| Clam Cyclina sinensis is an important economical tidal flat shellfish for farming in China.Affected by environmental factors such as breeding density,residual bait and other factors,the production of C.sinensis may decrease,and the quality and efficiency may also decrease.Ammonia nitrogen is an important environmental stress factor in aquaculture,which may affect tidal flat shellfish.At present,most of the research on C.sinensis focus on the effect of environmental stress on the survival of C.sinensis and the molecular marker screening of immune-related genes in C.sinensis.Therefore,studying the changes of immune-related enzyme activity and genes expression in C.sinensis and understanding the response mechanism of C.sinensis under ammonia nitrogen stress may contribute to the sustainable development of C.sinensis aquaculture.The specific contents of this study are as follows:1.To evaluate the toxicity of ammonia nitrogen on C.sinensis and the recovery after stress.The experimental group was determined by using the TAN 48 h LC50of the C.sinensis obtained from the previous experiments,and the related non-specific immune enzyme activities in the hepatopancreatic tissue of the C.sinensis within 48 h of acute ammonia nitrogen stress and the recovery of 48 h after the stress,including alkaline Stress changes in phosphatase（AKP）,acid phosphatase（ACP）and lysozyme（LZM）.The results are as follows:With the increase of TAN stress concentration,the activity of AKP increased at first and then decreased after stress,while the activity of AKP after recovery from stress showed an upward trend.The AKP activity after stress recovery was significantly higher than that in the control group.The ACP activities of T1（20 mg/L）and T2（40 mg/L）after recovery from stress were higher than those of the control group,while those of T3（60 mg/L）,T4（80 mg/L）and T5（100 mg/L）ACP activity was significantly higher than that of the control group.The LZM activities of T1 and T2 after stress were significantly higher than those of the control group,while the LZM activities of T3,T4 and T5 after stress were significantly lower than those of the control group.In general,ACP and LZM in green clams stressed at low levels of TAN（≤40 mg/L）could be fully recovered to normal levels.However,the 48-hour recovery period appeared to be insufficient to compensate for the AKP,ACP and LZM activities in clams exposed to high levels of TAN（>40 mg/L）.In conclusion,we evaluated the effects of acute ammonia nitrogen stress and post-stress recovery on the non-specific immunity of C.sinensis,and the results may help reveal the potential relationship between immune responses and ammonia nitrogen toxicity.2.In this chapter,the activity of immune enzyme in the hepatopancreas of the C.sinensis at different times under long-term chronic ammonia nitrogen stress was detected to explore the toxicity of ammonia nitrogen to the C.sinensis.According to the TAN concentration obtained from the experiment in the previous chapter,the TAN concentration was set to 8.07 mg/L for 768h（32 d）.The hepatopancreas tissue samples were taken to analyse the changes in the enzyme activities of ACP,AKP,LZM and superoxide dismutase（SOD）.The results showed that with the prolongation of time,the overall ACP enzyme activity showed an upward trend,and at the 6th hour,the expression level decreased slightly,and decreased to the minimum value,and there was no significant difference compared with the control group（P>0.05）;At 768 h（32 d）,it increased to the maximum value,which was significantly different from the control group（P<0.05）.The AKP enzyme activity generally showed a trend of first increasing and then decreasing,and at the6th hour,the AKP enzyme activity significantly increased to the maximum value,which was significantly up-regulated compared with the control（P<0.05）.After 12 hours of continuous stress,the experimental group Compared with the first 6 hours,the expression level of AKP enzyme activity decreased,but it was still significantly higher than that of the control group（P<0.05）.（P<0.05）.The overall activity of lysozyme（LZM）showed a wave-shaped change trend.From 3 h to 192 h,the expression level of the experimental group was significantly higher than that of the control group,and both were significantly up-regulated compared with the control group（P<0.05）.The enzyme activity of the experimental group was the most significant（P<0.05）.The activity of superoxide dismutase（SOD）showed a trend of increasing first and then decreasing.At the 3rd hour,the expression level of the experimental group increased,but it was not significantly up-regulated compared with the control group（P>0.05）,and at the 6th hour The expression of SOD enzyme activity in the experimental group did not change significantly from that at the 3rd hour;until the 96th hour,the SOD activity reached the highest point,which was significantly different from the control group.In general,under the long-term ammonia nitrogen stress of 768 h（32 d）,the activities of ACP and AKP in the hepatopancreas of C.sinensis increased and changed significantly,while the long-term ammonia nitrogen stress induced the expression of LZM activity and SOD increased vitality.It shows that under prolonged ammonia nitrogen stress,the activity of immune enzymes of C.sinensis is affected by stimulation,and the ability of bacteriolysis and anti-oxidation is enhanced.In conclusion,this provides a theoretical basis for exploring the relationship between chronic ammonia nitrogen stress and the immune enzyme activity in C.sinensis.3.The purpose of the experiment in this chapter is to investigate