BACKGROUND
many biological systems rely on symbiotic interactions between different organisms.
one of the most dramatic examples is the coral reef ecosystem, which has at its heart a mutualistic partnership between corals and endosymbiotic, dinoflagellate algae.
the dinoflagellates are classified in a single genus, symbiodinium, but molecular methods have revealed a high genetic diversity in this genus  <cit> .
the onset of these symbioses has been shown to display flexibility, but a range of specificity, i.e.
from highly flexible to highly specific, is apparent during its maintenance  <cit> .
this process is likely to involve early recognition mechanisms  <cit>  and an evasion of the hosts' digestive and immune systems  <cit> , as well as adaptations to diverse ecological niches  <cit>  and physiological acclimation  <cit> .
there have also been controversial discussions of whether symbiodinium populations may shift toward more heat-tolerant types as a consequence of thermal stress  in order to adapt to environmental changes  <cit>  such as increasing seawater temperatures.
in light of global climate change, this subject, i.e.
cnidarian bleaching, has received much attention as devastating mass bleaching events have increased both in frequency and geographic extent  <cit> .
nonetheless, our knowledge of the underlying cellular and molecular mechanisms that facilitate the recognition between the partners, and determine the specificity, dynamics, and collapse of cnidarian-dinoflagellate symbioses, is limited.
the cellular and molecular interactions between host and symbiont cells are important targets for genetic and genomic dissection, but corals are notoriously difficult to work with.
for example, corals form large, slow-growing colonies that are difficult and costly to maintain in the laboratory, and their handling for microscopy and amenability to other cell biological, biochemical, and genetic methods is complicated by the calcareous skeleton precipitated by reef-building corals.
what is needed to make rapid advances in this field is a model system that possesses the key characteristics of coral symbiosis, but allows more facile laboratory investigation .
the sea anemone aiptasia represents a good candidate system  <cit> , as it possesses the same mutualistic relationship with symbiodinium spp., but lacks the calcareous skeleton that hinders cellular-level work.
it is widely distributed, and found in shallow tropical marine environments worldwide.
sequence characterized amplified region  data indicate that the vast majority of aiptasia worldwide , appear to be genetically homogeneous .
the one exception is a closely related, but genetically distinct, lineage potentially restricted to the florida keys.
data from the santos lab also indicate that natural populations of aiptasia from the florida keys preferentially host symbiodinium spp.
comprised of only clade a or both clades a and b, whereas those from the remaining global range host clade b exclusively.
typically considered a pest organism by seawater aquarists, aiptasia is hardy and proliferates rapidly by asexual reproduction.
individual polyps can be maintained in a symbiotic or aposymbiotic state , experimentally re-infected with a variety of symbiodinium strains  <cit> , and cultured at low cost  <cit> .
in fact, numerous studies have addressed symbiosis-related questions using a. pallida and its sister species a. pulchella by applying multiple tools ranging from microscopy to rna-interference methods  <cit> .
the generation of genomic resources for aiptasia would therefore greatly advance research addressing the understanding of symbioses at a molecular, cell-biological, and genomic level.
as a cost-effective alternative to sequencing the genome of an organism, the generation and analysis of expressed-sequence-tag  libraries provides an efficient method for discovering novel genes, estimating gene content, and approximating levels of gene expression.
once established, these resources can be utilized for comparative genomics studies or the construction of gene expression microarrays  <cit> .
among cnidarians, the extensive genomic resources now available for the non-symbiotic sea anemone nematostella vectensis have opened new perspectives on the study of basal metazoans  <cit> , and several est resources have been generated for symbiotic cnidarians  and symbiodinium spp.
<cit> .
however, to date, only one small-scale project has generated ests  for the symbiotic anemone aiptasia pulchella  <cit> .
in this study, we report the generation and analysis of  <dig>  high-quality ests from a symbiodinium clade a-hosting clonal population of aiptasia pallida that was likely derived from an individual originating from the florida keys lineage, which were processed through a software pipeline  <cit>  resulting in a user-friendly, queryable, web-accessible database named aiptasiabase.
a blastx-based approach was used to estimate the relative contributions of each partner to the mixed cdna library, and we were able to identify numerous genes involved in key processes of cnidarian-dinoflagellate symbioses.
