Histopathology And Skin MicroRNA Expression Profiling Of Carassius Auratus Infected With Aeromonas Hydrophila

Crucian carp(Carassius auratus)is one of the major freshwater aquaculture fish species in China.Aeromonas hydrophila is a typical pathogen of human,animal and fish,widely distributed in aquaculture water.Except for C.auratus infection lead to a large number of deaths,it also causes bacterial septicemia in the freshwater fish such as grass carp,silver carp and variegated carp.Fish skin plays an important role in the innate immune response to bacterial infection,but its molecular mechanism in mucosal immune response remain unclear.Therefore,the C.auratus induced by A.hydrophila infection was used as the animal model,and the histopathological observation,de novo sequencing library construction of skin transcriptome,and miRNA expression profile and target gene prediction analysis were investigated in this study.The main results are as follows:Firstly,Crucian carp C.auratus was infected with A.hydrophila by artificial intraperitoneal injection,the results showed bleeding at the base of the fin,congestion and hemorrhage on skin,hemorrhage,swelling and other septic lesions in internal organs.Furthermore,the skin,liver,and intestinal tissues were done by paraffin section and HE staining methods,the results of histopathological observation showed that the infected fish epidermal cuticle thinning,loose layer and dense layer separation,epithelial cell necrosis,nuclear enrichment and deep staining in skin;and intestinal tract rupture,mucosal epithelial cell necrosis in intestine,hepatic congestion and hemorrhage,hepatocyte edema enlargement and vacuolar degeneration in liver,renal parenchymal injury,renal tubule epithelial cell shedding and semi-inflammatory cell infiltration in kidney.Secondly,the transcriptome de novo library of healthy C.auratus skin was constructed by using the Illumina Hiseq2000 high-throughput sequencing analysis,the results showed that a total of 4915776 unigenes was obtained in skin,and the average length was 649 bp,and the N50 was 1407 bp.The NCBI nr database is annotated with 60824(25.16%)genes,and the total unigenes of transcripts was analyzed for Cyprinidae fish homologous species,in which 48379(79.54%)of the annotated genes were shown in the zebrafish(Brachidanio rerio)genome.In addition,nine types of mucins were identified in the C.auratus skin sequencing analysis,indicating that MUC1,MUC2,MUC5 B,MUC5AC and MUC18 mucins are the main structural components of the skin mucosa;furtherly,based on the amino acid sequences of mucins from salmon,rainbow trout,zebrafish,frog,murine and human,multiple alignment analysis showed that the mucin Muc5 AC,Muc2 and Muc18 contained conserved domains such as C8,CT and Ig_PVR,respectively.And phylogenetic tree analysis showed that the mucin Muc5 AC and Muc2 were more closely related to secreted mucin,while Muc18 was closely related to membrane-bound mucin and naturally culsered into one group.Finally,microRNA expression profiling and target gene prediction analysis of three skin tissue samples of induced C.auratus by A.hydrophila infection during different time(ie.,control group mSC,6hpi mST1 and 12 hpi mST2 group),the results showed that the skin-related miRNA,rRNA,tRNA was 69444(13.39%),29550(5.70%)and 10704(2.06%),respectively.The differentially expressed miRNA analysis of induced skin showed that the 829 miRNAs were presented at 6h(mST1)and 856 at 12h(mST2).Of these,the differential coexpression of 53 miRNAs(P < 0.01)were found to be involved in the mucosal immune response process in fish skin,including miR-1688?miR-8089?miR-1908-3p?miR-122?miR-146a?let-7e,etc.And all 118111 target genes were obtained from target gene prediction of the known miRNAs,of which 884 and 877 were obtained at 6h(mST1)and 12h(mST2),respectively.Additionally,22 and 15 unknown miRNAs were obtained at 6h and 12 h,respectively,and its target genes were predicted to be 92783 and 77568 genes,respectively.Further enrichment analysis of the target genes for GO function and KEEG signaling pathway were performed,in which GO enrichment based on biological processes showed the mainly terms such as cytological processes,metabolic processes,biological regulation and stress response;KEGG enrichment analysis showed that the 35 immune-related signaling pathways were found,including MAPK,TLR,NLR,NF-kB,JAK-STAT,TCR,BCR,antigen processing and presentation,chemokines,apoptosis,complement system signaling pathways.The above results will provide a scientific reference for elucidating the mechanism of miRNA and its target genes involved in fish skin immune response.