The protegulum and juvenile shell of a Recent articulate brachiopod: patterns of growth and chemical composition

Patterns of shell formation and the chemical composition of the shell deposited during early post-larval life were investigated in laboratory-reared cultures of the Recent articulate brachiopod Terebraralia transversa (Sowerby). A non-hinged protegulum averaging 148 pm in length is secreted by the mantle within a day after larval metamorphosis. The inner surface of the protegulum exhibits finely granular, non-fibrous material. A rudimentary periostracum constitutes the outer layer of the primordial shell. and concentrically arranged growth lines are lacking. By four days post-metamorphosis, a brephic type of juvenile shell develops from periodic additions of shell material to the anterior and lateral edges of the protegulum. Imbricated secondary fibers occur throughout the inner layer of the newly formed juvenile shell, and a rudimentary hinge apparatus is present posteriorly. The external surface of the shell exhibits concentric growth lines anterior to the caudally situated protegulum, and unbranched punctae begin to form in the subperiostracal region of the shell. At 23 days post-metamorphosis, the shell weighs an average of 1.7 μg and measures 318 μm in length. Electron microprobe analyses reveal that the protegulum is calcified. Minor amounts of sulfur, magnesium, iron, chlorine, aluminum, and silicon are also present in protegula and juvenile shells. Based on electron diffraction data, the mineral phase of juvenile shells consists of calcite, and protegula also appear to contain calcite.     

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.     

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.     

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.     

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.     

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。     

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)     

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.     

The nature of siliceous mosaics forming the first shell of the brachiopod Discinisca

The juvenile shell of the brachiopod Discinisca consists of a mosaic of micrometer-sized siliceous tablets embedded in a chitinous substrate. The first-formed tablets are secreted on glycocalyx by a newly differentiated collective of outer epithelial cells. They are mainly rhombic but may also be ellipsoidal, discoidal, or deformed and sporadically overlap one another. On the surrounding juvenile shell, secreted by an incipient outer mantle lobe, the tablets are nearly all perfect rhombic plates in rhombic arrays. Their constant size, arrangement, and centripetal crystallization suggest intracellular assembly. The tablets, which are normally bilamellar, consist of discrete aggregates of crystalline spherules of silica in rhombic arrays within an organic matrix of fibrous protein and, presumably, a soluble polysaccharide(s). Mosaic secretion ceases at about the time when juveniles settle on the sea bed, which more or less coincides with the secretion of a ring of lamellae around the mosaic, induced by rapid advances and retractions of the outer mantle lobe prior to deposition of the organophosphatic mature shell. Energy dispersion X-ray analyses of pelagic and newly settled juveniles show that phosphatic secretion, even in the site of the first-formed outer epithelial collective, does not begin until all siliceous secretion has ceased.     

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.     

In vitro culture of glochidia from the freshwater mussel Anodonta cygnea

Abstract. Larvae of the freshwater swan mussel, Anodonta cygnea , were cultured in artificial media at the controlled temperature of 23°±2°C, with successful metamorphosis for the first time. The artificial medium contained a mixture of M199, common carp plasma, and antibiotics/antimycotics. Glochidia were reared to the juvenile stage in the medium after 10–11 d of culture. After 15 d of controlled feeding with phytoplankton, the juveniles showed an elongated shell with several growth lines. Larval survival was 34.3±9.3%, whereas the proportion undergoing metamorphosis was ≤60.8±4.2%. The ultrastructure of early developmental stages was observed by scanning electron microscopy, from the glochidial to the juvenile stage. Glochidia had a hooked shell, with two equal triangular valves formed by a calcareous layer with numerous pores and covered by a thin cuticle of chitin–keratin. The appearance of the complete foot within 11 d of in vitro culture was considered the final feature of metamorphosis to the juvenile stage. The main alteration during juvenile development was the formation, under the glochidial shell, of a new periostracum with growth lines. The prominent foot, gradually covered by long, dense cilia, showed rhythmical movements involved in the capture of particulate matter. Similarly, cilia and microvilli present in the mantle also performed the same role. Longer cilia, sparsely distributed in the mantle, may function as chemotactile sensors.     

Aragonitic dendritic prismatic shell microstructure in Thracia (Bivalvia,Anomalodesmata)

The shells of most anomalodesmatan bivalves are composed of an outer aragonitic layer of either granular or columnar prismatic microstructure, and an inner layer of nacre. The Thraciidae is one of the few anomalodesmatan families whose members lack nacreous layers. In particular, shells of members of the genus Thracia are exceptional in their possession of a very distinctive but previously unreported microstructure, which we term herein “dendritic prisms.” Dendritic prisms consist of slender fibers of aragonite which radiate perpendicular to, and which stack along, the axis of the prism. Here we used scanning and transmission electron microscopical investigation of the periostracum, mantle, and shells of three species of Thracia to reconstruct the mode of shell calcification and to unravel the crystallography of the dendritic units. The periostracum is composed of an outer dark layer and an inner translucent layer. During the free periostracum phase the dark layer grows at the expense of the translucent layer, but at the position of the shell edge, the translucent layer mineralizes with the units typical of the dendritic prismatic layer. Within each unit, the c‐axis is oriented along the prismatic axis, whereas the a‐axis of aragonite runs parallel to the long axis of the fibers. The six‐rayed alignment of the latter implies that prisms are formed by {110} polycyclically twinned crystals. We conclude that, despite its distinctive appearance, the dendritic prismatic layer of the shell of Thracia spp. is homologous to the outer granular prismatic or prismatic layer of other anomalodesmatans, while the nacreous layer present in most anomalodesmatans has been suppressed.     

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.     

Copillaria hepatica: Fine structure of egg shell

The structure of the Capillaria hepatica egg shell was studied with the electron microscope and correlated with light microscope histochemical observations. The shell is composed of fibrous and nonfibrous components, both of which stain for protein. The fibrous component, the major portion of the shell, consists of submicroscopic fibers. The nonfibrous component is located in the outer region of the shell but is not always visible; when present it has a reticulated appearance in electron micrographs. The fibrous component is divided into outer and inner regions. The outer region is composed of radially arranged pillars which are connected at their outer surface by a beam-like network and are anchored at the base to a compact inner region. The inner region consists of a series of concentrically arranged lamellae above which is located a nonlaminated region where the pillar bases originate. At each polar end of the shell is a single opening plugged with a material which contains acid mucopolysaccharide. The fine structure of the body of the plug is unresolvable with the electron microscope; its outer surface is impregnated with electron dense particles. Externally the shell is covered by a 250 Å thick continuous membrane which is in close opposition to the surrounding host tissue.     

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.     

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.     

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)     

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.