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Coral bleaching events are increasing with such frequency and intensity that many of the world’s reef-building corals are in peril. Some corals appear to be more resilient after bleaching but the mechanisms underlying their ability to recover from bleaching and persist are not fully understood. We used shotgun proteomics to compare the proteomes of the outer layer (OL) tissue and inner core (IC) tissue and skeleton compartments of experimentally bleached and control (i.e., non-bleached) colonies of Montipora capitata , a perforate Hawaiian species noted for its resilience after bleaching. We identified 2,361 proteins in the OL and IC compartments for both bleached and non-bleached individuals. In the OL of bleached corals, 63 proteins were significantly more abundant and 28 were significantly less abundant compared to the OL of non-bleached corals. In the IC of bleached corals, 22 proteins were significantly more abundant and 17 were significantly less abundant compared to the IC of non-bleached corals. Gene ontology (GO) and pathway analyses revealed metabolic processes that were occurring in bleached corals but not in non-bleached corals. The OL of bleached corals used the glyoxylate cycle to derive carbon from internal storage compounds such as lipids, had a high protein turnover rate, and shifted reliance on nitrogen from ammonia to nitrogen produced from the breakdown of urea and betaine. The IC of bleached corals compartmentalized the shunting of glucose to the pentose phosphate pathway. Bleached corals increased abundances of several antioxidant proteins in both the OL and IC compartments compared to non-bleached corals. These results highlight contrasting strategies for responding to bleaching stress in different compartments of bleached M. capitata and shed light on some potential mechanisms behind bleaching resilience.
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fmars-09-797517 February 8, 2022 Time: 15:26 # 1
published: 14 February 2022
doi: 10.3389/fmars.2022.797517
Edited by:
Davide Seveso,
University of Milano-Bicocca, Italy
Reviewed by:
Rowan H. McLachlan,
Oregon State University,
United States
Jeremie Vidal-Dupiol,
Institut Français de Recherche pour
l’Exploitation de la Mer (IFREMER),
Jeremy B. Axworthy
These authors have contributed
equally to this work and share last
Specialty section:
This article was submitted to
Aquatic Physiology,
a section of the journal
Frontiers in Marine Science
Received: 18 October 2021
Accepted: 11 January 2022
Published: 14 February 2022
Axworthy JB,
Timmins-Schiffman E, Brown T,
Rodrigues LJ, Nunn BL and
Padilla-Gamiño JL (2022) Shotgun
Proteomics Identifies Active Metabolic
Pathways in Bleached Coral Tissue
and Intraskeletal Compartments.
Front. Mar. Sci. 9:797517.
doi: 10.3389/fmars.2022.797517
Shotgun Proteomics Identifies Active
Metabolic Pathways in Bleached
Coral Tissue and Intraskeletal
Jeremy B. Axworthy1*, Emma Timmins-Schiffman2, Tanya Brown1, Lisa J. Rodrigues3,
Brook L. Nunn2and Jacqueline L. Padilla-Gamiño1
1School of Aquatic and Fishery Sciences, University of Washington, Seattle, WA, United States, 2Department of Genome
Sciences, University of Washington, Seattle, WA, United States, 3Department of Geography and the Environment, Villanova
University, Villanova, PA, United States
Coral bleaching events are increasing with such frequency and intensity that many of
the world’s reef-building corals are in peril. Some corals appear to be more resilient
after bleaching but the mechanisms underlying their ability to recover from bleaching
and persist are not fully understood. We used shotgun proteomics to compare the
proteomes of the outer layer (OL) tissue and inner core (IC) tissue and skeleton
compartments of experimentally bleached and control (i.e., non-bleached) colonies of
Montipora capitata, a perforate Hawaiian species noted for its resilience after bleaching.
We identified 2,361 proteins in the OL and IC compartments for both bleached and
non-bleached individuals. In the OL of bleached corals, 63 proteins were significantly
more abundant and 28 were significantly less abundant compared to the OL of non-
bleached corals. In the IC of bleached corals, 22 proteins were significantly more
abundant and 17 were significantly less abundant compared to the IC of non-bleached
corals. Gene ontology (GO) and pathway analyses revealed metabolic processes that
were occurring in bleached corals but not in non-bleached corals. The OL of bleached
corals used the glyoxylate cycle to derive carbon from internal storage compounds
such as lipids, had a high protein turnover rate, and shifted reliance on nitrogen
from ammonia to nitrogen produced from the breakdown of urea and betaine. The
IC of bleached corals compartmentalized the shunting of glucose to the pentose
phosphate pathway. Bleached corals increased abundances of several antioxidant
proteins in both the OL and IC compartments compared to non-bleached corals. These
results highlight contrasting strategies for responding to bleaching stress in different
compartments of bleached M. capitata and shed light on some potential mechanisms
behind bleaching resilience.
Keywords: coral bleaching, eco-physiology, metabolic pathways, calcification, glyoxylate, betaine, proteins,
perforate skeleton
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Axworthy et al. Proteomics Bleached Coral Tissue Compartments
Rising seawater temperatures threaten the future of coral reefs
and the ecosystem services they provide. Increased temperatures
can result in the breakdown of mutualistic symbiosis between
corals and Symbiodiniaceae (i.e., coral bleaching), causing a
severe energy deficit to the coral that impairs critical physiological
functions and can lead to mortality. However, if temperature
stress abates before the coral’s energy stores are consumed,
symbiosis can be restored, and the coral can recover. To
survive and recover from bleaching, corals must rely on
internal energy and nutrient stores and/or employ alternative
strategies to sustain themselves until the stressor abates and
their Symbiodiniaceae population can recover (Grottoli et al.,
2006;Rodrigues and Grottoli, 2007). For example, some corals
exploit energy from stored lipids following bleaching, and some
corals increase feeding on zooplankton to make up the energy
deficit (Grottoli et al., 2006;Rodrigues and Grottoli, 2007;
Ferrier-Pagès et al., 2010;Hughes and Grottoli, 2013). Bleaching
comes with physiological costs and important consequences for
coral fitness, such as decreased fecundity and skeletal growth,
shifts in metabolic activity, increased antioxidant response and
susceptibility to diseases and mortality (Lesser et al., 1990;Ward
et al., 2000;Rodrigues and Grottoli, 2007;Cantin et al., 2010;
Muller et al., 2018). While these processes are well documented,
and we are beginning to understand their underlying cellular
mechanisms, many knowledge gaps remain, including how a
coral’s proteome responds to bleaching stress and how variable
the response is in different parts of the colony. Identifying
such proteins and patterns can help to develop physiological
biomarkers of coral resilience and improve our mechanistic
understanding of recovery from bleaching.
The coral host has two distinct compartments: the tissue,
where cellular functions involved in the overall maintenance
and fitness of the coral occur; and the skeleton, which provides
structural foundation for individual corals and entire reef
ecosystems. The coral’s polyps and interconnecting tissue or
coenosarc between polyps make up the tissue fraction where
physiological functions such as feeding, nutrient acquisition
and reproduction occur, and it is in this compartment where
the majority of Symbiodiniaceae reside (Yost et al., 2013).
The coral’s skeleton consists of an inorganic aragonitic calcium
carbonate structure deposited by the coral tissue and an intra-
skeletal organic matrix, including sugars, lipids and proteins
(Isa and Okazaki, 1987;Dauphin, 2001;Puverel et al., 2005).
The interface between coral tissue and skeleton depends on a
coral’s skeletal architecture. In corals with a perforate (porous)
skeleton, tissue penetrates the cavities of the skeletal matrix.
In contrast, tissue does not typically penetrate imperforate
coral skeletons. It has been suggested that perforate corals are
more resistant to external stressors than imperforate corals
because they provide refuge for coral tissue and Symbiodiniaceae
(Brown et al., 1994;Santos et al., 2009;Yost et al., 2013).
Furthermore, tissue that penetrates perforate coral skeletons can
host Symbiodiniaceae which may enhance calcification (Pearse
and Muscatine, 1971;Gladfelter, 1983;Yost et al., 2013). These
benefits of having a perforate skeleton suggest that there may
be differences in cellular functions and biological processes that
occur between a corals outer tissue layer and intra-skeletal
tissue. However, to our knowledge, no studies have attempted
to uncover these potential differences. Nor has there been a
comparison of how these different compartments respond to
bleaching stress.
