Supplemental Information. Sex Peptide Receptor and Neuronal. TOR/S6K Signaling Modulate. Nutrient Balancing in Drosophila

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1 Current Biology, Volume 20 Supplemental Information Sex Peptide Receptor and Neuronal TOR/S6K Signaling Modulate Nutrient Balancing in Drosophila Carlos Ribeiro and Barry J. Dickson Table S1. Data for Assays Shown in Figure 1D Sex of flies Days on sucrose n (assays) Median yeast preference index 25th percentile 75th percentile Statistical significance Females NS NS p < p < p < p < Males NS NS NS p < p < p < Significance for the comparison to 0 days on sucrose was determined using the Kruskal-Wallis test followed by Dunn s multiple comparison test. When all datapoints for one condition were the same, Wilcoxon Signed Rank test was used. NS, p > 0.05.

2 Table S2. Data for Assays Shown in Figure 2 Sex of flies Days on sucrose and yeast extract n (assays) Median yeast preference index 25th percentile 75th percentile Statistical significance Females days yeast NS deprived p < p < Males days yeast p < 0.01 deprived p < p < Significance for the comparison to 0 days on sucrose and yeast extract was determined using the Kruskal-Wallis test followed by Dunn s multiple comparison test. When all datapoints for one condition were the same, Wilcoxon Signed Rank test was used. NS, p > 0.05.

3 Table S3. Genotype and Origin of Lines Used Line name Detailed genotype Source References elav-gal4 w[1118], P{w[+mC]=UAS-Dcr-2} / P{hs-hid}Y; +; Dickson laboratory [1-3] P{w[+mC]=GAL4-elav.L}3 nsyb-gal4 w[1118], P{w[+mC]=UAS-Dcr-2} / P{hs-hid}Y; +; P{w[+mC]=GAL4-nSyb} / TM3, Sb J. Simpson and Dickson laboratory w - w[1118] (VDRC RNAi library background) Vienna Drosophila RNAi Center [1] Y hs-hid (CS) + / P{hs-hid}Y (in Canton-S background) Dickson laboratory SPR - w[+], Df(1)Exel6234, P{w[+mC]=XP- Dickson laboratory [3] U}Exel6234/FM7c; +; + SPR - ; UAS- w[+], Df(1)Exel6234, P{w[+mC]=XP- Dickson laboratory [4] SPR U}Exel6234/FM7c; P{w[+mC]=UAS-SPR} / CyO; + SPR - ; ppk- w[+], Df(1)Exel6234, P{w[+mC]=XP- Dickson laboratory [4, 5] GAL4 U}Exel6234/FM7c; P{w[+mC]= GAL4-ppk} / CyO; + SP 0 +; +; SP[0] / TM3, Sb E. Kubli [6] Δ 130 +; +; Δ[130] / TM3, Sb E. Kubli [6] SP + +; +; SP[+] / TM3, Sb E. Kubli [6] ovod 1 ovo[d1], v[24] / C(1)DX, y[1], w[1], f[1]; +; + Bloomington stock center, stock number 1309 UAS-TSC1, y[1], w[1118], P{ry[+t7.2]=hsFLP}; +; T.P. Neufeld [7, 8] UAS-TSC2 P{w[+mC]=UAS-TSC1}A10, P{w[+mC]=UAS- TSC2}g1 UAS- y[1], w[1118], P{ry[+t7.2]=hsFLP}; +; T.P. Neufeld [8, 9] Rheb EP Rheb[EP loxP] / TM6B UAS-S6K WT y[1], w[1118], P{ry[+t7.2]=hsFLP}; P{w[+mC]=UAS- T.P. Neufeld [8, 10] S6K[WT]}4; + UAS- y[1], w[1118], P{ry[+t7.2]=hsFLP}; P{w[+mC]=UAS- T.P. Neufeld [8, 11] S6K T398E S6K[TE]}4; + UAS- p60 y[1], w[1118], P{ry[+t7.2]=hsFLP}; P{w[+mC]=UAS- T.P. Neufeld [8, 12] p60}ii; + UAS- y[1], w[1118], P{ry[+t7.2]=hsFLP}; P{w[+mC]=UASp110[wt]} T.P. Neufeld [8, 12] PI3K/p110 (Unknown chromosomal location) UAS- y[1], w[1118], P{ry[+t7.2]=hsFLP}; P{w[+mC]=UAS- T.P. Neufeld [8, 13] TOR TEDIIA TOR[TED]}IIA; + UAS- TOR TEDIIIA y[1], w[1118], P{ry[+t7.2]=hsFLP}; +; P{w[+mC]=UAS-TOR[TED]}IIIA T.P. Neufeld [8, 13]

