BACKGROUND
sea anemones, together with corals, jellyfish and hydras, belong to the cnidaria, which are basal to the eumetazoa and ancestral to the bilateria.
cnidaria are characterized by a sac-like body plan with a single oral opening surrounded by numerous tentacles.
as diploblastic animals, they are composed of only two embryonic tissue layers, the epiderm and the gastroderm .
many cnidarians harbour photosynthetically active unicellular algae within their gastrodermal cells.
in most cases, such symbiont algae are dinoflagellates from the genus symbiodinium, commonly referred to as zooxanthellae.
this association is a trophic endosymbiosis and is considered to be mutualistic because the zooxanthellae provide their cnidarian host with reduced organic carbon resulting from their photosynthetic activity  <cit>  while the host provides the zooxanthellae with inorganic carbon  <cit> , inorganic nitrogen  <cit>  and inorganic phosphate  <cit> , as well as a refuge from herbivory.
this simple mutual partnership has recently been revealed to be more complex, however, since the holobiont was found to be a dynamic assemblage of animal, zooxanthellae, endolithic algae and fungi, prokaryotes  and viruses  <cit> .
endosymbioses are thus highly complex associations, implying intimate interactions between host and symbionts as well as constraints, such as hyperoxic conditions generated by symbiont photosynthesis, and transfer of inorganic carbon to the symbiont  <cit> .
in recent decades, biochemical and physiological studies have highlighted numerous adaptations in cnidarian host tissues , such as the presence of natural sunscreens , remarkable antioxidant defences  <cit> , specific mechanisms of inorganic carbon absorption and concentration  <cit> , and mechanisms of inorganic nitrogen absorption  <cit> .
however, despite increasing knowledge about their physiological inter-relationship, very little is known about the molecular adaptations that have permitted this successful partnership.
the cnidarian-dinoflagellate endosymbiotic association is the very foundation of the highly productive and diversified coral reef ecosystem.
coral reefs are considered to host at least 30% of all known marine fauna  <cit> , like "oases" within marine nutrient-deprived deserts  <cit> , and play a crucial role in shaping tropical ecosystems.
coral reefs are, however, now also experiencing high levels of anthropogenically-induced stress .
such environmental perturbations, in addition to pathogens, contribute to the breakdown of symbiosis known as "coral bleaching", and even mortality  <cit> .
bleaching results in whitening of cnidarian symbiotic tissues, due either to a direct loss of dinoflagellates and/or a decrease in photosynthetic pigment concentration  <cit> .
mass bleaching events have been increasing in both frequency and severity since the 1980s  <cit> .
the most significant contributions to cnidarian molecular biology are the complete genome analysis of the starlet sea anemone nematostella vectensis  <cit>  and the hydra magnipapillata genome project  <cit> .
however, neither of these species is symbiotic.
in addition, phylogenetic studies suggest that n. vectensis and a. viridis might belong to different suborders , although both belong to the hexacorallia actiniaria  <cit> .
very few studies using high-throughput techniques have been published to date on symbiotic cnidarians.
most cdna libraries have been constructed from non-symbiotic cells and only limited est datasets were generated.
only three studies have been conducted on symbiotic anthozoans: acropora millepora, a reef building coral ; aiptasia pulchella, a tropical sea anemone ; and a comparative study on two scleractinian corals, montastrea faveolata  and acropora palmata .
all have shown that cnidarians are more similar to vertebrates, in terms of gene repertoire, composition and intron/exon structure, than the well known ecdysozoan model organisms, drosophila melanogaster and caenorhabditis elegans, where extensive gene loss has occurred  <cit> .
these est datasets have been used to develop small-scale microarrays for gene expression analysis in several coral-algal symbioses: acropora millepora, acropora palmata and montastrea faveolata  <cit> .
genomic information on zooxanthellae has been obtained by leggat et al  <cit>  who analysed  <dig>  ests of tropical symbiodinium  extracted from the coral acropora aspera.
another cdna library  was constructed from a cultured symbiodinium of clade a , originally isolated from the upside-down jellyfish cassiopea sp, and sequences were compared to those of clade c <dig>  <cit> .
additional metagenomic analyses were performed on the microbial community associated with the coral porites asteroids  <cit> , extending our knowledge on the organismal diversity of the holobiont, although no transcriptomic analyses were carried out.
to better characterize symbiotic relationships between cnidarian host and associated symbionts, we conducted a large-scale est study on a symbiotic sea anemone.
we chose the sea anemone anemonia viridis as our study species, in which the two tissue layers  can be easily separated.
a. viridis is the most abundant sea anemone of the mediterranean coasts and hosts the temperate symbiodinium sp.
of clade a, which has been suggested to be the dominant clade of zooxanthellae in the mediterranean sea  <cit> .
we prepared a single cdna library from whole specimens under several stress conditions, in order to maximize the presence of genes required for symbiosis.
