Chemical disinfection as a simple and reliable method to control the amphibian chytrid fungus at breeding points of endangered amphibians.

Chytridiomycosis caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd) is pushing amphibians towards extinction. Whilst mitigation methods were suggested a decade ago, we lack field trials testing their efficacy. We used the agrochemical fungicide, tebuconazole, to treat Bd infected breeding waterbodies of an endangered species that is highly susceptible to the fungus. Just two applications of tebuconazole led to a significant reduction in infection loads in the vast majority of sites, and at six sites the disinfection remained one/two-years post-application. Tebuconazole values drastically decreased in the waterbodies within a week after application, with no significant effects on their hydrochemical and hydrobiological characteristics. Although the use of chemicals in natural populations is undesirable, the growing existential threat to amphibians all over the world indicates that effective interventions in selected populations of endangered species are urgently needed.

Field-based molecular detection of Batrachochytrium dendrobatidis in critically endangered Atelopus toads and aquatic habitats in Ecuador.

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.

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

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

Batrachochytrium dendrobatidis strain affects transcriptomic response in liver but not skin in latitudinal populations of the common toad (Bufo bufo).

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.

FIRST RECORD OF BATRACHOCHYTRIUM DENDROBATIDIS IN THE NORTHERN NETHERLANDS.

Batrachochytrium dendrobatidis (Bd) infects amphibians and has been linked to the decline of hundreds of anuran amphibians all over the world. In the province of Groningen in the Netherlands, this fungal pathogen was not detected before this study. To determine whether Groningen was Bd-free, we surveyed 12 locations in this province in 2020 and 2021. Samples were then used to quantify the presence of Bd with a qPCR assay. In total, 2 out of 110 (∼0.02%) collected in 2020 and 11 out of 249 samples collected in 2021 tested positive for Bd. Infected amphibians were found in 4 out of the 12 sites, and the prevalence of Bd was estimated at 4% for both years combined. Our study provides the first record of Bd in Groningen, and we hypothesize that Bd is present throughout the Netherlands in regions currently considered "Bd-free." Furthermore, we warn scientists and policymakers to be apprehensive when calling a site free from Bd when sampling is limited or not recent.

The Binding, Infection, and Promoted Growth of Batrachochytrium dendrobatidis by the Ranavirus FV3.

Increasing reports suggest the occurrence of co-infection between Ranaviruses such as Frog Virus 3 (FV3) and the chytrid fungus Batrachochytrium dendrobatidis (Bd) in various amphibian species. However, the potential direct interaction of these two pathogens has not been examined to date. In this study, we investigated whether FV3 can interact with Bd in vitro using qPCR, conventional microscopy, and immunofluorescent microscopy. Our results reveal the unexpected ability of FV3 to bind, promote aggregation, productively infect, and significantly increase Bd growth in vitro. To extend these results in vivo, we assessed the impact of FV3 on Xenopus tropicalis frogs previously infected with Bd. Consistent with in vitro results, FV3 exposure to previously Bd-infected X. tropicalis significantly increased Bd loads and decreased the host's survival.

eDNA-based monitoring of Batrachochytrium dendrobatidis and Batrachochytrium salamandrivorans with ddPCR in Luxembourg ponds: taking signals below the Limit of Detection (LOD) into account.

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.

Genetic transformation of the frog-killing chytrid fungus Batrachochytrium dendrobatidis.

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.

The role of Culex territans mosquitoes in the transmission of Batrachochytrium dendrobatidis to amphibian hosts.

BACKGROUND: Mosquitoes are the deadliest organisms in the world, killing an estimated 750,000 people per year due to the pathogens they can transmit. Mosquitoes also pose a major threat to other vertebrate animals. Culex territans is a mosquito species found in temperate zones worldwide that feeds almost exclusively on amphibians and can transmit parasites; however, little is known about its ability to transmit other pathogens, including fungi. Batrachochytrium dendrobatidis (Bd) is a topical pathogenic fungus that spreads through contact. With amphibian populations around the world experiencing mass die-offs and extinctions due to this pathogen, it is critical to study all potential modes of transmission. Because Cx. territans mosquitoes are in contact with their hosts for long periods of time while blood-feeding, we hypothesize that they can transmit and pick up Bd. METHODS: In this study, we first assessed Cx. territans ability to transfer the fungus from an infected surface to a clean one under laboratory conditions. We also conducted a surveillance study of Bd infections in frogs and mosquitoes in the field (Mountain Lake Biological station, VA, USA). In parallel, we determined Cx. territans host preference via blood meal analysis of field caught mosquitoes. RESULTS: We found that this mosquito species can carry the fungus to an uninfected surface, implying that they may have the ability to transmit Bd to their amphibian hosts. We also found that Cx. territans feed primarily on green frogs (Rana clamitans) and bullfrogs (Rana catesbeiana) and that the prevalence of Bd within the frog population at our field site varied between years. CONCLUSIONS: This study provides critical insights into understanding the role of amphibian-biting mosquitoes in transmitting pathogens, which can be applied to disease ecology of susceptible amphibian populations worldwide.

