| Chinese mitten crab(Erioeheir sznensis)is one of the most important freshwater aquaculture species in China,and its mainly economic character is the size of crab.Molting is essential for growth and development of E.sinensis.For achievement of metamorphosis and growth,this process occurs several times during the life cycle of E.sinensis.As the juvenile crab uptakes water,the body rapidly expands during molting.However,the mechanism underlying the physiological reactions involved in molting process is to a great extent unknown.In this study,the high-throughput sequencing technology was used to construct the gill transcriptome and proteome database of E.sinensis in four molting stages(Intermolt(stage C),Premolt(stage D),Ecdysis(stage E)and Postmolt(stage A)),and the key genes of osmoregulation were found.In addition,the transcriptional expression pattern of osmoregulation of E.sinensis in different molting stages was constructed.Meanwhile,the osmoregulation mechanism of E.sinensis was studied by physiology and molecular biology.1.Relationship between hemolymph osmotic pressure and growth of E.sinensisThe molting cycle of E.sinensis was divided into four major stages basing on microscopically observed morphological features.The growth indexes of juvenile E.sinensis after molting were also measure in the assay.And the osmotic pressure of hemolymph in different molting stages was determined.(1)After molting,the growth of juvenile crab was jumping,with average body length,width and height increased by 5.565%,6.643%and 5.768 in male and 8.171%,9.956%and 8.006%in female,respectively.(2)It was found that the osmotic pressure in stage C and D was the highest in the molting cycle.In stage E,the osmotic pressure was significantly lower than stage C and D(P<0.05),and gradually increased in stage A.It is proved that osmoregulation of E.sinensis was initiated during the molting process,and leaded to the jumping growth of juvenile crab.2.Physiological changes associated with osmoregulation of E.sinensis in different molting stagesCompared with stage C,the concentration of Na in the hemolymph was increased significantly in stage D(P<0.05),and then decreased sharply in stage E(P<0.05).And Na+ concentration in stage A was the lowest during the molting cycle.The highest concentration of K+ was in stage A and stage D,and decreased markedly in stage E(p<0.05).K+ concentration was the lowest in stage C,which was considerably lower than that in other three molting stages.Cl-concentration in stage C was significantly lower than that in stages A,D and E.The concentration Ca2+ was significantly higher in stage D and E than in stage A and C.The concentration of Mg2+ of hemolymph was the highest in stage E,and decreased significantly in stage A.FAA total contents in the hemolymph of E.sinensis in stage A,C,D and E were 2.8830,1.4947,2.1104 and 3.7826 mM,respectively.The content of aspartic acid,serine,cysteine,lysine and arginine was higher in stage E than that in stage C and D.The results showed that Na+and Cl-were the main contributors to the formation of hemolymph osmotic pressure.And both ions and free amino acids were involved in the regulation of hemolymph osmotic pressure.3.Transcriptomic and proteomic anaysis of osmoregulation in E.sinensisIn the laboratory,we performed Illumina RNA sequencing of E.sinensis gills,generating 112,406,504?98,690,970?90,634,634 and 115,833,814 raw reads in stage A,C,D and E,respectively.Comparative transcription analysis showed that 2,751 genes were significantly differentially expressed in stage A compared with stage C,including 1,475 upregulated and 1,276 downregulated genes.And 97 genes were significantly differentially expressed in stage C compared with stage D,including 65 upregulated and 32 downregulated genes.In addition,2,925 genes were significantly differentially expressed in stage D compared with stage E,including 1,352 upregulated and 1,573 downregulated genes.And 324 genes were significantly differentially expressed in stage E compared with stage A,including 171 upregulated and 153 downregulated genes.DEGs were significantly enriched in glucagon signaling pathway,which indicated that osmoregulation of E.sinensis might be regulated by the neuroendocrine system.In addition,DEGs were also significantly enriched in pathways related to osmoregulation,such as aldosterone synthesis and secretion,phosphatidylinositol signaling system and so on.At the same time,genes related to osmoregulation such as PLCP,CALM and PKC were also obtained.In the assay of labeling and quantitating of proteome in E.sinensis gills in four molting stages,total of 2,321 proteins was obtained.Comparative proteomic analysis showed that 141 proteins were differently expressed in C vs.A,including 61 upregulated and 80 downregulated proteins.And 42 proteins were differently expressed in D vs.C,including 22 upregulated and 20 downregulated proteins.In addition,176 proteins were differently expressed in E vs.D,including 124 upregulated and 52 downregulated proteins.Moreover,49 proteins were differently expressed in E vs.A,including 35 upregulated and 14 downregulated proteins.Osmoregulation DEPs were related to inorganic ions,substance transport and metabolic,such as CLCA4 and MRC.4.Preliminary study on osmoregulation mechanism of E.sinensisBasing on the results of transcriptome and proteome analysis,genes related osmoregulation of E.sinensis in different molting stages were selected,including HTR2,CALM,PDE4,ADCY8,ATP1 A,NKAIN,CLCA4,ABCB8,SLC17A5 and AQPN.The mRNA expression of ATPIA,CLCA4,ABCB8,ADCY8,CALM and HTR2 was the highest in stage D,and the expression of SLC17A5 was the highest in stage E,while the expression of AQPN was the lowest in stage E.Na+/K+-ATPase activity was significantly higher in stage D than that in other three stages(P<0.05).The osmoregulation model of E.sinensis was constructed,basing on the studies of physiological changes in hemolymph,transcriptional expression level of candidate genes,and DEGs and DEPs function of transcriptome and proteome.And the osmoregulation mechanism of E.sinensis was preliminarily explained from the physiological and molecular levels.Biogenic amine receptor,proteins related to signal transduction,ion transport,water transport and FAA metabolism were confirmed as important factors on osmoregulation of E.sinensis. |
PDF Full Text Request