Limnol. Oceanogr., 59(3), 2014, 758–768
2014, by the Association for the Sciences of Limnology and Oceanography, Inc.
doi:10.4319/lo.2014.59.3.0758
E
Effects of environmental factors on dimethylated sulfur compounds and their potential
role in the antioxidant system of the coral holobiont
E. S. M. Deschaseaux,1,2,3,* G. B. Jones,1,2 M. A. Deseo,4 K. M. Shepherd,4 R. P. Kiene,5,6
H. B. Swan,1,7 P. L. Harrison,1,2 and B. D. Eyre 1,3
1 School
of Environment, Science and Engineering, Southern Cross University, Lismore, New South Wales, Australia
Ecology Research Centre, Southern Cross University, Lismore, New South Wales, Australia
3 Centre for Coastal Biogeochemistry, Southern Cross University, Lismore, New South Wales, Australia
4 Southern Cross Plant Science, Southern Cross University, Lismore, New South Wales, Australia
5 Department of Marine Sciences, University of South Alabama, Alabama
6 Dauphin Island Sea Lab, Dauphin Island, Alabama
7 National Measurement Institute, Chemical and Biological Metrology, North Ryde, New South Wales, Australia
2 Marine
Abstract
Biogenic dimethylsulfide (DMS) and its main precursors, dimethylsulfoniopropionate (DMSP) and dimethylsulfoxide (DMSO), are potential scavengers of reactive oxygen species in marine algae, and these dimethylated
sulfur compounds (DSC) could take part in the algal antioxidant system. In this study, a link between the DSC
production and the antioxidant capacity (AOC) of Acropora aspera reef coral was investigated under a range of
environmental factors (temperature, light, salinity, and air exposure) that can lead to oxidative stress in the coral
holobiont. Enhanced DMS(P)(O) production occurred under experimental conditions, indicating that DSC are
potential biomarkers of stress level in coral tissue. Differences in concentrations and partitioning as a response to
different treatments suggest that DSC production and turnover undergo different biochemical pathways
depending on the type and severity of environmental stress. Osmotic pressure and light depletion led to an upregulation of the coral AOC that was correlated with a significant increase in DMSO : DSC ratio. These results,
combined with a positive correlation between the AOC and DMSO concentrations under these two treatments,
suggest that the DMSP-based antioxidant system is involved in the overall antioxidant regulation of the coral
holobiont. Enhanced DMS production coupled with an increased DMS : DSC ratio under increased temperature
indicated that thermal stress triggers DMS formation in coral tissue. Considering the role that DMS can have in
both climate regulation and the DMSP-based antioxidant system, our findings highlight the need to further
examine the fate of DSC in coral reef environments under scenarios of increasing sea surface temperatures.
reactive oxygen species (ROS) in marine algae (Sunda et al.
2002). Due to its biochemical property as a dipolar, aprotic,
hydroscopic substance, DMSO is known to permeate easily
through membranes of healthy cells, which is possibly the
reason why DMSO is usually found in lower intracellular
concentrations than DMSP (Hatton and Wilson 2007).
Similarly, being uncharged, volatile, and more hydrophobic, DMS is believed to diffuse through cell membranes
more easily than both DMSP and DMSO (Sunda et al.
2002).
Particular interest has been given to DMS over the past
two decades because this sulfur compound may exert a
negative feedback effect on global warming (Charlson et al.
1987); however, this is debated (Quinn and Bates 2011).
According to the Charlson, Lovelock, Andreae, and
Warren (CLAW) hypothesis, DMS is oxidized to non–
sea-salt sulfate aerosols that can contribute to the
formation of cloud condensation nuclei, increasing the
albedo of tropospheric clouds over the ocean and locally
lowering solar radiation and sea surface temperatures
(Charlson et al. 1987). In parallel, a wide range of
biological functions has been proposed for DMSP, and/or
DMSO, such as osmoregulation (Reed 1983), thermoregulation (Kirst et al. 1991), chemo-attraction (DeBose et al.
2008), and chemoprotection against grazers (Wolfe et al.
1997). Both DMS and its precursors are suspected to act as
Dimethylsulfide (DMS) is the most abundant volatile
sulfur compound in seawater and the largest input of
biogenically derived sulfur into the marine boundary layer
(Andreae and Crutzen 1997). Its main precursor, dimethylsulfoniopropionate (DMSP), is mostly synthesized by
photosynthetic marine algae; however, it has recently been
demonstrated that both the endosymbiotic algae and
animal host in corals produce this sulfur compound (Raina
et al. 2013). DMSP is a zwitterion that does not easily
permeate through cell membranes during normal metabolism, but it can diffuse into the surrounding media when
there is a loss of membrane integrity through grazing or cell
lysis (Dacey and Blough 1987). DMSP can be enzymatically cleaved into DMS by DMSP-lyases that have been
found in various organisms, including various types of
marine algae and bacteria (Stefels 2000), making DMSP a
considerable source of volatile sulfur and labile carbon for
the microbial food web.
