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Tributyltin (TBT) is extensively applied as an antifouling mediator, and it is well-known as an endocrine disrupting chemical. However, it remains elusive whether TBT can affect the development of pubertal Leydig cells (LCs), as a part of its endocrine mechanism of action. In this study, male Sprague Dawley rats (35-day-old) orally received TBT (0, 1, or 5 mg/kg) for 24 days. Immature LCs (ILCs) were separated from 35-day-old rats, and the cells were exposed to TBT at various concentrations (0, 0.05, 0.5, 1, or 5 μM) for 24 h. In vivo TBT treatment decreased the number of LCs and inhibited androgen production (2.92 ± 0.44, 1.16 ± 0.29, and 0.67 ± 0.10 ng/ml after treatment with TBT at 0, 1, and 5 mg/kg, respectively). In vitro TBT treatment reduced androgen production, cell viability, and cell cycle progression, while increased the levels of reactive oxygen species (ROS) along with subsequent apoptosis, particularly at the concentration of 5 μM. According to in vitro and in vivo findings, TBT downregulated the expression levels of genes that could control steroidogenesis (Hsd17b3, Scarb1, Hsd3b1, and Star), and decreased protein levels due to potential reduction of NR5A1 (Nr5a1). In summary, TBT inhibited cholesterol transport and activities of certain steroidogenic enzymes, thereby leading to the reduction of androgen production.

Estuaries are resistome hotspots owing to resistome accumulation and propagation at these locations from surrounding rivers, yet the large-scale biogeographic pattern of resistome, especially biocide and metal resistance genes (BMRGs) and its driving mechanisms in estuarine waters remains to be elucidated. Here, a metagenomics-based approach was firstly used to investigate resistome and mobilome profiles in waters from 30 subtropical estuaries, South China. The Pearl River estuaries had a higher diversity and abundance of antibiotic resistance genes (ARGs), BMRGs, and mobile genetic elements (MGEs) when compared with estuaries from east and west regions. Genes resistant to multiple antibiotics, metals, and biocides were the most abundant gene types in the resistome. The abundance of MGEs (e.g., intI1, IS91, and tnpA) was highly associated with the total abundance of resistance genes, suggesting their utility as potential indicators for quantitative estimations of the resistome contamination. Further, MGEs contributed more than bacterial communities in shaping the resistome in subtropical estuaries. Physicochemical factors (e.g., pH) regulated MGE composition and stochastic assembly, which mediated the co-selection of ARGs and BMRGs via horizontal gene transfer. Our findings have important implications and provide a reference on the management of ARGs and BMRGs in subtropical estuarine ecosystems.

In this study, shell growth, shell microstructure, and expression levels of shell matrix protein genes (aspein, n16, and nacrein) that play a key role in the CaCO3 crystal polymorphism (calcite and aragonite) of the shell were investigated in the pearl oyster Pinctada fucata at pH 8.10, 7.70, and 7.40. We found that the shell length and total weight index did not vary significantly between oysters reared at pH 8.10 and 7.70, but was significantly lower at pH 7.40. Calcium content and shell hardness were not significantly different between pH 8.10 and 7.70, but were significantly different at pH 7.40. At pH 7.40, the shell exhibited a poorly organized nacreous microstructure, and showed an apparent loss of structural integrity in the nacreous layer. The prismatic layer appeared morphologically dissimilar from the samples at pH 8.10 and 7.70. The internal layer was corroded and had dissolved. At pH 7.40, the expression levels of nacrein, aspein, and n16 decreased on day 1, and remained low between days 2 and 42. The expression levels of these genes were significantly lower at pH 7.40 than at pH 8.10 and 7.70 during days 2-42. These results suggest that ocean acidification will have a limited impact on shell growth, calcification, and associated gene expression levels at a pH of 7.70, which is projected to be reached by the end of the century. The negative effects were found on calcification and gene expression occurred at the lowest experimental pH (7.40).