RESULTS
est library construction and assembly
a total of  <dig>  cdna clones were bi-directionally sequenced, resulting in  <dig>  raw chromatograms, which served as input for the processing pipeline.
after base calling by phred  <cit> , lucy  <cit>  discarded  <dig>  low-quality sequences, short or insert-less sequences, and vector or polya-only sequences.
an additional  <dig> sequences were removed by seqclean  <cit> , leaving  <dig>  high-quality ests  for further processing .
assembly of these ests by cap <dig>  <cit>  resulted in the generation of  <dig>  contigs, which ranged from  <dig> to  <dig>  bp in length and contained  <dig> –  <dig> ests .
together with the remaining  <dig>  singletons, a total of  <dig>  unique sequences  were generated.
because of the possibility that two  uniseqs originated from the same transcript, we also estimated the number of unique genes  in our dataset by assembling only the reverse reads of the directionally cloned cdnas.
the resulting estimate of  <dig>  unigenes compared to the  <dig>  uniseqs is likely to reflect the large average size  of inserts in the cdna library; thus, in many cases, uniseqs represent the 3' and 5' ends of genes for which the central parts were not captured due to sanger-sequencing length limitations .
in addition, different splice variants or alleles of the same gene may have contributed to the excess of uniseqs over unigenes.
detailed pre-assembly statistics are summarized in additional file 1: quality control and assembly statistics.
previously, a small-scale est project was conducted in order to compare the abundance of transcripts between symbiotic and aposymbiotic aiptasia pulchella polyps  <cit> .
the present study included bi-directional sequencing, and the total number of ests is more than  <dig> times larger than in the earlier study.
therefore, the availability of almost  <dig>  uniseqs for about  <dig>  unigenes represents a rich transcriptomic resource, previously unavailable at this scale, for a symbiotic anemone.
annotation of unique sequences and implementation of aiptasiabase
all uniseqs were assigned putative identities based on blastx hits  to the uniprot knowledgebase databases swissprot and trembl  <cit> .
about 37% and ~63% of the uniseqs found hits in swissprot and trembl, respectively, leaving ~36% of the uniseqs without similarities to known proteins.
because the trembl database contains protein sequences based on conceptual translations of all nucleotide sequence entries in embl/genbank/ddbj, we chose to annotate the uniseqs according to the curated swissprot entries.
assignments of gene ontologies  could be made for about one third of uniseqs in each of the go categories: molecular function, biological process, and cellular component.
because our cdna library represents the symbiotic, adult life-history stage of a. pallida, the go resource generated in this study sets the stage for statistical assessments of over- or under-representation of specific go-categories in libraries obtained from anemones under different conditions such as life-history stages, symbiotic state , or environmental conditions .
in addition to blast and go annotations, all uniseqs were screened for single-nucleotide polymorphisms  and simple-sequence repeats , providing resources for the investigation of gene polymorphisms between individuals and/or populations.
the prediction of open reading frames within uniseqs also provided the basis for domain annotations at the protein level.
about 25% of uniseqs matched a protein domain entry in the pfam database  <cit> .
one of the primary challenges of sequencing ests from a mixed transcriptome originating from two or more partners is to assign sequences to the proper genome of origin.
taking a bioinformatic approach to this problem, we constructed taxon-specific databases representing either "cnidaria-only" or "alveolata-only"  sequences from genbank, and then performed blastx-searches against those databases as well as the complete non-redundant database .
we then employed a best-blastx-hit  approach  to estimate the numbers of ests that originated from a. pallida and symbiodinium spp., respectively, at various levels of confidence .
irrespective of the confidence level, about one quarter of ests had no blastx-hit .
at the different levels of confidence,  <dig> – 70% and  <dig>  –  <dig> % were predicted to originate from the anemone and the symbiodinium genomes, respectively  distribution and assignment).
the relatively small fraction of symbiodinium ests could be expected given that symbiodinum spp.
are spatially restricted to the endodermal tissue layer of the host and that no special effort was made to disrupt the algal cell walls during the preparation of the rna .
furthermore, the number of uniseqs without a significant blastx hit may be higher for symbiodinium transcripts.
however, the uncertainty about the origin of non-annotated sequences represents a current limitation to our approach.
ongoing and future genome-sequencing projects for symbiotic cnidarians and their dinoflagellate endosymbionts should soon become available and help to uncover the origins of sequences without currently known homologs in other organisms.
this will provide an interesting opportunity to revisit our data set to look further at these perhaps taxonomically restricted genes.
