Myriad Mapping of nanoscale minerals reveals calcium carbonate hemihydrate in forming nacre and coral biominerals.

Calcium carbonate (CaCO3) is abundant on Earth, is a major component of marine biominerals and thus of sedimentary and metamorphic rocks and it plays a major role in the global carbon cycle by storing atmospheric CO2 into solid biominerals. Six crystalline polymorphs of CaCO3 are known-3 anhydrous: calcite, aragonite, vaterite, and 3 hydrated: ikaite (CaCO3·6H2O), monohydrocalcite (CaCO3·1H2O, MHC), and calcium carbonate hemihydrate (CaCO3·½H2O, CCHH). CCHH was recently discovered and characterized, but exclusively as a synthetic material, not as a naturally occurring mineral. Here, analyzing 200 million spectra with Myriad Mapping (MM) of nanoscale mineral phases, we find CCHH and MHC, along with amorphous precursors, on freshly deposited coral skeleton and nacre surfaces, but not on sea urchin spines. Thus, biomineralization pathways are more complex and diverse than previously understood, opening new questions on isotopes and climate. Crystalline precursors are more accessible than amorphous ones to other spectroscopies and diffraction, in natural and bio-inspired materials.

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.

Pervasive tandem duplications and convergent evolution shape coral genomes.

BACKGROUND: Over the last decade, several coral genomes have been sequenced allowing a better understanding of these symbiotic organisms threatened by climate change. Scleractinian corals are reef builders and are central to coral reef ecosystems, providing habitat to a great diversity of species. RESULTS: In the frame of the Tara Pacific expedition, we assemble two coral genomes, Porites lobata and Pocillopora cf. effusa, with vastly improved contiguity that allows us to study the functional organization of these genomes. We annotate their gene catalog and report a relatively higher gene number than that found in other public coral genome sequences, 43,000 and 32,000 genes, respectively. This finding is explained by a high number of tandemly duplicated genes, accounting for almost a third of the predicted genes. We show that these duplicated genes originate from multiple and distinct duplication events throughout the coral lineage. They contribute to the amplification of gene families, mostly related to the immune system and disease resistance, which we suggest to be functionally linked to coral host resilience. CONCLUSIONS: At large, we show the importance of duplicated genes to inform the biology of reef-building corals and provide novel avenues to understand and screen for differences in stress resilience.

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.

An alternative and effective method for extracting skeletal organic matrix adapted to the red coral Corallium rubrum.

Skeleton formation in corals is a biologically controlled process in which an extracellular organic matrix (OM) is entrapped inside the calcified structure. The analysis of OM requires a time-consuming and tedious extraction that includes grinding, demineralization, multiple rinsing and concentration steps. Here we present an alternative and straightforward method for the red coral Corallium rubrum that requires little equipment and saves steps. The entire skeleton is directly demineralized to produce a tractable material called ghost, which is further rinsed and melted at 80°C in water. The comparative analysis of the standard and alternative methods by electrophoresis, western blot, and FTIR of C. rubrum OM, shows that the 'alternative OM' is of higher quality. Advantages and limitations of both methods are discussed.

Investigating calcification-related candidates in a non-symbiotic scleractinian coral, Tubastraea spp.

In hermatypic scleractinian corals, photosynthetic fixation of CO2 and the production of CaCO3 are intimately linked due to their symbiotic relationship with dinoflagellates of the Symbiodiniaceae family. This makes it difficult to study ion transport mechanisms involved in the different pathways. In contrast, most ahermatypic scleractinian corals do not share this symbiotic relationship and thus offer an advantage when studying the ion transport mechanisms involved in the calcification process. Despite this advantage, non-symbiotic scleractinian corals have been systematically neglected in calcification studies, resulting in a lack of data especially at the molecular level. Here, we combined a tissue micro-dissection technique and RNA-sequencing to identify calcification-related ion transporters, and other candidates, in the ahermatypic non-symbiotic scleractinian coral Tubastraea spp. Our results show that Tubastraea spp. possesses several calcification-related candidates previously identified in symbiotic scleractinian corals (such as SLC4-γ, AMT-1like, CARP, etc.). Furthermore, we identify and describe a role in scleractinian calcification for several ion transporter candidates (such as SLC13, -16, -23, etc.) identified for the first time in this study. Taken together, our results provide not only insights about the molecular mechanisms underlying non-symbiotic scleractinian calcification, but also valuable tools for the development of biotechnological solutions to better control the extreme invasiveness of corals belonging to this particular genus.

Biomineralization: Integrating mechanism and evolutionary history.

