Structure of an amorphous calcium carbonate phase involved in the formation of Pinctada margaritifera shells.

Some mollusc shells are formed from an amorphous calcium carbonate (ACC) compound, which further transforms into a crystalline material. The transformation mechanism is not fully understood but is however crucial to develop bioinspired synthetic biomineralization strategies or accurate marine biomineral proxies for geoscience. The difficulty arises from the simultaneous presence of crystalline and amorphous compounds in the shell, which complicates the selective experimental characterization of the amorphous fraction. Here, we use nanobeam X-ray total scattering together with an approach to separate crystalline and amorphous scattering contributions to obtain the spatially resolved atomic pair distribution function (PDF). We resolve three distinct amorphous calcium carbonate compounds, present in the shell of Pinctada margaritifera and attributed to: interprismatic periostracum, young mineralizing units, and mature mineralizing units. From this, we extract accurate bond parameters by reverse Monte Carlo (RMC) modeling of the PDF. This shows that the three amorphous compounds differ mostly in their Ca-O nearest-neighbor atom pair distance. Further characterization with conventional spectroscopic techniques unveils the presence of Mg in the shell and shows Mg-calcite in the final, crystallized shell. In line with recent literature, we propose that the amorphous-to-crystal transition is mediated by the presence of Mg. The transition occurs through the decomposition of the initial Mg-rich precursor into a second Mg-poor ACC compound before forming a crystal.

Comparative transcriptomics identifies genes underlying growth performance of the Pacific black-lipped pearl oyster Pinctada margaritifera.

BACKGROUND: In bivalves, the rate at which organisms grow is a major functional trait underlying many aspects of their commercial production. Growth is a highly polygenic trait, which is typically regulated by many genes with small to moderate effects. Due to its complexity, growth variability in such shellfish remains poorly understood. In this study, we aimed to investigate differential gene expression among spat of the pearl oyster Pinctada margaritifera with distinct growth phenotypes. RESULTS: We selected two groups of P. margaritifera spat belonging to the same F2 cohort based on their growth performance at 5.5 months old. Transcriptome profile analysis identified a total of 394 differentially expressed genes between these Fast-growing (F) and Slow-growing (S) phenotypes. According to functional enrichment analysis, S oysters overexpressed genes associated with stress-pathways and regulation of innate immune responses. In contrast, F oysters up-regulated genes associated with cytoskeleton activity, cell proliferation, and apoptosis. Analysis of genome polymorphism identified 16 single nucleotide polymorphisms (SNPs) significantly associated with the growth phenotypes. SNP effect categorization revealed one SNP identified for high effect and annotated for a stop codon gained mutation. Interestingly, this SNP is located within a gene annotated for scavenger receptor class F member 1 (SRF1), which is known to modulate apoptosis. Our analyses also revealed that all F oysters showed up-regulation for this gene and were homozygous for the stop-codon mutation. Conversely, S oysters had a heterozygous genotype and a reduced expression of this gene. CONCLUSIONS: Altogether, our findings suggest that differences in growth among the same oyster cohort may be explained by contrasted metabolic allocation between regulatory pathways for growth and the immune system. This study provides a valuable contribution towards our understanding of the molecular components associated with growth performance in the pearl oyster P. margaritifera and bivalves in general.

Diversification of von Willebrand Factor A and Chitin-Binding Domains in Pif/BMSPs Among Mollusks.

Pif is a shell matrix protein (SMP) identified in the nacreous layer of Pinctada fucata (Pfu) comprised two proteins, Pif97 and Pif 80. Pif97 contains a von Willebrand factor A (VWA) and chitin-binding domains, whereas Pif80 can bind calcium carbonate crystals. The VWA domain is conserved in the SMPs of various mollusk species; however, their phylogenetic relationship remains obscure. Furthermore, although the VWA domain participates in protein-protein interactions, its role in shell formation has not been established. Accordingly, in the current study, we investigate the phylogenetic relationship between PfuPif and other VWA domain-containing proteins in major mollusk species. The shell-related proteins containing VWA domains formed a large clade (the Pif/BMSP family) and were classified into eight subfamilies with unique sequential features, expression patterns, and taxa diversity. Furthermore, a pull-down assay using recombinant proteins containing the VWA domain of PfuPif 97 revealed that the VWA domain interacts with five nacreous layer-related SMPs of P. fucata, including Pif 80 and nacrein. Collectively, these results suggest that the VWA domain is important in the formation of organic complexes and participates in shell mineralisation.

