abstract · web viewdimethylsulphoniopropionate, superoxide dismutase and glutathione as stress...
TRANSCRIPT
Dimethylsulphoniopropionate, superoxide dismutase and glutathione as stress response
indicators in three corals under short-term hyposalinity stress
Stephanie G. Gardner1, Daniel A. Nielsen1, Olivier Laczka1, Ronald Shimmon2, Victor H. Beltran3,
Peter J. Ralph1, Katherina Petrou1
1Plant Functional Biology & Climate Change Cluster (C3), University of Technology Sydney, NSW,
Australia
2School of Chemistry and Forensic Science, University of Technology Sydney, NSW, Australia
3Symbiont culture facility (SCF), Australian Institute of Marine Science (AIMS), Townsville, QLD,
Australia
Corresponding author:
Katherina Petrou
1
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
Abstract
Corals are amongst the most active producers of dimethylsulphoniopropionate (DMSP), a key
molecule in marine sulphur cycling, yet the specific physiological role of DMSP in corals remains
elusive. Here we examine the oxidative stress response of three coral species (Acropora millepora,
Stylophora pistillata and Pocillopora damicornis) and explore the antioxidant role of DMSP and its
breakdown products under short-term hyposalinity stress. Symbiont photosynthetic activity declined
with hyposalinity exposure in all three reef-building corals. This corresponded with the up-regulation
of superoxide dismutase (SOD) and glutathione (GSx) in the animal host of all three species. For the
symbiont component, there were differences in antioxidant regulation, demonstrating differential
responses to oxidative stress between the Symbiodinium sub-clades. Of the three coral species
investigated, only A. millepora provided any evidence of the role of DMSP in the oxidative stress
response. Our study reveals variability in antioxidant regulation in corals and highlights the influence
life history traits and the subcladal differences can have on coral physiology. Our data expands on the
emerging understanding of the role of DMSP in coral stress regulation and emphasises the importance
of exploring both the host and symbiont responses for defining the threshold of the coral holobiont to
hyposalinity stress.
Key words
Dimethylsulphoniopropionate (DMSP), reactive oxygen species (ROS), hyposalinity, Acropora
millepora, Stylophora pistillata, Pocillopora damicornis
2
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
Introduction
Dimethylsulphoniopropionate (DMSP) represents a major fraction of organic sulphur within marine
systems 1, 2 and is produced by many macroalgae and microalgal species, including dinoflagellates
from the genus Symbiodinium. Scleractinian or reef-building corals, which comprise a symbiosis
between an animal host (Cnidarian phylum) and a symbiotic dinoflagellate algae (Symbiodinium), are
among the largest producers of DMSP 3, 4. However, the underlying physiological function(s) and
regulation of DMSP in corals is still unknown 5, 6. In marine algae, DMSP has been proposed to
function as an osmolyte 7, a cryoprotectant 8, 9, an overflow mechanism for intracellular sulphur 10, a
herbivore deterrent 11, 12, as well as a chemical attractant, acting as a foraging cue for herbivorous
fishes 13, phytoplankton 14, bacteria 15, 16 and sea birds 17. It has also been suggested to form an antiviral
defence mechanism 18 and most recently been shown to act as a trigger for dinoflagellate parasitoid
activation 19. However, following the work of Sunda et al. 20, over the last decade there has been a
strong interest in the role DMSP may play in alleviating cellular oxidative stress 5, 6, 20, 21 and the
biochemical processes thought to be involved with coral bleaching and antioxidant quenching from
DMSP explored 22.
Oxidative stress refers to the production and accumulation of reduced oxygen intermediates such as
superoxide radicals (O2-), singlet oxygen (1O2), hydrogen peroxide (H2O2), and hydroxyl radicals
(OH-) which can damage lipids, proteins, and DNA 23, 24. Oxidative stress plays a crucial role in coral
bleaching whereby stress-induced damage to the photosynthetic machinery of the algal symbiont
results in over production of oxygen radicals, which in turn damages the animal host. As such, the
Oxidative Theory of Coral Bleaching proposes that the expulsion of the symbionts from the host is a
corals final defence against oxidative stress 25. This has been validated by studies that have measured
increasing antioxidant activity in corals under environmental stress (hyposalinity, increased
temperature and high light 26), utilising a number of enzymatic and non-enzymatic antioxidants such
as superoxide dismutase (SOD) and glutathione (GSx) to protect against the damaging effects of
3
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
reactive oxygen species (ROS). The first line of defence, or primary antioxidant against ROS, is
usually SOD, converting superoxide anions into hydrogen peroxide and oxygen 23 close to the site of
production, while the glutathione system is tightly linked to the Foyer-Halliwell-Asada cycle (or
ascorbate-glutathione pathway) to regenerate ascorbate peroxidase, an enzyme responsible for
scavenging hydrogen peroxide 27. Reduced glutathione (GSH) is a key antioxidant in animal tissues
and generally represents 90-95% of the total glutathione (GSx) pool inside cells 28. Together SOD and
GSx form an effective antioxidant system in corals.
Like SOD and GSx, DMSP and its breakdown products (dimethylsulphide (DMS), acrylate,
dimethylsulphoxide (DMSO) and methanesulphonic acid (MSNA)) can readily scavenge hydroxyl
radicals and other ROS 20. Upon reacting with ROS, DMS and DMSP are oxidised to form DMSO.
Therefore, if the DMSP-based antioxidant system were to have a significant role as an antioxidant
under increased oxidative stress, DMSO should increase 21 , meaning DMSP can be a very sensitive
indicator of coral stress 29.
Coral reefs can experience extreme changes in salinity of varying duration following heavy rainfall
events, which may lead to bleaching and mortality 30. Hyposaline conditions can induce an oxidative
stress response in both the host and its algal symbionts 30. Under future climate change scenarios, the
intensity and frequency of storms, bringing heavy rainfall, are on the rise and this puts additional
strain on the existing antioxidant systems to quench the build-up of ROS. Here we measure the
physiological and biochemical stress response of three reef-building corals Acropora millepora,
Stylophora pistillata and Pocillopora damicornis to short term (24 h) hyposalinity stress. We
investigate the stress response metabolites involved in quenching oxidative stress in corals, by
targeting specific antioxidants (SOD and GSx) in both the host and the symbiont and measure coral
holobiont DMSP (combined host and symbiont) and its oxidised product, DMSO. The findings of this
4
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
study have allowed us to determine whether DMSP has a functional role in the antioxidant stress
response of corals.
