Developmental gene expression profiles of the human pathogen Schistosoma japonicum
Developmental gene expression profiles of the human pathogen Schistosoma japonicum
Geoffrey N Gobert 1
Luke Moertel 1
Paul J Brindley 0
Donald P McManus 1
0 Department of Microbiology, Immunology & Tropical Medicine, George Washington University Medical Center , Washington, DC 20037 , USA
1 Division of Infectious Diseases & Immunology, Queensland Institute of Medical Research , Brisbane, Queensland 4029 , Australia
Background: The schistosome blood flukes are complex trematodes and cause a chronic parasitic disease of significant public health importance worldwide, schistosomiasis. Their life cycle is characterised by distinct parasitic and free-living phases involving mammalian and snail hosts and freshwater. Microarray analysis was used to profile developmental gene expression in the Asian species, Schistosoma japonicum. Total RNAs were isolated from the three distinct environmental phases of the lifecycle - aquatic/snail (eggs, miracidia, sporocysts, cercariae), juvenile (lung schistosomula and paired but pre-egg laying adults) and adult (paired, mature males and eggproducing females, both examined separately). Advanced analyses including ANOVA, principal component analysis, and hierarchal clustering provided a global synopsis of gene expression relationships among the different developmental stages of the schistosome parasite. Results: Gene expression profiles were linked to the major environmental settings through which the developmental stages of the fluke have to adapt during the course of its life cycle. Gene ontologies of the differentially expressed genes revealed a wide range of functions and processes. In addition, stage-specific, differentially expressed genes were identified that were involved in numerous biological pathways and functions including calcium signalling, sphingolipid metabolism and parasite defence. Conclusion: The findings provide a comprehensive database of gene expression in an important human pathogen, including transcriptional changes in genes involved in evasion of the host immune response, nutrient acquisition, energy production, calcium signalling, sphingolipid metabolism, egg production and tegumental function during development. This resource should help facilitate the identification and prioritization of new anti-schistosome drug and vaccine targets for the control of schistosomiasis.
Schistosomiasis afflicts ~200 million people in 76
countries . The disease is caused by infection with blood
flukes of the genus Schistosoma and depending on the
invading species, is characterised clinically by chronic
hepatic and intestinal fibrosis, portal hypertension,
anaemia and calcification of the urinary tract. The parasitic
worms have a complex developmental cycle that involves
infection of freshwater intermediate molluscan hosts and
the blood stream of mammals (Figure 1). Schistosome
infection results from direct contact with fresh water
contaminated by free-swimming larval forms of the parasite
known as cercariae. Cercariae penetrate human skin, shed
their tails, releasing schistosomula which enter capillaries
and lymphatic vessels en route to the lungs. After several
days, the male and female juvenile worms migrate to the
portal venous system, where they mature and unite. Adult
worm pairs then migrate to the veins of the intestines, in
the case of Schistosoma mansoni and S. japonicum, or the
bladder with S. haematobium. Egg production commences
four to six weeks after infection and continues for the life
of the worm – usually three to five years. Eggs pass from
the lumen of blood vessels into adjacent tissues, and
many then pass through the intestinal or bladder mucosa
and are shed with the faeces or urine. The life cycle is
completed when the eggs hatch, releasing miracidia that, in
turn, infect specific freshwater snails. After two asexual
generations within the snail, mother followed by
daughter sporocysts, cercariae are released.
Schistosomes undergo dramatic changes in gross
morphology and cellular composition and specialization
during their transition from free-swimming cercariae in
freshwater to mature adult worms, which reside in the
ostensibly inimical environment of the mammalian host
blood stream . In previous microarray studies, several
FThigeucroem1plex lifecycle of Schistosoma japonicum involves distinct free-living and parasitic stages (see text for details)
The complex lifecycle of Schistosoma japonicum involves distinct free-living and parasitic stages (see text for
details). The numbers indicate the seven developmental stages investigated by microarray and real time PCR analysis.
hundred cDNAs from S. japonicum and S. mansoni [3,4] or
a few thousand  oligonucleotides based on S. mansoni
sequences, were used to identify sex-, developmental
stage- and strain-specific genes, providing a clear
indication of the value and power of microarray analysis for
studying the biology of schistosomes. A subsequent study
 examined 3,088 contigs or singletons across seven life
cycle stages of S. mansoni early liver worms, adult worms,
eggs, daughter sporocysts, cercariae, and day 2 and day 7
schistosomula produced by mechanical transformation of
cercariae followed by in vitro culture. A new 22,575 feature
60-mer microarray was deployed to investigate gene
expression patterns between and within discrete Chinese
and Philippine strains of S. japonicum  and to
demonstrate stage-associated gene expression between mature
adult worms and lung schistosomula from amplified S.
japonicum mRNAs . Recently,  investigated gene
expression profiles in cercariae, sporocysts, mechanically
transformed schistosomula and paired male and female
adult worms of S. mansoni. Other methods have also been
successfully employed to investigate gene expression
changes in schistosomes, including the use of serial
analysis of gene expression (SAGE) to study S. mansoni . In
the present study, we deployed a custom designed
oligonucleotide microarray to profile gene expression
throughout the development of S. japonicum, the Asian blood
fluke, with hybridizations of RNAs from seven
developmental stages of the parasite – lung stage schistosomula,
4-wk-old immature female and male worms, sexually
mature male and female worms, eggs, miracidia,
sporocysts and cercariae. The findings of this study establish
baseline transcriptional information of the
developmental biology of this important human pathogen, and
identified stage-specific, differentially expressed genes
involved in numerous biological pathways and functions
including calcium signalling, sphingolipid metabolism
and parasite defence.
