Spectroscopic studies for identifying the chemical states of the periostracum of the Corbicula species in Lake Biwa

Corbicula clam shells consist of thin periostracum and calcareous layers made of calcium carbonate (CaCO₃). Depending on habitat conditions, the shell exhibits various colorations, such as yellow, brown, and black. The chemical state of the periostracum of the Corbicula species in Lake Biwa was studied by X-ray absorption fine structure (XAFS) and Raman scattering spectroscopies. Fe K-edge X-ray absorption near edge structure (XANES) revealed that the Fe³⁺ intensity increases as the color of the shell changes from yellow to black. Raman spectra suggested that quinone-based polymers cover the yellow shell, and the black shell is further covered by dihydroxyphenylalanine (DOPA) rings of amino acid derivatives. From Fe K-edge extended X-ray absorption fine structure (EXAFS), it was found that Fe³⁺ in the periostracum was surrounded by five to six oxygen atoms with an average Fe-O ligand distance of 2.0 Å. Accordingly, a tris-DOPA-Fe³⁺ complex is formed, which is responsible for the periostracum’s black color.     

Morphological studies on the calcification process in the fresh-water mussel Amblema

Light microscopy, transmission electron microscopy, scanning electron microscopy, various histochemical procedures for the localization of mineral ions, and analytical electron microscopy have been used to investigate the mechanisms inherent at the mantle edge for shell formation and growth in Amblema plicata perplicata, Conrad. The multilayered periostracum, its component laminae formed from the epithelia lining either the periostracal groove or the outer mantle epithelium (of the periostracal cul de sac), appears to play the major regulatory and organizational role in the formation of the component mineralized layers of the shell. Thus, the inner layer of the periostracum traps and binds calcium and subsequently gives rise to matricial proteinaceous fibrils or lamellar extensions which serve as nucleation templates for the formation and orientation of the crystalline subunits (rhombs) in the forming nacreous layer. Simultaneously, the middle periostracal layer furnishes or provides the total ionic calcium pool and the matricial organization necessary for the production of the spherical subunits which pack the matricial ‘bags’ of the developing prismatic layer. The outer periostracal layer appears to be a supportive structure, possibly responsible for the mechanical deformations which occur in the other laminae of the periostracum. The functional differences in the various layers of the periostracum are related to peculiar morphological variables (foliations, vacuolizations, columns) inherent in the structure and course of this heterogeneous (morphologically and biochemically) unit. From this study, using the dynamic mantle edge as a morphological model system, we have been able to identify at least six interrelated events which culminate in the production of the mature mineralized shell layers (nacre, prisms) at the growing edge of this fresh-water mussel.     

A Novel Acidic Matrix Protein,PfN44, Stabilizes Magnesium Calcite to Inhibit the Crystallization of Aragonite

Magnesium is widely used to control calcium carbonate deposition in the shell of pearl oysters. Matrix proteins in the shell are responsible for nucleation and growth of calcium carbonate crystals. However, there is no direct evidence supporting a connection between matrix proteins and magnesium. Here, we identified a novel acidic matrix protein named PfN44 that affected aragonite formation in the shell of the pearl oyster Pinctada fucata. Using immunogold labeling assays, we found PfN44 in both the nacreous and prismatic layers. In shell repair, PfN44 was repressed, whereas other matrix proteins were up-regulated. Disturbing the function of PfN44 by RNAi led to the deposition of porous nacreous tablets with overgrowth of crystals in the nacreous layer. By in vitro circular dichroism spectra and fluorescence quenching, we found that PfN44 bound to both calcium and magnesium with a stronger affinity for magnesium. During in vitro calcium carbonate crystallization and calcification of amorphous calcium carbonate, PfN44 regulated the magnesium content of crystalline carbonate polymorphs and stabilized magnesium calcite to inhibit aragonite deposition. Taken together, our results suggested that by stabilizing magnesium calcite to inhibit aragonite deposition, PfN44 participated in P. fucata shell formation. These observations extend our understanding of the connections between matrix proteins and magnesium.     

