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

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

Multiple functions of thymosin ß4 in the pearl oyster Pinctada fucata suggest its multiple potential roles in artificial pearl culture.

Thymosin ß4 is a multifunctional protein in vertebrates that participates in physiological processes, such as wound healing, immune response, cell proliferation and migration. We assessed the multifarious roles of this small peptide in Pinctada fucata, an oyster commonly used in pearl culture in China. Our results showed that when P. fucata was challenged by bacterial pathogens or LPS, the relative expression level of Pfthymosin ß4 mRNA was significantly up-regulated, suggesting its involvement in immune response of the animal. Recombinant Pfthymosin ß4 (rPfthymosin ß4) was produced and showed in vitro different antibacterial activities against different pathogenic bacteria; the inhibitory effect of rPfthymosin ß4 on bacterial growth was relatively stronger in the broth culture than agar culture. The overexpression of Pfthymosin ß4 in Escherichia coli BL21(DE3) cells could improve their resistance to Cu2+, Zn2+, Cd2+, and H2O2, suggesting that Pfthymosin ß4 is likely involved with antioxidant. rPfthymosin ß4 also significantly promoted the proliferation and migration of mouse aortic vascular smooth muscle cells as indicated by MTT assay and cell scratch assay, respectively. In addition, chemically synthesized or recombinant Pfthymosin ß4 could transiently increase the circulating total hemocytes counts but down-regulated by RNAi in P. fucata. Taking together above results and previous studies suggested that Pfthymosin ß4 is potentially able to promote wound healing through enhancing antibacterial activity and antioxidant capacity, promotion of cell proliferation and migration, and increase of circulating hemocytes in P. fucata due to nucleus implantation injury. Thus, the future of recombinant Pfthymosin ß4 should be promising in the culture of pearls in P. fucata.

Transcriptome analysis of the immune reaction of the pearl oyster Pinctada fucata to xenograft from Pinctada maxima.

The pearl oyster Pinctada maxima exhibits great difficulty to culture pearls through nuclear insertion with an allograft, but it is easy for P. fucata to culture pearls after allografting. If P. fucata could be used as a surrogate mother to culture P. maxima pearls, it would benefit the pearl culture industry of P. maxima. However, this is blocked by the immune rejection of P. fucata against P. maxima mantle grafts. In this study, the immune responses of P. fucata hemocyte to allograft and xenograft were investigated after transplantation by transcriptome analysis. In total, 107.93 Gb clean reads were produced and assembled using the reference genome of P. fucata. Gene Ontology Term enrichment and KEGG enrichment analyses indicated that apoptosis, hippo signaling pathway, oxidation-reduction, MAPK signaling pathway, ribosome, protein processing in endoplasmic reticulum, purine metabolism, NF-kappa B signaling pathway, oxidative phosphorylation, Ras signaling pathway, and ubiquitin mediated proteolysis were involved in response to transplantation. Many genes related to oxidation-reduction reactions, the MAPK signaling pathway, and apoptosis were identified by comparison of the allograft group and the xenograft group at 0 h, 6 h, 12 h, 24 h, 48 h, 72 h, and 96 h post-transplantation. Among them, the expression levels of NADH dehydrogenase, succinate dehydrogenase and other dehydrogenases were increased significantly in the xenograft groups compared with allograft groups at 0 h post transplantation, indicating that a respiratory burst of neutrophils occurred immediately after xenograft transplantation. Additionally, HSP70 was highly expressed from 0 h to 96 h in the xenograft groups, indicating an oyster immune response to the xenograft. The genes enriched in the ribosome and hippo-signaling pathways were also identified, and expression patterns of these DEGs were different as compared between transplantation and control groups. Finally, altered expression levels of 10 randomly selected immune-related DEGs were confirmed by quantitative real-time PCR. These results indicated that oxidation-reduction is likely the key factor responsible for immune rejection to transplantation. The findings should provide some new insight into the molecular mechanism of immune rejection of the host against xenograft, and thus benefit to development of immunosuppressive reagents to facilitate effective xenograft pearling.

Serum immune response of pearl oyster Pinctada fucata to xenografts and allografts.

