Are novel or locally adapted pathogens more devastating and why? Resolving opposing hypotheses.

There is a rich literature highlighting that pathogens are generally better adapted to infect local than novel hosts, and a separate seemingly contradictory literature indicating that novel pathogens pose the greatest threat to biodiversity and public health. Here, using Batrachochytrium dendrobatidis, the fungus associated with worldwide amphibian declines, we test the hypothesis that there is enough variance in "novel" (quantified by geographic and phylogenetic distance) host-pathogen outcomes to pose substantial risk of pathogen introductions despite local adaptation being common. Our continental-scale common garden experiment and global-scale meta-analysis demonstrate that local amphibian-fungal interactions result in higher pathogen prevalence, pathogen growth, and host mortality, but novel interactions led to variable consequences with especially virulent host-pathogen combinations still occurring. Thus, while most pathogen introductions are benign, enough variance exists in novel host-pathogen outcomes that moving organisms around the planet greatly increases the chance of pathogen introductions causing profound harm.

Centrifugation is an effective and inexpensive way to determine Batrachochytrium dendrobatidis quantity in water samples with low turbidity.

Batrachochytrium dendrobatidis (Bd) is a pathogenic chytrid fungus that is particularly lethal for amphibians. Bd can extirpate amphibian populations within a few weeks and remain in water in the absence of amphibian hosts. Most efforts to determine Bd presence and quantity in the field have focused on sampling hosts, but these data do not give us a direct reflection of the amount of Bd in the water, which are useful for parameterizing disease models, and are not effective when hosts are absent or difficult to sample. Current methods for screening Bd presence and quantity in water are time, resource, and money intensive. Here, we developed a streamlined method for detecting Bd in water with low turbidity (e.g., water samples from laboratory experiments and relatively clear pond water from a natural lentic system). We centrifuged water samples with known amounts of Bd to form a pellet and extracted the DNA from that pellet. This method was highly effective and the resulting concentrations across all tested treatments presented a highly linear relationship with the expected values. While the experimentally derived values were lower than the inoculation doses, the values were highly correlated and a conversion factor allows us to extrapolate the actual Bd concentration. This centrifuge-based method is effective, repeatable, and would greatly expand the domain of tractable questions to be explored in the field of Bd ecology. Importantly, this method increases equity in the field, because it is time- and cost-efficient and requires few resources.

Pseudacris regilla metamorphs acquire resistance to Batrachochytrium dendrobatidis after exposure to the killed fungus.

The pathogenic fungus Batrachochytrium dendrobatidis (Bd) is associated with drastic global amphibian declines. Prophylactic exposure to killed zoospores and the soluble chemicals they produce (Bd metabolites) can induce acquired resistance to Bd in adult Cuban treefrogs Osteopilus septentrionalis. Here, we exposed metamorphic frogs of a second species, the Pacific chorus frog Pseudacris regilla, to one of 2 prophylactic treatments prior to live Bd exposures: killed Bd zoospores with metabolites, killed zoospores alone, or a water control. Prior exposure to killed Bd zoospores with metabolites reduced Bd infection intensity in metamorphic Pacific chorus frogs by 60.4% compared to control frogs. Interestingly, Bd intensity in metamorphs previously exposed to killed zoospores alone did not differ in magnitude relative to the control metamorphs, nor to those treated with killed zoospores plus metabolites. Previous work indicated that Bd metabolites alone can induce acquired resistance in tadpoles, and so these findings together indicate that it is possible that the soluble Bd metabolites may contain immunomodulatory components that drive this resistance phenotype. Our results expand the generality of this prophylaxis work by identifying a second amphibian species (Pacific chorus frog) and an additional amphibian life stage (metamorphic frog) that can acquire resistance to Bd after metabolite exposure. This work increases hopes that a Bd-metabolite prophylaxis might be widely effective across amphibian species and life stages.

Variability in environmental persistence but not per capita transmission rates of the amphibian chytrid fungus leads to differences in host infection prevalence.

