An endogenous DNA virus in an amphibian-killing fungus associated with pathogen genotype and virulence.

The global panzootic lineage (GPL) of the pathogenic fungus Batrachochytrium dendrobatidis (Bd) has caused severe amphibian population declines, yet the drivers underlying the high frequency of GPL in regions of amphibian decline are unclear. Using publicly available Bd genome sequences, we identified multiple non-GPL Bd isolates that contain a circular Rep-encoding single-stranded (CRESS)-like DNA virus, which we named Bd DNA virus 1 (BdDV-1). We further sequenced and constructed genome assemblies with long read sequences to find that the virus is integrated into the nuclear genome in some strains. Attempts to cure virus-positive isolates were unsuccessful; however, phenotypic differences between naturally virus-positive and virus-negative Bd isolates suggested that BdDV-1 decreases the growth of its host in vitro but increases the virulence of its host in vivo. BdDV-1 is the first-described CRESS DNA mycovirus of zoosporic true fungi, with a distribution inversely associated with the emergence of the panzootic lineage.

Coevolution of a generalist pathogen with many hosts: the case of the amphibian chytrid Batrachochytrium dendrobatidis.

Generalist pathogens maintain infectivity in numerous hosts; how this broad ecological niche impacts host-pathogen coevolution remains to be widely explored. Batrachochytrium dendrobatidis (Bd) is a highly generalist pathogenic fungus that has caused devastating declines in hundreds of amphibian species worldwide. This review examines amphibian chytridiomycosis host-pathogen interactions and available evidence for coevolution between Bd and its numerous hosts. We summarize recent evidence showing that Bd genotypes vary in geographic distribution and virulence, and that amphibian species also vary in Bd susceptibility according to their geographic distribution. How much variation can be explained by phenotypic plasticity or genetic differences remains uncertain. Recent research suggests that Bd genotypes display preferences for specific hosts and that some hosts are undergoing evolution as populations rebound from Bd outbreaks. Taken together, these findings suggest the potential for coevolution to occur and illuminate a path for addressing open questions through integrating historical and contemporary genetic data.

Host immune responses to enzootic and invasive pathogen lineages vary in magnitude, timing, and efficacy.

Infectious diseases of wildlife continue to pose a threat to biodiversity worldwide, yet pathogens are far from uniform in virulence or host disease outcome. Within the same pathogen species, virulence can vary considerably depending on strain or lineage, in turn eliciting variable host responses. One pathogen that has caused extensive biodiversity loss is the amphibian-killing fungus, Batrachochytrium dendrobatidis (Bd), which is comprised of a globally widespread hypervirulent lineage (Bd-GPL), and multiple geographically restricted, enzootic lineages. Whereas host immunogenomic responses to Bd-GPL have been characterized in a number of amphibian species, immunogenomic responses to geographically restricted, enzootic Bd lineages are less clear. To examine lineage-specific host immune responses to Bd, we exposed a species of pumpkin toadlet, Brachycephalus pitanga, which is endemic to Brazil's Southern Atlantic Forest, to either the Bd-GPL or the enzootic Bd-Asia-2/Brazil (hereafter Bd-Brazil) lineage. Using temporal samples from early, mid, and late infection stages, we quantified functional immunogenomic responses over the course of infection using differential gene expression tests and coexpression network analyses. Host immune responses varied significantly with Bd lineage. Relative to controls, toadlet responses to Bd-Brazil were weak at early infection (25 genes significantly differentially expressed), peaked by mid-stage infection (414 genes), and were nearly fully resolved by late-stage infection (nine genes). In contrast, responses to Bd-GPL were magnified and delayed; toadlets significantly differentially expressed 111 genes early, 87 genes at mid-stage infection, and 726 genes by late-stage infection relative to controls. Given that infection intensity did not vary between mid- and late-stage disease in either Bd-Brazil or Bd-GPL treatments, this suggests that pumpkin toadlets may be at least partially tolerant to the enzootic Bd-Brazil lineage. In contrast, late-stage immune activation against Bd-GPL was consistent with immune dysregulation previously observed in other species. Our results demonstrate that both the timing of immune response and the particular immune pathways activated are specific to Bd lineage. Within regions where multiple Bd lineages co-occur, and given continued global Bd movement, these differential host responses may influence not only individual disease outcome, but transmission dynamics at the population and community levels.

Habitat fragmentation in the Brazilian Atlantic Forest is associated with erosion of frog immunogenetic diversity and increased fungal infections.