Methods
Sample collection, maintenance and experimental design
Colonies of Acropora millepora, Stylophora pistillata, and Pocillopora damicornis were obtained
from Heron Island lagoon in the southern Great Barrier Reef, Australia (< 2 m depth, 152o06’E,
20o29’S) and maintained in a flow-through aquaria system under shaded light (pH 8.2, daily
maximum 381.9 ± 18.9 µmol photons m-2 s-1) for 2 d at 20.8 ± 1.1 oC. Four replicate colonies from
each species were broken into 32 fragments (3 cm length each) and maintained in the flow-through
system for 24 h prior to experiments with constant aeration. Coral fragments from each colony were
secured with plasticine into plastic racks with a randomised design and one rack was placed into each
treatment tank (four biological replicates per treatment tank). While our treatment tanks were not
replicated, the effect of pseudoreplication was minimised by maintaining a high water-volume-to-
coral-biomass ratio and ensuring fragments were incubated for a minimal amount of time (max 24 h).
Furthermore, the central and upright placement of the coral fragments within aquaria meant that the
flow, temperature and light field were homogenous within treatments, ensuring identical treatment
conditions experienced by all coral fragments. Additionally, temperature, light and pH were
monitored in all aquaria to ensure that salinity was the only variable (between treatments) to influence
the biology. The treatment tanks were set up in a shaded flow-through aquaria with constant flow of
lagoon seawater around the tanks to maintain the temperature in the tanks at that of the lagoon water
(20.8 ± 1.1 oC). The ambient light intensity was measured every five minutes using an integrating
light sensor (Odyssey, Dataflow Systems Pty Limited, New Zealand).
5
100
101
102
103
104
105
106
107
108
109
110
111
112
113
114
115
116
117
118
119
120
121
122
123
Treatment salinities were reached by linear dilutions with dH2O over 6 time points (6 h) to reach
target salinities of 24, 20 and 16 psu for A. millepora, 24, 22 and 20 for S. pistillata and 28, 22 and 20
for P. damicornis. Coral fragments were left for 24 h at target salinity before data collection.
Salinities were chosen based on 24 h preliminary experiments measuring the photosynthetic health of
the coral fragments at various salinities (MiniPAM, Walz GmbH, Effeltrich, Germany), where lethal
was considered the salinity that resulted in a maximum quantum yield of PSII (FV/FM) < 0.2 and sub-
lethal < 0.4. After 24 h exposure to treatment salinities, coral fragments were either frozen in liquid
nitrogen and stored at -80 oC until further analysis or used immediately to measure photosynthetic
activity and respiration. Water for total alkalinity measurements was sampled from each treatment
tank after the 24 h salinity stress, fixed with 0.02 % HgCl2 and stored at 4 °C in amber glass bottles.
Physiological condition under short term hyposalinity stress
To determine the physiological health of the symbionts under hyposalinity stress, we measured
variable chlorophyll a fluorescence using a Pulse Amplitude Modulated (PAM) fluorometer (Imaging
PAM, Max/K, RGB, Walz GmbH, Effeltrich, Germany). Coral fragments (n = 4) were placed in a
large wide-mouthed beaker containing seawater of the corresponding salinity and dark-adapted for 10
min. Following dark-adaptation, minimum fluorescence (FO) was recorded before application of a
saturating pulse of light (saturating pulse width = 0.8 s; saturating pulse intensity > 3,000 μmol
photons m-2 s-1), where maximum fluorescence (FM) was determined. From these two parameters the
FV/FM was calculated as FV/FM = (FM - FO)/ FM 28. Following FV/FM we performed a steady-state light
curves (SSLC) with nine light levels (56, 111, 186, 281, 396, 531, 611, 701, 926 μmol photons m -2 s-1)
applied for 3 min each before recording the light-adapted minimum (FT) and maximum fluorescence
(FM') values.
Coral fragments were used to measure respiration rates in the dark and light (170 µmol photons m -2 s-
1, below the minimum saturating irradiance). Briefly, fragments were placed into perspex chambers in
6
124
125
126
127
128
129
130
131
132
133
134
135
136
137
138
139
140
141
142
143
144
145
146
147
148
149
their respective salinities and left to acclimate for 5 min. An oxygen microsensor (Unisense A/S,
Denmark) was inserted into the lid of the chambers to monitor oxygen concentrations over 10 min
before the light was switched on for a further 10 min. The oxygen microsensor was calibrated
according to the manufacturer protocol. Respiration rates were normalised to surface area and gross
photosynthetic rate normalise to chlorophyll a (see supplementary methods).
Host and symbiont enzyme activity
Two key antioxidants, superoxide dismutase (SOD) and total glutathione (GSx), were selected to
determine the antioxidant response of the host and symbiont. Cells from the coral fragments were
extracted in 5 ml filtered seawater (FSW) using a Waterpik 31. Tissue suspensions were concentrated
using a centrifuge at ~3,600 g for 10 min at 4 oC. The supernatant was then used for host enzymatic
and antioxidant analysis while the remaining algal pellet was immediately frozen and stored at -80 oC.
To measure the enzymatic activity of the symbiont cells, frozen pellets were resuspended in 2 ml
FSW and centrifuged at ~3,600 g for 10 min at 4 oC before the supernatant was removed, and this
was repeated three times to remove all host tissue. Washed pellets were resuspended in 2 ml FSW,
and cells were ruptured three times per sample under pressure (800 psi) using a French Press. The
suspension was then centrifuged at ~3,600 g for 10 min at 4 oC to pellet the broken cell walls and the
supernatant was analysed using enzymatic assays (see below) and for the detection of total protein.
Total protein analysis was conducted using the Pierce™ BCA Protein Assay Kit (Thermo Scientific,
USA) to normalise the enzyme activity data. Total SOD (including Cu/Zn and Mn) was measured
using a Superoxide Dismutase (SOD) activity determination kit (SOD-560, Applied Bioanalytical
Labs) according to manufacturer’s guidelines. Total glutathione (GSx) (reduced (GSH) + oxidised
(GSSG)) was measured using a Glutathione Assay Kit (CS0260, Sigma Aldrich) as described by the
manufacturer.