Substantial gene expression shifts during larval transitions in the pearl oyster Pinctada margaritifera.

Early development stages in marine bivalve are critical periods where larvae transition from pelagic free-life to sessile mature individuals. The successive metamorphosis requires the expression of key genes, the functions of which might be under high selective pressure, hence understanding larval development represents key knowledge for both fundamental and applied research. Phenotypic larvae development is well known, but the underlying molecular mechanisms such as associated gene expression dynamic and molecular cross-talks remains poorly described for several nonmodel species, such as P. margaritifera. We designed a whole transcriptome RNA-sequencing analysis to describe such gene expression dynamics following four larval developmental stages:  d-shape, Veliger, Umbo and Eye-spot. Larval gene expression and annotated functions drastically diverge. Metabolic function (gene expression related to lipid, amino acid and carbohydrate use) is highly upregulated in the first development stages, with increasing demand from  d-shape to umbo. Morphogenesis and larval transition are partly ordered by Thyroid hormones and Wnt signaling. While larvae shells show some similar characteristic to adult shells, the cause of initialization of biomineralization differ from the one found in adults. The present study provides a global overview of Pinctada margaritifera larval stages transitioning through gene expression dynamics, molecular mechanisms and ontogeny of biomineralization, immune system, and sensory perception processes.

Structural and Functional Analysis of the Amorphous Calcium Carbonate-Binding Protein Paramyosin in the Shell of the Pearl Oyster, Pinctada fucata.

Amorphous calcium carbonate (ACC) is an important precursor phase for the formation of aragonite crystals in the shells of Pinctada fucata. To identify the ACC-binding protein in the inner aragonite layer of the shell, extracts from the shell were used in the ACC-binding experiments. Semiquantitative analyses using liquid chromatography-mass spectrometry revealed that paramyosin was strongly associated with ACC in the shell. We discovered that paramyosin, a major component of the adductor muscle, was included in the myostracum, which is the microstructure of the shell attached to the adductor muscle. Purified paramyosin accumulates calcium carbonate and induces the prism structure of aragonite crystals, which is related to the morphology of prism aragonite crystals in the myostracum. Nuclear magnetic resonance measurements revealed that the Glu-rich region was bound to ACC. Activity of the Glu-rich region was stronger than that of the Asp-rich region. These results suggest that paramyosin in the adductor muscle is involved in the formation of aragonite prisms in the myostracum.

The role of Pm-miR-184-3p in regulating the immune response in the pearl oyster Pinctada fucata martensii.

microRNAs are a class of non-coding RNAs with post-transcriptional regulatory functions in eukaryotes. In our previous study, miR-184-3p was identified in the hemocyte transcriptome of Pinctada fucata martensii (Pm-miR-184-3p), and its expression was shown to be up-regulated following transplantation surgery; however, its role in regulating transplantation immunity has not yet been clarified. Here, the role of Pm-miR-184-3p in regulating the immune response of P. f. martensii was studied. The expression of Pm-miR-184-3p increased following the stimulation of pathogen-associated molecular patterns, and Pm-miR-184-3p overexpression increased the activity of antioxidant-related enzymes, such as superoxide dismutase and catalase. Transcriptome analysis obtained 1096 differentially expressed genes (DEGs) after overexpression of Pm-miR-184-3p, and these DEGs were significantly enriched in conserved pathways such as the Cell cycle pathway and NF-kappa B signaling pathway, as well as GO terms including base excision repair, cell cycle, and DNA replication, suggesting that Pm-miR-184-3p could enhance the inflammation process. Target prediction and dual luciferase analysis revealed that pro-inflammatory related genes Pm-TLR3 and Pm-FN were the potential target of Pm-miR-184-3p. We speculate that Pm-miR-184-3p may utilize negative regulation of target genes to delay the activation of corresponding immune pathways, potentially preventing excessive inflammatory responses and achieving a delicate balance within the organism. Overall, Pm-miR-184-3p play a key role in regulating cellular responses to transplantation. Our findings provide new insights into the immune response of P. f. martensii to transplantation.

