Preliminary investigation on the effect of Vibrio splendidus stimulation on the intestinal flora of Strongylocentrotus intermedius.

To better understand the effect of Vibrio splendidus infection on Strongylocentrotus intermedius, 16S rRNA sequencing was carried out to investigate the intestinal flora of S. intermedius stimulated by 0 CFU/mL (Con), 1.5 × 107 CFU/mL (Vib1) and 1.5 × 108 CFU/mL (Vib2) concentrations of V. splendidus. The results showed that there was significant difference in intestinal flora diversity between Con group and Vib1 group, but no significant difference between Con group and Vib2 group. However, there were significant differences in the composition of intestinal flora among all groups. Bacteroidota, Proteobacteria and Firmicutes were the dominant phylum in the Con group. The abundance of Bacteroidota and Firmicutes decreased and Proteobacteria increased in Vib1 and Vib2 groups. The relative abundance of the potential probiotic bacteria Muribaculaceae and Alloprevotella was significantly lower in the Vib1 and Vib2 groups. In addition, the opportunistic pathogen Desulfovibrio was found in Vib1 and Vib2 groups. It is evident that V. splendidus infection not only alters the composition of the microbial community in the intestinal tract of S. intermedius, but may also lead to the production of opportunistic pathogens, which could be potentially harmful to the health of S. intermedius. The results of this study provide a foundation for exploring the diseases caused by V. splendidus stimulation leading to an imbalance in the intestinal flora of S. intermedius, and contribute to our further understanding of the role of Vibrio on the health of S. intermedius.

Molecular confirmation and functional study of signal transducer and activator of transcription genes in the Pacific oyster Crassostrea gigas.

The JAK (Janus kinase)-STAT (Signal transducer and activator of transcription) is a well-known functional signaling pathway that plays a key role in several important biological activities such as apoptosis, cell proliferation, differentiation, and immunity. However, limited studies have explored the functions of STAT genes in invertebrates. In the present study, the gene sequences of two STAT genes from the Pacific oyster (Crassostrea gigas), termed CgSTAT-Like-1 (CgSTAT-L1) and CgSTAT-Like-2 (CgSTAT-L2), were obtained using polymerase chain reaction (PCR) amplification and cloning. Multiple sequence comparisons revealed that the sequences of crucial domains of these proteins were conserved, and the similarity with the protein sequence of other molluscan STAT is close to 90 %. The phylogenetic analyses indicated that CgSTAT-L1 and CgSTAT-L2 are novel members of the mollusk STAT family. Quantitative real-time PCR results implied that CgSTAT-L1 and CgSTAT-L2 mRNA expression was found in all tissues, and significantly induced after challenge with lipopolysaccharide (LPS), peptidoglycan (PGN), or poly(I:C). After that, dual-luciferase reporter assays denoted that overexpression of CgSTAT-L1 and CgSTAT-L2 significantly activated the NF-κB signaling, and, interestingly, the overexpressed CgSTAT proteins potentiated LPS-induced NF-κB activation. These results contributed a preliminary analysis of the immune-related function of STAT genes in oysters, laying the foundation for deeper understanding of the function of invertebrate STAT genes.

Molecular cloning and functional study of a Rel homologous gene in sea urchin Strongylocentrotus intermedius.

NF-κB (Nuclear factor-kappa B) family proteins are versatile transcription factors that play crucial regulatory roles in cell development, growth, apoptosis, inflammation, and immune response. However, there is limited research on the function of these key genes in echinoderms. In this study, an NF-κB family gene (SiRel) was identified in sea urchin Strongylocentrotus intermedius. The gene has an open reading frame length of 1809 bp and encodes for 602 amino acids. Domain prediction results revealed that the N-terminal of SiRel protein encodes a conserved Rel homology domain (RHD), including the RHD-DNA binding domain and the RHD-dimerization domain. Multiple sequence comparison results showed that the protein sequences of these two domains were conserved. Phylogenetic analysis indicated that SiRel clustered with Strongylocentrotus purpuratus p65 protein and Rel protein of other echinoderms. Results from quantitative real-time PCR demonstrated detectable SiRel mRNA expression in all tested sea urchin tissues, with the highest expression level found in the gills. And SiRel mRNA expression levels were significantly induced after LPS (Lipopolysaccharide) and poly(I:C) (Polyinosinic:polycytidylic acid) stimulation. In addition, SiRel protein expression can be found in cytoplasm and nucleus of HEK293T cells. Co-immunoprecipitation results showed that SiRel could interact with sea urchin IκB (Inhibitor of NF-κB) protein. Western blotting and dual-luciferase reporter gene assay results indicated that overexpression of SiRel in HEK293T cells could impact the phosphorylation levels of JNK (c-Jun N-terminal kinase) and Erk1/2 (Extracellular signal-regulated kinases1/2) and activate interleukin-6 (IL-6), activating protein 1 (AP-1), interferon (IFN)α/ß/γ, and signal transducer and activator of transcription 3 (STAT3) reporter genes in HEK293T cells. In conclusion, this study reveals that SiRel plays an important role in the innate immune response of sea urchins and enriches our understanding of comparative immunology theory.

