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.

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.

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.

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.

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.

Immunomodulatory effects of decitabine in pearl oyster Pinctada fucata martensii.

Decitabine (DAC), an inhibitor of DNA methyltransferase, is typically used to reverse DNA methylation and is considered an epigenetic modifying drug. DNA methylation is crucial to the regulation of gene expression without altering genetic information. Our previous research showed that the DNA methylation levels of many immune-related genes changed after the pre-grafting condition in pearl production. In the present study, we evaluated the DNA methylation level and analyzed transcriptome, enzyme, and antimicrobial activities after DAC treatment to evaluate the effect of DAC on DNA methylation and immune system of pearl oyster Pinctada fucata martensii. Results showed that DAC significantly decreased the level of global DNA methylation in the hemocytes of the pearl oysters. Transcriptome analysis obtained 577 differentially expressed genes (DEGs) between the control and DAC treatment group. The DEGs were mainly enriched in the following pathways: "Relaxin signaling pathway," "Cytosolic DNA-sensing pathway," "Platelet activation," and "Peroxisome," and related genes were overexpressed after DAC treatment. DAC treatment resulted in a substantial increase in the levels of serum superoxide dismutase, interleukin-17, phenol oxidase, tumor necrosis factor, and antimicrobial activity, compared with the control. These results suggested that DAC can alter DNA methylation level, activate immune-related genes, and improve the level of humoral immunity in pearl oysters, thereby increasing our understanding of the mechanism underlying DNA methylation in immune regulation.

Globular C1q domain-containing protein from Pinctada fucata martensii participates in the immune defense process.

The globular C1q domain-containing (C1qDC) protein can recognize a variety of ligands, such as pathogen-associated molecular patterns, and plays an important role in the innate immune response. Our previous studies showed that a novel globular C1q domain-containing protein (PmC1qDC-1) is involved in the damage repair process of pearl oyster shells. However, the function of PmC1qDC-1 in pearl oyster innate immunity remains unknown. In the present study, the high-level structural analysis showed that PmC1qDC-1 was a spherical structure composed of 10 strands and was similar to the AiC1qDC-2 of bay scallop (Argopecten irradians). In situ hybridization indicated that PmC1qDC-1 had strong fluorescence signal in gills. Furthermore, the mRNA expression of PmC1qDC-1 was highly induced at 6-48 h in gill after lipopolysaccharide, peptidoglycan and polyinosinic-polycytidylic acid stimulation. Additionally, we obtained the recombinant protein of PmC1qDC-1 (rPmC1qDC-1) and found that rPmC1qDC-1 had antibacterial activity against Gram-negative (i.e., Pseudomonas aeruginosa, Vibrio parahaemolyticus, Escherichia coli, and Aeromonas hydrophila) and Gram-positive (i.e., Staphylococcus aureus and Bacillus subtilis) bacteria. These results indicated that PmC1qDC-1 might play an important role in the immune response against bacteria and viruses. This study provides clues for further studying the immune defense of Pinctada fucata martensii against pathogens and exploring the evolution of the classic pathway of complement system.

Characterization of cyclin dependent kinase-7 and its differential response to grafting challenge in the black shell colored selected line of pearl oyster Pinctada fucata martensii.

Cyclin dependent kinase-7 (Cdk-7) is a protein kinase associated with regulating the cell cycle, cell differentiation and proliferation, apoptosis and inflammatory response. This study characterized the full cDNA sequence of Cdk-7 in Pinctada fucata martensii (PmCdk-7). A full length sequence of 1473bp with an open reading frame (ORF) of 915bp and encodes a 304aa, 5'-UTR of 58bp and a 3'-UTR of 500bp was obtained. The construed amino acid sequence of PmCdk-7 comprised of a Serine/Threonine protein kinases, catalytic (S_TKc) domain with a protein kinases ATP-binding region signature (14-38aa) and the serine/Threonine protein kinases active-site signature (129-141aa) within the domain. Tissue distribution analysis revealed a high relative mRNA expression of PmCdk-7 within haemocytes. Following the insertion operation (grafting), the relative expression levels of PmCdk-7 in the haemocyte was expressed differentially among the studied groups; the black shell colored selected line (BS) and the control group (CG). High expression was recorded between 12 h and 5d with a peak at 3d suggesting a heightened level of DNA replication and inflammatory response during the pearl-sac formation and this expression was higher in BS than CS showcasing, the heightened immune capacity of BS to grafting operation. Immune stimulation experiment with bacterial endotoxin and a viral mimic revealed PmCdk-7 response to pathogenic stress. The results from our study showed that PmCdk-7 performs a vital function during the cell cycle by aiding DNA replication and also aid response to inflammations generated due to the incision from the grafting operation and long exposure to immune-stimulants (pathogens).

