l-Stereoselective amino acid amidase with broad substrate specificity from Brevundimonas diminuta: characterization of a new member of the leucine aminopeptidase family

Brevundimonas diminuta TPU 5720 produces an amidase acting l-stereoselectively on phenylalaninamide. The enzyme (LaaA_Bd) was purified to electrophoretic homogeneity by ammonium sulfate fractionation and four steps of column chromatography. The final preparation gave a single band on SDS-PAGE with a molecular weight of ≈53,000. The native molecular weight of the enzyme was about 288,000 based on gel filtration chromatography, suggesting that the enzyme is active as a homohexamer. It had maximal activity at 50°C and pH 7.5. LaaA_Bd lost its activity almost completely on dialysis against potassium phosphate buffer (pH 7.0), and the amidase activity was largely restored by the addition of Co²⁺ ions. The enzyme was, however, inactivated in the presence of ethylenediaminetetraacetic acid even in the presence of Co²⁺, suggesting that LaaA_Bd is a Co²⁺-dependent enzyme. LaaA_Bd had hydrolyzing activity toward a broad range of l-amino acid amides including l-phenylalaninamide, l-glutaminamide, l-leucinamide, l-methioninamide, l-argininamide, and l-2-aminobutyric acid amide. Using information on the N-terminal amino acid sequence of the enzyme, the gene encoding LaaA_Bd was cloned from the chromosomal DNA of the strain and sequenced. Analysis of 4,446 bp of the cloned DNA revealed the presence of seven open-reading frames (ORFs), one of which (laaA _Bd ) encodes the amidase. LaaA_Bd is composed of 491 amino acid residues (calculated molecular weight 51,127), and the deduced amino acid sequence exhibits significant similarity to that of ORFs encoding hypothetical cytosol aminopeptidases found in the genomes of Caulobacter crescentus, Bradyrhizobium japonicum, Rhodopseudomonas palustris, Mesorhizobium loti, and Agrobacterium tumefaciens, and leucine aminopeptidases, PepA, from Rickettsia prowazekii, Pseudomonas putida ATCC 12633, and Escherichia coli K-12. The laaA _Bd gene modified in the nucleotide sequence upstream from its start codon was overexpressed in an E. coli transformant. The activity of the recombinant LaaA_Bd in cell-free extracts of the E. coli transformant was 25.9 units mg⁻¹ with l-phenylalaninamide as substrate, which was 50 times higher than that of B. diminuta TPU 5720.     

Amphibian‐killing chytrid in Brazil comprises both locally endemic and globally expanding populations

Chytridiomycosis, caused by the fungus Batrachochytrium dendrobatidis (Bd), is the emerging infectious disease implicated in recent population declines and extinctions of amphibian species worldwide. Bd strains from regions of disease‐associated amphibian decline to date have all belonged to a single, hypervirulent clonal genotype (Bd‐GPL). However, earlier studies in the Atlantic Forest of southeastern Brazil detected a novel, putatively enzootic lineage (Bd‐Brazil), and indicated hybridization between Bd‐GPL and Bd‐Brazil. Here, we characterize the spatial distribution and population history of these sympatric lineages in the Brazilian Atlantic Forest. To investigate the genetic structure of Bd in this region, we collected and genotyped Bd strains along a 2400‐km transect of the Atlantic Forest. Bd‐Brazil genotypes were restricted to a narrow geographic range in the southern Atlantic Forest, while Bd‐GPL strains were widespread and largely geographically unstructured. Bd population genetics in this region support the hypothesis that the recently discovered Brazilian lineage is enzootic in the Atlantic Forest of Brazil and that Bd‐GPL is a more recently expanded invasive. We collected additional hybrid isolates that demonstrate the recurrence of hybridization between panzootic and enzootic lineages, thereby confirming the existence of a hybrid zone in the Serra da Graciosa mountain range of Paraná State. Our field observations suggest that Bd‐GPL may be more infective towards native Brazilian amphibians, and potentially more effective at dispersing across a fragmented landscape. We also provide further evidence of pathogen translocations mediated by the Brazilian ranaculture industry with implications for regulations and policies on global amphibian trade.     

