BACKGROUND
schistosomiasis, the most widespread trematode infection, is estimated to infect around  <dig> million people in  <dig> countries of the developing world, leading to chronic debilitating disease and up to  <dig>  <dig> deaths per year  <cit> .
the freshwater snail biomphalaria glabrata is an intermediate host for schistosoma mansoni, a digenean parasite that causes human intestinal schistosomiasis.
this medically relevant gastropod is a member of one of the largest invertebrate phyla, the mollusca, which are lophotrochozoans, a lineage of animal evolution distinct from ecdysoans, represented by present model invertebrates such as caenorhabditis and drosophila.
many of the genomic studies in molluscs have focussed on bivalves owing to the importance of these organisms in aquaculture and fisheries and to their role in marine environmental science  <cit> , while in gastropods, expressed sequence tag  studies have been carried out in lymnaea stagnalis  <cit>  and b. glabrata  <cit> .
characterizing genes and biochemical pathways central to immunity and defence in gastropods is predicted to reveal innovative data, significant due to the medical and economic importance of intermediate host snails in parasite transmission, and b. glabrata is poised to become the next invertebrate model organism.
indeed, advances towards the ultimate goal of obtaining the b. glabrata genome sequence  <cit>  include the complete mitochondrial genome  <cit> , the development of a bac library for genome sequencing  <cit>  and complementary gene discovery projects  <cit> .
interactions between snails and schistosomes are complex and there exists an urgent need to elucidate pathways involved in snail-parasite relationships as well as to identify those factors involved in the intricate balance between the snail internal defence system  and trematode infectivity mechanisms that determine the success or failure of an infection .
molluscs appear to lack an adaptive immune system like that found in vertebrates and, instead, are considered to use various innate mechanisms involving cell-mediated and humoral reactions  that interact to recognize and eliminate invading pathogens or parasites in incompatible or resistant snails .
however, a diverse family of fibrinogen related proteins  containing immunoglobulin-like domains has been discovered in b. glabrata and may play a role in snail defence  <cit> .
circulating haemocytes  in the snail haemolymph are known to aggregate in response to trauma, phagocytose small particles  and encapsulate larger ones, such as parasites.
final killing is effected by haemocyte-mediated cytotoxicity mechanisms involving non-oxidative and oxidative pathways, including lysosomal enzymes and reactive oxygen/nitrogen intermediates  <cit> .
certain alleles of cytosolic copper/zinc superoxide dismutase  have been associated with resistance  <cit>  also suggesting these processes are important in the snail ids.
compatible snail-trematode infections may reflect the parasite's capacity to avoid or interfere with the innate response of the snail.
the term 'resistant' can be applied to those individuals within a single snail species that are able to evade infection by a species or strain of schistosome that is normally capable of parasitizing that species of snail.
identification of specific molecules mediating the defensive events in snail intermediate hosts, in particular those differentially expressed in resistant/incompatible snails, is expected to reveal much about the pathways and processes involved.
previous studies of differential gene expression in resistant and susceptible snail lines have used a number of different techniques including differential display  <cit>  and suppression subtractive hybridization   <cit>  to identify in each case, some differentially expressed genes.
the application of cdna microarray technology allows large-scale analysis of differential gene expression, using large numbers of sequenced cdna clones, which will enable a more detailed examination of the b. glabrata transcriptome.
this paper describes the construction of the first b. glabrata microarray using previously sequenced orestes clones  <cit>  as well as new sequences from ssh libraries enriched for differentially expressed genes, and demonstrates its use in detecting differentially expressed transcripts in the haemocytes of parasite-challenged resistant and susceptible snail lines, with the aim of identifying strain-specific transcripts potentially involved in snail internal defence.
RESULTS
sequence analysis of ssh and orestes clones for microarray fabrication
to initiate large scale analysis of differential gene expression in b. glabrata, a cdna microarray was constructed from  <dig> previously sequenced pcr-amplified orestes clones  <cit>  and, in addition to these,  <dig> new sequenced clones from existing ssh libraries  <cit> .
it also contains transcripts derived from our previous expression studies  <cit> .
the orestes clones were from the first  <dig> bg orestes libraries  <cit> , available at the time of printing and were derived from  <dig> different tissues , from the two snail strains used in this experiment, nhm <dig>  and nhm <dig> .
all the available est clones were printed, including those from tissues other than haemocytes, since it could not be ascertained a priori that none of these would be involved in snail defence, or that transcripts originally derived from tissues other than haemocytes would definitely not be expressed in the haemocytes.
the analyzed ests were screened to remove duplicates within each library.
in addition to the clones derived from the orestes libraries,  <dig> haemocyte and haemopoietic organ  ssh libraries  were available for sequencing and clone selection.
two to three  <dig> well plates of clones were sequenced from each library.
a total of  <dig> sequences were obtained , of which just over 2%  had been previously sequenced from b. glabrata.
over 35% had no known identity , either having no match in publicly available sequence databases, or matching a protein or other est that had no known identity.
after removal of duplicate, short  and ribosomal sequences,  <dig> were available for printing on the microarray.
analysis of the types of sequences obtained from the b. glabrata ssh libraries identified with blast searches of genbank.
the available ests from ssh and orestes  were compared to genbank and where possible gene ontology was assigned.
the assignments in all three categories, molecular function, biological process and cellular component show no obvious differences in the sequence types obtained from the two different methods .
an analysis of metabolic pathways using the kyoto encyclopaedia of genes and genomes  showed that, generally, genes from similar pathways were identified from both orestes and ssh, for instance, genes implicated in the immune system showed similar numbers in each pathway from both techniques.
however, there were many more genes involved in oxidative phosphorylation sequenced from ssh  when compared to orestes  and the same was seen with the numbers of ribosomal proteins from ssh  compared to orestes .
expression profiling by microarray analysis
the microarray was used for a direct comparison of mrna from haemocytes of parasite-exposed resistant and susceptible snails.
haemocytes sampled over the first  <dig> h post parasite exposure were compared to investigate differences between snail lines after parasite exposure during the period when the haemocytes are thought to respond and encapsulate the parasite in resistant snails  <cit> .