The mantle piece from the donor pearl oyster would be rejected by the immune system of recipient oyster in pearl culture practice, especially in the case that the donor and receptor are different species. Thus, investigation of the immune response of recipient oyster to grafted mantle pieces, particularly to xenografts, is of importance in creating xenograft transplantation technology for pearl culture industry. The humoral immune responses of P. fucata to allograft (mantle piece of P. fucata) and xenografts (mantle pieces of P. maxima and P. margaritifera, respectively) were studied in this paper. The oysters receiving no transplantations were served as the control group. The serum was collected from recipient P. fucata at 1 d, 2 d, 3 d, 4 d, 5 d, 7 d, 9 d, 11 d, 13 d, and 15 d, respectively after transplantation, and the serum antibacterial activity, lysozyme activity (LZM), alkaline phosphatase (AKP), acid phosphatase (ACP), total antioxidant capacity (TAC), and agglutination to rabbit red blood cells were investigated. The result indicated that serum of both the experimental groups and the control group can agglutinate rabbit red blood cells, with variation between groups and between time points, respectively. The antibacterial activity in the experimental group was significantly higher than that in the control group at 2-4 d, but lower at 5-11 d and returned back to normal at 15 d, with significant differences among experimental groups (P < 0.05). The LZM in the experimental group was significantly higher than that in the control group at 3-7 d, with significant differences in bacteriolytic activity among various groups (P < 0.05). Both the ACP and AKP activity levels in the experimental groups were higher than those in the control group at 2-9 d, with significant differences among various groups at 3-9 d (P < 0.05). The TAC level in the experimental groups was higher than that in the control group at 1-7 d, with significant differences among various groups at 4-7 d (P < 0.05). The above results indicated that all of the humoral immune factors investigated showed immune responses to both allografts and xenografts, with no specific to any of them. Thus, it is necessary to further screen immune rejection factors specific to xenografts, including cellular immune components.

High carotenoids content can enhance resistance of selected Pinctada fucata families to high temperature stress.

Carotenoids are a class of natural antioxidants widely found in aquatic, and they have significant effects on the growth, survival, and immunity of these organisms. To investigate the mechanisms of carotenoids in high temperature resistance, we observed the immune response of selected pearl oyster Pinctada fucata (Akoya pearl oyster) families with different carotenoids contents to high temperature stress. The results indicated that the survival rate (SR) of P. fucata decreased significantly with increase in temperature from 26 °C to 34 °C and with the decrease of total carotenoids content (TCC); when the TCC was higher, the SR tended to be higher. TCC and total antioxidant capacity (TAC) decreased significantly at 30 °C with increasing stress time. Correlation analysis indicated that TAC was positively and linearly correlated with TCC, and SR was S-type correlated with TCC and TAC. Immune analysis indicated that levels of superoxide dismutase (SOD), catalase (CAT), and malondialdehyde (MDA) in selected families (with higher TCC) under temperature stress (at 30 °C) were generally significantly lower than in the control group (with lowest TCC) and from 0 to 96 h, the levels of each of these substances varied significantly. Levels of SOD, CAT, and MDA within each family first rose from 0 to 3 h, then decreased to their lowest point after 24 h, and then rose again to their highest levels at 96 h. When TCC was higher, the levels of SOD, CAT, and MDA tended to be lower. These findings indicated that carotenoids play an important role in improving survival rates of P. fucata under high temperature stress by enhancing animals' antioxidant system, and could serve as an index for breeding stress-resistant lines in selective breeding practices.

Differentially expressed immune-related genes in hemocytes of the pearl oyster Pinctada fucata against allograft identified by transcriptome analysis.

The pearl oyster Pinctada fucata is commonly cultured for marine pearls in China. To culture pearls, a mantle piece from a donor pearl oyster is grafted with a nucleus into a receptor. This transplanted mantle piece may be rejected by the immune system of the recipient oyster, thus reducing the success of transplantation. However, there have been limited studies about the oyster's immune defense against allograft. In this study, hemocyte transcriptome analysis was performed to detect the immune responses to allograft in P. fucata at 0 h and 48 h after a transplant. The sequencing reaction produced 92.5 million reads that were mapped against the reference genome sequences of P. fucata. The Gene Ontology (GO) annotation and the Kyoto Encyclopedia of Genes and Genomes (KEGG) were used to identify all immune-related differentially expressed genes (DEGs). Compared with patterns at 0 h, a total of 798 DEGs were identified, including 410 up-regulated and 388 down-regulated genes at 48 h. The expression levels of interleukin receptor and toll-like receptor in hemocytes were increased significantly 48 h post-transplant, indicating that the oyster immune response was induced. Finally, altered levels of 18 randomly selected immune-related DEGs were confirmed by quantitative real-time PCR (qRT-PCR). Our results provide the basis for further analysis of the immune rejection of allotransplantation.

