Genetic basis of stony coral biomineralization: History,trends and future prospects

Despite their simple body plan, stony corals (order Scleractinia, phylum Cnidaria) can produce massive and complex exoskeletal structures in shallow, tropical and subtropical regions of Earth’s oceans. The species-specific macromorphologies of their aragonite skeletons suggest a highly coordinated biomineralization process that is rooted in their genomes, and which has persisted across major climatic shifts over the past 400 + million years. The mechanisms by which stony corals produce their skeletons has been the subject of interest for at least the last 160 years, and the pace of understanding the process has increased dramatically in the past decade since the sequencing of the first coral genome in 2011. In this review, we detail what is known to date about the genetic basis of the stony coral biomineralization process, with a focus on advances in the last several years as well as ways that physical and chemical tools can be combined with genetics, and then propose next steps forward for the coming decade.     

Perspectives in coral reef hydrodynamics

Knowledge of skeletogenesis in scleractinian corals is central to reconstructing past ocean and climate histories, assessing and counteracting future climate and ocean acidification impacts upon coral reefs, and determining the taxonomy and evolutionary path of the Scleractinia. To better understand skeletogenesis and mineralogy in extant scleractinian corals, we have investigated the nature of the initial calcium carbonate skeleton deposited by newly settling coral recruits. Settling Acropora millepora larvae were sampled daily for 10 days from initial attachment, and the carbonate mineralogy of their newly deposited skeletons was investigated. Bulk analyses using Raman and infrared spectroscopic methods revealed that the skeletons were predominantly comprised of aragonite, with no evidence of calcite or an amorphous precursor phase, although presence of the latter cannot be discounted. Sensitive selected area electron diffraction analyses of sub-micron areas of skeletal regions further consolidated these data. These findings help to address the uncertainty surrounding reported differences in carbonate mineralogy between larval and adult extant coral skeletons by indicating that skeletons of new coral recruits share the same aragonitic mineralogy as those of their mature counterparts. In this respect, we can expect that skeletogenesis in both larval and mature growth stages of scleractinian corals will be similarly affected by ocean acidification and predicted environmental changes.     

The skeletal organic matrix from Mediterranean coral Balanophyllia europaea influences calcium carbonate precipitation

Scleractinian coral skeletons are made mainly of calcium carbonate in the form of aragonite. The mineral deposition occurs in a biological confined environment, but it is still a theme of discussion to what extent the calcification occurs under biological or environmental control. Hence, the shape, size and organization of skeletal crystals from the cellular level through the colony architecture, were attributed to factors as diverse as mineral supersaturation levels and organic mediation of crystal growth. The skeleton contains an intra-skeletal organic matrix (OM) of which only the water soluble component was chemically and physically characterized. In this work that OM from the skeleton of the Balanophyllia europaea, a solitary scleractinian coral endemic to the Mediterranean Sea, is studied in vitro with the aim of understanding its role in the mineralization of calcium carbonate. Mineralization of calcium carbonate was conducted by overgrowth experiments on coral skeleton and in calcium chloride solutions containing different ratios of water soluble and/or insoluble OM and of magnesium ions. The precipitates were characterized by diffractometric, spectroscopic and microscopic techniques. The results showed that both soluble and insoluble OM components influence calcium carbonate precipitation and that the effect is enhanced by their co-presence. The role of magnesium ions is also affected by the presence of the OM components. Thus, in vitro, OM influences calcium carbonate crystal morphology, aggregation and polymorphism as a function of its composition and of the content of magnesium ions in the precipitation media. This research, although does not resolve the controversy between environmental or biological control on the deposition of calcium carbonate in corals, sheds a light on the role of OM, which appears mediated by the presence of magnesium ions.     

