Bioinformatics characteristics and expression analysis of IL-8 and IL-10 in largemouth bass (Micropterus salmoides) upon Nocardia seriolae infection.

IL-8 and IL-10 are crucial inflammatory cytokines that participate in defending host cells against infections. To demonstrate the function of the two interleukin genes in largemouth bass (Micropterus salmoides), we initially cloned and identified the cDNA sequences of il-8 and il-10 in largemouth bass, referred to as Msil-8 and Msil-10, respectively. The open reading frame (ORF) of Msil-8 was 324 bp in length, encoding 107 amino acids, while the ORF of Msil-10 consisted of 726 bp and encoded 241 amino acids. Furthermore, the functional domains of the SCY domain in MsIL-8 and the IL-10 family signature motif in MsIL-10 were highly conserved across vertebrates. Additionally, both MsIL-8 and MsIL-10 showed close relationships with M. dolomieu. Constitutive expression of Msil-8 and Msil-10 was observed in various tissues, with the highest level found in the head kidney. Subsequently, largemouth bass were infected with Nocardia seriolae via intraperitoneal injection to gain a further understanding of the function of these two genes. Bacterial loads were initially detected in the foregut, followed by the midgut, hindgut, and liver. The mRNA expression of Msil-8 was significantly down-regulated after infection, especially at 2 days post-infection (DPI), with a similar expression to Msil-10. In contrast, the expression of Msil-8 and Msil-10 was significantly upregulated in the foregut at 14 DPI. Taken together, these results reveal that the function of IL-8 and IL-10 was likely hindered by N. seriolae, which promoted bacterial proliferation and intercellular diffusion.

A comparative study on the immune response in the head and trunk kidney of yellow catfish infected with Edwardsiella ictaluri.

The teleost kidneys are anatomically divided into head kidney and trunk kidney, each performing distinct physiological functions. Although previous research has elucidated the role of the head kidney in immune responses, there is a paucity of literature on the comparative studies of the head and trunk kidney response to bacterial infection. Therefore, an Edwardsiella ictaluri infection model of yellow catfish was constructed to investigate and compare the immune responses between the two kidney types. The findings indicated that E. ictaluri infection induced significant pathological changes in both the head and trunk kidney. Despite variances in structure, both the head and trunk kidney of yellow catfish exhibit robust immune responses following E. ictaluri infection. Unexpectedly, the up-regulation level of IgM was found to be higher in the trunk kidney compared to the head kidney. Additionally, both the IgM+ and IgD+ B cells were increased after bacterial infection. This research elucidates the parallels and distinctions in immune functions between both the head and trunk kidney in fish, enriching the immune theory of the fish kidney, and also providing a theoretical basis for the immune response of teleost kidney against bacterial infections.

Molecular characterization and expression analyses of five genes involved in the MyD88-dependent pathway of yellow catfish (Pelteobagrus fulvidraco) responding to challenge of Aeromonas hydrophila.

MyD88-dependent pathway mediated by Toll-like receptor is one of the vital ways activating immune responses. In order to identify the role of MyD88-dependent signaling pathway in yellow catfish, the Pf_MyD88, Pf_IRAK4, Pf_IRAK1, Pf_TRAF6 and Pf_NFκB1 (p105) (Pf: abbreviation of Pelteobagrus fulvidraco) were cloned and characterized respectively. The Pf_MyD88, Pf_IRAK4, Pf_IRAK1 and Pf_TRAF6 were all highly conserved among species and showed the highest homology to that of Pangasianodon hypophthalmus. Pf_NFκB1 showed the highest homology to that of Ictalurus punetaus. All of the five genes showed similar expression patterns in various tissues, with the highest expression level in the liver. These genes also showed similar expression levels in different embryonic development stages, except Pf_IRAK4. The higher expression level was detected from fertilized eggs to 1 day post hatching (dph), lower expression from 3 dph to 30 dph. After stimulation of inactivated Aeromonas hydrophila, the mRNA expressions of Pf_MyD88, Pf_IRAK4, Pf_IRAK1, Pf_TRAF6 and Pf_NFκB1 were significantly increased at 24 h in the liver, spleen, head kidney and trunk kidney, suggesting that all the five genes were involved in the innate immune response of yellow catfish. These results showed that MyD88-dependent signaling pathway plays important roles for disease defensing in the innate immune response. Meanwhile, inactivated A. hydrophila can cause strong innate immune response, which provides theoretical bases for the application of inactivated vaccines in defense against bacterial diseases of teleost.

