Adaptation Of Gut Microbes To Seasonal Changes Of Diet In Farmland Frogs(Fejervarya Multistriata)

Ecological adaptation is a process where organisms vary their morphology,structure and physiological and biochemical characteristics to adapt to the environmental fluctuation based on the process of long-term natural selection.This process is ubiquitous in kingdom.When the environment changes ocurr,animals adopt corresponding strategies to adapt to the fluctuating environment to ensure their survival and reproduction.For example,animals adapt to new dietary niches by adopting strategiesby altering the composition of their gut microbes.The research on the adaptive mechanism of intestinal microbes may provide scientific basis for animal protection.However,in the study of animal adaptive evolution or the evaluation of the relationship between microorganisms and host adaptability,amphibians are a good species to study evolution,as they are amphibious and sensitive to the environment.It is of great significance to study the adaptive strategy mechanism of amphibians in the fluctuating environment with extreme weather increasing,human activities increasing and amphibians decreasing.Therefore,Fejervarya limnocharis,in this article,was selected as the research object for their character of widely distribution and toleration to human disturbance.We collected 170 F.limnocharis of different seasons(spring,summer,autumn and pre-hibernation)and sampled stomach and intestinal contents,combining with stomach content analysis,16 S rRNA high-throughput sequencing on the IonS5 TMXL plateform,bioinformatics technology and Tax4 Fun function prediction analysis,to study the seasonal dietary changes of F.limnocharis;to study the seasonal varities of intestinal flora structure and diversity in F.limnocharis;and to analyze the correlation between seasonal feeding habits and intestinal microorganisms of F.limnocharis.Here,we aimed to explore,in terms of anuran intestinal microbes,the adaptive mechanism in response to environmental changes.The results are as follows:According to the analysis of stomach contents,F.limnocharis mainly preyed on coleoptera,araneae,isopoda,centipedes and diptera,but showed different feeding habits in different seasons and sexes.For instance,niche width(B): in autumn > pre-hibernation > spring > summer;in spring,male frog > female frog;in summer,male frog > female frog;and in autumn,the males < the females.Food diversity(H): autumn > spring > pre-hibernation = summer;spring,male frog < female frog;summer,male frog > female frog;autumn,male frog < female frog.However,Anosim analysis expressed that the intramural differences in different seasons and between males and females in summer were smaller than the inter-group differences.Kruskal-wallis and wilcoxon rank-sum test found that there was no significant difference in food contents,niche width and food diversity.A total of 12842042 high-quality sequences and 18874 OTUs were obtained in this experiment.These sequences could be identified to 2 boundaries,71 phylum,83 classes,187 orders,381 families and 1301 genera.The microflora is mainly distributed in Proteobacteria,Firmicutes and Bacteroidetes.While at genus level,Stenotrophomonas,Faecalitalea and Parabacteroides were the dominant flora.In F.limnocharis of different seasons,there were 3,969 core OTUs mainly from Proteobacteria,Firmicutes,Actinobacteria,Bacteroidetes and Planctomycetes.In addition,there were 671 special OTUs in spring,5050 in summer,1198 in autumn and 1189 Pre-hibernation.Interesting,in summer,both the number of unique OTUs and the total OTUs are the most abundant.Wilcox rank-sum test and T test revealed the differences of intestinal microbial structure and diversity in different groups were significant.In the phylum level,Proteobacteria dominated in the spring,while Firmicutes dominated in the summer,autumn and pre-hibernation.In terms of genus level,in spring samples,Stenotrophomonas was the absolute dominant bacteria,while in the summer,autumn and pre-hibernation groups,the majority was unidentified Clostridiales.The composition and diversity of intestinal microorganisms between males and females of F.multistriata in different seasons were different,but the difference was not significant.Anosim analysis revealed that only in autumn the intra-group differences between males and females were smaller than the inter-group differences(r>0).The t-test of intestinal microbial composition of male and female frogs in autumn revealed significant differences in 1 order,5 families,3 genera and 1 species.The results of Tax4 Fun function prediction analysis showed that there were significant differences in function among seasons,and mainly in metabolic function.The intestinal microbe of F.limnocharis had certain chitin-degrading ability and resistance ability.The abundance of chitin-degrading enzyme and antibiotic gene was the highest in pre-hibernation.The functions of male and female frogs in autumn revealed that both male and female frogs were mainly metabolized,and there were significant differences in Nucleotide metabolism.The total abundance of chitinase and antibiotic genes in female frogs was higher than that in male frogs,among which the abundance of Putative chitinase and Bifunction chitinase/lysozyme genes,Vancomycin resistance,Tetracycline biosynthesis and Clavulanic acid biosynthesis genes were significantly different.UniFrac PCoA combined with Procrustes analysis and Monte Carlo test showed that food type was significantly correlated with intestinal microbial structure.To sum up,the study showed that the dietary difference of F.limnocharis was not significant both between different seasons and between male and female.The composition,diversity and function of intestinal microorganisms in different seasons were significantly different.There was no significant difference in the microbial diversity between male and female frogs,but in composition and function.Gut microbiomes of F.limnocharis fluctuate seasonally responds to food.Furthermore,the significant relationship between the composition and function of intestinal microbiota and the seasonal variation of food showed that the composition and function of intestinal microorganisms in F.limnocharis changed with seasonal food.