BACKGROUND
schistosomiasis afflicts ~ <dig> million people in  <dig> countries  <cit> .
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 .
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  <cit> .
in previous microarray studies, several hundred cdnas from s. japonicum and s. mansoni  <cit>  or a few thousand  <cit>  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  <cit>  examined  <dig>  contigs or singletons across seven life cycle stages of s. mansoni early liver worms, adult worms, eggs, daughter sporocysts, cercariae, and day  <dig> and day  <dig> schistosomula produced by mechanical transformation of cercariae followed by in vitro culture.
a new  <dig>  feature 60-mer microarray was deployed to investigate gene expression patterns between and within discrete chinese and philippine strains of s. japonicum  <cit>  and to demonstrate stage-associated gene expression between mature adult worms and lung schistosomula from amplified s. japonicum mrnas  <cit> .
recently,  <cit>  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  to study s. mansoni  <cit> .
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.
RESULTS
filtering of microarray data to identify stage enriched genes
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  <dig>  probes  on the schistosome microarray .
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  <dig>  probes .
next, anova applied to the data identified  <dig>  probes  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 ≥  <dig> fold  in each lifecycle stage; these combined lists consist of  <dig>  probes/ <dig> genes represent stage-enriched gene transcripts.
these lists comprised of a subset of  <dig>  unique genes or contigs, and were subjected to further annotation or in silico characterisation.
the numbers of  <dig> fold or  <dig> fold or higher enriched genes for each individual developmental stage or lifecycle grouping is summarised in table  <dig> .
examples of differentially expressed genes for each lifecycle stage are presented in table  <dig>
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  <dig> value in parenthesis in the total row, refers to the number of non-redundant genes enriched 2-fold or higher.
hierarchal clustering to provide overview of developmental changes
hierarchal clustering was employed to investigate gene expression profiles in each developmental stage.
figure  <dig> 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  <dig>  up-regulation is shown in red, down-regulation in green, whereas the black lines indicate absence of up- or down-regulation.
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  among the ~ <dig>  probes examined.
for example, considerable enrichment of gene expression was evident in the egg compared with the miracidial stage .
among the developmental stages, four prominent clusters of up-regulated genes were evident these were in  aquatic and snail stages,  adult female worms ,  adult female worms , and  adult males.
blast2go and gene ontology analysis to provide overview of functions and process
the  <dig>  genes that were identified as being enriched  <dig> fold in any given lifecycle stage were subjected to protein translational blast and gene ontology  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  <dig>  this information was used to supplement the already known gos based on nucleotide sequence previously published  <cit> .
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  <dig> 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 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  <cit>  and s. mansoni  <cit> .
examples of kegg pathways identified by blast2go analysis, ie pathways that contain multiple genes involved in metabolism/anabolism are presented in supplementary figures 2- <dig>  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 , sarcoplasmic reticulum atpase calcium pump , and sphingomyelin phosphodiesterase .
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  <dig> microarray hybridisations performed.
a subset of  <dig> differentially expressed genes, representative for each of the life cycle stages examined, were subjected to verification by real time pcr .
the majority of genes that were shown differentially expressed by the microarray analysis correlated well with the results obtained by real time pcr.
figure  <dig> outlines the  <dig> genes examined by real time pcr  and presents also the corresponding microarray signal detected .
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 sj <dig>  which was enriched  in cercariae, eggs , miracidia  and sporocysts ; it was also enriched in lung stage schistosomula .
a homologue  of the schistosoma mansoni calcium-binding calmodulin was also upregulated in several stages, including eggs , miracidia  and sporocysts .
genes encoding putative dynein proteins were differentially expressed in the aquatic/snail stages including sm <dig>  which was upregulated in eggs , miracidia  and sporocysts ; sj <dig>   mirrored this expression pattern with gene expression enriched in eggs , miracidia  and sporocysts .
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 contig <dig> and contig <dig> encoding cytochrome c oxidase subunit i and cytochrome c oxidase  <dig>  respectively.
enrichment of these two genes occurred in eggs , miracidia , sporocysts  and cercariae .