Bladder cancer (BC), as one of the most common cancers around the world, begins in the inner side of the bladder and is inclined to spread to the remaining parts of the body. Extensive documents have shown that long noncoding RNAs function as stimuli in various cancer types. With regard to LINC00649, there is limited investigation on its role previously. In our research, we discovered that LINC00649 was considerably highly expressed in BC cells and the lack of LINC00649 would cause inactivity in cellular proliferation, migration, and invasion. miR-16-5p turned out to be competitively incorporated by LINC00649 in the upstream or JARID2 downstream. In BC cells, LINC00649 was found to bind with miR-16-5p to increase the expression of JARID2. Overly expressed JARID2 was found to reverse the LINC00649 shortage-mediated suppressive impacts on the cellular process of BC cells. Concisely, it was the first study on the molecular mechanism of LINC00649 in BC. This work detected that LINC00649 enhanced cell proliferation, migration, and invasion of BC cells by acting as a sponge of miR-16-5p and upregulating JARID2, providing novel insight into understating BC.

Whiteflies, Bemisia tabaci (Hemiptera), are pests causing economic damage to many crops, capable of transmitting hundreds of plant vector-borne viruses. They are believed to secrete salivary protein effectors that can improve vector colonization and reproductive fitness in host plants. However, little is known about effector biology and the precise mechanism of action of whitefly effectors. Here, we report a functional screening of B. tabaci salivary effector proteins (Bsp) capable of modulating plant innate immunity triggered by plant endogenous pattern peptide Pep1. Four immunity suppressors and two elicitors were identified. Bsp9, the most effective immunity suppressor, was further identified to directly interact with an immunity regulator WRKY33. We provide evidence that Bsp9 may suppress plant immune signalling by interfering with the interaction between WRKY33 and a central regulator in the MAPK cascade. The interference by Bsp9 therefore reduces plant resistance to whitefly by inhibiting activation of WRKY33-regulated immunity-related genes. Further detailed analysis based on transgenic plants found that whitefly effector Bsp9 could promote whitefly preference and performance, increasing virus transmission. This study enriches our knowledge on insect effector biology. This article is part of the theme issue ‘Biotic signalling sheds light on smart pest management’.

The signal transducers and activators of the transcription (STAT) family play an important role in regulatory and cellular functions by regulating the expression of a variety of genes, including cytokines and growth factors. In the present study, a Pinctada fucata STAT protein, termed PfSTAT, was described. The deduced amino acid sequence of PfSTAT contains the conserved STAT_bind domain and the SH2 domain, and the additional Bin/Amphiphysin/Rvs (BAR) domain, but does not have STAT_alpha and STAT_int domains. Multiple sequence alignments revealed that PfSTAT showed relatively low identity with vertebrate and other invertebrate STATs, and phylogenetic analysis indicated that the evolution of STAT may have been more complex and ancient. Gene expression analysis revealed that PfSTAT is involved in the immune response to polyinosinic-polycytidylic acid (poly I:C) stimulation and in the nucleus insertion operation. This study contributes to a better understanding of PfSTAT in protecting the pearl oyster from disease or injury caused by grafting.

Extracellular signal-regulated kinases (ERKs) are conserved and related with protein-serine/threonine kinases that participate in the regulation of multiple biological processes, such as cell survival, cell differentiation, proliferation, metabolism, and inflammation. However, little is known about the roles of this kinase in the pearl oyster. In this study, we cloned and identified an ERK homolog from Pinctada fucata (PfErk). Furthermore, we have unraveled its expressional kinetics after lipopolysaccharide (LPS) and polyinosinic-epolycytidylic acid (poly I:C) immune challenge. Pferk harbored a 5' untranslated region (UTR) of 12 bp, a coding sequence of 1074 bp, and a 3' UTR of 882 bp. The putative peptide comprised a predicted molecular mass of 41.19 kDa, with a theoretical pI of 6.15. Sequence analysis showed that it possesses one STK catalytic domain and a conserved His-Arg-Asp (HRD) domain. In addition, a canonical Thr-Glu-Tyr (TEY) dual phosphorylation motif and an ATRW substrate binding site were also identified in the coding protein. Homology assessment of PfErk showed high similarity to Homo sapiens ERK. Phylogenetic analysis supported a close evolutionary relationship with molluscan orthologs. The expression patterns of Pferk were observed in seven different tissues of pearl oyster, with highest expression in the mantle and lowest expression in the digestive gland. Pferk mRNA expression levels were detected at developmental stages, with the highest expression in D-shaped larvae, followed by the 32-cell stage. The mRNA expression of Pferk was upregulated significantly in P. fucata mantle primary cells and mantle tissue after LPS and poly (I:C) treatment, and PfErk phosphorylation levels were activated by LPS and poly (I:C) challenges. Overall, our results suggested that PfErk may play important roles in pearl oyster innate immunity, and provided a new understanding of mantle immunity in the pearl oyster.

