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Identification of genes for synthesis of the blue pigment, biliverdin IX[alpha], in the blue coral Heliopora coerulea.

Abstract. Heliopora coerulea is the only species in the subclass Octocorallia that has a crystalline aragonite skeleton. The skeleton has been reported to contain the blue pigment, biliverdin IX[alpha], which is formed by heme oxygenase (HO) during heme decomposition. There is little information regarding gene expression in H. coerulea; therefore, the biosynthesis pathway for biliverdin IX[alpha] is poorly understood. To identify the genes related to heme synthesis and degradation, metatranscripts of H. coerulea and its symbiont Symbiodinium spp. were sequenced and separated from the host- and symbiont-derived sequences. From the metatranscriptome analyses, all genes for heme synthesis and three HOs were isolated from the host and symbiont. From our phylogenetic and amino acid analysis, we noted that one of the HO iso-forms in the host coral was predicted to possess HO activity. However, biliverdin reductase, which reduces biliverdin to bilirubin, was not identified in the present study. Similarly, biliverdin reductase was not identified in the transcripts of the red coral Corallium rubrum, a species that also belongs to Octocorallia. However, genes related to heme synthesis and HO were found in C. rubrum. We speculate that Heliopora coerulea can produce biliverdin and accumulate it in the skeleton, while red corals and other Octocorallia species cannot. Further information from molecular studies of H. coerulea will provide insights into the synthesis of biliverdin IX[alpha], the blue pigment in the hard crystalline aragonite skeleton, and will be fundamental to future ecological and physiological studies.

Introduction

The blue coral Heliopora coerulea (Pallas, 1766) is a "living fossil" species whose morphology differs slightly from that of the lower cretaceous Heliopora japonica (Eguchi, 1948). The most prominent feature of H. coerulea is its blue, hard, crystalline aragonite skeleton, because Heliopora is more closely related to soft coral species (Octocorallia) than to hard coral species (Hexacorallia). Heliopora coerulea is widely distributed in the Indo-Western Pacific, but some of its habitats have been lost due to climate change. Thus far, H. coerulea is the only surviving species in the family Helio-poridae (Zann and Bolton, 1985). While it has been suggested that the blue skeleton of Heliopora originated from biliverdin IX[alpha] (Rudiger et al., 1968), which occurs as a result of heme degradation, to our knowledge there is no evidence that biliverdin IX[alpha] is synthesized in Heliopora.

Heme synthesis is one of the essential metabolic pathways for the prosthetic group of hemoglobin, myoglobin, cytochrome P450, catalase, and peroxidase (Rodgers, 1999). The heme synthetic pathway involves up to eight enzymes and eight reactants in three steps (Fig. 1 ). For the first step in heme synthesis, 5-aminolevulinic acid (ALA) is synthesized by two pathways, called the Shemin pathway and the C5 pathway. In the Shemin pathway, which is found in animals and fungi as well as a-proteobacteria (Heinemann et al., 2008), ALA is formed from Succinyl-Coenzyme A (Succinyl-CoA) and glycine with elimination of C[O.sub.2] (Kikuchi et al., 1958; Fig. 1 ). The C5 pathway, which is found in bacteria, Ar-chaea, and plants, synthesizes the C5 skeleton of glutamate as the first substrate in the plastids (Beale and Castelfranco, 1973; Fig. 1). In the second step, coproporphyrinogen III is formed from the synthesized ALA, following four reactions in the cytoplasm (Fig. 1). In the final step, the synthesized coproporphyrinogen III is transported to the mitochondria and is converted into heme after undergoing three reactions (Fig. 1). In plants, all of the heme synthesis processes occur in the plastids (Mochizuki et al., 2010). To control surplus heme, ALA synthase and glutamyl-fRNA reductase are inhibited by negative feedback and are rate-limiting enzymes (Meskauskiene and Apel, 2002; Furuyama et al., 2007). Heme is degraded to biliverdin IX[alpha], F[e.sup.2+], and carbon monoxide by heme oxygenase (HO) (Montellano, 2000). The biliverdin IX[alpha] is further reduced by biliverdin reductase, thus forming bilirubin (Baranano et al., 2002). In H. caerulea, the genes related to heme synthesis and degradation have not yet been identified.