Transcriptome analysis revealed the function of five tandemly duplicated nAChRs in the transplantation immunity in pearl oyster Pinctada fucata martensii.

nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels that play an important role in the homeostatic regulation of physiological functions. Our previous studies showed that nAChRs in the genome of pearl oyster Pinctada fucata martensii (PmnAChRs) were expanded through tandem duplication. This study aimed to analyze the function of five tandemly duplicated PmnAChRs in the transplantation immunity in P. f. martensii. Transcriptome analysis reveals that the differentially expressed genes (DEGs) shared between PmnAChR-RNAi and the control group were functionally involved in Signal transduction, Immune system et al., and most of the related genes were down-regulated in the PmnAChR-RNAi group. The different copies of PmnAChR may regulate transplantation immunity through various pathways, such as Wnt, protein digestion and absorption, Hippo, and gap junction pathway. The inflammation factor interleukin-17 (IL-17) and tumor necrosis factor-alpha (TNF-α) were down-regulated in PmnAChR-1, 4, 5-RNAi group, and the serum from the pearl oysters in the PmnAChR-1-4-RNAi group could promote the proliferation of the Vibrio harveyi, indicating the immunosuppressive function after down-regulation of PmnAChRs. The different responses of antioxidant enzymes and diverse signal pathways after down-regulation of PmnAChRs suggested that the five tandemly duplicated PmnAChRs may cooperate with different α type PmnAChRs and constitute the functional ion channel in the membrane. Results of this study not only provide insight for the effective regulation of the transplantation immunity, but also provide a theoretical reference for the study of the adaptive evolutionary mechanism of repeating genes.

Identification and functional characterization of a superoxide dismutase (CuZnSOD) from Pinctada fucata martensii.

Copper/zinc superoxide dismutase (Cu/Zn-SOD) can effectively eliminate reactive oxygen species (ROS)，avoid damage from O2 to the body, and maintain O2 balance. In this study, multi-step high-performance liquid chromatography (HPLC), combined with Mass Spectrometry (MS), was used to isolate and identify Cu/Zn-SOD from the serum of Pinctada fucata martensii (P. f. martensii) and was designated as PmECSOD. With a length of 1864 bp and an open reading frame (ORF) of 1422 bp, the cDNA encodes a 473 amino acid protein. The PmECSOD transcript was detected in multiple tissues by quantitative real-time PCR (qRT-PCR), with its highest expression level being in the gills. Additionally, the temporal expression of PmECSOD mRNA in the hemolymph was highest at 48 h after in vivo stimulation with Escherichia coli and Micrococcus luteus. The results from this study provide a valuable base for further exploration of molluscan innate immunity and immune response.

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.

Prokaryotic Expression, Purification, and Biological Properties of a Novel Bioactive Protein (PFAP-1) from Pinctada fucata.