Heterogeneities in infections among host populations may arise through differences in environmental conditions through two mechanisms. First, environmental conditions may alter host exposure to pathogens via effects on survival. Second, environmental conditions may alter host susceptibility, making infection more or less likely if contact between a host and pathogen occurs. Further, host susceptibility might be altered through acquired resistance, which hosts can develop, in some systems, through exposure to dead or decaying pathogens and their metabolites. Environmental conditions may alter the rates of pathogen decomposition, influencing the likelihood of hosts developing acquired resistance. The present study primarily tests how environmental context influences the relative contributions of pathogen survival and per capita transmission on host infection prevalence using the amphibian chytrid fungus (Batrachochytrium dendrobatidis; Bd) as a model system. Secondarily, we evaluate how environmental context influences the decomposition of Bd because previous studies have shown that dead Bd and its metabolites can illicit acquired resistance in hosts. We conducted Bd survival and infection experiments and then fit models to discern how Bd mortality, decomposition and per capita transmission rates vary among water sources [e.g. artificial spring water (ASW) or water from three ponds]. We found that infection prevalence differed among water sources, which was driven by differences in mortality rates of Bd, rather than differences in per capita transmission rates. Bd mortality rates varied among pond water treatments and were lower in ASW compared to pond water. These results suggest that variation in Bd infection dynamics could be a function of environmental factors in waterbodies that result in differences in exposure of hosts to live Bd. In contrast to the persistence of live Bd, we found that the rates of decomposition of dead Bd did not vary among water sources, which may suggest that exposure of hosts to dead Bd or its metabolites might not commonly vary among nearby sites. Ultimately, a mechanistic understanding of the environmental dependence of free-living pathogens could lead to a deeper understanding of the patterns of outbreak heterogeneity, which could inform surveillance and management strategies.

Batrachochytrium dendrobatidis (Bd) exposure damages gill tissue and inhibits crayfish respiration.

Batrachochytrium dendrobatidis (Bd) is a pathogenic fungus known to infect amphibians and crayfish. In crayfish, Bd causes gill tissue damage, and in some cases, mortality. Most research has focused on the amphibian-Bd system, so to date, little is known about the effects of Bd on the crayfish host. Here, we studied the effects of sublethal exposure to Bd and the metabolites produced by Bd on crayfish Procambarus alleni survival, gill damage, and oxygen consumption (as a proxy for mass-specific metabolic rate). Oxygen consumption increased 24 h post-exposure to live Bd, indicative of a stress response, followed by a decrease in oxygen consumption over time (χ21 = 6.39, p = 0.012). There was no difference in response when comparing the crayfish exposure to live Bd and Bd-metabolites alone (χ21 = 2.70, p = 0.101), indicating that the metabolites may have been the causative agent responsible for the response. Additionally, oxygen consumption decreased with gill damage (tissue recession) in Bd-exposed individuals. We found that high doses of Bd cause outright mortality in crayfish, and we show here that sublethal Bd-induced inhibition of oxygen consumption could negatively impact crayfish in the field, possibly reducing their overall fitness. More research is needed to understand this understudied host-parasite system. It is essential that we incorporate the disease dynamics associated with Bd and crayfish in conservation disease models, as this is the only way to develop comprehensive community-based models.

Amphibians acquire resistance to live and dead fungus overcoming fungal immunosuppression.

Emerging fungal pathogens pose a greater threat to biodiversity than any other parasitic group, causing declines of many taxa, including bats, corals, bees, snakes and amphibians. Currently, there is little evidence that wild animals can acquire resistance to these pathogens. Batrachochytrium dendrobatidis is a pathogenic fungus implicated in the recent global decline of amphibians. Here we demonstrate that three species of amphibians can acquire behavioural or immunological resistance to B. dendrobatidis. Frogs learned to avoid the fungus after just one B. dendrobatidis exposure and temperature-induced clearance. In subsequent experiments in which B. dendrobatidis avoidance was prevented, the number of previous exposures was a negative predictor of B. dendrobatidis burden on frogs and B. dendrobatidis-induced mortality, and was a positive predictor of lymphocyte abundance and proliferation. These results suggest that amphibians can acquire immunity to B. dendrobatidis that overcomes pathogen-induced immunosuppression and increases their survival. Importantly, exposure to dead fungus induced a similar magnitude of acquired resistance as exposure to live fungus. Exposure of frogs to B. dendrobatidis antigens might offer a practical way to protect pathogen-naive amphibians and facilitate the reintroduction of amphibians to locations in the wild where B. dendrobatidis persists. Moreover, given the conserved nature of vertebrate immune responses to fungi and the fact that many animals are capable of learning to avoid natural enemies, these results offer hope that other wild animal taxa threatened by invasive fungi might be rescued by management approaches based on herd immunity.

