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Many endangered amphibian species survive in captive breeding facilities, but there have been few attempts to reintroduce captive-born individuals to rebuild wild populations. We conducted a soft-release trial of limosa harlequin frogs, Atelopus limosus, which are highly susceptible to the amphibian chytrid fungus Batrachochytrium dendrobatidis (Bd), to understand changes associated with the transition from captivity to the wild. Specifically, we assessed changes in body condition, skin-associated bacterial communities and disease status after release. Frogs were housed individually in field mesocosms and monitored for 27 days. Body condition did not significantly change in the mesocosms, and was similar to, or higher than, that of wild conspecifics at day 27. The skin bacteria of captive-born frogs, based on 16S rRNA gene amplicons, became similar to that of wild conspecifics after 27 days in mesocosms. Prevalence of Bd in wild conspecifics was 13–27%, and 15% of the A. limosus in mesocosms became infected with Bd, but no mortality of infected frogs was observed. We conclude that mesocosms are suitable for systematically and repeatedly monitoring amphibians during release trials, and that body condition, the skin microbiome, and Bd status can all change within one month of placement of captive-born individuals back into the wild. [ABSTRACT FROM AUTHOR]

Captive breeding programs are a valuable conservation strategy, particularly when integrated with research goals. Panamanian Harlequin frogs (genus Atelopus) serve as a case study for integrating captive breeding and research goals because they have experienced drastic chytridiomycosis-related declines and have large captive populations. Captive breeding efforts in Panama and the United States established secure ex-situ populations of Atelopus certus , A. glyphus , A. limosus , A. varius , and A. zeteki. Atelopus chiriquiensis is presumed to be extinct with no captive populations. The status of one undescribed species, Atelopus aff. limosus , has not been evaluated and no secure captive population has yet been established. Captive breeding efforts that produce a surplus of Atelopus are an important resource for research into collections management, disease mitigation, and adaptive management approaches for Atelopus reintroduction efforts. We reevaluated all Panamanian Atelopus species through the IUCN Redlist and compiled occurrence records for Panamanian Atelopus species to create a historical distribution map. We model Atelopus habitat suitability using Maxent and found annual mean air temperature to be the best predictor of Atelopus occurrence. The model will improve our knowledge of their likely spatial distribution and guide future conservation and reintroduction efforts. The recent proliferation of molecular tools, climate models, bio-banking, and reproductive technologies position us to address multiple applied and basic evolutionary questions such as: What factors cause differential disease outcomes? Do persisting populations have heritable traits associated with improved survivorship? Are there climatic refugia from disease? Ultimately, the answers to these questions will help us develop applied solutions and facilitate the reestablishment of self-sustaining wild populations. • Atelopus species have experienced severe chytridiomycosis-related declines. • This case study documents the wild and captive status of Atelopus in Panama. • Habitat models improve historical distribution maps and guide future efforts. • Captive breeding efforts prevent extinctions and are a valuable research resource. [ABSTRACT FROM AUTHOR]

Amphibians around the world are declining from threats that cannot currently be mitigated, making it impossible to safeguard some species in their natural habitats. Amphibians in the mountainous neotropics are one example where severe disease-related declines prompted calls for the establishment of captive assurance colonies to avoid extinctions. We surveyed experts in Panamanian amphibians to determine the probability of avoiding chytridiomycosis-related extinctions using captive breeding programs. We ranked Panamanian amphibian species by perceived susceptibility to chytridiomycosis, then calculated the likelihood of avoiding extinction as the product of three probabilities, which include (1) finding sufficient founder animals, (2) successfully breeding these species in captivity and (3) becoming extinct in the wild. The likelihood of finding enough animals to create a captive founding population was low for many rare species, especially for salamanders and caecilians. It was also low for frogs which were once regularly encountered, but have already disappeared including Atelopus chiriquiensis, Craugastor emcelae, C. obesus, C. punctariolus, C. rhyacobatrachus, Ecnomiohyla rabborum, Isthmohyla calypsa and Oophaga speciosa. Our results indicate that captive breeding could improve the odds of avoiding extinction for species that have severely declined or are likely to decline due to chytridiomycosis including Atelopus certus, A. glyphus, A. limosus, A. varius, A. zeteki, Anotheca spinosa, Gastrotheca cornuta, Agalychnis lemur and Hemiphractus fasciatus. Priority species that experts predicted were highly susceptible to chytridiomycosis that might also benefit from ex situ management include Craugastor tabasarae, C. azueroensis, C. evanesco, Strabomantis bufoniformis and Colostethus panamansis. In spite of high levels of uncertainty, this expert assessment approach allowed us to refine our priorities for captive amphibian programs in Panama and identify priority conservation actions with a clearer understanding of the probability of success. [ABSTRACT FROM AUTHOR]

