Molecular pathology of skeletal growth anomalies in the brain coral Platygyra carnosa: A meta-transcriptomic analysis

MPB-08508; No of Pages 8 Marine Pollution Bulletin xxx (2017) xxx–xxx

Contents lists available at ScienceDirect

Marine Pollution Bulletin journal homepage: www.elsevier.com/locate/marpolbul

Molecular pathology of skeletal growth anomalies in the brain coral Platygyra carnosa: A meta-transcriptomic analysis Yu Zhang a,1, Jin Sun b,c,1, Huawei Mu b, Janice C.Y. Lun d, Jian-Wen Qiu b,⁎ a Shenzhen Key Laboratory of Marine Bioresource and Eco–environmental Science, Guangdong Engineering Research Center for Marine Algal Biotechnology, College of Life Sciences and Oceanography, Shenzhen University, Shenzhen, China b Department of Biology, Hong Kong Baptist University, Hong Kong, China c Division of Life Sciences, The Hong Kong University of Science and Technology, Hong Kong, China d Agriculture, Fisheries and Conservation Department, The Government of the Hong Kong Special Administrative Region, China

a r t i c l e

i n f o

Article history: Received 24 September 2016 Received in revised form 17 March 2017 Accepted 22 March 2017 Available online xxxx Keywords: Coral Coral disease Coral health Coral tumor Gene expression Transcriptome

a b s t r a c t Coral skeletal growth anomaly (GA) is a common coral disease. Although extensive ecological characterizations of coral GA have been performed, the molecular pathology of this disease remains largely unknown. We compared the meta-transcriptome of normal and GA-affected polyps of Platygyra carnosa using RNA-Seq. Approximately 50 million sequences were generated from four pairs of normal and GA-affected tissue samples. There were 109 differentially expressed genes (DEGs) in P. carnosa and 31 DEGs in the coral symbiont Symbiodinium sp. These differentially expressed host genes were enriched in GO terms related to osteogenesis and oncogenesis. There were several differentially expressed immune genes, indicating the presence of both bacteria and viruses in GA-affected tissues. The differentially expressed Symbiodinium genes were enriched in reproduction, nitrogen metabolism and pigment formation, indicating that GA affects the physiology of the symbiont. Our results have provided new insights into the molecular pathology of coral GA. © 2017 Elsevier Ltd. All rights reserved.

1. Introduction Coral skeletal growth anomaly (GA) is a common coral disease discovered in a wide variety of coral species, including Platygyra pini and Platygyra sinensis in the Great Barrier Reef (Loya et al., 1984), Montipora informis (Yamashiro et al., 2000; Yamashiroa et al., 2001; Yasuda and Hidaka, 2012), Porites australiensis (Yasuda and Hidaka, 2012), and Acropora cytherea (Irikawa et al., 2011) in Okinawa; Pavona clavus in Costa Rica (Gateño et al., 2003); and Montipora capitata (Burns and Takabayashi, 2011) and Porites compressa (Breitbart et al., 2005; Domart-Coulon et al., 2006; Stimson, 2011) in Hawaii. They are characterized by an increased polyp growth rate, resulting in rough circular protuberances in the affected area of the colony (Loya et al., 1984). Coral GA is sometimes referred to as “coral tumor” due to its apparent similarity with the abnormal cell growth in mammalian neoplasm. However, the use of “coral tumor” is arguable given that a previous study did not show evidence of molecular and cellular oncogenesis in corals (Spies and Takabayashi, 2013). Coral GA has several common features. The colonies affected by GA are usually found in the shallow water where photo-oxidative and thermal stresses are high (Domart-Coulon et al., 2006; Stimson, 2011). On ⁎ Corresponding author. E-mail address: [email protected] (J.-W. Qiu). 1 These authors contributed equally to this study.

unit area basis, GA-affected tissues usually have fewer symbiotic zooxanthellae (Gateño et al., 2003) and fewer polyps compared to normal tissues (Work et al., 2008). The polyps in GA-affected tissues are enlarged and contain more somatic cells than adjacent tissues (Work and Rameyer, 2005). The skeleton of GA-affected corals is very active in vertical growth, with unchanged calcification rate and enlarged gastrovascular canal system (Domart-Coulon et al., 2006). Moreover, GAaffected coral populations usually have low fecundity and are more vulnerable to environmental stress, as compared to their normal counterparts (Stimson, 2011; Yasuda and Hidaka, 2012). Previous studies have revealed that the prevalence of coral GA is positively correlated with local/regional human population density, indicating that anthropogenic activities are a potential inducer of GA (Aeby et al., 2011). In addition, coral GA and bleaching are related, as bleached Porites colonies have a higher percent occurrence of GA than non-bleached colonies (McClanahan et al., 2009). Therefore, multiple human and climate related stressors could interact to affect the development of coral GA. Although many histological and ecological studies have been conducted to characterize coral GA, only in few studies of few coral species has the molecular pathology of GA been examined. For example, Domart-Coulon et al. (2006) used ELISA and found overexpression of 4 stress-related proteins (MutY, HSP90a1, GRP75 and metallothionein) in the GA-affected tissues compared to adjacent apparently normal and reference tissues of Porites compressa. Yasuda and Hidaka (2012) used TUNEL and BrdU assays and found that apoptotic

http://dx.doi.org/10.1016/j.marpolbul.2017.03.047 0025-326X/© 2017 Elsevier Ltd. All rights reserved.

Please cite this article as: Zhang, Y., et al., Molecular pathology of skeletal growth anomalies in the brain coral Platygyra carnosa: A metatranscriptomic analysis, Marine Pollution Bulletin (2017), http://dx.doi.org/10.1016/j.marpolbul.2017.03.047

