Biochemical Analysis And Non-invasive Detection Techniques Study Of Larimichthys Crocea Under High Temperature Stress

Large yellow croaker(Larimichthys crocea)is one of the important marine economic fishes in China.In the course of aquaculture,large yellow croaker will be affected by various adverse environmental factors,which often trigger the stress response,and may cause death in severe cases.Therefore,it is necessary to monitor the health status regularly of large yellow croaker in the aquaculture process.At present,the health monitoring of large yellow croaker adopts direct observation the body surface and assisted with blood biochemical indicators detection.As the process of blood collection often causes serious injury or even death to the fish,it is urgent to develop non-invasive detection methods.High temperature is an important stressor in the process of large yellow croaker aquaculture,which often leads to slow growth and lower immunity.Therefore,it is necessary to monitor the health status of large yellow croaker in high temperature season.In this experiment,the content of cortisol in the serum,skin mucus and surrounding water of the large yellow croaker under acute high temperature stress were studied.Correlation analysis of cortisol in serum,fish skin mucus and aquaculture water was carried out.Meanwhile,malondialdehyde(MDA)content,immunoglobulin M(IgM)content and alkaline phosphatase(AKP)activity in serum and skin mucus were measured under acute high temperature stress.Correlation analysis of the same indicators in serum and skin mucus were performed.By slowly increasing the temperature(rising from 25℃ to 35℃),individuals with thermal-tolerant and thermal-sensitive are selected.Proteomics analysis was carried out on the skin mucus of selected thermal-tolerant and thermal-sensitive group in large yellow croaker.The main results as follows:(1)Under the condition of acute high temperature stress(30℃),with the prolongation of stress time,the content of cortisol in serum,skin mucus and in the surrounding water all showed a same trend,i.e.first increasing and then decreasing,and all reached a peak at 1.5 h.Furthermore,the trends concerning the cortisol content in different matrixes were highly consistent.The cortisol content in the three matrixes showed extreme significant correlation between each other(P<0.01).The results show that the traditional blood test can be replaced by detecting the concentration of cortisol in the skin mucus of the yellow croaker or surrounding water.(2)Under the condition of acute high temperature stress(30℃),with the prolongation of stress time,,the MDA and IgM content in the serum and fish skin mucus both increased first and then decreased,while the AKP activity in the serum and skin mucus decreased first and then increased.The MDA,IgM content,and AKP activity in the skin mucus changed more rapidly than those in the serum.For the MDA,IgM,and AKP content,the Pearson correlation coefficients between the serum and skin mucus were 0.586,0.762,and 0.792,respectively,all with a significance level of P<0.01.(3)TMT(tandem mass tag)technology were used to analyze the skin mucus proteome of the large yellow croaker in thermal-tolerant and thermal-sensitive group.The fold changes≥ 1.2(up-regulation 1.2-fold or down-regulation 0.83-fold,P<0.05)was selected as differential proteins criterion.Apply PRM(quantitative proteomics parallel reaction monitoring)technology to verify randomly selected differential proteins.A total of 3069 proteins and 396 differentially abundant proteins(DAPs)were identified in this study,including 215 up-regulated proteins and 181 down-regulated proteins.The PRM technology was used to verify the random selected 10 differential proteins.The general trend of the quantitative results of TMT and PRM was consistent(r=0.805,P<0.01),which showed that TMT’s result of omics data was reliable.Using bioinformatics analysis,it was found that calreticulin,macrophage receptor,lily-type lectin,pyruvate kinase,heat shock protein 70 and heat shock protein 60 were significantly up-regulated in the heat-resistant group(P<0.05).These results indicated that they may play an important role in the mechanism of high temperature adaptation and can be used as candidate biomarkers for the high temperature breeding in large yellow croaker.