Prandiology of Drosophila and the CAFE assay

Abstract

Studies of feeding behavior in genetically tractable invertebrate model systems have been limited by the lack of proper methodology. We introduce the Capillary Feeder (CAFE), a method allowing precise, real-time measurement of ingestion by individual or grouped fruit flies on the scale of minutes to days. Using this technique, we conducted the first quantitative analysis of prandial behavior in Drosophila melanogaster. Our results allow the dissection of feeding into discrete bouts of ingestion, defining two separate parameters, meal volume and frequency, that can be uncoupled and thus are likely to be independently regulated. In addition, our long-term measurements show that flies can ingest as much as 1.7x their body mass over 24 h. Besides the study of appetite, the CAFE can be used to monitor oral drug delivery. As an illustration, we used the CAFE to test the effects of dietary supplementation with two compounds, paraquat and ethanol, on food ingestion and preference. Paraquat, a prooxidant widely used in stress tests, had a strong anorexigenic effect. In contrast, in a feeding preference assay, ethanol-laced food, but not ethanol by itself, acted as an attractant.

Fig. 1.

The CAFE assay. (A) Schematic diagram. Liquid food, topped with an oil layer to minimize evaporation, is introduced via a glass capillary held in place by a pipette tip. The pierced bottom of the inner chamber provides humidity. (B) Fly feeding from the capillary. To facilitate visualization, a red dye has been added to the medium and can be seen in the proboscis and abdomen of the fly.

Fig. 2.

Prandial behavior analyzed in the CAFE. (A) Intake by three individually housed male flies fed 5% sucrose + 5% yeast extract measured in 10-min intervals. A vertical rise flanked by two intervals of no intake was defined as a meal. (B) Intake by individual flies fed 5% sucrose. (C) Meal volume and frequency can be decoupled by modulating nutrient conditions. On 5% sucrose, average meal size increases, whereas meal frequency is unchanged; 5% sucrose + 5% yeast extract: n = 10 flies, 52 meals; 5% sucrose: n = 4 flies, 18 meals. All values are given as averages ± SE. ∗, P ≤ 0.01, two-tailed t test.

Fig. 3.

Measurement of long-term food consumption in the CAFE. (A) Cumulative ingestion by 17 individual male flies over 5 days. Average consumption = 1.5 ± 0.04 μl per day per fly. (B) The number of animals per chamber does not influence individual feeding rate. One, two, four, or eight flies were housed per CAFE. Average consumption was 2.0 ± 0.02, 2.1 ± 0.1, 2.3 ± 0.1, and 2.0 ± 0.1 μl per day per fly, respectively (R² > 0.98 for each linear fit; ANOVA P = 0.24). (C) The number of capillaries per chamber does not affect food intake. One, two, or three capillaries were used per CAFE. Three flies were housed per chamber. Average consumption was 1.5 ± 0.03, 1.3 ± 0.04, and 1.3 ± 0.1 μl per day per fly, respectively (R² > 0.98; ANOVA P = 0.25). (D) Capillary depth has no effect on food ingestion. Four flies were used per CAFE, with the capillary tip placed 4 mm, 6.5 mm, or 16.5 mm below the top of the chamber, respectively (Fig. 1A). Average consumption was 2.2 ± 0.2, 1.9 ± 0.1, and 1.8 ± 0.1 μl per day per fly, respectively (R² > 0.99; ANOVA P = 0.26). In all experiments, 5% sucrose + 5% autolyzed yeast extract was served. All values are given as averages ± SE.

Fig. 4.

Dietary paraquat inhibits food intake. (A) Ingestion of a 5% sucrose solution with or without 20 mM paraquat over a 12-h period (n = 5 flies per condition. (B) Paraquat inhibits meal size. Consumption was recorded every 10 min during the first 6 h of the long-term experiment shown in A. All values are given as averages ± SE. ∗, P < 0.01, two-tailed t test.

Fig. 5.

Serving ethanol in the CAFE. (A) A dietary ethanol supplement has a modest, inhibitory effect on long-term food intake. Flies were fed 5% sucrose + 5% autolyzed yeast extract medium alone or supplemented with 1%, 5%, or 15% (vol/vol) ethanol. Average consumption was 1.7 ± 0.07, 1.7 ± 0.03, 1.4 ± 0.1, and 1.1 ± 0.01 μl per day per fly, respectively (R² > 0.97 for each linear fit; ANOVA P = 0.018; n = 8 flies per condition). (B) In the absence of food medium, ingestion of either plain water or ethanol is remarkably low. For 24 h, flies were offered a choice between two capillaries, one containing pure water and the other containing one of three concentrations of ethanol: 1% (dotted line), 10% (dashed line), or 50% (solid line). Maximum ingestion was <0.07 μl per day per fly with 1% ethanol (n = 12 animals per condition). (C) Desiccation stimulates water consumption. Flies were deprived of food and water for 24 h in either a humidified or nonhumidified CAFE and then provided with plain water in regular humidified conditions. (D) Given a choice between food (5% sucrose + 5% autolyzed yeast extract) with and without a 15% ethanol supplement, flies showed a strong preference for the ethanol-laced regimen (n = 8 animals per condition). All values are given as averages ± SE. ∗, P < 0.05; ∗∗, P < 0.01, two-tailed t test.