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.     

STRUCTURE OF THE CONCHIOLIN CASES OF THE PRISMS IN MYTILUS EDULIS LINNE

The prisms in the shell of Mytilus edulis Linné are calcite needles. Their small size and their thin conchiolin cases distinguish them from the prisms of many other species of mollusks. These Mytilus prisms have been studied with the electron microscope. The material consisted of positive replicas of surfaces of the prismatic layer, etched with chelating agents, and of preparations of tubular cases from decalcified prisms which were compared with the conchiolin from decalcified mother-of-pearl of the same species. In the replicas, the cases appear as thin pellicles in the intervals between the prism crystals. Both the prism cases and the nacreous conchiolin, disintegrated by exposure to ultrasonic waves and sedimented on supporting films, appear in the form of tightly meshed, reticulated sheets, described as "tight pelecypod pattern" in former studies on nacreous conchiolin of Mytilus. The results show that in the shell of this species the same conchiolin structure is associated with aragonite in mother-of-pearl and with calcite in the prismatic layer.     

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.     

SHELL MICROSTRUCTURE OF GASTROPODS FROM LAKE TANGANYIKA,AFRICA: ADAPTATION,CONVERGENT EVOLUTION,AND ESCALATION

Gastropod shells from Lake Tanganyika, with their heavy calcification, coarse noded ribbing, spines, apertural lip thickening and repair scars, resemble marine shells more closely than they resemble other lacustrine shells. This convergence between Tanganyikan and marine gastropod shells, however, is not just superficial. Scanning electron microscope (SEM) studies reveal that the Tanganyikan shells are primarily layers of crossed-lamellar crystal architecture (that is, needle-like aragonite crystals arranged into laths that are packed into sheets such that the aragonite needles of adjacent laths are never parallel). The number of crossed-lamellar layers can vary from one to four between different Tanganyikan gastropod species. In species with two or more crossed-lamellar layers, the orientation of the lamellae is offset by approximately 90° between the different layers. The number of crossed-lamellar layers in the shell wall is positively correlated with shell strength and with predation resistance. Three and four crossed-lamellar layers in the shell wall evolved several times independently within the endemic thiarid gastropod radiation in Lake Tanganyika. Repeated origins of three and four crossed-lamellar layers suggest that they may be specific adaptations by Tanganyikan gastropods to strengthen their shells as a defense against shell-crushing predators.     

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.     

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.     

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.     

Patterns of Expression in the Matrix Proteins Responsible for Nucleation and Growth of Aragonite Crystals in Flat Pearls of Pinctada fucata

The initial growth of the nacreous layer is crucial for comprehending the formation of nacreous aragonite. A flat pearl method in the presence of the inner-shell film was conducted to evaluate the role of matrix proteins in the initial stages of nacre biomineralization in vivo. We examined the crystals deposited on a substrate and the expression patterns of the matrix proteins in the mantle facing the substrate. In this study, the aragonite crystals nucleated on the surface at 5 days in the inner-shell film system. In the film-free system, the calcite crystals nucleated at 5 days, a new organic film covered the calcite, and the aragonite nucleated at 10 days. This meant that the nacre lamellae appeared in the inner-shell film system 5 days earlier than that in the film-free system, timing that was consistent with the maximum level of matrix proteins during the first 20 days. In addition, matrix proteins (Nacrein, MSI60, N19, N16 and Pif80) had similar expression patterns in controlling the sequential morphologies of the nacre growth in the inner-film system, while these proteins in the film-free system also had similar patterns of expression. These results suggest that matrix proteins regulate aragonite nucleation and growth with the inner-shell film in vivo.     

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.     

几种生物CaCO3霰石结晶的取向性

CaCO3结晶广泛分布于生物界,其主要结晶形式为方解石、霰石及球霰石。用X－射线衍射法对三角帆蚌及合浦珍珠母贝的珍珠层、墨鱼骨和大黄鱼耳石的CaCO3结晶进行测定,发现各样品均有一定取向性,以三角帆蚌和合浦珍珠母贝珍珠层的取向性为最强,墨鱼骨的取向性次之,大黄鱼耳石的取向性最小,以上材料粉末样的衍射分析表明,各样品对应d值间差异极小,均为X射线衍射卡（5－0453）所表征的CaCO3霰石结构。     

几种生物CaCO_3霰石结晶的取向性

ＣａＣＯ３结晶广泛分布于生物界,其主要结晶形式为方解石、霰石及球霰石。用Ｘ－射线衍射法对三角帆蚌及合浦珍珠母贝的珍珠层、墨鱼骨和大黄鱼耳石的ＣａＣＯ３结晶进行测定,发现各样品均有一定取向性,以三角帆蚌和合浦珍珠母贝珍珠层的取向性为最强,墨鱼骨的取向性次之,大黄鱼耳石的取向性最小,以上材料粉末样的衍射分析表明,各样品对应ｄ值间差异极小,均为Ｘ射线衍射卡（５—０４５３）所表征的ＣａＣＯ３霰石结构。     

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.     

