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.     

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.     

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.     

Characterization of a Monoclonal Antibody Directed against Mytilus spp Larvae Reveals an Antigen Involved in Shell Biomineralization

The M22.8 monoclonal antibody (mAb) developed against an antigen expressed at the mussel larval and postlarval stages of Mytilus galloprovincialis was studied on adult samples. Antigenic characterization by Western blot showed that the antigen MSP22.8 has a restricted distribution that includes mantle edge tissue, extrapallial fluid, extrapallial fluid hemocytes, and the shell organic matrix of adult samples. Other tissues such as central mantle, gonadal tissue, digestive gland, labial palps, foot, and byssal retractor muscle did not express the antigen. Immunohistochemistry assays identified MSP22.8 in cells located in the outer fold epithelium of the mantle edge up to the pallial line. Flow cytometry analysis showed that hemocytes from the extrapallial fluid also contain the antigen intracellularly. Furthermore, hemocytes from hemolymph have the ability to internalize the antigen when exposed to a cell-free extrapallial fluid solution. Our findings indicate that hemocytes could play an important role in the biomineralization process and, as a consequence, they have been included in a model of shell formation. This is the first report concerning a protein secreted by the mantle edge into the extrapallial space and how it becomes part of the shell matrix framework in M. galloprovincialis mussels.     

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.     

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.     

The biology and functional morphology of Arca noae (Bivalvia: Arcidae) from the Adriatic Sea, Croatia, with a discussion on the evolution of the bivalve mantle margin

In the Croatian Adriatic, Arca noae occurs from the low intertidal to a depth of 60 m; it can live for > 15 years and is either solitary or forms byssally attached clumps with Modiolus barbatus. The shell is anteriorly foreshortened and posteriorly elongate. The major inhalant flow is from the posterior although a remnant anterior stream is retained. There are no anterior but huge posterior byssal retractor muscles and both anterior and posterior pedal retractors. The ctenidia are of Type B(1a) and the ctenidial–labial palp junction is Category 3. The ctenidia collect, filter and undertake the primary sorting of potential food in the inhalant water. The labial palps are small with simple re‐sorting tracks on the ridges of their inner surfaces. The ciliary currents of the mantle cavity appear largely concerned with the rejection of particulate material. The mantle margin comprises an outer and an (either) inner or middle fold. The outer fold is divided into outer and inner components that secrete the shell and are photo‐sensory, respectively. The latter bears a large number of pallial eyes, especially posteriorly. The inner/middle mantle fold of A. noae, possibly representative of simpler, more primitive conditions, may have differentiated into distinct folds in other recent representatives of the Bivalvia.     

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.     

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.     

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)     

Endocochliate embryo model in the Mesozoic Ammonitida

An endocochliate embryo model for the Mesozoic Ammonitida is proposed based on scanning electron microscopy of the ammonitella (= embryonic shell) stage of well‐preserved Japanese Cretaceous specimens belonging to nine species of five superfamilies. As in other specimens described previously, the ammonitella wall succeeding from the initial chamber ("protoconch") in the species examined consists of the inner prismatic, middle subprismatic and outer prismatic layers, with minute tubercles resting on the outer. Developmental patterns of these structures and their comparison with primary shell formation in modern Nautilus and Spirula suggest that the outer thin prismatic layer with microtubercles was secreted by the exterior epithelium after the completion of the main ammonitella wall by the interior shell gland. Thus, the early ammonite embryo might have had an endocochliate structural plan as in coleoids, and at the time of hatching the overlying mantle epithelium had shifted anteriorly to become an ectocochliate condition.     

Cloning,differential tissue expression of a novel hcApo gene,and its correlation with total carotenoid content in purple and white inner-shell color pearl mussel Hyriopsis cumingii

As a molecular carrier and storage protein, apolipoprotein (Apo) mediates the intracellular uptake of lipids, proteins, vitamins and carotenoids. In this study, we identified a novel Apo gene, designated hcApo, from the freshwater pearl mussel Hyriopsis cumingii. The complete hcApo cDNA consists of 4104 nucleotides with an open reading frame encoding 1155 amino acid residues. The hcApo protein contains a conserved lipoprotein N-terminal domain (LPD-N) that is a characteristic of the large lipid transfer protein (LLTP) superfamily. The hcApo mRNA is constitutively expressed in a wide range of tissues with the highest expression level in the liver. Moreover, differential expression analysis revealed that the hcApo gene is more highly expressed in the liver, kidney, mantle and gill of purple line mussels compared to white line mussels. In situ hybridization investigations of the precise expression site of hcApo mRNA in the mantle showed that hcApo mRNA is specifically expressed in the outer epithelial cells of the middle fold and the inner epithelial cells of the outer fold of the mantle, as well as throughout the outer epithelium of the outer fold and ventral mantle. Another very important finding is that significantly positive correlation existed between the hcApo gene expression level and the total carotenoid content in purple line mussels. These findings may provide a better understanding of the roles of hcApo in the molecular mechanisms of shell formation and coloring of H. cumingii.     

