Pearl Farming Micro-Nanoplastics Affect Oyster Physiology and Pearl Quality.

Pearl farming is crucial for the economy of French Polynesia. However, rearing structures contribute significantly to plastic waste, and the widespread contamination of pearl farming lagoons by microplastics has raised concerns about risks to the pearl industry. This study aimed to evaluate the effects of micro-nanoplastics (MNPs, 0.4-200 µm) on the pearl oyster (Pinctada margaritifera) over a 5-month pearl production cycle by closely mimicking ecological scenarios. MNPs were produced from weathered plastic pearl farming gear and tested at environmentally relevant concentrations (0.025 and 1 µg L-1) to decipher biological and functional responses through integrative approaches. The significant findings highlighted the impacts of MNPs on oyster physiology and pearl quality, even at remarkably low concentrations. Exposure to MNPs induced changes in energy metabolism, predominantly driven by reduced assimilation efficiency of microalgae, leading to an alteration in gene expression patterns. A distinct gene expression module exhibited a strong correlation with physiological parameters affected by MNP conditions, identifying key genes as potential environmental indicators of nutritional-MNP stress in cultured oysters. The alteration in pearl biomineralization, evidenced by thinner aragonite crystals and the presence of abnormal biomineral concretions, known as keshi pearls, raises concerns about the potential long-term impact on the Polynesian pearl industry.

Industrial manufacturing method and characterization of Ayurvedic marine drugs: mother pearl, cowry, coral and pearl.

Introduction: Ayurvedic marine drugs derived from mollusc shells and coral are regularly used by Ayurvedic physicians to treat several disease conditions like acid peptic disease, irritable bowel syndrome, osteoporosis, etc. However, standard operating procedures for manufacturing these drugs and their complete characterization have not been published in the Ayurvedic Formulary and Ayurvedic Pharmacopeia of India to date. Methods: Present study describes the traditional manufacturing process and thorough characterization using classical and advanced analytical tools. The raw materials characters, in-process parameters, and finished product specifications have been elaborated to develop monographs. Especially, the identity and purity of raw coral and pearl were checked by Raman Spectroscopy and Energy Dispersive X-ray Fluorescence analysis. Results: In the finished product analysis, the X-Ray Diffraction study revealed that incineration after trituration with Aloe barbadensis leaf pulp or rose water converted the aragonite phase of calcium carbonate into calcite phase in mother pearl, cowry, and pearl while the calcite form of raw coral was retained. The prominent bands around 1390, 870, and 712 cm-1 detected by Fourier Transform-Infrared Spectroscopy and mass loss between 39-44% (w/w) revealed by thermogravimetric analysis confirmed the carbonate form of these calcium-based drugs. The finished products were very fine grayish-white powders constituted by irregularly shaped nano-micro particulate calcium carbonate exhibiting particle size between 600 nm (D10 value) to 1.2 µm (D90 value). Conclusion: The quality control and assurance achieved in this study may be further utilized by the pharmaceutical industries to manufacture quality marine drugs and conduct efficacy studies.

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.

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.

Identification of novel adhesive proteins in pearl oyster by proteomic and bioinformatic analysis.

Byssuses, which are proteinaceous fibers secreted by mollusks, are remarkable underwater adhesives. Although mussel adhesives are well known, much less is known about the byssal proteins of pearl oysters especially in the adhesive regions. In this study, adhesive proteins from the pearl oyster Pinctada fucata were studied in depth by transcriptomics and proteomics approaches. In total, 16 novel proteins were identified including a von Willebrand factor type A domain-containing protein, a thrombospondin-1-like protein, tyrosinase, mucin-like proteins, protease inhibitors, and Pinctada unannotated foot protein 3 (PUF3) to PUF6. Interestingly, PUF3-6 are enriched with glycine, serine, and PXG (X = F/Y/W/K/L) motifs and are highly expressed in the foot. The identification of byssal proteins of the pearl oyster is a key step for understanding byssus formation and may inspire the synthesis of novel adhesives for underwater use and the development of anti-biofouling strategies.

Identification of a long non-coding RNA (LncMSEN2) from pearl oyster and its potential roles in exoskeleton formation and LPS stimulation.

