Molecular detection of <i>Batrachochytrium dendrobatidis</i> (Chytridiomycota) and culturable skin bacteria associated with three critically endangered species of <i>Atelopus</i> (Anura: Bufonidae) in Ecuador

Chytridiomycosis is a fungal disease responsible for massive amphibian die-offs worldwide, caused by the fungus <i>Batrachochytrium dendrobatidis</i> (Bd). Potential symbiotic relationships between frogs and the bacteria residing on their skin-referred to as skin-bacteria-may inhibit Bd growth, aiding in resistance to this lethal disease. This research had three main objectives: (1) to detect the presence of Bd in native populations of <i>Atelopus balios, A. bomolochos</i>, and <i>A. nanay</i> in the central Andes and coastal southern regions of Ecuador; (2) to identify the culturable skin-bacteria; and (3) to analyze differences among the bacterial communities in the three <i>Atelopus</i> species studied. Skin swabs were collected from two populations of <i>A. balios</i> (107-203 m a.s.l.) and one population each of <i>A. bomolochos</i> and <i>A. nanay</i> (3,064-3,800 m a.s.l.). These swabs served two purposes: first, to detect Bd using conventional PCR; and second, to isolate culturable bacteria, which were characterized through DNA sequencing, molecular phylogeny, and community composition similarity analysis (Jaccard index). Results showed that Bd was present in all species, with positive Bd PCR amplification found in 11 of the 12 sampled amphibians. The culturable skin-bacteria were classified into 10 genera: <i>Pseudomonas</i> (31.4%), <i>Stenotrophomonas</i> (14.3%), <i>Acinetobacter</i> (11.4%), <i>Serratia</i> (11.4%), <i>Aeromonas</i> (5.7%), <i>Brucella</i> (5.7%), <i>Klebsiella</i> (5.7%), <i>Microbacterium</i> (5.7%), <i>Rhodococcus</i> (5.7%), and <i>Lelliottia</i> (2.9%). The Jaccard index revealed that bacterial genera were least similar in <i>A. bomolochos</i> and <i>A. balios</i> (<i>J</i> = 0.10), while the highest similarity at the genus level was between <i>A. bomolochos</i> and <i>A. nanay</i> (<i>J</i> = 0.33). At the clade-species level, only <i>A. bomolochos</i> and <i>A. nanay</i> show common bacteria (<i>J</i> = 0.13). Culturable bacterial communities of specimens diagnosed as Bd positive (<i>n</i> = 10) or Bd negative (<i>n</i> = 1) share a J value of 0.1 at genus and 0.04 at species-clade level. The prevalence of Bd and the composition of cutaneous bacteria could be influenced by Bd reservoirs, <i>Atelopus</i> biology, and intrinsic environmental conditions. This research contributes to understanding the relationship between endangered Andean species and Bd, and explores the potential use of native skin-bacteria as biocontrol agents against Bd.