Proteomics is a powerful tool that has advantages over other
“omics” technologies for exploring how an organism responds
to stress. While genomics and epigenomics provide the blueprint
for all potential proteins, revealing the metabolic flexibility of an
organism, they do not reveal metabolic pathways that are actively
responding to the environment at a time of interest. In contrast,
transcriptomics reveals which genes are transcribed in response
to a specific stress, yet it has been demonstrated to be a magnified
response that does not often correlate to protein translation
(Gygi et al., 1999;Maier et al., 2009;Mayfield et al., 2016;
Mayfield, 2020). As a result, there are challenges with relating how
genes and transcripts equate to biological processes occurring in
cells at the time of collection. High-throughput discovery-based
proteomics circumvents these challenges by directly identifying
and quantifying proteins in the cells at the time of collection,
which are closer to the realized function of mRNA and have
been shown to be environmentally sensitive (Pandey and Mann,
2000;Tomanek, 2014;Stuhr et al., 2018). Recent advances in
proteomics allow for the identification of potentially thousands
of proteins by coupling liquid chromatography with tandem mass
spectrometry, i.e., “shotgun” proteomics.
In this study, we used shotgun proteomics to explore how
a reef-building perforate coral, Montipora capitata, responds at
the proteomic level to experimental bleaching and to investigate
whether the outer layer (OL) tissue and inner core (IC) intra-
skeletal tissue and skeleton compartments respond to bleaching
differently. By examining proteins from these two distinct
compartments, we aimed to uncover connections between
metabolic functions of the OL that support overall maintenance
of the coral, and of the IC that support skeletal processes.
Specifically, we aimed to address these questions: (1) Are there
differential abundances of metabolic proteins in the OL and
IC? and (2) Which metabolic pathways differ between these
different coral compartments in response to bleaching? We
hypothesized that the proteomes of coral OL and IC reflect
different functions specific to these two compartments, and that
the proteomes and metabolic pathways of coral’s OL and IC
respond differently to bleaching.
Species and Experimental Design
Montipora capitata is locally abundant, dominant reef-building
coral in Hawai
i, United States. It is a small-polyp species (ca.
0.8 mm) that grows in branching and plating forms, or it can
exhibit both morphologies in a single colony. Only the branching
form was collected for this study. M. capitata was chosen because
it has a highly perforate skeleton, and because it is often noted
for its resilience (Ritson-Williams and Gates, 2020) indicated by
its shift from auto- to heterotrophy (Grottoli et al., 2006) and
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Axworthy et al. Proteomics Bleached Coral Tissue Compartments
its ability to reproduce following thermally induced bleaching
(Cox, 2007).
Between August 22–25, 2017, 10 colonies of Montipora
capitata with a diameter of 24 cm were collected from the
inner lagoon of K¯
ohe Bay, surrounding the Hawai
i Institute
of Marine Biology (HIMB, 21.428N, 157.792W). Individual
colonies were collected more than 3 m apart and although no
genetic analyses were conducted, they were considered to be
different genets. Upon collection, each colony was halved using a
hammer and chisel to produce two genetically identical colonies
(ramets). The two halves from each colony were randomly
assigned to one of three outdoor flow-through tanks for the
ambient temperature control group or one of three tanks for the
bleaching treatment group. Colonies were allowed to acclimate
for 7–10 days prior to temperature adjustments. The thermal
stress treatment started in September during the period when
coral bleaching events may occur. Maximum monthly mean
temperature for the main Hawaiian Islands is 27C and occurs
between August–September (NOAA Coral Reef Watch, 2018).
On September 1, the temperature in the bleaching treatment
tanks was elevated (600 W Titanium Aquarium Heater, Bulk Reef
Supply, United States) 0.6C day1over 4 days, reaching a mean
(±SD) temperature of 30.4 ±0.5C (range: 29.4–31C). The
control tanks had a temperature of 28 ±0.9C. On September 26,
the heat in the bleaching treatment tanks was gradually decreased
to ambient levels by September 29. Twenty-four hours later, two
small fragments (ca. 2 cm) were sampled from each colony, one
to quantify chlorophyll aand Symbiodiniaceae density, and one
for proteomics. Samples were cut from the colonies using toe-nail
clippers (Revlon Inc., United States) and were immediately frozen
in liquid nitrogen. They were stored at 80C at HIMB until
they were shipped on dry ice to the University of Washington,
WA, United States.
All tanks were maintained with a volume of 400 L of ambient
sand-filtered seawater from K¯
ohe Bay. Throughout the
experiment, corals were randomly rotated among tanks weekly
to minimize any tank effects. Water was circulated using 100 W
submersible pumps (RioR
26HF HyperFlow Water Pump 6019
LPH, TAAM, United States). Mean daytime photosynthetic active
radiation (PAR) levels in the tanks were 584 µmol photons
m2s1, and mean PAR at 12:00 was 1,249 µmol photons
m2s1, measured using a waterproof PAR logger (OdysseyR
Dataflow Systems Limited, NZ). Corals were not given any
supplemental food during the experiment.
Bleaching Status
Bleaching was measured by quantifying chlorophyll aand
Symbiodiniaceae densities. Chlorophyll awas extracted by first
grinding each sample in separate glass mortar and pestle.
Extractions were then conducted for two consecutive 24 h-
periods with fresh 100% acetone used at the start of each period.
The two-part extraction allowed us to extract total chlorophyll
per coral fragment. At the end of each 24-h period absorbances
were measured at 630, 663, and 750 nm. Equations from
Jeffrey and Humphrey (1975) were used to calculate chlorophyll
concentration for each period and summed to determine total
chlorophyll per sample. Symbiodiniaceae were separated from
FIGURE 1 | Sampling method for protein extractions from bleached and
non-bleached Montipora capitata colonies. A small cross-section (5 mm
thick) was removed from one branch of each coral fragment at least 1 cm
from the tip. The cross-section was laid flat, and a thin layer of tissue
(1–2 mm) was removed from four sides using a sterilized razor blade
producing an outer layer (OL) of tissue and an inner core (IC) of intra-skeletal
tissue and skeleton. The cross-section diagram shows the composition of our
two sample types as the tissue of M. capitata penetrates the perforate
skeleton (coral tissue indicated in brown and skeleton indicated in white).
ground coral tissue by centrifugation (two times for 5 min at
4,000 rpm). Symbiodiniaceae pellets were resuspended in filtered
seawater with 1% formalin and 2–3 drops of Lugol’s iodine,
then homogenized using a Tissue TearerTM (Model# 985–370).
Three subsamples were counted using a hemocytometer and
the mean was determined. Chlorophyll aand Symbiodiniaceae
densities were standardized to grams of ash-free dry weight (gdw)
of coral tissue.
Coral branches for proteomics were sub-sectioned into two
sample types: an outer layer (OL) of coral tissue and an
inner core (IC) consisting of intra-skeletal tissue and skeleton
(Figure 1). Samples were collected at least 1 cm from the tip
of the fragment. A cross-section, about 5 mm thick, was cut
from the fragment, then the OL and IC were separated using
a razor blade. An effort was made to collect OL samples of
tissue from the oral surface of the coral to the base of the
polyps where tissue meets the surface of the skeleton. The
remaining skeleton and intra-skeletal tissue material made up
the IC samples. All samples were crushed with a metal spatula,
placed in 1.5 ml microcentrifuge tubes and frozen at 80C until
proteomic analyses.
To lyse cells, coral samples were sonicated in a 100 µl
solution of 50 mM sodium bicarbonate (NH4HCO3) with 6
M urea three times (for 15 s then placed on ice for 20–30 s
each time), using a titanium micro-probe sonicator (Branson
250 Sonifier; 20 kHz), then flash frozen in a dry ice bath
for 30 s and stored at 80C. Samples were thawed on ice
and centrifuged at 4C at 5,000 rpm for 10 min to separate
and remove solid skeletal material to prevent particles from
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Axworthy et al. Proteomics Bleached Coral Tissue Compartments
clogging the chromatography system and from disrupting pH
balance during the digestion step. Protein concentrations were
quantified from the supernatant in triplicate using a BCA assay
(PierceTM), following the manufacturer’s instructions for limited
sample size (microplate procedure). Samples from one coral
colony yielded extremely low protein concentration and were
removed from further analyses. For each sample, 50 µg of
protein were aliquoted into a new microcentrifuge tube, then
brought to a total volume of 100 µl using a solution of 50 mM
NH4HCO3with 6 M urea. Samples were frozen at 80C until
further processing.
