Quantification of cytosolic 'free' calcium in isolated coral cells with confocal microscopy.

Despite its prominent role as an intracellular messenger in all organisms, cytosolic free calcium ([Ca2+]i) has never been quantified in corals or cnidarians in general. Ratiometric calcium dyes and cell imaging have been key methods in successful research on [Ca2+]i in model systems, and could be applied to corals. Here, we developed a procedure to quantify [Ca2+]i in isolated cells from the model coral species Stylophora pistillata using Indo-1 and confocal microscopy. We quantified [Ca2+]i in coral cells with and without intracellular dinoflagellate symbionts, and verified our procedure on cultured mammalian cells. We then used our procedure to measure changes in [Ca2+]i in coral cells exposed to a classic inhibitor of [Ca2+]i regulation, thapsigargin, and also used it to record elevations in [Ca2+]i in coral cells undergoing apoptosis. Our procedure paves the way for future studies into intracellular calcium in corals and other cnidarians.

Testing hypotheses on the calcification in scleractinian corals using a spatio-temporal model that shows a high degree of robustness.

Calcification in photosynthetic scleractinian corals is a complicated process that involves many different biological, chemical, and physical sub-processes that happen within and around the coral tissue. Identifying and quantifying the role of separate processes in vivo or in vitro is difficult or not possible. A computational model can facilitate this research by simulating the sub-processes independently. This study presents a spatio-temporal model of the calcification physiology, which is based on processes that are considered essential for calcification: respiration, photosynthesis, Ca2＋-ATPase, carbonic anhydrase. The model is used to test different hypotheses considering ion transport across the calicoblastic cells and Light Enhanced Calcification (LEC). It is also used to quantify the effect of ocean acidification (OA) on the Extracellular Calcifying Medium (ECM) and ATP-consumption of Ca2＋-ATPase. It was able to reproduce the experimental data of three separate studies and finds that paracellular transport plays a minor role compared to transcellular transport. In the model, LEC results from increased Ca2＋-ATPase activity in combination with increased metabolism. Implementing OA increases the concentration of CO2 throughout the entire tissue, thereby increasing the availability of CO3- in the ECM. As a result, the model finds that calcification becomes more energy-demanding and the calcification rate increases.

From particle attachment to space-filling coral skeletons.

Reef-building corals and their aragonite (CaCO3) skeletons support entire reef ecosystems, yet their formation mechanism is poorly understood. Here we used synchrotron spectromicroscopy to observe the nanoscale mineralogy of fresh, forming skeletons from six species spanning all reef-forming coral morphologies: Branching, encrusting, massive, and table. In all species, hydrated and anhydrous amorphous calcium carbonate nanoparticles were precursors for skeletal growth, as previously observed in a single species. The amorphous precursors here were observed in tissue, between tissue and skeleton, and at growth fronts of the skeleton, within a low-density nano- or microporous layer varying in thickness from 7 to 20 µm. Brunauer-Emmett-Teller measurements, however, indicated that the mature skeletons at the microscale were space-filling, comparable to single crystals of geologic aragonite. Nanoparticles alone can never fill space completely, thus ion-by-ion filling must be invoked to fill interstitial pores. Such ion-by-ion diffusion and attachment may occur from the supersaturated calcifying fluid known to exist in corals, or from a dense liquid precursor, observed in synthetic systems but never in biogenic ones. Concomitant particle attachment and ion-by-ion filling was previously observed in synthetic calcite rhombohedra, but never in aragonite pseudohexagonal prisms, synthetic or biogenic, as observed here. Models for biomineral growth, isotope incorporation, and coral skeletons' resilience to ocean warming and acidification must take into account the dual formation mechanism, including particle attachment and ion-by-ion space filling.

Faster Crystallization during Coral Skeleton Formation Correlates with Resilience to Ocean Acidification.

