Mechanism of Chinese sturgeon IFN-γ inhibition on Mycobacterium marinum (Acipenser sinensis).

IFN-γ plays a crucial role in both innate and adaptive immune responses and is a typical Th1 cytokine that promotes Th1 response and activates macrophages. When macrophages were incubated with IFN-γ, their phagocytosis ratio against Mycobacterium marinum increased significantly, as observed under fluorescence microscopy. The macrophages engulfed a large number of M. marinum. The proliferative ability of macrophages treated with IFN-γ was significantly weaker on the 4th and 7th day after phagocytosis and subsequent re-infection with marine chlamydia (P < 0.001). This suggests that IFN-γ enhances the phagocytosis and killing ability of macrophages against M. marinum. IFN-γ protein also significantly increased the production of reactive oxygen species (H2O2) and nitric oxide (NO) by macrophages. Additionally, the expression levels of toll-like receptor 2 (tlr2) and caspase 8 (casp8) were significantly higher in macrophages after IFN-γ incubation compared to direct infection after 12 h of M. marinum stimulation. Apoptosis was also observed to a higher degree in IFN-γ incubated macrophage. Moreover, mRNA expression of major histocompatibility complex (MHC) molecules produced by macrophages after IFN-γ incubation was significantly higher than direct infection. This indicates that IFN-γ enhances antigen presentation by upregulating MHC expression. It also upregulates tlr2 and casp8 expression through the TLR2 signaling pathway to induce apoptosis in macrophages. The pro-inflammatory cytokine showed an initial increase followed by a decline, suggesting that IFN-γ enhances the immune response of macrophages against M. marinum infection. On the other hand, the anti-inflammatory cytokine showed a delayed increase, significantly reducing the expression of pro-inflammatory cytokines. The expression of both cytokines balanced each other and together regulated the inflammatory reaction against M. marinum infection.

Functional characterization of group â ¡ interferon, IFNf in the acipenseriform fish, Chinese sturgeon (Acipenser sinensis).

Teleost fish possess a diversity of type Ⅰ interferons (IFNs) repertoire, which play a crucial role in antiviral and antimicrobial immune responses. In our previous study, IFNe1-3 and IFNb were identified and cloned from Chinese sturgeon (Acipenser sinensis), an acipenseriform fish. However, the absence of Chinese sturgeon genome data has left the question of whether there are other type Ⅰ IFN members in this species unresolved. In this study, we have identified and characterized a novel IFN, IFNf in Chinese sturgeon (AsIFNf). Bioinformatics analysis revealed that the AsIFNf contains a unique disulfide bond (2 cysteines) located in the second exon and fifth exon region, distinguishing it from other reported teleost type I IFNs. Meanwhile, qPCR results showed that AsIFNf mRNA was detectable in all examined tissues and up-regulated in the spleen or kidney in response to poly I: C, Citrobacter freundii, and Spring Viremia of Carp Virus (SVCV), but not by LPS. Furthermore, compared to recombinant AsIFNe2 protein (rAsIFNe2), rAsIFNf exhibited a stronger protective effect on Chinese sturgeon fin cells against SVCV and also induced higher expression of antiviral genes Mx and viperin. Importantly, AsIFNf displayed characteristics similar to antimicrobial peptides (AMPs) with a positive charge and demonstrated a broad spectrum of antimicrobial activity in vitro. These findings provide a theoretical foundation for understanding the primitive structure and function of interferon, as well as deepening our comprehension of the innate immune system and disease defense in the endangered Chinese sturgeon.

Pathogenicity of Plesiomonas shigelloides and Citrobacter freundii isolated from the endangered Chinese sturgeon (Acipenser sinensis).

Chinese sturgeon (Acipenser sinensis) is an indigenous species of China and is listed as a critically endangered species. Recently, second filial generations of Chinese sturgeon in the Yangtze River Fisheries Research Institute suffered from a severe disease. In this study, two kinds of pathogenic bacteria were isolated from diseased sturgeon and identified as Plesiomonas shigelloides and Citrobacter freundii, based on 16S rDNA gene sequence alignment analysis. Antimicrobial susceptibility testing showed that P. shigelloides was resistant to ampicillin, penicillin, midecamycin, oxacillin, and clindamycin; and sensitive to tocefatriaxone, piperacillin, cefoperazone, cefazolin, and ciprofloxacin. C. freundii was resistant to ampicillin, penicillin, midecamycin, oxacillin, and clindamycin; and sensitive to chloramphenicol, cefuroxime, norfloxacin, ciprofloxacin, and ceftazidime. The median lethal dose (LD50) values of P. shigelloides and C. freundii were 4.50 × 103 colony forming units (CFU)/g and 3.20 × 103 CFU/g, respectively. Clinical symptoms of challenged sturgeons were the same as those of naturally infected sturgeons. Histopathological examination disclosed severe damage in the viscera of P. shigelloides and C. freundii-infected sturgeons. This is the first report suggesting that P. shigelloides infection is associated with mortality of Chinese sturgeon. The results of this study revealed the pathogenesis and severe pathogenicity of P. shigelloides and C. freundii in cultured Chinese sturgeon, and offer insights into the prevention and treatment of bacterial infection caused by P. shigelloides and C. freundii in cultured sturgeons.

