| The oriental river prawn,Macrobrachium nipponense,is one of the most important commercial aquaculture species in China and Southeast Asia.M.nipponense commonly occurs in estuaries and freshwater habitats with low salinity.As China is one of the countries with a large area of saline water in the world,a large proportion of saline water is not effectively utilized,and M.nipponense is capable of surviving ranges of salinity between 7 and 20 ppt.Therefore,the salinity tolerance of M.nipponense can be fully utilized for culture in brackish or saline waters,which provides an important way for the efficient utilization of saline water in China.In this paper,we first investigated the response mechanism of M.nipponense under shortterm high salinity stress by transcriptomic as well as metabolomic approaches;and further investigated the function of the s PLA2 gene family,a key gene obtained by the integrated analysis of the proteome and transcriptome.Then,we analyzed the physiological and biochemical changes of M.nipponense by means of growth traits data,histological section observation,and physiological and biochemical index detection under long-term different salinity stress.Finally,the molecular response mechanisms of M.nipponense under long-term different salinity stress were investigated by transcriptomic and proteomic approaches,and the results are as follows.1.Integrated transcriptomic and metabolic analysis response in gills,hepatopancreas,and muscle metabolism in M.nipponense in response toacute high salinity stressTo elucidate the gene expression pattern under salinity stress,the numbers of DEGs of the four-time point treatments(12,24,36,and 48 h)were compared with the control group(0h)respectively.In the hepatopancreas,632 DEGs were found to be differentially expressed with the fold change(FC)of all the four-time points(FC ≥ 2,P < 0.05).In the gills,836 DEGs were found to be differentially expressed in four treatment time points.In the muscles,there were 99 DEGs were significantly differentially expressed.Generally,a total of 16735,23013,and 21241 DEGs were detected in the hepatopancreas,gills,and muscles,respectively.The networks of molecular interactions were identified by mapping the DEGs to the KEGG pathway.Compared with the control group of hepatopancreas,seven KEGG pathways were significantly enriched in the treatment group,including the PPAR signaling pathway,antigen processing and presentation,carbohydrate digestion and absorption,protein digestion and absorption,biosynthesis of unsaturated fatty acids,and lysine degradation.In addition,six KEGG pathways were enriched in the gills,including protein digestion and absorption,lysosome,and oxidative phosphorylation.In the muscles,six pathways were enriched,including arginine biosynthesis,arginine biosynthesis,c GMP-PKG signaling pathway,and D-Glutamine and D-glutamate metabolism.To investigate the differences in metabolic levels in acute salinity,metabolite profiles in three different tissues were analyzed using LC-MS.In total,1432metabolites(589 negative and 843 positive metabolites)were identified successfully after qualitative and quantitative analysis.To elucidate the expression of metabolites under acute high salinity stress,the number of differentially expressed metabolites in the four treatment groups(12,24,36,and 48 h)was compared with the control group.A total of 16,31,and 37 metabolites obtained by LC-MS from gills,hepatopancreas,and muscle,respectively,of M.nipponense were found to be common and differentially expressed at the four-time points under the experimental conditions.From the defined results,the acute salinity significantly affected the experimental animals and revealed subtle differences between biological replicates,which were related to natural differences among individuals.Most of these metabolites were classified as “glycerophosphocholines”,“amino acids,peptides,and analogs”,and“carbohydrates and carbohydrate conjugates”.Further study identified the top 20 significantly different metabolites pathways in KEGG analysis.In the gills,the metabolites were significantly enriched in riboflavin metabolism and tyrosine metabolism,biotin metabolism galactose metabolism,and phenylalanine,tyrosine,and tryptophan biosynthesis.In hepatopancreas,the metabolites were significantly enrichened in arachidonic acid metabolism,sphingolipid metabolism,oxidative phosphorylation,glycerophospholipid metabolism,riboflavin metabolism,and fatty acid metabolism.In muscle,the metabolites were significantly enrichened in onecarbon pool by folate,sphingolipid metabolism,lysine biosynthesis arachidonic acid metabolism,and glycine,serine,and threonine metabolism.Through an integrated analysis of the transcriptome and metabolome,16 pathways were identified in three tissues of M.nipponense.In addition,24 metabolites and 34 related DEGs were recorded.Based on the results,a pathway model for the metabolomic response to acute high salinity stress in M.nipponense was proposed.2.Genome-wide identification and expression analysis of the s PLA2 genefamily,a gene related to acute hypersalinity stress tolerance in M.nipponenseThe s PLA2 genes were significantly differentially expressed under acute salinity stress by integrated transcriptome and metabolome analysis of the M.nipponense.A total of seven members of the s PLA2 gene family were distributed on six chromosomes of M.nipponense.s PLA2 gene family members ranged in amino acid length from 148 to 425.A total of 10 motifs were obtained by MEME prediction.The number of motifs in the s PLA2 gene family ranged from 1 to 10.Motif 1 was present in all s PLA2 gene