details can be found in additional file 2: flow diagram illustrating bbh appraoch and additional file 3: detailed est  distribution and assignment.
using an est-processing software   <cit> , we stored all ests, uniseqs, and annotations in a queryable database named aiptasiabase , which is accessible through the url: .
in addition to the results generated by the software, we have included the annotation of uniseqs according to kegg, which provides a convenient way to explore pathway components that were identified in this study.
analysis of the most highly abundant transcripts
we identified the contigs containing the greatest numbers of ests, which we used as a proxy for the most abundant transcripts.
although the numbers of ests in contigs that are predicted to originate from symbiodinium were too low to be analyzed , many of the most abundant host-derived transcripts represented genes that are involved in the processes of protein biosynthesis, extracellular-matrix formation, and oxidative-stress response .
a blastx  search in swissprot .
contig numbering is according to aiptasiabase_v <dig>
b contigs  <dig> and  <dig> are likely to originate from the same transcript.
c contigs  <dig> and  <dig> are likely to originate from the same transcript.
d best blast search hit  in nr: von willebrand factor-like protein  .
e sequencing error artifact.
f best blast search hit  in nr: mesoglein variant  <dig>  .
kuo et al.
reported that the most highly expressed gene in symbiotic a. pulchella was ferritin , whereas we found only  <dig> ests  that represented this gene.
although differences in the preparations of the cdna libraries  and sequencing depths  pose an obstacle for a direct comparison, the discrepancy in the numbers of ferritin transcripts appears to be noteworthy.
in a recent study that investigated the effect of increased temperature and uv levels on the symbiotic anemone anthopleura elegantissima, richier et al.
observed a more than 17-fold up-regulation of ferritin expression upon thermal stress, but not uv stress  <cit> .
given this observation, it seems possible that the anemones in the study by kuo et al.
were under elevated thermal stress at the time of sampling, which, taken together with the methodological differences mentioned above, makes any further comparative analyses unfeasible.
this result has important implications, i.e., how culturing conditions of organisms as well as methodological differences between studies may have an impact on the transcriptome, and by extension, the interpretation of gene expression analyses.
the highly abundant sequences with the highest uncertainties for correct annotation , apolipophorin and the cub and zona pellucida-like domain-containing protein  <dig>  were further scrutinized by similarity searches in additional databases.
these searches revealed that the best hit for cub and zona pellucida-like domain-containing protein  <dig> in the genbank non-redundant database  was mesoglein, a protein that is proposed to be a structural element of the extracellular matrix of the mesoglea in the jellyfish aurelia aurita  <cit> .
the sequence annotated as apolipophorin contains a von willebrand factor type-d domain, and was reported to be involved in forming lipoprotein particles that bind lipoproteins and lipids  <cit> .
two other highly abundant sequences had no homologs among previously characterized proteins, suggesting that they are novel, and a third contig with no blastx-hit was identified as an artifact due to misassembled sequences.
these results illustrate some of the caveats to automated sequence assembly and annotation and highlight the necessity for corroboration after automated sequence processing when focusing on single genes or groups of genes of interest.
candidate genes with potential relevance to cnidarian-dinoflagellate symbioses
we generated a candidate gene list of groups containing uniseqs that are likely to be of relevance to cnidarian-dinoflagellate symbioses .
among these, the cellular antioxidant-response system could be most comprehensively reconstructed .
genes related to the innate immune system and sugar-binding proteins gave rise to a partial gene inventory .
other genes that are likely to play a role in the cellular events surrounding the breakdown of symbiosis  were also identified.
stress-induced photoinhibition and damage to algal photosystem ii are thought to be responsible for an increased production of reactive oxygen species  <cit>  and consequently, diffusion of hydrogen peroxide  through the membranes into the host cells  <cit> .
the detoxification of h2o <dig> requires the activity of catalase or other peroxidases.
superoxide dismutase , which catalyzes the reduction of superoxide to h2o <dig>  as well as glutathione peroxidase , which uses glutathione to detoxify h2o <dig>  were both not found among the sequenced ests.
one possibility is that the abundance of sod transcripts in host cells was low, and the generation of superoxide spatially limited .
in this case, superoxide may have been efficiently eliminated within the symbiodinium cells, while excess h2o <dig> that was not detoxified , could have diffused into the host cytosol and been reduced to h2o and o <dig> by catalase.
alternatively, methodological factors such as insert-size selection or general rna processing may have prevented the detection of sod.
other genes that had previously been reported in the context of cnidarian-dinoflagellate symbioses  were also not detected, perhaps for same reasons as discussed above for sod.