2

Y. Zhang et al. / Marine Pollution Bulletin xxx (2017) xxx–xxx

pathways were suppressed and cell proliferation was promoted in GAaffected corals. Spies and Takabayashi (2013) examined the expression of five genes including three (Galaxin, murine double minute 2, tumor necrosis factor, tyrosine protein kinase and βγ-crystallin) that are associated with oncogenesis in human in GA-affected M. capitata using quantitative reverse transcriptase PCR. They found that the expression patterns of these coral genes were inconsistent with the expression patterns of homologous genes in human neoplastic diseases showing similar morphological symptoms, indicating that calcification is likely not enhanced in coral GA and that coral GA is not a malignant neoplasm. These studies have provided some insights into the molecular pathology of coral GAs, but lacked the throughput required to systematically investigate this complex biological process, which are likely mediated by multiple genes and metabolic pathways. The only large-scale comparison of normal and GA-affected tissues is Frazier (2016), which revealed consistent expression patterns between several M. capitata genes (i.e., tumor necrosis factor receptor-associated factors 3, 5 and 6, deleted in malignant brain tumors 1, and bone morphogenetic protein 1) and their homologous genes in human neoplastic diseases, indicating that coral GA is a malignant neoplasm. Platygyra carnosa, previous incorrectly written as Platygyra carnosus without considering the need for agreement in gender between genus and species names in a few papers (e.g., Chan et al., 2005; Chiu et al., 2012; Dumont et al., 2013; Wong et al., 2016), is a common species along the coasts of the South China Sea (Veron, 2000). In southern China including Hong Kong, this species is a dominant structure-building massive coral (Qiu et al., 2014), which can grow to 2 m above volcanic rocks in protected bays. Skeletal GA is common in Platygyra carnosa in Hong Kong, and previous studies have characterized the composition of bacterial communities associated with the GA-affected and apparently healthy tissues (Chiu et al., 2012; Ng et al., 2015). In a previous study, we sequenced the transcriptome of P. carnosa, with the aim of generating a high-coverage genomic database (PcarnBase, http://www.comp. hkbu.edu.hk/~db/PcarnBase/index.php) for future analysis of molecular mechanisms of coral diseases and stress responses in this species (Sun et al., 2013), which is among a clade of scleractinian corals with very few genomic resources. In the present study, we took advantage of PcarnBase, and used a different set of samples to compare the GA-affected and apparently normal tissues of P. carnosa at the gene expression level for the first time. We analyzed the gene expression by transcriptome sequencing using comparative RNA-Seq, a high throughput technique that has been used in studies of genome-wide coral gene expression in recent years (e.g., Meyer et al., 2011; Barshis et al., 2013, 2014; Mayfield et al., 2014; Daniels et al., 2015; Wright et al., 2015; Mayfield et al., 2016). Our purpose was to obtain a systematic view of the complex network of genes that might be involved in GA formation of P. carnosa. 2. Material and methods 2.1. Sample collection Platygyra carnosa was collected from Sharp Island, Hong Kong (22.366°N, 114.299°E) by SCUBA from water depths of 2–3 m on 19 July 2012 at around 11 am. Several environmental factors obtained from PM3, a government water quality monitoring site located in the middle of Port Shelter (EPD, 2013), approximately 1 km of the coral sampling site, are listed as follows: 6.3 mg/L (dissolved oxygen), 31.6 psu (salinity), 26.7 °C (temperature), 1.7 NTU (turbidity), 0.31 mg/L (total nitrogen), and 0.02 mg/L (total phosphorous) (data collected on 28 July 2012). Coral samples were collected from four colonies of P. carnosa, with one sample of GA-affected tissue and one sample of apparently healthy tissue from each colony (Fig. 1). The samples, each containing three to five polyps, were collected after hammering a steel core into roughly 2 cm of the skeleton, and immediately transported to a boat by divers and frozen using dry ice in the field. Within 2 h,

the samples were transported to Hong Kong Baptist University and stored at −80 °C until further analysis. 2.2. RNA extraction, cDNA synthesis and Illumina sequencing The Platygyra carnosa polyps with tissue and skeleton were frozen using liquid nitrogen and ground into powder using a pre-chilled mortar and pestle. TRIzol® reagent (Invitrogen, CA, USA) was then added to extract the RNA following Sun et al. (2013). Contaminating DNA was removed using Turbo DNA-free kit (Ambion, Austin, TX, USA). RNA quality and quantity were assessed using a Bioanalyzer 2100 (Agilent Technologies, CA, USA). The library was constructed using a TruSeq DNA sample preparation kit (Illumina). In brief, approximately 20 μg of total RNA from each sample was used for mRNA enrichment using the PolyATract® mRNA Isolation System (Ambion, Austin, TX, USA), which targets the 3′ poly(A) + region present in mature eukaryotic mRNAs. Short mRNA fragments (approximately 200 bp) were generated by mixing the mRNA with a fragmentation buffer. Randomized hexamer– primers were used to synthesize the first strand cDNA, and a buffer consisting of dNTPs, RNase H and DNA polymerase I was then used to synthesize the second strand. Short fragments were purified using QiaQuick polymerase chain reaction (PCR) extraction kit (Qiagen, Valencia, CA, USA). Sequencing adaptors were ligated to the end of the cDNA after end reparation. The cDNA was sequentially amplified by PCR and single-ended sequenced on half lane of an Illumina HiSeq2000 (Illumina, San Diego, CA, USA). 2.3. Bioinformatics analyses A homology based method was used to distinguish the transcripts from Platygyra carnosa and its dinoflagellate endosymbionts (Symbiodinium). First, protein sequences of Acropora digitifera, the only species of coral whose whole genome has been sequenced, were downloaded according to Shinzato et al. (2011). The deduced protein sequences of Symbiodinium transcriptome (Bayer et al., 2012), the protein sequences of Symbiodinium minutum (Shoguchi et al., 2013), and the protein sequences of Symbiodinium kawagutii (Lin et al., 2015) were also downloaded. The A. digitifera protein sequences and the deduced Symbiodinium protein sequences were appended to the downloaded sequences from NCBI's non-redundant protein database. BLASTx was performed to search our P. carnosa transcripts (Sun et al., 2013) against this new database with an E-value threshold of 1e-5. Only sequences having the best match to a metazoan protein, including both the A. digitifera and other metazoan sequences from NCBI, were considered as real P. carnosa transcripts. Sequences having the best match to a Symbiodinium protein were considered as Symbiodinium transcripts. The remaining transcripts were loosely grouped as “Others”, which possibly were of bacterial, fungi, and viral origins. Reads were cleaned to remove adaptors and low quality reads using Trimmomatic v0.33 (Bolger et al., 2014) with the settings of “LEADING:15, TRAILING:15, SLIDINGWINDOW:4:20, and MINLEN:40”. The clean reads were deposited in NCBI's Sequence Read Archive (http:// www.ncbi.nlm.nih.gov/Traces/sra) with accession number SRP087599. Gene quantification was performed by using kallisto (Bray et al., 2016; Sun et al., 2017) against the classified references of the P. carnosa and Symbiodinium transcripts, respectively. The bootstrap value was set as 100, and the estimated average length and standard deviation of the RNA-Seq library insert size was set as 200 bp and 20 bp, respectively. Only transcripts supported by at least 10 reads per million in at least one sample were kept (Meyer et al., 2011). An inverted beta-binomial (ibb) test specifically designed for paired samples was used to identify the differentially expressed genes (DEGs) between normal and GA samples (Pham and Jimenez, 2012), with the P-values adjusted using a false discovery rate cut off of 0.1 (Daniels et al., 2015). In the ibb-test, genes that were specifically expressed in one group only (GA polyps or healthy polyps) had a fold change of “100,000” or “−100,000”.

