Phosphatic Shell Formation in Atremate Brachiopods

The atremate brachiopods are unique in that they possess shellsof calcium phosphate. In Lingula adamsi and Gloltidia pyramidata,the shell mineral is (CO₃ + F)-containing apatite and is crystallo-chemicallysimilar but not identical to the mineral francolite. The shellof Glottidia consists of a thin periostracum, a mineralizedthick primary layer, and alternating mineralized layers andless mineralized chitin layers. The basic unit of the crystalsis the spherulite. Proteinaceous and glycosaminoglycan (GAG)matrices are present in the primary and mineralized layers.The GAGS in the chitin layer are morphologically different fromthose of the other layers. The GAGS are intimately associatedwith the apatite crystals. Shell formation appears to be mediated by three different typesof cells in the outer epithelium. The cells primarily involvedin the mineral formation are characterized by many vacuoleswith electron-dense granular inclusions containing Ca, P, andS. The connective tissue at the anterior edge of the mantlealso contains fine granules with Ca, P, and S. Those granulesare considered to be a mineral reserve for shell formation.Some problems of the mechanisms of shell formation are discussed.     

Functional Morphology of Perihelia conradi Relative to Shell-Penetration

The pholad, Penitella conradi, is found along the Californiacoast in the calcareous shell of the abalone, Haliotis rufescens.These pholads penetrate the abalone shell, and when they breakthrough the inside of the shell they cease boring, secrete acallum, and then become sexually mature. The normal adult isa stenomorphic form,defined by Bartsch as an animal whose sexualmaturity is induced by over-crowding or insufficient substratumin which to bore. In the case of P. conradi, sexual maturityis always induced by the spatial limits of the substratum, thatis, the relatively thin abalone shell. The role of mechanical abrasion by the valves of P. conradiis minor. Experiments indicate that the teeth of P. conradiare worn at a greater rate than the polished shell of the abalone. The boring process in P. conradi proceeds mainly by chemicaldissolution of the calcareous substrate. The pathway of thesolvents is unknown. It may be through the organic matrix, orthe solvent may react directly with the crystals. Mechanicalabrasion helps to remove loosened crystals and/or organic matrixwhich are then carried to the exterior by the ciliary currentsflowing in through the pedal gape and out through the exhalentsiphon.     

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.     

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.     

Microstructure and crystallographic-texture of giant barnacle (Austromegabalanus psittacus) shell

Barnacle shell is a very complex and strong composite bioceramic composed of different structural units which consist of calcite 15 microcrystals of very uniform size. In the study reported herein, the microstructural organization of these units has been examinated in detail with optical and scanning electron microscopy, and X-ray diffraction techniques. These analyses showed that the external part of the shell has a massive microstructure consisting of randomly oriented crystals. Toward the interior, the shell became organized in mineral layers separated by thin organic sheets. Each of these mineral layers has a massive microstructure constituted by highly oriented calcite microcrystals with their c-axes aligned [(001) fibre texture] perpendicular to the organic sheets and the shell surface. Interestingly, in another structural unit, the shell shield, the orientation of the c-axis calcite crystals shifts from being perpendicular to being parallel to the shell surface across its thickness. This study provides evidence that the organic matrix is responsible for the organization of the shell mineral and exterts strong a strict control on the polymorphic type, size and orientation of shell-forming crystals.     

Mineral phase in linguloid brachiopod shell: Lingula adamsi

The linguloid brachiopod shell family has been the focus of several studies because of the similarity in the composition of the mineral phase of these shells to that of human bone. However, ultrastructural features of Lingula shells have not yet been fully demonstrated at high magnification using Transmission Electron Microscopy (TEM) and Electron Diffraction. Ultrastructural characterization of the mineral phase in Lingula shells will improve our understanding of the biomineralization processes and mineral/organic interaction in more complex systems such as in bone or in other human mineralized tissues. In this study, the mineral phase of Lingula adamsi was characterized using a combination of ultrastructural and crystallographic techniques. The results showed that L. adamsi shells consist of apatite crystals of varying size, shape, and orientation in different areas of the shell. The c-axis of apatite was parallel to the shell surface and crystals were organized in different laminae. Compared to trabecular bovine bone, L. adamsi shells demonstrated a higher crystallinity and a lower amount of carbonate and organic compounds. This study therefore demonstrated how dissimilar organic matrix between L. adamsi shell and trabecular bone can modify the ultrastructural characteristics of apatite crystals in these two biomineralized tissues.     