Enzymatic digestions of the 50 µg protein lysate aliquots
were performed following Nunn et al. (2015). Briefly, samples
were reduced with T tris(2-carboxyethyl)phosphine, alkylated
with iodoacetamide, and diluted with a 4:1 ratio of ammonium
bicarbonate to methanol prior to enzymatic digestion with
Promega Trypsin (1:20; enzyme: protein) overnight at 37C.
To stop the digestion, each sample’s pH was modified with
10% formic acid to a pH 2. Samples were evaporated
to near dryness (<20 µl) in a speed vacuum (CentriVapR
Refrigerated Centrifugal Concentrator Model 7310021) prior to
desalting. To remove buffer salts, urea and other non-peptide
molecules prior to mass spectrometry analysis, samples were
desalted with MicroSpinTM Columns (Nest Group) following the
manufacturer’s instructions. The resulting peptide samples were
evaporated to near dryness, then reconstituted in 5% acetonitrile
(ACN) with 0.1% formic acid to final concentration of 0.5 µg
µl1and stored at 80C prior to analysis.
Liquid chromatography tandem mass spectrometry was
performed on Thermo Fisher QExactive according to Nunn
et al. (2015). Liquid chromatography was performed using a
28 cm, 75 µm i.d., fused silica capillary column (Magic C18AQ,
100 Å, 5; Michrom, Bioresources, CA) with a 4 cm, 100 mm
i.d., precolumn (Magic C18AQ, 100 Å, 5; Michrom). Peptide
samples were randomized in the autosampler and loaded on the
precolumn for 10 min with 5% ACN, followed by elution onto
the analytical column using a 90 min gradient of 5–35% ACN
with 0.1% formic acid. Top 20 data dependent acquisition (DDA)
was used to collect tandem mass spectrometry data. MS1 data
was collected on the mass range of 400–1,400 m/z and collision
energy was set to 25. The 20 most intense ions with +2 to +4
charge states were selected for collision induced fragmentation
and subsequent data acquisition in the MS2 using a dynamic
exclusion of 10 s. The column was then washed for 10 min with
80% ACN and 0.1% formic acid and equilibrated for 10 min
in 5% ACN and 0.1% formic acid prior to the next sample.
Quality control standards were introduced every five runs to
monitor chromatography and MS performance and visualized
using Skyline (MacLean et al., 2010).
To generate a high quality protein database, the
transcriptome of M. capitata (Frazier et al., 2017;
GSE97888_Montiporacapitata_transcriptome.fasta) was
translated using Transdecoder v 2.0.1 (Haas et al., 2013).
Peptide tandem mass spectra were searched against the resulting
protein database of M. capitata concatenated with 50 common
contaminant proteins (Mellacheruvu et al., 2013). Spectra were
searched using Comet version 2017.01 rev. 4 (Eng et al., 2013).
Search parameters included a concatenated decoy search, peptide
mass tolerance of 10 ppm, trypsin as the search enzyme, oxidized
methionine as a variable modification (+15.9949 Da), alkylated
cysteine (57.021464 Da), and up to three missed cleavages.
PeptideProphet (Keller et al., 2002) and ProteinProphet
(Nesvizhskii et al., 2003) were used to assign probabilities to
peptide and protein identifications. Adjusted normalized spectral
abundance factor (ADJNSAF) for each sample was determined
using Abacus (Fermin et al., 2011). Abacus parameters included
minimum PeptideProphet score “maxIniProbTH” = 0.99
and “miniProbTH” = 0.50, and a combined ProteinProphet
score >0.88 (FDR of 0.01) (Supplementary File 1). For all
downstream analyses, the dataset included only proteins with
at least 2 unique peptides across all samples and proteins
identified at FDR <0.01. The mass spectrometry proteomics
data have been deposited to the ProteomeXchange Consortium
via the PRIDE [1] partner repository with the dataset identifier
Data Analysis
To investigate the role of proteins identified by the proteomics
experiments we used Gene Ontology (GO) and Kyoto
Encyclopedia of Genes and Genomes (KEGG) (Kanehisa
and Goto, 2000) pathway analyses. To recover GO terms
for each protein, protein sequences were BLASTed against
the UniProtKB Swiss-Prot non-redundant protein database
(downloaded 10.15.2018). Top results are reported with cutoff
E-value <1E-10. Due to a low annotation rate retrieved from
the UniProtKB database (49%), a second BLAST analysis
was performed against the National Center for Biotechnology
Information (NCBI) nr database on proteins that lacked
detailed UniProt annotations. KEGG ID numbers were retrieved
from BlastKoala (Kanehisa et al., 2016) using M. capitata
predicted protein sequence data. GO terms and KEGG data
were used to organize proteins into functional categories
(e.g., carbon metabolism, response to oxidative stress, etc.;
Supplementary File 2).
Proteomics differences among all sample types were visualized
using non-metric multidimensional scaling (NMDS) plots based
on log (base 10) transformed adjusted normalized spectral
abundance factor (ADJNSAF) values. NMDS was performed with
distance = “bray,” trymax = 100 and autotransform = FALSE,
using the vegan package in R (Oksanen et al., 2020). Bray-Curtis
dissimilarity was used to account for a high amount of zero values
in the dataset. Analysis of similarity (ANOSIM) was used to test
for significant differences among all sample types and between
the following comparisons: bleached outer layer (BOL) vs. non-
bleached outer layer (NBOL), bleached inner core (BIC) vs.
non-bleached inner core (NBIC), and NBOL vs. NBIC. Statistical
differences of protein abundances between sample types were
determined using Qspec, a program that computes differential
protein abundance (Choi et al., 2015). Analyses were performed
on (n= 9) paired samples (ramets) using the qspec-paired
command (burn-in = 2,000, iterations = 10,000, normalized = 1)
for the following comparisons: BOL vs. NBOL, BIC vs. NBIC, and
NBOL vs. NBIC. Qspec was performed using raw spectral counts
with a sum of at least two unique peptides for each protein in each
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Axworthy et al. Proteomics Bleached Coral Tissue Compartments
comparison. Qspec output included a protein-length corrected
log-fold change analysis (base 2) and a false discovery rate (FDR)-
corrected z-score based on the posterior distribution of the
LFC parameter, guided by the FDR estimated by a well-known
Empirical Bayes method. Differential expression was reported
using a LFC | 0.5| and z-score | 2| (Supplementary File 3).
Enrichment analysis of biological processes were compared
between NBOL and NBIC using MetaGOmics ver. 0.1.1 (Riffle
et al., 2017). MetaGOmics quantifies functional differences
among treatments based on peptide, not protein, abundance
using GO. The M. capitata proteome identified in this study
was set as the background reference. Then, for each treatment
group spectral counts were pooled across all samples (n= 9)
and compared against the reference. GO terms were considered
significant when Laplace corrected LFC >| 0.5| and when Laplace
corrected and Bonferroni corrected p-value <0.01. The Laplace
correction adds one to every spectral count for each GO term
to account for spectral counts that equal zero when calculating
log fold ratios, while the Bonferroni correction controls for type
I errors due to multiple hypothesis testing (Riffle et al., 2017).
As above, GO term data were used to organize proteins into
functional categories.
Bleaching Status
Photobiological data confirmed that the bleaching treatment
corals were bleached (Figure 2). Symbiodiniaceae density
(number of cells gdw1) decreased 71% in bleached corals
compared to non-bleached control corals (Wilcoxon rank sum
test, W= 8, p-value = 0.002756). Chlorophyll aconcentration (µg
gdw1) decreased 78% in bleached corals compared to non-
bleached corals (T-test, t=8.0304, df = 13.91, p-value = 1.368e-
Proteomic Results Across All Sample
Proteomic analyses revealed a total of 2,361 proteins that were
identified across all treatments and sample types. Overall, 90%
(2,128) of the proteins identified in this study were annotated
with either National Center for Biotechnology Information
(NCBI) (n= 2,082) or UniProtKB IDs (n= 1,167) and 81%
(1,918) were associated with GO terms. Of all 2,361 proteins,
71% (1,687) had associated KEGG terms. This high percentage
of annotated proteins allowed for more detailed analysis of active
metabolic pathways utilized in each sample type across both
treatments. About 70% (1,660 proteins) of all proteins were found
in every sample group and the number of unique proteins per
treatment or sample type ranged from 0.5–2%, with the highest
number of unique proteins observed in the OL of bleached corals
(Figure 3A). NMDS ordination of protein profiles of all sample
types show a distinction between the OL and IC compartments
but not between bleached and non-bleached samples for either
OL or IC (Figure 3B). ANOSIM revealed that there was a
significant difference of protein profiles among all sample types
(R= 0.5701; p= 9.999e-05).
