Effects of captivity and artificial breeding on microbiota in feces of the red-crowned crane (Grus japonensis)

Abstract

Reintroduction of the threatened red-crowned crane has been unsuccessful. Although gut microbiota correlates with host health, there is little information on gut microbiota of cranes under different conservation strategies. The study examined effects of captivity, artificial breeding and life stage on gut microbiota of red-crown cranes. The gut microbiotas of wild, captive adolescent, captive adult, artificially bred adolescent and artificially bred adult cranes were characterized by next-generation sequencing of 16S rRNA gene amplicons. The gut microbiotas were dominated by three phyla: Firmicutes (62.9%), Proteobacteria (29.9%) and Fusobacteria (9.6%). Bacilli dominated the 'core' community consisting of 198 operational taxonomic units (OTUs). Both captivity and artificial breeding influenced the structures and diversities microbiota of the gut. Especially, wild cranes had distinct compositions of gut microbiota from captive and artificially bred cranes. The greatest alpha diversity was found in captive cranes, while wild cranes had the least. According to the results of ordination analysis, influences of captivity and artificial breeding were greater than that of life stage. Overall, captivity and artificial breeding influenced the gut microbiota, potentially due to changes in diet, vaccination, antibiotics and living conditions. Metagenomics can serve as a supplementary non-invasive screening tool for disease control.

Figure 1. Relative abundances of the most abundant phyla in feces of red crown cranes.

The 5 most abundant phyla (>1% of the total sequences) are presented. Samples are grouped according to the breeding type and age range, and arranged by the relative abundance of the most dominant phylum, Firmicutes.

Figure 2. Association network of core OTUs collapsed at the genus level.

Shape of node, class; color of node, order; red dashed edges, mutual exclusivity; blue edge, co-occurrence. The widths of the lines represent the relative abundances of OTUs.

Figure 3. PCoA ordination of unweighted UniFrac distances revealing the influence of captivity, artificial breeding and life stage on community structuring.

Distances among centroids of three groups were 0.404 (artificially bred to captive group), 0.448 (artificially bred to wild group) and 0.450 (captive to wild group). The influence of captivity and artificial breeding were greater than that of life stage (the distance between centroids of adolescent and adult cranes: 0.189).

Figure 4. Comparisons of phylogenetic diversity and Shannon index among groups.

The Kruskal-Wallis test was performed, followed by post hoc Mann-Whitney U tests. Significance was determined at P values < 0.001 (***), < 0.01 (**), and < 0.05 (*).

Figure 5. Patterns of abundance and mean relative abundances of the most abundant, assignable genera varied among groups.

Criteria for abundant assignable genera are mean relative abundance > 1% and with genus annotation (annotation confidence score > 60%). Key hubs (determined by network analysis) were in blue, and Campylobacter (mutually exclusive with key hubs) were in red. Mean relative abundance was tested with ANOVA, P values < 0.05, followed by post hoc Tukey-Kramer tests among groups. P values were corrected using the Benjamin-Hochberg FDR method.