Novel,panzootic and hybrid genotypes of amphibian chytridiomycosis associated with the bullfrog trade

Global amphibian declines are linked with the presence of specific, highly virulent genotypes of the emerging fungal disease chytridiomycosis caused by Batrachochytrium dendrobatidis (Bd) known as the global panzootic lineage (Bd‐GPL). The global trade in amphibians for human consumption is suspected to have facilitated emergence of the disease, but evidence to support this is largely lacking. Here, we investigated the role the Lithobates catesbeianus (North American bullfrog) trade in spreading Bd genotypes by comparing strains associated with L. catesbeianus to a global panel using 36 sequenced loci from multiple chromosomal regions. Most bullfrogs were infected with Bd‐GPL genotypes, but we also detected novel, highly divergent Bd genotypes (Bd‐Brazil) from a live bullfrog in a US market and from native Brazilian anurans in the Atlantic Forest where bullfrogs are widely farmed. Sexual reproduction was also detected for the first time in Bd in the form of a hybrid genotype between the Bd‐GPL and Bd‐Brazil lineages in the Atlantic Forest. Despite the demonstration that ribosomal RNA types in Bd fail to undergo concerted evolution (over 20 sequence types may be found in a single strain), the Bd‐GPL and Bd‐Brazil lineages form largely separate clusters of related internal transcribed spacer (ITS) RNA sequences. Using ITS sequences, we then demonstrate the presence of Bd‐Brazil in Japan, primarily on invasive L. catesbeianus. The finding that Bd is capable of sexual reproduction between panzootic and endemic genotypes emphasizes the risk of international wildlife trade as a source of additional Bd epizootics owing to hybridization.     

Host size influences the effects of four isolates of an amphibian chytrid fungus

Understanding factors that influence host–pathogen interactions is key to predicting outbreaks in natural systems experiencing environmental change. Many amphibian population declines have been attributed to an amphibian chytrid fungus, Batrachochytrium dendrobatidis (Bd). While this fungus is widespread, not all Bd‐positive populations have been associated with declines, which could be attributed to differences in pathogen virulence or host susceptibility. In a laboratory experiment, we examined the effects of Bd isolate origin, two from areas with Bd‐associated amphibian population declines (El Copé, Panama, and California, USA) and two from areas without Bd‐related population declines (Ohio and Maine, USA), on the terrestrial growth and survival of American toad (Anaxyrus americanus) metamorphs reared in larval environments with low or high intraspecific density. We predicted that (1) Bd isolates from areas experiencing declines would have greater negative effects than Bd isolates from areas without declines, and (2) across all isolates, growth and survival of smaller toads from high‐density larval conditions would be reduced by Bd exposure compared to larger toads from low‐density larval conditions. Our results showed that terrestrial survival was reduced for smaller toads exposed to Bd with variation in the response to different isolates, suggesting that smaller size increased susceptibility to Bd. Toads exposed to Bd gained less mass, which varied by isolate. Bd isolates from areas with population declines, however, did not have more negative effects than isolates from areas without recorded declines. Most strikingly, our study supports that host condition, measured by size, can be indicative of the negative effects of Bd exposure. Further, Bd isolates’ impact may vary in ways not predictable from place of origin or occurrence of disease‐related population declines. This research suggests that amphibian populations outside of areas experiencing Bd‐associated declines could be impacted by this pathogen and that the size of individuals could influence the magnitude of Bd's impact.     

Widespread Occurrence of the Chytrid Fungus Batrachochytrium dendrobatidis on Oregon Spotted Frogs (Rana pretiosa)

The pathogen Batrachochytrium dendrobatidis (Bd) has been associated with amphibian declines in multiple continents, including western North America. We investigated Bd prevalence in Oregon spotted frog (Rana pretiosa), a species that has declined across its range in the Pacific Northwest. Polymerase chain reaction analysis of skin swabs indicated that Bd was prevalent within populations (420 of 617 juvenile and adults) and widespread among populations (36 of 36 sites) where we sampled R. pretiosa in Oregon and Washington. We rarely detected Bd in R. pretiosa larvae (2 of 72). Prevalence of Bd in postmetamorphic R. pretiosa was inversely related to frog size. We found support for an interactive effect of elevation and sampling date on Bd: prevalence of Bd generally increased with date, but this effect was more pronounced at lower elevations. We also found evidence that the body condition of juvenile R. pretiosa with Bd decreased after their first winter. Our data indicate that some Oregon spotted frog populations are currently persisting with relatively high Bd prevalence, but the risk posed by Bd is unknown.     

