The outer mantle epithelium of Haliotis tuberculata (Gastropoda Haliotidae): an ultrastructural and histochemical study using lectins

The mantle of molluscs has been the subject of many studies as it is the organ that forms the shell. Microscopic studies in particular focus on the outer mantle epithelium, but few studies address this epithelium in a histochemical way. In this study, the outer mantle epithelium in adult specimens of Haliotis tuberculata is studied, that is, in specimens involved in maintaining and repairing the shell rather than in generating it. The epithelial cells are studied by scanning (SEM) and transmission electron microscopy (TEM), and by histochemical techniques, including the use of lectins for their biochemical characterization. The epithelium is composed of pigmented epidermal cells with small microvilli and junctional complexes. It furthermore contains a few ciliated cells, as well as two types of secretory cells which differ in the ultrastructural appearance of their secretory granules and their glycoconjugate content. Histochemical study shows secretory cells containing sulphated glycoconjugates such as glycosaminoglycans or mucins rich in N‐acetylgalactosamine and N‐glycoproteins rich in fucose. Furthermore, the apical regions of the epidermal cells are positive for lectins that label fucose, mannose and N‐acetylglucosamine. The role of epithelial cells in the synthesis of structural components of the shell is discussed.     

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.     

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.     

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.     

An ultrastructural study of the mantle of the barnacle, Elminius modestus Darwin in relation to shell formation

The fine structure of the mantle and shell of the barnacle, Elminius modestus Darwin has been examined by electron microscopy. The epithelial cells along the outer face of the mantle differ in size, shape, and organelle complexity according to the different components of the shell they secrete. The shell consists of a non-calcareous basis and calcareous mural and opercular plates which are connected by a flexible opercular hinge. Both the basis and opercular hinge are composed of two main units: an outer cuticulin layer and a lamellate component of well ordered arched fibrils. During the deposition of the latter structures morphological changes in the cells occur which may be correlated with the moulting cycle. Preliminary results show that the calcareous plates are covered by an outer epicuticle, which is bordered by a cuticulin layer; the inner calcareous component, consists of an orderly arrangement of organic matrix envelopes within which crystals may be initiated.

The cells lining the inner surface of the mantle are uniform in appearance with a thin cuticle at their free surface which lines the body cavity. The latter structure of the cuticle and manner of its deposition are similar to those of the basis and opercular hinge. Separating the outer and inner mantle epithelial cells is connective tissue which comprises several differing cell types. The possibilities are discussed of the rôle these cells may play in shell deposition. The modes by which underlying cells secrete the different shell components and the cuticle lining the inner face of the mantle, are also discussed.     

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。     

Shell formation and calcium transport in the barnacle Chthamalus fragilis

The mantle epithelium of the barnacle Chthamalus fragilis (Darwin) exhibits several ultrastructural features which may serve to regulate the calcification process. At the basis-mural plate and intermural plate junctions where rapid shell growth occurs, cells are characterized by long apical cytoplasmic projections and large intercellular spaces. These features may increase the functional surface area of the epithelium and enable more rapid deposition of calcium. The cells underlying the general shell surfaces contain numerous electron-dense inclusion bodies and show frequent cellular disintegration near the growing shell interface. Release of the granular contents of these inclusion bodies has been observed in both disintegrating and non-disintegrating cells. X-ray microanalysis revealed significantly higher calcium levels in the inclusion bodies than in the surrounding cytoplasm. This suggests a calcium transport role for these inclusion bodies. Cellular debris produced as a result of the disintegration of the mantle cells near the shell may play some role in the formation of the organic matrix of the shell. The presence of large numbers of mitochondria and well-developed apical microvilli in the cells of the inner mantle epithelium suggest that these cells serve to transport calcium into the mantle from the ambient sea water.     

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.     

Epithelial moulds on the shells of the early Palaeozoic brachiopod Lingulella

A fine hexagonal network of microscopic ridges has been discovered on the internal shell surface of the cosmopolitan Lower Palaeozoic inarticulate genus Lingulella. The micro-ornamentation has been recognised on all well-preserved specimens examined, from Cambrian and Ordovician successions in Ireland, North America, Gt. Britain, Spitzbergen, and China. Examination of the mantle of the related Recent genus Lingula revealed that the outer epithelial cells are arranged in a hexagonal close-packed pattern comparable in size and form to the micro-ornamentation in the fossil shells. This fact, along with the discovery of a polygonal pattern on some organic layers in the Lingula shell, suggests that the micro-ornamentation in fossil Lingulella is the mould of the outer epithelium responsible for shell secretion. No trace of epithelial moulds has yet been found in specimens of Lingula from younger Palaeozoic successions. This difference may prove to be a useful diagnostic feature in distinguishing linguloid inarticulates from one another. Epithelial moulds , Lingulella, Palaeoglossa, Lingula, Cambrian, Ordovician, Recent.     

