A next-generation microarray further reveals stage-enriched gene expression pattern in the blood fluke Schistosoma japonicum
Cai et al. Parasites & Vectors
A next-generation microarray further reveals stage-enriched gene expression pattern in the blood fluke Schistosoma japonicum
Pengfei Cai 0 1 3
Shuai Liu 1 3
Xianyu Piao 1 3
Nan Hou 1 3
Hong You 0
Donald P. McManus 0
Qijun Chen 1 2 3
0 Molecular Parasitology Laboratory, QIMR Berghofer Medical Research Institute , Queensland , Australia
1 MOH Key Laboratory of Systems Biology of Pathogens, Institute of Pathogen Biology, Chinese Academy of Medical Sciences & Peking Union Medical College , Beijing , People's Republic of China
2 Key Laboratory of Zoonosis, Shenyang Agriculture University , Shenyang , People's Republic of China
3 MOH Key Laboratory of Systems Biology of Pathogens, Institute of Pathogen Biology, Chinese Academy of Medical Sciences & Peking Union Medical College , Beijing , People's Republic of China
Background: Schistosomiasis is caused by infection with blood flukes of the genus Schistosoma, and ranks, in terms of disability-adjusted life years (DALYs), as the third most important neglected tropical disease. Schistosomes have several discrete life stages involving dramatic morphological changes during their development, which require subtle gene expression modulations to complete the complex life-cycle. Results: In the current study, we employed a second generation schistosome DNA chip printed with the most comprehensive probe array for studying the Schistosoma japonicum transcriptome, to explore stage-associated gene expression in different developmental phases of S. japonicum. A total of 328, 95, 268 and 532 mRNA transcripts were enriched in cercariae, hepatic schistosomula, adult worms and eggs, respectively. In general, genes associated with transcriptional regulation, cell signalling and motor activity were readily expressed in cercariae; the expression of genes involved in neuronal activities, apoptosis and renewal was modestly upregulated in hepatic schistosomula; transcripts involved in egg production, nutrition metabolism and glycosylation were enriched in adult worms; while genes involved in cell division, microtubule-associated mobility, and host-parasite interplay were relatively highly expressed in eggs. Conclusions: The study further highlights the expressional features of stage-associated genes in schistosomes with high accuracy. The results provide a better perspective of the biological characteristics among different developmental stages, which may open new avenues for identification of novel vaccine candidates and the development of novel control interventions against schistosomiasis.
Schistosoma japonicum; Microarray; Gene profiling; Stage-enriched expression; Developmental biology
Schistosomiasis, a debilitating and chronic disease caused
by infection with blood flukes (digenetic trematodes) of
the genus Schistosoma, remains one of the most
significant parasitic diseases worldwide, afflicting more than 230
million people, with about 800 million exposed to the risk
of the infection [1, 2]. Schistosomiasis caused about 3.31
million DALYs in 2010, exceeded only by intestinal
nematode infections and leishmaniasis, in the list of global
neglected tropical diseases . Schistosoma mansoni, S.
haematobium and S. japonicum are the three main species
of clinical relevance. Currently, there are no practical
anti-schistosome vaccines available. The repeated use
of a single effective drug, praziquantel, is required for
schistosomiasis treatment, while a variety of morbidity
management strategies have been adopted for control
of the disease [4, 5].
The schistosome life-cycle involves an aquatic snail as
an intermediate host and a mammal as definitive host
. Schistosome cercariae are shed from infected snails
under a light stimulus and are released into water
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resources. The free-swimming cercariae infect a
mammalian host by skin contact. After skin penetration, the
larvae lose their tails and transform into schistosomula.
Once entering into capillaries or lymphatic vessels, they
are carried to the heart and lungs within 3–5 days
depending on the species. The lung-stage schistosomula
continue migration to the hepatic portal system at about
14-days post-infection, where the juveniles pair up and
become sexually mature. Then the schistosomes in
copula migrate to the mesenteric veins (S. mansoni and S.
japonicum) or the pelvic venous plexus (S.
haematobium), where the female worms lay eggs intravascularly,
with varied patency periods among the species. Some
eggs are lodged in tissues causing disease whereas others
enter the intestine or bladder and are excreted from the
host. The mature eggs hatch under favourable conditions
to release miracidia which penetrate a snail host and
develop asexually into mother and then daughter sporocysts,
within which cercariae are produced, which are then
released from the snail and continue the life-cycle.
The availability of schistosome transcriptome [7, 8]
and genome sequences [9–11] for the three major
Schistosoma spp., provides an invaluable resource to profile
gene expression across different developmental stages
and between the sexes. In this respect, high-throughput
technologies, such as microarrays [12–18], serial analysis
of gene expression (SAGE) [19–21], digital gene
expression (DGE) , and, more recently, RNAseq [23, 24]
have been employed in the analysis of gene profiling in
schistosomes. These pioneering investigations have
provided unique information on developmental-enriched,
gender-biased, tissue-specific, strain-specific and
hostassociated gene expression features within schistosomes
[12, 14, 25–28], revealing critical insight on the biology
of these parasites. With respect to using microarray
platforms, the interpretation of microarray experiment
depends on the quality of genetic information contained in
the collection of DNA templates employed for probe
design. The first-generation of schistosome cDNA chips
were printed based on EST transcripts, so that the data
obtained from these chip experiments resulted in a poor
interpretation due to the problems in annotating these
ESTs [12–14]. We considered it essential to generate a
second generation DNA microarray with a well-curated
probe design, based on both transcriptomic and genomic
sequences, in order to increase our understanding of
We have constructed a second generation schistosome
DNA chip printed with the most comprehensive
coverage of probes, designed based on S. japonicum and S.
mansoni genomic and transcriptomic sequences for
transcriptomic studies [29–31]. Here, we have identified
stage-enriched transcripts in cercariae, hepatic
schistosomula, adult worms and eggs using this next-generation
DNA microarray. This study presents a comprehensive
view of the expression features of stage-enriched genes for
four developmental phases of S. japonicum, and provides
novel insights on schistosome developmental biology.
Schistosoma japonicum-infected snails (Oncomelania
hupensis) were purchased from Hunan Institute of
Parasitic Diseases, Yueyang, China. Cercariae were shed from
these snails under light stimulation and were collected.
Hepatic schistosomula at 14 days post-infection (p.i.)
were perfused from S. japonicum-infected New Zealand
rabbits via the vascular system. Mixed adult worms were
perfused from S. japonicum-infected rabbits at 6 weeks
p.i. Schistosome eggs were purified from liver tissues of
infected rabbits (6 weeks p.i.) by enzyme digestion .
All parasite samples (except eggs) were soaked in
RNAlater (Ambion, CA, USA), and stored at -80 °C until
total RNA extraction. Total RNA from eggs was isolated
immediately after purification.
Total RNA isolation
Total RNA samples were isolated from S. japonicum
cercariae, hepatic schistosomula, adult worms and eggs using
RNeasy Mini kits (QIAGEN, GmbH, Hilden, Germany)
according to the manufacturer’s instructions. Potential
contaminating genomic DNA was removed from RNA
samples using a Turbo DNA-free kit (Ambion, CA, USA).
The quantity of RNA in each sample was assessed by a
NanoDropND-1000 spectrophotometer (NanoDrop
Technologies, Wilmington, DE). The integrity of total RNA in
each sample was checked by denaturing agarose gel
electrophoresis (Additional file 1: Figure S1).
Microarray construction and hybridization and
subsequent data analysis
A schistosome genome-wide microarray was employed
for profiling the gene expression in S. japonicum
cercariae, hepatic schistosomula, adult worms and eggs.
The details regarding the design and construction of the
microarray, the hybridization method, and feature
extraction have been reported [29–33]. For each target
sequence, 3 or 4 pairs of complementary oligonucleotide
probes (forward and reverse, 60-mer) were designed (in
total 145,000 probes). Probes with random sequences
were printed as negative controls (background signal),
while eight spike-RNA probes from the intergenic
sequence of yeast were used as hybridization controls.