Similar to other coral species, Heliopora coerulea interacts with photosynthetic dinoflagellates of the genus Symbiodinium, commonly called zooxanthellae (symbiont) (Pochon and Gates, 2010). The symbionts are harbored in their host vacuolar membrane (symbiosome) of gastrodermal cells (Davy et al., 2012). The photosynthetic products of the symbiont are essential to the survival of the host coral. Elevated seawater temperatures and high irradiance can damage the chloroplasts of the symbiont. Under such conditions, the symbiont produces reactive oxygen species (ROS), which oxidize membranes, denature proteins, and damage nucleic acids (Weis, 2008). Thus, ROS produced by the symbiont disrupt the coral symbiosis and lead to coral bleaching. Although heme is needed for apoprotein as the prosthetic group, free heme can be a source of ROS and is toxic (Kumar and Bandyopadhyay, 2005). Heme oxygenase is important for the removal of ROS produced by free heme. In humans, expression of HO is one of the indicators of response to oxidative stress. One of the HO isoforms, HO-1, can be upregulated approximately a hundred-fold in response to oxidative stress (Maines, 1988). In the coral Acropora grandis, expression of HO also has been induced by heat and oxidative stress (Fang et al., 1997). However, information on the role of physical activity of HO in H. coerulea is limited.

In the present study, we conducted what is, to our knowledge, the first metatranscriptome analysis of Heliopora coerulea and its symbionts, by RNAseq using Illumina sequencing (Illumina, San Diego. CA). This transcript information is useful and fundamental to ecological and physiological studies; we identified genes related to heme synthesis and degradation. Functions of HO in H. coerulea and their symbionts were predicted based on phylogenetic and amino acid sequence analyses. Based on these findings, we discuss whether H. coerulea can produce the blue pigment, biliverdin IX[alpha], and provide transcript information about H. coerulea.

Materials and Methods

Animal sampling

Heliopora coerulea were collected from Sekisei Lagoon at Ishigaki Island, Okinawa prefecture. Japan (24[degrees]15'28.3"N, 124[degrees]07'47.8"E). Samples were collected at a depth of 2.5 m by scuba diving in May 2014, for which we obtained Okinawa Prefecture permission (No. 26-27). Collected fragments of H. coerulea from a single colony were immediately immersed in RNAlater (Thermo Fisher Scientific, Waltham, MA), left overnight at 4 [degrees]C, and then stored at-80 [degrees]C until use.

RNA extraction, library construction, sequencing, and de novo assembly

Three small fragments from a single colony of Heliopora coerulea (2-cm diameter) that were immersed in RNMater were pulverized using a mortar and pestle. Total RNA was extracted using a TRIzol plus RNA Purification Kit (Thermo Fisher Scientific), and any contaminating DNA in the total RNA was digested using PureLink DNase (Thermo Fisher Scientific) according to the manufacturer's instructions. The concentration and quality of purified total RNA were determined using a Qubit RNA HS Assay Kit (Thermo Fisher Scientific) and Bioanalyzer 2100 with a RNA 6000 Nano Kit (both from Agilent Technologies, Santa Clara, CA) according to the manufacturer's instructions.

Three libraries were constructed using 1 [micro]g of the total RNA in accordance with TruSeq RNA Sample Prep ver. 2 (LS) protocol (Illumina, Inc.). Complementary DNA (cDNA) was synthesized using SuperScript III Reverse Transcriptase (Thermo Fisher Scientific). The cDNA libraries were sequenced with 150 base pairs (bp) of the paired-end by NextSeq 500 (Illumina, Inc.) at the Research Center for Bioinformatics and Biosciences of the National Research Institute of Fisheries Science, Yokohama, Japan.

Adaptor sequences and low-quality ends with a Phred quality value < 30 (QV30) (base calling program for DNA sequencing; Phred, CodonCode Corp., Centerville, MA) in the reads were trimmed using Trimmomatic (Bolger et ai, 2014). Sequence length and quality were confirmed using FastQC (Andrews, 2010). The remaining paired-end reads were assembled using Trinity with command options "-JM = 40G," "-CPU = 28," and "-min_kmer_cov=2" (Grabherr et al., 2011); the other options used were the default options.