Figures S1-S6; Tables S1-S3

Myostatin (MSTN or growth differentiation factor-8) is considered a negative regulator of muscle growth and development. In this study, we cloned and characterized the full-length MSTN cDNA from Pinctada fucata, and named it as the Pf-MSTN cDNA. The single nucleotide polymorphisms (SNPs) in Pf-MSTN cDNA were then screened and genotyped. The full-length Pf-MSTN cDNA was 2644 bp, including an open reading frame of 1248 bp encoding 415 amino acids which contained typical structural characteristics shared by all members of the transforming growth factor-β (TGF-β) superfamily including an N-terminal signal peptide, a propeptide domain, and a TGF-β superfamily bioactive domain. The Pf-MSTN mRNA was detected in all tested tissues, with the highest mRNA levels observed in the adductor muscle, indicating that Pf-MSTN may play a major role in this tissue. Furthermore, by sequencing and alignment, 32 SNP loci were identified in Pf-MSTN cDNA. Genotyping 50 individuals from a common breeding stock revealed that 21 of these 32 loci were polymorphic. The minor allele frequency was in the range of 0.0400–0.4800, and the polymorphism information content value varied from 0.0739 to 0.3750. The observed and expected heterozygosity ranged from 0.0200 to 1.0000 and from 0.0776 to 0.5051, respectively. These SNPs identified in Pf-MSTN will be useful for future studies investigating their utility in marker-assisted selection for P. fucata breeding.

The mitogen-activated protein kinase kinase 4 (MKK4) is a key component of the c-Jun N-terminal kinase (JNK) signaling pathway and regulates multiple cellular activities. However, little is known about the roles of this kinase in pearl oyster. In this study, we identified an MKK4 homologue in Pinctada fucata by using a transcriptome database. Sequence analysis and protein structure prediction showed that PfMKK4 is highly conserved to MKK4 from other vertebrate and invertebrate species. Phylogenetic analysis revealed that PfMKK4 has the closest relationship with that from Crassostrea gigas. QPCR was used to investigate expression profiles in different healthy adult tissues and developmental stages of P. fucata. We found that PfMKK4 was ubiquitously expressed in all tissues and developmental stages examined except for in D-shaped larvae. Gene expression analysis suggested that PfMKK4 is involved in the response to the nucleus insertion operation. Lipopolysaccharide (LPS) and polyinosinic:polycytidylic acid [poly(I:C)] stimulation in vivo reduced PfMKK4 mRNA expression at 6 h, 48 h and 48 h, 72 h, respectively. LPS and poly(I:C) induced PfMKK4 phosphorylation in a primary mantle cell culture. These results contribute to better understanding of the potential role played by PfMKK4 in protecting the pearl oyster from injury caused by grafting or disease.

The platelet-derived growth factor/vascular endothelial growth factor (PDGF/VEGF, PVF) family of proteins have been implicated in a wide range of biological functions in vertebrates, including cell proliferation, cell differentiation, cell migration, neural development and especially angiogenesis/vasculogenesis. In this study, a PVF gene, belonging to the PDGF/VEGF family, was cloned and characterized from Pinctada fucata. It contained an ORF of 1110 bp encoding a putative protein of 369 amino acids. The deduced amino acid sequence presented the typical structural features of PDGF family members and the N-terminal signal peptide for secretion. Comparative phylogenetic analysis revealed that PfPVF shows relatively high identity with other invertebrate PVF homologues. Furthermore, gene expression analysis revealed that PfPVF is involved in not only the nucleus grafting operation and but also the response to immune stimulation. The study may help to increase understanding of the functions of molluscan PVF.

