Supplementary Figure 1. Flies form water-reward memory only in the thirsty state

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1 2 3 4 5 6 7 Supplementary Figure 1. Flies form water-reward memory only in the thirsty state Thirsty but not sated wild-type flies form robust 3 min memory. For the thirsty group, the flies were water-deprived for 16 h before conditioning. For the sated group, the 16-h water-deprived flies were allowed to drink water for 30 min before conditioning. Each value represents mean ± SEM (N = 8 for each bar). *P < 0.05; t-test. 8 1

9 10 11 12 13 14 15 Supplementary Figure 2. Different training procedures produce similar memory performances Water paired with the second odor presentation during training (black bar; CS-/CS+) produced similar 3 min memory performance compared to that in the group in which water was paired with the first odor during training (gray bar: CS+/CS-). Each value represents mean ± SEM (N = 8 for each bar). NS, not significant (P > 0.05); t-test. 16 2

17 18 19 20 21 22 23 Supplementary Figure 3. Water deprivation for 16 h and 24 h before training produces similar memory performances Both 16- and 24-h water-deprivation states before conditioning produced similar learning, STM, and LTM performances in wild-type flies. Each value represents mean ± SEM (N = 8, 8, 6, 6, 8, 8 from left to right bars). NS, not significant (P > 0.05); t- test. 24 3

25 26 Supplementary Figure 4. GFP expression patterns driven by GAL4 lines used in 27 this study 28 (a) All MB neurons under R13F02-GAL4 expression. (b f) PAM neurons under DDC- 29 GAL4 (b), R48B04-GAL4 (c), VT19841-GAL4 (d), VT6554-GAL4 (e), and VT8167-30 GAL4 (f) expression. (g) MB α β neurons under VT57244-GAL4 expression. (h) MB 31 αβ neurons under C739-GAL4 expression. (i) MBs γ neurons under VT44966-GAL4 32 expression. (j) MB α β neurons under VT30604-GAL4 expression. (k,l) MBs γ 33 neurons under R16A06-GAL4 (k) and 5HT1B-GAL4 (l) expression. (m) MB αβ 34 neurons under VT49246-GAL4 expression. (n p) MB-M6 neurons under R27G01-35 GAL4 (n), E1255-GAL4 (o), and VT57242-GAL4 (p) expression. (q,r) MB-V3 36 neurons under G0239-GAL4 (q) and MB082C-GAL4 (r) expression. Brain neuropils 37 were counterstained with anti-dlg immunostaining (magenta). Scale bar represents 4

38 39 40 41 42 43 44 45 46 47 48 49 50 51 50 μm. Genotypes: (1) +/UAS-mCD8::GFP; R13F02-GAL4/UAS-mCD8::GFP, (2) DDC-GAL4/+; +/UAS-mCD8::GFP; +/UAS-mCD8::GFP, (3) +/UAS-mCD8::GFP; R48B04-GAL4/UAS-mCD8::GFP, (4) +/UAS-mCD8::GFP; VT19841-GAL4/UASmCD8::GFP, (5) +/UAS-mCD8::GFP; VT6554-GAL4/UAS-mCD8::GFP, (6) +/UASmCD8::GFP; VT8167-GAL4/UAS-mCD8::GFP, (7) +/UAS-mCD8::GFP; VT57244- GAL4/UAS-mCD8::GFP, (8) C739-GAL4/UAS-mCD8::GFP; +/UAS-mCD8::GFP, (9) +/UAS-mCD8::GFP; VT44966-GAL4/UAS-mCD8::GFP, (10) +/UASmCD8::GFP; VT30604-GAL4/UAS-mCD8::GFP, (11) +/UAS-mCD8::GFP; R16A06- GAL4/UAS-mCD8::GFP, (12) 5HT1B-GAL4/UAS-mCD8::GFP; +/UAS-mCD8::GFP, (13) +/UAS-mCD8::GFP; VT49246-GAL4/UAS-mCD8::GFP, (14) +/UASmCD8::GFP; R27G01-GAL4/UAS-mCD8::GFP, (15) E1255-GAL4/+; +/UASmCD8::GFP; +/UAS-mCD8::GFP, (16) +/UAS-mCD8::GFP; VT57242-GAL4/UASmCD8::GFP, (17) G0239-GAL4/UAS-mCD8::GFP; +/UAS-mCD8::GFP, and (18) R40B08-GAL4AD/UAS-mCD8::GFP; R23C06-GAL4BD/UAS-mCD8::GFP. 52 5