FIGURE 2 | Photobiological measurements of experimental corals. Bars
indicate the mean and error bars indicate 1 standard error, for
(A) Symbiodiniaceae density (number of cells gdw-1) and (B) chlorophyll a
concentration (µg gdw-1).
Comparison of Protein Profiles From the
Outer Layer and Inner Core of
Non-bleached Corals
To gain a general understanding of the overall protein differences
between M. capitata OL and IC, we compared the proteomes of
the OL and IC of non-bleached corals. Across the two sample
types, we identified 2,242 proteins. ANOSIM revealed that there
was a significant difference of protein profiles between the OL
and IC of non-bleached samples (R= 0.5936; p= 0.001). Further
analysis revealed that 187 proteins were significantly more
abundant in the OL and 132 proteins were more abundant in the
IC (Supplementary File 3). Based on enrichment analysis, the OL
of non-bleached M. capitata was enriched in biological processes
involved in anatomical development, carbohydrate metabolism,
cell differentiation, endocytosis, lipid metabolism, protein
metabolism, RNA processing and small molecule processing
(Figure 4A and Supplementary File 4). Peroxidasin (PER2;
m.25079; LFC3.7) was detected at the highest abundance
in the OL and is part of cellular response to oxidative
stress. Several other proteins related to carbon and nitrogen
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Axworthy et al. Proteomics Bleached Coral Tissue Compartments
metabolism were also present in high abundances such as GDP-
mannose 4,6 dehydratase (GMD; m.26053; LFC1.2), pyruvate
dehydrogenase (PDH; m.4559; LFC1.1), aconitate hydratase
(ACN; m.20259; LFC0.7), and isocitrate dehydrogenase (IDH;
m.11767; LFC0.7) (Figure 4B). The IC was characterized by
an enrichment in GO biological processes involved in cellular
structure and adhesion, carbohydrate metabolism, immune
response, lipid metabolism, oxidoreductase activity and response
to stimulus (Figure 4A and Supplementary File 4). The protein
identified in the OL to have the highest fold change difference
compared to the IC was a lipase-related protein (LIP1; m.21324,
LFC +2.1) that is involved in lipid metabolism (Figure 4B).
Von Willebrand factor type A (VWA; m.31548, LFC +1.5),
an important adhesion structural protein, was also present at
significantly high concentration in the IC (Figure 4B).
Comparison of Bleaching Effects on the
Outer Layer Proteome
In the OL of bleached and non-bleached M. capitata, 2,281
proteins were identified. ANOSIM did not reveal a significant
difference of protein profiles between OL of bleached and
non-bleached corals (R= 0.05316; p= 0.159). Qspec analysis
of differential abundances between the OL of bleached and
non-bleached corals revealed 63 significant proteins at higher,
and 28 at lower, abundance in bleached corals compared
to non-bleached corals (Supplementary File 3). Biological
enrichment analysis of GO terms identified that the OL of
bleached corals have a higher abundance of proteins involved
in carbon metabolism, lipid metabolism, nitrogen metabolism,
protein metabolism, response to oxidative stress, RNA processing
and cellular signaling and transport than non-bleached corals
(Figures 5A,6). Proteins with the highest log-fold change
included calumenin (CALU; m.10914, LFC +1.8), betaine-
homocysteine S-methyltransferase (BHMT; m.1453, LFC +1.6),
peroxidasin (PER1; m.28022, LFC +1.5), urease (URE; m.24102,
LFC +1.4), and catalase (CAT; m.4762, LFC +1) (Figures 5A,
6). Key cellular pathways that increased in the OL of bleached
corals include the glyoxylate cycle, fatty acid beta oxidation, beta
alanine metabolism, protein degradation and synthesis, betaine
degradation and urea degradation. GO terms associated with less
abundant proteins in the OL of bleached corals are associated
with biological processes involved in lipid metabolism, amino
acid synthesis, protein metabolism, RNA processing, cellular
signaling and transport, and cellular structure (Figures 5A,
6). Proteins that were significantly lower in abundance in the
OL of bleached corals included cholesterol transporter (CHLT;
m.15955, LFC 1.8), glutamine synthetase (GS; m.30399, LFC
1.6), phospholipase B (PLIP; m.27749, LFC 1.1), and mucin
protein (MUC; m.29491, LFC 1) (Figures 5A,6).
Comparison of Bleaching Effects on the
Inner Core Proteome
In the IC of bleached and non-bleached M. capitata, 2,181
proteins were identified. ANOSIM did not reveal a significant
difference of protein profiles between the IC of bleached and
non-bleached corals (R=0.04287; p= 0.636). Qspec analysis
of relative abundances of the proteins detected in the IC revealed
22 proteins at significantly higher abundance and 17 at lower
abundance in the IC of bleached corals compared to non-
bleached corals. Associated GO terms indicate that proteins at
higher abundance in the IC of bleached corals are involved
in biological functions including carbon metabolism, protein
metabolism, response to oxidative stress and RNA processing
(Figures 5B,6). The top 5 proteins with the highest positive log-
fold change in the IC of bleached corals included peroxidasin
(PER2; m.25079, LFC 1.9), ependymin (EPDR; m.10937, LFC 1),
aldehyde dehydrogenase (ALDH; m.27710, LFC 1), calumenin
(CALU; m.10914, LFC 0.9), and stomatin (STOM; m.21902,
LFC 0.8) (Figures 5B,6). Additionally, a protein involved in
the pentose phosphate pathway was also represented in this
dataset with significantly increased abundance (Figures 5B,6).
Biologically enriched GO terms associated with the IC of non-
bleached corals were involved in nitrogen metabolism, cellular
signaling and cellular structure (Figures 5B,6). The proteins that
were lower in abundance in the IC of bleached corals compared to
the IC of non-bleached corals included glutamine synthetase (GS;
m.30399, LFC 1.9), peroxidasin (PER3; m.6107, LFC 1.2), and
mucin (MUC; m.29491, LFC 0.8) (Figures 5B,6).
Proteomics has proven to be a valuable tool for uncovering
active cellular processes and elucidating the response of different
biological compartments of symbiotic cnidarians in response
to stress (Weston et al., 2015;Ricaurte et al., 2016;Cziesielski
et al., 2018;Hernández-Elizárraga et al., 2019;Mayfield, 2020;
Mayfield et al., 2021;Petrou et al., 2021). Our high-throughput
discovery-based proteomic analyses of the OL and IC of
bleached and non-bleached corals support our hypothesis that
proteomic signatures differ between M. capitata OL and IC,
reflecting different biological functions associated with these two
compartments. Additionally, comparisons of bleached and non-
bleached proteomes support our hypothesis that M. capitata
OL and IC respond in different metabolic ways to bleaching
stress. We observed bleaching induced differences in carbon
metabolism, response to oxidative stress, protein turnover and
nitrogen metabolism between corals’ OL and IC compartments.
Our results corroborate several previous transcriptomics studies
(DeSalvo et al., 2010, 2012;Barshis et al., 2013;Kenkel et al.,
2013;Polato et al., 2013;Aguilar et al., 2019) by providing
direct evidence of protein translation in response to bleaching
stress. To our knowledge, our analysis was the first to compare
the simultaneous bleaching proteomic response in both OL and
IC. Our results highlight the differential metabolic response
between OL and IC in M. capitata that may reflect the
compartmentalization of specific physiological functions and
energy acquisition and allocation in each compartment.
Biological Functions of (Non-bleached)
Outer Layer and Inner Core
Among our four sample types, the most significant proteomic
difference was found when comparing the OL and IC (see
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FIGURE 3 | Global results of proteomics analysis of bleached and non-bleached Montipora capitata outer layer (OL) tissue and inner core (IC) intra-skeletal tissue
and skeleton. (A) Venn diagram depicts number of proteins identified in each sample type, and (B) non-metric dimensional scaling (NMDS) plot of all treatments.
BOL, bleached outer layer; NBOL, non-bleached outer layer; NBIC, bleached inner core; BIC, bleached inner core.
