Molluscan Biomineralization Research Articles

The adult shell matrix protein repertoire of the marine snail Crepidula is dominated by conserved genes that are also expressed in larvae

Mollusca is a morphologically diverse phylum, exhibiting an immense variety of calcium carbonate structures. Proteomic studies of adult shells often report high levels of rapidly-evolving, ‘novel’ shell matrix proteins (SMPs), which are hypothesized to drive shell diversification. However, relatively little is known about the phylogenetic distribution of SMPs, or about the function of individual SMPs in shell construction. To understand how SMPs contribute to shell diversification a thorough characterization of SMPs is required. Here, we build tools and a foundational understanding of SMPs in the marine gastropod species Crepidula fornicata and Crepidula atrasolea because they are genetically-enabled mollusc model organisms. First, we established a staging system of shell development in C. atrasolea for the first time. Next, we leveraged previous findings in C. fornicata combined with phylogenomic analyses of 95 metazoan species to determine the evolutionary lineage of its adult SMP repertoire. We found that 55% of C. fornicata’s SMPs belong to molluscan orthogroups, with 27% restricted to Gastropoda, and only 5% restricted at the species level. The low percentage of species-restricted SMPs underscores the importance of broad-taxon sampling and orthology inference approaches when determining homology of SMPs. From our transcriptome analysis, we found that the majority of C. fornicata SMPs that were found conserved in C. atrasolea were expressed in both larval and adult stages. We then selected a subset of SMPs of varying evolutionary ages for spatial-temporal analysis using in situ hybridization chain reaction (HCR) during larval shell development in C. atrasolea. Out of the 18 SMPs analyzed, 12 were detected in the larval shell field. These results suggest overlapping larval vs. adult SMP repertoires. Using multiplexed HCR, we observed five SMP expression patterns and three distinct cell populations within the shell field. These patterns support the idea that modular expression of SMPs could facilitate divergence of shell morphological characteristics. Collectively, these data establish an evolutionary and developmental framework in Crepidula that enables future comparisons of molluscan biomineralization to reveal mechanisms of shell diversification.

Deciphering the molecular toolkit: regulatory elements governing shell biomineralization in marine molluscs.

The intricate process of shell biomineralization in marine molluscs is governed by a complex interplay of regulatory elements, encompassing secretomes, transporters, and noncoding RNA. This review delves into recent advancements in understanding these regulatory mechanisms, emphasizing their significance in elucidating the functions and evolutionary dynamics of the molluscan shell biomineralization process. Central to this intricate orchestration are secretomes with diverse functional domains, selectively exported to the extrapallial space, which directly regulate crystal growth and morphology. Transporters are crucial for substrate transportation in the calcification and maintenance of cellular homeostasis. Beyond proteins and transporters, noncoding RNA molecules are integral components influencing shell biomineralization. This review underscores the nonnegligible roles played by these genetic elements at the molecular level. To comprehend the complexity of biomineralization in mollusc, we explore the origin and evolutionary history of regulatory elements, primarily secretomes. While some elements have recently evolved, others are ancient genes that have been co-opted into the biomineralization toolkit. These elements undergo structural and functional evolution through rapidly evolving repetitive low-complexity domains and domain gain/loss/rearrangements, ultimately shaping a distinctive set of secretomes characterized by both conserved features and evolutionary innovations. This comprehensive review enhances our understanding of molluscan biomineralization at the molecular and genetic levels.

Exoskeleton protein repertoires in decapod crustaceans revealed distinct biomineralization evolution with molluscs

Crustaceans are the champions of mineral mobilization and deposition in the animal kingdom due to their unique ability to rapidly and periodically mineralize and demineralize their exoskeletons. They are commonly covered with mineralized exoskeletons for protection and regularly molt throughout their lives. Mineralized crustacean exoskeletons are formed under the control of macromolecules especially matrix proteins but the types of matrix proteins are understudied compared to those in molluscan shells. This gap hinders our understanding of their evolutionary paths compared with those of molluscs. Here, we comprehensively analyzed matrix proteins in the exoskeleton of two crabs, one shrimp, and one crayfish and resulted in a major improvement (∼10-fold) in the identification of biomineralization proteins compared to conventional methods for decapod crustaceans. By a comparison with well-studied molluscan biomineralization proteins, we found that decapod crustaceans evolved novel proteins to form mineralized exoskeletons while sharing some proteins with those of molluscs. Our study sheds light on their evolution and adaption to different environment for exoskeleton formation and provides a foundation for further studies of mineralization in crustaceans under normal and climate-changed conditions. SignificanceMost crustaceans have mineralized exoskeletons as protection. How they form these hierarchical structures is still unclear. This is due partially to the understudied matrix proteins in the minerals. This study filled such a gap by using proteomic analysis of matrix proteins from four decapod crustacean exoskeletons. Many novel proteins were discovered which enabled a solid comparison with those of molluscs. By comparison, we proposed that crustaceans evolved novel proteins to form mineralized exoskeletons while sharing some proteins with those of molluscs. This is useful for us to understand the evolution of two major biomineralized phylum.

