A new model for periostracum and shell formation in Unionidae (Bivalvia, Mollusca)

The periostracum in Unionidae consists of two layers. The outer one is secreted within the periostracal groove, while the inner layer is secreted by the epithelium of the outer mantle fold. The periostracum reaches its maximum thickness at the shell edge, where it reflects onto the shell surface. Biomineralization begins within the inner periostracum as fibrous spheruliths, which grow towards the shell interior, coalesce and compete mutually, originating the aragonitic outer prismatic shell layer. Prisms are fibrous polycrystalline aggregates. Internal growth lines indicate that their growth front is limited by the mantle surface. Transition to nacre is gradual. The first nacreous tablets grow by epitaxy onto the distal ends of prism fibres. Later growth proceeds onto previously deposited tablets. Our model involves two alternative stages. During active shell secretion, the mantle edge extends to fill the extrapallial space and the periostracal conveyor belt switches on, with the consequential secretion of periostracum and shell. During periods of inactivity, only the outer periostracum is secreted; this forms folds at the exit of the periostracal groove, leaving high-rank growth lines. Layers of inner periostracum are added occasionally to the shell interior during prolonged periods of inactivity in which the mantle is retracted.     

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

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

Periostracal mineralization in the gastrochaenid bivalve Spengleria

We investigated the spikes on the outer shell surface of the endolithic gastrochaenid bivalve genus Spengleria with a view to understand the mechanism by which they form and evaluate their homology with spikes in other heterodont and palaeoheterodont bivalves. We discovered that spike formation varied in mechanism between different parts of the valve. In the posterior region, spikes form within the translucent layer of the periostracum but separated from the calcareous part of the shell. By contrast those spikes in the anterior and ventral region, despite also forming within the translucent periostracal layer, become incorporated into the outer shell layer. Spikes in the posterior area of Spengleria mytiloides form only on the outer surface of the periostracum and are therefore, not encased in periostracal material. Despite differences in construction between these gastrochaenid spikes and those of other heterodont and palaeoheterodont bivalves, all involve calcification of the inner translucent periostracal layer which may indicate a deeper homology.     

THE ANATOMY OF CALLOCARDIA HUNGERFORDI (BIVALVIA: VENERIDAE) AND THE ORIGIN OF ITS SHELL CAMOUFLAGE

Callocardia hungerfordi (Veneridae: Pitarinae) lives in subtidalmuds (220 to 240m C.D.) and is covered by a dense mat of mudthat, effectively, camouflages the shell. The periostracum is two layered. The inner layer is thick andpleated, the outer thin and perforated. From the outer surfaceof the inner layer develop numerous, delicate (0.5 mm in diameter),calcified, periostracal needles. These penetrate the outer periostracum.Mucus produced from sub-epithelial glands in the inner surfaceof the mantle, slides over the cuticle-covered epithelium ofthe inner and outer surfaces of the inner fold and the innersurface of the middle mantle fold to coat the outer surfaceof the periostracum and its calcified needles. Increased productionat some times produces solidified strands of mucus which bindmud and detrital material into their fabric to create the shellcamouflage. Calcified periostracal needles have been identified in othervenerids, including some members of the Pitarinae, but how theyare secreted and how the covering they attract is producedand, thus, how the whole structure functions, has not been explained. (Received 7 December 1998; accepted 5 February 1999)     

The ontogeny of shell secretion in Terebratalia transversa (Brachiopoda, Articulata). II. Formation of the protegulum and juvenile shell