<dig>   <dig> ests were generated and a total of  <dig>  uniseqs were identified, assigned a putative origin and annotated.
this large collection of uniseqs provides the better characterized transcriptomic knowledge of symbiosis in cnidarians.
the data will further our comprehension of such relationships and contribute to functional genomic surveys.
RESULTS
ests generation and analysis
a cdna library was made from both symbiotic  and non-symbiotic  tissues of the sea anemone anemonia viridis .
in order to maximize the diversity of genes expressed under both normal and stress conditions within the symbiotic association, sea anemones were subjected to different environmental conditions before rna extraction and cdna library construction .
out of  <dig>  sequenced clones, a total of  <dig>  readable sequences were produced, corresponding to a sequencing success of 82%, and  <dig>  high quality ests were generated .
a. viridis repetitive sequences were identified and masked using repeatmasker before assembly.
trimmed masked ests were further subjected to cluster analysis using the tigr-tgicl pipeline with default parameters  <cit> .
a total of  <dig>  ests were assembled into  <dig>  contigs  while  <dig>  sequences remained as singletons.
the mean trimmed est sequence length was  <dig> bp, but sequences could be assembled into contigs of up to  <dig> kb .
most contigs  were composed of three ests or less, with an average length of  <dig> bp, suggesting that they covered only parts of the transcript sequences.
apart from the functional genomics resources available for the reef-building corals acropora palmata and montastrea faveolata  <cit> , most cnidarian genomic studies have been performed on non-symbiotic tissues .
to our knowledge, our study provides the largest est collection from a symbiotic cnidarian.
the most abundant transcripts  are listed in table  <dig>  most of these correspond to ribosomal proteins, or proteins involved in basic cellular processes .
however, it should be highlighted that two of these highly abundant transcripts had no similarity with any available sequence, either in uniprot or nr databases, while two other large clusters represent proteins that might be involved in stress responses .
ferritins are ubiquitously expressed proteins that have a central role in normal iron homoeostasis, as well as during oxidant stress, by reducing the participation of iron in free-radical-generating reactions  <cit> .
ferritin genes have also been identified as being highly expressed in acropora est datasets and one has been shown to be under positive selection  <cit> .
microarray experiments, designed to identify genes differentially expressed in response to elevated temperature in anthopleura elegantissima  <cit>  or a. viridis , revealed that ferritin genes might be induced in response to increases in reactive oxygen species following thermal stress.
repeated elements
based on reverse transcriptase domain search,  <dig> transposable elements  were identified .
among these,  <dig> were of prokaryotic origin  and  <dig> were of metazoan origin.
while  <dig> of them were almost identical to n. vectensis transposable elements,  <dig> only had slight similarity to n. vectensis sequences , and  <dig> showed no similarity to n. vectensis sequences but had been previously identified in other metazoa.
self blastn analysis on our est dataset also identified another abundant set of repeated elements.
over  <dig>  repetitive sequences were identified, with  <dig> repeated more than  <dig> times in our uniseqs dataset.
a typical sequence would be around  <dig> bp length, harbouring 40– <dig> bp inverted terminal repeats at its extremities.
these repeats often formed very long and stable hairpins, depending on their orientation .
they were found in  <dig> % of uniseqs and were overrepresented within contigs .
when open reading frame could be identified, these a. viridis repeated elements were always mapped in the 3'utrs of the protein coding genes.
using a blastn analysis, none of these sequences were found either in the genome of n. vectensis or in the large est datasets of hydra magnipapillata, and search in repbase did not show any homologs.
however, members of these a. viridis repeated elements could be found in the limited est datasets available for the symbiotic coral acropora millepora and the sea anemone anthopleura elegantissima.
further structural and functional characterisations of these repeated elements are currently being made.
comparative analysis to related taxa
because the cdna library was made from symbiotic tissue, we expected to find ests related both to the cnidarian host and to its dinoflagellate symbionts.
the analysis pipeline used in this study is presented in figure  <dig>  sequences were compared with swissprot  and uniprot kb   databases using blastx with a cutoff e-value of <  <dig> - <dig> to retrieve functional annotations.
of the assembled dataset,  <dig>  uniseqs had putative similarities while  <dig>  had no similarity to any sequences in the chosen databases.
sequences were also compared with ncbi-indexed prokaryotic nucleic sequences , using blastn with a stringent e-value of <  <dig> - <dig> to assess the proportion of prokaryotic sequences.
finally, a specific search of the uniseqs for virus proteins returned  <dig> hits.