Multi-omic insights into the formation and evolution of a novel shell microstructure in oysters

BackgroundMolluscan shell, composed of a diverse range of architectures and microstructures, is a classic model system to study the relationships between molecular evolution and biomineralized structure formation. The shells of oysters differ from those of other molluscs by possessing a novel microstructure, chalky calcite, which facilitates adaptation to the sessile lifestyle. However, the genetic basis and evolutionary origin of this adaptive innovation remain largely unexplored.ResultsWe report the first whole-genome assembly and shell proteomes of the Iwagaki oyster Crassostrea nippona. Multi-omic integrative analyses revealed that independently expanded and co-opted tyrosinase, peroxidase, TIMP genes may contribute to the chalky layer formation in oysters. Comparisons with other molluscan shell proteomes imply that von Willebrand factor type A and chitin-binding domains are basic members of molluscan biomineralization toolkit. Genome-wide identification and analyses of these two domains in 19 metazoans enabled us to propose that the well-known Pif may share a common origin in the last common ancestor of Bilateria. Furthermore, Pif and LamG3 genes acquire new genetic function for shell mineralization in bivalves and the chalky calcite formation in oysters likely through a combination of gene duplication and domain reorganization.ConclusionsThe spatial expression of SMP genes in the mantle and molecular evolution of Pif are potentially involved in regulation of the chalky calcite deposition, thereby shaping the high plasticity of the oyster shell to adapt to a sessile lifestyle. This study further highlights neo-functionalization as a crucial mechanism for the diversification of shell mineralization and microstructures in molluscs, which may be applied more widely for studies on the evolution of metazoan biomineralization.

Isotope systematics of subfossil, historical, and modern Nautilus macromphalus from New Caledonia.

Cephalopod carbonate geochemistry underpins studies ranging from Phanerozoic, global-scale change to outcrop-scale paleoecological reconstructions. Interpreting these data hinges on assumed similarity to model organisms, such as Nautilus, and generalization from other molluscan biomineralization processes. Aquarium rearing and capture of wild Nautilus suggest shell carbonate precipitates quickly (35 μm/day) in oxygen isotope equilibrium with seawater. Other components of Nautilus shell chemistry are less well-studied but have potential to serve as proxies for paleobiology and paleoceanography. To calibrate the geochemical response of cephalopod δ15Norg, δ13Corg, δ13Ccarb, δ18Ocarb, and δ44/40Cacarb to modern anthropogenic environmental change, we analyzed modern, historical, and subfossil Nautilus macromphalus from New Caledonia. Samples span initial human habitation, colonialization, and industrial pCO2 increase. This sampling strategy is advantageous because it avoids the shock response that can affect geochemical change in aquarium experiments. Given the range of living depths and more complex ecology of Nautilus, however, some anthropogenic signals, such as ocean acidification, may not have propagated to their living depths. Our data suggest some environmental changes are more easily preserved than others given variability in cephalopod average living depth. Calculation of the percent respired carbon incorporated into the shell using δ13Corg, δ13Ccarb, and Suess-effect corrected δ13CDIC suggests an increase in the last 130 years that may have been caused by increasing carbon dioxide concentration or decreasing oxygen concentration at the depths these individuals inhabited. This pattern is consistent with increasing atmospheric CO2 and/or eutrophication offshore of New Caledonia. We find that δ44/40Ca remains stable across the last 130 years. The subfossil shell from a cenote may exhibit early δ44/40Ca diagenesis. Questions remain about the proportion of dietary vs ambient seawater calcium incorporation into the Nautilus shell. Values of δ15N do not indicate trophic level change in the last 130 years, and the subfossil shell may show diagenetic alteration of δ15N toward lower values. Future work using historical collections of Sepia and Spirula may provide additional calibration of fossil cephalopod geochemistry.

