Microbial community composition differences between freshly dissected gut regions and cloacal swabs
Using amplicon sequencing of the 16S rRNA gene, we acquired 1,055,295 (mean ± SE: 17,300 ± 2,798) sequences from freshly dissected gut regions and cloacal swabs, which classified into 2,189 operational taxonomic units (OTUs) at the 97% similarity level (Additional file 1: Table S1). Bacterial OTU richness (One-way ANOVA: F6,54 = 2.556, p = 0.029: Fig. 1a) and Chao1 richness estimates (One-way ANOVA: F6,54 = 2.445, p = 0.0365; Fig. 1b) only differed significantly between cloacal swabs and the cloacal region (Fig. 1a, b). Shannon’s diversity index (One-way ANOVA: F6,54 = 1.059, p = 0.398; Fig. 1c) and Simpson’s inverse diversity index (One-way ANOVA: F6,54 = 1.376, p = 0.241; Fig. 1d) did not differ between gut sections and cloacal swabs. Overall, 84.1% of the total number of OTUs belonged to the phylum Firmicutes, while Proteobacteria accounted for 7.5%, Bacteroidetes 3.8% and Actinobacteria 2.3%. There was a relative decrease of Proteobacteria in the midgut sections (middle of small intestine: 2.2%, ileum: 2.5%, large intestine: 4.8%) compared to the stomach (9.1%), the cloaca (15.2%) and the cloacal swabs (32.6%). Compared to the different digestive tract regions, cloacal swabs represented the diversity of the bacterial communities in the whole digestive tract at the phylum level (Fig. 2a). The twenty-five most common OTUs accounted for 89.5% ± 1.3% (mean ± SE) of the sequences, and cloacal swabs qualitatively represented these major bacterial OTUs from the different digestive tract regions (Fig. 2b).
We found a significant difference in bacterial community composition between gut sections (including cloacal swabs) [PERMANOVA999permutations (Bray-Curtis): F6,54 = 1.82, R2 = 0.1682, p = 0.001]. Community composition did not differ significantly between digestive tract regions; however, cloacal swab bacterial communities differed significantly from four out of six gut regions (Table 1). These community-level differences remained present even after merging results from different gut compartments into three major gut regions (the stomach, the midgut [the small intestine and the cecum], and the hindgut [the large intestine and the cloaca]; additional file 2: Table S2 and Fig. 3). To qualitatively compare the bacterial communities, we conducted PERMANOVA analyses using the Jaccard distance matrix (based on presence/absence data). Even though the analyses revealed significant qualitative differences in microbial communities among gut sections (including cloacal swabs) [PERMANOVA999permutations (Jaccard): F6,54 = 1.44, R2 = 0.1383, p = 0.001], the pairwiseAdonis showed that swabs differed significantly from the large intestine and the ilium (additional file 2: Table S3).
Table 1. The results of pairwiseAdonis analyses (Bray-Curtis distances) between gut microbial communities of different regions of the digestive tract, including cloacal swabs.
Comparisons
|
F
|
R2
|
P adjusted
|
Swabs vs. Cloaca
|
3.576
|
0.1517
|
0.0420*
|
Swabs vs. Large Intestine
|
3.809
|
0.1599
|
0.0210*
|
Swabs vs. Ilium
|
4.164
|
0.1723
|
0.0210*
|
Swabs vs. Middle of Small Intestine
|
2.182
|
0.1137
|
0.0630
|
Swabs vs. Beginning of Small intestine
|
1.779
|
0.1128
|
0.3990
|
Swabs vs. Stomach
|
2.425
|
0.1187
|
0.0210*
|
Cloaca vs. Large Intestine
|
0.6861
|
0.0367
|
1
|
Cloaca vs. Ilium
|
1.042
|
0.0547
|
1
|
Cloaca vs. Middle of Small Intestine
|
1.569
|
0.0946
|
1
|
Cloaca vs. Beginning of Small intestine
|
2.024
|
0.1443
|
0.5880
|
Cloaca vs. Stomach
|
1.783
|
0.1003
|
0.9240
|
Large Intestine vs. Ilium
|
0.5702
|
0.0307
|
1
|
Large Intestine vs. Middle of Small Intestine
|
1.421
|
0.0865
|
1
|
Large Intestine vs. Beginning of Small intestine
|
1.720
|
0.1254
|
1
|
Large Intestine vs. Stomach
|
1.440
|
0.0826
|
1
|
Ilium vs. Middle of Small Intestine
|
0.9653
|
0.0605
|
1
|
Ilium vs. Beginning of Small intestine
|
1.720
|
0.1254
|
0.5880
|
Ilium vs. Stomach
|
1.391
|
0.0799
|
0.9240
|
Middle of Small Intestine vs. Beginning of Small intestine
|
0.8934
|
0.0903
|
1
|
Middle of Small Intestine vs. Stomach
|
0.6036
|
0.0444
|
1
|
Beginning of Small intestine vs. Stomach
|
0.8747
|
0.0804
|
1
|
Microbial community composition differences between fresh and alcohol specimens
We generated 1,904,813 16S rRNA amplicon sequences (mean ± SE: 29,048 ± 3,057) from the gut sections of bird alcohol specimens, which classified into 2,933 OTUs (Additional file 1: Table S1). Similar to the fresh samples, microbial communities of alcohol specimens were dominated by Firmicutes (88.0%) followed by Proteobacteria (6.16%). OTU richness, Chao1 richness estimate, Shannon’s and inverse Simpson’s diversity indices did not differ in any of the six gut regions between freshly directed samples, and both two-weeks and two-months old alcohol specimens (Fig. 4 and Additional file 2: Table S4). Overall, phylum-level relative abundances of Bacteroidetes and Actinobacteria decreased in multiple gut sections between fresh and two months old alcohol specimens (Fig. 5a). Despite the high individual variation in gut microbiomes, the 15 most common bacterial genera per gut section were present in both fresh and alcohol specimens (Fig. 5b). The bacterial community compositions of different gut regions did not differ significantly between fresh and alcohol specimens, except between the fresh and two-month old (alcohol specimen) ileal microbiota (Table 2 and Additional file 3: Fig. S1).
