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Fig. 2 Effects of reactive oxygen species on behavioral rhythms and locomotion patterns in zebrafish larvae The effects of H₂O₂ and DPI treatments on the locomotor activity of 4 dpf zebrafish larvae were evaluated under light‒dark (LD) and dark‒dark (DD) conditions. (A) Locomotor activity of 4 dpf zebrafish larvae (n = 16) treated with H₂O₂ and DPI continuously monitored for 3 days under LD conditions. Line plots show the average distance moved every 10 min per group, with shaded areas representing confidence intervals. (B) Total distance moved over 3 days across different treatment groups. (C) Comparison of average distances moved under LD and DD conditions. (D) Proportion of time spent in three locomotion modes (inaduration, duration, and larduration) under the LD and DD conditions. (E) Locomotor activity of zebrafish larvae (n = 16) treated with H₂O₂ and DPI and monitored for 3 days under DD conditions. Line plots show the average distance moved every 10 min per group, with shaded areas representing confidence intervals. (F) Polynomial fitting analysis of average locomotor distances under DD conditions. (G) Average locomotor distances over 3 days in different treatment groups. (H) Scatter plots of the mean phase analysis across groups. (I) Scatter plots of the mean period analysis showing locomotor cycles in different groups. (J) Phase ranking of locomotor activity across groups over 3 days. (K) Proportions of total activity time spent in three locomotion modes (inaduration, duration, and larduration) over 3 days. Statistical significance was determined via t tests, with significance levels indicated as follows: ns, p > 0.05; ∗∗, p < 0.01. (LD: light‒dark; DD: dark‒dark).