Study On Response Mechanisms Of Marsupenaeus Japonicus Under High Temperature Stress

The kuruma shrimp Marsupenaeus japonicus is one of the most important commercial shrimp species with high aquaculture interest cultured in China.The farming industry of M.japonicus has been deeply obstructed by the mass mortality during hot summer months and the limitation of optimum culture season and region.A comprehensive understanding of response mechanisms of M.japonicus under high temperature stress will enrich our knowledge on the mechanisms underlying the response of aquatic organisms to high temperature stress as well as provide a theoretical foundation for generating effective strategies to promote the survival of M.japonicus during hot summer months.However,few studies have focused on illustrating the response of M.japonicus to high temperature stress,and the mechanisms underlying the summer mortality syndrome of M.uaponicus still remains largely unclear.In this eontext,the response mechanisms of M.japonicus under high temperature stress were elucidated by the transcriptome and miRNA expression profiles analysis,combining with the excavation and function analysis of heat shock response and antioxidant response genes as well as enzymological and histological studies.The present study focused on the heat shock response,antioxidant response,immune response,metabolism alterations,oxidative injury and histopathological changes to elucidate the response mechanisms of M.japonicus under high temperature stress and investigate the potential mechanisms underlying the summer mortality syndrome in M.japonicus.The main results are as follows:1.Transcriptome analysis of response mechanisms of M.japonicus under high temperature stressGene expression patterns in hepatopancreas and gill of M.japonicus at different high temperature stress stages were determined via comparative transcriptome analysis.DEGs expression profiles of M.japonicus under high temperature stress were constructed,generating a total of 95,763 unigenes.A total of 301 and 1136 DEGs were detected in the hepatopancreas and gill at 3 h HS,respectively.Meanwhile,443 and 1228 DEGs were identified in the hepatopancreas and gill at 96 h HS,respectively.The functional annotation of DEGs showed that the transcription of various heat shock proteins and antioxidant enzymes were significantly increased in either the hepatopancreas or gill of M.japonicus under high temperature stress.However,the expression levels of numerous immune molecules,e.g.,crustin,a-2 macroglobulin homolog,anti-lipopolysaccharide factor,prophenoloxidase activating factor,prophenoloxidase b,C-type lectin and peroxinectin were significantly decreased in either the hepatopancreas or gill of M.japonicus under high temperature stress.Additionally,the KEGG pathway analysis of DEGs showed that the transcription levels of several key rate-limiting enzymes in glycometabolism pathways,such as phosphenolpynuvate carboxykinase.?-glucosidase and glycogen phosphorylase,were significantly down-regulated in the hepatopancreas of M.japonicus under high temperature stress.Furthermore,the transcriptional levels of several critical enzymes and components in the TCA cycle and electron transport chain,including citrate synthase,isocitrate dehydrogenase,succinate dehydrogenase,cytochrome c and ATP synthase were down-regulated in the gill of M.japonicus under high temperature stress.The suppression of the immune capacity as well as nutrient and energy metabolisms under high temperature stress might contribute to the mass mortality of M.japonicus during hot summer months.2.MicroRNAs expression profiles analysis of M.japonicus under high temperature stressIn addition to the construction of DEGs expression profiles,microRNAs expression profiles in the hepatopancreas and gill of M.japonicus under high temperature stress were analyzed.In total,26 known miRNAs and 53 novel miRNAs were identified.The length distribution showed that miRNA of 21 to 23 nts represented the majority of miRNA.MiRNAs with RPM>100 were selected for DEMs identification and follow-up function analysis.A total of 15 miRNAs(RPM>100)were differentially expressed in either hepatopancreas or gill under heat stress.The results of target genes prediction and functional annotation of DEMs indicated that a broad sphere of biological processes,including signal