The shells of pearl oysters, Pinctada fucata, are composed of calcite and aragonite and possess remarkable mechanical properties. These shells are formed under the regulation of macromolecules, especially shell matrix proteins (SMPs). Identification of diverse SMPs will lay a foundation for understanding biomineralization process. Here, we identified 72 unique SMPs using liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of proteins extracted from the shells of P. fucata combined with a draft genome. Of 72 SMPs, 17 SMPs are related to both the prismatic and nacreous layers. Moreover, according to the diverse domains found in the SMPs, we hypothesize that in addition to controlling CaCO3 crystallization and crystal organization, these proteins may potentially regulate the extracellular microenvironment and communicate between cells and the extracellular matrix (ECM). Immunohistological localization techniques identify the SMPs in the mantle, shells and synthetic calcite. Together, these proteomic data increase the repertoires of the shell matrix proteins in P. fucata and suggest that shell formation in P. fucata may involve tight regulation of cellular activities and the extracellular microenvironment.

Interactive effects of ocean acidification and ocean warming on marine calcifiers vary among species, but little is known about the underlying mechanisms. The present study investigated the combined effects of seawater acidification and elevated temperature (ambient condition: pH 8.1 × 23 °C, stress conditions: pH 7.8 × 23 °C, pH 8.1 × 28 °C, and pH 7.8 × 28 °C, exposure time: two months) on the transcriptome and biomineralization of the pearl oyster Pinctada fucata, which is an important marine calcifier. Transcriptome analyses indicated that P. fucata implemented a compensatory acid-base mechanism, metabolic depression and positive physiological responses to mitigate the effects of seawater acidification alone. These responses were energy-expensive processes, leading to decreases in the net calcification rate, shell surface calcium and carbon content, and changes in the shell ultrastructure. Elevated temperature (28 °C) within the thermal window of P. fucata did not induce significant enrichment of the sequenced genes and conversely facilitated calcification, which was detected to alleviate the negative effects of seawater acidification on biomineralization and the shell ultrastructure. Overall, this study will help elucidate the mechanisms by which pearl oysters respond to changing seawater conditions and predict the effects of global climate change on pearl aquaculture.

Ocean acidification and global warming have been shown to significantly affect the physiological performances of marine calcifiers; however, the underlying mechanisms remain poorly understood. In this study, the transcriptome and biomineralization responses of Pinctada fucata to elevated CO2 (pH 7.8 and pH 7.5) and temperature (25 °C and 31 °C) are investigated. Increases in CO2 and temperature induced significant changes in gene expression, alkaline phosphatase activity, net calcification rates and relative calcium content, whereas no changes are observed in the shell ultrastructure. “Ion and acid-base regulation” related genes and “amino acid metabolism” pathway respond to the elevated CO2 (pH 7.8), suggesting that P. fucata implements a compensatory acid-base mechanism to mitigate the effects of low pH. Additionally, “anti-oxidation”-related genes and “Toll-like receptor signaling”, “arachidonic acid metabolism”, “lysosome” and “other glycan degradation” pathways exhibited responses to elevated temperature (25 °C and 31 °C), suggesting that P. fucata utilizes anti-oxidative and lysosome strategies to alleviate the effects of temperature stress. These responses are energy-consuming processes, which can lead to a decrease in biomineralization capacity. This study therefore is important for understanding the mechanisms by which pearl oysters respond to changing environments and predicting the effects of global climate change on pearl aquaculture.

In bivalves, the mantle tissue secretes organic matrix and inorganic ions into the extrapallial space (EPS) to form the shells. In addition, more and more evidences indicate the participation of hemocytes in shell mineralization, but no direct evidence has been reported that verifies the presence of hemocytes in the EPS, and their exact roles in biomineralization remain uncertain. Here, we identified hemocytes from the EPS of Pinctada fucata. Numerous components involved in cellular and humoral immunity were identified by proteome analysis, together with several proteins involved in calcium metabolism. The hemocytes exerted active phagocytosis and significantly upregulated the expression of immune genes after immune stimulation. A group of granulocytes were found to contain numerous calcium-rich vesicles and crystals, which serve as a calcium pool. During shell regeneration, some genes involved in calcium metabolism are upregulated. Strikingly, most of the shell matrix proteins were absent in the hemocytes, suggesting that they might not be solely responsible for directing the growth of the shell. Taken together, our results provided comprehensive information about the function of hemocytes in immunity and shell formation.

