The protective effects of vitamin C on apoptosis, DNA damage and proteome of pufferfish (Takifugu obscurus) under low temperature stress.

The aim of this study was to investigate the protective effects of vitamin C on apoptosis, DNA damage and proteome of pufferfish under low temperature stress. Six diets were formulated to contain 2.60, 48.90, 95.50, 189.83, 382.40, 779.53mg/kg vitamin C. After 8-week feeding trial, fish were exposed to low temperature challenge. The results showed that pufferfish receiving vitamin C diet exhibited a significant decrease in ROS production (48.9-189.83mg/kg vitamin C diet groups), cytoplasmic free-Ca2+ concentration (48.9-779.53mg/kg vitamin C diet groups), apoptotic cell ratio (95.5-779.53mg/kg vitamin C diet groups) and DNA damage (189.83-779.53mg/kg vitamin C diet groups) under low temperature stress in comparison with those of control. We also investigated the alteration in protein expression under low temperature stress by a comparative proteomic analysis. The results demonstrated that 24 protein spots showed significantly differential expression in the cold-stress-treated group compared with those of the control group, and 5 protein spots were successfully identified. Furthermore, comparative proteomic analysis revealed that vitamin C could increase expressed proteins related to energy metabolism, immune responses and cytoskeleton. These findings would be helpful to understand the protective effects of vitamin C against cold stress.

Effect of dietary astaxanthin on the growth performance, non-specific immunity, and antioxidant capacity of pufferfish (Takifugu obscurus) under high temperature stress.

The present study was conducted to investigate the effects of astaxanthin on growth performance, biochemical parameters, ROS production, and immune-related gene expressions of the pufferfish (Takifugu obscurus) under high temperature stress. The experimental basal diets supplemented with astaxanthin at the rates of 0 (control), 20, 40, 80, 160, and 320 mg kg-1 were fed to fish for 8 weeks. The results showed that the fish fed diet with 80, 160, and 320 mg kg-1 astaxanthin significantly improved weight gain and specific growth rate. Furthermore, fish fed the moderate dietary astaxanthin increased plasma alkaline phosphatase activities, and decrease plasma aspartate aminotransferase and alanine aminotransferase activities. After the feeding trial, the fish were exposed to high temperature stress for 48 h. The results shown that astaxanthin could suppress ROS production induced by high temperature stress. Meanwhile, compared with the control group, the astaxanthin groups increased SOD, CAT, and HSP70 mRNA levels under high temperature stress. These results showed that the basal diet supplemented with 80-320 mg kg-1 astaxanthin could enhance growth, nonspecific immune responses, and antioxidant defense system and improve resistance against high temperature stress 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.

Identification, characterization and expression analysis of tumor suppressor protein p53 from pufferfish (Takifugu obscurus) after the Vibrio alginolyticus challenge.

The tumor suppressor protein p53 plays a critical role in cell cycle, apoptosis and DNA repair. In this study, the full-length pufferfish p53 (Pf-p53) was obtained, containing an open reading frame of 1095 bp, a 5'UTR of 157 bp and a 3'UTR of 285 bp with a poly (A) tail. The Pf-p53 encoded a polypeptide of 364 amino acids with a theoretical isoelectric point of 8.03 and predicted molecular weight of 40.6 kDa. Pf-p53 was ubiquitously expressed in various tissues with a high-level expression in kidney, liver and gill. Vibrio alginolyticus challenge could induce ROS production and disrupt Ca2+ homeostasis, subsequently leading to the induction of DNA damage and apoptosis, while the Vibrio alginolyticus-induced oxidative stress can also increase the non-specific immunity. The pufferfish challenged with Vibrio alginolyticus showed a sharp increase of Pf-p53 transcript in liver. Subcellular localization analysis revealed that Pf-p53 was primarily localized in nucleus. Furthermore, overexpression of Pf-p53 in Hela cells could inhibit cell proliferation and the transcriptional activities of the NF-ĸB promoter. Taken together, our results indicated that Pf-p53 may play an important role in the immune response to Vibrio alginolyticus challenge.

High temperature induces apoptosis and oxidative stress in pufferfish (Takifugu obscurus) blood cells.