Carbonic anhydrases in anthozoan corals—A review

Coral reefs are among the most biologically diverse and economically important ecosystems on the planet. The deposition of massive calcium carbonate skeletons (biomineralization or calcification) by scleractinian corals forms the coral reef framework/architecture that serves as habitat for a large diversity of organisms. This process would not be possible without the intimate symbiosis between corals and photosynthetic dinoflagellates, commonly called zooxanthellae. Carbonic anhydrases play major roles in those two essential processes of coral’s physiology: they are involved in the carbon supply for calcium carbonate precipitation as well as in carbon-concentrating mechanisms for symbiont photosynthesis. Here, we review the current understanding of diversity and function of carbonic anhydrases in corals and discuss the perspective of theses enzymes as a key to understanding impacts of environmental changes on coral reefs.     

The impact of reduced pH on the microbial community of the coral Acropora eurystoma

Rising concentrations of atmospheric carbon dioxide are acidifying the world''s oceans. Surface seawater pH is 0.1 units lower than pre-industrial values and is predicted to decrease by up to 0.4 units by the end of the century. This change in pH may result in changes in the physiology of ocean organisms, in particular, organisms that build their skeletons/shells from calcium carbonate, such as corals. This physiological change may also affect other members of the coral holobiont, for example, the microbial communities associated with the coral, which in turn may affect the coral physiology and health. In the present study, we examined changes in bacterial communities in the coral mucus, tissue and skeleton following exposure of the coral Acropora eurystoma to two different pH conditions: 7.3 and 8.2 (ambient seawater). The microbial community was different at the two pH values, as determined by denaturing gradient gel electrophoresis and 16S rRNA gene sequence analysis. Further analysis of the community in the corals maintained at the lower pH revealed an increase in bacteria associated with diseased and stressed corals, such as Vibrionaceae and Alteromonadaceae. In addition, an increase in the number of potential antibacterial activity was recorded among the bacteria isolated from the coral maintained at pH 7.3. Taken together, our findings highlight the impact that changes in the pH may have on the coral-associated bacterial community and their potential contribution to the coral host.     

The calcifying interface in a stony coral primary polyp: An interplay between seawater and an extracellular calcifying space

Stony coral exoskeletons build the foundation for the most biologically diverse marine ecosystems on Earth, coral reefs, which face major threats due to many anthropogenic–related stressors. Therefore, understanding coral biomineralization mechanisms is crucial for coral reef management in the coming decades and for using coral skeletons in geochemical studies. This study combines in–vivo imaging with cryo-electron microscopy and cryo–elemental mapping to gain novel insights into the biological microenvironment and the ion pathways that facilitate biomineralization in primary polyps of the stony coral Stylophora pistillata. We document increased tissue permeability in the primary polyp and a highly dispersed cell packing in the tissue directly responsible for producing the coral skeleton. This tissue arrangement may facilitate the intimate involvement of seawater at the mineralization site, also documented here. We further observe an extensive filopodial network containing carbon-rich vesicles extruding from some of the calicoblastic cells. Single-cell RNA-Sequencing data interrogation supports these morphological observations by showing higher expression of genes involved in filopodia and vesicle structure and function in the calicoblastic cells. These observations provide a new conceptual framework for resolving the ion pathway from the external seawater to the tissue-mineral interface in stony coral biomineralization processes.     

Slow‐flow habitats as refugia for coastal calcifiers from ocean acidification

The pH of the oceans’ surface water is dropping, termed ocean acidification (OA), and the 0.4 unit reduction in pH by 2100 is projected to negatively impact benthic coastal organisms that produce calcium carbonate “skeletons.” Research has focussed on identifying species that are susceptible to OA, but there is an urgent need to discover refuge habitats that will afford protection to vulnerable species. The susceptibility of calcium carbonate skeletons to dissolution by OA depends on the pH at their surface, and this is controlled by the interaction between seawater velocity and organismal metabolism. This perspective considers how seawater velocity modifies the responses of calcifying organisms (seaweed, shellfish, and tropical corals) to OA through its action on controlling diffusion boundary layer thickness and thereby the pH and calcium carbonate saturation state (Ω) at the organisms’ surface. Evidence is presented to support the idea that slow‐flow habitats, such as wave‐sheltered bays or the within canopies of seaweed/seagrass beds, might provide inexpensive refugia from OA for vulnerable coastal calcifiers.     