In this study, we examined the effect of elevated temperature on the expression patterns of genes, i.e., nacrein, irr, n16, n19, and hsp70 in the pearl oyster Pinctada fucata. The experiment was carried out at 4 temperatures, i.e., 20A degrees C (ambient, control), 24, 28A degrees C, and 32A degrees C. The expression levels of target genes in P. fucata were assayed at 0, 6, 24, 48, and 96 h via real-time polymerase chain reaction. Results showed that the expression levels of nacrein and irr had no significant variations among different time points below 28A degrees C, but significantly increased over time at 32A degrees C. The expression levels of n16 and n19 did not change markedly at 20A degrees C. The former increased significantly at 6 h and 24 h while the latter substantially decreased during 6-96 h at 24, 28 and 32A degrees C. Among different temperatures, the level of n16 was significantly lower at 20A degrees C than at other temperatures during 6-96 h, and the level of n19 significantly varied among different temperatures at 48 h and 96 h. The expression level of hsp70 was significantly higher at 32A degrees C than at 20, 24 and 28A degrees C at 24 h. These results demonstrated that elevated temperature impacted the physiological processes of P. fucata and potentially influenced its adaptability to thermal stress.

In this study, we screened 103,050 gene sequences of Pinctada fucata and identified 1450 simple sequence repeats (SSRs) distributing in 1355 sequences, in which 97 have sufficient flanking sequences for primer design, and 25 novel microsatellite loci were stable and polymorphic with Na which varied from 2 to 6. Six microsatellite loci were found in the open reading frame of their corresponding sequences. With the 25 SSR loci, five breeding populations and a wild population, a total of 240 individuals from wild population (W), three consecutive selected populations (G1, G2, and G3), and two backcross populations (Do, Dr) (40 individuals per population) were assayed. In these populations, we detected 108 alleles of 111 bp to 362 bp. Each population had mean Ho of 0.5718-0.7366 and mean He of 0.5830-0.6954. Except the Fst = 0.0697 (W vs. G3), pairwise Fst values ranged from 0.0131 to 0.0473, the genetic identity coefficients varied from 0.8688 to 0.9663, and genetic distance ranged from 0.0343 to 0.1307, all of these suggested that the breeding populations had genetic differentiation but at a low level generally. Besides, all the genetic index values, backcross populations (Do, Dr) were higher than corresponding G3 values, indicating that backcross could make the offspring incline to the recurrent parents.

Background Mollusca is the second largest phylum in nature. The shell of molluscs is a remarkable example of a natural composite biomaterial. Biomineralization and how it affects mollusks is a popular research topic. The BMP-2 signaling pathway plays a canonical role in biomineralization. SMAD4 is an intracellular transmitter in the BMP signaling pathway in mammals, and some genomic data show SMAD4’s involvment in BMP signaling in invertbrates, but whether SMAD4 plays a conservative role in pearl oyster, Pinctada fucata, still need to be tested. Results In this study, we identified a SMAD4 gene (hereafter designated PfSMAD4) in pearl oyster Pinctada fucata. Bioinformatics analysis of PfSMAD4 showed high identity with its orthologs. PfSMAD4 was located in the cytoplasm in immunofluorescence assays and analyses of PfSMAD4 mRNA in tissues and developmental stages showed high expression in ovaries and D-shaped larvae. An RNA interference experiment, performed by PfSMAD4 double-stranded RNA (dsRNA) injection, demonstrated inhibition not only of nacre growth but also organic sheet formation with a decrease in PfSMAD4 expression. A knockdown experiment using PfBMP2 dsRNA showed decreased PfBMP2 and PfSMAD4 mRNA and irregular crystallization of the nacreous layer using scanning electron microscopy. In co-transfection experiments, PfBMP2-transactivated reporter constructs contained PfSMAD4 promoter sequences. Conclusions Our results suggest that PfSMAD4 plays a role in biomineralization and can transduce BMP signals in P. fucata. Our data provides important clues about the molecular mechanisms that regulate biomineralization in pearl oyster.