Dimethylsulfoxide (DMSO) is another biogenic precursor of DMS when it is reduced by marine algae and sulfatereducing bacteria through a poorly understood reduction
pathway that might depend on reductases (Spiese et al.
2009). DMSO is also an end-product of DMS oxidation by
* Corresponding author: [email protected]
758
Dimethylated sulfur compounds in corals
antioxidants, because all three dimethylated sulfur compounds (DSC) can actively scavenge ROS (Sunda et al.
2002), and particularly hydroxyl radical (OHN), which is the
most harmful ROS known (Gill and Tuteja 2010). In all
likelihood, DMSP serves multiple physiological functions
(Stefels 2000) that are still not completely understood.
During normal metabolism, phototrophs produce ROS,
through both cellular respiration and photosynthesis,
which makes these organisms much more susceptible to
oxidative damage than aerobic heterotrophs (Gill and
Tuteja 2010). These ROS are normally in balance with a
range of antioxidants that scavenge and convert them to
nonreactive species (Csaszar et al. 2009). However, this
balance can shift toward oxidative stress under environmental stress, either through an excessive accumulation of
ROS or a depletion in antioxidants that can cause damage
to proteins, lipids, carbohydrates, deoxyribonucleic acid,
and the entire photosynthetic system, ultimately leading to
cell death (Martindale and Holbrook 2002; Gill and Tuteja
2010). Increased DMSP production has been associated
with increased ROS levels in Symbiodinium (McLenon and
DiTullio 2012) and increased ascorbate peroxidase activity
in marine algae (Sunda et al. 2002), the main antioxidant in
chloroplasts, which is a major site for ROS generation.
DMSP and DMSO concentrations were also positively
correlated with the antioxidant beta-carotene in upwelled
seawater (Riseman and DiTullio 2004), supporting the
antioxidant hypothesis for DMS and its precursors. This
DMSP-based antioxidant system is now thought to play an
important role in the complex antioxidant system of marine
algae, with each compound possibly acting at various rates
and reacting preferentially with different ROS (Sunda et al.
2002).
The coral host, its associated microbial community, and
its endosymbiotic algae, whose density averages several
millions of cells per square centimeter of coral tissue (Fitt et
al. 2000), constitute the coral holobiont, with each partner
being a substantial source of DSC in corals (Raina et al.
2009; Fischer and Jones 2012; Raina et al. 2013). This study
concentrated on the production of DSC under potential
environmental stressors (elevated temperature, direct sunlight, reduced salinity, air exposure, and light depletion) in
Acropora aspera, a common branching reef-building coral of
the Great Barrier Reef (GBR), Australia. The antioxidant
capacity (AOC) was examined as a whole rather than
targeting specific antioxidants, with the aim to understand
the role that DSC play in the complex antioxidant system of
the coral holobiont. Since an up-regulation of the antioxidant system is expected in acclimating organisms under
oxidative stress (Lesser and Shick 1989), stress exposure
should lead to an increase in the AOC in coral tissue. In
parallel, if the DMSP-based antioxidant system contributes
to the coral AOC, then DMS and DMSP are expected to
quickly oxidize to DMSO through the scavenging of ROS
under oxidative stress (Sunda et al. 2002). However,
oxidative stress and production of ROS can also vary
greatly with respect to the type of treatment, its dosage, and/
or the duration of exposure (Martindale and Holbrook
2002). Therefore, the extent of environmental stress and the
time of exposure are important factors to consider.
759
Methods
Coral collection and experimental design—Three colonies
of A. aspera were collected at low tide (low vs. high depth
range: 0.2–3.1 m) within the scientific research zone of the
Heron Island reef flat (23u26946.190S/151u54946.350E) using
a hammer and chisel and were transported to the Heron
Island Research Station (HIRS) in Nally bins that were
filled with seawater collected in situ. Colonies were
immediately nubbinized to fragments approximately 4 cm
long, which were randomly cut using hand shears. A first
mix of 12 nubbins (4 per coral colony) were immediately
snap frozen in liquid nitrogen and kept at 280uC as
untreated controls. The remaining nubbins were left to
acclimate at the HIRS in an outdoor flow-through seawater
holding tank for 40 h before the start of the experiment.
