A scanning electron microscopic study of the inorganic and organic matrices comprising the mature shell of Amblema, a fresh-water mollusc

The scanning electron microscope has been used to describe the morphology of the mature shell in a fresh-water bivalve. The structure of the organic and inorganic components within the nacre, the myostracum, and the prismatic layer is described. A transitional or intermediate zone, interposed between the prismatic layer and the nacre, was identified. In demineralized samples, the organic component of the nacre was found to consist of parallel matricial sheets interconnected by irregular transverse bridges. The structure of the mineral component of the nacre was found to vary with the method of specimen preparation. With polished-etched samples, brick-like units were seen. When shells were simply broken and fixed in osmium, the layers of nacreous material consisted of fusing rhomboidal crystals of aragonite which demonstrated subconchoidal fractures. On the inner surface of the shell, the rhomboidal crystals showed an apparent spiral growth pattern. The myostracum was characterized by regions of modified nacreous structure consisting of enlarged aragonite crystals with a pyramidal morphology. The peripheral aspect of the muscle scars was characterized by rhomboidal crystals, the latter fusing to form the typical nacreous laminae. The uniqueness of the anterior adductor scar is exemplified by the presence of pores, each pore walled by pyramidal units, for the insertion of adductor fibres. In most regions of the shell, the prismatic layer consisted of one prism unit thickness with a height of approximately 225–250 μm. However, in two specialized regions of the shell, this layer was seen to consist of multiple layers of stacked prisms. The organic matrices of the prismatic layer are arranged in a honeycomb-like arrangement and packed with mineralized spherical subunits.     

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.     

Mineralogy of shells from two freshwater snails Belgrandiella fontinalis and B kuesteri

X-ray powder diffraction (XRD) was used to study the mineral composition of shells of snails Belgrandiella fontinalis and Belgrandiella kuesteri collected from three freshwater springs in northeastern Slovenia. The fractions of aragonite, calcite, dolomite and quartz in particular shells were determined. The analysed shells consisted of two or more distinct inorganic layers. The outer shell layer for both species and all sampling localities contained aragonite. The outer layer of B. fontinalis collected at one locality, also contained a small fraction of calcite ( approximately 1 molar%) besides the dominant aragonite. Calcite was identified in the inner layer(s) of both species (2 to 3 molar%), while quartz was found only in B. kuesteri (5-7 molar%). However, both species sampled at one locality showed the presence of dolomite (approx. 20 molar%) in the inner layer(s). The presence of dolomite in the shells of adult gastropods and even molluscs is unusual. A possible formation mechanism and specific ecological factor that could influence the precipitation of dolomite in the shells of different Belgrandiella species is discussed.     

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.     

Structure and composition of the boundary zone between aragonitic crossed lamellar and calcitic prism layers in the shell of Concholepas concholepas (Mollusca,Gastropoda)

Mollusc shells are composed of two or three layers. The main layers are well‐studied, but the structural and chemical changes at their boundaries are usually neglected. A microstructural, mineralogical, and biochemical study of the boundary between the inner crossed lamellar and outer prismatic layers of the shell of Concholepas concholepas showed that this boundary is not an abrupt transition. Localized structural and chemical analyses showed that patches of the inner aragonitic crossed lamellar layer persist within the outer calcitic prismatic layer. Moreover, a thin aragonitic layer with a fibrous structure is visible between the two main layers. A three‐step biomineralization process is proposed that involves changes in the chemical and biochemical composition of the last growth increments of the calcite prisms. The changes in the secretory process in the mantle cells responsible for the shell layer succession are irregular and discontinuous.     

A study of the egg shells of the Falconiformes

A study of a selection of egg shells of the Falconiformes has been made similar to the earlier ones on ratites, the Anatidae and the Sphenisciformes. Chemical analyses, and histological and plastic embedding techniques were used.
The main part of the shell in all species studied consists of large crystals running through the shell. There was no layer of fine vertical crystals above this and no cover, and even the cuticle was not very pronounced. Histological studies showed no major differences, except that some shells had vacuoles in the outer layers. All such shells also gave an unetched outer layer when plastic embedded radial sections were studied and thin sections showed spaces between and within crystals. These spaces in the outer layers of the shell were of taxonomic interest for they were not present in the Cathartidae, the Falconidae and Sagittarius serpentarius.
Pore channels appeared to be much sparser than in other orders so far studied and all pores were single. Pigment was present on the surface of some shells, but it was also found in different layers of the shell right down to the cone layer and, in one case, had leaked through on to the membrane.
There were significant relationships between total and soluble shell nitrogen which divided the Falconidae from most of the Accipitridae but left Pernis and Pandion in an intermediate position.     

