Variation in phenotype and virulence among enzootic and panzootic amphibian chytrid lineages

The Global Panzootic Lineage of the fungus Batrachochytrium dendrobatidis (Bd-GPL) is threatening amphibians worldwide. In contrast, four lineages (Bd-Brazil, Bd-CH, Bd-Cape, and Bd-Korea) that diverged early in the history of Bd have not yet been directly linked to amphibian declines. Bd likely evolves in response to strong selective pressure imposed by hosts and the environment, leading to differences among pathogen phenotypes and genotypes that may directly affect virulence. Here, we report on variation in phenotype, genotype, and virulence of Bd-Brazil and Bd-GPL. Specifically, we (i) used a controlled infection experiment to compare virulence between one Bd-Brazil and three Bd-GPL isolates on a North American amphibian host (Lithobates sylvaticus), (ii) tested for relative phenotypic and genotypic differentiation among Bd isolates from Brazil, and (iii) tested for possible correlations between environmental variables and Bd phenotypes. We found substantial variation in virulence among Bd-GPL isolates and found that our Bd-Brazil isolate showed virulence comparable to an average North American Bd-GPL. North American hosts infected with a Bd-GPL isolate from Panama did not show significant mortality. Bd phenotypes varied significantly across sampling locations; these phenotypes were neither spatially clustered nor correlated with any environmental variables. Additionally, we found a surprising lack of correlation between genotypic divergence and zoospore and zoosporangium sizes in our sample. Although Bd-Brazil was less virulent infecting L. sylvaticus than one Bd-GPL isolate, this endemic lineage still caused ∼50% mortality in our experimental North American hosts. This indicates that Bd-Brazil has the potential to kill amphibians if introduced to naïve wild populations. Our findings underscore that characterizing virulence of multiple Bd isolates and lineages is important for understanding the evolutionary history and diversity of Bd.     

Correlates of virulence in a frog-killing fungal pathogen: evidence from a California amphibian decline

The fungal pathogen Batrachochytrium dendrobatidis (Bd) has caused declines and extinctions in amphibians worldwide, and there is increasing evidence that some strains of this pathogen are more virulent than others. While a number of putative virulence factors have been identified, few studies link these factors to specific epizootic events. We documented a dramatic decline in juvenile frogs in a Bd-infected population of Cascades frogs (Rana cascadae) in the mountains of northern California and used a laboratory experiment to show that Bd isolated in the midst of this decline induced higher mortality than Bd isolated from a more stable population of the same species of frog. This highly virulent Bd isolate was more toxic to immune cells and attained higher density in liquid culture than comparable isolates. Genomic analyses revealed that this isolate is nested within the global panzootic lineage and exhibited unusual genomic patterns, including increased copy numbers of many chromosomal segments. This study integrates data from multiple sources to suggest specific phenotypic and genomic characteristics of the pathogen that may be linked to disease-related declines.     

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.     

Local phenotypic variation in amphibian-killing fungus predicts infection dynamics

Environmental factors can limit the distribution of organisms if they are not able to respond through phenotypic plasticity or local adaptation. Batrachochytrium dendrobatidis (Bd) is a broadly distributed pathogen, which shows spatially patterned genotypic and phenotypic variation; however, information on the functional consequences of this variation on disease dynamics in natural hosts is limited. We genotyped and quantified variation in Bd phenotypes across an elevational gradient and quantified host infection dynamics at each site. All Bd strains were members of the global panzootic lineage yet differed in phenotype. We hypothesize that this phenotypic variance results from adaptive processes due to the interaction between pathogen, hosts, and environment. We detected a correlation between zoospore and zoosporangia sizes and a positive association between zoosporangia size and Bd prevalence. Given that Bd phenotype predicted disease status in our wild populations, we developed an index to identify critical environments where the fungus could be more deleterious.     

