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Because invertebrates lack acquired immunity, they rely primarily on the innate immune system to defend themselves against viral and bacterial infections. SVWC, also called Vago, is a class of small-molecule proteins characterized by a single von Willebrand factor C-domain and appears to be restricted to arthropods. It has been reported that SVWC is involved in antiviral immunity in invertebrates, but whether it is involved in antimicrobial immunity and the mechanism of its involvement in antimicrobial immunity remains unclear. In this study, we identified a novel SVWC gene in Eriocheir sinensis and named it EsSVWC. EsSVWC was found to respond positively to bacterial stimulation and to regulate the expression of related antimicrobial peptides (AMPs). The EsSVWC protein recognized and bound to a variety of pathogen-associated molecular patterns (PAMPs) but did not exhibit direct bactericidal effects. Thus, the EsSVWC protein in crabs helps resist bacterial infection and improve survival rates. In summary, EsSVWC may regulate the innate immune system of crabs in response to microbial invasion in an indirect manner. • We identified and characterized a new SVWC gene, EsSVWC , in Eriocheir sinensis. • EsSVWC regulates antimicrobial peptide expression in response to bacterial stress. • EsSVWC binds to bacteria by recognizing pathogen-associated molecular patterns. • EsSVWC does not have a direct bactericidal or bacterial growth inhibitory effect. • EsSVWC is indirectly involved in the innate antibacterial response in E. sinensis. [ABSTRACT FROM AUTHOR]

Bivalves have evolved effective strategies to combat different pathogens in the environment. They rely on innate immunity to deal with the invasion of various bacteria, viruses, and other microorganisms. However, the molecular mechanisms underlying the responses remain largely unknown. Herein, we constructed 21 transcriptomes of the hemocytes after lipopolysaccharide (LPS), peptidoglycan (PGN) and polyinosinic-polycytidylic acid (poly(I:C)) stimulation to investigate the molecular mechanisms underlying adaptations and plastic responses to different pathogen-related molecular patterns (PAMPs) in pearl oyster Pinctada fucata martensii. Transcriptome analysis revealed 1986–3427 responsive genes enriched in the major immune and cell cycle-related pathways at different times after PAMP stimulation, and the expression patterns of genes under these pathways are complex and diverse. Moreover, "lysosomes" were enriched 6 h after LPS and PGN stimulation, while "peroxisomes" were only enriched in poly(I:C) group. These results suggest different response strategies of pearl oyster to different PAMPs. Furthermore, we identified 261 pattern-recognition receptors (PRRs) including 4 retinoic acid-inducible gene I-like receptors, 38 NOD-like receptors, 83 Toll-like receptors, and 136 C-type lectins in the genome of P. f. martensii. The diverse expression patterns of these PRRs after different PAMP stimulation indicated that pearl oyster evolved complex and specific recognition systems due to tandem repeat and diverse domain combination, which may help pearl oyster cope with the different pathogens in the environment. The present study improved our understanding of the molecular response of pearl oyster to different PAMP stimulation. • Immune and cell cycle-related pathways were enriched after each pathogen-related molecular pattern (PAMP) stimulation. • Pearl oyster have different response strategies to different PAMPs. • 261 pattern recognition receptors (PRRs) were identified in Pinctada fucata martensii. • The expression patterns of PRRs after PAMPs stimulation are diverse. • Pearl oyster evolved complex and specific recognition systems due to tandem repeat and diverse domain combinations of RPPs. [ABSTRACT FROM AUTHOR]

CL-11 (Collectin-11, also known as Collectin kidney-1 or CL-K1) is a member of collectin family that works as a pattern recognition molecule (PRM) and participating in lectin-complement pathway in host defense against pathogens. We identified the CL-11 homologue SsCL-11 in black rockfish (Sebastes schlegelii) and investigated the functional characteristics in this study. The SsCL-11 has conserved protein modules, i.e. an N-terminal hydrophobic region, a collagen-like region, an α-helical neck region and a carbohydrate recognition domain (CRD). SsCL-11 has varying degrees of expressions in difference tissues, among which the highest expression is observed in liver. It also shows induced expressions in immune-related tissues following Aeromonas salmonicida (A. salmonicida) infection. In addition, SsCL-11 exhibits binding abilities to different kinds of carbohydrates, pathogen-associated molecular patterns (PAMPs) and bacteria. It exhibits comparatively strong binding to l -fucose, d -mannose, and d -glucose, which is consistent with the functional EPN motif in its CRD. SsCL-11 also shows agglutinating effects on various bacteria in the presence of Ca2+. Furthermore, SsCL-11 is confirmed to be a secretory lectin and can form multimers. These findings collectively demonstrate that SsCL-11 can function as a recognition molecule in pathogen resistance in black rockfish, which will promote our understanding of immunological roles of fish collectins. • SsCL-11 is a secretory protein. • SsCL-11 can bind carbohydrates, PAMPs and pathogenic bacteria in a dose-dependent manner. • SsCL-11 can agglutinate bacteria in the presence of Ca2+. • SsCL-11 can form multimers. [ABSTRACT FROM AUTHOR]

Peptidoglycan recognition proteins (PGRPs) belong to the pattern recognition receptor (PRR) family and are conserved from insects to mammals. PGRPs show specific binding abilities to peptidoglycans (PGNs) in various microbes. In this study, molecular and functional analyses of PGRP-SC2 from Amphiprion clarkii (AcPGRP-SC2) were conducted. The 492 bp ORF of AcPGRP-SC2 encoded a protein of 164 amino acids with a molecular weight of 17.58 kDa and pI of 8.9. The PGRP superfamily domain was identified from the protein sequence of AcPGRP-SC2 and sequence similarities were observed with homologous proteins. Quantitative polymerase chain reaction (qPCR) analysis revealed that AcPGRP-SC2 transcripts were ubiquitously expressed in all tested tissues, with high levels in the skin, and transcript expression was significantly modulated by immune stimulation with lipopolysaccharide (LPS), Polyinosinic:polycytidylic acid (poly I:C), and Vibrio harveyi post-immune challenge. Recombinant AcPGRP-SC2 with the maltose-binding protein fusion (rAcPGRP-SC2) was used to evaluate LPS-, PGN-, and bacterial-binding activities and to conduct bacterial agglutination assays, and the results demonstrated that AcPGRP-SC2 exhibited bacterial recognition, binding, and colonization abilities to a range of Gram-positive and Gram-negative bacterial strains. Moreover, rAcPGRP-SC2-pre-treated Fat Head Minnow (FHM) cells exhibited significant upregulation in NF-ĸB1 , NF-ĸB2 , and stat3 expression upon treatment with killed bacteria. Taken together, our findings suggest that AcPGRP-SC2 plays an important role in the immune response against microbial pathogens in A. clarkii. • Peptidoglycan recognition protein SC2 from Amphiprion clarkii (AcPGRP-SC2) was identified and characterized. • The expression of AcPGRP-SC2 was ubiquitous in Amphiprion clarkii tissues and was modulated upon immune challenges. • rAcPGRP-SC2 demonstrated the detectable LPS-, PGN-, and bacterial-binding activity and bacterial agglutination. • The bacterial-induced NF-κB1, NF-κB2, and stat3 expressions were significantly upregulated by rAcPGRP-SC2 in FHM cells. [ABSTRACT FROM AUTHOR]

Polyinosinic-polycytidylic acid (poly I:C) is a synthetic analog of double-stranded RNA (dsRNA) that activates anti-infective innate immunity. The underlying mechanisms are identified as targeting pattern recognition receptors and Th1-inducing. However, whether poly I:C manipulates metabolism to implement this anti-infective function is unknown. Here, GC-MS based metabolomics was used to characterize metabolic profiles induced by different doses of poly I:C. Analysis on the dose-dependent metabolomes shows that elevation of the TCA cycle and malate with the increasing dose of ploy I:C forms the most characteristic feature of the poly I:C stimulation. Exogenous malate activates the TCA cycle and elevates survival of zebrafish infected with Vibrio alginolyticus , which is related to the elevated expression of il-1b, il-6, il-8, tnf-a , and c3b. These results reveal a previously unknown regulation of poly I:C that boosts the TCA cycle to enhance innate immunity against bacterial infection. • Metabolic profiles induced by different doses of poly I:C. was characterized. • Elevated TCA cycle and malate forms the most characteristic feature. • Malate boosts TCA cycle and elevates zebrafish survival post bacterial infection. • This is related to the elevated expression of il-1b, il-6, il-8, tnf-a , and c3b. • These reveal a metabolic modulation of poly I:C against bacterial infection. [ABSTRACT FROM AUTHOR]

The claudin family of proteins are pivotal components of tight junction (TJ) participating in the epithelial barrier function in fish. Our previous studies indicated that one of the claudins, claudin-4-like (OmCLDN4L) was differentially expressed in rainbow trout (Oncorhynchus mykiss) spleen post infection of Flavobacterium psychrophilum , which is the causative pathogen of bacterial coldwater disease (BCWD). However, little is known about the function of OmCLDN4L in rainbow trout against bacterial infection. In the present study, the OmCLDN4L was identified and functionally characterized from rainbow trout. The OmCLDN4L has an open reading frame (ORF) of 668 bp, encoding a 22.86 kDa four-transmembrane protein with function of bicellular tight junction and apical tight junction. OmCLDN4L has the highest similarity with CLDN28a, CLDN28b and CLDN30 in amino acid sequence. Phylogenetic analysis showed that all of CLDN4 and CLDN4-like from fish clustered together but diverged from their counterparts in mammals, with main differences lying in their N-terminus. RT-qPCR results indicated that OmCLDN4L was constitutively expressed in all tissues investigated under healthy conditions, primarily in mucus, liver, skin and intestine. The expression of OmCLDN4L in rainbow trout intestine was slightly down-regulated at day 1 while up-regulated at day 3 and day 7 post F. psychrophilum infection, with the similar profiling of CLDN30 and CLDN10e. The expression level of inflammatory cytokines TNF-α , IL4/13A , IL-6 and pattern recognition receptor TLR-2 showed the same trend with OmCLDN4L in the intestine at day 3 and day 7 post F. psychrophilum infection. Collectively, these findings demonstrate that OmCLDN4L participates in the immune response to bacterial infection, offering new insights into the molecular mechanism of intestinal barrier in rainbow trout against F. psychrophilum infection. • OmCLDN4L was identified and functionally characterized from rainbow trout. • OmCLDN4L was constitutively expressed in all tissues investigated, primarily in mucus, liver, skin and intestine. • The expression of OmCLDN4L was induced in rainbow trout intestine post F. psychrophilum invasion. • OmCLDN4L has the similar expression profiling with CLDN30 and CLDN10e in rainbow trout. • OmCLDN4L and its related genes were up-regulated in rainbow trout post F. psychrophilum infection. [ABSTRACT FROM AUTHOR]