Transcriptome Sequencing Reveals the Antiviral Innate Immunity by IFN-γ in Chinese Sturgeon Macrophages.

To further study the biological function of interferon-gamma (IFN-γ) in the Chinese sturgeon (Acipenser sinensis), we conducted a transcriptome analysis of primary macrophages induced by IFN-γ using Illumina sequencing technology. We obtained 88,879 unigenes, with a total length of 93,919,393 bp, and an average length of 1,057bp. We identified 8,490 differentially expressed genes (DEGs) between the untreated and IFN-γ-treated macrophages, with 4,599 upregulated and 3,891 downregulated. Gene ontology (GO) analysis showed that 4,044 DEGs were enriched in the biological, cellular components, and molecular function categories. Kyoto Encyclopedia of Genes and Genomes (KEGG) identified 278 immunity-related pathways enriched for the DEGs. According to the GO enrichment results, eight key immunity-related genes were screened for verification using qPCR. Results indicate that IFN-γ can activate macrophage Interferon Regulatory Factors (IRFs) and type I interferon (IFN-I), activate RIG-I-like and Toll-like receptor-related pathways, and improve the antiviral ability of macrophages in Chinese sturgeon.

FoxO3 Modulates LPS-Activated Hepatic Inflammation in Turbot (Scophthalmus maximus L.).

In mammals, forkhead box O3 (foxo3) plays important roles in liver immune system. The foxo3 can regulate cell cycle, DNA repair, hypoxia, apoptosis and so on. However, as such an important transcription factor, few studies on foxo3 in fish have been reported. The present study characterized the foxo3 in turbot (Scophthalmus maximus L.). Lipopolysaccharide (LPS) incubated in vitro (hepatocytes) and injected in vivo (turbot liver) were used to construct inflammatory models. The foxo3 was interfered and overexpressed to investigate its functions in liver inflammation. The open reading frame (ORF) of foxo3 was 1998 bp (base pair), encoding 665 amino acids. Sequence analysis showed that foxo3 of turbot was highly homologous to other fishes. Tissue distribution analysis revealed that the highest expression of foxo3 was in muscle. Immunofluorescence result showed that foxo3 was expressed in cytoplasm and nucleus. Knockdown of foxo3 significantly increased mRNA levels of tumor necrosis factor-α (tnf-α), interleukin-1ß (il-1ß), interleukin-6 (il-6), myeloid-differentiation factor 88 (myd88), cd83, toll-like receptor 2 (tlr-2) and protein level of c-Jun N-terminal kinase (JNK) in sifoxo3 + LPS (siRNA of foxo3+ LPS) group compared with NC + LPS (negative control + LPS) group in turbot hepatocytes. Overexpressed foxo3 significantly decreased mRNA levels of tnf-α, il-6, nuclear transcription factor-kappa B (nf-κb), cd83, tlr-2 and the protein level of JNK in vitro. In vivo analysis, foxo3 knockdown significantly increased levels of GOT in serum after LPS injection compared with NC+LPS group. Overexpressed foxo3 significantly decreased levels of GPT and GOT in pcDNA3.1-foxo3+LPS group compared with pcDNA3.1+LPS group in vivo. Foxo3 knockdown significantly increased mRNA levels of tnf-α, il-1ß, il-6, nf-κb, myd88 and protein level of JNK in vivo in sifoxo3+LPS group compared with NC+LPS group in turbot liver. Overexpressed foxo3 significantly decreased mRNA levels of il-1ß, il-6, myd88, cd83, jnk and protein level of JNK in pcDNA3.1-foxo3+LPS group compared with pcDNA3.1+LPS group in turbot liver. The results indicated that foxo3 might modulate LPS-activated hepatic inflammation in turbot by decreasing the proinflammatory cytokines, the levels of GOT and GPT as well as activating JNK/caspase-3 and tlr-2/myd88/nf-κb pathways. Taken together, these findings indicated that FoxO3 may play important roles in liver immune responses to LPS in turbot and the research of FoxO3 in liver immunity enriches the studies on immune regulation, and provides theoretical basis and molecular targets for solving liver inflammation and liver injury in fish.