Ultrastructure of the metanephridia of Terebratulina retusa and Crania anomala (Brachiopoda)

Adult specimens of Terebratulina retusa and Crania anomala have one pair of metanephridia. Each metanephridium is composed of a ciliated nephridial funnel (nephrostome) and an outleading nephridial canal, thus, these organs are open ducts connecting the metacoel of the animal with the outer medium. In both species, the inner side of a nephrostome is lined by a columnar monociliated epithelium which contacts the coelothel within one of the two ileoparietal bands. The coelothel contains basal filaments (in C. anomala these are definite myofilaments). The canal epithelium also consists of monociliated columnar cells which differ from the nephrostome epithelial cells in size and some cell components. Within the nephropore, the canal epithelium makes contact with the so-called inner mantle epithelium which lines the mantle cavity. The nephrostome epithelial cells and the canal epithelial cells never contain any contractile filaments. There are always continuous transitions between these different epithelia and distinct borders cannot be observed. The present results, especially in comparison to Phoronida, do not contradict the hypothesis of a coelothelially derived nephridial funnel and an ectodermal nephridial duct in Brachiopoda. But with regard to the differences between Phoronida and Brachiopoda (larval protonephridia and podocytes in the adults are unknown in Brachiopoda), further investigations have to be done to test the hypothesis of such heterogeneously assembled metanephridia.     

Calcium localization in the shell-forming tissue of the freshwater snail,Biomphalaria glabrata: a comparative study of various methods for localizing calcium

Summary The routes calcium might take across the mantle to the shell have been investigated with various electron-microscopical techniques in the freshwater snailBiomphalaria glabrata (Planorbidae, Basommatophora).In chemically-fixed tissue, calcium was precipitated with a tannic acid-antimonate technique in predominantly the intercellular spaces of the outer mantle epithelium and the interstitium below it. Some vacuoles of the outer mantle epithelium and one type of mucus cell in the inner mantle epithelium also contained precipitate. The presence of calcium in the precipitates was proved by electron energy loss spectroscopy combined with electron spectroscopic imaging. Incubation with lead acetate and uranyl acetate revealed binding-sites for calcium in the intercellular spaces of the epithelia interstitium and the mucus cells of the inner mantle epithelium. Precipitates were also seen after all incubations in the calcium spherites of the connective tissue.The concentrations of calcium and other elements were analysed in freeze-dried ultrathin sections of cryofixed mantle tissue by means of energy-dispersive X-ray microanalysis. Only in mitochondria of the musculature could high amounts of calcium and phosphorous be detected.     

大珠母贝外套膜表皮细胞的超微结构

利用透射电系统地观察大珠母贝的外套有皮细胞，结果表明，细胞可分为５种，即柱状表皮细胞、凸细胞、电子透明大粒细胞、电子稠密粒细胞和电子透明小粒细胞。它们在不同区域的分布、形态和数量变化与外套膜的功能分化密切相关，尤其是与贝壳组分的分泌有关。结缔组织中也分布着许多闰细胞和电子稠密粒细胞，它们可作变形运动进入表皮层。     

The chiton gill: Ultrastructure in Chiton olivaceus (Mollusca,Polyplacophora)

Gills of Chiton olivaceus, a primitive mollusc, are relatively simple in their structure and ultrastructure but are well adapted to a life in the intertidal zone. In contrast to some other molluscs, there is no differentiation of the gill epithelium into functional regions other than respiratory ones. Ciliation of the epithelium in certain areas may optimize water flow from the outer to the inner part of the mantle cavity. The hemolymph sinuses are oriented so that hemolymph flows in the opposite direction. Interstitial cells link epithelial cells with nerve endings. Muscle cells as well as the collagenous matrix in the connective tissue differ within the main gill axis and the lateral lamellae. The life cycle of immunoactive cells within the connective tissue and the hemolymph is described.     

Fine Structure of the Midgut and Hindgut in Lepidocampa weberi (Insecta,Diplura)

The fine structure of the alimentary canal, especially the midgut and hindgut of Lepidocampa weberi (Diplura: Campodeidae) is described. The general organization of the canal is similar to that of Campodea. The midgut epithelium is composed of columnar apical microvillated cells. Each nucleus contains a single intranuclear crystal. Close to the pyloric region, the posterior midgut cells are devoid of microvilli and intranuclear crystals. There is no special pyloric chamber as in Protura or pyloric cuticular ring as in Collembola but a morphological transformation from midgut to hindgut cells. Eight globular Malpighian papillae, consisting of distal microvillated cells and flat proximal cells, open into the gut lumen via ducts formed by hindgut cells. The structure of the hindgut is complicated and can be divided into three segments. The anterior hindgut cells have an irregular shape and compact cytoplasm. A striking interdigitation between the large bottle-shaped epithelial cells and longitudinal muscle cells occurs in the middle segment of the hindgut. The thick cuticle gives rise to long spikes projecting into the gut lumen. The posterior hindgut cells possess the morphological features for water reabsorption. Some hypotheses are advanced about the function of the different regions of the gut.     

