Outbreaks of emerging infectious diseases are influenced by local biotic and abiotic factors, with host declines occurring when conditions favour the pathogen. Deterioration in the population of the micro-endemic Tanzanian Kihansi spray toad (Nectophrynoides asperginis) occurred after the construction of a hydropower dam, implicating habitat modification in this species decline. Population recovery followed habitat augmentation; however, a subsequent outbreak of chytridiomycosis caused by Batrachochytrium dendrobatidis (Bd) led to the spray toad\'s extinction in the wild. We show using spatiotemporal surveillance and mitogenome assembly of Bd from archived toad mortalities that the outbreak was caused by invasion of the BdCAPE lineage and not the panzootic lineage BdGPL. Molecular dating reveals an emergence of BdCAPE across southern Africa overlapping with the timing of the spray toad\'s extinction. That our post-outbreak surveillance of co-occurring amphibian species in the Udzungwa Mountains shows widespread infection by BdCAPE yet no signs of ill-health or decline suggests these other species can tolerate Bd when environments are stable. We conclude that, despite transient success in mitigating the impact caused by dams\' construction, invasion by BdCAPE caused the ultimate die-off that led to the extinction of the Kihansi spray toad.

RNA interference (RNAi) has not been tested in the pandemic amphibian pathogen, Batrachochytrium dendrobatidis, but developing this technology could be useful to elucidate virulence mechanisms, identify therapeutic targets, and may present a novel antifungal treatment option for chytridiomycosis. To manipulate and decipher gene function, rationally designed small interfering RNA (siRNA) can initiate the destruction of homologous messenger RNA (mRNA), resulting in the \"knockdown\" of target gene expression. Here, we investigate whether siRNA can be used to manipulate gene expression in B. dendrobatidis via RNAi using differing siRNA strategies to target genes involved in glutathione and ornithine synthesis. To determine the extent and duration of mRNA knockdown, target mRNA levels were monitored for 24-48 h after delivery of siRNA targeting glutamate-cysteine ligase, with a maximum of ~56% reduction in target transcripts occurring at 36 h. A second siRNA design targeting glutamate-cysteine ligase also resulted in ~53% knockdown at this time point. siRNA directed toward a different gene target, ornithine decarboxylase, achieved 17% reduction in target transcripts. Although no phenotypic effects were observed, these results suggest that RNAi is possible in B. dendrobatidis, and that gene expression can be manipulated in this pathogen. We outline ideas for further optimization steps to increase knockdown efficiency to better harness RNAi techniques for control of B. dendrobatidis.

Chytridiomycosis is a devastating disease and is a key cause of amphibian population declines around the world. Despite active research on this amphibian disease system for over 2 decades, we still do not have treatment methods that are safe and that can be broadly used across species. Here, we show evidence that voriconazole is a successful method of treatment for 1 species of amphibian in captivity and that this treatment could offer benefits over other treatment options like heat or itraconazole, which are not able to be used for all species and life stages. We conducted 2 treatments of chytridiomycosis using voriconazole. The treatment was effective and resulted in 100% pathogen clearance, and mortality ceased. Additionally, treating frogs with voriconazole requires less handling than treatment methods like itraconazole and requires no specialized equipment, like heat treatment. We highlight that clinical treatment trials should be conducted to identify an optimum dosage and treatment time and that trials should test whether this treatment is safe and effective for tadpoles and other species.

