TOPOGRAPHY OF THE ORGANIC COMPONENTS IN MOTHER-OF-PEARL

1. The topography of the organic components (conchiolin) has been investigated on positive, postshadow-cast, formvar, and carbon replicas of mother-of-pearl from shells of a Cephalopod, of two Gastropods, and of six Pelecypods. All these shells are characterized by a true nacreous inner shell layer. 2. The material included normal shell surfaces, fragments of cleavage obtained by fracture, and surfaces polished tangentially and transversally to the inner surface of the shells. Replicas of these surfaces were prepared before and after etching of graded heaviness, induced by a chelating agent (sequestrene NA 2, titriplex III). Micrographs of the successive steps of the process of corrosion have been recorded. 3. Corrosion unmasked, on the nacreous surfaces, organic membranes or sheets, running as continuous formations in between adjacent mineral lamellae, and separating the individual crystals of aragonite which are aligned in rows and constitute each lamella. 4. The interlamellar sheets of material exhibit a reticulated structure, which is especially visible in preparations orientated tangentially to the lamellae and to the tabular surface of the aragonite crystals. The pattern of this lace-like structure, different in the various species studied, appeared in the same species as closely similar to that reported previously in leaflets of thoroughly decalcified mother-of-pearl, dissociated by ultrasonic waves. The present results support former conclusions with regard to the existence of taxonomic differences between Cephalopods, Gastropods, and Pelecypods in the morphological organization of the organic phase within mother-of-pearl.     

Topography of the organic components in mother-of pearl

1. The topography of the organic components (conchiolin) has been investigated on positive, postshadow-cast, formvar, and carbon replicas of mother-of-pearl from shells of a Cephalopod, of two Gastropods, and of six Pelecypods. All these shells are characterized by a true nacreous inner shell layer. 2. The material included normal shell surfaces, fragments of cleavage obtained by fracture, and surfaces polished tangentially and transversally to the inner surface of the shells. Replicas of these surfaces were prepared before and after etching of graded heaviness, induced by a chelating agent (sequestrene NA 2, titriplex III). Micrographs of the successive steps of the process of corrosion have been recorded. 3. Corrosion unmasked, on the nacreous surfaces, organic membranes or sheets, running as continuous formations in between adjacent mineral lamellae, and separating the individual crystals of aragonite which are aligned in rows and constitute each lamella. 4. The interlamellar sheets of material exhibit a reticulated structure, which is especially visible in preparations orientated tangentially to the lamellae and to the tabular surface of the aragonite crystals. The pattern of this lace-like structure, different in the various species studied, appeared in the same species as closely similar to that reported previously in leaflets of thoroughly decalcified mother-of-pearl, dissociated by ultrasonic waves. The present results support former conclusions with regard to the existence of taxonomic differences between Cephalopods, Gastropods, and Pelecypods in the morphological organization of the organic phase within mother-of-pearl.     

Aragonitic dendritic prismatic shell microstructure in Thracia (Bivalvia,Anomalodesmata)

The shells of most anomalodesmatan bivalves are composed of an outer aragonitic layer of either granular or columnar prismatic microstructure, and an inner layer of nacre. The Thraciidae is one of the few anomalodesmatan families whose members lack nacreous layers. In particular, shells of members of the genus Thracia are exceptional in their possession of a very distinctive but previously unreported microstructure, which we term herein “dendritic prisms.” Dendritic prisms consist of slender fibers of aragonite which radiate perpendicular to, and which stack along, the axis of the prism. Here we used scanning and transmission electron microscopical investigation of the periostracum, mantle, and shells of three species of Thracia to reconstruct the mode of shell calcification and to unravel the crystallography of the dendritic units. The periostracum is composed of an outer dark layer and an inner translucent layer. During the free periostracum phase the dark layer grows at the expense of the translucent layer, but at the position of the shell edge, the translucent layer mineralizes with the units typical of the dendritic prismatic layer. Within each unit, the c‐axis is oriented along the prismatic axis, whereas the a‐axis of aragonite runs parallel to the long axis of the fibers. The six‐rayed alignment of the latter implies that prisms are formed by {110} polycyclically twinned crystals. We conclude that, despite its distinctive appearance, the dendritic prismatic layer of the shell of Thracia spp. is homologous to the outer granular prismatic or prismatic layer of other anomalodesmatans, while the nacreous layer present in most anomalodesmatans has been suppressed.     