Helical microstructures in molluscan biomineralization are a biological example of close packed helices that may form from a colloidal liquid crystal precursor in a twist–bend nematic phase

We demonstrate that nature has produced a close-packed helical twisted filamentous material in the biomineralization of the mollusc. In liquid crystals, twist-bend nematics have been predicted and observed. We present and analyse evidence that the helical biomineral microstructure of mollusc shells may be formed from such a liquid-crystal precursor.

Nautilus pompilius

Nautilus pompilius, a ‘living fossil’, has many ancestral features, such as an external chambered shell and pinhole eye, despite its long evolutionary history, thus providing an excellent prototypical model for studying cephalopod evolution. It also has a compact, minimalist genome with fewer genes and slower evolutionary rates in both noncoding and coding regions compared with its sister coleoid genomes. The genome retained a conserved molluscan biomineralization toolkit; relevant proteins such as shell matrix proteins, Sushi/SCR/CCP, laminin, and chitin-binding domains show high similarity with proteins in other mollusks and have massive lineage-specific expanded repetitive low-complexity domains, which co-contribute to the production of aragonite crystals, a major component of the nautilus shell.

The genome of Nautilus pompilius illuminates eye evolution and biomineralization

Nautilus is the sole surviving externally shelled cephalopod from the Palaeozoic. It is unique within cephalopod genealogy and critical to understanding the evolutionary novelties of cephalopods. Here, we present a complete Nautilus pompilius genome as a fundamental genomic reference on cephalopod innovations, such as the pinhole eye and biomineralization. Nautilus shows a compact, minimalist genome with few encoding genes and slow evolutionary rates in both non-coding and coding regions among known cephalopods. Importantly, multiple genomic innovations including gene losses, independent contraction and expansion of specific gene families and their associated regulatory networks likely moulded the evolution of the nautilus pinhole eye. The conserved molluscan biomineralization toolkit and lineage-specific repetitive low-complexity domains are essential to the construction of the nautilus shell. The nautilus genome constitutes a valuable resource for reconstructing the evolutionary scenarios and genomic innovations that shape the extant cephalopods.

Sticky problems: extraction of nucleic acids from molluscs

Traditional molecular methods and omics-techniques across molluscan taxonomy increasingly inform biology of Mollusca. Recovery of DNA and RNA for such studies is challenged by common biological properties of the highly diverse molluscs. Molluscan biomineralization, adhesive structures and mucus involve polyphenolic proteins and mucopolysaccharides that hinder DNA extraction or copurify to inhibit enzyme-catalysed molecular procedures. DNA extraction methods that employ the detergent hexadecyltrimethylammoniumbromide (CTAB) to remove these contaminants importantly facilitate molecular-level study of molluscs. Molluscan pigments may stain DNA samples and interfere with spectrophotometry, necessitating gel electrophoresis or fluorometry for accurate quantification. RNA can reliably be extracted but the ‘hidden break’ in 28S rRNA of molluscs (like most protostomes) causes 18S and 28S rRNA fragments to co-migrate electrophoretically. This challenges the standard quality control based on the ratio of 18S and 28S rRNA, developed for deuterostome animals. High-AT content in molluscan rRNA prevents the effective purification of polyadenylated mRNA. Awareness of these matters aids the continuous expansion of molecular malacology, enabling work also with museum specimens and next-generation sequencing, with the latter imposing unprecedented demands on DNA quality. Alternative methods to extract nucleic acids from molluscs are available from literature and, importantly, from communications with others who study the molecular biology of molluscs. This article is part of the Theo Murphy meeting issue ‘Molluscan genomics: broad insights and future directions for a neglected phylum’.

Computationally predicted gene regulatory networks in molluscan biomineralization identify extracellular matrix production and ion transportation pathways.

MotivationThe molecular processes regulating molluscan shell production remain relatively uncharacterized, despite the clear evolutionary and societal importance of biomineralization.ResultsHere we built the first computationally predicted gene regulatory network (GRN) for molluscan biomineralization using Antarctic clam (Laternula elliptica) mantle gene expression data produced over an age-categorized shell damage-repair time-course. We used previously published in vivo in situ hybridization expression data to ground truth gene interactions predicted by the GRN and show that candidate biomineralization genes from different shell layers, and hence microstructures, were connected in unique modules. We characterized two biomineralization modules of the GRN and hypothesize that one module is responsible for translating the extracellular proteins required for growing, repairing or remodelling the nacreous shell layer, whereas the second module orchestrates the transport of both ions and proteins to the shell secretion site, which are required during normal shell growth, and repair. Our findings demonstrate that unbiased computational methods are particularly valuable for studying fundamental biological processes and gene interactions in non-model species where rich sources of gene expression data exist, but annotation rates are poor and the ability to carry out true functional tests are still lacking.Availability and implementationThe raw RNA-Seq data is freely available for download from NCBI SRA (Accession: PRJNA398984), the assembled and annotated transcriptome can be viewed and downloaded from molluscDB (ensembl.molluscdb.org) and in addition, the assembled transcripts, reconstructed GRN, modules and detailed annotations are all available as Supplementary Files.Supplementary information Supplementary data are available at Bioinformatics online.

