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.

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.

Effects of titanium dioxide nanoparticle exposure on the gut microbiota of pearl oyster (Pinctada fucata martensii).

With the advancement of nanotechnology and the growing utilization of nanomaterials, titanium dioxide (TiO2) has been released into aquatic environments, posing potential ecotoxicological risks to aquatic organisms. In this study, the toxicological effects of TiO2 nanoparticles were investigated on the intestinal health of pearl oyster (Pinctada fucata martensii). The pearl oysters were subjected to a 14-day exposure to 5-mg/L TiO2 nanoparticle, followed by a 7-day recovery period. Subsequently, the intestinal tissues were analyzed using 16S rDNA high-throughput sequencing. The results from LEfSe analysis revealed that TiO2 nanoparticle increased the susceptibility of pearl oysters to potential pathogenic bacteria infections. Additionally, the TiO2 nanoparticles led to alterations in the abundance of microbial communities in the gut of pearl oysters. Notable changes included a decrease in the relative abundance of Phaeobacter and Nautella, and an increase in the Actinobacteria, which could potentially impact the immune function of pearl oysters. The abundance of Firmicutes and Bacteroidetes, as well as the expression of genes related to energy metabolism (AMPK, PK, SCS-1, SCS-2, SCS-3), were down-regulated, suggesting that TiO2 nanoparticles exposure may affect the digestive and energy metabolic functions of pearl oysters. Furthermore, the short-term recovery of seven days did not fully restore these levels to normal. These findings provide crucial insights and serve as an important reference for understanding the toxic effects of TiO2 nanoparticles on bivalves.

The physiological responses to titanium dioxide nanoparticles exposure in pearl oysters (Pinctada fucata martensii).

To evaluate the physiological responses to titanium dioxide nanoparticles exposure in pearl oysters (Pinctada fucata martensii), pearl oysters were exposed for 14 days to different levels (0.05, 0.5, and 5 mg/L) of nano-TiO2 suspensions, while a control group did not undergo any nano-TiO2 treatment. And then recovery experiments were performed for 7 days without nano-TiO2 exposure. At days 1, 3, 7, 14, 17, and 21, hepatopancreatic tissue samples were collected and used to examine the activities of protease, amylase, lipase, catalase (CAT), glutathione peroxidase (GPx), superoxide dismutase (SOD), lysozyme (LYS), alkaline phosphatase (AKP), and acid phosphatase (ACP). The microstructure of the nacreous layer in shell was also analyzed by scanning electron microscopy. Results showed that pearl oysters exposed to 5 mg/L of TiO2 nanoparticles had significantly lower protease, amylase, and lipase activities and significantly higher CAT, SOD, GPx, LYS, ACP, and AKP activities than control pearl oysters did even after 7-day recovery (P-values <0.05). Pearl oysters exposed to 0.5 mg/L or 0.05 mg/L of TiO2 nanoparticles had lower protease, amylase, and lipase activities and higher CAT, SOD, GPx, LYS, ACP, and AKP activities than control pearl oysters did during the exposure period. After 7-day recovery, no significant differences in protease, lipase, SOD, GPx, CAT, ACP, AKP, or LYS activities were observed between pearl oysters exposed to 0.05 mg/L of TiO2 nanoparticles and control pearl oysters (P-values >0.05). In the period from day 7 to day 14, indistinct and irregular nacreous layer crystal structure in shell was observed. This study demonstrates that TiO2 nanoparticles exposure influences the levels of digestion, immune function, oxidative stress, and biomineralization in pearl oysters, which can be partially and weakly alleviated by short-term recovery. These findings contribute to understanding the mechanisms of action of TiO2 nanoparticles in bivalves. However, studies should evaluate whether a longer recovery period can restore to their normal levels in the future.

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.

Phosphoproteomics changes due to allograft-induced stress responses of Pinctada fucata martensii.

