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.

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.

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.

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.

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.

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.

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.

Purification and Characterization of the Lecithin-Dependent Thermolabile Hemolysin Vhe1 from the Vibrio sp. Strain MA3 Associated with Mass Mortality of Pearl Oyster (Pinctada fucata).

In a previous study, we isolated a Vibrio sp. strain MA3 and its virulence factor, a hemolysin encoded by vhe1. This strain is associated with mass mortalities of the pearl oyster Pinctada fucata. In the present study, the vhe1 gene from strain MA3 was cloned and its encoded product was purified and characterized. Our results show that the vhe1 gene encodes a protein of 417 amino acids with an estimated molecular mass of 47.2 kDa and a pI of 5.14. The deduced protein, Vhe1, was found to contain the conserved amino acid sequence (GDSL motif) of the hydrolase/esterase superfamily and five conserved blocks characteristic of SGNH hydrolases. A BLAST homology search indicated that Vhe1 belongs the lecithin-dependent hemolysin/thermolabile hemolysin (LDH/TLH) family. In activity analyses, the optimal temperature for both the hemolytic and phospholipase activities of Vhe1 was 50 °C. Vhe1 hemolytic activity and phospholipase activity were highest at pH 8.5 and pH 8.0, respectively. However, both enzymatic activities sharply decreased at high temperature (> 50 °C) and pH < 7.0. Compared with previously reported hemolysins, Vhe1 appeared to be more thermal- and pH-labile. Both its hemolytic activity and phospholipase activity were significantly inhibited by CuCl2, CdCl2, ZnCl2, and NiCl2, and slightly inhibited by MnCl2 and CoCl2. Vhe1 showed higher phospholipase activity toward medium-chain fatty acids (C8-C12) than toward shorter- and longer-chain fatty acids. These results accumulate knowledge about the LDH/TLH of V. alginolyticus, which detailed characterization has not been reported, and contribute to solving of the mass mortality of pearl oyster.

Development and evaluation of a rapid, specific, and sensitive loop-mediated isothermal amplification assay to detect Tenacibaculum sp. strain pbs-1 associated with black-spot shell disease in Akoya pearl oysters.

Black-spot shell disease decreases pearl quality and threatens pearl oyster survival. Establishment of a rapid, specific, and sensitive assay to detect Tenacibaculum sp. strain Pbs-1 associated with black-spot shell disease is of commercial importance. We developed a rapid, specific, and highly sensitive loop-mediated isothermal amplification (LAMP) assay to detect Tenacibaculum sp. Pbs-1 in Akoya pearl oysters Pinctada fucata. A set of five specific primers (two inner, two outer, and a loop) were designed based on the 16S-23S internal spacer region of strain Pbs-1. The optimum reaction temperature was 63 °C, and concentrations of the inner and loop primers were 1.4 and 1.0 µM, respectively. The LAMP product can be detected using agarose gel electrophoresis, and the color change in the reaction tube can be detected visually (by the naked eye) following the addition of malachite green. Our assay proved to be specific for strain Pbs-1, with no cross-reactivity with five other species of Tenacibaculum. The detection limit of the LAMP assay at 35 min is 50 pg, and at 60 min it is 5 fg. We evaluated the LAMP assay using diseased and healthy pearl oysters. The results demonstrate the suitability and simplicity of this test for rapid field diagnosis of strain Pbs-1.

Cloning and functional analysis of a trypsin-like serine protease from Pinctada fucata martensii.

