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Despite growing knowledge, much remains unknown regarding how signaling within neural networks translate into specific behaviors. To pursue this quest, we need better understanding of the behavioral output under different experimental conditions. Zebrafish is a key model to study the relationship between network and behavior and illumination is a factor known to influence behavioral output. By only assessing behavior under dark or light conditions, one might miss behavioral phenotypes exclusive to the neglected illumination setting. Here, we identified locomotor behavior, using different rearing regimes and experimental illumination settings, to showcase the need to assess behavior under both light and dark conditions. Characterization of free-swimming zebrafish larvae, housed under continuous darkness or a day/night cycle, did not reveal behavioral differences; larvae were most active during light conditions. However, larvae housed under a day/night cycle moved a shorter distance, had lower maximum velocity and maximum acceleration during the startle response under light conditions. Next, we explored if we could assess behavior under both dark and light conditions by presenting these conditions in sequence, using the same batch of larvae. Our experiments yielded similar results as observed for naïve larvae: higher activity during light conditions, regardless of order of illumination (i.e. dark-light or light-dark). Finally, we conducted these sequenced illumination conditions in an experimental setting by characterizing behavioral phenotypes in larvae following neuromast ablation. Depending on the illumination during testing, the behavioral phenotype following ablation was characterized differently. In addition, the results indicate that the order in which the light and dark conditions are presented has to be considered, as habituation may occur. Our study adds to existing literature on illumination-related differences in zebrafish behavior and emphasize the need to explore behavioral phenotypes under both light and dark condition to maximize our understanding of how experimental permutations affect behavior.

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