Molecular characteristics, polymorphism and expression analysis of mhc â ¡ in yellow catfish(pelteobagrus fulvidraco)responding to Flavobacterium columnare infection.

The major histocompatibility complex (MHC) is an important component of the immune system of vertebrates, which plays a vital role in presenting extrinsic antigens. In this study, we cloned and characterized the mhc ⅡA and mhc ⅡB genes of yellow catfish Pelteobagrus fulvidraco. The open reading frames (ORFs) of mhc ⅡA and mhc ⅡB genes were 708 bp and 747bp in length, encoding 235 and 248 amino acids, respectively. The structure of mhc ⅡA and mhc ⅡB includes a signal peptide, an α1/ß1 domain, an α2/ß2 domain, a transmembrane region and a cytoplasmic region. Homologous identity analysis revealed that both mhc ⅡA and mhc ⅡB shared high protein sequence similarity with that of Chinese longsnout catfish Leiocassis longirostris. mhc ⅡA and mhc ⅡB showed similar expression patterns in different tissues, with the higher expression level in spleen, head kidney and gill and lower expression in liver, stomach, gall bladder and heart. The mRNA expression level of mhc ⅡA and mhc ⅡB in different embryonic development stages also showed the similar trends. The higher expression was detected from fertilized egg to 32 cell stage, low expression from multicellular period to 3 days post hatching (dph), and then the expression increased to a higher level from 4 dph to 14 dph. The mRNA expression levels of mhc ⅡA and mhc ⅡB were significantly up-regulated not only in the body kidney and spleen, but also in the midgut, hindgut, liver and gill after challenge of Flavobacterium columnare. The results suggest that Mhc Ⅱ plays an important role in the anti-infection process of yellow catfish P. fulvidraco.

Molecular characterization and expression analysis of IL-22 and its two receptors genes in yellow catfish (Pelteobagrus filvidraco) in response to Edwardsiella ictaluri challenge.

Interleukin (IL)-22, as a member of the interleukin (IL)-10 family, is an important mediator between the immune cells and epithelial tissues during infection and inflammation. This study reported the characterization and mRNA expression patterns of Pf_IL-22 gene and its cell surface-associated receptors Pf_IL-22RA1 and soluble Pf_IL-22RA2 genes in yellow catfish (Pelteobagrus filvidraco). The open reading frames (ORFs) of the Pf_IL-22, Pf_IL-22RA1 and Pf_IL-22RA2 genes were 546 bp, 1740 bp and 690 bp in length, encoding 181, 579 and 229 amino acids, respectively. Alignments of the deduced amino acid sequences present that the Pf_IL-22 has a conserved IL-10 family signature motif, and the Pf_IL-22RA1 and Pf_IL-22RA2 have two conserved fibronectin type-III domains. Quantitative real-time PCR (qPCR) analyses showed that the Pf_IL-22 and Pf_IL-22RA1 mRNAs were highly expressed in mucosal tissues such as the fin, gill, intestine, skin mucus and stomach, and were weakly expressed in the kidney, liver and head kidney of adult yellow catfish, indicating that the Pf_IL-22 transcripts may be mainly produced by mucosal immune cells/tissues in healthy yellow catfish. The mRNA expression levels of the Pf_IL-22RA2 gene were high in the muscle and liver, and were relatively low in the spleen and kidney. The mRNA expression levels of the Pf_IL-22 and its two receptor genes were significantly up-regulated in both mucosal tissues (gill, hindgut, and skin mucus) and systemic immune tissues (spleen, head kidney and blood) after Edwardsiella ictaluri challenge. These results indicated that the Pf_IL-22 and its two receptors genes might play an important role in the innate immune defense against bacterial invasion.

