A C-type lectin with a single carbohydrate-recognition domain (CRD) containing unique QPN/WDD motifs from Tegillarca granosa is involved in the innate immune defense.

C-type lectins (CTLs), a superfamily of Ca2+-dependent carbohydrate-recognition proteins, serve as pattern recognition receptors (PRRs) in the immune response of many species. However, little is currently known about the CTLs of the commercially and ecologically important bivalve species, blood clam (Tegillarca granosa). In this study, a CTL (designated as TgCTL-1) with a single carbohydrate-recognition domain (CRD) containing unique QPN/WDD motifs was identified in the blood clam through transcriptome and whole-genome searching. Multiple alignment and phylogenetic analysis strongly suggested that TgCTL-1 was a new member of the CTL superfamily. Expression analysis demonstrated that TgCTL-1 was highly expressed in the hemocytes and visceral mass of the clam under normal condition. In addition, the expression of TgCTL-1 was shown to be significantly up-regulated upon pathogen challenge. Moreover, the recombinant TgCTL-1 (rTgCTL-1) displayed agglutinating and binding activities against both the gram-positive and gram-negative bacteria tested in a Ca2+-dependent manner. Furthermore, it was found that the in vitro phagocytic activity of hemocytes was significantly enhanced by rTgCTL-1. In general, our results showed that TgCTL-1 was an inducible acute-phase secretory protein, playing crucial roles in recognizing, agglutinating, and binding to pathogenic bacteria as well as modulating phagocytic activity of hemocytes in the innate immune defense of blood clam.

Identification, characterization, and agglutinating activity of a novel C-type lectin domain family 3 member B (CLEC3B) discovered in golden pompano, Trachinotus ovatus.

The lectins are a large family of carbohydrate-binding proteins that play important roles in the innate immune response of various organisms. Although C-type lectin domain family 3 member B (CLEC3B), an important member of C-type lectin, has been well documented in humans and several other higher vertebrates, little is currently known about this molecule in economically important marine fish species. In this study, through transcriptomic and BLAST screening, a novel CLEC3B gene was identified in the golden pompano (Trachinotus ovatus). The T. ovatus CLEC3B (ToCLEC3B) was subsequently characterized by bioinformatic analysis and compared with those reported in other species. In addition, the expression patterns of ToCLEC3B in different tissues under normal condition and at different times post pathogen challenge were assessed. Furthermore, the agglutinating activity of ToCLEC3B with and without Ca2+ against different bacteria and blood cells of donor species were verified using the recombinant T. ovatus CLEC3B (rToCLEC3B). Our results demonstrated that ToCLEC3B is a Ca2+-dependent galactose-binding lectin with a single copy of carbohydrate recognition domain (CRD). Similar to CLEC3B reported in other species, the CRD domain of ToCLEC3B consists of two α-helices, six ß-sheets, and four loops, forming two Ca2+- and a galactose-binding sites. According to the phylogenetic analysis, the ToCLEC3B was highly similar (similarity at 95.00%) to that of its relative, the greater amberjack (Seriola dumerili). The expression of ToCLEC3B was detected in all tissues examined under normal condition and was significantly up-regulated by injection of pathogenic microbes. In addition, the rToCLEC3B exhibited strong agglutinating activity against different bacteria and blood cells of donor species in the presence of Ca2+. Our results indicate that ToCLEC3B is a constitutive and inducible acute-phase immune factor in the host's innate immune response of T. ovatus.

Assessment of Nonalcoholic Fatty Liver Disease Symptoms and Gut-Liver Axis Status in Zebrafish after Exposure to Polystyrene Microplastics and Oxytetracycline, Alone and in Combination.