4 Table S4. Genotypes of Flies Used Name of flies Detailed genotype Figures standard background for behavioural experiments w[1118], P{w[+mC]=UAS-Dcr-2} / w[1118]; +; P{w[+mC]=GAL4- elav.l}3/ + 1, 2, 3A, 3E, S1A, S1B SPR - w[+], Df(1)Exel6234, P{w[+mC]=XP-U}Exel6234; +; + 3B and 3C SPR-; ppk-gal4 w[+], Df(1)Exel6234, P{w[+mC]=XP-U}Exel6234; P{w[+mC]= GAL4-3D ppk} / +; + SPR-; UAS-SPR w[+], Df(1)Exel6234, P{w[+mC]=XP-U}Exel6234; P{w[+mC]=UAS- 3D SPR} / +; + SPR-; ppk-gal4 / w[+], Df(1)Exel6234, P{w[+mC]=XP-U}Exel6234; P{w[+mC]= GAL4-3D UAS-SPR ppk} / P{w[+mC]=UAS-SPR}; + SP 0 +; +; SP[0] / Δ[130] 3E and S3B SP + +; +; SP[+] / Δ[130] 3E and S3B Canton-S / ovod 1 ovo[d1], v[24] / +; +; + 3F nsyb-gal4 / + TSC1, UAS-TSC2 Rheb S6K WT S6K T398E PI3K/p110 p60 TOR TEDIIA nsyb-gal4/ UAS- TOR TEDIIIA w[1118], P{w[+mC]=UAS-Dcr-2} / w[1118]; +; P{w[+mC]=GAL4- nsyb} / + +; P{w[+mC]=UAS-TSC1}A10, P{w[+mC]=UAS-TSC2}g1 / P{w[+mC]=GAL4-nSyb} y[1], w[1118], P{ry[+t7.2]=hsFLP}I / w[1118], P{w[+mC]=UAS-Dcr-2}; +; Rheb[EP loxP] / P{w[+mC]=GAL4-nSyb} P{w[+mC]=UAS-S6K[WT]}4 / +; P{w[+mC]=GAL4-nSyb} / + P{w[+mC]=UAS-S6K[TE]}4 / +; P{w[+mC]=GAL4-nSyb} / + P{w[+mC]=UAS-p110[wt]} (Unknown chromosomal location) / + ; P{w[+mC]=GAL4-nSyb} / + P{w[+mC]=UAS- p60}ii I +; P{w[+mC]=GAL4-nSyb} / + P{w[+mC]=UAS-TOR[TED]}IIA / +; P{w[+mC]=GAL4-nSyb} / + +; P{w[+mC]=GAL4-nSyb} / P{w[+mC]=UAS-TOR[TED]}IIIA 4A, 4B, 4D and S4 4A 4B 4C 4C 4D 4D S4 S4

5 Table S5. Genotype and Origin of Wild-Type Lines Used Line name Source Collection details Canton-S Bloomington stock center, stock number 1 Canton, Ohio, USA VAG1 UC San Diego Drosophila species stock center, Athens, Greece (1965) stock number BER1 UC San Diego Drosophila species stock center, Bermuda (1954) stock number Wild 5B Bloomington stock center, stock number 3886 Red Top Mountain, Georgia, USA (1966) Ica Ken-ting Oahu UC San Diego Drosophila species stock center, stock number UC San Diego Drosophila species stock center, stock number UC San Diego Drosophila species stock center, stock number Ica, Peru (1956) Ken-ting, Taiwan (1968) Oahu, Hawaii, USA (1955)

6 Figure S1.

7 Figure S1. The Feeding Switch Is Yeast Dependent and Is Observed across Multiple Wild-Type Strains (A and B) Protocol and yeast preference indices for 3-days yeast-deprived females (A) and 10-days yeast deprived males (B) tested with blue medium with and without yeast. Arrows indicate the timepoint of the assay. (C and D) Food choice assays of females from various wild-type lines after 0 and 3 days of yeast deprivation (C) and males from various wild-type lines after 0, 3 and 10 days of yeast deprivation. Significance was tested using the Mann-Whitney test in (A, B and C) and using the Kruskal-Wallis test followed by Dunn s multiple comparison test in (D). NS, **p < 0.01 and ***p <

8 Figure S2. Recovery from Yeast Deprivation in Various Wild-Type Strains Food choice of 3-day yeast-deprived females (A) and 10-day yeast-deprived males (B) following recovery from yeast deprivation by feeding for 3 days on sucrose (suc) and yeast extract (ye). **p < 0.01 and ***p < 0.001, Mann-Whitney test. When all datapoints for one condition were the same, Wilcoxon Signed Rank test was used.