Molecular characterization and expression analysis of three TLR genes in yellow catfish (Pelteobagrus fulvidraco): Responses to stimulation of Aeromonas hydrophila and TLR ligands.

Toll-like receptors (TLRs) are one of the most extensively researched pattern recognition receptors (PRRs) and play an important role in the innate immune system. In this study, partial cDNA sequences of the Pf_TLR18 and Pf_TLR19 genes and complete cDNA sequence of the Pf_TLR21 gene were cloned from yellow catfish (Pelteobagrus fulvidraco). The open reading frames (ORFs) of the Pf_TLR18, Pf_TLR19 and Pf_TLR21 genes were 1956 bp, 2262 bp and 2949 bp in length, encoding 651, 753 and 982 amino acids, respectively. The Pf_TLR18 and Pf_TLR19 consist of leucine-rich repeats (LRRs), a transmembrane domain and a Toll/interleukin-I receptor domain, and the Pf_TLR21 only has LRRs and TIR domain. Homologous identity revealed that the Pf_TLR18, Pf_TLR19 and Pf_TLR21 genes have high nucleotide and protein sequence similarity with channel catfish, especially the TIR domains that exhibited the greatest conservation compared to channel catfish. Ontogenetic expression analyses indicated that the mRNA expressions of the Pf_TLR18, Pf_TLR19 and Pf_TLR21 genes could be detected from fertilized eggs to 30 day post-hatching and they exhibited different variation trends after hatching. The three TLR genes were expressed in various tissues, but they were mostly highly expressed in the spleen. The mRNA expression levels of the three genes were up-regulated in the spleen, head kidney, trunk kidney, liver and blood after challenge of killed Aeromonas hydrophila. In addition, the expressions of the three TLR genes were induced to up-regulate in isolated peripheral blood lymphocytes of yellow catfish after stimulation with lipopolysaccharides (LPS), peptidoglycan (PGN) and polyinosinic-polycytidylic acid (Poly I:C). Our findings indicate that the three TLR genes may play a potential role in the host defense against pathogenic microbes. These results will provide valuable information to better understand the function of TLR genes in the innate immune system of yellow catfish.

Expression analysis of nine Toll-like receptors in yellow catfish (Pelteobagrus fulvidraco) responding to Aeromonas hydrophila challenge.

Toll-like receptors (TLRs) are important components of pattern recognition receptors (PRRs), which play significant roles in innate immunity to defense against pathogen invasion. Many TLRs have been found in teleosts, but there are no reports about cloning and expression of TLR genes in yellow catfish (Pelteobagrus fulvidraco). In this study, we analyzed the sequence characters and the relative mRNA expression levels of nine TLRs (TLR1, TLR2, TLR3, TLR4-1, TLR5, TLR7, TLR8-2, TLR9 and TLR22) in different tissues of yellow catfish. The results showed that all nine TLR genes are highly expressed in head kidney, trunk kidney, spleen and liver, all of which are related to host immunity. Subsequently we used Aeromonas hydrophila as a stimulating agent to detect the expression profiles of these nine TLRs in the liver, spleen, trunk kidney and head kidney of yellow catfish at different time points after injection with killed Aeromonas hydrophila. All nine TLRs responded to A. hydrophila challenge with tissue-specific patterns in different immune tissues. The kinetics of up- or down-regulation of these nine TLRs exhibited a similar trend, rising to an elevated level at first and then falling to the basal level, but the peak value differed at different time points in different tissues. The expression levels of the TLR3, TLR4-1, TLR9 and TLR22 genes were significantly up-regulated after bacterial challenge in the liver, spleen, head kidney and trunk kidney. The relatively high expression of TLR genes in the immune tissues in response to the A. hydrophila challenge indicated that TLRs may play important roles in the innate immune response against gram-negative bacteria in yellow catfish.

