Neal T. Halstead

The Fungicide Chlorothalonil Is Nonlinearly Associated with Corticosterone Levels, Immunity, and Mortality in Amphibians

Background: Contaminants have been implicated in declines of amphibians, a taxon with vital systems similar to those of humans. However, many chemicals have not been thoroughly tested on amphibians or do not directly kill them. Objective: Our goal in this study was to quantify amphibian responses to chlorothalonil, the most commonly used synthetic fungicide in the United States. Methods: We reared Rana sphenocephala (southern leopard frog) and Osteopilus septentrionalis (Cuban treefrog) in outdoor mesocosms with or without 1 time (1×) and 2 times (2×) the expected environmental concentration (EEC) of chlorothalonil (~ 164 μg/L). We also conducted two dose–response experiments on O. septentrionalis, Hyla squirella (squirrel treefrog), Hyla cinerea (green treefrog), and R. sphenocephala and evaluated the effects of chlorothalonil on the stress hormone corticosterone. Results: For both species in the mesocosm experiment, the 1× and 2× EEC treatments were associated with > 87% and 100% mortality, respectively. In the laboratory experiments, the approximate EEC caused 100% mortality of all species within 24 hr; 82 μg/L killed 100% of R. sphenocephala, and 0.0164 μg/L caused significant tadpole mortality of R. sphenocephala and H. cinerea. Three species 
showed a nonmonotonic dose response, with low and high concentrations causing significantly greater mortality than did intermediate concentrations or control treatments. For O. septentrionalis, corticosterone exhibited a similar nonmonotonic dose response and chlorothalonil concentration was inversely associated with liver tissue and immune cell densities (< 16.4 μg/L). Conclusions: Chlorothalonil killed nearly every amphibian at the approximate EEC; at concentrations to which humans are commonly exposed, it increased mortality and was associated with elevated corticosterone levels and changes in immune cells. Future studies should directly quantify the effects of chlorothalonil on amphibian populations and human health.

Agrochemicals increase trematode infections in a declining amphibian species

Global amphibian declines have often been attributed to disease, but ignorance of the relative importance and mode of action of potential drivers of infection has made it difficult to develop effective remediation. In a field study, here we show that the widely used herbicide, atrazine, was the best predictor (out of more than 240 plausible candidates) of the abundance of larval trematodes (parasitic flatworms) in the declining northern leopard frog Rana pipiens. The effects of atrazine were consistent across trematode taxa. The combination of atrazine and phosphate—principal agrochemicals in global corn and sorghum production—accounted for 74% of the variation in the abundance of these often debilitating larval trematodes (atrazine alone accounted for 51%). Analysis of field data supported a causal mechanism whereby both agrochemicals increase exposure and susceptibility to larval trematodes by augmenting snail intermediate hosts and suppressing amphibian immunity. A mesocosm experiment demonstrated that, relative to control tanks, atrazine tanks had immunosuppressed tadpoles, had significantly more attached algae and snails, and had tadpoles with elevated trematode loads, further supporting a causal relationship between atrazine and elevated trematode infections in amphibians. These results raise concerns about the role of atrazine and phosphate in amphibian declines, and illustrate the value of quantifying the relative importance of several possible drivers of disease risk while determining the mechanisms by which they facilitate disease emergence.

Biodiversity inhibits parasites: Broad evidence for the dilution effect

Significance The dilution effect hypothesis suggests that diverse ecological communities limit disease spread via several mechanisms. Therefore, biodiversity losses could worsen epidemics that harm humans and wildlife. However, there is contentious debate over whether the hypothesis applies broadly, especially for parasites that infect humans. We address this fundamental question with a formal meta-analysis of >200 assessments relating biodiversity to disease in >60 host–parasite systems. We find overwhelming evidence of dilution, which is independent of host density, study design, and type and specialization of parasites. A second analysis identified similar effects of diversity in plant–herbivore systems. Thus, biodiversity generally decreases parasitism and herbivory. Consequently, human-induced declines in biodiversity could increase human and wildlife diseases and decrease crop and forest production. 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.

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.

Parasitism in a community context: trait-mediated interactions with competition and predation.