the effect of Vibrio parahaemolyticus infection on the non-specific immunity of C.sinensis under ammonia nitrogen stress.Immunoenzyme activities and non-specific immune-related genes（（Interleukin-17-3,IL-17-3）and interferon-induced helicase C domain-containing protein 1,IFITM1）)expression.The results are as follows,the ACP enzyme activity in the hepatopancreas of the C.sinensis showed a trend of first decreasing and then increasing.At 6 h,it decreased to the lowest value,but the difference was not significant（P>0.05）;at 12 h,it rose to the highest value,with significant difference（P<0.05）,the experimental group was significantly increased at48 h,96 h and 120 h,and was significantly up-regulated compared with the control group（P<0.05）.The AKP enzyme activity generally showed an upward trend,and the AKP enzyme activity in the experimental group increased significantly at 12 h,48 h and 96 h,and increased to the maximum at 96 h,which was significantly up-regulated compared with the control group（P<0.05）.The LZM enzyme activity generally showed a wave-shaped change trend.At the 6th hour,the LZM enzyme activity in the experimental group was significantly increased,and it was significantly up-regulated compared with the control（P<0.05）.at 96 h,the expression was most significant（P<0.05）;after 144 h（7 d）of continuous stress,the enzyme activity of LZM in the experimental group was still significantly higher than that in the control group（P<0.05）.The SOD enzyme activity generally showed an upward trend.The SOD enzyme activity in the control group and the experimental group was at the 48th hour,and the expression level of the experimental group increased to the maximum value,which was significantly up-regulated compared with the control group（P<0.05）;and the second most significant change was at the120th hour,the overall expression at each time point was significantly different from the control group,indicating that the enzyme activity of SOD changed strongly.In terms of the temporal expression levels of IL-17-3 and IFITM1 genes,there were differences between the overall and the control group with the prolongation of time.It was higher than the control group,and there was a significant difference（P<0.05）,among which the expression level was the highest at 48 h.The expression level of IFITM1 m RNA changed significantly from 6 h（P<0.05）,and reached the highest level at 48 h（P<0.05）;in general,except for 3 h,the group was significantly up-regulated compared with the control group（P<0.05）.In general,after the infection of Vibrio parahaemolyticus,the ACP activity in the hepatopancreas of C.sinensis was significantly increased at 12 h,48 h,96 h and 120 h（P<0.05）,and the AKP activity was increased at 12 h,48h and 96 h were significantly up-regulated（P<0.05）,the wave-shaped changes of LZM activity and the significant up-regulation of SOD.Combined with the changes in the activities of three hydrolases and one antioxidant enzyme,the infection of Vibrio can lead to a strong impact on the humoral immunity of the hepatopancreas of C.sinensis.In addition,under the infection of Vibrio parahaemolyticus,both IL17-3 and IFITM1 genes were expressed,and the overall expression was significantly up-regulated（P<0.05）,indicating that these two genes are involved in the immunity defensive answer in the hepatopancreas of C.sinensis.4.In this chapter,in order to explore the functions of IL-17-3 and IFITM1 genes related to the non-specific immunity of C.sinensis under ammonia nitrogen stress,the full-length c DNA sequences of the two genes were cloned by RACE technology and real-time fluorescence quantitative expression was detected to evaluate ammonia nitrogen toxicity and immunity.genetic relationship.The results are as follows:The full-length c DNA sequence of the IL-17-3gene is 1027 bp in total,including 603 bp open reading frame,encoding 200 amino acids.The phylogenetic tree results show that the IL-17-3 gene has a high homology with other lamellibranchial species,indicating its evolutionary conservation.The full-length c DNA sequence of the IFITM1 gene is 2434 bp in total,including 2301 bp open reading frame,encoding a total of 714 amino acids.The phylogenetic tree results show that the phylogenetic tree has a high homology and evolutionary conservation.Fluorescence quantitative results showed that under long-term chronic ammonia nitrogen stress,the expression level of IL-17-3gene in C.sinensis was the highest at 6 h,which was significantly higher than that of the control group（P<0.05）,while the expression levels at other time points were higher than those of the control group,but not significant（P<0.05）.The expression level of IFITM1 gene in C.sinensis was the highest at 192 h,which was significantly higher than that in the control group（P<0.05）.In general,the IL17-3 and IFITM1 genes cloned in the hepatopancreas tissue of C.sinensis in this experiment are both involved in the immune response of C.sinensis under the environment of ammonia nitrogen stress,indicating that long-term ammonia nitrogen stress affects the cells in the hepatopancreas of C.sinensis,the immune-related IL17-3 and IFITM1genes play a regulatory role,and the accumulation of ammonia concentration may cause cell damage in C.sinensis,and the non-specific immune expression of the two disease resistance genes provides the basis for the toxicity mechanism of C.sinensis,and also provides a basis for the toxicity mechanism of the two disease resistance genes.It provides a reference to further explore the content of non-specific immunity of C.sinensis. |
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