FIGURE 4 | Volcano plots of (A) associated gene ontology (GO) terms derived from MetaGOmics and (B) proteins identified in non-bleached outer layer (OL) tissue
and inner core (IC) intra-skeletal tissue and skeleton of Montipora capitata. Points represent the magnitude of the log2fold change and the -log(p-value) or z-score
for each GO term or protein, respectively. Dashed lines indicate significance thresholds. Select proteins that are discussed in the text are indicated by their
abbreviated protein name.
Figure 3B). Biological enrichment analysis of GO terms using
MetaGOmics highlight how the coral OL is characterized by
routine cellular activities such as metabolic processes, cellular
maintenance, and growth, while the IC was significantly enriched
in proteins related to structural maintenance, cell adhesion,
immune and stimulus response, as well as carbon and lipid
metabolism. These differences in biological processes suggest that
there are compartmentalized pathways between the OL and IC,
with IC proteins playing a larger role in skeletal formation than
OL proteins. Previous proteomic studies of coral skeleton report
the presence of structural and cell adhesion proteins that were
also found in this study in the M. capitata IC compartment,
including von Willebrand factor type A (VWA), hemicentin
(HMCN), and collagens (COL) (Drake et al., 2013;Ramos-
Silva et al., 2013). The von Willebrand factor type A containing
proteins have been suggested to play a role in connecting the
layer of cells at the interface of the skeleton organic matrix to the
skeleton, and collagen has been suggested to serve as a site for
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Axworthy et al. Proteomics Bleached Coral Tissue Compartments
FIGURE 5 | Volcano plots of proteins identified in non-bleached and bleached (A) outer layer (OL) tissue and (B) inner core (IC) intra-skeletal tissue and skeleton of
Montipora capitata. Points represent the magnitude of the log2fold change and the z-score for each protein. Dashed lines indicate significance thresholds. Select
proteins that are discussed in the text are indicated by their abbreviated protein name.
proteins to bind where minerals, such as calcium carbonate, can
nucleate (Drake et al., 2013).
A considerable amount of redundancy was revealed between
the OL and IC of M. capitata as 2,000 proteins were detected
with similar abundances between the two compartments, which
is likely due in part to our sampling method of a perforate coral.
In corals with a perforate skeleton, tissue penetrates the complex
matrix of crevices and cavities and can host Symbiodiniaceae
that can drive biological activity (Pearse and Muscatine, 1971;
Gladfelter, 1983;Yost et al., 2013). In contrast, the skeleton of
imperforate corals is more dense and typically does not contain
coral tissue. Evidence suggests that perforate corals are more
robust to stressful events than imperforate corals (Krupp et al.,
1992;Jokiel et al., 1993;Santos et al., 2009), which may be due
to higher protein and Symbiodiniaceae densities in their deep
tissue compared to imperforate corals (Schlöder and D’Croz,
2004). It has also been suggested that perforate skeletons can
provide refuge for deep coral tissue and Symbiodiniaceae that
reside deep in the skeleton during stressful conditions (Santos
et al., 2009). Given that the IC samples collected here contain both
coral tissue and skeleton, it is likely that there is some overlap in
metabolic processes and pathways that occur between the OL and
IC compartments, which would explain some of the similarity in
proteins between them. Furthermore, proteins from tissue within
the skeleton that do not play a role in calcification may have been
at a higher abundance and were more likely to be sampled with
our mass spectrometry methods than proteins that are directly
involved in calcification, such as carbonic anhydrase, calcium ion
pumps or the coral acid rich proteins identified by other studies
(Drake et al., 2013;Ramos-Silva et al., 2013).
Molecular Responses to Bleaching
Carbon Metabolism
Carbon is a critical nutrient for corals that is primarily provided
in the form of glucose-based photosynthate that is passed
to the host from Symbiodiniaceae (Muscatine et al., 1981).
As corals bleach and the association with Symbiodiniaceae is
weakened and carbon-based photosynthate decreases, it has been
proposed that corals maintain their energetic needs through
the degradation of stored lipids (Grottoli and Rodrigues, 2011).
Here, we provide direct evidence of modifications to carbon
metabolic pathways through quantitative analyses of proteins
that are differentially abundant in bleached corals compared to
non-bleached corals. Our results reveal mitigation strategies for
bleached corals to generate the required energy needed to persist
after bleaching stress.
In the OL of bleached M. capitata, increased abundance of
isocitrate lyase (ISL) suggests an increased capacity to produce
glyoxylate. Typically produced in the glyoxylate cycle, glyoxylate
is a two-carbon metabolite and a precursor to glucose and many
other C-storage molecules. The glyoxylate cycle, a variation of the
Krebs cycle, uses acetyl-CoA as a source of carbon and conserves
carbon by bypassing two decarboxylation steps of the Krebs
Cycle (Kornberg and Krebs, 1957). Although it was once thought
to only occur in plants and bacteria, recent transcriptomics
studies have shown that glyoxylate cycle enzymes also occur in
corals (DeSalvo et al., 2010;Kenkel et al., 2013;Polato et al.,
2013). Bleached corals may upregulate the glyoxylate cycle, or
the production of glyoxylate, to metabolize energy in stored
lipids, breaking down triacylglycerol into their component fatty
acids for release of acetyl-CoA, when carbohydrate supply is low
(Polato et al., 2013;Wright et al., 2015;Petrou et al., 2021). Malate
synthetase (MS), the other key enzyme of the glyoxylate cycle that
converts glyoxylate and acetyl-CoA to malate, was present in the
proteomes identified in this study, but no significant difference
was detected between bleached and non-bleached corals. The
same trend of increased ISL abundance and no increase in
MS abundance has been reported for Porites asteroides (Kenkel
et al., 2013) and Acropora millepora (Petrou et al., 2021). We
also detected three key enzymes involved in lipid metabolism
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FIGURE 6 | Heatmap of differentially abundant proteins in non-bleached and bleached Montipora capitata outer layer (OL) tissue and inner core (IC) intra-skeletal
tissue and skeleton. Cell shading represents the mean NSAF value for each treatment normalized by the row mean. Rows are clustered using the “correlation”
method of the pheatmap function in R. The dendrogram was set to cut 5 distinct clusters. The row annotations (categories) represent broad biological function
categories based on GO and KEGG terms associated with each protein. NBOL, non-bleached outer layer; BOL, bleached outer layer; NBIC, non-bleached inner
core; BIC, bleached inner core.
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Axworthy et al. Proteomics Bleached Coral Tissue Compartments
in higher abundance in the OL of bleached corals compared to
the OL of non-bleached corals: very long-chain specific acyl-
CoA dehydrogenase (ACADVL), medium chain specific acyl-
CoA dehydrogenase (ACADM) and acetyl-CoA acetyltransferase
(ACAT1). The presence of these enzymes further supports
the hypothesis that bleached corals utilize the glyoxylate cycle
to extract energy from lipids when Symbiodiniaceae-derived
photosynthate is decreased (Figure 7).
Malonate-semialdehyde dehydrogenase (ALDH) was another
enzyme that was significantly increased in bleached corals and
can provide an additional source of acetyl-CoA that could be used
in the glyoxylate cycle. ALDH is involved in several metabolic
pathways including carbon metabolism, beta-alanine metabolism
and propanoate metabolism where it catalyzes the conversion
of malonate semialdehyde (3-oxopropanoate) to acetyl-CoA.
Enzymes associated with the glyoxylate cycle and fatty acid beta-
oxidation were higher in the OL of bleached corals than the OL
of non-bleached corals. However, ALDH was more abundant
in both the OL and IC of bleached corals compared to these
compartments in non-bleached corals (Figure 5).
An enzyme involved in the pentose phosphate pathway (PPP),
transketolase (TKT), was lower in the IC of non-bleached corals
compared to the OL of non-bleached corals, but higher in
the IC of bleached corals than the IC of non-bleached corals,
suggesting that this pathway is initially prioritized in the OL
but becomes more essential in the IC during bleaching. The
PPP helps maintain carbon homeostasis, generates precursors
to nucleotides, and produces reducing molecules (e.g., NADPH)
to fight oxidative stress. This pathway has been elevated in
stressed cnidarians suffering from disease (Wright et al., 2015;
Garcia et al., 2016) as well as thermal stress (Oakley et al.,
2017;Fonseca et al., 2019). Hernández-Elizárraga et al. (2019)
found proteomic evidence that bleached fire coral, Millepora
complanata, redirected carbohydrate flux from glycolysis to the
PPP, which would help the coral alleviate oxidative stress through
the production of NADPH. Additionally, the generation of
nucleotides could assist with multiple other cellular processes.