Predation and competition can induce important density- and trait-mediated effects on species, with implications for community stability. However, interactions of these factors with parasitism remain understudied. Here we investigate interactions among competition, predation and parasitism by crossing tadpole density (Bufo americanus), presence of a caged predator (Notophthalmus viridescens), and Echinostoma trivolvis trematodes, experimentally partitioning their effects on tadpole exposure and susceptibility to infection. Predation did not affect E. trivolvis infection but accelerated tadpole development and growth, and decreased activity. The presence of E. trivolvis caused the opposite effects on these three responses and reduced tadpole survival. High conspecific density reduced tadpole survival, growth, and development, and increased tadpole activity. Effects of predation and parasitism on activity were only evident at low tadpole density. High-density mesocosms also had twice the number of E. trivolvis infections as low-density mesocosms, despite a lack of evidence for stress-induced immunomodulation. Instead, this effect was explained by high density delaying tadpole development, which increased both the duration of exposure to cercariae and susceptibility to infection, because tadpoles spent more time in highly susceptible early stages. These results highlight the importance of accounting for trait-mediated effects, host plasticity, and exposure vs. susceptibility in parasite ecology.

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 groups (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.

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.

Predator diversity, intraguild predation, and indirect effects drive parasite transmission

Significance Humans are altering biodiversity globally and infectious diseases are on the rise; thus, there is considerable interest in understanding how changes to biodiversity affect disease risk. We show that the diversity of predators that consume parasites was the best negative predictor of infections in frogs, suggesting that predation on parasites can be an important mechanism of disease reduction. Follow-up experiments, field data, and mathematical models revealed that intraguild predators, predators that consume both hosts and parasites, decrease macroparasite infections per host less than predators that only consume parasites, representing a general trait of predators that predicts their ability to reduce disease. Consequently, managing assemblages of non-intraguild and intraguild predators is an underutilized tool to minimize human and wildlife diseases. Humans are altering biodiversity globally and infectious diseases are on the rise; thus, there is interest in understanding how changes to biodiversity affect disease. Here, we explore how predator diversity shapes parasite transmission. In a mesocosm experiment that manipulated predator (larval dragonflies and damselflies) density and diversity, non-intraguild (non-IG) predators that only consume free-living cercariae (parasitic trematodes) reduced metacercarial infections in tadpoles, whereas intraguild (IG) predators that consume both parasites and tadpole hosts did not. This likely occurred because IG predators reduced tadpole densities and anticercarial behaviors, increasing per capita exposure rates of the surviving tadpoles (i.e., via density- and trait-mediated effects) despite the consumption of parasites. A mathematical model demonstrated that non-IG predators reduce macroparasite infections, but IG predation weakens this “dilution effect” and can even amplify parasite burdens. Consistent with the experiment and model, a wetland survey revealed that the diversity of IG predators was unrelated to metacercarial burdens in amphibians, but the diversity of non-IG predators was negatively correlated with infections. These results are strikingly similar to generalities that have emerged from the predator diversity–pest biocontrol literature, suggesting that there may be general mechanisms for pest control and that biocontrol research might inform disease management and vice versa. In summary, we identified a general trait of predators—where they fall on an IG predation continuum—that predicts their ability to reduce infections and possibly pests in general. Consequently, managing assemblages of predators represents an underused tool for the management of human and wildlife diseases and pest populations.

Comparative toxicities of organophosphate and pyrethroid insecticides to aquatic macroarthropods.

As agricultural expansion and intensification increase to meet the growing global food demand, so too will insecticide use and thus the risk of non-target effects. Insecticide pollution poses a particular threat to aquatic macroarthropods, which play important functional roles in freshwater ecosystems. Thus, understanding the relative toxicities of insecticides to non-target functional groups is critical for predicting effects on ecosystem functions. We exposed two common macroarthropod predators, the crayfish Procambarus alleni and the water bug Belostoma flumineum, to three insecticides in each of two insecticide classes (three organophosphates: chlorpyrifos, malathion, and terbufos; and three pyrethroids: esfenvalerate, λ-cyhalothrin, and permethrin) to assess their toxicities. We generated 150 simulated environmental exposures using the US EPA Surface Water Contamination Calculator to determine the proportion of estimated peak environmental concentrations (EECs) that exceeded the US EPA level of concern (0.5×LC50) for non-endangered aquatic invertebrates. Organophosphate insecticides generated consistently low-risk exposure scenarios (EECs<0.5×LC50) for both P. alleni and B. flumineum. Pyrethroid exposure scenarios presented consistently high risk (EECs>0.5×LC50) to P. alleni, but not to B. flumineum, where only λ-cyhalothrin produced consistently high-risk exposures. Survival analyses demonstrated that insecticide class accounted for 55.7% and 91.1% of explained variance in P. alleni and B. flumineum survival, respectively. Thus, risk to non-target organisms is well predicted by pesticide class. Identifying insecticides that pose low risk to aquatic macroarthropods might help meet increased demands for food while mitigating against potential negative effects on ecosystem functions.