this approach was designed to identify large and significant differences in gene expression between the two snail lines, although small, transient rna expression changes might not be identified.
since very small amounts of rna were available from the haemocytes, independent smart amplifications  <cit>  and labelling reactions were carried out and four technical replicate hybridizations performed.
smart amplification has been shown to have a higher true-positive rate than global amplification, but has a lower absolute discovery rate, and a systematic compression of observed expression ratio  <cit> .
analysis of duplicate spots on each array showed good correlation of normalized mean pixel intensity ratios  demonstrating that the hybridizations were consistent within each array .
the data were screened to remove weak signals below the level of background hybridization in both channels based on negative control vector levels of hybridization.
comparisons were made between technical replicate arrays, of mean  normalized mean pixel intensity ratios obtained for each clone, using the data that passed the filtering and background  threshold .
correlation coefficients for each array comparison were high, ranging from  <dig>  to  <dig> , showing good agreement in gene expression values between array hybridizations, suggesting that smart amplification of the cdna was not affecting representation of transcripts.
the amplified cdna demonstrated a good degree of hybridization reproducibility and a low level of variation between technical replicates, giving confidence to the assignment of differentially expressed strain-associated transcripts.
to identify genes showing differential expression between the resistant and susceptible snail haemocytes, 99% confidence intervals were calculated based on the remaining data, but excluding data from ssh clones.
these were not included in calculating significance intervals, since the 99% confidence intervals are based on the assumption that the clones are normally distributed; however since the ssh clones were enriched for differential expression this assumption is not true for these clones.
clones that fell outside the 99% confidence interval in  <dig> or more of the  <dig> replicates were designated differentially expressed .
a total of  <dig> differentially expressed transcripts were identified,  <dig> were from the resistant haemocytes and only  <dig> were from the susceptible haemocytes.
all of these identified genes showed an average  of between  <dig> and  <dig>  fold difference in expression between resistance and susceptible snails .
nine candidates,  <dig> susceptible-associated transcripts and  <dig> resistant-associated transcripts, were selected for confirmation of differential expression with qpcr using unamplified cdna .
in each case the qpcr result confirmed the result found from the microarray.
the difference in expression of all of these genes was much greater than that indicated in the array; in many cases the transcript was barely detectable in the strain which showed less expression from the microarray.
this confirms the suggestion that smart amplification compresses the observed expression ratio and may also suggest that the fold difference in gene expression observed from the microarray underestimates the true difference in expression of transcripts.
genes identified as differentially expressed between resistant and susceptible snail lines, ranked in order of mean  fold difference in expression, based on normalized mean intensity ratios.
negative fold difference values indicate resistant-associated clones.
asource indicates whether the clone was from a suppression subtractive hybridization  library or open reading frame est  library.
bthe name, organism, accession number and e-value given for the best match with genbank using blastx.
* chosen for qpcr
^ also confirmed from ssh experiment
1– <dig> clusters of overlapping sequences
all the identified differentially expressed genes were originally sequenced from haemocyte libraries ; although many other transcripts were present in the samples, they were not differentially expressed.
just over a quarter  of the differentially expressed genes were from orestes and the remaining from ssh.
clustering the identified differentially expressed sequences revealed  <dig> clusters, one with  <dig> sequences and  <dig> with two , leaving  <dig> unique sequences or clusters.
one of the clusters contained two susceptible specific sequences leaving  <dig> unique susceptible and  <dig> resistant specific sequences.
all of the susceptible-specific transcripts had no database matches and were derived from ssh.
they included  <dig> sequences  that had previously been identified as differentially expressed between resistant and susceptible snail haemocytes by screening macroarrays of ssh clones and confirmed with rt-pcr  <cit> .
annotation of identified sequences
sixty of the  <dig> sequences or clusters did not identify other genbank sequences in blastx searches  and were categorized as genes with unknown function.
the functions of the other identified genes were more closely examined using gene ontology and kegg pathway analysis.
gene ontologies were assigned for  <dig> genes of the  <dig> differentially expressed transcripts , including, among other genes ; titin , importin  <dig> , copine  <dig> , elongation factor 1α  and ef- <dig>  and the stress response protein hsp <dig> .
kegg pathway analysis identified  <dig> enzymes with  <dig> clones in a number of metabolic pathways , including malate dehydrogenase  involved in carbohydrate metabolism; six genes involved in oxidative phosphorylation in energy metabolism and six clones involved in amino acid metabolism including two clones homologous to ornithine decarboxylase  <dig>  .
although both sequences identified odc <dig> in blast searches, they did not cluster at the nucleotide level, despite identifying overlapping protein sequence and are likely to be paralogous.
two genes coding for odc <dig> have been found in both xenopus laevis  <cit>  and drosophila melanogaster  <cit> .
six genes involved in translation were identified as well as the ubiquitin conjugating enzyme, ube2d/e , involved in folding, sorting and degradation.
we also identified one sequence with a weak homology to cathepsin l cysteine protease , involved in degradation of exogenous and endogenous proteins in lysosomes.
the function of these genes and their potential involvement in the snail ids is discussed further below.
pathways identified by genes found only in the parasite-exposed resistant snails.
discussion
a b. glabrata cdna microarray was successfully constructed consisting of  <dig> clones from headfoot, ovotestis, haemopoeitic organ and haemoctyes of two different strains of b. glabrata.
screening the array with rna from haemocytes of schistosome-exposed resistant and susceptible snail strains identified  <dig> differentially expressed genes or gene clusters.
there was little difference between technical replicates indicating that there were no sampling effects from amplifying small amounts of starting rna, giving confidence in the assignment of strain-associated differential expression.
examination of the expression of  <dig> selected genes using real-time rt-pcr on unamplified cdna confirmed differential expression in each case.
most of the genes identified  were present only in the resistant snail line.
that only a few differentially expressed genes were identified in susceptible snails may be due to the failure of the snail's defence system to recognize the parasite, possibly because s. mansoni sporocyts may mimic snail moieties  or because they may rapidly acquire molecules from the host with which to disguise themselves  <cit> .
alternatively, s. mansoni sporocysts may actively suppress a humoral response in susceptible hosts.