Impacts of thermal mismatches on chytrid fungus Batrachochytrium dendrobatidis prevalence are moderated by life stage, body size, elevation and latitude.

Global climate change is increasing the frequency of unpredictable weather conditions; however, it remains unclear how species-level and geographic factors, including body size and latitude, moderate impacts of unusually warm or cool temperatures on disease. Because larger and lower-latitude hosts generally have slower acclimation times than smaller and higher-latitude hosts, we hypothesised that their disease susceptibility increases under 'thermal mismatches' or differences between baseline climate and the temperature during surveying for disease. Here, we examined how thermal mismatches interact with body size, life stage, habitat, latitude, elevation, phylogeny and International Union for Conservation of Nature (IUCN) conservation status to predict infection prevalence of the chytrid fungus Batrachochytrium dendrobatidis (Bd) in a global analysis of 32 291 amphibian hosts. As hypothesised, we found that the susceptibility of larger hosts and hosts from lower latitudes to Bd was influenced by thermal mismatches. Furthermore, hosts of conservation concern were more susceptible than others following thermal mismatches, suggesting that thermal mismatches might have contributed to recent amphibian declines.

An interaction between climate change and infectious disease drove widespread amphibian declines.

Climate change might drive species declines by altering species interactions, such as host-parasite interactions. However, few studies have combined experiments, field data, and historical climate records to provide evidence that an interaction between climate change and disease caused any host declines. A recently proposed hypothesis, the thermal mismatch hypothesis, could identify host species that are vulnerable to disease under climate change because it predicts that cool- and warm-adapted hosts should be vulnerable to disease at unusually warm and cool temperatures, respectively. Here, we conduct experiments on Atelopus zeteki, a critically endangered, captively bred frog that prefers relatively cool temperatures, and show that frogs have high pathogen loads and high mortality rates only when exposed to a combination of the pathogenic chytrid fungus (Batrachochytrium dendrobatidis) and high temperatures, as predicted by the thermal mismatch hypothesis. Further, we tested various hypotheses to explain recent declines experienced by species in the amphibian genus Atelopus that are thought to be associated with B. dendrobatidis and reveal that these declines are best explained by the thermal mismatch hypothesis. As in our experiments, only the combination of rapid increases in temperature and infectious disease could account for the patterns of declines, especially in species adapted to relatively cool environments. After combining experiments on declining hosts with spatiotemporal patterns in the field, our findings are consistent with the hypothesis that widespread species declines, including possible extinctions, have been driven by an interaction between increasing temperatures and infectious disease. Moreover, our findings suggest that hosts adapted to relatively cool conditions will be most vulnerable to the combination of increases in mean temperature and emerging infectious diseases.

Metabolites produced by Batrachochytrium dendrobatidis alter development in tadpoles, but not growth or mortality.

The mass decline of amphibian populations poses a serious threat to global biodiversity and ecosystem stability. The pathogenic fungus Batrachochytrium dendrobatidis (Bd) has contributed to the extirpation and extinction of hundreds of amphibian species worldwide. Bd produces potentially damaging metabolites during the host infection process that may affect amphibian growth and development, even in the absence of infection. In this experiment, Cuban tree frog Osteopilus septentrionalis tadpoles and adults were exposed once to either artificial spring water (ASW) or Bd metabolites (n = 31 tadpoles per treatment and n = 30 and 20 adults per treatment, respectively). Tadpoles exposed to Bd metabolites alone developed faster than those exposed to ASW; however, there was no difference in tadpole length, weight change, or mortality between treatments. Despite the faster developmental speed, metabolite exposure did not reduce tadpole weight or length (compared at Gosner stages 27, 29, and 31). There was no effect of treatment on adult size or mortality. These results indicate that both tadpole and adult O. septentrionalis do not appear to be negatively impacted by exposure to non-infectious Bd-contaminated water. In fact, tadpoles developed faster when exposed to metabolites and were of equal size as those in their stage cohort, implying a potential long-term benefit if faster development allows them to leave Bd-infected waters sooner.

Biodiversity inhibits parasites: Broad evidence for the dilution effect.