transduction,anti-stress response,ribosomal biogenesis,transcription,RNA processing and modification and lipid metabolism,were subject to miRNA dependent regulation under high temperature stress.Futhermore,the WSSV genes targeted by shrimp miRNAs were also identified by using the genome data of WSSV.A total of 30 miRNAs with potential anti-WSSV function were obtained.3.Antioxidant response and histopathological changes in M.japonicus under high temperature stressBased on the DEGs expression profiles,molecular cloning and expression analysis of antioxidant enzymes genes as well as studies on antioxidant enzymes activities changes and the histopathology alterations were conducted to elucidate the antioxidant response and histological injury of M.japonicus under high temperature stress.In this study,the full-length cDNA sequences of MjSeGpx and MjGSTMu in M.japonicus were cloned.The deduced amino acid sequences of MjSeGpx and MjGSTMu contained typical structures and function domains of Se-Gpx and GSTMu,respectively.Tissue distribution analysis showed that MjseGpx and MjGSTMu were ubiquitously expressed in hepatopancreas,gill,hemocytes,stomach,intestine,heart,muscle and eyestalk.The expression level of MjSeGpx was significantly higher in hepatopancreas and hemocytes,and the MjGSTMu was predominantly expressed in hepatopancreas.In general,the transcriptional levels of MjSeGpx and MjGSTMu increased under high temperature stress and recovered to basal levels with the time prolong.When exposed to water temperature of 32? and 34?,the mRNA expression levels of MjSeGpx and MjGSTMu in hepatopancreas all peaked at 12 h and the transcriptional levels of MjSeGpx and MjGSTMu in gill reached a maximum at 72 h and 48 h,respectively.Meanwhile,the CAT activity in hepatopancreas and gill of shrimp exposed to 32? and 34? showed a significant increment,and the increase speed and peak activity exhibited a tendency of positive correlation with the temperature.In addition,the MDA content significantly increased 2.30-fold at 24 h in the hepatopancreas of M.japonicus exposed to 34?.Although the MDA content in the gill of 32? and 34? exposed shrimp elevated at 6 h,it's still remain at relatively low levels.Histopathology alterations appeared in the hepatopancreas of M.japonicus exposed to water temperature of 320C and 340C for 24 h and 48 h,including inflated tubules,abnormal lumen,collision between adjacent tubules and blurred boundaries between tubules4.Expression and function analysis of heat shock proteins and heat shock factor in M kaponicusHeat shock proteins(HSPs)and heat shock factor(HSF)were representative factors in the high temperature stress response.In the current study,the full-length cDNA sequences of MjHSPIO and MjHSF1 in M.japonicus were cloned,and the significance of' MjHSPlO and MjHSF1 in the heat shock response were elucidated The deduced amino acid sequences of MjHSPlO and MjHSFl exhibited the conserved structures and function features of HSPlO and HSFl,respectivelyTissue distribution analysis showed that MjHSPIO and MjHSFI were ubiquitously expressed in hepatopancreas,gill,hemocytes,stomach,intestine,heart,muscle and eyestalk.High temperature stress induced a significant expression increment of MjHSP10 MjHSF1MjHSP60MjHSP70 and MjHSP90,and the up-regulated levels exhibited a tendency of positively correlating with temperature The four HSPs exhibited different expression profiles in response to high temperature stress.The expression levels of MjHSPlO,MjHSP60 and MjHSP90 kept up-regulated throughout the high temperature stress period or mcreased at most time point under high temperature stress,whereas MjHSP70 merely elevated at one or two time points with relatively lower peak level.Additionally,the transcription of MjHSFI showed relatively low up-regulation levels under high temperature stress.The expression level of MjHSFI in hepatopancreas only up-regulated under long-term(48 h)stress and the transcriptional level of MjHSFI in gill merely increased under acute(340C)stress Expression of MjHSFl in hemocytes was silenced by RNAi at 24 h and 48 h post dsMjHSFI injection and resulted in down-regulation of MjHSPIO at 48 h post dsMjHSFl injection.Finally,GST pull-down assay showed that there was an interaction between MjHSPIO and DNA binding domain of MjHSFI,which may suppress the DNA binding activity of MjHSF1.