Pioneer access of the foam nest bacterial community of Leptodactylidae frogs and its biotechnological potential.

Many anuran amphibians deposit their eggs in foam nests, biostructures that help protect the eggs and tadpoles from predators. Currently, there are no other identification and description studies of the cultivable microbiota role in the nests of the Leptodactylid frogs such as Physalaemus cuvieri, Leptodactylus vastus and Adenomera hylaedactyla. This study aimed to isolate and identify the culturable bacteria from these three anuran species' nests, as well as to prospect enzymes produced by this microbiota. Foam nests samples and environmental samples were diluted and viable cell count was determined. Bacterial morphotypes from foam nest samples were isolated through spread plate technique. Isolates' DNAs were extracted followed by rRNA 16S gene amplification and Sanger sequencing. To evaluate their enzymatic potential, the isolates were cultured in ATGE medium supplemented with starch (0.1% w/v), gelatin (3% w/v) and skimmed milk (1% w/v), to verify amylase and protease activity. A total of 183 bacterial morphotypes were isolated, comprising 33 bacterial genera. Proteobacteria phylum was the most abundant in all the three nests (79%). The genera Pseudomonas and Aeromonas were the most abundant taxon in P. cuvieri and L. vastus. In A. Hylaedactyla, were Enterobacter and Bacillus. Regarding enzymatic activities, 130 isolates displayed protease activity and 45 isolates were positive for amylase activity. Our results provide unprecedented information concerning culturable bacterial microbiota of the foam nests of the Leptodactylid frogs, as well as their potential for biomolecules of biotechnological interest.

Widespread occurrence of the amphibian chytrid panzootic lineage in Uruguay is constrained by climate.

The amphibian chytrid fungus Batrachochytrium dendrobatidis (Bd) causes chytridiomycosis, a disease among the main causes of amphibian declines worldwide. However, Bd studies on Neotropical amphibians from temperate areas are scarce. We present a comprehensive survey of Bd in Uruguay, in temperate central eastern South America, carried out between 2006 and 2014. Skin swabs of 535 specimens of 21 native and exotic frogs were tested by PCR. We used individual-level data to examine the relationship between infection, climatic variables, and their effects on body condition and the number of prey items found in stomach contents. Infection was widespread in free-ranging anurans with an overall prevalence of 41.9%, detected in 15 native species, wild American bullfrogs Aquarana catesbeiana, and captive specimens of Ceratophrys ornata and Xenopus laevis. Three haplotypes of the Bd ITS region were identified in native amphibians, all belonging to the global panzootic lineage (BdGPL), of which only one was present in exotic hosts. Despite high infection frequencies in different anurans, we found no evidence of morbidity or mortality attributable to chytridiomycosis, and we observed no discernible impact on body condition or consumed prey. Climatic conditions at the time of our surveys suggested that the chance of infection is associated with monthly mean temperature, mean humidity, and total precipitation. Temperatures below 21°C combined with moderate humidity and pronounced rainfall may increase the likelihood of infection. Multiple haplotypes of BdGPL combined with high frequencies of infection suggest an enzootic pattern in native species, underscoring the need for continued monitoring.

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.

Chytrid fungus in amphibians from the lowland Brazilian Amazon.

Infectious diseases are one of the main threats to biodiversity. The fungus Batrachochytrium dendrobatidis (Bd) is associated with several amphibian losses around the globe, and environmental conditions may dictate the success of pathogen spread. The Brazilian Amazon has been considered climatically unsuitable for chytrid fungus, but additional information on Bd dynamics in this ecoregion is still lacking. We sampled 462 amphibians (449 anurans, 4 caudatans and 9 caecilians), representing 57 species from the Brazilian Amazon, and quantified Bd infections using qPCR. We tested whether abiotic variables predicted the risk of Bd infections, and tested for relationships between biotic variables and Bd. Finally, we experimentally tested the effects of Bd strains CLFT 156 and CLFT 102 (from the southern and northern Atlantic Forest, respectively) on Atelopus manauensis. We detected higher Bd prevalence than those previously reported for the Brazilian Amazon, and positive individuals in all 3 orders of amphibians sampled. Both biotic and abiotic predictors were related to prevalence, and no variable explained infection load. Moreover, we detected higher Bd prevalence in forested than open areas, while the host's reproductive biology was not a factor. We detected higher mortality in the experimental group infected with CLFT 156, probably because this strain was isolated from a region characterized by discrepant climatic conditions (latitudinally more distant) when compared with the host's sampling site in Amazon. The lowland Brazilian Amazon is still underexplored and future studies targeting all amphibian orders are essential to better understand Bd infection dynamics in this region.

