Microstructure and crystallographic-texture of giant barnacle (Austromegabalanus psittacus) shell

Barnacle shell is a very complex and strong composite bioceramic composed of different structural units which consist of calcite 15 microcrystals of very uniform size. In the study reported herein, the microstructural organization of these units has been examinated in detail with optical and scanning electron microscopy, and X-ray diffraction techniques. These analyses showed that the external part of the shell has a massive microstructure consisting of randomly oriented crystals. Toward the interior, the shell became organized in mineral layers separated by thin organic sheets. Each of these mineral layers has a massive microstructure constituted by highly oriented calcite microcrystals with their c-axes aligned [(001) fibre texture] perpendicular to the organic sheets and the shell surface. Interestingly, in another structural unit, the shell shield, the orientation of the c-axis calcite crystals shifts from being perpendicular to being parallel to the shell surface across its thickness. This study provides evidence that the organic matrix is responsible for the organization of the shell mineral and exterts strong a strict control on the polymorphic type, size and orientation of shell-forming crystals.     

The Micro/Nanostructure Characteristics and the Mechanical Properties of Hemifusus tuba Conch Shell

<正> The mollusk shell mobilizes calcium from environment for skeletal mineralization.This occurs through synthesizing solidsin solution in the presence of organic molecules of specific interior regions of the conch shell.The ultrastructure and microhardnessof the Hemifusus tuba conch shell living in the Huang/Bo sea area are investigated in the paper.It is shown that thecomposition and microstructure of the mollusk shell vary in different positions.The prodissoconch shell consists only of aragonitewith the crossed-lamellar microstructure.While the spiral shell and the body shell of the Hemifusus tuba conch shell arecomposed of one calcite layer and several aragonite layers.The calcite layer consists of cylindrical grains,but the aragonitelayers are crossed-lamellar ultrastructure at three size scales.The minimum structure size (the third-order lamella) is at about20 nm - 80 nm.The margin of shell aperture is only composed of calcite with cylindrical grains.This natural optimization of theshell microstructure is intimately due to the growth of the Organic matrix.At different positions the microhardness of molluscshell is different due to different crystal structures and crystal arrangements.The growth process of shells allows a constantrenewal of the material,thus enabling their functional adaptation to external environments.     

Calcite-specific coupling protein in barnacle underwater cement

The barnacle relies for its attachment to underwater foreign substrata on the formation of a multiprotein complex called cement. The 20 kDa cement protein is a component of Megabalanus rosa cement, although its specific function in underwater attachment has not, until now, been known. The recombinant form of the protein expressed in bacteria was purified in soluble form under physiological conditions, and confirmed to retain almost the same structure as that of the native protein. Both the protein from the adhesive layer of the barnacle and the recombinant protein were characterized. This revealed that abundant Cys residues, which accounted for 17% of the total residues, were in the intramolecular disulfide form, and were essential for the proper folding of the monomeric protein structure. The recombinant protein was adsorbed to calcite and metal oxides in seawater, but not to glass and synthetic polymers. The adsorption isotherm for adsorption to calcite fitted the Langmuir model well, indicating that the protein is a calcite-specific adsorbent. An evaluation of the distribution of the molecular size in solution by analytical ultracentrifugation indicated that the recombinant protein exists as a monomer in 100 mm to 1 m NaCl solution; thus, the protein acts as a monomer when interacting with the calcite surface. cDNA encoding a homologous protein was isolated from Balanus albicostatus, and its derived amino acid sequence was compared with that from M. rosa. Calcite is the major constituent in both the shell of barnacle base and the periphery, which is also a possible target for the cement, due to the gregarious nature of the organisms. The specificity of the protein for calcite may be related to the fact that calcite is the most frequent material attached by the cement.     