Leucine-rich repeat (LRR)-only proteins have been proved to be involved in the innate immune responses as they could mediate protein-protein or protein-ligand interactions. In the present study, a novel LRR-only protein (Lv LRRop-1) was identified and characterized from Pacific white shrimp Litopenaeus vannamei. The complete cDNA sequence of Lv LRRop-1 contains an open reading frame (ORF) of 1488 bp, which encoded a polypeptide of 495 amino acids with a predicted molecular mass of 55.67 kDa and a calculated theoretical isoelectric point of 6.435. There are five LRR motifs, six LRR_TYP motifs in the protein sequence of Lv LRRop-1 with consensus signature sequences of LxxLxxLxLxxNxL. The Lv LRRop-1 mRNA transcripts could be detected in all the tested tissues, including eyestalk, gill, gonad, heart, hemocytes, hepatopancreas, intestine, muscle, nerve and stomach, especially highest in hemocytes and hepatopancreas. The mRNA transcripts of Lv LRRop-1 increased within the first 6 h in hemocytes and hepatopancreas after Vibrio parahaemolyticus or white spot syndrome virus (WSSV) challenges. The recombinant Lv LRRop-1 could bind four typical pathogen-associated molecular patterns (PAMPs), including lipopolysaccharide (LPS), peptidoglycan (PGN), glucan (GLU) and polycytidine-polycytidylic acid (poly IC), in a dose-dependent manner, and inhibit the growth of bacteria Micrococcus luteu s. These data indicated that Lv LRRop-1 could play a pivotal role in the innate immune response of shrimps as a kind of pattern recognition receptor (PRR). • Molecular characterization of a novel LRR-only protein in Litopenaeus vannamei • Its expression profiles and pattern recognition ligands spectrum were investigated • It could inhibit the growth of bacteria Micrococcus luteu s [ABSTRACT FROM AUTHOR]

Lectins are important pattern recognition receptors (PRRs) and their immunological action is related to the recognition of glycans present in the pathogen cells surface. The lectins described for Litopenaeus vannamei are divided into C-type, L-type and galectin, which are mainly expressed in hepatopancreas and hemocytes. They are involved in several immune response pathways, such as phagocytosis, hemocytes recruitment, prophenoloxidase activation, and gene regulation. Although lectins have multiple immune functions, most experimental challenges focus only on WSSV and Vibrio sp. This article is a detailed review on the role of lectins in L. vannamei immune system, bringing together information on molecular structure, temporal and special expression and immune function, highlighting the wide participation of these molecules in shrimp innate immune system. • All known Litopenaeus vannamei lectins can be classified into three categories. • L. vannamei lectins are mainly expressed in hepatopancreas, gills and hemolymph. • Many L. vannamei lectins are upregulated after pathogen challenges. • L. vannamei lectins are involved in several immune pathways. • L. vannamei lectins are involved in response against bacteria and viruses. [ABSTRACT FROM AUTHOR]

Galectins are β-galactoside sugar binding proteins which function as important pattern recognition receptors (PRRs) in innate immunity. Here, we identified a galectin-9 gene from koi carp (Cyprinus carpio), named kGal-9. The ORF of kGal-9 is 963 bp in length, which encodes a polypeptide of 320 amino acids without either signal peptide. The predicted molecular weight is 36.25 kDa, and the isoelectric point is 8.3. Multiple sequence alignment showed that the putative kGal-9 contains two carbohydrate recognition domains (CRD), which are conserved in Galectin-9s. The phylogenetic tree showed that kGal-9 clustered to Galectin-9s from other teleosts, and shared the highest identity of 87.5% with Qihe crucian (Carassius auratus). kGal- 9 mRNA was abundant in head kidney, gills, and gut, but low in liver and muscle. Further, the expression level of kGal-9 in the head kidney and liver increased significantly after Aeromonas veronii (abbreviated A.v) infection. Unexpectedly, kGal-9 showed a remarkable downregulation in the spleen at various time points post A.v infection. Intramuscular injection of pckGal-9 not merely reduced the bacterial load of spleen tissue, but also improved the survival rate of koi carp post A.v challenge. Besides, administration of pckGal-9 stimulated the expression of several immuno-related genes including proinflammatory cytokines (IL-1β , IL-6), anti-inflammatory cytokine (IL-10), complement components (C4 , C9), with fluctuation in spleen and head kidney. Taken together, the obtained results suggest that kGal-9 occupies an important role in innate immunity and defense against bacterial infection in koi carp. • Galectin-9 was characterized in koi carp, and the eukaryotic expression plasmid pckGal-9 was constructed. • kGal-9 was abundant in head kidney, gill, and intestine and up-regulated in head kidney and liver by bacteria A.v infection. • IAdministration of pckGal-9 reduced the splenic bacterial load, but also improved the survival rate of koi post A.v challenge. • Injection of pckGal-9 stimulated the expression of IL-1β, IL -6, IL -10, C4, C9 , with fluctuation in spleen and head kidney. [ABSTRACT FROM AUTHOR]

Ficolin, a member of the fibrinogen-related proteins family (FREPs), functions as a pattern recognition receptor (PRR) in vertebrates and in invertebrates as a novel lectin. In this study, we discovered the Ficolin homolog of Chinese mitten crab (Eriocheir sinensis), which we named Es Ficolin. The obtained sequence showed that it has a highly conserved C-terminal fibrinogen-related domain (FReD) and a coiled-coil structure for trimer formation. Es Ficolin was up-regulated in hemocytes after being stimulated by bacteria. Recombinant Es Ficolin protein binds to gram-negative and gram-positive bacteria and agglutinates bacteria through pathogen-associated molecular patterns. In-depth study found that recombinant Es Ficolin could effectively remove bacteria and showed direct antibacterial activity. Es Ficolin could also promote the phagocytosis of hemocytes to enhance bacterial clearance. These findings suggest that Es Ficolin plays an important role in the crab antibacterial immune response. • A Chinese mitten crab ficolin homolog, Es Ficolin, was identified. • Es Ficolin was upregulated in hemocytes after bacterial infection. • Es Ficolin binds and agglutinates bacteria via pathogen-associated molecular patterns. • Es Ficolin showed direct antibacterial activity via hemocyte-induced phagocytosis. • Es Ficolin has an important function in the crab antibacterial immune response. [ABSTRACT FROM AUTHOR]

The LGP2 (Laboratory of Genetics and Physiology 2) protein is a member of the retinoic acid-inducible gene I (RIG-I)-like receptor (RLRs) family, which is a class of antiviral pattern recognition receptors located in the cytoplasm. However, few studies have investigated the function of LGP2 in invertebrates. In this study, the complete coding sequence of the LGP2 gene of the Pacific oyster , Crassostrea gigas, was obtained and named CgLGP2-like. Sequence analysis revealed that CgLGP2-like encodes 803 amino acids, and the encoded protein contains a DEXDc, HELICc, and C-terminal regulatory domains. Multiple sequence alignment demonstrated that the sequences of these key protein functional domains were relatively conserved. Phylogenetic analysis revealed that CgLGP2-like was a new member of the animal LGP2 family. Quantitative real-time PCR results showed that CgLGP2-like mRNA was expressed in all tested oyster tissues, with the highest expression observed in the labial palpus and digestive glands. CgLGP2-like expression in gill tissues was significantly induced after the poly(I:C) challenge. Furthermore, multiple IRF and NF-κB binding sites were identified in the CgLGP2-like promoter region, which may be one of the reasons why CgLGP2-like responds to poly(I:C) stimulation. Finally, the results of dual-luciferase reporter gene assays revealed that overexpression of CgLGP2-like may have a regulatory effect on the human IFN , AP-1 , and oyster CgIL-17 genes in HEK293T cells. Overall, our results preliminarily elucidate the immune functions of invertebrate LGP2 protein and provide valuable information for the development of comparative immunology. • CgLGP2-like was identified and characterized in the Pacific oyster. • CgLGP2-like expression was induced by poly(I:C) challenge. • CgLGP2-like may have a regulatory effect on the human IFN , AP-1 , and oyster CgIL-17 genes. [ABSTRACT FROM AUTHOR]

The sea cucumber Apostichopus japonicus is one of the most dominant and economically important aquaculture species in China. Saponin, which possesses notable biological and pharmacological properties, is a key determinant of the nutritional and health value of A. japonicus. In the present study, we amplified the full-length cDNA of a phosphomevalonate kinase (PMK) gene (named AjPMK) using rapid amplification of cDNA ends (RACE). Subsequently, we engineered a recombinant AjPMK (rAjPMK) protein and assessed its enzymatic activity by enzyme-linked immunosorbent assay (ELISA). Proteins that interact with rAjPMK were screened and identified via pull-down assay combined with liquid chromatography with tandem mass spectrometry (LC-MS/MS). We found that the full-length cDNA of AjPMK contained 1354 bp and an open reading frame (ORF) of 612 bp. The AjPMK protein was predicted not to contain a signal peptide but to contain a phosphonolate kinase domain seen in higher eukaryotes and a P-loop with a relatively conserved nucleoside triphosphate hydrolase domain. The molecular weight of the AjPMK protein was estimated to be 23.81 kDa, and its isoelectric point was predicted to be 8.72. Phylogenetic analysis showed that AjPMK had a closer evolutionary relationship with genes from starfish than with those of other selected species. Besides, we found that rAjPMK synthesized mevalonate-5-diphosphate, interacted either directly or indirectly with crucial pattern recognition receptors (PRRs) and was regulated by immune-related processes, including antioxidative reactions, stress resistance responses and enzyme hydrolysis. Moreover, AjPMK also interacted with farnesyl pyrophosphate synthase, an enzyme reported to be involved in saponin biosynthesis. Together, our findings implied that AjPMK may be directly involved in saponin biosynthesis and the regulation of various innate immune processes. • A phosphomevalonate kinase (PMK) gene was identified in sea cucumber Apostichopus japonicus. • AjPMK is involved in sea cucumber saponin biosynthesis. • AjPMK might be regulated by multiple immune processes. [ABSTRACT FROM AUTHOR]

C-type lectin (CTL) is an important pattern recognition receptor that play vital functions in the innate immunity. Many soluble CTLs in crustacean participate in the inhibition or promotion of white spot syndrome virus (WSSV) infection. However, whether transmembrane CTLs participate in WSSV infection in crustacean remains unknown. In the present study, four spliced isoforms of a transmembrane CTL (designated as PcTlec) from Procambarus clarkii were identified for the first time. The genome structure of PcTlec contains eight exons, six known introns, and one unknown intron. PcTlec-isoform1 is produced by intron retention, whereas PcTlec-isoform3 and PcTlec-isoform4 are produced by exon skipping. All of them contain the transmembrane domain and characteristic carbohydrate recognition domain (CRD). Four PcTlec isoforms were mainly expressed in the hepatopancreas, stomach, and intestine. After WSSV challenge, the expression levels of PcTlec-isoform1-4 in the intestine were upregulated. The knockdown of the region shared by four PcTlec isoforms evidently decreased the expression of WSSV envelope protein VP28 and the copies of viral particles. A recombinant protein (rPcTlec-CRD) containing the CRD that was shared by four PcTlec isoforms was acquired by procaryotic expression system. The injection of purified rPcTlec-CRD protein evidently increased the VP28 expression and WSSV copies during viral infection. Moreover, rPcTlec-CRD could directly bind to WSSV and interact with VP28 protein. These findings indicate that new-found transmembrane CTL isoforms in P. clarkii may act as viral receptors that facilitate WSSV infection. This study contributes to the recognition and understanding of the functions of transmembrane CTLs in crustacean in the infection of host by WSSV. • Four spliced isoforms of a transmembrane CTL were identified in P. clarkii. • The expressions of four PcTlec isoforms were increased after WSSV challenge. • Knockdown of PcTlec remarkably decreased the VP28 expression and virus copies. • Injection of rPcTlec-CRD evidently increased the VP28 expression and virus copies. • rPcTlec-CRD could directly bind to WSSV and interact with VP28. [ABSTRACT FROM AUTHOR]