Functional annotation and separation of the host and symbiont sequences

Open reading frames (ORFs) over 150 bp were extracted and translated into amino acid sequences using TransDecoder software (Haas et al., 2013). Heliopora coerulea is considered diploid; therefore, transcripts derived from two haplo-types possibly were contained in the transcriptome assembly. To remove the redundant transcript sequences, the ORFs of 95% homologous amino acid sequences were clustered using the CD-HIT program with the "-c 0.95" command option (Li and Godzik, 2006); the other options used were the default options.

To separate the host and symbiont proteins, we used the method from previous studies, such as Karako-Lampert et al. (2014) and Shinzato et ai (2014). The clustered amino acid sequences of ORFs were utilized for a homology search, using the BLASTP program against the following databases: protein sequences of Nematostella vectensis (24,780 sequences, Putnam et al., 2007); Acropora digitifera (26,275 sequences, Shinzato et al., 2011 ); Hydra vulgaris (21,990 sequences. Chapman et al., 2010); Corallium rubrum (200.589 sequences, extracted using TransDecoder software, Pratlong et al., 2015); Symbiodinium minutum in clade B (47,014 sequences, Shoguchi et al., 2013); Syinbiodinium spp. in clade C1 (43,592 sequences, accession ID MMETSP1367, Keeling et al., 2014); and Syinbiodinium spp. in clade C15 (35,777 sequences, accession ID MMETSP1370, Keeling et al., 2014). The threshold of sequence homology was defined as having an e-value of [less than or equal to] [le.sup.~5]. Each BLASTP result was merged and sorted by bit score, and the topi gene (of the BLAST hit) result for each ORF was extracted. When the top1 gene hit of ORF was similar to cnidarian or Syinbiodinium sequences, the ORF was assigned as the host or symbiont-derived sequence, respectively. Guanine and cytosine (GC) content of the nucleic acid sequences derived from the host and symbiont were calculated by Perl script (i.e., the authors' custom script) and plotted in R (R Core Team, 2016).

For protein annotation, the clustered amino acid sequences of ORFs were utilized for a homology search against the database of non-redundant proteins (nr database) of the National Center for Biotechnology Information (NCBI), and the UniRef90 database (Suzek et al., 2015), using the BLASTP program. The threshold of sequence homology was defined as having an e-value of [less than or equal to] [le.sup.-5]. The ORFs were annotated based on their similarity to sequences in the nr and UniRef90 databases. The gene ontology (GO) numbers, which are shared with accession numbers used in UniRef90 (Camon et al., 2004; Suzek et al., 2015), were assigned from the best hits of the BLASTP results against UniRef90. To identify the proteins related to heme synthesis and degradation pathways, the Enzyme Commission (EC) number was obtained from the GO numbers by Blast2GO software (Conesa et al., 2005). Protein domains in the extracted ORFs of Heliopora coerulea and Corallium rubrum were examined against the pfam-A database using the hmmsearch command in the HMMER program (Eddy, 1998); the threshold of the domain search was defined as having an e-value of [less than or equal to] [le.sup.-5].

Maximum likelihood phylogenetic analysis

To classify the Syinbiodinium clade of the Heliopora coerulea symbiont, a maximum likelihood (ML) phylogenetic tree was constructed using nucleic acid sequences of chloro-plast large subunit ribosomal DNA (cp23S). All cp23S genes of clade A-I of Syinbiodinium refer to Pochon et al. (2014) and were retrieved from the nucleotide (nt) database of the NCBI. For the functional position of HO, amino acid sequences of HO from cnidarians, teleosts, human, insects, polychaeta, ascidians, and Syinbiodinium were retrieved from the nr database of the NCBI.

Multiple-sequence alignments were created using Clustal Omega software (Sievers et al., 2011), and all gaps were automatically trimmed with trimAl software (Capella-Gutierrez et al., 2009). Best-fit models of evolution and ML analysis were performed using MEGA software ver. 6.06 (Tamura et al., 2013). Bootstrap values were obtained from 500 resampling sequence alignments.

