Molecular Mechanism Of Osmoregulation In Coilia Nasus Based On Multi-Omics

1.Whole-genome resequencing of three Coilia nasus population reveals genetic variations in genes related to immune,vision,migration,and osmoregulationCoilia nasus can be classified into the anadromous population(AP),landlocked population(LP),and sea population(SP).However,they have never been systematically studied.Whole-genome resequencing of AP,LP,and SP were performed to enrich the understanding of different populations of C.nasus.At the genome level,3176204,3307069,and 3207906 single nucleotide polymorphisms(SNPs)and 1892068,2002912,and 1922168 insertion/deletion polymorphisms(InDels)were generated in AP,LP,and SP,respectively.Selective sweeping analysis showed that 1022 genes were selected in AP vs LP;983 genes were selected in LP vs SP;116 genes were selected in AP vs SP.Among them,selected genes related to immune,vision,migration,and osmoregulation were identified.Furthermore,their expression profiles were detected by quantitative real-time PCR.Expression levels of selected genes related to immune,and vision in LP were significantly lower than AP and SP.Selected genes related to migration in AP were expressed significantly more highly than LP.Expression levels of selected genes related to osmoregulation were also detected.The expression of NKAa and NKCC1 in LP were significantly lower than SP,while expression of NCC,SLC4A4,NHE3,and V-ATPase in LP was significantly higher than SP.Combined to life history of C.nasus populations,our results revealed that the molecular mechanisms of their differences of immune,vision,migration,and osmoregulation.Our findings will provide a further understanding of different populations of C.nasus and will be beneficial for wild C.nasus protection.2.Full-length transcriptomic analysis reveals osmoregulatory mechanisms in Coilia nasus eyes and gills reared under hypotonic and hyperosmotic environmentsOxford Nanopore Technologies(ONT)sequencing was performed in C.nasus eyes and gills under hypotonic and hyperosmotic environments for the first time.22.5G and 23.8G clean reads,and 26884 and 27659 full-length non-redundant sequences were generated in eyes and gills via ONT sequencing,respectively.AS events,APA,TF,and LncRNA were identified.In eyes,46 up-regulated DEGs and 28 down-regulated DEGs were identified in hypotonic environment.In hypertonic environment,190 up-regulated DEGs and 182 downregulated DEGs were identified.These DEGs were associated with immune,metabolism,and transport responses.The expression of these DEGs indicated that apoptosis and inflammation were triggered during hypotonic and hyperosmotic environments.Polyamines metabolism and transport of Na+and Cl-from inter-cellular to extra-cellular were activated in hypotonic environment.In hyperosmotic environment,amino acids metabolism and transport of myo-inositol and Na+from extra-cellular to inter-cellular were activated,while Cl-transport was inhibited.Moreover,different transcript isoforms generated from the same gene performed different expression patterns during hypotonic and hypertonic environments.In gills,58 up-regulated DEGs and 36 down-regulated DEGs were identified in hypotonic environment.429 up-regulated DEGs and 480 down-regulated DEGs were identified in hyperosmotic environment.These DEGs were associated with metabolism,cell cycle,and transport.The analysis of these DEGs indicated that carbohydrate and fatty acid metabolism were activated to provide energy for cell cycle and transport in hypotonic and hypertonic environments.Cell cycle was also activated.Polyamines metabolism,ion absorption and water transport from extra-cellular to intra-cellular were promoted,while ion secretion was inhibited in hypotonic environment.Glutamine,alanine,proline,and inositol metabolism were activated.Ion absorption and water transport from intra-cellular to extra-cellular were inhibited in hyperosmotic environment.Moreover,different transcript isoforms generated from the same gene performed different expression patterns during hypotonic and hypertonic environments.These findings will be beneficial to understand osmoregulatory mechanism of Coilia nasus.3.Genome-wide identification of the NHE gene family in Coilia nasus and its effect on osmoregulationNa+/H+exchanger(NHE)gene family is a group of transmembrane proteins regulating intracellular pH,ion transport,and ammonia excretion.In the present study,ten NHEs,the entire NHE gene family,were identified from Coilia nasus genome and systemically analyzed via phylogenetic,structural,and synteny analysis.Different expression patterns of C.nasus NHEs in multiple tissues indicated that expression profiles of NHE genes displayed tissue-specific.Expression patterns of C.nasus NHEs were related to ammonia excretion during multiple embryonic development stages.To explore the potential functions on salinity challenge and ammonia stress,expression levels of ten NHEs were detected in C.nasus gills under hypotonic environment,hypertonic environment,and ammonia stress.Expression levels of all NHEs were upregulated during hypotonic environment,while they were downregulated during hypertonic environment.NHE2 and NHE3 displayed higher expression levels in C.nasus larvae and juvenile gills under ammonia stress.Our finding will provide insight into effects of C.nasus NHE gene family on ion transport and ammonia tolerance and be beneficial for healthy aquaculture of C.nasus.4.The effect of RNA interference and overexpression of NHE gene on osmoregulationThis study investigated the effects of RNA interference and overexpression of zebrafish NHE3b gene on its ion channel gene expression and antioxidant activity to evaluate the function of NHE gene in C.nasus osmoregulation.The results showed that during the period of hypotonic environment,the expression of NCC and AQP3 in the RNAi-hypotonic group was significantly higher than that of the hypotonic group,and the CAT,SOD and GSH-Px activities and MDA levels of the RNAi-hypotonic group were significantly higher than those of the hypotonic group;During the period of hypertonic environment,the expression of NKA and CFTR in the overexpression hypertonic group were significantly higher than that of the hypertonic group,and the CAT and SOD activities and MDA levels of the overexpressionhypertonic group were significantly higher than those of the hypertonic group.These results indicate that under salinity stress,when NHE genes are regulated(interference or overexpression),the body regulates osmotic pressure by up-regulating other ion channels,but it cannot alleviate oxidative stress.This indicates that NHE genes play an important role in salinity stress.