Shade cloths attenuating sunlight by approximately 50%
were placed over the holding tank during hours of direct
sunlight in order to regulate ultraviolet (UV) exposure. A
mix of four nubbins per colony were randomly assigned to
five experimental treatments that aimed to mimic acute
environmental stress: T1, elevated temperature (increased
from 28uC to 31uC over 4 d and maintained at 31uC for 3 d);
T2, direct sunlight exposure (daily maximum of approximately 350 mmol photons m22 s21 for 7 d); T3, reduced
salinity (27% for 2 d); T4, short-term air exposure
mimicking low tide effects (exposure to air for 5 h); and
T5, light depletion (daily maximum of approximately
5 mmol photons m22 s21 for 3 d; Table 1). Two sets of 12
nubbins (4 per colony) were kept as experimental controls
under monitored control conditions (28uC, 35%, and
approximate daily maximum of 150 mmol photons
m22 s21) in either flow-through seawater holding tanks
(as representative controls for T2, T4, and T5) or aerated
enclosed tanks kept in flow-through water baths (as
representative controls for T1 and T3) for the entire time
of the experiment (7 d). Light and temperature were
monitored every 10 min using Hobo data loggers (Onset),
and salinity was measured using a portable Vital Sine
refractometer (SR-6). At the end of each experimental
treatment, nubbins were immediately snap frozen in liquid
nitrogen and kept at 280uC for future tissue extraction.
Tissue extraction and sample preservation—Frozen tissue
was extracted by air blasting in 10 mL of 75 mmol L21
sodium phosphate buffer (pH 7.4; 16.7% NaH2PO4 Sigma
Aldrich S-8282 and 83.3% Na2HPO4 Sigma Aldrich S7907), using an air gun connected to an air cylinder because
this was the most appropriate method for antioxidant
extraction from coral tissue (Deschaseaux et al. 2013).
Because DMSP and DMSO are enzyme regulated and
DMS is a highly volatile compound, it is possible that the
balance of DSC in the samples could have been altered
during the extraction procedure, which likely released
enzymes. However, since all samples were extracted and
preserved in the same way throughout the experiment,
concentrations among treatments should be comparable.
Tissue lysates were transferred into centrifuge tubes,
manually shaken for homogenization, and kept on ice
while subsampled. A 1 mL aliquot was sonicated for 5 min
760
Deschaseaux et al.
Table 1. Summary of measured light, temperature, and salinity levels over time for the different experimental treatments and
unexposed controls. PAR 5 photosynthetic active radiation (mmol photons m22 s21).
Parameters
Collection
day
Acclimatization
day
Day 1
Day 2
Day 3
Day 4
Day 5
Day 6
Day 7
Elevated temperature
Light (PAR)
Temperature (uC)
Salinity (%)
400
28
35
150
28
35
150
28
35
150
29
35
150
30
35
150
31
35
150
31
35
150
31
35
150
31
35
Direct sunlight
Light (PAR)
Temperature (uC)
Salinity (%)
400
28
35
150
28
35
350
28
35
350
28
35
350
28
35
350
28
35
350
28
35
350
28
35
350
28
35
Reduced salinity
Light (PAR)
Temperature (uC)
Salinity (%)
400
28
35
150
28
35
150
28
27
150
28
27
Air exposure
Light (PAR)
Temperature (uC)
Salinity (%)
400
28
35
150
28
35
150
5 h air exposure
Light depletion
Light (PAR)
Temperature (uC)
Salinity (%)
400
28
35
150
28
35
5
28
35
Unexposed controls
Light (PAR)
Temperature (uC)
Salinity (%)
400
28
35
in a chilled water bath and was centrifuged at 10,000 g for
15 min; supernatant was subdivided into two 500 mL
subsamples for AOC and protein content measurements.
Another 1 mL subsample was diluted to 5 mL for symbiont
density and cell size measurements. Another 3 mL subsample was diluted to 30 mL with MilliQ water and was
divided into three 10 mL aliquots. One aliquot was handfiltered through Whatman Grade GF/F glass fiber filters
for chlorophyll a (Chl a) content analysis, and filters were
frozen at 280uC. The second 10 mL aliquot was purged
with high purity N2 for 10 min at 100 mL min21 to remove
free DMS derived from coral tissue, whereas the third
10 mL aliquot was left unpurged. One milliliter of
10 mol L21 NaOH was added to both purged and
unpurged aliquots (final pH . 14), and vials were
immediately sealed with gas-tight septa impermeable to
DMS (Agilent Technologies, Polytetrafluoroethylene and
Silicone septa, 5183-4477) and were crimp capped. Purged
aliquots contained only DMSP-derived DMS that was
generated during the alkaline treatment, whereas unpurged
aliquots contained both free and DMSP-derived DMS.
Tissue DMS concentrations were, therefore, obtained by
calculating the difference between purged and unpurged
aliquots.
Analysis of sulfur compounds—Purged and unpurged
samples were analyzed for total DMSP and DMS content
in coral tissue within 2 weeks of sample preservation.
5
28
35
5
28
35
Analysis was performed on a gas chromatograph (GC;
Agilent Technologies 6890N) with a mass selective (MS)
detector (Agilent Technologies 5973N) operated in scan
mode coupled with a Gerstel multipurpose sampler (MPS
2XL) configured for headspace analysis with a 2.5 mL
syringe. The syringe temperature was set at 95uC, and the
injection volume used was 1000 mL, with an injection speed
of 500 mL s21. The GC injector temperature was set at
280uC, and the injection was made with a split ratio of
25 : 1. DMS was eluted using a capillary column (5%
Phenyl Polysilphenylene-siloxane, BPX5, 50 m, 0.22 mm 3
1 mm film thickness, Scientific Glass Engineering) with high
purity helium (He) as the carrier gas at a constant flow rate
of 1.1 mL min21. The GC oven temperature was
programmed from 35uC (held for 8 min) to 180uC at a
ramp rate of 80uC per min and was held at 180uC for 2 min.
