Dye-Based Fly Feeding Assay

Agar-based food medium containing dye (dissolved in media prior to solidifying) was poured into plastic feeder caps (Fig. 1A) and allowed to cool at room temperature to solidify, placed in a humidified plastic box, and stored at 4 °C overnight. The following day, feeder caps containing media were warmed to room temperature for 1 h, inverted and placed in the open end of vials containing adult flies (Fig. 1B). The feeder caps used in these studies hold ~4.5 mL of medium (many-fold more than flies consume), have flanges that prevent them from falling into the vials and fit in the vials used so that condensation does not build-up, yet flies cannot escape. Adult flies (typically 15/vial, but see Fig. 6) in the vials consumed medium from the feeder caps (the only food source) and then excreted waste over time (Fig. 1C). A single feeder cap was used in each vial over the duration of each experiment. Feeder caps were discarded at the conclusion of feeding (Fig. 1D). The dye inside the flies (internal dye, INT) was collected via homogenization of animals in 1.5 ml of water followed by centrifugation to pellet debris. The dye excreted by flies on the walls of the vials (excreted vial dye, ExVial) was collected by addition of 3 ml of water to vials followed by vortexing (Fig. 1D). Definitions of abbreviations for all Con-Ex measures are provided in Table S1. Absorbance of the INT and ExVial dye in water extracts was determined in a spectrophotometer (Pharmacia Biotech Ultraspec 2000) (Fig. 1E) at wavelengths appropriate for each dye (Blue 1, 630 nm; xylene cyanol, 615 nm; Red 40, 504 nm; Green 5, 608 nm; Blue 2, 608 nm; Red 4, 500 nm; Red 6, 442 nm; Yellow 5, 425 nm). Absorbance values were converted to volumes of medium consumed by interpolation from standard curves of pure dyes (Fig. 1F). Extracts of flies fed medium without dye controlled for background absorbance. The standard amount of time flies were allowed to consume-excrete in the vials was 24 h, but the consumption-excretion time was varied in some studies as described. When used, starvation of flies was achieved by housing them in empty vials for 17 h. In studies that assessed mating status on Con-Ex, virgin flies were collected under brief CO₂ and then housed at 15 flies per bottle in the presence of 15 males (mated) or in the absence of males (virgin) for 2 d prior to the initiation of Con-Ex. Flies in all studies had a single water/food source (the feeder cap) and were housed undisturbed at 25 °C and 65% relative humidity under a 12-hour light/dark cycle while consuming media from feeder caps.