Water temperature is an important environmental factor in aquaculture farming that affects the survival and growth of organisms. The change in culture water temperature may not only modify various chemical and biological processes but also affect the status of fish populations. In previous studies, high temperature induced apoptosis and oxidative stress. However, the precise mechanism and the pathways that are activated in fish are still unclear. In the present study, we investigated the effects of high temperature (34°C) on the induction of apoptosis and oxidative stress in pufferfish (Takifugu obscurus) blood cells. The data showed that high temperature exposure increased oxygen species (ROS), cytoplasmic free-Ca(2+) concentration and cell apoptosis. To test the apoptotic pathway, the expression pattern of some key apoptotic related genes including P53, Bax, caspase 9 and caspase 3 were examined. The results showed that acute high temperature stress induced up-regulation of these genes, suggesting that the p53-Bax pathway and the caspase-dependent apoptotic pathway could be involved in apoptosis induced by high temperature stress. Furthermore, the gene expression of antioxidant enzymes (Cu/Zn-SOD, Mn-SOD, CAT, GPx, and GR) and heat shock proteins (HSP90 and HSP70) in the blood cells were induced by high temperature stress. Taken together, our results showed that high temperature-induced oxidative stress may cause pufferfish blood cells apoptosis, and cooperatively activated p53-Bax and caspase-dependent apoptotic pathway.

Identification, characterization and functional analysis of anti-apoptotic protein BCL-2-like gene from pufferfish, Takifugu obscurus, responding to bacterial challenge.

Apoptosis plays a crucial role in many biological processes, including development, cellular homeostasis and immune responses. The BCL-2 family is a key regulator of the mitochondrial response to apoptotic signals in the intrinsic pathway. In this study, we identified and characterized the cDNA and expression pattern of pufferfish BCL-2 (PfBCL-2). The full-length cDNA of PfBCL-2 was 1412 bp with an open reading frame of 657 bp encoding a putative protein of 219 amino acids (Accession no: KP898414). The calculated molecular mass of the PfBCL-2 was 24.2 kDa with a predicted isoelectric point of 5.27. The deduced PfBCL-2 protein exhibited four highly conserved BCL-2 homology domains, suggesting that PfBCL-2 may play a similar role in the apoptotic-signaling pathway as in other species. Real-time PCR results showed that PfBCL-2 transcript was expressed in a wide range of tissues but exhibited the greatest level of expression in blood. Transcriptional responses of PfBCL-2 exhibited different spatial and temporal expression profiles in liver and blood after bacterial infection. PfBcl-2 transcript was significantly up-regulated in liver at 6, 12, 24 and 48 h (with maximum induction at 48 h) and was up-regulated in blood at 3, 6, 12 and 24 h (with maximum induction at 12 h). Meanwhile, recombinant PfBCL-2 fused with His6 tag was efficiently expressed in Escherichia coli BL21 (DE3) and purified using Ni-nitrilotriacetic acid resin. Western blot analysis indicated that its protein level appeared to be elevated during the initial bacterial infection. These results suggest that PfBCL-2 plays important roles in immune responses against bacteria challenge.

Effects of ammonia exposure on apoptosis, oxidative stress and immune response in pufferfish (Takifugu obscurus).

Ammonia is one of major environmental pollutants in the freshwater aquatic system that affects the survival and growth of organisms. In the present study, we investigated the effects of ammonia exposure on apoptosis, oxidative stress and immune response in pufferfish (Takifugu obscurus). Fish were exposed to various concentrations of ammonia (0, 1.43, 3.57, 7.14mM) for 72h. The date showed that ammonia exposure could induce intracellular reactive oxygen species (ROS), interrupt intracellular Ca(2+) (cf-Ca(2+)) homeostasis, and subsequently lead to DNA damage and cell apoptosis. To test the apoptotic pathway, the expression patterns of some key apoptotic related genes including P53, Bax Bcl2, Caspase 9, Caspase 8 and Caspase 3 in the liver were examined. The results showed that ammonia stress could change these genes transcription, associated with increasing of cell apoptosis, suggesting that the P53-Bax-Bcl2 pathway and caspase-dependent apoptotic pathway could be involved in cell apoptosis induced by ammonia stress. In addition, ammonia stress could induced up-regulation of inflammatory cytokines (BAFF, TNF-α, IL-6 and IL-12) transcription, indicating that innate immune system play important roles in ammonia-induced toxicity in fish. Furthermore, the gene expressions of antioxidant enzymes (Mn-SOD, CAT, GPx, and GR) and heat shock proteins (HSP90 and HSP70) in the liver were induced by ammonia stress, suggesting that antioxidant system and heat shock proteins tried to protect cells from oxidative stress and apoptosis induced by ammonia stress. Our results will be helpful to understand the mechanism of aquatic toxicology induced by ammonia in fish.

