Extra high superoxide dismutase in host tissue is associated with improving bleaching resistance in “thermal adapted” and Durusdinium trenchii-associating coral

Global warming threatens reef-building corals with large-scale bleaching events; therefore, it is important to discover potential adaptive capabilities for increasing their temperature resistance before it is too late. This study presents two coral species (Platygyra verweyi and Isopora palifera) surviving on a reef having regular hot water influxes via a nearby nuclear power plant that exhibited completely different bleaching susceptibilities to thermal stress, even though both species shared several so-called “winner” characteristics (e.g., containing Durusdinium trenchii, thick tissue, etc.). During acute heating treatment, algal density did not decline in P. verweyi corals within three days of being directly transferred from 25 to 31 °C; however, the same treatment caused I. palifera to lose < 70% of its algal symbionts within 24 h. The most distinctive feature between the two coral species was an overwhelmingly higher constitutive superoxide dismutase (ca. 10-fold) and catalase (ca. 3-fold) in P. verweyi over I. palifera. Moreover, P. verweyi also contained significantly higher saturated and lower mono-unsaturated fatty acids, especially a long-chain saturated fatty acid (C22:0), than I. palifera, and was consistently associated with the symbiotic bacteria Endozoicomonas, which was not found in I. palifera. However, antibiotic treatment and inoculation tests did not support Endozoicomonas having a direct contribution to thermal resistance. This study highlights that, besides its association with a thermally tolerable algal symbiont, a high level of constitutive antioxidant enzymes in the coral host is crucial for coral survivorship in the more fluctuating and higher temperature environments.

Regulation of the Coral-Associated Bacteria and Symbiodiniaceae in Acropora valida Under Ocean Acidification

Ocean acidification is one of many stressors that coral reef ecosystems are currently contending with. Thus, understanding the response of key symbiotic microbes to ocean acidification is of great significance for understanding the adaptation mechanism and development trend of coral holobionts. Here, high-throughput sequencing technology was employed to investigate the coral-associated bacteria and Symbiodiniaceae of the ecologically important coral Acropora valida exposed to different pH gradients. After 30 days of acclimatization, we set four acidification gradients (pH 8.2, 7.8, 7.4, and 7.2, respectively), and each pH condition was applied for 10 days, with the whole experiment lasting for 70 days. Although the Symbiodiniaceae density decreased significantly, the coral did not appear to be bleached, and the real-time photosynthetic rate did not change significantly, indicating that A. valida has strong tolerance to acidification. Moreover, the Symbiodiniaceae community composition was hardly affected by ocean acidification, with the C1 subclade (Cladocopium goreaui) being dominant among the Symbiodiniaceae dominant types. The relative abundance of the Symbiodiniaceae background types was significantly higher at pH 7.2, indicating that ocean acidification might increase the stability of the community composition by regulating the Symbiodiniaceae rare biosphere. Furthermore, the stable symbiosis between the C1 subclade and coral host may contribute to the stability of the real-time photosynthetic efficiency. Finally, concerning the coral-associated bacteria, the stable symbiosis between Endozoicomonas and coral host is likely to help them adapt to ocean acidification. The significant increase in the relative abundance of Cyanobacteria at pH 7.2 may also compensate for the photosynthesis efficiency of a coral holobiont. In summary, this study suggests that the combined response of key symbiotic microbes helps the whole coral host resist the threats of ocean acidification.

Intra-genomic variation in symbiotic dinoflagellates: Recent divergence or natural hybridization?