DMS was identified by reference to the mass spectra library
database (National Institute of Standards and Technology,
NIST98), and data were processed using ChemStation
software (version D.02.00.275, Agilent Technologies).
DMSP?HCl standards (certified reference material WR002,
purity 90.3 6 1.8% mass fraction, National Measurement
Institute, Sydney, Australia) and blanks were prepared with
the same proportions of phosphate buffer, MilliQ water, and
NaOH as for DMS and DMSP samples.
DMSO concentrations were measured from prepurged
aliquots that were diluted 10- or 100-fold into 75 mmol L21
sodium phosphate buffer (pH 7.4) and were purged with N2
Dimethylated sulfur compounds in corals
for 10 min to remove the DMS that was generated during
the alkaline treatment of DMSP samples. Two hundred
microliters of 20% w : w TiCl3 (Sigma Aldrich) was added
to 1 mL of diluted subsample in glass vials, which were
immediately sealed with gas-tight septa (VWR International, 16171-651) and crimp capped before being heated in a
50uC water bath for 1 h (Kiene and Gerard 1994). After
samples had cooled down to room temperature, headspace
analysis was performed by direct injection on a Shimadzu
GC (model 2014) fitted with a flame photometric detector
(FPD) set at 175uC, and a 20 cm 3 0.32 cm Teflon
fluorinated ethylene propylene column packed with Chromosil 330. The GC oven was used isothermally at 60uC
with a He carrier flow of 25 mL min21. DMSO data were
processed using the GCsolution software (version 2.32.00,
LabSolutions, Shimadzu). DMSO standards (Fisher Scientific BP321) and blanks were prepared in the same
proportions of phosphate buffer, MilliQ water, and NaOH
as used for the samples.
AOC and protein content—Samples for AOC and protein
concentrations were analyzed using the oxygen radical
absorbance capacity (ORAC) assay (Ou et al. 2001) and
Biuret assay (Dustin 1950), respectively, as described in
Deschaseaux et al. (2013). Trolox ([6]-6-hydroxy-2,5,7,8tetramethylchromane-2-carboxylic acid, Fluka Chemika
56510, 100 mmol L21) was used as a reference standard
in the ORAC assay, and AOC values were expressed per
micromole of Trolox equivalent (TE) and were standardized to protein content in each sample.
Cellular parameters and surface area measurements—
Eight replicate cell counts of the diluted tissue homogenates
were performed on a hemocytometer using a compound
microscope (Olympus CX21) at a magnification of 1003
for symbiont density measurements, and the diameters of
20 random cells were measured at a magnification of 2003
using a stage micrometer. Filters for Chl a analysis were
homogenized and extracted overnight at 220uC in 90%
acetone. Samples were thawed, sonicated, and centrifuged
for 15 min. Absorbance of the acetone extract was
measured by spectrophotometry (GBC Scientific Equipment, UV visible 916 spectrophotometer) at 664, 665, and
750 nm wavelengths before and after adding 0.1 mol L21
HCl. Chl a concentration was determined using the
equation from Standard Methods (Clesceri et al. 1998).
Remaining nubbin skeletons used for coral tissue extracts
were cleaned of skeleton debris and remaining tissue in
concentrated sodium hypochlorite and were left to dry for
several hours. The surface area of each bleached nubbin
was measured using the hot wax method (Chancerelle
2000).
Statistical treatment and analysis of variates—Variates
were tested for normality, and data were transformed when
the assumption of normality was violated. A Student’s ttest was performed to compare the values obtained for
cellular parameters, DSC, and AOC between untreated
controls and experimental controls. No significant difference was found in the AOC levels, Chl a content, cell
761
density, cell size, and DMS, DMSP, and DMSO concentrations (p . 0.05) between unexposed and experimental
controls. As such, only values of unexposed controls are
presented because they provide a more accurate view of
natural levels.
A one-way ANOVA was fitted to the dataset using
Bonferroni adjustments for pairwise comparisons between
treatments. Pearson’s correlation coefficients and corresponding p values were established for each treatment
between AOC and DMSO as well as between DMSO and
reduced sulfur compounds using a two-tailed test.
The effective sample size of this study may be smaller
than the 72 samples employed in the analysis because it is
unknown which nubbins were collected from which
colonies. Because the resulting p values from the analyses
are likely to be smaller by an unknown amount, our
approach was to interpret the results of the analyses very
conservatively, using a stricter probability criterion for
significance of p # 0.01, and to calculate standard error
based on the number of colonies (n 5 3). Averaged values
are reported as mean 6 standard error (SE) throughout the
manuscript.