Results

Functional annotation and separation of the host and symbiont sequences

Sequencing of the cDNA libraries of Heliopora coerulea yielded a total of 34,202,929 Illumina, 150-bp, paired-end reads. Raw sequence data were submitted to the DNA Data Bank of Japan (DDBJ) Sequence Read Archive under accession number DRA005137. After trimming the adapter and low-quality sequences, the remaining reads were assembled into 169,850 contigs by Trinity (Grabherr et al., 2011; Table 1), and ORFs were extracted from 106,750 assembled contigs (62.8% of the total contig) by TransDecoder (Fig. 2A). In the extracted ORFs, 90,309 assembled contigs (53.2%) were clustered sequences (Table 2; Fig. 2A).

Because total RNA was extracted from an adult H. coerulea colony, the host and symbiont transcripts were mixed in the transcriptome assembly. The host and symbiont-derived sequences were assigned to 23,037 (13.6%) and 52,544 contigs (30.9%), respectively (Table 2; Fig. 2A). In the host-derived sequences, 90.2% were similar to Corallium rubrum (20,785 contigs), 5.1% to Acropora digitifera (1172 contigs), 2.9% to Nematostella vectensis (668 contigs), and 1.8% to Hydra vulgaris (412 contigs; Fig. 2A). The symbiont-derived sequences were 24.6% of Symbiodinium minutum clade B (12,917 contigs), 66.7% of Symbiodinium spp. clade C1 (35,052 contigs), and 8.7% of Symbiodinium spp. clade C15 (4575 contigs; Fig. 2A). The protein identity of each organism in the top1 hit was distributed from 24% to 100% in Corallium rubrum, from 22.3% to 90.1% in Acropora digitifera, from 20.4% to 98.5% in Nematostella vectensis, from 22.3% to 69.2% in Hydra vulgaris, from 22.5% to 100% in Symbiodinium minutum clade B, from 94.8% to 100% in Symbiodinium spp. clade C1, and from 62% to 100% in Symbiodinium spp. clade C15 (Fig. 2B). The GC content of the host-derived sequences, in most cases, existed in about 40%, while GC content of the symbiont was in about 54% (Fig. 2C).

A total of 49,670 (55.0%) and 48,671 (53.9%) of all contigs showed significant similarities (e-value [less than or equal to] [le.sup.-5]) to protein sequences in the nr and UniRef90 databases, respectively (Table 2). The annotated contig of the host-derived sequences was 19,737 (85.7% in nr) and 19,131 (83.0% in UniRef90); the annotated contig of the symbiont was 28,504 (54.2% in nr) and 28,162 (53.6% in UniRef90; Table 2). A total of 4246 GO numbers were assigned to 25,911 contigs (28.7%), and 856 Enzyme Commission (EC) numbers were assigned to 8735 contigs (9.7%) from the GO numbers (Table 2). In the protein domain search analysis, 5703 domains were found in 38,922 ORFs (43.1%; Table 2). All contigs assigned to the host- and symbiont-derived sequences were submitted to the DDBJ Transcriptome Shotgun Assembly database under accession numbers IABP01000001-IABP01075583. The results of homology and domain search are shown in Supplementary Table 1 (view online).

Classification of Symbiodinium clades using chloroplast large subunit ribosomal DNA

One cp23S gene sequence (c36813_gl_il; accession no. IABP01075583) of Symbiodinium in Heliopora caerulea was obtained from the transcript assembly. Using 462 bp of the DNA alignment, a ML tree was calculated based on the Tamura 3-parameter model (Tamura. 1992; Fig. 3). A discrete gamma distribution was applied to model evolutionary rate differences among sites (5 categories [+G, parameter =0.3044]). The cp23S gene of the H. caerulea symbiont formed a clade with clade C of Symbiodinium (bootstrap value of 99%; Fig. 3).

Identification of enzymes for heme synthesis pathway and degradation

Forty-two contigs related to heme synthesis and degradation were identified from the assigned EC numbers, pfam ID, and annotated description (Fig. 1; Supplementary Tables 1 and 2, view online). Within the ALA synthesis pathways, the host expressed ALA synthase, while the symbiont expressed glutamyl-tRNA synthetase, glutamyl-tRNA reductase, and glutamate-1-semialdehyde 2, 1-aminomutase (Fig. 1; Supplementary Table 2, view online). All enzymes synthesizing all intermediates from ALA to protoheme were identified from the host and symbiont (Fig. 1 ; Supplementary Table 2, view online).

