BACKGROUND
in recent years, large scale sequencing studies have benefitted from the advance of high-throughput sequencing technologies that have resulted in substantial improvement in sequencing efficiency.
additionally, increases in the length and quality of sequencing reads have improved assemblies of sequenced genomes and transcriptomes.
sequencing is a powerful technique allowing for the rapid generation of transcriptome assemblies for any species of interest.
transcriptome sequencing measures expressed sequences only, thus does not have some of the challenges in dna sequencing   <cit> .
de novo transcriptome assembly removes the need for a reference genome in quantitative rna-seq experiments, allowing for the rapid and accurate quantification of transcript abundance in a given biological sample.
these aspects are especially useful in studies for organisms with limited genomic resources.
exemplary is the application of de novo transcriptome sequencing to a large range of organisms: vertebrates, e.g.
brown trout   <cit> , invertebrates e.g.
sea louse   <cit> , oriental fruit flies   <cit>  and the pollen beetles   <cit> , fungi   <cit>  and other microorganisms.
despite the rapid advances in sequence capabilities and in bioinformatics resources for generating high quality assemblies , de novo transcriptome studies in poorly characterized taxonomic groups continue to be challenging because of difficulties with annotation.
this is due to the lack of information available on the genes of interest in closely related organisms.
the subphylum crustacea represents one such taxonomic group for which limited information exists.
the ensembl genome database for metazoan species contains mainly diptera , nematoda  and hymenoptera , but information on only a single crustacean: the common water flea, daphnia pulex  <cit> .
furthermore, the number of ncbi entrez records in the invertebrate taxonomic branch shows huge under-representation of crustaceans.
in total, there are approximately  <dig> , <dig> nucleotide sequences in the subphylum crustacea; in comparison the order hymenoptera which alone contains almost  <dig> , <dig> nucleotide sequences .
consequently, subtaxa within the subphylum crustacea contain less information: decapoda  have a total of  <dig>  nucleotide and  <dig>  protein sequences available.
the european shore crab , c. maenas, is a keystone species in the european marine environment and is the only crustacean on the global invasive species database  <cit> , with invasions into australia, south africa and the united states  <cit> .
in such locations, c. maenas threatens local fishing industries, for example the destruction of the soft-shell clam  fishery in new england  <cit> .
c. maenas is also an important study species for biomonitoring and ecotoxicology  <cit> .
the species has been used in monitoring for heavy metal contamination  <cit> , metal toxicity studies , and more recently in exposures studies with nanomaterials  <cit>  and microplastics  <cit> .
pathological studies are a new area wherein c. maenas could play a role.
a study investigating infection of crustaceans with white spot syndrome virus , recognized as the most significant pathogen affecting global shrimp aquaculture, showed that c. maenas are relatively resistant to the virus .
despite its importance in these research areas, and its biological significance in the environment, the available molecular resources for c. maenas are extremely limited.
to date, sequence data for this species comprises approximately  <dig>  est sequences and several hundred nucleotide and protein sequences  <cit> .
given the ecological importance of c. maenas, together with its wider general utility for research purposes, we aimed to sequence, assemble and annotate a shore crab transcriptome.
we further set out to establish the relative expression profiles of all sequenced transcripts in different body tissues and organs, and to characterize immune pathways against those known for other invertebrates as a resource for future investigations on the response of this host to pathogens.
RESULTS
rna sequencing and assembly
twelve sequence libraries corresponding to  <dig> pooled tissue samples from adult male and female c. maenas were sequenced on an illumina hiseq  <dig> platform and yielded a total of  <dig> , <dig> paired reads across all tissues.
after removal of low quality reads through quality filtering, there were  <dig> , <dig> remaining paired reads.
on average  <dig>  ±  <dig>  million read pairs were obtained for each tissue and the distribution of the reads per pooled transcript sample is presented in table  <dig>  the filtered rna-seq data were used for de novo transcriptome assembly using the trinity pipeline with default parameters.
the assembled transcriptome encompassed  <dig> ,469 bp distributed over  <dig>  loci, represented by  <dig>  transcripts .
the transcript lengths had a median of 380 bp and a mean of 992 bp , and ranged between 201 bp and  <dig>  bp .
the transcriptome n <dig> was calculated to be  <dig>  bp.
<dig>  % of the read pairs could be mapped back to the de novo assembled transcriptome using the bowtie <dig> aligner.table  <dig> number of read pairs obtained for each crab tissue before and after removal of adapter sequences and quality filtering
a total of  <dig> out of the  <dig> highly conserved eukaryotic “core” genes were identified completely  and  <dig> genes  partially in the transcriptome by the cegma pipeline  <cit> , indicating that the transcriptome contains a near complete set of core eukaryotic genes.
transcriptome characterization
several approaches were taken to annotate the assembled transcripts.
firstly, the transcript sequences were compared to existing c. maenas est sequences in the ncbi database using blastn.
in total,  <dig>  sequences  showed high similarity to  <dig>  est sequences .
this indicates that the majority of transcripts in the assembly were previously un-reported for c. maenas.
a broader sequence homology search was performed using blastx against the ncbi non-redundant nr protein database and hits were found for  <dig>   of the transcripts using an e-value threshold of 1e- <dig>  open reading frames were identified in  <dig>   of transcripts and the majority of the predicted peptides , corresponding to  <dig>  % of all predicted peptides were annotated using the uniprot/swissprot database .
