Comparison of the soluble matrices of the calcitic prismatic layer of Pinna nobilis (Mollusca,Bivalvia, Pteriomorpha)

The calcitic prisms of the outer layer of the shell of Pinna nobilis, surrounded by thick organic walls, contain a soluble intracrystalline matrix. The structure and composition of the outer interprismatic walls are not well known. The current viewpoint is they are composed of an insoluble matrix. Another thick organic structure, the interlamellar sheet of the nacreous layer, is composed of insoluble and soluble matrices. The composition of two sets of soluble organic matrices from the calcitic layer of Pinna nobilis, extracted with and without the organic walls are compared. According to the various analyses (SEM and AFM, UV and FTIR spectrometry, HPLC, electrophoreses, XANES), the main characteristics of the two matrices are similar, but not identical. Thus, the organic walls contain soluble components. However, the three-layered structure of the interlamellar sheet of the nacreous layer has not been observed.     

Flexible nacre in the nautiloid Isorthoceras, with remarks on the evolution of cephalopod nacre

Secondarily phosphatized shells of Isorthoceras sociale (Hall) from the Upper Ordovician Maquokcta shale, Iowa, U.S.A., often have their original ultrastructurc preserved. Scanning Electron Microscope study reveals that the nacreous layer is similar to that in the shells of Recent Nautilus , except that the nacreous tablets usually have a much larger diameter, and that numerous vertical interspaces arc left between adjacent stacks of tablets. Because of the latter condition, the nacre in A sociale is porous, whereas it is compact in Nautilus. The pores in the nacre were probably filled with a non-mincralizcd organic matrix. The high porosity and high content of organic matrix made the nacreous layer of I. sociale much more flexible and considerably less strong than that in Nautilus. As a consequence, the shells of I. sociale , and probably also of other orthoconic cephalopods, were more flexible and could not withstand such high hydrostatic pressures as the shells of Nautilus. During evolution, the porosity of the nacreous layer became completely reduced. As a result, the mechanical properties of the nacre changed towards a lower flexibility and a higher tensile strength. Cephalopoda, Nautiloidea, nacre, ultrastructurc, phosphatization, Upper Ordovician.     

Mollusk shell formation: mapping the distribution of organic matrix components underlying a single aragonitic tablet in nacre

Control over mineral formation in mollusk shells is exerted by the macromolecules of the organic matrix. Using histochemical methods, we mapped the carboxylates and sulfates of proteins and polysaccharides on the surfaces of decalcified interlamellar matrices from the nacreous shell layer of the cephalopod Nautilus pompilius, expanding upon an earlier study by Crenshaw and Ristedt [Crenshaw, M.A., Ristedt, H., 1976. The histochemical localization of reactive groups in septal nacre from Nautilus pompilius. In: Watabe, N., Wilbur, K.M. (Ed.), The Mechanisms of Mineralization in the Invertebrates and Plants. University of South Carolina Press, Colombia, pp. 355-367]. We observed four different zones underlying a single crystal: (1) a central spot rich in carboxylates; (2) a central ring-shaped area rich in sulfates; (3) an area between the central nucleation region and the imprint periphery containing carboxylates, and (4) the intertabular matrix, rich in carboxylates and sulfates. We also mapped matrix functional groups on the nacreous matrix surfaces of the bivalve Atrina rigida, but did not identify well-defined zones. Immuno-mapping of the constituents of the aragonite-nucleating protein fraction from Atrina nacre showed that these macromolecules are located both in the intertabular matrix and in the center of the crystal imprints for both Atrina and Nautilus matrix surfaces. Their presence at the latter location is consistent with their purported role in aragonite nucleation. The observed differentiation in the distribution of matrix components and their functional groups shows that the different stages of single crystal growth are highly controlled by the matrix.     

Identification of chitin in the prismatic layer of the shell and a chitin synthase gene from the Japanese pearl oyster, Pinctada fucata

The shell of the Japanese pearl oyster, Pinctada fucata, consists of two layers, the prismatic layer on the outside and the nacreous layer on the inside, both of which comprise calcium carbonate and organic matrices. Previous studies indicate that the nacreous organic matrix of the central layer of the framework surrounding the aragonite tablet is beta-chitin, but it remains unknown whether organic matrices in the prismatic layer contain chitin or not. In the present study, we identified chitin in the prismatic layer of the Japanese pearl oyster, Pinctada fucata, with a combination of Calcofluor White staining with IR and NMR spectral analyses. Furthermore, we cloned a cDNA encoding chitin synthase (PfCHS1) that produces chitin, contributing to the formation of the framework for calcification in the shell.     

