Metabolite profiling of symbiont and host during thermal stress and bleaching in the coral <Emphasis Type="Italic">Acropora aspera</Emphasis>

Rising seawater temperatures pose a significant threat to the persistence of coral reefs. Despite the importance of these systems, major gaps remain in our understanding of how thermal stress and bleaching affect the metabolic networks that underpin holobiont function. We applied gas chromatography–mass spectrometry (GC–MS) metabolomics to detect changes in the intracellular free metabolite pools (polar and semi-polar compounds) of in hospite dinoflagellate symbionts and their coral hosts (and any associated microorganisms) during early- and late-stage thermal bleaching (a reduction of approximately 50 and 70% in symbiont density, respectively). We detected characteristic changes to the metabolite profiles of each symbiotic partner associated with individual cellular responses to thermal, oxidative and osmotic stress, which progressed with the severity of bleaching. Alterations were also indicative of changes to energy-generating and biosynthesis pathways in both partners, with a shift to the increased catabolism of lipid stores. Specifically, in symbiont intracellular metabolite pools, we observed accumulations of multiple free fatty acids, plus the chloroplast-associated antioxidant alpha-tocopherol. In the host, we detected a decline in the abundance of pools of multiple carbohydrates, amino acids and intermediates, in addition to the antioxidant ascorbate. These findings further our understanding of the metabolic changes that occur to symbiont and host (and its associated microorganisms) during thermal bleaching. These findings also provide further insight into the largely undescribed roles of free metabolite pools in cellular homeostasis, signalling and acclimation to thermal stress in the cnidarian–dinoflagellate symbiosis.     

Apoptosis and autophagy as mechanisms of dinoflagellate symbiont release during cnidarian bleaching: every which way you lose

Cnidarian bleaching results from the breakdown in the symbiosis between the host cnidarian and its dinoflagellate symbiont. Coral bleaching in recent years has increasingly caused degradation and mortality of coral reefs on a global scale. Although much is understood about the environmental causes of bleaching, the underlying cellular mechanisms of symbiont release that drive the process are just beginning to be described. In this study, we investigated the roles of two cellular pathways, host cell apoptosis and autophagy, in the bleaching process of the symbiotic anemone Aiptasia pallida. Host cell apoptosis was experimentally manipulated using gene knockdown of an anemone caspase by RNA interference, chemical inhibition of caspase using ZVAD-fmk and an apoptosis-inducer wortmannin. Autophagy was manipulated by chemical inhibition using wortmannin or induction using rapamycin. The applications of multiple single treatments resulted in some increased bleaching in anemones under control conditions but no significant drop in bleaching in individuals subjected to a hyperthermic stress. These results indicated that no single pathway is responsible for symbiont release during bleaching. However, when multiple inhibitors were applied simultaneously to block both apoptosis and autophagy, there was a significant reduction in bleaching in heat-stressed anemones. Our results allow us to formulate a model for cellular processes involved in the control of cnidarian bleaching where apoptosis and autophagy act together in a see-saw mechanism such that if one is inhibited the other is induced. Similar interconnectivity between apoptosis and autophagy has previously been shown in vertebrates including involvement in an innate immune response to pathogens and parasites. This suggests that the bleaching response could be a modified immune response that recognizes and removes dysfunctional symbionts.     

Differential nitric oxide synthesis and host apoptotic events correlate with bleaching susceptibility in reef corals