relative contribution is shown in figure  <dig>  first of all, a relatively high proportion of sequences  remained that showed no significant similarities to previously described genes and were therefore considered as 'unknown'.
this is somewhat comparable with results obtained from other cnidaria, acropora palmata, acropora millepora, montastrea faveolata, and nematostella vectensis  <cit> .
most of the uniseqs identified in symbiodinium sp were also of unknown origin  <cit> .
metazoan hits were found for  <dig> % of uniseqs .
among these, most of the annotated uniseqs  matched with nematostella vectensis predicted proteins.
however, we also identified sequences that were clearly from the host , but these had no significant similarity to predicted proteins of n. vectensis.
three of these  were studied in more detail .
pcr and sequencing were first performed on genomic dna from both a. viridis epiderm and in vitro cultured symbiodinium, which confirmed the animal origin of these sequences .
the first sequence studied is related to the ependymin glycoprotein family , which has been well described in vertebrates due to its involvement in the regeneration processes.
ependymins are secretory proteins that can bind calcium and that were found predominantly in the cerebrospinal fluid of teleost fish.
a bound form has been described, associated with the extracellular matrix.
recent data demonstrated that these proteins are also present in non-vertebrate deuterostomes and protostomes  <cit> , and that positive selection may have shaped their evolution.
figure 4b illustrates an amino acid alignment of a. viridis merp sequences to homologous proteins found in publicly available databases .
the presence of ependymin proteins in basal organisms clearly indicates for the first time that this protein family is far older than previously thought .
in addition, there are at least  <dig> distinct merp homologues in a. viridis, all of animal origin.
as this is never the case in other phyla , we could hypothesize that merp are important for symbiosis in cnidarians.
the second sequence is related to a copper-dependent ferroxidase family protein  involved in copper detoxification, which has been described both in eukaryotes  and prokaryotes.
nevertheless, no significant similarity was found in d. melanogaster or in c. elegans genomes, nor in any available protostome database, suggesting that this has been lost during protostome evolution.
the third sequence is related to a prokaryotic amine-oxidase  family protein, which has also been identified in the genome of c. elegans and other caenorhabditidae but not in any other animal sequence database.
such species-specific occurrence could be explained by the presence of a similar prokaryote in both nematoda and a. viridis associated flora  or a parallel gene transfer event during their evolution.
however, molecular phylogenetic analyses  showed that cnidarian and rhabditidae nematode sequences cluster to outside other prokaryote representative gene sequences .
the latter rather suggest that amine-oxidase genes were maintained in these taxa but lost in other metazoa.
quite a large proportion of uniseqs  were uniquely shared with prokaryotes, of which proteobacteria was the most prominent bacterial group .
the holobiont has already been described as a dynamic assemblage, made up of the animal host, zooxanthellae, endolithic algae and fungi, bacteria, and archea  <cit> .
such "prokaryotic sequences" could therefore be assigned to the sea anemone-associated flora.
in addition, a small but significant number of a. viridis sequences, recognized as being of prokaryotic origin based on blast analysis, had already been identified in the n. vectensis genome.
such genes are clearly similar in sequence to prokaryotic homologs, although they contain introns.
in n. vectensis and a. millepora they were proposed as "ancient genes", conserved in cnidarians but lost in other animal genomes  <cit> .
although our results are in line with this interpretation, comparative genomic studies among cnidarians, such as a. viridis, could help to identify the most probable evolutionary scenario between maintenance of "non-metazoan" genes in cnidarians or lateral gene transfer events, followed by rapid intron acquisition.
surprisingly, only a small fraction of our dataset could be assigned to unicellular eukaryote sequences .
two well accepted explanations have been proposed: i) poor representation of dinoflagellate sequences in databases, leading to wrong assignment after blast analysis; and ii) technical bias due to the symbiodinium cell wall impairing complete rna extraction with standard methods, thus leading to an under-representative number of cdnas in our library.
gc content was calculated for all uniseqs to better assign a source to the transcripts .
figure  <dig> represents the distribution of gc content in the three major taxa , as well as for sequences without blastx hits .
two distinct distribution curves were observed, with the metazoan ests averaging 40% gc, while bacterial ests averaged 63%.
despite our small number of symbiodinium sequences, their gc content  agreed with that already published for dinoflagellates, which have a relatively high gc content compared with other eukaryotes: triplett et al  <cit>  calculated a gc content of 64% for heterocapsa pygmaea and an est analysis of alexandrium tamarense estimated that coding region gc-content was  <dig> %, whereas gc-content in the 3'-utr was slightly less, at  <dig> %  <cit> .
gc-content analysis of our unknown sequences revealed that ests were clustered around two peaks corresponding to 35% and 58% gc content for the larger and smaller peaks respectively.
these data strongly suggest that most of the unknown sequences are likely to be of animal origin.
