Xiaoling Liu

Analysis of the transcriptomic profilings of Mandarin fish (Siniperca chuatsi) infected with Flavobacterium columnare with an emphasis on immune responses

Flavobacterium columnare (FC) is the causative pathogen of columnaris which has caused great economic loss in fish culture worldwide, including in Mandarin fish (Siniperca chuatsi) culture. In the present study, the transcriptomic profiles of the head kidneys from FC-infected and non-infected Mandarin fish were obtained using HiSeq™ 2000 (Illumina). Totally 31,168 unigenes with high quality were obtained. Genes involved in protein folding, metabolism and energy, immune responses, oxidoreductase activity, cell growth and death were identified as enriched classes. 1019 differently expressed genes between the two groups were identified, including 603 up-regulated and 416 down-regulated genes. 27 differently expressed immune related genes were scrutinized, including 17 up-regulated and 10 down-regulated genes. Six of the differently expressed genes were further validated by qRT-PCR. The roles of the immune related genes were discussed. Identification of the host genes in response to FC infection will shed a new light on the prevention of columnaris.

Oligochitosan stimulated phagocytic activity of macrophages from blunt snout bream (Megalobrama amblycephala) associated with respiratory burst coupled with nitric oxide production

The immunostimulating effects of oligochitosan have been proven in several fish, however, the mechanisms underlying the stimulation are not characterized. In the present study, the effects of oligochitosan were investigated using macrophages isolated from blunt snout bream (Megalobrama amblycephala). The results showed that the phagocytic activity of the macrophages was enhanced by the addition of oligochitosan in vitro and in vivo. The two of the most important antimicrobial pathways of macrophages, NADPH oxidase and iNOS pathways were included for further studies. The amounts of superoxide anion and the mRNAs of the five subunits of NADPH oxidase genes were significantly enhanced in the oligochitosan-treated macrophages and macrophages isolated from fish fed with feed containing oligochitosan. In addition, the NO production, iNOS activity and iNOS gene expression were all significantly increased in the presence of oligochitosan. Furthermore, the mRNA levels of the TNF-α and IL-1β were also significantly increased in the macrophages derived from fish fed with oligochitosan. In conclusion, the stimulation effects of oligochitosan on the phagocytic activity of the fish macrophages were associated with respiratory burst coupled with nitric oxide production.

Mannose receptor mediated phagocytosis of bacteria in macrophages of blunt snout bream (Megalobrama amblycephala) in a Ca2+-dependent manner

Mannose receptor (MR) is an important pattern-recognition receptor in macrophages and plays a critical role in immune responses. It is has been reported that mammalian macrophages are able to engulf a wide range of microorganisms mediated by Ca(2+)-dependent MR binding to terminal mannose residues which are frequently found on the pathogen surfaces. However, little is known about the MR-mediated phagocytosis in macrophages of fish. In this report, the distributions of MR in the macrophage and head kidney tissue from blunt snout bream were examined using MaMR specific antibody generated in our lab. Mannan and MaMR specific antibody inhibition experiments results collectively showed that MR was involved in the GFP-expressed E. coli engulfed in the macrophages, resulting in respiratory burst, nitric oxide production as well as inflammatory cytokines secretion, and the MaMR-mediated phagocytosis was Ca(2+)-dependent. These results will shed a new light on the immune functions of teleost MRs.

Molecular Cloning and Functional Characterization of Mannose Receptor in Zebra Fish (Danio rerio) during Infection with Aeromonas sobria.

Mannose receptor (MR) is a member of pattern-recognition receptors (PRRs), which plays a significant role in immunity responses. Much work on MR has been done in mammals and birds while little in fish. In this report, a MR gene (designated as zfMR) was cloned from zebra fish (Danio rerio), which is an attractive model for the studies of animal diseases. The full-length cDNA of zfMR contains 6248 bp encoding a putative protein of 1428 amino acids. The predicted amino acid sequences showed that zfMR contained a cysteine-rich domain, a single fibronectin type II (FN II) domain, eight C-type lectin-like domains (CTLDs), a transmembrane domain and a short C-terminal cytoplasmic domain, sharing highly conserved structures with MRs from the other species. The MR mRNA could be detected in all examined tissues with highest level in kidney. The temporal expression patterns of MR, IL-1β and TNF-α mRNAs were analyzed in the liver, spleen, kidney and intestine post of infection with Aeromonas sobria. By immunohistochemistry assay, slight enhancement of MR protein was also observed in the spleen and intestine of the infected zebra fish. The established zebra fish-A. sobria infection model will be valuable for elucidating the role of MR in fish immune responses to infection.