Protein phosphorylation modifications are post-translational modifications (PTMs) that play important roles in signal transduction and immune regulation. Implanting a spherical nucleus into a recipient shellfish is critical in marine pearl aquaculture. Protein phosphorylation may be important in the immune responses of Pinctada fucata martensii after nucleus implantation, but their involvement in regulation remains unclear. Here, phosphoproteomics of P. f. martensii gill tissues was conducted 12 h after nuclear implantation using label-free data-independent acquisition (DIA) with LC-MS/MS. Among the 4024 phosphorylated peptides with quantitative information, 181 were up-regulated and 148 were down-regulated. Functional enrichment analysis of these differentially expressed phosphorylated proteins (DEPPs) revealed significant enrichment in functions related to membrane trafficking, exosomes, cytoskeleton, and signal transduction mechanisms. Further, 16 conserved motifs were identified among the DEPPs, including the RSphP, SphP, RSphA, RSphE, PTphP, and ATphP motifs that were significantly conserved, and which may be related to specific kinase recognition. Parallel response monitoring (PRM) analysis validated the abundances of 12 DEPPs from the proteomics, indicating that the phosphoproteomics analyses were robust. 12 DEPPs were selected from the proteomics results through Quantitative real-time PCR (qPCR) technology, and verification analysis was conducted at the gene level. The study suggests that kinases such as MAPKs, Akt, and CK2 may regulate the phosphorylation of related proteins following nuclear implantation. Furthermore, the important signaling pathways of Rap 1, IL-17A, and NF-κB, which are influenced by phosphorylated or dephosphorylated proteins, are found to be involved in this response. Overall, this study revealed the protein phosphorylation responses after nucleus implantation in P. f. martensii, helping to elucidate the characteristics and mechanisms of immune regulation responses in P. f. martensii, in addition to promoting a further understanding of protein phosphorylation modification functions in P. f. martensii.

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.

Transcriptome and exosome proteome analyses provide insights into the mantle exosome involved in nacre color formation of pearl oyster Pinctada fucata martensii.

Color polymorphisms in molluscan shells play an important economic in the aquaculture industry. Among bivalves, shell color diversity can reflect properties such as growth rate and tolerance. In pearl oysters, the nacre color of the donor is closely related to the pearl color. Numerous genes and proteins involved in nacre color formation have been identified within the exosomes of the mantle. In this study, we analyzed the carotenoids present in the mantle of gold- and silver-lipped pearl oysters, identifying capsanthin and xanthophyll as crucial pigments contributing to coloration. Transcriptome analysis of the mantle revealed several differentially expressed genes (DEGs) involved in color formation, including ferric-chelate reductase, mantle genes, and larval shell matrix proteins. We also isolated and identified exosomes from the mantles of both gold- and silver-lipped strains of the pearl oyster Pinctada fucata martensii, revealing the extracellular transition mechanism of coloration-related proteins. From these exosomes, we obtained a total of 1223 proteins, with 126 differentially expressed proteins (DEPs) identified. These proteins include those associated with carotenoid metabolism and Fe(III) metabolism, such as apolipoproteins, scavenger receptor proteins, ß,ß-carotene-15,15'-dioxygenase, ferritin, and ferritin heavy chains. This study may provide a new perspective on the nacre color formation process and the pathways involved in deposition within the pearl oyster P. f. martensii.

Whole-genome bisulfite sequencing reveals the function of DNA methylation in the allotransplantation immunity of pearl oysters.

Introduction: In the pearl culture industry, a major challenge is the overactive immunological response in pearl oysters resulting from allotransplantation, leading to shell-bead rejection and death. To better understand the molecular mechanisms of postoperative recovery and the regulatory role of DNA methylation in gene expression, we analyzed the changes in DNA methylation levels after allotransplantation in pearl oyster Pinctada fucata martensii, and elucidated the regulatory function of DNA methylation in promoter activity of nicotinic acetylcholine receptor (nAChR) gene. Methods: We constructed nine DNA methylomes at different time points after allotransplantation and used bisulfite genomic sequencing PCR technology (BSP) to verify the methylation status in the promoter of nAChR. We performed Dual luciferase assays to determine the effect of the dense methylation region in the promoter on transcriptional activity and used DNA pull-down and mass spectrometry analysis to assess the capability of transcription factor binding with the dense methylation region. Result: The DNA methylomes reveal that CG-type methylation is predominant, with a trend opposite to non-CG-type methylation. Promoters, particularly CpG island-rich regions, were less frequently methylated than gene function elements. We identified 5,679 to 7,945 differentially methylated genes (DMGs) in the gene body, and 2,146 to 3,385 DMGs in the promoter at each time point compared to the pre-grafting group. Gene ontology and pathway enrichment analyses showed that these DMGs were mainly associated with "cellular process", "Membrane", "Epstein-Barr virus infection", "Notch signaling pathway", "Fanconi anemia pathway", and "Nucleotide excision repair". Our study also found that the DNA methylation patterns of the promoter region of nAChR gene were consistent with the DNA methylomics data. We further demonstrated that the dense methylation region in the promoter of nAChR affects transcriptional activity, and that the methylation status in the promoter modulates the binding of different transcription factors, particularly transcriptional repressors. Conclusion: These findings enhance our understanding of the immune response and regulation mechanism induced by DNA methylation in pearl oysters after allotransplantation.

