Similar construction of spicules and shell plates: Implications for the origin of chiton biomineralization.

The hard shells of mollusks are products of biomineralization, a distinctive feature of the Cambrian explosion. Despite our understanding of shell structure and mechanical properties, their origin remains mysterious. In addition to their shell plates, most chitons have calcium deposits on their girdles. However, the similarity of these two mineralized structures still needs to be determined, limiting our comprehension of their origins. In our study, we analyzed the matrix proteins in the spicules of chiton (Acanthopleura loochooana) and compared them with the matrix proteins in the shells of the same species. Proteomics identified 96 unique matrix proteins in spicules. Comparison of biomineralization-related matrix proteins in shell plates and spicules revealed shared proteins, including carbonic anhydrases, tyrosinase-hemocyanin, von Willebrand factor type A, cadherin, and glycine-rich unknown proteins. Based on similarities in key matrix proteins, we propose that spicules and shell plates originated from a common mineralization system in their ancestral lineage, suggesting the existence of a common core or toolkit of matrix proteins among calcifying organisms. SIGNIFICANCE: In this study, we try to understand the types and diversity of matrix proteins in the biomineralization of chiton shell plates and spicules. Through a comparative analysis, we seek insights into the core biomineralization toolkit of ancestral mollusks. To achieve this, we conducted LC-MS/MS and RT-qPCR analyses to identify the types and relative expression levels of matrix proteins in both shell plates and spicules. The analysis revealed 96 matrix proteins in the spicules. A comparison of biomineralization-related matrix proteins in shell plates and spicules from the same species revealed shared proteins including many unknown proteins unique to chitons. Blast searching reveals a universal conservation of these proteins among other chitons. Hence, we propose that spicules and shell plates originated from a common mineralization system in their ancestral lineage. Our work provides a molecular basis for studying biomineralization in polyplacophoran mollusks and understanding biomineralization evolution. In addition, it identifies potential matrix proteins that could be applied to control crystal growth.

Oxidation and cross-link of tyrosine-rich proteins are involved in the periostracum formation of the green mussel Perna viridis (Linnaeus).

Ocean acidification causes severe shell dissolution and threats the survival of marine molluscs. The periostracum in molluscs consists of macromolecules such as proteins and polysaccharides, and protects the inner shell layers from dissolution and microbial erosion. Moreover, it serves as the primary template for shell deposition. However, the chemical composition and formation mechanism of the periostracum is largely unknown. In this study, we applied transcriptomic, proteomics, physical, and chemical analysis to unravel the mysteries of the periostracum formation in the green mussel Perna viridis Linnaeus. FTIR analysis showed that the periostracum layer was an organic membrane mainly composed of polysaccharides, lipids, and proteins, similar to that of the shell matrix. Interestingly, the proteomic study identified components enriched in tyrosine and some enzymes that evolved in tyrosine oxidation, indicating that tyrosine oxidation might play an essential role in the periostracum formation. Moreover, comparative transcriptomics suggested that tyrosine-rich proteins were intensively synthesized in the periostracum groove. After being secreted, the periostracum proteins were gradually tanned by oxidation in the seawater, and the level of crosslink increased significantly as revealed by the ATR-FTIR. Our present study sheds light on the chemical composition and putative tanning mechanism of the periostracum layer in bivalve molluscs. SIGNIFICANCE: The periostracum layer, plays an essential role in the initiation of shell biomineralization, the protection of minerals from dissolution for molluscs and especially ocean acidification conditions in the changing global climate. However, the molecular mechanism underlying the periostracum formation is not fully understood. In this study, we revealed that the oxidation and cross-link of tyrosine-rich proteins by tyrosinase are involved in periostracum formation in the green mussel Perna viridis. This study provides some insights into the first step of mussel shell formation and the robust adaptation of P. viridis to diverse habitats. These findings also help to reveal the potential acclimation of bivalves to the projected acidifying seawater.

Proteomic analysis of shell matrix proteins from the chiton Acanthopleura loochooana.