<p>The perpetuity of coral reefs will ultimately depend on the ability of corals to adapt to changing conditions. Inter-specific hybridization can provide the raw genetic material necessary for adaptation, and stimulate macro-evolutionary leaps during periods of environmental upheaval. Though well-documented in corals, hybridization has yet to be identified in their dinoflagellate symbionts (genus Symbiodinium), despite growing evidence of sexual reproduction in this genus. The integral roles that these symbiotic algae play in coral productivity, reef accretion and ‘coral bleaching’ emphasize the need to better understand their short-term evolutionary potential. In this thesis, I develop new molecular and statistical methodology, and combine lab- and field-based analysis to explore the potential for hybridization between divergent Symbiodinium taxa. To screen for putative Symbiodinium hybrids, intra-genomic variation was examined within individual symbionts isolated from the reef-building coral Pocillopora damicornis at Lord Howe Island (Australia). A nested quantitative PCR (qPCR) assay was developed to quantify polymorphic internal transcribed spacer 2 (ITS2) sequences within the genome of each symbiont cell. Three genetically distinct Symbiodinium populations were detected co-existing within the symbiont consortium of P. damicornis. Mixed populations of ‘pure’ Symbiodinium types C100 and C109 coexisted with a population of cells hosting co-dominant C100 and C109 ITS2 repeats. Genetically heterogeneous Symbiodinium cells were more common than homogeneous symbionts in four of the six colonies analysed, with a maximum proportional abundance of 89%. Morphological, functional and ecological attributes of heterogeneous Symbiodinium cells were characterized to assess their candidacy as putative hybrids. The proportional abundance of genetically heterogeneous symbionts was spatially and temporally conserved within colonies, indicating a lack of competition between Symbiodinium populations. However, this abundance ratio varied considerably between colonies separated by metres to tens of metres, and to a greater extent between sites isolated by hundreds to thousands of metres. The local thermal maximum emerged as a significant predictor of the proportional abundance of genetically heterogeneous Symbiodinium cells, suggesting that the distribution of these ‘putative hybrids’ is influenced by a reduced affinity for thermal stress. Genetically heterogeneous Symbiodinium cells were around 50% larger (by volume) than homogeneous cells, occupied tissue of the coral host at reduced densities, and showed relatively poor light-harvesting efficiency. Colonies hosting a higher proportion of these symbionts suffered a reduction in overall photosynthetic performance (maximum gross photosynthesis normalised to respiration; P:R) at the ambient temperature of 25 °C. This disparity was maintained when the temperature was elevated to simulate the maximum experienced within the LHI lagoon (29 °C). Under these stressful conditions, colonies dominated by putative Symbiodinium hybrids were only marginally capable of net oxygen production. The influence of putative Symbiodinium hybrids on the growth and survival of P. damicornis was tested by reciprocally transplanting coral colonies between reef sites featuring distinct temperature regimes. Neither calcification nor mortality was influenced by the proportional abundance of genetically heterogeneous cells in the symbiont consortium. This uncoupling of symbiont performance and host fitness may be explained by stochastic events such as predation and disease, which substantially increase variation in growth and mortality in field experiments. Alternatively, it may represent some unknown benefit associated with hosting hybrid symbionts, belying their relatively poor photosynthetic performance, and explaining the widespread abundance of these heterogeneous Symbiodinium cells on the Lord Howe Island reef. Our inability to maintain many clade C Symbiodinium types in culture prevents direct observations of hybridization between C100 and C109. Unequivocal evidence of this phenomenon will therefore likely remain elusive until high-resolution, single-copy nuclear markers can be developed, since the incomplete displacement of ancestral polymorphisms can leave a similar genomic signature to that of hybridization. However, this study serves to provide an initial proof-of-principle for hybridization between divergent Symbiodinium taxa. In doing so, it highlights the need to better understand the evolutionary processes underpinning coral- and symbiont-adaptation in a changing climate.</p>

Intra-genomic variation in symbiotic dinoflagellates: Recent divergence or natural hybridization?