The cytology of the testaceous rhizopod Lesquereusia spiralis (Ehrenberg) Penard. I. Ultrastructure and shell formation

Ultrastructure and shell formation in the testaceous ameba, Lesquereusia spiralis, were investigated with both scanning and transmission electron microscopy and X-ray microanalysis. The nucleus, surrounded by a fibrous lamina, contains multiple nucleoli. The cytoplasm, containing a well developed granular endoplasmic reticulum, also contains remnants of starch granules in stages of digestion. Spherical aggregates of ribosome-like particles may be seen. Golgi complexes seem to produce both a nonordered fibrous material and an electron dense vesicle. Only the latter appears to bleb off from the Golgi complex. X-ray microanalysis demonstration of silicon in Golgi vesicles and in some dense vesicles suggests that the fibrous component of the cisternae may take up and concentrate silica to form the electron-dense component of the vesicles. Membrane-bound siliceous crystals are often seen adjacent to the Golgi, suggesting either a Golgi origin or platelet formation in vesicles after release from the Golgi complex. Both electron-dense bodies and siliceous platelets are released from the cell by a process similar to apocrine secretion and may be seen outside the cell in route to the shell during shell morphogenesis. Shell development involves fusion of electron-dense bodies to form a matrix, positioning of siliceous platelets in this matrix parallel to the shell surface, and development of a system of matrix chambers. A particulate glycoconjugate is released to the shell surface upon rupture of the matrix chamber.     

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.     

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.     

Studies on Shell Formation : VII. The Submicroscopic Structure of the Shell of the Oyster Crassostrea virginica

The submicroscopic structure of the growing surface of the shell of the oyster, Crassostrea virginica, was studied by means of shadowed replicas. The outer edge of the prismatic region consists of a fine grained matrix enclosing crystals, the surfaces of which show a finely pebbled structure. Crystal size varies continously from 0.01 µ to 8 µ. The matrix surface shows no evidence of fibrous structure. The outer portions of the prismatic region exhibit a tile-like arrangement of large crystals separated by granular matrix 0.02 to 0.08 µ in thickness. The exposed crystal surfaces have indentations of varying form which appear as roughly parallel grooves spaced at intervals of approximately 0.3 µ. The final form of this region is believed to result from the random distribution of crystal seeds, which grow without orientation and through coalescence and growth come into contact, producing polygonal areas. The crystal arrangement of the nacreous region is one of overlapping rows of crystals in side to side contact, and with one end of each crystal free, permitting continued increase in length. Crystal angles and plane indices are presented.     

STUDIES ON SHELL FORMATION : IX. An Electron Microscope Study of Crystal Layer Formation in the Oyster

Details of crystal growth in the calcitostracum of Crassostrea virginica have been studied with the purpose of analyzing the formation of the overlapping rows of oriented tabular crystals characteristic of this part of the shell. Crystal elongation, orientation, and dendritic growth suggest the presence of strong concentration gradients in a thin layer of solution in which crystallization occurs. Formation of the overlapping rows can be explained by three processes observed in the shell: a two-dimensional tree-like dendritic growth in which one set of crystal branchings creeps over an adjacent set of branchings; three-dimensional dendritic growth; and growth by dislocation of crystal surfaces. Multilayers of crystals may thus be formed at one time. This is favored by infrequent secretion of a covering organic matrix which would inhibit crystal growth. The transitional zone covering the outer part of the calcitostracum and the inner part of the prismatic region is generally characterized by aggregates of small crystals with definite orientation. Growth in this zone appears to take place in a relatively homogeneous state of solution without strong concentration gradients. Thin membranes and bands of organic matrix were commonly observed in the transitional zone bordering the prismatic region. The membrane showed a very fine oriented network pattern.     

Two forms of apatite deposited during mineralization of the hen tendon.

Old hen tendon provides a model suitable for the study of calcification in an extracellular matrix. In the present study, we observed the mineralizing substances of hen tendon by scanning electron microscopy of plasma-osmium-coated specimens and by transmission electron microscopy of those processed by a plasma-polymerization film replica method. The mineralizing front area revealed a number of elliptical particles fused to each other and forming rod-like structures oriented parallel to collagen fibrils. The area of advanced mineralization possessed non-mineralizing cavities, in which tendon cells were likely to exist. At this site, we recognized a second form of mineral structure, one in which the crystals had a scale-like morphology and were deposited onto the major first-form mineral component. This crystal form was similar to hydroxyapatite synthesized under wet reaction conditions. These findings strongly suggest that the second form of mineral formed independent of collagen fibrils existed together with the predominant, collagen-dependent form of mineral. We speculate that cell membranes and an extremely slow mineralization process may contribute to the formation of this form of mineral during the mineralization process in the hen tendon.     

MECHANICAL AND MORPHOLOGICAL PROPERTIES OF THE SHELL OF SCROBICULARIA PLANA (DA COSTA) UNDER NORMAL AND STRESS CONDITIONS

Scrobicularia plana reacts to salinity stress by closing itsvalves and respiring anaerobically. The acidic products of respirationare buffered by calcium carbonate removed from the shell. Shellmass decreases by 15% over a period of three weeks, and thereis a similar reduction in shell strength. Erosion of the shell surface, indicating buffering, takes placequickly, pits can be seen in the valves of shells at the endof six hours of normal intertidal emersion. (Received 10 February 1982;     

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.     