A scanning electron microscopic study of the inorganic and organic matrices comprising the mature shell of Amblema, a fresh-water mollusc

The scanning electron microscope has been used to describe the morphology of the mature shell in a fresh-water bivalve. The structure of the organic and inorganic components within the nacre, the myostracum, and the prismatic layer is described. A transitional or intermediate zone, interposed between the prismatic layer and the nacre, was identified. In demineralized samples, the organic component of the nacre was found to consist of parallel matricial sheets interconnected by irregular transverse bridges. The structure of the mineral component of the nacre was found to vary with the method of specimen preparation. With polished-etched samples, brick-like units were seen. When shells were simply broken and fixed in osmium, the layers of nacreous material consisted of fusing rhomboidal crystals of aragonite which demonstrated subconchoidal fractures. On the inner surface of the shell, the rhomboidal crystals showed an apparent spiral growth pattern. The myostracum was characterized by regions of modified nacreous structure consisting of enlarged aragonite crystals with a pyramidal morphology. The peripheral aspect of the muscle scars was characterized by rhomboidal crystals, the latter fusing to form the typical nacreous laminae. The uniqueness of the anterior adductor scar is exemplified by the presence of pores, each pore walled by pyramidal units, for the insertion of adductor fibres. In most regions of the shell, the prismatic layer consisted of one prism unit thickness with a height of approximately 225–250 μm. However, in two specialized regions of the shell, this layer was seen to consist of multiple layers of stacked prisms. The organic matrices of the prismatic layer are arranged in a honeycomb-like arrangement and packed with mineralized spherical subunits.     

Nautilin-63, a novel acidic glycoprotein from the shell nacre of Nautilus macromphalus

In molluscs, and more generally in metazoan organisms, the production of a calcified skeleton is a complex molecular process that is regulated by the secretion of an extracellular organic matrix. This matrix constitutes a cohesive and functional macromolecular assemblage, containing mainly proteins, glycoproteins and polysaccharides that, together, control the biomineral formation. These macromolecules interact with the extruded precursor mineral ions, mainly calcium and bicarbonate, to form complex organo-mineral composites of well-defined microstructures. For several reasons related to its remarkable mechanical properties and to its high value in jewelry, nacre is by far the most studied molluscan shell microstructure and constitutes a key model in biomineralization research. To understand the molecular mechanism that controls the formation of the shell nacreous layer, we have investigated the biochemistry of Nautilin-63, one of the main nacre matrix proteins of the cephalopod Nautilus macromphalus. After purification of Nautilin-63 by preparative electrophoresis, we demonstrate that this soluble protein is glycine-aspartate-rich, that it is highly glycosylated, that its sugar moieties are acidic, and that it is able to bind chitin in vitro. Interestingly, Nautilin-63 strongly interacts with the morphology of CaCO(3) crystals precipitated in vitro but, unexpectedly, it exhibits an extremely weak ability to inhibit in vitro the precipitation of CaCO(3) . The partial resolution of its amino acid sequence by de novo sequencing of its tryptic peptides indicates that Nautilin-63 exhibits short collagenous-like domains. Owing to specific polyclonal antibodies raised against the purified protein, Nautilin-63 was immunolocalized mainly in the intertabular nacre matrix. In conclusion, Nautilin-63 exhibits 'hybrid' biochemical properties that are found both in the soluble and insoluble proteins, rendering it difficult to classify according to the standard view on nacre proteins. DATABASE: The protein sequences of N63 appear on the UniProt Knowledgebase under accession number P86702.     

