Study On The Molecular Mechanism Of Salinity Adaptation Of The Elvers Of The Flower Eel (Anguilla Marmorata)

Marbled eel(Anguilla marmorata)belonging to Anguilliformes,is China's second-grade protected wildlife.They are distributed in the seas and estuaries near Hainan,Fujian and Guangdong provinces in China.In recent years,the breeding of A.marmorata has been developed at a fast speed.However,due to their own habits and the lack of relevant research,the artificial breeding of eel is greatly restricted by the fact that most eel larva can only be captured in natural waters and their survival rate in breeding industry is rather low.As a typical catadromous fish,studies on the osmoregulation mechanisms of tropical marbled eel have not received sufficient attention.Additionally,there are not adequate researches conducted from the perspective of the molecular level and protein data and the osmotic regulation mechanisms that help tropical marbled eel migrate from the salty water to the fresh water remain largely unclear.In this study,we attempt to explore the salinity adaptation regulation mechanisms of tropical marbled eel by means of transcriptome high-throughput sequencing and proteomic sequencing.The present study focuses on the following two aspects:the level of RNA transcription and the level of protein expression.At the same time,special attention is paid to finding out the important roles that the three key genes(Hsp90,NKCC1 and HSD11B2)play in the salinity adaptation regulation mechanisms of tropical marbled eel.1.Transcriptome analysis of salinity change in Anguilla marmorataWith the help of high-throughput Illumina platform,the mixed RNA of elvers'gill,intestine,kidney at three salinity levels(FW,salinity 0 ppt;BW,salinity 10 ppt;SW,salinity 25 ppt)were sequencing with 100 bp depth of double end.Sequencing results show that approximately 33 million total reads can be obtained from each group.Compared with Anguilla anguilla transcriptome reference sequence,altogether 65536 contigs are identified after the De novo assembly,of which 45976 can be annotated.All compared with FW group,the GO annotation results of SW and BW group are different.The contigs number is larger in SW GO terms than which in BW GO terms.The amount of RNA expression is calculated by using the FPKM value.Compared with FW group,1511 genes in BW group are up-regulated and 238 genes in BW group are down-regulated.Compared with FW group,2208 genes in SW group are up-regulated and 335 genes in SW group are down-regulated.A total of 133 pathways have been identified in the transcriptome.Pathways that have annotated more relative genes are Purine metabolism,Phosphatidylinositol signaling system and Inositol phosphate metabolism,which contains some genes related to osmoregulation.Seven genes(AQP3,NHE8,Hsp90,Hsp70,Hsc70,NBCe3 and HSD11B2)relevant to osmoregulation in the transcriptome are amplified by RACE method.The mRNA expression of each gene at three salinity levels was monitored by qPCR.The results reveal that five genes(AQP3,NHE8,Hsp90,Hsp70 and NBCe3)shown significant changes of mRNA expression.2.iTRAQ proteomic analysis of salinity change in gill from Anguilla marmorataIn the proteome analysis of the gill tissue at three different salinity levels,a total of 1937 proteins were identified by using iTRAQ technique,of which 1560 proteins were quantified.Compared with the protein expression level in the gill of marbled eel in FW group,336 proteins were up-regulated in SW group and 33 proteins were up-regulated in BW group.The 336 up-regulated proteins contain the Na+/K-A-TPase,V-type proton ATPase,sodium-potassium-chloride co-transporter(NKCC),heat shock protein 90(Hsp90),etc.Differentially expressed proteins were enriched in different KEGG pathways.Oxidative phosphorylation pathway and seleno-compound metabolism pathway involve ATP synthesis and decomposition.Ribosome pathway reflects a lot of protein expression.Even more remarkable is the tight junction pathway and cardiac muscle contraction pathway that may be linked with the ion transport in gill cells.It is hoped that these results can provide new interacting protein and metabolic pathways for the osmoregulation of A.marmorata.3.Heat shock protein 90(Hsp90)Expression of Anguilla marmorata adaption to salinity changeThe expression of mRNA and protein of Hsp90 in the gill tissue were tested after elvers were transferred to two different salinity environments.The results revealed that there exist significant differences in the development tendency of mRNA and protein expression between the gill tissue transferred to the brackish water and the sea water.Nevertheless,at each salinity level,there is a link between the expression quantity of mRNA and protein during different time periods.This result shows that Hsp90 may be involved in the regulation process in the cell and play an important role in gill's adaptation to the salinity change.The rise of Hsp90 occur after the elvers stay in the sea water for 24h,which suggests that the change of Hsp90 does not happen soon after the change of salinity in the environment of high salinity.4.Na-K-2Cl coordinated transport protein 1(NKCC1)Expression of Anguilla marmorata adaption to salinity changeThe expression of mRNA and protein of NKCC1 in the gill tissue were tested after elvers were transferred to two different salinity environments.The results revealed that the expression of NKCC1 mRNA reached the very significant peak after elvers stayed in the brackish water for 6h and 48h respectively.As for the sea water group,the expression of NKCC1 mRNA reached the very significant peak which is higher than that in the brackish water after elvers stayed in the sea water for 6h.The tendency of the protein expression is similar to that of mRNA in the brackish water,while the protein expression is relatively low after elvers are transferred to the sea water.As a result,it can be inferred that the salinity adaptation mechanisms that NKCC1 is involved in have changed in the high-salinity environment.In other words,A.marmorata and other euryhaline fish have different NKCC1 regulation mechanisms when they adapt to the high-salinity environment.5.11-beta dehydrogenase II(HSD11B2)Expression of Anguilla marmorata adaption to salinity changeThe expression of mRNA and protein of HSD11B2 in the gill tissue were tested after elvers were transferred to two different salinity environments.The results indicated that the expression of HSD11B2 mRNA in gill reached the very significant peak after elvers stayed in the brackish water for 12h and then it gradually declined to the level that is below the level of the control group after elvers stayed in the brackish water for 96h,After elvers were transferred to the sea water,the expression of HSD11B2 mRNA and protein keeps at a low level in every period.It can be speculated that,in the brackish water,HSD11B2 may not regulate the ability to adapt to salinity change by affecting cortisol's activity.When reacting to high salinity levels,elvers are likely to reduce the expression level of HSD11B2 to participate in the salinity adaptation.