D. melanogaster wild-type Canton Special (CS) and mutant flies were raised on standard medium at 18°C and 60% humidity in a 12-hour light/dark cycle. UAS-SPRRNAi1 and UAS-SPRRNAi2 were obtained from the Vienna Drosophila Resource Center (VDRC; SPR-RNAi1, ID v330048 and SPR-RNAi2, ID v106804). UAS-MIPRNAi1 and UAS-MIPRNAi2 were obtained from the Bloomington Drosophila Stock Center (BDSC; MIP-RNAi1, ID 41680 and MIP-RNAi2, ID 26246). UAS-DncRNAi was generated as previously described (36). Gal4 drivers for expressing the genes of interest in the SPN include GH298-Gal4, VT026326-Gal4 (VDRC, ID 201794), and VT057280-Gal4 (VDRC, ID 200916) (22). For expression in SPSN, VT003280-Gal4 (VDRC, ID 200327) was used (14). MIP-Gal4 from the Korea Drosophila Stock Centre was provided by Y.-J. Kim. SP mutant males were obtained by crossing SP0/TM3,Sb flies to Δ130/TM3,Sb; these flies are referred to as SP0 throughout the text and figures (9). Only females were used for the behavioral and imaging experiments. For the memory assay, virgin female and mated control groups were generated by collecting virgins in groups of 30 to 40 from unanesthetized freshly hatched fly cultures. Seven to 10 CS wild-type or SP0 mutant males were added to half of the groups, and experiments were then performed with 2- to 3-day-old female flies. If indicated, RNAi expression was specifically induced in adults using the TARGET system (37). To achieve RNAi induction, flies were kept at 30°C for 2 to 3 days before conditioning; in addition, flies for LTM were maintained at 30°C until the memory assay. For the imaging experiments, virgin females were collected in groups of 10 from unanesthetized freshly hatched fly cultures. Five CS wild-type or SP0 mutant males were added to half of the groups, and experiments were then performed with 2-day-old female flies.

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