Long non-coding RNAs (lncRNAs) play regulatory roles in various biological processes, including exoskeleton formation and immune response. The exoskeleton-based mantle-shell defense system is an important defense mechanism in shellfish. In this study, we found a novel lncRNA, herein formally named, LncMSEN2, from the pearl oyster Pinctada fucuta martensii, and its sequence was validated via polymerase chain reaction (PCR). LncMSEN2 was highly expressed in mantle tissues, especially in the central region (P < 0.05), and was also expressed in the pearl sac as detected by quantitative real-time PCR. In situ hybridization experiments revealed that LncMSEN2 had a strong positive signal in the inner and outer epidermal cells of the mantle pallial and central regions. RNA interference experiments showed that interference of LncMSEN2 expression with dsRNA in mantle tissues led to an abnormal crystal structure of the nacre. In addition, LncMSEN2 expression significantly increased 6 h after lipopolysaccharide stimulation in mantle tissues (P < 0.05). These results indicated that LncMSEN2 may be a novel regulator of the mantle-shell defense system of pearl oyster.

Breaking the long-standing morphological paradigm: Individual prisms in the pearl oyster shell grow perpendicular to the c-axis of calcite.

Cross-sections of calcitic prismatic layers in mollusk shells, cut perpendicular to growth direction, reveal well-defined polygonal shapes of individual "grains" clearly visible by light and electron microscopy. For several kinds of shells, it was shown that the average number of edges in an individual prism approaches six during the growth process. Taking into account the rhombohedral symmetry of calcite, often presented in hexagonal axes, all this led to the long-standing opinion that calcitic prisms grow along the c-axis of calcite. In this paper, using X-ray diffraction and electron backscatter diffraction (EBSD), we unambiguously show that calcitic prisms in pearl oyster Pinctada margaritifera predominantly grow perpendicular to the c-axis. The obtained results imply that the hexagon-like habitus of growing crystallites may be not necessarily connected to calcite crystallography and, therefore, other factors should be taken into consideration. We analyze this phenomenon by comparing the organic contents in Pinctada margaritifera and Pinna nobilis shells, the later revealing regular growth of calcitic prisms along the c-axis.

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.

HSP40 gene family in pearl oyster Pinctada fucata martensii: Genome-Wide identification and function analysis.

HSP40, also called DnaJ, functions as a molecular chaperone by binding to Hsp70 and plays critical roles in the growth, development, and response to heat stress. However, this gene family is rarely reported in pearl oyster. In this study, 31 putative HSP40 genes from Pinctada fucata martensii (PmHSP40) were identified through bioinformatics methods and classified into three groups according to the presence of the complete three domains (J, G/F zinc finger domain, and cysteine rich domain). Further analysis showed that the PmHSP40 genes are highly diverse in sequence, domain structure, and tissue and development expression profile, implying diversified functions. In addition, one highly induced PmHSP40 in low-temperature (PmHSP40LT) was cloned, and its function in temperature response was explored. PmHSP40LT has a full length of 1741 bp, containing 1059 bp ORF, 152 bp 5'UTR, and a 507 bp 3'UTR, and encodes 352 amino acids. PmHSP40LT expression was significantly induced at low (17 °C) and high temperature (32 °C) at 6 h, 1 d, and 3 d relative to the control group. Thus, PmHSP40LT possibly participates in response to high and low temperatures in pearl oyster. In conclusion, all these results provide a comprehensive basis for the further analysis of PmHSP40 function in pearl oysters.

Genome and transcriptome analyses providing insight into the immune response of pearl oysters after allograft and xenograft transplantations.

The immune response after allograft or xenograft transplantation in the pearl oyster is a major factor that cause its nucleus rejection and death. To determine the mechanism underlying the immune response after allograft and xenograft transplantations in the pearl oyster Pinctada fucata martensii, we constructed two sets of transcriptomes of hemocytes at different times (6 and 12 h; 1, 3, 6, 12, and 30 d) after allograft and xenograft transplantations, in which the xenografted mantle tissue was from Pinctada maxima. The transcriptomic analysis reveals many genes are involved in the immune response to transplantation, such as transient receptor potential cation channel (TRP), calmodulin (CaM), DNA replication-related genes, and sugar and lipid metabolism-related genes. The expression of these identified genes was higher in the host pearl oyster transplanted with xenograft than that by allograft. The histological analysis of the pearl sac also confirmed that many hemocytes were still gathered around the transplanted nucleus, and no pearl sac was formed in the host pearl oysters at 30 d after xenograft transplantation. The genomic analysis indicated that pearl oysters evolved many copies of genes, such as TRP, CaM, and GST, to sense and cope with the immune response after transplantation. "Ribosome" and "Cytosolic DNA-sensing pathway" were specifically induced in the xenograft group, whereas "Notch signaling pathway" specifically responded to the allograft transplantation. These results can improve our understanding of the mechanism underlying the immune response of pearl oysters after allograft and xenograft transplantations.