Microarrays were printed in a 12 × 135 K feature format
(Roche NimbleGen) representing 41,982 features. cDNA
was labelled with a fluorescent dye (Cy3-dCTP) using a
cRNA Amplification and Labelling Kit (CapitalBio,
Beijing, China) . Hybridization was performed using
three biological replicates for all samples by CapitalBio,
Beijing, China. Procedures for array hybridization,
washing, scanning, and data acquisition were performed
according to the NimbleGen Arrays User’s Guide. The
arrays were scanned using a MS200 scanner (NimbleGen
Systems) at 2-μm resolution, and NimbleScan software
(NimbleGen) was employed to extract fluorescent
intensity raw data from the scanned images. Normalized gene
expression data were generated using the Robust
Multichip Average (RMA) algorithm [35, 36]. Outlier probes
were identified and their contribution was reduced at
the reported gene expression level . The expression
value of a gene is a weighted average of all forward or
reverse probe sets when both background correction
and quantile normalization are performed.
Bioinformatics analysis on stage-enriched mRNA and EST
mRNA and EST transcripts highly enriched in cercariae,
hepatic schistosomula, adult worms and eggs of S.
japonicum were retrieved from the NCBI database (http://
www.ncbi.nlm.nih.gov/sites/batchentrez) based on
foldchange (FC = the mean intensity/the median of the mean
intensity values of the four developmental stages) values.
(FC ≥ 2 for both forward and reverse probe sets, and three
biological replicates were used for each stage). Student’s
ttest was used to determine differentially expressed genes
between one particular stage and any of the other three
stages [28, 30] (P < 0.05). Heat maps were constructed
based on the transformed log2FC values (forward probe
sets) using HemI 1.0 software . Blast2GO was used to
annotate the four gene sets functionally . A
comprehensive re-annotation was performed against these gene
sets using the BLASTx algorithm, with the annotation of
S. mansoni, S. haematobium and Clonorchis sinensis
homologues as a reference. For possible improved
annotation, potential conserved protein domains were searched
against genes annotated as hypothetical protein or
unknown in the NCBI CDD database (v3.14) .
Quantitative real-time PCR
A total of 20 stage-enriched genes were selected for
validation using qRT-PCR as described . One microgram
total RNA samples were reverse transcribed into
firststrand cDNA using a SuperScript III Reverse
Transcriptase Kit (Invitrogen, Carlsbad, CA, USA) with oligo (dT)
15 primer. The cDNA products were diluted 20-fold with
nuclease-free water before undertaking the qPCR. Each
25 μl PCR reaction contained 12.5 μl of 2 × Brilliant II
SYBR Green QPCR Master Mix (Agilent, USA), 1 μl
cDNA, 1 μl of the forward and reverse primer pair
(Additional file 2: Table S1), and 10.5 μl of sterile water.
PCR cycling conditions were as follows: 95 °C for
10 min, followed by 40 cycles of 30 s denaturation at
95 °C and 1 min annealing and extension at 60 °C. A
dissociation step (95 °C for 15 s, 60 °C for 1 min, 95 °C
for 15 s and 60 °C for 15 s) was performed to confirm
the amplification specificity for each gene. 26S
proteasome non-ATPase regulatory subunit 4 (PSMD4)
[29, 40] was employed as a house-keeping gene in the
assays. PCR reactions were performed in technical
triplicates on the 7300 Real-Time PCR system (Applied
Biosystems). The relative expression level of each gene
was analysed using SDS 1.4 software (Applied
Biosystems). Correlations between the microarray and qPCR
results for 20 stage-enriched genes were analysed with
the Spearman’s rho.
Results and discussion
Global view of stage-enriched mRNA transcripts in S.
By employing a microarray with the most comprehensive
probe coverage design to date, signal intensities from
3571, 1014, 1728 and 3381 sequences were found to be
enriched (FC of mean of intensity value to the median of
the mean of intensity values of the four stages ≥ 2) in
cercariae, hepatic schistosomula, adult worms and eggs,
respectively. Based on the initial screening, we further
retrieved a total of 1768 potential mRNA transcripts and
470 expressed sequence tags (ESTs) associated with
developmental stages from the NCBI database (Additional
file 3: Table S2). The gene collection was further filtered
by requiring FC values from both forward and reverse
probe sets ≥ 2. This filtration finally retained 328, 95, 268
and 532 mRNA transcripts highly enriched in cercariae,
hepatic schistosomula, adult worms and eggs, respectively
(Additional files 4, 5, 6 and 7: Tables S3–S6), which
contrasted with 128, 31, 83 and 84 ESTs, respectively,
highly enriched in these four stages (Additional files 8, 9,
10 and 11: Tables S7–S10). However, the percentage of
genes that were annotated as hypothetical protein or
unknown (23.57% in the mRNA data in contrast to 69.01%
in the EST data), highlights the utility of the second
generation S. japonicum DNA chip in profiling gene expression
in this parasite.
We observed that more mRNA transcripts were
enriched in the egg stage than in the other stages, with a
stronger biased expression (higher FC value) (Figs 1 and
2a-d). For example, 46.1% egg-enriched mRNA
transcripts showed a strong biased expression (FC > 10); this
number decreased to 22.0% in adult worms, and further
dropped to only 3.0 and 1.1% in cercariae and hepatic
schistosomula, respectively. A similar tendency was
observed when analysing the stage-enriched EST
transcripts (Additional file 12: Figure S2). In regards to
fluorescence intensity, 13.4, 8.42, 25.0 and 27.5% mRNA
transcripts enriched in cercariae, hepatic schistosomula,
Fig. 1 Heatmap for mRNA transcripts enriched in cercariae, hepatic
schistosomula, adult worms and eggs. A total of 328, 95, 268 and
532 mRNA transcripts were enriched in cercariae, hepatic schistosomula,
adult worms and eggs, respectively. The heatmap was created by HemI
1.0 based on the transformed data of log2 FC values. The data are based
on the mean of weighted signal intensity values of forward probe sets
(three biological replicates)
adult worms and eggs, respectively showed an average
intensity value > 10,000 (Fig. 2e-h).
Comparing the results with previous transcriptome data
A complete and accurate comparison of the results
obtained in the current study with data from previous
reports is hindered due to the following reasons. Firstly,
the annotation of stage-enriched genes was not ideal in
previous reports due to the fact that EST sequences were
used for probe design coupled with less sequence
homology information from other trematode species being
available. Secondly, the annotation for the same gene
may not have been unique. Thirdly, the screening
criteria for stage-enriched genes may have varied among
different studies. Nevertheless, we compared our data
with these from previous Schistosoma transcriptome
data [7, 13, 14, 23, 28] by manual checking. Globally,
about 4.57, 10.07 and 12.97% genes enriched in
cercariae, adult worms and eggs, respectively, were
reported in previous studies (Additional files 4, 5 and 7:
Tables S3, S4, S6). With respect to hepatic schistosomula
(14 days p.i.), to our knowledge the only other relevant
investigation on this particular stage was carried out on
S. mansoni by Fitzpatrick et al. , but no enriched
gene clustering was evident in that study. This was
probably due to the fact a large number (15) of distinct stages
were analysed , and this has made comparison with
our data for hepatic schistosomula difficult.
qPCR validation of the expression pattern of
A subset of 20 representative stage-enriched genes was
selected for qRT-PCR validation (Figs 3 and 4a-d). Most
genes were associated with important biological functions
in each of the parasite forms. The expression of these
genes at the four developmental stages validated by
qRTPCR analysis significantly correlated with the results
obtained by microarray: for cercariae-enriched genes
selected, r(30) = 0.8959, P < 0.0001 (Fig. 4e); for hepatic
schistosomula-enriched genes selected, r(30) = 0.7375,
P < 0.0001 (Fig. 4f ); for adult-enriched genes selected,
r(20) = 0.9082, P < 0.0001 (Fig. 4g); for egg-enriched
genes selected, r(21) = 0.8983, P < 0.0001 (Fig. 4h).
