Unusual micrometric calcite-aragonite interface in the abalone shell Haliotis (Mollusca, Gastropoda).

Species of Haliotis (abalone) show high variety in structure and mineralogy of the shell. One of the European species (Haliotis tuberculata) in particular has an unusual shell structure in which calcite and aragonite coexist at a microscale with small patches of aragonite embedded in larger calcitic zones. A detailed examination of the boundary between calcite and aragonite using analytical microscopies shows that the organic contents of calcite and aragonite differ. Moreover, changes in the chemical composition of the two minerals seem to be gradual and define a micrometric zone of transition between the two main layers. A similar transition zone has been observed between the layers in more classical and regularly structured mollusk shells. The imbrication of microscopic patches of aragonite within a calcitic zone suggests the occurrence of very fast physiological changes in these taxa.

Control of aragonite deposition in colonial corals by intra-skeletal macromolecules.

Scleractinian coral skeletons are composed mainly of aragonite in which a small percentage of organic matrix (OM) molecules is entrapped. It is well known that in corals the mineral deposition occurs in a biological confined nucleation site, but it is still unclear to what extent the calcification is controlled by OM molecules. Hence, the shape, size and organization of skeletal crystals from the fiber level through the colony architecture, were also attributed to factors as diverse as nucleation site mineral supersaturation and environmental factors in the habitat. In this work the OMs were extracted from the skeleton of three colonial corals, Acropora digitifera, Lophelia pertusa and Montipora caliculata. A. digitifera has a higher calcification rate than the other two species. OM molecules were characterized and their CaCO3 mineralization activity was evaluated by experiments of overgrowth on coral skeletons and of precipitation from solutions containing OM soluble and insoluble fractions and magnesium ions. The precipitates were characterized by spectroscopic and microscopic techniques. The results showed that the OM molecules of the three coral share similar features, but differ from those associated with mollusk shells. However, A. digitifera OM shows peculiarities from those from L. pertusa and M. caliculata. The CaCO3 overgrowth and precipitation experiments confirm the singularity of A. digitifera OM molecules as mineralizers. Moreover, their comparison indicates that only specific molecules are involved in the polymorphism control and suggests that when the whole extracted materials are used the OM's main effect is on the control of particles' shape and morphology.

Synchrotron-based photoelectron spectroscopy provides evidence for a molecular bond between calcium and mineralizing organic phases in invertebrate calcareous skeletons.

Organic compounds have been extracted from calcium carbonate skeletons produced by three invertebrate species belonging to distinct phyla. The soluble parts of these skeleton matrices were isolated and analysed by synchrotron-based X-ray spectroscopy (XPS). The presence of calcium associated with these organic materials was revealed in every sample studied, with important variations in Ca 2p binding energy from species to species. Measured Ca 2p binding energy values are more related to compositional diversity of the mineralizing matrices of the skeletons, whose taxonomic dependence has long been established, than to the Ca carbonate polymorph selected to build the skeletal units. This suggests a physical bond between species-specific mineralizing organic assemblages and the associated calcium. Remarkably, the binding energy of 2p electrons in calcium associated with mineralizing matrices is consistently higher than Ca 2p values obtained in purely mineral carbonate (both calcite and aragonite). The ability both to identify and measure the effect of organic matrices on their mineral counterpart in calcareous biominerals opens a new perspective for a functional approach to the biomineralization process.

Prism substructures in the shell of Pinna nobilis (Linnaeus, 1758), Mollusca - Evidence for a three-dimensional pulsed-growth model.

In the shells of the Pelecypods belonging to the Pinnidae family, the calcareous prismatic units of the outer layer are long-standing references for biomineralization studies. To elucidate how the mechanism of prism formation enables both shell elongation and thickness increase, a top-down structural analysis of these classical "simple prisms" has been carried out, taking advantage of shell sampling on actively mineralizing animals. Particular attention was paid to the morphological and structural patterns of the calcareous units sequentially produced at the margins of the growth lamellae. This pre-prismatic part of the shell allows for studying the mineralizing stages not taken into account in prism reconstructions based on samples taken from older areas of the shell. Examination of the microstructural sequence shows that within the actively mineralizing area of the shell, a step-by-step structuring process is continuously running, providing a renewed view of prism formation as it makes obvious the progressive occurrence of their specific patterns. Given the critically endangered status of the species, a better knowledge of the mineralization process associated to shell growth may become handy for future studies aimed at understanding the health status of individuals based on their shell records.

Structure and composition of the nacre-prisms transition in the shell of Pinctada margaritifera (Mollusca, Bivalvia).

