Multi-omics Joint Analysis Reveals The Response Mechanism Of The Intestine Of Marsupenaeus Japonicus Under Decapod Iridescent Virus 1 Infection

The intestine is not only an important immune organ,but also an important digestive organ of shrimp,which plays an important role in maintaining homeostasis.As a complex ecosystem,intestine and its symbiotic microorganisms can resist pathogens through mechanical barriers,immune barriers and biological barriers.Similar to other invertebrates,shrimp lacks specific immunity and cannot obtain antibodies through vaccination and other means.Therefore,adding microecological agents and immune enhancers to water or feed to improve the innate immunity of the shrimp intestines has become the best way to prevent shrimp viral diseases.However,most of the current studies on shrimp intestinal immune response under viral infections mainly rely on single omics for analysis,and lack of multi-omics systematic research.Decapod iridescent virus 1（DIV1）is a highly lethal virus newly discovered in shrimp,which seriously threatens the healthy development of shrimp aquaculture.At present,there are few studies on the molecular mechanism of shrimp infection with DIV1.On this basis,this study took Marsupenaeus japonicus as the research object,and analyzed the comprehensive response of M.japonicus intestine under DIV1 infection from the aspects of physiology and biochemistry,RNA-Seq,mi RNA-Seq,metabolome and microbiome.The main research contents and results are as follows:Through the method of artificial injection,it had been determined that M.japonicus is the host of DIV1.The M.japonicus infected with DIV1 showed empty stomach and intestine,yellow hepatopancreas,red body and soft shell.In addition,the toxicity of DIV1 to M.japonicus was measured through LD₅₀test,and the half lethal concentrations of 36 hpi,48 hpi,60 hpi,70 hpi and 84 hpi were obtained.Through testing the activities of immune and digestive enzyme of M.japonicus infected with DIV1,it was found that the activities of immune enzyme were generally inhibited after DIV1 infection and the ROS sacvenging ability was weakened,which meant that the intestinal immune function was impaired.In addition,α-amylase and lipase activity increased significantly in all tissues,which may be used as a diagnostic marker for DIV1 infection in M.japonicus.mRNA-Seq on the intestine was used to study the effect of DIV1 infection on m RNA expression in M.japonicus.A total of 1,976 DEGs were identified,including1,234 up-regulated genes and 742 down-regulated genes.Through the KEGG pathway enrichment analysis of DEGs,it was found that 4 marker pathways of Warburg effect,2 pathways related to vitamin metabolism and 2 pathways of glycosaminoglycan synthesis were significantly enriched.The results showed that DIV1 infection can induce the Warburg effect to provide energy and raw materials for its successful replication.DEGs in the intestine can affect the immune function of the intestinal mucosa by regulating the metabolism of vitamin A and vitamin C,regulating the activity of immune enzymes and the production of ROS,and participated in the host’s immune response against DIV1 by regulating the glycosaminoglycan biosynthesis.miRNA-Seq on the intestine was used to study the effect of DIV1 infection on mi RNA expression in M.japonicus.A total of 32 DEMs were identified,including 19up-regulated mi RNAs and 13 down-regulated mi RNAs.Through the KEGG enrichment analysis of the target genes of DEMs,it was found that 3 immune-related pathways,1 glycosaminoglycan synthesis pathway,and 3 pathways related to the intestinal mucosal barrier function were significantly enriched.The results showed that DIV1 infection can cause the the permeability of intestinal epithelial cells to increase,and the intestinal mucosal barrier function was destroyed,which caused bacterial invasion and infection.In addition,DEMs in intestine can participate in the host immune response to against DIV1 by regulating immune-related pathways and glycosaminoglycan biosynthesis.Through the method of LC-MS/MS,the intestinal metabolites of M.japonicus infected and uninfected with DIV1 were analyzed.A total of 41 and 24 differential metabolites were identified in the positive ion mode and negative ion mode,respectively.Among them,7 fatty acid lipid metabolites changed significantly,and 2Warburg effect marker metabolites increased significantly,including Arachidonic acid,Eicosapentaenoic acid,Docosapentaenoic acid,9-oxo-10（e）,12（e）-octadecadienoic acid,Gamma-linolenic acid,8（s）-hydroxy-（5z,9e,11z,14z）eicosatetraenoic acid,16-hydroxyhexadecanoic acid,L-（+）-lactic acid and Pyruvic acid.These nine metabolites can be used as diagnostic marker metabolites of DIV1 infection.In the pathway enrichment,it was found that 3 pathways related to amino acid metabolism and 4 pathways related to vitamin metabolism were significantly enriched.It showed that DIV1 infection reshaped the lipid metabolism of the host’s intestine,and influenced the host’s energy metabolism and amino acid metabolism through the Warburg effect to provide energy and raw materials for its own replication.In addition,DIV1 infection had also caused a significant impact on the intestinal vitamin metabolism,which affected the immune function of the intestine of shrimp.Through the method of 16S rDNA high-throughput sequencing,the intestinal microbiome of M.japonicus infected and uninfected with DIV1 was analyzed.The results showed that the abundance of Vibrioceae was significantly increased after DIV1 infection,suggesting that shrimp intestinal barrier function was impaired.It was worth noting that the abundance of Photobacterium increased significantly after M.japonicus infected with DIV1,while the abundance of Corynebacterium decreased significantly.These two types of bacteria can be used as the marker microorganism to judge DIV1 infection.PICRUSt function prediction results indicated that intestinal microbiota play an important role in carbohydrate metabolism,vitamin metabolism and amino acid metabolism.Through a joint analysis of the miRNA-Seq and mRNA-Seq,a total of 21 DMEs were negatively correlated with 194 DEGs,and there are 223 correlation numbers.Through the KEGG function enrichment analysis of differentially co-expressed negatively related genes,it is found that a total of 5 DEMs were involved in the regulation of Toll and Imd signaling pathways,and by affecting the expression of caspase 4,transcription factor ATF-2（ATF2）,dual oxidase（DUOX）,immune deficiency（IMD）and Ankyrin to promote cell apoptosis,ROS production,and regulated the synthesis of AMPs,thereby responding to the immune response of DIV1infection.In addition,pathways related to vitamin A metabolism,vitamin C metabolism,and glycosaminoglycan synthesis were also significantly enriched in joint analysis,which confirmed the results of transcriptome and mi RNA sequencing.Through a joint analysis of the metabolome and microbiome,the results show that the intestinal microbiota was highly correlated with the pathways of differential metabolite,and DIV1 infection showed a significant impact on the intestinal microbiota and metabolites.A canonical correlation analysis was performed on the nine marker differential metabolites which screened in the metabolome and the microbial groups whose relative abundance was greater than 0.5%in all samples.The results indicate that the intestinal microbiota is involved in the remodeling of lipid metabolism caused by DIV1 infection.The significant increased in the abundance of Photobacterium and Vibrio promoted the Warburg effect and its related metabolites,thereby promoting the successful replication of the DIV1.