| The Yangtze finless porpoise(YFP,Neophocaena asiaeorientalis)mainly inhabits the middle and lower reaches of the Yangtze River and contiguous Poyang and Dongting Lakes and tributary tailbacks in China.In recent decades,with the rapid social and economic development of the Yangtze River basin,the intensity of human activities has increased drastically,leading to the continuous deterioration of the ecological environment and the serious decline of aquatic biological resources.As an important indicator of the health of the Yangtze River ecosystem,there are only more than 1000 YFPs left and the population is critically endangered.As an aquatic mammal,Yangtze finless porpoise is constantly exposed to risks such as environmental pollutants and pathogenic microorganisms in water,and its immune system faces many challenges.However,the immune system characteristics and immune adaptive mechanisms of the YFPs are still unclear.Blood analysis is a basic means to monitor and evaluate the health and physiological status of aquatic wild animals.By analyzing the abundance of transcripts in blood,we can effectively monitor the immune levels and obtain rich immune response related genes and pathways.In this study,we collected blood samples from YFPs at different ages,sexes and habitat types,and analyzed the blood physiological and biochemical parameters.The blood transcriptome library of YFPs was constructed by RNA-Seq method,and high-throughput sequencing was carried out to reveal the expression patterns and differential expression level of blood genes,and to clarify the alterations of immune adaptive processes dominated by differentially expressed genes.In addition,the Yangtze finless porpoise has a unique evolutionary history,with its ancestors returning from land to sea and then from sea to freshwater.In this process,whether the complex aquatic environment drives the evolution and adaptation of immune genes.In this study,we used the complement gene family as an entry point,combined with bioinformatics analysis,to explore the evolutionary model of the complement gene family from the sequence level,with the aim of providing basic information for a comprehensive understanding of the immune system of Yangtze finless porpoise and its adaptations in diverse environments.The main findings of this paper are as follows.1.Forty-six samples of healthy YFPs were collected from the natural and ex-situ waters of the lower Yangtze River in Anhui Province.After grouping according to age,sex and habitat,the blood routine,blood biochemistry and serum immunological indices of YFPs were analyzed.In terms of age,the values of RBC,HGB and ALP in adult and old groups were significantly lower than those in young group(P < 0.05);The values of LYMPH and NEUT in the old group were significantly lower than those in the young group(P < 0.05),while the values of TBIL and ALB were significantly higher than those in the young group(P < 0.05);The EOS value of adult group was significantly higher than that of old group(P < 0.05).In terms of sex,male YFPs had significantly higher values of WBC,NEUT and ALB indicators(P< 0.05),while females had significantly higher values of MCH indicators(P < 0.05).In terms of habitat,LYMPH,GLOB,GGT,UA,TG,TNF-α and IL-6 were significantly higher in natural waters(P < 0.05),while RBC,HGB,HCT,MCHC and CO2 were significantly higher in ex-situ waters(P < 0.05).These findings suggest that the blood routine,blood biochemistry and serum immunological indices of YFP are affected by age,sex and habitat.2.Nine blood tissues of YFPs at different ages(three each from young,adult and old)were sequenced and analyzed using the BGISEQ-500 sequencing platform.A total of 623 million clean reads were obtained from 9 samples and further assembled into15904 unigenes,accounting for 72.2% of the total annotated genes in the genome.There were 74 up-regulated genes and 821 down regulated genes in the old vs adult comparison.The expression of genes related to innate and adaptive immune system was higher in the adult group,including complement components,major histocompatibility complexes,interleukins,TNF receptors,chemokines and cytokines.There were 135 up-regulated genes and 879 down regulated genes in the old vs young comparison.The expression of genes related to the innate immune system was higher in the young group,and the toll like receptor signaling pathway was significantly enriched.In the adult vs young comparison,there were 195 up-regulated genes and 220 down regulated genes.The expression of genes related to adaptive immunity and Fox O signaling pathway was higher in the adult group,while the expression of genes related to PI3K-Akt signaling pathway was lower.A total of 478 conserved miRNAs and 576 predicted miRNAs were detected by miRNA screening.A total of 545 differentially expressed miRNAs(DEMs)were obtained by differential expression analysis.In the comparison group of old vs adult,old vs young and adult vs young,81,94 and 122 DEMs were up-regulated and 92,80 and 76 DEMs were down regulated,respectively.Based on the combined analysis of DEGs and DEMs,the negative regulatory relationships of 326,316 and 211 on miRNA-mRNA were predicted in the comparison groups of old vs adult,old vs young and adult vs young respectively.This study showed that the innate immunity of YFP has obvious advantages in the early stage,while the adaptive immunity