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.

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.

Selection of an anti-pathogen skin microbiome following prophylaxis treatment in an amphibian model system.

With emerging diseases on the rise, there is an urgent need to identify and understand novel mechanisms of prophylactic protection in vertebrate hosts. Inducing resistance against emerging pathogens through prophylaxis is an ideal management strategy that may impact pathogens and their host-associated microbiome. The host microbiome is recognized as a critical component of immunity, but the effects of prophylactic inoculation on the microbiome are unknown. In this study, we investigate the effects of prophylaxis on host microbiome composition, focusing on the selection of anti-pathogenic microbes contributing to host acquired immunity in a model host-fungal disease system, amphibian chytridiomycosis. We inoculated larval Pseudacris regilla against the fungal pathogen Batrachochytrium dendrobatidis (Bd) with a Bd metabolite-based prophylactic. Increased prophylactic concentration and exposure duration were associated with significant increases in proportions of putatively Bd-inhibitory host-associated bacterial taxa, indicating a protective prophylactic-induced shift towards microbiome members that are antagonistic to Bd. Our findings are in accordance with the adaptive microbiome hypothesis, where exposure to a pathogen alters the microbiome to better cope with subsequent pathogen encounters. Our study advances research on the temporal dynamics of microbiome memory and the role of prophylaxis-induced shifts in microbiomes contributing to prophylaxis effectiveness. This article is part of the theme issue 'Amphibian immunity: stress, disease and ecoimmunology'.

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.

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.

Early-life exposure to Ivermectin alters long-term growth and disease susceptibility.

Ivermectin is a broad-spectrum antiparasitic medicine, which is often used as a treatment for parasites or as a prophylaxis. While studies have looked at the long-term effects of Ivermectin on helminths, studies have not considered the long-term impacts of this treatment on host health or disease susceptibility. Here, we tracked the effects of early life Ivermectin treatment in Cuban tree frogs (Osteopilus septentrionalis) on growth rates, mortality, metabolically expensive organ size, and susceptibility to Batrachochytrium dendrobatidis (Bd) infection. One year after exposure, there was no effect of Ivermectin exposure on frog mass (X21 = 0.904, p = 0.34), but when tracked through the exponential growth phase (~2.5 years) the Ivermectin exposed individuals had lower growth rates and were ultimately smaller (X21 = 7.78, p = 0.005; X21 = 5.36, p = 0.02, respectively). These results indicate that early life exposure is likely to have unintended impacts on organismal growth and potentially reproductive fitness. Additionally, we exposed frogs to Bd, a pathogenic fungus that has decimated amphibian populations globally, and found early life exposure to Ivermectin decreased disease susceptibility (disease load: X21 = 17.57, p = 0.0002) and prevalence (control: 55%; Ivermectin: 22%) over 2 years after exposure. More research is needed to understand the underlying mechanism behind this phenomenon. Given that Ivermectin exposure altered disease susceptibility, proper controls should be implemented when utilizing this drug as an antiparasitic treatment in research studies.

A meta-analysis reveals temperature, dose, life stage, and taxonomy influence host susceptibility to a fungal parasite.

Complex ecological relationships, such as host-parasite interactions, are often modeled with laboratory experiments. However, some experimental laboratory conditions, such as temperature or infection dose, are regularly chosen based on convenience or convention, and it is unclear how these decisions systematically affect experimental outcomes. Here, we conducted a meta-analysis of 58 laboratory studies that exposed amphibians to the pathogenic fungus Batrachochytrium dendrobatidis (Bd) to understand better how laboratory temperature, host life stage, infection dose, and host species affect host mortality. We found that host mortality was driven by thermal mismatches: hosts native to cooler environments experienced greater Bd-induced mortality at relatively warm experimental temperatures and vice versa. We also found that Bd dose positively predicted Bd-induced host mortality and that the superfamilies Bufonoidea and Hyloidea were especially susceptible to Bd. Finally, the effect of Bd on host mortality varied across host life stages, with larval amphibians experiencing lower risk of Bd-induced mortality than adults or metamorphs. Metamorphs were especially susceptible and experienced mortality when inoculated with much smaller Bd doses than the average dose used by researchers. Our results suggest that when designing experiments on species interactions, researchers should carefully consider the experimental temperature, inoculum dose, and life stage, and taxonomy of the host species.

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.

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.

Agrochemicals increase risk of human schistosomiasis by supporting higher densities of intermediate hosts.

Schistosomiasis is a snail-borne parasitic disease that ranks among the most important water-based diseases of humans in developing countries. Increased prevalence and spread of human schistosomiasis to non-endemic areas has been consistently linked with water resource management related to agricultural expansion. However, the role of agrochemical pollution in human schistosome transmission remains unexplored, despite strong evidence of agrochemicals increasing snail-borne diseases of wildlife and a projected 2- to 5-fold increase in global agrochemical use by 2050. Using a field mesocosm experiment, we show that environmentally relevant concentrations of fertilizer, a herbicide, and an insecticide, individually and as mixtures, increase densities of schistosome-infected snails by increasing the algae snails eat and decreasing densities of snail predators. Epidemiological models indicate that these agrochemical effects can increase transmission of schistosomes. Identifying agricultural practices or agrochemicals that minimize disease risk will be critical to meeting growing food demands while improving human wellbeing.

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.

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.

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.

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.

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.

Transition of chytrid fungus infection from mouthparts to hind limbs during amphibian metamorphosis.

The chytrid fungus, Batrachochytrium dendrobatidis (Bd), is implicated in worldwide amphibian declines. Bd has been shown to qualitatively transition from the mouthparts of tadpoles to the hindlimbs during metamorphosis, but we lack evidence of consistency in the timing of this transition across amphibian species. We also do not have predictive functions for the abundance of Bd in mouthparts and limbs as tadpoles develop or for the relationship between keratin and Bd abundance. Hence, researchers presently have little guidance on where to sample developing amphibians to maximize Bd detection, which could affect the accuracy of prevalence and abundance estimates for this deadly pathogen. Here, we show consistency in the timing of the transition of Bd from mouthparts to hind limbs across two frog species (Osteopilus septentrionalis and Mixophyes fasciolatus). Keratin and Bd simultaneously declined from the mouthparts starting at approximately Gosner stage 40. However, keratin on the hindlimbs began to appear at approximately stage 38 but, on average, Bd was not detectable on the hindlimbs until approximately stage 40, suggesting a lag between keratin and Bd arrival. Predictive functions for the relationships between developmental stage and keratin and developmental stage and Bd for mouthparts and hind limbs are provided so that researchers can optimize sampling designs and minimize erroneous conclusions associated with missing Bd infections or misestimating Bd abundance.