CONCLUSIONS
by analyzing > <dig>  high-quality ests and generating a comprehensive database for the user community, we have provided a foundation of transcriptomic resources for a symbiotic anemone that is becoming an important model system for studying coral-dinoflagellate symbioses.
the set of sequences identified constitutes a rich source of candidate genes that are likely to be involved in processes related to the onset, maintenance, and breakdown of symbiosis.
in this context, we were able to reconstruct the oxidative-stress response, which we also found to be prominent during basal transcription.
at the current depth of sequencing, we have identified two problems, namely  that some transcripts are represented by two  contigs and  that we lack information on transcripts of low abundance.
these issues will be addressed in the near future by using  <dig> sequencing, which, for example, has been successfully applied to sequence the coral larval transcriptome of acropora millepora at  <dig> × coverage  <cit> .

 many biological systems rely on symbiotic interactions between different organisms. one of the most dramatic examples is the coral reef ecosystem, which has at its heart a mutualistic partnership between corals and endosymbiotic, dinoflagellate algae. the dinoflagellates are classified in a single genus, symbiodinium, but molecular methods have revealed a high genetic diversity in this genus. the onset of these symbioses has been shown to display flexibility, but a range of specificity, i.e. from highly flexible to highly specific, is apparent during its maintenance. this process is likely to involve early recognition mechanisms and an evasion of the hosts' digestive and immune systems, as well as adaptations to diverse ecological niches and physiological acclimation. there have also been controversial discussions of whether symbiodinium populations may shift toward more heat-tolerant types as a consequence of thermal stress in order to adapt to environmental changes such as increasing seawater temperatures. in light of global climate change, this subject, i.e. cnidarian bleaching, has received much attention as devastating mass bleaching events have increased both in frequency and geographic extent. nonetheless, our knowledge of the underlying cellular and molecular mechanisms that facilitate the recognition between the partners, and determine the specificity, dynamics, and collapse of cnidarian-dinoflagellate symbioses, is limited. the cellular and molecular interactions between host and symbiont cells are important targets for genetic and genomic dissection, but corals are notoriously difficult to work with. for example, corals form large, slow-growing colonies that are difficult and costly to maintain in the laboratory, and their handling for microscopy and amenability to other cell biological, biochemical, and genetic methods is complicated by the calcareous skeleton precipitated by reef-building corals. what is needed to make rapid advances in this field is a model system that possesses the key characteristics of coral symbiosis, but allows more facile laboratory investigation. the sea anemone aiptasia represents a good candidate system, as it possesses the same mutualistic relationship with symbiodinium spp., but lacks the calcareous skeleton that hinders cellular-level work. it is widely distributed, and found in shallow tropical marine environments worldwide. sequence characterized amplified region data indicate that the vast majority of aiptasia worldwide, appear to be genetically homogeneous. the one exception is a closely related, but genetically distinct, lineage potentially restricted to the florida keys. data from the santos lab also indicate that natural populations of aiptasia from the florida keys preferentially host symbiodinium spp. comprised of only clade a or both clades a and b, whereas those from the remaining global range host clade b exclusively. typically considered a pest organism by seawater aquarists, aiptasia is hardy and proliferates rapidly by asexual reproduction. individual polyps can be maintained in a symbiotic or aposymbiotic state, experimentally re-infected with a variety of symbiodinium strains, and cultured at low cost. in fact, numerous studies have addressed symbiosis-related questions using a. pallida and its sister species a. pulchella by applying multiple tools ranging from microscopy to rna-interference methods. the generation of genomic resources for aiptasia would therefore greatly advance research addressing the understanding of symbioses at a molecular, cell-biological, and genomic level. as a cost-effective alternative to sequencing the genome of an organism, the generation and analysis of expressed-sequence-tag libraries provides an efficient method for discovering novel genes, estimating gene content, and approximating levels of gene expression. once established, these resources can be utilized for comparative genomics studies or the construction of gene expression microarrays. among cnidarians, the extensive genomic resources now available for the non-symbiotic sea anemone nematostella vectensis have opened new perspectives on the study of basal metazoans, and several est resources have been generated for symbiotic cnidarians and symbiodinium spp.. however, to date, only one small-scale project has generated ests for the symbiotic anemone aiptasia pulchella. in this study, we report the generation and analysis of high-quality ests from a symbiodinium clade a-hosting clonal population of aiptasia pallida that was likely derived from an individual originating from the florida keys lineage, which were processed through a software pipeline resulting in a user-friendly, queryable, web-accessible database named aiptasiabase. a blastx-based approach was used to estimate the relative contributions of each partner to the mixed cdna library, and we were able to identify numerous genes involved in key processes of cnidarian-dinoflagellate symbioses.  