Research On Immune Adaptive Response Of Coilia Nasus Infected With Nematode In The Yangtze River Based On Multi-Omics Techniques

Coilia nasus is the most important species for fishing and consumption in the lower reaches of the Yangtze River,which is the only existing diadromous fish with a steady fishing season.The C.nasus resource is being diminished under the double pressures from the over-fishing and the aquatic environmental pollution.The annual catch now is only about 2% of the historical maximum.The present study systematically investigated the infection feature of nematode and its community structure.Based on simplified genome technology and Otolith fingerprint element technique,the C.nasus who have higher homogeneity in genetic background and close habitat resume were selected as study samples.Integrated transcriptome and metabolome techniques were used to study the variation of immune-related genes and metabolites after the infection of Anisakidae,and then reveal the immune signaling pathways associated with the infection of Anisakidae,and finally to investigate the immunological adaptive response after the migratory group of C.nasus infected with Anisakidae.Combined with the population ecology of C.nasus,the understanding of interaction between C.nasus and Anisakidae will be useful for future research on population recruitment of C.nasus.The main research content is as follows:Investigation on the nematode infection of Coilia nasus.Seven investigation sections were set up from the Yangtze River estuary to Poyang Lake,695 samples of C.nasus were collected during its fishing season.7271 nematodes were detected in 541 samples and the infection rate of the whole samples is 77.84%,the average infection intensity is 13.44±30.68(units),the average infection abundance is 10.46±27.36(units).A significant difference(p<0.05)is found in the average infection intensity and average infection abundance among investigation sections.1160 randomly selected nematodes were used for species identification based on molecular identification techniques,6 species belonged to 2 genera are identified as Anisakis pegreffii,A.simplex,Hysterothylacium aduncum,H.fabri,H.sinens,H.amoyense.The dominant species is A.pegreffii,which is accounted for 86.9% of the total biomass.ITS sequence analysis results show that the population diversity of A.nematophila is low,with a haplotype diversity(Hd)of 0.174,a nucleotide diversity(π)of 0.00028.There is no obvious genetic differentiation among investigation sections(Fst<0.05),and total gene flow is 75.74.Besides,sufficient gene exchange is observed among groups.Research on the genetic background of the sampling group of Coilia nasus.A total of 165 Coilia nasus samples were sequenced using 2b-RAD technology.After screening and clustering analysis,373,244 tags were obtained,with an average number of Unique tags of 289,602.The specific ratio variation of Unique tags is 73.77% to 80.25%.The filtered Enzyme Reads were matched to the reference sequence by SOAP software,and maximum likelihood method(ML)was used to carry out SNP labeling typing.A total of 36,975 SNP markers were obtained,among which 26 were highly polymorphic SNP(PIC>0.5)sites.The forms of A/G and C/T is 10013 and 12881 respectively,which are the main types in 36469 dimorphic SNP.The percentage of the two types compared to total SNP is 27.08% and 34.84%.The specific value of transition to transversion is 1.69.The observed heterozygosity(Ho)ranges from 0.1286-0.1408,while,the maximum and minimum of expected heterozygosity(He)is 0.1340 and 0.1446 respectively among four groups.In addition,the genetic differentiation coefficient(Fst)is all lower than 0.005,suggesting that there is no significantly genetic differentiation occurred among sample groups,and together with frequent communication,higher heterozygosity and multiple genetic diversity.Through this screening and verification,we can ensure that the experimental samples of C.nasus used in the study of omics have similar genetic backgrounds,thus eliminating the interference of germplasm differences on the analysis results.Study on the habitat resume of the sampling group of Coilia nasus.Otolith fingerprinting technique was used to invert the habitat resume of 81 C.nasus samples,and totally three ecological types were found.Among them,91.36% of the samples are diadromous fishes,6.17% are estuarine fishes,and 2.47% are freshwater fishes.In terms of the relationship between Anisakidaes and the habitat resume,we found that all the infected C.nasus samples were diadromous fishes.However,the results indicated that diadromous fishes were not always infected with nematode,and all of freshwater fishes were not infected with nematodes.Therefore,the screening and verification in this study can ensure the similar migratory resumes of C.nasus samples used in the omics research,so as to eliminate the interference of C.nasus samples in the results due to the differences in habitat resume.Transcriptome analysis of liver tissue in Anisakidae-infected Coilia nasus.High-throughput RNA sequencing technology was used to investigate gene expression profiling of Anisakidae infection in C.nasus.After removing low-quality and short reads,a final set of 62,604 unigenes were obtained.A total of 545 genes were significantly up-regulated and 416 were significantly down-regulated in the extracts of liver from C.nasus.The RNA-seq data is well supported by the qRT-PCR confirmation allowing it to provide the basis for the following analysis.The differentially expressed genes were annotated by GO and KEGG pathway database.Our results show activation of antigen processing and presentation,initiation of the T cell receptor signaling pathway,which indicate perturbations to the host immune system In the antigen processing and presentation signaling pathway,MHCⅡ,TCR,CTSL and CIITA were found to be interrelated.The expression of six genes(TCR,CD3 D,LCP2,ITK,IL10 and p38)were participated in T cell receptor signaling pathway.The results of this study provide support for the further understanding of the interaction mechanism between the Anisakidae and the C.nasus.Metabolome analysis of serum in Anisakida-infected Coilia nasus.We performed untargeted metabolomic analysis of serum from Anisakidae-infected C.nasus using GC-MS.A total of 65 metabolites were identified,28 metabolites were significantly up-regulated and 37 were significantly down-regulated.The multivariate statistical analysis(PLS-DA and OPLS-DA)showed that the infection of Anisakidae had a significant effect on the metabolic profiling of serum in C.nasus.The metabolites and their involved metabolic pathways were analyzed in the serum of the C.nasus.A variety of metabolites and genes involved in the regulation of related metabolites have shown changes in the amount of expression in the pathways of the Glycerolipid,Biotin,Pentose phosphate pathway,beta-Alanine,d-Glutamine and d-Glutamate metabolism,Pyrimidine,and TCA cycle.In addition,the expression of DAG was significantly increased,and it plays an important role in the critical immune pathways required for T cell activation.