Structure of an amorphous calcium carbonate phase involved in the formation of Pinctada margaritifera shells.

Some mollusc shells are formed from an amorphous calcium carbonate (ACC) compound, which further transforms into a crystalline material. The transformation mechanism is not fully understood but is however crucial to develop bioinspired synthetic biomineralization strategies or accurate marine biomineral proxies for geoscience. The difficulty arises from the simultaneous presence of crystalline and amorphous compounds in the shell, which complicates the selective experimental characterization of the amorphous fraction. Here, we use nanobeam X-ray total scattering together with an approach to separate crystalline and amorphous scattering contributions to obtain the spatially resolved atomic pair distribution function (PDF). We resolve three distinct amorphous calcium carbonate compounds, present in the shell of Pinctada margaritifera and attributed to: interprismatic periostracum, young mineralizing units, and mature mineralizing units. From this, we extract accurate bond parameters by reverse Monte Carlo (RMC) modeling of the PDF. This shows that the three amorphous compounds differ mostly in their Ca-O nearest-neighbor atom pair distance. Further characterization with conventional spectroscopic techniques unveils the presence of Mg in the shell and shows Mg-calcite in the final, crystallized shell. In line with recent literature, we propose that the amorphous-to-crystal transition is mediated by the presence of Mg. The transition occurs through the decomposition of the initial Mg-rich precursor into a second Mg-poor ACC compound before forming a crystal.

Amorphous-to-crystal transition in the layer-by-layer growth of bivalve shell prisms.

Biomineralization integrates complex physical and chemical processes bio-controlled by the living organisms through ionic concentration regulation and organic molecules production. It allows tuning the structural, optical and mechanical properties of hard tissues during ambient-condition crystallisation, motivating a deeper understanding of the underlying processes. By combining state-of-the-art optical and X-ray microscopy methods, we investigated early-mineralized calcareous units from two bivalve species, Pinctada margaritifera and Pinna nobilis, revealing chemical and crystallographic structural insights. In these calcite units, we observed ring-like structural features correlated with a lack of calcite and an increase of amorphous calcium carbonate and proteins contents. The rings also correspond to a larger crystalline disorder and a larger strain level. Based on these observations, we propose a temporal biomineralization cycle, initiated by the production of an amorphous precursor layer, which further crystallizes with a transition front progressing radially from the unit centre, while the organics are expelled towards the prism edge. Simultaneously, along the shell thickness, the growth occurs following a layer-by-layer mode. These findings open biomimetic perspectives for the design of refined crystalline materials. STATEMENT OF SIGNIFICANCE: Calcareous biominerals are amongst the most present forms of biominerals. They exhibit astonishing structural, optical and mechanical properties while being formed at ambient synthesis conditions from ubiquitous ions, motivating the deep understanding of biomineralization. Here, we unveil the first formation steps involved in the biomineralization cycle of prismatic units of two bivalve species by applying a new multi-modal non-destructive characterization approach, sensitive to chemical and crystalline properties. The observations of structural features in mineralized units of different ages allowed the derivation of a temporal sequence for prism biomineralization, involving an amorphous precursor, a radial crystallisation front and a layer-by-layer sequence. Beyond these chemical and physical findings, the herein introduced multi-modal approach is highly relevant to other biominerals and bio-inspired studies.

Monitoring of coastal pollution using shell alterations in the false limpet Siphonaria pectinata.

Lipid peroxidation level (LPO), shell biometry, shape, elemental content, and microstructure were studied in three populations of Siphonaria pectinata in the complex lagoon-channel of Bizerte across a coastal pollution gradient (northern Tunisia). LPO was found in higher concentrations in harbour populations, and shells had centred apex and were flattened. Shells were also thicker, particularly in the inner layer, with many fibrous inter-beds formed. Difference in crystallization pattern was observed in numerous shells from all three populations, being more common in harbours. From the control station to the contaminated stations, shell elemental changes were observed, with a decrease in Ca, P, Sr, and S and an increase in Cl, Cd, Cu, Fe, and K. All of these findings suggested that shell alterations could be used as a good biomarker for coastal contamination.

Insights into the MALDI Process after Matrix Deposition by Sublimation Using 3D ToF-SIMS Imaging.