Future research should investigate potential links between the
pentose phosphate pathway and coral calcification.
Response to Oxidative Stress
Oxidative stress, due to production of excess reactive oxygen
species (ROS), has been implicated as a key underlying
mechanism in the breakdown of coral-algal symbiosis leading
to bleaching (Lesser et al., 1990;Lesser, 1997;Downs et al.,
2002). Under environments favoring physiological homeostasis,
ROS produced during metabolic processes of the coral host,
Symbiodiniaceae, and in the chloroplasts of Symbiodiniaceae,
are kept in check by cellular antioxidants. Under environmental
stress, however, this tightly regulated process can become
unbalanced resulting in excess ROS, leading to cellular damage
and bleaching (Lesser et al., 1990;Downs et al., 2002). One
possible mechanism for a coral’s resilience to bleaching may be
explained by the timing, magnitude, and types of antioxidants
regulated during stress (Gardner et al., 2017).
Several enzymes involved in oxidative stress response were
at higher abundance in both the OL and IC of bleached
M. capitata compared to samples from non-bleached corals.
This observation is consistent with proteomics (e.g., Weston
et al., 2015;Cziesielski et al., 2018;Petrou et al., 2021) and
transcriptomics (e.g., DeSalvo et al., 2008, 2010;Voolstra et al.,
2009) studies that have demonstrated increased antioxidant
activity in response to thermal stress in cnidarians. Multiple
catalases (CAT) were elevated in the OL and IC of bleached
corals. This antioxidant enzyme is an efficient scavenger of
hydrogen peroxide and is commonly observed in high quantities
in bleached corals (Lesser et al., 1990;Seneca et al., 2010;Krueger
et al., 2015;Gardner et al., 2017). Peroxidasins (PER1, PER2,
PER3) that also neutralize hydrogen peroxide were detected
at significantly higher abundances in both compartments from
bleached corals. In coral embryos, significant upregulation of
peroxidasin genes, a multi-domain peroxidase, has been observed
after 48 h of thermal stress (Voolstra et al., 2009), while in
adult corals it was downregulated after 11 days of thermal stress
exposure (DeSalvo et al., 2008). Here we report that peroxidases
are present in an adult coral following nearly 1 month of
thermal stress. Peroxidasin has been proposed as a biomarker
for heat stress in coral embryos and adults (Voolstra et al.,
2009) and may be important for resilience to high temperatures
(Barshis et al., 2013).
Another enzyme higher in the OL of bleached corals, gamma-
glutamyltranspeptidase 1 (GGT1), likely plays an indirect role
in oxidative stress response through its role in the glutathione
cycle. Glutathione, in its reduced form, is important for various
metabolic processes where it can neutralize ROS and help break
down toxins (Mailloux et al., 2013;Bachhawat and Yadav, 2018).
GGTs break down oxidized glutathione that has been transported
out of the cell into glutamate and cysteine-glycine. Cysteine-
glycine can then be transported back into the cell to produce
more glutathione in its reduced form, ready to neutralize ROS
(Mailloux et al., 2013;Bachhawat and Yadav, 2018).
Protein Turnover
Environmental stressors that have the potential to induce
cellular damage, or significantly alter homeostatic metabolisms,
drive high protein turnover rates (Downs et al., 2002;Oakley
et al., 2017). Several pathways, including protein degradation,
translation, and protein folding, play key roles in the turnover
of proteins and maintaining proteostasis. Significantly higher
abundances of proteins involved in each of those pathways were
quantified in bleached corals compared to non-bleached controls,
indicating a significantly higher level of protein turnover. In
the OL of bleached corals, protein degradation was indicated by
higher abundance of two proteins associated with proteasomes,
the protein complexes responsible for degrading extraneous
or damaged proteins: 26S proteasome regulatory subunit 6A-B
(PSMC6A-B) and proteasome subunit beta type-7-like (PSMB7).
The observed increase in proteasome components in this study is
consistent with transcriptomic (Traylor-Knowles et al., 2017) and
proteomic (Petrou et al., 2021) research investigating short term
heat stress in corals.
Multiple chaperone subunits of the T-complex protein Ring
Complex (TRiC: TCPE, TCPEt, TCPZ), as well as calnexin
(CANX) were in high abundance in the OL of bleached corals,
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Axworthy et al. Proteomics Bleached Coral Tissue Compartments
FIGURE 7 | Lipid degradation and the glyoxylate cycle. Increased abundance of acyl-CoA dehydrogenases (ACAD) in the outer layer (OL) tissue of bleached coral
indicated the breakdown of lipids that produce acetyl CoA (blue arrows). Acetyl CoA is the primary source of carbon in the glyoxylate cycle (red arrows). The
glyoxylate cycle, a modified version of the Kreb’s cycle (black arrows), utilizes isocitrate lyase (ISL) and malate synthetase (MS) and can produce additional
intermediate molecules of the Krebs cycle that can be used to generate glucose. ISL was significantly elevated in the OL of bleached corals while MS was elevated
but not significantly. Created with
suggesting that bleached corals were undergoing endoplasmic
reticulum (ER) stress. ER stress occurs due to accumulation
of unfolded or misfolded proteins and initiates the unfolded
protein response which, under prolonged or severe stress,
can lead to cell death (Xu et al., 2005). The TRiC assists
the folding of up to 10% of eukaryotic cytosolic proteins
involved in a broad range of functions (Yam et al., 2008). It
is suggested to be well-equipped for folding complex, slow-
folding proteins that are prone to aggregation (Yam et al.,
2008;Gestaut et al., 2019), making it critical for maintaining
proteostasis and reducing ER stress. Calnexin is recognized
for its role in processing glycoproteins in the ER where it
binds to unfolded or misfolded proteins and prohibits their
release (Ou et al., 1993). Eight different calumenins (CALU)
were in higher abundance in bleached corals, including five
in the OL (CALU1-5) and three in the IC (CALU2, CALU3,
CALU4). Although its role as a chaperone in the ER has not
been directly confirmed, increased abundance of calumenin
resulted in down-regulation of proteins involved in ER stress
reduction, suggesting it functions similarly to chaperones (Lee
et al., 2013). Chaperones, most notably heat shock proteins, are
commonly detected in heat-stressed and bleached corals (Black
et al., 1995;Brown et al., 2002;Barshis et al., 2013;Weston
et al., 2015;Ricaurte et al., 2016;Traylor-Knowles et al., 2017;
Seveso et al., 2020) because of their role in stress response.
However, no heat shock proteins were identified at significant
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Axworthy et al. Proteomics Bleached Coral Tissue Compartments
levels in this study. Few coral bleaching studies have reported the
presence of the chaperone proteins detected in the current study
(Bellantuono et al., 2012;Maor-Landaw et al., 2014;Chakravarti
et al., 2020).
The increased abundances of proteins related to high protein
turnover in our bleached samples, one day after the heat stress
was removed, may be the result of a rapid adjustment to a change
in the coral’s environment (e.g., a recovery phase) via alteration
of its proteome. High rates of protein turnover have been
associated with an organism’s ability to acclimatize (Hawkins,
1991). For corals it has been suggested that slow growth rates
and high metabolic rates correlate to high protein turnover
and acclimatization potential (Gates and Edmunds, 1999). An
analysis of protein turnover rates in bleached and healthy states
between M. capitata and other species would allow more direct
testing of the hypothesis that protein turnover is directly linked
to thermal acclimation.
Nitrogen Metabolism
Nitrogen (N) is a critical element used for synthesizing
nucleotides, amino acids, proteins and other molecules in both
the coral host and Symbiodiniaceae. Living in oligotrophic,
tropical waters, the coral holobiont has adapted to efficiently
acquire N from the surrounding environment via heterotrophy,
or assimilation of dissolved organic and inorganic N by the
host and the Symbiodiniaceae (Grover et al., 2006, 2008;
Pernice et al., 2012). Although ammonia is the primary N-based
metabolite (Grover et al., 2008), corals also have the enzymatic
capacity for transport and assimilation of nitrate, dissolved
free amino acids and, to a much lesser degree, urea from the
surrounding water, providing them with a range of mechanisms
to adapt to changing nutrient conditions (Grover et al., 2008).