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  <dig>  and a putative ferredoxin reductase .
transporters were also enriched including contig <dig> , contig <dig> , tc <dig> , contig <dig>  and contig <dig> .
multiple ribosomal genes were over expressed  reflecting the high protein requirements of the female worm associated with egg production.
notable genes upregulated in adult male s. japonicum were those involved in molecular transport including contig <dig>  and contig <dig> .
other genes enriched in adult males included the endopeptidases cathepsin b  and cathepsin f ; components of vesicle mediated transport for both endocytosis-clathrin coat assembly protein ap <dig> , adaptin-related protein  <dig>  and exocytosis-syntaxin  <dig> .
genes involved in carbohydrate metabolism and glycolysis, including glycosyltransferase  <dig>  and pyruvate dehydrogenase , were also enriched.
further examples of enriched genes for each of the lifecycle stages examined are presented in table  <dig>
comparison of lifecycle stages
principal component analysis  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  exhibited similarities in gene expression .
in general, the developmental stage expression profile, based on pca, clustered into three major groupings:  adult males,  adult females, and  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 pre-egg laying to egg-laying status at four to six weeks post-infection of the mammalian host.
by contrast, the schistosomulum  and miracidium  did not display obvious associations or grouping with any of the other stages.
gene expression profiles for  <dig> 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 .
this was also the case when pools of biological replicates of adult male worms were analysed  .
after filtering the data initially for flags,  <dig>  probes were further filtered for anova.
at the default settings, no genes were identified as being significantly variable  between biological replicates; only  <dig> genes were identified as variable after increasing the error cut-off rate to 18% .
this indicated that a very low level of variation in gene expression occurred between biological replicates resulting from parasite samples obtained from separate infections.
discussion
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  <cit> .
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  and intermediate  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 microarray analysis.
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 ≥  <dig> fold  in each lifecycle stage; these combined lists consist of  <dig>  probes/ <dig> 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  <cit> , and the selection of a fold change of ≥  <dig> can be related back to previous experiments with the same chip  <cit> , thus providing confidence in our findings.
as illustrated in figures  <dig> and  <dig>  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  <dig>   <dig>   <dig>  and  <dig> week old adult parasites, although there was an apparent change in expression from week four  to week six onwards .
this similarity in gene expression likely reflects parasitism of the mammalian blood vessels by the schistosome and the discrete differentiation and development of the males and females  <cit> .
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 livers.
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 sm <dig> antigen  [contig <dig> , dynein  <dig> kda light chain flagellar outer arm   and tc <dig> cathepsin b <dig> isotype  <dig>  was evident in adult male worms.
similar enrichment of these genes was shown in sporocysts and adults of s. mansoni  <cit>  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  was possible.
additional file  <dig> shows that of the  <dig> tc probes which had data from s. japonicum and s. mansoni, only  <dig> probes could be correlated as up-regulated to the same lifecycle stages for both species .
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 .
a brief description of some of the other genes developmentally expressed in the various lifecycle stages of s. japonicum follows.
sm <dig> is an anti-inflammatory glycoprotein first identified in adult s. mansoni  <cit>  that was shown by immunoelectron microscopy to be localised in cercariae in the acetabular glands and in subtegumental cell bodies packed with membranous vesicles  <cit> .
sm <dig> acts as a lipid binding protein that is taken up by mammalian cells via endocytosis and acts within the cytoplasm to induce apoptosis  <cit> .
il-1a is stimulated by sm <dig> 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  <cit> .
sm <dig> may aid s. mansoni in penetration of the mammalian host  <cit>  and our results support this hypothesis as the transcript of the s. japonicum homologue  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 , miracidium , sporocyst , and lung stage schistosomulum .
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  <cit> .
the high levels of sj <dig> 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  in the adult male  and the lung schistosomulum .