as the gc content of 3' utrs is usually lower than that of 5' utrs or cdss, these results are also consistent with our hypothesis that these sequences are mainly 3' utr , which also explains their low level of annotation.
on the other hand, the smaller peak  could be attributed to dinoflagellate sequences, although no dinoflagellate genome is currently available to test this, and the most closely related available genomes are those of apicomplexans , which are also compact genomes of parasitic protozoans.
to gain some insight into genes potentially involved in symbiosis among cnidaria, we performed comparative blast analyses on the subpart of a. viridis sequences that were of animal origin .
they were subjected to tblastx  against cnidarian est sequences available in ncbi-dbest .
a parallel blastx  was performed against the n. vectensis predicted proteins dataset.
table  <dig> gives the number of positive blast hits  obtained from a. viridis sequences against ests from selected cnidarian species.
this comparison highlighted two species with a high number of homologous sequences, the sea anemone m. senile and the symbiotic stony corals a. millepora and a. palmata.
in the figure  <dig> venn diagram we compared a. viridis with two non symbiotic species , and the symbiotic acropora species:  <dig>  sequences were common to the four species, while only  <dig> sequences  were specific to the symbiotic species, which probably contain candidate genes required for the symbiosis.
sequences identified as metazoan origin  were compared to cnidarian sequences available in databases.
1predicted proteins dataset from jgi
functional classification of ests
finally, this set of uniseqs was annotated using similarity searches in both nucleotide and protein databases, as well as domain searches.
gene ontology terms were then assigned automatically using customized scripts based on interproscan search results.
we also submitted n. vectensis predicted proteins to the same interproscan analysis for a comparative approach.
to homogenize annotation level we only kept the root domain, using the hierarchical domain organization available from ebi.
interpro protein domains were identified in 40% of proteins.
based on similarity searches , we assigned putative taxa  to each unique sequence.
figure  <dig> represents the distribution of assigned gene ontology terms.
comparison of domain occurrence in a. viridis with that in n. vectensis demonstrated that our library is highly representative of the actinaria transcriptome.
we also identified increased abundance of a number of molecular functions in a. viridis compared with n. vectensis: translation regulator activity , oxidoreductase activity , antioxidant activity , and structural molecule activity .
it is noteworthy that some of these molecular functions are involved in symbiotic interactions with zooxanthellae, such as protein binding  <cit> , nutrient transport and antioxidant activity  <cit> .
for example, a. viridis counters the oxidative stress produced by the photosynthetic activity of its symbionts by using a great diversity of antioxidant defences  <cit> .
some biological processes were also increased in the a. viridis dataset compared with n. vectensis: secretion  and catabolism .
the cytoplasmic components were also found to be more highly represented in a. viridis compared with n. vectensis .
all these differences between the present a. viridis dataset and the n. vectensis genome may reflect the crucial role of trophic exchanges between the sea anemone and its dinoflagellate symbionts, as well as specific host adaptations.
CONCLUSIONS
this large est collection has provided high quality data on all aspects of a temperate symbiotic cnidarian, particularly with regard to coding sequences and regulation features.
for example, we identified many novel repeated elements  in 3'utrs, suggesting an invasion of most animal sequences by some specific re families.
it will be interesting to further investigate their potential biological role, particularly on gene regulation.
phylogenetic origin and functional classification of the holobiont sequences allowed the identification of several symbiotic candidate genes.
these data are now being used to develop a dedicated microarray that will provide a valuable resource for future studies on symbiotic interactions in a. viridis.
furthermore, these data also have shown the importance of symbiodinium symbionts as well as the associated flora of the three major prokaryotic species.
some sequences will be further analysed from the new perspective of gene transfer between host and symbiont.
the relatively low abundance of sequences from symbiodinium was attributed to experimental bias; a new ongoing sequencing project should fill this gap.
finally, these data from a temperate zone cnidarian provide novel molecular insights that will complement those obtained from tropical anthozoans.
this dataset is valuable resource that will be of great help for comparative genomics and evolutionary studies.

