A dynamic humidity arena to explore humidity-related behaviours in insects.

Humidity is a critical environmental factor influencing the behaviour of terrestrial organisms. Despite its significance, the neural mechanisms and behavioural algorithms governing humidity sensation remain poorly understood. Here, we introduce a dynamic humidity arena that measures the displacement and walking speed of insects responding to real-time changes in relative humidity (RH). This arena operates in a closed-loop mode, adjusting humidity based on the insect's position with 0.2% RH resolution, allowing the insect to choose its optimal humidity. It can also be set to maintain a specific RH, simulating an open-loop condition to observe insect behaviour at constant humidity levels. Using the dynamic humidity arena, we found that desiccated and starved Drosophila melanogaster search for a RH of around 65-70% at 23°C, whereas sated flies show no unique preference for any RH. If the desiccated and starved flies are rehydrated, their searching behaviour is abolished, suggesting that desiccation has a great impact on the measured response. In contrast, mutant flies with impaired humidity sensing, due to a non-functional ionotropic receptor (Ir)93a, show no preference for any RH level irrespective of being desiccated and starved or sated. These results demonstrate that the dynamic humidity arena is highly sensitive and precise in capturing the nuanced behaviours associated with hydration status and RH preference in D. melanogaster. The dynamic humidity arena is easily adaptable to insects of other sizes and offers a foundation for further research on the mechanisms of hygrosensation, opening new possibilities for understanding how organisms perceive and respond to humidity in their environment.