Please cite this article as: Zhang, Y., et al., Molecular pathology of skeletal growth anomalies in the brain coral Platygyra carnosa: A metatranscriptomic analysis, Marine Pollution Bulletin (2017), http://dx.doi.org/10.1016/j.marpolbul.2017.03.047

Y. Zhang et al. / Marine Pollution Bulletin xxx (2017) xxx–xxx

3

Fig. 1. Platygyra carnosa. Photographs showing a colony with skeletal growth anomaly (GA) affected area outlined by a red dash line (a), vertical profile of normal polyps (b), and GA affected polyps (c). Scale for (b) and (c): 0.5 mm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

To identify the dinoflagellate endosymbionts in the coral samples, the Illumina sequences from each sample was assembled separately using Trinity (version: r20131110) (Haas et al., 2013) with default settings. Three common marker genes that were used for Symbiodinium clade identification, internal transcribed spacer region of nuclear rRNA (ITS), chloroplast 23S rRNA (cp23S), and cytochrome c oxidase I (COX1) (Pochon et al., 2012), were searched against the assembled transcriptome of each sample. The DEGs were categorized and plotted using WEGO (Web Gene Ontology Annotation Plot, http://wego.genomics.org.cn) with default settings. In addition, the DEGs were analyzed for GO term enrichment using GOEAST (Gene Ontology Enrichment Analysis Software Toolkit, http://omicslab.genetics.ac.cn/GOEAST/index. php) with the Hypergeometric Statistical Test method. GO categories with P-value b 0.01 were considered significant (Zheng and Wang, 2008). 2.4. Real-time PCR on selected genes Total RNA from normal and GA-affected coral polyps of another three colonies was extracted using TRIzol reagent as described (Sun et al., 2013). DNA was removed using a Turbo DNA-free kit (Ambion, Austin, TX, USA). After quality check by using agarose gel electrophoresis, cDNA was synthesized using M-MLV reverse transcriptase (USB, Cleveland, OH, USA) and randomized hexamer–primer. Eight DEGs were

randomly chosen to determine the correspondence between gene expression measured by RNA-Seq and qPCR. Primers for qPCR were designed using Primer Premier 5 (Supplementary Table S1). Real-time PCR was performed in triplicate on a MxPro-Mx3000P machine (Agilent) using the iTaq™ Fast SYBR Green Supermix kit (Bio-Rad). A one-cycle melting step was used to check the specificity of the primers against the target genes. An actin gene (Unigene46441_Mix) was used as the internal standard because its expression level remained almost unchanged between the two treatments (fold change ± S.D. = 1.04 ± 0.13). Quantification was based on the 2−ΔΔCT method (Livak and Schmittgen, 2001).

Table 1 Overall statistics of sequence data, and number of reads (unit: million reads) mapped to the coral Platygyra carnosa and dinoflagellate Symbiodinium sp. in four pairs of normal (N) and skeletal tissue anomaly (STA) affected tissue samples. Sequences

Raw reads Clean reads Mapped reads (coral) Mapped reads (algae)

Healthy

Diseased

N1

N2

N3

N4

STA1

STA2

STA3

STA4

6.17 5.92 2.11 1.39

6.23 5.98 1.61 2.06

6.06 5.83 1.81 1.74

6.05 5.70 1.69 1.92

5.93 5.69 1.64 1.63

6.23 6.02 1.93 1.62

6.16 5.93 1.72 2.00

5.84 5.50 1.47 1.83

Please cite this article as: Zhang, Y., et al., Molecular pathology of skeletal growth anomalies in the brain coral Platygyra carnosa: A metatranscriptomic analysis, Marine Pollution Bulletin (2017), http://dx.doi.org/10.1016/j.marpolbul.2017.03.047

4

Y. Zhang et al. / Marine Pollution Bulletin xxx (2017) xxx–xxx

3. Results The Illumina sequencing run generated approximately 6 million reads per sample (Table 1). Mapping to the databases led to the discovery of 18,271 P. carnosa genes (Supplementary Table S2), and 24,328 Symbiodinium genes (Supplementary Table S3). Among the coral genes, 109 (ca. 0.59%) were significantly differentially expressed between GA-affected and normal tissues, with 61 genes being up-regulated and 48 genes being down-regulated. There was significant positive correlation between the expression levels determined by RNA-Seq and qPCR (r = 0.853, P b 0.001) (Supplementary Table S1). The WEGO analysis showed that the 109 DEGs of Platygyra carnosa were assigned to biological process (BP), cellular component (CC), and molecular function (MF), with more genes involved in BP than the other two GO categories (Fig. 2), which was probably due to a bias in GO databases which have more annotated genes for BP than CC or MF. The BP was dominated by cellular process, metabolic process/macromolecule metabolic process and developmental process. The MF was dominated by binding and catalytic activities. The CC was dominated by cell and cell part (Fig. 2). The GOEAST analysis showed that 32 GO terms were enriched (Table 2). Among them were GO terms associated with cell differentiation (GO:0030154), osteoblast differentiation (GO:0001649), ossification (GO:0001503), branching morphogenesis of an epithelial tube (GO: 0048754), regulation of tissue remodeling (GO:0034103) and sterol metabolic process (GO:0016125). Among the 24,328 Symbiodinium genes, 31 (ca. 0.13%) were significantly differentially expressed between GA-affected and normal tissues (Supplementary Table S2). Of these DEGs in Symbiodinium, 16 genes were up-regulated and 15 genes were down-regulated. The WEGO

analysis showed that the dominant BPs were cellular process, metabolic process, developmental process and reproductive process. The MF was dominated by binding and catalytic and the CC was dominated by cell and cell part (Fig. 3). An GOEAST analysis of the 31 Symbiodinium DEGs showed that only five GO cellular components were enriched, which included two related to reproduction (GO:0000003, 0022414), two related to organic nitrogen metabolism (GO:000678, 1901565), and one related to metabolism of plant pigments (GO:0033013) (Table 2). Transcripts assembled using Trinity for each sample were analyzed for similarity with the ITS, cp23S and COX1 gene sequences of various Symbiodinium strains. The results showed that the Symbiodinium sequences from all of the eight samples had the highest similarity (96.2% to 99.4%) to Symbiodinium clade C1, which is in line with a recent genotyping investigation showing that all of the 10 P. carnosa colonies examined in Hong Kong waters in that study hosted this genotype (Wong et al., 2016). 4. Discussion Our comparative analysis showed that there were substantial differences in gene expression between the healthy and GA-affected tissues in both the P. carnosa and Symbiodinium compartments of the symbiotic relationship. Nevertheless, only a small percentage of genes were differentially expressed (i.e., 109 in the coral host and 31 in the symbiont), and there were much fewer genes differentially expressed in Symbiodinium sp. than in its coral host, which is consistent with a few studies showing coral diseases caused more DEGs in the host than in the algal symbiont (Libro et al., 2013; Barshis et al., 2014; Daniels et

Fig. 2. Gene Ontology (GO) distribution of annotated differentially expressed coral genes between normal and GA-affected tissues of Platygyra carnosa. The categorization was based on their biological process, cellular component and molecular function. The upper and lower abscissa indicates the percentage and the number of annotated genes, respectively.

Please cite this article as: Zhang, Y., et al., Molecular pathology of skeletal growth anomalies in the brain coral Platygyra carnosa: A metatranscriptomic analysis, Marine Pollution Bulletin (2017), http://dx.doi.org/10.1016/j.marpolbul.2017.03.047

Y. Zhang et al. / Marine Pollution Bulletin xxx (2017) xxx–xxx

5

Table 2 Significantly enriched Gene Ontology (GO) biological process terms for differentially expressed genes in Platygyra carnosa and Symbiodinium. GO ID

Term

Level

No. genes in gene group

No. genes in transcriptome

P value

Platygyra carnosa GO:1901615 GO:0003002 GO:0007389 GO:0030326 GO:0035113 GO:0007369 GO:0044093 GO:0001667 GO:0016125 GO:0001503 GO:0001649 GO:0030154 GO:0001763 GO:0048754 GO:0060429 GO:0061138 GO:0002791 GO:0010817 GO:0032880 GO:0046883 GO:0050708 GO:0050796 GO:0051046 GO:0051223 GO:0070201 GO:0090087 GO:0090276 GO:1,903,530 GO:0019827 GO:0098727 GO:0009725 GO:0034103