Most molluscs have mineralized shells to protect themselves. Although the remarkable mechanical properties of shells have been well-studied, the origin of shells is still elusive. Chitons are unique in molluscs because they are shelly Aculifera which diverged from Conchifera (comprising all the shell-bearing classes of molluscs) in the early pre-Cambrian. We developed a method to extract shell proteins from chiton shell plates (removing embedded soft tissues) and then compared the shell proteome to that of Conchifera groups. Sixteen shell matrix proteins from Acanthopleura loochooana were identified by proteomics, in which Nacrein-like, Pif-like proteins, and protocadherin were found. Additional evidences from shell proteome in another species Chiton densiliratus and comparative sequence alignment in five chitons supported a conserved biomineralization toolkit in chitons. Our findings shed light on the evolution of mineralization in chitons and pose a hypothesis that ancestral molluscs have already evolved biomineralization toolkits, which may facilitate the formation of mineralized shells.

Characterization of the shell proteins in two freshwater snails Pomacea canaliculata and Cipangopaludina chinensis.

Uncovering the molecular mechanism of shell formation not only reveals the evolution of molluscs but also lay a foundation for shell-inspired biomaterial synthesis. Shell proteins are the key macromolecules of the organic matrices that guide the calcium carbonate deposition during shell mineralization and have thus been intensively studied. However, previous studies on shell biomineralization have mainly focused on marine species. In this study, we compared the microstructure and shell proteins in the apple snail Pomacea canaliculata which is an alien species that has invaded Asia, and a freshwater snail Cipangopaludina chinensis which is native to China. The results showed that although the shell microstructures were similar in these two snails, the shell matrix in C. chinensis contained more polysaccharides. Moreover, the compositions of shell proteins were quite different. While the shared 12 shell proteins (including PcSP6/CcSP9, Calmodulin-A, and proline-rich protein) were supposed to play key roles in shell formation, the differential proteins were mainly immune components. The presence of chitin in both shell matrices and the chitin-binding domains containing PcSP6/CcSP9 underpinned the relevance of chitin as a major fraction in gastropods. Interestingly, carbonic anhydrase was absent in both snail shells, suggesting that freshwater gastropods might have unique pathways to regulate the calcification process. Our study suggested that shell mineralization might be very different in freshwater and marine molluscs, and therefore, the field should pay more attention to the freshwater species to achieve a more comprehensive insight into biomineralization.

Direct control of shell regeneration by the mantle tissue in the pearl oyster Pinctada fucata.

Molluscs rapidly repair the damaged shells to prevent further injury, which is vital for their survival after physical or biological aggression. However, it remains unclear how this process is precisely controlled. In this study, we applied scanning electronic microscope and histochemical analysis to examine the detailed shell regeneration process in the pearl oyster Pinctada fucata. It was found that the shell damage caused the mantle tissue to retract, which resulted in relocation of the partitioned mantle zones with respect to their correspondingly secreting shell layers. As a result, the relocated mantle tissue dramatically altered the shell morphology by initiating de novo precipitation of prismatic layers on the former nacreous layers, leading to the formation of sandwich-like "prism-nacre-prism-nacre" structure. Real-time PCR revealed the up-regulation of the shell matrix protein genes, which was confirmed by the thermal gravimetric analysis of the newly formed shell. The increased matrix secretion might have led to the change of CaCO3 precipitation dynamics which altered the mineral morphology and promoted shell formation. Taken together, our study revealed the close relationship between the physiological activities of the mantle tissue and the morphological change of the regenerated shells.

Rngtt governs biliary-derived liver regeneration initiation by transcriptional regulation of mTORC1 and Dnmt1 in zebrafish.