Coral Skeletons Defend against Ultraviolet Radiation

Background

Many coral reef organisms are photosynthetic or have evolved in tight symbiosis with photosynthetic symbionts. As such, the tissues of reef organisms are often exposed to intense solar radiation in clear tropical waters and have adapted to trap and harness photosynthetically active radiation (PAR). High levels of ultraviolet radiation (UVR) associated with sunlight, however, represent a potential problem in terms of tissue damage.

Methodology/Principal Findings

By measuring UVR and PAR reflectance from intact and ground bare coral skeletons we show that the property of calcium carbonate skeletons to absorb downwelling UVR to a significant extent, while reflecting PAR back to the overlying tissue, has biological advantages. We placed cnidarians on top of bare skeletons and a UVR reflective substrate and showed that under ambient UVR levels, UVR transmitted through the tissues of cnidarians placed on top of bare skeletons were four times lower compared to their counterparts placed on a UVR reflective white substrate. In accordance with the lower levels of UVR measured in cnidarians on top of coral skeletons, a similar drop in UVR damage to their DNA was detected. The skeletons emitted absorbed UVR as yellow fluorescence, which allows for safe dissipation of the otherwise harmful radiation.

Conclusions/Significance

Our study presents a novel defensive role for coral skeletons and reveals that the strong UVR absorbance by the skeleton can contribute to the ability of corals, and potentially other calcifiers, to thrive under UVR levels that are detrimental to most marine life.     

Morphology,microstructure, crystallography,and chemistry of distinct CaCO3 deposits formed by early recruits of the scleractinian coral Pocillopora damicornis

Scleractinian corals begin their biomineralization process shortly after larval settlement with the formation of calcium carbonate (CaCO₃) structures at the interface between the larval tissues and the substrate. The newly settled larvae exert variable degrees of control over this skeleton formation, providing an opportunity to study a range of biocarbonate structures, some of which are transient and not observed in adult coral skeletons. Here we present a morphological, structural, crystallographic, and chemical comparison between two types of aragonite deposits observed during the skeletal development of 2‐days old recruits of Pocillopora damicornis: (1) Primary septum and (2) Abundant, dumbbell‐like structures, quasi‐randomly distributed between initial deposits of the basal plate and not present in adult corals—At the mesoscale level, initial septa structures are formed by superimposed fan‐shaped fasciculi consisting of bundles of fibers, as also observed in adult corals. This organization is not observed in the dumbbell‐like structures. However, at the ultrastructural level there is great similarity between septa and dumbbell components. Both are composed of <100 nm granular units arranged into larger single‐crystal domains.Chemically, a small difference is observed between the septae with an average Mg/Ca ratio around 11 mmol/mol and the dumbbell‐like structures with ca. 7 mmol/mol; Sr/Ca ratios are similar in the two structures at around 8 mmol/mol—Overall, the observed differences in distribution, morphology, and chemistry between septa, which are highly conserved structures fundamental to the architecture of the skeleton, and the transient, dumbbell‐like structures, suggest that the latter might be formed through less controlled biomineralization processes. Our observations emphasize the inherent difficulties involved in distinguishing different biomineralization pathways based on ultrastructural and crystallographical observations. J. Morphol. 276:1146–1156, 2015. © 2015 Wiley Periodicals, Inc.     

