In vitro regulation of CaCO(3) crystal polymorphism by the highly acidic molluscan shell protein Aspein

Biominerals, especially molluscan shells, generally contain unusually acidic proteins. These proteins are believed to function in crystal nucleation and inhibition. We previously identified an unusually acidic protein Aspein from the pearl oyster Pinctada fucata. Here we show that Aspein can control the CaCO(3) polymorph (calcite/aragonite) in vitro. While aragonite is preferentially formed in Mg(2+) -rich solutions imitating the extrapallial fluids of marine molluscs, Aspein exclusively induced calcite precipitation. Our results suggest that Aspein is involved in the specific calcite formation in the prismatic layer. Experiments using truncated Aspein demonstrated that the aspartic acid rich domain is crucial for the calcite precipitation.     

A Novel Acidic Matrix Protein,PfN44, Stabilizes Magnesium Calcite to Inhibit the Crystallization of Aragonite

Magnesium is widely used to control calcium carbonate deposition in the shell of pearl oysters. Matrix proteins in the shell are responsible for nucleation and growth of calcium carbonate crystals. However, there is no direct evidence supporting a connection between matrix proteins and magnesium. Here, we identified a novel acidic matrix protein named PfN44 that affected aragonite formation in the shell of the pearl oyster Pinctada fucata. Using immunogold labeling assays, we found PfN44 in both the nacreous and prismatic layers. In shell repair, PfN44 was repressed, whereas other matrix proteins were up-regulated. Disturbing the function of PfN44 by RNAi led to the deposition of porous nacreous tablets with overgrowth of crystals in the nacreous layer. By in vitro circular dichroism spectra and fluorescence quenching, we found that PfN44 bound to both calcium and magnesium with a stronger affinity for magnesium. During in vitro calcium carbonate crystallization and calcification of amorphous calcium carbonate, PfN44 regulated the magnesium content of crystalline carbonate polymorphs and stabilized magnesium calcite to inhibit aragonite deposition. Taken together, our results suggested that by stabilizing magnesium calcite to inhibit aragonite deposition, PfN44 participated in P. fucata shell formation. These observations extend our understanding of the connections between matrix proteins and magnesium.     

A Comparative Study of the Shell Matrix Protein Aspein in Pterioid Bivalves

Aspein is one of the unusually acidic shell matrix proteins originally identified from the pearl oyster Pinctada fucata. Aspein is thought to play important roles in the shell formation, especially in calcite precipitation in the prismatic layer. In this study, we identified Aspein homologs from three closely related pterioid species: Pinctada maxima, Isognomon perna, and Pteria penguin. Our immunoassays showed that they are present in the calcitic prismatic layer but not in the aragonitic nacreous layer of the shells. Sequence comparison showed that the Ser-Glu-Pro and the Asp-Ala repeat motifs are conserved among these Aspein homologs, indicating that they are functionally important. All Aspein homologs examined share the Asp-rich D-domain, suggesting that this domain might have a very important function in calcium carbonate formation. However, sequence analyses showed a significantly high level of variation in the arrangement of Asp in the D-domain even among very closely related species. This observation suggests that specific arrangements of Asp are not required for the functions of the D-domain.     

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 comparative study of the ultrastructure and mineralogy of calcified land snail eggs (Pulmonata: Stylommatophora)

This study indicates that eggs containing calcium carbonate crystals occur in at least 36 of the 65 known families of the land snails (class Gastropoda: order Stylommatophora). Eggs from 22 of these families were available for examination. The x-ray diffraction data, available for the first time for 21 of these families, shows that these egg shells are all made of calcite only, or of a combination of calcite with smaller amounts of aragonite. All of the snail (body) shells examined were made of aragonite only. This is the first ultrastructural investigation of these egg shells, and it indicates that the eggs exhibit enough structural diversity to allow identification of parental animals to genus, and often to species level solely on the basis of egg shell ultrastructure. All of the calcified eggs may be divided into two groups: (1) partly calcified, with discrete crystals of CaCo₃ dispersed in the jelly layer, and (2) heavily calcified, with a hard, brittle egg shell made of fused crystals of CaCO₃ much like an avian egg. Both types of calcified eggs occur in oviparous as well as in ovoviviparous snails. Because of the wide distribution of calcified eggs in the Stylommatophora, and because of the occurrence of heavily calcified eggs in ancient families such as Partulidae, Endodontidae, and Zonitidae, the calcified egg is viewed as a primitive land snail trait associated with terrestrial adaptation. The function of the calcified egg shell, in addition to mechanical support of egg contents, is to supply the developing embryo with enough calcium to form the embryonic shell by the time of hatching.     