A newly identified scallop MyD88 interacts with TLR and functions in innate immunity.

Myeloid differentiation factor-88 (MyD88) is a key adaptor of the toll-like receptor (TLR) signaling pathway and plays a crucial role in innate immune signal transduction in animals. However, the MyD88-mediated signal transduction mechanism in shellfish has not been well studied. In this study, a new MyD88 gene, CfMyD88-2, was identified in the Zhikong scallop, Chlamys farreri. The 1779 bp long open reading frame encodes 592 amino acids. The N-terminus of CfMyD88-2 contains a conserved death domain (DD), followed by a TIR (TLR/Interleukin-1 Receptor) domain. The results of the multi-sequence comparison showed that the TIR domain sequences were highly conserved. Phylogenetic analysis revealed that CfMyD88-2 was first associated with Mizuhopecten yessoensis MyD88-4 and Argopecten irradians MyD88-4. CfMyD88-2 mRNA was expressed in all scallop tissues, as detected by qRT-PCR, and the expression level was the highest in the mantle and hepatopancreas. In addition, CfMyD88-2 mRNA expression significantly increased after pathogen-associated molecular patterns (PAMPs, such as lipopolysaccharide, peptidoglycan, or polyinosinic-polycytidylic acid) stimulation. The results of the co-immunoprecipitation experiments in HEK293T cells showed that both CfMyD88-1 and CfMyD88-2 interacted with the TLR protein of scallops, suggesting the existence of more than one functional TLR-MyD88 signaling axis in scallops. Dual luciferase reporter gene assays indicated that the overexpressed CfMyD88-2 in HEK293T cells activated interferon (IFN) α, IFN-ß, IFN-γ, and NF-κB reporter genes, indicating that the protein has multiple functions. The results of the subcellular localization experiment uncovered that CfMyD88-2 was mainly localized in the cytoplasm of human cells. In summary, the novel identified CfMyD88-2 can respond to the challenge of PAMPs, participate in TLR immune signaling, and may activate downstream effector genes such as NF-κB gene. These research results will be useful in advancing the theory of innate immunity in invertebrates and provide a reference for the selection of disease-resistant scallops in the future.

A scallop IκB protein involved in innate immunity acts as a key regulator of NF-κB.

Chlamys farreri, a commercially important bivalve mollusk, is extensively cultivated in China. In recent years, the frequent occurrence of diseases has led to significant mortality in scallop farms. Despite this, our understanding of scallop's innate immune mechanisms remains limited. The NF-κB signaling pathway plays a crucial role in various biological processes, including cellular, developmental, and immune defense mechanisms. Inhibitors of NF-κB (IκB) proteins block the nuclear localization and DNA binding of NF-κB, thereby inhibiting its activity. However, the role of these proteins in invertebrates is not well understood. In this study, we identified a new homolog of the IκB gene in C. farreri, named CfIκB1. The open reading frame of CfIκB1 spans 1089 bp, encoding 362 amino acids. Through sequence comparison and phylogenetic analysis, CfIκB1 was classified as a member of the invertebrate IκB family. Quantitative real-time PCR revealed that CfIκB1 transcripts are present in all examined tissues, with the highest expression observed in hemocytes. Expression levels were significantly upregulated following exposure to lipopolysaccharide, peptidoglycan, and polyinosinic:polycytidylic acid. Co-immunoprecipitation studies confirmed that CfIκB1 interacts with NF-κB family proteins CfRel-1 and CfRel. Dual-luciferase reporter assays demonstrated that CfIκB1 inhibits CfRel-dependent activation of NF-κB, ISRE, IFNß, and AP-1. These findings suggest that CfIκB1 plays a crucial role in regulating NF-κB activity, which is integral to the innate immunity of C. farreri. This research enhances our understanding of the innate immune system in invertebrates and provides a theoretical basis for developing disease-resistant scallops at the molecular level.

