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Supplementary Information
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Supplementary Methods
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(a) Illumina-based transcriptome sequencing and library construction
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Sequencing was conducted using RNA extractions from dissected whole midgut
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regions (M1-M4) of five symbiotic and aposymbiotic bugs, respectively, fed on their
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natural diet of cottonseeds, resulting in two pooled samples. Prior to sequencing, the
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extracted RNA was exposed to a poly-A enrichment strategy. RNA sequencing was
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performed by a commercial service provider (Fasteris; http://www.fasteris.com) using
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5 μg total RNA (per sample) on the HiSeqTM 2000 Sequencing System from Illumina
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(http://www.illumina.com/), utilizing the paired read 100 bp technology. The reads
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were trimmed according to the sequencing quality test using Timmomatic [1]. The
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leading and trailing bases of each read were cut off if the quality values were below
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the default threshold. Additionally, reads were discarded if they were shorter than 30
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base pairs.
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Following quality checks, the trimmed reads were assembled de novo into contigs
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using Trinity [2]. The minimal contig length was set to 200 and the k-mer length to 25
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base pairs. The read libraries of symbiotic and aposymbiotic bugs were pooled and
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assembled into a single backbone. After the assembly with Trinity, the contigs were
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clustered by CD-HIT EST according to their sequence similarity to remove potential
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duplicates. Sequences with more than 99% sequence similarity to other contigs were
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subsequently collapsed. For the assignment of expression values to each
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constructed transcript in the respective library, the original reads were mapped back
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to the respective backbone assembly using the algorithm Bowtie2 [3]. The generated
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output was processed using SAMtools [4] to create BAM files and asses the
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coverage depth as the number of reads mapped to each transcript. The files were
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parsed with the custom script nucdepth.R (supplementary materials) using the R
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package Rsamtools [5] for further analysis. The correction for biases due to the
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different depths of sequencing across treatments and due to different transcript sizes
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were addressed using the RPKM (reads per kilobase of transcript per million of
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mapped reads) transformation to obtain estimates of relative expression levels.
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Homology searches (BLASTx and BLASTn) of unique sequences and functional
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annotation
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(http://www.blast2go.de).
were
determined
using
the
BLAST2GO
software
suite
v2.4.1
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(b) Validation of host gene expression with quantitative PCR
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Quantitative PCRs (qPCRs) for the candidate host genes involved in B vitamin
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metabolism were conducted across (i) aposymbiotic and symbiotic bugs reared on
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their natural diet of cotton seeds (to confirm the transcriptome sequencing results),
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and (ii) across the four experimental treatments of aposymbiotic and symbiotic bugs
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reared on a complete and vitamin-deficient artificial diet to examine if similar
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expression patterns are observed under controlled conditions with vitamin availability
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as the only nutritional difference between dietary treatments. Primers were designed
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based on the candidate gene sequences available from the transcriptome, and
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checked for specificity in vitro using capillary sequencing of amplified PCR products
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(Table S2).
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The qPCR reactions were performed using a RotorGene®-Q cycler (Qiagen, Hilden,
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Germany), with the same individual cDNA extracts used for the diagnostic PCR
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screens. The final reaction volume of 25 µl included the following components: 1 µl of
2
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cDNA template, 2.5 µl of each primer (10 µM), 6.5 µl of autoclaved distilled H 2O, and
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12.5 µl of SYBR Green Mix (Qiagen, Hilden, Germany).
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Conditions for qPCR were optimized using a VWR® Gradient Thermocycler (VWR,
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Radnor, PA, USA) at various annealing temperatures (60-68 °C). Standard curves for
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absolute quantification in the qPCR (10-fold dilution series from 1 ng/µl to 10-6 ng/µl)
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were generated using purified PCR products for all primer pairs after measuring the
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PCR product concentrations using a NanoDropTM1000 spectrophotometer (Peqlab).
