Wei-Na Wang

Oxidative stress, DNA damage and antioxidant enzyme gene expression in the Pacific white shrimp, Litopenaeus vannamei when exposed to acute pH stress.

The ROS production, the percentage of dead and damaged haemocytes, the DNA Olive Tail Moment (OTM) value and the gene expression of manganese superoxide dismutase (MnSOD), catalase (CAT), glutathione peroxidase (GPx) and thioredoxin (TRx), were studied in the Pacific white shrimp, Litopenaeus vannamei, when exposed to acute pH stress. The increased ROS production in haemocytes and the increased OTM value in both the haemocytes and the hepatopancreas cells suggest that oxidative damage occurred in shrimp exposed to pH 5.6 and pH 9.3, with apoptosis, mainly being associated with excess Ca(2+)influx and changes in cell viability. Acid and alkaline pH-induced DNA damage was time dependent in the haemocytes and the hepatopancreas cells. The concentration of intracellular free calcium [Ca(2+)] (i) after different pH treatments increased significantly over time, reaching its highest concentration after 12 h, but decreasing gradually to normal levels after 24 h. The [Ca(2+)] (i) content in shrimp cells when exposed to pH 9.3 and pH 5.6 for 12 h had increased by 58%-81%, compared with exposure to pH 7.4 (control). In addition, the gene expression of cMnSOD, CAT, GPx and TRx in the hepatopancreas of L. vannamei was induced by acid and alkaline pH stress, although there were differences in the expression response with respect to the duration of induction and the different pH treatments (acid or alkaline). Our results show that acidic or alkaline-induced oxidative stress may cause DNA damage, and cooperatively activate expression of CAT, GPx and TRx mRNA. Calcium ions appear to be important in mediating shrimp responses to pH stress.

Expression of HSP60 and HSP70 in white shrimp, Litopenaeus vannamei in response to bacterial challenge.

In the present study, cDNA encoding a heat shock protein 60 (LvHSP60) gene in Litopenaeus vannamei was cloned using a combination of homology and rapid amplification of cDNA end (RACE) methods. The full length of the LvHSP60 cDNA was found to be 2379bp, with a 1737bp open reading frame. The translated amino acid sequence consisted of 579 residues with a calculated molecular mass of 60.8kD and an isoelectronic point (pI) of 5.97. Comparison of the deduced amino acid sequence showed that it has high identity (85-89%) with HSP60/chaperonins from insects and mammals. Quantitative real-time PCR and Western blot analysis were carried out to investigate the expression patterns and distribution profiles of LvHSP60 before and after stimulation with the Gram-positive bacterium Staphylococcus aureus and the Gram-negative bacterium Vibrio alginolyticus. LvHSP60 mRNA was found to be both constitutive and inducible, and was highly expressed in haemocytes and almost all tissues examined, including muscle, stomach, heart, hepatopancreas and gill tissue, but it was less strongly expressed in the intestine. The expression analysis revealed that LvHSP60 was significantly up-regulated in the gills, hepatopancreas and haemocytes after bacterial challenge. Transcription of LvHSP70 was also induced in haemocytes and the hepatopancreas after different bacteria injection. Subsequent flow cytometry analysis showed that the concentration of Ca(2+) ions increased significantly within bacteria-challenged haemocytes by 1.5h after injection. The results indicate that LvHSP60 and LvHSP70 may play important roles in mediating the immune responses of L. vannamei to bacterial challenge, and that the Ca(2+) signalling transduction pathway may be involved in the initiation of the shrimps immune responses in early stages of infection.

Oxidative stress, DNA damage and osmolality in the Pacific white shrimp, Litopenaeus vannamei exposed to acute low temperature stress

To evaluate the genotoxic, physiological and immunological effects of short-term acute low temperature stress on the Pacific white shrimp, Litopenaeus vannamei, we rapidly transferred shrimp from tanks at 23±2 °C to aquaria at the same temperature (controls) or 12±2 °C for 12 h. Changes in the shrimp hemocyte respiratory burst activity and DNA damage were examined during and after exposure to the temperature stress using flow cytometry and the comet assay, respectively. We also monitored changes in the total hemocyte count, malondialdehyde levels, total protein concentration and osmolality in shrimp plasma. The results show that hemocyte respiratory burst activity, malondialdehydes levels and hemocyte DNA damage in the plasma all increased significantly after exposure to 12±2 °C for 3 h. In contrast, total hemocyte count, total protein concentration and osmolality in the plasma decreased compared to the controls. We conclude that acute low temperature can induce oxidative stress, DNA damage, lipid peroxidation and changes in osmolality in L. vannamei.

