BACKGROUND
the hard clam, mercenaria mercenaria, is an ecologically- and economically-important marine bivalve species that thrives along the northeastern coasts of the united states and maritime canada.
in the past few decades, the hard clam industry has been severely impacted by a protistan parasite called qpx , which is responsible for mortality episodes in both wild and cultured clam populations .
qpx is believed to be an opportunistic pathogen and has been detected in a wide variety of environmental substrates including sediments, marine aggregates and other organic matrices .
interestingly, previous reports highlight the ability of qpx to sustain very low abundance in clams without causing disease outbreaks until it encounters hosts with reduced immunity or following shifts of environmental conditions that add to the virulence of the parasite, under which conditions qpx can take advantage to establish infection sometimes leading to severe clam mortality events  <cit> .
lesions caused by qpx, usually associated with the presence of nodules, are commonly found in pallial tissues, such as alongside the inner rim of the mantle or at the base of the siphon  <cit> .
these places are widely considered as the portal of entry for qpx cells acquired from the surrounding environment during suspension-feeding  <cit> .
the qpx nodules represent inflammatory masses containing both parasite cells and abundant clam hemocytes, resulting from a series of comprehensive host immune responses induced by the infection that leads to massive focal hemocyte infiltration, parasite encapsulation and partial necrosis of the affected area  <cit> .
like other invertebrates, the hard clam lacks the specific immune responses and their defense mechanism mainly relies on the effectors of innate immunity, which is mediated by circulating hemocytes and highly diversified humoral antimicrobial factors.
these cellular and humoral immune components work in a synergistic way to initiate the recognition, segregation and ultimately elimination of pathogens and other non-self entities  <cit> .
the launching of innate immune responses involves myriad cellular and humoral events modulated not only at the infection sites  but also at a larger, whole-organism scale .
in general, the focal response represents the alterations driven by direct host-pathogen interactions at the infection sites where direct cell-cell  interactions mediate the response; while the systemic response reflects overall modifications within the host as a result of the ongoing infection and is mainly associated with dynamic changes of circulating hemocytes and their secreted immune mediators.
most of the previous investigations have solely focused on the systemic response of m. mercenaria against qpx during the infection events, where changes in cellular and humoral immune parameters  production, etc.)
as well as expression of a limited number of immune-related genes in tissues and circulating hemocytes were assessed .
in contrast, no previous studies have focused on the characterization of clam focal response at the infection sites.
given the fact that qpx disease is usually focal with formation of well-delimited lesions, the study of clam immune responses at the infection site in the lesions per se is of specific value as it provides insights to better characterize cellular interactions between the hard clam and qpx upon their contact.
in this framework, qpx disease in clams offer a unique opportunity to contrast focal and systemic responses against microbial diseases in invertebrates allowing for a more comprehensive understanding of defense strategies used by these animals to fend microbial attacks.
our study aimed to characterize the gene regulation features of m. mercenaria during qpx infection by profiling the transcripts at the infection lesion and compare focal clam responses with systemic responses detected in healthy tissues from infected clams .
this study allowed the identification of factors involved in the interactions with the parasite as well as molecular pathways activated by the host to neutralize qpx.
RESULTS
illumina sequencing and de novo transcriptome assembly
the main objective of this study was to identify molecular features of m. mercenaria in response to qpx infection and to compare the immune-related pathways involved in the lesion-specific focal response with the whole-organism scale systemic response.
the high-throughput illumina rna sequencing and de novo assembly employed in this investigation allowed the construction of the transcriptome in the absence of m. mercenaria genome information.
a total of  <dig> , <dig> of 100 bp raw reads were generated from the illumina paired-end sequencing with about  <dig> to  <dig> millions paired-end reads generated from each of the  <dig> sequenced libraries .
the short read sequences generated from this rnaseq project have been deposited at the ncbi short read archive database under the sra accession number srp <dig>  trimming and filtering procedures yielded  <dig> , <dig> clean reads that were used for the de novo assembly of consensus transcriptome based on all sequenced rna libraries in order to maximize the diversity of transcripts.
this allowed  <dig>  to  <dig>  % of the reads from the  <dig> libraries be used for the transcriptome assembly.
a total of  <dig>  transcripts ranging from  <dig> to  <dig>  bp with average size of  <dig>  bp and median size of 835 bp were produced from the assembly after low fpkm and rare isoforms filtering.
the size distribution of all the de novo assembled transcripts is shown in fig.
1b.
once the transcriptome was constructed, the  <dig> libraries were individually remapped to the  <dig>  transcripts and resulted with  <dig>  to  <dig>  % of reads remapping.
theses counting data were then used for de analysis.table  <dig> rna samples for rna-seq libraries.
each pool is made with equal amounts of rna from  <dig> individual clams.
pools a and b were derived from the same clams
fig.
<dig>
m. mercenaria de novo assembled transcriptome summary.
a transcriptome sequencing, assembly and annotation overview.
b assembled transcripts size distribution.
c distribution of the top  <dig> species with most homologues to m. mercenaria.
transcripts were searched using blastx against ncbi nr database with a cutoff value of e < 10e-03
transcriptome functional annotation
the transcriptome annotation performed using blast2go returned a total of  <dig>  transcripts  with significant blastx homology matches to other sequences in ncbi nr database  .
not surprisingly, the top  <dig> species that had the most similarity to m. mercenaria sequences were mollusks with available genomes and included the pacific oyster, crassostrea gigas , followed by the owl limpet lottia gigantea  and the california sea slug aplysia californica  .
kegg orthology  terms were assigned to  <dig>  sequences and reference pathways were mapped to the kegg database based on the assigned ko terms .
a total of  <dig>  sequences were identified to match to at least one conserved protein domain in the interpro database .
gene ontology  assignments were used to classify functions of the predicted clam proteins.
based on sequence similarity ,  <dig>  sequences were assigned to at least one go annotation .
as summarized in fig.
<dig>  a total of  <dig> ,  <dig>  and  <dig>  sequences were respectively categorized into the three main categories: biological process, cellular component, and molecular function at the second functional annotation level.
the most dominant terms presented in the three categories are the “cellular process”, “metabolic process”, “binding”, “catalytic activity”, “cell”, and “organelle”.
very few transcripts were clustered into “rhythmic process”, “cell killing”, “protein tag”, “channel regulator activity”, “nucleoid” or “virion”.
it is noticeable that a good fraction of transcripts were clustered into the immune-related categories of response to stimulus , immune system process  and biological adhesion .
those transcripts were of special interest given that they might be involved in the m. mercenaria defense and resistance toward qpx infection.fig.
<dig> gene ontology  annotations of the m. mercenaria transcriptome.
go terms were identified by blast2go and the results were summarized in three main go categories: biological process , cellular component , molecular function  at level-2
a significant portion  of m. mercenaria transcripts did not match any blastx hit in ncbi nr database, in agreement with previous transcriptomic studies in mollusks .
most of the unannotated transcripts may represent transcripts spanning untranslated mrna regions, or transcripts containing only non-conserved protein domains  <cit> .
identification of differentially expressed transcripts
the generated transcriptome was used as a reference for downstream investigations of global gene expression in the three different tissues of interest  to identify genes associated with m. mercenaria’s focal and systemic immune response against qpx.
a gene-isoform relationship was estimated using rsem over trinity output isoforms.
results showed that about 43 %  of all the transcripts had  <dig> isoform, 19 %  had  <dig> isoforms and 38 %  had  <dig> isoforms, suggesting extensive isoform diversity in m. mercenaria transcriptome.
transcript isoform variation could affect mrna stability, localization and translation, as well as the production of protein variants that differ in localization or function  <cit> .
by comparing the number of transcripts expressed in each sample, the contribution of specific samples to the analysis can be estimated.
the highest number of expressed transcripts was found in the nodules of infected clams, which were closely followed by that found in the healthy clam samples .
the lowest number of expressed transcripts came from non-nodule samples of qpx infected clams, with about  <dig>  less transcripts expressed than the other two samples.
read coverage, which is critical in accurate determination of fold change, averaged  <dig>   <dig> and  <dig> reads per transcript for nodule, non-nodule and healthy tissue samples, respectively .fig.
