Reciprocal changes in calcification of the gastrolith and cuticle during the molt cycle of the red claw crayfish Cherax quadricarinatus

Mobilization of calcium during the molt cycle from the cuticle to transient calcium deposits is widely spread in crustaceans. The dynamics of calcium transport to transient calcium deposits called gastroliths and to the cuticle over the course of the molt cycle were studied in the crayfish Cherax quadricarinatus. In this species, calcium was deposited in the gastroliths during premolt and transported back to the cuticle during postmolt, shown by digital X-ray radiograph analysis. The predominant mineral in the crayfish is amorphous calcium carbonate embedded in an organic matrix composed mainly of chitin. Scanning electron micrographs of the cuticle during premolt showed that the endocuticle and parts of the exocuticle were the source of most of the labile calcium, while the epicuticle did not undergo degradation and remained mineralized throughout the molt cycle. The gastroliths are made of concentric layers of amorphous calcium carbonate intercalated between chitinous lamella. Measurements of pH and calcium levels during gastrolith deposition showed that calcium concentrations in the gastroliths, stomach, and muscle were about the same (10 to 11 mmol l(-1)). On the other hand, pH varied greatly, from 8.7+/-0.15 in the gastrolith cavity through 7.6+/-0.2 in muscle to 6.9+/-0.5 in the stomach.     

Molding mineral within microporous hydrogels by a polymer-induced liquid-precursor (PILP) process

Natural biominerals often have exquisite morphologies, where the cells exercise a high degree of crystallographic control through secretion of biological macromolecules and regulation of ion transport. One important example is the sea urchin spine. It has recently been shown to be formed through deposition of a transient amorphous calcium carbonate (ACC) precursor phase that later transforms to single-crystalline calcite, ultimately forming an elaborate three-dimensional microporous calcium carbonate structure with interconnected pores. Macromolecules associated with the mineral phase are thought to play a key role in regulating this transformation. The work described here mimics this type of morphological control by "molding" an amorphous calcium carbonate precursor within a porous poly(2-hydroxyethyl methacrylate) (PHEMA) hydrogel that has been prepared as a negative replica from the void space of an urchin spine. Using an acidic biomimetic polymer as a process-directing agent, we show that polyaspartic acid induces amorphous calcium carbonate (ACC) nanoparticles, which have fluidic character and therefore are able to infiltrate the PHEMA hydrogel replica and coalesce into the convoluted morphology that replicates the original microporous structure of the sea urchin spine. By "molding" calcium carbonate into a complex morphology at room temperature, using a precursor process that is induced by a biomimetic acidic macromolecule, the PILP process is a useful in vitro model for examining different aspects of the amorphous-to-crystalline transformation process that is apparently used by a variety of biomineralizing organisms. For example, although we were able to replicate the overall morphology of the spine, it had polycrystalline texture; further studies with this system will focus on controlling the nucleation event, which may help to elucidate how such a convoluted structure can be prepared with single-crystalline texture via an amorphous precursor. Through a better understanding of the mechanisms used by organisms to regulate crystal properties, such biomimetic processes can lead to the synthesis of materials with superior electronic, mechanical, and optical properties.     

Biomineralizations: insights and prospects from crustaceans

For growing, crustaceans have to molt cyclically because of the presence of a rigid exoskeleton. Most of the crustaceans harden their cuticle not only by sclerotization, like all the arthropods, but also by calcification. All the physiology of crustaceans, including the calcification process, is then linked to molting cycles. This means for these animals to find regularly a source of calcium ions quickly available just after ecdysis. The sources of calcium used are diverse, ranging from the environment where the animals live to endogenous calcium deposits cyclically elaborated by some of them. As a result, crustaceans are submitted to an important and energetically demanding calcium turnover throughout their life. The mineralization process occurs by precipitation of calcium carbonate within an organic matrix network of chitin-proteins fibers. Both crystalline and stabilized amorphous polymorphs of calcium carbonate are found in crustacean biominerals. Furthermore, Crustacea is the only phylum of animals able to elaborate and resorb periodically calcified structures. Notably for these two previous reasons, crustaceans are more and more extensively studied and considered as models of choice in the biomineralization research area.     

