Marine Pollution Bulletin 164 (2021) 111982

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Marine Pollution Bulletin

journal homepage: www.elsevier.com/locate/marpolbul

A proteomic analysis of skeletal tissue anomaly in the brain coral

Platygyra carnosa
Yue-Him Wong a, Yu Zhang b, c, *, Janice C.Y. Lun d, Jian-Wen Qiu c, e, f, **
a
Institute for Advance Study, Shenzhen University, Shenzhen, China
b
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
c
Southern Marine Science and Engineering Guangdong Laboratory (Guangzhou), Guangzhou, China
d
Agriculture, Fishery and Conservation Department, The Government of the Hong Kong Special Administrative Region, China
e
Department of Biology, Hong Kong Baptist University, Hong Kong, China
f
HKBU Institute of Research and Continuing Education, Shenzhen, China

A R T I C L E I N F O A B S T R A C T

Keywords: Coral skeletal growth anomaly (GA) is a common coral disease. It has been considered as a pathological condition
Coral disease comparable to abnormal tissue growth in mammals, but little is known about the molecular changes underlying
Coral growth anomaly coral GA. To investigate the molecular pathology of GA, we compared the proteome between normal and GA-
Coral tumor
affected tissues of the brain coral Platygyra carnosa using iTRAQ-labeling and LC-MS/MS, which quantified
iTRAQ
818 proteins and identified 117 differentially expressed proteins (DEPs). GO analyses revealed DEPs that might
Proteomics
be related to GA included “translational elongation”, “proteasome core complex”, “amine metabolic processes”
and “lysosome”. Several proteins implicated in calcification and fluorescence were differentially expressed at
both protein and mRNA level. Protein-protein interaction network suggested possible involvement of TNF re­
ceptor signaling in GA. Overall, our results provided novel insights into the molecular pathology of coral GA,
which will pave the way for determination of the causative agent(s) of this coral disease.

1. Introduction
Coral diseases are considered one of the major causes of coral reefs
decline (Bruckner and Hill, 2009). Coral skeletal growth anomaly (GA)
is one of the major coral diseases (Work et al., 2015). GA refers to
abnormal development of coral skeleton and associated tissue, resulting
in major differences in skeleton structure and elements, tissue size,
shape and color when compared with the healthy skeleton and tissue.
GAs in different coral species share several common characteristics,
including 1) protuberant and porous skeleton due to abnormal
arrangement of skeletal element and tissue structure (Zhang et al.,
2017); 2) very active and irregular vertical skeletal growth compared to
adjacent healthy colonies/tissues (Yasuda et al., 2012); 3) tissue dis­
colouration due to reduced density of zooxanthellae (Gateño et al.,
2003); and 4) reduced photosynthetic capability (Burns and Takabaya­
shi, 2011). As such, occurrence of GA can have negative effect on the
structural integrity of coral skeleton, nutrition production and uptake,
fecundity, reproduction, and the overall fitness of the affected colonies
(Stimson, 2011; Yasuda and Hidaka, 2012).
Since its first documentation of GA on Madrepora kauaiensis in 1965
(Squires, 1965), GA has been reported in at least 27 Indo-Pacific species
and 16 Caribbean species from 21 genera of scleractinian corals (Gateño
et al., 2003; Sutherland et al., 2004; Burns et al., 2011; Irikawa et al.,
2011; Hussain et al., 2016). The prevalence of GA has been associated
with bleaching intensity and local anthropogenic stressors such as high
phosphorus concentration (McClanahan et al., 2009), as well as global
stressors such as sea-surface temperature anomalies (Bruno et al., 2007).
Hence, GA constitutes a threat to coral reef systems, especially to those
reefs that are in proximity to highly populated cities.
Previous studies of coral GA have mainly covered the changes in
coral morphology (Peters et al., 1986; Work et al., 2008), histopathology
(Peters et al., 1986; Yamashiro et al., 2000; Gateño et al., 2003; Domart-
Coulon et al., 2006), physiology (Gateño et al., 2003) and ecology (Aeby
et al., 2011). A model has been proposed to link environmental pollution

* Corresponding author.
** Correspondence to: J.-W. Qiu, Department of Biology, Hong Kong Baptist University, Hong Kong, China.
E-mail addresses: biozy@szu.edu.cn (Y. Zhang), qiujw@hkbu.edu.hk (J.-W. Qiu).

