Biologically controlled mineralization in the hypercalcified sponge Petrobiona massiliana (Calcarea, Calcaronea)

Hypercalcified sponges, endowed with a calcium carbonate basal skeleton in addition to their spicules, form one of the most basal metazoan group engaged in extensive biomineralization. The Mediterranean species Petrobiona massiliana was used to investigate biological controls exerted on the biomineralization of its basal skeleton. Scanning and transmission electron microscopy (SEM, TEM) confirmed that basopinacocytes form a discontinuous layer of flattened cells covering the skeleton and display ultrastructural features attesting intense secretory activity. The production of a highly structured fibrillar organic matrix framework by basopinacocytes toward the growing skeleton was highlighted both by potassium pyroantimonate and ruthenium red protocols, the latter further suggesting the presence of sulfated glycosaminoglycans in the matrix. Furthermore organic material incorporated into the basal skeleton was shown by SEM and TEM at different structural levels while its response to alcian blue and acridine orange staining might suggest a similar acidic and sulfated chemical composition in light microscopy. Potassium pyroantimonate revealed in TEM and energy electron loss spectroscopy (EELS) analysis, heavy linear precipitates 100-300 nm wide containing Ca(2+) and Mg(2+) ions, either along the basal cell membrane of basopinacocytes located toward the decalcified basal skeleton or around decalcified spicules in the mesohyl. Based on the results of the previous mineralogical characterization and the present work, an hypothetical model of biomineralization is proposed for P. massiliana: basopinacocytes would produce an extracellular organic framework that might guide the assemblage of submicronic amorphous Ca- and Mg-bearing grains into higher structural units.     

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.     

Biomineralization in newly settled recruits of the scleractinian coral Pocillopora damicornis

Calcium carbonate biomineralization of scleractinian coral recruits is fundamental to the construction of reefs and their survival under stress from global and local environmental change. Establishing a baseline for how normal, healthy coral recruits initiate skeletal formation is, therefore, warranted. Here, we present a thorough, multiscale, microscopic and spectroscopic investigation of skeletal elements deposited by Pocillopora damicornis recruits, from 12 h to 22 days after settlement in aquarium on a flat substrate. Six growth stages are defined, primarily based on appearance and morphology of successively deposited skeletal structures, with the following average formation time‐scales: A (<24 h), B (24–36 h), C (36–48 h), D (48–72 h), E (72–96 h), and F (>10 days). Raman and energy dispersive X‐ray spectroscopy indicate the presence of calcite among the earliest components of the basal plate, which consist of micrometer‐sized, rod‐shaped crystals with rhomboidal habit. All later CaCO₃ skeletal structures are composed exclusively of aragonite. High‐resolution scanning electron microscopy reveals that, externally, all CaCO₃ deposits consist of <100 nm granular units. Fusiform, dumbbell‐like, and semispherulitic structures, 25–35 µm in longest dimension, occur only during the earliest stages (Stages A–C), with morphologies similar to structures formed abiotically or induced by organics in in vitro carbonate crystallization experiments. All other skeletal structures of the basal plate are composed of vertically extending lamellar bundles of granules. From Stage D, straight fibrils, 40–45 nm in width and presumably of organic composition, form bridges between these aragonitic bundles emerging from the growing front of fusing skeletal structures. Our results show a clear evolution in the coral polyp biomineralization process as the carbonate structures develop toward those characterizing the adult skeleton. J. Morphol. 275:1349–1365, 2014. © 2014 Wiley Periodicals, Inc.     