Amphibian Hymenochirus boettgeri as an experimental model for infection studies with the chytrid fungus Batrachochytrium dendrobatidis.

Model organisms are crucial in research as they can provide key insights applicable to other species. This study proposes the use of the amphibian species Hymenochirus boettgeri, widely available through the aquarium trade, as a model organism for the study of chytridiomycosis, a disease caused by the fungus Batrachochytrium dendrobatidis (Bd) and linked to amphibian decline and extinction globally. Currently, no model organisms are used in the study of chytridiomycosis, particularly because of the lack of availability and nonstandardized methods. Thus, laboratories around the world use wild local species to conduct Bd infection experiments, which prevents comparisons between studies and reduces reproducibility. Here, we performed a series of Bd infection assays that showed that H. boettgeri has a dose- and genotype-dependent response, can generalize previous findings on virulence estimates in other species, and can generate reproducible results in replicated experimental conditions. We also provided valuable information regarding H. boettgeri husbandry, including care, housing, reproduction, and heat treatment to eliminate previous Bd infections. Together, our results indicate that H. boettgeri is a powerful and low-ecological-impact system for studying Bd pathogenicity and virulence.

Comparison of methods for determining Batrachochytrium dendrobatidis zoospore viability.

The emerging fungal pathogen Batrachochytrium dendrobatidis (Bd) threatens hundreds of amphibian species globally. During laboratory-based experiments it is often essential to quantify live Bd cells, but a comparison of the effectiveness of methods for counting and assessing the viability of the infectious zoospore life stage has not been done. A direct comparison of staining methods that assess viability will ensure that the most accurate and efficient method is used. Here, we compared the use of 2 relatively cheap common stains, trypan blue and methylene blue, and assessed their accuracy and precision for estimating the viability of Bd zoospores during both manual counting and colorimetric assays. We stained known proportions of killed Bd zoospores (0, 0.25, 0.50, 0.75, and 1.00) with each stain and estimated the proportion of stained (dead) and unstained (viable) cells in each sample using both manual counting and colorimetric assays. Trypan blue was found to be a much more effective stain than methylene blue for both microscopy and colorimetric assays. Additionally, counting zoospores via microscopy was both a more accurate and precise technique. We recommend using manual counts via microscopy using the trypan blue stain for assessing Bd zoospore viability.

Divergent population responses following salamander mass mortalities and declines driven by the emerging pathogen Batrachochytrium salamandrivorans.

Understanding wildlife responses to novel threats is vital in counteracting biodiversity loss. The emerging pathogen Batrachochytrium salamandrivorans (Bsal) causes dramatic declines in European salamander populations, and is considered an imminent threat to global amphibian biodiversity. However, real-life disease outcomes remain largely uncharacterized. We performed a multidisciplinary assessment of the longer-term impacts of Bsal on highly susceptible fire salamander (Salamandra salamandra) populations, by comparing four of the earliest known outbreak sites to uninfected sites. Based on large-scale monitoring efforts, we found population persistence in strongly reduced abundances to over a decade after Bsal invasion, but also the extinction of an initially small-sized population. In turn, we found that host responses varied, and Bsal detection remained low, within surviving populations. Demographic analyses indicated an ongoing scarcity of large reproductive adults with potential for recruitment failure, while spatial comparisons indicated a population remnant persisting within aberrant habitat. Additionally, we detected no early signs of severe genetic deterioration, yet nor of increased host resistance. Beyond offering additional context to Bsal-driven salamander declines, results highlight how the impacts of emerging hypervirulent pathogens can be unpredictable and vary across different levels of biological complexity, and how limited pathogen detectability after population declines may complicate surveillance efforts.

