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The chytrid Batrachochytrium dendrobatidis (Bd) is a widespread fungus causing amphibian declines across the globe. Although data on Bd occurrence in Eastern Europe are scarce, a recent species distribution model (SDM) for Bd reported that western and north-western parts of Ukraine are highly suitable to the pathogen. We verified the SDM-predicted range of Bd in Ukraine by sampling amphibians across the country and screening for Bd using qPCR. A total of 446 amphibian samples (tissue and skin swabs) from 11 species were collected from 36 localities. We obtained qPCR-positive results for 33 samples including waterfrogs (Pelophylax esculentus complex) and fire- and yellow-bellied toads (Bombina spp.) from 8 localities. We found that Bd-positive localities had significantly higher predicted Bd habitat suitability than sites that were pathogen-free. Amplification and sequencing of the internal transcribed spacer (ITS) region of samples with the highest Bd load revealed matches with ITS haplotypes of the globally distributed BdGPL strain, and a single case of the BdASIA-2/BdBRAZIL haplotype. We found that Bd was non-randomly distributed across Ukraine, with infections present in the western and north-central forested peripheries of the country with a relatively cool, moist climate. On the other hand, our results suggest that Bd is absent or present in low abundance in the more continental central, southern and eastern regions of Ukraine, corroborating the model-predicted distribution of chytrid fungus. These areas could potentially serve as climatic refugia for Bd-susceptible amphibian hosts.

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., (© 2024 The Author(s). Journal of Basic Microbiology published by Wiley‐VCH GmbH.)

Parasitism is the most common lifestyle on Earth and has emerged many times independently across the eukaryotic tree of life. It is frequently found among chytrids (Chytridiomycota), which are early-branching unicellular fungi that feed osmotrophically via rhizoids as saprotrophs or parasites. Chytrids are abundant in most aquatic and terrestrial environments and fulfil important ecosystem functions. As parasites, they can have significant impacts on host populations. They cause global amphibian declines and influence the Earth's carbon cycle by terminating algal blooms. To date, the evolution of parasitism within the chytrid phylum remains unclear due to the low phylogenetic resolution of rRNA genes for the early diversification of fungi, and because few parasitic lineages have been cultured and genomic data for parasites is scarce. Here, we combine transcriptomics, culture-independent single-cell genomics and a phylogenomic approach to overcome these limitations. We newly sequenced 29 parasitic taxa and combined these with existing data to provide a robust backbone topology for the diversification of Chytridiomycota. Our analyses reveal multiple independent lifestyle transitions between parasitism and saprotrophy among chytrids and multiple host shifts by parasites. Based on these results and the parasitic lifestyle of other early-branching holomycotan lineages, we hypothesise that the chytrid last common ancestor was a parasite of phytoplankton., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

The chytrid fungus Batrachochytrium salamandrivorans [Bsal] is causing declines in the amphibian populations. After a decade of mapping the pathogen in Europe, where it is causing dramatic outbreaks, and North America, where its arrival would affect to the salamander's biodiversity hotspot, little is known about its current status in Asia, from presumably is native. Japan has several species considered as potential carriers, but no regulation is implemented against Bsal spreading. Previous Bsal known presence detected various cases on the Okinawa Island, southwestern Japan. Previous studies on its sister species, B. dendrobatidis presented a high genomic variation in this area and particularly on Cynops ensicauda. Here, we have done the largest monitoring to date in Japan on the Cynops genus, focusing on Okinawa Island and updating its distribution and providing more information to unravel the still unknown origin of Bsal. Interestingly, we have provided revealing facts about different detectability depending on the used molecular techniques and changes in its Japanese distribution. All in all, the Bsal presence in Japan, together with its low variability in the sequenced amplicons, and the lack of apparent mortalities, may indicate that this part of Asia has a high diversity of chytrids., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Lastra González et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

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., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

We identified two new parasite species of Chytridiomycota isolated during blooms of the dinoflagellate Alexandrium minutum in the coastal Mediterranean Sea. Light and electron microscopy together with molecular characterization of the nuclear 18S, ITS, and 28S rDNA regions led to their identification as two new species, Dinomyces gilberthii and Paradinomyces evelyniae, both belonging to the family Dinomycetaceae, order Rhizophydiales. Dinomyces gilberthii differs from the previously described D. arenysensis by the presence of discharge papillae and the development of a drop-shaped sporangium. Paradinomyces evelyniae differs from the previously described P. triforaminorum by the prominent lipid globule present in early sporangia and by the pointed end producing a rhizoid. The two chytrids differed in their geographical distribution. Dinomyces gilberthii was detected in several Mediterranean habitats, including harbours and beaches, and was particularly prevalent during summer dinoflagellate blooms. Its widespread occurrence in coastal ecosystems suggested a high level of adaptability to this environment. Paradinomyces evelyniae had a more restricted distribution in the coastal-marine environment, occurring in harbour sediments and only occasionally in the water column during winter and early spring. Paradinomyces evelyniae has previously been detected in the Baltic Sea, suggesting that its distribution encompasses contrasting coastal environments, although its presence is rare., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier GmbH.. All rights reserved.)

Batrachochytrium dendrobatidis (Bd) is a lethal fungal species that parasitizes vertebrates and is associated with the worldwide decline of amphibian populations. The development of sensitive, rapid detection methods, particularly DNA-based techniques, is critical for effective management strategies. This study evaluates the efficacy of DNA extraction and a portable PCR device in a mountable field laboratory setup for detecting Bd near the habitats of three critically endangered Atelopus toad species in Ecuador. We collected skin swabs from Atelopus balios, A. nanay, and A. bomolochos, and environmental DNA (eDNA) samples from streams in Andean and coastal regions of Ecuador. For eDNA, a comparison was made with duplicates of the samples that were processed in the field and in a standard university laboratory. Our findings revealed Bd detection in eDNA and swabs from 6 of 12 water samples and 10 of 12 amphibian swab samples. The eDNA results obtained in the field laboratory were concordant with those obtained under campus laboratory conditions. These findings highlight the potential of field DNA-based monitoring techniques for detecting Bd in amphibian populations and their aquatic habitats, particularly in remote areas. Furthermore, this research aligns with the National Action Plan for the Conservation of Ecuadorian Amphibians and contributes to the global effort to control this invasive and deadly fungus., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Riascos-Flores et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

An amphibian emerging infectious disease (EID), chytridiomycosis, caused by Batrachochytrium dendrobatidis (Bd), originated in Asia but primarily led to declines and extinctions in amphibian populations outside of Asia. Host major histocompatibility complex (MHC) molecules exhibit high polymorphism, and the evolution of MHC can be influenced by recombination and pathogens. Previous studies have indicated that host MHC class II is associated with Bd resistance. In this study, I conducted recombination and selection tests on functional MHC IIß1 alleles from an Asian Bd-resistant anuran species (Bufo gargarizans) and an Australasian Bd-susceptible species (Litoria caerulea). Recombination at the same site was identified in both species, supporting the hypothesis that recombination contributes to MHC IIß1 diversity in amphibians. Positive selection was observed in MHC IIß1 alleles in both species. In L. caerulea, at least four amino acid sites were identified under significant positive selection in the MHC IIß1, whereas these sites were either negatively selected or conserved in B. gargarizans. This suggests these sites might be selected for Bd resistance. Hydrophobicity was detected in certain amino acid sites relating to Bd resistance, suggesting this physicochemical property may be a factor selected to counteract Bd infection. These findings of this study provide an evolutionary basis for understanding how amphibian MHC IIß1 may undergo selection in response to chytrid infection., Competing Interests: Declaration of competing interest The author declares no competing interests., (Copyright © 2023. Published by Elsevier B.V.)

