First evidence of ranavirus in native and invasive amphibians in Colombia.

Ranaviruses can cause mass mortality events in amphibians, thereby becoming a threat to populations that are already facing dramatic declines. Ranaviruses affect all life stages and persist in multiple amphibian hosts. The detrimental effects of ranavirus infections to amphibian populations have already been observed in the UK and in North America. In Central and South America, the virus has been reported in several countries, but the presence of the genus Ranavirus (Rv) in Colombia is unknown. To help fill this knowledge gap, we surveyed for Rv in 60 species of frogs (including one invasive species) in Colombia. We also tested for co-infection with Batrachochytrium dendrobatidis (Bd) in a subset of individuals. For Rv, we sampled 274 vouchered liver tissue samples collected between 2014 and 2019 from 41 localities covering lowlands to mountaintop páramo habitat across the country. Using quantitative polymerase chain reaction (qPCR) and end-point PCR, we detected Rv in 14 individuals from 8 localities, representing 6 species, including 5 native frogs of the genera Osornophryne, Pristimantis and Leptodactylus, and the invasive American bullfrog Rana catesbeiana. Bd was detected in 7 of 140 individuals, with 1 co-infection of Rv and Bd in an R. catesbeiana specimen collected in 2018. This constitutes the first report of ranavirus in Colombia and should set off alarms about this new emerging threat to amphibian populations in the country. Our findings provide some preliminary clues about how and when Rv may have spread and contribute to understanding how the pathogen is distributed globally.

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

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.

The Influence of Habitat and Phylogeny on the Skin Microbiome of Amphibians in Guatemala and Mexico.

Microbial symbionts are increasingly recognized as playing a critical role in organismal health across a wide range of hosts. Amphibians are unique hosts in that their skin helps to regulate the exchange of water, ions, and gases, and it plays an active role in defense against pathogens through the synthesis of anti-microbial peptides. The microbiome of amphibian skin includes a diverse community of bacteria known to defend against pathogens, including the global pandemic lineage of Batrachochytrium dendrobatidis associated with mass amphibian die-offs. The relative influence of host phylogeny and environment in determining the composition of the amphibian skin microbiome remains poorly understood. We collected skin swabs from montane amphibians in Mexico and Guatemala, focusing on two genera of plethodontid salamanders and one genus of frogs. We used high throughput sequencing to characterize the skin bacterial microbiome and tested the impact of phylogeny and habitat on bacterial diversity. Our results show that phylogenetic history strongly influences the diversity and community structure of the total bacterial microbiome at higher taxonomic levels (between orders), but on lower scales (within genera and species), the effect of habitat predominates. These results add to a growing consensus that habitat exerts a strong effect on microbiome structure and composition, particularly at shallow phylogenetic scales.

Prodigiosin, Violacein, and Volatile Organic Compounds Produced by Widespread Cutaneous Bacteria of Amphibians Can Inhibit Two Batrachochytrium Fungal Pathogens.

Symbiotic bacteria can produce secondary metabolites and volatile compounds that contribute to amphibian skin defense. Some of these symbionts have been used as probiotics to treat or prevent the emerging disease chytridiomycosis. We examined 20 amphibian cutaneous bacteria for the production of prodigiosin or violacein, brightly colored defense compounds that pigment the bacteria and have characteristic spectroscopic properties making them readily detectable, and evaluated the antifungal activity of these compounds. We detected violacein from all six isolates of Janthinobacterium lividum on frogs from the USA, Switzerland, and on captive frogs originally from Panama. We detected prodigiosin from five isolates of Serratia plymuthica or S. marcescens, but not from four isolates of S. fonticola or S. liquefaciens. All J. lividum isolates produced violacein when visibly purple, while prodigiosin was only detected on visibly red Serratia isolates. When applied to cultures of chytrid fungi Batrachochytrium dendrobatidis (Bd) and B. salamandrivorans (Bsal), prodigiosin caused significant growth inhibition, with minimal inhibitory concentrations (MIC) of 10 and 50 µM, respectively. Violacein showed a MIC of 15 µM against both fungi and was slightly more active against Bsal than Bd at lower concentrations. Although neither violacein nor prodigiosin showed aerosol activity and is not considered a volatile organic compound (VOC), J. lividum and several Serratia isolates did produce antifungal VOCs. White Serratia isolates with undetectable prodigiosin levels could still inhibit Bd growth indicating additional antifungal compounds in their chemical arsenals. Similarly, J. lividum can produce antifungal compounds such as indole-3-carboxaldehyde in addition to violacein, and isolates are not always purple, or turn purple under certain growth conditions. When Serratia isolates were grown in the presence of cell-free supernatant (CFS) from the fungi, CFS from Bd inhibited growth of the prodigiosin-producing isolates, perhaps indicative of an evolutionary arms race; Bsal CFS did not inhibit bacterial growth. In contrast, growth of one J. lividum isolate was facilitated by CFS from both fungi. Isolates that grow and continue to produce antifungal compounds in the presence of pathogens may represent promising probiotics for amphibians infected or at risk of chytridiomycosis. In a global analysis, 89% of tested Serratia isolates and 82% of J. lividum isolates were capable of inhibiting Bd and these have been reported from anurans and caudates from five continents, indicating their widespread distribution and potential for host benefit.