Biomineralization by photosynthetic organisms: Evidence of coevolution of the organisms and their environment?

Biomineralization is widespread among photosynthetic organisms in the ocean, in inland waters and on land. The most quantitatively important biogeochemical role of land plants today in biomineralization is silica deposition in vascular plants, especially grasses. Terrestrial plants also increase the rate of weathering, providing the soluble substrates for biomineralization on land and in water bodies, a role that has had global biogeochemical impacts since the Devonian. The dominant photosynthetic biomineralizers in today's ocean are diatoms and radiolarians depositing silica and coccolithophores and foraminifera depositing calcium carbonate. Abiotic precipitation of silica from supersaturated seawater in the Precambrian preceded intracellular silicification dominated by sponges, then radiolarians and finally diatoms, with successive declines in the silicic acid concentration in the surface ocean, resulting in some decreases in the extent of silicification and, probably, increases in the silicic acid affinity of the active influx mechanisms. Calcium and bicarbonate concentrations in the surface ocean have generally been supersaturating with respect to the three common calcium carbonate biominerals through geological time, allowing external calcification as well as calcification in compartments within cells or organisms. The forms of calcium carbonate in biominerals, and presumably the evolution of the organisms that produce them, have been influenced by abiotic variations in calcium and magnesium concentrations in seawater, and calcium carbonate deposition has probably also been influenced by carbon dioxide concentration whose variations are in part biologically determined. Overall, there has been less biological feedback on the availability of substrates for calcification than is the case for silicification.     

Multitalentierte Biominerale

Biominerals and their applications in medicine and technology Biomineral forming systems are widespread in nature, but have gained more attention only in recent years with regard to the development of new materials and their application in medicine and technology. A major factor were, in particular, the development of the modern nanotechnology and the discovery that the main classes of biominerals, silica, calcium carbonate and calcium phosphate/hydroxyapatite, can be formed by an enzymatic mechanism. This allows the biocatalytic production both of organic‐inorganic hybrid materials with new property combinations and of defined structures from these biominerals. These applications range from optics to surface coatings, cell encapsulation, core shell materials and implants.     

Monohydrocalcite in calcareous corpuscles of Mesocestoides corti

Mesocestoides corti (syn. vogae), as many other cestode platyhelminthes, contains abundant mineralized structures called calcareous corpuscles. These concretions may constitute as much as 40% of the dry weight of the organisms, but their function remains poorly understood. In this work, we reviewed the mineral composition of the calcareous corpuscles of M. corti. X-ray diffraction pattern showed that the major mineral component of the corpuscles is a hydrated form of calcium carbonate, monohydrocalcite, also confirmed by infrared spectrometry. The baseline shift of the X-ray diffraction spectra suggested the presence of amorphous calcium carbonate, accordingly to previous reports, and an organic matrix was confirmed by FTIR. Monohydrocalcite is a rare mineral unusually found in biominerals. Although the significance of monohydrocalcite in biominerals has not been determined, the knowledge of corpuscles composition is of relevance to establish their function and for the elucidation of the mechanisms involved in mineralization processes.     

Biomineralization of the spicules of sea urchin embryos

The formation of calcareous skeletal elements by various echinoderms, especially sea urchins, offers a splendid opportunity to learn more about some processes involved in the formation of biominerals. The spicules of larvae of euechinoids have been the focus of considerable work, including their developmental origins. The spicules are composed of a single optical crystal of high magnesium calcite and variable amounts of amorphous calcium carbonate. Occluded within the spicule is a proteinaceous matrix, most of which is soluble; this matrix constitutes about 0.1% of the mass. The spicules are also enclosed by an extracellular matrix and are almost completely surrounded by cytoplasmic cords. The spicules are deposited by primary mesenchyme cells (PMCs), which accumulate calcium and secrete calcium carbonate. A number of proteins specific, or highly enriched, in PMCs, have been cloned and studied. Recent work supports the hypothesis that proteins found in the extracellular matrix of the spicule are important for biomineralization.     