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The following cycling parameters were used: 95°C for 10 min., followed by 45 cycles
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of 68ºC for 30 s, 72ºC for 20 s, and 95ºC for 15 s. Subsequently, a melting curve
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analysis was conducted by increasing the temperature from 60ºC to 95ºC within 20
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min. Six replicates of one of the standard concentrations were used, for each primer
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pair and concentration, for the configuration and calibration of the standard curve.
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The resulting averages were then utilized to correct for possible errors in the DNA
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concentration measurements. Based on the standard curve, absolute copy numbers
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were calculated. The linear correlation coefficient (R2) of all the measured genes
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ranged from 0.981 to 1.000. According to the slopes of the standard curves,
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amplification efficiencies of the standards utilized in our study ranged from
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93%~102%, (based on the formula E = 10 1/-slope -1).
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Gene copy numbers estimated from qPCRs were first normalized against the 60S
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ribosomal protein L13a (Table S3), then compared using the Mann-Whitney U-test
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and Kruskal-Wallis tests with Dunn posthoc tests, respectively, to asses levels of
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significance (P < 0.05) across treatments.
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Supplementary References
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1. Lohse M, Bolger AM, Nagel A, Fernie AR, Lunn JE, Stitt M, Usadel B. 2012
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RobiNA: a user-friendly, integrated software solution for RNA-Seq-based
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transcriptomics. Nucleic Acids Res. 40, W622-627. (doi:10.1093/nar/gks540).
76
2. Grabherr MG, Haas BJ, Yassour M, Levin JZ, Thompson DA, Amit I, Adiconis
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X, Fan L, Raychowdhury R, Zeng Q, et al. 2011 Full-length transcriptome
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assembly from RNA-Seq data without a reference genome. Nat. Biotechnol.
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29(7), 644-652. (doi:10.1038/nbt.1883).
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3. Langmead B, Salzberg SL. 2012 Fast gapped-read alignment with Bowtie 2.
Nat. Methods 9(4), 357-359. (doi:10.1038/nmeth.1923).
4. Huang Y, Niu B, Gao Y, Fu L, Li W. 2010 CD-HIT Suite: a web server for
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clustering and comparing biological sequences. Bioinformatics 26(5), 680-682.
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(doi:10.1093/bioinformatics/btq003).
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5. Delhomme N, Padioleau I, Furlong EE, Steinmetz LM. 2012 easyRNASeq: a
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bioconductor package for processing RNA-Seq data. Bioinformatics 28(19),
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2532-2533. (doi:10.1093/bioinformatics/bts477).
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Supplementary tables and figures
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Table S1: Summary of all B vitamin related transporter and intracellular processing isoforms
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detected in the transcriptome of symbiotic and aposymbiotic D. fasciatus reared on
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cottonseeds. Transcripts with Aposymbiotic/Symbiotic fold change > 2 are marked with an
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asterisk.
Seq. Name
Seq. Description
Symbiotic
Aposymbiotic
Fold
(RPKM)
(RPKM)
Aposymbiotic/Symbiotic
0.9
2.0
2.3*
Intracellular Processing
Dfas-06032
Thiamine pyrophosphokinase
(TPK) (B1)
Dfas-08844
Riboflavin kinase (RFK) (B2)
23.5
41.6
1.8
Dfas-46813
Nicotinamide mononucleotide
9.8
11.6
1.2
2.9
4.9
1.7
adenylyltransferase (NMNAT)
(B3)
Dfas-29450
Pantothenate kinase (PANK)
(B5)
Dfas-50613
Pyridoxal kinase (PK) (B6)
13.6
19.7
1.5
Dfas-11244
Biotin-protein lyase (BPL) (B7)
0.3
0.8
2.4*
Dfas-40090
Dihydrofolate reductase
6.2
9.6
1.6
257.8
237.3
0.9
1.4
3.4
2.5*
5.5
25.7
4.7*
6.3
32.1
5.1*
(DHFR) (B9)
Dfas-21030
Transcobalamine 2 (TCII) (B12)
Transport
Dfas-27947
Proton-coupled folate
transporter (PCFT) (B9)
Dfas-08740
Thiamine alkaline phosphatase
(ALKP) (B1)
Dfas-21251
Thiamine transporter 2 (THTR2)
(B1)
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100
5
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Table S2: Summary of all stress-related isoforms detected in the transcriptome of symbiotic
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and
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Aposymbiotic/Symbiotic fold change > 2 are marked with an asterisk.
aposymbiotic
Seq. Name
D.
fasciatus
Seq. Description
reared
on
cottonseeds.