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  <cit> .
the highly developed musculature of the adult male  <cit>  supports the female adult worm in copula.
the localisation of paramyosin on the surface of the lung schistosomulum  <cit>  was the first indication of the developmental significance of the expression of this protein.
immunological and biochemical findings  <cit>  have further emphasised the importance of paramyosin in protecting schistosomes from immune attack by binding proteins of the complement pathway  <cit> .
the antioxidant, thioredoxin is present in cercariae, schistosomula, eggs and both male and female adults of s. mansoni  <cit> .
thioredoxin is secreted by eggs of s. mansoni  <cit> , and likely is implicated in granuloma formation within the liver of the mammalian host  <cit> .
our gene microarrays revealed that s. japonicum thioredoxin  expression was up-regulated in the egg , thereby confirming its importance in this stage and also the adult male .
the antioxidant properties of thioredoxin would be expected to protect the schistosome from reactive oxygen species released by host inflammatory responses
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  <cit> .
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  <cit> .
the current microarray results support these observations but also showed enrichment of calponin  in lung schistosomula  and the aquatic/snail stages .
this likely reflects general upregulation of muscle-related genes as schistosomes have significantly increased muscle deposition during development  <cit> .
annexins bind phospholipids in a calcium-dependent manner and are thought to reduce inflammation via the suppression of phospholipase a <dig> in a similar manner to glucocorticoids  <cit> .
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  <cit> .
annexins are a well known component of the adult schistosome tegument as shown by surface biotinylation and proteomics studies  <cit> .
our microarray and real time pcr analysis revealed that annexin  expression was raised in lung schistosomula  and adult males  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 response  <cit> .
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  <cit> , 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  <cit> .
this is supported by the findings presented here as both the microarray and real time pcr analysis showed highest expression  of the potassium channel gene  in adult male s. japonicum, which has an extensively developed musculature  <cit> .
sm <dig>  has sequence similarity to dynein light chains , it is one of a family of ef-hand containing parasite proteins and is also a major non-parasite allergen  <cit> .
little is known of the function of sm <dig>  but its transcript was originally shown to be present in the sporocyst, mechanically transformed schistosomulum and adult s. mansoni  <cit> .
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  <cit> .
here, the results of the developmental expression studies on the s. japonicum homologue, sj <dig> , mirrored in part, the previous studies on sm <dig>  but transcripts for sj <dig>   were enriched predominantly in the egg , miracidium  and sporocyst  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 sj <dig> , as determined by the microarray analysis and real time pcr was sm <dig> , being upregulated in eggs , miracidia  and sporocysts .
this gene encodes another dlc that has been localised to the tegument  <cit>  and is strongly immunogenic  <cit> .
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 sm <dig> were found in miracidia which utilise cilia for locomotion.
while sm <dig> 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  systems  <cit> .
sm <dig> has homology to members of the lc <dig> family of dlcs, of which one member, dynll <dig>  is up-regulated in mammalian testes and lung, two tissues which have considerable numbers of cilia or flagella present  <cit> .
the s. japonicum transcript encoding the homologue of sperm associated antigen  <dig>   is likely a component of tubulin structures and was enriched in both the egg , the adult stages  and to a lesser extent, juvenile males .
spag <dig> is the murine orthologue of chlamydomonas pf <dig>  a gene that is a component of the flagella central apparatus  <cit> , 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  <cit> .
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  <cit> .
regions of the adult s. mansoni tegument have been identified as detergent-insoluble glycosphingolipid-enriched membrane domains  and contain multiple caveolae structures  <cit> .
we detected three potential homologs represented by two microarray probes, related to the sphingolipid metabolism pathway  that were up regulated in adult males and females, lung schistosomula and eggs  of s. japonicum .
glycosphingolipids have been identified as components of schistosome eggs  <cit>  and are selectively bound by the antigen presenting cells of the liver by liver/lymph node specific icam-3-grabbing non-integrins  <cit> .
a further role for sphingolipids in lung schistosomula has been previously suggested by el ridi and tallima  <cit>  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  .
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  <cit> .
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 .
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  <cit> .
surprisingly these ppp genes were not up regulated in the juvenile stages  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  <cit> .
notably, one of these trypanosome drug targets is phosphogluconate dehydrogenase, a gene that was up-regulated in adult female s. japonicum .
six genes of the calcium signalling pathway  were up-regulated in the adult male of s. japonicum, as well as in the egg, miracidium, sporocyst and cercaria aquatic/snail stages  .
elevated levels of sarco-endoplasmic reticulum atpase  in adult male worms probably reflect their requirements for increased muscle mass and the recognised importance of calcium in muscle physiology  <cit> .
