Developing the anemone Aiptasia as a tractable model for cnidarian-dinoflagellate symbiosis: the transcriptome of aposymbiotic A. pallida
© Lehnert et al.; licensee BioMed Central Ltd. 2012
Received: 3 February 2012
Accepted: 22 June 2012
Published: 22 June 2012
Coral reefs are hotspots of oceanic biodiversity, forming the foundation of ecosystems that are important both ecologically and for their direct practical impacts on humans. Corals are declining globally due to a number of stressors, including rising sea-surface temperatures and pollution; such stresses can lead to a breakdown of the essential symbiotic relationship between the coral host and its endosymbiotic dinoflagellates, a process known as coral bleaching. Although the environmental stresses causing this breakdown are largely known, the cellular mechanisms of symbiosis establishment, maintenance, and breakdown are still largely obscure. Investigating the symbiosis using an experimentally tractable model organism, such as the small sea anemone Aiptasia, should improve our understanding of exactly how the environmental stressors affect coral survival and growth.
We assembled the transcriptome of a clonal population of adult, aposymbiotic (dinoflagellate-free) Aiptasia pallida from ~208 million reads, yielding 58,018 contigs. We demonstrated that many of these contigs represent full-length or near-full-length transcripts that encode proteins similar to those from a diverse array of pathways in other organisms, including various metabolic enzymes, cytoskeletal proteins, and neuropeptide precursors. The contigs were annotated by sequence similarity, assigned GO terms, and scanned for conserved protein domains. We analyzed the frequency and types of single-nucleotide variants and estimated the size of the Aiptasia genome to be ~421 Mb. The contigs and annotations are available through NCBI (Transcription Shotgun Assembly database, accession numbers JV077153-JV134524) and at http://pringlelab.stanford.edu/projects.html.
The availability of an extensive transcriptome assembly for A. pallida will facilitate analyses of gene-expression changes, identification of proteins of interest, and other studies in this important emerging model system.
KeywordsAnemone Cnidaria Coral Dinoflagellate Neuropeptide Single-nucleotide variant Symbiosis Transcriptome
Coral reefs are global resources of great ecological, economic, and aesthetic value. The success of corals in their typically nutrient-poor environments is due largely to their symbiosis with dinoflagellates of the genus Symbiodinium. These algae inhabit the symbiosome (a vacuole derived from the early endosome) in gastrodermal cells of the host [1–4] and transfer up to 95% of their photosynthetically fixed carbon to the host . Reef-building corals have recently declined worldwide, with pollution, disease, destructive fishing practices, increased sea-surface temperatures, and ocean acidification all implicated as contributory factors. Some of these environmental changes affect the symbiotic relationship between algae and host and can lead to dramatic and potentially lethal “bleaching” events, during which the algae are lost and the host may die. Bleaching events have become more frequent over the past 20 years.
Much recent research in coral biology has focused on the effects of stresses – particularly high temperature and lowered pH – on the coral holobiont (the community of living organisms making up a healthy coral), as well as on which genetic and molecular factors of the host and alga lead to differential stress responses and resilience [6–13]. However, these efforts have been impeded by the lack of an experimentally tractable system for studies of the establishment, maintenance, and breakdown of the symbiosis. Corals themselves present major logistical difficulties for laboratory investigation. They grow slowly and are difficult and costly to maintain, their calcareous skeletons make many biochemical and cell biological techniques difficult, and it can be difficult to obtain sufficient biomass to do high-throughput experiments. In addition, samples collected from the wild can have heterogeneous genetic backgrounds, causing difficulties in the application and interpretation of gene-expression studies.
To circumvent these difficulties, we and others are developing the small sea anemone Aiptasia as a model system for studies of dinoflagellate-cnidarian symbiosis [14, 15]. Like corals, Aiptasia is an anthozoan (a Class in the Phylum Cnidaria) and maintains intracellular symbiotic dinoflagellates closely related to those in corals. However, unlike corals, Aiptasia is extremely hardy, grows and reproduces rapidly via asexual reproduction in the laboratory (allowing the generation of large clonal populations), and lacks a calcareous skeleton. The lack of skeletal deposition makes Aiptasia an unsuitable model for this aspect of coral biology but greatly facilitates other studies of cell biology and biochemistry. Additionally, Aiptasia can exist in an aposymbiotic (dinoflagellate-free) state or host a variety of Symbiodinium types (although not all), allowing facile studies of symbiosis specificity [14, 16, 17]. We have recently developed a protocol for the year-round induction of spawning and larvae production in laboratory-raised Aiptasia, which should free a variety of studies from dependence on the seasonal coral reproductive cycle and potentially open the door to genetic analysis.