Organic hydroxy compound metabolic process Regionalization Pattern specification process Embryonic limb morphogenesis Embryonic appendage morphogenesis Gastrulation positive regulation of molecular function Ameboidal-type cell migration Sterol metabolic process Ossification Osteoblast differentiation Cell differentiation Morphogenesis of a branching structure Branching morphogenesis of an epithelial tube Epithelium development Morphogenesis of a branching epithelium Regulation of peptide secretion Regulation of hormone levels Regulation of protein localization Regulation of hormone secretion Regulation of protein secretion Regulation of insulin secretion Regulation of secretion Regulation of protein transport Regulation of establishment of protein localization Regulation of peptide transport Regulation of peptide hormone secretion Regulation of secretion by cell Stem cell population maintenance Maintenance of cell number Response to hormone Regulation of tissue remodeling

1 5 5 6 6 5 1 3 4 2 3 2 4 6 1 5 3 1 2 5 4 6 2 3 2 2 6 3 4 1 2 2

3 3 3 2 2 3 3 2 2 2 2 6 2 2 4 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 3 2

39 57 73 21 21 33 67 17 14 17 6 288 18 15 103 18 8 23 18 8 8 8 13 16 16 8 8 12 6 6 74 3

0.0016 0.0048 0.0095 0.0069 0.0069 0.0010 0.0075 0.0045 0.0031 0.0045 0.0005 0.0076 0.0051 0.0035 0.0034 0.0051 0.0010 0.0082 0.0051 0.0010 0.0010 0.0010 0.0026 0.0040 0.0040 0.0010 0.0010 0.0022 0.0005 0.0005 0.0099 0.0001

Symbiodinium GO:0000003 GO:0022414 GO:0006778 GO:0033013 GO:1901565

Reproduction Reproductive process Porphyrin-containing compound metabolic process Tetrapyrrole metabolic process Organonitrogen compound catabolic process

1 2 5 5 3

2 2 1 1 1

53 47 6 6 6

0.0021 0.0016 0.0076 0.0076 0.0076

al., 2015; Wright et al., 2015). It should be noted that, as in most RNASeq experiments, the few replicates might have resulted in a low statistical power to detect DEGs. In addition, while we aimed to control Type I error, applying false positive corrections in transcriptome-wide data could lead to some false negative results. Therefore, the number of DEGs detected in our study should be conservative. Nevertheless, our results clearly indicated that GA affects several important physiological processes in the symbiont. For instance, it is known that nitrogen limitation will slow down the transition from G1 to S phase in symbiotic dinoflagellates, thereby reducing their cell growth (Yellowlees et al., 2008). The enrichment of two GO terms related to organic nitrogen metabolism in Symbiodinium in our study thus indicates disruption of nitrogen metabolism in the symbiont, which may have consequences on its growth. Indeed, the enrichment of two reproduction related GO terms among the Symbiodinium DEGs in P. carnosa indicates that GA affects the reproduction in the symbiont, which is consistent with the result of a study showing GA-affected coral tissue had a lower density of Symbiodinium cells than the healthy tissue (Burns et al., 2013). In addition, enrichment of the tetrapyrrole metabolic process indicates the impact of GA on pigment production in the symbiont, which is consistent with the lighter color of GA-affected tissue when compared with the normal tissue. Since GA affected more coral host genes than symbiont genes, our discussion below is focused on the coral host. Among the host DEGs are GO terms related to osteogenesis, oncogenesis and pathogen attack, providing evidence to show the conserved pathways involved in bone pathology, tumor formation and pathogen defense between corals and mammals.

4.1. Osteogenesis related genes Two skeletal genesis related ontology terms, osteoblast differentiation (GO:001649) and ossification (GO:001503), were enriched in our GOEAST analysis, indicating the involvement of osteogenesis related genes in GA formation. Among the DEGs are five genes that have been reported to be associated with osteogenesis, including genes encoding low-density lipoprotein receptor related protein 5 (LRP5), bone morphogenetic protein 1 (BMP1), complement component C3 (C3), Rho guanine nucleotide exchange factor (GEF) and heparin sulfate proteoglycan (HSPG). Among them, two LRP5 genes were down-regulated in the GA-affected P. carnosa tissue. LRP5 plays a crucial role in bone homeostasis through regulating the Wnt/B-cathenin pathway as its malfunctioning can cause either the osteoporosis pesudoglioma syndrome (OPPG) or the high bone mass (HBM) kindred in humans (Lara-Castillo and Johnson, 2015), which is characterized by low and high bone density, respectively. In M. capitata, LRP5 expression was also down-regulated in the GA-affected tissue (Frazier, 2016), supporting the involvement of this gene in causing the reduced skeletal density in different coral species affected by GA (Yamashiro et al., 2000; Burns et al., 2013). BMP1 was detected in the normal tissue but not in GA-affected P. carnosa tissue. In humans, BMP1 regulates bone and cartilage development through modulating the normal assembly of extracellular matrix and normal cleavage of procollagen (Tabas et al., 1991). Knockdown of histone H3K9 acetyltransferase PCAF, a BMP activating factor, significantly reduced the bone formation (Zhang et al., 2016). In M. capitata, BMP1 expression was down-regulated in the GA-affected tissue

Please cite this article as: Zhang, Y., et al., Molecular pathology of skeletal growth anomalies in the brain coral Platygyra carnosa: A metatranscriptomic analysis, Marine Pollution Bulletin (2017), http://dx.doi.org/10.1016/j.marpolbul.2017.03.047

6

Y. Zhang et al. / Marine Pollution Bulletin xxx (2017) xxx–xxx

Fig. 3. Gene Ontology (GO) distribution of annotated differentially expressed dinoflagellate endosymbionts (Symbiodinium) genes between normal and GA-affected tissues of Platygyra carnosa. The categorization was based on their biological process, cellular component, and molecular function. The upper and lower indicates abscissa the percentage and the number of annotated genes, respectively.

(Frazier, 2016), indicating this gene is essential for the skeletal formation in different species of corals. HSPG has been reported to interact with fibroblast growth factor to control human osteogenesis (Jackson et al., 2006). Complement component C3 is significantly down-regulated in humans with osteoporosis (Kuo et al., 2014). GEF is a protein regulating the function of osteoblasts and osteoclasts, and knock out of this gene will lead to significantly reduced bone mass in mice (Huang et al., 2014). The differential expression of HSPG in GA-affected P. carnosa tissue indicates its active roles in GA formation. 4.2. Oncogenesis related genes Six P. carnosa DEGs have been reported to be associated with oncogenesis in mammals: fibroblast growth factor receptor (FGFR), deleted in malignant brain tumors 1 (DMBT1), Hedgehog (Hh), inositol hexakisphosphate kinase-2 (IP6K2), inositol-tetrakisphosphate kinase1 (IP4K1), and notum. FGFRs are a family of proteins which regulate a wide variety of biological processes, such as cell proliferation, survival and differentiation (Haugsten et al., 2010). Mutation of FGFR genes and imbalanced FGFR signaling have been reported to promote cancer development and progression in different parts of the human body (Pollock et al., 2007; Zhou et al., 2015). In P. carnosa, FGFR2 was only detected in the normal tissue, indicating down-regulation of FGFR2 expression could have contributed to the formation of coral skeletal anomalies. Deleted in malignant brain tumors 1 (DMBT1), is a glycoprotein containing multiple scavenger receptor cysteine-rich (SRCR) domains separated by SRCR-interspersed domains. DMBT1 has immune function, as