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  <cit>  which was highly upregulated in the egg and miracidium and also in the sporocyst of s. japonicum .
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.
CONCLUSIONS
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 schistosome-induced 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.

 schistosomiasis afflicts ~ million people in 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. 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 hundred cdnas from s. japonicum and s. mansoni 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 contigs or singletons across seven life cycle stages of s. mansoni early liver worms, adult worms, eggs, daughter sporocysts, cercariae, and day and day schistosomula produced by mechanical transformation of cercariae followed by in vitro culture. a new 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 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 genes 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 probes on the schistosome microarray. 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 probes. next, anova applied to the data identified probes 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 ≥ fold in each lifecycle stage; these combined lists consist of probes/ genes represent stage-enriched gene transcripts. these lists comprised of a subset of unique genes or contigs, and were subjected to further annotation or in silico characterisation. the numbers of fold or fold or higher enriched genes for each individual developmental stage or lifecycle grouping is summarised in table. examples of differentially expressed genes for each lifecycle stage are presented in table 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 value in parenthesis in the total row, refers to the number of non-redundant genes enriched 2-fold or higher. hierarchal clustering to provide overview of developmental changes hierarchal clustering was employed to investigate gene expression profiles in each developmental stage. figure 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 up-regulation is shown in red, down-regulation in green, whereas the black lines indicate absence of up- or down-regulation. 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 among the ~ probes examined. for example, considerable enrichment of gene expression was evident in the egg compared with the miracidial stage. among the developmental stages, four prominent clusters of up-regulated genes were evident these were in aquatic and snail stages, adult female worms, adult female worms, and adult males. blast2go and gene ontology analysis to provide overview of functions and process the genes that were identified as being enriched fold in any given lifecycle stage were subjected to protein translational blast and gene ontology 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 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 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 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. mansoni. examples of kegg pathways identified by blast2go analysis, ie pathways that contain multiple genes involved in metabolism/anabolism are presented in supplementary figures 2- 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, sarcoplasmic reticulum atpase calcium pump, and sphingomyelin phosphodiesterase. 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 microarray hybridisations performed. a subset of differentially expressed genes, representative for each of the life cycle stages examined, were subjected to verification by real time pcr. the majority of genes that were shown differentially expressed by the microarray analysis correlated well with the results obtained by real time pcr. figure outlines the genes examined by real time pcr and presents also the corresponding microarray signal detected. 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 sj which was enriched in cercariae, eggs, miracidia and sporocysts; it was also enriched in lung stage schistosomula. a homologue of the schistosoma mansoni calcium-binding calmodulin was also upregulated in several stages, including eggs, miracidia and sporocysts. genes encoding putative dynein proteins were differentially expressed in the aquatic/snail stages including sm which was upregulated in eggs, miracidia and sporocysts; sj mirrored this expression pattern with gene expression enriched in eggs, miracidia and sporocysts. 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 contig and contig encoding cytochrome c oxidase subunit i and cytochrome c oxidase respectively. enrichment of these two genes occurred in eggs, miracidia, sporocysts and cercariae. 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 and a putative ferredoxin reductase. transporters were also enriched including contig, contig, tc, contig and contig. multiple ribosomal genes were over expressed reflecting the high protein requirements of the female worm associated with egg production. notable genes upregulated in adult male s. japonicum were those involved in molecular transport including contig and contig. other genes enriched in adult males included the endopeptidases cathepsin b and cathepsin f; components of vesicle mediated transport for both endocytosis-clathrin coat assembly protein ap, adaptin-related protein and exocytosis-syntaxin. genes involved in carbohydrate metabolism and glycolysis, including glycosyltransferase and pyruvate dehydrogenase, were also enriched. further examples of enriched genes for each of the lifecycle stages examined are presented in table comparison of lifecycle stages principal component analysis 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 exhibited similarities in gene expression. in general, the developmental stage expression profile, based on pca, clustered into three major groupings: adult males, adult females, and 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 pre-egg laying to egg-laying status at four to six weeks post-infection of the mammalian host. by contrast, the schistosomulum and miracidium did not display obvious associations or grouping with any of the other stages. gene expression profiles for 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. this was also the case when pools of biological replicates of adult male worms were analysed. after filtering the data initially for flags, probes were further filtered for anova. at the default settings, no genes were identified as being significantly variable between biological replicates; only genes were identified as variable after increasing the error cut-off rate to 18%. this indicated that a very low level of variation in gene expression occurred between biological replicates resulting from parasite samples obtained from separate infections. discussion 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 and intermediate 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 microarray analysis. 