Studies of the dinoflagellate-cnidarian symbiosis can take advantage of genomics approaches. For example, gene-expression studies should help to elucidate how symbiotic cnidarians respond to various stressors, whereas comparative genomics approaches using sequence data from cnidarians that are not symbiotic with dinoflagellates should help us understand how these symbioses evolved. Genomic and transcriptomic resources for cnidarians are beginning to accumulate rapidly, thanks to the advent of new sequencing technologies. Recently, the genome of Acropora digitifera, a common Indo-Pacific coral, was sequenced and assembled . In addition, the genomes of two non-symbiotic cnidarians, the anemone Nematostella vectensis (an anthozoan) and the more distantly related Hydra magnipapillata (in Class Hydrozoa), have been sequenced [20, 21]. Small, Sanger-sequenced EST datasets are available for several species of corals and anemones [15, 22, 23], as are larger 454-sequenced datasets for several corals [24–26].
As a step in the development of Aiptasia as a model system, we have performed a detailed analysis of the transcriptome of the aposymbiotic animals. Unlike previous transcriptomes in the field of symbiotic cnidarian biology, these data are derived from a clonal and easily distributed strain of anemone, greatly facilitating a straightforward comparison of experimental results between different laboratories.
Aiptasia strain and culture
All animals used were from clonal population CC7 , which in spawning experiments typically behaves as a male (hundreds of spawns have produced sperm compared to three occasions on which individual polyps have produced eggs) . The stock cultures were grown in a circulating artificial seawater (ASW) system at ~25°C with 20–40 μmol photons m-2 s-1 of photosynthetically active radiation (PAR) on an ~12 h light : 12 h dark cycle and fed freshly hatched brine-shrimp nauplii approximately twice per week. Aposymbiotic animals were generated by several repetitions of the following process: cold-shocking by addition of 4°C ASW and incubation at 4°C for 4 h, followed by 1–2 days of treatment at ~25°C in ASW containing the photosynthesis inhibitor diuron (Sigma-Aldrich) at 50 μM. After recovery for several weeks in ASW in the light (~12:12 light:dark) at ~25°C, putatively aposymbiotic anemones were inspected by fluorescence microscopy to confirm the complete absence of dinoflagellates (whose bright chlorophyll autofluorescence is conspicuous when they are present) and were then cultured in separate tanks as described for the stock culture above.
RNA extraction and sequencing
Separate populations of animals were exposed to various conditions prior to RNA isolation in an attempt to maximize the diversity of genes expressed. Whole, medium-sized (~1 cm long) anemones were collected in three pools: (i) ~20 animals grown in control conditions; (ii) animals (2–3 per concentration and time point) exposed to bacterial lipopolysaccharide [LPS (Sigma, cat. no. L2880), which is commonly used to induce a strong innate immune response in other organisms] at 1, 10, or 100 μg/μl for 6 or 24 h; (iii) animals (2–3 per treatment) that had been exposed to a single treatment [elevated light (~250 μmol photons m-2 s-1) for 3 h; dark for 3 h; cold shock at 4°C for 4 h; heat shock at 37°C for 4 h; ultraviolet illumination for several minutes; starvation for one week; hyperosmolarity (1.5x normal salt concentration) or hypoosmolarity (0.3x normal salt concentration) for 30 min; exposure to 10 μM or 100 μM of the 20 standard amino acids or the sugars sucrose and D-glucose for 1 h]. Treated animals were stored in RNALater (Ambion, cat. no. AM7021) at −20°C for later processing.
We extracted total RNA from the anemones in each pool by homogenization in TRIzol reagent (Invitrogen, cat. no. 15596–026) following the manufacturer’s protocol and using the alternative high-salt method of RNA precipitation recommended by Invitrogen to reduce proteoglycan and polysaccharide contamination. We enriched for polyadenylated RNA using the MicroPoly(A) Purist kit (Ambion, cat. no. 1919) and then fragmented the RNA using divalent cations [5 min at 94°C in the reverse-transcriptase first-strand buffer supplied with SuperScript III reverse transcriptase (Invitrogen, cat. no. 18080044)]. cDNA was synthesized using random-hexamer primers (Invitrogen, cat. no. N8080127), ligated to Illumina PE Adaptors, size-selected, amplified, and size-selected a second time. Libraries with different insert sizes (ca. 200, 400, and 600 bp) were synthesized for each pool. Clustering and sequencing were performed by the Stanford Center for Genomics and Personalized Medicine using an Illumina Genome Analyzer IIx (GAIIX) sequencer.