Filter-feeding bivalves, such as the Mytilus species, are exposed to different types of bacteria in the surrounding waters, in particular of the Vibrio genus. Mussels lack an adaptive immune system and hemocytes can recognize pathogen-associated molecular patterns (PAMPs) via pattern recognition receptors (PRRs) to activate intracellular signaling pathways to trigger the antimicrobial effectors synthesis. Among the areas of bivalve immunity that deserve study include the role of hemocyte subpopulations. Since little information are available on immune responses at the tissue level to human pathogenic vibrios commonly detected in coastal waters involved in seafood-borne diseases, in this work, immunological parameters of the hemocytes from the Mediterranean mussel M. galloprovincialis were evaluated in response to in vivo challenge with Vibrio splendidus. The histological approach has been first used in order to identify the hemocytes recruitment at the infection site and the morphological change of muscular fibers. In addition, using immunolabeling with specific antibody we detected the production of molecules involved in the inflammatory activated cascade: Toll-like receptors 4 (TLR4), the myeloid differentiation factor 88 (MyD88), the Allograft inflammatory factor-1 (AIF-1) and the ribonucleases RNASET2, belonging to the T2 family, that in vertebrates are involved in the recruitment and activation of macrophages. Our results indicate the activation of TLR4 during bacterial infection preparatory to the recruitment of the MyD88 adapter with a putative role in recognition and intracellular signalling. Furthermore, the data presented in this work suggest that challenging with Gram-negative bacteria causes a massive migration of AIF-1+ hemocytes and that the ribonuclease RNASET2 could play a key role in the recruitment of these activated hemocytes. Our approach is useful for further understanding the complex molecular defence mechanisms of the host in invertebrates, especially in relation to the need to develop methods to evaluate the immunological response of bivalve molluscs used in aquaculture. [Display omitted] • Recruitment of hemocytes occurred in mussel M. galloprovincialis in response to Vibrio splendidus bacterial challenge. • Pattern-recognition receptors initiate TLR4 and Myd88 cascades, with a putative role in recognition and intracellular signalling. • The AIF-1 and RNASET2 production significantly increase in the context of bacterial infection. [ABSTRACT FROM AUTHOR]

As lower vertebrates, fish have both innate and adaptive immune systems, but the role of the adaptive immune system is limited, and the innate immune system plays an important role in the resistance to pathogen infection. C-type lectins (CLRs) are one of the major pattern recognition receptors (PRRs) of the innate immune system. CLRs can combine with pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs) to trigger NF-κB signaling pathway and exert immune efficacy. In this study, Ssclec12b and Ssclec4e of the C-type lectins, were found to be significantly up-regulated in the transcripts of Sebastes schlegelii macrophages stimulated by bacteria. The identification, expression and function of these lectins were studied. In addition, the recombinant proteins of the above two CLRs were obtained by prokaryotic expression. We found that rSsCLEC12B and rSsCLEC4E could bind to a variety of bacteria in a Ca2+-dependent manner, and promoted the agglutination of bacteria and blood cells. rSsCLEC12B and rSsCLEC4E assisted macrophages to recognize PAMPs and activate the NF-κB signaling pathway, thereby promoting the expression of inflammatory factors (TNF-α , IL-1β , IL-6 , IL-8) and regulating the early immune inflammation of macrophages. These results suggested that SsCLEC12B and SsCLEC4E could serve as PRRs in S. schlegelii macrophages to recognize pathogens and participate in the host antimicrobial immune process, and provided a valuable reference for the study of CLRs involved in fish innate immunity. • The expression changes of Ssclec12b and Ssclec4e before and after bacterial stimulation were investigated in immunologic tissues. • The bacterial binding activity and the Ca2+-dependent bacterial agglutination activity of rSsCLEC12B and rSsCLEC4E had been demonstrated, which proved the important role in the process of S. schlegelii resisting pathogen infection. • The expression of p65 , TRAF3 , TAK1 and NIK1 of NF-κB signaling pathway was significantly up-regulated in macrophages infected with A. johnsonii. The addition of rSsCLEC12B and rSsCLEC4E further increased the expression of the above four genes. • rSsCLEC12B and rSsCLEC4E significantly increased the expression of pro-inflammatory factors in macrophages. [ABSTRACT FROM AUTHOR]

C-type lectins (CTLs) are glycan-binding pattern recognition receptors (PRRs) that can bind to carbohydrates on pathogen surfaces, triggering immune responses in shrimp innate immunity. In this study, a unique Ca2+-inhibited CTL named Fc Lec was identified and characterized in Chinese shrimp Fenneropenaeus chinensis. The full-length cDNA sequence of Fc Lec was 976 bp (GenBank accession number KU361826), with a 615 bp open reading frame (ORF) encoding 204 amino acids. Fc Lec possesses a C-type lectin-like domain (CTLD) containing four conserved cysteines (Cys105, Cys174, Cys192, and Cys200) and two sugar-binding site structures (QPD and LNP). The tertiary structure of Fc Lec deduced revealed three α-helices and eight β-pleated sheets. The mRNA expression levels of Fc Lec in hemocytes and the hepatopancreas were markedly elevated after stimulation with Vibrio anguillarum and white spot syndrome virus (WSSV). The recombinant Fc Lec protein exhibited Ca2+-independent hemagglutination and bacterial agglutination, but these activities were observed only in the presence of EDTA to chelate metal ions. These findings suggest that Fc Lec plays important and functionally distinct roles in the shrimp's innate immune response to bacteria and viruses, enriching the current understanding of the relationship between CTL activity and Ca2+ in invertebrates. [Display omitted] • A novel CTL, Fc Lec, were identified from F. chinensis. • Fc Lec plays distinct and significant roles in the innate immune system's reaction to microbial assaults. • Fc Lec exhibits Ca2+-independent and even Ca2+-inhibited activity. [ABSTRACT FROM AUTHOR]

Moritella viscosa (M. viscosa) and sea lice (Lepeophtheirus salmonis) are severe pathogens that primarily infect the skin of Atlantic salmon (Salmo salar), which cause significant economic losses in the farming industry. However, the pathogenesis and molecular mechanisms underlying the host's immune defence at the post-transcriptional level remain unclear. Alternative splicing (AS) is an evolutionarily conserved post-transcriptional mechanism that can greatly increase the richness of the transcriptome and proteome. In this study, transcriptomic data derived from skin tissues of Atlantic salmon after M. viscosa and sea lice infections were used to examine the AS profiles and their differential expression patterns. In total, we identified 33,044 AS events (involving 13,718 genes) in the control (CON) group, 35,147 AS events (involving 14,340 genes) in the M. viscosa infection (MV) group, and 30,364 AS events (involving 13,142 genes) in the sea lice infection (LC) group, respectively. Among the five types of AS identified in our study (i.e., SE, A5SS, A3SS, MXE, and RI), SE was the most prevalent type in all three groups (i.e., CON, MV, and LC groups). Decreased percent-spliced-in (PSI) levels were observed in SE events under both MV- and LC-infected conditions, suggesting that MV or LC infection elevated exon-skipping isoforms and promoted the selection of shorter transcripts in numerous DAS genes. In addition, most of the differential AS genes were found to be associated with pathways related to mRNA regulation, epithelial or muscle development, and immune response. These findings provide novel insights into the role of AS in host-pathogen interactions and represent the first comparative analysis of AS in response to bacterial and parasitic infections in fish. • This study represented the first comparative analysis of the AS patterns in response to bacterial and parasitic infections in Atlantic salmon. • We found that the AS patterns could be modulated in a pathogen-specific way.. • Decreased PSI levels in SE events suggested that MV and LC infection elevated exon-skipping isoforms and promoted the selection of shorter transcripts in numerous DAS genes. [ABSTRACT FROM AUTHOR]

Toll-like receptors (TLRs) are one of the extensively studied pattern recognition receptors (PRRs) and play crucial roles in the immune responses of vertebrates and invertebrates. In this study, 14 TLR genes were identified from the genome-wide data of Octopus sinensis. Protein structural domain analysis showed that most TLR proteins had three main structural domains: extracellular leucine-rich repeats (LRR), transmembrane structural domains, and intracellular Toll/IL-1 receptor domain (TIR). The results of subcellular localization prediction showed that the TLRs of O. sinensis were mainly located on the plasma membrane. The results of quantitative real-time PCR (qPCR) showed that the detected TLR genes were differentially expressed in the hemolymph, white bodies, hepatopancreas, gills, gill heart, intestine, kidney, and salivary gland of O. sinensis. Furthermore, the present study investigated the expression changes of O. sinensis TLR genes in hemolymph, white bodies, gills, and hepatopancreas in different phases (6 h, 12 h, 24 h, 48 h) after stimulation with PGN, poly(I: C) and Vibrio parahaemolyticus. The expression of most of the TLR genes was upregulated at different time points after infection with pathogens or stimulation with PAMPs, a few genes were unchanged or even down-regulated, and many of the TLR genes were much higher after V. parahaemolyticus infection than after PGN and poly(I:C) stimulation. The results of this study contribute to a better understanding of the molecular immune mechanisms of O. sinensis TLRs genes in resistance to pathogen stimulation. • Fourteen TLR genes were identified from the whole genome data of Octopus sinensis. • The 14 TLRs of O. sinensis were expressed to varying degrees in eight tissues. • After Vibrio , PGN, and poly(I: C) stimulation, most of TLR s were up-regulated in examined tissues. • 4.The expression of many TLR s after Vibrio infection was higher than those stimulated by PGN and poly(I: C). [ABSTRACT FROM AUTHOR]

C-type lectins (CTLs) execute critical functions in multiple immune responses of crustaceans as a member of pattern recognition receptors (PRRs) family. In this study, a novel CTL was identified from the exoskeleton of the oriental river prawn Macrobrachium nipponense (Mn Lec3). The full-length cDNA of Mn Lec3 was 1150 bp with an open reading frame of 723 bp, encoding 240 amino acids. Mn Lec3 protein contained a signal peptide and one single carbohydrate-recognition domain (CRD). Mn Lec3 transcripts were widely distributed at the exoskeleton all over the body. Significant up-regulation of Mn Lec3 in exoskeleton after Aeromonas hydrophila challenged suggested the involvement of Mn Lec3 as well as the possible function of the exoskeleton in immune response. In vitro tests with recombinant Mn Lec3 protein (r Mn Lec3) manifested that it had polysaccharide binding activity, a wide spectrum of bacterial binding activity and agglutination activity only for tested Gram-negative bacteria (Escherichia coli , Vibrio anguillarum and A. hydrophila). Moreover, r Mn Lec3 significantly promoted phagocytic ability of hemocytes against A. hydrophila in vivo. What's more, Mn Lec3 interference remarkably impaired the survivability of the prawns when infected with A. hydrophila. Collectively, these results ascertained that Mn Lec3 derived from exoskeleton took an essential part in immune defense of the prawns against invading bacteria as a PRR. • A novel CTL (Mn Lec3) was identified from the exoskeleton of the oriental river prawn Macrobrachium nipponense. • Mn Lec3 plays a vital role in the immune response of the prawn. • 3.The exoskeleton might be more active in the immune response of the prawn by generating immunologic factors. [ABSTRACT FROM AUTHOR]

