Global Expression Analysis Revealed Novel Gender-Specific Gene Expression Features in the Blood Fluke Parasite Schistosoma japonicum
et al. (2011) Global Expression Analysis Revealed Novel Gender-Specific Gene Expression Features in the Blood Fluke
Parasite Schistosoma japonicum. PLoS ONE 6(4): e18267. doi:10.1371/journal.pone.0018267
Global Expression Analysis Revealed Novel Gender- Specific Gene Expression Features in the Blood Fluke Parasite Schistosoma japonicum
Xianyu Piao 0
Pengfei Cai 0
Shuai Liu 0
Nan Hou 0
Lili Hao 0
Fan Yang 0
Heng Wang 0
Jianwei Wang 0
Qi Jin 0
Qijun Chen 0
Erika Martins Braga, Universidade Federal de Minas Gerais, Brazil
0 1 Laboratory of Parasitology, Institute of Pathogen Biology/Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College , Beijing , China , 2 State Key Laboratory for Molecular Virology and Genetic Engineering, Institute of Pathogen Biology, Chinese Academy of Medical Sciences and Peking Union Medical College , Beijing , China , 3 Key Laboratory of Zoonosis, Ministry of Education, Institute of Zoonosis, Jilin University , Changchun , China , 4 College of Life Science and Technology, Southwest University of Nationalities , Chengdu, Sichuan , China
Background: Schistosoma japonicum is one of the remarkable Platyhelminths that are endemic in China and Southeast Asian countries. The parasite is dioecious and can reside inside the host for many years. Rapid reproduction by producing large number of eggs and count-react host anti-parasite responses are the strategies that benefit long term survival of the parasite. Praziquantel is currently the only drug that is effective against the worms. Development of novel antiparasite reagents and immune-prevention measures rely on the deciphering of parasite biology. The decoding of the genomic sequence of the parasite has made it possible to dissect the functions of genes that govern the development of the parasite. In this study, the polyadenylated transcripts from male and female S. japonicum were isolated for deep sequencing and the sequences were systematically analysed. Results: First, the number of genes actively expressed in the two sexes of S. japonicum was similar, but around 50% of genes were biased to either male or female in expression. Secondly, it was, at the first time, found that more than 50% of the coding region of the genome was transcribed from both strands. Among them, 65% of the genes had sense and their cognate antisense transcripts co-expressed, whereas 35% had inverse relationship between sense and antisense transcript abundance. Further, based on gene ontological analysis, more than 2,000 genes were functionally categorized and biological pathways that are differentially functional in male or female parasites were elucidated. Conclusions: Male and female schistosomal parasites differ in gene expression patterns, many metabolic and biological pathways have been identified in this study and genes differentially expressed in gender specific manner were presented. Importantly, more than 50% of the coding regions of the S. japonicum genome transcribed from both strands, antisense RNA-mediated gene regulation might play a critical role in the parasite biology.
Funding: This study was carried out with support to Q. Chen from grants (2007IPB002, 2007IPB13) to invited professor of the Institute of Pathogen Biology (IPB),
Chinese Academy of Medical Sciences (CAMS) and Jilin University, China; the National Science and Technology (S&T) Key project (2008zc1004-011) and the Young
Distinguished Scientist Grant (30625029) of the Chinese Natural Scientific Foundation. The funders had no role in study design, data collection and analysis,
decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
Human schistosomiasis, the second only to malaria in term of
morbidity and mortality, is caused by infections of Schistosoma
species depending on the endemic region of the parasites . S.
japonicum is the causative agent of schistosomiasis perturbing
millions of people in several East and Southeast Asian countries.
Though schistosomal parasites are sensitive to the treatment of
praziquantel, high re-infection rates in both human and animals
plus the requirement of frequent administration still limit the
overall success of chemotherapy. More therapeutic targets are to
be defined for an optimal treatment as well as disease prevention.