Reef-building corals act as long-term sink for microplastic

The pollution of the marine environment with microplastics is pervasive. However, microplastic concentrations in the seawater are lower than the number of particles entering the oceans, suggesting that plastic particles accumulate in environmental sinks. Yet, the exact long-term sinks related to the “missing plastic” phenomenon are barely explored. Sediments in nearshore biogenic habitats are known to trap large amounts of microplastics, but also the three-dimensional structures of coral reefs might serve as unique, living long-term sinks. The main framework builders, reef-building corals, have been shown to ingest and overgrow microplastics, potentially leading to a deposition of particles in reef structures. However, little is known about the number of deposited particles and the underlying processes determining the permanent deposition in the coral skeletons. To test whether corals may act as living long-term sink for microplastic, we exposed four reef-building coral species to polyethylene microplastics (200 particles L^?1) in an 18-month laboratory experiment. We found microplastics in all treatment specimens, with low numbers of particles trapped in the coral tissue (up to 2 particles per cm²) and much higher numbers in the skeleton (up to 84 particles per cm³). The numbers of particles accumulated in the coral skeletons were mainly related to coral growth (i.e., skeletal growth in volume), suggesting that deposition is a regularly occurring stochastic process. We estimate that reef-building corals may remove 0.09%–2.82% of the bioavailable microplastics from tropical shallow-reef waters per year. Our study shows for the first time that microplastic particles accumulate permanently in a biological sink, helping to explain the “missing plastic” phenomenon. This highlights the importance of coral reefs for the ecological balance of the oceans and reinforces the need to protect them, not only to mitigate the effects of climate change but also to preserve their ecosystem services as long-term sink for microplastic.     

Multi-scale mineralogical characterization of the hypercalcified sponge Petrobiona massiliana (Calcarea, Calcaronea)

The massive basal skeleton of a few remnant living hypercalcified sponges rediscovered since the 1960s are valuable representatives of ancient calcium carbonate biomineralization mechanisms in basal Metazoa. A multi-scale mineralogical characterization of the easily accessible Mediterranean living hypercalcified sponge belonging to Calcarea, Petrobiona massiliana (Vacelet and Lévi, 1958), was conducted. Oriented observations in light and electron microscopy of mature and growing areas of the Mg-calcite basal skeleton were combined in order to describe all structural levels from the submicronic to the macroscopic scale. The smallest units produced are ca. 50–100 nm grains that are in a mushy amorphous state before their crystallization. Selected area electron diffraction (SAED) further demonstrated that submicronic grains are assembled into crystallographically coherent clusters or fibers, the latter are even laterally associated into single-crystal bundles. A model of crystallization propagation through amorphous submicronic granular units is proposed to explain the formation of coherent micron-scale structural units. Finally, XRD and EELS analyses highlighted, respectively, inter-individual variation of skeletal Mg contents and heterogeneous spatial distribution of Ca ions in skeletal fibers. All mineralogical features presented here cannot be explained by classical inorganic crystallization principles in super-saturated solutions, but rather underlined a highly biologically regulated formation of the basal skeleton. This study extending recent observations on corals, mollusk and echinoderms confirms that occurrence of submicronic granular units and a possible transient amorphous precursor phase in calcium carbonate skeletons is a common biomineralization strategy already selected by basal metazoans.     

Polyp oriented modelling of coral growth

The morphogenesis of colonial stony corals is the result of the collective behaviour of many coral polyps depositing coral skeleton on top of the old skeleton on which they live. Yet, models of coral growth often consider the polyps as a single continuous surface. In the present work, the polyps are modelled individually. Each polyp takes up resources, deposits skeleton, buds off new polyps and dies. In this polyp oriented model, spontaneous branching occurs. We argue that branching is caused by a so called “polyp fanning effect” by which polyps on a convex surface have a competitive advantage relative to polyps on a flat or concave surface. The fanning effect generates a more potent branching mechanism than the Laplacian growth mechanism that we have studied previously (J. Theor. Biol. 224 (2003) 153). We discuss the application of the polyp oriented model to the study of environmentally driven morphological plasticity in stony corals. In a few examples we show how the properties of the individual polyps influence the whole colony morphology. In our model, the spacing of polyps influences the thickness of coral branches and the overall compactness of the colony. Density variations in the coral skeleton may also be important for the whole colony morphology, which we address by studying two variants of the model. Finally, we discuss the importance of small scale resource translocation in the coral colony and its effects on the morphology of the colony.     

