Associate Professor Bill Leggat

Outbreaks of coral disease are often associated with global and local stressors like changes in temperature and poor water quality. A severe coral disease outbreak was recorded in the primary reef-building taxa Montipora spp. in a high-latitude lagoon at Norfolk Island following heat stress and pollution events in 2020. Disease signs suggest the occurrence of a Montiporid White Syndrome with four distinct phases and maximum measured tissue loss of 329 mm-2 day-1. In December 2020 and April 2021, 60% of the Montipora community were impacted and disease severity increased by 54% over this period. Spatial patterns in prevalence indicate the disease is associated with exposure to poor water quality in addition to size class of coral colonies. High prevalence levels make this event comparable to some of the most severe coral disease outbreaks recorded to date demonstrating the vulnerability of this system to combined impacts of warming and pollution.

Generalist coral species may play an important role in predicting, managing, and responding to the growing coral reef crisis as sea surface temperatures are rising and reef wide bleaching events are becoming more common. Pocilloporids are amongst the most widely distributed and studied of generalist corals, characterized by a broad geographic distribution, phenotypic plasticity, and tolerance of sub-optimal conditions for coral recruitment and survival. Emerging research indicates that microbial communities associated with Pocilloporid corals may be contributing to their persistence on coral reefs im- pacted by thermal stress; however, we lack detailed information on shifts in the coral-bacterial symbiosis during bleaching events across many of the reef habitats these corals are found. Here, we characterized the bacterial communities of healthy and bleached Pocillopora damicornis corals during the bleaching events that occurred during the austral summer of 2020 on Heron Island, on the southern Great Barrier Reef, and the austral summer of 2019 on Lord Howe Island, the most southerly coral reef in Australia. Regardless of reef location, significant differences in and diversities, core bacterial community, and inferred functional profile of the bleached microbiome of P. damicornis were not detected. Consistent with previous reports, patterns in the Pocilloporid coral microbiome, including no increase in pathogenic taxa or evidence of dysbiosis, are conserved during bleaching responses. We hypothesize that the resilience of holobiont interactions may aid the Pocilloporids to survive Symbiodiniaceae loss and contribute to the success of Pocilloporids.

Coral disease prevalence has significantly increased under a changing climate, impacting coral community structure and functionality. The impacts and ecology of coral diseases are unclear in most high-latitude reefs (coral reefs above 28° north and below 28° south). High-latitude locations are vulnerable to climate change; therefore, identifying diseases and developing region-specific baselines are important for local management. We report the first coral disease findings at the UNESCO World Heritage listed Lord Howe Island Marine Park (31.5°S, 159°E), the southernmost coral reef system. This study assessed coral disease prevalence during November 2018, March 2019 and October 2019. Surveys from three lagoonal reefs identified four coral diseases: white syndrome, skeletal eroding band, growth anomalies and endolithic hypermycosis impacting six coral taxa (Acropora, Isopora, Montipora, Pocillopora, Porites and Seriatopora). Overall, disease prevalence was 5 ± 1% and significantly differed between time and site. Disease prevalence was highest in November 2018 (10 ± 1%) and significantly lower during March 2019 (5 ± 1%), coinciding with a bleaching event. White syndrome was the most prevalent disease (4 ± 1%) with 83 colonies of six taxa affected, predominately Isopora. Acroporids recorded the highest disease susceptibility, with three of the four diseases observed. Documenting baseline coral disease prevalence and monitoring throughout a bleaching event assists our understanding of disease ecology dynamics under current climate change impacts at high-latitude reefs.

Coral bleaching events continue to drive the degradation of coral reefs worldwide, causing a shift in the benthic community from coral- to algae-dominated ecosystems. Critically, this shift may decrease the capacity of degraded coral reef communities to maintain net positive accretion during warming-driven stress events (e.g., reef-wide coral bleaching). Here we measured rates of net ecosystem calcification (NEC) and net ecosystem production (NEP) on a degraded coral reef lagoon community (coral cover <10% and algae cover >20%) during a reef-wide bleaching event in February 2020 at Heron Island on the Great Barrier Reef. We found that during this bleaching event, rates of NEP and NEC across replicate transects remained positive and did not change in response to bleaching. Repeated benthic surveys over a period of 20d indicated an increase in the percent area of bleached coral tissue, corroborated by relatively low Symbiodiniaceae densities (1/40.6×106cm-2) and dark-adapted photosynthetic yields in photosystem II of corals (1/40.5) sampled along each transect over this period. Given that a clear decline in coral health was not reflected in the overall NEC estimates, it is possible that elevated temperatures in the water column that compromise coral health enhanced the thermodynamic favorability for calcification in other ahermatypic benthic calcifiers. These data suggest that positive NEC on degraded reefs may not equate to the net positive accretion of a complex, three-dimensional reef structure in a future, warmer ocean. Critically, our study highlights that if coral cover continues to decline as predicted, NEC may no longer be an appropriate proxy for reef growth as the proportion of the NEC signal owed to ahermatypic calcification increases and coral dominance on the reef decreases.

One of the most widespread coral diseases linked to anthropogenic activities and recorded on reefs worldwide is characterized by anomalous growth formations in stony corals, referred to as coral growth anomalies (GAs). The biological functions of GA tissue include limited reproduction, reduced access to resources, and weakened ability to defend against predators. Transcriptomic analyses have revealed that, in some cases, disease progression can involve host genes related to oncogenesis, suggesting that the GA tissues may be malignant neoplasms such as those developed by vertebrates. The number of studies reporting the presence of GAs in common reef-forming species highlights the urgency of a thorough understanding of the pathology and causative factors of this disease and its parallels to higher organism malignant tissue growth. Here, we review the current state of knowledge on the etiology and holobiont features of GAs in reef-building corals.

Coral reefs are restricted to warm waters, but are increasingly threatened by coral bleaching induced by sustained high sea surface temperatures. Coral endosymbiont thermal resilience has been proposed to depend, at least in part, on the lipid composition of their thylakoid membranes, which influences photosynthetic performance under sub- and super-optimal thermal conditions in photosynthetic organisms. Dinoflagellate symbionts of high-latitude coral reefs experience large seasonal changes in temperature, requiring a wide range of thermal tolerance, and so the thermal responses of their membrane lipids are of particular interest. Using gas chromatography¿mass spectrometry, we investigated the composition and response to high- and low-temperature stress of thylakoid fatty acids of dinoflagellate symbionts isolated from corals of Lord Howe Island, the world¿s southernmost coral reef. We detected genotype-specific differences in the quality of thylakoid fatty acids of two Cladocopium ITS2 consortia/genotypes, C100/118 and C111*, common local symbionts of the corals Pocillopora damicornis and Porites heronensis. The capability to adjust thylakoid fatty acid composition in response to temperature differed between distinct Cladocopium genotypes, and between the same Cladocopium consortium (C100/118) in the same coral species from different locations. Fatty acid adjustments were highly similar in response to short-term cold and heat stresses, with substantial increases in long-chain polyunsaturated fatty acids and a corresponding increase in the ratio of unsaturated to saturated fatty acids, but these changes did not correlate with the quantum yield of photosystem II. The response of thylakoid fatty acid composition to changes in temperature was a function of symbiont genotype, coral host species and, potentially, environmental history. Our data suggest the existence of common responses to high- and low-temperature stresses and that thylakoid fatty acid saturation is an unreliable predictor of photosystem efficiency under thermal stress in dinoflagellate symbionts.

Coral bleaching has increasingly impacted reefs worldwide over the past four decades. Despite almost 40 years of research into the mechanistic, physiological, ecological, biophysical and climatic drivers of coral bleaching, metrics to allow comparison between ecological observations and experimental simulations still do not exist. Here we describe a novel metric ¿ experimental Degree Heating Week (eDHW) ¿ with which to standardise the persistently variable thermal conditions employed across experimental studies of coral bleaching by modify the widely used Degree Heating Week (DHW) metric used in ecological studies to standardise cumulative heat loading.

The Symbiodiniaceae are a taxonomically and functionally diverse family of marine dinoflagellates. Their symbiotic relationship with invertebrates such as scleractinian corals has made them the focus of decades of research to resolve the underlying biology regulating their sensitivity to stressors, particularly thermal stress. Research to-date suggests that Symbiodiniaceae stress sensitivity is governed by a complex interplay between phylogenetic dependent and independent traits (diversity of characteristics of a species). Consequently, there is a need for datasets that simultaneously broadly resolve molecular and physiological processes under stressed and non-stressed conditions. Therefore, we provide a dataset simultaneously generating transcriptome, metabolome, and proteome data for three ecologically important Symbiodiniaceae isolates under nutrient replete growth conditions and two temperature treatments (ca. 26 °C and 32 °C). Elevated sea surface temperature is primarily responsible for coral bleaching events that occur when the coral-Symbiodiniaceae relationship has been disrupted. Symbiodiniaceae can strongly influence their host¿s response to thermal stress and consequently it is necessary to resolve drivers of Symbiodiniaceae heat stress tolerance. We anticipate these datasets to expand our understanding on the key genotypic and functional properties that influence the sensitivities of Symbiodiniaceae to thermal stress.