<p>The perpetuity of coral reefs will ultimately depend on the ability of corals to adapt to changing conditions. Inter-specific hybridization can provide the raw genetic material necessary for adaptation, and stimulate macro-evolutionary leaps during periods of environmental upheaval. Though well-documented in corals, hybridization has yet to be identified in their dinoflagellate symbionts (genus Symbiodinium), despite growing evidence of sexual reproduction in this genus. The integral roles that these symbiotic algae play in coral productivity, reef accretion and ‘coral bleaching’ emphasize the need to better understand their short-term evolutionary potential. In this thesis, I develop new molecular and statistical methodology, and combine lab- and field-based analysis to explore the potential for hybridization between divergent Symbiodinium taxa. To screen for putative Symbiodinium hybrids, intra-genomic variation was examined within individual symbionts isolated from the reef-building coral Pocillopora damicornis at Lord Howe Island (Australia). A nested quantitative PCR (qPCR) assay was developed to quantify polymorphic internal transcribed spacer 2 (ITS2) sequences within the genome of each symbiont cell. Three genetically distinct Symbiodinium populations were detected co-existing within the symbiont consortium of P. damicornis. Mixed populations of ‘pure’ Symbiodinium types C100 and C109 coexisted with a population of cells hosting co-dominant C100 and C109 ITS2 repeats. Genetically heterogeneous Symbiodinium cells were more common than homogeneous symbionts in four of the six colonies analysed, with a maximum proportional abundance of 89%. Morphological, functional and ecological attributes of heterogeneous Symbiodinium cells were characterized to assess their candidacy as putative hybrids. The proportional abundance of genetically heterogeneous symbionts was spatially and temporally conserved within colonies, indicating a lack of competition between Symbiodinium populations. However, this abundance ratio varied considerably between colonies separated by metres to tens of metres, and to a greater extent between sites isolated by hundreds to thousands of metres. The local thermal maximum emerged as a significant predictor of the proportional abundance of genetically heterogeneous Symbiodinium cells, suggesting that the distribution of these ‘putative hybrids’ is influenced by a reduced affinity for thermal stress. Genetically heterogeneous Symbiodinium cells were around 50% larger (by volume) than homogeneous cells, occupied tissue of the coral host at reduced densities, and showed relatively poor light-harvesting efficiency. Colonies hosting a higher proportion of these symbionts suffered a reduction in overall photosynthetic performance (maximum gross photosynthesis normalised to respiration; P:R) at the ambient temperature of 25 °C. This disparity was maintained when the temperature was elevated to simulate the maximum experienced within the LHI lagoon (29 °C). Under these stressful conditions, colonies dominated by putative Symbiodinium hybrids were only marginally capable of net oxygen production. The influence of putative Symbiodinium hybrids on the growth and survival of P. damicornis was tested by reciprocally transplanting coral colonies between reef sites featuring distinct temperature regimes. Neither calcification nor mortality was influenced by the proportional abundance of genetically heterogeneous cells in the symbiont consortium. This uncoupling of symbiont performance and host fitness may be explained by stochastic events such as predation and disease, which substantially increase variation in growth and mortality in field experiments. Alternatively, it may represent some unknown benefit associated with hosting hybrid symbionts, belying their relatively poor photosynthetic performance, and explaining the widespread abundance of these heterogeneous Symbiodinium cells on the Lord Howe Island reef. Our inability to maintain many clade C Symbiodinium types in culture prevents direct observations of hybridization between C100 and C109. Unequivocal evidence of this phenomenon will therefore likely remain elusive until high-resolution, single-copy nuclear markers can be developed, since the incomplete displacement of ancestral polymorphisms can leave a similar genomic signature to that of hybridization. However, this study serves to provide an initial proof-of-principle for hybridization between divergent Symbiodinium taxa. In doing so, it highlights the need to better understand the evolutionary processes underpinning coral- and symbiont-adaptation in a changing climate.</p>

Bleaching of the Cnidarian-Dinoflagellate Symbiosis: Aspects of Innate Immunity and the Role of Nitric Oxide