Effect of acute ammonia exposure on expression of GH/IGF axis genes GHR1, GHR2 and IGF-1 in pufferfish (Takifugu obscurus).

Waterborne ammonia has become a persistent pollutant of aquatic habitats. The exposure to ammonia stress can reduce growth in a wide range of aquatic organisms. To assess the effect of ammonia exposure on the growth hormone/insulin-like growth factors (GH/IGF) axis, we identified and characterized GHR1, GHR2 and IGF-1 from pufferfish. Comparative analysis showed that these genes shared high identity and similarity with corresponding genes in other fish species. The transcripts of these genes were widely expressed in all tested tissues. The highest level of GHR1 mRNA was found in the brain, whereas GHR2 and IGF-1 mRNA levels were the highest in the liver. Following acute ammonia exposure (100 mg/L total ammonia-nitrogen), GHR2 expression in the liver did not change at 6 h and then significantly decreased at 12, 24 and 48 h, whereas GHR1 and IGF-1 expressions were significantly down-regulated at 6, 12, 24 and 48 h, respectively. These results indicated that ammonia stress decreased the expression of GH/IGF axis genes, which might have negative effect on the growth and development of pufferfish.

Effects of dietary amylose/amylopectin ratio on growth performance, feed utilization, digestive enzymes, and postprandial metabolic responses in juvenile obscure puffer Takifugu obscurus.

The effect of dietary amylose/amylopectin (AM/AP) ratio on growth, feed utilization, digestive enzyme activities, plasma parameters, and postprandial blood glucose responses was evaluated in juvenile obscure puffer, Takifugu obscurus. Five isonitrogenous (430 g kg(-1) crude protein) and isolipidic (90 g kg(-1) crude lipid) diets containing an equal starch level (250 g kg(-1) starch) with different AM/AP ratio diets of 0/25, 3/22, 6/19, 9/16 and 12/13 were formulated. Each experimental diet was fed to triplicate groups (25 fish per tank), twice daily during a period of 60 days. After the growth trial, a postprandial blood response test was carried out. Fish fed diet 6/19 showed best growth, feed efficiency and protein efficiency ratio. Hepatosomatic index, plasma total cholesterol concentration, liver glycogen and lipid content, and gluconokinase, pyruvate kinase and fructose-1,6-bisphosphatase activities were lower in fish fed highest AM/AP diet (12/13) than in fish fed the low-amylose diets. Activities of liver and intestinal trypsin in fish fed diet 3/22 and diet 6/19 were higher than in fish fed diet 9/16 and diet 12/13. Activities of liver and intestinal amylase and intestinal lipase, and starch digestibility were negatively correlated with dietary AM/AP ratio. Fish fed diet 3/22 and diet 6/19 showed higher plasma total amino acid concentration than fish fed the other diets, while plasma urea nitrogen concentration and activities of alanine aminotransferase and aspartate aminotransferase showed the opposite trend. Equal values were found for viscerosomatic index and condition factor, whole body and muscle composition, plasma high-density and low-density lipoprotein cholesterol concentrations, and activities of lipase and hexokinase and glucose-6-phosphatase in liver. Postprandial plasma glucose and triglyceride peak value of fish fed diet 12/13 were lower than in fish fed the low-amylose diets, and the peak time of plasma glucose was later than in fish fed the other diets. Plasma glucose and triglyceride concentrations showed a significant difference at 2 and 4 h after a meal and varied between dietary treatments. According to regression analysis of weight gain against dietary AM/AP ratio, the optimum dietary AM/AP ratio for maximum growth of obscure puffer was 0.25. The present result indicates that dietary AM/AP ratio could affect growth performance and feed utilization, some plasma parameters, digestive enzyme as well as hepatic glucose metabolic enzyme activities in juvenile obscure puffer.

Haemocyte apoptosis of the tiger shrimp Penaeus monodon exposed to cadmium.

