Beyond bold versus shy: Zebrafish exploratory behavior falls into several behavioral clusters and is influenced by strain and sex

Consistent individual differences in exploratory behavior have been found across a range of taxa, including zebrafish, and are thought to contribute to evolutionary fitness. Animals that explore more of a novel environment, and visit areas of high predation risk, are considered bold, whereas animals with the opposite pattern of behavior are considered shy. Here, we examined whether this bimodal characterization of bold versus shy adequately captures the breadth of exploratory behavior exhibited by zebrafish or if, instead, behavior falls into multiple distinct behavioral subtypes. To identify the presence of behavioral subtypes, we applied unsupervised machine learning to behaviors extracted from three-dimensional swim traces from over 400 adult zebrafish across four strains (AB, TL, TU, and WIK) and both sexes. We found that behavior stratified into four distinct clusters. These included previously described bold and shy behavior as well as two new behavioral types: wall-huggers and active explorers. Consistent with prior work that found individual differences to be stable across time and influenced by biological factors like genetics and sex, the behavioral subtypes we identified were stable for up to 10 weeks and were influenced by the strain and sex of the animals. Taken together, our work suggests that individual differences in zebrafish exploratory behavior goes beyond bold versus shy and exhibits greater complexity than is typically assumed.