it can bind to pathogens and interacts with immune proteins (Ligtenberg et al., 2007). Loss of DMBT1 sequence in chromosome has been associated with the progression of human cancers, and tissues of various cancers have been found to show low expression of DMBT1 gene (Somerville et al., 1998; Wu et al., 1999; Mollenhauer et al., 2001). Consistent with the pattern in M. capitata detected by Frazier (2016), GA-affected P. carnosa tissue showed significant down-regulation of DMBT1, indicating the low activity of this gene is a sign of the disruption of the immune system and GA formation in both species of corals. The Hedgehog (Hh) pathway is an important pathway regulating repair response through compensatory proliferation. In FGFR3-deficient mice, inhibition of Hh signaling can attenuate chondroma-like lesion (osteochondromas) and basal cell carcinoma caused by FGFR3 deletion and the subsequent enhanced Hh expression (Zhou et al., 2015). Uncontrolled activation of Hh signaling was associated with oncogenesis (Pasca di and Hebrok, 2003). In P. carnosa, expression of Hh was significantly upregulated in the GA-affected tissue, indicating GA progression is accompanied by a high level of cell proliferation and low level of apoptosis – signs of high oncogenic activity, in agreement with a previous report showing increased proliferation and reduced apoptosis levels in growth anomalies of the scleractinian corals Porites australiensis and Montipora informis (Yasuda and Hidaka, 2012). Inositol hexakisphosphate kinase-2 (IP6K2) is a proapoptotic protein functioning through inhibiting the expression of prearrest gene targets (Koldobskiy et al., 2010). IP6K activity is increased during cell death, while silencing and knockout of IP6K2 decreases and diminishes cell death, respectively (Nagata et al., 2005). However, a more recent study showed that IP6K2 can function as a positive regulator of Hh signaling, and unregulated activation of Hh signaling is associated with the formation of several cancers (Sarmah and Wente, 2010). In the current study, expression level of IP6K2 in the GA-affected tissue of P. carnosa is much higher than that of the normal tissue, suggesting a high carcinogenesis-like activity. Nevertheless, in view of the diverse effects of IP6K2 in tumor formation, its exact function in coral GA should be further determined. Notum is an extracellular Wnt-specific deacylase and the only secreted feedback antagonist that suppresses Wnt signaling (Kakugawa et al., 2015). Down-regulation of Notum can activate the Wnt pathway, leading to the inhibition of cytochrome C release, activation of caspase and apoptosis, and consequently carcinogenesis in humans (PećinaŠlaus, 2010). Expression of Notum was down-regulated in the GA-affected tissue of P. carnosa, suggesting a higher Wnt activity and lower apoptotic level. Support for this hypothesis comes from the up-regulation of inositol-tetrakisphosphate, a caspase and apoptosis related gene that has been reported to be a negative regulator of apoptosis (Sun et al., 2003), indicating a lower apoptotic activity in tissues exhibiting GA. These results are in agreement with the above mentioned cellular kinetics in GA-affected tissue of scleractinian corals (Yasuda and Hidaka, 2012). 4.3. Pathogens of coral skeletal tissue anomalies Although little is known about the causes of coral skeletal growth anomaly (Burns et al., 2013), there is evidence that biotic factors, such as parasitic crustaceans (Grygier and Cairns, 1996), bacteria (DomartCoulon et al., 2006), and parasitic algae (Work et al., 2008) are involved the GA formation in a variety of coral species. A recent study showed that, the incidence of GA in Porites has stronger correlation with human population size than other predictor variables tested (Aeby et al., 2011). Based on the culture-based strain isolation methods and 16S rRNA analysis, Chiu et al. (2012) detected only minor bacterial community differences between normal and GA-affected tissues of Platygyra carnosa. Using a more sensitive 16S rRNA pyrosequencing technique, however, Ng et al. (2015) detected several potentially pathogenic bacteria that might be involved in the formation of GA in P. carnosa.

Please cite this article as: Zhang, Y., et al., Molecular pathology of skeletal growth anomalies in the brain coral Platygyra carnosa: A metatranscriptomic analysis, Marine Pollution Bulletin (2017), http://dx.doi.org/10.1016/j.marpolbul.2017.03.047

Y. Zhang et al. / Marine Pollution Bulletin xxx (2017) xxx–xxx

Nevertheless, it is unknown whether the differences in bacterial community structure were the cause or outcome of coral GA, and whether viruses are also involved in this disease. Our meta-transcriptomic analysis of both P. carnosa and its symbiotic Symbiodinium sp. has provided some clues about the involvement of both viruses and bacteria in the formation of GA. Bactericidal permeability-increasing protein (BPI) is an endogenous protein commonly found in various human tissues including the mucosal epithelia; it can bind to lipopolysaccharides produced by Gram-negative bacteria, and has a potent antibiotic activity against bacteria (Canny et al., 2002). Its downregulation in the GA-affected tissue of P. carnosa could indicate that the disease compromises the bacterial killing capacity in the coral. On the other hand, RIKEN cDNA C330046G03 is a mammalian protein found in epithelial cells and T cells that are targets during primary chickenpox virus infection. It can tightly bind to ORF65 protein of the virus, and the up-regulation of this gene promotes viral replication (Niizuma et al., 2003). In GA-affected P. carnosa tissue, RIKEN cDNA C330046G03 was significantly up-regulated, indicating viral infection. ADP-ribosylation factor 1 (Arf1) is a small GTPase involved in intracellular trafficking and organelle structure. Inhibition of Arf1 activation reduces the RNA level and production of viral particles in the infectious hepatitis C virus (Matto et al., 2011). In the marine shrimp Exopalaemon carinicauda, expression level of Arf1 was significantly up-regulated upon Vibrio parahaemolyticus and Whitespot Syndrome Baculovirus complex (WSSV) challenges (Duan et al., 2016), suggesting the involvement of Arf1 in immune responses to both viruses and bacteria. In our study, the expression of Arf1 was significantly upregulated in GA-affected tissue of P. carnosa, which is in agreement with the previous studies and suggesting the existence of viral and/or bacterial infection. Autophagy (ATG) related proteins are multifunctional proteins that participate in autophagy and phagocytosis in responses to the attack of viruses/protozoan parasites (Bestebroer et al., 2013). They regulate the formation of viral replication complexes (Hwang et al., 2012) and are involved in inflammatory immune response (Bestebroer et al., 2013). In P. carnosa, expression of ATG9a was down-regulated in GA-affected tissue, indicating possible virus infection. This result is consistent with Daniels et al. (2015), which considered up-regulation of an autophagy inhibitor in the coral Orbicella faveolata affected by the White Plague Disease as a strategy of the host to reduce viral replication. 5. Concluding remarks We have successfully identified a set of differential regulated molecular biomarkers by comparing the transcriptional expression between normal and skeletal GA- affected tissues in Platygyra carnosa. Our results revealed that the differentially expressed host genes are enriched in GO terms related to osteogenesis and oncogenesis, indicating GA is a malignant neoplasm and providing evidence to support the use of the term coral tumor to describe this disease. Several differentially expressed immune genes indicate the presence of both bacteria and viruses in GA-affected tissues. The differentially expressed Symbiodinium genes are involved in reproduction, nitrogen metabolism and pigment formation, indicating GA has affected several aspects of the symbiont's physiology. Our results have provided new insights into the molecular pathology of coral GA, and candidate genes for functional studies aiming to reveal the mechanisms of GA formation. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.marpolbul.2017.03.047. Acknowledgements This paper was the product of a collaborative study between Hong Kong Baptist University and the Agriculture, Fisheries and Conservation Department of Hong Kong SAR Government. The data analysis and paper writing were partially supported by Scientific and Technical Innovation Council of Shenzhen and Guangdong Natural Science Foundation