Putative functions predicted by GO analysis
We analysed the potential biological functions of the
stage-enriched genes in S. japonicum using GO
classification  (Fig. 5, Additional files 13, 14, 15 and 16:
Tables S11–S14). Of the biological process categories,
the most highly enriched GO terms were organic
substance metabolic process, single-organism cellular process,
primary metabolic process and cellular metabolic process
for cercariae, adult worms and eggs; the first three of these
GO terms and regulation of cellular process were the most
highly enriched GO terms for hepatic schistosomula. The
percentages of genes involved in regulation of cellular
process, cellular response to stimulus, and single organism
signaling were higher in cercariae and schistosomula than
those in adults and eggs. Of the molecular function
categories, the percentages of genes involved in ion,
heterocyclic compound and organic cyclic compound, small
molecule and carbohydrate derivative binding were higher
in cercariae and schistosomula than in adults and eggs. A
higher percentage of genes related to protein binding,
signaling receptor activity and receptor activity were
observed in schistosomula, while the GO term extracellular
matrix structural constituent was only evident for this
stage. In addition, a higher percentage of genes involved in
hydrolase activity were assigned to adult worms. In the
Fig. 2 Bias ratio and signal intensity analysis of stage-associated genes. Scatter plot showing the distribution of the bias ratio and fluorescence
intensity for mRNA transcripts enriched in cercariae (a), hepatic schistosomula (b), adult worms (c), and eggs (d). The y-axis corresponds to bias
ratios (FC value) and the x-axis corresponds to the fluorescence intensities, both of which are log10-transformed. Pie diagrams representing the
percentage of stage-enriched mRNA transcripts in cercariae (e), hepatic schistosomula (f), adult worms (g), and eggs (h) showed by different
cellular component categories, gene products localised to
intracellular, intracellular part and intracellular organelle
were more abundant in cercariae, while gene products
localised to intrinsic component of membrane were more
enriched in the other three stages. Further, genes with GO
terms of protein complex, cell periphery, plasma
membrane, plasma membrane part and proteinaceous
extracellular matrix were relatively enriched in hepatic
schistosomula. In addition, the GO term cilium was
present only in the egg stage.
The top 25 genes enriched in S. japonicum cercariae,
hepatic schistosomula, adult worms and eggs
The top 25 highly stage-associated genes in cercariae,
hepatic schistosomula, adult worms and eggs were analysed
(Table 1). Collectively, the upregulated expression of these
genes in cercariae indicates that signal transduction
(ribosomal protein S6 kinase beta-2 ), vesicular trafficking
(calcium-dependent secretion activator  and small
GTPase Rab-protein 11 ) and energy metabolism
(AMP deaminase  and 5′-AMP-activated protein
kinase ) and transcriptional regulation (krueppel-like
factor 11, homeobox protein SMOX-1, and retinoid X
receptor RXR-2) are active processes in this stage.
The over-expression of the top 25 genes in hepatic
schistosomula appears to reflect a diversity of
physiological activities, including transcriptional (homeobox
protein engrailed-like SMOX-2 [47, 48], serum and
glucocorticoid-regulated kinase 1 (SGK1)  and
nuclear receptor subfamily 4 group A [50, 51]) and
neuronal (protocadherin FAT4 , Aromatic-L-amino-acid
decarboxylase  and delphilin ) activities,
together with tegumental integrity (annexin A3 [55, 56]),
skeletal morphogenesis (protocadherin FAT4 ) and
endosome-to-Golgi retrieval (vacuolar protein
sortingassociated protein 29 ).
In mixed adult worms, genes encoding a number of
trematode eggshell synthesis (TES) domain-containing
proteins, DDR48 stress proteins, an asparagine-rich
antigen Pfa35-2, two distinct tyrosinase homologues, cadherin,
female-specific protein 800 and a prostatic
sperminebinding protein are listed in the top 25 enriched mRNA
transcripts (Table 1). Most of these genes are
femalebiased expressed genes  with potential molecular
functions in egg production .
In the egg stage, genes encoding a glutenin high
molecular weight subunit DX5, egg protein CP1531, two
histidine-rich glycoproteins, three ribonucleases, two
tetraspanins, three venom allergen-like (VAL) proteins and
cell wall integrity and stress response component 1 are
present in the top 25 upregulated mRNA transcripts
(Table 1). Notably, it has been shown that T2 ribonuclease
omega-1 in soluble egg antigen is a major Th2 polarizing
component, which is capable of regulating inflammasome
Fig. 3 Twenty stage-enriched genes selected for qPCR validation. The heat map illustrates the hierarchical clustering of 20 stage-enriched genes
based on the transformed data of log2 FC value of the three biological replicates. Abbreviations: C, cercariae; S, hepatic schistosomula; A, adult
worms; E, eggs
activity . It has been shown previously that VAL-5 is
mainly present in the egg, miracidium and sporocyst
developmental stages .
Genes enriched in cercariae
Interestingly, a group of genes encoding transcription
factors, i.e. homeobox protein SMOX-1 (AY915497),
bhlhzip transcription factor max/bigmax (FN314500),
pre-B-cell leukemia transcription factor 2 (AY809282),
transcription factor 25 (AY808969), 20 (AY813668),
BTF3 (EZ000130), TFIID subunit 3 (AY812404) and 7
(FN317813), IIIB subunit (AY812330), LIM/homeobox
protein (AY915618) and transcriptional repressor
NFX1(AY813973) were actively transcribed in cercariae
(Additional file 4: Table S3), indicating gene
transcription may not be as silent as previously suggested in this
stage. It has been shown that the highest ratio of
miRNAs, the critical post-transcriptional regulators, in the
total small RNA population was observed in cercariae
compared with other different developmental stages of
S. japonicum [32, 63], leading us to hypothesise that a
specific group of genes may be actively transcribed in
this aquatic stage. In addition, miRNAs may inhibit the
translation of a subset of these transcripts, forming a
repertoire of genes that make schistosomula ready to
adapt to subsequent intra-mammalian life. Further,
there is epigenetic control of gene expression in S.
mansoni cercariae . Overall, these observations
indicate that active transcriptional regulation occurs at
different layers in cercariae to subtly control gene
expression in this stage.
We also observed that an extensive gene panel
involved in cellular signalling transduction, i.e. F-box
protein 25/32 (EZ000162), dual specificity
mitogenactivated protein kinase 2 (AY815572),
Serine/threonine kinase NLK (FN317434), Rho GTPase-activating
protein 39 (FN317833), GDP/GTP exchange factor
Sec2p domain containing protein (FN317362),
Rhoassociated protein kinase 1 (FN330915), mitogen-activated
protein kinase 3 (EZ000180), Ran binding protein 9-related
protein (AY812647), GTP-binding protein 2 (FN317377),
NF-kappa-B inhibitor-interacting Ras-like protein 1
(AY812481), son of sevenless (AY915633), MAP kinase
(AY594257), C-Jun-amino-terminal kinase-interacting
protein 4 (AY808598), and regulator of G-protein signaling 7
(AY810841), were over-expressed in cercariae (Additional
file 4: Table S3). These results support recent finding that
three signaling pathways, extracellular signal-regulated
Fig. 4 qPCR validation of stage-enriched genes. The expression of 5 selected genes enriched in cercariae (a), hepatic schistosomula (b), adult
worms (c), and eggs (d), respectively, was quantified by qRT-PCR analysis. The PSMD4 gene was used for internal normalization among the four
developmental stages. The highest expression level in one particular stage was set as 1. The error bars represent the standard deviation for three
technical replicates. Correlations between the microarray and qPCR results for the selected genes enriched in cercariae (e), hepatic schistosomula
(f), adult worms (g), and eggs (h), were analysed using Spearman’s rho
kinase (ERK), p38 mitogen-activated protein kinase
(MAPK), and protein kinase C (PKC), are modulated in
cercariae in response to light and temperature cues as well
as the skin fatty acid linoleic acid (LA) and are important
in host penetration mechanisms .
In line with, and expanding on, previous transcriptional
studies on schistosomes [13, 14, 66], genes encoding an
array of cytoskeleton motor proteins, including dynein
light intermediate chain 1, cytosolic (AY809199),
troponins (FN317001 and AY809606), tensin-1 (AY809674),
villin (AY808977), myosin light chain kinase, actin-related
protein 5 (FN326677), dynamin (AY809889), catenin beta
(AY814842), coronin (AY814365), dynein light chain
Tctex-type 1 (AY811669) and alpha-actinin (FN326862)
(Additional file 4: Table S3) were more highly expressed in
cercariae than the other stages evaluated. Transcripts
encoding LIM or PDZ domain-containing proteins, which
contribute to cytoskeletal organisation, such as LIM/
homeobox protein (AY915618), actin binding LIM protein
1 (AY813306), four and a half LIM domains protein 2
(FN317368), and PDZ and LIM domain protein 7
(FN317962) (Additional file 4: Table S3), were also
enriched in cercariae. Proteomic studies also revealed that
cytoskeleton-related proteins are abundant in schistosome
cercariae . Together, these data indicate modulated
signalling and motor activities and rigid transcriptional
Fig. 5 GO analysis of mRNA transcripts enriched in the four developmental stages of S. japonicum. The Blast2Go program defined the GO terms
into three categories: biological processes (a), molecular functions (b) and cellular component (c). The y-axis shows the ratio of the number of
mapped genes versus total number of genes in each cognate stage identified as a function of all available GO terms. The x-axis shows GO terms
at the 3rd level
regulation are the most important biological events in
cercariae, which enable them to seek, invade and adapt to a
suitable definitive host.
