| The intestinal flora plays an important role in vertebrate growth and development,immune response and other life activities.Both endogenous and exogenous factors affect intestinal flora community composition and diversity.Endogenous factors include age,genetics,sex,etc,and exogenous factors include environment,food,etc.With the rapid development of modern molecular biology techniques,the study of bird gut flora is increasing,but compared with the diversity of birds（more than 10,000species）,research on the diversity of wild bird gut flora and the affecting factors are still relatively limited.In this paper,yellow-rumped flycatcher（Ficedula zanthopygia）nestlings was used as research objects,and non-invasive technique was used to collect fresh feces and nest material samples from chicks（3,6,9 and 12 days old）and adults,and PCR amplification and high-throughput sequencing of the V3-V4 high variation region of bacterial 16S r RNA was performed in all samples to clarify the composition of the intestinal flora of F.zanthopygia by analyzing the structure and function of microbial communities.I investigated the effects of age,sex,nest environment and parents on the intestinal flora composition and diversity of nestlings.I also investigated the effects of age,sex,nesting environment and parents on the composition and diversity of the intestinal flora of the nestlings to elucidate the diversity,dynamics and potential functions of the intestinal flora of the nestlings.The main results were as follows:（1）The dominant intestinal flora of F.zanthopygia were Firmicutes（relative abundance:34.64-48.69%）,Proteobacteria（24.04-47.45%）and Actinobacteria（22.52-29.08%）.There was no significant difference between the dominant phylum in nestlings and adults（Kruskal-Wallis test:P>0.05）,individual genera had significant differences before different ages,for example,Rhodococcus spp.were significantly enriched in 3-day-old chicks.Alpha diversity PD index（Kruskal-Wallis test:H=15.764,P<0.01）,ACE index（Kruskal-Wallis test:H=40.547,P<0.001）and Chao1 index（Kruskal-Wallis test:H=38.371,P<0.001）of F.zanthopygia were highly significantly different at different day old stages,and there were highly significantly differences in the structure of the intestinal flora（Adonis:P<0.001）.（2）No significant difference in gut fiora Alpha diversity between male and female（Female:16,Male:19）of F.zanthopygia chicks（Kruskal-Wallis test:P>0.05）,Beta diversity was also not significantly different（Adonis:P>0.05）.And the LEFSe analysis revealed that the intestinal flora of male and female nestlings differed at the genus level in different day-old stages,with a significantly higher abundance of Ureaplasma spp.in 3-day-old males（LDA>4.5,P<0.05）,and in 12-day-old females the abundance of Lactococcus spp.was significantly higher（LDA>4.0,P<0.05）in3-day-old males and 12-day-old females.（3）There was no significant difference in the diversity of Alpha intestinal flora among the nestlings（Kruskal-Wallis test:P>0.05）,indicating that the nestlings from different nests had similar diversity and abundance.However,principal coordinate analysis showed that the intestinal flora structure was significantly different between nests at 3 days of age（Adonis:P<0.01）and very significantly different between nests at 6,9 and 12 days of age（Adonis:P<0.001）,and the intestinal flora of chicks from the same nest was more similar than that of chicks from different nests.（4）The common flora of nestling and nest material ectomycorrhizal bacteria was greater than that of adult intestinal flora,and the number of common flora increased as the nestlings lived longer in the nest.Among the four day-old stages of nestlings,the Shannon index（Kruskal-Wallis test:H=14.1,P<0.01）,Chao1 index（Kruskal-Wallis test:H=13.492,P<0.01）and ACE index（Kruskal-Wallis test:H=13.789,P<0.01）of intestinal flora were significantly differed from nesting material only at 3 days of age.The differences in the flora structure of intestinal flora and ectomycorrhizal bacteria in chicks and adults were highly significant（Adonis,P<0.001）.（5）The difference in intestinal flora diversity was mainly influenced by factors of nest environment and adults together at 10.21%(PERMANOVA:Pseudo-F1,83=2.3705,R2=0.1021),followed by the day-age factor at 5.6%(Pseudo-F1,83=1.9668,R2=0.0562)and the sex factor at 0.91%(Pseudo-F1,83=1.1949,R2=0.0091)to the least extent.（6）Functional predictions showed that the most abundant functional groups in F.zanthopygia were carbohydrate metabolism,amino acid transport,membrane transport,energy metabolism and metabolism of cofactors and vitamins.The secondary metabolic pathways of the chick’s intestinal flora were significantly different at each day of age,for example,the abundance of membrane transport and signal transduction pathways were significantly higher at 6 days of age than at other.The abundance of membrane transport and signal transduction pathways was significantly higher at 6 days of age than at other stages（LDA>3,P<0.05）.This study clarified the community composition of the intestinal flora of F.zanthopygia,and confirmed that the intestinal flora of the nestlings had high diversity and richness,and the intestinal flora diversity of the nestlings differed significantly among different day-old stages,and there was no significant difference in the intestinal flora of the nestlings between different sexes,and the nest environment and the adults had a greater influence on the intestinal flora diversity.In addition,F.zanthopygia showed differences in functional abundance at different day-old stages.This study enriches the study of intestinal flora diversity and its influencing factors in wild birds,and deepens the understanding of symbiotic mechanisms in wild birds and their intestinal flora. |
