Transcriptomic Analysis Of Grass Carp And Spinibarbus Hollandi Infected By Ichthyophthirius Multifiliis And Expression Analysis Of Complement Component C3

Ichthyophthirius multifiliis is a protozoan ciliate that causes "white spot disease" in freshwater fish.Spinibarbus hollandi belongs to Cyprinidae and Barbinae.It was found in breeding and experiment that it is less susceptible to Ichthyophthirius multifiliis than grass carp(Ctenopharyngodon Idella).In this study,10,000 theronts/tails were used to infect grass carp(n=30)and S.hollandi(n=30).All grass carp died12 days after infection,and the survival rate of S.hollandi was above 80%.This study used transcriptomics,bioinformatics and fluorescence in situ hybridization to compare and analyze the DEGs in five tissues of gill,skin,liver,spleen,and head kidney in grass carp and S.hollandi after infected by I.multifiliis 48 hours.Comparison of amino acid sequences of C3 gene of grass carp and S.hollandi.And the expression location of C3 gene in gill tissues after infection with I.multifiliis.To study the immune response of these two kinds of fishes with different susceptibility to I.multifiliis,provide reference materials for bony fishes to prevention and control I.multifiliis.The main findings are as follows:1.Grass carp transcriptome sequencing obtained a total of 254,969,114 Clean Reads,and the Mapped Ratio compared to the grass carp reference genome was 92.96%.There is a total of 12,185 DEGs in five tissues,of which 4,088 are significantly upregulated and 8,097 are significantly down-regulated.Using GO classification and KEGG pathway enrichment analysis,105 significantly up-regulated immune-related genes were obtained.Inflammation-related genes CD40,CD80,TNF-α,TNFSF13,TNFRSF9,TNFRSF19 L,TNFRSF1A,CXCL8,CXCL11,CXCR4,LTA,TLR-4,IL-1β,IL-12 A,IL-8,IL-6,IL-11 is enriched in the cytokine-cytokine receptor interaction pathway and the Toll-like receptor pathway.Immune-related genes IRF3,IRF7,P38,RIG-I,MHC-I,MHC-II,CTSS,CTSL are enriched in RIG-I-like receptor signaling pathway and phagosome pathway.Apoptosis-related genes CDK4,CDK1,GADD45 are enriched in p53 signaling pathway.The number of immune-related DEGs in grass carp skin is the largest,followed by spleen and head kidney.48 hours after grass carp was infected by I.multifiliis,the inflammatory cytokines were greatly up-regulated.The inflammatory storm may cause the death of grass carp.2.S.hollandi transcriptome sequencing obtained a total of 234,327,582 Clean Reads,assembled into 100,713 unigenes,with an average length of 910 bp and an N50 length of 1,919 bp.There is a total of 15,386 DEGs in the five tissues,of which 5,665 are significantly up-regulated and 9,721 are significantly down-regulated.Using GO classification and KEGG pathway enrichment analysis,46 significantly up-regulated immune-related genes were obtained.The complement pathway genes MBL,C1 q A,C2,C3,and C9 are enriched in the complement and coagulation cascade pathway.Immunerelated genes THBS2 S,MRC,Ig H,MHC-I,CTSS,CTSL,ACP5,F2 R,SH3GL,CDC42 are enriched in the phagosome pathway,lysosome pathway and endocytosis pathway.Inflammation-related genes CXCL12,IL-10,IL-12Rβ,TNFRSF11β are enriched in the cytokine-cytokine receptor interaction pathway.48 hours after S.hollandi was infected by I.multifiliis,the immunoglobulin heavy chain gene Ig H was up-regulated,and the complement genes(MBL,C1 q A,C2,C,C9)were up-regulated in the gills and head kidney,and the complement pathway was comprehensive.Activation,the innate immune response and the adaptive immune response work together to increase the survival rate of the S.hollandi infected by I.multifiliis.3.Grass carp and S.holland have different amino acid positions in the C3 gene amino acid sequence ANATO domain and C3 thioester domain.The expression of C3 gene in the tissues of S.holland from high to low is liver,spleen,intestine,head kidney,blood and gills.There is almost no expression in skin,muscle,brain,and heart.The C3 gene is expressed in large amounts in the gill tissue of the S.holland was infected by I.multifiliis.It circulates from the gill arch through the gill blood to the gill filament vascular tract,and finally covers all the gill fragments.After the grass carp was infected by I.multifiliis,only a small amount of C3 gene was expressed in the gill arch.There is almost no C3 gene expression in the gill tissues of healthy grass carp and S.holland.The extensive inflammatory reaction in the body of grass carp infected with I.multifiliis may be the cause of the death of grass carp.The complement pathway of S.hollandi is fully activated after being infected by I.multifiliis,and the complement genes of the gills are up-regulated.It was the first line of defense to resist the invasion of I.multifiliis.