A microstructural, mineralogical, and chemical study of the nacre-prisms boundary in the shells of Pinctada margaritifera shows that this boundary is not an abrupt transition, but that there exists a distinct fibrous layer with clear topographic structures and evidence of growth lines. A three-step biomineralization process is proposed that involves changes in the chemical and biochemical composition of the last growth increments of the calcite prisms, formation of the fibrous layer, and development of regular tablets in the nacreous layer.

Soluble organic matrices of aragonitic skeletons of Merulinidae (Cnidaria, Anthozoa).

Our interpretation of the overall taxonomy and evolution of the Scleractinia, the most important reef builders in tropical areas, has long depended exclusively on morphology of the calcareous skeletons. The reported series of physical and biochemical characterizations of skeletons and the mineralizing matrices extracted from the skeletons allow, for the first time, the level of biochemical diversity among corallites of the same family to be estimated. Similarities and differences observed in the micro- and nanostructures of the skeletons reflect those of the soluble organic matrices. Sulphur is mainly associated with sulphated acidic sugars. The role of sulphated sugars on the biomineralization processes is still underestimated. The resulting data suggest that environmental conditions may act on the mineralization process through the detailed compositions of the mineralizing matrices.

Isotropic microscale mechanical properties of coral skeletons.

Scleractinian corals are a major source of biogenic calcium carbonate, yet the relationship between their skeletal microstructure and mechanical properties has been scarcely studied. In this work, the skeletons of two coral species:solitary Balanophyllia europaea and colonial Stylophora pistillata, were investigated by nanoindentation. The hardness HIT and Young's modulus E(IT) were determined from the analysis of several load-depth data on two perpendicular sections of the skeletons: longitudinal (parallel to the main growth axis) and transverse. Within the experimental and statistical uncertainty,the average values of the mechanical parameters are independent on the section's orientation. The hydration state of the skeletons did not affect the mechanical properties. The measured values, EIT in the 76-77 GPa range, and H(IT) in the 4.9­5.1 GPa range, are close to the ones expected for polycrystalline pure aragonite. Notably, a small difference in H(IT) is observed between the species. Different from corals, single-crystal aragonite and the nacreous layer of the seashell Atrina rigida exhibit clearly orientation-dependent mechanical properties. The homogeneous and isotropic mechanical behaviour of the coral skeletons at the microscale is correlated with the microstructure,observed by electron microscopy and atomic force microscopy, and with the X-ray diffraction patterns of the longitudinal and transverse sections.

Gains and losses of coral skeletal porosity changes with ocean acidification acclimation.

Ocean acidification is predicted to impact ecosystems reliant on calcifying organisms, potentially reducing the socioeconomic benefits these habitats provide. Here we investigate the acclimation potential of stony corals living along a pH gradient caused by a Mediterranean CO2 vent that serves as a natural long-term experimental setting. We show that in response to reduced skeletal mineralization at lower pH, corals increase their skeletal macroporosity (features >10 µm) in order to maintain constant linear extension rate, an important criterion for reproductive output. At the nanoscale, the coral skeleton's structural features are not altered. However, higher skeletal porosity, and reduced bulk density and stiffness may contribute to reduce population density and increase damage susceptibility under low pH conditions. Based on these observations, the almost universally employed measure of coral biomineralization, the rate of linear extension, might not be a reliable metric for assessing coral health and resilience in a warming and acidifying ocean.

Suppression of skeletal growth in scleractinian corals by decreasing ambient carbonate-ion concentration: a cross-family comparison.

Biogenic calcification is influenced by the concentration of available carbonate ions. The recent confirmation of this for hermatypic corals has raised concern over the future of coral reefs because [CO(3)(2-)] is a decreasing function of increasing pCO(2) in the atmosphere. As one of the overriding features of coral reefs is their diversity, understanding the degree of variability between species in their ability to cope with a change in [CO(3)(2-)] is a priority. We cultured four phylogenetically and physiologically different species of hermatypic coral (Acropora verweyi, Galaxea fascicularis, Pavona cactus and Turbinaria reniformis) under 'normal' (280 micromol kg(-1)) and 'low' (140 micromol kg(-1)) carbonate-ion concentrations. The effect on skeletogenesis was investigated quantitatively (by calcification rate) and qualitatively (by microstructural appearance of growing crystalline fibres using scanning electron microscopy (SEM)). The 'low carbonate' treatment resulted in a significant suppression of calcification rate and a tendency for weaker crystallization at the distal tips of fibres. However, while the calcification rate was affected uniformly across species (13-18% reduction), the magnitude of the microstructural response was highly species specific: crystallization was most markedly affected in A. verweyi and least in T. reniformis. These results are discussed in relation to past records and future predictions of carbonate variability in the oceans.