三角帆蚌Hc-GSK3β基因鉴定及内壳色性状相关SNP筛选

为了探究三角帆蚌(Hyriopsis cumingii)糖原合成激酶-3β(GSK3β)基因对壳色的影响,研究采用RACE技术获得Hc-GSK3β基因cDNA全长1867 bp,其中包含1261 bp的ORF区编码420个氨基酸, ORF中含有一个S＿TKc结构域,该结构域序列高度保守。组织差异表达分析发现Hc-GSK3β基因在紫色蚌鳃、斧足、内脏团和边缘膜组织中表达量高于白色蚌的表达量(P<0.05),且在斧足和边缘膜表达差异水平达到极显著(P<0.01),而在紫色蚌闭壳肌组织中表达量显著低于白色蚌(P<0.05)。原位杂交(ISH)实验结果显示在三角帆蚌外套膜的外褶、中褶、內褶、背膜区和腹膜区均有阳性信号产生,且在外褶的信号表达较强烈。该基因经重测序比较,共鉴定出6个SNP位点,其中在C+185A位点的CA基因型在紫色蚌的分布频率显著高于白色三角帆蚌(P<0.05);在紫色蚌中, T+341G位点TT基因型三角帆蚌内壳颜色参数b值显著低于TG基因型(P<0.05)。研究表明, Hc-GSK3β基因参与了三角帆蚌壳色形成,筛选的SNP标记可用于三角帆蚌壳...     

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.     

马氏珠母贝(Pinctada fucata martensii)PmHEX基因的功能研究

几丁质是软体动物贝壳有机框架的重要成分,其代谢在贝壳矿化中发挥重要作用。β-N-乙酰-己糖胺酶(HEX, EC3.2.1.52)是几丁质代谢的关键水解酶。为了探究马氏珠母贝β-N-乙酰-己糖胺酶(Pm HEX)(登录号:MF555152)在贝壳形成中的作用,本研究利用原位杂交(ISH)技术检测Pm HEX基因在外套膜的定位,结果显示Pm HEX的mRNA主要分布于外侧褶的外上皮细胞、中褶的内侧上皮细胞和内褶上皮细胞。利用RNAi技术抑制Pm HEX表达后,Pm HEX在边缘区和套膜区的表达量均显著下调;SEM观察发现实验组的棱柱层和珍珠层的微观结构都出现不同程度的紊乱。综上所述,Pm HEX可能通过影响几丁质代谢,参与马氏珠母贝贝壳棱柱层和珍珠层的矿化过程。     

The ontogeny of shell secretion in Terebratalia transversa (Brachiopoda, Articulata). I. Development of the mantle

The morphology of the mantle in free-swimming and metamorphosing larvae of the articulate brachiopod Terebratalia transversa has been examined by scanning and transmission electron microscopy. The mantle begins to form approximately 2 days after fertilization and subsequently develops into a skirtlike lobe that encircles the middle region of the larval body. A simple epithelium covers both the outer surface of the mantle lobe and the inner side situated next to the pedicle lobe of the larva. During metamorphosis, the mantle lobe is everted over the anterior end of the larva. Thus, the epithelium covering the outer part of the mantle lobe in the larva subsequently becomes the inner epithelium of the juvenile mantle. Similarly, the inner epithelium of the larval mantle lobe represents the future outer epithelium of the juvenile mantle. In free-swimming larvae, the prospective outer mantle epithelium contains two types of cells, called "lobate" and "vesicular" cells. Lobate cells initially deposit a thin layer of amorphous material, and vesicular cells produce ovoid multigranular bodies. Following settlement at about 5 days postfertilization, the vesicular cells secrete an electron-dense sheet that constitutes the basal layer of the developing periostracum. Within several hours to a day thereafter, reversal of the mantle lobe is rapidly effected, apparently by contractions of the pedicle adjustor muscles.     

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.     

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 MANTLE AND SHELL OF SOLEMYA PARKINSONI (PROTOBRANCHIA: BIVALVIA)

The shell of Solemya exhibits considerable flexibility which is further enhanced by the marked extension of the periostracum beyond the calcareous portions of the valves. This fcature, more than any other, has made possible the habit, unique among bivalves, of burrowing deep within the substrate without direct contact with the water above. The inner calcareous layer of tho valves is restricted to a small area near the umbones while the outer calcareous layer is thin and contains a high proportion of organic material. The shell conchiolin consists mainly of protein, varying in composition, but much of it strengthcned by quinone-tanning, and in ccrtain regions probably by the presence of appreciable quantities of chitin. The ligament, although superficially resembling an amphidetic structure, is opisthodetic, the extcnsion anterior to the umbones consisting of anterior outer layer only.
The mantle is characterized by an extension of the outer fold of the mantle margin which has effected equally both the inner and outer surfaces of this fold. The secretory epithelium and the modified pallial musculature, contraction of which results in the intucking and plaiting of the periostracum, is dcscribed. Simple tubular oil glands open at the mantlo margin and are responsible for the water-repellent nature of the periostracum.
The form of the mantlelshell and that of the enclosed body are discussed and compared with those of other bivalves in which elongation of the mantle/shell is achieved in a different way. It is concluded that the mantlelshell of Solemya is of little value in determining its relationships, and that the greatly elongatod ligament, the edentulous hinge and the flexible shell are all adaptations to a specialized mode of life.