furthermore, conserved pfam domains were assigned to  <dig>   of the peptides and  <dig>   of these peptides appeared to contain signal peptides  as determined by signalp.
transcriptome annotation details can be found in additional files  <dig> and  <dig> table  <dig> number of annotated transcripts and open reading frames  using different annotation methods and sequence databases
transcriptome functional annotation
gene ontology  terms were assigned to  <dig>   of the annotated transcripts and  <dig>  % of the annotated predicted peptides  by blast2go  <cit> .
the most common go terms were protein binding , cytoplasm , nucleus , plasma membrane  and membrane .
the most common annotations for the three gene ontology trees are presented in table  <dig>  and a full list of transcript annotations is available in additional file  <dig> table  <dig> identification, sequence similarity and gene ontology annotation statistics of peptide sequences in the transcriptome
taxonomy
the blastx output was used as input for megan <dig> to illustrate the taxonomic origin of blast hits for the transcriptome in a phylogenetic tree.
a partially collapsed phylogenetic tree is presented in fig.
<dig>  the taxon with the largest number of sequence homologies was the pancrustacean taxon wherein  <dig>  c. maenas transcripts showed similarity.
within this taxon, transcripts were split between the crustacean and hexapoda taxa.
since c. maenas is a crustacean species it is expected that a large proportion of transcripts show similarity to sequences derived from this taxon.
however, due to the limitations in crustacean genomic resources a significant proportion of transcripts mapped to related sequences in the hexapoda taxon instead .
furthermore, it can be seen that a variety of sequences were derived from micro-organisms , which may correspond to transcripts originating from micro-organisms living within the c. maenas hosts, and/or may reflect contamination of kits and samples with environmental micro-organisms  <cit> .
to remove these potential contaminating transcripts from the transcriptome we filtered the transcriptome for sequences that mapped to the metazoan taxon.
following the application of this filtering step, a transcriptome encompassing  <dig>  transcripts was retained and used in subsequent analysis.fig.
<dig> taxonomic classifications of carcinus maenas transcripts.
partially collapsed phylogenetic tree produced by megan <dig>  numbers illustrate the number of transcripts representing each taxa.
within the metazoan taxon, the pancrustacea represented the largest taxonomic group
differential gene expression
transcript expression in the twelve tissue types was estimated by the rsem program  <cit> .
next, differentially expressed transcripts were identified through comparing gene expression profiles of each sampled tissue to the others.
the number of differentially expressed  transcripts for the various tissues ranged between  <dig>  in gill and  <dig>  in hepatopancreas .
all tissues showed enrichment for gene ontology  terms; the top five for every tissue are listed in table  <dig> .
the enriched go terms often reflected the function of the tissue e.g.
structural constituent of cuticle in eggs, angiogenesis in haemolymph and sarcolemma in muscle.
in several tissues the link to function is not very clear in the top five, but becomes apparent in other enriched terms.
for example, in the eye, phototransduction  and detection of light stimulus  were over-represented; contractile fibre  and sarcomere  were enriched in the heart tissue and finally, the epidermis and ovary tissues yielded only three enriched annotations .table  <dig> differentially expressed transcripts in specific tissues
immune pathway characterization in c. maenas
application of c. maenas as a model organism to study crustacean infectious diseases requires insight in the organism's immune system.
since crustaceans do not have adaptive immune systems, innate immune strategies will predominate in this organism when responding to pathogenic insults.
we investigated the presence of several innate immune system pathways in the c. maenas transcriptome and mapping the transcripts to pathways in the kegg database.
in total  <dig>   of transcripts were annotated to a kegg orthology group .
the kegg server  <cit>  allows mapping of the present orthology groups to pathways in the kegg database and visualization of presence/absence of their components.
li et al.
<dig> characterized a selection of innate immune pathways in the hepatopancreas transcriptome of the mitten crab eriocheir sinensis, including the rnai pathway, toll-like receptor pathway, immune deficiency  pathway, the jak-stat and mitogen activated protein kinase  signalling pathways  <cit> .
we characterized the same pathways in the c. maenas transcriptome with additions including the endocytosis pathway.
the latter is not directly related to the immune response but many viruses utilize its machinery to gain entry to host cells  <cit> .
its characterization can thus be important for investigations of viral infections.
pathogen associated molecular pattern recognition
the first stage in immune defence is the identification of invading pathogens by an organism.
in this process a distinction between cells from the organism itself and those of the invading pathogens needs to occur.
to achieve this, the innate immune system employs a group of pattern recognition receptors  that are able to recognize pathogen associated molecular patterns .
examples of pamps include lipopolysaccharides, peptidoglycans and β- <dig> -glucans  <cit>  and groups of prrs include gram-negative binding proteins , peptidoglycan recognition proteins , thioester containing proteins and lectins  <cit> .
upon successful pathogen recognition, prrs initiate immune responses.
c. maenas transcripts that show sequence similarity to known prr groups are shown in table  <dig>  representatives of most groups of prr have counterparts in the c. maenas transcriptome as identified through sequence similarity, often to sequences derived from organisms that are closely related to c. maenas.
one group that is not represented are the pgrps, this has also been reported in other crustacean species  <cit> .
down syndrome cell adhesion molecule  is a pamp recognition protein that has been hypothesized to be involved in immune memory .
this gene can produce many isoforms, and initial findings suggested that it played an important role in the development of the nervous system in invertebrates where dscam isoforms aid in the discrimination of neuritis  <cit> .