Potato wart disease is caused by the obligate fungal pathogen Synchytrium endobioticum. DNA extraction from compost, purified spores and crude wart tissue derived from tuber galls of infected potatoes often results in low S. endobioticum DNA concentration or highly contaminated with DNA coming from other microorganisms and the potato host. Therefore, Illumina sequencing of these samples generally results in suboptimal recovery of the nuclear genome sequences of S. endobioticum. A hybridization-based target enrichment protocol was developed to strongly enhance the recovery of S. endobioticum DNA while off-target organisms DNA remains uncaptured. The design strategy involved creating a set of 180,000 molecular baits targeting both gene and non-gene regions of S. endobioticum. The baits were applied to whole genome amplified DNA samples of various S. endobioticum pathotypes (races) in compost, from purified spores and crude wart tissue samples. This was followed by Illumina sequencing and bioinformatic analyses. Compared to non-enriched samples, target enriched samples: 1) showed a significant increase in the proportion of sequenced bases mapped to the S. endobioticum nuclear genome, especially for crude wart tissue samples; 2) yielded sequencing data with higher and better nuclear genome coverage; 3) biased genome assembly towards S. endobioticum sequences, yielding smaller assembly sizes but higher representation of putative S. endobioticum contigs; 4) showed an increase in the number of S. endobioticum genes detected in the genome assemblies. Our hybridization-based target enrichment protocol offers a valuable tool for enhancing genome sequencing and NGS-based molecular detection of S. endobioticum, especially in difficult samples., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Nguyen et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The chytrid fungus Batrachochytrium dendrobatidis (Bd) was discovered in 1998 as the cause of chytridiomycosis, an emerging infectious disease causing mass declines in amphibian populations worldwide. The rapid population declines of the 1970s-1990s were likely caused by the spread of a highly virulent lineage belonging to the Bd-GPL clade that was introduced to naïve susceptible populations. Multiple genetically distinct and regional lineages of Bd have since been isolated and sequenced, greatly expanding the known biological diversity within this fungal pathogen. To date, most Bd research has been restricted to the limited number of samples that could be isolated using culturing techniques, potentially causing a selection bias for strains that can grow on media and missing other unculturable or fastidious strains that are also present on amphibians. We thus attempted to characterize potentially non-culturable genetic lineages of Bd from distinct amphibian taxa using sequence capture technology on DNA extracted from host tissue and swabs. We focused our efforts on host taxa from two different regions that likely harbored distinct Bd clades: (1) wild-caught leopard frogs (Rana) from North America, and (2) a Japanese Giant Salamander (Andrias japonicus) at the Smithsonian Institution's National Zoological Park that exhibited signs of disease and tested positive for Bd using qPCR, but multiple attempts failed to isolate and culture the strain for physiological and genetic characterization. We successfully enriched for and sequenced thousands of fungal genes from both host clades, and Bd load was positively associated with number of recovered Bd sequences. Phylogenetic reconstruction placed all the Rana-derived strains in the Bd-GPL clade. In contrast, the A. japonicus strain fell within the Bd-Asia3 clade, expanding the range of this clade and generating additional genomic data to confirm its placement. The retrieved ITS locus matched public barcoding data from wild A. japonicus and Bd infections found on other amphibians in India and China, suggesting that this uncultured clade is widespread across Asia. Our study underscores the importance of recognizing and characterizing the hidden diversity of fastidious strains in order to reconstruct the spatiotemporal and evolutionary history of Bd. The success of the sequence capture approach highlights the utility of directly sequencing pathogen DNA from host tissue to characterize cryptic diversity that is missed by culture-reliant approaches., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Batrachochytrium dendrobatidis (Bd) is a fungal pathogen that has decimated amphibian populations worldwide for several decades. We examined the changes in gene expression in response to Bd infection in two populations of the common toad, Bufo bufo, in a laboratory experiment. We collected B. bufo eggs in southern and northern Sweden, and infected the laboratory-raised metamorphs with two strains of the global panzoonotic lineage Bd-GPL. Differential expression analysis showed significant differences between infected and control individuals in both liver and skin. The skin samples showed no discernible differences in gene expression between the two strains used, while liver samples were differentiated by strain, with one of the strains eliciting no immune response from infected toads. Immune system genes were overexpressed in skin samples from surviving infected individuals, while in liver samples the pattern was more diffuse. Splitting samples by population revealed a stronger immune response in northern individuals. Differences in transcriptional regulation between populations are particularly relevant to study in Swedish amphibians, which may have experienced varying exposure to Bd. Earlier exposure to this pathogen and subsequent adaptation or selection pressure may contribute to the survival of some populations over others, while standing genetic diversity in different populations may also affect the infection outcome., (© 2024. The Author(s).)

Batrachochytrium dendrobatidis ( Bd ), a causative agent of chytridiomycosis, is decimating amphibian populations around the world. Bd belongs to the chytrid lineage, a group of early-diverging fungi that are widely used to study fungal evolution. Like all chytrids, Bd develops from a motile form into a sessile, growth form, a transition that involves drastic changes in its cytoskeletal architecture. Efforts to study Bd cell biology, development, and pathogenicity have been limited by the lack of genetic tools with which to test hypotheses about underlying molecular mechanisms. Here, we report the development of a transient genetic transformation system for Bd . We used electroporation to deliver exogenous DNA into Bd cells and detected transgene expression for up to three generations under both heterologous and native promoters. We also adapted the transformation protocol for selection using an antibiotic resistance marker. Finally, we used this system to express fluorescent protein fusions and, as a proof of concept, expressed a genetically encoded probe for the actin cytoskeleton. Using live-cell imaging, we visualized the distribution and dynamics of polymerized actin at each stage of the Bd life cycle, as well as during key developmental transitions. This transformation system enables direct testing of key hypotheses regarding mechanisms of Bd pathogenesis. This technology also paves the way for answering fundamental questions of chytrid cell, developmental, and evolutionary biology., Competing Interests: Competing interests statement:The authors declare no competing interest.

Background: Batrachochytrium dendrobatidis (Bd) and Batrachochytrium salamandrivorans (Bsal) are two pathogenic fungi that are a significant threat to amphibian communities worldwide. European populations are strongly impacted and the monitoring of the presence and spread of these pathogens is crucial for efficient decision-making in conservation management., Results: Here we proposed an environmental DNA (eDNA) monitoring of these two pathogenic agents through droplet digital PCR (ddPCR) based on water samples from 24 ponds in Luxembourg. In addition, amphibians were swabbed in eight of the targeted ponds in order to compare the two approaches at site-level detection. This study allowed the development of a new method taking below-Limit of Detection (LOD) results into account thanks to the statistical comparison of the frequencies of false positives in no template controls (NTC) and below-LOD results in technical replicates. In the eDNA-based approach, the use of this method led to an increase in Bd and Bsal detection of 28 and 50% respectively. In swabbing, this resulted in 8% more positive results for Bd. In some samples, the use of technical replicates allowed to recover above-LOD signals and increase Bd detection by 35 and 33% respectively for eDNA and swabbing, and Bsal detection by 25% for eDNA., Conclusions: These results confirmed the usefulness of technical replicates to overcome high levels of stochasticity in very low concentration samples even for a highly sensitive technique such as ddPCR. In addition, it showed that below-LOD signals could be consistently recovered and the corresponding amplification events assigned either to positive or negative detection via the method developed here. This methodology might be particularly worth pursuing in pathogenic agents' detection as false negatives could have important adverse consequences. In total, 15 ponds were found positive for Bd and four for Bsal. This study reports the first record of Bsal in Luxembourg., (© 2023. The Author(s).)