https://doi.org/10.1016/j.marpolbul.2021.111982
Received 30 April 2020; Received in revised form 19 December 2020; Accepted 20 December 2020
Available online 28 January 2021
0025-326X/© 2021 Elsevier Ltd. All rights reserved.
Y.-H. Wong et al.
and reduced coral health to GA (McClanahan et al., 2009), but the cause
effect relationship between these environmental factors and GA devel­
opment has not been confirmed. Recent molecular analyses revealed
differential expression of stress-related proteins (Domart-Coulon et al.,
2006), oncogenesis-related genes (Spies and Takabayashi, 2013) and
immune system genes (Zhang et al., 2017; Frazier et al., 2017) associ­
ated with coral GA. Significantly higher activity of the key immunity
enzyme phenoloxidase (PO) was detected in GA affected tissue of
Acropora hyacinthus, suggesting that GA triggers immune responses
(Palmer and Baird, 2018). In independent transcriptomic analyses of GA
in Platygyra carnosa and Montipora capitata, genes implicated in immune
response and oncogenesis were up-regulated in GA affected tissue
(Zhang et al., 2017; Frazier et al., 2017). These molecular data explored
the potential underlying mechanism of GA, but mainly at the gene
expression (mRNA) level.
Comparing to gene expression, protein expression is more directly
relevant to biological functions because gene transcripts may experience
different degrees of post-translational regulation. Given that mRNA and
protein expression do not always show a high level of correlation (de
Sousa Abreu et al., 2009; Vogel and Marcotte, 2012), protein expression
data are necessary to fill the missing gap between gene expression and
biological function. However, no high-throughput proteomic study of
coral GA has been conducted.
The brain coral Platygyra carnosa (previous incorrectly written as
Platygyra carnosus in a few papers) is a common reef-building coral in the
Indo-Pacific, including the South China Sea (SCS) and Red Sea. It is one
of the dominant species along the northern shorelines of the SCS, often
forming major structural frameworks (Veron, 2000; Qiu et al., 2014). In
contrast to the brown color of normal colony, GA affected P. carnosa
tissue shows discoloration, as well as active and irregular vertical skel­
eton growth (see Fig. 1 in Zhang et al., 2017).
In the current study, GA affected and apparently healthy tissues of
P. carnosa were analyzed using an iTRAQ-coupled LC-MS/MS approach
(Han et al., 2013). The iTRAQ proteomic results were then compared
with the published transcriptomic dataset of the same species (Zhang
et al., 2017). Through comparative transcriptomic and proteomic
analysis, we attempted to provide a comprehensive understanding of the
molecular pathology of coral GA, as well as the possible causes of this
disease.
2. Material and methods
2.1. Sample collection
The procedures of sample collection are identical to those described
in Zhang et al. (2017). In brief, coral samples were collected from Sharp
Island, Hong Kong (22.366◦ N, 114.299◦ E) in 2011 by SCUBA. Four
colonies of P. carnosa at 2–3 m water depth were sampled. From each
colony, one sample of GA affected tissue and one sample of apparently
healthy (normal) tissue were collected using a chisel and hammer. The
samples were kept on dry ice in the field, transported to the laboratory at
Hong Kong Baptist University and stored at − 80 ◦ C until use.
2.2. Protein extraction and iTRAQ labeling
An algal-free protein extraction method (Weis and Levine, 1996) was
applied in protein extraction. Each sample, including tissue and skel­
eton, was frozen in liquid nitrogen in a pre-chilled mortar. A homoge­
nization buffer consisting of 40 mmol− 1 Tris HCl, 10 mmol l− 1 EDTA and
protease inhibitor at pH 7.4 was added. The sample was ground to
powder using a pestle. The homogenate was centrifuged at 3000 ×g for
5 min, and then the supernatant was centrifuged at 15,000 ×g for 10 min
in order to pellet the algae from the symbiotic homogenate. After that,
proteins from the supernatant were precipitated by adding four volumes
of pre-chilled acetone, followed by centrifugation at 13,000 rpm for 15
min at 4 ◦ C. The protein pellet was then dissolved in 8 M urea solution,
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Marine Pollution Bulletin 164 (2021) 111982
and protein concentration was determined using a RC-DC kit (Bio-Rad,
Hercules, CA, USA).
Approximately 200 μg of protein from each sample was reduced with
5 mM triscarboxyethyl phosphine hydrochloride (TCEP) for 60 min at
60 ◦ C, then alkylated with 10 mM methylethanethiosulfonate (MMTS)
for 20 min at room temperature. Each sample was diluted seven-fold
with 50 mM triethylammunium bicarbonate (TEAB), then digested
with sequencing grade trypsin (Promega, Madison, WI, USA) for 16 h at
37 ◦ C in a 1:50 trypsin-to-protein mass-ratio. The protein digest was
desalted using a Sep-Pak C18 cartridge (Waters, Milford, MA, USA) and
dried in a SpeedVac (Thermo Electron, Waltham, MA, USA).
The digested samples were labeled with iTRAQ reagents as described
by Sun et al. (2013a). In brief, the samples were reconstituted in 30 mL
of dissolution buffer and mixed with 70 mL of ethanol-suspended iTRAQ
reagents, with one iTRAQ reporter tag per protein sample. Two normal
coral samples were labeled with the reporter tags 114 and 115,
respectively; and the corresponding two GA affected coral samples were
labeled with the reporter tags 116 and 117, respectively. Two experi­
mental runs were conducted, and each run had two normal and two GA-
affected samples. Labeling reactions were carried out at room temper­
ature for 90 min. The labeled samples from each experimental run were
mixed in a single tube and dried in a SpeedVac.
2.3. 2D-LC-MS/MS analysis
The dried iTRAQ labeled samples were reconstituted with 100 mL
buffer A (10 mM KH2PO4, pH 3.0, ACN/H2O 25/75 (v/v)) and loaded
into a PolySULFOETHYL A column (200 mm length, 4.6 mm id, 200-Å
pore size, 5 mm particle size) (PolyLC, Columbia, MD, USA) coupled to
an Alliance 2695 HPLC System (Waters, Milford, MA, USA). Each sample
was fractionated using a gradient of 100% buffer A for 5 min, 5–30%
buffer B (10 mM KH2PO4, pH 3.0, 500 mM KCl, ACN/H2O 25/75 (v/v))
for 40 min, 30–100% buffer B for 5 min, and finally 100% buffer B for 5
min, at a constant flow rate of 1 mL/min for a total of 55 min. The eluted
fractions were monitored using an UV detector at 214 nm wavelength,
and collected at 1 min intervals. Consecutive fractions with low peak
intensity were combined, resulting in a total of 22 fractions. The frac­
tionated samples were dried in a SpeedVac, reconstituted in 0.1% TFA
and desalted using Sep-Pak C18 cartridges. Each fraction was recon­
stituted in 0.1% formic acid and analyzed using a nanoflow UPLC
(nanoAcquity™, Waters) coupled with an ESI-hybrid Q-TOF (Pre­
mierTM, Waters) tandem mass spectrometer as previously described
(Sun et al., 2012).
2.4. Database search and bioinformatics analyses
The raw MS/MS data files were converted into pkl data format using
Proteinlynx Global Server 2.2.5 (Waters Corp., Milford, MA). The data
were searched against PcanBase, the P. carnosa transcriptome translated
protein database (Sun et al., 2013a) using Mascot version 2.3.0 (Matrix
Sciences, Ltd., London, UK). Only data that were identified as P. carnosa
proteins were retained. Data normalization was applied based on the
summed intensity. The false discovery rate was determined as 1% using
a “target-decoy” database searching strategy (Sun et al., 2013b). The
intensity of the iTRAQ reporter of each protein was determined and
normalized with a “median” method. Proteins that were significantly
different between the normal and GA-affected tissues were determined
by running paired Student’s t-test on the log transformed data. Differ­
entially expressed proteins (DEPs) were defined as those with a p-value
less than 0.05, after correction with the Benjamini & Hochberg method
(Benjamini and Hochberg, 1995), and a differential expression of over
1.5 times. To visualize the expression pattern of DEP among the four
healthy and four disease samples, a heat map was generated by applying
the maximum linkage clustering method and Pearson distance similarity
metric method using the Heml toolkit version 1.0.3.3) (Deng et al.,
2014).
Y.-H. Wong et al.
2.5. Functional annotation and enrichment analyses
The DEPs were searched against the NCBI non-redundant database to
obtain accession numbers of best matches. The DEPs and differentially
expressed gene (DEGs) sequences (translated protein sequence) were
submitted to KOBAS BLAST search against the Homo sapiens and
Acropora digitifera genomes, using the annotation function of KOBAS 3.0
(available at http://kobas.cbi.pku.edu.cn). The resultant Uniprot IDs
were categorized using the PANTHER Classification System v 8.1
(available at http://www.PANTHERdb.org/) (Mi et al., 2013), which
reports the functional categories in GO-slim Molecular Functions, GO-
slim Biological Processes, GO-slim Cellular Component, Protein Class
and PANTHER pathways. The human genome annotation of DEPs and
DEGs were further searched against the KEGG pathway using the Gene-
List Enrichment function of KOBAS. Enrichment analyses were per­
formed using the hypergeometric test/Fisher’s exact statistical test, with
the Benjamini & Hochberg FDR correction method applied to reduce
Type-1 error (Xie et al., 2011). The Benjamini p-value cutoff for
enrichment was set at 0.001. GO enrichment analysis was performed
using BiNGO (Maere et al., 2005). The DEPs annotated using the human
dataset were submitted as the test dataset and the whole proteome was
used as the reference dataset. The criteria of GO enrichment analyses
were the same as for KEGG pathways except the Benjamini p-value
cutoff for enrichment was set at 0.005. Network visualization of the
enriched GO terms was performed using Cytoscape v3.7.2 (Shannon
et al., 2003).
2.6. Comparison of protein and mRNA expression levels
Data from the previous transcriptomic analysis (Zhang et al., 2017)
and the current proteomic analysis were used in this comparison. The
mRNA and protein expression values were log10 transformed and
normalized according to the mean expression value in each sample.
Linear regression, correlation coefficient and the associated p-value
were computed using the funciton “ggscatter” in the R package
“ggpubr”.
2.7. Protein-protein interaction (PPI) network analysis
The annotated DEPs were converted into human gene symbols and
then submitted to Bioprofiling.de (Antonov, 2011) for network analysis
(available at http://www.bioprofiling.de/). The result from PPI-spider
module, which mapped the submitted gene list against a reference PPI
network database containing 7980 PPI (Antonov, 2011) was adopted.
Sub-network model having an enrichment p-value below 0.05 was
downloaded as a “.xgmml” file and visualized using Cytoscape v3.7.2
(Shannon et al., 2003). The inferred PPI sub-network model was pre­
sented in the y-layer organic layout. In the current study, the D2 PPI
spider network model inferred from the DEPs was selected. The D2
model allowed 1 missing protein in the PPI network. A “missing protein”
was defined as the missing protein in an interacting pair that was part of
the inferred PPI network model. Mapped DEPs were presented as rect­
angle nodes and “missing proteins”, proteins that have physical in­
teractions with at least one of the mapped DEPs, was presented as
triangle nodes in the inferred PPI spider network. Each edge represents
the existence of physical interactions between a protein pair with
reference to the human PPI network (see Supplementary Table 3).
3. Results
3.1. Functional annotation of the proteome
The proteomic analysis resulted in the identification and quantifi­
cation of 818 P. carnosa proteins. Among them, 693 and 794 P. carnosa
proteins exhibited significant homology (e-value <1e-5) to at least one
sequence in the human genome or the stony coral Acropora digitifera
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Marine Pollution Bulletin 164 (2021) 111982
genome. Based on gene annotation against the human genome, 604 and
603 proteins were mapped to the Gene Ontology (GO) framework and
PANTHER protein class annotation framework, respectively. The top
five GOslim Biological Process (GOBP) terms were “cellular process”
(GO:0009987), “metabolic process” (GO:0008152), “biological regula­
tion” (GO:0065007), “cellular component organization or biogenesis”
(GO:0071840), and “localization” (GO:0051179) (Fig. 1a). The top five
Molecular Function (GOMF) terms were “catalytic activity”
(GO:0003824), “binding” (GO:0005488), “structural molecule activity”
(GO:0005198), “molecular function regulator” (GO:0098772), and
“transporter activity” (GO:0005215) (Fig. 1a). The top five Cellular
Component (GOCC) terms were “cell” (GO:0005623), “cell part”
(GO:0044464), “organelle” (GO:0043226), “protein-containing com­
plex” (GO:0032991) and “membrane” (GO:0016020). The top five
PANTHER protein classes were “metabolite interconversion enzyme”
(PC00262), “protein modifying enzyme” (PC00260), “cytoskeletal pro­
tein” (PC00085), “translational protein” (PC00263), and “membrane
traffic protein” (PC00150) (Fig. 1b). These top protein classes consti­
tuted 47.3% of the annotated proteins in the proteome, therefore might
represent the main biological processes and functions of the coral.
3.2. GO enrichment analyses of differentially expressed proteins (DEPs)
Analysis of the proteomic data detected 117 proteins that were
differentially expressed (Benjamin p-value <0.05) between GA affected
and normal tissues, among which 49 proteins were up-regulated, while
68 were down-regulated in GA affected tissue (Supplementary Fig. 1 and
Supplementary Table 1). A heatmap and clustering dendrogram analysis
of DEPs showed that the replicates of each experimental group (114 and
115 labeled with normal tissues, while 116 and 117 labeled with GA
affected tissue) clustered together, indicating high reproducibility of
these biological replicates (Fig. 2).
Despite the limitation of only 117 DEPs being available, GO
enrichment analysis revealed over-representation of GOBP, GOMF and
GOCC relating to de novo synthesis (GOBP: “translational elongation”;
GOMF: “structural constituent of ribosome”, “mRNA binding”; GOCC:
“ribosome”, “cytosolic large ribosomal subunit”, “cytosolic small ribo­
somal subunit”), amino acid metabolism (GOBP: “amine metabolic
process”, “cellular amino acid and derivative metabolic process”), pro­
tein metabolism (GOBP: “protein metabolic process”) and protein
degradation (GOMF: “dipeptidase activity”, “threonine-type endopep­
tidase activity”; GOCC: “lysosome”, “lytic vacuole”, “proteasome core
complex, alpha-subunit complex”) (Fig. 3). Details of the statistic of the
enrichment analysis and the protein annotations relate to the enriched
GO terms are shown in Supplementary Table 2.
DEPs relate to the GOBP terms “translational elongation”, “structural
constituent of ribosome” and the GOCC term “ribosome” were 13 ribo­
somal proteins, in which 12 of them were up-regulated; DEPs relate to
“proteasome core complex” were four 20S proteasome subunit proteins
which were all up-regulated; DEPs relate to the GOCC term “lysosome”
include multiple digestive enzymes which were all down-regulated;
DEPs relate to “cellular amino acid and derivative metabolic process”
include 11 metabolic proteins, in which 10 of them were down-
regulated (Table 1, Supplementary Table 3).
3.2.1. DEPs implicated in immune defense and apoptosis
Immune responsive proteins such as TNF receptor associated factors
(TRAFs) and Macrophage mannose receptor 1-like protein have been
known to be involved in coral GA formation (Frazier et al., 2017). In our
study, C-type mannose-binding lectin or mannose receptor (C-type
MBL), which is considered a pattern recognition protein in the innate
immune system (McGreal et al., 2004), was down-regulated. TNF re­
ceptor associated protein 1 (TRAP1), also known as HSP75, which is
known to be involved in death signaling in damaged cells and hence part
of the immune system, was up-regulated. Apoptosis-inducing factor 1
(AIF1), a mitochondrial protein implicated in caspase-independent
Y.-H. Wong et al. Marine Pollution Bulletin 164 (2021) 111982

