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Fig. 1 CO2 Evokes a Slow Avoidance Behavior in Larval Zebrafish (A) Schematic illustration of the experimental setup. (B and C) Avoidance response is evoked by CO2, but not by other chemical stimuli. Vehicle control (black, n = 6), low CO2 (60–80 mg/L, pink, n = 24), middle CO2 (170–250 mg/L, orange, n = 24), high CO2 (700–780 mg/L, red, n = 20), HCl (blue, n = 10), skin extract (gray, n = 11), and taurocholic acid (yellow, n = 13). (B) Avoidance latencies after the stimulus onset. The cutoff time was set to 15 s (dotted line), and the avoidance latency of non-responded fish was scored as 15 s in statistical analysis. Wilcoxon signed-rank sum test for comparisons between vehicle and CO2 stimulations. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001. (C) Avoidance probabilities upon the application of various chemicals. Open, light-shaded, and dark-shaded bars represent percentages of fish that showed an avoidance response within 5, 10, and 15 s after the stimulus onset, respectively. (D) Top: representative time-lapse images of CO2-evoked avoidance behavior. Still images were taken from a movie at 10 frames/s. The asterisk represents the time point of avoidance onset (time 0). The first image was taken at the time of CO2 presentation (4,200 ms before the avoidance onset). Scale bar, 1 mm. Middle: time-extended images of an initial turn in the CO2-evoked avoidance behavior. Still images were taken from the same movie at 250 frames/s. Bottom: representative fast-start escape behavior in response to a touch stimulus onto the left head. Still images were taken from a movie at 250 frames/s. (E) Avoidance trajectories of larval zebrafish elicited by CO2 (left) and touch (right). Arrows indicate the stimulus positions. Individual fish trajectories are color coded (n = 6). (F) Quantification of avoidance kinematics in response to CO2 (red) and touch (green). Left: avoidance directionality (in degrees). Middle: change in heading angle (in degrees). Right: swim velocity (in millimeters per second). Boxplots show median, quartiles (box), and range (whiskers) (n = 6 each). Paired t test; ∗p < 0.05. See also Figure S1 and Movies S1, S2, S3, S4, and S5.