Figure 7. Avoidance behavior in the five-fish bouncing ball assay.

7 dpf larvae, five per well, were imaged without visual stimuli (Control) or in the presence of various stimuli, including a bouncing ball (BB) and/or a stationary ball. A) Control: larvae without visual stimuli were distributed evenly in the upper and lower halves of the well. Blue BB: larvae exposed to a blue bouncing ball in the upper half of the well spent significantly more time in the bottom half of the well. Red BB: larvae exposed to a red bouncing ball in the upper half of the well, spent significantly more time in the bottom half of the well. Blue Ball: larvae did not display a significant response to a stationary blue ball. Red Ball: larvae did not display a significant response to a stationary red ball. Blue BB and Blue Ball: larvae accumulated down in the dish when exposed to a blue bouncing ball in the upper half of the dish and a blue stationary ball in the bottom half of the dish. Red BB and Red Ball: larvae accumulated down in the dish when exposed to a red bouncing ball in the upper half of the dish and a red stationary ball in the bottom half of the dish. The larvae were imaged for one hour using a 60 second interval between images. B) The experiment with the red bouncing ball in the upper half of the dish and the stationary red ball in the bottom half of the dish was analyzed over time in 1 minute intervals (n=12 wells). The dotted line indicates the expected 50% down in a random distribution. C) Larvae were first imaged for 30 minutes without visual stimuli and then for 30 minutes with visual stimuli. Similar to the results shown above, larvae exposed to a red bouncing ball (BB) and a red stationary ball showed a significant response. In contrast, simultaneous blinking balls (SIM) or alterating blinking balls (ALT) did not show a significant response. The larvae were imaged for one hour total, using a 30 second interval between images.