Fig. 1. Functional categories of 818 proteins quantified by iTRAQ labeling. The proteins were annotated with human genome and the categories were determined
using PANTHER. a) Gene ontology (GO-slim) Biological Process, Molecular Function and Cellular Component; b) PANTHER protein classes.

Fig. 2. Heatmap and clustering dendrogram of differentially expressed coral proteins between the healthy tissue and GA affected tissue. Columns labeled with 114
and 115 represents healthy tissues labeled with iTRAQ 114 and 115 isotopes, while 116 and 117 represents GA affected tissue labeled with iTRAQ 116 and 117
isotopes. DEPs are shown in rows, with values normalized to the mean iTRAQ quantification value of each DEP.

4
Y.-H. Wong et al.
Fig. 3. Gene Ontology (GO) functional enrichment analyses of 117 differentially expressed coral proteins. Each node represents an enriched GO term and edge
represents the linkage between different nodes in the hierarchical framework of GO. The terminal nodes in each GO hierarch network are highlighted and their
enrichment p-value are shown in Supplementary Table 1.
apoptosis (Cande et al., 2004) and was down-regulated. Superoxide
dismutase 2 (SOD2) which metabolize reactive oxygen species and re­
lates to apoptosis, was down-regulated in GA affected tissue (Table 1,
Supplementary Table 1).
3.2.2. DEPs implicated in calcification
Based on literature on calcification (Ramos-Silva et al., 2013; Drake
et al., 2013b; Mass et al., 2014), six of the DEPs discovered in this study
have been implicated in calcification or coral skeleton organic matrix.
These calcification proteins included calreticulin (CRT), which was up-
regulated; two collagen alpha (KO definition: collagen, type VI, alpha;
collagen, type VI, alpha), which were both down-regulated; mucin-2-
like (KO definition: mucin-5B), which was down-regulated; osteoclast-
stimulating factor (OSF), which was up-regulated; myosin-2 essential
light chain-like which was down-regulated (Table 1, Supplementary
Table 1).
3.2.3. DEPs implicated in stress response
Heat shock proteins (HSPs) have been known as molecular chap­
erons that help repair misfolded proteins (Wandinger et al., 2008). They
have been known to be involved in coral GA formation (Domart-Coulon
et al., 2006). Our list of P. carnosa DEPs included five HSPs, including
HSP 90-alpha (HSP90AA1), 10 kDa HSP (HSPE1), HSP70 A-8 (HSPA8),
HSP 75 kDa (HSP75) and heat shock cognate 71 kDa protein-like
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Marine Pollution Bulletin 164 (2021) 111982
(HSPA8/HSC71). Except HSPE1, all of the above DEPs were up-
regulated in GA affected tissue (Table 1, Supplementary Table 1) and
were associated with GO Biological Process (GOBP) terms related to
stress, including response to oxidative stress (GO:0006979), response to
stress (GO:0006950) and response to osmotic stress (GO:0006970). Note
that HSP75 and HSPE1 are also known as TNF receptor associated
protein 1 (TRAP1) and Chaperonin (CPN), respectively (Table 1, Sup­
plementary Table 1).
3.2.4. Fluorescent proteins
Four DEPs were annotated as green or red FP (Table 1, Supplemen­
tary Table 1). These FPs were all up-regulated in the GA affected tissue.
Nevertheless, no GO term was assigned to the FPs, probably because the
GO annotations are mainly based on human proteins.
Overall, DEPs related to translation, lysosome, calcification,
apoptosis, immune system and fluorescent proteins were found to be
differentially expressed. These DEPs were likely involved in coloration
change, abnormal skeleton growth and indicated a sign of stress in GA
affected tissue. Some of the genes corresponding these DEPs were also
found to be differentially expressed in two previous transcriptomic
studies of coral GA (Frazier et al., 2017; Zhang et al., 2017).
Y.-H. Wong et al. Marine Pollution Bulletin 164 (2021) 111982

Table 1
Highlighted differentially expressed proteins involved in growth anomaly of Platygyra carnosa.
ID Fold_change ± S. DEP ko_definition Nr-annotation Gene symbol
E.

Ribosomal proteins
Unigene45048_Mix 0.99 ± 0.07 Up large subunit ribosomal protein L10e Ribosomal protein L10e RPL10
Unigene45164_Mix 0.71 ± 0.09 Up large subunit ribosomal protein LP2 Ribosomal protein, large, P2 RPLP2
Unigene45920_Mix 0.74 ± 0.14 Up – Ribosomal protein rps20 RPS20
Unigene46070_Mix 0.82 ± 0.47 Up – Ribosomal protein rpl13a RPL13A
Unigene46102_Mix 0.69 ± 0.15 Up – 40S ribosomal protein S3-like RPS3
Unigene46813_Mix 0.78 ± 0.16 Up – Ribosomal protein L23 RPL23
Unigene47018_Mix 0.29 ± 0.09 Up – Ribosomal protein L17 RPL17-
C18orf32
Unigene47022_Mix 1.05 ± 0.04 Up – Ribosomal L4/L1 RPL4
Unigene47179_Mix 0.81 ± 0.36 Up – 40S ribosomal protein S8-like RPS8
Unigene47523_Mix 0.6 ± 0.01 Up – 60S ribosomal protein L6 RPL6
Unigene47638_Mix 0.99 ± 0.3 Up – Ribosomal protein L31e RPL31
Unigene47708_Mix 0.62 ± 0.15 Up – Ribosomal protein S4, X-linked-like RPS4X
Unigene47721_Mix 1.37 ± 0.09 Up – 40S ribosomal protein S14 RPS14