Towards a better understanding of allograft-induced stress response in the pearl oyster Pinctada fucata martensii: Insights from iTRAQ-based comparative proteomic analysis.

Implantation of a spherical nucleus into a recipient oyster is a critical step in artificial pearl production. The implanted nucleus is known to trigger cellular stress responses at several levels, yet the molecular mechanism underpinning physiological adaptation of the pearl oysters to nucleus implantation is still poorly understood. In this study, we took advantage of the iTRAQ-based proteomics and LC-MS/MS approach to look into allograft induced gene regulation at the protein expression level in the pearl oyster Pinctada fucata martensii, across a period of 30 days following nucleus implantation. A wide variety of proteins, including a group of immune-related proteins such as E3 ubiquitin-ligase and heat shock proteins, exhibited differential expression in response to the surgical operation. Further comparisons between different sampling points revealed that GO terms including "translation" and "oxidation-reduction process" and KEGG pathways including "glycolysis/gluconeogenesis" and "pyruvate metabolism" were significantly enriched at several time points, indicating the important roles of these molecular events in the stress response of pearl oysters to nucleus implantation. In addition, considerable discrepancy between protein expression level and gene transcript abundancy was identified, as only a few genes showed at least 2-fold expression changes at both proteomic and transcriptomic levels. The result implies that post-transcriptional gene regulation for the key proteins may represent an important aspect of allograft-induced stress response in the pearl oysters. Taken together, the data obtained would contribute to a deeper understanding of the molecular mechanisms enabling stress adaptation of the pearl oysters in response to nucleus implantation.

Comprehensive transcriptome analysis reveal key molecular events in the pearl oyster after pre-grafting conditioning.

Pre-grafting conditioning is a crucial procedure before transplant surgery during pearl production. To investigate the molecular response of the pearl oyster Pinctada fucata martensii to conditioning, we constructed two hemocyte transcriptomes from pearl oysters with and without conditioning. A total of 134,222,686 raw reads were generated and assembled using the reference genome of the pearl oyster. Transcriptome analysis revealed 3,074 differentially expressed genes (DEGs). Gene ontology and pathway enrichment analyses revealed that these DEGs were mainly associated with "microtubule-based process", "regulation of actin cytoskeleton", and "cell cycle". All related genes were over-expressed in pearl oysters after conditioning. Some nucleotide-binding oligomerization domain-like receptors (NLR), toll-like receptor, myd88, proinflammatory cytokine interleukin-17 (IL-17), and apoptosis-related genes were highly expressed in pearl oysters after conditioning, indicating that conditioning induced the immune response of pearl oysters. "Fatty acid biosynthesis" (FA biosynthesis) was included in the enriched terms, and all eight FA synthase genes in this pathway were highly induced after conditioning. Four tandemly duplicated arginine kinase genes (PmAK) were found in the genome of P. f. martensii, gene structure and sequence analysis indicated PmAK genes were more diverse compared with that from human and zebra fish. The four tandemly duplicated PmAKs were highly up-regulated after conditioning. These findings will help to elucidate the responding molecular events after conditioning and explain the high pearl oyster survival rate with conditioning after transplantation, thereby providing useful information in perfecting the conditioning method to improve pearl oyster survival rate after transplantation.

Purification, Characterization and Evaluation of Inhibitory Mechanism of ACE Inhibitory Peptides from Pearl Oyster (Pinctada fucata martensii) Meat Protein Hydrolysate.