Infectious diseases of humans, wildlife, and domesticated species are increasing worldwide, driving the need to understand the mechanisms that shape outbreaks. Simultaneously, human activities are drastically reducing biodiversity. These concurrent patterns have prompted repeated suggestions that biodiversity and disease are linked. For example, the dilution effect hypothesis posits that these patterns are causally related; diverse host communities inhibit the spread of parasites via several mechanisms, such as by regulating populations of susceptible hosts or interfering with parasite transmission. However, the generality of the dilution effect hypothesis remains controversial, especially for zoonotic diseases of humans. Here we provide broad evidence that host diversity inhibits parasite abundance using a meta-analysis of 202 effect sizes on 61 parasite species. The magnitude of these effects was independent of host density, study design, and type and specialization of parasites, indicating that dilution was robust across all ecological contexts examined. However, the magnitude of dilution was more closely related to the frequency, rather than density, of focal host species. Importantly, observational studies overwhelmingly documented dilution effects, and there was also significant evidence for dilution effects of zoonotic parasites of humans. Thus, dilution effects occur commonly in nature, and they may modulate human disease risk. A second analysis identified similar effects of diversity in plant-herbivore systems. Thus, although there can be exceptions, our results indicate that biodiversity generally decreases parasitism and herbivory. Consequently, anthropogenic declines in biodiversity could increase human and wildlife diseases and decrease crop and forest production.

The thermal mismatch hypothesis explains host susceptibility to an emerging infectious disease.

Parasites typically have broader thermal limits than hosts, so large performance gaps between pathogens and their cold- and warm-adapted hosts should occur at relatively warm and cold temperatures, respectively. We tested this thermal mismatch hypothesis by quantifying the temperature-dependent susceptibility of cold- and warm-adapted amphibian species to the fungal pathogen Batrachochytrium dendrobatidis (Bd) using laboratory experiments and field prevalence estimates from 15 410 individuals in 598 populations. In both the laboratory and field, we found that the greatest susceptibility of cold- and warm-adapted hosts occurred at relatively warm and cool temperatures, respectively, providing support for the thermal mismatch hypothesis. Our results suggest that as climate change shifts hosts away from their optimal temperatures, the probability of increased host susceptibility to infectious disease might increase, but the effect will depend on the host species and the direction of the climate shift. Our findings help explain the tremendous variation in species responses to Bd across climates and spatial, temporal and species-level variation in disease outbreaks associated with extreme weather events that are becoming more common with climate change.

Light and noise pollution interact to disrupt interspecific interactions.

Studies on the consequences of urbanization often examine the effects of light, noise, and heat pollution independently on isolated species providing a limited understanding of how these combined stressors affect species interactions. Here, we investigate how these factors interact to affect parasitic frog-biting midges (Corethrella spp.) and their túngara frog (Engystomops pustulosus) hosts. A survey of túngara frog calling sites revealed that frog abundance was not significantly correlated with urbanization, light, noise, or temperature. In contrast, frog-biting midges were sensitive to light pollution and noise pollution. Increased light intensity significantly reduced midge abundance at low noise levels. At high noise intensity, there were no midges regardless of light level. Two field experiments controlling light and noise levels to examine attraction of the midges to their host and their feeding behavior confirmed the causality of these field patterns. These findings demonstrate that both light and noise pollution disrupt this host-parasite interaction and highlight the importance of considering interactions among species and types of pollutants to accurately assess the impacts of urbanization on ecological communities.

A pesticide paradox: fungicides indirectly increase fungal infections.

There are many examples where the use of chemicals have had profound unintended consequences, such as fertilizers reducing crop yields (paradox of enrichment) and insecticides increasing insect pests (by reducing natural biocontrol). Recently, the application of agrochemicals, such as agricultural disinfectants and fungicides, has been explored as an approach to curb the pathogenic fungus, Batrachochytrium dendrobatidis (Bd), which is associated with worldwide amphibian declines. However, the long-term, net effects of early-life exposure to these chemicals on amphibian disease risk have not been thoroughly investigated. Using a combination of laboratory experiments and analysis of data from the literature, we explored the effects of fungicide exposure on Bd infections in two frog species. Extremely low concentrations of the fungicides azoxystrobin, chlorothalonil, and mancozeb were directly toxic to Bd in culture. However, estimated environmental concentrations of the fungicides did not reduce Bd on Cuban tree frog (Osteopilus septentrionalis) tadpoles exposed simultaneously to any of these fungicides and Bd, and fungicide exposure actually increased Bd-induced mortality. Additionally, exposure to any of these fungicides as tadpoles resulted in higher Bd abundance and greater Bd-induced mortality when challenged with Bd post-metamorphosis, an average of 71 d after their last fungicide exposure. Analysis of data from the literature revealed that previous exposure to the fungicide itraconazole, which is commonly used to clear Bd infections, made the critically endangered booroolong frog (Litoria booroolongensis) more susceptible to Bd. Finally, a field survey revealed that Bd prevalence was positively associated with concentrations of fungicides in ponds. Although fungicides show promise for controlling Bd, these results suggest that, if fungicides do not completely eliminate Bd or if Bd recolonizes, exposure to fungicides has the potential to do more harm than good. To ensure that fungicide applications have the intended consequence of curbing amphibian declines, researchers must identify which fungicides do not compromise the pathogen resistance mechanisms of amphibians.

