2.5. Motility assays using the wMicroTracker

The wMicroTracker (InVivo Biosystems; Fig. 1C) is a LED-based assay system that allows drug effects on nematode motility to be recorded over time (Liu et al., 2019; Risi et al., 2019). As with the electrophysiological assays, wMicroTracker experiments were performed using Day-1 adult C. elegans. Prior to the experiment, worms were rinsed from NGM agar plates into a 15 ml conical reaction vessel containing M9 + 0.001% Triton-X (Sigma-Aldrich, St. Louis; MO USA). The worms were allowed to sink to the bottom and the supernatant was replaced three times by fresh M9 + 0.001% Triton-X. Approximately 70 worms in 100 μl M9 + 0.001% Triton-X were pipetted into each well of a flat-bottom 96-well microtiter plate (flat bottom, Greiner Bio-One; Germany). All drugs were applied in six different concentrations (including a solvent-only control) with four replicates per plate (final ML concentrations: 0.1 μM, 0.33 μM, 1 μM, 3.3 μM, 10 μM; final LEV concentrations: 3.3 μM, 10 μM, 33 μM, 100 μM, 330 μM). To ensure a final solvent concentration of 0.1%, drugs were pre-diluted in M9. The experiment was repeated three times independently. All wMicroTracker recordings were 4 h in duration (Fig. 1Cii) and motility values for different experimental groups were analyzed in 30-min time bins.