The mature skeletons of hard corals, termed stony or scleractinian corals, are made of aragonite (CaCO3). During their formation, particles attaching to the skeleton's growing surface are calcium carbonate, transiently amorphous. Here we show that amorphous particles are observed frequently and reproducibly just outside the skeleton, where a calicoblastic cell layer envelops and deposits the forming skeleton. The observation of particles in these locations, therefore, is consistent with nucleation and growth of particles in intracellular vesicles. The observed extraskeletal particles range in size between 0.2 and 1.0 µm and contain more of the amorphous precursor phases than the skeleton surface or bulk, where they gradually crystallize to aragonite. This observation was repeated in three diverse genera of corals, Acropora sp., Stylophora pistillata─differently sensitive to ocean acidification (OA)─and Turbinaria peltata, demonstrating that intracellular particles are a major source of material during the additive manufacturing of coral skeletons. Thus, particles are formed away from seawater, in a presumed intracellular calcifying fluid (ICF) in closed vesicles and not, as previously assumed, in the extracellular calcifying fluid (ECF), which, unlike ICF, is partly open to seawater. After particle attachment, the growing skeleton surface remains exposed to ECF, and, remarkably, its crystallization rate varies significantly across genera. The skeleton surface layers containing amorphous pixels vary in thickness across genera: ∼2.1 µm in Acropora, 1.1 µm in Stylophora, and 0.9 µm in Turbinaria. Thus, the slow-crystallizing Acropora skeleton surface remains amorphous and soluble longer, including overnight, when the pH in the ECF drops. Increased skeleton surface solubility is consistent with Acropora's vulnerability to OA, whereas the Stylophora skeleton surface layer crystallizes faster, consistent with Stylophora's resilience to OA. Turbinaria, whose response to OA has not yet been tested, is expected to be even more resilient than Stylophora, based on the present data.

The Evolution of Calcification in Reef-Building Corals.

Corals build the structural foundation of coral reefs, one of the most diverse and productive ecosystems on our planet. Although the process of coral calcification that allows corals to build these immense structures has been extensively investigated, we still know little about the evolutionary processes that allowed the soft-bodied ancestor of corals to become the ecosystem builders they are today. Using a combination of phylogenomics, proteomics, and immunohistochemistry, we show that scleractinian corals likely acquired the ability to calcify sometime between ∼308 and ∼265 Ma through a combination of lineage-specific gene duplications and the co-option of existing genes to the calcification process. Our results suggest that coral calcification did not require extensive evolutionary changes, but rather few coral-specific gene duplications and a series of small, gradual optimizations of ancestral proteins and their co-option to the calcification process.

Telomere dysfunction is associated with dark-induced bleaching in the reef coral Stylophora pistillata.

Telomere DNA length is a complex trait controlled by both multiple loci and environmental factors. A growing number of studies are focusing on the impact of stress and stress accumulation on telomere length and the link with survival and fitness in ecological contexts. Here, we investigated the telomere changes occurring in a symbiotic coral, Stylophora pistillata, that has experienced continuous darkness over 6 months. This stress condition led to the loss of its symbionts in a similar manner to that observed during large-scale bleaching events due to climate changes and anthropogenic activities, threatening reef ecosystems worldwide. We found that continuous darkness was associated with telomere length shortening. This result, together with a phylogenetic analysis of the telomere coral proteins and a transcriptome survey of the continuous darkness condition, paves the way for future studies on the role of telomeres in the coral stress response and the importance of environmentally induced telomere shortening in endangered coral species.

A role for primary cilia in coral calcification?

Cilia are evolutionarily conserved organelles that extend from the surface of cells and are found in diverse organisms from protozoans to multicellular organisms. Motile cilia play various biological functions by their beating motion, including mixing fluids and transporting food particles. Non-motile cilia act as sensors that signal cells about their microenvironment. In corals, cilia have been described in some of the cell layers but never in the calcifying epithelium, which is responsible for skeleton formation. In the present study, we used scanning electron microscopy and immunolabelling to investigate the cellular ciliature of the different tissue layers of the coral Stylophora pistillata, with a focus on the calcifying calicoblastic ectoderm. We show that the cilium of the calcifying cells is different from the cilium of the other cell layers. It is much shorter, and more importantly, its base is structurally distinct from the base observed in cilia of the other tissue layers. Based on these structural observations, we conclude that the cilium of the calcifying cells is a primary cilium. From what is known in other organisms, primary cilia are sensors that signal cells about their microenvironment. We discuss the implications of the presence of a primary cilium in the calcifying epithelium for our understanding of the cellular physiology driving coral calcification and its environmental sensitivity.