 sea anemones, together with corals, jellyfish and hydras, belong to the cnidaria, which are basal to the eumetazoa and ancestral to the bilateria. cnidaria are characterized by a sac-like body plan with a single oral opening surrounded by numerous tentacles. as diploblastic animals, they are composed of only two embryonic tissue layers, the epiderm and the gastroderm. many cnidarians harbour photosynthetically active unicellular algae within their gastrodermal cells. in most cases, such symbiont algae are dinoflagellates from the genus symbiodinium, commonly referred to as zooxanthellae. this association is a trophic endosymbiosis and is considered to be mutualistic because the zooxanthellae provide their cnidarian host with reduced organic carbon resulting from their photosynthetic activity while the host provides the zooxanthellae with inorganic carbon, inorganic nitrogen and inorganic phosphate, as well as a refuge from herbivory. this simple mutual partnership has recently been revealed to be more complex, however, since the holobiont was found to be a dynamic assemblage of animal, zooxanthellae, endolithic algae and fungi, prokaryotes and viruses. endosymbioses are thus highly complex associations, implying intimate interactions between host and symbionts as well as constraints, such as hyperoxic conditions generated by symbiont photosynthesis, and transfer of inorganic carbon to the symbiont. in recent decades, biochemical and physiological studies have highlighted numerous adaptations in cnidarian host tissues, such as the presence of natural sunscreens, remarkable antioxidant defences, specific mechanisms of inorganic carbon absorption and concentration, and mechanisms of inorganic nitrogen absorption. however, despite increasing knowledge about their physiological inter-relationship, very little is known about the molecular adaptations that have permitted this successful partnership. the cnidarian-dinoflagellate endosymbiotic association is the very foundation of the highly productive and diversified coral reef ecosystem. coral reefs are considered to host at least 30% of all known marine fauna, like "oases" within marine nutrient-deprived deserts, and play a crucial role in shaping tropical ecosystems. coral reefs are, however, now also experiencing high levels of anthropogenically-induced stress. such environmental perturbations, in addition to pathogens, contribute to the breakdown of symbiosis known as "coral bleaching", and even mortality. bleaching results in whitening of cnidarian symbiotic tissues, due either to a direct loss of dinoflagellates and/or a decrease in photosynthetic pigment concentration. mass bleaching events have been increasing in both frequency and severity since the 1980s. the most significant contributions to cnidarian molecular biology are the complete genome analysis of the starlet sea anemone nematostella vectensis and the hydra magnipapillata genome project. however, neither of these species is symbiotic. in addition, phylogenetic studies suggest that n. vectensis and a. viridis might belong to different suborders, although both belong to the hexacorallia actiniaria. very few studies using high-throughput techniques have been published to date on symbiotic cnidarians. most cdna libraries have been constructed from non-symbiotic cells and only limited est datasets were generated. only three studies have been conducted on symbiotic anthozoans: acropora millepora, a reef building coral; aiptasia pulchella, a tropical sea anemone; and a comparative study on two scleractinian corals, montastrea faveolata and acropora palmata. all have shown that cnidarians are more similar to vertebrates, in terms of gene repertoire, composition and intron/exon structure, than the well known ecdysozoan model organisms, drosophila melanogaster and caenorhabditis elegans, where extensive gene loss has occurred. these est datasets have been used to develop small-scale microarrays for gene expression analysis in several coral-algal symbioses: acropora millepora, acropora palmata and montastrea faveolata. genomic information on zooxanthellae has been obtained by leggat et al who analysed ests of tropical symbiodinium extracted from the coral acropora aspera. another cdna library was constructed from a cultured symbiodinium of clade a, originally isolated from the upside-down jellyfish cassiopea sp, and sequences were compared to those of clade c. additional metagenomic analyses were performed on the microbial community associated with the coral porites asteroids, extending our knowledge on the organismal diversity of the holobiont, although no transcriptomic analyses were carried out. to better characterize symbiotic relationships between cnidarian host and associated symbionts, we conducted a large-scale est study on a symbiotic sea anemone. we chose the sea anemone anemonia viridis as our study species, in which the two tissue layers can be easily separated. a. viridis is the most abundant sea anemone of the mediterranean coasts and hosts the temperate symbiodinium sp. of clade a, which has been suggested to be the dominant clade of zooxanthellae in the mediterranean sea. we prepared a single cdna library from whole specimens under several stress conditions, in order to maximize the presence of genes required for symbiosis. ests were generated and a total of uniseqs were identified, assigned a putative origin and annotated. this large collection of uniseqs provides the better characterized transcriptomic knowledge of symbiosis in cnidarians. the data will further our comprehension of such relationships and contribute to functional genomic surveys.  