Pearl oysters have been found to secrete nacre and form pearls with good quality and significant commercial interest. However, the transcriptomic and genomic resources for pearl oysters are still limited. To improve this situation, transcriptome sequencing was conducted from four species of pearl oysters with Illumina HiSeq™ 2000. There were four gigabase-scale transcriptomes for four species of pearl oysters, ∼26.3 million reads with ∼2.37 gigabase base pairs (Gbp) in Pinctada fucata, ∼26.5 million reads with ∼2.39 Gbp in Pinctada margaritifera, ∼27.0 million reads with ∼2.43 Gbp in Pinctada maxima, and ∼25.9 million reads with ∼2.33 Gbp in Pteria penguin, respectively. After sequence assembly and blastx alignment, the numbers of annotated unigenes ≥200 bp were 33,882 in P. fucata, 30,666 in P. margaritifera, 26,420 in P. maxima, and 29,928 in P. penguin. Based on these annotated unigenes among four species of pearl oysters, CDSs were extracted and predicted and furthermore, analyses of GO and KEGG assignments were performed. In addition, 60 putative genes of growth factors and their receptors from four species of pearl oysters were predicted. This study established an excellent resource for gene discovery and expression in pearl oysters, but also offered a significant platform for functional genomics and comparative genomic studies for mollusks.

TRAF3 is a highly versatile regulator that negatively regulates JNK and alternative nuclear factor-κB signalling, but positively controls type I interferon production. To investigate TRAF3 function in innate immune responses among invertebrate especially mollusk, we characterized TRAF3 (PfTRAF3) from pearl oyster Pinctada fucata, one of the most important bivalve mollusks for seawater pearl production. PfTRAF3 cDNA is 2261 bp with an open reading frame of 1623 bp encoding a putative protein of 541 amino acids. The deduced PfTRAF3 contains a RING finger domain, two TRAF domains with zinc finger domains and a conserved C-terminal meprin and TRAF homology (MATH) domain. Comparison and phylogenetic analysis revealed that PfTRAF3 from mollusk shared a higher identity with Ciona intestinalis TRAF3 from urochordata, Branchiostoma belcheri TRAF3 from cephalochordate, and even TRAF3 from vertebrate than with insect homologues. Furthermore, gene expression analyses suggested that PfTRAF3 was involved in the immune response to Vibrio alginolyticus.

Galectin is one important member of pattern recognition proteins that plays a pivotal role in regulating innate immune response of invertebrates. In this study, we cloned the promoter sequence of a tandem-repeat galectin from the pearl oyster Pinctada fucata (P. fucata). The quantitative real-time PCR analysis revealed that galectin mRNA expression in mantle tissues were firstly up-regulated from time points of 2 h-24 h, and then down-regulated from time points of 24 h-168 h after mantle injury. The genome methylation level of mantle tissue was inversely related to galectin mRNA expression (Pearson correlation: -0.554, P: 0.154). The "CpG4-6" methylation level in promoter region of galectin was significant positive correlated with the mRNA expression (Pearson correlation: 0.313, P: 0.049). The results indicated that galectin gene may be involved in immune response in mantle wound healing process of P. fucata, and DNA methylation may be a regulation factor of gene expression.