in either case the transcripts identified in the resistant snails are genes, either constitutively over-expressed in resistant snails, not suppressed in resistant snails, or activated when the snail does recognize the parasite's presence and responds.
of the  <dig> genes, or gene clusters, identified in susceptible snails, two  had been previously identified and confirmed as differentially expressed in our earlier experiments  <cit>  suggesting that they are consistently differentially expressed by susceptible snails.
since none has a known function it is difficult to speculate on their possible role.
over half  of the genes identified with higher expression levels in the resistant snails have no known function.
although many of these may play a significant role in defence, further characterization of their function is required to ascertain what that role might be in the snail ids.
of the ests with homology to known genes, the observed differential expression of genes involved in energy metabolism, in particular oxidative phosphorylation, amino acid metabolism and genetic translation, indicates a general increase in cellular activity, consistent with generating the necessary components for mounting a defensive response.
for example, ef-1α  and ef- <dig> , as well as several other proteins involved in protein synthesis, were identified, including crooked neck-like  <dig> protein  involved in pre mrna splicing.
differential expression of these types of transcripts suggests an increase in general cell activity, with increased production of new proteins in response to infection in the resistant compared to the susceptible line.
two paralogous ornithine decarboxylase  odc <dig> gene fragments  were identified as differentially expressed in resistant snails.
odc is the first and rate-limiting enzyme in the polyamine biosynthesis pathway, which decarboxylates l-ornithine  to form putrescine.
polyamines  regulate gene expression, modulate cell signalling and are required for normal cell proliferation, important in inflammatory and infection processes  <cit> .
polyamines have been described as primordial stress molecules with defensive functions against diverse stresses  <cit> , including protecting cells from dna strand breakage induced by reactive oxygen species  and functioning directly as free radical scavengers  <cit> .
generation of ros has been shown to play a role in sporocyst killing by molluscan haemocytes in incompatible snail-trematode systems  <cit> .
production of polyamines could protect the resistant snail's own cells from the damaging effects of ros.
the identification of odc in resistant snails may also imply activation of arginine metabolic pathways, which play an important role in inflammation and wound healing  <cit> .
increased levels of odc in resistant snails indicate production of the substrate l-ornithine, inferring the depletion of l-arginine by arginase activity and subsequent inhibition of nitric oxide  preventing damage to host cells.
l-arginine  is the substrate for both arginase  and nitric oxide synthase  .
in inflammatory diseases, it is thought that no production from l-arg is involved in the initial early host response creating an overall cytotoxic environment, whilst l-ornithine production from l-arg is involved in healing, promoting cell growth and proliferation  <cit> .
in its role as host defender, no regulates inflammatory responses and acts as an effector molecule of haemocyte cytotoxicity against invaders , whilst at the same time when produced in excessive amounts, no is cytotoxic not only to the invading schistosome but also to the snail hosts own cells.
hence, infected snail hosts must strive to find a balance between anti-schistosome and cytotoxic effects of no towards its own cells.
no has been shown to mediate host-protective responses in a variety of parasitic infections  <cit>  including the killing of s. mansoni sporocysts by haemocytes from resistant b. glabrata  <cit> .
differential expression of odc in resistant snails may suggest an involvement in snail defence by scavenging the damaging free radicals produced during the production of ros by haemocytes for cytotoxic killing of sporocysts; in regulating production of cytotoxic no by depleting the competing substrate; or, with the production of polyamines leading to dna protection and cell proliferation, aiding wound healing following miracidial penetration.
also identified as differentially expressed in the resistant snail line, hsp <dig>  was previously identified, using differential display, as upregulated only in the resistant snails after parasite exposure  <cit> , verifying our original suggestion that upregulation of hsp <dig> is an expected response to infection, and is absent from the susceptible snails.
the induction of heat shock or stress proteins represents a homeostatic defence mechanism of cells to metabolic and environmental insults, and this response has previously been demonstrated in molluscs, for example in oyster haemocytes in response to environmental stimulus  <cit> .
experiments with mollusc haemocytes derived from crassostrea gigas and haliotis tuberculata demonstrated that the hsp <dig> gene promoter is induced by noradrenaline and α-adrenergic stimulations  <cit>  showing that the response in these cells is integrated with neuroendocrine signalling pathways.
several proteins potentially involved in cell signalling were also identified in the resistant snails only.
one transcript  was found to be homologous to titin , which is a giant springlike protein responsible for passive tension generation and for positioning of the thick filament at the centre of vertebrate striated muscle sacromeres.
this protein has multiple elastic and signalling functions derived from a complex subdomain structure, including a series of immunoglobulin  domains.
the kinase domain of titin initiates a signal transduction cascade that controls sarcomere assembly, protein turnover, and transcriptional control in response to mechanical changes in vertebrates .
smaller but related molecules have been identified in invertebrate striated or smooth muscles, variously named mini-titins, projectins or twitchins , depending on their origin  <cit> .
the expression of this gene in resistant snails may indicate a signalling or muscle response to parasite infection that is not initiated in susceptible snails.
in addition to titin, a cytoplasmic intermediate filament  protein  was identified only in the resistant snails.
rather than merely providing a cellular framework, recent research has demonstrated that ifs are dynamic, motile elements of the cytoskeleton in vertebrate cells  <cit> .
an if protein has previously been identified in b. glabrata, using a comparative proteomic approach, as differentially expressed in snails resistant to e. caproni  <cit>  and it was suggested that this gene could be a candidate to explain differences in susceptibility/resistance, considering the major role of haemocyte mobility and adherence capabilities in defence to the parasite.
interestingly, uninfected susceptible snails demonstrated higher levels of the if gene transcript; however post-exposure to the parasite the resistant snails showed a significant increase in transcript levels, while susceptible snails had a decrease.
our sequence does not identify this transcript either at the nucleotide or protein level and demonstrates close homology  to a neuronal if, neurofilament protein nf <dig> from helix aspersa  <cit>  one of the type iv ifs.
it has also been suggested that some ifs participate in signalling processes by providing a scaffold to bring together activated map kinases  with other molecules  <cit> .
