We investigated intestinal integrity using the Smurf assay (Regan et al., 2016; Rera et al., 2012). Experimental flies were grouped in vials (25 flies per vial and 12 replicates per each diet) 2–3 days following eclosion. Following exposure to a choice or fixed diet for 12 days (food was changed every 2–3 days and survivorship of w1118 males on choice diet was roughly 70%), flies were transferred to S30 (30% [w/v] sucrose) food with 2.5% (w/v) blue dye (FD and C Blue No. 1; Spectrum Chemical) for 24 hr. A fly was counted as a Smurf when blue was observed outside of the digestive tract (Regan et al., 2016; Rera et al., 2012). The proportion of Smurf flies in each replicate was calculated and used as a single observation for statistical analysis.

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