Results
Chl a, symbiont density, and cell size—Symbiont density
decreased significantly under elevated temperature (p ,
0.001) and direct sunlight (p , 0.001; Fig. 1A). Although
the amount of Chl a per cell did not vary among treatments
(p . 0.01, data not shown), Chl a concentration per square
cm of coral surface area decreased significantly under
reduced salinity (p # 0.01) and light depletion (p # 0.01;
Fig. 1A). Symbiont cells shrank significantly when corals
were exposed to reduced salinity (p , 0.001), air (p # 0.01),
and light depletion (p # 0.01; Fig. 1B).
DSC—Concentrations of DMS, DMSP, and DMSO
were normalized to Chl a, protein concentrations, coral
surface area, zooxanthellae cell number, and cell volume
(Table 2). However, the p values reported in the text are
those obtained for concentrations expressed per mg of
protein because protein concentration is the most representative proxy for live tissue, including the host, symbiont,
and microbial fractions that constitute the coral holobiont.
DMSP levels were significantly greater under elevated
temperature (p , 0.001), direct sunlight (p , 0.001), and air
exposure (p # 0.01; Table 2). However, the DMSP : DSC
ratio under direct sunlight (78% 6 1%) and air exposure
(71% 6 7%) was similar to the controls (72% 6 3%; p .
0.01), whereas it was significantly reduced under elevated
temperature (39% 6 5%), reduced salinity (40% 6 5%),
and light depletion (51% 6 3%; p , 0.001; Fig. 2). DMS
concentrations increased significantly under elevated temperature (p , 0.001) and direct sunlight exposure (p ,
0.001; Table 2); however, the DMS : DSC ratio exclusively
increased under thermal stress (36% 6 4%) compared to
the controls (6% 6 2%; Fig. 2). DMS was undetectable in
coral tissue under reduced salinity, air exposure, and light
depletion (Table 2; Fig. 2); and, therefore, both DMS
concentrations and the DMS : DSC ratio were found to
762
Deschaseaux et al.
Fig. 1. (A) Symbiont density and Chl a concentration along with (B) cell diameter for each
treatment. Error bars 5 SE, n 5 3. An asterisk indicates significant differences relative to controls.
decrease significantly under these three treatments compared to the controls (p , 0.001). DMSO concentrations
significantly increased as a response to stress exposure,
regardless of the experimental treatment (p , 0.001;
Table 2). However, the DMSO : DSC ratio only increased
under reduced salinity (60% 6 5%) and light depletion
(49% 6 3%), compared to a DMSO : DSC ratio of 22% 6
2% in the controls (Fig. 2).
Correlations between DMSO, DMSP, and DMS—A
positive correlation was found between DMS and DMSO
concentrations under elevated temperature (r 5 0.762, p #
0.01; Fig. 3A). DMSP and DMSO concentrations were
positively correlated under direct sunlight (r 5 0.827, p ,
0.001), reduced salinity (r 5 0.936, p , 0.001), air exposure
(r 5 0.984, p , 0.001), and light depletion (r 5 0.938, p ,
0.001; Fig. 3B–E). DMS concentrations fell below the
detection limit under reduced salinity, air exposure, and
light depletion (Fig. 2; Table 2), so no relationship could be
established between DMS and DMSO for those treatments.
AOC and DMSO—Significantly greater AOC was found
in corals that were exposed to reduced salinity (p , 0.001)
and light depletion (p , 0.001; Fig. 4). Coral AOC was
correlated with DMSO concentrations under direct sunlight exposure (r 5 0.856, p , 0.001), reduced salinity (r 5
0.777, p # 0.01), and light depletion (r 5 0.838, p , 0.001;
Fig. 5A–C).
Discussion
DSC as biomarkers of environmental stress in corals—
DMS, DMSP, and DMSO were found at different
concentrations in coral tissue depending upon the environmental treatment to which corals were exposed (Table 2).
Specifically, DMSO concentrations significantly increased
under experimental conditions regardless of the treatment.
This response suggests that ecological and physical factors,
such as temperature, light availability, salinity, and
exposure to air at low tides, influence the production and
turnover of these sulfur compounds in the coral holobiont,
making DSC substantial biomarkers of stress exposure in
corals, with DMSO being the most indicative of all because
it significantly increased under each treatment.
DMSP concentrations increased significantly in nubbins
of A. aspera that were exposed to elevated temperature,
direct sunlight, and air, with the greatest concentration
being recorded under UV and tidal stress (Table 2).
Maximum intracellular concentrations under direct sunlight as opposed to low intracellular DMSP concentrations
under light depletion suggest that DMSP production in the
coral holobiont is intimately linked to photosynthesis in the
Dimethylated sulfur compounds in corals
763
Table 2. DMS, DMSP, and DMSO concentrations across
different normalizations for each experimental treatment and
controls. Concentrations are reported as mean 6 SE, with the
exception of DMS concentrations that were below the detection
limit (BDL). Values in bold are those that were significantly
different relative to the controls (p , 0.01).
