Effect Of Crowding Stress On The Immune Response In Turbot(Scophthalmus Maximus)Infected With Attenuated Edwardsiella Tarda

In commercial aquaculture,overcrowding has negative effects on the growth,health welfare,and immune response in farmed fish.As a chronic stressor in teleosts,overcrowding may increase the incidence of physical injuries and susceptibility to diseases,as well as suppressing the immune response.Many studies have demonstrated the influence of a high stocking density on various physiological and biochemical parameters,including its adverse effects on the growth performance in cultured flatfish.By increasing the production of relative oxygen species,a high stocking density can also affect the level of oxidative stress and alter cellular functions.Cortisol is generally used as the primary stress response to indicate the health status of fish.A high stocking density can trigger significant changes in the cortisol levels in many speciesand there is a direct relationship between the stocking density and cortisol concentration.In addition,cortisol appears to be effective as an immunosuppressive agent with complex actions on immune cells.Increasing the cortisol level decreases the number of lymphocytes,induces apoptosis in B cells,and suppresses the activity of complement in teleosts.Edwardsiellosis is a devastating fish disease that causes extensive losses in many important fish,including turbot(S.maximus),tilapia(Oreochromis niloticus),carp(Cyprinus carpio),and mullet(Liza macrolepis).Immunoprophylaxis is a useful method for inhibiting outbreaks of this disease and it has become an important part of fish farming.The feasibility of using a live attenuated vaccine against edwardsiellosis has been demonstrated in turbot based on immersion or intraperitoneal injection routes.However,different vaccination strategies and environmental factors greatly influence the effectiveness of vaccines for immunoprophylaxis.If fish undergo stress in adverse aquatic settings,vaccinations may attenuate the effects of immunoprophylaxis,and attenuated vaccines can even become more aggressive and even recover their virulence.Thus,it is necessary to confirm whether a live attenuated vaccine is still efficacious under crowding stress conditions.In the present study,we aimed to determine the effects of crowding stress on modulating the immune response in vaccinated fish.The kinetics of surface immunoglobulin-positive(sIg+)cells and specific IgM antibody were employed to evaluate the effects of crowding stress on the immune response in turbot when exposed to attenuated Edwardsiella tarda in this study.1.The effects of crowding stress on immunity index of turbotIn this study,the response of crowding stress were studied though the change of the immune and stress index in blood,spleen,head kidney,gills and skin of turbot which were inoculated with attenuated Edwardsiella tarda vaccine in the different stocking initial density(low density:5 kg/m2,middle density:10 kg/m2,high density:20 kg/m2).It is showed that:The immune indexes of Serum interleukin(1L-1?),complement(C3),immunoglobulin M(IgM,lysozyme(LZM)and tumor necrosis factor(TNF-?)in low density breed were significant higher than that in middle or high density breeding.MHCII gene expression in high density breeding was higher than that in medium or low density in gill;IL-1? expression in low density breeding was higher than that existed in medium or high density;The expression of TNF-? in LD group skin is higher than MD and HD.Above all,in order to improve the immune and reduce stress of turbot,it is suggested to adopt low density breeding.2.Effect of crowding stress on the special immune response in turbotThe aim of this study was to evaluate the immune responses in turbot,Scophthalmus maximus,when subjected to crowding stress under low density(LD;5 kg/m2),medium density(MD;10 kg/m2),and high density(HD;20 kg/m2)conditions for 8 weeks,while also treated with attenuated Edwardsiella tarda(0.1 ml/fish).After vaccination,we measured the dynamics of surface immunoglobulin-positive(slg+)cells in the blood,spleen,and pronephros,as well as the serum antibody and cortisol levels to evaluate the immune responses.The results showed that the time of the peak value for the percentage of sIg+ cells in blood leucocytes(BL),spleen leucocytes(SL),and pronephros leucocytes(PL)occurred during the sixth week in the HD group,which was delayed by week compared with the other groups.In addition,the peak values for sIg+ cells in BL,SL,and PL from the HD group(41.37±3.18%in BL,29.6 ± 2.34%in SL,22.37±1.76%in PL,with the peak values in the sixth week)were lower than those from the MD(46.73±4.12%in BL,33.28±2.68%-in SL,24.57±2.32%in PL,with the peak values in the fifth week,P<0.05)and LD groups(53.14 ± 3.25%in BL,38.36 ±3.54%in SL,28.13 ±3.45%in PL,with the peak values in the fifth week,P<0.05).In addition,the specific immunoglobulin M(IgM antibody levels increased from the first week in all groups and reached a peak in the fifth week in the LD and MD groups,but in the sixth week in the HD group.The serum cortisol levels were greater in the HD group compared with the other groups in the last 3 or 4 weeks.These results show that stocking turbot at a LD obtained the most effective immunization,and thus we conclude that crowding stress may reduce the ability to deal with immune challenge.3.The spleen of turbot transcriptome sequencing and analysisScophthalmus maximus is an important economical cold-water fish species in China,but it is susceptible to various pathogens when suffer the overcrowding stress in commercial aquaculture.In order to clearly elucidate the immune response mechanism,in this study,we have analyzed the transcriptome of S.maximum spleen after challenge with the attenuated Edwardsiella tarda under two different densities:high density(HD,20kg/m2),low density(LD,5kg/m2),using next generation sequencing technology(RNA-seq).A total of 447 million reads were assembled into 41,136 unigenes with an average