Long‐term endemism of two highly divergent lineages of the amphibian‐killing fungus in the Atlantic Forest of Brazil

The recent global spread of the amphibian‐killing fungus [Batrachochytrium dendrobatidis (Bd)] has been closely tied to anthropogenic activities; however, regional patterns of spread are not completely understood. Using historical samples, we can test whether Bd was a spreading or endemic pathogen in a region within a particular time frame, because those two disease states provide different predictions for the regional demographic dynamics and population genetics of Bd. Testing historical patterns of pathogen prevalence and population genetics under these predictions is key to understanding the evolution and origin of Bd. Focusing on the Atlantic Forest (AF) of Brazil, we used qPCR assays to determine the presence or absence of Bd on 2799 preserved postmetamorphic anurans collected between 1894 and 2010 and used semi‐nested PCRs to determine the frequency of rRNA ITS1 haplotypes from 52 samples. Our earliest date of detection was 1894. A mean prevalence of 23.9% over time and spatiotemporal patterns of Bd clusters indicate that Bd has been enzootic in the Brazilian AF with no evidence of regional spread within the last 116 years. ITS1 haplotypes confirm the long‐term presence of two divergent strains of Bd (BdGPL and Bd‐Brazil) and three spatiotemporally broad genetic demes within BdGPL, indicating that Bd was not introduced into southeast Brazil by the bullfrog trade. Our data show that the evolutionary history and pathogen dynamics of Bd in Brazil is better explained by the endemic pathogen hypothesis.     

Prevalence and genetic diversity of Batrachochytrium dendrobatidis in Central African island and continental amphibian communities

The fungal pathogen Batrachochytrium dendrobatidis (Bd) infects hundreds of amphibian species and is implicated in global amphibian declines. Bd is comprised of several lineages that differ in pathogenicity, thus, identifying which Bd strains are present in a given amphibian community is essential for understanding host–pathogen dynamics. The presence of Bd has been confirmed in Central Africa, yet vast expanses of this region have not yet been surveyed for Bd prevalence, and the genetic diversity of Bd is largely unknown in this part of the world. Using retrospective surveys of museum specimens and contemporary field surveys, we estimated the prevalence of Bd in Central African island and continental amphibian assemblages, and genotyped strains of Bd present in each community. Our sampling of museum specimens included just a few individuals collected in the Gulf of Guinea archipelago prior to 1998, yet one of these individuals was Bd‐positive indicating that the pathogen has been on Bioko Island since 1966. We detected Bd across all subsequent sample years in our study and found modest support for a relationship between host life history and Bd prevalence, a positive relationship between prevalence and host community species richness, and no significant relationship between elevation and prevalence. The Global Panzootic Lineage (BdGPL) was present in all the island and continental amphibian communities we surveyed. Our results are consistent with a long‐term and widespread distribution of Bd in amphibian communities of Gabon and the Gulf of Guinea archipelago.     

Global and endemic Asian lineages of the emerging pathogenic fungus Batrachochytrium dendrobatidis widely infect amphibians in China