A microstructural study of individual nacre tablet of Pinctada maxima.

Nacre tablets from the shell of Pinctada maxima were studied with SEM, TEM and STEM. The systematic nanolath morphology on the (001) surface of nacre tablets was observed after acidic etching and mechanical polishing. The nanolaths were along the [100] crystallographic orientation of aragonite crystal. The (010) and (100) cross section surfaces of the nacre tablets showed nanolath and nanograin morphologies, respectively, which was consistent with [100] crystallographic orientation of nanolath on the (001) surface. Sheet-like defects with low mass density were observed on the (001) plane inside nacre tablets and were considered to be the cause of nanolath morphology revealed on the surfaces by acidic etching and mechanical polishing. On the other hand, large block [110] twins that divide the nacre tablets into two sectors were identified. The implication of these twins on the understanding to the crystallization mechanism of nacre tablets was discussed.

Model of shrimp pond-mediated spatiotemporal dynamic distribution of antibiotic resistance genes in the mangrove habitat of a subtropical gulf.

Aquacultures are the main reason for the environmental selection of antibiotic resistance genes (ARGs), resulting in the enrichment of ARGs. As a filter, a marine mangrove ecosystem can reduce antimicrobial resistance (AMR) or eliminate ARGs; however, its elimination mechanism remains unclear. This study investigated the spatiotemporal dynamic distribution of ARGs in two different types of mangrove habitats (shrimp ponds and virgin forests), within a subtropical gulf located in the Beibu Gulf, China, during dry and wet seasons by using metagenomics and real time quantitative polymerase chain reaction (RT-qPCR) analysis. As the key environmental factors, sulfide, salinity, and mobile genetic elements significantly were found to contribute to ARGs distribution, respectively. Wet and dry seasons influenced the dispersal of ARGs but did not affect the microbial community structure. Three potential biomarkers, TEM-116, smeD, and smeE, played key roles in seasonal differences. The key different genes in the biological relevance of absolute abundance were demonstrated by RT-qPCR. Co-occurrence network analysis indicated that high-abundance ARGs were distributed in a modular manner. For the first time, a risk index weighted by risk rank (RIR) was proposed and used to quantify the human risk of ARGs in the mangrove metagenome. The shrimp ponds during the wet season showed the highest RIR detected. In addition to offering a perspective on reducing AMR in mangrove wetlands, this study constructed the first spatiotemporal dynamic model of ARGs in the Beibu Gulf, China and contributed to revealing the global spread of ARGs. Meanwhile, this study proposes a new pipeline for assessing the risk of ARGs, while also exploring the concept of "One Health."

Sulfur metabolism in subtropical marine mangrove sediments fundamentally differs from other habitats as revealed by SMDB.

Shotgun metagenome sequencing provides the opportunity to recover underexplored rare populations and identify difficult-to-elucidate biochemical pathways. However, information on sulfur genes, including their sequences, is scattered in public databases. Here, we introduce SMDB ( https://smdb.gxu.edu.cn/ )-a manually curated database of sulfur genes based on an in-depth review of the scientific literature and orthology database. The SMDB contained a total of 175 genes and covered 11 sulfur metabolism processes with 395,737 representative sequences affiliated with 110 phyla and 2340 genera of bacteria/archaea. The SMDB was applied to characterize the sulfur cycle from five habitats and compared the microbial diversity of mangrove sediments with that of other habitats. The structure and composition of microorganism communities and sulfur genes were significantly different among the five habitats. Our results show that microorganism alpha diversity in mangrove sediments was significantly higher than in other habitats. Genes involved in dissimilatory sulfate reduction were abundant in subtropical marine mangroves and deep-sea sediments. The neutral community model results showed that microbial dispersal was higher in the marine mangrove ecosystem than in others habitats. The Flavilitoribacter of sulfur-metabolizing microorganism becomes a reliable biomarker in the five habitats. SMDB will assist researchers to analyze genes of sulfur cycle from the metagenomic efficiently.