Transcripts
with
Symbiotic
Aposymbiotic
Fold
(RPKM)
(RPKM)
Aposymbiotic/Symbiotic
432.19
1,162.36
2.69*
2.40
2.51
1.05
Dfas-25287
Heat shock protein 70
Dfas-38373
Stress-activated protein kinase jnk
Dfas-54540
Heat shock protein 90
107.91
227.00
2.10*
Dfas-38957
Heat shock protein 70
27.80
14.23
0.51
Dfas-28415
Heat shock protein 70
3.27
4.21
1.28
Dfas-40833
Heat shock protein 90
135.47
381.08
2.81*
Dfas-08437
Related to glyoxal oxidase precursor
2980.39
3231.26
1.08
Dfas-41199
Related to glyoxal oxidase precursor
171.70
338.03
1.97
Dfas-11475
Related to glyoxal oxidase precursor
211.52
412.40
1.95
Dfas-19692
Peroxiredoxin 6
733.49
668.13
0.91
Dfas-10108
Ccaat enhancer-binding
59.72
63.70
1.07
Dfas-47994
Superoxide dismutase 2
16.61
17.92
1.08
Dfas-48940
Superoxide dismutase
220.71
165.31
0.75
Dfas-23888
Superoxide dismutase
0.87
2.75
3.15*
Dfas-22754
Superoxide dismutase
2.40
3.39
1.41
Dfas-52644
Mitochondrial manganese superoxide
36.37
43.21
1.19
0.40
0
N/A
161.79
122.79
0.76
dismutase
Dfas-39352
Tryparedoxin peroxidase
Dfas-12213
Peroxiredoxin- mitochondrial-like
Dfas-01237
Peroxiredoxin 4
0.82
2.21
2.70*
Dfas-36450
Thioredoxin peroxidase 2-like
0.31
2.10
6.75*
Dfas-39210
Thioredoxin family trp26
5.07
6.73
1.33
Dfas-38112
Venom allergen 5-like
0.33
3.38
10.13*
Dfas-35010
Trypsin-like protease
1.96
0
N/A
Dfas-31914
Glucose transporter type 1-like
0.94
1.33
1.41
Dfas-19251
Solute carrier family facilitated glucose
25.94
63.95
2.47*
transporter member 8
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Table S3. Composition of the artificial diet. To generate the vitamin-deficient diet, the B
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vitamin solution was substituted with water.
Component
Amount
soybean protein
15.0 g
potato starch
7.5 g
dextrose
7.5 g
sucrose
2.5 g
cellulose
12.5 g
B vitamin stock solution
15.0 ml
thiamine (B1)
(0.25 g)
riboflavin (B2)
(0.5 g)
nicotinamide (B3)
(1 g)
calcium pantothenate (B5) (1 g)
pyradoxine (B6)
biotin (B7)
folic acid (B9)
(0.25 g)
(0.02 ml)
(0.25 g)
cobalamin (B12)
water
(1g)
(ad 1000 ml)
soybean oil
20 ml
wheat germ
10 g
water
25 ml
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Table S4: Summary of all primer pairs utilized in this study.
Primer
Primer Sequence
Orientation
Target Gene
TPK fwd
TCTTTCCGAAGGATTTGGTG
Fwd.
Thiamine pyrophosphokinase (TPK)
TPK rev
TTTCGGGACAAATTCGAGAG
Rev.