Fish gastro-intestinal system harbors diverse microbiomes that affect the host's digestion, nutrition, and immunity. Despite the great taxonomic diversity of fish, little is understood about fish microbiome and the factors that determine its structure and composition. Damselfish are important coral reef species that play pivotal roles in determining algae and coral population structures of reefs. Broadly, damselfish belong to either of two trophic guilds based on whether they are planktivorous or algae-farming. In this study, we used 16S rRNA gene sequencing to investigate the intestinal microbiome of 5 planktivorous and 5 algae-farming damselfish species (Pomacentridae) from the Great Barrier Reef. We detected Gammaproteobacteria ASVs belonging to the genus Actinobacillus in 80% of sampled individuals across the 2 trophic guilds, thus, bacteria in this genus can be considered possible core members of pomacentrid microbiomes. Algae-farming damselfish had greater bacterial alpha-diversity, a more diverse core microbiome and shared 35 ± 22 ASVs, whereas planktivorous species shared 7 ± 3 ASVs. Our data also highlight differences in microbiomes associated with both trophic guilds. For instance, algae-farming damselfish were enriched in Pasteurellaceae, whilst planktivorous damselfish in Vibrionaceae. Finally, we show shifts in bacterial community composition along the intestines. ASVs associated with the classes Bacteroidia, Clostridia, and Mollicutes bacteria were predominant in the anterior intestinal regions while Gammaproteobacteria abundance was higher in the stomach. Our results suggest that the richness of the intestinal bacterial communities of damselfish reflects host species diet and trophic guild.

The limestone skeleton of Scleractinian corals is a complex and intricate environment consisting of an array of ecological microniches, which harbour a vast microbial community. In addition, recent studies have demonstrated that endolithic microbes play a variety of important ecological roles. Here, we use a combination of metabarcoding of the small subunit rRNA genes, microscopy and spectrophotometry to characterize the endolithic community of the coral Isopora palifera, one of the most common reef builders of the Great Barrier Reef. While a previous study suggested that the Isopora skeleton was dominated by anoxygenic phototrophs, our data show an abundance of chlorophyll a, highlighting the presence of oxygenic photosynthetic endolithic microbes. Proteobacteria, Bacteriodetes, Actinobacteria and Spirochaetes were consistently found, and the bacterial community was similar in shallow and deeper skeletal micro-samples. The micro-eukaryotic community was dominated by endolithic green algae, and the protist Labyrynthula, found at previously unreported high relative abundance.

There is a growing interest in the endolithic microbial biofilms inhabiting skeletons of living corals because of their contribution to coral reef bioerosion and the reputed benefits they provide to live coral hosts. Here, we sought to identify possible correlations between coral interspecific patterns in skeletal morphology and variability in the biomass of, and chlorophyll concentrations within, the endolithic biofilm. We measured five morphological characteristics of five coral species and the biomasses/chlorophyll concentrations of their endolithic microbiome, and we compare interspecific patterns in these variables. We propose that the specific density of a coral's skeleton and its capacity for capturing and scattering incident light are the main correlates of endolithic microbial biomass. Our data suggest that the correlation between light capture and endolithic biomass is likely influenced by how the green microalgae (obligatory microborers) respond to skeletal variability. These results demonstrate that coral species differ significantly in their endolithic microbial biomass and that their skeletal structure could be used to predict these interspecific differences. Further exploring how and why the endolithic microbiome varies between coral species is vital in defining the role of these microbes on coral reefs, both now and in the future. IMPORTANCE Microbial communities living inside the skeletons of living corals play a variety of important roles within the coral meta-organism, both symbiotic and parasitic. Properly contextualizing the contribution of these enigmatic microbes to the life history of coral reefs requires knowledge of how these endolithic biofilms vary between coral species. To this effect, we measured differences in the morphology of five coral species and correlate these with variability in the biomass of the skeletal biofilms. We found that the density of the skeleton and its capacity to trap incoming light, as opposed to scattering it back into the surrounding water, both significantly correlated with skeletal microbial biomass. These patterns are likely driven by how dominant green microalgae in the endolithic niche, such as Ostreobium spp., are responding to the skeletal morphology. This study highlights that the structure of a coral's skeleton could be used to predict the biomass of its resident endolithic biofilm.

Climate change is increasing the frequency of marine heatwaves around the world, causing widespread degradation of coral reefs. Endolithic microalgae inhabiting the coral skeleton have been highlighted as potentially important mediators of the consequences of heatwaves on coral reefs. These microalgae often bloom during heat stress due to greater light availability, theoretically delaying coral starvation by providing photoassimilates. However, these microalgae also dissolve coral skeletons at an accelerated rate during marine heatwaves, affecting the structural complexity of the reef. Despite their ecological role, no studies have examined endolithic algal blooms during a natural bleaching event. We quantified blooms of endolithic microalgae in the skeletons of lagoon corals bleaching on Heron Island in the austral summer of 2020. At the peak of heat stress, 20-30% of bleached corals across 9 genera at 3 sites had blooms. They were predominantly seen in branching Acropora spp. (37.8, 65.7 and 66.7% at three sites), which are primary reef builders at Heron Island. At the end of the bleaching event, the overall prevalence varied between 5 and 42%, and nearly all blooms were observed in acroporids. The relative high frequency of these blooms highlights the ongoing need to understand the role of these microbes during coral bleaching events.

Coral bleaching events in the marine environment are now occurring globally, and the frequency and severity of these events are increasing. Critically, these events can cause the symbiosis between Symbiodiniaceae and their coral hosts to break down, but how the microbial community within the coral responds to bleaching is still equivocal. We investigated the impact of thermal stress exposure on the meta-organism responses of the generalist scleractinian coral species Pocillopora damicornis. Using mesocosms to recreate warming scenarios previously observed at Heron Island, we show that P. damicornis symbiont densities and photophysiological parameters declined at a similar rate under thermal stress regardless of the length of pre-bleaching thermal stress, defined here as temperatures above the monthly maximum mean (MMM) for Heron Island but below the local bleaching threshold (MMM + 2°C). However, we find that the P. damicornis microbiome remains stable over time regardless of the degree of thermal stress and the accumulation of pre-bleaching thermal stress. Our study therefore suggests that while P. damicornis is physiologically impacted by bleaching temperatures, the microbial community identified through 16S rRNA sequencing remains unchanged at the ASV level throughout bleaching. Understanding the capacity of a generalist species to withstand bleaching events is imperative to characterizing what coral species will exist on coral reefs following disturbances, as it has been suggested that the success of environmental generalist species may simplify community structure and lead to changes in biodiversity following environmental disturbance.

Coral bleaching has impacted reefs worldwide and the predictions of near-annual bleaching from over two decades ago have now been realized. While technology currently provides the means to predict large-scale bleaching, predicting reef-scale and within-reef patterns in real-time for all reef users is limited. In 2020, heat stress across the Great Barrier Reef underpinned the region's third bleaching event in 5 years. Here we review the heterogeneous emergence of bleaching across Heron Island reef habitats and discuss the oceanographic drivers that underpinned variable bleaching emergence. We do so as a case study to highlight how reef end-user groups who engage with coral reefs in different ways require targeted guidance for how, and when, to alter their use of coral reefs in response to bleaching events. Our case study of coral bleaching emergence demonstrates how within-reef scale nowcasting of coral bleaching could aid the development of accessible and equitable bleaching response strategies on coral reefs. Also see the video abstract here: https://youtu.be/N9Tgb8N-vN0.

The effects of thermal anomalies on tropical coral endosymbiosis can be mediated by a range of environmental factors, which in turn ultimately influence coral health and survival. One such factor is the water flow conditions over coral reefs and corals. Although the physiological benefits of living under high water flow are well known, there remains a lack of conclusive experimental evidence characterizing how flow mitigates thermal stress responses in corals. Here we use in situ measurements of flow in a variety of reef habitats to constrain the importance of flow speeds on the endosymbiosis of an important reef building species under different thermal regimes. Under high flow speeds (0.15 m s-1) and thermal stress, coral endosymbionts retained photosynthetic function and recovery capacity for longer compared to low flow conditions (0.03 m s-1). We hypothesize that this may be due to increased rates of mass transfer of key metabolites under higher flow, putatively allowing corals to maintain photosynthetic efficiency for longer. We also identified a positive interactive effect between high flow and a pre-stress, sub-lethal pulse in temperature. While higher flow may delay the onset of photosynthetic stress, it does not appear to confer long-term protection; sustained exposure to thermal stress (eDHW accumulation equivalent to 4.9°C weeks) eventually overwhelmed the coral meta-organism as evidenced by eventual declines in photo-physiological function and endosymbiont densities. Investigating flow patterns at the scale of metres within the context of these physiological impacts can reveal interesting avenues for coral reef management. This study increases our understanding of the effects of water flow on coral reef health in an era of climate change and highlights the potential to learn from existing beneficial bio-physical interactions for the effective preservation of coral reefs into the future.