Effects of ambient UVB light on Pacific oyster Crassostrea gigas mantle tissue based on multivariate data.

Ambient ultraviolet radiation (UVB) from solar and artificial light presents serious environmental risks to aquatic ecosystems. The Pacific oyster, Crassostrea gigas, perceives changes in the external environment primarily through its mantle tissue, which contains many nerve fibers and tentacles. Changes within the mantles can typically illustrate the injury of ambient UVB. In this study, a comprehensive analysis of phenotypic, behavioral, and physiological changes demonstrated that extreme UVB radiation (10 W/m²) directly suppressed the behavioral activities of C. gigas. Conversely, under ambient UVB radiation (5 W/m²), various physiological processes exhibited significant alterations in C. gigas, despite the behavior remaining relatively unaffected. Using mathematical model analysis, the integrated analysis of the full-length transcriptome, proteome, and metabolome showed that ambient UVB significantly affected the metabolic processes (saccharide, lipid, and protein metabolism) and cellular biology processes (autophagy, apoptosis, oxidative stress) of the C. gigas mantle. Subsequently, using Procrustes analysis and Pearson correlation analysis, the association between multi-omics data and physiological changes, as well as their biomarkers, revealed the effect of UVB on three crucial biological processes: activation of autophagy signaling (key factors: Ca2+, LC3B, BECN1, caspase-7), response to oxidative stress (reactive oxygen species, heat shock 70, cytochrome c oxidase), and recalibration of energy metabolism (saccharide, succinic acid, translation initiation factor IF-2). These findings offer a fresh perspective on the integration of multi-data from non-model animals in ambient UVB risk assessment.

Nonylphenol displays immunotoxicity by triggering hemocyte extracellular traps in Manila clam via ROS burst, ERK pathway and glycolysis.

Nonylphenol (NP), an endocrine disruptor, has been demonstrated to be a harmful environmental contaminant and toxic to organisms. In this study, to address concerns regarding the immunotoxicity of NP, we treated clam Ruditapes philippinarum hemocytes with NP in vitro and explored the underlying mechanisms of NP-induced extracellular traps (ETs). NP could induce the formation of hemocytes ETs in a dose-dependent manner. Transcriptomics analysis revealed changes of signaling pathway involved in immunity and energy metabolism in hemocytes after NP stimulation. In this process, both reactive oxygen species (ROS) and myeloperoxidase (MPO) were up-regulated. Moreover, mitogen-activated protein kinase (MAPK) signaling pathway was proved to be activated in the formation of NP-induced ETs, manifested as enhanced phosphorylation of extracellular signal-regulated kinase (ERK) but not p38 or c-Jun N-terminal kinase (JNK). In the presence of U0126, an ERK phosphorylation inhibitor, the NP-induced expression of NADPH oxidase enzyme (NOX) was significantly decreased, which further alleviated the ROS production and ultimately limited the release of ETs. NP exposure increased glucose uptake, along with enhanced activities of glycolysis-related enzymes such as hexokinase (HK) and pyruvate kinase (PK). After inhibiting glycolysis by the inhibitor 2-DG, the formation of NP-induced ETs was significantly suppressed. ERK could regulate mTOR signaling and the PI3K/AKT pathway, potentially directing ETs formation by orchestrating the glycolysis through the activation of key transcription factors c-Myc and HIF-1α. Collectively, the results preliminary confirm that the ERK-NOX-ROS axis and glycolysis are involved in NP-induced ETs formation, contributing to the cellular immunotoxicity in clam.

CfIRF8-like interacts with the TBK1/IKKε family protein and regulates host antiviral innate immunity.

