Widespread Elevational Occurrence of Antifungal Bacteria in Andean Amphibians Decimated by Disease: A Complex Role for Skin Symbionts in Defense Against Chytridiomycosis

Abstract

Emerging infectious disease is a growing threat to global health, and recent discoveries reveal that the microbiota dwelling on and within hosts can play an important role in health and disease. To understand the capacity of skin bacteria to protect amphibian hosts from the fungal disease chytridiomycosis caused by Batrachochytrium dendrobatidis (Bd), we isolated 192 bacterial morphotypes from the skin of 28 host species of frogs (families Bufonidae, Centrolenidae, Hemiphractidae, Hylidae, Leptodactylidae, Strabomantidae, and Telmatobiidae) collected from the eastern slopes of the Peruvian Andes (540-3,865 m a.s.l.) in the Kosñipata Valley near Manu National Park, a site where we previously documented the collapse of montane frog communities following chytridiomycosis epizootics. We obtained isolates through agar culture from skin swabs of wild frogs, and identified bacterial isolates by comparing 16S rRNA sequences against the GenBank database using BLAST. We identified 178 bacterial strains of 38 genera, including 59 bacterial species not previously reported from any amphibian host. The most common bacterial isolates were species of Pseudomonas, Paenibacillus, Chryseobacterium, Comamonas, Sphingobacterium, and Stenotrophomonas. We assayed the anti-fungal abilities of 133 bacterial isolates from 26 frog species. To test whether cutaneous bacteria might inhibit growth of the fungal pathogen, we used a local Bd strain isolated from the mouthparts of stream-dwelling tadpoles (Hypsiboas gladiator, Hylidae). We quantified Bd-inhibition in vitro with co-culture assays. We found 20 bacterial isolates that inhibited Bd growth, including three isolates not previously known for such inhibitory abilities. Anti-Bd isolates occurred on aquatic and terrestrial breeding frogs across a wide range of elevations (560-3,695 m a.s.l.). The inhibitory ability of anti-Bd isolates varied considerably. The proportion of anti-Bd isolates was lowest at mid-elevations (6%), where amphibian declines have been steepest, and among hosts that are highly susceptible to chytridiomycosis (0-14%). Among non-susceptible species, two had the highest proportion of anti-Bd isolates (40 and 45%), but one common and non-susceptible species had a low proportion (13%). In conclusion, we show that anti-Bd bacteria are widely distributed elevationally and phylogenetically across frog species that have persisted in a region where chytridiomycosis emerged, caused a devastating epizootic and continues to infect amphibians.

Keywords: 16S rRNA gene; amphibian declines; amphibian skin bacteria; antifungal bacteria; elevational gradient; montane diversity gradient; neotropical; tropical Andes.

FIGURE 1

Representative amphibian hosts and their habitats sampled for this study: (A,B) Hypsiboas gladiator is non-susceptible to chytridiomycosis and lays aquatic eggs in streamside basins along montane streams in the cloud forest; (C,D) Psychrophrynella usurpator is non-susceptible and lays terrestrial eggs that undergo direct development under mosses in the high-Andean grassland; (E,F) Telmatobius marmoratus is highly susceptible to chytridiomycosis and lays aquatic eggs in small, high-Andean streams. Photographs by A. Catenazzi.

FIGURE 2

Change in the composition of frog skin bacterial isolates arranged by phyla along the elevational gradient in the Kosñipata Valley near Manu National Park, SE Peru.

FIGURE 3

(A) Variation in proportion of anti-Bd bacterial isolates in Bd-susceptible and non-susceptible amphibian hosts. In susceptible species, days to death after Bd infection are calculated from survival analysis (Catenazzi et al., 2017). Number of tested bacterial morphotypes reported above each data point. (B) Average strength (±SE) of Bd inhibition of anti-Bd isolates (as measured by the relative distance from the streak of the query bacterium to point of 50% of max Bd growth; see Materials and Methods) ranked by inhibitory strength (from strongest to weakest inhibitor) in three Bd-susceptible hosts (in red), three non-susceptible hosts (in blue), and three hosts of unknown Bd susceptibility (i.e., not tested, in gray). Bacterial strains vary by frog host species.

FIGURE 4

Change in proportion of anti-Bd bacterial strains within phyla (bars) and across all phyla (circles; numbers indicate host sample size) along the elevational gradient in the Kosñipata Valley near Manu National Park, SE Peru. Diamonds indicate disease (Bd) prevalence with Bayes credible intervals in amphibian hosts, and the shaded area indicates elevations at which at least 35% of amphibian species have disappeared following the Bd epizootics (Catenazzi et al., 2011).

FIGURE 5

Variation in average strength (±SE) of Bd inhibition of anti-Bd isolates (ranked from strongest to weakest inhibitor) across host reproductive modes.