Wildlife diseases are a major global threat to biodiversity. Boreal toads (Anaxyrus [Bufo] boreas) are a state-endangered species in the southern Rocky Mountains of Colorado and New Mexico, and a species of concern in Wyoming, largely due to lethal skin infections caused by the amphibian chytrid fungus Batrachochytrium dendrobatidis (Bd). We performed conservation and landscape genomic analyses using single nucleotide polymorphisms from double-digest, restriction site-associated DNA sequencing in combination with the development of the first boreal toad (and first North American toad) reference genome to investigate population structure, genomic diversity, landscape connectivity and adaptive divergence. Genomic diversity (π = 0.00034-0.00040) and effective population sizes (N e = 8.9-38.4) were low, likely due to post-Pleistocene founder effects and Bd-related population crashes over the last three decades. Population structure was also low, likely due to formerly high connectivity among a higher density of geographically proximate populations. Boreal toad gene flow was facilitated by low precipitation, cold minimum temperatures, less tree canopy, low heat load and less urbanization. We found >8X more putatively adaptive loci related to Bd intensity than to all other environmental factors combined, and evidence for genes under selection related to immune response, heart development and regulation and skin function. These data suggest boreal toads in habitats with Bd have experienced stronger selection pressure from disease than from other, broad-scale environmental variations. These findings can be used by managers to conserve and recover the species through actions including reintroduction and supplementation of populations that have declined due to Bd., (© 2023 The Authors. Molecular Ecology published by John Wiley & Sons Ltd.)

Amphibian chytridiomycosis, caused by Batrachochytrium dendrobatidis (Bd), emerged from Asia and spread globally. By comparing functional MHC IIß1 alleles from an Asian Bd-resistant anuran species (Bufo gargarizans) with those of an Australasian Bd-susceptible species (Litoria caerulea), we identified MHC genotypes associated with Bd resistance. These alleles encode a glycine deletion (G90β1) and adjacent motifs in the deepest pathogen-derived peptide-binding groove. Every Bd-resistant individual, but no susceptible individuals, possessed at least one allele encoding the variant. We detected trans-species polymorphism at the end of the MHC IIβ1 sequences. The G90β1 deletion was encoded by different alleles in the two species, suggesting it may have evolved independently in each species rather than having been derived from a common ancestor. These results are consistent with a scenario by which MHC adaptations that confer resistance to the pathogen have evolved by convergent evolution. Immunogenetic studies such as this are critical to ongoing conservation efforts., Competing Interests: Declaration of Competing Interest The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Environmental DNA analyses of fungal communities typically reveal a much larger diversity than can be ascribed to known species. Much of this hidden diversity lies within undescribed fungal lineages, especially the early diverging fungi (EDF). Although these EDF often represent new lineages even at the phylum level, they have never been cultured, making their morphology and ecology uncertain. One of the methods to characterize these uncultured fungi is a single-cell DNA sequencing approach. In this study, we established a large data set of single-cell sequences of EDF by manually isolating and photographing parasitic fungi on various hosts such as algae, protists, and micro-invertebrates, combined with subsequent long-read sequencing of the ribosomal DNA locus (rDNA). We successfully obtained rDNA sequences of 127 parasitic fungal cells, which clustered into 71 phylogenetic lineages belonging to seven phylum-level clades of EDF: Blastocladiomycota, Chytridiomycota, Aphelidiomycota, Rozellomycota, and three unknown phylum-level clades. Most of our single cells yielded novel sequences distinguished from both described taxa and existing metabarcoding data, indicating an expansive and hidden diversity of parasitic taxa of EDF. We also revealed an unexpected diversity of endobiotic Olpidium -like chytrids and hyper-parasitic lineages. Overall, by combining photographs of parasitic fungi with phylogenetic analyses, we were able to better understand the ecological function and morphology of many of the branches on the fungal tree of life known only from DNA sequences. IMPORTANCE Much of the diversity of microbes from natural habitats, such as soil and freshwater, comprise species and lineages that have never been isolated into pure culture. In part, this stems from a bias of culturing in favor of saprotrophic microbes over the myriad symbiotic ones that include parasitic and mutualistic relationships with other taxa. In the present study, we aimed to shed light on the ecological function and morphology of the many undescribed lineages of aquatic fungi by individually isolating and sequencing molecular barcodes from 127 cells of host-associated fungi using single-cell sequencing. By adding these sequences and their photographs into the fungal tree, we were able to understand the morphology of reproductive and vegetative structures of these novel fungi and to provide a hypothesized ecological function for them. These individual host-fungal cells revealed themselves to be complex environments despite their small size; numerous samples were hyper-parasitized with other zoosporic fungal lineages such as Rozellomycota., Competing Interests: The authors declare no conflict of interest.

Skin microbial communities are an essential part of host health and can play a role in mitigating disease. Host and environmental factors can shape and alter these microbial communities and, therefore, we need to understand to what extent these factors influence microbial communities and how this can impact disease dynamics. Microbial communities have been studied in amphibian systems due to skin microbial communities providing some resistance to the amphibian chytrid fungus, Batrachochytrium dendrobatidis . However, we are only starting to understand how host and environmental factors shape these communities for amphibians. In this study, we examined whether amphibian skin bacterial communities differ among host species, host infection status, host developmental stage, and host habitat. We collected skin swabs from tadpoles and adults of three Ranid frog species ( Lithobates spp.) at the Mianus River Gorge Preserve in Bedford, New York, USA, and used 16S rRNA gene amplicon sequencing to determine bacterial community composition. Our analysis suggests amphibian skin bacterial communities change across host developmental stages, as has been documented previously. Additionally, we found that skin bacterial communities differed among Ranid species, with skin communities on the host species captured in streams or bogs differing from the communities of the species captured on land. Thus, habitat use of different species may drive differences in host-associated microbial communities for closely-related host species., Competing Interests: Christopher Nagy is the Director of Research & Education at the Mianus River Gorge Preserve., (© 2023 Gajewski et al.)

Genetic variation in the major histocompatibility complex (MHC) may be associated with resistance to the amphibian chytrid fungus Batrachochytrium dendrobatidis (Bd). The pathogen originated in Asia, then spread worldwide, causing amphibian population declines and species extinctions. We compared the expressed MHC IIβ1 alleles of a Bd-resistant species, Bufo gargarizans, from South Korea with those of a Bd-susceptible Australasian species, Litoria caerulea. We found at least six expressed MHC IIβ1 loci in each of the two species. Amino acid diversity encoded by these MHC alleles was similar between species, but the genetic distance of those alleles known for potential broader pathogen-derived peptide binding was greater in the Bd-resistant species. In addition, we found a potentially rare allele in one resistant individual from the Bd-susceptible species. Deep next-generation sequencing recovered approximately triple the genetic resolution accessible from traditional cloning-based genotyping. Targeting the full MHC IIβ1 enables us to better understand how host MHC may adapt to emerging infectious diseases., Competing Interests: Declaration of Competing Interest The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

The key role of symbiotic skin bacteria communities in amphibian resistance to emerging pathogens is well recognized, but factors leading to their dysbiosis are not fully understood. In particular, the potential effects of population translocations on the composition and diversity of hosts' skin microbiota have received little attention, although such transfers are widely carried out as a strategy for amphibian conservation. To characterize the potential reorganization of the microbiota over such a sudden environmental change, we conducted a common-garden experiment simulating reciprocal translocations of yellow-spotted salamander larvae across three lakes. We sequenced skin microbiota samples collected before and 15 days after the transfer. Using a database of antifungal isolates, we identified symbionts with known function against the pathogen Batrachochytrium dendrobatidis, a major driver of amphibian declines. Our results indicate an important reorganization of bacterial assemblages throughout ontogeny, with strong changes in composition, diversity and structure of the skin microbiota in both control and translocated individuals over the 15 days of monitoring. Unexpectedly, the diversity and community structure of the microbiota were not significantly affected by the translocation event, thus suggesting a strong resilience of skin bacterial communities to environmental change-at least across the time-window studied here. A few phylotypes were more abundant in the microbiota of translocated larvae, but no differences were found among pathogen-inhibiting symbionts. Taken together, our results support amphibian translocations as a promising strategy for this endangered animal class, with limited impact on their skin microbiota., (© 2023 The Authors. Molecular Ecology published by John Wiley & Sons Ltd.)