The recent decoding of the genome sequences of the two most
pathogenic parasites, S. mansoni and S. japonicum, has paved a
pivotal way for a systematic dissection of the parasite biology
The genome of S. japonicum harbors in 8 pairs of chromosomes
with an estimated 397 Mb containing 13,469 protein-coding
sequences , which accounts for 4% of the genome. In the
nonprotein coding regions, approximately 40% is composed of
repeated sequences including transposable elements (TE). Recent
study indicated that the transcripts of TE could be processed into
small RNAs (endogenous siRNA), which fulfilled regulatory
functions from the maintenance of genome stability to
stagespecific gene activation or silencing [5,6,7]. Genomic variation
such as single nucleotide polymorphism (SNP) has been noticed
but its biological significance remains to be further studied [8,9].
The availability of the genome sequences of several schistosomal
parasites plus the free-living Schmidtea mediterranea have paved the
way for deep functional analysis on the genomes and the encoding
biology of the pathogenic parasites [10,11,12]. Primary analyses
have revealed remarked features of both parasite biology and
hostparasite interaction [10,11,12]. Genomic sequencing project has
revealed that S. japonicum has abandoned more than 1,000 protein
coding domains as compared to the free living worm Caenorhabditis
elegans, indicating the parasite has gained the ability to exploit host
factors for its development . For example, several signal
transduction pathways (including those for Wnt, Notch,
Hedgehog, and transforming growth factor b (TGF-b) found in human)
are also present in the parasite . These include endogenous
hormones such as insulin, epidermal growth factor (EGF)-like and
fibroblast growth factors (FGF)-like peptides. Predicted
components of the RasRafMAPK and TGF-bSMAD signaling
pathways (including FGF and EGF receptors) share high sequence
identity with their mammalian orthologs, indicating that
schistosomes, in addition to utilizing their own signaling pathways,
exploit host endocrine signals for their own development
Schistosomal parasites are featured with very complicated
developmental and biological cycles. They are the first group of
organisms that are dioecious with marked differences in sexual
dimorphism and biology , which are controlled by genetic as
well as epigenetic regulation factors. Studies on stage- and
genderspecific expression profiles with parasites of various developmental
stages have been carried out with different methodological
approaches, from manual sequencing of expression sequence tag
(EST) to full-length cDNA cloning, microarray hybridization, and
random sequencing [15,16,17,18,19,20,21]. The valuable data
obtained from the genomic and post-genomic studies has
facilitated tremendously in understanding parasite biology as well
as parasite-host interactions (for review, see refs 10, 11, 12).
While the stage-specific transcriptomic information of S.
japonicum keeps increasing, investigation with specific perspectives
on the differences of genome-wide transcriptions of the male and
female parasites has mainly been based on the availability of the
genomic sequence which has been far from a complete
assembly. In this study, by using the high through-put
RNAseq techniques, we successfully explored the transcriptomes of
male and female schistosomal parasites. The data revealed novel
features of gender-specific expression and gene regulation
Libraries of sequence tags from male and female adult
worms of S. japonicum
In this study, we determined and compared transcriptomes of
male and female adult worms of S japonicum. DGE (Digital Gene
Expression) libraries were made, using RNA with a PolyA tail at
the 39-end of each template, for both genders, and all
polyadenylated RNA was sequenced using Solexa (Illumina) high
through-put technology (Figure 1). The two libraries (male and
female adult worms) contained 3,705,287 and 3,672,014 unfiltered
tags. After removal of tags containing ambiguous base calls and
adaptor tags, there were 3,660,835 (male) and 3,693,835 (female)
clean tags and the number of distinct tags in the two libraries of
male and female was 219,628 and 213,310, respectively (Table 1).