In cases of end-stage liver diseases, the proliferation of existing hepatocytes is compromised, a feature of human chronic liver disease, in which most hepatocytes are dysfunctional. So far, liver transplantation represents the only curative therapeutic solution for advanced liver diseases, and the shortage of donor organs leads to high morbidity and mortality worldwide. The promising treatment is to prompt the biliary epithelial cells (BECs) transdifferentiation. However, the critical factors governing the initiation of BEC-derived liver regeneration are largely unknown. The zebrafish has advantages in large-scale genetic screens to identify the critical factors involved in liver regeneration. Here, we combined N-ethyl-N-nitrosourea screen, positional cloning, transgenic lines, antibody staining, and in situ hybridization methods and identified a liver regeneration defect mutant ( lrd ) using the zebrafish extensive liver injury model. Through positional cloning and genomic sequencing, we mapped the mutation site to rngtt . Loss of rngtt leads to the defects of BEC dedifferentiation, bipotential progenitor cell activation, and cell proliferation in the initiation stage of liver regeneration. The transdifferentiation from BECs to hepatocytes did not occur even at the late stage of liver regeneration. Mechanically, Rngtt transcriptionally regulates the attachment of mRNA cap to mTOR complex 1 (mTORC1) components and dnmt1 to maintain the activation of mTORC1 and DNA methylation in BECs after severe liver injury and prompt BEC to hepatocyte conversion. Furthermore, rptor and dnmt1 mutants displayed the same liver regeneration defects as rngtt mutation. In conclusion, our results suggest Rngtt is a new factor that initiates BEC-derived liver regeneration.

Proteomics of Shell Matrix Proteins from the Cuttlefish Bone Reveals Unique Evolution for Cephalopod Biomineralization.

In contrast to the external shells in bivalves and gastropods, most cephalopods are missing this external protection. The cuttlefish, belonging to class cephalopod, has an internal biomineralized structure made of mainly calcium carbonate for controlling buoyancy. However, the macromolecules, especially proteins that control cuttlebone mineral formation, are not sufficiently understood, limiting our understanding of the evolution of this internal shell. In this study, we extracted proteins from the cuttlebone of pharaoh cuttlefish Sepia pharaonis and performed liquid chromatography-tandem mass spectrometry to identify the shell matrix proteins (SMPs). In total, 41 SMPs were identified. Among them, hemocyanin, an oxygen-carrying protein, was the most abundant SMP. By comparison with SMPs of other marine biominerals, hemocyanin, apolipophorin, soul domain proteins, transferrin, FL-rich, and enolase were found to be unique to the cuttlebone. In contrast, typical SMPs of external shells such as carbonic anhydrase complement control protein, fibronectin type III, and G/A-rich proteins were lacking from the cuttlebone. Furthermore, the cluster analysis of biomineral SMPs suggests that the SMP repertoire of the cuttlebone does not resemble that of other species with external shells. Taken together, this study implies a potential relationship of the cuttlefish internal shell with other internal biominerals, which highlights a unique shell evolutionary pathway in invertebrates.

Anti-Fouling Performance of Hydrophobic Hydrogels with Unique Surface Hydrophobicity and Nanoarchitectonics.

Hydrogel is a kind of soft and wet matter, which demonstrates favorable fouling resistance owing to the hydration anti-adhesive surfaces. Different from conventional hydrogels constructed by hydrophilic or amphiphilic polymers, the recently invented "hydrophobic hydrogels" composed of hydrophobic polymers exhibit many unique properties, e.g., surface hydrophobicity and high water content, suggesting promising applications in anti-fouling. In this paper, a series of hydrophobic hydrogels were prepared with different chemical structures and water content for anti-fouling investigations. The hydrophobic hydrogels showed high static water contact angles (WCAs > 90°), indicating remarkable surface hydrophobicity, which is abnormal for conventional hydrogels. Compared with the conventional hydrogels, all the hydrophobic hydrogels exhibited less than 4% E. coli biofilm coverage, showing a contrary trend of anti-fouling ability to the water content inside the polymer. Typically, the poly(2-(2-ethoxyethoxy)ethyl acrylate) (PCBA) and poly(tetrahydrofurfuryl acrylate) (PTHFA) hydrogels with relatively high surface hydrophobicity showed as low as 5.1% and 2.4% E. coli biofilm coverage even after incubation for 7 days in bacteria suspension, which are about 0.32 and 0.15 times of that on the hydrophilic poly(N,N-dimethylacrylamide) (PDMA) hydrogels, respectively. Moreover, the hydrophobic hydrogels exhibited a similar anti-adhesion ability and trend to algae S. platensis. Based on the results, the surface hydrophobicity mainly contributes to the excellent anti-fouling ability of hydrophobic hydrogels. In the meantime, the too-high water content may be somehow detrimental to anti-fouling performance.

