Pearl Oyster Pinctada Fucata Martensii Research Articles

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 1,096 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.

Effects of polyvinyl chloride microplastic on pearl oyster (Pinctada fucata martensii)

Microplastics (MPs) have recently attracted attention as an emerging pollutant. To investigate the effects of microplastics on the pearl oyster Pinctada fucata martensii (P. f. martensii) polyvinyl chloride microplastics (PVC-MPs) were used. We aimed to investigate the effect of PVC-MPs on the oxidation and reduction system by using the superoxide dismutase (SOD), malondialdehyde (MDA), reduced glutathione (GSH), and total antioxidant capacity (T-AOC); the immune system using acid phosphatase (ACP) and alkaline phosphatase (AKP); and the digestive system using amylase and protease as biomarkers. P. f. martensii were exposed to PVC-MPs at 0.15 mg/L, 1.5 mg/L, and 15 mg/L. Exposure to PVC-MPs increased the SOD activity and MDA content in the 15 mg/L dose group in 15 d compared to the control group. When compared with the control and other groups, MDA content and GSH content in the 0.15 mg/L group were higher on day 10. The T-AOC and AKP in all treatment groups were lower than the control group throughout the experiment, except in 1.5 mg/L and 15 mg/L groups in 15 d. The ACP activity increased with time except in the 0.15 mg/L group. The amylase and protease activity had an opposite pattern with amylase activity decreasing with time except in the 1.5 mg/L group in 15 d while protease increased with time except in the 0.15 mg/L group in 15 d group. The results indicate that PVC-MPs exposure induced oxidative stress, distorted the immune system, and digestive functions of P. f. martensii. The results help to understand the effects of MPs on the physiological responses of P. f. martensii.

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, β,β-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.

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. 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. 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. 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.

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.

Increased histone H3 acetylation inhibit the inflammatory response and activate the serum immunity of Pearl oyster Pinctada fucata martensii

To produce cultured pearls, a mantle graft with a nucleus is transplanted into a host pearl oyster, this process is called “transplantation”. The immune response of pearl oyster after transplantation is a major factor that leads to nucleus rejection and death. Butyrate is a histone deacetylase (HDAC) inhibitor which can inhibit the deacetylation process of histones and effectively reduce the inflammatory response. To clarify the function of histone acetylation in immune response after transplantation, butyrate (10 mmol/L) was used for the treatment of pearl oysters before transplantation. Results showed that the proportion of histone H3 acetylation of the hemocytes was significantly increased after butyrate treatment before transplantation (BH group) compared with the control group at 6–24 h. Transcriptome analysis showed that butyrate treatment activated the “lysosome”, inhibited cell migration and cell proliferation at 6 and 12 h, respectively, and activated the intracellular immune recognition response of pearl oyster at 24 h after transplantation. The apoptosis detection revealed no significant difference in the proportion of apoptotic cells between the control and BH group. Moreover, butyrate treatment increased the activity of some immune-related enzymes in the serum of pearl oyster 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.

Seasonal Variation of Biochemical Composition and Non-Volatile Taste Active Compounds in Pearl Oyster Pinctada fucata martensii from Two Selective Strains

Recently, a new pearl oyster Pinctada fucata martensii strain has begun to be cultured as seafood. In the present study, the seasonal variation (February and June) in biochemical composition and flavor compounds in two P. f. martensii strains (strain for pearl production was abbreviated to PP, and seafood was abbreviated to PE) were detected to compare the nutritional and flavor differences between them, and to provide a reference for the seasonal preference of consumers for eating P. f. martensii. The ratio of soft tissues in PE-Feb was significantly higher than that in PP-Feb (p < 0.05). The contents of ash, crude protein, and crude lipid were higher in PP than those in PE in the same season, while significantly higher contents of glycogen in the PE strain were observed compared to the PP strain in the same season (p < 0.05). The major amino acids (such as Glu and Asp) and PUFA (such as DHA and EPA) were almost the same in two P. f. martensii strains in the same season, while the contents of these nutrients were significantly higher in February compared to June (p < 0.05). Taurine content in PE-Feb was the highest (19.58 mg/g wet weight), followed by PP-Jun, PP-Feb, and PE-Jun. The umami and sweet FAA contents of the same P. f. martensii strain in February were significantly higher than those in June (p < 0.05). The AMP content in PP-Jun was the highest (64.17 mg/100 g wet weight), followed by PP-Feb, PE-Jun, and PE-Feb. Succinic acid was the major organic acid, and its content in February was significantly higher than in June (p < 0.05). The betaine content in PP-Feb was the highest (23.02 mg/g of wet weight), followed by PE-Feb (20.43 mg/g of wet weight), PP-Jun (16.28 mg/g of wet weight), and PE-Jun (12.33 mg/g of wet weight), and significant differences were observed among these four groups (p < 0.05). In conclusion, the edible P. f. martensii strain harvest in February is rich in protein, glycogen, PUFA (DHA and EPA), taurine, succinic acid, and betaine, which could provide healthy nutrition and a good flavor for humans.

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.