Nitrogen is transferred between the host and Symbiodiniaceae
(Rahav et al., 1989;Atkinson et al., 1994;Hoegh-Guldberg and
Williamson, 1999), however, when symbiosis breaks down due
to prolonged thermal stress, mutualistic N-exchange decreases,
demanding the coral host independently acquire N from the
environment or intracellularly recycle it. Here we show that
bleached M. capitata switches its dominant N- acquisition
strategy from ammonia uptake to the degradation of urea and
betaine (Figure 8).
Enzymes involved in the typical pathway of ammonia
assimilation decreased in bleached corals. Both the coral host and
Symbiodiniaceae are able to assimilate ammonia via catalysis by
glutamine synthetase (GS) or glutamine dehydrogenase in the
host (Yellowlees et al., 1994;Wang and Douglas, 1998;Su et al.,
2018) and via the glutamine synthetase/glutamine:2-oxoglutarate
aminotransferase (GS/GOGAT) cycle in Symbiodiniaceae (D’Elia
et al., 1983;Roberts et al., 2001). In bleached corals, GS
was present at significantly lower abundances in both the OL
and the IC compared to non-bleached corals, suggesting an
alternate route for N acquisition must be utilized (Figures 5,6).
This result is consistent with recent studies in corals (Petrou
et al., 2021;Rädecker et al., 2021), however, increased GS
activity has been reported for other cnidarians (Wang and
Douglas, 1998;Lipschultz and Cook, 2002;Oakley et al.,
2016). Both Petrou et al. (2021) and Rädecker et al. (2021)
FIGURE 8 | Nitrogen (N) source pathways in the outer layer (OL) tissue of
bleached Montipora capitata. Decreased abundance of glutamine synthetase
(GS) suggests a decrease in ammonium assimilation from surrounding
seawater. An increased abundance of urease (URE) suggests an increase in
assimilation of urea from surrounding seawater that is broken down by URE to
ammonia and carbon dioxide. Increased abundances of dimethylglycine
dehydrogenase (DMGDH) and betaine–homocysteine S-methyltransferase
(BHMT) indicate an increase in the betaine degradation pathway. Created with
also observed concurrent increases in glutamate dehydrogenase
(GDH) in bleached corals, which catabolizes glutamate to
α-ketoglutarate, an intermediate of the Krebs Cycle. Thus,
GDH indicates the degradation of amino acids for use as
energy while producing ammonia as a byproduct. While we
did not observe any change in GDH in bleached corals in
this study, we propose two alternative nitrogen sources in
bleached corals.
Urease (URE), an enzyme that catalyzes the conversion of
urea to ammonia and carbon dioxide, provides another metabolic
pathway for acquiring nitrogen and was significantly more
abundant in the OL of bleached corals than non-bleached
corals. Urea is thought to be an important source of nitrogen
for corals (Grover et al., 2006;Crandall and Teece, 2012)
because they can easily assimilate it from the surrounding
environment where it accumulates from anthropogenic runoff
and is a naturally produced metabolite from a variety of biological
sources (Conover and Gustavson, 1999;Lomas et al., 2002;
Glibert et al., 2006;McDonald et al., 2006). Grover et al.
(2008), however, calculated that urea only constitutes about
three percent of nitrogen uptake in Stylophora pistillata. Corals
may also produce urea on their own, but the mechanism
remains unclear because the complete enzymatic toolkit of the
urea cycle has yet to be observed in corals (Streamer, 1980).
Genes encoding four of the five enzymes of the urea cycle
are present in the M. capitata genome, including: carbamoyl
phosphate synthetase I (CPS1), ornithine transcarbamylase
(OTC), argininosuccinate synthase (ASS) and argininosuccinate
lyase (ASL), but the fifth enzyme, arginase (ARG1), has not been
identified. Three of those enzymes, OTC, ASS, and ASL, were
detected in the proteome identified in this study. Regardless,
the elevated level of urease provides direct evidence that
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Axworthy et al. Proteomics Bleached Coral Tissue Compartments
bleached corals rely on urea as a source of nitrogen, which
may help to counteract the effects of decreased GS activity
described above.
Here, we reveal that a critical source of nitrogen in bleached
corals likely comes from the intracellular degradation of glycine
betaine. Two enzymes involved in betaine degradation pathway,
betaine-homocysteine S-methyltransferase (BHMT) and
dimethylglycine dehydrogenase (DMGDH), were significantly
more abundant in the OL of bleached corals than non-bleached
corals. Enzymatic components of this pathway have been
observed in multiple marine organisms, including marine
invertebrates and corals (DeSalvo et al., 2012;Aguilar et al.,
2019;Sproles et al., 2019). Glycine betaine is an important
osmolyte because of its role in counteracting osmotic
and other abiotic stressors (Rathinasabapathi, 2000;Jahn
et al., 2006) and may have been produced in abundance
in response to bleaching (DeSalvo et al., 2012). Recently,
however, betaine has been proposed as a major source of
nitrogen in reef-building corals where it can account for
up to 16% of total nitrogen biomass (Ngugi et al., 2020).
A recent metabolomics study on the bleaching history of
M. capitata demonstrates that betaine-lipids are depleted
in bleached samples, whereas historically non-bleached
M. capitata is enriched in this metabolite (Roach et al.,
2021). These proteomic profiles on the same coral species
complement the metabolomic profiles of bleached vs. non-
bleached M. capitata and provide direct evidence of active
molecular pathways utilized by bleached corals to acquire N
from stored betaine-lipids, potentially leading to its long-term
persistence on reefs.
Through analyzing the proteomes of the OL and IC of
bleached and non-bleached M. capitata, we investigated:
(1) whether there were differential abundances of metabolic
proteins in the OL and IC compartments, and (2) if metabolic
pathways differ between these two compartments in response
to bleaching. We identified several metabolic pathways and
biological processes that are used in specific compartments
or shared across compartments following bleaching stress.
These included different strategies for metabolizing carbon,
lipids and nitrogen by each compartment and high oxidative
stress response and protein turnover in both compartments
following bleaching. Our findings suggest that some molecular
responses to bleaching are compartmentalized, which may
be the most efficient way to continue functions specific
to each compartment following bleaching. Future research
should assess whether these compartmentalized responses to
bleaching are linked to compartment-specific physiological
functions. For example, does energy produced from lipids
via the glyoxylate cycle help fuel reproductive output in
the coral OL compartment? Or are there links between
the products of the pentose phosphate pathway, such as
NADPH and nucleotide generation, and calcification?
Additionally, employing these compartmentalized strategies
may convey resilience to bleaching and studies targeting
these pathways, such as the glyoxylate cycle, the pentose
phosphate pathway, protein turnover, urea assimilation
and betaine degradation, should be undertaken to better
understand their influence on coral resilience. Answers to
questions like these will further our understanding of how and
where distinct physiological functions in corals are affected
by thermal stress and bleaching. Furthermore, elucidating
the precise role of these metabolic pathways and cellular
responses to bleaching could provide management with
molecular-based tools, such as biomarkers, for conservation
and restoration.
Data are available via ProteomeXchange with
identifier PXD021243.
JA performed the experiments, analyzed the data, and wrote the
manuscript. ET-S performed the experiments, analyzed the data,
and edited the manuscript. TB performed the experiments and
edited the manuscript. LR and JP-G designed, performed the
experiments, and edited the manuscript. BN designed, performed
the experiments, and wrote the manuscript. All authors
contributed to the article and approved the submitted version.
This work was supported in part by the University of
Washington’s Proteomics Resource (UWPR95794), NSF IOS-IEP
1655682 awarded to JP-G and BN, NSF IOS-IEP 1655888 to LR,
and NSF GFRP DGE1762114 awarded to JA.
We offer our warmest gratitude to the Gates Coral Lab for
hosting and supporting us during this experiment at the Hawai
Institute of Marine Biology. We thank Brenner Wakayama, Gavin
Kreitman, Melissa Jaffe and Sean Frangos for help with collecting
and culturing the experimental corals. We thank Callum
Backstrom for assistance with processing Symbiodiniaceae and
chlorophyll data. We also thank Hyungwon Choi for assistance
with statistical software.
