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1 Supplementary Methods 2 3 (a) Chemically-defined diets 4 The complete diet was formulation A of ref [1] with 0.5 M sucrose and 0.15 M amino acids, 5 comprising equimolar concentrations of nonessential and essential amino acids. The essential 6 amino acids were: 14.3 mM arginine, 8.7 mM histidine, 8.7 mM isoleucine, 8.7 mM leucine, 8.7 7 mM lysine, 8.7 mM threonine, 8.7 mM valine, 2.9 mM methionine, 2.9 mM phenylalanine and 8 2.9 mM tryptophan. The nonessential amino acids were: 16.5 mM glutamine, 14.3 mM 9 asparagine, 14.3 mM aspartic acid, 8.4 mM glutamic acid, 5.7 mM alanine, 5.7 mM proline, 5.7 10 mM serine, 2.7 mM cysteine, 1.2 mM glycine and 0.6 mM tyrosine. Individual omission of 11 essential amino acids yielded diets with total amino acid concentrations of the diets were 135.7– 12 147.1 mM. Aphids were administered to the diets as described in [1]. 13 14 (b) Budget Analysis 15 Two budget analyses were conducted: to quantify the net production of EAAs by Buchnera in 16 aphids reared on diets from which the EAA of interest was omitted: and to determine the 17 contribution of Buchnera to the increase in protein-EAA content of symbiotic aphids on the 18 control diet containing all EAAs. 19 To determine EAA production by aphids on diets lacking that EAA, 25 replicate 2-day-old 20 symbiotic and aposymbiotic aphids for each test diet were individually weighed on a Mettler 21 UltraBalance to 1 g precision, transferred to individual diet cages and re-weighed at day-7. The 22 protein content per aphid was calculated from protein density (mg protein g-1 weight), quantified 23 in separate sets of 2-day-old and 7-day-old symbiotic and aposymbiotic aphids, using the 24 Coomassie Brilliant Blue Protein Microassay of BioRad (catalogue number 500-0201), with 25 bovine serum albumin as standard (40–480 mg ml-1). Protein growth of each aphid was 26 determined as the difference in protein content between day-2 and day-7. Published data on the 27 amino acid content of aphid protein [2] were used to quantify the contribution of the EAA of 28 interest to the protein growth. The protein growth of aposymbiotic aphids (without Buchnera) 29 was attributed to endogenous reserves (e.g. incorporation of the EAA from the FAA pool into 30 protein) (Supplementary Table 1a). The protein growth of symbiotic aphids (with Buchnera) 31 was attributed to both the endogenous reserves and Buchnera-derived EAAs, and the Buchnera 32 contribution was calculated as the difference between the contribution of the EAA to protein 33 growth in symbiotic and aposymbiotic aphids (Supplementary Table 1b). 34 The Buchnera contribution to protein-EAA growth of the aphids on the control diet was 35 inferred from the difference between inputs (ingested from the diet and endogenous reserves) 36 and outputs (protein growth and elimination via honeydew). Aphids with greater outputs than 37 inputs of an EAA were interpreted to derive supplementary EAA from the Buchnera. 38 The food ingested by the aphids between day-2 and day-7 was determined by the radiolabelled 39 inulin technique, in which the diet is supplemented with a known concentration of the non- 40 permeant polysaccharide inulin, and the volume ingested is calculated from the amount of inulin 41 recovered from the honeydew [3, 4]. Ten 2-day-old aphids were transferred individually to a 42 Perspex ring (3.5 cm diam., 0.5 cm height) with the complete diet containing 16 µCi [14C] inulin 43 ml-1 (Sigma). Honeydew produced was deposited onto a 3.5 cm circle of absorbent paper (Nuc- 44 wipes, National Diagnostic) placed under each ring. The paper circle was transferred to 5 ml 45 Ecoscint (National Diagnostic) and counted in a scintillation counter (Beckman LS6500), with a 46 preset 14C window and quench curve. Counts for negative controls (mean of three replicate 47 aphids and paper circles on non-radioactive diets) were subtracted from the experimental values. 