Ultrastructural studies of the mantle and the periostracal lamina in the manila clam, Ruditapes philippinarum

The four folds of the mantle and the periostracal lamina of R. philippinarum were studied using light, transmission and scanning electron microscopy to determine the histochemical and ultrastructural relationship existing between the mantle and the shell edge. The different cells lining the four folds, and in particular those of the periostracal groove, are described in relation to their secretions. The initial pellicle of the periostracum arises in the intercellular space between the basal cell and the first intermediate cell. In front of the third cell of the inner surface of the outer fold, the periostracal lamina is composed of two major layers; an outer electron-dense layer or periostracum and an inner electron-lucent fibrous layer or fibrous matrix. The role and the fate of these two layers differ; the outer layer will recover the external surface of the shell and the inner layer will contribute to shell growth.     

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.     

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.     

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 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.     

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.     

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.     

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.     

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)     

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.     

THE PALLIAL EYES OF CTENOIDES FLORIDANUS (BIVALVIA: LIMOIDEA)

The structure of the pallial eye in the Limidae has neverbeen elucidated properly, largely because they are difficultto see among the mass of surrounding mantle tentacles and becausethey are few, small, and lose their pigmentation when preserved.Possibly two eye types are present, simple cup-shaped receptorsin species of Lima, like those seen in the Arcoida, and morecomplex invaginated ones in Ctenoides. The pallial eyes (;18on both lobes) of Ctenoides floridanus are formed by invaginationof the middle mantle fold at the periostracal groove, so thatall its contained structures are derived from the outer andlight is perceived through the inner epithelia of this fold.The eye comprises a simple multicellular lens and a photoreceptiveepithelium beneath it of lightly pigmented cells and alternatingvacuolated, support cells. In some species of the Arcoidea, Limopsoidea and Pterioidea, pallialeyes occur on the outer mantle fold and thus beneath the periostracum(and shell). The pallial eyes of Ctenoides floridanus and otherpterioideans, e.g. species of the Pectinidae, occur on the middlefold and may thus have improved vision. In the Cardiodea, Tridacniidaeand Laternulidae (Anomalodesmata) pallial eyes occur on theinner folds. There is thus a loose phylogenetic trend, in which Ctenoidesis a critical link, of increasing eye sophistication correlatedwith the historical age of the clades possessing them. (Received 16 November 1999; accepted 20 January 2000)     

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.     

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 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.     

Direct innervation of the mantle edge gland by neurosecretory axons in Helisoma duryi (Mollusca: Pulmonata)

Summary The mantle edge gland of Helisoma duryi is innervated by neurosecretory axons from the pallial nerves. Synaptoid contacts occur between axons and gland cells, and there is ultrastructural evidence for the release of neurosecretory material. The mantle edge gland contributes to the deposition of periostracum during shell formation, and direct neurosecretory innervation may control shell growth and regeneration.Supported by a National Research Council of Canada Grant (A-4673) and Negotiated Grant D-61     

Ontogeny of the Molluscan Shell Field: a Review

In the gastropod, scaphopod, lamellibranch, and cephalopod gastrulae a thickened portion of the posttrochal region is referred to as the embryonic shell field. It invaginates and gives rise to the shell gland. In species with an at least temporarily external shell, the shell gland evaginates and again forms a shell field. In lamellibranchs, the shell field grows into two halves connected by the ligament-secreting isthmus. In polyplacophorans plate fields are produced without invagination. Slugs and endocochleate cephalopods overgrow the embryonic shell field to form an internal shell sac. The calcified part of the shell is secreted by the flattened central region. The periostracum has its origin in the permanently thickened peripheral region of the shell field. In many forms, this region is depressed in a periostracal groove. If the shell is external, the central region of flattened cells, the mantle roof, along with the two or three marginal folds of the free mantle edge and, in species with internal shell, the shell sac are parts of the mantle. The shell field descends from the first somatoblasts. Either of 2 d or 2 c alone is able to form the shell field. There are arguments that the formation of the embryonic shell field is not autonomic, but induced by the entoderm during a period of contact. The shell gland and the shell field grow by mitotic cell divisions. Cells secreting organic material are highly prismatic, have a well developed ergastoplasm and large dictyosornes, and contain much peroxidase. The secretion of calcium manifests itself in very flat cells, rich in alkaline phosphatase and glycogen. The shell gland and the rosette of ectocochleate conchifera together are homologous to the proximal part of the shell sac in slugs and endocochleate cephalopods.     

A hairy business—Periostracal hair formation in two species of helicoid snails (Gastropoda,Stylommatophora, Helicoidea)

In molluscs, the calcareous shell is covered externally by a thin organic layer, the periostracum. The periostracum of some pulmonate species is of special taxonomic interest because it bears distinct microscale architectures. Where and how these structures are formed is as yet unknown. Using histological sections through their shells, gelatin cuts, and live observations I studied the pattern by which the periostracal hair‐like projections in two helicoid land snail species are secreted and evenly arranged on the shell. The results indicate a complex mechanism: a hair is formed in the periostracal groove independently of the periostracum, after which it is attached to the edge of the shell, drawn out of the tissue, and finally swivelled to the upper side of the periostracum. Upon further growth of the periostracum, the hairs are finally fixed upright on the shell. J. Morphol. 2011. © 2011 Wiley‐Liss, Inc.     

Arenophilic mantle glands in the Laternulidae (Bivalvia: Anomalodesmata) and their evolutionary significance

The mantle margins of several anomalodesmatans bear multicellular arenophilic glands, the mucoid secretions of which attach sand grains and other foreign particles to the outer surface of the periostracum. These glands have been recorded for many of the anomalodesmatan families and are used as a key morphological character in recent attempts to unravel the evolutionary relationships within the Anomalodesmata. The glands occur in Laternula elliptica, L. truncata, L. boschasina and L. marilina, discharging from the top of muscular papillae at the distal tip of the siphons. The secretions are laid down as threads organized in longitudinal lines along the length of the periostracum that covers the siphonal walls. This is the first record of arenophilic mantle glands in members of the Laternulidae, a finding that not only broadens our current knowledge of the family's morphology, but assists in the reconstruction of anomalodesmatan evolutionary history.     

Light and electron microscope localization of beta-glucuronidase activity in the stomach and digestive gland of the marine gastropod Littorina littorea

Summary An azo dye technique was used to investigate localization of the acid hydrolase,-glucuronidase, at light and electron microscope level in the stomach and digestive gland of the marine periwinkleLittorina littorea. Activity for-glucuronidase was located principally within digestive cells of the digestive gland and also associated with the microvillous border and epithelial cells lining the stomach. At the light microscope level all digestive tubules showed activity which appeared essentially restricted to the large heterolysosomes of the digestive cells. However not all digestive cells showed activity. In the electron microscope, reaction product was apparent in all types of macrovesicle in the digestive cells although not all stained positively. Heterophagosomes typically showed reaction product around their periphery or associated with the electron opaque contents. Activity was commonly seen around the apical edge of heterolysosomes where merging of heterophagosomes into heterolysosomes was apparent. Reaction product was commonly located within small electron lucent vesicles which lined the internal membrane of the heterolysosomes but sometimes also associated with flocculent, electron opaque contents. In the stomach dense clusters of reaction product were visible in lysosomes in the basal region of the epithelial cells and in the large granular inclusions of the secretory cells.