Fig. 3. Characterization of Complex I ROS-induced avoidance behavior in C. elegans.

a, b Light-induced Complex I ROS increases phototaxis avoidance behavior C. elegans. a Schematic illustration of phototaxis experimental procedure. Wild-type (N2) and nuo-1::Supernova worms were transferred to the center of the seeded plate with half of the plate shaded (dark). Worms were then illuminated (GYX, 0.78 mW/mm²) for 120 min and (b) the number of worms in the light and dark sections were scored and the phototaxis index was calculated. Data are mean ± SD. N = 3 independent experiments, each containing 25–80 animals, **p < 0.01 (unpaired, two-tailed t-test). c–g Light-induced Complex I ROS increases locomotion in C. elegans. (c) Schematic illustration of experimental procedure. C. elegans were acclimated to an unseeded plate and body bends were counted pre- and post-illumination. Wild-type (N2) and nuo-1::Supernova worms were individually transferred to unseeded plates and body bends were scored with and without light (MVX, 5.6 mW/mm²) for 15 s on (d) light titration of whole locomotion (MVX, 0, 0.9, 2.5, 5.6 mW/mm²); (e) forward movement; (f) Omega turn and (g) backward movement. Data are mean ± SD. N = 15 independent animals across 3 technical replicates, **p < 0.01 vs no light (Two-way ANOVA, Sidak’s multiple comparisons). h Reversibility and plasticity of light-induced complex I ROS. nuo-1::Supernova worms were transferred to unseeded plates and body bends were scored with light (MVX, 5.6 mW/mm²) for 15 s. For steady light, the light source was maintained and body bends were scored every 15 s for 60 s. For the reversal assay, the light source was removed and body bends were scored every 15 s for 60 s. For plasticity, the light was removed for 60 s, then reintroduced and body bends were scored for 15 s. Data are mean ± SD. N = 15 independent animals across 3 technical replicates, **p < 0.01 (Two-way ANOVA, Sidak’s multiple comparisons). i Aging abolished complex I ROS induced increase in locomotion. Staged Wild-type (N2) and nuo-1::Supernova worms L4, Day 1, Day 3 and Day 5 were individually transferred to unseeded plates and body bends scored with and without light (MVX, 5.6 mW/mm²) for 15 s. Data are mean ± SD. N = 15 independent animals across 3 technical replicates, **p < 0.01 vs wild-type (Three-way ANOVA, Tukey’s multiple comparisons).