Mengqiang Wang

A primitive Toll-like receptor signaling pathway in mollusk Zhikong scallop Chlamys farreri

As a member of pattern-recognition receptors (PRRs), the Toll-like receptor (TLR) and its signaling pathway play pivotal roles in recognizing various pathogen-associated molecular patterns (PAMPs), and buildup the front-line against invading pathogens. In the present study, the sequence features and mRNA expression profiles of five key genes involved in TLR signal pathway were characterized, and their functions in the immune responses were also investigated in order to validate the TLR signaling pathway and its potential roles in the immune defense of Zhikong scallop Chlamys farreri. These five genes, including CfTLR, CfMyD88, CfTRAF6, CfIκB and CfNFκB, exhibited significant similarity with their homologues from other model organisms, and contained the typical motifs. A strong interaction between the TIR domain from CfTLR and CfMyD88 protein was revealed via ELISA assays. The mRNA transcripts of these five genes were all up-regulated after LPS stimulation, indicating that they were involved in the immune response against LPS. When CfTLR expression was inhibited by RNAi technology, the mRNA expression level of CfMyD88, CfTRAF6, CfIκB, CfNFκB and G-type lysozyme were all decreased, while those of superoxide dismutase and catalase were increased. After Listonella anguillara challenge, the apoptosis level of those CfTLR-suppressed scallops was significantly lower than that in control groups (p<0.05) at the beginning of bacteria challenge, while the cumulative mortality was significantly higher than that of control groups (p<0.05). These results collectively favored that a rather canonical MyD88-dependent TLR pathway existed in scallop and this pathway was involved in immune signaling to active the diverse downstream reaction including anti-oxidant, anti-bacteria and apoptosis.

The immune system and its modulation mechanism in scallop.

Scallops are a cosmopolitan family of bivalves, and some of them are highly prized as dominant aquaculture species. In the past decades, there have been increasing studies on the basic biology and immunology of scallops, and this review summarizes the research progresses of immune system and its modulation mechanism in scallop. As invertebrate, scallops lack adaptive immunity and they have evolved an array of sophisticated strategies to recognize and eliminate various invaders by employing a set of molecules and cells. It is evident that basic immune reactions such as immune recognition, signal transduction, and effector synthesis involved in immune response are accomplished in a variety of ways. They rely upon an extensive repertoire of phagocytosis, apoptosis and encapsulation of the circulating hemocytes for eliminating invasive pathogens, as well as the production of immune effectors that are active against a large range of pathogens or sensitive for the environmental stress. Furthermore, the molecular constitutions, metabolic pathways and immunomodulation mechanisms of the primitive catecholaminergic, cholinergic, enkephalinergic system and NO system in scallop are also discussed, which can be taken as an entrance to better understand the origin and evolution of the neuroendocrine-immune regulatory network in lower invertebrates.

A novel C-type lectin from crab Eriocheir sinensis functions as pattern recognition receptor enhancing cellular encapsulation.

C-type lectins are a large family of Ca²⁺-dependent carbohydrate binding proteins which play crucial roles to recognize and eliminate pathogens in innate immunity. In the present study, a novel C-type lectin was identified from Eriocheir sinensis (designated as EsCTL). The full-length cDNA of EsCTL was of 789 bp with an open reading frame of 468 bp encoding a polypeptide of 156 amino acids with a signal sequence and single carbohydrate-recognition domain (CRD). The potential tertiary structure of the CRD adopted a typical double-loop structure with Ca²⁺-binding site 2 in the long loop region and two conserved disulfide bridges at the bases of the loops. An EPQ motif to determine carbohydrate binding specificity was identified in the CRD of EsCTL. The mRNA transcripts of EsCTL were mainly detected in hepatopancreas and its relative expression level in hemocytes was significantly up-regulated after the challenges of Vibrio anguillarum (P < 0.05) and Pichia pastoris (P < 0.05). The recombinant EsCTL protein (rEsCTL) could bind different PAMPs, including LPS, PGN, β-glucan, and polyI:C; and also bind various microorganisms including three Gram-positive bacteria, three Gram-negative bacteria and two yeasts. Moreover, rEsCTL could significantly enhance the in vitro encapsulation of crab hemocytes. All these results suggested that EsCTL functioned as an important PRR involved in immune defense against invading pathogen in crab.