The signal transducer and activator of transcription 2 (STAT2), a downstream factor of type I interferons (IFNs), is a key component of the cellular antiviral immunity response. However, the role of STAT2 in the upstream of IFN signaling, such as the regulation of pattern recognition receptors (PRRs), remains unknown. In this study, STAT2 homologue of black carp (Mylopharyngodon piceus) has been cloned and characterized. The open reading frame (ORF) of bcSTAT2 comprises 2523 nucleotides and encodes 841 amino acids, which presents the conserved structure to that of mammalian STAT2. The dual-luciferase reporter assay and the plaque assay showed that bcSTAT2 possessed certain IFN-inducing ability and antiviral ability against both spring viremia of carp virus (SVCV) and grass carp reovirus (GCRV). Interestingly, we detected the association between bcSTAT2 and bcRIG-I through co-immunoprecipitation (co-IP) assay. Moreover, when bcSTAT2 was co-expressed with bcRIG-I, bcSTAT2 obviously suppressed bcRIG-I-induced IFN expression and antiviral activity. The subsequent co-IP assay and immunoblotting (IB) assay further demonstrated that bcSTAT2 inhibited K63-linked polyubiquitination but not K48-linked polyubiquitination of bcRIG-I, however, did not affect the oligomerization of bcRIG-I. Thus, our data conclude that black carp STAT2 negatively regulates RIG-I through attenuates its K63-linked ubiquitination, which sheds a new light on the regulation of the antiviral innate immunity cascade in vertebrates. • bcSTAT2 possesses certain IFN-inducing ability and antiviral ability. • bcSTAT2 obviously inhibits bcRIG-I-induced IFN expression and antiviral activity. • bcSTAT2 restrains K63-linked polyubiquitination but not K48-linked polyubiquitination of bcRIG-I. [ABSTRACT FROM AUTHOR]

The family of TIR domain-containing receptors includes numerous proteins involved in innate immunity. In this study, a member of this family was characterized from the ovary of the oriental river prawn Macrobrachium nipponense and identified as interleukin-1 receptor (MnIL-1R). Meanwhile, to elucidate the conservation of IL-1R, its orthologous were identified in several crustacean species as well. In addition, the expression pattern of MnIL-1R in various adult tissues and post different pathogen-associated molecular patterns (PAMPs) challenge in ovary was analyzed with qRT-PCR technology. Finally, the roles of MnIL-1R in the ovary were analyzed by RNAi technology. The main results are as follows: (1) MnIL-1R comprises a 1785 bp ORF encoding 594 amino acids and is structurally composed of five domains: a signal peptide, two immunoglobulin (IG) domains, a transmembrane region, and a TIR-2 domain; (2) the TIR domain showed a high conservation among analyzed crustacean species; (3) MnIL-1R is widely detected in all tested tissues including ovary; (4) MnIL-1R showed a positive response to challenges with LPS, PGN, and polyI:C in the ovary; (5) its IG domain showed strong binding ability to LPS and PGN, confirming its role as a pattern recognition receptor; (6) the expression patterns of several members of the Toll signaling pathway (Myd88, TRAF-6, Dorsal, and Relish) was similar to that of MnIL-1R after challenges with LPS, PGN, and polyI:C in the ovary; (7) the silencing of MnIL-1R resulted in down-regulation of theses gene' (Myd88, TRAF-6, Dorsal, and Relish) expression level in the ovary. These results suggest that MnIL-1R can activate the Toll signaling pathway in the ovary by directly recognizing LPS and PGN through its IG domain, thereby contributing to the immune response in the ovary of M. nipponense. • The first report about interleukin-1 receptor (MnIL-1R) in Macrobrachium nipponense. • MnIL-1R was expressed in ovary and responded to LPS and PGN challenge in ovary. • MnIL-1R might be involved in ovary reproductive immunity through TLR signaling pathway. [ABSTRACT FROM AUTHOR]

C-type lectins (CTLs), as a member of the Ca2+-dependent carbohydrate recognition protein superfamily, play multiple roles in non-self recognition and the elimination of invading pathogens. In this study, a C-type lectin was identified and characterized from the Pacific abalone Haliotis discus hannai (designed as Hd Clec), and its open reading frame (ORF) encoded a polypeptide of 163 amino acids containing a typical signal peptide and only one carbohydrate-recognition domain (CRD). The deduced amino acid sequence of CRD in Hd Clec shared identities ranging from 22.4% to 39.8% with that of other identified CRDs of CTLs. A novel NPN motif was found in Ca2+-binding site 2 of Hd Clec. The mRNA transcripts of Hd Clec were detectable in all the examined tissues of non-stimulated abalones, with the highest expression in hepatopancreas (224.13-fold of that in gills). The expression of Hd Clec mRNA in hemocytes was significantly up-regulated after Vibrio harveyi challenge. Recombinant Hd Clec protein (r Hd Clec) could bind lipopolysaccharide (LPS) and peptidoglycan (PGN) in vitro in the presence of Ca2+. Coinciding with the PAMPs binding assay, r Hd Clec displayed broad agglutination activities towards Gram-negative bacteria V. splendidus , V. anguillarum , V. parahaemolyticus , V. harveyi , Escherichia coli , and Gram-positive bacteria Micrococcus luteus. Moreover, r Hd Clec could significantly elicit the chemotactic response of hemocytes in vitro. And the phagocytosis and encapsulation ability of hemocytes could be significantly enhanced by r Hd Clec. All these results showed that Hd Clec could function as pattern recognition receptors (PRRs) and further enhance the opsonization of hemocytes, which might play a crucial role in the innate immune responses of Pacific abalone. • A single-CRD C-type lectin (Hd CLec) with novel NPN motif was identified from abalone. • The mRNA of Hd CLec was significantly up-regulated post Vibrio harveyi challenge. • R Hd CLec displayed broad microbe agglutination spectrum. • R Hd CLec could elicit the chemotactic response of hemocytes in vitro. • R Hd CLec could function as an opsonin enhancing phagocytosis and encapsulation activities of hemocytes. [ABSTRACT FROM AUTHOR]

The pattern recognition receptors (PRRs) can recognize the conserved molecular structures of pathogens to active the innate immune responses, and subsequently induce the antigen-specific adaptive immune responses for the clearance of infected pathogen. Among the PRRs, Toll-like receptors (TLRs) are the first and best characterized PRRs across all the species. Among the TLR members, TLR7 showed significant conservation across the vertebrates, with the lowest rate of evolution for its LRR domains from primates to fishes. In the current study, one TLR7 (Sm TLR7) gene was captured in turbot, with a 3144 bp open reading frame (ORF), that encoding 1047 amino acid residues. Following multiple sequence comparison, Sm TLR7 was found to have the highest similarity and identity both to Paralichthys olivaceus with 91.9% and 85.9%, respectively. In phylogenetic analysis, Sm TLR7 was firstly clustered with Japanese flounder, and then clustered with fugu, rainbow trout, and zebrafish. In addition, Sm TLR7 was widely expressed in all the examined tissues with the highest expression level in spleen, followed by skin, while the lowest expression level was detected in blood. Following both Edwardsiella tarda and Vibrio anguillarum challenge, Sm TLR7 was significantly down-regulated in gill and intestine, and up-regulated in skin. Moreover, Sm TLR7 was significantly up-regulated in head kidney macrophages following LPS, LTA, PGN and polyI:C stimulation, as well as showed the strongest binding ability to LPS, followed by PGN, LTA, and polyI:C in a dose-dependent manner. Finally, following RNAi of Sm TLR7, MyD88 and IL-1β were slightly up-regulated, while TRAF6 and IL-8 were significantly down-regulated. The characterization of TLR7 can expand our understanding of the PRRs in teleost fishes, and eventually aid the exploration of interactions between host and pathogen. • The TLR7 gene from turbot exhibited highly similarities with that in other teleosts. • Turbot TLR7 was significantly regulated in mucosal tissues following different bacteria challenge. • Turbot TLR7 showed significantly strong responses to LPS, LTA, PGN and poly I:C stimulation. • Following RNAi of TLR7, TRAF6 and IL-8 were significantly down-regulated. [ABSTRACT FROM AUTHOR]

Retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) are a class of pattern recognition receptors located in the cytoplasm that play a key role in antiviral innate immunity in animals. However, few studies have been conducted on the function of RLR proteins in invertebrates. In this study, the complete coding sequence of the RLR gene of the Zhikong scallop, Chlamys farreri , was obtained and named CfRLR1 with an aim to study the response of CfRLR1 to polyinosinic:polycytidylic acid [poly (I:C)] stimulation and the interaction between the CfRLR1 and C. farreri mitochondrial antiviral signaling (MAVS) protein. Sequence analysis revealed that CfRLR1 encodes 1161 amino acids, and the encoded protein covers two tandem caspase activation and recruitment domains (CARDs), a helicase domain, and a C-terminal regulatory domain. Phylogenetic analysis revealed that CfRLR1 belongs to the RLR family of mollusks. Quantitative real-time polymerase chain reaction showed that CfRLR1 mRNA was expressed in all tested tissues, with its highest expression observed in feet and gill tissues. Furthermore, CfRLR1 expression in the gill tissues was significantly induced after the poly (I:C) challenge. Finally, the results of co-immunoprecipitation and yeast two-hybrid assays revealed that CfRLR1 can bind to the CfMAVS protein via CARD–CARD interactions. Overall, our results elucidate the immune function of invertebrate RLR proteins and provide valuable information on viral disease control for scallop farming. • CfRLR1 was identified and characterized in the Zhikong scallop. • CfRLR1 expression was induced by poly (I:C) challenge. • CfRLR1 interacts with CfMAVS via CARD-CARD interaction. [ABSTRACT FROM AUTHOR]

Toll-like receptors (TLRs) are a class of pattern recognition receptors (PRRs) that play a critical role in innate immune responses against pathogens. In the present study, a fish-specific TLR14 was identified and characterized from Monopterus albus (named Ma TLR14), which consisted of a 2658 bp open reading frame encoding a protein of 885 amino acids. Phylogenetic analysis revealed that Ma TLR14 belong to the TLR1 subfamily and shared the highest similarity to Paralichthys olivaceus TLR14. Immunohistochemistry assay showed that Ma TLR14 mainly located in intestinal epithelial cells of hindgut. Immunofluorescence revealed that Ma TLR14 largely localized to the intracellular region and partially co-localized with cell membrane of HeLa cells. The expression levels of Ma TLR14 were upregulated in the liver, spleen, foregut and hindgut post infection with Aeromonas hydrophila. When stimulated with LPS and Flagellin, the Ma TLR14 expression was elevated in isolated peripheral blood leukocytes. Further studies showed that recombinant Ma TLR14-LRR could bind to both the gram-negative and gram-positive bacteria and cause agglutination. Subsequently, the signaling pathway of Ma TLR14 was investigated. Confocal microscopy and co-immunoprecipitation assay demonstrated that Ma TLR14 recruited MyD88 as adaptor. When overexpressed, Ma TLR14 augmented the expression of TRAF6 and phosphorylation of ERK and p65, activated NF-κB and AP-1 and elicited the expression of il-6 and tnf-α. Collectively, Ma TLR14 plays an important role in the microorganism recognition and signaling transduction. • Ma TLR14 is expressed in the intestinal epithelium of hindgut and mainly localized to internal cellular compartments. • Ma TLR14 is upregulated at mRNA expression level after bacterial and PAMPs stimulation. • Ma TLR14 can bind gram-negative bacteria as well as gram-positive bacteria through the LRR domain. • Ma TLR14 significantly induces the expression of tnf-α and il-6 via NF-κB and MAPK pathways. [ABSTRACT FROM AUTHOR]