<dig> number of transcripts expressed and reads coverage in each sample.
a the x-axis indicates the sample .
the y-axis indicates the number of transcripts expressed in the samples.
b summary statistics of the reads coverage in each sample
statistical analysis by deseq identified  <dig>  differentially expressed  transcripts from the pair-wise comparisons | > <dig>  adjusted p-value < <dig> ) between clam tissue samples .
in nodules, a total of  <dig> transcripts, including  <dig> over-expressed and  <dig> under-expressed transcripts were identified as compared to non-nodule samples of qpx infected clams.
compared to tissues from healthy clams,  <dig>  de transcripts were identified in nodules with  <dig> over-expressed and  <dig> under-expressed transcripts.
similarly,  <dig>  de transcripts were obtained from the comparison between healthy and non-nodule clam tissues, of which  <dig> and  <dig>  were over- and under-expressed, respectively .
a total of  <dig>  of these de transcripts had protein homologs found in ncbi nr database by blastx searches , which were further examined for their putative functions during m. mercenaria immune response toward qpx.
annotated transcripts were subsequently grouped into curated categories according to their biological functions based on the gene ontology  terms and literature searches highlighting immune functions.
de transcript sets were further examined in reference to the assigned ko terms for the analysis of pathways regulation.
significantly enriched kegg pathways were identified via the fisher’s exact test   <cit> .fig.
<dig> heatmap  and number of differentially expressed transcripts  across all samples | >2).
over-expressed transcripts are shown in red in a and are enumerated in b. under-expressed transcripts are shown in green in a and are enumerated in c. replicate biological samples are displayed in a .
for b and c: the cross-shaded areas inside each bar represent the number of transcripts with higher  or lower  expression levels in a condition as compared to the other two
here we specifically focus on de transcripts drawn from comparisons between “nodule vs. non-nodule” and “non-nodule vs. healthy” tissue samples, which were respectively considered to reflect the transcriptomic changes caused by “focal” and “systemic” immune response of m. mercenaria toward qpx infection, respectively.
the overview of de transcripts drawn from these two responses is presented in fig.
<dig> and additional files  <dig> and  <dig>
differential expression of immune related transcripts
focal response
clam focal response reflected the alterations caused by direct clam-qpx interaction at the infection site .
qpx nodules are inflammatory masses resulting from massive hemocytes infiltration and encapsulation of parasite cells  <cit> .
this process largely relies on the motility and adhesion properties of hemocytes, thus allowing these cells to migrate throughout the circulatory system and recruit to the infection site.
hemocytes can sense stimuli in host tissues through an array of cell surface receptors, and use these cues to adjust their behavior accordingly  <cit> .
the activation of hemocytes requires the binding of specific ligands to the cell surface receptors, which subsequently initiate the transduction of extracellular signals into the cytoplasm via a variety of signaling pathways, thus inducing a series of hemocyte-mediated immune response such as phagocytosis, encapsulation , ros production, as well as secretion of immune effectors and cytokines .
a collection of de transcripts involved in these defense processes was identified during focal response, suggesting that strong and comprehensive host-pathogen interactions were taking places inside the qpx lesions .
a large fraction of the de transcripts of focal response was annotated as receptors or molecules with receptor activities, which putatively contribute to the host defense against qpx as  cell surface receptors expressed on hemocytes that mediate the recognition and phagocytosis or encapsulation of foreign entities through microbe associated molecular patterns ;  signaling receptors activating intracellular signaling cascades or  the soluble bridging molecules mediating the linkage between mamps and hemocytes  <cit> .
among those receptors, most are identified as pathogen pattern recognition receptors , which include the c-type lectins , the scavenger receptors  and the toll-like receptors .table  <dig> transcripts with annotated functions  related to immune recognition, signaling and regulation that were differentially expressed during m. mercenaria focal response against qpx.
additional information on these transcripts is given in additional file  <dig>  “inf” designates an infinite fold change calculated for focal response as the expression of that transcript in non-nodule tissue was equal to 0
the c-type mannose receptor- <dig>  identified during the focal response  is a member of the c-type lectins  superfamily, a large group of ca2+-dependent carbohydrate-binding proteins that play crucial roles in innate immunity.
ctls recognize pathogens and facilitate their phagocytosis  <cit>  or encapsulation .
mrcs are also key regulators of inflammatory responses and contribute to the removal of harmful inflammatory agents  <cit> .
the 16-fold over-expression of mrc <dig> during the focal response suggested that active hemocyte encapsulation and local inflammation was induced by qpx at the infection lesions, which is consistent with the results of histopathological observations  <cit> .
another over-expressed ctl member, the perlucin-like protein, has been previously shown to trigger immune response in manila clams during microbial infection  <cit> .
scavenger receptors  were also among the strongly over-expressed transcripts in infection foci .
these included somatomedin-b and thrombospondin type- <dig> domain-containing , insulin-related peptide receptor, hemicentin- <dig>  lysyl oxidase-like protein  <dig> and mam domain-containing glycosylphosphatidylinositol anchor protein  <dig>  srs are structurally diverse prrs that share the common function of recognizing oxidized or acetylated low-density lipoprotein   <cit> .
they contribute to innate immunity by recognizing mamps and mediating non-opsonic phagocytosis  <cit> .
they are extensively found on immune cells and are able to interact with both modified-host components and exogenous ligands, which makes srs a key component in host defense, apoptosis, inflammation and lipoprotein homeostasis .
for example, scallop srs bind not only to acetylated ldl but also to mamp including lipopolysaccharides , peptidoglycans , mannan and zymosan particles  <cit> .
the sea urchin genome encodes approximately  <dig> genes consisting of one or more scavenger receptor cysteine-rich  domains  <cit> , and the members of this gene family exhibit dynamic shifts in transcription after immune challenge  <cit> .
our results also show an over-expression of tlr- <dig> and toll-8/tollo in nodules , which is in agreement with previous investigations showing up-regulation of tlrs in m. mercenaria mantle following qpx challenge  <cit> .
tlrs are among the most ancient and conserved prrs.
they are expressed by immune cells and interact with a large variety of mamps.
bivalve tlrs have been characterized in the oyster c. gigas and the scallop c. farreri where they exhibited significant response to lps stimulation  <cit> .
transcriptional modulation of tlrs has also been reported in ruditapes philippinarum and mya arenaria following mamps stimulation and bacterial challenge  <cit> .
interestingly, toll- <dig>  has been shown to participate in drosophila epithelial immunity where it mediates host cells communication that subsequently activates systemic immune responses  <cit> .
this suggests that the toll pathway could be one of the crucial pivoting links that allow coordination between focal and systemic immune components during infection.
the qpx nodules are formed as the result of granulomatous inflammation, which is a chronic inflammatory reaction characterized by focal accumulation of activated immune cells to isolate the invading agent  <cit> .
the formation of granuloma requires local recruitment of hemocytes at the site of infection to execute extracellular defense processes around the invaders  <cit> .
an array of transcripts associated with cell migration, adhesion and proliferation was regulated in nodules, including g-protein coupled receptors  and integrins families .
gpcrs regulate inflammatory response via binding to chemokines and chemoattractants, thus activating pathways mediating hemocyte migration and adhesion  <cit> .
they also activate transcription factors in immune cells, thus modulating the synthesis and secretion of certain pro- or anti-inflammatory substances  <cit> .
on the other hand, integrins represent a major group of cell adhesion mediators  <cit> .
they not only modulate the cell-cell and cell-extracellular matrix adhesion, but also affect multiple signal transduction cascades regulating cell survival and proliferation  <cit> .
overexpression of gpcrs and integrins in nodules suggests their role in hemocytes adhesion and aggregation associated with the formation of granuloma  <cit> .
several enzymes regulating ros production were also over-expressed during focal response .
these included a dual oxidase, which is a key component mediating host-microbe interactions in mucosa  <cit> .
dual oxidase regulates oxidative burst and ros production in the gill muscosa of the shrimp marsupenaeus japonicus, favoring shrimp survivorship during viral infections  <cit> .
interestingly, transcripts of dual oxidase were only expressed in nodules, suggesting this enzyme was induced upon direct clam-qpx interactions as a part of the mantle mucosa-related immune response.
other transcripts associated with oxidation-reduction processes also exhibited somewhat nodule-exclusive pattern, including the allene oxide synthase-lipoxygenase , lysyl oxidase-like protein , ww domain-containing oxidoreductase , c-terminal binding protein , isocitrate dehydrogenase  and methylenetetrahydrofolate reductase .
these molecules are important for maintaining the redox homeostasis of extracellular environment as they are key regulators for oxi-reduction reactions.
over-expression of these transcripts in nodules suggests the need for the host to timely balance out excessive ros and other toxic intermediates produced during interaction with qpx.
in addition to redox-regulation, many of these molecules also take part in the immune modulation indirectly.
for example, aosl play a role in coral immunity by controlling the production of the inflammation regulator arachidonic acid during apoptosis  <cit> , and loxl acts both as a scavenger receptor and regulator for extracellular matrix remodeling that initiate hemocyte migration and tissue regeneration  <cit> , while wwox was shown to promote proliferation of immune cells through inhibition of their apoptosis  <cit> .