Regulation of coral calcification by the acid-base sensing enzyme soluble adenylyl cyclase.

Coral calcification is intricately linked to the chemical composition of the fluid in the extracellular calcifying medium (ECM), which is situated between the calcifying cells and the skeleton. Here we demonstrate that the acid-base sensing enzyme soluble adenylyl cyclase (sAC) is expressed in calcifying cells of the coral Stylophora pistillata. Furthermore, pharmacological inhibition of sAC in coral microcolonies resulted in acidification of the ECM as estimated by the pH-sensitive ratiometric indicator SNARF, and decreased calcification rates, as estimated by calcein labeling of crystal growth. These results indicate that sAC activity modulates some of the molecular machinery involved in producing the coral skeleton, which could include ion-transporting proteins and vesicular transport. To our knowledge this is the first study to directly demonstrate biological regulation of the alkaline pH of the coral ECM and its correlation with calcification.

Paracellular transport to the coral calcifying medium: effects of environmental parameters.

Coral calcification relies on the transport of ions and molecules to the extracellular calcifying medium (ECM). Little is known about paracellular transport (via intercellular junctions) in corals and other marine calcifiers. Here, we investigated whether the permeability of the paracellular pathway varied in different environmental conditions in the coral Stylophora pistillata Using the fluorescent dye calcein, we characterised the dynamics of calcein influx from seawater to the ECM and showed that increases in paracellular permeability (leakiness) induced by hyperosmotic treatment could be detected by changes in calcein influx rates. We then used the calcein-imaging approach to investigate the effects of two environmental stressors on paracellular permeability: seawater acidification and temperature change. Under conditions of seawater acidification (pH 7.2) known to depress pH in the ECM and the calcifying cells of S. pistillata, we observed a decrease in half-times of calcein influx, indicating increased paracellular permeability. By contrast, high temperature (31°C) had no effect, whereas low temperature (20°C) caused decreases in paracellular permeability. Overall, our study establishes an approach to conduct further in vivo investigation of paracellular transport and suggests that changes in paracellular permeability could form an uncharacterised aspect of the physiological response of S. pistillata to seawater acidification.

Comparison of the Sulfonamide Inhibition Profiles of the α-Carbonic Anhydrase Isoforms (SpiCA1, SpiCA2 and SpiCA3) Encoded by the Genome of the Scleractinian Coral Stylophora pistillata.

The ubiquitous metalloenzymes carbonic anhydrases (CAs, EC 4.2.1.1) are responsible for the reversible hydration of CO2 to bicarbonate (HCO3-) and protons (H⁺). Bicarbonate may subsequently generate carbonate used in many functional activities by marine organisms. CAs play a crucial role in several physiological processes, e.g., respiration, inorganic carbon transport, intra and extra-cellular pH regulation, and bio-mineralization. Multiple transcript variants and protein isoforms exist in the organisms. Recently, 16 α-CA isoforms have been identified in the coral Stylophora pistillata. Here, we focalized the interest on three coral isoforms: SpiCA1 and SpiCA2, localized in the coral-calcifying cells; and SpiCA3, expressed in the cytoplasm of the coral cell layers. The three recombinant enzymes were heterologously expressed and investigated for their inhibition profiles with sulfonamides and sulfamates. The three coral CA isoforms differ significantly in their susceptibility to inhibition with sulfonamides. This study provides new insights into the coral physiology and the comprehension of molecular mechanisms involved in the bio-mineralization processes, since CAs interact with bicarbonate transporters, accelerating the trans-membrane bicarbonate movement and modulating the pH at both sides of the plasma membranes.