Trypsin-like serine proteases (TLSs) play various roles in dietary protein digestion, hemolymph coagulation, antimicrobial peptide synthesis, and, in particular, the rapid immune pathways activated in response to pathogen detection. The cultured pearl industry, of which Pinctada fucata martensii is one of the most important species, is plagued by disease, thus leading to large economic losses. Herein, the molecular mechanisms underlying the innate immune response of P.f. martensii were explored. First, immune effector molecules from the P.f. martensii genome were screened and a TLS-like gene encoding a protein with a trypsin domain, herein designated as PmTLS, was identified. A multi-sequence alignment indicated a low sequence homology between PmTLS and other mollusk TLS-like proteins. Furthermore, a neighbor-joining phylogenetic analysis indicated that PmTLS has the closest genetic relationship to a Crassostrea gigas TLS. Additionally, real-time quantitative PCR (qPCR) analysis showed that PmTLS mRNA is constitutively expressed in all of the 6 examined P.f. martensii tissues, with significantly higher expression noted in hemocytes relative to the other tissues examined (p < 0.05). P.f. martensii samples were then challenged with various pathogen-associated molecular patterns (PAMPs), including lipopolysaccharide, peptidoglycan, and polyinosinic acid. In the challenge groups, PmTLS was significantly upregulated in hemocytes at 48 h post-challenge when compared to the unchallenged controls. Furthermore, treatment with recombinant PmTLS (rPmTLS) also significantly inhibited the growth of most of the examined gram-negative bacteria tested in vitro (p < 0.05), but it had little effect on the growth of the examined gram-positive bacteria. When examining morphological changes via transmission electron microscopy, rPmTLS treated bacteria exhibited morphological changes such as plasma wall separation. Thus, rPmTLS appears to play a bactericidal role by destroying bacterial cell membranes or cell walls, which subsequently leads to a release of the cellular contents and cell death. The findings presented herein have enabled further characterization of the immune defense mechanisms in P.f. martensii and may lead to improved disease control methods for the pearl cultivation industry.

Characterization of transcription factor activator pretein-1 (AP-1) and its association with cold tolerance in Pinctada fucata martensii.

AP-1 is an important transcription factor for cell proliferation/differentiation and animal immunity/development; however, its role in research in shellfish is poorly understood. Here, the cDNA of AP-1 gene from Pinctada fucata martensii was characterized. Its expression was detected in all six examined tissues, and a high level was observed in the gill and hepatopancreas. Analysis of the developmental transcriptomes showed that the PmAP-1 gene expression levels were high during D-stage larval and spat stages. The gene also exhibited a significantly high expression under cold tolerance stress. SNP analysis of the exon region and 5' flanking region of PmAP-1 revealed 19 SNPs of which 8 showed significant differences between cold tolerance selection line and base stock. Furthermore, three haplotypes generated by the SNPs of PmAP-1 were significantly associated with cold tolerance, respectively.These results suggest that the PmAP-1 gene plays an important role in the response of P. f. martensii to low temperature stress. These SNPs and haplotypes of PmAP-1 may be related to the cold tolerance of P. f. martensii, and could be candidate markers potentially for further selective breeding.

Lysine acetylation plays a role in the allograft-induced stress response of the pearl oyster (Pinctada fucata martensii).

Implanting a spherical nucleus into a recipient oyster is a critical step in artificial pearl production using the pearl oyster Pinctada fucata martensii. However, little is known about the role of post-translational modifications (PTMs) in the response of the pearl oyster to this operation. Lysine acetylation, a highly conserved PTM, may be an essential adaptive strategy to manage multiple biotic or abiotic stresses. We conducted the first lysine acetylome analysis of the P. f. martensii gill 12 h after nucleus implantation, using tandem mass tags (TMT) labeling and Kac affinity enrichment. We identified 2443 acetylated sites in 1301 proteins, and 1511 sites on 895 proteins were quantitatively informative. We found 25 conserved motifs from all of the identified lysine sites, particularly motifs Kac H, Kac S, and Kac Y were strikingly conserved, of which Kac Y, Kac H, Y Kac, Kac K, Kac *K, Kac R, and Kac F which have been observed in other species and are therefore highly conserved. We identified 58 sites that were significantly differently acetylated in P. f. martensii in response to allograft (|fold change|>1.2, P-value ≤ 0.05); 38 newly acetylated and 20 deacetylated. According to GO functional analysis, subcellar location, and KOG classIfication, these proteins were divided into four categories: cytoskeleton, response to stimulus, metabolism, and other. The differentially acetylated proteins (DAPs) enriched pathways include aminoacyl-tRNA biosynthesis, salmonella infection, and longevity regulating pathway-worm-Caenorhabditis elegans (nematode). Parallel reaction-monitoring (PRM) validation of the differential acetylation of 10 randomly selected differentially acetylated sites from the acetylome analysis. These results indicated that our acetylome analysis results were sufficiently reliable and reproducible. These results provide an essential resource for in-depth exploration of the stress responses and adaptation mechanisms associated with lysine acetylation in marine invertebrates and P. f. martensii.

Comparative proteomics and transcriptomics illustrate the allograft-induced stress response in the pearl oyster (Pinctada fucata martensii).