For heme degradation, three host proteins and one symbiont protein were identified as heme oxygenase (HO) (Fig. 1 ; Supplementary Table 2, view online). The three host HOs, c49658_gl_il, c50650_gl_i2, and c79749_g1_il, were designated hcHO-1, hcHO-2, and hcHO-3, respectively. Even though hcHO-3 was not a focus of this study, the amino acid sequence of hcHO-3 was too short for comparison of the phylogenetic positions and the active site. The ML tree of HO, using 186 amino acid lengths in the alignment, was calculated based on the Le and Gascuel (2008) model. A discrete gamma distribution was applied to model evolutionary rate differences among sites (5 categories [+G, parameter =1.4035]). The ML tree showed that two HOs of Heliopora caerulea were separated into two clades (bootstrap value of 100%; clades 1 and 2 in Fig. 4). Host heme oxygenase hcHO-2 formed a clade with Hexacorallia (bootstrap value of 44%; see white arrowhead in clade 1 of Fig. 4) and with teleost fishes and Homo sapiens (bootstrap value of 100%; clade 1 in Fig. 4); hcHO-1 formed a clade with Ciona intestinalis and Capitella teleta (bootstrap value of 59%; see black arrowhead in clade 2 of Fig. 4).

The amino acid residue at the 140th position (Asp 140) in human HO-1 is important for catalytic activity (Schuller et al., 1999; Lightning et al., 2001). The amino acid residue at the 140th position in hcHO-1 was Leu; however, it was Asp (Fig. 5) at the 140th position in hcHO-2 and symbiont (c9041_gl_il).

Discussion

In this study, we first conducted a metatranscriptome analysis of the Heliopora coerulea holobiont, and then separated the host- and their symbiont-derived sequences. We successfully identified the heme synthesis and degradation enzymes from the host and symbiont-expressed genes. We speculate that the lack of biliverdin reductase and a mechanism to accumulate biliverdin may be the reason for the blue coloration of the skeleton of H. coerulea.

We separated transcript sequences into host- and symbiont-derived sequences confidently based on a homology search in the seven databases. The GC content of these sequences indicated two major peaks, about 40% and 54% (Fig. 2C), further confirming the reliability of sequence separation. In the Pontes australiensis holobiont, two GC content peaks of approximately 40% and 53% were detected as the host- and symbiont-derived transcript sequences, respectively (Shinzato et al., 2014). In addition, GC content of the exons in the genome of Acropora digitifera was about 39% (Shinzato et al., 2011), while that of Symbiodinium minutum was 51% (Sho-guchi et al., 2013). Thus, the GC contents of the two separated sequences derived from the host Heliopora coerulea and the symbiont were similar to those of other hard-coral species.

Although 23,037 contigs of host-derived sequences accounted for 13.6% of all transcripts (Fig. 2A), greater than 83% of host transcripts showed significant similarities (e-value[less than or equal to] [le.sup.)-5] to protein in the nr and UniRef90 databases (Table 2). In the genome of Acropora digitifera and Nema-tostella vectensis, the numbers of predicted genes were 23,668 (Shinzato et al., 2011) and 27.273 (Putnam et al., 2007), respectively. The 26,658 sequences in Pontes australiensis were predicted as host transcripts (Shinzato et al., 2014). Therefore, we believe that detection of the expressed genes in Heliopora caerulea reached a threshold level. Conversely. the symbiont-derived sequences accounted for 30.9% of all transcripts, which was twice as many as what was identified in the host-derived sequences (Fig. 2A). However, in contrast to the host-derived sequences, approximately 54% of the symbiont-derived sequences showed significant similarities (e-value [less than or equal to] [le.sup.-5]) to protein in the nr and UniRef90 databases (Table 2). Although a domain search conducted by pfam detected the domain region in the symbiont-derived sequences, which showed no significant similarities to protein in either database (Supplementary Table 1, view online), it was suggested that the functional proteins that have no similarities to known proteins were probably included in these sequences.