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.     

贝壳的有机质及生物矿化机制分析

以腹足纲贝壳香螺壳为研究对象,用弱酸去钙法进行蛋白提取,采用280纳米(A280)光吸收法测定蛋白含量,并通过聚丙烯酰胺(SDS-PAGE)凝胶电泳法对蛋白按照分子量大小的区别进行分离.实验结果表明香螺壳中蛋白含量和种类较少,其文石层比方解石层蛋白含量高的多,总量分别为0.89%和0.0533%;文石层分离出五种分子量的可溶性蛋白和四种分子量的不可溶性蛋白;而方解石层中分离出三种分子量的可溶性蛋白和三种分子量的不可溶性蛋白,且分子量不相同.正是这少量的蛋白质对贝壳的生物矿化过程和不同晶型的形成起着决定性作用.     

Mollusc larval shell formation: amorphous calcium carbonate is a precursor phase for aragonite

The larval shells of the marine bivalves Mercenaria mercenaria and Crassostrea gigas are investigated by polarized light microscopy, infrared spectroscopy, Raman imaging spectroscopy, and scanning electron microscopy. Both species contain similar shell ultrastructures. We show that larval shells contain amorphous calcium carbonate (ACC), in addition to aragonite. The aragonite is much less crystalline than non-biogenic aragonite. We further show that the initially deposited mineral phase is predominantly ACC that subsequently partially transforms into aragonite. The postset juvenile shell, as well as the adult shell of Mercenaria also contains aragonite that is less crystalline than non-biogenic aragonite. We conclude that ACC fulfills an important function in mollusc larval shell formation. It is conceivable that ACC may also be involved in adult shell formation.     

Shell microstructure and mineralogy of the Littorinidae: ecological and evolutionary significance

An examination of the shell microstructure and mineralogy of species from 30 of the 32 genera and subgenera of the gastropod family Littorinidae shows that most species have a shell consisting of layers of aragonitic crossed-lamellar structure, with minor variations in some taxa. However, Pellilitorina, Risellopsis and most species of Littorina have partly or entirely calcitic shells. In Pellilitorina the shell is made entirely of calcitic crossed-foliated structure, while in the other two genera there is only an outer calcitic layer of irregular-prismatic structure. A cladistic analysis shows that the calcitic layers have been independently evolved in at least three clades. The calcite is found only in the outermost layers of the shell and in species inhabiting cooler waters of both northern and southern hemispheres. Calcium carbonate is more soluble in cold than warm water and, of the two polymorphs, calcite is about 35% less soluble than aragonite. We suggest that calcitic shell layers are an adaptation of high latitude littorinids to resist shell dissolution.     

Shell mineralogical trends in epifaunal Mesozoic bivalves and their relationship to seawater chemistry and atmospheric carbon dioxide concentration

The Late Triassic-Early Jurassic change from aragonite- to calcite-facilitating conditions in the oceans, which was caused by a decrease of the Mg²⁺/Ca²⁺ ratio of seawater in combination with an increase of the partial pressure of carbon dioxide, also affected the shell mineralogy of epifaunal bivalves. In the “calcite sea” of the Jurassic and Cretaceous, the most diverse and abundant families of epifaunal bivalves had largely calcitic shells. Some of them, such as the Inoceramidae, acquired this shell mineralogy earlier in Earth's history but did not significantly diversify until the onset of “calcite sea” conditions. Others, however, replaced aragonite by calcite in their shell at the beginning of the Jurassic, as shown for the Ostreidae, Gryphaeidae, Pectinidae, Plicatulidae, and Buchiidae. In these families, replacement of aragonite by calcite took place in the middle and inner layer of the shell and was not associated with changes in morphology and life habit. It is therefore proposed that lower metabolic costs rather than higher resistance against dissolution or advantageous physical properties triggered the calcite expansion in their shells. This explanation fits well the observation that clades of thin-shelled bivalves were less affected by the change of seawater chemistry. Thick-shelled clades, by contrast, may suffer a severe decline in diversity until they adapt their shell mineralogy, as demonstrated by the Hippuritoida: The diversity of the Megalodontoidea, which failed to adapt their shell mineralogy to “calcite sea” conditions, dramatically decreased at the end of the Triassic, whereas their descendents became dominant carbonate producers during the Late Mesozoic after they acquired a calcitic outer shell layer in the Late Jurassic. These examples indicate that changes in the seawater chemistry and in the partial pressure of carbon dioxide are factors that influence the diversity of carbonate-secreting animals, and, as in the case of the decline of the Megalodontoidea, may contribute to mass extinctions.     

