| Amphibians worldwide are currently experiencing severe population declines and extinctions attributable to habitat alteration,climate change,invasive species,chemical contaminants,and especially infectious diseases.Captive breeding and reintroduction are important methods for amphibian conservation,but the prevalence of disease and the low survival rate of captivity have become barriers to conservation efforts.Brown frog culture can protect wildlife conservation and provide frog products,but the development and benefit of brown frog culture industry is very poor,and the high incidence of diseases is the main problem of brown frog farming.Resident microbiota plays an important role in maintaining of host health,mitigation of diseases,improvement of captivity conditions and success rate of re-introductions,and management of invasive species.To understand the formation mechanism of gut microbiota in amphibians,and to analyze the composition and function of amphibian gut microbiota,and its response to the environment became particularly important for the protection and breeding of amphibians.To date,however,there has been very little research on host genetics,habitat,seasonal variations,food composition and captive factors on amphibian gut microbiota.There is a lack of comprehensive,systematic research specifically targeting amphibian gut microbiota.In this study,R.amurensis and R.dybowskii were selected,combined with the research techniques and research methods of modern molecular biology,bioinformatics,microecology and aquaculture,to study the effects of individual development,host genetics,habitat,seasonal variations,captive,feeding stage,health status and antibiotic intervention on the gut microbiota of brown frogs.To study the changes of gut microbiota of brown frogs in natural overwintering pool during hibernation,and to study the time dynamics of the similarity of gut microbiota of R.amurensis and R.dybowskii during hibernation under experimental wintering conditions;to study the changes of gut microbiota similarity of R.amurensis and R.dybowskii during hibernation;to compare and analyze the changes of similarity between the gut microbiota and skin microbiota of R.dybowskii in different seasons.The purpose of this study was to reveal the interaction between microbial communities,hosts and environmental factors,to analyze the dynamic development and stability of amphibian gut microbiota,and to explore the mechanism of amphibian health maintenance and adaptive evolution.The main research results of this thesis are as follows:(1)The composition of the intestinal microbial community of R.dybowskii varies across development.In this study,we sampled the gut bacterial communities during R.dubowskii development,including tadpole,metamorphosis,subadults(just metamorphosed,frogs in summer),adults(frogs in summer,autumn,and hibernation).We investigated whether the gut microbiota structure differs according to host developmental stage and whether the gut microbiota composition and alpha diversity are related to food supply or the ambient temperature via high-throughput Illumina sequencing.The results show that the structure of the gut microbiota significantly varied during different developmental stages.The LEf Se analysis identified eight significantly differentiated phyla: Cyanobacteria(T group),Proteobacteria(M group),Fusobacteria(F group),Firmicutes(As group),Actinobacteria(Aa group),Verrucomicrobia(Aa group),Tenericutes(Aa group),and Bacteroidetes(Ah group).Across all frog developmental stages,10 OTUs were present in more than 90% of the samples.Food supply and body weight were significantly correlated with the Sobs,ACE,Chao and PD index values,and canonical correspondence analysis of the tested samples showed that environmental factors(body weight,food supply and temperature)had significant effects on the frog gut microbial composition.Our results indicate that R.dubowskii gut microbiota assemble into distinct communities throughout development,and that th ese communities are closely associated with variations in environmental factors.(2)Similarity time variations and changes in the similarity of two Rana species during hibernation.The gut microbiota in the early and late hibernation of R.amurensis and R.dybowskiis was compared under the condition of natural overwintering,and the results showed that there were significant differences in gut microbiota composition between R.amurensis and R.dybowskii in the early stage and final stage of hibernation.The samples of R.amurensis and R.dybowskii were taken in October(early stage),December(middle stage),February(late stage)and April(final stage)under the experimental wintering conditions to compare the similarity time variations and the similarity of gut microbiota of two hosts during hibernation.The similarity time variations showed that the gut microbiota composition was significantly different between the early and middle,middle and late stages of R.amurensis and R.dybowski,however,the difference between late and final stage was not significant.The similarity of two hosts showed that the gut microbiota composition of R.amurensis and R.dybowskii was significantly different in the early and middle stage,and the difference of gut microbiota composition of R.amurensis and R.dybowskii was not significant in late stage and final stage.