The efficacy and pharmacokinetics of terbinafine against the frog-killing fungus (Batrachochytrium dendrobatidis).

Captive and wild amphibians are under threat of extinction from the deadly fungal pathogen Batrachochytrium dendrobatidis (Bd). The antifungal drug terbinafine (TBF) is used by pet owners to treat Bd-infected frogs; however, it is not widely used in academic or zoological institutions due to limited veterinary clinical trials. To assess TBF's efficacy, we undertook treatment trials and pharmacokinetic studies to investigate drug absorption and persistence in frog skin; and then we correlated these data to the minimal lethal concentrations (MLC) against Bd. Despite an initial reduction in zoospore load, the recommended treatment (five daily 5 min 0.01% TBF baths) was unable to cure experimentally infected alpine tree frogs and naturally infected common eastern froglets. In vitro and in vivo pharmacokinetics showed that absorbed TBF accumulates in frog skin with increased exposure, indicating its suitability for treating cutaneous pathogens via direct application. The MLC of TBF for zoosporangia was 100 µg/ml for 2 h, while the minimal inhibitory concentration was 2 µg/ml, suggesting that the drug concentration absorbed during 5 min treatments is not sufficient to cure high Bd burdens. With longer treatments of five daily 30 min baths, Bd clearance improved from 12.5% to 50%. A higher dose of 0.02% TBF resulted in 78% of animals cured; however, clearance was not achieved in all individuals due to low TBF skin persistence, as the half-life was less than 2 h. Therefore, the current TBF regime is not recommended as a universal treatment against Bd until protocols are optimized, such as with increased exposure frequency.

Transcriptome analyses of immune tissues from three Japanese frogs (genus Rana ) reveals their utility in characterizing major histocompatibility complex class II.

BACKGROUND: In Japan and East Asia, endemic frogs appear to be tolerant or not susceptible to chytridiomycosis, a deadly amphibian disease caused by the chytrid fungus Batrachochytridium dendrobatidis (Bd). Japanese frogs may have evolved mechanisms of immune resistance to pathogens such as Bd. This study characterizes immune genes expressed in various tissues of healthy Japanese Rana frogs. RESULTS: We generated transcriptome data sets of skin, spleen and blood from three adult Japanese Ranidae frogs (Japanese brown frog Rana japonica, the montane brown frog Rana ornativentris, and Tago's brown frog Rana tagoi tagoi) as well as whole body of R. japonica and R. ornativentris tadpoles. From this, we identified tissue- and stage-specific differentially expressed genes; in particular, the spleen was most enriched for immune-related genes. A specific immune gene, major histocompatibility complex class IIB (MHC-IIB), was further characterized due to its role in pathogen recognition. We identified a total of 33 MHC-IIB variants from the three focal species (n = 7 individuals each), which displayed evolutionary signatures related to increased MHC variation, including balancing selection. Our supertyping analyses of MHC-IIB variants from Japanese frogs and previously studied frog species identified potential physiochemical properties of MHC-II that may be important for recognizing and binding chytrid-related antigens. CONCLUSIONS: This is one of the first studies to generate transcriptomic resources for Japanese frogs, and contributes to further understanding the immunogenetic factors associated with resistance to infectious diseases in amphibians such as chytridiomycosis. Notably, MHC-IIB supertyping analyses identified unique functional properties of specific MHC-IIB alleles that may partially contribute to Bd resistance, and such properties provide a springboard for future experimental validation.

Characterization of MHC class IA in the endangered southern corroboree frog.

Southern corroboree frogs (Pseudophryne corroboree) have declined to near extinction in the wild after the emergence of the amphibian chytrid fungus Batrachochytrium dendrobatidis in southeastern Australia in the 1980s. A major captive breeding and reintroduction program is underway to preserve this iconic species, but improving resistance to B. dendrobatidis would help the wild population to be self-sustaining. Using 3' and 5' rapid amplification of complementary DNA ends (RACE), we characterized the major histocompatibility complex (MHC) class IA locus in this species. We then used sequences generated from RACE to design primers to amplify the peptide-binding region (PBR) of this functional genetic marker. Finally, we analysed the diversity, phylogeny, and selection patterns of PBR sequences from four P. corroboree populations and compared this with other amphibian species. We found moderately high MHC class IA genetic diversity in this species and evidence of strong positive and purifying selection at sites that are associated with putative PBR pockets in other species, indicating that this gene region may be under selection for resistance to Bd. Future studies should focus on identifying alleles associated with Bd resistance in P. corroboree by performing a Bd laboratory challenge study to confirm the functional importance of our genetic findings and explore their use in artificial selection or genetic engineering to increase resistance to chytridiomycosis.