Changes in coral microbial communities in response to a natural pH gradient

Surface seawater pH is currently 0.1 units lower than pre-industrial values and is projected to decrease by up to 0.4 units by the end of the century. This acidification has the potential to cause significant perturbations to the physiology of ocean organisms, particularly those such as corals that build their skeletons/shells from calcium carbonate. Reduced ocean pH could also have an impact on the coral microbial community, and thus may affect coral physiology and health. Most of the studies to date have examined the impact of ocean acidification on corals and/or associated microbiota under controlled laboratory conditions. Here we report the first study that examines the changes in coral microbial communities in response to a natural pH gradient (mean pH_T 7.3–8.1) caused by volcanic CO₂ vents off Ischia, Gulf of Naples, Italy. Two Mediterranean coral species, Balanophyllia europaea and Cladocora caespitosa, were examined. The microbial community diversity and the physiological parameters of the endosymbiotic dinoflagellates (Symbiodinium spp.) were monitored. We found that pH did not have a significant impact on the composition of associated microbial communities in both coral species. In contrast to some earlier studies, we found that corals present at the lower pH sites exhibited only minor physiological changes and no microbial pathogens were detected. Together, these results provide new insights into the impact of ocean acidification on the coral holobiont.     

An evolutionary fast‐track to biocalcification

The ability to construct mineralized shells, spicules, spines and skeletons is thought to be a key factor that fuelled the expansion of multicellular animal life during the early Cambrian. The genes and molecular mechanisms that control the process of biomineralization in disparate phyla are gradually being revealed, and it is broadly recognized that an insoluble matrix of proteins, carbohydrates and other organic molecules are required for the initiation, regulation and inhibition of crystal growth. Here, we show that Astrosclera willeyana, a living representative of the now largely extinct stromatoporid sponges (a polyphyletic grade of poriferan bauplan), has apparently bypassed the requirement to evolve many of these mineral‐regulating matrix proteins by using the degraded remains of bacteria to seed CaCO₃ crystal growth. Because stromatoporid sponges formed extensive reefs during the Paelozoic and Mesozoic eras (fulfilling the role that stony corals play in modern coral reefs), and fossil evidence suggests that the same process of bacterial skeleton formation occurred in these stromatoporid ancestors, we infer that some ancient reef ecosystems might have been founded on this microbial–metazoan relationship.     

Specific Expression of BMP2/4 Ortholog in Biomineralizing Tissues of Corals and Action on Mouse BMP Receptor

Bone morphogenetic proteins (BMPs) are members of the transforming growth factor β superfamily, and have been identified by their ability to induce bone formation in vertebrates. The biomineral-forming process, called biomineralization, is a widespread process, present in all kingdoms of living organisms and among which stony corals are one of the major groups of calcifying animals. Here, we report the presence of a BMP2/4 ortholog in eight species of adult corals. The synthesis of such a protein by the calcifying epithelium of corals suggests that coral BMP2/4 plays a role in skeletogenesis, making BMP the first common protein involved in biomineralization among Eumetazoans. In addition we show that recombinant coral BMP2/4 is able to inhibit human BMP2-induced osteoblastic differentiation in mesenchymal C2C12 cells. We suggest that this inhibition results from a competition between coral BMP2/4 and human BMP2, indicating conservation of binding affinity of BMP and its receptor during evolution from corals to vertebrates. Further studies are needed to understand interactions between coral BMP2/4 and its receptors, and, thus, the action of BMP2/4 in adult corals. Nucleotide sequence of the coral BMP2/4 genes cloned in this study is available in the GenBank under the accession number EU785981 (Stylophora pistillata) and EU785982 (Acropora sp.).     

Effects of variations in carbonate chemistry on the calcification rates of Madracis auretenra (= Madracis mirabilis sensu Wells, 1973): bicarbonate concentrations best predict calcification rates