other molecules potentially involved in signaling processes in the cell are importin  <dig> and copine  <dig>  a gene fragment homologous to importin  <dig>  was identified in the resistant snails.
this protein functions as a nuclear import cofactor, and in drosophila, has been implicated in the control of multiple signal transduction pathways, including the direct nuclear import of the activated  form of map kinase   <cit> .
s. mansoni excretory secretory products  and whole sporocysts have been shown to affect erk signalling in the haemocytes of susceptible snails, but not resistant ones  <cit>  suggesting that the disruption of erk signalling in haemocytes facilitates s. mansoni survival within susceptible b. glabrata.
another gene, copine  <dig> , implicated in membrane trafficking  <cit>  and signal transduction  <cit>  was identified in the resistant snails.
copine  <dig> is a calcium-dependent membrane binding protein which, in mammals, has been shown to regulate the nf kappa b transcriptional responses  <cit> .
in arabidopsis thaliana, copine  <dig> is suggested to play a role in plant disease resistant responses, possibly as a suppressor of defense responses including the hypersensitive cell death response  <cit> .
we identified two hydrolytic enzymes in haemocytes from the resistant snail line, elastase  and cathepsin l-like protease precursor , also known as cysteine proteinase.
higher levels of cysteine proteinase activity have previously been observed in hepatopancreas extracts from resistant  b. glabrata when compared to susceptible  snails  <cit> .
cathepsin l, cathepsin b and elastase were also identified among other hydrolytic enzymes from a hepatopancreas est library derived from resistant snails and cathepsin b demonstrated greater up-regulation in resistant snails compared to susceptible snails upon parasite exposure  <cit> .
in invertebrates, cysteine proteases play a major role in the lysosomal proteolytic system, responsible for intracellular protein degradation  <cit> .
this role, in the lysis of phagocytosed particles, may be significant in the breakdown of encapsulated sporocysts or phagocytosed parasite components.
in addition to a role as a scavenger for the clearance of unwanted proteins, this protease plays an important role in antigen processing and presentation in mammalian immune systems  <cit>  and lysosomal proteases have been implicated in innate immunity in insect haemocytes  <cit> .
genome-wide analysis of immune challenged drosophila revealed elevated expression of cathepsin l when infected with either gram-positive bacteria or fungi  <cit> .
similarly, cathepsin l was highly expressed in wssv  resistant shrimp, suggesting that it is involved in defence responses  <cit> .
a cathepsin l-like gene  has previously been identified in b. glabrata susceptible to echinostoma caproni infection  <cit> .
our sequence  does not cluster with this and identifies  a cathepsin-like domain 3' to sequence identified by the other gene fragment, suggesting that the ests may be two non-overlapping parts of the same gene.
the discrepancy between the observed changes in gene expression may be due to the different response elicited by two different parasite species.
s. mansoni fails to produce a response in susceptible snails as they fail to recognise the presence of the parasite, while in echinostome infections a defence response may be mounted by the snail, but is interfered with by the parasite  <cit> .
differential expression of ubiquitin conjugating enzyme  e <dig>   was detected in exposed resistant snails and may indicate removal of the phagocytosed sporocyst.
ubiquitination plays an important role in various cellular functions including apoptosis, cell cycle progression, transcription and endocytosis  <cit> .
a major role is regulating the half-life of proteins by targeting them for 26s proteasomal degradation, removing denatured, damaged or improperly translated proteins.
in this study we compared haemocytes obtained from resistant and susceptible snail strains  <dig> to  <dig> h after exposure to s. mansoni, and have found differentially expressed transcripts potentially involved in a range of responses from signalling and inflammation responses through to lysis of proteinacous products  and processing/degradation of such targeted products by ubiquitination.
in future, examination of biological replicates will increase the confidence that the transcripts identified in this study are truly significant in the snail ids, in addition to demonstrating the amplification techniques utilized in this study are robust.
examination of a series of narrower time slots will enable us to unravel the sequence of processes involved and highlight genes initiating the cascade in resistant  hosts.
a simultaneous comparison of both parasite-exposed and unexposed snails from both snail lines will also determine if the basis of the snail's resistance is due to an underlying difference in gene expression between the strains, or from differences in their response to the parasite.
such comparisons may also be significant in determining why susceptible snails do not respond to infection, either by non-recognition of invading parasite or by active suppression of innate response by the parasite, which may be indicated if s. mansoni-exposure results in significantly more down-regulated genes in susceptible snails.
at present we can only speculate on the function in b. glabrata of the genes identified.
future expression experiments involving rnai or in situ hybridization may elucidate their role in resistance.
CONCLUSIONS
in conclusion, the advent of the first cdna microarray for b. glabrata leads the way for detailed analysis of the b. glabrata transcriptome and can be developed further to include more cdnas as these become available.
the array described here is particularly suited for analysis of snail haemocytes and their role in the snail ids since it contains a large proportion of genes sequenced from haemocytes.
despite the current limited size of the array, prior enrichment for differentially expressed genes using the ssh approach has enabled us to identify a number of genes and pathways differentially expressed in the resistant snail line and potentially involved in the defence of resistant snails to schistosome infection.
these include hydrolytic enzymes such as elastase and the cysteine protease, cathepsin l; ornithine decarboxylase, involved in the production of polyamines; hsp70; potential signalling molecules importin  <dig> and copine  <dig> and transcription enzymes such as ef-1α and ef- <dig>  continued development of this array has great potential for examining and understanding the functions of the b. glabrata transcriptome.