[FIGURE 4 OMITTED]
Figure 5. Alignment of the active site in heme oxygenase (HO).
Accession numbers are shown in parentheses. Proteins with an asterisk
were retrieved from websites cited in (*) (Shinzato et al., 2011):
(**) (Traylor-Knowles et al., 2011): and (***) (Pratlong et al., 2015).

                                                    140

Homo sapiens HO-1 (P09601)                        G  D  L  S  G  G
Homo sapiens HO-2 (P30519)                        G  D  L  S  G  G
Ciona intestinalis (XP 009857539)                 A  I  F  S  G  G
Capitella teleta (ELU13716)                       A  I  L  A  G  G
Capitella teleta (ELU15215)                       A  I  T  G  G  G
Heliopora coerulea (hcHO-1 )                      G  L  M  A  G  G
Heliopora coerulea (hcHO-2)                       G  D  L  S  G  G
Corallium rubrum (Contig 21997 (***))             G  D  L  S  G  G
Corallium rubrum (Contig_31015 (***))             A  L  M  A  G  G
Corallium rubrum (Contig 18303 (***))             A  L  M  A  G  G
Acropora digirifera (aug_v2a.01584.t1 (*))        G  D  L  S  G  G
Pocillopora damicornis (bu 91849.1 C60182 (**))   G  D  L  S  G  G
Nematostella vectensis (XP 001629679)             G  D  L  S  G  G
Symbiodinium minutum (symbB.v1 .2.032139.t1 (*))  G  D  L  S  G  G
Symbiodinium sp. in H. coerulea (c9041_g1_i1)     G  D  L  S  G  G


A total of 66.7% of symbiont-derived sequences were homologous to proteins of clade C1 in Symbiodinium spp., and the identity of amino acid sequences was over 94.8% (Fig. 2B). In the transcript assembly, a contig of the internal transcribed spacer 2 (ITS2) locus was isolated and was homologous to clade C of Symbiodinium spp. (c24746_g1_i1; accession no. IABP01075582); and the ML tree of cp23S transcript in H. coerulea reliably formed a clade with clade C of Symbiodinium (Fig. 3). Therefore, H. coerulea probably harbors clade C of Symbiodinium spp.

All genes related to heme synthesis were found in the transcripts of Heliopora coerulea and their symbiont, suggesting that the H. coerulea holobiont can synthesize heme. In ALA synthesis, the host expressed ALA synthase and the symbiont expressed glutamyl-tRNA synthetase, glutamyl-tRNA reductase, and glutamate-1-semialdehyde 2.1-aminomutase, which clarified the finding that the host and symbiont had Shemin and C5 pathways, respectively. Heme oxygenase, which was also found in the transcripts of the host and symbiont, is important for producing biliverdin. Therefore, the functional phylogenetic positions of these HOs were compared to those of HOs from various animal species (Fig. 4). Host heme oxygenase hcHO-2 formed a clade with human HOs, the activities of which have been measured (Davydov et al., 2016), while hcHO-1 formed another clade (Fig. 4). It was suggested that the function of hcHO-1 possibly differed from that of hcHO-2 because the 140th amino acid residue in hcHO-1 was replaced by Leu (Fig. 5). Human HO-1, when replaced by Leu 140, did not form biliverdin, and it had peroxidase activity (Lightning et al., 2001). Thus, hcHO-1 may have peroxidase activity rather than biliverdin-forming activity, while hcHO-2 and symbiont HO (c9041_g1_i1) may play a role in biliverdin formation and are expressed in response to heat and oxidative stress (Maines, 1988; Fang et al., 1997).

Biliverdin reductase, which reduces biliverdin to bilirubin, was not identified in the host or the symbiont transcripts (Fig. 1). However, transcriptome analysis of the Heliopora coerulea holobiont was conducted in only a single colony and from a single time point. Therefore, it is possible that biliverdin reductase was not identified because the gene repertoire was incomplete. In the case of the transcriptome analysis of Corallium rubrum (Pratlong et al., 2015), 12 individuals at two locations were sampled and analyzed. However, in the present study the biliverdin reductase family region (pfam ID: Biliv-reduc_cat, PF09166) was not identified in the C. rubrum transcripts by the pfam search, even though all enzymes for heme synthesis and HOs were found by homology search against Heliopora coerulea (Fig. 1; Supplementary Table 2. view online). This result suggested that heme synthesis and degradation probably occurred; however, biliverdin may not be decomposed in Octocorallia, and only H. coerulea can accumulate biliverdin in its skeleton. If so, comparison of skeletal proteins in Octocorallia should be made.