Chronic effects of tributyltin on multiple biomarkers responses in juvenile common carp, Cyprinus carpio.

In this study, the chronic toxic effects of tributyltin (TBT), an antifouling paints commonly present in surface and ground water, on morphological indices, reactive oxygen species (ROS) generation, and ATPase activity and heat shock protein (Hsp) 70 protein in tissues (liver, gill, and white muscle) of common carp were investigated. Fish were exposed at sublethal concentrations of TBT (75 ng/L, 0.75 µg/L, and 7.5 µg/L) for 60 days. When compared with the control, there was significant lower condition factor in fish exposed at the higher concentration of TBT. ROS levels in three tissues increased significantly at higher TBT concentrations (0.75 and 7.5 µg/L). The hepatic antioxidant enzymes (total antioxidative capacity and superoxide dismutase) activities were induced at higher concentrations (0.75 µg/L) of TBT. When compared with the hepatic antioxidant enzymes activities in fish exposed to 0.75 µg/L of TBT, there was a decreasing trend in those exposed to TBT with a concentration of 7.5 µg/L. However, all the antioxidant enzymes activities were significantly inhibited in gill and muscle of fish exposed to higher TBT concentrations (0.75 and 7.5 µg/L). Moreover, there was significant lower Na-K-ATPase in three tissues after long-term exposure to higher concentration of TBT, but a significant higher Hsp70 protein levels was observed. In short, environmental concentrations of TBT could not induce obvious impacts on fish, but long-term exposure to higher concentrations of TBT could affect seriously the health status of fish. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 937-944, 2016.

Physiological and molecular responses in brain of juvenile common carp (Cyprinus carpio) following exposure to tributyltin.

Tributyltin (TBT), as antifouling paints, is widely present in aquatic environment, but little is known regarding the toxicity of TBT on fish brain. In this study, the effects of exposure to TBT on the antioxidant defense system, Na(+) -K(+) -ATPase activity, neurological enzymes activity and Hsp 70 protein level in brain of juvenile common carp (Cyprinus carpio) were studied. Fish were exposed to sublethal concentrations of TBT (5, 10 and 20 µg/L) for 7 days. Based on the results, with increasing concentrations of TBT, oxidative stress was apparent as reflected by the significant higher levels of oxidative indices, as well as the significant inhibition of all antioxidant enzymes activities. Besides, the activities of Acetylcholinesterase (AChE), Monoamine oxidases (MAO) and Na(+) -K(+) -ATPase were significantly inhibited after exposure to TBT with higher concentrations. In addition, the levels of Hsp 70 protein were evaluated under TBT stress with dose-depended manner. These results suggest that selected physiological responses in fish brain could be used as potential biomarkers for monitoring residual organotin compounds present in aquatic environment.

Molecular Characterization and Expression Analyses of the Complement Component C8α, C8ß and C9 Genes in Yellow Catfish (Pelteobagrus fulvidraco) after the Aeromonas hydrophila Challenge.

The complement components C8α, C8ß and C9 have important roles in the innate immune system against invading microorganisms. Partial cDNA sequences of the Pf_C8α, Pf_C8ß and Pf_C9 genes (Pf: abbreviation of Pelteobagrus fulvidraco) were cloned from yellow catfish. The Pf_C8α, Pf_C8ß and Pf_C9 genes showed the greatest amino acid similarity to C8α (54%) and C8ß (62%) of zebrafish and to C9 (52%) of grass carp, respectively. Ontogenetic expression analyses using real-time quantitative PCR suggested that the three genes may play crucial roles during embryonic and early larval development. The mRNA expressions of the three genes were all at the highest levels in liver tissue, and at lower or much lower levels in 16 other tissues, demonstrating that the liver is the primary site for the protein synthesis of Pf_C8α, Pf_C8ß and Pf_C9. Injection of Aeromonas hydrophila led to up-regulation of the three genes in the spleen, head kidney, kidney, liver and blood tissues, indicating that the three genes may contribute to the host's defense against invading pathogenic microbes. An increased understanding of the functions of the Pf_C8α, Pf_C8ß and Pf_C9 genes in the innate immunity of yellow catfish will help enhance production of this valuable freshwater species.

