Effects Of Salinity On Growth And Osmotic Gene Expression In Spotted Scat(Scatophagus Argus)

Spotted scat(Scatophagus argus)is a euryhaline marine species which can tolerate a wide range of environmental salinity,which make it a ideal model organism for studying in growth development and stress genes response.In addition,this species is extensively reared in coastal areas of Guangdong Province,China,providing considerable commercial values to the local aquaculture development.Salinity is a vital parameter that affect fish development and growth.Salinity is one of the challenges that teleosts will face in both freshwater(FW)and seawater(SW).Salinity fluctuations within a certain range will have an impact on the growth of fish,specifically reflected in the change of standard metabolic rate,osmoregulatic ability,hormonal stimulation etc.Slightly changes in environmental salinity negatively influenced body growth,challenge an creature to maintain homeostasis.Hormones play a crucially role in maintaining the physiological equilibrium and regulate growth of teleosts in a stable condition during fluctuation in the environmental salinity.Numerous studies have shown that the growth of teleosts is mainly regulated by the GH/IGF-I axis,in which growth hormone(GH)plays a major role.The GH/IGF-I axis is mainly composed of growth hormone,growth hormone receptor(GHR)and insulin-like growth factor I(IGF-I).In fish,GH is produced in the anterior pituitary gland and exerts its function by binding to growth hormone receptor(GHR),and finally promote the synthesis of IGF-I in the liver.Cortisol plays an important physiological role in the adaptation of teleosts to seawater with different salinity.In order to evaluate the effects of different environmental salinity on the growth of S.argus,specimens were reared for 5 months(from April to August,2015)at 3 different environmental salinity(2±0.5,25±2.2 and 35±3.1)in the wild.In this study,we cloned full-length cDNAs of IGF-I and GHR in S.argus,and detected the expression patterns of GH,GHR and IGF-I in different tissues of S.argus by semi-quantitative method.Hypophyseal GH,hepatic GHR and IGF-I expression levels were measured in the three rearing salinities tested by RT-PCR,the serum level of GH was also acquired.The total length,body weight and specific growth rate(SGR)were recorded.The average length,average weight and SGR of each group of experimental fish per month were calculated.The HPI axis plays a major role when teleosts were subjected to environmental salinity stress.Cortisol is an important component of the HPI axis.It is taken that cortisol,performs both glucocorticoid and mineralocorticoid actions through specific receptors,such as glucocorticoid receptor(GR)and mineralocorticoid receptor(MR).However,the effects of cortisol on saltwater stress response in S.argus and the regulatory mechanisms of GR and MR during different salinity treatments is poorly understood.To address this,The full length sequences of cortisol receptor GR and MR were cloned by molecular biology technology.In this study,S.argus were temporarily cultured for 7 days under different salinity(FW,BW,SW,HSW).The activity of NKA enzyme in gill with different salinity was measured.The osmotic pressure of serum under different salinity was measured by osmotic pressure meter.Serum cortisol was detected by ELISA.RT-PCR was used to detect the expression levels of GR and MRmRNA in gills and kidneys.Meanwhile,in situ hybridization technique was used to detect the location of GR and MR in the gill and kidney of S.argus.The main results of this study are below:1.Full-length of Sa-GHR cDNA were 2689 bp,containing a 1914-bp open reading frame(ORF),encoding a protein with 667 amino acids.The deduced protein contained 8 characteristic cysteine residues(Cys49,Cys59,Cys92,Cys102,Cys116,Cys225,Cys378,Cys394),7 tyrosine residues(Tyr79,Tyr115,Tyr326,Tyr331,Tyr456,Tyr568,Tyr585),3 N-glycosylation sites(Asn68,Asn107,Asn149).As for the similarity GHR amino acids to other fishes,this was 82%to Epinephelus coioides.The full-length of Sa-IGF-I was 2890 bp,containing a 588-bp open reading frame(ORF)with a 186 amino acids.The deduced protein contained 11 characteristic cysteine residues and 2 tyrosine residues.Biologically closest to Larimichthys crocea.2.RT-PCR results showed that at the same salinity,the pituitary GH mRNA expression level did not change significantly within the sampling time range.In different salinity groups,GH mRNA in pituitary were significantly different and negatively correlated with salinity.The expression levels of GHR and IGF-ImRNA in liver were positively correlated with salinity,with the most significant expression in the group with salinity of 35,followed by the group with salinity of 25,and the lowest expression level in the group with salinity of 2.The expression levels of GHR and IGF-I in liver gradually increased from April to July,and presented a downward trend in August,suggesting that the expression levels of GHR and IGF-ImRNA may also be affected by weather temperature.ELISA results showed that GH content in serum was proportional to salinity,with the lowest content in the group with salinity of 2 and the highest content in the group with salinity of 35.In the group with salinity of 2,serum GH did not change significantly from April to June,increased significantly in July,and then decreased in August,In the group with salinity of 25,the serum GH level did not change significantly from April to June,and the serum GH level began to increase from June,and the increase trend continued until August.Serum GH content in the group with salinity of 35 samples showed a significant upward trend from April to July(P<0.05),and decreased in August.The SGR values were 0.68±0.12,0.44±0.08,0.23±0.06 g/d respectively.There were significant differences in body mass index between the groups,indicating that not all energy was used for body growth,and most energy consumption was in osmotic regulation.When fish are exposed to osmotic conditions,they tend to spend less energy on osmotic regulation,allocating the extra energy to body growth.According to the results of the physical data of the fish,it can be seen that the fish in the group with salinity of 2 had the fastest growth rate,the whole length,and the average weight were the highest,followed by the group with salinity of 25,and the fish in the group with salinity of 35 had the slowest growth rate.3.The activity of NKA enzyme in gill and the osmotic pressure of serum were positively correlated with salinity.RT-PCR results showed that the expressions of MR and GR in gills and kidneys were similar,with the most significant expression in the BW group,the lowest expression in the FW group,and expression in the SW and HSW groups were almost the same.The in situ hybridization results showed that GR and MR hybridization signals were detected in the gill epithelial cells and in the proximal renal tubules.In addition,the expression levels of GR and MR mRNA in seawater were significantly higher than those in fresh water.In general,the presence of GR and MR in gills and kidneys demonstrates that they play an important role in osmotic regulation and homeostasis in S.argus,and the roles of both are similar.