Geometrical and crystallographic constraints determine the self-organization of shell microstructures in Unionidae (Bivalvia: Mollusca)

Unionid shells are characterized by an outer aragonitic prismatic layer and an inner nacreous layer. The prisms of the outer shell layer are composed of single-crystal fibres radiating from spheruliths. During prism development, fibres progressively recline to the growth front. There is competition between prisms, leading to the selection of bigger, evenly sized prisms. A new model explains this competition process between prisms, using fibres as elementary units of competition. Scanning electron microscopy and X-ray texture analysis show that, during prism growth, fibres become progressively orientated with their three crystallographic axes aligned, which results from geometric constraints and space limitations. Interestingly transition to the nacreous layer does not occur until a high degree of orientation of fibres is attained. There is no selection of crystal orientation in the nacreous layer and, as a result, the preferential orientation of crystals deteriorates. Deterioration of crystal orientation is most probably due to accumulation of errors as the epitaxial growth is suppressed by thick or continuous organic coats on some nacre crystals. In conclusion, the microstructural arrangement of the unionid shell is, to a large extent, self-organized with the main constraints being crystallographic and geometrical laws.     

Shell structure of two polar pelagic molluscs, Arctic Limacina helicina and Antarctic Limacina helicina antarctica forma antarctica

The purpose of this study was to investigate shell growth performance in two thin-shelled pelagic gastropods from cold seawater habitats. The shells of Arctic Limacina helicina and Antarctic Limacina helicina antarctica forma antarctica are very thin, approximately 2–9 μm for shells of 0.5–6 mm in diameter. Many axial ribbed growth lines were observed on the surface of the shell of both Limacina species. Distinct axial ribs were observed on the outermost whorl, while weak or no rib-like structures were observed on the inner whorls in the larger shell of L. helicina antarctica forma antarctica. For L. helicina, no ribs were observed on small individuals with three whorls, while larger individuals had distinct ribs on the outer whorls. Shell microstructure was examined in both species. There is an inner crossed-lamellar and extremely thin outer prismatic layer in small individuals of both species, and a distinct thick inner prismatic layer was observed beneath the crossed-lamellar layer in large Antarctic individuals. Various orientations of the crossed-lamellar structure were observed in one individual. Shell structure appeared to be different between the Antarctic and Arctic species and among shells of different size.     

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.     

Stratified mixed-culture biofilm model for anaerobic digestion

Development of a novel two-layer anaerobic biofilm model is based on substrate utilization kinetics and mass transport. The model is applied to steady-state conditions for a fixed-film anaerobic reactor. The microbial film is considered to consist of two distinct biofilm layers, one adjacent to the second, with an acidogenic bacteria biofilm forming the outer layer and a methanogenic film the inner one. The model assumes that sugars are only metabolized by the first layer and converted into volatile fatty acids (VFA), while fatty acids are taken up only by the inner layer. The model is able to predict both substrate flux net uptake and methane production for steady-state conditions. The results of modeling agree with methane production experimental data published elsewhere. Further, the model shows why layered fixed-film reactors can withstand high and inhibitory concentrations of volatile fatty acids as well as severe overloading without failure.     

Nanoscale structure and mechanical behavior of growth lines in shell of abalone Haliotis gigantea

In the natural world, bottom-up hierarchical construction of complex structures results in materials with remarkable properties. A well known example is the nacre of mollusk shells, commonly called "mother of pearl", whose excellent strength and toughness has been the subject of research for many decades. A significant discovery has been the presence of periodic layers called "growth lines". These are thin distinct layers within the bulk of the shell which form periodically, with their structure affected by environmental changes. Studies of their formation and behavior offer valuable insight into the architecture of seashells. In this work, the structure and mechanical behavior of growth lines in shells of abalone Haliotis gigantea were investigated using electron microscopy and nanoindentation. Growth lines form directly out of nacre into layers of blocks and irregular particles. In comparison to nacre, they have basic structures, form rapidly, and are harder, which suggest that they serve a protective role during lifecycle transitions. This exemplifies how natural structures are able to closely control growth architecture in order to form different structures for different functions, all from the same base materials.     