THE STRUCTURE AND ORGANIZATION OF, AND THE RELATIONSHIP BETWEEN THE ORGANIC MATRIX AND THE INORGANIC CRYSTALS OF EMBRYONIC BOVINE ENAMEL

Electron microscope and electron diffraction studies of developing embryonic bovine enamel have revealed the organization of the organic matrix and the inorganic crystals. The most recently deposited inorganic crystals located at the ameloblast-enamel junction are thin plates, approximately 1300 A long, 400 A wide, and 19 A thick. During maturation of the enamel, crystal growth occurs primarily by an increase in crystal thickness. Statistical analyses failed to show a significant change in either the width or the length of the crystals during the period of maturation studied. Even in the earliest stages of calcification, the crystals are organized within the prisms so that their long axes (c-axes) are oriented parallel to the long axes of the prisms but randomly distributed about their long axes. With maturation of the enamel, the crystals become more densely packed and more highly oriented within the prisms. The organic matrix in decalcified sections of enamel is strikingly similar in its over-all organization to that of the fully mineralized tissue. When viewed in longitudinal prism profiles, the intraprismatic organic matrix is composed of relatively thin dense lines, approximately 48 A wide, which are relatively parallel to each other and have their fiber axes parallel to the long axes of the prisms within which they are located. Many of these dense lines, which have the appearance of thin filaments, are organized into doublets, the individual 48 A wide filaments of the doublets being separated by approximately 120 A. When observed in oblique prism profiles, the intraprismatic organic matrix is likewise remarkably similar in general orientation and organization to that of the fully mineralized tissue. Moreover, the spaces between adjacent doublets or between single filaments have the appearance of compartments. These compartments, more clearly visualized in cross- or near cross-sectional prism profiles, are oval or near oval in shape. Therefore, the appearance of the intraprismatic organic matrix (in longitudinal, oblique, and cross-sectional prism profiles) indicates that it is organized into tubular sheaths which are oriented with their long axes parallel to the long axes of the prisms in which they are located, but randomly oriented about their own long axes, an orientation again remarkably "blue printing" that of the inorganic crystals. The predominant feature of the walls of the tubular sheaths, when viewed in cross- or near cross-section, is that of continuous sheets, although in many cases closely packed dot-like structures of approximately 48 A were also observed, suggesting that the wall of the sheaths consists of a series of closely packed filaments. The 48 A wide dense lines (filaments) representing the width of the sheath wall were resolved into two dense strands when viewed in longitudinal prism profiles. Each strand was 12 A wide and was separated by a less electron-dense space 17 A wide. The intraprismatic organic matrix is surrounded by a prism sheath which corresponds in mineralized sections to the electron-lucent uncalcified regions separating adjacent prisms. Structurally, the prism sheaths appear to consist of filaments arranged in basket-weave fashion.     

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.     

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.     

A Comparative Study of the Shell Matrix Protein Aspein in Pterioid Bivalves

Aspein is one of the unusually acidic shell matrix proteins originally identified from the pearl oyster Pinctada fucata. Aspein is thought to play important roles in the shell formation, especially in calcite precipitation in the prismatic layer. In this study, we identified Aspein homologs from three closely related pterioid species: Pinctada maxima, Isognomon perna, and Pteria penguin. Our immunoassays showed that they are present in the calcitic prismatic layer but not in the aragonitic nacreous layer of the shells. Sequence comparison showed that the Ser-Glu-Pro and the Asp-Ala repeat motifs are conserved among these Aspein homologs, indicating that they are functionally important. All Aspein homologs examined share the Asp-rich D-domain, suggesting that this domain might have a very important function in calcium carbonate formation. However, sequence analyses showed a significantly high level of variation in the arrangement of Asp in the D-domain even among very closely related species. This observation suggests that specific arrangements of Asp are not required for the functions of the D-domain.     