Angiotensin-I-converting enzyme (ACE) inhibitory peptides derived from natural products have shown a blood pressure lowering effect with no side effects. In this study, two novel ACE inhibitory peptides (His-Leu-His-Thr, HLHT and Gly-Trp-Ala, GWA) were purified from pearl oyster (Pinctada fucata martensii) meat protein hydrolysate with alkaline protease by ultrafiltration, polyethylene glycol methyl ether modified immobilized metal ion affinity medium, and reverse-phase high performance liquid chromatography. Both peptides exhibited high ACE inhibitory activity with IC50 values of 458.06 ± 3.24 µM and 109.25 ± 1.45 µM, respectively. Based on the results of a Lineweaver-Burk plot, HLHT and GWA were found to be non-competitive inhibitor and competitive inhibitor respectively, which were confirmed by molecular docking. Furthermore, the pearl oyster meat protein hydrolysate exhibited an effective antihypertensive effect on SD rats. These results conclude that pearl oyster meat protein is a potential resource of ACE inhibitory peptides and the purified peptides, HLHT and GWA, can be exploited as functional food ingredients against hypertension.

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.

Regulation of IL-17 by lncRNA of IRF-2 in the pearl oyster.

Long noncoding RNAs (lncRNAs), once thought to be nonfunctional, have recently been shown to participate in the multilevel regulation of transcriptional, posttranscriptional and epigenetic modifications and to play important roles in various biological processes, including immune responses. However, the expression and roles of lncRNAs in invertebrates, especially nonmodel organisms, remain poorly understood. In this study, by comparing a transcriptome to the PfIRF-2 genomic structure, we identified lncIRF-2 in the PfIRF-2 genomic intron. The results of the RNA interference (RNAi) and the nucleus grafting experiments indicated that PfIRF-2 might have a negative regulatory effect on lncIRF-2, and PfIRF-2 and lncIRF-2 may have a positive regulatory effect on PfIL-17. Additionally, lncIRF-2, PfIRF-2 and PfIL-17 were involved in responses to the nucleus graft. These results will enhance the knowledge of lncIRF-2, IRF-2, and IL-17 functions in both pearl oysters and other invertebrates.

Deep transcriptome profiling sheds light on key players in nucleus implantation induced immune response in the pearl oyster Pinctada martensii.

Immunological rejection of the pearl oysters following nucleus implantation is a major issue limiting the successful rate of cultured pearls. To date, the molecular mechanism of immune tolerance during pearl formation in the pearl oysters is still largely unknown. Through the RNA sequencing platform and comparative transcriptomic analysis, we investigated the chronic gene expression changes at seven time points (0, 5, 10, 15, 20, 30, 60 days post implantation or dpi) over a period of 60 days following nucleus implantation in the pearl oyster Pinctada martensii. A total of 81,390 unique transcripts (or unigenes) with a combined length of 96.8 million bp and a N50 value of 2227 bp were obtained. When compared with sequences in the nr, nt, Swiss-Prot, KEGG, COG and GO databases, 36,380 unigenes can find homologous genes. Pairwise comparison of gene expression among all the samples showed that the largest number (or 6846) of differentially expressed genes was observed at 10 dpi. The number then decreased to below 5000 at 15, 20 and 30 dpi and increased again to 6679 at 60 dpi. PCA analysis further showed that the seven time points can be roughly divided into four groups. Comparative transcriptomic analysis between the four groups identified a variety of genes showing differential expression at different time points, including many immune-related genes such as those encoding for toll-like receptor, lectin, scavenger receptor, and peroxidase. In addition, GO and KEGG enrichment analysis revealed that these differentially expressed genes were mainly associated with metabolism, ribosome function, immune response, signaling transduction, and cytoskeleton organization. Notably, two KEGG pathways, namely "cell adhesion molecules" and "primary immunodeficiency" were significantly enriched during the whole process. This finding indicates that genes in these pathways are likely to play critical roles in the immune tolerance of the pearl oysters. To conclude, the data obtained contribute to a better understanding of the molecular mechanisms of nucleus implantation induced immune response in the pearl oysters, and will facilitate the development of effective measures to improve the performance of pearl culture.

Recombinant production of a shell matrix protein in Escherichia coli and its application to the biomimetic synthesis of spherulitic calcite crystals.