Proteasome
Unigene136513_Mix 0.61 ± 0.3 Up 20S proteasome subunit beta 3 Unknown (protein for MGC:99279) PSMB3
Unigene67813_Mix 0.77 ± 0.29 Up 20S proteasome subunit alpha 7 Proteasome alpha 3 subunit-like PSMA3
Unigene68261_Mix 0.7 ± 0.31 Up 20S proteasome subunit alpha 1 Similar to prosomal P27K protein PSMA6
Unigene68413_Mix 0.69 ± 0.31 Up 20S proteasome subunit alpha 6 Proteasome subunit alpha type-1-like PSMA1

Lysosomal proteins
CL2385. − 0.44 ± 0.19 Down Niemann-Pick C2 protein Epididymal secretory protein E1-like isoform 1 NPC2
Contig1_Mix
Unigene16943_Mix − 0.43 ± 0.04 Down Hexosaminidase Beta-hexosaminidase beta chain HEXB
Unigene44973_Mix − 0.46 ± 0.13 Down Alpha 1,3-glucosidase Glycoside hydrolase GAA
Unigene125928_Mix − 0.5 ± 0.18 Down Cathepsin X Cathepsin Z precursor CTSZ/CTSX
Unigene47212_Mix − 0.55 ± 0.28 Down – Ependymin-like protein EPDR1
Unigene136444_Mix − 0.48 ± 0.26 Down Lysosomal alpha-mannosidase Lysosomal alpha-mannosidase MAN2B1
Unigene116638_Mix − 0.45 ± 0.18 Down Beta-glucuronidase Beta-glucuronidase-like GUSB
Unigene135446_Mix − 0.47 ± 0.16 Down N-acetylglucosamine-6-sulfatase N-acetylglucosamine-6-sulfatase precursor (G6S) GNS
(Glucosamine-6-sulfatase)

Immune system and apoptosis related

CL176.Contig1_Mix − 0.45 ± 0.05 Down Superoxide dismutase, Fe-Mn family Superoxide dismutase SOD2
Unigene30536_Mix* 0.85 ± 0.04 Up TNF receptor-associated protein 1 Heat shock protein 75 kDa, mitochondrial TRAP1/HSP75
Unigene145775_Mix − 0.6 ± 0.15 Down Mannose receptor, C type C-type MBL-2 protein FCER2
Unigene34204_Mix − 0.63 ± 0.14 Down programmed Cell death 8 Apoptosis-inducing factor 1, mitochondrial AIFM1

Proteins implicated in calcification

CL466.Contig1_Mix 0.68 ± 0.38 Up Calreticulin Calreticulin CALR
Unigene14534_Mix − 0.55 ± 0.06 Down Discoidin domain receptor family Similar to collagen, type XXVIII DDR2
member 1
Unigene18363_Mix 0.84 ± 0.2 Up Tankyrase Osteoclast-stimulating factor OSTF1
Unigene32215_Mix − 0.72 ± 0.16 Down Mucin-5B Mucin-2-like, partial MUC5B
Unigene66416_Mix − 0.42 ± 0.14 Down Collagen, type VI, alpha Collagen alpha-5(VI) chain-like COL6A6
Unigene68607_Mix − 0.53 ± 0.09 Down collagen, type XII, alpha GF11504 COL22A1

Stress-related heat shock proteins

Unigene139105_Mix 0.59 ± 0.12 Up Heat shock 70 kDa protein 1/8 Heat shock cognate 71 kDa protein-like HSPA8
Unigene30536_Mix* 0.85 ± 0.04 Up TNF receptor-associated protein 1 Heat shock protein 75 kDa, mitochondrial TRAP1
Unigene34513_Mix − 0.67 ± 0.31 Down Chaperonin GroES 10 kDa heat shock protein HSPE1/CPN10
Unigene46718_Mix 1.4 ± 0.08 Up – Heat shock protein HSP 90-alpha HSP90AA1
Unigene47110_Mix 0.63 ± 0.09 Up – Heat shock cognate 71 kDa protein-like HSPA8

Fluorescent protein
Unigene116047_Mix 0.84 ± 0.31 Up – Green fluorescent protein –
Unigene19745_Mix 1.17 ± 0.1 Up – Fluorescent protein –
Unigene45390_Mix − 0.36 ± 0.06 Down – Green fluorescent protein –
Unigene45392_Mix 0.44 ± 0.1 Up – Red fluorescent GFP-like protein –

3.3. Proteomic vs. transcriptomic dataset
To investigate the post-transcriptional regulation in GA affected
tissue, the published transcriptome dataset (Zhang et al., 2017) was
compared with the current proteomic dataset. The correlations between
mRNA and protein expression were weak yet positive (R = 0.22 in
normal tissue; R = 0.23 in GA affected tissue) and statistically significant
(p = 3e-10 in normal tissue; p = 6.9e-10 in GA affected tissue) (Sup­
plementary Fig. 1). Three DEGs reported previously corresponded to
DEPs in the current proteomic study (Supplementary Table 1). Two of
them were annotated as fluorescent proteins and were consistently up-
regulated in GA affected tissue at both the protein and mRNA levels,
indicating these proteins were actively involved in the GA formation at
both the transcript and protein levels. On the other hand, a DEP anno­
tated as “myosin-2 essential light chain-like” was consistently down-
regulated at both the protein and mRNA levels. Comparison of the
result of GO enrichment analysis further demonstrate the qualitative
differences between DEG and DEP. GO enrichment analysis of DEG
revealed enrichment of GO terms relating to cellular developmental
processes such as “Regulation of tissue remodeling”, “Osteoblast dif­
ferentiation”, “Gastrulation” (refer to Table 2 in Zhang et al., 2017)
while for DEPs, enrichment of GO terms relating to housekeeping
functions such as “translational elongation” and terms relating to
metabolism such as “cellular amino acid and derivative metabolic

6
Y.-H. Wong et al. Marine Pollution Bulletin 164 (2021) 111982

process” (Supplementary Table 2).
3.4. Protein-protein interaction (PPI) network analysis of DEPs
With proper statistical controls, PPI network analysis enables visu­
alization of PPI networks that are closely associated with the identified
proteins (Antonov et al., 2009) and could provide extra insights into the
underlying biological phenomenon. In order to perform PPI network
analysis associated with the GA induced proteomic changes, the DEPs
were firstly annotated to the human genome. Out of the 117 DEPs, 95
were annotated and 55 were mapped to the human reference PPI
network. The D2 PPI spider network model (allowed 1 missing protein in
the PPI network) inferred from DEPs, covered 40 proteins with a p-value
<0.005. The inferred network model as shown in Fig. 4 composed of 74
nodes and 82 edges. Interacting protein pairs in the inferred PPI spider
network are shown in Supplementary Table 3.
Out of 40 mapped DEPs, 11 were ribosomal proteins (RP) that were
functionally related to the term “translational elongation” (as red rect­
angle nodes in Fig. 4); four proteasome proteins (PSMs) that were
related to the term “negative regulation of ubiquitin-protein ligase ac­
tivity involved in mitotic cell cycle” (as green rectangle nodes in Fig. 4);
three HSPs that were related to “response to unfolded protein” (as light
blue rectangle nodes in Fig. 4). Note that all these DEPs were up-
regulated in GA affected tissue.
It is worth to note that four proteins related to “response to DNA
damage stimulus” were reported as “missing points” in the inferred PPI
network but were not detected as DEPs. These missing proteins were
Tank-binding kinase 1 (TBK1), Tumor Necrosis Factor (TNF) receptor
associated factor 1 (TRAF1), TNF receptor type 1-associated DEATH
domain protein (TRADD) and receptor interacting Serine/Theronine
Kinase 2 (RIPK2). TBK1 reportedly interact with the GAPDH and RP
small subunit 8 (RPS8); TRAF4 reportedly interact with GAPDH and
RPS3; while TRAF4 and TRADD were reportedly interacting with the up-
regulated RP small subunit S3 (RPS3). Other non-DEP nodes in the
inferred PPI network were related to “positive regulation of i-kappab
kinase(IkB)/nf-kappab(NFkB) cascade”, “mRNA processing”, “cell cycle
arrest” and “regulation of growth”.
Overall, our PPI network analysis highlights the involvement of in
protein translation, protein degradation and stress responses in coral
GA, as well as disruption of the TNF “death” signaling cascade in the GA
affected tissue.
4. Discussion
4.1. Changes in the amino acid metabolism and lysosomal pathway
Significant down-regulation of metabolic pathway proteins, espe­
cially proteins relating to amino acid biosynthesis and lysosomal

Fig. 4. Protein-protein interaction (PPI) network model inferred from 117 differentially expressed coral proteins. In this model one missing protein between each
node pair (interacting protein pair) in the reference global PPI network was allowed. The sub-network enrichment p-value was <0.005. Rectangle nodes represent
differentially expressed proteins between normal and GA affected tissue. Triangle nodes represent missing genes that are present in the reference global PPI network
but missing in the list of DEP. Genes belonging to different functional categories are colored according to the diagnostic chart in the figure. (For interpretation of the
references to color in this figure, the reader is referred to the web version of this article.)