Glutathione S-transferase in the white shrimp Litopenaeus vannamei: Characterization and regulation under pH stress

We first expressed a Mu-class GST from white shrimp Litopenaeus vannamei in Escherichia coli, and then characterized the purified recombinant enzyme with respect to the effects of pH, temperature on its catalytic (1-chloro-2, 4-dinitrobenzene-glutathione conjugation) activity. We also analyzed its expression profile in L. vannamei tissues, and assessed changes in Mu-GST expression, GST activity profiles and mortality rates following exposure of white shrimp to low and high pH (5.6 and 9.3, respectively). Realtime-PCR analysis showed that Mu-GST transcripts were expressed in all examined L. vannamei tissues, but were most abundant in the hepatopancreas. At low pH Mu-GST transcript levels in the hepatopancreas were highest after 12 h, and then declined to their original levels after 24 h. After 12 h they were also upregulated in haemocytes, but downregulated in the gills, and unchanged in the stomach following exposure to pH stress. Western blot analyses confirmed that the Mu-GST protein was strongly expressed in the hepatopancreas after 12 h at low pH and remain unchanged in the stomach after exposure to pH stress. pH-Related changes in GST activities in the shrimp hepatopancreas were similar to those displayed by the Mu-GST mRNA and protein profiles. In addition, the mortality of L. vannamei was higher at high pH than at low pH. These results suggest that L. vannamei Mu-GST expression is stimulated by acidic pH and that it may play important roles in detoxification of xenobiotics and antioxidant defenses.

Effects of cadmium on respiratory burst, intracellular Ca2+ and DNA damage in the white shrimp Litopenaeus vannamei.

Acute effects of heavy metal ions on shrimp have been an area of intense study worldwide. However, the molecular mechanism by which cadmium-induced injury occurs remains largely unclear, and methods for mitigating toxicity in vivo have rarely been reported. In this study, the changes in respiratory burst and intracellular free calcium in haemocytes of pacific white shrimp, Litopenaeus vannamei, after exposure to Cd(2+) (CdCl(2)) were examined using flow cytometry. Meanwhile, DNA damage and repair in haemocytes and hepatopancreas cells were studied using the comet assay. Respiratory burst generation, intracellular Ca(2+) concentration ([Ca(2+)]i) and DNA damage in haemocytes and hepatopancreas cells all exhibited a dose-dependent increase and a time-dependent change after treatment with Cd(2+) compared with controls. These results indicate that Cd can induce oxidative stress and DNA damage in the shrimp L. vannamei. Moreover, the results also demonstrate that these parameters can be used as sensitive indicators of exposure to this genotoxicant.

Acute acidic exposure induces p53-mediated oxidative stress and DNA damage in tilapia (Oreochromis niloticus) blood cells

Acid rain and inputs of acidic effluent can result in increased acidity in aquatic ecosystems, where it is known to have a significant impact and possibly, to cause the decline of some populations of aquatic organisms. In previous studies, intracellular acid-induced oxidative stress has been shown to cause DNA damage, and cooperatively activate the expression of the p53 gene. The acute effects of acidic environments on shrimp and fish have been widely studied. However, the molecular mechanism of acid-induced injury remains largely unknown. In this study, we examined the cellular responses of tilapia to acidic exposure-induced oxidative stress and antioxidant enzyme gene expression. Furthermore, we determined how acute acid stress activates the ATM-p53 signal pathway. We measured the upregulation of reactive oxygen species (ROS) production, the intracellular Ca(2)(+) concentration ([Ca(2)(+)](i)), the tail DNA values, the malondialdehyde (MDA) level in the blood cells and the percentage of dead and damaged blood cells. Our results suggest that oxidative stress and DNA damage occurred in tilapia in conditions where the pH was 5.3. Apoptosis was detected by Hoechst staining, which was mainly associated with changes in cell viability. The parameters that we measured were related to acid-induced DNA damage, and all parameters changed in the blood cells through time. The effects of acute acid exposure (pH 5.3) on the expression of ATM, p53, p21, Bax, manganese superoxide dismutase (MnSOD), glutathione peroxidase (GPx) were investigated in tilapia blood cells. The results showed that acute acid stress induced upregulation of ATM, p53 and p21, associated with increasing of DNA damage and apoptosis in blood cells. Additionally, the expression of Bax was slightly increased. Moreover, consensus p53-binding sequences were identified in tilapia MnSOD and GPx gene promoter regions and increased levels of ROS in the blood cells coincided with increased mRNA expression of p53, MnSOD and GPx. Therefore, it suggests that acid exposure-induced oxidative stress may cause DNA damage or apoptosis, and cooperatively activate ATM-p53 pathway, which may lead to the activation of p21 and regulate transcription of MnSOD and GPx.

