Evaluation Of The Oxidative Status Of Pacific Oyster Crassostrea Gigas Under High Temperature Stress

The pacific oyster Crassostrea gigas is an important mariculture shellfish in China.In recent years,with the expansion of aquaculture scale and global climate changes,mass mortality events in summer of C.gigas have occurred frequently,which seriously restricts the sustainable development of the aquaculture industry.Previous studies have shown that high temperature is one of the key factors leading to the mass mortality of C.gigas in summer.High temperature stress often induces oxidative stress and oxidative damage in C.gigas,and even leads to mass mortality in summer,causing serious economic losses.Therefore,it is of great theoretical significance and application value to analyze the changes of oxidative stress related indicators of C.gigas under high temperature stress and evaluate the oxidative stress status.In this study,we studied the regulation of antioxidant enzymes genes in the process of oxidative stress in C.gigas by bioinformatics and real-time fluorescence quantitative PCR,and analyzed the changes of oxidative stress related indicators in the process of indoor high temperature stress and culture.To construct an evaluation formula to comprehensively evaluate the oxidative stress of C.gigas during indoor high temperature stress and culture.Key findings of the study are as follows:1.Identification and function of antioxidant enzyme family genesUsing genomic and transcriptome data,gene identification,evolution,and expression analysis of the superoxide dismutase（SOD）,peroxiredoxin（Prx）,and glutathione peroxidase（glutathione peroxidase,GPX）in Crassostrea gigas were peformed.The mRNA expression level of GPx gene family in C.gigas showed significant changes under high temperature,Micrococcus luteus and Vibrio alginolyticus stress.The mRNA expression level of CgGPx-1 in Crassostrea gigas showed significant changes under high temperature,Micrococcus luteus and Vibrio alginolyticus stress,which were 1.22-fold,2.03-fold and 1.63-fold of the control group,respectively.RNA interference（RNAi）for CgGPx-1 was performed in C.gigas and the C.gigas was exposed to high temperature at 30℃for 48 h.The results showed that the GPx activity of theRNAi group（siRNA）was significantly decreased to 0.42-fold of the control group（p<0.01）.The total antioxidant capacity（T-AOC）was significantly decreased to 0.69-fold of the control group（p<0.01）,and the malondialdehyde（MDA）content was significantly increased to 1.73-fold of the control group（p<0.01）.Gill tissue sections showed tissue damage such as obvious swelling and cilia shedding.2.Changes of oxidative stress-related indicators of C.gigas under indoor high temperature stressC.gigas was exposed to different temperature（18℃,20℃,22℃,24℃,26℃,28℃,30℃,32℃,and 34℃）for 12 h,and compared with the control group（16℃）.The changes in oxidative stress-related indicators of gill tissue were analyzed.The SOD activity of C.gigas varied from118.33 U/mgprot to 257.14 U/mgprot under high temperature stress,with a significant decrease and an increase in the 28℃and 30℃groups.GPx（glutathione peroxidase,GPx）activity ranged from 126.35 U/mgprot to 261.41 U/mgprot,with a maximum of 261.41 U/mgprot at 30℃and a minimum of 132.54 U/mgprot at 32℃.The glutathione（GSH）content varied from 22.67μmol/gprot to 81.61μmol/gprot,and decreased to 45.34μmol/gprot and increased to 77.29μmol/gprot at 28℃and 30℃,respectively.Catalase（CAT）activity and peroxidase（POD）activity decreased significantly in the 28℃treatment group,which were 1.17 U/mgprot and 6.23 U/mgprot,respectively.MDA content showed an upward trend under high temperature stress,and the 30℃treatment group was significantly increased to 1.76-fold of the control group（p<0.05）.The changes of T-AOC under high temperature stress were significantly different.The mRNA expression levels of oxidative stress-related genes were further analyzed.The results showed that the mRNA expression level of CgNrf2 in the 30℃treatment group was decreased to 0.77-fold of the control group,and the 32℃treatment group was significantly increased to 3.19-fold of the control group（p<0.05）.The mRNA expression level of CgKeap1 in the 18-28℃treatment group was higher than that in the control group,the highest was 2.80-fold of the control group（p<0.05）,and the lowest was 0.63-fold of the control group in the 30℃treatment group.The mRNA expression level of CgPKC in the 24℃treatment group reached the minimum,which was 0.29-fold