The association of efficacy with PD-1/PD-L1 inhibition and tumor mutational burden in advanced nonsmall cell lung cancer: A PRISMA-guided literature review and meta-analysis.

BACKGROUND: Tumor mutation burden (TMB) has been reported to emerge as an independent biomarker of response to identify patients who would achieve benefit from immune checkpoint inhibitors. However, it still remains controversy that whether TMB can be a robust biomarker of response to programmed death-1 (PD-1)/programmed death-ligand 1 (PD-L1) inhibition. We performed this meta-analysis to assess the relationship between TMB and the efficacy with PD-1/PD-L1 inhibition in advanced nonsmall cell lung cancer (NSCLC). METHODS: Following the recommendations of the PRISMA statement, electronic databases literature search was done on the published articles till March 2021, including Pubmed, Embase, and Cochrane library databases. Studies were selected that focused on comparing the efficacy of TMB-high group and TMB-low group in NSCLC patients received with immune checkpoint inhibitors. Meta-analysis Revman 5.3 software was utilized to calculate the pooled outcomes. RESULTS: A systematic literature search was conducted 8 articles, including 11 comparative articles. Findings of our studies shown that patients with TMB-high group was associated with better clinical outcomes than TMB-low group, including progression-free survival (odds ratio [OR], 0.38; 95% confidence interval [CI], 0.29-0.49; P < .00001), complete response (OR, 4.71; 95% CI, 2.32-9.57; P < .0001), durable clinical benefit (OR, 3.76; 95% CI, 2.38-5.96; P < .00001) and the objective response rate (OR, 3.14; 95% CI, 1.83-5.37; P < .0001). While, it failed to predict overall survival benefits (OR, 0.74; 95% CI, 0.45-1.20; P = .22). CONCLUSIONS: Our study found that NSCLC with high TMB who benefit from immunotherapy. The findings suggest that TMB could associated with a greater predictive power of response. Possibly a more TMB-oriented prediction model might gain more benefits from PD-1/PD-L1 inhibitors.

Identification and Characterization of miRNAs and Their Predicted mRNAs in the Larval Development of Pearl Oyster Pinctada fucata.

As an important economic shellfish, the pearl oyster, Pinctada fucata, and its larvae are an ideal model for studying molecular mechanisms of larval development in invertebrates. Larval development directly affects the quantity and quality of pearl oysters. MicroRNAs (miRNAs) may play important roles in development, but the effects of miRNA expression on P. fucata early development remain unknown. In this study, miRNA and mRNA transcriptomics of seven different P. fucata developmental stages were analyzed using Illumina RNA sequencing. A total of 329 miRNAs, including 87 known miRNAs and 242 novel miRNAs, and 33,550 unigenes, including 26,333 known genes and 7217 predicted new genes, were identified in these stages. A cluster analysis showed that the difference in the numbers of miRNAs was greatest between fertilized eggs and trochophores. In addition, the integrated mRNA transcriptome was used to predict target genes for differentially expressed miRNAs between adjacent developmental stages, and the target genes were subjected to a gene ontology enrichment analysis. Using the gene ontology annotation, 100 different expressed genes and 95 differentially expressed miRNAs were identified as part of larval development regulation. Real-time PCR was used to identify eight mRNAs and three miRNAs related to larval development. The present findings will be helpful for further clarifying the regulatory mechanisms of miRNA in invertebrate larval development.

cDNA cloning and characterization of a new member of the tumor necrosis factor receptor family gene from scallop, Chlamys farreri.