length of 1274 bp and the N50 size of 2295 bp.Blast the 41,136 genes with the NCBI Nr database,22806 unigenes were annotated,account for 55.44%of the all genes.Then the 41,136 genes blast with Swissprot,KOG and KEGG database,22931 unigenes were annotated,account for 55.74%of the all genes.In order to further understand the transcriptome data integrity,the 41136 were annotated by COG database.41136 Unigenes be annotations to the 26 different functions in the family,by comparing found involved in Signal transduction mechannisms functional genes have a maximum of 7901 Unigenes,followed by General function prediction only features 6112 Unigenes,then Posttranslation modification function 3216 Unigenes involved in order to further the Unigenes description,GO for Unigenes for comments.Article 41136 Unigenes were annotation to the GO database components,biological process,and molecular function of cells in three types of 55 category.23 classes,including biological processes for the cellular components of the 20 classes,molecular function of the 12 classes.In the process of biology about cellular process Unigenes up to 6462,accounting for 15.7%.Cellular components of the cell and the cell part of article 4630 Unigenes,accounted for 11.3%of the total.Molecular features of bing class has 7120 Unigenes,accounted for up to 17.3%.Analysis of differential gene expression between HD and LD infected samples revealed 397 differential expressed genes(DEGs)in spleen,including 146 significantly up-regulated unigenes(fold change>2)and 251 significantly down-regulated unigenes(fold change>2).To validate the RNA-seq data and observe gene expression,the expression levels of 14 IRDs were detected by qPCR.The results indicated that the qPCR data presented a positive line correlation with RNA-seq data.Enrichment and pathway analysis of the differentially expressed genes showed the centrality of the pathogen attachment and recognition,antioxidant/apoptosis,mucus barrier modification and immune activation/inflammation in the pathogen entry and host immune responses.Our results can help to understand the mechanisms of turbot host defense,and may also provide foundation to identify the biomarkers for future selection of disease resistant brood stock and evaluation of disease prevention and treatment options.4.The transcriptome analysis different expression genes of turbot head kidneyAfter the transcriptome sequencing,raw reads data from each samples obtained by filtering,first remove the belt joint(adopter)reads,then remove the uncertain accounted for more than 10%of the base information reads,finally get rid of low quality of the clean reads reads.Will clean reads for assembly,high density group got 40762 unigenes and low density group get 40843 unigenes.Group with a low density as a benchmark,the use of RPKA value significance difference(P<0.05)gene expression between groups,and analysis of the data statistics through 2-fold method.A total of 758 differentially expressed genes were got,in which 100 genes rise significantly,658 genes significantly lower.All the differentially expressed genes(DGEs)by GO analysis,these genes are now owned by a biological process,cellular and molecular components function under the three categories of 42 branches.Biological process group contains 19 branches,of which the cellular process,single-organism process and metabolic process have highest proportion were 23.48%,20.32%and 17.94%,respectively;In the cell group has 13 branches,the cell,cell part and organelle accounted for the highest,respectively,15.17%,15.17%and 11.74%;There are 10 branches in molecular function,which binding,catalytic activity and molecular transducer activity accounted for the highest,respectively,30.21%,18.47%and 3.43%.Enrichment of differentially expressed genes GO function analysis shows that under crowding stress fish body metabolism and function of biological control.Through KEGG pathway annotations analysis helps us to understand the gene product,biological functions,metabolic pathway,and the enzyme catalysis.The differentially expressed gene sequences were blastx with KEGG database,the results showed that 758 differentially expressed genes involved in Metabolism of KEGG,based Information Processing,Environmental Information Processing,Cellular the Processes and the Organismal Systems,five types of 125 pathways.Among them the significantly different pathways(p<0.05)had Vascular smooth muscle contraction,cGMP-PKG signaling pathway,Rapl signaling pathways and so on.The p<0.01 significant differences pathways have six,respectively,focal adhesion,adherens junction,FoxO signaling pathways,Melanogenesis,ErbB signaling pathways and the Hedgehog signaling pathway.For turbot spleen transcriptome sequencing,will be benefit for turbot immune omics related research work.5.Turbot IL4/13 identification and expression analysisIn this study,we cloned and characterized cDNA sequences of IL4/13 from turbot spleen.The full length cDNA of IL4/13 was 1333 bp long and had an open reading frame of 435 bp encoding a predicted polypeptide of 144 amino acid residues.The front-end 21 amino acids are signal peptide and the rest of the 123 amino acids are mature peptide via SignalP 4.1 forecast.The construction of the evolutionary tree,the result shows that turbot IL-4/13 genes and bass IL-4/13-3 together as one.The expression analysis of IL-4/13 in turbot heart,spleen,intestine,head kidney,skin,muscle,brain,liver and gills in expression,found that in the heart,spleen,intestine and head kidney are higher expression.After immunization found the expression of IL-4/13 in each group were significantly increased.The results shown that IL-4/13 in turbot play an important role in the immune response;But among different density groups,there are no significant difference in the same organization,indicating that crowding stress does not significantly affect IL-4/13 gene expression.These results provided basic information for further studies on the role of IL-4/13in turbot immune respone.