in addition, icd, mthfr and cytochrome p <dig> are major detoxification enzymes  <cit> .
in fact, immune cells and their secreted effectors require the proper redox state in the extracellular environments to exert their immune functions, which makes the maintenance of redox homeostasis essential for persistent and effective host defense  <cit> .
this is particularly true in the case of qpx disease where the neutralization of parasites depends on extracellular killing pathways  <cit> .table  <dig> transcripts with putative functions  related to immune effectors that were differentially expressed during m. mercenaria focal response against qpx.
additional information on these transcripts is given in additional file  <dig>  “inf” designates an infinite fold change calculated for focal response as the expression of that transcript in non-nodule tissue was equal to 0
apoptosis is an essential host mechanism to effectively remove damaged and infected cells without causing inflammatory destructions to surrounding tissues  <cit> .
interestingly, apoptosis seems to be largely inhibited during m. mercenaria focal response, as shown by the under-expression of pro-apoptosis transcripts .
for example, the tumor necrosis factor -like protein and 3-hydroxy-3-methylglutaryl-coenzyme a  reductase-like protein were significantly under-expressed in nodules.
similarly, the pro-apoptotic p53-induced protein and solute carrier family  <dig> member protein were also under-expressed in nodules.
meanwhile, inhibitor of apoptosis protein  was over-expressed in nodules.
iaps regulate immune cell expansion and survival in highly inflammatory environments in mammals  <cit>  and they may share similar function in clams by preventing hemocytes from death during interaction with qpx.
in fact, ros production during parasite killing may trigger apoptotic cell death in molluscs  <cit> , and proper control of apoptosis mechanisms is required to maintain cellular homeostasis during immune response.
this suspected inhibition of host apoptosis is supported by the above-mentioned over-expression of integrins, as these were shown to protect cells from apoptosis and induce anti-apoptotic pathways during cell adhesion and spreading in the snails lymnaea stagnalis  <cit>  and b. glabrata  <cit> .
infection and tissue injury trigger host immune responses via immune signaling pathways  <cit> , by activating transcription factors and initiating the production of immune effectors and regulators.
immune signaling pathways identified in mollusks include toll, mapk/jnk and jak/stat signaling pathways  <cit> .
during m. mercenaria focal response to qpx, a variety of transcripts encoding kinases and phosphatases were over-expressed , suggesting the involvement of mapk and other kinase-mediated cascades in regulating the focal inflammatory response  <cit> , whereas the under-expression of ef-hand domain containing protein and calmodulin may indicate the suppression of calcium-regulated pathways  <cit> .
overexpression of e <dig> ubiquitin-protein ligase and its upstream regulator cop <dig> signalosome suggests the activation of the damage surveillance ubiquitin/proteasome pathway .
in parallel, the over-expression of rho gtpase and rho kinase suggests the induction of anti-apoptotic rho-mediated signaling pathway  <cit>  and reinforce the idea that apoptotic inhibition is extensively initiated by m. mercenaria to help fight qpx.
however, a very limited number of de transcripts was detected in relation to those conventional signaling pathways of innate immunity, such as the complement pathway and the toll/tlr pathway.
only  <dig> transcript encoding macrophage-expressed gene protein  <dig> , a putative member of the complement pathway  <cit> , was differentially expressed in qpx nodules.
as for the toll/tlr pathway, only a few receptors were identified  but none of their downstream components.
interestingly, components of the notch and the wnt signaling pathways were over-expressed during focal response .
these included two putative notch family members, the neurogenic locus notch protein and the mediator protein nicastrin, and the tyrosin-protein kinase ryk which belongs to the wnt pathway.
wnt signaling pathway regulates many cellular immune processes and is evolutionarily conserved across taxa  <cit> .
wnt signaling has been intensively exploited for its regulatory functions during wound healing and tissue regeneration  <cit> , so its over-expression may be related to wound healing to repair damage resulting from tissue digestion by the parasite or tissue necrosis.
the notch signaling network was scarcely explored in bivalves even though it has been reported to be associated with several aspects of immune response in mammals  <cit> , especially in regulating granulomatous reactions to foreign bodies  <cit> .
as an evolutionarily conserved pathway involved in modulating the intercellular signaling, the notch pathway presumably shares an equally important role in m. mercenaria by modulating the formation of granuloma.
notch signaling triggers macrophage expression of genes involved in pro-inflammatory responses  <cit> , but can suppress inflammation responses triggered by canonical tlr cascade  <cit> , in agreement with our observations.
these tightly regulated mechanisms ensure tailored immune responses against different pathogens and are crucial for the host to achieve high immune efficiency while avoiding excessive immune activation and self-inflicted damages.
several proteases  were also differentially regulated during m. mercenaria focal response against qpx.
these proteases mostly belong to the serine and metallo protease families and were generally over-expressed in qpx nodules .
proteases serve as key immune modulators partially through their ability to digest and remodel the extracellular matrix and tissues associated with hemocyte activation  <cit> .
commonly associated with lysosomes and granules of inflammatory cells, serine proteases participate in immune regulation either directly by degradation of pathogens or indirectly through activation of cell surface receptors and signal molecules .
the function of metalloproteases in immune regulation is even more diverse, acting as immune effectors, signal transducers, and mediators of immune cell development and migration  <cit> .
metalloproteases are also known to be involved in many pro-inflammatory pathways, particularly in the notch pathway where they act as a type of downstream element to notch  <cit> .
at the same time, immune effectors with known universal protease inhibitor activities, such as alpha2-macroglobulin  and thioester-containing protein , were also collectively overexpressed in qpx nodules .
the α 2 m superfamily inhibits peptidases of diverse origins  <cit> .
the simultaneous over-expression of proteases and protease inhibitors may reflect a finely adjusted defense response of m. mercenaria to maintain homeostasis and regulate self- and pathogen-derived proteases, as shown in other host-pathogen systems  <cit> , including bivalves  <cit> .
proteases have been identified as major virulence factors of qpx  <cit> , and are thought to degrade host proteinaceous immune effectors and hydrolyze host tissues to fulfill nutritional requirements.
therefore, inhibition of pathogen proteases contribute to host protection, and was shown to represent a determinant factor for resistance against infectious diseases in bivalves .
in parallel, teps have been extensively studied in mollusk immunity  <cit> , and beside their function as protease inhibitors, they also play a role as prrs or opsonins to facilitate microbial phagocytosis and encapsulation.
consistent over-expression of teps was noted in this study in agreement with findings following experimental infection with qpx  <cit> .
these results support a critical role of teps in clam immune response against qpx either via the protease-inhibitor activity of these proteins, or by mediating parasite encapsulation, or both.
in addition, several metal ion transporters were over-expressed in nodules , including the putative copper ion binding protein ceruloplasmin precursor, the transferrin enzyme ferric-chelate reductase and the divalent metal transporter .
these molecules contribute to host defense by controlling the supply of essential micronutrients in the vicinity of infection sites thus reducing parasite survival  <cit>  and favoring the production of antimicrobial factors  <cit> .
it is noteworthy to point out that some of the focally over-expressed transcripts might be partly driven by the dramatic increase of hemocyte proportion within tissues in the vicinity of infection foci as compared to the surrounding host tissues  <cit> .
this would be especially the case for transcripts known to be highly expressed in hemocytes, such as the cell surface prrs, secreted humoral immune effectors, cell signal transducers and enzymes associated with ros production.
systemic response
significant transcriptomic regulations were observed during m. mercenaria systemic immune response against qpx, with a total of  <dig>  de transcripts, which is about two times the number of focal de transcripts .
however, only about one third of the de transcripts  were over-expressed in response to qpx infection, the larger remaining part  represented significantly under-expressed transcripts , possibly due to the chronic stress imposed by the infection.
transcriptome-wide depression has been demonstrated in many marine invertebrates as the result of pathogenic or environmental stress .
a considerable number of systemically under-expressed transcripts was related to metabolism and biomineralization, which could be the result of host resource allocation during on-going infection.
the chronic inflammation induced by infection likely created extra energy demands, which require resources being allocated from other physiological processes, such as growth and reproduction, to immune processes which is critical for the survivorship of the host.
in fact, slow growth and lower tissue conditions are frequently observed in qpx-infected m. mercenaria  <cit> , and similar energy trade-offs existed between immune defense and other energy expenditure pathways .
interestingly, a suite of transcripts over-expressed during focal response was significantly under-expressed in non-nodule tissues as compared to healthy clams , which included several immune effectors and mediators associated with nodule formation and focal inflammation .
