Supplementary methods
(a) Metabolite analyses
To determine ammonia in aphid tissues and amino acids in haemolymph, each preparation was brought to
20 μl with PBS, hand-homogenized with 20 μl 40 mM HCl, incubated on ice for 30 minutes, and
centrifuged at 18000 g for 15 min at 4oC. For experiments on the ammonia content of host cell
preparations, the 10 μl supernatant of each sample in the timecourse was combined with 10 μl 40 mM
HCl, without dilution. For all samples, the supernatant was filtered through a 0.45 μm filter plate
(Millipore) by centrifugation at 1500 g for 10 min, and 2.5-5 μl filtrate was derivatized with AccQ Tag
(Waters), following manufacturer’s protocol, and injected into Waters Acquity UPLC with PDA detector
and AccQ-Tag Ultra 2.1 x 100 mm column. The 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, 78.8% A 21.2% B; 7.74-8.04 min, 40.4% A 59.6% B; 8.048.64 min, 10% A 90% B; 8.05-8.64 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 between each time point), where A is 90% AccQ-Taq Ultra Eluent A in water,
and B is Accq-Taq Ultra Eluent B. Ammonia and amino acids were determined by comparison to
standards, 1, 25,50 and 100 pmol ammonia and protein-amino acids μl-1 (Waters amino acid hydrolysate
standard #088122, supplemented with asparagine, tryptophan and glutamine).
(b) RNAseq
The whole body (WB) samples comprised 20 aphids; and host cell samples (HC) were the
Buchnera-free portion of bacteriocytes, which was the supernatant obtained from dissection of
bacteriocytes dissected from 1,000 aphids, homogenization in 240 μl lysis buffer [35 mM Tris
pH 7.5, 200 mM sucrose, 6 μl RNaseOut (Invitrogen)] and centrifugation at 1479 g at 4˚C for 5
min. The RNA samples obtained with the RNeasy Mini kit (Qiagen), following manufacturers’
instructions, were treated with DNase (DNA-Free kit, Ambion) to remove genomic DNA, and the
purity was checked with an Agilent 2100 Bioanalyzer.
The RNA samples were subjected to polyA+ RNA purification, RNA fragmentation, cDNA
synthesis, barcode-adaptor ligation, PCR amplification, and size selection according to the
instruction of Illumina mRNA sequencing sample preparation kit (Illumina). The standard PE
adaptor (Illumina) was used for the HC sample and an in-house barcode-adaptor (GAACT)
prepared by Cornell University Life Sciences Core Laboratories Center was used for the WB
sample. The two RNAseq libraries were sequenced in a single lane for 86 bp by Illumina
Genome Analyzer II platform.
Illumina sequence reads were quality checked by the Illumina package programs, Firecrest,
Bustard and Gerald, binned by barcode, and then trimmed of the barcode and adaptor sequences.
Fastx-Toolkit (http://hannonlab.cshl.edu/fastx_toolkit/) was used to generate 36 bp reads, to
enable comparison with the published data set (Hansen and Moran 2011). The RPKM values of
genes (with R2 > 0.99 in all pairwise comparisons) did not vary with read lengths 36 bp, 50 bp
and 80 bp.
Bioinformatics programs, TopHat (Trapnell et al. 2009) and Cufflinks (Trapnell et al. 2010),
were applied for mapping sequence reads into reference sequences and for computing expression
scores in RPKM (reads per kilobase of transcript per million mapped reads) (Mortazavi et al.
2008). TopHat was used to map quality-filtered sequence reads to pea aphid OGS 1.0 mRNAs
(Official Gene Set, representing 34,821 non redundant mRNAs) (Legeai et al. 2010) plus 13
protein-coding mitochondrial genes. The RPKM value of each gene transcript was estimated by
Cufflinks based on the alignment resulting from TopHat and a GTF file providing coordinates of
gene transcript boundaries. To detect genes with differential expression between the HC and WB
samples, criteria of FDR ≤ 0.01 (estimated by Cuffdiff) and difference in RPKM that is both ≥
two-fold and ≥10 units were applied. To compare our data with a published transcriptome study
of pea aphid bacteriocytes (Hansen and Moran 2011), the latter dataset was re-processed using
our analysis pipeline to obtain RPKM values of genes and to detect genes with differential
expression.
(c) Construction of Buchnera metabolic model iSM199
The metabolic model iSM199 was constructed using new information to improve the previous Buchnera
model iSM197 (MacDonald et al. 2011). Two reaction-specific modifications were made: the gene,
ahpC, encoding alkyl hydroperoxide reductase which can reoxidise significant amounts of NADPH (Cha
et al. 1995), replaced a dummy reaction in iSM197, to equilibrate NADPH and NADP pools; and the
reaction D-alanine-D-alanine ligase mediated by DdlB is included, following evidence for ddlB
expression (Poliakov et al. 2011), such that D-Ala (and not D-Ala-D-Ala) is taken up by Buchnera. In
addition, the amino acid stoichiometries of the Buchnera biomass reaction were recalculated, using data
from the experimentally determined quantitative proteome of Buchnera (Poliakov et al. 2011), to produce
a biologically realistic simulation of the amino acid (AA) contribution to Buchnera protein. Specifically
the AA stoichiometries in the biomass reaction (File S1) were computed from the relative abundance
(NadjSPC) of each protein in the Buchnera proteome (Poliakov et al. 2011) and the AA composition of
each protein retrieved from BuchneraBase (www.buchnera.org). These sequences were analysed
by ProtParam (web.expasy.org/protparam/) to obtain the amino acid content of each protein. The
%-abundance of each amino acid in the proteome was calculated by summing the product of its
abundance in each protein and the abundance of that protein in the proteome. Amino acid
stoichiometries from the biomass reaction of the iAF1260 metabolic network of Escherichia coli
(Feist et al. 2007) were summed and percentages calculated for each of the twenty protein amino
acids. Percentage amino acid abundances calculated from the Buchnera proteome were divided
by percentage abundances calculated from the E. coli biomass reaction, and then multiplied by
the amino acid stoichiometries from the E. coli biomass reaction to obtain the recalculated
stoichiometries for the Buchnera biomass reaction.
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