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.     

Crystallographic characterization of the crossed lamellar structure in the bivalve Meretrix lamarckii using electron beam techniques

To understand the formation mechanism of crossed lamellar structures in molluskan shells, the crystallographic structural features in the shell of a bivalve, Meretrix lamarckii, were investigated using scanning electron microscopy, electron backscattered diffraction, and transmission electron microscopy with a focused ion beam sample preparation technique. Approximately 0.5 μm-thick lamellae (the second-order units) are piled up obliquely toward the growth direction to form the first-order unit and the obliquity is inverted between adjacent units along the shell thickness direction. The first-order units originate around the center of the shell, initially growing parallel to the shell and subsequently curving toward the inner or outer surfaces. The lamellae consist of aragonite granular and columnar layers, which group together to adopt the same crystal orientation forming crystallographic units (crystallites). Multiple {1 1 0} twins are common both in the granular and columnar layers. The crystallite c-axis is parallel to the columns and is inclined at angles 0–50° from the lamellar normal (dispersing among individual lamellae), toward the shell growth direction. Probably, the directions of the a- and b-axes are random in the lamellae, showing no specific orientation.     

Biomineralization in the presence of calcium-binding phosphoprotein particles

The innermost shell lamella, which coats the inner surface of the shells in the estuarine clam Rangia cuneata, is a dynamic structure with a variable composition. In some populations the lamella is a phosphoprotein-rich structure devoid of crystalline mineral, and in others it is a glucosamine-rich structure often containing barite (BaSO4) inclusions. Mineral depositions was artificially stimulated in Rangia containing glucosamine-rich lamellae by scratching the inner shell surface. After stimulation, the lamellae were transformed into phosphoprotein-rich structures in which aragonite (CaCO3) was deposited. The mineral grew in spherulitic and dumbbell-shaped clusters characteristic of aragonite precipitated from strictly inorganic solutions. This study demonstrates that phosphoprotein particles accumulate in the innermost shell lamella during stimulated biomineralization but neither inhibit mineral deposition nor influence the crystal habits. Since phosphoprotein particles are high capacity calcium-binding proteins, they may be the source and transport vehicle for the calcium ions utilized in shell mineralization.     

Electron probe analysis of strontium in mussel (Bivalvia,Mytilidae) shells: Feasibility of estimating water temperature

Electron microprobe step-scan analyses across the inner nacreous layer of a sectionedMytilus edulis shell revealed no long-term periodic (e.g., seasonal) variation in the concentration of strontium. Similarly, no significant difference was found between a specimen sampled in February (water temperature = 1.3 °C) and one sampled in August (water temperature = 18.0 °C) with regard to the concentration of strontium within the most recently deposited aragonite. Correlation of the amount of strontium within various nacreous regions of the shells of living or fossil mytilids with water temperatures (present or past) is probably not possible through the use of an electron probe, at least to the extent that strontium variation within the nacre ofMytilus edulis is representative of that in nacreous layers of all mytilids.     

Mineralogy of shells from two freshwater snails Belgrandiella fontinalis and B kuesteri

X-ray powder diffraction (XRD) was used to study the mineral composition of shells of snails Belgrandiella fontinalis and Belgrandiella kuesteri collected from three freshwater springs in northeastern Slovenia. The fractions of aragonite, calcite, dolomite and quartz in particular shells were determined. The analysed shells consisted of two or more distinct inorganic layers. The outer shell layer for both species and all sampling localities contained aragonite. The outer layer of B. fontinalis collected at one locality, also contained a small fraction of calcite ( approximately 1 molar%) besides the dominant aragonite. Calcite was identified in the inner layer(s) of both species (2 to 3 molar%), while quartz was found only in B. kuesteri (5-7 molar%). However, both species sampled at one locality showed the presence of dolomite (approx. 20 molar%) in the inner layer(s). The presence of dolomite in the shells of adult gastropods and even molluscs is unusual. A possible formation mechanism and specific ecological factor that could influence the precipitation of dolomite in the shells of different Belgrandiella species is discussed.     

Irregular calcareous sheets preserved in a conch of the Cretaceous ammonoid Hypophylloceras from Hokkaido,Japan

The mantle tissue is essential for understanding the diverse ecology and shell morphology of ammonoid cephalopods. Here, we report on irregular calcareous sheets in a well-preserved shell of a Late Cretaceous phylloceratid ammonoid Hypophylloceras subramosum from Hokkaido, Japan, and their significance for repairing the conch through the mantle inside the body chamber. The sheets are composed of nacreous layers arranged parallel to the irregularly distorted outer whorl surface. The nacreous sheets formed earlier are unevenly distributed and attached to the outer shell wall locally, whereas the last formed sheet covers a wide area of the outer shell wall. The absence of any interruption of ribbing around the irregular area suggests that these sheets were secreted inside the body chamber from the inner mantle. Gross morphological and X-ray computed tomography observations revealed that the spacing of septal formation was not affected by this event. The complex structure of the irregular sheets suggests a highly flexible mantle inside the body chamber.