Pinctada fucata meat is the main by-product of the pearl harvesting industry. It is rich in nutrition, containing a lot of protein and peptides, and holds significant value for both medicine and food. In this study, a new active protein was discovered and expressed heterogeneously through bioinformatics analysis. It was then identified using Western blot, molecular weight, and mass spectrometry. The antibacterial activity, hemolysis activity, antioxidant activity, and Angiotensin-Converting Enzyme II (ACE2) inhibitory activity were investigated. An unknown functional protein was screened through the Uniprot protein database, and its primary structure did not resemble existing proteins. It was an α-helical cationic polypeptide we named PFAP-1. The codon-optimized full-length PFAP-1 gene was synthesized and inserted into the prokaryotic expression vector pET-30a. The induced expression conditions were determined with a final isopropyl-ß-d-thiogalactoside (IPTG) concentration of 0.2 mM, an induction temperature of 15 °C, and an induction time of 16 h. The recombinant PFAP-1 protein, with low endotoxin and sterility, was successfully prepared. The recombinant PFAP-1 protein exhibited strong antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA) in vitro, and the diameter of the inhibition zone was 15.99 ± 0.02 mm. Its minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were 37.5 µg/mL and 150 µg/mL, respectively, and its hemolytic activity was low (11.21%) at the bactericidal concentration. The recombinant PFAP-1 protein significantly inhibited the formation of MRSA biofilm and eradicated MRSA biofilm. It also demonstrated potent 1,1-diphenyl-2-picryl-hydrazyl radical (DPPH) scavenging activity with a half-maximal inhibitory concentration (IC50) of 40.83 µg/mL. The IC50 of ACE2 inhibition was 5.66 µg/mL. Molecular docking results revealed that the optimal docking fraction of PFAP-1 protein and ACE2 protein was -267.78 kcal/mol, with a confidence level of 0.913. The stable binding complex was primarily formed through nine groups of hydrogen bonds, three groups of salt bridges, and numerous hydrophobic interactions. In conclusion, recombinant PFAP-1 can serve as a promising active protein in food, cosmetics, or medicine.

In Silico Identification and Molecular Mechanism of Novel Tyrosinase Inhibitory Peptides Derived from Nacre of Pinctada martensii.

Pearl and nacre powders have been valuable traditional Chinese medicines with whitening properties for thousands of years. We utilized a high-temperature and high-pressure method along with compound enzyme digestion to prepare the enzymatic hydrolysates of nacre powder of Pinctada martensii (NP-PMH). The peptides were identified using LC-MS/MS and screened through molecular docking and molecular dynamics simulations. The interactions between peptides and tyrosinase were elucidated through enzyme kinetics, circular dichroism spectropolarimetry, and isothermal titration calorimetry. Additionally, their inhibitory effects on B16F10 cells were explored. The results showed that a tyrosinase-inhibitory peptide (Ala-His-Tyr-Tyr-Asp, AHYYD) was identified, which inhibited tyrosinase with an IC50 value of 2.012 ± 0.088 mM. The results of the in vitro interactions showed that AHYYD exhibited a mixed-type inhibition of tyrosinase and also led to a more compact enzyme structure. The binding reactions of AHYYD with tyrosinase were spontaneous, leading to the formation of a new set of binding sites on the tyrosinase. The B16F10 cell-whitening assay revealed that AHYYD could reduce the melanin content of the cells by directly inhibiting the activity of intracellular tyrosinase. Additionally, it indirectly affects melanin production by acting as an antioxidant. These results suggest that AHYYD could be widely used as a tyrosinase inhibitor in whitening foods and pharmaceuticals.

A Natural Bioactive Peptide from Pinctada fucata Pearls Can Be Used as a Potential Inhibitor of the Interaction between SARS-CoV-2 and ACE2 against COVID-19.