48 Preliminary experiments confirmed that the radioactivity in the aphid carcass was consistently 49 <10% of the radioactivity recovered from the honeydew, confirming that the inulin is not 50 assimilated. For the outputs, protein growth of aphids on the complete diet was calculated from 51 the weight gain and protein density of the aphids at day-2 and day-7 (as described above for the 52 budget analysis of aphids on diets lacking individual EAAs); and the amount of each EAA 53 released in the honeydew was determined from the EAA concentration in honeydew droplets and 54 total volume of ingested diet. 55 This analysis makes various simplifying assumptions, as follows. (1) no difference between 56 symbiotic and aposymbiotic aphids in turnover rate of each protein molecule and utilization of 57 EAAs for processes other than protein synthesis (e.g. utilization as respiratory fuel, synthesis of 58 serotonin from tryptophan, dopamine and melanin from tyrosine). Deviation from this 59 assumption would tend to change (decrease or increase) the contribution of endogenous reserves 60 to observed protein growth of symbiotic aphids. (2) The endogenous reserves contributing to 61 protein-EAAs is equivalent between 2-day-old symbiotic and aposymbiotic aphids. The 62 dominant source of EAAs incorporated into protein is the free amino acid pool, and this does not 63 differ significantly between the two aphid treatments, validating this assumption. (3) Ingested 64 EAAs are assimilated into protein with 100% efficiency. This assumption tends to overestimates 65 EAA input from food because the assimilation efficiency of ingested EAAs is <100%; and 66 underestimate outputs because the conversion efficiency to protein is <100%. Consequently, this 67 analysis may underestimate the contribution of Buchnera to the EAA budget of aphids on the 68 complete diet. Comprehensive data are not available to quantify the magnitude to this bias. 69 Where studied, EAA assimilation efficiency by symbiotic aphids is >90% and alternative fates of 70 EAAs (e.g. as respiratory fuel, FAA pool) are quantitatively small [2, 5, 6], suggesting that this 71 bias is small. 72 73 (c) Quantitative Proteomics 74 The proteome was obtained for three independent biological replicates of bacteriocytes dissected 75 from 7-day-old aphids reared on the complete diet and diets that lacked one of cysteine, 76 isoleucine, leucine, lysine methionine, phenylalanine/tyrosine or valine. Each lane of the SDS- 77 PAGE gel of bacteriocyte proteins was cut into 10 slices, and the proteins were reduced, 78 alkylated and digested with trypsin and peptide extracted as in [7, 8]. The extracted peptides 79 were analyzed by nanoLC-LTQ-Orbitrap (Thermo Electron) mass spectrometry using data 80 dependent acquisition and dynamic exclusion. Peptide extracts were loaded on a guard column 81 (LC Packings; MGU-30-C18PM), followed by separation on a PepMap C18 reverse-phase nano 82 column (LC Packings nan75-15-03-C18PM), using 90-min gradients with 95% water, 5% 83 acetonitrile (ACN), 0.1% FA (solvent A), and 95% ACN, 5% water, 0.1% FA (solvent B) at a 84 flow rate of 200 nl/min. The acquisition cycle consisted of a survey MS scan in the Orbitrap with 85 a set mass range from 350 m/z to 1800 m/z at the highest resolving power (100,000), followed 86 by 5 data-dependent MS/MS scans acquired in the LTQ. Dynamic exclusion was used with the 87 following parameters: exclusion size 500, repeat count 2, repeat duration 30 s, exclusion time 88 180 s, exclusion window ± 6 ppm. Target values were set at 5x105 and 104 for the survey and 89 Tandem MS scans, respectively, and the maximum ion accumulation times were set at 200 ms in 90 both cases. Regular scans were used both for the precursor and tandem MS without averaging. 91 The precursor isolation window was set at 2 m/z with mono-isotopic peak selection, and the 92 FTMS preview option was used. In total 240 MS runs were carried out, with extensive blanks 93 between each sample to avoid carry-over of peptides that could bias quantification. 