Comprehensive comparison of thirteen kinds of cytokine receptors from the endangered fish Chinese sturgeon (Acipenser sinensis).

The interferon receptor system in teleost fish is more complex than that in mammals. In the present study, we identified 13 cytokine receptor genes (10 interferon receptor genes and 3 IL10R2-like genes) from Chinese sturgeon (Acipenser sinensis) using RNA-sequencing. Sequence analysis indicated that these receptors had conserved domains, including signal peptides, FNⅢ, and transmembrane domains. Phylogenetic analysis suggested that they belonged to the cytokine receptor family. In the present study, we named them IFNAR1-like (CRFB5a, CRFB5b), IFNAR2-like (CRFB3a, CRFB3b), IFNGR1-like (IFNGR1), IFNGR2-like (CRFB6a, CRFB6b/IFNGR2-1, CRFB6c/IFNGR2-2, CRFB6d/IFNGR2-3, CRFB6e/IFNGR2-4) and IL10R2-like (CRFB4a, CRFB4b, CRFB4c), respectively. Constitutive expression analysis revealed that these receptor genes had potential functions in immune and non-immune tissue compartments. After stimulating with Poly (I:C), the expression fold changes of CRFB3a, CRFB4a, CRFB4b, CRFB5b, and CRFB6e/IFNGR2-4 in Chinese sturgeon were higher than those of other receptor genes, which revealed that these five genes had important functions in the immune process to resist virus invasion in the host. After stimulating with IFN gamma, the expression fold changes of CRFB3a, CRFB4a, and CRFB6b/IFNGR2-1 were higher than those other receptor genes. Based on other teleost fish interferon receptor models, we speculated that IFNAR1-like (CRFB5a, CRFB5b) and IFNAR2-like (CRFB3a, CRFB3b), comprised Chinese sturgeon type Ⅰ IFN receptors; and IFNGR1-like (IFNGR1) and IFNGR2-like (CRFB6/IFNGR2) comprised Chinese sturgeon type Ⅱ IFN receptors.

Host-microbiota interactions and responses to grass carp reovirus infection in Ctenopharyngodon idellus.

Gut microbiota could facilitate host to defense diseases, but fish-microbiota interactions during viral infection and the underlying mechanism are poorly understood. We examined interactions and responses of gut microbiota to grass carp reovirus (GCRV) infection in Ctenopharyngodon idellus, which is the most important aquaculture fish worldwide. We found that GCRV infection group with serious haemorrhagic symptoms (G7s) showed considerably different gut microbiota, especially with an abnormally high abundance of gram-negative anaerobic Cetobacterium somerae. It also showed the lowest (p < 0.05) alpha-diversity but with much higher ecological process of homogenizing dispersal (28.8%), confirming a dysbiosis of the gut microbiota after viral infection. Interestingly, signaling pathways of NOD-like receptors (NLRs), toll-like receptors (TLRs), and lipopolysaccharide (LPS) stimulation genes were significantly (q-value < 0.01) enriched in G7s, which also significantly (p < 0.01) correlated with the core gut microbial genera of Cetobacterium and Acinetobacter. The results suggested that an expansion of C. somerae initiated by GCRV could aggravate host inflammatory reactions through the LPS-related NLRs and TLRs pathways. This study advances our understanding of the interplay between fish immunity and gut microbiota challenged by viruses; it also sheds new insights for ecological defense of fish diseases with the help of gut microbiota.

Five subfamilies of ß-defensin genes are present in salmonids: Evolutionary insights and expression analysis in Atlantic salmon Salmo salar.

ß-defensins (BD) are the largest family of vertebrate defensins with potent antimicrobial, chemotactic and immune-regulatory activities. Four BD genes (BD1-4) have been cloned previously in rainbow trout but none have been reported in other salmonids. In this study seven BD genes (BD1a-b, 2-4, 5a-b) are characterised in Atlantic salmon and additional BD genes (BD1b and BD5) in rainbow trout. Bioinformatic analysis revealed up to seven BD genes in the genomes of other salmonids that belong to five subfamilies (BD1-5) due to whole genome duplications. BD1-2 and BD4-5 are also present in basal teleosts but only BD1 and/or BD5 are present in advanced teleosts due to loss of one chromosomal locus. BD3 is salmonid specific. Fish BD have a unique three-coding exon structure. Fish BD are highly divergent between subfamilies but conserved within each subfamily. Atlantic salmon BD genes are differentially expressed in tissues, often with low level expression in systemic immune organs (head kidney and spleen) yet with at least one BD gene highly expressed in mucosal tissues, heart, blood and liver. This suggests an important role of these BD genes in innate immunity in mucosa, liver and blood in Atlantic salmon.

