Download Supplementary Methods (a) Chemically

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Supplementary Methods
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(a) Chemically-defined diets
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The complete diet was formulation A of ref [1] with 0.5 M sucrose and 0.15 M amino acids,
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comprising equimolar concentrations of nonessential and essential amino acids. The essential
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amino acids were: 14.3 mM arginine, 8.7 mM histidine, 8.7 mM isoleucine, 8.7 mM leucine, 8.7
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mM lysine, 8.7 mM threonine, 8.7 mM valine, 2.9 mM methionine, 2.9 mM phenylalanine and
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2.9 mM tryptophan. The nonessential amino acids were: 16.5 mM glutamine, 14.3 mM
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asparagine, 14.3 mM aspartic acid, 8.4 mM glutamic acid, 5.7 mM alanine, 5.7 mM proline, 5.7
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mM serine, 2.7 mM cysteine, 1.2 mM glycine and 0.6 mM tyrosine. Individual omission of
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essential amino acids yielded diets with total amino acid concentrations of the diets were 135.7–
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147.1 mM. Aphids were administered to the diets as described in [1].
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(b) Budget Analysis
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Two budget analyses were conducted: to quantify the net production of EAAs by Buchnera in
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aphids reared on diets from which the EAA of interest was omitted: and to determine the
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contribution of Buchnera to the increase in protein-EAA content of symbiotic aphids on the
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control diet containing all EAAs.
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To determine EAA production by aphids on diets lacking that EAA, 25 replicate 2-day-old
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symbiotic and aposymbiotic aphids for each test diet were individually weighed on a Mettler
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UltraBalance to 1 g precision, transferred to individual diet cages and re-weighed at day-7. The
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protein content per aphid was calculated from protein density (mg protein g-1 weight), quantified
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in separate sets of 2-day-old and 7-day-old symbiotic and aposymbiotic aphids, using the
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Coomassie Brilliant Blue Protein Microassay of BioRad (catalogue number 500-0201), with
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bovine serum albumin as standard (40–480 mg ml-1). Protein growth of each aphid was
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determined as the difference in protein content between day-2 and day-7. Published data on the
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amino acid content of aphid protein [2] were used to quantify the contribution of the EAA of
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interest to the protein growth. The protein growth of aposymbiotic aphids (without Buchnera)
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was attributed to endogenous reserves (e.g. incorporation of the EAA from the FAA pool into
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protein) (Supplementary Table 1a). The protein growth of symbiotic aphids (with Buchnera)
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was attributed to both the endogenous reserves and Buchnera-derived EAAs, and the Buchnera
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contribution was calculated as the difference between the contribution of the EAA to protein
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growth in symbiotic and aposymbiotic aphids (Supplementary Table 1b).
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The Buchnera contribution to protein-EAA growth of the aphids on the control diet was
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inferred from the difference between inputs (ingested from the diet and endogenous reserves)
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and outputs (protein growth and elimination via honeydew). Aphids with greater outputs than
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inputs of an EAA were interpreted to derive supplementary EAA from the Buchnera.
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The food ingested by the aphids between day-2 and day-7 was determined by the radiolabelled
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inulin technique, in which the diet is supplemented with a known concentration of the non-
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permeant polysaccharide inulin, and the volume ingested is calculated from the amount of inulin
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recovered from the honeydew [3, 4]. Ten 2-day-old aphids were transferred individually to a
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Perspex ring (3.5 cm diam., 0.5 cm height) with the complete diet containing 16 µCi [14C] inulin
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ml-1 (Sigma). Honeydew produced was deposited onto a 3.5 cm circle of absorbent paper (Nuc-
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wipes, National Diagnostic) placed under each ring. The paper circle was transferred to 5 ml
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Ecoscint (National Diagnostic) and counted in a scintillation counter (Beckman LS6500), with a
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preset 14C window and quench curve. Counts for negative controls (mean of three replicate
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aphids and paper circles on non-radioactive diets) were subtracted from the experimental values.
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Preliminary experiments confirmed that the radioactivity in the aphid carcass was consistently
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<10% of the radioactivity recovered from the honeydew, confirming that the inulin is not
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assimilated. For the outputs, protein growth of aphids on the complete diet was calculated from
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the weight gain and protein density of the aphids at day-2 and day-7 (as described above for the
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budget analysis of aphids on diets lacking individual EAAs); and the amount of each EAA
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released in the honeydew was determined from the EAA concentration in honeydew droplets and
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total volume of ingested diet.