The Supplementary Material for this article can be found online
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Frontiers in Marine Science | 16 February 2022 | Volume 9 | Article 797517
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Coral reefs across the globe are threatened by warming oceans. The last few years have seen the worst mass coral bleaching events recorded, with more than one quarter of all reefs irreversibly impacted. Considering the widespread devastation, we need to increase our efforts to understanding the physiological and metabolic shifts underlying the breakdown of this important symbiotic ecosystem. Here, we investigated the proteome (PRIDE accession # PXD011668) of both host and symbionts of the reef-building coral Acropora millepora exposed to ambient (~ 28 °C) and elevated temperature (~ 32 °C for 2 days, following a five-day incremental increase) and explored associated biomolecular changes in the symbiont, with the aim of gaining new insights into the mechanisms underpinning the collapse of the coral symbiosis. We identified 1,230 unique proteins (774 host and 456 symbiont) in the control and thermally stressed corals, of which 107 significantly increased and 125 decreased in abundance under elevated temperature relative to the control. Proteins involved in oxidative stress and proteolysis constituted 29% of the host proteins that increased in abundance, with evidence of impairment to endoplasmic reticulum and cytoskeletal regulation proteins. In the symbiont, we detected a decrease in proteins responsible for photosynthesis and energy production (33% of proteins decreased in abundance), yet minimal signs of oxidative stress or proteolysis. Lipid stores increased > twofold despite reduction in photosynthesis, suggesting reduced translocation of carbon to the host. There were significant changes in proteins related to symbiotic state, including proteins linked to nitrogen metabolism in the host and the V-ATPase (-0.6 fold change) known to control symbiosome acidity. These results highlight key differences in host and symbiont proteomic adjustments under elevated temperature and identify two key proteins directly involved in bilateral nutrient exchange as potential indicators of symbiosis breakdown.
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Unlike most parts of the world, coral reefs of Taiwan’s deep south have generally been spared from climate change-induced degradation. This has been linked to the oceanographically unique nature of Nanwan Bay, where intense upwelling occurs. Specifically, large-amplitude internal waves cause shifts in temperature of 6–9 °C over the course of several hours, and the resident corals not only thrive under such conditions, but they have also been shown to withstand multi-month laboratory incubations at experimentally elevated temperatures. To gain insight into the sub-cellular basis of acclimation to upwelling, proteins isolated from reef corals (Seriatopora hystrix) featured in laboratory-based reciprocal transplant studies in which corals from upwelling and non-upwelling control reefs (<20 km away) were exposed to stable or variable temperature regimes were analyzed via label-based proteomics (iTRAQ). Corals exposed to their “native” temperature conditions for seven days (1) demonstrated highest growth rates and (2) were most distinct from one another with respect to their protein signatures. The latter observation was driven by the fact that two Symbiodiniaceae lipid trafficking proteins, sec1a and sec34, were marginally up-regulated in corals exposed to their native temperature conditions. Alongside the marked degree of proteomic “site fidelity” documented, this dataset sheds light on the molecular mechanisms underlying acclimatization to thermodynamically extreme conditions in situ.
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The Hawaiian Islands are at the northern edge of coral reef distributions, and corals found there are exposed to large seasonal temperature changes. Historically, coral bleaching in the Hawaiian Islands was extremely rare and had only occurred in 1996. However, in the summers of both 2014 and 2015, successive bleaching events occurred in Kāne‘ohe Bay, O‘ahu. Seawater temperatures were above 28 °C for approximately 1 month in 2014 and 3 months in 2015 and peaked above 30 °C in both years. Patterns of bleaching did not vary among the three sites within Kāne‘ohe Bay. Severe bleaching and paling covered 77 and 55% of reefs in 2014 and 2015, respectively. Different species showed a range of susceptibility with 80–100% of Pocillopora spp. bleaching in both years, but less than 50% bleaching of Porites compressa and Montipora capitata in Kāne‘ohe Bay. Less than 1% of the encrusting coral Leptastrea purpurea colonies bleached in both years. Sixty individual colonies of P. compressa and M. capitata and 28 colonies of Pocillopora damicornis were tagged and monitored for rates of bleaching, recovery and mortality throughout the two-year period. Most of the colonies that bleached recovered their symbionts within 3–4 months, though P. compressa visually recovered more rapidly than M. capitata and P. damicornis. Cumulatively, 19% of P. damicornis, 10% of M. capitata and no P. compressa died by May 2016. Partial mortality within a colony did not occur in 2014, but impacted 13% of the colonies in 2015, with P. damicornis and M. capitata having higher rates of partial mortality than P. compressa. Relatively, low susceptibility in the dominant species and low rates of mortality combined with rapid rates of recovery show coral resilience to anomalously high temperatures in Kāne‘ohe Bay, O‘ahu.
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The osmolyte glycine betaine (GB) ranks among the few widespread biomolecules in all three domains of life. In corals, tissue concentrations are substantially higher than in the ambient seawater. However, the synthetic routes remain unresolved, questioning whether intracellular GB originates from de novo synthesis or heterotrophic input. Here we show that the genomic blueprint of coral metaorganisms encode the biosynthetic and degradation machinery for GB. Member organisms also adopted the prokaryotic high-affinity carrier-mediated uptake of exogenous GB, rendering coral reefs potential sinks of marine dissolved GB. The machinery metabolizing GB is highly expressed in the coral model Aiptasia and its microalgal symbionts, signifying GB’s role in the cnidarian-dinoflagellate symbiosis. We estimate that corals store between 10⁶–10⁹ grams of GB-bound nitrogen globally, representing about 16% of their nitrogen biomass. Our findings provide a framework for further mechanistic studies addressing GB’s role in coral biology and reef ecosystem nitrogen cycling.
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Small increases in ocean temperature can disrupt the obligate symbiosis between corals and dinoflagellate microalgae, resulting in coral bleaching. Little is known about the genes that drive the physiological and bleaching response of algal symbionts to elevated temperature. Moreover, many studies to‐date have compared highly divergent strains, making it challenging to accredit specific genes to contrasting traits. Here we compare transcriptional responses at ambient (27°C) and bleaching‐relevant (31°C) temperatures in a monoclonal, wild‐type (WT) strain of Symbiodiniaceae to those of a selected‐strain (SS), derived from the same monoclonal culture and experimentally evolved to elevated temperature over 80 generations (2.5 years). Thousands of genes were differentially expressed at a log fold‐change of >8 between the WT and SS over a 35‐day temperature treatment period. At 31 °C, WT cells exhibited a temporally unstable transcriptomic response upregulating genes involved in the universal stress response such as molecular chaperoning, protein repair, protein degradation and DNA repair. Comparatively, SS cells exhibited a temporally stable transcriptomic response and downregulated many stress response genes that were upregulated by the WT. Among the most highly upregulated genes in the SS at 31°C were algal transcription factors and a gene likely of bacterial origin that encodes a type II secretion system protein, suggesting interactions with bacteria may contribute to the increased thermal tolerance of the SS. Genes and functional pathways conferring thermal tolerance in the SS could be targeted in future genetic engineering experiments designed to develop thermally resilient algal symbionts for use in coral restoration and conservation.
Full-text available
Coral bleaching represents the most serious threat to contemporary coral reefs. In response, focus is being laid on understanding the cellular processes involved in the response of corals to the environmental stresses and the molecular mechanisms that determine the bleaching patterns. In the present study, a component of the cellular stress response such as the expression of the heat shock proteins (Hsps) was analyzed following the coral bleaching event which occurred in the central Red Sea (Saudi Arabia) in 2015. During this event, corals of different species, growth forms and sites showed variable bleaching susceptibility. In particular, we investigated the expression of Hsp70, Hsp60 and Hsp32 in both healthy and bleached colonies belonging to four different coral species (Goniopora lobata, Porites lobata, Seriatopora hystrix and Stylophora pistillata), in order to explore the intra- and inter-specific modulation of these biomarkers as well as the existence of spatial patterns of Hsp expression. In healthy colonies, the level of all the biomarkers was significantly different among the different species, although within each species it remained similar regardless of the distance from the shore. All the coral species showed a significant modulation of the Hsp expression in response to bleaching, whose typology and amplitude were species-specific. In all the species, Hsp70 and Hsp60 showed a coordinated dual expression, which, in response to bleaching resulted in an up-regulation in G. lobata and P. lobata and in a down-regulation in S. hystrix and S. pistillata. Hsp32 was up-regulated in all four species following bleaching, indicative of elevated oxidative stress. Overall, the protein expression profiles of each species contribute to assess the role of Hsps in regulating the susceptibility to thermal stresses of the various coral taxa of the Red Sea.