7
150
151
152
153
154
155
156
157
158
159
160
161
162
163
164
165
166
167
168
169
170
171
172
173
174
Determination of intracellular DMSP and DMSO using NMR
Single pulse 1H NMR (nuclear magnetic resonance spectroscopy) enables a precise and quantitative
determination of the amount of molecular compounds in sample mixtures 32, and consequently was
used to quantify coral holobiont DMSP and DMSO in this study (herein referred to as intracellular
DMSP/O, or the combined measurement from the host and the symbiont). Sample extractions were
based on methods from Tapiolas et al. 33. The dried extracts were resuspended in 1 ml of deuterium
oxide (D2O) containing 0.05% 3-(Trimethylsilyl) propionic-2,2,3,3-d4 acid sodium salt (TSP) (both
from Sigma Aldrich), sonicated for 10 s to solubilise the compounds and then centrifuged for 20 min
at ~3,600 g at 4 oC. A 750 μL aliquot of the particulate free extract was transferred into a 5 mm
Wilmad NMR tube (Z566373, Sigma Aldrich) and analysed immediately. 1H NMR spectra were
recorded on an Agilent 500 MHz NMR spectrometer (Agilent Technologies). Spectra were acquired
using the VnmrJ software (version 4.2, Agilent Technologies, USA), with a sweep width of 8012.8
Hz, a 60° pulse to maximize sensitivity, a relaxation delay of 1 s, acquisition time of 4.089 s and 256
acquisition scans. The concentrations of DMSP and DMSO were determined by comparing the signal
intensities of well-resolved non-exchangeable protons ((CH3)2SCH2CH2CO2 at δ2.92 ppm for DMSP
and (CH ₃ )₂SO at δ2.73 ppm for DMSO) against the intensity of the reference signal (through signal
integration) (example spectra see Figure S2). Signals were confirmed with the addition of known
concentrations of DMSP (Serial #326871, Research Plus, Inc, USA) and DMSO (Sigma Aldrich) to
the samples and resulted in an increase of the signal intensity at the corresponding values. Sample
concentrations were calculated using the concentration of the standard D2O/TSP and the area of the
integration under the standard and respective sample signals. The concentration was then normalised
to the number of protons and molar mass for both the standard solvent and samples and then
normalised to surface area (see supplementary methods).
8
175
176
177
178
179
180
181
182
183
184
185
186
187
188
189
190
191
192
193
194
195
196
197
198
Data analysis
Data were analysed using GraphPad Prism v.6 (GraphPad Software, Inc., California). Given the lack
of sample independence, the univariate data were analysed using a non-parametric Kruskal-Wallis test
to determine differences (α = 0.05) in the medians of the responses between salinity treatments,
assuming no natural a priori ordering 34. Where differences between treatments were significant, a
Dunn’s test was used to identify which sample medians differed. Averaged values are reported as
mean ± standard error (SE) throughout the manuscript unless otherwise stated. Principle Component
Analyses (PCAs) of the physiological and biochemical data were performed using PRIMER v.7
(PRIMER-E Ltd, United Kingdom).
Results
Hyposalinity stress leads to a decline in host and symbiont activity
Chlorophyll a fluorescence revealed a reduction in photosynthetic efficiency of the symbionts with a
decline in the relative electron transport rates (rETR) between the controls and the lowest salinity for
each species (Figure 1a, b and c). The light utilisation efficiency (α) declined significantly for
P.damicornis (H(3) = 12.09, p = <0.001), maximum relative electron transport rate (rETRMAX) and
minimum saturating irradiance (EK) for all three species declined significantly with decreasing salinity
(Table S1). Similarly, there was a significant decline (p ≤ 0.001) in the maximum quantum yield of
PSII (FV/FM) for all species under hyposaline conditions (Figure 1d, e and f). Congruent with the
fluorescence data, gross photosynthesis declined with salinity in all species (Figure 1g, h and i; dark
grey bars). There was no difference in the respiration rates for A. millepora (Figure 1g; light grey
bars); however, at a salinity of 22, the respiration rate for S. pistillata was significantly lower (H(3) =
11.03, p = 0.002; Figure 1h), while for P. damicornis, the respiration rates were significantly lower
only at the lowest salinity treatment (H(3) = 7.257, p = 0.048; Figure 1i).
9
199
200
201
202
203
204
205
206
207
208
209
210
211
212
213
214
215
216
217
218
219
220
221
222
223
Symbiodinium subclades were coral species-specific, with C3 dominating in Acropora millepora, C8a
in Stylophora pistillata and C1 in Pocillopora damicornis. While hyposaline conditions resulted in
measurable physiological responses, Symbiodinium cell density and chlorophyll a did not change
between salinity treatments suggesting no significant loss of Symbiodinium cells (Figure S1a-f). An
increase in host caspase 3-like activity (a measure of the initiation of a cell death response) was
however recorded in P. damicornis at the lowest salinity (H(2) = 8.867, p = 0.01, Figure S1i).
Freshwater dilutions resulted in altered seawater carbonate chemistry, with a significant decrease in
total alkalinity (H(2) = 11.57, p = 0.041), CO32- (H(2) = 15.04, p = 0.01) and ΩCa (H(2) = 14.80, p =
0.01) between the controls and the lowest salinities (Table S2).
Hyposalinity results in differential oxidative stress in the host and symbiont
There was an increase in SOD under salinity stress in the host of A. millepora and P. damicornis
(Figure 2a-c; light grey bars), where concentrations of SOD were lowest in S. pistillata and highest in
P. damicornis. A. millepora showed the highest fold increase in host SOD (20 times) (Figure 2d),
compared with smaller increases of 1.4 and 8.5 for S. pistillata and P. damicornis, respectively
(Figure 2e-f). For the algal component, SOD concentrations increased significantly with hyposalinity
in A. millepora (H(2) = 7.692, p = 0.026; Figure 2a; dark grey bars), whilst no statistical difference or
change was detected in S. pistillata or P. damicornis (Figure 2b-c). Host glutathione (GSx) increased
with decreasing salinity in all three species (A. millepora H(2) = 11.17, p = < 0.001, Figure 3a; light
grey bars, S. pistillata H(2) = 10.67, p = < 0.001, Figure 3b and P. damicornis H(2) = 8.758, p = 0.009,
Figure 3c). In A. millepora, the GSx pool was highly dynamic, with the lowest base level of GSx
(117.75 ± 17.4 U/mg protein) at a salinity of 35, yet the greatest relative change (15-fold increase)
with decreasing salinity (Figure 3d; H(2) = 8.909, p = < 0.001). In contrast, there was no difference
between salinity treatments for S. pistillata or P. damicornis (Figure 3e-f). Interestingly, there was a
complete reverse response in the algal GSx, where concentrations increased significantly for both S.
pistillata (H(2) = 8.717, p = 0.014) and P. damicornis (H(2) = 9.577, p = 0.003), but no change was
10
224
225
226
227
228
229
230
231
232
233
234
235
236
237
238
239
240
241
242
243
244
245
246
247
248
249
detected in A. millepora (Figure 3a-c; dark grey bars). The relative change in algal GSx was a 6-fold
increase for P. damicornis (Figure 3f; dark grey circles), compared with only a 3-fold increase for S.
pistillata (Figure 3e).