in fact, maintaining high levels of these focally-induced molecules could be costly and dangerous as some are toxic to both the parasite and the host, so their production must be restrained within areas where they can directly exert defense function, and reduced outside the infection foci to minimize risks of undesirable effects on the host  <cit> .
on the other hand, systemically over-expressed transcripts included stress proteins and other soluble immune factors such as lysozyme , lectins , amp , proteases  and ferric-chelate reductase .
over-expression of host stress proteins, such as heat shock proteins  and universal stress protein  was also noted, in agreement with observations made during infection in other bivalve species .
increased levels of stress proteins provide host cells with protection against incorrect protein folding caused by infection, inflammation, oxidative stress and other destructive events  <cit> .
the systemic over-expression of soluble immune effectors  may help maintaining comparatively high immune capacity to prevent the spread of qpx  throughout the host.
in addition, transcripts of anti-apoptotic factors  were also over-expressed during the systemic response, indicating that anti-apoptotic processes noted during focal response are not limited to the infection foci.table  <dig> selected transcripts with annotated functions  related to immune response that were over-expressed during m. mercenaria systemic response against qpx.
additional information on these transcripts is given in additional file  <dig>  “inf” designates an infinite fold change calculated for focal response as the expression of that transcript in non-nodule tissue was equal to 0
pathway alterations during m. mercenaria’s response to qpx
transcriptomic alterations during both focal and systemic response discussed above were also highlighted in the pathway enrichment analysis of the de transcripts.
this analysis aims at extracting an overview of phenotypic changes on the underlying functional level, to reduce the complexity of biological information given by the long lists of de genes/transcripts  <cit> .
the kegg pathways of focal adhesion , ecm-receptor interaction , notch signaling pathway  and apoptosis  were significantly over-represented during both focal and systemic response , even though fold enrichment were generally higher during the focal response.
other immune-related pathways particularly enriched during the focal response included regulation of actin cytoskeleton , cell adhesion molecules , the leukocyte transendothelial migration , complement and coagulation cascade  and wnt signaling pathway .
these pathways are critically involved in the immune cells activation during migration, attachment and parasite encapsulation, which serve as the underlying mechanisms for nodule formation and qpx killing.
on the other hand, basic metabolic pathways such as the citrate cycle  and pyruvate metabolism were specially enriched during the systemic response.
these alterations were largely in accordance with the under-expression of metabolism-associated de transcripts in infected tissue compared to the healthy tissue , possibly reflecting changes in the energy allocation strategy during infection as discussed above.fig.
<dig> significantly enriched kegg pathways in m. mercenaria derived from the differentially expressed  genes during focal  and systemic  response against qpx.
the kegg pathways having significant enrichment  are presented, and the bar shows the x-fold enrichment of each kegg pathway.
pathways involved in immune response are marked with red dots while metabolism-related pathways are marked with green dots
interdependence of kegg pathways widely exist and most of these are interrelated with each other via shared components, forming a signaling network to allow for pathway crosstalk.
to investigate these interactions, we extracted the de transcripts shared by multiple enriched pathways and constructed a sketch of the hypothetical pathways network that are significantly altered by qpx infection .
in this framework, m. mercenaria response to qpx infection was initiated upon the sensing of danger signals via cell membrane receptors.
the signals subsequently transmitted down through the mapk, wnt and notch pathways and triggered the production of a series of host defense factors as the end results.
in parallel, activation of pathways regulating actin cytoskeleton and leukocyte transendothelial migration facilitated the recruitment of hemocytes to the infection area to build a barrier of cellular defense against the parasite.
recruited hemocytes then attached to and encapsulated qpx cells as suggested by the modulation of focal adhesion and ecm receptor interaction pathways.
these cellular activities were performed under a tight regulation of the apoptosis pathway to determine cell fates, resulting in either the survival or death of m. mercenaria cells.fig.
<dig> overview of immune-related enriched pathways of differentially expressed  transcripts during m. mercenaria response to qpx.
a schematic diagram of enriched pathways and their interactions.
only pathway components encoded by de transcripts  are presented within each enriched kegg pathway .
arrows display possible interactions  between pathway components.
b overview of de transcripts expression associated with enriched pathways in nodule, non-nodule and healthy m. mercenaria tissues.
the red and green heatmap values indicate log <dig> fold of relative expression levels for individual transcripts.
arp2/3: actin related protein 2/ <dig> complex; ctbp: c-terminal binding protein; camk2: calcium/calmodulin-dependent protein kinase  ii; cycd: cyclin d1; ecm: von willebrand factor; endo-g: endonuclease g; fak: focal adhesion kinase; fyn: tyrosine-protein kinase; hes1: hairy and enhancer of split 1; iap: baculoviral iap repeat-containing protein, ciaps; itga: intergrin alpha; itgb: intergrin beta; mlcp: serine/threonine-protein phosphatase pp <dig> catalytic subunit; nrxn: neurexin; ncstn: nicastrin; rac: ras-related c <dig> botulinum toxin substrate; rock: rho-associated protein kinase; rtk: proto-oncogene tyrosine-protein kinase; sele: selectin, endothelial cell; selp: selectin, platelet; trail: tumor necrosis factor ligand superfamily member 10); wnt: wingless-type mmtv integration site family
distinctive transcriptomic pattern of healthy clams
a suite of transcripts  exhibited higher transcription levels in healthy clams as compared to diseased clams .
a considerable fraction of these transcripts were related to metabolic processes, nucleic acids binding and transcriptional regulation.
the over-expressed immune-related transcripts identified in healthy clams are of particular interest as they may be involved in m. mercenaria resistance towards qpx.
for example, an antimicrobial protein  was exclusively identified in healthy clams with almost no detection in diseased clams.
in addition, the highest expression levels of a serine protease inhibitor were also observed in healthy clams.
a serine protease inhibitor has been linked to oyster  resistance against the protozoan parasite perkinsus marinus .
therefore, the high expression of the serine protease inhibitor in healthy clams supports its involvement in m. mercenaria’s resistance against qpx, likely by inhibiting the activity of parasite proteases.
moreover, a pathogen recognition protein  was significantly higher in healthy clams as compared to diseased animals, which may also contribute to clam resistance against qpx by promoting microbial recognition and encapsulation  <cit> .
previous studies demonstrated that clam genetic background affects m. mercenaria resistance toward qpx  <cit> , therefore immune-related transcripts specifically associated with healthy clams represent excellent candidates as molecular markers for further research on screening and breeding qpx-resistant m. mercenaria strains.table  <dig> selected transcripts with annotated functions  related to immune response that were differentially expressed in naïve m. mercenaria as compared to qpx infected individuals.
additional information on these transcripts is given in additional file  <dig>  “inf” designates an infinite fold change calculated for focal response as the expression of that transcript in non-nodule tissue was equal to 0
on the other hand, a subset of transcripts  exhibited lowest expression levels in healthy clams.
these include stress protein hsp  <dig> and usp, as well as the detoxification molecule cytochrome p <dig>  which together highlight the stress experienced by clams as the result of qpx infection .
in addition, other immune related transcripts including a protease , a protease inhibitor , a c-type lectin , and molecules involved in tissue regeneration and cell signaling, were also under-expressed in healthy clams as compared to infected individuals, suggesting their role in fighting the infection.
CONCLUSIONS
this is one of the first studies contrasting focal and systemic immune responses to infections in invertebrates using high-throughput sequencing.
resulting transcriptome represents a significant addition to the so far limited public genomic information available for m. mercenaria.
the transcriptomic profiles of healthy and infected clams reflected complex interactions between the host immune system and the pathogen leading to molecular changes at both the infection foci and the systemic level.
in general, the systemic responses of m. mercenaria reflected prevailing transcriptomic suppression accompanied with a contrasting over-expression of stress proteins and soluble antimicrobial effectors; whereas the focal response highlighted cell-cell interactions between hemocytes and the parasite that typically result in local inflammation, extracellular degradation, encapsulation, granuloma formation, and wound repair.
what needs to be kept in mind is that the regulation of these genes can be the result of either an effective immune response or a symptom of a future death.
in fact, several apoptotic and anti-apoptotic genes were regulated.
this highlights a fine adjustment of m. mercenaria defense mechanisms to precisely adapt to the infection development .
in addition, the identification of immune-related transcripts that were particularly associated with healthy clams offered new perspectives on the molecular features putatively involved in clam resistance against qpx.