Filtering of microarray data to identify stage enriched
All microarray hybridisations were performed in triplicate
with normalisations and filtering of data performed using
GeneSpring GX. Normalised data were filtered for each
replicated data point for each of the 38,444 probes
(19,222 contigs) on the schistosome microarray
(Additional file 1). Data points were filtered to preserve signals
that were flagged during the feature extraction process as
Present or Marginal in all hybridisations; this resulted in
the retention of 7,132 probes (4,371 contigs). Next,
ANOVA applied to the data identified 6,465 probes
(4,104 contigs) differentially expressed due to
transcriptional changes throughout the S. japonicum lifecycle.
A final cut-off was applied to the microarray data
generating lists of genes that were ≥ 2 fold (relative to the median
intensity) in each lifecycle stage; these combined lists
consist of 4,443 probes/3263 genes represent stage-enriched
gene transcripts. These lists comprised of a subset of 1,782
unique genes or contigs, and were subjected to further
annotation or in silico characterisation. The numbers of 2
fold or 5 fold or higher enriched genes for each individual
developmental stage or lifecycle grouping is summarised
in Table 1 (individual lists are available as Additional file
2). Examples of differentially expressed genes for each
lifecycle stage are presented in Table 2.
Hierarchal clustering to provide overview of
Hierarchal clustering was employed to investigate gene
expression profiles in each developmental stage. Figure 2
is a heat-map that presents the hierarchal clustering
performed on genes, identified by ANOVA, with clustering
for both gene and developmental stages; in Figure 2,
upregulation is shown in red, down-regulation in green,
whereas the black lines indicate absence of up- or
downregulation. As could be predicted, based on the marked
changes in morphology and the discrete environmental
changes encountered by the different developmental
stages, there were clear differences in transcription (up or
down regulation) among the ~6,500 probes examined.
For example, considerable enrichment of gene expression
was evident in the egg compared with the miracidial stage
(Figure 2; Table 1). Among the developmental stages, four
prominent clusters of up-regulated genes were evident
these were in (1) aquatic and snail stages, (2) adult female
worms [with corresponding down-regulation in males],
(3) adult female worms [with no down-regulation in
males], and (4) adult males.
Blast2Go and gene ontology analysis to provide overview
of functions and process
The 1,782 genes that were identified as being enriched 2
fold in any given lifecycle stage were subjected to protein
translational blast and gene ontology (GO) batch
analysis. This presented a further overview of the gene
ontologies that are modulated during the S. japonicum lifecycle;
two of the three major categories, Biological Process and
Molecular Function, are presented in Additional file 3.
This information was used to supplement the already
known GOs based on nucleotide sequence previously
published . To gain a more complete overview of the
GO categories that are modulated during the S. japonicum
lifecycle we used the software ErmineJ to correlate this
extended list of GOs with microarray fold change data.
Presented in Figure 3 are the prominent GOs associated
with the lifecycle. In the Biological Processes category
were developmentally expressed genes involved in
cellular or primary metabolism, cellular communication and
Adult Males Combined weeks 6–7
All Males Parasites + juvenile
Adult Females combined weeks 6–7
All Females Parasites + juvenile
Normalised Intensity/Median Intensity of All Stages
2 fold 5 fold
The intensity for each development stage was normalised to the median intensity of all the developmental stages examined for each gene, thus
providing a relative fold change. The number of probes or genes enriched 2- or 5-fold is presented. The 1782 value in parenthesis in the Total row,
refers to the number of non-redundant genes enriched 2-fold or higher.
biosynthesis, while outstanding among the Molecular
Function category were developmentally regulated genes
involved in the binding of proteins, nucleotides, nucleic
acids and enzymes such as hydrolases and transferases.
During development of S. japonicum genes in the GO
category of Biological Processes were most frequently
involved in metabolism whereas those categorised within
Molecular Functions were most frequently involved in
binding and enzyme activity. These trends were, in
general, similar to the GO findings observed previously in
EST transcriptome studies of both S. japonicum  and S.
Examples of KEGG pathways identified by BLAST2GO
analysis, ie pathways that contain multiple genes involved
in metabolism/anabolism are presented in
Supplementary Figures 2-4. Genes within the Pentose Phosphate,
Calcium Signalling and Sphingolipid Metabolism pathways
were expressed at higher levels in several of the S.
japonicum lifecycle stages. These included genes encoding
fructose-bisphosphate aldolase (Contig8868), sarcoplasmic
reticulum ATPase calcium pump (Contigs 3653, 4586),
and sphingomyelin phosphodiesterase (Contig7899).
Microarray findings are supported by real time PCR
NADH-ubiquinone reductase was selected as a
housekeeping gene for real time PCR analyses as this gene was
constant in all 39 microarray hybridisations performed. A
subset of 10 differentially expressed genes, representative
for each of the life cycle stages examined, were subjected
to verification by real time PCR (Figure 4). The majority of
genes that were shown differentially expressed by the
microarray analysis correlated well with the results
obtained by real time PCR. Figure 4 outlines the 10 genes
examined by real time PCR (bar graphs) and presents also
the corresponding microarray signal detected (underlying
heat map bar).
Genes within clusters identified by hierarchal clustering
Examples of genes upregulated in the aquatic/snail
lifecycle stages included a gene encoding an anti-inflammatory
protein Sj16 (Contig7172) which was enriched (11-fold
upregulated) in cercariae, eggs (49-fold upregulated),
miracidia (38-fold upregulated) and sporocysts (13-fold
upregulated); it was also enriched in lung stage
schistosomula (13-fold upregulated). A homologue
(Contig8558) of the Schistosoma mansoni calcium-binding
calmodulin was also upregulated in several stages,
including eggs (99 fold), miracidia (73 fold) and sporocysts (4
fold). Genes encoding putative dynein proteins were
differentially expressed in the aquatic/snail stages including
Sm10 (Contig714) which was upregulated in eggs
(32fold), miracidia (20-fold) and sporocysts (80-fold);
Sj21.7 (Contig2186) mirrored this expression pattern
with gene expression enriched in eggs (24-fold), miracidia
(17-fold) and sporocysts (5.6-fold). Genes involved in
mitochondrial-linked energy metabolism were
upregulated predominantly in eggs and the motile cercariae with
other aquatic/snail stages also enriched for these genes.