Ghrelin, neuropeptide Y (NPY) and cholecystokinin (CCK) in blunt snout bream (Megalobrama amblycephala): cDNA cloning, tissue distribution and mRNA expression changes responding to fasting and refeeding.

Blunt snout bream (Megalobrama amblycephala Yih, 1955) is an endemic freshwater fish in China for which the endocrine mechanism of regulation of feeding has never been examined. Ghrelin, neuropeptide Y (NPY) and cholecystokinin (CCK) play important roles in the regulation of fish feeding. In this study, full-length cDNAs of ghrelin, NPY and CCK were cloned and analyzed from blunt snout bream. Both the ghrelin and NPY genes of blunt snout bream had the same amino acid sequences as grass carp, and CCK also shared considerable similarity with that of grass carp. The three genes were expressed in a wide range of adult tissues, with the highest expression levels of ghrelin in the hindgut, NPY in the hypothalamus and CCK in the pituitary, respectively. Starvation challenge experiments showed that the expression levels of ghrelin and NPY mRNA increased in brain and intestine after starvation, and the expression levels of CCK decreased after starvation. Refeeding could bring the expression levels of the three genes back to the control levels. These results indicated that the feeding behavior of blunt snout bream was regulated by the potential correlative actions of ghrelin, NPY and CCK, which contributed to the defense against starvation. This study will further our understanding of the function of ghrelin, NPY and CCK and the molecular mechanism of feeding regulation in teleosts.

Responses of the hepatic glutathione antioxidant defense system and related gene expression in juvenile common carp after chronic treatment with tributyltin.

Recently, residual organotin compounds have generally been recognised as relevant sources of aquatic environmental pollutants. However, the effects of these contaminants on the glutathione (GSH)-antioxidant system of fishes have not been adequately studied. In the current study, the chronic effects of tributyltin (TBT) found within antifouling paints for ships, on the GSH antioxidant system and related gene expression in the liver of juvenile common carp (Cyprinus carpio) were investigated. Fishes were exposed to sub-lethal concentrations of TBT (75 ng/L, 0.75 and 7.5 µg/L) for 15, 30 and 60 days. GSH levels and GSH-related enzymes activities, including glutathione peroxidase (GPx), glutathione reductase (GR) and glutathione S-transferase (GST), were quantified in the fish liver. The levels of malondialdehyde were also measured as a marker of oxidative damage. In addition, the expression levels of gstp1, gr and gpx1 in common carp chronically exposed to TBT were determined. The results of the current study indicate that chronic exposure of TBT results in reactive oxygen species stress in the liver of common carp, and mRNA expression levels are more sensitive than related enzyme levels. In short, the measured GSH-related indices could potentially be used as molecular indicators for monitoring organotin compounds in the aquatic environment.

Molecular responses in digestive tract of juvenile common carp after chronic exposure to sublethal tributyltin.