Disentangling host,pathogen, and environmental determinants of a recently emerged wildlife disease: lessons from the first 15 years of amphibian chytridiomycosis research

The amphibian fungal disease chytridiomycosis, which affects species across all continents, recently emerged as one of the greatest threats to biodiversity. Yet, many aspects of the basic biology and epidemiology of the pathogen, Batrachochytrium dendrobatidis (Bd), are still unknown, such as when and from where did Bd emerge and what is its true ecological niche? Here, we review the ecology and evolution of Bd in the Americas and highlight controversies that make this disease so enigmatic. We explore factors associated with variance in severity of epizootics focusing on the disease triangle of host susceptibility, pathogen virulence, and environment. Reevaluating the causes of the panzootic is timely given the wealth of data on Bd prevalence across hosts and communities and the recent discoveries suggesting co‐evolutionary potential of hosts and Bd. We generate a new species distribution model for Bd in the Americas based on over 30,000 records and suggest a novel future research agenda. Instead of focusing on pathogen “hot spots,” we need to identify pathogen “cold spots” so that we can better understand what limits the pathogen''s distribution. Finally, we introduce the concept of “the Ghost of Epizootics Past” to discuss expected patterns in postepizootic host communities.     

Genetic characterization of chytrids isolated from larval amphibians collected in central and east Texas

Chytridiomycosis, an emerging infectious disease caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd), has caused amphibian population declines worldwide. Bd was first described in the 1990s and there are still geographic gaps in the genetic analysis of this globally distributed pathogen. Relatively few genetic studies have focused on regions where Bd exhibits low virulence, potentially creating a bias in our current knowledge of the pathogen's genetic diversity. Disease-associated declines have not been recorded in Texas (USA), yet Bd has been detected on amphibians in the state. These strains have not been isolated and characterized genetically; therefore, we isolated, cultured, and genotyped Bd from central Texas and compared isolates to a panel of previously genotyped strains distributed across the Western Hemisphere. We also isolated other chytrids from east Texas not known to infect amphibians. To identify larval amphibian hosts, we sequenced part of the COI gene. Among 37 Bd isolates from Texas, we detected 19 unique multi-locus genotypes, but found no genetic structure associated with host species, Texas localities, or across North America. Isolates from central Texas exhibit high diversity and genetically cluster with BdGPL isolates from the western U.S. that have caused amphibian population declines. This study genetically characterizes isolates of Bd from the south central U.S. and adds to the global knowledge of Bd genotypes.     

Pathogen pollution and the emergence of a deadly amphibian pathogen

Imagine a single pathogen that is responsible for mass mortality of over a third of an entire vertebrate class. For example, if a single pathogen were causing the death, decline and extinction of 30% of mammal species (including humans), the entire world would be paying attention. This is what has been happening to the world's amphibians – the frogs, toads and salamanders that are affected by the chytrid fungal pathogen, Batrachochytrium dendrobatidis (referred to as Bd), which are consequently declining at an alarming rate. It has aptly been described as the worst pathogen in history in terms of its effects on biodiversity (Kilpatrick et al. 2010). The pathogen was only formally described about 13 years ago (Longcore et al. 1999), and scientists are still in the process of determining where it came from and investigating the question: why now? Healthy debate has ensued as to whether Bd is a globally endemic organism that only recently started causing high mortality due to shifting host responses and/or environmental change (e.g. Pounds et al. 2006) or whether a virulent strain of the pathogen has rapidly disseminated around the world in recent decades, affecting new regions with a vengeance (e.g. Morehouse et al. 2003; Weldon et al. 2004; Lips et al. 2008). We are finally beginning to shed more light on this question, due to significant discoveries that have emerged as a result of intensive DNA‐sequencing methods comparing Bd isolates from different amphibian species across the globe. Evidence is mounting that there is indeed a global panzootic lineage of Bd (BdGPL) in addition to what appear to be more localized endemic strains (Fisher et al. 2009; James et al. 2009; Farrer et al. 2011). Additionally, BdGPL appears to be a hypervirulent strain that has resulted from the hybridization of different Bd strains that came into contact in recent decades, and is now potentially replacing the less‐virulent endemic strains of the pathogen (Farrer et al. 2011). In a new study published in this issue of Molecular Ecology, Schloegel et al. (2012) identify an additional unique Bd lineage that is endemic to the Atlantic Brazilian rainforests (Bd‐Brazil) and provide striking evidence that the Bd‐Brazil lineage has sexually recombined with the BdGPL lineage in an area where the two lineages likely came into contact as a result of classic anthropogenically mediated ‘pathogen pollution’(see below). Fungal pathogens, including Bd, have the propensity to form recombinant lineages when allopatric populations that have not yet formed genetic reproductive barriers are provided with opportunities to intermingle, and virulent strains may be selected for because they tend to be highly transmissible (Fisher et al. 2012). As Schloegel et al. (2012) point out, the demonstrated ability for Bd to undergo meiosis may also mean that it has the capacity to form a resistant spore stage (as yet undiscovered), based on extrapolation from other sexually reproducing chytrids that all have spore stages.     

