To discriminate the effects of antibiotics vs microbiome on the expression profile in the axenic state, we raised axenic flies for 5 d on antibiotic containing food, then aseptically transferred the axenic flies to sterile food without antibiotic in the laminar hood and grew these flies an additional five days without antibiotic, flipping once to new food. In parallel, 5-day-old axenic flies were transferred to food vials that had previously been contaminated with normal Drosophila flora by being used to transiently culture conventionally raised flies. These flies were also grown for five days, flipping once to new, contaminated vials. The absence of bacteria in flies raised on non-antibiotic food, and its presence in flies restored to microbe-contaminated food, was verified by PCR (Figure S9). Finally, 10-day-old flies were frozen using liquid nitrogen, and qPCR was performed using RNA from head samples and probes listed in Table S15.