7
Y.-H. Wong et al.
proteins suggested the disruption of metabolism in the GA affected tis­
sue. The lysosomal pathway is usually involved in catabolism and
recycling of material taken up by endocytosis (review by Watts, 2012).
The down-regulation of metabolic pathway proteins and lysosomal
proteins in the GA affected tissue could indicate malnutrition and
disruption of protein catabolism. Interestingly, in M. capitata, down-
regulation of major metabolic proteins was not reported in the GA
affected tissue. Instead, lysosomal acid lipase was reportedly up-
regulated in the GA affected tissue in M. capitata (Frazier et al., 2017).
4.2. Immune system components
In the current study, TRAF1 and TRADD which interact with the
receptor of the signaling molecule TNF (TNF receptor), were reported as
“missing proteins” in the PPI network inferred from the DEPs. According
to the reference (human) PPI network, two up-regulated DEPs, GAPDH
and RPS3 interact with TRAF4 and TRADD, respectively. TRAP1 (also
known as HSPE1), which also interacts with TNF receptor, was also up-
regulated. Activation of TNF receptor can directly lead to cell death via
apoptosis or indirectly lead to cell survival and inflammatory responses
through activation of downstream transcription factors (Schultz and
Harringto Jr., 2003). Up-regulation of proteins associated with TNF
signaling were often associated with GA and other coral diseases. For
example, TNF receptor associated factor 4 (TRAF4) was up-regulated in
GA lesion affected Montipora capitata tissue (Frazier et al., 2017). Hence,
the up-regulation of TRAP1/HSPE1, GAPDH and RPS3, which directly
or indirectly associating with TNF receptor signaling, suggested acti­
vation of TNF receptor in GA affected tissue in P. carnosa and was
consistent with the activation of immune system in corals affected by
different diseases and environmental stresses.
4.3. Cell apoptosis
One of the possible outcomes of TNF receptor activation is cell death
via apoptosis. Apoptosis, or programmed cell death, which serves as a
homeostatic mechanism to maintain cell populations in tissues and is
part of the immune system (Norbury and Zhivotovsky, 2004). The
Apoptosis Inducible Factor (AIF) is a mitochondrial protein that can
trigger caspase independent apoptosis. AIF could reportedly interact
with TRADD and is hence one of the downstream components of TNF
signaling. While PPI network analysis related the DEPs with proteins
involved in TNF receptor activation, AIF was down-regulated. In addi­
tion, none of the apoptotic caspase proteins were detected in the dif­
ferential expression analysis. Lysosomal proteins, which could also
involve in the degradation process during apoptosis, were also down-
regulated. Overall, our proteomic data suggested that apoptosis was
unlikely to be triggered in GA affected tissue.
Reactive oxygen species (ROS) stress is another major factor that
could directly lead to cell apoptosis. However, SOD2, which neutralize
the toxic and is reportedly up-regulated in response to ROS stress, was
down-regulated in GA affected tissue. This suggested that GA affected
tissue was not suffering from ROS stress.
Lysosomes also play an important role in the progression of
apoptosis. Proteases from the endosomal/lysosomal system are involved
in the execution of apoptosis, especially in pathological conditions
(Guicciardi et al., 2004). The down-regulation of lysosomal proteins
indirectly suggested that phagocytosis and apoptosis were unlikely to be
higher in GA affected tissue.
In coral, apoptotic genes were found to be up-regulated in
A. cervicornis suffering from White Band Disease (WBD) (Libro et al.,
2013). However, apoptotic genes expression was seldom reported in GA
affected coral tissue. Previous report has shown increased proliferation
and reduced apoptosis levels in GAs of Porites australiensis and Montipora
informis (Yasuda and Hidaka, 2012). Hence, our results supported that
cell apoptosis was not a major cellular event of GA affected tissue.
8
Marine Pollution Bulletin 164 (2021) 111982
4.4. Translational activities, molecular chaperone, and protein
degradation
One of the major findings in the current study is the up-regulation of
proteins involved in translational activities, molecular chaperone, and
protein degradation. Interestingly, some of these proteins have been
considered as house-keeping proteins that are constituently expressed.
For instance, expression of ribosomal proteins (RPs) was reported to be
maintained at a constant level in the scleractinian coral Acropora mil­
lepora undergoing natural bleaching (Seneca et al., 2010). As such, RP
was used as the internal control genes for standardization in semi-
quantitative gene expression analysis in certain studies (Seneca et al.,
2010; Rodriguez-Lanetty et al., 2008). However, RPs in the staghorn
coral Acropora cervicornis were down-regulated in White Band Disease
(WBD)-resistant corals but up-regulated in WBD-susceptible corals
(Libro and Vollmer, 2016). Hence, there could be remarkable variations
in RP gene or protein expressions in coral tissue affected by different
types of coral diseases.
Up-regulation of chaperone proteins HSPs could be linked to the up-
regulation of RPs, due to the cooperative role of HSPs in the translation
machinery (Vabulas et al., 2010). HSPs play a vital role in assisting the
correct folding of nascent polypeptides and proteins (Miyata et al.,
2013). They usually have multiple targets/clients and their expressions
are essential for adaptive responses to stress (Wandinger et al., 2008).
For instance, the 90 kDa heat shock protein (HSP90) has more than 200
client proteins (Trepel et al., 2010) and has been adopted as a general
stress-indicator in different taxa. In the current study, chaperone pro­
teins, including heat shock proteins (HSPs) 90-alpha, 75 kDa HSP, heat
shock cognate 71 kDa protein-like were up-regulated in GA affected
tissue. HSPs were also observed in Porites compressa GA affected tissue
(Domart-Coulon et al., 2006). Hence, the up-regulation of HSPs may
indicate that GA affected coral tissue in P. carnosa showed signs of stress.
Stress responses are often linked to ubiquitin–proteasome protein
degradation pathway, because proteins damaged by acute stress have to
either be rescued by chaperones via refolding or be rapidly degraded
(Aiken et al., 2011). Hence, the up-regulation of 20S proteasome subunit
proteins, which are part of the proteasome toolkit responsible for pro­
tein degradation, could be linked to the up-regulation of RPs and HSPs.
However, it is still unclear if the response in GA-affected tissues is a
consequence of internal changes such as abnormality in cellular activ­
ities or as a result of exposure to external stresses.
4.5. Calcification
Abnormal skeleton growth is the major symptom of GA affected
tissue. In P. carnosa, protuberant porous skeleton, active vertical skeletal
growth, and low bone mass were observed in GA affected tissue, indi­
cating indicates dysregulation in skeleton formation. Our proteomic
analysis revealed four of the six DEPs implicated in calcification were
down-regulated. Three of these down-regulated are collagens, which
were reportedly the major proteins of the organic matrix layer in coral
skeleton, molluscan shells and human bones (Drake et al., 2013a; Levi-
Kalisman et al., 2001; Keogh et al., 2010). In addition, carbonic anhy­
drase, one of the essential enzymes for calcification, was not differen­
tially expressed in GA affected tissue on neither mRNA nor protein level.
Gene expression of several types of collagens was also lower in GA
affected tissue of M. capitata compared to normal tissues (Frazier et al.,
2017). Five genes that have been reported to be associated with osteo­
genesis, including genes encoding low-density lipoprotein receptor
related protein 5 (LRP5), bone morphogenetic protein 1 (BMP1), com­
plement component C3 (C3), Rho guanine nucleotide exchange factor
(GEF) and heparin sulfate proteoglycan (HSPG) were reportedly down-
regulated in GA affected tissue or only detected in normal tissue in the
previous transcriptomic analysis (Zhang et al., 2017). On the other hand,
OSF, a negative regulator of calcification in sponge and human (Müller
et al., 2012; Reddy et al., 1998; Vermeren et al., 2017), was up-
Y.-H. Wong et al. Marine Pollution Bulletin 164 (2021) 111982

