(A) Schematic of the experimental procedure. Embryos were kept under constant darkness (solid black bar) or a 14/10 h day/night cycle (striped bar) until the behavioral experiments. Each behavioral section had two components; free-swimming for 10 min under light (open circle; white) or dark (solid circle; black) conditions after a 50 min acclimation period under same illumination condition, and a set of startle responses (280 ms) where a sequence of three taps was executed with 6-minute interval; arrows designate timing of taps. (B, C) Both larvae housed under continuous darkness or a day/night cycle showed significantly more activity during free-swimming when tested under light conditions, compared to dark test conditions. (D, E) For larvae housed under continuous darkness we observed no differences in startle behavior, but larvae housed under a day/night cycle showed significantly lower activity during startle behavior when tested under light conditions, compared to dark test conditions. Free-swimming: n = 23–24 per group, startle response: n = 18–21 per group. Data is presented in scatterplots showing individual values and group mean or median. Significance: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.

(A) Schematic of the experimental procedure. Embryos were kept under constant darkness (solid black bar) until the behavioral experiments. Each behavioral section had two components; free-swimming for 10 min under light (open circles; white) or dark (solid circles; black) conditions after a 50 min acclimation period under same illumination condition, and a set of startle responses (280 ms) where a sequence of three taps was executed with 6-minute interval; arrows designate timing of taps. For the sequential illumination test, larvae were given a 20-min acclimation period prior to assessing behavior under the illumination condition opposite to initial one. (B, C) Regardless of the order of illumination, larvae showed significantly more activity during free-swimming when tested under light conditions, compared to dark test conditions. (D, E) In terms of startle response behavior, almost all parameters decreased in second condition (regardless of illumination) representing potential habituation effect. Free-swimming: n = 76–86 per group, startle response: n = 85–87 per group. Data is presented in scatterplots showing individual values and group mean or median. Analysis was performed with paired t-tests. Significance: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.

(A, B, C, D) Neomycin treated larvae (triangles) had less response to tapping stimulus during 280 ms under both light (open triangles; white) or dark (solid triangles; black) regardless of order. (E) Heatmap representation of other analyzed parameters indicated the differences between control and treated group were evident under light. n = 23–24 per group. Data is presented in scatterplots showing individual values and group mean or median. Significance: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.