7

(grant no. 827000012, JCYJ20150625102622556, 2014A030310230). The transcriptome sequencing was conducted at Beijing Genomics Institute (BGI), Shenzhen, China. References Aeby, G.S., Williams, G.J., Franklin, E.C., Haapkyla, J., Harvell, C.D., Neale, S., Page, C.A., Raymundo, L., Vargas-Ángel, B., Willis, B.L., Work, T.M., Davy, S.K., 2011. Growth anomalies on the coral genera Acropora and Porites are strongly associated with host density and human population size across the Indo-Pacific. PLoS One 6, e16887. Barshis, D.J., Ladner, J.T., Oliver, T.A., Seneca, F.O., Traylor-Knowles, N., Palumbi, S.R., 2013. Genomic basis for coral resilience to climate change. Proc. Natl. Acad. Sci. 110, 1387–1392. Barshis, D.J., Ladner, J.T., Oliver, T.A., Palumbi, S.R., 2014. Lineage-specific transcriptional profiles of Symbiodinium spp. unaltered by heat stress in a coral host. Mol. Biol. Evol. 31, 1343–1352. Bayer, T., Aranda, M., Sunagawa, S., Yum, L.K., Desalvo, M.K., Lindquist, E., Coffroth, M.A., Voolstra, C.R., Medina, M., 2012. Symbiodinium transcriptomes: genome insights into the dinoflagellate symbionts of reef-building corals. PLoS One 7, e35269. Bestebroer, J., V'kovski, P., Mauthe, M., Reggiori, F., 2013. Hidden behind autophagy: the unconventional roles of ATG proteins. Traffic 14:1029–1041. http://dx.doi.org/10. 1111/tra.12091. Bolger, A.M., Lohse, M., Usadel, B., 2014. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30, 2114–2120. Bray, N.L., Pimentel, H., Melsted, P., Pachter, L., 2016. Near-optimal probabilistic RNA-seq quantification. Nat. Biotechnol. 34, 525–527. Breitbart, M., Bhagooli, R., Griffin, S., Johnston, I., Rohwer, F., 2005. Microbial communities associated with skeletal tumors on Porites compressa. FEMS Microbiol. Lett. 243, 431–436. Burns, J.H., Takabayashi, M., 2011. Histopathology of growth anomaly affecting the coral, Montipora capitata: implications on biological functions and population viability. PLoS One 6, e28854. Burns, J.H.R., Gregg, T.M., Takabayashi, M., 2013. Does coral disease affect Symbiodinium? Investigating the impacts of growth anomaly on symbiont photophysiology. PLoS One 8, e72466. Canny, G., Levy, O., Furuta, G.T., Narravula-Alipati, S., Sisson, R.B., Serhan, C.N., Colgan, S.P., 2002. Lipid mediator-induced expression of bactericidal/permeability-increasing protein (BPI) in human mucosal epithelia. Proc. Natl. Acad. Sci. 99, 3902–3907. Chan, A.L.K., Choi, C.L.S., McCorry, D., Chan, K.K., Lee, M.W., Ang, P.O., 2005. Field Guide to Hard Corals of Hong Kong. Agriculture, Fisheries and Conservation Department, Hong Kong SAR Government, Hong Kong. Chiu, J.M., Li, S., Li, A., Po, B., Zhang, R., Shin, P.K., Qiu, J.W., 2012. Bacteria associated with skeletal tissue growth anomalies in the coral Platygyra carnosus. FEMS Microbiol. Ecol. 79, 380–391. Daniels, C., Baumgarten, S., Yum, L.K., Michell, C.T., Bayer, T., Arif, C., Roder, C., Weil, E., Voolstra, C.R., 2015. Metatranscriptome analysis of the reef-building coral Orbicella faveolata indicates holobiont response to coral disease. Front. Mar. Sci. 2, 62. Domart-Coulon, I.J., Traylor-Knowles, N., Peters, E., Elbert, D., Downs, C., Price, K., Stubbs, J., McLaughlin, S., Cox, E., Aeby, G., Brown, P.R., Ostrander, G.K., 2006. Comprehensive characterization of skeletal tissue growth anomalies of the finger coral Porites compressa. Coral Reefs 25, 531–543. Duan, Y., Li, J., Zhang, Z., Li, J., Liu, P., 2016. Characterization of ADP ribosylation factor 1 gene from Exopalaemon carinicauda and its immune response to pathogens challenge and ammonia-N stress. Fish Shellfish Immunol. 55, 123–130. Dumont, C.P., Lau, D.C.C., Astudillo, J.C., Fong, K.F., Chak, S.T.C., Qiu, J.W., 2013. Coral bioerosion by the sea urchin Diadema setosum in Hong Kong: susceptibility of different coral species. J. Exp. Mar. Biol. Ecol. 441, 71–79. EPD (Environmental Protection Department), 2013. Marine Water Quality in Hong Kong in 2012. Environmental Department, The Government of the Hong Kong Special Administrative Region. Frazier, M., 2016. Impact of the Growth Anomaly Disease on Gene Expression in the Coral, Montipora capitata, at Waiʻōpae, Hawaiʻi. (MSc Thesis). University of Hawaiʻi at Hilo. Gateño, D., Leon, A., Barki, Y., Rinkevich, B., 2003. Skeletal tumor formations in the massive coral Pavona clavus. Mar. Ecol. Prog. Ser. 258, 97–108. Grygier, M.J., Cairns, S.D., 1996. Suspected neoplasms in deep-sea corals (Scleractinia: Oculinidae: Madrepora spp.) reinterpreted as galls caused by Petrarca madreporae n. sp. (Crustacea: Ascothoracida: Petrarcidae). Dis. Aquat. Org. 24, 61–69. Haas, B.J., Papanicolaou, A., Yassour, M., Grabherr, M., Blood, P.D., Bowden, J., Couger, M.B., Eccles, D., Li, B., Lieber, M., MacManes, M.D., Ott, M., Orvis, J., Pochet, N., Strozzi, F., Weeks, N., Westerman, R., William, T., Dewey, C.N., Henschel, R., LeDuc, R.D., Friedman, N., Regev, A., 2013. De novo transcript sequence reconstruction from RNA-seq using the Trinity platform for reference generation and analysis. Nat. Protoc. 8, 1494–1512. Haugsten, E.M., Wiedlocha, A., Olsnes, S., Wesche, J., 2010. Roles of fibroblast growth factor receptors in carcinogenesis. Mol. Cancer Res. 8, 1439–1452. Huang, S., Eleniste, P.P., Wayakanon, K., Mandela, P., Eipper, B.A., Mains, R.E., Allen, M.R., Bruzzaniti, A., 2014. The Rho-GEF Kalirin regulates bone mass and the function of osteoblasts and osteoclasts. Bone 60, 235–245. Hwang, S., Maloney, N., Bruinsma, M., Goel, G., Duan, E., Zhang, L., Shrestha, B., Diamond, M., Dani, A., Sosnovtsev, S., Green, K., Lopez-Otin, C., Xavier, R., Thackray, L., Virgin, H., 2012. Nondegradative role of Atg5-Atg12/Atg16L1 autophagy protein complex in antiviral activity of interferon gamma. Cell Host Microbe 11, 397–409. Irikawa, A., Casareto, B.E., Suzuki, Y., Agostini, S., Hidaka, M., van Woesik, R., 2011. Growth anomalies on Acropora cytherea corals. Mar. Pollut. Bull. 62, 1702–1707.