Regulation of DMSP is species-specific
Concentrations of intracellular DMSP decreased with decreasing salinity for A. millepora (H(2) =
9.974, p = < 0.001, Figure 4a; light grey bars) while no change was detected in the other two species
(Figure 4b-c). A significant decrease in DMSP compared with the control salinity was measured for
A. millepora (H(2) = 7.2, p = 0.004, Figure 4d; light grey circles), with no relative change detected in
S. pistillata or P. damicornis (Figure 4e-f). The concentration of DMSO did not change across
treatment and species; however, DMSO concentrations in A. millepora were 3 times higher than the
other species (Figure 4a-c; dark grey bars). The ratio of DMSO:DMSP (indicative of the conversion
of DMSP to DMSO, potentially mediated by reactive oxygen species) increased significantly in A.
millepora exposed to lower salinities (H(3) = 8.096, p = 0.03, Figure 4g), with no change in the ratio
for the other two coral species (Figure 4h-i).
Principal component analysis (PCA) of nine physiological and biochemical parameters collected
showed a clear clustering of samples by salinity groups along the PC1 axis (Figure S3a). Species
separation was primarily driven by a difference in DMSO, respiration and host GSx in A. millepora,
while P. damicornis expressed higher activity in the algae GSx (PC2; Figure S3b).The first principal
component, PC1, was associated with gross primary productivity (GPP; eigenvalue 0.446), host GSx
(-0.414), host SOD (−0.367), algae GSx (-0.348) and DMSP (0.337) explaining 38% of the variation.
PC2 explained 17.1% of the variation and was strongly associated with algae SOD (-0.547), FV/FM (-
0.477) and DMSO (-0.429), but also co-associated with PC1 for host SOD (-0.353). The third
component, PC3, was strongly associated with respiration (-0.717), and co-associated with PC2 for
11
250
251
252
253
254
255
256
257
258
259
260
261
262
263
264
265
266
267
268
269
270
271
272
273
274
DMSO (-0.326) and PC1 for host GSx (-0.368) (Figure S3b). Combined, PC1, 2 and 3 explained
68.3% of the total variation in the data.
Discussion
Corals are major contributors to biogenic sulphur on the reef, yet the physiological role of DMSP in
corals has only started to be investigated 3, 6, 22, 35-37. Damage to algal symbionts is often thought to be
the first step in a sequence of events that occurs during coral bleaching, i.e., expulsion of
Symbiodinium, and this is characterised by changes to the photosynthetic health and symbiont
metabolites 24, 25. Here we explored the antioxidant role of DMSP and its breakdown products in corals
under short-term hyposalinity stress, in search of evidence to support the hypothesis that DMSP acts
as a secondary antioxidant system (second to the enzymatic and non-enzymatic antioxidants
investigated in this study) for coping with oxidative stress in corals. The reduction in photosynthetic
efficiency and oxygen production observed in this study reveals that the change in salinity negatively
affected the photosynthetic performance of the algae, which is consistent with previous observations
of reduced FV/FM 30, 38, 39 and photosynthetic rates 40-42 under hyposalinity stress. Interestingly, of the
three coral species, A. millepora was least affected by hyposalinity, yet was exposed to the lowest
salinities used in the study (16 psu). This contrasted greatly with the response in S. pistillata and P.
damicornis at a salinity of 20, where photosynthetic and respiratory activities were greatly reduced by
the hypo-osmotic conditions, suggesting the importance of the role of the host in coral health and
regulation 43. Ecological niche adaptation could explain differences in salinity stress responses
between the three species, as A. millepora generally inhabits shallower waters (0-15m) than S.
pistillata or P. damicornis (0-19m) 44. This would expose it to higher fluctuations in salinity than the
deeper water species, potentially accounting for the regulatory response of A. millepora to
hyposalinity stress.
12
275
276
277
278
279
280
281
282
283
284
285
286
287
288
289
290
291
292
293
294
295
296
297
298
299
Separating the responses of the host from the symbiont is important when trying to understand
physiological processes and stress responses in endosymbiosis 45, 46. While photosynthetic activity can
be attributed to the symbionts alone, respiration takes place in both the host and the symbiont and can
only be measured intact at the holobiont level. Therefore, the respiration data in this study is
representative of the coral holobiont (comprising the animal host, symbiotic dinoflagellate algae and
the associated bacteria 47). The unaltered respiration rates of A. millepora suggests that the holobiont
activity was largely unaffected by the short-term hyposaline stress and given the strong decline in
photosynthetic activity in S. pistillata, the relatively small drop in respiration rate was likely a result
of the drop in Symbiodinium activity. In the case of P. damicornis however, where the reduction in
respiration rate was most significant, a concomitant increase in caspase 3-like activity in the host was
observed. Caspase 3-like activity can be used as a proxy for apoptosis and thus provides evidence for
a decline in the health of the host tissue 48, suggesting that in this case, both the symbionts and the host
were directly affected by the change in salinity.
The ability to activate antioxidants and enzymes under stress is the fundamental mechanism that
allows organisms to cope with ROS 49, where ultimately, the response to oxidative stress depends
upon the type and degree of stress and the severity of oxidative damage 50. Through teasing apart the
antioxidant response of the host from that of its symbionts, we were able to show differential host and
symbiont antioxidant regulation. The host showed greater upregulation of superoxide dismutase
(SOD) for A. millepora and P. damicornis, while the symbionts were responsible for the highest fold
increase for glutathione (GSx) in P. damicornis. The larger relative change in host antioxidants in all
three species demonstrates the importance of regulation by this partner to the health of the coral
holobiont; while the difference in antioxidant regulation of the symbionts between coral species is
indicative of subclade-specific differences in ROS quenching. This variability in the oxidative stress
response of the symbionts may be linked to their genetic diversity 51. It has been suggested that
differences in antioxidant defences between Symbiodinium clades and subclades contribute to the
13
300
301
302
303
304
305
306
307
308
309
310
311
312
313
314
315
316
317
318
319
320
321
322
323
324
325
varying susceptibility between different coral species, or even between populations of the same
species 28 and that this diverse expression of functional genes, such as enzymatic antioxidants, is
potentially a result of horizontal gene transfer 52. Of the three species investigated in this study, all
hosted Symbiodinium within clade C, with different dominant subclades found in each. The dominant
subclade in A. millepora was C3 and this generalist symbiont commonly dominates in the Indo-
Pacific 53. According to the results of this study, this clade appears to rely on SOD as its primary line
of defence against ROS. While both C1 and C3 are classified as generalist symbiont types, C8a is a
more recently divergent lineage from C1 and has only been identified in S. pistillata from the Great
Barrier Reef 54. S. pistillata and P. damicornis may instead rely on the overall antioxidant function of
glutathione, which is localised in all cell compartments (as a non-enzymatic antioxidant), as it was up-
regulated in both components. The specificity of the host-symbiont relationship likely plays a key role
in the resilience or susceptibility of the coral holobiont 47.
Similar to the antioxidant function, we found differences in DMSP regulation between coral species.