Immunomodulatory effects of one novel microRNA miR-63 in pearl oyster Pinctada fucata martensii.

Novel microRNA miR-63 (novel-miR-63) from pearl oyster Pinctada fucata martensii (Pm-novel-miR-63) is a species-specific miRNA. Our previous research has shown that the expression of Pm-novel-miR-63 was significantly downregulated at 24 h after nucleus transplantation. In this study, we analyzed the function and regulatory role of Pm-novel-miR-63 in the immune response of pearl oysters. The results showed that Pm-novel-miR-63 expression increased after the stimulation of pathogen associated molecular patterns at 6-12 h, and the activity of immune and antioxidant enzymes in the serum decreased after Pm-novel-miR-63 overexpression. Transcriptome analysis revealed that Pm-novel-miR-63 participated in regulating transplantation immunity through the Notch and mRNA surveillance signaling pathways. Target prediction and dual luciferase analysis revealed that Pm-GDP-FucTP, Pm-CysLTR2, and Pm-RLR were the target genes of Pm-novel-miR-63. These results suggested that Pm-novel-miR-63 participated in regulating the immune response in pearl oysters and can serve as a new interference target to reasonably control excessive immune rejection in pearl culture.

miR-10a-3p Participates in Nacre Formation in the Pearl Oyster Pinctada fucata martensii by Targeting NPY.

MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression via the recognition of their target messenger RNAs. MiR-10a-3p plays an important role in the process of ossification. In this study, we obtained the precursor sequence of miR-10a-3p in the pearl oyster Pinctada fucata martensii (Pm-miR-10a-3p) and verified its sequence by miR-RACE technology, and detected its expression level in the mantle tissues of the pearl oyster P. f. martensii. Pm-nAChRsα and Pm-NPY were identified as the potential target genes of Pm-miR-10a-3p. After the over-expression of Pm-miR-10a-3p, the target genes Pm-nAChRsα and Pm-NPY were downregulated, and the nacre microstructure became disordered. The Pm-miR-10a-3p mimic obviously inhibited the luciferase activity of the 3' untranslated region of the Pm-NPY gene. When the interaction site was mutated, the inhibitory effect disappeared. Our results suggested that Pm-miR-10a-3p participates in nacre formation in P. f. martensii by targeting Pm-NPY. This study can expand our understanding of the mechanism of biomineralization in pearl oysters.

Identification and comparative analysis of miRNA transcriptomes after allograft and xenograft transplantation in Pinctada fucata martensii.

Effective immune regulation after transplantation during pearl production is crucial for the cultivation of high-quality pearls. MicroRNAs (miRNAs) play an important role in a variety of physiological processes. To understand the regulatory rules of miRNAs after transplantation in Pinctada funcata martensii, we constructed 13 miRNA transcriptomes, including the control group (Con), allograft (Al), and xenograft (Xe) transplantation at six time points (6, 12, and 24 h and 3, 6, and 12 days), in which the xenografted mantle tissue was from Pinctada maxima. We identified 159 differentially expressed miRNAs (DEMs) and found that these DEMs showed high expression at 12 h, 24 h, and 3 days after transplantation. A total of 130 DEMs, such as Let-7, were present in the Al and Xe groups; miR-34 and 16 other DEMs were specifically present in the Al group; miR-216b and 13 other DEMs were specifically present in the Xe group. Compared with the Con group, the target genes of DEMs in the Al group were significantly enriched in protein complex, cytoskeleton, and macromolecular complex, and the Xe group was significantly enriched in ribonucleoside metabolic process, nucleoside binding, and cell division. Compared with the Al group, the target genes in the Xe group were significantly enriched in response to DNA damage stimulation. Overall, multiple pathways associated with cellular activity were enriched in higher numbers of genes in the Xe group than in the Al group. These findings enriched the information on immune regulatory mechanisms at the expression level of miRNAs in P. f. martensii after transplantation.