Corals at the world's southernmost coral reef of Lord Howe Island (LHI) experience large temperature and light fluctuations and need to deal with periods of cold temperature (<18°C), but few studies have investigated how corals are able to cope with these conditions. Our study characterized the response of key photophysiological parameters, as well as photoacclimatory and photoprotective pigments (chlorophylls, xanthophylls, and ß-carotene), to short-term (5-d) cold stress (~15°C; 7°C below control) in three LHI coral species hosting distinct Symbiodinium ITS2 types, and compared the coral¿symbiont response to that under elevated temperature (~29°C; 7°C above control). Under cold stress, Stylophora sp. hosting Symbiodinium C118 showed the strongest effects with regard to losses of photochemical performance and symbionts. Pocillopora damicornis hosting Symbiodinium C100/C118 showed less severe bleaching responses to reduced temperature than to elevated temperature, while Porites heronensis hosting Symbiodinium C111* withstood both reduced and elevated temperature. Under cold stress, photoprotection in the form of xanthophyll de-epoxidation increased in unbleached P.¿heronensis (by 178%) and bleached Stylophora sp. (by 225%), while under heat stress this parameter increased in unbleached P.¿heronensis (by 182%) and in bleached P.¿damicornis (by 286%). The xanthophyll pool size was stable in all species at all temperatures. Our comparative study demonstrates high variability in the bleaching vulnerability of these coral species to low and high thermal extremes and shows that this variability is not solely determined by the ability to activate xanthophyll de-epoxidation.

Endosymbioses between dinoflagellate algae (Symbiodinium sp.) and scleractinian coral species form the foundation of coral reef ecosystems. The coral symbiosis is highly susceptible to elevated temperatures, resulting in coral bleaching, where the algal symbiont is released from host cells. This experiment aimed to determine the transcriptional changes in cultured Symbiodinium, to better understand the response of cellular mechanisms under future temperature conditions. Cultures were exposed to elevated temperatures (average 31° C) or control conditions (24.5° C) for a period of 28 days. Whole transcriptome sequencing of Symbiodinium cells on days 4, 19, and 28 were used to identify differentially expressed genes under thermal stress. A large number of genes representing 37.01% of the transcriptome (~23,654 unique genes, FDR < 0.05) with differential expression were detected at no less than one of the time points. Consistent with previous studies of Symbiodinium gene expression, fold changes across the transcriptome were low, with 92.49% differentially expressed genes at =2-fold change. The transcriptional response included differential expression of genes encoding stress response components such as the antioxidant network and molecular chaperones, cellular components such as core photosynthesis machinery, integral light-harvesting protein complexes and enzymes such as fatty acid desaturases. Differential expression of genes encoding glyoxylate cycle enzymes were also found, representing the first report of this in Symbiodinium. As photosynthate transfer from Symbiodinium to coral hosts provides up to 90% of a coral¿s daily energy requirements, the implications of altered metabolic processes from exposure to thermal stress found in this study on coral-Symbiodinium associations are unknown and should be considered when assessing the stability of the symbiotic relationship under future climate conditions.

Background: Symbiosis is a phenomenon that allows organisms to colonise a wide range of environments and occupy a variety of ecological niches in marine environments. Large benthic foraminifera (LBF) are crucial marine calcifiers that rely on photo-endosymbionts for growth and calcification, yet the influence of environmental conditions in shaping their interactions with prokaryotic and eukaryotic associates is poorly known. Results: Here, we used next-generation sequencing to identify eukaryotic photosynthesizing and prokaryotic microbes associated with the common LBF Amphistegina lobifera across a physio-chemical gradient on the Great Barrier Reef (GBR). We collected samples from three reef sites located in the inner-, mid- and outer-shelf regions of the northern section of the GBR. Results showed the consistent presence of Bacillaryophyta as the main eukaryotic taxa associated with A. lobifera across all reef sites analysed; however, the abundance and the diversity of prokaryotic organisms varied among reef sites. Inner-shelf specimens showed the highest diversity of prokaryote associates, with a total of 231 genotypes in their core microbiome. A total of 30 taxa were identified in the core microbiome across all reef sites. Within these taxa, Proteobacteria was the most abundant bacteria present. The presence of groups such as Actinobacteria was significantly correlated with inner-shelf populations, whereas the abundance of Bacteroidetes and Firmicutes was associated with A. lobifera collected from mid- and outer-shelf reef sites. Conclusions: We found that benthic foraminifera form stable and persistent symbiosis with eukaryotic partners, but flexible and site-specific associations with prokaryotic microbes that likely influence the ecological success of these crucial calcifying organisms on the GBR.

Elevated sea surface temperatures (SSTs) are linked to an increase in the frequency and severity of bleaching events due to temperatures exceeding corals' upper thermal limits. The temperatures at which a breakdown of the coral-Symbiodinium endosymbiosis (coral bleaching) occurs are referred to as the upper thermal limits for the coral species. This breakdown of the endosymbiosis results in a reduction of corals' nutritional uptake, growth, and tissue integrity. Periods of elevated sea surface temperature, thermal stress and coral bleaching are also linked to increased disease susceptibility and an increased frequency of storms which cause injury and physical damage to corals. Herein we aimed to determine the capacity of corals to regenerate and recover from injuries (removal of apical tips) sustained during periods of elevated sea surface temperatures which result in coral stress responses, but which do not result in coral bleaching (i.e., sub-bleaching thermal stress events). In this study, exposure of the species Acropora aspera to an elevated SST of 32 °C (2 °C below the bleaching threshold, 34 °C) was found to result in reduced fluorescence of green fluorescent protein (GFP), reduced skeletal calcification and a lack of branch regrowth at the site of injury, compared to corals maintained under ambient SST conditions (26 °C). Corals maintained under normal, ambient, sea surface temperatures expressed high GFP fluorescence at the injury site, underwent a rapid regeneration of the coral branch apical tip within 12 days of sustaining injury, and showed extensive regrowth of the coral skeleton. Taken together, our results have demonstrated that periods of sustained increased sea surface temperatures, below the corals' bleaching threshold but above long-term summertime averages, impair coral recovery from damage, regardless of the onset or occurrence of coral bleaching.

Coral bleaching events threaten the sustainability of the Great Barrier Reef (GBR). Here we show that bleaching events of the past three decades have been mitigated by induced thermal tolerance of reef-building corals, and this protective mechanism is likely to be lost under near-future climate change scenarios. We show that 75% of past thermal stress events have been characterized by a temperature trajectory that subjects corals to a protective, sub-bleaching stress, before reaching temperatures that cause bleaching. Such conditions confer thermal tolerance, decreasing coral cell mortality and symbiont loss during bleaching by over 50%. We find that near-future increases in local temperature of as little as 0.5°C result in this protective mechanism being lost, which may increase the rate of degradation of the GBR.

For ecosystems vulnerable to environmental change, understanding the spatiotemporal stability of functionally crucial symbioses is fundamental to determining the mechanisms by which these ecosystems may persist. The coral Pachyseris speciosa is a successful environmental generalist that succeeds in diverse reef habitats. The generalist nature of this coral suggests it may have the capacity to form functionally significant microbial partnerships to facilitate access to a range of nutritional sources within different habitats. Here, we propose that coral is a metaorganism hosting three functionally distinct microbial interactions: a ubiquitous core microbiome of very few symbiotic host-selected bacteria, a microbiome of spatially and/or regionally explicit core microbes filling functional niches (<100 phylotypes), and a highly variable bacterial community that is responsive to biotic and abiotic processes across spatial and temporal scales (>100,000 phylotypes). We find that this coral hosts upwards of 170,000 distinct phylotypes and provide evidence for the persistence of a select group of bacteria in corals across environmental habitats of the Great Barrier Reef and Coral Sea. We further show that a higher number of bacteria are consistently associated with corals on mesophotic reefs than on shallow reefs. An increase in microbial diversity with depth suggests reliance by this coral on bacteria for nutrient acquisition on reefs exposed to nutrient upwelling. Understanding the complex microbial communities of host organisms across broad biotic and abiotic environments as functionally distinct microbiomes can provide insight into those interactions that are ubiquitous niche symbioses and those that provide competitive advantage within the hosts¿ environment.

Coral reef success is largely dependent on the symbiosis between coral hosts and dinoflagellate symbionts belonging to the genus Symbiodinium. Elevated temperatures can result in the expulsion of Symbiodinium or loss of their photosynthetic pigments and is known as coral bleaching. It has been postulated that the expression of light-harvesting protein complexes (LHCs), which bind chlorophylls (chl) and carotenoids, are important in photobleaching. This study explored the effect a sixteen-day thermal stress (increasing daily from 25-34 °C) on integral LHC (chlorophyll a-chlorophyll c 2-peridinin protein complex (acpPC)) gene expression in Symbiodinium within the coral Acropora aspera. Thermal stress leads to a decrease in Symbiodinium photosynthetic efficiency by day eight, while symbiont density was significantly lower on day sixteen. Over this time period, the gene expression of five Symbiodinium acpPC genes was quantified. Three acpPC genes exhibited up-regulated expression when corals were exposed to temperatures above 31.5 °C (acpPCSym-1:1, day sixteen; acpPCSym-15, day twelve; and acpPCSym-18, day ten and day sixteen). In contrast, the expression of acpPCSym-5:1 and acpPCSym-10:1 was unchanged throughout the experiment. Interestingly, the three acpPC genes with increased expression cluster together in a phylogenetic analysis of light-harvesting complexes.

