The ‘Shellome’ of the Crocus Clam Tridacna crocea Emphasizes Essential Components of Mollusk Shell Biomineralization

Molluscan shells are among the most fascinating research objects because of their diverse morphologies and textures. The formation of these delicate biomineralized structures is a matrix-mediated process. A question that arises is what are the essential components required to build these exoskeletons. In order to understand the molecular mechanisms of molluscan shell formation, it is crucial to identify organic macromolecules in different shells from diverse taxa. In the case of bivalves, however, taxon sampling in previous shell proteomics studies are focused predominantly on representatives of the class Pteriomorphia such as pearl oysters, edible oysters and mussels. In this study, we have characterized the shell organic matrix from the crocus clam, Tridacna crocea, (Heterodonta) using various biochemical techniques, including SDS-PAGE, FT-IR, monosaccharide analysis, and enzyme-linked lectin assay (ELLA). Furthermore, we have identified a number of shell matrix proteins (SMPs) using a comprehensive proteomics approach combined to RNA-seq. The biochemical studies confirmed the presence of proteins, polysaccharides, and sulfates in the T. crocea shell organic matrix. Proteomics analysis revealed that the majority of the T. crocea SMPs are novel and dissimilar to known SMPs identified from the other bivalve species. Meanwhile, the SMP repertoire of the crocus clam also includes proteins with conserved functional domains such as chitin-binding domain, VWA domain, and protease inhibitor domain. We also identified BMSP (Blue Mussel Shell Protein, originally reported from Mytilus), which is widely distributed among molluscan shell matrix proteins. Tridacna SMPs also include low-complexity regions (LCRs) that are absent in the other molluscan genomes, indicating that these genes may have evolved in specific lineage. These results highlight the diversity of the organic molecules – in particular proteins – that are essential for molluscan shell formation.

Stable isotopes in bivalves as indicators of nutrient source in coastal waters in the Bocas del Toro Archipelago, Panama

To examine N-isotope ratios (15N/14N) in tissues and shell organic matrix of bivalves as a proxy for natural and anthropogenic nutrient fluxes in coastal environments,Pinctada imbricata,Isognomon alatus, andBrachidontes exustusbivalves were live-collected and analyzed from eight sites in Bocas del Toro, Panama. Sites represent a variety of coastal environments, including more urbanized, uninhabited, riverine, and oceanic sites. Growth under differing environmental conditions is confirmed byδ18O values, with open ocean Escudo de Veraguas shells yielding the highest averageδ18O (−1.0‰) value and freshwater endmember Rio Guarumo the lowest (−1.7‰). At all sites there is no single dominant source of organic matter contributing to bivalveδ15N andδ13C values. Bivalveδ15N andδ13C values likely represent a mixture of mangrove and seagrass N and C, although terrestrial sources cannot be ruled out. Despite hydrographic differences between end-members, we see minimalδ15N andδ13C difference between bivalves from the river-influenced Rio Guarumo site and those from the oceanic Escudo de Veraguas site, with no evidence for N from open-ocean phytoplankton in the latter. Populated sites yield relative15N enrichments suggestive of anthropogenic nutrient input, but lowδ15N values overall make this interpretation equivocal. Lastly,δ15N values of tissue and shell organic matrix correlate significantly for pterioideansP. imbricataandI. alatus. Thus for these species, N isotope studies of historical and fossil shells should provide records of ecology of past environments.