Read filtering and transcriptome assembly
To minimize redundancy in the dataset, we used the Fulcrum program to collapse duplicate reads and return a single representative read with improved quality scores for each “read family” . Reads were then filtered for quality and length. Briefly, reads were trimmed such that no nucleotide had a quality score less than 10 and no ambiguous nucleotides (N’s) remained. Any read shorter than 45 bp was then discarded. The remaining reads were combined into files based on the insert size of the library (irrespective of the prior biological treatment) and assembled using an additive multiple-k-mer (35, 39, 43, 47, 51, 55, 59, 63, and 67) approach [28, 29] with the Velvet/Oases assembler (Velvet version 1.1.04 and Oases version 0.1.21) .
Oases assembled many contigs that formed “hairpins”, suggesting mis-assembly caused by the presence of palindromic or near-palindromic sequences in the reads. (This problem appears to have been solved in more recent versions of Oases that were released after our study was completed .) We identified these hairpin-containing sequences and split each of them into two separate contigs. The contigs resulting from the individual assemblies were then assembled together with the original Illumina reads using a k-mer length of 67 with the conserveLong option turned on. Both the output from this final assembly and the combined contigs from each individual assembly were merged into a single file, new hairpins were identified and processed as described above, and identical contigs were collapsed into single representatives using cd-hit-est . The resulting contigs were assembled using CAP3 (requiring ≥50-bp overlap with ≥90% identity to join two contigs) to join overlapping contigs and reduce redundancy in the transcriptome dataset . Contigs shorter than 200 bp were discarded as likely to be uninformative.
In order to assign putative functional roles to the transcripts, we aligned them to the NCBI non-redundant protein database (nr) using the blastx program from the standalone BLAST 2.2.25+ software suite with an e-value cutoff of 1e-3 . Predicted protein sequences were searched for specific domains using Interproscan . The blastx and Interproscan outputs were imported using the Blast2GO software package  and used to assign Gene Ontology (GO) terms to the predicted proteins .
Validation of contigs by alignment with paired-end Sanger reads
As one approach to contig validation, we aligned a set of paired-end Sanger-sequenced ESTs  to our transcriptome assembly using BLAT (minimum percent identity 90%) . We counted the number of times the best alignments of a pair of forward and reverse Sanger reads were to the same contig but with the expected opposite orientation.
Genomic DNA extraction and sequencing
Genomic DNA was isolated from medium-sized aposymbiotic anemones by incubating the whole animals at 55 °C for 4 h in lysis buffer (100 mM NaCl, 50 mM Tris pH 8.0, 50 mM EDTA, 1% SDS) to which Proteinase K had been added to a final concentration of 0.77 μg/μl. The resulting solution was extracted twice with equal volumes of buffer-saturated phenol (Invitrogen, cat. no. 15513–039) and once with an equal volume of phenol/chloroform/isoamyl alcohol (25:24:1). The genomic DNA was then precipitated by ethanol, resuspended in 100 μl of TE buffer, and sheared using a Covaris Adaptive Focused Acoustics machine, following the manufacturer’s instructions, to a target size of 400 bp (10% Duty Cycle, 4 Intensity, 200 cycles per burst, 55 seconds). End-repair and adapter ligation were performed following Illumina’s instructions, and two lanes were sequenced using an Illumina HiSeq system by the Stanford Center for Genomics and Personalized Medicine.
Heterozygous SNV detection
Putative single-nucleotide variants (SNVs) were detected using CLC Genomics Workbench version 4.6 (CLC bio). Fulcrum-collapsed (see above) and quality-filtered HiSeq genomic reads were mapped against the transcriptome. After an optimal alignment was generated, it was considered valid if 40% of the read aligned with ≥96% agreement (at least 34 of 35 base-pairs for the average post-trimming read length of 88-bp). We used 40% rather than something higher because the 100-bp reads could overlap exon-intron boundaries, and we do not yet have a good estimate of average exon size in Aiptasia. The 40% criterion should prevent intron sequence in the read from disallowing a valid match while still providing sufficient specificity. If a given site had a minimum of 10x coverage and ≥35% of the reads at that site contained the alternative base, we classified that base as an SNV.