OBJECTIVES: To overcome the limited production capability of shell matrix proteins and efficiently conduct in vitro CaCO3 biomineralization studies, a putative recombinant shell matrix protein was prepared and characterized. RESULTS: A glycine-rich protein (GRP_BA) was found in Pinctada fucata as a putative shell matrix protein (NCBI reference sequence; BAA20465). It was genetically redesigned for the production in Escherichia coli. The recombinant protein was obtained in a 400 ml shake-flask culture at approx. 30 mg l(-1) with a purity of >95 %. It efficiently formed a complex with Ca(2+). Ca(2+)-induced agglomeration was like other calcification-related proteins. Spherulitic calcite micro-particles, 20-30 µm diam. with rosette- and sphere-like structures were synthesized in the presence of the recombinant shell protein, which could be formed by stacking and/or aggregation of calcite nanograins and the bound protein. CONCLUSIONS: Recombinant production of a shell matrix protein could overcome potential difficulties associated with the limited amount of protein available for biomineralization studies and provide opportunities to fabricate biominerals in practical aspects.

Cloning and gene expression of signal transducers and activators of transcription (STAT) homologue provide new insights into the immune response and nucleus graft of the pearl oyster Pinctada fucata.

The signal transducers and activators of the transcription (STAT) family play an important role in regulatory and cellular functions by regulating the expression of a variety of genes, including cytokines and growth factors. In the present study, a Pinctada fucata STAT protein, termed PfSTAT, was described. The deduced amino acid sequence of PfSTAT contains the conserved STAT_bind domain and the SH2 domain, and the additional Bin/Amphiphysin/Rvs (BAR) domain, but does not have STAT_alpha and STAT_int domains. Multiple sequence alignments revealed that PfSTAT showed relatively low identity with vertebrate and other invertebrate STATs, and phylogenetic analysis indicated that the evolution of STAT may have been more complex and ancient. Gene expression analysis revealed that PfSTAT is involved in the immune response to polyinosinic-polycytidylic acid (poly I:C) stimulation and in the nucleus insertion operation. This study contributes to a better understanding of PfSTAT in protecting the pearl oyster from disease or injury caused by grafting.

Haemocyte persistence after grafting for pearl production in Pinctada margaritifera (Linnaeus, 1758).

The grafting process used for pearl production in pearl oysters triggers a significant haemocyte response which has an influence on the quality of pearls formed. One hundred and ten selected healthy adult Pinctada margaritifera were grafted for pearl production. Beginning two days after grafting, oysters were sacrificed regularly until the 48th day and the pearl-sacs of sampled oysters were sectioned for histological analysis. The level of haemocytes present in the pearl-sacs decreased overtime with the samples from day 2 showing the highest levels. Haemocyte levels also varied between samples from a particular day. The exact cause(s) of varying levels of haemocyte accumulation during pearl-sac development in P. margaritifera is not known. However, it is reasonable to assume that haemocyte production is positively related to the degree of damage caused to host oyster tissues during the grafting procedure. While haemocytes have an important wound healing role in pearl oysters, excessive haemocyte presence may be detrimental to maximizing pearl quality.

Nuclear factor of activated T cells (NFAT) in pearl oyster Pinctada fucata: molecular cloning and functional characterization.

Nuclear factor of activated T cells (NFAT) plays an important role in nonimmune cells and also in T cells and many other cells of the immune system, by regulating the expression of a variety of genes involved in the immune response, organ development, developmental apoptosis and angiogenesis. In the present study, the NFAT homology gene, PfNFAT, from the pearl oyster Pinctada fucata was cloned and its genomic structure and promoter were analyzed. PfNFAT encodes a putative protein of 1226 amino acids, and contains a highly conserved Rel homology region (RHR) with DNA-binding specificity, and a regulatory domain (NFAT homology region, NHR) containing a potent transactivation domain (TAD). The PfNFAT gene consists of 12 exons and 11 introns, and its promoter contains potential binding sites for transcription factors such as NF-κB (Nuclear factor κB), STATx (signal transducer and activator of transcription), AP-1 (activator protein-1) and Sox-5/9 (SRY type HMG box-5/9), MyoD (Myogenic Differentiation Antigen) and IRF (Interferon regulatory factor). Comparison and phylogenetic analysis revealed that PfNFAT shows high identity with other invertebrate NFAT, and clusters with the NFAT5 subgroup. Furthermore, gene expression analysis revealed that PfNFAT is involved in the immune response to lipopolysaccharide (LPS) and Polyinosinic-polycytidylic acid (poly I:C) stimulation and in the nucleus inserting operation. The study of PfNFAT may increase understanding of molluscan innate immunity.