The Chytridiomycota is a phylum of zoosporic eufungi that inhabit terrestrial, freshwater, and oceanic habitats. Within the phylum, the Rhizophydiales contains several monotypic families theorized to hold a diverse assemblage of fungi yet to be discovered and properly described. Based on morphology alone, many species in this order are difficult or impossible to identify. In this study, we isolated three chytrids from northern Thailand. Phylogenetic analyses placed the isolates in three monotypic genera within Rhizophydiales. Intrageneric genetic distances in the internal transcribed spacer (ITS) ranged between 1.5 and 8.5%. Angulomyces solicola sp. nov. is characterized by larger sporangia, spores, and fewer discharge papilla than A.argentinensis; Gorgonomyces thailandicus sp. nov. has larger zoospores and fewer discharge papillae in culture compared to G. haynaldii; Terramyces chiangraiensis sp. nov. produces larger sporangia than T. subangulosum. We delimited species of Angulomyces, Gorgonomyces and Terramyces using a tripartite approach that employed phylogeny, ITS genetic distances and Poisson tree processes (PTP). Results of these approaches suggest more than one species in each genus. This study contributes to the knowledge of chytrids, an understudied group in Thailand and worldwide., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

The origins and evolution of virulence in amphibian-infecting chytrids Batrachochytrium dendrobatidis ( Bd ) and Batrachochytrium salamandrivorans ( Bsal) are largely unknown. Here, we use deep nanopore sequencing of Bsal and comparative genomics against 21 high-quality genome assemblies that span the fungal Chytridiomycota. We discover that Bsal has the most repeat-rich genome of the Chytridiomycota, comprising 40.9% repetitive elements; this genome has expanded to more than 3× the length of its conspecific Bd , with autonomous and fully functional LTR/Gypsy elements contributing significantly to the expansion. The M36 metalloprotease virulence factors are highly expanded ( n = 177) in Bsal , most of which (53%) are flanked by transposable elements, suggesting they have a repeat-associated expansion. We find enrichment upstream of M36 metalloprotease genes of three novel repeat families belonging to the repeat superfamily of LINEs that are implicated with gene copy number variations. Additionally, Bsal has a highly compartmentalized genome architecture, with virulence factors enriched in gene-sparse/repeat-rich compartments, while core conserved genes are enriched in gene-rich/repeat-poor compartments. Genes upregulated during infection are primarily found in the gene-sparse/repeat-rich compartment in both Bd and Bsal . Furthermore, genes with signatures of positive selection in Bd are enriched in repeat-rich regions, suggesting these regions are a cradle for the evolution of chytrid pathogenicity. These are the hallmarks of two-speed genome evolution, and this study provides evidence of two-speed genomes in an animal pathogen, shedding light on the evolution of fungal pathogens of vertebrates driving global declines and extinctions.

The amphibian chytrid fungus (Bd) has caused declines and some extinctions of amphibian populations worldwide. Early and accurate Bd detection is essential for management of susceptible anurans. We analyzed the effectiveness of in situ DNA extraction with a handheld mobile quantitative PCR (qPCR) thermocycler to detect Bd on frog skin swabs and in water samples using environmental DNA (eDNA). We collected duplicate eDNA samples and skin swabs from 3 Bd-positive Rana sierrae populations. We processed one set of samples using a field protocol (a handheld thermocycler) and the other half using a standard lab protocol. We detected Bd DNA in all R. sierrae swabbed using both the field and lab protocols. We also detected Bd DNA in eDNA samples at all sites, although the field and lab protocols failed to detect Bd eDNA at separate singular sites; results from the field and lab eDNA protocol did not match. The probability of detecting Bd DNA in the technical replicates was lower for the field protocol than the lab protocol, suggesting the field protocol has lower sensitivity and may not detect low quantities of DNA. Our results suggest that the field extraction protocol using a handheld qPCR platform is a promising tool for rapid detection of Bd in susceptible amphibian populations, yielding accurate results in less than 60 min. However, the applied field protocol may be prone to false negatives when analyzing low-quantity DNA samples such as eDNA water samples or frog swabs with low pathogen loads.

The interactions of parasitic fungi with their phytoplankton hosts in the marine environment are mostly unknown. In this study, we evaluated the diversity of Chytridiomycota in phytoplankton communities dominated by dinoflagellates at several coastal locations in the NW Mediterranean Sea and demonstrated the most prominent interactions of these parasites with their hosts. The protist community in seawater differed from that in sediment, with the latter characterized by a greater heterogeneity of putative hosts, such as dinoflagellates and diatoms, as well as a chytrid community more diverse in its composition and with a higher relative abundance. Chytrids accounted for 77 amplicon sequence variants, of which 70 were found exclusively among different blooming host species. The relative abundance of chytrids was highest in samples dominated by the dinoflagellate genera Ostreopsis and Alexandrium, clearly indicating the presence of specific chytrid communities. The establishment of parasitoid-host co-cultures of chytrids and dinoflagellates allowed the morphological identification and molecular characterization of three species of Chytridiomycota, including Dinomyces arenysensis, as one of the most abundant environmental sequences, and the discovery of two other species not yet described., (© 2022 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd.)

Loss of the flagellum was an important step in the evolution of fungi. The flagellated fungi of the phylum Olpidiomycota are the closest relative of the non-flagellated terrestrial fungi. There are genes encoding proteins, the occurrence of which shows a strong correlation with the incidence of the flagellum. One of these gene/protein families is "TPPP-like proteins" whose main feature is the presence of the p25alpha domain. The functional link between TPPP and flagellum has also been shown. Most of the phyla of flagellated fungi have been known to contain TPPP-like proteins but Olpidiomycota was an exception. This study demonstrates that Olpidium bornovanus , similarly to some fungi of Chytridiomycota and Blastocladiomycota, has a "fungal-type" TPPP characterized by the presence of two (a complete and an incomplete) p25alpha domains.

Symbiotic interactions can determine the evolutionary trajectories of host species, influencing genetic variation through selection and changes in demography. In the context of strong selective pressures such as those imposed by infectious diseases, symbionts providing defences could contribute to increase host fitness upon pathogen emergence. Here, we generated genome-wide data of an amphibian species to find evidence of evolutionary pressures driven by two skin symbionts: a batrachochytrid fungal pathogen and an antifungal bacterium. Using demographic modelling, we found evidence of decreased effective population size, probably due to pathogen infections. Additionally, we investigated host genetic associations with infection status, antifungal bacterium abundance and overall microbiome diversity using structural equation models. We uncovered relatively lower nucleotide diversity in infected frogs and potential heterozygote advantage to recruit the candidate beneficial symbiont and fight infections. Our models indicate that environmental conditions have indirect effects on symbiont abundance through both host body traits and microbiome diversity. Likewise, we uncovered a potential offsetting effect among host heterozygosity-fitness correlations, plausibly pointing to different ecological and evolutionary processes among the three species due to dynamic interactions. Our findings revealed that evolutionary pressures not only arise from the pathogen but also from the candidate beneficial symbiont, and both interactions shape the genetics of the host. Our results advance knowledge about multipartite symbiotic relationships and provide a framework to model ecological and evolutionary dynamics in wild populations. Finally, our study approach can be applied to inform conservation actions such as bioaugmentation strategies for other imperilled amphibians affected by infectious diseases., (© 2022 John Wiley & Sons Ltd.)

