Allura red ac dye

We performed the Capillary Feeding (CAFE) assay to quantify food intake after starvation. The experimental set-up was described in [19 (link)]. Assays were carried at 16–18 o’clock using 4-d old adult flies pre-starved for 2 hours in tubes with 1% agar in a 25°C incubator. Male and female adult flies were measured separately. During the assay, the flies were fed on 5% sucrose containing 0.6 g/l Allura Red AC dye (Sigma-Aldrich), for 2 hours at 25°C without any disturbance. 10 tubes containing 4 flies each were measured in one day. These experiments were repeated on four subsequent days, each time using fresh 4-d old flies (n = 5). Tubes without flies were used as blank controls.

Two-choice feeding preference assay was performed as described44 (link). In brief, flies were kept on standard medium for 3–5 days after eclosion before they were offered the choice between sucrose medium (100 mM) containing a red dye and a yeast medium (5%, rich in protein) with a blue dye for 3 hours. Flies were scored according to the color of their abdomen. The “yeast preference index” was calculated and each experiment was repeated 3 times and each included 13–23 flies.
Quantification of sucrose intake was determined using the Capillary Feeding (CAFE) assay as described59 (link). Males were kept on standard medium for 3–5 days after eclosion and then transferred to vials with 1% agar (to allow access to water) and a glass capillary containing 5% sucrose and 0.6 g/l Allura Red AC dye (Sigma-Aldrich) for 2 hours at 25 °C and 60% humidity. Blank controls without flies were included in each experiment to determine evaporation.