Incapacitating effects of fungal coinfection in a novel pathogen system

As globalization lowers geographic barriers to movement, coinfection with novel and enzootic pathogens is increasingly likely. Novel and enzootic pathogens can interact synergistically or antagonistically, leading to increased or decreased disease severity. Here we examine host immune responses to coinfection with two closely related fungal pathogens: Batrachochytrium dendrobatidis (Bd) and Batrachochytrium salamandrivorans (Bsal). Both pathogens have had detrimental effects on amphibian populations, with Bd now largely enzootic, while Bsal is currently spreading and causing epizootics. Recent experimental work revealed that newts coinfected with Bd and Bsal had significantly higher mortality than those infected with either pathogen alone. Here we characterize host immunogenomic responses to chytrid coinfection relative to single infection. Across several classes of immune genes including pattern recognition receptors, cytokines, and MHC, coinfected host gene expression was weakly upregulated or comparable to that seen in single Bd infection, but significantly decreased when compared to Bsal infection. Combined with strong complement pathway downregulation and keratin upregulation, these results indicate that coinfection with Bd and Bsal compromises immune responses active against Bsal alone. As Bsal continues to invade naïve habitats where Bd is enzootic, coinfection will be increasingly common. If other Bd‐susceptible species in the region have similar responses, interactions between the two pathogens could cause severe population and community‐level declines.     

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.     

Expression Profiling the Temperature-Dependent Amphibian Response to Infection by Batrachochytrium dendrobatidis

Amphibians are experiencing a panzootic of unprecedented proportions caused by the emergence of Batrachochytrium dendrobatidis (Bd). However, all species are not equally at risk of infection, and risk is further modified by environmental variables, specifically temperature. In order to understand how, and when, hosts mount a response to Bd we analysed infection dynamics and patterns of gene expression in the model amphibian species Silurana (Xenopus) tropicalis. Mathematical modelling of infection dynamics demonstrate the existence of a temperature-dependent protective response that is largely independent of the intrinsic growth-rate of Bd. Using temporal expression-profiling by microarrays and qRT-PCR, we characterise this response in the main amphibian lymphoid tissue, the spleen. We demonstrate that clearance of Bd at the host-optimal temperature is not clearly associated with an adaptive immune response, but rather is correlated with the induction of components of host innate immunity including the expression of genes that are associated with the production of the antimicrobial skin peptide preprocareulein (PPCP) as well as inflammatory responses. We find that adaptive immunity appears to be lacking at host-optimal temperatures. This suggests that either Bd does not stimulate, or suppresses, adaptive immunity, or that trade-offs exist between innate and adaptive limbs of the amphibian immune system. At cold temperatures, S. tropicalis loses the ability to mount a PPCP-based innate response, and instead manifests a more pronounced inflammatory reaction that is characterised by the production of proteases and higher pathogen burdens. This study demonstrates the temperature-dependency of the amphibian response to infection by Bd and indicates the influence that changing climates may exert on the ectothermic host response to pathogens.     