The granulin/epithelin precursor (GEP) encodes a glycoprotein precursor which exhibits pleiotropic tissue growth factor activity with multiple functions. Here, GEP was isolated and its role in the shell biomineralization process of the pearl oyster Pinctada fucata was investigated. Three forms of GEP mRNA were isolated from the pearl oyster (designated PfGEP-1, PfGEP-2 and PfGEP-3). Genomic DNA flanking the splicing region of the PfGEP variants was sequenced and it was found that PfGEP-2 splices out Exon 4, whereas PfGEP-3 splices out Exon 3 compared to PfGEP-1. PfGEP-1 (1505 amino acids) consists of 18 granulin domains, whereas PfGEP-2 (1459 amino acids) and PfGEP-3 (1471 amino acids) consist of 17.5 granulin domains, respectively. Analyses of PfGEP-1 and PfGEP-3 mRNA showed differential patterns in the tissues and developmental stages. Western blotting results showed that the three splice variants can translate to proteins in HEK293T cells. A knockdown experiment using PfGEP dsRNA showed decreased PfGEP-1/PfGEP-3 and PfMSX mRNA, and irregular crystallization of the nacreous layer using scanning electron microscopy. In luciferase assays, co-transfection of PfGEP-1 could activate as well as repress luciferase expression of the reporter plasmid driven by the PfMSX promoter, whereas PfGEP-3 stimulated the expression, elucidating the molecular mechanisms involved in the correlation between PfGEP and PfMSX. These results suggested that GEP variants might function differently during the biomineralization process, which provides new knowledge on the mechanism regulating nacre formation.

The impact of postoperative complications (POCs) on long-term survival outcomes following hepatic resection for colorectal liver metastasis (CRLM) is in controversy. The aim of the present meta-analysis was to systematically evaluate the POC effect on overall survival (OS) and disease-free survival (DFS) in patients undergoing hepatic resection for CRLM. We conducted a systematic review and meta-analysis of all observational studies to evaluate the POC effect on OS and DFS in patients undergoing hepatic resection for CRLM. A search for all major databases and relevant journals from inception to January 2014 without restriction on languages or regions was performed. POCs were extracted and graded according to a validated system of classification. Outcome measures were postoperative 1-, 2-, 3-, and 10-year OSs and DFSs. Both random-effects and fixed-effect models were used to pool the hazard ratios (HRs) of the survival outcomes. Test of heterogeneity was performed with the Q statistic. A total of 2370 patients were included in the meta-analysis. Both 5- and 10-year postoperative OSs showed significant decreases in patients with POCs (HR = 1.52; 95 % CI 1.27–1.83; P

Interleukin 17 (IL-17) is an important pro-inflammatory cytokine and plays critical roles in the immune response to pathogens and in the pathogenesis of inflammatory and autoimmune diseases. Despite its important functions, the origin and evolution of IL-17 in animal phyla have not been characterized. As determined in this study, the distribution of the IL-17 family among 10 invertebrate species and 7 vertebrate species suggests that the IL-17 gene may have originated from Nematoda but is absent from Saccoglossus kowalevskii (Hemichordata) and Insecta. Moreover, the gene number, protein length and domain number of IL-17 differ widely. A comparison of IL-17-containing domains and conserved motifs indicated somewhat low amino acid sequence similarity but high conservation at the motif level, although some motifs were lost in certain species. The third disulfide bond for the cystine knot fold is formed by two cysteine residues in invertebrates, but these have been replaced by two serine residues in Chordata and vertebrates. One third of invertebrate IL-17 proteins were found to have no predicted signal peptide. Furthermore, an analysis of phylogenetic trees and exon-intron structures indicated that the IL-17 family lacks conservation and displays high divergence. These results suggest that invertebrate IL-17 proteins have undergone complex differentiation and that their members may have developed novel functions during evolution.

Nuclear factor of activated T cells (NFAT) plays an important role in nonimmune cells and also in T cells and many other cells of the immune system, by regulating the expression of a variety of genes involved in the immune response, organ development, developmental apoptosis and angiogenesis. In the present study, the NFAT homology gene, PfNFAT, from the pearl oyster Pinctada fucata was cloned and its genomic structure and promoter were analyzed. PfNFAT encodes a putative protein of 1226 amino acids, and contains a highly conserved Rel homology region (RHR) with DNA-binding specificity, and a regulatory domain (NFAT homology region, NHR) containing a potent transactivation domain (TAD). The PfNFAT gene consists of 12 exons and 11 introns, and its promoter contains potential binding sites for transcription factors such as NF-κB (Nuclear factor κB), STATx (signal transducer and activator of transcription), AP-1 (activator protein-1) and Sox-5/9 (SRY type HMG box-5/9), MyoD (Myogenic Differentiation Antigen) and IRF (Interferon regulatory factor). Comparison and phylogenetic analysis revealed that PfNFAT shows high identity with other invertebrate NFAT, and clusters with the NFAT5 subgroup. Furthermore, gene expression analysis revealed that PfNFAT is involved in the immune response to lipopolysaccharide (LPS) and Polyinosinic-polycytidylic acid (poly I:C) stimulation and in the nucleus inserting operation. The study of PfNFAT may increase understanding of molluscan innate immunity.