increases first and then declines with age,and the changes of miRNAs profile are related to the effect of age on immune function.3.mRNA and micro RNA sequencing were performed on blood samples from 3female and 3 male YFPs.A total of 474 million clean reads were obtained from six samples and further assembled into 15878 unigenes,accounting for 72.1% of the total annotated genes in the genome.KEGG analysis revealed that 1534 unigenes were annotated to an immune system related category and significantly enriched in 20 common immune pathways.A total of 539 differentially expressed genes(DEGs)were identified in male and female blood transcriptome,of which 299 were female preferred genes and240 were male preferred genes.GO and KEGG enrichment analysis revealed that female blood genes were significantly related to energy metabolism,while male blood genes were significantly related to cell growth.In addition,pathway enrichment analysis also revealed that Fox O and Hippo immune-related signaling pathways were activated in the blood of females.A total of 457 conserved miRNAs and 528 predicted miRNAs were detected by miRNA screening.A total of 160 differentially expressed miRNAs(DEMs)were obtained by differential expression analysis,of which 97 DEMs were up-regulated in female YFPs and 63 DEMs were up-regulated in male YFPs.Based on the combined analysis of DEGs and DEMs,45 negative miRNA-mRNA regulatory relationships were predicted,including 13 DEMs and 37 immune-related DEGs.This study suggested that female porpoises may have stronger immunity and the ability to maintain homeostasis,and micro RNAs can participate in the amphoteric immune system of porpoises by regulating gene expression.4.mRNA and micro RNA sequencing were performed on blood samples of 3collected from natural waters and 3 from ex-situ waters.A total of 424 million clean reads were obtained from six samples,and further assembled into 15878 unigenes,accounting for 71.6% of the total genome annotated genes.KEGG analysis revealed 1527 unigenes were annotated to an immune system related category and 103 important immune-related gene were identified.1051 DEGs were identified in the two habitats,of which 745 were up-regulated in natural waters and 306 were up-regulated in ex-situ waters.Forty DEGs were enriched in immune pathways,and most of them were up-regulated.Pathway enrichment analysis found that DEGs were significantly enriched in 7 immune-related pathways,among which the complement and coagulation cascade pathway was the most enriched.A total of 468 conserved miRNAs and 576 predicted miRNAs were detected by miRNA screening.A total of 189 differentially expressed miRNAs(DEMs)were obtained by differential expression analysis,of which 75 DEMs were up-regulated in natural waters and 114 DEMs were up-regulated in ex-situ waters.Based on the combined analysis of DEGs and DEMs,46 negative miRNA-mRNA regulatory relationships were predicted,including 18 DEMs and 43 immune-related DEGs.This study suggested that porpoises living in natural waters may have stronger immunity and micro RNAs may be involved in the immune adaptation of porpoises to different habitats by regulating gene expression.5.A total of 52 complement genes were identified from the genome of Yangtze finless porpoise,including 8 pattern recognitions,15 proteases,12 complement components,6 receptors and 11 regulators.Many complement genes evolved into multiple copies,and the location of these genes on the chromosomes suggests that tandem replication may account for the large number of gene copies.Complement genes are randomly distributed on 20 chromosome segments,with intron numbers ranging from 1to 41.Phylogenetic analysis of 18 representative mammals showed that cetaceans and other mammals were clustered into one branch,and YFPs and cetaceans were clustered into one branch,which were consistent with the status of species evolution.In addition,all complement genes are clustered into one branch with homologous genes,indicating that they are highly conserved in the process of evolution.The site model analysis revealed that C3,C4,C5,C6,C8,C9 and CR2 were positively selected,suggesting that adaptive evolution of complement genes occurred in mammalian evolution.In addition,branch model and branch-site model analysis revealed that C4,C7 and C9 were positively selected on the evolutionary branch of YFP,suggesting that the complement gene family of Yangtze finless porpoise has undergone adaptive evolution from terrestrial to aquatic.In this study,we explored the physiological and biochemical level and gene expression regulation mechanism of YFPs from three dimensions of age,sex and habitat through blood physiological and biochemical indexes and blood transcriptome,so as to provide a resource basis for a comprehensive understanding of the immune system of YFPs.On the other hand,a molecular evolutionary analysis of the important innate immune gene family Complement was conducted to lay the foundation for analyzing the evolutionary pattern of immune adaptations.This study provides a new perspective from the perspective of immunity to interpret the changes of physiological processes under the influence of different age and sex,and the adaptability of YFPs to diverse habitat.It also provides a reference for the protection of Yangtze finless porpoise. |
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