The decline in intracellular DMSP in response to hyposalinity stress was only evident in A. millepora,
indicative of a tightly regulated system. It was the only coral species to show significant DMSP loss
to the surrounding seawater in response to hypo-osmotic stress and equally the species most resistant
to lowered salinities. In contrast, S. pistillata and P. damicornis showed no regulation of DMSP with
changing salinity, suggesting it was less effective in regulating the corals osmotic potential, as
indicated by the greater physiological stress. With respect to its role in oxidative stress, increased
DMSP production is usually associated with increased ROS levels in Symbiodinium 6, 21, 29. The lack of
upregulation of DMSP production, irrespective of the increased antioxidant activity measured here,
suggests that the corals primary response to hyposaline conditions was to reduce its osmotic potential.
Furthermore, given that DMSP is energetically expensive to produce 55, if cellular activity of the coral
is reduced or compromised, de novo synthesis of DMSP may have been limited. The formation of
DMSO constitutes an additional shield against oxidative stress, because it can be further oxidized in
14
326
327
328
329
330
331
332
333
334
335
336
337
338
339
340
341
342
343
344
345
346
347
348
349
350
351
the presence of ROS 21, and as such, it has been suggested to be an effective biomarker of stress
exposure in corals 29. Evidence for the use of DMSP as an antioxidant through the increased
conversion of DMSP to DMSO was also only apparent in A. millepora, resulting in an increased ratio
of DMSO:DMSP (Figure 4g). Similar to this, a previous study found increased DMSO concentrations
relative to other dimethylated sulphur compounds (including DMSP, DMS and acrylate) under
reduced salinity in Acropora aspera 21. The multivariate analyses highlighted DMSO as the main
driver of the differences detected between species and identified physiological and biochemical shifts
in response to hyposalinity in the host and symbiont that varied between species. These responses
demonstrate that protective mechanisms, including enzymatic (SOD) and non-enzymatic scavenging
(DMSO and GSx), contribute to the onset and impact of hyposaline stress in the corals investigated.
Our results, together with another study on Acropora aspera 21, suggest that the DMSP-based
antioxidant system may play an important role in the overall response to oxidative stress in Acroporid
coral species, lending support to the notion that the antioxidant function of DMSP might be linked to
the generalist symbionts associated with Acropora spp., or perhaps a result of their symbiont
acquisition strategy.
Symbiont specificity is correlated with a host’s transmission mode, where corals that acquire
symbionts from their parents (vertical transmission) may acquire more specialist or tightly coupled
symbionts, while those that acquire symbionts from environmental reservoirs (horizontal
transmission) may be dominated by more opportunistic generalist symbiont types 56. The three species
investigated in this study have different life history traits which align with the divergence in
antioxidant strategies measured. A. millepora, which showed the most differentiated responses,
acquires its symbionts through horizontal transmission 57. In contrast, S. pistillata and P. damicornis,
which showed similarities in their responses to hyposalinity, vertically transmit their symbionts that
are permanently incorporated into the maternal germline 43, 58. However, to what extent the varying
15
352
353
354
355
356
357
358
359
360
361
362
363
364
365
366
367
368
369
370
371
372
373
374
375
376
levels of specificity of the symbioses are reflected in enzymatic antioxidant capacity during stress is
unknown 52.
Little is currently known about the ROS scavenging capacity of different Symbiodinium subclades 24, 59
and the knowledge about the role of glutathione in the antioxidant system of Symbiodinium is still
fragmentary 60; however, we do know that the intracellular location of ROS formation dictates which
antioxidant might increase at the site of production 61. Similarly, molecules such as DMS can diffuse
through biological membranes and reach any cellular compartment 20, 23 thereby interacting with the
same ROS molecules. Thus, the possible role of alternative molecules such as DMS in antioxidant
function may be masked by the complex and differential expression of the different antioxidant
systems 62. Furthermore, it has been shown that clade D symbionts from A. millepora maintain higher
concentrations of DMSP compared with clade C under stress22. Since different subclades of
Symbiodinium sp. appear to produce DMSP and DMS at different rates and concentrations, processes
that select for specific subclades within the Symbiodinium community structure, such as environment
or host specificity 63, will inevitably affect the routes by which DMSP and DMS are produced 62. The
observed differences in the DMSO:DMSP ratio could be explained by the presence of genes in the A.
millepora genome orthologous to the newly discovered algal DMSP lyase 64. These genes are
responsible for the breakdown of DMSP into DMS and acrylate, therefore the presence of these genes
would increase the DMS available to be oxidised.
Conclusion
The species in this study used different regulatory mechanisms to cope with oxidative stress in
response to short-term hyposalinity stress. We found hyposalinity induced a differential physiological
and oxidative-stress response in the host compared with the symbiont, as well as between coral
species. Although we found upregulation of both SOD and GSx in the host tissues of all species,
differences in antioxidant regulation of the three dominant Symbiodinium subclades were detected. 16
377
378
379
380
381
382
383
384
385
386
387
388
389
390
391
392
393
394
395
396
397
398
399
400
401
402
We found SOD to be the major ROS quencher in A. millepora, whereas GSx was dominated the
response in the symbionts of S. pistillata and P. damicornis. Our data revealed A. millepora was the
only species to regulate intracellular DMSP concentrations with hypo-osmotic stress. It was also the
only coral species to exhibit a DMSP-based antioxidant system via the increased conversion of DMSP
to DMSO under increased oxidative stress. Our results emphasise the importance of differentiating the
response of the host from that of the algal symbiont to better understand cellular-stress physiology in
symbiotic partnerships. The differences in the holobiont regulation suggests that awareness of life-
history characteristics is essential for understanding cellular responses to stress and thereby
developing a better sense of species-specific susceptibility and resilience.
Data accessibility. All data are available through Dryad: doi:10.5061/dryad.4mr3c
Competing interests. We have no competing interests
Authors’ contributions. SGG, KP designed the experiments, SGG, DAN and KP performed
experiments and analysed data, SGG and OL completed colourimetric assay kit experimentation and
analysis, SGG and RS performed NMR analysis, SGG, DAN and KP wrote the manuscript, VHB
carried out sequence alignments and all authors gave final approval for publication.
Acknowledgements. We thank Sutinee Sinutok for help processing total alkalinity samples and
Heron Island Research Station staff. Additionally, we value the input of Jean-Baptiste Raina, Matthew
Nitschke, Mathieu Pernice and Catalina Aguilar Hurtado for their discussion and comments on the
manuscript. Corals were collected under the Great Barrier Reef Marine Park Authority permits
G11/34670.1 and G09/31733.1 issued to PJR.
Funding. This research was supported by the Plant Functional Biology and Climate Change Cluster
(C3) and the School of Life Sciences, at the University of Technology Sydney. SGG was supported
by an Australian Postgraduate Award (APA). KP was supported by a UTS Chancellors Postdoctoral
Fellowship.