Ladderlectin is a pattern recognition receptor (PRR) in fish that is critical for rapid detection of bacteria in vitro , but the immunological function of ladderlectin in vivo is essentially unknown. In this study, we examined the expression and function of a ladderlectin homologue (WR-ladderlectin) from hybrid crucian carp. WR-ladderlectin contains 157 amino acids and possesses the conserved C-type lectin domain. WR-ladderlectin is mainly expressed in the intestine and is upregulated by bacterial infection. Recombinant WR-ladderlectin (rWR-ladderlectin) agglutinated Aeromonas hydrophila and Escherichia coli. rWR-ladderlectin also bound the A. hydrophila and E. coli in a protein dose-dependent manner. As well as its ability to bind bacterial cells, rWR-ladderlectin displayed apparent bactericidal activity against A. hydrophila and E. coli in vitro. When introduced in vivo , rWR-ladderlectin induced significant expression of the antimicrobial molecules and tight junctions in the intestine. In addition, rWR-ladderlectin prevented significant decrease in the length of intestine villus and enhanced the host's resistance to bacterial infection. These results indicate that WR-ladderlectin is a classic pattern recognition molecule that protects intestinal mucosal barrier against bacterial infection. • mRNA expression of WR-ladderlectin was significantly induced by bacterial challenge. • WR-ladderlectin exhibited bactericidal activity to Aeromonas hydrophila and Escherichia coli. • WR-ladderlectin could enhance intestinal barrier and resistance against bacterial infection. [ABSTRACT FROM AUTHOR]

Matrix metalloproteinases (MMPs) are an important family of proteinases involved in various physiological processes and associated with the immune response. However, the role of MMPs in the immune response remains unclear. To explore the possible role of MMPs in innate immunity, this study selected the MMP-16 gene encoding peptidoglycan (PGN) binding domain identified in the sea cucumber Apostichopus japonicus (named AjMMP-16, GenBank accession No. AQT26486) for microbial polysaccharide-induced transcriptional expression analysis by quantitative real-time PCR, correlation analysis with nine representative genes from A. japonicus immune pathways in microbial polysaccharide-induced transcriptional expression by using Pearson's correlation test, and prokaryotic recombinant expression. Next, its recombinant protein was employed for microbial polysaccharide-binding analysis with ELISA and bacterial binding analysis with the indirect immunofluorescence method. The results showed that AjMMP-16 was significantly induced by diaminopimelic acid (DAP)-type PGN, lipopolysaccharide, mannan, and β-1,3-glucan and was closely correlated with myeloid differentiation factor 88 (MyD88) in microbial polysaccharide-induced transcriptional expression. In addition, recombinant AjMMP-16 bound to lysine-type PGN, DAP-type PGN, lipopolysaccharide, mannan, β-1,3-glucan, Vibrio splendidus , Pseudoalteromonas nigrifaciens , Shewanella baltica , Bacillus cereus , Escherichia coli , and Staphylococcus aureus. These results suggest that AjMMP-16 might act as a pattern recognition receptor in innate immunity and play an important role in initiating the MyD88-dependent Toll-like receptor signaling pathway. • AjMMP-16 was able to be induced by diverse microbial polysaccharides. • AjMMP-16 might be associated with MyD88-dependent TLR signaling pathway. • AjMMP-16 had broad-spectrum pathogen recognition ability. [ABSTRACT FROM AUTHOR]

Galectin-8 is a typical β-galactoside binding lectin, which primarily functions as a pattern recognition receptor and/or danger receptor that is engaged in pathogen recognition by the host innate immune system. Although several fish galectins have been identified, the role of galectin-8 in teleost immunity is still not fully understood. In this study, molecular, transcriptional, and immune-related functions of galectin-8 (EaGal8) from red-spotted grouper (Epinephelus akaara) were analyzed. The open reading frame of EaGal8 comprised 960 bp encoding 319 amino acids of a ∼35 kDa protein, composed of the N- and C-terminal carbohydrate recognition domains joined by a short hinge peptide. Phylogenetic analysis revealed that EaGal8 was closely related to the Epinephelus lanceolatus galectin-8-like protein. Although EaGal8 showed ubiquitous tissue expression, the highest expression level was observed in the blood. Immunostimulants, including lipopolysaccharide, poly(I:C), and nervous necrosis virus, significantly upregulated the EaGal8 transcription level in a time-dependent manner (p < 0.05). Furthermore, recombinant EaGal8 (rEaGal8) showed a binding affinity toward seven different carbohydrates in a concentration-dependent manner. In addition, rEaGal8 caused strong agglutination of fish red blood cells and several gram-positive and gram-negative bacteria, including Streptococcus iniae , Streptococcus parauberis , Lactococcus garvieae , Escherichia coli , Edwardsiella tarda , Vibrio alginolyticus , Vibrio parahaemolyticus, and Pseudomonas aeruginosa. For the first time in teleosts, we report the wound healing ability of galectin-8 in this study. At low concentrations, rEaGal8 showed potential wound healing responses in FHM cells, in vitro. Thus, this study reinforces the role of EaGal8 in innate immune responses against bacterial and viral infections and wound healing in red-spotted grouper. • Galectin-8 gene was identified in red-spotted grouper (EaGal8). • EaGal8 was highly expressed in blood, brain, and kidney tissues. • EaGal8 mRNA expression level was significantly upregulated after immune challenges including LPS, poly I:C and NNV. • rEaGal8 could agglutinate fish RBCs and gram (+) and gram (−) marine bacterial strains. • rEaGal8 revealed wound healing activity in FHM cells. [ABSTRACT FROM AUTHOR]

Neocaridina denticulate sinensis is a promising crustacean model species due to its merits in raising and breeding. However, its molecular responses to copper remains largely unknown. In the present research, RNA-seq was used to mine the alteration in transcriptome of N. denticulate sinensis hepatopancreas under copper exposure. A total of 16,423 DEGs was identified between control and Cu2+ treatment groups. GO enrichment analysis of all DEGs suggested down-regulated genes exceeded up-regulated genes in all the significantly enriched terms, except for RNA polymerase III complex (GO:0005666). KEGG analysis showed Cu exposure only induced two significantly enriched pathways, including Phagosome (ko04145) and Pathogenic Escherichia coli infection (ko05130). Besides, pattern recognition receptors as Toll , lectin B , CTL1 and SRB , AMPs as crustin type I , lysozyme , and NOS were down-regulated after Cu2+ exposure, while hemocyanin , MT , HSP70 and HSP90 were significantly up-regulated, implying these molecules may play vital role in Cu2+ detoxification of N. denticulate sinensis. Our results here provide research direction of heavy metal detoxification of N. denticulate sinensis , simultaneously enriched its genomic information. [Display omitted] • Transcriptome data of Neocaridina denticulate sinensis response to copper exposure. • 23,266 unigenes were assembled and annotated. • 16,423 DEGs were identified and functional enrichment analysis was carried out. • Eight DEGs were validated by qRT-PCR. [ABSTRACT FROM AUTHOR]

Fibrinogen-related proteins (FREPs) are a family of glycoproteins that contain a fibrinogen-like (FBG) domain. Many members of FREPs have been shown to play an important role in innate immune response in both vertebrates and invertebrates. Here we reported the immune functional characterization of ANGPT4, member of FREPs, in zebrafish Danio rerio. Quantitative real time PCR showed that the expression of zebrafish ANGPT4 gene is up-regulated by the challenge with lipoteichoic acid (LTA) or lipopolysaccharides (LPS), hinting its involvement in innate immune response. The recombinant ANGPT4 (rANGPT4) could bind to both gram-positive bacteria Staphylococcus aureus and Bacillus subtilis and the gram-negative bacteria Escherichia coli and Aeromonas hydrophila as well as the pathogen-associated molecular patterns (PAMPs) on the bacterial surfaces including LTA, LPS and peptidoglycan (PGN), suggesting it capable of identifying pathogens via LTA, LPS and PGN. In addition, rANGPT4 also displayed strong bacteriolytic activities against both gram-positive and -negative bacteria tested via inducing membrane depolarization and intracellular ROS production. Moreover, the bacterial clearance assay in vivo showed that the rANGPT4 could also accelerate the clearance of bacteria in zebrafish embryos/larvae. Finally, we showed that the eukaryotically expressed recombinant ANGPT4 maintained antibacterial activity and binding activity to bacteria and LTA, LPS and PGN. All these suggested that ANGPT4 could not only capable of recognizing pathogens via LTA, LPS and PGN, but also capable of killing the Gram-positive and Gram-negative bacteria, in innate immune response. This work also provides further information to understand the biological roles of FREPs and the innate immunity in vertebrates. • Zebrafish angpt4 was markedly up-regulated following challenge with LTA and LPS. • Zebrafish recombinant ANGPT4 (rANGPT4) showed affinity to bacteria, LPS, LTA and PGN. • Zebrafish rANGPT4 showed strong bactericidal activity. • Zebrafish rANGPT4 disrupts the bacterial membrane by membrane depolarization and intracellular ROS production. [ABSTRACT FROM AUTHOR]

Cystatins comprise a vast superfamily of evolutionary conserved proteins, predominantly recognized for their roles as endogenous inhibitors by regulating the activity of cysteine proteases. Emerging lines of research evidence also provides insight into their alternative roles in a spectrum of biological and pathological processes, including neurodegenerative disorders, tumor progression, inflammatory diseases, and immune response. Nowadays, various type-1 cystatins (stefins) have been demonstrated among a variety of discovered vertebrate groups, while little is known about the related homologue in cephalochordate amphioxus, which are repositioned at the base of the chordate phylum. In the present study, a single type-1 cystatin homologue in Branchiostoma japonicum was first successfully cloned and designated as Bjcystatin-1. The deduced Bjcystatin-1 protein is structurally characterized by the presence of typical wedge-shaped cystatin features, including the 'QxVxG' and 'Px' motif, as well as the conserved N-terminal glycine residue. Phylogenomic analyses utilizing different cystatin counterparts affirmed the close evolutionary relationship of Bjcystatin-1 and type-1 cystatin homologue. Bjcystatin-1 was predominantly expressed in the gills and hind-gut in a tissue-specific pattern, and its expression was remarkably up-regulated in response to challenge with bacteria or their signature molecules LPS and LTA, suggesting the involvement in immune response. Additionally, the recombinant Bjcystatin-1 (rBjcystatin-1) protein showed significant inhibitory activity towards papain and binding ability to LPS and LTA, indicating its hypothesized role as a pattern recognition receptor in immune response. Subcellular localization results also showed that Bjcystatin-1 was located in the cytoplasm and nucleus, and its overexpression could attenuate the activation of LPS-induced nuclear transcription factors NF-κB. Taken together, our study suggests that amphioxus Bjcystatin-1 acts as a dual role in protease inhibitor and an immunocompetent factor, providing new insights into the immune defense effect of type-1 cystatin in amphioxus. • Amphioxus is immunocompetent to bacteria or PAMPs (LPS and LTA) challenge via regulating the expression of cystatin-1 gene. • Overexpression of cystatin-1 could inhibit LPS-induced nuclear transcription factors NF-κB activation. • Amphioxus Cystatin-1 protein acts as a dual role in protease inhibitor and an immunocompetent factor in amphioxus. [ABSTRACT FROM AUTHOR]

