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The skin microbiome is an important part of amphibian immune defenses and protects against pathogens such as the chytrid fungus Batrachochytrium dendrobatidis (Bd), which causes the skin disease chytridiomycosis. Alteration of the microbiome by anthropogenic factors, like pesticides, can impact this protective trait, disrupting its functionality. Chlorothalonil is a widely used fungicide that has been recognized as having an impact on amphibians, but so far, no studies have investigated its effects on amphibian microbial communities. In the present study, we used the amphibian Lithobates vibicarius from the montane forest of Costa Rica, which now appears to persist despite ongoing Bd-exposure, as an experimental model organism. We used 16S rRNA amplicon sequencing to investigate the effect of chlorothalonil on tadpoles’ skin microbiome. We found that exposure to chlorothalonil changes bacterial community composition, with more significant changes at a higher concentration. We also found that a larger number of bacteria were reduced on tadpoles’ skin when exposed to the higher concentration of chlorothalonil. We detected four presumed Bd-inhibitory bacteria being suppressed on tadpoles exposed to the fungicide. Our results suggest that exposure to a widely used fungicide could be impacting host-associated bacterial communities, potentially disrupting an amphibian protective trait against pathogens.

Amphibians are globally threatened by emerging infectious diseases, and ranaviruses are among the most concerning pathogens to threaten species in the wild. We sampled for ranaviruses in wild amphibians at 8 sites in Costa Rica, spanning broad climatic zones and taxonomic associations. Seven of these sites are inhabited by highly threatened amphibian species that persist at low global population sizes after population declines due to amphibian chytridiomycosis. One of the surveyed sites is occupied by an introduced amphibian species, which is relatively rare in Central America but may be an important pathway for long-distance transport of ranaviruses. We detected ranavirus using quantitative polymerase chain reaction in 16.3% of the 243 individuals and among 5 of our 8 sites, but not at the site with the introduced species. Infection prevalence varied among species and sites, but not with mean annual temperature or mean annual precipitation. Infection intensity did not vary with species, site, temperature, or precipitation. Our results show that ranavirus infection is spatially widespread in Costa Rica, affecting a broad range of host species, and occurs across climatic zones—though we encountered no mortality or morbidity in our sampled species. Ranaviruses are known to cause intermittent mass mortality in amphibian populations, and the threatened species sampled here are likely vulnerable to population impacts from emerging ranaviruses. Therefore, we believe the potential impacts of ranaviruses on amphibian populations in tropical regions have likely been underestimated, and that they should be viewed as a potential major stressor to threatened amphibians in tropical regions.

Amphibians have declined around the world in recent years, in parallel with the emergence of an epidermal disease called chytridiomycosis, caused by the chytrid fungus Batrachochytrium dendrobatidis (Bd). This disease has been associated with mass mortality in amphibians worldwide, including in Costa Rica, and Bd is considered an important contributor to the disappearance of this group of vertebrates. While many species are susceptible to the disease, others show tolerance and manage to survive infection with the pathogen. We evaluated the pathogen Bd circulating in Costa Rica and the capacity of amphibian skin bacteria to inhibit the growth of the pathogen in vitro. We isolated and characterized – genetically and morphologically – several Bd isolates from areas with declining populations of amphibians. We determined that the circulating chytrid fungus in Costa Rica belongs to the virulent strain Bd-GPL-2, which has been related to massive amphibian deaths worldwide; however, the isolates obtained showed genetic and morphological variation. Furthermore, we isolated epidermal bacteria from 12 amphibian species of surviving populations, some in danger of extinction, and evaluated their inhibitory activity against the collection of chytrid isolates. Through bioassays we confirmed the presence of chytrid-inhibitory bacterial genera in Costa Rican amphibians. However, we observed that the inhibition varied between different isolates of the same bacterial genus, and each bacterial isolation inhibited fungal isolation differently. In total, 14 bacterial isolates belonging to the genera Stenotrophomonas , Streptomyces , Enterobacter , Pseudomonas and Klebsiella showed inhibitory activity against all Bd isolates. Given the observed variation both in the pathogen and in the bacterial inhibition capacity, it is highly relevant to include local isolates and to consider the origin of the microorganisms when performing in vivo infection tests aimed at developing and implementing mitigation strategies for chytridiomycosis.

Global amphibian biodiversity has declined dramatically in the past 4 decades, and many amphibian species have declined to near extinction as a result of emergence of the amphibian chytrid fungus, Batrachochytrium dendrobatidis (Bd). However, persistent or recovering populations of several amphibian species have recently been rediscovered, and such populations may illustrate how amphibian species that are highly susceptible to chytridiomycosis may survive in the presence of Bd. We conducted field surveys for Bd infection in 7 species of Costa Rican amphibians (all species that have declined to near extinction but for which isolated populations persist) to characterize infection profiles in highly Bd-susceptible amphibians post-decline. We found highly variable patterns in infection, with some species showing low prevalence (~10%) and low infection intensity and others showing high infection prevalence (>80%) and either low or high infection intensity. Across sites, infection rates were negatively associated with mean annual precipitation, and infection intensity across sites was negatively associated with mean average temperatures. Our results illustrate that even the most Bd-susceptible amphibians can persist in Bd-enzootic ecosystems, and that multiple ecological or evolutionary mechanisms likely exist for host-pathogen co-existence between Bd and the most Bd-susceptible amphibian species. Continued monitoring of these populations is necessary to evaluate population trends (continuing decline, stability, or population growth). These results should inform efforts to mitigate impacts of Bd on amphibians in the field.