Tetraclita ehsani sp. n. (Cirripedia, Tetraclitidae), a common intertidal barnacle from the Gulf of Oman, Iran

A new species of intertidal acorn barnacle Tetraclita ehsanisp. n. was identified from the Iranian coast in the Gulf of Oman. Tetraclita ehsanisp. n. inhabits low exposed rocky shores and also attaches to shells of molluscs and the barnacle Megabalanus species. Parietes of Tetraclita ehsani ranged from white to pink which is different from Tetraclita serrata (in South African waters), which has green parietes. Morphology of the tergum and cirrus III of Tetraclita ehsanisp. n. is distinctive from other described West Indian Ocean species which have pink or white parietes (Tetraclita rufotincta, Tetraclita achituvi and Tetraclita reni). The tergum of Tetraclita ehsani is very narrow and the basal margin is slightly concave or straight, in contrast to Tetraclita rufotincta and Tetraclita reni, in which the tergum are board and with a very concave basal margin. Cirrus I anterior ramus of both Tetraclita ehsani and Tetraclita reni is antenniform and thus differing from the cirrus I of Tetraclita rufotincta (see Chan et al. 2009). Cirrus III of Tetraclita ehsanisp. n. is non-antenniform and lacks multicuspidate type setae, which is different from Tetraclita reni by having an antenniform cirrus III and with multicuspidate setae.     

Nano-cluster composite structure of calcitic sponge spicules--a case study of basic characteristics of biominerals

Spicules of calcareous sponges are elaborately shaped skeletal elements that nonetheless show characteristics of calcite single-crystals. Our atomic force microscopic and transmission electron microscopic investigation of the triradiate spicules of the sponge Pericharax heteroraphis reveals a nano-cluster structure with mostly well-aligned small crystal domains and pockets with accumulated domain misalignments. Combined high-resolution and energy-filtering transmission electron microscopy revealed carbon enrichments located in between crystal domain boundaries, which strongly suggests an intercalated network-like proteinaceous organic matrix. This matrix is proposed to be involved in the nano-clustered calcite precipitation via a transient phase that may enable a 'brick-by-brick' formation of composite and yet single-crystalline spicules with elaborate morphologies. This composite cluster structure reduces the brittleness of the material by dissipating strain energy and deflecting crack propagation from the calcite cleavage planes, but the lattice symmetry and anisotropic growth properties of calcite still play a major role in the morphogenesis of these unusual calcite single-crystals. Our structural, crystallographic, textural, and chemical analysis of sponge spicules corroborates the view that nano-clustered crystal growth, induced by organic matrices, is a basic characteristic of biomineralisation that enables the production of composite materials with elaborate morphologies.     

Characterization of a novel shell matrix protein with vWA domain from Mytilus coruscus

ABSTRACT

Mollusk shell is a product of biomineralization with excellent mechanical properties, and the shell matrix proteins (SMPs) have important functions in shell formation. A vWA domain-containing protein (VDCP) was identified from the shell of Mytilus coruscus as a novel shell matrix protein. The VDCP gene is expressed at a high level in specific locations in the mantle and adductor muscle. Recombinant VDCP (rVDCP) showed abilities to alter the morphology of both calcite and aragonite, induce the polymorph change of calcite, bind calcite, and decrease the crystallization rate of calcite. In addition, immunohistochemistry analyses revealed the specific location of VDCP in the mantle, the adductor muscle, and the myostracum layer of the shell. Furthermore, a pull-down analysis revealed eight protein interaction partners of VDCP in shell matrices and provided a possible protein–protein interaction network of VDCP in the shell.     

Are environmental conditions recorded by the organic matrices associated with precipitated calcium carbonate in cyanobacterial microbialites?

The amino acid composition of organic matrices associated with calcium carbonate precipitates in microbialites built by different Phormidium species (cyanobacteria) has been compared for samples recovered in lagoonal settings from two regions of the Southern Tropical Pacific separated by more than 4000 km: New Caledonia (Nouméa lagoon) and French Polynesia (Tikehau atoll). Calcium carbonate precipitation in these microbial structures was observed mainly in the interior of the domes and clearly separated from the photosynthetically active surface layer. This study focuses on the hydrolysable amino acid composition of the associated organic matrices that are typically rich in cysteine, leucine, alanine and arginine in New Caledonia, whereas they are particularly rich in dicarboxylic amino acids in French Polynesia. This striking difference is seemingly related to different environmental conditions that characterize the two reef settings. The high cysteine content suggests an origin from metallothioneins produced by the cyanobacteria and/or by epiphytic diatoms that were observed on the top layer, as the result of the input of metals from terrestrial origin in the Nouméa lagoon. In addition, we analysed the bulk organic matter of the photosynthetically active surface layer and of the interior of the domes. The former showed remarkable variations of amino acid composition throughout the year 2001, which may potentially reflect the impact of climatological events (e.g. cyclones) and/or a much stronger seasonality in New Caledonia than in French Polynesia. Although the mechanisms behind the differences remain elusive, our study clearly shows that environmental conditions can be reflected by amino acid compositions, particularly for the organic matrices associated with carbonate precipitates.     