The clean tags were mapped onto the S. japonicum genome of
SGST (http://lifecenter.sgst.cn) and the relationship between
sequence tags and genes was then built up. For genes with
multitags, the total distinct expressed tags were taken into account as the
gene expression value. Most of the tags were from highly expressed
genes (Figure 2 and Table S1). The redundancy for Sjc-F and
SjcM was respectively 94.2% and 94.1% which indicated the
sequencing quantity should be enough for both libraries
(Table 1). Of the 360,955 unique tags, 71,983 can be found in
both libraries. Male and female specific tags accounted for 3.85%
and 4.35% respectively. The number of clean distinct tags was
141,327 and 147,645 in Sjc-F and Sjc-M, respectively (Table 1).
As shown in figure 2, the most abundant tags (63%) were single
copy and tags with more than 10 copies accounted only around
3% in both female and male worms (Figure 2). All sequence data
has been deposited in the database (http://www.ncbi.nlm.nih.
gov/geo/info/faq.html#seq) with an accession number of
Genes differentially expressed in male and female
Tags that could specifically match to the reference genes of S
japonicum generated expression data of 9,239 genes, accounted for
73% of genes in the annotated genome which was estimated to
have 13,469 genes in the genome . A total of 4,732 (35%)
distinct genes were found differentially expressed between male
and female, of which 2,545 genes up-regulated and 2,187 genes
down-regulated in male versus female adult worms (Figure 3A and
Table S2). Genes showed significant differences in expression were
those coding proteins with functions associated with biological
process, cellular component or molecular functions (Table S2).
Genes related to the function of genetic information processing
which was more biased to the female parasite, while genes with
function related to interaction with host (environmental
information processing) were more active in the male parasites. To
evaluate whether the number of sequencing tags that could reflect
the patterns of differentially expressed genes between male and
female parasites, transcripts of 6 genes of AMP-activated kinase,
eggshell protein 1 precursor, an unknown gene (Sjc_0024870),
dynein light chain, paramyosin, and tropnin were analyzed by
quantitative PCR. The results from quantitative PCR correlated
with the number of sequence tags that were significantly different
between male and female parasite (Figure 3B).
Half of the coding regions in the genome of S. japonicum
was transcribed from both strands
When mapping the sequence tags to the genome we found that,
of the genes (9,239) with unambiguous tags detected, 7,261 genes
have tags transcribed from both sense and antisense strands. Thus
nearly 50% of the genes annotated in the genome of S. japonicum
were found transcribed from both strands. Of these genes, 5,487
genes had tags corresponding to sense strands more than that from
antisense strands, and 1411 genes had more tags from the
antisense strands than that from the sense strands. While 363
genes have equal number of tags generated from both strands
(Figure 4A, Table S3).
Further comparative analysis on the sequence tags between
male and female parasites revealed that 3,963 tags from sense
strand were significantly different in copy number between male
and female parasites. Of which, 2,562 tags had antisense and their
cognate sense transcripts co-expressed (higher levels of sense tags
also yield higher antisense tags counts, Figure 4B), 1,401 tags had
no matched antisense tags. There were 2,528 antisense tags which
were differentially expressed in the two sexes of the parasite, of
which 1,704 had sense counterparts co-expressed and 824 was
discordant with the sense strand. 1,851 genes had differentially
Distinct reads represent the number of distinct sequence reads in the two
libraries, Sjc-F and Sjc-M. Sex-specific reads represent number of sequence
reads specific to female (Sjc-F) or male (Sjc-M) parasite. The numbers of the
distinct reads from the two libraries that matched to the genomic sequences
were listed. The redundancy of the two libraries was calculated according to the
formula (Redundancy = 100-(Total Clean Distinct Tags/Total Tags x 100).
expressed tags from both sense and antisense strands, with 1,300
tags were co-expressed, and 551 tags were discordant (Figure 4B).
