Dual Gene Repertoires for Larval and Adult Shells Reveal Molecules Essential for Molluscan Shell Formation.

Molluscan shells, mainly composed of calcium carbonate, also contain organic components such as proteins and polysaccharides. Shell organic matrices construct frameworks of shell structures and regulate crystallization processes during shell formation. To date, a number of shell matrix proteins (SMPs) have been identified, and their functions in shell formation have been studied. However, previous studies focused only on SMPs extracted from adult shells, secreted after metamorphosis. Using proteomic analyses combined with genomic and transcriptomic analyses, we have identified 31 SMPs from larval shells of the pearl oyster, Pinctada fucata, and 111 from the Pacific oyster, Crassostrea gigas. Larval SMPs are almost entirely different from those of adults in both species. RNA-seq data also confirm that gene expression profiles for larval and adult shell formation are nearly completely different. Therefore, bivalves have two repertoires of SMP genes to construct larval and adult shells. Despite considerable differences in larval and adult SMPs, some functional domains are shared by both SMP repertoires. Conserved domains include von Willebrand factor type A (VWA), chitin-binding (CB), carbonic anhydrase (CA), and acidic domains. These conserved domains are thought to play crucial roles in shell formation. Furthermore, a comprehensive survey of animal genomes revealed that the CA and VWA-CB domain-containing protein families expanded in molluscs after their separation from other Lophotrochozoan linages such as the Brachiopoda. After gene expansion, some family members were co-opted for molluscan SMPs that may have triggered to develop mineralized shells from ancestral, nonmineralized chitinous exoskeletons.

Functional shell matrix proteins tentatively identified by asymmetric snail shell morphology.

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.

Phylogenetic comparisons reveal mosaic histories of larval and adult shell matrix protein deployment in pteriomorph bivalves.

Molluscan shells are organo-mineral composites, in which the dominant calcium carbonate is intimately associated with an organic matrix comprised mainly of proteins and polysaccharides. However, whether the various shell matrix proteins (SMPs) date to the origin of hard skeletons in the Cambrian, or whether they represent later deployment through adaptive evolution, is still debated. In order to address this issue and to better understand the origins and evolution of biomineralization, phylogenetic analyses have been performed on the three SMP families, Von Willebrand factor type A (VWA) and chitin-binding domain-containing protein (VWA-CB dcp), chitobiase, and carbonic anhydrase (CA), which exist in both larval and adult shell proteomes in the bivalves, Crassostrea gigas and Pinctada fucata. In VWA-CB dcp and chitobiase, paralogs for larval and adult SMPs evolved before the divergence of these species. CA-SMPs have been taken as evidence for ancient origins of SMPs by their presumed indispensable function in biomineralization and ubiquitous distribution in molluscs. However, our results indicate gene duplications that gave rise to separate deployments as larval and adult CA-SMPs occurred independently in each lineage after their divergence, which is considerably more recent than hitherto assumed, supporting the "recent heritage and fast evolution" scenario for SMP evolution.