A simple high throughput assay to evaluate water consumption in the fruit fly

Abstract

Water intake is essential for survival and thus under strong regulation. Here, we describe a simple high throughput system to monitor water intake over time in Drosophila. The design of the assay involves dehydrating fly food and then adding water back separately so flies either eat or drink. Water consumption is then evaluated by weighing the water vessel and comparing this back to an evaporation control. Our system is high throughput, does not require animals to be artificially dehydrated, and is simple both in design and implementation. Initial characterisation of homeostatic water consumption shows high reproducibility between biological replicates in a variety of experimental conditions. Water consumption was dependent on ambient temperature and humidity and was equal between sexes when corrected for mass. By combining this system with the Drosophila genetics tools, we could confirm a role for ppk28 and DopR1 in promoting water consumption, and through functional investigation of RNAseq data from dehydrated animals, we found DopR1 expression in the mushroom body was sufficient to drive consumption and enhance water taste sensitivity. Together, we provide a simple high throughput water consumption assay that can be used to dissect the cellular and molecular machinery regulating water homeostasis in Drosophila.

Figure 1

Water consumption assay. (a) Schematic diagram of assay. (b) Water consumption using non-dehydrated and dehydrated food. (c) To facilitate visualisation of consumed water, blue dye has been added to the water and can be seen in the abdomen of the fly. (d) The number of flies per chamber does not influence individual water consumption. Five, ten, fifteen, or twenty files were tested per assay for 3 days. (e) Temperature regulates water consumption in flies. The water consumption assay was tested in either 18 °C or 25 °C conditions for 3 days. (f) Desiccation stimulates water consumption in flies. The water consumption assay was tested in either low humidity (55%) or high humidity (70%) conditions for 3 days. All data represented mean ± S.E.M (n = 3–6). Student’s t-test, **p < 0.01; ***p < 0.001; n.s., not significant.

Figure 2

(a) Daily water consumption in male and female flies without body weight normalisation. (b) Daily water consumption in male and female flies with body weight normalisation. (c) Salt stimulates water consumption in flies. All data represented mean ± S.E.M (n = 3–6). Student’s t-test, **p < 0.01; ***p < 0.001; n.s., not significant.

Figure 3

The water taste receptor ppk28 regulates water consumption. (a) Water consumption of control (elav-Gal4 > w ¹¹¹⁸) and elav-Gal4 > ppk28 RNAi flies. (b) Water consumption and (c) food intake of w ¹¹¹⁸ control and ppk28 mutant flies (ppk28 ^Δ). All data represented mean ± S.E.M (n = 3–6). Student’s t-test, **p < 0.01; ***p < 0.001; n.s., not significant.

Figure 4

The dopamine receptor DopR1 regulates water consumption. (a) RNA Seq reads of DopR1 transcripts obtained from the heads of dehydrated or control flies. (b) DopR1 mRNA levels were analysed by RT-qPCR. (c) Water consumption of w ¹¹¹⁸ control and DopR1 mutant flies (dumb ²). (d) Food consumption of w ¹¹¹⁸ control and DopR1 mutant flies (dumb ²) highlights a specific defect in water consumption. (e) Proboscis extension response (PER) of w ¹¹¹⁸ control and DopR1 mutant flies to H₂O and 5% sucrose solution (f) L-dopa (3 mg/ml) pre-treatment increases the PER response to water in w ¹¹¹⁸ control but not DopR1 mutant flies. Sucrose responses are similar between treatments and genotypes indicating a water specific defect. For PER response, each independent trial consisted of ≥ 10 animals. (g) Water consumption of control (UAS-DopR1 RNAi/  +), elav-Gal4 > DopR1 RNAi and MB247-Gal4 > DopR1 RNAi flies. (h) Water consumption of control (w ¹¹¹⁸), DopR1 mutant (elav-Gal4/ + ; dumb ²/dumb ²) and rescue (elav-Gal4 > UAS-DopR1; dumb ²/dumb ² and MB247-Gal4 > UAS-DopR1; dumb ²/dumb ²). All data represented mean ± S.E.M (n = 3–6). Student’s t-test or one-way ANOVA followed by Tukey’s post hoc test, **p < 0.01, ***p < 0.001; n.s., not significant.

Figure 5

DopR1 is required for water taste. Extracellular bristle recordings of w ¹¹¹⁸ control, ppk28 mutant (ppk28 ^Δ), DopR1 mutant (dumb ² and elav-Gal4/ + ;;dumb ²/dumb ²) and rescue (elav-Gal4 > UAS-DopR1; dumb ²/dumb ² and MB247-Gal4 > UAS-DopR1; dumb ²/dumb ²) flies after stimulation with water or 40 mM sucrose. (a) Representative spikes showing response to water (1 mM KCl) or 40 mM sucrose. (b,c) Mean total spike number for the first second of response to (b) water or (c) sucrose is shown. 3 to 5 L-type labellar bristles were recorded per animal. All data represent mean ± S.E.M (n = 9–17 animals). One-way ANOVA followed by Tukey’s post hoc test, **p < 0.01, ***p < 0.001; n.s., not significant.