Please cite this article as: Zhang, Y., et al., Molecular pathology of skeletal growth anomalies in the brain coral Platygyra carnosa: A metatranscriptomic analysis, Marine Pollution Bulletin (2017), http://dx.doi.org/10.1016/j.marpolbul.2017.03.047

8

Y. Zhang et al. / Marine Pollution Bulletin xxx (2017) xxx–xxx

Jackson, R.A., Nurcombe, V., Cool, S.M., 2006. Coordinated fibroblast growth factor and heparan sulfate regulation of osteogenesis. Gene 379, 79–91. Kakugawa, S., Langton, P.F., Zebisch, M., Howell, S.A., Chang, T.H., Liu, Y., Feizi, T., Bineva, G., O'Reilly, N., Snijders, A.P., Jones, E.Y., Vincent, J.P., 2015. Notum deacylates Wnt proteins to suppress signalling activity. Nature 519, 187–192. Koldobskiy, M.A., Chakraborty, A., Werner Jr., J.K., Snowman, A.M., Juluri, K.R., Vandiver, M.S., Kim, S., Heletz, S., Snyder, S.H., 2010. p53-Mediated apoptosis requires inositol hexakisphosphate kinase-2. Proc. Natl. Acad. Sci. 107, 20947–20951. Kuo, S.J., Wang, F.J., Sheen, J.M., Yu, H.R., Wu, S.L., Ko, J.Y., 2014. Complement component C3: serologic signature for osteogenesis imperfecta: analysis of a comparative proteomic study. J. Formos. Med. Assoc. 114, 943–949. Lara-Castillo, N., Johnson, M.L., 2015. LRP receptor family member associated bone disease. Rev. Endocr. Metab. Disord. 16, 141–148. Libro, S., Kaluziak, S.T., Vollmer, S.V., 2013. RNA-seq profiles of immune related genes in the staghorn coral Acropora cervicornis infected with white band disease. PLoS One 8, e81821. Ligtenberg, A.J.M., Veerman, E.C., Nieuw Amerongen, A.V., Mollenhauer, J., 2007. Salivary agglutinin/glycoprotein-340/DMBT1: a single molecule with variable composition and with different functions in infection, inflammation and cancer. Biol. Chem. 388, 1275–1289. Lin, S., Cheng, S., Song, B., Zhong, X., Lin, X., Li, W., Li, L., Zhang, Y., Zhang, H., Ji, Z., Cai, M., Zhuang, Y., Shi, X., Lin, L., Wang, L., Wang, Z., Liu, X., Yu, S., Zeng, P., Hao, H., Zou, Q., Chen, C., Li, Y., Wang, Y., Xu, C., Meng, S., Xu, X., Wang, J., Yang, H., Campbell, D.A., Sturm, N.R., Dagenais-Bellefeuille, S., Morse, D., 2015. The Symbiodinium kawagutii genome illuminates dinoflagellate gene expression and coral symbiosis. Science 350, 691–694. Livak, K.J., Schmittgen, T.D., 2001. Analysis of relative gene expression data using realtime quantitative PCR and the 2(−Delta Delta C(T)) method. Methods 25, 402–408. Loya, Y., Bull, G., Puchon, M., 1984. Tumor formations in scleractinian corals. Helgoländer Meeresun. 37, 99–112. Matto, M., Sklan, E.H., David, N., Melamed-Book, N., Casanova, J.E., Glenn, J.S., Aroeti, B., 2011. Role for ADP ribosylation factor 1 in the regulation of hepatitis C virus replication. J. Virol. 85, 946–956. Mayfield, A.B., Wang, Y.B., Chen, C.S., Lin, C.Y., Chen, S.H., 2014. Compartment-specific transcriptomics in a reef-building coral exposed to elevated temperatures. Mol. Ecol. 23, 5816–5830. Mayfield, A.B., Wang, Y.B., Chen, C.S., Chen, S.H., Lin, C.Y., 2016. Dual-compartmental transcriptomic + proteomic analysis of a marine endosymbiosis exposed to environmental change. Mol. Ecol. 25, 944–5958. McClanahan, T.R., Weil, E., Maina, J., 2009. Strong relationship between coral bleaching and growth anomalies in massive Porites. Glob. Chang. Biol. 15, 1804–1816. Meyer, E., Aglyamova, G.V., Matz, M.V., 2011. Profiling gene expression responses of coral larvae (Acropora millepora) to elevated temperature and settlement inducers using a novel RNA-Seq procedure. Mol. Ecol. 20, 3599–3616. Mollenhauer, J., Herbertz, S., Helmke, B., Kollender, G., Krebs, I., Madsen, J., Holmskov, U., Sorger, K., Schmit, L., Wiemann, S., Otto, H.F., Grone, H.-J., Pousta, A., 2001. Deleted in malignant brain tumors 1 is a versatile mucin-like molecule likely to play a differential role in digestive tract cancer. Cancer Res. 61, 8880–8886. Nagata, E., Luo, H.R., Saiardi, A., Bae, B.I., Suzuki, N., Snyder, S.H., 2005. Inositol Hexakisphosphate Kinase-2, a physiologic mediator of cell death. J. Biol. Chem. 280, 1634–1640. Ng, J.C.Y., Chan, Y., Tun, H.M., Leung, F.C.C., Shin, P.K.S., Chiu, J.M.Y., 2015. Pyrosequencing of the bacteria associated with Platygyra carnosus corals with skeletal growth anomalies reveals differences in bacterial community composition in apparently healthy and diseased tissues. Front. Microbiol. 6, 1142. Niizuma, T., Zerboni, L., Sommer, M.H., Ito, H., Hinchliffe, S., Arvin, A.M., 2003. Construction of varicella-zoster virus recombinants from parent Oka cosmids and demonstration that ORF65 protein is dispensable for infection of human skin and T cells in the SCID-hu mouse model. J. Virol. 77, 6062–6065. Pasca di, M.M., Hebrok, M., 2003. Hedgehog signalling in cancer formation and maintenance. Nat. Rev. Cancer 3, 903–911. Pećina-Šlaus, N., 2010. Wnt signal transduction pathway and apoptosis: a review. Cancer Cell Int. 10, 22. Pham, T.V., Jimenez, C.R., 2012. An accurate paired sample test for count data. Bioinformatics 28, i596–i602. Pochon, X., Putnam, H.M., Burki, F., Gates, R.D., 2012. Identifying and characterizing alternative molecular markers for the symbiotic and free-living dinoflagellate genus Symbiodinium. PLoS One 7, e29816. Pollock, P.M., Gartside, M.G., Dejeza, L.C., Powell, M.A., Mallon, M.A., Davies, H., Mohammadi, M., Futreal, P.A., Stratton, M.R., Trent, J.M., Goodfellow, P.J., 2007. Frequent activating FGFR2 mutations in endometrial carcinomas parallel germline

