Microbiota and skin defense peptides may facilitate coexistence of two sympatric Andean frog species with a lethal pathogen

Abstract

Management of hyper-virulent generalist pathogens is an emergent global challenge, yet for most disease systems we lack a basic understanding as to why some host species suffer mass mortalities, while others resist epizootics. We studied two sympatric species of frogs from the Colombian Andes, which coexist with the amphibian pathogen Batrachochytrium dendrobatidis (Bd), to understand why some species did not succumb to the infection. We found high Bd prevalence in juveniles for both species, yet infection intensities remained low. We also found that bacterial community composition and host defense peptides are specific to amphibian life stages. We detected abundant Bd-inhibitory skin bacteria across life stages and Bd-inhibitory defense peptides post-metamorphosis in both species. Bd-inhibitory bacteria were proportionally more abundant in adults of both species than in earlier developmental stages. We tested for activity of peptides against the skin microbiota and found that in general peptides did not negatively affect bacterial growth and in some instances facilitated growth. Our results suggest that symbiotic bacteria and antimicrobial peptides may be co-selected for, and that together they contribute to the ability of Andean amphibian species to coexist with the global pandemic lineage of Bd.

Fig. 1

Alpha diversity (species richness) of bacterial communities in each species, life stage, and pond water. The asterisk indicates significant differences between the water and amphibian hosts

Fig. 2

Principal coordinate analysis of beta diversity of skin microbial communities for each amphibian species differentiated by life stage. Each point represents the skin bacterial community of an individual frog. Symbol and color indicate the life stage and species. Weighted Unifrac distance metric was used in beta diversity analyses

Fig. 3

Relative abundance of bacterial phyla by species and life stage. a Relative abundance of bacterial 16S sequences obtained through 16S sequencing of microbiota in two amphibian hosts across life stages and in pond water samples. b Relative abundance of isolates recovered through culturable-dependent techniques. The letters along the bottom indicate life stage, A—adult, J—juvenile, T—tadpole. Numbers inside the bars denoted the number of samples per category

Fig. 4

MALDI-TOF mass spectrometry profiles of skin peptides. a Dendropsophus labialis–—tadpole, b Dendropsophus labialis—adult, c Rheobates palmatus—tadpole, and d Rheobates palmatus—adult. Note that the noise in (a) and (c) (spectra from tadpole samples) indicate a lack of peptide signals

Fig. 5

Variation among frogs in the effect of their skin antimicrobial peptides on Bd growth. Peptide samples were collected from Dendropsophus labialis adults (black circles, N = 15), D. labialis tadpoles (white circles, N = 1), Rheobates palmatus adults (black triangles, N = 13), R. palmatus tadpoles (white triangles, N = 6). Vertical bars indicated ± 1 standard error. Positive control (black diamond) and negative control (white diamond) are included. The dotted line represents the growth of Bd in the absence of peptides

Fig. 6

Effect of antimicrobial peptides on bacterial growth. Each panel shows the proportion of affected bacteria when exposed to peptides from four different sources (adults and juveniles of Dendropsophus labialis and Rheobates palmatus). Bars represent the source of bacteria. Light gray represents the proportion of bacteria that were not significantly affected by peptides. Dark gray represents the proportion of bacteria whose growth was significantly enhanced by peptides. Black represents the proportion of bacteria that were significantly inhibited by peptides. White spaces indicate that this test was not performed