The interferon regulatory factor (IRF) family, a class of transcription factors with key functions, are important in host innate immune defense and stress response. However, further research is required to determine the functions of IRFs in invertebrates. In this study, the coding sequence of an IRF gene was obtained from the Zhikong scallop (Chlamys farreri) and named CfIRF8-like. The open reading frame of CfIRF8-like was 1371 bp long and encoded 456 amino acids. Protein domain prediction revealed a typical IRF domain in the N-terminus of the CfIRF8-like protein and a typical IRF3 domain in the C-terminus. Multiple sequence alignment confirmed the conservation of the amino acid sequences of these two functional protein domains. Phylogenetic analysis showed that CfIRF8-like clustered with mollusk IRF8 proteins and then clustered with vertebrate IRF3, IRF4, and IRF5 subfamily proteins. Quantitative real-time PCR detected CfIRF8-like mRNA in all tested scallop tissues, with the highest expression in the gills. Simultaneously, the expression of CfIRF8-like transcripts in gills was significantly induced by polyinosinic-polycytidylic acid challenge. The results of protein interaction experiments showed that CfIRF8-like could directly bind the TBK1/IKKε family protein of scallop (CfIKK2) via its N-terminal IRF domain, revealing the presence of an ancient functional TBK1/IKKε-IRF signaling axis in scallops. Finally, dual-luciferase reporter assay results showed that the overexpression of CfIRF8-like in human embryonic kidney 293T cells could specifically activate the interferon ß promoter of mammals and the interferon-stimulated response element promoter in dose-dependent manners. The findings of this preliminary analysis of the signal transduction and immune functions of scallop CfIRF8-like protein lay a foundation for an in-depth understanding of the innate immune function of invertebrate IRFs and the development of comparative immunology. The experimental results also provide theoretical support for the breeding of scallop disease-resistant strains.

In vitro study of deltamethrin-induced extracellular traps in hemocytes of Ruditapes philippinarum.

Deltamethrin (DLM), a broad-spectrum pesticide, has been proven to have toxic effects on aquatic organisms. Here, we detected the formation of extracellular traps (ETosis) formation in Manila clam (Ruditapes philippinarum) hemocytes stimulated by three concentrations of DLM (0.01, 0.1 and 1 µg/mL) in vitro, and explored the underlying mechanisms induced by this pesticide. Extracellular DNA structure observation and quantitative results indicated that DLM exposure could obviously induce hemocytes ETosis, especially under high concentration of DLM induction. Moreover, DLM increased the levels of myeloperoxidase (MPO) and reactive oxygen species (ROS) in a dose-dependent manner, and enhanced the mRNA expression of several ROS-related genes. DPI (NADPH oxidase inhibitor) and ABAH (MPO inhibitor) could substantially inhibit DLM-induced extracellular traps (ETs), suggesting that the induced ETs release was caused by the induction of the ROS burst and MPO production. In addition, three concentrations of DLM-induced ETs were also accompanied by mitochondrial dysfunction, such as increasing the production of mitochondrial ROS, leading to a decrease in mitochondrial membrane potential (MMP) and activation of mitochondrial permeability transition pore (MPTP). Taken together, these results will shed new light on the immunotoxicity of DLM in clams and perhaps lays the foundation for health assessment in bivalves.

Transcriptional expression analysis reveals multiple effects of nonylphenol exposure on scallop immune system.

Nonylphenol (NP) is an endocrine disruptor and environmental hormone representing alkylphenol compounds. Marine mollusks are an important source of protein for people worldwide. Many researchers have begun to study the effect of NP on marine mollusks immune system in view of its toxicity; however, the underlying molecular mechanisms require in-depth analysis. In this study, we focused on the transcriptional expression change of immune-related genes and antioxidant enzymes activities variation after NP exposure in a marine bivalve mollusk, Chlamys farreri, to explore the immunomodulatory capacity of NP in marine mollusks. We identified MAVS (Mitochondrial antiviral signaling protein), a key adaptor molecule in the RLR (RIG-I like receptor) pathway, and studied the expression of multiple immune-related genes in response to different concentrations of NP. The key genes involved in RLR/TLR (Toll like receptor) innate immune pathway, apoptosis, and cellular antioxidation mechanism were investigated. Changes in the enzymatic activities of scallop antioxidant enzymes after NP exposure were also examined. The results revealed that the genes expression and the antioxidant enzymes activities show significant changes, thus proving that NP stimulation affects the scallop immune system. Our research results demonstrate the immunomodulatory capacity of NP in marine bivalve mollusks and lay the foundation for further in-depth analysis of the molecular mechanism of NP toxicity.

Regulation of Tyrosinase Gene Expression by Retinoic Acid Pathway in the Pacific Oyster Crassostrea gigas.