Identification and characterization of three CXC chemokines in Asian swamp eel (Monopterus albus) uncovers a third CXCL11_like group in fish.

Chemokines direct cell migration in development and immune defense, and bridge between innate and adaptive immune responses. The chemokine gene family has been rapidly evolving and has undergone species/lineage-specific expansion. Mammals possess inflammatory CXC chemokines CXCL1-8/15 and CXCL9-11 sub-groups, and homeostatic CXCL12-14, 16-17. Orthologues of mammalian CXCL12-14, three chemokines related to CXCL1-8/15 (CXCL8_L1-3), two chemokines related to CXC9-11 (CXCL11_L1-2), and five fish-specific chemokines (CXCL_F1-5) have been described in teleosts. In this study, we reported three novel CXC chemokines in Asian swamp eel Monopterus albus, a commercially important freshwater fish species in China. Two of them belong to the fish-specific CXCL_F2 group, named CXCL_F2a/b, that share 89.5% amino acid identity. The other (CXCL11_L3) belongs to a third CXCL11_L related to the mammalian CXCL9-11 subfamily found only in percomorph fish species, and is the only CXCL9-11 related molecules in this lineage. Mammalian CXCL9-11 attract Th1 cells, and block the migration of Th2 cells in an immune response. This study suggests that all major lineages of teleosts have a CXCL9-11 related chemokine that will aid future functional investigation of CXCL11_L in fish. Cxcl_f2a is highly expressed constitutively in the skin of swamp eels that may attract immune cells to protect the skin in the absence of scales. Cxcl11_l3 and cxcl_f2b are highly expressed in immune tissues/organs and are up-regulated by the viral mimic poly I:C, but not bacterial infection in vivo, suggesting their role in anti-viral defense. The two cxcl_f2 paralogues are differentially expressed and modulated, indicating sub- and/or neo-functionalization.

Sequence characterization and expression pattern analysis of six kinds of IL-17 family genes in the Asian swamp eel (Monopterus albus).

Interleukin-17 (IL-17) is an important cytokine that plays a critical role in the inflammatory response and host defense against extracellular pathogens. In the present study, six novel IL-17 family genes (MaIL-17) were identified by analyzing Asian swamp eel (Monopterus albus) genome. Sequence analysis revealed that the MaIL-17 family genes shared similar features, comprising a signal peptide, an IL-17 superfamily region, and four conserved cysteines. Phylogenetic analysis showed that the MaIL-17 genes were clustered together with their corresponding IL-17 genes from other species. The similarity and identity of all IL-17 family genes indicated that the MaIL-17 genes are conserved among teleosts, while Ma-IL-17D is more conserved than the other Ma-IL-17s. Except for MaIL-17A/F3 and MaIL-17D, all MaIL-17s shared the same genomic structure as the genes from other fish, namely three exons and two introns. The MaIL-17s showed conserved synteny among fish, and we found that the MaIL-17D locus has a more conserved syntenic relationship with the loci from other fish and humans. These results demonstrated that MaIL-17D and human IL-17D might have evolved from a common ancestral gene and subsequently diverged. The analysis of swamp eel reference genes revealed that EEF1A1 (encoding eukaryotic translation elongation factor 1 alpha 1) was an ideal reference gene for accurate real-time qRT-PCR normalization in the swamp eel. The MaIL-17 genes are widely distributed throughout tissues, suggesting that MaIL-17s carry out their biological functions in immune and non-immune tissues compartments. The transcript of Ma-IL17s exhibited different fold changes in head kidney cells in response to Aeromonas veronii phorbol 12-myristate 13-acetate (PMA) and polyinosinic:polycytidylic acid (poly I:C) challenge, showing that MaIL-17A/F1 has stronger antiviral activities compared with other MaIL-17 family genes, and that MaIL-17A/F3 and MaIL-17A/F2 possess stronger effects against extracellular pathogens compared with the others; however, MaIL-17C2 and MaIL-17D may play vital roles during pathogen infection. The differential immune responses of these genes to Aeromonas veronii, PMA and poly I:C implied distinct mechanisms of host defense against extracellular pathogens.

Sequence analysis and characterization of type I interferon and type II interferon from the critically endangered sturgeon species, A. dabryanus and A. sinensis.