Intrinsically Disordered and Pliable Starmaker-Like Protein from Medaka (Oryzias latipes) Controls the Formation of Calcium Carbonate Crystals

Fish otoliths, biominerals composed of calcium carbonate with a small amount of organic matrix, are involved in the functioning of the inner ear. Starmaker (Stm) from zebrafish (Danio rerio) was the first protein found to be capable of controlling the formation of otoliths. Recently, a gene was identified encoding the Starmaker-like (Stm-l) protein from medaka (Oryzias latipes), a putative homologue of Stm and human dentine sialophosphoprotein. Although there is no sequence similarity between Stm-l and Stm, Stm-l was suggested to be involved in the biomineralization of otoliths, as had been observed for Stm even before. The molecular properties and functioning of Stm-l as a putative regulatory protein in otolith formation have not been characterized yet. A comprehensive biochemical and biophysical analysis of recombinant Stm-l, along with in silico examinations, indicated that Stm-l exhibits properties of a coil-like intrinsically disordered protein. Stm-l possesses an elongated and pliable structure that is able to adopt a more ordered and rigid conformation under the influence of different factors. An in vitro assay of the biomineralization activity of Stm-l indicated that Stm-l affected the size, shape and number of calcium carbonate crystals. The functional significance of intrinsically disordered properties of Stm-l and the possible role of this protein in controlling the formation of calcium carbonate crystals is discussed.     

Neuroendocrine Control Mechanisms in Shell Formation

The brain of Helisoma duryi contains several neurodendocrinecentres. Factors) present in the cerebral ganglia are thoughtto be involved in normal shell growth while neurosecretory substancespresent in the visceral ganglion are involved in the repairof damaged shell. In Lymnaea stagnalis a growth hormone is producedby the cerebral ganglion which stimulates periostracum formationand the calcification of the inner shell layer. The second effectis thought to occur through the action of a mantle edge calciumbinding protein. In Helisoma, mantle collar is able to produce the periostracumin vitro. The presence of brain from a fast growing donor increasesthe amount of periostracum produced by a mantle collar froma slow growing animal. This effect is further enhanced by theremoval of the lateral lobes. The periostracum produced by fastgrowing animals has a higher glycine content than that producedby slow growing snails. The presence of dorsal epithelial tissueenhances the incorporation of calcium into periostracum formedin vitro. These findings suggest that a single factor is present in thebrain of fast growing Helisoma which modulates shell formationrates in vivo and periostracum formation in vitro.     

Magnetic resonance microscopy of collagen mineralization

A model mineralizing system was subjected to magnetic resonance microscopy to investigate how water proton transverse (T₂) relaxation times and magnetization transfer ratios can be applied to monitor collagen mineralization. In our model system, a collagen sponge was mineralized with polymer-stabilized amorphous calcium carbonate. The lower hydration and water proton T₂ values of collagen sponges during the initial mineralization phase were attributed to the replacement of the water within the collagen fibrils by amorphous calcium carbonate. The significant reduction in T₂ values by day 6 (p < 0.001) was attributed to the appearance of mineral crystallites, which were also detected by x-ray diffraction and scanning electron microscopy. In the second phase, between days 6 and 13, magnetic resonance microscopy properties appear to plateau as amorphous calcium carbonate droplets began to coalesce within the intrafibrillar space of collagen. In the third phase, after day 15, the amorphous mineral phase crystallized, resulting in a reduction in the absolute intensity of the collagen diffraction pattern. We speculate that magnetization transfer ratio values for collagen sponges, with similar collagen contents, increased from 0.25 ± 0.02 for control strips to a maximum value of 0.31 ± 0.04 at day 15 (p = 0.03) because mineral crystals greatly reduce the mobility of the collagen fibrils.     

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.     

Otolin-1, an otolith- and otoconia-related protein,controls calcium carbonate bioinspired mineralization

BackgroundOtoliths and otoconia are calcium carbonate biomineral structures that form in the inner ear of fish and humans, respectively. The formation of these structures is tightly linked to the formation of an organic matrix framework with otolin-1, a short collagen-like protein from the C1q family as one of its major constituents.MethodsIn this study, we examined the activity of recombinant otolin-1 originating from Danio rerio and Homo sapiens on calcium carbonate bioinspired mineralization with slow-diffusion method and performed crystals characterization with scanning electron microscopy, two-photon excited fluorescence microscopy, confocal laser scanning microscopy and micro-Raman spectroscopy.ResultsWe show that both proteins are embedded in the core of CaCO₃ crystals that form through the slow-diffusion mineralization method. Both of them influence the morphology but do not change the polymorphic mineral phase. D.rerio otolin-1 also closely adheres to the crystal surface.General significanceThe results suggest, that otolin-1 is not a passive scaffold, but is directly involved in regulating the morphology of the resulting calcium carbonate biocrystals.     