mutations associated with craniosynostosis and skeletal dysplasiasyndromes. Oncogene 26, 7158–7162. Qiu, J.W., Lau, D.C.C., Cheung, C.C., Chow, W.K., 2014. Community-level destruction of hard corals by the sea urchin Diadema setosum. Mar. Pollut. Bull. 85, 783–788. Sarmah, B., Wente, S.R., 2010. Inositol hexakisphosphate kinase-2 acts as an effector of the vertebrate Hedgehog pathway. Proc. Natl. Acad. Sci. 107, 19921–19926. Shinzato, C., Shoguchi, E., Kawashima, T., Hamada, M., Hisata, K., Tanaka, M., Fujie, M., Fujiwara, M., Koyanagi, R., Ikuta, T., Fujiyama, A., Miller, D.J., Satoh, N., 2011. Using the Acropora digitifera genome to understand coral responses to environmental change. Nature 476, 320–323. Shoguchi, E., Shinzato, C., Kawashima, T., Gyoja, F., Mungpakdee, S., Koyanagi, R., Takeuchi, T., Hisata, K., Tanaka, M., Fujiwara, M., Hamada, M., Seidi, A., Fujie, M., Usami, T., Goto, H., Yamasaki, S., Arakaki, N., Suzuki, Y., Sugano, S., Toyoda, A., Kuroki, Y., Fujiyama, A., Medina, M., Coffroth, M.A., Bhattacharya, D., Satoh, N., 2013. Draft assembly of the Symbiodinium minutum nuclear genome reveals dinoflagellate gene structure. Curr. Biol. 23, 1399–1408. Somerville, R.P.T., Shoshan, Y., Eng, C., Barnett, G., Miller, D., Cowell, J.K., 1998. Molecular analysis of two putative tumor suppressor genes, PTEN and DMBT, which have been implicated in glioblastoma multiforme disease progression. Oncogene 17, 1755–1757. Spies, N.P., Takabayashi, M., 2013. Expression of galaxin and oncogene homologs in growth anomaly in the coral Montipora capitata. Dis. Aquat. Org. 104, 249–256. Stimson, J., 2011. Ecological characterization of coral growth anomalies on Porites compressa in Hawai'i. Coral Reefs 30, 133–142. Sun, Y., Mochizuki, Y., Majerus, P.W., 2003. Inositol 1,3,4-trisphosphate 5/6-kinase inhibits tumor necrosis factor-induced apoptosis. J. Biol. Chem. 278, 43645–43653. Sun, J., Chen, Q., Lun, J.C., Xu, J., Qiu, J.W., 2013. PcarnBase: development of a transcriptomic database for the brain coral Platygyra carnosus. Mar. Biotechnol. 15, 244–251. Sun, J., Zhang, Y., Xu, T., Zhang, Y., Mu, H., Zhang, Y., Lan, Y., Fields, C.J., Hui, J.H.L., Zhang, W., Li, R., Nong, W., Cheung, F.K.M., Qiu, J.-W., Qian, P.-Y., 2017. Adaptation to deepsea chemosynthetic environments as revealed by mussel genomes. Nat. Ecol. Evol. 1, 127. Tabas, J.A., Zasloff, M., Wasmuth, J.J., Emanuel, B.S., Altherr, M.R., McPherson, J.D., Wozney, J.M., Kaplan, F.S., 1991. Bone morphogenetic protein: chromosomal localization of human genes for BMP1, BMP2A, and BMP3. Genomics 9, 283–289. Veron, J., 2000. Corals of the World. Australian Institute of Marine Science, Townsville, Australia. Wong, J.C.Y., Thompson, P., Xie, J.Y., Qiu, J.-W., Baker, D.M., 2016. Symbiodinium clade C generality among common scleractinian corals in subtropical Hong Kong. Reg. Stud. Mar. Sci. http://dx.doi.org/10.1016/j.rsma.2016.02.005. Work, T.M., Rameyer, R.A., 2005. Characterizing lesions in corals from American Samoa. Coral Reefs 24, 384–390. Work, T.M., Aeby, G.S., Coles, S.L., 2008. Distribution and morphology of growth anomalies in Acropora from across the Indo-Pacific. Dis. Aquat. Org. 78, 255–264. Wright, R.M., Aglyamova, G.V., Meyer, E., Matz, M.V., 2015. Gene expression associated with white syndromes in a reef building coral, Acropora hyacinthus. BMC Genomics 16, 371. Wu, W., Kemp, B.L., Proctor, M.L., Wu, W., Kemp, B.L., Proctor, M.L., Gazdar, A.F., Minna, J.D., Hong, W.K., Mao, L., 1999. Expression of DMBT1, a candidate tumor suppressor gene, is frequently lost in lung cancer. Cancer Res. 59, 1846–1851. Yamashiro, H., Yamamoto, M., van Woesik, R., 2000. Tumor formation on the coral Montipora informis. Dis. Aquat. Org. 41, 211–217. Yamashiroa, H., Okub, H., Onagab, K., Iwasakib, H., Takarab, K., 2001. Coral tumors store reduced level of lipids. J. Exp. Mar. Biol. Ecol. 265, 171–179. Yasuda, N., Hidaka, M., 2012. Cellular kinetics in growth anomalies of the scleractinian corals Porites australiensis and Montipora informis. Dis. Aquat. Org. 102, 1–11. Yellowlees, D., Rees, T.A.V., Leggat, W., 2008. Metabolic interactions between algal symbionts and invertebrate hosts. Plant Cell Environ. 31, 679–694. Zhang, P., Liu, Y., Jin, C., Zhang, M., Lv, L., Zhang, X., Liu, H., Zhou, Y., 2016. Histone h3k9 acetyltransferase pcaf is essential for osteogenic differentiation through bone morphogenetic protein signaling and may be involved in osteoporosis. Stem Cells 34, 2332–2341. Zheng, Q., Wang, X.J., 2008. GOEAST: a web-based software toolkit for Gene Ontology enrichment analysis. Nucleic Acids Res. 36, W358–W363. Zhou, S., Xie, Y., Tang, J., Huang, J., Huang, Q., Xu, W., Wang, Z., Luo, F., Wang, Q., Chen, H., Du, X., Shen, Y., Chen, D., Chen, L., 2015. FGFR3 deficiency causes multiple chondroma-like lesions by upregulating hedgehog signaling. PLoS Genet. 11, e1005214.

Please cite this article as: Zhang, Y., et al., Molecular pathology of skeletal growth anomalies in the brain coral Platygyra carnosa: A metatranscriptomic analysis, Marine Pollution Bulletin (2017), http://dx.doi.org/10.1016/j.marpolbul.2017.03.047