Declines of amphibian populations have been a worldwide issue of concern for the scientific community during the last several decades. Efforts are being carried out to elucidate factors related to this phenomenon. Among these factors, pathogens, climate change, and environmental pollution have been suggested as possible causes. Regarding environmental pollutants, some pesticides are persistent in the environment and capable of being transported long distances from their release point. In Costa Rica, some pesticides have been detected in protected areas, at locations where amphibian populations have declined. Information about toxicity of pesticides used in Costa Rican agriculture to amphibians is still scarce, particularly for native species.Toxicity tests with chlorothalonil, a fungicide intensively used in Costa Rica, were carried out exposing tadpoles of three Costa Rican native species: Agalychnis callidryas, Isthmohyla pseudopuma, and Smilisca baudinii in order to evaluate acute and chronic toxicity as well as the biomarkers cholinesterase activity (ChE), glutathione-S transferase activity (GST), and lipid peroxidation (LPO).96-h LC50: 26.6 (18.9-35.8) μg/L to A. callidryas, 25.5 (21.3-29.7) μg/L to I pseudopuma and 32.3 (26.3-39.7) μg/L to S. baudinii were determined for chlorothalonil. These three species of anurans are among the most sensitive to chlorothalonil according to the literature. Besides, GST was induced in S. baudinii after exposure to sub-lethal concentrations of chlorothalonil while evisceration occurred in S. baudinii and A. callidryas tadpoles exposed to lethal concentrations of the fungicide. Chronic exposure to sub-lethal concentrations accelerated development in S. baudinii and caused lesions in tail of S. baudinii and I. pseudopuma tadpoles. Our results demonstrate that chlorothalonil is highly toxic to native amphibian species and that low concentrations can cause biochemical responses related to phase II of biotransformation and effects on development.

We report the rediscovery of a declared extinct frog,Craugastor escoces, at the edge of a protected forest in the Juan Castro Blanco National Park, Alajuela, Costa Rica. This species, which is endemic to Costa Rica, had not been observed since 1986. The rediscovery of this species comes after 30 years without being seen and 12 years of being declared extinct. The site where we found the species is located in the west of the central mountain region of Costa Rica, which is >15 km away from its known distribution. Our finding adds another species to the list of the few rediscovered frogs declared extinct. Knowing thatC. escocescan still be found triggers the need for conservation strategies to help the species survive.

Emerging infectious diseases are a growing threat to biodiversity worldwide. Outbreaks of the infectious disease chytridiomycosis, caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd), are implicated in the decline and extinction of numerous amphibian species. In Costa Rica, a major decline event occurred in 1987, more than two decades before this pathogen was discovered. The loss of many species in Costa Rica is assumed to be due to Bd-epizootics, but there are few studies that provide data from amphibians in the time leading up to the proposed epizootics. In this study, we provide new data on Bd infection rates of amphibians collected throughout Costa Rica, in the decades prior to the epizootics. We used a quantitative PCR assay to test for Bd presence in 1016 anuran museum specimens collected throughout Costa Rica. The earliest specimen that tested positive for Bd was collected in 1964. Across all time periods, we found an overall infection rate (defined as the proportion of Bd-positive individuals) of 4%. The number of infected individuals remained relatively low across all species tested and the range of Bd-positive specimens was shown to be geographically constrained up until the 1980s; when epizootics are hypothesized to have occurred. After that time, infection rate increased three-fold, and the range of specimens tested positive for Bd increased, with Bd-positive specimens collected across the entire country. Our results suggest that Bd dynamics in Costa Rica are more complicated than previously thought. The discovery of Bd's presence in the country preceding massive declines leads to a number of different hypotheses: 1) Bd invaded Costa Rica earlier than previously known, and spread more slowly than previously reported; 2) Bd invaded multiple times and faded out; 3) an endemic Bd lineage existed; 4) an earlier Bd lineage evolved into the current Bd lineage or hybridized with an invasive lineage; or 5) an earlier Bd lineage went extinct and a new invasion event occurred causing epizootics. To help visualize areas where future studies should take place, we provide a Bd habitat suitability model trained with local data. Studies that provide information on genetic lineages of Bd are needed to determine the most plausible spatial-temporal, host-pathogen dynamics that could best explain the epizootics resulting in amphibian declines in Costa Rica and throughout Central America.