Crystalline structure,orientation and nucleation of the nacreous tablets in the cephalopod <Emphasis Type="Italic">Nautilus</Emphasis>

The nacreous tablets in the Nautilus shell have similar crystalline structure as the tablets in the gastropod Gibbula shell. Etching with Mutvei’s solution reveals that each tablet is composed of vertical crystalline columns that are structurally similar to the acicular crystallites in the outer spherulitic-prismatic layer of the shell wall. The columns are attached to each other to form numerous vertical crystalline lamellae, oriented parallel to the longitudinal axis of the tablet. It is still unknown whether or not the orientation of the vertical lamellae corresponds to that of the crystallographic a- or b-axis. The orientation of the crystalline lamellae in the adjacent tablets is parallel in some nacreous laminae, but random in other laminae. Similar large variation was found in the nacreous tablets of the gastropod and bivalve shells. The nucleation sites of the nacreous tablets are predominantly situated on the peripheral portion of the upper surface of the preceding tablet, both in the shell wall and septa. The “aragonite-nucleating proteins” in the central portion of the crystal imprints of the organic interlamellar sheets, described by several writers, have therefore a negative correlation with the nucleation sites of the nacreous tablets.     

Electron diffraction of mollusc shell organic matrices and their relationship to the mineral phase

Electron diffraction patterns showing orientation of the chitin and protein constituents of the insoluble organic matrix of mollusc shell nacreous layers have been obtained, using low dose conditions and samples cooled to −100°C. Diffraction patterns of the aragonite crystals were also observed. In a gastropod and a bivalve the spatial relationship between the organic matrix constituents and the aragonite crystallographic axes were shown to be the same as was previously observed for a cephalopod using X-ray diffraction, supporting the notion that mineral crystal growth occurs epitaxially upon a matrix template.     

Geometrical and crystallographic constraints determine the self-organization of shell microstructures in Unionidae (Bivalvia: Mollusca)

Unionid shells are characterized by an outer aragonitic prismatic layer and an inner nacreous layer. The prisms of the outer shell layer are composed of single-crystal fibres radiating from spheruliths. During prism development, fibres progressively recline to the growth front. There is competition between prisms, leading to the selection of bigger, evenly sized prisms. A new model explains this competition process between prisms, using fibres as elementary units of competition. Scanning electron microscopy and X-ray texture analysis show that, during prism growth, fibres become progressively orientated with their three crystallographic axes aligned, which results from geometric constraints and space limitations. Interestingly transition to the nacreous layer does not occur until a high degree of orientation of fibres is attained. There is no selection of crystal orientation in the nacreous layer and, as a result, the preferential orientation of crystals deteriorates. Deterioration of crystal orientation is most probably due to accumulation of errors as the epitaxial growth is suppressed by thick or continuous organic coats on some nacre crystals. In conclusion, the microstructural arrangement of the unionid shell is, to a large extent, self-organized with the main constraints being crystallographic and geometrical laws.     

A possible mechanism for the formation of annual growth lines in bivalve shells

We report a unique shell margin that differed from the usual shell structure of Pinctada fucata. We observed empty organic envelopes in the prismatic layer and the formation of the nacreous layer in the shell margin. All the characteristics of the growing margin indicated that the shell was growing rapidly. To explain this anomaly, we propose the concept of “jumping development”. During jumping development, the center of growth in the bivalve shell jumps forward over a short time interval when the position of the mantle changes. Jumping development explains the unusual structure of the anomalous shell and the development of annual growth lines in typical shells. Annual growth lines are the result of a discontinuity in the shell microstructure induced by jumping development.     

Abalone nacre insoluble matrix induces growth of flat and oriented aragonite crystals

Mollusc shell formation takes place in a preformed extracellular matrix, composed of insoluble chitin, coated with proteins and dissolved macromolecules. The water-soluble matrix is known to have a strong influence on the growth of CaCO(3), whereas the role of the insoluble matrix on mineralization is unclear. Therefore, we mineralized the EDTA (ethylenediaminetetraacetic acid) insoluble organic matrix of abalone nacre with a modified double-diffusion set-up, where the diffusing solutions were constantly renewed. Control experiments were performed with cellulose and chitosan foils. The mineralized matrices/foils were analyzed with SEM. We show that the insoluble matrix of abalone nacre induces the growth of flat and roughly polygonal CaCO(3) crystals. In some of the experiments with the insoluble matrix, the growth of three-dimensional parallel sheets of densely packed platelets inside the insoluble matrix was observed. XRD on these samples revealed that they consist of oriented aragonite.     