Tumor necrosis factors receptor (TNFR) is a superfamily of proteins derived mainly from vertebrates. It plays significant role in diverse physiological and pathological events such as inflammation, apoptosis, autoimmunity and organogenesis. The gene of a new member of TNFR family, designated as CfTNFR2, was cloned and characterized from scallop, Chlamys farreri. The full-length cDNA of CfTNFR2 consisted of 1,501 nucleotides with a poly (A) tail, encoding a polypeptide of 378 amino acids with the estimated molecular mass of 42.70 kDa and predicted isoelectric point of 4.79. The characteristic motifs of the TNFR family proteins, such as three TNFR homology domains (also called CRD domains) and a death domain, were identified in CfTNFR2. Significantly, the deduced amino acid sequence of CfTNFR2 was closely homologous with mammalian osteoprotegerins showing approximately 37% identity. However, it shared only 11% amino acids identity with CfTNFR1, another TNFR homolog previously identified from the candidate scallop species, indicating that CfTNFR2 is a new molluscan TNFR protein. The spatial expression of CfTNFR2 in the tissues of the healthy and bacterial challenged scallops was detected by real-time PCR. CfTNFR2 mRNA was expressed constitutively in all selected tissues such as mantle, gill, gonad, hepatopancreas and hemocyte, among which gill and mantle displayed comparatively higher expression levels. Upon Listonella anguillarum challenge, CfTNFR2 expression was found to be remarkably up-regulated, especially in the tissues of gill (15.9-fold) and mantle (8.0-fold). The results reveal that CfTNFR2 is a constitutive and inducible acute-phase protein apparently involved in immune defense. The presence of CfTNFR2 (present study) and CfTNFR1 (previously identified from our lab) encouraged us to suggest that multiple members of TNFR family exist in mollusk, and the findings would help us to get better understanding on the evolutionary origin and functions of this protein family in mollusks.

The mitochondrial genome of Erronea caurica (Cypraeidae).

The mitochondrial genome of Erronea caurica from the South China Sea has been determined (GenBank Accession No. MT522622), which was the second report of mitochondrial genome in the superfamily Cypraeoidea. It is 16,053 bp long and consists of 21 tRNA genes, 2 rRNA genes, 13 protein-coding genes, and 1 control region. As previously reported mitochondrial genome in Cypraeoidea, all protein-coding genes of E. caurica use a typical start codon (ATN) and a complete stop codon (TAA or TAG). Phylogenetic tree demonstrated that E. caurica belongs to the family Cypraeoidea and closer to the superfamily Tonnoidea.

SEM observation of early shell formation and expression of biomineralization-related genes during larval development in the pearl oyster Pinctada fucata.

Shell formation of Pinctada fucata in larval development stages plays a crucial role in their survival. Scanning electron microscopy (SEM) was used to observe the morphological changes during larval development. We found that the early shell forms soon after enlargement of the blastopore at the anterior end of the trochophore stage and the complete shell forms in the spats stage, required for metamorphosis of P. fucata. Based on our transcriptome data of trochophore, D-shaped, umbonal, eyespots and spats stages, including the whole process of shell formation, 93 differentially expressed biomineralization-related genes were identified, of which 25 genes were unique to P. fucata, 30 were identical to genes in pacific oyster, and the remaining genes were annotated to other species. Two-dimensional and three-dimensional principal components analysis (PCA) showed that different developmental stages were significantly different, with the early two stages exhibiting a larger difference compared with the next stages. The 93 genes were sorted into 20 trends with three trends being significantly enriched: an initial increase and then a decrease, a monotonic decrease, and a monotonic increase. Gene expression patterns changed with regulatory function during shell formation. Almost all the biomineralization-related genes were up-regulated in the D-shaped stage, but only five genes were up-regulated in that stage but down-regulated in the remaining stages. There were also 11 genes up-regulated in the last three stages, and a total of 24 genes showed high expression level during the last four stages. The 55 genes selected for shell incision experiment sorted into five trends and most genes presented differences in expression between 24 h and other time points. Considering all these results, there is a correlation with the morphological change and the expression of biomineralization-related genes during larval developmental stages, especially of differently expressed genes.

Identification and Differential Expression of Biomineralization Genes in the Mantle of Pearl Oyster Pinctada fucata.