The frequent occurrence of viral infections poses a serious threat to human life. Identifying effective antiviral components is urgent. In China, pearls have been important traditional medicinal ingredients since ancient times, exhibiting various therapeutic properties, including detoxification properties. In this study, a peptide, KKCH, which acts against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was derived from Pinctada fucata pearls. Molecular docking showed that it bound to the same pocket of the SARS-CoV-2 S protein and cell surface target angiotensin-converting enzyme II (ACE2). The function of KKCH was analyzed through surface plasmon resonance (SPR), Enzyme-Linked Immunosorbent Assays, immunofluorescence, and simulation methods using the SARS-CoV-2 pseudovirus and live virus. The results showed that KKCH had a good affinity for ACE2 (KD = 6.24 × 10-7 M) and could inhibit the binding of the S1 protein to ACE2 via competitive binding. As a natural peptide, KKCH inhibited the binding of the SARS-CoV-2 S1 protein to the surface of human BEAS-2B and HEK293T cells. Moreover, viral experiments confirmed the antiviral activity of KKCH against both the SARS-CoV-2 spike pseudovirus and SARS-CoV-2 live virus, with half-maximal inhibitory concentration (IC50) values of 398.1 µM and 462.4 µM, respectively. This study provides new insights and potential avenues for the prevention and treatment of SARS-CoV-2 infections.

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.

Evolution of Epidermal Growth Factor (EGF)-like and Zona Pellucida Domains Containing Shell Matrix Proteins in Mollusks.

Several types of shell matrix proteins (SMPs) have been identified in molluskan shells. Their diversity is the consequence of various molecular processes, including domain shuffling and gene duplication. However, the evolutionary origin of most SMPs remains unclear. In this study, we investigated the evolutionary process EGF-like and zona pellucida (ZP) domains containing SMPs. Two types of the proteins (EGF-like protein (EGFL) and EGF-like and ZP domains containing protein (EGFZP)) were found in the pearl oyster, Pinctada fucata. In contrast, only EGFZP was identified in the gastropods. Phylogenetic analysis and genomic arrangement studies showed that EGFL and EGFZP formed a clade in bivalves, and their encoding genes were localized in tandem repeats on the same scaffold. In P. fucata, EGFL genes were expressed in the outer part of mantle epithelial cells are related to the calcitic shell formation. However, in both P. fucata and the limpet Nipponacmea fuscoviridis, EGFZP genes were expressed in the inner part of the mantle epithelial cells are related to aragonitic shell formation. Furthermore, our analysis showed that in P. fucata, the ZP domain interacts with eight SMPs that have various functions in the nacreous shell mineralization. The data suggest that the ZP domain can interact with other SMPs, and EGFL evolution in pterimorph bivalves represents an example of neo-functionalization that involves the acquisition of a novel protein through gene duplication.

The first reporting of prevalence Vibrio species and expression of HSP genes in rayed pearl oyster (Pinctada radiata) under thermal conditions.

The main goal of the present study was to evaluate the influence of thermal exposure on Vibrio population and HSP genes expression (HSP 90, HSP70, and HSP20) in rayed pearl oyster (P. radiata). To this end, the oysters were reared for 30 days at temperatures of 22 °C (control), 25 °C, 27 °C, and 29 °C. The results showed that five dominate Vibrio strains including Vibrio hepatarius, V. harveyi, V. alginolyticus, V. parahaemolyticus, and V. rotiferianus were identified. The highest population of V. parahaemolyticus, V. alginolyticus, and V. harveyi, was found in 29οC group. According to real-time PCR, mantle exhibited the highest expression levels of HSP20, HSP70, and HSP90 genes. A higher level of HSP20 expression was observed at high temperatures (25 °C, 27 °C, and 29 °C) in the gonad and mantle compared to the control group (22 °C) while decrease in HSP90 expression level was recorded in 25 °C, 27 °C, and 29 °C groups. HSP20 expression level in adductor muscle was remarkably down-regulated in 27 °C and 29 °C groups. In this tissue, HSP70 was detected at highest levels in the 29οC group. In mantle, HSP90 gene expression was lowest at 22 °C water temperature. Several Vibrio strains have been identified from pearl Gulf oyster that haven't been previously reported. The identification of dominant Vibrio species is essential for epidemiological management strategies to control and prevent Vibrio outbreaks in pearl oyster farms. The expression pattern of HSP genes differs in rayed pearl oyster tissues due to differences in their thermal tolerance capability and physiological and biological characteristics. The present study provides useful molecular information for the ecological adaptation of rayed pearl oysters after exposure to different temperature levels.