Structural relationship between interlamellar organic sheets and nacreous tablets in gastropods and the cephalopodNautilus

The nacreous tablets in gastropods and the cephalopodNautilus are composed of three calcareous layers: a principal, thick, finely granular layer and two thin, coarse-granular layers, one covering the upper surface of the principal layer and another the lower surface of this layer. The granules on the surface layers inNautilus differ from those in gastropods by their much more elongated shape and larger size. The central portion of the nacreous tablet of gastropods andNautilus is more or less elevated forming the central elevation. The granules on this portion usually are larger, irregularly shaped and more crowded than on the main, peripheral portion of the tablet. The untreated, dry interlamellar organic sheets on upper surfaces of immature nacreous tablets are uncalcified and elastic. Narrow thicker parts of the sheet, the trabeculae, Surround large intertrabecular spaces where the sheet is thin. In places it can be observed that each calcareous granula on the surface layer of the nacreous tablet is situated within the intertrabecular space. The size, shape and distribution of the intertrabecular Spaces correspond those of the surface granules. No mineral bridges were observed between the consecutive nacreous tablets.      

Characteristics of shell microstructure and growth analysis of the Antarctic bivalve <Emphasis Type="Italic">Laternula elliptica</Emphasis> from Lützow-Holm Bay,Antarctica

Growth performance of the Antarctic bivalve Laternula elliptica was examined both by shell microstructural observation and by applying a fluorescent substance, tetracycline, as a shell growth marker. The shell was composed of two calcareous layers: the thick outer layer was homogeneous or granular in structure and the thin inner layer was nacreous. The architecture of Antarctic L. elliptica was different from that of temperate L. marilina, and the ratio of thickness between the outer and inner layers appeared to be different. The growth rate of the nacreous layer was analyzed to be very low. High correlations were found between the major axis of chondrophore and both shell length and shell dry weight, respectively. It is suggested that the chondrophore is an appropriate growth indicator, and combining the information of growth increments with the fluorescent method may be useful in estimating the bivalve growth performance in the Antarctic sea.     

Ultrastructural Characteristics of the Nacre in Some Gastropods

The nacreous layer in Gibbula, Calliostoma, Trochus and Haliotis is described on the basis of scanning electron microscopic studies. The central part of each nacreous tablet contains a significant amount of calcified organic matrix which is insoluble in a chromium sulphate and a 25% glutaraldehyde solution. In most cases, the tablet is subdivided by radial vertical organic membranes into a varying number (2 to 50) of crystalline sectors. These sectors represent polysynthetically twinned crystal individuals which form cyclic or interpenetrant twins. The nacreous tablets in gastropods are compared with those in bivalves, and with the non-biogenic aragonite. The mechanical properties of the nacre, and the effects of the interlamellar conchiolin membranes upon the nucleation of the tablets, are discussed.     

Proteomics Analysis of the Nacre Soluble and Insoluble Proteins from the Oyster Pinctada margaritifera

Shell nacre is laid upon an organic cell-free matrix, part of which, paradoxically, is water soluble and displays biological activities. Proteins in the native shell also constitute an insoluble network and offer a model for studying supramolecular organization as a means of self-ordering. Consequently, difficulties are encountered in extraction and purification strategies for protein characterization. In this work, water-soluble proteins and the insoluble conhiolin residue of the nacre of Pinctada margaritifera matrix were analyzed via a proteomics approach. Two sequences homologous to nacre matrix proteins of other Pinctada species were identified in the water-soluble extract. One of them is known as a fundamental component of the insoluble organic matrix of nacre. In the conchiolin, the insoluble residue, four homologs of Pinctada nacre matrix proteins were found. Two of them were the same as the molecules characterized in the water-soluble extract. Results established that soluble and insoluble proteins of the nacre organic matrix share constitutive material. Surprisingly, a peptide in the conchiolin residue was found homologous to a prismatic matrix protein of Pinctada fucata, suggesting that prismatic and nacre matrices may share common proteins. The insoluble properties of shell matrix proteins appear to arise from structural organization via multimerization. The oxidative activity, found in the water-soluble fraction of the nacre matrix, is proposed as a leading process in the transformation of transient soluble proteins into the insoluble network of conchiolin during nacre growth.     