To estimate the percentage of false positives among our SNV calls, we amplified genomic DNA for some of them using primers to the flanking sequences and sequenced the products using the Sanger method. We identified SNVs as positions in otherwise high-quality chromatograms where there were peaks for two different bases.
Estimation of genome size
Genome size was estimated by using a slightly modified version of the protocol outlined by Hu et al.. We aligned two lanes of HiSeq genomic data (see above) against the assembled transcriptome using BLAT. We determined the number of bases in each read that aligned with the corresponding contig from the top hit that had no alignment gaps; where multiple hits with equal scores existed, the first hit listed was used. The numbers of aligned bases were summed for all genomic reads mapping to a given contig and divided by the contig length, giving each contig in the transcriptome an average coverage. The modal coverage of the entire contig dataset was then used to estimate the depth to which the genome had been sequenced. The total amount of sequence in the genomic reads was then divided by the estimated sequencing depth to obtain the genome size.
Results and discussion
Sequencing and assembly of the transcriptome
Properties of the libraries and sequencing runs used for transcriptome analysis
Population from which mRNA was derived
Approximate library insert length (bp)
Number of GAIIx cycles (bp)
Number of GAIIx lanes sequenced
Amount of sequence (Gb)
Summary of the aposymbiotic Aiptasia transcriptome assembly
Total number of contigs
Total base-pairs in contigs
Contig size range
200–13,061 bp a
Median contig length
Mean contig length
Validation and functional annotation
We used several approaches to validate the transcriptome assembly. First, we compared it to a set of 4,833 pairs of Sanger-sequence reads from a cDNA library derived from mRNA isolated from symbiotic anemones . In the preparation of this library, an effort was made to obtain full-length cDNAs, which were also size-selected to enrich for longer species (average size ≅ 1.95 kb); thus, it should be enriched for full-length or near-full-length transcripts. When we aligned these ESTs to our transcriptome assembly using BLAT, 73% (7,091) of the Sanger reads mapped to the transcriptome with identity ≥90%. The remaining 27% are likely to be sequences from Symbiodinium, from genes that are expressed only at low levels in aposymbiotic Aiptasia, or from other organisms that were present in the culture used to prepare the library for Sanger sequencing. Of the 755 Sanger read-pairs in which each read mapped to one and only one contig, 73% (551) mapped to the same contig in opposite directions. Of the additional 1,520 read-pairs with valid alignments in which one or both reads aligned to more than one contig, for 82% (1,239) there was at least one contig to which both reads aligned with opposite orientations. These data suggest that even among long transcripts, which are more likely to be fragmented in our assembly, many are represented by full-length or near-full-length contigs.
Length dependence of BLAST alignment success
Contig size range
Total number of contigs in size range
Number of contigs in size range with BLAST alignments (e-value ≤ 1e-10)
% of contigs in size range with BLAST alignments (e-value ≤ 1e-10)
200 bp – 599 bp
600 bp – 999 bp
1000 bp – 1399 bp
≥ 1400 bp
Estimating transcriptome completeness by comparison to Nematostella a
Number of predicted proteins assigned to this pathway in Nematostella
Number of orthologs in Aiptasia transcriptome
Average% of Nematostella coding sequence covered by best alignment
Average predicted amino acid similarity (%)b
Glycolysis and gluconeogenesis
Amino-sugar and nucleotide-sugar metabolism
Regulation of autophagy
Valine, leucine, and isoleucine degradation
Fatty acid biosynthesis
Completeness of transcripts and sequence conservation for some proteins involved in cellular spatial organization
Query protein (GenBank Accession Number)
Amino acids in query sequence
Amino acids of query covered by best BLAST hit(s)
% amino-acid sequence identity (number of gaps)
Length of Aiptasia contig (bp)
Positions in contig covered by best BLAST hit
Mouse Cdc42 (P60766)
Mouse cyto-plasmic actin 1 (P60710)
Mouse tubulin α1B (P05213)
Mouse tubulin β5 (P99024)
Mouse septin-2 (P42208) a
Mouse kinesin 1 heavy chain (Q61768)
Mouse myosin 8 (P13542)
Mouse dynein heavy chain 1 (Q9JHU4)
In summary, although we are undoubtedly lacking the sequences (or at least lacking complete sequences) for some transcripts that are expressed only at low levels, in particular cell types, during particular stages of development, or under conditions to which we did not expose the anemones, it appears that the transcriptome described here contains at least partial sequences (and many full-length sequences) for the large majority of transcripts expressed in adult, aposymbiotic anemones. It will be particularly interesting to see how many additional transcripts are identified when the transcriptome of symbiotic anemones is examined.
