Alterations in hematological and biochemical parameters, oxidative stress, and immune response in Takifugu rubripes under acute ammonia exposure.

Ammonia is a major pollutant in aquatic environments and poses a considerable threat to the survival of fish. In this study, we investigated the toxic effects of ammonia on the hematological and biochemical parameters, oxidative stress, and immune responses in Takifugu rubripes. Juvenile T. rubripes (average weight 246.17 ± 3.54 g) were exposed to different concentrations of ammonia (0, 5, 50, 100, and 150 mg/L) for 96 h. The results showed that the hematological parameters (hemoglobin, hematocrit, red blood cell, and white blood cell count) were significantly reduced in response to ammonia exposure. Of the plasma components, such as serum total protein, albumin, glucose, glutamic-oxalacetic transaminase, and glutamic-pyruvic transaminase, were significantly altered in response to ammonia exposure. Additionally, the levels of superoxide dismutase (SOD), catalase (CAT), glutathione (GSH), and glutathione peroxidase (GPx) were increased after exposure to low concentration ammonia exposure. However, when fish were exposed to a high concentration of ammonia, these parameters showed the opposite trend, suggesting that an increase in antioxidant enzymes during the early stages of ammonia exposure may contribute to the removal of the induced reactive oxygen species (ROS) and protect the cells from oxidative damage. However, as the ammonia concentration and exposure time increased, the overproduction of ROS accelerated the depletion of antioxidant enzymes. Ammonia exposure significantly increased the expression of heat shock proteins (HSP70 and HSP90). Ammonia poisoning elevated gene expressions of TLR-3, TNF-α, IL-6, IL-12, and IL-1ß in the gills, causing an inflammatory response. Our findings provide new insights into the mechanisms involved in ammonia-induced aquatic toxicology in marine fish, which may aid in their captive management.

Quantitative proteomic analysis in serum of Takifugu rubripes infected with Cryptocaryon irritans.

Cryptocaryon irritans can cause cryptocaryonosis (white spot disease) in marine fish but the pathogenesis of the disease is unclear. In this work, we used high-throughput proteomics to identify differentially expressed proteins in the serum of Takifugu rubripes challenged with C. irritans. By using quantitative proteomic assays combined with Tandem Mass Tag-labeled quantitative proteomic analysis, we identified a total of 2088 differentially abundant proteins (1706 proteins were quantified, p < 0.05, fold-change threshold ≥ 2), including 21 up-regulated and 44 down-regulated. Combined with STRING-based functional analysis, we ultimately obtained eight proteins including glucokinase-like, integrin beta-1-like isoform X2, H4, H2A.V, histone H1-like, histone H2AX-like, histone H2B 1/2-like and myosin-9 isoform X1, which could be considered as potential biomarkers for T. rubripes immune responses. Eight proteins that were selected to validate significant differentially expressed genes at the proteomic level were consistent with qPCR at the transcriptomic level. The proteins identified in our work may serve as candidates for elucidating the molecular mechanism of cryptocaryonosis in T. rubripes. Our collective findings could provide new insights into searching for disease-specific targets and biomarkers, which may be effective indicators of C. irritans infection in T. rubripes.

Impact of nitrite exposure on plasma biochemical parameters and immune-related responses in Takifugu rubripes.

Nitrite is a major environmental pollutant in aquatic environments that negatively affects aquatic species. In this study, we investigated the impact of nitrite exposure on plasma biochemical parameters and immune responses in Takifugu rubripes. Fish were exposed to various concentrations of nitrite (0, 0.5, 1, 3, and 6 mM) for 96 h. After 0, 12, 24, 48, and 96 h of exposure, fish blood samples were collected to assay the levels of total protein (TP), albumin (Alb), glutamic-oxaloacetic transaminase (GOT), glutamic-pyruvic transaminase (ALT), complement C3 (C3), complement C4 (C4), immunoglobulin (IgM), and lysozyme activity (LZM). The gills were sampled to analyze the mRNA levels of heat shock protein 70 (hsp70), heat shock protein 90 (hsp90), tumor necrosis factor α (tnf-α), B-cell activating factor (baff), interleukin-6 (il-6), and interleukin-12 (il-12). Levels of GOT, ALT, C3, and C4 were significantly enhanced in the high nitrite concentration group (3 and 6 mM), whereas those of TP, Alb, LZM, and IgM decreased significantly with the same treatments. Nitrite significantly upregulated hsp70, hsp90, tnf-α, il-6, il-12, and baff mRNA levels after 96 h of exposure. These results indicated that nitrite exposure altered the blood physiological status and immune system response, resulting in dysfunction and immunotoxicity in T. rubripes. Furthermore, our results reveal the possible mechanism of aquatic-nitrite-induced toxicity in fish.

