In molluscs, shell matrix proteins are associated with biomineralization, a biologically controlled process that involves nucleation and growth of calcium carbonate crystals. Identification and characterization of shell matrix proteins are important for better understanding of the adaptive radiation of a large variety of molluscs. We searched the draft genome sequence of the pearl oyster Pinctada fucata and annotated 30 different kinds of shell matrix proteins. Of these, we could identified Perlucin, ependymin-related protein and SPARC as common genes shared by bivalves and gastropods; however, most gastropod shell matrix proteins were not found in the P. fucata genome. Glycinerich proteins were conserved in the genus Pinctada. Another important finding with regard to these annotated genes was that numerous shell matrix proteins are encoded by more than one gene; e.g., three ACCBP-like proteins, three CaLPs, five chitin synthase-like proteins, two N16 proteins (pearlins), 10 N19 proteins, two nacreins, four Pifs, nine shematrins, two prismalin-14 proteins, and 21 tyrosinases. This diversity of shell matrix proteins may be implicated in the morphological diversity of mollusc shells. The annotated genes reported here can be searched in P. fucata gene models version 1.1 and genome assembly version 1.0 ( http://marinegenomics.oist.jp/pinctada_fucata ). These genes should provide a useful resource for studies of the genetic basis of biomineralization and evaluation of the role of shell matrix proteins as an evolutionary toolkit among the molluscs.

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.

Over the past decade, many skeletal matrix proteins that are possibly related to calcification have been reported in various calcifying animals. Molluscs are among the most diverse calcifying animals and some gastropods have adapted to terrestrial ecological niches. Although many shell matrix proteins (SMPs) have already been reported in molluscs, most reports have focused on marine molluscs, and the SMPs of terrestrial snails remain unclear. In addition, some terrestrial stylommatophoran snails have evolved an additional unique calcified character, called a “love dart,” used for mating behavior. We identified 54 SMPs in the terrestrial snail Euhadra quaesita, and found that they contain specific domains that are widely conserved in molluscan SMPs. However, our results also suggest that some of them possibly have evolved independently by domain shuffling, domain recruitment, or gene co-option. We then identified four dart matrix proteins, and found that two of them are the same proteins as those identified as SMPs. Our results suggest that some dart matrix proteins possibly have evolved by independent gene co-option from SMPs during dart evolution events. These results provide a new perspective on the evolution of SMPs and “love darts” in land snails.

Despite being a member of the shelled mollusks (Conchiferans), most members of extant cephalopods have lost their external biomineralized shells, except for the Nautiloids. Here, we report the result of our study to identify major Shell Matrix Proteins and their domains in the Nautiloid Nautilus pompilius, in order to gain a general insight into the evolution of Conchiferan Shell Matrix Proteins. In order to do so, we conducted transcriptomics of the mantle, and proteomics of the shell of N. pompilius simultaneously. Analyses of obtained data identified 61 distinct shell-specific sequences. Of the successfully annotated 27 sequences, protein domains were predicted in 19. Comparative analysis of Nautilus sequences with four Conchiferans for which Shell Matrix Protein data were available (the pacific oyster, the pearl oyster, the limpet, and the Euhadra snail) revealed that three proteins and six domains of the shell proteins are conserved in all Conchiferans. Interestingly, when the terrestrial Euhadra snail was excluded, another five proteins and six domains were found to be shared among the four marine Conchiferans. Phylogenetic analyses indicated that most of these proteins and domains were present in the ancestral Conchiferan, but employed in shell formation later and independently in most clades. Although further studies utilizing deeper sequencing techniques to obtain genome and full-length sequences, and functional analyses, must be done in the future, our results here provide important pieces of information for the elucidation of the evolution of Conchiferan shells at the molecular level.