Turritelline mass accumulations from the Lower Miocene of southern Germany: implications for tidal currents and nutrient transport within the North Alpine Foreland Basin

The mass occurrence of turritelline gastropod shells from the Lower Miocene of southern Germany allows for detailed studies of their palaeoecology, transport mechanisms, preservation potentials and the reconstruction of nutrient regimes. Changes in the fabric of the gastropod-dominated beds are used to reconstruct a generally deepening environment corresponding to the Lower Miocene transgression within the Upper Molasse Sea of the North Alpine Foreland Basin. The sedimentary succession ranges from chaotically arranged, densely packed and near-shore transported; wave-influenced deposits showing bimodal shell orientations; more widely dispersed shells showing a uni-directional orientation; and dispersed shells showing diverse orientations. The shells often show damage to the apex and aperture though it is not clear whether this is due to predation events, pagurisation or abrasion due to transport. An outstanding feature is the replacement of aragonite shells by calcite leading to internal vugs as well as modulating the outer shell surface morphology. The high density of turritelline gastropods indicates a nutrient-rich palaeoenvironment at the northern edge of the Molasse Sea.     

Ubiquitylation functions in the calcium carbonate biomineralization in the extracellular matrix

Mollusks shell formation is mediated by matrix proteins and many of these proteins have been identified and characterized. However, the mechanisms of protein control remain unknown. Here, we report the ubiquitylation of matrix proteins in the prismatic layer of the pearl oyster, Pinctada fucata. The presence of ubiquitylated proteins in the prismatic layer of the shell was detected with a combination of western blot and immunogold assays. The coupled ubiquitins were separated and identified by Edman degradation and liquid chromatography/mass spectrometry (LC/MS). Antibody injection in vivo resulted in large amounts of calcium carbonate randomly accumulating on the surface of the nacreous layer. These ubiquitylated proteins could bind to specific faces of calcite and aragonite, which are the two main mineral components of the shell. In the in vitro calcium carbonate crystallization assay, they could reduce the rate of calcium carbonate precipitation and induce the calcite formation. Furthermore, when the attached ubiquitins were removed, the functions of the EDTA-soluble matrix of the prismatic layer were changed. Their potency to inhibit precipitation of calcium carbonate was decreased and their influence on the morphology of calcium carbonate crystals was changed. Taken together, ubiquitylation is involved in shell formation. Although the ubiquitylation is supposed to be involved in every aspect of biophysical processes, our work connected the biomineralization-related proteins and the ubiquitylation mechanism in the extracellular matrix for the first time. This would promote our understanding of the shell biomineralization and the ubiquitylation processes.     

Biomineralization: functions of calmodulin-like protein in the shell formation of pearl oyster

Calmodulin-like protein (CaLP) was believed to be involved in the shell formation of pearl oyster. However, no further study of this protein was ever performed. In this study, the in vitro crystallization experiment showed that CaLP can modify the morphology of calcite. In addition, aragonite crystals can be induced in the mixture of CaLP and a nacre protein (at 16 kDa), which was detected and purified from the EDTA-soluble matrix of nacre. These results agreed with that of immunohistological staining in which CaLP was detected not only in the organic layer sandwiched between nacre (aragonite) and the prismatic layer (calcite), but also around the prisms of the prismatic layer. Take together, we concluded that (1) CaLP, as a component of the organic layer, can induce the nucleation of aragonite through binding with the 16-kDa protein, and (2) CaLP may regulate the growth of calcite in the prismatic layer.     

Direct observation of the transition from calcite to aragonite growth as induced by abalone shell proteins

The mixture of EDTA-soluble proteins found in abalone nacre are known to cause the nucleation and growth of aragonite on calcite seed crystals in supersaturated solutions of calcium carbonate. Past atomic force microscope studies of the interaction of these proteins with calcite crystals did not observe this transition because no information about the crystal polymorph on the surface was obtained. Here we have used the atomic force microscope to directly observe changes in the atomic lattice on a calcite seed crystal after the introduction of abalone shell proteins. The observed changes are consistent with a transition to (001) aragonite growth on a (1014) calcite surface.     