est library construction and assembly a total of cdna clones were bi-directionally sequenced, resulting in raw chromatograms, which served as input for the processing pipeline. after base calling by phred, lucy discarded low-quality sequences, short or insert-less sequences, and vector or polya-only sequences. an additional sequences were removed by seqclean, leaving high-quality ests for further processing. assembly of these ests by cap resulted in the generation of contigs, which ranged from to bp in length and contained – ests. together with the remaining singletons, a total of unique sequences were generated. because of the possibility that two uniseqs originated from the same transcript, we also estimated the number of unique genes in our dataset by assembling only the reverse reads of the directionally cloned cdnas. the resulting estimate of unigenes compared to the uniseqs is likely to reflect the large average size of inserts in the cdna library; thus, in many cases, uniseqs represent the 3' and 5' ends of genes for which the central parts were not captured due to sanger-sequencing length limitations. in addition, different splice variants or alleles of the same gene may have contributed to the excess of uniseqs over unigenes. detailed pre-assembly statistics are summarized in additional file 1: quality control and assembly statistics. previously, a small-scale est project was conducted in order to compare the abundance of transcripts between symbiotic and aposymbiotic aiptasia pulchella polyps. the present study included bi-directional sequencing, and the total number of ests is more than times larger than in the earlier study. therefore, the availability of almost uniseqs for about unigenes represents a rich transcriptomic resource, previously unavailable at this scale, for a symbiotic anemone. annotation of unique sequences and implementation of aiptasiabase all uniseqs were assigned putative identities based on blastx hits to the uniprot knowledgebase databases swissprot and trembl. about 37% and ~63% of the uniseqs found hits in swissprot and trembl, respectively, leaving ~36% of the uniseqs without similarities to known proteins. because the trembl database contains protein sequences based on conceptual translations of all nucleotide sequence entries in embl/genbank/ddbj, we chose to annotate the uniseqs according to the curated swissprot entries. assignments of gene ontologies could be made for about one third of uniseqs in each of the go categories: molecular function, biological process, and cellular component. because our cdna library represents the symbiotic, adult life-history stage of a. pallida, the go resource generated in this study sets the stage for statistical assessments of over- or under-representation of specific go-categories in libraries obtained from anemones under different conditions such as life-history stages, symbiotic state, or environmental conditions. in addition to blast and go annotations, all uniseqs were screened for single-nucleotide polymorphisms and simple-sequence repeats, providing resources for the investigation of gene polymorphisms between individuals and/or populations. the prediction of open reading frames within uniseqs also provided the basis for domain annotations at the protein level. about 25% of uniseqs matched a protein domain entry in the pfam database. one of the primary challenges of sequencing ests from a mixed transcriptome originating from two or more partners is to assign sequences to the proper genome of origin. taking a bioinformatic approach to this problem, we constructed taxon-specific databases representing either "cnidaria-only" or "alveolata-only" sequences from genbank, and then performed blastx-searches against those databases as well as the complete non-redundant database. we then employed a best-blastx-hit approach to estimate the numbers of ests that originated from a. pallida and symbiodinium spp., respectively, at various levels of confidence. irrespective of the confidence level, about one quarter of ests had no blastx-hit. at the different levels of confidence, – 70% and – % were predicted to originate from the anemone and the symbiodinium genomes, respectively distribution and assignment). the relatively small fraction of symbiodinium ests could be expected given that symbiodinum spp. are spatially restricted to the endodermal tissue layer of the host and that no special effort was made to disrupt the algal cell walls during the preparation of the rna. furthermore, the number of uniseqs without a significant blastx hit may be higher for symbiodinium transcripts. however, the uncertainty about the origin of non-annotated sequences represents a current limitation to our approach. ongoing and future genome-sequencing projects for symbiotic cnidarians and their dinoflagellate endosymbionts should soon become available and help to uncover the origins of sequences without currently known homologs in other organisms. this will provide an interesting opportunity to revisit our data set to look further at these perhaps taxonomically restricted genes. details can be found in additional file 2: flow diagram illustrating bbh appraoch and additional file 3: detailed est distribution and assignment. using an est-processing software, we stored all ests, uniseqs, and annotations in a queryable database named aiptasiabase, which is accessible through the url:. in addition to the results generated by the software, we have included the annotation of uniseqs according to kegg, which provides a convenient way to explore pathway components that were identified in this study. analysis of the