dscam isoforms were later found to be able to recognise pathogens, aiding in phagocytosis  <cit> .
in concordance with this hypothesis, the c. maenas dscam gene appears to encode many isoforms, and in total  <dig> transcripts with significant similarity to dscam sequences in ncbi were found in the transcriptome.table  <dig>
carcinus maenas pathogen associated molecular pattern recognition genes
the immune responses initiated by these prrs can occur at a transcriptional level, e.g.
activation of toll and imd can aid in phagocytosis e.g.
dscam binding, or can initiate proteolytic cascades leading to melanization.
toll-like receptor pathway
the toll receptor pathway is a signalling route that responds to the presence of pamps by ultimately activating nf-κb  <cit> .
in mammals, toll-like receptors  bind to pamps resulting in dimerization.
upon forming dimers, the tlrs recruit myd <dig> and subsequently irak kinases.
after irak kinases activate traf <dig>  its binding to tak <dig> and ikkβ ultimately frees nf-κb to diffuse into the nucleus  <cit> .
in invertebrates, such as d. melanogaster, the mechanism is slightly different, and instead of directly binding pamps, tlrs respond to the toll ligand spätzle  <cit> .
the kegg database contains a version of the toll-like receptor pathway which was used to visualize the coverage of this pathway in the c. maenas transcriptome .
homologues were found for most of the components in the paths from tlr to nf-κb and activator protein- <dig> .
since kegg is targeted towards vertebrate genes and pathways, a characterization of an invertebrate toll signalling pathway was also performed .
components of the d. melanogaster toll signalling pathway were taken from li et al.
<cit>  and kingsolver et al.
<cit>  and investigated for presence and expression in the assembled transcriptome.
transcripts with significant sequence similarity to most of the toll pathway components were found in the transcriptome .
tube, an irak homolog, was not identified in the c. maenas transcriptome.
successfully identified transcripts were found to be expressed across all tissues , and the median expression values varied from  <dig>  fpkm for myd <dig> to  <dig>  fpkm for toll.fig.
<dig> toll-like receptor signalling pathway coverage.
the toll-like receptor signalling pathway in the kegg database.
proteins in the pathway are depicted by boxes while arrows depict signalling routes.
pathway components with homologues in the carcinus maenas transcriptome are highlighted in pink
imd pathway
the imd pathway is also activated upon pathogen recognition, in particular by gram-negative bacteria.
similar to toll-like receptors, the binding of peptidoglycan by pgrps leads to dimerization  <cit> .
after the dimerization, the signal is transmitted through imd, as well as fadd and dredd.
activation of dredd leads to poly-ubiquitination of imd  <cit> , binding of tak <dig> and assembly of the ikk complex.
relish phosphorylation is promoted by ikk, and an event followed by cleavage of relish by dredd cause translocation of the n-terminal end to the nucleus where it regulates the expression of effector molecules  <cit> .
since the kegg database does not contain the imd pathway, the kegg tnf-signalling pathway was used instead.
as for the toll-like receptor pathway, homologues also were found for most constituents of the tnf-signalling pathway .
manual identification of imd pathway components derived from kingsolver et al.
<cit>  showed that fadd was the only absent component in the c. maenas transcriptome .
imd itself was only expressed in three out of twelve tissues  whereas the rest of the imd pathway was expressed across all tissue types .fig.
<dig> tnf signalling pathway.
overview of the kegg tnf signalling pathway, components depicted as in fig.
<dig>  components with homologues in the carcinus maenas transcriptome are highlighted in pink
jak-stat signalling pathway
the jak-stat signalling pathway mediates the response to chemical messenger molecules like cytokines.
it has been shown that stat signalling is activated upon wssv infection in shrimp  <cit> .
jak tyrosine kinases bind to cytokine receptors and upon ligand binding they phosphorylate tyrosine residues on those receptors  <cit> .
stat is able to bind and subsequently be phosphorylated by jak  <cit> .
following phosphorylation, stat forms dimers, translocates to the nucleus and organizes the response to the signalling molecule by altering gene expression  <cit> .
inhibitors of jak-stat signalling are present at several stages and include dominant negative co-receptors, prevention of stat recruitment by socs  and protein inhibitors of activated stat   <cit> .
the kegg reference pathway and coverage in the transcriptome are presented in fig.
<dig>  most of the components of the jak-stat pathway have a homologue in the c. maenas transcriptome.
the pathway in fig.
<dig> shows that only the cytokine receptor was not identified by the kegg annotation.
however one transcript  showed highly significant sequence homology to the cytokine receptor of harpegnathos saltator  and the domeless receptor of tribolium castaneum .fig.
<dig> jak-stat signalling pathway.
overview of the kegg jak-stat signalling pathway.
components with homologues in the carcinus maenas transcriptome are highlighted in pink
response proteins
the signalling cascade through the imd, toll and jak-stat pathways results in a transcriptional immune response mediated by transcription factors like stat and nf-kb.
one part of this immune response includes antimicrobial peptides  and lysozyme), which have evolved to attack pathogens  <cit> .
in addition to antimicrobial peptides, the innate immune system also employs nitric oxide as a defensive molecule.
nitric oxide is an important redox activated signalling molecule and can be produced in large concentrations by nitric oxide synthase  <dig> , an enzyme synthesized as a response to prr activation  <cit> .
response proteins identified in the c. maenas transcriptome are listed in table  <dig> along with their target pathogen type, as described in tassanakajon et al.
