Study On The Salinity Acclimation Regulation Mechanism Of Trachinotus Ovatus

Trachinotus ovatus(T.ovatus)is a pelagic and warm water fish.Due to its fast growth,delicious meat and high nutritional value,it has become one of the major aquaculture species in southern China.Extreme weather,such as rainstorms and typhoons,often leads to a sharp drop in salinity in ponds and bays,severely affecting the growth and survival of T.ovatus.It is very important to understand the osmoregulatory mechanism of T.ovatus.In this study,the transcriptome sequencing technique was used to analyze the gill of T.ovatus under chronic salinity stress,and significant differentially expressed genes and metabolic pathways were obtained.Under the acute salinity stress,osmotic pressure,Na+/Cl-ion concentration,cortisol(COR)and prolactin(PRL)in the serum were determined in this study.Na+/K+-ATPase was also measured in gill.Finally,the NKA?1,NKCC1 a,and AQP1 a gene sequences were screened from salinity-related significant differentially expressed genes in transcriptome data(unpublished),and their effects on the osmotic regulation of T.ovatus under acute salinity stress were studied.The main results are as follows:1.The transcriptome database related to oval salinity adaptation was constructed using transcriptome sequencing technology.A total of 410,071,902 raw reads were obtained.Filtration and removal of low-quality reads yielded high-quality 405,814,464 reads.The resulting Unigene was compared to the database,annotating 38,437 Unigene,but 37,892 were not annotated.For GO analysis,Unigene was assigned to 53 GO categories,of which 23 biological processes,18 cell components,and 12 molecular functions.Based on the KEGG pathway analysis of the resulting Unigene,a total of 22,232 Unigenes were identified and 229 metabolic pathways were included in the five major pathways of KEGG.These five categories of pathways contain 32 sub-pathways,and Unigene is significantly involved in signal transduction(3,532),translation(1,627),transport and catabolism(1,600),endocrine system(1,543)and carbohydrate metabolism(1,321).By further studying the transcriptome data and analyzing the existing fish salinity-related metabolic pathways,we classified the differential genes associated with salinity adaptation and osmoregulation in the T.ovatus transcriptome data into four categories.Energy production and metabolism,signal transduction,immune response and transport and catabolic pathways.Ten genes were selected from the genes that were significantly differently expressed and verified by real-time quantitative PCR(q RT-PCR).The results showed that the expression trends of the 10 genes were consistent with the transcriptome data,which further proved the reliability of the data.2.In the acute salinity stress,serum osmolality and Na+/Cl-concentrations show a similar decrease trend at the beginning 2 h.After 8 h,the serum osmolality and Na+/Clconcentrations all began to rise and trends to be stable.However,the sharp declines serum osmolality and Na+/Cl-concentrations found in fresh water(FW)group,moreover,the zero survival rate was also observed in FW group.These results revealed that T.ovatus could maintain osmotic balance in salt content from 10‰ to 40‰,but the capacity is not enough to withstand full-strength FW.After the acute salinity stress,the serum COR concentrations increased among different degrees.In particular,it significantly increased in 2 to 4 h.The increase in COR concentration can activate the activity of NKA enzyme,promote the transport of ion channels,and maintain the intracellular homeostasis.When transferred to hypotonic,the PRL concentration was increased in 10‰ sea water(SW)and 20‰ SW groups.However,in 40‰ SW group,the PRL concentration remained at a relatively stable level below the control group.This also proves that PRL is a freshwateradapting hormone,which plays an important role in preventing the loss of ionic flow and regulating osmotic pressure in hypo-osmolality environment.NKA activity of T.ovatus was effected after acute stress.In 10‰ SW and 40‰ SW groups,NKA activity is exhibited higher than control group.Interestingly,compared with 10‰ SW and 40‰ SW groups,NKA activity of 20‰ SW group was the lowest.3.The NKA?1,NKCC1 a and AQP1 a gene sequences were obtained in transcriptome data of the T.ovatus.The full length of NKA?1 was 3,631 bp.The open reading frame(ORF)was 3,072 bp,encoding 1,024 amino acids;NKCC1a,4,269 bp in length,including ORF 3,432 bp,encoding 1,143 amino acids;the length of AQP1 a was 1,078 bp,and the ORF was 786 bp,encodes 261 amino acids.q RT-PCR showed that these three genes were widely distributed in 10 tissues tested(spleen,kidney,stomach,muscle,skin,gonad,gill,brain,liver,and intestines),with higher expression levels in osmoregulatory organ(gill,kidney,and intestine).This shows that the three genes may play an important role in the osmoregulatory organ.Under the acute salinity stress,the genes of NKA ?1,NKCC1 a and AQP1 a showed similar results,responded rapidly to salinity change,and changed drastically within 2-4 h after low or high salinity stress.Different expression levels in the gill,kidney,and intestine are different in hyperosmolar and hypotonic conditions,which may be related to the functional properties and response time of the tissue,and may also be related to sensitivity to salinity.