Background

Detection of water and regulation of water intake are essential requirements for terrestrial animals to maintain proper osmotic homeostasis (Bourque, 2008; Cameron et al., 2010). Acute loss of one-tenth of body water can result in death in humans, while chronic water imbalance causes multiple disorders and accelerates the aging process (Minassian et al., 1989; Dmitrieva and Burg, 2011 and 2015; Hbibi et al., 2012; Kim et al., 2013). Water-seeking behavior has been well studied in a number of animal groups, but the neural circuits and molecular mechanisms involved in water-seeking are still poorly understood. The experimental method described below could be used to study the genetic mechanisms and neural circuits that regulate hygrotactic behavior, drinking motivation, thirst perception and water sensation in Drosophila.

Preference for specific relative humidities in flies is well established (Perttunen and Erkkila, 1952). Previous studies on humidity sensation in adult flies have generally used a binary-choice chamber (T-maze) (Sayeed and Benzer, 1996; Liu et al., 2007; Montell, 2008). In this design, dry and moist air are circulated to produce and maintain steep humidity gradients (Russell and Pierce-Shimomura, 2014). Recently, Stensmyr and colleagues devised a behavioral assay in which flies were given a choice between different levels of relative humidity (RH) (Enjin et al., 2016). These authors took advantage of the hygroscopic features of super-saturated salt solutions (Winston and Bates, 1960) to create niches with different RH levels in a multi-well plate. Super-saturated solutions of LiCl, NaCl, and KH₂PO₄ generated stable humidities of 20%, 70%, and 85% RH, respectively. Using this method, they showed that the preference for a specific humidity range depends on specialized neurons (Enjin et al., 2016). Furthermore, using a similar experimental protocol in which flies were allowed to choose between 20%, 70%, and 85% RH, Garrity and colleagues identified that IR93a, an ionotropic receptor, is important for hygrosensation in Drosophila (Knecht et al., 2016).

Here, we present a novel assay that we developed to study hygrotactic behaviors in dehydrated flies that have been deprived of water for several hours. A continuous humidity gradient was first established in a chamber with an inaccessible water source. Then, the characteristic behavior of dehydrated flies to rapidly aggregate near a water source was observed. The simplicity of this assay makes it useful for screening a large number of fly strains to investigate hygrotactic behaviors, drinking motivation, thirst perception, and water sensation. This new assay has helped us to identify the synaptic output from the mushroom-body α/β surface and posterior neurons as the critical step for both hygrotactic behavior and water sensation (Ji and Zhu, 2015).