Calcium carbonate (CaCO3) biomineralizing organisms have played major roles in the history of life and the global carbon cycle during the past 541 Ma. Both marine diversification and mass extinctions reflect physiological responses to environmental changes through time. An integrated understanding of carbonate biomineralization is necessary to illuminate this evolutionary record and to understand how modern organisms will respond to 21st century global change. Biomineralization evolved independently but convergently across phyla, suggesting a unity of mechanism that transcends biological differences. In this review, we combine CaCO3 skeleton formation mechanisms with constraints from evolutionary history, omics, and a meta-analysis of isotopic data to develop a plausible model for CaCO3 biomineralization applicable to all phyla. The model provides a framework for understanding the environmental sensitivity of marine calcifiers, past mass extinctions, and resilience in 21st century acidifying oceans. Thus, it frames questions about the past, present, and future of CaCO3 biomineralizing organisms.

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.

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.

Effects of Ocean Acidification on Resident and Active Microbial Communities of Stylophora pistillata.

Ocean warming and ocean acidification (OA) are direct consequences of climate change and affect coral reefs worldwide. While the effect of ocean warming manifests itself in increased frequency and severity of coral bleaching, the effects of ocean acidification on corals are less clear. In particular, long-term effects of OA on the bacterial communities associated with corals are largely unknown. In this study, we investigated the effects of ocean acidification on the resident and active microbiome of long-term aquaria-maintained Stylophora pistillata colonies by assessing 16S rRNA gene diversity on the DNA (resident community) and RNA level (active community). Coral colony fragments of S. pistillata were kept in aquaria for 2 years at four different pCO2 levels ranging from current pH conditions to increased acidification scenarios (i.e., pH 7.2, 7.4, 7.8, and 8). We identified 154 bacterial families encompassing 2,047 taxa (OTUs) in the resident and 89 bacterial families including 1,659 OTUs in the active communities. Resident communities were dominated by members of Alteromonadaceae, Flavobacteriaceae, and Colwelliaceae, while active communities were dominated by families Cyclobacteriacea and Amoebophilaceae. Besides the overall differences between resident and active community composition, significant differences were seen between the control (pH 8) and the two lower pH treatments (7.2 and 7.4) in the active community, but only between pH 8 and 7.2 in the resident community. Our analyses revealed profound differences between the resident and active microbial communities, and we found that OA exerted stronger effects on the active community. Further, our results suggest that rDNA- and rRNA-based sequencing should be considered complementary tools to investigate the effects of environmental change on microbial assemblage structure and activity.

New Insights From Transcriptomic Data Reveal Differential Effects of CO2 Acidification Stress on Photosynthesis of an Endosymbiotic Dinoflagellate in hospite.

Ocean acidification (OA) has both detrimental as well as beneficial effects on marine life; it negatively affects calcifiers while enhancing the productivity of photosynthetic organisms. To date, many studies have focused on the impacts of OA on calcification in reef-building corals, a process particularly susceptible to acidification. However, little is known about the effects of OA on their photosynthetic algal partners, with some studies suggesting potential benefits for symbiont productivity. Here, we investigated the transcriptomic response of the endosymbiont Symbiodinium microadriaticum (CCMP2467) in the Red Sea coral Stylophora pistillata subjected to different long-term (2 years) OA treatments (pH 8.0, 7.8, 7.4, 7.2). Transcriptomic analyses revealed that symbionts from corals under lower pH treatments responded to acidification by increasing the expression of genes related to photosynthesis and carbon-concentrating mechanisms. These processes were mostly up-regulated and associated metabolic pathways were significantly enriched, suggesting an overall positive effect of OA on the expression of photosynthesis-related genes. To test this conclusion on a physiological level, we analyzed the symbiont's photochemical performance across treatments. However, in contrast to the beneficial effects suggested by the observed gene expression changes, we found significant impairment of photosynthesis with increasing pCO2. Collectively, our data suggest that over-expression of photosynthesis-related genes is not a beneficial effect of OA but rather an acclimation response of the holobiont to different water chemistries. Our study highlights the complex effects of ocean acidification on these symbiotic organisms and the role of the host in determining symbiont productivity and performance.

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.

Intracellular pH regulation: characterization and functional investigation of H+ transporters in Stylophora pistillata.

BACKGROUND: Reef-building corals regularly experience changes in intra- and extracellular H+ concentrations ([H+]) due to physiological and environmental processes. Stringent control of [H+] is required to maintain the homeostatic acid-base balance in coral cells and is achieved through the regulation of intracellular pH (pHi). This task is especially challenging for reef-building corals that share an endosymbiotic relationship with photosynthetic dinoflagellates (family Symbiodinaceae), which significantly affect the pHi of coral cells. Despite their importance, the pH regulatory proteins involved in the homeostatic acid-base balance have been scarcely investigated in corals. Here, we report in the coral Stylophora pistillata a full characterization of the genomic structure, domain topology and phylogeny of three major H+ transporter families that are known to play a role in the intracellular pH regulation of animal cells; we investigated their tissue-specific expression patterns and assessed the effect of seawater acidification on their expression levels. RESULTS: We identified members of the Na+/H+ exchanger (SLC9), vacuolar-type electrogenic H+-ATP hydrolase (V-ATPase) and voltage-gated proton channel (HvCN) families in the genome and transcriptome of S. pistillata. In addition, we identified a novel member of the HvCN gene family in the cnidarian subclass Hexacorallia that has not been previously described in any species. We also identified key residues that contribute to H+ transporter substrate specificity, protein function and regulation. Last, we demonstrated that some of these proteins have different tissue expression patterns, and most are unaffected by exposure to seawater acidification. CONCLUSIONS: In this study, we provide the first characterization of H+ transporters that might contribute to the homeostatic acid-base balance in coral cells. This work will enrich the knowledge of the basic aspects of coral biology and has important implications for our understanding of how corals regulate their intracellular environment.