53 54 55 56 57 58 59 60 61 62 Supplementary Figure 5. The expression patterns of PAM-GAL4 lines used in this study Horizontal MB lobes innervation of DDC-GAL4 (a), R48B04-GAL4 (b), VT19841- GAL4 (c), and VT6554-GAL4 (d) fly brains were revealed using a UASmCD8::GFP;UAS-mCD8::GFP reporter. The MB neuropil structures were labeled with DLG antibody (magenta). Scale bars represent 20 μm. Genotypes: (1) DDC- GAL4/+; +/UAS-mCD8::GFP; +/UAS-mCD8::GFP/+, (2) +/UAS-mCD8::GFP; R48B04-GAL4/UAS-mCD8::GFP, (3) +/UAS-mCD8::GFP; VT19841-GAL4/UASmCD8::GFP, (4) +/UAS-mCD8::GFP; VT6554-GAL4/UAS-mCD8::GFP. 63 64 6

65 66 67 68 69 70 71 72 73 74 Supplementary Figure 6. Morphology and innervation patterns of individual dopaminergic PAM-β 1 neurons in relation to MB volume model (a) The somas of PAM-β 1 are TH-immunopositive. The brain was labeled with anti- TH antibody (magenta). Scale bars represent 20 μm. (b1 c2) Gross morphology and innervation patterns of a single PAM-β 1 neuron in the context of an MB volume model. Individual image of a single PAM-β 1 neuron was derived from FLP-out labeling of VT8167-GAL4. Arrowheads indicate the somas of PAM-β 1. The MB neuropil structures were labeled with DLG antibody (magenta). Genotype: hs-flp/+; +/+; VT8167-GAL4/UAS >rcd2,y+>mcd8::gfp. Scale bars represent 20 μm. 75 7

76 77 78 79 80 81 82 83 84 85 86 87 88 89 Supplementary Figure 7. Water-reward LTM requires reinforcing dopamine from PAM-β 1 neurons (a) The expression pattern of VT8167-GAL4-driven GFP in PAM-β 1 neurons in the adult fly brain. (b) VT8167-GAL4-driven GFP expression in the dopaminergic PAMβ 1 neurons was removed using an overlapping R58E02-GAL80 transgene. Scale bar, 50 μm. Genotypes: (1) +/UAS-mCD8::GFP; VT8167-GAL4/+ and (2) +/UASmCD8::GFP; VT8167-GAL4/R58E02-GAL80. (c) The LTM defect in the VT8167- GAL4 > UAS-shi ts flies were rescued by removing the expression in dopaminergic PAM-β 1 neurons using an overlapping R58E02-GAL80 transgene. Each value represents mean ± SEM (N = 8 for each bar). *P < 0.05; ANOVA followed by Tukey s test. Genotypes: (1) +/UAS-shi ts(jfrc100) ; +/+, (2) +/+; VT8167-GAL4/+, (3) +/UAS-shi ts(jfrc100) ; VT8167-GAL4/+, (4) +/UAS-shi ts(jfrc100) ; R58E02-GAL80/+, and (5) +/UAS-shi ts(jfrc100) ; VT8167-GAL4/R58E02-GAL80. 8