Chytridiomycosis, caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd), is an emergent infectious disease partially responsible for worldwide amphibian population declines. The spread of Bd along highland habitats (> 500 meters above sea level, m a.s.l.) of Costa Rica and Panamá is well documented and has been linked to amphibian population collapses. In contrast, data are scarce on the prevalence and dispersal of Bd in lowland habitats where amphibians may be infected but asymptomatic. Here we describe the spread (2009 to 2014) of Bd across lowland habitats east of the Panamá Canal (< 500 m a.s.l.) with a focus on the Túngara frog (Physalaemus [Engystomops] pustulosus), one of the most common and abundant frog species in this region. Highland populations in western Panamá were already infected with Bd at the start of the study, which was consistent with previous studies indicating that Bd is enzootic in this region. In central Panamá, we collected the first positive samples in 2010, and by 2014, we detected Bd from remote sites in eastern Panamá (Darién National Park). We discuss the importance of studying Bd in lowland species, which may serve as potential reservoirs and agents of dispersal of Bd to highland species that are more susceptible to chytridiomycosis. [ABSTRACT FROM AUTHOR]

Species losses are predicted to simplify food web structure, and disease-driven amphibian declines in Central America offer an opportunity to test this prediction. Assessment of insect community composition, combined with gut content analyses, was used to generate periphyton-insect food webs for a Panamanian stream, both pre- and postamphibian decline. We then used network analysis to assess the effects of amphibian declines on food web structure. Although 48% of consumer taxa, including many insect taxa, were lost between pre- and post-amphibian decline sampling dates, connectance declined by less than 3%. We then quantified the resilience of food web structure by calculating the number of expected cascading extirpations from the loss of tadpoles. This analysis showed the expected effects of species loss on connectance and linkage density to be more than 60% and 40%, respectively, than were actually observed. Instead, new trophic linkages in the post-decline food web reorganized the food web topology, changing the identity of ''hub'' taxa, and consequently reducing the effects of amphibian declines on many food web attributes. Resilience of food web attributes was driven by a combination of changes in consumer diets, particularly those of insect predators, as well as the appearance of generalist insect consumers, suggesting that food web structure is maintained by factors independent of the original trophic linkages. [ABSTRACT FROM AUTHOR]

For many threatened vertebrates, captivity may be the only option for species survival. Maintaining species in captivity prior to reintroduction presents many challenges, including the need to preserve genetic diversity and mitigation of disease risks. Recent studies suggest that captivity can alter the suite of symbiotic microbes that play important roles in host health. The Panamanian golden frog (Atelopus zeteki) has not been seen in its native habitat in Panamá since 2009. Along with habitat loss and illegal collecting, the lethal disease chytridiomycosis, caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd), is responsible for the severe decline of this species. Prior to the spread of Bd into golden frog habitat, conservation organizations collected golden frogs and placed them in captive survival assurance colonies. The skin of amphibians is host to a diverse resident bacterial community, which acts as a defense mechanism in some amphibians to inhibit pathogens. We characterized the cutaneous bacterial community from wild and F1 captive golden frogs originating from the same population with Illumina sequencing to assess how long-term captivity has affected this community. We found that species richness, phylogenetic diversity, and community structure of the skin microbiota was significantly different between wild and captive golden frogs. However, after approximately eight years of living in captivity, the offspring of the original captive golden frogs still shared 70% of their microbial community with wild frogs. These results demonstrate that host-associated microbial communities can be significantly altered by captive management, but most of the community composition can be preserved. [ABSTRACT FROM AUTHOR]