Optimized Sensory Units Integrated in the Chiton Shell.

The first step for animals to interact with external environment is to sense. Unlike vertebrate animals with flexibility, it is challenging for ancient animals that are less flexible especially for mollusca with heavy shells. Chiton, as an example, has eight overlapping shells covering almost the whole body, is known to incorporate sensory units called aesthetes inside the shell. We used micro-computed tomography combined with quantitative image analysis to reveal the optimized shell geometry to resist force and the aesthetes' global distribution at the whole animal levels to facilitate sense from diverse directions both in the seawater and air. Additionally, shell proteomics combined with transcriptome reveals shell matrix proteins responsible for shell construction and potentially sensory function, highlighting unique cadherin-related proteins among mollusca. Together, this multi-level evidence of sensory units in the chiton shell may shed light on the formation of chiton shells and inspire the design of hard armor with sensory function.

Mantle tissue in the pearl oyster Pinctada fucata secretes immune components via vesicle transportation.

Molluscan bivalves secrete shell matrices into the extrapallial space (EPS) to guide the precipitation of rigid shells. Meanwhile, immune components are present in the EPS and shell matrices, which are pivotal in resistant to invaded pathogens, thus ensuring the shell formation process. However, the origin of these components remains unclear. In this study, we revealed numerous vesicles were secreted from the outer mantle epithelial cells by using light and electron microscopes. The secreted vesicles were isolated by gradient centrifugation and confirmed by transmission electron microscopy. Proteomics analysis showed that the secreted vesicles were composed of cytoplasmic and immune components, most of which do not have signal peptides, indicating that they were secreted by a non-classical pathway. Moreover, real-time PCR revealed that some immune components were highly expressed in the mantle tissue, compared to the hemocytes. FTIR analysis verified the presence of lipids in the shell matrices, indicating that the vesicles have integrated into the shell layers. Taken together, our results suggested that mantle epithelial cells secreted some important immune components into the EPS via secreted vesicle transportation, thus cooperating with the hemocytes to play a vital role in immunity during shell formation.

Heterogeneous distribution of shell matrix proteins in the pearl oyster prismatic layer.

Shell formation in molluscan bivalves is regulated by organic matrices composed of biological macromolecules, but how these macromolecules assemble in vitro remains elusive. Prismatic layer in the pearl oyster Pinctada fucata consists of polygonal prisms enveloped by thick organic matrices. In this study, we found that the organic matrices were heterogeneously distributed, with highly acidic fractions (EDTA-soluble and EDTA-insoluble) embedded inside the prism columns, while basic EDTA-insoluble faction as inter-column framework enveloping the prisms. The intra-column matrix was enriched in aspartic acid whereas the inter-column matrix was enriched in glycine, tyrosine and phenylalanine. Moreover, the intra-column matrix contained sulfo group further contributing to its acidic property. Proteomics data showed that the intra-column proteins mainly consisted of acidic proteins, while some typical matrix proteins were absent. The absent matrix proteins such as shematrin family and KRMP family were highly basic and contained aromatic amino acids, suggesting that electric charge and hydrophobic effect might play a role in the matrix heterogeneity. Interestingly, chitin metabolism related proteins were abundant in the inter-column matrix, which may be involved in reconstructing the prism organic matrix. Overall, our study suggests that each single prism grew in an enclosed organic envelope and the organic matrix undergoes rearrangement, thus leading to the peculiar growth of the prismatic layer.

Transition from horizontal expansion to vertical growth in the oyster prismatic layer.