The fine structure of the shell and underlying mantle in young juveniles of the articulate brachiopod Terebratalia transversa has been examined by electron microscopy. The first shell produced by the mantle consists of a nonhinged protegulum that lacks concentric growth lines. The protegulum is secreted within a day after larval metamorphosis and typically measures 140-150 micron long. A thin organic periostracum constitutes the outer layer of the protegulum, and finely granular shell material occurs beneath the periostracum. Protegula resist digestion in sodium hypochlorite and are refractory to sectioning, suggesting that the subperiostracal portion of the primordial shell is mineralized. The juvenile shell at 4 days postmetamorphosis possesses incomplete sockets and rudimentary teeth that consist of nonfibrous material. The secondary layer occuring in the inner part of the juvenile shell contains imbricated fibers, whereas the outer portion of the shell comprises a bipartite periostracum and an underlying primary layer of nonfibrous shell. Deposition of the periostracum takes place within a slot that is situated between the so-called lobate and vesicular cells of the outer mantle lobe. Vesicular cells deposit the basal layer of the periostracum, while lobate cells contribute materials to the overlying periostracal superstructure. Cells with numerous tonofibrils and hemidesmosomes differentiate in the outer mantle epithelium at sites of muscle attachments, and unbranched punctae that surround mantle caeca develop throughout the subperiostracal portion of the shell. Three weeks after metamorphosis, the juvenile shell averages about 320 micron in length and is similar in ultrastructure to the shells secreted by adult articulates.     

An electron microscope study of the formation of the periostracum in the freshwater bivalve, Anodonta cygnea

The periostracum and cells lining the periostracal groove of Anodonta cygnea L. have been studied at the electron microscope level. The cells lining the inner face of the outer fold differ in fine structural details, five cell types being recognized. Along the length of the outer surface of the middle fold, to which the periostracum is closely applied, only two cell types are evident. At the base of the periostracal groove the two epithelia are separated by a bulbous region containing a group of basal cells which initiate the periostracum. The periostracum, which is homogenously electron-lucid, originates in the intercellular space between a basal cell and the first cell of the middle fold. It increases in thickness in the periostracal groove due to the secretory activity of the different outer fold cells. The cells of the middle fold do not appear to be involved in periostracum formation.     

The relationship of the mantle and shell of the Polyplacophora in comparison with that of other Mollusca

The structure and growth of the polyplacophoran shell, characteristically consisting of eight plates surrounded by a girdle, is examined in the light of current views on the relationships of mantle and shell in the Bivalvia. The periostracum and outer and inner calcareous layers of the shell of the latter group are homologous with the cuticle, tegmentum and articulamentum respectively of the shell of the Polyplacophora. The margin of the mantle consists of a large marginal fold, which secretes the cuticular girdle, and a small accessory fold bearing mucous cells. These are functionally comparable with all three folds of the mantle margin found in other molluscs, although anatomically the marginal fold of the chitons probably represents only the inner surface of the outer fold of the mantle margin.
The cuticle not only forms the girdle, which bears calcified spines or spicules, but also extends between the shell plates. The principal part of the cuticle consists largely of mucopolysaccharide material but there is also a thin discrete inner region which is similar chemically to the periostracum of other molluscs. The cuticle, possibly without spines, probably covered the entire dorsal surface of a primitive placophoran and beneath this, plates developed. As these grew the cuticle became worn away except marginally and between the plates. It is suggested that a covering of mucus over the visceropallium may have been the forerunner of the molluscan shell and the possible evolutionary relationships of the shell throughout the Mollusca are discussed.     

The relationship of the mantle and shell of the Polyplacophora in comparison with that of other Mollusca

The structure and growth of the polyplacophoran shell, characteristically consisting of eight plates surrounded by a girdle, is examined in the light of current views on the relationships of mantle and shell in the Bivalvia. The periostracum and outer and inner calcareous layers of the shell of the latter group are homologous with the cuticle, tegmentum and articulamentum respectively of the shell of the Polyplacophora. The margin of the mantle consists of a large marginal fold, which secretes the cuticular girdle, and a small accessory fold bearing mucous cells. These are functionally comparable with all three folds of the mantle margin found in other molluscs, although anatomically the marginal fold of the chitons probably represents only the inner surface of the outer fold of the mantle margin.
The cuticle not only forms the girdle, which bears calcified spines or spicules, but also extends between the shell plates. The principal part of the cuticle consists largely of mucopolysaccharide material but there is also a thin discrete inner region which is similar chemically to the periostracum of other molluscs. The cuticle, possibly without spines, probably covered the entire dorsal surface of a primitive placophoran and beneath this, plates developed. As these grew the cuticle became worn away except marginally and between the plates. It is suggested that a covering of mucus over the visceropallium may have been the forerunner of the molluscan shell and the possibleevolutionary relationships of the shell throughout the Mollusca are discussed.     