Populations of native Panamanian golden frogs ( Atelopus zeteki) have collapsed due to a recent chytridiomycosis epidemic. Reintroduction efforts from captive assurance colonies are unlikely to be successful without the development of methods to control chytridiomycosis in the wild. In an effort to develop a protective treatment regimen, we treated golden frogs with Janthinobacterium lividum, a skin bacterium that has been used to experimentally prevent chytridiomycosis in North American amphibians. Although J. lividum appeared to colonize A. zeteki skin temporarily, it did not prevent or delay mortality in A. zeteki exposed to Batrachochytrium dendrobatidis, the causative agent of chytridiomycosis. After introduction of J. lividum, average bacterial cell counts reached a peak of 1.7 × 10 cells per frog ~2 weeks after treatment but declined steadily after that. When J. lividum numbers declined to ~2.8 × 10 cells per frog, B. dendrobatidis infection intensity increased to greater than 13,000 zoospore equivalents per frog. At this point, frogs began to die of chytridiomycosis. Future research will concentrate on isolating and testing antifungal bacterial species from Panama that may be more compatible with Atelopus skin. [ABSTRACT FROM AUTHOR]

The pathogenic chytrid fungus, Batrachochytrium dendrobatidis, has been implicated as the main driver of many enigmatic amphibian declines in neotropical sites at high elevation. Batrachochytrium dendrobatidis is thought to be a waterborne pathogen limited by temperature, and the extent to which it persists and causes disease in amphibians at lower elevations in the neotropics is not known. It also is unclear by what mechanism(s) B. dendrobatidis has emerged as a pathogenic organism. To test whether B. dendrobatidis is limited by elevation in Panamá, we sought to determine the prevalence and intensity of B. dendrobatidis in relation to anuran abundance and diversity using quantitative PCR (qPCR) analyses. Sites were situated at varying elevations, from 45 to 1215 m, and were at varying stages of epizootic amphibian decline, including pre-epizootic, mid-epizootic, 2 years post-epizootic, and 10 years post-epizootic. Batrachochytrium dendrobatidis was found in all sites regardless of elevation or stage of epizootic decline. Levels of prevalence and infection intensity were comparable across all sites except at the mid-epizootic site, where both prevalence and intensity were significantly higher than at other sites. Symptoms of chytridiomycosis and corresponding declines in amphibian populations were variably seen at all elevations along a post-epizootic gradient. Because it is inherently difficult to prove a negative proposition, it can neither be proven that B. dendrobatidis is truly not present where it is not detected nor proven that it is only recently arrived where it is detected. Thus, there will always be doubts about whether B. dendrobatidis is enzootic or invasive. In any case, our results, coupled with current knowledge, suggest most clearly that the disease, chytridiomycosis, may be novel and invasive, and that the pathogen, B. dendrobatidis either is, or is becoming, globally ubiquitous. [ABSTRACT FROM AUTHOR]

Amphibian populations around the world are experiencing unprecedented declines attributed to a chytrid fungal pathogen, Batrachochytrium dendrobatidis. Despite the severity of the crisis, quantitative analyses of the effects of the epidemic on amphibian abundance and diversity have been unavailable as a result of the lack of equivalent data collected before and following disease outbreak. We present a community-level assessment combining long-term field surveys and DNA barcode data describing changes in abundance and evolutionary diversity within the amphibian community of El Cope. Panama. following a disease epidemic and mass-mortality event. The epidemic reduced taxonomic, lineage, and phylogenetic diversity similarly. We discovered that 30 species were lost, including five undescribed species, representing 41% of total amphibian lineage diversity in El Cope. These extirpations represented 33% of the evolutionary history of amphibians within the community, and variation in the degree of population loss and decline among species was random with respect to the community phylogeny. Our approach provides a fast, economical, and informative analysis of loss in a community whether measured by species or phylogenetic diversity. [ABSTRACT FROM AUTHOR]

Chytridiomycosis is a globally emerging disease of amphibians and the leading cause of population declines and extirpations at species-diverse montane sites in Central America. We continued long-term monitoring efforts for the presence of the fungal pathogen Batrachochytrium dendrobatidis ( Bd) and for amphibian populations at two sites in western Panama, and we began monitoring at three new sites to the east. Population declines associated with chytridiomycosis emergence were detected at Altos de Campana National Park. We also detected Bd in three species east of the Panama Canal at Soberanía National Park, and prevalence data suggests that Bd may be enzootic in the lowlands of the park. However, no infected frogs were found further east at Tortí (prevalence <7.5% with 95% confidence). Our results suggest that Panama’s diverse and not fully described amphibian communities east of the canal are at risk. Precise predictions of future disease emergence events are not possible until factors underlying disease emergence, such as dispersal, are understood. However, if the fungal pathogen spreads in a pattern consistent with previous disease events in Panama, then detection of Bd at Tortí and other areas east of the Panama Canal is imminent. Therefore, development of new management strategies and increased precautions for tourism, recreation, and biology are urgently needed. [ABSTRACT FROM AUTHOR]