Seawater acidification and warming resulting from anthropogenic production of carbon dioxide are increasing threats to marine ecosystems. Previous studies have documented the effects of either seawater acidification or warming on marine calcifiers; however, the combined effects of these stressors are poorly understood. In our study, we examined the interactive effects of elevated carbon dioxide partial pressure (P CO2 ) and temperature on biomineralization and amino acid content in an ecologically and economically important mussel, Mytilus edulis. Adult M. edulis were reared at different combinations of P CO2 ( pH 8.1 and 7.8) and temperature (19, 22 and 25°C) for 2 months. The results indicated that elevated P CO2 significantly decreased the net calcification rate, the calcium content and the Ca/Mg ratio of the shells, induced the differential expression of biomineralization-related genes, modified shell ultrastructure and altered amino acid content, implying significant effects of seawater acidification on biomineralization and amino acid metabolism. Notably, elevated temperature enhanced the effects of seawater acidification on these parameters. The shell breaking force significantly decreased under elevated P CO2 , but the effect was not exacerbated by elevated temperature. The results suggest that the interactive effects of seawater acidification and elevated temperature on mussels are likely to have ecological and functional implications. This study is therefore helpful for better understanding the underlying effects of changing marine environments on mussels and other marine calcifiers.

Biominerals are highly ordered crystals mediated by organic matters especially proteins in organisms. However, how specific proteins are distributed inside biominerals are not well understood. In the present study, we use fluorescein isothiocyanate (FITC) to label extracted proteins from the shells of bivalve Pinctada fucata. By confocal laser scanning microscopy (CLSM), we observe a heterogeneous distribution of dye-labelled proteins inside synthetic calcite at the microscale. Proteins from the prismatic calcite layers accumulate at the edge of crystals while proteins from the nacreous aragonite layers accumulate at the center of crystals. Raman and X-ray powder diffraction show that both the proteins cannot alter the crystal phase. Scanning electron microscope demonstrates both proteins are able to affect the crystal morphology. This study may provide a direct approach for the visualization of protein distributions in crystals by small-molecule dye-labelled proteins as the additives in the crystallization process and improve our understanding of intracrystalline proteins distribution in biogenic calcites.

Biomineralization, including shell formation, is dedicatedly regulated by matrix proteins. PfY2, a matrix protein detected in the ethylene diamine tetraacetic acid (EDTA)-soluble fraction from both prismatic layer and nacreous layer, was discovered by our group using microarray. It may play dual roles during biomineralization. However, the molecular mechanism is still unclear. In this research, we studied the function of PfY2 on crystallization in vivo and in vitro, revealing that it might be a negative regulator during shell formation. Notching experiment indicated that PfY2 was involved in shell repairing and regenerating process. Repression of PfY2 gene affected the structure of prismatic and nacreous layer simultaneously, confirming its dual roles in shell formation. Recombinant protein rPfY2 significantly suppressed CaCO3 precipitation rate, participated in the crystal nucleation process, changed the morphology of crystals and inhibited the transformation of amorphous calcium carbonate (ACC) to stable calcite or aragonite in vitro. Our results may provide new evidence on the biomineralization inhibition process.

Limpets are marine mollusks that use mineralized teeth, one of the hardest and strongest biomaterials, to feed on algae on intertidal rocks. However, most of studies only focus on the ultrastructure and chemical composition of the teeth while the molecular information is largely unknown, limiting our understanding of this unique and fundamental biomineralization process. The study investigates the microstructure, proteomics, and crystallization in the teeth of limpet Cellana toreuma. It is found that the limpets formed alternatively tricuspid teeth and unicuspid teeth. Small nanoneedles are densely packed at the tips or leading regions of the cusps. In contrast, big nanoneedles resembling chemically synthesized goethite are loosely packed in the trailing regions of the cusps. Proteins extracted from the whole radula, such as ferritin, peroxiredoxin, arginine kinase, GTPase-Rabs, and clathrin, are identified by proteomics. A goethite-binding experiment coupled with proteomics and RNA-seq highlights six chitin-binding proteins (CtCBPs). Furthermore, the extracted proteins from the cusps of radula or the framework chitin induce packing of crystals and possibly affect crystal polymorphs in vitro. This study provides insight into the unique biomineralization process in the limpet teeth at the molecular levels, which may guide biomimetic strategies aimed at designing hard materials at room temperature.