Chytrid fungus Batrachochytrium dendrobatidis has nonamphibian hosts and releases chemicals that cause pathology in the absence of infection.

Batrachochytrium dendrobatidis, a pathogenic chytrid fungus implicated in worldwide amphibian declines, is considered an amphibian specialist. Identification of nonamphibian hosts could help explain the virulence, heterogeneous distribution, variable rates of spread, and persistence of B. dendrobatidis in freshwater ecosystems even after amphibian extirpations. Here, we test whether mosquitofish (Gambusia holbrooki) and crayfish (Procambarus spp. and Orconectes virilis), which are syntopic with many amphibian species, are possible hosts for B. dendrobatidis. Field surveys in Louisiana and Colorado revealed that zoosporangia occur within crayfish gastrointestinal tracts, that B. dendrobatidis prevalence in crayfish was up to 29%, and that crayfish presence in Colorado wetlands was a positive predictor of B. dendrobatidis infections in cooccurring amphibians. In experiments, crayfish, but not mosquitofish, became infected with B. dendrobatidis, maintained the infection for at least 12 wk, and transmitted B. dendrobatidis to amphibians. Exposure to water that previously held B. dendrobatidis also caused significant crayfish mortality and gill recession. These results indicate that there are nonamphibian hosts for B. dendrobatidis and suggest that B. dendrobatidis releases a chemical that can cause host pathology, even in the absence of infection. Managing these biological reservoirs for B. dendrobatidis and identifying this chemical might provide new hope for imperiled amphibians.

Temperature variability and moisture synergistically interact to exacerbate an epizootic disease.

Climate change is altering global patterns of precipitation and temperature variability, with implications for parasitic diseases of humans and wildlife. A recent study confirmed predictions that increased temperature variability could exacerbate disease, because of lags in host acclimation following temperature shifts. However, the generality of these host acclimation effects and the potential for them to interact with other factors have yet to be tested. Here, we report similar effects of host thermal acclimation (constant versus shifted temperatures) on chytridiomycosis in red-spotted newts (Notophthalmus viridescens). Batrachochytrium dendrobatidis (Bd) growth on newts was greater following a shift to a new temperature, relative to newts already acclimated to this temperature (15°C versus 25°C). However, these acclimation effects depended on soil moisture (10, 16 and 21% water) and were only observed at the highest moisture level, which induced greatly increased Bd growth and infection-induced mortality. Acclimation effects were also greater following a decrease rather than an increase in temperature. The results are consistent with previous findings that chytridiomycosis is associated with precipitation, lower temperatures and increased temperature variability. This study highlights host acclimation as a potentially general mediator of climate-disease interactions, and the need to account for context-dependencies when testing for acclimation effects on disease.

Batrachochytrium dendrobatidis in natural and farmed Louisiana crayfish populations: prevalence and implications.

The pathogenic chytrid fungus Batrachochytrium dendrobatidis (Bd) has been linked to global declines and extinctions of amphibians, making it one of the most devastating wildlife pathogens known. Understanding the factors that affect disease dynamics in this system is critical for mitigating infection and protecting threatened species. Crayfish are hosts of this pathogen and can transmit Bd to amphibians. Because they co-occur with susceptible amphibian communities, crayfish may be important alternative hosts for Bd. Understanding the prevalence and seasonal dynamics of crayfish infections is of agricultural and ecological interest in areas where crayfish are farmed and traded for human consumption. We conducted a survey of Bd in farmed and natural crayfish (Procambarus spp.) populations in Louisiana, USA. We found that Bd prevalence and infection intensity was low in both farmed and native populations and that prevalence varied seasonally in wild Louisiana crayfish. This seasonal pattern mirrors that seen in local amphibians. As crayfish are an important globally traded freshwater taxon, even with low prevalence, they could be an important vector in the spread of Bd.