Aim Panzootic chytridiomycosis caused by the chytrid fungus Batrachochytrium dendrobatidis (Bd) is the proximate cause of rapid amphibian declines across diverse biomes. While the origin of Bd remains unclear, increasingly the global trade in amphibians is associated with the spread of the infection. Global samples of Bd genotypes from previously unsampled regions are essential to test this hypothesis. In this paper, we present a study of the prevalence and phylogeny of Bd in both invasive and native amphibian species in markets and in the wild in ten provinces of China. Location China. Method We used a nested PCR assay to amplify the ribosomal internal transcribed spacer region of Bd followed by sequencing. Result Our results showed 246 of 2734 amphibians testing positive for Bd, with 157 positive samples in the wild (7.6%) and 89 in markets (13.5%). 30 haplotypes of Bd were identified, including 20 first detections. Introduced Lithobates catesbeianus had the highest prevalence of infection and the largest number of Bd haplotypes in both the wild and markets. Phylogenetic analysis based on 73 haplotypes (57 from Asia and 16 from other continents) showed that a unique, well‐supported, basal haplotype is present in Asia. Phylogeographical analyses revealed that some geographical structure exists amongst a subset of global haplotypes. Main conclusions Strains of the basal haplotype infected Babina pleuraden, an amphibian that is endemic to China, and Andrias japonicus, endemic to Japan, showing that Southeast Asia harbours a novel endemic lineage of amphibian‐associated Bd. Our data suggest that Bd in Asia pre‐dates the expansion of a globalized lineage of Bd, a finding that is indicative of a broader association of amphibians and chytrids than has previously been recognized. More genetic data from Bd isolates are needed to reveal the phylogenetic relationship of Bd in China compared to that found elsewhere.     

What Drives Chytrid Infections in Newt Populations? Associations with Substrate,Temperature, and Shade

The pathogenic chytrid fungus Batrachochytrium dendrobatidis (Bd) is considered responsible for the population declines and extinctions of hundreds of amphibian species worldwide. The panzootic was likely triggered by human-assisted spread, but once the pathogen becomes established in a given region, its distribution is probably determined by local drivers. To assess the relative importance of potential drivers of infection in red-spotted newts (Notophthalmus viridescens), we measured Bd levels in 16 populations throughout central Pennsylvania. Infected individuals were detected in all but four populations, indicating that Bd is widespread in this region. We quantified local factors hypothesized to influence Bd, and found that infection levels were best predicted by the proportion of the pond substrate consisting of leaf litter or vegetation, along with a significant effect of water temperature. Bd infection in amphibians is temperature-dependent, and one possible explanation of the apparent substrate effect is that tree cover and vegetation provide shade, reducing the availability of shallow, warm-water patches in which newts might reduce or clear Bd infections. Alternatively, leaf litter and emergent vegetation might increase Bd infection more directly, perhaps by providing substrates for environmental growth of the fungus. We also observed a curvilinear relationship between Bd load and snout-vent length (a proxy for age), hinting that newts might develop acquired resistance to Bd infection. Though correlational, these results add to a growing body of evidence suggesting that environmental temperature is an important driver of Bd infection dynamics.     

A tale of two lineages: unexpected,long‐term persistence of the amphibian‐killing fungus in Brazil

For the past 17 years, scientists have been compiling a list of amphibian species susceptible to infection by the amphibian‐killing chytrid fungus, Batrachochytrium dendrobatidis (Bd), all over the world, with >500 species infected on every continent except Antarctica (Olson et al. 2013 ). Where Bd has been found, the impacts on amphibians has been one of two types: either Bd arrives into a naïve amphibian population followed by a mass die‐off and population declines (e.g. Lips et al. 2006 ), or Bd is present at some moderate prevalence, usually infecting many species but at apparently nonlethal intensities for a long time. In this issue of Molecular Ecology, Rodriguez et al. ( 2014 ) discover that the Atlantic Coastal Forest of Brazil is home to two Bd lineages: the Global Pandemic Lineage (Bd‐GPL) – the strain responsible for mass die‐offs and population declines – and a lineage endemic to Brazil (Bd‐Bz). Even more surprising was that both lineages have been present in this area for the past 100 years, making these the oldest records of Bd infecting amphibians. The team also described a moderate but steady prevalence of ~20% across all sampled anuran families for over 100 years, indicating that Brazil has been in an enzootic disease state for over a century. Most amphibians were infected with Bd‐GPL, suggesting this lineage may be a better competitor than Bd‐Bz or may be replacing the Bd‐Bz lineage. Rodriguez et al. ( 2014 ) also detected likely hybridization of the two Bd lineages, as originally described by Schloegel et al. ( 2012 ).     