94 Peak lists (.mgf format) were generated using DTA supercharge (v1.19) software 95 (http://msquant.sourceforge.net/) and searched with Mascot v2.2 (Matrix Science) against a 96 combined database containing the aphid genome with 34834 protein-coding gene models 97 (http://www.aphidbase.com/aphidbase), the Buchnera genome with 587 protein coding genes 98 (including 14 predicted pseudogenes and plasmid genes) [9], 187 sequences for known 99 contaminants (e.g. keratin, trypsin) and concatenated with a decoy database where all the 100 sequences were reversed; in total, this database contained 71,216 sequences. For off-line 101 calibration, a preliminary search was conducted with the precursor tolerance window set at ±30 102 ppm. Peptides with the ion scores above 40 were chosen as benchmarks to determine the offset 103 for each LC-MS/MS run. This offset was then applied to adjust precursor masses in the peak lists 104 of the respective .mgf file for recalibration using a Perl script. The recalibrated peak lists were 105 searched against the assembled Buchnera/aphid database. Each of the peak lists were searched 106 using Mascot v2.2 (maximum p-value of 0.01) for full tryptic peptides using a precursor ion 107 tolerance window set at ±6 ppm, variable methionine oxidation and fixed cysteine carbamido- 108 methylation and maximally one missed cleavage allowed. The maximum fragment ion tolerance 109 (MS/MS) was 0.8 Da. Minimal ion score threshold was chosen such that a peptide false 110 discovery rate (FDR) below 1% was achieved. Using an in-house written filter, the search results 111 were further filtered as follows: For identification with two or more peptides, the minimum ion 112 score threshold was set to 30. For protein identification based on a single peptide, the minimum 113 ion score threshold was set to 33, and the mass accuracy of the precursor ion was required to be 114 within ±3 ppm. The peptide false discovery rate (FDR) was calculated as: 2 × (decoy hits) / 115 (target + decoy hits) and below 1%. The FDR of proteins identified with two or more peptides 116 was zero. Peptides with less than seven amino acids were discarded. Mass spectrometry data 117 matched to identified aphid and Buchnera proteins can be viewed in the Plant Proteome 118 DataBase (PPDB) at http://ppdb.tc.cornell.edu/ under experimental ids #1136-1143 (Repl 1), 119 #1150-1157 (Repl. 2), 1158-1165 (Repl 3). To reduce false positive identifications, proteins 120 identified by only a single spectrum across all experiments or identified by a single peptide 121 shorter than 10 amino acids and containing isoleucine and/or leucine were removed. 122 Several aphid genes have more than one gene model, and in such cases the protein form with 123 the highest number of matched spectra was selected; if two gene models had the same number of 124 matched spectra, the model with higher alphanumerical order was selected. For quantification, 125 each protein accession was scored for total spectral counts (SPC), unique SPC (uniquely 126 matching to an accession) and adjusted SPC [7]. The latter assigns shared peptides to accessions 127 in proportion to their relative abundance using unique spectral counts for each accession as a 128 basis. To calculate the relative abundance for each protein sample type (per gel lane), the total 129 adjSPC was divided by the predicted protein length, yielding the spectral abundance factor 130 (SAF). The SAF values were then normalized to the total SAF of proteins identified in the gel 131 lane, yielding normalized spectral abundance factors (NSAFs). The normalized adjSPC 132 (NadjSPC) for each protein was calculated through division of adjSPC by the sum of all adjSPC 133 values for the proteins from that gel lane. NadjSPC provides a relative protein abundance 134 measure by mass, whereas NSAF estimates relative protein concentration within a particular 135 sample. Proteins that shared more than ~80% of their matched adjusted peptides with other 136 proteins across the complete dataset were grouped into clusters by generating a similarity matrix 137 through calculation of the dice coefficient between each pair of identified proteins as described 138 in [7]. In all analyses the group was represented by a single member of the group with the 139 highest value of adjSPC across all experiments and highest alphabetical order. 