Transcriptomic analysis of liver from grass carp (Ctenopharyngodon idellus) exposed to high environmental ammonia reveals the activation of antioxidant and apoptosis pathways

Abstract High concentration of ammonia in aquatic system leads to detrimental effects on the health of aquatic animals. However, the mechanism underlying ammonia‐induced toxicity is still not clear. To better understand the mechanism of ammonia toxicity effects on fish, juvenile grass carp was employed in the present study. RNA high‐throughput sequencing technique was applied to analyze the total RNAs extracted from the liver of fish after 8 h post exposure to the water containing 2 mM NH4HCO3 which experimentally mimicked the high environmental ammonia (HEA). A total of 49,971,114 and 53,826,986 clean reads were obtained in control and 2 mM HEA group, respectively, in which there were 911 differently expressed genes (DEGs) including 563 up‐regulated and 348 down‐regulated genes. In addition, 10 DEGs were validated by quantitative PCR. These DEGs were involved in several pathways related with oxidative stress or apoptosis. Further analysis on oxidative stress, histopathology and cellular apoptosis in grass carp liver after HEA exposure revealed interesting findings. Increased reactive oxygen species (ROS) content and superoxide dismutase (SOD) activity together with the decreased catalase (CAT) activity were detected, which may be effected by DEGs and related pathways such as FOXO signaling pathway. The histopathology and TUNEL assays results confirmed that apoptosis was induced in liver when fish had suffered HEA. Combined with the results of transcriptomic experiments, c‐Myc‐Bax‐Caspase9 apoptosis pathway could be involved in grass carp liver apoptosis induced by ammonia stress. HighlightsRNA‐sequencing was first applied to study the hepatotoxicity induced by ammonia.DEGs and phenomena about oxidative stress and apoptosis were observed in the fish.Pathways related to oxidative stress including FOXO pathway were identified.c‐Myc‐Bax‐Caspase9 apoptosis pathway was activated in fish liver by HEA exposure.

Contribution of nuclease to the pathogenesis of Aeromonas hydrophila

Aeromonas hydrophila is a gram-negative bacterium that is widely distributed in aquatic environments and can cause septicemia in both fish and humans. However, the underlying mechanisms leading to severe infection are not well understood. In this study, an A. hydrophila nuclease (ahn) deletion mutant was constructed to investigate its contribution to pathogenesis. This mutant did not differ from the wild-type strain in terms of its growth or hemolytic phenotype. However, the ahn-deficient mutant was more susceptible to being killed by fish macrophages and mouse blood in vitro. Furthermore, evidence obtained using both fish and murine infection models strongly indicated that the inactivation of Ahn impaired the ability of A. hydrophila to evade innate immune clearance in vivo. More importantly, the virulence of the mutant was attenuated in both fish and mice, with reductions in dissemination capacities and mortality rates. These findings implicate Ahn in A. hydrophila virulence, with important functions in evading innate immune defenses.

Phylogenetic position of the freshwater fish trypanosome, Trypanosoma ophiocephali (Kinetoplastida) inferred from the complete small subunit ribosomal RNA gene sequence

The complete small subunit rRNA (SSrRNA) gene sequence (2,142 nucleotides) of the freshwater fish trypanosome Trypanosoma ophiocephali Chen (1964) was determined. The phylogenetic analysis deduced using neighbor-joining, maximum parsimony, and Bayesian methods demonstrated the existence of an “aquatic clade”. T. ophiocephali was revealed to be a member of the freshwater fish trypanosomes and form the sister species with Trypanosoma siniperca and Trypanosoma sp. Carpio with high bootstrap values (98% MP, 100% NJ, 100% Bay). The high similarity of SSrRNA gene sequences and morphometric characters showed that T. ophiocephali, T. siniperca and T. sp. Carpio probably were the same species. The phylogenetic trees further suggested that Chinese freshwater fish trypanosome might be paraphyletic, and fish trypanosomes should have low host specificity.

Distribution of mannose receptor in blunt snout bream ( Megalobrama amblycephala ) during the embryonic development and its immune response to the challenge of Aeromonas hydrophila