OCCURRENCE OF PATHOGENIC CHYTRID FUNGI BATRACHOCHYTRIUM SALAMANDRIVORANS AND BATRACHOCHYTRIUM DENDROBATIDIS IN THE HONG KONG NEWT (PARAMESOTRITON HONGKONGENSIS) AND OTHER WILD AND IMPORTED AMPHIBIANS IN A SUBTROPICAL ASIAN REGION.

One of the major threats for the massive loss in global amphibian diversity is chytridiomycosis, caused by chytrid fungi Batrachochytrium dendrobatidis (Bd) and B. salamandrivorans (Bsal). Following its discovery in 2013, Bsal has emerged as a severe threat to the global survival of urodelans. In 2018, a study reported a high prevalence of Bsal (65.6%) in the Hong Kong newts (Paramesotriton hongkongensis, Near Threatened) of a southern China population adjacent to Hong Kong (HK). Uncertainty regarding the Bsal infection status of P. hongkongensis inhabiting HK raised deep concern over the risk of introducing Bsal from that population. We screened the skin swabs from wild individuals of P. hongkongensis, 15 sympatric amphibian species, and 16 imported amphibian species in HK for chytrids. We found that both Bsal and Bd occur in low prevalences in P. hongkongensis (Bsal 1.7%, 5/293; Bd 0.34%, 1/293), Hong Kong cascade frog, Amolops hongkongensis, family Ranidae (Bsal only, 5.26%, 1/19), and Asian common toad, Duttaphrynus melanostictus, family Bufonidae (Bsal only, 5.88%, 1/17), populations of HK, with infected individuals being asymptomatic, suggesting a potential role of these species as reservoirs of Bsal. Conversely, Bd, but not Bsal, was present on 13.2% (9/68) of imported amphibians, indicating a high chytrid introduction risk posed by international amphibian trade. Long-term surveillance of the presence of Bd and Bsal in wild and captive amphibians would be advisable, and we recommend that import and export of nonnative chytrid carriers should be prevented, especially to those regions with amphibian populations naïve to Bd and Bsal.

Comparative genomics of bacteria from amphibian skin associated with inhibition of an amphibian fungal pathogen, Batrachochytrium dendrobatidis.

Chytridiomycosis, caused by Batrachochytrium dendrobatidis (Bd), is a skin disease associated with worldwide amphibian declines. Symbiotic microbes living on amphibian skin interact with Bd and may alter infection outcomes. We completed whole genome sequencing of 40 bacterial isolates cultured from the skin of four amphibian species in the Eastern US. Each isolate was tested in vitro for the ability to inhibit Bd growth. The aim of this study was to identify genomic differences among the isolates and generate hypotheses about the genomic underpinnings of Bd growth inhibition. We identified sixty-five gene families that were present in all 40 isolates. Screening for common biosynthetic gene clusters revealed that this set of isolates contained a wide variety of clusters; the two most abundant clusters with potential antifungal activity were siderophores (N=17 isolates) and Type III polyketide synthases (N=22 isolates). We then examined various subsets of the 22 isolates in the phylum Proteobacteria for genes encoding specific compounds that may inhibit fungal growth, including chitinase and violacein. We identified differences in Agrobacterium and Sphingomonas isolates in the chitinase genes that showed some association with anti-Bd activity, as well as variation in the violacein genes in the Janthinobacterium isolates. Using a comparative genomics approach, we generated several testable hypotheses about differences among bacterial isolates from amphibian skin communities that could contribute to variation in the ability to inhibit Bd growth. Further work is necessary to explore and uncover the various mechanisms utilized by amphibian skin bacterial isolates to inhibit Bd.

KEEPING THE HEAT ON: WEIGHTED SURVEILLANCE FOR CHYTRID FUNGUS (BATRACHOCHYTRIUM DENDROBATIDIS) IN DIXIE VALLEY TOADS (ANAXYRUS [= BUFO] WILLIAMSI).