Shell microstructures of the helcionelloid mollusc Anabarella australis from the lower Cambrian (Series 2) Xinji Formation of North China

Although various types of shell microstructures are documented from Cambrian molluscs, the precise organization and mineralogical composition of Terreneuvian molluscs are relatively unknown. Anabarella was one of the first helcionellid molluscs to appear in the Terreneuvian, with the genus surviving until the third epoch of the Cambrian. Here, shell microstructures of Anabarella australis have been studied based on new collections from the lower Cambrian (Series 2) Xinji Formation of the North China Block. Results show that A. australis has a laminar inner shell layer that consists of crossed foliated lamellar microstructure (CFL). Nacreous, crossed-lamellar and foliated aragonite microstructures previously documented in the older (Terreneuvian) species A. plana are here revised as preservational artefacts of the CFL layers. This complex skeletal organization of Anabarella suggests that mechanisms of molluscan biomineralization evolved very rapidly. Morphologically, specimens from the Chaijiawa section show a pattern of distinct ‘pseudo-dimorphism’ as external coatings are identical to Anabarella, while associated internal moulds are similar to the helcionelloid genus Planutenia. In contrast, internal moulds from the Shangzhangwan section show considerable morphological variation owing to preservational bias and show greater similarities to specimens from South Australia, Northeast Greenland and Germany. These observations demonstrate that the extensive morphological variation seen in the internal moulds of the cosmopolitan genus Anabarella are primarily preservational artefacts and are unlikely to represent the real intra- and interspecific variability of the animal. In these cases, Planutenia is here confirmed to be a subjective synonym of Anabarella.

Gene structure and domain architecture in the biomineralizing protein Lustrin A from the abalone Haliotis rufescens

Among the proteins that play a role in molluscan biomineralization, Lustrin A was the first to be investigated and it remains the most-well studied. It is composed of many repetitive domains, which appear to be present in all members of the family Haliotidae. For the first time we have sequenced the complete gene (34,093 bp), coding for the cDNA of Haliotis rufescens Lustrin A, using long PCRs and cloning. It encompasses 34 exons and 33 introns, including 10 minisatellite-rich regions present in eight of these introns. The corresponding glycine and serine-rich (GS) domain and the C-terminus-WAP domain are well delimited by phase 1 introns, while phase 0 introns separate the Cysteine-rich domains. Within these domains, the Hr_Lustrin gene contains 10 phase 1 introns. No codon bias was detected in the nucleotide composition. We discuss the observe intron specificity together with the absence of exons corresponding to putative spacer regions or basic domains. We also compared H. rufescens Lustrin A with the H. tuberculata Lustrin A gene partial sequence.

Fluid-flow-templated self-assembly of calcium carbonate tubes in the laboratory and in biomineralization: The tubules of the watering-pot shells, Clavagelloidea

We show with laboratory experiments that self-assembled mineral tube formation involving precipitation around a templating jet of fluid — a mechanism well-known in the physical sciences from the tubular growth of so-called chemical gardens — functions with carbonates, and we analyse the microstructures and compositions of the precipitates. We propose that there should exist biological examples of fluid-flow-templated tubes formed from carbonates. We present observational and theoretical modelling evidence that the complex structure of biomineral calcium carbonate tubules that forms the ‘rose’ of the watering-pot shells, Clavagelloidea, may be an instance of this mechanism in biomineralization. We suggest that this is an example of self-organization and self-assembly processes in biomineralization, and that such a mechanism is of interest for the production of tubes as a synthetic biomaterial. Statement of SignificanceThe work discussed in the manuscript concerns the self-assembly of calcium carbonate micro-tubes and nano-tubes under conditions of fluid flow together with chemical reaction. We present the results of laboratory experiments on tube self-assembly together with theoretical calculations. We show how nature may already be making use of this process in molluscan biomineralization of the so-called watering-pot shells, and we propose that we may be able to take advantage of the formation mechanism to produce synthetic biocompatible micro- and nano-tubes.