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.

Molecular cloning, characterization and expression analysis of tumor suppressor protein p53 from orange-spotted grouper, Epinephelus coioides in response to temperature stress.

The tumor suppressor protein p53 is a critical component of cell cycle checkpoint responses. It upregulates the expression of cyclin-dependent kinase inhibitors in response to DNA damage and other cellular perturbations, and promotes apoptosis when DNA repair pathways are overwhelmed. In the present study, the cDNA of p53 from the orange-spotted grouper (Epinephelus coioides) (Ec-p53) was cloned by the combination of homology cloning and rapid amplification of cDNA ends (RACE) approaches. The full-length cDNA of Ec-p53 was of 1921 bp, including an open reading frame (ORF) of 1143 bp encoding a polypeptide of 380 amino acids with predicted molecular weight of 42.3 kDa and theoretical isoelectric point of 7.0. Quantitative real-time PCR (qRT-PCR) assays revealed that Ec-p53 was ubiquitously expressed in all the examined tissues but with high levels in intestine and liver of the orange-spotted grouper. In addition, we measured the DNA damage and apoptosis in the blood cells and the percentage of dead and damaged blood cells. Our results suggest that oxidative stress and DNA damage occurred in grouper in conditions where the temperature was 15 ± 0.5 °C. Furthermore, qRT-PCR and western blot confirmed that low temperature stress induced upregulation of Ec-p53 in the mRNA and protein levels. These results suggest that low temperature-induced oxidative stress may cause DNA damage or apoptosis, and cooperatively stimulate the expression of Ec-p53, which plays a critical role in immune defense and antioxidant responses.

Molecular characterization and functional analysis of a complement \{C3\} molecule in the orange-spotted grouper (Epinephelus coioides)

Complement component C3 is a key molecule in the complement system whose activation is essential for all the important functions performed by this system. In this study, we examined a temperature-subtracted library from the orange-spotted grouper (Epinephelus coioides), and identified a sequence with high similarity to other complement C3 proteins. Rapid amplification of the cDNA ends (RACE) yielded the full open reading frame of this protein, and subsequent analysis indicated that the Ec-C3 (E. coioides-C3) gene encodes a protein of 1657 amino acid residues with a molecular mass of 184.56 kDa. The deduced amino acid sequence showed that Ec-C3 has conserved residues and domains known to be critical for C3 function. RT-PCR assays showed that under normal physiological conditions Ec-C3 mRNA is most strongly expressed in the liver, but relatively high levels were also found in many other tissues, including intestine, muscles, gills, hemocytes, heart, spleen, head, kidney and brain. Further analysis of Ec-C3 gene expression in liver tissue by quantitative real-time PCR demonstrated that Ec-C3 transcript levels increased when the fish were exposed to both pH and temperature stress, but the time when its expression level peaked differed under these stresses. The results show that Ec-C3 mRNA expression in the orange-spotted grouper is influenced by pH and temperature stress and that Ec-C3 may play an important role in antioxidation mechanisms.

LvDJ-1 plays an important role in resistance against Vibrio alginolyticus in Litopenaeus vannamei

DJ-1 was first identified as an oncogene that transformed mouse NIH3T3 cells in cooperation with activated Ras. It has since exhibited a variety of functions in a range of organisms. In this study, the DJ-1 gene in Litopenaeus vannamei (LvDJ-1) was identified and characterized. A recombinant protein LvDJ-1 was produced in Pichia pastoris. LvDJ-1 expression in vivo was knocked down by dsRNA-mediated RNA interference (RNAi), which led to significantly decreased levels of LvDJ-1 mRNA and protein. When the L. vannamei were challenged with RNAi and Vibrio alginolyticus, the transcription and expression of copper zinc superoxide dismutase (LvCZSOD) in the hepatopancreas were dramatically lower in shrimp with knocked down LvDJ-1 than in controls. Transcription and expression of P53 (LvP53) were significantly higher in shrimp lacking LvDJ-1 than in controls. Hepatopancreas samples were analyzed using real time polymerase chain reaction and Western blot. Moreover, blood samples from the shrimp, assessed with flow cytometry, showed significant increases in respiratory burst and apoptosis in those lacking LvDJ-1 compared to the controls. Cumulative mortality in the shrimp lacking LvDJ-1 was significantly different from that in the control group after challenge with V. alginolyticus. Altogether, the results prove that LvDJ-1 regulates apoptosis and antioxidant activity, and that these functions play an important role in L. vannamei resistance against V. alginolyticus.