Presence of invasive American bullfrogs may reduce infectious disease in a native frog species.

Amphibians breeding in aquatic environments may encounter a myriad of threats during their life cycle. One species known to prey on native amphibians in aquatic habitats is the invasive North American bullfrog Lithobates catesbeianus, which, besides being a voracious predator and competitor, often acts as a pathogen carrier and disease superspreader because it tolerates high infection loads of the frog-killing fungus Batrachochytrium dendrobatidis (Bd). Here, we hypothesized that the presence of the bullfrogs in microcosms should either (1) decrease Bd disease severity in native frog species by discouraging them from using the aquatic environment, or (2) increase the mortality of the native species. We tested these 2 mutually exclusive hypotheses by co-housing the snouted treefrog Scinax x-signatus (native to our study area) with L. catesbeianus in the laboratory, exposing them to Bd, and using qPCR analysis to quantify the resulting Bd infection loads in the native frogs. Our experiment had the following replicated treatments: (1) native-only treatment (3 individuals of S. x-signatus), (2) native-predominant treatment (2 S. x-signatus + 1 L. catesbeianus), and (3) exotic-predominant treatment (1 S. x-signatus + 2 L. catesbeianus). We found that Bd infection loads in the native S. x-signatus were highest in the native-only treatment, and lowest in the exotic-predominant treatment, indicating that bullfrogs may discourage native frogs from occupying the aquatic habitat, thus reducing encounter rates between native frogs and the waterborne pathogen. This effect could be driven by the bullfrogs' predatory behavior and their high philopatry to aquatic habitats. Our results highlight that predation risk adds to the complexity of host-species interactions in Bd epidemiology.

Interpopulation differences in male reproductive effort drive the population dynamics of a host exposed to an emerging fungal pathogen.

Compensatory recruitment is a key demographic mechanism that has allowed the coexistence of populations of susceptible amphibians with Batrachochytrium dendrobatidis (Bd), a fungus causing one of the most devastating emerging infectious disease ever recorded among vertebrates. However, the underlying processes (e.g. density-dependent increase in survival at early life stages, change in reproductive traits) as well as the level of interpopulation variation in this response are poorly known. We explore potential mechanisms of compensatory recruitment in response to Bd infection by taking advantage of an amphibian system where male reproductive traits are easy to quantify in free-living populations. The Southern Darwin's frog Rhinoderma darwinii is a vocal sac-brooding species that exhibits a high susceptibility to lethal Bd infection. Using a 7-year capture-recapture study at four populations with contrasting Bd infection status (one high prevalence, one low prevalence and two Bd-free populations), we evaluated whether Bd-positive populations exhibited a higher adult recruitment and a higher male reproductive effort than Bd-negative populations. We also estimated population growth rates to explore whether recruitment compensated for the negative impacts of Bd on the survival of adults. In addition, we evaluated a potential demographic signal of compensatory recruitment (i.e. positive relationship between the proportion of juveniles and Bd prevalence) in response to Bd infection using raw count data from 13 R. darwinii populations. The high Bd prevalence population exhibited the highest male reproductive effort and the highest recruitment among the four monitored populations. This led to a growing population during the study period despite high mortality of adult hosts. In contrast, males from the population with low Bd prevalence had a low reproductive effort and this population, which had the lowest adult recruitment, was declining during the study period despite adults having a higher survival in comparison to the high Bd prevalence population. We also found a demographic signal of compensatory recruitment in response to Bd infection in our broader analysis of 13 R. darwinii populations. Our study underlines the importance of interpopulation variation in life-history strategies on the fate of host populations after infectious disease emergence. Our results also suggest that an increase in reproductive effort can be one of the processes underlying compensatory recruitment in populations of Bd-susceptible amphibians.