Description of a new species of Thais (Mollusca: Neogastropoda: Muricidae) from Taiwan, based on morphological and allozyme analyses

Thais keluo sp. nov. is described from intertidal shores of southwest Taiwan. The new species is differentiated from five other closely related species, namely T. bitubercularis (Lamarck), T. jubilaea Tan and Sigurdsson, T. clavigera (Küster), T. luteostoma (Holten) and T. rufotincta Tan and Sigurdsson, all of which occur in the South China Sea, on the basis of shell, radula and penis morphology. Thais keluo is also distinguished from the latter three species based on allozyme electrophoresis. The shell of T. keluo is characterized by four raised, spiral bands on the last whorl, one or two small, oblique columellar plica(e) on the inner lip, a finely crenate, thin, narrow, reddish-brown outer lip edge and four white, papillate denticles inside the outer lip of the aperture. In males, the penis is curved with a long, simple flagellum. The UPGMA cluster analysis based on 9 enzyme loci revealed that T. luteostoma is more closely related to T. clavigera than to T. keluo n.sp. The Nei's genetic distance (D) obtained between the new species and T. clavigera/T. luteostoma was 0.31, while T. clavigera and T. luteostoma were separated by a distance of 0.16. Thais rufotincta was separated from the other species by a distance of 0.78. In contrast, phylogenetic analysis of morphological data by maximum parsimony suggested that T. luteostoma was more closely related to T. keluo than to T. clavigera. However, both analyses indicated the close relationship amongst T. clavigera, T. luteostoma and the new species in relation to T. rufotincta.     

In situ chemical speciation of sulfur in calcitic biominerals and the simple prism concept

The microstructure and composition of two mollusc shells were investigated using a combination of light microscopy, SEM, EPMA, and XANES. The shells of Pinna and Pinctada are composed of calcite prisms separated by organic walls. The prismatic units of Pinna are monocrystalline, and those of Pinctada are polycrystalline with internal organic radial membranes. High-spatial-resolution XANES maps for the different S species across adjacent prisms show that sulfate is the principal component in both the intraprismatic organic matrices and the outer membranes. Additionally, these maps confirm that the inner structures of the prismatic units are different for both genera. In many ways, the prisms of Pinna and Pinctada are different and invalidate the "simple prism" concept.     

The Test Structure and Composition of the Foraminifer Rosalina floridana*

SYNOPSIS. The composition of the test of Rosalina floridana (Cushman) was examined histochemically, and its structure was studied with the electron microscope by means of thin sections and carbon replicas. The test is composed of a thick organic lining overlain by one or more calcite layers bounded above and below by thin membranes. The membranes are fused to organic pore processes composed of coarse fibers that penetrate the calcite layers. The ***lining, consisting of coarse fibers matted into a laminated sheet, is considered a strengthening element of the test. The membranes covering each calcite layer are composed of fine, headed fibrils which in aggregate have a striated pattern; they are thought to be the crystal-nucleating agent during calcification and to form a protective covering for the previously deposited calcite layers. The pore processes, which are devoid of an internal entrance for cytoplasm, are considered to be points of attachment for the membranes; they tie the organic test components into a unified whole. The calcite layers and the chambers lack this unity, being separated from each other and from the preceding chambers by membranes so that there are no calcite-to-calcite boundaries between them. An organic, sievelike structure of undetermined function has been found in the foramina of chambers near the prolocular region of the test. Histochemical methods show that the lining contains proteins, polysaccharides, and unidentified substances; the membranes and the pore processes stain as a protein-polysaccharide complex free of other substances.     

Mechanical characterization of an unusual elastic biomaterial from the egg capsules of marine snails (Busycon spp.)