In the present study, we identify three type I interferon (IFN) genes (Ad/AsIFNe1-3) and a type II IFN gene (Ad/AsIFNγ) from the Dabry's sturgeon (Acipenser dabryanus) and the Chinese sturgeon (Acipenser sinensis). Sequence analysis revealed that Ad/AsIFNe1-3 and Ad/AsIFNγ contain several conserved characteristics, including signal peptides, interferon alpha, beta, and delta (IFabd) domains, and N-glycosylation sites. Ad/AsIFNe1-3 belongs to the type I IFN group I subgroup, possessing two conserved cysteines residues (C1 and C3), and Ad/AsIFNγ contained a conserved nuclear localization sequence (NLS) motif. Ad/AsIFNe1-3 and Ad/AsIFNγ contain signature motifs indicative of their corresponding IFN group. The Ad/AsIFNe1-3 and Ad/AsIFNγ genes were found to consist of 5 exons/4 introns and 4 exons/3 introns, respectively. These IFNs were separated by four phase 0 introns (type I IFN) and three phase 0 introns (type II IFN). The sequences of IFNe1-3 and IFNγ from the Dabry's sturgeon and the Chinese sturgeon were closely aligned, suggested that these two species are closely related. Phylogenetic analysis revealed that Ad/AsIFNe1-3 and Ad/AsIFNγ clustered together with the corresponding homologous proteins from other fish species. AdIFNe1-3 were found to be high expressed in early embryonic development, suggesting that AdIFNe1-3 might indicate maternal transmission, while AdIFNγ may not mediate embryonic development. Tissue distribution analysis revealed that AdIFNe1-3 and AdIFNγ carry out biological functions in immune and non-immune tissues compartments. AdIFNe1-3 and AdIFNγ can be stimulated by polyinosinic-polycytidylic acid (poly I:C) and lipopolysaccharides (LPS). AdIFNe1-3 have stronger antiviral activity than AdIFNγ, and AdIFNγ has a stronger antibacterial activity than AdIFNe1-3. The differential responses of these genes to poly I:C and LPS suggest differences in the mechanisms of defense against viruses and bacteria.

Molecular characterization and expression analysis of Asian swamp eel (Monopterus albus) CXC chemokine receptor (CXCR) 1a, CXCR1b, CXCR2, CXCR3a, CXCR3b, and CXCR4 after bacteria and poly I:C challenge.

The CXC chemokine receptors (CXCRs) play critical roles in innate and adaptive immune systems. In this study, six Asian swamp eel (Monopterus albus) CXCRs (MaCXCR1-4) were identified and their molecular characterization and expression patterns were analyzed. The open reading frames (ORFs) of MaCXCR1a, MaCXCR1b, MaCXCR2, MaCXCR3a, MaCXCR3b, and MaCXCR4 were 1074 bp (base pairs), 1080 bp, 1125 bp, 1146 bp, 1083 bp, and 1140 bp, and encoded proteins of 357 aa (amino acids), 359 aa, 374 aa, 381 aa, 360 aa, and 379 aa, respectively. All these CXCRs have seven conserved transmembrane domains and four cysteines (with the exception of MaCXCR3b). Multiple sequence alignment revealed that the MaCXCRs possess a typical G-protein receptor family 1 signature and a DRY motif. There are also one to four potential N-glycosylation sites in the extracellular regions of the MaCXCRs, mainly distributed in the N-terminus and extracellular hydrophilic loop (ECL) 2 region. Phylogenetic analysis demonstrated that the MaCXCRs were clustered together with homologous proteins from other fish. Taken together with the amino acid identity and similarity analysis, these results suggested that the MaCXCRs are conserved with other homologous genes, in which CXCR4 is more conserved than CXCR1-3. The MaCXCRs loci showed conserved synteny among teleost fish, and we found that human CXCR1 shares a common ancestor with fish CXCR1a. MaCXCRs were constitutively expressed in a wide range of tissues (especially in immune-related tissues) with different expression levels, suggesting that the MaCXCRs have different roles in un-stimulated tissues, and may play vital roles under normal conditions. MaCXCRs showed different fold changes in the spleen after Aeromonas veronii and polyinosinic-polycytidylic acid (poly I:C) challenge, which suggested that MaCXCR1a and MaCXCR3a have longer antiviral activities compared with their antibacterial functions, and that MaCXCR1b possesses stronger antiviral than antibacterial activity. MaCXCR4 may play vital roles during bacterial and viral infection; however, MaCXCR2 has relatively small effect in antibacterial and antiviral responses. The differential responses of these genes to bacteria and poly I:C implied the differences in the mechanisms of defense against viruses and bacteria.

Transcriptome analysis of the critically endangered Dabry's sturgeon (Acipenser dabryanus) head kidney response to Aeromonas hydrophila.