17
403
404
405
406
407
408
409
410
411
412
413
414
415
416
417
418
419
420
421
422
423
424
425
426
427
References
[1] Kiene RP, Linn LJ, González J, Moran MA & Bruton JA. 1999 Dimethylsulfoniopropionate and methanethiol are important precursors of methionine and protein-sulfur in marine bacterioplankton. Appl. Environ. Microbiol. 65, 4549-4558.[2] Yoch DC. 2002 Dimethylsulfoniopropionate: Its sources, role in the marine food web, and biological degradation to dimethylsulfide. Appl. Environ. Microbiol. 68, 5804–5815. (doi:10.1128/AEM.68.12.5804–5815.2002).[3] Broadbent AD, Jones GB & Jones RJ. 2002 DMSP in corals and benthic algae from the Great Barrier Reef. Estuarine, Coastal and Shelf Science 55, 547-555. (doi:10.1006/ecss.2002.1021).[4] Broadbent AD & Jones GB. 2004 DMS and DMSP in mucus ropes, coral mucus, surface films and sediment pore waters from coral reefs in the Great Barrier Reef. Mar. Freshw. Res. 55, 849-855. (doi:10.1071/MF04114).[5] Yost DM, Jones RJ & Mitchelmore CL. 2010 Alterations in dimethylsulfoniopropionate (DMSP) levels in the coral Montastraea franksi in response to copper exposure. Aquat Toxicol 98, 367-373. (doi:10.1006/ecss.2002.1021).[6] McLenon AL & DiTullio GR. 2012 Effects of increased temperature on dimethylsulfoniopropionate (DMSP) concentration and methionine synthase activity in Symbiodinium microadriaticum. Biogeochemistry 110, 17–29. (doi:10.1007/s10533-012-9733-0).[7] Kirst GO, Thiel C, Wolff H, Nothnagel J, Wanzek M & Ulmke R. 1991 Dimethylsulfoniopropionate (DMSP) in icealgae and its possible biological role. Mar. Chem. 35, 381-388. (doi:10.1016/S0304-4203(09)90030-5).[8] Karsten U, Kück K, Vogt C & Kirst GO. 1996 Dimethylsulfoniopropionate production in phototrophic organisms and its physiological functions as a cryoprotectant. In Biological and Environmental Chemistry of DMSP and Related Sulfonium Compounds (eds. RP Kiene, PT Visscher, MD Keller & GO Kirst), pp. 143-153. New York, Springer US.[9] Trevena AJ, Jones GB, Wright SW & van den Enden RL. 2000 Profiles of DMSP, algal pigments, nutrients and salinity in pack ice from eastern Antarctica. J. Sea Res. 43, 265-273. (doi:10.1016/S1385-1101(00)00012-5).[10] Stefels J. 2000 Physiological aspects of the production and conversion of DMSP in marine algae and higher plants. J. Sea Res. 43, 183-197. (doi:1385-1101/00/$).[11] Wolfe GV & Steinke M. 1997 Grazing-activated chemical defence in a unicellular marine alga. Nature 387, 894.[12] Strom S, Wolfe GV, Holmes J, Stecher H, Shimeneck C, Lambert S & Moreno E. 2003 Chemical defense in the microplankton I: Feeding and growth rates of heterotrophic protists on the DMS-producing phytoplankter Emiliania huxleyi. Limnol. Oceanogr. 48, 217–229.[13] DeBose JL, Lema SC & Nevitt GA. 2008 Dimethylsulfoniopropionate as a foraging cue for reef fishes. Science 319, 1356. (doi:10.1126/science.1151109).[14] Vila-Costa M, Simo R, Harada H, Gasol JM, Slezak D & Kiene RP. 2006 Dimethylsulfoniopropionate uptake by marine phytoplankton. Science 314, 652-654. (doi:10.1126/science.1131043).[15] Miller TR & Belas R. 2004 Dimethylsulfoniopropionate metabolism by Pfiesteria-associated Roseobacter spp. Appl. Environ. Microbiol. 70, 3383-3391.[16] Seymour JR, Simó R, Ahmed T & Stocker R. 2010 Chemoattraction to dimethylsulfoniopropionate throughout the marine microbial food web. Science 329, 342-345. (doi:10.1126/science.1188418).[17] Nevitt GA. 2008 Sensory ecology on the high seas: the odor world of the procellariiform seabirds. J. Exp. Biol. 211, 1706-1713. (doi:10.1242/jeb.015412).
18
428
429
430431432433434435436437438439440441442443444445446447448449450451452453454455456457458459460461462463464465466467468469470471472473474475
[18] Evans C, Malin G, Wilson WH & Liss PS. 2006 Infectious titers of Emiliania huxleyi virus 86 are reduced by exposure to millimolar dimethylsulfide and acrylic acid. Limnol. Oceanogr. 51, 2468–2471.[19] Garcés E, Alacid E, Reñé A, Petrou K & Simó R. 2013 Host-released dimethylsulphide activates the dinoflagellate parasitoid Parvilucifera sinerae. ISME 7, 1065–1068. (doi:10.1038/ismej.2012.173).[20] Sunda W, Kieber DJ, Kiene RP & Huntsman S. 2002 An antioxidant function for DMS and DMSP in marine algae. Nature 418, 317-320.[21] Deschaseaux ESM, Jones GB, Deseo MA, Shepherd KM, Kiene RP, Swan HB, Harrison PL & Eyre BD. 2014 Effects of environmental factors on dimethylated sulfur compounds and their potential role in the antioxidant system of the coral holobiont. Limnol. Oceanogr. 59, 758-768. (doi:10.4319/lo.2014.59.3.0758).[22] Jones G & King S. 2015 Dimethylsulphoniopropionate (DMSP) as an Indicator of Bleaching Tolerance in Scleractinian Corals. Journal of Marine Science and Engineering 3, 444.[23] Lesser MP. 2006 Oxidative stress in marine environments: Biochemistry and physiological ecology. Annu. Rev. Physiol. 68, 253–278. (doi:doi: 10.1146/annurev.physiol.68.040104.110001).[24] Lesser MP. 2011 Coral bleaching: Causes and mechanisms. In Coral Reefs: An Ecosystem in Transition (eds. Z Dubinsky & N Stambler), pp. 405-419, 1st ed. New York, Springer [25] Downs CA, Fauth JE, Halas JC, Dustan P, Bemiss J & Woodley CM. 2002 Oxidative stress and seasonal coral bleaching. Free Radic. Biol. Med. 33, 533-543.[26] Hoegh-Guldberg O. 1999 Climate change, coral bleaching and the future of the world's coral reefs. Mar. Freshw. Res. 50, 839-866.[27] Foyer CH & Noctor G. 2011 Ascorbate and glutathione: The heart of the redox hub. Plant Physiology 155, 2-18. (doi:10.1104/pp.110.167569).[28] Krueger T, Becker S, Pontasch S, Dove S, Hoegh-Guldberg O, Leggat W, Fisher PL & Davy SK. 2014 Antioxidant plasticity and thermal sensitivity in four types of Symbiodinium sp. J. Phycol. 50, 1035-1047. (doi:10.1111/jpy.12232).[29] Jones GB, Fischer E, Deschaseaux ESM & Harrison PL. 2014 The effect of coral bleaching on the cellular concentration of dimethylsulphoniopropionate in reef corals. J. Exp. Mar. Biol. Ecol. 460, 19-31. (doi:http://dx.doi.org/10.1016/j.jembe.2014.06.003).