| Salinity is a major environmental factor that exerts intense pressure on marine fish,affecting their survival,metabolism,and distribution.Physiological adjustment to salinity fluctuation requires the ability to regulate body fluid homeostasis in relation to the environment via osmoregulation to control their osmotic pressure.Salinity adaptation of marine fish involves complex physiological traits,metabolic pathways,and molecular and gene networks in osmoregulatory organs.To begin addressing this issue,euryhaline fish such as Scatophagus argus offer a valuable source for osmoregulatory studies.Expression profiles at different salinity levels were characterized using RNA-sequencing in S.argus,and an integrated approach of combining molecular tools with physiological and biochemical techniques are employed to reveal renal osmoregulatory mechanisms in vivo and in vitro.We compared the renal transcriptomes of S.argus from hypo-saline stress group(0‰,FW),hyper-saline stress group(50‰,HW)and control group(25‰)to investigate the potential osmoregulatory mechanisms.In total,19,012 and 36,253 differentially expressed genes(DEGs)were obtained from the FW group and HW group,respectively.Based on functional classification of DEGs,the renal dopamine system-induced Na~+transport was demonstrated to play a fundamental role in osmoregulation,and many candidate genes associated with dopamine system were identified for the first time in this species.Changes in environmental salinity affected renal dopamine release/reuptake by regulating the expression of genes related to dopamine reuptake(dat and nkaα1),vesicular traffic-mediated dopamine release(pink1,lrrk2,ace,and apn),DAT phosphorylation(CaMKIIαand pkcβ)and internalization(akt1).This transcriptional regulation ensured appropriate extracellular dopamine abundance in the spotted scat kidney.Finally,fluctuation in extracellular dopamine had a direct influence on Na~+/K~+-ATPase(NKA)expression and activity,which is associated with Na~+homeostasis.This transcriptomic data provides insights into the molecular basis of renal osmoregulation in S.argus.Importantly,we reveal the mechanism of renal dopamine system-induced Na~+transport is essential in fish osmoregulation.Our results provide a solid basis for future studies on osmoregulatory mechanisms in euryhaline fish.As an important osmotic pressure regulator,the gill has been widely studied,but most of the studies focus on the gill tissue,and there are few studies on the cell level in vitro.The establishment of gill cell can provide an important experimental platform to reveal the osmoregulatory mechanism.This study aimed to establish the branchial cell line of S.argus.The conditions for primary culture and subculture were optimized,and cell characteristics were analyzed.The optimum growth performance was observed in the branchial cell line of S.argus at 28℃in L-15 which contained 20%fetal bovine serum(FBS),and could be successfully subcultured in 2-4 days.The population doubling time(PDT)of SG(abbreviation for the branchial cell line established in present study)cell line was 40.8h.87%SG cells could be resuscitated after cryopreservation in liquid nitrogen.To adapt to the water environment,branchial cells could maintain homeostasis through the osmotic stress response mechanism.SG cells were grown in culture media at different osmotic pressures,and cell proliferation and morphological changes were observed in this study.SG cells could proliferate during hypotonic and hyperosmotic stress(150 and600 mOsmol/kg),and the proliferation rate under hypotonic stress was 1.5times higher than that of hyperosmotic stress.It was observed that the volume of SG cell expanded a in the hypotonic medium and shrank in the hyperosmotic condition.Our results indicated that SG cells have strong osmotic tolerance,with a high adaptation in the hypoosmotic environment.The establishment of SG cell line provides basic experimental materials for the study of osmotic pressure response mechanism. |
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