We conducted a mark-recapture study of three subpopulations of Craugastor punctariolus at a mid-elevation site in central Panama between 1999 and 2005. The study spans a period over which the pathogenic fungus Batrachochytrium dendrobatidis (Bd) was absent from the site, invaded the site and decimated all amphibian populations, and now persists. We quantified natural demographic parameters prior to and during an event of mass mortality due to chytridiomycosis caused by Bd. Prior to the event of mass mortality, all three subpopulations of C. punctariolus were large (19–68 animals/200m), showed a stable age-size distribution, and had high survival. All age-size classes of this species co-occurred on boulder clumps along streams, and adults showed high site fidelity and were likely territorial. Following detection of Bd at this site in late September 2004, four dead C. punctariolus were found infected with Bd and all three subpopulations completely disappeared from this site within 2months. The association of all age-size classes with microhabitats appropriate for survival and growth of Bd likely contributed to the rapid and severe degree of decline of this species at this site. These data provide insight into the patterns and mechanisms of decline within a species due to Bd. [Copyright &y& Elsevier]

Pathogens rarely cause extinctions of host species, and there are few examples of a pathogen changing species richness and diversity of an ecological community by causing local extinctions across a wide range of species. We report the link between the rapid appearance of a pathogenic chytrid fungus Batrachochytrium dendrobatidis in an amphibian community at El Copé, Panama, and subsequent mass mortality and loss of amphibian biodiversity across eight families of frogs and salamanders. We describe an outbreak of chytridiomycosis in Panama and argue that this infectious disease has played an important role in amphibian population declines. The high virulence and large number of potential hosts of this emerging infectious disease threaten global amphibian diversity. [ABSTRACT FROM AUTHOR]

Chytridiomycosis is an emerging infectious disease of amphibians caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd), which has led to devastating declines in amphibian populations worldwide. Current theory predicts that Bd infections are maintained through both reproduction on the host's skin and reinfection from sources outside of the host. To investigate the importance of external reinfection on pathogen burden, we infected captive-bred individuals of the highly susceptible Panamanian Golden Frog, Atelopus glyphus, and wild-caught glass frogs, Espadarana prosoblepon, with Bd. We housed the animals in one of three treatments: individually, in heterospecific pairs, and in conspecific pairs. For 8 weeks, we measured the Bd load and shedding rate of all frogs. We found that Atelopus had high rates of increase in both Bd load and shedding rate, but pathogen growth rates did not differ among treatments. The infection intensity of Espadarana co-housed with Atelopus was indistinguishable from those housed singly and those in conspecific pairs, despite being exposed to a large external source of Bd zoospores. Our results indicate that Bd load in both species is driven by pathogen replication within an individual, with reinfection from outside the host contributing little to the amplification of host fungal load.

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 introduction of next-generation sequencing has allowed for greater understanding of community composition of symbiotic microbial communities. However, determining the function of individual members of these microbial communities still largely relies on culture-based methods. Here, we present results on the phylogenetic distribution of a defensive functional trait of cultured symbiotic bacteria associated with amphibians. Amphibians are host to a diverse community of cutaneous bacteria and some of these bacteria protect their host from the lethal fungal pathogen Batrachochytrium dendrobatidis (Bd) by secreting antifungal metabolites. We cultured over 450 bacterial isolates from the skins of Panamanian amphibian species and tested their interactions with Bd using an in vitro challenge assay. For a subset of isolates, we also completed coculture experiments and found that culturing isolates with Bd had no effect on inhibitory properties of the bacteria, but it significantly decreased metabolite secretion. In challenge assays, approximately 75% of the bacterial isolates inhibited Bd to some extent and these inhibitory isolates were widely distributed among all bacterial phyla. Although there was no clear phylogenetic signal of inhibition, three genera, Stenotrophomonas, Aeromonas and Pseudomonas, had a high proportion of inhibitory isolates (100%, 77% and 73%, respectively). Overall, our results demonstrate that antifungal properties are phylogenetically widespread in symbiotic microbial communities of Panamanian amphibians and that some functional redundancy for fungal inhibition occurs in these communities. We hope that these findings contribute to the discovery and development of probiotics for amphibians that can mitigate the threat of chytridiomycosis., (© 2015 John Wiley & Sons Ltd.)