<p>Driven by global warming and the increasing frequency of high temperature anomalies, the collapse of the cnidarian-dinoflagellate symbiosis (known as "bleaching" due to the whitening of host tissues) is contributing to worldwide coral reef decline. Much is known about the consequences of bleaching, but despite over 20 years of effort, we still know little about the physiological mechanisms involved. This is particularly true when explaining the differential susceptibility of coral hosts and their algal partners (genus Symbiodinium) to rising temperatures. Work carried out over the past 10 years suggests that bleaching may represent an innate immune-like host response to dysfunctional symbionts. This response involves the synthesis of nitric oxide (NO), a signalling molecule widely dispersed throughout the tree of life and implicated in diverse cellular phenomena. However, the source(s) of NO in the cnidarian-dinoflagellate association have been the subject of debate, and almost nothing is known of the capacity for differential NO synthesis among different host species or symbiont types. The aim of this study was to elucidate the role of NO in the temperature-induced breakdown of the cnidarian-dinoflagellate symbiosis and to assess differences in NO-mediated physiology at the level of both symbiont and host. The specific objectives were (i) to quantify NO synthesis in different types of symbiotic dinoflagellates, (ii) to determine a role for NO in the collapse of the cnidarian-dinoflagellate symbiosis, (iii) to confirm whether NO itself - as opposed to its more reactive derivatives - is capable of mediating cnidarian bleaching, and (iv) to measure the synthesis of NO and the regulation of associated pathways in different reef corals undergoing bleaching. This thesis demonstrates that both partners of the symbiosis have a capacity for synthesising NO when stimulated by elevated temperature. However, their contributions to NO synthesis in the intact symbiosis may not be equal, as heightened symbiont NO production invariably occurred after that of the host, and at a time when bleaching had already commenced. Closer examination of host-derived NO in the model anemone Aiptasia pulchella revealed that the compound most likely mediates bleaching through apoptotic-like cell death pathways, as either removing NO or inhibiting the activity of an important apoptosis-regulating enzyme could alleviate bleaching. NO's involvement in thermal bleaching also seems to be independent of its conversion to more toxic radicals such as peroxynitrite (ONOO-), which, although present at elevated temperature, had little influence on symbiont loss in A. pulchella. [...] As is the case in a wide variety of animal-microbe interactions, NO appears to mediate the cnidarian-dinoflagellate symbiosis by influencing the activity of host apoptotic-like pathways. Interestingly, the activation of these host responses at elevated temperature may occur before the dinoflagellate becomes photosynthetically compromised. As such, the model of bleaching as simply a response to symbiont photoinhibition could require modification. Furthermore, the differential sensitivity of symbiont types to NO, coupled with the differential regulation of NO-synthetic and apoptotic pathways in the host, could contribute to corals' varying bleaching susceptibilities. This thesis provides vital insights into the cell biology of the coral-dinoflagellate symbiosis and the events underpinning its breakdown during temperature stress. It also encourages a greater research emphasis on understanding physiological responses at the level of the coral host as well as during the early stages of a bleaching event.</p>

Bleaching of the Cnidarian-Dinoflagellate Symbiosis: Aspects of Innate Immunity and the Role of Nitric Oxide

<p>Driven by global warming and the increasing frequency of high temperature anomalies, the collapse of the cnidarian-dinoflagellate symbiosis (known as "bleaching" due to the whitening of host tissues) is contributing to worldwide coral reef decline. Much is known about the consequences of bleaching, but despite over 20 years of effort, we still know little about the physiological mechanisms involved. This is particularly true when explaining the differential susceptibility of coral hosts and their algal partners (genus Symbiodinium) to rising temperatures. Work carried out over the past 10 years suggests that bleaching may represent an innate immune-like host response to dysfunctional symbionts. This response involves the synthesis of nitric oxide (NO), a signalling molecule widely dispersed throughout the tree of life and implicated in diverse cellular phenomena. However, the source(s) of NO in the cnidarian-dinoflagellate association have been the subject of debate, and almost nothing is known of the capacity for differential NO synthesis among different host species or symbiont types. The aim of this study was to elucidate the role of NO in the temperature-induced breakdown of the cnidarian-dinoflagellate symbiosis and to assess differences in NO-mediated physiology at the level of both symbiont and host. The specific objectives were (i) to quantify NO synthesis in different types of symbiotic dinoflagellates, (ii) to determine a role for NO in the collapse of the cnidarian-dinoflagellate symbiosis, (iii) to confirm whether NO itself - as opposed to its more reactive derivatives - is capable of mediating cnidarian bleaching, and (iv) to measure the synthesis of NO and the regulation of associated pathways in different reef corals undergoing bleaching. This thesis demonstrates that both partners of the symbiosis have a capacity for synthesising NO when stimulated by elevated temperature. However, their contributions to NO synthesis in the intact symbiosis may not be equal, as heightened symbiont NO production invariably occurred after that of the host, and at a time when bleaching had already commenced. Closer examination of host-derived NO in the model anemone Aiptasia pulchella revealed that the compound most likely mediates bleaching through apoptotic-like cell death pathways, as either removing NO or inhibiting the activity of an important apoptosis-regulating enzyme could alleviate bleaching. NO's involvement in thermal bleaching also seems to be independent of its conversion to more toxic radicals such as peroxynitrite (ONOO-), which, although present at elevated temperature, had little influence on symbiont loss in A. pulchella. [...] As is the case in a wide variety of animal-microbe interactions, NO appears to mediate the cnidarian-dinoflagellate symbiosis by influencing the activity of host apoptotic-like pathways. Interestingly, the activation of these host responses at elevated temperature may occur before the dinoflagellate becomes photosynthetically compromised. As such, the model of bleaching as simply a response to symbiont photoinhibition could require modification. Furthermore, the differential sensitivity of symbiont types to NO, coupled with the differential regulation of NO-synthetic and apoptotic pathways in the host, could contribute to corals' varying bleaching susceptibilities. This thesis provides vital insights into the cell biology of the coral-dinoflagellate symbiosis and the events underpinning its breakdown during temperature stress. It also encourages a greater research emphasis on understanding physiological responses at the level of the coral host as well as during the early stages of a bleaching event.</p>

