Fenarimol

The pesticides Maneb, Diazinon and Fenarimol were purchased from Sigma-Aldrich (St. Louis, MO) were dissolved in dimethyl sulfoxide (DMSO; 0.1 M) prior to exposure. Gravid nematodes were lysed in an alkaline hypochlorite solution in order to generate age-matched embryos synchronized populations ^{29 (link)}. The embryos were cultured on 10-cm NGM plates without bacteria for 1 day at 20°C to generate a large pool of synchronized L1-stage worms. The L1-stage worm population was transferred to 10-cm NGM plates seeded with OP50 bacteria and cultured for 65 hr at 15°C to generate a large pool of L4-stage worms. After quantification under the microscope of the number of worms in population samples, 500 worms were transferred to 2-mL tubes with M9 buffer with bacteria, to which the pesticides were subsequently added at a final concentration of 100 μM. Each experiment contained a negative control of 0.1% DMSO. The worms were incubated with shaking on a nutator for 24 hr at 20°C. After the incubation the synchronized population of gravid adult worms settled by gravity and washed with M9 buffer before using it for further analysis.