Dabry's sturgeon (Acipenser dabryanus), as a living fossil, is considered a critically endangered aquatic animal in China. To date, the immune system of this species remains largely unknown, with limited available sequence information. In addition, increasing incidence of bacterial pathogenic diseases has been reported. Hence, the present study aimed to characterize comprehensively transcriptome profile of the head kidney from Dabry's sturgeon infected with Aeromonas hydrophila using Illumina platform. Over 42 million high-quality reads were obtained and de novo assembled into a final set of 195240 unique transcript fragments (unigenes), with an average length of 564 bp. Approximately 41702 unigenes were annotated in the NR NCBI database. Dabry's sturgeon unigenes had the highest number of hits with 14365 (34.45%) to Lepisosteus oculatus. The 195240 unigenes were assigned to three Gene Ontology (GO) categories: biological process, cellular component, and molecular function. Among them, 27770 unigenes were clustered into 26 Eukaryotic Orthologous Group (KOG) functional categories, and 36031 unigenes were mapped to 335 known Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. After A. hydrophila administration, 1728 differentially expressed unigenes were identified, including 980 upregulated and 748 downregulated unigenes. Further KEGG enrichment analysis of these unigenes identified 16 immune-related pathways, including the Toll-like receptor signaling pathway, chemokine signaling pathway, complement and coagulation pathway, RIG-I-like receptor signaling pathway, and NOD-like receptor signaling pathway. 20 DEGs were selected and their expression patterns are largely consistent with the transcriptome profile analysis, which clearly validated the reliability of the DEGs in transcriptome analysis. This work revealed novel gene expression patterns of Dabry's sturgeon host defense and contributes to a better understanding of the immune system and defense mechanisms of Dabry's sturgeon in response to bacterial infection. The results provide valuable references for studies in sturgeons that lack complete genomic sequences, and could also be helpful for the analyzing evolution among cartilaginous and teleost fish.

Interleukin (IL)-2 Is a Key Regulator of T Helper 1 and T Helper 2 Cytokine Expression in Fish: Functional Characterization of Two Divergent IL2 Paralogs in Salmonids.

Mammalian interleukin (IL)-2 is a cytokine centrally involved in the differentiation and survival of CD4+ T helper subsets and CD4+ T regulatory cells and in activation of cytotoxic effector lymphocytes. In bony fish, IL2 orthologs have been identified with an additional divergent IL2-Like gene on the same locus present in several fish species. We report here two divergent IL2 paralogs, IL2A and IL2B, in salmonids that originated from the whole genome duplication event in this fish lineage. The salmonid IL2 paralogs differ not only in sequence but also in exon sizes. The IL-2 isoforms that are encoded have disparate pI values and may have evolved to preferentially bind specific IL-2 receptors. Rainbow trout IL2 paralogs are highly expressed in thymus, spleen, gills, kidney and intestine, important tissues/organs in fish T cell development and function. Their expression in peripheral blood leukocytes (PBL) is low constitutively but can be upregulated by the mixed leukocyte reaction, by the T cell mitogen phytohemagglutinin and by signal mimics of T cell activation (phorbol 12-myristate 13-acetate and calcium ionophore). Both trout IL-2 isoforms promoted PBL proliferation and sustained high-level expression of CD4 and CD8, suggesting that trout IL-2 isoforms are T cell growth/survival factors mainly expressed by activated T cells. The recombinant proteins for these two trout IL2 paralogs have been produced in E. coli and possess shared but also distinct bioactivities. IL-2A, but not IL-2B, induced IL12P35A1 and CXCR1 expression in PBL. IL-2B had a stronger effect on upregulation of the T helper 1 (Th1) cytokine interferon-γ (IFNγ) and could sustain CD8α and CD8ß expression levels. Nevertheless, both cytokines upregulated key Th1 (IFNγ1, IFNγ2, TNFα2 and IL12) and T helper 2 (Th2) cytokines (IL4/13B1 and IL4/13B2), cytokine and chemokine receptors and the antimicrobial peptide cathelicidin-1 but had limited effects on T helper 17 cytokines and TGFß1 in PBL. They could also enhance PBL phagocytosis. These results suggest, for the first time in fish, that IL-2 isoforms may have an important role in regulating Th1 and Th2 cell development, and innate and adaptive host defenses in fish, and shed light on lineage-specific expansion, evolution, and functional diversification of IL2 in vertebrates.

Molecular characterization of three toll-like receptors (TLR21, TLR22, and TLR25) from a primitive ray-finned fish Dabry's sturgeon (Acipenser dabryanus).