Morphological studies on the mantle of the fresh-water mussel Amblema (UNIONIDAE): Scanning electron microscopy

The scanning electron microscope has been used to describe the surface morphology of the mantle in mantle-shell preparations from the fresh-water mussel Amblema. In some regions (adductor muscle insertions), the mantle is firmly attached to the shell. In other areas (along the main course of the mantle), transient adhesions between the outer mantle epithelial cells and the nacre appear to temporally further compartmentalize the extrapallial fluid possibly as a prerequisite for the initial crystallization phenomenon. At the mantle edge, as well as at the isthmus, the periostracum was seen to extrude from the periostracal groove. At the siphonal edge, peculiar fingerlike processes were identified; these may represent primitive photoreceptors. The epithelial cells of the outer mantle epithelium are all microvillated whereas those of the inner mantle epithelium are both microvillated and ciliated. Specific differences in surface morphology are described for various regions of the outer mantle epithelium. These may be related to precise regionalized functional differences of this tissue. Several functions of the mantle, in addition to shell formation, and based on its various morphologies, are also discussed.     

Morphological studies on the periostracum of the fresh-water mussel Amblema (uniondae): Light microscopy, transmission electron microscopy, and scanning electron microscopy

The structure of the periostracum in the fresh-water mussel Amblema has been described using light microscopy, transmission elec;ron microscopy, and scanning electron microscopy. The structure and evolutive course of the periostracum was studied along its entire length, from the periostracal groove until it forms the tough outer covering of the shell. At least five structurally and functionally distinct regions were identified. In addition, the periostracum itself was seen to be a multilayered structure consisting of three major layers which are themselves subdivided into minor layers. From these morphological observations, a regulatory role for the various periostracal layers in mineral trapping, nucleation, and the subsequent formation of the prismatic and nacreous layers of the shell can be postulated.     

Functional morphology of the mantle of Nautilus pompilius (Mollusca, Cephalopoda)

This study presents histological and cytological findings on the structural differentiation of the mantle of Nautilus pompilius in order to characterize the cells that are responsible for shell formation. The lateral and front mantle edges split distally into three folds: an outer, middle, and inner fold. Within the upper part of the mantle the mantle edge is divided into two folds only; the inner fold disappears where the hood is attached to the mantle. At the base of the outer fold of the lateral and front mantle edge an endo-epithelial gland, the mantle edge gland, is localized. The gland cells are distinguished by a distinct rough endoplasmic reticulum and by numerous secretory vesicles. Furthermore, they show a strong accumulation of calcium compounds, indicating that the formation of the shell takes place in this region of the mantle. Numerous synaptic contacts between the gland cells and the axons of the nerve fibers reveal that the secretion in the area of the mantle edge gland is under nervous control. The whole mantle tissue is covered with a columnar epithelium having a microvillar border. The analyses of the outer epithelium show ultrastructural characteristics of a transport active epithelium, indicating that this region of the mantle is involved in the sclerotization of the shell. Ultrastructural findings concerning the epithelium between the outer and middle fold suggest that the periostracum is formed in this area of the mantle, as it is in other conchiferan molluscs.     

Differentiation and Growth of the Brachiopod Mantle

Cell differentiation in the mantle edge of Notosaria, Thecidelhnaand Glottidia, representing respectively, the impunctate andpunctate calcareous articulate and chitinophosphatic inarticulatebrachiopods, is described. Comparison of electron micrographssuggests that outer epithelium which secretes periostracum andmineral shell, is separated from inner epithelium by a bandof "lobate" cells, of variable width, exuding an impersistentmucopolysaccharide film or pellicle. The lobate cells alwaysoccupy the same relative position on the inner surface of theouter mantle lobe; but the outer epithelium is commonly connectedwith the inner surface of the periostracum by papillae and protoplasmicstrands which persist during mineral deposition and ensure thatboth shell and attached mantle remain in situ relative to theoutwardly expanding inner surface of the outer mantle lobe.In the prototypic brachiopod, the lobate cells are likely atfirst to have occupied the hinge of the mantel fold but laterto have been displaced into their present position by the rigidoutward growing edge of the mineral shell.     