Recurrent disturbances on coral reefs that cause injuries, like predation and storm damage, and elevated seawater temperatures reduce coral fitness and immunocompetence. An effective immune response is essential to prevent disease and enhance colony survival. To evaluate how elevated seawater temperatures affect the coral immune response following injury, fragments of Acropora aspera were exposed to ambient (27¿29°C) or elevated (32¿33.5°C) seawater temperatures for 8¿days and subsequently experimentally injured. Expression patterns for 15 immune genes 24¿h post-injury revealed that most genes involved in the Toll-like receptor pathway were unaffected by elevated seawater temperatures. Exceptions to this pattern were cFos and cJun, which were upregulated and likely played a role in repair processes, and TRAF-6 and NF¿B, which were downregulated suggesting reduced immune function. Components of the complement system were upregulated (millectin, C3) or downregulated (Bf, Tx60, apextrin) in corals at high temperatures. However, corals that also sustained injury, showed normal Tx60 and apextrin expression, suggesting roles in the wounding response. Overall, basal expression levels of immune genes are sufficient to mount a response to injury in the short term, and the immune response of A. aspera following injury is not significantly affected by minor elevations in seawater temperatures.

White syndrome has been described as one of the most prolific diseases on the Great Barrier Reef. Previously, apoptotic cell death has been described as the mechanism driving the characteristic rapid tissue loss associated with this disease, but the molecular mechanisms controlling apoptotic cell death in coral disease have yet to be investigated. In situ methods were used to study the expression patterns of 2 distinct regulators of apoptosis in Acropora hya cinthus tissues undergoing white syndrome and apoptotic cell death. Apoptotic genes within the Bcl-2 family were not localized in apparently healthy coral tissues. However, a Bcl-2 family member (bax-like) was found to localize to cells and tissues affected by white syndrome and those with morphological evidence for apoptosis. A potential up-regulation of pro-apoptotic or bax-like gene expression in tissues with apoptotic cell death adjacent to disease lesions is consistent with apoptosis being the primary cause of rapid tissue loss in coral affected by white syndrome. Pro-apoptotic (bax-like) expression in desmocytes and the basal tissue layer, the calicodermis, distant from the disease lesion suggests that apoptosis may also underlie the sloughing of healthy tissues associated with the characteristic, rapid spread of tissue loss, evident of this disease. This study also shows that in situ hybridisation is an effective tool for studying gene expression in adult corals, and wider application of these methods should allow a better understanding of many aspects of coral biology and disease pathology.

Increasing physical damage on coral reefs from predation, storms and anthropogenic disturbances highlights the need to understand the impact of injury on the coral immune system. In this study, we examined the regulation of the coral immune response over 10 days following physical trauma artificially inflicted on in situ colonies of the coral Acropora aspera, simultaneously with bacterial colonization of the lesions. Corals responded to injury by increasing the expression of immune system-related genes involved in the Toll-like and NOD-like receptor signalling pathways and the lectin-complement system in three phases (<2, 4 and 10 days post-injury). Phenoloxidase activity was also significantly upregulated in two phases (<3 and 10 days post-injury), as were levels of non-fluorescent chromoprotein. In addition, green fluorescent protein expression was upregulated in response to injury from 4 days post-injury, while cyan fluorescent protein expression was reduced. No shifts in the composition of coral-associated bacterial communities were evident following injury based on 16S rRNA gene amplicon pyrosequencing. Bacteria-specific fluorescence in situ hybridization also showed no evidence of bacterial colonization of the wound or regenerating tissues. Coral tissues showed near-complete regeneration of lesions within 10 days. This study demonstrates that corals exhibit immune responses that support rapid recovery following physical injury, maintain coral microbial homeostasis and prevent bacterial infestation that may compromise coral fitness.

Mass coral bleaching due to thermal stress represents a major threat to the integrity and functioning of coral reefs. Thermal thresholds vary, however, between corals, partly as a result of the specific type of endosymbiotic dinoflagellate (Symbiodinium sp.) they harbour. The production of reactive oxygen species (ROS) in corals under thermal and light stress has been recognised as one mechanism that can lead to cellular damage and the loss of their symbiont population (Oxidative Theory of Coral Bleaching). Here, we compared the response of symbiont and host enzymatic antioxidants in the coral species Acropora millepora and Montipora digitata at 28. °C and 33. °C. A. millepora at 33. °C showed a decrease in photochemical efficiency of photosystem II (PSII) and increase in maximum midday excitation pressure on PSII, with subsequent bleaching (declining photosynthetic pigment and symbiont density). M. digitata exhibited no bleaching response and photochemical changes in its symbionts were minor. The symbiont antioxidant enzymes superoxide dismutase, ascorbate peroxidase, and catalase peroxidase showed no significant upregulation to elevated temperatures in either coral, while only catalase was significantly elevated in both coral hosts at 33. °C. Increased host catalase activity in the susceptible coral after 5. days at 33. °C was independent of antioxidant responses in the symbiont and preceded significant declines in PSII photochemical efficiencies. This finding suggests a potential decoupling of host redox mechanisms from symbiont photophysiology and raises questions about the importance of symbiont-derived ROS in initiating coral bleaching.

Despite being one of the simplest metazoans, corals harbor some of the most highly diverse and abundant microbial communities. Differentiating core, symbiotic bacteria from this diverse hostassociated consortium is essential for characterizing the functional contributions of bacteria but has not been possible yet. Here we characterize the coral core microbiome and demonstrate clear phylogenetic and functional divisions between the micro-scale, niche habitats within the coral host. In doing so, we discover seven distinct bacterial phylotypes that are universal to the core microbiome of coral species, separated by thousands of kilometres of oceans. The two most abundant phylotypes are co-localized specifically with the corals' endosymbiotic algae and symbiont-containing host cells. These bacterial symbioses likely facilitate the success of the dinoflagellate endosymbiosis with corals in diverse environmental regimes.

Some animals have the remarkable capacity to acclimate across generations to projected future climate change; however, the underlying molecular processes are unknown. We sequenced and assembled de novo transcriptomes of adult tropical reef fish exposed developmentally or transgenerationally to projected future ocean temperatures and correlated the resulting expression profiles with acclimated metabolic traits from the same fish. We identified 69 contigs representing 53 key genes involved in thermal acclimation of aerobic capacity. Metabolic genes were among the most upregulated transgenerationally, suggesting shifts in energy production for maintaining performance at elevated temperatures. Furthermore, immune- and stress-responsive genes were upregulated transgenerationally, indicating a new complement of genes allowing the second generation of fish to better cope with elevated temperatures. Other differentially expressed genes were involved with tissue development and transcriptional regulation. Overall, we found a similar suite of differentially expressed genes among developmental and transgenerational treatments. Heat-shock protein genes were surprisingly unresponsive, indicating that short-term heat-stress responses may not be a good indicator of long-term acclimation capacity. Our results are the first to reveal the molecular processes that may enable marine fishes to adjust to a future warmer environment over multiple generations.

Background: The diversity of the symbiotic dinoflagellate Symbiodinium sp., as assessed by genetic markers, is well established. To what extent this diversity is reflected on the amino acid level of functional genes such as enzymatic antioxidants that play an important role in thermal stress tolerance of the coral-Symbiodinium symbiosis is, however, unknown. Here we present a predicted structural analysis and phylogenetic characterization of the enzymatic antioxidant repertoire of the genus Symbiodinium. We also report gene expression and enzymatic activity under short-term thermal stress in Symbiodinium of the B1 genotype. Results: Based on eight different ITS2 types, covering six clades, multiple protein isoforms for three of the four investigated antioxidants (ascorbate peroxidase [APX], catalase peroxidase [KatG], manganese superoxide dismutase [MnSOD]) are present in the genus Symbiodinium. Amino acid sequences of both SOD metalloforms (Fe/Mn), as well as KatG, exhibited a number of prokaryotic characteristics that were also supported by the protein phylogeny. In contrast to the bacterial form, KatG in Symbiodinium is characterized by extended functionally important loops and a shortened C-terminal domain. Intercladal sequence variations were found to be much higher in both peroxidases, compared to SODs. For APX, these variable residues involve binding sites for substrates and cofactors, and might therefore differentially affect the catalytic properties of this enzyme between clades. While expression of antioxidant genes was successfully measured in Symbiodinium B1, it was not possible to assess the link between gene expression and protein activity due to high variability in expression between replicates, and little response in their enzymatic activity over the three-day experimental period. Conclusions: The genus Symbiodinium has a diverse enzymatic antioxidant repertoire that has similarities to prokaryotes, potentially as a result of horizontal gene transfer or events of secondary endosymbiosis. Different degrees of sequence evolution between SODs and peroxidases might be the result of potential selective pressure on the conserved molecular function of SODs as the first line of defence. In contrast, genetic redundancy of hydrogen peroxide scavenging enzymes might permit the observed variations in peroxidase sequences. Our data and successful measurement of antioxidant gene expression in Symbiodinium will serve as basis for further studies of coral health.