Genes enriched in hepatic schistosomula
On invading a mammalian host, schistosomes have
evolved several mechanisms to adapt to, and survive in,
the hostile host environment; in particular, they develop
a unique syncytial tegument, as well as mechanisms of
antigenic mimicry , immune modulation  and
evasion [69, 70]. In this study, we found extracellular
matrix constituents, that are located in the tegumental
protein assemblage, were enriched in hepatic
schistosomula. These collagen components included, for example,
collagen alpha-1(V) chain (AY810683, AY811988, and
AY815998), alpha-1(IV) chain (AY809845), alpha-1(XXIV)
chain (AY814344), alpha-2(I) chain (AY810097, FN313634)
and alpha-2(V) chain (AY813923) (Additional file 5:
Table S4). This observation raises the possibility that
collagen components may form a protective barrier on
Table 1 The top 25 genes enriched in S. japonicum cercariae, hepatic schistosomula, mixed adult worms and eggs
NCBI Nucleotide NCBI Protein Annotation
Enriched in cercariae
AY811679.1 AAX27568.2 Tegumental antigen
AY812964.1 AAW24696.1 Lysophosphatidic acid phosphatase type 6
AY808793.1 AAX24682.2 Krueppel-like factor 11
AY814888.1 AAP06195.1 Hypothetical protein
AY915869.1 AAX31090.1 UPF0506 domain containing protein
AY811006.1 AAX26895.2 Putative sodium-dependent transporter
FN319257.1 CAX74986.1 Ribosomal protein S6 kinase beta-2
AY813254.1 CAX83692.1 Gag-Pol polyprotein
AY812158.1 AAX28047.2 Calcium-dependent secretion activator
FN327240.1 CAX82964.1 UPF0364 protein
FN319112.1 CAX74840.1 Anti-inflammatory protein 16
AY809199.1 AAX25088.2 Dynein light intermediate chain 1 cytosolic
AY815066.1 AAW26798.1 Calpain
FN314407.1 CAX70140.1 Rab-protein 11
AY813232.1 AAW24964.1 DM9 domain-containing protein
AY915497.1 AAX30718.2 Homeobox protein SMOX-1
AY813605 AAW25337.1 Hypothetical protein
FN319705.1 CAX75429.1 THO complex subunit 1
AY813585.1 AAW25317.1 Hypothetical protein
AY811834.1 AAX27723.2 AMP deaminase
AY813088.1 AAW24820.1 Hypothetical protein
FN314484 CAX70217.1 Hypothetical protein
AY811464.1 ABA40369.1 5′-AMP-activated protein kinase subunit gamma-1
EU046089.1 AAW25910.1 Cercarial stage-specific protein Sj20H8
AY808884.1 AF129816_1 Retinoid X receptor RXR-2
Enriched in hepatic schistosomula
AY809629.1 AAX25518.2 Hypothetical protein
AY810683 AAX26572.2 Putative collagen alpha-1(V) chain precursor
AY815366.1 AAW27592.1 Alpha-ketoglutarate-dependent dioxygenase alkB 6
AY813429.1 AAW25161.1 Hypothetical protein
AY810949.1 AAX26838.2 Homeobox protein engrailed-like SMOX-2
EZ000055.1 ACE06835.1 Vacuolar protein sorting-associated protein 29
AY810397.1 AAX26286.2 Protocadherin Fat 4
AY811075.1 AAX26964.2 Hypothetical protein
AY815532.1 AAW27264.1 Hypothetical protein
AY814356 AAW26088.1 RhoGAP domain containing protein
AY811025.1 AAX26914.2 Serine/threonine-protein kinase Sgk1
AY809477.1 AAX25366.2 SAM and SH3 domain-containing protein 1
FN314446.1 CAX70179.1 Annexin A3 (Annexin III)
AY814048.1 AAW25780.1 Basic proline-rich protein-like isoform
AY808501.1 AAR28090.2 Nuclear receptor subfamily 4 group A
AY809584.1 AAX25473.2 Hypothetical protein
AY812287.1 AAX28176.2 Run domain Beclin-1 interacting and cysteine-rich containing protein
Table 1 The top 25 genes enriched in S. japonicum cercariae, hepatic schistosomula, mixed adult worms and eggs (Continued)
AY813648.1 AAW25380.1 Hypothetical protein 3.439
AY915540.1 ABA40872.1 Leishmanolysin-like peptidase 3.419
AY812557.1 AAX28446.2 Aromatic-L-amino-acid decarboxylase 3.335
AY808377.1 AAX24266.2 Regulator of G-protein signaling 3 3.250
FN313634.1 CAX69368.1 Collagen alpha-2(I) chain 3.244
AY813683.1 AAW25415.1 Delphilin 3.240
AY812144.1 AAX28033.2 Hypothetical protein 3.212
AY813563 AAW25295.1 Hypothetical protein 3.203
Enriched in mixed adult worms
FN314868.1 CAX70600.1 Asparagine-rich antigen Pfa35-2
EZ000096 ACE06876.1 Putative eggshell protein precursor
FN314999 CAX70731.1 TES domain containing protein
AY813556.1 AAW25288.1 Hypothetical protein
AY814029 AAW25761.2 Stress protein DDR48 (DNA damage-responsive protein 48)
FN313935.1 CAX69669.1 Stress protein DDR48 (DNA damage-responsive protein 48)
FN317103 CAX72834.1 Stress protein DDR48 (DNA damage-responsive protein 48)
FN313912 CAX69646.1 TES domain containing protein
FN313715.1 CAX69449.1 TES domain containing protein
AY812810.1 AAW24542.1 Histidine-rich glycoprotein precursor
FN315504.1 CAX71236.1 TES domain containing protein
AY815518 AAW27250.1 TES domain containing protein
FN314997 CAX70729.1 TES domain containing protein
AY813405 AAW25137.1 TES domain containing protein
AY815264.1 AAW26996.1 Tyrosinase 1
AY812315.1 AAX28204.2 Hypothetical protein
FN330801 CAX83018.1 Stress protein DDR48 (DNA damage-responsive protein 48)
AY814142.1 AAW25874.1 Putative FAM75 family member
AY812904 AAW24636.1 Tyrosinase 2
FN315510.1 CAX71242.1 Hypothetical protein
AY814814 AAW26546.1 Cadherin
AY815418 AAW27150.1 Female-specific protein 800
FN316955 CAX72686.1 Prostatic spermine-binding protein precursor
AY222885 AAP05897.1 Stress protein DDR48 (DNA damage-responsive protein 48)
FN314903.1 CAX70635.1 Hypothetical protein
Enriched in eggs
FN317800 CAX73529.1 Glutenin high molecular weight subunit DX5
FN319280 CAX75008.1 Tetraspanin 22
FN322023.1 CAX77751.1 Histidine-rich glycoprotein
FN324495.1 CAX80219.1 Hypothetical protein
FN326817 CAX82541.1 Histidine-rich glycoprotein
FN317759.1 CAX73488.1 Similar to venom allergen-like (VAL) 25 protein
FN324480.1 CAX80126.1 Hypothetical protein
FN321785 CAX77509.1 Ribonuclease T2
FN321171.1 CAX76897.1 Hypothetical protein
FN324498.1 CAX80222.1 Hypothetical protein
Table 1 The top 25 genes enriched in S. japonicum cercariae, hepatic schistosomula, mixed adult worms and eggs (Continued)
CIA30 domain containing protein
Cell wall integrity and stress response component 1
GLIPR1-like protein 1/venom allergen-like protein 5
GLIPR1-like protein 1/venom allergen-like protein 5
the worm surface, which may help the schistosomula
evade host attack.
Schistosomula undertake a lengthy migration in the
mammalian host to the portal venous system, where
they mature into adult worms and pair. This migration
is closely associated with locomotion activity controlled
by the neuronal system. The data presented here show
that neuronal activities may be particularly active in
hepatic schistosomula, which could be linked to the fact that
responses to environmental cues from the host and the
subsequent control of mobility are required to guarantee
that they reach their destination . A cohort of genes
involved in neuronal activities in this stage includes
netrin receptor unc5B (AY915275), nephrin (AY809045),
caskin 2 (AY812623), spondin-1 (AY812421), as well as
the previously described genes protocadherin FAT4,
aromatic-L-amino-acid decarboxylase and delphilin.