Histone deacetylase inhibitor butyrate inhibits the cellular immunity and increases the serum immunity of pearl oyster Pinctada fucata martensii.

Histone acetylation is a dynamic epigenetic modification and sensitive to the changes in extracellular environment. Butyrate, a histone deacetylase inhibitor, can inhibit the deacetylation process of histones. In this study, we found that the acetylation level of H3 was enhanced at 12 h after lipopolysaccharide (LPS) stimulation and increased at 6 h after combining treatment with LPS and butyrate in pearl oyster Pinctada fucata martensii. Transcriptome analysis indicated that butyrate counter-regulated 29.95%-36.35% of the genes repressed by LPS, and these genes were mainly enriched in the "cell proliferation" and "Notch signaling pathway". Meanwhile, butyrate inhibited the up-regulation of 31.54%-54.96% of the genes induced by LPS, and these genes were mainly enriched in "Notch signaling pathway", "cell proliferation", "NF-kappa B signaling pathway", "TNF signaling pathway", "apoptosis", "NOD-like receptor signaling pathway", "RIG-I-like receptor signaling pathway" and "cytosolic DNA-sensing pathway". Gene expression analysis showed that butyrate downregulated most of cell proliferation, immune-related genes effected by LPS. The activities of LAP, LYS, ACP, ALP, and GSH-Px were up-regulated at 6 h after combining treatment with LPS and butyrate, suggesting that butyrate could activate serum immune-related enzymes in pearl oyster. These results can improve our understanding of the function of histone deacetylase in the immune response of pearl oyster and provide references for an in-depth study of the functions of histone deacetylase in mollusks.

Integrated transcriptomic and metabolomic analysis sheds new light on adaptation of Pinctada fucata martensii to short-term hypoxic stress.

Analyses of the transcriptome and metabolome were conducted to clarify alterations of key genes and metabolites in pearl oysters following exposure to short-term hypoxic treatment. We totally detected 209 DEGs between the control and hypoxia groups. Enrichment analysis indicated the enrichment of GO terms including "oxidation-reduction process", "ECM organization", "chaperone cofactor-dependent protein refolding", and "ECM-receptor interaction" KEGG pathway by the DEGs. In addition, between the two groups, a total of 28 SDMs were identified, which were implicated in 13 metabolic pathways, such as "phenylalanine metabolism", "D-amino acid metabolism", and "aminoacyl-tRNA biosynthesis". Results suggest that pearl oysters are exposed to oxidative stress and apoptosis under short-term hypoxia. Also, pearl oysters might adapt to short-term hypoxic treatment by increasing antioxidant activity, modulating immune and biomineralization activities, maintaining protein homeostasis, and reorganizing the cytoskeleton. The results of our study help unveil the mechanisms by which pearl oysters respond adaptively to short-term hypoxia.

Genome-wide association study analysis to resolve the key regulatory mechanism of biomineralization in Pinctada fucata martensii.

Biomineralization-controlled exo-/endoskeleton growth contributes to body growth and body size diversity. Molluscan shells undergo ectopic biomineralization to form the exoskeleton and biocalcified "pearl" involved in invading defence. Notably, exo-/endoskeletons have a common ancestral origin, but their regulation and body growth are largely unknown. This study employed the pearl oyster, Pinctada fucata marntensii, a widely used experimental model for biomineralization in invertebrates, to perform whole-genome resequencing of 878 individuals from wild and breeding populations. This study characterized the genetic architecture of biomineralization-controlled growth and ectopic biomineralization. The insulin-like growth factor (IGF) endocrine signal interacted with ancient single-copy transcription factors to form the regulatory network. Moreover, the "cross-phylum" regulation of key long noncoding RNA (lncRNA) in bivalves and mammals indicated the conserved genetic and epigenetic regulation in exo-/endoskeleton growth. Thyroid hormone signal and apoptosis regulation in pearl oysters affected ectopic biomineralization in pearl oyster. These findings provide insights into the mechanism underlying the evolution and regulation of biomineralization in exo-/endoskeleton animals and ectopic biomineralization.

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.

Gene cloning and functional study of PmKSPI from Pinctada fucata martensii.