Mantle histology and histochemistry of three pearl oysters: Pinctada persica,Pinctada radiata and Pteria penguin

Shells in pearl oysters are produced by the mantle which is also used as a graft in pearl operations. Here, we investigate the mantle structure of a new pearl oyster species of the Persian Gulf, Pinctada persica, and compare it to two other pearl-producing species, Pinctada radiata and Pteria penguin. The anterior, ventral and posterior segments of the mantle edge of each valve were fixed, and tissue sections were stained with haematoxylin and eosin. A new pentachrome method and PAS-alcian blue staining were used to characterise the different mucosubstances. The mantle edges were found to have an outer, middle and inner fold, which have different morphology in each species. The mantle edge is lined by cuboidal and columnar epithelia, and interspersed among these epithelial cells we found mucous cells and cells that contained brown granules. The outer and middle folds of the two Pinctada species show different shapes to that of Pteria penguin. Most of the mucous cells in the mantle contain acidic mucosubstances and small amounts of mixed acidic-neutral mucosubstances were observed in the middle and inner fold of Pinctada persica. This study reveals that the mantle edges of the three species possess similar cellular structure, but vary in the shape of the folds.     

Calcineurin Plays an Important Role in the Shell Formation of Pearl Oyster (Pinctada fucata)

Calcineurin (CN) is a multifunctional protein involved in many important physiological processes in mammalians, but the function of CN in mollusks is still largely unknown. In the present study, through the shell regeneration system, the changes of enzymatic activity of CN were determined in the process of shell regeneration in pearl oyster Pinctada fucata. CN was activated immediately and continuously in the shell regeneration process. The speed of shell regeneration was measured and the ultrastructure of inner shell surface was observed by scanning electron microscopy after inhibiting CN by intramuscular injection of immunosuppresant cyclosporine A (CsA). The results showed that the speed of shell regeneration was delayed and the morphology of calcite and aragonite in the inner shell surface became abnormal when CN was inhibited by CsA. Meanwhile, RT-PCR analysis revealed that the expression of P. fucata BMP-2 in mantle tissue decreased with CsA injection. In vitro secretion level of proteoglycans (PGs) in primary cultures of mantle cells was also decreased when mantle cells were exposed to CsA. Taken together, our results, for the first time, show that CN is involved in the shell formation through regulating the expression of Pf-BMP-2 in mantle tissue, which controls the secretion of PGs/GAGs of the mantle epithelial cells.     

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.     

Zona Localization of Shell Matrix Proteins in Mantle of <Emphasis Type="Italic">Haliotis tuberculata</Emphasis> (Mollusca,Gastropoda)

Organic matrix from molluscan shells has the potential to regulate calcium carbonate deposition and crystallization. Control of crystal growth thus seems to depend on control of matrix protein secretion or activation processes in the mantle cells, about which little is known. Biomineralization is a highly orchestrated biological process. The aim of this work was to provide information about the source of shell matrix macromolecule production, within the external epithelium of the mantle. An in vivo approach was chosen to describe the histologic changes in the outer epithelium and in blood sinus distribution, associated with mantle cells implicated in shell matrix production. Our results characterized a topographic and time-dependent zonation of matrix proteins involved in shell biomineralization in the mantle of Haliotis.     

Shell repair process in the green ormer Haliotis tuberculata: a histological and microstructural study

In the present paper, juvenile and adult shells of the green ormer Haliotis tuberculata ('Oreille de Saint-Pierre') were perforated in a zone close to the shell edge and the shell repair process was followed at two levels: (1) by observing the histology of the calcifying mantle in the repair zone and (2) by analyzing with SEM the microstructure of the shell repair zone. Histological data clearly show the presence of calcium carbonate granules into the connective tissues, but not in the epithelial cells. This suggests that calcium carbonate granules are synthesized by sub-epithelial cells and actively transported through the epithelium to the repair zone, via a process which may be similar to that described by Mount et al. [Mount, A.S., Wheeler, A.P., Paradkar, R.P., Snider, D., 2004. Hemocyte-mediated shell mineralization in the eastern oyster. Science 304, 297-300]. Furthermore, SEM observations show that the repair zone exhibits different stratified microstructures (spherulitic, thin prismatic, blocklike, sub-nacreous, nacreous, foliated-like), some of which are not continuous (i.e. lenticular) along the repair zone. This suggests a complex secreting regime of the calcifying mantle and an elaborate geometry of the epithelium involved in shell repair.