Biomineralization plasticity and environmental heterogeneity predict geographical resilience patterns of foundation species to future change

Although geographical patterns of species' sensitivity to environmental changes are defined by interacting multiple stressors, little is known about compensatory processes shaping regional differences in organismal vulnerability. Here, we examine large‐scale spatial variations in biomineralization under heterogeneous environmental gradients of temperature, salinity and food availability across a 30° latitudinal range (3,334 km), to test whether plasticity in calcareous shell production and composition, from juveniles to large adults, mediates geographical patterns of resilience to climate change in critical foundation species, the mussels Mytilus edulis and M. trossulus. We find shell calcification decreased towards high latitude, with mussels producing thinner shells with a higher organic content in polar than temperate regions. Salinity was the best predictor of within‐region differences in mussel shell deposition, mineral and organic composition. In polar, subpolar, and Baltic low‐salinity environments, mussels produced thin shells with a thicker external organic layer (periostracum), and an increased proportion of calcite (prismatic layer, as opposed to aragonite) and organic matrix, providing potentially higher resistance against dissolution in more corrosive waters. Conversely, in temperate, higher salinity regimes, thicker, more calcified shells with a higher aragonite (nacreous layer) proportion were deposited, which suggests enhanced protection under increased predation pressure. Interacting effects of salinity and food availability on mussel shell composition predict the deposition of a thicker periostracum and organic‐enriched prismatic layer under forecasted future environmental conditions, suggesting a capacity for increased protection of high‐latitude populations from ocean acidification. These findings support biomineralization plasticity as a potentially advantageous compensatory mechanism conferring Mytilus species a protective capacity for quantitative and qualitative trade‐offs in shell deposition as a response to regional alterations of abiotic and biotic conditions in future environments. Our work illustrates that compensatory mechanisms, driving plastic responses to the spatial structure of multiple stressors, can define geographical patterns of unanticipated species resilience to global environmental change.     

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.     

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.     

Structure and composition of the boundary zone between aragonitic crossed lamellar and calcitic prism layers in the shell of Concholepas concholepas (Mollusca,Gastropoda)

Mollusc shells are composed of two or three layers. The main layers are well‐studied, but the structural and chemical changes at their boundaries are usually neglected. A microstructural, mineralogical, and biochemical study of the boundary between the inner crossed lamellar and outer prismatic layers of the shell of Concholepas concholepas showed that this boundary is not an abrupt transition. Localized structural and chemical analyses showed that patches of the inner aragonitic crossed lamellar layer persist within the outer calcitic prismatic layer. Moreover, a thin aragonitic layer with a fibrous structure is visible between the two main layers. 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. The changes in the secretory process in the mantle cells responsible for the shell layer succession are irregular and discontinuous.     

CONVERGENT SHELL MORPHOLOGY IN INTERTIDAL GASTROPODS

The functional morphology of shell infrastructure in 2 speciesof intertidal trochid was compared with that in 2 species ofnerite. The shell of Monodonta constrictais typical of the majorityof trochids. The shell is composed of 4 layers: a distal layer(calcite), anouter prismatic layer (aragonite), a nacreous layer(aragonite), and an oblique prismatic layer (aragonite). Monodontalabio lacks a distal layer and an oblique prismatic layer. Theoblique prismatic layer is replaced by an inner prismatic layerwhich forms an apertural ridge as a result of deposition andresorption. The shells of Nerita versicolor and N. tessellataconsistof 3 layers: an outer prismatic layer (calcite), a crossedlamellar layer (aragonite), and a complex crossed lamellar layer(aragonite). The complex crossed lamellar layer is covered withinclined platelets which superficially resemble the surfaceof the ique prismatic layer of trochids. In addition, the complexcrossed lamellar layer forms an apertural ridge which is similarin appearance to that of Monodonta labio. The outer surfaceof the mantle of Nerita versicolor and N. tessellata is throwninto a series of large folds which lie in contact with the inclinedplatelets of the omplex crossed lamellar layer. The interactionof the mantle folds with the inclined platelets is thought toserve as a rachet mechanism to aid in extension of themantle;a similar function has previously been proposed for trochids.The apertural ridges of Monodonta labio and Nerita are thoughtto prevent excessive desiccation when these gastropodsare exposedat low tide. ¹Contribution No. 56 of the Tallahassee, Sopchoppy & GulfCoast Marine Biological Association (Received 6 July 1979;     

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.     