90 91 92 93 94 95 96 97 98 99 100 101 102 103 Supplementary Figure 8. Significant implanted memories are formed in watersated DDC-GAL4/UAS-TrpA1 and R48B04-GAL4/UAS-TrpA1 flies (a) Pairing the odor presentation with TrpA1-mediated activation of the DDC dopaminergic neurons during memory acquisition resulted in significant learning, STM, and LTM in water-sated flies. Genotypes: (1) DDC-GAL4/+; +/+; +/+, (2) +/+; +/UAS-TrpA1, and (3) DDC-GAL4/+; +/+; +/UAS-TrpA1. Each value represents mean ± SEM (N =12, 12, 12, 6, 6, 6, 8, 8, and 8 from left to right bars). *P < 0.05; ANOVA followed by followed by Tukey s test. (b) Pairing the odor presentation with TrpA1-mediated activation of the R48B04 dopaminergic neurons during memory acquisition resulted in significant learning and STM in water-sated flies. Genotypes: (1) +/+; R48B04-GAL4/+, (2) +/+; +/UAS-TrpA1, and (3) +/+; R48B04-GAL4/UAS-TrpA1. Each value represents mean ± SEM (N = 6, 6, 6, 8, 8, and 8 from left to right bars). *P < 0.05; ANOVA followed by Tukey s test. 104 9

105 106 107 108 109 110 111 112 113 114 115 Supplementary Figure 9. Normal odor acuity and water preference in thirsty dumb 2 flies (a b) Odor acuity and water preference controls for the DopR1 rescue experiment. All DopR1 mutant flies showed normal odor acuity to OCT and MCH and water preference compared with wild-type controls (+/+). Genotypes: (1) +/+; +/+, (2) +/+; VT57244-GAL4,dumb 2 /VT57244-GAL4,dumb 2, (3) C739-GAL4/C739-GAL4; dumb 2 /dumb 2, (4) +/+; VT44966-GAL4,dumb 2 /VT44966-GAL4,dumb 2, (5) +/+; dumb 2 /dumb 2. Each value represents mean ± SEM (N = 8, 8, 8, 8, 6, 8, 8, 10, 8, and 6 form left to right bars in (a), P > 0.05, ANOVA; N = 8, 8, 8, 7, and 8 form left to right bars in (b), P > 0.05, ANOVA). 116 117 118 10

119 120 121 122 123 124 125 126 127 128 Supplementary Figure 10. Expression patterns and behavioral controls of MB- GAL4 lines used in this study (a f) GFP expression patterns driven by GAL4 lines used in this study. (a, b) MB α β neurons under VT30604-GAL4 (a) and VT57244-GAL4 (b) expression. (c, d) MB αβ neurons under C739-GAL4 (c) and VT49246-GAL4 (d) expression. (e, f) MB γ neurons under R16A06-GAL4 (e) and 5HT1B-GAL4 (f) expression. The MB neuropil structures were labeled with anti-dlg antibody (magenta). Scale bars represent 20 μm. (g) The VT57244-GAL4 > UAS-shi ts, VT30604-GAL4 > UAS-shi ts, C739-GAL4 > UAS-shi ts, VT49246-GAL4 > UAS-shi ts, 5HT1B-GAL4 > UAS-shi ts, and R16A06-11

129 130 131 132 133 134 135 136 137 138 GAL4 > UAS-shi ts flies showed a normal water-preference response at 32 C restrictive temperature compared with their internal controls. Each value represents mean ± SEM (N = 30, 6, 6, 6, 6, 16, 16, 16, 16, 8, 8, 8, and 8 from left to right bars). P > 0.05; ANOVA. (h k) The R16A06-GAL4 > UAS-shi ts, 5HT1B-GAL4 > UAS-shi ts, C739-GAL4 > UAS-shi ts, and VT49246-GAL4 > UAS-shi ts flies showed normal odor acuity to OCT or MCH at 32 C restrictive temperature compared with their internal controls. Each value represents mean ± SEM (N = 8 for each bar in (h), P > 0.05, ANOVA; N = 8 for each bar in (i), P > 0.05, ANOVA; N = 10, 8, 8, 9, 8, and 8 from left to right bars in (j), P > 0.05; ANOVA; N = 10, 8, 8, 10, 8, and 8 from left to right bars in (k), P > 0.05, ANOVA). 12

139 140 141 142 143 144 145 146 147 148 Supplementary Figure 11. Expression patterns and behavioral controls of MB- M6 GAL4 lines used in this study (a) E1255-GAL4 and R27G01-GAL4 drive reporter gene expression in the same MB- M6 neurons. Arrowheads indicate the somas of MB-M6. The MB neuropil structures were labeled with anti-dlg antibody (magenta). Scale bars represent 20 μm. (b) E1255-GAL4 and VT57242-GAL4 drive reporter gene expression in the same MB-M6 neurons. The MB neuropil structures were labeled with anti-dlg antibody (magenta). Arrowheads indicate the somas of MB-M6. Scale bar represents 20 μm. (c) The expression pattern of VT57242-GAL4. The MB neuropil structures were labeled with 13