A new type of six-bar swaying machine was put forward, which is an ingenious combination of plane multi-bar mechanism and high pressure oil cylinder. Preliminary analysis shows that this machine has many advantages, such as the torque produced by its unit weight, its small size, its light deadweight, etc. Thus it can be applied to situations that need swaying mechanism with low rotational speed and great torque. Firstly, the mechanism composition and working principle of the swaying machine were introduced. Secondly, parameterized modeling of the mechanism was carried out by utilizing software ADAMS. Then kinematic analysis and kinetic analysis were completed by using ADAMS. Finally, key dimensions were adjusted according to kinetic analysis. These tasks are believed to be beneficial to the development of the novel transmission.

Interferon regulatory factors (IRFs) control many facets of the innate and adaptive immune responses, regulate the development of the immune system itself and involve in reproduction and morphogenesis. In the present study, the IRF-2 homology gene, PfIRF-2 from pearl oyster Pinctada fucata was cloned and its genomic structure and promoter were analyzed. PfIRF-2 encodes a putative protein of 350 amino acids, and contains a highly conserved N-terminal DNA-binding domain and a variable C-terminal regulatory domain. Comparison and phylogenetic analysis revealed that PfIRF-2 shared a relatively higher identity with other mollusk but relatively lower identity with vertebrate IRF-2, and was clustered with IRF-1 subfamily composed of IRF-2 and IRF-1. Furthermore, gene expression analysis revealed that PfIRF-2 involved in the immune response to LPS and poly(I:C) stimulation. Immunofluorescence assay showed that the expressed PfIRF-2 was translocated into the nucleus and dual-luciferase reporter assays indicated that PfIRF-2 could involved and activate interferon signaling or NF-κB signal pathway in HEK293 cells. The study of PfIRF-2 may help better understand the innate immune in mollusk.

IL-17 from pearl oyster Pinctada fucata, one of mollusk, was identified and characterized, and its genomic structure and promoter were analyzed. The full-length cDNA of P. fucata IL-17 (PfIL-17) is 907 bp with an open reading frame of 585 bp encoding a putative protein of 194 amino acids. The deduced PfIL-17 contains a 19 amino acid signal peptide and a conserved IL-17 domain. Multiple sequence alignments and phylogenetic analysis revealed that PfIL-17 has lower similarity with other invertebrate IL-17 and was clustered with CgIL-17, but not clustered with other invertebrate IL-17. Gene expression analysis indicated that PfIL-17 took part in the immune response to LPS and poly(I:C) stimulation, and dual-luciferase reporter assays showed that PfIL-17 could active vertebrate target genes containing the NF-kappa B binding site and involve NF-kappa B signal pathway in HEK293 cells. Combined with the results mentioned above, it is suggested that PfIL-17 might involve and activate NF-kappa B signal pathway against extracellular pathogens. (C) 2013 Elsevier Ltd. All rights reserved.

Cytokine-induced suppressor of cytokine signaling (SOCS) family acts as a negative regulator of cytokine receptor signaling to control excessive cytokine effects and inhibit a variety of signal transduction pathways, particularly the Janus kinases/signal transducers and activators of transcription (JAK/STAT) pathway. In present study, SOCS-2 homolog (PfSOCS-2) from pearl oyster Pinctada fucata was cloned and its gene has no intron. Multiple sequence alignments and phylogenetic analysis showed that PfSOCS-2 was clustered with other mollusk SOCS-2. LPS or polyI:C challenge and gene expression analysis revealed that PfSOCS-2 involved the innate immune response against bacterial and viral infections and that induction of PfSOCS-2 was varied with the different challenge stimulations. Furthermore, Dual-luciferase reporter assays showed that PfSOCS-2 involved in the regulation of vertebrate target genes containing the IFN-stimulated response element or NF-kappa B binding site in vitro. These results indicated that SOCS-2 from P. fucata plays a regulatory role against the stimulation. (c) 2013 Elsevier Ltd. All rights reserved.

