Shao-An Liao

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.

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.

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.

Effects of different dietary lipid level on the growth, survival and immune-relating genes expression in Pacific white shrimp, Litopenaeus vannamei

Five feeding trials based on the isonitrogenous and isoenergetic experimental diets containing 34% protein, 6%, 8%, 10%, 12% or 14% lipid respectively in the circulating water culture system for both 30 and 60 days were conducted to investigate the effect of the dietary lipid level on the growth and immunity in white shirmp, Litopenaeus vannamei adults. The body weight and specific growth rate of white shrimp in different treatments indicated that shrimps fed the diet of 12% lipid level for 30d and 10% lipid level for 60d had the best developmental status. The ability of respiratory burst in hemocytes was improved as the increase of dietary lipid level. The transcripts of LGBP and pPO were sensitive to the dietary lipid in hemocyte and hepatopancreas respectively. The activities of CAT, GPx and AKP were increased to a certain extend according to dietary lipid level. Qualification of MDA showed the lowest level in the sample subjected to 12% lipid level diet, indicating an optimal utilization of the dietary lipid and an efficient clearance of MDA in vivo. These results suggested that dietary lipid level of 10-12% significantly tunes the growth and enhance the immune abilities mainly via ROS pathway of L. vannamei.

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.

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.

Microbial diversity of mangrove sediment in Shenzhen Bay and gene cloning, characterization of an isolated phytase-producing strain of SPC09 B. cereus

Phytases hydrolyze phytate to release inorganic phosphate, which decreases the requirement for phosphorus in fertilizers for crops and thus reduces environmental pollutants. This study analyzed microbial communities in rhizosphere sediment, collected in September 2012 from Shenzhen Bay, Guangdong, China, using high-throughput pyrosequencing; the results showed that the dominant taxonomic phyla were Chloroflexi, Firmicutes, and Proteobacteria, and the proportion of the beneficial bacteria, Bacillus, was 4.95 %. Twenty-nine culturable, phytase-producing bacteria were isolated, their phosphorus solubilization capacity was analyzed, and they were taxonomically characterized. Their phylogenetic placement was determined using 16S ribosomal RNA (rRNA) gene sequence analysis. The result shows that most of the isolates are members of the order Bacillales, although seven strains of Enterobacteriales, two strains of Pseudomonadales, and one strain of Oceanospirillales were also identified. The phytase gene was cloned from SPC09, Bacillus cereus, which showed the highest phosphorus solubilizing ability among the isolated strains. The gene encoded a primary translation product of 335 amino acids. A construct including the 1005-nt ORF fragment, Bc-phy, was transformed into Escherichia coli. The recombinant phytase was produced and purified, which revealed the temperature optima at 60 °C and pH optima at 6.5. The assessment by quantitative PCR (qPCR) showed an abundance of bacteria containing the Bc-phy gene; the level was generally higher in the mangrove forest than in the tidal flats and in surface soil compared to bottom soil, and the highest value was obtained in June. Herein, we report on the cloning, characterization, and activity of a novel phytase isolated from a mangrove system.

Identification and characterization of a B-type mannose-binding lectin from Nile tilapia (Oreochromis niloticus) in response to bacterial infection

Lectins are a group of carbohydrate-binding proteins, which play an important role in innate immune system against pathogen infection. In this study, a B-type mannose-binding lectin (OnBML) was identified from Nile tilapia (Oreochromis niloticus), and characterized at expression patterns against bacterial infection and capability to promote phagocytosis by macrophages. The open reading frame of OnBML is 354 bp of nucleotide sequence encoding polypeptides of 117 amino acids. The deduced protein is highly homologous to other teleost BMLs, containing two repeats of the conserved mannose-binding motif QXDXNXVXY. Expression of OnBML was widely exhibited in all examined tissues, with the most abundance in spleen and following gill, peripheral blood, and head kidney. The OnBML expressions were significantly up-regulated following two major bacterial infections including a Gram-positive bacterium (Streptococcus agalactiae) and a Gram-negative bacterium (Aeromonas hydrophila) in vivo and in vitro. Recombinant OnBML protein possessed capacities of mannose-binding and calcium-dependent agglutination to S. agalactiae and A. hydrophila, and promoted the phagocytosis by macrophages. Taken together, the present study indicated that OnBML is likely to get involved in host defense against bacterial infection in Nile tilapia.

High-efficiency inorganic nitrogen removal by newly isolated Pannonibacter phragmitetus B1

An aerobic heterotrophic nitrogen removal bacterium strain, B1, was isolated from aquaculture water and identified as Pannonibacter phragmitetus (99% similarity) by 16S rRNA sequencing analysis. When ammonium, nitrite or nitrate was the sole nitrogen source, with an initial nitrogen concentration of 14 mg/L, the nitrogen removal efficiencies were 98.66%, 99.96% and 98.73%, respectively, and the corresponding maximum removal rates reached as high as 1.16, 0.77 and 0.81 mg/L/h, respectively. In the presence of NH4+-N, the removal efficiency of 56 mg/L NO2--N within 27 h increased by 83.50%, and the corresponding removal rate reached as high as 1.72 mg/L/h. Additionally, different carbon sources (dl-malic acid, sucrose, sodium citrate, and glucose) could be utilized in nitrogen removal. Sequence amplification indicates that the denitrification genes nirK, norB and narG are present in strain B1. All results demonstrate that strain B1 has high promise for future applications of removing inorganic nitrogen from wastewater.