Coral bleaching poses a threat to coral reefs worldwide. As a consequence of the temperature-induced breakdown in coral–dinoflagellate symbiosis, bleaching can have extensive effects on reef communities. However, our understanding of bleaching at a cellular level is limited, and this is particularly true regarding differential susceptibility among coral species. Recent work suggests that bleaching may represent a host innate immune-like response to symbiont dysfunction that involves synthesis of the signalling compound nitric oxide (NO) and the induction of host apoptotic-like cell death. In this study, we examined the activity of apoptosis-regulating enzymes alongside oxidised NO accumulation (a proxy for NO synthesis) in the reef corals Acropora millepora, Montipora digitata, and Pocillopora damicornis during experimental thermal stress. P. damicornis was the most sensitive species, suffering mortality (tissue sloughing) after 5 days at 33 °C but non-lethal bleaching after 9 days at 31.5 °C. A. millepora bleached at 33 °C but remained structurally intact, while M. digitata showed little evidence of bleaching. P. damicornis and A. millepora both exhibited evidence of temperature-induced NO synthesis and, after 5 days of heating, levels of oxidised NO in both species were fivefold higher than in controls maintained at 28.5 °C. These responses preceded bleaching by a number of days and may have occurred before symbiont dysfunction (measured as chlorophyll a degradation and oxidised NO accumulation). In A. millepora, apparent NO synthesis correlated with the induction of host apoptotic-like pathways, while in P. damicornis, the upregulation of apoptotic pathways occurred later. No evidence of elevated NO production or apoptosis was observed in M. digitata at 33 °C and baseline activity of apoptosis-regulating enzymes was negligible in this species. These findings provide important physiological data in the context of the responses of corals to global change and suggest that early events in the host may be important in the collapse of the coral–dinoflagellate symbiosis.     

Oxidative stress causes coral bleaching during exposure to elevated temperatures

 Elevated temperatures and solar ultraviolet (UV) radiation have been implicated as recent causes for the loss of symbiotic algae (i.e., bleaching) in corals and other invertebrates with photoautotrophic symbionts. One hypothesized mechanism of coral bleaching involves the production of reduced oxygen intermediates, or toxic oxygen, in the dinoflagellate symbionts and host tissues that subsequently causes cellular damage and expulsion of symbionts. Measurements of photosynthesis in the Caribbean coral Agaricia tenuifolia, taken during temperature-induced stress and exposure to full solar radiation, showed a decrease in photosynthetic performance followed by bleaching. Exposure of corals to exogenous antioxidants that scavenge reactive oxygen species during temperature-induced stress improves maximum photosynthetic capacity to rates indistinguishable from corals measured at the ambient temperature of their site of collection. Additionally, these antioxidants prevent the coral from “ bleaching ” and affect the mechanism of symbiont loss from the coral host. These observations confirm a role for oxidative stress, whether caused by elevated temperatures or exposure to UV radiation, in the bleaching phenomenon. Accepted: 18 October 1996     

Host Coenzyme Q Redox State Is an Early Biomarker of Thermal Stress in the Coral Acropora millepora

Bleaching episodes caused by increasing seawater temperatures may induce mass coral mortality and are regarded as one of the biggest threats to coral reef ecosystems worldwide. The current consensus is that this phenomenon results from enhanced production of harmful reactive oxygen species (ROS) that disrupt the symbiosis between corals and their endosymbiotic dinoflagellates, Symbiodinium. Here, the responses of two important antioxidant defence components, the host coenzyme Q (CoQ) and symbiont plastoquinone (PQ) pools, are investigated for the first time in colonies of the scleractinian coral, Acropora millepora, during experimentally-induced bleaching under ecologically relevant conditions. Liquid chromatography-mass spectrometry (LC-MS) was used to quantify the states of these two pools, together with physiological parameters assessing the general state of the symbiosis (including photosystem II photochemical efficiency, chlorophyll concentration and Symbiodinium cell densities). The results show that the responses of the two antioxidant systems occur on different timescales: (i) the redox state of the Symbiodinium PQ pool remained stable until twelve days into the experiment, after which there was an abrupt oxidative shift; (ii) by contrast, an oxidative shift of approximately 10% had occurred in the host CoQ pool after 6 days of thermal stress, prior to significant changes in any other physiological parameter measured. Host CoQ pool oxidation is thus an early biomarker of thermal stress in corals, and this antioxidant pool is likely to play a key role in quenching thermally-induced ROS in the coral-algal symbiosis. This study adds to a growing body of work that indicates host cellular responses may precede the bleaching process and symbiont dysfunction.     

Thermal Stress Promotes Host Mitochondrial Degradation in Symbiotic Cnidarians: Are the Batteries of the Reef Going to Run Out?