Although the precise functions of these genes in
schistosomes remain unknown, there is evidence from other
studies that at least three are involved in axon guidance.
In mammals, it has been shown that the unc5B receptor,
interacting with netrin-1, activates the downstream
signal transduction pathway that mediates axon guidance
. A caskin ortholog in Drosophila is a cytoplasmic
adaptor protein, which has been shown to mediate Lar
signal transduction motor axon guidance . Similarly,
spondin-1 is an extracellular matrix protein, and
previous research showed that its C. elegans ortholog
functions in axon guidance and fasciculation in motoneurons
. Also, the expression of nephrin homologues has
been observed in the central nervous system of
mammals, and nephrin may potentially interact with
glutamate receptors [74, 75].
In multicellular organisms, apoptosis is a highly
controlled cellular process of programmed cell death which
plays a key role in maintaining cell populations during an
organism’s life-cycle. The apoptosis pathway has been
suggested as a potential intervention target in schistosomes
. The activities of two central proteolytic enzymes
involved in the apoptosis process, caspase-3 and -7, were
shown to peak in S. japonicum schistosomula (14 days
p.i.) . The upregulated expression of caspase 7
(AY813428) in hepatic schistosomula was confirmed in
this study (Additional file 5: Table S4). It is of note that a
cohort of planarian neoblast-like cells with self-renewal
function has been identified in S. mansoni, with a
potential role in renewal of the tegument . In this
respect, fibroblast growth factor receptor 2, a crucial
gene for the maintenance of neoblast-like cell
population in schistosomes , was enriched in hepatic
schistosomula (Additional file 5: Table S4), emphasising
the requirement for rapid tegumental renewal during
this period of fast-growth.
Genes enriched in adult worms
One of the major biological roles of adult worms is to
produce a large number of eggs, a key process in the
schistosome life-cycle. As earlier mentioned, within the top 25
adult-enriched genes, most are associated with egg
production. However, two pre-requisites for egg production
are mating and nutrient acquisition. In fulfilment of the
former process, the gene encoding gynecophoral canal
protein has been shown upregulated in adults, with a
dramatic bias towards male worms . In regards to
nutrient uptake, and consistent with a previous study ,
over-expression of a number of ‘blood processing’
proteases in adult worms was also revealed here. For
instance, cathepsin family members, i.e. cathepsin C
(FN315267), cathepsin D2-like (AY812817), cathepsin
Blike (AY814095), cathepsin L (FN313884) and cathepsin
L-like isoforms (AY222874, FN314782, and FN314778),
and aminopeptidase N (FN317672) were readily
identified as adult worm-enriched genes (Additional file 6:
Table S5). In addition, saposin B domain-containing
proteins (FN314931, FN315898 and FN314355), which
have been proposed as being involved in nutrient
acquisition by disrupting the membrane of red blood cells to
release haemoglobin , were highly expressed in
In schistosomes, glycosylation is a complex process
which plays a crucial role in their biology, particularly
in terms of immune modulation . A subset of
transcripts involved glycosylation in was enriched in adult
worms of S. japonicum. These genes included
beta1,4-galactosyltransferase 4 (AY813412),
glycosyltransferase 1 domain-containing protein 1 (FN319898),
GDP-fucose protein O-fucosyltransferase 2 (AY810860),
beta-1,3-galactosyltransferase 5 (AY814132),
glycoproteinN-acetylgalactosamine 3-beta-galactosyltransferase 1
(AY809881), glycoprotein 3-alpha-L-fucosyltransferase
A (FN317387), alpha-1,3-mannosyl-glycoprotein
2-betaN-acetylglucosaminyltransferase (AY812621), and
alphaL-fucosidase-like protein (FN317475) (Additional file 6:
Table S5). However, given the inherent complexity of
glycosylation and that multiple glycosyltransferases
responsible for similar molecular functions are present in
the Schistosoma genomes [81, 82], it is difficult to
conclude that the global level of glycosylation or the
expression of specific glycans is higher in adults than in the
other stages examined here.
Genes enriched in eggs
Globally, genes associated with the egg stage are involved
in a diversity of biological functions, which may be the
result of using samples for analysis that comprise a mixture
of immature and mature eggs. In addition to anticipated
genes encoding egg proteins, immunogenic miracidial
antigens and major egg antigens, a number of genes involved
in the cell cycle and proliferation, including meiosis
expressed protein 1 (FN317540), meiosis-specific nuclear
structural protein 1 (AY810474), mitogen-activated
protein kinase 15 (FN317209), putative chromosome
segregation protein SMC (AY812773), different isoforms of
leishmanolysin-like peptidase (AY811259, FN317512,
AY810562 and FN319863) and probably protein VHS3
(FN330961), placenta-specific gene 8 protein (FN317134),
placental protein 25 homolog (FN317187) and
centrosomal protein of 162 kDa (AY810094), were upregulated in
eggs (Additional file 7: Table S6). These transcripts may
be enriched in immature eggs, hinting that active cell
division is essential for embryonic development.
Further, a group of transcripts encoding tubulin and
microtubule-associated motor proteins, i.e. tubulin alpha
(FN317215), tubulin beta (FN320386), tubulin beta-2C
chain (FN320061), cytoplasmic dynein light chain 1
(FN317588) and 2 (AY914882), dynein light chain 1,
axonemal (FN317727), inner dynein arm light chain, axonemal
(FN317915), outer dynein arm protein 1 (AY813443),
dynein heavy chain 5, axonemal (AY810177), as well as the
ciliary and flagellar microtubule components, i.e. tektins
(AY814061, AY914954, FN317819 and FN314465), dynein
intermediate chain 3 (AY810742) and outer dense fibre
protein 3-B (FN318315) were over-expressed in eggs
(Additional file 7: Table S6). These transcriptional
differences may reflect the fact that a miracidium is
enclosed in the eggshell of the mature egg, and once
the egg is released into the external environment and
contacts freshwater, a high level of movement is
required for the larva to hatch and escape from the eggs
, and to seek the snail intermediate host in order to
establish an infection.
Though the miracidium is enclosed by an eggshell, an
active parasite-host interplay takes place via pores in
the egg . On one hand, nutrients (e.g. iron, amino
acid and lipid) are acquired by eggs from the host, a
process supported by the upregulation of genes
involved in transport and exchange activities, such as
putative sodium-dependent transporter (FN318875),
sodium/hydrogen exchanger (AY815720), sodium/calcium
exchanger (FN318247), large neutral amino acids
transporter small subunit 2 (FN327074), Y + L amino acid
transporter 2 (FN313722), high-affinity choline
transporter 1 (FN317430), iron channels (i.e. voltage-gated
hydrogen channel (FN318209), two pore calcium channel
protein 2 (FN326741), and TWiK family of potassium
channels protein (AY813707), and lipid metabolism (i.e.
fatty acid-binding protein (FN318753) (Additional file 7:
Table S6). On the other hand, it has been shown that
major egg products from S. mansoni such as ribonuclease
omega-1, kappa 5 (FN329842) and IPSE/alpha-1 are
released into host tissues and modulate host immune
responses [84–87]. In this study, S. japonicum homologues
of ribonuclease omega-1 (FN330952) and kappa 5
(FN321248) were also enriched in the egg stage, although
as yet, no homologue of IPSE/alpha-1 has been identified
in this schistosome species.
In this study, we present the most comprehensive
transcriptomic profile to date of four stage-associated genes
in S. japonicum based on a next-generation DNA chip.
The study has revealed the key biological and
physiological features of the four development stages: cercariae,
hepatic schistosomula, adult parasites and eggs. Overall,
this study adds new insights on the developmental
biology of S. japonicum which further the discovery of novel
intervention targets against this persistent parasite and
the disease it causes.