The effect of long-term exposure to tributyltin (TBT) on the intestine-related biochemical biomarkers in common carp was investigated in this study. Fish were exposed at sub-lethal concentrations of TBT (75 ng/L, 0.75 and 7.5 µg/L) for 60 days. Multiple biomarkers were measured, including digestive enzymes (trypsin, lipase and amylase), antioxidant responses (malondialdehyde (MDA) and total antioxidative capacity (T-AOC)), RNA/DNA ratio and the expression of digestive-related genes (try, lipc and amy). TBT exposure at 0.75 and 7.5 µg/L led to significantly inhibited activities of all digestive enzymes. At higher concentration of TBT, oxidative stress was apparent as reflected by the significant higher MDA content in the fish intestine, associated with an inhibition of T-AOC activities. After 60 days, the RNA/DNA ratio in fish intestine was significantly lower in groups exposed to TBT at higher concentrations (0.75 and 7.5 µg/L). In addition, the expression levels of try, lipc and amy in intestine of all treated fish were inhibited, even at the environmental concentration (75 ng/L). Our results suggest that long-term exposure to TBT could result in different responses of intestine-related biochemical biomarkers in fish, which could be used as new potential indicators for monitoring residual TBT present in aquatic environment.

Dietary Curcumin Supplementation Could Improve Muscle Quality, Antioxidant Enzyme Activities and the Gut Microbiota Structure of Pelodiscus sinensis.

This experiment aimed to assess the impact of different dietary curcumin (CM) levels on growth, muscle quality, serum-biochemical parameters, antioxidant-enzyme activities, gut microbiome, and liver transcriptome in Chinese soft-shelled turtles (Pelodiscus sinensis). Five experimental diets were formulated to include graded levels of curcumin at 0 (control, CM0), 0.5 (CM0.5), 1 (CM1), 2 (CM2) and 4 g/kg (CM4). Each diet was randomly distributed to quadruplicate groups of turtles (164.33 ± 5.5 g) for 6 weeks. Our findings indicated that dietary curcumin supplementation did not have a significant influence on growth performance (p > 0.05); however, it significantly improved the muscular texture profiles (p < 0.05). Serum total superoxide dismutase (SOD), liver catalase (CAT), and total antioxidant capacity (T-AOC) activities increased significantly as dietary curcumin levels rose from 0.5 to 4 g/kg (p < 0.05). Dietary curcumin supplementation improved gut microbiota composition, as evidenced by an increase in the proportion of dominant bacteria such as Lactobacillus and Flavobacterium. Liver transcriptome analysis revealed that curcumin altered metabolic pathways in the liver. In conclusion, based on the evaluation of the activities of SOD in serum and CAT in liver under current experimental design, it was determined that the appropriate dietary curcumin supplementation for Chinese soft-shelled turtles is approximately 3.9 g/kg.

Comparative Study on Immune Function of the Head and Trunk Kidney in Rainbow Trout Responding to IHNV Infection.

A teleost's kidney was divided into head kidney and trunk kidney. The head kidney is an important lymphatic organ, while the trunk kidney mainly performs osmotic pressure regulation and excretion functions. Previous studies have shown that the teleost's head kidney exerts a strong immune response against pathogen invasion, while the mechanism of immune response in the trunk kidney is still rarely reported. Therefore, in this study, we established an Infectious hematopoietic necrosis virus (IHNV) immersion infection model to compare the similarities and differences of immune response mechanisms between the head kidney and trunk kidney against viral infection. The results showed that IHNV infection causes severe tissue damage and inflammatory reaction in the head and trunk kidney, triggers a series of interferon cascade reactions, and produces strong immune response. In addition, the transcriptome data showed that the head kidney and trunk kidney had similar immune response mechanisms, which showed that the NOD-like receptor signaling pathway and Toll-like receptor signaling pathway were activated. In conclusion, despite functional differentiation, the teleost's trunk kidney still has a strong immune response, especially the interferon-stimulated genes, which have stronger immune response in the trunk kidney than in the head kidney when responding to IHNV infection. This study contributes to a more comprehensive understanding of the teleost immune system and enriches the theory of kidney immunity in teleosts.

Molecular and functional characterization of teleost-specific Interleukin-17N in yellow catfish (Pelteobagrus fulvidraco).

