Flies were grown and maintained on standard cornmeal/molasses/yeast/agar media at 25°C and 70% humidity on a 12:12 L:D cycle with lights-on at 9:00 AM. Behavioral characterization was performed on wild-type Canton-S (CS) flies recently isogenized for the 2nd and 3rd chromosomes. All GAL4 and UAS lines42 were backcrossed for at least 5 generations to a w-Berlin strain, recently isogenized for the 2nd and 3rd chromosome. No significant differences were found between CS and Berlin backgrounds in conditioned preference (Wilcoxon two-sample: 30 min memory Z(1,16)=-0.05, p=0.96, 24 hr memory Z(1,16)=-0.17, p=0.86). Lines 4-59 and 5-66a were obtained from our P[GAL4GawB] screen collection (see Supplementary Fig. 5b,c for expression). UAS-shi was obtained from S. Sweeney. sca1 and scaBP2 were obtained from Bloomington Stock Center and UAS-sca from N. Baker. The screen was carried out using 160 P[GAL4] homozygous viable strains in a w- Berlin genetic background (carrying the GawB element)42 with previously characterized expression in the mushroom body. 5-120 was backcrossed for 5 generations onto a newly isogenized w- Berlin background and re-tested.