<cit> .
neither penaeidins  <cit>  nor stylicins  <cit>  were identified for c. maenas and we hypothesise that both are probably limited to penaeid shrimp species.
the antimicrobial arsenal of c. maenas includes alf, lysozyme, crustins, carcinin and inducible nitric oxide synthase.
it is possible that the c. maenas transcriptome also contains novel anti-microbial peptides but to identify them will require exposure studies to trigger their activation.table  <dig>
carcinus maenas immune system response proteins
melanization pathway
the c. maenas innate immune system also contains a more direct response to pathogen infection in the form of the melanization pathway.
activated within minutes after infection, melanization damages and encapsulates invading pathogens with melanin  <cit> .
the production of melanin from phenols and quinones generates reactive oxygen species that are damaging to the pathogen.
synthesis of quinones is catalyzed by the phenol oxidase  enzyme.
po is readily available as a precursor  that is activated through proteolysis, ensuring a fast response time.
recognition of pamps by prrs leads to activation of a serine protease cascade that ends with the activation of po  <cit> .
the proteolytic cascade is regulated by serpins that act as serine protease inhibitors  <cit> .
members of the melanization pathway as described in tang  <dig>  <cit>  and transcripts with significant sequence similarity are listed in additional file  <dig>  the upstream proteases of propo: mp <dig>  sp <dig> and the activating enzyme ppae and prophenoloxidase itself are identified.
transcripts coding for the transcription factors serpent and lozenge, controlling the expression of propo  <cit> , and peroxinectin, a protein that is associated with the propo pathway and aids in cellular adhesion of haemocytes to pathogens  <cit>  were also found.
the expression of propo varied across tissues , and was particularly high in the hepatopancreas and ovary.fig.
<dig> melanization pathway expression.
expression of melanization pathway components in twelve carcinus maenas tissues.
the expression values are presented in fpkm, values of  <dig> are coloured white and values over  <dig> fpkm are binned together
rnai pathway
rna interference  is one of the major antiviral pathways within the invertebrate innate immune system  <cit> .
the pathway produces small interfering rna molecules  from virus derived dsrna  <cit> .
in short, dsrna is recognized by dicer proteins that subsequently cleave it to  <dig> nucleotide  sirnas.
sirnas are loaded into the risc complex, which utilizes argonaute  protein to cleave viral rnas targeted by the sirna, and thus silencing expression  <cit> .
the rnai pathway can also be employed to silence specific genes in cells and forms the basis of antiviral immunity strategies, a topic explored in la fauce et al.
<dig>  <cit> .
identification of components of the rnai pathway was based on those listed in wang et al.
<dig>  <cit> , results are shown in table  <dig>  d. melanogaster has distinct functions for dicer- <dig> and dicer- <dig>  the first being involved in the mirna pathway and the latter in sirna  <cit> .
both dicer- <dig> and dicer- <dig> were identified in c. maenas suggesting that a similar division of tasks could exist in this organism.table  <dig>
carcinus maenas rnai pathway components
endocytosis pathway
the endocytosis pathway plays a crucial role in viral challenges.
whereas some viruses are able to enter the cytosol directly, the majority require uptake via endocytosis  <cit> .
viral particles can enter endosomes via various endocytotic mechanisms .
decreasing ph in the endosome environment is a cue to the viral particles, which then penetrate into the cytosol  <cit> .
this indicates that there are important interactions between components of the endocytosis pathway and viral proteins, e.g.
cellular rab <dig> can interact with the vp <dig> protein of the white spot syndrome virus  <cit> .
therefore, information on the sequences and expression of the c. maenas endocytic system may aid in the study of viral infection.
the mechanisms of endocytosis, maturation of endosomes and related signalling molecules are depicted in the kegg pathway shown in fig.
<dig>  the number of identified components demonstrates that c. maenas contains an endocytic system that closely resembles this canonical kegg pathway.
the kegg annotation did not yield transcripts similar to caveolin, an important constituent of caveolar-mediated endocytosis.
however a tblastn search of ncbi caveolin protein sequences in the transcriptome identified similarity between ‘comp141181_c0_seq1’ and caveolin-3-like isoform x <dig> .
expression of components of the endocytosis pathway is visualized in fig.
<dig>  and most of these components were expressed across all tissues.
the muscle tissue showed an endocytosis expression profile that differs from the other tissues.fig.
<dig> endocytosis pathway.
overview of the kegg endocytosis pathway.
components with homologues in the carcinus maenas transcriptome are highlighted in pink
fig.
<dig> endocytosis pathway expression.
expression of endocytosis pathway components in twelve carcinus maenas tissues.
the expression values are in fpkm, values of  <dig> are coloured white and values over  <dig> fpkm are binned together
CONCLUSIONS
we produced an assembled transcriptome for c. maenas that consists of  <dig>  loci and  <dig>  transcripts and provides a significant molecular resource for wide studies into both basic and applied biology for this species.
comparisons run in the ncbi-nr database showed 30 % of c. maenas transcripts had significant homology against known sequences, but a large number were novel transcripts that have yet to be characterized.
expression analysis revealed tissues and organ transcript specificity that mapped with gene ontology annotations relating to specific tissue/organ-related functions.
of particular relevance for studies into pathogenesis and disease, we identified the presence of a series of known targets and functional pathways including the rnai pathway, toll-like receptor signalling, imd and jak-stat pathways that form part of their innate immune system.

