The Role Of Intestinal Flora And Tryptophan Metabolism In The Occurrence And Development Of Hematopoietic Necrosis Of Crucian Carp

In recent years,crucian carp hematopoietic necrosis disease caused by carp herpesvirus type ?(Cy HV-2)has seriously endangered goldfish and crucian carp aquaculture in many countries around the world,causing huge economic losses.The morbidity and mortality of the disease are high,and there is currently no effective control method.The gut microbial community has been a research hotspot of microorganisms in recent years.It plays an important role in maintaining the health of the body and can participate in the regulation of host metabolism,immunity,growth and development.However,little is known about the correlation between the gut microbiota of the gibel carp and Cy HV-2 infection.This study used Illumina Mi Seq sequencing technology and liquid chromatography-mass spectrometry(LC-MS)to analyze the gut microbiota composition and metabolite profiles of Cy HV-2-infected gibel carp,and used R language Pearson Correlation analysis between gut microbiota and metabolome.According to the results of gut microbiota and metabolome analysis,the Ah R(Aryl hydrocarbon Receptor)pathway downstream of tryptophan metabolism was taken as an example to further verify the effect of the Ah R pathway on the process of Cy HV-2 infection in the heterotrophic silver crucian carp.To clarify the gut microbiota and the metabolites are closely related to Cy HV-2 infection,which provides valuable theoretical guidance and practical reference for the prevention and control of hematopoietic necrosis in crucian carp.The main findings are as follows:1.The results of 16 s RNA sequencing showed that at the phylum level,the gut microbiota of the gibel carp were dominated by Proteobacteria,Fusobacterium,Firmicutes and Bacteroidetes.However,the relative abundances of Proteobacteria and Firmicutes were significantly increased in the Cy HV-2-infected group compared with the control group,while the abundance of Fusobacterium decreased(p < 0.05);The genera with relatively high relative abundance at the genus level were Aeromonas,Cetobacter,Flavobacterium,Shewanella,etc.Cy HV-2 infection altered the gut microbiota structure,with a significant decrease in the relative abundance of Cetobacter and a significant increase in the relative abundance of Aeromonas(p <0.05).Therefore,it is speculated that Cy HV-2 infection affects the physical barrier of the intestinal tract of the gibel carp after Cy HV-2 infection.After Cy HV-2 infection,the intestinal barrier of the gibel carp was destroyed,the tight junction between intestinal epithelial cells was incomplete,the gap became wider,and the distribution of intestinal villi was uneven or even shed.The relative expressions of physical barrier function-related genes,including CLDN(Claudin),VPF(Vascular permeability factor),OCLD(Occludin),ET(Endothelin),ZO(Zonula occludens)and TF(Tissue factor)were down-regulated by 26.54,9.08,16.99,30.98,14.42 and 14.69,respectively;2.Twenty-four identifiable and significantly altered metabolites were found in the gut of the gibel carp following Cy HV-2 infection.Among them,The concentrations of 8 metabolites of Norizalpinin,6-(Dibromomethylene)-17beta-hydroxy-androst-4-en-3-one propiote,1-Deoxy-D-altro-heptulose 7-phosphate,Ellagic acid,D-Maltose,Alanyl-Valine,N-a-Acetyl-L-arginine and Phosphophosphite increased,and the concentrations of other 16 metabolites decreased;KEGG annotation indicated that 11 signaling pathways showed significant changes after Cy HV-2 infection(p < 0.05).Among them,the most distinct signaling pathways are tryptophan metabolism,starch and sucrose metabolism,carbohydrate digestion and absorption,metabolic pathways and taste transduction.The results of the correlation analysis of gut microbiota and metabolome showed that there were 7 metabolites that were significantly correlated with Aeromonas.Among them,glycyrrhetic acid,sedoheptulose 1-phosphate,donepezil and 2-oxyarginine were positively correlated with Aeromonas,and 1-ketose and 8-iso-15-keto-PGE2 were negatively correlated with it.18 metabolites were clearly related to Cetobacter,including adenosine monophosphate,4-ketocyclophosphamide,Lyso PC(24:0),D-glucono,cis-aconitate,cis-4-Carboxymethylenebut-2-en-4-olide,4-Pyridoxate,4-hydroxybenzaldehyde,pyrrolidonecarboxylic acid,indoleacetaldehyde,13-oxo-ODE,L-cysteine,ketoleucine,4-hydroxyphenylpyruvic acid,deoxyribose 5-phosphate,stearoyl sphingomyelin,phytosphingosine and 2-oxoarginine.3.Taking tryptophan metabolism as an example,the effect of its changes on the virus infection process was verified in reverse.The results showed that Cy HV-2 infection significantly up-regulated the relative expressions of Ah R1 and Ah R2 downstream of tryptophan metabolism in the head kidney of gibel carp.FICZ(6-Formylindolo[3,2-b]carbazole)can effectively activate the Ah R pathway in gibel carp,and can inhibit the proliferation of Cy HV-2 in vivo by activating the Ah R pathway.This inhibitory effect may be achieved by increasing the expression of antiviral genes and enhancing the Th2 immune response.In conclusion,Cy HV-2 infection significantly disrupted gut microbial ecology and induced the proliferation of the opportunistic pathogen Aeromonas.These changes disrupt the metabolic function of the gut microbiome,resulting in abnormal gut permeability.Therefore,controlling Cy HV-2 infection by modulating the gut microbiome may become an effective measure.Activation of the Ah R pathway has a certain inhibitory effect on Cy HV-2 infection.Further in-depth exploration of its impact mechanism may provide a new target for the treatment of Cy HV-2 virus infection,and provide new ideas for the immune prevention and control of the gibel carp.