Units
DMS
DMSP
DMSO
5.7962.09
6.3962.31
2.0061.00
3.3761.30
5.0661.88
78.5610.9
97.4638.5
24.165.72
42.666.02
63.968.35
24.262.86
28.466.48
7.5461.69
13.462.17
20.063.14
Elevated temperature
nmol (mg protein)21
1120±156
nmol cm22
927±211
nmol (mg Chl a)21
587±186
fmol cell21
1120±225
mmol (L cell volume)21 1480±323
1250±219
941±121
580±95.3
1280±354
1680±452
796±82.3
649±142
400±103
801±150
1050±195
Direct sunlight
nmol (mg protein)21
nmol cm22
nmol (mg Chl a)21
fmol cell21
mmol (L cell volume)21
88.3629.0
68.1621.7
45.6622.0
84.6627.3
130640.6
2330±168
1820±168
1090±336
2180±169
3270±296
567±38.2
442±33.2
262±73.5
534±50.9
797±72.3
BDL
3256183
41.1613.5
28.269.24
39.3614.0
77.0630.7
439±226
60.2613.8
40.4±9.85
56.9±17.7
111±39.0
BDL
2120±1020
1220±657
623±387
960±455
1710±837
616±252
340±170
172±99.8
272±118
479±215
BDL
118625.7
31.865.02
20.766.18
22.963.00
39.765.75
108±23.6
30.164.40
20.967.46
21.963.15
38.066.03
Controls
nmol (mg protein)21
nmol cm22
nmol (mg Chl a)21
fmol cell21
mmol (L cell volume)21
Reduced salinity
nmol (mg protein)21
nmol cm22
nmol (mg Chl a)21
fmol cell21
mmol (L cell volume)21
Air exposure
nmol (mg protein)21
nmol cm22
nmol (mg Chl a)21
fmol cell21
mmol (L cell volume)21
Light depletion
nmol (mg protein)21
nmol cm22
nmol (mg Chl a)21
fmol cell21
mmol (L cell volume)21
symbiotic microalgae. A loss of symbiotic zooxanthellae in
response to increased temperature and direct sunlight
(Fig. 1A) indicated that bleaching occurred under these
two treatments (Brown 1997). Together, these results
suggest that DMSP production is significantly enhanced
during bleaching of reef-building corals. This observation is
particularly interesting considering that endosymbiotic
zooxanthellae, whose density is reduced during bleaching,
are likely the dominant source of DMSP in the coral
holobiont. High DMSP concentrations associated with
bleaching could reflect a change in the relative abundance
of clades through either switching or shuffling (Baker
2003), with the newly dominant clade being a greater
producer of DMSP than the clade it replaced (Steinke et al.
Fig. 2. Partitioning of relative DMS, DMSP, and DMSO
concentrations in the coral holobiont expressed in ratios of
DMS(P)(O) : DSC (%) for each treatment.
2011). However, this study did not investigate clade
genotyping.
DMSP concentration did not vary significantly under
reduced salinity and light depletion compared to controls
(Table 2). Considering the osmotic properties of DMSP
and the photosynthetic requirement for its synthesis (Stefels
2000), low concentrations were expected under these two
treatments. Results suggest that DMSP might have been
expelled extracellularly in order to reestablish cellular
osmotic balance as a response to reduced salinity and that
DMSP formation might have been inhibited under light
depletion. Cell shrinkage under reduced salinity (Fig. 1B)
provided evidence for a loss of cell integrity, which could
have facilitated the diffusion of osmolytes such as DMSP
through cell membranes. Similarly, decreased Chl a
concentration per coral surface area under light depletion
(Fig. 1A) suggested a lower yield for photosynthesis and,
therefore, for DMSP synthesis for this treatment.
DMS concentrations increased in response to elevated
temperature and direct sunlight, although DMS concentration under thermal stress was more than 10 times that
recorded under direct UV exposure (Table 2). Similarly,
DMS as a fraction of total DSC in the coral tissue mainly
increased as a response to elevated temperature (Fig. 2).
Together, these results suggest that a rise in temperature
and direct sunlight exposure trigger DMS production in the
coral holobiont, with temperature being a strong driver of
DMS formation. Under reduced salinity, air exposure, and
light depletion, DMS levels fell below the detection limit
(Fig. 2; Table 2), indicating either (1) enhanced DMS
consumption or loss from coral tissue or (2) inhibited
DMS production, with the former suggesting either DMS
oxidation to DMSO (Sunda et al. 2002) or direct DMS
discharge to the surrounding media. The increase in
DMSO : DSC ratio under reduced salinity and light
depletion (Fig. 2) suggests that DMS was preferentially
764
Deschaseaux et al.
