Skin bacterial diversity of Panamanian frogs is associated with host susceptibility and presence of Batrachochytrium dendrobatidis

Abstract

Symbiotic bacteria on amphibian skin can inhibit growth of the fungus Batrachochytrium dendrobatidis (Bd) that has caused dramatic population declines and extinctions of amphibians in the Neotropics. It remains unclear how the amphibians' skin microbiota is influenced by environmental bacterial reservoirs, host-associated factors such as susceptibility to pathogens, and pathogen presence in tropical amphibians. We sampled skin bacteria from five co-occurring frog species that differ in Bd susceptibility at one Bd-naive site, and sampled one of the non-susceptible species from Bd-endemic and Bd-naive sites in Panama. We hypothesized that skin bacterial communities (1) would be distinct from the surrounding environment regardless of the host habitat, (2) would differ between Bd susceptible and non-susceptible species and (3) would differ on hosts in Bd-naive and Bd-endemic sites. We found that skin bacterial communities were enriched in bacterial taxa that had low relative abundances in the environment. Non-susceptible species had very similar skin bacterial communities that were enriched in particular taxa such as the genera Pseudomonas and Acinetobacter. Bacterial communities of Craugastor fitzingeri in Bd-endemic sites were less diverse than in the naive site, and differences in community structure across sites were explained by changes in relative abundance of specific bacterial taxa. Our results indicate that skin microbial structure was associated with host susceptibility to Bd and might be associated to the history of Bd presence at different sites.

Figure 1

Frog skin and perch bacterial communities of five frog species at Sapo (a) Percentage of shared and unique OTUs of each frog species and perch samples. (b) Alpha diversity (Shannon) of each host and its respective perch. T-test results are shown at the bottom of each paired comparison. (c) Beta diversity of hosts and perch samples. Principal coordinate analysis of Bray Curtis distances. Ellipses show confidence Intervals (CI) of 95% for each sample type. (d) Relative abundances of OTUs on each frog species and its respective perch samples. Red dots show OTUs with a total relative abundance >0.1%. Kendall's ranked correlations, R_Tau, and P-values are shown to the right of each plot. Photo credits: Brian Gratwicke (A. certus, E. prosoblepon, C. panamansis, C. fitzingeri) and Brad Wilson (S. bufoniformis).

Figure 2

Skin bacterial community structure of five frog species at Sapo. (a) Relative abundance of shared and unique OTUs on each host species. (b) Alpha diversity (Shannon) of five frog species. Different letters (a, b and c) signify statistically significant differences among hosts, as indicated by the Tukey's post hoc tests. (c) Beta diversity of five host species. Principal coordinate analysis of Bray Curtis distances. Ellipses show confidence Intervals (CI) of 95% for each host species. (d) UPGMA and heatmap of bacterial genera with relative abundances >0.1% across five hosts species and their perch. Rows are bacterial genera. Columns are samples. Colors indicate taxa with a higher (red) or lower (blue) relative abundance in each sample. P, perch samples; AC, A. certus; EP, E. prosoblepon; CP, C. panamansis; CF, C. fitzingeri; SB, S. bufoniformis. Photo credits: Brian Gratwicke (A. certus, E. prosoblepon, C. panamansis, C. fitzingeri) and Brad Wilson (S. bufoniformis).

Figure 3

Skin bacterial community structure of C. fitzingeri. (a) Alpha diversity (Shannon) of C. fitzingeri across four regions (ANOVA F_(9,68) =7.279, P=4.5 × 10⁻⁴). Different letters (a and b) signify statistically significant differences among hosts, as indicated by the Tukey's post hoc tests. (b and c) Beta diversity of C. fitzingeri across four sites: Gamboa, Soberania, Mamoni and Sapo. Principal coordinate analyses are based on Bray Curtis distances. (d) Stacked bar chart of mean relative abundances of bacterial taxa (genus level) of C. fitzingeri across sites. Colored bars (legend) indicate the nine most abundant taxa. (e) Map of the four study sites in Panama.

Figure 4

Bacterial OTUs that differ between Bd-endemic and Bd-naive sites based on LEFfSe analysis. (a) OTUs with the highest linear discriminant analysis (LDA) scores from Bd-endemic sites and the Bd-naive site. Bars on graph indicate LDA values. Asterisks indicate taxa that are shown in (b) and (c). (b) Four representative OTUs with high relative abundances in the Bd-endemic sites. (c) Four representative OTUs with high relative abundances in the Bd-naive site. OTUs chosen for (b) and (c) represent examples of OTUs with the following attributes: (1) high LDA scores, (2) they span different taxonomic groups and (3) OTUs that were identified in only one of the groups (infected or naive). Bars in (b) and (c) correspond to individual frogs and colors correspond to the four sites: Sapo, Gamboa, Mamoni and Soberania.