In mammals, six interleukin-17 (IL-17) genes, as potent pro-inflammatory cytokines, all accelerate the inflammatory responses. In teleosts, seven IL-17 genes have been found in various species, but little is known about the function of teleost-specific IL-17N. In this study, teleost IL-17N and IL-17A/F2 genes all had six conserved cysteine residues forming three intrachain disulfide bridges, the length of three exons of teleost IL-17N gene was similar to that of teleost IL-17A/F2 gene, and the neighbor-joining (NJ) phylogenetic tree showed that teleost IL-17N was clustered with vertebrate IL-17A/F, implying that teleost IL-17N gene may be a paralog of teleost IL-17A/F gene. Pelteobagrus fulvidraco (Pf) IL-17N gene was highly expressed in the blood, brain and kidney of healthy yellow catfish. Pf_IL-17N transcript and protein were notably up-regulated in the spleen, head kidney, gill and kidney detected after Edwardsiella ictaluri infection. Lipopolysaccharides (LPS), polyinosinic-polycytidylic acid (Poly I:C) and peptidoglycan (PGN) also remarkably induced the expression of Pf_IL-17N in the isolated peripheral blood leucocytes (PBLs) of yellow catfish. These results reveal that Pf_IL-17N may play important roles in preventing the invasion of pathogens. Furthermore, the recombinant (r) Pf_IL-17N protein could significantly induce the mRNA expressions of inflammatory cytokines, chemokines and antimicrobial peptide genes in yellow catfish in vivo and in vitro, and it also notably promoted the phagocytosis of myeloid cells in the PBLs and the chemotaxis of the PBLs and gill leucocytes (GLs) in yellow catfish. Besides, though the rPf_IL-17N protein could induce and aggravate inflammation infiltration in the kidney of yellow catfish, it did not effectively and notably increase the survival rate of yellow catfish after E. ictaluri infection. Furthermore, the rPf_IL-17N protein could induce the mRNA expressions of nuclear factor kappa-B (NF-κB) and mitogen-activated protein kinase (MAPK) signal pathways related genes, and the inhibitor of NF-κB and MAPK signal pathways could restrain the rPf_IL-17N protein-induced inflammatory response. This study provides crucial evidence that the Pf_IL-17N may mediate inflammatory response to eliminate invasive pathogens.

Characterization, expression and function analysis of pfTLR5S and pfTLR5M in yellow catfish (Pelteobagrus fulvidraco) responding to bacterial challenge.

Toll-Like Receptors (TLRs) are important pattern recognition receptors, playing critical roles in the early innate immune response to defensing against pathogen invasion. In this study, we found both soluble form TLR5 (pfTLR5S) and membrane form TLR5 (pfTLR5M) in yellow catfish Pelteobagrus fulvidraco. The open reading frames (ORFs) of pfTLR5M and pfTLR5S genes were 2655 bp and 1947 bp in length, encoding 884 and 648 amino acids, respectively. pfTLR5M was composed of thirteen LRR domains, one TIR domain and one transmembrane domain. However, pfTLR5S have only fifteen LRR domains, without any TIR domain and transmembrane domain. Both pfTLR5M and pfTLR5S genes had the highest expression in liver, especially for pfTLR5S, which showed a noticeable high expression in liver. We also compared the relative mRNA expression levels of pfTLR5M and pfTLR5S in digestive and immune-related tissues after challenge of three different bacteria. In addition, we also found that pfTLR5S can interact with pfTLR5M, and inhibit the expression of pfTLR5M protein, while induced the expression of downstream proinflammatory factors, such as TNFα and IL8. These results revealed that both pfTLR5M and pfTLR5S play important and different roles in defensing against the invasion of flagellated bacteria, and they may function by binding to each other.

Blood brain barrier permeability and immune function of brain in rainbow trout responding to IHNV infection.