Additional file 1: Figure S1. Denaturing agarose gel electrophoresis of
RNA samples isolated from different developmental stages (1, cercariae; 2,
hepatic schistosomula; 3, adult worms; 4, eggs); one of three biological
replicates for each stage are presented. (TIF 82 kb)
Additional file 3: Table S2. Retrieval of S. japonicum stage-enriched
genes from the NCBI database based on the DNA chip results. (XLSX 9 kb)
Additional file 4: Table S3. Information on mRNA transcripts enriched
in cercariae (forward probe). (XLSX 188 kb)
Additional file 5: Table S4. Information on mRNA transcripts enriched
in hepatic schistosomula (forward probe). (XLSX 64 kb)
Additional file 6: Table S5. Information on mRNA transcripts enriched
in mixed adult worms (forward probe). (XLSX 150 kb)
Additional file 7: Table S6. Information on mRNA transcripts enriched
in eggs (forward probe). (XLSX 287 kb)
Additional file 8: Table S7. Information on EST transcripts enriched in
cercariae (forward probe). (XLSX 81 kb)
Additional file 9: Table S8. Information on EST transcripts enriched in
hepatic schistosomula (forward probe). (XLSX 25 kb)
Additional file 10: Table S9. Information on EST transcripts enriched in
mixed adult worms (forward probe). (XLSX 51 kb)
Additional file 11: Table S10. Information on EST transcripts enriched
in eggs (forward probe). (XLSX 51 kb)
Additional file 12: Figure S2. Heatmap for EST transcripts enriched in
cercariae, hepatic schistosomula, adult worms and eggs. The heatmap
was created by HemI 1.0 based on the transformed data of log2 FC value.
The data are based on the mean of weighted signal intensity value of
forward probe sets (three biological replicates). (TIF 110 kb)
Additional file 13: Table S11. Detailed GO annotation for mRNA
transcripts enriched in cercariae. (XLSX 26 kb)
Additional file 14: Table S12. Detailed GO annotation for mRNA
transcripts enriched in hepatic schistosomula. (XLSX 18 kb)
Additional file 15: Table S13. Detailed GO annotation for mRNA
transcripts enriched in mixed adult worms. (XLSX 22 kb)
Additional file 16: Table S14. Detailed GO annotation for mRNA
transcripts enriched in eggs. (XLSX 24 kb)
CDS: Coding DNA sequences; DALYs: Disability adjusted life years; DGE: Digital
gene expression; ERK: Extracellular signal-regulated kinase; ESTs: Expressed
sequence tags; FC: Fold-changes; LAPs: Hydrolysis of lysophosphatidic acids;
MAPK: Mitogen-activated protein kinase; PKC: Protein kinase C; SAGE: Serial
analysis of gene expression; TES: Trematode eggshell synthesis;
UTRs: Untranslated regions; VAL: Venom allergen-like
This study was supported by the National Natural Science Foundation of
China (Grant No. 81270026), the National S & T Major Program (Grant No.
2012ZX10004-220), the Special Fund for Health Research in the Public Interest
(Grant No. 201202019), and the Program for Changjiang Scholars and
Innovative Research Team in University (IRT13007). DPM is a NHMRC Senior
Principal Research Fellow and Senior Scientist at QIMR Berghofer Medical
Research Institute. The funders had no role in study design, data collection and
analysis, decision to publish, or preparation of the manuscript.
Availability of data and materials
Raw data and the normalized data have been deposited at the public
domain Gene Expression Omnibus under the accession number for the
platform GPL18617 and series GSE57143.
PC and QC conceived the project and designed the strategy. PC, SL, XP and
NH carried out the experiments. PC, SL, DPM and QC analysed the data. PC,
HY, DPM and QC wrote the manuscript. All authors read and approved the
Ethics approval and consent to participate
All procedures performed on animals within this study were conducted
following animal husbandry guidelines of the Chinese Academy of Medical
Sciences and with permission from the Experimental Animal Committee
(Institute of Pathogen Biology, CAMS) with Ethical Clearance Number
1. Steinmann P , Keiser J , Bos R , Tanner M , Utzinger J. Schistosomiasis and water resources development: systematic review, meta-analysis, and estimates of people at risk . Lancet Infect Dis . 2006 ; 6 : 411 - 25 .
2. Weerakoon KG , Gobert GN , Cai P , McManus DP . Advances in the diagnosis of human schistosomiasis . Clin Microbiol Rev . 2015 ; 28 : 939 - 67 .
3. Hotez PJ , Alvarado M , Basanez MG , Bolliger I , Bourne R , Boussinesq M , et al. The global burden of disease study 2010: interpretation and implications for the neglected tropical diseases . PLoS Negl Trop Dis . 2014 ; 8 : e2865 .
4. Mutapi F , Rujeni N , Bourke C , Mitchell K , Appleby L , Nausch N , et al. Schistosoma haematobium treatment in 1-5 year old children: safety and efficacy of the antihelminthic drug praziquantel . PLoS Negl Trop Dis . 2011 ; 5 : e1143 .
5. Chen MG . Assessment of morbidity due to Schistosoma japonicum infection in China . Infect Dis Poverty . 2014 ; 3 : 6 .
6. Cai P , Gobert GN , You H , McManus DP . The Tao survivorship of schistosomes: implications for schistosomiasis control . Int J Parasitol . 2016 ; 46 : 453 - 63 .
7. Hu W , Yan Q , Shen DK , Liu F , Zhu ZD , Song HD , et al. Evolutionary and biomedical implications of a Schistosoma japonicum complementary DNA resource . Nat Genet . 2003 ; 35 : 139 - 47 .
8. Verjovski-Almeida S , DeMarco R , Martins EA , Guimaraes PE , Ojopi EP , Paquola AC , et al. Transcriptome analysis of the acoelomate human parasite Schistosoma mansoni . Nat Genet . 2003 ; 35 : 148 - 57 .
9. Berriman M , Haas BJ , LoVerde PT , Wilson RA , Dillon GP , Cerqueira GC , et al. The genome of the blood fluke Schistosoma mansoni . Nature . 2009 ; 460 : 352 - 8 .
10. The Schistosoma japonicum Genome Sequencing and Functional Analysis Consortium. The Schistosoma japonicum genome reveals features of hostparasite interplay . Nature . 2009 ; 460 : 345 - 51 .
11. Young ND , Jex AR , Li B , Liu S , Yang L , Xiong Z , et al. Whole-genome sequence of Schistosoma haematobium . Nat Genet . 2012 ; 44 : 221 - 5 .
12. Waisberg M , Lobo FP , Cerqueira GC , Passos LK , Carvalho OS , Franco GR , et al. Microarray analysis of gene expression induced by sexual contact in Schistosoma mansoni . BMC Genomics . 2007 ; 8 : 181 .
13. Jolly ER , Chin CS , Miller S , Bahgat MM , Lim KC , DeRisi J , et al. Gene expression patterns during adaptation of a helminth parasite to different environmental niches . Genome Biol . 2007 ; 8 : R65 .
14. Gobert GN , Moertel L , Brindley PJ , McManus DP . Developmental gene expression profiles of the human pathogen Schistosoma japonicum . BMC Genomics . 2009 ; 10 : 128 .
15. Chai M , McManus DP , McInnes R , Moertel L , Tran M , Loukas A , et al. Transcriptome profiling of lung schistosomula, in vitro cultured schistosomula and adult Schistosoma japonicum . Cell Mol Life Sci . 2006 ; 63 : 919 - 29 .
16. Moertel L , McManus DP , Piva TJ , Young L , McInnes RL , Gobert GN . Oligonucleotide microarray analysis of strain- and gender-associated gene expression in the human blood fluke, Schistosoma japonicum . Mol Cell Probes . 2006 ; 20 : 280 - 9 .
17. Fitzpatrick JM , Johansen MV , Johnston DA , Dunne DW , Hoffmann KF . Gender-associated gene expression in two related strains of Schistosoma japonicum . Mol Biochem Parasitol . 2004 ; 136 : 191 - 209 .
18. Parker-Manuel SJ , Ivens AC , Dillon GP , Wilson RA . Gene expression patterns in larval Schistosoma mansoni associated with infection of the mammalian host . PLoS Negl Trop Dis . 2011 ; 5 : e1274 .
19. Ojopi EP , Oliveira PS , Nunes DN , Paquola A , DeMarco R , Gregorio SP , et al. A quantitative view of the transcriptome of Schistosoma mansoni adultworms using SAGE . BMC Genomics . 2007 ; 8 : 186 .
20. Williams DL , Sayed AA , Bernier J , Birkeland SR , Cipriano MJ , Papa AR , et al. Profiling Schistosoma mansoni development using serial analysis of gene expression (SAGE) . Exp Parasitol . 2007 ; 117 : 246 - 58 .
21. Cogswell AA , Kommer VP , Williams DL . Transcriptional analysis of a unique set of genes involved in Schistosoma mansoni female reproductive biology . PLoS Negl Trop Dis . 2012 ; 6 : e1907 .
22. Piao X , Cai P , Liu S , Hou N , Hao L , Yang F , et al. Global expression analysis revealed novel gender-specific gene expression features in the blood fluke parasite Schistosoma japonicum . PLoS One . 2011 ; 6 : e18267 .