family amino acid sequences.s PLA2 showed tissue-specific expression patterns in various tissues.s PLA2 is expressed to varying degrees in all three tissues(hepatopancreas,gill,and muscle).Through the detection of two members of the s PLA2 family(M.n PLA2-1-3 and M.n PLA2g12-1),it was found that the expression level was high in hepatopancreas.The expression of M.n PLA2-1-3 in the three tissues was slightly higher than that of M.n PLA2g12-1.The expression of M.n PLA2-1-3 and M.n PLA2g12-1 basically maintained a process of rising first and then falling.In addition,the expression of M.n PLA2-1-3 and M.n PLA2g12-1 was higher in hepatopancreas than in muscle and gill tissue.3.Changes in growth traits,histology,and physiological and biochemicalindicators of M.nipponense under long-term different salinity stressThe results of the body weight and length performance of M.nipponense exposed to different salinity gradients were summarised.The results illustrated that long-term salinity stress 14 ppt(0.436 ± 0.356 g)and 21 ppt(0.307 ± 0.124 g)inhibited weight development,compared with the control group(0.259 ± 0.081)and 0ppt group(0.676 ± 0.499)(p < 0.05).Besides,the length of M.nipponense in 7 ppt(2.890 ± 0.514 cm)was significantly higher than in 21 ppt(2.485 ± 0.389 cm)(p <0.05).However,there was no significant difference between the 0 ppt(2.921 ± 0.576cm)and 7 ppt groups(p > 0.05).Similarly,the salinity stress significantly affected the length of M.nipponense in the 14 ppt and 21 ppt(p > 0.05).Additionally,the body length of M.nipponense in the 7ppt was not significantly different from 0 ppt(p >0.05).Histological differences of the gills and hepatopancreas of M.nipponense in 4salinity gradients(0 ppt,7 ppt,14 ppt,and 21 ppt)were identified by histological analysis.To elucidate the activities of digestive enzymes under salinity fluctuation,trypsin,lipase,and amylase were applied in this study.To determine the antioxidant system alternation,the SOD,ACP,AKP,GPX,GSH,and MDA were calculated.To determine the antioxidant system alternation,the activities of HK,GLU,and TG were calculated.The HK activity was first decreased at 7 ppt and then continuedly to increase to the maximum level at 21 ppt(p < 0.05).Besides,the Glu activity kept decreasing from 0 ppt to 21 ppt(p < 0.05).Eventually,the TG activity firstly decreased from 7 ppt to 14 ppt.It then increased at 21 ppt(p < 0.05).4.Integrated analysis of the transcriptome and proteome profiles of orientalriver prawn Macrobrachium nipponense under long-term salinity exposureTo detect the salinity gradient-dependent expression of M.nipponense under long-term salinity stress exposure,high-throughput m RNA sequencing was performed on three tissue samples(gills,hepatopancreas,and muscle)from five different salinity gradients(0,7,14,and 21 ppt).Three biological repetitions were performed on three tissues(hepatopancreas,gills,and muscle)in four different salinity gradients(0,7,14,and 21 ppt),respectively.11356,2227,and 1819 DEGs were identified in gills,hepatopancreas,and muscle,respectively.Several differentially expressed genes(DEGs)were compared with the control group in each of the three treatment groups(7,14,21 ppt)to determine how gene expression changes under salinity fluctuation(0ppt).Generally,a total of 722(588 up-and 134 down-regulated),70(28 up-and 42down-regulated),and 98(38 up-and 60 down-regulated)DEGs were detected in the gills,hepatopancreas,and muscle,respectively.Based on comparisons between DEGs and KEGG pathways,molecular interaction networks within cells and variants specific to organisms were derived.In gills,there were significant enrichments in four KEGG pathways across different salinity gradients,including cell cycle,cellular senescence,vitamin digestion and absorption,and progesterone-mediated oocyte maturation.In hepatopancreas,insect hormone biosynthesis and vibrio cholerae infection were significantly enriched.Eventually,there were five KEGG pathways enriched considerably in muscle,including the AMPK signaling pathway,PPAR signaling pathway,cardiac muscle contraction,biosynthesis of unsaturated fatty acid,and amino sugar ad nucleotide sugar metabolism.To elucidate the proteomic expression pattern under salinity stress,the TMT was utilized to evaluate the molecular changes in M.nipponense.Compared with the 0 ppt group,a total of 439 and 230 DEPs were identified in gills and hepatopancreas in M.nipponense,respectively.Besides,203(104 up-and 99 down-expressed),112(83 upand 29 down-expressed),and 299(250 up-and 49 down-expressed)DEPs were obtained at the salinity of 7,14,and 21 ppt in gills,respectively.Notably,38 DEPs were kept differentially expressed in 3 treatment gradients in gills.Additionally,85(43 up-and 42 down-expressed),140(104 up-and 36 down-expressed),and 103(72up-and 31 down-expressed)DEPs were obtained at the salinity of 7,14,and 21 ppt in hepatopancreas,respectively.18 DEPs were kept differentially expressed in 3treatment gradients in hepatopancreas.As a result of an integrated analysis of the transcriptome and proteome,92 pathways were identified in two tissues of M.nipponens.In addition,16 up and down-regulated proteins and related DEGs were recorded through KEGG enrichment analysis.In gills,14 DEGs and DEPs were detected in 7 ppt,14 ppt,and 21 ppt groups.In hepatopancreas,6 DEGs and DEPs were found in the 14 ppt and 21 ppt groups.Obviously,no DEG and DEP were identified in the 7ppt group of hepatopancreas.Additionally,AP3 D and ETHE1 a were significantly up-regulated in7 ppt and 21 ppt groups,compared with the 0 ppt group in gills. |
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