Human-induced environmental changes have been linked directly with loss of biodiversity. Coral reefs, which have been severely impacted by anthropogenic activities over the last few decades, exemplify this global problem and provide an opportunity to develop research addressing key knowledge gaps through "omics"-based approaches. While many stressors, e.g., global warming, ocean acidification, overfishing, and coastal development have been identified, there is an urgent need to understand how corals function at a basic level in order to conceive strategies for mitigating future reef loss. In this regard, availability of fully sequenced genomes has been immensely valuable in providing answers to questions of organismal biology. Given that corals are metaorganisms comprised of the coral animal host, its intracellular photosynthetic algae, and associated microbiota (i.e., bacteria, archaea, fungi, viruses), these efforts must focus on entire coral holobionts. The Reef Future Genomics 2020 (ReFuGe 2020) Consortium has formed to sequence hologenomes of 10 coral species representing different physiological or functional groups to provide foundation data for coral reef adaptation research that is freely available to the research community.

Warmer than average summer sea surface temperature is one of the main drivers for coral bleaching, which describes the loss of endosymbiotic dinoflagellates (genus: Symbiodinium) in reef-building corals. Past research has established that oxidative stress in the symbiont plays an important part in the bleaching cascade. Corals hosting different genotypes of Symbiodinium may have varying thermal bleaching thresholds, but changes in the symbiont's antioxidant system that may accompany these differences have received less attention. This study shows that constitutive activity and up-regulation of different parts of the antioxidant network under thermal stress differs between four Symbiodinium types in culture and that thermal susceptibility can be linked to glutathione redox homeostasis. In Symbiodinium B1, C1 and E, declining maximum quantum yield of PSII (Fv/Fm) and death at 33°C were generally associated with elevated superoxide dismutase (SOD) activity and a more oxidized glutathione pool. Symbiodinium F1 exhibited no decline in Fv/Fm or growth, but showed proportionally larger increases in ascorbate peroxidase (APX) activity and glutathione content (GSx), while maintaining GSx in a reduced state. Depressed growth in Symbiodinium B1 at a sublethal temperature of 29°C was associated with transiently increased APX activity and glutathione pool size, and an overall increase in glutathione reductase (GR) activity. The collapse of GR activity at 33°C, together with increased SOD, APX and glutathione S-transferase activity, contributed to a strong oxidation of the glutathione pool with subsequent death. Integrating responses of multiple components of the antioxidant network highlights the importance of antioxidant plasticity in explaining type-specific temperature responses in Symbiodinium.

Climate change is projected to increase ocean temperatures by at least 2°C, and levels of pH by ~0.2 units (ocean acidification, OA) by the end of this century. While the effects of these stressors on marine organisms have been relatively well explored in isolation, possible interactions between temperature and OA have yet to be thoroughly investigated. OA at levels projected to occur within this century has few direct ecological effects on the early life history stages of corals. In contrast, temperature has pronounced effects on many stages in the early life history of corals. Here, we test whether temperature might act in combination with OA to produce a measurable ecological effect on fertilization, development, larval survivorship or metamorphosis of 2 broadcast spawning species, Acropora millepora and A. tenuis, from the Great Barrier Reef. We used 4 treatments: control, high temperature (+2°C), high partial pressure of CO2 (pCO2) (700 µatm) and a combination of high temperature and high pCO2, corresponding to the current levels of these variables and the projected values for the end of this century under the IPCC A2 scenario. We found no consistent effect of elevated pCO2 on fertilization, development, survivorship or metamorphosis, neither alone nor in combination with temperature. In contrast, a 2°C rise in temperature increased rates of development, but otherwise had no consistent effect on fertilization, survivorship or metamorphosis. We conclude that OA is unlikely to be a direct threat to the early life history stages of corals, at least in the near future. In contrast, rising sea temperatures are likely to affect coral population dynamics by increasing the rate of larval development with resulting changes in patterns of connectivity. © Inter-Research 2013.

Until recently, research into the consequences of oceanic uptake of CO 2 for corals focused on its effect on physiological processes, in particular, calcification. However, events early in the life history of corals are also likely to be vulnerable to changes in ocean chemistry caused by increases in the atmospheric concentration of CO2 (ocean acidification). We tested the effect of reduced pH on embryonic development, larval survivorship and metamorphosis of 3 common scleractinian corals from the Great Barrier Reef. We used 4 treatment levels of pH, corresponding to the current level of ocean pH and 3 values projected to occur later this century. None of the early life-history stages we studied were consistently affected by reduced pH. Our results suggest that there will be no direct ecological effects of ocean acidification on the early life-history stages of reef corals, at least in the near future. © Inter-Research 2013 · www.int-res.com.

Metabolomics and in particular, nontargeted metabolomics, has become a popular technique for the study of biological samples as it provides considerable amounts of information on extractable metabolites and is ideal for studying the metabolic response of an organism to stressors in its environment. One such organism, the symbiotic hard coral, presents its own complexity when considering a metabolomics approach in that it forms intricate associations with an array of symbiotic macro- and microbiota. While not discounting the importance of these many associations to the function of the coral holobiont, the coral-Symbiodinium relationship has been the most studied to date and as such, is the primary focus of this extraction protocol. This protocol provides details for the sample collection, extraction, and measurement of hard coral holobiont metabolites using both 1 H nuclear magnetic resonance (NMR) spectroscopy and liquid chromatography coupled with mass spectrometry (LC-MS). Using this nontargeted metabolomics approach, the holobiont metabolism can be investigated for perturbations resulting from either (1) natural or anthropogenic environmental challenges, (2) the controlled application of stressors, and (3) differences between phenotypes or species. Consequently, this protocol will benefit both environmental and natural products based research of hard coral and their algal symbionts. Every effort has been made to provide the reader with all the details required to perform this protocol, including many of the costly and time consuming "pitfalls" or "traps" that were discovered during its development. As a result, this protocol can be confidently accomplished by those with less experience in the extraction and analysis of symbiotic hard coral, requiring minimal user input whilst ensuring reproducible and reliable results using readily available lab ware and reagents. © 2013 Springer Science+Business Media, LLC.

Metabolomics and in particular, nontargeted metabolomics, has become a popular technique for the study of biological samples as it provides considerable amounts of information on extractable metabolites and is ideal for studying the metabolic response of an organism to stressors in its environment. One such organism, the symbiotic hard coral, presents its own complexity when considering a metabolomics approach in that it forms intricate associations with an array of symbiotic macro- and microbiota. While not discounting the importance of these many associations to the function of the coral holobiont, the coral-Symbiodinium relationship has been the most studied to date and as such, is the primary focus of this extraction protocol. This protocol provides details for the sample collection, extraction, and measurement of hard coral holobiont metabolites using both ¹H nuclear magnetic resonance (NMR) spectroscopy and liquid chromatography coupled with mass spectrometry (LC-MS). Using this nontargeted metabolomics approach, the holobiont metabolism can be investigated for perturbations resulting from either (1) natural or anthropogenic environmental challenges, (2) the controlled application of stressors, and (3) differences between phenotypes or species. Consequently, this protocol will benefit both environmental and natural products based research of hard coral and their algal symbionts. Every effort has been made to provide the reader with all the details required to perform this protocol, including many of the costly and time consuming "pitfalls" or "traps" that were discovered during its development. As a result, this protocol can be confidently accomplished by those with less experience in the extraction and analysis of symbiotic hard coral, requiring minimal user input whilst ensuring reproducible and reliable results using readily available lab ware and reagents. © 2013 Springer Science+Business Media, LLC.

Symbiotic dinoflagellates are unicellular photosynthetic algae that live in mutualistic symbioses with many marine organisms. Within the transcriptome of coral endosymbionts Symbiodinium sp. (type C3), we discovered the sequences of two novel and highly polymorphic hemoglobin-like genes and proposed their 3D protein structures. At the protein level, four isoforms shared between 87 and 97% sequence identity for Hb-1 and 78-99% for Hb-2, whereas between Hb-1 and Hb-2 proteins, only 15-21% sequence homology has been preserved. Phylogenetic analyses of the dinoflagellate encoding Hb sequences have revealed a separate evolutionary origin of the discovered globin genes and indicated the possibility of horizontal gene transfer. Transcriptional regulation of the Hb-like genes was studied in the reef-building coral Acropora aspera exposed to elevated temperatures (6-7°C above average sea temperature) over a 24-h period and a 72-h period, as well as to nutrient stress. Exposure to elevated temperatures resulted in an increased Hb-1 gene expression of 31% after 72¿h only, whereas transcript abundance of the Hb-2 gene was enhanced by up to 59% by both 1-day and 3-day thermal stress conditions. Nutrient stress also increased gene expression of Hb-2 gene by 70%. Our findings describe the differential expression patterns of two novel Hb genes from symbiotic dinoflagellates and their polymorphic nature. Furthermore, the inducible nature of Hb-2 gene by both thermal and nutrient stressors indicates a prospective role of this form of hemoglobin in the initial coral-algal responses to changes in environmental conditions. This novel hemoglobin has potential use as a stress biomarker. © 2013 The Authors. Ecology and Evolution.

