Determination Of The Pathogen Load After Pseudomonas Plecoglossicida Infection And Transcriptomic Analysis In Larimichthys Crocea

In recent years,the development of aquaculture industry of the large yellow croaker(Larimichthys crocea)is heavily damaged by visceral white-spots syndrome caused by Pseudomonas plecoglossicida.In this study we established a real-time quantitative PCR method to detect the relative pathogen load of P.plecoglossicida in the large yellow croaker.And this method was further used to measure disease-resistance ability of 400 individuals of large yellow croakers after artificial challenge with P.plecoglossicida.RNA extracted from spleen of 10 highand 10 low-pathogen carrying fish was sequenced on an Illumina HiSeq platform,and differential expression genes were screened from differential expression analysis and the hub genes were identified from WGCNA analysis.Furthermore,the expression pattern of several differentially expressed genes were followed and examined by real-time fluorescent quantitative PCR in the spleen of the chanllenged large yellow croaker.The main findings of this work are as followed:(1)Specific primers were designed based on the single copy gene gdf-8 of the large yellow croaker and the gyrB gene of P.plecogloss’icida,and a real-time quantitative PCR method to detect the relative pathogen load of P.plecoglossicida was established.With this method,we detected the P.plecoglossicida load of the challenged large yellow croaker,and the phenotype value was expressed as relative bacterial load.The relative bacterial load in the spleen of 400 large yellow croakers was between 5.04×10-7～0.482.The correlation between the relative bacterial load and their body weight was analyzed.The Pearson correlation was-0.035 and P value was 0.487,indicating that there was no significant correlation between body weight and bacterial load.(2)Transcriptomic analysis was performed on 20 large yellow croakers carrying the highest and lowest amount of bacteria selected from the 400 challenged large yellow croakers.In total,17,090 transcripts were assembled.892 differentially expressed genes were screened by differential expression analysis at the criteria of p(adj)<0.001 and |log2Foldchange|≥ 2.There were 769 differentially expressed genes annotated in the UniProt databases,in which 487 genes were up-regulated and 282 genes were down-regulated in the susceptible group compared to resistant group.Five hub genes were identified via WGCNA analysis.We found that these genes are mainly related to immunity and metabolism,such as cytokines including cxcr3，cxcr4，cxcl8,ccl4,ccl8,ccl19,il-1b,il-6,il-10,il-12b,ifna,which can provide a theoretical basis for subsequent gene function research and breeding.(3)The real-time fluorescence quantitative method were used for detecting the relative pathogen load in the spleen of large yellow croaker in different time course after artificial infections by P.plecoglossicida.The expression of differentially expressed gene cd200,ccl4,aqp4,and sema3d were examined in the spleen of the infection process by qRT-PCR Technology.The expression of cd200 and ccl4 genes in spleen tissue was increasing with the relative pathogen load.The expression of aqp4 and sema3d gene in the spleen increased significantly after 3 hours of infection,while it showed down-regulation after 6-48 hours infection in the overall amount of expression in the spleen,but the expression was higher than control group at 0 hour.The expression was stable after 3-6 hours infection.The results of this work provided valuable information for the study on improving the resistance of large yellow croaker to visceral white-spots syndrome.