Estimation of SNV frequency
SNV and indel distributions in Aiptasia
Frequency in Transcriptome
A/G Transition SNV
C/T Transition SNV
A/C Transversion SNV
G/C Transversion SNV
A/T Transversion SNV
G/T Transversion SNV
Insertion or Deletion
8,691 a [1, 5,773; 2, 1,289; 3, 862; 4, 372; 5, 165; 6,143; 7, 57; 8, 30]
To evaluate the reliability of our SNV calls, we designed primers to nine contigs in our assembly based on the following criteria. (1) The top BLAST hit was to a cnidarian, so we could be confident that we were looking at an Aiptasia-derived contig. (2) The predicted SNV was not located so close to an end of the contig that it would be within 40 bp of the primer that we were using to amplify (as this could have led to confusion from low-quality sequence near the primer sites). (3) The variant was a simple base-pair change rather than an indel (as these would have been undetectable by our method of inquiry). (4) Contigs with multiple SNVs were preferred as this enabled to us perform more tests with fewer primers.
For the nine contigs, we created 12 primer pairs that would amplify regions containing a total of 17 putative SNVs. Of these 12 primer pairs, eight produced clear PCR products with single bands, encompassing a total of 11 putative SNVs. Six of these bands had the predicted sizes, and two were larger (~400 instead of 250 bp and ~1600 instead of 576 bp), presumably indicating the presence of introns. The remaining four primer pairs presumably either needed additional optimization of the PCR reactions to ensure specificity or represented regions in which the exons were separated by introns that were too long for amplification under standard PCR conditions. All eight of the PCR products were sequenced using the same primers as used for the PCR, and the SNV was considered to be validated when there were dual peaks matching the reference and variant calls at the specified location surrounded by otherwise high-quality peaks. All 11 of the SNVs tested were validated in this test, suggesting that there is only a low false-positive rate for our larger set of SNV calls.
Estimation of genome size
We aligned ~10.1 Gb of genomic reads to the transcriptome assembly (see Methods), and estimated a modal coverage of ~24x per contig. Thus, we estimate a genome size of 10,100 Mb/24 = 421 Mb. Nematostella and Acropora digitifera, the closest relatives of Aiptasia whose genomes have been sequenced, have genome sizes of ~450 Mb and ~420 Mb, respectively [19, 20]. Given its apparently modest size, the Aiptasia genome could be readily sequenced using the currently available technologies.
Identification of possible neuropeptide precursors
We have assembled and characterized a reference transcriptome for adult, aposymbiotic Aiptasia pallida using the Illumina sequencing platform. We have used this resource to detect SNVs in our clonal population of anemones, estimate the genome size, and identify possible neuropeptide-encoding genes. This transcriptome will enable future studies to explore the changes in gene expression that accompany the association with dinoflagellate endosymbionts, determine how the symbiotic partners respond to a variety of stressors, further test the applicability of this model system to corals, and complete the assembly and annotation of the Aiptasia genome (for which the transcriptomic data will be essential). The contigs and their associated annotations are available through NCBI (Transcription Shotgun Assembly database, accession numbers JV077153-JV134524) and at http://pringlelab.stanford.edu/projects.html. The limitations of the current assembly should diminish in updated versions that incorporate additional sequence data, particularly those from symbiotic animals and from different developmental stages. Updated assemblies will be made available through both the NCBI site and our lab website.
Genome Analyzer IIx
Photosynthetically active radiation
Expressed sequence tag.
This study was funded by the Gordon and Betty Moore Foundation (grant #2629), a National Science Foundation Graduate Research Fellowship to EML, and National Institutes of Health Training Grant HG000044 support to EML and MSB. Annotation was performed on the Bio-X2 cluster, which is funded by NSF award CNS-0619926. We thank Carlo Caruso for coordinating the project during its early stages and Jodi Schwarz, Shini Sunagawa, Morgan Mouchka, Virginia Weis, Emilie-Fleur Dicks, and Camille Paxton for providing feedback that allowed us to improve the final project.
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