Shell nacre ultrastructure and depressurisation dissolution in the deep-sea hydrothermal vent mussel Bathymodiolus azoricus

This study describes the micro-morphological features of the shell nacre in the vent mytilid Bathymodiolus azoricus collected along a bathymetric gradient of deep-sea hydrothermal vents of the mid-Atlantic ridge (MAR). Pressure-dependent crystallisation patterns were detected in animals subjected to post-capture hydrostatic simulations. We provide evidence for the following: (1) shell micro morphology in B. azoricus is similar to that of several vent and cold-seep species, but the prismatic shell layers may vary among bathymodiolids; (2) nacre micro-morphology of mussels from three vent sites of the MAR did not differ significantly; minor differences do not appear to be related to hydrostatic pressure, but rather to calcium ion availability; (3) decompression stress may cause drop off in pH of the pallial fluid that damages nascent crystals, and in a more advanced phase, the aragonite tablets as well as the continuous layer of mature nacre; and (4) adverse effects of decompression on calcium salt deposition in shells was diminished by re-pressurisation of specimens. The implications of the putative influence of hydrostatic pressure on biomineralisation processes in molluscs are discussed. An erratum to this article can be found at     

Mucosal folding in biologic vessels

A two-layer model is used to simulate the mechanical behavior of an airway or other biological vessel under external compressive stress or smooth muscle constriction sufficient to cause longitudinal mucosal buckling. Analytic andfinite element numerical methods are used to examine the onset of buckling. Post-buckling solutions are obtained by finite element analysis, then verified with large-scale physical model experiments. The two-layer model provides insight into how the stiffness of a vessel wall changes due to changes in the geometry and intrinsic material stiffnesses of the wall components. Specifically, it predicts that the number of mucosal folds in the buckled state is diminished most by increased thickness of the inner collagen-rich layer, and relatively little by increased thickness of the outer submucosal layer. An increase in the ratio of the inner to outer material stiffnesses causes an intermediate reduction in the number of folds. Results are cast in a simple form that can easily be used to predict buckling in a variety of vessels. The model quantitatively confirms that an increase in the thickness of the inner layer leads to a reduction in the number of mucosal folds, and further, that this can lead to increased vessel collapse at high levels of smooth muscle constriction.     

Molecular phylogeny of protobranch bivalves and systematic implications of their shell microstructure

Higher systematics and evolutionary history of Protobranchia, a subclass of Bivalvia, have long been controversial due to paucity of prominent shell characters and difficulties in collecting live material for diverse taxa. Here, we evaluate the reliability of shell microstructure for protobranch higher systematics by reconstructing a molecular phylogeny of the subclass. Relationships were assessed using the nuclear (18S rRNA, 28S rRNA and histone H3) and mitochondrial (16S rRNA and cytochrome c oxidase subunit 1) gene sequences from 89 in-group species. Maximum likelihood reconstruction with the nuclear markers recognized five superfamilies (Nuculoidea, Solemyoidea, Manzanelloidea, Nuculanoidea and Sareptoidea) as the in-group clades of the monophyletic Protobranchia. Sareptoidea is herein redefined to comprise Sarepta and Setigloma in the sole family Sareptidae, whereas Pristigloma and its monotypic Pristiglomidae are transferred from this superfamily to Nuculanoidea, both in the order Nuculanida. Mapping of shell microstructure characters on the tree confirmed their conservativeness at superfamily level when only living species were taken into account. The Nuculoidea have shells with the outer prismatic and middle/inner nacreous structures; Solemyoidea are characterized by either the radially elongate simple prismatic structure or the reticulate structure in the outer shell layer; Manzanelloidea, Nuculanoidea and Sareptoidea have shells of homogeneous, fibrous prismatic and/or fine complex crossed lamellar structures, all of which lack large structural units. Our Bayesian time calibration, on the contrary, suggested frequent loss of nacre in the Paleozoic and Mesozoic history of Protobranchia, at least once each in Nuculoidea, Manzanelloidea, Solemyoidea and Sareptoidea in the Paleozoic, and perhaps multiple times in Nuculanoidea by the Mesozoic.     