Sex-Chromosome Homomorphy in Palearctic Tree Frogs Results from Both Turnovers and X-Y Recombination.

Contrasting with birds and mammals, poikilothermic vertebrates often have homomorphic sex chromosomes, possibly resulting from high rates of sex-chromosome turnovers and/or occasional X-Y recombination. Strong support for the latter mechanism was provided by four species of European tree frogs, which inherited from a common ancestor (∼ 5 Ma) the same pair of homomorphic sex chromosomes (linkage group 1, LG1), harboring the candidate sex-determining gene Dmrt1. Here, we test sex linkage of LG1 across six additional species of the Eurasian Hyla radiation with divergence times ranging from 6 to 40 Ma. LG1 turns out to be sex linked in six of nine resolved cases. Mapping the patterns of sex linkage to the Hyla phylogeny reveals several transitions in sex-determination systems within the last 10 My, including one switch in heterogamety. Phylogenetic trees of DNA sequences along LG1 are consistent with occasional X-Y recombination in all species where LG1 is sex linked. These patterns argue against one of the main potential causes for turnovers, namely the accumulation of deleterious mutations on nonrecombining chromosomes. Sibship analyses show that LG1 recombination is strongly reduced in males from most species investigated, including some in which it is autosomal. Intrinsically low male recombination might facilitate the evolution of male heterogamety, and the presence of important genes from the sex-determination cascade might predispose LG1 to become a sex chromosome.

Major histocompatibility complex selection dynamics in pathogen-infected túngara frog (Physalaemus pustulosus) populations.

Pathogen-driven selection can favour major histocompatibility complex (MHC) alleles that confer immunological resistance to specific diseases. However, strong directional selection should deplete genetic variation necessary for robust immune function in the absence of balancing selection or challenges presented by other pathogens. We examined selection dynamics at one MHC class II (MHC-II) locus across Panamanian populations of the túngara frog, Physalaemus pustulosus, infected by the amphibian chytrid fungus Batrachochytrium dendrobatidis (Bd). We compared MHC-II diversity in highland túngara frog populations, where amphibian communities have experienced declines owing to Bd, with those in the lowland region that have shown no evidence of decline. Highland region frogs had MHC variants that confer resistance to Bd. Variant fixation appeared to occur by directional selection rather than inbreeding, as overall genetic variation persisted in populations. In Bd-infected lowland sites, however, selective advantage may accrue to individuals with only one Bd-resistance allele, which were more frequent. Environmental conditions in lowlands should be less favourable for Bd infection, which may reduce selection for specific Bd resistance in hosts. Our results suggest that MHC selection dynamics fluctuate in túngara frog populations as a function of the favourability of habitat to pathogen spread and the vulnerability of hosts to infection.

Advances in Managing Chytridiomycosis for Australian Frogs: Gradarius Firmus Victoria.

Extensive knowledge gains from research worldwide over the 25 years since the discovery of chytridiomycosis can be used for improved management. Strategies that have saved populations in the short term and/or enabled recovery include captive breeding, translocation into disease refugia, translocation from resistant populations, disease-free exclosures, and preservation of disease refuges with connectivity to previous habitat, while antifungal treatments have reduced mortality rates in the wild. Increasing host resistance is the goal of many strategies under development, including vaccination and targeted genetic interventions. Pathogen-directed strategies may be more challenging but would have broad applicability. While the search for the silver bullet solution continues, we should value targeted local interventions that stop extinction and buy time for evolution of resistance or development of novel solutions. As for most invasive species and infectious diseases, we need to accept that ongoing management is necessary. For species continuing to decline, proactive deployment and assessment of promising interventions are more valid than a hands-off, do-no-harm approach that will likely allow further extinctions.

Comparative analysis of amphibian genomes: An emerging resource for basic and applied research.

