Amphibian granular glands provide a wide range of compounds on the skin that 
defend against pathogens and predators. We identified three bufadienolides-the 
steroid-like compounds arenobufagin, gamabufotalin, and telocinobufagin-from the 
boreal toad, Anaxyrus boreas, through liquid chromatography mass spectrometry 
(LC/MS). Compounds were detected both after inducing skin gland secretions and 
in constitutive mucosal rinses from toads. We described the antimicrobial 
properties of each bufadienolide against Batrachochytrium dendrobatidis (Bd), an 
amphibian fungal pathogen linked with boreal toad population declines. All three 
bufadienolides were found to inhibit Bd growth at similar levels. The maximum Bd 
inhibition produced by arenobufagin, gamabufotalin, and telocinobufagin were 
approximately 50%, in contrast to the complete Bd inhibition shown by 
antimicrobial skin peptides produced by some amphibian species. In addition, 
skin mucus samples significantly reduced Bd viability, and bufadienolides were 
detected in 15 of 62 samples. Bufadienolides also appeared to enhance growth of 
the anti-Bd bacterium Janthinobacterium lividum, and thus may be involved in 
regulation of the skin microbiome. Here, we localized skin bacteria within the 
mucus layer and granular glands of toads with fluorescent in situ hybridization. 
Overall, our results suggest that bufadienolides can function in antifungal 
defense on amphibian skin and their production is a potentially convergent trait 
similar to antimicrobial peptide defenses found on the skin of other species. 
Further studies investigating bufadienolide expression across toad populations, 
their regulation, and interactions with other components of the skin mucosome 
will contribute to understanding the complexities of amphibian immune defense.