Biomimetic materials inspired by biominerals have substantial applications in various fields. The prismatic layer of bivalve molluscs has extraordinary flexibility compared to inorganic CaCO3. Previous studies showed that in the early stage, minerals expanded horizontally and formed prism domains as a Voronoi division, while the evolution of the mature prisms were thermodynamically driven, which was similar to grain growth. However, it was unclear how the two processes were correlated during shell formation. In this study, we used scanning electronic microscopy and laser confocal scanning microscopy to look into the microstructure of the columnar prismatic layer in the pearl oyster Pinctada fucata. The Dirichlet centers of the growing domains in mature prisms were calculated, and the corresponding Voronoi division was reconstructed. It was found that the domain pattern did not fit the Voronoi division, indicating the driving forces of the mature prisms evolution and the initiation stage were different. During the transition from horizontal expansion to vertical growth, the minerals broke through the inner periostracum and squeezed out the organic materials to the inter-prism space. Re-arrangement of the organic framework pattern was driven by elastic relaxation at the vertices, indicating the transition process was thermodynamically driven. Our study provided insights into shell growth in bivalves and pave the way to synthesize three-dimensional material biomimetically.

Generation of Chicken IgY against SARS-COV-2 Spike Protein and Epitope Mapping.

This new decade has started with a global pandemic of COVID-19 caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), precipitating a worldwide health crisis and economic downturn. Scientists and clinicians have been racing against time to find therapies for COVID-19. Repurposing approved drugs, developing vaccines and employing passive immunization are three major therapeutic approaches to fighting COVID-19. Chicken immunoglobulin Y (IgY) has the potential to be used as neutralizing antibody against respiratory infections, and its advantages include high avidity, low risk of adverse immune responses, and easy local delivery by intranasal administration. In this study, we raised antibody against the spike (S) protein of SARS-CoV-2 in chickens and extracted IgY (called IgY-S) from egg yolk. IgY-S exhibited high immunoreactivity against SARS-CoV-2 S, and by epitope mapping, we found five linear epitopes of IgY-S in SARS-CoV-2 S, two of which are cross-reactive with SARS-CoV S. Notably, epitope SIIAYTMSL, one of the identified epitopes, partially overlaps the S1/S2 cleavage region in SARS-CoV-2 S and is located on the surface of S trimer in 3D structure, close to the S1/S2 cleavage site. Thus, antibody binding at this location could physically block the access of proteolytic enzymes to S1/S2 cleavage site and thereby impede S1/S2 proteolytic cleavage, which is crucial to subsequent virus-cell membrane fusion and viral cell entry. Therefore, the feasibility of using IgY-S or epitope SIIAYTMS-specific IgY as neutralizing antibody for preventing or treating SARS-CoV-2 infection is worth exploring.

Shell repair and the potential microbial causal in a shell disease of the pearl oyster Pinctada fucata.

The pearl oyster Pinctada fucata is famous for producing luxurious pearls. As filter feeders, they are confronted with various infectious microorganisms. Despite a long history of aquaculture, diseases in P. fucata are not well studied, which limits the development of the pearl industry. We report here a shell disease in P. fucata and a study of the shell repair processes. Scanning electron microscopy (SEM) revealed that the nacreous layer gradually recovered from disordered CaCO3 deposition, accompanied by a polymorphic transition from a calcite-aragonite mixture to an aragonite-dominant composition, as revealed by X-ray diffraction analysis. SEM also showed that numerous microbes were embedded in the abnormal shell layers. Similar indications were induced by a high concentration of microbes injected into the extrapallial space, suggesting the potential pathogenic effect of uncontrolled microbes. Furthermore, hemocytes were found to participate in pathogens resistance and might promote shell repair. These results further our understanding of pathogen-host interactions in pearl oysters and have implications for biotic control in pearl aquaculture.

Cloning, characterization and functional analysis of an Alveoline-like protein in the shell of Pinctada fucata.

Shell matrix proteins (SMPs) have important functions in biomineralization. In the past decades, the roles of SMPs were gradually revealed. In 2015, our group identified 72 unique SMPs in Pinctada fucata, among which Alveoline-like (Alv) protein was reported to have homologous genes in Pinctada maxima and Pinctada margaritifera. In this study, the full-length cDNA sequence of Alv and the functional analysis of Alv protein during shell formation were explored. The deduced protein (Alv), which has a molecular mass of 24.9 kDa and an isoelectric point of 11.34, was characterized, and the functional analyses was explored in vivo and in vitro. The Alv gene has high expression in mantle and could response to notching damage. The functional inhibition of Alv protein in vivo by injecting recombinant Alv (rAlv) antibodies destroyed prism structure but accelerated nacre growth. Western blot and immunofluorescence staining showed that native Alv exists in the EDTA-insoluble matrix of both prismatic and nacreous layers and has different distribution patterns in the inner or outer prismatic layer. Taken together, the characterization and functional analyses of matrix protein Alv could expand our understanding of basic matrix proteins and their functions during shell formation.