Habitat fragmentation and infectious diseases threaten wildlife globally, but the interactions of these threats are poorly understood. For instance, while habitat fragmentation can impact genetic diversity at neutral loci, the impacts on disease-relevant loci are less well-studied. We examined the effects of habitat fragmentation in Brazil's Atlantic Forest on amphibian genetic diversity at an immune locus related to antigen presentation and detection (MHC IIB Exon 2). We used a custom high-throughput assay to sequence a fragment of MHC IIB and quantified Batrachochytrium dendrobatidis (Bd) infections in six frog species in two Atlantic Forest regions. Habitat fragmentation was associated with genetic erosion at MHC IIB Exon 2. This erosion was most severe in forest specialists. Significant Bd infections were detected only in one Atlantic Forest region, potentially due to relatively higher elevation. In this region, forest specialists showed an increase in both Bd prevalence and infection loads in fragmented habitats. Reduced population-level MHC IIB diversity was associated with increased Bd infection risk. On the individual level, MHC IIB heterozygotes exhibited a trend toward reduced Bd infection risk, although this was marginally non-significant. Our results suggest that habitat fragmentation increases Bd infection susceptibility in amphibians, mediated at least in part through erosion of immunogenetic diversity. Our findings have implications for management of fragmented populations in the face of emerging infectious diseases.

Geography, Host Genetics, and Cross-Domain Microbial Networks Structure the Skin Microbiota of Fragmented Brazilian Atlantic Forest Frog Populations.

The host-associated microbiome plays a significant role in health. However, the roles of factors such as host genetics and microbial interactions in determining microbiome diversity remain unclear. We examined these factors using amplicon-based sequencing of 175 Thoropa taophora frog skin swabs collected from a naturally fragmented landscape in southeastern Brazil. Specifically, we examined (1) the effects of geography and host genetics on microbiome diversity and structure; (2) the structure of microbial eukaryotic and bacterial co-occurrence networks; and (3) co-occurrence between microeukaryotes with bacterial OTUs known to affect growth of the fungal pathogen Batrachochytrium dendrobatidis (Bd). While bacterial alpha diversity varied by both site type and host MHC IIB genotype, microeukaryotic alpha diversity varied only by site type. However, bacteria and microeukaryote composition showed variation according to both site type and host MHC IIB genotype. Our network analysis showed the highest connectivity when both eukaryotes and bacteria were included, implying that ecological interactions may occur among domains. Lastly, anti-Bd bacteria were not broadly negatively co-associated with the fungal microbiome and were positively associated with potential amphibian parasites. Our findings emphasize the importance of considering both domains in microbiome research and suggest that for effective probiotic strategies for amphibian disease management, considering potential interactions among all members of the microbiome is crucial.

Globally invasive genotypes of the amphibian chytrid outcompete an enzootic lineage in coinfections.

Competition between genotypes is likely to be a key driver of pathogen evolution, particularly following a geographical invasion by distant strains. Theory predicts that competition between disease strains will result in the most virulent strain persisting. Despite its evolutionary implications, the role of strain competition in shaping populations remains untested for most pathogens. We experimentally investigated the in vivo competitive differences between two divergent lineages of the amphibian-killing chytrid fungus ( Batrachochytrium dendrobatidis, Bd). These Bd lineages are hypothesized to have diverged in allopatry but been recently brought back into secondary contact by human introduction. Prior studies indicate that a panzootically-distributed, global lineage of Bd was recently introduced into southern Brazil, and is competitively excluding enzootic lineages in the southern Atlantic Forest. To test for differences in competitive ability between invasive and enzootic Brazilian Bd isolates, we coinfected a model host frog system which we developed for this study ( Hymenochirus curtipes). We tracked isolate-specific zoospore production over the course of the coinfection experiment with chip-based digital PCR (dPCR). The globally invasive panzootic lineage had a competitive advantage in spore production especially during the first one to four weeks of infection, and on frogs that eventually succumbed to Bd infection. Our study provides new evidence that competitive pressure resulting from the human movement of pathogen strains can rapidly alter the genetics, community dynamics and spatial epidemiology of pathogens in the wild.

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.

Partitioning the net effect of host diversity on an emerging amphibian pathogen.

The 'dilution effect' (DE) hypothesis predicts that diverse host communities will show reduced disease. The underlying causes of pathogen dilution are complex, because they involve non-additive (driven by host interactions and differential habitat use) and additive (controlled by host species composition) mechanisms. Here, we used measures of complementarity and selection traditionally employed in the field of biodiversity-ecosystem function (BEF) to quantify the net effect of host diversity on disease dynamics of the amphibian-killing fungus Batrachochytrium dendrobatidis (Bd). Complementarity occurs when average infection load in diverse host assemblages departs from that of each component species in uniform populations. Selection measures the disproportionate impact of a particular species in diverse assemblages compared with its performance in uniform populations, and therefore has strong additive and non-additive properties. We experimentally infected tropical amphibian species of varying life histories, in single- and multi-host treatments, and measured individual Bd infection loads. Host diversity reduced Bd infection in amphibians through a mechanism analogous to complementarity (sensu BEF), potentially by reducing shared habitat use and transmission among hosts. Additionally, the selection component indicated that one particular terrestrial species showed reduced infection loads in diverse assemblages at the expense of neighbouring aquatic hosts becoming heavily infected. By partitioning components of diversity, our findings underscore the importance of additive and non-additive mechanisms underlying the DE.