Genetic variation of Batrachochytrium dendrobatidis is linked to skin bacterial diversity in the Pacific treefrog Hyliola regilla (hypochondriaca).

Symbiotic bacterial communities are crucial to combating infections and contribute to host health. The amphibian skin microbiome plays an important role in protecting their hosts against pathogens such as Batrachochytrium dendrobatidis (Bd), one of the causative agents of chytridiomycosis, which is responsible for dramatic amphibian population declines worldwide. Although symbiotic skin bacteria are known to inhibit Bd growth, an understanding of the relationship between Bd genetic variability, environmental conditions, and skin bacterial communities is limited. Therefore, we examined the associations between Bd infection load, Bd genetic diversity and skin bacterial communities in five populations of Hyliola regilla (hypochondriaca) from environmentally contrasting sites in Baja California, Mexico. We observed differences in Bd genetics and infection load among sites and environments. Genetic analysis of Bd isolates revealed patterns of spatial structure corresponding to the five sites sampled. Amphibian skin bacterial diversity and community structure differed among environments and sites. Bacterial community composition was correlated with Bd genetic differences and infection load, with specific bacterial taxa enriched on infected and un-infected frogs. Our results indicate that skin-associated bacteria and Bd strains likely interact on the host skin, with consequences for microbial community structure and Bd infection intensity.

Cutaneous Bacteria of Confiscated Telmatobius culeus in Lima, Peru.

The Lake Titicaca frog is endangered due to threats such as water pollution, introduced species, and overharversting for markets, where people consume them as frog juice. This study, conducted June to November 2012, aimed to determinate the bacteria microflora living on the skin of frogs confiscated from the La Parada market, Lima, Peru, and housed individually in the Laboratory of Wildlife at the Faculty of Veterinary Medicine and Zootechnic of the Universidad Peruana Cayetano Heredia in Lima, Peru. Samples collected with sterile swabs and cultured on blood, tryptic soy, and MacConkey agars were investigated using commercially available test kits, to investigate the commonly encountered bacterial and potentially zoonotic microorganisms associated with their consumption. We found three species of zoonotic concern in the genus Vibro: Vibrio alginolyticus, Vibro cholerae, and Vibro fluvialis. Other Gram-negative species cultured included two different colonies of Aeromonas hydrophila, or Aeromonas caviae or Aeromonas sobria; Pseudomona luteola; one example of Weeksella virosa or Empedobacter brevis; and Citrobacter freundii. Gram-positive bacteria detected were Staphylococcus spp., Micrococcus spp., and Erysipelothrix rhusiopathiae. We recommend against the consumption of this frog due to the pathogens it may carry that could cause serious illness among consumers and in vendors who handle animals.

Niche Contraction of an Endangered Frog Driven by the Amphibian Chytrid Fungus.

Introduced pathogens can alter the geographic distribution of susceptible host species. For example, Batrachochytrium dendrobatidis (Bd) is a fungal pathogen that has been linked to the global decline and extinction of numerous amphibian species during the last four decades. A growing number of studies have described the distribution of Bd and susceptible hosts across the globe; however, knowledge on how Bd may shape the climatic niche of susceptible species is still missing. We estimated the effect of Bd on the geographic distribution and niche dynamics of the critically endangered lowland robber frog (Craugastor ranoides) in Costa Rica. We found a reduction of 98% in the geographic range of this species by 1995, following the epizootic outbreaks of Bd that affected Costa Rica in the 1980 and early 1990s. We also quantified niche contraction and found that the species is currently restricted to dry and warm environments that have been considered unsuitable for Bd. Our results contribute to the understanding of how emerging pathogens shape the climatic niches and geographic distribution of susceptible species.

Skin microbiome correlates with bioclimate and Batrachochytrium dendrobatidis infection intensity in Brazil's Atlantic Forest treefrogs.

