| Bats(Chiroptera)are hosts of various pathogens and major epidemic sources.With the human exploiting,their natural habitats are losing,the contacting between them and human are increasing,their pathogens are spreading and transferring to others.Therefore,the diversity of pathogenic microorganisms in bat habitats urgently needs to be explored.Meanwhile,ecological environment changes also caused by the loss of natural habitat,excessive use of fossil fuels and various wildlife epidemics also affect the fitness characteristics related to immune resistance in the host,bringing certain selection pressure to the survival of bats.Therefore,it is urgent and important to study the impact of pathogen diversity on bat immunerelated genes and adaptive evolution.The greater horseshoe bat(Rhinolophus ferrumequinum)is widely found in heterogeneous landscapes,which makes them as the natural reservoir for a variety of pathogens.R.ferrumequinum is also highly resistant to Pseudogymnoascus destructans(Pd),which is the pathogenic fungus of white-nose syndrome,suggesting a potential relationship between autoimmunity and pathogens.The skin microorganisms of R.ferrumequinum are affected by environmental microorganisms,which suggested the interaction between R.ferrumequinum and its ecological environment habitat.So far,the relationship between immune genes and various pathogens and their evolutionary driving forces remains unclear.Therefore,the major histocompatibility complex(MHC),a highly polymorphic adaptive immune gene,was selected to study the animal adaptive evolution and host-pathogen interaction in R.ferrumequinum.Thus,we integrated data from multiple pathogens(the environmental virome of the bat habitat,the16 S r RNA environmental bacteria,and Pd load of R.ferrumequinum)as well as microsatellite and climate factors to study the influence of multi-pathogen stress on MHC genes and their adaptive evolution mechanism.Firstly,in order to study the diversity of pathogens in bat habitats,viral metagemomics sequencing technology was used to reveal the composition,abundance and spatial changes of soil viruses in bat-dwelling caves habitat.In addition,phage host prediction and 16 S r RNA environmental bacterial data were combined to explore the interactions between viruses and bacteria.The results showed a total of 31 families of viruses were obtained,and alpha diversity,beta diversity and relative abundance of soil virus family were all significant differences between 6 different bat cave-dwelling habitats,indicating the habitat specificity of soil viruses in the bat cave-dewling habitats.It was found that the composition of bats’ habitat viruses was44-63% similar to the virus family of R.ferrumequinum in previous study.Zoonotic risk viruses of 6 family and 48 species were also found in bats’ habitat.In addition,1-14 predicted hosts were found for each viral contig,and there was a significant correlation between viruses and bacteria in alpha diversity and relative species abundance at the family level,indicating the interaction between viruses and bacteria.Secondly,to investigate the effects of environmental viruses,bacteria and climate on MHC class Ⅰ polymorphism of R.ferrumequinum,the study found a significant negative correlation between virus and bacteria alpha diversity and precipitation in the wettest month.There was also a significant correlation between MHC class I allele frequency and the relative abundance of four virus families and ten bacterial families.Structural equation modeling analysis further revealed causal relationships,indicating that precipitation in the coldest season significantly negatively affected virus diversity,which in turn significantly negatively affected the frequency of the MHC class I allele(Hap＿7)in the R.ferrumequinum.The frequency of the MHC class I allele was also negatively affected by bacterial alpha diversity,but not by interactions between viruses and bacteria.The results of the study suggest that the MHC class I gene of the R.ferrumequinum is driven by environmental pathogen selection pressure,and that environmental climate affects host immune gene polymorphism by regulating pathogen diversity.Then,combined with MHC class Ⅱ genes that mainly deal with bacterial and fungal pathogens,the effects of habitat bacteria and Pd load on MHC class Ⅱ genes were investigated.The results showed that the alpha diversity of environmental bacteria was negatively correlated with the MHC class Ⅱ polymorphism at the population level,and was significantly negatively correlated with the family species abundance of potential pathogenic bacteria of bats,suggesting that the MHC class Ⅱ gene may be resistant to the infection of environmental bacteria;The results of the generalized linear mixing effect model analysis showed that the Pd load and infection status were affected by the MHC class Ⅱ supertype(ST3)and the interaction between ST3 and sex(male).These results indicated that MHC class Ⅱ gene had the ability to resist Pd and also indicated that individual immune genetic makeup may be related to the life history strategies and reproductive tradeoffs of male R.ferrumequinum.It was also found that there was a negative correlation between MHC class Ⅱ polymorphism and the Pd prevalence,and the MHC class Ⅱ polymorphism was only affected by the temperature of bat habitat,but not related to the Pd load and infection status.This not only further indicated that MHC classⅡ gene could resist Pd infection,but also indicated that the diversity of MHC class Ⅱ was affected by environmental temperature.Finally,in addition to the selection pressure of pathogens,MHC polymorphism are also affected by demographic processes and local adaptation caused by heterogeneity of ecological environment.Therefore,we combined neutral microsatellite loci,MHC class Ⅱ locus,and climatic factors to unravel the mechanisms affecting the diversity and genetic differentiation of MHC genes in R.ferrumequinum.The results showed that 71% of the MHC genetic differentiation among populations was higher than the microsatellite genetic distance,suggesting that the MHC class Ⅱ gene was driven by the diversifying selection.Although significant correlation between MHC genetic distance and microsatellite genetic distance was found,indicating that random effects on MHC class Ⅱ genes.However,the partial mantel test showed that there was still a significant correlation between the genetic differentiation of MHC and geographical distance when controlling the genetic distance of neutral markers,indicating that the diversifying selection played a major role in MHC class Ⅱ genes.In addition,the results of multiple regression analysis showed that MHC diversity was significantly correlated with temperature and precipitation.The multinomial logarithm model further showed that MHC polymorphism was affected by BIO2,BIO3 and BIO15.The phylogenetic generalized least squares model and partial mantel test were used to rule out the phylogeographic structure of R.ferrumequinum.The results were consistent with the results of the multicomponent logarithm model,indicating that MHC class Ⅱ genes are affected by local adaptation driven by environmental climate.In conclusion,this paper elucidates the effects of multiple pathogen pressures and climate on MHC gene polymorphism in natural populations,and provides case support for understanding the adaptive evolution of immune gene MHC in non-model animal groups in natural populations,and provides a new perspective for studying host-pathogen coevolution.In addition.It also revealed the diversity of viruses in bat habitat,which provided warning significance for protecting bat habitat from human infestation and preventing zoonotic diseases. |
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