The modulation of catecholamines to the immune response against bacteria Vibrio anguillarum challenge in scallop Chlamys farreri

Catecholamines are pivotal signal molecules in the neuroendocrine-immune regulatory network, and implicated in the modulation of immune response. In the present study, the activities of some immune-related enzymes and the concentration of catecholamines were determined in circulating haemolymph of scallops Chlamys farreri after bacteria Vibrio anguillarum challenge. The activities of superoxide dismutase (SOD), catalase (CAT) and lysozyme (LYZ) increased significantly and reached 610 U mg(-1) at 12 h, 37.6 U mg(-1) at 6 h and 261.5 U mg(-1) at 6 h after bacteria challenge, respectively. The concentration of norepinephrine, epinephrine and dopamine also increased significantly and reached 114.9 ng mL(-1) at 12 h, 86.9 ng mL(-1) at 24 h and 480.4 pg mL(-1) at 12 h after bacteria challenge, respectively. Meanwhile, the activities of these immune-related enzymes in haemolymph were monitored in those scallops which were challenged by bacteria V. anguillarum and stimulated simultaneously with norepinephrine, epinephrine and adrenoceptor antagonist. The injection of norepinephrine and epinephrine repressed significantly the induction of bacteria challenge on the activities of immune-related enzymes, and they were reduced to about half of that in the control groups. The blocking of α and β-adrenoceptor by antagonist only repressed the increase of CAT and LYZ activities significantly, while no significant effect was observed on the increase of SOD activities. The collective results indicated that scallop catecholaminergic neuroendocrine system could be activated by bacteria challenge to release catecholamines after the immune response had been triggered, and the immune response against bacteria challenge could been negatively modulated by norepinephrine, epinephrine, and adrenoceptor antagonist. This information is helpful to further understand the immunomodulation of catecholamines in scallops.

Immune response and energy metabolism of Chlamys farreri under Vibrio anguillarum challenge and high temperature exposure

The complex interactions among host, pathogen and environment are believed to be the main causes for the mass mortality of cultured scallops during summer period. In the present study, the temporal variations of immune and energy parameters of Chlamys farreri under Vibrio anguillarum challenge, higher temperature (29°C) exposure as well as their combined treatment were investigated in order to better understand the energetic mechanisms of scallop immune defense. After the treatments, the superoxide anion level, the activities of superoxide dismutase (SOD) and acid phosphatase, as well as heat shock protein 70 expression level in the hemolymph of scallops increased substantially within 48 h. And as time progressed, the malondialdehyde content in the serum of scallops in the higher temperature treated and the combined stress treated groups were significantly increased, while the SOD activity was significantly depressed (96 h, P<0.05). After 3 h, a significant decline (P<0.05) in glycogen reserves was observed in the examined tissues of all the scallops in the bacteria challenged, higher temperature treated and the combined stress treated groups. The cellular energy allocation (CEA) in the examined tissues dropped considerably when the treatments lasted 48 h. There was a significant decline in the CEA and a significant increase in the energy consumption in the examined tissues compared with other treatments when the scallops were exposed to the combined stress for 96 h (P<0.05). All the results demonstrated that the antioxidant systems and acute phase response system in scallops were not enough to wholly repair oxidative damage caused by higher temperature and the combined stress with bacteria challenge, and glycogen reserved in relative tissues were mobilized to meet the increased energy demands during the process of immune defense. Immune defense against the combined stress imposed greater costs on scallops energy expenditure than either stressor alone, and CEA could be a useful tool to evaluate energetic allocation. The information provided valuable insights into possible mechanisms of scallop mass mortalities during summer period.