regulated. Hence, both transcriptome and proteomic analyses hint to a
reduction in calcification in GA affected tissue.
The calcium binding protein calreticulin, which plays an important
role in Ca2+ absorption from seawater (Wilbur and Saleuddin, 1983),
was the only up-regulated protein implicated in calcification. However,
calreticulin is also associated with intracellular Ca2+ signaling pathway
and whether this differential expression was involved in calcification in
GA affected tissue remains unclear.
The down-regulation of major calcification genes could have
contributed to the formation of protuberant porous skeleton in GA
affected tissue. The down-regulation of calcification genes in P. carnosa
GA affected tissue suggested reduced calcification comparing to the
unaffected tissue. Hence, accelerated vertical skeleton growth was not
accompanied by increased calcification, which may result in a reduction
of skeleton density and a porous skeleton anatomy (as shown in Fig. 1 in
Zhang et al., 2017). Additional experiments, such as analysis of the
elemental differences and proteomic profiling of the organic matrix
proteins in GA affected tissue and normal tissues, would be promising in
further revealing the molecular mechanism of abnormal skeleton
growth.
4.6. Fluorescence proteins
In addition to abnormal skeleton growth, pigmentation alteration is
another common symptom of GA. Up-regulation of FPs was consistently
detected in this proteomic study and in the previous transcriptomic
study (Zhang et al., 2017). Increased expression of FPs in GA affect
tissues might be correlated with the coloration alteration in GA affected
P. carnosa tissues (Fig. 1 in Zhang et al., 2017). In corals, Green fluo­
rescent proteins (GFPs) and GFP-like proteins are the major FPs. The
variations in the absorption spectrum of GFP or GFP-like proteins will
affect the pigmentation of coral tissue. GFP or GFP-like proteins absorb
high energy (low wavelength) light wave and offer photoprotection to
coral tissues (Smith et al., 2013; DeSalvo et al., 2008). GFP or GFP-like
proteins have been suggested to contribute to the coloration changes of
different corals species in response to wounding and infestation by
epibionts/parasites (Bonesso et al., 2017). In Porites lobata, high
abundance of GFPs and high phenoloxidase activity were observed in
the proliferating regions that exhibit accelerated growth (D’Angelo
et al., 2012). GFP-like proteins could also serve as a superoxide radical
quencher or oxidant and protect coral tissues from reactive oxidant
stress (Bou-Abdallah et al., 2006; Palmer et al., 2009). Interestingly, our
dataset also hinted to the up-regulation of certain immune system
components, such as a number of TNF receptor associating proteins.
SOD2, a detoxification enzyme that metabolizes reactive oxygen species
(Chen et al., 2009), was also up-regulated, suggesting the need for
metabolism of ROS in GA-affected tissue. Hence, coloration change in
GA affected tissue, which is likely the result of differential expression of
FPs, could be associated with the intrinsic protection mechanism.
4.7. Comparison with literature on coral diseases
The development of GA may indicate compromised coral health
conditions. We summarized the key findings of the current study and
those from a number of gene expression and biochemical studies on the
coral diseases, A. cervicornis (Libro et al., 2013), A. millepora (Palmer
et al., 2008), Montipora capitata (Spies and Takabayashi, 2013; Frazier
et al., 2017) and Porites compressa (Domart-Coulon et al., 2006) to
facilitate comparison between the molecular pathology of different
diseases and bleaching conditions on different coral species (Table 2).
A common finding among the gene expression studies of coral dis­
eases and bleaching is the differential expression, mostly up-regulation,
of immune genes, such as a number of TNF receptor associating proteins
including TRAF4. Other consensuses include the up-regulation of genes
relate to cell differentiation, osteoblast, development. In addition to
discovering the changes in immune related genes associated with GA
formation, our proteomic analysis also revealed the differential
expression of molecular chaperones (such as HSP90), ribosomal proteins
and lysosomal proteins.
In general, both “omic” studies on P. carnosa GA suggested a
consistent trend in different biological processes, such as the reduction
of calcification and changes in fluorescent proteins, which were also
reported in the study of GA in other coral species. We believe, for future
studies, multi-omics approaches that integrate genomic, transcriptomic,

Table 2
Gene expression studies on the molecular biology of coral diseases/bleaching.
Species Disease/episode Technique Major findings Reference

Acropora millepora non-normally Phenoloxidase (PO) Up-regulation of PO activity in areas of non-normal pigmentation in Palmer et al. (2008)
& Porites sp. pigmented tissues activity assay A. millepora
Increased melanin production in pigmented Porites tissues
Acropora Growth anomalies Phenoloxidase (PO) Higher total potential PO activity Palmer & Baird
hyacinthus (GA lesion tissue) activity assay (2018)
Montipora capitata Growth anomalies Quantitative reverse Down-regulation: tyrosine protein kinase (TPK), βγ-crystallin (BGC) Spies and
(GA lesion tissue) transcriptase PCR No change: Galaxin, murine double minute 2 (MDM2), tumor necrosis factor Takabayashi (2013)
(TNF)
Acropora White Band Disease RNAseq Differential expression: macrophage-mediated pathogen recognition and Libro et al. (2013)
cervicornis (WBD) ROS production, two hallmarks of phagocytosis, apoptosis and calcium
homeostasis
No change: Toll-like receptors (TLR), Complement, and prophenoloxydase
pathways
Up-regulation: allene oxide synthase-lipoxygenase
Acropora White syndromes RNAseq Activation of several innate immunity pathways Wright et al. (2015)
hyacinthus Activation of tissue repair pathways
Reduction of calcification
Switch towards lipid storage
Montipora capitata Growth anomalies RNAseq Differential expression: morphogenesis, organogenesis, and immune Frazier et al. (2017)
(GA lesion tissue) response
No change: cell cycle control, metabolism, DNA repair pathways
Platygyra canosa Growth anomalies RNAseq (meta- Differential gene expression in Zhang et al. (2017)
transtranscriptome) HOST: (GOBP terms) osteogenesis, oncogenesis and immune genes
Holobiont: (GOBP terms) reproduction, nitrogen metabolism and pigment
formation
Platygyra canosa Growth anomalies iTRAQ labeling, shotgun Up-regulation: ribosomal proteins, heat shock proteins (HSPs) 90-alpha, This study
proteomics HSP-75 kDa HSP 71 kDa-like, down-regulation: no change:
Porites compressa Growth anomalies ELISA Up-regulation: MutY, HSP90a1, GRP75 and metallothionein Domart-Coulon
et al. (2006)