Identification of different biological or metabolic
pathways between male and female parasites
Gene categorization based on potential functions of the coded
proteins was performed. Sequence tags from 2,148 genes can be
categorized into different functions or biological pathways
(Figure 5, Table 2, and Table S4). Of which, 940 genes related
to metabolic pathways, 475 genes were with functions related to
genetic information processing, 495 genes were related to
responses to environmental changes, and 958 genes were related
to cellular processing (Figure 5A).
Genes with differential expression patterns between male and
female parasites were also identified (Table S5, S6), of the 940
genes with functions associated to metabolism, 230 genes were
upregulated and 238 genes are down-regulated in male compared to
female parasites. Of the 475 genes with functions related to genetic
information processing, 98 genes were up-regulated and 168 genes
were down-regulated in the male parasite. 102 genes related to
environmental information processing were up-regulated and 65
genes were down regulated in male parasites. 168 genes function
in cellular processing were more active in male parasites, while 185
genes were more silent than female counterpart (Figure 5B).
Among the metabolic pathways identified in the parasites, the
expression of 5 genes related to the xenobiotic metabolism was
found up-regulated in female parasites (Table S5, S6).
The draft genomic sequence of S. japonicum has been available
, but functional determination of genes related to important
biological significance will likely rely on the analysis of mRNA
transcripts and the encoded proteins, since the multi-cellular
nature of the pathogen and its specific structure of tegument has
made it difficult to carry out genetic manipulation directly on the
parasite . In this study, by combining the powerful Digital
Gene Expression (DGE)-tag and high through-put RNA-seq
technique , the global transcriptomes of male and female S.
japonicum were obtained and compared. DGE offers distinct
advantages over other methods (such as array-based
geneexpression analysis systems) for transcriptomic studies. First, it
has a better coverage and an ability to measure low-abundance
genes, find unknown transcripts with minimal background noise
for increased sensitivity. Secondly, as demonstrated in Figure 1,
all sequence tags were anchored on a chip matrix at the 39 side
before sequencing, thus only the cDNA strand (complimentary to
the polyA-tailed RNA template) was sequenced. The advantages
of this approach are that most adenylated transcripts can be
obtained and the step of cDNA cloning is not needed. Further,
the rationale in tag preparation was that the restriction enzyme
(NlaIII) would cleave at the 39 most CATG site, thus the 39 UTR
(Un-translating region) information will be critical for the
following tag annotation. To avoid false positive of CATG site,
we used 3 kb as the cutoff value to define the 39 UTR of the
selected RNA templates. The CATG cleavage sites were
identified in the gene accompanied with 3 kb potential 39 UTR
using in-house perl script. Thus, contrast to normal EST
sequencing which mainly obtains sequence information close to
the 59 end of the templates, the DGE method explored here could
target the mRNA sequences which were more likely in
fulllength. Though deep (or random) sequencing can generate
genome-wide transcriptome information, it does not discriminate
strand-specific transcription. Further, all sequence tags were
mapped to the protein-coding genes with non-coding sequences
dismissed, thus small transcripts such as pre-microRNAs and
transcripts from non-coding regions were not included in the
The number of sequence tags identified in male and female
parasites was similar (Figure 2 and Table S1). However, around
one third of genes in the genome were found with bias in
preferential expression between male and female. Interestingly, the
number of genes with preferential expression in male and female
parasite was similar (Figure 3A and Table S2). The differences in
gene expression between male and female parasites were related to
the function of genetic information processing which was more
biased to the female parasite, which was likely due to the
production of eggs. While genes with function related to
interaction with host (environmental information processing) were
more active in the male parasites, this was presumably due to the
physiological character of male parasite which was much larger
than the female and most of its surface was exposed to the host
while female parasite was held in the cavity of the male. Further,
previous studies with microarray identified around 1,000 genes
that were differentially expressed in either male or female parasite
[19,24]. The reason that low numbers of genes identified in early
studies was likely due to the in-availability of a complete genome
sequence when the studies were performed. The advantage of the
current study is that the readout does not depend on the genome
sequence. Thus the number of genes identified with differential
expression in male and female parasite was more than that with
other approaches [19,22,24].