Community ecology theory predicts the effects of agrochemical mixtures on aquatic biodiversity and ecosystem properties.

Ecosystems are often exposed to mixtures of chemical contaminants, but the scientific community lacks a theoretical framework to predict the effects of mixtures on biodiversity and ecosystem properties. We conducted a freshwater mesocosm experiment to examine the effects of pairwise agrochemical mixtures [fertiliser, herbicide (atrazine), insecticide (malathion) and fungicide (chlorothalonil)] on 24 species- and seven ecosystem-level responses. As postulated, the responses of biodiversity and ecosystem properties to agrochemicals alone and in mixtures was predictable by integrating information on each functional group's (1) sensitivity to the chemicals (direct effects), (2) reproductive rates (recovery rates), (3) interaction strength with other functional groups (indirect effects) and (4) links to ecosystem properties. These results show that community ecology theory holds promise for predicting the effects of contaminant mixtures on biodiversity and ecosystem services and yields recommendations on which types of agrochemicals to apply together and separately to reduce their impacts on aquatic ecosystems.

Early-life exposure to a herbicide has enduring effects on pathogen-induced mortality.

Exposure to stressors at formative stages in the development of wildlife and humans can have enduring effects on health. Understanding which, when and how stressors cause enduring health effects is crucial because these stressors might then be avoided or mitigated during formative stages to prevent lasting increases in disease susceptibility. Nevertheless, the impact of early-life exposure to stressors on the ability of hosts to resist and tolerate infections has yet to be thoroughly investigated. Here, we show that early-life, 6-day exposure to the herbicide atrazine (mean ± s.e.: 65.9±3.48 µg l(-1)) increased frog mortality 46 days after atrazine exposure (post-metamorphosis), but only when frogs were challenged with a chytrid fungus implicated in global amphibian declines. Previous atrazine exposure did not affect resistance of infection (fungal load). Rather, early-life exposure to atrazine altered growth and development, which resulted in exposure to chytrid at more susceptible developmental stages and sizes, and reduced tolerance of infection, elevating mortality risk at an equivalent fungal burden to frogs unexposed to atrazine. Moreover, there was no evidence of recovery from atrazine exposure. Hence, reducing early-life exposure of amphibians to atrazine could reduce lasting increases in the risk of mortality from a disease associated with worldwide amphibian declines. More generally, these findings highlight that a better understanding of how stressors cause enduring effects on disease susceptibility could facilitate disease prevention in wildlife and humans, an approach that is often more cost-effective and efficient than reactive medicine.

Nonmonotonic and monotonic effects of pesticides on the pathogenic fungus Batrachochytrium dendrobatidis in culture and on tadpoles.

Pesticides and the pathogenic fungus Batrachochytrium dendrobatidis (Bd) co-occur and are implicated in the global decline of amphibians, a highly threatened taxon. Here, we investigated the effects of ecologically relevant concentrations of chlorothalonil and atrazine, two of the most commonly used, immunomodulatory pesticides in the United States, on tadpole (Osteopilus septentrionalis) survival and Bd growth. Tadpole survival was unaffected by the pesticides but was reduced by Bd. Atrazine monotonically (i.e., consistently increasing or decreasing) reduced Bd in culture and on tadpoles, and every concentration tested (0.0106-106 µg/L) significantly reduced Bd growth compared to controls. Chlorothalonil had a nonmonotonic (i.e., nonlinear) effect on Bd growth both in culture and on tadpoles, where low (0.0176-1.76 µg/L) and high (32-176 µg/L) concentrations inhibited Bd growth significantly more than did intermediate concentrations (8.2-17.6 µg/L) and controls. To our knowledge, this is one of only a handful of studies to document a nonmonotonic dose response of a nonvertebrate (Bd) to a pesticide. Although both pesticides reduced Bd growth on frogs, neither cleared the infection entirely, and because we know little about the long-term effects of the pesticides on hosts, we do not recommend using these chemicals to control Bd.