Effects Of Ph, Ammonia Stress On Apoptosis And Antioxidant System Of Chinese Shrimp, Fenneropenaeus Chinensis

Chinese shrimp, Fenneropenaeus chinensis, is an important mariculture species in China and has many advantages, such as wild adaptability, fast growth, low temperature resistance and rich in nutrition. However, with the constant expansion of shrimp culture, deteriorated pond environment due to management subsequently resulted in the increased incidences of stress-induced physiology reaction and diseases of shrimp, or even seriously affected the survival rate of farming shrimp. pH and ammonia are both important factors to measure water quality. Good water environment is a key to acquire success in shimp culture. In order to reveal the stress mechanism on pH and ammonia stress of Chinese shrimp, we do the study of pH and ammonia stress on apoptosis and antioxidant system of Chinese shrimp. This will give a theoretical foundation for stress-resistance traits breeding and healthy culture. The study consists of two parts:The first part: Molecular clonging and characterization of apoptosis factor Caspase gene from Chinese shrimp Fenneropenaeus chinensis. In the present study, a Caspase gene was cloned from the Chinese shrimp, Fenneropenaeus chinensis (GenBank No. GU597089). The full-length of Caspase was of 1329 bp with 972 bp ORF, 109 bp of the 5′-untranslated region, 248 bp of the 3′-untranslated region and a poly(A) tail and a putative polyadenylation signal (AATAAA). The entire open reading frame encoded a deduced protein of 323 aminoacids with the putative initiation methioine codon (ATG). The calculated molecular mass and pI of Caspase protein was 36.0 kDa and 6.27 respectively. The deduced aminoacid sequence of Caspase contained a potential active site (QACRG pentapeptide) conserved in most Caspases and two profile hits (p20 and p10 domain profile). No putative signal peptide aminoacids was present in the Caspase sequence. The deduced protein Caspase domain contais two conserved residue sites: a histidine150 residue and a cysteine198 residue which play important to catalysis the activity of Caspase. Sequence conparision showed that the Caspase of F. chinensis shares 83%, 82% and 76% identity with that of Penaeus monodon Caspase, Fenneropenaeus merguiensis Caspase, Litopenaeus vannamei Caspase-3.The expression of Caspase gene in different tissues of Chinese shrimp after exposure to pH 7.0 and 9.0 stress was analyzed by Real-time PCR. The results showed that the number of Caspase transcripts in the pH 7.0 group increased gradually in the haemocytes during the the first 12h, then decreased to their lowest level by 48h. Following this, expression of the transcripts increased again to their highest level at 96h. The levels at the end of the experiment were significantly lower than those in the control group (P<0.05). Levels of the Caspase transcript in the pH 9.0 group also increased in the haemocytes at 3h, then decreased slightly by 24 h (P<0.05). The highest levels were observed after 120 h. The Caspase expression in the gill of pH 7.0 was almost higher than in the control and reached to its highest level at 148 h, except at 48 and 120 h. The Caspase transcript levels were significantly lower at 3 h in the pH 9.0 group compared to the control group (P<0.05), but then gradually increased up to 24 h before decreasing untile 48 h. The Caspase gene expression was lower in the pH 7.0 than in the control group untile the end of the experiment, except at 3 h and 96 h. The transcript levels in the hepatopancreas of the pH 9.0 group increased after 3 h, but then decreased and were slightly lower than the control group at 12 h. The transcript levels peaked at 120 h and were higher than the control group from 24 h to 148 h. The pattern of Caspase transcript expression was similar at the pH 7.0 and 9.0 groups in the muscle tissue. Levels increased slightly 3 h post-stress, and then decreased significantly from 12 h to 48 h. Following this, levels increased again and peaked at 96 h. At the end of experiment, the expression of Caspase transcripts was significantly lower after 120 h and was lower than the control group by 148 h. The number of Caspase transcripts in lymphoid organ of shrimp in the pH 7.0 and 9.0 groups increased by 3 h then decreased to baseline by 12 h. Levels increased up to 24 h. Thereafter, the expressions were almost always higher than in the control group. The expression of Caspase in stomach was also lower after 3 h and 12 h in the pH 7.0 group, but had increased by 24 h. Levels then decreased and were at their lowest by 72 h. The highest expression levels were observed by 148 h. The expression of Caspase decreased significantly by 3 h in the stomach tissue of shrimp in the pH 9.0. Levels then increased groudually between 12 h and 72 h, and decreased again up to 96 h before peaking at 120.TUNEL analysis results showed that apoptosis began to appear in the hepatopancreas cells of Chinese shrimp exposure to pH 7.0 and 9.0 for 12 h. The amount of apoptosis seems positively correlated with length of exposure to the pH stressor. Conversely, Brownish nuclei (TUNEL-positive) representing fragmented DNA were not observed in the control (Ph 8.2) and negative control. These results suggested that both high and low pH stress induced apoptosis in hepatopancreas cells of F. chinensis.The second part: The recombinant expression, purification and polyclonal antibody of Chinese shrimp Caspase. Specific primers were designed according to the opean reading frame (ORF) sequence of Caspase cDNA of Chinese