Confronting inconsistencies in the amphibian‐chytridiomycosis system: implications for disease management

Chytridiomycosis, caused by the pathogenic fungus Batrachochytrium dendrobatidis (Bd), is one of the largest threats to wildlife and is putatively linked to the extirpation of numerous amphibians. Despite over a decade of research on Bd, conflicting results from a number of studies make it difficult to forecast where future epizootics will occur and how to manage this pathogen effectively. Here, we emphasize how resolving these conflicts will advance Bd management and amphibian conservation efforts. We synthesize current knowledge on whether Bd is novel or endemic, whether amphibians exhibit acquired resistance to Bd, the importance of host resistance versus tolerance to Bd, and how biotic (e.g. species richness) and abiotic factors (e.g. climate change) affect Bd abundance. Advances in our knowledge of amphibian–chytrid interactions might inform the management of fungal pathogens in general, which are becoming more common and problematic globally.     

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.     

Endemic and introduced haplotypes of Batrachochytrium dendrobatidis in Japanese amphibians: sink or source?

The global emergence of the amphibian chytrid pathogen Batrachochytrium dendrobatidis (Bd) is one of the most compelling, and troubling, examples of a panzootic. Only discovered in 1998, Bd is now recognized as a proximate driver of global declines in amphibian diversity and is now widely acknowledged as a key threatening process for this ancient class of vertebrates. Moreover, Bd has become a member of a small group of highly virulent multihost pathogens that are known to have had effects on entire vertebrate communities and the ecosystem‐level effects of Bd‐driven amphibian declines are starting to emerge as a consequence of regional decreases in amphibian diversity. Despite the speed at which this species of aquatic chytrid has become a focus of research efforts, major questions still exist about where Bd originated, how it spreads, where it occurs and what are Bd’s effects on populations and species inhabiting different regions and biomes. In this issue, Goka et al. (2009 ) make an important contribution by publishing the first nationwide surveillance for Bd in Asia. Although previous data had suggested that amphibians in Asia are largely uninfected by Bd, these surveys were limited in their extent and few firm conclusions could be drawn about the true extent of infection. Goka et al. herein describe a systematic surveillance of Japan for both native and exotic species in the wild, as well as amphibians housed in captivity, using a Bd‐specific nested PCR reaction on a sample of over 2600 amphibians. Their results show that Bd is widely prevalent in native species across Japan in at least three of the islands that make up the archipelago, proving for the first time that Asia harbours Bd.     

Known and estimated distribution in Mexico of Batrachochytrium dendrobatidis,a pathogenic fungus of amphibians

Chytridiomycosis caused by fungus Batrachochytrium dendrobatidis (Bd) is one of the decline global causes of amphibians. Currently, it is distributed throughout a broad range of climates and ecosystems around the world. An epidemic wave of chytridiomycosis began in North America, resulting in population decline and local extinction of many species, reconfiguring species composition of amphibian communities in the Americas. In Mexico, Bd has caused an amphibian population decrease, and its potential distribution area has not been determined. We reviewed the number of species infected, obtaining Bd frequency of infection by land use and vegetation type, and by elevation range. We examined the known distribution of Bd, estimated the potential distribution, and obtained the bioclimate variables relevant for Bd. Our results indicate that in Mexico, Bd has been detected in 78 species of amphibians in 10 families, from 29 different land use and vegetation types, with cloud forest having the highest number of cases (139) and infected species (15). Bd occurs over an elevation range of 1–3,300 m asl and is most frequent at 1,200–1,500 m asl (36%). In addition to the regions previously described as suitable for Bd, our model included desert, coastal, and tropical forest regions, revealing an increase in the area where amphibians could be at risk of infection. Distribution of Bd is mainly associated with temperature of the wettest quarter and potential evapotranspiration of the warmer quarter. We offer an estimate of the ideal conditions for Bd in Mexico, also information for future studies on Bd and the conservation of amphibians. Abstract in Spanish is available with online material.     