Microboring in a Freshwater Fluvial Unionid Bivalve Substrate

Samples of the unionid bivalve Elliptio complanata were collected from the channel of the freshwater Saint John River, from Fredericton, New Brunswick, Canada. Scanning electron microscopy imaging of prepared shell samples revealed an assemblage of microborings. No borings are noted on the periostracum or prismatic shell layers. Boring structures are instead confined to the underlying nacreous aragonitic shell material, together with its associated organic conchiolin layers. Three main styles of boring are encountered, encompassing both predominantly surficial structures and penetrative tubular borings. Surficial structures are represented by a polygonal network on an exposed conchiolin shell layer. The penetrative borings take two forms, one being simple unbranched tubes, steeply aligned (perpendicular to the shell surface) and traversing the full thickness of the nacreous shell layer. The other penetrative boring style, again occurring within the nacreous layer, comprises a complex irregular network of randomly oriented rarely branching tubular borings. Borings generally display diameters of micron scale. Biofilm and extracellular polymeric substances, with bacterial, diatomaceous and filamentous components are also observed, often displaying a close association with both the borings and the conchiolin layers within the shell. The formation of the borings may be attributed to cyanobacteria, cyanophyte or fungal progenitors.     

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.     

SHELL STRUCTURES OF THE RECENT VETIGASTROPODA

A cladistic analysis of the shell microstructures of RecentVetigastropoda demonstrates, it is more parsimonious to assumecrossed lamellar shell structures, rather than nacreous, areplesiomorphic. The clade Vetigastropoda is characterized byhaving intersected crossed platy shell structure. The analysissuggests resolution of the internal phylogeny of the Vetigastropoda,particularly separation of a crown group with nacreous shells,composed of Haliotidae, Pleurotomariidae, Seguenziidae, Stomatellidae,Trochidae, and Turbinidae, separated from an unresolved gradewith cross lamellar structures, Fissurellidae, Osteopeltidae,and Pseudococculin-idae. It is also suggested, that Phasianellidaeis neither part of nor sister taxon to Turbinidae.     

N40, a novel nonacidic matrix protein from pearl oyster nacre, facilitates nucleation of aragonite in vitro

A novel nonacidic matrix protein from pearl oyster nacre has been purified by cation-exchange chromatography. It was designated N40 for the nacreous protein of approximately 40 kDa. On the basis of the extraction method (with Tris-buffered Milli-Q water) and amino acid compositions (Gly- and Ala-rich), N40 was inferred to be a conventional "insoluble matrix protein". Crystallization experiments showed that N40 could facilitate the nucleation of aragonite drastically. So far, among the macromolecules that have been purified from the shell, N40 is an exclusive protein that can nucleate aragonite by itself, without the need for adsorption to a substrate. Thus, the present study has proposed the possibility that the nonacidic shell protein (maybe a conventional "insoluble framework protein") can also directly participate in aragonite nucleation and even act as a nucleation site. It is a valuable supplement to the classic biomineralization theory, in which the soluble acidic proteins of the shell are generally believed to function as a nucleation site.     

Macromolecular structure of the organic framework of nacre in Haliotis rufescens: implications for growth and mechanical behavior

We have performed a macromolecular structural analysis of the interlamellar and intertabular parts of the organic framework of the nacreous part of the shell of Haliotis rufescens, including the identification of structural chitin. Using histochemical optical microscopy we have mapped the locations of carboxylates and sulfates of proteins and chitin on the surfaces and within the core of the interlamellar layers and the intertabular matrix that together form the external organic matrix of composite nacre. This extends the earlier work of Nudelmann et al. [Nudelman, F., Gotliv, B.A., Addadi, L. and Weiner, S. 2006. Mollusk shell formation: mapping the distribution of organic matrix components underlying a single aragonite tablet in nacre. J. Struct. Biol. 153, 176–187] and Crenshaw and Ristedt [Crenshaw, M.A., Ristedt, H. 1976. The histochemical localization of reactive groups in septal nacre from Nautilus pompilius. In: Omori, M., Watabe, N. (Eds.) The Mechanisms of Biomineralization in Animals and Plants. Tokai University Press, Toyko] on Nautilus pompilius. Our mapping identifies distinct regions, defined by the macromolecular groups, including what is proposed to be the sites of CaCO₃ nucleation and that play a key role in nacre growth. Using AFM scanning probe microscopy we have identified a fibrous core within the framework that we associate with chitin. The structural picture that is evolved is then used to develop a simple structural model for the organic framework which is shown to be consistent with mechanical property measurements. The role of the intracrystalline matrix within the nacre tablets in mediating nacre’s mechanical response is noted within the framework of our model.