Examples of enriched mitochondrial genes included
Contig8754 and Contig7759 encoding cytochrome c
oxidase subunit I and cytochrome c oxidase 3, respectively.
Nucleotide Description/Protein Homology
S. mansoni calcium-binding protein/calmodulin
S. mansoni anti-inflammatory protein 16/SJCHGC05573
solute carrier family 37 (glycerol-3-phosphate transporter)
S. mansoni eukaryotic translation initiation factor 2
A. thaliana dynamin-like protein 6 (ADL6).
,/insulin-like peptide receptor ilp-r
S. mansoni for Sm10 protein/dynein light chain 2
S. mansoni calponin homolog,/SJCHGC02977
solute carrier family 7 (cationic amino acid)
cytochrome c oxidase subunit i
S. japonicum for
serine-enzyme/---NA--SJCHGC01839/glioma pathogenesis-related protein
S. japonicum for
serine-enzyme/---NA--putative ferredoxin reductase; Mesorhizobium loti/
SJCHGC00284/epididymal secretory protein e1
S. japonicum clone ZZD10/SJCHGC06317/cd63 antigen
solute carrier family 7 (cationic amino acidy+ system)member 8
valosin containing protein
adenylosuccinate synthase like 1
DNA mismatch repair protein MSH2 – African clawed frog
novel transmembrane amino acid transporter protein
Enrichment of these two genes occurred in eggs (3 & 5.6
fold), miracidia (2.2 & 2.3 fold), sporocysts (2.7 & 1.2
fold) and cercariae (3.4 & 3.1 fold).
Adult female S. japonicum expressed high levels of a suite
of genes reflecting well recognised biological functions
associated with this stage. These included genes involved
in iron metabolism including ferric-chelate reductase 1
FHiigeurarrech2al clustering by gene and developmental stage
Hierarchal clustering by gene and developmental stage. Four clusters of up-regulated genes were identified: 1,
Aquatic/snail stages; 2 and 3, Adult female worms; 4. Adult male worms. Gene expression is shown in the heat map as
up-regulated (Red) down-regulated (Green) or no change (Black). E, eggs; M, miracidia; S, sporocysts; C, cercariae; L, lung
schistosomula; F4, juvenile females; M4, juvenile males; F6, F6.5, F7, adult female worms analysed at 6, 6.5 and 7 weeks post-cercarial
challenge; M6, M6.5, M7, adult male worms analysed at 6, 6.5 and 7 weeks post-cercarial challenge.
uFGsieginnugerEeOr m3ntionleoJgsyo(ftGwOar)eanalysis showing the correlation between GO categories and microarray expression data calculated
Gene Ontology (GO) analysis showing the correlation between GO categories and microarray expression data
calculated using ErmineJ software. The GO annotations with parent-child analysis are presented on the left.
Contributions to each of the categories from each lifecycle stage (2-fold or higher gene expression) are shaded. The overall number of
genes in each category represented on the microarray and the correlation p-value associated with the entire lifecycle are
shown on the right. NA, the parent category is significant but a child category is not. Genes expressed in adult parasites from
VFiagliudarteio4n of selected transcripts in different developmental stages of S. japonicum
Validation of selected transcripts in different developmental stages of S. japonicum. The real time PCR analysis is
presented as bar graphs and is shown in copy number for each gene and stage. The corresponding microarray gene expression
data are presented below the bar graphs as heat maps, with up-regulated genes shown in red, down-regulated genes shown in
green and unchanged genes shown in black. E, eggs; M, miracidia; S, sporocysts; C,: cercariae; L, lung schistosomula; F4, juvenile
females; M4, juvenile males; F6, F6.5, F7, adult female worms analysed at 6, 6.5 and 7 weeks post-cercarial challenge; M6, M6.5,
M7, adult male worms analysed at 6, 6.5 and 7 weeks post-cercarial challenge.
(Contig7830, 3.9 fold) and a putative ferredoxin
reductase (TC9476, 134 fold). Transporters were also enriched
including Contig5142 (solute carrier family 7, 11.8 fold),
Contig7944 (sodium-dependent amino acid transporter,
3.8 fold), TC8509 (solute carrier family 25, 2.7 fold),
Contig5284 (solute carrier family member b1, 2.6 fold)
and Contig3928 (novel protein vertebrate udp-galactose
transporters, 2.5 fold). Multiple ribosomal genes were
over expressed (Table 2) reflecting the high protein
requirements of the female worm associated with egg
Notable genes upregulated in adult male S. japonicum
were those involved in molecular transport including
Contig4443 (solute carrier family 1, 6 fold) and
Contig8527 (glucose transporter protein GTP1, solute
carrier family 2, 2.4 fold). Other genes enriched in adult
males included the endopeptidases cathepsin B
(Contig7921, 4.6 fold) and cathepsin F (Contig8927, 4.8
fold); components of vesicle mediated transport for both
endocytosis-clathrin coat assembly protein ap19
(Contig6302, 2.4 fold), adaptin-related protein 2
(TC7859, 2.6 fold) and exocytosis-syntaxin 16
(Contig7586, 1.5 fold). Genes involved in carbohydrate
metabolism and glycolysis, including glycosyltransferase
1 (Contig8718, 4.1 fold) and pyruvate dehydrogenase
(Contig4093, 2.7 fold), were also enriched. Further
examples of enriched genes for each of the lifecycle stages
examined are presented in Table 2.
Comparison of lifecycle stages
Principal component analysis (PCA) was employed to
establish relationships of the gene expression profiles
among the different developmental stages of S. japonicum.