 in recent years, large scale sequencing studies have benefitted from the advance of high-throughput sequencing technologies that have resulted in substantial improvement in sequencing efficiency. additionally, increases in the length and quality of sequencing reads have improved assemblies of sequenced genomes and transcriptomes. sequencing is a powerful technique allowing for the rapid generation of transcriptome assemblies for any species of interest. transcriptome sequencing measures expressed sequences only, thus does not have some of the challenges in dna sequencing. de novo transcriptome assembly removes the need for a reference genome in quantitative rna-seq experiments, allowing for the rapid and accurate quantification of transcript abundance in a given biological sample. these aspects are especially useful in studies for organisms with limited genomic resources. exemplary is the application of de novo transcriptome sequencing to a large range of organisms: vertebrates, e.g. brown trout, invertebrates e.g. sea louse, oriental fruit flies and the pollen beetles, fungi and other microorganisms. despite the rapid advances in sequence capabilities and in bioinformatics resources for generating high quality assemblies, de novo transcriptome studies in poorly characterized taxonomic groups continue to be challenging because of difficulties with annotation. this is due to the lack of information available on the genes of interest in closely related organisms. the subphylum crustacea represents one such taxonomic group for which limited information exists. the ensembl genome database for metazoan species contains mainly diptera, nematoda and hymenoptera, but information on only a single crustacean: the common water flea, daphnia pulex. furthermore, the number of ncbi entrez records in the invertebrate taxonomic branch shows huge under-representation of crustaceans. in total, there are approximately, nucleotide sequences in the subphylum crustacea; in comparison the order hymenoptera which alone contains almost, nucleotide sequences. consequently, subtaxa within the subphylum crustacea contain less information: decapoda have a total of nucleotide and protein sequences available. the european shore crab, c. maenas, is a keystone species in the european marine environment and is the only crustacean on the global invasive species database, with invasions into australia, south africa and the united states. in such locations, c. maenas threatens local fishing industries, for example the destruction of the soft-shell clam fishery in new england. c. maenas is also an important study species for biomonitoring and ecotoxicology. the species has been used in monitoring for heavy metal contamination, metal toxicity studies, and more recently in exposures studies with nanomaterials and microplastics. pathological studies are a new area wherein c. maenas could play a role. a study investigating infection of crustaceans with white spot syndrome virus, recognized as the most significant pathogen affecting global shrimp aquaculture, showed that c. maenas are relatively resistant to the virus. despite its importance in these research areas, and its biological significance in the environment, the available molecular resources for c. maenas are extremely limited. to date, sequence data for this species comprises approximately est sequences and several hundred nucleotide and protein sequences. given the ecological importance of c. maenas, together with its wider general utility for research purposes, we aimed to sequence, assemble and annotate a shore crab transcriptome. we further set out to establish the relative expression profiles of all sequenced transcripts in different body tissues and organs, and to characterize immune pathways against those known for other invertebrates as a resource for future investigations on the response of this host to pathogens.  rna sequencing and assembly twelve sequence libraries corresponding to pooled tissue samples from adult male and female c. maenas were sequenced on an illumina hiseq platform and yielded a total of, paired reads across all tissues. after removal of low quality reads through quality filtering, there were, remaining paired reads. on average  ±  million read pairs were obtained for each tissue and the distribution of the reads per pooled transcript sample is presented in table  the filtered rna-seq data were used for de novo transcriptome assembly using the trinity pipeline with default parameters. the assembled transcriptome encompassed,469 bp distributed over loci, represented by transcripts. the transcript lengths had a median of 380 bp and a mean of 992 bp, and ranged between 201 bp and  bp. the transcriptome n was calculated to be  bp.  % of the read pairs could be mapped back to the de novo assembled transcriptome using the bowtie aligner.table number of read pairs obtained for each crab tissue before and after removal of adapter sequences and quality filtering a total of out of the highly conserved eukaryotic “core” genes were identified completely and genes partially in the transcriptome by the cegma pipeline, indicating that the transcriptome contains a near complete set of core eukaryotic genes. transcriptome characterization several approaches were taken to annotate the assembled transcripts. firstly, the transcript sequences were compared to existing c. maenas est sequences in the ncbi database using blastn. in total, sequences showed high similarity to est sequences. this indicates that the majority of transcripts in the assembly were previously un-reported for c. maenas. a broader sequence homology search was performed using blastx against the ncbi non-redundant nr protein database and hits were found for of the transcripts using an e-value threshold of 1e- open reading frames were identified in of transcripts and the majority of the predicted peptides, corresponding to  % of all predicted peptides were annotated using the uniprot/swissprot database. furthermore, conserved pfam domains were assigned to of the peptides and of these peptides appeared to contain signal peptides as determined by signalp. transcriptome annotation details can be found in additional files and table number of annotated transcripts and open reading frames using different annotation methods and sequence databases transcriptome functional annotation gene ontology terms were assigned to of the annotated transcripts and  % of the annotated predicted peptides by blast2go. the most common go terms were protein binding, cytoplasm, nucleus, plasma membrane and membrane. the most common annotations for the three gene ontology trees are presented in table  and a full list of transcript annotations is available in additional file table identification, sequence similarity and gene ontology annotation statistics of peptide sequences in the