Daily locomotor rhythms and ecdysial stage-dependent behaviors in the tardigrade Hypsibius exemplaris

Tardigrades, key ecdysozoan organisms, have poorly characterized daily behavioral rhythms linked to their molting (ecdysial) cycle. We continuously tracked the locomotor activity of the model tardigrade Hypsibius exemplaris using infrared videography and deep learning-based automated tracking under three photic regimes: a light/dark cycle, constant darkness following entrainment, and constant darkness without prior entrainment. We identified a robust endogenous circadian-like rhythm in locomotor activity that persisted in constant darkness, independent of photic input. Although the light/dark cycle enhanced the rhythm amplitude and the overall animal activity, it was not required for rhythmicity. Individual phases varied widely, indicating a lack of population-level synchronization. Furthermore, we developed a behavioral classification system based on activity level, which enables categorizing distinct behaviors. Behavioral patterns were modulated by distinct ecdysial stages, with quiescence gating premolt and time-of-day dependent patterns in feeding and wandering. These findings provide evidence consistent with an endogenous, circadian-like timing system in tardigrades that integrates daily activity with molting cycles and establish H. exemplaris as a promising model for studying ecdysozoan chronobiology.