The PCA demonstrated clearly that stages that were
temporally related (e.g. sporocyst to cercaria) exhibited
similarities in gene expression (Figure 5). In general, the
developmental stage expression profile, based on PCA,
clustered into three major groupings: (1) adult males, (2)
adult females, and (3) aquatic and snail stages comprising
eggs, sporocysts and cercariae. Furthermore, a clear
change of transcript profile was evident within the male
and female adult worms, as the females mature from
preegg laying to egg-laying status at four to six weeks
postinfection of the mammalian host. By contrast, the
schistosomulum (mammalian lung) and miracidium (ciliated,
free-living aquatic larva) did not display obvious
associations or grouping with any of the other stages.
Gene expression profiles for 7 week-old adult males of S.
japonicum examined by PCA and ANOVA analysis
revealed limited variation between biological replicates, ie
worms obtained and examined from independent
infections at different times (not shown). This was also the case
when pools of biological replicates of adult male worms
were analysed (n = 4) (not shown). After filtering the data
initially for flags, 10,765 probes were further filtered for
ANOVA. At the default settings, no genes were identified
as being significantly variable (p-value = 0.05) between
biological replicates; only 27 genes were identified as
variable after increasing the error cut-off rate to 18% (the rate
at which the identified genes would be expected to pass
the restriction by chance). This indicated that a very low
level of variation in gene expression occurred between
biological replicates resulting from parasite samples
obtained from separate infections.
Microarrays provide a powerful platform to monitor
developmental changes in the transcriptome of an
organism and a foundation for studies of gene regulation and
proteomics analysis . This study provides and
compares the baseline transcriptional levels of the majority of
genes in discrete developmental stages of Schistosoma
japonicum including those within the definitive (eggs, lung
schistosomula, juvenile and adult male and female
worms) and intermediate (sporocysts) hosts as well as the
aquatic, free-swimming miracidia and cercariae. Gene
transcription studies of many metazoan parasites,
including the schistosomes, have been disadvantaged because
these pathogens cannot be maintained in culture and/or
are only available in limited quantities for analysis. The
deployment of gene microarrays can help obviate these
challenges since only small quantities of scarce microbial
tissues are required for preparation of targets for
Our findings present the first global overview of
differential gene expression among the major developmental
stages of this schistosome. The final fold cut-off was
applied to the microarray data, thus generating lists of
genes that were ≥ 2 fold (relative to the median intensity)
in each lifecycle stage; these combined lists consist of
4,443 probes/3263 genes representing stage-enriched
gene transcripts. We choose this arbitrary level due to its
general acceptance in many other similar microarray
studies. However it is known that statistical variability is
primarily chip specific [14,15], and the selection of a fold
change of ≥ 2 can be related back to previous experiments
with the same chip , thus providing confidence in our
As illustrated in Figures 2 and 3, similarities and
relationships were apparent in the gene expression of several of
the developmental stages. In particular, gene expression
profiles were more similar within the 4, 6, 6.5 and 7 week
old adult parasites, although there was an apparent
change in expression from week four (pre-egg laying in
female worms) to week six onwards (after the
commencement of egg release). This similarity in gene expression
PFmrieginnuctriapelasl5tcaogemspoofnSe.njatpaonnailcyusmis aonf atlhyeseAdNOVA showing an overview of differentially expressed genes in the different
developPrincipal component analysis of the ANOVA showing an overview of differentially expressed genes in the
different developmental stages of S. japonicum analysed. E, eggs; M, miracidia; S, sporocysts; C,: cercariae; L, lung
schistosomula; F4, juvenile females; M4, juvenile males; F6, F6.5, F7, adult female worms analysed at 6, 6.5 and 7 weeks post-cercarial
challenge; M6, M6.5, M7, adult male worms analysed at 6, 6.5 and 7 weeks post-cercarial challenge.
likely reflects parasitism of the mammalian blood vessels
by the schistosome and the discrete differentiation and
development of the males and females [4,7,16,17]. The
eggs, sporocysts and the cercariae also shared similarities
in gene expression profiles. These similarities likely reflect
adaptations to the aquatic environment and parasitism of
the snail host, but are somewhat surprising in regards to
the eggs, given that these were recovered from mouse
It is not straightforward to compare the present findings
with the earlier reports of microarray-based gene
expression in schistosomes given that many of the previous
investigations focused on S. mansoni. Nonetheless, we
have been able to make some general comparisons,
including demonstration of the sex-specific nature of
some important developmental changes in S. japonicum.
For example, upregulated gene transcription for
S-transferase SM28 antigen (TC10486) [Contig6578
(glutathione s-transferase, 1.7–2 fold), dynein 8 kDa light chain
flagellar outer arm (TC8189) (8 kDa outer arm dynein
light chain 1.8–2.3 fold) and TC10493 Cathepsin B1
isotype 1 (3.2–4.0 fold) was evident in adult male worms.
Similar enrichment of these genes was shown in
sporocysts and adults of S. mansoni  but, as we separately
investigated the sexes of adult S. japonicum, we were able
to show that this up-regulation was male-specific.
However, a direct correlation of small subset of probes
originally designed from S. mansoni EST and present on
both microarrays (TC probes) was possible. Additional
file 4 shows that of the 20 TC probes which had data from
S. japonicum and S. mansoni, only 5 probes could be
correlated as up-regulated to the same lifecycle stages for both
species (shaded in the Table). This comparison highlights
the difficulty of inter-species comparisons using
microarray technology, a limitation that is compounded by the
lack of complete genomes for both S. japonicum and S.
mansoni and the absence of comprehensive
bioinformatics comparisons at the genomic level. However, the
current study of S. japonicum has provided a more
comprehensive overview of the developmental biology of
schistosomes in terms of the coverage of the
transcriptome through the size of the microarray used, and also the
number of lifecycle stages examined including separate
adult sexes, in vivo derived lung schistosomula, miracidia
and the juvenile pre-egg producing parasite. Additionally
the study has also used analysis methods to cluster
multiple genes to KEGG pathways not applied previously in
schistosome microarray studies. This analysis has further
highlighted stage specific functions and provides insights
into transcriptional changes of schistosome genes
involved in evasion of the host immune response,
nutrient acquisition, energy production, calcium signalling,
sphingolipid metabolism, egg production and tegumental
function during development (as is discussed further
A brief description of some of the other genes
developmentally expressed in the various lifecycle stages of S.