The superfamily of light-harvesting complex (LHC) proteins is comprised of proteins with diverse functions in light-harvesting and photoprotection. LHC proteins bind chlorophyll (Chl) and carotenoids and include a family of LHCs that bind Chl a and c. Dinophytes (dinoflagellates) are predominantly Chl c binding algal taxa, bind peridinin or fucoxanthin as the primary carotenoid, and can possess a number of LHC subfamilies. Here we report 11 LHC sequences for the chlorophyll a-chlorophyll c2-peridinin protein complex (acpPC) subfamily isolated from Symbiodinium sp. C3, an ecologically important peridinin binding dinoflagellate taxa. Phylogenetic analysis of these proteins suggests the acpPC subfamily forms at least three clades within the Chl a/c binding LHC family; Clade 1 clusters with rhodophyte, cryptophyte and peridinin binding dinoflagellate sequences, Clade 2 with peridinin binding dinoflagellate sequences only and Clades 3 with heterokontophytes, fucoxanthin and peridinin binding dinoflagellate sequences. © 2012 Boldt et al.

Dinoflagellate symbionts of the genus Symbiodinium are integral to the success of the coral holobiont (a coral host and the microbial community it harbours), however despite their importance we currently have a very limited knowledge of the genes which they possess and their genomic organisation. Analysis shows that the number of expressed sequence tags (genes that are expressed) available for Symbiodinium (7964) is less than 1/10 of those available for the scleractinian coral host (103,434). This lack of DNA sequence information limits the functional genomic studies that can be undertaken from the symbiont perspective. In addition these sequences are from only three Symbiodinium types (C3, A1, A3) and do not represent the large diversity of clades and subclades seen. Here we summarise our current understanding of the Symbiodinium genomic content with reference to our knowledge of other dinoflagellates. The genetic information of Symbiodinium is encompassed in the nuclear, plastid and mitochondrial genomes. As is the case with other dinoflagellates these three genomes are significantly different from the "general" phototrophic eukaryote. Firstly the nuclear genome of dinoflagellates is extremely large, utilises modified DNA bases not normally found in eukaryotes, and tandem repeat regions seem to contain the most highly expressed genes. Meanwhile the plastid genome, which normally contains between 40 and 250 genes in other eukaryotes, has been reduced to 18 genes encoded in "minicircles." Finally the dinoflagellate mitochondrial genome only encodes for 2 or 3 proteins instead of the normal 40-50 in other eukaryotes. While we have some knowledge of Symbiodinium genome structure, little is known about its transcriptome. With the advent of inexpensive high throughput sequencing technologies, our understanding of the Symbiodinium genome will rapidly increase and we will begin to be able to look into the responses of these important single celled organisms. © 2011 Elsevier B.V.

The success of any symbiosis under stress conditions is dependent upon the responses of both partners to that stress. The coral symbiosis is particularly susceptible to small increases of temperature above the long term summer maxima, which leads to the phenomenon known as coral bleaching, where the intracellular dinoflagellate symbionts are expelled. Here we for the first time used quantitative PCR to simultaneously examine the gene expression response of orthologs of the coral Acropora aspera and their dinoflagellate symbiont Symbiodinium. During an experimental bleaching event significant up-regulation of genes involved in stress response (HSP90 and HSP70) and carbon metabolism (glyceraldehyde-3-phosphate dehydrogenase, a-ketoglutarate dehydrogenase, glycogen synthase and glycogen phosphorylase) from the coral host were observed. In contrast in the symbiont, HSP90 expression decreased, while HSP70 levels were increased on only one day, and only the a-ketoglutarate dehydrogenase expression levels were found to increase. In addition the changes seen in expression patterns of the coral host were much larger, up to 10.5 fold, compared to the symbiont response, which in all cases was less than 2-fold. This targeted study of the expression of key metabolic and stress genes demonstrates that the response of the coral and their symbiont vary significantly, also a response in the host transcriptome was observed prior to what has previously been thought to be the temperatures at which thermal stress events occur. © 2011 Leggat et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Apoptotic cell death has been implicated in coral bleaching but the molecules involved and the mechanisms by which apoptosis is regulated are only now being identified. In contrast the mechanisms underlying apoptosis in higher animals are relatively well understood. To better understand the response of corals to thermal stress, the expression of coral homologs of six key regulators of apoptosis was studied in Acropora aspera under conditions simulating those of a mass bleaching event. Significant changes in expression were detected between the daily minimum and maximum temperatures. Maximum daily temperatures from as low as 3°C below the bleaching threshold resulted in significant changes in both pro-and anti-apoptotic gene expression. The results suggest that the control of apoptosis is highly complex in this eukaryote-eukaryote endosymbiosis and that apoptotic cell death cascades potentially play key roles tipping the cellular life/death balance during environmental stress prior to the onset of coral bleaching.

Reef-building corals are representatives of one of the earliest diverging metazoan lineages and are experiencing increases in bleaching events (breakdown of the coral-. Symbiodinium symbiosis) and disease outbreaks. The present study investigates the roles of two pattern recognition proteins, the mannose binding lectin Millectin and a complement factor C3-like protein (C3-Am), in the coral Acropora millepora. The results indicate that the innate immune functions of these molecules are conserved and arose early in evolution. C3-Am is expressed in response to injury, and may function as an opsonin. In contrast, Millectin expression is up-regulated in response to lipopolysaccharide and peptidoglycan. These observations, coupled with localization of Millectin in nematocysts in epidermal tissue, and reported binding of pathogens, are consistent with a key role for the lectin in innate immunity. Furthermore, Millectin was consistently detected binding to the symbiont Symbiodinium in vivo, indicating that the Millectin function of recognition and binding of non-self-entities may have been co-opted from an ancient innate immune system into a role in symbiosis. © 2010 Elsevier Ltd.

Symbioses play an important role within the marine environment. Among the most well known of these symbioses is that between coral and the photosynthetic dinoflagellate, Symbiodinium spp. Understanding the metabolic relationships between the host and the symbiont is of the utmost importance in order to gain insight into how this symbiosis may be disrupted due to environmental stressors. Here we summarize the metabolites related to nutritional roles, diel cycles and the common metabolites associated with the invertebrate-Symbiodinium relationship. We also review the more obscure metabolites and toxins that have been identified through natural products and biomarker research. Finally, we discuss the key role that metabolomics and functional genomics will play in understanding these important symbioses. © 2010 by the authors; licensee MDPI.

The larvae of most coral species spend some time in the plankton, floating just below the surface and hence exposed to high levels of ultraviolet radiation (UVRThe high levels of UVR are potentially stressful and damaging to DNA and other cellular components, such as proteins, reducing survivorship. Consequently, mechanisms to either shade (prevent) or repair damage potentially play an important role. In this study, the role of photoreactivation in the survival of coral planulae was examined. Photoreactivation is a light-stimulated response to UV-damaged DNA in which photolyase proteins repair damaged DNA. Photoreactivation rates, as well as the localization of photolyase, were explored in planulae under conditions where photoreactivation was or was not inhibited. The results indicate that photoreactivation is the main DNA repair pathway in coral planulae, repairing UV-induced DNA damage swiftly (K=1.75h -1 and a half-life of repair of 23 min), with no evidence of any light-independent DNA repair mechanisms, such as nucleotide excision repair (NER), at work. Photolyase mRNA was localized to both the ectoderm and endoderm of the larvae. The amount of cell death in the coral planulae increased significantly when photoreactivation was inhibited, by blocking photoreactivating light. We found that photoreactivation, along with additional UV shielding in the form of five mycosporine-like amino acids, are sufficient for survival in surface tropical waters and that planulae do not accumulate DNA damage despite being exposed to high UVR.

Dinoflagellates are ubiquitous marine and freshwater protists. The endosymbiotic relationship between dinoflagellates of the genus Symbiodinium (also known as zooxanthellae) and corals forms the basis of coral reefs. We constructed and analyzed a cDNA library from a cultured Symbiodinium species clade A (CassKB8). The majority of annotated ESTs from the Symbiodinium sp. CassKB8 library cover metabolic genes. Most of those belong to either carbohydrate or energy metabolism. In addition, components of extracellular signal transduction pathways and genes that play a role in cell-cell communication were identified. In a subsequent analysis, we determined all orthologous cDNA sequences between this library (1,484 unique sequences) and a library from a Symbiodinium species clade C (C3) (3,336 unique sequences) that was isolated directly from its symbiotic host. A set of 115 orthologs were identified between Symbiodinium sp. CassKB8 and Symbiodinium sp. C3. These orthologs were subdivided into three groups that show different characteristics and functions: conserved across eukaryotes (CE), dinoflagellate-specific (DS) and Symbiodinium-specific (SS). Orthologs conserved across eukaryotes are mainly comprised of housekeeping genes, photosynthesis-related transcripts and metabolic proteins, whereas the function for most of the dinoflagellate-specific orthologs remains unknown. A dN/dS analysis identified the highest ratio in a Symbiodinium-specific ortholog and evidence for positive selection in a dinoflagellate-specific gene. Evolution of genes and pathways in different dinoflagellates seems to be affected by different lifestyles, and a symbiotic lifestyle may affect population structure and strength of selection. This study is the first evolutionary comparative analysis of orthologs from two coral dinoflagellate symbionts. © 2008 Elsevier Inc. All rights reserved.