Characterization of crystalline structures in Opuntia ficus-indica

This research studies the crystalline compounds present in nopal (Opuntia ficus-indica) cladodes. The identification of the crystalline structures was performed using X-ray diffraction, scanning electron microscopy, mass spectrometry, and Fourier transform infrared spectroscopy. The crystalline structures identified were calcium carbonate (calcite) [CaCO₃], calcium-magnesium bicarbonate [CaMg(CO₃)₂], magnesium oxide [MgO], calcium oxalate monohydrate [Ca(C₂O₄)•(H₂O)], potassium peroxydiphosphate [K₄P₂O₈] and potassium chloride [KCl]. The SEM images indicate that calcite crystals grow to dipyramidal, octahedral-like, prismatic, and flower-like structures; meanwhile, calcium-magnesium bicarbonate structures show rhombohedral exfoliation and calcium oxalate monohydrate is present in a drusenoid morphology. These calcium carbonate compounds have a great importance for humans because their bioavailability. This is the first report about the identification and structural analysis of calcium carbonate and calcium-magnesium bicarbonate in nopal cladodes, as well as the presence of magnesium oxide, potassium peroxydiphosphate and potassium chloride in these plants. The significance of the study of the inorganic components of these cactus plants is related with the increasing interest in the potential use of Opuntia as a raw material of products for the food, pharmaceutical, and cosmetic industries.     

Mineral Deposition in Bacteria-Filled and Bacteria-Free Calcium Bodies in the Crustacean Hyloniscus riparius (Isopoda: Oniscidea)

Crustacean calcium bodies are epithelial sacs which contain a mineralized matrix. The objectives of this study were to describe the microscopic anatomy of calcium bodies in the terrestrial isopod Hyloniscus riparius and to establish whether they undergo molt-related structural changes. We performed 3D reconstruction of the calcium bodies from paraffin sections and analyzed their structure with light and electron microscopy. In addition, we analyzed the chemical composition of their mineralized matrices with micro-Raman spectroscopy. Two pairs of these organs are present in H. riparius. One pair is filled with bacteria while the other pair is not. In non-molting animals, the bacteria-filled calcium bodies contain apatite crystals and the bacteria-free calcium bodies enclose CaCO₃-containing concretions with little organic matrix. During preparation for molt, an additional matrix layer is deposited in both pairs of calcium bodies. In the bacteria-filled calcium bodies it contains a mixture of calcium carbonate and calcium phosphate, whereas only calcium carbonate is present in bacteria-free calcium bodies. After ecdysis, all mineral components in bacteria-free calcium bodies and the additional matrix layer in bacteria-filled calcium bodies are completely resorbed. During calcium resorption, the apical surface of the calcium body epithelium is deeply folded and electron dense granules are present in spaces between epithelial cells. Our results indicate that the presence of bacteria might be linked to calcium phosphate mineralization. Calcium bodies likely provide a source of calcium and potentially phosphate for the mineralization of the new cuticle after molt. Unlike other terrestrial isopods, H. riparius does not form sternal CaCO₃ deposits and the bacteria-free calcium bodies might functionally replace them in this species.     

Genesis of amorphous calcium carbonate containing alveolar plates in the ciliate Coleps hirtus (Ciliophora,Prostomatea)

In the protist world, the ciliate Coleps hirtus (phylum Ciliophora, class Prostomatea) synthesizes a peculiar biomineralized test made of alveolar plates, structures located within alveolar vesicles at the cell cortex. Alveolar plates are arranged by overlapping like an armor and they are thought to protect and/or stiffen the cell. Although their morphology is species-specific and of complex architecture, so far almost nothing is known about their genesis, their structure and their elemental and mineral composition. We investigated the genesis of new alveolar plates after cell division and examined cells and isolated alveolar plates by electron microscopy, energy-dispersive X-ray spectroscopy, FTIR and X-ray diffraction. Our investigations revealed an organic mesh-like structure that guides the formation of new alveolar plates like a template and the role of vesicles transporting inorganic material. We further demonstrated that the inorganic part of the alveolar plates is composed out of amorphous calcium carbonate. For stabilization of the amorphous phase, the alveolar vesicles, the organic fraction and the element phosphorus may play a role.     