Retinoic acid (RA) plays important roles in various biological processes in animals. RA signaling is mediated by two types of nuclear receptors, namely retinoic acid receptor (RAR) and retinoid x receptor (RXR), which regulate gene expression by binding to retinoic acid response elements (RAREs) in the promoters of target genes. Here, we explored the effect of all-trans retinoic acid (ATRA) on the Pacific oyster Crassostera gigas at the transcriptome level. A total of 586 differentially expressed genes (DEGs) were identified in C. gigas upon ATRA treatment, with 309 upregulated and 277 downregulated genes. Bioinformatic analysis revealed that ATRA affects the development, metabolism, reproduction, and immunity of C. gigas. Four tyrosinase genes, including Tyr-6 (LOC105331209), Tyr-9 (LOC105346503), Tyr-20 (LOC105330910), and Tyr-12 (LOC105320007), were upregulated by ATRA according to the transcriptome data and these results were verified by real-time quantitative polymerase chain reaction (RT-qPCR) analysis. In addition, increased expression of Tyr (a melanin-related TYR gene in C. gigas) and Tyr-2 were detected after ATRA treatment. The yeast one-hybrid assay revealed the DNA-binding activity of the RA receptors CgRAR and CgRXR, and the interaction of CgRAR with RARE present in the Tyr-2 promoter. These results provide evidence for the further studies on the role of ATRA and the mechanism of RA receptors in mollusks.

Identification, antibacterial activities and action mode of two macins from manila clam Venerupis philippinarum.

In this study, two macins were identified from clam Venerupis philippinarum (designated as VpMacin-1 and VpMacin-2). They showed 64.71% similarity with each other. The highest mRNA expression of VpMacin-1 and VpMacin-2 was detected in gills and hepatopancreas, respectively, in non-stimulated clams, and their expression could be induced significantly in hemocytes after Vibrio anguillarum infection. Silencing of VpMacin-1 and VpMacin-2 led to 22% and 49% mortality 6 days post infection. Escherichia coli cells were killed by recombinant protein rVpMacin-1 and rVpMacin-2 within 1000 and 400 min, respectively, at a concentration of 1.0 × MIC. Compared with rVpMacin-1, rVpMacin-2 not only showed higher broad-spectrum antimicrobial activities towards Vibrio strains, but possessed stronger abilities to inhibit the formation of bacterial biofilm. Both membrane integrity and electrochemical assay indicated that rVpMacins were capable of causing bacterial membrane permeabilization, especially for rVpMacin-2. Besides, rVpMacin-1 significantly induced both phagocytic (0.1 and 1.0 × MIC, p < 0.05) and chemotactic effects (0.1 × MIC, p < 0.01) of hemocytes, while there was no significant increase for rVpMacin-2. Overall, our results suggested that VpMacin-1 and VpMacin-2 play important roles in host defense against invasive pathogens.

Antibacterial activity and mechanism of a type-I ubiquitin from the clam Ruditapes philippinarum.

In the present study, a ubiquitin (designated as RpUbi) was identified and characterized from clam Ruditapes philippinarum. Phylogenetic analysis strongly suggested that RpUbi was a member of the ubiquitin family. In non-stimulated clams, RpUbi transcripts were constitutively expressed in all examined tissues, especially in the gills and hemocytes. After Vibrio anguillarum challenge, expression of RpUbi mRNA in hemocytes was significantly up-regulated. Recombinant RpUbi (rRpUbi) showed high antibacterial activity against Gram-positive and Gram-negative bacteria. Notably, membrane integrity and electrochemical assay indicated that rRpUbi could invade the inner layer. Moreover, DNA migration could be inhibited by rRpUbi in a concentration-dependent manner. In general, our results suggested that RpUbi played an important role in host defense against invading bacteria, perhaps through a DNA-binding process.

Antibacterial activities and mechanisms of action of a defensin from manila clam Ruditapes philippinarum.

Defensins represent an evolutionary ancient family of antimicrobial peptides, which played an undeniably important role in host defense. In the present study, a defensin isoform was identified and characterized from manila clam Ruditapes philippinarum (designed as Rpdef1α). Multiple alignments and phylogenetic analysis suggested that Rpdef1α belonged to the defensin family. Quantitative RT-PCR and immunohistochemical analysis revealed that Rpdef1α transcripts and the encoding peptide were dominantly expressed in the tissues of gills and mantle. After Vibrio anguillarum challenge, the Rpdef1α transcripts were significantly up-regulated in gills of clams. In addition, rRpdef1α not only showed broad-spectrum antimicrobial activities towards Vibrio species, but also inhibited the formation of bacterial biofilms. Knockdown of Rpdef1α transcripts caused significant increase in the cumulative mortality of manila clams post V. anguillarum challenge. Membrane integrity, scanning electron microscopy analysis and electrochemical assay indicated that rRpdef1α was capable of causing bacterial membrane permeabilization and then resulted in cell death. Moreover, phagocytosis and chemotactic ability of hemocytes could be significantly enhanced after incubation with rRpdef1α. Overall, these results suggested that Rpdef1α could act as both antibacterial agent and opsonin to defend against the invading microorganisms in manila clam R. philippinarum.