Egg capsule material serves as a putative protection mechanism for developing snail embryos facing the perils of the marine environment. We conducted the first quantitative study of this acellular structural protein with the goals of characterizing its chemical and mechanical properties and the relationship of these properties to its biological protective function. We have found that this protein polymer exhibits long-range elasticity with an interesting recoverable yield evidenced by an order of magnitude decrease in elastic modulus (apparent failure) that begins at 3%-5% strain. This material differs significantly from other common structural proteins such as collagen and elastin in mechanical response to strain. Qualitative similarities in stress/strain behavior to keratin, another common structural protein, are more than coincidental when composition and detailed mechanical quantification are considered. This suggests the possibility of alpha-helical structure and matrix organization that might be similar in these two proteins. Indeed, the egg capsule protein may be closely related to vertebrate keratins such as intermediate filaments. We conclude that while this material's bimodal tensile properties may serve as useful protection against the impact loading egg capsules encounter in the intertidal zone, the full biological importance of these capsules is not known.     

Speciation and the establishment of zonation in an intertidal barnacle: specific settlement vs. selection

The tropical barnacle Tetraclita forms a belt on hard substrates in the intertidal zone of the Red Sea. Based on morphological data, three distinct species were suggested to exist, occupying different vertical levels - T. barnesorum, T. rufotincta and T. achituvi. In this study we used molecular (12S mitochondrial ribosomal DNA) and ecological data to examine whether this morphological variability reflects genetic differences, or is a result of environmental factors. Adults and spats, collected from settlement plates, were censused and screened genotypically using single strand conformation polymorphism (SSCP) analysis, and settlement dynamics was recorded. We provide evidence for the existence of only two distinct species, and point out both phenotypic plasticity and convergence within and between the proposed species. Cyprids of T. achituvi settle specifically at the lower part of the Tetraclita belt, and feature one phenotype. In contrast, T. rufotincta, occupying the upper and middle portions of the Tetraclita belt, settles throughout the range, shows phenotypic plasticity (three variants), and presumably undergoes selection at the lower part. Thus, the vertical zonation of Tetraclita is produced by the combination of pre-settlement and post-settlement factors, in T. achituvi and T. rufotincta, respectively. The examined system may offer a model in which to study the mechanisms underlying sympatric speciation.     

Comparison of the soluble matrices of the calcitic prismatic layer of Pinna nobilis (Mollusca,Bivalvia, Pteriomorpha)

The calcitic prisms of the outer layer of the shell of Pinna nobilis, surrounded by thick organic walls, contain a soluble intracrystalline matrix. The structure and composition of the outer interprismatic walls are not well known. The current viewpoint is they are composed of an insoluble matrix. Another thick organic structure, the interlamellar sheet of the nacreous layer, is composed of insoluble and soluble matrices. The composition of two sets of soluble organic matrices from the calcitic layer of Pinna nobilis, extracted with and without the organic walls are compared. According to the various analyses (SEM and AFM, UV and FTIR spectrometry, HPLC, electrophoreses, XANES), the main characteristics of the two matrices are similar, but not identical. Thus, the organic walls contain soluble components. However, the three-layered structure of the interlamellar sheet of the nacreous layer has not been observed.     

Microstructure of matrix and mineral components of eggshells from White Leghorn chickens (Gallus gallus)

The avian eggshell is a composite structure of organic matrix and mineral (calcium carbonate) that is rapidly and sequentially fabricated in the oviduct in <24 hr. The eggshell is an excellent vehicle for the study of biomineralization processes and the role of the organic matrix in the mineral-matrix composite. The organic matrix components of eggshells from White Leghorn chickens (Gallus gallus) were examined by transmission electron microscopy (TEM) and optical microscopy. The mineral phase was analyzed by TEM, scanning electron microscopy (SEM), X-ray compositional microanalysis, and electron diffraction. Ultrastructural examination of the matrices within the calcified eggshell reveals a complex architecture that differs within each of the major zones of the eggshell: the shell membranes, the mammillary zone, the palisade region, and the cuticle. The mammillary layer consists of the calcium reserve assembly (CRA) and crown region, each with a unique substructure. TEM images show that the matrix of the CRA consists of a dense, flocculent material partially embedded within the outer shell membrane (a mostly noncalcified region of the shell). The mantle of the collagen fibers of the shell membranes is rich in polyanions (cuprolinic blue-positive), as is the CRA matrix. The CRA is capped by a centrally located calcium reserve body sac (CRB sac) that contains numerous 300–400 nm, electron-dense, spherical vesicles. Directly above the CRB sac is a zone of matrix consisting of stacks of interconnected vesicles (similar in morphology to CRA vesicles) that are interspersed with a granular material. The palisade region, the largest of the mineralized zones, contains hollow vesicles ∼450 nm (s.d. = 75 nm) in diameter, with a crescent-shaped, electron-dense fringe. An interconnecting matrix material is also found between the vesicles in the palisades region. The cuticle is composed of two layers, a mineralized inner layer and an outer layer consisting of only organic matrix. The bulk of the mineral within the eggshell is calcite, with small amounts of needlelike hydroxyapatite in the inner cuticle and occasionally, vaterite micro crystals found at the base of the palisade (cone) region. The well-crystallized calcite crystals within the palisade are columnar, typically ∼20 μm wide by 100–200 μm long; aside from numerous entrapped vesicles and occasional dislocations, they are relatively defect-free. The bulk of the matrix found in the palisade and crown regions are thought to be residual components of the rapid mineralization process. The unique matrix structure within the CRB corresponds to the region of preferentially solubilized calcite used by the developing embryo and the hydroxyapatite found in the inner cuticle may play a role in the cessation of mineral growth. © 1996 Wiley-Liss, Inc.     