ests generation and analysis a cdna library was made from both symbiotic and non-symbiotic tissues of the sea anemone anemonia viridis. in order to maximize the diversity of genes expressed under both normal and stress conditions within the symbiotic association, sea anemones were subjected to different environmental conditions before rna extraction and cdna library construction. out of sequenced clones, a total of readable sequences were produced, corresponding to a sequencing success of 82%, and high quality ests were generated. a. viridis repetitive sequences were identified and masked using repeatmasker before assembly. trimmed masked ests were further subjected to cluster analysis using the tigr-tgicl pipeline with default parameters. a total of ests were assembled into contigs while sequences remained as singletons. the mean trimmed est sequence length was bp, but sequences could be assembled into contigs of up to kb. most contigs were composed of three ests or less, with an average length of bp, suggesting that they covered only parts of the transcript sequences. apart from the functional genomics resources available for the reef-building corals acropora palmata and montastrea faveolata, most cnidarian genomic studies have been performed on non-symbiotic tissues. to our knowledge, our study provides the largest est collection from a symbiotic cnidarian. the most abundant transcripts are listed in table most of these correspond to ribosomal proteins, or proteins involved in basic cellular processes. however, it should be highlighted that two of these highly abundant transcripts had no similarity with any available sequence, either in uniprot or nr databases, while two other large clusters represent proteins that might be involved in stress responses. ferritins are ubiquitously expressed proteins that have a central role in normal iron homoeostasis, as well as during oxidant stress, by reducing the participation of iron in free-radical-generating reactions. ferritin genes have also been identified as being highly expressed in acropora est datasets and one has been shown to be under positive selection. microarray experiments, designed to identify genes differentially expressed in response to elevated temperature in anthopleura elegantissima or a. viridis, revealed that ferritin genes might be induced in response to increases in reactive oxygen species following thermal stress. repeated elements based on reverse transcriptase domain search, transposable elements were identified. among these, were of prokaryotic origin and were of metazoan origin. while of them were almost identical to n. vectensis transposable elements, only had slight similarity to n. vectensis sequences, and showed no similarity to n. vectensis sequences but had been previously identified in other metazoa. self blastn analysis on our est dataset also identified another abundant set of repeated elements. over repetitive sequences were identified, with repeated more than times in our uniseqs dataset. a typical sequence would be around bp length, harbouring 40– bp inverted terminal repeats at its extremities. these repeats often formed very long and stable hairpins, depending on their orientation. they were found in % of uniseqs and were overrepresented within contigs. when open reading frame could be identified, these a. viridis repeated elements were always mapped in the 3'utrs of the protein coding genes. using a blastn analysis, none of these sequences were found either in the genome of n. vectensis or in the large est datasets of hydra magnipapillata, and search in repbase did not show any homologs. however, members of these a. viridis repeated elements could be found in the limited est datasets available for the symbiotic coral acropora millepora and the sea anemone anthopleura elegantissima. further structural and functional characterisations of these repeated elements are currently being made. comparative analysis to related taxa because the cdna library was made from symbiotic tissue, we expected to find ests related both to the cnidarian host and to its dinoflagellate symbionts. the analysis pipeline used in this study is presented in figure sequences were compared with swissprot and uniprot kb databases using blastx with a cutoff e-value of < - to retrieve functional annotations. of the assembled dataset, uniseqs had putative similarities while had no similarity to any sequences in the chosen databases. sequences were also compared with ncbi-indexed prokaryotic nucleic sequences, using blastn with a stringent e-value of < - to assess the proportion of prokaryotic sequences. finally, a specific search of the uniseqs for virus proteins returned hits. relative contribution is shown in figure first of all, a relatively high proportion of sequences remained that showed no significant similarities to previously described genes and were therefore considered as'unknown'. this is somewhat comparable with results obtained from other cnidaria, acropora palmata, acropora millepora, montastrea faveolata, and nematostella vectensis. most of the uniseqs identified in symbiodinium sp were also of unknown origin. metazoan hits were found for % of uniseqs. among these, most of the annotated uniseqs matched with nematostella vectensis predicted proteins. however, we also identified sequences that were clearly from the host, but these had no significant similarity to predicted proteins of n. vectensis. three of these were studied in more detail. pcr and sequencing were first performed on genomic dna from both a. viridis epiderm and in vitro cultured symbiodinium, which confirmed the animal origin of these sequences. the first sequence studied is related to the ependymin glycoprotein family, which has been well described in vertebrates due to its involvement in the regeneration processes. ependymins are secretory proteins that can bind calcium and that were found predominantly in the cerebrospinal fluid of teleost fish. a bound form has been described, associated with the extracellular matrix. recent data demonstrated that these proteins are also present in non-vertebrate deuterostomes and protostomes, and that positive selection may have shaped their evolution. figure 4b illustrates an amino acid alignment of a. viridis merp sequences to homologous proteins found in publicly available databases. the presence of ependymin proteins in basal organisms clearly indicates for the first time that this protein family is far older than previously thought. in addition, there are at least distinct merp homologues in a. viridis, all of animal origin. as this is never the case in other phyla, we could hypothesize that merp are important for symbiosis in cnidarians. the