9
Y.-H. Wong et al.
proteomics and metabolomic analyses could provide the most compre­
hensive and interactive view of the molecular pathology of coral GA.
4.8. Possible causative factors of GA
Coral GAs have been suggested to be associated with different fac­
tors, and our data demonstrated that stress responsive proteins RPs and
HSPs were up-regulated in GA affected tissue in P. carnosa. Up-
regulation of RPs and HSPs are implicated in both alteration of
intrinsic mechanisms (such as tumor-like condition) (Xu et al., 2016;
Trepel et al., 2010) and invasion of micro-organisms or viruses (Dutta
et al., 2009; Campos et al., 2017). Hence, whether the stress response in
GA affected tissue was initiated by internal stresses such as dysregula­
tion of cell cycle due to tumor-like mutations or external factors such as
pollution and infection by microorganisms remain unclear.
Nonetheless, C-type MBL, which is considered the pattern recogni­
tion protein, and lysosomal proteins, which are involved in the degra­
dation of engulfed materials during phagocytosis, were all down-
regulation (review by Watts, 2012). These data overall suggested that
the stress condition in GA is unlikely to be triggered by microbial
infections.
5. Concluding remarks
Our proteomic analyses revealed up-regulation of stress responsive
proteins and down-regulation of apoptotic proteins, lysosomal proteins,
proteins implicated in calcification and amino acid metabolic proteins in
GA affected tissue of P. carnosa. Up-regulation of fluorescent proteins at
both the mRNA and protein level could be relating to the coloration
changes in GA. In addition to calcification and immune related genes,
our differential protein expression analysis indicated that structural and
housekeeping genes, were also involved in GA formation. Finally, our
data do not support the hypothesis of bacterial infection as a causative
factor in GA formation.
CRediT authorship contribution statement
Yue-Him Wong: Conceptualization, Formal analysis, Funding
acquisition, Investigation, Methodology, Project administration, Super­
vision, Validation, Writing – original draft, Writing – review & editing.
Yu Zhang: Conceptualization, Formal analysis, Funding acquisition,
Investigation, Methodology, Project administration, Supervision, Vali­
dation, Writing – original draft, Writing – review & editing. Janice C.Y.
Lun: Conceptualization, Resources. Jian-Wen Qiu: Conceptualization,
Data curation, Funding acquisition, Investigation, Project administra­
tion, Supervision, Writing – review & editing.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgements
We thank Dr. Jin Sun for his contribution to the early stage of this
study. J.W.Q. was supported by the Southern Marine Science and En­
gineering Guangdong Laboratory (Guangzhou) (GML2019ZD0404,
L2019005), Environment and Conservation Fund (2017–03) and Gen­
eral Research Fund of Hong Kong (12102018). Y. Z. was supported by a
seed funding from the Scientific and Technical Innovation Council of
Shenzhen Government (Grant No. 827000012), and Natural Science
Foundation of Guangdong Province (Grant No. 2020A1515011117). Y.
H. W. was supported by a seed funding from the Shenzhen University
(Grant No. 2110380) and the National Natural Science Foundation of
China (41906091).
10
Marine Pollution Bulletin 164 (2021) 111982
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.marpolbul.2021.111982.
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.
Aiken, C.T., Kaake, R.M., Wang, X., Huang, L., 2011. Oxidative stress-mediated
regulation of proteasome complexes. Mol. Cell. Proteomics 10, R110.006924.
Antonov, A.V., 2011. BioProfiling. de: analytical web portal for high-throughput cell
biology. Nucleic Acids Res. 39(suppl_2), W323-327.
Antonov, A.V., Dietmann, S., Rodchenkov, I., Mewes, H.W., 2009. PPI spider: a tool for
the interpretation of proteomics data in the context of protein–protein interaction
networks. Proteomics 9, 2740–2749.
Benjamini, Y., Hochberg, Y., 1995. Controlling the false discovery rate: a practical and
powerful approach to multiple testing. J. Roy. Stat. Soc. B. Met. 57, 289–300.
Bonesso, J.L., Leggat, W., Ainsworth, T.D., 2017. Exposure to elevated sea-surface
temperatures below the bleaching threshold impairs coral recovery and regeneration
following injury. PeerJ. 5, e3719.
Bou-Abdallah, F., Chasteen, N.D., Lesser, M.P., 2006. Quenching of superoxide radicals
by green fluorescent protein. BBA-Gen. Subjects 1760, 1690–1695.
Bruckner, A.W., Hill, R.L., 2009. Ten years of change to coral communities off Mona and
Desecheo Islands, Puerto Rico, from disease and bleaching. Dis. Aquat. Org. 87,
19–31.
Bruno, J.F., Selig, E.R., Casey, K.S., Page, C.A., Willis, B.L., et al., 2007. Thermal stress
and coral cover as drivers of coral disease outbreaks. PLoS Biol. 5, 1220–1227.
Burns, J.H.R., 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., Rozet, N.K., Takabayashi, M., 2011. Morphology, severity, and
distribution of growth anomalies in the coral, Montipora capitata, at Wai’pae,
Hawai’i. Coral Reefs 30, 819–826.
Campos, R.K., Wong, B., Xie, X., Lu, Y.F., Shi, P.Y., Pompon, J., Garcia-Blanco, M.A.,
Bradrick, S., 2017. RPLP1 and RPLP2 are essential flavivirus host factors that
promote early viral protein accumulation. J. Virol. 91, e01706–e01716.
Cande, C., Vahsen, N., Garrido, C., Kroemer, G., 2004 Jun 1. Apoptosis-inducing factor
(AIF): caspase-independent after all. Cell Death Differ. 11 (6), 591.
Chen, Y., Azad, M.B., Gibson, S.B., 2009. Superoxide is the major reactive oxygen species
regulating autophagy. Cell Death Differ. 16, 1040–1052.
D’Angelo, C., Smith, E.G., Oswald, F., Burt, J., Tchernov, D., Wiedenmann, J., 2012.
Locally accelerated growth is part of the innate immune response and repair
mechanisms in reef-building corals as detected by green fluorescent protein (GFP)-
like pigments. Coral Reefs 31 (4), 1045–1056.
Deng, W., Wang, Y., Liu, Z., Cheng, H., Xue, Y., 2014. HemI: a toolkit for illustrating
heatmaps. PLoS One 9, e111988.
DeSalvo, M.K., Voolstra, C.R., Sunagawa, S., Schwarz, J.A., Stillman, J.H., Coffroth, M.
A., Szmant, A.M., Medina, M., 2008. Differential gene expression during thermal
stress and bleaching in the Caribbean coral Montastraea faveolata. Mol. Ecol. 17,
3952–3971.
Domart-Coulon, I.J., Traylor-Knowles, N., Peters, E., Elbert, D., Downs, C.A., Price, K.,
Stubbs, Joanne, McLaughlin, Shawn, Cox, Evelyn, 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.
Drake, J.L., Mass, T., Haramaty, L., Zelzion, E., Bhattacharya, D., Falkowski, P.G., 2013a.
Proteomic analysis of skeletal organic matrix from the stony coral Stylophora
pistillata. Proc. Natl. Acad. Sci. U. S. A. 110, 3788–3793.
Drake, J.L., Mass, T., Haramaty, L., Zelzion, E., Bhattacharya, D., Falkowski, P.G., 2013b.
Reply to Ramos-Silva et al.: regarding coral skeletal proteome. Proc. Natl. Acad. Sci.
U. S. A. 110, E2147–E2148.
Dutta, D., Bagchi, P., Chatterjee, A., Nayak, M.K., Mukherjee, A., Chattopadhyay, S.,
et al., 2009. The molecular chaperone heat shock protein-90 positively regulates
rotavirus infection. Virol. 391, 325–333.
Frazier, M., Helmkampf, M., Bellinger, M.R., Geib, S.M., Takabayashi, M., 2017. De novo
metatranscriptome assembly and coral gene expression profile of Montipora capitata
with growth anomaly. BMC Genomics 18, 710.
Gateño, D., Leon, A., Barki, Y., Cortes, J., Rinkevich, B., 2003. Skeletal tumor formations
in the massive coral Pavona clavus. Mar. Ecol. Prog. Ser. 258, 97–108.
Guicciardi, M.E., Leist, M., Gores, G.J., 2004. Lysosomes in cell death. Oncogene 23,
2881–2890.
Han, Z., Sun, J., Zhang, Y., He, F., Xu, Y., Matsumura, K., He, L.S., Qiu, J.W., Qian, P.Y.,
2013. iTRAQ-based proteomic profiling of the barnacle Balanus amphitrite in
response to the antifouling compound meleagrin. J. Proteome Res. 12 (5),
2090–2100.
Hussain, A.De.K., Thomas, L., Nagesh, R., Mote, S., Ingole, B., 2016. Prevalence of
skeletal tissue growth anomalies in a scleractinian coral: Turbinaria mesenterina of
Malvan Marine Sanctuary, eastern Arabian Sea. Dis. Aquat. Org. 121, 79–83.
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.
Y.-H. Wong et al.
Keogh, M.B., O’Brien, F.J., Daly, J.S., 2010. A novel collagen scaffold supports human
osteogenesis—applications for bone tissue engineering. Cell Tissue Res. 340,