The improved energy metabolism and blood oxygen-carrying capacity for pufferfish, Takifugu fasciatus, against acute hypoxia under the regulation of oxygen sensors.

Hypoxia frequently occurs in aquatic ecosystem, which is influenced by salinity, water temperature, weather, and surface water runoff. In order to shed further light on the evolutionary and adaptive mechanisms in fish under hypoxic condition, the impact of acute hypoxia (1.63 ± 0.2 mg/L) and reoxygenation (7.0 ± 0.3 mg/L) on oxygen sensors, energy metabolism, and hematological indices was evaluated in Takifugu fasciatus. Data from transcriptional level analysis show that the expressions of genes related to oxygen sensors (HIF-1α, PHD2, and VHL) were upregulated in the brain and liver under hypoxia and recovered under reoxygenation. The upregulation of GLUT2, VEGF-A, and EPO in conjugation with VEGF-A protein and hematological indices conferred the rapid adjustments of cellular glucose uptake and blood oxygen-carrying capacities in pufferfish. Higher levels of glycolysis-related mRNAs (HK, PGK1, and PGAM2), HK activity, and proteins (PGK1 and PGAM2) were detected in the brain and liver under hypoxic condition compared with control. Interestingly, the expression of MDH1 at the mRNA, enzyme activity, and protein levels was significantly increased in the brain at 0 or 2 h and in the liver at 8 h under hypoxic condition. In addition, although the enzyme activity and mRNA expression of LDH in the brain were not significantly changed, a persistent upregulation was observed in the liver during hypoxia exposure. This study demonstrated that pufferfish could counterpoise the energetic demands and hematological functional properties evoked by oxygen sensors after hypoxia. Our findings provided new insights into the molecular regulatory mechanism of hypoxia in pufferfish.

Effects of high temperature on biochemical parameters, oxidative stress, DNA damage and apoptosis of pufferfish (Takifugu obscurus).

Water temperature is an important environmental factor that affects physiology and biochemical activities of fish. In this study, we investigated of high temperature on biochemical parameters, oxidative stress, DNA damage and apoptosis of pufferfish. Thermal stress could significantly increase the levels of AST, ALT, LDH, GLU and TG, whereas the levels of ALP and TP decrease significantly. In addition, thermal stress also decreased total blood cell count, inhibited cell viability, and subsequently lead to DNA damage and apoptosis. The mRNA levels of p53, caspase-9 and caspase-3 were up-regulated under thermal stress. These results suggested that caspase-dependent and p53 signaling pathways could play important roles in thermal stress-induced apoptosis in fish. Furthermore, the gene expression of SOD, CAT, HSP90 and C3 were induced by thermal stress. This study provides new insights into the mechanism whereby thermal stress affects physiological responses and apoptosis in pufferfish.

Immune and physiological responses of pufferfish (Takifugu obscurus) under cold stress.

Low temperature is an important environmental factor in aquaculture farming that affects the survival and growth of organisms. In the present study, we investigated the effects of low temperature on biochemical parameters, oxidative stress and apoptosis in pufferfish. In the stress group, water temperature decreased from 25 °C to 13 °C at a rate of 1 °C/1 h. Fish blood and liver were collected to assay biochemical parameters, oxidative stress and expression of genes at 25 °C, 21 °C, 17 °C, 13 °C and 13 °C for 24 h. The results showed that low temperature could decrease total blood cell count, inhibit cell viability, and subsequently lead to DNA damage. Biochemical parameters such as plasma protein and ALP significantly declined in fish under low temperature, while a significant increase in AST, ALT, LDH and glucose was observed. The gene expression of antioxidant enzymes (SOD and CAT), HSP90 and C3 were induced by low temperature stress. Furthermore, the gene expression of apoptotic related genes including P53, caspase-9 and caspase-3 were up-regulated, suggesting that caspase-dependent pathway could play important roles in low temperature-induced apoptosis in fish. This study may provide baseline information about how cold stress affects the physiological responses and apoptosis in fish.