First Record of Batrachochytrium dendrobatidis Infecting Four Frog Families from Peninsular Malaysia

The fungal pathogen Batrachochytrium dendrobatidis (Bd) infects amphibians on every continent where they occur and is linked to the decline of over 200 amphibian species worldwide. At present, only three published Bd surveys exist for mainland Asia, and Bd has been detected in South Korea alone. In this article, we report the first survey for Bd in Peninsular Malaysia. We swabbed 127 individuals from the six amphibian families that occur on Peninsular Malaysia, including two orders, 27 genera, and 47 species. We detected Bd on 10 out of 127 individuals from four of five states and five of 11 localities, placing the 95% confidence interval for overall prevalence at 4–14%. We detected no variation in Bd prevalence among regions, elevations, or taxonomic groups. The infection intensity ranged from 1 to 157,000 genome equivalents. The presence of Bd infections in native species without clinical signs of disease suggests that Bd may be endemic to the region. Alternately, Bd may have been introduced from non-native amphibians because of the substantial amphibian food trade in Peninsular Malaysia. Under both scenarios, management efforts should be implemented to limit the spread of non-native Bd and protect the tremendous amphibian diversity in Peninsular Malaysia.     

Species distribution models predict the geographic expansion of an enzootic amphibian pathogen

Globally, numerous amphibian species have declined due to the introduction of the chytrid fungus Batrachochytrium dendrobatidis (Bd). However, the understanding of the spatiotemporal dynamics remains incomplete. Therefore, estimating the current geographic distribution of Bd is urgently needed, especially in countries like Costa Rica, where susceptible species are still recovering from Bd‐driven declines. We conducted model tuning and spatial analysis to compare the habitat suitability for epizootic and enzootic Bd in Costa Rica and to identify data‐deficient regions, opportunistic sampling, and Bd hotspots. Our dataset combined two methods of detection (histology and PCR methods) for a total of 451 Bd‐positive records from 34 localities. We found that the distribution of enzootic Bd in Costa Rica increased 60% since previous estimates in the early 2000s and extended to highlands and dry lowlands that were considered unsuitable for Bd. We also found that Bd is common across protected lands (80%) and within the herpetological provinces containing the highest amphibian richness and endemism in Costa Rica. Opportunistic sampling of Bd has focused on sites where epizootics occurred with the strongest intensity, leading to deficient or absent sampling across the Talamanca Range, the Nicoya Peninsula, and the northern lowlands. Our results showed that PCR increased the power of Bd detection in lowlands and favored the identification of Bd hotspots across the Caribbean side of Costa Rica. Our results add to the understanding of disease spread during enzootics and can be used to identify new hotspots for disease to mitigate future outbreaks of this pathogen. Abstract in Spanish is available with online material     

Experimental evolution alters the rate and temporal pattern of population growth in Batrachochytrium dendrobatidis,a lethal fungal pathogen of amphibians

Virulence of infectious pathogens can be unstable and evolve rapidly depending on the evolutionary dynamics of the organism. Experimental evolution can be used to characterize pathogen evolution, often with the underlying objective of understanding evolution of virulence. We used experimental evolution techniques (serial transfer experiments) to investigate differential growth and virulence of Batrachochytrium dendrobatidis (Bd), a fungal pathogen that causes amphibian chytridiomycosis. We tested two lineages of Bd that were derived from a single cryo‐archived isolate; one lineage (P10) was passaged 10 times, whereas the second lineage (P50) was passaged 50 times. We quantified time to zoospore release, maximum zoospore densities, and timing of zoospore activity and then modeled population growth rates. We also conducted exposure experiments with a susceptible amphibian species, the common green tree frog (Litoria caerulea) to test the differential pathogenicity. We found that the P50 lineage had shorter time to zoospore production (T_min), faster rate of sporangia death (d_s), and an overall greater intrinsic population growth rate (λ). These patterns of population growth in vitro corresponded with higher prevalence and intensities of infection in exposed Litoria caerulea, although the differences were not significant. Our results corroborate studies that suggest that Bd may be able to evolve relatively rapidly. Our findings also challenge the general assumption that pathogens will always attenuate in culture because shifts in Bd virulence may depend on laboratory culturing practices. These findings have practical implications for the laboratory maintenance of Bd isolates and underscore the importance of understanding the evolution of virulence in amphibian chytridiomycosis.     