Amphibians are the most threatened group of vertebrates and are in dire need of conservation intervention to ensure their continued survival. They exhibit unique features including a high diversity of reproductive strategies, permeable and specialized skin capable of producing toxins and antimicrobial compounds, multiple genetic mechanisms of sex determination and in some lineages, the ability to regenerate limbs and organs. Although genomic approaches would shed light on these unique traits and aid conservation, sequencing and assembly of amphibian genomes has lagged behind other taxa due to their comparatively large genome sizes. Fortunately, the development of long-read sequencing technologies and initiatives has led to a recent burst of new amphibian genome assemblies. Although growing, the field of amphibian genomics suffers from the lack of annotation resources, tools for working with challenging genomes and lack of high-quality assemblies in multiple clades of amphibians. Here, we analyse 51 publicly available amphibian genomes to evaluate their usefulness for functional genomics research. We report considerable variation in genome assembly quality and completeness and report some of the highest transposable element and repeat contents of any vertebrate. Additionally, we detected an association between transposable element content and climatic variables. Our analysis provides evidence of conserved genome synteny despite the long divergence times of this group, but we also highlight inconsistencies in chromosome naming and orientation across genome assemblies. We discuss sequencing gaps in the phylogeny and suggest key targets for future sequencing endeavours. Finally, we propose increased investment in amphibian genomics research to promote their conservation.

The Amphibian Genomics Consortium: advancing genomic and genetic resources for amphibian research and conservation.

Amphibians represent a diverse group of tetrapods, marked by deep divergence times between their three systematic orders and families. Studying amphibian biology through the genomics lens increases our understanding of the features of this animal class and that of other terrestrial vertebrates. The need for amphibian genomic resources is more urgent than ever due to the increasing threats to this group. Amphibians are one of the most imperiled taxonomic groups, with approximately 41% of species threatened with extinction due to habitat loss, changes in land use patterns, disease, climate change, and their synergistic effects. Amphibian genomic resources have provided a better understanding of ontogenetic diversity, tissue regeneration, diverse life history and reproductive modes, antipredator strategies, and resilience and adaptive responses. They also serve as essential models for studying broad genomic traits, such as evolutionary genome expansions and contractions, as they exhibit the widest range of genome sizes among all animal taxa and possess multiple mechanisms of genetic sex determination. Despite these features, genome sequencing of amphibians has significantly lagged behind that of other vertebrates, primarily due to the challenges of assembling their large, repeat-rich genomes and the relative lack of societal support. The emergence of long-read sequencing technologies, combined with advanced molecular and computational techniques that improve scaffolding and reduce computational workloads, is now making it possible to address some of these challenges. To promote and accelerate the production and use of amphibian genomics research through international coordination and collaboration, we launched the Amphibian Genomics Consortium (AGC, https://mvs.unimelb.edu.au/amphibian-genomics-consortium) in early 2023. This burgeoning community already has more than 282 members from 41 countries. The AGC aims to leverage the diverse capabilities of its members to advance genomic resources for amphibians and bridge the implementation gap between biologists, bioinformaticians, and conservation practitioners. Here we evaluate the state of the field of amphibian genomics, highlight previous studies, present challenges to overcome, and call on the research and conservation communities to unite as part of the AGC to enable amphibian genomics research to "leap" to the next level.

Conserved Evolution of MHC Supertypes among Japanese Frogs Suggests Selection for Bd Resistance.

The chytrid fungus Batrachochytrium dendrobatidis (Bd) is a major threat to amphibians, yet there are no reports of major disease impacts in East Asian frogs. Genetic variation of the major histocompatibility complex (MHC) has been associated with resistance to Bd in frogs from East Asia and worldwide. Using transcriptomic data collated from 11 Japanese frog species (one individual per species), we isolated MHC class I and IIb sequences and validated using molecular cloning. We then compared MHC from Japanese frogs and other species worldwide, with varying Bd susceptibility. Supertyping analysis, which groups MHC alleles based on physicochemical properties of peptide binding sites, identified that all examined East Asian frogs contained at least one MHC-IIb allele belonging to supertype ST-1. This indicates that, despite the large divergence times between some Japanese frogs (up to 145 million years), particular functional properties in the peptide binding sites of MHC-II are conserved among East Asian frogs. Furthermore, preliminary analysis using NetMHCIIpan-4.0, which predicts potential Bd-peptide binding ability, suggests that MHC-IIb ST-1 and ST-2 have higher overall peptide binding ability than other supertypes, irrespective of whether the peptides are derived from Bd, other fungi, or bacteria. Our findings suggest that MHC-IIb among East Asian frogs may have co-evolved under the same selective pressure. Given that Bd originated in this region, it may be a major driver of MHC evolution in East Asian frogs.