In Brazil's Atlantic Forest (AF) biodiversity conservation is of key importance since the fungal pathogen Batrachochytrium dendrobatidis (Bd) has led to the rapid loss of amphibian populations here and worldwide. The impact of Bd on amphibians is determined by the host's immune system, of which the skin microbiome is a critical component. The richness and diversity of such cutaneous bacterial communities are known to be shaped by abiotic factors which thus may indirectly modulate host susceptibility to Bd. This study aimed to contribute to understanding the environment-host-pathogen interaction determining skin bacterial communities in 819 treefrogs (Anura: Hylidae and Phyllomedusidae) from 71 species sampled across the AF. We investigated whether abiotic factors influence the bacterial community richness and structure on the amphibian skin. We further tested for an association between skin bacterial community structure and Bd co-occurrence. Our data revealed that temperature, precipitation, and elevation consistently correlate with richness and diversity of the skin microbiome and also predict Bd infection status. Surprisingly, our data suggest a weak but significant positive correlation of Bd infection intensity and bacterial richness. We highlight the prospect of future experimental studies on the impact of changing environmental conditions associated with global change on environment-host-pathogen interactions in the AF.

Microbiological and cytological characterization of coelomic fluid from three captive endangered amphibian Gastrotheca species with edema syndrome: preliminary analysis.

OBJECTIVE: Edema syndrome is highly prevalent but under researched in captive frogs around the world. The objective of the present study was to characterize at a basic microbiological and cytological level of the bacteria of the edema fluid of 20 individuals of the genus Gastrotheca to determine the presence of possible anaerobic and aerobic bacteria. RESULTS: Fourteen types of bacteria were identified in the edema fluid, 12 of them at the species level (Pasteurella haemolytica, Hafnia alvei, Enterobacter agglomerans, Aeromonas hydrophila, Pseudomonas fluorescens, Burkholderia pseudomallei, Salmonella arizonae, Enterobacter gergoviae, Enterobacter sakazakii, Yersinia enterocolitica, Klebsiella oxytoca, and Klebsiella ozaenae) and two at the genus level (Enterococcus spp. and Streptococcus spp.). The most frequently identified cells were lymphocytes (37.7% in females and 46.4% in males), erythrocytes (23.5% in females and 17.5% in males) and neutrophils (4.2% in females and 2.8% in males). Finally, no relationship was found between the data obtained and the sex of the individuals studied.

Widespread chytrid infection across frogs in the Peruvian Amazon suggests critical role for low elevation in pathogen spread and persistence.

Outbreaks of emerging infectious diseases are becoming more frequent as climate changes wildlife communities at unprecedented rates, driving population declines and raising concerns for species conservation. One critical disease is the global pandemic of chytridiomycosis in frogs, which can be caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd). Although there is clear evidence for Bd-induced mortality across high-elevation frog communities, little attention is given to the role of lowlands in Bd's persistence and spread because low elevations are assumed to be too warm to harbor significant levels of Bd. Here, we report widespread Bd infection across 80 frog species from three sites in the lowland Peruvian Amazon, an area with no documented Bd-related amphibian declines. Despite observing no clinical signs of infection in the field, we found that 24-46% of individuals were infected per site (up to ≈105,000 zoospore equivalents per frog) by three Bd strains from the global pandemic lineage (Bd-GPL). We also found collection site and seasonal effects to be only weak predictors of Bd prevalence and load, with lower elevation and drier habitats marginally decreasing both prevalence and load. We found no further effect of host phylogeny, ecotype, or body size. Our results showing high and widespread prevalence across a lowland tropical ecosystem contradict the expectations based on the global pattern of pathogenicity of Bd that is largely restricted to higher elevations and colder temperatures. These findings imply that the lowlands may play a critical role in the spread and persistence of Bd over time and space.

Low-load pathogen spillover predicts shifts in skin microbiome and survival of a terrestrial-breeding amphibian.