This study investigated the effect of ambient Cadmium (Cd) on haemocyte apoptosis of the shrimp, Penaeus monodon. Cellular response was determined in Cd-exposed (0, 0.05, 0.5 and 5 mg L(-1)) shrimp. Results showed that 0.05 mg L(-1) Cd(2+) had no significant effect on the haemocyte parameters during the 48 h exposure. Cadmium at doses of 0.5 and 5 mg L(-1) depressed the total haemocyte count (THC), and increased reactive oxygen species (ROS) production and apoptosis ratio in haemocytes. Esterase activity increased in shrimp exposed to 0.5 mg L(-1) Cd(2+) for 6 h, and decreased to the initial level later. Depressed esterase activity could be observed in shrimp after 24 and 48 h exposure to 5 mg L(-1) Cd(2+). These results demonstrated that Cd(2+) modified esterase activity and induced ROS generation, which led to haemocyte apoptosis and THC reduction. Oxidative stress is one of the induction mechanisms for Cd-caused apoptosis of shrimp haemocytes.

Trascriptome analysis of the Pacific white shrimp Litopenaeus vannamei exposed to nitrite by RNA-seq.

In the present study, transcriptome of nitrite-exposed Litopenaeus vannamei was performed using a newly developed high-throughput sequencing technology (Illumina RNA-seq). As many as 42,336 unigenes were generated with 561 bp of average length and 736 bp of unigene N50 after filtering and assembly. These unigenes from the de novo assembly were further annotated using BLAST and BLAST2GO softwares. A total of 23,532 unigenes were unambiguous alignments to the reference when BLAST against non-redundant protein sequence (Nr), non-redundant nucleotide (Nt), Swiss-Prot, Gene Ontology database (GO), Clusters of Orthologous Groups (COG) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases available at NCBI. Numerous candidate genes associated with immune response, detoxification, apoptosis pathway were identified. Ten candidate genes related to immune responses and apoptosis were selected for validating the results of assembly and annotation by real-time quantitative PCR. Results revealed that the expressions of all these ten genes were up-regulated after nitrite exposure. Combining to our previous study, we speculate that all these selected genes may be involved in the response to nitrite stress. The study shows a systematic overview of the transcriptome analysis in L. vannamei, and provides valuable gene information for studying molecular mechanisms under nitrite exposure.

Gene expression of apoptosis-related genes, stress protein and antioxidant enzymes in hemocytes of white shrimp Litopenaeus vannamei under nitrite stress.

Apoptotic cell ratio and mRNA expression of caspase-3, cathepsin B (CTSB), heat shock protein 70 (HSP70), manganese superoxide dismutase (MnSOD), catalase (CAT), glutathione peroxidase (GPx) and thioredoxin (TRx) in hemocytes of white shrimp Litopenaeus vannamei exposed to nitrite-N (20 mg/L) was investigated at different stress time (0, 4, 8, 12, 24, 48 and 72 h). The apoptotic cell ratio and mRNA expression level of CTSB were significantly increased in shrimp exposed to nitrite-N for 48 and 72 h. Caspase-3 mRNA expression level significantly increased by 766.50% and 1811.16% for 24 and 48 h exposure, respectively. HSP70 expression level significantly increased at 8 and 72 h exposure. MnSOD mRNA expression in hemocytes up-regulated at 8 and 48 h, while CAT mRNA expression level increased at 24 and 48 h. GPx expression showed a trend that increased first and then decreased. Significant increases of GPx expression were observed at 8 and 12 h exposure. Expression level of TRx reached its highest level after 48 h exposure. These results suggest that nitrite exposure induces expression of apoptosis-related genes in hemocytes, and subsequently caused hemocyte apoptosis. Meanwhile, expression levels of HSP70 and antioxidant enzymes up-regulated to protect the hemocyte against nitrite stress.

Measurement of intracellular nitric oxide (NO) production in shrimp haemocytes by flow cytometry.