Sea shell diversity and rapidly evolving secretomes: insights into the evolution of biomineralization.

An external skeleton is an essential part of the body plan of many animals and is thought to be one of the key factors that enabled the great expansion in animal diversity and disparity during the Cambrian explosion. Molluscs are considered ideal to study the evolution of biomineralization because of their diversity of highly complex, robust and patterned shells. The molluscan shell forms externally at the interface of animal and environment, and involves controlled deposition of calcium carbonate within a framework of macromolecules that are secreted from the dorsal mantle epithelium. Despite its deep conservation within Mollusca, the mantle is capable of producing an incredible diversity of shell patterns, and macro- and micro-architectures. Here we review recent developments within the field of molluscan biomineralization, focusing on the genes expressed in the mantle that encode secreted proteins. The so-called mantle secretome appears to regulate shell deposition and patterning and in some cases becomes part of the shell matrix. Recent transcriptomic and proteomic studies have revealed marked differences in the mantle secretomes of even closely-related molluscs; these typically exceed expected differences based on characteristics of the external shell. All mantle secretomes surveyed to date include novel genes encoding lineage-restricted proteins and unique combinations of co-opted ancient genes. A surprisingly large proportion of both ancient and novel secreted proteins containing simple repetitive motifs or domains that are often modular in construction. These repetitive low complexity domains (RLCDs) appear to further promote the evolvability of the mantle secretome, resulting in domain shuffling, expansion and loss. RLCD families further evolve via slippage and other mechanisms associated with repetitive sequences. As analogous types of secreted proteins are expressed in biomineralizing tissues in other animals, insights into the evolution of the genes underlying molluscan shell formation may be applied more broadly to understanding the evolution of metazoan biomineralization.

Genetic structure and polymorphisms of the N16 gene in Pinctada fucata

The molluscan shell is a composite of inorganic crystals comprising calcium carbonate and a minute amount of organic matrix. The organic matrix (OM) is intimately involved in every step of shell formation and has consequently received much attention in recent years. However, most of the deposited information has resulted from cDNA analysis, with little analysis of the genome, including the presence and effects of polymorphic genes encoding OM proteins.The current study aimed to clarify the genome structure of the N16 gene from the Japanese pearl oyster, Pinctada fucata, with particular reference to polymorphisms. The N16 gene was analyzed using PCR and DNA sequencing. 23 polymorphic variants were identified from 28 individuals. The variants were analyzed for their relationship to shell formation. All the variations detected by genomic PCR appeared in the cDNAs, implying that all the polymorphisms were transcribed and translated into N16 proteins. Additional genome analysis revealed at least two N16 genes, which were sequentially positioned, each of them comprising four exons and three introns.Further analyses of the transcriptional regulation and function of the N16 genes may provide new insights into their role in molluscan biomineralization.

The Mining of Pearl Formation Genes in Pearl Oyster Pinctada fucata by cDNA Suppression Subtractive Hybridization

Recent researches revealed the regional preference of biomineralization gene transcription in the pearl oyster Pinctada fucata: it transcribed mainly the genes responsible for nacre secretion in mantle pallial, whereas the ones regulating calcite shells expressed in mantle edge. This study took use of this character and constructed the forward and reverse suppression subtractive hybridization (SSH) cDNA libraries. A total of 669 cDNA clones were sequenced and 360 expressed sequence tags (ESTs) greater than 100bp were generated. Functional annotation associated 95 ESTs with specific functions, and 79 among them were identified from P. fucata at the first time. In the forward SSH cDNA library, it recognized mass amount of nacre protein genes, biomineralization genes dominantly expressed in the mantle pallial, calcium-ion-binding genes, and other biomineralization-related genes important for pearl formation. Real-time PCR showed that all the examined genes were distributed in oyster mantle tissues with a consistence to the SSH design. The detection of their RNA transcripts in pearl sac confirmed that the identified genes were certainly involved in pearl formation. Therefore, the data from this work will initiate a new round of pearl formation gene study and shed new insights into molluscan biomineralization.