transcriptome taxonomy the blastx output was used as input for megan to illustrate the taxonomic origin of blast hits for the transcriptome in a phylogenetic tree. a partially collapsed phylogenetic tree is presented in fig.  the taxon with the largest number of sequence homologies was the pancrustacean taxon wherein c. maenas transcripts showed similarity. within this taxon, transcripts were split between the crustacean and hexapoda taxa. since c. maenas is a crustacean species it is expected that a large proportion of transcripts show similarity to sequences derived from this taxon. however, due to the limitations in crustacean genomic resources a significant proportion of transcripts mapped to related sequences in the hexapoda taxon instead. furthermore, it can be seen that a variety of sequences were derived from micro-organisms, which may correspond to transcripts originating from micro-organisms living within the c. maenas hosts, and/or may reflect contamination of kits and samples with environmental micro-organisms. to remove these potential contaminating transcripts from the transcriptome we filtered the transcriptome for sequences that mapped to the metazoan taxon. following the application of this filtering step, a transcriptome encompassing transcripts was retained and used in subsequent analysis.fig. taxonomic classifications of carcinus maenas transcripts. partially collapsed phylogenetic tree produced by megan numbers illustrate the number of transcripts representing each taxa. within the metazoan taxon, the pancrustacea represented the largest taxonomic group differential gene expression transcript expression in the twelve tissue types was estimated by the rsem program. next, differentially expressed transcripts were identified through comparing gene expression profiles of each sampled tissue to the others. the number of differentially expressed transcripts for the various tissues ranged between in gill and in hepatopancreas. all tissues showed enrichment for gene ontology terms; the top five for every tissue are listed in table . the enriched go terms often reflected the function of the tissue e.g. structural constituent of cuticle in eggs, angiogenesis in haemolymph and sarcolemma in muscle. in several tissues the link to function is not very clear in the top five, but becomes apparent in other enriched terms. for example, in the eye, phototransduction and detection of light stimulus were over-represented; contractile fibre and sarcomere were enriched in the heart tissue and finally, the epidermis and ovary tissues yielded only three enriched annotations.table differentially expressed transcripts in specific tissues immune pathway characterization in c. maenas application of c. maenas as a model organism to study crustacean infectious diseases requires insight in the organism's immune system. since crustaceans do not have adaptive immune systems, innate immune strategies will predominate in this organism when responding to pathogenic insults. we investigated the presence of several innate immune system pathways in the c. maenas transcriptome and mapping the transcripts to pathways in the kegg database. in total of transcripts were annotated to a kegg orthology group. the kegg server allows mapping of the present orthology groups to pathways in the kegg database and visualization of presence/absence of their components. li et al. characterized a selection of innate immune pathways in the hepatopancreas transcriptome of the mitten crab eriocheir sinensis, including the rnai pathway, toll-like receptor pathway, immune deficiency pathway, the jak-stat and mitogen activated protein kinase signalling pathways. we characterized the same pathways in the c. maenas transcriptome with additions including the endocytosis pathway. the latter is not directly related to the immune response but many viruses utilize its machinery to gain entry to host cells. its characterization can thus be important for investigations of viral infections. pathogen associated molecular pattern recognition the first stage in immune defence is the identification of invading pathogens by an organism. in this process a distinction between cells from the organism itself and those of the invading pathogens needs to occur. to achieve this, the innate immune system employs a group of pattern recognition receptors that are able to recognize pathogen associated molecular patterns. examples of pamps include lipopolysaccharides, peptidoglycans and β- -glucans and groups of prrs include gram-negative binding proteins, peptidoglycan recognition proteins, thioester containing proteins and lectins. upon successful pathogen recognition, prrs initiate immune responses. c. maenas transcripts that show sequence similarity to known prr groups are shown in table  representatives of most groups of prr have counterparts in the c. maenas transcriptome as identified through sequence similarity, often to sequences derived from organisms that are closely related to c. maenas. one group that is not represented are the pgrps, this has also been reported in other crustacean species. down syndrome cell adhesion molecule is a pamp recognition protein that has been hypothesized to be involved in immune memory. this gene can produce many isoforms, and initial findings suggested that it played an important role in the development of the nervous system in invertebrates where dscam isoforms aid in the discrimination of neuritis. dscam isoforms were later found to be able to recognise pathogens, aiding in phagocytosis. in concordance with this hypothesis, the c. maenas dscam gene appears to encode many isoforms, and in total transcripts with significant similarity to dscam sequences in ncbi were found in the transcriptome.table carcinus maenas pathogen associated molecular pattern recognition genes the immune responses initiated by these prrs can occur at a transcriptional level, e.g. activation of toll and imd can aid in phagocytosis e.g. dscam binding, or can initiate proteolytic cascades leading to melanization. toll-like receptor pathway the toll receptor pathway is a signalling route that responds to the presence of pamps by ultimately activating nf-κb. in mammals, toll-like receptors bind to pamps resulting in dimerization. upon forming dimers, the tlrs recruit myd and subsequently irak kinases. after irak kinases activate traf its binding to tak and ikkβ ultimately frees nf-κb to diffuse into the nucleus. in invertebrates, such as d. melanogaster, the mechanism is slightly different, and instead of directly binding pamps, tlrs respond to the toll ligand spätzle. the kegg database contains a version of the toll-like receptor pathway which was used to visualize the coverage of this pathway in the c. maenas transcriptome. homologues were found for most of the components in the paths from tlr to nf-κb and