Chytridiomycosis, an infectious skin disease caused by the chytrid fungi, Batrachochytrium dendrobatidis and B. salamandrivorans, poses a significant threat to amphibian biodiversity worldwide. Antifungal bacteria found on the skin of chytrid-resistant amphibians could potentially provide defense against chytridiomycosis and lower mortality rates among resistant individuals. The Hong Kong newt (Paramesotriton hongkongensis) is native to East Asia, a region suspected to be the origin of chytrids, and has exhibited asymptomatic infection, suggesting a long-term coexistence with the chytrids. Therefore, the skin microbiota of this resistant species warrant investigation, along with other factors that can affect the microbiota. Among the 149 newts sampled in their natural habitats in Hong Kong, China, putative antifungal bacteria were found in all individuals. There were 314 amplicon sequence variants distributed over 25 genera of putative antifungal bacteria; abundant ones included Acinetobacter, Flavobacterium, and Novosphingobium spp. The skin microbiota compositions were strongly influenced by the inter-site geographical distances. Despite inter-site differences, we identified some core skin microbes across sites that could be vital to P. hongkongensis. The dominant cores included the family Comamonadaceae, family Chitinophagaceae, and class Betaproteobacteria. Moreover, habitat elevation and host sex also exhibited significant effects on skin microbiota compositions. The antifungal bacteria found on these newts offer an important resource for conservation against chytridiomycosis, such as developing probiotic treatments for susceptible species.

Phylosymbiosis is an association between host-associated microbiome composition and host phylogeny. This pattern can arise via the evolution of host traits, habitat preferences, diets, and the co-diversification of hosts and microbes. Understanding the drivers of phylosymbiosis is vital for modelling disease-microbiome interactions and manipulating microbiomes in multi-host systems. This study quantifies phylosymbiosis in Appalachian salamander skin in the context of infection by the fungal pathogen Batrachochytrium dendrobatidis (Bd), while accounting for environmental microbiome exposure. We sampled ten salamander species representing >150M years of divergence, assessed their Bd infection status, and analysed their skin and environmental microbiomes. Our results reveal a significant signal of phylosymbiosis, whereas the local environmental pool of microbes, climate, geography, and Bd infection load had a smaller impact. Host-microbe co-speciation was not evident, indicating that the effect stems from the evolution of host traits influencing microbiome assembly. Bd infection is correlated with host phylogeny and the abundance of Bd-inhibitory bacterial strains, suggesting that the long-term evolutionary dynamics between salamander hosts and their skin microbiomes affect the present-day distribution of the pathogen, along with habitat-linked exposure risk. Five Bd-inhibitory bacterial strains showed unusual generalism: occurring in most host species and habitats. These generalist strains may enhance the likelihood of probiotic manipulations colonising and persisting on hosts. Our results underscore the substantial influence of host-microbiome eco-evolutionary dynamics on environmental health and disease outcomes.

North American salamanders are threatened by intercontinental spread of chytridiomycosis, a deadly disease caused by the fungal pathogen Batrachochytrium salamandrivorans (Bsal). To predict potential dispersal of Bsal spores to salamander habitats, we evaluated the capacity of soil microbial communities to resist invasion. We determined the degree of habitat invasibility using soils from five locations throughout the Great Smoky Mountains National Park, a region with a high abundance of susceptible hosts. Our experimental design consisted of replicate soil microcosms exposed to different propagule pressures of the non-native pathogen, Bsal, and an introduced but endemic pathogen, B. dendrobatidis (Bd). To compare growth and competitive interactions, we used quantitative PCR, live/dead cell viability assays, and full-length 16S rRNA sequencing. We found that soil microcosms with intact bacterial communities inhibited both Bsal and Bd growth, but inhibitory capacity diminished with increased propagule pressure. Bsal showed greater persistence than Bd. Linear discriminant analysis (LDA) identified the family Burkolderiaceae as increasing in relative abundance with the decline of both pathogens. Although our findings provide evidence of environmental filtering in soils, such barriers weakened in response to pathogen type and propagule pressure, showing that habitats vary their invasibility based on properties of their local microbial communities.