Fugu (Takifugu rubripes) serum GlcNAc-binding lectin is a kalliklectin but has different properties from those of a reported homologue.

A 40-kDa lectin with N-acetyl-d-glucosamine-binding ability was purified from the sera of fugu (Takifugu rubripes) by affinity chromatography and subsequent gel filtration. N-terminal amino acid sequencing, in silico cloning using the fugu genome database and cDNA cloning demonstrated that this lectin is a homologue of kalliklectin, a novel lectin that was previously found in the flathead teleost Platycephalus indicus and has structural similarity to mammalian plasma kallikreins and coagulation factor XI. This is the second report of a kalliklectin, but the fugu kalliklectin differs in its sugar-binding spectra, intersubunit association and tissue distribution from the previously identified flathead kalliklectin. These findings indicate that kalliklectins vary in properties among fish species.

Identification of perfluorooctane sulfonate binding protein in the plasma of tiger pufferfish Takifugu rubripes.

It is well known that perfluorooctane sulfonate (PFOS) preferentially accumulates in the plasma of wildlife and humans. Although earlier studies have suggested that this was due to binding of PFOS to a plasma protein, definite characterization of the protein in in vivo exposure studies was not conducted thus far. In this study, we conducted both in vitro and in vivo experiments to identify PFOS binding protein in the plasma of fish. For the in vivo studies, PFOS was administered intraperitoneally to tiger pufferfish, Takifugu rubripes, and the plasma was separated by ammonium sulfate fractionation. High concentrations of PFOS were found in the 65-70 percent ammonium sulfate fraction (190ng/mL). After SDS-PAGE and N-terminal amino acid sequence analysis, the PFOS-binding protein was identified as an apolipoprotein A-I, which was confirmed on the basis of a significant correlation to the PFOS concentration in each fraction. The plasma samples fractionated by ammonium sulfate from untreated pufferfish were subjected to PFOS binding assay by the equilibrium dialysis method. The results further confirmed that the 60-65 percent ammonium sulfate fraction showed a high PFOS-binding ratio, similar to that found from in vivo studies. We demonstrated that PFOS is likely bound to an apolipoprotein A-I in the plasma of tiger pufferfish in in vivo and in vitro studies.

Serum GlcNAc-binding IgM of fugu (Takifugu rubripes) suppresses the growth of fish pathogenic bacteria: a novel function of teleost antibody.

N-acetyl-d-glucosamine (GlcNAc) is one of the components of peptidoglycan, a biopolymer in the bacterial cell wall. We purified a novel GlcNAc-binding protein, designated as fGBP-78, from sera of fugu (Takifugu rubripes). The fGBP-78 is a heteromer of 78- and 25-kDa subunits. Moreover, fGBP-78 exerted remarkable inhibitory effects on the growth of both Gram-positive and Gram-negative bacteria, including ones virulent for marine fish species as well as non-pathogenic Escherichia coli. These results suggest that fGBP-78 contributes to bacterial clearance in fugu. Furthermore, the nanoLC-MS/MS and Western blotting analyses reveal that the 78-kDa subunit is the fugu IgM heavy chain. In addition, the molecular mass of the other subunit (25 kDa) was equal to that of the Ig light chain. Overall, results indicate that fGBP-78 is an IgM molecule presumably acts as a natural antibody. This paper reports a novel function of teleost IgM as a significant suppresser against bacterial growth.

RT-PCR- and MALDI-TOF mass spectrometry-based identification and discrimination of isoforms homologous to pufferfish saxitoxin- and tetrodotoxin-binding protein in the plasma of non-toxic cultured pufferfish (Takifugu rubripes).

Four genes of Takifugu rubripes, tentatively designated Tr1-Tr4, encoding homologs of pufferfish saxitoxin- and tetrodotoxin-binding protein, were identified by BLAST search and 3'-RACE. RT-PCR and MALDI-TOF mass spectrometry allowed the identification and discrimination of Tr isoforms from the non-toxically cultured specimens. The expression of Tr1 and Tr3 mRNAs exclusively in the liver and the presence of their products as 120-kDa plasma proteins were confirmed.

