We used the protocol described previously (Shell et al., 2018). Experimental flies were co-housed in vials (10 flies per vial, 8–10 replicates for each group) for 2–3 days following eclosion and then sorted into individual sex cohorts. After exposure to the choice or fixed diet for 12 days (food was changed every 2–3 days), flies were transferred to the choice or fixed diet with 1% (w/v) FD and C Blue No. 1 in either (1) only the S10 (10% [w/v] sucrose) food in the choice diet; (2) only the Y10 (10% [w/v] yeast) food in the choice diet; or (3) food in both wells in the fixed diet SY10 (10% [w/v] sucrose and 10% [w/v] yeast). Vials were discarded if one or more dead flies were observed after the 24 hr feeding period. Excreted dye (ExVial) was collected by addition of 3 ml of Milli-Q water of vials followed by vortexing. Concentration of the ExVial dye in water extracts was determined by absorbance at 630 nm, which was used to infer macronutrient consumption.
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