Scavenger receptor class B type I (SR-BI) in Ruditapes philippinarum: A versatile receptor with multiple functions.

In the present study, a scavenger receptor class B type I (designed as RpSR-BI) was cloned and characterized from manila clam Ruditapes philippinarum. The full-length cDNA of RpSR-BI was of 2000 bp, containing an open reading frame (ORF) of 1515 bp. Multiple alignments and phylogenetic analysis strongly suggested that RpSR-BI was a member of the scavenger receptors family. The mRNA transcript of RpSR-BI was constitutively expressed in all tested tissues, and mainly expressed in hepatopancreas and hemocytes. Generally, Vibrio anguillarum or Micrococcus luteus challenge induced the expression of RpSR-BI transcripts in hemocytes of manila clams. Recombinant protein of RpSR-BI (rRpSR-BI) could bind lipopolysaccharides, peptidoglycan and glucan, but not chitin in vitro. Coinciding with the PAMPs binding assay, a broad agglutination spectrum was displayed by rRpSR-BI including Gram-positive bacteria and Gram-negative bacteria. Moreover, rRpSR-BI could enhance the phagocytosis and chemotaxis of hemocytes. These results showed that RpSR-BI functioned as a pattern recognition receptor (PRR) with distinct recognition spectrum, and also as an opsonin involved in the innate immune response of R. philippinarum.

A peptidoglycan recognition protein involved in immune recognition and immune defenses in Ruditapes philippinarum.

Peptidoglycan recognition proteins (PGRPs) are important pattern recognition receptors in the innate immune system of invertebrates. In the study, a short PGRP (designed as RpPGRP) was identified and characterized from the manila clam Ruditapes philippinarum. The open reading frame of RpPGRP encoded a polypeptide of 249-amino acids with a calculated molecular mass of 27.2 kDa and an isoelectric point of 6.62. Multiple alignments and phylogenetic analysis strongly suggested that RpPGRP was a new member of the PGRP superfamily. In non-stimulated clams, RpPGRP exhibited different tissue expression pattern, and highly expressed in hepatopancreas and hemocytes. Expression of RpPGRP transcripts was significantly up-regulated in hemocytes of clams post Vibrio anguillarum or Micrococcus luteus challenge. The recombinant RpPGRP (rRpPGRP) exhibited high affinity to PGN, LPS and zymosan in a concentration-dependent manner. With a broad spectrum of bacterial binding activities, rRpPGRP exhibited strong agglutination activity to Escherichia coli, Vibrio splendidus, V. anguillarum and M. luteus. Furthermore, rRpPGRP exhibited Zn2+-dependent amidase activity and catalyzed the degradation of insoluble PGN. Especially, rRpPGRP exhibited significant antibacterial activity against E. coli and M. luteus. Moreover, the biofilm formation of E. coli could be inhibited after rRpPGRP incubation in the presence of Zn2+. This inhibitory effect of rRpPGRP might attribute to its amide bactericidal activity. Taken together, rRpPGRP played important roles in PGRP-mediated immune defense mechanisms, especially by recognizing antigens and eliminating bacteria.

Molecular cloning and characterization of FADD from the manila clam Ruditapes philippinarum.

Fas-associated protein with death domain (FADD) is an essential element in cell death, and also implicates in cell cycle progression, inflammation and innate immunity. In the study, an FADD (designated as RpFADD) was identified and characterized from manila clam, Ruditapes philippinarum. Multiple alignments and phylogenetic analysis strongly suggested that RpFADD was a new member of the FADD family. The RpFADD transcripts were constitutively expressed in a wide range of tissues, and dominantly expressed in hemocytes. After challenged with Vibrio anguillarum or Micrococcus luteus, the expression level of RpFADD transcripts was significantly induced and reached the maximum level at 72 h and 48 h, respectively. Knockdown of RpFADD down-regulated the transcript levels of RpIKK, RpTAK1 and RpNF-κB with the exception of RpIκB. Moreover, RpFADD primarily localized in the cell cytoplasm, and its over-expression promoted the apoptosis of HeLa cells. These results revealed that RpFADD perhaps regulated the NF-κB signaling pathways positively, which provided a better understanding of RpFADD in innate immunity.