Ultrastructure and Cytochemistry of Periostracum and Mantle Edge of Biomphalaria glabrata (Gastropoda,Basommatophora)

Abstract Three layers of different electron density can be distinguished in the periostracum. Periostracal units of up to 900 nm length are merged into the outer fibrous layer and binding of gold-labelled lectin-WGA indicates the presence of chitin because it is labile to chitinase treatment. The periostracum is formed by the epithelia of the groove and the belt at the mantle edge. The distal and basal epithelium of the groove consists mainly of type A cells with an extended Golgi apparatus and apical vesicles. The presence of peroxidase and phenol oxidase indicates a function in tanning of the periostracum. In the proximal epithelium of the groove, type B cells with protruding apices add more material for periostracum formation. Type C cells secrete single periostracal units which are formed within single vesicles or larger vacuoles. Type D cells secrete electron-dense vesicles which also contain WGA-positive material. The distal cells of the belt are characterized by predominating strands of the rER while subapical vacuoles, to some of which WGA binds, dominate in the cells of the central part. In the belt, phenol oxidase and peroxidase can be localized in cisternae of the rER and the Golgi apparatus. Numerous control incubations indicate that, indeed, two different enzymes are localized.     

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.     

淡水贝类贝壳多层构造形成研究

对几种淡水贝（包括蚌、螺）进行形态及组织学观察，并通过实验方法重现贝壳三种物质，即：角质、棱柱质、珍珠质的生成过程，结果表明：外套膜外表皮细胞是由相同类型细胞组成，这些相同细胞在不同的作用条件下形成贝壳多层构造。     

Embryonic development of the shell in biomphalaria glabrata (Say).

During embryogenesis of the fresh water snail Biomphalaria glabrata (Say) (Pulmonata, Basommatophora) shell formation has been studied by light and electron microscopical techniques. The shell field invagination (SFI), the secretion of the first shell layers, the development of the shell-forming mantle edge gland and spindle formation have been investigated. During embryonic development at 28 degrees C environmental temperature, the shell field invaginates after 35 h. After 40 h the SFI is closed apically by cellular protrusions and scale-like precursors of the periostracum. The first electron translucent layer of the periostracum stems from electron dense vesicles of the cells which lie at the opening of the SFI. A second electron dense layer appears some hours afterwards. When the shell appears birefringent in the polarizing microscope (45 h of development) calcium can be detected in it using energy dispersive x-ray analysis. As calcification occurs the intercrystalline matrix appears under the periostracum and the SFI begins to open. In embryos of 60 h the mantle cavity appears at the left caudal side. When the mantle edge groove develops (65 h of development) lamellate units are added to the outer layer of the periostracum, but no distinct lamellar layer is formed in B. glabrata. In addition to the lamellar cell and the periostracum cell, a secretory cell can be observed in the developing groove. After 65 h of development, spindle formation starts and the shell begins to coil in a left hand spiral. After 5 days of development the embryos are ready to leave the egg capsules.     