Comparison of aragonitic molluscan shell proteins

Acidic macromolecules, as a nucleation factor for mollusc shell formation, are a major focus of research. It remains unclear, however, whether acidic macromolecules are present only in calcified shell organic matrices, and which acidic macromolecules are crucial for the nucleation process by binding to chitin as structural components. To clarify these questions, we applied 2D gel electrophoresis and amino acid analysis to soluble shell organic matrices from nacre shell, non-nacre aragonitic shell and non-calcified squid shells. The 2D gel electrophoresis results showed that the acidity of soluble proteins differs even between nacre shells, and some nacre (Haliotis gigantea) showed a basic protein migration pattern. Non-calcified shells also contained some moderately acidic proteins. The results did not support the correlation between the acidity of soluble shell proteins and shell structure.     

The preservation of ostracod shells in the siliceous chalk of the Danish-Polish Furrow (Baltic Sea)

Summary The number, morphological details and structure of ostracod shells washed from chalk by mechanical disaggregation differ from those in siliceous chalk treated by hydrofluoric acid (HF, conc.). The shells studied are from erratic material (Ger. “Schollen”, “Geschiebe”) of young Quaternary deposits from Nossentin near Malchow/Mecklenburg, NE Germany; Wicko (Vietzig) near Miedzyzdroje (Misdroy), Isle of Wolin/Baltic Sea, NW Poland (Upper Turonian) and from outcropping chalk (Lower Maastrichtian) of the Island of Rügen/Baltic Sea, NE Germany as well as erratic boulders (Upper Maastrichtian), NE Germany. The differences in the number of shells and their state of preservation are primarily caused by biotic and nonbiotic influences after death, e.g. loss of organic substance and shell deformation by low pressure. Greater destruction of the shell and its sculpture is caused by pressure (sedimentary compaction), recrystallization, sparitization and accretine crystallization of the shell calcite during diagenesis. The silification process or origin of flint in chalk is caused by weakly acidic environmental conditions (pH<5) before sedimentary compaction. It is indicated by dispersed pyrite and by well-preserved siliceous radiolarian skeletons. Finely-foliated opal-CT crystals start growing between the crystallites of the ostracod shell, later on forming lepispheres. Silification is early diagenetic, as verified by the well-preserved shell sculpture and structure and the undisturbed bioturbate structure in siliceous nodules.     

Structure and composition of the septal nacreous layer of Nautilus macromphalus L. (Mollusca, Cephalopoda)

The nacreous layer of Mollusca is the best-known aragonitic structure and is the usual model for biomineralization. However, data are based on less than 10 species. In situ observations of the septal nacreous layer of the cephalopod Nautilus shell has revealed that the tablets are composed of acicular laths. These laths are composed of round nanograins surrounded by an organic sheet. No hole has been observed in the decalcified interlamellar membranes. A set of combined analytical data shows that the organic matrices extracted from the nacreous layer are glycoproteins. In both soluble and insoluble matrices, S amino acids are rare and the soluble organic matrices have a higher sulfated sugar content than the insoluble matrices. It is possible that the observed differences in the structure and composition of the nacreous layers of the outer wall and septa of the Nautilus shell have a dual origin: evolution and functional adaptation. However, we have no appropriate data as yet to answer this question.     

