| Nile tilapia(Oreochromis niloticus)belongs to the Cichlaidae family of Perciformes.Because of its excellent growth performance,strong resistance to disease and nutrient richness,O.niloticus is widely breed.O.niloticus is able to tolerate a wide range of salt and even can survive in seawater after domestication.The aquaculture of O.niloticus in brackish water provides a new direction to expand the production of tilapia.However,high salinity farming can raise many problems of growth,proliferation,development and other aspects.Up to date,a great deal of research has been devoted to exploring the physiological and biochemical changes in the process of salinity acclimation of O.niloticus,but no literature has been able to reveal the overall response at molecular level and the energy metabolism under salinity acclimation.In this paper,to demonstrate the role of energy change sensor of AMPK,we reported the sequences of adenylate-activated protein kinase a subunits(AMPKa),bioinformatics analysis,the response to acute salinity stress,and supplemented with tilapia content change of energy materials to support AMPK's function.In addition,the transcriptome responses of liver of O.niloticus acclimated in different salinity levels were also studied.Through the analysis of transcriptome,we mainly focused on cholesterol-related pathways,and to explore whether the different levels of cholesterol in the diets would alleviate the response to salinity stress and provide a reference to solve the salinity stress on O.niloticus.The main results are followed:1.AMPK expression and the content of energy materials in response to acute hyper-salinity stress in O.niloticusAdenosine monophosphate-activated protein kinase(AMPK),as an energy sensor,has aroused widespread interest in mammalian studies,in which thea catalytic subunit plays a decisive role in AMPK activity.In order to study the basic information and determine the role of energy-changing sensor of AMPK in fish,the nucleotide sequences of AMPKa in O.niloticus were studied by segment cloning,bioinformatics and real-time fluorescence quantitative PCR to obtain the sequence characteristics and organizational distribution;lively O.niloticus(10.92±1.25g)were used to carry out acute 16 psu salinity stress experiment with the control group of 0 psu and were sacrificed at 0 h,6 h,12 h,24 h,48 h and 96 h.The contents of glucose,lactic acid and triglyceride in serum were detected by related kits.The contents of glycogen,lactic acid,soluble protein and the activity of AMPK in liver and muscle were detected.The results showed that AMPKa subunit contained two subtypes:al(1753bp)and a2(2563bp),encoding 571 and 557 amino acids,respectively,and the molecular mass was 64 kDa and 52 kDa,respectively.AMPKal and AMPKa2 share the key structural features of the N-terminal Thr172 site consistent with the mammals.In addition,AMPKa subunits have 81.3%-98.1%homology with the AMPKa subunits of other fish species and vertebrates and are widely distributed in tissues.The results of hyper-salinity stress study showed that the activity of AMPK in liver in the brankish water group was significantly higher than that in freshwater group at 6h and 24h(P<0.05),and the activity of AMPK increased with the increase of time,reached the peak at 24h,and then decreased and recovered to the level at 0 h(P<0.05).At 48 h,the activity of AMPK in muscle was significantly higher than that in freshwater group(P<0.05).The overall trend of the activity of AMPK in muscle was the same in treatment and control group,which showed the trend of increasing first and decreasing secondly.The content of glucose in serum was significantly higher in the experimental group than in the control group at 12 h(P<0.05)and the lactic acid level at 96 h was significantly higher than that in the control group(P<0.05).There was no significant difference in triglyceride content between the two groups,but decreased with time(P<0.05).In the muscle,the content of lactic acid in the treatment group was significantly higher than that in the control group at 48h and 96h(P<0.05).There was no significant difference in the content of muscle glycogen in the muscle group,but decreased significantly with the prolongation of time(P<0.05).The soluble protein concentration in the muscle of the treated group was significantly higher than that of the control group at 12h(P<0.05).The content of glycogen in liver decreased significantly with the increasing time(P<0.05).The concentration of lactic acid was significantly higher than that of the control group at 6h,12h,24h and 96h,and the concentration of lactic acid increased with the increase of time(P<0.05).This research laid the foundation for further study of the roles of AMPK in fish.Meanwhile,it proven that AMPK could play an important role in energy metabolism in acute salinity stress and also provides a basis for further study of energy metabolism under salinity stress.2.Energy metabolism and liver transcriptome response of O.niloticus under chronic hyper-salinity conditionThe aim of this study was to investigate the content changes of energy materials and the transcriptome in liver of O.niloticus after acclimation of chronic hyper-salinity.The same sized O.niloticus juveniles(0.88±0.04 g)were randomly divided into 3 groups,each group of three parallels,respectively,they were cultured under 0 psu,8 psu and 16 psu environment for 8 weeks.Total RNA was sequenced using RNA-Seq method.Based on the change of gene expressions,the differential genes unions from