Landscape epidemiology of Batrachochytrium dendrobatidis in central California

Amphibian chytridiomycosis (caused by Batrachochytrium dendrobatis; Bd) was first identified in 1998 and has since been implicated in numerous amphibian declines worldwide. Most researchers have since investigated broad‐scale geographic and taxonomic occurrences of the pathogen in tropical lotic or cool montane systems. In this study, we analyzed how environmental factors, land use practices, and landscape structure may affect the dynamics of the pathogen's distribution in a landscape dominated by lentic systems within a region of Mediterranean climate. We quantified the occurrence of Bd testing the six resident amphibian species that occur in 54 isolated perennial and ephemeral ponds in central California between May and June annually from 2004 to 2007. The geographic distribution of Bd within the landscape varied markedly between years. Inter‐annual variation in climate affected the pond landscape structure indicating that climate conditions indirectly influence the distribution of the pathogen. Fourteen ponds, 12 perennial and 2 ephemeral, were positive for Bd≥3 yr of the study and were treated as Bd hotpots for comparative purposes. Occurrences of Bd within the landscape were spatially autocorrelated and ponds within ~1000–1500 m of Bd hotspots were more likely to test positive. Local land use, (presence/absence of grazing or recreational activity and developed lands), did not influence Bd status of a pond, indicating that the most likely means of Bd transmission between ponds may be waterfowl and/or amphibians.     

Historical dynamics of Batrachochytrium dendrobatidis in Amazonia

The Amazon forest is known for its astonishing amphibian diversity, yet the potential distribution and underlying impacts of the most important amphibian pathogen is unknown for most of Amazonia. In this retrospective survey of preserved Leptodactylus frogs, collected over a 119 yr period, we used quantitative PCR to detect the fungal pathogen Batrachochytrium dendrobatidis (Bd) and performed spatial scan analyses to identify spatiotemporal clusters of Bd. We also quantified the potential effect of environmental factors on the likelihood of Bd occurrence and generated an updated suitability map for Bd in the Amazon that included our retrospective sampling. We detected Bd in lowland Amazon as early as 1935, in the state of Pará, Brazil, and we found low prevalence (～ 3.8%) over time. We identified two statistically significant spatiotemporal clusters of Bd: a recent and narrow cluster in the Amazon River delta and a spatiotemporally broad cluster in the southern edge of Amazon and Brazilian savanna. Furthermore, we found an increase in Bd‐positive samples in the southwestern Amazon after the 1990s, coinciding with reported amphibian declines in neighboring high elevation sites on Andean slopes of Peru. Spatial regressions indicated that higher human interference, higher precipitation, and lower temperatures were significant predictors of Bd occurrence. Environmental niche modeling predicted some narrow areas of suitable climates along the Amazon's periphery and generally low climatic suitability for Bd in the central Amazon; although, we found clusters of Bd‐positive samples with unexpectedly high infection loads in areas of predicted low suitability. Our study indicates that accelerated human development may put Amazonian amphibians at risk from Bd introductions, and it highlights the potential need to monitor Bd dynamics near Amazonian port cities.     

Reduced skin bacterial diversity correlates with increased pathogen infection intensity in an endangered amphibian host

The fungal pathogen Batrachochytrium dendrobatidis (Bd) infects the skin of amphibians and has caused severe declines and extinctions of amphibians globally. In this study, we investigate the interaction between Bd and the bacterial skin microbiome of the endangered Sierra Nevada yellow‐legged frog, Rana sierrae, using both culture‐dependent and culture‐independent methods. Samples were collected from two populations of R. sierrae that likely underwent Bd epizootics in the past, but that continue to persist with Bd in an enzootic disease state, and we address the hypothesis that such “persistent” populations are aided by mutualistic skin microbes. Our 16S rRNA metabarcoding data reveal that the skin microbiome of highly infected juvenile frogs is characterized by significantly reduced species richness and evenness, and by strikingly lower variation between individuals, compared to juveniles and adults with lower infection levels. Over 90% of DNA sequences from the skin microbiome of highly infected frogs were derived from bacteria in a single order, Burkholderiales, compared to just 54% in frogs with lower infection levels. In a culture‐dependent Bd inhibition assay, the bacterial metabolites we evaluated all inhibited the growth of Bd. Together, these results illustrate the disruptive effects of Bd infection on host skin microbial community structure and dynamics, and suggest possible avenues for the development of anti‐Bd probiotic treatments.