The pearl oyster Pinctada fucata has great potential as a model system for lophotrochozoan developmental biology research. Pinctada fucata is an important commercial resource, and a significant body of primary research on this species has emphasized its basic aquaculture biology such as larval biology and growth, aquaculture, pearl formation and quality improvement, shell formation, and biomineralization. Recently, a draft genome sequence of this species was published, and many experimental resources are currently being developed, such as bioinformatics tools, embryo and larva manipulation methods, gene knockdown technique, etc. In this paper, we report the results from our genomic survey pertaining to gene families that encode developmental signaling ligands (Fgf, Hedgehog, PDGF/VEGF, TGFβ, and Wnt families). We found most of the representative genes of major signaling pathways involved in axial patterning, as well as copies of the signaling molecule paralogs. Phylogenetic character mapping was used to infer a possible evolutionary scenario of the signaling molecules in the protostomes, and to reconstruct possible copy numbers of signaling molecule-coding genes for the ancestral protostome. Our reconstruction suggests that P. fucata retains the ancestral protostome gene complement, providing further justifications for the use of this taxon as a model organism for developmental genomics research.

BackgroundThe calcitic brachipod shells contain proteins that play pivotal roles in shell formation and are important in understanding the evolution of biomineralization. Here, we performed a large-scale exploration of shell matrix proteins in the brachiopod Laqueus rubellus.ResultsA total of 40 proteins from the shell were identified. Apart from five proteins, i.e., ICP-1, MSP130, a cysteine protease, a superoxide dismutase, and actin, all other proteins identified had no homologues in public databases. Among these unknown proteins, one shell matrix protein was identified with a domain architecture that includes a NAD(P) binding domain, an ABC-type transport system, a transmembrane region, and an aspartic acid rich region, which has not been detected in other biominerals. We also identified pectin lyase-like, trypsin inhibitor, and saposin B functional domains in the amino acid sequences of the shell matrix proteins. The repertoire of brachiopod shell matrix proteins also contains two basic amino acid-rich proteins and proteins that have a variety of repeat sequences.ConclusionsOur study suggests an independent origin and unique mechanisms for brachiopod shell formation.Electronic supplementary materialThe online version of this article (doi:10.1186/s12953-015-0077-2) contains supplementary material, which is available to authorized users.

Despite being a member of the shelled mollusks (Conchiferans), most members of extant cephalopods have lost their external biomineralized shells, except for the basally diverging Nautilids. Here, we report the result of our study to identify major Shell Matrix Proteins and their domains in the Nautilid Nautilus pompilius, in order to gain a general insight into the evolution of Conchiferan Shell Matrix Proteins. In order to do so, we performed a multiomics study on the shell of N. pompilius, by conducting transcriptomics of its mantle tissue and proteomics of its shell matrix. Analyses of obtained data identified 61 distinct shell-specific sequences. Of the successfully annotated 27 sequences, protein domains were predicted in 19. Comparative analysis of Nautilus sequences with four Conchiferans for which Shell Matrix Protein data were available (the pacific oyster, the pearl oyster, the limpet and the Euhadra snail) revealed that three proteins and six protein domains were conserved in all Conchiferans. Interestingly, when the terrestrial Euhadra snail was excluded, another five proteins and six protein domains were found to be shared among the four marine Conchiferans. Phylogenetic analyses indicated that most of these proteins and domains were probably present in the ancestral Conchiferan, but employed in shell formation later and independently in most clades. Even though further studies utilizing deeper sequencing techniques to obtain genome and full-length sequences, and functional analyses, must be carried out in the future, our results here provide important pieces of information for the elucidation of the evolution of Conchiferan shells at the molecular level.

Molluscan shell matrix proteins (SMPs) are essential in biomineralization. Here, we identify potentially important SMPs by exploiting the asymmetric shell growth in snail, Lymnaea stagnalis. Asymmetric shells require bilaterally asymmetric expression of SMP genes. We examined expression levels of 35,951 transcripts expressed in the left and right sides of mantle tissue of the pond snail, Lymnaea stagnalis. This transcriptome dataset was used to identify 207 SMPs by LC-MS/MS. 32 of the 207 SMP genes show asymmetric expression patterns, which were further verified for 4 of the 32 SMPs using quantitative PCR analysis. Among asymmetrically expressed SMPs in dextral snails, those that are more highly expressed on the left side than the right side are 3 times more abundant than those that are more highly expressed on the right than the left, suggesting potentially inhibitory roles of SMPs in shell formation. The 32 SMPs thus identified have distinctive features, such as conserved domains and low complexity regions, which may be essential in biomineralization.