Implantation of a spherical nucleus into a recipient oyster is a critical step in artificial pearl production. However, the molecular mechanisms underlying the response of the pearl oyster to this operation are poorly understood. In this research, we used transcriptomic and proteomic analyses to examine allograft-induced changes in gene/protein expression patterns in Pinctada fucata martensii 12 h after nucleus implantation. Transcriptome analysis identified 688 differential expression genes (DEGs) (FDR<0.01 and |fold change) ＞ 2). Using a 1.2-fold increase or decrease in protein expression as a benchmark for differentially expressed proteins (DEPs), 108 DEPs were reliably quantified, including 71 up-regulated proteins (DUPs) and 37 down-regulated proteins (DDPs). Further analysis revealed that the GO terms, including "cellular process", "biological regulation" and "metabolic process" were considerably enriched. In addition, the transcriptomics analysis showed that "Neuroactive ligand-receptor interaction", "NF-kappa B signaling pathway", "MAPK signaling pathway", "PI3K-Akt signaling pathway', "Toll-like receptor signaling pathway", and "Notch signaling pathway" were significantly enriched in DEGs. The proteomics analysis showed that "ECM-receptor interaction", "Human papillomavirus infection", and "PI3K-Akt signaling pathway" were significantly enriched in DEPs. The results indicate that these functions could play an important role in response to pear oyster stress at nucleus implantation. To assess the potential relevance of quantitative information between mRNA and proteins, using Ward's hierarchical clustering analysis clustered the protein/gene expression patterns across the experimental and control samples into six groups. To investigate the biological processes associated with the protein in each cluster, we identified the significantly enriched GO terms and KEGG pathways in the proteins in each cluster. Gene set enrichment analysis (GSEA) was used to reveal the potential protein or transcription pathways associated with the response to nuclear implantation. Thus, the study of P. f. martensii is essential to enhance our understanding of the molecular mechanisms involved in pearl biosynthesis and the biology of bivalve molluscs.

Exploring crucial molecular events in pearl oyster after pre-grafting conditioning by genome-wide bisulfite sequencing for DNA methylation analysis.

Pre-grafting condition is an important method to promote recovery from transplant surgery during pearl production. In the present study, we constructed two DNA methylomes from pearl oysters with and without conditioning to investigate the molecular mechanism of the pearl oyster Pinctada fucata martensii underlying the pre-grafting condition. A total of 4,594,997 and 4,930,813 methyl CG in the control (Con) and pre-grafting group (PT) were detected, resulting in the whole genome methylation profile and methylation pattern in P. f. martensii. Results reveal that the promoter, especially the CpG island-rich region, was more infrequently methylated than the gene function elements in P. f. martensii. A total of 51,957 differently methylated regions (DMRs) between Con and PT were obtained, including 3789 DMR in the promoter and 16,021 in the gene body. Based on gene ontology and pathway enrichment analyses, these DMRs were mainly related to "cellular process", "metabolic process", "Epstein-Barr virus infection", and "Fanconi anemia pathway". The methylation site in the promoter region may be associated with the promoter activity and transcription factor binding. These results help our understanding of the mechanism of pre-grafting condition, thereby providing key information in guiding to improve the conditioning methods for enhanced pearl oyster survival rate after transplantation.

Comprehensive analysis of the lncRNAs, mRNAs, and miRNAs implicated in the immune response of Pinctada fucata martensii to Vibrio parahaemolyticus.

Non-coding RNAs (ncRNAs) have been implicated in a variety of biological processes. However, most ncRNAs are of unknown function and are as-yet unannotated. The immune-related functions of ncRNAs in the pearl oyster Pinctada fucata martensii were explored based on transcriptomic differences in the expression levels of long non-coding RNAs (lncRNAs), microRNAs (miRNAs), and messenger RNAs (mRNAs) in the hemocytes of P.f. martensii after challenge by the pathogenic bacterium Vibrio parahaemolyticus. Across the challenged and control pearl oysters, 144 miRNAs and 14,571 lncRNAs were identified. In total, 13,375 ncRNAs were differentially expressed between the challenged and control pearl oysters; in the challenged pearl oysters as compared to the controls, 15 miRNAs and 5147 lncRNAs were upregulated, while 51 miRNAs and 8162 lncRNAs were downregulated. The sequencing results were validated using quantitative real-time polymerase chain reaction (qRT-PCR) analysis. GO and KEGG pathway analysis showed that genes targeted by the differentially expressed ncRNAs were associated with the vascular endothelial growth factor (VEGF) signaling pathway and the nuclear factor kappa-B (NF-κB) signaling pathway. An lncRNA-mRNA-miRNA network that was developed based on the transcriptomic results of this study suggested that lncRNAs may compete with miRNAs for mRNA binding sites. This study may provide a useful framework for the detection of additional novel ncRNAs, as well as new insights into the pathogenic mechanisms underlying the response of P.f. martensii to V. parahaemolyticus.