activator protein-. since kegg is targeted towards vertebrate genes and pathways, a characterization of an invertebrate toll signalling pathway was also performed. components of the d. melanogaster toll signalling pathway were taken from li et al. and kingsolver et al. and investigated for presence and expression in the assembled transcriptome. transcripts with significant sequence similarity to most of the toll pathway components were found in the transcriptome. tube, an irak homolog, was not identified in the c. maenas transcriptome. successfully identified transcripts were found to be expressed across all tissues, and the median expression values varied from fpkm for myd to fpkm for toll.fig. toll-like receptor signalling pathway coverage. the toll-like receptor signalling pathway in the kegg database. proteins in the pathway are depicted by boxes while arrows depict signalling routes. pathway components with homologues in the carcinus maenas transcriptome are highlighted in pink imd pathway the imd pathway is also activated upon pathogen recognition, in particular by gram-negative bacteria. similar to toll-like receptors, the binding of peptidoglycan by pgrps leads to dimerization. after the dimerization, the signal is transmitted through imd, as well as fadd and dredd. activation of dredd leads to poly-ubiquitination of imd, binding of tak and assembly of the ikk complex. relish phosphorylation is promoted by ikk, and an event followed by cleavage of relish by dredd cause translocation of the n-terminal end to the nucleus where it regulates the expression of effector molecules. since the kegg database does not contain the imd pathway, the kegg tnf-signalling pathway was used instead. as for the toll-like receptor pathway, homologues also were found for most constituents of the tnf-signalling pathway. manual identification of imd pathway components derived from kingsolver et al. showed that fadd was the only absent component in the c. maenas transcriptome. imd itself was only expressed in three out of twelve tissues whereas the rest of the imd pathway was expressed across all tissue types.fig. tnf signalling pathway. overview of the kegg tnf signalling pathway, components depicted as in fig.  components with homologues in the carcinus maenas transcriptome are highlighted in pink jak-stat signalling pathway the jak-stat signalling pathway mediates the response to chemical messenger molecules like cytokines. it has been shown that stat signalling is activated upon wssv infection in shrimp. jak tyrosine kinases bind to cytokine receptors and upon ligand binding they phosphorylate tyrosine residues on those receptors. stat is able to bind and subsequently be phosphorylated by jak. following phosphorylation, stat forms dimers, translocates to the nucleus and organizes the response to the signalling molecule by altering gene expression. inhibitors of jak-stat signalling are present at several stages and include dominant negative co-receptors, prevention of stat recruitment by socs and protein inhibitors of activated stat. the kegg reference pathway and coverage in the transcriptome are presented in fig.  most of the components of the jak-stat pathway have a homologue in the c. maenas transcriptome. the pathway in fig.  shows that only the cytokine receptor was not identified by the kegg annotation. however one transcript showed highly significant sequence homology to the cytokine receptor of harpegnathos saltator and the domeless receptor of tribolium castaneum.fig. jak-stat signalling pathway. overview of the kegg jak-stat signalling pathway. components with homologues in the carcinus maenas transcriptome are highlighted in pink response proteins the signalling cascade through the imd, toll and jak-stat pathways results in a transcriptional immune response mediated by transcription factors like stat and nf-kb. one part of this immune response includes antimicrobial peptides and lysozyme), which have evolved to attack pathogens. in addition to antimicrobial peptides, the innate immune system also employs nitric oxide as a defensive molecule. nitric oxide is an important redox activated signalling molecule and can be produced in large concentrations by nitric oxide synthase, an enzyme synthesized as a response to prr activation. response proteins identified in the c. maenas transcriptome are listed in table  along with their target pathogen type, as described in tassanakajon et al.. neither penaeidins nor stylicins were identified for c. maenas and we hypothesise that both are probably limited to penaeid shrimp species. the antimicrobial arsenal of c. maenas includes alf, lysozyme, crustins, carcinin and inducible nitric oxide synthase. it is possible that the c. maenas transcriptome also contains novel anti-microbial peptides but to identify them will require exposure studies to trigger their activation.table carcinus maenas immune system response proteins melanization pathway the c. maenas innate immune system also contains a more direct response to pathogen infection in the form of the melanization pathway. activated within minutes after infection, melanization damages and encapsulates invading pathogens with melanin. the production of melanin from phenols and quinones generates reactive oxygen species that are damaging to the pathogen. synthesis of quinones is catalyzed by the phenol oxidase enzyme. po is readily available as a precursor that is activated through proteolysis, ensuring a fast response time. recognition of pamps by prrs leads to activation of a serine protease cascade that ends with the activation of po. the proteolytic cascade is regulated by serpins that act as serine protease inhibitors. members of the melanization pathway as described in tang and transcripts with significant sequence similarity are listed in additional file the upstream proteases of propo: mp sp and the activating enzyme ppae and prophenoloxidase itself are identified. transcripts coding for the transcription factors serpent and lozenge, controlling the expression of propo, and peroxinectin, a protein that is associated with the propo pathway and aids in cellular adhesion of haemocytes to pathogens were also found. the expression of propo varied across tissues, and was particularly high in the hepatopancreas and ovary.fig. melanization pathway expression. expression of melanization pathway components in twelve carcinus maenas tissues. the expression values are presented in fpkm, values of are coloured white and values over fpkm are binned together rnai pathway rna interference is one of the major antiviral pathways within the invertebrate innate immune system. the pathway produces small interfering rna molecules from virus derived dsrna. in short, dsrna is recognized by dicer proteins that subsequently cleave it to nucleotide sirnas. sirnas are loaded into the risc