Dabry's sturgeon (Acipenser dabryanus) is a useful model for the study of fish evolution, as it is one of the most primitive actinopterygian species. However, studies of the immune system of this fish are limited. Here, we identified three toll-like receptors (adaTLR21, adaTLR22, and adaTLR25) from Dabry's sturgeon. The three sturgeon TLRs had characteristic TLR features, including a signal peptide, several leucine rich repeat (LRR) domains, a transmembrane domain, and a Toll/interleukin-1 receptor (TIR) domain. Although the predicted amino acid sequences encoded by the sturgeon adaTLR21, adaTLR22, and adaTLR25 had somewhat low levels of sequence identity and similarity with TLRs from other fish species, the three sturgeon TLRs fell in well-supported clades with other teleost TLRs in our neighbor-joining phylogenetic tree. Real-time quantitative PCR showed that the three sturgeon TLRs were ubiquitously expressed in all examined tissues from healthy adult sturgeon, but that their expression patterns varied greatly among the different tissues. The three sturgeon TLRs were also expressed across all embryonic developmental stages that were examined, but their expression levels differed between developmental stages. All three TLRs were upregulated in head-kidney primary leucocytes following lipopolysaccharide (LPS) and polyinosinic: polycytidylic acid (polyI:C) stimulation. To the best of our knowledge, this is the first characterization of these three TLRs in Darby's sturgeon. Our results provide a framework for further studies of TLR ligand specificity and signaling pathways in sturgeon, and increase our understanding of the functional evolution of TLRs in vertebrates.

Evolution, expression, and characterisation of liver-expressed antimicrobial peptide genes in ancient chondrostean sturgeons.

Liver-expressed antimicrobial peptide 2 (leap-2) is an evolutionarily ancient molecule that acts as the key component in vertebrate innate immunity against invading pathogens. Leap-2 has been identified and characterised in several teleosts, but not yet in chondrosteans. Herein, the complete coding sequences of leap-2b and leap-2c were identified from expressed sequence tags (ESTs) isolated from Dabry's sturgeon (Acipenser dabryanus) and Chinese sturgeon (A. sinensis), designated as adleap-2b, adleap-2c, asleap-2b, and asleap-2c, respectively. Adleap-2b and adleap-2c sequences share 98% and 100% sequence identity with asleap-2b, and asleap-2c, respectively. Sequence alignment revealed that all four genes contain four cysteine residues, conserved in all fish leap-2 homologs, that form two disulfide bonds. Comparative analysis of the exon-intron structure revealed a three exon/two intron structure for that leap-2 genes in animals, but intron 1 is much longer in sturgeons than in other species. The adleap-2c gene was expressed mainly in the liver of Dabry's sturgeon, and transcription of adleap-2c was significantly up-regulated (p < 0.05) in the liver and midkidney in response to Aeromonas hydrophila challenge. These results suggest adleap-2c may contribute to the defence against pathogenic bacterial invasion. The findings further our understanding of the function of adleap-2c and the molecular mechanism of innate immunity in chondrosteans.

Molecular characterization and expression analysis of TLR1 and TLR4 from the endangered fish Dabry's sturgeon (Acipenser dabryanus).

Toll-like receptors (TLRs) are important pattern recognition receptors (PRRs) of teleost innate immune system. However, information about TLRs is absent in Dabry's sturgeon (Acipenser dabryanus), one of the most primitive actinopterygii species. In the present study, the full lengths of adaTLR1 and adaTLR4 were cloned from Dabry's sturgeon using RT-PCR and RACE-PCR. The obtained adaTLR1 was 2957 bp in length, encoding a putative protein of 767 amino acids and adaTLR4 cDNA was 2902 bp in length, encoding a putative protein of 830 aa. Both adaTLR1 and adaTLR4 possessed several typical TLRs motifs, including signal peptides, leucine-rich repeat (LRR) motifs, a transmembrane domain and a TIR motifs. In addition, adaTLR4 contained three conserved boxes in its TIR motif, involving in TLRs signal transduction. A proline, important for LPS recognition of mammalian TLR4, was also found in adaTLR4. Physicochemical features of adaTLR1 and adaTRL4 were also analyzed. Quantitative realtime PCR showed that both transcripts were ubiquitously expressed in all 11 normal tissues selected, but they exhibited different expression patterns, with adaTLR1 highly expression in heart and adaTLR4 highly in skin. Further, adaTLR1 and adaTLR4 were up-regulated in the primary head-kidney leucocytes following LPS and polyI:C stimulation, indicating that both genes involved in the sturgeon immune response to LPS and polyI:C. To our best knowledge, this was the first report of these genes in sturgeon and these results provided the basis for further elucidating the ligand specificity and signaling pathway of fish TLRs.