The byssal-producing glands and proteins of the silverlip pearl oyster Pinctada maxima (Jameson, 1901).

Pinctada maxima are most well known for their production of high-quality natural pearls. They also generate another natural material, the byssus, an adhesive thread critical for steadfast attachment underwater. Herein, P. maxima byssal threads were analysed via proteotranscriptomics to reveal 49 proteins. Further characterisation was undertaken on five highly expressed genes: glycine-rich thread protein (GRT; also known as PUF3), apfp1/perlucin-like protein (Pmfp1); peroxidase; thrombospondin 1, and Balbiani ring 3 (BR3), which showed localised tissue expression. The spatial distribution of GRT and Pmfp1 via immunodetection combined with histology helped to identify glandular regions of the foot that contribute to byssal thread production: the byssal gland, the duct gland, and two thread-forming glands of basophilic and acidophilic serous-like cells. This work advanced primary knowledge on the glands involved in the creation of byssal threads and the protein composition of the byssus for P. maxima, providing a platform for the design of marine biopolymers.

Isolation and characterization of a Vibrio sp. strain MA3 associated with mass mortalities of the pearl oyster Pinctada fucata.

In the summers of 2019 and 2020, a previously undescribed disease occurred in both juvenile and adult shellfish, causing mass mortalities in cultured pearl production, characterized by the major symptom of extreme atrophy of the soft tissues, including the mantle. However, the causative organism was uncertain. We isolated Vibrio sp. strain MA3 from the mantles of diseased pearl oysters Pinctada fucata. Analyses of 16S rRNA gene and DNA gyrase sequence homologies and its biochemical and morphological characteristics suggested that strain MA3 is a new strain of Vibrio alginolyticus. In addition, a hemolysin gene (Vhe1) of strain MA3 was detected as one of the virulence factors, and the complete sequence was determined. BLAST searches showed that Vhe1 shares 99.8% nucleotide sequence identity with Vibrio alginolyticus strain A056 lecithin-dependent hemolysin (ldh) gene, complete cds. Experimental infection of healthy oysters via injection with strain MA3 indicated it could cause high mortalities of the typically affected oysters from which the strain was isolated. These results suggest that the newly isolated Vibrio sp. strain MA3 is a putative causal agent of the recent disease outbreaks in Akoya pearl oysters.

Purification, crystallization and X-ray analysis of Pf-SCP (sarcoplasmic Ca-binding protein), related to storage and transport of calcium in mantle of Pinctada fucata.

Pf-SCP, a 21 kDa protein with two EF-hand motifs and a phosphorylation site, was identified from mantle tissue and binds to calcium ions and transports calcium components from cell to the shell of Pinctada fucata. To reveal the molecular basis of the calcium binding activity of Pf-SCP, we expressed the recombinant protein of full-length Pf-SCP in Escherichia coli. Recombinant Pf-SCP (rPf-SCP) purified by Ni affinity chromatography and size exclusion chromatography appeared as a single band on SDS-PAGE. The circular dichroism spectroscopy showed that the α-helix content decreased when rPf-SCP interacted with both calcium ions and calcium carbonate. Western blotting and immunostaining verified the Pf-SCP expression in the shell and localization most in the mantle epithelial cells. To further understand the structural and functional regulation of Pf-SCP by calcium ions and calcium carbonate, the crystallization experiments of rPf-SCP in the presence of calcium ions were performed. A crystal of rPf-SCP obtained in the presence of calcium ions diffracted X-rays up to a resolution of 1.8 Å. The space group of the crystal is C2 with unit cell parameters of a = 96.828 Å, b = 55.906 Å, c = 102.14 Å and ß = 90.009°, indicating that three molecules of rPf-SCP are contained in an asymmetric unit as estimated at the value of the Matthews coefficient. These results suggest that Pf-SCP may play a role in calcium ions transportation and shell mineralization by concentrating calcium ions inside the mantle epithelial cells and interacting with calcium carbonate molecules.