As an ancient species with both conservation and commercial value, Sturgeon's inflammatory regulation mechanism is a research point. Nucleotide-binding and oligomerization domain-containing proteins 1 and 2 (NOD1/2) are classical intracellular pattern recognition receptors (PRRs) in immunity of anti-bacterial infection. However, the characterization and function of NOD1/2 in Sturgeon are still unclear. In this study, we analyzed the synteny relationship of NOD1/2 genes between Acipenser ruthenus and representative fishes at the genome-level. Results showed that the Ar NOD2 collinear genes pair was present in all representative fishes. The duplicated Ar NOD1/2 genes were under purifying selection during evolution as indicated by their Ka/Ks values. To explore the function of NOD1/2, we further investigated their expression patterns and the effects of pathogenic infection, PAMPs treatment, and siRNA interference in Acipenser baerii , the sibling species of A. ruthenus. Results showed that both Ab NOD1/2 were expressed at early developmental stages and in different tissues. Pathogenic infection in vivo and PAMPs treatment in vitro demonstrated that Ab NOD1/2 could respond to pathogen stimulation. siRNA interference with Ab NOD1/2 inhibited expression levels of RIPK2 and inflammatory cytokines compared to the control group after iE-DAP or MDP treatment. This study hinted that the Ab NOD1/2 could stimulate the inflammatory cytokines response during evolutionary processes. • ● Ar NOD1 and Ar NOD2 genes were under purifying selection during evolution. • ● Ab NOD1 and Ab NOD2 were expressed at early developmental stages and in different tissues. • Ab NOD1 and Ab NOD2 could respond to pathogen by mediate expression levels of inflammatory cytokines. [ABSTRACT FROM AUTHOR]

As a lymphocyte-specific surface receptor belonging to the cysteine-rich superfamily of scavenger receptors, CD6 acts as a pattern recognition receptor for microbial components and is involved in the regulation of inflammatory responses. However, the characteristics and functions of CD6 molecules in lower vertebrates represented by teleost fish are unknown. In this study, a CD6 homolog (designated OnCD6) was characterized from Nile tilapia (Oreochromis niloticus), and establishing its role as a PRRs that participates in immune recognition. OnCD6 contains an open reading frame of 1872 bp that encodes a peptide of 623 amino acids, and contains two conserved SR domain. Multiple sequence alignment revealed that OnCD6 shares a relatively high level of identity with those of other species. Transcriptional expression analysis revealed that OnCD6 was constitutively expressed in immunes tissues such as head kidney and thymus. The expression level of OnCD6 in mainly immune tissues were found significantly upregulated after the injection of Streptococcus agalactiae (S. agalactiae). Moreover, OnCD6 protein was located in the head kidney and brain, mainly over the plasma membrane of lymphocytes in these immune tissues. In vitro experiments showed that CD6 extracellular protein bound to and aggregated several Gram-positive and -negative bacterial strains through the recognition of bacterial surface conserved components LPS and LTA etc. In vivo experiments demonstrated that overexpression OnCD6 before S. agalactiae challenge significantly improved tilapia survival, and this was concomitant with reduced bacterial load and pro-inflammatory cytokines (IL-1β and TNF-α). Taken together, our results illustrated the function of CD6 molecular pattern recognition receptors (PRRs) is conserved and plays an important role in antibacterial infection. • OnCD6 was identified from Nile tilapia, Oreochromis niloticus. • OnCD6 was mainly expressed in immune tissues and induced by the challenge of S. agalactiae. • In vitro , rOnCD6E exhibited binding activities to a wide spectrum of microbes and PAMPs. • In vivo , overexpression OnCD6 before S. agalactiae challenge significantly improved tilapia survival. [ABSTRACT FROM AUTHOR]

C-type lectins (CTLs) function as pattern recognition receptors (PRRs) by recognizing invading microorganisms, thereby triggering downstream immune events against infected pathogens. In this study, a novel CTL containing a low-density lipoprotein receptor class A (LDLa) domain was obtained from Litopenaeus vannamei , designed as LvLDLalec. Stimulation by the bacterial pathogen Vibrio anguillarum (V. anguillarum) resulted in remarkable up-regulation of LvLDLalec, as well as release of LvLDLalec into hemolymph. The rLvLDLalec protein possessed broad-spectrum bacterial binding and agglutinating activities, as well as hemocyte attachment ability. Importantly, LvLDLalec facilitated the bacterial clearance in shrimp hemolymph and protected shrimp from bacterial infection. Further studies revealed that LvLDLalec promoted hemocytes phagocytosis against V. anguillarum and lysosomes were involved in the process. Meanwhile, LvLDLalec participated in humoral immunity through activating and inducing nuclear translocation of Dorsal to regulate phagocytosis-related genes and antimicrobial peptides (AMPs) genes, thereby accelerated the removal of invading pathogens in vivo and improved the survival rate of L. vannamei. These results unveil that LvLDLalec serves as a PRR participate in cellular and humoral immunity exerting opsonin activity to play vital roles in the immune regulatory system of L. vannamei. • LvLDLalec is activated by V. anguillarum and promotes bacterial clearance. • LvLDLalec binds to the surface of hemocytes to promote phagocytosis and lysis of ingested V. anguillarum in phagosomes. • LvLDLalec regulates the expression of immune-related genes by promoting the nuclear translocation of Dorsal. • LvLDLalec protectes shrimp from infection from V. anguillarum to improve the survival rate of L. vannamei. [ABSTRACT FROM AUTHOR]

DM9-containing protein in invertebrates functions as pattern recognition receptor (PRR) to play significant roles in innate immunity. In the present study, a novel DM9-containg protein (defined as Es DM9CP-1) was identified from the Chinese mitten crab Eriocheir sinensis. Es DM9CP-1 is composed of 330 amino acids containing a Methyltransf_FA domain and two tandem DM9 repeats. The deduced amino acid sequence of Es DM9CP-1 shared low similarity with the previously identified DM9CPs from other species, and it was closely clustered with Platyhelminthes DM9CPs and then assigned into the branch of invertebrate DM9CPs in the unrooted phylogenetic tree. The mRNA transcripts of Es DM9CP-1 were highly expressed in haemocytes, gill, and heart. After Aeromonas hydrophila stimulation, the expression levels of Es DM9CP-1 mRNA in haemocytes increased significantly at 3 h (3.88-fold, p < 0.05) and 6 h (2.71-fold, p < 0.05), compared with that of PBS group, respectively. Es DM9CP-1 protein was mainly distributed in the cytoplasm and membrane of haemocytes. The recombinant Es DM9CP-1 protein (r Es DM9CP-1) exhibited binding affinity to MAN, PGN, LPS and Poly (I:C), and also to Gram-positive bacteria (Staphylococcus aureus , Micrococcus luteus and Bacillus subtilis), Gram-negative bacteria (Escherichia coli , A. hydrophila and Vibrio splendidus) and fungi (Pichia pastoris and Metschnikowia bicuspidata) in a Ca2+-dependent manner. It was able to agglutinate A. hydrophila , S. aureus , M. luteus , M. bicuspidata and P. pastoris , and inhibit the growth of A. hydrophila and M. bicuspidate. These results suggested that Es DM9CP-1 in crab not only functioned as a PRR, but also agglutinated and inhibited the growth of microbes. • Es DM9CP-1 was able to bind various PAMPs and microbes. • Es DM9CP-1 could agglutinate and inhibit the growth of various microbes. • Es DM9CP-1 functioned as a multipotent PRR in the form of homodimers. [ABSTRACT FROM AUTHOR]

C-type lectins (CTLs) are important immune molecules in innate immune, which participate in non-self recognition and clearance of pathogens. Here, a new CTL with two distinct C-type lectin domains (CTLDs) from Pacific white shrimp Penaeus vannamei , designated as PvMR1 was identified. The obtained PvMR1 coding sequence (CDS) was 1044 bp long encoding a protein with 347 amino acids. PvMR1 had two CTLD, a conserved mannose-specific EPN motif and a galactose-specific QPD motif, clustering into the same branch as the crustacean CTLs. PvMR1 was widely distributed in shrimp tissues with the highest transcription level in the hepatopancreas, with significantly induced mRNA expression on the hepatopancreas and intestines after immune challenge with Vibrio anguillarum. In vitro assays with recombinant PvMR1 (rPvMR1) protein revealed that it exhibited a wide range of antimicrobial activity, bacterial binding ability, and bacterial agglutination activity in a Ca2+-independent manner. Moreover, PvMR1 promoted bacterial phagocytosis in hemocytes. Furthermore, rPvMR1 treatment could significantly enhance the bacterial clearance in hemolymph and greatly improved the survival of shrimp under V. anguillarum infection in vivo. These results collectively suggest that PvMR1 plays an important role in antibacterial immune response of P. vannamei. • In this study, a new CTL with two distinct C-type lectin domains (CTLDs) from Penaeus vannamei , designated as PvMR1 was identified. • The observations indicated that PvMR1 plays an important role in innate immune responses of Penaeus vannamei. • Considering that PvMR1 functioned as a pattern recognition receptor that participates in innate immunity, we suggest that consideration for the strategies used to control Penaeus vannamei aquaculture disease. [ABSTRACT FROM AUTHOR]

Galectin-1 (also known as galecin-2), one member of galectins family, has multiple functions as a pattern recognition receptor (PRR) in innate immune defense system. In the present study, LcGal-1, a prototype galectin, was identified and function investigated in large yellow croaker (Larimichthys crocea). LcGal-1 consists of one carbohydrate recognition domain (CRD), which contains two carbohydrate binding motifs HFNPR and WG-E-R. LcGal-1 had a ubiquitous tissues profile with the highest and lowest expression in spleen and muscle, respectively. Moreover, it was in cytoplasm and nucleus of head-kidney cells in large yellow croaker. RT-qRCR showed that P. plecoglossicida induced LcGal-1 up-regulated expression in liver and gills, and the results were validated by immunohistochemistry analysis. Additionally , the recombinant LcGal-1 (rLcGal-1) showed agglutinate activity on erythrocytes, and the histidine (His) in the HFNPR motif was a key locus to the activity. The agglutination effect of rLcGal-1 on erythrocytes could be inhibited by LPS, α-lactase and d -galactose. The rLcGal-1 was able to bind and agglutinate Gram+ and Gram-bacteria, and damage bacterial membrane as confirmed by PI staining and SEM observation. Transcriptome analysis showed that the overexpressed LcGal-1 in HEK 293T cells could induce 176 DGEs, including 172 boosting genes and 4 falling genes. Collectively, LcGal-1 was a key immune gene involved in the recognition, conjunction, and elimination of pathogens in L. crocea , as well as multiple physiological and pathological regulatory processes. • The His in the HFNPR motif is a key locus to the LcGal-1 hemagglutination. • The rLcGal-1 could bind and agglutinate bacteria, and damage bacterial membrane. • The overexpressed LcGal-1 induced 176 DGEs (172 up- and 4 down-regulated genes). [ABSTRACT FROM AUTHOR]