Syn-Vivo Bioerosion of Nautilus by Endo- and Epilithic Foraminiferans (New Caledonia and Vanuatu)

A variety of syn-vivo bioerosion traces produced by foraminiferans is recorded in shells of Nautilus sampled near New Caledonia and Vanuatu. These are two types of attachment scars of epilithic foraminiferans and two forms of previously undescribed microborings, a spiral-shaped and a dendritic one, both most likely being the work of endolithic ''naked'' foraminiferans. Scanning electron microscopy of epoxy-resin casts of the latter revealed that these traces occur in clusters of up to many dozen individuals and potentially are substrate-specific. The foraminiferan traces are the sole signs of bioerosion in the studied Nautilus conchs, and neither traces of phototrophic nor other chemotrophic microendoliths were found. While the complete absence of photoautotrophic endoliths would be in good accordance with the life habit of Nautilus, which resides in aphotic deep marine environments and seeks shallower waters in the photic zone for feeding only during night-time, the absence of any microbial bioerosion may also be explained by an effective defence provided by the nautilid periostracum. Following this line of reasoning, the recorded foraminiferan bioerosion traces in turn would identify their trace makers as being specialized in their ability to penetrate the periostracum barrier and to bioerode the shell of modern Nautilus.     

Temperature effects on calcification rate and skeletal deposition in the temperate coral, Plesiastrea versipora (Lamarck)

The geographic range of the coral, Plesiastrea versipora (Lamarck, 1816), extends into temperate waters outside the southern limit for hermatypic corals. In the present study, calcification in Plesiastrea collected from Port Phillip Bay, Victoria was examined over the coral's normal annual temperature range (10-21 °C), which is well below the normal optimum for coral calcification in tropical corals (25-28 °C). Calcification rate in Plesiastrea was considerably lower than in reef corals, but showed a similar pattern in temperature responses, with a trend towards higher rates at ∼18 °C. The light/dark calcification ratio was markedly lower than that in tropical corals. Autoradiography showed that calcification occurred primarily by deposition of calcium carbonate at the upper surfaces of the septo-costae. Scanning electron microscopy (SEM) showed that skeletal deposition in Plesiastrea had a temperature-dependent diel pattern. In the light, calcium carbonate was deposited as small spheroidal crystals and, at higher temperatures, small needle-shaped crystals. In the dark, calcium carbonate deposition appeared to be in the form of an amorphous sheet-like cementation. Compared with other scleractinian corals, calcification rate in Plesiastrea was relatively slow and showed different patterns of skeletal deposition.     

A histological and ultrastructural study of the cells of the mantle edge of a marine bivalve, Cerastoderma edule

The cells of the mantle edge of Cerastoderma edule are described after light and electron microscopical observations. Histochemical tests for calcium in the mantle edge and digestive gland (Dahl, 1952; McGee-Russell, 1958) and analytical electron microscopy of the mantle edge of C. edule both failed to show calcium. Similar results were obtained for Mytilus edulis and Chlamys opercularis. However, calcium was detected in the digestive gland of the terrestrial gastropod Helix aspersa. The outer secretory fold of the mantle edge is composed of tall columnar cells. These cells have highly convoluted lateral cell membranes with which many mitochondria are closely associated. These features are indicative of an ion pump which could move calcium from the mantle space to the extrapallial cavity (compare with Bubel's findings, 1973b). There are many features of the cells lining the periostracal groove of C. edule that have not been reported previously (e.g. Bubel, 1973b) and which are now discussed. The periostracal sheet arises within a line of basal cells in the fundus of the periostracal groove. Within these cells the periostracum in section has a spiral form. It is suggested that the newly formed periostracum adheres to the microvillous border through secretions produced from the middle fold cells lining the groove. During its passage along the groove the periostracum is gradually thickened by secretions from the outer fold cells.     