Gender-specific transcriptome analysis revealed that more than
2,000 genes were potentially involved in metabolic pathways or
biological functions (Figure 5 and Table S4). Among the
metabolic pathways identified in the parasites, the expression of
genes related to the xenobiotic metabolism was found more
interesting. Xenobiotic metabolism reactions often function in
detoxifying poisonous compounds . The reactions contain
three phases. In phase I, enzymes such as cytochrome P450
oxidases introduce reactive or polar groups into xenobiotics. These
modified compounds are then conjugated to polar compounds in
phase II reactions. These reactions are catalyzed by transferase
enzymes such as glutathione S-transferases (GST). In phase III, the
conjugated xenobiotics are recognized by efflux transporters and
Biosynthesis of secondary metabolites
Genetic information processing
Folding, sorting and degradation
Signaling molecules and interaction
Cell growth and death
pumped out of cells . Proteins encoded by these genes are
likely involved in fertilization or egg production in the female
parasite. Studies on S. mansoni has reported functions of P450 and
GST in the parasite . However, this is the first report which
reveals more complete connection of the enzymes in the
xenobiotic metabolism pathway in S. japonicum. So far, GST has
been regarded as a best candidate for development of
antifecundity vaccine for japonicum schistosomiasis . In light of
the components identified in the pathways related to the
reproduction of the parasite, more molecules such as P450
homologue might be potential candidate in the vaccine
development. Further, genes with functions related to the pairing of the
two sexes were found differentially expressed. Male parasite
expressed more genes related to WNT (originally been identified
as a recessive mutation affecting wing and haltere development in
Drosophila melanogaster) signaling pathway which might be beneficial
for embryo development in female parasites. Interestingly, genes
encoded actin proteins were found more active in female parasites
than male parasites, whether this related to the egg-shedding
function or the pairing of the two sexes remains further
elucidation. Furthermore, the axon guidance pathway was found
more active in female than male. Compounds targeting these
pathways may effectively block parasite development and reduce
pathological reaction in the liver of the host.
The discovery of tremendous antisense transcripts from the
coding region is remarkable. The estimated number of
proteincoding genes in the S. japonicum genome is 13,469, while 7,261 genes
were found transcribed from both strands. To our knowledge, this is
the first observation in S. japonicum that more than 50% of the
protein-coding genes were bi-directionally transcribed. It much be
pointed out that previous studies in S. mansoni using a microarray
already found bi-directional transcription in 7% of the active no
match genes . Thus bi-directional transcription is likely a
common feature in schistosomal parasites. Though most of the
transcripts were from sense strands of the genes, more than 1,000
genes were found to have more antisense than sense transcripts and
around 500 genes were transcribed symmetrically. Since the RNA
templates were selected based on the poly-A tail, thus the antisense
transcripts were likely polyadenylated. It cannot be ruled out that
some of the anti-sense RNAs may encode proteins, but it is unlikely
that all polyadenylated antisense RNAs do so. Recent study on the
antisense transcripts in human found that the pseudogenes could be
sources of natural antisense transcripts . Transcripts from
pseudogenes form hybrids with that of parental genes, which will be
further processed into regulatory endogenous siRNAs. Though it
could not be ruled out that such a mechanism also existed in S.
japonicum, it is unlikely that the parasite harbors so many
pseudogenes in the genome, as antisense transcripts complementary
to more than half of the protein-coding genes were detected. Thus,
some of the antisense transcripts must be a result of bi-directional
transcription, at least in the adult worms. The mechanism behind
the bi-directional transcription is still not known; but, with the
discovery of NAT (natural antisense transcripts)-derived
endogenous siRNAs in the parasite , it can be hypothesized that some, if
not all, sense and antisense RNA hybrids are the sources of
NATderived endo-siRNAs . However, it is also possible that some of
the antisense transcripts exerted post-transcriptional regulation
through direct hybridization with the mRNA templates.