Inhibitory bacteria reduce fungi on early life stages of endangered Colorado boreal toads (Anaxyrus boreas)

Increasingly, host-associated microbiota are recognized to mediate pathogen establishment, providing new ecological perspectives on health and disease. Amphibian skin-associated microbiota interact with the fungal pathogen, Batrachochytrium dendrobatidis (Bd), but little is known about microbial turnover during host development and associations with host immune function. We surveyed skin microbiota of Colorado''s endangered boreal toads (Anaxyrus boreas), sampling 181 toads across four life stages (tadpoles, metamorphs, subadults and adults). Our goals were to (1) understand variation in microbial community structure among individuals and sites, (2) characterize shifts in communities during development and (3) examine the prevalence and abundance of known Bd-inhibitory bacteria. We used high-throughput 16S and 18S rRNA gene sequencing (Illumina MiSeq) to characterize bacteria and microeukaryotes, respectively. Life stage had the largest effect on the toad skin microbial community, and site and Bd presence also contributed. Proteobacteria dominated tadpole microbial communities, but were later replaced by Actinobacteria. Microeukaryotes on tadpoles were dominated by the classes Alveolata and Stramenopiles, while fungal groups replaced these groups after metamorphosis. Using a novel database of Bd-inhibitory bacteria, we found fewer Bd-inhibitory bacteria in post-metamorphic stages correlated with increased skin fungi, suggesting that bacteria have a strong role in early developmental stages and reduce skin-associated fungi.     

Diversity in growth patterns among strains of the lethal fungal pathogen <Emphasis Type="Italic">Batrachochytrium dendrobatidis</Emphasis> across extended thermal optima

The thermal sensitivities of organisms regulate a wide range of ecological interactions, including host–parasite dynamics. The effect of temperature on disease ecology can be remarkably complex in disease systems where the hosts are ectothermic and where thermal conditions constrain pathogen reproductive rates. Amphibian chytridiomycosis, caused by the pathogen Batrachochytrium dendrobatidis (Bd), is a lethal fungal disease that is influenced by temperature. However, recent temperature studies have produced contradictory findings, suggesting that our current understanding of thermal effects on Bd may be incomplete. We investigated how temperature affects three different Bd strains to evaluate diversity in thermal responses. We quantified growth across the entire thermal range of Bd, and beyond the known thermal limits (T _max and T _min). Our results show that all Bd strains remained viable and grew following 24 h freeze (?12 °C) and heat shock (28 °C) treatments. Additionally, we found that two Bd strains had higher logistic growth rates (r) and carrying capacities (K) at the upper and lower extremities of the temperature range, and especially in low temperature conditions (2–3 °C). In contrast, a third strain exhibited relatively lower growth rates and carrying capacities at these same thermal extremes. Overall, our results suggest that there is considerable variation among Bd strains in thermal tolerance, and they establish a new thermal sensitivity profile for Bd. More generally, our findings point toward important questions concerning the mechanisms that dictate fungal thermal tolerances and temperature-dependent pathogenesis in other fungal disease systems.     

Amphibian Symbiotic Bacteria Do Not Show a Universal Ability To Inhibit Growth of the Global Panzootic Lineage of Batrachochytrium dendrobatidis

Microbiomes associated with multicellular organisms influence the disease susceptibility of hosts. The potential exists for such bacteria to protect wildlife from infectious diseases, particularly in the case of the globally distributed and highly virulent fungal pathogen Batrachochytrium dendrobatidis of the global panzootic lineage (B. dendrobatidis GPL), responsible for mass extinctions and population declines of amphibians. B. dendrobatidis GPL exhibits wide genotypic and virulence variation, and the ability of candidate probiotics to restrict growth across B. dendrobatidis isolates has not previously been considered. Here we show that only a small proportion of candidate probiotics exhibited broad-spectrum inhibition across B. dendrobatidis GPL isolates. Moreover, some bacterial genera showed significantly greater inhibition than others, but overall, genus and species were not particularly reliable predictors of inhibitory capabilities. These findings indicate that bacterial consortia are likely to offer a more stable and effective approach to probiotics, particularly if related bacteria are selected from genera with greater antimicrobial capabilities. Together these results highlight a complex interaction between pathogens and host-associated symbiotic bacteria that will require consideration in the development of bacterial probiotics for wildlife conservation. Future efforts to construct protective microbiomes should incorporate bacteria that exhibit broad-spectrum inhibition of B. dendrobatidis GPL isolates.     