The effect of captivity on the skin microbial symbionts in three Atelopus species from the lowlands of Colombia and Ecuador.

Many amphibian species are at risk of extinction in their natural habitats due to the presence of the fungal pathogen Batrachochytrium dendrobatidis (Bd). For the most highly endangered species, captive assurance colonies have been established as an emergency measure to avoid extinction. Experimental research has suggested that symbiotic microorganisms in the skin of amphibians play a key role against Bd. While previous studies have addressed the effects of captivity on the cutaneous bacterial community, it remains poorly studied whether and how captive conditions affect the proportion of beneficial bacteria or their anti-Bd performance on amphibian hosts. In this study we sampled three amphibian species of the highly threatened genus, Atelopus, that remain in the wild but are also part of ex situ breeding programs in Colombia and Ecuador. Our goals were to (1) estimate the diversity of culturable bacterial assemblages in these three species of Atelopus, (2) describe the effect of captivity on the composition of skin microbiota, and (3) examine how captivity affects the bacterial ability to inhibit Bd growth. Using challenge assays we tested each bacterial isolate against Bd, and through sequencing of the 16S rRNA gene, we identified species from thirteen genera of bacteria that inhibited Bd growth. Surprisingly, we did not detect a reduction in skin bacteria diversity in captive frogs. Moreover, we found that frogs in captivity still harbor bacteria with anti-Bd activity. Although the scope of our study is limited to a few species and to the culturable portion of the bacterial community, our results indicate that captive programs do not necessarily change bacterial communities of the toad skins in a way that impedes the control of Bd in case of an eventual reintroduction.

DNA Barcoding Survey of Anurans across the Eastern Cordillera of Colombia and the Impact of the Andes on Cryptic Diversity.

Colombia hosts the second highest amphibian species diversity on Earth, yet its fauna remains poorly studied, especially using molecular genetic techniques. We present the results of the first wide-scale DNA barcoding survey of anurans of Colombia, focusing on a transect across the Eastern Cordillera. We surveyed 10 sites between the Magdalena Valley to the west and the eastern foothills of the Eastern Cordillera, sequencing portions of the mitochondrial 16S ribosomal RNA and cytochrome oxidase subunit 1 (CO1) genes for 235 individuals from 52 nominal species. We applied two barcode algorithms, Automatic Barcode Gap Discovery and Refined Single Linkage Analysis, to estimate the number of clusters or "unconfirmed candidate species" supported by DNA barcode data. Our survey included ~7% of the anuran species known from Colombia. While barcoding algorithms differed slightly in the number of clusters identified, between three and ten nominal species may be obscuring candidate species (in some cases, more than one cryptic species per nominal species). Our data suggest that the high elevations of the Eastern Cordillera and the low elevations of the Chicamocha canyon acted as geographic barriers in at least seven nominal species, promoting strong genetic divergences between populations associated with the Eastern Cordillera.

Surviving chytridiomycosis: differential anti-Batrachochytrium dendrobatidis activity in bacterial isolates from three lowland species of Atelopus.

In the Neotropics, almost every species of the stream-dwelling harlequin toads (genus Atelopus) have experienced catastrophic declines. The persistence of lowland species of Atelopus could be explained by the lower growth rate of Batrachochytrium dendrobatidis (Bd) at temperatures above 25 °C. We tested the complementary hypothesis that the toads' skin bacterial microbiota acts as a protective barrier against the pathogen, perhaps delaying or impeding the symptomatic phase of chytridiomycosis. We isolated 148 cultivable bacterial strains from three lowland Atelopus species and quantified the anti-Bd activity through antagonism assays. Twenty-six percent (38 strains representing 12 species) of the bacteria inhibited Bd growth and just two of them were shared among the toad species sampled in different localities. Interestingly, the strongest anti-Bd activity was measured in bacteria isolated from A. elegans, the only species that tested positive for the pathogen. The cutaneous bacterial microbiota is thus likely a fitness-enhancing trait that may (adaptation) or not (exaptation) have appeared because of natural selection mediated by chytridiomycosis. Our findings reveal bacterial strains for development of local probiotic treatments against chytridiomycosis and also shed light on the mechanisms behind the frog-bacteria-pathogen interaction.