Transcriptomic plasticity and symbiont shuffling underpin Pocillopora acclimatization across heat-stress regimes in the Pacific Ocean

The characterization of adaptation and acclimation capacities of coral holobionts is crucial for anticipating the impact of global climate change on coral reefs. Understanding the extent to which the coral host and its photosymbionts contribute to adaptive and/or plastic responses in the coral metaorganism is equally important. In this study, we highlight new and complex links between coral genomes, transcriptomes, and environmental features in Pocilloporid corals at basin-wide scale. We analyzed metagenomic and metatranscriptomic sequence data from Pocillopora colonies sampled from 11 islands across the Pacific Ocean in order to investigate patterns of gene expression in both the host and photosymbiont across an environmental gradient. Single nucleotide polymorphism (SNP) analysis partitioned coral hosts and algal photosymbionts into five genetic lineages each. We observed strong host-symbiont fidelity across environments except at islands where recent and/or historical heat stress may have induced a symbiont shift towards more heat-tolerant lineages in some colonies. Host gene expression profiles were strongly segregated by genetic lineage and environment, and were significantly correlated with several historical sea surface temperature (SST) traits. Symbiont expression profiles were less dependent on environmental context than the host and were primarily driven by algal genotype. Overall, our results suggest a three-tiered strategy underpinning thermal acclimatization in Pocillopora holobionts with 1) host-photosymbiont fidelity, 2) host transcriptomic plasticity, and 3) photosymbiont shuffling playing progressive roles in response to elevated SSTs. Our data provide a reference for the biological state of coral holobionts across the Indo-Pacific and demonstrate the power of disentangling environmental and genetic effects to provide new insights into corals′ capacities for acclimatization and adaptation under environmental change.

Multi-omic characterization of the thermal stress phenome in the stony coral Montipora capitata

Background Corals, which form the foundation of biodiverse reef ecosystems, are under threat from warming oceans. Reefs provide essential ecological services, including food, income from tourism, nutrient cycling, waste removal, and the absorption of wave energy to mitigate erosion. Here, we studied the coral thermal stress response using network methods to analyze transcriptomic and polar metabolomic data generated from the Hawaiian rice coral Montipora capitata. Coral nubbins were exposed to ambient or thermal stress conditions over a 5-week period, coinciding with a mass spawning event of this species. The major goal of our study was to expand the inventory of thermal stress-related genes and metabolites present in M. capitata and to study gene-metabolite interactions. These interactions provide the foundation for functional or genetic analysis of key coral genes as well as provide potentially diagnostic markers of pre-bleaching stress. A secondary goal of our study was to analyze the accumulation of sex hormones prior to and during mass spawning to understand how thermal stress may impact reproductive success in M. capitata. Methods M. capitata was exposed to thermal stress during its spawning cycle over the course of 5 weeks, during which time transcriptomic and polar metabolomic data were collected. We analyzed these data streams individually, and then integrated both data sets using MAGI (Metabolite Annotation and Gene Integration) to investigate molecular transitions and biochemical reactions. Results Our results reveal the complexity of the thermal stress phenome in M. capitata, which includes many genes involved in redox regulation, biomineralization, and reproduction. The size and number of modules in the gene co-expression networks expanded from the initial stress response to the onset of bleaching. The later stages involved the suppression of metabolite transport by the coral host, including a variety of sodium-coupled transporters and a putative ammonium transporter, possibly as a response to reduction in algal productivity. The gene-metabolite integration data suggest that thermal treatment results in the activation of animal redox stress pathways involved in quenching molecular oxygen to prevent an overabundance of reactive oxygen species. Lastly, evidence that thermal stress affects reproductive activity was provided by the downregulation of CYP-like genes and the irregular production of sex hormones during the mass spawning cycle. Overall, redox regulation and metabolite transport are key components of the coral animal thermal stress phenome. Mass spawning was highly attenuated under thermal stress, suggesting that global climate change may negatively impact reproductive behavior in this species.