149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 anti-dlg antibody (magenta). Arrowheads indicate somas. Scale bar represents 20 μm. (d) Blocking the output from MB-M6 during retrieval in flies carrying VT57242- GAL4 > UAS-shi ts disrupted LTM. Each value represents mean ± SEM (N = 8 for each bar). *P < 0.05; ANOVA followed by Tukey s test. (e) Normal odor acuity to OCT or MCH and normal water preference at 32 C restrictive temperature in R27G01-GAL4 > UAS-shi ts flies. Each value represents mean ± SEM (N = 8 for each bar in odor avoidance, P > 0.05, ANOVA; N = 8 for each bar in water preference assays, P > 0.05, ANOVA). (f) Normal odor acuity to OCT or MCH and normal water preference at 32 C restrictive temperature in E1255-GAL4 > UAS-shi ts flies. Each value represents mean ± SEM (N = 7, 6, 8, 7, 8, and 7 from left to right bars in odor avoidance assay, P > 0.05, ANOVA; N = 8 for each bar in water preference assay, P > 0.05, ANOVA). (g) Blocking synaptic output from MB-M6 neurons during retrieval did not affect STM. Each value represents mean ± SEM (N = 10 for each bar). P > 0.05; ANOVA. (h) Constitutive silencing of VGlut in MB-M6 neurons disrupted LTM. Each value represents mean ± SEM (N = 8 for each bar). *P < 0.05; ANOVA followed by Tukey s test. (i) Genetic silencing of VGlut in MB-M6 neurons did not affect odor acuity to OCT or MCH, or water preference in thirsty flies. Each value represents mean ± SEM (N = 6 for each bar in odor avoidance, P > 0.05, ANOVA; N = 6 for each bar in water preference assays, P > 0.05, ANOVA). 168 169 14

170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 Supplementary Figure 12. Behavioral controls of MB-V3 GAL4 lines used in this study (a) Normal odor acuity to OCT or MCH and normal water preference at 32 C restrictive temperature in G0239-GAL4 > UAS-shi ts flies. Each value represents mean ± SEM (N = 8, 7, 7, 6, 6, and 7 form left to right bars in odor avoidance assay, P > 0.05, ANOVA; N = 6 for each bar in water preference assay, P > 0.05, ANOVA). (b) Blocking synaptic output from MB-V3 neurons during retrieval did not affect STM. Each value represents mean ± SEM (N = 8 for each bar). P > 0.05; ANOVA. (c) Constitutive silencing of ChAT in MB-V3 neurons affected LTM. Each value represents mean ± SEM (N = 8 for each bar). *P < 0.05; ANOVA followed by Tukey s test. (d) Genetic silencing of ChAT in MB-V3 neurons did not affect odor acuity to OCT or MCH, or water preference. Each value represents mean ± SEM (N = 6 for each bar in odor avoidance, P > 0.05, ANOVA; N = 6 for each bar in water preference assays, P > 0.05, ANOVA). 185 15