NF-kappa B transcription factors play central roles in many important physiological and pathological processes including innate immune responses. Here we report the cloning of an NF-kappa B transcription factor. PfRelish from pearl oyster Pinctada fucata, one of the most important bivalve mollusks for seawater pearl production. PfRelish full-length cDNA is 3916 bp with an open reading frame of 3558 bp encoding a putative protein of 1186 amino acids. The deduced PfRelish contains a N-terminal RHD, a nucleus localization signal, an I kappa B-like domain with six ankyrin repeats and a death domain at the C-terminus, which is similar to class I NF-kappa B transcription factors. Comparison and phylogenetic analysis revealed that class I NF-kappa Bs in mollusks including PfRelish might have most distant relationship to the arthropod Relish. Further expression analysis showed that PfRelish was apparently upregulated after Vibrio alginolyticus injection, which suggested that PfRelish was involved in the immune response to V. alginolyticus. (C) 2012 Elsevier Ltd. All rights reserved.

Oceanic uptake of anthropogenic carbon dioxide results in decrease in seawater pH and increase in temperature. In this study, we demonstrated the synergistic effects of elevated seawater temperature and declined seawater pH on gene expression patterns of aspein, calmodulin, nacrein, she-7-F10 and hsp70 in the pearl oyster Pinctada fucata. Under 'business-as-usual' scenarios, four treatments were examined: (1) ambient pH (8.10) and ambient temperature (27 °C) (control condition), (2) ambient pH and elevated temperature (+3 °C), (3) declined pH (7.70) and ambient temperature, (4) declined pH and elevated temperature. The results showed that under warming and acidic seawater conditions, expression of aspein and calmodulin showed no significant differences among different time point in condition 8.10 T. But the levels of aspein and calmodulin in conditions 8.10 T+3, 7.70 T and 7.70 T+3, and levels of nacrein, she-7-F10 in all the four treatments changed significantly. Low pH and pH × temperature interaction influenced the expression of aspein and calmodulin significantly after hours 48 and 96. Significant effects of low pH and pH × temperature interaction on the expression of nacrein were observed at hour 96. The expression level of she-7-F10 was affected significantly by pH after hours 48 and 96. The expression of hsp70 was significantly affected by temperature, pH, temperature × pH interaction at hour 6, and by temperature × pH interaction at hour 24. This study suggested that declined pH and pH × temperature interaction induced down regulation of calcification related genes, and the interaction between declined seawater pH and elevated temperature caused up regulation of hsp70 in P. facata. These results demonstrate that the declined seawater pH and elevated temperature will impact the physiological process, and potentially the adaptability of P. fucata to future warming and acidified ocean.

We reported pearl oyster Pinctada fucata cDNA and genomic characterization of a new homeobox-containing protein, PfMSX. The PfMSX gene encodes a transcription factor that was localized to the nucleus. Analyses of PfMSX mRNA in tissues and developmental stages showed high expressions in mantle or D-shaped larvae. In electrophoretic mobility shift assays (EMSAs) PfMSX binded to MSX consensus binding sites in the 5' flanking region of the Pif promoter. In co-transfection experiment PfMSX transactivated reporter constructs containing Pif promoter sequences, and mutation of the MSX-binding sites attenuated transactivation. A knockdown experiment using PfMSX dsRNA showed decreased Pif mRNA and unregular crystallization of the nacreous layer using scanning electron microscopy. Our results suggested that PfMSX was a conserved homeodomain transcription factor gene, which can activate Pif gene expression through MSX binding site, and was then involved in the mineralization process in pearl oyster Pinctada fucata. Our data provided important clues about mechanisms regulating biomineralization in pearl oyster.