Table 2. Results of Adonis (bold) and pairwiseAdonis (Bray-Curtis distances) analyses of gut microbial communities of digestive tract regions of fresh (0D), two-weeks old (2W), and two-months (2M) old alcohol specimen. * indicates significant differences.
|
Comparison
|
F
|
R2
|
P adjust
|
Cloaca (df2,18)
|
|
1.144
|
0.1251
|
0.268
|
Pair-wise Adonis
|
0D vs. 2W
|
1.035
|
0.0794
|
1
|
2W vs. 2M
|
1.173
|
0.1435
|
0.720
|
0D vs. 2M
|
1.479
|
0.1185
|
0.477
|
Large Intestine (df2,18)
|
|
1.144
|
0.1251
|
0.245
|
Pair-wise Adonis
|
0D vs. 2W
|
0.9088
|
0.0653
|
1
|
2W vs. 2M
|
1.614
|
0.1873
|
0.270
|
0D vs. 2M
|
0.0866
|
0.0866
|
0.993
|
Ilium (df2,18)
|
|
1.579
|
0.1649
|
0.088
|
Pair-wise Adonis
|
0D vs. 2W
|
1.326
|
0.0925
|
0.549
|
2W vs. 2M
|
0.7268
|
0.0941
|
1
|
0D vs. 2M
|
2.247
|
0.1577
|
0.108
|
Middle of Small Intestine (df2,14)
|
|
2.562
|
0.2992
|
0.003*
|
Pair-wise Adonis
|
0D vs. 2W
|
1.441
|
0.1379
|
0.420
|
2W vs. 2M
|
3.815
|
0.3527
|
0.06
|
0D vs. 2M
|
3.097
|
0.2791
|
0.021*
|
Beginning of Small Intestine (df2,9)
|
|
1.253
|
0.2637
|
0.219
|
Pair-wise Adonis
|
0D vs. 2W
|
1.782
|
0.2627
|
0.492
|
2W vs. 2M
|
0.8224
|
0.1705
|
1
|
0D vs. 2M
|
1.197
|
0.1931
|
0.867
|
Stomach (df2,17)
|
|
1.239
|
0.1418
|
0.198
|
Pair-wise Adonis
|
0D vs. 2W
|
1.035
|
0.0794
|
1
|
2W vs. 2M
|
1.173
|
0.1435
|
0.720
|
0D vs. 2M
|
1.479
|
0.1185
|
0.477
|
Multiple DeSeq2 comparisons of microbial communities of freshly dissected and comparable gut sections in differently-aged alcohol specimens revealed only relatively few significantly differentially abundant bacterial genera (Fig. 6 and Additional file 4: Table S5). Only 72 out of 431 genera that were identified to genus-level were significantly differentially abundant in all gut regions between fresh and two-weeks old alcohol specimens, with each gut compartment accounting for in average 14.33 (SE ± 3.09) differentially abundant genera. Between fresh and two-months old alcohol gut sections, total of 55 taxa that identified to genus level were differentially abundant (mean differentially abundant genera per gut section ± SE: 15.16 ± 3.71). Of all differentially abundant genera, only Bacillus significantly increased in relative abundance in all gut sections of two-months old alcohol specimens compared to fresh samples, and the genus Macrococcus was more abundant in fresh compared to the two-months old samples in all gut sections except the large intestine (Fig. 6).