23. Anderson L , Amaral MS , Beckedorff F , Silva LF , Dazzani B , Oliveira KC , et al. Schistosoma mansoni egg, adult male and female comparative gene expression analysis and identification of novel genes by RNA-Seq . PLoS Negl Trop Dis . 2015 ; 9 : e0004334 .
24. Picard MA , Boissier J , Roquis D , Grunau C , Allienne JF , Duval D , et al. Sex-biased transcriptome of Schistosoma mansoni: host-parasite interaction, genetic determinants and epigenetic regulators are associated with sexual differentiation . PLoS Negl Trop Dis . 2016 ; 10 :e0004930.
25. Jones MK , Higgins T , Stenzel DJ , Gobert GN . Towards tissue specific transcriptomics and expression pattern analysis in schistosomes using laser microdissection microscopy . Exp Parasitol . 2007 ; 117 : 259 - 66 .
26. Gobert GN , McManus DP , Nawaratna S , Moertel L , Mulvenna J , Jones MK . Tissue specific profiling of females of Schistosoma japonicum by integrated laser microdissection microscopy and microarray analysis . PLoS Negl Trop Dis . 2009 ; 3 : e469 .
27. Nawaratna SS , McManus DP , Moertel L , Gobert GN , Jones MK . Gene Atlasing of digestive and reproductive tissues in Schistosoma mansoni . PLoS Negl Trop Dis . 2011 ; 5 : e1043 .
28. Fitzpatrick JM , Peak E , Perally S , Chalmers IW , Barrett J , Yoshino TP , et al. Anti-schistosomal intervention targets identified by life-cycle transcriptomic analyses . PLoS Negl Trop Dis . 2009 ; 3 : e543 .
29. Liu S , Cai P , Hou N , Piao X , Wang H , Hung T , et al. Genome-wide identification and characterization of a panel of house-keeping genes in Schistosoma japonicum . Mol Biochem Parasitol . 2012 ; 182 : 75 - 82 .
30. Liu S , Cai P , Piao X , Hou N , Zhou X , Wu C , et al. Expression profile of the Schistosoma japonicum degradome reveals differential protease expression patterns and potential anti-schistosomal intervention targets . PLoS Comput Biol . 2014 ; 10 :e1003856.
31. Liu S , Zhou X , Piao X , Wu C , Hou N , Chen Q. Comparative analysis of transcriptional profiles of adult Schistosoma japonicum from different laboratory animals and the natural host, water buffalo . PLoS Negl Trop Dis . 2015 ; 9 : e0003993 .
32. Cai P , Piao X , Hao L , Liu S , Hou N , Wang H , et al. A deep analysis of the small noncoding RNA population in Schistosoma japonicum eggs . PLoS One . 2013 ; 8 : e64003 .
33. Wu C , Hou N , Piao X , Liu S , Cai P , Xiao Y , et al. Non-immune immunoglobulins shield Schistosoma japonicum from host immunorecognition . Sci Rep . 2015 ; 5 : 13434 .
34. Li DD , Zhao LX , Mylonakis E , Hu GH , Zou Y , Huang TK , et al. In vitro and in vivo activities of pterostilbene against Candida albicans biofilms . Antimicrob Agents Chemother . 2014 ; 58 : 2344 - 55 .
35. Irizarry RA , Hobbs B , Collin F , Beazer-Barclay YD , Antonellis KJ , Scherf U , et al. Exploration , normalization, and summaries of high density oligonucleotide array probe level data . Biostatistics . 2003 ; 4 : 249 - 64 .
36. Irizarry RA , Bolstad BM , Collin F , Cope LM , Hobbs B , Speed TP . Summaries of Affymetrix GeneChip probe level data . Nucleic Acids Res . 2003 ; 31 :e15.
37. Deng W , Wang Y , Liu Z , Cheng H , Xue Y. HemI: a toolkit for illustrating heatmaps . PLoS One . 2014 ; 9 : e111988 .
38. Gotz S , Garcia-Gomez JM , Terol J , Williams TD , Nagaraj SH , Nueda MJ , et al. High-throughput functional annotation and data mining with the Blast2GO suite . Nucleic Acids Res . 2008 ; 36 : 3420 - 35 .
39. Marchler-Bauer A , Derbyshire MK , Gonzales NR , Lu S , Chitsaz F , Geer LY , et al. CDD: NCBI's conserved domain database . Nucleic Acids Res . 2015 ; 43 : D222 - 6 .
40. Cai P , Piao X , Hou N , Liu S , Wang H , Chen Q. Identification and characterization of argonaute protein, Ago2 and its associated small RNAs in Schistosoma japonicum . PLoS Negl Trop Dis . 2012 ; 6 : e1745 .
41. Ashburner M , Ball CA , Blake JA , Botstein D , Butler H , Cherry JM , et al. Gene ontology: tool for the unification of biology. The Gene Ontology Consortium . Nat Genet . 2000 ; 25 : 25 - 9 .
42. Valovka T , Verdier F , Cramer R , Zhyvoloup A , Fenton T , Rebholz H , et al. Protein kinase C phosphorylates ribosomal protein S6 kinase betaII and regulates its subcellular localization . Mol Cell Biol . 2003 ; 23 : 852 - 63 .
43. Daily NJ , Boswell KL , James DJ , Martin TF . Novel interactions of CAPS (Ca2 +-dependent activator protein for secretion) with the three neuronal SNARE proteins required for vesicle fusion . J Biol Chem . 2010 ; 285 : 35320 - 9 .
44. Ligasova A , Bulantova J , Sebesta O , Kasny M , Koberna K , Mikes L. Secretory glands in cercaria of the neuropathogenic schistosome Trichobilharzia regenti - ultrastructural characterization, 3-D modelling, volume and pH estimations . Parasit Vectors . 2011 ; 4 : 162 .
45. Morisaki T , Gross M , Morisaki H , Pongratz D , Zollner N , Holmes EW . Molecular basis of AMP deaminase deficiency in skeletal muscle . Proc Natl Acad Sci U S A . 1992 ; 89 : 6457 - 61 .
46. Neumann D , Woods A , Carling D , Wallimann T , Schlattner U. Mammalian AMP-activated protein kinase: functional, heterotrimeric complexes by coexpression of subunits in Escherichia coli . Protein Expr Purif . 2003 ; 30 : 230 - 7 .
47. Morgan R. Engrailed : complexity and economy of a multi-functional transcription factor . FEBS Lett . 2006 ; 580 : 2531 - 3 .
48. Webster PJ , Mansour TE . Conserved classes of homeodomains in Schistosoma mansoni, an early bilateral metazoan . Mech Dev . 1992 ; 38 : 25 - 32 .
49. Mansley MK , Watt GB , Francis SL , Walker DJ , Land SC , Bailey MA , et al. Dexamethasone and insulin activate serum and glucocorticoid-inducible kinase 1 (SGK1) via different molecular mechanisms in cortical collecting duct cells . Physiol Rep . 2016 ; 4 : e12792 .
50. Wu W , LoVerde PT . Nuclear hormone receptors in parasitic helminths . Mol Cell Endocrinol . 2011 ; 334 : 56 - 66 .
51. Wu W , Loverde PT . Schistosoma mansoni: identification of SmNR4A, a member of nuclear receptor subfamily 4 . Exp Parasitol . 2008 ; 120 : 208 - 13 .
52. Badouel C , Zander MA , Liscio N , Bagherie-Lachidan M , Sopko R , Coyaud E , et al. Fat1 interacts with Fat4 to regulate neural tube closure, neural progenitor proliferation and apical constriction during mouse brain development . Development . 2015 ; 142 : 2781 - 91 .
53. Brun L , Ngu LH , Keng WT , Ch'ng GS , Choy YS , Hwu WL , et al. Clinical and biochemical features of aromatic L-amino acid decarboxylase deficiency . Neurology . 2010 ; 75 : 64 - 71 .
54. Miyagi Y , Yamashita T , Fukaya M , Sonoda T , Okuno T , Yamada K , et al. Delphilin: a novel PDZ and formin homology domain-containing protein that synaptically colocalizes and interacts with glutamate receptor delta 2 subunit . J Neurosci . 2002 ; 22 : 803 - 14 .
55. Tararam CA , Farias LP , Wilson RA , Leite LC . Schistosoma mansoni Annexin 2: molecular characterization and immunolocalization . Exp Parasitol . 2010 ; 126 : 146 - 55 .