The mutualistic relationship between corals and their unicellular dinoflagellate symbionts (Symbiodinium sp.) is a fundamental component within the ecology of coral reefs. Thermal stress causes the breakdown of the relationship between corals and their symbionts (bleaching). As with other organisms, this symbiosis may acclimate to changes in the environment, thereby potentially modifying the environmental threshold at which they bleach. While a few studies have examined the acclimation capacity of reef-building corals, our understanding of the underlying mechanism is still in its infancy. The present study focused on the role of recent thermal history in influencing the response of both corals and symbionts to thermal stress, using the reef-building coral Acropora aspera. The symbionts of corals that were exposed to 31°C for 48 h (pre-stress treatment) 1 or 2 weeks prior to a 6-day simulated bleaching event (when corals were exposed to 34°C) were found to have more effective photoprotective mechanisms. These mechanisms included changes in non-photochemical quenching and xanthophyll cycling. These differences in photoprotection were correlated with decreased loss of symbionts, with those corals that were not prestressed performing significantly worse, losing over 40% of their symbionts and having a greater reduction in photosynthetic efficiency. These results are important in that they show that thermal history, in addition to light history, can influence the response of reef-building corals to thermal stress and therefore have implications for the modeling of bleaching events. However, whether acclimation is capable of modifying the thermal threshold of corals sufficiently to cope as sea temperatures increase in response to global warming has not been fully explored. Clearly increases in sea temperatures that extend beyond 1-2°C will exhaust the extent to which acclimation can modify the thermal threshold of corals.

Thermal stress causes the coral-dinoflagellate symbiosis to disassociate and the coral tissues to whiten. The onset and occurrence of this coral bleaching is primarily defined via the dinoflagellate responses. Here we demonstrate that thermal stress responses occur in the coral host tissues in the days before the onset of coral bleaching. The observed sequence of thermal responses includes reductions in thickness of coral tissue layers and apoptosis of the cells prior to reductions in symbiont density. In the days before the onset of coral bleaching the outer coral tissue layer (epithelium) thickness reduces and apoptosis occurs within the gastrodermis. Two days following this, coinciding with an initial reduction of symbiont density (by approximately 25%), gastrodermal thickness decreased and apoptosis of host cells was identified in the epithelium. This was eventually followed by large reduction in symbiont density (by approximately 50%) consistent with coral bleaching. Both pro-apoptotic and anti-apoptotic genes are identified in the reef building coral Acropora aspera, demonstrating the necessary pathways are present for fine control of host apoptosis. Our study shows that defining periods of host stress based on the responses defined by dinoflagellate symbiont underestimates the importance of early cellular events and the cellular complexity of coral host. © 2008 Elsevier B.V.

The cells and tissues of many marine invertebrates and their associated flora contain fluorescent pigments and proteins, many of which have been utilized commercially and provide marker molecules in other systems for fluorescence imaging technology. However, in the study of marine invertebrates and their symbioses these naturally occurring molecules have been seen to limit or confound fluorescence microscopy analyses. Here we demonstrate the endogenous fluorescence associated with two marine invertebrates (coral and foraminifera) and describe how these qualities can be utilized in fluorescence microanalyses. Understanding and imaging the diversity of fluorescent molecules provide insight into how fluorescence microscopy techniques can now be applied to these complex systems. © 2008 The Authors.

Some invertebrates have enlisted autotrophic unicellular algae to provide a competitive metabolic advantage in nutritionally demanding habitats. These symbioses exist primarily but not exclusively in shallow tropical oceanic waters where clear water and low nutrient levels provide maximal advantage to the association. Mostly, the endosymbiotic algae are localized in host cells surrounded by a host-derived membrane (symbiosome). This anatomy has required adaptation of the host biochemistry to allow transport of the normally excreted inorganic nutrients (CO2, NH3 and PO43-) to the alga. In return, the symbiont supplies photosynthetic products to the host to meet its energy demands. Most attention has focused on the metabolism of CO2 and nitrogen sources. Carbon-concentrating mechanisms are a feature of all algae, but the products exported to the host following photosynthetic CO2 fixation vary. Identification of the stimulus for release of algal photosynthate in hospite remains elusive. Nitrogen assimilation within the symbiosis is an essential element in the host's control over the alga. Recent studies have concentrated on cnidarians because of the impact of global climate change resulting in coral bleaching. The loss of the algal symbiont and its metabolic contribution to the host has the potential to result in the transition from a coral-dominated to an algal-dominated ecosystem. © 2008 The Authors.

Corals form the framework of the world's coral reefs and are under threat from increases in disease and bleaching (symbiotic dysfunction), yet the mechanisms of pathogen and symbiont recognition remain largely unknown. Here we describe the isolation and characterisation of an ancient mannose-binding lectin in the coral Acropora millepora, which is likely to be involved in both processes. The lectin ('Millectin') was isolated by affinity chromatography and was shown to bind to bacterial pathogens as well as coral symbionts, dinoflagellates of the genus Symbiodinium. cDNA analysis of Millectin indicate extensive sequence variation in the binding region, reflecting its ability to recognise various mannose-like carbohydrate structures on non-self cells, including symbionts and pathogens. This is the first mannose-binding lectin to show extensive sequence variability as observed for pattern recognition proteins in other invertebrate immune systems and, given that invertebrates rely on non-adaptive immunity, is a potential keystone component of coral defence mechanisms. © 2008 Elsevier Ltd. All rights reserved.

Dinoflagellates (Symbiodinium sp. Freud.) are an obligatory endosymbiont of the reef-building corals. Recent changes to the environment surrounding coral reefs (e.g., global warming) have demonstrated that this endosymbiotic relationship between corals and Symbiodinium is particularly sensitive to environmental changes. Therefore, understanding gene expression patterns of Symbiodinium is critical to understanding why coral reefs are susceptible to global climate change. This study identified 1456 unique expression sequence tags (ESTs) generated for Symbiodinium (clade C3) from the staghorn coral Acropora aspera following exposure to a variety of stresses. Of these, only 10% matched previously reported dinoflagellate ESTs, suggesting that the conditions used in the construction of the library resulted in a novel transcriptome. The function of 561 (44%) of these ESTs could be identified. The majority of these genes coded for proteins involved in posttranslational modification, protein turnover, and chaperones (12.3%); energy production and conversion (12%); or an unknown function (18.6%). The most common transcript found was a homologue to a bacterial protein of unknown function. This algal protein is targeted to the chloroplast and is present in those phototrophs that acquired plastids from the red algal lineage. An additional 48 prokaryote-like proteins were also identified, including the first glycerol-phosphate:phosphate antiporter from dinoflagellates. A protein with similarity to the fungi-archael-bacterial heme catalase peroxidases was also found. A variety of stress genes, in particular heat-shock proteins and proteins involved in ubiquitin cascades, were also identified. This study is the first transcriptome from the unicellular component of a eukaryote-eukaryote symbiosis. © 2007 Phycological Society of America.

Hundreds of species of reef-building corals spawn synchronously over a few nights each year, and moonlight regulates this spawning event. However, the molecular elements underpinning the detection of moonlight remain unknown. Here we report the presence of an ancient family of blue-light-sensing photoreceptors, cryptochromes, in the reef-building coral Acropora millepora. In addition to being cryptochrome genes from one of the earliest-diverging eumetazoan phyla, cry1 and cry2 were expressed preferentially in light. Consistent with potential roles in the synchronization of fundamentally important behaviors such as mass spawning, cry2 expression increased on full moon nights versus new moon nights. Our results demonstrate phylogenetically broad roles of these ancient circadian clock-related molecules in the animal kingdom.

The prevalence of coral-associated fungi was four times higher in diseased Acropora formosa colonies than in healthy colonies. Since taxonomically related fungal species were isolated from diseased and healthy colonies, we suggest that their association with coral may be constitutive but that their abundance is dependent on coral health. Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Understanding whether or not corals have the flexibility to change their complement of symbionts to adapt to changing climate is an important goal in reef ecology studies. While host fidelity to a single clade of symbiont is the dominant pattern in present-day corals, current estimates of flexibility are unreliable, because few studies have measured it rigorously and with adequately sensitive genetic techniques. Furthermore, flexibility must be explored at the level of the subclade, because generalisations of physiological performance among clades of Symbiodinium are not valid. In addition, we should not necessarily expect to see shifts among symbionts without distinct and enduring changes in environmental conditions. The few biogeographical comparisons available suggest that when corals encounter a new environment they have the flexibility to acquire different symbionts. Flexibility in the acquisition of symbionts is common at the time of infection, which in most corals occurs at, or shortly after, settlement. Consequently, flexibility is likely to be a feature of the life history of all species that must reacquire symbionts in each new generation. © Inter-Research 2007.