Pathogenicity of Vibrio tapetis,the etiological agent of brown ring disease in clams

Brown ring disease (BRD) causes high mortalities in the introduced Manila clam Ruditapes philippinarum cultured in western Europe. The etiological agent of BRD, Vibrio tapetis, adheres to and disrupts the production of the periostracal lamina, causing the anomalous deposition of periostracum around the inner shell. Because the primary sign of BRD is found outside the soft tissues, the processes leading to death are not as obvious as those for internal pathogens. This study was designed to evaluate the pathogenicity of V. tapetis, in an attempt to help explain the mechanisms of mortality. We found high mortalities (up to 100%) for clams following the inoculation of V. tapetis into the extrapallial space (between mantle and inner shell) or the posterior adductor muscle of healthy R. philippinarum. Microscopy and immunological detection methods showed that the pathogen was rapidly eliminated from tissues and hemolymph of animals that survived the inoculation. In clams that died, the bacteria were found to have proliferated, resulting in severe tissue disruption. Bacteria were able to penetrate into tissues from the extrapallial space through the external epithelium of the mantle. In contrast, no mortalities were observed following injection of V. tapetis in the native European clam Ruditapes decussatus, which is resistant to BRD. This clam rapidly eliminated the bacterium from hemolymph and soft tissues. Clam mortality associated with BRD in the field is likely to result from the penetration of V. tapetis into the clam's extrapallial space through the disrupted periostracal lamina and from there into the soft tissues through the irritated mantle epithelium. Some bacteria also penetrate through the digestive epithelia. In either case, bacteria proliferate rapidly in the soft tissues, causing severe damage and subsequent death.     

Tube construction in the watering pot shell Brechites vaginiferus (Bivalvia; Anomalodesmata; Clavagelloidea)

The bizarre watering pot shells of the clavagellid bivalve Brechites comprise a calcareous tube encrusted frequently with sand grains and other debris, the anterior end of which terminates in a convex perforated plate (the ‘watering pot’). It has not proved easy to understand how such extreme morphologies are produced. Previously published models have proposed that the tube and ‘watering pot’ are formed separately, outside the periostracum, and fuse later. Here we present the results of a detailed study of the structure and repair of the tubes of Brechites vaginiferus which suggest that these models are not correct. Critical observations include the fact that the external surface of the tube and ‘watering pot’ are covered by a thin organic film, on to the inner surface of which the highly organized aragonite crystals are secreted. There is no evidence of a suture between the tube and the ‘watering pot’ or that the periostracum of the juvenile shell passes through the wall of the tube. Live individuals of B. vaginiferus are able to repair substantial holes in the tube or ‘watering pot’ by laying down a new organic film followed by subsequent calcareous layers. Brechites vaginiferus displays Type C mantle fusion, with the result that the whole animal is encased by a continuous ring of mantle and periostracum, thereby making it possible to secrete a continuous ‘ring’ of shell material. On the basis of these observations we suggest that watering pot shells are not extra‐periostracal but are the product of simple modification of ‘normal’ shell‐secreting mechanisms.     

REMOTE BIOMINERALIZATION IN DIVARICATE RIBS OF STRIGILLA AND SOLECURTUS (TELLINOIDEA: BIVALVIA)

Deposits composed of aragonite prisms, which were formed afterthe outer shell layer, have been found at the posterior steepslopes of divaricate ribs in two species of Strigilla and anothertwo of Solecurtus. These prisms have their axes oriented perpendicularto the outer shell surface and differ in morphology from fibresof the surface-parallel composite prisms forming the outer shell.They display crystalline features indicating that, unlike crystalsforming the outer shell surface, their growth front was free,unconstrained by the mantle or periostracum. These particulardeposits are called free-growing prisms (FGPs). In these generathe periostracum is clearly not the substrate for biomineralizationand, upon formation, does not adhere to the steep slope of ribs,but detaches at the rib peak and reattaches towards the posterior,just beyond the foot of the posterior scarps of ribs. In thisway, a sinus or open space developed between the internal surfaceof the periostracum and the outer shell surface along each steeprib slope. These spaces could remain filled with extrapallialfluid after the mantle advances beyond that point during shellsecretion. FGPs grow within this microenvironment, out of contactwith the mantle. Other species with divaricate ribs do not developFGPs simply because the periostracum adheres tightly to both ribslopes (which are never so steep as in Solecurtus and Strigilla).FGPs constitute one of the rare cases of remote biomineralizationin which aragonite is produced and direct contact with the mantlenever takes place. (Received 22 November 1999; accepted 20 February 2000)     

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

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