To clarify whether Heliopora coerulea can produce the blue pigment, biliverdin IX[alpha], we first examined a metatran-scriptome analysis of the H. coerulea holobiont and isolated genes related to heme synthesis and degradation. Our findings suggested that H. coerulea can synthesize biliverdin IX[alpha]. However, the accumulation mechanism of biliverdin IX[alpha] in its skeleton still needs to be elucidated. The transcript information of the H. coerulea holobiont will advance our understanding of the mechanism of blue pigment synthesis, and will help answer why it is that only Heliopora coerulea retains a unique, blue, hard crystalline aragonite skeleton in the skeletal evolution of Octocorallia.

Acknowledgments

We are grateful to Mr. Mitsuhiro Ueno for sample collection. We also thank Dr. Issei Nishiki for helping with the NGS experiment. Computations were partially performed on the NIG supercomputer at ROIS National Institute of Genetics. The present study was supported by the Environment Research and Technology Development Fund (4-1304) of the Ministry of the Environment, Japan.

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YUKI HONGO (1,*), NINA YASUDA (2) AND SATOSHI NAGAI (1)

(1) Research Center for Bioinformatics and Biosciences, National Research Institute of Fisheries Science, Japan Fisheries Research and Education Agency, 2-12-4 Fukuura, Kanazawa, Yokohama, Kanagawa 236-8648, Japan; and; (2) Organization for Promotion of Tenure Track, University of Miyazaki, 1-1 Gakuenkibanadai-Nishi, Miyazaki, Miyazaki 889-2192, Japan

Received 13 October 2016; Accepted 1 March 2017; Published online 15 May 2017.

(*) To whom correspondence should be addressed. E-mail: hongoy@affrc.go.jp

The authors declare they have no competing interests.

YH and SN conceived and designed the experiments; YH performed all experiments. YH. NY. and SN analyzed all data. All of the data and analyzed results were examined carefully and discussed by all authors. All authors have read and approved the final manuscript.

Abbreviations: ALA, 5-aminolevulinic acid; contig (from contiguous), a group of DNA segments that overlap and. as one, depict a consensus region of DNA; DDBJ, DNA Data Bank of Japan; GO, gene ontology; hcHO-1, 2, 3, three isoforms of heme oxygenase in Heliopora coerulea; HO. heme oxygenase; ROS. reactive oxygen species.
Table 1
Summary of sequence assembly

                               Total contig

Number of contigs                   169,850
Total bases (bp)                169,902,610
Longest contig length (bp)           25,008
Shortest contig length (bp)             201
Average of contig length (bp)         1.000
Median of contig length (bp)            571
N50                                   1,708
N90                                     382

bp, base pair; N50 and N90. minimum contig length such that the sum of
contigs of equal length or longer is at least 50% and 90% of the total
length of all contigs, respectively

Table 2
Summary of open reading frame (ORF) extraction, homology, and domain
search, and separation of the host and symbiont sequences

                  Extracted  Blast hit   Blast hit against
                    ORFs     against nr      UniRef90

All contigs        90,309      49,670         48,671
Host contigs       23,037      19,737         19,131
Symbiont contigs   52,544      28,504         28,162

                                    Domain against
                   GO nos.   EC nos.      pfamA

All contigs         25,911    8735        38,922
Host contigs      9884        2889        14,596
Symbiont contigs    15,264    5571        23,327

Contigs, a group of DNA segments that overlap and together depict a
consensus region of DNA; EC, Enzyme Commission; GO, gene ontology; nr.
NCBI's database of non-redundant proteins; ORFs. open reading frames;
pfamA, pfam protein domain database; UniRef90. database of clustered
sequences from UniProt Knowledgebase.
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Author:Hongo, Yuki; Yasuda, Nina; Nagai, Satoshi
Publication:The Biological Bulletin
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Date:Apr 1, 2017
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