Protonography and anion inhibition profile of the α-carbonic anhydrase (CruCA4) identified in the Mediterranean red coral Corallium rubrum.

CruCA4 is a secreted isoform of the α-carbonic anhydrase (CA, EC 4.2.1.1) family, which has been identified in the octocoral Corallium rubrum. This enzyme is involved in the calcification process leading to the formation of the coral calcium carbonate skeleton. We report here experiments performed on the recombinant CruCA4 with the technique of protonography that can be used to detect in a simple way the enzyme activity. We have also investigated the inhibition profile of CruCA4 with one major class of CA inhibitors, the inorganic anions. A range of weak and moderate inhibitors have been identified having KI in the range of 1-100 mM, among which the halides, pseudohalides, bicarbonate, sulfate, nitrate, nitrite, and many complex inorganic anions. Stronger inhibitors were sulfamide, sulfamate, phenylboronic acid, phenylarsonic acid, and diethylditiocarbamate, which showed a better affinity for this enzyme, with KI in the range of 75 µM-0.60 mM. All these anions/small molecules probably coordinate to the Zn(II) ion within the CA active site as enzyme inhibition mechanism.

Coral host cells acidify symbiotic algal microenvironment to promote photosynthesis.

Symbiotic dinoflagellate algae residing inside coral tissues supply the host with the majority of their energy requirements through the translocation of photosynthetically fixed carbon. The algae, in turn, rely on the host for the supply of inorganic carbon. Carbon must be concentrated as CO2 in order for photosynthesis to proceed, and here we show that the coral host plays an active role in this process. The host-derived symbiosome membrane surrounding the algae abundantly expresses vacuolar H(+)-ATPase (VHA), which acidifies the symbiosome space down to pH ∼ 4. Inhibition of VHA results in a significant decrease in average H(+) activity in the symbiosome of up to 75% and a significant reduction in O2 production rate, a measure of photosynthetic activity. These results suggest that host VHA is part of a previously unidentified carbon concentrating mechanism for algal photosynthesis and provide mechanistic evidence that coral host cells can actively modulate the physiology of their symbionts.

Comparison of the Anion Inhibition Profiles of the α-CA Isoforms (SpiCA1, SpiCA2 and SpiCA3) from the Scleractinian Coral Stylophora pistillata.

Carbonic anhydrases (CAs, EC 4.2.1.1) are widespread metalloenzymes used by living organisms to accelerate the CO2 hydration/dehydration reaction at rates dramatically high compared to the uncatalyzed reaction. These enzymes have different isoforms and homologues and can be found in the form of cytoplasmic, secreted or membrane-bound proteins. CAs play a role in numerous physiological processes including biomineralization and symbiosis, as is the case in reef-building corals. Previously, molecular and biochemical data have been obtained at the molecular level in the branching coral Stylophora pistillata for two coral isoforms which differ significantly in their catalytic activity and susceptibility to inhibition with anions and sulfonamides. More recently it has been determined that the genome of S. pistillata encodes for 16 CAs. Here, we cloned, expressed, purified and characterized a novel α-CA, named SpiCA3, which is cytoplasmic and ubiquitously expressed in all the cell layers including the calcifying cells. SpiCA3 is the most effective CA among the coral isoforms investigated and the most efficient catalyst known up to date in Metazoa. We also investigated the inhibition profiles of SpiCA3 and compared it with those obtained for the two other isoforms in the presence of inorganic anions and other small molecules known to interfere with metalloenzymes. These results suggest that S. pistillata has adapted its CA isoforms to achieve the physiological functions in different physicochemical microenvironments.

Computing the carbonate chemistry of the coral calcifying medium and its response to ocean acidification.