japonicum follows. Sm16 is an anti-inflammatory
glycoprotein first identified in adult S. mansoni  that was
shown by immunoelectron microscopy to be localised in
cercariae in the acetabular glands and in subtegumental
cell bodies packed with membranous vesicles . Sm16
acts as a lipid binding protein that is taken up by
mammalian cells via endocytosis and acts within the cytoplasm to
induce apoptosis . IL-1a is stimulated by Sm16 which
via keratinocytes leads to a decrease in the number of
lymphoproliferative cells that are recruited, presumably
during the penetration of the mammalian host, by the
invading skin schistosomula . Sm16 may aid S.
mansoni in penetration of the mammalian host  and our
results support this hypothesis as the transcript of the S.
japonicum homologue (Sj16; Contig7172) was 11-fold
enriched in cercariae. However, the microarray and/or
real-time PCR analysis indicated that even higher levels of
the gene were expressed in the egg (49-fold upregulated),
miracidium (38-fold upregulated), sporocyst (13-fold
upregulated), and lung stage schistosomulum (13-fold
upregulated). This expression profile suggests an
additional intra-mammalian role for the product of this gene
perhaps as a defence mechanism against the host immune
system in both the invading cercariae, the lung
schistosomulum and the egg, which during the chronic stage of
infection induces granuloma formation and fibrogenesis
. The high levels of Sj16 expression in the miracidium
and sporocyst may reflect a role in the penetration of the
molluscan host and in protection of the sporocyst from
snail innate immunity.
The microarray and real time PCR analysis revealed
elevated levels of expression of paramyosin (Contig6538) in
the adult male (microarray 2.5–3-fold upregulated) and
the lung schistosomulum (real time PCR 3.7-fold
upregulated). These profiles correlate with the well documented
functions for paramyosin in schistosomes as a structural
component of smooth muscle fibres and as an
immunomodulator of the host immune response through its
binding to the immunoglobulin Fc region and its
inhibition of complement activation [23-25]. The highly
developed musculature of the adult male  supports the
female adult worm in copula. The localisation of
paramyosin on the surface of the lung schistosomulum  was
the first indication of the developmental significance of
the expression of this protein. Immunological and
biochemical findings [24,27] have further emphasised the
importance of paramyosin in protecting schistosomes
from immune attack by binding proteins of the
complement pathway .
The antioxidant, thioredoxin is present in cercariae,
schistosomula, eggs and both male and female adults of S.
mansoni . Thioredoxin is secreted by eggs of S. mansoni
, and likely is implicated in granuloma formation
within the liver of the mammalian host . Our gene
microarrays revealed that S. japonicum thioredoxin
(Contig3028) expression was up-regulated in the egg
(2.4fold), thereby confirming its importance in this stage and
also the adult male (2.6–2.7-fold). The antioxidant
properties of thioredoxin would be expected to protect the
schistosome from reactive oxygen species released by host
Calponin is a calcium binding protein that inhibits the
ATPase activity of smooth muscle myosin necessary for
long-term contractions. It is expressed predominantly
within the muscle fibres of adult male and female S.
japonicum . Further localization studies have indicated that
expression also occurs within the stratified muscle of the
tail and smooth muscle of the head region of cercariae
. The current microarray results support these
observations but also showed enrichment of calponin
(Contig7933) in lung schistosomula (5.4-fold) and the
aquatic/snail stages (egg, 12.8-fold; miracidium,
18.5fold; sporocyst, 6.2-fold; cercariae, 3.4-fold). This likely
reflects general upregulation of muscle-related genes as
schistosomes have significantly increased muscle
deposition during development .
Annexins bind phospholipids in a calcium-dependent
manner and are thought to reduce inflammation via the
suppression of phospholipase A2 in a similar manner to
glucocorticoids . Other predominant functions of
annexins include the organisation of cellular structures,
signal transduction, cellular growth and modulation of
intracellular calcium while acting as atypical calcium
channels . Annexins are a well known component of
the adult schistosome tegument as shown by surface
biotinylation and proteomics studies . Our microarray and
real time PCR analysis revealed that annexin
(Contig8017) expression was raised in lung
schistosomula (1.3–1.4-fold) and adult males (2-8-3.3-fold) of S.
japonicum. Both of these lifecycle stages would benefit
from the anti-inflammatory properties of annexin due to
the immune response generated in the lungs, and the
relatively large number of tegumental proteins present on
the adult male parasite that may incite an immune
In contrast to what is currently known regarding calcium
channels in schistosomes, including sub-unit structure,
cellular distribution and interactions with the
anti-schistosomal drug praziquantel , there is limited
knowledge of the biology of potassium channels in these
worms. What is known is that they are localised to the
nervous system and musculature of adult S. mansoni .
This is supported by the findings presented here as both
the microarray and real time PCR analysis showed highest
expression (12 fold enriched) of the potassium channel
gene (Contig1637) in adult male S. japonicum, which has
an extensively developed musculature .