 the hard clam, mercenaria mercenaria, is an ecologically- and economically-important marine bivalve species that thrives along the northeastern coasts of the united states and maritime canada. in the past few decades, the hard clam industry has been severely impacted by a protistan parasite called qpx, which is responsible for mortality episodes in both wild and cultured clam populations. qpx is believed to be an opportunistic pathogen and has been detected in a wide variety of environmental substrates including sediments, marine aggregates and other organic matrices. interestingly, previous reports highlight the ability of qpx to sustain very low abundance in clams without causing disease outbreaks until it encounters hosts with reduced immunity or following shifts of environmental conditions that add to the virulence of the parasite, under which conditions qpx can take advantage to establish infection sometimes leading to severe clam mortality events. lesions caused by qpx, usually associated with the presence of nodules, are commonly found in pallial tissues, such as alongside the inner rim of the mantle or at the base of the siphon. these places are widely considered as the portal of entry for qpx cells acquired from the surrounding environment during suspension-feeding. the qpx nodules represent inflammatory masses containing both parasite cells and abundant clam hemocytes, resulting from a series of comprehensive host immune responses induced by the infection that leads to massive focal hemocyte infiltration, parasite encapsulation and partial necrosis of the affected area. like other invertebrates, the hard clam lacks the specific immune responses and their defense mechanism mainly relies on the effectors of innate immunity, which is mediated by circulating hemocytes and highly diversified humoral antimicrobial factors. these cellular and humoral immune components work in a synergistic way to initiate the recognition, segregation and ultimately elimination of pathogens and other non-self entities. the launching of innate immune responses involves myriad cellular and humoral events modulated not only at the infection sites but also at a larger, whole-organism scale. in general, the focal response represents the alterations driven by direct host-pathogen interactions at the infection sites where direct cell-cell interactions mediate the response; while the systemic response reflects overall modifications within the host as a result of the ongoing infection and is mainly associated with dynamic changes of circulating hemocytes and their secreted immune mediators. most of the previous investigations have solely focused on the systemic response of m. mercenaria against qpx during the infection events, where changes in cellular and humoral immune parameters production, etc.) as well as expression of a limited number of immune-related genes in tissues and circulating hemocytes were assessed. in contrast, no previous studies have focused on the characterization of clam focal response at the infection sites. given the fact that qpx disease is usually focal with formation of well-delimited lesions, the study of clam immune responses at the infection site in the lesions per se is of specific value as it provides insights to better characterize cellular interactions between the hard clam and qpx upon their contact. in this framework, qpx disease in clams offer a unique opportunity to contrast focal and systemic responses against microbial diseases in invertebrates allowing for a more comprehensive understanding of defense strategies used by these animals to fend microbial attacks. our study aimed to characterize the gene regulation features of m. mercenaria during qpx infection by profiling the transcripts at the infection lesion and compare focal clam responses with systemic responses detected in healthy tissues from infected clams. this study allowed the identification of factors involved in the interactions with the parasite as well as molecular pathways activated by the host to neutralize qpx.  illumina sequencing and de novo transcriptome assembly the main objective of this study was to identify molecular features of m. mercenaria in response to qpx infection and to compare the immune-related pathways involved in the lesion-specific focal response with the whole-organism scale systemic response. the high-throughput illumina rna sequencing and de novo assembly employed in this investigation allowed the construction of the transcriptome in the absence of m. mercenaria genome information. a total of, of 100 bp raw reads were generated from the illumina paired-end sequencing with about to millions paired-end reads generated from each of the sequenced libraries. the short read sequences generated from this rnaseq project have been deposited at the ncbi short read archive database under the sra accession number srp trimming and filtering procedures yielded, clean reads that were used for the de novo assembly of consensus transcriptome based on all sequenced rna libraries in order to maximize the diversity of transcripts. this allowed to  % of the reads from the libraries be used for the transcriptome assembly. a total of transcripts ranging from to  bp with average size of  bp and median size of 835 bp were produced from the assembly after low fpkm and rare isoforms filtering. the size distribution of all the de novo assembled transcripts is shown in fig. 1b. once the transcriptome was constructed, the libraries were individually remapped to the transcripts and resulted with to  % of reads remapping. theses counting data were then used for de analysis.table rna samples for rna-seq libraries. each pool is made with equal amounts of rna from individual clams. pools a and b were derived from the same clams fig. m. mercenaria de novo assembled transcriptome summary. a transcriptome sequencing, assembly and annotation overview. b assembled transcripts size distribution. c distribution of the top species with most homologues to m. mercenaria. transcripts were searched using blastx against ncbi nr database with a cutoff value of e < 10e-03 transcriptome functional annotation the transcriptome annotation performed using blast2go returned a total of transcripts with significant blastx homology matches to other sequences in ncbi nr database. not surprisingly, the top species that had the most similarity to m. mercenaria sequences were mollusks with available genomes and included the pacific oyster, crassostrea gigas, followed by the owl limpet lottia gigantea and the california sea slug aplysia californica. kegg orthology terms were assigned to sequences and reference pathways were mapped to the kegg database based on the assigned ko terms. a total of sequences were identified to match to at least one conserved protein domain in the interpro database. gene ontology assignments were used to classify functions of the predicted clam proteins. based on sequence similarity, sequences were assigned to at least one go annotation. as summarized in fig.  a total of, and sequences were respectively categorized into the three main categories: biological process, cellular component, and molecular function at the second functional annotation level. the most dominant terms presented in the three categories are the “cellular process”, “metabolic process”, “binding”, “catalytic activity”, “cell”, and “organelle”. very few transcripts were clustered into “rhythmic process”, “cell killing”, “protein tag”, “channel regulator activity”, “nucleoid” or “virion”. it is noticeable that a good fraction of transcripts were clustered into the immune-related categories of response to stimulus, immune system process and biological adhesion. those transcripts were of special interest given that they might be involved in the m. mercenaria defense and resistance toward qpx infection.fig. gene ontology annotations of the m. mercenaria transcriptome. go terms were identified by blast2go and the results were summarized in three main go categories: biological process, cellular component, molecular function at level-2 a significant portion of m. mercenaria transcripts did not match any blastx hit in ncbi nr database, in agreement with previous transcriptomic studies in mollusks. most of the unannotated transcripts may represent transcripts spanning untranslated mrna regions, or transcripts containing only non-conserved protein domains. identification of differentially expressed transcripts the generated transcriptome was used as a reference for downstream investigations of global gene expression in the three different tissues of interest to identify genes associated with m. mercenaria’s focal and systemic immune response against qpx. a gene-isoform relationship was estimated using rsem over trinity output isoforms. results showed that about 43 % of all the transcripts had isoform, 19 % had isoforms and 38 % had isoforms, suggesting extensive isoform diversity in m. mercenaria transcriptome. transcript isoform variation could affect mrna stability, localization and translation, as well as the production of protein variants that differ in localization or function. by comparing the number of transcripts expressed in each sample, the contribution of specific samples to the analysis can be estimated. the highest number of expressed transcripts was found in the nodules of infected clams, which were closely followed by that found in the healthy clam samples. the lowest number of expressed transcripts came from non-nodule samples of qpx infected clams, with about less transcripts expressed than the other two samples. read coverage, which is critical in accurate determination of fold change, averaged and reads per transcript for nodule, non-nodule and healthy tissue samples, respectively.fig. number of transcripts expressed and reads coverage in each sample. a the x-axis indicates the sample. the y-axis indicates the number of transcripts expressed in the samples. b summary statistics of the reads coverage in each sample statistical analysis by deseq identified differentially expressed transcripts from the pair-wise comparisons | > adjusted p-value < ) between clam tissue samples. in nodules, a total of transcripts, including over-expressed and under-expressed transcripts were identified as compared to non-nodule samples of qpx infected clams. compared to tissues from healthy clams, de transcripts were identified in nodules with over-expressed and under-expressed transcripts. similarly, de transcripts were obtained from the comparison between healthy and non-nodule clam tissues, of which and were over- and under-expressed, respectively. a total of of these de transcripts had protein homologs found in ncbi nr database by blastx searches, which were further examined for their putative functions during m. mercenaria immune response toward qpx. annotated transcripts were subsequently grouped into curated categories according to their biological functions based on the gene ontology terms and literature searches highlighting immune functions. de transcript sets were further examined in reference to the assigned ko terms for the analysis of pathways regulation. significantly enriched kegg pathways were identified via the fisher’s exact test.fig. heatmap and number of differentially expressed transcripts