The amphibian skin pathogen Batrachochytrium dendrobatidis (Bd) has caused an ongoing biodiversity crisis, including in the locally endangered Colorado boreal toad (Anaxyrus boreas boreas). Although researchers have investigated the bacteria living on amphibian skin and how they interact with Bd, there is less information about fungal community members. This study describes (1) the diversity of culturable fungi from boreal toad skin, (2) which subset of these isolates is Bd-inhibitory, and (3) how Bd affects these isolates\' growth and morphology. Most isolates were from the orders Capnodiales, Helotiales, and Pleosporales. Of 16 isolates tested for Bd-inhibition, two from the genus Neobulgaria and three from Pseudeurotium inhibited Bd. Fungal growth in co-culture with Bd varied with weak statistical support for Neobulgaria sp. (isolate BTF_36) and cf Psychrophila (isolate BTF_60) (p-values = 0.076 and 0.092, respectively). Fungal morphology remained unchanged in co-culture with Bd, however, these results could be attributed to low replication per isolate. Nonetheless, two fungal isolates\' growth may have been affected by Bd, implying that fungal growth changes in Bd co-culture could be a variable worth measuring in the future (with higher replication). These findings add to the sparse but growing literature on amphibian-associated fungi and suggest further study may uncover the relevance of fungi to amphibian health and Bd infection.

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.

Amphibian populations are undergoing extensive declines globally. The fungal disease chytridiomycosis, caused by the pathogenic fungus Batrachochytrium dendrobatidis (Bd), is a primary contributor to these declines. The amphibian metamorphic stages (Gosner stages 42-46) are particularly vulnerable to a range of stressors, including Bd. Despite this, studies that explicitly examine host response to chytridiomycosis throughout the metamorphic stages are lacking. We aimed to determine how Bd exposure during the larval stages impacts metamorphic development and infection progression in the endangered Fleay\'s barred frog (Mixophyes fleayi). We exposed M. fleayi to Bd during pro-metamorphosis (Gosner stages 35-38) and monitored infection dynamics throughout metamorphosis. We took weekly morphological measurements (weight, total body length, snout-vent-length and Gosner stage) and quantified Bd load using qPCR. While we observed minimal impact of Bd infection on animal growth and development, Bd load varied throughout ontogeny, with an infection load plateau during the tadpole stages (Gosner stages 35-41) and temporary infection clearance at Gosner stage 42. Bd load increased exponentially between Gosner stages 42 and 45, with most exposed animals becoming moribund at Gosner stage 45, prior to the completion of metamorphosis. There was variability in infection outcome of exposed individuals, with a subgroup of animals (n = 5/29) apparently clearing their infection while the majority (n = 21/29) became moribund with high infection burdens. This study demonstrates the role that metamorphic restructuring plays in shaping Bd infection dynamics and raises the concern that substantial Bd-associated mortality could be overlooked in the field due to the often cryptic nature of these latter metamorphic stages. We recommend future studies that directly examine the host immune response to Bd infection throughout metamorphosis, incorporating histological and molecular methods to elucidate the mechanisms responsible for the observed trends.

Batrachochytrium dendrobatidis (Bd), the causative agent of the devastating global amphibian disease chytridiomycosis, was not projected to threaten amphibians in hot and arid regions due to its sensitivity to heat and desiccation. However, Bd is being detected more frequently than ever in hot and arid regions of Australia and the USA, challenging our current understanding of the environmental tolerances of the pathogen under natural conditions. We surveyed for Bd in an extremely hot and arid portion of the Sonoran Desert, where the pathogen is not projected to occur, and related presence and prevalence of the pathogen to local environmental conditions. We collected eDNA samples from isolated desert water sites including six tinajas and 13 catchments in June and August of 2020 and swabbed a total of 281 anurans of three species (red-spotted toad Anaxyrus punctatus, Couch\'s spadefoot Scaphiopus couchii, and the Sonoran Desert toad Incillius alvarius) across five catchments and six tinajas from June to September of 2020. Overall, Bd occurred at 68.4% of sites, despite extreme heat and aridity routinely exceeding tolerances established in laboratory studies. Average summer maximum air and water temperatures were 40.7°C and 30.7°C, respectively, and sites received an average of just 16.9 mm of precipitation throughout the summer monsoon season. Prevalence was low (5.7%) across species and life stage. Our results demonstrate that Bd is capable of persisting and infecting amphibians beyond its projected range, indicating a need to account for higher thermal tolerances when quantifying risk of Bd presence and infection.