The Microstructure, Proteomics and Crystallization of the Limpet Teeth.

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.

Hemocytes in the extrapallial space of Pinctada fucata are involved in immunity and biomineralization.

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.

Amorphous calcium carbonate: A precursor phase for aragonite in shell disease of the pearl oyster.

Amorphous calcium carbonate (ACC) has long been shown to act as an important constituent or precursor phase for crystalline material in mollusks. However, the presence and the role of ACC in bivalve shell formation are not fully studied. In this study, we found that brown deposits containing heterogeneous calcium carbonates were precipitated when a shell disease occurred in the pearl oyster Pinctada fucata. Calcein-staining of the brown deposits indicated that numerous amorphous calcium deposits were present, which was further confirmed by Fourier-transform infrared spectroscopy (FTIR), Raman spectrum and X-ray difraction (XRD) analyses. So we speculate that ACC plays an important role in rapid calcium carbonate precipitation during shell repair process in diseased oysters.

The receptor genes PfBMPR1B and PfBAMBI are involved in regulating shell biomineralization in the pearl oyster Pinctada fucata.

Mounting evidence suggests that TGFß/BMP signaling pathway is most likely involved in shell biomineralization in molluscs, but the function of pathway receptors is poorly studied. Here, we cloned and identified two homologous BMP receptor genes, PfBMPR1B and PfBAMBI, from the pearl oyster Pinctada fucata. Real-time quantitative PCR and in situ hybridization revealed that these genes were expressed in mantle edge and pallial, specifically located at the outer epithelia. Knockdown of PfBMPR1B by RNA interference (RNAi) significantly decreased the expression levels of matrix protein (MP) genes and induced the abnormal ultrastructure of prismatic and nacreous layers. Conversely, knockdown of PfBAMBI significantly increased the expression levels of a portion of MP genes and induced the overgrowth of nacreous layer crystals. In the RNAi and shell notching experiments, MP gene expressions were competitively regulated by PfBMPR1B and PfBAMBI. In addition, the receptor inhibitor LDN193189 reduced the expression levels of MP genes in mantle primary cells and larvae, and induced abnormal D-shaped shell formation during larval development. Collectively, these results clearly show that PfBMPR1B and PfBAMBI are involved in regulating shell biomineralization in P. fucata. Our study therefore provides the direct evidence that BMP receptors participate in mollusc biomineralization.

Hemocytes participate in calcium carbonate crystal formation, transportation and shell regeneration in the pearl oyster Pinctada fucata.

In this study, light microscope, scanning and transmission electron microscope, hematoxylin-eosin and fluorescent staining, and mass spectrometry methods were employed to observe the calcium carbonate (CaCO3) crystal formation, hemocyte release and transportation, and hemocyte distribution at the shell regeneration area and to analyse the proteome of hemocytes in the pearl oyster, Pinctada fucata. The results indicated that intracellular CaCO3 crystals were observed in circulating hemocytes in P. fucata, implying that there was a suitable microenvironment for crystal formation in the hemocytes. This conclusion was further supported by the proteome analysis, in which various biomineralization-related proteins were detected. The crystal-bearing hemocytes, mainly granulocytes, may be released to extrapallial fluid (EPF) by the secretory cavities distributed on the outer surface of the mantle centre. These granulocytes in the EPF and between the regenerated shells were abundant and free. In the regenerated prismatic layer, the granulocytes were fused into each column and fragmented with the duration of shell maturation, suggesting the direct involvement of hemocytes in shell regeneration. Overall, this study provided evidence that hemocytes participated in CaCO3 crystal formation, transportation and shell regeneration in the pearl oyster. These results are helpful to further understand the exact mechanism of hemocyte-mediated biomineralization in shelled molluscs.