Characterization of the exoskeleton of the Antarctic king crab Paralomis birsteini

Ocean acidification is projected to inhibit the biogenic production of calcium carbonate skeletons in marine organisms. Antarctic waters represent a natural environment in which to examine the long‐term effects of carbonate undersaturation on calcification in marine predators. King crabs (Decapoda: Anomura: Lithodidae), which currently inhabit the undersaturated environment of the continental slope off Antarctica, are potential invasives on the Antarctic shelf as oceanic temperatures rise. Here, we describe the chemical, physical, and mechanical properties of the exoskeleton of the deep‐water Antarctic lithodid Paralomis birsteini and compare our measurements with two decapod species from shallow water at lower latitudes, Callinectes sapidus (Brachyura: Portunidae) and Cancer borealis (Brachyura: Cancridae). In Paralomis birsteini, crabs deposit proportionally more calcium carbonate in their predatory chelae than their protective carapaces, compared with the other two crab species. When exoskeleton thickness and microhardness were compared between the chelae and carapace, the magnitude of the difference between these body regions was significantly greater in P. birsteini than in the other species tested. Hence, there appeared to be a greater disparity in P. birsteini in overall investment in calcium carbonate structures among regions of the exoskeleton. The imperatives of prey consumption and predator avoidance may be influencing the deposition of calcium to different parts of the exoskeleton in lithodids living in an environment undersaturated with respect to calcium carbonate.     

Intrinsically Disordered and Pliable Starmaker-Like Protein from Medaka (Oryzias latipes) Controls the Formation of Calcium Carbonate Crystals

Fish otoliths, biominerals composed of calcium carbonate with a small amount of organic matrix, are involved in the functioning of the inner ear. Starmaker (Stm) from zebrafish (Danio rerio) was the first protein found to be capable of controlling the formation of otoliths. Recently, a gene was identified encoding the Starmaker-like (Stm-l) protein from medaka (Oryzias latipes), a putative homologue of Stm and human dentine sialophosphoprotein. Although there is no sequence similarity between Stm-l and Stm, Stm-l was suggested to be involved in the biomineralization of otoliths, as had been observed for Stm even before. The molecular properties and functioning of Stm-l as a putative regulatory protein in otolith formation have not been characterized yet. A comprehensive biochemical and biophysical analysis of recombinant Stm-l, along with in silico examinations, indicated that Stm-l exhibits properties of a coil-like intrinsically disordered protein. Stm-l possesses an elongated and pliable structure that is able to adopt a more ordered and rigid conformation under the influence of different factors. An in vitro assay of the biomineralization activity of Stm-l indicated that Stm-l affected the size, shape and number of calcium carbonate crystals. The functional significance of intrinsically disordered properties of Stm-l and the possible role of this protein in controlling the formation of calcium carbonate crystals is discussed.     

Amorphous and crystalline calcium carbonate distribution in the tergite cuticle of moulting Porcellio scaber (Isopoda, Crustacea)

The main mineral components of the isopod cuticle consists of crystalline magnesium calcite and amorphous calcium carbonate. During moulting isopods moult first the posterior and then the anterior half of the body. In terrestrial species calcium carbonate is subject to resorption, storage and recycling in order to retain significant fractions of the mineral during the moulting cycle. We used synchrotron X-ray powder diffraction, elemental analysis and Raman spectroscopy to quantify the ACC/calcite ratio, the mineral phase distribution and the composition within the anterior and posterior tergite cuticle during eight different stages of the moulting cycle of Porcellio scaber. The results show that most of the amorphous calcium carbonate (ACC) is resorbed from the cuticle, whereas calcite remains in the old cuticle and is shed during moulting. During premoult resorption of ACC from the posterior cuticle is accompanied by an increase within the anterior tergites, and mineralization of the new posterior cuticle by resorption of mineral from the anterior cuticle. This suggests that one reason for using ACC in cuticle mineralization is to facilitate resorption and recycling of cuticular calcium carbonate. Furthermore we show that ACC precedes the formation of calcite in distal layers of the tergite cuticle.     

