| It has a strong ability to regulate osmotic pressure is essential to survive for euryhaline.Many studies on osmoregulation mechanisms of euryhaline have been studied,but limitation.The turbot Scophthalmus maximus has evolved extensive physiological ability to adapt to multiple environmental salinities,and are an excellent model species to study the physiological adaptations of flounder associated with osmoregulatory plasticity.In present study,comparative transcriptomic analysis revealed firstly mechanisms of divergence in osmotic regulation of the turbot,which also showed that myo-inositol metabolism and the phosphatidylinositol signaling system associated with myo-inositol act on multiple patterns of osmotic regulation.Then,the roles of myo-inositol,myo-inositol biosynthesis pathway,and PI3K-AKT signaling pathway which is the core component of the phosphatidylinositol signaling system on osmoregulation of turbot have been investigated.Comparative transcriptomic analysis reveals mechanisms of divergence in osmotic regulation of the turbot.The morphological changes of the kidney indicated the adaptability difference and similarity of turbot to salinity stress.Identify transcriptome-wide differences between low salinity seawater(LSW,salinity 5)-and high salinity seawater(HSW,salinity 50)-acclimated kidneys of turbot to decipher the osmotic regulation mechanism.We identified 688 differentially expressed genes(DEGs)and2,441 DEGs in the LSW-and HSW-acclimated kidneys compared with seawater-acclimated kidneys of turbot,respectively.We investigated three patterns(common,low-salinity specific,and high-salinity specific)of gene regulation to salinity stress that involved in ion channels and transporters,functions of calcium regulation,organic osmolytes,energy demand,cell cycle regulation,and cell protection.Additionally,protein-protein interaction(PPI)analysis of DEGs suggested the presence of a frequent functional interaction pattern and that crucial genes in the PPI network are involved in hyper-osmotic regulation.Based on the analysis of comparative transcriptome data and related literature reports,we conclude that the mechanisms responsible for osmotic regulation and its divergence in turbot are related to various genes that are involved in canonical physiological functions.In addition,we found that myo-inositol metabolism and the phosphatidylinositol signaling system associated with myo-inositol act on multiple patterns of osmotic regulation on turbot.Osmoregulation by the myo-inositol biosynthesis pathway in turbot and its regulation by anabolite and c-Myc.The MIPS and IMPA1 genes were sequenced in turbot and found to be highly conserved in phylogenetic evolution,especially within the fish species tested.The results of multiple sequence analysis in the current study showed that MIPS shared maximum identity with L.crocea MIPS protein(85.69%),and the IMPA1of turbot shared a maximum identify with the tilapia(Oreochromis mossambicus)IMPA1protein(87.41%).The results of the matching prediction indicated that MPIS is homo-tetramer and IMPA1 was a homodimer.Under salinity stress in turbot,both MIPS and IMPA1 showed adaptive expression,a turning point in the level of expression occurred at12 h in all tissues tested.We performed an RNAi assay mediated by long fragment ds RNA prepared by transcription in vitro.The findings demonstrated that knockdown of the MIB pathway weakened the function of gill osmotic regulation,and may induce a compensation response in the kidney and gill to maintain physiological function.Even though the gill and kidney conducted stress reactions or compensatory responses to salinity stress,this inadequately addressed the consequences of MIB knockdown.Therefore,the survival time of turbot under salinity stress after knockdown was obviously less than that under seawater,especially under low salt stress.Pearson's correlation analysis between gene expression and dietary myo-inositol concentration indicated that the MIB pathway had a remarkable negative feedback control,and the dynamic equilibrium mediated by negative feedback on the MIB pathway played a crucial role in osmoregulation in turbot.An RNAi assay with c-Myc in vivo and the use of a c-Myc inhibitor(10058-F4)in vitro demonstrated that c-Myc was likely to positively regulate the MIB pathway in turbot.myo-inositol facilitates salinity tolerance by modulating multiple physiological functions in turbot.A significant increase in the myo-inositol content of the gill of turbot exposed to salinity stress was detected using enzyme-linked immunosorbent assay(ELISA).myo-inositol supplementation provided by immersion or inclusion in the diet significantly increased survival time under salinity stress,with the exception of immersion at salinity 0.Transcriptome data indicated that myo-inositol increased physiological capacities related to the steroid biosynthetic process,steroid metabolic process,circadian rhythm,tryptophan metabolism,metabolism of xenobiotics by cytochrome P450,oxidoreductase activity,iron ion binding,and heme binding in turbot.Furthermore,q PCR results showed that myo-inositol strengthened osmotic regulation represented by expression of ion channel genes of turbot under salinity stress by stimulating the activities of steroid reductase and antioxidase,modulating immune function,and inhibiting the cell cycle and energy metabolism.GO and KEGG enrichment analyses of differentially expressed genes,PPI analysis,and q PCR data showed that the signaling pathways associated with steroids including the steroid biosynthetic process,steroid metabolic process,and steroid hormone biosynthesis,mediated by myo-inositol occupy a central place in osmoregulation in turbot.myo-inositol enhances the low-salinity tolerance of turbot by modulating cortisol synthesis.In this part of study,Na~+-K~+-ATPase activity,expression of ion channel genes,and survival time under low salinity were analyzed after dietary delivery of exogenous myo-inositol,cortisol,and metyrapone(a cortisol inhibitor)to study the role of myo-inositol on osmoregulation.Dietary myo-inositol resulted the remarkable regular changes in gene expression and Na~+-K~+-ATPase activity,and prolonged the survival time of turbot under low salinity.Cortisol concentration regulated by dietary myo-inositol were remarkably correlated with Na~+-K~+-ATPase activity,expression of partial ion channel genes,and survival time.The optimal concentrations of dietary cortisol stimulated gill Na~+-K~+-ATPase activity and increased the expression of ion channel genes to enhance low-salinity tolerance represented by longer survival time under low salinity.In the context of cortisol suppression,myo-inositol failed to increase the survival time of turbot under low salinity.And the significantly or remarkably correlations were identified between cortisol concentration and Na~+-K~+-ATPase activity,expression of partial ion channel genes,and survival time of turbot.Therefore,myo-inositol enhances the low salinity tolerance of turbot by modulating cortisol synthesis.Response of the PI3K-AKT signaling pathway to low salinity and the effect of its inhibition mediated by wortmannin on ion channels in turbot.In this study,gill and kidney transcriptome data demonstrated that the PI3K-AKT signaling pathway plays an important role in the stress response of the turbot when exposed to low salinity.Low-salinity stress led to down-regulation of gene expression,protein content,and phosphorylation of AKT1 at Thr308 of the PI3K-AKT signaling pathway.However,the abundance and phosphorylation of AKT1 protein at Ser473 and Thr308 were detected only in the gill and not in the kidney.Inhibition of the PI3K-AKT signaling pathway mediated by wortmannin showed that wortmannin remarkably inhibited PI3K gene expression and protein synthesis as well as phosphorylation of AKT1 at Thr308 with or without low-salinity stress.In the context of pathway inhibition,suppressed expression levels of ion channel genes demonstrated that the PI3K-AKT signaling pathway acts on osmoregulation by positively regulating ion transport. |
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