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Figure S1: Schematic of two-choice CAFE assay (not to scale). A single male fly was housed in inner vial, with water in outer vial, to keep a high humidity inside. Two kinds of liquid food were provided to the fly through two capillaries separately. One contains 5% sucrose and 5% yeast extract, which is represented by green color. The other contains 5% sucrose, 5% yeast extract and ethanol in a certain concentration, which is represented by red color. The two capillaries were replaced every 24 h with their locations were exchanged.

Figure S2: Ethanol is not an efficient energy source for Drosophila. w1118 mutants in a CS genetic background did not survive as long on agar as flies fed with 1% ethanol. Consistent with the data for wild-type CS, the ethanol fed flies did not survive for very long. Hence, ethanol can be used as a food substrate by these flies, but not efficiently. Each data point is mean ± SEM.

Figure S3: Ethanol preference in the CAFE assay does not rely on gustatory or olfactory attraction. (a) The PER index of CS flies was not different between liquid food without ethanol and liquid food with 5%, 10% or 15% ethanol, which suggested that ethanol was not an appetitive gustatory cue. (b) The orco2 mutant ethanol preference to 5%, 10% or 15% ethanol is not significantly different from the ethanol preferences of CS. (c) The ethanol preferences of lush1 to 5%, 10% or 15% ethanol are not significantly different from the preferences of CS. Panels (b) and (c) suggest that ethanol preference on Drosophila is not due to olfactory attraction of ethanol. Data are mean ± SEM.

Figure S4: The decreased ethanol preference in rut2080 is not due to decreased food consumption. (a) In the CAFE assay, rut2080 consumed significantly less food than CS at each ethanol concentration. (b) This defect in food consumption was not increased significantly by the post-developmental expression of a wild-type rut cDNA in the nervous system with the elav-GeneSwitch driver. However, the same treatment (RU486 feeding) induced a higher ethanol preference than the vehicle-feeding group (Fig. 2). (c and d) The defect in food consumption was not rescued by the rutabaga expression driven by the OK107 or c772 Gal4 driver. However, this defect of ethanol preference in rut2080 was rescued by OK107 or c772 driven rutabaga expression in mushroom body (Fig. 4). Panels (b), (c) and (d) indicated that the rut2080 ethanol preference phenotype is independent of the total food consumption phenotype. Data are mean ± SEM. N.S., no significance. &ast;P < 0.05 and &ast;&ast;P < 0.01. Because the negative control rut2080; +; 238y and rut2080; c305a/+; MB247/+ genotype displayed no difference with CS in food consumption (data not shown), these results cannot indicted whether the two phenotypes are independent each other in rut2080 or not.

Figure S5: The expression of rutabaga in the α′/β′ lobe neurons alone is not sufficient for a full rescue of the rut2080 ethanol preference phenotype. (a) The expression of UAS-rut driven by the c305a α′/β′ Gal4 drive was not sufficient to fully rescue the rut2080 ethanol preference phenotype. The rut2080; c305a/+; UAS-rut ethanol preference phenotype was not significantly different than CS, and was significantly higher than one control, rut2080; UAS-rut/+ genotype. However, because it was not significantly different than the rut2080; c305a/+ genotype control, it is still a question whether the rutabaga expression in α′/β′ lobe is required for ethanol preference or not. Data are mean ± SEM. N.S., no significance. (b) c305a Gal4 drives the GFP expression in the α′/β′ lobe mushroom body neurons. Scale bar: 100 µm.

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