Sm21.7 has sequence similarity to dynein light chains
(DLCs), it is one of a family of EF-hand containing
parasite proteins and is also a major non-parasite allergen
. Little is known of the function of Sm21.7 but its
transcript was originally shown to be present in the
sporocyst, mechanically transformed schistosomulum and
adult S. mansoni . Subsequently, it has been localised
to the adult worm tegument, it is expressed in eggs, adults
and cercariae and is known to stimulate the mammalian
immune system since it is constantly released by eggs
trapped in liver granulomas . Here, the results of the
developmental expression studies on the S. japonicum
homologue, Sj21.7, mirrored in part, the previous studies
on Sm21.7 but transcripts for Sj21.7 (Contig2186) were
enriched predominantly in the egg (24-fold), miracidium
(17-fold) and sporocyst (5.6-fold) with limited
expression in the intra-mammalian stages, possibly indicating
differences in functional expression between the two
schistosome species. Another gene exhibiting a similar
expression profile to Sj21.7, as determined by the
microarray analysis and real time PCR was Sm10 (Contig714),
being upregulated in eggs (32-fold), miracidia (20-fold)
and sporocysts (80-fold). This gene encodes another DLC
that has been localised to the tegument  and is
strongly immunogenic . The microarray analysis
suggests that the DLCs of S. japonicum may have a greater
requirement for microtubule motility during the aquatic
developmental stages compared with the
intra-mammalian stages. This observation is particularly pertinent since
the highest expression levels of Sm10 were found in
miracidia which utilise cilia for locomotion. While Sm10 has
not yet been immunolocalised in the miracidium stage,
there is evidence for a role of dyneins in the cilia and
flagella of other mammalian and non-mammalian (C.
elegans) systems . Sm10 has homology to members of
the LC8 family of DLCs, of which one member, DYNLL1,
is up-regulated in mammalian testes and lung, two tissues
which have considerable numbers of cilia or flagella
The S. japonicum transcript encoding the homologue of
sperm associated antigen 6 (Spag6) (Contig2404) is likely
a component of tubulin structures and was enriched in
both the egg (2.4-fold), the adult stages (1.6–2.6 fold)
and to a lesser extent, juvenile males (1.5-fold). Spag6 is
the murine orthologue of Chlamydomonas PF16, a gene
that is a component of the flagella central apparatus ,
so the enrichment of this gene in adult male S. japonicum
likely reflects a role in reproduction, probably in sperm
function. Its involvement in eggs is less clear but may
reflect a structural role associated with microtubules and
development of the miracidium.
Sphingolipids represent a class of fatty acids that have
been associated with protection of the cell surface against
environmental stress . This protection is provided
through both mechanical strengthening and enhanced
chemical resistance to the apical membrane of cells.
Sphingolipids have also been implicated in schistosomes
in the formation of caveolae in the apical surface .
Regions of the adult S. mansoni tegument have been
identified as detergent-insoluble glycosphingolipid-enriched
membrane domains (DIG) and contain multiple caveolae
structures . We detected three potential homologs
represented by two microarray probes, related to the
Sphingolipid Metabolism Pathway (SMP) that were up
regulated in adult males and females, lung schistosomula
and eggs (Contig7899 lung schistosomula 2.5 fold, adult
males 3.4 fold; Contig7987 eggs 3.1 fold, adult females
3.0 fold) of S. japonicum (Additional file 5).
Glycosphingolipids have been identified as components of
schistosome eggs  and are selectively bound by the antigen
presenting cells of the liver by liver/lymph node specific
ICAM-3-grabbing non-integrins . A further role for
sphingolipids in lung schistosomula has been previously
suggested by El Ridi and Tallima  who demonstrated
that an equilibrium of these lipids is critical for the
selective absorption of small molecules across the lipid bilayer
while also acting as a defence mechanism through the
exclusion of antibody binding to the schistosome surface.
Six differentially expressed genes were identified as
components of the Pentose Phosphate Pathway (PPP)
(Additional file 6). These included genes encoding isomerases,
gluconate dehydrogenases and proteins involved in
fructose phosphorylation. The primary role of the PPP is
anabolic rather than catabolic resulting in the production of
nucleotides, RNA and DNA, and NADPH for reductive
biosynthesis [50,51]. In addition, the PPP is an alternative
to glycolysis for oxidation of glucose and energy
production. Elevated expression of these genes was observed in
eggs, miracidia, sporocysts, cercariae and adult female
schistosomes, likely reflecting increased activity of this
pathway (Contig6630 egg 7.9 fold, miracidium 2.7 fold,
sporocyst 2.1 fold; Contig8868 egg 2.0 fold; TC7683
cercariae 2.1 fold; Contig6298 egg 2.4 fold; Contig8062
adult female 2.4 fold). Up-regulation of components of
this pathway may reflect increased metabolism in the
aquatic lifecycle stages where considerable tissue/cellular
remodelling is required. Up-regulation of PPP
components in the adult female of S. japonicum mirrors the
extensive egg production and considerable cellular
synthesis that are prominent biological features of this stage
. Surprisingly these PPP genes were not up regulated
in the juvenile stages (lung schistosomula; liver stage/
paired but immature male/female worms) which develop
significantly in size and complexity. Further
characterisation of PPP components as potential anti-schistosome
drug targets may be rewarding, especially as the pathway
has been targeted for drug development against
trypanosomes and Leishmania [53,54]. Notably, one of these
trypanosome drug targets is phosphogluconate
dehydrogenase, a gene that was up-regulated in adult female S.
japonicum (Contig8062; 2.4 fold).
Six genes of the Calcium Signalling Pathway (CSP) were
up-regulated in the adult male of S. japonicum, as well as
in the egg, miracidium, sporocyst and cercaria aquatic/
snail stages (Contig7484 adult male 8.9 fold; Contig3653
cercaria 2.3 fold; Contig4586 adult male 3.1 fold; TC7426
egg 2.3 fold; Contig8713 egg 2.03 fold; Contig8558 egg
99.1 fold, miracidium 73.2 fold, sporocyst 4.0 fold)
(Additional file 7). Elevated levels of sarco-endoplasmic
reticulum ATPase (Contig4586 2.7–3.1 fold) in adult
male worms probably reflect their requirements for
increased muscle mass and the recognised importance of
calcium in muscle physiology . Of the other
up-regulated CSP genes, those in the egg may be necessary for the
translation of environmental signals to this stage,
especially those required to initiate hatching following its exit
from the mammalian host and its entry into fresh water.