The class A scavenger receptors play important roles in innate immunity and are distributed on plasma membrane of macrophages and other cell types. Notably, the class A scavenger receptor 4 (SCARA4) contains a typical C-type (calcium-dependent) lectin domain, which belongs to the collectin family of pattern recognition receptors and is involved in the immune response against infection. Here, one turbot SCARA4 gene was identified with a 2,292 bp open reading frame (ORF) encoding 763 amino acid residues. Multiple sequence analysis and phylogenetic analysis confirmed that Sm SCARA4 gene was more close to that of P. olivaceus. Gene structure and syntenic analysis showed conserved exon/intron organization pattern and syntenic pattern across selected vertebrate species. Tissue distribution analysis showed Sm SCARA4 was expressed in all the tested healthy tissues with the relative high expression levels in skin, gill and spleen. Following both E. tarda and V. anguillarum challenge in vivo , Sm SCARA4 was significantly repressed in gill and intestine. Remarkably, Sm SCARA4 showed the strongest binding ability to LPS and strongest upregulation in turbot head kidney macrophages in response to LPS. Knockdown and overexpression of Sm SCARA4 revealed its interactions with the two pro-inflammatory cytokines, TNF-α and IL-1β. Finally, repression of Sm SCARA4 via combined treatment of LPS and overexpression of Sm SCARA4 construct in turbot head kidney macrophages further indicated an inhibitory role of Sm SCARA4 in LPS-stimulated inflammation. Taken together, turbot Sm SCARA4 plays an important role in turbot immunity, especially in the mucosa-related systems; Sm SCARA4 possesses strong binding specificity to LPS, and exerts protective roles in response to LPS infection by reducing the release of pro-inflammatory cytokines. The mechanisms of inhibitory role of Sm SCARA4 in LPS-elicited inflammation await further investigation. • Gene structure and syntenic analysis of SCARA4 showed conserved patterns across selected vertebrate species. • Sm SCARA4 was expressed in all the tested healthy tissues with the relative high expression levels in skin, gill and spleen. • Following both E. tarda and V. anguillarum challenge, Sm SCARA4 was significantly repressed in gill and intestine. • Sm SCARA4 showed the strongest binding ability to LPS and strongest upregulation in turbot head kidney macrophages in response to LPS. • Knockdown and overexpression of Sm SCARA4 revealed its interactions with TNF-α and IL-1β. [ABSTRACT FROM AUTHOR]

The lack of high-throughput sequencing data makes the research progress of Heliocidaris crassispina slow. Therefore, we used PacBio single-molecule real-time sequencing to generate the first full-length transcriptome. Here, 31,181 isoforms were obtained, with an average length of 2383.20 and a N50 length of 2732 bp. Meanwhile, 764 alternative splicing (AS) events, 5098 long-noncoding RNAs (LncRNAs), 6978 simple sequence repeats (SSRs), and 950 hypothetical transcript factors (TFs) were identified. Moreover, five key innate immune pattern recognition receptors (PRRs), including toll-like receptor (TLR), NACHT domain and leucine-rich repeat (NLR), scavenger receptor cysteine-rich (SRCR), peptidoglycan recognition proteins (PGRP), and gram-negative binding proteins (GNBP), were searched in the transcriptome. In addition, 37 isoforms enriched in KEGG and GO immune systems were also detected. The study provid abundant data support for the current research on H. crassispina. • The first full-length transcriptome of sea urchin H. crassispina has been constructed. • A total of 31,181 isoforms with a N50 length of 2732 bp were obtained. • 764 AS events, 5098 LncRNAs, 6978 SSRs, and 950 TFs were identified in the transcriptome. • The multiplicity of key innate immune PRRs were identified in the study, including TLR, NLR, SRCR, PGRP, and GNBP. [ABSTRACT FROM AUTHOR]

Vibrio parahaemolyticus is an important marine pathogen that cause inflammation even death in teleost. It has brought huge economic losses to aquaculture and serious threats to the sustainable development of marine fisheries. Here, we isolated the DNA, RNA, and total flagellin from V. parahaemolyticus , and obtained the primary spleen and head kidney cells (including leukocytes) from Tetraodon nigroviridis. V. parahaemolyticus DNA, RNA, and total flagellin were used to treat the T. nigroviridis primary cells described above. The results show that the nitric oxide (NO) production and respiratory burst response were significantly induced after stimulation with V. parahaemolyticus total flagellin in T. nigroviridis head kidney and spleen cells. And total flagellin could promote the gene expression and protein production of IL-1β in T. nigroviridis leukocytes. T. nigroviridis TLR5M (Tn TLR5M) and TLR5S (Tn TLR5S) ORF sequences were obtained as the main recognition receptor for flagellin. Real-time fluorescent quantitative PCR (qRT-PCR) was performed to detect the expression of pattern recognition receptor Tn TLR5M and Tn TLR5S, the important signal molecule of inflammatory system Tn MyD88 and Tn TRAF6, and inflammatory cytokines Tn IL-1β and Tn IFN-γ2. The results show that there were a significant upregulation after challenge with V. parahaemolyticus total flagellin. We further demonstrated that the total flagellin of V. parahaemolyticus could activate the luciferase activity of the NF-κB reporter gene mediated by Tn TLR5M. Overall, our results suggest that V. parahaemolyticus total flagellin activated the NO production, respiratory burst response, and inflammatory cytokines expressions, such as Tn IL-1β and Tn IFN-γ2, through the Tn TLR5M–NF–κB signaling pathway in T. nigroviridis. • The main components activate fish inflammatory response in V. parahaemolyticus was determined. • Both TLR5M and TLR5S of T. nigroviridis were identified to have the highest expression in the spleen. • TLR5M is the main receptor in response to V. parahaemolyticus flagellin in T. nigroviridis. • After Vp Flagellin challenge, the TLR5-triggered NF-κB signal pathway was activated. [ABSTRACT FROM AUTHOR]

Toll-like receptors (TLRs) are evolutionarily conserved proteins of pattern recognition receptors (PRRs) and play a crucial role in innate immune systems recognition of conserved pathogen-related molecular samples (PAMPs). We identified and characterized TLR18 from Nile tilapia (Oreochromis niloticus), OnTLR18 , to elucidate its role in tissue expression patterns, modulation of gene expression after microbial challenge and TLR ligands, subcellular localization in fish and human cells, and the possible effectors TLR18 induces in a melanomacrophage-like cell line (tilapia head kidney (THK) cells). OnTLR18 expression was detected in all tissues examined, with the highest levels in the intestine and the lowest in the liver. OnTLR18 transcript was up-regulated in immune-related organs after bacterial and polyinosinic-polycytidylic acid (poly I:C) challenges and in the THK cells after lipopolysaccharide (LPS) stimulation. In transfected THK and human embryonic kidney (HEK) 293 cells, OnTLR18 localizes in the intracellular compartment. OnMyD88 and OnTRIF, but not OnTIRAP, were co-immunoprecipitated with OnTLR18, suggesting that the former two molecules are recruited by OnTLR18 as adaptors. The constitutively active form of OnTLR18 induced the production of pro-inflammatory cytokines, type I interferon (IFN), and antimicrobial peptides such as tumor necrosis factor α , interferon (IFN) d2.13 , tilapia piscidin (TP) 2, TP3, TP4, and hepcidin in THK cells. Our results suggest that OnTLR18 plays an important role in innate immunity through initiating nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and IFN signaling pathways via OnMyD88 and OnTRIF and induces the production of various effectors in melanomacrophages. • OnTLR18 was cloned and characterized from Nile tilapia. • The expression of OnTLR18 was induced after bacterial injection and LPS stimulation. • OnTLR18 was localized to internal cellular compartments. • OnTLR18 recruited MyD88 and TRIF as adaptors. • Overexpression of truncated OnTLR18 promotes immune-related transcription factors. [ABSTRACT FROM AUTHOR]

Bathymodiolinae mussels are dominant species in cold seeps and hydrothermal vents and could harbor endosymbionts in gill bacteriocytes. However, mechanisms underlying the symbiosis have remained largely undisclosed for years. In the present study, the global expression pattern of immune-related genes and miRNAs were surveyed in Gigantidas platifrons during bacterial challenges using enriched symbiotic methane oxidation bacteria MOBs or nonsymbiotic Vibrio. As a result, multiple pattern recognition receptors were found differentially expressed at 12 h and 24 h post bacteria challenges and distinctly clustered between stimulations. Dozens of immune effectors along with signal transducers were also modulated simultaneously during MOB or Vibrio challenge. A total of 459 miRNAs were identified in the gill while some were differentially expressed post MOB or nonsymbiotic bacteria challenge. A variety of immune-related genes were annotated as target genes of aforesaid differentially expressed miRNAs. As a result, biological processes including the immune recognition, lysosome activity and bacteria engulfment were suggested to be dynamically modulated by miRNAs in either symbiotic or nonsymbiotic bacteria challenge. It was suggested that G. platifrons mussels could maintain a robust immune response against invading pathogens while establishing symbiosis with chemosynthetic bacteria with the orchestra of immune-related genes and miRNAs. • Expression pattern of miRNAs/genes were profiled after MOB or Vibrio challenge. • PRRs and other immune genes were found distinctly clustered between the challenges. • A total of 459 miRNAs were identified in gill tissue of the deep-sea mussel. • miRNAs targeting apoptosis and phagocytosis were found responsive to challenges. • The orchestra of genes and miRNAs advantaged the immune response of deep-sea mussel. [ABSTRACT FROM AUTHOR]

Toll-like receptors (TLRs) are an important class of molecules involved in non-specific immunity, and they are also the bridge connecting between non-specific immunity and specific immunity. As a vital member of TLR family TLR9 can be activated by bacterial DNA and induce the production of inflammatory cytokines. In this study, a full length of TLR9 homologue of 3677 bp in Nibea albiflora (named as NaTLR9, GenBank accession no: MN125017.1) was characterized, and its ORF was 3180 bp encoding 1059 amino acid residues with a calculated molecular weight of 121.334 kDa (pI = 6.29). Several leucine-rich repeated sequences (LRR domain) and conservative TIR domain were found in NaTLR9, which was mainly expressed in dendritic cells and macrophages. The phylogenetic and synteny analysis further revealed high sequence identity of NaTLR9 with its counterparts of other teleost, confirming their correct nomenclature and conservative during evolution as an important pattern recognition receptor. The NaTLR9-TIR-pEGFP-N1 fusion protein showed green fluorescence and mainly distributed in the cytoplasm. After co-transfection of NaTLR9-TIR-pEGFP-N1 and NaMyD88-pDsRED-Monomer-N1, green fluorescence obviously overlapped with red and changed into yellowish-green, which suggested that there might be the interaction between homologous NaTLR9-TIR and MyD88. Based on this result the pCDNA3.1-NaTLR9-TIR-flag and pcMV-NaMyD88-TIR-Myc plasmids were co-transfected into 293T cells for the immunoprecipitation test. According to Western blot, TLR9 and MyD88 protein could interact with each other. Furthermore, NaTLR9 was ubiquitously expressed in all the investigated tissues, most abundantly in head kidney, followed by stomach, spleen, liver and gill, but lower in muscle. The vitro immune stimulation experiments revealed that Pseudomonas plecoglossicida and polyinosinic-polycytidylic acid [Poly (I:C)] induced higher levels of NaTLR9 mRNA expression with the peaks of 9.52 times at 2 h and 39.91 times at 24 h compared with the control group respectively. The functional domains (LRRs and TIR, named NaTLR9-TIR and NaTLR9-LRR respectively) of NaTLR9 were expressed and purified, the recombinant proteins both could bind three kinds of typical aquatic pathogenic bacteria (Vibrio. parahaemolyticus, Vibrio alginolyticus, and Vibrio harveyi), which showed that NaTLR9 could couple to bacteria by its function domains. The aforementioned results indicated that NaTLR9 played a significant role in the defense against pathogenic bacteria infection in innate immune response of sciaenidae fish, which may provide some further understandings of the regulatory mechanisms in the teleostean innate immune system. • A TLR9 homologue was identified from Nibea albiflora (NaTLR9). • The functional domains in NaTLR9 were highly conserved and located in cytoplasm. • There was the interaction between NaTLR9 and MyD88 for Toll/NF-κB pathway. • NaTLR9 could be significantly induced in response to bacteria and viruses infection. • NaTLR9 could couple to the pathogenic bacteria. [ABSTRACT FROM AUTHOR]