RFK fwd
GATGGGCGAAACTTGAAGAA
Fwd.
RFK rev
AGGGTTCCAACCAACACTCA
Rev.
NMNAT fwd
CTGGGAATGCAGTCAGGAAA
Fwd.
Nicotinamide mononucleotide
NMNAT rev
TGGCTCGTCATTCTCATTCA
Rev.
adenylyltransferase (NMNAT)
PANK fwd
GGAGACAGCACAAGCTGGAC
Fwd.
Pantothenate kinase (PANK)
PANK rev
ACCGCTCTACCCATTCCTTC
Rev.
PK fwd
ATAGCGCTCCATGCTTCATC
Fwd.
PK rev
GATCCGGTAATGGGTGACAA
Rev.
BPL fwd
GGATGTGCTACGTTTTCTCTCC
Fwd.
BPL rev
TAAGGCCTCAAGTCCGTGTT
Rev.
DHFR fwd
GAGTGTCTGGATAATCGGAGGA
Fwd.
DHFR rev
TCTTCTTGGACTTCCGTTGG
Rev.
TCII fwd
CTTTAAGAAGCCGGCAACAG
Fwd.
TCII rev
TTGACAGGCATAAGGGTCGT
Rev.
ALKP fwd
GACATATGCGGCGAACAAAC
Rev.
ALKP rev
GTCGGGCCTCTTGTTTAAGG
Fwd.
THTR2 fwd
GCTTCGACAAGTCCATTCCA
Rev.
THTR2 rev
GATGTTCTGGTGGGCGTTAG
Fwd.
PCFT fwd
AGACGAGGCAAACTGTTCCA
Rev.
Proton-coupled folate transporter
PCFT rev
GGCGTCTTCTCTGTGCTGTT
Fwd.
(PCFT)
GLUT8 fwd
AGGGTGGAAGGTTTGCTTCT
Fwd.
Glucose transporter (GLUT8)
GLUT8 rev
GAAAGCCCTAATGGTGCTGA
Rev.
Hsp70 fwd
GGATGCCGGTACAATTTCTG
Fwd.
Hsp70 rev
GGTTCCACCACCAAGATCAA
Rev.
RPL13A fwd
CGAGGATAAGACGGAACTTGG
Rev.
RPL13A rev
CATGAAGGCTATGGGTCTGG
Fwd.
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8
Riboflavin kinase (RFK)
Pyridoxal kinase (PK)
Biotin-protein lyase (BPL)
Dihydrofolate reductase (DHFR)
Transcobalamine 2 (TCII)
Thiamine alkaline phosphatase (ALKP)
Thiamine transporter 2 (THTR2)
Heat shock protein (Hsp) 70
60S ribosomal protein L13a
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Figure Captions
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Figure S1. Midgut transcriptome of cottonseed-fed symbiotic and aposymbiotic D. fasciatus.
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(a) Illumina transcriptome sequencing and assembly statistics. (b) Histogram of contig
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lengths as assembled by Trinity. (c) Summary of the BLAST and GO annotation results. (d)
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Heatmap of the normalized expression (RPKM) of all differentially expressed transcripts. For
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each row, the expression was centered according to the mean and standard deviation. The
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row dendrogram represents the Pearson correlation of all genes.
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Figure S2. Differential expression of non-vitamin transporter and housekeeping genes by
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qPCR for symbiotic and aposymbiotic D. fasciatus reared on a complete or vitamin deficient
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artificial diet (normalized to the 60S ribosomal protein L13a). (a) Non-B vitamin transporter
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genes. (b) Elongation factor 1 alpha (EF1a). Shading of boxes signifies the experimental
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treatments (see legend). Lines represent medians, boxes comprise the 25–75 percentiles,
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and whiskers denote the range. Significant differences were assessed based on the
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normalized expression in reference to the 60S ribosomal protein L13a with the Kruskal-Wallis
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test and Dunn posthoc tests.
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