The symbiotic relationship between cnidarians and their dinoflagellate symbionts, Symbiodinium spp, which underpins the formation of tropical coral reefs, can be destabilized by rapid changes to environmental conditions. Although some studies have concluded that a breakdown in the symbiosis begins with increased reactive oxygen species (ROS) generation within the symbiont due to a decoupling of photosynthesis, others have reported the release of viable symbionts via a variety of host cell derived mechanisms. We explored an alternative model focused upon changes in host cnidarian mitochondrial integrity in response to thermal stress. Mitochondria are often likened to being batteries of the cell, providing energy in the form of ATP, and controlling cellular pathway activation and ROS generation. The overall morphology of host mitochondria was compared to that of associated symbionts under an experimental thermal stress using confocal and electron microscopy. The results demonstrate that hyperthermic stress induces the degradation of cnidarian host mitochondria that is independent of symbiont cellular deterioration. The potential sites of host mitochondrial disruption were also assessed by measuring changes in the expression of genes associated with electron transport and ATP synthesis using quantitative RT-PCR. The primary site of degradation appeared to be downstream of complex III of the electron transport chain with a significant reduction in host cytochrome c and ATP synthase expression. The consequences of reduced expression could limit the capacity of the host to mitigate ROS generation and maintain both organelle integrity and cellular energy supplies. The disruption of host mitochondria, cellular homeostasis, and subsequent cell death irrespective of symbiont integrity highlights the importance of the host response to thermal stress and in symbiosis dysfunction that has substantial implications for understanding how coral reefs will survive in the face of climate change.     

Investigating the causes and consequences of symbiont shuffling in a multi-partner reef coral symbiosis under environmental change

Dynamic symbioses may critically mediate impacts of climate change on diverse organisms, with repercussions for ecosystem persistence in some cases. On coral reefs, increases in heat-tolerant symbionts after thermal bleaching can reduce coral susceptibility to future stress. However, the relevance of this adaptive response is equivocal owing to conflicting reports of symbiont stability and change. We help reconcile this conflict by showing that change in symbiont community composition (symbiont shuffling) in Orbicella faveolata depends on the disturbance severity and recovery environment. The proportion of heat-tolerant symbionts dramatically increased following severe experimental bleaching, especially in a warmer recovery environment, but tended to decrease if bleaching was less severe. These patterns can be explained by variation in symbiont performance in the changing microenvironments created by differentially bleached host tissues. Furthermore, higher proportions of heat-tolerant symbionts linearly increased bleaching resistance but reduced photochemical efficiency, suggesting that any change in community structure oppositely impacts performance and stress tolerance. Therefore, even minor symbiont shuffling can adaptively benefit corals, although fitness effects of resulting trade-offs are difficult to predict. This work helps elucidate causes and consequences of dynamism in symbiosis, which is critical to predicting responses of multi-partner symbioses such as O. faveolata to environmental change.     

Regulation of apoptotic mediators reveals dynamic responses to thermal stress in the reef building coral Acropora millepora

Background

Mass coral bleaching is increasing in scale and frequency across the world''s coral reefs and is being driven primarily by increased levels of thermal stress arising from global warming. In order to understand the impacts of projected climate change upon corals reefs, it is important to elucidate the underlying cellular mechanisms that operate during coral bleaching and subsequent mortality. In this respect, increased apoptotic cell death activity is an important cellular process that is associated with the breakdown of the mutualistic symbiosis between the cnidarian host and their dinoflagellate symbionts.

Methodology/Principal Findings

The present study reports the impacts of different stressors (colchicine and heat stress) on three phases of apoptosis: (i) the potential initiation by differential expression of Bcl-2 members, (ii) the execution of apoptotic events by activation of caspase 3-like proteases and (iii) and finally, the cell disposal indicated by DNA fragmentation in the reef building coral Acropora millepora. In corals incubated with colchicine, an increase in caspase 3-like activity and DNA fragmentation was associated with a relative down-regulation of Bcl-2, suggesting that the initiation of apoptosis may be mediated by the suppression of an anti-apoptotic mechanism. In contrast, in the early steps of heat stress, the induction of caspase-dependent apoptosis was related to a relative up-regulation of Bcl-2 consecutively followed by a delayed decrease in apoptosis activity.