The skeletome of the red coral Corallium rubrum indicates an independent evolution of biomineralization process in octocorals.

BACKGROUND: The process of calcium carbonate biomineralization has arisen multiple times during metazoan evolution. In the phylum Cnidaria, biomineralization has mostly been studied in the subclass Hexacorallia (i.e. stony corals) in comparison to the subclass Octocorallia (i.e. red corals); the two diverged approximately 600 million years ago. The precious Mediterranean red coral, Corallium rubrum, is an octocorallian species, which produces two distinct high-magnesium calcite biominerals, the axial skeleton and the sclerites. In order to gain insight into the red coral biomineralization process and cnidarian biomineralization evolution, we studied the protein repertoire forming the organic matrix (OM) of its two biominerals. RESULTS: We combined High-Resolution Mass Spectrometry and transcriptome analysis to study the OM composition of the axial skeleton and the sclerites. We identified a total of 102 OM proteins, 52 are found in the two red coral biominerals with scleritin being the most abundant protein in each fraction. Contrary to reef building corals, the red coral organic matrix possesses a large number of collagen-like proteins. Agrin-like glycoproteins and proteins with sugar-binding domains are also predominant. Twenty-seven and 23 proteins were uniquely assigned to the axial skeleton and the sclerites, respectively. The inferred regulatory function of these OM proteins suggests that the difference between the two biominerals is due to the modeling of the matrix network, rather than the presence of specific structural components. At least one OM component could have been horizontally transferred from prokaryotes early during Octocorallia evolution. CONCLUSION: Our results suggest that calcification of the red coral axial skeleton likely represents a secondary calcification of an ancestral gorgonian horny axis. In addition, the comparison with stony coral skeletomes highlighted the low proportion of similar proteins between the biomineral OMs of hexacorallian and octocorallian corals, suggesting an independent acquisition of calcification in anthozoans.

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.

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.

Longevity strategies in response to light in the reef coral Stylophora pistillata.

Aging is a multifactorial process that results in progressive loss of regenerative capacity and tissue function while simultaneously favoring the development of a large array of age-related diseases. Evidence suggests that the accumulation of senescent cells in tissue promotes both normal and pathological aging. Oxic stress is a key driver of cellular senescence. Because symbiotic long-lived reef corals experience daily hyperoxic and hypoxic transitions, we hypothesized that these long-lived animals have developed specific longevity strategies in response to light. We analyzed transcriptome variation in the reef coral Stylophora pistillata during the day-night cycle and revealed a signature of the FoxO longevity pathway. We confirmed this pathway by immunofluorescence using antibodies against coral FoxO to demonstrate its nuclear translocation. Through qPCR analysis of nycthemeral variations of candidate genes under different light regimens, we found that, among genes that were specifically up- or downregulated upon exposure to light, human orthologs of two "light-up" genes (HEY1 and LONF3) exhibited anti-senescence properties in primary human fibroblasts. Therefore, these genes are interesting candidates for counteracting skin aging. We propose a large screen for other light-up genes and an investigation of the biological response of reef corals to light (e.g., metabolic switching) to elucidate these processes and identify effective interventions for promoting healthy aging in humans.

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.

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.

Ubiquitous macropinocytosis in anthozoans.

Transport of fluids, molecules, nutrients or nanoparticles through coral tissues are poorly documented. Here, we followed the flow of various tracers from the external seawater to within the cells of all tissues in living animals. After entering the general coelenteric cavity, we show that nanoparticles disperse throughout the tissues via the paracellular pathway. Then, the ubiquitous entry gate to within the cells' cytoplasm is macropinocytosis. Most cells form large vesicles of 350-600 nm in diameter at their apical side, continuously internalizing their surrounding medium. Macropinocytosis was confirmed using specific inhibitors of PI3K and actin polymerization. Nanoparticle internalization dynamics is size dependent and differs between tissues. Furthermore, we reveal that macropinocytosis is likely a major endocytic pathway in other anthozoan species. The fact that nearly all cells of an animal are continuously soaking in the environment challenges many aspects of the classical physiology viewpoints acquired from the study of bilaterians.