most highly abundant transcripts we identified the contigs containing the greatest numbers of ests, which we used as a proxy for the most abundant transcripts. although the numbers of ests in contigs that are predicted to originate from symbiodinium were too low to be analyzed, many of the most abundant host-derived transcripts represented genes that are involved in the processes of protein biosynthesis, extracellular-matrix formation, and oxidative-stress response. a blastx search in swissprot. contig numbering is according to aiptasiabase_v b contigs and are likely to originate from the same transcript. c contigs and are likely to originate from the same transcript. d best blast search hit in nr: von willebrand factor-like protein. e sequencing error artifact. f best blast search hit in nr: mesoglein variant. kuo et al. reported that the most highly expressed gene in symbiotic a. pulchella was ferritin, whereas we found only ests that represented this gene. although differences in the preparations of the cdna libraries and sequencing depths pose an obstacle for a direct comparison, the discrepancy in the numbers of ferritin transcripts appears to be noteworthy. in a recent study that investigated the effect of increased temperature and uv levels on the symbiotic anemone anthopleura elegantissima, richier et al. observed a more than 17-fold up-regulation of ferritin expression upon thermal stress, but not uv stress. given this observation, it seems possible that the anemones in the study by kuo et al. were under elevated thermal stress at the time of sampling, which, taken together with the methodological differences mentioned above, makes any further comparative analyses unfeasible. this result has important implications, i.e., how culturing conditions of organisms as well as methodological differences between studies may have an impact on the transcriptome, and by extension, the interpretation of gene expression analyses. the highly abundant sequences with the highest uncertainties for correct annotation, apolipophorin and the cub and zona pellucida-like domain-containing protein were further scrutinized by similarity searches in additional databases. these searches revealed that the best hit for cub and zona pellucida-like domain-containing protein in the genbank non-redundant database was mesoglein, a protein that is proposed to be a structural element of the extracellular matrix of the mesoglea in the jellyfish aurelia aurita. the sequence annotated as apolipophorin contains a von willebrand factor type-d domain, and was reported to be involved in forming lipoprotein particles that bind lipoproteins and lipids. two other highly abundant sequences had no homologs among previously characterized proteins, suggesting that they are novel, and a third contig with no blastx-hit was identified as an artifact due to misassembled sequences. these results illustrate some of the caveats to automated sequence assembly and annotation and highlight the necessity for corroboration after automated sequence processing when focusing on single genes or groups of genes of interest. candidate genes with potential relevance to cnidarian-dinoflagellate symbioses we generated a candidate gene list of groups containing uniseqs that are likely to be of relevance to cnidarian-dinoflagellate symbioses. among these, the cellular antioxidant-response system could be most comprehensively reconstructed. genes related to the innate immune system and sugar-binding proteins gave rise to a partial gene inventory. other genes that are likely to play a role in the cellular events surrounding the breakdown of symbiosis were also identified. stress-induced photoinhibition and damage to algal photosystem ii are thought to be responsible for an increased production of reactive oxygen species and consequently, diffusion of hydrogen peroxide through the membranes into the host cells. the detoxification of h2o requires the activity of catalase or other peroxidases. superoxide dismutase, which catalyzes the reduction of superoxide to h2o as well as glutathione peroxidase, which uses glutathione to detoxify h2o were both not found among the sequenced ests. one possibility is that the abundance of sod transcripts in host cells was low, and the generation of superoxide spatially limited. in this case, superoxide may have been efficiently eliminated within the symbiodinium cells, while excess h2o that was not detoxified, could have diffused into the host cytosol and been reduced to h2o and o by catalase. alternatively, methodological factors such as insert-size selection or general rna processing may have prevented the detection of sod. other genes that had previously been reported in the context of cnidarian-dinoflagellate symbioses were also not detected, perhaps for same reasons as discussed above for sod.  by analyzing > high-quality ests and generating a comprehensive database for the user community, we have provided a foundation of transcriptomic resources for a symbiotic anemone that is becoming an important model system for studying coral-dinoflagellate symbioses. the set of sequences identified constitutes a rich source of candidate genes that are likely to be involved in processes related to the onset, maintenance, and breakdown of symbiosis. in this context, we were able to reconstruct the oxidative-stress response, which we also found to be prominent during basal transcription. at the current depth of sequencing, we have identified two problems, namely that some transcripts are represented by two contigs and that we lack information on transcripts of low abundance. these issues will be addressed in the near future by using sequencing, which, for example, has been successfully applied to sequence the coral larval transcriptome of acropora millepora at × coverage. 