Conclusions/Significance

In the light of these results, we propose a model of heat stress in coral hosts whereby increasing temperatures engage activation of caspase 3-dependent apoptosis in cells designated for termination, but also the onset of a delayed protective response involving overexpression of Bcl-2 in surviving cells. This mitigating response to thermal stress could conceivably be an important regulatory mechanism for cell survival in corals exposed to sudden environmental changes.     

<Superscript>13</Superscript>C metabolomics reveals widespread change in carbon fate during coral bleaching

Introduction

Rising seawater temperatures are threatening the persistence of coral reefs; where above critical thresholds, thermal stress results in a breakdown of the coral-dinoflagellate symbiosis and the loss of algal symbionts (coral bleaching). As symbiont-derived organic products typically form a major portion of host energy budgets, this has major implications for the fitness and persistence of symbiotic corals.

Objectives

We aimed to determine change in autotrophic carbon fate within individual compounds and downstream metabolic pathways in a coral symbiosis exposed to varying degrees of thermal stress and bleaching.

Methods

We applied gas chromatography–mass spectrometry coupled to a stable isotope tracer (¹³C), to track change in autotrophic carbon fate, in symbiont and host individually, following exposure to elevated water temperature.

Results

Thermal stress resulted in partner-specific changes in carbon fate, which progressed with heat stress duration. We detected modifications to carbohydrate and fatty acid metabolism, lipogenesis, and homeostatic responses to thermal, oxidative and osmotic stress. Despite pronounced photodamage, remaining in hospite symbionts continued to produce organic products de novo and translocate to the coral host. However as bleaching progressed, we observed minimal ¹³C enrichment of symbiont long-chain fatty acids, also reflected in ¹³C enrichment of host fatty acid pools.

Conclusion

These data have major implications for our understanding of coral symbiosis function during bleaching. Our findings suggest that during early stage bleaching, remaining symbionts continue to effectively translocate a variety of organic products to the host, however under prolonged thermal stress there is likely a reduction in the quality of these products.

     

A profile of an endosymbiont-enriched fraction of the coral Stylophora pistillata reveals proteins relevant to microbial-host interactions

This study examines the response of Symbiodinium sp. endosymbionts from the coral Stylophora pistillata to moderate levels of thermal "bleaching" stress, with and without trace metal limitation. Using quantitative high throughput proteomics, we identified 8098 MS/MS events relating to individual peptides from the endosymbiont-enriched fraction, including 109 peptides meeting stringent criteria for quantification, of which only 26 showed significant change in our experimental treatments; 12 of 26 increased expression in response to thermal stress with little difference affected by iron limitation. Surprisingly, there were no significant increases in antioxidant or heat stress proteins; those induced to higher expression were generally involved in protein biosynthesis. An outstanding exception was a massive 114-fold increase of a viral replication protein indicating that thermal stress may substantially increase viral load and thereby contribute to the etiology of coral bleaching and disease. In the absence of a sequenced genome for Symbiodinium or other photosymbiotic dinoflagellate, this proteome reveals a plethora of proteins potentially involved in microbial-host interactions. This includes photosystem proteins, DNA repair enzymes, antioxidant enzymes, metabolic redox enzymes, heat shock proteins, globin hemoproteins, proteins of nitrogen metabolism, and a wide range of viral proteins associated with these endosymbiont-enriched samples. Also present were 21 unusual peptide/protein toxins thought to originate from either microbial consorts or from contamination by coral nematocysts. Of particular interest are the proteins of apoptosis, vesicular transport, and endo/exocytosis, which are discussed in context of the cellular processes of coral bleaching. Notably, the protein complement provides evidence that, rather than being expelled by the host, stressed endosymbionts may mediate their own departure.     

