Immobilization-free chemotaxis analysis reveals the novel behavioral mode of leaving in Caenorhabditis elegans

Animals balance staying in a favorable environment with exploring new ones. In C. elegans chemotaxis, the process by which worms migrate toward an attractant has been extensively studied. However, what happens after they reach it remains largely unexplored, partly because conventional assays immobilize worms at the point of arrival. Here, we quantitatively analyzed chemotactic behavior upon reaching an attractive odor source using an immobilization-free chemotaxis assay. We observed that 62% animals left the isoamyl alcohol region after initially approaching it, a behavior we termed "leaving behavior." Quantitative analysis revealed that leaving behavior represents a distinct locomotor state compared with free-moving, high-concentration odor avoidance, and approach behavior. To test whether leaving behavior is related to olfactory adaptation, we analyzed mutants in adaptation-related genes. The proportion of leaving behavior was significantly increased in egl-4 loss-of-function mutants compared with wild-type animals, whereas arr-1 mutants showed no significant difference. These results suggest that egl-4 negatively regulates leaving behavior, suggesting a role for this kinase in stabilizing post-arrival behavioral states beyond its known function in olfactory adaptation. Our findings indicate that chemotaxis involves dynamic behavioral transitions even after reaching an attractant, consistent with an exploration-exploitation trade-off framework.