We detected the fungal assemblages present in lake sediments on James Ross Island, Antarctica, using DNA metabarcoding. A total of 132 amplicon sequence variants (ASVs) were assigned, dominated by taxa of the phyla Ascomycota, Basidiomycota, Mortierellomycota and Mucoromycota. The less common phyla Chytridiomycota, Rozellomycota, Monoblepharomycota, Basidiobolomycota, Aphelidiomycota and the fungus-like Straminopila were also detected. Fungal sp. 1, Fungal sp. 2, Spizellomycetales sp. 1, Rozellomycotina sp. 1, Talaromyces rubicundus and Betamyces sp. dominated the assemblages. In general, the assemblages displayed high diversity and richness, and moderate dominance. Saprophytic, pathogenic and symbiotic fungi were detected. The metabarcoding data indicated that Antarctic lakes may represent a hotspot of fungal diversity in Antarctica. The sediments of these lakes may accumulate different fungal fragments and active fungal mycelia and their propagules, deposited over long periods of time. Lakes in the Antarctic Peninsula region are sensitive environments threatened by the effects of regional climatic changes. The abundance of sequences of little-known Rozellomycota and Chytridiomycota (Spizellomycetales) taxa in these ecosystems highlights the need for further studies to identify if they are metabolically active in the sediments and whether they have potentially pathogenic capabilities., (Copyright © 2022 British Mycological Society. Published by Elsevier Ltd. All rights reserved.)

Temporal changes and transmission patterns in host-associated microbial communities have important implications for host health. The diversity of amphibian skin microbial communities is associated with disease outcome in amphibians exposed to the fungal pathogen Batrachochytrium dendrobatidis (Bd). To successfully develop conservation strategies against Bd, we need a comprehensive understanding of how skin microbes are maintained and transmitted over time within populations. We used 16S rRNA sequence analysis to compare Epipedobates anthonyi frogs housed with one conspecific to frogs housed singly at four time points over the course of 1 year. We found that both α and β diversity of frog skin bacterial communities changed significantly over the course of the experiment. Specifically, we found that bacterial communities of cohabitating frogs became more similar over time. We also observed that some bacterial taxa were differentially abundant between frogs housed singly and frogs housed with a conspecific. These results suggest that conspecific contact may play a role in mediating amphibian skin microbial diversity and that turnover of skin microbial communities can occur across time. Our findings provide rationale for future studies exploring horizontal transmission as a potential mechanism of host-associated microbial maintenance in amphibians.

Zoosporic fungi of the phylum Chytridiomycota (chytrids) regularly dominate pelagic fungal communities in freshwater and marine environments. Their lifestyles range from obligate parasites to saprophytes. Yet, linking the scarce available sequence data to specific ecological traits or their host ranges constitutes currently a major challenge. We combined 28 S rRNA gene amplicon sequencing with targeted isolation and sequencing approaches, along with cross-infection assays and analysis of chytrid infection prevalence to obtain new insights into chytrid diversity, ecology, and seasonal dynamics in a temperate lake. Parasitic phytoplankton-chytrid and saprotrophic pollen-chytrid interactions made up the majority of zoosporic fungal reads. We explicitly demonstrate the recurrent dominance of parasitic chytrids during frequent diatom blooms and saprotrophic chytrids during pollen rains. Distinct temporal dynamics of diatom-specific parasitic clades suggest mechanisms of coexistence based on niche differentiation and competitive strategies. The molecular and ecological information on chytrids generated in this study will aid further exploration of their spatial and temporal distribution patterns worldwide. To fully exploit the power of environmental sequencing for studies on chytrid ecology and evolution, we emphasize the need to intensify current isolation efforts of chytrids and integrate taxonomic and autecological data into long-term studies and experiments., (© 2022. The Author(s).)

While some pathogens are limited to single species, others can colonize many hosts, likely contributing to the emergence of novel disease outbreaks. Despite this biodiversity threat, traits associated with host niche expansions are not well understood in multihost pathogens. Here, we aimed to uncover functional machinery driving multihost invasion by focusing on Batrachochytrium dendrobatidis (Bd), a pathogen that infects the skin of hundreds of amphibians worldwide. We performed a meta-analysis of Bd gene expression using data from published infection experiments and newly generated profiles. We analysed Bd transcriptomic landscapes across the skin of 14 host species, reconstructed Bd isolates phylogenetic relationships, and inferred the origin and evolutionary history of differentially expressed genes under a phylogenetic framework comprising other 12 zoosporic fungi. Bd displayed plastic infection strategies when challenged by hosts with different disease susceptibility. Our analyses identified sets of differentially expressed genes under host environments with similar infection outcome. We stressed nutritional immunity and gene silencing as important processes required to overcome challenging skin environments in less susceptible hosts. Overall, Bd genes expressed during amphibian skin exploitation have arisen mainly via gene duplications with great family expansions, increasing the gene copy events previously described for this fungal species. Finally, we provide a comprehensive gene data set that can be used to further examine eco-evolutionary hypotheses for this host-pathogen system. Our study supports the idea that host environments exert contrasting selective pressures, such that gene expression plasticity could be one of the evolutionary keys leading to the success of multihost pathogens., (© 2022 John Wiley & Sons Ltd.)

Molecular technologies have revolutionized the field of wildlife disease ecology, allowing the detection of outbreaks, novel pathogens, and invasive strains. In particular, metabarcoding approaches, defined here as tools used to amplify and sequence universal barcodes from a single sample (e.g., 16S rRNA for bacteria, ITS for fungi, 18S rRNA for eukaryotes), are expanding our traditional view of host-pathogen dynamics by integrating microbial interactions that modulate disease outcome. Here, I provide an analysis from the perspective of the field of amphibian disease ecology, where the emergence of multi-host pathogens has caused global declines and species extinctions. I reanalyzed an experimental mesocosm dataset to infer the functional profiles of the skin microbiomes of coqui frogs (Eleutherodactylus coqui), an amphibian species that is consistently found infected with the fungal pathogen Batrachochytrium dendrobatidis and has high turnover of skin bacteria driven by seasonal shifts. I found that the metabolic activities of microbiomes operate at different capacities depending on the season. Global enrichment of predicted functions was more prominent during the warm-wet season, indicating that microbiomes during the cool-dry season were either depauperate, resistant to new bacterial colonization, or that their functional space was more saturated. These findings suggest important avenues to investigate how microbes regulate population growth and contribute to host physiological processes. Overall, this study highlights the current challenges and future opportunities in the application of metabarcoding to investigate the causes and consequences of disease in wild systems., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Society for Integrative and Comparative Biology.)

Many endangered amphibian species survive in captive breeding facilities, but there have been few attempts to reintroduce captive-born individuals to rebuild wild populations. We conducted a soft-release trial of limosa harlequin frogs, Atelopus limosus, which are highly susceptible to the amphibian chytrid fungus Batrachochytrium dendrobatidis (Bd), to understand changes associated with the transition from captivity to the wild. Specifically, we assessed changes in body condition, skin-associated bacterial communities and disease status after release. Frogs were housed individually in field mesocosms and monitored for 27 days. Body condition did not significantly change in the mesocosms, and was similar to, or higher than, that of wild conspecifics at day 27. The skin bacteria of captive-born frogs, based on 16S rRNA gene amplicons, became similar to that of wild conspecifics after 27 days in mesocosms. Prevalence of Bd in wild conspecifics was 13-27%, and 15% of the A. limosus in mesocosms became infected with Bd, but no mortality of infected frogs was observed. We conclude that mesocosms are suitable for systematically and repeatedly monitoring amphibians during release trials, and that body condition, the skin microbiome, and Bd status can all change within one month of placement of captive-born individuals back into the wild.