 schistosomiasis, the most widespread trematode infection, is estimated to infect around million people in countries of the developing world, leading to chronic debilitating disease and up to deaths per year. the freshwater snail biomphalaria glabrata is an intermediate host for schistosoma mansoni, a digenean parasite that causes human intestinal schistosomiasis. this medically relevant gastropod is a member of one of the largest invertebrate phyla, the mollusca, which are lophotrochozoans, a lineage of animal evolution distinct from ecdysoans, represented by present model invertebrates such as caenorhabditis and drosophila. many of the genomic studies in molluscs have focussed on bivalves owing to the importance of these organisms in aquaculture and fisheries and to their role in marine environmental science, while in gastropods, expressed sequence tag studies have been carried out in lymnaea stagnalis and b. glabrata. characterizing genes and biochemical pathways central to immunity and defence in gastropods is predicted to reveal innovative data, significant due to the medical and economic importance of intermediate host snails in parasite transmission, and b. glabrata is poised to become the next invertebrate model organism. indeed, advances towards the ultimate goal of obtaining the b. glabrata genome sequence include the complete mitochondrial genome, the development of a bac library for genome sequencing and complementary gene discovery projects. interactions between snails and schistosomes are complex and there exists an urgent need to elucidate pathways involved in snail-parasite relationships as well as to identify those factors involved in the intricate balance between the snail internal defence system and trematode infectivity mechanisms that determine the success or failure of an infection. molluscs appear to lack an adaptive immune system like that found in vertebrates and, instead, are considered to use various innate mechanisms involving cell-mediated and humoral reactions that interact to recognize and eliminate invading pathogens or parasites in incompatible or resistant snails. however, a diverse family of fibrinogen related proteins containing immunoglobulin-like domains has been discovered in b. glabrata and may play a role in snail defence. circulating haemocytes in the snail haemolymph are known to aggregate in response to trauma, phagocytose small particles and encapsulate larger ones, such as parasites. final killing is effected by haemocyte-mediated cytotoxicity mechanisms involving non-oxidative and oxidative pathways, including lysosomal enzymes and reactive oxygen/nitrogen intermediates. certain alleles of cytosolic copper/zinc superoxide dismutase have been associated with resistance also suggesting these processes are important in the snail ids. compatible snail-trematode infections may reflect the parasite's capacity to avoid or interfere with the innate response of the snail. the term'resistant' can be applied to those individuals within a single snail species that are able to evade infection by a species or strain of schistosome that is normally capable of parasitizing that species of snail. identification of specific molecules mediating the defensive events in snail intermediate hosts, in particular those differentially expressed in resistant/incompatible snails, is expected to reveal much about the pathways and processes involved. previous studies of differential gene expression in resistant and susceptible snail lines have used a number of different techniques including differential display and suppression subtractive hybridization to identify in each case, some differentially expressed genes. the application of cdna microarray technology allows large-scale analysis of differential gene expression, using large numbers of sequenced cdna clones, which will enable a more detailed examination of the b. glabrata transcriptome. this paper describes the construction of the first b. glabrata microarray using previously sequenced orestes clones as well as new sequences from ssh libraries enriched for differentially expressed genes, and demonstrates its use in detecting differentially expressed transcripts in the haemocytes of parasite-challenged resistant and susceptible snail lines, with the aim of identifying strain-specific transcripts potentially involved in snail internal defence.  sequence analysis of ssh and orestes clones for microarray fabrication to initiate large scale analysis of differential gene expression in b. glabrata, a cdna microarray was constructed from previously sequenced pcr-amplified orestes clones and, in addition to these, new sequenced clones from existing ssh libraries. it also contains transcripts derived from our previous expression studies. the orestes clones were from the first bg orestes libraries, available at the time of printing and were derived from different tissues, from the two snail strains used in this experiment, nhm and nhm. all the available est clones were printed, including those from tissues other than haemocytes, since it could not be ascertained a priori that none of these would be involved in snail defence, or that transcripts originally derived from tissues other than haemocytes would definitely not be expressed in the haemocytes. the analyzed ests were screened to remove duplicates within each library. in addition to the clones derived from the orestes libraries, haemocyte and haemopoietic organ ssh libraries were available for sequencing and clone selection. two to three well plates of clones were sequenced from each library. a total of sequences were obtained, of which just over 2% had been previously sequenced from b. glabrata. over 35% had no known identity, either having no match in publicly available sequence databases, or matching a protein or other est that had no known identity. after removal of duplicate, short and ribosomal sequences, were available for printing on the microarray. analysis of the types of sequences obtained from the b. glabrata ssh libraries identified with blast searches of genbank. the available ests from ssh and orestes were compared to genbank and where possible gene ontology was assigned. the assignments in all three categories, molecular function, biological process and cellular component show no obvious differences in the sequence types obtained from the two different methods. an analysis of metabolic pathways using the kyoto encyclopaedia of genes and genomes showed that, generally, genes from similar pathways were identified from both orestes and ssh, for instance, genes implicated in the immune system showed similar numbers in each pathway from both techniques. however, there were many more genes involved in oxidative phosphorylation sequenced from ssh when compared to orestes and the same was seen with the numbers of ribosomal proteins from ssh compared to orestes. expression profiling by microarray analysis the microarray was used for a direct comparison of mrna from haemocytes of parasite-exposed resistant and susceptible snails. haemocytes sampled over the first h post parasite exposure were compared to investigate differences between snail lines after parasite exposure during the period when the haemocytes are thought to respond and encapsulate the parasite in resistant snails. this approach was designed to identify large and significant differences in gene expression between the two snail lines, although small, transient rna expression changes might not be identified. since very small amounts of rna were available from the haemocytes, independent smart amplifications and labelling reactions were carried out and four technical replicate hybridizations performed. smart amplification has been shown to have a higher true-positive rate than global amplification, but has a lower absolute discovery rate, and a systematic compression of observed expression ratio. analysis of duplicate spots on each array showed good correlation of normalized mean pixel intensity ratios demonstrating that the hybridizations were consistent within each array. the data were screened to remove weak signals below the level of background hybridization in both channels based on negative control vector levels of hybridization. comparisons were made between technical replicate arrays, of mean normalized mean pixel intensity ratios obtained for each clone, using the