In situ distribution and characterization of the organic content of the oyster shell Crassostrea gigas (Mollusca, Bivalvia).

Cultivation of commercial oysters is now facing the possible influence of global change in sea water composition, commonly referred to as "ocean acidification". In order to test the potential consequence of the predicted environmental changes, a cultivation experiment was carried out. The left and right valves of the oyster shell Crassostrea gigas differ in their structure; moreover, lenses of non compact layers are irregular. The shell layers of juvenile C. gigas are studied using a variety of highly spatially resolved techniques to establish their composition and structure. Our results confirm the presence of three different calcitic structural types. The role of the lenses of chalky layers is not yet deciplered. Despite a common mineralogy, the elemental composition of the layers differs. The sulphur aminoacids and sulphated polysaccharide contents of the intracrystalline and intercrystalline matrices differ, as well as those of the structural types. The possible different sensitivity of these structures to environmental changes is still unknown.

High resolution electron backscatter diffraction (EBSD) data from calcite biominerals in recent gastropod shells.

Electron backscatter diffraction (EBSD) is a microscopy technique that reveals in situ crystallographic information. Currently, it is widely used for the characterization of geological materials and in studies of biomineralization. Here, we analyze high resolution EBSD data from biogenic calcite in two mollusk taxa, Concholepas and Haliotis, previously used in the understanding of complex biomineralization and paleoenvironmental studies. Results indicate that Concholepas has less ordered prisms than in Haliotis, and that in Concholepas the level of order is not homogenous in different areas of the shell. Overall, the usefulness of data integration obtained from diffraction intensity and crystallographic orientation maps, and corresponding pole figures, is discussed as well as its application to similar studies.

Shell layers of the black-lip pearl oyster Pinctada margaritifera: matching microstructure and composition.

The nacre-prism transition of the mollusc shell Pinctada margaritifera was studied using scanning electron microscopy, electron probe micro-analysis (EPMA) and time-of-flight secondary ion mass spectrometry (TOF-SIMS). Mineralogical change is correlated with a change in organic matrix. Previous analyses had shown that sugars were involved in the transition layer (fibrous aragonite). The new Energy Dispersive Spectrometry (EDS) and TOF-SIMS maps show that the modifications at the layer boundary are complex, and that proteins and lipids are also involved. Detailed TOF-SIMS maps show that the thick organic envelopes surrounding the prisms, and between the prisms and the fibrous aragonitic layer, are not composed by regular layers, but are a patchwork of various molecules. The amino acid compositions of the nacreous and prismatic layer are compared thanks to the TOF-SIMS localized analyses.

Occurrence and diversity of lipids in modern coral skeletons.

Coral skeletons are composite acellular structures, in which organic macromolecules are intimately associated with mineral phases. Previous studies focussed on proteins and sugars of the soluble organic matrices extracted from the skeletons. Here we report the occurrence of diverse lipids which were extracted from the aragonitic skeletons of seven modern coral species. Using thin layer chromatography, we show that these lipids differ in quantity and composition between the species. Higher proportions of sterols and sterol esters in skeleton extracts as compared to a much higher abundance of waxes and triglycerides in previously studied extracts from scleractinian soft tissues suggest a specific, although not yet determined, role in biomineralization. The occurrence of intraskeletal lipids along with other organic components should also be taken into account when using coral skeletons as bone allografts, as well as in fossilization processes.

Structural, mineralogical, and biochemical diversity in the lower part of the pearl layer of cultivated seawater pearls from Polynesia.

A series of Polynesian pearls has been investigated with particular attention to the structural and compositional patterns of the early developmental stages of the pearl layer. These initial steps in pearl formation bear witness of the metabolic changes that have occurred during the pearl-sac formation. The resulting structurally and biochemically complex structures have been investigated using a variety of techniques that provide us with information concerning both mineral phases and the organic components. Results are discussed with respect to our understanding of the biomineralization mechanisms, as well as for the grafting process.

Subdaily growth patterns and organo-mineral nanostructure of the growth layers in the calcitic prisms of the shell of Concholepas concholepas Bruguière, 1789 (Gastropoda, Muricidae).

Fluorochrome marking of the gastropod Concholepas concholepas has shown that the prismatic units of the shell are built by superimposition of isochronic growth layers of about 2 mum. Fluorescent growth marks make it possible to establish the high periodicity of the cyclic biomineralization process at a standard growth rhythm of about 45 layers a day. Sulphated polysaccharides have been identified within the growth layers by using synchrotron radiation, whereas high resolution mapping enables the banding pattern of the mineral phase to be correlated with the layered distribution of polysaccharides. Atomic force microscopy has shown that the layers are made of nanograins densely packed in an organic component.

