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

Abstract

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.

Fig. 1.

Repertoires of larval and adult SMPs differ, but they share some functional domains. Numbers of shared and unshared shell matrix proteins among larval, adult nacreous, and adult prismatic shells in P. fucata (A), and between larval and adult shells in C. gigas (B). Comparisons of functional domains found in larval and adult SMPs of P. fucata (C) and C. gigas (D). Functional domains in larval (E) and adult (F) SMPs of the two species were also compared. Domains shared by both larvae and adults of two species are colored in red.

Fig. 2.

Stage-specific expression of larval and adult SMP genes in P. fucata. The heatmap shows expression levels of identified SMP genes at different developmental stages and different regions of mantle tissues. Colored bars on the right indicate the presence of the SMPs in larval, adult nacre, and adult prism shells. The gene expression pattern is congruent with localization of corresponding SMPs in the shells.

Fig. 3.

VWA and chitin-binding domain-containing protein (VWA–CB dcp) is essential for molluscan shell biomineralization. (A) Numbers of VWA–CB dcp genes in protostomian animal genomes. The phylogeny of Spiralia, with Ecdysozoa as an out group, is based on Luo et al. (2018). VWA–CB dcps are found only in Mollusca (purple) and Lophotrochozoa (yellow). (B) Conserved domain architecture of VWA–CB domain-containing SMPs in P. fucata, C. gigas, and L. gigantea. One VWA–CB dcp found in the brachiopod (L. anatina) genome is shown for comparison. Red and cyan circles on the left indicate larval and adult SMPs, respectively. BMSPs are found both in P. fucata and C. gigas larval proteomes. See supplementary table S3, Supplementary Material online for species name abbreviations.

Fig. 4.

Molecular phylogeny of protostomian alpha carbonic anhydrase (α-CA) shows lineage-specific expansion of the gene family and its recruitment for shell formation in molluscs. Red and cyan circles indicate larval and adult SMPs, respectively. Colored branches indicate that the cluster is exclusively dominated by α-CA genes of specific taxonomic clades (yellow: Bivalvia, green: Gastropoda). We analyzed protein sequences containing CA domains and having more than 100 amino acids. The tree was constructed using the ML method with the LG model.