Skin microbiomes of frogs vary among body regions, revealing differences that reflect known patterns of chytrid infection

Abstract

Introduction: The amphibian skin microbiome is an important line of defense against pathogens including the deadly chytrid fungus, Batrachochytrium dendrobatidis (Bd). Bd is known to preferentially infect ventral skin surfaces and feet of host amphibians, often leaving dorsal surfaces like the back uninfected. Within-individual variation in infection distribution across the skin, therefore, may relate to differences in microbiomes among skin regions. However, microbiome heterogeneity within amphibian individuals remains poorly characterized.

Methods: We utilized 16S rRNA gene amplicon sequencing to compare microbiomes of 10 body regions from nine captive Rana sierrae individuals and their tank environments. These individuals were naive to Bd, allowing us to assess whether microbiomes differed among body regions prior to any impacts that may be caused by infection.

Results: We found that frog skin and tank environments harbored distinct microbial communities. On frog skin, the bacterial families Burkholderiaceae (phylum Proteobacteria) and Rubritaleaceae (phylum Verrucomicrobia) were dominant, driven in large part by relative abundances of undescribed members of these families that were significantly higher on frogs than in their environment. Within individuals, we detected differences between microbiomes of body regions where Bd infection would be expected compared to regions that infrequently experience infection. Notably, putative Bd-inhibitory relative abundance was significantly higher on body regions where Bd infection is often localized.

Discussion: These findings suggest that microbiomes in certain skin regions may be predisposed for interactions with Bd. Further, our results highlight the importance of considering intraindividual heterogeneities, which could provide insights relevant to predicting localized interactions with pathogens.

Keywords: Batrachochytrium dendrobatidis; Rana sierrae; amphibian; captivity; microbiome; skin; within-individual heterogeneity.

Figure 1

Diagram of Rana sierrae body regions sampled in this study. Body regions sampled are encircled in dashed lines. Ventral surfaces sampled are indicated with purple text and arrows. Dorsal surfaces sampled are indicated with orange text and arrows. Feet surfaces sampled are indicated with blue text and arrows. For limbs and feet, both the right and left were sampled. Forefeet were only sampled on the ventral surface, whereas hindfeet samples were collected from both the ventral and dorsal surfaces. Frogs sampled ranged from 46.4 to 54.4 mm snout-to-vent length (SVL) and from 9.5 to 17.8 g in weight.

Figure 2

Alpha diversity-based comparisons of sample groupings. Within-sample diversity in terms of observed richness (A,C) and Shannon diversity (B,D). (A,B) Frog and tank environment samples with boxplots and points colored by the type of sample substrate. Panel background shading differentiates frog samples from tank environment samples. (C,D) Frog samples with boxplots and points colored by frog body region. Panel background shading differentiates groups of body regions sampled (dorsal, ventral, and feet). (A–D) Results of Kruskal-Wallis tests are shown. Post hoc Dunn test results are displayed as significance bars where applicable (*p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001).

Figure 3

Beta diversity-based comparisons of sample groupings. (A–C) Microbial community structure of frog and tank environment samples with points colored and shaped by the type of sample substrate. (D–F) Microbial community structure of frog samples with points colored and shaped by body region. (A,D) PCoA visualizations of unweighted UniFrac distances. (B,E) PCoA visualizations of weighted UniFrac distances. (C,F) PCoA visualizations of Bray-Curtis dissimilarities.

Figure 4

Mean relative abundances of bacterial phyla and families. Stacked bar chart for frog, rock perch, tank wall, tank water, and underwater rock samples was generated using microshades v. 1.11 (Dahl et al., 2022). Taxa are colored by phylum, and families within each phylum are colored with unique shades of the associated phylum color. Phyla and families are ordered so that higher mean relative abundance groups appear lower in stacked bars. Blastocatellaceae belong to the phylum Acidobacteria. Clostridiaceae and Bacillaceae belong to the phylum Firmicutes.

Figure 5

Relative abundances of putative Bd-inhibitory taxa among frog body regions. Values were calculated based on our strict subset of 29 frog-associated ASVs that shared 100% sequence identity with taxa known to inhibit Bd growth (Woodhams et al., 2015). Boxplots and points are colored by frog body region. Panel background shading differentiates groups of body regions sampled (dorsal, ventral, and feet). Results of Kruskal-Wallis tests are shown. Post hoc Dunn test results are displayed as significance bars where applicable (*p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001).