Mucosal defenses are crucial in animals for protection against pathogens and predators. Host defense peptides (antimicrobial peptides, AMPs) as well as skin-associated microbes are key components of mucosal immunity, particularly in amphibians. We integrate microbiology, molecular biology, network-thinking, and proteomics to understand how host and microbially derived products on amphibian skin (referred to as the mucosome) serve as pathogen defenses. We studied defense mechanisms against chytrid pathogens, Batrachochytrium dendrobatidis (Bd) and B. salamandrivorans (Bsal), in four salamander species with different Batrachochytrium susceptibilities. Bd infection was quantified using qPCR, mucosome function (i.e., ability to kill Bd or Bsal zoospores in vitro ), skin bacterial communities using 16S rRNA gene amplicon sequencing, and the role of Bd-inhibitory bacteria in microbial networks across all species. We explored the presence of candidate-AMPs in eastern newts and red-backed salamanders. Eastern newts had the highest Bd prevalence and mucosome function, while red-back salamanders had the lowest Bd prevalence and mucosome function, and two-lined salamanders and seal salamanders were intermediates. Salamanders with highest Bd infection intensity showed greater mucosome function. Bd infection prevalence significantly decreased as putative Bd-inhibitory bacterial richness and relative abundance increased on hosts. In co-occurrence networks, some putative Bd-inhibitory bacteria were found as hub-taxa, with red-backs having the highest proportion of protective hubs and positive associations related to putative Bd-inhibitory hub bacteria. We found more AMP candidates on salamanders with lower Bd susceptibility. These findings suggest that salamanders possess distinct innate mechanisms that affect chytrid fungi. IMPORTANCE How host mucosal defenses interact, and influence disease outcome is critical in understanding host defenses against pathogens. A more detailed understanding is needed of the interactions between the host and the functioning of its mucosal defenses in pathogen defense. This study investigates the variability of chytrid susceptibility in salamanders and the innate defenses each species possesses to mediate pathogens, thus advancing the knowledge toward a deeper understanding of the microbial ecology of skin-associated bacteria and contributing to the development of bioaugmentation strategies to mediate pathogen infection and disease. This study improves the understanding of complex immune defense mechanisms in salamanders and highlights the potential role of the mucosome to reduce the probability of Bd disease development and that putative protective bacteria may reduce likelihood of Bd infecting skin.

The putatively positive association between host genetic diversity and the ability to defend against pathogens has long attracted the attention of evolutionary biologists. Chytridiomycosis, a disease caused by the chytrid fungus Batrachochytrium dendrobatidis (Bd), has emerged in recent decades as a cause of dramatic declines and extinctions across the amphibian clade. Bd susceptibility can vary widely across populations of the same species, but the relationship between standing genetic diversity and susceptibility has remained notably underexplored so far. Here, we focus on a putatively Bd-naive system of two mainland and two island populations of the common toad (Bufo bufo) at the edge of the species' range and use controlled infection experiments and dd-RAD sequencing of >10 000 SNPs across 95 individuals to characterize the role of host population identity, genetic variation and individual body mass in mediating host response to the pathogen. We found strong genetic differentiation between populations and marked variation in their susceptibility to Bd. This variation was not, however, governed by isolation-mediated genetic erosion, and individual heterozygosity was even found to be negatively correlated with survival. Individual survival during infection experiments was strongly positively related to body mass, which itself was unrelated to population of origin or heterozygosity. Our findings underscore the general importance of context-dependency when assessing the role of host genetic variation for the ability of defence against pathogens., (© 2022 The Authors. Journal of Evolutionary Biology published by John Wiley & Sons Ltd on behalf of European Society for Evolutionary Biology.)

Potato wart disease is considered one of the most important quarantine pests for cultivated potato and is caused by the obligate biotrophic chytrid fungus Synchytrium endobioticum. This review integrates observations from early potato wart research and recent molecular, genetic, and genomic studies of the pathogen and its host potato. Taxonomy, epidemiology, pathology, and formation of new pathotypes are discussed, and a model for molecular S. endobioticum-potato interaction is proposed., Taxonomy: Currently classified as kingdom: Fungi, phylum: Chytridiomycota, class: Chytridiomycetes, order: Chytridiales, family: Synchytriaceae, genus: Synchytrium, species: Synchytrium endobioticum, there is strong molecular support for Synchytriaceae to be transferred to the order Synchytriales., Hosts and Disease Symptoms: Solanum tuberosum is the main host for S. endobioticum but other solanaceous species have been reported as alternative hosts. It is not known if these alternative hosts play a role in the survival of the pathogen in (borders of) infested fields. Disease symptoms on potato tubers are characterized by the warty cauliflower-like malformations that are the result of cell enlargement and cell multiplication induced by the pathogen. Meristematic tissue on tubers, stolons, eyes, sprouts, and inflorescences can be infected while the potato root system seems to be immune., Pathotypes: For S. endobioticum over 40 pathotypes, which are defined as groups of isolates with a similar response to a set of differential potato varieties, are described. Pathotypes 1(D1), 2(G1), 6(O1), and 18(T1) are currently regarded to be most widespread. However, with the current differential set other pathogen diversity largely remains undetected., Pathogen-Host Interaction: A single effector has been described for S. endobioticum (AvrSen1), which is recognized by the potato Sen1 resistance gene product. This is also the first effector that has been described in Chytridiomycota, showing that in this fungal division resistance also fits the gene-for-gene concept. Although significant progress was made in the last decade in mapping wart disease resistance loci, not all resistances present in potato breeding germplasm could be identified. The use of resistant varieties plays an essential role in disease management., (© 2022 The Authors. Molecular Plant Pathology published by British Society for Plant Pathology and John Wiley & Sons Ltd.)

Host-pathogen specificity can arise from certain selective environments mediated by both the host and pathogen. Therefore, understanding the degree to which host species identity is correlated with pathogen genotype can help reveal historical host-pathogen dynamics. One animal disease of particular concern is chytridiomycosis, typically caused by the global panzootic lineage of the amphibian chytrid fungus (Batrachochytrium dendrobatidis, Bd), termed Bd-GPL. This pathogen lineage has caused devastating declines in amphibian communities around the world. However, the site of origin for the common ancestor of modern Bd-GPL and the fine-scale transmission dynamics of this lineage have remained a mystery. This is especially the case in North America where Bd-GPL is widespread, but disease outbreaks occur sporadically. Herein, we use Bd genetic data collected throughout the United States from amphibian skin swabs and cultured isolate samples to investigate Bd genetic patterns. We highlight two case studies in Pennsylvania and Nevada where Bd-GPL genotypes are strongly correlated with host species identity. Specifically, in some localities bullfrogs (Rana catesbeiana) are infected with Bd-GPL lineages that are distinct from those infecting other sympatric amphibian species. Overall, we reveal a previously unknown association of Bd genotype with host species and identify the eastern United States as a Bd diversity hotspot and potential site of origin for Bd-GPL., Competing Interests: The authors have declared that no competing interests exist.