A series of proteins are involved in shell formation of the pearl oyster Pinctada fucata, but the involved mechanisms and the relative expression levels of these proteins have not been elucidated. In this study, we sequenced and characterized the transcriptome of P. fucata mantle tissue. A total of 100,679 unique transcripts were assembled, 43687 Unigenes were annotated, and 48654 CDSs were determined. Of these, GO annotated 16353 Unigenes, COG defined 11585 unigenes into 25 categories, and KEGG sorted 25053 unigenes into 258 pathways. In total, 67 biomineralization-related genes were identified, of which 23 genes were newly described in P. fucata. These genes included ones that expressed shell matrix proteins, regulatory factors, and uncharacterized genes. Differential expression of these 67 genes and 9 other biomineralization-related genes was confirmed using qPCR. Of the 8 nacreous layer-related genes, MSI60 (774.00) was expressed at a much higher level than the others. KRMP2-4 and MSI31 were the most highly expressed of the 13 prismatic layer-related genes and KRMP2 was expressed at nearly 10000 times of the level of the 18S gene. For genes related to both layers, shematrin 2 (3977.84), nacrein (2404.75), PFMG 10 (2113.93), and PFMG 4 (1015.89) were highly expressed, and ferritin-like protein (877.54) and PFMG 8 (516.48) were highly expressed among the 16 undefined genes. The expression levels of regulation factors were generally low, and the highest level was 324.09 (EF-hand) and the lowest occurred in the BMP and wnt families. The expression levels of the prismatic matrix proteins were much higher than those of nacreous ones, consistent with a thicker prismatic layer. MSI60 and nacrein are likely the main components of the nacreous layer, and KRMP2-4, MSI31, shematrin 2, and PFMG 10 gene products are the main components of the prismatic layer. This is the first report of transient expression levels of a large number of biomineralization-related genes at the same time in mantle tissue of P. fucata. These findings provide a novel perspective to understand the molecular mechanisms of shell formation and will be beneficial to genetic improvement of P. fucata for the production of high-quality pearls as well.

Molecular characterization and expression analysis of chitinase from the pearl oyster Pinctada fucata.

Chitinase is an enzyme that plays an important role in the chitin metabolism of a wide range of organisms. However, the function of chitinase in the pearl oyster Pinctada fucata is yet to be determined. In this study, a chitinase gene (named PfChi1) was cloned from P. fucata and its expression profiles were investigated. The full-length cDNA of PfChi1 was 2743bp and consisted of a 2187-bp open reading frame encoding 728 amino acid residues, a 47-bp 5'-untranslated region (UTR), and a 509-bp 3'-UTR. Similar to other known chitinases, the PfChi1 protein is composed of an N-terminal leading signal peptide, a catalytic domain, a linker region, and a C-terminal chitin-binding domain. The results of qRT-PCR showed that PfChi1 was expressed in a wide range of tissues with relatively high levels in the mantle, muscle, gill, and gonad, and relatively low levels in hemocytes, the intestine, and the digestive gland (P<0.05). In situ hybridization showed that PfChi1 was mainly expressed in the mantle edge, particularly in the outer epithelial cells of the inner fold, whereas few hybridization signals were detected in the inner epithelial cells of the middle fold. A shell damage experiment indicated that PfChi1 transcript levels were up-regulated significantly (P<0.05) at 24h after shell damage and decreased gradually thereafter, followed by shell regeneration, indicating that PfChi1 is involved in shell formation. In addition, PfChi1 expression was higher in trochophore larvae than in other developmental stages (P<0.05), indicating a possible association with the formation of prodissoconch shells. To the best of our knowledge, this study is the first to report the potential biomineralization function of a chitinase in P. fucata.

Characterization of transcriptome and identification of biomineralization genes in winged pearl oyster (Pteria penguin) mantle tissue.

The winged pearl oyster Pteria penguin is a commercially important marine pearl oyster species, with pearls that are quite different from those of other pearl oysters. Among such species, mantle tissue is the main organ responsible for shell and pearl formation, a biomineralization process that is regulated by a series of genes, most of which remain unknown. In this study, we sequenced and characterized the transcriptome of P. penguin mantle tissue using the HiSeq 2000 sequencing platform. A total of 93,204 unique transcripts were assembled from 51,580,076 quality reads, with a mean length of 608bp, and 40,974 unigenes were annotated. The sequence data enabled the identification of 79,702 potential single nucleotide polymorphism loci and 4345 putative simple sequence repeat loci. A total of 71 unique transcripts were identified homologous to known biomineralization genes, including mantle gene, nacrein, pearlin, pif, chitinase, and shematrin, of which only 3 were previously reported in P. penguin. qPCR analysis indicated that 10 randomly selected biomineralization genes were much more highly expressed in mantle tissue than in the other tissues. In addition, 30 unique sequences were identified as highly expressed, with FPKM values of >3000, and most of these were biomineralization-related genes, including shematrin family genes, a jacalin-related lectin synthesis gene, calponin-2, and paramyosin. These findings will be useful for future studies of biomineralization in P. penguin, as well as in other Pteria species.