Nevertheless, the finding in this study has opened up new avenue for
dissection of parasite biology regarding the function of antisense
RNA-dependent gene regulation.
In this study, transcripts of 73% of the genes in S. japonicum
genome was identified by high-through-put sequencing, of which,
35% (4,732/13,469) was preferential expressed in either male or
female parasite. More than 900 genes involved in metabolic and
biological pathways were identified and genes that were
differentially expressed in gender specific manner were analyzed.
Further, polyadenylated antisense RNAs were mapped to more
than 50% of the coding regions in S. japonicum genome, indicating
bi-directional transcription were common, at least in adult worm
stage of the parasite. Antisense-mediated gene regulation might
play a critical role in the parasite biology.
Parasites and RNA purification
S. japonicum-infected snails were collected from the endemic area
in Jiangxi province. Cercarie were released from the snails in room
temperature (around 25 degree) under a lamp. One New Zealand
white female rabbit (5 month old) was infected with 1500-2000
cercarie for 42 days. Mature adult parasites were harvested from
the infected rabbit by flushing the blood vessels with PBS as
described earlier [5,30]. Male and female parasites were manually
separated and total RNA from the parasites was purified with
Trizol reagent (Invitrogen, CA, USA) as described [5,30].
Generation of expression tags of male and female
parasites for sequencing
Messenger RNA from male and female S. japonicum parasite was
selectively purified from total RNA using oligo-(dT) conjugated
magnetic beads (DynabeadsH, Invitrogen). Complementary DNA
(cDNA) was synthesized guided by oligo-(dT) as a primer.
Sequencing tags were generated as illustrated in Fig. 1. Briefly,
double stranded cDNA sample was digested with the endonuclease
NlaIII that recognizes the CATG sites on cDNAs. After cleavage,
the 39-regions of the cDNAs attached on the magnetic beads were
selected. The first sequencing adapter (Illumina adapter 1) 
was added to the 59 ends of each fragment which was further
digested with MmeI, an enzyme cuts 17 bp downstream of the
CATG site. After removing 39 fragments with magnetic beads
precipitation, Illumina adapter 2 was introduced at 39 ends of the
tags to generate tag library with different adapters at both ends.
The fragments were PCR amplified and the 85 base strips were
purified by 6% TBE PAGE Gel electrophoresis and sequenced
with the Solexa high-throughput sequencing technology. The
advantage of this approach is that transcripts from both strands
(sense and anti-sense) can be targeted and sequenced.
After removing the low quality and adaptor tags, the clean
sequence tags were mapped onto the gene reference tag data set
and the relationship between sequence tags and genes were then
built up. For genes with multi-tags, the total distinct expressed tags
were taken into account as the gene expression value. For tags that
mapped to different genes, the mean value of tag number was used
as the expression level for each gene.
Reads with CATG site were selected and mapped to the
genome sequences. Sequences that with complete match to the
genome sequences were further analyzed for differential
expression. We employed IDEG6 (http://telethon.bio.unipd.it/bioinfo/
IDEG6/) to identify differentially expressed mRNAs based on
their relative abundance which was reflected by total count of
individual sequence read between the two libraries. The general
Chi test was employed which has been proved to be one of the
most efficient tests . Finally, genes with a P value , = 0.05
were deemed to be significantly different between the two libraries.
Gene sequences were firstly blasted with Kyoto Encyclopedia of
Genes and Genomes database (KEGG, release 50) (http://
nematode.net/cgi-bin/keggview.cgi and http://www.nematode.
net/FTP/index.php) with E values , = 1e-10 . The KO
information was retrieved from blast result using which the
possible pathway information for each gene could be identified.
Domain information was annotated by InterProScan and
functional assignments were mapped onto Gene Ontology (GO).