Wildlife disease dynamics are strongly influenced by the structure of host communities and their symbiotic microbiota. Conspicuous amphibian declines associated with the waterborne fungal pathogen Batrachochytrium dendrobatidis (Bd) have been observed in aquatic-breeding frogs globally. However, less attention has been given to cryptic terrestrial-breeding amphibians that have also been declining in tropical regions. By experimentally manipulating multiple tropical amphibian assemblages harbouring natural microbial communities, we tested whether Bd spillover from naturally infected aquatic-breeding frogs could lead to Bd amplification and mortality in our focal terrestrial-breeding host: the pumpkin toadlet Brachycephalus pitanga. We also tested whether the strength of spillover could vary depending on skin bacterial transmission within host assemblages. Terrestrial-breeding toadlets acquired lethal spillover infections from neighbouring aquatic hosts and experienced dramatic but generally non-protective shifts in skin bacterial composition primarily attributable to their Bd infections. By contrast, aquatic-breeding amphibians maintained mild Bd infections and higher survival, with shifts in bacterial microbiomes that were unrelated to Bd infections. Our results indicate that Bd spillover from even mildly infected aquatic-breeding hosts may lead to dysbiosis and mortality in terrestrial-breeding species, underscoring the need to further investigate recent population declines of terrestrial-breeding amphibians in the tropics.

Viscosin-like lipopeptides from frog skin bacteria inhibit Aspergillus fumigatus and Batrachochytrium dendrobatidis detected by imaging mass spectrometry and molecular networking.

Amphibian populations worldwide have declined and in some cases become extinct due to chytridiomycosis, a pandemic disease caused by the fungus Batrachochytrium dendrobatidis; however, some species have survived these fungal epidemics. Previous studies have suggested that the resistance of these species is due to the presence of cutaneous bacteria producing antifungal metabolites. As our understanding of these metabolites is still limited, we assessed the potential of such compounds against human-relevant fungi such as Aspergillus. In this work we isolated 201 bacterial strains from fifteen samples belonging to seven frog species collected in the highlands of Panama and tested them against Aspergillus fumigatus. Among the 29 bacterial isolates that exhibited antifungal activity, Pseudomonas cichorii showed the greatest inhibition. To visualize the distribution of compounds and identify them in the inhibition zone produced by P. cichorii, we employed MALDI imaging mass spectrometry (MALDI IMS) and MS/MS molecular networking. We identified viscosin and massetolides A, F, G and H in the inhibition zone. Furthermore, viscosin was isolated and evaluated in vitro against A. fumigatus and B. dendrobatidis showing MIC values of 62.50 µg/mL and 31.25 µg/mL, respectively. This is the first report of cyclic depsipeptides with antifungal activity isolated from frog cutaneous bacteria.

Antibacterial activity of the skin secretion of Phyllomedusa azurea (Anura: Hylidae) from the Central Brazil Cerrado / Actividad antibacterial de secreciones de la piel de Phyllomedusa azurea (Anura: Hylidae) en Cerrado, Brasil centro

Abstract Phyllomedusa azurea is a frog species well distributed geographically in South America, including Brazilian biomes as Pantanal and Cerrado. Compared with other anurans from the Phyllomedusinae family, there are few reports on the bioactive potential of skin-derived molecules from this species. In this perspective, the aim of the present study was to evaluate the in vitro antibacterial activity of skin secretion of P. azurea by detection of Minimum Inhibitory Concentration (MIC) of the growth of bacterial indicator strains and to determine if occurs a changing in the bacterial cell envelope permeability. The MIC determination was carried out by the microdilution plate method. The absorbance was measured and analyzed statistically using the t-test to compare two groups (0.05 % of significance). The impact of the crude extract on cell envelope permeability of Staphylococcus aureus ATCC 25923 was conducted by the crystal violet assay, and the absorbance was measured spectrophotometry followed by the calculation of the crystal violet uptake percentage. The specific MIC for S. aureus ATCC 25923 and Escherichia coli ATCC 25922 was 31.25 µg/mL, while for Bacillus subtilis ATCC 6633 was 125 µg/mL and Pseudomonas aeruginosa ATCC 27853 was 250 µg/mL. The treatment with crescent concentrations of frog skin secretion increased the crystal violet uptake by S. aureus ATCC 25923 cells, suggesting an action on the cell plasma membrane. The results demonstrated that the skin secretion of P. azurea presents antibacterial activity and merit further investigations to characterize the bioactive molecules.(AU)