[30] Downs CA, Kramarsky-Winter E, Woodley CM, Downs A, Winters G, Loya Y & Ostrander GK. 2009 Cellular pathology and histopathology of hypo-salinity exposure on the coral Stylophora pistillata. Sci. Total Environ. 407, 4838–4851.[31] Deschaseaux ESM, Deseo MA, Shepherd KM, Jones GB & Harrison PL. 2013 Air blasting as the optimal approach for the extraction of antioxidants in coral tissue. J. Exp. Mar. Biol. Ecol. 448, 146-148. (doi:doi:10.1016/j.jembe.2013.07.002).[32] Malz F & Jancke H. 2005 Validation of quantitative NMR. J. Pharm. Biomed. Anal. 38, 813–823. (doi:10.1016/j.jpba.2005.01.043).[33] Tapiolas DM, Raina JB, Lutz A, Willis BL & Motti CA. 2013 Direct measurement of dimethylsulfoniopropionate (DMSP) in reef-building corals using quantitative nuclear magnetic resonance (qNMR) spectroscopy. J. Exp. Mar. Biol. Ecol. 443, 85–89.[34] Sokal R & Rohlf F. 2000 Biometry: The principles and practice of statistics in biological research. . 4th Edition ed. New York, W.H. Freeman and company.[35] Broadbent A & Jones G. 2006 Seasonal and diurnal cycles of dimethylsulfide, dimethylsulfoniopropionate and dimethylsulfoxide at One Tree Reef lagoon. Environmental Chemistry 3, 260-267. (doi:10.1071/EN06011).[36] Van Alsytne K, Dominique VJ & Muller-Parker G. 2009 Is dimethylsulfoniopropionate (DMSP) produced by the symbionts or the host in an anemone-zooxanthella symbiosis. Coral Reefs 28, 167-176. (doi:10.1007/s00338-008-0443-y).[37] Raina J, Dinsdale EA, Willis BL & Bourne DG. 2010 Do the organic sulfur compounds DMSP and DMS drive coral microbial associations. Trends Microbiol. 18, 101-108. (doi:doi:10.1016/j.tim.2009.12.002).
19
476477478479480481482483484485486487488489490491492493494495496497498499500501502503504505506507508509510511512513514515516517518519520521522523524525526527
[38] Kerswell AP & Jones RJ. 2003 Effects of hypo-osmosis on the coral Stylophora pistillata: nature and cause of ‘low-salinity bleaching’. Mar. Ecol. Prog. Ser. 253, 145-154.[39] Chartrand KM, Durako MJ & Blum JE. 2009 Effect of hyposalinity on the photophysiology of Siderastrea radians. Marine Biology 156, 1691–1702. (doi:10.1007/s00227-009-1204-3).[40] Muthiga NA & Szmant AM. 1987 The effects of salinity stress on the rates of aerobic respiration and photosynthesis in the hermatypic coral Siderastrea siderea. Biological Bulletin 173, 539-551.[41] Moberg F, Nystrom M, Kautsky N, Tedengren M & Jarayabhand P. 1997 Effects of reduced salinity on the rates of photosynthesis and respiration in the hermatypic corals Porites lutea and Pocillopora damicornis. Mar. Ecol. Prog. Ser. 157, 53-59.[42] Titlyanov EA, Tsukahara J, Tytlianov TL, Leletkin VA, Van Woesik R & Yamazato K. 2000 Zooxanthellae population density and physiological state of the coral Stylophora pistillata during starvation and osmotic shock. Symbiosis 28, 303–322.[43] Baird AH, Bhagooli R, Ralph PJ & Takahashi S. 2008 Coral bleaching: the role of the host. Trends in Ecology and Evolution 24, 16-20.[44] Tonk L, Bongaerts P, Sampayo EM & Hoegh-Guldberg O. 2013 SymbioGBR: a web-based database of Symbiodinium associated with cnidarian hosts on the Great Barrier Reef. BMC Ecol. 13, 1-9. (doi:10.1186/1472-6785-13-7).[45] Gates RD, Baghdasarian G & Muscatine L. 1992 Temperature Stress Causes Host Cell Detachment in Symbiotic Cnidarians: Implications for Coral Bleaching. The Biological Bulletin 182, 324-332.[46] Ainsworth TD, Hoegh-Guldberg O, Heron SF, Skirving WJ & Leggat W. 2008 Early cellular changes are indicators of pre-bleaching thermal stress in the coral host. J. Exp. Mar. Biol. Ecol. 364, 63–71.[47] Thompson JR, Rivera HE, Closek CJ & Medina M. 2015 Microbes in the coral holobiont: partners through evolution, development, and ecological interactions. Frontiers in Cellular and Infection Microbiology 4, 176. (doi:10.3389/fcimb.2014.00176).[48] Kvitt H, Rosenfeld H, Zandbank K & Tchernov D. 2011 Regulation of apoptotic pathways by Stylophora pistillata (Anthozoa, Pocilloporidae) to survive thermal stress and bleaching. PLoS ONE 6, e28665. (doi:10.1371/journal.pone.0028665).[49] Finkel T & Holbrook NJ. 2000 Oxidants, oxidative stress and the biology of ageing. Nature 408, 239-247. (doi:10.1038/35041687).[50] Martindale JL & Holbrook NJ. 2002 Cellular response to oxidative stress: signaling for suicide and survival. J. Cell. Physiol. 192, 1-15. (doi:10.1002/jcp.10119).[51] LaJeunesse TC, Loh WKW, van Woesik R, Hoegh-Guldberg O, Schmidt GW & Fitt WK. 2003 Low symbiont diversity in southern Great Barrier Reef corals, relative to those of the Caribbean. Limnol. Oceanogr. 48, 2046–2054.[52] Krueger T, Fisher PL, Becker S, Pontasch S, Dove S, Hoegh-Guldberg O, Leggat W & Davy SK. 2015 Transcriptomic characterization of the enzymatic antioxidants FeSOD, MnSOD, APX and KatG in the dinoflagellate genus Symbiodinium. BMC Evol. Biol. 15. (doi:10.1186/s12862-015-0326-0).[53] Sampayo EM, Franceschinis L, Hoegh-Guldberg O & Dove S. 2007 Niche partitioning of closely related symbiotic dinoflagellates. Mol. Ecol. 16, 3721–3733. (doi:10.1111/j.1365-294X.2007.03403.x).[54] Tonk L, Sampayo EM, LaJeunesse TC, Schrameyer V & Hoegh-Guldberg O. 2014 Symbiodinium (Dinophyceae) diversity in reef-invertebrates along an offshore to inshore reef gradient near Lizard Island, Great Barrier Reef. J. Phycol. 50, 552-563. (doi:10.1111/jpy.12185).[55] Keller MD, Kiene RP, Matrai PA & Bellows WK. 1999 Production of glycine betaine and dimethylsulfoniopropionate in marine phytoplankton. I. Batch cultures. Marine Biology 135, 237-248.[56] Fabina NS, Putnam HM, Franklin EC, Stat M, Gates RD & Ferse SCA. 2012 Transmission mode predicts specificity and interaction patterns in coral-symbiodinium networks. PLoS ONE 7, 1-9. (doi:10.1371/journal.pone.0044970).