Critical to determining vulnerability or resilience of reef corals to Ocean Acidification (OA) is a clearer understanding of the extent to which corals can control carbonate chemistry in their Extracellular Calcifying Medium (ECM) where the CaCO3 skeleton is produced. Here, we employ a mathematical framework to calculate ECM aragonite saturation state (Ωarag.(ECM)) and carbonate system ion concentration using measurements of calcification rate, seawater characteristics (temperature, salinity and pH) and ECM pH (pH(ECM)). Our calculations of ECM carbonate chemistry at current-day seawater pH, indicate that Ωarag.(ECM) ranges from ∼10 to 38 (mean 20.41), i.e. about 5 to 6-fold higher than seawater. Accordingly, Dissolved Inorganic Carbon (DIC) and Total Alkalinity (TA) were calculated to be around 3 times higher in the ECM than in seawater. We also assessed the effects of acidification on ECM chemical properties of the coral Stylophora pistillata. At reduced seawater pH our calculations indicate that Ωarag.(ECM) remains almost constant. DIC(ECM) and TA(ECM) gradually increase as seawater pH declines, reaching values about 5 to 6-fold higher than in seawater, respectively for DIC and TA. We propose that these ECM characteristics buffer the effect of acidification and explain why certain corals continue to produce CaCO3 even when seawater chemistry is less favourable.

Kinetic properties and affinities for sulfonamide inhibitors of an α-carbonic anhydrase (CruCA4) involved in coral biomineralization in the Mediterranean red coral Corallium rubrum.

We report the kinetic properties and sulfonamide inhibition profile of an α-carbonic anhydrase (CA, EC 4.2.1.1), named CruCA4, identified in the red coral Corallium rubrum. This isoform is involved in the biomineralization process leading to the formation of a calcium carbonate skeleton. Experiments performed on the recombinant protein show that the enzyme has a "moderate activity" level. Our results are discussed compared to values obtained for other CA isoforms involved in biomineralization. This is the first study describing the biochemical characterization of an octocoral CA.

Activation Profile Analysis of CruCA4, an α-Carbonic Anhydrase Involved in Skeleton Formation of the Mediterranean Red Coral, Corallium rubrum.

CruCA4, a coral α-carbonic anhydrase (CA, EC 4.2.1.1) involved in the biomineralization process of the Mediterranean red coral, Corallium rubrum, was investigated for its activation with a panel of amino acids and amines. Most compounds showed considerable activating properties, with a rather well defined structure-activity relationship. The most effective CruCA4 activators were d-His, 4-H2N-l-Phe, Histamine, Dopamine, Serotonin, 1-(2-Aminoethyl)-piperazine, and l-Adrenaline, with activation constants in the range of 8-98 nM. Other amines and amino acids, such as d-DOPA, l-Tyr, 2-Pyridyl-methylamine, 2-(2-Aminoethyl) pyridine and 4-(2-Aminoethyl)-morpholine, were submicromolar CruCA4 activators, with KA ranging between 0.15 and 0.93 µM. Since it has been shown that CA activators may facilitate the initial phases of in-bone mineralization, our study may be relevant for finding modulators of enzyme activity, which can enhance the formation of the red coral skeleton.

Structural molecular components of septate junctions in cnidarians point to the origin of epithelial junctions in eukaryotes.

Septate junctions (SJs) insure barrier properties and control paracellular diffusion of solutes across epithelia in invertebrates. However, the origin and evolution of their molecular constituents in Metazoa have not been firmly established. Here, we investigated the genomes of early branching metazoan representatives to reconstruct the phylogeny of the molecular components of SJs. Although Claudins and SJ cytoplasmic adaptor components appeared successively throughout metazoan evolution, the structural components of SJs arose at the time of Placozoa/Cnidaria/Bilateria radiation. We also show that in the scleractinian coral Stylophora pistillata, the structural SJ component Neurexin IV colocalizes with the cortical actin network at the apical border of the cells, at the place of SJs. We propose a model for SJ components in Cnidaria. Moreover, our study reveals an unanticipated diversity of SJ structural component variants in cnidarians. This diversity correlates with gene-specific expression in calcifying and noncalcifying tissues, suggesting specific paracellular pathways across the cell layers of these diploblastic animals.