data that passed the filtering and background threshold. correlation coefficients for each array comparison were high, ranging from to, showing good agreement in gene expression values between array hybridizations, suggesting that smart amplification of the cdna was not affecting representation of transcripts. the amplified cdna demonstrated a good degree of hybridization reproducibility and a low level of variation between technical replicates, giving confidence to the assignment of differentially expressed strain-associated transcripts. to identify genes showing differential expression between the resistant and susceptible snail haemocytes, 99% confidence intervals were calculated based on the remaining data, but excluding data from ssh clones. these were not included in calculating significance intervals, since the 99% confidence intervals are based on the assumption that the clones are normally distributed; however since the ssh clones were enriched for differential expression this assumption is not true for these clones. clones that fell outside the 99% confidence interval in or more of the replicates were designated differentially expressed. a total of differentially expressed transcripts were identified, were from the resistant haemocytes and only were from the susceptible haemocytes. all of these identified genes showed an average of between and fold difference in expression between resistance and susceptible snails. nine candidates, susceptible-associated transcripts and resistant-associated transcripts, were selected for confirmation of differential expression with qpcr using unamplified cdna. in each case the qpcr result confirmed the result found from the microarray. the difference in expression of all of these genes was much greater than that indicated in the array; in many cases the transcript was barely detectable in the strain which showed less expression from the microarray. this confirms the suggestion that smart amplification compresses the observed expression ratio and may also suggest that the fold difference in gene expression observed from the microarray underestimates the true difference in expression of transcripts. genes identified as differentially expressed between resistant and susceptible snail lines, ranked in order of mean fold difference in expression, based on normalized mean intensity ratios. negative fold difference values indicate resistant-associated clones. asource indicates whether the clone was from a suppression subtractive hybridization library or open reading frame est library. bthe name, organism, accession number and e-value given for the best match with genbank using blastx. * chosen for qpcr ^ also confirmed from ssh experiment 1– clusters of overlapping sequences all the identified differentially expressed genes were originally sequenced from haemocyte libraries; although many other transcripts were present in the samples, they were not differentially expressed. just over a quarter of the differentially expressed genes were from orestes and the remaining from ssh. clustering the identified differentially expressed sequences revealed clusters, one with sequences and with two, leaving unique sequences or clusters. one of the clusters contained two susceptible specific sequences leaving unique susceptible and resistant specific sequences. all of the susceptible-specific transcripts had no database matches and were derived from ssh. they included sequences that had previously been identified as differentially expressed between resistant and susceptible snail haemocytes by screening macroarrays of ssh clones and confirmed with rt-pcr. annotation of identified sequences sixty of the sequences or clusters did not identify other genbank sequences in blastx searches and were categorized as genes with unknown function. the functions of the other identified genes were more closely examined using gene ontology and kegg pathway analysis. gene ontologies were assigned for genes of the differentially expressed transcripts, including, among other genes; titin, importin, copine, elongation factor 1α and ef- and the stress response protein hsp. kegg pathway analysis identified enzymes with clones in a number of metabolic pathways, including malate dehydrogenase involved in carbohydrate metabolism; six genes involved in oxidative phosphorylation in energy metabolism and six clones involved in amino acid metabolism including two clones homologous to ornithine decarboxylase. although both sequences identified odc in blast searches, they did not cluster at the nucleotide level, despite identifying overlapping protein sequence and are likely to be paralogous. two genes coding for odc have been found in both xenopus laevis and drosophila melanogaster. six genes involved in translation were identified as well as the ubiquitin conjugating enzyme, ube2d/e, involved in folding, sorting and degradation. we also identified one sequence with a weak homology to cathepsin l cysteine protease, involved in degradation of exogenous and endogenous proteins in lysosomes. the function of these genes and their potential involvement in the snail ids is discussed further below. pathways identified by genes found only in the parasite-exposed resistant snails. discussion a b. glabrata cdna microarray was successfully constructed consisting of clones from headfoot, ovotestis, haemopoeitic organ and haemoctyes of two different strains of b. glabrata. screening the array with rna from haemocytes of schistosome-exposed resistant and susceptible snail strains identified differentially expressed genes or gene clusters. there was little difference between technical replicates indicating that there were no sampling effects from amplifying small amounts of starting rna, giving confidence in the assignment of strain-associated differential expression. examination of the expression of selected genes using real-time rt-pcr on unamplified cdna confirmed differential expression in each case. most of the genes identified were present only in the resistant snail line. that only a few differentially expressed genes were identified in susceptible snails may be due to the failure of the snail's defence system to recognize the parasite, possibly because s. mansoni sporocyts may mimic snail moieties or because they may rapidly acquire molecules from the host with which to disguise themselves. alternatively, s. mansoni sporocysts may actively suppress a humoral response in susceptible hosts. in either case the transcripts identified in the resistant snails are genes, either constitutively over-expressed in resistant snails, not suppressed in resistant snails, or activated when the snail does recognize the parasite's presence and responds. of the genes, or gene clusters, identified in susceptible snails, two had been previously identified and confirmed as differentially expressed in our earlier experiments suggesting that they are consistently differentially expressed by susceptible snails. since none has a known function it is difficult to speculate on their possible role. over half of the genes identified with higher expression levels in the resistant snails have no known function. although many of these may play a significant role in defence, further characterization of their function is required to ascertain what that role might be in the snail ids. of the ests with homology to known genes, the observed differential expression of genes involved in energy metabolism, in particular oxidative phosphorylation, amino acid metabolism and genetic translation, indicates a general increase in cellular activity, consistent with generating the necessary components for mounting a defensive response. for example, ef-1α and ef-, as well as several other proteins involved in protein synthesis, were identified, including crooked neck-like protein involved in pre mrna splicing. differential expression of these types of transcripts suggests an increase in general cell activity, with increased production of new proteins in response to infection in the resistant compared to the susceptible line. two paralogous ornithine decarboxylase odc gene fragments were identified as differentially expressed in resistant snails. odc is the first and rate-limiting enzyme in the polyamine biosynthesis pathway, which decarboxylates l-ornithine to form putrescine. polyamines regulate gene expression, modulate