Genetic approaches for increasing fitness in endangered species.

The global rate of wildlife extinctions is accelerating, and the persistence of many species requires conservation breeding programs. A central paradigm of these programs is to preserve the genetic diversity of the founder populations. However, this may preserve original characteristics that make them vulnerable to extinction. We introduce targeted genetic intervention (TGI) as an alternative approach that promotes traits that enable species to persist in the face of threats by changing the incidence of alleles that impact on fitness. The TGI toolkit includes methods with established efficacy in model organisms and agriculture but are largely untried for conservation, such as synthetic biology and artificial selection. We explore TGI approaches as a species-restoration tool for intractable threats including infectious disease and climate change.

Response to Comment on "Amphibian fungal panzootic causes catastrophic and ongoing loss of biodiversity".

Lambert et al question our retrospective and holistic epidemiological assessment of the role of chytridiomycosis in amphibian declines. Their alternative assessment is narrow and provides an incomplete evaluation of evidence. Adopting this approach limits understanding of infectious disease impacts and hampers conservation efforts. We reaffirm that our study provides unambiguous evidence that chytridiomycosis has affected at least 501 amphibian species.

Amphibian fungal panzootic causes catastrophic and ongoing loss of biodiversity.

Anthropogenic trade and development have broken down dispersal barriers, facilitating the spread of diseases that threaten Earth's biodiversity. We present a global, quantitative assessment of the amphibian chytridiomycosis panzootic, one of the most impactful examples of disease spread, and demonstrate its role in the decline of at least 501 amphibian species over the past half-century, including 90 presumed extinctions. The effects of chytridiomycosis have been greatest in large-bodied, range-restricted anurans in wet climates in the Americas and Australia. Declines peaked in the 1980s, and only 12% of declined species show signs of recovery, whereas 39% are experiencing ongoing decline. There is risk of further chytridiomycosis outbreaks in new areas. The chytridiomycosis panzootic represents the greatest recorded loss of biodiversity attributable to a disease.

Selective constraint acting on TLR2 and TLR4 genes of Japanese Rana frogs.

Toll-like receptors (TLRs) are an important component of innate immunity, the first line of pathogen defence. One of the major roles of TLRs includes recognition of pathogen-associated molecular patterns. Amphibians are currently facing population declines and even extinction due to chytridiomycosis caused by the Batrachochytrium dendrobatidis (Bd) fungus. Evidence from other vertebrates shows that TLR2 and TLR4 are involved in innate immunity against various fungi. Such genes therefore may play a functional role in amphibian-chytridiomycosis dynamics. Frogs from East Asia appear to be tolerant to Bd, so we examined the genetic diversity that underlies TLR2 and TLR4 from three Japanese Ranidae frog species, Rana japonica, R. ornativentris and R. tagoi tagoi (n = 5 per species). We isolated 27 TLR2 and 20 TLR4 alleles and found that these genes are evolutionarily conserved, with overall evidence supporting purifying selection. In contrast, site-by-site analysis of selection identified several specific codon sites under positive selection, some of which were located in the variable leucine rich repeat domains. In addition, preliminary expression levels of TLR2 and TLR4 from transcriptome data showed overall low expression. Although it remains unclear whether infectious pathogens are a selective force acting on TLRs of Japanese frogs, our results support that certain sites in TLRs of these species may have experienced pathogen-mediated selection.

Development and worldwide use of non-lethal, and minimal population-level impact, protocols for the isolation of amphibian chytrid fungi.