Eggshell formation during prolonged gravidity of the tuatara Sphenodon punctatus

Scanning electron microscopy (SEM) was used to examine the process of shell formation in tuatara. Tuatara carry eggs in the oviducts for ∼ 7–8 mo before nesting, a period of gravidity more than three times as long as in any other oviparous reptile. Our aim was to determine whether shell formation occurred rapidly after ovulation, or whether it occurred gradually throughout gravidity. Eggs were obtained from females in early gravidity (May, ∼ 1 mo after ovulation), midgravidity (August and September, 4–5 mo after ovulation), and late gravidity, immediately prior to nesting (December, 8 mo after ovulation). The shell membrane (fibrous layer) was well formed by May, but calcification of the outer surface had only just begun. Vertical columns of calcium carbonate were embedded in the shell membrane and appeared to erupt through the outer surface between early and midgravidity. Changes in the appearance of the outer calcareous layer were evident as gravidity progressed. In all shells, calcium carbonate was present as calcite. The appearance of the inner boundary (innermost layer of eggshell) was variable; some shells had a smooth and amorphous inner boundary as previously reported for tuatara and other reptiles, whereas other shells had an inner boundary composed of small spherical granules on the inner surface of which small calcareous spicules were scattered. A previously published model of the process of shell formation in tuatara eggshells is refined in light of our observations. We interpret the ability of female tuatara to shell their eggs gradually during winter as further evidence of their unusual physiological tolerance of cold conditions. © 1996 Wiley-Liss, Inc.     

隆线溞[Daphnia(Ctenodaphnia)carinata]卵鞍的超微结构

在不利的环境条件下,枝角类中有一部分种类可以形成卵鞍(ephippium),内含休眠卵。本文应用扫描电镜和透射电镜对隆线溞的卵鞍进行了超微结构的研究。研究表明:卵鞍外面大部分略呈浅的蜂窝状,内面则排布着多数卵石状小突起。卵鞍分为内外两层,两层的超微结构截然不同;各层又可分为三小层。     

A scanning and transmission electron microscopic study of the membranes of chicken egg.

Questions regarding the structure of the inner and outer shell membranes of the chicken egg were addressed in this study by correlating observations from light microscopy and scanning and transmission electron microscopy. The egg membrane had a limiting membrane, which measured .9 to .15 microns in thickness and appeared to be a continuous and an impervious layer, but the shell membrane did not. Under the SEM, each membrane was seen to be made up of several fibre layers. In the tear preparations viewed under the SEM two layers were observed in the egg membranes and three to five layers in the shell membrane, with an apparent plane of cleavage between each layer. Each fibre was made up of a central core and an outer mantle layers. The central core was perforated by channels which measured .08 to 1.11 microns in diameter and ran longitudinally along the length of the fibre. Between the mantle layer and the fibre core was a gap or cleft measuring between .03 to .07 microns. The diameter of the fibres of the inner layer of the egg membrane ranged between .08 to .64 microns, whereas those of the outer layer of the same membrane ranged from .05 to 1.11 microns. Fibres in the shell membrane ranged from .11 to 4.14 microns diameter.     

Palaeobiology and taxonomy ofPachyperna laverdana Oppenheim,an Eocene bivalve of Mesozoic heritage

Pachyperna laverdana is a large Eocene bivalve characterized by an extremely thick shell wall. Rediscovery of the type locality(Pernabank Auctt.) after more than a century has made it possible to collect abundant material which is used here to provide a better morphological definition of the taxon. In particular, indication is given of its broad intraspecific variability mostly due to the gregareous habit (ecomorphism) and by change of mode of life through ontogenesis. As regards the latter factor, functional analysis of the shell suggests that in its early juvenile stages the bivalve was an epibyssate, pleurothetic form, attached to hard substrata. Then, it moved to soft-bottom substrates, where a “heavy-weight” strategy was developed to compensate for a weak byssal attachment. In the adult stage, it may be considered a reclining, orthothetic form. The shell is made up of a thin outer layer formed by simple prismatic calcite and by thick, aragonitic, irregular fibrous-prismatic inner layers, both with a well marked periodicity of growth. Mechanical, functional and systematic significance of shell microstructures are discussed. The diagnosis of the genusPachyperna is herewith emended.     

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.     

Egg envelopes of Streptocephalus dichotomus Baird: A structural and histochemical study

The origin, ultrastructure and histochemical properties of the egg membranes in the South Indian anostracan, Streptocephalus dichotomus have been studied. The egg morphology and the ultrastructure of the tertiary membrane of this phyllopod crustacean have been examined both by scanning and transmission electron microscopy. Scanning electron microscopic observations on the egg surface reveal the characteristic ridges on the egg surface with pores. Similarly, the tertiary egg shell of S. dichotomus consists of two distinct layers, an outer cortex and an inner alveolar layer. There are specific differences in the structure and in the relationship of one layer to the other. The alveolar layer is characterised by large lipid droplets and an alveolar mesh. These two layers termed as tertiary layers are secreted by maternal shell glands. The outer tertiary egg layers are phenolically tanned, the precursor materials for tanning being derived from shell gland secretions.