LncRNA7467 participated in shell biomineralization in pearl oyster Pinctada fucata martensii

Long noncoding RNAs (LncRNAs) are reported to have multiple roles in various biological processes, including epigenetic regulation, transcriptional regulation and post-transcriptional regulation. However, the current studies for lncRNAs are mainly limited to mammalian species, and the investigations of functional roles for lncRNAs in bivalve species are still lacking. Here, we identified a novel lncRNA named lncRNA7467 from pearl oyster Pinctada fucata martensii, located in chromosome 9 at 14962920–14963218 bp. Intriguingly, lncRNA7467 showed the highest expression level in mantle tissue among tested tissues, and responded to repair and regeneration of the injured shell. After knockdown gene expression by injecting lncRNA7467-specific double-stranded RNA, the inner surface of the prismatic layer changed significantly, and then the N44 gene was correspondingly inhibited. In addition, lncRNA7467 mainly distributed in the cytoplasm, and it mays primarily play a role in post-transcriptional regulation. These works indicated that lncRNA7467 was involved in the biomineralization process in pearl oyster 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.

Yellow Pigmentation Analysis of Pearl Oyster Pinctada fucata martensii Shells through in Situ Micro-Raman Spectroscopy

Yellow shell morphotypes of the pearl oyster Pinctada fucata martensii are rare and intriguing, whereas polyene pigments in mineralized shells from yellow pearl oysters have not been identified. An in situ micro-Raman spectroscopy and scanning electron microscopy investigation were performed on yellow and brown shell pearl oysters. Carotenoids or polyene pigments were found in both prismatic and nacreous layer from yellow pearl oysters. The peaks of pigments in the yellow prismatic surface were at wavenumbers of 1,280.8, 1,318.7, 1,146.1, and 1,577.2 cm–1, whereas those in the nacreous layer were at 1,135.3 and 1,530.6 cm–1. In brown pearl oysters, carotenoids related vibration peaks were not found in the prismatic layer, whereas slight spectra in the nacreous layer at wavenumbers of 1,136.3 and 1,531.4 cm–1 were attributed to polyene pigments. Pigments in the prismatic and nacreous layers of the yellow shell were not the same, and the former showed shorter polyene backbones and more complex molecular structures. This research provided characteristic Raman spectra for brown and yellow shell phenotypes of pearl oysters and can contribute to explore the connection between polyene pigments and biomineralization.

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.

Long non-coding RNA LncMPEG1 responds to multiple environmental stressors by affecting biomineralization in pearl oyster Pinctada fucata martensii

Marine environmental change directly affects bivalve growth and survival. Exoskeleton formation, the main energy dissipation in the physiological metabolism, typically reflects the body growth of the bivalve. However, how bivalves regulate the biomineralization of the exoskeleton under environmental stressors is not yet clear. Long non-coding RNA regulates various life processes through complex mechanisms in vertebrates and invertebrates. In this research, we cloned the complete sequence of a mantle-specific expressed long non-coding RNA (designated as LncMPEG1) from a pearl oyster, Pinctada fucata martensii. A quantitative real-time PCR analysis showed that LncMPEG1 expression was significantly high in early umbo larvae and juveniles, which would be the critical periods of shell development. LncMPEG1 was identified in the outer epithelium of the middle fold from the mantle edge, mantle pallial, and mantle center by using in situ hybridization. Additionally, the expression of LncMPEG1 was stimulated by shell damage, alien invasion, heat and cold temperature stress, and hypoxia stress. In the mantle, a decreased in LncMPEG1 expression was detected by RNA interference, which can cause the irregular growth of crystals on the inner surface of the prismatic layer and nacre in the shells. Therefore, we propose that LncMPEG1 could be a key regulator in biomineralization and responds to environmental stress in the mantle .

Molecular cloning, characterization, and expression of two 5-HTRs from the pearl oyster Pinctada fucata martensii

The receptors of serotonin, also known as 5-hydroxytryptamine receptor (5-HTR) can mediate regulatory metamorphosis processes in a variety of mollusks. Studying the mechanisms of metamorphosis of the pearl oyster is significant to elucidate breeding, resource recovery and marine pearl production. In this study, two 5-HTR genes from Pinctada fucata martensii (Pm5-HTR2 and Pm5-HTR4) were cloned. A total of 1623 bp open reading frame was identified in Pm5-HTR2, and a 1185 bp open reading frame was detected in Pm5-HTR4; these open reading frames encoded a 540-residue polypeptide and a 394-residue polypeptide, respectively. We also conducted a domain analysis, which indicated that Pm5-HTR2 and Pm5-HTR4 contained a seven-transmembrane domain and revealed that the receptors had high similarity to Crassostrea gigas 5-HTR2 (54.62%) and 5-HTR4 (66.23%). Sequence analysis demonstrated conserved advanced structure and motifs (the DRY/ERY and NPXXY motifs). The expression pattern analysis revealed high expression levels of Pm5-HTR2 and Pm5-HTR4 during the developmental stages. ISH analysis showed that Pm5-HTR2 was primarily expressed in the FE, B, T, EU, and EL stages and Pm5-HTR4 was mainly expressed in the FE, B, T, D, EU, and EL stages. These results suggest that 5-HTRs may play key roles in P. f. martensii larval metamorphosis.

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.