Resumen P. azurea es una especie de rana bien distribuida geográficamente en América del Sur, que incluye biomas brasileños como Pantanal y Cerrado. En comparación con otros anuros de Phyllomedusinae, existen pocos informes sobre el potencial bioactivo de las moléculas derivadas de la piel de esta especie. En esta perspectiva, el objetivo del presente estudio fue evaluar la actividad antibacteriana in vitro de la secreción de la piel de P. azurea mediante la detección de la Concentración Inhibitoria Mínima (CIM) del crecimiento de cepas indicadoras bacterianas y determinar si ocurre un cambio en la permeabilidad de la envoltura celular bacteriana. La determinación de MIC se llevó a cabo mediante el método de la placa de microdilución. La absorbancia se midió y se analizó estadísticamente mediante la prueba t para comparar dos grupos (0.05 de significancia). El impacto del extracto crudo sobre la permeabilidad de la envoltura celular de Staphylococcus aureus ATCC 25923 se realizó mediante el ensayo de cristal violeta, y se midió la absorbancia mediante espectrofotometría seguida del cálculo del porcentaje de absorción de violeta cristal. La CIM específica para S. aureus ATCC 25923 y Escherichia coli ATCC 25922 fue 31.25 μg / ml, mientras que para Bacillus subtilis ATCC 6633 de 125 μg / ml y Pseudomonas aeruginosa ATCC 27853 de 250 μg / ml. El tratamiento con concentraciones crecientes de secreción de piel de rana aumentó la absorción de violeta cristal por las células de S. aureus ATCC 25923, sugiriendo una acción sobre la membrana plasmática de la célula. Los resultados demostraron que la secreción de la piel de P. azurea presenta actividad antibacteriana y amerita más investigaciones para caracterizar las moléculas bioactivas.(AU)

Community richness of amphibian skin bacteria correlates with bioclimate at the global scale.

Animal-associated microbiomes are integral to host health, yet key biotic and abiotic factors that shape host-associated microbial communities at the global scale remain poorly understood. We investigated global patterns in amphibian skin bacterial communities, incorporating samples from 2,349 individuals representing 205 amphibian species across a broad biogeographic range. We analysed how biotic and abiotic factors correlate with skin microbial communities using multiple statistical approaches. Global amphibian skin bacterial richness was consistently correlated with temperature-associated factors. We found more diverse skin microbiomes in environments with colder winters and less stable thermal conditions compared with environments with warm winters and less annual temperature variation. We used bioinformatically predicted bacterial growth rates, dormancy genes and antibiotic synthesis genes, as well as inferred bacterial thermal growth optima to propose mechanistic hypotheses that may explain the observed patterns. We conclude that temporal and spatial characteristics of the host's macro-environment mediate microbial diversity.

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.

External Reinfection of a Fungal Pathogen Does not Contribute to Pathogen Growth.

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.

Occurrence of Batrachochytrium dendrobatidis in anurans of the Mediterranean region of Baja California, México.

Chytridiomycosis is caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd) and is regarded as one of the most significant threats to global amphibian populations. In México, Bd was first reported in 2003 and has now been documented in 13 states. We visited 33 localities and swabbed 199 wild-caught anurans from 7 species (5 native, 2 exotic) across the Mediterranean region of the state of Baja California. Using quantitative PCR, Bd was detected in 94 individuals (47.2% of samples) at 25 of the 33 survey localities for 5 native and 1 exotic frog species. The exotic Xenopus laevis was the only species that tested completely negative for Bd. We found that remoteness, distance to agricultural land, and elevation were the best positive predictors of Bd presence. These are the first Bd-positive results for the state of Baja California, and its presence should be regarded as an additional conservation threat to the region's native frog species.

Shifts in disease dynamics in a tropical amphibian assemblage are not due to pathogen attenuation.

Infectious diseases rarely end in extinction. Yet the mechanisms that explain how epidemics subside are difficult to pinpoint. We investigated host-pathogen interactions after the emergence of a lethal fungal pathogen in a tropical amphibian assemblage. Some amphibian host species are recovering, but the pathogen is still present and is as pathogenic today as it was almost a decade ago. In addition, some species have defenses that are more effective now than they were before the epidemic. These results suggest that host recoveries are not caused by pathogen attenuation and may be due to shifts in host responses. Our findings provide insights into the mechanisms underlying disease transitions, which are increasingly important to understand in an era of emerging infectious diseases and unprecedented global pandemics.