Non-detection of mycoviruses in amphibian chytrid fungus (Batrachochytrium dendrobatidis) from Australia

Mycoviruses may influence the pathogenicity of disease-causing fungi. Although mycoviruses have been found in some chytrid fungi, limited testing has not detected them in Batrachochytrium dendrobatidis (Bd), the cause of the devastating amphibian disease, chytridiomycosis. Here we conducted a survey for mycovirus presence in 38 Bd isolates from Australia (n = 31), Brazil (n = 5) and South Korea (n = 2) with a combination of modern high-throughput sequencing and conventional dsRNA cellulose chromatography. Mycoviruses were not detected in any isolates. This result was unexpected, given the long evolutionary history of Bd, as well as the high prevalence of mycoviruses in related fungal species. Given our widespread sampling in Australia and the limited number of Bd introductions, we suggest that mycoviruses are uncommon or absent from Australian Bd. Testing more isolates from regions where Bd originated, as well as regions with high diversity or low fungal virulence may identify mycoviruses that could aid in disease control.     

<Emphasis Type="Italic">Batrachochytrium dendrobatidis</Emphasis> and the Decline and Survival of the Relict Leopard Frog

Epizootic disease caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd) is a major driver of amphibian declines, yet many amphibians declined before the pathogen was described. The Relict Leopard Frog, Rana onca (=Lithobates onca), was nearly extinct, with the exception of populations within a few geothermal springs. Growth of Bd, however, is limited by high water temperature, and geothermal springs may have provided refuge during outbreaks of chytridiomycosis. We conducted field surveys and laboratory experiments to assess the susceptibility of R. onca to Bd. In the field, we found Bd at one of the two areas where remnant populations of R. onca still occur, but not in the other. In the laboratory, we infected juvenile frogs from these two areas with two hypervirulent Bd isolates associated with declines in other ranid species. In our experiments, these Bd isolates did not affect survivorship of R. onca and most infections (64%) were cleared by the end of the experiments. We propose that R. onca either has inherent resistance to Bd or has recently evolved such resistance. These results may be important for conservation efforts aimed at establishing new populations of R. onca across a landscape where Bd exists. Resistance, however, varies among life stages, and we also did not assess Bd from the local environment. We caution that the resistance we observed for young frogs under laboratory conditions may not translate to the situation for R. onca in the wild.     

Chytridiomycosis and Amphibian Population Declines Continue to Spread Eastward in Panama

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.     

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.     

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.     

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.     

Spatiotemporal adaptive evolution of an MHC immune gene in a frog-fungus disease system

Genetic diversity of major histocompatibility complex (MHC) genes is linked to reduced pathogen susceptibility in amphibians, but few studies also examine broad spatial and temporal patterns of MHC and neutral genetic diversity. Here, we characterized range-wide MHC diversity in the Northern leopard frog, Rana pipiens, a species found throughout North America that is experiencing disease-related declines. We used previously sequenced neutral markers (mitochondrial DNA and microsatellites), sequenced an expressed MHC class IIß gene fragment, and measured infection prevalence and intensity of the global fungal pathogen Batrachochytrium dendrobatidis (Bd) across 14 populations. Four populations were sampled across two decades, enabling temporal comparisons of selection and demography. We recovered 37 unique MHC alleles, including 17 that were shared across populations. Phylogenetic and population genetic patterns between MHC and neutral markers were incongruent, and five MHC codon positions associated with peptide binding were under positive selection. MHC heterozygosity, but not neutral marker heterozygosity, was a significant factor explaining spatial patterns of Bd prevalence, whereas only environmental variables predicted Bd intensity. MHC allelic richness (AR) decreased significantly over time but microsatellite-based AR did not, highlighting a loss of functional immunogenetic diversity that may be associated with Bd selective pressures. MHC supertype 4 was significantly associated with an elevated risk of Bd infection, whereas one supertype 2 allele was associated with a nearly significant reduced risk of Bd. Taken together, these results provide evidence that positive selection contributes to MHC class IIß evolution in R. pipiens and suggest that functional MHC differences across populations may contribute to disease adaptation.Subject terms: Genetic variation, Immunogenetics