To expand on this, it is known that schistosome egg
hatching requires appropriate regulation of calcium through
calmodulin  which was highly upregulated in the egg
and miracidium and also in the sporocyst of S. japonicum
(Contig8558; egg, 71–99 fold; miracidium, 48–73 fold;
sporocyst, 3–4 fold upregulated). Other genes within the
CSP, especially those shown to be up-regulated within the
egg of S. japonicum, may also encode components critical
for hatching and/or other important biological functions
and are worthy of further study.
This report presents global gene expression patterns for
the Asian blood fluke S. japonicum during its
developmental cycle. Individual genes, gene ontologies and pathways
that are modulated during the parasite lifecycle were also
profiled. Using advanced filtering analyses including
ANOVA, principal component analysis, and hierarchal
clustering, we have provided a synopsis of relationships of
gene expression among seven developmental stages of the
schistosome. This represents the most comprehensive
report to date on genes analysed for stage-specific
expression in S. japonicum, indeed, in schistosomes in general,
and should lead to a better understanding of development
and differentiation in these parasites, their interaction
with their mammalian and molluscan hosts, and how
they modify gene regulation to adapt to a complex
developmental cycle involving free-living and parasitic phases.
The work may also provide new insights on
schistosomeinduced pathogenesis and has implications for
developing new interventions for future schistosomiasis control.
Indeed, the analyses revealed that gene expression profiles
are linked to the major environmental settings through
which the fluke progresses – from free-living aquatic
miracidium, to parasitism of the snail by the sporocyst, to
parasitism of the blood of the mammal by the maturing
and adult schistosomes. As well as facilitating future
research on gene regulation, at large, for a broad range of
organisms with similar complex processes of
development and differentiation, this resource should facilitate
identification and prioritization of new intervention
targets for the control and treatment of schistosomiasis.
Lifecycle maintenance and collection of S. japonicum life
Oncomelania hupensis hupensis snails, infected with a
Chinese mainland field isolate (from Anhui Province) of S.
japonicum, were kindly provided by the National Institute
of Parasitic Diseases, CDC, Shanghai. All samples of
isolated parasite stages were resuspended in 1 ml of PBS and
stored at -80°C until the total RNA was isolated. Viable
schistosome eggs were obtained from infected mouse
livers by digestion with collagenase B . Eggs were used
either for total RNA extraction or the production of
miracidia. Miracidia were hatched and isolated as described
. Miracidia were either stored or used immediately for
the production of sporocysts by incubation in MEMSE-j
medium and maintenance in hypoxia chambers for 48 h
under an atmosphere of 5%O2, 5% CO2, 90%N2 at 27°C
[58,59]. Cercariae were obtained from surface water after
being shed from infected snails exposed for 3 h to a bright
Lung schistosomula were isolated using modifications of
a published procedure . Approximately 1000
cercariae, pooled from several infected Oncomelania snails, were
used to challenge female BALB/c mice. Three days later the
lungs were removed, minced and incubated in RPMI at
37°C for 3 h on a rocker-shaker. The lung tissue solution
was sieved and schistosomula were removed using a
finetipped glass pipette. Adult worm pairs were perfused and
separated by sex from female BALB/c mice challenged
percutaneously with 30 cercariae of S. japonicum  at 4
(immature worms) and 6, 6.5 and 7 (mature,
egg-producing worms) weeks post-infection.
Total RNA isolation
Total RNA was isolated from parasites/free-living stages as
described , with care being taken to ensure that all
RNA samples were of high quality and quantity as
assessed by Nanodrop ND-1000 spectrophotometer
(A260/A280 nm ≥1.7 in nuclease-free water) and a
The design and construction of the schistosome
microarray  used in this study was facilitated by the
availability of transcriptome data for S. japonicum  and S.
mansoni  which added approximately 160,000 new
schistosome ESTs (Expressed Sequence Tag) to GenBank.
A large proportion of the EST sequences generated still
require characterisation as only 45% of the S. mansoni and
65% of the S. japonicum ESTs showed similarity to
sequences already in GenBank. Nevertheless, the two
datasets likely represent the majority of the transcriptome
of these two schistosome species . The microarray
comprises 19,222 target sequences printed twice from two
independent probe designs, including 12,166 probes
derived from S. mansoni contiguous sequences (contigs)
and 7,056 probes derived from S. japonicum contigs.
Variations to this initial design included the use of a second,
independently formulated probe for each contig. Further
details of the microarray design and the raw data from this
study are presented in Supplementary Tables 1 and 2.
Supplementary Information has been submitted at
GEOGene Expression Omnibus, http://
www.ncbi.nlm.nih.gov/geo/ accession numbers
Microarray hybridisation and feature extraction
A 300 ng aliquot of total RNA from each developmental
stage was used to synthesise fluorophore-labelled cRNA
using Cyanine 3-CTP (CY3c) as described (One-Color
Microarray-Based Gene Expression Analysis Protocol;
Version 5.5, February 2007 Agilent). Samples were purified
using the Qiagen RNeasy kit. Cyanine-labelled cRNA
samples were examined at A260 and A550 using a ND-1000
spectrophotometer to determine yield, concentration,
amplification efficiency and abundance of cyanine
fluorophore. Once the concentration had been determined,
1.65 μg aliquots of CY3c were placed in a fresh tube
together with the fragmentation mix (Agilent
Technologies, Santa Clara, USA) and incubated for 30 min at 60°C.