Members of the histone-derived antimicrobial peptide family from the pearl oyster Pinctada fucata martensii: Inhibition of bacterial growth.

Because it is difficult to isolate standard antimicrobial peptides (AMPs) using traditional biochemical approaches, we designed, synthesized, and evaluated a series of structurally altered histone-derived AMPs (HDAPs) from the pearl oyster Pinctada fucata martensii using molecular cloning approaches. Four histone-homolog genes (PmH2A, PmH2B, PmH3, and PmH4-1) were identified, of which PmH2A and PmH2B had yet to be described. PmH2A and PmH2B were therefore cloned using Rapid Amplification of cDNA Ends (RACE) and characterized. Constitutive PmH2A and PmH2B mRNA expression was detected in all six pearl oyster tissues tested, with comparatively greater transcript abundance in the gonads. Because α-helical content, hydrophilicity index, and the presence of a proline hinge may be the three important factors influencing the antimicrobial efficacy of HDAPs, we synthesized a series of eight N- and C-terminally truncated or amino acid-substituted synthetic candidate HDAP analogs derived from PmH2A, PmH2B, PmH3, and PmH4-1. Only the PmH2A- and PmH4-derived AMPs inhibited bacterial growth. The PmH2A-derived AMPs were α-helical proteins, while the PmH4-derived AMPs were extended strand/random coil proteins. Our results suggested that having an α-helical structure was particularly important for the antibacterial efficacy of the PmH2A-derived peptides; amphipathic structures (hydrophilic index, 0.3 to -0.3) may enhance the antimicrobial function of both the PmH2A- and PmH4-derived peptides. The high antibacterial efficacy of one of the HDAP analogs studied, PmH2A-AMP (5-13) [KLLK]3, indicated that this protein may represent a promising candidate for the treatment of bacterial infections in aquaculture mollusk species. This first study of HDAPs from the pearl oyster P. f. martensii provides new insights into the design and function of highly effective antimicrobial peptides.

Toll-like receptor may be involved in acquired immune response in pearl oyster Pinctada fucata.

The increasing experimental evidence suggests that there are some forms of specific acquired immunity in invertebrates, in which Toll-like receptors (TLRs) play vital roles in activating innate and adaptive immunity and have been comprehensively investigated in mammalian species. Yet, the immune mechanisms underlying TLR mediation in mollusks remain obscure. In this study, we identified a TLR13 gene in the pearl oyster Pinctada fucata for the first time and named it PfTLR13 which consists of a 5'-untranslated terminal region (5'-UTR) of 543 bp, an open reading frame (ORF) of 2667 bp, and a 3'-UTR of 729 bp. We found that PfTLR13 mRNA was expressed in all tissues examined, with the highest level in the gills. The expression of PfTLR13 in the gills of oysters exposed to Vibrio alginolyticus or pathogen-associated molecular patterns (PAMPs) (including LPS, PGN, and poly(I:C)) was significantly higher than in the control group. Interestingly, the immune response to the first stimulation was weaker than the response to the second stimulation, suggesting that the primary stimulation may lead to immune priming of TLR in pearl oysters, similar to acquired immunity in vertebrates. Furthermore, we found that PfTLR13 expression was differentially associated with allograft and xenograft in the pearl oyster P. fucata, with the highest expression levels observed at 12 h post-allograft and 24 h post-xenograft. Overall, our findings provide new insights into the immune mechanisms underlying TLR mediation in mollusks and suggest that PfTLR13 may play a crucial role in the specific acquired immunity of pearl oysters.

Defence mechanisms of Pinctada fucata martensii to Vibrio parahaemolyticus infection: Insights from proteomics and metabolomics.