cell signalling and are required for normal cell proliferation, important in inflammatory and infection processes. polyamines have been described as primordial stress molecules with defensive functions against diverse stresses, including protecting cells from dna strand breakage induced by reactive oxygen species and functioning directly as free radical scavengers. generation of ros has been shown to play a role in sporocyst killing by molluscan haemocytes in incompatible snail-trematode systems. production of polyamines could protect the resistant snail's own cells from the damaging effects of ros. the identification of odc in resistant snails may also imply activation of arginine metabolic pathways, which play an important role in inflammation and wound healing. increased levels of odc in resistant snails indicate production of the substrate l-ornithine, inferring the depletion of l-arginine by arginase activity and subsequent inhibition of nitric oxide preventing damage to host cells. l-arginine is the substrate for both arginase and nitric oxide synthase. in inflammatory diseases, it is thought that no production from l-arg is involved in the initial early host response creating an overall cytotoxic environment, whilst l-ornithine production from l-arg is involved in healing, promoting cell growth and proliferation. in its role as host defender, no regulates inflammatory responses and acts as an effector molecule of haemocyte cytotoxicity against invaders, whilst at the same time when produced in excessive amounts, no is cytotoxic not only to the invading schistosome but also to the snail hosts own cells. hence, infected snail hosts must strive to find a balance between anti-schistosome and cytotoxic effects of no towards its own cells. no has been shown to mediate host-protective responses in a variety of parasitic infections including the killing of s. mansoni sporocysts by haemocytes from resistant b. glabrata. differential expression of odc in resistant snails may suggest an involvement in snail defence by scavenging the damaging free radicals produced during the production of ros by haemocytes for cytotoxic killing of sporocysts; in regulating production of cytotoxic no by depleting the competing substrate; or, with the production of polyamines leading to dna protection and cell proliferation, aiding wound healing following miracidial penetration. also identified as differentially expressed in the resistant snail line, hsp was previously identified, using differential display, as upregulated only in the resistant snails after parasite exposure, verifying our original suggestion that upregulation of hsp is an expected response to infection, and is absent from the susceptible snails. the induction of heat shock or stress proteins represents a homeostatic defence mechanism of cells to metabolic and environmental insults, and this response has previously been demonstrated in molluscs, for example in oyster haemocytes in response to environmental stimulus. experiments with mollusc haemocytes derived from crassostrea gigas and haliotis tuberculata demonstrated that the hsp gene promoter is induced by noradrenaline and α-adrenergic stimulations showing that the response in these cells is integrated with neuroendocrine signalling pathways. several proteins potentially involved in cell signalling were also identified in the resistant snails only. one transcript was found to be homologous to titin, which is a giant springlike protein responsible for passive tension generation and for positioning of the thick filament at the centre of vertebrate striated muscle sacromeres. this protein has multiple elastic and signalling functions derived from a complex subdomain structure, including a series of immunoglobulin domains. the kinase domain of titin initiates a signal transduction cascade that controls sarcomere assembly, protein turnover, and transcriptional control in response to mechanical changes in vertebrates. smaller but related molecules have been identified in invertebrate striated or smooth muscles, variously named mini-titins, projectins or twitchins, depending on their origin. the expression of this gene in resistant snails may indicate a signalling or muscle response to parasite infection that is not initiated in susceptible snails. in addition to titin, a cytoplasmic intermediate filament protein was identified only in the resistant snails. rather than merely providing a cellular framework, recent research has demonstrated that ifs are dynamic, motile elements of the cytoskeleton in vertebrate cells. an if protein has previously been identified in b. glabrata, using a comparative proteomic approach, as differentially expressed in snails resistant to e. caproni and it was suggested that this gene could be a candidate to explain differences in susceptibility/resistance, considering the major role of haemocyte mobility and adherence capabilities in defence to the parasite. interestingly, uninfected susceptible snails demonstrated higher levels of the if gene transcript; however post-exposure to the parasite the resistant snails showed a significant increase in transcript levels, while susceptible snails had a decrease. our sequence does not identify this transcript either at the nucleotide or protein level and demonstrates close homology to a neuronal if, neurofilament protein nf from helix aspersa one of the type iv ifs. it has also been suggested that some ifs participate in signalling processes by providing a scaffold to bring together activated map kinases with other molecules. other molecules potentially involved in signaling processes in the cell are importin and copine a gene fragment homologous to importin was identified in the resistant snails. this protein functions as a nuclear import cofactor, and in drosophila, has been implicated in the control of multiple signal transduction pathways, including the direct nuclear import of the activated form of map kinase. s. mansoni excretory secretory products and whole sporocysts have been shown to affect erk signalling in the haemocytes of susceptible snails, but not resistant ones suggesting that the disruption of erk signalling in haemocytes facilitates s. mansoni survival within susceptible b. glabrata. another gene, copine, implicated in membrane trafficking and signal transduction was identified in the resistant snails. copine is a calcium-dependent membrane binding protein which, in mammals, has been shown to regulate the nf kappa b transcriptional responses. in arabidopsis thaliana, copine is suggested to play a role in plant disease resistant responses, possibly as a suppressor of defense responses including the hypersensitive cell death response. we identified two hydrolytic enzymes in haemocytes from the resistant snail line, elastase and cathepsin l-like protease precursor, also known as cysteine proteinase. higher levels of cysteine proteinase activity have previously been observed in hepatopancreas extracts from resistant b. glabrata when compared to susceptible snails. cathepsin l, cathepsin b and elastase were also identified among other hydrolytic enzymes from a hepatopancreas est library derived from resistant snails and cathepsin b demonstrated greater up-regulation in resistant snails compared to susceptible snails upon parasite exposure. in invertebrates, cysteine proteases play a major role in the lysosomal proteolytic system, responsible for intracellular protein degradation. this role, in the lysis of phagocytosed particles, may be significant in the breakdown of encapsulated sporocysts or phagocytosed parasite components. in addition to a role as a scavenger for the clearance of unwanted proteins, this protease plays an important role in antigen processing and presentation in mammalian immune systems and lysosomal proteases have been implicated in innate immunity in insect haemocytes. genome-wide analysis of immune challenged drosophila revealed elevated expression of cathepsin l when infected with either gram-positive bacteria or fungi. similarly, cathepsin l was highly expressed in wssv resistant shrimp, suggesting that it is involved in defence responses. a cathepsin l-like gene has previously been identified in b. glabrata susceptible to echinostoma caproni infection. our sequence does not cluster with this and identifies a cathepsin-like domain 3' to sequence identified