Corals in the hottest reefs in the world exhibit symbiont fidelity not flexibility

Reef‐building corals are at risk of extinction from ocean warming. While some corals can enhance their thermal limits by associating with dinoflagellate photosymbionts of superior stress tolerance, the extent to which symbiont communities will reorganize under increased warming pressure remains unclear. Here we show that corals in the hottest reefs in the world in the Persian Gulf maintain associations with the same symbionts across 1.5 years despite extreme seasonal warming and acute heat stress (≥35°C). Persian Gulf corals predominantly associated with Cladocopium (clade C) and most also hosted Symbiodinium (clade A) and/or Durusdinium (clade D). This is in contrast to the neighbouring and milder Oman Sea, where corals associated with Durusdinium and only a minority hosted background levels of Cladocopium. During acute heat stress, the higher prevalence of Symbiodinium and Durusdinium in bleached versus nonbleached Persian Gulf corals indicates that genotypes of these background genera did not confer bleaching resistance. Within symbiont genera, the majority of ITS2 rDNA type profiles were unique to their respective coral species, confirming the existence of host‐specific symbiont lineages. Notably, further differentiation among Persian Gulf sites demonstrates that symbiont populations are either isolated or specialized over tens to hundreds of kilometres. Thermal tolerance across coral species was associated with the prevalence of a single ITS2 intragenomic sequence variant (C3gulf), definitive of the Cladocopium thermophilum group. The abundance of C3gulf was highest in bleaching‐resistant corals and at warmer sites, potentially indicating a specific symbiont genotype (or set of genotypes) that may play a role in thermal tolerance that warrants further investigation. Together, our findings indicate that co‐evolution of host–Symbiodiniaceae partnerships favours fidelity rather than flexibility in extreme environments and under future warming.     

The role of zooxanthellae in the thermal tolerance of corals: a 'nugget of hope' for coral reefs in an era of climate change

The ability of coral reefs to survive the projected increases in temperature due to global warming will depend largely on the ability of corals to adapt or acclimatize to increased temperature extremes over the next few decades. Many coral species are highly sensitive to temperature stress and the number of stress (bleaching) episodes has increased in recent decades. We investigated the acclimatization potential of Acropora millepora, a common and widespread Indo-Pacific hard coral species, through transplantation and experimental manipulation. We show that adult corals, at least in some circumstances, are capable of acquiring increased thermal tolerance and that the increased tolerance is a direct result of a change in the symbiont type dominating their tissues from Symbiodinium type C to D. Our data suggest that the change in symbiont type in our experiment was due to a shuffling of existing types already present in coral tissues, not through exogenous uptake from the environment. The level of increased tolerance gained by the corals changing their dominant symbiont type to D (the most thermally resistant type known) is around 1-1.5 degrees C. This is the first study to show that thermal acclimatization is causally related to symbiont type and provides new insight into the ecological advantage of corals harbouring mixed algal populations. While this increase is of huge ecological significance for many coral species, in the absence of other mechanisms of thermal acclimatization/adaptation, it may not be sufficient to survive climate change under predicted sea surface temperature scenarios over the next 100 years. However, it may be enough to 'buy time' while greenhouse reduction measures are put in place.     

Incorporating adaptive responses into future projections of coral bleaching

Climate warming threatens to increase mass coral bleaching events, and several studies have projected the demise of tropical coral reefs this century. However, recent evidence indicates corals may be able to respond to thermal stress though adaptive processes (e.g., genetic adaptation, acclimatization, and symbiont shuffling). How these mechanisms might influence warming‐induced bleaching remains largely unknown. This study compared how different adaptive processes could affect coral bleaching projections. We used the latest bias‐corrected global sea surface temperature (SST) output from the NOAA/GFDL Earth System Model 2 (ESM2M) for the preindustrial period through 2100 to project coral bleaching trajectories. Initial results showed that, in the absence of adaptive processes, application of a preindustrial climatology to the NOAA Coral Reef Watch bleaching prediction method overpredicts the present‐day bleaching frequency. This suggests that corals may have already responded adaptively to some warming over the industrial period. We then modified the prediction method so that the bleaching threshold either permanently increased in response to thermal history (e.g., simulating directional genetic selection) or temporarily increased for 2–10 years in response to a bleaching event (e.g., simulating symbiont shuffling). A bleaching threshold that changes relative to the preceding 60 years of thermal history reduced the frequency of mass bleaching events by 20–80% compared with the ‘no adaptive response’ prediction model by 2100, depending on the emissions scenario. When both types of adaptive responses were applied, up to 14% more reef cells avoided high‐frequency bleaching by 2100. However, temporary increases in bleaching thresholds alone only delayed the occurrence of high‐frequency bleaching by ca. 10 years in all but the lowest emissions scenario. Future research should test the rate and limit of different adaptive responses for coral species across latitudes and ocean basins to determine if and how much corals can respond to increasing thermal stress.     