Survival of pearl oysters is not only challenged by coastal pollution, but also pathogen infection that may eventually incur substantial economic losses in the pearl farming industry. Yet, whether pearl oysters can defend themselves against pathogen infection through molecular mechanisms remains largely unexplored. By using iTRAQ proteomic and metabolomic analyses, we analysed the proteins and metabolites in the serum of pearl oysters (Pinctada fucata martensii) when stimulated by pathogenic bacteria (Vibrio parahaemolyticus). Proteomic results found that a total of 2,242 proteins were identified in the experimental (i.e., Vibrio-stimulated) and control groups, where 166 of them were differentially expressed (120 upregulated and 46 downregulated in the experimental group). Regarding the immune response enrichment results, the pathway of signal transduction was significantly enriched, such as cytoskeleton and calcium signalling pathways. Proteins, including cathepsin L, heat shock protein 20, myosin and astacin-like protein, also contributed to the immune response of oysters. Pathogen stimulation also altered the metabolite profile of oysters, where 49 metabolites associated with metabolism of energy, fatty acids and amino acids were found. Integrated analysis suggests that the oysters could respond to pathogen infection by coordinating multiple cellular processes. Thus, the proteins and metabolites identified herein not only represent valuable genetic resources for developing molecular biomarkers and genetic breeding research, but also open new avenues for studies on the molecular defence mechanisms of pearl oysters to pathogen infection.

Optimising flow-cytometry methods for marine mollusc haemocytes using the pearl oyster Pinctada maxima as a model.

Flow-cytometry has become increasingly popular to assess the haemocytes morphology and functions of marine molluscs. Indeed, haemocytes are the first line of defence of the immune system in molluscs and are used as a proxy for oyster health. Authors publishing in the field of flow-cytometry and molluscs health seemed to utilise the same methods for all model species used, independently of their geographical location in the world (temperate, tropical, etc.). Hence, this paper dived into flow-cytometry methodology and investigated if using different plates, different thresholds, different incubation times and temperatures as well as different fluorochromes concentrations affected the results. This study revealed that the cell count did not change when using different thresholds on the FSC-H parameter of the instrument but was affected by the plate type, the temperature of incubation, and the time of incubation. Indeed, non-adherent plates yielded the highest cell count and lower cell counts were associated with a higher temperature and a longer time of incubation. Furthermore, the haemocytes functions such as the phagocytosis, the lysosomal content, the intracellular oxidative activity, and the mitochondria activity were also affected by the temperature and the time of incubation. An increase in the phagocytosis capacity, lysosomal content and mitochondria activity was observed with a higher temperature. At the exception of the phagocytosis rate, all the other parameters such as the phagocytosis capacity, the intracellular oxidative activity, and the lysosomal content increased with a longer incubation time. We also showed that it is best to optimise the amount of fluorochromes used to avoid unnecessary background or non-specific staining.

Preliminary investigation of the immune activity of PmH2A-derived antimicrobial peptides from the pearl oyster Pinctada fucata martensii.

Antimicrobial peptides (AMPs) play important roles in the immune defense against pathogenic microorganisms. For instance, histone 2A (H2A)-derived AMPs is an antimicrobial peptide involved in the host's innate immune defense and immunoregulation. AMPs have been isolated from the pearl oyster Pinctada fucata martensii but their role in host defense remains poorly understood. To elucidate the structural features of P. f. martensii H2A (PmH2A)-derived AMPs and their potential immune functions, we synthesized a series of laboratory-designed synthetic analogs of PmH2A and examined their antimicrobial properties, as well as their mechanisms of action. This analysis revealed inhibitory effects on the growth of Gram-positive and Gram-negative bacteria. Further assessment by transmission electron microscopy (TEM) of two of the three peptides, PmH2A-AMP and PmH2A-AMP(5-13)[KLLK]3, confirmed that it exerted an anti-bacterial activity through membrane lysis. Finally, we found that the hemocytes and gills of P. f. martensii released antimicrobial H2A histones in response to LPS exposure, mimicking tissue damage and infection. This immune response is reminiscent of the neutrophil extracellular traps (NETs) recently described in oysters. Thus, the LPS challenge is sufficient to induce histone-derived peptide accumulation in pearl oyster P.f. martensii.