with the advent of next-generation sequencing methods, the transcriptomic inventory of a. pallida and its symbiont, and thus the extent of aiptasiabase, should expand dramatically in the near future. in order to expedite this field of research, we generated and analyzed a collection of expressed sequence tags from the sea anemone aiptasia pallida and its dinoflagellate symbiont, a system that is gaining popularity as a model to study cellular, molecular, and genomic questions related to cnidarian-dinoflagellate symbioses. we have established the first large-scale transcriptomic resource for aiptasia pallida and its dinoflagellate symbiont. the molecular mechanisms underlying the establishment, maintenance, and breakdown of the symbiotic partnership are, however, not well understood. these data provide researchers with tools to study questions related to cnidarian-dinoflagellate symbioses on a molecular, cellular, and genomic level. all ests and uniseqs along with annotation results and other tools have been made accessible through the implementation of a publicly accessible database named aiptasiabase. furthermore, we were able to identify groups of genes that are known or likely to play a role in cnidarian-dinoflagellate symbioses, including oxidative stress pathways that emerged as a prominent biological feature of this transcriptome. this groundwork represents a crucial step towards the establishment of a tractable model system that can be utilized to better understand cnidarian-dinoflagellate symbioses. this may reflect low levels of gene expression in the symbionts, low coverage of alveolate genes in the sequence databases, a small number of symbiont cells relative to the total cellular content of the anemones, or failure to adequately lyse symbiont cells. a set of unique sequences comprising clusters of or more ests and unclustered ests was generated by analyzing high-quality ests from a mixed host/symbiont cdna library. using a blast-based approach to predict which unique sequences derived from the host versus symbiont genomes, we found that the contribution of the symbiont genome to the transcriptome was surprisingly small.
 the most diverse marine ecosystems, coral reefs, depend upon a functional symbiosis between cnidarian hosts and unicellular dinoflagellate algae. the molecular mechanisms underlying the establishment, maintenance, and breakdown of the symbiotic partnership are, however, not well understood. efforts to dissect these questions have been slow, as corals are notoriously difficult to work with. in order to expedite this field of research, we generated and analyzed a collection of expressed sequence tags from the sea anemone aiptasia pallida and its dinoflagellate symbiont, a system that is gaining popularity as a model to study cellular, molecular, and genomic questions related to cnidarian-dinoflagellate symbioses.  a set of unique sequences comprising clusters of or more ests and unclustered ests was generated by analyzing high-quality ests from a mixed host/symbiont cdna library. using a blast-based approach to predict which unique sequences derived from the host versus symbiont genomes, we found that the contribution of the symbiont genome to the transcriptome was surprisingly small. this may reflect low levels of gene expression in the symbionts, low coverage of alveolate genes in the sequence databases, a small number of symbiont cells relative to the total cellular content of the anemones, or failure to adequately lyse symbiont cells. furthermore, we were able to identify groups of genes that are known or likely to play a role in cnidarian-dinoflagellate symbioses, including oxidative stress pathways that emerged as a prominent biological feature of this transcriptome. all ests and uniseqs along with annotation results and other tools have been made accessible through the implementation of a publicly accessible database named aiptasiabase.  we have established the first large-scale transcriptomic resource for aiptasia pallida and its dinoflagellate symbiont. these data provide researchers with tools to study questions related to cnidarian-dinoflagellate symbioses on a molecular, cellular, and genomic level. this groundwork represents a crucial step towards the establishment of a tractable model system that can be utilized to better understand cnidarian-dinoflagellate symbioses. with the advent of next-generation sequencing methods, the transcriptomic inventory of a. pallida and its symbiont, and thus the extent of aiptasiabase, should expand dramatically in the near future. 