How corals made rocks through the ages

Hard, or stony, corals make rocks that can, on geological time scales, lead to the formation of massive reefs in shallow tropical and subtropical seas. In both historical and contemporary oceans, reef‐building corals retain information about the marine environment in their skeletons, which is an organic–inorganic composite material. The elemental and isotopic composition of their skeletons is frequently used to reconstruct the environmental history of Earth's oceans over time, including temperature, pH, and salinity. Interpretation of this information requires knowledge of how the organisms formed their skeletons. The basic mechanism of formation of calcium carbonate skeleton in stony corals has been studied for decades. While some researchers consider coral skeletons as mainly passive recorders of ocean conditions, it has become increasingly clear that biological processes play key roles in the biomineralization mechanism. Understanding the role of the animal in living stony coral biomineralization and how it evolved has profound implications for interpreting environmental signatures in fossil corals to understand past ocean conditions. Here we review historical hypotheses and discuss the present understanding of how corals evolved and how their skeletons changed over geological time. We specifically explain how biological processes, particularly those occurring at the subcellular level, critically control the formation of calcium carbonate structures. We examine the different models that address the current debate including the tissue–skeleton interface, skeletal organic matrix, and biomineralization pathways. Finally, we consider how understanding the biological control of coral biomineralization is critical to informing future models of coral vulnerability to inevitable global change, particularly increasing ocean acidification.     

Amorphous calcium carbonate in the shells of adult Unionoida

Shells of adult individuals from two different bivalve families, Hyriopsis cumingii and Diplodon chilensis patagonicus, were studied by Micro-Raman spectroscopy and Focussed Ion Beam-assisted TEM. The shells contain amorphous calcium carbonate in a zone at the interface between the periostracum and the prismatic layer. In this area, the initial prism structures protrude from the inner periostracum layer and it is demonstrated that these structures systematically consist of highly disordered and amorphous calcium carbonate. Within this zone, ordered and disordered areas are intermingled discounting the existence of a crystallization front and favouring models of domainal crystallization processes via so-called mesocrystals. These observations are the first documentation of the use of amorphous calcium carbonate as a precursor phase by adult mollusc species and lend further support to hypotheses postulating widespread use of amorphous phases as building material of skeletal tissue in biology.     

Skeletal development inAcropora cervicornis

Scanning electron microscopy, field studies using dyes which become incorporated into the skeleton of living corals as time markers, and petrographic and mineralogic techniques were used to describe the diel pattern of calcium carbonate accretion in the extending axial corallite ofAcropora cervicornis. The axial corallite extends by the formation of randomly oriented fusiform crystals at the distal tip of the branch. Morphological and mineralogical characteristics suggest that these might be calcite crystals. They form a framework upon which needle-like aragonite crystals (initially small tufts) begin to grow. As the needles elongate, groups of them form well defined bundles, fasciculi, which compose the primary skeletal elements. There is a diel pattern in the deposition of the skeleton. At night (1800–0600 hours) the distal spines are pointed and composed primarily of fusiform crystals. During the day (0600–1800 hours) mineral accretion occurs on all surfaces of the skeleton, apparently by epitaxial growth on the aragonite needles of the fasciculi.     

Developmental biology meets materials science: Morphogenesis of biomineralized structures

Biomineralization is the process by which metazoa form hard minerals for support, defense, and feeding. The minerals so formed, e.g., teeth, bones, shells, carapaces, and spicules, are of considerable interest to chemists and materials scientists. The cell biology underlying biomineralization is not well understood. The study of the formation of mineralized structures in developing organisms offers opportunities for understanding some intriguing aspects of cell and developmental biology. Five examples of biomineralization are presented: (1) the formation of siliceous spicules and frustules in sponges and diatoms, respectively; (2) the structure of skeletal spicules composed of amorphous calcium carbonate in some tunicates; (3) the secretion of the prism and nacre of some molluscan shells; (4) the development of skeletal spicules of sea urchin embryos; and (5) the formation of enamel of vertebrate teeth. Some speculations on the cellular and molecular mechanisms that support biomineralization, and their evolutionary origins, are discussed.     

Biomineralization of the spicules of sea urchin embryos

The formation of calcareous skeletal elements by various echinoderms, especially sea urchins, offers a splendid opportunity to learn more about some processes involved in the formation of biominerals. The spicules of larvae of euechinoids have been the focus of considerable work, including their developmental origins. The spicules are composed of a single optical crystal of high magnesium calcite and variable amounts of amorphous calcium carbonate. Occluded within the spicule is a proteinaceous matrix, most of which is soluble; this matrix constitutes about 0.1% of the mass. The spicules are also enclosed by an extracellular matrix and are almost completely surrounded by cytoplasmic cords. The spicules are deposited by primary mesenchyme cells (PMCs), which accumulate calcium and secrete calcium carbonate. A number of proteins specific, or highly enriched, in PMCs, have been cloned and studied. Recent work supports the hypothesis that proteins found in the extracellular matrix of the spicule are important for biomineralization.     