Microstructure and chemical composition of giant avian eggshells.

The microstructure and composition of the layers of two giant avian eggshells were investigated using a combination of scanning electron microscopy, electron probe microanalyses, and X-ray absorption near-edge structure spectroscopy (XANES). The two species have some similarities and differences in their microstructure and composition; the composition is not homogeneous throughout the eggshell thickness. XANES studies show that sulfur is associated with amino acids in the inner organic membranes, whereas in the mineralised layers the sulfur is mainly associated with sulfated polysaccharides. These results are similar to those obtained on chicken eggshells, and confirm the active role of sulfated acidic polysaccharides in biomineralisation processes of carbonate skeletons.

The two-step mode of growth in the scleractinian coral skeletons from the micrometre to the overall scale.

It has been known since the 19th century that coral skeletons are built of aragonite crystals with taxonomy-linked arrangements, but the way by which each coral species controls this crystallization process remains an unsolved question. The problem became still more intriguing when it was shown that isotopic compositions of coral aragonite were subject to taxonomy-linked influences (the "vital effect"). On the other hand, presence of an organic component in coral skeletons is also long known, but localization of these compounds is admittedly restricted to particular structures called "centres of calcifications." Fibres, the largely predominant part of the coral skeletons, are usually considered as purely mineral units. In this paper, it is shown that in both "centres of calcification" and fibres, organic compounds are associated with the mineral material at a deep structural level. A series of variously scaled observations and localized measurements allow recognition of the presence of an organic component at the nanometre scale. Far from being a freely operating process, crystallization of coral fibres is thus permanently controlled by the polyp basal ectoderm through a cyclic two-step process acting at the micrometre-scale. The biomineralization cycle begins by secretion of a proteoglycan matrix. As the composition of these sugars-proteins assemblages has been shown taxonomy dependent, the hypothesis can be made that multiple and long recognized specificities of coral skeletons are linked to this biochemically driven crystallization process. Additionally, this new concept of the biomineralization process in coral skeletons provides us with an access to the long term evolution of the Scleractinia. Remarkably, results of a skeleton-based approach using microstructural criteria (i.e., the spatial relationships of "centres of calcification" and the three-dimensional arrangements of fibres), are consistent with a molecular phylogenetic analysis carried out on the same species. Clearly, at the overall ontogenic level, the two-step growth mode of coral skeletons is also a valuable tool to reconstruct the evolutionary history of Scleractinia.

Stable isotopes (delta13C and delta15N) of organic matrix from coral skeleton.

The evolutionary success of reef-building corals in nutrient-poor tropical waters is attributed to endosymbiotic dinoflagellates. The algae release photosynthetic products to the coral animal cells, augment nutrient flux, and enhance the rate of coral calcification. Natural abundance of stable isotopes (delta13C and delta18O) provides answers to modern and paleobiological questions about the effect of photosymbiosis on sources of carbon and oxygen in coral skeletal calcium carbonate. Here we compare 17 species of symbiotic and nonsymbiotic corals to determine whether evidence for photosymbiosis appears in stable isotopes (delta13C and delta15N) of an organic skeletal compartment, the coral skeletal organic matrix (OM). Mean OM delta13C in symbiotic and nonsymbiotic corals was similar (-26.08 per thousand vs. -24.31 per thousand), but mean OM delta15N was significantly depleted in 15N in the former (4.09 per thousand) relative to the latter (12.28 per thousand), indicating an effect of the algae on OM synthesis and revealing OM delta15N as a proxy for photosymbiosis. To answer an important paleobiological question about the origin of photosymbiosis in reef-building corals, we applied this proxy test to a fossil coral (Pachythecalis major) from the Triassic (240 million years ago) in which OM is preserved. Mean OM delta15N was 4.66 per thousand, suggesting that P. major was photosymbiotic. The results show that symbiotic algae augment coral calcification by contributing to the synthesis of skeletal OM and that they may have done so as early as the Triassic.

In situ chemical speciation of sulfur in calcitic biominerals and the simple prism concept.

The microstructure and composition of two mollusc shells were investigated using a combination of light microscopy, SEM, EPMA, and XANES. The shells of Pinna and Pinctada are composed of calcite prisms separated by organic walls. The prismatic units of Pinna are monocrystalline, and those of Pinctada are polycrystalline with internal organic radial membranes. High-spatial-resolution XANES maps for the different S species across adjacent prisms show that sulfate is the principal component in both the intraprismatic organic matrices and the outer membranes. Additionally, these maps confirm that the inner structures of the prismatic units are different for both genera. In many ways, the prisms of Pinna and Pinctada are different and invalidate the "simple prism" concept.