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

Individual differences in exploratory behavior have been found across a range of taxa 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 behavioral pattern are shy. Here, we determined whether this bimodal characterization of bold versus shy adequately captures the breadth of behavioral variation in zebrafish or if there are more than these two subtypes. To identify behavioral categories, we applied unsupervised machine to 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: previously described bold and shy behavior and two new behavioral types we call wall-huggers and active explorers. Clusters were stable across time and influenced by strain and sex where we found that TLs were shy, female TU fish were bold, male TU fish were active explorers, and male ABs were wall-huggers. Our work suggests that zebrafish exploratory behavior has greater complexity than previously recognized and lays the groundwork for the use of zebrafish in understanding the biological basis of individual differences in behavior.