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This analysis makes various simplifying assumptions, as follows. (1) no difference between
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symbiotic and aposymbiotic aphids in turnover rate of each protein molecule and utilization of
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EAAs for processes other than protein synthesis (e.g. utilization as respiratory fuel, synthesis of
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serotonin from tryptophan, dopamine and melanin from tyrosine). Deviation from this
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assumption would tend to change (decrease or increase) the contribution of endogenous reserves
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to observed protein growth of symbiotic aphids. (2) The endogenous reserves contributing to
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protein-EAAs is equivalent between 2-day-old symbiotic and aposymbiotic aphids. The
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dominant source of EAAs incorporated into protein is the free amino acid pool, and this does not
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differ significantly between the two aphid treatments, validating this assumption. (3) Ingested
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EAAs are assimilated into protein with 100% efficiency. This assumption tends to overestimates
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EAA input from food because the assimilation efficiency of ingested EAAs is <100%; and
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underestimate outputs because the conversion efficiency to protein is <100%. Consequently, this
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analysis may underestimate the contribution of Buchnera to the EAA budget of aphids on the
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complete diet. Comprehensive data are not available to quantify the magnitude to this bias.
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Where studied, EAA assimilation efficiency by symbiotic aphids is >90% and alternative fates of
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EAAs (e.g. as respiratory fuel, FAA pool) are quantitatively small [2, 5, 6], suggesting that this
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bias is small.
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(c) Quantitative Proteomics
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The proteome was obtained for three independent biological replicates of bacteriocytes dissected
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from 7-day-old aphids reared on the complete diet and diets that lacked one of cysteine,
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isoleucine, leucine, lysine methionine, phenylalanine/tyrosine or valine. Each lane of the SDS-
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PAGE gel of bacteriocyte proteins was cut into 10 slices, and the proteins were reduced,
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alkylated and digested with trypsin and peptide extracted as in [7, 8]. The extracted peptides
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were analyzed by nanoLC-LTQ-Orbitrap (Thermo Electron) mass spectrometry using data
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dependent acquisition and dynamic exclusion. Peptide extracts were loaded on a guard column
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(LC Packings; MGU-30-C18PM), followed by separation on a PepMap C18 reverse-phase nano
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column (LC Packings nan75-15-03-C18PM), using 90-min gradients with 95% water, 5%
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acetonitrile (ACN), 0.1% FA (solvent A), and 95% ACN, 5% water, 0.1% FA (solvent B) at a
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flow rate of 200 nl/min. The acquisition cycle consisted of a survey MS scan in the Orbitrap with
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a set mass range from 350 m/z to 1800 m/z at the highest resolving power (100,000), followed
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by 5 data-dependent MS/MS scans acquired in the LTQ. Dynamic exclusion was used with the
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following parameters: exclusion size 500, repeat count 2, repeat duration 30 s, exclusion time
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180 s, exclusion window ± 6 ppm. Target values were set at 5x105 and 104 for the survey and
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Tandem MS scans, respectively, and the maximum ion accumulation times were set at 200 ms in
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both cases. Regular scans were used both for the precursor and tandem MS without averaging.
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The precursor isolation window was set at 2 m/z with mono-isotopic peak selection, and the
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FTMS preview option was used. In total 240 MS runs were carried out, with extensive blanks
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between each sample to avoid carry-over of peptides that could bias quantification.