Molecular characterization and expression analysis of the myostatin gene and its association with growth traits in Noble scallop (Chlamys nobilis).

Myostatin (MSTN), also called growth and differentiation factor-8 (GDF-8), is a member of the transforming growth factor-ß (TGF-ß) superfamily and an inhibitor of muscle differentiation and growth. In this report, we identified and characterized a MSTN gene (CnMSTN) from the scallop Chlamys nobilis. The open reading frame of CnMSTN was 1374bp in length, encoding 457 amino acids. The structure of CnMSTN included a putative signal peptide, a TGF-ß propeptide domain, and a conserved TGF-ß domain. Phylogenetic analysis showed that the CnMSTN gene was clustered in the same subgroup with the MSTN gene found in Mollusca. Quantitative real-time PCR showed that the CnMSTN gene was widely expressed in all tissues tested, with the highest expression level observed in the adductor muscle. Six single nucleotide polymorphisms (SNPs) were identified in the promoter region, but no SNP was detected in the exon regions. Association analysis showed that SNP g.-579A/C had significant effects on body mass, soft-tissue mass, and adductor muscle mass. The CC and AC genotypes of g.-579A/C had significantly higher growth trait values than that of genotype AA (P<0.05). These results suggest that CnMSTN could be used as a candidate gene for the selective breeding of C. nobilis.

Genomic structure of the α-amylase gene in the pearl oyster Pinctada fucata and its expression in response to salinity and food concentration.

Amylase is one of the most important digestive enzymes for phytophagous animals. In this study, the cDNA, genomic DNA, and promoter region of the α-amylase gene of the pearl oyster Pinctada fucata were cloned by using reverse transcription-polymerase chain reaction (RT-PCR), rapid amplification of cDNA ends, and genome-walking methods. The full-length cDNA sequence was 1704bp long and consisted of a 5'-untranslated region of 17bp, a 3'-untranslated region of 118bp, and a 1569-bp open reading frame encoding a 522-aa polypeptide with a 20-aa signal peptide. Sequence alignment revealed that P. fucata α-amylase (Pfamy) shared the highest identity (91.6%) with Pinctada maxima. The phylogenetic tree showed that it was closely related to P. maxima, based on the amino acid sequences. The genomic DNA was 10850bp and contained nine exons, eight introns, and a promoter region of 3932bp. Several transcriptional factors such as GATA-1, AP-1, and SP1 were predicted in the promoter region. Quantitative RT-PCR assay indicated that the relative expression level of Pfamy was significantly higher in the digestive gland than in other tissues (gonad, gills, muscle, and mantle) (P<0.001). The expression level at salinity 27‰ was significantly higher than that at other salinities (P<0.05). Expression reached a minimum when the algal food concentration was 16×10(4)cells/mL, which was significantly lower than the level observed at 8×10(4)cells/mL and 20×10(4) cells/mL (P<0.05). Our findings provide a genetic basis for further research on Pfamy activity and will facilitate studies on the growth mechanisms and genetic improvement of the pearl oyster P. fucata.

The complete nucleotide sequence of Malabar grouper (Epinephelus malabaricus) mitochondrial genome.

In this study, we reported the complete mitochondrial DNA sequence of the Epinephelus malabaricus. The full-length of the mitochondrial genome consisted of a 16,423 bp fragment, with the base composition of A (28.70%), T (26.55%), G (15.92%) and C (28.83%). It contained 2 rRNA genes, 13 protein-coding genes, 22 tRNA genes and a major non-coding control region (D-loop region). The composition and order of these genes were identical to most of other vertebrates. All the protein initiation codons were ATG, except that COX1 began with GTG, ATP-6 and ND6 was not determined, respectively. The complete mitogenome of the Epinephelus malabaricus provided an important data set for the study in genetic mechanism of the hybridization.