Jian-An Xian

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.

Phagocytic activity, respiratory burst, cytoplasmic free-Ca2+ 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.

Apoptosis of tiger shrimp (Penaeus monodon) haemocytes induced by Escherichia coli lipopolysaccharide.

This study was aimed at investigating the toxicity mechanism of lipopolysaccharide (LPS) on Penaeus monodon haemocytes at a cellular level. Reactive oxygen species (ROS) production, nitric oxide (NO) production, non-specific esterase activity, cytoplasmic free-Ca(2+) (CF-Ca(2+)) concentration, DNA damaged cell ratio and apoptotic cell ratio of in vitro LPS-treated haemocytes were measured by flow cytometry. Two concentrations of Escherichia coli LPS (5 and 10 μg mL(-1)) were used. Results showed that ROS production, NO production and CF-Ca(2+) concentration were significantly induced in the LPS-treated haemocytes. Ratio of DNA damaged cell and apoptotic cell increased caused by LPS, while esterase activity increased at the initial 60 min and dropped later. The initial increase in esterase activity suggested that LPS activated the release of esterase, and the later decrease might result from apoptosis. These results indicated that LPS would induce oxidative stress on shrimp haemocytes, and cause Ca(2+) release, DNA damage and subsequently cell apoptosis. This process of ROS/RNS-induced Ca(2+)-mediated apoptosis might be one of the toxicity mechanisms of LPS on shrimp haemocytes.

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.