Die Mikro- und Ultrastruktur abgedeckter Hohlelemente und die Conellen des Ammoniten-Gehäuses

Hollow floored spines in the shell ofKosmoceras (Kosmoceras) spinosum (Sow.) and the hollow floored keel ofEleganticeras elegantulum (Young & Bird) have been studied with the scanning electron microscope. In both cases the shell wall is complete in so far as it consists of the outer prismatic layer, the nacreous layer and at least the distal zones of the inner prismatic layer. Both types of hollow shell elements are separated from the lumen of the whorl by a floor which is made up by the proximal zones of the inner prismatic layer. This explains why conellae occur, with preference, along the floors of hollow spines and keels. The origin of primary aragonitic conellae and of secondary calcitic conellae is discussed as well as their dependence on structural properties of the corresponding shell layer, which is the inner prismatic layer. An attempt is made to reconstruct the way of formation of the floored hollow spines and the floored hollow keels by the mantle epithelium.     

Studies on Shell Formation : VIII. Electron Microscopy of Crystal Growth of the Nacreous Layer of the Oyster Crassostrea virginica

Electron microscope observations have been made by means of the replica method on growth processes of calcite crystals of the nacreous layer of the shell of the oyster, Crassostrea virginica. Layer formation is initiated by the secretion of a conchiolin matrix and the deposition of rounded crystal seeds on or in this material. In some areas crystal seeds are elongate and within a given area show a similar orientation, probably due to slower deposition. The seeds appear to increase in size by dendritic growth, and smaller seeds become incorporated into larger ones which come into contact to form a single layer. With further growth, crystals overlap, forming a step-like arrangement. The direction of growth is frequently different in neighboring regions. Crystal seeds deposited on crystal surfaces are usually elongate and oriented. Well developed crystals have a tabular idiomorphic form and are parallel in their growth. Rounded and irregular crystals were also observed. The crystals show reticular structure with units of the order of 100 A and striations corresponding with the rhombohedral axes of the crystals. The role of the mantle is discussed in relation to the growth patterns of crystals and shell structure.     

Expression of genes responsible for biomineralization of Pinctada fucata during development

This study compares the expression levels of nacrein, N16, MSI60, Prismalin-14, aspein and MSI31 genes during the ontogeny of Pinctada fucata. Several novel findings were obtained: 1) The early calcitic prismatic layer was distinguished as a thin membrane-like structure. 2) Initial formation of the nacreous layer started from the mantle pallial region at the age of 31 days. 3) 18S rRNA of P. fucata was determined to be more suitable as a real-time PCR reference gene compared with GAPDH and β-actin genes. 4) A relationship was recognized between the expression levels of the above six organic matrix genes and biomineralization of the larval shell. The lack of calcite in the shells of the veliger and pediveliger stages, when MSI31 and Prismalin-14 genes were expressed, makes a role of polymorph control by these genes less likely. The hypothetical involvement of N16 and MSI60 proteins in aragonitic nacreous layer formation was corroborated by the expression levels of N16 and MSI60 genes during ontogeny. Our results are important with respect to the control of CaCO₃ crystal polymorphism and shell microstructures in P. fucata.     

Breaking the long-standing morphological paradigm: Individual prisms in the pearl oyster shell grow perpendicular to the c-axis of calcite

Cross-sections of calcitic prismatic layers in mollusk shells, cut perpendicular to growth direction, reveal well-defined polygonal shapes of individual “grains” clearly visible by light and electron microscopy. For several kinds of shells, it was shown that the average number of edges in an individual prism approaches six during the growth process. Taking into account the rhombohedral symmetry of calcite, often presented in hexagonal axes, all this led to the long-standing opinion that calcitic prisms grow along the c-axis of calcite. In this paper, using X-ray diffraction and electron backscatter diffraction (EBSD), we unambiguously show that calcitic prisms in pearl oyster Pinctada margaritifera predominantly grow perpendicular to the c-axis. The obtained results imply that the hexagon-like habitus of growing crystallites may be not necessarily connected to calcite crystallography and, therefore, other factors should be taken into consideration. We analyze this phenomenon by comparing the organic contents in Pinctada margaritifera and Pinna nobilis shells, the later revealing regular growth of calcitic prisms along the c-axis.