Larval photomotor response assay

To determine if zinc deficiency and arsenic exposure alters the activity of embryos following light and dark stimuli the photomotor response assay was performed. Embryos at 120 hpf were tested in the 96-well exposure plates by placing them into the Viewpoint Zebrabox HD (software version 3.2, Viewpoint Life Sciences, Lyon, France) and measuring locomotor activity using the tracking setting during 3 minute periods of alternating light and dark for a total of 24 minutes. The integration time was set to 6 seconds to increase statistical power. The total movement (swim distance) in response to the multiple light-dark transition was tracked by an HD camera at 30 frames / s [41]. Any dead or malformed animals at 120 hpf were excluded from the data analysis and 232–329 embryos were analyzed. Raw data files were processed using custom R scripts to average the total distance traveled for each integration time point, and then the area under the curve was computed [41]. The overall area under the curve was compared to the control (zinc adequate, no arsenic) using a Kolmogorov-Smirnov test (p<0.01).