| Water temperature is a key environmental factor,which directly or indirectly affecting the reproduction,development,growth,metabolism and other physiological processes of fish.Following the changing climates,extreme weather often leads to sudden changes in water temperature,thus causing temperature stress on fish,which threatens their survival and growth,reduces the economic benefits of aquaculture,and may even cause great economic losses.In present study,we obtained the complete CDS of AMPK family genes,then assessed the effect of temperature stress on the level of adenine nucleotides,content of energy reserves,fatty acid composition,and the mRNA expression and activity of AMPK and fatty acid metabolism related genes in Onychostoma macrolepis.This study focused on the molecular mechanism of energy homeostasis and fatty acid metabolism in O.macrolepis under temperature stress,which aiming to provide some meritorious for the establishment and optimization of aquaculture techniques that can alleviate the negative impacts of temperature stress on fish in the future.Trial 1 Cloning and bioinformatics analysis of the AMPK family genes in O.macrolepis.The complete CDS of AMPKα1(MG720299.1),AMPKα2(MG720300.1),AMPKβ1(MG720301.1),AMPKβ2(MG720302.1),AMPKγ1(MG720303.1),AMPKγ2(MG720304.1)and AMPKγ3(MG720305.1)in O.macrolepis were 1713 bp,1659 bp,783 bp,801 bp,993 bp,1605 bp and 1014 bp,which encoding peptides of 570,552,260,266,330,534 and 337 amino acids,respectively.These amino acid sequences retained the high similarity with those of other fish,such as Cyprinidae,including common carp and zebrafish,especially Sinocyclocheilus grahami,a native species in China(92.44%~97.95%),and covered pretty conserved structural domains.Furthermore,the mRNA of AMPK family genes were highly expressed in the heart,liver and muscle of O.macrolepis,which might result from the more vigorous energy metabolism in these tissues.Trial 2 Cold stress promoted the energy consumption and fatty acid β-oxidation while supressed fatty acid synthesis and modified the fatty acid composition of O.macrolepis.We determined the level of adenine nucleotides,content of triglycerides(TG),non-esterified fatty acid(NEFA),total cholesterol(T-CHO),glucose(GLU)and total protein(TP)in the serum,liver and muscle,the fatty acid composition,the mRNA expression levels and activity of AMPKα in the liver and muscle of O.macrolepis with cold stress(10°C)at different time(0h,1h,3h,6h,12 h,24h,48h).The results showed that the AMP/ATP value significantly increased(P<0.05)in the serum and muscle,and the AEC value was lower than 0.75 in the serum and liver.The GLU content increased firstly,then gradually decreased in the serum and liver.And the TG content decreased significantly after 3h(P<0.05),while T-CHO and TP contents didn’t change significantly in the serum.And the NEFA content gradually decreased over time in the liver.The monounsaturated fatty acid(MUFA)gradually decreased,while n-3 series highly unsaturated fatty acid(n-3 HUFA)increased over time in the muscle.At the same time,the mRNA expression level of AMPKα2 increased gradually in the liver,and the mRNA expression levels of AMPKα1 and AMPKα2 increased significantly at 48h(P<0.05)in the muscle,while their activities changed without significance.The mRNA expression level of ATGL increased,but no significance,ACC2 and CPT1 A mRNA expression levels significantly increased(P<0.05)in the liver,while the mRNA expression levels of ACC1 and FAS significantly decreased in the liver and muscle(P<0.05).This study suggested the continuous energy consumption then the AMPK could be activated in O.macrolepis under cold stress(10°C).And the energy reserves could be efficiently possessed the allocation and subsequent mobilization,especially enhancing fatty acid oxidation for energy and supressing fatty acid synthesis to maintaining the energy homeostasis.In addition,the decline of MUFA suggested that it might preferentially supply energy via β-oxidation,while the increase of n-3 HUFA over time might be explained that its important role in maintaining the fluidity and ensuring the normal function of cell membrane.Trial 3 Thermal stress promoted lipolysis and fatty acid β-oxidation while supressed fatty acid synthesis and affected fatty acid composition in O.macrolepis.We determined the content of NEFA,the fatty acid composition in the tissues,and the mRNA expression of fatty acid metabolism related genes in O.macrolepis experiencing thermal stress(30°C)at different time(0h,1h,3h,6h,12 h,24h,48h).Under thermal stress,the NEFA content firstly increased,and gradually decreased with the prolongation of exposure in the liver and muscle of O.macrolepis.The saturated fatty acid(SFA)decreased significantly at 12h(P<0.05),and the n-3 HUFA decreased,mainly due to the decrease in the proportion of docosahexaenoic acid(DHA)in the muscle.And the mRNA expression levels of ATGL and HSL increased,and the mRNA expression levels of CPT1 A and PPARα also significantly increased,while the mRNA expression levels of FAS and SREBP-1 significantly decreased(P<0.05)in the liver.This study suggested that thermal stress could promote lipolysis and enhance fatty acid β-oxidation,which providing more energy.At the same time,fatty acid synthesis was weakened,thereby reducing energy consumption.The fatty acid composition was also altered,especially n-3 HUFA reduced over time in the O.macrolepis under thermal stress.Overall,we obtained the complete CDS of AMPK family genes in O.macrolepis and found that the O.macrolepis exposed to temperature stress constantly expended energy then the energy homeostasis was disrupted.The AMPK could be activated and then enhanced the lipolysis and fatty acid β-oxidation for energy supply,while inhibited the fatty acid synthesis by regulating the fatty acid metabolism related genes in O.macrolepis,thereby reducing energy consumption.Besides,the adaptive modifications of fatty acid composition also were beneficial to respond temperature stress for fish. |