Molecular analysis of two genes, DD9A and B, which are expressed during the postmolt stage in the decapod crustacean Penaeus japonicus

In decapod crustaceans, deposition of calcium carbonate crystals (calcification) in the exoskeleton takes place during the postmolt phase of the molt cycle. In an attempt to identify proteins which regulate the calcification process, the differential display technique was used to identify genes which were specifically expressed in the integument during the postmolt stage in the penaeid prawn Penaeus japonicus. One of the genes thus identified, named DD9A, was expressed in the epithelial cells of the tail fan. DD9A encoded a putative precursor of a secreted protein of 113 amino acids which exhibited sequence similarities to a group of crustacean and insect cuticular proteins, suggesting that DD9A was a protein component of the exoskeleton. Another gene, DD9B, which was also transcribed specifically during the postmolt period was identified based on its sequence similarity to DD9A. Potential roles of the DD9A protein in the calcification of the exoskeleton will be discussed.     

The Biomineralization Proteome: Protein Complexity for a Complex Bioceramic Assembly Process

There are over 62 different biominerals on Earth and a diverse array of organisms that generate these biominerals for survival. This review will introduce the process of biomineralization and the current understanding of the molecular mechanisms of mineral formation, and then comparatively explore the representative secretomes of two well‐documented skeletal systems: vertebrate bone (calcium phosphate) and invertebrate mollusk shell (calcium carbonate). It is found that both skeletal secretomes have gross similarities and possess proteins that fall into four functional categories: matrix formers, nucleation assisters, communicators, and remodelers. In many cases the mineral‐associated matrix former and nucleation assister sequences in both skeletal systems are unique and possess interactive conserved globular domains, intrinsic disorder, post‐translational modifications, sequence redundancy, and amyloid‐like aggregation‐prone sequences. Together, these molecular features create a protein‐based environment that facilitates mineral formation and organization and argue in favor of conserved features that evolve from the mollusk shell to bone. Interestingly, the mollusk shell secretome appears to be more complex compared to that of bone tissue, in that there are numerous protein subcategories that are required for the nucleation and organization of inner (nacre) and outer (prismatic) calcium carbonate regions of the shell. This may reflect the organizational and material requirements of an exoskeletal protective system.     

Complex biomineralization pattern in cactaceae

The infrared spectroscopic investigation of biominerals isolated from different Cactaceae species belonging to the Opuntioideae subfamily shows the presence of a very complex mineral composition, including whewellite (monohydrated calcium oxalate), opal (SiO2) and calcite (CaCO3). This is the first report on the presence of a calcium carbonate in these types of plants.     

Purification and structural determination of a phosphorylated peptide with anti-calcification and chitin-binding activities in the exoskeleton of the crayfish, Procambarus clarkii

Organic matrices in calcified hard tissues have been considered to control calcification. A matrix peptide, designated CAP-1, was extracted and purified by anion-exchange and reverse-phase high performance liquid chromatographies from the exoskeleton of the crayfish, Procambarus clarkii. The amino acid sequence of CAP-1 was determined by mass spectral and sequence analyses of the intact peptide and its enzymatically digested peptides. CAP-1 consisted of 78 amino acid residues, including a phosphoserine residue, and was rich in acidic amino acid residues. CAP-1 had a Rebers-Riddiford consensus sequence, which is conserved in cuticle proteins from many arthropods. CAP-1 inhibited precipitation of calcium carbonate in an in vitro anticalcification assay dose-dependently, and completely inhibited it at 3 x 10(-7) M. CAP-1 also showed chitin-binding ability, indicating that this molecule was bifunctional and played an important role in formation of the exoskeleton.     

Mutated <Emphasis Type="Italic">otopetrin 1</Emphasis> affects the genesis of otoliths and the localization of Starmaker in zebrafish

Otoliths in bony fishes and otoconia in mammals are composite crystals consisting of calcium carbonate and proteins. These biominerals are part of the gravity and linear acceleration detection system of the inner ear. Mutations in otopetrin 1 have been shown to result in lack of otoconia in tilted and mergulhador mutant mice. The molecular function of Otopetrin 1, a novel protein that contains ten predicted transmembrane domains, however, has remained elusive. Here we show that a mutation in the orthologous gene in zebrafish is responsible for the complete absence of otoliths in backstroke mutants. We examined the localization of Starmaker, a secreted protein that is highly abundant in otoliths in backstroke mutants. Starmaker protein accumulated within cells of the otic epithelium, indicating a possible defect in secretion. Our data suggest that Otopetrin 1 in zebrafish may be involved in the protein trafficking of components required for formation of biominerals in the ear.     