BACKGROUND: Environmental pollution may give rise to the incidence and progression of nonalcoholic fatty liver disease (NAFLD), the most common cause for chronic severe liver lesions. Although knowledge of NAFLD pathogenesis is particularly important for the development of effective prevention, the relationship between NAFLD occurrence and exposure to emerging pollutants, such as microplastics (MPs) and antibiotic residues, awaits assessment. OBJECTIVES: This study aimed to evaluate the toxicity of MPs and antibiotic residues related to NAFLD occurrence using the zebrafish model species. METHODS: Taking common polystyrene MPs and oxytetracycline (OTC) as representatives, typical NAFLD symptoms, including lipid accumulation, liver inflammation, and hepatic oxidative stress, were screened after 28-d exposure to environmentally realistic concentrations of MPs (0.69mg/L) and antibiotic residue (3.00µg/L). The impacts of MPs and OTC on gut health, the gut-liver axis, and hepatic lipid metabolism were also investigated to reveal potential affecting mechanisms underpinning the NAFLD symptoms observed. RESULTS: Compared with the control fish, zebrafish exposed to MPs and OTC exhibited significantly higher levels of lipid accumulation, triglycerides, and cholesterol contents, as well as inflammation, in conjunction with oxidative stress in their livers. In addition, a markedly smaller proportion of Proteobacteria and higher ratios of Firmicutes/Bacteroidetes were detected by microbiome analysis of gut contents in treated samples. After the exposures, the zebrafish also experienced intestinal oxidative injury and yielded significantly fewer numbers of goblet cells. Markedly higher levels of the intestinal bacteria-sourced endotoxin lipopolysaccharide (LPS) were also detected in serum. Animals treated with MPs and OTC exhibited higher expression levels of LPS binding receptor (LBP) and downstream inflammation-related genes while also exhibiting lower activity and gene expression of lipase. Furthermore, MP-OTC coexposure generally exerted more severe effects compared with single MP or OTC exposure. DISCUSSION: Our results suggested that exposure to MPs and OTC may disrupt the gut-liver axis and be associated with NAFLD occurrence. https://doi.org/10.1289/EHP11600.

Impacts of dietary supplementation of bamboo vinegar and charcoal powder on growth performance, intestinal morphology, and gut microflora of large-scale loach Paramisgurnus dabryanus.

Although accumulating data demonstrate that dietary supplementation of bamboo vinegar and charcoal powder (BVC) can significantly strengthen the immunity and boost the growth of domestic animals, its application potency still awaits verification in commercial fish species such as the large-scale loach Paramisgurnus dabryanus. The impacts of 90-day dietary supplementation of 1% and 2% BVC on survival and growth performance of the loach, and on the intestinal morphological characteristics and gut microflora were analyzed. Our data show that the large-scale loach supplied with BVC at the experimental doses had significant higher survival rates and better growth performance (indicated by greater weight gain (1.13-1.14 times), higher specific growth rate (1.04 times), and lower feed conversion ratio (0.88-0.89 times)) compared to that of the control (P<0.05). Histological examination revealed significant longer villus (3.22-5.54 times), deeper crypt (1.77-1.87 times), and thicker muscle (1.59-3.17 times) in the intestines of large-scale loach fed with BVC (P<0.05). Furthermore, we found that the gut microflora consisted of significantly fewer proportions of potential pathogenic bacterial species (Aeromonas veronii and Escherichia coli), but significantly greater proportions of beneficial microbes (Lactococus raffinolactis and Faecalibacterium prausnitzii). Therefore, dietary intake of BVC can promote intestinal tract development and optimize gut microflora, by which the survival and growth of large-scale loach may be improved.

Gamma-aminobutyric acid (GABA) suppresses hemocyte phagocytosis by binding to GABA receptors and modulating corresponding downstream pathways in blood clam, Tegillarca granosa.

Although accumulating data demonstrated that gamma-aminobutyric acid (GABA), an inhibitory neurotransmitter, plays an important regulatory role in immunity of vertebrates, its immunomodulatory function and mechanisms of action remain poorly understood in invertebrates such as bivalve mollusks. In this study, the effect of GABA on phagocytic activity of hemocytes was evaluated in a commercial bivalve species, Tegillarca granosa. Furthermore, the potential regulatory mechanism underpinning was investigated by assessing potential downstream targets. Data obtained demonstrated that in vitro GABA incubation significantly constrained the phagocytic activity of hemocytes. In addition, the GABA-induced suppression of phagocytosis was markedly relieved by blocking of GABAA and GABAB receptors using corresponding antagonists. Hemocytes incubated with lipopolysaccharides (LPS) and GABA had significant higher K+-Cl- cotransporter 2 (KCC2) content compared to the control. In addition, GABA treatment led to an elevation in intracellular Cl-, which was shown to be leveled down to normal by blocking the ionotropic GABAA receptor. Treatment with GABAA receptor antagonist also rescued the suppression of GABAA receptor-associated protein (GABARAP), KCC, TNF receptor associated factor 6 (TRAF6), inhibitor of nuclear factor kappa-B kinase subunit alpha (IKKα), and nuclear factor kappa B subunit 1 (NFκB) caused by GABA incubation. Furthermore, incubation of hemocytes with GABA resulted in a decrease in cAMP content, an increase in intracellular Ca2+, and downregulation of cAMP-dependent protein kinase (PKA), calmodulin kinase II (CAMK2), calmodulin (CaM), calcineurin (CaN), TRAF6, IKKα, and NFκB. All these above-mentioned changes were found to be evidently relieved by blocking the metabotropic G-protein-coupled GABAB receptor. Our results suggest GABA may play an inhibitory role on phagocytosis through binding to both GABAA and GABAB receptors, and subsequently regulating corresponding downstream pathways in bivalve invertebrates.