Evolutionary and functional analysis of MyD88 genes in pearl oyster Pinctada fucata martensii.

Myeloid differentiation factor 88 (MyD88) is an adapter protein that links toll-like receptor and interleukin 1 receptor-mediated signal transduction. In this study, we identified 20 MyD88 genes from eight mollusk genomes and found that MyD88 was expanded in bivalves. This expansion tends to be tandem duplication. Phylogenetic analysis suggested that the tandem duplication of MyD88 was formed before bivalve differentiation. All of the identified MyD88 contained both of death domain (DD) and toll/interleukin-1 receptor (TIR) domain, and 13 mollusks MyD88 have low complexity regions (LCRs), which were not found in the MyD88 from humans and zebrafish. The genomic structure showed that most of the mollusk MyD88 (14 of 19) contained five conserved introns, four of which were found in humans and zebrafish. Furthermore, the cDNA full length of PfmMyD88-2 (one of the two identified MyD88 in Pincatada fucata martensii) was obtained with 1591 bp, including 260 bp of 5'UTR, 257 bp of 3'UTR, and 1077 bp of open reading frame encoding 358 amino acids. Quantitative real-time PCR analysis demonstrated that PfmMyD88-2 mRNA was widely expressed in all detected tissues. The highest expression level was in the gills and followed by hepatopancreas and feet. After lipopolysaccharide stimulation, PfmMyD88-2 expression level increased and reached the highest level at 12 h and then gradually declined to the normal level. Over-expression of PfmMyD88-2 in HEK293T increased the luciferase activity of the pNF-κB-Luc reporter. We also identified that PfmmiR-4047 could regulate the expression of PfmMyD88-2. These results help us elucidate the mechanism underlying mollusk immune response.

Survival, retention rate and immunity of the black shell colored stocks of pearl oyster Pinctada fucata martensii after grafting operation.

We have developed a black shell colored selected line observed to have higher survival ability. In this study, to understand its immune capacity, total carotenoid content (TCC) of the black shell colored line (BG) and the control group (CG) were compared. Survival and retention rates, immunity and antioxidant capacity of BG were compared relative to CG at different times after grafting operation. The results showed that BG had significantly larger TCC than CG (P < 0.05). BG had significantly higher survival and retention rates than CG on days 7, 30 and 360 after grafting (P < 0.05). On days 360, BG had significantly larger pearl thickness than CG (P < 0.05). BG exhibited increased ACP, AKP, SOD, CAT, TAOC and LZ activity than the CG on 0 h, 12 h, 1 d, 3 d, 5 d, 7 d and 30 d after grafting. BG had higher expression levels of Fascin, SOD, CDK-7, CDAP-1, IRAK-1, α2m, GST-1, TRAF-3 and Caspase-2 than CG. The results suggested that BG had higher immune competence and pearl production performances, which is promising to improve pearl quality and production.

Response of pearl oyster Pinctada fucata martensii to allograft-induced stress from lipid metabolism.

The pearl oyster, Pinctada fucata martensii, produces high-quality pearls. During pearl production, excess immune and inflammatory response after transplantation will lead to nucleus rejection, pearl sac formation failure, and death of the host pearl oyster. The hemocyte transcriptome and fatty acid (FA) contents in the adductor muscle before and after transplantation were analyzed to investigate the response of pearl oyster P. f. martensii to allograft-induced stress from lipid metabolism. The hemocyte transcriptome analysis detected 193 lipid metabolism-related genes, such as the elongation of very long-chain FA protein 5, acyl-CoA 6-desaturase, cytochrome P450, phospholipase A2, glycerol-3-phosphate O-acyltransferase, and prostaglandin-H2 d-isomerase. Pathway enrichment analyses revealed that these genes were mainly involved in the "biosynthesis of unsaturated FAs," "FA biosynthesis," "ARA metabolism," and "glycerolipid metabolism." An analysis of FA contents in the adductor muscle indicated no significant difference in the contents of lauric acid, myristic acid, pentadecanoic acid, palmitic acid, palmitoleic acid, heptadecanoic acid, stearic acid, oleic acid, vaccenic acid, linoleic acid, arachidic acid, α-linolenic acid, eicosadienoic acid, docosadienoic acid, and 11,14,17-eicosatrienoic acid. However, ARA, DHA, and EPA in the adductor muscle after transplantation were significantly greater than those processed without grafting surgery. These results suggest that pearl oysters require more polyunsaturated FAs (PUFAs) to regulate their inflammatory and immune response after transplantation. However, their ability to biosynthesize unsaturated FAs is limited. Given these results, the addition of PUFA-containing diets or selection of a line with strong ability to biosynthesize unsaturated FAs may be valuable for pearl oyster recovery after transplantation.