Physiological data and models of coral calcification indicate that corals utilize a combination of seawater bicarbonate and (mainly) respiratory CO₂ for calcification, not seawater carbonate. However, a number of investigators are attributing observed negative effects of experimental seawater acidification by CO₂ or hydrochloric acid additions to a reduction in seawater carbonate ion concentration and thus aragonite saturation state. Thus, there is a discrepancy between the physiological and geochemical views of coral biomineralization. Furthermore, not all calcifying organisms respond negatively to decreased pH or saturation state. Together, these discrepancies suggest that other physiological mechanisms, such as a direct effect of reduced pH on calcium or bicarbonate ion transport and/or variable ability to regulate internal pH, are responsible for the variability in reported experimental effects of acidification on calcification. To distinguish the effects of pH, carbonate concentration and bicarbonate concentration on coral calcification, incubations were performed with the coral Madracis auretenra (= Madracis mirabilis sensu Wells, 1973) in modified seawater chemistries. Carbonate parameters were manipulated to isolate the effects of each parameter more effectively than in previous studies, with a total of six different chemistries. Among treatment differences were highly significant. The corals responded strongly to variation in bicarbonate concentration, but not consistently to carbonate concentration, aragonite saturation state or pH. Corals calcified at normal or elevated rates under low pH (7.6–7.8) when the seawater bicarbonate concentrations were above 1800 μm . Conversely, corals incubated at normal pH had low calcification rates if the bicarbonate concentration was lowered. These results demonstrate that coral responses to ocean acidification are more diverse than currently thought, and question the reliability of using carbonate concentration or aragonite saturation state as the sole predictor of the effects of ocean acidification on coral calcification.     

The Rugosa–Scleractinia gap re-examined through microstructural and biochemical evidence: A tribute to H.C. Wang

More than sixty years ago, H.C. Wang carried out an extensive study of skeletal microstructures of the Paleozoic corals and concluded that a “direct descent” may have existed between the two coral suborders: the Paleozoic Rugosa and the younger Scleractinia that had been established as distinct phyla by Haeckel (1896). Skeletal microstructures and three-dimensional reconstructions of walls and septa have revealed remarkable similarities between some Permian and Triassic corals, but it is only during the recent years that significant relationships were established between the structural properties of coral skeletons and their control by the biological process. Supported by recent genetic studies of calcareous biomineralization among various invertebrate phyla, the Wang's opinion now appears a reasonable working hypothesis.     

Aragonite precipitation by "proto-polyps" in coral cell cultures

The mechanisms of coral calcification at the molecular, cellular and tissue levels are poorly understood. In this study, we examine calcium carbonate precipitation using novel coral tissue cultures that aggregate to form "proto-polyps". Our goal is to establish an experimental system in which calcification is facilitated at the cellular level, while simultaneously allowing in vitro manipulations of the calcifying fluid. This novel coral culturing technique enables us to study the mechanisms of biomineralization and their implications for geochemical proxies. Viable cell cultures of the hermatypic, zooxanthellate coral, Stylophora pistillata, have been maintained for 6 to 8 weeks. Using an enriched seawater medium with aragonite saturation state similar to open ocean surface waters (Ω(arag)~4), the primary cell cultures assemble into "proto-polyps" which form an extracellular organic matrix (ECM) and precipitate aragonite crystals. These extracellular aragonite crystals, about 10 μm in length, are formed on the external face of the proto-polyps and are identified by their distinctive elongated crystallography and X-ray diffraction pattern. The precipitation of aragonite is independent of photosynthesis by the zooxanthellae, and does not occur in control experiments lacking coral cells or when the coral cells are poisoned with sodium azide. Our results demonstrate that proto-polyps, aggregated from primary coral tissue culture, function (from a biomineralization perspective) similarly to whole corals. This approach provides a novel tool for investigating the biophysical mechanism of calcification in these organisms.     

On the celestite-secreting Acantharia and their effect on seawater strontium to calcium ratios

Significant variations in the Sr/Ca in waters from the eastern Indian Ocean in the vicinity of Australia, both from the surface and from shallow depth profiles, are documented. The strontium sulfate-secreting protozoans Acantharia, which are common in the upper 400 m of the oceans, especially at low latitudes, contribute substantially to changes in the Sr/Ca of oceanic waters by extracting Sr for the formation of their skeletons. Below such depths, these organisms dissolve and the Sr/Ca of seawater regains its ‘conservative’ nature. This mechanism accounts for some of the variability of the Sr/Ca near the surface of the ocean, but this may still be found at depth as surface waters become entrained at greater depths. It is argued here that the noted Sr/Ca variations may explain discrepancies between coral data sets from different parts of the oceans, and calls for caution when reconstructing sea-surface temperatures from the Sr/Ca of corals.