across all samples | >2). over-expressed transcripts are shown in red in a and are enumerated in b. under-expressed transcripts are shown in green in a and are enumerated in c. replicate biological samples are displayed in a. for b and c: the cross-shaded areas inside each bar represent the number of transcripts with higher or lower expression levels in a condition as compared to the other two here we specifically focus on de transcripts drawn from comparisons between “nodule vs. non-nodule” and “non-nodule vs. healthy” tissue samples, which were respectively considered to reflect the transcriptomic changes caused by “focal” and “systemic” immune response of m. mercenaria toward qpx infection, respectively. the overview of de transcripts drawn from these two responses is presented in fig.  and additional files and differential expression of immune related transcripts focal response clam focal response reflected the alterations caused by direct clam-qpx interaction at the infection site. qpx nodules are inflammatory masses resulting from massive hemocytes infiltration and encapsulation of parasite cells. this process largely relies on the motility and adhesion properties of hemocytes, thus allowing these cells to migrate throughout the circulatory system and recruit to the infection site. hemocytes can sense stimuli in host tissues through an array of cell surface receptors, and use these cues to adjust their behavior accordingly. the activation of hemocytes requires the binding of specific ligands to the cell surface receptors, which subsequently initiate the transduction of extracellular signals into the cytoplasm via a variety of signaling pathways, thus inducing a series of hemocyte-mediated immune response such as phagocytosis, encapsulation, ros production, as well as secretion of immune effectors and cytokines. a collection of de transcripts involved in these defense processes was identified during focal response, suggesting that strong and comprehensive host-pathogen interactions were taking places inside the qpx lesions. a large fraction of the de transcripts of focal response was annotated as receptors or molecules with receptor activities, which putatively contribute to the host defense against qpx as cell surface receptors expressed on hemocytes that mediate the recognition and phagocytosis or encapsulation of foreign entities through microbe associated molecular patterns; signaling receptors activating intracellular signaling cascades or the soluble bridging molecules mediating the linkage between mamps and hemocytes. among those receptors, most are identified as pathogen pattern recognition receptors, which include the c-type lectins, the scavenger receptors and the toll-like receptors.table transcripts with annotated functions related to immune recognition, signaling and regulation that were differentially expressed during m. mercenaria focal response against qpx. additional information on these transcripts is given in additional file “inf” designates an infinite fold change calculated for focal response as the expression of that transcript in non-nodule tissue was equal to 0 the c-type mannose receptor- identified during the focal response is a member of the c-type lectins superfamily, a large group of ca2+-dependent carbohydrate-binding proteins that play crucial roles in innate immunity. ctls recognize pathogens and facilitate their phagocytosis or encapsulation. mrcs are also key regulators of inflammatory responses and contribute to the removal of harmful inflammatory agents. the 16-fold over-expression of mrc during the focal response suggested that active hemocyte encapsulation and local inflammation was induced by qpx at the infection lesions, which is consistent with the results of histopathological observations. another over-expressed ctl member, the perlucin-like protein, has been previously shown to trigger immune response in manila clams during microbial infection. scavenger receptors were also among the strongly over-expressed transcripts in infection foci. these included somatomedin-b and thrombospondin type- domain-containing, insulin-related peptide receptor, hemicentin- lysyl oxidase-like protein and mam domain-containing glycosylphosphatidylinositol anchor protein srs are structurally diverse prrs that share the common function of recognizing oxidized or acetylated low-density lipoprotein. they contribute to innate immunity by recognizing mamps and mediating non-opsonic phagocytosis. they are extensively found on immune cells and are able to interact with both modified-host components and exogenous ligands, which makes srs a key component in host defense, apoptosis, inflammation and lipoprotein homeostasis. for example, scallop srs bind not only to acetylated ldl but also to mamp including lipopolysaccharides, peptidoglycans, mannan and zymosan particles. the sea urchin genome encodes approximately genes consisting of one or more scavenger receptor cysteine-rich domains, and the members of this gene family exhibit dynamic shifts in transcription after immune challenge. our results also show an over-expression of tlr- and toll-8/tollo in nodules, which is in agreement with previous investigations showing up-regulation of tlrs in m. mercenaria mantle following qpx challenge. tlrs are among the most ancient and conserved prrs. they are expressed by immune cells and interact with a large variety of mamps. bivalve tlrs have been characterized in the oyster c. gigas and the scallop c. farreri where they exhibited significant response to lps stimulation. transcriptional modulation of tlrs has also been reported in ruditapes philippinarum and mya arenaria following mamps stimulation and bacterial challenge. interestingly, toll- has been shown to participate in drosophila epithelial immunity where it mediates host cells communication that subsequently activates systemic immune responses. this suggests that the toll pathway could be one of the crucial pivoting links that allow coordination between focal and systemic immune components during infection. the qpx nodules are formed as the result of granulomatous inflammation, which is a chronic inflammatory reaction characterized by focal accumulation of activated immune cells to isolate the invading agent. the formation of granuloma requires local recruitment of hemocytes at the site of infection to execute extracellular defense processes around the invaders. an array of transcripts associated with cell migration, adhesion and proliferation was regulated in nodules, including g-protein coupled receptors and integrins families. gpcrs regulate inflammatory response via binding to chemokines and chemoattractants, thus activating pathways mediating hemocyte migration and adhesion. they also activate transcription factors in immune cells, thus modulating the synthesis and secretion of certain pro- or anti-inflammatory substances. on the other hand, integrins represent a major group of cell adhesion mediators. they not only modulate the cell-cell and cell-extracellular matrix adhesion, but also affect multiple signal transduction cascades regulating cell survival and proliferation. overexpression of gpcrs and integrins in nodules suggests their role in hemocytes adhesion and aggregation associated with the formation of granuloma. several enzymes regulating ros production were also over-expressed during focal response. these included a dual oxidase, which is a key component mediating host-microbe interactions in mucosa. dual oxidase regulates oxidative burst and ros production in the gill muscosa of the shrimp marsupenaeus japonicus, favoring shrimp survivorship during viral infections. interestingly, transcripts of dual oxidase were only expressed in nodules, suggesting this enzyme was induced upon direct clam-qpx interactions as a part of the mantle mucosa-related immune response. other transcripts associated with oxidation-reduction processes also exhibited somewhat nodule-exclusive pattern, including the allene oxide synthase-lipoxygenase, lysyl oxidase-like protein, ww domain-containing oxidoreductase, c-terminal binding protein, isocitrate dehydrogenase and methylenetetrahydrofolate reductase. these molecules are important for maintaining the redox homeostasis of extracellular environment as they are key regulators for oxi-reduction reactions. over-expression of these transcripts in nodules suggests the need for the host to timely balance out excessive ros and other toxic intermediates produced during interaction with qpx. in addition to redox-regulation, many of these molecules also take part in the immune modulation indirectly. for example, aosl play a role in coral immunity by controlling the production of the inflammation regulator arachidonic acid during apoptosis, and loxl acts both as a scavenger receptor and regulator for extracellular matrix remodeling that initiate hemocyte migration and tissue regeneration, while wwox was shown to promote proliferation of immune cells through inhibition of their apoptosis. in addition, icd, mthfr and cytochrome p are major detoxification enzymes. in fact, immune cells and their secreted effectors require the proper redox state in the extracellular environments to exert their immune functions, which makes the maintenance of redox homeostasis essential for persistent and effective host defense. this is particularly true in the case of qpx disease where the neutralization of parasites depends on extracellular killing pathways.table transcripts with putative functions related to immune effectors that were differentially expressed during m. mercenaria focal response against qpx. additional information on these transcripts is given in additional file “inf” designates an infinite fold change calculated for focal response as the expression of that transcript in non-nodule tissue was equal to 0 apoptosis is an essential host mechanism to effectively remove damaged and infected cells without causing inflammatory destructions to surrounding tissues. interestingly, apoptosis seems to be largely inhibited during m. mercenaria focal response, as shown by the under-expression of pro-apoptosis transcripts. for example, the tumor necrosis factor -like protein and 3-hydroxy-3-methylglutaryl-coenzyme a reductase-like protein were significantly under-expressed in nodules. similarly, the pro-apoptotic p53-induced protein and solute carrier family member protein were also under-expressed in nodules. meanwhile, inhibitor of apoptosis protein was over-expressed in nodules. iaps regulate immune cell expansion and survival in highly inflammatory environments in mammals and they may share similar function in clams by preventing hemocytes from death during interaction with qpx. in fact, ros production during parasite killing may trigger apoptotic cell death in molluscs, and proper control of apoptosis mechanisms is required to maintain cellular homeostasis during immune response. this suspected inhibition of host apoptosis is supported by the above-mentioned over-expression of integrins, as these were shown to protect cells from apoptosis and induce anti-apoptotic pathways during cell adhesion and spreading in the snails lymnaea stagnalis and b. glabrata. infection and tissue injury trigger host immune responses via immune signaling pathways, by activating