Then, the samples were combined with 2× Gene
Expression hybridization Buffer HI-RPM, mixed and applied to
a gasket slide that was pre-positioned in a hybridisation
chamber (Agilent). The microarray slide was placed probe
side towards the target. The chamber was assembled and
placed in a hybridisation oven and incubated for 17 h at
65°C. After hybridisation the chamber was opened and
the microarray slides were washed using the standard
Agilent protocol (Agilent Technologies, USA) and scanned
on an Agilent microarray scanner at 550 nm. Microarray
hybridisations were performed in triplicate for all S.
japonicum samples; additionally, biological replicates of adult
male worms (collected at week 7) from separate infections
were collected to determine any variation in gene
expression. Microarray slides were scanned using an Agilent
Microarray Scanner (B version). The "tag image format
files" (tiff) produced by the scanner were loaded into the
image analysis program Feature Extraction 22.214.171.124
(Agilent Technologies, USA) to establish standardised data for
statistical analysis. All microarray slides were checked for
background evenness by viewing the tiff image on Feature
Feature extracted data were analysed using GENESPRING
software, version 7.3.1 (Agilent Technologies/Silicon
Genetics, Redwood City, CA). Microarray data were
normalised using the genespring normalisation scenario for
"Agilent FE one-color" which including "Data
Transformation: Set measurements less than 5.0 to 5.0", "Per
Chip: Normalise to 50th percentile" and "Per Gene:
Normalise to median".
Data sets were further analysed using published
procedures  that consisted of methods related to one-colour
experiments and utilised gProcessedSignal values
determined using Agilent's Feature Extraction software
including aspects of signal/noise ratio, spot morphology and
homogeneity. ProcessedSignal represents signal after
localised background subtraction and includes
corrections for surface trends. Features were deemed Absent
when the processed signal intensity was less than two fold
the value of the processed signal error value. Features were
deemed Marginal when the measured intensity was at a
saturated value or if there was a substantial amount of
variation in the signal intensity within the pixels of a
particular feature. Features that were not Absent or Marginal
were deemed Present. Data points were included only if
Present or Marginal and probes were retained if all data
points were Present. Analysis of Variance (ANOVA) was
applied to this data using genes with statistically
significant differences when grouped by lifecycle stage;
parametric test, variances not assumed equal (Welch ANOVA);
pvalue cutoff 0.05; multiple testing correction: Benjamini
and Hochberg False Discovery Rate . About 5.0% of
the identified genes would be expected to pass the
restriction by chance. Principal Component Analysis (PCA) was
run on conditions ie for each lifecycle stage, using mean
centering and scaling.
Protein blast and gene ontology analysis using Blast2Go
Batch BlastX (6 frame translation protein homology) was
performed at http://www.blast2go.de on al contigs. The
Blast2go website was used to complement gene ontology
and identify multiple genes within the Kyoto
Encyclopedia of Genes and Genomes (KEGG) metabolic pathway
maps http://www.genome.jp/kegg/. Gene ontology
correlations with relative gene expression values were made
using ErmineJ software . Correlations for Biological
Processes and Molecular Functions were made against
microarray data using maximum gene dataset of 100,
minimum dataset of 5 and maximum iterations of
Real time PCR
Gene expression patterns of a subset of genes were
validated using real time PCR. Complementary DNA was
synthesised from total RNA using a Qiagen QuantiTect whole
transcriptome kit. Forward and reverse primers were
designed from S. japonicum contigs. All cDNA samples
synthesised from aliquots of the same total RNA used for
the microarray hybridizations were diluted to 50 ng/μl,
and quantified using a Nanodrop ND-1000
spectrophotometer. Subsequently aliquots of 1 μl were combined
with 10 μl of SYBERs Green, 3 μl of water and 2 μl (5
pmol) of the forward and reverse primers in a 0.1 ml tube.
All reactions were performed on a Rotor-Gene (3000) real
time PCR and analyzed by Rotor Gene 6 Software. In
order to minimise indiscriminate binding of
doublestranded DNA, which can produce readings in the "no
template" controls, separate reverse transcription and PCR
steps were included. NADH-ubiquinone reductase was
employed as a control (house-keeping) gene in the real
time PCR studies . Primer sets used are shown in
Additional file 8.
Supplementary Information has been submitted at GEO,
accession numbers GPL7160, GSE12704. http://
ANOVA: Analysis of variance; CSP: Calcium Signalling
Pathway; DLC: dynein light chain; EST: Expressed
Sequence Tag; GO: Gene Ontology; GEO: Gene
Expression Omnibus; KEGG: Kyoto Encyclopedia of Genes and
Genomes; PCA: Principal Component Analysis; PPP:
Pentose Phosphate Pathway; SAGE: serial analysis of gene
expression; Sj: Schistosoma japonicum; Sm: Schistosoma
mansoni, SMP: Sphingolipid Metabolism Pathway.
GG designed the study, analysed data and wrote the
manuscript. DM and PB designed the study and wrote the
manuscript. LM performed the microarray and real time
Additional file 1
Supplementary Figure 1.
Filtering of data from triplicate hybridisation of 38,444 probes to
schistosome transcripts representing 19,222 genes. After filtering for
"flagged" genes against all hybridisations, 7,132 probes were left,
representing 4,371 genes; a final ANOVA of this dataset retained 6,465 probes
and 4,104 genes. E, eggs; M, miracidia; S, sporocysts; C, cercariae; L,
lung schistosomula; F4, juvenile females; M4, juvenile males; F6, F6.5,
F7, adult female worms analysed at 6, 6.5 and 7 weeks post-cercarial
challenge; M6, M6.5, M7, adult male worms analysed at 6, 6.5 and 7
weeks post-cercarial challenge.
Click here for file
Additional file 2
Supplementary Table 1.
Complete list of differentially expressed S. japonicum genes after
filtering for flags. Genes enriched 2 fold or higher (sorted in decreasing
order of expression) for each of the lifecycle stages are shown on different
sheets for the egg, Miracidium, Sporocyst, Cercaria, Lung
Schistosomulum, Juvenile Female (F4), Juvenile Male (M4), Adult Female
(combined weeks 6–7), Adult Male (combined weeks 6–7).
Click here for file
Additional file 3
Additional file 4
Additional file 5
Additional file 6
Additional file 7
Additional file 8
We thank Mary Duke for maintenance of the S. japonicum lifecycle at QIMR.
This research was supported by the National Health and Medical Research
Council (NHMRC) of Australia and the Wellcome Trust (UK).
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