log(agonist) vs. normalized response function. Statistical comparisons of the best fit values were made with F-tests using GraphPad Prism software.
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