140 141 (d) UPLC analysis of free amino acids in aphid honeydew 142 The free amino acid (FAA) content of the bacteriocytes and whole body samples of 7-day-old 143 aphids, honeydew produced by 6-7-day-old aphids, and to quantify methionine release from 144 Buchnera preparations was analyzed using the AccQ Tag derivatization kit (Waters) by UPLC 145 with PDA detector (Waters Acquity). The aphid samples were homogenized in ice-cold PBS. To 146 collect honeydew, 15 replicate groups of 10 aphids were fed on complete diet for 4 days (from 147 day-2 to day-6). Each diet cage was then suspended above a dish of water-saturated mineral oil 148 for 24 h (day 6 – day 7), and the honeydew droplets were then collected from the oil, flash- 149 frozen and stored at -80oC. 150 Each sample was combined with an equal volume of 40 mM HCl, incubated on ice for 30 151 minutes, and centrifuged at 18000 g for 10 minutes at 4˚C. The supernatant was filtered through 152 a 0.45 μm filter plate (Millipore) by centrifugation at 1500 g for 10 min, the filtrate (2.5 μl) was 153 derivatized with AccQ Tag (Waters), following manufacturer’s protocol, and injected into 154 Waters Acquity UPLC with PDA detector and AccQ-Tag Ultra 2.1 x 100 mm column. The 155 gradient was: 0-0.54 min, 99.9% A 0.1% B; 0.54-5.74 min, 90.9% A and 9.1% B; 5.74-7.74 min, 156 78.8% A 21.2% B; 7.74-8.04 min, 40.4% A 59.6% B; 8.04-8.64 min, 10% A 90% B; 8.05-8.64 157 min 10% A 90% B; 8.64-8.73 min 99.9% A 0.1% B; 8.73-9.50 min, 99.9% A 0.1% B (linear 158 between each time point), where A is 10% AccQ-Taq Ultra Eluent A in water, and B is Accq- 159 Taq Ultra Eluent B. Amino acids were determined by comparison to standards: 1, 5, 10, 50 and 160 100 pmol amino acids μl-1 (Waters amino acid hydrolysate standard #088122, supplemented with 161 asparagine, tryptophan, homocysteine, cystathionine and glutamine). 162 163 References 164 1 Prosser, W.A., and Douglas, A.E. 1992 A test of the hypotheses that nitrogen Is upgraded and 165 166 167 168 169 170 171 recycled in an aphid (Acyrthosiphon pisum) symbiosis. J. Insect Physiol. 38, 93-99. 2 Douglas, A.E., Minto, L.B., and Wilkinson, T.L. 2001 Quantifying nutrient production by the microbial symbionts in an aphid. J. Exp. Biol. 204, 349-358. 3 Wright, J.P., Fisher, D.B., and Mittler, T.E. 1985 Measurement of aphid feeding rates on artificial diets using H-3-inulin. Ent. Exp. Appl. 37, 9-11. 4 Karley, A.J., Douglas, A.E., and Parker, W.E. 2002 Amino acid composition and nutritional quality of potato leaf phloem sap for aphids. J. Exp. Biol. 205, 3009-3018. 172 5 Febvay, G., Rahbe, Y., Rynkiewicz, M., Guillaud, J., and Bonnot, G. 1999 Fate of dietary 173 sucrose and neosynthesis of amino acids in the pea aphid, Acyrthosiphon pisum, reared on 174 different diets. J. Exp. Biol. 202, 2639-2652. 175 6 Wilkinson, T.L., Minto, L.B., and Douglas, A.E. 2001 Amino acids as respiratory substrates in 176 aphids: an analysis of Aphis fabae reared on plants and diets. Physiol.Entomol. 26, 225-228. 177 7 Poliakov, A., Russell, C.W., Ponnala, L., Hoops, H.J., Sun, Q., Douglas, A.E., and van Wijk, 178 K.J. 2011 Large-scale label-free quantitative proteomics of the pea aphid-Buchnera symbiosis. 179 Mol. Cell Proteomics 10, M110 007039. 180 8 Zybailov, B., Friso, G., Kim, J., Rudella, A., Rodriguez, V.R., Asakura, Y., Sun, Q., and van 181 Wijk, K.J. 2009 Large scale comparative proteomics of a chloroplast Clp protease mutant 182 reveals folding stress, altered protein homeostasis, and feedback regulation of metabolism. 183 Mol. Cell Proteomics 8, 1789-1810. 184 9 Shigenobu, S., Watanabe, H., Hattori, M., Sakaki, Y., and Ishikawa, H. 2000 Genome 185 sequence of the endocellular bacterial symbiont of aphids Buchnera sp. APS. Nature 407, 81- 186 86.