Biominerals and waxes of <Emphasis Type="Italic">Calamagrostis epigejos</Emphasis> and <Emphasis Type="Italic">Phragmites australis</Emphasis> leaves from post-industrial habitats

Vascular plants are able to conduct biomineralization processes and collect synthesized compounds in their internal tissues or to deposit them on their epidermal surfaces. This mechanism protects the plant from fluctuations of nutrient levels caused by different levels of supply and demand for them. The biominerals reflect both the metabolic characteristics of a vascular plant species and the environmental conditions of the plant habitat. The SEM/EDX method was used to examine the surface and cross-sections of the Calamagrostis epigejos and Phragmites australis leaves from post-industrial habitats (coal and zinc spoil heaps). The results from this study have showed the presence of mineral objects on the surfaces of leaves of both grass species. The calcium oxalate crystals, amorphous calcium carbonate spheres, and different silica forms were also found in the inner tissues. The high variety of mineral forms in the individual plants of both species was shown. The waxes observed on the leaves of the studied plants might be the initializing factor for the crystalline forms and structures that are present. For the first time, wide range of crystal forms is presented for C. epigejos. The leaf samples of P. australis from the post-industrial areas showed an increased amount of mineral forms with the presence of sulfur.     

The Molecular Evolution of the Pif Family Proteins in Various Species of Mollusks

Various novel proteins have been identified from many kinds of mollusk shells. Although such matrix proteins are believed to play important roles in the calcium carbonate crystal formation of shells, no common proteins that interact with calcium carbonate or that are involved in the molecular mechanisms behind shell formation have been identified. Pif consists of two proteins, Pif 80 and Pif 97, which are encoded by a single mRNA. Pif 80 was identified as a key acidic protein that regulates the formation of the nacreous layer in Pinctada fucata, while Pif 97 has von Willebrand factor type A (VWA) and chitin-binding domains. In this study, we identified Pif homologues from Pinctada margaritifera, Pinctada maxima, Pteria penguin, Mytilus galloprovincialis, and in the genome database of Lottia gigantea in order to compare their primary protein sequences. The VWA and chitin-binding domains are conserved in all Pif 97 homologues, whereas the amino acid sequences of the Pif 80 regions differ markedly among the species. Sequence alignment revealed the presence of a novel significantly conserved sequence between the chitin-binding domain and the C-terminus of Pif 97. Further examination of the Pif 80 regions suggested that they share a sequence that is similar to the laminin G domain. These results indicate that all Pif molecules in bivalves and gastropods may be derived from a common ancestral gene. These comparisons may shed light on the correlation between molecular evolution and morphology in mollusk shell microstructure.     

A hairy business—Periostracal hair formation in two species of helicoid snails (Gastropoda,Stylommatophora, Helicoidea)

In molluscs, the calcareous shell is covered externally by a thin organic layer, the periostracum. The periostracum of some pulmonate species is of special taxonomic interest because it bears distinct microscale architectures. Where and how these structures are formed is as yet unknown. Using histological sections through their shells, gelatin cuts, and live observations I studied the pattern by which the periostracal hair‐like projections in two helicoid land snail species are secreted and evenly arranged on the shell. The results indicate a complex mechanism: a hair is formed in the periostracal groove independently of the periostracum, after which it is attached to the edge of the shell, drawn out of the tissue, and finally swivelled to the upper side of the periostracum. Upon further growth of the periostracum, the hairs are finally fixed upright on the shell. J. Morphol. 2011. © 2011 Wiley‐Liss, Inc.     

Comparative Characterization of the Microbial Diversities of an Artificial Microbialite Model and a Natural Stromatolite

Microbialites are organosedimentary structures that result from the trapping, binding, and lithification of sediments by microbial mat communities. In this study we developed a model artificial microbialite system derived from natural stromatolites, a type of microbialite, collected from Exuma Sound, Bahamas. We demonstrated that the morphology of the artificial microbialite was consistent with that of the natural system in that there was a multilayer community with a pronounced biofilm on the surface, a concentrated layer of filamentous cyanobacteria in the top 5 mm, and a lithified layer of fused oolitic sand grains in the subsurface. The fused grain layer was comprised predominantly of the calcium carbonate polymorph aragonite, which corresponded to the composition of the Bahamian stromatolites. The microbial diversity of the artificial microbialites and that of natural stromatolites were also compared using automated ribosomal intergenic spacer analysis (ARISA) and 16S rRNA gene sequencing. The ARISA profiling indicated that the Shannon indices of the two communities were comparable and that the overall diversity was not significantly lower in the artificial microbialite model. Bacterial clone libraries generated from each of the three artificial microbialite layers and natural stromatolites indicated that the cyanobacterial and crust layers most closely resembled the ecotypes detected in the natural stromatolites and were dominated by Proteobacteria and Cyanobacteria. We propose that such model artificial microbialites can serve as experimental analogues for natural stromatolites.