shimp. Prokaryotic expression vector pET30a-CAS was contructed and transformed into the host E. coli BL21 (DE) pLysS. SDS-PAGE analysis results showed that a band of approximately 50 kDa corresponding to the His-tag Caspase fusion protein was observed after IPTG induction. Peptide fragments of the recombinant Caspase matched with the known aminoacid sequence of Caspase based on the comparison of peptide mass fingerprint using the MALDI-TOF-MS analytical system. The recombinant Caspase protein was purified using TALON Resin affinity chromatography. A polyclonal antiserum against Caspase was obtained from rabbits by injection of the purified fusion protein in complete Freund's adjuvant and incomplete Freund's adjuvant. To characterize the anti-Caspase serum, total protein extract of hemolymph, haemocytes, gill, muscle, lymphoid organ, heart, stomach and hepatopancreas were subject to western blot analysis. The results showed that Caspase was present in all tissues, whereas no target band was detected from the hemolymph.The third part: Effects of pH stress on antioxidant system and HSP90 gene expression of F. chinensis. The activity of T-AOC, anti-superoxide anion, catalase (CAT) and the gene expression of catalase (CAT), peroxiredoxin (Prx) , HSP90 were analyzed in the gill, hepatopancreas, muscle and haemocytes of Chinese shrimp, Fenneropenaeus chinensis, after exposure to pH 7.0 and 9.0 stress. The activity of T-AOC, anti-superoxide anion, CAT and the gene expression of CAT in different tissues of Chinese shrimps were increased from 12 h to 24 h after pH stress. However, they were inhibited with the extending periods of pH stress time. The gene expression of Prx in heaptopancreas and muscle was gradually increased, but it was decreased in gill and haemocytes with prolonging pH stress time. The enzyme activities and gene expression of antioxidant system in shrimp gills of pH 9.0 group were reached to its highest level, which were earlier than other tissues in pH 7.0 and 9.0 group. The antioxidant enzyme activities in shrimp hepatopancreas of pH 7.0 group changed faster than other tissues'antioxidant enzyme activities. The gene expression of HSP90 in hepatopancreas and muscle of neutral stress group was significantly lower than the control group (P<0.05), but it was significantly higher than the control group in hepatopancreas of alkaline pH stress group (P<0.05). The gene expression of HSP90 in muscle of alkaline pH stress group reached to its highest level at 72h and was significantly lower at other sampled times. The results indicate that the antioxidant enzyme activities and gene expression of Chinese shrimp were induced between 3 h to 24 h after pH stress which is the response to oxidative stress, while they were inhibited with the extending pH stress time. It was showed that long time neutral and alkaline-induced oxidative stress probably caused the antioxidant defense system injury in Chinese shrimp. Gills and hepatopancreas could be as sensitive tissues to resist high and low pH stress respectively. The fouth part: The effect of ammonia stress on antioxidant system, haemocyte nitrogenous contents and HSP90 gene expression in Chinese shrimp. In order to investigate the mechanism of ammonia stress on Chinese shrimp, the concentration of haemocyte ammonia, urea, the activity of T-AOC, anti-superoxide anion, Na⁺ K⁺-ATPase,Ca²⁺ Mg²⁺-ATPase and the gene expression of catalase (CAT), peroxiredoxin (Prx), Caspase, HSP90 were analyzed in the gill, hepatopancreas, muscle and haemocytes of Chinese shrimp, Fenneropenaeus chinensis, after exposure to different ammonia concentrations (0mg/L, 2mg/L, 4mg/L, 6mg/L, 8mg/L) stress. The actual ammonia concentration of water was measured at the same time of sampling, and then calculated the concentration of ammonia molecules. The results showed at: (1)When the actual ammonia concentration of water was from 0.220mg/L to 13.449mg/L, the contents of haemocyte ammonia in Chinese shrimp were significantly increased. And the contents of haemocyte ammonia was decreased to its lowest level at 24-48h, reached to its highest level at 72-96, but they were always significantly higher than control group (P<0.05). (2) When the actual ammonia concentration of water was from 0.220mg/L to 9.446mg/L, the contents of haemocyte urea in Chinese shrimp were increased gradually and significantly higher than control group (P<0.05). They reached to their highest level at 6h; The activity of Na⁺ K⁺-ATPase, Ca²⁺ Mg²⁺-ATPase and HSP90 gene expression were obviously increased and they were shown the same trend. However, when the actual ammonia concentration of water was from 9.446mg/L to 13.449mg/L, the contents of haemocyte urea and the activity of Na⁺ K⁺-ATPase, Ca²⁺ Mg²⁺-ATPase and HSP90 gene expression were all decreased. This indicated that the high ammonia inhibited the haemocyte synthesis of urea and the activity of ATPase. (3) As the actual ammonia concentration of water was from 0.303mg/L to 8.625mg/L, the activity of T-AOC, anti-superoxide anion and CAT gene expression were increased and reached to their highest level at 6h or 96h; Prx gene expression was reached to its highest level at 72-96h. However, the antioxidant enzyme activities and gene expression of Chinese shrimp were inhibited with the content of ammonia in water larger than 8.625mg/L. (4) Caspase gene expression in ammonia stress showed similar with its in pH stress, it reached to highest level at 72-96h. This indicated that high content of ammonia could induce high expression of Caspase gene, which may cause the apoptosis of Chinese shrimp.