Imaging mass spectrometry (IMS) has become a powerful tool to characterize the spatial distribution of biomolecules in thin tissue sections. In the case of matrix-assisted laser desorption ionization (MALDI) IMS, homogeneous matrix deposition is critical to produce high-quality ion images, and sublimation in particular has shown to be an excellent matrix deposition method for the imaging of lipids. Matrix deposition by sublimation is, however, a completely solvent-free system, which ought to prevent the mixing of matrix and analytes thought to be necessary for successful MALDI. Using 3D time-of-flight secondary ion imaging mass spectrometry, we have studied the matrix-tissue interface in 3D with high resolution to understand the MALDI process of lipids after matrix deposition by sublimation. There is a strong indication that diffusion is the process by which lipids migrate from the tissue to the matrix layer. We show that triacylglycerols and phospholipids have a delayed migratory trend as compared to diacylglycerols and monoacylglycerols, which is dependent on time and matrix thickness. Additional experiments show that a pure lipid's capacity to migrate into the matrix is dependent on its fluidity at room temperature. Furthermore, it is shown that cholesterol can only migrate in the presence of a (fluid) lipid and appears to fluidize lipids, which could explain its colocalization with the diacylglycerols and monoacylglycerols in the matrix.

Hierarchical architecture of the inner layers of selected extant rhynchonelliform brachiopods.

In spite of several attempts for a best knowledge of the phylum, brachiopods remain, compared with molluscs, among those least analysed in terms of biomineralization. The lack of economic impact for extant species is probably liable for that situation. Much attention has been on the microstructure of calcite biomaterials (rhynchonelliforms and craniiforms). Here, we emphasize the sub-micrometric structure of selected examples of rhynchonelliform shells using Atomic Force Microscopy (AFM) to complement Scanning Electron Microscopy (SEM) and Energy Dispersive X-ray (EDX) analyses. The hierarchical organization of the shell layers (secondary and/or tertiary elements) is highlighted for species non-yet observed from this point of view, and is compared to a few already mentioned in the literature. Previous analysis revealed that granules are composed of a complex aggregation of sub-units in intimate relation with an intracrystalline matrix. Their shape, size and probably early orientation depend on the species as well as age and living environments of the specimens studied. The control of the inorganic part of the composite fibrous elements is constrained by the deposition of nearly arched shape or polygonal protein membranes at the inner boundary of the primary layer, prior to the deposition of the first granules, membranes becoming proteinaceous sheathes progressively enshrining fibres. The diverse orientations of the granules in fibrous neighbours thus further increase arguments in favour of the tendency to improve the shell strength.

Limpet Shells from the Aterian Level 8 of El Harhoura 2 Cave (Témara, Morocco): Preservation State of Crossed-Foliated Layers.

The exploitation of mollusks by the first anatomically modern humans is a central question for archaeologists. This paper focuses on level 8 (dated around ∼ 100 ka BP) of El Harhoura 2 Cave, located along the coastline in the Rabat-Témara region (Morocco). The large quantity of Patella sp. shells found in this level highlights questions regarding their origin and preservation. This study presents an estimation of the preservation status of these shells. We focus here on the diagenetic evolution of both the microstructural patterns and organic components of crossed-foliated shell layers, in order to assess the viability of further investigations based on shell layer minor elements, isotopic or biochemical compositions. The results show that the shells seem to be well conserved, with microstructural patterns preserved down to sub-micrometric scales, and that some organic components are still present in situ. But faint taphonomic degradations affecting both mineral and organic components are nonetheless evidenced, such as the disappearance of organic envelopes surrounding crossed-foliated lamellae, combined with a partial recrystallization of the lamellae. Our results provide a solid case-study of the early stages of the diagenetic evolution of crossed-foliated shell layers. Moreover, they highlight the fact that extreme caution must be taken before using fossil shells for palaeoenvironmental or geochronological reconstructions. Without thorough investigation, the alteration patterns illustrated here would easily have gone unnoticed. However, these degradations are liable to bias any proxy based on the elemental, isotopic or biochemical composition of the shells. This study also provides significant data concerning human subsistence behavior: the presence of notches and the good preservation state of limpet shells (no dissolution/recrystallization, no bioerosion and no abrasion/fragmentation aspects) would attest that limpets were gathered alive with tools by Middle Palaeolithic (Aterian) populations in North Africa for consumption.

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.

Crystallization in organo-mineral micro-domains in the crossed-lamellar layer of Nerita undata (Gastropoda, Neritopsina).

Crossed-lamellar shell microstructure consists of a sophisticated arrangement of interspersed lamellae, which is very commonly found in Gastropoda or Bivalvia shell layers. Its smallest constitutive microstructural units are usually described as sub-micrometric fibers, or rods, and form very ordered and regular patterns. Atomic force microscopy (AFM) and transmission electron microscopy (TEM) imaging confirms the presence of even smaller building units in the form of organo-mineral granules, and we further investigate their internal structure within aragonite crossed-lamellar internal layer of Nerita undata (Gastropoda, Neritopsina) shell. Their coalescence may have controlled anisotropically the propagation of the crystallographic coherence through this complex microstructure, as suggested by the propagation of the microtwinning pattern between neighboring granules.

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.