Viral infection of the central nervous system (CNS) is often associated with blood-brain barrier (BBB) disruption. Mammals have developed complicated and efficient immune strategies to protect the BBB. However, the immune defense of brain and BBB permeability changes are not well-understood in teleost during virus invading. In this study, we constructed an infectious hematopoietic necrosis virus (IHNV) immersion infected rainbow trout model. After IHNV infection, pathological changes occurred in the brain, and MPO and ROS activities were significantly increased. In addition, the expression levels of BBB permeability-related genes were also changed. Transcriptome analysis showed that immune-related genes and signaling pathways in the brain were activated after IHNV infection. These results showed that the permeability of BBB increased significantly after IHNV infection, thus activating immune related factors and cells to enter the CNS through blood circulation to resist pathogenic infection.

Anemia induced by repeated exposure to cyanobacterial extracts with explorations of underlying mechanisms.

Hematological abnormalities or derangements have been demonstrated in patients suffering form microcystins (MCs) in hemodialysis unit in Caruaru, Brazil, 1996. While experimental study on hematological effect of microcystins has been rare and the underlying mechanisms are still puzzling. In the present study, microcystins were repeatedly intraperitoneally injected with a dose of 6 µg/kg/day in rabbits (Oryctolagus cuniculus) for 14 days, and the prolonged effects of extracted microcystins on hematotoxicology were investigated. Significant decreases were observed in the hematological indices red blood cell counts, hematocrit, hemoglobin, and platelet count, while an obvious anemia occurred in rabbits after 14-day exposure. Moreover, red blood cell volume distribution width, mean corpuscular volume, and mean corpuscular hemoglobin did not vary significantly, indicating that rabbits suffered from normocytic anemia. In bone marrow, on the 14th day after toxin exposure, the frequency of micronucleus increased significantly, and the viability of bone marrow cells decreased markedly compared with the control. Serum erythropoietin levels declined on the 7th and 14th day, which suggested that the ability to regulate differentiation and maturation of erythrocytes was impaired. These results indicate that repeated exposure of microcystins can result in normocyte anemia, and the bone marrow injures and the sharp decreases of erythropoietin levels were responsible for the anemia.

Expression and Function Analysis of Interleukin-17A/F1, 2, and 3 Genes in Yellow Catfish (Pelteobagrus fulvidraco): Distinct Bioactivity of Recombinant IL-17A/F1, 2, and 3.

In mammals, Interleukin-17 cytokine family plays critical roles in both acute and chronic inflammatory responses. In fish species, three Interleukin-17A/F (IL-17A/F) genes have been identified to be homologous to mammalian IL-17A and IL-17F, but little is known about their functional activity. In this study, Pf_IL-17A/F1, 2 and 3 genes were cloned from yellow catfish (Pelteobagrus fulvidraco) and they differed in protein structure and exon length, implying that they may have divergent bioactivity. Real-time quantitative PCR analyses revealed that three Pf_IL-17A/F genes were highly expressed in blood and mucosal tissues (skin+mucus and gill) from healthy adult fish. The mRNA expressions of Pf_IL-17A/F1, 2 and 3 genes were significantly up-regulated in the gill, skin+mucus, head kidney and spleen after challenge with Edwardsiella ictaluri and in the isolated peripheral blood leucocytes (PBLs) of yellow catfish after stimulation with phytohaemagglutinin (PHA), lipopolysaccharides (LPS), peptidoglycan (PGN) and polyinosinic-polycytidylic acid (Poly I:C). These results indicate that Pf_IL-17A/F1, 2 and 3 genes may play a vital role in the regulation of immune against pathogens. Additionally, the recombinant (r) Pf_IL-17A/F1, 2 and 3 proteins significantly induced the mRNA expressions of proinflammatory cytokines, chemokines and antibacterial peptides genes, and the rPf_IL-17A/F 2 and 3 proteins promoted phagocytosis of PBLs more powerfully than the rPf_IL-17A/F1. Furthermore, the rPf_IL-17A/F1, 2 and 3 proteins might activate the NF-κB and MAPK signal pathways by IL-17RA, ACT1, TRAF6, TRAF2, TRAF5 and TAK1, indicating that the three Pf_IL-17A/F proteins may play different roles in promoting inflammatory response.