by the other gene fragment, suggesting that the ests may be two non-overlapping parts of the same gene. the discrepancy between the observed changes in gene expression may be due to the different response elicited by two different parasite species. s. mansoni fails to produce a response in susceptible snails as they fail to recognise the presence of the parasite, while in echinostome infections a defence response may be mounted by the snail, but is interfered with by the parasite. differential expression of ubiquitin conjugating enzyme e was detected in exposed resistant snails and may indicate removal of the phagocytosed sporocyst. ubiquitination plays an important role in various cellular functions including apoptosis, cell cycle progression, transcription and endocytosis. a major role is regulating the half-life of proteins by targeting them for 26s proteasomal degradation, removing denatured, damaged or improperly translated proteins. in this study we compared haemocytes obtained from resistant and susceptible snail strains to h after exposure to s. mansoni, and have found differentially expressed transcripts potentially involved in a range of responses from signalling and inflammation responses through to lysis of proteinacous products and processing/degradation of such targeted products by ubiquitination. in future, examination of biological replicates will increase the confidence that the transcripts identified in this study are truly significant in the snail ids, in addition to demonstrating the amplification techniques utilized in this study are robust. examination of a series of narrower time slots will enable us to unravel the sequence of processes involved and highlight genes initiating the cascade in resistant hosts. a simultaneous comparison of both parasite-exposed and unexposed snails from both snail lines will also determine if the basis of the snail's resistance is due to an underlying difference in gene expression between the strains, or from differences in their response to the parasite. such comparisons may also be significant in determining why susceptible snails do not respond to infection, either by non-recognition of invading parasite or by active suppression of innate response by the parasite, which may be indicated if s. mansoni-exposure results in significantly more down-regulated genes in susceptible snails. at present we can only speculate on the function in b. glabrata of the genes identified. future expression experiments involving rnai or in situ hybridization may elucidate their role in resistance.  in conclusion, the advent of the first cdna microarray for b. glabrata leads the way for detailed analysis of the b. glabrata transcriptome and can be developed further to include more cdnas as these become available. the array described here is particularly suited for analysis of snail haemocytes and their role in the snail ids since it contains a large proportion of genes sequenced from haemocytes. despite the current limited size of the array, prior enrichment for differentially expressed genes using the ssh approach has enabled us to identify a number of genes and pathways differentially expressed in the resistant snail line and potentially involved in the defence of resistant snails to schistosome infection. these include hydrolytic enzymes such as elastase and the cysteine protease, cathepsin l; ornithine decarboxylase, involved in the production of polyamines; hsp70; potential signalling molecules importin and copine and transcription enzymes such as ef-1α and ef- continued development of this array has great potential for examining and understanding the functions of the b. glabrata transcriptome. 
we have developed a element cdna microarray for b. glabrata containing clones from orestes libraries, suppression subtractive hybridization libraries and clones identified in previous expression studies. snail haemocyte rna, extracted from parasite-challenged resistant and susceptible snails, to h post-exposure to s. mansoni, was hybridized to the custom made cdna microarray and differentially expressed genes or gene clusters were identified, resistant-associated and susceptible-associated. quantitative pcr analysis verified the cdna microarray results for representative transcripts. production of the first cdna microarray for profiling gene expression in b. glabrata provides a foundation for expanding our understanding of pathways and genes involved in the snail internal defence system. examining differential gene expression between s. mansoni-exposed schistosome-resistant and susceptible snail lines will identify genes and pathways that may be involved in snail defences. biomphalaria glabrata is an intermediate snail host for schistosoma mansoni, one of the important schistosomes infecting man. resistant strain-specific expression of genes implicated in innate immunity of invertebrates was identified, including hydrolytic enzymes such as cathepsin l, a cysteine proteinase involved in lysis of phagocytosed particles; metabolic enzymes such as ornithine decarboxylase, the rate-limiting enzyme in the production of polyamines, important in inflammation and infection processes, as well as scavenging damaging free radicals produced during production of reactive oxygen species; stress response genes such as hsp70; proteins involved in signalling, such as importin and copine cytoplasmic intermediate filament protein and transcription enzymes such as elongation factor 1α and ef- we demonstrate resistant strain-specific expression of genes potentially associated with the snail ids, ranging from signalling and inflammation responses through to lysis of proteinacous products and processing/degradation of these targeted products by ubiquitination. differentially expressed genes were annotated and clustered using gene ontology terminology and kyoto encyclopaedia of genes and genomes pathway analysis.
 biomphalaria glabrata is an intermediate snail host for schistosoma mansoni, one of the important schistosomes infecting man. b. glabrata/s. mansoni provides a useful model system for investigating the intimate interactions between host and parasite. examining differential gene expression between s. mansoni-exposed schistosome-resistant and susceptible snail lines will identify genes and pathways that may be involved in snail defences.  we have developed a element cdna microarray for b. glabrata containing clones from orestes libraries, suppression subtractive hybridization libraries and clones identified in previous expression studies. snail haemocyte rna, extracted from parasite-challenged resistant and susceptible snails, to h post-exposure to s. mansoni, was hybridized to the custom made cdna microarray and differentially expressed genes or gene clusters were identified, resistant-associated and susceptible-associated. quantitative pcr analysis verified the cdna microarray results for representative transcripts. differentially expressed genes were annotated and clustered using gene ontology terminology and kyoto encyclopaedia of genes and genomes pathway analysis. 61% of the identified differentially expressed genes have no known function including the susceptible strain-specific transcripts. resistant strain-specific expression of genes implicated in innate immunity of invertebrates was identified, including hydrolytic enzymes such as cathepsin l, a cysteine proteinase involved in lysis of phagocytosed particles; metabolic enzymes such as ornithine decarboxylase, the rate-limiting enzyme in the production of polyamines, important in inflammation and infection processes, as well as scavenging damaging free radicals produced during production of reactive oxygen species; stress response genes such as hsp70; proteins involved in signalling, such as importin and copine cytoplasmic intermediate filament protein and transcription enzymes such as elongation factor 1α and ef-  production of the first cdna microarray for profiling gene expression in b. glabrata provides a foundation for expanding our understanding of pathways and genes involved in the snail internal defence system. we demonstrate resistant strain-specific expression of genes potentially associated with the snail ids, ranging from signalling and inflammation responses through to lysis of proteinacous products and processing/degradation of these targeted products by ubiquitination. 