Exploring Metabolic Response And Antioxidant Responses Of Discus Fish(Symphysodon Spp.)Exposed To Cold Stress

Discus fish(Symphysodon spp.)belonging to the family Cichlidae(Teleostei)is naturally distributed in Amazon River basin of South America.With elegant posture and gorgeous changing color,the fish is one of the most highly praised ornamental fish and honored as the “King of tropical fish”.Discus fish is an important tropical ornamental fish cultured in Asia with a high and consistent demand for import and export.The water temperature control in the import and export process mainly depends on heating and heat preservation,hence the discus fish are vulnerable to cold stress and then death which leading to huge losses.The aim of this study was to explore the metabolic and antioxidant response to low temperature stress of discus fish,which would provid suggestions for the breeding of cold tolerance strains and for the management of their rearing and transporting temperature1.Exploring antioxidant responses of discus fish(Symphysodon spp.)exposed to acute cold stressHere,a comparative analysis of two species of discus fish,i.e.,S.haraldi and S.aequifasciatus,based on several antioxidant indexes was conducted.We set up a continuous sequence of three temperature programs,namely cooling(28 °C to 14 °C;-1 °C/h),cold maintenance(14 °C for 12 h)and recovery(14 °C to 28 °C;+1 °C/h).In S.aequifasciatus,most of the hardiness indexes were seriously affected by low temperature,and significantly up-regulated,such as superoxide dismutase(SOD),glutathione peroxidase(GPx),glutathione reductase(GR),glutathione-S transferase(GST)activities and reactive oxygen species(ROS),Malondialdehyde(MDA)and protein Carbonylation(PC)contents.But in S.haraldi,GSH activity was the only index sustained up-regulated throughout the treatment,indicating that cold stress resulted in more seriously oxidative stress in S.aequifasciatus than in S.haraldi.Subordinate function(SF)combined with principal component analysis(PCA)showed that the cold hardiness of S.haraldi during cold treatment was in the order of cooling > cold maintenance ? recovery,but the cold hardiness of S.aequifasciatus during cold treatment was in the order of cold maintenance > cooling > recovery.Specifically,the lowest cold hardiness was observed in S.aequifasciatus during recovery.These results showed S.haraldi has stronger cold resistance than S.aequifasciatus,exhibited significant interspecific variability under cold stress.2.Adaptive mechanism of discus fish under chronic low temperature stress.A 28-day experiment was conducted to investigate the temporal variations in cellular energy allocation(CEA),oxygen consumption rate(OCR)and activities of energy metabolism related enzymes in discus fish(S.aequifasciatus)exposed to decreased water temperatures.The steady temperature decrease(1 °C/day)from control(28 °C)to low temperatures(24 or 20 °C)significantly reduced the initial energy reserves(mainly lipids and carbohydrates)while significantly increased the energy consumption(respiratory electron transport system(ETS)activity)in the muscle tissue.Accordingly,on day 0 the CEA was slightly reduced with decreased temperatures.After 28 days of cold acclimation,however,the initial differences in energy reserves,ETS activity and CEA between treatments were decreased.Concurrently,the OCR was reduced with decreased temperatures,suggesting a decreased metabolic rate.During cold acclimation,however,activities of hepatic alkaline phosphatase(ALP),lactate dehydrogenase(LDH),alanine aminotransferase(ALT)and aspartate aminotransferase(AST)were generally increased with decreased temperatures,indicating a compensation for the decreased metabolic rate.Linear discriminant analysis(LDA)showed that the fish exposed to 24 °C only on day 14 was separated from the initial day 0 while those exposed to 20 °C on both days 14 and 28 were separated from the initial day 0,with LDH and ALP activities being the two most discriminant factors.Moreover,after cold acclimation the fish exposed to 24 °C showed comparable growth rates with those exposed to 28 °C.These findings reveal that S.aequifasciatus could recover from the initial energy disturbances resulted from decreased temperatures.The enhanced metabolic activity in response to 24 °C was well within the homeostatic range.It might be promising to culture the juvenile S.aequifasciatus at water temperature as low as 24 °C.Further,by using conventional biochemical assays and gas chromatography timeof-flight mass spectrometry metabolomics,we investigated the physiological responses of S.aequifasciatus gills exposed for 30 days to two temperature regimes: 28 °C and 20 °C.Low temperature resulted in elevated production of reactive oxygen species but not increased malondialdehyde.This might be partially related to protective responses in the antioxidant system,revealed by increased activities of SOD and GPx,and level of GSH,compensating for the depletion of CAT activity.A total of 35 metabolites were identified as potential biomarkers of cold stress,showing themost influenced pathways including starch and sucrosemetabolism,pentose phosphate pathway,glycerolipid metabolism,sphingolipid metabolism,glutathione metabolism,and arginine and proline metabolism.Moreover,the activation of glutathione metabolism agreed with the increased GSH level detected by biochemical assays.Overall,the results of this study suggest that low temperature can activate a protective antioxidant defence response and modify the metabolic pathways in gills of S.aequifasciatus,providing insights into the physiological regulation in response to cold stress in this tropical fish.