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

Alessandro Catenazzi,David Burkart,Joseph Townsend,Nathan D. Hooven,Vance T. Vredenburg,Sandra V. Flechas

doi:10.3389/fmicb.2018.00465

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.

Highlights

Emerging infectious disease is a growing threat to global health and is identified as a major factor involved in the current biodiversity crisis (Daszak et al, 1999)
Culture-based methods can provide accurate information on the functions of a specific bacterium (Becker et al, 2015b), and relevance of these functions to its symbiotic considering how little is known about amphibian skin bacteria in the tropical Andes (Bresciano et al, 2015; Burkart et al, 2017), the overarching goal of this study is to examine the role of skin bacteria, and their anti-Batrachochytrium dendrobatidis (Bd) abilities, in protecting amphibian hosts from disease at our study site
The number of operative taxonomic units (OTUs) varied with host sample size (F1,26 = 366.3, p < 0.001), and after taking into account this sampling effect, there was no change in the average number of OTUs per host across elevation (F1,5 < 0.01, p = 0.977)

Summary

Introduction

Emerging infectious disease is a growing threat to global health and is identified as a major factor involved in the current biodiversity crisis (Daszak et al, 1999). To understand the role of skin bacteria in protecting amphibians from chytridiomycosis, we document the distribution of amphibian skin bacteria with antifungal properties in host communities along the eastern slopes of the tropical Andes where amphibian populations have collapsed after Bd epizootics (Catenazzi et al, 2011) This region has the highest amphibian species richness on Earth (Hutter et al, 2017), and along our elevational gradient amphibian richness changes from more than 60 species in the Andean foothills to six species of amphibians in the high-elevation grasslands (Catenazzi et al, 2013). In our previous work (Burkart et al, 2017), we showed that culturable skin bacteria inhibiting Bd growth (antiBd isolates) is linked to resistance to chytridiomycosis in highelevation frogs We extend this approach to the broader elevational gradient, including elevations where species declines have been steepest from 1,250 to 1,750 m a.s.l. We extend this approach to the broader elevational gradient, including elevations where species declines have been steepest from 1,250 to 1,750 m a.s.l. (Catenazzi et al, 2011), and relate the presence of anti-Bd bacteria on frogs to host susceptibility to chytridiomycosis (Catenazzi et al, 2017)

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