Ultrastructure of Protein Crystals in Potato and Tomato Trichomes

Large protein crystals were located in the leaf and stem trichomesof Solanum tuberosum L. and Lycopersicon esculentum Mill. Inpotato the crystals ranged from 1.05 to 4.5 µm (average2.3 µm) on a side and in tomato they ranged from 1.16to 3.5 µm (average 2.7 µm) on a side. The proteinnature of the crystals was determined by histochemical stainingwith Coumassie brilliant blue R250 and aniline blue black. Thecrystalline structure of the inclusions was observed in ultrathinsections using electron microscopy. In potato, in cleared areasof the cytoplasm, ribosomes were observed scattered among proteinfilaments. The filaments were approximately 7 nm in diameter.Morphologically similar crystals were observed in the tomatotrichomes but the protein filaments were smaller (approximately4 nm in diameter). Protein crystals were also observed in palisadeand spongy parenchyma and epidermal leaf cells in tomato. Protein crystals, trichomes, potato, Solanum tuberosum L., tomato, Lycopersicon esculentum Mill., ultrastructure     

Amelogenesis: Transformation of a protein-mineral matrix into tooth enamel

During enamel formation, the organic enamel protein matrix interacts with calcium phosphate minerals to form elongated, parallel, and bundled enamel apatite crystals of extraordinary hardness and biomechanical resilience. The enamel protein matrix consists of unique enamel proteins such as amelogenin, ameloblastin, and enamelin, which are secreted by highly specialized cells called ameloblasts. The ameloblasts also facilitate calcium and phosphate ion transport toward the enamel layer. Within ameloblasts, enamel proteins are transported as a polygonal matrix with 5 nm subunits in secretory vesicles. Upon expulsion from the ameloblasts, the enamel protein matrix is re-organized into 20 nm subunit compartments. Enamel matrix subunit compartment assembly and expansion coincide with C-terminal cleavage by the MMP20 enamel protease and N-terminal amelogenin self-assembly. Upon enamel crystal precipitation, the enamel protein phase is reconfigured to surround the elongating enamel crystals and facilitate their elongation in C-axis direction. At this stage of development, and upon further amelogenin cleavage, central and polyproline-rich fragments of the amelogenin molecule associate with the growing mineral crystals through a process termed “shedding”, while hexagonal apatite crystals fuse in longitudinal direction. Enamel protein sheath-coated enamel “dahlite” crystals continue to elongate until a dense bundle of parallel apatite crystals is formed, while the enamel matrix is continuously degraded by proteolytic enzymes. Together, these insights portrait enamel mineral nucleation and growth as a complex and dynamic set of interactions between enamel proteins and mineral ions that facilitate regularly seeded apatite growth and parallel enamel crystal elongation.     

SYSTEMATIC REVISION OF PATELLOIDA PYGMAEA (DUNKER, 1860) (GASTROPODA: LOTTIIDAE), WITH A DESCRIPTION OF A NEW SPECIES

Patelloida pygmaea (Dunker) and its closely allied species,P. heroldi (Dunker) and P. conulus (Dunker) have caused nomenclaturalconfusion because of their variable shell morphology and distinctivehabitats. According to current nomenclature, these species ofPatelloida have been synonymized and treated as one specieswith two ecological forms. Patelloida pygmaea lives on the shellof Crassostrea gigas (Ostreidae), P. pygmaea form heroldi occurson intertidal rocks on sheltered shores and P. pygmaea formconulus is found on the shell of Batillaria multiformis (Batillariidae).Their taxonomic relationships and possible species status are,however, unclear. Using two mitochondrial genes (fragments ofCOI and 16S ribosomal RNA; total 1192 sites), we analysed 88specimens of P. pygmaea, P. pygmaea form heroldi and P. pygmaeaform conulus from 37 localities in East Asia. In the resultingmolecular phylogenetic trees, the specimens of Patelloida fallinto four clades with high bootstrap probabilities; these cladescorrespond taxonomically to P. pygmaea, P. conulus, P. heroldiand a fourth previously unrecognized taxon, Patelloida ryukyuensisn. sp., described here. A minimum-spanning network for 29 uniquemitochondrial COI haplotypes obtained from 45 specimens in thesame bay in central Japan form three distinct clusters, consistingof P. pygmaea, P. conulus and P. heroldi, respectively. Thissuggests that reproductive isolation has been established betweeneach group. A detailed examination of radular and shell morphologiesof the four taxa clarifies the morphological distinction betweenthese species. The four species form a rather young clade inthe genus Patelloida that diverged during the Pliocene. Theyprovide an example of habitat segregation in a restricted marineenvironment. (Received 21 December 2004; accepted 11 March 2005)     

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.     

Oriented crystallization of octacalcium phosphate into beta-chitin scaffold

Composites of beta-chitin with octacalcium phosphate (OCP) or hydroxylapatite (HAP) were prepared by precipitation of the mineral into a chitin scaffold by means of a double diffusion system. The beta-chitin was obtained from the pen of the Loligo sp. squid. Only oriented precipitation of OCP was observed. The OCP crystals with the usual form of (001) blades grow inside chitin layers preferentially oriented with the [100] faces parallel to the surface of the squid pen and were more stable to the hydrolysis to HAP with respect to that precipitated in solution. Reasons are given why mechanical factors are thought to be the predominant cause for the orientation of the OCP crystals with the a-axis almost normal to the chitin fibers. We conclude that in these in vitro experiments the compartmentalized space in the chitin governs the orientation of the crystals, even if epitaxial factors may play a role in the nucleation processes.