An integrin from oyster Crassostrea gigas mediates the phagocytosis toward Vibrio splendidus through LPS binding activity

Integrins are a family of cell adhesion molecules which play important roles in the regulation of cell adhesion, migration, proliferation, apoptosis and phagocytosis. In the present study, the immune function of an integrin from the oyster Crassostrea gigas (designated CgIntegrin) was characterized to understand the regulatory mechanism of hemocyte phagocytosis toward different microbes. The full-length cDNA of CgIntegrin was 2571 bp with an open reading frame (ORF) of 2397 bp, encoding a polypeptide of 799 amino acids. The mRNA transcripts of CgIntegrin were predominantly detected in hemocytes, gonad and adductor muscle, while lowly in hepatopancreas, mantle and gill. The mRNA expression level was up-regulated at 6 h post lipopolysaccharide (LPS) stimulation (p < 0.01), while no significant change was observed after peptidoglycan (PGN) stimulation. The oyster hemocytes with relative high CgIntegrin expression level exhibited different phagocytic abilities towards different microorganism and particles, such as Gram-positive bacteria Vibrio splendidus, Gram-negative bacteria Staphylococcus aureus and latex beads. Moreover, the phagocytic rate towards V. splendidus was significantly decreased after the blockade of CgIntegrin using the polyclonal antibody. The recombinant CgIntegrin (rCgIntegrin) displayed agglutinating activity towards V. splendidus but not S. aureus and Y. lipolytica. It also exhibited a higher binding affinity towards LPS (compared to rTrx group) in a dose-dependent manner with the apparent dissociation constant (Kd) of 5.53 × 10(-6) M. The results indicated that CgIntegrin served as a pattern recognition receptor with LPS binding activity, which could directly bind to V. splendidus and enhance the phagocytosis of oyster hemocytes.

Maternal transfer of immunity in scallop Chlamys farreri and its trans-generational immune protection to offspring against bacterial challenge

Maternal immunity plays a crucial role in protecting the offspring at early stages of life and contributes a trans-generational effect on the offsprings phenotype. In the present study, maternal transfer of immunity and its trans-generational effect on offspring in scallop Chlamys farreri were investigated. The proteins including CfLGBP, CfLBP/BPI, CfLYZ and CfCu/Zn-SOD existed in the scallop eggs with high level while CfLec-3 was not detected. In contrast, the mRNA levels of these proteins were extremely low except that of CfCu/Zn-SOD. The protein extracts of scallop eggs exhibited remarkable agglutination activity and bactericidal effect against gram-negative bacteria Escherichia coli and Vibro anguillarum, and fungi Pichia pastoris. When the maternal scallops were stimulated with heat-killed V. anguillarum, the mRNA levels of CfLBP/BPI and CfLYZ in their offspring were expressed significant higher in D-shaped larvae. Furthermore, the protein levels of CfLBP/BPI and CfCu/Zn-SOD in the offspring of maternal immune stimulation group were higher than that of control at almost all the developmental stages, while the level of CfLec-3 and CfLYZ was higher than that of control just in eggs or trochophore, respectively. A significant enhancement of Cu/Zn-SOD and antibacterial activities was also observed in eggs, 4-cell embryos and trochophore of offspring from immune stimulated mother scallops. Moreover, the mortality of offspring from the immune stimulated mother scallops was significantly lower than that of control after bacterial challenge, especially in trochophore. The results indicated that scallop eggs or embryos received maternal derived immune competence to defense against the invading pathogens, and the maternal scallops received an immune stimulation endowed their offspring with a trans-generational immune capability to protect them against infections effectively.

The phenoloxidase activity and antibacterial function of a tyrosinase from scallop Chlamys farreri