Barnacle growth rate on artificial substrate in the Salton Sea,California

The Salton Sea is one of the few saline, inland lakes in the world with a population of barnacles, Balanus amphitrite. It is also one of California’s most impaired water bodies due to excessive nutrient loading which leads to phytoplankton blooms and low dissolved oxygen. Currently, B. amphitrite growth is limited due to lack of hard substrate in and around the Sea. We have hypothesized that artificial substrate could support the growth of B. amphitrite and their filter-feeding would lead to improved water quality. Periodic harvesting of the barnacles would result in the permanent removal of nitrogen and phosphorus from the Sea. A 44-day in-situ experiment was carried out in the Salton Sea to assess the rate of barnacle growth and phosphorus and nitrogen sequestration on burlap sheets suspended vertically from a floating line. Burlap panels were collected weekly and the barnacles analyzed for Ca, total-P, inorganic-P, total-N, total-C, CaCO₃, and organic matter content. After 44 days of growth, the barnacle mats weighed 7.4 kg m⁻² on a dry weight basis, with 80% of the mass as shell material. The nutrient sequestration was 9.4 g P m⁻² and 100 g N m⁻². Approximately half of the P was inorganic and appears to be coprecipitated with the calcium carbonate shell material. Results indicate that harvesting barnacles grown on artificial substrate in the Salton Sea would not be an effective method for removing N or P from the lake because of the relative proportions of shell material and organic material. Guest editor: S. H. Hurlbert The Salton Sea Centennial Symposium. Proceedings of a Symposium Celebrating a Century of Symbiosis Among Agriculture, Wildlife and People, 1905–2005, held in San Diego, California, USA, March 2005     

Magnesium and sulphur in the calcite shells of two brachiopods, Terebratulina retusa and Novocrania anomala

This study determines the distribution of magnesium and sulphur in the shells of two species of brachiopod from the same environment to highlight environmental and biological influences on shell composition. In Terebratulina retusa there are differences in magnesium concentration between the primary layer and the outer and inner regions of the secondary layer. In contrast, Novocrania anomala has a shell composed of high magnesium calcite and there is no significant difference in magnesium concentration between the primary and the secondary shell layers. Sulphur provides an indication of the distribution of sulphated organic matrix within the shells of T. retusa and N. anomala . In T. retusa the distribution of magnesium and sulphur correlates across the shell; however, there is no evidence for a relationship between magnesium and sulphur distribution in N. anomala . The relationship between magnesium and sulphur in T. retusa indicates that a proportion of the magnesium content of the shell is associated with the sulphated fraction of the organic matrix. In these two species of brachiopod, from the same environment, magnesium and organic concentration and distribution are very different, emphasizing the importance of fully understanding the factors that control biomineral composition before the application of these biominerals to environmental studies.     

AN EARLY CAMBRIAN ORGANOPHOSPHATIC BRACHIOPOD WITH CALCITIC GRANULES

Abstract:  The linguliform brachiopod Eoobolus from the Early Cambrian Mural Formation (Jasper National Park, Canadian Rocky Mountains) exhibits various calcitic features in its otherwise apatitic shell. It is argued here that the decomposition of the organic matter within the shell led to a microenvironment similar to those resulting in the phosphatization of soft tissues. This diagenetic regime encouraged the initial precipitation of apatite cements followed by calcite cements. By fully coating primary structures early apatite cements separate primary structures from the later precipitation of calcite cement. Round calcareous grains, about 3  µ m in size, that occur in the centre of apatite botryoids must therefore represent original components of the shell. The equivalent pits of such calcareous granules are seen in the larval shells of many Palaeozoic linguliform brachiopods. This suggests that mixed organophosphatic-calcareous shells were relatively common at that time but that they have been overlooked owing to the obliteration of original calcareous structures by traditional acid preparation methods for the extraction of phosphatic fossils. The Eoobolus shell structure is intermediate between purely organophosphatic and calcitic shells. Although one such genus is not sufficient to reconstruct the ancestral composition of the brachiopod shell, it provides a means of recognizing other transitional forms that are needed to understand fully the shift in shell mineralogy.