ABSTRACT The mannose receptor (MR) is a type I transmembrane protein. Its ectodomain has eight C‐type lectin‐like domains, which are able to recognize and mediate the phagocytosis of a wide range of pathogens. Comprehensive studies have revealed that mammalian MR is widely distributed in the mononuclear phagocyte system (MPS, previously known as the reticuloendothelial system) and play a key role both in the physiological clearance and cell activation. Hitherto, neither the MR distribution, nor the function of clearance and cell activation has been investigated in fish. In the previous study, we have reported the full‐length cDNA of blunt snout bream MR, analyzed its structure and relative mRNA expression during embryogenesis and in the liver, head kidney, spleen and intestine of fish after stimulation with killed Aeromonas hydrophila. In the present study, we developed a rabbit polyclonal antibody against MR and undertook a systematic survey of the expression of MR at the protein level by immunohistochemistry. To get more information about MR function, the mRNA expression of MR, pro‐inflammatory factor TNF‐&agr; and anti‐inflammatory factor ARG2 genes was measured by qRT‐PCR in the liver, head kidney, and spleen after A. hydrophila challenge. We first observed MR expression in the yolk sac at the fertilized egg stage and possibly MR was expressed by early macrophages. We also showed the MR distribution in head kidney, body kidney, spleen, liver, intestine, muscle, brain, heart, and gills. Following A. hydrophila challenge the MR immunoreactive cells became more widespread in head kidney and spleen, which are the major reticuloendothelial systems of fish. The quantitative studies at mRNA levels showed that there exists a high correlation between MR expression and immune cytokine expressions after bacteria challenge. HIGHLIGHTSDistribution of fish MR from early embryogenesis through to adulthood in Megalobrama amblycephala.Modulation of MR expression after challenge with Aeromonas hydrophila.The expression of MR, TNF‐&agr; and ARG2 in the tissues of M. amblycephala after A. hydrophila challenge.

Hepcidin protects grass carp (Ctenopharyngodon idellus) against Flavobacterium columnare infection via regulating iron distribution and immune gene expression

ABSTRACT Columnaris disease (CD) caused by Flavobacterium columnare (F. columnare) is lack of knowledge on effective treatment measures. Bacterial pathogens require iron as an essential nutrient to infect the host. While hepcidin acts as a master regulator in iron metabolism, its contribution to host defense is emerging as complex and multifaceted. In vitro, recombinant Ctenopharyngodon idellus (C. idellus) hepcidin (CiHep) and synthetic CiHep both showed the ability to increase the expression of hepcidin and ferritin in C. idellus kidney cells, especially the recombinant CiHep. In vivo, recombinant CiHep improved the survival rate of C. idellus challenged with F. columnare. In addition, the fish fed diet containing recombinant CiHep (group H‐1) had a higher survival rate than other pretreatment groups. The study showed that recombinant CiHep regulated iron metabolism causing iron redistribution, decreasing serum iron levels and increasing iron accumulation in the hepatopancreas. Moreover, the expression of iron‐related genes was upregulated in various degrees at a different time except for group H‐1. Immune‐related genes were also evaluated, showing higher expression in the groups pretreated with CiHep at an early stage of infection. Of note, a clear upregulation of more immune genes occurred in the groups pretreated with recombinant CiHep than that pretreated with synthetic CiHep in the late stage of infection. In conclusion, the recombinant CiHep has a protective effect on the host response to bacterial pathogens. We speculate that hepcidin protects C. idellus against F. columnare infection via regulating the iron distribution and immune gene expression. HighlightsRecombinant CiHep improves the survival rate of grass carp infected with F. columnare.CiHep regulates the iron distribution to establish the protective effects.CiHep modulates the expression of immune genes in grass carp.

Chemotactic effect of β-defensin 1 on macrophages in Megalobrama amblycephala

ABSTRACT Besides their function as a physical barrier against pathogens, &bgr;‐defensins possess the ability to induce direct or indirect chemotaxis in leukocytes of mammals. However little is known about the ability of defensins to guide the migration of macrophages in fish. The objective of our study was to investigate whether &bgr;‐defensin 1 (maBD1) can recruit leukocytes (specifically macrophages) in vivo and in vitro in a farmed cyprinid fish Megalobrama amblycephala. The M. amblycephala &bgr;‐defensin 1 (maBD1) gene was amplified from the head‐kidney transcriptome. Synthetic maBD1 polypeptide (as well as its N‐terminus half, but not the C‐terminus half) was capable of inducing the migration of leukocytes (specifically macrophages) at concentrations from 26.0&mgr;g/mL to 52.0&mgr;g/mL in head kidney tissue in vitro. When injected intraperitoneally in vivo, the number of leukocytes in the peritoneal cavity was in positive correlation with the maBD1 concentration. maBD1 also induced the expression of two proinflammatory cytokines (IL‐1beta and TNF‐alpha) in spleen, head and body kidney, and hepatopancreas. These results strongly indicate that BD1 has a chemoattractant capacity for macrophages, as well as the ability to modulate the expression of proinflammatory cytokines in fish. HIGHLIGHTSM. amblycephala macrophages were identified via the relative expression of CD68.&bgr;‐defensin 1 induced the migration of macrophages.&bgr;‐defensin 1 enhanced the inflammatory response by stimulating the expression of proinflammatory cytokines.