transcription factors and initiating the production of immune effectors and regulators. immune signaling pathways identified in mollusks include toll, mapk/jnk and jak/stat signaling pathways. during m. mercenaria focal response to qpx, a variety of transcripts encoding kinases and phosphatases were over-expressed, suggesting the involvement of mapk and other kinase-mediated cascades in regulating the focal inflammatory response, whereas the under-expression of ef-hand domain containing protein and calmodulin may indicate the suppression of calcium-regulated pathways. overexpression of e ubiquitin-protein ligase and its upstream regulator cop signalosome suggests the activation of the damage surveillance ubiquitin/proteasome pathway. in parallel, the over-expression of rho gtpase and rho kinase suggests the induction of anti-apoptotic rho-mediated signaling pathway and reinforce the idea that apoptotic inhibition is extensively initiated by m. mercenaria to help fight qpx. however, a very limited number of de transcripts was detected in relation to those conventional signaling pathways of innate immunity, such as the complement pathway and the toll/tlr pathway. only transcript encoding macrophage-expressed gene protein, a putative member of the complement pathway, was differentially expressed in qpx nodules. as for the toll/tlr pathway, only a few receptors were identified but none of their downstream components. interestingly, components of the notch and the wnt signaling pathways were over-expressed during focal response. these included two putative notch family members, the neurogenic locus notch protein and the mediator protein nicastrin, and the tyrosin-protein kinase ryk which belongs to the wnt pathway. wnt signaling pathway regulates many cellular immune processes and is evolutionarily conserved across taxa. wnt signaling has been intensively exploited for its regulatory functions during wound healing and tissue regeneration, so its over-expression may be related to wound healing to repair damage resulting from tissue digestion by the parasite or tissue necrosis. the notch signaling network was scarcely explored in bivalves even though it has been reported to be associated with several aspects of immune response in mammals, especially in regulating granulomatous reactions to foreign bodies. as an evolutionarily conserved pathway involved in modulating the intercellular signaling, the notch pathway presumably shares an equally important role in m. mercenaria by modulating the formation of granuloma. notch signaling triggers macrophage expression of genes involved in pro-inflammatory responses, but can suppress inflammation responses triggered by canonical tlr cascade, in agreement with our observations. these tightly regulated mechanisms ensure tailored immune responses against different pathogens and are crucial for the host to achieve high immune efficiency while avoiding excessive immune activation and self-inflicted damages. several proteases were also differentially regulated during m. mercenaria focal response against qpx. these proteases mostly belong to the serine and metallo protease families and were generally over-expressed in qpx nodules. proteases serve as key immune modulators partially through their ability to digest and remodel the extracellular matrix and tissues associated with hemocyte activation. commonly associated with lysosomes and granules of inflammatory cells, serine proteases participate in immune regulation either directly by degradation of pathogens or indirectly through activation of cell surface receptors and signal molecules. the function of metalloproteases in immune regulation is even more diverse, acting as immune effectors, signal transducers, and mediators of immune cell development and migration. metalloproteases are also known to be involved in many pro-inflammatory pathways, particularly in the notch pathway where they act as a type of downstream element to notch. at the same time, immune effectors with known universal protease inhibitor activities, such as alpha2-macroglobulin and thioester-containing protein, were also collectively overexpressed in qpx nodules. the α 2 m superfamily inhibits peptidases of diverse origins. the simultaneous over-expression of proteases and protease inhibitors may reflect a finely adjusted defense response of m. mercenaria to maintain homeostasis and regulate self- and pathogen-derived proteases, as shown in other host-pathogen systems, including bivalves. proteases have been identified as major virulence factors of qpx, and are thought to degrade host proteinaceous immune effectors and hydrolyze host tissues to fulfill nutritional requirements. therefore, inhibition of pathogen proteases contribute to host protection, and was shown to represent a determinant factor for resistance against infectious diseases in bivalves. in parallel, teps have been extensively studied in mollusk immunity, and beside their function as protease inhibitors, they also play a role as prrs or opsonins to facilitate microbial phagocytosis and encapsulation. consistent over-expression of teps was noted in this study in agreement with findings following experimental infection with qpx. these results support a critical role of teps in clam immune response against qpx either via the protease-inhibitor activity of these proteins, or by mediating parasite encapsulation, or both. in addition, several metal ion transporters were over-expressed in nodules, including the putative copper ion binding protein ceruloplasmin precursor, the transferrin enzyme ferric-chelate reductase and the divalent metal transporter. these molecules contribute to host defense by controlling the supply of essential micronutrients in the vicinity of infection sites thus reducing parasite survival and favoring the production of antimicrobial factors. it is noteworthy to point out that some of the focally over-expressed transcripts might be partly driven by the dramatic increase of hemocyte proportion within tissues in the vicinity of infection foci as compared to the surrounding host tissues. this would be especially the case for transcripts known to be highly expressed in hemocytes, such as the cell surface prrs, secreted humoral immune effectors, cell signal transducers and enzymes associated with ros production. systemic response significant transcriptomic regulations were observed during m. mercenaria systemic immune response against qpx, with a total of de transcripts, which is about two times the number of focal de transcripts. however, only about one third of the de transcripts were over-expressed in response to qpx infection, the larger remaining part represented significantly under-expressed transcripts, possibly due to the chronic stress imposed by the infection. transcriptome-wide depression has been demonstrated in many marine invertebrates as the result of pathogenic or environmental stress. a considerable number of systemically under-expressed transcripts was related to metabolism and biomineralization, which could be the result of host resource allocation during on-going infection. the chronic inflammation induced by infection likely created extra energy demands, which require resources being allocated from other physiological processes, such as growth and reproduction, to immune processes which is critical for the survivorship of the host. in fact, slow growth and lower tissue conditions are frequently observed in qpx-infected m. mercenaria, and similar energy trade-offs existed between immune defense and other energy expenditure pathways. interestingly, a suite of transcripts over-expressed during focal response was significantly under-expressed in non-nodule tissues as compared to healthy clams, which included several immune effectors and mediators associated with nodule formation and focal inflammation. in fact, maintaining high levels of these focally-induced molecules could be costly and dangerous as some are toxic to both the parasite and the host, so their production must be restrained within areas where they can directly exert defense function, and reduced outside the infection foci to minimize risks of undesirable effects on the host. on the other hand, systemically over-expressed transcripts included stress proteins and other soluble immune factors such as lysozyme, lectins, amp, proteases and ferric-chelate reductase. over-expression of host stress proteins, such as heat shock proteins and universal stress protein was also noted, in agreement with observations made during infection in other bivalve species. increased levels of stress proteins provide host cells with protection against incorrect protein folding caused by infection, inflammation, oxidative stress and other destructive events. the systemic over-expression of soluble immune effectors may help maintaining comparatively high immune capacity to prevent the spread of qpx throughout the host. in addition, transcripts of anti-apoptotic factors were also over-expressed during the systemic response, indicating that anti-apoptotic processes noted during focal response are not limited to the infection foci.table selected transcripts with annotated functions related to immune response that were over-expressed during m. mercenaria systemic response against qpx. additional information on these transcripts is given in additional file “inf” designates an infinite fold change calculated for focal response as the expression of that transcript in non-nodule tissue was equal to 0 pathway alterations during m. mercenaria’s response to qpx transcriptomic alterations during both focal and systemic response discussed above were also highlighted in the pathway enrichment analysis of the de transcripts. this analysis aims at extracting an overview of phenotypic changes on the underlying functional level, to reduce the complexity of biological information given by the long lists of de genes/transcripts. the kegg pathways of focal adhesion, ecm-receptor interaction, notch signaling pathway and apoptosis were significantly over-represented during both focal and systemic response, even though fold enrichment were generally higher during the focal response. other immune-related pathways particularly enriched during the focal response included regulation of actin cytoskeleton, cell adhesion molecules, the leukocyte transendothelial migration, complement and coagulation cascade and wnt signaling pathway. these pathways are critically involved in the immune cells activation during migration, attachment and parasite encapsulation, which serve as the underlying mechanisms for nodule formation and qpx killing. on the other hand, basic metabolic pathways such as the citrate cycle and pyruvate metabolism were specially enriched during the systemic response. these alterations were largely in accordance with the under-expression of metabolism-associated de transcripts in infected tissue compared to the healthy tissue, possibly reflecting changes in the energy allocation strategy during infection as discussed above.fig. significantly enriched kegg pathways in m. mercenaria derived from the differentially expressed genes during focal and systemic response against qpx. the kegg pathways having significant enrichment are presented, and the bar shows the x-fold