186 Supplementary Note 1 187 188 189 190 191 Genotypes Figure 1. Genotypes: (1) +/+, (2) ppk28 mutant, (3) P{EP} cerg6085, (4) PBac{WH}teq f01792, (5) rsh 1, (6) +/UAS-mCD8-GFP; R13F02-GAL4/UASmCD8::GFP, (7) +/+; R13F02-GAL4/+, (8) UAS-dCREB2-b/+; +/tub-gal80 ts, and (9) UAS-dCREB2-b/+; R13F03-GAL4/tub-GAL80 ts. 192 193 194 195 196 197 Figure 2. Genotypes: (1) DDC-GAL4/+; +/+; +/+, (2) +/+; +/UAS-shi ts, (3) DDC- GAL4/+; +/+; +/UAS-shi ts, (4) +/+; R48B04-GAL4/+, (5) +/+; R48B04-GAL4/UASshi ts, (6) +/+; VT19841-GAL4/+, (7) +/+; VT19841-GAL4/UAS-shi ts, (8) +/+; VT6554-GAL4/+, (9) +/+; VT6554-GAL4/UAS-shi ts, (10) +/UAS-mCD8::GFP; VT8167-GAL4/UAS-mCD8::GFP, (11) +/+; VT8167-GAL4/+, and (12) +/+; VT8167-GAL4/UAS-shi ts. 198 199 200 Figure 3. Genotypes: (1) +/+; +/UAS-shi ts, (2) +/+; VT8167-GAL4/+, (3) +/+; VT8167-GAL4/UAS-shi ts, (4) +/+; VT6554-GAL4/+, (5) +/+; VT6554-GAL4/UAS- shi ts. 201 202 203 204 205 206 Figure 4. Genotypes: (1) DDC-GAL4/+; +/+; +/+, (2) +/+; +/UAS-TrpA1, (3) DDC-GAL4/+; +/+; +/UAS-TrpA1, (4) +/+; R48B04-GAL4/+, (5) +/+; R48B04- GAL4/UAS-TrpA1, (6) +/+; VT8167-GAL4/+, (7) +/+; VT8167-GAL4/UAS-TrpA1, (8) +/+; +/+, (9) +/+; VT57244-GAL4,dumb 2 /VT57244-GAL4,dumb 2, (10) C739- GAL4/C739-GAL4; dumb 2 /dumb 2, (11) +/+; VT44966-GAL4,dumb 2 /VT44966- GAL4,dumb 2, (12) +/+; dumb 2 /dumb 2. 16

207 208 Figure 5. Genotypes: (1) DDC-GAL4/+; +/+; +/UAS-GCaMP6m and (2) +/+; VT8167-GAL4/UAS-GCaMP6m. 209 210 211 212 213 214 Figure 6. Genotypes: (1) +/+; VT30604-GAL4/+, (2) +/+; +/UAS-shi ts, (3) +/+; VT30604-GAL4/UAS-shi ts, (4) +/+; VT57244-GAL4/+, (5) +/+; VT57244- GAL4/UAS-shi ts, (6) +/+; R16A06-GAL4/+, (7) +/+; R16A06-GAL4/UAS-shi ts, (8) 5HT1B-GAL4/+; +/+, (9) 5HT1B-GAL4/+; +/UAS-shi ts, (10) +/+; VT49246-GAL4/+, (11) +/+; VT49246-GAL4/UAS-shi ts, (12) C739-GAL4/+; +/+, and (13) C739- GAL4/+; +/UAS-shi ts. 215 216 217 218 219 220 221 222 223 224 225 Figure 7. Genotypes: (1) +/UAS-mCD8::GFP; R27G01-GAL4/UAS-mCD8::GFP, (2) E1255-GAL4/+; +/ UAS-mCD8::GFP; +/UAS-mCD8::GFP, (3) +/+; R27G01- GAL4/+, (4) +/+; +/UAS-shi ts, (5) +/+; R27G01-GAL4/UAS-shi ts, (6) E1255- GAL4/+; +/+; +/+, (7) E1255-GAL4/+; +/+; +/UAS-shi ts, (8) +/UAS-VGlut RNAi ; +/tub-gal80 ts, (9) +/UAS-VGlut RNAi ; R27G01-GAL4/tub-GAL80 ts, (10) G0239- GAL4/UAS-mCD8::GFP; +/UAS-mCD8::GFP, (11) R40B08-GAL4AD/UASmCD8::GFP; R23C06-GAL4BD/UAS-mCD8::GFP, (12) G0239-GAL4/+; +/+, (13) G0239-GAL4/+; +/UAS-shi ts, (14) R40B08-GAL4AD /+; R23C06-GAL4BD/+, (15) R40B08-GAL4AD/+; R23C06-GAL4BD/UAS-shi ts, (16) G0239-GAL4/+; +/tub- GAL80 ts, (17) +/+; +/UAS-ChAT RNAi, and (18) G0239-GAL4/+; tub-gal80 ts /UAS- ChAT RNAi. 226 227 17

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