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 ≥ fold in each lifecycle stage; these combined lists consist of probes/ 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, and the selection of a fold change of ≥ can be related back to previous experiments with the same chip, thus providing confidence in our findings. as illustrated in figures and 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 and week old adult parasites, although there was an apparent change in expression from week four to week six onwards. this similarity in gene expression likely reflects parasitism of the mammalian blood vessels by the schistosome and the discrete differentiation and development of the males and females. 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 livers. 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 sm antigen [contig, dynein kda light chain flagellar outer arm and tc cathepsin b isotype 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 was possible. additional file shows that of the tc probes which had data from s. japonicum and s. mansoni, only probes could be correlated as up-regulated to the same lifecycle stages for both species. 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. a brief description of some of the other genes developmentally expressed in the various lifecycle stages of s. japonicum follows. sm 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. sm 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 sm 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. sm may aid s. mansoni in penetration of the mammalian host and our results support this hypothesis as the transcript of the s. japonicum homologue 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, miracidium, sporocyst, and lung stage schistosomulum. 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 sj 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 in the adult male and the lung schistosomulum. 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. 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 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 expression was up-regulated in the egg, thereby confirming its importance in this stage and also the adult male. the antioxidant properties of thioredoxin would be expected to protect the schistosome from reactive oxygen species released by host inflammatory responses 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 in lung schistosomula and the aquatic/snail stages. 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 a 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 expression was raised in lung schistosomula and adult males 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 response. 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 of the potassium channel gene in adult male s. japonicum, which has an extensively developed musculature. sm has sequence similarity to dynein light chains, 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 sm 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, sj, mirrored in part, the previous studies on sm but transcripts for sj were enriched predominantly in the egg, miracidium and sporocyst 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 sj, as determined by the microarray analysis and real time pcr was sm, being upregulated in eggs, miracidia and sporocysts. 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 sm were found in miracidia which utilise cilia for locomotion. while sm 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 systems. sm has homology to members of the lc family of dlcs, of which one member, dynll is up-regulated in mammalian testes and lung, two tissues which have considerable numbers of cilia or flagella present. the s. japonicum transcript encoding the homologue of sperm associated antigen is likely a component of tubulin structures and was enriched in both the egg, the adult stages and to a lesser extent, juvenile males. spag is the murine orthologue of chlamydomonas pf 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 and contain multiple caveolae structures. we detected three potential homologs represented by two microarray probes, related to the sphingolipid metabolism pathway that were up regulated in adult males and females, lung schistosomula and eggs of s. japonicum. 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. 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. 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. 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 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. notably, one of these trypanosome drug targets is phosphogluconate dehydrogenase, a gene that was up-regulated in adult female s. japonicum. six genes of the calcium signalling pathway were up-regulated in the adult male of s. japonicum, as well as in the egg, miracidium, sporocyst and cercaria aquatic/snail stages. elevated levels of sarco-endoplasmic reticulum atpase 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. 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 schistosome-induced 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. 
gene ontologies of the differentially expressed genes revealed a wide range of functions and processes. 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. 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. microarray analysis was used to profile developmental gene expression in the asian species, schistosoma japonicum. 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. 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. their life cycle is characterised by distinct parasitic and free-living phases involving mammalian and snail hosts and freshwater.
 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, juvenile and adult. 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.  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.  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. 