Parasitic chytrid fungi have emerged as a significant threat to amphibian species worldwide, necessitating the development of techniques to isolate these pathogens into culture for research purposes. However, early methods of isolating chytrids from their hosts relied on killing amphibians. We modified a pre-existing protocol for isolating chytrids from infected animals to use toe clips and biopsies from toe webbing rather than euthanizing hosts, and distributed the protocol to researchers as part of the BiodivERsA project RACE; here called the RML protocol. In tandem, we developed a lethal procedure for isolating chytrids from tadpole mouthparts. Reviewing a database of use a decade after their inception, we find that these methods have been applied across 5 continents, 23 countries and in 62 amphibian species. Isolation of chytrids by the non-lethal RML protocol occured in 18% of attempts with 207 fungal isolates and three species of chytrid being recovered. Isolation of chytrids from tadpoles occured in 43% of attempts with 334 fungal isolates of one species (Batrachochytrium dendrobatidis) being recovered. Together, these methods have resulted in a significant reduction and refinement of our use of threatened amphibian species and have improved our ability to work with this group of emerging pathogens.

Recent Asian origin of chytrid fungi causing global amphibian declines.

Globalized infectious diseases are causing species declines worldwide, but their source often remains elusive. We used whole-genome sequencing to solve the spatiotemporal origins of the most devastating panzootic to date, caused by the fungus Batrachochytrium dendrobatidis, a proximate driver of global amphibian declines. We traced the source of B. dendrobatidis to the Korean peninsula, where one lineage, BdASIA-1, exhibits the genetic hallmarks of an ancestral population that seeded the panzootic. We date the emergence of this pathogen to the early 20th century, coinciding with the global expansion of commercial trade in amphibians, and we show that intercontinental transmission is ongoing. Our findings point to East Asia as a geographic hotspot for B. dendrobatidis biodiversity and the original source of these lineages that now parasitize amphibians worldwide.

Introduced bullfrogs are associated with increased Batrachochytrium dendrobatidis prevalence and reduced occurrence of Korean treefrogs.

Bullfrogs, Lithobates catesbeianus, have been described as major vectors of the amphibian chytrid fungus, Batrachochytrium dendrobatidis (Bd). Bd is widespread throughout the range of amphibians yet varies considerably within and among populations in prevalence and host impact. In our study, the presence of L. catesbeianus is correlated with a 2.5 increase in Bd prevalence in treefrogs, and the endangered Dryophytes suweonensis displays a significantly higher Bd prevalence than the more abundant D. japonicus for the 37 sites surveyed. In addition, the occurrence of L. catesbeianus was significantly correlated with a decrease in presence of D. suweonensis at sites. We could not determine if it is the presence of bullfrogs as competitors or predators that is limiting the distribution of D. suweonensis or whether this is caused by bullfrogs acting as a reservoir for Bd. However, L. catesbeianus can now be added to the list of factors responsible for the decline of D. suweonensis populations.

Correction: Introduced bullfrogs are associated with increased Batrachochytrium dendrobatidis prevalence and reduced occurrence of Korean treefrogs.

[This corrects the article DOI: 10.1371/journal.pone.0177860.].

Swabbing often fails to detect amphibian Chytridiomycosis under conditions of low infection load.

The pathogenic chytrid fungus, Batrachochytrium dendrobatidis (denoted Bd), causes large-scale epizootics in naïve amphibian populations. Intervention strategies to rapidly respond to Bd incursions require sensitive and accurate diagnostic methods. Chytridiomycosis usually is assessed by quantitative polymerase chain reaction (qPCR) amplification of amphibian skin swabs. Results based on this method, however, sometimes yield inconsistent results on infection status and inaccurate scores of infection intensity. In Asia and other regions where amphibians typically bear low Bd loads, swab results are least reliable. We developed a Bd-sampling method that collects zoospores released by infected subjects into an aquatic medium. Bd DNA is extracted by filters and amplified by nested PCR. Using laboratory colonies and field populations of Bombina orientalis, we compare results with those obtained on the same subjects by qPCR of DNA extracted from swabs. Many subjects, despite being diagnosed as Bd-negative by conventional methods, released Bd zoospores into collection containers and thus must be considered infected. Infection loads determined from filtered water were at least 1000 times higher than those estimated from swabs. Subjects significantly varied in infection load, as they intermittently released zoospores, over a 5-day period. Thus, the method might be used to compare the infectivity of individuals and study the periodicity of zoospore release. Sampling methods based on water filtration can dramatically increase the capacity to accurately diagnose chytridiomycosis and contribute to a better understanding of the interactions between Bd and its hosts.