Identification, characterization, and antimicrobial activity of a novel big defensin discovered in a commercial bivalve mollusc, Tegillarca granosa.

Molluscs, the second largest animal phylum on earth, primarily rely on cellular and humoral immune responses to fight against pathogen infection. Although antimicrobial peptides (AMPs) such as big defensin play crucial roles in the humoral immune response, it remains largely unknown in the ecological and economic important blood clam (Tegillarca granosa). In this study, a novel big defensin gene (TgBD) was identified in T. granosa through transcripts and whole genome searching. Bioinformatic analyses were conducted to explore the molecular characteristics of TgBD, and comparisons of TgBD with those reported in other molluscs were performed by multiple alignments and phylogenetic analysis. In addition, the expression patterns of TgBD in various tissues and upon bacterial challenge were investigated while the antimicrobial activity of synthetic N-terminal domain of TgBD was confirmed in vitro by radial diffusion experiment. Results obtained showed TgBD had an open reading frame (ORF) of 369 bp, encoding a prepropeptide containing a signal peptide and a propeptide. Similar to big defensins reported in other species, TgBD consists of a hydrophobic N-terminal domain containing ß1-α1-α2-ß2 folds and a cysteine-rich cationic C-terminal domain with three disulfide bonds between C1-C5, C2-C4, and C3-C6. Phylogenetic analysis showed that TgBD shared 76.80% similarity to its close relative ark shell (Scapharca broughtoni). In addition, TgBD expression was observed in all tissues investigated under normal conditions and was significantly induced by injection of Vibrio parahaemolyticus. Furthermore, synthetic N-terminal peptide of TgBD exhibited strong antimicrobial activity against Gram-positive bacteria tested. Our results indicated that TgBD is a constitutive and inducible acute phase AMP, which provides a universal and prompt protection for T. granosa.

Modulatory function of calmodulin on phagocytosis and potential regulation mechanisms in the blood clam Tegillarca granosa.

Unlike vertebrate species, invertebrates lack antigen-antibody mediated immune response and mainly rely on haemocyte phagocytosis to fight against pathogen infection. Recently, studies conducted in model vertebrates demonstrated that the multifunctional protein calmodulin (CaM) plays an important role in regulating immune responses. However, the intrinsic relation between CaM and phagocytosis process remains poorly understood in invertebrate species such as bivalve mollusks. Therefore, in the present study, the immunomodulatory function of CaM on haemocyte phagocytosis was verified in the blood clam, Tegillarca granosa, using the CaM-specific inhibitor N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7). Results obtained show that CaM inhibition significantly suppressed the phagocytic activity of haemocytes. In addition, CaM inhibition constrained intracellular Ca2+ elevation, hampered actin cytoskeleton assembly, suppressed calcineurin (CaN) activity, and disrupted NF-κB activation in haemocytes upon LPS induction. Furthermore, expression of seven selected genes from the actin cytoskeleton regulation- and immune-related pathways were significantly downregulated whereas those of CaM and CaN from the Ca2+-signaling pathway were significantly upregulated by in vitro incubation of haemocytes with W-7. For the first time, the present study demonstrated that CaM play an important role in phagocytosis modulation in bivalve species. In addition, the intracellular Ca2+ and downstream Ca2+-signaling-, actin cytoskeleton regulation-, and immune-related pathways offer candidate routes through which CaM modulates phagocytosis.

Acetylcholine suppresses phagocytosis via binding to muscarinic- and nicotinic-acetylcholine receptors and subsequently interfering Ca2+- and NFκB-signaling pathways in blood clam.

Though immunomodulation via cholinergic neurotransmitter acetylcholine (ACh), an important part of neuroendocrine-immune (NEI) regulatory network, has been well established in vertebrate species, the mechanisms remain poorly understood in invertebrates. In the present study, the immunomodulatory effect of ACh on haemocyte phagocytosis was investigated in an invertebrate bivalve species, Tegillarca granosa. Data obtained showed that in vitro ACh incubation suppressed phagocytic activity of haemocytes along with a significant elevation in intracellular Ca2+. In addition, the expressions of genes from Ca2+ signaling pathway were significantly induced whereas those from NF-κB signaling pathway were significantly down-regulated by ACh incubation. Furthermore, these adverse impacts of ACh were significantly relieved by the blocking of muscarinic acetylcholine receptors (mAChRs) or nicotinic acetylcholine receptors (nAChRs) using corresponding antagonists. Our study suggests that ACh suppresses phagocytosis via binding to both mAChRs and nAChRs, which disrupts intracellular Ca2+ homeostasis and subsequently interferes with downstream Ca2+ and NF-κB signaling pathways.