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Dimethylsulphoniopropionate, superoxide dismutase and glutathione as stress response indicators in three corals under short-term hyposalinity stressStephanie G. Gardner1, Daniel A. Nielsen1, Olivier?Laczka1, Ronald Shimmon2, Victor H. Beltran3, Peter J. Ralph1, Katherina Petrou11Plant Functional Biology & Climate Change Cluster (C3), University of Technology Sydney, NSW, Australia 2School of Chemistry and Forensic Science, University of Technology Sydney, NSW, Australia3Symbiont culture facility (SCF), Australian Institute of Marine Science (AIMS), Townsville, QLD, AustraliaCorresponding author:Katherina PetrouKatherina.Petrou@uts.edu.auAbstractCorals 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 wordsDimethylsulphoniopropionate (DMSP), reactive oxygen species (ROS), hyposalinity, Acropora millepora, Stylophora pistillata, Pocillopora damicornisIntroductionDimethylsulphoniopropionate (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 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 study have allowed us to determine whether DMSP has a functional role in the antioxidant stress response of corals.MethodsSample collection, maintenance and experimental designColonies 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 ?mol 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). 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 stressTo 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 ?mol photons m-2 s-1, below the minimum saturating irradiance). Briefly, fragments were placed into perspex chambers in 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 activityTwo 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. Determination of intracellular DMSP and DMSO using NMRSingle 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?)?SO 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). Data analysisData 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). ResultsHyposalinity stress leads to a decline in host and symbiont activityChlorophyll 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). 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 ?Ca (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 symbiontThere 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 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-specificConcentrations 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 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.DiscussionCorals 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. 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 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 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 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. ConclusionThe 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. 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.4mr3cCompeting interests. We have no competing interestsAuthors’ 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). 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Letters and symbols indicate significant differences at α = 0.05. Averages (± SE) shown (n = 4).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).FiguresFigure 1Figure 2Figure 3Figure 4 ................
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