Developmental characteristics of pearl oyster Pinctada fucata martensii: insight into key molecular events related to shell formation, settlement and metamorphosis.

BACKGROUND: Marine bivalves undergo complex development processes, such as shell morphology conversion and changes of anatomy and life habits. In this study, the transcriptomes of pearl oyster Pinctada fucata martensii and Pacific oyster Crassostrea gigas at different development stages were analyzed to determine the key molecular events related to shell formation, settlement and metamorphosis. RESULT: According to the shell matrix proteome, biomineralization-related genes exhibited a consensus expression model with the critical stages of shell formation. Differential expression analysis of P. f. martensii, revealed the negative regulation and feedback of extracellular matrixs as well as growth factor pathways involved in shell formation of larvae, similar to that in C. gigas. Furthermore, neuroendocrine pathways in hormone receptors, neurotransmitters and neuropeptide receptors were involved in shell formation, settlement and metamorphosis. CONCLUSION: Our research demonstrated the main clusters of regulation elements related to shell formation, settlement and metamorphosis. The regulation of shell formation and metamorphosis could be coupled forming the neuroendocrine-biomineralization crosstalk in metamorphosis. These findings could provide new insights into the regulation in bivalve development.

Effect of vitamin D3 on immunity and antioxidant capacity of pearl oyster Pinctada fucata martensii after transplantation: Insights from LC-MS-based metabolomics analysis.

Postoperative care is a critical step of pearl culture that ultimately determines culture success. To determine the effect of dietary vitamin D3 (VD3) levels on immunity and antioxidant capacity of pearl oyster Pinctada fucata martensii during postoperative care and explore the mechanisms behind this phenomenon, five isonitrogenous and isolipidic experimental diets were formulated by adding different levels of dietary VD3 (0, 500, 1000, 3000, and 10000 IU/kg), and the diets were fed to five experimental groups (EG1, EG2, EG3, EG4, and EG5) in turn and cultured indoors. The control group (CG) was cultured in the natural sea. Pearl oysters that were 1.5 years old were subjected to nucleus insertion. After culturing for 30 days, EG3 exhibited significantly higher survival rates than those in CG and EG5 (P < 0.05). Moreover, EG3 exhibited the highest activities of alkaline phosphatase, acid phosphatase, catalase, superoxide dismutase, and lysozyme. However, EG5 achieved the highest activities of glutathione peroxidase. Metabolomics-based profiling of pearl oysters fed with high levels of dietary VD3 (EG5) and optimum levels of dietary VD3 (EG3) revealed 76 significantly differential metabolites (SDMs) (VIP > 1 and P < 0.05). Pathway analysis indicated that SDMs were involved in 21 pathways. Furthermore, integrated key metabolic pathway analysis suggested that pearl oysters in EG5 regulated the pentose phosphate pathway, glutathione metabolism, sphingolipid metabolism, and arachidonic acid metabolism in response to stress generated from excessive VD3. These findings had significant implications on strengthening the future development and application of VD3 in aquaculture of pearl oyster P. f. martensii.

The global effects of PmRunt co-located and co-expressed with a lincRNA lncRunt in pearl oyster Pinctada fucata martensii.

Runt related transcription factors as trans-acting elements play critical roles in the developmental control of cell fate, hematopoiesis, bone formation and cancers. In previous study, the homologue of runt related transcription factor PmRunt has been identified from pearl oyster Pinctada fucata martensii and considered to play an important role in nacre formation. In this study, we used the same samples to perform RNA-seq to detect the global effects after the decrease of PmRunt expression. The transcription levels of several nacre shell matrix protein (NSMP) genes were significantly changed and the potential compensatory effect could happen internal gene families. Downregulation of PmRunt could also influence the biosynthesis of NSMPs through affecting amino acid metabolism, translation, protein processing and export. The inhibition of PmRunt also possibly affected the expression of caspases, IAPs and C1qs that related to apoptosis and immune. In addition, PmRunt highly expressed at 12 h and 12 d after transplantation in hemolymph, which was corresponded to transplantation immunity immune response and the morphology of pearl sac, suggested the cross-talk of biomineralization-immune regulation in hemocytes. Furthermore, a lincRNA (LncRunt) that co-located with PmRunt was identified and showed a significantly relative expression with PmRunt, which suggested the potential regulation. Therefore, these findings provided new idea to find the regulation targets of runt-related transcription factors and offers evidence of lncRNAs in potential biomineralization-immune regulation.

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

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

Evolution and function analysis of interleukin-17 gene from Pinctada fucata martensii.