Pre-settlement events play an important role in determining larval success in marine invertebrates with bentho-pelagic life histories, yet the consequences of these events typically are not well understood. The purpose of this study was to examine the pre-settlement impacts of different seawater temperatures on the size and population density of dinoflagellate symbionts in brooded larvae of the Caribbean coral Porites astreoides. Larvae were collected from P. astreoides at 14-20m depth on Conch Reef (Florida) in June 2002, and incubated for 24 h at 15 temperatures spanning the range 25.1°-30.0°C in mean increments of 0.4 ± 0.1°C (± SD). The most striking feature of the larval responses was the magnitude of change in both parameters across this 5°C temperature range within 24 h. In general, larvae were largest and had the highest population densities of Symbiodinium sp. between 26.4°-27.7°C, and were smallest and had the lowest population densities at 25.8°C and 28.8°C. Larval size and symbiont population density were elevated slightly (relative to the minimal values) at the temperature extremes of 25.1°C and 30°C. These data demonstrate that coral larvae are highly sensitive to seawater temperature during their pelagic phase, and respond through changes in size and the population densities of Symbiodinium sp. to ecologically relevant temperature signals within 24 h. The extent to which these changes are biologically meaningful will depend on the duration and frequency of exposure of coral larvae to spatiotemporal variability in seawater temperature, and whether the responses have cascading effects on larval success and their entry to the post-settlement and recruitment phase. © 2005 American Microscopical Society, Inc.

This report describes the presence of a unique dual domain carbonic anhydrase (CA) in the giant clam, Tridacna gigas. CA plays an important role in the movement of inorganic carbon (Ci) from the surrounding seawater to the symbiotic algae that are found within the clam's tissue. One of these isoforms is a glycoprotein which is significantly larger (70 kDa) than any previously reported from animals (generally between 28 and 52 kDa). This a-family CA contains two complete carbonic anhydrase domains within the one protein, accounting for its large size; dual domain CAs have previously only been reported from two algal species. The protein contains a leader sequence, an N-terminal CA domain and a C-terminal CA domain. The two CA domains have relatively little identity at the amino acid level (29%). The genomic sequence spans in excess of 17 kb and contains at least 12 introns and 13 exons. A number of these introns are in positions that are only found in the membrane attached/secreted CAs. This fact, along with phylogenetic analysis, suggests that this protein represents the second example of a membrane attached invertebrate CA and it contains a dual domain structure unique amongst all animal CAs characterized to date. © 2005 FEBS.

Bleaching (loss of symbiotic dinoflagellates) is known to significantly decrease the fitness of symbiotic marine invertebrates resulting in reduced growth, fecundity and survival. This report is the first to quantify the effects of bleaching on inorganic carbon (C1) and ammonium flux, fixation and export of photosynthate to the host, in this case the giant clam Tridacna gigas. The 1998 bleaching event was found to decrease the zooxanthellae population 30-fold when comparing bleached to non-bleached clams. This resulted in significant increases in haemolymph C1 and decreases in haemolymph pH and glucose concentration, the predominant photosynthate exported from zooxanthellae in this symbiosis. There was also a decrease in the expression levels of host carbonic anhydrase, an enzyme involved in C1 transport to the zooxanthellae, and although host glutamine synthase levels were unaffected, the clams ability to assimilate ammonium was eliminated in bleached individuals, suggesting that photosynthate from the zooxanthellae is required for ammonium assimilation. In an artificial bleaching experiment haemolymph Ci (r2 = 0.97), pH (r 2 = 0.94) and glucose levels (r2 = 0.95) were correlated to zooxanthellae numbers during both bleaching and recovery. Recovery of the zooxanthellae population, was enhanced four-fold by the addition of organic and inorganic nutrients, as were related haemolymph characteristics. These results highlight the profound physiological changes that occur in symbiotic organisms during and after a bleaching event.

The concentration of glutamine in Tridacna gigas haemolymph increased > 35-fold following exposure to sea water supplemented with ammonium (20 µM), but no increase was observed with nitrate (20 µM). Lack of a diel cycle, no decrease in haemolymph glucose levels, the expression patterns of glutamine synthetase in zooxanthellae and host, and the lack of glutamine release in response to nitrate supplementation all support the proposition that the increase in haemolymph glutamine is a product of the host and not the zooxanthellae. Unlike ammonium, nitrate accumulates rapidly in the haemolymph. It has no effect on the concentration of glutamine in the haemolymph, but there is an increase in arginine, histidine and lysine in the haemolymph, suggesting the release of these essential amino acids from zooxanthellae. Glutamine synthetase (GS) activity decreased markedly in the gill and less so in the mantle over a period of 6 d exposure to elevated ammonium (20 µM). In contrast, GS activity in zooxanthellae doubled. The response of zooxanthellae in situ was confirmed by incubating freshly isolated zooxanthellae for 4 d in ammonium, which resulted in a ten-fold increase in GS activity. Comparison of the in situ response of zooxanthellae with that obtained in vitro indicates that the symbionts are likely to be exposed to ammonium concentrations lower than that found in the haemolymph.

The presence of a carbon-concentrating mechanism in the symbiotic dinoflagellate Symbiodinium sp. was investigated. Its existence was postulated to explain how these algae fix inorganic carbon (C(i)) efficiently despite the presence of a form II Rubisco. When the dinoflagellates were isolated from their host, the giant clam (Tridacna gigas), CO2 uptake was found to support the majority of net photosynthesis (45%-80%) at pH 8.0; however, 2 d after isolation this decreased to 5% to 65%, with HCO3- uptake supporting 35% to 95% of net photosynthesis. Measurements of intracellular C(i) concentrations showed that levels inside the cell were between two and seven times what would be expected from passive diffusion of C(i) into the cell. Symbiodinium also exhibits a distinct light-activated intracellular carbonic anhydrase activity. This, coupled with elevated intracellular C(i) and the ability to utilize both CO2 and HCO3- from the medium, suggests that Symbiodinium sp. does possess a carbon-concentrating mechanism. However, intracellular C(i) levels are not as large as might be expected of an alga utilizing a form II Rubisco with a poor affinity for CO2.

To evaluate the impact of elevated nutrients on reef organisms symbiotic with zooxanthellae, giant clams Tridacna maxima were exposed daily to increased ammonia and phosphate (N, P, N+P) in their natural reef environment for 3 to 6 mo. The results strongly corroborate the major responses of the symbiotic association to nutrient enrichment previously observed (with T. gigas) under controlled outdoor conditions. Exposure of the clams to elevated N (10 µM) increased zooxanthellae density, reduced zooxanthellae size, down-regulated N uptake by zooxanthellae freshly isolated from their hosts, and reduced glutamate in the clam haemolymph, with increased pools of some free amino acids (methionine, tyrosine) in the zooxanthellae. These results confirm that the zooxanthellae in giant clams are N limited in situ and have free access to inorganic N from the sea water. There is also corroborating evidence that the zooxanthellae are P limited in situ as well, possibly due to host interference. While the N:P ratios of the animal host reflected ambient N and P concentrations in the sea water, those of the zooxanthellae did not. Regardless of P exposure (2 µM P) of the clams, zooxanthellae N:P ratios were consistently high (>30:1) and phosphate concentrations in the clam haemolymph bathing the zooxanthellae tube system consistently low (< 0.1 µM). These field findings, consistent with previous laboratory observations, confirm the limiting roles of both N and P in the giant clam-zooxanthellae symbiosis. That significant changes occurred earlier and at lower nutrient loading compared to some reef organisms investigated within the same experimental framework further demonstrates organism-level responses of a potential bio-indicator of the early onset of eutrophication in reef waters.

Dinoflagellates exist in symbiosis with a number of marine invertebrates including giant clams, which are the largest of these symbiotic organisms. The dinoflagellates (Symbiodinium sp.) live intercellularly within tubules in the mantle of the host clam. The transport of inorganic carbon (Ci) from seawater to Symbiodinium (=zooxanthellae) is an essential function of hosts that derive the majority of their respiratory energy from the photosynthate exported by the zooxanthellae. Immunolocalisation studies show that the host has adapted its physiology to acquire, rather than remove CO2, from the haemolymph and clam tissues. Two carbonic anhydrase (CA) isoforms (32 and 70 kDa) play an essential part in this process. These have been localised to the mantle and gill tissues where they catalyse the interconversion of HCO3- to CO2, which then diffuses into the host tissues. The zooxanthellae exhibit a number of strategies to maximise Ci acquisition and utilisation. This is necessary as they express a form II Rubisco that has poor discrimination between CO2 and O2. Evidence is presented for a carbon concentrating mechanism (CCM) to overcome this disadvantage. The CCM incorporates the presence of a light-activated CA activity, a capacity to take up both HCO3- and CO2, an ability to accumulate an elevated concentration of Ci within the algal cell, and localisation of Rubisco to the pyrenoid. These algae also express both external and intracellular CAs, with the intracellular isoforms being localised to the thylakoid lumen and pyrenoid. These results have been incorporated into a model that explains the transport of Ci from seawater through the clam to the zooxanthellae.