Introduced fungal pathogens have caused declines and extinctions of naïve wildlife populations across vertebrate classes. Consequences of introduced pathogens to hosts with small ranges might be especially severe because of limited redundancy to rescue populations and lower abundance that may limit the resilience of populations to perturbations like disease introduction. As a complement to biosecurity measures to prevent the spread of pathogens, surveillance programs may enable early detection of pathogens, when management actions to limit the effects of pathogens on naïve hosts might be most beneficial. We analyzed surveillance data for the endangered and narrowly endemic Dixie Valley toad (Anaxyrus [= Bufo] williamsi) from two time periods (2011-2014 and 2019-2021) to estimate the minimum detectable prevalence of the amphibian fungal pathogen Batrachochytrium dendrobatidis (Bd). We assessed if detection efficiency could be improved by using samples from both Dixie Valley toads and co-occurring introduced American bullfrogs (Lithobates catesbeianus) and literature-derived surveillance weights. We further evaluated a weighted surveillance design to increase the efficiency of surveillance efforts for Bd within the toad's small (<6 km2) range. We found that monitoring adult and larval American bullfrogs would probably detect Bd more efficiently than monitoring Dixie Valley toads alone. Given that no Bd was detected, minimum detectable prevalence of Bd was <3% in 2011-2014, and <5% (Dixie Valley toads only) and <10% (American bullfrogs only) in 2019-2021. Optimal management for Bd depends on the mechanisms underlying its apparent absence from the range of Dixie Valley toads, but a balanced surveillance scheme that includes sampling American bullfrogs to increase the likelihood of detecting Bd, and adult Dixie Valley toads to ensure broad spatial coverage where American bullfrogs do not occur, would probably result in efficient surveillance, which might permit timely management of Bd if it is detected.

The two chytrid pathogens of amphibians in Eurasia-climatic niches and future expansion.

BACKGROUND: Climate affects the thermal adaptation and distribution of hosts, and drives the spread of Chytridiomycosis-a keratin-associated infectious disease of amphibians caused by the sister pathogens Batrachochytrium dendrobatidi (Bd) and B. salamandrivorans (Bsal). We focus on their climate-pathogen relationships in Eurasia, the only region where their geographical distributions overlap. Eurasia harbours invaded and native areas of both pathogens and the natural habitats where they co-exist, making it an ideal region to examine their environmental niche correlations. Our understanding of how climate change will affect their distribution is broadened by the differences in climate correlates and niche characteristics between Bd and Bsal in Asia and Europe. This knowledge has potential conservation implications, informing future spread of the disease in different regions. RESULTS: We quantified the environmental niche overlap between Bd and Bsal in Eurasia using niche analyses. Results revealed partial overlap in the niche with a unique 4% of non-overlapping values for Bsal, suggesting segregation along certain climate axes. Bd tolerates higher temperature fluctuations, while Bsal requires more stable, lower temperature and wetter conditions. Projections of their Realized Climatic Niches (RCNs) to future conditions show a larger expansion of suitable ranges (SRs) for Bd compared to Bsal in both Asia and Europe, with their centroids shifting in different directions. Notably, both pathogens' highly suitable areas in Asia are expected to shrink significantly, especially under the extreme climate scenarios. In Europe, they are expected to expand significantly. CONCLUSIONS: Climate change will impact or increase disease risk to amphibian hosts, particularly in Europe. Given the shared niche space of the two pathogens across available climate gradients, as has already been witnessed in Eurasia with an increased range expansion and niche overlap due to climate change, we expect that regions where Bsal is currently absent but salamanders are present, and where Bd is already prevalent, may be conducive for the spread of Bsal.

Drivers of Batrachochytrium dendrobatidis infection load, with evidence of infection tolerance in adult male toads (Bufo spinosus).

Chytridiomycosis is affecting hundreds of amphibian species worldwide, but while in tropical areas, adult individuals have been the focus of most investigations, the exact role played by infection intensity of breeding adults is not well understood in temperate areas. We conducted mark-recapture-capture surveys during spiny common toad breeding seasons from 2006 to 2018 at the site of the first recorded outbreak of chytridiomycosis in Europe, the Peñalara Massif (Sierra de Guadarrama National Park, central Spain), and collected infection samples and several variables related to the reproductive effort of male individuals. We used general linear mixed models to evaluate the contribution of study variables on the infection loads of adult male toads exhibited at their capturing date. We also analysed the differences on several male characteristics between the pond with the largest breeding population against the rest of the ponds. We found that the duration of time spent in the waterbody and the condition of the host predicted infection loads. Animals of good physical condition, that spent longer in water, have higher infection levels than individuals with the opposite set of traits. The pond supporting the largest breeding population housed smaller male toads and in poorer condition. Our results are consistent with a shift in reproductive strategy in response to infection and potentially a strategy of tolerance, rather than resistance to infection. These findings have applications for disease mitigation and theoretical implications related to the trade-offs made and the evolution of traits in response to the disease.