20
528529530531532533534535536537538539540541542543544545546547548549550551552553554555556557558559560561562563564565566567568569570571572573574575576577
[57] Nitschke MR, Davy SK, Cribb TH & Ward S. 2015 The effect of elevated temperature and substrate on free-living Symbiodinium cultures. Coral Reefs 34, 161–171. (doi:10.1007/s00338-014-1220-8).[58] Stat M, Loh WKW, Hoegh-Guldberg O & Carter DA. 2008 Symbiont acquisition strategy drives host–symbiont associations in the southern Great Barrier Reef. Coral Reefs 27, 763–772. (doi:10.1007/s00338-008-0412-5).[59] Suggett DJ, Warner ME, Smith DJ, Davey P, Hennige S & Baker NR. 2008 Photosynthesis and production of hydrogen peroxide by Symbiodinium (pyrrhophyta) phylotypes with different thermal tolerances. J. Phycol. 44, 948-956.[60] Kramer WE, Caamano-Ricken I, Richter C & Bischof K. 2012 Dynamic regulation of photoprotection determines thermal tolerance of two phylotypes of Symbiodinium clade A at two photon fluence rates. Photochem. Photobiol. 88, 398-413. (doi:10.1111/j.1751-1097.2011.01048.x).[61] Janknegt PJ, De Graaff M, Van De Poll WH, Visser RJW, Rijstenbil JW & Buma AGJ. 2009 Short-term antioxidative responses of 15 microalgae exposed to excessive irradiance including ultraviolet radiation. Eur. J. Phycol. 44, 525-539. (doi:10.1080/09670260902943273).[62] Steinke M, Brading P, Kerrison P, Warner ME & Suggett D, J. 2011 Concentrations of dimethylsulfoniopropionate and dimethyl sulfide are strain-specific in symbiotic dinoflagellates (Symbiodinium sp. Dinophyceae). J. Phycol. 47, 775-783. (doi:DOI: 10.1111/j.1529-8817.2011.01011.x).[63] LaJeunesse TC, Pettay DT, Sampayo EM, Phongsuwan N, Brown B, Obura DO, Hoegh-Guldberg O & Fitt WK. 2010 Long-standing environmental conditions, geographic isolation and host–symbiont specificity influence the relative ecological dominance and genetic diversification of coral endosymbionts in the genus Symbiodinium. J. Biogeogr. 37, 785-800. (doi:10.1111/j.1365-2699.2010.02273.x).[64] Alcolombri U, Ben-Dor S, Feldmesser E, Levin Y, Tawfik DS & Vardi A. 2015 Identification of the algal dimethyl sulfide–releasing enzyme: A missing link in the marine sulfur cycle. Science 348, 1466-1469. (doi:10.1126/science.aab1586).
Figure Legends
Figure 1. Photophysiology for Acropora millepora, Stylophora pistillata and Pocillopora damicornis
showing; a-c) relative electron transport rate (rETR), d-f) maximum quantum yield of photosystem II
(FV/FM) and g-i) gross photosynthetic rate (dark grey bars) and respiration (light grey bars). Letters
and symbols indicate significant differences at α = 0.05. Averages (± SE) shown (n = 4).
21
578579580581582583584585586587588589590591592593594595596597598599600601602603604605
606
607
608
609
610
611
612
613
614
615
616
Figure 2. Superoxide dismutase (SOD) activity in a) Acropora millepora, b) Stylophora pistillata and
c) Pocillopora damicornis in the host (light grey bars) and algal components (dark grey bars). The
relative change in superoxide dismutase as fold change from the control (dotted line) for d) Acropora
millepora, e) Stylophora pistillata and f) Pocillopora damicornis in the host (light grey circles) and
algal components (dark grey circles). Letters and symbols indicate significant differences at α = 0.05.
Averages (± SE) are shown (n = 3-4). Data for A. millepora algae SOD was log10+1 transformed.
Figure 3. Glutathione (GSx) activity in a) Acropora millepora, b) Stylophora pistillata and c)
Pocillopora damicornis in the host (light grey bars) and algal components (dark grey bars). The
relative change in glutathione as fold change from the control (dotted line) for d) Acropora millepora,
e) Stylophora pistillata and f) Pocillopora damicornis in the host (light grey circles) and algal
components (dark grey circles). Letters and symbols indicate significant differences at α = 0.05.
Averages (± SE) are shown (n = 2-4). Data for P. damicornis host GSx was log10+1 transformed.
Figure 4. Concentrations of intracellular DMSP (light grey bars) and DMSO (dark grey bars) for a)
Acropora millepora, b) Stylophora pistillata and c) Pocillopora damicornis. The relative change in
intracellular DMSP (light grey circles) and DMSO (dark grey circles) from the control (dotted line)
for d) Acropora millepora, e) Stylophora pistillata and f) Pocillopora damicornis. The ratio of
DMSO:DMSP (black squares) for g) Acropora millepora, h) Stylophora pistillata and i) Pocillopora
damicornis. Letters indicate significant differences at α = 0.05. Averages (± SE) are shown (n = 3-4).
22
617
618
619
620
621
622
623
624
625
626
627
628
629
630
631
632
633
634
635
636
637
638
639
640
641
642
Figures
23
643
644
645
646
647
648
649
650
651
652
653
654
655
656
657
658
659
660
661
662
Figure 1
24
663
664
665
Figure 2
25
666
667
668
Figure 3
26
669
670
671
Figure 4
27
672
673