Fig. 3. Significant correlations between log-transformed DMSO concentrations and logtransformed DMS or DMSP concentrations under (A) elevated temperature, (B) direct sunlight,
(C) reduced salinity, (D) air exposure, and (E) light depletion.
oxidized to DMSO under these treatments. On the other
hand, the steady DMSO : DSC ratio under air exposure
(Fig. 2) could indicate inhibited DMS production or DMS
tissue-to-air exchange. The latter interpretation coincides
with observations of enhanced atmospheric DMS emissions
in coral reefs following low tide (Jones and Trevena 2005;
Jones et al. 2007; Swan et al. 2012).
Disparities observed in DSC partitioning and concentrations between treatments are most probably due to
complex biochemical interactions that take place in the
coral holobiont under environmental stress (Lesser 2006;
Thurber et al. 2009). As previously mentioned, such
biochemical processes could involve photosynthesis, bleaching, or symbiont recombination but could also implicate
coral-associated bacteria that constitute a determinant
factor in the coral sulfur cycle during both basal conditions
and stress exposure (Bourne et al. 2008; Raina et al. 2009). It
is also important to acknowledge that the treatments used in
this study should be considered acute stressors that might
occur more frequently and over longer periods of time under
Dimethylated sulfur compounds in corals
Fig. 4. AOC of the coral holobiont for each treatment. Error bars 5 SE, n 5 3. An asterisk
indicates significant differences relative to controls.
Fig. 5. Significant correlations between log-transformed AOC and log-transformed DMSO
concentrations under (A) direct sunlight, (B) reduced salinity, and (C) light depletion.
765
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Deschaseaux et al.
future climate change scenarios. The production and
consumption of DSC are likely changing at variable rates
and amplitude over time and in response to the severity of
these diverse factors. However, the implication of changes
we observed in the production of DSC under stress remains
speculative at this stage.
The DMSP antioxidant system in the antioxidant response
of the coral holobiont—The response to oxidative stress
depends upon the type of treatment and the severity of
oxidative damage that has been caused to the organism
(Martindale and Holbrook 2002). The AOC was similar to
that of unexposed controls under elevated temperature,
direct sunlight, and air exposure, environmental factors to
which reef-building corals are naturally exposed (Jimenez et
al. 2012; Fig. 4). In contrast, under reduced salinity and light
depletion, the AOC increased significantly (Fig. 4), indicating oxidative stress and ROS accumulation under these two
treatments (Lesser and Shick 1989). Cell shrinkage under
reduced salinity (Fig. 1B) and decreased Chl a concentration
per coral surface area under light depletion (Fig. 1A)
confirmed physiological impairments under these treatments.
Increased AOC levels under reduced salinity and light
depletion (Fig. 4) correlated with a significant increase in
the DMSO : DSC ratio in these two treatments (Fig. 2),
suggesting that DMSO production in coral tissue is
intimately linked to the up-regulation of the coral AOC.
This observation was further supported by positive correlations between the AOC and DMSO concentrations under
reduced salinity and light depletion (Fig. 4B,C). Because
DMSO can result from DMS and DMSP oxidation through
the scavenging of ROS (Sunda et al. 2002), our results
collectively suggest that the DMSP-based antioxidant
system plays a substantial role in the overall antioxidant
response of the coral holobiont to oxidative stress.
Although DMSP and DMSO concentrations were
significantly higher under direct sunlight than in the
controls (Table 2), the relative partitioning of DSC was
similar to that of controls (Fig. 2). Similarly, the AOC
obtained under direct sunlight did not vary from the
controls (Fig. 4). However, a positive correlation was
established between the AOC and DMSO concentrations
for this treatment (Fig. 5A). These results suggest that
DMSO production may be involved in the maintenance of
the antioxidant system of the coral holobiont during
photosynthesis, when high production of ROS, and
particularly singlet oxygen and superoxide radicals, occurs
(Gill and Tuteja 2010). The oxidation of reduced sulfur
compounds and especially DMS, which is highly reactive
toward singlet oxygen (Sunda et al. 2002), can be expected
during photosynthesis. Considering that DMSO might
accumulate at higher cellular concentrations than DMS
(Sunda et al. 2002), this result also suggests that DMSO
formation during photosynthesis will constitute a second
shield against oxidative stress because it can be further
oxidized in the presence of ROS.
The relative speciation of DSC—The low permeability of
cell membranes to DMSP makes this compound a relatively
stable biomarker to measure in coral tissue under normal
metabolism compared to both DMS and DMSO, which
should freely pass through cell membranes (Sunda et al. 2002).
Due to these differences in properties, DSC most likely operate
at different levels within cell compartments and membranes,
with their relative abundance reflecting the needs of the cells
and the ability for these compounds to be generated in areas
where ROS are synthesized or accumulated. However, during
environmental stress leading to cell impairment, cell loss
(bleaching) and/or symbiont recombination, the relative
cellular permeability of DSC becomes compromised (Dacey
and Blough 1987). Moreover, because the coral holobiont
comprises coral tissue, endosymbiotic cells, and a vast
microbial community, with each partner potentially being
involved in the production, consumption, and turnover of
DSC (Raina et al. 2013), this adds to the difficulty in trying to
understand the DMSP-based cycle in corals.