WEGO was employed to do GO classification and draw GO tree
Verification of gender-specific transcripts by real-time
Total RNA of S. japonicum (adult male and female worms) was
extracted using Trizol reagent (Invitrogen, CA, USA). The RNAs
were dissolved in diethylpyrocarbonate (DEPC)-treated water and
reverse transcribed with 200 U SuperScriptTM III Reverse
Transcriptase (Invitrogen) according to the manufacturers
instruction. The following primers were designed as forward and reverse
primers based on the female, male specific tags and a-tubulin gene
(endogenous control): AMP-activated kinase F:
TATTTTCAT-39. Eggshell protein 1 precursor F:
59-CACACATTACGATATTACAGTGAGATG-39. Unknown (Sjc_0024870: S.
japonicum expressed protein, putative mRNA) F:
59-ACCCGAATATCGTGAAACAGA39. Dynein light chain F:
59-GCTGCAATGGCTATGGATAAA39, R: 59-TCCACGATCTTCCAGTGAGA-39. Paramyosin F:
59-TCTCCTCCTCCAACTGAA-39. Tropnin F: 59-CGATGGAAAGTCTGAAGC-39, R:
59-ACGTTCCCCTCTACGAAA-39. a-tubulin F:
We used a-tubulin transcript as the endogenous control.
Quantitative RT-PCR was conducted in triplicate and each
reaction underwent 40 amplification cycles using an Applied
Biosystems 7300 real-time PCR system (Applied Biosystems,
Foster City, USA) with cDNA equivalent to 15 ng of total RNA,
200 mM of primers and 12.5 ml SYBR Green PCR Master Mix
(ABI, USA) adjusted to final volume of 25 ml with DEPC-treated
water. Dissociation curves were generated for each sample to
verify the amplification of a single PCR product. The Relative
expression was analyzed using the SDS 1.4 software (Applied
Biosystems, Foster City, USA). Due to the fact that the
transcription of a-tubulin gene in male was 2 times higher than
in female, a step of normalization was included in the final
Table S1 Description of the libraries generated with sequence
tags from male and female S. japonicum. The first column (Class)
defined the sequence classes. In the columns of Sjc-F and Sjc-M, #
represents the number of tags; % represents the percentage of
clean tags with different copy numbers in the total clean tag pools
of female and male parasite respectively.
Table S2 Genes showed significant differences in expression in
female versus male parasite and the biological functions associated.
The function of genes identified were classified into general (First
Class) and more defined (Second Class). The number of genes
up(# of Up), down-regulated (# of Down) as well as the contig
names were listed.
Table S3 Tags mapped to either sense, antisense strand or both
stands of the genes identified. The first column is the gene name,
the second column Both means gene expressed in both strand. #
of Detected is the number of tags detected by sequencing. Total
Express means total times of detected tags including both female
and male. Sjc-F Expression and Sjc-M Expression means total
times of detected tags in female and male respectively. Total
TPM means total times of detected tags per million, and Sjc-F
TPM, Sjc-M TPM means total times of detected tags per million
in female and male respectively. M-F means difference between
the TPM value of Sjc-M and Sjc-F. Up in the Mark column means
the TPM value of Sjc-M is higher than that of Sjc-F. The last
column Tags represents tag positions in genome, for example.
Y means the tag is distinct. The numbers represent the position
of the CATG from the 39 end of the gene, total TPM and the
TPMs of the same tag in Sjc-F and Sjc-F respectively.
Table S4 Tags mapped to genes involved in metabolic and other
biological functions. The first column lists the metabolic pathways
and classified biological functions identified. The second column
represents the number of genes involved and the thirst column
represents the contig names.
We are very grateful to the farmers who helped to collect the snails that
infected with S. japonicum.
Conceived and designed the experiments: XP QC. Performed the
experiments: XP PC SL NH LH. Analyzed the data: XP QC. Contributed
reagents/materials/analysis tools: FY HW JW QJ. Wrote the paper: XP
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