second sequence is related to a copper-dependent ferroxidase family protein involved in copper detoxification, which has been described both in eukaryotes and prokaryotes. nevertheless, no significant similarity was found in d. melanogaster or in c. elegans genomes, nor in any available protostome database, suggesting that this has been lost during protostome evolution. the third sequence is related to a prokaryotic amine-oxidase family protein, which has also been identified in the genome of c. elegans and other caenorhabditidae but not in any other animal sequence database. such species-specific occurrence could be explained by the presence of a similar prokaryote in both nematoda and a. viridis associated flora or a parallel gene transfer event during their evolution. however, molecular phylogenetic analyses showed that cnidarian and rhabditidae nematode sequences cluster to outside other prokaryote representative gene sequences. the latter rather suggest that amine-oxidase genes were maintained in these taxa but lost in other metazoa. quite a large proportion of uniseqs were uniquely shared with prokaryotes, of which proteobacteria was the most prominent bacterial group. the holobiont has already been described as a dynamic assemblage, made up of the animal host, zooxanthellae, endolithic algae and fungi, bacteria, and archea. such "prokaryotic sequences" could therefore be assigned to the sea anemone-associated flora. in addition, a small but significant number of a. viridis sequences, recognized as being of prokaryotic origin based on blast analysis, had already been identified in the n. vectensis genome. such genes are clearly similar in sequence to prokaryotic homologs, although they contain introns. in n. vectensis and a. millepora they were proposed as "ancient genes", conserved in cnidarians but lost in other animal genomes. although our results are in line with this interpretation, comparative genomic studies among cnidarians, such as a. viridis, could help to identify the most probable evolutionary scenario between maintenance of "non-metazoan" genes in cnidarians or lateral gene transfer events, followed by rapid intron acquisition. surprisingly, only a small fraction of our dataset could be assigned to unicellular eukaryote sequences. two well accepted explanations have been proposed: i) poor representation of dinoflagellate sequences in databases, leading to wrong assignment after blast analysis; and ii) technical bias due to the symbiodinium cell wall impairing complete rna extraction with standard methods, thus leading to an under-representative number of cdnas in our library. gc content was calculated for all uniseqs to better assign a source to the transcripts. figure represents the distribution of gc content in the three major taxa, as well as for sequences without blastx hits. two distinct distribution curves were observed, with the metazoan ests averaging 40% gc, while bacterial ests averaged 63%. despite our small number of symbiodinium sequences, their gc content agreed with that already published for dinoflagellates, which have a relatively high gc content compared with other eukaryotes: triplett et al calculated a gc content of 64% for heterocapsa pygmaea and an est analysis of alexandrium tamarense estimated that coding region gc-content was %, whereas gc-content in the 3'-utr was slightly less, at %. gc-content analysis of our unknown sequences revealed that ests were clustered around two peaks corresponding to 35% and 58% gc content for the larger and smaller peaks respectively. these data strongly suggest that most of the unknown sequences are likely to be of animal origin. as the gc content of 3' utrs is usually lower than that of 5' utrs or cdss, these results are also consistent with our hypothesis that these sequences are mainly 3' utr, which also explains their low level of annotation. on the other hand, the smaller peak could be attributed to dinoflagellate sequences, although no dinoflagellate genome is currently available to test this, and the most closely related available genomes are those of apicomplexans, which are also compact genomes of parasitic protozoans. to gain some insight into genes potentially involved in symbiosis among cnidaria, we performed comparative blast analyses on the subpart of a. viridis sequences that were of animal origin. they were subjected to tblastx against cnidarian est sequences available in ncbi-dbest. a parallel blastx was performed against the n. vectensis predicted proteins dataset. table gives the number of positive blast hits obtained from a. viridis sequences against ests from selected cnidarian species. this comparison highlighted two species with a high number of homologous sequences, the sea anemone m. senile and the symbiotic stony corals a. millepora and a. palmata. in the figure venn diagram we compared a. viridis with two non symbiotic species, and the symbiotic acropora species: sequences were common to the four species, while only sequences were specific to the symbiotic species, which probably contain candidate genes required for the symbiosis. sequences identified as metazoan origin were compared to cnidarian sequences available in databases. 1predicted proteins dataset from jgi functional classification of ests finally, this set of uniseqs was annotated using similarity searches in both nucleotide and protein databases, as well as domain searches. gene ontology terms were then assigned automatically using customized scripts based on interproscan search results. we also submitted n. vectensis predicted proteins to the same interproscan analysis for a comparative approach. to homogenize annotation level we only kept the root domain, using the hierarchical domain organization available from ebi. interpro protein domains were identified in 40% of proteins. based on similarity searches, we assigned putative taxa to each unique sequence. figure represents the distribution of assigned gene ontology terms. comparison of domain occurrence in a. viridis with that in n. vectensis demonstrated that our library is highly representative of the actinaria transcriptome. we also identified increased abundance of a