Kunitz-type serine protease inhibitors (KSPI) are a family of serine protease inhibitors (SPIs) and are extensively found in animals, plants, and microbes. SPI can inhibit proteases that may be harmful or unwanted to its cells. Here, a four-domain Kunitz-type SPI, PmKSPI, was cloned by RACE in the pearl oyster Pinctada fucata martensii. The full-length cDNA sequence of PmKSPI was 1318 bp, including the 5' UTR (25 bp), the 3' UTR (96 bp) and ORF (1197 bp). Homology analysis indicated that PmKSPI had the highest resemblance (30.14%) with its homolog in Crassostrea gigas. Phylogenetic analysis revealed that PmKSPI clustered with homologs in other molluscs. We found that PmKSPI mRNA expression in P. f. martensii was distributed in all six tissues, with the highest level in the mantle, and almost no expression in other tissues. After PAMPs challenge, expression of PmKSPI mRNA in the mantle was significantly up-regulated. The recombinant protein rPmKSPI significantly inhibited the growth of 5 kinds of Gram-negative bacteria but had little effect on Gram-positive bacterial activity. Transmission electron microscopy showed that plasmolysis occurred in two Gram-negative bacteria species when treated with rPmKSPI. rPmKSPI may thus have a bactericidal effect by destroying the bacterial cell membrane or cell walls and releasing its contents. Therefore, our results suggest that PmKSPI is tightly associated with the immunological defence of P. f. martensii.

Transcriptome analysis reveals the diverse response of pearl oyster Pinctada fucata martensii after different PAMP stimulation.

Bivalves have evolved effective strategies to combat different pathogens in the environment. They rely on innate immunity to deal with the invasion of various bacteria, viruses, and other microorganisms. However, the molecular mechanisms underlying the responses remain largely unknown. Herein, we constructed 21 transcriptomes of the hemocytes after lipopolysaccharide (LPS), peptidoglycan (PGN) and polyinosinic-polycytidylic acid (poly(I:C)) stimulation to investigate the molecular mechanisms underlying adaptations and plastic responses to different pathogen-related molecular patterns (PAMPs) in pearl oyster Pinctada fucata martensii. Transcriptome analysis revealed 1986-3427 responsive genes enriched in the major immune and cell cycle-related pathways at different times after PAMP stimulation, and the expression patterns of genes under these pathways are complex and diverse. Moreover, "lysosomes" were enriched 6 h after LPS and PGN stimulation, while "peroxisomes" were only enriched in poly(I:C) group. These results suggest different response strategies of pearl oyster to different PAMPs. Furthermore, we identified 261 pattern-recognition receptors (PRRs) including 4 retinoic acid-inducible gene I-like receptors, 38 NOD-like receptors, 83 Toll-like receptors, and 136 C-type lectins in the genome of P. f. martensii. The diverse expression patterns of these PRRs after different PAMP stimulation indicated that pearl oyster evolved complex and specific recognition systems due to tandem repeat and diverse domain combination, which may help pearl oyster cope with the different pathogens in the environment. The present study improved our understanding of the molecular response of pearl oyster to different PAMP stimulation.

Lipidomic insights into the immune response and pearl formation in transplanted pearl oyster Pinctada fucata martensii.

During pearl culture, the excess immune responses may induce nucleus rejection and death of pearl oysters after transplantation. To better understand the immune response and pearl formation, lipidomic analysis was applied to investigate changes in the serum lipid profile of pearl oyster Pinctada fucata martensii following transplantation. In total, 296 lipid species were identified by absolute quantitation. During wound healing, the content of TG and DG initially increased and then decreased after 3 days of transplantation with no significant differences, while the level of C22:6 decreased significantly on days 1 and 3. In the early stages of transplantation, sphingosine was upregulated, whereas PC and PUFAs were downregulated in transplanted pearl oyster. PI was upregulated during pearl sac development stages. GP and LC-PUFA levels were upregulated during pearl formation stage. In order to identify enriched metabolic pathways, pathway enrichment analysis was conducted. Five metabolic pathways were found significantly enriched, namely glycosylphosphatidylinositol-anchor biosynthesis, glycerophospholipid metabolism, alpha-linolenic acid metabolism, linoleic acid metabolism and arachidonic acid metabolism. Herein, results suggested that the lipids involved in immune response, pearl sac maturation, and pearl formation in the host pearl oyster after transplantation, which might lead to an improvement in the survival rate and pearl quality of transplanted pearl oyster.

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.