Studies On The Immunity Mechanism Of Nile Tilapia(Oreochromis Niloticus)Against Gyrodactylus Cichlidarum Infection

Nile tilapia,Oreochromis niloticus,being native to Africa,has been introduced and become an important aquaculture species worldwide.It was first introduced to China in 1978,and is now the main freshwater aquaculture species in south China.Frequent outbreaks of diseases,however,have always restricted the development of Nile tilapia culture.Monogeneans belonging to Monogenea of Phylum Platyhelminthes are common pathogens of cultured tilapia,which may cause mass mortality of juvenile fish.In this study,multiple infection experiments and transcriptome sequencing were used to analyze the expression changes of immunity related genes of tilapia.According to the result of transcriptome,the expressions of MHC(major histocompatibility complex)family genes after Gyrodactylus cichlidarum infection were verified in detail.By analyzing the polymorphism and allele composition of MHC II ? and MHC ? ? in O.niloticus of different susceptibility to Gyrodactylus cichlidarum,the associations between MHC alleles diversity and resistance/susceptibility to monogenean were detected and confirmed by independent infection experiments,the main results are as follows.Based on 76 samples sequenced by Second-generation transcriptome sequencing and one sample by Third-generation sequencing,the full length transcript of Nile tilapia was obtained for the first time by third-generation sequencing,a total of 18.32 Gb clean data was produced from 7 cells,containing 485040 Reads of Insert with 256476 nonchimeric full-length sequences included.After clustering,74013 consensus isoforms were obtained,including 56926 high-quality transcripts corrected for non-full-length sequences.After removal of low-quality and redundancy sequences by comparing to Second-generation data,a total of 16239 gene loci were detected from 32551 transcripts by alternative splicing analysis,including 4259 new gene loci and 26405 new transcripts.A total of 36338 SSRs,17614 ORF sequences and 4111 lncRNA were predicted by sequence structure analysis of newly discovered transcripts,and functional annotation of 23048 new transcripts was completed.Compared with the control group,the expression level of immune-related genes in tilapia started to change on the second day(early stage of infection)after Gyrodactylus cichlidarum infection,the most upregulated genes(1443)are on the fifth day after infection(the peak of infection),and the least up-regulated genes(169)are on the 10 th day after infection(regression stage of infection).Among the three immune tissues of tilapia,the liver had the most differentially expressed genes,followed by the tail fin,and the least in the spleen.A total of 13642 differentially expressed genes were detected in tilapia of different susceptibility.The significantly up-regulated genes included MHC genes,ubiquitin carboxy terminal hydrolase,complement genes,Toll-like receptors,chemokines,T cell receptors,interleukin and other genes related to immune stress which were significantly enriched in antigen processing and presentation pathway,Toll-like receptors signaling pathways,Cell adhesion molecules CAMs pathway,Herpes simplex virus 1 infection pathway,RIG-I-like receptor signaling pathway,Cytokine-cytokine receptor interaction pathway,JAK-STAT signaling pathway,calcium signaling pathway and Intestinal immune network for IgA production pathway.In addition,MHC genes were enriched in many immue pathways.The significantly down-regulated genes included collagen,laminin subunit,matrix-remodeling-associated protein,myosin-binding protein,insulin-like growth factor-binding protein,G-protein coupled receptor,ergosterol biosynthetic protein and acetoacetyl-CoA synthetase,which are related to the physiological activities of tilapia including growth,movement,cell signal transduction and sterols synthesis.The full length of three MHC ? class genes OnBf,OnC2 and OnC4 were firstly identified from Oreochromis niloticus.The full-length cDNA of OnBf comprises an open reading frame(ORF)of 2055 bp encoding a polypeptide of 684 amino acids,the open reading frame of OnC2 cDNA is 2385 bp encodes a protein of 794 amino acids,and the full-length cDNA of OnC4 is comprised of an open reading frame of 5082 bp encoding a polypeptide of 1693 amino acids;OnBf,OnC2 and OnC4 had higher levels of expression in the liver,stomach and spleen than in the other tissues examined.The infection experiment showed that the Gyrodactylus cichlidarum had significant effects on tilapia after 6 days of infection,and some fish showed symptoms such as skin eruptions,swimming slowly and incomplete fins;more parasites were distributed on the fins than on the body surface and the parasite abundance of the tail fin was highest;in the process of infection,the prevalence and mean intensity showed similar dynamic pattern,firstly increased until day 10,then decreased.Quantitative real-time PCR(qPCR)analysis revealed that,after Gyrodactylus cichlidarum infection,the expression levels of MHC genes were upregulated significantly in contrast with the control group,especially MHC ? class genes,in general accordance with dynamics of prevalence and mean intensity,each showing an initial increase and subsequent decrease.These results suggested that Gyrodactylus cichlidarum infection can induce an immune response via the up-regulated expressions of MHC genes against the parasite.In this study,tilapias were divided into susceptible group,moderate group and resistant group by two successive Gyrodactylus cichlidarum infections of increasing intensity.The polymorphism of MHC II ? and MHC II ? are all very high in Oreochromis niloticus,and the polymorphism of resisitant group is higher than susceptible group;a total of 50 alleles of MHC II ? and 40 alleles of MHC II ? were identified,named as Orni-DAA*0101-5001 and Orni-DAB*1001-4901.The association analysis revealed that alleles Orni-DAA*1001,Orni-DAA*3601,OrniDAB*1401,Orni-DAB*1801,Orni-DAB*2901(P < 0.05),Orni-DAA*1601 and Orni-DAA*1801(P < 0.01)might be associated with resistance to monogenean,while OrniDAA*0701,Orni-DAA*1901,Orni-DAB*0901,Orni-DAB*0601,Orni-DAB*0701,Orni-DAB*3501(P < 0.05),Orni-DAA*0101 and Orni-DAA*0201(P < 0.01)alleles might be associated with its susceptibility.An additional independent infection experiment confirmed that the alleles Orni-DAA*0201,Orni-DAA*0701,OrniDAB*0901 and Orni-DAB*3501 were significantly associated with susceptibility(P < 0.05),while Orni-DAA*3601 and Orni-DAB*1401 highly associated with resistance(P < 0.05)to monogenean Gyrodactylus cichlidarum.The results suggested that MHC alleles could be used as markers in molecular-assisted selection of O.niloticus to enhance disease(parasite)resistance.