Background: The fungal pathogen Batrachochytrium dendrobatidis (Bd) threatens amphibian biodiversity and ecosystem stability worldwide. Amphibian skin microbial community structure has been linked to the clinical outcome of Bd infections, yet its overall functional importance is poorly understood., Methods: Microbiome taxonomic and functional profiles were assessed using high-throughput bacterial 16S rRNA and fungal ITS2 gene sequencing, bacterial shotgun metagenomics and skin mucosal metabolomics. We sampled 56 wild midwife toads (Alytes obstetricans) from montane populations exhibiting Bd epizootic or enzootic disease dynamics. In addition, to assess whether disease-specific microbiome profiles were linked to microbe-mediated protection or Bd-induced perturbation, we performed a laboratory Bd challenge experiment whereby 40 young adult A. obstetricans were exposed to Bd or a control sham infection. We measured temporal changes in the microbiome as well as functional profiles of Bd-exposed and control animals at peak infection., Results: Microbiome community structure and function differed in wild populations based on infection history and in experimental control versus Bd-exposed animals. Bd exposure in the laboratory resulted in dynamic changes in microbiome community structure and functional differences, with infection clearance in all but one infected animal. Sphingobacterium, Stenotrophomonas and an unclassified Commamonadaceae were associated with wild epizootic dynamics and also had reduced abundance in laboratory Bd-exposed animals that cleared infection, indicating a negative association with Bd resistance. This was further supported by microbe-metabolite integration which identified functionally relevant taxa driving disease outcome, of which Sphingobacterium and Bd were most influential in wild epizootic dynamics. The strong correlation between microbial taxonomic community composition and skin metabolome in the laboratory and field is inconsistent with microbial functional redundancy, indicating that differences in microbial taxonomy drive functional variation. Shotgun metagenomic analyses support these findings, with similar disease-associated patterns in beta diversity. Analysis of differentially abundant bacterial genes and pathways indicated that bacterial environmental sensing and Bd resource competition are likely to be important in driving infection outcomes., Conclusions: Bd infection drives altered microbiome taxonomic and functional profiles across laboratory and field environments. Our application of multi-omics analyses in experimental and field settings robustly predicts Bd disease dynamics and identifies novel candidate biomarkers of infection. Video Abstract., (© 2022. The Author(s).)

Host-associated microbial biofilms can provide protection against pathogen establishment. In many host-microbe symbioses (including, but not limited to humans, plants, insects, and amphibians), there is a correlation between host-associated microbial diversity and pathogen infection risk. Diversity may prevent infection by pathogens through sampling effects and niche complementarity, but an alternative hypothesis may be that microbial biomass is confounded with diversity and that host-associated biofilms are deterring pathogen establishment through space preemption. In this study, we use the amphibian system as a model for host-microbe-pathogen interactions to ask two questions: (i) is bacterial richness confounded with biofilm thickness or cell density, and (ii) to what extent do biofilm thickness, cell density, and bacterial richness each deter the establishment of the amphibian fungal pathogen Batrachochytrium dendrobatidis? To answer these questions, we built a custom biofilm microcosm that mimics the host-environment interface by allowing nutrients to diffuse out of a fine-pore biofilm scaffolding. This created a competitive environment in which bacteria and the fungal pathogen compete for colonization space. We then challenged bacterial biofilms ranging in community richness, biofilm thickness, bacterial cell density, and B. dendrobatidis (also known as Bd )-inhibitory metabolite production with live B. dendrobatidis zoospores to determine how B. dendrobatidis establishment success on membranes varies. We found that biofilm thickness and B. dendrobatidis-inhibitory isolate richness work in complement to reduce B. dendrobatidis establishment success. This work underscores that physical aspects of biofilm communities can play a large role in pathogen inhibition, and in many studies, these traits are not studied. IMPORTANCE Our finding highlights the fact that diversity, as measured through 16S rRNA gene sequencing, may obscure the true mechanisms behind microbe-mediated pathogen defense and that physical space occupation by biofilm-forming symbionts may significantly contribute to pathogen protection. These findings have implications across a wide range of host-microbe systems since 16S rRNA gene sequencing is a standard tool used across many microbial systems. Further, our results are potentially relevant to many host-pathogen systems since host-associated bacterial biofilms are ubiquitous.

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., (© 2021 Society for Applied Microbiology and John Wiley & Sons Ltd.)

While emerging fungi threaten global biodiversity, the paucity of fungal genome assemblies impedes thoroughly characterizing epidemics and developing effective mitigation strategies. Here, we generate de novo genomic assemblies for six outbreaks of the emerging pathogen Batrachochytrium salamandrivorans (Bsal). We reveal the European epidemic currently damaging amphibian populations to comprise multiple, highly divergent lineages demonstrating isolate-specific adaptations and metabolic capacities. In particular, we show extensive gene family expansions and acquisitions, through a variety of evolutionary mechanisms, and an isolate-specific saprotrophic lifecycle. This finding both explains the chytrid's ability to divorce transmission from host density, producing Bsal's enigmatic host population declines, and is a key consideration in developing successful mitigation measures., (© 2021. The Author(s).)

Host-associated microbes can interact with macro-organisms in a number of ways that affect host health. Few studies of host-associated microbiomes, however, focus on fungi. In addition, it is difficult to discern whether a fungal organism found in or on an ectotherm host is associating with it in a durable, symbiotic interaction versus a transient one, and to what extent the habitat and host share microbes. We seek to identify these host-microbe interactions on an amphibian, the Colorado boreal toad (Anaxyrus boreas boreas). We sequenced the ITS1 region of the fungal community on the skin of wild toads (n = 124) from four sites in the Colorado Rocky Mountains, across its physiologically dynamic developmental life stages. We also sampled the common habitats used by boreal toads: water from their natal wetland and aquatic pond sediment. We then examined diversity patterns within different life stages, between host and habitat, and identified fungal taxa that could be putatively host-associated with toads by using an indicator species analysis on toad versus environmental samples. Host and habitat were strikingly similar, with the exception of toad eggs. Post-hatching toad life stages were distinct in their various fungal diversity measures. We identified eight fungal taxa that were significantly associated with eggs, but no other fungal taxa were associated with other toad life stages compared with their environmental habitat. This suggests that although pre- and post-metamorphic toad life stages differ from each other, the habitat and host fungal communities are so similar that identifying obligate host symbionts is difficult with the techniques used here. This approach does, however, leverage sequence data from host and habitat samples to predict which microbial taxa are host-associated versus transient microbes, thereby condensing a large set of sequence data into a smaller list of potential targets for further consideration., Competing Interests: The authors have declared that no competing interests exist.

Chytridiomycosis is a fungal disease caused by the pathogens, Batrachochytrium dendrobatidis (Bd) and B. salamandrivorans (Bsal), which has caused declines in amphibian populations worldwide. Asia is considered as a coldspot of infection, since adult frogs are less susceptible to Bd-induced mortality or morbidity. Using the next-generation sequencing approach, we assessed the cutaneous bacterial community composition and presence of anti-Bd bacteria in six frog species from India using DNA isolated from skin swabs. All the six frog species sampled were tested using nested PCR and found Bd negative. We found a total of 551 OTUs on frog skin, of which the bacterial phyla such as Proteobacteria (56.15% average relative abundance) was dominated followed by Actinobacteria (21.98% average relative abundance) and Firmicutes (13.7% average relative abundance). The contribution of Proteobacteria in the anti-Bd community was highest and represented by 175 OTUs. Overall, the anti-Bd bacterial community dominated (51.7% anti-Bd OTUs) the skin microbiome of the frogs. The study highlights the putative role of frog skin microbiome in affording resistance to Bd infections in coldspots of infection., (© 2021. The Author(s).)

Emerging infectious diseases are a pressing threat to global biological diversity. Increased incidence and severity of novel pathogens underscores the need for methodological advances to understand pathogen emergence and spread. Here, we use genetic epidemiology to test, and challenge, key hypotheses about a devastating zoonotic disease impacting amphibians globally. Using an amplicon-based sequencing method and non-invasive samples we retrospectively explore the history of the fungal pathogen Batrachochytrium dendrobatidis ( Bd ) in two emblematic amphibian systems: the Sierra Nevada of California and Central Panama. The hypothesis in both regions is the hypervirulent Global Panzootic Lineage of Bd ( Bd GPL) was recently introduced and spread rapidly in a wave-like pattern. Our data challenge this hypothesis by demonstrating similar epizootic signatures can have radically different underlying evolutionary histories. In Central Panama, our genetic data confirm a recent and rapid pathogen spread. However, Bd GPL in the Sierra Nevada has remarkable spatial structuring, high genetic diversity and a relatively older history inferred from time-dated phylogenies. Thus, this deadly pathogen lineage may have a longer history in some regions than assumed, providing insights into its origin and spread. Overall, our results highlight the importance of integrating observed wildlife die-offs with genetic data to more accurately reconstruct pathogen outbreaks.