(3)Seasonal hibernation effects on the similarities between the skin microbiota and gut microbiota of R.dybowskii.In this study,the gut and skin microbiota of R.dybowskii in summer and winter were investigated via high-throughput Illumina sequencing.The results indicate that the alpha diversity of the microbiota in the winter gut group(GW)and the winter cutaneous group(CW)was lower than that of the summer gut group(GS)and the summer cutaneous group(CS).In both summer and winter,the microbial composition differed significantly between the gut and skin.Seasonal change and water-land habitat change affect the skin and gut microbiotas of R.dybowskii.The pairwise distances between the intestinal and skin microbiotas were greater in winter than in summer.The similarities between the gut and skin microbiotas of R.dybowskii differed between summer and winter.(4)Effects of host and habitat on gut microbiota of brown frogs.In this study,the differences of gut microbiota of two hosts(R.amurensis and R.dybowskii)under two habitats(stream and static water)were compared,and the results showed that the analysis of gut microbiota was influenced by host and habitat type based on Bray-Curtis and unweight Uni Frac distance,and their interaction did not have no effect.The analysis based on weight Uni Frac distance shows that gut microbiota structure is influenced by host species,while habitat type and interaction have no effect.Through ANOSIM analysis and Adonis analysis based on Bray-Curtis distance and unweight Uni Frac distance,the differences of gut microbiota composition of different habitats and frogs were compared,and the results were only based on Bray-Curtis distance,the difference of gut microbiota composition between R.dybowskii and R.amurensis(am L vs.dy L)under the condition of static water was not significant.(5)Effects of captive and growth stages on the gut microbiota of R.dybowskii.We performed a comprehensive analysis using high-throughput sequencing technology and functional predictions and observed general changes in the gut microbiota of frogs in different growth stages and growth environments.There were no significant differences in microbial richness and diversity between the different growth stages of captive frogs,or between wild and captive frogs.There were significant differences in the gut microbiota community structure of R.dybowskii in different growth stages of captive frogs and between wild and captive R.dybowskii.The relative abundance of several red-legsyndrome-related pathogens increased significantly in captive frogs compared with that in wild frogs.To our knowledge,this is the first study of the effects of individual seasons and captivity on the gut microbiota of frogs.(6)Effects of short-term antibiotic exposure on gut microbiota of R.dybowskii.Samples of gentamicin bath for 7 days,samples of 7 days of withdrawal after medicine bath,and samples of control group were compared with each other.The results showed that gentamicin bath significantly changed the alpha diversity of gut microbiota,changed the composition of gut microbiota,and the gut microbiota was still similar to the gut microbiota of gentamicin bath group after 7 days of recovery.Gentamicin baths significantly reduced the relative abundance of red-leg-syndromerelated pathogens.(7)Comparison of gut microbiota of diarrhea R.dybowskii and healthy R.dybowskii.Comparing diarrhea with healthy R.dybowskii samples,the results showed that the alpha diversity of diarrhea brown frog was significantly higher than that of healthy R.dybowskii,and the gut microbiota composition of diarrhea R.dybowskii and healthy R.dybowskii was significantly different.Lef Se analysis showed that Actinobacteria were enriched in the diarrhea group and Bacteroidetes in the health group.In summary,host development,host genetic and habitat affect the gut microbiota of brown frog.The culture,culture stage,antibiotic bath and diarrhea affect the gut microbiota of brown frog.The Similarity time variations and the similarity of two hosts were not similar to similar during hibernation.The composition of skin and gut microbiota of brown frog in winter and summer was significantly different.In winter and summer,there was a significant difference in the similarity between skin and gut microbiota of R.dybowskii.This study can not only evaluate the health status of R.dybowskii and R.amurensis,but also provide important suggestions for culture brown frogs and the protection of wild populations. |
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