Tyrosinase (TYR), also known as monophenol monooxygenase, is a ubiquitous binuclear copper-containing enzyme which catalyzes the hydroxylation of phenols to catechols and the oxidation of catechols to quinones. In the present study, the cDNA of a tyrosinase (CfTYR) was identified from scallop Chlamys farreri, which encoded a polypeptide of 486 amino acids. The CfTYR mRNA transcripts were expressed in all the tested tissues, including haemocytes, adductor muscle, kidney, hepatopancreas, gill, gonad and mantle, with the highest level in mantle. The expression level of CfTYR mRNA in haemocytes decreased significantly during 3-6 h after LPS stimulation, and reached the lowest level at 6 h (0.05-fold, P < 0.05). Then, it began to increase at 12 h (0.32-fold, P > 0.05), and reached the highest level at 24 h (2.91-fold, P < 0.05). At 3 h after LPS stimulation, the phenoloxidase activity catalyzing L-dopa and dopamine in haemolymph increased significantly to 53.13 and 40.36 U mg(-1) respectively, but it decreased to 10.82 U mg(-1) and even undetectable level after CfTYR activity was inhibited. Furthermore, the antibacterial activity of haemolymph against Escherichia coli was also increased significantly at 3 h after LPS stimulation, but it decreased significantly when the haemolymph was treated by TYR inhibitor. The recombinant protein of the mature CfTYR peptide expressed in the in vitro Glycoprotein Expression Kit displayed phenoloxidase activity of 64.36 ± 5.51 U mg(-1) in the present of trypsinase and Cu(2+). These results collectively suggested that CfTYR was a homologue of tyrosinase in scallop C. farreri with the copper-dependence phenoloxidase activity, and it could be induced after immune stimulation and mediate immune response for the elimination of invasive pathogens in scallop.

A novel scavenger receptor-cysteine-rich (SRCR) domain containing scavenger receptor identified from mollusk mediated PAMP recognition and binding

Scavenger receptors (SRs) are significant endocytic receptors contributing to constant internal environment. SR-cysteine-rich (SRCR) domain-containing SR is the most important class of SRs which has been so far reported exclusively in mammals and birds. In the present study, a novel SRCR domain-containing SR (CfSR) was firstly identified from scallop Chlamys farreri. The full-length cDNA of CfSR was of 2639 bp encoding a polypeptide of 804 amino acids with a signal peptide, six SRCR domains, a UPAR-like domain and a ShK toxin-like domain. All the SRCR domains contain highly conserved six cysteine residues to form three pairs of intradomain disulfide, among which SRCR-D5 was assumed to participate in ligand-binding. An attachment site of sequence CTTPLCN was found in UPAR-like domain, indicating CfSR was an anchor protein. This prediction was confirmed by its localization on the outer surface of hemocytes with immunofluorescence assay. The mRNA expression of CfSR was up-regulated significantly by the stimulations of lipopolysaccharides, peptidoglycan and β-glucan. A truncated CfSR (from V⁴⁵⁶ to T⁸⁰⁴) including SRCR-D5 was recombined and expressed in Escherichia coli, and the recombined protein displayed unique broad ligand-binding properties not only for acetylated low density lipoprotein (Ac-LDL) and dextran sulfate, but also for various pathogen associated molecular patterns, such as LPS, PGN, mannan and zymosan. All the results indicated that CfSR, the most primitive SR identified to date, was a versatile PRR involved in immune recognition, and the existence of functional SR might trace back to at least mollusk phylum.

Protective immunity induced by CpG ODNs against white spot syndrome virus (WSSV) via intermediation of virus replication indirectly in Litopenaeus vannamei

The worldwide shrimp culture is beset with diseases mainly caused by white spot syndrome virus (WSSV) and suffered huge economic losses, which bring out an urgent need to develop the novel strategies to better protect shrimps against WSSV. In the present study, CpG-rich plasmid pUC57-CpG, plasmid pUC57 and PBS were employed to pretreat shrimps comparatively to evaluate the protective effects of CpG ODNs on shrimps against WSSV. The survival rates, WSSV copy numbers, and antiviral associated factors (Dicer, Argonaute, STAT and ROS) were detected in Litopenaeus vannamei. There were higher survival proportion, lower WSSV copy numbers, and higher mRNA expression of Dicer and STAT in pUC57-CpG-pretreatment shrimps than those in pUC57- and PBS-pretreatment shrimps after WSSV infection. The Argonaute mRNA expression in pUC57-CpG-, pUC57- and PBS-pretreatment shrimps after WSSV infection was significantly higher than that of shrimps post PBS stimulation on the first day. The ROS levels in pUC57-CpG-pretreatment shrimps post secondary stimulation of PBS were significantly higher than those post WSSV infection on the first day. These results together demonstrated that pUC57-CpG induced partial protective immunity in shrimps against WSSV via intermediation of virus replication indirectly and could be used as a potential candidate in the development of therapeutic agents for disease control of WSSV in L. vannamei.