number of molecular functions in a. viridis compared with n. vectensis: translation regulator activity, oxidoreductase activity, antioxidant activity, and structural molecule activity. it is noteworthy that some of these molecular functions are involved in symbiotic interactions with zooxanthellae, such as protein binding, nutrient transport and antioxidant activity. for example, a. viridis counters the oxidative stress produced by the photosynthetic activity of its symbionts by using a great diversity of antioxidant defences. some biological processes were also increased in the a. viridis dataset compared with n. vectensis: secretion and catabolism. the cytoplasmic components were also found to be more highly represented in a. viridis compared with n. vectensis. all these differences between the present a. viridis dataset and the n. vectensis genome may reflect the crucial role of trophic exchanges between the sea anemone and its dinoflagellate symbionts, as well as specific host adaptations.  this large est collection has provided high quality data on all aspects of a temperate symbiotic cnidarian, particularly with regard to coding sequences and regulation features. for example, we identified many novel repeated elements in 3'utrs, suggesting an invasion of most animal sequences by some specific re families. it will be interesting to further investigate their potential biological role, particularly on gene regulation. phylogenetic origin and functional classification of the holobiont sequences allowed the identification of several symbiotic candidate genes. these data are now being used to develop a dedicated microarray that will provide a valuable resource for future studies on symbiotic interactions in a. viridis. furthermore, these data also have shown the importance of symbiodinium symbionts as well as the associated flora of the three major prokaryotic species. some sequences will be further analysed from the new perspective of gene transfer between host and symbiont. the relatively low abundance of sequences from symbiodinium was attributed to experimental bias; a new ongoing sequencing project should fill this gap. finally, these data from a temperate zone cnidarian provide novel molecular insights that will complement those obtained from tropical anthozoans. this dataset is valuable resource that will be of great help for comparative genomics and evolutionary studies. 
a single cdna library was constructed from symbiotic tissue of sea anemones a. viridis in various environmental conditions. altogether, these results provide new molecular insights that could be used as a starting-point for further functional genomics studies. to better characterize symbiotic relationships between a cnidarian host and its dinoflagellate symbionts, we conducted a large-scale est study on a symbiotic sea anemone, anemonia viridis, in which the two tissue layers can be easily separated. we identified genes of animal origin that have no homolog in the non-symbiotic starlet sea anemone nematostella vectensis genome, but in other symbiotic cnidarians, and may therefore be involved in the symbiosis relationship in a. viridis. comparison of protein domain occurrence in a. viridis with that in n. vectensis demonstrated an increase in abundance of some molecular functions, such as protein binding or antioxidant activity, suggesting that these functions are essential for the symbiotic state and may be specific adaptations. the comparison with the closest available genome, the sea anemone n. vectensis, as well as with est datasets from other symbiotic cnidarians provided a set of candidate genes involved in symbiosis-related molecular crosstalks. their immense ecological success is due to a symbiotic association between cnidarian hosts and unicellular dinoflagellate algae, known as zooxanthellae. this large dataset of sequences provides a valuable resource for future studies on symbiotic interactions in cnidaria. sequences were analysed and sorted according to their putative origin.
 coral reef ecosystems are renowned for their diversity and beauty. their immense ecological success is due to a symbiotic association between cnidarian hosts and unicellular dinoflagellate algae, known as zooxanthellae. these algae are photosynthetic and the cnidarian-zooxanthellae association is based on nutritional exchanges. maintenance of such an intimate cellular partnership involves many crosstalks between the partners. to better characterize symbiotic relationships between a cnidarian host and its dinoflagellate symbionts, we conducted a large-scale est study on a symbiotic sea anemone, anemonia viridis, in which the two tissue layers can be easily separated.  a single cdna library was constructed from symbiotic tissue of sea anemones a. viridis in various environmental conditions. we generated high quality ests, which were assembled into unique sequences. sequences were analysed and sorted according to their putative origin. we identified many new repeated elements in the 3'utr of most animal genes, suggesting that these elements potentially have a biological role, especially with respect to gene expression regulation. we identified genes of animal origin that have no homolog in the non-symbiotic starlet sea anemone nematostella vectensis genome, but in other symbiotic cnidarians, and may therefore be involved in the symbiosis relationship in a. viridis. comparison of protein domain occurrence in a. viridis with that in n. vectensis demonstrated an increase in abundance of some molecular functions, such as protein binding or antioxidant activity, suggesting that these functions are essential for the symbiotic state and may be specific adaptations.  this large dataset of sequences provides a valuable resource for future studies on symbiotic interactions in cnidaria. the comparison with the closest available genome, the sea anemone n. vectensis, as well as with est datasets from other symbiotic cnidarians provided a set of candidate genes involved in symbiosis-related molecular crosstalks. altogether, these results provide new molecular insights that could be used as a starting-point for further functional genomics studies. 