A macin identified from Venerupis philippinarum: Investigation on antibacterial activities and action mode.

In the present study, a macin was cloned and characterized from clam Venerupis philippinarum (designed as VpMacin). The full-length cDNA of VpMacin was of 579 bp, encoding a peptide of 87 amino acids with the predicted molecular weight of 9.7 kDa. Analysis of the conserved domain suggested that VpMacin was a new member of the macin family. In non-stimulated clams, VpMacin transcripts exhibited different tissue expression pattern, and highly expressed in the tissues of gills and hepatopancreas. Generally, the temporal expression of VpMacin transcripts was significantly induced in hemocytes of clams post Vibrio anguillarum challenge. Moreover, the recombinant VpMacin protein (rVpMacin) showed obvious antimicrobial activities against Gram-positive and Gram-negative bacteria. After incubated with 40 µM rVpMacin, all detected Escherichia coli could be killed within 60 min. Membrane integrity analysis revealed that rVpMacin could increase the membrane permeability of bacteria and then resulted in cell death. Overall, our results suggested that VpMacin had an important function in host defense against invasive pathogens.

A bactericidal permeability-increasing protein (BPI) from manila clam Ruditapes philippinarum: Investigation on the antibacterial activities and antibacterial action mode.

Bactericidal permeability-increasing protein (BPI) is an antimicrobial protein with potent endotoxin-neutralising activity and plays a crucial role in innate immunity against bacterial infection. In the present study, a bpi (designed as rpbpi) was identified and characterized from manila clam Ruditapes philippinarum. Multiple alignments and phylogenetic analysis suggested that rpbpi was a new member of the bpis family. In non-stimulated clams, rpbpi transcripts were ubiquitously expressed in all tested tissues with the highest expression level in hemocytes. After Vibrio anguillarum challenge, the expression levels of rpbpi mRNA in hemocytes were up-regulated significantly at 3 h and 48 h compared with that in the control, which were 4.01- and 19.10-fold (P < 0.05), respectively. The recombinant RpBPI (rRpBPI) showed high antibacterial activities against Gram-negative bacteria Escherichia coli and V. anguillarum, but not Staphylococcus aureus. Moreover, membrane integrity analysis revealed that rRpBPI increased the membrane permeability of Gram-negative bacteria, and then resulted in cell death. Overall, our results suggested that RpBPI played an important role in the elimination of invaded bacteria through membrane-disruptive activity.

Two macrophage migration inhibitory factors (MIFs) from the clam Ruditapes philippinarum: Molecular characterization, localization and enzymatic activities.

Macrophage migration inhibitory factor (MIF) is an evolutionarily ancient cytokine-like factor and plays a critical role in both innate and adaptive immunity. In the present study, two MIFs (designed as RpMIF-1 and RpMIF-2, respectively) were identified and characterized from the clam Ruditapes philippinarum by rapid amplification of cDNA ends (RACE) approaches. The full-length cDNA of RpMIF-1 and RpMFI-2 consisted of 531 and 722 nucleotides, encoding a polypeptide of 113 and 114 amino acid residues, respectively. Multiple alignments and phylogenetic analysis revealed that both RpMIF-1 and RpMIF-2 belonged to the MIF family. The conserved catalytic-site Pro2 for tautomerase activity was identified in the deduced amino acid sequences of RpMIFs. Both RpMIF-1 and RpMIF-2 transcripts were constitutively expressed in examined tissues of R. philippinarum with dominant expression in hepatopancreas, gills and hemocytes. Immunolocalization analysis showed that RpMIF-1 and RpMIF-2 proteins were expressed in examined tissues with the exception of adductor muscle and foot. After Vibrio anguillarum and Micrococcus luteus challenge, the mRNA expression of RpMIFs was significantly modulated in hemocytes, gills and hepatopancreas. Recombinant RpMIF-1 and RpMIF-2 proteins possessed significant tautomerase activity and oxidoreductase activity, indicating that these two proteins was perhaps involved in inflammatory responses. In summary, our results suggested that RpMIF-1 and RpMIF-2 played an important role in the innate immunity of R. philippinarum.