Under elevated temperature, the high DMS concentration was likely sourced from DMSP, as indicated by a
significant decrease in the DMSP : DSC ratio and a
significant increase in DMS : DSC under this treatment
(Fig. 2). This result suggests that increasing temperature
triggers enhanced DMSP-lyase activity in the coral holobiont. However, DMS concentration under this treatment
could also reflect a snapshot of accumulated DMS within the
holobiont. The positive correlation that was observed
between DMS and DMSO under elevated temperature
(Fig. 3A) suggests that some of this DMS was probably
converted to DMSO by ROS or other oxidation pathways.
However, it might also indicate that part of the DMS came
from DMSO reduction (Spiese et al. 2009). However, without
turnover data we cannot draw categorical conclusions about
the source and fate of DMS under increased temperature.
DMSO and DMSP concentrations were correlated under
most variables (Fig. 3B–E), suggesting that DMSP is the
main source of DMSO in coral tissue under environmental
stress. However, direct oxidation of DMSP to DMSO is
chemically unlikely to bypass DMS formation (Van Alstyne
et al. 2001; Wolfe et al. 2002). These correlations, therefore,
most likely indicate DMSP cleavage into DMS, which was
then oxidized to DMSO. Since DMS is highly reactive
toward OHN (Sunda et al. 2002), it is unlikely to accumulate
in coral tissue under enhanced oxidative stress and excessive
ROS production but is likely to be quickly oxidized to
DMSO and other oxidized sulfur species. This possibility is
supported by the absence of DMS under reduced salinity, air
exposure, and light depletion (Fig. 2; Table 2).
Levels of DMSP measured under thermal, UV, and air
exposure in this study were among the highest concentrations yet reported in corals (Broadbent et al. 2002; Yost et
al. 2012), especially when expressed per symbiont cell or
cell volume (Table 2). However, under environmental
stress, corals tend to produce copious amounts of mucus
(visual observations), which has been shown to contain the
highest concentrations of DMS and DMSP in the marine
environment (Broadbent and Jones 2004). Hence, data
obtained in these treatments most likely represent accumulated DMSP in mucus, symbiont cells, and coral tissue and
are, therefore, more accurate when normalized to protein
or square centimeter of coral surface area than to cell, cell
volume, or Chl a content.
Dimethylated sulfur compounds in corals
Implications for the Great Barrier Reef—About 10% of
the DMS that is biosynthesized in the ocean is emitted to the
atmosphere (Malin et al. 1992), possibly leading to an
increase in cloud cover and a negative feedback effect on sea
surface temperature and incident solar radiation (Charlson
et al. 1987); however, this mechanism is much more complex
than first outlined in the CLAW hypothesis (Quinn and
Bates 2011). In the present study, increased temperature was
found to be an important driver of DMS production in the
coral holobiont, as indicated by a substantial increase in
DMS concentrations under thermal stress (Table 2), reaching a DMS : DSC ratio of 36% 6 4% compared to 6% 6 2%
in the controls (Fig. 2). This finding could have direct
relevance to the ongoing debate over whether coral reefs
under thermal stress could contribute to a local feedback
effect on climate (Deschaseaux et al. 2012), because
enhanced coral DMS production could be expected to lead
to enhanced DMS discharge into the water column,
increasing the dissolved pool for potential atmospheric
emissions. Whereas, in this study, no measurements were
made of dissolved or gaseous DMS, chamber experiments
conducted on Acropora formosa (Jones et al. 2007) and
Acropora intermedia (Fischer and Jones 2012) showed a
decrease in both dissolved and atmospheric DMS as a result
of temperature increase. Further experiments are, therefore,
needed to understand the fate of enhanced DMS production
in coral tissue under scenarios of increasing sea surface
temperature and to understand their effect on atmospheric
DMS production derived from the coral holobiont.
Acknowledgments
This project was partly funded by the Marine Ecology
Research Centre (Southern Cross University [SCU], Lismore),
the Australian Institute for Marine Science (Townsville), and the
Australian Research Council Discovery grant DP0878683 awarded to Bradley D. Eyre. We are grateful to Southern Cross Plant
Science (SCU, Lismore) for providing access to equipment and
facilities and to SCU for a travel bursary to visit the Dauphin
Island Sea Lab (Alabama), where the analysis of dimethylsulfoxide samples was conducted. That work was partially supported
by a National Science Foundation grant Ocean Sciences-0928968.
We thank the Great Barrier Reef Marine Park Authority for the
permit to collect coral specimens from Heron Island, Heron Island
Research Station staff for assistance, and Lyndon Brooks (SCU)
for statistical advice. We also thank the reviewers for providing
useful suggestions that have helped improve the manuscript.
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Associate editor: Thomas R. Anderson
Received: 04 September 2013
Accepted: 02 January 2014
Amended: 10 January 2014