freshwater to saline water were classified into three categories:the constant change category,change-then-stable category and stable-then-changecategory.In the constant change category,steroid biosynthesis,steroid hormone biosynthesis,fat digestion and absorption,complement and coagulation cascades were significantly affected by salinity indicating the pivotal roles of sterol-related pathways in response to salinity stress.In the change-then-stable category,ribosomes,oxidative phosphorylation,signaling pathways for peroxisome proliferator activated receptors,and fat digestion and absorption changed significantly with increasing salinity,showing sensitivity to salinity variation in the environment and a responding threshold to salinity change.In the stable-then-change category,protein export,protein processing in endoplasmic reticulum,tight junction,thyroid hormone synthesis,antigen processing and presentation,glycolysis/gluconeogenesis and glycosaminoglycan biosynthesis-keratin sulfate were the significantly changed pathways,suggesting that these pathways were lesss ensitive to salinity variation.This study reveals fundamental mechanism of the molecular response to salinity adaptation in O.niloticus,and provides a general guidance to understand saline acclimation in O.niloticus.Besides,the contents of glucose,lactic acid and triglyceride in serum were measured,and the contents of glycogen,lactic acid and soluble protein in liver and muscle were detected in 0 psu and 16 psu groups.The results showed that the lactic acid level in serum,muscle and liver of 16 psu group was significantly higher than that in the 0 psu group(P<0.05)and the content of glycogen was significantly lower than that of the control group(P<0.05).The above results suggested that during the long-term salinity stress,O.niloticus require more energy to maintain normal physiological balance.Although the energy metabolism of tilapia has been re-entered the equilibrium state,but incomplete aerobic breathing still kept the fish at a high level of lactic acid.The mRNA of AMPK didn't change significantly which proven that AMPK could only play roles in short-term energy change and have no function after reaching energy balance.3.Effects of dietary cholesterol level on growth,antioxidant capacity and HPI axis of O.niloticus under chronic hyper-salinity stressAccording to the results of transcriptomics,this study selected the cholesterol as the additive and to explore whether additional cholesterol supplementation had a mitigating effect on salinity stress in O.niloticus and the regulation pathways.Healthy O.niloticus(2.20±0.12g)were randomly assigned to six groups,each with three parallels within 29 fish.O.niloticus were feeded with purified diets with a cholesterol content of 0%,0.4%,0.8%,1.2%,1.6%and 2.4%for 8 weeks,respectively.Recorded the daily diet,measured the levels of cholesterol,superoxide dismutase(SOD),glutathione(GSH)and low density lipoprotein in serum and liver and tested the mRNA level of 11 ?-hydroxy steroid dehydrogenation(11P-HSD2)and 20?-hydroxy steroid dehydrogenase 2(20?-HSD2)and cholesterol side chain cleavage enzyme P450(P450scc)in head kidney,and the mRNA levels of glucocorticoid receptor 1(GR1)and GR2B in liver by real-time fluorescence quantitative(RT-qPCR).The activity of Na+/K+-ATPase in gill and the concentration of cortisol in serum were measured by specific kits.The results showed the weight gain rate at cholesterol concentration of 0.4%was significantly higher than that in other groups(P<0.05),and the feed coefficient was significantly lower than that of other groups P<0.05).The levels of cholesterol and serum LDL in serum and liver increased with the increase of cholesterol in the diet(P<0.05).GSH increased with the increase of cholesterol,SOD decreased with the increase of cholesterol(P<0.05).The content of GSH in the liver decreased with the increase of cholesterol(P<0.05)and the content of SOD had no significant change.There was no significant difference in the content of P450scc in the head kidney of the other groups compared with the treatment group with cholesterol content of 0%.The mRNA level of 11?-HSD2 was the highest in the 0%group and decreased significantly with the increase of cholesterol concentration(P<0.05).The mRNA level of 20?-HSD2 was significantly higher than that of other groups at the concentration of 0.4%and 1.2%in cholesterol group(P<0.05).When the concentration of cholesterol was 0.8%,the mRNA contents of GR1 and GR2 were the highest(P<0.05).The activity of Na+/K+-ATPase in the gill of the second branchial gland showed a tendency to increase first and then decrease with the increase of cholesterol content.The activity of Na+/K+-ATPase in 0.4%group was significantly higher than that in other groups(P<0.05).The concentration of cortisol in serum increased with the increase of cholesterol level in the feed,but when the cholesterol content was more than 0.8%,the concentration of cortisol in serum remained steady(P>0.05).The above results indicated that under salt stress,cholesterol is not a necessity for the survival of O.niloticus,but can be used as a diet nutrient additive.Cholesterol is involved in regulating the ability of O.niloticus to adapt to the brackish water environment through the HPI axis.Low concentrations of cholesterol will have a positive effect on the fish while high concentrations cause negative effects.Dietary cholesterol content of 0.4%-0.8%is most conducive to tilapia culture under salt water. |
PDF Full Text Request