Amorphous Calcium Carbonate Precipitation by Cellular Biomineralization in Mantle Cell Cultures of Pinctada fucata

The growth of molluscan shell crystals is generally thought to be initiated from the extrapallial fluid by matrix proteins, however, the cellular mechanisms of shell formation pathway remain unknown. Here, we first report amorphous calcium carbonate (ACC) precipitation by cellular biomineralization in primary mantle cell cultures of Pinctada fucata. Through real-time PCR and western blot analyses, we demonstrate that mantle cells retain the ability to synthesize and secrete ACCBP, Pif80 and nacrein in vitro. In addition, the cells also maintained high levels of alkaline phosphatase and carbonic anhydrase activity, enzymes responsible for shell formation. On the basis of polarized light microscopy and scanning electron microscopy, we observed intracellular crystals production by mantle cells in vitro. Fourier transform infrared spectroscopy and X-ray diffraction analyses revealed the crystals to be ACC, and de novo biomineralization was confirmed by following the incorporation of Sr into calcium carbonate. Our results demonstrate the ability of mantle cells to perform fundamental biomineralization processes via amorphous calcium carbonate, and these cells may be directly involved in pearl oyster shell formation.     

Soluble proteins control growth of skeleton crystals in three coralline demosponges

Summary Calcified demosponges (coralline sponges, sclero-sponges), the first metazoa producing a carbonate skeleton, used to be important reef building organisms in the past. The relatives of this group investigated here,Spirastrella (Acanthochaetetes) wellsi, Astrosclera willeyana andVaceletia cf.crypta, are restricted to cryptic niches of modern Pacific coral reefs and may be considered as “living fossils’. They are characterized by a basic biologically controlled metazoan biomineralization process. Each of the investigated taxa forms its calcareous basal skeleton in a highly specialized way. Moreover, each taxon secretes distinct Ca²⁺-binding macromolecules which were entrapped within the calcium carbonate crystals during skeleton formation. Therefore these Ca²⁺-binding macromolecules were also described as intracrystalline macromolecules. When isolated and separated by SDS polyacrylamide gel electrophoresis, the organic skeleton matrix of the three species revealed to be composed of a respective distinct array of EDTA-soluble proteins. A single protein of 41 kDa was detected inS. wellsi, two proteins of 38 and 120 kDa inA. willeyana, and four proteins of 18 kDa, 30 kDa, 33 kDa, and 37 kDa inVaceletia sp. When run on IEF gel, the Ca²⁺-binding proteins gave staining bands at pH values between 5.25 and 5.65. As proved by anin vitro mineralization assay, the extracted proteins effectively inhibit CaCO₃ and SrCO₃ precipitation, respectively, in a saturated solution. Biochemical properties and behavior of the extracted proteins strongly suggest that they are involved in crystal nucleation and skeleton carbonate formation within the calcified sponges studied here.     

厚壳贻贝一种新型贝壳胶原蛋白的重组表达与功能分析

贝壳是一种具有优异力学性能的生物硬组织,贝壳基质蛋白质对贝壳的形成具有重要意义。厚壳贻贝(Mytilus coruscus)贝壳中发现一种类似胶原蛋白质的新型贝壳基质蛋白质,命名为collagen-like protein 2（CLP-2）。然而,该蛋白质的结构与功能以及对贝壳形成的影响机制尚不清楚。为此,本研究对CLP 2开展了序列分析;进一步采取密码子优化结合原核重组表达策略,开展了CLP-2的重组表达;在此基础上分析了重组CLP-2对酸钙结晶的诱导、结晶速率抑制以及碳酸钙结合能力。对CLP-2的序列分析结果表明,该蛋白质序列中含有信号肽及两个Von Willebrand factor A（VWA）结构域。CLP-2在数据库中尚无高同源性蛋白质存在,表明这是一种较为新颖的贝壳基质蛋白。所获得的重组CLP-2对碳酸钙体外结晶表现出明显的诱导作用,扫描电镜以及傅里叶红外光谱结果表明,重组CLP-2可诱导碳酸钙晶体的形貌由立方体形转化为球形,并在高浓度下进一步转化为哑铃形;同时,重组CLP-2可促使碳酸钙晶体的晶型由方解石型向文石型转化;重组CLP-2在体外具有碳酸钙晶体结合作用;此外,重组CLP-2能显著抑制碳酸钙晶体的结晶速度（P<0.01）,并具有浓度依赖性。上述结果表明,厚壳贻贝贝壳CLP-2蛋白质在贝壳,特别是文石型肌棱柱层的生物矿化过程中具有重要作用。上述研究为深入了解贻贝贝壳的形成机制,以及胶原类蛋白质对生物矿化过程的影响奠定了基础。     