Identification,distribution, and quantification of biominerals in a deciduous forest

Biomineralization is a common process in most vascular plants, but poorly investigated for trees. Although the presence of calcium oxalate and silica accumulation has been reported for some tree species, the chemical composition, abundance, and quantification of biominerals remain poorly documented. However, biominerals may play important physiological and structural roles in trees, especially in forest ecosystems, which are characterized by nutrient‐poor soils. In this context, our study aimed at investigating the morphology, distribution, and relative abundance of biominerals in the different vegetative compartments (foliage, branch, trunk, and root) of Fagus sylvatica L. and Acer pseudoplatanus L. using a combination of scanning electron microscopy and tomography analyses. Biomineral crystallochemistry was assessed by X‐ray diffraction and energy‐dispersive X‐ray analyses, while calcium, silicon, and oxalic acid were quantified in the compartments and at the forest scale. Our analyses revealed that biominerals occurred as crystals or coating layers mostly in bark and leaves and were identified as opal, whewellite, and complex biominerals. In both tree species, opal was mostly found in the external tissues of trunk, branch, and leaves, but also in the roots of beech. In the stand, opal represents around 170 kg/ha. Whewellite was found to suit to conductive tissues (i.e., axial phloem parenchyma, vascular bundles, vessel element) in all investigated compartments of the two tree species. The shape of whewellite was prismatic and druses in beech, and almost all described shapes were seen in sycamore maple. Notably, the amount of whewellite was strongly correlated with the total calcium in all investigated compartments whatever the tree species is, suggesting a biologic control of whewellite precipitation. The amount of whewellite in the aboveground biomass of Montiers forest was more important than that of opal and was around 1170 kg/ha. Therefore, biominerals contribute in a substantial way to the biogeochemical cycles of silicon and calcium.     

Purification and Structural Determination of a Phosphorylated Peptide with Anti-calcification and Chitin-binding Activities in the Exoskeleton of the Crayfish,Procambarus clarkii

Organic matrices in calcified hard tissues have been considered to control calcification. A matrix peptide, designated CAP-1, was extracted and purified by anionexchange and reverse-phase high performance liquid chromatographies from the exoskeleton of the crayfish, Procambarus clarkii. The amino acid sequence of CAP-1 was determined by mass spectral and sequence analyses of the intact peptide and its enzymatically digested peptides. CAP-1 consisted of 78 amino acid residues, including a phosphoserine residue, and was rich in acidic amino acid residues. CAP-1 had a RebersRiddiford consensus sequence, which is conserved in cuticle proteins from many arthropods. CAP-1 inhibited precipitation of calcium carbonate in an in vitro anticalcification assay dose-dependently, and completelyinhibited it at 3×10^-7 M. CAP-1 also showed chitinbinding ability, indicating that this molecule was bifunctional and played an important role in formation of the exoskeleton.     

Characterization of the calcium biomineral in the radular teeth of Chiton pelliserpentis

The radula in a group of molluscan invertebrates, the chitons (Polyplacophora), is a ribbon-like apparatus used for feeding and which bears a series of distinctive mineralized teeth called the major lateral teeth. While some chiton species deposit only iron biominerals in these teeth, many others deposit both iron and calcium. In this study, the calcium biomineral in the teeth of one of the latter types of species, the Australian east-coast chiton, Chiton pelliserpentis, has been isolated and examined for the first time. Spectroscopic and crystallographic techniques have identified the biomineral as a carbonate-substituted apatite with significant fluoride substitution also likely. Fourier-transform infrared and laser Raman spectroscopy indicated that the carbonate content was less than that of either bovine tibia cortical bone or human tooth enamel. X-ray diffraction analysis showed the biomineral to be poorly crystalline due to small crystal size and appreciable anionic substitution. The lattice parameters were calculated to be a=9.382?Å and c=6.883?Å, which are suggestive of a fluorapatite material. It is postulated that structural and biochemical differences in the tooth organic matrix of different chiton species will ultimately determine if the teeth become partly calcified or iron mineralized only.