SHELL MICROSTRUCTURE OF GASTROPODS FROM LAKE TANGANYIKA,AFRICA: ADAPTATION,CONVERGENT EVOLUTION,AND ESCALATION

Gastropod shells from Lake Tanganyika, with their heavy calcification, coarse noded ribbing, spines, apertural lip thickening and repair scars, resemble marine shells more closely than they resemble other lacustrine shells. This convergence between Tanganyikan and marine gastropod shells, however, is not just superficial. Scanning electron microscope (SEM) studies reveal that the Tanganyikan shells are primarily layers of crossed-lamellar crystal architecture (that is, needle-like aragonite crystals arranged into laths that are packed into sheets such that the aragonite needles of adjacent laths are never parallel). The number of crossed-lamellar layers can vary from one to four between different Tanganyikan gastropod species. In species with two or more crossed-lamellar layers, the orientation of the lamellae is offset by approximately 90° between the different layers. The number of crossed-lamellar layers in the shell wall is positively correlated with shell strength and with predation resistance. Three and four crossed-lamellar layers in the shell wall evolved several times independently within the endemic thiarid gastropod radiation in Lake Tanganyika. Repeated origins of three and four crossed-lamellar layers suggest that they may be specific adaptations by Tanganyikan gastropods to strengthen their shells as a defense against shell-crushing predators.     

Shell microstructure in the Permian nonmarine bivalve Palaeomutela Amalitzky: Revision of the generic diagnosis

The microstructure of aragonitic and calcitic shells of the genus Palaeomutela Amalitzky, 1891 is examined. The aragonitic shell consists of three main layers, each is distinguished by certain crossed lamellar microstructure: comarginal, radial, and complex. As aragonite is recrystallized into pelitic calcite, microstructural shell features are preserved. Many species of Palaeomutela from localities of different age display the same microstructural pattern, which is possible to regard as a character of generic rank.     

Studies of Biominerals Relevant to the Search for Life on Mars

The evidence of the water erosion on Mars is particularly interesting since present climatic conditions are such that liquid water cannot exist at the surface. But, if water was present on the planet in the past, there may have been life, too. Since the discovery of carbonates on Mars also may have very important implications on the possibility that life developed there, we are studying minerals that can have biotic or abiotic origin: calcite (CaCO₃) and aragonite, a metastable state of calcite. We have analysed biomineral aragonite, in the form of recent sea shells, as well as crystals of mineral aragonite. Infrared spectroscopy in the 2–25 μm wavelength range reveals that, after thermal processing, the biotic samples have a different spectral behaviour from the abiotic ones. As a result, it is possible to distinguish abiotic mineral aragonite from aragonite of recent biological origin. Obviously, if life existed in the past on the Red Planet, we could expect to find “ancient” biotic carbonates, which should therefore be investigated, in order to search for a way of discriminating them from abiotic minerals. For this reason, at the beginning we have considered samples of crushed fossil shells of aragonite composition. Afterwards, in order to take into account that fossilization processes almost always produce a transformation of metastable form (aragonite) into more stable form (calcite), we also studied samples of mineral calcite and different types of fossils completely transformed into calcite. All these biotic fossil samples show the same spectral behaviour as the fresh biotic material after thermal annealing at 485°C. Instead, the calcite behaves like abiotic aragonite. Furthermore, it is known that seashells and other biominerals are formed through an intimate association of inorganic materials with organic macromolecules. The macromolecules control the nucleation, structure, morphology, crystal orientation and spatial confinement of the inorganic phase: this differentiates biominerals from minerals. Analysing the aragonite or calcite fossils with a Scanning Electron Microscope, we found that the fossilization process did not modify the structure of the biominerals which maintain their microscopic characteristics. Looking at the morphology of fossil biominerals, it is evident that the crystals are arranged in complex architectures compared with the compact structure of the mineral crystals. In conclusion, the properties and structure of the biominerals are different from those of the minerals. The rapid increase of the crystalline structure developed under biotic conditions makes these minerals less resistant to thermal treatments, compared with samples of abiotic origin. This result holds both for recent shells as well as all fossil samples. The spectroscopic behaviour of all analysed calcium carbonates of biotic origin is different from that of the abiotic one. Therefore, the infrared spectroscopy is a valid technique to discern the origin of the samples and a powerful tool for analysing in-situ and “sample-return” Mars missions specimens. Also Optical and Scanning Electron Microscopy can be useful to support this type of studies. ^*Presented at: National Workshop on Astrobiology: Search for Life in the Solar System, Capri, Italy, 26 to 28 October, 2005     

Calcite and aragonite in the egg shell of Chelonia mydas L.

The structure of the calcified layer of egg shells from farm-reared and wild turtles has been examined using scanning electron microscopy and infrared analysis. Farm-reared egg shells contained discrete morphologically distinct regions of blocks of calcite and spherulites of aragonite. In contrast, the egg shells from feral populations consisted only of the spherulites. Differences in the impurity contents of the calcite and aragonite structures have been observed. SEM revealed a thin cuticular membrane.