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Peak lists (.mgf format) were generated using DTA supercharge (v1.19) software
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(http://msquant.sourceforge.net/) and searched with Mascot v2.2 (Matrix Science) against a
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combined database containing the aphid genome with 34834 protein-coding gene models
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(http://www.aphidbase.com/aphidbase), the Buchnera genome with 587 protein coding genes
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(including 14 predicted pseudogenes and plasmid genes) [9], 187 sequences for known
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contaminants (e.g. keratin, trypsin) and concatenated with a decoy database where all the
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sequences were reversed; in total, this database contained 71,216 sequences. For off-line
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calibration, a preliminary search was conducted with the precursor tolerance window set at ±30
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ppm. Peptides with the ion scores above 40 were chosen as benchmarks to determine the offset
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for each LC-MS/MS run. This offset was then applied to adjust precursor masses in the peak lists
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of the respective .mgf file for recalibration using a Perl script. The recalibrated peak lists were
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searched against the assembled Buchnera/aphid database. Each of the peak lists were searched
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using Mascot v2.2 (maximum p-value of 0.01) for full tryptic peptides using a precursor ion
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tolerance window set at ±6 ppm, variable methionine oxidation and fixed cysteine carbamido-
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methylation and maximally one missed cleavage allowed. The maximum fragment ion tolerance
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(MS/MS) was 0.8 Da. Minimal ion score threshold was chosen such that a peptide false
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discovery rate (FDR) below 1% was achieved. Using an in-house written filter, the search results
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were further filtered as follows: For identification with two or more peptides, the minimum ion
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score threshold was set to 30. For protein identification based on a single peptide, the minimum
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ion score threshold was set to 33, and the mass accuracy of the precursor ion was required to be
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within ±3 ppm. The peptide false discovery rate (FDR) was calculated as: 2 × (decoy hits) /
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(target + decoy hits) and below 1%. The FDR of proteins identified with two or more peptides
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was zero. Peptides with less than seven amino acids were discarded. Mass spectrometry data
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matched to identified aphid and Buchnera proteins can be viewed in the Plant Proteome
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DataBase (PPDB) at http://ppdb.tc.cornell.edu/ under experimental ids #1136-1143 (Repl 1),
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#1150-1157 (Repl. 2), 1158-1165 (Repl 3). To reduce false positive identifications, proteins
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identified by only a single spectrum across all experiments or identified by a single peptide
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shorter than 10 amino acids and containing isoleucine and/or leucine were removed.
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Several aphid genes have more than one gene model, and in such cases the protein form with
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the highest number of matched spectra was selected; if two gene models had the same number of
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matched spectra, the model with higher alphanumerical order was selected. For quantification,
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each protein accession was scored for total spectral counts (SPC), unique SPC (uniquely
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matching to an accession) and adjusted SPC [7]. The latter assigns shared peptides to accessions
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in proportion to their relative abundance using unique spectral counts for each accession as a
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basis. To calculate the relative abundance for each protein sample type (per gel lane), the total
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adjSPC was divided by the predicted protein length, yielding the spectral abundance factor
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(SAF). The SAF values were then normalized to the total SAF of proteins identified in the gel
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lane, yielding normalized spectral abundance factors (NSAFs). The normalized adjSPC
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(NadjSPC) for each protein was calculated through division of adjSPC by the sum of all adjSPC
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values for the proteins from that gel lane. NadjSPC provides a relative protein abundance
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measure by mass, whereas NSAF estimates relative protein concentration within a particular
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sample. Proteins that shared more than ~80% of their matched adjusted peptides with other
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proteins across the complete dataset were grouped into clusters by generating a similarity matrix
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through calculation of the dice coefficient between each pair of identified proteins as described
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in [7]. In all analyses the group was represented by a single member of the group with the
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highest value of adjSPC across all experiments and highest alphabetical order.
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(d) UPLC analysis of free amino acids in aphid honeydew
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The free amino acid (FAA) content of the bacteriocytes and whole body samples of 7-day-old
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aphids, honeydew produced by 6-7-day-old aphids, and to quantify methionine release from
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Buchnera preparations was analyzed using the AccQ Tag derivatization kit (Waters) by UPLC
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with PDA detector (Waters Acquity). The aphid samples were homogenized in ice-cold PBS. To
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collect honeydew, 15 replicate groups of 10 aphids were fed on complete diet for 4 days (from
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day-2 to day-6). Each diet cage was then suspended above a dish of water-saturated mineral oil
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for 24 h (day 6 – day 7), and the honeydew droplets were then collected from the oil, flash-
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frozen and stored at -80oC.
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Each sample was combined with an equal volume of 40 mM HCl, incubated on ice for 30
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minutes, and centrifuged at 18000 g for 10 minutes at 4˚C. The supernatant was filtered through
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a 0.45 μm filter plate (Millipore) by centrifugation at 1500 g for 10 min, the filtrate (2.5 μl) was
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derivatized with AccQ Tag (Waters), following manufacturer’s protocol, and injected into
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Waters Acquity UPLC with PDA detector and AccQ-Tag Ultra 2.1 x 100 mm column. The
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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,
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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
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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
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between each time point), where A is 10% AccQ-Taq Ultra Eluent A in water, and B is Accq-
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Taq Ultra Eluent B. Amino acids were determined by comparison to standards: 1, 5, 10, 50 and
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100 pmol amino acids μl-1 (Waters amino acid hydrolysate standard #088122, supplemented with
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asparagine, tryptophan, homocysteine, cystathionine and glutamine).
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