Humidity as a potential zeitgeber for circadian entrainment of insect systems

Humidity levels, like light and temperature, fluctuate daily yet are less predictable; however, whether humidity can entrain circadian clocks and synchronize animal behaviors with environmental variations remains unknown. Here, we investigate the circadian humidity entrainment in various insects across multiple orders. Insect species respond to humidity cycles with distinct patterns, some active during either wet/dry periods or at the arid-humid transition. When the humidity cue is removed, most species continue to show rhythmic activity associated with the previous arid-humid (AH) cycles. Fruit flies shift their activity accordingly when humidity cycles are altered and remain in the new rhythms under the following free-running conditions (FRC; constant humidity, HH). Moreover, Drosophila clock and hygrosensation mutants have lower rhythmic activity during AH and a significant reduction in rhythms after humidity entrainment (FRC with HH), indicating that core clock components and hygrosensors are essential for humidity-dependent circadian entrainment. Our findings provide strong evidence that humidity is likely to serve as a potential zeitgeber for circadian entrainment in most, but not all, insect systems and should have broad applicability and importance across animal systems. While light and temperature act as the primary zeitgebers, understanding the mechanisms of humidity entrainment will help us better interpret the behavioral patterns of terrestrial animals, particularly small insects susceptible to dehydration.