Amphibian populations around the world have been affected by two pathogenic fungi within the phylum Chytridiomycota. Batrachochytrium dendrobatidis (Bd) has infected hundreds of species and led to widespread declines and some species extinctions. Batrachochytrium salamandrivorans (Bsal) has devastated some native European salamanders, especially the iconic fire salamanders (Salamandra salamandra). Comparative genomic studies show that Bd is more diverse and widespread than previously thought, and global lineages occur together allowing for the development of hybrid lineages. New studies raise the concern of greater pathogenesis if both Bd and Bsal infect the same host. Although amphibians possess robust immune defenses, co-infected and many single-infected hosts seem unable to mount effective immune responses. A strong defense may actually be harmful. Analysis of Bd and Bsal secretions documents small metabolites that signal high density to limit their growth and to suppress adaptive immune defenses, thus enabling a stealth presence in the skin compartment., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Molecular and ultrastructural investigations of the Chytridiomycota during the last 20 years have led to the separation of new orders, including the Rhizophydiales. Most species in this order are morphologically similar, possessing monocentric, eucarpic, spherical thalli. Here, based on analysis of nuc 18S and 28S rDNA, we add the new genus and species Polyrhizophydium stewartii to the order. This saprobe of moribund aquatic plant leaves is the first known rhizomycelial species in the order. In our molecular phylogeny, P. stewartii groups with the amphibian pathogens Batrachochytriuim dendrobatidis and B. salamandrivorans , making it of particular interest to investigators studying evolutionary pathways associated with host-switching and morphological adaptation.

Amphibian skin bacteria may confer protection against the fungus Batrachochytrium dendrobatidis (Bd), but responses of skin bacteria to different Bd lineages are poorly understood. The global panzootic lineage (Bd-GPL) has caused amphibian declines and extinctions globally. However, other lineages are enzootic (Bd-Asia-2/Brazil). Increased contact rates between Bd-GPL and enzootic lineages via globalization pose unknown consequences for host-microbiome-pathogen dynamics. We conducted a laboratory experiment and used 16S rRNA amplicon-sequencing to assess: (i) whether two lineages (Bd-Asia-2/Brazil and Bd-GPL) and their recombinant, in single and mixed infections, differentially affect amphibian skin bacteria; (ii) and the changes associated with the transition to laboratory conditions. We determined no clear differences in bacterial diversity among Bd treatments, despite differences in infection intensity. However, we observed an additive effect of mixed infections on bacterial alpha diversity and a potentially antagonistic interaction between Bd genotypes. Additionally, observed changes in community composition suggest a higher ability of Bd-GPL to alter skin bacteria. Lastly, we observed a drastic reduction in bacterial diversity and a change in community structure in laboratory conditions. We provide evidence for complex interactions between Bd genotypes and amphibian skin bacteria during coinfections, and expand on the implications of experimental conditions in ecological studies., (© The Author(s) 2021. Published by Oxford University Press on behalf of FEMS.)

Systematics of Chytridiales has been deeply influenced by analyses of molecular loci and zoospore ultrastructure. Even though the Chytridiales is the largest order within Chytridiomycota, Brazilian isolates of this clade have been poorly integrated. Here, we isolated seven species and documented their morphology, including zoospore ultrastructure for Siphonaria aurea , and phylogenetic positions for all based on analyses of nuc 18S and 28S rDNA. Phylogenetic results support the placement of these species in Chytriomycetaceae and Chytridiaceae, with two new species described, Rhizidium crepaturum and Siphonaria aurea , and Rodmanochytrium sphaericum recorded for the first time from Brazil.

Global amphibian populations are facing a novel threat, chytridiomycosis, caused by the fungus Batrachochytrium dendrobatidis (Bd), which is responsible for the severe decline of a number of species across several continents. Chytridiomycosis in Asia is a relatively recent discovery yet there have been no reports on Bd-presence in Bangladeshi amphibians. We conducted a preliminary study on 133 wild frogs from seven sites in Bangladesh between April and July 2018. Nested PCR analysis showed 20 samples (15.04%) and 50% of the tested taxa (9 species from 6 genera and 4 families) as Bd-positive. Eight of the nine species are discovered as newly infected hosts. Analysis of Bd-positive samples shows prevalence does not significantly vary among different land cover categories, although the occurrence is higher in forested areas. The prevalence rate is similar in high and low disturbed areas, but the range of occurrence is statistically higher in low disturbance areas. Maximum entropy distribution modeling indicates high probabilities of Bd occurrence in hilly and forested areas in southeast and central-north Bangladesh. The Bd-specific ITS1-5.8S-ITS2 ribosomal gene sequence from the Bd-positive samples tested is completely identical. A neighbor-joining phylogenetic tree reveals that the identified strain shares a common ancestry with strains previously discovered in different Asian regions. Our results provide the first evidence of Bd-presence in Bangladeshi amphibians, inferring that diversity is at risk. The effects of environmental and climatic factors along with quantitative PCR analysis are required to determine the infection intensity and susceptibility of amphibians in the country., (© 2021. EcoHealth Alliance.)

Microbiome-pathogen interactions are increasingly recognized as an important element of host immunity. While these host-level interactions will have consequences for community disease dynamics, the factors which influence host microbiomes at larger scales are poorly understood. We here describe landscape-scale pathogen-microbiome associations within the context of post-epizootic amphibian chytridiomycosis, a disease caused by the panzootic chytrid fungus Batrachochytrium dendrobatidis. We undertook a survey of Neotropical amphibians across altitudinal gradients in Ecuador ~30 years following the observed amphibian declines and collected skin swab-samples which were metabarcoded using both fungal (ITS-2) and bacterial (r16S) amplicons. The data revealed marked variation in patterns of both B. dendrobatidis infection and microbiome structure that are associated with host life history. Stream breeding amphibians were most likely to be infected with B. dendrobatidis. This increased probability of infection was further associated with increased abundance and diversity of non-Batrachochytrium chytrid fungi in the skin and environmental microbiome. We also show that increased alpha diversity and the relative abundance of fungi are lower in the skin microbiome of adult stream amphibians compared to adult pond-breeding amphibians, an association not seen for bacteria. Finally, stream tadpoles exhibit lower proportions of predicted protective microbial taxa than pond tadpoles, suggesting reduced biotic resistance. Our analyses show that host breeding ecology strongly shapes pathogen-microbiome associations at a landscape scale, a trait that may influence resilience in the face of emerging infectious diseases., (© 2021 The Authors. Molecular Ecology published by John Wiley & Sons Ltd.)

The zoosporic obligate endoparasites, Olpidium, hold a pivotal position to the reconstruction of the flagellum loss in fungi, one of the key morphological transitions associated with the colonization of land by the early fungi. We generated genome and transcriptome data from non-axenic zoospores of Olpidium bornovanus and used a metagenome approach to extract phylogenetically informative fungal markers. Our phylogenetic reconstruction strongly supported Olpidium as the closest zoosporic relative of the non-flagellated terrestrial fungi. Super-alignment analyses resolved Olpidium as sister to the non-flagellated terrestrial fungi, whereas a super-tree approach recovered different placements of Olpidium, but without strong support. Further investigations detected little conflicting signal among the sampled markers but revealed a potential polytomy in early fungal evolution associated with the branching order among Olpidium, Zoopagomycota and Mucoromycota. The branches defining the evolutionary relationships of these lineages were characterized by short branch lengths and low phylogenetic content and received equivocal support for alternative phylogenetic hypotheses from individual markers. These nodes were marked by important morphological innovations, including the transition to hyphal growth and the loss of flagellum, which enabled early fungi to explore new niches and resulted in rapid and temporally concurrent Precambrian diversifications of the ancestors of several phyla of fungi.