Biomineralizations: insights and prospects from crustaceans

For growing, crustaceans have to molt cyclically because of the presence of a rigid exoskeleton. Most of the crustaceans harden their cuticle not only by sclerotization, like all the arthropods, but also by calcification. All the physiology of crustaceans, including the calcification process, is then linked to molting cycles. This means for these animals to find regularly a source of calcium ions quickly available just after ecdysis. The sources of calcium used are diverse, ranging from the environment where the animals live to endogenous calcium deposits cyclically elaborated by some of them. As a result, crustaceans are submitted to an important and energetically demanding calcium turnover throughout their life. The mineralization process occurs by precipitation of calcium carbonate within an organic matrix network of chitin-proteins fibers. Both crystalline and stabilized amorphous polymorphs of calcium carbonate are found in crustacean biominerals. Furthermore, Crustacea is the only phylum of animals able to elaborate and resorb periodically calcified structures. Notably for these two previous reasons, crustaceans are more and more extensively studied and considered as models of choice in the biomineralization research area.     

THE EVOLUTION OF BONE

Vertebrates are practically unique among the Metazoa in their possession of a skeleton made from calcium phosphate rather than calcium carbonate. Interpretation of the origin of a phosphatic skeleton in early vertebrates has previously centered primarily on systemic requirements for phosphate and/or calcium storage or excretion. These interpretations afford no anatomical or physiological advantage(s) that would not have been equally valuable to many invertebrates. We suggest the calcium phosphate skeleton is distinctly advantageous to vertebrates because of their relatively unusual and ancient pattern of activity metabolism: intense bursts of activity supported primarily by rapid intramuscular formation of lactic acid. Bursts of intense activity by vertebrates are followed by often protracted periods of marked systemic acidosis. This postactive acidosis apparently generates slight skeletal dissolution, associated with simultaneous vascular hypercalcemia. A variety of apparently unrelated histological features of the skeleton in a number of vertebrates may minimize this postactive hypercalcemia. We present new data that suggest that postactive skeletal dissolution would be significantly exacerbated if bone were composed of calcium carbonate rather than calcium phosphate. The former is far less stable both in vivo and in vitro than is calcium hydroxyapatite, under both resting and postactive physiological conditions.     

Intracellular amorphous Ca-carbonate and magnetite biomineralization by a magnetotactic bacterium affiliated to the Alphaproteobacteria

Bacteria synthesize a wide range of intracellular submicrometer-sized inorganic precipitates of diverse chemical compositions and structures, called biominerals. Their occurrences, functions and ultrastructures are not yet fully described despite great advances in our knowledge of microbial diversity. Here, we report bacteria inhabiting the sediments and water column of the permanently stratified ferruginous Lake Pavin, that have the peculiarity to biomineralize both intracellular magnetic particles and calcium carbonate granules. Based on an ultrastructural characterization using transmission electron microscopy (TEM) and synchrotron-based scanning transmission X-ray microscopy (STXM), we showed that the calcium carbonate granules are amorphous and contained within membrane-delimited vesicles. Single-cell sorting, correlative fluorescent in situ hybridization (FISH), scanning electron microscopy (SEM) and molecular typing of populations inhabiting sediments affiliated these bacteria to a new genus of the Alphaproteobacteria. The partially assembled genome sequence of a representative isolate revealed an atypical structure of the magnetosome gene cluster while geochemical analyses indicate that calcium carbonate production is an active process that costs energy to the cell to maintain an environment suitable for their formation. This discovery further expands the diversity of organisms capable of intracellular Ca-carbonate biomineralization. If the role of such biomineralization is still unclear, cell behaviour suggests that it may participate to cell motility in aquatic habitats as magnetite biomineralization does.Subject terms: Phylogenetics, Biodiversity, Biogeochemistry, Water microbiology     