Analysis of the expression patterns of the cytokine receptor family B (CRFB) and interferon gamma receptor (IFNGR) in Dabry's sturgeon (Acipenser dabryanus).

Teleost fish have more complex interferon receptor systems than mammals. In the present study, genes encoding four cytokine receptor family B (CRFBs) and two interferon gamma receptors (IFNGRs) in Dabry's sturgeon (Acipenser dabryanus) were identified by RNA-sequencing. Sequence analysis revealed that the Dabry's sturgeon CRFBs and IFNGRs contained several conserved characteristics features, including signal peptides and a transmembrane domain. Phylogenetic analysis suggested that they belong to the CRFB3, CRFB5, and IFNGR protein families, and were named CRFB3a, CRFB3b, CRFB5a, CRFB5b, IFNGR1, and IFNGR2. The expression patterns of the CRFB and IFNGR genes were investigated in Dabry's sturgeon. The expression levels of CRFB5a, CRFB5b, and IFNGR1 showed no significant changes, suggesting that those genes do not mediate embryonic development. By contrast, the high expression levels of CRFB3a, CRFB3b, and IFNGR2 in the fertilized egg, 16-cell phase, and initial blastula stage implied the existence of maternally expression in the oocyte and association with embryonic development. Tissue distribution analysis revealed that the CRFB and IFNGR proteins have potential functions in immune and non-immune tissue compartments. Comprehensive analysis in Dabry's sturgeon revealed that the expression fold changes of CRFB3a, CRFB3b, CRFB5a, and CRFB5b in Dabry's sturgeon stimulated with poly I:C were higher than those in fish administrated with lipopolysaccharide (LPS). Conversely, the fold changes IFNGRs mRNA levels stimulated with LPS were higher than those in fish administrated with poly I: C. CRFB5a and IFNGR2 genes showed the earliest responses to the poly I: C, and the CRFB5a and IFNGR1 genes showed the earliest responses to LPS. These results implied that CRFB5a has important role in the IFN immune response. Our findings indicated that the Dabry's sturgeon CRFB and IFNGR genes have important functions in antiviral and antibacterial immune responses. The differential responses of these genes to poly I: C and LPS implied differences in the defense mechanisms against viruses and bacteria.

Characterization and expression analysis of g- and c-type lysozymes in Dabry's sturgeon (Acipenser dabryanus).

Dabry's sturgeon (Acipenser dabryanus) is mainly distributed in the upper Yangtze River. Although extensively farmed, little information is available on its innate immune system. In this study, we conducted de novo transcriptome assembly of the head kidney to create a comprehensive dataset for A. dabryanus. A total of 51,324,686 high quality reads were obtained from head kidney cDNA library by the Illumina sequencing platform and 131,261 unigenes were determined to contain complete ORFs. The complete coding sequences of g- and c-type lysozymes were identified from unigenes, and designated as ADLysG and ADLysC. Aeromonas hydrophila infection of Dabry's sturgeon caused a significant increase (P < 0.05) in blood for both lysozyme types, confirming their active defensive role against bacterial infections. This research provides the first characterization of these enzymes in an ancestral chondrostean. These data suggest that ADLysG and ADLysC have the potential for immune defense system against bacterial infection.

Molecular characterization and expression analysis of cathepsin C in Chinese giant salamander (Andrias davidianus) after Aeromonas hydrophila infection

Background: Cathepsin C (CTSC) (dipeptidyl peptidase I, DPPI), is a member of the papain superfamily of cysteine proteases and involves in a variety of host reactions. However, the information of CTST in Chinese giant salamander (Andrias davidianus), an amphibian species with important evolutionary position and economic values, remained unclear. Results: The full-length salamander CTSC cDNA contained a 96 bp of 5'-UTR, a 1392 bp of ORF encoding 463 amino acids, and a 95 bp of 3'-UTR. The salamander CTSC possessed several sequence features similar to other reported CTSCs such as a signal peptide, a propeptide and a mature peptide. The active site triad of Cys, His and Asn were also found existing in salamander CTSC. Salamander CTSC mRNA was constitutively expressed in all the examined tissues with significantly variant expression level. The highest expression of CTSC was in intestine, followed with stomach, spleen, lung and brain. Following Aeromonas hydrophila infection for 12 h, salamander CTSC was significantly up-regulated in several tissues including lung, spleen, brain, kidney, heart, stomach and skin. Conclusion: CTSC plays roles in the immune response to bacterial infection, which provided valuable information for further studying the functions of CTSC in salamander.