Water Pollution Increases the Risk of Chytridiomycosis in Mexican Amphibians.

Chytridiomycosis is affecting amphibians worldwide, causing the decline and extinction of several amphibian populations. The disease is caused by the fungus Batrachochytrium dendrobatidis (Bd), a multihost pathogen living in freshwater habitats. While several environmental factors have been associated with the prevalence of Bd and its virulence, the effects of water quality on the pathogen are not clear yet. Some evidence suggests that water pollution may reduce amphibians' immune response and increase prevalence of Bd. To explore this hypothesis, we analyzed the relationship between water quality and the presence of Bd by using spatial data mining of 150 geolocations of Bd in amphibians from 9 families where Bd positive specimens have been previously reported, and water quality in 4,202 lentic and lotic water bodies in Mexico from 2010 to 2021. Our model showed that in the 3 main families where Bd was recorded, its presence is high in locations with low water quality, i.e., water polluted likely contaminated with urban and industrial waste. Using this model, we inferred areas suitable for Bd in Mexico; mainly in poorly studied areas along the gulf and on the pacific slope. We further argue that actions to reduce water pollution should become an integral part of public policies to prevent the spread of Bd and protect amphibians from this deadly pathogen.

Effects of Herbicides and the Chytrid Fungus Batrachochytrium dendrobatidis on the growth, development and survival of Larval American Toads (Anaxyrus americanus).

Pesticides and pathogens adversely affect amphibian health, but their interactive effects are not well known. We assessed independent and combined effects of two agricultural herbicides and the fungal pathogen Batrachochytrium dendrobatidis (Bd) on the growth, development and survival of larval American toads (Anaxyrus americanus). Wild-caught tadpoles were exposed to four concentrations of atrazine (0.18, 1.8, 18.0, 180 µg/L) or glyphosate (7, 70, 700, 7000 µg a.e./L), respectively contained in Aatrex® Liquid 480 (Syngenta) or Vision® Silviculture Herbicide (Monsanto) for 14 days, followed by two doses of Bd. At day 14, atrazine had not affected survival, but it non-monotonically affected growth. Exposure to the highest concentration of glyphosate caused 100% mortality within 4 days, while lower doses had an increasing monotonic effect on growth. At day 65, tadpole survival was unaffected by atrazine and the lower doses of glyphosate. Neither herbicide demonstrated an interaction effect with Bd on survival, but exposure to Bd increased survival among both herbicide-exposed and herbicide-control tadpoles. At day 60, tadpoles exposed to the highest concentration of atrazine remained smaller than controls, indicating longer-term effects of atrazine on growth, but effects of glyphosate on growth disappeared. Growth was unaffected by any herbicide-fungal interaction but was positively affected by exposure to Bd following exposure to atrazine. Atrazine exhibited a slowing and non-monotonic effect on Gosner developmental stage, while exposure to Bd tended to speed up development and act antagonistically toward the observed effect of atrazine. Overall, atrazine, glyphosate and Bd all showed a potential to modulate larval toad growth and development.

FUNGAL METABOLITES PROVIDE PRE-EXPOSURE PROTECTION BUT NO POSTEXPOSURE BENEFIT OR HARM AGAINST BATRACHOCHYTRIUM DENDROBATIDIS.

Disease control tools are needed to mitigate the effect of the fungal pathogen Batrachochytrium dendrobatidis (Bd) on amphibian biodiversity loss. In previous experiments, Bd metabolites (i.e., noninfectious chemicals released by Bd) have been shown to induce partial resistance to Bd when administered before live pathogen exposure and therefore have potential as an intervention strategy to curb Bd outbreaks. In the wild, however, amphibians inhabiting Bd-endemic ecosystems may have already been exposed to or infected with Bd before metabolite administration. It is therefore critical to evaluate the efficacy and safety of Bd metabolites applied postexposure to live Bd. We tested whether Bd metabolites administered postexposure would induce resistance, exacerbate infections, or have no effect. The results confirmed that Bd metabolites applied before pathogen exposure significantly reduced infection intensity, but Bd metabolites applied after pathogen exposure neither protected against nor exacerbated infections. These results reveal the importance of timing the application of Bd metabolites early in the transmission season for Bd-endemic ecosystems and emphasize that Bd metabolites prophylaxis may be a useful tool in captive reintroduction campaigns where Bd threatens the success of re-establishing endangered amphibian populations.