C-type lectins (CTL) are a large group of pattern-recognition proteins and to play important roles in glycoprotein metabolism, multicellular integration, and immunity. Based on their overall domain structure, they can be classified as different groups that possess different physiological functions. A typical C-type lectin (named as Om Lec1) was identified from the fish, Onychostoma macrolepis , an important cultured fish in China. Open reading frame of Om Lec1 contains a 570 bp, encoding a protein of 189 amino acids that includes a signal peptide and a single carbohydrate-recognition domain. The phylogenetic analysis showed that Om Lec1 could be grouped with C-type lectin from other fish. Om Lec1 was expressed in all the tissues in our study, and the expression level was highest in liver. And its relative expression levels were significantly upregulated following infection with Aeromonas hydrophila. The recombinant Om Lec1 protein (r Om Lec1) could agglutinate some Gram-negative bacteria and Gram-positive bacteria in vitro in the presence of Ca2+, showing a typical Ca2+-dependent carbohydrate-binding protein. Furthermore, r Om Lec1 purified from E. coli BL21 (DE3), strongly bound to LPS and PGN, as well as all tested bacteria in a Ca2+-dependent manner. These results indicate that Om Lec1 plays a central role in the innate immune response and as a pattern recognition receptor that recognizes diverse pathogens among O. macrolepis. • A typical C-type lectin with a EPN/WND motif (Om Lec1) was identified and characterized from Onychostoma macrolepis. • The expression levels of Om Lec1 gene were significantly up-regulated after pathogen challenge. • Recombinant Om Lec1 can agglutinates various bacteria in vitro in the presence of Ca2+. • Recombinant Om Lec1 could bind some saccharides such as d -mannose, LPS and PGN. [ABSTRACT FROM AUTHOR]

C-type lectins (CTLs) are important pathogen pattern recognition receptors that recognize carbohydrate structures. In present study, a C-type lectin domain family 4 member E-like gene from turbot, which tentatively named SmCLEC4E-like (SmCLEC4EL), was identified, and the expressional and functional analyses were performed. In our results, SmCLEC4EL showed conserved synteny with CLEC4E-like genes from several fish species in genome, and possessed a typical type II transmembrane CTL architecture: an N-terminal intracellular region, a transmembrane domain and a C-terminal extracellular region which contained a predicted carbohydrate recognition domain (CRD). In addition, SmCLEC4EL exhibited the highest expression level in spleen in healthy fish, and showed significantly induced expression in mucosal tissues, intestine and skin, under bacteria challenge. Finally, the recombinant SmCLEC4EL protein combined with LPS, PGN, LTA and five different kinds of bacteria in a dose-dependent manner, and agglutinated these bacteria strains in the presence of calcium. These findings collectively demonstrated that SmCLEC4EL, a calcium-dependent CTL, could function as a pattern recognition receptor in pathogen recognition and participate in host anti-bacteria immunity. • SmCLEC4EL showed conserved synteny to its counterparts in other vertebrates. • SmCLEC4EL was ubiquitously expressed in turbot tissues. • SmCLEC4EL showed different expression patterns following different bacterial challenge. • SmCLEC4EL had strong binding ability to LPS, LTA, PGN, and five different kinds of bacteria in a dose-dependent manner. [ABSTRACT FROM AUTHOR]

TLRs are the first and best-characterized pattern recognition receptors conserved across all the species. Different from mammals, the TLRs in teleost fishes are very diversified due to various evolutionary mechanisms. Here, we characterized one TLR1 gene in turbot, with a 2,415 bp open reading frame (ORF), that encoding 804 amino acid residues, and have the highest similarity and identity both to Paralichthys olivaceus with 88.9% and 79.9%. In phylogenetic analysis, it was firstly clustered with P. olivaceus , and then clustered with Takifugu rubripes. TLR1 was widely expressed in all the examined healthy tissues with the highest expression level in spleen, followed by head-kidney. In addition, it was significantly regulated in gill, skin and intestine following Edwardsiella tarda and Vibrio anguillarum challenge with different expression patterns. In in vitro stimulation with pathogen-associated molecular patterns, TLR1 showed significantly strong and elevated responses to LPS, but only responded to LTA and Poly(I:C) at the highest evaluated concentration, while no response was detected using PGN stimulation. Moreover, in subcellular localization analysis, TLR1 was distributed in the cytoplasm, membrane and nucleus. Taken together, TLR1 played vital roles for host immune response to bacterial infection, only with strong binding ability to LPS and involved in the production of inflammatory cytokines. However, the specific ligand for TLR1 and its functional association with other TLRs should be further characterized in fish species. • The TLR1 gene from turbot exhibited highly similarities with that in other teleosts. • Turbot TLR1 was widely expressed in all the examined tissues and significantly regulated in mucosal tissues following different bacteria challenge. • Turbot TLR1 showed much more significantly strong responses to LPS than LTA, poly I:C and PGN stimulation.. • Turbot TLR1 was distributed in the cytoplasm, membrane and nucleus. [ABSTRACT FROM AUTHOR]

The family of fibrinogen-related proteins (FREPs) is a group of proteins with fibrinogen-like (FBG) domains, which play important roles as pattern recognition receptors (PRRs) in the innate immune responses. In the present study, a fibrinogen-like protein was identified from the oyster Crassostrea gigas (defined as Cg FREP1). The open reading frame of Cg FREP1 was of 966 bp that encoded a predicted polypeptide of 321 amino acids comprising a signal peptide and a fibrinogen-like domain. The mRNA expression of Cg FREP1 was detected in all the examined tissues. The recombinant Cg FREP1 (r Cg FREP1) displayed binding activities to lipopolysaccharide (LPS), mannose (MAN), as well as Gram-positive bacteria (Micrococcus luteus and Staphylococcus aureus) and Gram-negative bacteria (Vibrio splendidus and Escherichia coli). The r Cg FREP1 displayed the agglutinating activity towards M. luteus , V. splendidus and E. coli in the presence of Ca2+. r Cg FREP1 was able to enhance the phagocytic activity of haemocytes towards V. splendidus , and exhibited binding activity to the CUB domain of Cg MASPL-1. These results suggest that Cg FREP1 not only serves as a PRR to recognize and agglutinate different bacteria but also mediates the haemocytes phagocytosis towards V. splendidus. • The mRNA expression of Cg FREP1 was able to be detected in all the examined tissues. • The Cg FREP1 displayed the binding activity to different PAMPs and bacteria. • Cg FREP1 was able to interact with the CUB domain of Cg MASPL-1. • The phagocytic activity of haemocytes was enhanced after V. splendidus incubated with Cg FREP1. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) are a class of highly conserved, endogenous non-coding single-stranded small RNA molecules with a length of 18–25 nucleotides. MiRNAs can negatively regulate the target gene through complementary pairing with the mRNA. It has been more than 20 years since the discovery of miRNA molecules, and many achievements have been made in fish research. This paper reviews the research progress in the regulation of fish nutrition and immunity by miRNAs in recent years. MiRNAs regulate the synthesis of long-chain polyunsaturated fatty acids, and are involved in the metabolism of glucose, lipids, as well as cholesterol in fish. Moreover, miRNAs play various roles in antibacterial and antiviral immunity of fish. They can promote the immune response of fish, but may also participate in the immune escape mechanism of bacteria or viruses. One important aspect of miRNAs regulation on fish immunity is mediated by targeting pattern recognition receptors and downstream signaling factors. Together, current results indicate that miRNAs are widely involved in the complex regulatory network of fish. Further studies on fish miRNAs may deepen our understanding of the regulatory network of fish nutrition and immunity, and have the potential to promote the development of microRNA-based products and detection reagents that can be applied in aquaculture industry. [ABSTRACT FROM AUTHOR]

Galectin-8 gene belongs to the agglutinin family, which can specifically recognize β -galactoside bonds and play essential roles in many biological processes. In this study, we researched the sequence characteristics and immune-related function of Galectin-8 gene in Japanese flounder Paralichthys olivaceus , named PoGalectin-8. The results showed that the open reading frame of PoGalectin-8 was 891 bp, which encoding a protein with 296 amino acid residues and containing typical HXNPR and WGXEE motifs in the N-terminal and C-terminal CRD domains. Sequence alignment showed that PoGalectin-8 was conserved in different aquatic animals and exhibited the highest similarity (95.27%) with Seriola dumerili. PoGalectin-8 expressed in all detected tissues and exhibited the highest expression level in spleen, followed by skin and kidney. After infected by Edwardsiella tarda , the expression of PoGalectin-8 was down-regulated in the spleen and skin tissues of P. olivaceus. Further to study its immune-related functions, the recombinant PoGalectin-8 (rPoGalectin-8) was expressed and purified. The rPoGalectin-8 can specifically bind to lipopolysaccharide and peptidoglycan, the main components of cell walls from Gram-negative and Gram-positive bacteria. Bacteria binding and the microbial agglutinating experiments showed that the rPoGalectin-8 could bind and agglutinate all examined Gram-positive and Gram-negative bacteria. This study implied that PoGalectin-8 , as a pattern recognition receptor, may play important roles during immune responses against bacterial infection, which laid a foundation for further functional identification of Galectin-8 in aquatic animal immunity. • Galectin-8 from Japanese flounder (PoGalectin-8) were identified and characterized. • PoGalectin-8 was ubiquitously expressed in all tested tissues. • PoGalectin-8 was down-regulated after challenged with E. tarda. • The rPoGalectin-8 showed high affinity to lipopolysaccharide and peptidoglycan. • The rPoGalectin-8 could bind and agglutinate Gram-positive and -negative bacteria. [ABSTRACT FROM AUTHOR]

The Manila clam (Ruditapes philippinarum), one of the major marine aquaculture species in China, is susceptible to infection with the pathogen Vibrio , which results in massive mortality and economic losses. Toll-like receptors (TLRs) are significant pattern recognition receptors (PRRs) of innate immunity that are involved in immune regulation against pathogenic invasion. Molecular characterization of Manila clam TLRs and investigations of their immune functions are essential to prevent and control Vibrio infection. In the present research, eight cDNA sequences of R. philippinarum TLRs (RpTLRs) were identified from previous transcriptome libraries and then classified into four groups, namely, P-TLR (one sequence), V-TLR (one sequence), Ls-TLR (two sequences) and sP-TLR (four sequences), based on the corresponding LRR domain arrangement of their protein structures within the typical TLR motifs. A selective pressure test firstly suggested that the molluscan P-TLR, V-TLR, Ls-TLR and sP-TLR families underwent positive selection, and different numbers of positive selection sites (PSSs) were identified in different domains of the four types of RpTLRs, as determined by PAML and analysis of website data. These findings indicated that the evolution of RpTLRs may be associated with their immune recognition and function. Furthermore, tissue-specific expression analysis showed that all RpTLRs were ubiquitously expressed in all test tissues and were dominant in hemocytes. Quantitative real-time PCR revealed that the cDNA expression of all eight RpTLRs was upregulated after injection with Vibrio anguillarum (P < 0.01) in R. philippinarum hemocytes, revealing that these RpTLRs play important roles in responding to pathogenic stimulation. In summary, these findings provide a foundation for future investigations of the molecular classification and evolutionary patterns of Toll-like receptors in invertebrates, and the innate immune responses of TLR signaling pathways in Mollusca. [Display omitted] • P-TLR, V-TLR,Ls-TLR and sP-TLR genes were identified and characterized from Ruditapes philippinarum transcriptome. • The four families of molluscan TLRs underwent positive selection, and PSSs of them are located in different domain. • RpTLRs dominantly exist in hemocytes and significantly up-regulated by Vibrio anguillarum stimulation. [ABSTRACT FROM AUTHOR]