56. Leow CY , Willis C , Osman A , Mason L , Simon A , Smith BJ , et al. Crystal structure and immunological properties of the first annexin from Schistosoma mansoni: insights into the structural integrity of the schistosomal tegument . FEBS J . 2014 ; 281 : 1209 - 25 .
57. Mao Y , Kuta A , Crespo-Enriquez I , Whiting D , Martin T , Mulvaney J , et al. Dchs1-Fat4 regulation of polarized cell behaviours during skeletal morphogenesis . Nat Commun . 2016 ; 7 : 11469 .
58. Fuse A , Furuya N , Kakuta S , Inose A , Sato M , Koike M , et al. VPS29-VPS35 intermediate of retromer is stable and may be involved in the retromer complex assembly process . FEBS Lett . 2015 ; 589 : 1430 - 6 .
59. Cai P , Liu S , Piao X , Hou N , Gobert GN , McManus DP , et al. Comprehensive transcriptome analysis of sex-biased expressed genes reveals discrete biological and physiological features of male and female Schistosoma japonicum . PLoS Negl Trop Dis . 2016 ; 10 :e0004684.
60. Fitzpatrick JM , Hirai Y , Hirai H , Hoffmann KF. Schistosome egg production is dependent upon the activities of two developmentally regulated tyrosinases . FASEB J . 2007 ; 21 : 823 - 35 .
61. Ferguson BJ , Newland SA , Gibbs SE . Tourlomousis P , Fernandes dos Santos P , Patel MN , et al. The Schistosoma mansoni T2 ribonuclease omega-1 modulates inflammasome-dependent IL-1beta secretion in macrophages . Int J Parasitol . 2015 ; 45 : 809 - 13 .
62. Chalmers IW , McArdle AJ , Coulson RM , Wagner MA , Schmid R , Hirai H , et al. Developmentally regulated expression, alternative splicing and distinct subgroupings in members of the Schistosoma mansoni venom allergen-like (SmVAL) gene family . BMC Genomics . 2008 ; 9 : 89 .
63. Cai P , Hou N , Piao X , Liu S , Liu H , Yang F , et al. Profiles of small non-coding RNAs in Schistosoma japonicum during development . PLoS Negl Trop Dis . 2011 ; 5 : e1256 .
64. Roquis D , Lepesant JM , Picard MA , Freitag M , Parrinello H , Groth M , et al. The epigenome of Schistosoma mansoni provides insight about how cercariae poise transcription until infection . PLoS Negl Trop Dis . 2015 ; 9 : e0003853 .
65. Ressurreicao M , Kirk RS , Rollinson D , Emery AM , Page NM , Walker AJ . Sensory protein kinase signaling in Schistosoma mansoni cercariae: host location and invasion . J Infect Dis . 2015 ; 212 : 1787 - 97 .
66. Han ZG , Brindley PJ , Wang SY , Chen Z. Schistosoma genomics: new perspectives on schistosome biology and host-parasite interaction . Annu Rev Genomics Hum Genet . 2009 ; 10 : 211 - 40 .
67. Liu M , Ju C , Du XF , Shen HM , Wang JP , Li J , et al. Proteomic analysis on cercariae and schistosomula in reference to potential proteases involved in host invasion of Schistosoma japonicum larvae . J Proteome Res . 2015 ; 14 : 4623 - 34 .
68. Jenkins SJ , Hewitson JP , Jenkins GR , Mountford AP . Modulation of the host's immune response by schistosome larvae . Parasite Immunol . 2005 ; 27 : 385 - 93 .
69. Cai P , Bu L , Wang J , Wang Z , Zhong X , Wang H. Molecular characterization of Schistosoma japonicum tegument protein tetraspanin-2: sequence variation and possible implications for immune evasion . Biochem Biophys Res Commun . 2008 ; 372 : 197 - 202 .
70. Zhang W , Li J , Duke M , Jones MK , Kuang L , Zhang J , et al. Inconsistent protective efficacy and marked polymorphism limits the value of Schistosoma japonicum tetraspanin-2 as a vaccine target . PLoS Negl Trop Dis . 2011 ; 5 : e1166 .
71. Bradford D , Cole SJ , Cooper HM . Netrin-1: diversity in development . Int J Biochem Cell Biol . 2009 ; 41 : 487 - 93 .
72. Weng YL , Liu N , DiAntonio A , Broihier HT . The cytoplasmic adaptor protein Caskin mediates Lar signal transduction during Drosophila motor axon guidance . J Neurosci . 2011 ; 31 : 4421 - 33 .
73. Woo WM , Berry EC , Hudson ML , Swale RE , Goncharov A , Chisholm AD . The C. elegans F-spondin family protein SPON-1 maintains cell adhesion in neural and non-neural tissues . Development . 2008 ; 135 : 2747 - 56 .
74. Li M , Armelloni S , Ikehata M , Corbelli A , Pesaresi M , Calvaresi N , et al. Nephrin expression in adult rodent central nervous system and its interaction with glutamate receptors . J Pathol . 2011 ; 225 : 118 - 28 .
75. Putaala H , Soininen R , Kilpelainen P , Wartiovaara J , Tryggvason K. The murine nephrin gene is specifically expressed in kidney, brain and pancreas: inactivation of the gene leads to massive proteinuria and neonatal death . Hum Mol Genet . 2001 ; 10 : 1 - 8 .
76. Lee EF , Young ND , Lim NT , Gasser RB , Fairlie WD . Apoptosis in schistosomes: toward novel targets for the treatment of schistosomiasis . Trends Parasitol . 2014 ; 30 : 75 - 84 .
77. Han H , Peng J , Gobert GN , Hong Y , Zhang M , Han Y , et al. Apoptosis phenomenon in the schistosomulum and adult worm life cycle stages of Schistosoma japonicum . Parasitol Int . 2013 ; 62 : 100 - 8 .
78. Pearson MS , Loukas A. The parasite's new clothes . Elife . 2016 ; 5 : e12473 .
79. Collins 3rd JJ , Wang B , Lambrus BG , Tharp ME , Iyer H , Newmark PA. Adult somatic stem cells in the human parasite Schistosoma mansoni . Nature . 2013 ; 494 : 476 - 9 .
80. Don TA , Bethony JM , Loukas A. Saposin-like proteins are expressed in the gastrodermis of Schistosoma mansoni and are immunogenic in natural infections . Int J Infect Dis . 2008 ; 12 : e39 - 47 .
81. Mickum ML , Prasanphanich NS , Heimburg-Molinaro J , Leon KE , Cummings RD . Deciphering the glycogenome of schistosomes . Front Genet . 2014 ; 5 : 262 .
82. Smit CH , van Diepen A , Nguyen DL , Wuhrer M , Hoffmann KF , Deelder AM , et al. Glycomic analysis of life stages of the human parasite Schistosoma mansoni reveals developmental expression profiles of functional and antigenic glycan motifs . Mol Cell Proteomics . 2015 ; 14 : 1750 - 69 .
83. Jones MK , Bong SH , Green KM , Holmes P , Duke M , Loukas A , et al. Correlative and dynamic imaging of the hatching biology of Schistosoma japonicum from eggs prepared by high pressure freezing . PLoS Negl Trop Dis . 2008 ; 2 : e334 .
84. Steinfelder S , Andersen JF , Cannons JL , Feng CG , Joshi M , Dwyer D , et al. The major component in schistosome eggs responsible for conditioning dendritic cells for Th2 polarization is a T2 ribonuclease (omega-1) . J Exp Med . 2009 ; 206 : 1681 - 90 .
85. Meyer NH , Mayerhofer H , Tripsianes K , Blindow S , Barths D , Mewes A , et al. A Crystallin fold in the Interleukin-4-inducing principle of Schistosoma mansoni eggs (IPSE/alpha-1) mediates IgE binding for antigen-independent basophil activation . J Biol Chem . 2015 ; 290 : 22111 - 26 .
86. Schramm G , Mohrs K , Wodrich M , Doenhoff MJ , Pearce EJ , Haas H , et al. Cutting edge: IPSE/alpha-1, a glycoprotein from Schistosoma mansoni eggs, induces IgE-dependent, antigen-independent IL-4 production by murine basophils in vivo . J Immunol . 2007 ; 178 : 6023 - 7 .
87. Schramm G , Hamilton JV , Balog CI , Wuhrer M , Gronow A , Beckmann S , et al. Molecular characterisation of kappa-5, a major antigenic glycoprotein from Schistosoma mansoni eggs . Mol Biochem Parasitol . 2009 ; 166 : 4 - 14 .