complex, which utilizes argonaute protein to cleave viral rnas targeted by the sirna, and thus silencing expression. the rnai pathway can also be employed to silence specific genes in cells and forms the basis of antiviral immunity strategies, a topic explored in la fauce et al.. identification of components of the rnai pathway was based on those listed in wang et al., results are shown in table  d. melanogaster has distinct functions for dicer- and dicer- the first being involved in the mirna pathway and the latter in sirna. both dicer- and dicer- were identified in c. maenas suggesting that a similar division of tasks could exist in this organism.table carcinus maenas rnai pathway components endocytosis pathway the endocytosis pathway plays a crucial role in viral challenges. whereas some viruses are able to enter the cytosol directly, the majority require uptake via endocytosis. viral particles can enter endosomes via various endocytotic mechanisms. decreasing ph in the endosome environment is a cue to the viral particles, which then penetrate into the cytosol. this indicates that there are important interactions between components of the endocytosis pathway and viral proteins, e.g. cellular rab can interact with the vp protein of the white spot syndrome virus. therefore, information on the sequences and expression of the c. maenas endocytic system may aid in the study of viral infection. the mechanisms of endocytosis, maturation of endosomes and related signalling molecules are depicted in the kegg pathway shown in fig.  the number of identified components demonstrates that c. maenas contains an endocytic system that closely resembles this canonical kegg pathway. the kegg annotation did not yield transcripts similar to caveolin, an important constituent of caveolar-mediated endocytosis. however a tblastn search of ncbi caveolin protein sequences in the transcriptome identified similarity between ‘comp141181_c0_seq1’ and caveolin-3-like isoform x. expression of components of the endocytosis pathway is visualized in fig.  and most of these components were expressed across all tissues. the muscle tissue showed an endocytosis expression profile that differs from the other tissues.fig. endocytosis pathway. overview of the kegg endocytosis pathway. components with homologues in the carcinus maenas transcriptome are highlighted in pink fig. endocytosis pathway expression. expression of endocytosis pathway components in twelve carcinus maenas tissues. the expression values are in fpkm, values of are coloured white and values over fpkm are binned together  we produced an assembled transcriptome for c. maenas that consists of loci and transcripts and provides a significant molecular resource for wide studies into both basic and applied biology for this species. comparisons run in the ncbi-nr database showed 30 % of c. maenas transcripts had significant homology against known sequences, but a large number were novel transcripts that have yet to be characterized. expression analysis revealed tissues and organ transcript specificity that mapped with gene ontology annotations relating to specific tissue/organ-related functions. of particular relevance for studies into pathogenesis and disease, we identified the presence of a series of known targets and functional pathways including the rnai pathway, toll-like receptor signalling, imd and jak-stat pathways that form part of their innate immune system. 
to address this resource shortfall we produced a c. maenas transcriptome, enabled by the progress in next-generation sequencing technologies, and applied this to assemble information on the innate immune system in this species. we isolated and pooled rna for twelve different tissues and organs from c. maenas individuals and sequenced the rna using next generation sequencing on an illumina hiseq platform. we have produced an assembled transcriptome for c. maenas that provides a significant molecular resource for wide ranging studies in this species. however, the genomic resources for this organism are still sparse, limiting research progress in these fields. the european shore crab, carcinus maenas, is used widely in biomonitoring, ecotoxicology and for studies into host-pathogen interactions. analysis of the transcriptome has revealed the presence of a series of known targets and functional pathways that form part of their innate immune system and illustrate tissue specific differences in their expression patterns. based on sequence homology to immune system components in other organisms, we show both the presence of transcripts for a series of known pathogen recognition receptors and response proteins that form part of the innate immune system, and transcripts representing the rnai, toll-like receptor signalling, imd and jak/stat pathways. there were differential patterns of expression for between and transcripts across tissues and organs with over-represented gene ontology terms relating to their specific function.
 the european shore crab, carcinus maenas, is used widely in biomonitoring, ecotoxicology and for studies into host-pathogen interactions. it is also an important invasive species in numerous global locations. however, the genomic resources for this organism are still sparse, limiting research progress in these fields. to address this resource shortfall we produced a c. maenas transcriptome, enabled by the progress in next-generation sequencing technologies, and applied this to assemble information on the innate immune system in this species.  we isolated and pooled rna for twelve different tissues and organs from c. maenas individuals and sequenced the rna using next generation sequencing on an illumina hiseq platform. after de novo assembly a transcriptome was generated encompassing transcripts. the transcripts were filtered, annotated and characterised using a variety of tools and databases. there were differential patterns of expression for between and transcripts across tissues and organs with over-represented gene ontology terms relating to their specific function. based on sequence homology to immune system components in other organisms, we show both the presence of transcripts for a series of known pathogen recognition receptors and response proteins that form part of the innate immune system, and transcripts representing the rnai, toll-like receptor signalling, imd and jak/stat pathways.  we have produced an assembled transcriptome for c. maenas that provides a significant molecular resource for wide ranging studies in this species. analysis of the transcriptome has revealed the presence of a series of known targets and functional pathways that form part of their innate immune system and illustrate tissue specific differences in their expression patterns. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. issue-copyright-statement© the author 2015 