Seasonal and ontogenetic variation of skin microbial communities and relationships to natural disease dynamics in declining amphibians

Abstract

Recently, microbiologists have focused on characterizing the probiotic role of skin bacteria for amphibians threatened by the fungal disease chytridiomycosis. However, the specific characteristics of microbial diversity required to maintain health or trigger disease are still not well understood in natural populations. We hypothesized that seasonal and developmental transitions affecting susceptibility to chytridiomycosis could also alter the stability of microbial assemblages. To test our hypothesis, we examined patterns of skin bacterial diversity in two species of declining amphibians (Lithobates yavapaiensis and Eleutherodactylus coqui) affected by the pathogenic fungus Batrachochytrium dendrobatidis (Bd). We focused on two important transitions that affect Bd susceptibility: ontogenetic (from juvenile to adult) shifts in E. coqui and seasonal (from summer to winter) shifts in L. yavapaiensis. We used a combination of community-fingerprinting analyses and 16S rRNA amplicon sequencing to quantify changes in bacterial diversity and assemblage composition between seasons and developmental stages, and to investigate the relationship between bacterial diversity and pathogen load. We found that winter-sampled frogs and juveniles, two states associated with increased Bd susceptibility, exhibited higher diversity compared with summer-sampled frogs and adult individuals. Our findings also revealed that hosts harbouring higher bacterial diversity carried lower Bd infections, providing support for the protective role of bacterial communities. Ongoing work to understand skin microbiome resilience after pathogen disturbance has the potential to identify key taxa involved in disease resistance.

Keywords: 16S amplicon sequencing; Eleutherodactylus coqui; Lithobates yavapaiensis; community fingerprinting; dysbiosis; host–pathogen dynamics.

Figure 1.

Mean interspecific differences in alpha diversity estimated by ARISA in E. coqui (open circles) and L. yavapaiensis (grey circles). (a) OTU richness, (b) Shannon's diversity and (c) Pielou's evenness.

Figure 2.

NMDS plots using Bray–Curtis distance matrices generated with ARISA show differences in community composition (beta diversity) by (a) species: E. coqui versus L. yavapaiensis, (b) life stage in E. coqui: juveniles versus adults and (c) season in L. yavapaiensis: summer versus winter. Venn diagrams show the number of shared and unique OTUs in each comparison.

Figure 3.

Relative abundance of 16S V4 amplicons by major phyla after rarefaction at 5000 reads per individual.

Figure 4.

Relative abundance and distribution of 16S V4 Proteobacteria phylotypes by class after rarefaction at 5000 reads per individual. Venn diagram shows the total number of unique or shared phylotype sequences by species, whereas the pie charts represent the distribution of reads by class. We considered % unique as the total number of unique Proteobacteria reads for each species divided by total number of sequences (% unique _Ly=5483/15 000; % unique _Ec=8325/95 000). Similarly, we considered % shared as the total number of Proteobacteria reads shared between species divided by the total number of sequences (% shared _Ly=3401/15 000; % shared _Ec= 62 014/95 000).

Figure 5.

Correlations of Bd load (number of zoospore genomic equivalents) in E. coqui versus Proteobacteria (a) phylotype richness, (b) Shannon's diversity, (c) phylogenetic diversity, (d) dominance and (e) evenness. Photo of E. coqui by Alberto L. López-Torres.