Viral-Like Particles Are Associated With Endosymbiont Pathology in Florida Corals Affected by Stony Coral Tissue Loss Disease

Stony coral tissue loss disease (SCTLD) was first documented in 2014 near the Port of Miami, Florida, and has since spread north and south along Florida’s Coral Reef, killing large numbers of more than 20 species of coral and leading to the functional extinction of at least one species, Dendrogyra cylindrus. SCTLD is assumed to be caused by bacteria based on presence of different molecular assemblages of bacteria in lesioned compared to apparently healthy tissues, its apparent spread among colonies, and cessation of spread of lesions in individual colonies treated with antibiotics. However, light microscopic examination of tissues of corals affected with SCTLD has not shown bacteria associated with tissue death. Rather, microscopy shows dead and dying coral cells and symbiotic dinoflagellates (endosymbionts) indicating a breakdown of host cell and endosymbiont symbiosis. It is unclear whether host cells die first leading to death of endosymbionts or vice versa. Based on microscopy, hypotheses as to possible causes of SCTLD include infectious agents not visible at the light microscopy level or toxicosis, perhaps originating from endosymbionts. To clarify this, we examined corals affected with SCTLD and apparently healthy corals using transmission electron microscopy. Endosymbionts in SCTLD-affected and apparently healthy corals consistently had varying degrees of pathology associated with elongated particles compatible in morphology with filamentous positive single-stranded RNA viruses of plants termed anisometric viral-like particles (AVLP). There was apparent progression from early to late replication of AVLP in the cytoplasm of endosymbionts adjacent to or at times within chloroplasts, with morphologic changes in chloroplasts consistent with those seen in plant cells infected by viruses. Coral host cell pathology appeared limited to massive proliferation and lysis of mucus cells. Based on these findings, we hypothesize that SCTLD is a viral disease of endosymbionts leading to coral host death. Efforts to confirm the presence of a virus associated with SCTLD through other means would be appropriate. These include showing the presence of a virus through molecular assays such as deep sequencing, attempts to grow this virus in the laboratory through culture of endosymbionts, localization of virus in tissue sections using immunohistochemistry or in situ hybridization, and experimental infection of known-virus-negative corals to replicate disease at the gross and microscopic level.

Microbiome restructuring: dominant coral bacterium Endozoicomonas species display differential adaptive capabilities to environmental changes

Bacteria in the coral microbiome play a crucial role in determining coral health and fitness, and the coral host often restructures its microbiome composition in response to external factors. An important but often neglected factor determining this microbiome restructuring is the capacity of microbiome members to adapt to a new environment. To address this issue, we examined how the microbiome structure of Acropora muricata corals changed over 9 months following a reciprocal transplant experiment. Using a combination of metabarcoding, genomics, and comparative genomics approaches, we found that coral colonies separated by a small distance harbored different dominant Endozoicomonas related phylotypes belonging to two different species, including a novel species, Candidatus Endozoicomonas penghunesis 4G, whose chromosome level (complete) genome was also sequenced in this study. Furthermore, the two dominant Endozoicomonas species showed varied adaptation capabilities when coral colonies were transplanted in a new environment. The differential adaptation capabilities of dominant members of the microbiome can a) provide distinct advantages to coral hosts when subjected to changing environmental conditions and b) have positive implications for future reefs.