Coral Carbonic Anhydrases: Regulation by Ocean Acidification.

Global change is a major threat to the oceans, as it implies temperature increase and acidification. Ocean acidification (OA) involving decreasing pH and changes in seawater carbonate chemistry challenges the capacity of corals to form their skeletons. Despite the large number of studies that have investigated how rates of calcification respond to ocean acidification scenarios, comparatively few studies tackle how ocean acidification impacts the physiological mechanisms that drive calcification itself. The aim of our paper was to determine how the carbonic anhydrases, which play a major role in calcification, are potentially regulated by ocean acidification. For this we measured the effect of pH on enzyme activity of two carbonic anhydrase isoforms that have been previously characterized in the scleractinian coral Stylophora pistillata. In addition we looked at gene expression of these enzymes in vivo. For both isoforms, our results show (1) a change in gene expression under OA (2) an effect of OA and temperature on carbonic anhydrase activity. We suggest that temperature increase could counterbalance the effect of OA on enzyme activity. Finally we point out that caution must, thus, be taken when interpreting transcriptomic data on carbonic anhydrases in ocean acidification and temperature stress experiments, as the effect of these stressors on the physiological function of CA will depend both on gene expression and enzyme activity.

Impact of seawater acidification on pH at the tissue-skeleton interface and calcification in reef corals.

Insight into the response of reef corals and other major marine calcifiers to ocean acidification is limited by a lack of knowledge about how seawater pH and carbonate chemistry impact the physiological processes that drive biomineralization. Ocean acidification is proposed to reduce calcification rates in corals by causing declines in internal pH at the calcifying tissue-skeleton interface where biomineralization takes place. Here, we performed an in vivo study on how partial-pressure CO(2)-driven seawater acidification impacts intracellular pH in coral calcifying cells and extracellular pH in the fluid at the tissue-skeleton interface [subcalicoblastic medium (SCM)] in the coral Stylophora pistillata. We also measured calcification in corals grown under the same conditions of seawater acidification by measuring lateral growth of colonies and growth of aragonite crystals under the calcifying tissue. Our findings confirm that seawater acidification decreases pH of the SCM, but this decrease is gradual relative to the surrounding seawater, leading to an increasing pH gradient between the SCM and seawater. Reductions in calcification rate, both at the level of crystals and whole colonies, were only observed in our lowest pH treatment when pH was significantly depressed in the calcifying cells in addition to the SCM. Overall, our findings suggest that reef corals may mitigate the effects of seawater acidification by regulating pH in the SCM, but they also highlight the role of calcifying cell pH homeostasis in determining the response of reef corals to changes in external seawater pH and carbonate chemistry.

Spatial variability of and effect of light on the cœlenteron pH of a reef coral.

Coral reefs, the largest bioconstruction on Earth, are formed by calcium carbonate skeletons of corals. Coral skeleton formation commonly referred to as calcification occurs in a specific compartment, the extracellular calcifying medium (ECM), located between the aboral ectoderm and the skeleton. Calcification models often assume a direct link between the surrounding seawater and the ECM. However, the ECM is separated from the seawater by several tissue layers and the cœlenteron, which contains the cœlenteric fluid found in both polyps and cœnosarc (tissue connecting the polyps). Symbiotic dinoflagellate-containing cells line the cœlenteron and their photosynthetic activity contributes to changes in the chemistry of the cœlenteric fluid, particularly with respect to pH. The aim of our study is to compare cœlenteron pH between the cœnosarc and polyps and to compare areas of high or low dinoflagellate density based on tissue coloration. To achieve this, we use liquid ion exchange (LIX) pH microsensors to profile pH in the cœlenteron of polyps and the cœnosarc in different regions of the coral colony in light and darkness. We interpret our results in terms of what light and dark exposure means for proton gradients between the ECM and the coelenteron, and how this could affect calcification.