Deep Sequencing of ESTs from Nacreous and Prismatic Layer Producing Tissues and a Screen for Novel Shell Formation-Related Genes in the Pearl Oyster

BackgroundDespite its economic importance, we have a limited understanding of the molecular mechanisms underlying shell formation in pearl oysters, wherein the calcium carbonate crystals, nacre and prism, are formed in a highly controlled manner. We constructed comprehensive expressed gene profiles in the shell-forming tissues of the pearl oyster Pinctada fucata and identified novel shell formation-related genes candidates.Principal FindingsWe employed the GS FLX 454 system and constructed transcriptome data sets from pallial mantle and pearl sac, which form the nacreous layer, and from the mantle edge, which forms the prismatic layer in P. fucata. We sequenced 260477 reads and obtained 29682 unique sequences. We also screened novel nacreous and prismatic gene candidates by a combined analysis of sequence and expression data sets, and identified various genes encoding lectin, protease, protease inhibitors, lysine-rich matrix protein, and secreting calcium-binding proteins. We also examined the expression of known nacreous and prismatic genes in our EST library and identified novel isoforms with tissue-specific expressions.ConclusionsWe constructed EST data sets from the nacre- and prism-producing tissues in P. fucata and found 29682 unique sequences containing novel gene candidates for nacreous and prismatic layer formation. This is the first report of deep sequencing of ESTs in the shell-forming tissues of P. fucata and our data provide a powerful tool for a comprehensive understanding of the molecular mechanisms of molluscan biomineralization.

N-linked oligosaccharide in MSP-1 and its implication for scallop calcification

We speculated the structure of the N-linked oligosaccharides enzymatically released from the organic matrix (OM) component in the foliated layer of Patinopecten yessoensis. The 80 kDa component of the soluble OM was detected by lectin blotting and was identified as MSP-1 using liquid chromatography/mass spectrometry (LC/MS/MS). LC/MS/MS analysis of the N-glycan liberated from MSP-1 detected a hybrid-type N-glycan, which contained sulfite and sialic acid at its terminus based on the characteristic Y ions. The data strongly imply that MSP-1, a sulfated OM glycoprotein, participates in molluscan biomineralization by creating a favorable environment for calcium ion uptake through sulfite acid and sialic acid. Further analyses of oligosaccharides linked to the OM components in wide variety of species and shell microstructures may definitely contribute in elucidation of molluscan biomineralization at the molecular level.

Isolation and Characterization of the N-linked Oligosaccharides in Nacrein from Pinctada fucata

We analyzed the structure of the N-linked oligosaccharides enzymatically liberated from the organic matrix (OM) component in the nacreous layer of Japanese pearl oyster: Pinctada fucata. The lectin-blot analysis of the soluble OM after separation by SDS-PAGE, four components, with sizes of approximately 55 kDa, 35 kDa, 25 kDa, and 21 kDa were detected with GNA lectin, which recognized terminal mannose of high mannose and hybrid types of N-glycan. The 55-kDa component of the soluble OM detected by lectin blotting was identified as nacrein by using liquid chromatography/mass spectrometry (LC/MS). LC/MS analysis of the N-glycan liberated from nacrein detected a hybrid-type N-glycan, which contained sulfite and sialic acid at its terminus. The data strongly imply that nacrein, a sulfated OM glycoprotein, participates in molluscan biomineralization by creating a favorable environment for calcium ion uptake through sulfonic acid and sialic acid.

Molluscan biomineralization: The proteinaceous shell constituents of Pinna nobilis L.

The shell of molluscs is a remarkable example of a natural composite biomaterial, synthesized at ambient temperature. Consequently, many consider it as a model for trying to develop at little cost new biomimetic materials of superior mechanical properties. The peculiar resistance of shells to fracture lies in an organic matrix, which is closely associated with the mineral phase. This matrix regulates the crystal growth, by allowing nucleation of the crystals only where appropriate, by favoring crystal elongation in privileged directions, and by inhibiting their growth. This matrix is a mixture of glycoproteins and polysaccharides, the primary structure and function of which are poorly known. Pinna nobilis, the Mediterranean fan mussel, is one of the few molluscs for which molecular data on shell proteins are available. In the present paper, we review what is known, at molecular level, on the macromolecular constituents of the shell of P. nobilis by describing three proteins, the characterization of which is still going on: the first one, mucoperlin, is specific of the nacreous layer . The two others were obtained from the calcitic prismatic layer and one of them was localized by immunogold and further tested to check its ability to modify the shapes of CaCO3 crystals. The data shown here, together with data obtained from the pearl oyster or the abalone, put into question some common ideas about molluscan mineralization.