Interleukin-17 (IL-17) is a proinflammatory cytokine that plays an important role in immune responses. In this study, we identified 57 IL-17 genes from the genomes of six marine invertebrates, including Pinctada fucata martensii, Crassostrea gigas, Lottia gigantea, Capitella teleta, Mizuhopecten yessoensis, and Mytilus galloprovincialis. Phylogenetic analysis showed that all invertebrate IL-17 genes were clustered into one group, implying that invertebrate IL-17 evolved from one common ancestral gene. From the extron-intron analysis, we found many intronless IL-17 genes in mollusks, which may be caused by retroposition. Tissue and development transcriptomic analysis showed that the expression of PmIL-17 was tissue and developmental stage-specific. Moreover, we cloned the full length of the IL-17-2 gene from P. f. martensii (PmIL-17-2) and explored its function in the immune response. The full-length cDNA of PmIL-17-2 is 719 bp, containing an open reading frame of 564 bp, a 5' -untranslated region (UTR) of 31 bp, and a 3' -UTR of 124 bp with a 30 bp poly (A) tail. PmIL-17-2 had a strong response to lipopolysaccharide (LPS), indicating that the PmIL-17-2 participates in innate immune responses. In situ hybridization of hemocytes showed that PmIL-17-2 was mainly produced by granulosa cells, and the number of the stained granulosa increased after LPS stimulation. These results lay the foundation for the research of IL-17 family in marine invertebrates.

Adaptive response of pearl oyster Pinctada fucata martensii to low water temperature stress.

The pearl oyster Pinctada fucata martensii is a warm-water shellfish that is sensitive to cold environments. To investigate its potential adaptation to low-temperature stress, the selected line (SL) and based population (BP) were sampled to undergo transcriptome sequence. Results of transcriptome analysis showed 572 significant differentially expressed genes. The typical HSP70 and HSP40 exhibited the polar expression model in the two groups. Meanwhile, the related genes that involved in energy release mediated by oxidative phosphorylation and the biosynthesis of unsaturated fatty acid were increased in the SL. The apparent enrichment of different expressed genes in amino acid metabolism indicated that the small molecule system with amino acids was one of the main regulator for low-temperature stress. The different expressions of immune-related and lysosome protein encoding genes also reflected the variation of immunity in the two groups and indicated that it could affect the adaptation ability in different temperature. In addition, the similar trends of different expression of typical genes between two groups were obtained by using RNA-seq and qRT-PCR. These results suggested that multi-system adjustments are involved in the processes of low water temperature stress in pearl oyster, providing insights into the response systems of shellfish to acclimatise with ambient environment change.

Identification of EGFR in pearl oyster (Pinctada fucata martensii) and correlation analysis of its expression and growth traits.

Marine pearl production is directly influenced by the growth speed of Pinctada fucata martensii. However, the slow growth rate of this organism remains the main challenge in aquaculture production. Epidermal growth factor receptor (EGFR), an important receptor of tyrosine kinases in animals, plays versatile functions in development, growth and tissue regeneration. In this study, we described the characteristic and function of an EGFR gene identified from P. f. martensii (PmEGFR). PmEGFR possesses a typical EGFR structure and is expressed in all studied tissues, with the highest expression level in adductor muscle. PmEGFR expression level is significantly higher in the fast-growing group than that in the slow-growing one. Correlation analysis represents that shell height and shell weight show positive correlation with PmEGFR expression (p < 0.05), and total weight and tissue weight exhibit positive correlation with it (p < 0.01). This study indicates that PmEGFR is a valuable functional gene associated with growth traits.

Pax3 Gene Regulated Melanin Synthesis by Tyrosinase Pathway in Pteria penguin.

The paired-box 3 (Pax3) is a transcription factor and it plays an important part in melanin synthesis. In this study, a new Pax3 gene was identified from Pteria penguin (Röding, 1798) (P. penguin) by RACE-PCR (rapid-amplification of cDNA ends-polymerase chain reaction) and its effect on melanin synthesis was deliberated by RNA interference (RNAi). The cDNA of PpPax3 was 2250 bp long, containing an open reading fragment of 1365 bp encoding 455 amino acids. Amino acid alignment and phylogenetic tree showed PpPax3 shared the highest (69.2%) identity with Pax3 of Mizuhopecten yessoensis. Tissue expression profile showed that PpPax3 had the highest expression in mantle, a nacre-formation related tissue. The PpPax3 silencing significantly inhibited the expression of PpPax3, PpMitf, PpTyr and PpCdk2, genes involved in Tyr-mediated melanin synthesis, but had no effect on PpCreb2 and an increase effect on PpBcl2. Furthermore, the PpPax3 knockdown obviously decreased the tyrosinase activity, the total content of eumelanin and the proportion of PDCA (pyrrole-2,3-dicarboxylic acid) in eumelanin, consistent with influence of tyrosinase (Tyr) knockdown. These data indicated that PpPax3 played an important regulating role in melanin synthesis by Tyr pathway in P. penguin.

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

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