Purification and characterization of tributyltin-binding protein of tiger puffer, Takifugu rubripes.

We successfully purified Trub.TBT-bpα, a tributyltin (TBT) binding protein (bp) of the tiger puffer, Takifugu rubripes. Tiger puffer was injected intraperitoneally with TBT (1.0mg/kg body weight) and Trub.TBT-bpα was purified from serum by ammonium sulfate fractionation, gel filtration chromatography and polyacrylamide gel electrophoresis. Gel electrophoresis revealed that the Trub.TBT-bpα has a molecular mass of approximately 48.5kDa and contains at least 40% N-glycan. The deduced 212 amino acid sequence of the protein showed the highest identity (41%, 212 amino acid overlap and E-value: 9e-42) with TBT-binding protein type 1 (TBT-bp1) of Paralichthys olivaceus (Japanese flounder). Analysis of the gene structure of Trub.TBT-bpα suggests that this protein belongs to the lipocalin superfamily, which may be important in the accumulation and elimination of TBT. Phylogenetic analysis suggests that functionalization of TBT-bps has occurred during evolution, and that the functions of this group of proteins might be important for fish survival.

Characterization of CD8+ leukocytes in fugu (Takifugu rubripes) with antiserum against fugu CD8alpha.

We have investigated the characteristics of CD8+ leukocytes by using an anti-CD8alpha antiserum raised in mouse by DNA-immunization. The magnetically sorted CD8alpha+ peripheral blood leukocyte (PBL) population comprised lymphocytes/thrombocytes and monocytes, whereas CD8alpha- PBLs consisted of lymphocytes/thrombocytes, monocytes, and neutrophils. Expression analysis demonstrated that both groups of cells expressed the CD3epsilon and TCRalpha genes. The CD8alpha and CD8beta genes were detected only in CD8alpha+ cells, whereas expression of CD4 and immunoglobulin light chain (IgL) was observed only in CD8alpha- cells. These results suggest that fugu CD8alpha+ leukocytes contain CD8+ T cells, but not CD4+ T cells or B cells. Furthermore, mitogenesis of the CD8+ lymphocyte/thrombocyte population was induced by phytohemaglutinin stimulation, suggesting that fish CD8+ lymphocytes/thrombocytes (probably CD8+ T cells) have characteristics similar to mammalian CD8+ T cells. Neutrophils and monocytes/macrophages infiltrating a subcutaneous inflammatory site expressed only CD8alpha, but not CD8beta, CD4, TCRalpha, or IgL. This result suggests that similar to mammalian dendritic cells, fugu monocytes/macrophages express CD8alpha.

Molecular evolution of the vertebrate blood coagulation network.

In mammalian blood coagulation 5 proteases, factor VII (FVII), factor IX (FIX), factor X (FX), protein C (PC) and prothrombin act with two cofactors factor V and factor VIII to control the generation of fibrin. Biochemical evidence and molecular cloning data have previously indicated that blood coagulation involving tissue factor, prothrombin and fibrinogen is present in all vertebrates. Using degenerate RT-PCR we have isolated and characterized novel cDNAs with sequence identity to the blood coagulation serine proteases and cofactors from chicken and the puffer fish (Fugu rubripes). Sequence alignments, phylogenetic and comparative sequence analysis all support the existence of the Gla-EGF1-EGF2-SP domain serine proteases FVII, FIX, FX, PC and the A1-A2-B-A3-C1-C2 domain protein cofactors FV and FVIII in these species. These results strongly suggest that the blood coagulation network is present in all jawed vertebrates and evolved before the divergence of tetrapods and teleosts over 430 million years ago; and that vertebrate blood coagulation may have benefited from two rounds of gene or whole genome duplication. Sequences identified in Fugu coding for additional FVII-like, FIX-like and PC-like sequences support the possibility of further tandem and large-scale duplications in teleosts. Comparative sequence analyses of amino acid residues in the active site region suggest these additional sequences have evolved new and as yet unknown functions.