Video tracking and data analysis

During behavioral testing, two apparatuses (totally 8 square tanks) were recorded simultaneously. One infrared camera (Panasonic CCTV camera Model No. WV-BP334) was set above each apparatus. A 8-min recording was made through the camera positioned above the tanks. The videos were recorded by Noldus MPEG Recorder 2.1 as digital video files. At the end of the 8-min recording, larval zebrafish were gathered in a dish to be euthanized with ice.

The position of the zebrafish larvae was traced using Ethovision XT 5.0 to analyze the digital video files. The output parameters included duration in the light zone or the dark zone, and swim velocity. For the light/dark choice assay, percentage of time spent in light/dark side and choice index were calculated over the 8-min recording period. Up to 10–15 % of samples were excluded as software failed to trace larvae due to high degree freezing and/or movement close to the walls of the chambers. Statistical analyses and graph generation were performed using the Graphpad Prism 5.0. Choice indices are presented as scattered plots showing individual sample values and error bars representing mean and 95 % CI. ANOVA followed by the Dunnett’s post hoc test was used to compare data for each stressor treatment with the control (no stressor treatment). Student t-tests (two tailed, unpaired) were performed for each treatment group compared to the control. For effect size, cohen’s d was calculated using d = 2t/√(df)