Micropearls and other intracellular inclusions of amorphous calcium carbonate: an unsuspected biomineralization capacity shared by diverse microorganisms

An unsuspected biomineralization process, which produces intracellular inclusions of amorphous calcium carbonate (ACC), was recently discovered in unicellular eukaryotes. These mineral inclusions, called micropearls, can be highly enriched with other alkaline-earth metals (AEM) such as Sr and Ba. Similar intracellular inclusions of ACC have also been observed in prokaryotic organisms. These comparable biomineralization processes involving phylogenetically distant microorganisms are not entirely understood yet. This review gives a broad vision of the topic in order to establish a basis for discussion on the possible molecular processes behind the formation of the inclusions, their physiological role, the impact of these microorganisms on the geochemical cycles of AEM and their evolutionary relationship. Finally, some insights are provided to guide future research.     

The Roles of Mg over the Precipitation of Carbonate and Morphological Formation in the Presence of Arthrobacter sp. Strain MF-2

Microbial mineralization of carbonate is a research subject widely studied in the past decades. The magnesium ions (Mg²⁺), present in water systems, are a key determinant in biomineralization process of carbonate and they are widely found in calcium-based biominerals as an accessory component. However, the crystallization mechanism and morphological change of carbonate polymorphs in the presence of Mg²⁺ ions has not been clarified sufficiently. In this report, a series of culture experiments were performed for 50 days using the Arthrobacter sp. strain MF-2 in a M2 culture medium using Mg/Ca molar ratios (R) of 0, 1.5, 3, 6, 9, and 12 in solution. And the roles of Mg²⁺ ions on the crystal growth and morphological change of biogenic carbonate were investigated. Experimental results show: (1) MF-2 could induce aragonite, high-Mg calcite, and Ca-dolomite formation in M2 culture media with different R values. The increased stability of amorphous calcium carbonate suggests Mg²⁺ ions inhibit carbonate crystallization. (2) The mineral morphologies were varied (rod-shaped, dumbbell-shaped, cauliflower-like, spherical, etc.) in the medium with R = 1.5, but they became simple (spherical and lamellar) in high Mg²⁺ concentrations (Mg > 0.15 M, R > 3). (3) The increased ionic strength of Mg²⁺ ions in the environment has an influence on the polymorphs and morphologies of carbonate formed by controlling the metabolism of strain MF-2 and the activity of carbonic anhydrase.     

A unique skeletal microstructure of the deep‐sea micrabaciid scleractinian corals

Micrabaciids are solitary, exclusively azooxanthellate deep‐sea corals belonging to one of the deepest‐living (up to 5,000 m) scleractinian representatives. All modern micrabaciid taxa (genera: Letepsammia, Rhombopsammia, Stephanophyllia, Leptopenus) have a porous and often very fragile skeleton consisting of two main microstructural components known also from other scleractinians: rapid accretion deposits and thickening deposits. However, at the microstructural level, the skeletal organization of the micrabaciids is distinctly different from that of other scleractinians. Rapid accretion deposits consist of alternations of superimposed “microcrystalline” (micrometer‐sized aggregates of nodular nanodomains) and fibrous zones. In contrast to all shallow‐water and sympatric deep‐water corals so far described, the thickening deposits of micrabaciids are composed of irregular meshwork of short (1–2 μm) and extremely thin (ca. 100–300 nm) fibers organized into small, chip‐like bundles (ca. 1–2 μm thick). Longer axes of fiber bundles are usually subparallel to the skeletal surfaces and oriented variably in this plane. The unique microstructural organization of the micrabaciid skeleton is consistent with their monophyletic status based on macromorphological and molecular data, and points to a diversity of organic matrix‐mediated biomineralization strategies in Scleractinia. J. Morphol.,2011. © 2010 Wiley‐Liss, Inc.