9 Figure S3. Mating Status and Feeding Decisions in Canton-S Females (A) Food choices of mated and virgin Canton-S females. ***p < 0.001, Mann-Whitney test. When all datapoints for one condition were the same, Wilcoxon Signed Rank test was used. (B) Female Canton-S feeding decisions prior to mating, and upon mating with SP 0 and control (SP + ) males. **p < 0.01 and ***p < 0.001, Kruskal-Wallis test followed by Dunn s multiple comparison test.

10 Figure S4. Dominant-Negative TOR Affects Feeding Decisions Food choices of male and females overexpressing a dominant-negative form of TOR in the nervous system. UAS-TOR TEDIIa and UAS-TOR TEDIIIa are independent transgenic insertions. **p < 0.01, ***p < 0.001, Mann-Whitney test.

11 Supplemental References 1. Dietzl, G., Chen, D., Schnorrer, F., Su, K.C., Barinova, Y., Fellner, M., Gasser, B., Kinsey, K., Oppel, S., Scheiblauer, S., et al. (2007). A genome-wide transgenic RNAi library for conditional gene inactivation in Drosophila. Nature 448, Luo, L., Liao, Y.J., Jan, L.Y., and Jan, Y.N. (1994). Distinct morphogenetic functions of similar small GTPases: Drosophila Drac1 is involved in axonal outgrowth and myoblast fusion. Genes & development 8, Yapici, N., Kim, Y.J., Ribeiro, C., and Dickson, B.J. (2008). A receptor that mediates the postmating switch in Drosophila reproductive behaviour. Nature 451, Hasemeyer, M., Yapici, N., Heberlein, U., and Dickson, B.J. (2009). Sensory neurons in the Drosophila genital tract regulate female reproductive behavior. Neuron 61, Grueber, W.B., Ye, B., Moore, A.W., Jan, L.Y., and Jan, Y.N. (2003). Dendrites of distinct classes of Drosophila sensory neurons show different capacities for homotypic repulsion. Curr Biol 13, Liu, H., and Kubli, E. (2003). Sex-peptide is the molecular basis of the sperm effect in Drosophila melanogaster. Proceedings of the National Academy of Sciences of the United States of America 100, Tapon, N., Ito, N., Dickson, B.J., Treisman, J.E., and Hariharan, I.K. (2001). The Drosophila tuberous sclerosis complex gene homologs restrict cell growth and cell proliferation. Cell 105, Scott, R.C., Schuldiner, O., and Neufeld, T.P. (2004). Role and regulation of starvation-induced autophagy in the Drosophila fat body. Developmental cell 7, Bassuk, A.G., Wallace, R.H., Buhr, A., Buller, A.R., Afawi, Z., Shimojo, M., Miyata, S., Chen, S., Gonzalez-Alegre, P., Griesbach, H.L., et al. (2008). A homozygous mutation in human PRICKLE1 causes an autosomal-recessive progressive myoclonus epilepsy-ataxia syndrome. American journal of human genetics 83, Stocker, H., Radimerski, T., Schindelholz, B., Wittwer, F., Belawat, P., Daram, P., Breuer, S., Thomas, G., and Hafen, E. (2003). Rheb is an essential regulator of S6K in controlling cell growth in Drosophila. Nature cell biology 5, Barcelo, H., and Stewart, M.J. (2002). Altering Drosophila S6 kinase activity is consistent with a role for S6 kinase in growth. Genesis 34, Weinkove, D., Neufeld, T.P., Twardzik, T., Waterfield, M.D., and Leevers, S.J. (1999). Regulation of imaginal disc cell size, cell number and organ size by Drosophila class I(A) phosphoinositide 3- kinase and its adaptor. Curr Biol 9, Hennig, K.M., and Neufeld, T.P. (2002). Inhibition of cellular growth and proliferation by dtor overexpression in Drosophila. Genesis 34,