A flow cytometric method to measure the production of intracellular nitric oxide (NO) was adapted for use with shrimp haemocytes. We applied fluorescent probe 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate (DAF-FM DA) for NO detection in haemocytes from the tiger shrimp Penaeus monodon, and used flow cytometry to quantify fluorescence intensity in individual haemocyte. The optimized protocol for intracellular NO analysis consists to incubate haemocytes with DAF-FM DA at 10 µM for 60 min to determine the mean fluorescence intensity. Result showed that NO was also produced in the untreated shrimp haemocytes. NO level in granular cells and semigranular cells were much higher than that in hyaline cells. Defined by different characteristic of NO content, three subsets of haemocytes were observed. Zymosan A at dose of 10 or 100 particles per haemocyte triggered higher DAF-FM fluorescence intensity in granular and semigranular cells, than PMA that had no significant impact on all three cell types. These results indicate that granular and semigranular cells are the primary cells for NO generation. Cytochalasin B significantly inhibited the NO level induced by zymosan A. NG-Monomethyl-L-arginine (L-NMMA) and diphenylene iodonium chloride (DPI) significantly suppressed the DAF-FM fluorescence in haemocytes, but apocynin could not modulate it, indicating that the DAF-FM fluorescence was closely related to the activity of NO-synthase pathway. The NO donor sodium nitroprusside (SNP) improved the DAF-FM fluorescence in haemocytes, while the NO scavenger C-PTIO (2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide) significantly decreased the fluorescence, demonstrating that the fluorescence intensity of DAF-FM is mainly dependent on the intracellular NO level.

In vitro toxicity of nitrite on haemocytes of the tiger shrimp, Penaeus monodon, using flow cytometric analysis.

This study investigated the in vitro effects of nitrite on reactive oxygen species (ROS) production, NO production, esterase activity and cell apoptosis of Penaeus monodon haemocytes. Haemocytes were in vitro exposed to different dose of nitrite (0, 0.1, 0.5, 1, 5 and 10 µM). Cellular responses of nitrite-treated haemocytes were determined by flow cytometry. The results revealed that haemocytes treated by nitrite in vitro showed conspicuous time- and dose-dependent decreases in ROS and NO production as well as esterase activity. Additionally, 0.1 and 0.5 µM nitrite did not affect the apoptotic cell ratio during the 3h experimental time, while significant increases in apoptotic cells were observed after haemocyte exposure to nitrite at 1 µM for 3h, and at 5 or 10 µM for 1h. These results indicated that nitrite suppresses cellular functions, including production of ROS and NO, and activity of esterase. Cell apoptosis of haemocytes would be induced by extracellular nitrite as doses exceed 1 µM.

Phagocytic activity, respiratory burst, cytoplasmic free-Ca(2+) concentration and apoptotic cell ratio of haemocytes from the black tiger shrimp, Penaeus monodon under acute copper stress.

The aim of this study was to investigate the cellular toxicity of copper-induced injury to the black tiger shrimp Penaeus monodon. The 24h, 48h, 72h and 96h LC(50) (median lethal concentration) of Cu(2+) on P. monodon (11.63+/-1.14g) were found to be 3.49, 1.54, 0.73 and 0.40mgL(-1), respectively. Total haemocyte count (THC), phagocytic activity, respiratory burst (RB), cytoplasmic free-Ca(2+) (cf-Ca(2+)) concentration and apoptotic cell ratio of shrimp were determined after exposure to different concentrations of Cu(2+) (0, 0.05, 0.5, 1.5 and 3.5mgL(-1)) for 0, 6, 12, 24 and 48h. There was no significant effect on the analytic indicator of shrimp exposed to 0.05mgL(-1) Cu(2+). THC decreased after Cu-exposure to 0.5mgL(-1) for 48h, 1.5mgL(-1) for 24h and 3.5mgL(-1) for 12h. Phagocytic activity decreased in P. monodon following 48h exposure to 3.5mgL(-1) Cu(2+). RB was induced after 6h exposure to 0.5, 1.5 and 3.5mgL(-1) Cu(2+). cf-Ca(2+) concentration increased after 48h exposure to 0.5mgL(-1) Cu(2+), and 12h exposure to 1.5 and 3.5mgL(-1) Cu(2+). The percentage of apoptotic cells increased to 9.5%, 16.3% and 18.6% respectively following 48h exposure to 0.5, 1.5 and 3.5mgL(-1) Cu(2+). These results indicate that Cu can induce oxidative stress, elevation of cf-Ca(2+) and cell apoptosis, and inhibit phagocytic activity in the shrimp P. monodon, and the lethal injury of Cu(2+) to P. monodon may be mainly due to the sharp reduction of THC caused by ROS-induced apoptosis.