Dopaminergic System In Response To The Environmental Salinity Change And Influence On The Gonadogenesis In The Spotted Scat (Scatophagus Argus)

Scatophagus argus,a euryhaline fish,is notable for its ability to tolerate a wide range of environmental salinities,especially for its tolerance to a rapid,marked reduction in salinity.Therefore,S.argus is a good model for studying the molecular mechanisms mediating osmoregulation.The spotted scat has been popularized in South China in recent years as a new resource for marine aquaculture due to its high environmental stress tolerance.Salinity is one of the main environmental factors exerting selective pressure on aquatic organisms.1.In the present study we demonstrate the role of dopamine(DA)via its receptor in mediating the salinity tolerance in the brain of the spotted scat.Dopamine is mainly synthesized and secreted by dopaminergic cells located in the central nervous system.The concentrations of DA,precursor of DA(L-Dopa)and its metabolites in brain and plasma were measured by LC-MS/MS technology.During hypo-salinity acclimation,the content of L-Dopa and DA in brain and Homovanillic acid(HVA)in plasma were significantly increased(*p<0.05;**p<0.01).The significantly decrease of L-Dopa in brain was observed at hyper-salinity groups,while DA was not detected(*p<0.05;**p<0.01).Furthermore,the spotted scat brain from control group was perfused for immunohistochemistry.Quantitative reverse transcriptase-polymerase chain reaction(RT-qPCR)was used to determine dopamine D1-like receptors change in expression during abrupt FW shock.In immunofluorescent staining(IF)methods showed that the protein expression of SaDRD1 and SaDRD5 was labeled in striatum of the spotted scat brain.Using RT-qPCR,we found that both SaDRD1 and SaDRD5 mRNA expression were significantly increased during 7 days of abrupt FW shock,and then the expression of Sa DRD5 dropped to undetectable levels at 7 days post transfer(dpt).Subsequently,the protein expression levels of dopamine D1-like receptors were verified in the spotted scat brain by western blotting and showed patterns that was consistent with the RT-qPCR.Hypo-salinity shock produced a specific increase in the expression of Sa DRD1 and SaDRD5 in brain,suggesting that these two receptor subtypes are upregulated by sodium/osmolar output.Finally,the functional changes are reflected in the decreased protein expression of Na+/K+-ATPase(NKA),while the increased mRNA level of nka was observed during abrupt FW shock.In summary,we demonstrate that the changes of environmental salinity affected the synthesis and mentalism of DA in the spotted scat brain,and the dopaminergic system responds to hypoosmotic shock via dopamine D1-like receptors activation,which leads to inhibition of NKA activity/expression and maintenance of osmotic balance in brain.Our results provide novel insight into the molecular mechanisms that underlie salinity tolerance in teleost.2.The characterization of the expression and regulation of osmoregulation-related genes in the salinity-sensitive organ of S.argus is of great interest to aquaculturists.We used a comparative approach to investigate the regulation mechanisms of long-term salinity acclimation.Total RNA was isolated from kidney,and equal quantities of RNA from three groups were pooled to construct two cDNA libraries(hypo-and hyper-salinity acclimation).Using the Illumina paired-end sequencing technology,nearly 575.3 million clean reads in length of 150 bp were generated and then assembled into 186,397 contigs,of which 43,933 were filtered with an average length of 2,022 bp after the elimination of redundancies.Of these,14,259 transcripts(32.45% of the total)were successfully annotated against NCBI-NR database.GO,KEGG pathway mapping and KOG analysis were performed to predict potential genes and their functions.Based on our sequence analysis and published documents,many candidate genes of renal dopaminergic system with fundamental roles in the spotted scat kidney associated with osmoregulation were identified for the first time in this species.This informative transcriptome provides valuable new data to increase genomic resources of S.argus.The future studies of corresponding gene functions will be very useful for the management of reproduction,growth and disease control in the spotted scat aquaculture breeding programs.Our results provide a solid basis from which to conduct future studies on the osmoregulatory mechanisms in the euryhaline fish.3.Dopamine is an important regulator of renal natriuresis and is critical for the adaptation of many animals to changing environmental salinity.However,the molecular mechanisms through which dopamine promotes this adaptation remain poorly understood.We studied the effects of dopamine on renal hypo-osmoregulation in the euryhaline fish S.argus during abrupt transfer from seawater(SW)to freshwater(FW).Following the transfer,serum dopamine concentration was decreased,and dopamine activated expression of the dopamine receptor 1(designated SaDRD1)in the kidney,triggering the osmoregulatory signaling cascade.SaDRD1 protein is expressed in the renal proximal tubule cells in vivo,and is localized to the cell membrane of renal primary cells in vitro.Knockdown of SaDRD1 mRNA by siRNA significantly increased Na+/K+-ATPase(NKA)activity in cultured renal primary cells in vitro,suggesting that expression of SaDRD1 may oppose the activity of NKA.We demonstrate that exogenous dopamine enhances the response of NKA to hyposaline stress after transferring primary renal cells from isosmotic medium to hypoosmotic medium.Our results indicate that dopamine regulation via SaDRD1 ignited the renal dopaminergic system to balance the osmotic pressure through inhibiting NKA activity,providing a new perspective on the hyposaline adaptation of fish.4.The spotted scat(S.argus),as an important mariculture species,that females exhibit higher growth rates and larger sizes compared to males.Males and females share the same genome,thus,phenotypic divergence are potentially driven by differential gene expression and sex-specific regulation.To gain a global view of molecular mechanisms whereby gene expression may influence sexual dimorphism in the spotted scat,we performed a large-scale transcriptome study.In this study,we employed Illumina paired-end sequencing technology and obtained a total of 9.44×108 reads from three developing stages(stage II,III & IV)of male and female S.argus.Through annotation to the NCBI database,we obtained 33,520 unigenes.Analysis of the gonad transcriptome revealed many genes differing in expression between sexes.This resource allowed us to identify candidate sex determination gene,sex-specific gene expression,and permitted quantitative analysis of differential gene expression.