of the control group（p<0.05）.The mRNA expression level of CgMaf-k in the 28℃treatment group reached the maximum,which was 8.88-fold of the control group（p<0.05）.In conclusion,different temperature stress at 18-34℃could cause changes in antioxidant enzyme activities and increases in MDA content in C.gigas.Under the high temperature stress at 28℃,antioxidant enzyme activities significantly decreased,MDA content significantly increased,and the mRNA expression levels of CgNrf2 oxidative stress regulatory pathway genes were significantly changed.3.Changes in oxidative stress related indexes of C.gigas during aquaculture periodIn this study,from April to October 2021,63 second-instar C.gigas were collected from the Zhuanghe aquaculture area.The correlation between water quality factors in the breeding area and the changes of oxidative stress-related indicators was analyzed.The results showed that SOD activity increased to 204.35 U/mgprot in May,which was significantly higher than that in April（p<0.05）.CAT activity increased from May to July,and reached a maximum of 2.05 U/mgprot in July,which was significantly higher than that in April（p<0.05）.GSH content showed an upward trend from April to July,and reached a maximum of 84.56μmol/gprot in July,which was higher than that in April,and showed a downward trend from August to October,and reached a minimum of 13.00μmol/gprot in October,which was significantly lower than that in April（p<0.05）.MDA content decreased first and then increased from April to October,and reached the minimum of 3.41nmol/mgprot in August,which was significantly lower than that in April（p<0.05）.No significant changes in T-AOC were detected from April to October.Water temperature increased from 8.3℃to 23.9℃from April to August,which was negatively correlated with MDA content（r=-0.743,p<0.001）.These results indicated that the activities of antioxidant enzymes and MDA content of cultivated C.gigas decreased significantly in summer.The increase of water temperature in summer may be an important reason for the change of oxidative stress in cultivated C.gigas.4.Comprehensive evaluation of oxidative stress status of C.gigasPrincipal component analysis（PCA）and factor analysis（FA）were used to analyze the data matrix of oxidative stress related indicators of C.gigas during indoor high temperature stress and aquaculture period,and to construct an evaluation formula for oxidative stress.The oxidative stress scores were also analyzed.The results showed that during the process of 18-34℃indoor high temperature stress,indoor oxidative stress scores(OSS_in)ranged from 15.07 to 60.21,and the OSS_in in the high temperature stress group was significantly higher than that in the control group.OSS_in decreased sharply to 22.26 at 28℃,which was significantly higher than that at 26℃（p<0.05）,and increased rapidly to 51.35 at 30℃.From April to October,outdoor oxidative stress scores(OSS_out)increased first and then decreased,with a range of 20.73 to 40.99,and OSS_outincreased to a maximum of 40.99 in July.It dropped to a minimum of 20.73 in August.In conclusion,the oxidative stress of C.gigas changed significantly when exposed to high temperature at 28℃for 12 h and during July and August.In summary,through the identification of antioxidant enzyme family genes and antioxidant function study,it was found that CgGPx-1 gene may play an important role in the antioxidant response of C.gigas to high temperature stress.Based on the changes and comprehensive evaluation of oxidative stress-related indicators in C.gigas under indoor high temperature stress,it was found that the activities of antioxidant enzymes significantly decreased,the content of MDA significantly increased,and the mRNA expression levels of oxidative stress-related genes such as CgNrf2 changed significantly after 12 h of high temperature stress at 28℃.The oxidative stress of C.gigas changed significantly.Based on the changes and comprehensive evaluation of oxidative stress related indicators in the cultivation process of C.gigas,it was found that the activities of antioxidant enzymes changed significantly,the content of MDA decreased significantly,and the oxidative stress state changed significantly in the summer of July and August.The results provided theoretical basis and technical support for further elucidating the changes of oxidative stress in C.gigas under different temperature stresses,the mass mortality of C.gigas in summer,and screening evaluation indicators for the health status of C.gigas.