| Rainbow trout(Oncorhynchus mykiss)is one of the most economically valuable and widely farmed cold water fish species in the world.However,the increase in water temperature caused by global warming severely restricts the development of rainbow trout aquaculture.Therefore,it is of great significance to understand its adaptive mechanism to heat stress and reduce the negative effects of heat stress on the breeding of this species.In this study,rainbow trout with good health status and the same genetic background were taken and exposed to high temperatures at 20°C and 24°C,then returned to normal temperature.We used biochemical indicators,metabolomics,and transcriptomics methods to determine the effects of heat stress on biochemical markers,gene and metabolic expression profiles in the liver of rainbow trout.The main results are as follows:1.Effects of heat stress on the biochemical indexes of rainbow troutThe activities of aspartate aminotransferase(AST)and alanine aminotransferase(ALT)in the serum of rainbow trout at 24℃ were significantly increased(P<0.05);in the recovery stage,AST activity decreased(P<0.05)which was not significantly different from the control group(14℃),the activity of ALT remained unchanged.The content of total bilirubin(TBil)increased significantly at 24℃(P<0.05),and then fell back to the level of the control group.Compared with the control group,alkaline phosphatase(AKP)showed no significant change at 20℃,but decreased significantly at 24℃(P<0.05),and there was an upward trend in the recovery phase.Lysozyme(LZM)showed a significant increase(P<0.05)at 24℃ and followed by a significant decrease(P<0.05)during the recovery phase.At 20℃,the liver total superoxide dismutase(T-SOD)activity of rainbow trout under heat stress was significantly higher than that of the control group(P<0.05);it showed a downward trend at 24℃;in the recovery stage,the T-SOD activity increased significantly which was significantly different from the control group and the heat stress group(P<0.05).There was no significant difference in catalase(CAT)activity during the heat stress.Glutathione peroxidase(GSH-PX)did not change significantly at 20℃ before the significant increase at 24℃(P<0.05);it had a significant down-regulation during the recovery phase and remained at a relatively high level.Malondialdehyde(MDA)content in rainbow trout liver was significantly increased(P<0.05)at 24℃ and decreased to control level during the recovery phase.2.Effects of heat stress on the metabolomics of rainbow troutWe explored the effects of heat stress and recovery on the liver metabolic profile of rainbow trout by non-targeted metabolomics technology.Unsupervised pattern recognition method principal component analysis(PCA)and supervised pattern recognition method orthogonal partial least squares discriminant analysis(OPLS-DA)were used to cluster the samples,and it was found that there was a clear tendency for different groups to separate.The identified metabolites were screened by the criteria of variable importance in projection(VIP)>1 and P<0.05.There are 115(14℃ VS 20℃: 52up-regulated and 63 down-regulated),130(14℃ VS 24℃: 57 up-regulated and 73 downregulated),121(14℃ VS R14℃: 67 up-regulated,54 down-regulated)differentially expressed metabolites were obtained respectively,of which 66 differentially expressed metabolites were co-expressed.These metabolites were further imported into Metabo Analyst 5.0 for analysis and were found to be significantly associated with linoleic acid metabolism,α-linolenic acid metabolism,glycerophospholipid metabolism,glutathione metabolism,and sphingolipid metabolism pathways during both heat stress and temperature recovery.3.Effects of heat stress on the transcriptomics of rainbow troutChanges in gene expression profiles in rainbow trout liver at 24℃ were detected by RNA-Seq technology.A total of 45.49 Gb clean data was acquired from 6 liver samples,and the clean data of each sample was over 6.38 Gb,and the percentage of Q30 bases was over 91.73.A total of 40,576 expressed genes were detected in this analysis,including39,468 known genes and 1,108 new genes;a total of 85,890 expressed transcripts,including 75,567 known transcripts and 10,323 new transcripts.Taking |log2FC|≥1 and P-adjust<0.05 as the threshold,a total of 916 differentially expressed genes were screened,of which 481 were up-regulated and 435 were down-regulated.Functional annotation was done to the differentially expressed genes.Cluster of orthologous groups(COG)showed that a total of 21 categories were annotated,of which posttranslational modifications,protein turnover,and chaperones were most annotated;three categories and 21 classes were annotated by Gene ontology(GO)and mainly focus on the categories of cellular processes,binding,and catalytic activity.Five categories and 32 classes were annotated by Kyoto encyclopedia of genes and genomes(KEGG),in which more differential genes were annotated to signal transduction and immune system.Taking P-adjust<0.05 as standard,GO and KEGG enrichment analysis was applied to acquire a deeper understanding of the hepatic metabolic pathways of rainbow trout under heat stress,and multiple immune and metabolic pathways are found to be involved such as glutathione metabolism pathway,antigen processing and presentation,IL-17 signaling pathway,complement and coagulation cascade,etc.The above results showed that the combination of biochemical indicators,metabolomics,and transcriptomics methods provide strong evidence that heat stress induces oxidative stress and immune responses in rainbow trout liver.It is worth noting that the genes(GPx4),proteins(GSH-Px)and metabolites(glutathione and oxidized glutathione)of the glutathione metabolic pathway were significantly expressed under heat stress,indicating that it is the key metabolic pathways in the rainbow trout liver in response to heat stress,and confirming that heat stress has activated the antioxidant system of rainbow trout.Furthermore,it has laid a solid scientific and theoretical basis for the sustainable development of the industry and the breeding of rainbow trout species with heat resistant property. |