Resistance to thermal stress in corals without changes in symbiont composition

Discovering how corals can adjust their thermal sensitivity in the context of global climate change is important in understanding the long-term persistence of coral reefs. In this study, we showed that short-term preconditioning to higher temperatures, 3°C below the experimentally determined bleaching threshold, for a period of 10 days provides thermal tolerance for the symbiosis stability between the scleractinian coral, Acropora millepora and Symbiodinium. Based on genotypic analysis, our results indicate that the acclimatization of this coral species to thermal stress does not come down to simple changes in Symbiodinium and/or the bacterial communities that associate with reef-building corals. This suggests that the physiological plasticity of the host and/or symbiotic components appears to play an important role in responding to ocean warming. The further study of host and symbiont physiology, both of Symbiodinium and prokaryotes, is of paramount importance in the context of global climate change, as mechanisms for rapid holobiont acclimatization will become increasingly important to the long-standing persistence of coral reefs.     

Thermally tolerant corals have limited capacity to acclimatize to future warming

Thermal stress affects organism performance differently depending on the ambient temperature to which they are acclimatized, which varies along latitudinal gradients. This study investigated whether differences in physiological responses to temperature are consistent with regional differences in temperature regimes for the stony coral Oculina patagonica. To resolve this question, we experimentally assessed how colonies originating from four different locations characterized by >3 °C variation in mean maximum annual temperature responded to warming from 20 to 32 °C. We assessed plasticity in symbiont identity, density, and photosynthetic properties, together with changes in host tissue biomass. Results show that, without changes in the type of symbiont hosted by coral colonies, O. patagonica has limited capacity to acclimatize to future warming. We found little evidence of variation in overall thermal tolerance, or in thermal optima, in response to spatial variation in ambient temperature. Given that the invader O. patagonica is a relatively new member of the Mediterranean coral fauna, our results also suggest that coral populations may need to remain isolated for a long period of time for thermal adaptation to potentially take place. Our study indicates that for O. patagonica, mortality associated with thermal stress manifests primarily through tissue breakdown under moderate but prolonged warming (which does not impair symbiont photosynthesis and, therefore, does not lead to bleaching). Consequently, projected global warming is likely to cause repeat incidents of partial and whole colony mortality and might drive a gradual range contraction of Mediterranean corals.     

Heterotrophy mitigates the response of the temperate coral Oculina arbuscula to temperature stress

Anthropogenic increases in atmospheric carbon dioxide concentration have caused global average sea surface temperature (SST) to increase by approximately 0.11°C per decade between 1971 and 2010 – a trend that is projected to continue through the 21st century. A multitude of research studies have demonstrated that increased SSTs compromise the coral holobiont (cnidarian host and its symbiotic algae) by reducing both host calcification and symbiont density, among other variables. However, we still do not fully understand the role of heterotrophy in the response of the coral holobiont to elevated temperature, particularly for temperate corals. Here, we conducted a pair of independent experiments to investigate the influence of heterotrophy on the response of the temperate scleractinian coral Oculina arbuscula to thermal stress. Colonies of O. arbuscula from Radio Island, North Carolina, were exposed to four feeding treatments (zero, low, moderate, and high concentrations of newly hatched Artemia sp. nauplii) across two independent temperature experiments (average annual SST (20°C) and average summer temperature (28°C) for the interval 2005–2012) to quantify the effects of heterotrophy on coral skeletal growth and symbiont density. Results suggest that heterotrophy mitigated both reduced skeletal growth and decreased symbiont density observed for unfed corals reared at 28°C. This study highlights the importance of heterotrophy in maintaining coral holobiont fitness under thermal stress and has important implications for the interpretation of coral response to climate change.