169–177.
Levi-Kalisman, Y., Falini, G., Addadi, L., Weiner, S., 2001. Structure of the nacreous
organic matrix of a bivalve mollusk shell examined in the hydrated state using cryo-
TEM. J. Struct. Biol. 135, 8–17.
Libro, S., Vollmer, S.V., 2016. Genetic signature of resistance to white band disease in the
Caribbean staghorn coral Acropora cervicornis. PLoS One 11, e0146636.
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.
Maere, S., Heymans, K., Kuiper, M., 2005. BiNGO: a Cytoscape plugin to assess
overrepresentation of gene ontology categories in biological networks.
Bioinformatics. 21, 3448–3449.
Mass, T., Drake, J.L., Peters, E.C., Jiang, W., Falkowski, P.G., 2014. Immunolocalization
of skeletal matrix proteins in tissue and mineral of the coral Stylophora pistillata.
Proc. Natl. Acad. Sci. U. S. A. 111, 12728–12733.
McClanahan, T.R., Weil, E., Maina, J., 2009. Strong relationship between coral bleaching
and growth anomalies in massive Porites. Glob. Change Biol. 15, 1804–1816.
McGreal, E.P., Martinez-Pomares, L., Gordon, S., 2004. Divergent roles for C-type lectins
expressed by cells of the innate immune system. Mol. Immunol. 41, 1109–1121.
Mi, H., Muruganujan, A., Casagrande, J.T., Thomas, P.D., 2013. Large-scale gene
function analysis with the PANTHER classification system. Nat. Protoc. 8,
1551–1566.
Miyata, Y., Nakamoto, H., Neckers, L., 2013. The therapeutic target Hsp90 and cancer
hallmarks. Curr. Pharm. Design 19, 347–365.
Müller, W.E., Wang, X., Grebenjuk, V.A., Korzhev, M., Wiens, M., Schlossmacher, U.,
Schröder, H.C., 2012. Common genetic denominators for Ca++-based skeleton in
Metazoa: role of osteoclast-stimulating factor and of carbonic anhydrase in a
calcareous sponge. PLoS One 7, e34617.
Norbury, C.J., Zhivotovsky, B., 2004. DNA damage-induced apoptosis. Oncogene. 23,
2797–2808.
Palmer, C.V., Baird, A.H., 2018. Coral tumor-like growth anomalies induce an immune
response and reduce fecundity. Dis. Aquat. Org. 130 (1), 77–81.
Palmer, C.V., Mydlarz, L.D., Willis, B.L., 2008. Evidence of an inflammatory-like
response in non-normally pigmented tissues of two scleractinian corals. Proc. R. Soc.
London. Ser. B. 275, 2687–2693.
Palmer, C.V., Modi, C.K., Mydlarz, L.D., 2009. Coral fluorescent proteins as antioxidants.
PLoS One 4, e7298.
Peters, E.C., Halas, J.C., McCarty, H.B., 1986. Calicoblastic neoplasms in Acropora
palmata, with a review of reports on anomalies of growth and form in corals. J. Natl.
Cancer Inst. 76, 895–912.
Qiu, J.W., Lau, D.C., Cheang, C.C., Chow, W.K., 2014. Community-level destruction of
hard corals by the sea urchin Diadema setosum. Mar. Pollut. Bull. 85 (2), 783–788.
Ramos-Silva, P., Kaandorp, J., Huisman, L., Marie, B., Zanella-Cléon, I., Guichard, N.,
Miller, D.J., Marin, F., 2013. The skeletal proteome of the coral Acropora millepora:
the evolution of calcification by co-option and domain shuffling. Mol. Biol. Evol. 30,
2099–2112.
Reddy, S., Devlin, R., Menaa, C., Nishimura, R., Choi, S.J., Dallas, M., Yoneda, T.,
Roodman, G.D., 1998. Isolation and characterization of a cDNA clone encoding a
novel peptide (OSF) that enhances osteoclast formation and bone resorption. J. Cell.
Physiol. 177, 636–645.
Rodriguez-Lanetty, M., Phillips, W.S., Dove, S., Hoegh-Guldberg, O., Weis, V.M., 2008.
Analytical approach for selecting normalizing genes from a cDNA microarray
platform to be used in q-RT-PCR assays: a cnidarian case study. J. Biochem. Bioph.
Meth. 70, 985–991.
Schultz, D.R., Harringto, Jr. W.J., 2003. Apoptosis: programmed cell death at a
molecular level. In Seminars in arthritis and rheumatism (Vol. 32, No. 6, pp. 345-
369). WB Saunders.
Seneca, F., Foret, S., Ball, E., Smith-Keune, C., Miller, D., van Oppen, M.J.H., 2010.
Patterns of gene expression in a Scleractinian coral undergoing natural bleaching.
Mar. Biotechnol. 12, 594–604.
Shannon, P., Markiel, A., Ozier, O., Baliga, N.S., Wang, J.T., Ramage, D., Amin, N.,
Schwikowski, B., Ideker, T., 2003. Cytoscape: a software environment for integrated
models of biomolecular interaction networks. Genome Res. 13, 2498–2504.
11
Marine Pollution Bulletin 164 (2021) 111982
Smith, E.G., D’Angelo, C., Salih, A., Wiedenmann, J., 2013. Screening by coral green
fluorescent protein (GFP)-like chromoproteins supports a role in photoprotection of
zooxanthellae. Coral Reefs 32, 463–474.
de Sousa Abreu, R., Penalva, L.O., Marcotte, E.M., Vogel, C., 2009. Global signatures of
protein and mRNA expression levels. Mol. BioSyst. 5, 1512–1526.
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.
Squires, D.F., 1965. Neoplasia in a coral? Science 148, 503–505.
Stimson, J., 2011. Ecological characterization of coral growth anomalies on Porites
compressa in Hawai‘i. Coral Reefs 30, 133–142.
Sun, J., Mu, H.W., Zhang, H.M., Chandramouli, K.H., Qian, P.Y., Wong, C.K.C., Qiu, J.W.,
2013a. Understanding the regulation of estivation in a freshwater snail through
iTRAQ-based comparative proteomics. J. Proteome Res. 12, 5271–5280.
Sun, J., Chen, Q., Lun, J.C.Y., Xu, J., Qiu, J.W., 2013b. PcarnBase: development of a
transcriptomic database for the brain coral Platygyra carnosus. Mar. Biotechnol. 15,
244–251.
Sun, J., Wang, M., Wang, H., Zhang, H., Zhang, X., Thiyagarajan, V., Qian, P.Y., Qiu, J.
W., 2012. De novo assembly of the transcriptome of an invasive snail and its multiple
ecological applications. Mol. Ecol. Resour. 12, 1133–1144.
Sutherland, K.P., Porter, J.W., Torres, C., 2004. Disease and immunity in Caribbean and
Indo-Pacific zooxanthellate corals. Mar. Ecol. Prog. Ser. 266, 273–302.
Trepel, J., Mollapour, M., Giaccone, G., Neckers, L., 2010. Targeting the dynamic HSP90
complex in cancer. Nat. Rev. Cancer 10, 537–549.
Vabulas, R.M., Raychaudhuri, S., Hayer-Hartl, M., Hartl, F.U., 2010. Protein folding in
the cytoplasm and the heat shock response. CSH Perspect. Biol. 2, a004390.
Vermeren, M., Lyraki, R., Wani, S., Airik, R., Albagha, O., Mort, R., Hildebrandt, F.,
Hurd, T., 2017. Osteoclast stimulation factor 1 (Ostf1) KNOCKOUT increases
trabecular bone mass in mice. Mamm. Genome 28, 498–514.
Veron, J., 2000. Corals of the World. Australian Institute of Marine Science, Townsville.
Vogel, C., Marcotte, E.M., 2012. Insights into the regulation of protein abundance from
proteomic and transcriptomic analyses. Nat. Rev. Gen. 13, 227–232.
Wandinger, S.K., Richter, K., Buchner, J., 2008. The Hsp90 chaperone machinery. J. Biol.
Chem. 283, 18473–18477.
Watts, C., 2012. The endosome–lysosome pathway and information generation in the
immune system. Biochimica et Biophysica Acta (BBA)-Proteins and Proteomics. Jan
1;1824(1):14-21.
Weis V.M., Levine R.P., 1996. Differential protein profiles reflect the different lifestyles
of symbiotic and aposymbiotic Anthopleura elegantissima, a sea anemone from
temperate waters. Journal of Experimental Biology. Apr 1;199(4):883-92.
Wilbur, K.M., Saleuddin A.S., 1983. Shell formation. In: vol. 6, The mollusca Jan 1 (pp.
235-287). Academic Press.
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.
Work, T.M., Kaczmarsky, L.T., Peters, E.C., 2015. Skeletal growth anomalies in corals. In:
Woodley, C.M., et al. (Eds.), Diseases of Coral. John Wiley & Sons, New Jersey.
https://doi.org/10.1002/9781118828502.ch20.
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 (1), 371.
Xie, C., Mao, X., Huang, J., Ding, Y., Wu, J., Dong, S., Kong, L., Gao, G., Li, C.Y., Wei, L.,
2011. KOBAS 2.0: a web server for annotation and identification of enriched
pathways and diseases. Nucleic Acids Res. 39 (Suppl. 2), W316–W322.
Xu, X., Xiong, X., Sun, Y., 2016. The role of ribosomal proteins in the regulation of cell
proliferation, tumorigenesis, and genomic integrity. Sci. China Life Sci.59, 656-672.
Yamashiro, H., Yamamoto, M., van Woesik, R., 2000. Tumor formation on the coral
Montipora informis. Dis. Aquat. Org. 41, 211–217.
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.
Yasuda, N., Nakano, Y., Yamashiro, H., Hidaka, M., 2012. Skeletal structure and
progression of growth anomalies in Porites australiensis in Okinawa. Japan. Dis.
Aquat. Org. 97, 237–247.
Zhang, Y., Sun, J., Mu, H., Lun, J.C., Qiu, J.W., 2017. Molecular pathology of skeletal
growth anomalies in the brain coral Platygyra carnosa: a meta-transcriptomic
analysis. Mar. Pollut. Bull. 124, 660–667.