enrichment of each kegg pathway. pathways involved in immune response are marked with red dots while metabolism-related pathways are marked with green dots interdependence of kegg pathways widely exist and most of these are interrelated with each other via shared components, forming a signaling network to allow for pathway crosstalk. to investigate these interactions, we extracted the de transcripts shared by multiple enriched pathways and constructed a sketch of the hypothetical pathways network that are significantly altered by qpx infection. in this framework, m. mercenaria response to qpx infection was initiated upon the sensing of danger signals via cell membrane receptors. the signals subsequently transmitted down through the mapk, wnt and notch pathways and triggered the production of a series of host defense factors as the end results. in parallel, activation of pathways regulating actin cytoskeleton and leukocyte transendothelial migration facilitated the recruitment of hemocytes to the infection area to build a barrier of cellular defense against the parasite. recruited hemocytes then attached to and encapsulated qpx cells as suggested by the modulation of focal adhesion and ecm receptor interaction pathways. these cellular activities were performed under a tight regulation of the apoptosis pathway to determine cell fates, resulting in either the survival or death of m. mercenaria cells.fig. overview of immune-related enriched pathways of differentially expressed transcripts during m. mercenaria response to qpx. a schematic diagram of enriched pathways and their interactions. only pathway components encoded by de transcripts are presented within each enriched kegg pathway. arrows display possible interactions between pathway components. b overview of de transcripts expression associated with enriched pathways in nodule, non-nodule and healthy m. mercenaria tissues. the red and green heatmap values indicate log fold of relative expression levels for individual transcripts. arp2/3: actin related protein 2/ complex; ctbp: c-terminal binding protein; camk2: calcium/calmodulin-dependent protein kinase ii; cycd: cyclin d1; ecm: von willebrand factor; endo-g: endonuclease g; fak: focal adhesion kinase; fyn: tyrosine-protein kinase; hes1: hairy and enhancer of split 1; iap: baculoviral iap repeat-containing protein, ciaps; itga: intergrin alpha; itgb: intergrin beta; mlcp: serine/threonine-protein phosphatase pp catalytic subunit; nrxn: neurexin; ncstn: nicastrin; rac: ras-related c botulinum toxin substrate; rock: rho-associated protein kinase; rtk: proto-oncogene tyrosine-protein kinase; sele: selectin, endothelial cell; selp: selectin, platelet; trail: tumor necrosis factor ligand superfamily member 10); wnt: wingless-type mmtv integration site family distinctive transcriptomic pattern of healthy clams a suite of transcripts exhibited higher transcription levels in healthy clams as compared to diseased clams. a considerable fraction of these transcripts were related to metabolic processes, nucleic acids binding and transcriptional regulation. the over-expressed immune-related transcripts identified in healthy clams are of particular interest as they may be involved in m. mercenaria resistance towards qpx. for example, an antimicrobial protein was exclusively identified in healthy clams with almost no detection in diseased clams. in addition, the highest expression levels of a serine protease inhibitor were also observed in healthy clams. a serine protease inhibitor has been linked to oyster resistance against the protozoan parasite perkinsus marinus. therefore, the high expression of the serine protease inhibitor in healthy clams supports its involvement in m. mercenaria’s resistance against qpx, likely by inhibiting the activity of parasite proteases. moreover, a pathogen recognition protein was significantly higher in healthy clams as compared to diseased animals, which may also contribute to clam resistance against qpx by promoting microbial recognition and encapsulation. previous studies demonstrated that clam genetic background affects m. mercenaria resistance toward qpx, therefore immune-related transcripts specifically associated with healthy clams represent excellent candidates as molecular markers for further research on screening and breeding qpx-resistant m. mercenaria strains.table selected transcripts with annotated functions related to immune response that were differentially expressed in naïve m. mercenaria as compared to qpx infected individuals. additional information on these transcripts is given in additional file “inf” designates an infinite fold change calculated for focal response as the expression of that transcript in non-nodule tissue was equal to 0 on the other hand, a subset of transcripts exhibited lowest expression levels in healthy clams. these include stress protein hsp and usp, as well as the detoxification molecule cytochrome p which together highlight the stress experienced by clams as the result of qpx infection. in addition, other immune related transcripts including a protease, a protease inhibitor, a c-type lectin, and molecules involved in tissue regeneration and cell signaling, were also under-expressed in healthy clams as compared to infected individuals, suggesting their role in fighting the infection.  this is one of the first studies contrasting focal and systemic immune responses to infections in invertebrates using high-throughput sequencing. resulting transcriptome represents a significant addition to the so far limited public genomic information available for m. mercenaria. the transcriptomic profiles of healthy and infected clams reflected complex interactions between the host immune system and the pathogen leading to molecular changes at both the infection foci and the systemic level. in general, the systemic responses of m. mercenaria reflected prevailing transcriptomic suppression accompanied with a contrasting over-expression of stress proteins and soluble antimicrobial effectors; whereas the focal response highlighted cell-cell interactions between hemocytes and the parasite that typically result in local inflammation, extracellular degradation, encapsulation, granuloma formation, and wound repair. what needs to be kept in mind is that the regulation of these genes can be the result of either an effective immune response or a symptom of a future death. in fact, several apoptotic and anti-apoptotic genes were regulated. this highlights a fine adjustment of m. mercenaria defense mechanisms to precisely adapt to the infection development. in addition, the identification of immune-related transcripts that were particularly associated with healthy clams offered new perspectives on the molecular features putatively involved in clam resistance against qpx. 
transcripts were assembled and the expression levels were compared between nodule and healthy tissues from infected clams, and between these and tissues from healthy clams. it is also one of the first studies contrasting focal and systemic responses to infections in invertebrates using high-throughput sequencing. a total of transcripts were identified as differentially expressed in different tissues. qpx infection establishes in pallial organs with the lesions typically characterized as nodules, which represent inflammatory masses formed by hemocyte infiltration and encapsulation of parasites. qpx infection is known to induce host changes on both the whole-organism level and at specific lesion areas, which imply systemic and focal defense responses, respectively. our study is the first attempt to thoroughly characterize m. mercenaria transcriptome and identify molecular features associated with qpx infection. among transcripts significantly modulated upon host-pathogen interactions, those involved in non-self recognition, signal transduction and defense response were over-represented. results identified the molecular signatures of clam systemic and focal defense responses, to collectively mediate immune processes such as hemocyte recruitment and local inflammation. rna-seq was performed using illumina hiseq to characterize m. mercenaria focal and systemic immune responses to qpx. results allowed the identification of host immune factors implicated in the systemic and focal responses against qpx and unraveled the pathways involved in parasite neutralization. de novo assembly reconstructed a consensus transcriptome of sequences that was functionally-annotated. these investigations improve our understanding of bivalve immunity and provide molecular targets for probing the biological bases of clam resistance towards qpx.
 the hard clam mercenaria mercenaria is an important seafood species widely exploited along the eastern coasts of the united states and play a crucial role in coastal ecology and economy. severe hard clam mortalities have been associated with the protistan parasite qpx. qpx infection establishes in pallial organs with the lesions typically characterized as nodules, which represent inflammatory masses formed by hemocyte infiltration and encapsulation of parasites. qpx infection is known to induce host changes on both the whole-organism level and at specific lesion areas, which imply systemic and focal defense responses, respectively. however, little is known about the molecular mechanisms underlying these alterations.  rna-seq was performed using illumina hiseq to characterize m. mercenaria focal and systemic immune responses to qpx. transcripts were assembled and the expression levels were compared between nodule and healthy tissues from infected clams, and between these and tissues from healthy clams. de novo assembly reconstructed a consensus transcriptome of sequences that was functionally-annotated. a total of transcripts were identified as differentially expressed in different tissues. results allowed the identification of host immune factors implicated in the systemic and focal responses against qpx and unraveled the pathways involved in parasite neutralization. among transcripts significantly modulated upon host-pathogen interactions, those involved in non-self recognition, signal transduction and defense response were over-represented. alterations in pathways regulating hemocyte focal adhesion, migration and apoptosis were also demonstrated.  our study is the first attempt to thoroughly characterize m. mercenaria transcriptome and identify molecular features associated with qpx infection. it is also one of the first studies contrasting focal and systemic responses to infections in invertebrates using high-throughput sequencing. results identified the molecular signatures of clam systemic and focal defense responses, to collectively mediate immune processes such as hemocyte recruitment and local inflammation. these investigations improve our understanding of bivalve immunity and provide molecular targets for probing the biological bases of clam resistance towards qpx. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords hard clammercenaria mercenariaqpxrnaseqimmune responsefocalsystemichttp://dx.doi.org/ /100000192national oceanic and atmospheric administrationna07oar4170010allam bassem http://dx.doi.org/ /100000076directorate for biological sciencesios 1050596allam bassem issue-copyright-statement© the author 2016 