Studies On Intestinal Barrier In Response To Aflatoxin B1 And Vibrio Parahaemolyticus E1 Infection On Litopenaeus Vannamei

Litopenaeus vannamei were challenged by aflatoxin B1 and Vibrio parahaemolyticus E1 respectively,and changes of microstructure and ultrastructure as well as expression level were determined to investigate the mechanism of shrimp intestinal barrier responding to toxin and vibrio infection.In order to determine the impact of Aflatoxin B1(AFB1)on the intestinal mucosa of Litopenaeus vannamei,shrimps were fed with normal diet and diet containing 15mg/kg of AFB1 for 12 days,and the tissues of intestine were collected at the 2rd,4th,8th and 12 th day of the experiment.The relative expression of genes in mTOR signal pathway including eif4 ebp,eif4e1a,eif4e2 and p70s6 k,the immune-related genes including Dorsal,Relish and pro PO and mucin-like PM gene were quantified with RT-qPCR.The changes of intestinal morphology were explored under microscope.The results showed that,the expression of genes in mTOR signal pathway were affected by AFB1,eif4 ebp gene were up-regulated since the 2rd day,and decreased after reaching its peak at the 8th day;eif4e2 and eif4e1 a gene were both inhibited at the 8th and 12 th day;p70s6k gene were down-regulated from the 2rd to 8th day,and climbed to the initial level at the 12 th day.The immune response of the intestine was also stimulated by AFB1,Dorsal and Relish gene were induced significantly after fed with AFB1;proPO gene was up-regulated at the 4th and 8th day,following by a fall to the initial level at the 12 th day;and the mucin-like PM gene was also significantly induced at the 2th,4th and 8th day.The morphology of shrimp gut was changed by AFB1,the nuclear of epithelial cells swelled,the clear margin of chromatin was no longer present,and the ecclasis of epithelial cell layer occurred.In conclusion,the function of intestinal mucosal barrier was affected by AFB1,not only the mechanical barrier was damaged but also the chemical and immunologic barrier were impaired.Transcriptome expression,microstructure and ultrastructure were analyzed to get a better understanding of the mechanism of shrimp intestinal barrier responding to vibrio infection.A total of 164420 unigenes were obtained from RNAseq data after quality control and assembly,and 29.52% of the unigenes were annotated to at least one of the 7 databases(Nr,Nt,GO,KOG,PAMF,SwissProt and KEGG).4646 differentially expressed genes(DEGs)were determined by DEseq,and 2469 were upregulated while the other 2177 were downregulated.Genes related to intestinal barrier were determined involving in various functions of mechanical barrier,chemical barrier and immunologic barrier,including peritrophin,cytoskeleton and cell junction,pattern recognition receptors,antimicrobial peptide and immune signaling pathways,serine protease/ protease inhibitor and proPO system,apoptosis and phagocytosis,antioxidant and ROS system.Fifteen differentially expressed genes were selected for validation by RTqPCR and the results were consistent with the RNA-seq data.Microstructure and ultrastructure were significantly changed after VPE1 challenge.The epithelial layers detached from the basement membrane.The structure of cell junction was destroyed,nucleus pycnosis and secondary lysosome were also observed.Some of the cells lost their microvilli,cells containing bacterial cells and apoptotic bodies were occasionally seen.These results showed functions of mechanical barrier,chemical barrier and immunologic barrier involved in the response of intestinal barrier to the vibrio infection,and played significant roles in the defense of intestinal barrier.Our data contribute to improve the current understanding of host-Vibrio interaction,providing an abundant source for identification of novel genes.